Advances in Botanical Research, Volume 46

Nitric Oxide and Plant Growth Promoting Rhizobacteria: Common Features Influencing Root Growth and Development

´ NICA CREUS,* CELESTE MOLINA‐FAVERO,* CECILIA MO { ´ MARIA LUCIANA LANTERI, NATALIA CORREA‐ARAGUNDE,{ MARI´A CRISTINA LOMBARDO,{,{ CARLOS ALBERTO BARASSI* AND LORENZO LAMATTINA{

*Unidad Integrada Balcarce, INTA‐Facultad de Cs. Agrarias, Universidad Nacional de Mar del Plata, CC 276, 7620 Balcarce, Argentina { Instituto de Investigaciones Biolo´gicas, Universidad Nacional de Mar del Plata, CC 1245, 7600 Mar del Plata, Argentina { Departamento de Biologı´a, Universidad Nacional de Mar del Plata, CC 1245, 7600 Mar del Plata, Argentina

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . II. NO Is a Regulator of Root Growth and Developmental Processes . . . . . . . . A. NO Induces Adventitious Root Formation ................................ B. NO and Lateral Root Development: NO Is Downstream Auxin in Triggering LRD ............................ C. General Features Associated to Root Hair Formation................... D. The Effects of PGPR on Root Architecture ............................... III. Perspectives. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Advances in Botanical Research, Vol. 46 Incorporating Advances in Plant Pathology Copyright 2008, Elsevier Ltd. All rights reserved.

2 3 3 5 6 10 21 23 24

0065-2296/08 $35.00 DOI: 10.1016/S0065-2296(07)46001-3

2

C. MOLINA‐FAVERO ET AL.

ABSTRACT Nitric oxide (NO) is a gas produced by prokaryotes and eukaryotes as part of their N metabolism that profoundly influences the physiology of the cells. In plants, the biological implications of NO as a signal molecule modulating physiological responses have been elucidated in the last decade. The NO action as an intermediary in auxin‐ regulated signaling cascades influencing root growth and developmental processes is probably one of the most important functions in plant biology. Here we describe the signaling pathways and the cellular messengers involved in the NO induction of adventitious root formation, lateral root development, and root hair formation. We also review the first evidence supporting the NO role in the induction of adventitious and lateral root development by plant growth promoting rhizobacteria (PGPR). Finally, it is presented and discussed as an overview of the putative and potential biosynthetic pathways of NO and their close dependence on the diVerent N sources in PGPR.

ABBREVIATIONS AR BNF CDPK cGMP CDK Nas ½Ca2þ cyt CPTIO Nir DAF‐2 DA EPR IAA LR LRD Nar MAPK NO Nor Nos NOS PCIB Nap PGPR RHF Trp

adventitious root biological nitrogen fixation Ca2þ‐dependent protein kinase cyclic guanosine 30 50 ‐monophosphate cyclin‐dependent kinase cytoplasmic assimilatory nitrate reductase cytosolic Ca2þ concentration 2‐(4‐carboxyphenyl)‐4,4,5,5‐tetramethylimidazoline‐1‐oxyl‐3‐oxide dissimilative nitrite reductase 4,5‐diaminofluorescein diacetate electron paramagnetic resonance indole‐3‐acetic acid lateral root lateral root development membrane‐bound nitrate reductase mitogen‐activated protein kinase nitric oxide nitric oxide reductase nitrous oxide reductase nitric oxide synthase p‐chlorophenoxy isobutyric acid periplasmic nitrate reductase plant growth promoting rhizobacteria root hair formation tryptophan

I. INTRODUCTION One important goal to improve agricultural performance and increase food production is to attain high yields, even at low soil fertility or without intensive fertilization. To achieve this goal, the control of processes that

NO AND PLANT GROWTH PROMOTING RHIZOBACTERIA

3

determine root architecture and physiology appears to be central. Roots are dynamic anchorages of plants. They not only support the whole plant architecture, but also its entire physiological activity. Greater adventitious rooting, increased number of lateral roots (LRs), and higher length and density of root hairs are targets of many research projects in plant biology. Some of the most complex physical, chemical, and biological interactions experienced by plants are those that occur between roots and their surroundings. Signals derived from changes in the soil environment trigger selective root and shoot responses. In this scenario, the interrelationships established between roots and the biotic components of the rhizosphere would have a strong impact on plant growth. Undoubtedly, there are numerous processes occurring in the rhizosphere and the signals that govern and orchestrate their dynamic are still hidden to our knowledge. The symbiotic and nonsymbiotic associations between organisms in the rhizosphere rely on interacting factors and chemical signals that operate on time and space scales. Among them, compounds of hormonal nature play major roles. To make the picture more complex, all these factors vary with water content, temperature, nutrients and soil structure, and others. Root‐colonizing bacteria are able to both suppress disease in host plants by the production of inhibitory compounds that inhibit soil pathogen growth and, at the same time, stimulate growth and defense responses in host plants. There are complex and multitargeted responses that are yet poorly understood since the knowledge of chemical signals involved in plant‐ microorganism association are largely unknown. In this chapter, we present a review of the available data that strongly support a central role for nitric oxide (NO) as a chemical signal involved in root growth and development and in the interaction of roots with the plant growth promoting rhizobacteria (PGPR) Azospirillum.

II. NO IS A REGULATOR OF ROOT GROWTH AND DEVELOPMENTAL PROCESSES A. NO INDUCES ADVENTITIOUS ROOT FORMATION

Auxin is known to be involved in the process of adventitious root (AR) formation for a long time, mainly in promoting the initiation of root primordia (Haissig and Davis, 1994). SteVens et al. (2006) showed that the development and emergence of root primordia are positively controlled by ethylene. AR formation can also be induced by sugars, temperature, and light conditions (Takahashi et al., 2003). By contrast, there are few reports regarding the inhibition of

4

C. MOLINA‐FAVERO ET AL.

AR formation. Kuroha et al. (2002) showed that exogenous treatment with gibberellins, cytokinins, and abscisic acid (ABA) results in an inhibitory eVect on AR formation in cucumber (Cucumis sativus) hypocotyls. Thus, data indicate that complex interactions between diVerent phytohormones take place in determining the timing and intensity of the AR formation process. The ability to form ARs is critical for plants that are propagated through vegetative cuttings and, as a consequence, problems associated with rooting of cuttings frequently result in significant economic losses (De Klerk et al., 1999). While the physiology of AR formation is reasonably well known, the genetic and molecular mechanisms involved are still poorly understood. During the last years, several observations support a link between auxin‐ and NO‐ dependent signaling pathways during AR formation in cucumber explants (Lanteri et al., 2006a; Pagnussat et al., 2002, 2003, 2004). The first evidence showed that auxin induces AR formation through an increase of the NO concentration at the base of cucumber hypocotyls (Pagnussat et al., 2002). The maximum NO concentration was 60 nmol per gram of fresh weight after 24 h of auxin treatment, measured by electron paramagnetic resonance (EPR; Pagnussat et al., 2002). Since this work, many reports have enlarged the knowledge of the NO actions in the network that controls root morphology and physiology (Lanteri et al., 2006b). As a result, we now know that components that were described as cellular messengers for NO in animal cells are also involved in NO‐regulated responses in plants (Lamattina and Polacco, 2007). It was demonstrated that auxin and NO trigger both cGMP‐dependent and cGMP‐independent pathways leading to AR formation (Lanteri et al., 2006a; Pagnussat et al., 2003, 2004). Cumulative evidence indicates that the NO‐dependent activation of the guanylate cyclase‐catalyzed synthesis of cGMP results in an increase in cytosolic Ca2þ concentration ð½Ca2þ cyt Þ through the release of Ca2þ from intracellular stores regulated by cADPR. The entrance of Ca2þ from the extracellular space and from Ca2þ channels triggered by IP3 would also contribute to this raise in ½Ca2þ cyt . As a consequence, Ca2þ‐dependent protein kinases (CDPKs) become activated (Lanteri et al., 2006a). Another line of evidence suggests a role for NO in the activation of a cGMP‐independent mitogen‐activated protein kinase (MAPK) signaling cascade (Pagnussat et al., 2004). Collectively, available data support the claim that, in cucumber, AR formation is controlled by a complex and intricate set of cellular messengers involving auxin, NO, cGMP, cADPR, IP3, Ca2þ, CDPKs, and MAPKs. Future analyses will have to be directed at the identification of the molecular mechanisms that characterize the interaction between the diVerent components of the signaling cascade. We have as yet no exact knowledge regarding the mechanism by which auxin increase NO level in cucumber hypocotyls and the specific NO source/s

NO AND PLANT GROWTH PROMOTING RHIZOBACTERIA

5

during AR formation. In a recent report, the histological distribution and the source of NO during AR formation in mung bean hypocotyl cuttings were investigated (She and Huang, 2004). It was concluded that the enzyme nitric oxide synthase (NOS) is responsible for the production of NO during this process analyzed by the NADPH‐diaphorase activity assay, commonly employed as a marker for NOS (She and Huang, 2004). Authors showed that NADPH‐diaphorase activity and the specific NO fluorescence detected by the probe 4,5‐diaminofluorescein diacetate (DAF‐2 DA) gradually increased during AR formation and were mainly distributed in the AR meristem (She and Huang, 2004). Taking into consideration that the activity of the Arabidopsis NOS1 gene (AtNOS1) is a matter of discussion since results from Zemojtel et al. (2006) raise critical questions regarding both the activity and function of AtNOS1, our understanding of the participation, occurrence, and putative function of NOS in plants is not yet complete. Therefore, it will be interesting to explore the involvement of other enzymatic and nonenzymatic sources of NO (reviewed in Sto¨hr and Stremlau, 2006) during AR formation.

B. NO AND LATERAL ROOT DEVELOPMENT: NO IS DOWNSTREAM AUXIN IN TRIGGERING LRD

The process of LR formation has been extensively studied in many plants. Diverse signals regulate LR formation, including environmental and intrinsic factors (Malamy, 2005). Among environmental signals, nutrients are one of the major regulators of lateral root development (LRD). The concentration and patchy distribution of the nutrients nitrate, phosphate, and sulphate in soils have been shown to regulate the spatial distribution, density, and length of LRs (Kutz et al., 2002; Linkohr et al., 2002; Zhang and Forde, 2000). Furthermore, novel reports suggest an osmotic regulation during LRD (Deak and Malamy, 2005; van der Weele et al., 2000). Among internal signals, even though many hormones have been involved in the regulation of LRD, auxin plays a major role in this process. In accordance with the process of adventitious rooting (Pagnussat et al., 2002, 2003), a link between auxin and NO was shown during LRD. The application of NO donors resulted in an increase of LR number in tomato (Correa‐Aragunde et al., 2004). The auxin‐induced LR formation could be repressed by the addition of 2‐(4‐carboxyphenyl)‐4,4,5,5‐tetramethylimidazoline‐1‐oxyl‐3‐oxide (CPTIO), a specific NO scavenger. In addition, the Arabidopsis mutant noa1, in which NO production is impaired (Crawford et al., 2006), failed to respond to auxin in LRD (C. D. Todd, N. Correa‐Aragunde, M. E. Hoyos, P. K. Dhanoa, C. Santa‐Catarina, L. Lamattina, R.T. Mullen,

6

C. MOLINA‐FAVERO ET AL.

E. I. Segal Floh, and J. C. Polacco, unpublished results). The available data suggest that NO acts downstream of auxin leading to LRD. 1. NO acts at earlier stages of LR formation through the activation of cell division in pericycle In spite of intensive studies on root growth and developmental processes, the control of LR initiation is a yet poorly understood mechanism. In Arabidopsis, LRs are formed from a subset of pericycle cells termed founder cells, which are adjacent to the two xylem poles. Once activated, founder cells undergo anticlinal divisions followed by radial expansion and subsequent periclinal division, giving rise to an LR primordium. The LR primordium grows up through the cortex and emerges from the parent root primarily by expansion of the preexisting cells rather than by cell division (Malamy and Benfey, 1997). The mechanism by which specific pericycle cells became founder cells is still unknown. Several observations suggest an NO role in early stages of LR initiation. Microscopically, detection of NO during LRD in tomato reveals an accumulation of NO during the first stages of LR primordium development. In addition, NO depletion results in a severe reduction of LR formation (Correa‐Aragunde et al., 2004). Reports have presented data supporting a role of NO in the stimulation of cell division (Correa‐Aragunde et al., 2006; Otvos et al., 2005). During LR initiation in tomato, NO induces the expression of the cell cycle regulatory genes CDKA1, CYCD3;1, and CYCA2;1 while the gene encoding the cyclin‐dependent kinase (CDK) inhibitor KRP2 is repressed. Moreover, the regulation of these cell cycle regulatory genes by auxin is NO dependent (Correa‐Aragunde et al., 2006). In agreement, similar results were shown in a cell culture system. NO can stimulate the activation of cell division and embryogenic cell formation in leaf protoplast‐derived cells of alfalfa (Otvos et al., 2005). Even though the participation of NO in the control of cell cycle progression was already demonstrated, it still remains to be elucidated the NO source/s and the specific target molecules regulated by NO leading to the activation of cell division. C. GENERAL FEATURES ASSOCIATED TO ROOT HAIR FORMATION

Root hairs are specialized cell types that function in root anchoring and for increasing the soil area exploitable by the plant (Peterson and Farquhar, 1996). By greatly increasing the total surface area of the root system, root hairs are believed to play an important role in the absorption of water and nutrients from the soil (Clarkson, 1985). In the root system of higher plants,

NO AND PLANT GROWTH PROMOTING RHIZOBACTERIA

7

the epidermis is composed of two cell types: (1) root hair cells or trichoblasts and (2) non‐hair cells or atrichoblasts. Trichoblast and atrichoblast show diVerent cellular characteristics in the meristematic root region, indicating that the cue of cellular specification must be operating during the first stage of root development (embryonic development). The identity of epidermal cells, as trichoblast or atrichoblast, when protodermic cells, is defined on entering the elongation phase. At this time, the fate of root epidermal cells is determined by their position with respect to the underlying cortical cells. Atrichoblasts are located over a periclinal (outer tangential) cell whereas trichoblasts are located over the clef of two cells formed by adjacent cortical cells (Dolan et al., 1994; Galway et al., 1994). The cortical cells might confer positional information to result in a precise pattern of cell fate (Gilroy and Jones, 2000). This patterning is characteristic in Arabidopsis roots, where files of trichoblasts alternate with files of atrichoblasts (Dolan et al., 1993), suggesting a noticeable cell‐to‐cell communication soon after diVerentiation. Root hair formation (RHF) can be analyzed in phases: cell fate specification, initiation, tip growth, and maturation. Although positional information is provided postembryonically, epidermic root cells are defined as atrichoblast or trichoblast due to genetic action. However, the last proportion of trichoblasts is determined by environmental factors and nutritional requirements. The environmental factors that influence RHF are temperature, pH, calcium, iron, and phosphorus availability, among others (Hofer, 1996). NO was reported to be involved in the regulation of RHF in Arabidopsis and lettuce (Lombardo et al., 2006).

1. NO regulates RHF As stated, NO aVects the morphology and developmental pattern of roots in a noticeable manner. NO is involved in the promotion of lateral and AR initiation in several plant species (Correa‐Aragunde et al., 2004; Pagnussat et al., 2002, 2004). NO was shown to be also involved at the initiation and the elongation processes of RHF (Lombardo et al., 2006). In lettuce, NO is a critical molecule in determining root hair diVerentiation and elongation, mediating an auxin‐triggered signaling cascade (Lombardo et al., 2006). In Arabidopsis, NO and auxins are mainly involved in the regulation of mechanisms controlling the elongation process (Lombardo et al., 2006; Pitts et al., 1998). Indeed, several auxin response mutants of Arabidopsis display a phenotype similar to that generated by NO depletion in which root hair elongation is the main process aVected during RHF (Pitts et al., 1998). Auxin treatment stimulates NO production in Arabidopsis roots and this NO production is mainly located in the root hair cell files (Lombardo et al.,

8

C. MOLINA‐FAVERO ET AL.

Fig. 1. Endogenous NO production during root hair development in tomato. Tomato roots (15‐day‐old) were loaded with 15 mM of the specific NO probe DAF‐2 DA for 1 h. Photographs show the presence of NO in diVerent stages of root hair development in tomato root. Bar ¼ 0.1 mm.

2006). Figure 1 shows a detail of NO localization in diVerent developmental stages of RHF in tomato roots. After initiation of root hairs, elongation proceeds by polarized expansion. This expansion involves tip growth and requires biosynthesis of new wall material, localized wall loosening, and the flux of vesicles from the endomembrane system to the growing tip. These processes are regulated by the activity of ion channels and by the cytoskeleton (Ryan et al., 2001). The available data indicate that a signaling network including changes in reactive oxygen species (ROS), phospholipids, and ½Ca2þ cyt operates during root hair initiation and tip growth (Foreman et al., 2003; Ohashi et al., 2003). A Ca2þ current enters the root hair cell exclusively at the apex (Jones et al., 1995; Schiefelbein et al., 1992; Wymer et al., 1997). This Ca2þ current is confined to the apical 20–50 mm of the root hair and depends critically on external pH and [Ca2þ]. A parallel gradient in ½Ca2þ cyt is observed in this region (Felle and Hepler, 1997; Wymer et al., 1997). The ½Ca2þ cyt at the apex is several‐fold greater than ½Ca2þ cyt in the basal region. These phenomena appear to be specifically associated with root hair elongation (White, 1998). A very recent report have also established that extracellular ATP is also playing a role in root hair growth (Kim et al., 2006). On the other hand, NADPH oxidase/RHD2 (ROOT HAIR DEFECTIVE 2) is a key enzyme that produces ROS as second messengers involved in intracellular signaling. Foreman et al. (2003) demonstrated that ROS activate a specific type (hyperpolarization activated) of Ca2þ channel localized on root hair tips. Neither the apical Ca2þ current (Schiefelbein et al., 1992) nor the gradient in ½Ca2þ cyt (Wymer et al., 1997) are observed in mature, nongrowing root cells or in root hairs of rhd2 Arabidopsis mutant. This mutant forms root hair bulges but no elongated root hairs (Foreman et al., 2003). Interestingly, the requirement of a high ½Ca2þ cyt at the root tip for maintaining its growth

NO AND PLANT GROWTH PROMOTING RHIZOBACTERIA

9

rate fits with the already established action of NO in modulating Ca2þ level in guard cells (Garcı´a‐Mata et al., 2003): (1) the elevation of ½Ca2þ cyt through the regulation of Ca2þ release from intracellular stores; and (2) the regulation of Ca2þ‐dependent ion channel activities. In guard cells, NO treatment increased ½Ca2þ cyt from 500 to 800 nM when cells were stimulated by –200 mV (Garcı´a‐ Mata et al., 2003). Additionally, as was previously stated, disruption in Ca2þ homeostasis was shown to severely aVect NO‐induced AR formation in cucumber (Lanteri et al., 2006a). Two other reports showed that NO induces ½Ca2þ cyt in Nicotiana cells (Lamotte et al., 2004, 2006). Authors demonstrated that cells challenged by cryptogein (Lamotte et al., 2004) or hyperosmotic stress (Lamotte et al., 2006) increased ½Ca2þ cyt in an NO‐dependent pathway. 2. Cross talk between NO and other plant hormones during RHF An interaction between NO and ethylene was reported during the maturation and senescence of plant tissues (Lamattina et al., 2003; Leshem et al., 1998), and an antagonistic action of both gases was suggested during senescence (Leshem et al., 1998). Lindermayr et al. (2006) showed that NO might influence ethylene production in plants by inhibiting methionine adenosyltransferase through S‐nitrosylation. In roots, ethylene is another hormone involved in the regulation of RHF. Ethylene acts as a positive regulator of root hair diVerentiation. Antagonists that block either the synthesis or the perception of ethylene inhibit the diVerentiation of root hairs (Tanimoto et al., 1995). The constitutive triple response (CTR1) gene encodes a Raf‐like protein kinase that negatively regulates the ethylene signal transduction pathway (Kieber et al., 1993). The ethylene‐dependent triple response Arabidopsis mutant ctr1 possesses ectopic root hairs on their atrichoblasts. Ethylene functions as a diVusible positive regulator and confers the ‘‘hair’’ character on cells overlying cortical, anticlinal cell walls. Ethylene may accumulate in the air spaces that are formed at the junction between trichoblasts and the underlying cortex. The location of trichoblast cells over these spaces may expose these cells to elevated levels of ethylene and thereby induce hairs preferentially in these cells (Dolan et al., 1994). It is possible that NO could be regulating the action of this hormone during RHF. On the other hand, Zhu et al. (2006) have demonstrated that jasmonic acid (JA) and methyl jasmonate (MeJA) promote RHF. They also concluded that ethylene is a prerequisite for JAs’ function since the eVect of JAs is abolished in the ethylene‐insensitive Arabidopsis mutants etr1‐1 and etr1‐3, or by inhibiting ethylene action (Agþ) or biosynthesis (AVG). Furthermore, it was found that inhibitors of JA biosynthesis ibuprofen and SHAM (a known inhibitor of lipooxygenase in jasmonate biosynthesis) repressed ACC‐driven or eto1‐1 (ethylene overproducing mutant)‐induced RHF (Zhu et al., 2006). Collectively, these data support a role for the interaction between JAs and

10

C. MOLINA‐FAVERO ET AL.

ethylene in the regulation of RHF in Arabidopsis. It remains to be elucidated in which part of the signaling cascade, which regulates RHF, occurs the cross talk between ethylene, JA, and NO. Finally, a role of microtubules during root hair initiation has been demonstrated (Samaj et al., 2004). A putative linkage between microtubules and NO during root hair initiation deserves to be analyzed. It is already known that cortical microtubules become randomized during initiation of LR primordia in pericycle cells (Baluska et al., 2000) as well as during root hair initiation in trichoblasts (Baluska et al., 2000; Van Bruaene et al., 2004). Since NO is involved in LRD (Correa‐Aragunde et al., 2004) and in RHF (Lombardo et al., 2006), it is also possible that NO could be involved in the randomization of cortical microtubules which have been shown to precede the dramatic switch in cell polarity during the morphogenetic events described above. NO has already been involved in microtubule configuration in neurons (He et al., 2002). Altogether, the advances of the knowledge concerning the NO functions in root growth and developmental processes indicate that it is a central signal molecule in the auxin transduction pathways leading to the determination of root morphology and physiology. D. THE EFFECTS OF PGPR ON ROOT ARCHITECTURE

Root is the organ through which the plant can sense and communicate with other living systems that inhabit the soil. It is accepted that root activity alters the habitat of microorganisms and these, in turn, could trigger changes in the overall plant behavior. Among microorganisms living in the rhizosphere, root colonizers that exert beneficial eVects on plant growth and development are referred to as PGPR (Kloepper, 1992). As a primary target, root is the organ that shows the first stimulating bacterial eVects. This was particularly remarkable in plants inoculated with Azospirillum spp. (Okon, 1985), the most studied rhizobacteria (Bashan et al., 2004). Indeed, field experiments performed with azospirilla‐inoculated crops have shown significantly increased yields accompanied by better water and mineral uptake and positive changes in the root morphology and growth (Creus et al., 2004; Dobbelaere et al., 2001; Okon and Labandera‐Gonza´lez, 1994; Sarig et al., 1988). An increase in the branching degree of roots, an improvement in the root architecture, and its associated enhanced capacity to explore soil in the quest for water would contribute to a better hydrated status of plants exposed to water deficit. It was reported that Azospirillum‐inoculated wheat seedlings subjected to osmotic stress developed a significant higher coleoptile and better water status than noninoculated seedlings (Alvarez et al., 1996; Creus et al., 1998). Taking into account that plant exposed to salt stress also suVers water

NO AND PLANT GROWTH PROMOTING RHIZOBACTERIA

11

deficit, when assayed, it was proved that Azospirillum‐inoculated wheat seedlings were able to survive when exposed to up to 320‐mM NaCl for 3 days (Creus et al., 1997). In salty soils or in those lacking enough water, the success of inoculation will be dependent on the seed capability to germinate under these stressing conditions. In field assays, Azospirillum’s eVects in mitigating water stress were observed in maize and wheat crops (Casanovas et al., 2003; Creus et al., 2004). Germination and growth under 80‐mM NaCl could be greatly improved in lettuce seeds inoculated with A. brasilense Sp245 (Barassi et al., 2006). The beneficial eVects that Azospirillum exerts on plants, whether they are achieved under normal or environmental stressing conditions, rely on molecular mechanisms that are poorly understood. Several mechanisms have been postulated to explain how PGPR enhances plant growth and development. These can be broadly distinguished as providing either direct or indirect growth stimulation (Glick, 1995). Direct mechanisms elicit growth promotion by bacterial determinants, while indirect ones result in skipping the plant from growth limitations imposed by pathogenic or nonpathogenic microorganisms (Ryu et al., 2004). Whatever the type of ecological relationship occurring between plant and rhizobacteria, the mechanisms that enable roots to interpret the innumerable signals they receive from the rhizosphere, including those produced by PGPR, and how those signals elicit plant growth promotion, are largely unknown. As mentioned above, the most studied PGPR is Azospirillum spp., included in the alpha subclass of Proteobacteria belonging to the IV rRNA superfamily (Xia et al., 1994). This group of free‐living microorganisms encompasses eight species, each one classified according to its particular biochemical and molecular characteristics (Bashan et al., 2004; Peng et al., 2006; Xie and Yokota, 2005). Since the genera can be found in a wide range of habitats associated to roots of both graminaceous as well as nongraminaceous species, it has been regarded as a general plant colonizer (Bashan and Holguin, 1997). Azospirillum can fix atmospheric N2 through nitrogenase complex, when the availability of N compounds and oxygen tension are low (Do¨bereiner and Day, 1976; Steenhoudt and Vanderleyden, 2000). Even though this characteristic could be extremely valuable in agriculture, field studies including those in which isotopic dilution techniques were used, failed to demonstrate a significant biological nitrogen fixation (BNF) in Azospirillum‐inoculated crops (Vande Broek et al., 2000). Even at the organism level, the growth promotion induced by the inoculation of axenic seedlings could not be ascribed to BNF (Bashan et al., 1989). One of the first observations regarding plant growth promotion activity exerted by Azospirillum was on root morphology (Okon, 1985). On inoculation,

12

C. MOLINA‐FAVERO ET AL.

the root displayed a significant increase in the number and the length of root hairs, the rate of appearance and number of LRs, the diameter and length of lateral and ARs, and the root surface area (Creus et al., 2005; Dobbelaere et al., 1999; Fallik et al., 1994; Kapulnik et al., 1985). Besides, Levanony and Bashan (1989) reported an increase in cell division in the root tips of inoculated wheat. Several reports showed that the inoculation of wheat or maize seedlings with Azospirillum cells resulted in an increased number of root hair showing a Y‐shaped deformation (Jain and Patriquin, 1984; Kapulnik et al., 1985; Patrikin et al., 1983; Zamudio and Bastarrachea, 1994). All these eVects are dependent on the plant species and cultivar inoculated and on the concentration of Azospirillum inoculum (Vande Broek et al., 2000). Inoculation of many diVerent plant species with Azospirillum in a range between 106 and 108 cells per seedling provoked root elongation (Creus et al., 1996; Kapulnik et al., 1985). However, higher concentrations of bacteria always result in an inhibition of root elongation (Harari et al., 1988). Thus, there exists a bacterial concentration that results optimum for triggering root elongation. The dose response of the root system to Azospirillum inoculation resembles the responses triggered by exogenous hormonal application. The production of phytohormones, namely auxins, cytokinins, and gibberellins, is the most commonly invoked mechanism of plant growth promotion exerted by PGPR (Garcı´a de Salamone et al., 2001). Among them, auxins are thought to play the major role. Even though it was suggested more than 60 years ago that rhizobacteria could produce auxins (Roberts and Roberts, 1939), it was only in the seventies that this assumption was proved (Barea and Brown, 1974; Brown, 1972; Tien et al., 1979). Nowadays it is well known that Azospirillum can synthesize indole‐3‐acetic acid (IAA) by at least three diVerent pathways. By means of in vivo labeling experiments, Prinsen et al. (1993) demonstrated the existence of one tryptophan (Trp)‐ independent pathway and two Trp‐dependent biosynthetic routes. The presence of Trp in culture medium strongly induces the Trp‐dependent pathways, resulting in a tenfold increase of the IAA levels. Although Trp‐independent IAA biosynthesis occurs in various plant species, Azospirillum is so far the only bacterium in which such an IAA biosynthetic pathway has been identified (Vande Broek et al., 2000). Therefore, this ability could be of biochemical and ecological significance, since some root exudate like those produced by maize lack Trp (Guckert, 1985). Controlled experiments in vitro showed that IAA content increased in roots and shoots of A. brasilense FT326‐inoculated tomato (Ribaudo et al., 2006). To evaluate the involvement of bacterial IAA in the promotion of root development, several investigations were conducted with mutant strains

NO AND PLANT GROWTH PROMOTING RHIZOBACTERIA

13

altered in IAA production. A. brasilense SpM7918, a very low‐IAA producer, showed a reduced ability to promote root system development in terms of both number and length of LRs and distribution of root hairs compared to the wild‐type (wt) strain Sp6 (Barbieri and Galli, 1993; Dobbelaere et al., 1999). Another mutant of A. brasilense with low production of phytohormones but high nitrogenase activity did not enhance root growth over uninoculated controls (Kundu et al., 1997). However, there are no reports showing to what extent IAA is produced in the rhizosphere by Azospirillum (Lambrecht et al., 2000; Steenhoudt and Vanderleyden, 2000). On the other hand, several authors have shown evidence of a lack of correlation between the capacity for IAA synthesis of Azospirillum and the eVects on root growth promotion (Bothe et al., 1992; Harari et al., 1988; Kapulnik et al., 1985). Nevertheless, the possibility that Azospirillum could not only produce IAA but also to enhance the endogenous IAA produced by the plant should not be excluded. Most studies on the mechanisms for plant growth promotion by PGPR have focused on bacterial traits without examining the host plant’s physiological responses (Bloemberg and Lugtenberg, 2001). Moreover, the role of chemical signals in mediating rhizospheric interactions is beginning to be understood (Bais et al., 2006). If a positive eVect of inoculation with Azospirillum sp. is expected, a successful colonization of roots followed by an appropriate bacterial cells location is needed. Using the green fluorescent protein to tag bacteria, Liu et al. (2003) confirmed that bacteria are established mainly on the root surface. Even though some strains of A. lipoferum and A. brasilense are capable of colonizing the inner part of the root, they always locate outside the plant cells in the apoplast and intercellular spaces. The fact that Azospirillum aVects plant cell metabolism from outside the cell suggests that the bacteria is capable of excreting and transmitting signals that are perceived by the plant cell wall and/or the plasma membrane. This interaction initiates a chain of events that results in the observed altered metabolism of inoculated plants. Since membranes are extremely sensitive to any change, they may serve as the precise indicator for Azospirillum activity at the cellular level (Bashan et al., 1992). 1. Azospirillum‐promoted root growth involves NO‐mediated actions Bloom et al. (2003) have reviewed the signals and molecules that are potentially involved in root development. Among them, nitrogen species as ammonium, nitrate, and NO were proposed to be implicated in root growth and proliferation. As stated above, it has been already demonstrated that NO functions as a signal molecule in the IAA‐induced signaling cascade leading to AR formation, LRD, and RHF (Correa‐Aragunde et al., 2004; Lombardo et al., 2006; Pagnussat et al., 2002). It is largely known that Azospirillum can

14

C. MOLINA‐FAVERO ET AL.

Fig. 2. A. brasilense produces NO. A. brasilense Sp245 was cultured in medium containing NH4Cl as N source. At late exponential phase, cells were pelleted, washed, and resuspended in buVer HEPES–NaOH 20 mM pH 7.8 in the absence (left) or presence (right) of the NO scavenger CPTIO at 0.5 mM. After 30 min of incubation, 15 mM of the NO‐specific probe DAF‐2 DA was added, and samples were incubated for two more hours. Washed bacteria were examinated by epifluorescence microscopy at 1000 magnification.

produce NO at low O2 pressure by denitrification (Hartmann and Zimmer, 1994). Creus et al. (2005) have reported the NO production by Azospirillum growing under aerobic conditions. Figure 2 shows cells of A. brasilense cultured in liquid medium supplemented with 0.1% (w/v) NH4Cl as N source. The green fluorescence is produced by the addition of the NO‐specific fluorescent probe DAF‐2 DA whereas the addition of CPTIO, an NO scavenger, diminished the fluorescence (Fig. 2). A concentration of 6.4 nmol of NO per gram of A. brasilense was quantified when bacterium reached the end of growing log phase (Creus et al., 2005). The remarkable analogies found between the experimental data concerning Azospirillum stimulation of plant root development and the capability of NO to act as a nontraditional plant growth regulator (Beligni and Lamattina, 2001) promoting AR formation, LRD, and RHF led us to explore whether the Azospirillum ability to promote root growth and modify root architecture relies on NO. Azospirillum‐inoculated tomato roots incubated with the NO‐specific fluorescent probe DAF‐2 DA displayed higher fluorescence intensity compared to noninoculated roots. Fluorescence was mainly located at the vascular tissues and subepidermal cells of roots (Creus et al., 2005). Moreover, the Azospirillum‐mediated induction of LRD appears to be NO dependent since treatment of inoculated seedlings with the NO scavenger CPTIO completely blocked this eVect (Creus et al., 2005). In addition, two other strategies were carried out to validate this result: (1) the use of a mutant strain of

NO AND PLANT GROWTH PROMOTING RHIZOBACTERIA

15

A. brasilense with reduced ability to synthesize IAA, but the same capacity for NO production; and (2) the addition of the auxin antagonist p‐chlorophenoxy isobutyric acid (PCIB), which competes with endogenous auxin. Inoculation of tomato seedlings with either the wt or the IAA‐deficient mutant of Azospirillum increased LR number and percentage of seedlings displaying AR formation with respect to the noninoculated ones. The wt strain induced higher LRD than the mutant strain, in agreement with previous findings in wheat inoculated with diVerent IAA‐impaired Azospirillum mutants (Barbieri and Galli, 1993; Dobbelaere et al., 1999). However, when NO was removed with the NO scavenger CPTIO, both lateral and AR formation were inhibited and attained to the noninoculated values, evidencing that NO is strongly involved in the Azospirillum‐induced root branching (C. Molina‐Favero, C. M. Creus, M. Simontacchi, S. Puntarulo, and L. Lamattina, unpublished results). Besides, the addition of PCIB to inoculated tomato seedlings decreased the percentage of seedlings with LRs (Creus et al., 2005). Altogether, these results suggest that auxins are involved but not exclusively in Azospirillum‐mediated eVects on roots. Indeed, data support that both auxins and NO have a role as cellular messengers in the interaction occurring in the rhizosphere between roots and PGPR. Whether the auxin synthesized by PGPR triggers an NO production in the bacterial cell and/or in the plant root remains to be elucidated. 2. NO sources in Azospirillum and other PGPR NO is a central component in the nitrogen cycle. It is produced and released by almost all soils, particularly those well fertilized (Sto¨hr and Ullrich, 2002). Several biological and chemical pathways are involved in regulating the NO steady state levels in soils, including denitrification, nitrogen mineralization (i.e., conversion of organic N into inorganic forms), dinitrogen fixation, and nitrification. In the interaction between plants and PGPR, these pathways can be accomplished alternatively or simultaneously according to the nutrient availability, physical conditions, and the organisms involved. In associative or symbiotic relationships between roots and microorganisms, it is likely that both partners contribute to NO production. In addition, it could be expected that either bacteria or plant could influence NO synthesis in the partner in a synergistic, compensatory, and/or complementary way. NO production in plants relies principally on nitrite reduction. This intermediary can be reduced enzymatically by a cytosolic nitrate reductase (Sto¨hr and Ullrich, 2002), the root mitochondria (Gupta et al., 2005), and a plasma membrane‐bound nitrite:NO reductase (Sto¨hr et al., 2001); and nonenzymatically in the apoplast at acidic pH values (Bethke et al., 2004) or by carotenoids in a reaction mediated by light (Cooney et al., 1994). The presence of an

16

C. MOLINA‐FAVERO ET AL.

NOS in plants has not been fully confirmed (Zemojtel et al., 2006), but there is biochemical and immunological evidence supporting it (Barroso et al., 1999; Jasid et al., 2006). In bacteria, there are also several NO‐producing pathways that share similar features to the plant pathways. Meyer and Sto¨hr (2002) suggested that NO might be one of the signals for the presence of nitrate in a given place. Likewise, the NO synthesis by PGPR from nitrate or ammonium may generate an NO gradient which could trigger specific signaling processes. All these considerations make NO a potential signal molecule in the bacterial plant root association. a. Denitrification. Denitrification is the stepwise dissimilative reduction of  nitrate ðNO 3 Þ to nitrite ðNO2 Þ, NO, nitrous oxide (N2O), and dinitrogen (N2) by the corresponding N oxides reductases. In this process, nitrate is used instead of oxygen as a final electron acceptor in respiration. This pathway allows denitrifiers to generate energy and to grow under low oxygen or anaerobic conditions (Zumft, 1997). Denitrification has been known for a long time, although it has been more recently accepted that NO is an obligatory intermediary (Ye et al., 1994). Several PGPR are able to denitrify, including species of genera such as Pseudomonas spp. and Bacillus spp. (Cutruzzola´, 1999). In the genus Azospirillum, most strains of A. lipoferum and A. brasilense are denitrifiers, but A. amazonense, A. irakense, and A. oryzae are unable to denitrify (Hartmann and Zimmer, 1994; Xie and Yokota, 2005). Anaerobic growth of A. brasilense in nitrate, nitrite, or nitrous oxide has been well established (Penteado Stephan et al., 1984; Zimmer et al., 1984). In contrast, A. brasilense cannot grow with NO as sole electron acceptor since its reduction does not generate a proton electrochemical gradient across the membrane (Voßwinkel et al., 1991). Denitrifiers are predominantly heterotrophic microorganisms and facultative anaerobes (Wrage et al., 2001). Thus, the easily decomposable matter provided by root exudates could increase the activity of denitrifiers in the rhizosphere. Besides, it has been reported that anoxic roots accumulate and excrete nitrite (Stoimenova et al., 2003), which may be further reduced to NO by denitrifiers. Though rhizosphere is not always an anoxic place, A. brasilense and other PGPR may inhabit microaerobic or anaerobic microsites in which the conditions for denitrification are given. The enzymes involved in denitrification are nitrate reductase, nitrite reductase, nitric oxide reductase (Nor), and nitrous oxide reductase (Nos). Three diVerent nitrate reductase activities can be found in bacteria. Azospirillum and other PGPR can perform all of these activities. First, the cytoplasmic soluble assimilatory nitrate reductase (Nas) reduces nitrate to nitrite which is further reduced by a nitrite reductase to ammonia in order to be incorporated into amino acids (Hartmann and Zimmer, 1994; Steenhoudt et al., 2001a).

NO AND PLANT GROWTH PROMOTING RHIZOBACTERIA

17

Second, the membrane‐bound nitrate reductase (Nar) allows PGPR to generate energy through reduction of nitrate to nitrite (Steenhoudt et al., 2001a). Finally, there is a periplasmic nitrate reductase (Nap), which is thought to function maintaining an optimal redox balance by dissipation of the reducing equivalent excess (Steenhoudt et al., 2001a,b). Both, Nap and Nar are dissimilatory enzymes that reduce nitrate to nitrite in the first step of denitrification. The three enzymes bind the molybdenum cofactor (Steenhoudt et al., 2001a). Dissimilative nitrite reductase (Nir) is considered the major known source of NO in bacteria. This is a periplasmic‐located enzyme that catalyzes the reduction of nitrite mainly to NO (Cutruzzola´ 1999) and, only in minor quantities, to N2O (Ye et al., 1994). Two distinct types of Nir have been found in denitrifiers: (1) a cytochrome cd1‐dNir, containing one heme c prosthetic group covalently linked to the polypeptide chain and one heme d1 noncovalently associated with the protein; and (2) a copper‐containing protein called Cu‐dNir, which is found in about one‐ fourth of the isolated denitrifiers (Ye et al., 1994). These diVerent Nir are never synthesized by the same organism (Zumft, 1997). Nor is a plasma membrane‐ bound enzyme that catalyzes the reduction of NO to N2O (Ye et al., 1994). Since the accumulation of NO can be lethal for bacteria (Ye et al., 1994), expression of Nir and Nor are tightly regulated (Tosques et al., 1996) and it has been suggested that both enzymes form a functional unit (Jetten et al., 1997). Two Nor have been isolated. Both enzymes are cytochrome complexes containing heme b and heme c (Ye et al., 1994). Finally, Nos is a periplasmic‐located copper‐containing enzyme that reduces N2O to N2 in the last step of denitrification (Jetten et al., 1997). Aerobic denitrification occurs when denitrification genes are activated at a high O2 level (Zumft, 1997). Steenhoudt et al. (2001b) have identified and characterized a Nap in A. brasilense Sp245, which is neither repressed nor inactivated by oxygen. In aerobically grown cultures of A. brasilense Sp245 with nitrate as the sole N source, a production of 120‐nmol NO per gram of bacteria was determined at the end of log phase of growth by EPR. The NO þ concentration was 25‐fold higher in NO 3 ‐than in NO4 ‐grown cultures (C. Molina‐Favero, C. M. Creus, M. Simontacchi, S. Puntarulo, and L. Lamattina, unpublished results). A Nap knockout mutant of A. brasilense Sp245 (strain Faj164; Steenhoudt et al., 2001b) produced only 5% of NO with respect to the wt level indicating that aerobic denitrification can be an important source of NO in this bacterium (C. Molina‐Favero, C. M. Creus, M. Simontacchi, S. Puntarulo, and L. Lamattina, unpublished results). Since the derived protein sequence of the A. brasilense Nap is highly homologous to the NapABC protein sequences of Escherichia coli, Pseudomonas sp. G‐179, Ralstonia eutropha, Rhodobacter sphaeroides, and Paracoccus denitrificans

18

C. MOLINA‐FAVERO ET AL.

(Steenhoudt et al., 2001b), the possibility of an aerobic synthesis of NO by these microorganisms cannot be excluded. NO production could be now considered as an advantage of the process of denitrification which was first described as undesirable in PGPR, since it may contribute to the loss of nitrogen available for plants (Paul and Clark, 1996). Supporting this suggestion, it has been reported that the root colonization ability by rhizobacteria and the plant growth‐stimulatory eVects are significantly diminished in the Sp245chl1 strain of A. brasilense, a mutant defective in both assimilatory and Nap activity (Boddey et al., 1986; Jetten et al., 1997). Before the finding of the signaling role of NO in plant development, it has been proposed that PGPR strains that can reduce nitrate to nitrite show a competitive advantage (Do¨bereiner and Pedrosa, 1987). During the nitrate respiration, part of the nitrite produced is excreted to the external medium (Bothe et al., 1981; Neuer et al., 1985; Zimmer et al., 1984). Moreover, classical tests for determining auxin eVects show that nitrite, in concentrations similar to those produced by nitrate respiration, can mimic the IAA‐ and the Azospirillum‐promoting eVects (Zimmer and Bothe, 1988; Zimmer et al., 1988). Authors also showed that the promoting eVects of nitrite could be enhanced by adding ascorbate. Regarding NO chemistry, this observation can be explained by the nonenzymatic reduction of nitrite by ascorbate at acidic pH (Weitzberg and Lundberg, 1998). Furthermore, A. brasilense can increase the proton eZux by root cells, making the external pH more acidic (Bashan et al., 1992) and therefore leading to NO formation in the apoplastic and intercellular space. b. Heterotrophic nitrification. Nitrification is the biological oxidation of ammonium to nitrate. The first step of the general pathway is the oxidation of ammonium to hydroxylamine (NH2OH), which is catalyzed by the enzyme ammonium monooxygenase. Next, hydroxylamine is oxidized to nitrite by hydroxylamine oxidoreductase. Finally, nitrite is oxidized to nitrate by nitrite oxidoreductase (Wrage et al., 2001). In this pathway, NO and N2O are produced in the reduction of NO 2 to N2 by chemical decomposition of NO2 or NH2OH (Anderson et al., 1993; Wrage et al., 2001). Nitrification was first described in autotrophic bacteria belonging to genera such as Nitrosomonas and Nitrobacter. The complete nitrification is accomplished in two steps by two diVerent groups of microorganisms, the NH3 oxidizers and the NO2 oxidizers (Wrage et al., 2001). These oxidations allow autotrophic bacteria to generate energy for CO2 fixation (Paul and Clark, 1996). Besides autotrophic nitrification, it has been recognized that heterotrophic nitrification is an important process in soils. This pathway is carried out by several fungi and heterotrophic bacteria (Paul and Clark, 1996). Autotrophic

NO AND PLANT GROWTH PROMOTING RHIZOBACTERIA

19

and heterotrophic nitrifications have the same substrates, intermediaries, and products, even though the enzymes involved may be diVerent in each route. Other important diVerences are that the heterotrophic nitrification is accomplished by a single organism and that energy is not produced during the process (Wrage et al., 2001). In addition to NHþ 4 , some heterotrophic nitrifiers are capable of producing nitrate by oxidation of organic amines or amides (Papen et al., 1989). Heterotrophic nitrification can be a significant source of NO from bacteria living in aerobic and microaerobic soil and water (Anderson et al., 1993; Papen et al., 1989). This process is connected with denitrification through its  products NO 2 and NO3 , and it has been demonstrated that both pathways could be performed simultaneously in some organisms (Wrage et al., 2001). Moreover, it is frequent that the heterotrophic nitrifiers would also be aerobic denitrifiers (Anderson et al., 1993; Steenhoudt et al., 2001a; Wrage et al., 2001). Heterotrophic nitrification has been proved in some PGPR strains of Pseudomonas sp. (Castignetti and Hollocher, 1984; Papen et al., 1989), Arthrobacter sp. (Verstraete and Alexander, 1972; Witzel and Overbeck, 1979), and Bacillus spp. (Lang and Jagnow, 1986). Aerobically grown cultures of A. brasilense are able to produce NO with ammonium as N source (Creus et al., 2005). When these cultures were supplemented with hydroxylamine, a fourfold increase in the rate of NO production was observed. This increase was dose dependent, being highest at 5‐mM hydroxylamine. Overall, these results suggest that A. brasilense possesses a heterotrophic nitrification‐like pathway (C. Molina‐Favero, A. Arruebarrena Di Palma, C. A. Barassi, L. Lamattina, and C. M. Creus, unpublished results). c. Nitric Oxide synthase. In the past, attention was focused on bacterial NO production by nitrification–denitrification related processes. However, it has now been established that some bacteria can also synthesize NO in a reaction catalyzed by an NOS. This enzyme converts, in presence of oxygen, L‐arginine to L‐citrulline and NO in a mechanism similar to that of eukaryotes (Adak et al., 2002a,b; Midha et al., 2005; Sari et al., 1998). Bacterial NOS can also oxidize N‐hydroxy‐L‐arginine (NOHA), which is the intermediary in the reaction of mammalian NOS (Chen and Rosazza, 1995; Sari et al., 1998). The first report on a bacterial NOS was published in 1994. In their work, Chen and Rosazza (1994, 1995) described an NOS activity in the genus Nocardia. Subsequently, NOS activity has been characterized in microorganisms such as Deinococcus radiodurans (Adak et al., 2002b), Rhodococcus spp. (Cohen and Yamasaki, 2003; Sari et al., 1998), Bacillus subtilis (Adak et al., 2002a), B. anthracis (Midha et al., 2005), Physarum polycefalum (Werner‐Felmayer et al., 1994), Staphylococcus aureus (Choi et al.,

20

C. MOLINA‐FAVERO ET AL.

1997; Hong et al., 2003), Salmonella typhimurium (Choi et al., 2000), Geobacillus stearothermophilus (Sudhamsu and Crane, 2006), and Streptomyces turgidiscabies (Kers et al., 2004), among others. The mammalian NOS is a dimeric protein formed by an N‐terminal oxygenase domain (NOSoxy) that binds protoporphyrin IX (heme), 6R‐tetrahydrobiopterin (H4B), and the substrate L‐arginine; and by a C‐terminal reductase domain (NOSred) that binds FMN, FAD, and NADPH (Stuehr, 1997). Studies of the sequence of bacterial NOS reveal the lack of an NOSred domain but show high homology with the mammalian NOS oxygenase domain. Bacterial NOS also shows similarities in key structural features involving the conformation of the active site, the heme environment and its interaction with substrates, cofactors, and coenzymes. Generally, bacterial NOS lacks an N‐terminal extension implicated in the dimer formation of mammalian isoforms, however alternative interactions allow bacterial NOS to form a dimer (Adak et al., 2002a,b; Bird et al., 2002; Midha et al., 2005). Despite the numerous studies on the role of NO in plant physiology and the recognized existence of NOS in bacteria, little is known about NOS‐mediated NO production and its function/s in PGPR. Some strains of Rhodococcus spp. and Nocardia spp. are able to nonpathogenically colonize the root apoplast and, the former, also the leaf apoplast in several plants. In these places, it is thought that they might benefit the plant by providing metabolites and/or outcompeting pathogens (Araujo et al., 2002; Cohen and Yamasaki, 2003; Conn and Franco, 2004). It was suggested that the activity of NOS in Rhodococcus sp. R312 could be involved in the regulation of the enzyme nitrile hydratase (Sari et al., 1998). In addition, Cohen and Yamasaki (2003) proposed that NOS could promote tolerance of Rhodococcus APG1 to oxidative stress. In Nocardia sp., NO produced by NOS could increase the levels of cyclic guanosine 30 ,50 ‐monophosphate (cGMP) by activation of guanylate cyclase. The function of cGMP remains to be determined in this bacterium (Son and Rosazza, 2000). As described before, cGMP mediates auxin responses leading to AR formation in plants (Pagnussat et al., 2003, 2004). In Bacillus sp., the role of NOS‐dependent NO production is unknown, neither in its own physiology nor in the interaction with plants. Nevertheless, a role for NOS could be hypothesized given that exogenously added NO can modify gene expression in B. subtilis (Moore et al., 2004; Nakano, 2002). Recently, evidence for an NOS‐like activity in A. brasilense has been found. Pure cultures of a wt strain or a Nap‐deficient mutant (strain Faj164; Steenhoudt et al., 2001b) showed a significant increase in NO production when culture mediums were supplemented with L‐arginine (Creus et al., 2005; C. Molina‐Favero, C. M. Creus, M. Simontacchi, S. Puntarulo, and L. Lamattina, unpublished results). However, this

NO AND PLANT GROWTH PROMOTING RHIZOBACTERIA

21

stimulation was insensitive to mammalian NOS inhibitors (Creus et al., 2005). Taking into account the location of PGPR in plant root tissues, the possibility that they could improve plant growth by an NOS‐related NO synthesis is open and further research is needed to determine the importance of this activity in the interaction. In addition to the occurrence of NOS in nonsymbiotic PGPR, an NOS‐like activity was detected in Lupinus albus nodules (Cueto et al., 1996). Moreover, working in the symbiosis between Medicago truncatula and Sinorhizobium meliloti, Baudouin et al. (2006) found that functional nodules synthesized NO by a mechanism that is neither related to denitrification nor nitrogen fixation. In both reports, mammalian NOS inhibitors were eVective in the inhibition of NO synthesis suggesting that an NOS‐like activity is the active pathway. It is still uncertain whether the plant or the bacteria carry out the NOS activity. The role of NO in the interaction is also unclear. Considering the evidence that involves both the inhibition of polar auxin transport during the first steps of nodulation (Mulder et al., 2005) and the requirement of NO in auxin‐induced root developmental processes (Correa‐Aragunde et al., 2004; Lombardo et al., 2006; Pagnussat et al., 2002, 2003), it was hypothesized that NO could have a signaling role in the establishment of legume–rhizobia interactions (Baudouin et al., 2007).

III. PERSPECTIVES NO is a gas with a broad chemistry that involves several reactive forms that could explain its versatility as an extensive signal molecule in intra‐ and intercellular communication (Lamattina et al., 2003). In 1992, NO was termed ‘‘Molecule of the year’’ by the magazine Science. Since then, a great amount of data is coming from studies on NO biology in plants (Lamattina and Polacco, 2007). In particular, the NO role in root growth and development is one of the most studied fields at the moment. We know that a close relationship and similarities exist between auxin’s actions and NO eVects in root responses. It would be extremely interesting to find at what step of auxin signaling pathway NO is acting, and what molecular mechanism/s and NO form/s are involved. It was demonstrated that when a plant senses a pathogenic bacteria it synthesizes microRNAs (miRNAs) that interfere with the production of specific proteins related with auxin signaling (Navarro et al., 2006). It was shown that repression of auxin signaling restricts bacterial growth, implicating auxin in disease susceptibility (Navarro et al., 2006). It would be interesting to know if a repression of auxin signaling occurs when nonpathogenic bacteria like PGPR

22

C. MOLINA‐FAVERO ET AL.

associate with plants. We could probably speculate that plant cells would not restrict PGPR growth by repression of auxin signaling, since most of the eVects in roots after PGPR colonization rely on auxin. Figure 3 shows a schematic model with proposed pathways for NO and auxin synthesis in Azospirillum and their possible eVects on roots. IAA is synthesized in these bacteria by diVerent Trp‐dependent and Trp‐independent pathways (Prinsen et al., 1993). Furthermore, NO is potentially produced by several reactions as a part of the nitrogen metabolism, including denitrification, heterotrophic nitrification, and NOS (Creus et al., 2005; Hartmann and Zimmer, 1994; C. Molina‐Favero, C. M. Creus, M. Simontacchi, S. Puntarulo, and L. Lamattina, unpublished results). During the PGPR– plant interaction, both IAA and NO of bacterial origin could reach plant cells and initiate the rooting processes. It is also possible that these signaling compounds, as well as nitrite and nitrate, could be freely interchanged between both partners of the association. In root cells, NO could be produced from nitrite, nitrate, and L‐arginine in enzymatic and nonenzymatic pathways (Sto¨hr and Stremlau, 2006). Moreover, it is known that auxins increase NO production triggering RHF, LRD, and AR formation (Correa‐Aragunde et al., 2004; Lombardo et al., 2006; Pagnussat et al., 2002). Several second messengers such as cGMP, Ca2þ, and MAPK were reported to be involved in these developmental processes (Lanteri et al., 2006a; Pagnussat et al., 2003, 2004). Taking into account the similarities that Azospirillum and NO display on influencing root growth, developmental, and physiological processes, it would be interesting to know if these eVects are exerted through the same second messengers. Even though a general picture can be depicted, several questions raise from experiments involving NO in the PGPR–root interaction and in the root developmental processes. It would be also valuable to find a more precise explanation of the roles of NO, produced by both bacteria and root cells, in the establishment of the association and in root branching, in order to correlate it with plant fitness. The understanding of how auxin induces NO synthesis in root cells and how plant modulates NO production in the microorganism would be major aims in the future research. Concerning this aspect, the use of genetic tools will be necessary to find PGPR strains and plants with lower and higher production of NO to study the mechanisms of NO synthesis and the ways by which NO modifies root architecture. The exploration of the tight link of NO in auxin‐modulated processes like root growth and development will surely be a matter of intense research in the next future.

NO AND PLANT GROWTH PROMOTING RHIZOBACTERIA

23

Fig. 3. Schematic model proposing NO synthesis and signaling pathways influencing root growth and developmental processes in the PGPR–root association. NO and IAA are produced by diVerent metabolic pathways in both PGPR and root cells. In the PGPR–root interaction, these signaling compounds can be exchanged between both partners. In root cells, IAA induces NO production by one or more hypothetical mechanisms. Then, NO acts as a messenger triggering a complex signaling network that leads to root branching and growth. The role of other hormones and cellular messengers are presented for LRD, AR formation, and suggested for RHF. Solid arrows indicate established pathways. Dashed arrows indicate pathways with supporting experimental evidence but not completely proved. Double gray arrows indicate unknown transport and/or diVusion processes. Abbreviations: aa, amino acids; cGMP, cyclic GMP; IAA, indole‐3‐acetic acid; IGP, indole‐3‐glycerol phosphate; L‐Arg, L‐arginine; MAPK, mitogen‐activated protein kinase; Trp, tryptophan.

ACKNOWLEDGMENTS We would like to thank Lic. Magdalena Graziano for the permission to include the photographs shown in the Figs. 1 and 2 of the present chapter. This work was supported by Agencia Nacional de Promocio´n Cientı´fica y Tecnolo´gica

24

C. MOLINA‐FAVERO ET AL.

(ANPCyT), Consejo Nacional de Investigaciones Cientı´ficas y Te´cnicas (CONICET), Fundacio´n Antorchas, and institutional grants from Universidad Nacional de Mar del Plata (UNMdP), Argentina. L.L. is a member of the Permanent Research StaV, M.C.L. is a technical assistant, and N.C.‐A. and M.L.L. are Postgraduate Fellows from CONICET, Argentina. C.M.C. and C.A.B. are Professors from the UNMdP. C.M.‐F is a Postgraduate Fellow from ANPCyT. L.L. is a fellow from J. S. Guggenheim Foundation.

REFERENCES Adak, S., Aulak, K. S. and Stuehr, D. J. (2002a). Direct evidence for nitric oxide production by a nitric‐oxide synthase‐like protein from Bacillus subtilis. The Journal of Biological Chemistry 277, 16167–16171. Adak, S., Bilwes, A. M., Panda, K., Hosfield, D., Aulak, K. S., McDonald, J. F., Tainer, J. A., GetzoV, E. D., Crane, B. R. and Stuehr, D. J. (2002b). Cloning, expression, and characterization of a nitric oxide synthase protein from Deinococcus radiodurans. Proceedings of the National Academy of Science United States of America 99, 107–112. Alvarez, M. I., Sueldo, R. J. and Barassi, C. A. (1996). EVect of Azospirillum on coleoptile growth in wheat seedlings under water stress. Cereal Research Communications 24, 101–107. Anderson, I. C., Poth, M., Homstead, J. and Burdige, D. (1993). A comparison of NO and N2O production by the autotrophic nitrifier Nitrosomonas europaea and the heterotrophic nitrifier Alcaligenes faecalis. Applied and Environmental Microbiology 59, 3525–3533. Araujo, W. L., Marcon, J., Maccheroni, W., Jr., van Elsas, J. D., van Vuurde, J. W. L. and Azevedo, J. L. (2002). Diversity of endophytic bacterial populations and their interaction with Xylella fastidiosa in citrus plants. Applied and Environmental Microbiology 68, 4906–4914. Bais, H. P., Weir, T. L., Perry, L. G., Gilroy, S. and Vivanco, J. M. (2006). The role of root exudates in rhizosphere interactions with plants and other organisms. Annual Review of Plant Biology 57, 233–266. Baluska, F., Salaj, J., Mathur, J., Braun, M., Jasper, F., Samaj, J., Chua, N. H., Barlow, P. W. and Volkmann, D. (2000). Root hair formation: F‐actin‐ dependent tip growth is initiated by local assembly of profilin‐supported F‐actin meshworks accumulated within expansin‐enriched bulges. Developmental Biology 227, 618–632. Barassi, C. A., Ayrault, G., Creus, C. M., Sueldo, R. J. and Sobrero, M. T. (2006). Seed inoculation with Azospirillum mitigates NaCl eVects on lettuce. Scientia Horticulturae 109, 8–14. Barbieri, P. and Galli, E. (1993). EVect on wheat root development of inoculation with an Azospirillum brasilense mutant with altered indole‐3‐acetic acid production. Research in Microbiology 44, 69–75. Barea, J. M. and Brown, M. E. (1974). EVects on plant growth produced by Azotobacter paspali related to synthesis of plant growth regulating substances. Journal of Applied Bacteriology 40, 583–593. Barroso, J. B., Corpas, F. J., Carreras, A., Sandalio, L. M., Valderrama, R., Palma, J. M., Lupianez, J. A. and del Rio, L. A. (1999). Localization of

NO AND PLANT GROWTH PROMOTING RHIZOBACTERIA

25

nitric‐oxide synthase in plant peroxisomes.The Journal of Biological Chemistry 274, 36729–36733. Bashan, Y. and Holguin, G. (1997). Azospirillum‐plant relationships: Environmental and physiological advances. Canadian Journal of Microbiology 43, 103–121. Bashan, Y., Alcaraz‐Melendez, L. and Toledo, G. (1992). Responses of soybean and cowpea root membranes to inoculation with Azospirillum brasilense. Symbiosis 13, 217–228. Bashan, Y., Holguin, G. and de‐Bashan, L. E. (2004). Azospirillum‐plant relationships: Physiological, molecular, agricultural, and environmental advances (1997– 2003). Canadian Journal of Microbiology 50, 521–577. Bashan, Y., Singh, C. S. and Levanony, H. (1989). Contribution of Azospirillum brasilense Cd to growth of tomato seedlings is not through nitrogen fixation. Canadian Journal of Botany 67, 2429–2434. Baudouin, E., Pauly, N. and Puppo, A. (2007). Nitric oxide in nitrogen‐fixing symbiosis. In ‘‘Nitric Oxide in Plant Growth, Development and Stress Physiology’’ (L. Lamattina and J. Polacco, eds.), pp. 173–186. Springer‐Verlag, Heidelberg. Baudouin, E., Pieuchot, L., Engler, G., Pauly, N. and Puppo, A. (2006). Nitric oxide is formed in Medicago truncatula‐Sinorhizobium meliloti functional nodules. Molecular Plant‐Microbe Interactions 19, 970–975. Beligni, M. V. and Lamattina, L. (2001). Nitric oxide: A non‐traditional regulator of plant growth. Trends in Plant Science 6, 508–509. Bethke, P. C., Badger, M. R. and Jones, R. L. (2004). Apoplastic synthesis of nitric oxide by plant tissues. The Plant Cell 16, 332–341. Bird, L. E., Ren, J., Zhang, J., Foxwell, N., Hawkins, A. R., Charles, I. G. and Stammers, D. K. (2002). Crystal structure of SANOS, a bacterial nitric oxide synthase oxygenase protein from Staphylococcus aureus. Structure 10, 1687–1696. Bloemberg, G. V. and Lugtenberg, B. J. J. (2001). Molecular basis of plant growth promotion and biocontrol by rhizobacteria. Current Opinion in Plant Biology 4, 343–350. Bloom, A. J., MeyerhoV, P. A., Taylor, A. R. and Rost, T. L. (2003). Root development and absorption of ammonium and nitrate from the rhizosphere. Journal of Plant Growth Regulation 21, 416–431. Boddey, R. M., Baldani, V. L. D., Baldani, J. I. and Do¨bereiner, J. (1986). EVect of inoculation of Azospirillum spp. on nitrogen accumulation by field grown wheat. Plant and Soil 95, 109–121. Bothe, H., Klein, B., Stephan, M. P. and Do¨bereiner, J. (1981). Transformations of inorganic nitrogen by Azospirillum spp. Archives of Microbiology 130, 96–100. Bothe, H., Ko¨rsgen, H., Lehmacher, T. and Hundeshagen, B. (1992). DiVerential eVects of Azospirillum, auxin and combined nitrogen on the growth of the roots of wheat. Symbiosis 13, 167–179. Brown, M. E. (1972). Plant growth substances produced by microorganisms of soil and rhizosphere. Journal of Applied Bacteriology 43, 443–451. Casanovas, E. M., Barassi, C. A., Andrade, F. H. and Sueldo, R. J. (2003). Azospirillum‐ inoculated maize plant responses to irrigation restraints imposed during flowering. Cereal Research Communications 31, 395–402. Castignetti, D. and Hollocher, T. C. (1984). Heterotrophic nitrification among denitrifiers. Applied and Environmental Microbiology 47, 620–623. Chen, Y. and Rosazza, J. P. N. (1994). A bacterial nitric oxide synthase from a Nocardia species. Biochemical and Biophysical Research Communications 203, 1251–1258.

26

C. MOLINA‐FAVERO ET AL.

Chen, Y. and Rosazza, J. P. N. (1995). Purification and characterization of nitric oxide synthase (NOSNoc) from a Nocardia species. Journal of Bacteriology 177, 5122–5128. Choi, D. W., Oh, H. Y., Hong, S. Y., Han, J. W. and Lee, H. W. (2000). Identification and characterization of nitric oxide synthase in Salmonella typhimurium. Archives of Pharmacal Research 23, 407–412. Choi, W. S., Chang, M. S., Han, J. W., Hong, S. Y. and Lee, H. W. (1997). Identification of nitric oxide synthase in Staphylococcus aureus. Biochemical and Biophysical Research Communications 237, 554–558. Clarkson, D. T. (1985). Factors aVecting mineral nutrient acquisition by plants. Annual Review of Plant Physiology 36, 77–115. Cohen, M. F. and Yamasaki, H. (2003). Involvement of nitric oxide synthase in sucrose‐enhanced hydrogen peroxide tolerance of Rhodococcus sp. strain APG1, a plant‐colonizing bacterium. Nitric Oxide 9, 1–9. Conn, V. M. and Franco, C. M. M. (2004). Analysis of the endophytic actinobacterial population in the roots of wheat (Triticum aestivum L.) by terminal restriction fragment length polymorphism and sequencing of 16s rRNA clones. Applied and Environmental Microbiology 70, 1787–1794. Cooney, R. V., Harwood, P. J., Custer, L. J. and Franke, A. A. (1994). Light‐ mediated conversion of nitrogen dioxide to nitric oxide by carotenoids. Environmental Health Perspectives 102, 460–462. Correa‐Aragunde, N., Graziano, M. and Lamattina, L. (2004). Nitric oxide plays a central role in determining lateral root development in tomato. Planta 218, 900–905. Correa‐Aragunde, N., Graziano, M., Chevalier, C. and Lamattina, L. (2006). Nitric oxide modulates the expression of cell cycle regulatory genes during lateral root formation in tomato. Journal of Experimental Botany 57, 581–588. Crawford, N. M., Galli, M., Tischner, R., Heimer, Y. M., Okamoto, M. and Mack, A. (2006). Response to Zemojtel et al. Plant nitric oxide synthase: Back to square one. Trends in Plant Science 11, 526–527. Creus, C. M., Sueldo, R. J. and Barassi, C. A. (1996). Azospirillum inoculation in pregerminating wheat seeds. Canadian Journal of Microbiology 42, 83–86. Creus, C. M., Sueldo, R. J. and Barassi, C. A. (1997). Shoot growth and water status in Azospirillum‐inoculated wheat seedlings grown under osmotic and salt stresses. Plant Physiology and Biochemistry 35, 939–944. Creus, C. M., Sueldo, R. J. and Barassi, C. A. (1998). Water relations in Azospirillum inoculated wheat seedlings under osmotic stress. Canadian Journal of Botany 76, 238–244. Creus, C. M., Sueldo, R. J. and Barassi, C. A. (2004). Water relations and yield in Azospirillum‐inoculated wheat exposed to drought in the field. Canadian Journal of Botany 82, 273–281. Creus, C. M., Graziano, M., Casanovas, E., Pereyra, M., Simontacchi, M., Puntarulo, S., Barassi, C. and Lamattina, L. (2005). Nitric oxide is involved in the Azospirillum brasilense‐induced lateral root formation in tomato. Planta 221, 297–303. Cueto, M., Herna´ndez‐Perera, O., Martı´n, R., Bentura, M. L., Rodrigo, J., Lamas, S. and Golvano, M. P. (1996). Presence of nitric oxide synthase activity in roots and nodules of Lupinus albus. FEBS Letters 398, 159–164. Cutruzzola´, F. (1999). Bacterial nitric oxide synthesis. Biochimica et Biophysica Acta 1411, 231–249. Deak, K. I. and Malamy, J. (2005). Osmotic regulation of root system architecture. Plant Journal 43, 17–28.

NO AND PLANT GROWTH PROMOTING RHIZOBACTERIA

27

De Klerk, G. J., Krieken, W. V. D. and Jong, J. (1999). The formation of adventitious roots: New concepts, new possibilities. In Vitro Cellular and Developmental Biology 35, 189–199. Dobbelaere, S., Croonenborghs, A., Thys, A., Vande Brooke, A. and Vanderleyden, J. (1999). Phytostimulatory eVect of Azospirillum brasilense wild type and mutant strains altered in IAA production on wheat. Plant and Soil 212, 155–164. Dobbelaere, S., Croonenborghs, A., Thys, A., Ptacek, D., Vanderleyden, J., Dutto, P., Labandera‐Gonzalez, C., Caballero‐Mellado, J., Aguirre, J. F., Kapulnik, Y., Brener, S., Burdman, S., et al. (2001). Responses of agronomically important crops to inoculation with Azospirillum. Australian Journal of Plant Physiology 28, 871–879. Do¨bereiner, J. and Day, J. M. (1976). Associative symbiosis in tropical grasses: Characterization of microorganisms and dinitrogen fixing sites. In ‘‘Proceedings of the First International Symposium on Nitrogen Fixation’’ (W. E. Newton and C. J. Nyman, eds.), pp. 518–538. Washington State University Press, Pullman, WA. Do¨bereiner, J. and Pedrosa, F. O. (1987). ‘‘Nitrogen‐Fixing Bacteria in Nonleguminous Crop P lants.’’ Science Tech. Publishers, Madison, WI. Dolan, L., Janmaat, K., Willemsen, V., Linstead, P., Poethig, S., Roberts, K. and Scheres, R. (1993). Cellular organization of the Arabidopsis thaliana root. Development 119, 71–84. Dolan, L., Duckett, C., Grierson, C., Linstead, P., Schneider, K., Lawson, E., Dean, C., Poethig, S. and Roberts, K. (1994). Clonal relations and patterning in the root epidermis of Arabidopsis. Development 120, 2465–2474. Fallik, E., Sarig, S. and Okon, Y. (1994). Morphology and physiology of plant roots associated with Azospirillum. In ‘‘Azospirillum/Plant Associations’’ (Y. Okon, ed.), pp. 75–85. CRC Press, Boca Raton, Florida. Felle, H. H. and Hepler, P. K. (1997). The cytosolic Ca2þ concentration gradient of Sinapsis alba root hairs as revealed by Ca2þ‐selective microelectrode tests and fura‐dextran ratio imaging. Plant Physiology 114, 39–45. Foreman, J., Demidchik, V., Bothwell, J. H. F., Mylona, P., Miedema, H., Torres, M. A., Linstead, P., Costa, S., Brownlee, C., Jones, J. D. G., Davies, J. M. and Dolan, L. (2003). Reactive oxygen species produced by NADPH oxidase regulate plant cell growth. Nature 422, 442–446. Galway, M. E., Masucci, J. D., Lloyd, A. M., Walbot, V., Davis, R. W. and Schiefelbein, J. W. (1994). The TTG gene is required to specify epidermal cell fate patterning in the Arabidopsis root. Developmental Biology 166, 740–754. Garcı´a de Salamone, I. E., Hynes, R. K. and Nelson, L. M. (2001). Cytokinin production by plant growth promoting rhizobacteria and selected mutants. Canadian Journal of Microbiology 47, 404–411. Garcı´a‐Mata, C., Gay, R., Sokolowski, S., Hills, A., Lamattina, L. and Blatt, M. R. (2003). Nitric oxide regulates Kþ channels in guard cells through a subset of ABA‐evoked signaling pathways. Proceedings of the National Academy of Sciences United States of America 100, 11116–11121. Gilroy, S. and Jones, D. (2000). Through form to function: Root hair development and nutrient uptake. Trends in Plant Science 5, 56–60. Glick, B. R. (1995). The enhancement of plant growth by free‐living bacteria. Canadian Journal of Microbiology 41, 109–117. Guckert, A. (1985). Root exudation and microbial activity at the soil‐root interface. Second IPI-ISS Workshop on Potassium, Nanjing, Academia Sinica, Nanjing, China, pp. 49–60.

28

C. MOLINA‐FAVERO ET AL.

Gupta, K. J., Stoimenova, M. and Kaiser, W. M. (2005). In higher plants, only root mitochondria, but not leaf mitochondria reduce nitrite to NO, in vitro and in situ. Journal of Experimental Botany 56, 2601–2609. Haissig, B. E. and Davis, T. D. (1994). An historical evaluation of adventitious rooting research to 1993. In ‘‘Biology of Adventitious Root Formation’’ (T. D. Davis and B. E. Haissig, eds.), pp. 275–331. Plenum Publishing Corporation, New York. Harari, A., Kigel, J. and Okon, Y. (1988). Involvement of IAA in the interaction between Azospirillum brasilense and Panicum miliaceum roots. Plant and Soil 110, 275–282. Hartmann, A. and Zimmer, W. (1994). Physiology of Azospirillum. In ‘‘Azospirillum/ Plant Association’’ (Y. Okon, ed.), pp. 15–39. CRC Press, Boca Raton, Florida. Hofer, R. M. (1996). Root hairs. In ‘‘Plants Roots: The Hidden Half’’ (Y. Waisel, A. Eshel and U. Kafkafi, eds.), pp. 111–126. Marcel Dekker, Inc., New York. Hong, I., Kim, Y. K., Choi, W. S., Seo, D. W., Yoon, J. W., Han, J. W., Lee, H. Y. and Lee, H. W. (2003). Purification and characterization of nitric oxide synthase from Staphylococcus aureus. FEMS Microbiology Letters 222, 177–182. Jain, D. K. and Patriquin, D. G. (1984). Root hair deformation, bacterial attachment, and plant growth in wheat‐Azospirillum associations. Applied and Environmental Microbiology 48, 1208–1213. Jasid, S., Simontacchi, M., Bartoli, C. G. and Puntarulo, S. (2006). Chloroplasts as a nitric oxide cellular source. EVect of reactive nitrogen species on chloroplastic lipids and proteins. Plant Physiology 142, 1246–1255. Jetten, M. S. M., Logemann, S., Muyzer, G., Robertson, L. A., de Vries, S., van Loosdrecht, M. C. M. and Kuenen, J. G. (1997). Novel principles in the microbial conversion of nitrogen compounds. Antonie van Leeuwenhoek 71, 75–93. Jones, D. L., ShaV, J. and Kochian, L. V. (1995). Role of calcium and other ions in directing root hair tip growth in Limnobium stoloniferum. Planta 197, 672–680. Kapulnik, Y., Okon, Y. and Henis, Y. (1985). Changes in root morphology of wheat caused by Azospirillum inoculation. Canadian Journal of Microbiology 31, 881–887. Kers, J. A., Wach, M. J., KrasnoV, S. B., Widom, J., Cameron, K. D., Bukhalid, R. A., Gibson, D. M., Crane, B. R. and Loria, R. (2004). Nitration of a peptide phytotoxin by bacterial nitric oxide synthase. Nature 429, 79–82. Kieber, J. J., Rothenberg, M., Roman, G., Feldmann, K. A. and Ecker, J. R. (1993). CTR1, a negative regulator of the ethylene response pathway in Arabidopsis, encodes a member of the raf family of protein kinases. Cell 72, 427–441. Kim, S. Y., Sivaguru, M. and Stacey, G. (2006). Extracellular ATP in plants. Visualization, localization, and analysis of physiological significance in growth and signaling. Plant Physiology 142, 984–992. Kloepper, J. W. (1992). Plant growth promoting rhizobacteria as biological control agents. In ‘‘Soil Microbial Ecology. Applications in Agricultural and Environmental Management’’ (F. B. Metting, Jr., ed.), pp. 255–274. Marcel Dekker, Inc., New York. Kundu, B. S., Sangwan, P., Sharma, P. K. and Nandwal, A. S. (1997). Response of pearl millet to phytohormones produced by Azospirillum brasilense. Indian Journal of Plant Physiology 2, 101–104. Kuroha, T., Kato, H., Asami, T., Yoshida, S., Kamada, H. and Satoh, S. (2002). A trans‐zeatin riboside in root xylem sap negatively regulates adventitious

NO AND PLANT GROWTH PROMOTING RHIZOBACTERIA

29

root formation on cucumber hypocotyls. Journal of Experimental Botany 53, 2193–2200. Kutz, A., Muller, A., Hennig, P., Kaiser, W. M., Piotrowski, M. and Weiler, E. W. (2002). A role for nitrilase 3 in the regulation of root morphology in sulphur‐starving Arabidopsis thaliana. The Plant Journal 30, 95–106. Lamattina, L. and Polacco, J. (2007). ‘‘Nitric Oxide in Plant Growth, Development and Stress Physiology,’’ p. 280. Springer‐Verlag, Germany. Lamattina, L., Garcia‐Mata, C., Graziano, M. and Pagnussat, G. (2003). Nitric oxide: The versatility of an extensive signal molecule. Annual Review of Plant Biology 54, 109–136. Lambrecht, M., Okon, Y., Vande Broek, A. and Vanderleyden, J. (2000). Indole‐3‐ acetic acid: A reciprocal signalling molecule in bacteria‐plant interactions. Trends in Microbiology 8, 298–300. Lamotte, O., Courtois, C., Dobrowolska, G., Besson, A., Pugin, A. and Wendehenne, D. (2006). Mechanisms of nitric‐oxide‐induced increase of free cytosolic Ca2þ concentration in Nicotiana plumbaginifolia cells. Free Radical Biology & Medicine 40, 1369–1376. Lamotte, O., Gould, K., Lecourieux, D., Sequeira‐Legrand, A., Lebrun‐Garcia, A., Durner, J., Pugin, A. and Wendehenne, D. (2004). Analysis of nitric oxide signaling functions in tobacco cells challenged by the elicitor cryptogein. Plant Physiology 135, 516–529. Lang, E. and Jagnow, G. (1986). Fungi of a forest soil nitrifying at low pH values. FEMS Microbiology Ecology 38, 257–265. Lanteri, M. L., Pagnussat, G. C. and Lamattina, L. (2006a). Calcium and calcium‐ dependent protein kinases are involved in nitric oxide‐ and auxin‐induced adventitious root formation in cucumber. Journal of Experimental Botany 57, 1341–1351. Lanteri, M. L., Graziano, M., Correa‐Aragunde, N. and Lamattina, L. (2006b). From cell division to organ shape: Nitric oxide is involved in auxin‐ mediated root development. In ‘‘Communication in Plants’’ (F. Baluska, S. Mancuso and D. Volkmann, eds.), pp. 123–136. Springer‐Verlag, Berlin, Heidelberg. Leshem, Y. Y., Wills, R. B. H. and Veng‐Va Ku, V. (1998). Evidence for the function of the free radical gas, nitric oxide (NO), as an endogenous maturation and senescence regulating factor in higher plants. Plant Physiology and Biochemistry 36, 825–833. Levanony, H. and Bashan, Y. (1989). Enhancement of cell division in wheat root tips and growth of root elongation zone induced by Azospirillum brasilense Cd. Canadian Journal of Botany 67, 2213–2216. Lindermayr, C., Saalbach, G., Bahnweg, G. and Durner, J. (2006). DiVerential inhibition of Arabidopsis methionine adenosyltransferases by protein S‐nitrosylation. Journal of Biological Chemistry 281, 4285–4291. Linkohr, B. I., Williamson, L. C., Fitter, A. H. and Leyser, H. M. (2002). Nitrate and phosphate availability and distribution have diVerent eVects on root system architecture of Arabidopsis. The Plant Journal 29, 751–760. Liu, Y., Chen, S. F. and Li, J. I. (2003). Colonization pattern of Azospirillum brasilense yu62 on maize roots. Acta Botanica Sinica 45, 748–752. Lombardo, M. C., Graziano, M., Polacco, J. C. and Lamattina, L. (2006). Nitric oxide functions as a positive regulator of root hair development. Plant Signaling & Behavior 1, 28–33. Malamy, J. E. (2005). Intrinsic and environmental response pathways that regulate root system architecture. Plant, Cell & Environment 28, 67–77.

30

C. MOLINA‐FAVERO ET AL.

Malamy, J. E. and Benfey, P. N. (1997). Organization and cell diVerentiation in lateral roots of Arabidopsis thaliana. Development 124, 33–44. Meyer, C. and Sto¨hr, C. (2002). Nitrate reductase and nitrite reductase. In ‘‘Advances in Photosynthesis: Photosynthxetic Nitrogen Assimilation and Associated Carbon Metabolism’’ (C. H. Foyer and G. Noctor, eds.), pp. 49–62. Kluwer Academic Publishers, Dordrecht. Midha, S., Mishra, R., Aziz, M. A., Sharma, M., Mishra, A., Khandelwal, P. and Bhatnagar, R. (2005). Cloning, expression, and characterization of recombinant nitric oxide synthase‐like protein from Bacillus anthracis. Biochemical and Biophysical Research Communications 336, 346–356. Moore, C. M., Nakano, M. M., Wang, T., Ye, R. W. and Helmann, J. D. (2004). Response of Bacillus subtilis to nitric oxide and the nitrosating agent sodium nitroprusside. Journal of Bacteriology 186, 4655–4664. Mulder, L., Hogg, B., Bersoult, A. and Cullimore, J. V. (2005). Integration of signalling pathways in the establishment of the legume‐rhizobia symbiosis. Physiologia Plantarum 123, 207–218. Nakano, M. (2002). Induction of ResDE‐dependent gene expression in Bacillus subtilis in response to nitric oxide and nitrosative stress. Journal of Bacteriology 184, 1783–1787. Navarro, L., Dunoyer, P., Jay, F., Arnold, B., Dharmasiri, N., Estelle, M., Voinnet, O. and Jones, J. D. G. (2006). A plant miRNA contributes to antibacterial resistance by repressing auxin signaling. Science 312, 436–439. Neuer, G., Kronemberg, A. and Bothe, H. (1985). Denitrification and nitrogen fixation by Azospirillum. III Properties of a wheat‐Azospirillum association. Archives of Microbiology 141, 364–370. Ohashi, Y., Oka, A., Rodrigues‐Pousada, R., Possenti, M., Ruberti, I., Morelli, G. and Aoyama, T. (2003). Modulation of phospholipid signaling by GLABRA2 in root‐hair pattern formation. Science 300, 1427–1430. Okon, Y. (1985). Azospirillum as a potential inoculant for agriculture. Trends in Biotechnology 3, 223–228. Okon, Y. and Labandera‐Gonza´lez, C. A. (1994). Agronomic applications of Azospirillum: An evaluation of 20 years worldwide field inoculation. Soil Biology and Biochemistry 26, 1591–1601. Otvos, K., Pasternak, T. P., Miskolczi, P., Domoki, M., Dorjgotov, D., Szucs, A., Bottka, S., Dudits, D. and Feher, A. (2005). Nitric oxide is required for, and promotes auxin‐mediated activation of, cell division and embryogenic cell formation but does not influence cell cycle progression in alfalfa cell cultures. The Plant Journal 43, 849–860. Pagnussat, G. C., Lanteri, M. L. and Lamattina, L. (2003). Nitric oxide and cyclic GMP are messengers in the IAA‐induced adventitious rooting process. Plant Physiology 132, 1241–1248. Pagnussat, G. C., Lanteri, M. L. and Lamattina, L. (2004). Nitric oxide mediates the indole acetic acid induction activation of a mitogen‐activated protein kinase cascade involved in adventitious root development. Plant Physiology 135, 279–286. Pagnussat, G. C., Simontacchi, M., Puntarulo, S. and Lamattina, L. (2002). Nitric oxide is required for root organogenesis. Plant Physiology 129, 954–956. Papen, H., von Berg, R., Hinkel, I., Thoene, B. and Rennenberg, H. (1989). Hetero trophic nitrification by Alcaligenes faecalis: NO 2 , NO3 , N2O, and NO production in exponentially growing cultures. Applied & Environmental Microbiology 55, 2068–2072. Patrikin, D. G., Do¨bereiner, J. and Jain, D. K. (1983). Sites and processes of association between diazotrophs and grasses. Canadian Journal of Microbiology 29, 900–915.

NO AND PLANT GROWTH PROMOTING RHIZOBACTERIA

31

Paul, E. A. and Clark, F. E. (1996). Ammonification and nitrification. In ‘‘Soil Microbiology and Biochemistry’’ (E. A. Paul and F. E. Clark, eds.), 2nd Edn., pp. 191–197. Academic Press, New York. Peng, G., Wang, H., Zhang, G., Hou, W., Liu, Y., Wang, E. T. and Tan, Z. (2006). Azospirillum melinis sp. nov., a group of diazotrophs isolated from tropical molasses grass. International Journal of Systematic and Evolutionary Microbiology 56, 1263–1271. Penteado Stephan, M., Zimmer, W. and Bothe, H. (1984). Denitrification of Azospirillum brasilense Sp7. II. Growth with nitrous oxide as respiratory electron acceptor. Archives of Microbiology 138, 212–216. Peterson, R. L. and Farquhar, M. L. (1996). Root hairs: Specialized tubular cells extending root surfaces. Botanical Research 62, 2–33. Pitts, R. J., Cernec, A. and Estelle, M. (1998). Auxin and ethylene promote root hair elongation in Arabidopsis. The Plant Journal 16, 553–560. Prinsen, E., Costacurta, A., Michiels, K., Vanderleyden, J. and Van Onckelen, H. (1993). Azospirillum brasilense indole‐3‐acetic acid biosynthesis: Evidence for a non‐tryptophan dependent pathway. Molecular Plant Microbe Interactions 6, 609–615. Ribaudo, C. M., Krumpholz, E. M., Cassa´n, F. D., Bottini, R., Cantore, M. L. and Cura´, J. A. (2006). Azospirillum sp. promotes root hair development in tomato plants through a mechanism that involves ethylene. Journal of Plant Growth Regulation 24, 175–185. Roberts, J. L. and Roberts, E. (1939). Auxin production by soil microorganism. Soil Science 48, 135–139. Ryan, E., Steer, M. and Dolan, L. (2001). Cell biology and generation of root hair formation in Arabidopsis thaliana. Protoplasma 215, 140–149. Ryu, C. M., Farag, M. A., Hu, C. H., Reddy, M. S., Kloepper, J. W. and Pare, R. W. (2004). Bacterial volatiles induce systemic resistance in Arabidopsis. Plant Physiology 134, 1017–1026. Samaj, J., Baluska, F. and Menzel, D. (2004). New signaling molecules regulating root hair tip growth. Trends in Plant Science 9, 217–220. Sari, M. A., Moali, C., Boucher, J. L., Jaouen, M. and Mansuy, D. (1998). Detection of a nitric oxide synthase possibly involved in the regulation of the Rhodococcus sp. R312 nitrile hydratase. Biochemical and Biophysical Research Communications 250, 364–368. Sarig, S., Blum, A. and Okon, Y. (1988). Improvement of the water status and yield of field‐grown grain sorghum (Sorghum bicolor) by inoculation with Azospirillum brasilense. The Journal of Agricultural Science 110, 271–277. Schiefelbein, J. W., Shipley, A. and Eowse, P. (1992). Calcium influx at the tip of growing root‐hair cells of Arabidopsis thaliana. Planta 187, 455–459. She, X. P. and Huang, A. X. (2004). Change of nitric oxide and NADPH‐diaphorase during the generation and the development of adventitious roots in mung bean hypocotyls cuttings. Acta Botanica Sindica 46, 1049–1055. Son, J. K. and Rosazza, J. P. N. (2000). Cyclic guanosine‐30 ,50 ‐monophosphate and biopteridine biosynthesis in Nocardia sp. Journal of Bacteriology 182, 3644–3648. Steenhoudt, O. and Vanderleyden, J. (2000). Azospirillum a free‐living nitrogen‐fixing bacterium closely associated with grasses: Genetic, biochemical and ecological aspects. FEMS Microbiology Reviews 24, 487–506. Steenhoudt, O., Ping, Z., Vande Broek, A. and Vanderleyden, J. (2001a). A spontaneous chlorate‐resistant mutant of Azospirillum brasilense Sp245 displays defects in nitrate reduction and plant root colonization. Biology and Fertility of Soils 33, 317–322.

32

C. MOLINA‐FAVERO ET AL.

Steenhoudt, O., Keijers, V., Okon, Y. and Vanderleyden, J. (2001b). Identification and characterization of a periplasmic nitrate reductase in Azospirillum brasilense Sp245. Archives of Microbiology 175, 344–352. SteVens, B., Wang, J. and Sauter, M. (2006). Interactions between ethylene, gibberellin and abscisic acid regulate emergence and growth rate of adventitious roots in deepwater rice. Planta 223, 604–612. Sto¨hr, C. and Ullrich, W. R. (2002). Generation and possible roles of nitric oxide in plant roots and their apoplastic space. Journal of Experimental Botany 53, 2293–2303. Sto¨hr, C. and Stremlau, S. (2006). Formation and possible roles of nitric oxide in plant roots. Journal of Experimental Botany 57, 463–470. Sto¨hr, C., Strube, F., Marx, G., Ullrich, W. R. and Rockel, P. (2001). A plasma membrane‐bound enzyme of tobacco roots catalyses the formation of nitric oxide from nitrite. Planta 212, 835–841. Stoimenova, M., Libourel, I. G. L., RatcliVe, R. G. and Kaiser, W. M. (2003). The role of root nitrate reduction in the anoxic metabolism of roots. II. Anoxic metabolism of tobacco roots with or without nitrate reductase activity. Plant and Soil 253, 155–167. Stuehr, D. J. (1997). Structure‐function aspects in the nitric oxide synthases. Annual Reviews of Pharmacological Toxicology 37, 339–359. Sudhamsu, J. and Crane, B. R. (2006). Structure and reactivity of a thermostable prokaryotic nitric‐oxide synthase that forms a long‐lived oxy‐heme complex. The Journal of Biological Chemistry 281, 9623–9632. Takahashi, F., Sato‐Nara, K., Kobayashi, K., Suzuki, M. and Suzuki, H. (2003). Sugar‐induced adventitious roots in Arabidopsis seedlings. Journal of Plant Research 116, 83–91. Tanimoto, M., Roberts, K. and Dolan, L. (1995). Ethylene is a positive regulator of root hair development in Arabidopsis thaliana. The Plant Journal 8, 943–948. Tien, T. M., Gaskin, M. H. and Hubbell, D. H. (1979). Plant growth substances produced by Azospirillum brasilense and their eVect on the growth of pearl millet (Pennisetum americanum L.). Applied and Environmental Microbiology 37, 1016–1024. Tosques, I. E., Shi, J. and Shapleigh, J. P. (1996). Cloning and characterization of nnrR, whose product is required for the expression of proteins involved in nitric oxide metabolism in Rhodobacter sphaeroides 2.4.3. Journal of Bacteriology 178, 4958–4964. Van Bruaene, N., Joss, G. and Van Oostveldr, P. (2004). Reorganization and in vivo dynamics of microtubules during Arabidopsis root hair development. Plant Physiology 136, 3905–3919. Vande Broek, A., Dobbelaere, S., Vanderleyden, J. and Van Dommeles, A. (2000). Azospirillum‐plant root interactions: Signaling and metabolic interactions. In ‘‘Prokaryotic Nitrogen Fixation: A Model System for the Analysis of a Biological Process’’ (E. W. Triplett, ed.), pp. 761–777. Horizon Scientific Press, Wymondham, UK. van der Weele, C. M., Spollen, W. G., Sharp, R. E. and Baskin, T. I. (2000). Growth of Arabidopsis thaliana seedlings under water deficit studied by control of water potential in nutrient‐agar media. Journal of Experimental Botany 51, 1555–1562. Verstraete, W. and Alexander, M. (1972). Heterotrophic nitrification by Arthrobacter sp. Journal of Bacteriology 110, 955–961.

NO AND PLANT GROWTH PROMOTING RHIZOBACTERIA

33

Voßwinkel, R., Neidt, I. and Bothe, H. (1991). The production and utilization of nitric oxide by a new, denitrifying strain of Pseudomonas aeruginosa. Archives of Microbiology 156, 62–69. Weitzberg, E. and Lundberg, J. O. N. (1998). Nonenzymatic nitric oxide production in humans. Nitric Oxide 2, 1–7. Werner‐Felmayer, G., Golderer, G., Werner, E. R., Grobner, P. and Wachter, H. (1994). Pteridine biosynthesis and nitric oxide synthase in Physarum polycephalum. Biochemical Journal 304, 105–111. White, P. (1998). Calcium channels in the plasma membrane of root cells. Annals of Botany 81, 173–183. Witzel, K. P. and Overbeck, H. J. (1979). Heterotrophic nitrification by Arthrobacter sp. (strain 9006) as influenced by diVerent cultural conditions, growth state and acetate metabolism. Archives of Microbiology 122, 137–143. Wrage, N., Velthof, G. L., van Beusichem, M. L. and Oenema, O. (2001). Role of nitrifier denitrification in the production of nitrous oxide. Soil Biology and Biochemistry 33, 1723–1732. Wymer, C. L., Bibikova, T. and Gilroy, S. (1997). Cytoplasmic free calcium distributions during the development of root hairs of Arabidopsis thaliana. The Plant Journal 12, 427–439. Xia, Y., Embley, T. M. and O’Donnell, A. G. (1994). Phylogenetic analysis of Azospirillum by direct sequencing of PCR‐amplified 16S rDNA. Systematic and Applied Microbiology 17, 197–201. Xie, C. H. and Yokota, A. (2005). Azospirillum oryzae sp. nov., a nitrogen‐fixing bacterium isolated from the roots of the rice plant Oryza sativa. International Journal of Systematic and Evolutionary Microbiology 55, 1435–1438. He, Y., Yu, W. and Baas, P. W. (2002). Microtubule reconfiguration during axonal retraction induced by nitric oxide. Journal of Neuroscience 22, 5982–5991. Ye, R. W., Averill, B. A. and Tiedje, J. M. (1994). Denitrification: Production and consumption of nitric oxide. Applied and Environmental Microbiology 60, 1053–1058. Zamudio, M. and Bastarrachea, F. (1994). Adhesiveness and root hair deformation capacity of Azospirillum strains for wheat seedlings. Soil Biology and Biochemistry 26, 791–797. Zemojtel, T., Frohlich, A., Palmieri, M. C., Kolanczyk, M., Mikula, I., Wyrwicz, L. S., Wanker, E. E., Mundlos, S., Vingron, M., Martasek, P. and Durner, J. (2006). Plant nitric oxide synthase: A never‐ending story? Trends in Plant Science 11, 524–525. Zhang, H. and Forde, B. G. (2000). Regulation of Arabidopsis root development by nitrate availability. Journal of Experimental Botany 51, 51–59. Zhu, C., Gan, L., Shen, Z. and Xia, K. (2006). Interaction between jasmonates and ethylene in the regulation of root hair development in Arabidopsis. Journal of Experimental Botany 57, 1299–1308. Zimmer, W. and Bothe, H. (1988). The phytohormonal interactions between Azospirillum and wheat. Plant and Soil 110, 239–247. Zimmer, W., Penteado‐Stephan, M. and Bothe, H. (1984). Denitrification by Azospirillum brasilense Sp 7. Archives of Microbiology 138, 206–211. Zimmer, W., Roeben, K. and Bothe, H. (1988). An alternative explanation for plant growth promotion by bacteria of the genus Azospirillum. Planta 176, 333–342. Zumft, W. G. (1997). Cell biology and molecular basis of denitrification. Microbiology and Molecular Biology Reviews 61, 533–616.

How the Environment Regulates Root Architecture in Dicots

MARIANA JOVANOVIC,* VALE´RIE LEFEBVRE,*,{ PHILIPPE LAPORTE,* SILVINA GONZALEZ‐RIZZO,* CHRISTINE LELANDAIS‐BRIE`RE,*,{ FLORIAN FRUGIER,* CAROLINE HARTMANN*,{ AND MARTIN CRESPI*

*Institut des Sciences du Ve´ge´tal, Centre National de la Recherche Scientifique, 91198 Gif sur Yvette, France { Universite´ Paris VII‐Denis Diderot, 2 place Jussieu, 75251 Paris Cedex 5, France

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . II. The Root System and the Model A. thaliana . . . . . . . . . . . . . . . . . . A. The RAM: Establishment and Patterning .................................. B. Radial Organization of Root Tissues........................................ C. LR Organogenesis .............................................................. III. Root Growth in the Soil Environment. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Endogenous Signals Regulating Root Growth ............................ B. The Peculiar Legume Root System and its Symbiotic Interactions ........................................................ IV. Changing Root Architecture: Adaptive Responses to the Soil Environment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Water Availability and the Osmotic Potential of the Medium .......... B. Water Excess and Adventitious Rooting ................................... C. Nutrient Availability ........................................................... D. Effects of Abiotic Stresses on Legume Roots .............................. V. Root Growth and Differentiation in Response to Environmental Conditions: Small Noncoding RNAs as New Posttranscriptional Regulators . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VI. Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Advances in Botanical Research, Vol. 46 Incorporating Advances in Plant Pathology Copyright 2008, Elsevier Ltd. All rights reserved.

36 38 39 41 41 42 42 46 47 48 51 52 54 55 59 61 61

0065-2296/08 $35.00 DOI: 10.1016/S0065-2296(07)46002-5

36

M. JOVANOVIC ET AL.

ABSTRACT The eYcient acquisition of soil resources (nutrients and water) through the root system is crucial for crop productivity. In order to adapt root growth to the soil environment, plants can optimize their root architecture by initiating primordia and influencing growth of primary roots or lateral roots (LRs). Root architecture results from the integration of genetic programs governing root growth patterns and environmental factors which aVect signaling pathways. We review here recent knowledge acquired mainly in Arabidopsis thaliana on primary root and LR development and the impact that diVerent environmental constraints (water, phosphate, nitrate, and sulfate) have on root growth and development. Since Arabidopsis is unable to develop specific organogenesis resulting from symbiotic interactions, we also discuss recent molecular data on the analysis of the nitrogen‐fixing symbiotic nodules and their influence on root architecture in legumes. Finally, molecular analysis of the role of noncoding RNAs in environmentally activated signaling pathways will be discussed. These RNAs are emerging as crucial regulators of diVerentiation and adaptation to environmental conditions.

ABBREVIATIONS ABA advR BR CC CK IC LR miRNA N nat‐siRNA npc RNA P QC QTL RAM ROS S siRNA tasiRNA

abscissic acid adventitious root brassinosteroids cortical cell cytokinin initial cell lateral root microRNA nitrogen natural antisense‐mediated siRNA nonprotein coding RNA phosphate quiescent center quantitative trait locus root apical meristem reactive oxygen species sulfur small interfering RNA trans‐acting siRNA

I. INTRODUCTION Plant development after germination is essentially derived from stem cells localized in two apical regions formed during embryogenesis, the shoot and root apical meristems. This particular characteristic allows plants, which are sessile organisms, to adapt their morphology and consequently organ development to environmental conditions. The root system, which

ROOT ARCHITECTURE AND ENVIRONMENT

37

shows indeterminate growth, plays a crucial role in the survival of land plants under a wide variety of conditions. It assures two main functions: the anchorage to the soil and the exploration thereof for water and mineral nutrients. The root system has therefore a major impact on crop yield and productivity (Lynch, 1995). Moreover, the root is a remarkable example of developmental plasticity: its spatial configuration (number and length of lateral organs), so‐called architecture, varies greatly, depending on the plant species, soil composition, and, particularly, on water and mineral nutrients availability. Thus, extensive morphological diVerences (in size, number, and distribution of lateral root organs) are observed in genetically identical plants cultivated under diVerent nutritional conditions (Lopez‐ Bucio et al., 2003). An optimal adaptation of root architecture to the soil allows plants to recover eYciently critical resources and increase their ecological fitness when these resources are limited. Understanding the molecular mechanisms governing such developmental plasticity is therefore likely to be crucial for crop improvement in sustainable agriculture. Root architecture is under the coordinated control of both genetic endogenous programs regulating growth and organogenesis and the action of abiotic and biotic environmental stimuli. The mature root system therefore results from the integration of intrinsic and extrinsic signals (Malamy, 2005). Their interactions however complicate the dissection of specific transduction pathways involved in root growth and development. Such complex traits likely depending on multiple genes may be eYciently analyzed through quantitative genetics. For instance, in the model plant Arabidopsis thaliana and in maize, a largely cultivated cereal species, quantitative trait loci (QTL) linked to root architecture have been identified (Mouchel et al., 2004; Tuberosa et al., 2002a,b). In this chapter, we discuss the influence of the soil environment on root growth and diVerentiation through its action on existing and de novo meristems. First, we will briefly describe the Arabidopsis model root system and its main features: the root apical meristem (RAM) and lateral roots (LRs). In the wild, plant roots are surrounded by microorganisms in the rhizosphere that can modify their architecture. Unfortunately, A. thaliana is not able to form symbioses, although root symbiotic associations are essential to more than 80% of higher plants (Hirsch and LaRue, 1997). Hence, a second part of this chapter will be dedicated to the symbiotic associations of legumes with bacteria, collectively called rhizobia. These bacteria modify the root system by inducing the formation of new meristems which form root nodules that are able to fix nitrogen (N). This allows legumes to grow in N‐poor soils (Crespi and Galvez, 2000; Stacey et al., 2006). In contrast, mycorrhizal associations between fungi and plant roots allowing the expansion of the

38

M. JOVANOVIC ET AL.

explored soil volume will not be discussed here as they do not imply the formation of new meristems. Recent reviews are available on this subject (Brachmann, 2006; Gianinazzi‐Pearson et al., 2007; Graham and Miller, 2005; Harrison, 2005; Wang and Qiu, 2006) as these associations may have been critical for the colonization of the land by plants early in evolution and have a major impact on root metabolism and architecture. Furthermore, we will not describe here the eVects of plant growth‐promoting rhizobacteria (PGPR) in promoting LR development since an excellent review in this issue is dedicated to this topic (Molina‐Farero et al., 2007). In a third part of this chapter, we will emphasize on the impact of certain soil resources and their availability on the modification of the root system growth. Finally, we will focus on the recent work on RNA‐mediated posttranscriptional regulation, which may be crucial in root diVerentiation, auxin signaling as well as biotic and abiotic interactions, to further apprehend the diverse mechanisms involved in the formation of a root system.

II. THE ROOT SYSTEM AND THE MODEL A. THALIANA Arabidopsis displays a typical allorhizic root system: the primary root is derived from the embryonic root and the development of LRs is initiated from a specific set of cells located in the pericycle of the primary root. Adventitious roots (advRs) can also appear, under particular culture conditions, diVerentiating from pericycle cells at the hypocotyl–root junction (Sorin et al., 2005). Arabidopsis roots, like most monocots and dicots, comprise three zones: (1) the distal root apex, consisting of the root cap that protects the underlying RAM, where cells divide actively; (2) an elongation zone above the RAM, where cells expand mainly in a longitudinal direction; and (3) a diVerentiation zone. Roots are composed of concentric cell layers originating from the RAM (Fig. 1). The Arabidopsis epidermal cell layer (the most external) presents a specific pattern of root hair distribution, with a defined alternation of atrichoblast and trichoblast cell files (corresponding to non‐hair‐forming and hair‐forming cells, respectively) (Dolan and Costa, 2001). The two inner layers, called cortex and endodermis, which envelop the stele, consist each of a single cell file (Benfey and Scheres, 2000). Even though the Arabidopsis root patterning is generally conserved, many variations in root anatomy exist. For example, in legume roots, the epidermal cell files show no specific root hair patterning and the cortex consists of three to five cell layers, usually defined as outer, middle, and inner cortex (Gage, 2004).

39

ROOT ARCHITECTURE AND ENVIRONMENT

Lateral root primordium

Lateral root initiation

Stele and vascular tissues Pericycle

Endodermis Cortex Epidermis

Root apical meristem (RAM) Cortex/ endodermis initial

Quiescent center (QC)

Epidermis/ lateral root cap initial

Lateral root cap Columella

Fig. 1.

Schematic representation of primary root cell lineage and LR formation. A. THE RAM: ESTABLISHMENT AND PATTERNING

The RAM of angiosperms comprises a slowly dividing quiescent center (QC), which is surrounded by mitotically active initial cells (ICs) that give rise to the diVerent cell types constitutive of root tissues and therefore could be considered as stem cells (Fig. 1; Benfey and Scheres, 2000). Plant and animal stem cells develop in a microenvironment, the stem cell niche, where they can be auto‐maintained in a nondiVerentiated state through the action of diverse

40

M. JOVANOVIC ET AL.

signals (Singh and Bhalla, 2006). In Arabidopsis, the QC consists of 4 cells surrounded by 1 IC layer, whereas in maize, 800–1200 cells may compose the QC, surrounded by several hundred ICs (Feldman, 1994). After each IC division, one daughter cell leaves the RAM, becomes isolated from the stem niche signal(s), and then starts diVerentiation. To better understand QC function, Nawy et al. (2005) used microarrays to determine the expression pattern of its four cells. They first generated a transgenic line expressing a marker under the control of a cis‐regulatory sequence belonging to the gene encoding the MADS box transcription factor AGL42, expressed in the QC. Using cell‐sorting of root protoplasts, cells expressing this construct were used in a transcription profiling experiment that demonstrated the enrichment of 290 genes belonging to 3 major functional categories: (1) hormonal signaling [auxin: 5 genes, gibberellin (GA): 3 genes, and brassinosteroid (BR): 1 gene]; (2) transcription factors (37 genes); and (3) metabolism (63 genes). The absence of phenotypes for mutants aVected in 11 of the QC‐enriched transcription factors suggests functional redundancies between them, likely to assure root growth and survival. RAM specification occurs very early in embryo development with diVerentiation of the hypophysis, the apical cell of the suspensor (Benfey and Scheres, 2000). Auxin appears to be essential for this process as many auxin‐related mutants, such as monopteros (mp), bodenlos (bdl), and auxin transport inhibitor resistant 1 (tir1) and related tir1/afb1–3 (auxin signaling F‐box gene 1, 2, and 3) quadruple mutant, are unable to specify the hypophysis and then to form the embryonic RAM. The auxin flux coming from the apical region of the embryo into the hypophysis leads to TIR1 (and related redundant AFBs) pathway activation and induction of auxin‐response genes such as PIN genes (coding for auxin eZux carriers), whose products will increase auxin transport and accumulation into the hypophysis to further diVerentiate this cell (Benkova et al., 2003). After division, the hypophysis generates the QC and part of the root cap. RAM diVerentiation is under auxin control and involves a complex network of interactions in order to maintain the stem cell niche in the distal part of the root (Aida et al., 2004). The RAM has two functions: (1) determination of the root patterning, through IC stereotyped divisions, leading to the formation of the diVerent root cell files and (2) auto‐maintenance of stem cells to allow later postembryonic root growth. Two GRAS transcription factors, SCARECROW (SCR) and SHORT ROOT (SHR), have been associated with RAM maintenance: indeed, root growth is delayed in scr and shr mutants due to the lack of one IC formation, leading to the absence of endodermal cell files (Di Laurenzio et al., 1996; Helariutta et al., 2000; Scheres et al., 1995). Although SHR proteins control SCR expression, QC function cannot be completely rescued when the SCR protein is overexpressed in an shr background. Levesque

ROOT ARCHITECTURE AND ENVIRONMENT

41

et al. (2006) have identified eight potential targets for SHR using microarrays analyses. Thus, SHR not only controls SCR, but certainly acts on other genes to regulate IC diVerentiation. B. RADIAL ORGANIZATION OF ROOT TISSUES

Arabidopsis roots can be viewed as a set of concentric cylinders. As mentioned earlier, the epidermal cells form trichoblasts and atrichoblasts. With respect to the position of the neighboring cortical cells (CCs), contact of one epidermal cell with only one CC would lead to an atrichoblast fate, while contact with two CCs would lead to a trichoblast fate (Berger et al., 1998). A whole regulatory network of transcription factors and, more recently, chromatin organization (at least at some loci like GLABRA 2 ) have been involved in signaling the positional information defined by CCs (Bernhardt et al., 2003; Costa and Shaw, 2006). The cortical and endodermis cell files originate from the asymmetrical division of a single IC. The SHR and SCR transcription factors are involved in this specification event, and SHR synthesized in the stele may diVuse into the endodermis to regulate SCR expression. This movement may be linked to cell specification in the radial axis of the root (Gallagher et al., 2004). C. LR ORGANOGENESIS

In dicots, the root system is constituted by the primary root and several orders of LRs, which are produced throughout the plant’s life. Root system architecture is dependent on the number and size of LRs. LR development (Fig. 1) can be divided in diVerent steps: primordium initiation and development, emergence, and meristem activation. LR initiation is the key element for LR development. It occurs strictly acropetally; for example, a primordium is always initiated in a more distal root portion relatively to already initiated LRs and de novo initiation is not possible between two LRs primordia or two mature LRs. Moreover, branching capacity may be accession specific (Dubrovsky et al., 2006). Pericycle founder cells, from which the LRs originate, are peculiar cells that retain the ability to dediVerentiate and divide—a characteristic of stem cells—even after leaving the RAM (Beeckman et al., 2001; DiDonato et al., 2004; Dubrovsky et al., 2000). This particular cell population accounts mainly for the extensive developmental plasticity of the root and may be responsive to both an endogenous control and environmental cues. How the competence of the founder cells is determined remains still unknown. In Arabidopsis, the root primordium originates from at least three founder

42

M. JOVANOVIC ET AL.

cells (Fig. 1) undergoing first anticlinal divisions in front of protoxylem poles (Malamy and Benfey, 1997a,b). This event is essential to LR initiation: alf4 mutant (aberrant lateral root formation 4), which is blocked in the LR initiation, has lost its capacity to maintain pericycle cells in a mitotically active state (DiDonato et al., 2004); this has been nicely shown using the CycB1;1 marker gene (only expressed around the G2/M cell cycle transition) (Fukaki et al., 2002). As well, the dominant mutation slr‐1 (solitary root‐1) aVected in IAA14 (a member of the AUX/IAA protein family) cannot develop LRs due to a lack of early cell divisions (Fukaki et al., 2002). Unlike Arabidopsis, LR primordia of other angiosperms arise from periclinal divisions, and sometimes in front of protophloem pole (Mallory et al., 1970). After the primordium has been formed inside the parental root, cell elongation is responsible for its emergence outward. The LR meristem seems identical to the embryonic RAM. Mutant analyses indeed revealed that abnormalities found in embryonic roots were also found in LR primordia (Helariutta et al., 2000; Wysocka‐Diller et al., 2000).

III. ROOT GROWTH IN THE SOIL ENVIRONMENT Root growth in the soil is regulated by endogenous signals that maintain RAM activity and patterning as well as contribute to the generation of new LRs. Among them, auxin plays a crucial role, although other hormones contribute to the overall root architecture. We will emphasize here on the role of hormone signals in this regulation based on molecular genetic studies mainly in Arabidopsis. However other signals, such as the redox status, may also play significant roles in root growth and development. For example, the RAM is highly sensitive to glutathione levels: in the root meristemless 1 mutant (rml1), which presents a short root phenotype, the mutated protein catalyzes the first step of glutathione biosynthesis, and the root growth defects have been correlated with a very low level of glutathione (Vernoux et al., 2000). Combined analyses of diVerent accessions or mutants aVected in root architecture under various environmental conditions allowed to identify several hormone signaling pathways and even QTL that regulate LR size and distribution (De Smet et al., 2006; Fitz Gerald et al., 2006; Loudet et al., 2005). A. ENDOGENOUS SIGNALS REGULATING ROOT GROWTH

Auxin, the major determinant of root growth, actively participates in embryonic and postembryonic root development as well as gravitropism. It can be synthesized in seedlings either in the aerial parts of the plant or at the tips of

ROOT ARCHITECTURE AND ENVIRONMENT

43

primary roots and LRs (Ljung et al., 2005). The auxin fluxes, which are under a variety of controls involving PIN as well as AUX1 influx carrier genes, converge to the RAM (Friml et al., 2006). In all the species studied so far, inhibition of auxin transport leads rapidly to a decrease in primary root growth (Blilou et al., 2005). In certain Arabidopsis pin mutants, auxin distribution is altered and root growth is slightly aVected, suggesting functional redundancies between PIN proteins (Friml et al., 2006). To further characterize the role of five of these genes during growth and root patterning, Blilou et al. (2005) used various combinations of double, triple, and quadruple pin mutants (pin1 to pin4 and pin7). This elegant work confirmed that PINs collectively control auxin distribution in the root and that the circulating flow of this hormone regulates meristem size. Moreover, this study showed that cell division and elongation are controlled by modulation of auxin distribution. The AUX1 (AUXIN RESISTANT 1) influx carrier is also involved in the regulation of auxin fluxes at the root tip and has been mainly described as critical in the root cap as well as in the epidermis to allow root gravitropic responses (Bennett et al., 1996; De Smet et al., 2007; Sieberer and Leyser, 2006; Swarup et al., 2005). The major role of auxin in LR initiation and development has been known for years; indeed, both an exogenous application or an endogenous overaccumulation of auxin via plant transformation cause an increase in LRs number (Boerjan et al., 1995; Celenza et al., 1995). Furthermore, a disturbed polar auxin transport between the stem and the primary root completely blocks the initiation of LRs (Reed et al., 1998). Several mutants altered in the transport, signaling, or homeostasis of auxin are also aVected in LR initiation and emergence (Casimiro et al., 2003; De Smet et al., 2006). For instance, the aux1 mutant is aVected in promotion of LR development and their positioning along the parental root (De Smet et al., 2007; Marchant et al., 2002). AUX1 action in LR cap and/or epidermis induces priming of pericycle cells in the meristem. Moreover, specific PIN members may be linked to LR organogenesis (Benkova et al., 2003). In addition, alf3 mutants (aberrant lateral root formation 3) do not seem able to activate the growth of the LR meristem. Although the function of ALF3 is not known, the wild‐type phenotype can be restored by an exogenous supply of auxin, suggesting a role for this gene in hormone production or accumulation (Celenza et al., 1995). Finally, AUX/IAAs (a 29 members’ multigene family) and ARFs (23 members) show a large diversity of expression patterns in diVerent root domains and root cell types, likely determining the global action of auxin on root development (Remington et al., 2004). SOLITARY‐ROOT/IAA14 as well as NPH4/ARF7 and ARF19 and their recently identified direct regulatory targets LBD16/ASL18 and LBD29/ASL16 (LATERAL ORGAN BOUNDARIES‐ DOMAIN/ASYMMETRIC LEAVES2‐LIKE) have been involved in the

44

M. JOVANOVIC ET AL.

control of LR initiation, and transcriptome analyses revealed that several ARFs and AUX/IAAs are among the earliest activated genes during LR initiation (Fukaki et al., 2005; Okushima et al., 2007; Vanneste et al., 2005; Wilmoth et al., 2005). AUX/IAA genes show a very early response to auxin and encode proteins present at low concentrations, with a short half‐life, generally localized in the nucleus where they act as negative regulators of auxin‐response genes (Abel et al., 1994). Notably, AUX/IAA can form heterodimers with ARFs, transcription factors that recognize, in a hormone‐independent manner, the auxin‐response elements (AREs) present in auxin‐inducible genes (Ulmasov et al., 1999). Indeed, BDL/IAA12 and MP/ARF5 antagonistic proteins have been shown to interact in vivo in the embryo (Berleth and Ju¨rgens, 1993; Hamman et al., 2002). AUX/IAA‐ARF dimers subsequently repress transcription of these genes. Fixation of auxin on the F‐box protein then stimulates interaction between SCFTIR E3 ubiquitin ligase complex and the AUX/ IAA, via the recognition of the ‘‘degron’’ motif. The ubiquitinated AUX/ IAA proteins are finally degraded by the 26S proteasome and the promoter‐ associated ARFs, thus relieved from inhibition, promote transcription of the downstream genes. TIR1, inside the SCFTIR complex, corresponds to one of the long‐awaited auxin receptors (Dharmasiri et al., 2005; Kepinski and Leyser, 2005). Recently, some ARFs and certain auxin‐related F‐box have been shown to be regulated by microRNAs (miRNAs) or tasiRNAs (trans‐acting siRNAs) posttranscriptional mechanisms, and this regulation is crucial for postembryonic root development (see Section V). BRs play multiple roles in cell elongation, senescence, photomorphogenesis, and stress responses in plants (Nemhauser and Chory, 2004). A link between auxin and BR signaling pathways has been described, and microarray data analysis also strongly suggests that both pathways converge to regulate the expression of similar target genes (Goda et al., 2004; Nemhauser et al., 2004). The nuclear protein BRX (BREVIS RADIX), which is involved in the regulation of transcription, seems to be one of the cross talk elements between these two hormonal pathways (Mouchel et al., 2004, 2006). The brx mutant is strongly aVected in its root growth, with few and small root cells as well as a smaller RAM than the wild type (Mouchel et al., 2004). As already mentioned, a reduced meristem size could be a consequence of an altered auxin transport (Blilou et al., 2005). Transcriptome analysis showed that up to 15% of the transcriptome is aVected in brx roots. Notably, the expression of three genes [PIN3, PIN4, and PGP4 (ATP‐BINDING CASSETTE P GLYCOPROTEIN)] involved in auxin flow at the root tip is reduced. Moreover, the transcripts corresponding to CONSTITUTIVE PHOTOMORPHOGENESIS AND DWARF (CPD) are barely detectable

ROOT ARCHITECTURE AND ENVIRONMENT

45

in the brx mutant roots. The CPD enzyme catalyzes a limiting step in the biosynthesis of brassinolide, the predominant BR in Arabidopsis. This study shows that during root growth, BRX is responsible for both BR level regulation and auxin signaling (Mouchel et al., 2006). Cytokinins (CKs) are involved in many developmental processes and in cell division control. CK synthesis occurs mainly in root tips, even though the expression of isopentenyltransferases (IPTs), a key biosynthesis enzyme, has been detected in other plant organs (Miyawaki et al., 2004). Overexpression of IPTs or CKX (CYTOKININ OXIDASE) involved in CK degradation leads to modifications in the CK pool, correlated with root developmental defects. CKX‐overexpressing plants have indeed an increased root length and more LRs (Werner et al., 2001, 2003). Recently, it has been observed that the CK receptor CRE1/AHK4 (CYTOKININ RESPONSE 1/ HISTIDINE KINASE 4) and many response regulator (RR) genes are mainly expressed in roots (Higuchi et al., 2004; Mason et al., 2004). A particular mutant allele aVecting the CRE1/HK4 gene, wooden leg (wol), showed a drastic short root phenotype associated with specific defects in phloem diVerentiation (Scheres et al., 1995). Triple mutants of the ahk2/ahk3/ahk4 CK receptors show a similar phenotype, whereas an ahk2/ahk3 mutant has increased root length and LR number (Higuchi et al., 2004; Nishimura et al., 2004; Riefler et al., 2006). These results suggest that apart from CRE1, other CK receptors may play overlapping functions in root growth. DiVerent combinations of mutants aVecting other CK signaling elements (AHP, for histidine phosphotransfer proteins and RRs) also confirmed the crucial role of CK in root architecture (both on primary root growth and LR formation), even though the precise developmental stage where they are involved remains to be determined (Ferreira and Kieber, 2005; Mason et al., 2005; Rashotte et al., 2006; To et al., 2004). CK eVects on meristematic activity and in vascular bundles diVerentiation may be responsible for the described defects in root architecture. Ethylene also plays a major role in root growth eventually through its interactions with auxin signaling (Souter et al., 2004; Stepanova et al., 2005). The ethylene overexpression 1 (eto1) mutant plants overaccumulate ethylene, have an increased sensitivity to ethylene, and display a shorter primary root than wild‐type plants. Analysis of polaris (pls) mutants, which also display a short root phenotype, has underlined a possible interplay between auxin and ethylene signaling pathways. The auxin‐regulated PLS gene encodes a 36‐amino acids‐long peptide, essential for proper auxin transport and thereby root growth. This peptide inhibits ethylene signaling, leading to an arrest of the cytoskeletal dynamics required for root growth (Chilley et al., 2006).

46

M. JOVANOVIC ET AL.

The role of other hormones such as abscisic acid (ABA) and GAs in root development will be described in relation to environmental stresses (see Section IV). B. THE PECULIAR LEGUME ROOT SYSTEM AND ITS SYMBIOTIC INTERACTIONS

In legumes, the soil environmental conditions together with the symbiotic interactions are the major determinants of root architecture. Legume roots can develop two types of secondary root organs: LRs and N‐fixing nodules. The latter organs result from the symbiotic interaction with soil bacteria collectively known as rhizobia. These bacteria colonize the root surface, attach to root hairs, and induce their deformation and curling as well as a series of rapid changes in root hair cells, such as calcium spiking, depolarization of the plasma membrane, and gene expression (Oldroyd and Downie, 2004). Concomitantly to rhizobial infection, pericycle cells are transiently stimulated for division. Then, cortex cells divide, usually in front of a protoxylem pole close to the infection point (Timmers et al., 1999). These actively dividing CCs form most of the nodule primordium, wherein large amounts of amyloplasts accumulate. At the root surface, rhizobia penetrate into root hairs through plant‐derived infection threads. Infection threads progress intracellularly through the outer cortex, ramify, and finally penetrate the nodule primordium cells. A diVerentiation process is then initiated heralded by cell enlargement in both partners. Bacteria diVerentiate into specific N‐fixing forms called bacteroids, surrounded by a peribacteroid membrane, which are released from infection threads into the cytoplasm of the enlarged plant cells forming symbiosomes. In parallel to bacteroid diVerentiation, the nodule primordium, comprising a persistent or transient meristem (according to the plant species), develops into a mature nodule (Brewin, 1991). The organogenesis of legume nodules requires a precise spatiotemporal expression of specific genes during the diVerent stages of the symbiotic interaction. Analyses of plant signaling pathways involved in the early stages of this developmental process have been carried out, mainly based on genetic approaches and high‐throughput gene expression studies (Stacey et al., 2006). A model for the early stages of the symbiotic interaction leading to nodule organogenesis has been proposed (Geurts et al., 2005). Nodules and LRs share several aspects of their development, even though they have divergent developmental origins (Hirsch and LaRue, 1997; Mathesius et al., 2000). LRs and nodule primordia are formed primarily from diVerent tissues, pericycle and cortex, respectively (Brewin, 1991; Hirsch, 1992). Thus, even though the same root tissue layers are involved, they have diVerent relative contributions

ROOT ARCHITECTURE AND ENVIRONMENT

47

to the respective primordia. Patterns of IC divisions are divergent between both lateral organs, and furthermore, legume nodules lack a root cap and have a peripheral vasculature. Several legume mutants aVected in genes with a dual function in nodule formation and root development were recently identified, such as latd/nip (lateral root organ‐defective) and several hypernodulating mutants (har1, hypernodulation and aberrant root formation; sunn, supernodule number; nts382, nitrate sensitive 382; skl, sickle), suggesting the existence of common regulatory pathways between these two root‐derived organogeneses (Bright et al., 2005; Day et al., 1986; Penmetsa and Cook, 1997; Penmetsa et al., 2003; Veereshlingam et al., 2004; Wopereis et al., 2000). Other mutants such as crinkle and astray are additionally aVected in other plant organs (Nishimura et al., 2002; Tansengco et al., 2003). The Medicago truncatula latd main root grows normally few days after germination, later it stops and a strong inhibition of LR formation is observed (Bright et al., 2005). The disorganized latd LRs lack a visible root cap and nodule primordia remain small, white, and undiVerentiated. The LATD gene seems therefore required for the function of three root‐derived meristems (e.g., primary root, LR, and symbiotic nodule). Hypernodulating or supernodulating mutants are aVected in autoregulation, a systemic feedback mechanism negatively controlling the final number of nodules formed in legume root systems (Caetano‐Anolle´s and GresshoV, 1991). These negative autoregulatory mechanisms may also aVect the regulation of other root meristems (primary roots and LRs) since LR density and certain hormonal responses related to LR formation are perturbed in at least some of these mutants such as har1 (Krusell et al., 2002; Wopereis et al., 2000). Consequently, the whole architecture of legume roots in symbiotic or nonsymbiotic growing conditions may be at least partially controlled by the same genes.

IV. CHANGING ROOT ARCHITECTURE: ADAPTIVE RESPONSES TO THE SOIL ENVIRONMENT Under natural culture conditions, modifications of soil composition occur generally in a slow and progressive manner, thus allowing plants to set up an adaptation strategy. Generally, after perception of abiotic stresses such as mineral deficiencies or water stress, both local and systemic signals maybe integrated in these adaptive responses. In contrast, the widespread experimental laboratory conditions usually rapidly impose a strong stress to the plant which produces major changes on gene expression. From these results, extrapolations to real field conditions need to be prudently analyzed.

48

M. JOVANOVIC ET AL.

The main abiotic stresses aVecting root architecture are water stress— water deficit or high water retention in the soil—and deficiencies in essential mineral nutrients such as phosphorus, nitrogen, and sulfur. To cope with these deprivations, plants increase their uptake ability, as they modify the nutrients solubilization in the soil by excreting organic compounds or enzymes, and also adapt their root architecture. New LR formation and/or LR growth as well as the diVerentiation/elongation of root hairs lead to a considerable increase of the overall absorption surface. In Arabidopsis, identification of mutants aVected either in the biosynthesis, perception, or signal transduction of hormones on one hand, and transcriptome studies on the other hand have shed light on hormone‐regulated target genes and developmental processes involved in root growth and development. Nevertheless, much less is known on the cross talk or overlap between these diVerent signaling pathways during adaptive developmental responses to the environment. Several excellent reviews, dealing with the modifications of Arabidopsis root architecture in response to environmental conditions, have been recently published (Lopez‐Bucio et al., 2003; Malamy, 2005). We will thus further discuss only recent relevant results in this research field. Between the application of a given stress and the following root morphological adaptations, early events such as modifications of gene expression can be monitored. For example, a deficiency in essential inorganic nutrients (phosphate, nitrate, and sulfate) induces genes encoding the corresponding high‐aYnity transporters (Lopez‐Bucio et al., 2003). In addition, reactive oxygen species (ROS) are produced; it is known that ROS act as signal molecules in all types of stresses. In fact, ROS fluctuations in time and space can be interpreted as signals to regulate growth, development, cell death, and stress responses (Foreman et al., 2003; Gechev et al., 2006). In fine, the particularity of the biological response (e.g., a modification in root architecture) to a given constraint appears to be dependent on numerous factors: the production site, nature and intensity of signals in response to stress (such as ROS), the developmental and nutritional state of the plant, and also the modifications undergone by the plant before the stress occurred [e.g., stress acclimation (Malamy, 2005; Mittler, 2006; Shin et al., 2005)]. A. WATER AVAILABILITY AND THE OSMOTIC POTENTIAL OF THE MEDIUM

Acidity and concentration of inorganic nutrients in the soil or sucrose concentration in vitro not only determine the osmotic potential of the substrate but also influence plant nutrition. All these parameters are rarely taken into account when plants are cultivated in vitro (e.g., in the presence of various concentrations of inorganic nutrients on a medium supplemented

ROOT ARCHITECTURE AND ENVIRONMENT

49

Auxin

ABA

ABI3

AUX1 and PINs

BRs PAT

Ethylene Adv roots

Initiation Cytokinin ?

Ethylene

Cytokinin ?

NitrateH

Adv roots

Osmotic potentialH

Primordium development

PhosphateL NitrateL

PhosphateH

Lateral root growth

NitrateH

Emergence and meristem activation

PhosphateH

Osmotic potential

Ethylene

Lateral root

Fig. 2. Environmental and endogenous factors aVecting LR development. H, high concentration; L, low concentration; BR, brassinolide; PAT, polar auxin transport (shoot root).

with 1–4.5% sucrose). In vitro, Arabidopsis roots are very sensitive to the osmotic potential of the medium; under certain conditions, the undergone osmotic stress resembles the one provoked by a water deficit (Deak and Malamy, 2005). LR formation is repressed by an osmotic stress, and a reverse correlation exists between the strength of the osmotic potential and LR growth. Osmotic potential is thought to aVect the number of fully developed LRs by acting on primordia development, emergence, and meristem activation rather than the initiation step (Fig. 2; Deak and Malamy, 2005).

50

M. JOVANOVIC ET AL.

However, van der Weele et al. (2000) reported a decrease in LR number correlated with a disturbed step of LR initiation during an osmotic stress caused by PEG. During a progressive drought stress, newly formed LRs exhibit a particular phenotype: roots are short, tuberized, do not form hairs, and accumulate starch (Vartanian, 1981). ABA plays a critical role in the plant response to water stress. In ABA‐ deficient mutants (aba), the root system is largely developed. In wild‐type plants, exogenous ABA treatments lead to a dormancy of the newly LRs formed, a phenomenon also noticed during water stress (Deak and Malamy, 2005; De Smet et al., 2003). This particular LR dormancy could have an essential adaptive role: to allow a rapid recovery of root growth and absorption functions once the environmental conditions are favorable again. The relationship between LR dormancy and tolerance has just been demonstrated using a genetic approach: the dig3 mutant (drought inhibition of lateral root growth 3), in which LR growth is not inhibited by ABA, is in fact much more sensitive to stress than the wild type (Xiong et al., 2006). Still, this mutant displays a classical response to osmotic stress, as marker genes (generally under the control of ABA) are correctly expressed. The DIG3 locus does not bear any known stress‐related gene, suggesting that DIG3 could be a component of a yet unknown regulation pathway. The same type of interrelation—growth inhibition by ABA and stress tolerance—has been observed in plants overexpressing the RGS1 protein (REGULATOR OF G‐PROTEIN SIGNALING) that intervenes in the G‐protein–mediated signal transduction pathway (Chen et al., 2006). Vartanian et al. described that aba1 and abi1 mutants display a decreased number of ‘‘short roots’’ compared to wild type in response to progressive drought stress. This indicates that ABA plays a promoting role in drought stress‐induced rhizogenesis, in other words blocks the expansion of the root system. However, no changes were found in abi2 and abi3 mutants (Vartanian et al., 1994). The involvement of ABA, the stress‐related hormone, in modifications of the root system further underlines its relationship with auxin signaling. The overlap between both signaling pathways had already been noticed while studying the abscisic acid insensitive 3 (abi3) mutant, which has a subtle LR phenotype and is less responsive to auxin treatments (Brady et al., 2003). This interdependence may be linked to the ability of the transcription factor ABI3, at least in common bean, to bind as eYciently to promoter sequences of both ABA‐ and auxin‐inducible genes (Nag et al., 2005). As well, the analysis of several ethylene mutants, in particular etr1 (ethylene response 1) and ein2 (ethylene insensitive 2), has shown that a functional ethylene signaling pathway is required for normal root growth in response to ABA (Beaudoin et al., 2000; Ghassemian et al., 2000).

ROOT ARCHITECTURE AND ENVIRONMENT

51

GAs may also be involved in osmotic stress responses. These hormones are known to stimulate plant growth via the degradation of DELLA proteins through the ubiquitination pathway (Fu and Harberd, 2003). These nuclear proteins are also involved in the attenuation of both shoot and root development in response to environmental stress. When four out of five DELLA genes (GAI, RGA, RGL1, and RGL2) are mutated in Arabidopsis, root elongation is almost no longer aVected by salt stress, demonstrating that GAs play a role in root growth under environmental constraints (Achard et al., 2006).

B. WATER EXCESS AND ADVENTITIOUS ROOTING

Like LRs, advRs develop on the hypocotyl from pericycle cells generally contiguous to xylem poles. The appearance of advRs is controlled by environmental conditions such as levels of water retention in the soil, light, and, for a few legume plants, phosphate (P) deficiency (King and Stimart, 1998; Miller et al., 2003). Auxin plays, as well, a preponderant role in the formation of this particular root type since the superroot 1 and 2 (sur) mutants, which spontaneously produce advR, overaccumulate auxin (Boerjan et al., 1995). However, in certain cases, a role of ethylene in this phenomenon cannot be excluded. Indeed, in water‐imbibed soils, this gas diVuses less eYciently and is more accumulated in immersed roots. This overaccumulation may block the auxin flow in specific cells and thus leads to advR formation (Aloni et al., 2006). The scaVold protein RACK1A (RECEPTOR FOR ACTIVATED C KINASE 1A) could also be a part of this signaling pathway as the corresponding mutant is highly impaired in adventitious and LR formation (Chen et al., 2006). The lack of RACK1A function may aVect many hormone signaling pathways in Arabidopsis, notably auxin sensitivity. Sorin et al. (2005) have correlated the inability of allelic series of ago1 mutants to form advRs with an accumulation of the auxin‐responsive factor ARF17. This gene is posttranscriptionally controlled by MIR160, a regulation that is perturbed in these ago1 mutants (Mallory et al., 2005). However, ago1 null mutants display strong pleiotropic phenotypes as AGO1 is a major player of the posttranscriptional regulation mediated by miRNAs in all tissues (see Section V). A multigenic control of adventitious rooting has been revealed by characterizing QTLs linked to this trait in Arabidopsis and several tree species (Han et al., 1994; King and Stimart, 1998; Marques et al., 1999).

52

M. JOVANOVIC ET AL. C. NUTRIENT AVAILABILITY

Plants have set diVerent strategies to cope with inorganic nutrient deficiencies. P deficiency induces P remobilization from macromolecules and/or modifications of root architecture in order to increase the plant’s uptake capacity (Raghothama, 1999). On a P‐deprived medium, Arabidopsis plants adapt their root architecture as the primary root growth stops and numerous new LRs emerge. In addition, numerous root hairs appear, their length being inversely correlated with the P concentration in the medium (Lopez‐Bucio et al., 2003). The high root hair number is linked to an increase in diVerentiation of epidermal cells into trichoblasts (Ma et al., 2001). Comparative analysis of biomasses after cultivating wild‐type and rhd2 (root hair deficient 2) mutant plants, unable to form root hairs, on a P‐deficient medium, has demonstrated a key role for root hairs in P uptake (Bates and Lynch, 2000). On a P‐rich medium, the primary root growth is maintained, whereas LR development is inhibited at the stage of primordium development (Fig. 1). A particular category of phospholipase D (PLD), called PLD, is a component of this diVerential regulation between primary roots and LRs. The PLD are indeed involved in the elongation of the primary root, the inhibition of LR elongation, and root hair initiation (Li et al., 2006; Ohashi et al., 2003). The main hormone influencing these morphological changes in response to P limitation is auxin as changes in its quantitative levels and distribution and/ or cell sensitivity to this hormone have been observed (Nacry et al., 2005). Ethylene and CKs could also play a significant role in signaling during P‐starvation responses at the whole plant level (Lopez‐Bucio et al., 2002; Martin et al., 2000). Indeed, some genes induced by a P deprivation are repressed by exogenous CK treatments (Martin et al., 2000). Moreover, several mutants insensitive to P deficiency and unable to regulate the P‐starvation responsive gene At4 are aVected either in AHK4/CRE1 or AHK3 CK receptors (Franco‐Zorrilla et al., 2002, 2005; Martin et al., 2000). A particular transcription factor called PHOSPHATE STARVATION RESPONSE1 (PHR1), regulated by sumoylation, is a key component of the P signaling pathway (Miura et al., 2005; Rubio et al., 2001). PHR1 regulates the expression of many genes specifically expressed under P deficiency, such as those involved in lipid or nucleic acids remobilization as well as MIR399 (see Section V; Bari et al., 2006). When N distribution in the soil is spatially unequal, plants set a diVerential root growth. In nitrate‐rich soils, LRs are initiated but blocked just before activation of the meristem, whereas in regions deprived of N source, LR growth is increased (Fig. 2; Linkohr et al., 2002). The LR growth arrest is

ROOT ARCHITECTURE AND ENVIRONMENT

53

much less drastic in ABA‐insensitive mutants, suggesting that nitrate‐induced meristem quiescence of LRs is mediated by ABA (Signora et al., 2001). Signaling pathways involved in nitrate‐regulated responses are being deciphered. Nitrate itself, and not one of its metabolites, is able to stimulate LR initiation. The nitrate transporter NRT2.1 could be either the sensor or a key component of the transduction pathway (Little et al., 2005; Malamy and Ryan, 2001). Recent analyses of atnrt2.1–1 mutant lines revealed that the amount of nitrate absorbed, and not its external concentration, governs the modifications of root architecture (Remans et al., 2006). The nitrate‐ inducible transcription factor ANR1, by feedback mechanisms, could be a regulator which determines the intensity of the LR response (Gan et al., 2005; Zhang and Forde, 1998). Finally, as for P deficiency, a transcription factor from the PHR family overexpressed under N deficiency plays presumably a key role in the whole plant response (Todd et al., 2004). Impact of N deficiency on root morphology is strongly modulated by the overall N status of the plant, implicating long‐range signaling in modifications of root architecture (Zhang et al., 1999). This could be a consequence of the interaction between nitrate and auxin biosynthesis or transport, as axr4 mutant is insensitive to the eVect of N on LR growth. Nitrate also induces CK accumulation in roots, which could account for part of the nitrate‐induced root growth inhibition (Horgan and Wareing, 1980). Furthermore, glutamate, a metabolite involved in N metabolism, is also able to modify the root architecture of Arabidopsis. Among several N metabolites, only L‐glutamate can inhibit the primary root growth and aVect LR development in vitro (Walch‐Liu et al., 2006). Hence, N deficiency modulates root architecture through a complex cross talk between hormone signals, N metabolites, and specific N‐regulated signaling pathways. Sulfur (S) uptake is essential for the biosynthesis of sulfured amino acids, cell metabolism, and stress responses (Kopriva and Rennenberg, 2004). In S‐deprived conditions, two types of LR modifications have been observed: either an increase in the number of LRs formed locally close to the root tip or a reduction in the overall number of LRs and primordia that emerge from the primary root (Dan et al., 2007; Kutz et al., 2002; Lopez‐Bucio et al., 2003; Nikiforova et al., 2003). A decrease in S uptake can always be linked to many metabolic modifications that strongly change the growth of the plant. An S‐responsive element (SURE) has been recently identified upstream of several genes encoding S transporters or involved in S uptake (Maruyama‐ Nakashita et al., 2005). However, no transcription factor able to bind to these sequences has been identified so far. Genes involved in S uptake such as the one encoding the high‐aYnity transporter, SULTR1;2 (SULPHATE TRANSPORTER 1;2), are strongly

54

M. JOVANOVIC ET AL.

regulated during an S deprivation (Maruyama‐Nakashita et al., 2004). CKs repress SULTR1;2 expression and alter the expression of S‐metabolism genes (Dan et al., 2007). Transcriptome analyses during S deprivation revealed changes in expression of several genes involved in auxin signal transduction (IAA9, IAA17, IAA18, and IAA28) or biosynthesis (NIT3) (Nikiforova et al., 2003). However, Kutz et al. (2002) did not detect any statistically significant modulation of auxin concentration between S‐deprived or control whole seedlings. On the contrary, a downregulation of DR5::GUS fusions has been observed, suggesting a decrease in auxin level or sensitivity, which is in agreement with the described decrease in LR number (Dan et al., 2007; Nikiforova et al., 2005). Finally, transcripts corresponding to jasmonic acid biosynthesis genes are accumulated during an S deprivation. Interestingly, jasmonic acid controls several key enzymes of S metabolism (Jost et al., 2005). The diVerent interactions between hormones and abiotic stresses or nutrient deficiencies are schematized in Fig. 2. D. EFFECTS OF ABIOTIC STRESSES ON LEGUME ROOTS

Several environmental factors such as nitrate or P availability or growth under abiotic stress conditions influence the development of root‐derived organs in legumes. The ability of legume roots to interact with symbiotic microorganisms constitutes an adaptation to specific nutrient starvation conditions (e.g., combined N for the N‐fixing symbiosis). Nitrate is particularly relevant for legume root architecture as its availability exerts complex eVects on root growth, LR formation, and symbiotic interactions (Dazzo and Brill, 1978; GresshoV, 1993). Indeed, nitrate deprivation represents the major environmental factor that regulates nodulation and most hypernodulating mutants such as har1 in Lotus japonicus, or several nts (nitrate tolerant symbiosis) mutants in G. max are also aVected in their nitrate regulation, suggesting that these two pathways are tightly interconnected (Carroll et al., 1985; Wopereis et al., 2000). As well, P availability aVects root development and nodulation (Pereira and Bliss, 1989). Among abiotic stresses, studies involving physiological, molecular, and functional data in legumes have been carried out mainly on salt stress. Increasing salt concentrations in soils leads to marked changes in the root growth pattern of legumes, and also aVects the symbiotic N fixation process. Legumes are very sensitive to salt levels in soils, whereas rhizobia are generally much more tolerant (up to 700‐mM NaCl) than their respective hosts (Arrese‐Igor et al., 1999; del Papa et al., 1999; Lanter et al., 1981; Singleton and Bohlool, 1984). DiVerent steps of the symbiotic interaction and nodule development are aVected by salt stress, leading to a reduction in nodule

ROOT ARCHITECTURE AND ENVIRONMENT

55

number and subsequent limited N fixation (Singleton and Bohlool, 1984). Reduced colonization and early rhizobial infection events (such as root hair curling, infection thread formation, and nodule initiation) are particularly sensitive to salt stress (Duzan et al., 2004). Genes encoding potential regulators of adaptive responses to osmotic and salt stresses, and more particularly putative transcription factors, have been identified (Hasegawa, 2000). In alfalfa, MsALFIN1 is a salt‐inducible transcript that encodes a zinc‐finger protein predominantly expressed in roots (Winicov, 1993). Overexpression of this putative transcription factor enhances root growth under control and saline conditions (Winicov, 2000). Another C2H2 zinc‐finger transcription factor (ZPT2–1) has been involved in the regulation of bacteroid diVerentiation in M. truncatula (Frugier et al., 1998, 2000). Its expression is induced by salt stress, and antisense transgenic lines are impaired in their ability to recover from a salt stress, suggesting that this transcription factor may be involved in nodule and root osmotolerance responses (Merchan et al., 2003). In several legume species, unlike Arabidopsis, ABA increases LR development (Liang et al., 2007). Several studies have also shown that exogenous ABA application inhibits nodule formation in various legumes (Suzuki et al., 2004). Observation of root hair infection events in Trifolium repens revealed that ABA blocks early infection events such as root hair deformation. Moreover, decreasing ABA levels by using specific inhibitors led to an increase in nodule number (Asami et al., 2003). Thus, ABA, similarly to abiotic stresses, could exert a negative control on nodule number and a positive one in LR formation in legumes. Indeed, latd mutants are defective in ABA responses and ABA controls root meristem function (Bright et al., 2005; Liang et al., 2007).

V. ROOT GROWTH AND DIFFERENTIATION IN RESPONSE TO ENVIRONMENTAL CONDITIONS: SMALL NONCODING RNAS AS NEW POSTTRANSCRIPTIONAL REGULATORS Regulatory pathways involved in growth and diVerentiation have been recently shown to be dependent on a myriad of small noncoding RNAs. miRNAs are noncoding 20‐ to 24‐nt‐long RNAs, initially discovered in Caenorhabditis elegans as temporal regulators of larvae diVerentiation, and more recently in mammals and plants (Lagos‐Quintana et al., 2001; Lee et al., 1993; Pasquinelli et al., 2000; Reinhart et al., 2000, 2002; Wightman et al., 1993). miRNAs are encoded by particular genes generally present, in

56

M. JOVANOVIC ET AL.

plants, in intergenic regions of the genome. Maturation of the primary transcript, generated by RNA polymerase II, requires the intervention of a particular type III RNase named DICER‐LIKE 1 (DCL1) which cuts twice on a hairpin‐structured double‐stranded RNA (Kurihara and Watanabe, 2004). The mature miRNA is then incorporated in a protein complex, the so‐called RISC (RNA‐induced silencing complex) that can recognize mRNAs partially complementary to the miRNA nucleotide sequence. This recognition event mediated by the RISC‐loaded miRNA leads to the cleavage (as it is generally the case in plants) or the translational inhibition of the target mRNA. Up to now, 43 miRNA families in 71 diVerent plant species have been defined using homology criteria (Zhang et al., 2006). Sequences of certain MIR families as well as their targets are highly conserved, suggesting that those MIRs may play the same function in diVerent species. Nevertheless, many other MIRs are specific to only one or few phylogenetically related species, indicating their rapid evolution. In plants, MIRs have been shown to play significant roles notably in the regulation of diVerentiation and in response to environmental conditions (Mallory and Vaucheret, 2006). Another class of small RNAs is the siRNAs (small interfering RNAs), initially identified in plants (Hamilton and Baulcombe, 1999). They intervene mainly in two processes: changes in chromatin conformation (e.g., through methylation) and destruction of foreign RNAs such as viral RNAs or aberrant transgene mRNAs (Voinnet, 2005). Plant siRNAs are 21‐ to 24‐nt RNAs generated from long perfectly matched double‐stranded RNAs by the action of DCL2 and DCL3 enzymes (Bouche et al., 2006). These siRNAs lead to the extinction, either posttranscriptionally (PTGS for posttranscriptional gene silencing) or transcriptionally (TGS for transcriptional gene silencing), of the gene from which the dsRNA originates. In plants, two other endogenous pathways leading to gene extinction have been described, one mediated by the tasiRNAs and one mediated by nat‐siRNA (natural antisense‐mediated siRNA) (Borsani et al., 2005; Mallory and Vaucheret, 2006). The 21‐nt‐long tasiRNAs are diVerent from the siRNAs due to their action in trans on a gene diVerent from the one encoding them (Peragine et al., 2004; Vazquez et al., 2004b). A long nonprotein‐coding RNA (npcRNA) is generated from a TAS locus, which is cleaved by the action of an miRNA on one or two sites (Axtell et al., 2006). The npcRNA cleavage products are recognized by an RNA‐dependent RNA polymerase (RDR6) and matured into a dsRNA, which becomes a substrate of DCL4 producing the 21‐nt tasiRNAs. The nat‐siRNA (only one has been described up to now) is generated in response to a salt stress in Arabidopsis and will be described later (see below) (Borsani et al., 2005).

ROOT ARCHITECTURE AND ENVIRONMENT

57

Mutants aVected in miRNA metabolism (biosynthesis, action, and transport as dcl1, ago1, hen1, hyl1, hst1, se) show pleiotropic phenotypes confirming the role of miRNAs in diverse developmental processes (Bollman et al., 2003; Chen et al., 2002; Jacobsen et al., 1999; Kidner and Martienssen, 2005; Vazquez et al., 2004a; Yang et al., 2006). miRNAs action is exerted directly on transcripts coding for genes involved in development (e.g., transcription factors), notably auxin signaling genes such as the ARF transcription factors (Teale et al., 2006). In Arabidopsis, MIR160 targets ARF10, ARF16, and ARF17 transcripts, whereas transcripts encoding ARF3 and ARF4 proteins are recognized by tasiRNAs derived from the TAS3 loci (see also Section IV; Fahlgren et al., 2006; Mallory et al., 2005; Rhoades et al., 2002; Wang et al., 2005; Williams et al., 2005a). Using experimental approaches that modify the miRNA pairing site in the target transcript without aVecting the encoded protein (known as miR‐resistant transcripts) and by overexpressing miRNAs (thus reducing target transcript levels), Sorin et al. (2005) and Mallory et al. (2005) demonstrated the involvement of MIR160 in the regulation of ARF17 transcripts during root development and branching. Furthermore, MIR160, through its action on ARF10 and ARF16 mRNAs, plays a primordial role in root cap diVerentiation (Wang et al., 2005). Indeed, constitutive expression of MIR160 inhibits the root cap cell diVerentiation and results in agravitropic roots. Additionally, mRNAs encoding the NAC1 transcription factor involved in late steps of auxin signal transduction pathway and LR formation are regulated by MIR164 (Xie et al., 2000). Overexpressing MIR164 (using an inducible promoter) or an MIR164‐resistant NAC1 mRNA leads to a significant decrease in LR number (Guo et al., 2005). Noteworthy, these experiments have been done using very high sucrose concentration which aVects root architecture (see Section IV) and a strong promoter mixing both the eVects of miRNA cleavage and the misregulation of NAC1 transcripts. These experiments suggest that the MIR164‐mediated regulation of NAC1 is involved in LR formation in Arabidopsis. Bioinformatic predictions on miRNA‐target interactions in plants suggest that miRNA‐mediated regulation may contribute to plant stress responses (Jones‐Rhoades and Bartel, 2004). The first observation that environmental conditions could aVect miRNA expression was done on Arabidopsis plants grown on a sulfate‐deprived medium. These plants overaccumulated MIR395 which targets several ATP sulfurylases (APS1, APS3, and APS4), leading to a drastic reduction of APS1 transcripts (Jones‐Rhoades and Bartel, 2004). Later on, several other miRNAs or siRNAs were shown to be regulated by abiotic stresses (cold, drought, and salt stresses) or ABA treatments (Sunkar and Zhu, 2004). For example, MIR399 plays a key role in P homeostasis in Arabidopsis (Bari et al., 2006; Chiou et al., 2006; Fujii et al., 2005).

58

M. JOVANOVIC ET AL.

MIR399 is strongly induced during P starvation, whereas the expression of one target transcript, UBC (for ubiquitin‐conjugating enzyme), is concomitantly reduced. In contrast to the majority of plant miRNAs, MIR399 does not bind a single site in the transcript‐coding region but several recognition sites present in the 50 UTR. Plants expressing an MIR399‐resistant UBC transcript show a reduced response of the primary root to low P concentrations. In addition, MIR399 overexpression leads to the disappearance of endogenous UBC transcripts and increased P accumulation in the plant. This demonstrates that the MIR399‐UBC pair plays a key role in the control of P homeostasis in Arabidopsis. Another well‐studied example is MIR398 that regulates mRNAs encoding a cytosolic (CSD1) or a chloroplastic (CSD2) form of a Cu/Zn superoxide dismutase (Sunkar et al., 2006). During an oxidative stress, MIR398 expression is reduced, whereas its target transcripts accumulate. This response likely allows plant cells to cope with ROS. Plants expressing an MIR398‐resistant CSD2 mRNA were more tolerant to an oxidative stress demonstrating the major role of CSD2 and its MIR398‐mediated regulation in plant stress responses. Considering that S and P deprivations through ROS action have major consequences in root architecture (see Section IV), we can speculate that miRNA‐mediated regulation could participate in root responses to these stresses. A new mechanism involving siRNAs in stress responses has been recently discovered in Arabidopsis (Borsani et al., 2005). Under salt stress conditions, a 24‐nt‐long siRNA could be detected, coming from two partially overlapping mRNAs that are in antisense configuration. A dsRNA (around 700 bp) is formed by complementarity between a constitutively expressed gene encoding a pyrroline‐5‐carboxylate dehydrogenase (P5CDH; involved in proline homeostasis) and an antisense stress‐inducible transcript, SRO5, of unknown function. This dsRNA is processed into so‐called nat‐siRNAs. The latter induces the cleavage of P5CDH transcripts, acting thus as true siRNAs and leading to a complete extinction of this gene under stress conditions. This SRO5‐mediated downregulation of P5CDH allows the accumulation of proline, an osmolyte known to be involved in stress responses. In Arabidopsis, the actual estimates of overlapping genes (potential antisense RNAs) being around 2000, such nat‐siRNA‐mediated regulation could have a strong impact on a variety of conditions including stress responses, hormone signaling, and diVerentiation processes. Nevertheless, other examples of nat‐siRNAs are needed to further support this particular regulation pathway. Regulatory RNAs not only aVect abiotic responses but are also involved in biotic interactions. During the compatible interaction between the pathogen P. syringae and Arabidopsis, an miRNA seems to participate in plant defense

ROOT ARCHITECTURE AND ENVIRONMENT

59

responses. Transcripts coding for particular category of auxin receptors (TIR1, AFB2, and AFB3) are negatively regulated by MIR393, which is induced by the bacterial elicitor flagellin. Since several pathogens produce auxin and this hormone may intervene in the infection process, the MIR393‐ mediated repression of hormone receptors may be linked to a natural ‘‘immune’’ response of the plant to control pathogen infection (Navarro et al., 2006). Knowing the major role of auxin in root development, MIR393 could also be involved in pathogen responses in roots. Other biotic interactions are beneficial for the plant as the mentioned symbiotic interaction between Rhizobium and legume plants. In M. truncatula, an HAP transcription factor has been shown to be essential for nodule diVerentiation and the corresponding mRNA is spatially controlled by MIR169 (Combier et al., 2006). Abolishment of this posttranscriptional regulation (using an MIR‐resistant version of the MtHAP2–1 mRNA) leads to delayed nodule development, likely due to misregulated meristematic activity. Due to the large diversity of these novel regulatory RNAs, we are only beginning to identify a wide variety of processes that may be controlled posttranscriptionally (Lu et al., 2005; Rajagopalan et al., 2006). Potential roles of miRNAs in root development or responses to abiotic stresses are summarized in Fig. 3.

VI. CONCLUDING REMARKS In contrast to animals, plants adapt to the environment by modulating their growth and diVerentiation. The meristematic cells integrate signals from the external conditions to regulate specific developmental responses and cope with environmental constraints. Both postembryonic development and response to environmental conditions require the activation of hormone‐ related signaling pathways. The appropriate developmental response to a given stress is therefore the result of the integration of many signals perceived by the plant and their cross talk with hormone action. Analyses are even more complicated when plants overcome a stress due to inorganic nutrient deficiencies such as phosphate, nitrate, and sulfate. These nutrients and/or their metabolites can act as signal molecules directly aVecting plant development or through interactions with hormonal signaling pathways. QTL approaches are likely to be very useful in the dissection of such pathways. Moreover, experimental procedures (e.g., culture conditions, nutrient concentrations) are variables between studies aiming to describe the same phenomenon, namely a nutrient deficiency or excess. One can thus only infer tendencies from the synthesis of the actual data. As it is already done for the

60

M. JOVANOVIC ET AL.

Soil environment

Biotic interactions

Abiotic stresses

MIR160 MIR164

ARF10, ARF16, NAC1

MIR393 MIR169

TIR1/AFB2-3 HAP2-1

MIR399 MIR395

UBC APS1

Lateral root organs

TAS3 ARF3, ARF4

RAM

Developmental adaptation

MIR 160 ARF17

Fig. 3. RNA‐mediated regulation of root architecture. Integration of riboregulation with environmental and endogenous signaling pathways. RAM, root apical meristem; gene names are mentioned in the text.

homologation of new crop cultivars or expression profiling via DNA arrays, meta‐analyses studies (e.g., comparing data from diVerent experimental conditions in diVerent laboratories using many mutants and/or genotypes) should be launched to define in an unambiguous manner the phenotypes linked to environmental modifications. The results obtained by Bray (2004)

ROOT ARCHITECTURE AND ENVIRONMENT

61

from the comparison of transcriptomic data on water stress experiments in Arabidopsis revealed the importance to analyze the overlap existing between diVerent studies. This may help to discriminate between specific targets and ‘‘noise’’ variation due to the environment in transcriptional profiles. These analyses need to be reinforced in the future so that large‐scale data obtained on model plants (as Arabidopsis or M. truncatula) can be translated in useful agronomic traits for crops. In addition to the diverse mechanisms implied in the regulation of root growth, which involve homeostasis and signaling pathways of several hormones, posttranscriptional regulation of developmental regulators mediated by noncoding RNAs is emerging as an important determinant of diVerentiation in eukaryotes. These novel regulatory mechanisms may be particularly relevant to adjust diVerentiation processes to the environmental conditions encountered during growth. In roots, developmental plasticity accounts mainly for the adaptation of root architecture to the soil conditions (involving parameters such as water and mineral levels or interactions with symbiotic microorganisms). Environmental responses may be integrated in the root system through the action of specific regulators, such as transcription factors, on primary root and LR developmental programs. As mRNAs encoding transcription factors seem privileged targets of miRNAs, temporal and spatial regulation of miRNA‐target transcription factor interactions may play significant roles in the adaptation of root architecture to the soil environment.

ACKNOWLEDGMENTS M.J. and P.L. are supported by the Ministe`re de l’Education Nationale, de l’Enseignement Supe´rieur et de la Recherche (MENR). S.G.R. was the recipient of a grant from Consejo Nacional de Ciencia y Tecnologia, Mexico. The support of the ‘‘Grain legume’’ and RIBOREG FP6 EEC project are also acknowledged.

REFERENCES Abel, S., Oeller, P. W. and Theologis, A. (1994). Early auxin‐induced genes encode short‐lived nuclear proteins. Proceedings of the National Academy of Sciences of the United States of America 91, 326–330. Achard, P., Cheng, H., De Grauwe, L., Decat, J., Schoutteten, H., Moritz, T., Van Der Straeten, D., Peng, J. and Harberd, N. P. (2006). Integration of plant responses to environmentally activated phytohormonal signals. Science 311, 91–94.

62

M. JOVANOVIC ET AL.

Aida, M., Beis, D., Heidstra, R., Willemsen, V., Blilou, I., Galinha, C., Nussaume, L., Noh, Y. S., Amasino, R. and Scheres, B. (2004). The PLETHORA genes mediate patterning of the Arabidopsis root stem cell niche. Cell 119, 109–120. Aloni, R., Aloni, E., Langhans, M. and Ullrich, C. I. (2006). Role of cytokinin and auxin in shaping root architecture: Regulating vascular diVerentiation, lateral root initiation, root apical dominance and root gravitropism. Annals of Botany (London) 97, 883–893. Arrese‐Igor, C., Gonza´lez, E. M., Gordon, A. J., Minchin, F. R., Ga´lvez, L., Royuela, M., Cabrerizo, P. M. and Aparicio‐Tejo, P. M. (1999). Sucrose synthase and nodule nitrogen fixation under drought and other environmental stresses. Symbiosis 27, 189–212. Asami, T., Sun‐Young, H., Kitahata, N., Saito, T., Kobayashi, M., Shinozaki, K., Nakano, T. and Nakashima, K. (2003). Drug design of abscisic acid biosynthesis inhibitor and its eVect on plants. Plant and Cell Physiology 45, 154. Axtell, M. J., Jan, C., Rajagopalan, R. and Bartel, D. P. (2006). A two‐hit trigger for siRNA biogenesis in plants. Cell 127, 565–577. Bari, R., Datt Pant, B., Stitt, M. and Scheible, W. R. (2006). PHO2, microRNA399, and PHR1 define a phosphate‐signaling pathway in plants. Plant Physiology 141, 988–999. Bates, T. R. and Lynch, J. P. (2000). Plant growth and phosphorus accumulation of wild type and two root hair mutants of Arabidopsis thaliana (Brassicaceae). American Journal of Botany 87, 958–963. Beaudoin, N., Serizet, C., Gosti, F. and Giraudat, J. (2000). Interactions between abscisic acid and ethylene signaling cascades. Plant Cell 12, 1103–1115. Beeckman, T., Burssens, S. and Inze, D. (2001). The peri‐cell‐cycle in Arabidopsis. Journal of Experimental Botany 52, 403–411. Benfey, P. N. and Scheres, B. (2000). Root development. Current Biology 10, R813–R815. Benkova, E., Michniewicz, M., Sauer, M., Teichmann, T., Seifertova, D., Jurgens, G. and Friml, J. (2003). Local, eZux‐dependent auxin gradients as a common module for plant organ formation. Cell 115, 591–602. Bennett, M. J., Marchant, A., Green, H. G., May, S. T., Ward, S. P., Millner, P. A., Walker, A. R., Schulz, B. and Feldmann, K. A. (1996). Arabidopsis AUX1 gene: A permease‐like regulator of root gravitropism. Science 273, 948–950. Berger, F., HaseloV, J., Schiefelbein, J. and Dolan, L. (1998). Positional information in root epidermis is defined during embryogenesis and acts in domains with strict boundaries. Current Biology 8, 421–430. Berleth, T. and Ju¨rgens, G. (1993). The role of the monopteros gene in organising the basal body region of the Arabidopsis embryo. Development 118, 575–587. Bernhardt, C., Lee, M. M., Gonzalez, A., Zhang, F., Lloyd, A. and Schiefelbein, J. (2003). The bHLH genes GLABRA3 (GL3) and ENHANCER OF GLABRA3 (EGL3) specify epidermal cell fate in the Arabidopsis root. Development 130, 6431–6439. Blilou, I., Xu, J., Wildwater, M., Willemsen, V., Paponov, I., Friml, J., Heidstra, R., Aida, M., Palme, K. and Scheres, B. (2005). The PIN auxin eZux facilitator network controls growth and patterning in Arabidopsis roots. Nature 433, 39–44. Boerjan, W., Cervera, M. T., Delarue, M., Beeckman, T., Dewitte, W., Bellini, C., Caboche, M., Van Onckelen, H., Van Montagu, M. and Inze, D. (1995). Superroot, a recessive mutation in Arabidopsis, confers auxin overproduction. Plant Cell 7, 1405–1419.

ROOT ARCHITECTURE AND ENVIRONMENT

63

Bollman, K. M., Aukerman, M. J., Park, M. Y., Hunter, C., Berardini, T. Z. and Poethig, R. S. (2003). HASTY, the Arabidopsis ortholog of exportin 5/ MSN5, regulates phase change and morphogenesis. Development 130, 1493–1504. Borsani, O., Zhu, J., Verslues, P. E., Sunkar, R. and Zhu, J. K. (2005). Endogenous siRNAs derived from a pair of natural cis‐antisense transcripts regulate salt tolerance in Arabidopsis. Cell 123, 1279–1291. Bouche, N., Lauressergues, D., Gasciolli, V. and Vaucheret, H. (2006). An antagonistic function for Arabidopsis DCL2 in development and a new function for DCL4 in generating viral siRNAs. The EMBO Journal 25, 3347–3356. Brachmann, A. (2006). Plant‐fungal symbiosis en gros and en detail. The New Phytologist 171, 242–246. Brady, S. M., Sarkar, S. F., Bonetta, D. and McCourt, P. (2003). The ABSCISIC ACID INSENSITIVE 3 (ABI3) gene is modulated by farnesylation and is involved in auxin signaling and lateral root development in Arabidopsis. The Plant Journal 34, 67–75. Bray, E. A. (2004). Genes commonly regulated by water‐deficit stress in Arabidopsis thaliana. Journal of Experimental Botany 55, 2331–2341. Brewin, N. J. (1991). Development of the legume root nodule. Annual Review of Cell Biology 7, 191–226. Bright, L. J., Liang, Y., Mitchell, D. M. and Harris, J. M. (2005). The LATD gene of Medicago truncatula is required for both nodule and root development. Molecular Plant Microbe Interactions 18, 521–532. Caetano‐Anolle´s, G. and GresshoV, P. M. (1991). Plant genetic control of nodulation. Annual Review of Microbiology 45, 345–382. Carroll, B. J., McNeil, D. L. and GresshoV, P. M. (1985). A supernodulation and nitrate‐tolerant symbiotic (nts) soybean mutant. Plant Physiology 78, 34–40. Casimiro, I., Beeckman, T., Graham, N., Bhalerao, R., Zhang, H., Casero, P., Sandberg, G. and Bennett, M. J. (2003). Dissecting Arabidopsis lateral root development. Trends in Plant Science 8, 165–171. Celenza, J. L., Jr., Grisafi, P. L. and Fink, G. R. (1995). A pathway for lateral root formation in Arabidopsis thaliana. Genes & Development 9, 2131–2142. Chen, J. G., Ullah, H., Temple, B., Liang, J., Guo, J., Alonso, J. M., Ecker, J. R. and Jones, A. M. (2006). RACK1 mediates multiple hormone responsiveness and developmental processes in Arabidopsis. Journal of Experimental Botany 57, 2697–2708. Chen, X., Liu, J., Cheng, Y. and Jia, D. (2002). HEN1 functions pleiotropically in Arabidopsis development and acts in C function in the flower. Development 129, 1085–1094. Chilley, P. M., Casson, S. A., Tarkowski, P., Hawkins, N., Wang, K. L., Hussey, P. J., Beale, M., Ecker, J. R., Sandberg, G. K. and Lindsey, K. (2006). The POLARIS peptide of Arabidopsis regulates auxin transport and root growth via eVects on ethylene signaling. Plant Cell 18, 3058–3072. Chiou, T. J., Aung, K., Lin, S. I., Wu, C. C., Chiang, S. F. and Su, C. L. (2006). Regulation of phosphate homeostasis by MicroRNA in Arabidopsis. Plant Cell 18, 412–421. Combier, J. P., Frugier, F., de Billy, F., Boualem, A., El‐Yahyaoui, F., Moreau, S., Vernie, T., Ott, T., Gamas, P., Crespi, M. and Niebel, A. (2006). MtHAP2– 1 is a key transcriptional regulator of symbiotic nodule development regulated by microRNA169 in Medicago truncatula. Genes & Development 20, 3084–3088.

64

M. JOVANOVIC ET AL.

Costa, S. and Shaw, P. (2006). Chromatin organization and cell fate switch respond to positional information in Arabidopsis. Nature 439, 493–496. Crespi, M. and Galvez, S. (2000). Molecular mechanisms in root nodule development. Journal of Plant Growth Regulation 19, 155–166. Dan, H., Yang, G. and Zheng, Z. L. (2007). A negative regulatory role for auxin in sulphate deficiency response in Arabidopsis thaliana. Plant Molecular Biology 63, 221–235. Day, D. A., Lambers, H., Bateman, J., Carroll, B. J. and GresshoV, P. M. (1986). Growth comparisons of a supernodulating soybean (Glycine max) mutant and its wild‐type parent. Physiologia Plantarum 68, 375–382. Dazzo, F. B. and Brill, W. J. (1978). Regulation by fixed nitrogen of host‐symbiont recognition in the rhizobium‐clover symbiosis. Plant Physiology 62, 18–21. De Smet, I., Signora, L., Beeckman, T., Inze, D., Foyer, C. H. and Zhang, H. (2003). An abscisic acid‐sensitive checkpoint in lateral root development of Arabidopsis. The Plant Journal 33, 543–555. De Smet, I., Vanneste, S., Inze, D. and Beeckman, T. (2006). Lateral root initiation or the birth of a new meristem. Plant Molecular Biology 60, 871–887. De Smet, I., Tetsumura, T., De Rybel, B., Frey, N. F., Laplaze, L., Casimiro, I., Swarup, R., Naudts, M., Vanneste, S., Audenaert, D., Inze, D., Bennett, M. J., et al. (2007). Auxin‐dependent regulation of lateral root positioning in the basal meristem of Arabidopsis. Development 134, 681–690. Deak, K. I. and Malamy, J. (2005). Osmotic regulation of root system architecture. The Plant Journal 43, 17–28. del Papa, M. F., Balague, L. J., Sowinski, S. C., Wegener, C., Segundo, E., Abarca, F. M., Toro, N., Niehaus, K., P hler, A., Aguilar,O. M., Martinez‐Drets, G. and Lagares, A. (1999). Isolation and characterization of alfalfa‐nodulating rhizobia present in acidic soils of central argentina and uruguay. Applied and Environmental Microbiology 65, 1420–1427. Dharmasiri, N., Dharmasiri, S. and Estelle, M. (2005). The F‐box protein TIR1 is an auxin receptor. Nature 435, 441–445. Di Laurenzio, L., Wysocka‐Diller, J., Malamy, J. E., Pysh, L., Helariutta, Y., Freshour, G., Hahn, M. G., Feldmann, K. A. and Benfey, P. N. (1996). The SCARECROW gene regulates an asymmetric cell division that is essential for generating the radial organization of the Arabidopsis root. Cell 86, 423–433. DiDonato, R. J., Arbuckle, E., Buker, S., Sheets, J., Tobar, J., Totong, R., Grisafi, P., Fink, G. R. and Celenza, J. L. (2004). Arabidopsis ALF4 encodes a nuclear‐localized protein required for lateral root formation. The Plant Journal 37, 340–353. Dolan, L. and Costa, S. (2001). Evolution and genetics of root hair stripes in the root epidermis. Journal of Experimental Botany 52, 413–417. Dubrovsky, J. G., Doerner, P. W., Colon‐Carmona, A. and Rost, T. L. (2000). Pericycle cell proliferation and lateral root initiation in Arabidopsis. Plant Physiology 124, 1648–1657. Dubrovsky, J. G., Gambetta, G. A., Hernandez‐Barrera, A., Shishkova, S. and Gonzalez, I. (2006). Lateral root initiation in Arabidopsis: Developmental window, spatial patterning, density and predictability. Annals of Botany (London) 97, 903–915. Duzan, H. M., Zhou, X., Souleimanov, A. and Smith, D. L. (2004). Perception of Bradyrhizobium japonicum nod factor by soybean [Glycine max (L.) Merr.] root hairs under abiotic stress conditions. Journal of Experimental Botany 55, 2641–2646.

ROOT ARCHITECTURE AND ENVIRONMENT

65

Fahlgren, N., Montgomery, T. A., Howell, M. D., Allen, E., Dvorak, S. K., Alexander, A. L. and Carrington, J. C. (2006). Regulation of AUXIN RESPONSE FACTOR3 by TAS3 ta‐siRNA aVects developmental timing and patterning in Arabidopsis. Current Biology 16, 939–944. Feldman, L. (1994). The maize roots. In ‘‘The Maize Handbook’’ (M. Freeling and V. Walbot, eds.), pp. 29–37. Springer, New York. Ferreira, F. J. and Kieber, J. J. (2005). Cytokinin signaling. Current Opinion in Plant Biology 8, 518–525. Fitz Gerald, J. N., Lehti‐Shiu, M. D., Ingram, P. A., Deak, K. I., Biesiada, T. and Malamy, J. E. (2006). Identification of quantitative trait loci that regulate Arabidopsis root system size and plasticity. Genetics 172, 485–498. Foreman, J., Demidchik, V., Bothwell, J. H., Mylona, P., Miedema, H., Torres, M. A., Linstead, P., Costa, S., Brownlee, C., Jones, J. D., Davies, J. M. and Dolan, L. (2003). Reactive oxygen species produced by NADPH oxidase regulate plant cell growth. Nature 422, 442–446. Franco‐Zorrilla, J. M., Martin, A. C., Solano, R., Rubio, V., Leyva, A. and Paz‐ Ares, J. (2002). Mutations at CRE1 impair cytokinin‐induced repression of phosphate starvation responses in Arabidopsis. The Plant Journal 32, 353–360. Franco‐Zorrilla, J. M., Martin, A. C., Leyva, A. and Paz‐Ares, J. (2005). Interaction between phosphate‐starvation, sugar, and cytokinin signaling in Arabidopsis and the roles of cytokinin receptors CRE1/AHK4 and AHK3. Plant Physiology 138, 847–857. Friml, J., Benfey, P., Benkova, E., Bennett, M., Berleth, T., Geldner, N., Grebe, M., Heisler, M., Hejatko, J., Jurgens, G., Laux, T., Lindsey, K., et al., (2006). Apical‐basal polarity: Why plant cells don’t stand on their heads. Trends in Plant Science 11, 12–14. Frugier, F., Kondorosi, A. and Crespi, M. (1998). Identification of novel putative regulatory genes induced during alfalfa nodule development with a cold‐ plaque screening procedure. Moleculor Plant Microbe Interactions 11, 358–366. Frugier, F., Poirier, S., Satiat‐Jeunemaitre, B., Kondorosi, A. and Crespi, M. (2000). A Kruppel‐like zinc finger protein is involved in nitrogen‐fixing root nodule organogenesis. Genes & Development 14, 475–482. Fu, X. and Harberd, N. P. (2003). Auxin promotes Arabidopsis root growth by modulating gibberellin response. Nature 421, 740–743. Fujii, H., Chiou, T. J., Lin, S. I., Aung, K. and Zhu, J. K. (2005). A miRNA involved in phosphate‐starvation response in Arabidopsis. Current Biology 15, 2038–2043. Fukaki, H., Tameda, S., Masuda, H. and Tasaka, M. (2002). Lateral root formation is blocked by a gain‐of‐function mutation in the SOLITARY‐ROOT/ IAA14 gene of Arabidopsis. The Plant Journal 29, 153–168. Fukaki, H., Nakao, Y., Okushima, Y., Theologis, A. and Tasaka, M. (2005). Tissue‐ specific expression of stabilized SOLITARY‐ROOT/IAA14 alters lateral root development in Arabidopsis. The Plant Journal 44, 382–395. Gage, D. J. (2004). Infection and invasion of roots by symbiotic, nitrogen‐fixing rhizobia during nodulation of temperate legumes. Microbiology and Molecular Biology Reviews 68, 280–300. Gallagher, K. L., Paquette, A. J., Nakajima, K. and Benfey, P. N. (2004). Mechanisms regulating SHORT‐ROOT intercellular movement. Current Biology 14, 1847–1851.

66

M. JOVANOVIC ET AL.

Gan, Y., Filleur, S., Rahman, A., Gotensparre, S. and Forde, B. G. (2005). Nutritional regulation of ANR1 and other root‐expressed MADS‐box genes in Arabidopsis thaliana. Planta 222, 730–742. Gechev, T. S., Van Breusegem, F., Stone, J. M., Denev, I. and Laloi, C. (2006). Reactive oxygen species as signals that modulate plant stress responses and programmed cell death. Bioessays 28, 1091–1101. Geurts, R., Fedorova, E. and Bisseling, T. (2005). Nod factor signaling genes and their function in the early stages of Rhizobium infection. Current Opinion in Plant Biology 8, 346–352. Ghassemian, M., Nambara, E., Cutler, S., Kawaide, H., Kamiya, Y. and McCourt, P. (2000). Regulation of abscisic acid signaling by the ethylene response pathway in Arabidopsis. Plant Cell 12, 1117–1126. Gianinazzi‐Pearson, V., Se´jalon‐Delmas, N., Genre, A., Jeandroz, S. and Bonfante, P. (2007). Plants and arbuscular mycorrhizal fungi: Cues and communication in the early steps of symbiotic interactions. Advances in Botanical Research 46, 181–207. Goda, H., Sawa, S., Asami, T., Fujioka, S., Shimada, Y. and Yoshida, S. (2004). Comprehensive comparison of auxin‐regulated and brassinosteroid‐ regulated genes in Arabidopsis. Plant Physiology 134, 1555–1573. Graham, J. H. and Miller, R. M. (2005). Mycorrhizas: Gene to function. Plant and Soil 274, 79–100. GresshoV, P. M. (1993). Molecular genetic analysis of nodulation genes in soybean. Plant Breeding Reviews 11, 275–318. Guo, H. S., Xie, Q., Fei, J. F. and Chua, N. H. (2005). MicroRNA directs mRNA cleavage of the transcription factor NAC1 to downregulate auxin signals for Arabidopsis lateral root development. Plant Cell 17, 1376–1386. Hamilton, A. J. and Baulcombe, D. C. (1999). A species of small antisense RNA in posttranscriptional gene silencing in plants. Science 286, 950–952. Hamman, T., Benkova, E., Ba¨urle, I., Kientz, M. and Ju¨rgens, G. (2002). The Arabidopsis BODENLOS gene encodes an auxin response protein inhibiting MONOPTEROS‐mediating embryo patterning. Genes & Development 16, 1610–1615. Han, K. H., Bradshaw, H. and Gordon, M. (1994). Adventitious root and shoot regeneration in vitro is under major gene control in an F2 family of hybrid poplar (Populus trichocarpa  P. deltoides). For Genet 1, 139–146. Harrison, M. J. (2005). Signaling in the arbuscular mycorrhizal symbiosis. Annual Review of Microbiology 59, 19–42. Hasegawa, P. M. (2000). Plant cellular and molecular responses to high salinity. Annual Review of Plant Physiology and Plant Molecular Biology 51, 463–499. Helariutta, Y., Fukaki, H., Wysocka‐Diller, J., Nakajima, K., Jung, J., Sena, G., Hauser, M. T. and Benfey, P. N. (2000). The SHORT‐ROOT gene controls radial patterning of the Arabidopsis root through radial signaling. Cell 101, 555–567. Higuchi, M., Pischke, M. S., Mahonen, A. P., Miyawaki, K., Hashimoto, Y., Seki, M., Kobayashi, M., Shinozaki, K., Kato, T., Tabata, S., Helariutta, Y., Sussman, M. R., et al. (2004). In planta functions of the Arabidopsis cytokinin receptor family. Proceedings of the National Academy of Sciences of the United States of America 101, 8821–8826. Hirsch, A. M. (1992). Developmental biology of legume nodulation. The New Phytologist 122, 211–237.

ROOT ARCHITECTURE AND ENVIRONMENT

67

Hirsch, A. M. and LaRue, T. A. (1997). Is the legume nodule a modified root or stem or an organ sui generis? Critical Reviews in Plant Sciences 16, 361–392. Horgan, J. M. and Wareing, P. F. (1980). Cytokinin and the growth responses of seedlings of Betula pendula to nitrogen and phosphorus deficiency. Journal of Experimental Botany 31, 525–532. Jacobsen, S. E., Running, M. P. and Meyerowitz, E. M. (1999). Disruption of an RNA helicase/RNAse III gene in Arabidopsis causes unregulated cell division in floral meristems. Development 126, 5231–5243. Jones‐Rhoades, M. W. and Bartel, D. P. (2004). Computational identification of plant microRNAs and their targets, including a stress‐induced miRNA. Molecular Cell 14, 787–799. Jost, R., Altschmied, L., Bloem, E., Bogs, J., Gershenzon, J., Hahnel, U., Hansch, R., Hartmann, T., Kopriva, S., Kruse, C., Mendel, R. R., Papenbrock, J., et al. (2005). Expression profiling of metabolic genes in response to methyl jasmonate reveals regulation of genes of primary and secondary sulfur‐ related pathways in Arabidopsis thaliana. Photosynthesis Research 86, 491–508. Kepinski, S. and Leyser, O. (2005). The Arabidopsis F‐box protein TIR1 is an auxin receptor. Nature 435, 446–451. Kidner, C. A. and Martienssen, R. A. (2005). The role of ARGONAUTE1 (AGO1) in meristem formation and identity. Developmental Biology 280, 504–517. King, J. J. and Stimart, D. P. (1998). Genetic analysis of variation for auxin‐induced adventitious root formation among eighteen ecotypes of Arabidopsis thaliana L. Heynh. The Journal of Heredity 89, 481–487. Kopriva, S. and Rennenberg, H. (2004). Control of sulphate assimilation and glutathione synthesis: Interaction with N and C metabolism. Journal of Experimental Botany 55, 1831–1842. Krusell, L., Madsen, L. H., Sato, S., Aubert, G., Genua, A., Szczyglowski, K., Duc, G., Kaneko, T., Tabata, S., de Bruijn, F., Pajuelo, E., Sandal, N., et al. (2002). Shoot control of root development and nodulation is mediated by a receptor‐like kinase. Nature 420, 422–426. Kurihara, Y. and Watanabe, Y. (2004). Arabidopsis micro‐RNA biogenesis through Dicer‐like 1 protein functions. Proceedings of the National Academy of Sciences of the United States of America 101, 12753–12758. Kutz, A., Muller, A., Hennig, P., Kaiser, W. M., Piotrowski, M. and Weiler, E. W. (2002). A role for nitrilase 3 in the regulation of root morphology in sulphur‐starving Arabidopsis thaliana. The Plant Journal 30, 95–106. Lagos‐Quintana, M., Rauhut, R., Lendeckel, W. and Tuschl, T. (2001). Identification of novel genes coding for small expressed RNAs. Science 294, 853–858. Lanter, D. J., Munns, D. N. and Clarkin, K. L. (1981). Salt response of chickpeas influenced by N supply. Agronomy Journal 96, 1–966. Lee, R. C., Feinbaum, R. L. and Ambros, V. (1993). The C. elegans heterochronic gene lin‐4 encodes small RNAs with antisense complementarity to lin‐14. Cell 75, 843–854. Levesque, M. P., Vernoux, T., Busch, W., Cui, H., Wang, J. Y., Blilou, I., Hassan, H., Nakajima, K., Matsumoto, N., Lohmann, J. U., Scheres, B. and Benfey, P. N. (2006). Whole‐genome analysis of the SHORT‐ROOT developmental pathway in Arabidopsis. PLoS Biology 4, e143. Li, M., Qin, C., Welti, R. and Wang, X. (2006). Double knockouts of phospholipases Dzeta1 and Dzeta2 in Arabidopsis aVect root elongation during phosphate‐limited growth but do not aVect root hair patterning. Plant Physiology 140, 761–770.

68

M. JOVANOVIC ET AL.

Liang, Y., Mitchell, D. M. and Harris, J. M. (2007). Abscissic acid rescues the root meristem defects of the Medicago truncatula latd mutant. Developmental Biology 304, 297–307. Linkohr, B. I., Williamson, L. C., Fitter, A. H. and Leyser, H. M. (2002). Nitrate and phosphate availability and distribution have diVerent eVects on root system architecture of Arabidopsis. The Plant Journal 29, 751–760. Little, D. Y., Rao, H., Oliva, S., Daniel‐Vedele, F., Krapp, A. and Malamy, J. E. (2005). The putative high‐aYnity nitrate transporter NRT2.1 represses lateral root initiation in response to nutritional cues. Proceedings of the National Academy of Sciences of the United States of America 102, 13693–13698. Ljung, K., Hull, A. K., Celenza, J., Yamada, M., Estelle, M., Normanly, J. and Sandberg, G. (2005). Sites and regulation of auxin biosynthesis in Arabidopsis roots. Plant Cell 17, 1090–1104. Lopez‐Bucio, J., Hernandez‐Abreu, E., Sanchez‐Calderon, L., Nieto‐Jacobo, M. F., Simpson, J. and Herrera‐Estrella, L. (2002). Phosphate availability alters architecture and causes changes in hormone sensitivity in the Arabidopsis root system. Plant Physiology 129, 244–256. Lopez‐Bucio, J., Cruz‐Ramirez, A. and Herrera‐Estrella, L. (2003). The role of nutrient availability in regulating root architecture. Current Opinion in Plant Biology 6, 280–287. Loudet, O., Gaudon, V., Trubuil, A. and Daniel‐Vedele, F. (2005). Quantitative trait loci controlling root growth and architecture in Arabidopsis thaliana confirmed by heterogeneous inbred family. Theoretical and Applied Genetics 110, 742–753. Lu, C., Tej, S. S., Luo, S., Haudenschild, C. D., Meyers, B. C. and Green, P. J. (2005). Elucidation of the small RNA component of the transcriptome. Science 309, 1567–1569. Lynch, J. (1995). Root architecture and plant productivity. Plant Physiology 109, 7–13. Ma, Z., Bielenberg, D. G., Brown, K. M. and Lynch, J. P. (2001). Regulation of root hair density by phosphorus availability in Arabidopsis thaliana. Plant, Cell & Environment 24, 459–467. Malamy, J. E. (2005). Intrinsic and environmental response pathways that regulate root system architecture. Plant, Cell & Environment 28, 67–77. Malamy, J. E. and Benfey, P. N. (1997a). Down and out in Arabidopsis: The formation of lateral roots. Trends in plant science 2, 390–396. Malamy, J. E. and Benfey, P. N. (1997b). Organization and cell diVerentiation in lateral roots of Arabidopsis thaliana. Development 124, 33–44. Malamy, J. E. and Ryan, K. S. (2001). Environmental regulation of lateral root initiation in Arabidopsis. Plant Physiology 127, 899–909. Mallory, A. C. and Vaucheret, H. (2006). Functions of microRNAs and related small RNAs in plants. Nat Genet 38(Suppl.), S31–S36. Mallory, A. C., Bartel, D. P. and Bartel, B. (2005). MicroRNA‐directed regulation of Arabidopsis AUXIN RESPONSE FACTOR17 is essential for proper development and modulates expression of early auxin response genes. Plant Cell 17, 1360–1375. Mallory, T. E., Chiang, S.‐H., Cutter, E. G. and GiVord, E. M. (1970). Sequence and pattern of lateral root formation in five selected species. American Journal of Botany 57, 800–809. Marchant, A., Bhalerao, R., Casimiro, I., Eklof, J., Casero, P. J., Bennett, M. and Sandberg, G. (2002). AUX1 promotes lateral root formation by facilitating

ROOT ARCHITECTURE AND ENVIRONMENT

69

indole‐3‐acetic acid distribution between sink and source tissues in the Arabidopsis seedling. Plant Cell 14, 589–597. Marques, C., Vasquez‐Kool, J., Carocha, V., Ferreira, J., O’Malley, D., Liu, B. and SederoV, R. (1999). Genetic dissection of vegetative traits in Eucalyptus tereticornis and E. globulus. Theoretical and Applied Genetics 99, 936–946. Martin, A. C., del Pozo, J. C., Iglesias, J., Rubio, V., Solano, R., de La Pena, A., Leyva, A. and Paz‐Ares, J. (2000). Influence of cytokinins on the expression of phosphate starvation responsive genes in Arabidopsis. The Plant Journal 24, 559–567. Maruyama‐Nakashita, A., Nakamura, Y., Yamaya, T. and Takahashi, H. (2004). Regulation of high‐aYnity sulphate transporters in plants: Towards systematic analysis of sulphur signalling and regulation. Journal of Experimenal Botany 55, 1843–1849. Maruyama‐Nakashita, A., Nakamura, Y., Watanabe‐Takahashi, A., Inoue, E., Yamaya, T. and Takahashi, H. (2005). Identification of a novel cis‐acting element conferring sulfur deficiency response in Arabidopsis roots. The Plant Journal 42, 305–314. Mason, M. G., Li, J., Mathews, D. E., Kieber, J. J. and Schaller, G. E. (2004). Type‐B response regulators display overlapping expression patterns in Arabidopsis. Plant Physiology 135, 927–937. Mason, M. G., Mathews, D. E., Argyros, D. A., Maxwell, B. B., Kieber, J. J., Alonso, J. M., Ecker, J. R. and Schaller, G. E. (2005). Multiple type‐B response regulators mediate cytokinin signal transduction in Arabidopsis. Plant Cell 17, 3007–3018. Mathesius, U., Weinman, J. J., Rolfe, B. G. and Djordjevic, M. A. (2000). Rhizobia can induce nodules in white clover by ‘‘hijacking’’ mature cortical cells activated during lateral root development. Moleculor Plant Microbe Interactions 13, 170–182. Merchan, F., Breda, C., Perez Hormaeche, J., Sousa, C., Kondorosi, A., Aguilar, O. M., Megias, M. and Crespi, M. (2003). A Kru¨ppel‐like transcription factor gene is involved in salt stress responses in Medicago spp. Plant and Soil 257, 1–9. Miller, C. R., Ochoa, I., Nielsen, K. L., Beck, D. and Lynch, J. P. (2003). Genetic variation for adventitious rooting in response to low phosphors availability: Potential utility for phosphorus acquisition from stratified soils. Functional Plant Biology 30, 973–985. Mittler, R. (2006). Abiotic stress, the field environment and stress combination. Trends in Plant Science 11, 15–19. Miura, K., Rus, A., Sharkhuu, A., Yokoi, S., Karthikeyan, A. S., Raghothama, K. G., Baek, D., Koo, Y. D., Jin, J. B., Bressan, R. A., Yun, D. J. and Hasegawa, P. M. (2005). The Arabidopsis SUMO E3 ligase SIZ1 controls phosphate deficiency responses. Proceedings of the National Academy of Sciences of the United States of America 102, 7760–7765. Miyawaki, K., Matsumoto‐Kitano, M. and Kakimoto, T. (2004). Expression of cytokinin biosynthetic isopentenyltransferase genes in Arabidopsis: Tissue specificity and regulation by auxin, cytokinin, and nitrate. The Plant Journal 37, 128–138. Molina‐Farero, C., Creus, C. M., Lanteri, M. L., Correa‐Aragunde, N., Lombardo, M. C., Barassi, C. A. and Lamattina, L. (2007). Nitric oxide and plant growth promoting rhizobacteria: Common features influencing root growth and development. Advances in Botanical Research 46, 1–23.

70

M. JOVANOVIC ET AL.

Mouchel, C. F., Briggs, G. C. and Hardtke, C. S. (2004). Natural genetic variation in Arabidopsis identifies BREVIS RADIX, a novel regulator of cell proliferation and elongation in the root. Genes & Development 18, 700–714. Mouchel, C. F., Osmont, K. S. and Hardtke, C. S. (2006). BRX mediates feedback between brassinosteroid levels and auxin signalling in root growth. Nature 443, 458–461. Nacry, P., Canivenc, G., Muller, B., Azmi, A., Van Onckelen, H., Rossignol, M. and Doumas, P. (2005). A role for auxin redistribution in the responses of the root system architecture to phosphate starvation in Arabidopsis. Plant Physiology 138, 2061–2074. Nag, R., Maity, M. K. and Dasgupta, M. (2005). Dual DNA binding property of ABA insensitive 3 like factors targeted to promoters responsive to ABA and auxin. Plant Molecular Biology 59, 821–838. Navarro, L., Dunoyer, P., Jay, F., Arnold, B., Dharmasiri, N., Estelle, M., Voinnet, O. and Jones, J. D. (2006). A plant miRNA contributes to antibacterial resistance by repressing auxin signaling. Science 312, 436–439. Nawy, T., Lee, J. Y., Colinas, J., Wang, J. Y., Thongrod, S. C., Malamy, J. E., Birnbaum, K. and Benfey, P. N. (2005). Transcriptional profile of the Arabidopsis root quiescent center. Plant Cell 17, 1908–1925. Nemhauser, J. L. and Chory, J. (2004). BRing it on: New insights into the mechanism of brassinosteroid action. Journal Experimental Botany 55, 265–270. Nemhauser, J. L., Mockler, T. C. and Chory, J. (2004). Interdependency of brassinosteroid and auxin signaling in Arabidopsis. PLoS Biology 2, E258. Nikiforova, V., Freitag, J., Kempa, S., Adamik, M., Hesse, H. and Hoefgen, R. (2003). Transcriptome analysis of sulfur depletion in Arabidopsis thaliana: Interlacing of biosynthetic pathways provides response specificity. The Plant Journal 33, 633–650. Nikiforova, V. J., Daub, C. O., Hesse, H., Willmitzer, L. and Hoefgen, R. (2005). Integrative gene‐metabolite network with implemented causality deciphers informational fluxes of sulphur stress response. Journal Experimental Botany 56, 1887–1896. Nishimura, C., Ohashi, Y., Sato, S., Kato, T., Tabata, S. and Ueguchi, C. (2004). Histidine kinase homologs that act as cytokinin receptors possess overlapping functions in the regulation of shoot and root growth in Arabidopsis. Plant Cell 16, 1365–1377. Nishimura, R., Ohmori, M. and Kawaguchi, M. (2002). The novel symbiotic phenotype of enhanced‐nodulating mutant of Lotus japonicus: Astray mutant is an early nodulating mutant with wider nodulation zone. Plant Cell Physiology 43, 853–859. Ohashi, Y., Oka, A., Rodrigues‐Pousada, R., Possenti, M., Ruberti, I., Morelli, G. and Aoyama, T. (2003). Modulation of phospholipid signaling by GLABRA2 in root‐hair pattern formation. Science 300, 1427–1430. Okushima, Y., Fukaki, H., Onoda, M., Theologis, A. and Tasaka, M. (2007). ARF7 and ARF19 regulate lateral root formation via direct activation of LBD/ ASL genes in Arabidopsis. Plant Cell 19, 118–130. Oldroyd, G. E. and Downie, J. A. (2004). Calcium, kinases and nodulation signalling in legumes. Nature Reviews Molecular Cell Biology 5, 566–576. Pasquinelli, A. E., Reinhart, B. J., Slack, F., Martindale, M. Q., Kuroda, M. I., Maller, B., Hayward, D. C., Ball, E. E., Degnan, B., Muller, P., Spring, J., Srinivasan, A., et al. (2000). Conservation of the sequence and temporal expression of let‐7 heterochronic regulatory RNA. Nature 408, 86–89. Penmetsa, R. V. and Cook, D. R. (1997). A legume ethylene‐insensitive mutant hyperinfected by its rhizobial symbiont. Science 275, 527–530.

ROOT ARCHITECTURE AND ENVIRONMENT

71

Penmetsa, R. V., Frugoli, J. A., Smith, L. S., Long, S. R. and Cook, D. R. (2003). Dual genetic pathways controlling nodule number in Medicago truncatula. Plant Physiology 131, 998–1008. Peragine, A., Yoshikawa, M., Wu, G., Albrecht, H. L. and Poethig, R. S. (2004). SGS3 and SGS2/SDE1/RDR6 are required for juvenile development and the production of trans‐acting siRNAs in Arabidopsis. Genes & Development 18, 2368–2379. Pereira, P. A. and Bliss, F. A. (1989). Selection of common bean (Phaseolus vulgaris L.) for Nitrogen fixation at diVerents levels of avaiable phosphorus under field and environmentally‐controlled conditions. Plant Soil 115, 75–82. Raghothama, K. G. (1999). Phosphate acquisition. Annual Review of Plant Physiology and Plant Molecular Biology 50, 665–693. Rajagopalan, R., Vaucheret, H., Trejo, J. and Bartel, D. P. (2006). A diverse and evolutionarily fluid set of microRNAs in Arabidopsis thaliana. Genes & Development 20, 3407–3425. Rashotte, A. M., Mason, M. G., Hutchison, C. E., Ferreira, F. J., Schaller, G. E. and Kieber, J. J. (2006). A subset of Arabidopsis AP2 transcription factors mediates cytokinin responses in concert with a two‐component pathway. Proceedings of the National Academy of Sciences of the United States of America 103, 11081–11085. Reed, R. C., Brady, S. R. and Muday, G. K. (1998). Inhibition of auxin movement from the shoot into the root inhibits lateral root development in Arabidopsis. Plant Physiology 118, 1369–1378. Reinhart, B. J., Slack, F. J., Basson, M., Pasquinelli, A. E., Bettinger, J. C., Rougvie, A. E., Horvitz, H. R. and Ruvkun, G. (2000). The 21‐nucleotide let‐7 RNA regulates developmental timing in Caenorhabditis elegans. Nature 403, 901–906. Reinhart, B. J., Weinstein, E. G., Rhoades, M. W., Bartel, B. and Bartel, D. P. (2002). MicroRNAs in plants. Genes & Development 16, 1616–1626. Remans, T., Nacry, P., Pervent, M., Girin, T., Tillard, P., Lepetit, M. and Gojon, A. (2006). A central role for the nitrate transporter NRT2.1 in the integrated morphological and physiological responses of the root system to nitrogen limitation in Arabidopsis. Plant Physiology 140, 909–921. Remington, D. L., Vision, T. J., Guilfoyle, T. J. and Reed, J. W. (2004). Contrasting modes of diversification in the Aux/IAA and ARF gene families. Plant Physiology 135, 1738–1752. Rhoades, M. W., Reinhart, B. J., Lim, L. P., Burge, C. B., Bartel, B. and Bartel, D. P. (2002). Prediction of plant microRNA targets. Cell 110, 513–520. Riefler, M., Novak, O., Strnad, M. and Schmulling, T. (2006). Arabidopsis cytokinin receptor mutants reveal functions in shoot growth, leaf senescence, seed size, germination, root development, and cytokinin metabolism. Plant Cell 18, 40–54. Rubio, V., Linhares, F., Solano, R., Martin, A. C., Iglesias, J., Leyva, A. and Paz‐ Ares, J. (2001). A conserved MYB transcription factor involved in phosphate starvation signaling both in vascular plants and in unicellular algae. Genes & Development 15, 2122–2133. Scheres, B., Di Laurenzio, L., Willemsen, V., Hauser, M. T., Janmaat, K., Weisbeek, P. and Benfey, P. (1995). Mutations aVecting the radial organisation of the Arabidopsis root display specific defects throughout the embryonic axis. Development 121, 53–62. Shin, R., Berg, R. H. and Schachtman, D. P. (2005). Reactive oxygen species and root hairs in Arabidopsis root response to nitrogen, phosphorus and potassium deficiency. Plant Cell Physiology 46, 1350–1357.

72

M. JOVANOVIC ET AL.

Sieberer, T. and Leyser, O. (2006). Plant science. Auxin transport, but in which direction? Science 312, 858–860. Signora, L., De Smet, I., Foyer, C. H. and Zhang, H. (2001). ABA plays a central role in mediating the regulatory eVects of nitrate on root branching in Arabidopsis. The Plant Journal 28, 655–662. Singh, M. B. and Bhalla, P. L. (2006). Plant stem cells carve their own niche. Trends in Plant Science 11, 241–246. Singleton, P. W. and Bohlool, B. (1984). EVect of salinity on nodule formation by soybean. Plant Physiology 74, 72–76. Sorin, C., Bussell, J. D., Camus, I., Ljung, K., Kowalczyk, M., Geiss, G., McKhann, H., Garcion, C., Vaucheret, H., Sandberg, G. and Bellini, C. (2005). Auxin and light control of adventitious rooting in Arabidopsis require ARGONAUTE1. Plant Cell 17, 1343–1359. Souter, M. A., Pullen, M. L., Topping, J. F., Zhang, X. and Lindsey, K. (2004). Rescue of defective auxin‐mediated gene expression and root meristem function by inhibition of ethylene signalling in sterol biosynthesis mutants of Arabidopsis. Planta 219, 773–783. Stacey, G., Libault, M., Brechenmacher, L., Wan, J. and May, G. D. (2006). Genetics and functional genomics of legume nodulation. Current Opinion in Plant Biology 9, 110–121. Stepanova, A. N., Hoyt, J. M., Hamilton, A. A. and Alonso, J. M. (2005). A link between ethylene and auxin uncovered by the characterization of two root‐ specific ethylene‐insensitive mutants in Arabidopsis. Plant Cell 17, 2230–2242. Sunkar, R. and Zhu, J. K. (2004). Novel and stress‐regulated microRNAs and other small RNAs from Arabidopsis. Plant Cell 16, 2001–2019. Sunkar, R., Kapoor, A. and Zhu, J. K. (2006). Posttranscriptional induction of two Cu/Zn superoxide dismutase genes in Arabidopsis is mediated by downregulation of miR398 and important for oxidative stress tolerance. Plant Cell 18, 2051–2065. Suzuki, A., Akune, M., Kogiso, M., Imagama, Y., Osuki, K., Uchiumi, T., Higashi, S., Han, S. Y., Yoshida, S., Asami, T. and Abe, M. (2004). Control of nodule number by the phytohormone abscisic acid in the roots of two leguminous species. Plant and Cell Physiology 45, 914–922. Swarup, R., Kramer, E. M., Perry, P., Knox, K., Leyser, H. M., HaseloV, J., Beemster, G. T., Bhalerao, R. and Bennett, M. J. (2005). Root gravitropism requires lateral root cap and epidermal cells for transport and response to a mobile auxin signal. Nature Cell Biology 7, 1057–1065. Tansengco, M. I., Hayashi, M., Kawaguchi, M., Imaizumi‐Anraku, H. and Murooka, Y. (2003). crinkle, a novel symbiotic mutant that aVects the infection thread growth and alters the root hair, trichome, and seed development in Lotus japonicus. Plant Physiology 131, 1054–1063. Teale, W. D., Paponov, I. A. and Palme, K. (2006). Auxin in action: Signalling, transport and the control of plant growth and development. Nature Reviews Molecular Cell Biology 7, 847–859. Timmers, A. C., Auriac, M. C. and Truchet, G. (1999). Refined analysis of early symbiotic steps of the Rhizobium‐Medicago interaction in relationship with microtubular cytoskeleton rearrangements. Development 126, 3617–3628. To, J. P., Haberer, G., Ferreira, F. J., Deruere, J., Mason, M. G., Schaller, G. E., Alonso, J. M., Ecker, J. R. and Kieber, J. J. (2004). Type‐A Arabidopsis response regulators are partially redundant negative regulators of cytokinin signaling. Plant Cell 16, 658–671.

ROOT ARCHITECTURE AND ENVIRONMENT

73

Todd, C. D., Zeng, P., Huete, A. M., Hoyos, M. E. and Polacco, J. C. (2004). Transcripts of MYB‐like genes respond to phosphorous and nitrogen deprivation in Arabidopsis. Planta 219, 1003–1009. Tuberosa, R., Salvi, S., Sanguineti, M. C., Landi, P., Maccaferri, M. and Conti, S. (2002a). Mapping QTLs regulating morpho‐physiological traits and yield: Case studies, shortcomings and perspectives in drought‐stressed maize. Annals of Botany (London) 89(Spec No), 941–963. Tuberosa, R., Sanguineti, M. C., Landi, P., Giuliani, M. M., Salvi, S. and Conti, S. (2002b). Identification of QTLs for root characteristics in maize grown in hydroponics and analysis of their overlap with QTLs for grain yield in the field at two water regimes. Plant Molecular Biology 48, 697–712. Ulmasov, T., Hagen, G. and Guilfoyle, T. J. (1999). Activation and repression of transcription by auxin‐response factors. Proceedings of the National Academy of Sciences of the United States of America 96, 5844–5849. van der Weele, C. M., Spollen, W. G., Sharp, R. E. and Baskin, T. I. (2000). Growth of Arabidopsis thaliana seedlings under water deficit studied by control of water potential in nutrient‐agar media. Journal of Experimental Botany 51, 1555–1562. Vanneste, S., De Rybel, B., Beemster, G. T., Ljung, K., De Smet, I., VanIsterdael, G., Naudts, M., Iida, R., Gruissem, W., Tasaka, M., Inze, D., Fukaki, H., et al. (2005). Cell cycle progression in the pericycle is not suYcient for SOLITARY ROOT/IAA14‐mediated lateral root initiation in Arabidopsis thaliana. Plant Cell 17, 3035–3050. Vartanian, N. (1981). Some aspects of structural and functional modifications induced by drought in root systems. Plant Soil 63, 83–92. Vartanian, N., Marcotte, L. and Giraudat, J. (1994). Drought rhizogenesis in Arabidopsis thaliana (diVerential responses of hormonal mutants). Plant Physiology 104, 761–767. Vazquez, F., Gasciolli, V., Crete, P. and Vaucheret, H. (2004a). The nuclear dsRNA binding protein HYL1 is required for microRNA accumulation and plant development, but not posttranscriptional transgene silencing. Current Biology 14, 346–351. Vazquez, F., Vaucheret, H., Rajagopalan, R., Lepers, C., Gasciolli, V., Mallory, A. C., Hilbert, J. L., Bartel, D. P. and Crete, P. (2004b). Endogenous trans‐acting siRNAs regulate the accumulation of Arabidopsis mRNAs. Molecular Cell 16, 69–79. Veereshlingam, H., Haynes, J. G., Penmetsa, R. V., Cook, D. R., Sherrier, D. J. and Dickstein, R. (2004). nip, a symbiotic Medicago truncatula mutant that forms root nodules with aberrant infection threads and plant defense‐like response. Plant Physiology 136, 3692–3702. Vernoux, T., Wilson, R. C., Seeley, K. A., Reichheld, J. P., Muroy, S., Brown, S., Maughan, S. C., Cobbett, C. S., Van Montagu, M., Inze, D., May, M. J. and Sung, Z. R. (2000). The ROOT MERISTEMLESS1/CADMIUM SENSITIVE2 gene defines a glutathione‐dependent pathway involved in initiation and maintenance of cell division during postembryonic root development. Plant Cell 12, 97–110. Voinnet, O. (2005). Induction and suppression of RNA silencing: Insights from viral infections. Nature Reviews Genetics 6, 206–220. Walch‐Liu, P., Ivanov, II., Filleur, S., Gan, Y., Remans, T. and Forde, B. G. (2006). Nitrogen regulation of root branching. Annals of Botany (London) 97, 875–881. Wang, B. and Qiu, Y. L. (2006). Phylogenetic distribution and evolution of mycorrhizas in land plants. Mycorrhiza 16, 299–363.

74

M. JOVANOVIC ET AL.

Wang, J. W., Wang, L. J., Mao, Y. B., Cai, W. J., Xue, H. W. and Chen, X. Y. (2005). Control of root cap formation by MicroRNA‐targeted auxin response factors in Arabidopsis. Plant Cell 17, 2204–2216. Werner, T., Motyka, V., Strnad, M. and Schmulling, T. (2001). Regulation of plant growth by cytokinin. Proceedings of the National Academy of Sciences of the United States of America 98, 10487–10492. Werner, T., Motyka, V., Laucou, V., Smets, R., Van Onckelen, H. and Schmulling, T. (2003). Cytokinin‐deficient transgenic Arabidopsis plants show multiple developmental alterations indicating opposite functions of cytokinins in the regulation of shoot and root meristem activity. Plant Cell 15, 2532–2550. Wightman, B., Ha, I. and Ruvkun, G. (1993). Posttranscriptional regulation of the heterochronic gene lin‐14 by lin‐4 mediates temporal pattern formation in C. elegans. Cell 75, 855–862. Williams, L., Carles, C. C., Osmont, K. S. and Fletcher, J. C. (2005a). A database analysis method identifies an endogenous trans‐acting short‐interfering RNA that targets the Arabidopsis ARF2, ARF3, and ARF4 genes. Proceedings of the National Academy of Sciences of the United States of America 102, 9703–9708. Wilmoth, J. C., Wang, S., Tiwari, S. B., Joshi, A. D., Hagen, G., Guilfoyle, T. J., Alonso, J. M., Ecker, J. R. and Reed, J. W. (2005). NPH4/ARF7 and ARF19 promote leaf expansion and auxin‐induced lateral root formation. The Plant Journal 43, 118–130. Winicov, I. (1993). cDNA encoding putative zinc finger motifs from salt‐tolerant alfalfa (Medicago sativa L.) cells. Plant Physiology 102, 681–682. Winicov, I. (2000). Alfin transcriptor factor overexpression enhances plants root growth under normal and saline conditions and improves salt tolerance in alfalfa. Planta 210, 416–422. Wopereis, J., Pajuelo, E., Dazzo, F. B., Jiang, Q., GresshoV, P. M., De, B. F. J., Stougaard, J. and Szczyglowski, K. (2000). Short root mutant of Lotus japonicus with a dramatically altered symbiotic phenotype. The Plant Journal 23, 97–114. Wysocka‐Diller, J. W., Helariutta, Y., Fukaki, H., Malamy, J. E. and Benfey, P. N. (2000). Molecular analysis of SCARECROW function reveals a radial patterning mechanism common to root and shoot. Development 127, 595–603. Xie, Q., Frugis, G., Colgan, D. and Chua, N. H. (2000). Arabidopsis NAC1 transduces auxin signal downstream of TIR1 to promote lateral root development. Genes & Development 14, 3024–3036. Xiong, L., Wang, R. G., Mao, G. and Koczan, J. M. (2006). Identification of drought tolerance determinants by genetic analysis of root response to drought stress and abscisic acid. Plant Physiology 142, 1065–1074. Yang, L., Liu, Z., Lu, F., Dong, A. and Huang, H. (2006). SERRATE is a novel nuclear regulator in primary microRNA processing in Arabidopsis. The Plant Journal 47, 841–850. Zhang, B., Pan, X., Cannon, C. H., Cobb, G. P. and Anderson, T. A. (2006). Conservation and divergence of plant microRNA genes. The Plant Journal 46, 243–259. Zhang, H. and Forde, B. G. (1998). An Arabidopsis MADS box gene that controls nutrient‐induced changes in root architecture. Science 279, 407–409. Zhang, H., Jennings, A., Barlow, P. W. and Forde, B. G. (1999). Dual pathways for regulation of root branching by nitrate. Proceedings of the National Academy of Sciences of the United States of America 96, 6529–6534.

Aquaporins in Plants: From Molecular Structure to Integrated Functions

OLIVIER POSTAIRE, LIONEL VERDOUCQ AND CHRISTOPHE MAUREL

Biochimie et Physiologie Mole´culaire des Plantes, SupAgro/CNRS/INRA/UM2 UMR 5004, 2 Place Viala, F‐34060 Montpellier Cedex 1, France

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . II. Significance of Aquaporin Molecular Structure for Transport Specificity and Gating . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Aquaporins Are Tetrameric Transmembrane Channels.................. B. Methods for Functional Characterization of Plant Aquaporins ........ C. Plant Aquaporins Are Not Just Water Channels.......................... D. Molecular and Structural Bases of Aquaporin Selectivity ............... E. Molecular Mechanisms of Aquaporin Gating ............................. III. Aquaporins in Roots . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Principles of Root Water Uptake ............................................ B. Aquaporin Expression in Roots.............................................. C. Measurements of Root Water Transport ................................... D. Evidence for Water Transport by Aquaporins in Roots ................. E. Effects of Stimuli on Root Water Transport ............................... F. Transport of Nutrients ........................................................ IV. Aquaporins in Leaves . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Aquaporin Expression in Leaves............................................. B. Measurements of Water Transport in Leaves.............................. C. Water Transport Pathways in Leaves ....................................... D. Functions of Aquaporins in Leaf Water Transport....................... E. Physiological Regulations of Kleaf ........................................... F. CO2 Transport ..................................................................

Advances in Botanical Research, Vol. 46 Incorporating Advances in Plant Pathology Copyright 2008, Elsevier Ltd. All rights reserved.

76 78 78 80 82 84 86 91 91 93 94 96 100 105 106 107 108 110 112 113 116

0065-2296/08 $35.00 DOI: 10.1016/S0065-2296(07)46003-7

76

O. POSTAIRE ET AL.

V. Aquaporins in Reproductive Organs. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Aquaporins in Flowers ........................................................ B. Aquaporins in Seeds ........................................................... VI. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

117 118 120 122 123

ABSTRACT Aquaporins belong to a superfamily of membrane channels with members in all living organisms. In plants, aquaporins mediate a large part of the cell‐to‐cell and intracellular water movements. The ability of certain plant aquaporins homologues to transport nutrient such as boron or gas such as CO2 has recently been demonstrated. This present chapter specifically examines how our current understanding of aquaporin structure and function can be integrated into whole plant physiology. Expression studies coupled with physiological and genetic analyses have allowed to delineate a variety of functions for aquaporins in roots, leaves, and during plant reproduction. In addition, a large variety of molecular and cellular mechanisms have been identified that lead to fine regulation of membrane water transport, during plant development, or in response to environmental stimuli. However, central physiological questions remain, such as the role of aquaporins in carbon assimilation, or in a hydraulic control of growth and cell movements.

ABBREVIATIONS CAM GFP gm Gs HPFM Kleaf Lpr MIP NIP Pf PIP SIP TIP

crassulacean acid metabolism green fluorescent protein conductance of mesophyll to CO2 stomatal conductance high pressure flow meter leaf hydraulic conductance root hydraulic conductivity major intrinsic protein nodulin26‐like intrinsic protein osmotic water permeability plasma membrane intrinsic protein water potential small basic intrinsic protein tonoplast intrinsic protein

I. INTRODUCTION Aquaporins belong to a superfamily of membrane channels named after its founding member, the major intrinsic protein (MIP) of lens fibers. MIP homologues have now been identified in all living organisms (Agre et al., 1998). In recent years, tremendous progress has been made in understanding their molecular structure and primary transport properties. CHIP28, an abundant protein in erythrocytes and kidney tubules was the first MIP to

AQUAPORINS IN PLANTS

77

be identified as a molecular water channel (Preston et al., 1992) and hence was renamed aquaporin‐1 (AQP1). In the following years, several other MIPs including plant
‐tonoplast intrinsic proteins (
‐TIP or TIP1;1) were also shown to transport substantial amounts of water and were also designated as aquaporins (Chrispeels and Agre, 1994). By contrast, other MIPs, such as bacterial glycerol facilitator GlpF, function as solute channels and have no or a weak water channel activity (Maurel et al., 1995). These MIPs have been named aquaglyceroporins (Agre et al., 1998). Inplants, aquaporins show a typically high isoform multiplicity. Plantgenome sequencing has identified 35 and 33 aquaporin homologues in Arabidopsis thaliana and rice, respectively (Johansson et al., 2000; Quigley et al., 2001; Sakurai et al., 2005). A similar number of homologues are found in maize (Chaumont et al., 2001). Sequence analysis also showed that plant aquaporins fall into four major homology subgroups that somehow reflect specific subcellular localizations (Fig. 1). For instance, the plasma membrane intrinsic proteins (PIPs; 13 homologues in Arabidopsis) and tonoplast intrinsic proteins (TIPs; 10 homologues in Arabidopsis) sit predominantly in the plasma membrane and in the tonoplast, respectively, hence their name (for review, see Maurel et al., 2002). A third subclass of Nodulin26‐like Intrinsic Proteins (NIPs) is formed by homologues of Nodulin‐26 (NOD26), an aquaporin of the peribacteroid membrane of symbiotic nitrogen‐fixing root nodules (Weig et al., 1997). NIPs are also present in non‐legume plants (nine homologues in Arabidopsis), and some of them have been localized in the plasma membrane of rice and Arabidopsis cells (Ma et al., 2006; Takano et al., 2006). The fourth subclass of so‐called Small basic Intrinsic Proteins (SIPs) comprises three homologues in Arabidopsis, which show preferential localization in the endoplasmic reticulum (Ishikawa et al., 2005). More than 10 years after their discovery in plants, it now appears that aquaporins mediate a large part of the cell‐to‐cell and intracellular water movements in these organisms (Maurel et al., 2002; Tyerman and Niemietz, 2002). Their characterization has pointed to a large variety of molecular and cellular mechanisms that lead to fine regulation of membrane water transport, during plant development or in response to environmental stimuli (Chaumont et al., 2005; Luu and Maurel, 2005). More recently, several plant aquaporin homologues have been associated to the transport of small neutral molecules, including gas or micronutrients. Therefore, the function of MIP channels in plants appears to go much beyond plant water relations (KaldenhoV and Fischer, 2006; Tyerman and Niemietz, 2002). In this present chapter, we want to summarize our current knowledge on the integrated function of aquaporin homologues in plants. For this, we will in the first place examine how novel insights were gained from enhanced knowledge of their structure–function relationship. Our aim is to show how this

78

O. POSTAIRE ET AL. hAQP4 hAQP1

hAQP2 hAQP5 hAQP0 hAQP6

PIPs

AtTIP5;1 AtTIP4;1 TIPs AtTIP3;2 AtTIP3;1 AtTIP1;3 AtTIP1;2

AtPIP1;1 AtPIP1;2 AtPIP1;3 AtPIP1;4 AtPIP1;5 AtPIP2;2 AtPIP2;3 AtPIP2;1 AtPIP2;4 AtPIP2;5 AtPIP2;6 AtPIP2;7 AtPIP2;8

AtTIP1;1 AtTIP2;1 AtTIP2;3 AtTIP2;2

AqpZ AtNIP7;1 AtNIP3;1

SoPIP2;1

AtNIP2;1

AtSIP1;1

NOD26 AtSIP1;2

AtNIP1;2 AtNIP1;1

SIPs

AtSIP2;1

AtNIP4;2

hAQP8 hAQP3 hAQP10 hAQP9 0.1

NIPs

AtNIP4;1 AtNIP6;1 AtNIP5;1 GlpF hAQP7

Aquaglyceroporins

Fig. 1. Sequence relationship between aquaporin homologues of Arabidopsis thaliana, human, and Escherichia coli. The amino acid sequences were aligned with ClustalW and a phylogenetic tree was constructed with TreeView. The tree illustrates the subdivision of the Arabidopsis aquaporin family in four subfamilies: PIPs, TIPs, NIPs, and SIPs. Two other well‐characterized plant aquaporins, SoPIP2;1 and NOD26 from spinach and soybean, respectively, are also included in the tree for reference. The two E. coli aquaporins (GlpF and AqpZ) are represented in blue, while the 11 human aquaporins (hAQP0 to hAQP10) are represented in red. Note that the human aquaporins group into two subfamilies. One of them comprises GlpF and corresponds to the aquaglyceroporin subfamily.

knowledge, combined to genetic and physiological evidences, now defines a large array of functions in roots, leaves, and during plant reproduction.

II. SIGNIFICANCE OF AQUAPORIN MOLECULAR STRUCTURE FOR TRANSPORT SPECIFICITY AND GATING A. AQUAPORINS ARE TETRAMERIC TRANSMEMBRANE CHANNELS

MIPs have emerged as a family of general interest among membrane proteins, as the atomic structures of AQP1 and GlpF were among the first to be solved for polytopic membrane proteins (Murata et al., 2000; Sui et al., 2001).

AQUAPORINS IN PLANTS

79

To date, 18 structures of aquaporin homologues are available. These structural studies have allowed to validate earlier structure–function analyses. Most importantly, they have also provided an exquisite understanding of water and solute permeation mechanisms through the aquaporin channel. Very recently, the first atomic structure of a plant aquaporin was solved (Tornroth‐Horsefield et al., 2006). Because, for the first time, an aquaporin structure was determined in both the open and closed states, this work gave unique insights into the gating mechanisms of aquaporins. Primary sequence analyses indicate that most MIPs consist of small (25–34 kDa), hydrophobic proteins (Chrispeels and Agre 1994; Zardoya and Villalba, 2001), with a typical symmetrical organization due to an early gene duplication. All MIPs comprise six transmembrane domains with the N‐ and C‐terminal ends of the protein being located in the cytoplasm. Early structure–function analyses of AQP1 in oocytes suggested an ‘‘hourglass‐fold’’ model for this aquaporin (Jung et al., 1994). In this model, two highly conserved loops (loops B and E), each of them carrying a conserved Asn‐Pro‐Ala (NPA) motif, deep into the pore from either side of the membrane (Chrispeels and Agre 1994; Jung et al., 1994). The NPA motif constitutes the most prominent signature sequence of aquaporins. Biochemical and functional studies also suggested that aquaporins assemble as homo‐tetramers. Unlike tetrameric potassium channels, the water transporting pore is not localized at the center of the aquaporin tetramer but instead one channel is formed at the center of each monomer (Jung et al., 1994). This typical arrangement was first visualized by freeze‐fracture studies (Verbavatz et al., 1993) and then by electron microscopy of two‐ dimensional crystals. A similar approach showed that plant TIPs and PIPs also exhibit a tetrameric organization (Daniels et al., 1999; Fotiadis et al., 2001). A major breakthrough in aquaporin research was the resolution by cryoe˚ atomic structure for human AQP1 (Murata lectron microscopy of a 3.8 A ˚ atomic structures for et al., 2000), and by X‐ray crystallography of 2.2 A bacterial GlpF (Fu et al., 2000) and bovine AQP1 (Sui et al., 2001). These pioneering studies confirmed the ‘‘hourglass‐fold’’ model and showed how each aquaporin monomer is folded according to a conserved structural core of six transmembrane ‐helices tilted along the plane of the membrane. These studies also revealed in great detail how each monomer contributes to form ˚ an individual, narrow, aqueous pathway through the membrane. The 2.1 A atomic structure of spinach SoPIP2;1 (formerly named PM28A) revealed a very similar ‘‘hourglass‐fold’’ that therefore has been conserved between 1.6 billion year distant proteins, with a root to mean square deviation of ˚ (Tornroth‐Horsefield et al., 2006). only 0.8 A

80

O. POSTAIRE ET AL. B. METHODS FOR FUNCTIONAL CHARACTERIZATION OF PLANT AQUAPORINS

Accurate measurements of water permeability at the level of isolated membranes, single cells, or whole organs have proved crucial to investigate the various aspects of plant aquaporin function, including their transport selectivity, the molecular and cellular mechanisms of their regulation, and their integrated function. These measurements can be made in plant materials or after expression of an individual aquaporin in a heterologous system. In many cases, the membrane water permeability of the cells or vesicles can be derived from the kinetics of volume adjustment in response to a rapid osmotic challenge. The kinetics of all of these objects are determined by both their intrinsic water permeability and their surface‐to‐volume ratio. The latter is inversely proportional to the object size and can span over four orders of magnitude from Xenopus oocytes (diameter
103 m) to membrane vesicles (diameter
107 m). 1. Stopped‐flow techniques These techniques allow to monitor fast (in the 0.1–1 s range) flow kinetics in small objects like isolated vesicles (Beuron et al., 1995; Verkman, 2000). Stopped‐flow techniques have proved useful to characterize the activity of plant aquaporins in their native membranes, after heterologous expression in yeasts, or after purification and reconstitution in proteoliposomes (Karlsson et al., 2003; Laize´ et al., 1995). Water permeability of the membrane vesicles is deduced from the kinetics of osmotic volume adjustments and from an independent size determination, usually made by electron microscopy or dynamic light scattering. Stopped‐flow techniques have also proved useful to elucidate various aspects of aquaporin transport selectivity. For instance, the membrane permeability to small neutral solutes, such as glycerol or urea, can be determined by iso‐ or hyper‐osmotic challenges in the presence of a concentration gradient for the solute (Gerbeau et al., 1999). Stopped‐flow spectrophotometry can also be used to monitor CO2 transport on vesicles loaded with exogenous carbonic anhydrase and pH‐dependent probes (Prasad et al., 1998). Although these measurements exhibit a great biophysical accuracy, there have been concerns that the water or solute transport properties of aquaporins may be altered during membrane isolation from plant materials. In studies on plasma membranes purified from Arabidopsis cell suspensions, Gerbeau et al. (2002) suggested that protection of aquaporins from dephosphorylation by protein phosphatases or from inhibition by divalent ions (see Section II.E.4) may be critical to maintain purified plasma membranes with their native water permeability.

AQUAPORINS IN PLANTS

81

2. Swelling and shrinking of plant protoplasts and vacuoles Kinetic size measurements on objects such as plant protoplasts or vacuoles are problematic because the rapid imposition of an osmotic gradient can be destructive because of the great fragility of these objects. Video‐microscopy has been used to record early changes in protoplast/vacuole volume after a quick transfer of the object in solutions of varying osmolalities (Ramahaleo et al., 1999). This transfer can be achieved by micromanipulation using a pipette or by immobilization in a microscopic observation chamber that supports a continuous perfusion. Using the latter technique, Moshelion et al. (2004) observed a delay of a few seconds in the swelling behavior of protoplasts subjected to a hypotonic challenge. This delay was tentatively explained as a complex adjustment of membrane water permeability during the course of cell swelling (Moshelion et al., 2004). 3. Cell pressure probe Cell pressure probe techniques allow to determine cell water relation parameters in intact plant tissues (Steudle, 1993). The instrument can be assimilated to a miniature syringe with an oil‐filled micropipette linked to a pressure transducer. The micropipette is used to impale a living cell. Displacement of the meniscus, formed at the tip of the pipette by the contact between the oil and the cell sap, is used to monitor volume exchanges between the probe and the cell. When the micropipette enters the cell, the turgor pressure shifts the meniscus and this shift can be compensated by applying a pressure in the instrument, which corresponds to the initial cell turgor. Osmotic or hydrostatic perturbations can thereafter be imposed by altering the osmolarity of the bathing solution or by imposing pressure shifts via the syringe. These maneuvers result in cell pressure relaxations, which provide crucial informations on the mechanical properties of the cell (cell wall elastic modulus) and its hydraulic conductivity. The membrane reflection coeYcients for diVerent solutes can also be determined. Over the past two decades, this technique has been extensively used to uncover a large variety of physiological regulations of membrane water transport in plants. More recently, the technique has been applied to the phenotypic characterization of aquaporin knock‐out mutants and has allowed a precise quantification of the contribution of a single aquaporin (AtPIP2;2) to water transport in root cortical cells (Javot et al., 2003). 4. Water measurement in heterologous systems Xenopus laevis oocytes represent one of the most favorable system for assaying the water and solute transport of cloned individual aquaporins. First, these cells are virtually devoid of endogenous water channels and have thus a

82

O. POSTAIRE ET AL.

low basal water permeability. Second, oocytes can eYciently express exogenous membrane proteins upon intracellular injection of in vitro transcribed complementary RNAs. Third, because of their large size, oocytes exhibit relatively slow swelling kinetics that can easily be followed by video microscopy. Historically, expression in oocytes has provided a central evidence to prove that MIPs cloned from animals, bacteria, or plants encoded functional water channels (Maurel et al., 1993, 1994; Preston et al., 1992). In addition, the capacity of aquaporins to transport solutes such as glycerol or urea can also be monitored in this system by the uptake of radio‐labeled molecules (Maurel et al., 1993). Luckily enough, many plant aquaporins, even those such as TIPs which are targeted to intracellular plant membranes, are at least in part routed to the oocyte surface and thus can be functionally characterized in these cells. In all plant species, PIP aquaporins can be subdivided into two typical subclasses, PIP1 and PIP2. Several laboratories have noticed that PIP1 homologues are recalcitrant to oocyte expression (Fetter et al., 2004). One reason is a deficient targeting of PIP1s to the cell surface (see Section II.E). Finally, plant aquaporins are regulated by a variety of cytoplasmic eVectors. Some of them can be eYciently altered in oocytes and therefore these cells have been instrumental for disecting the structural basis of aquaporin regulation by phosphorylation or by cytoplasmic pH (Johansson et al., 1998; Tournaire‐Roux et al., 2003). Aquaporins are among the most highly expressed membrane proteins in animals or plants and can easily be purified from their native organisms (for review, see Maurel et al., 2002). High‐expression levels of recombinant plant aquaporins can also be obtained in heterologous systems, such as yeasts (Daniels and Yeager, 2005; Karlsson et al., 2003). The purified proteins can then be inserted in artificial membranes (Daniels and Yeager, 2005; Dean et al., 1999; Karlsson et al., 2003). This so‐called proteoliposomes provide a unique system for studying the molecular and structural determinants involved in aquaporin gating. In particular, the specific intrinsic water permeability of a single aquaporin can be determined in an accurately controlled environment. Thus, this approach may be adequate for deciphering the molecular basis of PIP regulation by cytoplasmic calcium since oocytes or other living cells tightly control their cytoplasmic calcium concentration (see Section II.E.4). C. PLANT AQUAPORINS ARE NOT JUST WATER CHANNELS

It has been proposed that the great diversity of plant aquaporins may reflect, in addition to distinct subcellular localizations, a broad range of transport specificities (Tyerman and Niemietz, 2002). Functional expression in

AQUAPORINS IN PLANTS

83

Xenopus oocytes revealed that Arabidopsis TIP1;1 is only permeable to water (Maurel et al., 1993), whereas its tobacco homologue NtTIPa transports urea and glycerol in addition to water (Gerbeau et al., 1999). Other TIP homologues have recently been identified as permeable to urea through complementation of a yeast mutant defective in its corresponding endogenous transport system (Klebl et al., 2003; Liu et al., 2003). Solute transport is, however, not restricted to TIPs and other plant aquaporins of the PIP (Biela et al., 1999) and NIP subfamilies (Rivers et al., 1997; Weig and Jakob 2000) transport small neutral solutes in addition to water. In particular, functional reconstitution in proteoliposomes of soybean NOD26 has shown that this protein transports glycerol with a high eYciency and has a low intrinsic water permeability (Dean et al., 1999). Very recently, Lsi1, a AtNIP7;1 relative, was characterized from a rice mutant defective in silicon uptake and was shown to transport silicon in Xenopus oocytes (Ma et al., 2006). In an other study, it was shown that boron uptake in Arabidopsis plants is mediated in large part by NIP5;1 (Takano et al., 2006). Other molecules relevant to plant physiology, such as antimonite (Sanders et al., 1997) and hydrogen peroxide (H2O2) (Bienert et al., 2007; Henzler and Steudle, 2000), have also been shown to be transported by MIPs. The permeability of plant and animal aquaporins to reactive oxygen species, such as H2O2 (Bienert et al., 2007; Henzler and Steudle, 2000) or NO (Herrera et al., 2006), raises the exciting possibility that aquaporins may participate in cell‐signaling cascades, and therefore supports the general idea that aquaporins fulfill multiple functions, besides water and nutrient transport. In these respects, the capacity of certain aquaporins to transport gaseous compounds has recently raised a great interest. CO2 transport, in particular, may be one of most significant. It is important to note that CO2, similar to water, is a highly diVusive molecule that can freely pass the lipid bilayer of cell membranes. This property has long been considered as suYcient to account for the diVusion of the gas, from the stomatal chamber to mesophyll cell chloroplasts. The transport of CO2 by mammalian AQP1 has been unambiguously demonstrated after heterologous expression of the protein in oocytes (Cooper and Boron 1998; Nakhoul et al., 1998) and after functional reconstitution in proteoliposomes (Prasad et al., 1998). Recently, functional expression in oocytes of a tobacco PIP aquaporin, NtAQP1, has shown that this aquaporin also significantly transports gaseous CO2 (Uehlein et al., 2003). In addition, ammonia transport has been hypothesized for NOD26 (Niemietz and Tyerman, 2000) and demonstrated for several Arabidopsis and wheat TIP homologues (Jahn et al., 2004; Loque et al., 2005). Interestingly, some human aquaporins such as AQP1 and AQP6 can function as ionic channels provided that they are placed under very specific

84

O. POSTAIRE ET AL.

conditions, that is, acidic extracellular pH (Yasui et al., 1999) or stimulation by cGMP (Anthony et al., 2000). Similar behavior has not yet been identified in plant aquaporins. D. MOLECULAR AND STRUCTURAL BASES OF AQUAPORIN SELECTIVITY

The determination of atomic structures for human AQP1 (Murata et al., 2000), bacterial GlpF (Fu et al., 2000), and bovine AQP1 (Sui et al., 2001) has allowed to uncover fundamental mechanisms of transport selectivity in aquaporins. Two conserved structural motifs have been determined as crucial. One motif is formed by loops B and E that fold as half‐helices and dip into the center of the channel from opposite sides of the membrane, therefore gathering the two NPA motifs in close vicinity (Fig. 2). In this arrangement, the Asn residues of the two NPA motifs play a key role in the formation of

Apoplasm

Ar/R

Arg 231

Phe 87 His 216 Phe 51 Asn 228 Asn 107

NPA

Plasma membrane

C-ter N-ter

LoopD

Cytoplasm

Fig. 2. Structural model of Arabidopsis PIP2;1. A predictive structure (amino‐ acids 28–269) was obtained by homology modeling (using the Swiss‐Model server at http://www.expasy.org/swissmod/swiss‐model.html) based on the X‐ray structure of spinach SoPIP2;1 in its open conformation (PDB ID: 2B5F). The picture represents a single monomer with the six transmembrane domains and the two constrictions Ar/R and NPA shown. The Asn residue of the two NPA motifs and the residues forming the Ar/R motif are shown in red and yellow, respectively.

AQUAPORINS IN PLANTS

85

the pore selectivity filter. However, exceptions to the NPA aquaporin signature exist in plants, and some NIP and SIP isoforms show a substitution of at least one Ala residue by another residue (for review, see Ishibashi, 2006). In the canonical aquaporin structure, the positive electrostatic potential at the NPA constriction is supposed to prevent proton translocation through the pore (Chakrabarti et al., 2004; De Groot et al., 2003). This model is supported by molecular dynamic simulations on animal or plant aquaporins showing a 1808 dipole reorientation of the water molecule upon its passage (De Groot et al., 2001; Tajkhorshid et al., 2002; Tornroth‐Horsefield et al., 2006). This reorientation breaks the single file of water molecules and therefore blocks proton transfer through the channel. A second arrangement named Ar/R, because it is composed of aromatic (Ar) and polar (Arg or R) residues, forms a constriction in the extracellular half of the pore. This constriction is narrow for strictly water selective ˚ for mammalian AQP1, 2.4 A ˚ for aquaporins, with a diameter of 2.8 A ˚ bacterial AQPZ, and 2.1 A for plant SoPIP2;1 (Murata et al., 2000; Savage et al., 2003; Tornroth‐Horsefield et al., 2006). By contrast, glycerol facilita˚ in bacterial GlpF for instance tors display a wider constriction, of 3.3 A (Fu et al., 2000). Such channel widening is predicted to occur in all aquaporin homologues permeable to small solutes such as glycerol or urea. In addition, the positive charge of Arg in the Ar/R arrangement is thought to generate an electrostatic repulsion, which together with size restriction, would also act as an eYcient filter for protons and ions (for review, see Fujiyoshi et al., 2002). A model of polyol permeation in GlpF has proposed that the carbon backbone of the solute slides on a hydrophobic side of the pore, whereas its hydroxyl groups form hydrogen bonds with residues on the opposite side of the pore (Fu et al., 2000). This model explains why GlpF is permeable to linear polyols that have their OH groups lined up in the same direction with respect to the carbon backbone (i.e., glycerol, ribitol), whereas the channel protein is poorly permeable to asymmetrical stereoisomers, such as xylitol or D‐arabitol. Although most of the structural information available on aquaporins was obtained on water specific aquaporins, the overall molecular structure of plant aquaporins displaying non‐classical solute specificities, such as TIPs or NIPs, can be predicted from the available structures and can help identify the specificity determinants of these proteins. Again, these modeling approaches pointed to the importance of the Ar/R constriction (Wallace and Roberts, 2004). Structure–function analyses with Lotus japonicus LIMP2 aquaporin, a close homologue of soybean NOD26, confirmed that a single amino acid substitution in this domain can alter transport selectivity (Wallace et al., 2002). More recently, an elegant work, combining biochemical and structural approaches demonstrated that residues at the Ar/R constriction, and

86

O. POSTAIRE ET AL.

especially in helix two, play a critical role as a selectivity filter in the Arabidopsis aquaporin AtNIP6;1 (Wallace and Roberts, 2005). This study is especially relevant to a subgroup of NIPs (subgroup II). These plant MIPs seem to have evolved from multifunctional aquaglyceroporins and are now specialized in the transport of solutes such as silicon or boron (Ma et al., 2006; Takano et al., 2006; Wallace and Roberts, 2005). One striking example is rice Lsi1, which was genetically identified as a silicon channel after characterization of mutant plants defective in silicon uptake. The mutant Lsi1 allele carried a single Ala to Thr substitution in helix three. Molecular modeling showed that this substitution causes a severe conformational change of the Asn located in the first NPA motif, and therefore inactivates the channel (Ma et al., 2006). E. MOLECULAR MECHANISMS OF AQUAPORIN GATING

A current challenge in the study of membrane proteins, and especially channel proteins, is to understand the structural aspects of their gating properties. The aim is to describe a sequence of conformational changes that lead to pore opening or closing. While biochemical and physiological studies have indicated that plant aquaporins are regulated by numerous factors related to intracellular metabolic state or signaling, recent structural study has provided critical insights into the molecular events that underlie these regulations (Tornroth‐Horsefield et al., 2006). 1. Blockade by mercury Mercury has long been recognized as a common blocker of aquaporins and, despite its low specificity and cellular toxicity, has been extensively used in physiological studies that addressed the role of aquaporins in plants. Cys residues are known to be preferential targets for oxidation by mercury. In human AQP1, mercury acts at a unique Cys residue, which is located right at the Ar/R constriction, in front of His180 and Arg195. This site of action provides a clear mechanistic explanation to the blocking eVects of mercury (Sui et al., 2001). By contrast, the structural basis of inhibition of plant aquaporins is as yet unclear. In particular, there is no conserved cysteine residue in the pore of these proteins. However, introduction of one such residue in the pore‐forming loop B of AtPIP2;3, a mercury insensitive aquaporin isoform, was able to confer sensitivity to the blocker (Daniels et al., 1996). The structure of SoPIP2;1 revealed that a conserved Cys residue at the N‐terminal end of helix two can make a disulfide bridge between two adjacent monomers (Tornroth‐Horsefield et al., 2006) and may stabilize them. Therefore, this residue may be a target for mercury inhibition of PIPs. Silver

AQUAPORINS IN PLANTS

87

and gold ions have also been described as eYcient blockers (IC50 ¼ 1–10 mM) of aquaporins in membranes from roots, soybean nodules, and human red blood cells (Niemietz and Tyerman, 2002), but the molecular basis of this inhibition is still unknown. 2. Regulation by reactive oxygen species Direct gating of plant aquaporins by reactive oxygen species has been suggested from experiments made in Chara corallina internodal cells (Henzler et al., 2004) and more recently in corn roots (Ye and Steudle, 2006). Henzler et al. (2004) proposed that hydroxyl radicals, produced by a Fenton reaction from exogenously supplied H2O2, act on aquaporin gating either by direct oxidation of the aquaporins or by indirectly through lipid membrane oxidation and formation of secondary radicals. In addition, reactive oxygen species are also critical elements in cell signaling and may trigger a cascade of events ultimately leading to aquaporin downregulation in living cells (Y. Boursiac and C.M. unpublished results). 3. Regulation by osmotic and hydrostatic pressures Evidence exists that high concentrations of osmolytes act not only as a driving force for water transport but also can interfere with aquaporin gating. Niemietz and Tyerman (1997) reported that the osmotic water permeability of tonoplast vesicles isolated from wheat root was dependent on the strength of the imposed osmotic gradient. In another study, the hydraulic conductivity of C. corallina internodal cells was shown to be inhibited by high concentrations of high molecular weight, cell permeable solutes (Steudle and Tyerman, 1983; Ye et al., 2004). A proposed model for this regulation was that the osmolytes, present on either side of the membrane, were excluded from the channel pore due to their size. This resulted in tensions (negative pressures) inside the water‐filled pore and a collapse of the pore eventually leading to its closure (Ye et al., 2004). At variance with these ideas, molecular dynamics simulations have shown that the aquaporin structure (in this case human AQP1) was stable up to applied pressures of 200 MPa (Zhu et al., 2002, 2004). This suggested that aquaporins can tolerate extraordinary high hydrostatic and osmotic pressures. The cell hydrostatic pressure is another parameter that may regulate water channel opening. Pressure pulses of varying amplitudes, generated by a pressure probe device, diVerentially altered the hydraulic conductivity of young maize root cortical cells. Whereas medium‐sized pressure pulses (<0.2 MPa) caused a reversible inhibition of the hydraulic conductivity, larger pressure pulses induced changes that were not reversible except in

88

O. POSTAIRE ET AL.

the presence of ABA (Wan et al., 2004). The authors suggested that the high rate of water flow through the aquaporin, which was proportional to the pressure pulse, could cause conformational changes in the channel, and thereby induce its closure.

4. Regulation by protons and calcium The first evidence that plant aquaporins are regulated by pH was initially obtained in purified membrane vesicles or organelles. For instance, Amodeo et al. (2002) observed pH sensitivity of tonoplast water channels in Beta vulgaris storage roots, using swelling assays on isolated vacuoles. In another study, Gerbeau et al. (2002) used stopped flow spectrophotometry on plasma membrane vesicles purified from Arabidopsis suspension cells to show that protons can inhibit water channels with an half inhibition at pH 7.2–7.5. Expression of wild‐type and site‐directed mutant aquaporins in Xenopus oocytes provided further functional insights into this regulation. It was first showed that all Arabidopsis PIP isoforms investigated were blocked by intracellular protons (Tournaire‐Roux et al., 2003). In addition, a pH‐sensing His residue, which is specifically conserved in loop D on the cytosolic side of all PIPs, was identified as central for aquaporin gating. In particular, point mutations at this site resulted in active aquaporins that were insensitive to cytosolic pH (Tournaire‐Roux et al., 2003). A sensitivity of plasma membrane aquaporins to Ca2þ inhibition, with an IC50 of 75 mM, has been described in Arabidopsis plasma membrane (Gerbeau et al., 2002). A recent study on plasma membranes from B. vulgaris storage roots confirmed these observations (Alleva et al., 2006). In this study, however, both a high apparent aYnity component with an IC50 of 5 nM and a lower apparent aYnity component with an IC50 of 200 mM were identified. ˚ ) and in an The high‐resolution structures of SoPIP2;1 in a closed (2.1 A ˚ open (3.9 A) conformation has given critical clues to the plant aquaporin gating properties mentioned above (Tornroth‐Horsefield et al., 2006). These structures confirmed the key role of loop D in pH‐dependent channel gating. Moreover, this work showed that the binding of cadmium (Cd2þ) (assumed to be replaced by Ca2þ in vivo) in the vicinity of two conserved acidic residues at the N‐terminal part of the protein (Asp28, Glu31 in SoPIP2;1) can also gate the channel. Here, the divalent cation acts through a chain of ionic interactions and hydrogen bonds involving the loop B and the loop D, and the two acidic residues carried by the N‐terminal tail. While the physiological significance of PIP regulation by pH is fairly well understood, the significance of inhibition by divalent cation (Ca2þ) remains as yet unknown.

AQUAPORINS IN PLANTS

89

5. Posttranslational modifications: Phosphorylation and methylation In contrast to phosphorylation of mammalian aquaporins, which generally mediates membrane targeting of aquaporins (Nejsum et al., 2002; Procino et al., 2003, 2006), the reversible phosphorylation of plant aquaporins is thought to directly gate the pore itself. The first demonstration that phosphorylation regulates the activity of a plant aquaporin was obtained after expression of the seed specific TIP isoform from Phaseolus vulgaris (homologous to AtTIP3;1) in Xenopus oocytes (Maurel et al., 1995). Site‐directed mutation of serine residues within the protein allowed to show that three phosphorylation sites can regulate the protein water transport activity, with two of these sites being located on the N‐terminal tail of the protein and one on loop B. A similar approach was used with spinach SoPIP2;1 and showed that a similar Ser phosphorylation site in loop B, together with a major Ser phosphorylation site in the C‐terminal tail can regulate this aquaporin in oocytes (Johansson et al., 1998). Molecular dynamics simulations based on the molecular structure of SoPIP2;1 suggested that the opening of the pore induced by phosphorylation of loop B is dominant over the closure induced by cation binding (Tornroth‐Horsefield et al., 2006). This work also proposed an original model in which phosphorylation of the C‐terminal part of the protein can modulate pore opening by interaction of the phosphorylated residue (Ser274 for SoPIP2;1) with the loop D of an adjacent monomer. These models point to intricate mechanisms for AQP gating by multiple factors. Of course, these mechanisms will need to be further assessed through functional analysis of aquaporins carrying well‐defined point mutations. The phosphorylation of all plant aquaporins examined thus far seems to be mediated by calcium‐dependent protein kinases and can be modulated in response to environmental stimuli. For instance, phosphorylation of SoPIP2;1 was decreased in response to a hypertonic treatment on excised spinach leaves (Johansson et al., 1996). The biochemical characterization of two distinct kinase activities acting on the loop B or the C‐terminal tail of SoPIP2;1 should be instrumental for deciphering the signal transduction pathways that control the gating of this aquaporin (Sjo¨vall‐Larsen et al., 2006). In tulip flowers, a decrease in phosphorylation of PIPs was correlated with the cold‐induced closure of petals (Azad et al., 2004b). Finally, its was shown that phosphorylation of the C‐terminal tail of NOD26 in the peribacteroid membrane of symbiotic root nodules was enhanced by both drought and salinity stress (Guenther et al., 2003). While aquaporin phosphorylation has attracted much attention, it should be stressed that aquaporins can be posttranslationally modified and possibly regulated by other mechanisms. Although no such evidence is available in plants, ubiquitination of mammalian AQP1 controls the diVerential stability

90

O. POSTAIRE ET AL.

of the protein between normal and osmotic stress conditions (Leitch et al., 2001). In addition, N‐linked glycosylation of human AQP2 and plant vacuolar McTIP1;2 aquaporin seems to control the specific subcellular localization of these proteins (Hendriks et al., 2004; Vera‐Estrella et al., 2004). In the latter example, the appearance of a glycosylated form of McTIP1;2 in response to an osmotic stress correlated with the redistribution of the protein from tonoplast to other intracellular membrane fractions. Finally, highly resolution techniques, such as mass spectrometry, have assisted in the discovery of novel posttranslational modifications in aquaporins. In particular, methylation of two residues (Lys and Glu) in the N‐terminal tail of AtPIP2 aquaporins was recently described. While these decorations seem to have no eVect on the intrinsic water permeability of AtPIP2;1 (Santoni et al., 2006), it remains to be tested whether they interfere with the subcellular distribution of the protein. 6. Regulation by heterotetramer formation Structural models of mammalian AQP1, bacterial GlpF, and plant SoPIP2;1 have provided exquisite molecular details on how aquaporins spontaneously assemble as homo‐tetramers, each monomer delineating a functionally independent water channel (Fujiyoshi et al., 2002; Tornroth‐Horsefield et al., 2006). However, heteromers comprising members of the PIP1 and PIP2 subgroups may occur in maize or mimosa (Fetter et al., 2004; Temmei et al., 2005). This hypothesis is supported by the observation that, when independently expressed in oocytes, most PIP1 aquaporins, in contrast to PIP2 aquaporins, fail to increase the osmotic water permeability of the oocyte membrane. By contrast, coexpression of a seemingly inactive PIP1 with reduced amounts of a functional PIP2 led to a marked increase in permeability, which was proportional to the amounts of PIP1 expressed (Fetter et al., 2004; Temmei et al., 2005). This suggested that isoforms of the two subgroups interacted, probably as hetero‐tetramers. This idea was corroborated by microscopic observations of oocytes expressing PIP1 and/or PIP2 aquaporins fused to the green fluorescent protein (GFP). Physical interaction of ZmPIP1;2 and ZmPIP2;1 was also demonstrated by copurification experiments. Yet structural evidence that PIP1 and PIP2 assemble as hetero‐tetramers is still lacking. The idea that hetero‐tetramer formation of PIPs controls their expression at the cell surface is also consistent with early observations made in transgenic Arabidopsis plants showing that antisense inhibition of PIP1 or PIP2 aquaporin expression, independently or in combination, resulted in a similar reduction in root hydraulic conductance (Martre et al., 2002). All these evidences suggested that aquaporins of the PIP1 and PIP2 subgroups act in

91

AQUAPORINS IN PLANTS

a cooperative manner to form the same active water channels (Fetter et al., 2004; Martre et al., 2002; Temmei et al., 2005). The proposal that the C‐terminal tail of an individual PIP monomer can interact with the pore of an adjacent monomer may bring some structural clues to this mechanism (Tornroth‐Horsefield et al., 2006).

III. AQUAPORINS IN ROOTS A. PRINCIPLES OF ROOT WATER UPTAKE

Roots are organs specialized in water and nutrient absorption from the soil (Figs. 3–4). The rate of water uptake (Jv) is proportional to both the diVerence in water potential (
) between the soil and the xylem vessels and the hydraulic conductance (L) of the root system (Steudle, 1989): Jv ¼ L  DC L is determined by both the intrinsic root water permeability (root hydraulic conductivity, Lpr) and the exchange surface (S).
reflects the additive

Gas phase

−10 MPa < Ψatmosphere < −100 MPa

Transpiration Leaf water transport

Xylem transport Ψhypocotyl Ψroots

Liquid phase

Water potential (Ψ) gradient

Ψleaf

Water uptake

0 MPa < Ψsoil < −1.5 MPa

Fig. 3. Water flows in the soil–plant–atmosphere continuum. Water transport is driven by a gradient of decreasing water potentials and occurs in both liquid and gas phase. For all processes indicated, the rate of water flow (Jv) is determined by the water potential gradient (
) and the hydraulic conductance of the plant organ considered (K): Jv ¼ K
.

92

O. POSTAIRE ET AL.

Cell wall Cytosol Vacuole

Fig. 4. Water uptake by roots. The graph shows how water passes through concentric cell layers (epidermis, cortex, endodermis, and stele) before being loaded into the xylem vessels where it is pulled up by tensions. According to the composite model, water can flow along both the apoplastic and the cell‐to‐cell pathways. The latter comprises the symplastic and the transcellular paths. The contribution of plasma membrane and tonoplast aquaporins to the transcellular path is indicated.

contribution of hydrostatic (
P) and osmotic (
) driving forces. In the latter case, the diVerence in osmotic potential between the soil and the xylem vessels (
) is modulated by the reflection coeYcient (), which is equal to unity in hemipermeable barriers and smaller when the barrier is permeable to solutes. Therefore, water uptake can be modeled according the following equation: Jv ¼ Lpr SðDP  sDpÞ Although roots form highly branched tortuous organs, they can be schematically reduced to elemental cylindrical segments, in which water first flows radially from the soil into the xylem vessels (Fig. 4). Water is then transported axially along the vessels. Measurements of the hydraulic resistance of roots and comparison to the axial resistance calculated from vessel diameter using Poiseuille’s law have indicated that, in most plant species, axial water transport does not oppose a significant resistance within the root. This point was spectacularly exemplified by Steudle and Peterson (1998), who indicated that a cylindrical vessel with a diameter of 23 mm would require a length of 24 km to exhibit a resistance comparable with that of a single cell membrane.

AQUAPORINS IN PLANTS

93

Radial water transport in roots can be mediated through two parallel pathways (Fig. 4). The composite model of root water transport developed by Steudle and co‐workers (for review, see Steudle, 2000; Steudle and Peterson, 1998) has explored all physiological and biophysical significance of this dual configuration. First, the cell wall continuum defines the apoplastic pathway in which water flow is essentially driven by hydrostatic forces (i.e., pressures or tensions) (Steudle, 1994). Second, the cell‐to‐cell pathway includes water transport through the continuum of cytoplasms linked by plasmodesmata (symplastic path) and through cell membranes (transcellular path). Because cells and membranes create eYcient barriers against solute diVusion, both osmotic and hydrostatic forces are at work in the cell‐to‐cell pathway (Steudle, 1994; Steudle and Peterson, 1998). The respective contribution of the diVerent pathways is biophysically diYcult to establish, and it has been assumed for a long time that the apoplasm oVered the pathway of lesser resistance. The realization that aquaporins, by enhancing the water permeability of cells, can significantly decrease the resistance of the cell‐to‐cell pathway has changed our classical views on root water transport (Maurel and Chrispeels, 2001). Aquaporins have also provided solid molecular bases, with which to explore the amazing properties of roots to adjust their water transport properties (Lpr) in response to environmental stimuli. B. AQUAPORIN EXPRESSION IN ROOTS

Numerous data are available in the literature that describe in great detail the cell specific expression pattern of aquaporins in roots. These data have been reviewed in detail elsewhere (Javot and Maurel, 2002; Maurel et al., 2002) and will not be further commented here. Overall, they show that individual aquaporins can be detected in virtually all root tissues investigated including the root apical meristem and lateral root primordia, the elongation and diVerentiation zones, the epidermis and root hairs, the cortex and the vascular bundles including a variety of cell types such as tracheary elements, xylem parenchyma cells, phloem, phloem‐associated cells and cambium (for review, see Javot and Maurel, 2002). This variety of expression patterns is consistent with putative roles of aquaporins in root‐specific functions, that is, radial water transport, but also more general functions such as cell expansion or sap transport into and out of vascular tissues. Recently, a few proteomic and transcriptomic studies have provided complementary information and helped address expression of the whole aquaporin family (Alexandersson et al., 2005; Birnbaum et al., 2003; Hachez et al., 2006; Santoni et al., 2003). All these approaches have confirmed that roots express a large number of

94

O. POSTAIRE ET AL.

aquaporin isoforms, some of them being highly abundant. In particular, mass spectrometry analysis of plasma membranes purified from Arabidopsis roots has unambiguously established the expression of at least 6 PIP isoforms (PIP1;1 and/or PIP1;2, PIP1;5, PIP2;1, PIP2;2, PIP2;4, and PIP2;7) (Santoni et al., 2003). A first global view of cell‐specific expression of mRNAs (including those encoding aquaporins) within the Arabidopsis roots was provided by Birnbaum et al. (2003). Protoplasts were prepared by digestion of various root tissues and automatically sorted on the basis of expression of cell‐specific reporter genes. Hybridization to AVymetrix chips of cDNAs prepared from various cell fractions was used to generate an expression map comprising 15 root zones. Aquaporin expression was detected in all of these zones, and, for example, PIP1;1 was more expressed in the diVerentiated zone than in the apex or the elongation zone. Moreover, this isoform was strongly expressed in the stele, the endodermis, and the cortex but was barely expressed in the epidermis. Hachez et al. (2006) have developed a thorough analysis of plasma membrane aquaporin expression in maize primary root using quantitative RT‐PCR on microdissected root segments, in situ RT‐PCR and immuno‐localization. This study allowed to identify expression of two predominant isoforms (ZmPIP1;5, ZmPIP2;5) in the diVerentiated zone of the root. Strong expression of aquaporins, including ZmPIP2;5, was observed in the endodermis and exodermis, consistent with a controlling role of these two cell layers in radial water transport. A role of PIP2;5 in primary water uptake was further suggested by polar expression of this aquaporin on the external periclinal side of epidermal cells. C. MEASUREMENTS OF ROOT WATER TRANSPORT

The whole set of techniques developed to measure water transport at the cell and subcellular levels, that is, stopped‐flow spectrophotometry on isolated membrane vesicles, osmotic swelling of isolated protoplasts, and cell pressure probe techniques has been applied to root materials (see Section II.B). The techniques discussed below allow to measure water transport specifically at the whole root level. 1. Pressure chamber Pressure chambers have been instrumental to measure water potentials of plant organs. Pressure chambers can also be used to measure the hydrostatic hydraulic conductivity (Lpr) of excised roots (Boursiac et al., 2005; Markhart et al., 1979). Briefly, an excised root system is inserted in a chamber filled with soil or a bathing solution and the cut stem is threaded through and sealed to the chamber lid. A pneumatic pressure is delivered into the chamber to create

AQUAPORINS IN PLANTS

95

a hydrostatic pressure gradient across the root and to induce a water flow. The rate of sap flow is determined by collecting the expressed sap into a graduated capillary, whereas the surface area (or the mass) of the root is determined independently. Within certain pressure limits, the flow of sap exuding from the stem section is proportional to the hydrostatic pressure gradient present between the root solution and the xylem solution and the Lpr value is easily derived from the pressure‐to‐flow relationship and the root surface area (or mass). The eVects of osmotic gradients of varying amplitude on the balancing pressure required for null‐flow conditions, together with the diVerence in osmotic potential between the bathing solution and exuded sap, can be used to calculate the root reflection coeYcient (Boursiac et al., 2005; Steudle, 1989). 2. High pressure flow meter (HPFM) Here, a flow of sap is pushed, using a pump, within the excised root, through the stem section (Siefritz et al., 2002). Flow rates are measured for varying pressures, and the slope of the flow versus pressure relationship, divided by the root surface area, provides an Lpr value. Therefore, this and the pressure chamber technique rely on very similar principles, except that they work with flows of opposite directions. The two techniques have also raised the same types of criticism. First, the forced flow of water leads to tissue (air space) infiltration and therefore may change the respective contributions of the diVerent pathways. In addition, unstirred layers may lead to a biased measurement of Lpr. In particular, an intense water flow across root tissues may induce an accumulation or sweep away of solutes in the vicinity of membrane barriers and may build up local, artifactual osmotic gradients (Steudle, 1994). 3. Root pressure probe An excised root, maintained at atmospheric pressure, is tightly connected, via silicon seals, to a device that allows to measure and control the pressure within the xylem vessels (Steudle, 1993). In resting conditions, the basal root pressure (Proot) can be measured, and reflects the hydrostatic pressure developed through spontaneous osmotic transport of water across the root. By analogy to pressure probe measurements at the cell level, the whole root is considered as an elastic barrier that can support both hydrostatic and osmotic pressure gradients. Therefore, water flows can be induced by changes in either of the two forces and the relaxation over time of Proot is indicative of the root osmotic or hydrostatic hydraulic conductivities. In case of a permeable solute, the relaxation curve of Proot is biphasic and can be used to calculate the root permeability to the solute together with the root reflection coeYcient (Azaizeh and Steudle, 1991; Steudle, 1993).

96

O. POSTAIRE ET AL. D. EVIDENCE FOR WATER TRANSPORT BY AQUAPORINS IN ROOTS

1. Cell level The contribution of aquaporins to water transport in roots was first probed using mercury inhibition. Cell membrane fractionation and stopped‐flow measurements on isolated tonoplast vesicles revealed extremely high water permeability values (Pf > 500 mm s1) and strong inhibition by mercurials (>80%) in membranes from wheat roots and radish taproots (Niemietz and Tyerman, 1997; Ohshima et al., 2001). Water transport in intact vacuoles isolated from protoplasts of rape or red beet roots confirmed the idea that the tonoplast of most plant cells exhibits a high Pf (>200 mm s1) (Morillon and Lassalles 1999). Niemietz and Tyerman (1997) and Ohshima et al. (2001) have also measured the Pf of plasma membrane (PM) vesicles from wheat roots and radish taproots respectively, and have shown this to be several‐fold lower than the Pf of the corresponding tonoplast vesicles. This diVerence in water permeability between plasma membranes and endomembranes has also been observed in tobacco suspension cells, and its general significance in terms of cell osmoregulation has been discussed elsewhere (Maurel et al., 2002; Tyerman et al., 1999). We note that by contrast to the studies mentioned above, Alleva et al. (2006) found a high Pf (542  40 mm s1) and active water channels in plasma membranes vesicles prepared from red beet storage roots. In most cases, however, it has been assumed that plasma membranes represent the limiting barrier for transcellular water transport. The specific contribution of PIP1 or PIP2 aquaporins to water transport in Arabidopsis root cells has first been inferred from osmotic swelling assays in protoplasts prepared from wild‐type plants or plants expressing a PIP1;2 or a PIP2;3 antisense gene (Martre et al., 2002). Reduced expression of PIP1 or PIP2 aquaporins resulted in significant decreases in root protoplast water permeability. For instance, protoplasts isolated from control and PIP2;3 antisense plants had a mean hydraulic conductivity of 117  17  108 m s1 MPa1 and 4  1  108 m s1 MPa1, respectively. Cell pressure probe measurements in Arabidopsis roots, either wild‐type or carrying a T‐DNA insertion in the PIP2;2 gene, showed that this single aquaporin isoform contributes to about 25% of the cell hydraulic conductivity of cortical cells (Javot et al., 2003). 2. Organ level a. Mercury inhibition. Mercury inhibition has provided the first indication that water channels can represent a major path for root water uptake (Maggio and Joly, 1995). The significance and limitations of this approach have been discussed in detail elsewhere (Javot and Maurel, 2002). Briefly, eVects of HgCl2 on root water transport have been investigated in more

AQUAPORINS IN PLANTS

97

than 10 plant species. Although diVerences exist in the dose applied (106 to 103 M) and the duration of treatment (10 min to several hours), all studies pointed to a significant inhibition of Lpr by mercury, from 32% in Opuntia acanthocarpa to 90% in barley (Javot and Maurel, 2002). Although mercury is now a widely used inhibitor for water transport studies, we insist that this compound can react with exposed cysteine residues of any cellular proteins and therefore can have profound and toxic cellular eVects. For instance, mercury reduced the membrane electrical potential of wheat root cortex cells with a dose dependency similar to that for the inhibition of hydraulic conductivity (Zhang and Tyerman, 1999). Wan and Zwiazek (1999) observed that a long (>1 h) mercury treatment reduced both root cell respiration and stomatal conductance in aspen seedlings. These side eVects reflect an overall metabolic inhibition in mercury treated plants, with a possibly strong impact on their overall water and solute transport properties. To evaluate a possible role on solute pumping, several authors have checked that mercury treatment did not alter Kþ transport in the root sap (Carvajal et al., 1999; Maggio and Joly, 1995; Wan and Zwiazek, 1999). It is also important to assess that the primary eVects of mercury are mediated through an oxidation of cellular components (possibly aquaporins) and can rapidly be reversed by reducing agents such ‐mercaptoethanol or dithiothreitol. This mandatory control is missing in certain studies. b. Acid loading, a new procedure for aquaporin inhibition. Because all plant PIPs display the same Hþ‐gating properties (Tournaire‐Roux et al., 2003), cytosolic acidification can provide a powerful means for blocking aquaporins, specifically at the plasma membrane. This can be experimentally achieved by acid loading, that is, treating plant tissues with a weak acid, which diVuses trough the cell membrane preferentially in its neutral (acidic) form and therefore releases protons intracellularly. For instance, Tournaire‐Roux et al. (2003) observed that exposure of Arabidopsis roots to 20 mM of propionic acid/potassium propionate, pH 6.0, induced a rapid (t1/2 ¼ 3.7  0.3 min) and marked (71%  3%) decrease in Lpr. Remarkably, this inhibition was reversible upon removal of the propionic acid/potassium propionate from the bathing solution. Pressure probe measurements revealed that acid load treatments induced an even stronger inhibition of hydraulic conductivity in cortical cells providing evidence that eVects were truly exerted on membrane water transport (Tournaire‐Roux et al., 2003). The same study also established that respiratory poisons such as azide or cyanide, which had been previously identified as potent inhibitors of water transport in roots (Zhang and Tyerman, 1991) induced a marked drop in cytosolic pH and therefore blocked Lpr (Tournaire‐Roux et al., 2003).

98

O. POSTAIRE ET AL.

Here again, the eVects were reversible, upon washing the root with a control bathing solution, suggesting that the poison molecules rapidly diVused out of the cells and/or were eYciently metabolized. Altogether, the data indicated that water transport in the Arabidopsis root is largely dominated by the transcellular pathway (Tournaire‐Roux et al., 2003) (Fig. 5). In addition, acid load treatments were established as an eYcient procedure for inhibiting aquaporins in living plant tissues. This procedure is reversible and possibly less toxic than mercury treatment. c. Reverse genetics. Because of a lack of specific aquaporin inhibitor, reverse genetics remains a central approach to explore the physiological function of aquaporins. In principle, the function of specific isoforms or subgroups of aquaporins can be addressed.

HgCl2

100 80 60 40 20 0

Acid load

Inhibition of Lpr (%)

100 80 60 40 20 0

Azide

100 80 60 40 20 0

0

10 20 Time (min)

30

Fig. 5. Inhibition of water transport in the Arabidopsis root by aquaporin blockers. Excised roots were inserted into a pressure chamber and the rate of water flow at constant pressure (equivalent to Lpr) was measured over time. At time 0, roots were treated with 50 mM HgCl2, 20 mM propionic acid, pH ¼ 6.0, or 1 mM azide. The inhibition response, very similar between the three treatments, indicates that, in the Arabidopsis root, the cell‐to‐cell pathway is largely predominant.

AQUAPORINS IN PLANTS

99

Antisense inhibition of PIP aquaporin expression in Arabidopsis and tobacco has revealed the importance of this group of aquaporins in root water transport. For instance, expression of a PIP1 (NtAQP1) antisense gene in tobacco reduced Lpr by 58%, to an extent which was comparable to the reduction in Pf observed at the level of isolated root protoplasts (55%) (Siefritz et al., 2002). In Arabidopsis, expression of a PIP1;2 or a PIP2;3 antisense gene, individually or in combination, reduced the root hydraulic conductivity by 60, 47, and 68%, respectively. The finding that the simple and double antisense lines induced similar reductions in water permeability, both at the whole root and at the protoplast levels, was interpreted to mean that PIP1and PIP2 aquaporins cooperate to form the same active water channels in the plasma membrane (Martre et al., 2002). Both KaldenhoV et al. (1998) and Martre et al. (2002) also observed that antisense inhibition of PIP aquaporins in Arabidopsis lead to an increase in root mass by two‐ to fivefold, depending on the lines. Therefore, the reduced water permeability in the plasma membrane of root cells was compensated by an increase in the size of the root system. This morphological adjustment allowed the root conductance, in other words the overall capacity of the root to deliver water to the shoot, to be maintained. Knockout approaches in Arabidopsis have also revealed the contribution of a single aquaporin isoform (PIP2;2) to root water uptake (Javot et al., 2003). Whereas this aquaporin contributed to about 25% of cortical cell hydraulic conductivity, no significant contribution to the overall root hydrostatic hydraulic conductivity (Lpr), as measured with a pressure chamber, could be resolved. By contrast, free exudation by excised roots yielded sap of greater osmolality in mutant than in wild‐type plants (Javot et al., 2003). This reflected a reduced osmotic hydraulic conductivity in roots of the mutant plants. The overall data allowed to propose that PIP2;2 is an aquaporin specialized in osmotic fluid transport. This specific function may possibly be related to the preferential expression of this aquaporin in the inner root tissues. Interestingly, PIP2;2 and its close homologue PIP2;3 have likely evolved through a recent gene duplication and share 97% identity in their amino acid sequence. The detection of a phenotype in PIP2;2 knockout plants indicates that despite their high isoform multiplicity, plant aquaporins do not have fully overlapping functions (Javot et al., 2003). A reverse strategy has been to overexpress a single aquaporin gene in transgenic plants. Katsuhara et al. (2003) showed that expression of a barley PIP aquaporin in rice yielded an increase in Lpr by 140%. A note of caution should be taken when interpreting physiological changes due to ectopic expression of an aquaporin in another plant species, since crucial physiological regulations of the aquaporin of interest may not be maintained. A more

100

O. POSTAIRE ET AL.

consistent strategy was reported by Lian et al. (2004) who overexpressed in rice a PIP1 homologue from the same species, the transgene being placed under the control of a stress responsive promoter. Consistent with this, Lpr was similar between wild‐type and transformed plants in standard conditions, whereas, under water stress conditions, it was higher by 25% in the latter plants. E. EFFECTS OF STIMULI ON ROOT WATER TRANSPORT

The ability of plants to adjust their root water transport properties during development or in response to environmental stimuli has received much attention from plant physiologists. Long‐term adaptations can in part be accounted for by morphological changes, that is, overall changes in root architecture or diVerentiation of specialized cell types. For instance, drought can lead to the development of Casparian bands and suberin lamellae in the exodermis and endodermis and to the production of a suberized periderm outside the stele (see Zimmermann and Steudle, 1998, and references therein). It now appears that regulation of aquaporins also significantly contributes to the regulation of root water permeability and the characterization of short‐term eVects on root water transport of hormonal and environmental stimuli has allowed the eVects of aquaporin regulation to be dissociated from slower morphological changes. 1. Water stress In nature, drought, that is, a drop in soil water potential develops continuously over days. Martre et al. (2001) have performed a thorough analysis of water transport in the root of O. acanthocarpa and observed both deep anatomical changes and a significant decrease in Lpr after 45 days of soil drying. In addition, the Lpr of water stressed plants had become fully insensitive to inhibition by 50 mM HgCl2. North et al. (2004) reported that roots of Agave deserti that had been drought‐stressed for 10 days had a lower hydraulic conductivity than control roots grown in a moist soil. In addition, mercury inhibition of water transport was only found in control roots. These two reports provide some evidence that an inhibition of aquaporins contributed to the adaptation of roots to drought. Although the significance of drought‐induced inhibition of Lpr is not fully clear yet, this response has been interpreted as a strategy to avoid a backflow of water from the plant toward the dry soil. The eVects of drought can be mimicked in plants grown in hydroponic conditions and subjected to a hyperosmotic treatment (Boursiac et al., 2005; Shangguan et al., 2005). For example, treatment of sorghum seedlings by a polyethylene glycol (PEG) solution at 0.3 MPa reduced their Lpr by up to 70% in 30–60 h (Shangguan et al., 2005).

AQUAPORINS IN PLANTS

101

ABA is synthesized in response to drought and plays a central role in regulating the plant water status. ABA not only induces stomatal closure but also controls root water transport. For instance, treatment of maize roots with 1 mM ABA transiently increased the hydraulic conductivity of cortical cells by 27‐fold after 1 h and had no eVect after 2 h (Hose et al., 2000). An increase in Lpr by a factor of 3–4 was observed after 70–100 min of an ABA treatment. This strategy may allow the plant to take up residual soil water, before severe drought develops. The regulation of PIP aquaporin expression in response to water stress has been investigated in several plant species (for review, see Tyerman and Niemietz, 2002). In rice, for instance, water deficit, as induced by treatment with 20% PEG6000 for 10 h, resulted in an accumulation of PIP proteins in roots (Lian et al., 2004). A thorough RT‐PCR analysis of expression of all 13 PIP isoforms in the Arabidopsis root revealed that a hyperosmotic treatment (250 mM of mannitol, 12 h) induced an upregulation of some isoforms (PIP1;3, PIP1;4, PIP2;1, and PIP2;5), whereas transcripts of other isoforms (PIP1;5, PIP2;2, PIP2;3, and PIP2;4) were markedly downregulated (Jang et al., 2004). However, the relation between changes in aquaporin expression and water transport regulation has not been established in these studies. Plants with genetically altered expression of aquaporins have provided stronger direct evidences for a role of aquaporins during water stress. For instance, antisense inhibition of PIPs in tobacco and Arabidopsis resulted in a marked defect of the plants to adapt to water deprivation (Martre et al., 2002; Siefritz et al., 2002). Upon rewetting after drought, the signs of leaf wilting took longer to dissipate in PIP1 antisense than in control tobacco plants (Siefritz et al., 2002). This suggested that the capacity of the whole plant to take up and transport water was limiting in the antisense lines. Using a diVerent approach, Lian et al. (2004) have overexpressed in a lowland, drought sensitive rice cultivar, a PIP aquaporin (RCW3 or OsPIP1;3) that was specifically induced by drought in an upland, drought tolerant cultivar. Interestingly, this manipulation enhanced the ability of the first cultivar to avoid drought. While these observations suggest that an enhanced mobility of water within the plant body and possibly enhanced capacity to take up soil water are beneficial to plants under water stress, more work is needed to draw a clear picture of aquaporin function during water stress. 2. Salt stress Soil salinity exerts noxious eVects on plant yield in part by challenging the plant water status. One of the primary responses of plants to salt is inhibition of their root water uptake capacity. In many plant species, including paprika

102

O. POSTAIRE ET AL.

(Carvajal et al., 1999), melon (Carvajal et al., 2000), Arabidopsis (Boursiac et al., 2005), and tomato (Peyrano et al., 1997), salinization with 50–100 mM of NaCl reduced Lpr by 60% after 2–4 days. This response can be observed in a large variety of glycophytic or halophytic plant species. However, roots of certain species, such as tobacco, do not respond to a salt treatment (Tyerman et al., 1989). The inhibition of root water transport by salt can be counteracted by external calcium, but the molecular basis of these ameliorative eVects is not understood yet (Azaizeh and Steudle, 1991; Carvajal et al., 2000; Martinez‐Ballesta et al., 2000, 2006). The idea that the primary inhibition of Lpr by salt could be accounted for by aquaporin inhibition was raised from early experiments showing that the residual Lpr observed after salt treatment of paprika, melon, or Arabidopsis roots was insensitive to mercury (Carvajal et al., 1999, 2000; Martinez‐Ballesta et al., 2003). Microarrays experiments by Maathuis et al. (2003) also revealed that exposure for 6–24 h of Arabidopsis roots to salinity stress induced an overall reduction in aquaporin expression, whereas longer‐term treatments lead to enhanced aquaporin expression. By combining water transport assays and Northern blot analyses in Arabidopsis roots treated for 24 h with various salt concentrations, Martinez‐Ballesta et al. (2003) further showed a correlation between the extent of Lpr inhibition and the downregulation of PIP1 mRNAs. To investigate further the molecular and cellular mechanisms of aquaporin downregulation under salt stress, Boursiac et al. (2005) analyzed the short‐ term responses of Arabidopsis roots to salt. Kinetic analyses revealed that the decrease in Lpr induced by 100 mM of NaCl occurred rapidly with a half‐ time of about 45 min. All most abundant PIP and TIP transcripts were stably expressed until 2 h of treatment and showed a coordinated and marked decrease in abundance on the long term (6–24 h). ELISA and Western blot analyses using antibodies that specifically recognized aquaporins of the PIP1, PIP2, and TIP1 subclasses revealed a decrease in PIP1 abundance by 30–40% as soon as 30 min after salt exposure, whereas aquaporins of the other subclasses showed a reduced abundance only after 8 h. In addition, transgenic lines expressing various aquaporins fused to GFP revealed that TIP1;1 was partially relocalized into circular structures associated to the main vacuole and firstly described by Saito et al. (2002) as vacuolar bulbs. The labeling of intracellular structures by PIP–GFP fusions after 2–4 h of salt exposure also suggested that internalization of PIPs contributed to the reduced water permeability of the plasma membrane. Therefore, altered expression at diVerent levels of aquaporins in Arabidopsis roots can provide a basis to explain the inhibitory eVects of salt on Lpr, mostly in the long term. Other mechanisms such as altered aquaporin phosphorylation may contribute to the early (<2 h) eVects of salt.

AQUAPORINS IN PLANTS

103

Findings about the regulation of NOD26 in the peribacteroid membrane of nitrogen fixing symbiotic of soybean roots support these ideas. Guenther et al. (2003) showed that phosphorylation of the aquaporin C‐terminal tail, as probed with anti‐phosphopeptide antibodies was stimulated in response to drought and salt stress. This suggested a role for NOD26 in the osmoregulation of root nodule cells during water stress. The finding that pretreatment of melon protoplasts by okadaic acid, a potent inhibitor of phosphoprotein phosphatases 1 and 2A, counteracted the inhibition of protoplast Pf by salt stress provides another line of evidence for a role of protein phosphorylation during the regulation of water transport (Martinez‐Ballesta et al., 2000). 3. Anoxia Soil flooding or compaction reduces oxygen diVusion and can result in a severe hypoxic stress for plant roots. This physiological context can be mimicked by bubbling N2 instead of air in a root bathing solution or by treating roots by respiratory poisons such as azide or cyanide. In most plant species, these treatments lead to a marked inhibition of Lpr. For instance, oxygen depletion in sunflower roots reduced Lpr by 50% in <2 h, whereas treatment of red‐osier dogwood roots with 0.5 mM azide led to a decrease by 35% in both O2 uptake and Lpr after 2 h (Everard and Drew, 1989; Kamaluddin and Zwiazek, 2001). In Arabidopsis roots, hypoxic stress or treatment with 1 mM azide for 30 min reduced the Lpr by 49 and 87%, respectively (Tournaire‐Roux et al., 2003). Parallel in vivo 31P nuclear magnetic resonance measurements revealed that these treatments also resulted in a rapid drop in cytosolic pH by about 0.5 units. The mechanism of this cell acidosis is primarily due to a depletion in cellular ATP accompanied by an increase in cytosolic inorganic phosphate, which prevents Hþ‐ATPases to extrude cytosolic protons (Gout et al., 2001). As discussed above (see Section III.D.2b), the work by Tournaire‐Roux et al. (2003) has established how the inhibition of Lpr under anoxic stress can directly be accounted for the pH‐dependent closure of PIP aquaporins. 4. Light More than 30 years ago, Parsons and Kramer (1974) observed that cotton roots exhibited a diurnal cycling of their hydraulic conductivity that was minimal at night and was increased two‐ to threefold at midday. This kind of observation has now been extended to many other species including wheat (Carvajal et al., 1996), paprika pepper (Carvajal et al., 1999), tomato (Dell’Amico et al., 2001), L. japonicus (Henzler et al., 1999), and maize (Lopez et al., 2003). This phenomenon may provide a means for reducing tension in the root xylem and therefore avoiding cavitation during transpiration. A link between diurnal Lpr

104

O. POSTAIRE ET AL.

fluctuations and aquaporin functions has been established by several authors. For instance, Henzler et al. (1999) found that the diurnal rhythm of the hydraulic and osmotic conductivities of Lotus roots over a 24‐h period was tightly correlated to the abundance of PIP1 transcripts, as measured by Northern blots (Henzler et al., 1999). Similar evidence has been obtained in maize roots where the abundance of the four most highly expressed ZmPIP transcripts started to raise just before dawn and was maximal after 4 h of light (Lopez et al., 2003). Therefore, the expression of ZmPIP transcripts slightly preceded the increase in osmotic root water transport observed during the day. In addition, the abundance of PIP2 proteins was well correlated to the diurnal variations in root water transport (Lopez et al., 2003). 5. Nutrient status The plant nutrient status is another factor that governs the root hydraulic properties (Clarkson et al., 2000). For example, roots excised from maize or barley plants grown under nitrate or sulphate deprivation for 7 days and 4 days, respectively, exhibited a reduction by 50–80% in exudation rate or Lpr, respectively, with respect to roots from plants grown in nitrate or sulfate replete conditions (Hoarau et al., 1996; Karmoker et al., 1991). In wheat, a significant part of root water uptake is mediated through aquaporins, as suggested by the marked (66%) inhibition of Lpr by mercury (Carvajal et al., 1996). Under 5 or 7 days of a complete deprivation in nitrogen (N) or phosphorus (P), respectively, the Lpr was reduced by 80–85%. Interestingly, the residual root water transport activity had become fully insensitive to mercury and could be reversed to initial control activity in about one day, suggesting that the eVects of nutrient starvation were mediated through inhibition of aquaporin expression or activity (Carvajal et al., 1996). This idea is further substantiated by a study in cotton showing that the hydraulic conductivity of root cortical cells, as measured with a cell pressure probe, was decreased by 60 and 80% after 4 days of deprivation in P or N, respectively (Radin and Matthews, 1989). In a recent study, Shangguan et al. (2005) have investigated the interactions between water and P deprivation stresses in sorghum. It was found that the latter stress accentuated the inhibitory eVects of drought on Lpr and slowed the reversal of Lpr toward initial value that was normally observed in the 12 h following plant rewatering. These experiments support the idea that P‐starved plants were more susceptible to drought than P‐replete plants. 6. Low temperatures Studies in cucumber (Lee et al., 2004), tomato (Bloom et al., 2004), spinach (Fennell and Markhart, 1998), and maize (Aroca et al., 2001, 2005; Melkonian et al., 2004) have shown that plants respond to chilling by

AQUAPORINS IN PLANTS

105

decreasing their root hydraulic conductivity. In a chilling sensitive maize cultivar, for instance, the Lpr of 11‐day‐old plants was decreased by up to 80% after 6 h at 5 8C. The osmotic hydraulic conductivity was also decreased and reached a null value after 54 h of chilling treatment (Aroca et al., 2001). In all plant species investigated, chilling‐induced inhibition of root water transport resulted in plant water deficit, as shown by a rapid decrease of leaf relative water content (Aroca et al., 2001; Yu et al., 2006) or leaf water potential (Melkonian et al., 2004). The finding that chilling enhances H2O2 production in cucumber roots and that treatment of these roots by 2 mM of H2O2 (Lee et al., 2004) leads to a twofold inhibition of hydraulic conductivity of cortical cells has led to the idea that the primary eVects of chilling on Lpr may be mediated through an inhibition of root aquaporins by H2O2. However, the mechanisms that underlie this inhibition are not fully understood yet (see Section II.E.2). In maize, the ability of certain cultivars to resist to chilling is linked to their ability to restore a normal root osmotic conductivity under long‐term (3 days) exposure to cold. Aroca et al. (2005) observed a parallel increase in PIP protein abundance and phosphorylation. However, a similar response was also observed in a sensitive cultivar that showed sustained osmotic conductivity inhibition. This suggested that the aquaporin upregulation response was not suYcient in the latter plants and was probably dominated by other mechanisms. In particular, a more important membrane damage was observed in the sensitive cultivar. In rice, the recovery of root osmotic conductivity after a transient chilling treatment (7 8C, 24 h) was total for a resistant cultivar and partial for a sensitive cultivar (Yu et al., 2006). A stronger increase in specific OsPIP transcripts was observed during recovery in the resistant cultivar as compared to the sensitive one. However, no diVerence in protein abundance, as measured by Western blot, was detected between the two cultivars (Yu et al., 2006). F. TRANSPORT OF NUTRIENTS

Whereas the analysis of water transport in plants altered in aquaporin expression has revealed phenotypes of reduced amplitude (Javot et al., 2003; Martre et al., 2002; Siefritz et al., 2002), recent genetic analyses have provided compelling evidences about the role of NIP aquaporins in nutrient transport in plants. First, molecular characterization of lsi1, a rice mutant defective in silicon uptake, revealed that the corresponding locus encoded a plasma membrane localized NIP homologue expressed in root exodermis and endodermis (Ma et al., 2006). The role of Lsi1 was confirmed in rice plants that expressed

106

O. POSTAIRE ET AL.

a corresponding RNAi construct and showed a capacity to take up silicon reduced by 8‐ to 10‐fold. In addition, mRNA expression of Lsi1 in wild‐type plants was downregulated by continuous silicon supply. Therefore, Lsi1 represents the first silicon transporter identified in higher plants. It has close homologues in maize, and its characterization provides a unique opportunity to manipulate silicon transport and possibly stress resistance in crops. As discussed above (see Section II.C), functional expression in Xenopus oocytes has revealed a boron channel activity for AtNIP5;1 (Takano et al., 2006). A first hint at this function was obtained after observing that the abundance in AtNIP5;1 transcripts was reversibly increased by 12‐fold in response to boron deficiency. Promoter‐‐glucuronidase fusions showed NIP5;1 to be preferentially expressed under boron limitation in the root elongation zone and the root hair zone. The characterization of two independent Arabidopsis knockout lines allowed to clearly establish the physiological significance of boron transport by NIP5;1 (Takano et al., 2006). First, a reduction in root and shoot growth was observed at limiting boron concentration (<3 mM), exclusively in the mutant plants. These phenotypes can be associated with a failure of mutant plants to take up boron. Under low boron supply, mutant plants accumulated less boron than wild‐type plants, and the rate of boron uptake, as estimated using a [10B] isotope, was reduced by at least fivefold. Another class of boron transporters, whose founding member is BOR1, has been identified in plants. Whereas NIP5;1 can account for most of the primary uptake of boron from the soil, BOR1 seem to be rather involved in xylem loading and root‐to‐shoot translocation of boron (see Takano et al., 2006, and references therein).

IV. AQUAPORINS IN LEAVES Water transport in leaves occurs in both liquid and gas phases (Fig. 3). Water is lost by transpiration, that is, mainly by diVusion of water vapor through the stomata. This process results in a drop in leaf water potential that drives a flow of liquid water through the petiole and the inner leaf tissues to the cell surfaces that delimit the stomatal cavity (Fig. 6). This process requires water transport through the veins (vascular compartment) and eventually through the vascular bundles and the mesophyll, which together define the extravascular compartment (Fig. 6). Although the rate of water transport through transpiring plants is primarily controlled by their stomatal conductance (Gs), the hydraulic resistance of inner leaf tissues may be critical in determining the water potential gradients

107

AQUAPORINS IN PLANTS

Upper epidermis Vascular bundle Bundle sheath

Mesophyll

Stomatal cavity

Stomata Lower epidermis

Fig. 6. Water transport within an angiosperm leaf. The contributions of the vascular and the extravascular compartments are indicated. In the latter compartment, water flows through both the apoplastic and cell‐to‐cell pathways.

through the leaf. Also, stomatal regulation implies significant changes in guard cell volume. Therefore, a limiting role of transmembrane water transport can be expected in various cell types and physiological contexts in leaves. A. AQUAPORIN EXPRESSION IN LEAVES

The expression pattern of aquaporins in leaves can provide a first glance at their possible role in the leaf water relations. In a recent study, Alexandersson et al. (2005) have developed microarrays carrying aquaporin gene specific tags and were the first to monitor the expression profile of all 35 Arabidopsis aquaporins in rosette leaves, and compare it to that in other organs such as roots and flowers. Whereas some aquaporin isoforms were found to be specifically expressed in roots or flowers, none of the aquaporin transcripts detected in leaves was specific for this organ. In addition, the overall expression level of aquaporins was lower in leaves than in roots. For instance, with the exception of SIP1;1, expression in leaves of members of the NIP and SIP classes was barely detectable with this approach. By contrast, two TIP isoforms (TIP1;2 and TIP2;1) and three PIP isoforms (PIP1;2, PIP2;1, and PIP2;6) showed strong expression, whereas five additional PIP isoforms (PIP1;1, PIP1;4, PIP1;5, PIP2;2, and PIP2;7) had detectable but less abundant transcripts.

108

O. POSTAIRE ET AL.

A large number of other studies have provided a more accurate description of cell‐specific expression patterns of aquaporin isoforms. For instance, Fraysse et al. (2005) have analyzed the expression pattern in spinach leaves of two PIP1s, SoPIP1;1 and SoPIP1;2. Immunolocalization experiments revealed that the two aquaporins were specifically expressed in guard cells and phloem sieve elements, respectively. Expression of PIP and TIP aquaporins has also been reported in guard cells of Arabidopis, Vicia faba, and sunflower (KaldenhoV et al., 1995; Sarda et al., 1997; Sun et al., 2001). In the latter species, expression of a TIP transcript was under diurnal regulation and maximal during stomatal closure (Sarda et al., 1997). In leaves of Brassica napus, strong expression of aquaporins recognized by anti‐PIP1 and anti‐TIP1 antibodies was detected in bundle‐sheath cells (Frangne et al., 2001). Expression of two PIP isoforms has also been reported in the xylem parenchyma of walnut twigs (Sakr et al., 2003). In summary, expression of aquaporins seems to be lower in leaves than in roots. Yet expression has been detected in most leaf cell types of all herbaceous or woody species investigated. In particular, the various expression patterns suggest multiple functions of aquaporins in mediating water transport through the extravascular compartment during transpiration, in loading and unloading xylem and phloem sap, and during stomatal movement. Functional data in favor of some of these hypotheses have recently emerged and are discussed (see Sections IV.D and E). B. MEASUREMENTS OF WATER TRANSPORT IN LEAVES

Various techniques have been developed to measure leaf water transport at the cell and organ levels. The accuracy of these techniques is and will be crucial for exploring aquaporin function in leaves. 1. Cell level Most of generic techniques discussed above (see Sections II.B and III.C) and used for characterizing water transport in plant cells or isolated membranes have been applied to leaves. Pressure probe measurements, in particular, have been used to characterize the water transport properties of specific leaf cell types. For instance, Westgate and Steudle (1985) found high cell hydraulic conductivity values (0.3–2.5 106 m s1 MPa1) in the parenchymatous midrib tissues of Zea mays leaves. In Tradescantia virginiana leaves, epidermal cells had a hydraulic conductivity in the range of 107 m s1 MPa1, which was 10‐fold higher than in the corresponding mesophyll cells (Tomos et al., 1981; Zimmermann et al., 1980). Crassulacean acid metabolism (CAM) was developed by certain plant species to improve their water use eYciency.

AQUAPORINS IN PLANTS

109

Water transport in two CAM species, Kalanchoe daigremontiana and Graptopetalum paraguayens, has been characterized by pressure probe and stopped‐flow techniques, respectively (Ohshima et al., 2001; Steudle et al., 1980). Because these two studies revealed strikingly high and low water permeabilities, respectively, it has been diYcult to conclude whether CAM metabolism is associated with particular water transport properties. More recently, cell water transport has been characterized by means of swelling assays in protoplasts isolated from Arabidopsis, petunia, and tobacco leaves (Ding et al., 2004; Martre et al., 2002; Morillon and Chrispeels 2001; Ramahaleo et al., 1999). This approach, however, has been restricted to mesophyll protoplasts. 2. Organ level Numerous techniques have been developed to measure transpiration and stomatal conductance (Gs) in intact leaves or plants. The simplest method consists of measuring the overall loss of weight from a potted plant. More sophisticated techniques consist of measuring water vapor release by porometry or infra‐red gas exchange analysis. These techniques that primarily reflect the limiting role of stomata for transpiration will not be further addressed in this chapter. As explained above, transpiration also drives a flow of liquid water from the petiole to the stomatal chamber. As exemplified for water transport in roots (see Section III.A), this transport depends on the diVerence in water potential between the petiole and the stomatal cavity, on the one hand, and on the inner leaf hydraulic conductance (Kleaf), on the other hand. Therefore, Kleaf integrates the water conductance in series of the petiole, leaf veins, and extravascular tissues. Three major techniques have been developed to measure Kleaf in intact plants or excised leaves, the latter system providing the most accurate measurements. First, in the so‐called ‘‘vacuum pump method,’’ water is pulled through an excised leaf using a vacuum pump, while the flow rate is measured through the rate of water uptake into the petiole (Sack et al., 2002). The Kleaf is derived from flow measurements at varying vacuum pressures. It is noteworthy that, in these experiments, the leaf is carefully maintained at saturating water vapor. Therefore, the drop in atmospheric pressure can fully account for the drop in water potential across the leaf. Also, the possible resistance of stomata to water diVusion in gas phase, which normally dominates the process of transpiration, is close to zero because of the absence of a vapor pressure deficit. Thus, the measured Kleaf mostly reflects a liquid phase conductance of inner leaf tissues (Sack et al., 2002). A second method called the evaporative flux method relies on the relationship that exists between the flux of transpiration across the plant or an

110

O. POSTAIRE ET AL.

excised leaf and the corresponding drop in water potential (Martre et al., 2002; Sack et al., 2002). Briefly, the transpiration flow (E) throughout the whole plant is measured under steady‐state conditions, using gravimetric methods for instance. A leaf is then excised to measure the leaf water potential ( leaf). In parallel to this, a leaf of the plant that had been previously covered with a bag to locally prevent any transpiration is used to measure the stem water potential ( stem). The Kleaf can then be calculated as the ratio of E to ( stem  leaf). Finally, the high pressure flow meter (HPFM) method requires a flow of solution to be pushed using a pump, from the petiole throughout the leaf (Sack et al., 2002; Tyree et al., 2005). During the measurements, the leaf is submerged in a liquid solution and leaf airspaces rapidly become infiltrated. As for other methods, the leaf conductance can be deduced from the flow versus pressure relationship. Note that in these conditions, the technique assays the resistance of the inner leaf tissues and the stomata in series and therefore can theoretically underestimate the Kleaf. The assumption that stomata do not oVer any limiting resistance to water flows in a liquid phase is based on simple calculations using Poiseuille’s law. This has however been disputed, particularly in contexts where irradiance induces apparent changes in Kleaf (Sack et al., 2002; Tyree et al., 2005). For instance, Sack et al. (2002) have argued that the reduced aperture of stomata in Quercus rubra leaves at low irradiance could account for their reduced Kleaf. By characterizing the eVects of irradiance in various plant species, Tyree et al. (2005) found however that the extent of light‐induced increase in Kleaf was not always correlated to the accompanying increase in stomatal aperture. In addition, treatment of Juglans regia leaves with ABA induced a marked closure of stomata without any alteration in Kleaf (Tyree et al., 2005). Finally, the three methods discussed above have been validated through a comparative study showing that similar Kleaf values could be determined by these methods in each of six woody angiosperm species examined (Sack et al., 2002). C. WATER TRANSPORT PATHWAYS IN LEAVES

Sack et al. (2003) have shown that among 34 plant species, leaf hydraulic resistance (Rleaf ¼ 1/Kleaf) can account for at least one quarter of the whole plant water resistance and, therefore, can significantly contribute to the drop in water potential across the plant. In some species, leaves can contribute up to 80% of the whole plant resistance (Nardini et al., 2005). A recent survey of all data on leaf hydraulic properties existing in the literature (Sack and Holbrook, 2006) also showed a large variability in Kleaf values, from

AQUAPORINS IN PLANTS

111

0.76 mmol m2 s1 MPa1 in Adiantum lunulatum to 49 mmol m2 s1 MPa1 in Macaranga triloba. In general, Kleaf tends to be the lowest for conifers and pteridophytes, intermediate for woody angiosperms, and the highest for crop plants. The total leaf hydraulic resistance sums up the contribution of two compartments that act in series, in dicots especially: the vascular compartment that comprises all the veins and the extravascular compartment that consists of the vascular bundles together with the mesophyll (Sack and Holbrook, 2006) (Fig. 6). Although vein networks are very variable among tracheophyte species in terms of vein density, size, and arrangement, it is assumed that the bulk of transpired water always follows the path of lesser resistance down the vein network, through the midrib, the major vein, and finally the minor veins, and then exits the vessels (Sack and Holbrook, 2006). By analogy to what was previously discussed in roots, water can flow throughout the extravascular compartment via parallel apoplastic and cell‐to‐cell pathways (Figs. 3 and 6). Two diVerent approaches have been developed to quantify the partitioning of hydraulic resistance within a lamina, that is, the respective resistances of the vascular and extravascular compartments. One method is to determine how Rleaf is modified when the extravascular water pathway is progressively bypassed by cutting an increasing number of minor veins. DiVerent studies have applied this technique to woody (Acer saccharum, Q. rubra) and herbaceous (sunflower) leaves and observed that following up to several hundred cuts, the measured Rleaf progressively decreased and converged toward a stable value, the supposed vascular resistance (Nardini et al., 2005; Sack et al., 2004). Note that the procedure by which minor veins are cut is still a matter of debate. Alternatively, the disruption of all living structures of the leaf, after freezing or boiling the entire organ, reduces Rleaf to its vascular component (Cochard et al., 2004). However, this conclusion is only valid, provided that the treatments do not alter the xylem vessel diameter or the extensibility of the walls. A comparison of numerous plant species, all being grown in standard conditions, that is, with a nonlimiting supply of light and water, showed that the partitioning of hydraulic resistance in leaves is extremely variable between species. For instance, the hydraulic resistance of the extravascular compartment contributed to 26, 36, and 58% of Rleaf in red oak, sugar maple (Sack et al., 2004), or sunflower (Nardini et al., 2005), respectively. These data indicate that leaves can exhibit a large diversity of hydraulic architectures and, due to their branched network of minor veins, often oppose a significant vascular hydraulic resistance to water. However, leaves can also, similarly to roots, show a significant hydraulic resistance in extravascular tissues, this being possibly under the control of aquaporins.

112

O. POSTAIRE ET AL. D. FUNCTIONS OF AQUAPORINS IN LEAF WATER TRANSPORT

1. Functional evidence for aquaporins in leaf water transport a. EVects of mercury. EVects of mercury were first investigated in isolated protoplasts and, for instance, KaldenhoV et al. (1998) reported that exposure of Arabidopsis leaf protoplasts to 1 mM HgCl2 resulted in a fivefold reduction in their Pf. However, the low basal water permeability of protoplasts with respect to that of walled cells and the fact that leaf protoplasts quite exclusively originate from the mesophyll restrict the physiological significance of this and other similar studies (Ding et al., 2004). More recently, a few reports have addressed the contribution of aquaporins to water transport in whole leaves. Nardini et al. (2005) investigated the dose‐ dependent eVects of HgCl2 on the Kleaf of sunflower plants collected during the light period. They found that treatment of leaves for 1 h with 200 mM of HgCl2 induced a significant reduction of Kleaf by 33% (from 4  0.4  104 kg s1 m2 MPa1 to 2.5  0.3  104 kg s1 m2 MPa1) and increased the leaf extravascular resistance by about 100%. In view of the very high HgCl2 concentration used and the long time of exposure, it was critical to examine the toxicity of the mercury treatment. The authors found that mercury‐ induced reduction of Kleaf could somehow be reversed by subsequent treatment with 30 mM of mercaptoethanol for 1 h (Nardini et al., 2005). Altogether, the data point to a significant contribution of the cell‐to‐cell (aquaporin) pathway for extravascular transport in illuminated sunflower leaves. In another study, Aasamaa and Sober (2005) analyzed the eVect of a mercury treatment (1 mM of HgCl2) on leaf water transport in six temperate deciduous trees. More specifically, they measured the hydraulic conductance of the shoot, petiole, stem, and lamina during a seasonal course of growth. HgCl2 treatment was found to decrease the hydraulic conductance of leaf lamina (Klamina) in all species, and throughout the whole season. For example, Klamina in the growing period was decreased by about 40% for Populus tremula and 45% for Quercus robur. A conclusion similar to that drawn by Nardini et al. (2005) in sunflower was made for the six trees species investigated. However, extremely high concentrations of mercury were used, and their cellular toxicity was not investigated. b. EVects of temperature. Sack et al. (2004) took another approach to probe for the contribution of the cell‐to‐cell pathway and analyzed the eVects of temperature on Kleaf. Measurements were made on intact leaves, or on leaves whose minor veins, tertiary veins, or petiole had been cut to bypass the extravascular compartment. The eVects of temperature on water flow through intact leaves were significantly greater than those expected from

AQUAPORINS IN PLANTS

113

the eVects of temperature on the viscosity of water. By contrast, there was no such discrepancy in leaves with dissected veins suggesting that the temperature‐sensitive component was localized in the extravascular compartment. However, further interpretation was diYcult, since the eVects of temperature could not be analyzed through a single Arrhenius relationship. c. Genetic evidence. Contribution of aquaporins to liquid water transport in leaves has also been inferred from measurements made in transgenic Arabidopsis plants expressing PIP antisense genes (Martre et al., 2002). For instance, antisense genes for AtPIP1;2 or AtPIP2;3 or both reduced the Pf of mesophyll protoplasts by 5‐ to 20‐fold. However, the hydraulic conductivity of the rosette under transpiring conditions, as measured by the evaporative flux method, was similar in wild‐type and transgenic plants expressing either one or both antisens genes. These results suggest that mesophyll cells do not oppose any significant hydraulic resistance and that in the mesophyll, the apoplastic pathway is dominant. E. PHYSIOLOGICAL REGULATIONS OF KLEAF

The hydraulic resistance of veins can be altered through diVerent mechanisms. For instance, drought or chilling conditions induce cavitations and/or wall collapse in vessels, which create important barriers to leaf water transport. The xylem sap composition, and in particular its potassium concentration, determines hydrogel eVects, which interferes with the wall permeability of tracheids (Zwieniecki et al., 2001). Although of great importance for leaf water transport, these processes do not directly involve aquaporins. More recently, evidence has emerged that the extravascular path is also subject to important physiological regulations. The observations described below oVer interesting contexts in which to explore the contributing role of aquaporins to leaf hydraulic regulation. 1. Transpiration demand Morillon and Chrispeels (2001) investigated the Pf of Arabidopsis mesophyll protoplasts from plants grown under various transpiring regimes. In transpiring conditions, most of Pf values were low (<70 mm s1). By contrast, treatment of plants with 1 mM ABA during 24 h led to a significant increase in mean leaf protoplast Pf from 69 to 126 mm s1. This apparent increase in aquaporin activity was however not associated to any increase in PIP1 and PIP2 aquaporin expression, as evaluated by Western blotting. Direct application of ABA to protoplasts did not increase their Pf suggesting that the drop in transpiration flow due to ABA‐induced stomatal closure was

114

O. POSTAIRE ET AL.

the determining factor for increasing Pf. This idea was somehow confirmed in ABA‐insensitive mutants where a fourfold reduced transpiration due to an increase in air humidity from 45 to 85% significantly increased leaf protoplast Pf (Morillon and Chrispeels, 2001). The overall data suggested that the water permeability of Arabidopsis mesophyll cells would be tightly linked to the leaf transpiration regime. The significance of a high cell water permeability at low transpiration is not clear and may favor water potential equilibration within the leaf. By contrast, under transpiring conditions, mesophyll cells would have a low water permeability and the apoplastic pathway would predominate. 2. Light Changes in Kleaf due to changes in irradiance have now been reported in several plant species (Nardini et al., 2005; Sack et al., 2002; Tyree et al., 2005). In sunflower, for instance, Kleaf, as measured with the HPFM method (Nardini et al., 2005), was reduced by 30–40% during the night. Tyree et al. (2005) have analyzed the eVects of a transition between a very low (<10 mmol m2 s1) and a very high irradiance (>1000 mmol m2 s1) onto the Kleaf of 11 tropical plant species. Whereas 5 of them were insensitive to this treatment, 6 species showed an increase in Kleaf from 21% (Cordia alliodora) to 196% (Miconia argentea) in the 30 min following the transition to high light. Light‐induced increases in Kleaf have been interpreted as a mechanism to avoid cavitations in the xylem. Under high light, that is, under transpiring conditions, the leaf water potential can drop significantly and the xylem sap can be under great tensions. An increase in Kleaf would favor water supply, reduce the drop in water potential throughout the leaf and would maintain it above a critical vulnerability threshold where cavitation can be avoided (Tsuda and Tyree, 2000). The mechanisms that underlie the light‐dependent regulation of Kleaf have been addressed in a few recent reports. Nardini et al. (2005) observed that when sunflower plants were kept in the dark for several days, the Kleaf continued to oscillate in phase with their subjective period, meaning that these changes were driven by the circadian clock. The authors also found that the hydraulic conductivity of the veins remained unchanged, whereas the conductivity of the extravascular compartment increased by 62.5% during the transition from dark to light. The hypothesis that the increase in Kleaf would be mediated through aquaporin activation has found strong support in a study by Cochard et al. (2007) in walnut trees. The authors analyzed, using quantitative RT‐PCR, the abundance of two major PIP2 aquaporin transcripts during a transition from dark to high light and found a very good kinetic correlation between the increase in Kleaf and the increase in PIP2 aquaporin expression.

AQUAPORINS IN PLANTS

115

3. Water stress Nardini and Salleo (2005) have analyzed the eVects of long‐term water stresses on the leaf hydraulic architecture of developing sunflower plants. An increase in the hydraulic resistance of the veins was observed under mild ( soil ¼ 0.58 MPa) and strong ( soil ¼ 0.94 MPa) water stresses and could be explained by a reduced diameter of xylem vessels formed under drought. However, the overall Rleaf was unchanged or decreased under mild and strong water stress, respectively, suggesting that, superimposed on eVects on veins, water stress induced a progressive decrease in the hydraulic resistance of the extravascular compartment. The authors hypothesized that the latter eVect was due to activation of leaf aquaporins. Whereas they did not exhibit any change in water status in normal growth conditions, Arabidopsis plants expressing both a AtPIP1;2 or a AtPIP2;3 antisense transgene revealed interesting phenotypes during water stress (Martre et al., 2002). At the end of an 8‐day soil drying period, the leaf water potential of the antisense plants was significantly lower than that of control plants, indicating that the former plants were more aVected by water stress. The diVerence between the two plant genotypes was even stronger during rewatering, since antisense mutants could hardly restore the initial value of leaf water potential even after 4 days. The whole plant water conductance was similarly decreased by drought in the two genotypes but progressively restored on rewatering, exclusively in wild‐type plants. The mechanisms that underlie the recovery process are not elucidated yet and may involve proliferation of new roots, embolism repair, and/or aquaporin activation. Nevertheless, the overall data indicates that aquaporin function can be important to withstand a water stress and even more important during plant recovery. 4. Cold embolism repair During winter, freeze‐thaw cycles can induce the formation of air bubbles (embolism) within the xylem vessels of woody plants and therefore induce a significant decrease in vascular hydraulic conductance (Ameglio et al., 2001; Cochard and Tyree, 1990). DiVerent plant species may exhibit diVerent vulnerability to winter embolism, depending on the anatomy and diameter of their vessels. Yet plants have developed mechanisms for embolism repair (Holbrook and Zwieniecki, 1999). One strategy is to create a positive pressure in the lumen of the xylem vessels to chase out the air bubbles by an incoming flow of water. For this, parenchyma cells transform starch to sugar and extrude it to the xylem vessels leading to an increase in osmotic potential. This process pulls water from the surrounding cells into the vessels and push the air bubbles. Sakr et al. (2003) have proposed that, in walnut twigs,

116

O. POSTAIRE ET AL.

aquaporins can account for this kind of water transport. They have characterized two PIP2 aquaporins from a xylem cDNA library and followed their expression in xylem parenchyma cells from October to March. The abundance in transcripts of the two isoforms was strongly increased during winter and dramatically decreased in March. The abundance of the corresponding proteins in xylem tissues was consistently increased from January to February. Immunolocalization studies revealed that expression was the highest in parenchyma cells that are in direct contact with xylem vessels, probably to facilitate water influx into embolized vessels. Drought also induces embolism in vessels of woody plants. Therefore, a role of aquaporins in water‐stressed plants, for embolism repair during the night or on rewatering, can be expected. F. CO2 TRANSPORT

DiVusion of CO2 from the stomatal cavity to the stroma of chloroplasts is important in determining the concentration of CO2 that is available for fixation by ribulose bisphosphate carboxylase in mesophyll cells. Therefore, a limitation in this process may ultimately aVect the rate of carbon assimilation and possibly growth. It has also been observed in several plant species that the eYciency of CO2 transport, that is, the conductance of mesophyll to CO2 (gm), varies in response to environmental signals (Flexas et al., 2006, and references therein). However, the molecular basis for this phenomenon remains unclear. The finding that certain aquaporins can somehow mediate CO2 transport in vitro or after expression in Xenopus oocytes (Uehlein et al., 2003) has opened interesting perspectives into this area of research and has led several authors to directly investigate a role for aquaporins in CO2 diVusion in inner leaf tissues. In a first study, Terashima and Ono (2002) have analyzed the eVects of mercury on V. faba and P. vulgaris leaves. A combination of gas exchange and fluorescence measurements revealed that, at moderate mercury concentrations, the stimulation of photosynthesis by intercellular CO2 was reduced, whereas the dependency of photosynthesis on chloroplastic CO2 was unchanged. This was interpreted to mean that mercury blocked CO2 diVusion into the chloroplast without aVecting the CO2 fixation machinery. More specifically, treatment of V. faba leaves by 0.3 mM of HgCl2 decreased gm by 56%. However, due to the overall toxicity and lack of selectivity of mercury, this approach has remained insuYcient to unambiguously establish a physiological role of plant aquaporins in CO2 transport. Uehlein et al. (2003) have taken a genetic approach and used transgenic tobacco with altered expression in NtAQP1 due to expression of an antisense or a tetracycline‐inducible sense transgene. In these plants, the rate of [14C]

AQUAPORINS IN PLANTS

117

incorporation in leaf disks fed with 14CO2 was positively correlated to the level of NtAQP1 expression. Gas exchange measurements also showed that at ambient, that is, limiting CO2 (380 ppm), net photosynthesis was reduced in the antisense line, whereas it was enhanced in the overexpressing line. A thorough biophysical characterization of the same plant lines has recently been reported by Flexas et al. (2006). Values of gm were evaluated by two diVerent methods, gas exchange, and chlorophyll fluorescence, on the one hand, and online 13C discrimination on the other hand. Both methods unambiguously showed that leaf conductance to CO2 was altered in parallel to NtAQP1 expression. Overall, gm in the overexpressing line was twice as high as in the antisense line. This result was interpreted to mean that NtAQP1 functions as a CO2 channel in the mesophyll. However, two other major leaf parameters, that is, stomatal conductance (Gs) and maximal CO2 assimilation were also tightly linked to NtAQP1 expression. It has been argued that all these parameters are known to be coregulated and therefore any change in gm will result in an overall change in leaf properties. It may also be argued that, conversely, altered expression in NtAQP1 primarily alters stomatal behavior (Gs), as a result of altered leaf water relations, and therefore induces a change in gm. To a certain extent, similar questions had been raised by a previous study (Hanba et al., 2004) in which a barley aquaporin (HvPIP2;1) was overexpressed in transgenic rice. While a good correlation was found between gm and HvPIP2;1 expression, transgenic plants also showed an increase in Gs. In addition, fine alterations in leaf morphology were uncovered in transgenic plants. In particular, stomata were reduced in density and size, mesophyll cells were smaller and their walls were thicker. It remains unclear whether, in this study, these changes result from heterologous expression of a barley aquaporin in rice. Nevertheless, the notion that all these changes may directly or indirectly contribute to alteration in gm makes it diYcult to interpret the gas exchange properties of the transgenic plants with regard to CO2 transport by aquaporins.

V. AQUAPORINS IN REPRODUCTIVE ORGANS During their life cycle and especially during sexual reproduction, higher plants pass through highly desiccated forms that facilitate their dissemination and allow their resistance to adverse environmental conditions. In particular, pollen grains that carry the male gametes and seeds that are produced from fertilized flowers diVerentiate through well‐defined dehydration sequences to finally contain <30% water (Bots et al., 2005b). The subsequent germination of seeds and pollen grains is triggered by a rapid

118

O. POSTAIRE ET AL.

imbibition that precedes a dramatic growth of the embryo or pollen tube. All these events rely on precise water exchange processes and possibly involve the contribution of membrane water transport and aquaporins. A. AQUAPORINS IN FLOWERS

1. Aquaporin expression in flowers A number of studies have revealed that aquaporins are abundantly expressed in flowers (Alexandersson et al., 2005; Bots et al., 2005b; Dixit et al., 2001; Marin‐Olivier et al., 2000; O’Brien et al., 2002). Aquaporin microarrays experiments in Arabidopsis have shown that several PIP (PIP1;2, PIP2;1, PIP2;6, and PIP2;7) and TIP (TIP1;1, TIP1;2, and TIP3;1) isoforms are predominantly expressed in flowers (Alexandersson et al., 2005). Expression in wild potato Solanum chacoense of ScPIP2a, a flower‐specific PIP2 homologue, has been described in detail by O’Brien et al. (2002). The transcript levels of ScPIP2a were ninefold higher in reproductive tissues than in leaves with preferential expression in the anther, pistil, and young developing fruit. In Brassica oleracea, two PIP1 aquaporins that had been initially isolated from an anther and stigma cDNA library were shown to have abundant transcripts in petals, sepals, ovary, stigma, and anthers (Marin‐Olivier et al., 2000). 2. Functions of aquaporins in flowers A role for aquaporins during specific processes of plant reproduction, such as anther dehiscence, pollen recognition, pollen germination, and petal movement, has been inferred from a combination of gene expression and genetic studies. The dehydration of anthers results from water losses by evaporation or via osmotically driven water eZux through the vascular bundle (Bots et al., 2005b). Aquaporins may facilitate both of these processes by increasing water permeability of the anther cells. This idea was recently investigated in tobacco by Bots et al. (2005b). Western blot analyses have shown that anthers of tobacco flowers express aquaporins of both the PIP1 and PIP2 subclasses (Bots et al., 2005b). In this species, the dehydration of anthers starts between stages 7 and 8. During stage 8, the abundance of PIP2 proteins was increased and the proteins were the most abundant in the vascular bundle and connective tissues. Bots et al. (2005a) have introduced into transgenic tobacco an RNAi construct that targeted this aquaporin subclass. The percentage of flowers that contained fully dehisced anthers was reduced from 76% in wild‐type controls to 36–63% in transgenic lines that expressed the RNAi construct. The latter lines also exhibited a reduced rate of anther dehydration, as measured by in vivo 1H‐NMR. The data were interpreted to

AQUAPORINS IN PLANTS

119

mean that PIP2 aquaporins facilitate the dehydration and therefore the dehiscence of tobacco anthers. The role of aquaporins in the pistil may vary between species. In tobacco flowers, PIP1 aquaporins are expressed in the stigma, mostly at the latest stages of floral development, indicating that these aquaporins may be involved in pollen grain imbibition and/or pollen tube growth. The two functions are supported by in situ hybridization experiments showing a strong expression signal in the neck‐cells of the stigma and transmitting tissue of the style (Bots et al., 2005b). In B. oleracea, in situ hybridization revealed a strong expression of PIP1 homologues in the stigmatic cells underneath the papillae cell layer and in the style tissue (Marin‐Olivier et al., 2000). By contrast, no expression was detected in the papillar cells, that is, in the cells that are in contact with the pollen grains and directly supply water during their imbibition. Thus, in this species, aquaporins do not seem directly involved in pollen grain germination. In S. chacoense, expression of ScPIP2a in the pistil was found to be strongly increased after cross‐pollination, whereas it remained low after self‐incompatible pollination. Precise kinetic analyses suggested that the increase in ScPIP2a mRNA expression was actually triggered by fertilization. The abundance of ScPIP2a transcripts was also strongly correlated to the rate of style elongation, indicating a possible role for this aquaporin in cell expansion (O’Brien et al., 2002). Self‐incompatibility, a central process in sexual plant reproduction, is based on recognition of self‐incompatible pollens that leads to signaling cascades that will ultimately induce a blockade of pollen tube development. A mutation that prevents self‐incompatibility in Brassica has been associated to a genetic locus encoding MIP‐MOD, a PIP1 aquaporin homologue (Ikeda et al., 1997). This gene is expressed in the pistil, essentially in the stigma (papillar cells) and the distal portion of the style (Dixit et al., 2001). By reference to the studies discussed above, it is somewhat diYcult to explain how inactivation of a pistil aquaporin may favor pollen tube growth. Ikeda et al. (1997) have suggested that in self‐incompatible (wild‐type) plants, the activation of MIP‐MOD would redirect water flow away from the pollen tube. Although not yet established, this mechanism supports the general idea that regulation of water transport from the stigma to the pollen is a checkpoint in early events of pollination in crucifers. Recent work by Azad et al. (2004a) has pointed to an original function of aquaporins during tulip petal movements. In dark conditions, petals open at 20 8C and close at 5 8C. Petal opening is accompanied by water transport from the stem to the petals. Azad et al. (2004a) showed that the amount of titriated water ([3H2O]) taken up by the flower was grossly proportional to flower aperture and that once the flower was opened, the [3H2O] content remained

120

O. POSTAIRE ET AL.

constant. During petal closure, water content decreased rapidly. These authors also found that, during petal opening, a PIP homologue was phosphorylated by a Ca2þ‐dependent protein kinase activity and that dephosphorylation of this aquaporin was associated to petal closure. These results support the idea that reversible phosphorylation of PIPs provides a means for regulating water transport during temperature‐dependent tulip petal oscillation. B. AQUAPORINS IN SEEDS

Seed germination is triggered by tissue imbibition and, after an apparent lag phase, is followed by the elongation of the radicle and thereafter of the whole embryonic axis. More specifically, rupture of the maternal seed coat (testa) occurring in some species can be taken as the first external sign of initiation of germination, whereas subsequent rupture of the zygotic tissue, the endosperm, and radicle protrusion indicate the completion of the process. The primary imbibition of seeds, the subsequent lag phase, and finally embryo growth can be associated to triphasic kinetics of water uptake, which represent a landmark feature of germinating plant seeds (Bewley, 1997; Bewley and Black, 1994). Water transport in seeds has classically been described using gravimetric methods (Bewley and Black, 1994). More recently, magnetic resonance imaging revealed a precise spatial distribution of water in germinating seeds and strikingly diVerent patterns between species (Krishnan et al., 2004; Manz et al., 2005; McEntyre et al., 1998; Terskikh et al., 2005). In addition, rapid imbibition of seeds can cause mechanical injuries (Veselova et al., 2003). Thus, a tight control of water transport operates within seeds. The expression of aquaporins in developing seeds and during germination has been investigated in several plant species. Aquaporins of the TIP3 (‐TIP) subclass are seed‐specific and expressed in the membrane of protein storage vacuoles (Ho¨fte et al., 1992; Johnson et al., 1989; Oliviusson and Hakman 1995). Their expression strongly decreases during seedling establishment. Based on these expression properties, Maurel et al. (1997) have proposed a role for these aquaporins in cell osmoregulation and maturation of the vacuolar apparatus during late seed development and during the early stages of germination. Expression of the whole aquaporin family in dry and germinating seeds has recently been investigated in Arabidopsis by Vander Willigen et al. (2006). This study confirmed that TIP3 homologues represent together with TIP5;1 the most highly expressed aquaporins in dry seeds. Surprisingly, the abundance of all PIP mRNAs was very low. This expression profile was changed shortly after germination to a profile dominated by strong expression of aquaporins of the PIP1, PIP2, TIP1, and TIP2 subclasses.

121

AQUAPORINS IN PLANTS

% Radicle emergence

A role for aquaporins in controlling water transport across cell plasma membranes during seed germination has first found some support in a study by Gao et al. (1999) in B. napus. These authors observed that expression of a lowly abundant PIP2 aquaporin in dry seeds was markedly increased by priming and therefore was tightly linked to the eYciency of seeds to germinate under normal or stress conditions. There are very few functional evidences for a role of aquaporins in seeds, and all of them are based on the inhibitory eVects of mercurials. For instance, Veselova et al. (2003) have shown that the aquaporin blocker p‐chloromercuriphenylsulfonic acid significantly reduces the rate of pea seed imbibition. This idea is in accordance with the work by Schuurmans et al. (2003) that uncovered strong expression of a PIP1 homolog in dry and germinating pea seeds. Arabidopsis seeds are much smaller in size and show a faster rate of imbibition, with a half time of about 22 min. Treatment of Arabidopsis seeds by 5 mM HgCl2 did not delay this process (Vander Willigen et al., 2006). The idea that hydraulic factors, that is, tissue water transport, could be limiting for embryo growth was initially discarded based on general models of plant tissue expansion (Cosgrove, 1993). However, there have been recent concerns about the significance of growth‐induced water potential gradients indicating a possible involvement of aquaporins in cell expansion (Fricke, 2002; Tang and Boyer, 2002). In support of this, Fukuhara et al. (1999) observed that aquaporin expression in seeds of Mesembryanthemum crystallinum was associated with embryo growth and was retarded in dormant seeds. In addition, Vander Willigen et al. (2006) showed that mercury treatment (5 mM HgCl2) of Arabidopsis seeds delayed both testa rupture and radicle emergence and therefore altered very early steps of embryo growth (Fig. 7). These eVects could be fully reversed by the reducing molecule DTT

100 80 60 40 20 0

0

10

20

30

40

50

60

70

80

90

Time (h)

Fig. 7. EVects of mercury on the germination of Arabidopsis seeds. Germination curves of seeds grown in the absence of HgCl2 (circles), or in the presence of 5 mM HgCl2 (diamonds) or 5 mM HgCl2 þ 2 mM DTT (triangles).

122

O. POSTAIRE ET AL.

and were not associated to any morphological abnormality at the time of radicle emergence.

VI. CONCLUSION The existence of water channels in plants was largely unsuspected just 15 years ago (Maurel, 1997). Thus, the molecular characterization of aquaporins, in plants and other organisms, is relatively recent and has insuZated novel ideas into the field of water transport. The high resolution atomic structure of spinach SoPIP2;1 gives a striking illustration of the accuracy of knowledge gained into the molecular mechanisms of transport selectivity and gating of plant aquaporins (Tornroth‐Horsefield et al., 2006). Also, biochemical and functional studies have revealed that plant aquaporins can be more than water channels and can be permeated by a large variety of molecules, including gas and solutes of high physiological significance such as CO2, NH3, or H2O2. One aim of this present chapter was to put into perspective the significance of molecular and functional knowledge on plant aquaporins with respect to integrated physiological processes. Here, we showed that, although still rare, examples exist in which a continuum in knowledge between aquaporin molecular structures and whole plants was established. In particular, the conserved gating properties of PIP aquaporins by Hþ have been interpreted in terms of protein conformational changes and their significance with respect to the response of plant roots to anoxia has been established (Tornroth‐Horsefield et al., 2006; Tournaire‐Roux et al., 2003). Also, the deficiency of mutant rice plants to take up silicon was linked to an inactivating, point mutation in the vicinity of the pore of a NIP aquaporin homologue (Ma et al., 2006). By contrast, the gating of aquaporins by divalent ions including calcium has been established in vitro (Gerbeau et al., 2002), can be interpreted by molecular modeling (Tornroth‐Horsefield et al., 2006), but its significance in the plant remains largely conjectural. Understanding the integrated function of aquaporins, whether coupled or not to molecular approaches, remains a central objective. Recent studies have provided strong support to the initial assumption that aquaporins are central to water relations. They have also revealed unsuspected links to other great physiological functions. It is now fairly well established that aquaporins play a predominant role in root water uptake and in its regulation by environmental factors. A further dissection of aquaporin functions in the diVerentiated cell layers of the root, by gene expression mapping and reverse genetics, should provide a more precise interpretation of the biophysical and physiological properties of

AQUAPORINS IN PLANTS

123

this organ. Beyond this, and because of the knowledge already gained, roots will remain a unique system to explore general mechanisms of aquaporin regulation. Recent studies have also pointed to the leaf as another organ in which aquaporins mediate transcellular water transport and therefore confer highly regulated hydraulic properties. We realize that inhibition studies and molecular and cellular analyses of aquaporin regulation in this organ rely on progresses made in roots. Yet aquaporins in leaves raise very original issues, one of them being a role in CO2 transport. Also, the role of aquaporins in stomatal regulation has been barely examined and a gap in knowledge needs to be filled. The function of aquaporins in other organs such as seeds and flowers has remained much more conjectural, in particular because a biophysical description of water relations has been much more diYcult to establish than in roots or leaves. Nevertheless, molecular and genetic analyses have brought interesting insights and underscored an original role of aquaporins during tissue desiccation and imbibition. Central physiological questions also remain, beyond the dissection of aquaporin function in each single plant organ. One such question is to understand the hydraulic aspects of growth and cell movements and their links to aquaporins. This question has been punctually addressed in roots (Hukin et al., 2002), leaves (Siefritz et al., 2004), or seeds (Vander Willigen et al., 2006) and will require a more careful examination. A role of aquaporins in the transport of carbon assimilates and carbon metabolism has been suggested (Ma et al., 2004) but not evaluated in detail. Finally, aquaporins transport reactive oxygen species. The significance of this property in terms of cell signaling and response to oxidative stress remains largely unknown.

REFERENCES Aasamaa, K. and Sober, A. (2005). Seasonal courses of maximum hydraulic conductance in shoots of six temperate deciduous tree species. Functional Plant Biology 32, 1077–1087. Agre, P., Bonhivers, M. and Borgnia, M. J. (1998). The aquaporins, blueprints for cellular plumbing systems. Journal of Biological Chemistry 273, 14659–14662. Alexandersson, E., Fraysse, L., Sjovall‐Larsen, S., Gustavsson, S., Fellert, M., Karlsson, M., Johanson, U. and Kjellbom, P. (2005). Whole gene family expression and drought stress regulation of aquaporins. Plant Molecular Biology 59, 469–484. Alleva, K., Niemietz, C. M., Sutka, M., Maurel, C., Parisi, M., Tyerman, S. D. and Amodeo, G. (2006). Plasma membrane of Beta vulgaris storage root shows high water channel activity that is regulated by cytoplasmic pH and a dual

124

O. POSTAIRE ET AL.

range of calcium concentrations. Journal of Experimental Botany 57, 609–621. Ameglio, T., Ewers, F. W., Cochard, H., Martignac, M., Vandame, M., Bodet, C. and Cruiziat, P. (2001). Winter stem xylem pressure in walnut trees: EVects of carbohydrates, cooling and freezing. Tree Physiology 21, 387–394. Amodeo, G., Sutka, M., Dorr, R. and Parisi, M. (2002). Protoplasmic pH modifies water and solute transfer in Beta vulgaris root vacuoles. Journal of Membrane Biology 187, 175–184. Anthony, T. L., Brooks, H. L., Boassa, D., Leonov, S., Yanochko, G. M., Regan, J. W. and Yool, A. J. (2000). Cloned human aquaporin‐1 is a cyclic GMP‐gated ion channel. Molecular Pharmacology 57, 576–588. Aroca, R., Tognoni, F., Irigoyen, J. J., Sanchez‐Diaz, M. and Pardossi, A. (2001). DiVerent root low temperature response to two maize genotypes diVering in chilling sensitivity. Plant Physiology and Biochemistry 39, 1067–1073. Aroca, R., Amodeo, G., Fernandez‐Illescas, S., Herman, E. M., Chaumont, F. and Chrispeels, M. J. (2005). The role of aquaporins and membrane damage in chilling and hydrogen peroxide induced changes in the hydraulic conductance of maize roots. Plant Physiology 137, 341–353. Azad, A. K., Sawa, Y., Ishikawa, T. and Shibata, H. (2004a). Characterization of protein phosphatase 2A acting on phosphorylated plasma membrane aquaporin of tulip petals. Biosciences Biotechnology Biochemistry 68, 1170–1174. Azad, A. K., Sawa, Y., Ishikawa, T. and Shibata, H. (2004b). Phosphorylation of plasma membrane aquaporin regulates temperature‐dependent opening of tulip petals. Plant and Cell Physiology 45, 608–617. Azaizeh, H. and Steudle, E. (1991). EVects of salinity on water transport of excised maize (Zea mays L.) roots. Plant Physiology 97, 1136–1145. Beuron, F., Cahe´rec, F. L., Guillam, M. T., Cavalier, A., Garret, A., Tassan, J. P., Delamarche, C., Schultz, P., Mallouh, V., Rolland, J. P., Hubert, J. F. Gouranton, J. et al. (1995). Structural analysis of a MIP family protein from the digestive tract of Cicadella viridis. Journal of Biological Chemistry 270, 17414–17422. Bewley, J. D. (1997). Seed germination and dormancy. Plant Cell 9, 1055–1066. Bewley, J. D. and Black, M. (1994). ‘‘Seeds: Physiology of Development and Germination.’’ Plenum Press, New York. Biela, A., Grote, K., Otto, B., Hoth, S., Hedrich, R. and KaldenhoV, R. (1999). The Nicotiana tabacum plasma membrane aquaporin NtAQP1 is mercury‐ insensitive and permeable for glycerol. Plant Journal 18, 565–570. Bienert, G. P., Moller, A. L., Kristiansen, K. A., Schulz, A., Moller, I. M., Schjoerring, J. K. and Jahn, T. P. (2007). Specific aquaporins facilitate the diVusion of hydrogen peroxide across membranes. Journal of Biological Chemistry 282, 1183–1192. Birnbaum, K., Shasha, D. E., Wang, J. Y., Jung, J. W., Lambert, G. M., Galbraith, D. W. and Benfey, P. N. (2003). A gene expression map of the Arabidopsis root. Science 302, 1956–1960. Bloom, A. J., Zwieniecki, M. A., Passoura, J. B., Randall, L. B., Holbrook, N. M. and St. Clair, D. A. (2004). Water relations under root chilling in a sensitive and tolerant tomato species. Plant Cell and Environment 27, 971–979. Bots, M., Vergeldt, F., Wolters‐Arts, M., Weterings, K., Van As, H. and Mariani, C. (2005a). Aquaporins of the PIP2 class are required for eYcient anther dehiscence in tobacco. Plant Physiology 137, 1049–1056. Bots, M., Feron, R., Uehlein, N., Weterings, K., KaldenhoV, R. and Mariani, T. (2005b). PIP1 and PIP2 aquaporins are diVerentially expressed during

AQUAPORINS IN PLANTS

125

tobacco anther and stigma development. Journal of Experimental Botany 56, 113–121. Boursiac, Y., Chen, S., Luu, D. T., Sorieul, M., van den Dries, N. and Maurel, C. (2005). Early eVects of salinity on water transport in Arabidopsis roots– Molecular and cellular features of aquaporin expression. Plant Physiology. 139, 790–805. Carvajal, M., Cooke, D. T. and Clarkson, D. T. (1996). Responses of wheat plants to nutrient deprivation may involve the regulation of water‐channel function. Planta 199, 372–381. Carvajal, M., Martinez, V. and Alcaraz, C. F. (1999). Physiological function of water channels as aVected by salinity in roots of paprika pepper. Physiologia Plantarum 105, 95–101. Carvajal, M., Cerda, A. and Martinez, V. (2000). Does calcium ameliorate the negative eVect of NaCl on melon root water transport by regulating aquaporin activity? New Phytologist 145, 439–447. Chakrabarti, N., Tajkhorshid, E., Roux, B. and Pomes, R. (2004). Molecular basis of proton blockage in aquaporins. Structure 12, 65–74. Chaumont, F., Barrieu, F., Wojcik, E., Chrispeels, M. J. and Jung, R. (2001). Aquaporins constitute a large and highly divergent protein family in maize. Plant Physiology. 125, 1206–1215. Chaumont, F., Moshelion, M. and Daniels, M. J. (2005). Regulation of plant aquaporin activity. Biology of the Cell 97, 749–764. Chrispeels, M. J. and Agre, P. (1994). Aquaporins: Water channel proteins of plant and animal cells. Trends in Biochemical Sciences 19, 421–425. Clarkson, D. T., Carvajal, M., Henzler, T., Waterhouse, R. N., Smyth, A. J., Cooke, D. T. and Steudle, E. (2000). Root hydraulic conductance: Diurnal aquaporin expression and the eVects of nutrient stress. Journal of Experimental Botany 51, 61–70. Cochard, H. and Tyree, M. T. (1990). Xylem dysfunction in Quercus: Vessel sizes, tyloses, cavitation and seasonal changes in embolism. Tree Physiology 6, 393–407. Cochard, H., Nardini, A. and Coll, L. (2004). Hydraulic architecture of leaf blades: Where is the main resistance? Plant, Cell and Environment 27, 1257–1267. Cochard, H., Venisse, J. S., Barigah, T. S., Brunel, N., Herbette, S., Guilliot, A., Tyree, M. T. and Sakr, S. (2007). New insights into the understanding of variable hydraulic conductances in leaves. Evidence for a possible implication of plasma membrane aquaporins. Plant Physiology 143, 122–133. Cooper, G. J. and Boron, W. F. (1998). EVect of PCMBS on CO2 permeability of Xenopus oocytes expressing aquaporin 1 or its C189S mutant. American Journal of Physiology. Cell Physiology 44, C1481–C1486. Cosgrove, D. J. (1993). Water uptake by growing cells: An assessment of the controlling roles of wall relaxation, solute uptake and hydraulic conductance. International Journal of Plant Sciences 154, 10–21. Daniels, M. J. and Yeager, M. (2005). Phosphorylation of aquaporin PvTIP3;1 defined by mass spectrometry and molecular modeling. Biochemistry 44, 14443–14454. Daniels, M. J., Chaumont, F., Mirkov, T. E. and Chrispeels, M. J. (1996). Characterization of a new vacuolar membrane aquaporin sensitive to mercury at a unique site. Plant Cell 8, 587–599. Daniels, M. J., Chrispeels, M. J. and Yeager, M. (1999). Projection structure of a plant vacuole membrane aquaporin by electron cryo‐crystallography. Journal of Molecular Biology 294, 1337–1349.

126

O. POSTAIRE ET AL.

De Groot, B., Engel, A. and Grubmu¨ller, H. (2001). A refined structure of human aquaporin‐1. FEBS Letters 504, 206–211. De Groot, B. L., Frigato, T., Helms, V. and Grubmuller, H. (2003). The mechanism of proton exclusion in the aquaporin‐1 water channel. Journal of Molecular Biology 333, 279–293. Dean, R. M., Rivers, R. L., Zeidel, M. L. and Roberts, D. M. (1999). Purification and functional reconstitution of soybean Nodulin 26. An aquaporin with water and glycerol transport properties. Biochemistry 38, 347–353. Dell’Amico, J., Torrecillas, A., Rodriguez, P., Morales, D. and Sanchez‐Blanco, M. J. (2001). DiVerences in the eVects of flooding the soil early and late in the photoperiod on the water relations of pot‐grown tomato plants. Plant Science 160, 481–487. Ding, X., Iwasaki, I. and Kitagawa, Y. (2004). Overexpression of a lily PIP1 gene in tobacco increased the osmotic water permeability of leaf cells. Plant, Cell and Environment 27, 177–186. Dixit, R., Rizzo, C., Nasrallah, M. and Nasrallah, J. (2001). The brassica MIP‐MOD gene encodes a functional water channel that is expressed in the stigma epidermis. Plant Molecular Biology 45, 51–62. Everard, J. D. and Drew, M. C. (1989). Mechanisms controlling changes in water movement through the roots of Helianthus annuus L. During continuous exposure to oxygen deficiency. Journal of Experimental Botany 40, 95–104. Fennell, A. and Markhart, A. (1998). Rapid acclimation of root hydraulic conductivity to low temperature. Journal of Experimental Botany 49, 879–884. Fetter, K., Van Wilder, V., Moshelion, M. and Chaumont, F. (2004). Interactions between plasma membrane aquaporins modulate their water channel activity. Plant Cell 16, 215–228. Flexas, J., Ribas‐Carbo, M., Hanson, D. T., Bota, J., Otto, B., Cifre, J., McDowell, N., Medrano, H. and KaldenhoV, R. (2006). Tobacco aquaporin NtAQP1 is involved in mesophyll conductance to CO2 in vivo. Plant Journal 48, 427–439. Fotiadis, D., Jeno, P., Mini, T., Wirtz, S., Muller, S. A., Fraysse, L., Kjellbom, P. and Engel, A. (2001). Structural characterization of two aquaporins isolated from native spinach leaf plasma membranes. Journal of Biological Chemistry 276, 1707–1714. Frangne, N., Maeshima, M., SchaVner, A. R., Mandel, T., Martinoia, E. and Bonnemain, J. L. (2001). Expression and distribution of a vaculoar aquaporin in young and mature leaf tissues of Brassica napus in relation to water fluxes. Planta 212, 270–278. Fraysse, L. C., Wells, B., McCann, M. C. and Kjelbom, P. (2005). Specific plasma membrane aquaporins of the PIP1 subfamily are expressed in sieve elements and guard cells. Biology of the Cell 97, 519–534. Fricke, W. (2002). Biophysical limitation of cell elongation in cereal leaves. Annals of Botany 90, 157–167. Fu, D., Libson, A., Miercke, L. J. W., Weitzman, C., Nollert, P., Krucinski, J. and Stroud, R. M. (2000). Structure of a glycerol‐conducting channel and the basis for its selectivity. Science 290, 481–486. Fujiyoshi, Y., Mitsuoka, K., de Groot, B. L., Philippsen, A., Grubmu¨ller, H., Agre, P. and Engel, A. (2002). Structure and function of water channels. Current Opinion in Structural Biology 12, 509–515. Fukuhara, T., Kirch, H. H. and Bohnert, H. J. (1999). Expression of Vp1 and water channel proteins during seed germination. Plant, Cell and Environment 22, 417–424.

AQUAPORINS IN PLANTS

127

Gao, Y. P., Young, L., Bonham‐Smith, P. and Gusta, L. V. (1999). Characterization and expression of plasma and tonoplast membrane aquaporins in primed seed of Brassica napus during germination under stress conditions. Plant Molecular Biology 40, 635–644. Gerbeau, P., Guclu, J., Ripoche, P. and Maurel, C. (1999). Aquaporin Nt‐TIP a can account for the high permeability of tobacco cell vacuolar membrane to small neutral solutes. Plant Journal 18, 577–587. Gerbeau, P., Amodeo, G., Henzler, T., Santoni, V., Ripoche, P. and Maurel, C. (2002). The water permeability of Arabidopsis plasma membrane is regulated by divalent cations and pH. Plant Journal 30, 71–81. Gout, E., Boisson, A. M., Aubert, S., Douce, R. and Bligny, R. (2001). Origin of the cytoplasmic pH changes during anaerobic stress in higher plant cells. 13Carbon and 31phosphorous nuclear magnetic resonance studies. Plant Physiology 125, 912–925. Guenther, J. F., Chanmanivone, N., Galetovic, M. P., Wallace, I. S., Cobb, J. A. and Roberts, D. M. (2003). Phosphorylation of soybean Nodulin 26 on serine 262 enhances water permeability and is regulated developmentally and by osmotic signals. Plant Cell 15, 981–991. Hachez, C., Moshelion, M., Zelazny, E., Cavez, D. and Chaumont, F. (2006). Localization and quantification of plasma membrane aquaporin expression in maize primary root: A clue to understanding their role as cellular plumbers. Plant Molecular Biology 62, 305–323. Hanba, Y. T., Shibasaka, M., Hayashi, Y., Hayakawa, T., Kasamo, K., Terashima, I. and Katsuhara, M. (2004). Overexpression of the barley aquaporin HvPIP2;1 increases internal CO2 conductance and CO2 assimilation in the leaves of transgenic rice plants. Plant and Cell Physiology 45, 521–529. Hendriks, G., Koudijs, M., van Balkom, B. W., Oorschot, V., Klumperman, J., Deen, P. M. and van der Sluijs, P. (2004). Glycosylation is important for cell surface expression of the water channel aquaporin‐2 but is not essential for tetramerization in the endoplasmic reticulum. Journal of Biological Chemistry 279, 2975–2983. Henzler, T. and Steudle, E. (2000). Transport and metabolic degradation of hydrogen peroxide in Chara corallina: Model calculations and measurements with the pressure probe suggest transport of H2O2 across water channels. Journal of Experimantal Botany 51, 2053–2066. Henzler, T., Waterhouse, R. N., Smyth, A. J., Carvajal, M., Cooke, D. T., SchaVner, A. R., Steudle, E. and Clarkson, D. T. (1999). Diurnal variations in hydraulic conductivity and root pressure can be correlated with the expression of putative aquaporins in the roots of lotus japonicus. Planta 210, 50–60. Henzler, T., Ye, Q. and Steudle, E. (2004). Oxidative gating of water channels (aquaporins) in Chara by hydroxyl radicals. Plant, Cell and Environment 27, 1184–1195. Herrera, M., Hong, N. J. and Garvin, J. L. (2006). Aquaporin‐1 transports NO across cell membranes. Hypertension 48, 157–164. Hoarau, J., Barthes, L., Bousser, A., Dele´ens, A. and Prioul, J. L. (1996). EVect of nitrate on water transfer across roots of nitrogen pre‐starved maize seedlings. Planta 200, 405–415. Ho¨fte, H., Hubbard, L., Reizer, J., Ludevid, D., Herman, E. M. and Chrispeels, M. J. (1992). Vegetative and seed‐specific forms of tonoplast intrinsic protein in the vacuolar membrane of Arabidopsis thaliana. Plant Physiology 99, 561–570.

128

O. POSTAIRE ET AL.

Holbrook, N. M. and Zwieniecki, M. A. (1999). Embolism repair and xylem tension: Do we need a miracle? Plant Physiology 120, 7–10. Hose, E., Steudle, E. and Hartung, W. (2000). Abscisic acid and hydraulic conductivity of maize roots: A study using cell‐ and root‐pressure probes. Planta 211, 874–882. Hukin, D., Doering‐Saad, C., Thomas, C. R. and Pritchard, J. (2002). Sensitivity of cell hydraulic conductivity to mercury is coincident with symplasmic isolation and expression of plasmalemma aquaporin genes in growing maize roots. Planta 215, 1047–1056. Ikeda, S., Nasrallah, J. B., Dixit, R., Preiss, S. and Nasrallah, M. E. (1997). An aquaporin‐like gene required for the Brassica self‐incompatibility response. Science 276, 1564–1566. Ishibashi, K. (2006). Aquaporin subfamily with unusual NPA boxes. Biochimica Biophysica Acta 1758, 989–993. Ishikawa, F., Suga, S., Uemura, T., Sato, M. H. and Maeshima, M. (2005). Novel type aquaporin SIPs are mainly localized to the ER membrane and show cell‐ specific expression in Arabidopsis thaliana. FEBS Letters 579, 5814–5820. Jahn, T. P., Moller, A. L., Zeuthen, T., Holm, L. M., Klaerke, D. A., Mohsin, B., Kuhlbrandt, W. and Schjoerring, J. K. (2004). Aquaporin homologues in plants and mammals transport ammonia. FEBS Letters 574, 31–36. Jang, J. Y., Kim, D. G., Kim, Y. O., Kim, J. S. and Kang, H. (2004). An expression analysis of a gene family encoding plasma membrane aquaporins in response to abiotic stresses in Arabidopsis thaliana. Plant Molecular Biology 54, 713–725. Javot, H. and Maurel, C. (2002). The role of aquaporins in root water uptake. Annals of Botany 90, 301–313. Javot, H., Lauvergeat, V., Santoni, V., Martin‐Laurent, F., Guclu, J., Vinh, J., Heyes, J., Franck, K. I., SchaVner, A. R., Bouchez, D. and Maurel, C. (2003). Role of a single aquaporin isoform in root water uptake. Plant Cell 15, 509–522. Johansson, I., Larsson, C., Ek, B. and Kjellbom, P. (1996). The major integral proteins of spinach leaf plasma membranes are putative aquaporins and are phosphorylated in response to Ca2þ and apoplastic water potential. Plant Cell 8, 1181–1191. Johansson, I., Karlsson, M., Shukla, V. K., Chrispeels, M. J., Larsson, C. and Kjellbom, P. (1998). Water transport activity of the plasma membrane aquaporin PM28A is regulated by phosphorylation. Plant Cell 10, 451–460. Johansson, I., Karlsson, M., Johanson, U., Larsson, C. and Kjellbom, P. (2000). The role of aquaporins in cellular and whole plant water balance. Biochimica Biophysica Acta 1465, 324–342. Johnson, K. D., Herman, E. M. and Chrispeels, M. J. (1989). An abundant, highly conserved tonoplast protein in seeds. Plant Physiology 91, 1006–1013. Jung, J. S., Preston, G. M., Smith, B. L., Guggino, W. B. and Agre, P. (1994). Molecular structure of the water channel through aquaporin CHIP. The hourglass model. Journal of Biological Chemistry 269, 14648–14654. KaldenhoV, R. and Fischer, M. (2006). Functional aquaporin diversity in plants. Biochimica Biophysica Acta 1758, 1134–1141. KaldenhoV, R., Kolling, A., Meyers, J., Karmann, U., Ruppel, G. and Richter, G. (1995). The blue light‐responsive AthH2 gene of Arabidopsis thaliana is primarily expressed in expanding as well as in diVerentiating cells and encodes a putative channel protein of the plasmalemma. Plant Journal 7, 87–95.

AQUAPORINS IN PLANTS

129

KaldenhoV, R., Grote, K., Zhu, J. J. and Zimmermann, U. (1998). Significance of plasmalemma aquaporins for water‐transport in Arabidopsis thaliana. Plant Journal 14, 121–128. Kamaluddin, M. and Zwiazek, J. J. (2001). Metabolic inhibition of root water flow in red‐osier dogwood (Cornus stolonifera) seedlings. Journal of Experimental Botany 52, 739–745. Karlsson, M., Fotiadis, D., Sjovall, S., Johansson, I., Hedfalk, K., Engel, A. and Kjellbom, P. (2003). Reconstitution of water channel function of an aquaporin overexpressed and purified from Pichia pastoris. FEBS Letters 537, 68–72. Karmoker, J. L., Clarkson, D. T., Saker, L. R., Rooney, J. M. and Purves, J. V. (1991). Sulphate deprivation depresses the transport of nitrogen to the xylem and the hydraulic conductivity of barley (Hordeum vulgare L.) roots. Planta 185, 269–278. Katsuhara, M., Koshio, K., Shibasaka, M., Hayashi, Y., Hayakawa, T. and Kasamo, K. (2003). Over‐expression of a barley aquaporin increased the shoot/root ratio and raised salt sensitivity in transgenic rice plants. Plant and Cell Physiology 44, 1378–1383. Klebl, F., Wolf, M. and Sauer, N. (2003). A defect in the yeast plasma membrane urea transporter Dur3p is complemented by CpNIP1, a Nod26‐like protein from zucchini (Cucurbita pepo L.), and by Arabidopsis thaliana delta‐TIP or gamma‐TIP. FEBS Letters 547, 69–74. Krishnan, P., Joshi, D. K., Nagarajan, S. and Moharir, A. V. (2004). Characterisation of germinating and non‐germinating wheat seeds by nuclear magnetic resonance (NMR) spectroscopy. European Biophysical Journal 33, 76–82. Laize´, V., Rousselet, G., Verbavatz, J. M., Berthonaud, V., Gobin, R., Roudier, N., Abrami, L., Ripoche, P. and Tacnet, F. (1995). Functional expression of the human CHIP28 water channel in a yeast secretory mutant. FEBS Letters 373, 269–274. Lee, S. H., Singh, A. P. and Chung, G. C. (2004). Rapid accumulation of hydrogen peroxide in cucumber roots due to exposure to low temperature appears to mediate decreases in water transport. Journal of Experimental Botany 55, 1733–1741. Leitch, V., Agre, P. and King, L. S. (2001). Altered ubiquitination and stability of aquaporin‐1 in hypertonic stress. Proceedings of the National Academy of Sciences of USA 98, 2894–2898. Lian, H. L., Yu, X., Ye, Q., Ding, X. S., Kitagawa, Y., Kwak, S. S., Su, W. A. and Tang, Z. C. (2004). The role of aquaporin RWC3 in drought avoidance in rice. Plant and Cell Physiology 45, 481–489. Liu, L. H., Ludewig, U., Gassert, B., Frommer, W. B. and von Wiren, N. (2003). Urea transport by nitrogen‐regulated tonoplast intrinsic proteins in Arabidopsis. Plant Physiology 133, 1220–1228. Lopez, F., Bousser, A., SissoeV, I., Gaspar, M., Lachaise, B., Hoarau, J. and Mahe, A. (2003). Diurnal regulation of water transport and aquaporin gene expression in maize roots: Contribution of PIP2 proteins. Plant and Cell Physiology 44, 1384–1395. Loque, D., Ludewig, U., Yuan, L. and von Wiren, N. (2005). Tonoplast intrinsic proteins AtTIP2;1 and AtTIP2;3 facilitate NH3 transport into the vacuole. Plant Physiology 137, 671–680. Luu, D. T. and Maurel, C. (2005). Aquaporins in a challenging environment: Molecular gears for adjusting plant water status. Plant Cell and Environment 28, 85–96.

130

O. POSTAIRE ET AL.

Ma, J. F., Tamai, K., Yamaji, N., Mitani, N., Konishi, S., Katsuhara, M., Ishiguro, M., Murata, Y. and Yano, M. (2006). A silicon transporter in rice. Nature 440, 688–691. Ma, S., Quist, T. M., Ulanov, A., Joly, R. and Bohnert, H. J. (2004). Loss of TIP1;1 aquaporin in Arabidopsis leads to cell and plant death. Plant Journal 40, 845–859. Maathuis, F. J., Filatov, V., Herzyk, P., Krijger, G. C., Axelsen, K. B., Chen, S., Green, B. J., Li, Y., Madagan, K. L., Sanchez‐Fernandez, R., Forde, B. G., Palmgren, M. G., et al. (2003). Transcriptome analysis of root transporters reveals participation of multiple gene families in the response to cation stress. Plant Journal 35, 675–692. Maggio, A. and Joly, R. J. (1995). EVects of mercuric chloride on the hydraulic conductivity of tomato root systems. Evidence for a channel‐mediated water pathway. Plant Physiology 109, 331–335. Manz, B., Muller, K., Kucera, B., Volke, F. and Leubner‐Metzger, G. (2005). Water uptake and distribution in germinating tobacco seeds investigated in vivo by nuclear magnetic resonance imaging. Plant Physiology 138, 1538–1551. Marin‐Olivier, M., Chevalier, T., Fobis‐Loisy, I., Dumas, C. and Gaude, T. (2000). Aquaporin PIP genes are not expressed in the stigma papillae in Brassica oleracea. Plant Journal 4, 231–240. Markhart, A. H., Fiscus, E. L., Naylor, A. W. and Kramer, P. J. (1979). Effect of abscisic acid on root hydraulic conductivity. Plant Physiology 64, 611–614. Martinez‐Ballesta, M. C., Martinez, V. and Carvajal, M. (2000). Regulation of water channel activity in whole roots and in protoplasts from roots of melon plants grown under saline conditions. Australian Journal of Plant Physiology 27, 685–691. Martinez‐Ballesta, M. C., Aparicio, F., Pallas, V., Martinez, V. and Carvajal, M. (2003). Influence of saline stress on root hydraulic conductance and PIP expression in Arabidopsis. Journal of Plant Physiology 160, 689–697. Martinez‐Ballesta, M. C., Silva, C., Lopez‐Berenguer, C., Cabanero, F. J. and Carvajal, M. (2006). Plant aquaporins: New perspectives on water and nutrient uptake in saline environment. Plant Biology (Stuttgart) 8, 535–546. Martre, P., North, G. B. and Nobel, P. S. (2001). Hydraulic conductance and mercury‐sensitive water transport for roots of Opuntia acanthocarpa in relation to soil drying and rewetting. Plant Physiology 126, 352–362. Martre, P., Morillon, R., Barrieu, F., North, G. B., Nobel, P. S. and Chrispeels, M. J. (2002). Plasma membrane aquaporins play a significant role during recovery from water deficit. Plant Physiology 130, 2101–2110. Maurel, C. (1997). Aquaporins and water permeability of plant membranes. Annual Review of Plant Physiology and Plant Molecular Biology 48, 399–429. Maurel, C. and Chrispeels, M. J. (2001). Aquaporins. A molecular entry into plant water relations. Plant Physiology 125, 135–138. Maurel, C., Reizer, J., Schroeder, J. I. and Chrispeels, M. J. (1993). The vacuolar membrane protein
‐TIP creates water specific channels in Xenopus oocytes. EMBO Journal 12, 2241–2247. Maurel, C., Reizer, J., Schroeder, J. I., Chrispeels, M. J. and Saier, M. H. J. (1994). Functional characterization of the Escherichia coli glycerol facilitator, GlpF, in Xenopus oocytes. Journal of Biological Chemistry 269, 11869–11872. Maurel, C., Kado, R. T., Guern, J. and Chrispeels, M. J. (1995). Phosphorylation regulates the water channel activity of the seed‐specific aquaporin ‐TIP. EMBO Journal 14, 3028–3035.

AQUAPORINS IN PLANTS

131

Maurel, C., Chrispeels, M. J., Lurin, C., Tacnet, F., Geelen, D., Ripoche, P. and Guern, J. (1997). Function and regulation of seed aquaporins. Journal of Experimental Botany 48, 421–430. Maurel, C., Javot, H., Lauvergeat, V., Gerbeau, P., Tournaire, C., Santoni, V. and Heyes, J. (2002). Molecular physiology of aquaporins in plants. International Review of Cytology 215, 105–148. McEntyre, E., Ruan, R. and Fulcher, R. G. (1998). Comparison of water absorption patterns in two barley cultivars using magnetic resonance imaging. Cereal Chemistry 75, 792–795. Melkonian, J., Yu, L. X. and Setter, T. L. (2004). Chilling responses of maize (Zea mays L.) seedlings: Root hydraulic conductance, abscisic acid, and stomatal conductance. Journal of Experimental Botany 55, 1751–1760. Morillon, R. and Chrispeels, M. J. (2001). The role of ABA and the transpiration stream in the regulation of the osmotic water permeability of leaf cells. Proceedings of the National Academy of Sciences of USA 98, 14138–14143. Morillon, R. and Lassalles, J. P. (1999). Osmotic water permeability of isolated vacuoles. Planta 210, 80–84. Moshelion, M., Becker, D., Biela, A., Uehlein, N., Hedrich, R., Otto, B., Levi, H., Moran, N. and KaldenhoV, R. (2002). Plasma membrane aquaporins in the motor cells of Samanea saman: Diurnal and circadian regulation. Plant Cell 14, 727–739. Moshelion, M., Moran, N. and Chaumont, F. (2004). Dynamic changes in the osmotic water permeability of protoplast plasma membrane. Plant Physiology 135, 2301–2317. Murata, K., Mitsuoka, K., Hirai, T., Walz, T., Agre, P., Heymann, J. B., Engel, A. and Fujiyoshi, Y. (2000). Structural determinants of water permeation through aquaporin‐1. Nature 407, 599–605. Nakhoul, N. L., Davis, B. A., Romero, M. F. and Boron, W. F. (1998). EVect of expressing the water channel aquaporin‐1 on the CO2 permeability of Xenopus oocytes. American Journal of Physiology. Cell Physiology 43, C543–C548. Nardini, A. and Salleo, S. (2005). Water stress‐induced modifications of leaf hydraulic architecture in sunflower: Co‐ordination with gas exchange. Journal of Experimental Botany 56, 3093–3101. Nardini, A., Salleo, S. and Andri, S. (2005). Circadian regulation of leaf hydraulic conductance in sunflower (Helianthus annuus L. cv Margot). Plant, Cell and Environment 28, 750–759. Nejsum, L. N., Kwon, T. H., Jensen, U. B., Fumagalli, O., Frokiaer, J., Krane, C. M., Menon, A. G., King, L. S., Agre, P. C. and Nielsen, S. (2002). Functional requirement of aquaporin‐5 in plasma membranes of sweat glands. Proceedings of the National Academy of Sciences of USA 99, 511–516. Niemietz, C. M. and Tyerman, S. D. (1997). Characterization of water channels in wheat root membrane vesicles. Plant Physiology 115, 561–567. Niemietz, C. M. and Tyerman, S. D. (2000). Channel‐mediated permeation of ammonia gas through the peribacteroid membrane of soybean nodules. FEBS Letters 465, 110–114. Niemietz, C. M. and Tyerman, S. D. (2002). New potent inhibitors of aquaporins: Silver and gold compounds inhibit aquaporins of plant and human origin. FEBS Letters 531, 443–447. North, G. B., Martre, P. and Nobel, P. S. (2004). Aquaporins account for variations in hydraulic conductance for metabolically active root regions of Agave deserti in wet, dry and rewetted soil. Plant, Cell and Environment 27, 219–228.

132

O. POSTAIRE ET AL.

O’Brien, M., Bertrand, C. and Matton, D. P. (2002). Characterization of a fertilization‐induced and developmentally regulated plasma‐membrane aquaporin expressed in reproductive tissues, in the wild potato Solanum chacoense Bitt. Planta 215, 485–493. Ohshima, Y., Iwasaki, I., Suga, S., Murakami, M., Inoue, K. and Maeshima, M. (2001). Low aquaporin content and low osmotic water permeability of the plasma and vacular membranes of a CAM plant Graptopetalum paraguayense: Comparison with radish. Plant and Cell Physiology 42, 1119–1129. Oliviusson, P. and Hakman, I. (1995). A tonoplast intrinsic protein (TIP) is present in seeds, roots and somatic embryos of Norway spruce (Picea abies). Physiologia Plantarum 95, 288–295. Parsons, L. R. and Kramer, P. J. (1974). Diurnal cycling in root resistance to water movement. Physiologia Plantarum 30, 19–23. Peyrano, G., Taleisnik, E., Quiroga, M., de Forchetti, S. M. and Tigier, H. (1997). Salinity eVects on hydraulic conductance, lignin content and peroxydase activity in tomato roots. Plant Physiology and Biochemistry 35, 387–393. Prasad, G. V. R., Coury, L. A., Finn, F. and Zeidel, M. L. (1998). Reconstituted aquaporin 1 water channels transport CO2 across membranes. Journal of Biological Chemistry 273, 33123–33126. Preston, G. M., Carroll, T. P., Guggino, W. B. and Agre, P. (1992). Appearance of water channels in Xenopus oocytes expressing red cell CHIP28 protein. Science 256, 385–387. Procino, G., Carmosino, M., Marin, O., Brunati, A. M., Contri, A., Pinna, L. A., Mannucci, R., Nielsen, S., Kwon, T. H., Svelto, M. and Valenti, G. (2003). Ser‐256 phosphorylation dynamics of aquaporin 2 during maturation from the ER to the vesicular compartment in renal cells. FASEB Journal 17, 1886–1888. Procino, G., Caces, D. B., Valenti, G. and Pessin, J. E. (2006). Adipocytes support cAMP‐dependent translocation of aquaporin‐2 from intracellular sites distinct from the insulin‐responsive GLUT4 storage compartment. American Journal of Physiology. Renal Physiology 290, F985–F994. Quigley, F., Rosenberg, J. M., Shachar‐Hill, Y. and Bohnert, H. J. (2001). From genome to function: The Arabidopsis aquaporins. Genome Biology 3, 1–17. Radin, J. W. and Matthews, M. A. (1989). Water transport properties of cortical cells in roots of nitrogen‐ and phosphorus‐deficient cotton seedlings. Plant Physiology 89, 264–268. Ramahaleo, T., Morillon, R., Alexandre, J. and Lassalles, J. P. (1999). Osmotic water permeability of isolated protoplasts. Modifications during development. Plant Physiology 119, 885–896. Rivers, R. L., Dean, R. M., Chandy, G., Hall, J. E., Roberts, D. M. and Zeidel, M. L. (1997). Functional analysis of Nodulin 26, an aquaporin in soybean root nodule symbiosomes. Journal of Biological Chemistry 272, 16256–16261. Sack, L. and Holbrook, N. M. (2006). Leaf hydraulics. Annual Review of Plant Biology 57, 361–381. Sack, L., Melcher, P. J., Zwieniecki, M. A. and Holbrook, N. M. (2002). The hydraulic conductance of the angiosperm leaf lamina: A comparison of three measurement methods. Journal of Experimental Botany 53, 2177–2184. Sack, L., Cowan, P. D., Jaikumar, N. and Holbrook, N. M. (2003). The ‘hydrology’ of leaves: Co‐ordination of structure and function in temperate woody species. Plant, Cell and Environment 26, 1343–1356. Sack, L., Streeter, C. M. and Holbrook, N. M. (2004). Hydraulic analysis of water flow through leaves of sugar maple and red oak. Plant Physiology 134, 1824–1833.

AQUAPORINS IN PLANTS

133

Saito, C., Ueda, T., Abe, H., Wada, Y., Kuroiwa, T., Hisada, A., Furuya, M. and Nakano, A. (2002). A complex and mobile structure forms a distinct subregion within the continuous vacuolar membrane in young cotyledons of Arabidopsis. Plant Journal 29, 245–255. Sakr, S., Alves, G., Morillon, R., Maurel, K., Decourteix, M., Guilliot, A., Fleurat‐ Lessard, P., Julien, J. L. and Chrispeels, M. J. (2003). Plasma membrane aquaporins are involved in winter embolism recovery in walnut tree. Plant Physiology 133, 630–641. Sakurai, J., Ishikawa, F., Yamaguchi, T., Uemura, M. and Maeshima, M. (2005). Identification of 33 rice aquaporin genes and analysis of their expression and function. Plant and Cell Physiology 46, 1568–1577. Sanders, O. I., Rensing, C., Kuroda, M., Mitra, B. and Rosen, B. P. (1997). Antimonite is accumulated by the glycerol facilitator GlpF in Escherichia coli. Journal of Bacteriology 179, 3365–3367. Santoni, V., Vinh, J., Pflieger, D., Sommerer, N. and Maurel, C. (2003). A proteomic study reveals novel insights into the diversity of aquaporin forms expressed in the plasma membrane of plant roots. Biochemical Journal 372, 289–296. Santoni, V., Verdoucq, L., Sommerer, N., Vinh, J., Pflieger, D. and Maurel, C. (2006). Methylation of aquaporins in plant plasma membrane. Biochemical Journal 400, 189–197. Sarda, X., Tousch, D., Ferrare, K., Legrand, E., Dupuis, J. M., Casse‐Delbart, F. and Lamaze, T. (1997). Two TIP‐like encoding aquaporins are expressed in sunflower guard cells. Plant Journal 12(5), 1103–1111. Savage, D. F., Egea, P. F., Robles‐Colmenares, Y., O’Connell, J. D. and Stroud, R. M. (2003). Architecture and selectivity in aquaporins: 2.5 A X‐ray structure of aquaporin Z. PLoS Biology 1, E72. Schuurmans, J. A., van Dongen, J. T., Rutjens, B. P. W., Boonman, A., Pieterse, C. M. J. and Borstlap, A. C. (2003). Members of the aquaporin family in the developing pea seed coat include representatives of the PIP, TIP, and NIP subfamilies. Plant Molecular Biology 53, 633–645. Shangguan, Z. P., Lei, T. W., Shao, M. A. and Xue, Q. W. (2005). EVects of phosphorus nutrient on the hydraulic conductivity of sorghum (Sorghum vulgare Pers.) seedling roots under water deficiency. Journal of Integrative Plant Biology 47, 421–427. Siefritz, F., Tyree, M. T., Lovisolo, C., Schubert, A. and KaldenhoV, R. (2002). PIP1 plasma membrane aquaporins in tobacco: From cellular eVects to function in plants. Plant Cell 14, 869–876. Siefritz, F., Otto, B., Bienert, G. P., Van Der Krol, A. and KaldenhoV, R. (2004). The plasma membrane aquaporin NtAQP1 is a key component of the leaf unfolding mechanism in tobacco. Plant Journal 37, 147–155. Sjo¨vall‐Larsen, S., Alexandersson, E., Johansson, I., Karlsson, M., Johanson, U. and Kjellbom, P. (2006). Purification and characterization of two protein kinases acting on the aquaporin SoPIP2;1. Biochimica and Biophysica Acta 1758, 1157–1164. Steudle, E. (1989). Water flow in plants and its coupling to other processes: An overview? Methods in Enzymology 174, 183–225. Steudle, E. (1993). Pressure probe techniques: Basic principles and application to studies of water and solute relations at the cell, tissue and organ level. In ‘‘Water Deficits: Plant Responses from Cell to Community’’ (J. A. C. Smith and H. GriYths, eds.), pp. 5–35. Bios Scientific Publishers Ltd, Oxford, UK. Steudle, E. (1994). The regulation of plant water at the cell, tissue, and organ level: Role of active processes and of compartmentation. In ‘‘Flux Control in

134

O. POSTAIRE ET AL.

Biological Systems. From Enzymes to Populations and Ecosystems’’ (E. D. Schultze, ed.), pp. 237–299. Academic Press, San Diego, CA. Steudle, E. (2000). Water uptake by roots: EVects of water deficit. Journal of Experimental Botany 51, 1531–1542. Steudle, E. and Peterson, C. A. (1998). How does water get through roots. Journal of Experimental Botany 49, 775–788. Steudle, E. and Tyerman, S. D. (1983). Determination of permeability coeYcients, reflection coeYcients, and hydraulic conductivity of Chara corallina using the pressure probe: EVects of solute concentrations. Journal of Membrane Biology 75, 85–96. Steudle, E., Smith, J. A. C. and Lu¨ttge, U. (1980). Water‐relation parameters of individual mesophyll cells of the crassulacean acid metabolism plant Kalanchoe¨ daigremontiana. Plant Physiology 66, 1155–1163. Sui, H., Han, B., Lee, J., Walian, P. and Jap, B. (2001). Structural basis of water‐ specific transport through the AQP1 water channel. Nature 414, 872–878. Sun, M. H., Xu, W., Zhu, Y. F., Su, W. A. and Tang, Z. C. (2001). A simple method for in situ hybridization to RNA in guard cells of Vicia Faba L.: The expression of aquaporins in guard cells. Plant Molecular Biology Reporter 19, 129–135. Tajkhorshid, E., Nollert, P., Jensen, M., Miercke, L., O’Connell, J., Stroud, R. and Schulten, K. (2002). Control of the selectivity of the aquaporin water channel family by global orientational tuning. Science 296, 525–530. Takano, J., Wada, M., Ludewig, U., Schaaf, G., von Wiren, N. and Fujiwara, T. (2006). The Arabidopsis major intrinsic protein NIP5;1 is essential for eYcient boron uptake and plant development under boron limitation. Plant Cell 18, 1498–1509. Tang, A. C. and Boyer, J. S. (2002). Growth‐induced water potentials and the growth of maize leaves. Journal of Experimental Botany 53, 489–503. Temmei, Y., Uchida, S., Hoshino, D., Kanzawa, N., Kuwahara, M., Sasaki, S. and Tsuchiya, T. (2005). Water channel activities of Mimosa pudica plasma membrane intrinsic proteins are regulated by direct interaction and phosphorylation. FEBS Letters 579, 4417–4422. Terashima, I. and Ono, K. (2002). EVects of HgCl2 on CO2 dependence of leaf photosynthesis: Evidence indicating involvement of aquaporins in CO2 diVusion across the plasma membrane. Plant and Cell Physiology 43, 70–78. Terskikh, V. V., Feurtado, J. A., Ren, C., Abrams, S. R. and Kermode, A. R. (2005). Water uptake and oil distribution during imbibition of seeds of western white pine (Pinus monticola Dougl. ex D. Don) monitored in vivo using magnetic resonance imaging. Planta 221, 17–27. Tomos, A. D., Steudle, E., Zimmermann, U. and Schulze, E. D. (1981). Water relations of leaf epidermal cells of Tradescantia virginiana. Plant Physiology 68, 1135–1143. Tornroth‐Horsefield, S., Wang, Y., Hedfalk, K., Johanson, U., Karlsson, M., Tajkhorshid, E., Neutze, R. and Kjellbom, P. (2006). Structural mechanism of plant aquaporin gating. Nature 439, 688–694. Tournaire‐Roux, C., Sutka, M., Javot, H., Gout, E., Gerbeau, P., Luu, D. T., Bligny, R. and Maurel, C. (2003). Cytosolic pH regulates root water transport during anoxic stress through gating of aquaporins. Nature 425, 393–397. Tsuda, M. and Tyree, M. T. (2000). Plant hydraulic conductance measured by the high pressure flow meter in crop plants. Journal of Experimental Botany 51, 823–828.

AQUAPORINS IN PLANTS

135

Tyerman, S. D. and Niemietz, C. M. (2002). Plant aquaporins: Multifunctional water and solute channels with expanding roles. Plant Cell and Environment 25, 173–194. Tyerman, S. D., Oats, P., Gibbs, J., Dracup, M. and Greenway, H. (1989). Turgor‐ volume regulation and cellular water relations of Nicotiana tabacum roots grown in high salinities. Australian Journal of Plant Physiology 16, 517–531. Tyerman, S. D., Bohnert, H. J., Maurel, C., Steudle, E. and Smith, J. A. (1999). Plant aquaporins: Their molecular biology, biophysics and significance for plant water relations. Journal of Experimental Botany 50, 1055–1071. Tyree, M. T., Nardini, A., Salleo, S., Sack, L. and El Omari, B. (2005). The dependence of leaf hydraulic conductance on irradiance during HPFM measurements: Any role for stomatal response? Journal of Experimental Botany 56, 737–744. Uehlein, N., Lovisolo, C., Siefritz, F. and KaldenhoV, R. (2003). The tobacco aquaporin NtAQP1 is a membrane CO2 pore with physiological functions. Nature 425, 734–737. Vander Willigen, C., Postaire, O., Tournaire‐Roux, C., Boursiac, Y. and Maurel, C. (2006). Expression and inhibition of aquaporins in germinating Arabidopsis seeds. Plant and Cell Physiology 47, 1241–1250. Vera‐Estrella, R., Barkla, B. J., Bohnert, H. J. and Pantoja, O. (2004). Novel regulation of aquaporins during osmotic stress. Plant Physiology 135, 2318–2329. Verbavatz, J. M., Brown, D., Sabolı´c, I., Valenti, G., Ausiello, D. A., Vanhoek, A. N., Ma, T. and Verkman, A. S. (1993). Tetrameric assembly of CHIP28 water channels in liposomes and cell membranes. A freeze‐fracture study. Journal of Cell Biology 123, 605–618. Verkman, A. S. (2000). Water permeability measurement in living cells and complex tissues. Journal of Membrane Biology 173, 73–87. Veselova, T. V., Veselovskii, V. A., Usmanov, P. D., Usmanova, O. V. and Kozar, V. I. (2003). Hypoxia and imbibition injuries to aging seeds. Russian Journal of Plant Physiology 50, 835–842. Wallace, I. S. and Roberts, D. M. (2004). Homology modeling of representative subfamilies of Arabidopsis major intrinsic proteins. Classification based on the aromatic/arginine selectivity filter. Plant Physiology 135, 1059–1068. Wallace, I. S. and Roberts, D. M. (2005). Distinct transport selectivity of two structural subclasses of the nodulin‐like intrinsic protein family of plant aquaglyceroporin channels. Biochemistry 44, 16826–16834. Wallace, I. S., Wills, D. M., Guenther, J. F. and Roberts, D. M. (2002). Functional selectivity for glycerol of the nodulin 26 subfamily of plant membrane intrinsic proteins. FEBS Letters 523, 109–112. Wan, X., Steudle, E. and Hartung, W. (2004). Gating of water channels (aquaporins) in cortical cells of young corn roots by mechanical stimuli (pressure pulses): EVects of ABA and of HgCl2. Journal of Experimental Botany 55, 411–422. Wan, X. C. and Zwiazek, J. J. (1999). Mercuric chloride eVects on root water transport in aspen seedlings. Plant Physiology 121, 939–946. Weig, A., Deswarte, C. and Chrispeels, M. J. (1997). The major intrinsic protein family of Arabidopsis has 23 members that form three distinct groups with functional aquaporins in each group. Plant Physiology 114, 1347–1357. Weig, A. R. and Jakob, C. (2000). Functional identification of the glycerol permease activity of Arabidopsis thaliana NLM1 and NLM2 proteins by heterologous expression in Saccharomyces cerevisiae. FEBS Letters 481, 293–298. Westgate, E. and Steudle, E. (1985). Water transport in the midrib tissue of maize leaves: Direct measurement of the propagation of changes in cell turgor across a plant tissue. Plant Physiology 78, 183–191.

136

O. POSTAIRE ET AL.

Yasui, M., Hazama, A., Kwon, T. H., Nielsen, S., Guggino, W. B. and Agre, P. (1999). Rapid gating and anion permeability of an intracellular aquaporin. Nature 402, 184–187. Ye, Q. and Steudle, E. (2006). Oxidative gating of water channels (aquaporins) in corn roots. Plant and Cell Environment 29, 459–470. Ye, Q., Wiera, B. and Steudle, E. (2004). A cohesion/tension mechanism explains the gating of water channels (aquaporins) in Chara internodes by high concentration. Journal of Experimental Botany 55, 449–461. Yu, X., Peng, Y. H., Zhang, M. H., Shao, Y. J., Su, W. A. and Tang, Z. C. (2006). Water relations and expression analysis of plasma membrane intrinsic proteins in sensitive and tolerant rice during chilling and recovery. Cell Research 16, 599–608. Zardoya, R. and Villalba, S. (2001). A phylogenetic framework for the aquaporin family in eukaryotes. Journal of Molecular Evolution 52, 391–404. Zhang, W. H. and Tyerman, S. D. (1991). EVect of low O2 concentration and azide on hydraulic conductivity and osmotic volume of the cortical cells of wheat roots. Australian Journal of Plant Physiology 18, 603–613. Zhang, W. H. and Tyerman, S. D. (1999). Inhibition of water channels by HgCl2 in intact wheat root cells. Plant Physiology 120, 849–858. Zhu, F., Tajkhorshid, E. and Schulten, K. (2002). Pressure‐induced water transport in membrane channels studied by molecular dynamics. Biophysical Journal 83, 154–160. Zhu, F., Tajkhorshid, E. and Schulten, K. (2004). Theory and simulation of water permeation in aquaporin‐1. Biophysical Journal 86, 50–57. Zimmermann, H. M. and Steudle, E. (1998). Apoplastic transport across young maize roots: EVect of the exodermis. Planta 206, 7–19. Zimmermann, U., Hu¨sken, D. and Schulze, E. D. (1980). Direct turgor pressure measurements in diVerent leaf cells of Tradescantia virginiana. Planta 149, 445–453. Zwieniecki, M. A., Melcher, P. J. and Holbrook, M. (2001). Hydrogel control of xylem hydraulic resistance in plants. Science 291, 1059–1062.

Iron Dynamics in Plants

JEAN‐FRANC ¸ OIS BRIAT

Biochimie et Physiologie Mole´culaire des Plantes, Centre National de la Recherche Scientifique (UMR 5004), Institut National de la Recherche ´ cole Nationale Supe´rieure Agronomique, Universite´ Montpellier 2, E d’Agronomie, 2 Place Viala, F‐34060 Montpellier Cedex 2, France

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . II. Iron Mobilization After Germination. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Metabolic‐Induced Dynamics of Iron After Germination: An Example ..................................................................... B. Evidences for Seed Iron Mobilization After Germination ............... C. Molecular Aspects of Seed Iron Remobilization After Germination.............................................................. III. Iron Acquisition and Circulation During Vegetative Growth . . . . . . . . . . . . . . A. Plant/Soil/Microorganisms Interactions in the Rhizosphere............. B. Iron Uptake by Roots ......................................................... C. Long Distance Iron Circulation Within the Plant......................... D. Subcellular Compartmentation of Iron ..................................... E. Cross Talks Between Iron and Zinc Uptake and Compartmentation in Plants.................................................. IV. Iron and Photosynthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Photosynthesis Impairment due to Iron Deficiency ....................... B. Overview of Heme and Fe–S Cluster Biosynthesis in Chloroplasts .... V. Iron and Reproduction in Plants. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Iron During Flowering ........................................................ B. Iron Unloading and Storage in Seeds ....................................... VI. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Advances in Botanical Research, Vol. 46 Incorporating Advances in Plant Pathology Copyright 2008, Elsevier Ltd. All rights reserved.

138 140 140 141 141 143 143 146 153 155 157 158 158 160 164 164 165 167 168

0065-2296/08 $35.00 DOI: 10.1016/S0065-2296(07)46004-9

138

J.‐F. BRIAT

ABSTRACT Iron dynamics in plants start by complex interactions between plants and the rhizospheric microflora, which determine the amount of available iron for uptake by root transporters. Under iron‐deficient conditions, two types of high aYnity transport systems are activated, depending on the plant family considered. In nongrass plants, Fe(III)‐chelate reduction is followed by Fe(II) uptake, whereas in grasses, Fe(III) chelated to secreted phytosiderophores (PSs) is taken up by roots. Long distance allocation of iron between organs and tissues, as well as its subcellular compartmentation and remobilization, also involve various chelation and reduction activities, associated to transporters and to soluble proteins storing and buVering this metal. This iron traYcking at the whole plant, cellular, and subcellular levels, is a highly regulated process starting to be characterized at a molecular level. To maintain iron homeostasis is an important determinant to build up prosthetic groups such as heme and Fe–S clusters, and to assemble them into apoproteins. Such processes require complex protein machineries which are located in mitochondria and plastids. An essential and plant‐specific role of these iron dynamics is evidenced by the strong iron requirement for the photosynthetic reaction to take place.

I. INTRODUCTION Iron is an essential element for living organisms because of its physicochemical properties. It is able to form six coordinated links with electron donor atoms such as oxygen or nitrogen (Marschner, 1995). As a consequence, it is found associated to a huge range of metalloprotein active sites, under the form of various prosthetic groups including heme and [Fe–S] clusters. Among its properties, its existence under two redox forms, ferrous (Fe2þ) or ferric (Fe3þ), explains its activity in most of the basic redox reactions required in both production (photosynthesis) and consumption (respiration) of oxygen, consistent with its presence in many proteins of the mitochondrial and chloroplastic electron transfer chains. Iron is also involved in many enzymatic reactions required for nitrogen fixation, DNA, and plant hormones synthesis. At pH 7 in an oxygenated medium, soluble iron concentration is between 1011 and 1010 M (Lindsay and Schwab, 1987), whereas the required iron concentration for a plant in order to make its life cycle ranges from 109 to 108 M (Guerinot and Yi, 1994). Iron being essential to many cellular activities required for optimum growth and development, and, on the other hand, iron being insoluble in aerated soils at neutral or basic pH, occurrence of its deficiency is frequent. It deeply alters the morphology and physiology of plants. The most visible symptom is an interveinal yellowing of the leaves known as chlorosis, resulting from an alteration of both the structure and function of chloroplasts (Briat et al., 1995). Iron reactivity with oxygen has another negative consequence for plants. Iron‐mediated oxidative stress

IRON DYNAMICS IN PLANTS

139

through the Fenton reaction can lead to amino acid oxidation, lipid peroxidation, and DNA mutations, ultimately causing cell death (Briat, 2002). As a consequence of these drawbacks, plants have evolved mechanisms to control iron uptake, transport in various organs, and storage to ensure an optimal development by preventing both iron deficiency and toxicity (Colangelo and Guerinot, 2006). It appears that iron homeostasis in the various plant tissues during growth and development is a dynamic process resulting from an integrated regulation of the expression of the various genes encoding proteins acting in the transport, storage, and utilization of iron (Table I). The main objective of this chapter is to address these dynamics at the various steps of the life cycle of a plant, from seed to seed.

TABLE I Abbreviations of Discussed Genes NA and MA synthesis genes NAS Nicotianamine synthase NAAT Nicotianamine amino transferase DMAS Deoxymugineic acid synthase IDS2 Iron deficiency specific 2 Genes involved in iron uptake ZmYS1 Zea mays yellow stripe 1 FRO Ferric reductase oxidase IRT Iron regulated transporter ZIP Zinc iron protein Genes involved in long distance iron circulation AtYSL A. thaliana yellow stripe like IREG Iron regulated exporter from gut (ferroportin) ITP Iron transport protein FRD Ferric reductase deregulated OPT Oligopeptides transporter Genes involved in intracellular circulation DMT Divalent metal transporter Nramp Natural resistance associated macrophage protein CCC1 S. cerevisiae Ca2þ‐sensitive Cross complementer 1 AtVIT1 A. thaliana vacuolar iron transporter 1 (orthologous to ScCCC) ATM1 S. cerevisiae ABC transporter from mitochondria 1 STA1 Arabidopsis Mitochondrial ABC Transporter orthologous to ATM1 Genes involved in iron homeostasis regulation FIT1 Fe‐deficiency induced transcription factor 1 FRU Fer‐like iron deficiency induced transcription factor (continued)

140

J.‐F. BRIAT TABLE I (continued)

FER

Fe regulator (from tomato; orthologous to Arabidopsis FIT/FRU) OsIRO Oryzae sativa iron related transcription factor DwMYB2 Dendrobium hybrid Woo Leng myeloblastosis 2 BRZ Bronze DGL Degenerative leaves Genes involved in heme and Fe–S cluster synthesis AtFC A. thaliana ferrochelatase NIF Nitrogen fixation ISC Iron–sulfur cluster SUF Mobilization of sulfur HCF High chlorophyll fluorescence AtNAP A. thaliana nonintrinsic ABC protein LAF6 Long after far‐red 6 AtFH A. thaliana frataxin Photosynthetic genes RBCS Ribulose‐1,5‐biphophate carboxylase small subunit RBCL Ribulose‐1,5‐biphophate carboxylase large subunit PS Photosystem PSI‐K Photosystem I kinase LHC Light harvesting complex CRD1 Copper response defect Miscellaneous genes GFP Green fluorescent protein SDH Succinate dehydrogenase AtMTP3 A. thaliana metal tolerant protein 3

II. IRON MOBILIZATION AFTER GERMINATION Dry seeds contain iron in variable amounts (see Section V.B). This stored iron is important immediately after germination step, before the seedlings have developed their roots, enabling them to acquire minerals from the environment. In addition, iron is a key element for photosynthesis (see Section IV). Therefore, the mobilization of seed iron at the germination to the young seedlings is a major process for plants to successfully switch from autotrophy to heterotrophy.

A. METABOLIC‐INDUCED DYNAMICS OF IRON AFTER GERMINATION: AN EXAMPLE

Metabolic activity at, and immediately after, germination requires reprogramming of many activities, among which iron proteins are important. An example has been obtained by looking at the expression of the three nuclear genes SDH2‐1, SDH2‐2, and SDH2‐3, which encode the essential iron–sulfur subunit

IRON DYNAMICS IN PLANTS

141

of mitochondrial complex II in Arabidopsis thaliana (Elorza et al., 2006). These authors have shown that the SDH2‐3 gene is specifically expressed during seed development. SDH2‐3 transcripts appear during seed maturation, persist through dessication, are abundant in dry seeds, and markedly decline during germination. In contrast to SDH2‐3 transcripts, SDH2‐1 and SDH2‐2 transcripts are barely detected in dry seeds and increase during germination and postgerminative growth. These data strongly suggest that during germination the embryo‐specific SDH2‐3 is replaced by SDH2‐1 or SDH2‐2 in mitochondrial complex II. Therefore, Fe–S cluster biogenesis and assembly into these de novo synthesized apoproteins must occur, illustrating one aspect of the dynamics of iron during the germination process. B. EVIDENCES FOR SEED IRON MOBILIZATION AFTER GERMINATION

Indeed, more than 30 years ago, TiYn and Chaney (1973) produced soybean plants treated from anthesis to seed maturity with radiolabeled 59Fe. It enabled these authors to determine that seed coats accounted for 7% of dry seed weight and had Fe concentrations five times greater than the embryos. Cotyledons of two‐day‐old seedlings, contained 70% and radicle 5% of original seed Fe. These data clearly demonstrated that dry seed iron was remobilized for the needs of seedlings’ early development. At that time it was hypothesized that two iron fractions (in addition to Fe enzymes) were present at the early stages of seedling growth. The first one is the mobile chelated form(s) of iron, which is physiologically important for the developing seedling, and which was hypothesized to be Fe3þ‐citrate (TiYn and Chaney, 1973). However, confirmation that citrate is the major long distance shuttle during iron remobilization from stores during early growth is still lacking, and whether or not other chelates than iron‐citrate are involved in this process is still an unanswered question. The second, and the principal, iron fraction was supposed to be ferritin iron, since the iron storage protein ferritin was already known to be present in ungerminated pea cotyledons (Hyde et al., 1963). C. MOLECULAR ASPECTS OF SEED IRON REMOBILIZATION AFTER GERMINATION

1. Ferritins: Plastid iron storage proteins Ferritins are a class of universal 24‐mer multimeric proteins, present in all living kingdoms and able to accommodate few thousands of iron atoms in their central cavity (Harrison and Arosio, 1996; Lobreaux et al., 1992). In plants, these proteins are located in plastids, and they play various roles related to iron homeostasis during development (among which storage of a part of iron into seeds) or in response to environmental stresses (for review see

142

J.‐F. BRIAT

Briat et al., 2006). Hyde et al. (1963) proposed that ferritin reserves in the cotyledons could be mobilized for young seedlings axis development. Such a function is consistent with the observation that ferritin and iron concentration in pea cotyledons decrease progressively during the first week after germination. Ferritin in the embryo axis was processed, and disappeared, after germination, within the first 4 days of radicle and epicotyl growth (Lobreaux and Briat, 1991). This degradation of ferritin in vivo was marked by a shortening of a 28 kDa subunit, giving 26.5 and 25 kDa polypeptides, reminiscent of the radical damage occurring in pea seed ferritin during iron exchange in vitro (Laulhere et al., 1989). However, confirmation that seed ferritin iron is indeed a major iron source for seedling early development remains to be worked out. 2. Natural resistance associated macrophage protein (Nramp) 3–4 and vacuole iron transporter (VIT1): Iron vacuole transporters Other compartments than the plastids, where ferritins are localized, could also play a role in iron storage. Indeed, when nongreen cultured soybean cells were treated by 100 or 500 mM ferric citrate, an 11‐ and 28‐fold increase in the total intracellular iron concentration and a 30‐ and 60‐fold increase in the ferritin concentration, respectively, were observed (Lescure et al., 1990). However, the percentage of iron stored in the mineral core of ferritin remained constant whatever the ferric citrate concentration used to induce ferritin synthesis, and represented only 5% of the cellular iron. This led the authors to postulate that the vacuoles could play a major role in iron storage. Nramp proteins are a ubiquitous family of metal transporters that includes mammalian Nramp2/divalent cation transporter 1 (DCT1)/divalent metal ion transporter 1 (DMT1), which has a broad substrate range (Gunshin et al., 1997) and functions in intestinal Fe uptake (Cellier and Gros, 2004). Likewise, plant Nramp family members have been implicated in the transport of several divalent cations, including Fe (Bereczky et al., 2003; Curie et al., 2000; Kaiser et al., 2003; Thomine et al., 2000). In A. thaliana, AtNramp3 and AtNramp4 are induced under Fe deficiency. AtNramp3 and AtNramp4 exhibit the same expression patterns and they are both targeted to the vacuole membrane. Single nramp3 and nramp4 null mutants lack obvious phenotype, suggesting a functional redundancy. A major observation was recently reported showing an arrest in the germination of nramp3 nramp4 double mutants under low Fe nutrition (Lanquar et al., 2005). This defect was fully rescued by high Fe supply. A wild‐type Fe content was measured in the seeds of the double mutant, indicating that it was not aVected in metal storage. However, the use of electron microscopy coupled to iron imaging of inelastically scattered electrons enabled to show

IRON DYNAMICS IN PLANTS

143

that iron remained associated to the vacuole globoids in nramp3 nramp4 double mutant, whereas it is eYciently mobilized from the globoids in wild‐ type germinating seeds. These results indicate that vacuole globoids represent an important site for Fe storage in A. thaliana. They also suggest that AtNramp3 and AtNramp4 are required to retrieve globoid‐associated iron after germination in order to make stored iron available for seedling development, until eYcient systems for Fe acquisition from the soil take over. Whether this process is universal or not in the plant kingdom remains an open question, since a huge variability do exist between plant species in terms of ferritin iron content and globoids number (Lobreaux and Briat, 1991; Lott et al., 1984). However, consistent with a critical role of vacuole iron remobilization in Arabidopsis during the early steps of seedling growth, is the recent report that VIT1 required for iron loading into seed vacuoles, is essential for seedling development, as vit1‐1 seedlings grow poorly when iron is limiting (Kim et al., 2006).

III. IRON ACQUISITION AND CIRCULATION DURING VEGETATIVE GROWTH Once seedlings have developed enough, they can take up iron from their environment by their roots, and allocate it to the various organs and tissues of plants in order for them to grow in an optimal manner. A major parameter for iron nutrition concerns its bioavailability in the rhizosphere, which is the result of complex interactions between the soil, the microorganisms it contains, and the plant itself which can influence its environment. A. PLANT/SOIL/MICROORGANISMS INTERACTIONS IN THE RHIZOSPHERE

It is not the objective of this section to give an exhaustive review of our current knowledge about iron status in soil. However, most of the molecular studies concerning iron uptake by roots are performed under simplified conditions. They use plants grown hydroponically or in vitro on Petri dishes, with simplified medium in which iron is most often brought chelated to EDTA, ethylenediamine di‐(O‐hydroxyphenylacetic) acid (EDDHA), or citrate. It appears important, therefore, to draw the attention of the readers to upstream iron uptake mechanisms by plants. The bioavailability of this metal is a major parameter which takes place in a very complex environment, the rhizosphere, which has been defined as the part of soil surrounding the root, being directly or indirectly influenced by it, and bearing a strong microbial activity (Fig. 1).

144

J.‐F. BRIAT

Shoot

Rhizospheric interactions (soil/plant/microbes)

FRD3

Phloem

YSLs ? Fe(III)-ITP Fe(II)-NA

Fe(III)-citrate

Xylem

AtFRO6 ?

?

Pyoverdin Bioavailable iron

IRT1 YS1

Root

Fig. 1. Iron uptake from the rhizosphere and distribution to the various plant tissues. Iron availability to the roots depends on complex interactions in the rhizosphere between soil physicochemical properties, plant exudates, and microbes. Among them, bacteria produces siderophores, including the pyoverdins from Pseudomonas, who are likely to strongly influence iron plant nutrition. Once taken up by YS1 in grasses and the FRO/IRT system in nongrasses, long distance iron traYcking between plant organs and tissues occurs. Iron is loaded within the xylem sap where Fe(III) is chelated to organic acids, among which citrate is the major one, and is transported to the shoot part of the plant by the transpiration stream. The Ferric Reductase Defective 3 (FRD3) gene encodes an iron‐regulated root transmembrane protein of the MATE family, which is involved in citrate loading of the xylem. Unloading of the xylem has not been yet fully described at a molecular level, but could involve AtFRO6, a member of the Ferric Reductase Oxidase (FRO) family. Loading of the phloem, which contains iron transport peptides (ITP) chelating Fe(III) and NA chelating Fe(II) is an important process for iron delivery to sink tissues (roots, seeds, young leaves), and likely for long distance signaling and control of the root uptake system. YSL genes are good candidates to code for transporters involved in phloem loading (adapted from Briat, 2006).

1. Iron in soils Soils are usually described as a three‐phase system consisting of solid, soluble, and exchangeable phases. Each of these phases is connected by dissolution, exchange, and diVusion mechanisms that control movements of Fe. Iron availability is determined by both its absolute concentration and its partitioning between these three phases. Iron is the fourth most abundant element in the earth’s crust, found in most rocks as primary minerals in

IRON DYNAMICS IN PLANTS

145

ferromagnesium silicate forms like olivine, augite, hornblende, or biotite (Louet, 1986). Primary soil minerals are generally unstable in soils and slowly weather in the presence of water and oxygen, to release Fe(II) and Fe(III) ions. These iron ions will precipitate in presence of hydroxyl ions to give insoluble secondary minerals such as oxides (magnetite, hematite, ferrihydrite) and hydroxides (goethite, lepidocrocite), which are the two most abundant iron forms in soils. The concentration of iron in the soluble phase of soils is determined by the rate of dissolution versus precipitation of oxides and hydroxides according to the reaction Fe(OH)3$Fe(III) þ 3OH. The equilibrium state of this reaction is strongly dependent of the pH of the soil solution. Iron concentration in solution decreases when pH increases, reaching a minimum for pH values ranging from 7.4 to 8.5, corresponding to the pH of most of the cultivated soils (Lindsay and Schwab, 1982). Furthermore, iron solubility is also influenced by (1) redox conditions of the soil, (2) properties of the solid mineral: solubility increasing as particle sizes decrease, and (3) presence of organic matter: organic chelates such as bacterial siderophores (Neilands et al., 1987), plant root exudates (Takagi, 1976), or humic substances (Stevenson, 1994) increasing iron solubility. 2. Impact of plants and microbes on iron availability in soils As described above, iron availability in soils is dependent of redox and chelation events, with the passage from ferric iron to soluble ferrous iron, and the ability to form complexes or chelates with organics or mineral products. Chemical properties of the rhizosphere are known to diVer from those of uncultivated soil, as the result of various processes induced by plant roots and/or rhizosphere microbes (Hinsinger et al., 2005). In the rhizosphere, plant–microorganism interactions can be described as a feedback loop during which (1) plant roots secrete exudates that modify the rhizospheric environment; (2) these changes are sensed by the rhizospheric microbial populations; (3) selection of adapted microbial population to the new rhizospheric environment occurs, leading to a modification of the structure and activities of the microbial community; (4) in turn, these variations will influence plant growth and physiology, leading to changes in plant root exudates, and so on (Lemanceau et al., 2006). It is in this context of complex permanent dynamics that iron available to the root uptake system is produced. The stiV competition for iron which occurs between plants and microbes in the rhizosphere can be positive for plants. Contribution of soil microflora to plant iron nutrition has been recently documented (Masalha et al., 2000; Rroco et al., 2003). More specifically, possible positive eVects of pyoverdine, the major class of siderophores

146

J.‐F. BRIAT

produced by fluorescent Pseudomonas, on plant iron nutrition has been well documented. It was reported that pyoverdine could correct iron chlorosis of plants and improve their shoot iron content (Jurkevitch et al., 1988; Sharma et al., 2003). Iron chelated to the bacterial siderophore is potentially directly acquired by the plant (Bar‐Ness et al., 1991; Walter et al., 1994a), through a mechanism not yet characterized at a molecular level (Vansuyt et al., 2007). Other microbial siderophores such as ferrioxamine and rhodothorulic acid have also been reported to contribute to plant iron nutrition (Cline et al., 1984; Crowley et al., 1988; Ho¨rdt et al., 2000; Siebner‐Freibach et al., 2003; Wang et al., 1993; Yehuda et al., 1996). B. IRON UPTAKE BY ROOTS

Under iron suYcient condition, a low aYnity uptake system has been described in Saccharomyces cerevisiae involving the Fet4p transporter (Dix et al., 1994, 1997). Such system has not yet been reported in plants. Indeed, in these organisms, only high aYnity iron transport systems induced in response to iron deficiency have been documented in the last decade, by a wealth of cellular and molecular informations. It is possible that these systems also play a role in iron uptake under suYcient conditions. Importantly, iron uptake from the soil in response to deficiency conditions occurs through diVerent systems in grasses and in nongrass plants (Fig. 1). 1. In grasses In response to iron deficiency, grasses induce the synthesis of strong Fe(III) ligands belonging to the mugineic acids (MAs) family, their secretion in the rhizosphere, and the uptake by a specific transporter of the chelates they form with Fe(III) (Ma et al., 1995; Shojima et al., 1990; Takagi, 1976; Von Wiren et al., 1994). MAs are synthesized from a structurally related precursor called nicotianamine (NA), found in all plants, and result from the condensation of three S‐adenosyl methionine molecules. The fact that MAs have been described only in grasses is due to the existence of specific enzymes in these plant species, able to convert NA to MA. The nicotianamine amino transferase (NAAT) is one of these key enzymes which catalyzes the amino transfer of NA. NAAT activity is dramatically induced by Fe deficiency and suppressed by Fe resupply. Two NAAT cDNA from barley were identified and characterized (Takahashi et al., 1999). They encode proteins belonging to the aminotransferase family, sharing consensus sequences for the pyridoxal phosphate‐binding site. Another major enzyme in this pathway is the deoxymugineic acid synthase (DMAS). DMAS genes from rice, barley, wheat, and maize have been recently characterized. Their nucleotide sequences indicate

IRON DYNAMICS IN PLANTS

147

they encode predicted polypeptides ranging from 314 to 318 amino acids, belonging to the aldo‐keto reductase superfamily (Bashir et al., 2006). All of these proteins show DMA synthesis activity in vitro. Their enzymatic activities are highest at pH 8–9, consistent with the hypothesis that DMA could be synthesized in subcellular vesicles. Although no molecular determinants of the secretion pathway of MAs have been reported so far, the vesicle hypothesis is consistent with the results obtained from a rice cDNA microarray analysis (Negishi et al., 2002). In this study, transcript levels of genes for calmodulin, translation initiation factor 4A2, ras‐related small GTP‐binding protein (GTPase), and ADP‐ribosylation factor 1 (ARF1) increased both in response to iron deficiency conditions and before sunrise, and gradually decreased after sunrise. These diurnal changes in transcript levels support the idea that these genes could be involved in the diurnal secretion of MAs. Regulation of these genes, and unpublished results, led these authors to postulate that MAs would be synthesized in rER‐derived vesicles localized to the cell boundaries and secreted in the rhizosphere by a polar vesicle transport process, involving ARF and Rab1 GTPase proteins. Diurnally regulated transcription of calmodulin could serve this scenario through two alternative pathways: (1) by interactions of kinesin‐like calmodulin‐binding protein and cytoskeletal microtubules (Vos et al., 2000); or (2) by regulating the Kþ release that occurs simultaneously with MAs secretion, since MAs are secreted in the form of a monovalent anion via an anion channel using the Kþ gradient between the cytoplasm and the cell exterior (Sakaguchi et al., 1999). The Fe(III)–MA transport system, specific of the iron‐deficiency response in grasses, has been characterized by using maize as a model grass organism. The maize ys1 mutant carries a monogenic recessive mutation, responsible for a defect in the transport of the Fe(III)–MA through the root plasma membrane. In this mutant, MA synthesis and secretion is normal (Von Wiren et al., 1994). The maize YS1 gene has been cloned (Curie et al., 2001). It encodes a putative transmembrane protein containing 12 hydrophobic domains, and an E rich N‐terminal region containing a specific motif (REGLE) known for its interaction with Fe(III) (Fig. 1). It has been expressed in the fet3fet4 yeast mutant strain which is deficient in low and high aYnity iron transport activities, and bears a growth defect under low iron conditions. Under these conditions, ZmYS1 restored growth to this yeast mutant strain (Curie et al., 2001; Roberts et al., 2004). Electrophysiological analysis of ZmYS1 expressed in Xenopus oocytes demonstrated that Fe–PS transport depends on proton cotransport and the membrane potential (Schaaf et al., 2004). It allows ZmYS1‐mediated transport even at alkaline pH, and this could be an explanation why grasses are less susceptible to

148

J.‐F. BRIAT

chlorosis that nongrass plants. An intriguing output of this work was the discovery by sequence database mining that eight A. thaliana genes share important sequence similarities with maize YS1, although A. thaliana does not produce MAs. These genes were named AtYSLs, and it was hypothesized that they could be involved in metal–NA traYcking inside dicotyledonous plants (Curie et al., 2001). Recently, in barley, the HvYS1 gene was cloned and the protein it encodes had the same characteristic as ZmYS1, although it seemed to have a metal specificity restricted to Fe3þ; in addition, in situ hybridization analysis of iron‐deficient barley roots revealed that the HvYS1 mRNA was localized in epidermal root cells, as well as the protein as shown by immunohistological staining using an anti‐HvYS1 polyclonal antibody (Murata et al., 2006). 2. In nongrasses a. A. thaliana. In the nongrass model plant A. thaliana, iron deficiency induces acidification of the rhizosphere through an uncharacterized Hþ‐ ATPase. As evoked above, such an acidification helps iron oxide solubilization. Resulting Fe(III)‐chelates are then reduced by a specific Fe(III)‐chelate reductase to generate Fe(II) (Marschner, 1995). The Arabidopsis FRO2 gene encodes this protein. Its cDNA was cloned by a PCR approach using degenerated oligonucleotides derived from the yeast FRE reductase sequences (Robinson et al., 1999). The FRO2 gene is allelic to frd1‐1, one out of three Arabidopsis mutants (frd1‐1, frd1‐2, frd1‐3) that do not show induction of Fe(III)‐chelate reductase activity under iron‐deficient conditions. It confirms that iron must be reduced prior to its transport and that Fe3þ‐reduction can be uncoupled from proton release (Yi and Guerinot, 1996). FRO2 encodes a 725‐amino acid protein with eight putative transmembrane domains, and shares similarities with human phagocytic NADPH gp91phox oxydoreductase and with the yeast ferric chelate reductases. Like gp91phox and yeast FREp, FRO2 contains binding sites for heme and for nucleotide cofactors, consistent with its function in electron transfer from cytosolic NADPH to extracellular Fe3þ. The FRO2‐generated Fe2þ is then taken up by iron regulated transporter 1 (IRT1) which is the major root Fe(II) transporter activated under iron‐deficient conditions (Fig. 1). IRT1, is the founder of a new eukaryotic transporter family denominated zinc iron protein (ZIP), and exhibiting a large substrate specificity, since in addition to iron it is able to transport Zn, Cd, Co, and Mn, but not Cu (Eide et al., 1996; Guerinot 2000; Rogers et al., 2000). It is predicted to contain eight transmembrane domains, and it is located at the plasmalemma of root epidermal cells. It was shown to be essential for plant growth and development in Arabidopsis by the characterization of IRT1‐KO lines (Vert et al., 2002).

IRON DYNAMICS IN PLANTS

149

b. Tomato. Tomato is also a nongrass model plant used to study Fe uptake, likely because of the existence of mutants such as chloronerva and fer (Ling et al., 1996), which were known and characterized from a physiological point of view, well before A. thaliana was used in this field of research. Tomato genes and cDNAs belonging to the FRO and ZIP (IRT) family, and potentially involved in the root high aYnity iron transport system for Fe(II) uptake, have been isolated and characterized. LeFRO1 encodes an Fe(III)‐chelate reductase protein, based on its ability to increase such an enzymatic activity when expressed in yeast (Li et al., 2004). It is targeted to the plasma membrane, and its transcript is observed in roots, leaves, cotyledons, flowers, and young fruits. Furthermore, LeFRO1 gene expression is activated in response to iron deficiency in roots but not in leaves where it is constitutively expressed. Whether or not LeFRO1 is the AtFRO2 orthologue is unknown since no loss‐of‐function mutant is available for this gene. Concerning the transporter, two tomato IRT cDNA (LeIRT1 and LeIRT2) have been isolated from a library constructed from roots of iron‐deficient tomato plants, using the IRTI Arabidopsis iron transporter cDNA, as a probe (Eckhardt et al., 2001). When expressed in the yeast mutant fet3fet4, aVected in low and high aYnity iron transport, both LeIRT1 and LeIRT2 restored the growth of this strain on low iron, as well as iron uptake. The expression of these genes was analyzed at the transcript level. Both genes were predominantly expressed in roots, where transcription of LeIRT2 was unaVected by the iron status of the plant, in contrast to LeIRT1 expression which was strongly enhanced in response to iron deficiency (Bereczky et al., 2003; Eckhardt et al., 2001). Based on these results, the LeIRT1 gene can be postulated to be the orthologue of the AtIRT1 gene, but as for the LeFRO1 gene, functional genetic data are still lacking to prove it. c. Legumes. In legumes, iron uptake and homeostasis is recently receiving a particular attention because of the richness of their seed iron content, compared to cereals for example (Laulhere et al., 1988; Marentes and Grusak, 1998). This is an important property in the context of iron biofortification of animal and human food (Theil and Briat, 2004). Plants from the legume family also take up iron through Fe3þ reduction and Fe2þ transporters. For example, in pea, the FR01 gene encodes a protein 55% identical to Arabidopsis FRO2 (Waters et al., 2002) which displays a ferric chelate reductase activity when expressed in yeast, and which likely represents the pea reductase involved in root iron acquisition. The PsFRO1‐generated Fe(II) is then taken up by pea roots through a transporter encoded by the RIT1 gene which is upregulated in iron deficiency and encodes a protein 63% identical to AtIRT1. PsRIT1 complements both the fet3fet4 and zrt1zrt2

150

J.‐F. BRIAT

yeast mutants, thus potentially mediating high aYnity Fe and Zn uptake. In the model legume plant Medicago truncatula, six members of the ZIP gene family have been characterized (Lopez‐Millan et al., 2004). However, based on their expression pattern in roots and in leaves, and in response to various metal deficiencies, including iron, it is so far not easy to predict which of them could be the AtIRT1 orthologue. DiVerent from other dicotyledonous plants, legume plants specifically develop a symbiosis with some soil bacteria enabling nitrogen fixation. This symbiotic process takes place in specific root structures, the nodules, within the cortical cells of which bacteria evolve into bacteroids which are able to reduce atmospheric nitrogen into ammonia. This process requires some essential iron‐containing proteins such as nitrogenase and leghemoglobin (Tang et al., 1990). Iron uptake by the bacteroids has to be viewed as a potential iron uptake mechanism for legumes, since part of iron in soybean seed could originate from nodules (Burton et al., 1998). Iron uptake inside the nodule requires three activities. First, Fe(III)–organic acid chelates are transported across the peribacteroidal membrane to accumulate within the peribacteroidal space (Levier et al., 1996; Moreau et al., 1995) where they will be exchanged with bacterial‐type siderophores (Wittenberg et al., 1996). Second, Fe(III)‐chelate reductases are active at the peribacteroidal membrane and uptake of Fe(III) in isolated symbiosomes is stimulated by NADH (Levier et al., 1996). Third, Fe(II) is also transported across the peribacteroidal membrane (Moreau et al., 1998) likely through the GmDMT1 transporter (Kaiser et al., 2003), which belongs to the Nramp family of metal transporters. 3. Regulation of the expression of plant high aYnity iron transport systems a. Regulation of the IRT/FRO system in nongrasses plants. Regulation of the root high aYnity iron uptake system from Arabidopsis was investigated through monitoring FRO2 and IRT1 gene expression (Vert et al., 2003). Recovery from iron‐deficient conditions, and modulation of apoplastic iron pools indicated that iron itself plays a major role in the regulation of root iron deficiency responses at the mRNA and protein levels. Split‐root experiments showed that the expression of IRT1 and FRO2 could be controlled both by a local induction from the root iron pool, and through a systemic pathway involving an as yet uncharacterized shoot‐borne signal; both signals are integrated in order to tightly control production of the root iron uptake proteins. Very recently, interesting findings by Lucena et al. (2006) opened new insights in the regulation of the nongrass iron uptake system. These authors have shown that ethylene could be involved in the regulation of

IRON DYNAMICS IN PLANTS

151

IRT1 and FRO2. They propose that the iron that controls FRO2 and IRT1 expression would not be the total root iron pool, as suggested by Vert et al. (2003), but rather the phoem iron. This hypothesis is based on the fact that the frd3 Arabidopsis mutant (see Section C: Long distance iron circulation within the plant), which has constitutively upregulated FRO2 and IRT1 expression, is able to accumulate enough symplastic iron in its root when grown under Fe‐suYcient conditions, as suggested by the observation of ferritin accumulation in this genetic background (Green and Rogers, 2004). At the molecular level, a major breakthrough occurred when the tomato FER gene was cloned by map‐based cloning (Ling et al., 2002). The fer mutant fails to activate iron deficiency responses and the tomato LeIRT1 and LeFRO1 homologue genes of the Arabidopsis AtIRT1 and AtFRO2 genes, are not expressed in response to iron deficiency in this genetic background (Bereczky et al., 2003; Li et al., 2004). The FER gene encodes a transcription factor belonging to the basic helix–loop–helix (bHLH). It is targeted to plant nuclei, and when fused to the GAL4 DNA‐binding domain, it is able to transactivate the transcription of the lacZ reporter gene placed under the GAL4 cis‐acting responsive element in yeast (Brumbarova and Bauer, 2005). It therefore strongly suggests that FER, indeed acts as a transcription regulator in plant. The FER gene is itself controlled by the iron status of the plant. It is downregulated under high iron concentration (100 mM) in the culture medium, through both transcriptional and posttranscriptional pathways (Brumbarova and Bauer, 2005). Databases mining enabled to identify a FER gene homologue in the Arabidopsis genome, which was named BHLH029 or FRU, for FER‐like regulator of iron uptake (Jakoby et al., 2004). FRU is induced by iron deficiency, and fru mutant plants are chlorotic and unable to induce expression of the AtIRT1 and AtFRO2 genes in response to iron deficiency. Expression of FRU in transgenic fer tomato mutant plant complements this mutation, and restores all the iron deficiency responses, proving that the FRU gene is the orthologue of the FER gene (Yuan et al., 2005). At the same time and independently, using a diVerent approach, Colangelo and Guerinot (2004) identified the same bHLH transcription factor that they named FIT1 for Fe‐deficiency Induced Transcription Factor 1. FIT1 and FRU are therefore the same protein. FRU/FIT 1 is required for regulating the Fe(III) chelate reductase FRO2 at the level of steady state mRNA accumulation, and by controlling protein accumulation of the Fe(II) transporter IRT1. Microarray analysis of the fit1 mutant reveals that it is involved in the regulation of many genes implicated in iron homeostasis, as well as many novel genes.

152

J.‐F. BRIAT

In addition to the transcriptional regulation of IRT1 and FRO2 genes in response to iron deficiency, an additional control of the steady state amount of the proteins encoded by these genes occurs (Connolly et al., 2002, 2003). Indeed, transgenic Arabidopsis plants transformed with a p35SCAMV::IRT1 or a p35SCAMV::FRO2 construct overexpress IRT1 and FRO2 transcripts whatever the iron nutrition conditions of the plants may be, whereas the corresponding proteins are accumulated only under iron deficiency conditions. These results clearly indicate that IRT1 and FRO2 expression are also regulated at a posttransductional level. The molecular basis of this control has not been reported so far.

b. Regulation of iron uptake in grasses. The barley IDS2 gene is highly expressed in roots in response to iron deficiency and it is likely to encode a 2‐oxoglutarate‐dependent dioxygenase which hydroxylates the C‐3 positions of MA to deoxy‐MA. It is therefore an important gene for PS biosynthesis in grasses in response to iron limitation (Nakanishi et al., 2000). The barley IDS2 promoter was analyzed in transgenic tobacco (Kobayashi et al., 2003). Deletion analysis revealed that a region between 272 and 91 from the translational start site was necessary and suYcient to express the beta‐ glucuronidase (GUS) reporter gene in tobacco roots. Further deletions and linker scanning analysis of this region enabled to identify two cis‐acting elements named iron deficiency responsive elements 1 and 2 (IDE1 and IDE2), which synergistically induced Fe‐deficiency‐specific expression in tobacco roots. Sequences homologous to IDE1 are present in many promoters of genes regulated in response to iron deficiency, including those coding for NAAT, and NA synthase from barley and rice, and for A. thaliana FRO2 and IRT1. These results suggest that IDE1 and 2 could be important for the regulation of various iron‐responsive genes, both in grass and nongrass plants. No trans‐acting factor interacting with IDE1 and 2 have been reported so far. The question of whether or not a bHLH transcription factor, such as FIT/ FRU in Arabidopsis or FER in tomato, could be involved in the iron‐ regulated expression of genes in grass plants has recently been documented. A transcriptome analysis using a rice 22k oligo‐DNA microarray has revealed a putative bHLH transcription factor gene, named OsIRO2, strongly expressed in both roots and shoots, specifically in response to a Fe deficiency stress (Ogo et al., 2006). An in silico search revealed that IRO2 is highly conserved among graminaceous plants. The cis‐acting sequence bound by IRO2 was determined, and is found upstream of several genes known to be involved in Fe acquisition, such as OsNAS1, OsNAS3, OsIRT1, OsFDH, OsAPT1, and IDS3.

IRON DYNAMICS IN PLANTS

153

C. LONG DISTANCE IRON CIRCULATION WITHIN THE PLANT

Iron circulation to and from the various organs and tissues of plants require its loading in and unloading from the xylem and phloem saps. 1. Through the xylem Organic acids, and especially citrate, are the main metal chelators in the xylem, and this has been well documented (Cataldo et al., 1988; Ouerdane et al., 2006). Active root transporters are likely necessary to load iron from the root cortex cells to the xylem. However, such eZux iron transporters are still uncharacterized at the molecular level in plants. A mammalian duodenal protein encoded by the iron regulated exporter from gut 1 (IREG1) gene is an iron‐regulated transporter involved in the basolateral eZux of iron from epithelial cell to the circulation (McKie et al., 2000). IREG isologs being present in the Arabidopsis genome, they are therefore putative candidates for loading vessels with iron. However, it is unlikely for the only Arabidopsis gene, AtIREG2, characterized so far. When expressed in yeast, this gene exhibited no iron transport function, but enhanced tolerance to elevated concentrations of nickel at acidic pH (Schaaf et al., 2006). Consistent with these data obtained in a heterologous system, transgenic plants overexpressing AtIREG2 showed an increased tolerance to elevated concentrations of nickel, whereas T‐DNA insertion lines lacking AtIREG2 expression were more sensitive to nickel, particularly under iron deficiency, and accumulated less nickel in roots. AtIREG2 expression is coregulated with AtIRT1 by the transcription factor FRU/FIT1, and a role in vacuolar substrate transport was supported by localization of AtIREG2–green fluorescent protein (GFP) fusion proteins to the tonoplast in Arabidopsis suspension cells and root cells of intact plants. Besides IREG genes, the FRD3 gene could also play a role in xylem iron loading in Arabidopsis. FRD3 encodes a protein predicted to be a member of the multidrug and toxin eZux (MATE) family, and is expressed only in the root (Rogers and Guerinot, 2002). The frd3 mutant of Arabidopsis is chlorolotic and constitutively expresses its iron uptake responses. It has been recently published that ferric reductase deregulated 3 (FRD3) is likely to be a citrate eZuxer responsible for loading this Fe(III) chelator into the root xylem sap (Durrett et al., 2007; Fig. 1). Once in the leaves, Fe(III)‐citrate is likely to be the substrate of leaf ferric chelate reductase, since such an enzymatic activity has been described in leaf mesophyll cells (Bruggemann et al., 1993). Whether some of the FRO genes could be involved in this process in Arabidopsis (Robinson et al., 1999) remains to be clearly established. However, it is very likely, since

154

J.‐F. BRIAT

Feng et al. (2006) have reported that the AtFRO6 gene is expressed in leaves, where it is light regulated in a tissue‐ or cell diVerentiation‐specific manner to facilitate iron acquisition (Fig. 1). In pea, such a function could be achieved by the PsFRO1 gene which is expressed in leaves (Waters et al., 2002). Unloading of iron from the xylem is also very less documented. Recently, AtYSL2, one of the maize YS1 homologues in Arabidopsis has been characterized and could be a Fe(II)–NA transporter in nongrass plants (DiDonato et al., 2004). It is expressed in the vasculature of both roots and leaves, at the level of xylem‐associated cells, and its transcript amount decreases in response to iron deficiency. Based on imaging of AtYSL2::GFP in transgenic Arabidopsis plants, this transporter is located at the plasmalemma of xylem parenchyma cells, exclusively at the edges of these cells, and not at their apical or basal ends. Such a localization implies that AtYSL2 could move Fe(II)–NA complex laterally within the veins of both leaves and roots. The major physiological role for AtYSL2 would be, therefore, to take up iron that has arrived in tissues via xylem transport, thus moving it away from the xylem vessels. 2. Through the phloem Mobility of iron from source to sink tissues via the phloem sap is poorly documented. It is nevertheless well established that the phloem sap contains Fe (Stephan et al., 1994) arising from its mobilization in source organs (Grusak 1995). One of the molecules identified initially as a potential phloem metal‐transporter is the NA (Stephan and Scholz, 1993). However, according to a later work of the same group, NA would participate in the loading of iron into the phloem, but not in its transport (Schmidke et al., 1999). The OsYSL2 gene, 1 out of the 18 putative OsYSL rice genes is expressed in leaves in response to iron deficiency, at the level of the phloem vessels (Koike et al., 2004). OsYSL2 fused to the GFP localizes at the plasmalemma, and when expressed in Xenopus oocytes it is able to transport Fe(II)–NA but not Fe(III)– NA nor Fe(III) deoxy‐MA. It is therefore hypothesized that OSYSL2 is required in the long distance transport of Fe(II)–NA in the phloem. Iron has also been suggested to travel in the phloem of Ricinus communis in a ferric complex with a phloem protein named iron transport protein (ITP) (Krueger et al. 2002; Fig. 1). An ITP cDNA has been cloned and encodes a 96‐amino acid protein belonging to the late embryogenesis abundant (LEA) family. No transporter for ITP and/or ITP–Fe(III) loading or unloading of the phloem have been reported so far. 3. Regulation of long distance iron translocation The signaling pathways and regulatory molecules involved in the control of iron allocation in plants are unknown. The recent discovery that an orchid (Dendrobium hybrid) transcription factor of the R2R3 Myb family

IRON DYNAMICS IN PLANTS

155

(DwMYB2) induces iron deficiency when ectopically expressed in A. thaliana prompted the authors to characterize this DwMYB2 transgenic plant (Chen et al., 2006). They observed that the iron content in DwMYB2 transgenic root is twofold higher compared to that in wild‐type root, whereas the reverse characterizes the shoot. This imbalance of iron content between root and shoot suggests a defect in the translocation of iron from root to shoot in response to the expression of the orchid DwMYB2 in Arabidopsis. It is consistent with the fact that expression of known iron‐regulated genes is deregulated in DwMYB2 transgenic plants. The ferric‐chelate reductase gene, AtFRO2, and the iron transporter genes, AtIRT1 and AtIRT2, are upregulated in response to DwMYB2 expression, while other potential iron transporters such as AtIREG1, AtFRD3, and Nramp1 are downregulated. These observations have been done by overexpressing ectopically a heterologous transcription factor in Arabidopsis. Furthermore, no closely related orthologue of the orchid DwMYB2 gene in Arabidopsis can be simply identified by sequence comparison, ruling out the possibility to analyze a loss‐of‐function mutant. Nevertheless, Chen and coworkers observation opens the possibilty that one of the 125 known R2R3 Myb transcription factors from Arabidopsis could be involved in iron metabolism regulation, and more precisely in the control of iron long distance circulation between root and shoot. D. SUBCELLULAR COMPARTMENTATION OF IRON

Very little information is presently available concerning intracellular iron dynamics inside plant cells (Fig. 2). Iron uptake by mitochondria and chloroplast is necessary to furnish iron to the respiratory and photosynthetic apparatus. Chloroplasts iron uptake is light dependent, requires a Fe(III)‐ chelate reductase activity (Bughio et al., 1997), and occurs through an inward‐directed Fe(II) transport mediated by a potential‐stimulated uniport mechanism (Shingles et al., 2002). Among the ZIP, Nramp, yellow stripe like (YSL) (Curie and Briat, 2003), and still uncharacterized gene families, it is likely that some members will encode proteins involved in iron transport across the membranes of these various plant cell organelles. Concerning iron uptake by organelles, only one transporter has been recently shown to play such a role. The Arabidopsis orthologue of the yeast CC1 transporter is responsible for moving Fe into the plant vacuole (Grotz and Guerinot, 2006; Kim et al., 2006). In yeast, CCC1 (Ca2þ‐sensitive cross‐complementer) encodes a transporter that can mediate the transport of Fe and Mn into vacuoles (Li et al., 2001). Arapidopsis VIT1 is 58% similar to CCC1, and the expression of VIT1 in a ccc1 yeast mutant rescues the ability of the mutant to grow in the presence of high Fe.

156

J.‐F. BRIAT

VIT1 V

N

Fe(II)

NRAMP3/4

? Ferritin Heme (FC) and Fe–S (SUF system) biogenesis P

?

Frataxin Heme (FC) and Fe–S (NFU and ISC) biogenesis M

[Fe–S]

STA1

Fig. 2. Intracellular compartmentation of iron into plant cells. VIT1 is an iron transporter involved in loading iron into seed vacuoles (V). Iron stored within seed vacuoles is remobilized by Nramp3 and 4 transporters belonging to the Nramp family. The plastids (P), among which chloroplasts support photosynthesis activity, are iron rich. This plastid iron is acquired through a Fe(II) transporter, which has not been yet characterized at a molecular level. The iron storage protein ferritin is located within plastids, and plays an important role to buVer iron in its ferric form, avoiding its toxicity. It is unknown how iron is taken up by mitochondria (M), which contain frataxin as iron store. Both plastids and mitochondria support heme and Fe–S cluster synthesis in plant cells. FC is a key enzyme found in both compartments and responsible for inserting Fe(II) into protoporphyrin IX, the precursor of heme and chlorophyll. The Fe–S biogenesis machinery diVers between the two organelles. The plastids contain a complete bacterial SUF‐like system, whereas mitochondria posses a universal ISC/NFU system reminiscent of the yeast system. The STA1 ABC transporter, homologous to the yeast Atm1p transporter, is required to export [Fe–S] cluster from mitochondria to cytosol. N: nucleus (adapted from Briat, 2006).

Once iron is loaded inside organelles, if it is not used, it needs to be stored. Iron storage proteins, the ferritins, store and buVer iron inside the plastids (Briat et al., 1999), and these proteins are also likely to be present in the mitochondria (Zancani et al., 2004). Moving iron outside from cellular organelles has been documented at the molecular level in two cases. Remobilization of iron from vacuolar iron stores during germination require the Nramp3 and Nramp 4 transporters (see Section II.C.2, Lanquar et al., 2005). Also, iron eZux from Arabidopsis mitochondria, could occur through the protein encoded by the STA1 gene, a homologue of the yeast ATM1p (Kushnir et al., 2001). It encodes an

IRON DYNAMICS IN PLANTS

157

ABC‐transporter located at the mitochondrial inner membrane, and could be required to export Fe–S clusters from the mitochondrial matrix to the cytoplasm (Kispal et al., 1999; Lill and Kispal, 2000). E. CROSS TALKS BETWEEN IRON AND ZINC UPTAKE AND COMPARTMENTATION IN PLANTS

In Arabidopsis, the ferrous iron transporter IRT1 is also able to transport other metals, including zinc (Vert et al., 2002). Because accumulation of zinc in plants is toxic, it is therefore logical that IRT1 gene expression is repressed when high amounts of zinc are present in the medium (Connolly et al., 2002). The root ferric chelate reductase FRO2 is not known to play a role in zinc uptake or homeostasis. However, as for the IRT1 transcript, the FRO2 transcript is undetectable in roots when plants are grown on iron‐deficient medium supplemented with 500 M zinc, confirming that zinc, in addition to iron, mediates coordinate control of FRO2 and IRT1 transcript abundance (Connolly et al., 2003). The fact that the IRT1 RNA is present in the roots of p35SCAMV::IRT1 transgenic plants grown on iron‐deficient medium supplemented with 500 M Zn supports the hypothesis that the repression of IRT1 RNA levels in wild‐type plants in response to high zinc is mediated through the IRT1 promoter (Connolly et al., 2002). In case of iron deficiency, zinc uptake through IRT1 could be potentialy toxic and zinc excess has to be handled by plants. The AtMTP3 gene contributes to Zn tolerance in Arabidopsis, and its expression and function are integrated with those of the iron uptake machinery (Arrivault et al., 2006). When expressed in yeast, AtMTP3 enhances Zn tolerance and accumulation. In plants, its overexpression leads to an increased Zn tolerance, whereas MTP3 RNAi lines exhibit an increased sensitivity to Zn overload. AtMTP3 is strongly induced by Zn excess and by iron deficiency, as IRT1. It is expressed in roots and located at the tonoplast. The upregulation of MTP3 expression under Fe deficiency enables the root cells to detoxify the incoming Zn ions through IRT1, and their sequestration into the vacuoles of root cortex and epidermis cells restricts the movement of Zn to the shoot. In grasses, PS are secreted in response to Fe deficiency, and this plant family could therefore use this chelation strategy to take up Zn from the soil (Welch, 1995). Indeed, Zn–PS complexes have been demonstrated to be taken up by maize (Von Wiren et al., 1996), and it has been suggested that an increase in PS extrusion under Zn‐deficient conditions can occur (Cakmak et al., 1996). However, the reproducibility of this PS extrusion seems to be largely dependent on the growth conditions (Pedler et al., 2000), and Walter et al. (1994b) hypothesized that impairment of Fe transport from the roots to

158

J.‐F. BRIAT

the shoot under Zn‐deficient conditions results in Fe deficiency, leading to the secretion of MAs. Indeed by measuring the concentration of Fe, the pattern of ferritin expression, and the translocation of iron, Suzuki et al. (2006) concluded that Zn‐deficient barley plants are not deficient in Fe. Furthermore, more Zn(II)–DMA than Zn2þ is absorbed in Zn‐deficient barley plants, suggesting that deoxy‐MA secreted as a result of Zn deficiency is eVective in absorbing Zn from the soil. Conflicting results also concern the role that the Fe(III)–PS transporter YS1 could play in mediating the transport of Zn–PS complexes (Roberts et al., 2004; Schaaf et al., 2004).

IV. IRON AND PHOTOSYNTHESIS A. PHOTOSYNTHESIS IMPAIRMENT DUE TO IRON DEFICIENCY

Photosynthesis is one of the major plant‐specific process, and chloroplasts are the primary place of iron utilization in plants, since they contain 80% of a leaf cell iron content (Smith, 1984). In A. thaliana, 70% of the iron in green tissue is in chloroplasts, and 40% is found in the thylakoids (Shikanai et al., 2003). Indeed, the photosynthetic apparatus contains 21–22 iron atoms, making it one of the more iron demanding cellular system. It can explain why chloroplasts and the photosynthetic activity are among the major targets aVected by iron deficiency. The characteristic symptom of iron deficency in plants is chlorosis, an interveinal yellowing due to a decrease in chlorophyll content of plant aerial parts. Indeed, various steps of pigment metabolism are iron dependent. Iron is required to form the chlorophyll biosynthesis precursor aminolevulinic acid (Pushnik et al., 1984), and for the oxidation reactions leading coproporphyrinogene III to protoporphyrinogene IX and Mg‐protoporphyrin monomethyl ester to protochlorophyllide (Spiller et al., 1982). In addition, it has been nicely shown that the di‐iron enzyme CHL27 is involved in the synthesis of protochlorophyllide (Tottey et al., 2003). Iron deficiency also impacts chlorophyll degradation by increasing it (Terry and Zayed, 1995). The quantity of carotenoids decreases when iron is short (Val et al., 1987), likely due to the iron requirement of phytoene desaturase for its activity to convert phytoene into ‐carotene (Pascal et al., 1995). At an ultrastructural level, a decrease in thylakoid density is observed in chloroplasts from iron‐deficient plant cells (Briat et al., 1995; Stocking, 1975), correlated to a decrease in galactolipid content (Nishio et al., 1985). Furthermore, thylakoid proportion of unsaturated fatty acids diminishes (Abadia et al., 1988; Monge et al., 1993), likely because iron plays a role in

IRON DYNAMICS IN PLANTS

159

linoleic acid conversion to linolenic acid (Pascal and Dorne, 1994). In addition to a decrease in light absorption capacity due to a photosynthetic pigment diminution, and to a chloroplast structure alteration, iron deficiency decreases photochemical capacities. Indeed electron transport capacity through PS II and I are decreased, as well as contents in cytochrome b559, cytochrome f, P700, and ferredoxin (Sharma and Sanwal, 1992; Spiller and Terry, 1980; Stocking, 1975). Some stroma enzymes involved in CO2 fixation have their synthesis and/or activities altered in response to iron deficiency. Ribulose‐1,5‐bisphosphate carboxylase (RUBISCO) amount and activity is decreased (Arulanantham et al., 1990; Terry, 1980), as well as the abundance of rbcS and rbcL transcripts (Spiller et al., 1987; Winder and Nishio, 1995). Furthermore, ribulose‐1,5‐bisphosphate regeneration in the Calvin cycle is perturbed in iron‐deficient sugarbeet, because of a decrease in ribulose‐5‐phosphate kinase activity (Arulanantham et al., 1990). More recently, a thylakoid sugar beet proteome analysis revealed that iron deficiency induced significant changes in the polypeptide profile of the photosynthetic membranes (Andaluz et al., 2006). Indeed, the relative amounts of electron transfer protein complexes are reduced, whereas those of proteins involved in leaf carbon fixation are increased. The more advanced study of the impact of iron deficiency on the structure and function of the photosynthetic apparatus has been performed with the green unicellular algae Chamydomonas reinhardtii (Moseley et al., 2002a). Chlamydomonas cell responses to iron deficiency are comparable to those reported for vascular plants. A proteolytically induced loss of photosynthetic components including both photosystems and the cyt b6/f complex make the cells chlorotic. The antenna protein complexes are diVerentially aVected by iron deficiency. Specific components of light harvesting complex I (LHCI) are drastically reduced leading to an overall downregulation of LHCI, whereas light harvesting complex II (LHCII) overall abundance remains fairly constant. The loss of photosystem I (PSI) in response to iron shortage changes the type and quantity of LHCI polypeptides (Hippler et al., 2001; Naumann et al., 2005), highlighting an antenna remodeling program that could be required to bypass the light sensitivity resulting from PSI loss. This adaptation is a sequential process. It starts with uncoupling the antenna from the PSI core and is followed by specific degradation of LHCs and induction of new LHCs, prior to end with assembly of new antenna complexes in Fe‐deficient cells. The initial uncoupling of the LHCI antenna from PSI could be regulated via the photosystem I kinase (PSI‐K) subunit of PSI. PSI‐K accumulation is influenced by the activity of Crd1, a candidate

160

J.‐F. BRIAT

Fe‐containing enzyme in chlorophyll biosynthesis proposed as a key target of plastid Fe deficiency (Moseley et al., 2000, 2002b; Spiller et al., 1982).

B. OVERVIEW OF HEME AND FE–S CLUSTER BIOSYNTHESIS IN CHLOROPLASTS

The major role of iron in the photosynthetic activity is achieved by its association with metalloproteins of the thylakoid electron transfer chain. This association is achieved by two prosthetic groups: heme and iron–sulfur clusters. Their biosynthesis are therefore major components of the fate of iron dynamics in plants (Fig. 2). 1. Heme Heme and chlorophyll are synthesized in the common branched tetrapyrole pathway in plants and algae (Cornah et al., 2002). The terminal enzyme of heme biosynthesis is ferrochelatase (FC), which catalyzes the insertion of ferrous iron into protoporphyrin IX, the last common intermediate of heme and chlorophyll synthesis. FC is thus at the branchpoint between the two pathways, and is a key protein for iron utilization. As such, this enzyme is likely to be important in the regulation of this branchpoint, and may also play a role in the coordination of heme and apoprotein production. In Arabidopsis two FC genes have been isolated by functional complementation of a yeast mutant (Chow et al., 1998; Smith et al., 1994). The AtFCII gene is only expressed in aerial plant tissues and the AtFC‐I gene is expressed in all tissues of the plant (Chow et al., 1998; Singh et al., 2002). AtFC‐I and AtFC‐II are members of two distinct groups of FCs. AtFC‐I groups with barley FC and cucumber FC‐1, and AtFC‐II with cucumber FC‐2, and FCs from rice, potato, and cyanobacteria (Chow et al., 1998; Suzuki et al., 2002). Using in vitro import assays, type II FCs have been shown to be imported into isolated pea chloroplasts, but not into pea mitochondria, whereas all type I FCs can be imported into both organelles with similar eYciency (Chow et al., 1997; Suzuki et al., 2002). However, using a sensitive fluorimetric assay, FC activity was recently quantified in plastids and mitochondria from roots and from green or etiolated leaves of pea plants. More than 90% of the activity was measured associated with plastids, although FC was reproducibly detected in mitochondria, at levels greater than the contaminating plastid marker enzyme (Cornah et al., 2002). It indicates that plastids are the major site of heme biosynthesis in higher plant cells. In leaves it is consistent with the fact that heme in the photosynthetic cytochromes would constitute its major fraction within the cell. In roots, however, the major pool of heme

IRON DYNAMICS IN PLANTS

161

would be outside the plastids, and yet these organelles have some 15 times more FC activity than the mitochondria, evidencing a plastid heme export mechanism. 2. Fe–S clusters Electron flow in the thylakoids requires Fe–S clusters found in cytochrome b6/f complex, ferredoxin and photosystem I (Kapazoglou et al., 2000; Raven et al., 1999). In vivo biogenesis, assembly and repair of Fe–S cluster has been well documented in bacteria, where numerous proteins are required to achieve these processes. Three bacterial systems exist termed NIF (nitrogen fixation), ISC (iron–sulfur cluster), and SUF (mobilization of sulfur) (Loiseau et al., 2003; Takahashi and Tokumoto, 2002). In plants, an output of in vitro import studies of the ferredoxin precursor was to demonstrate the ability of chloroplasts to mature the imported apoprotein into a functional holo‐ferredoxin (Li et al., 1990). Fe–S biogenesis in intact (Takahashi et al., 1986) and lysed (Takahashi et al., 1991a,b) spinach chloroplasts was investigated, revealing the existence of an ATP‐ and light‐ derived NADPH–Fe–S assembly process in the stroma. It is very recently that the molecular actors involved in chloroplast Fe–S biogenesis have been characterized (for review see Balk and Lobre´aux, 2005). Arabidopsis plastids most probably contain a complete SUF system (Xu and Møller, 2004), and homologues of the six bacterial SUF proteins reside in the plastids, whereas the three IscU homologues and the IscS‐like desulfurase are localized to the mitochondria. Therefore, plants have the ability to synthesize Fe–S clusters both in mitochondria and plastids, as for heme biosynthesis. Based on current knowledge of the suf and isc gene products in bacteria, the plastids are likely to contain the Fe–S cluster assembly pathway that is more resistant to oxygen. Genetic approaches have recently enabled to link the chloroplast Fe–S biogenenesis/repair and photosynthesis. The Fe–S scaVold protein NFU2 is required for Fe–S cluster biogenesis of ferredoxin and PSI (Touraine et al., 2004; Yabe et al., 2004), and the universal P‐loop ATPase HCF101 is also necessary for assembly of PSI [4Fe–4S] clusters (Lezhneva et al., 2004). However, some proteins potentially playing a role in Fe–S biogenesis in plastids do not yet have a clear estabished function. For example, it has been proposed that the chloroplast localized IscA protein (SufA homologue) serves as a scaVold in chloroplast Fe–S cluster assembly (Abdel‐Ghany et al., 2005). However, an iscA null mutant bears any significant defect, either in normal plant growth or in biogenesis of major iron–sulfur proteins, indicating that this protein is not essential or redundant for these functions (Yabe and Nakai, 2006).

162

J.‐F. BRIAT

The Fe–S biogenesis machinery of chloroplast is likely to work as proteic complexes, the specificity and regulation of which being achieved through combinatorial interactions, most of them yet to be discovered. Such regulatory interactions have already been documented for the S production from cystein by the chloroplast desulfurase NifS‐like protein, necessary for Fe–S cluster biogenesis (Leon et al., 2002; Pilon‐Smits et al., 2002; Ye et al., 2005), and which is activated by the chloroplast SufE protein (Xu and Møller, 2006; Ye et al., 2006). Another example of such protein–protein interactions to form complexes involved in Fe–S cluster biogenesis comes from our current knowledge of the SUF‐like plastid system. AtNAP7 is a functional ABC/ ATPase and is a plastidic SufC‐like protein (Xu and Møller, 2004). As in bacteria, AtNAP7 is involved in the maintenance and repair of oxidatively damaged Fe–S clusters. Evidence for a SufCSufD complex in plastids has come from protein interaction studies revealing that AtNAP7 can interact with the SufD homologue AtNAP6 (Xu and Møller, 2004). More recently, an Arabidopsis homologue of SufB named AtNAP1 (also known as LAF6 or AtABC1) has been characterized (Xu et al., 2005), and shown to interact with AtNAP7 inside chloroplasts, suggesting the presence of an AtNAP1– AtNAP7–AtNAP6 (SufB–SufC–SufD) complex in Arabidopsis plastids. SufB and SufC may form subunits of an iron‐dependent ABC transporter protein in bacteria. It is therefore tempting to speculate that AtNAP1 and AtNAP7 may act as interacting ATPase subunits involved in iron transport into or within the chloroplast, and subsequently to sites of Fe–S biosynthesis and repair. In this context, and at the diVerence to prokaryotic SufB proteins, it is important to notice that AtNAP1 is an iron‐stimulated ATPase, exhibiting an optimum activity at physiological range of in planta iron concentrations. Furthermore, AtNAP1 transcription is downregulated in response to iron starvation in Arabidopsis (Xu et al., 2005). Therefore, AtNAP1 activity in response to iron is regulated both at the transcriptional and posttranslational level, consistent with the hypothesis that AtNAP1, in conjunction with AtNAP7, could act as a iron‐stimulated sensor fueling Fe–S assembly and repair in response to changes in iron levels in plastids (Xu et al., 2005). A putative role of AtNAP1 in regulating iron homesotasis is indeed consistent with the photomorphogenic phenotype and reduced chlorophyll content of AtNAP1‐deficient laf6 mutant seedlings (Møller et al., 2001). A defective Fe–S cluster formation in laf6 chloroplasts leads to increased iron levels. In turn, a shift in the equilibrium between the heme and chlorophyll pathways during tetrapyrrole biosynthesis may occur. Alternately, the phenotype of laf6 may also be because of tetrapyrrol biosynthetic enzymes requiring Fe–S clusters. Tetrapyrrole intermediates are known to be important for the

IRON DYNAMICS IN PLANTS

163

communication between chloroplasts and the nucleus. Therefore, disordered tetrapyrrole biosynthesis caused by abnormal iron homeostasis may explain the photomorphogenic phenotype of AtNAP1‐deficient plants (Møller et al., 2001).

3. Putative iron donors for heme and Fe–S biogenesis In plants, heme and Fe–S cluster biogenesis can take place in mitochondria and plastids. Two potential iron donors in these subcellular compartments are frataxin and/or ferritin. Frataxin is a highly conserved protein from bacteria to mammals that has been proposed to participate in iron–sulfur cluster assembly and mitochondrial iron homeostasis. The bacterial orthologue of frataxin, CyaY, can establish a specific interaction with IscS, a cysteine desulfurase participating in iron–sulfur cluster assembly. Biochemical analysis showed that the CyaY‐ Fe3þ protein is functional in vitro as an iron donor during [Fe–S] cluster assembly on the scaVold protein IscU in the presence of IscS and cysteine (Layer et al., 2006). These results point out the role of frataxin as iron donor for Fe–S cluster assembly in bacteria, and in yeast and mammals mitochondria (Chen et al., 2002; Huynen et al., 2001). In plants, the AtFH gene product has significant homology to other members of the frataxin family and has a potential N‐terminal targeting peptide for its mitochondrial localization. When expressed in yeast it can complement the yeast frataxin mutation. AtFH is expressed in many plant organs with high levels in flowers (Busi et al., 2004). Whether or not plant frataxin is directly involved as iron donor in Fe–S biogenesis in mitochondria remains to be determined. However, a very recent report strongly suggests that frataxin is an essential protein in plants, required for full activity of mitochondrial Fe–S proteins (Busi et al., 2006). Two atfh knockout mutants from Arabidopsis are embryo lethal. A knockdown mutant exhibiting reduced frataxin mRNA and protein levels has, however, enabled to adress the role of frataxin in Arabidopsis. Two Fe–S mitochondrial enzymes, aconitase and succinate dehydrogenase, have their activities reduced in this mutant, whereas, the activity of malate dehydrogenase, which does not contain a Fe–S moiety, remains unchanged, indicating a possible role for frataxin in Fe–S cluster biogenesis in plant mitochondria. Another potential iron donor for heme and Fe–S biogenesis is the iron storage protein ferritin. In plants, these proteins are mainly located within plastids, where they are supposed to play a key role in buVering iron (Briat et al., 1999). They can accomodate transiently few thousands of

164

J.‐F. BRIAT

iron atoms in a nontoxic form inside the central cavity defined by their 24 subunits proteic shell. Interestingly, plant ferritin could also be present in mitochondria (Zancani et al., 2004).

V. IRON AND REPRODUCTION IN PLANTS A. IRON DURING FLOWERING

Iron is known to be involved in the development of flower colors (Yoshida et al., 2006), but besides this ‘‘beautiful’’ property, iron homeostasis is a key determinent of flower development. The best evidence for such a role came from the study of tobacco plants overexpressing a barley nicotianamine aminotransferase (naat) gene (Takahashi et al., 2003). Because NAAT is the specific enzyme converting NA into MAs in grasses, the consequence of its expression in tobaccco is to induce a shortage in NA in these plants. As a consequencce iron (and other metals) homeostasis is deregulated, and few morphological and developmental consequences have been observed (Takahashi et al., 2003). The naat tobacco inflorescence developed marked morphological abnormalities. The flowers of wild‐type tobacco plants have five petals, and pollen is released before the flower opens. In contrast, naat tobacco plants produce two types of abnormally shaped flowers: one type at an early stage of flowering and a second type at a later stage. At an early flowering stage buds have an aberrant shape, and flowers have bigger and thicker sepals compared to those of the wild‐type plants. At a later stage flowers of naat tobacco plants have some similarities with flowers of wild‐ type tobacco under severe Fe deficiency. The sepals, like those of flowers at an ealier stage of development, are large and thick but have a normal shape. The sepals, stamen filaments, and petals are similar in color, and the anthers produced little pollen. Application of exogenous NA–Fe to naat tobacco plants reverses their flower morphological abnormalities and plenty of pollen is produced, indicating that NA and Fe are required for normal flower development. These observations lead to hypothesize that NA–Fe transport to the flower is an important parameter for normal flowering (Takahashi et al., 2003). It has been postulated that the YSLs genes could be responsible for NA–Fe transport (Curie et al., 2001), and it is supported by the demonstration that maize YS1 can indeed mediates NA–Fe transport (Schaaf et al., 2004). Concerning flowers, it has been recently reported (Le Jean et al., 2005; Waters et al., 2006) that in Arabidopsis the AtYSL1 gene was expressed in

IRON DYNAMICS IN PLANTS

165

the pedicel, sepals, and faintly in petals, as well as in immature anthers, in filaments of mature stamens, and in the vasculature of the pistil. The AtYSL3 gene is also expressed in floral tissues but only in anthers and pollen grains (Waters et al., 2006). A double knockout mutant ysl1ysl3 being able to reach the reproductive stage, it allows to observe that most of the flowers on the mutant plants do not develop siliques. Defective pollen is likely to be responsible for this altered fertility, because viable pollen development failed within the anthers of ysl1ysl3 mutant plants (Waters et al., 2006). These data are in favor of a role of YSL genes in flower development, likely through transport of NA–Fe (and other metals) in this organ. Other genes than the YSLs, also involved in iron and other metals transport, could be involved in flower development. For example, the Fe and Zn that are not complexed by NA could be transported to flower organs by IRT1 because an Arabidopsis IRT1 knockout mutant is sterile and an IRT1 promoter–GUS fusion showed GUS staining in the anther filament (Vert et al., 2002). Finally, some members of the oligopeptides transporter (OPT) gene family, closely phylogenetically related to the YSL genes, could also play a role in this organ development, based on the observation that iron limitation enhances expression of the AtOPT3 gene, which is expressed in pollen (Stacey et al., 2006). B. IRON UNLOADING AND STORAGE IN SEEDS

Iron deficiency is the principal cause of human anemia, and constitute the major nutritional disorder according to the World Health Organisation (http://www.who.int/nut/ida.htm). One of the recommendation of the WHO to tackle this public health problem is to obtain an enrichment of the bioavailable iron in edible parts of plants, which led to the concept of ‘‘Iron Biofortification.’’ Plant grains are one of the major source for mineral nutrients in the human diet, especially in poor countries where anemia is prevailing. However, our present ignorance of the mechanisms controlling plant iron homeostasis is a limit for improving plant iron content, and in particular in seeds. A major seed iron store is the iron storage protein, ferritin (Briat et al., 2006). This observation led few laboratories to overexpress this protein ectopically (Van Wuytswinkel et al., 1999), or under the control of seed‐ specific promoters (Drakakaki et al., 2005; Goto et al., 1999), in order to try to increase seed iron content. Indeed, by creating an ‘‘iron sink’’ leading to an upregulation of the root iron uptake system (Van Wuytswinkel et al., 1999), this approach was successful in increasing seed iron content

166

J.‐F. BRIAT

(Drakakaki et al., 2005; Goto et al., 1999). However, this increase is never higher than two to threefold than the iron content measured in control plants, and no linear relationship was obseved between the level of ferritin expression and the iron content (Qu et al., 2005). Furthermore, this increase in iron content turned out to be very dependant of the physicochemical properties of the soil used to grow the various transgenic plants (Vansuyt et al., 2000). These observations with plants overexpressing ferritin in seeds indicate that a limiting factor does exist for seed iron loading. Indeed, such a statement is in agreement with the fact that the brz pea mutant, which contain 50‐fold more iron in its leaves compared to wild‐type plant leaves, has the same seed iron content than wild‐type seeds (Grusak and Pezeshgi, 1996). However, seed Fe levels can be much higher in the dgl pea mutant, when compared to wild type (Marentes and Grusak, 1998). Recent studies of the YSL gene family, both in rice and Arabidopsis, indicate that some members could be involved in the loading of metals, among which iron, in the developing seeds. This is the case for the Fe–NA OsYSL2 transporter from rice, which is expressed in rice seeds, and could participate to iron seed loading (Koike et al., 2004). In Arabidopsis, AtYSL1 is necessary to determine the NA and iron content in seeds (Le Jean et al., 2005). Furthermore, the impossibilty to restore a wild‐type seed iron content in a ysl1 null mutant by treating it with Fe(III)–EDDHA strengthens the idea that iron speciation in specific chelates is an important parameter to achieve iron seed loading. Not only AtYSL1 is important for seed iron loading but AtYSL3 is also involved in this mechanism, and a double ysl1ysl3 mutant has an altered metal composition in its seeds (Waters et al., 2006). A natural variation of the seed iron content does exist between plant species. For example, cereals have a seed iron content lower than the one measured in grain legumes (Laulhere et al., 1988; Marentes and Grusak, 1998). Such a variability also exists within the same species. For example, the mean value of bean seed iron content is of 55 mg/kg, but some varieties identified by the International Center for Tropical Agriculture are up to 100 mg/kg (http://www.ciat.cgiar.org/beans/mineral_content05.htm). The important quantity of iron measured in grains of legumes compared to other plant species is correlated with an increase in root iron uptake at the early stage of seed development (Lobreaux and Briat, 1991). However, iron can also be remobilized from vegetative organs to seeds. For example in pea, 20–30% of the leaf iron can be translocated to seeds (Grusak, 1994; Hocking and Pate, 1978). In Arabidopsis, an increase in YSL1 and YSL3 gene expression is observed in senescing leaves, as well as a decrease in metal mobilization, including iron, during leaf senescence in the ysl1ysl3 mutant

IRON DYNAMICS IN PLANTS

167

(Waters et al., 2006). These observations led the authors to postulate that YSL family members could be important for recycling of metals from senescing tissues. More precisely, one of the suggested role of AtYSL1 and AtYSL3 gene products could be to translocate metals into vascular parenchyma cells for distribution away from interveinal regions towards phloem tissue in senescing leaves (Waters et al., 2006). In addition to leaf iron remobilization, the specific case of legumes has to be considered. Root nodules have an iron content much higher than other vegetative organs, and an active iron remobilization from senescent nodules to the seeds, could explain why the seed iron content in legumes is higher than the one in seeds of most of the other plant species. Consistent with such a hypothesis, it has been reported that part of seed iron in soybean could originate from nodules (Burton et al., 1998). At a subcellular level, the localization of iron to the provascular strands of the embryo is completely abolished when the vacuolar iron uptake transporter VIT1 is disrupted (Kim et al., 2006), evidencing the role of vacuole iron loading in seed iron content.

VI. CONCLUSION This last decade, a wealth of information describing iron transporters in plants has been obtained. It has enabled to characterize the high aYnity root iron uptake systems, both in grass and nongrass plants. These systems are very eYcient under iron starvation conditions. They could also play a role in the uptake of iron under Fe‐suYcient conditions. Indeed, fit1 or fer knockout mutants do not grow well under Fe‐suYcient conditions, which suggest that ITR1 and FRO2 are required for iron uptake. Furthermore, the IRT1/FRO2 uptake system is induced at diVerent levels depending on the Fe concentration in the external medium, and it is never totally ‘‘oV.’’ Finally, very little is known about iron uptake mechanisms under conditions prevailing within the rhizosphere. There is clearly a need of research in that direction. Although putative transporters involved in iron allocation within the plant have been described, we are still far from a knowledge of their respective roles in major physiological functions such as leaf metabolism and photosynthesis, nodule biology and nitrogen fixation, or iron storage in seeds. Also, a new challenge will be to understand how these activities are integrated at the whole plant level, and what are the signaling networks and regulatory molecules responsible for the control of iron dynamics in plants (Fig. 3).

168

J.‐F. BRIAT

Targets involved in iron utilization (FC, SUF, ISC, NFU,...) Targets involved in iron transport (IRT1, FRO2, YS1 and YSLs)

Iron homeostasis

Targets involved in iron storage (Ferritin, VIT1, NRAMPs)

Signalization regulation (FIT/FRU, FER,...) Genotype Iron status (quantity speciation, compartmentation) Environment (soil, light,…)

Fig. 3. Integration of iron dynamics in plants. The iron status of a plant results from the quantity, the speciation, and the compartmentation of iron. It is determined by the interaction between a given genotype and its environment among which soil and light parameters are crucial determinants. Sensing of the iron status will lead to its signaling in order to regulate target genes involved in the establishment of iron homeostasis. Among regulators, transcription factors belonging to the bHLH family, such as FIT/FRU and FER, have been characterized. Target genes of iron homeostasis can be classified in three groups: (1) genes involved in iron uptake and distribution throughout the plant (IRT1, FRO2, YS1 and YSLs); (2) genes involved in intracellular compartmentation, storage, and remobilization of iron; it can be transporters (VIT1, Nramps) or soluble iron storage proteins (ferritins); (3) genes involved in iron utilization to synthesize and assemble heme (FC) or Fe–S (SUF, ISC, NFU, . . .) prosthetic groups into proteins involved in many metabolic reactions including photosynthesis and respiration.

REFERENCES Abadia, A., Ambrad‐Bretteville, F., Re´my, R. and Tre´moile`res, A. (1988). Iron deficiency in pea leaves: EVect on lipid composition and synthesis. Physiologia Plantarum 72, 713–717. Abdel‐Ghany, S. E., Ye, H., Garifullina, G. F., Zhang, L., Pilon‐Smits, E. A. H. and Pilon, M. (2005). Iron‐sulfur cluster biogenesis in chloroplasts. Involvement of the scaVold protein CpIscA. Plant Physiology 138, 161–172. Andaluz, S., Lopez‐Millan, A. F., De Las Rivas, J., Aro, E. M., Abadia, J. and Abadia, A. (2006). Proteomic profiles of thylakoid membranes and changes in response to iron deficiency. Photosynthesis Research 89, 141–155. Arrivault, S., Senger, T. and Kramer, U. (2006). The Arabidopsis metal tolerance protein AtMTP3 maintains metal homeostasis by mediating Zn exclusion from the shoot under Fe deficiency and Zn oversupply. The Plant Journal 46, 861–879.

IRON DYNAMICS IN PLANTS

169

Arulanantham, A. R., Rao, I. M. and Terry, N. (1990). Limiting factors in photosynthesis. IV. Regeneration of ribulose 1,5‐bisphosphate limits photosynthesis at low photochemical capacity. Plant Physiology 93, 1465–1475. Balk, J. and Lobre´aux, S. (2005). Biogenesis of iron–sulfur proteins in plants. Trends in Plant Science 10, 324–331. Bar‐Ness, E., Chen, Y., Hadar, Y., Marschner, H. and Romheld, V. (1991). Siderophores of Pseudomonas putida as an iron source for dicot and monocot plants. Plant and Soil 130, 231–241. Bashir, K., Inoue, H., Nagasaka, S., Takahashi, M., Nakanishi, H., Mori, S. and Nishizawa, N. K. (2006). Cloning and characterization of deoxymugineic acid synthase genes from graminaceous plants. The Journal of Biological Chemistry 281(43), 32395–32402. Bereczky, Z., Wang, H. Y., Schubert, V., Ganal, M. and Bauer, P. (2003). DiVerential regulation of nramp and irt metal transporter genes in wild type and iron uptake mutants of tomato. The Journal of Biological Chemistry 278, 24697–24704. Briat, J. F. (2002). Metal ion‐activated oxidative stress and its control. In ‘‘Oxidative Stress in Plants’’ (D. Inze´ and M. Van Montagu, ed.), pp. 171–190. Taylor & Francis, London, NewYork. Briat, J. F. (2006). Cellular and whole organism aspects of iron transport and storage in plants. In ‘‘Molecular Biology of Metal Homeostasis and Detoxification from Microbes to Man’’ (L. W. J. Klomp, E. Martinoia and M. J. Tamas, eds.), vol. 14, pp. 193–213. Topics in Current Genetic Series, Springer, Berlin, Heidelberg. Briat, J. F., Fobis‐Loisy, I., Grignon, N., Lobre´aux, S., Pascal, N., Savino, G., Thoiron, S., von Wire´n, N. and Van Wuytswinkel, O. (1995). Cellular and molecular aspects of iron metabolism in plants. Biology of the Cell 84, 69–81. Briat, J. F., Lobreaux, S., Grignon, N. and Vansuyt, G. (1999). Regulation of plant ferritin synthesis: How and why. Cellular and Molecular Life Sciences 56, 155–166. Briat, J. F., Cellier, F. and Gaymard, F. (2006). Ferritins and iron accumulation in plant tissues. In ‘‘Iron Nutrition in Plants and Rhizospheric Microorganisms’’ (L. Barton and J. Abadia, eds.), pp. 345–361. Springer, Berlin, Heidelberg. Bruggemann, W., Maas‐Kantel, K. and Moog, P. R. (1993). Iron uptake by leaf mesophyll cells: The role of the plasma membrane‐bound ferric‐chelate reductase. Planta 190, 151–155. Brumbarova, T. and Bauer, P. (2005). Iron‐mediated control of basic helix‐loop‐helix protein FER, a regulator of iron uptake in tomato. Plant Physiology 137, 1018–1026. Bughio, N., Takahashi, M., Yoshimura, E., Nishizawa, N. K. and Mori, S. (1997). Light‐dependent iron transport into isolated barley chloroplasts. Plant and Cell Physiology 38, 101–105. Burton, J. W., Harlow, C. and Theil, E. C. (1998). Evidence for reutilization of nodule iron in soybean seed development. Journal of Plant Nutrition 21, 913–927. Busi, M. V., Zabaleta, E. J., Araya, A. and Gomez‐Casati, D. F. (2004). Functional and molecular characterization of the frataxin homolog from Arabidopsis thaliana. FEBS Letters 576, 141–144. Busi, M. V., Maliandi, M. V., Valdez, H., Clemente, M., Zabaleta, E. J., Araya, A. and Gomez‐Casati, D. F. (2006). Deficiency of Arabidopsis thaliana frataxin alters activity of mitochondrial Fe–S proteins and induces oxidative stress. The Plant Journal 48(6), 873–882 (doi: 10.1111/j.1365‐313X.2006.02923.x). ¨ ztu¨rk, L., Karanlik, S., Marschner, H. and Ekiz, H. (1996). Zinc‐eYcient Cakmak, I., O wild grasses enhance release of phytosiderophores under zinc deficiency. Journal of Plant Nutrition 19, 551–563.

170

J.‐F. BRIAT

Cataldo, D. A., McFadden, K. M., Garland, T. R. and Wildung, R. E. (1988). Organic constituents and complexation of nickel(II), iron(III), cadmium(II), and plutonium(IV) in soybean xylem exudates. Plant Physiology 86, 734–739. Cellier, M. and Gros, P. (2004). ‘‘The NRAMP Family.’’ Eurekah.com and Kluwer Academic/Plenum, New York. Chen, O. S., Hemenway, S. and Kaplan, J. (2002). Inhibition of Fe‐S cluster biosynthesis decreases mitochondrial iron export: Evidence that Yfh1p aVects Fe‐S cluster synthesis. Proceedings of the National Academy of Sciences USA 99, 12321–12326. Chen, Y. H., Wu, X. M., Ling, H. Q. and Yang, W. C. (2006). Transgenic expression of DwMYB2 impairs iron transport from root to shoot in Arabidopsis thaliana. Cell Research 16, 830–840. Chow, K.‐S., Singh, D. P., Roper, J. M. and Smith, A. G. (1997). A single precursor protein for ferrochelatase‐I from Arabidopsis is imported in vitro into both chloroplasts and mitochondria. The Journal of Biological Chemistry 272, 27565–27571. Chow, K.‐S., Singh, D. P., Walker, A. R. and Smith, A. G. (1998). Two diVerent genes encode ferrochelatase in Arabidopsis: Mapping, expression and subcellular targeting of the precursor proteins. The Plant Journal 15, 531–541. Cline, G. R., Reid, C. C. P., Powell, P. E. and Szaniszlo, P. J. (1984). EVects of hydroxamate siderophore on iron absorption by sunflower and sorghum. Plant Physiology 76, 36–39. Colangelo, E. P. and Guerinot, M. L. (2004). The essential basic helix‐loop‐helix protein FIT1 is required for the iron deficiency response. The Plant Cell 16, 3400–3412. Colangelo, E. P. and Guerinot, M. L. (2006). Put the metal to the petal: Metal uptake and transport throughout plants. Current Opinion in Plant Biology 9, 322–330. Connolly, E. L., Fett, J. P. and Guerinot, M. L. (2002). Expression of the IRT1 metal transporter is controlled by metals at the levels of transcript and protein accumulation. The Plant Cell 14, 1347–1357. Connolly, E. L., Campbell, N. H., Grotz, N., Prichard, C. L. and Guerinot, M. L. (2003). Overexpression of the FRO2 ferric chelate reductase confers tolerance to growth on low iron and uncovers posttranscriptional control. Plant Physiology 133, 1102–1110. Cornah, J. E., Singh, D. P., Roper, J. M. and Smith, A. G. (2002). Measurement of ferrochelatase activity using a novel assay suggests that plastids are the major site of haem biosynthesis in both photosynthetic and nonphotosynthetic cells of pea (Pisum sativum L.). The Biochemical Journal 362, 423–432. Crowley, D. E., Reid, C. C. P. and Szaniszlo, P. J. (1988). Utilization of microbial siderophores in iron acquisition by oat. Plant Physiology 87, 685–688. Curie, C. and Briat, J. F. (2003). Iron transport and signaling in plants. Annual Review of Plant Biology 54, 183–206. Curie, C., Alonso, J. M., Le Jean, M., Ecker, J. R. and Briat, J. F. (2000). Involvement of NRAMP1 from Arabidopsis thaliana in iron transport. The Biochemical Journal 347, 749–755. Curie, C., Panaviene, Z., Loulergue, C., Dellaporta, S. L., Briat, J. F. and Walker, E. L. (2001). Maize yellow stripe1 encodes a membrane protein directly involved in Fe(III) uptake. Nature 409, 346–349. DiDonato, R. J., Jr., Roberts, L. A., Sanderson, T., Eisley, R. B. and Walker, E. L. (2004). Arabidopsis yellow stripe‐like 2 (YSL2): A metal‐regulated gene encoding a plasma membrane transporter of nicotianamine‐metal complexes. The Plant Journal 39, 403–414.

IRON DYNAMICS IN PLANTS

171

Dix, D. R., Bridgham, J. T., Broderius, M. A., Byersdorfer, C. A. and Eide, D. J. (1994). The FET4 gene encodes the low aYnity Fe(II) transport protein of Saccharomyces cerevisiae. The Journal of Biological Chemistry 269, 26092–26099. Dix, D. R., Bridgham, J. T., Broderius, M. A., Byersdorfer, C. A. and Eide, D. J. (1997). Characterization of the FET4 protein of yeast. Evidence for a direct role in the transport of iron. The Journal of Biological Chemistry 272, 11770–11777. Drakakaki, G., Marcel, S., Glahn, R. P., Lund, E. K., Pariagh, S., Fischer, R., Christou, P. and Stoger, E. (2005). Endosperm‐specific co‐expression of recombinant soybean ferritin and Aspergillus phytase in maize results in significant increases in the levels of bioavailable iron. Plant Molecular Biology 59, 869–880. Durrett, T. P., Gassmann, W. and Rogers, E. E. (2007). The FRD3‐mediated eZux of citrate into the root vasculature is necessary for eYcient iron translocation. Plant Physiology 144(1), 197–205. Eckhardt, U., Mas Marques, A. and Buckhout, T. J. (2001). Two iron‐regulated cation transporters from tomato complement metal uptake‐deficient yeast mutants. Plant Molecular Biology 45, 437–448. Eide, D., Broderius, M., Fett, J. and Guerinot, M. L. (1996). A novel iron‐regulated metal transporter from plants identified by functional expression in yeast. Proceedings of the National Academy of Sciences USA 93, 5624–5628. Elorza, A., Roschzttardtz, H., Go´mez, I., Mouras, A., Holuigue, L., Araya, A. and Jordana, X. (2006). A nuclear gene for the iron–sulfur subunit of mitochondrial complex II is specifically expressed during Arabidopsis seed development and germination. Plant and Cell Physiology 47, 14–21. Feng, H., An, F., Zhang, S., Ji, Z., Ling, H. Q. and Zuo, J. (2006). Light‐regulated, tissue‐specific, and cell diVerentiation‐specific expression of the Arabidopsis Fe(III)‐chelate reductase gene AtFRO6. Plant Physiology 140, 1345–1354. Goto, F., Yoshihara, T., Shigemoto, N., Toki, S. and Takaiwa, F. (1999). Iron fortification of rice seed by the soybean ferritin gene. Nature Biotechnology 17, 282–286. Green, L. S. and Rogers, E. E. (2004). FRD3 controls iron localization in Arabidopsis. Plant Physiology 136, 2523–2531. Grotz, N. and Guerinot, M. L. (2006). Molecular aspects of Cu, Fe and Zn homeostasis in plants. Biochimica et Biophysica Acta 1763, 595–608. Grusak, M. A. (1994). Iron transport to developing ovules of Pisum sativum: Seed import characteristics and phloem iron‐loading capacity of sources regions. Plant Physiology 104, 649–655. Grusak, M. A. (1995). Whole‐root iron(III)‐reductase activity throughout the life cycle of iron‐grown Pisum sativum L. (Fabaceae): Relevance to the iron nutrition of developing seeds. Planta 197, 111–117. Grusak, M. A. and Pezeshgi, S. (1996). Shoot‐to‐root signal transmission regulates root Fe(III) reductase activity in the dgl mutant of pea. Plant Physiology 110, 329–334. Guerinot, M. L. (2000). The ZIP family of metal transporters. Biochimica et Biophysica Acta 1465, 190–198. Guerinot, M. L. and Yi, Y. (1994). Iron: Nutritious, noxious, and not readily available. Plant Physiology 104, 815–820. Gunshin, H., Mackenzie, B., Berger, U. V., Gunshin, Y., Romero, M. F., Boron, W. F., Nussberger, S., Gollan, J. L. and Hediger, M. A. (1997). Cloning and characterization of a mammalian proton‐coupled metal‐ion transporter. Nature 388, 482–488.

172

J.‐F. BRIAT

Harrison, P. M. and Arosio, P. (1996). The ferritins: Molecular properties, iron storage function and cellular regulation. Biochimica et Biophysica Acta 1275, 161–203. Hinsinger, P., Gobran, G. R., Gregory, P. J. and Wenzel, W. W. (2005). Rhizosphere geometry and heterogeneity arising from root‐mediated physical and chemical processes. The New Phytologist 168, 266–268. Hippler, M., Klein, J., Fink, A., Allinger, T. and Hoerth, P. (2001). Towards functional proteomics of membrane protein complexes: Analysis of thylakoid membranes from Chlamydomonas reinhardtii. The Plant Journal 28, 595–606. Hocking, P. J. and Pate, J. S. (1978). Mobilization of minerals to developing seeds of legumes. Annals of Botany 41, 1259–1278. Ho¨rdt, W., Ro¨hmeld, V. and Winkelmann, G. (2000). Fusarinines and dimerum acid, mono‐ and dihydroxamate siderophores from Penicillium chrysogenum, improve iron utilization by strategy I and strategy II plants. BioMetals 13, 37–46. Huynen, M. A., Snel1, B.,Bork,P. and Gibson, T. J. (2001). The phylogenetic distribution of frataxin indicates a role in iron‐sulfur cluster protein assembly. Human Molecular Genetics 10, 2463–2468. Hyde, B. B., Hodge, A. J., Kahn, A. and Birnstiel, M. L. (1963). Studies of phytoferritin. I. Identification and localization. The Journal of Ultrastructural Research 9, 248–258. Jakoby, M., Wang, H. Y., Reidt, W., Weisshaar, B. and Bauer, P. (2004). FRU (BHLH029) is required for induction of iron mobilization genes in Arabidopsis thaliana. FEBS Letters 577, 528–534. Jurkevitch, E., Hadar, Y. and Chen, Y. (1988). Involvement of bacterial siderophores in the remedy of lime induced chlorosis peanut. Soil Science Society of America Journal 52, 1032–1038. Kaiser, B. N., Moreau, S., Castelli, J., Thomson, R., Lambert, A., Bogliolo, S., Puppo, A. and Day, D. A. (2003). The soybean NRAMP homologue, GmDMT1, is a symbiotic divalent metal transporter capable of ferrous iron transport. The Plant Journal 35, 295–304. Kapazoglou, A., Mould, R. M. and Gray, J. C. (2000). Assembly of the Rieske iron‐ sulphur protein into the cytochrome bf complex in thylakoid membranes of isolated pea chloroplasts. European Journal of Biochemistry 267, 352–360. Kim, S. A., Punshon, T., Lanzirotti, A., Li, L., Alonso, J. M., Ecker, J. R., Kaplan, J. and Guerinot, M. L. (2006). Localization of iron in Arabidopsis seed requires the vacuolar membrane transporter VIT1. Science 314(5803), 1295–1298 (DOI: 10.1126/science.1132563). Kispal, G., Csere, P., Prohl, C. and Lill, R. (1999). The mitochondrial proteins Atm1p and Nfs1p are essential for biogenesis of cytosolic Fe/S proteins. The EMBO Journal 18, 3981–3989. Kobayashi, T., Nakayama, Y., Itai, R. N., Nakanishi, H., Yoshihara, T., Mori, S. and Nishizawa, N. K. (2003). Identification of novel cis‐acting elements, IDE1 and IDE2, of the barley IDS2 gene promoter conferring iron‐ deficiency‐inducible, root‐specific expression in heterogeneous tobacco plants. The Plant Journal 36, 780–793. Koike, S., Inoue, H., Mizuno, D., Takahashi, M., Nakanishi, H., Mori, S. and Nishizawa, N. K. (2004). OsYSL2 is a rice metal‐nicotianamine transporter that is regulated by iron and expressed in the phloem. The Plant Journal 39, 415–424. Krueger, C., Berkowitz, O., Stephan, U. W. and Hell, R. (2002). A metal‐binding member of the late embryogenesis abundant protein family transports iron

IRON DYNAMICS IN PLANTS

173

in the phloem of Ricinus communis L. The Journal of Biological Chemistry 277, 25062–25069. Kushnir, S., Babiychuk, E., Storozhenko, S., Davey, M. W., Papenbrock, J., De Rycke, R., Engler, G., Stephan, U. W., Lange, H., Kispal, G., Lill, R. and Van Montagu, M. (2001). A mutation of the mitochondrial ABC transporter Sta1 leads to dwarfism and chlorosis in the Arabidopsis mutant starik. The Plant Cell 13, 89–100. Lanquar, V., Lelievre, F., Bolte, S., Hames, C., Alcon, C., Neumann, D., Vansuyt, G., Curie, C., Schroeder, A., Kramer, U., Barbier‐Brygoo, H. and Thomine, S. (2005). Mobilization of vacuolar iron by AtNRAMP3 and AtNRAMP4 is essential for seed germination on low iron. The EMBO Journal 24, 4041–4051. Laulhere, J. P., Lescure, A. M. and Briat, J. F. (1988). Purification and characterization of ferritin from seeds of maize, pea and soyabean. Distribution in various pea organs. The Journal of Biological Chemistry 263, 10289–10294. Laulhere, J. P., Laboure, A. M. and Briat, J. F. (1989). Mechanism of the transition from plant ferritin to phytosiderin. The Journal of Biological Chemistry 264, 3629–3635. Layer, G., Ollagnier de Choudens, S., Sanakis, Y. and Fontecave, M. (2006). Iron‐ sulfur cluster biosynthesis: Characterization of Escherichia coli CYaY as an iron donor for the assembly of [2Fe‐2S] clusters in the scaVold IscU. The Journal of Biological Chemistry 281, 16256–16263. Le Jean, M., Schikora, A., Mari, S., Briat, J. F. and Curie, C. (2005). A loss‐of‐ function mutation in AtYSL1 reveals its role in iron and nicotianamine seed loading. The Plant Journal 44, 769–782. Lemanceau, P., OVre, P., Mougel, C., Gamalero, E., Dessaux, Y., Moenne‐ Loccoz, Y. and Berta, G. (2006). Microbial ecology of the rhizosphere. In ‘‘Microbiological Methods for Assessing Soil Quality’’ (J. Bloem, D. W. Hopkins and A. Benedetti, eds.), pp. 228–230. CABI publishing, Cambridge, Massachussets, USA. Leon, S., Touraine, B., Briat, J. F. and Lobreaux, S. (2002). The AtNFS2 gene from Arabidopsis thaliana encodes a NifS‐like plastidial cysteine desulphurase. The Biochemical Journal 366, 557–564. Lescure, A. M., Massenet, O. and Briat, J. F. (1990). Purification and characterization of an iron‐induced ferritin from soybean (Glycine max) cell suspensions. The Biochemical Journal 272, 147–150. Levier, K., Day, D. A. and Guerinot, M. L. (1996). Iron uptake by symbiosomes from soybean root nodules. Plant Physiology 111, 893–900. Lezhneva, L., Amann, K. and Meurer, J. (2004). The universally conserved HCF101 protein is involved in assembly of [4Fe‐4S]‐cluster‐containing complexes in Arabidopsis thaliana chloroplasts. The Plant Journal 7, 174–185. Li, H. M., Theg, S. M., Bauerle, C. M. and Keegstra, K. (1990). Metal‐ion‐center assembly of ferredoxin and plastocyanin in isolated chloroplasts. Proceedings of the National Academy of Sciences USA 87, 6748–6752. Li, L., Chen, O. S., Ward, D. M. and Kaplan, J. (2001). CCC1 is a transporter that mediates vacuolar iron storage in yeast. The Journal of Biological Chemistry 276, 29515–29519. Li, L., Cheng, X. and Ling, H. Q. (2004). Isolation and characterization of Fe(III)‐ chelate reductase gene LeFRO1 in tomato. Plant Molecular Biology 54, 125–136. Lill, R. and Kispal, G. (2000). Maturation of cellular Fe‐S proteins: An essential function of mitochondria. Trends in Biochemical Sciences 25, 352–356.

174

J.‐F. BRIAT

Lindsay, W. L. and Schwab, A. P. (1982). The chemistry of iron in soils and its availability to plants. Journal of Plant Nutrition 5, 821–840. Ling, H. Q., Pich, A., Scholz, G. and Ganal, M. W. (1996). Genetic analysis of two tomato mutants aVected in the regulation of iron metabolism. Molecular and General Genetics 252, 87–92. Ling, H. Q., Bauer, P., Bereczky, Z., Keller, B. and Ganal, M. (2002). The tomato fer gene encoding a bHLH protein controls iron‐uptake responses in roots. Proceedings of the National Academy of Sciences USA 99, 13938–13943. Lobreaux, S. and Briat, J. F. (1991). Ferritin accumulation and degradation in diVerent organs of pea (Pisum sativum) during development. The Biochemical Journal 274, 601–606. Lobreaux, S., Yewdall, S. J., Briat, J. F. and Harrison, P. (1992). Amino‐acid sequence and predicted three‐dimensional structure of pea seed (Pisum sativum) ferritin. The Biochemical Journal 288, 931–939. Loiseau, L., Ollagnier‐de‐Choudens, S., Nachin, L., Fontecave, M. and Barras, F. (2003). Biogenesis of Fe‐S cluster by the bacterial Suf system: SufS and SufE form a new type of cysteine desulfurase. The Journal of Biological Chemistry 278, 38352–38359. Lopez‐Millan, A. F., Ellis, D. R. and Grusak, M. A. (2004). Identification and characterization of several new members of the ZIP family of metal ion transporters in Medicago truncatula. Plant Molecular Biology 54, 583–596. Lott, J. N. A., Goodchild, D. J. and Graig, S. (1984). Studies of mineral reserves in pea (Pisum sativum) cotyledons using low‐water‐content procedures. Australian Journal of Plant Physiology 11, 459–469. Louet, E. (1986). Le fer. In ‘‘Les Oligoe´le´ments en Agriculture’’ (S. J. Markich, P. L. Brown and R. A. Jeffree, ed.), pp. 45–85. Nathan Ed., Paris. Lucena, C., Waters, B. M., Romera, F. J., Garcia, M. J., Morales, M., Alcantara, E. and Perez‐Vicente, R. (2006). Ethylene could influence ferric reductase, iron transporter, and Hþ‐ATPase gene expression by aVecting FER (or FER‐ like) gene activity. Journal of Experimental Botany 57, 4145–4154. Ma, J. F., Shinada, T., Matsuda, C. and Nomoto, K. (1995). Biosynthesis of phytosiderophores, mugineic acids, associated with methionine cycling. The Journal of Biological Chemistry 270, 16549–16554. Marentes, E. and Grusak, M. A. (1998). Iron transport and storage within the seed coat and embryo of developing seeds of pea (Pisum sativum L.). Seed Science Research 8, 367–375. Marschner, H. (1995). ‘‘Mineral Nutrition of Higher Plants.’’ Academic Press, London. Masalha, J., Kosegarten, H., Elmaci, O. and Mengel, K. (2000). The central role of microbial activity for iron acquisition in maize and sunflower. Biology and Fertility of Soils 30, 433–439. McKie, A. T., Marciani, P., Rolfs, A., Brennan, K., Wehr, K., Barrow, D., Miret, S., Bomford, A., Peters, T. J., Farzaneh, F., Hediger, M. A. Hentze, M. W. et al. (2000). A novel duodenal iron‐regulated transporter, IREG1, implicated in the basolateral transfer of iron to the circulation. Molecular Cell 5, 299–309. Møller, S. G., Kunkel, T. and Chua, N. H. (2001). A plastidic ABC protein involved in intercompartmental communication of light signaling. Genes and Development 15, 90–103. Monge, E., Perez, C., Pequerul, A., Madero, P. and Val, J. (1993). EVect of iron chlorosis on mineral nutrition and lipid composition of thylakoid biomembrane in Prunus persica (L.) Batsch. Plant and Soil 154, 97–102. Moreau, S., Meyer, J. M. and Puppo, A. (1995). Uptake of iron by symbiosomes and bacteroids from soybean nodules. FEBS Letters 361, 225–228.

IRON DYNAMICS IN PLANTS

175

Moreau, S., Day, D. A. and Puppo, A. (1998). Ferrous iron is transported across the peribacteroid membrane of soybean nodules. Planta 207, 83–87. Moseley, J., Quinn, J., Eriksson, M. and Merchant, S. (2000). The Crd1 gene encodes a putative di‐iron enzyme required for photosystem I accumulation in copper deficiency and hypoxia in Chlamydomonas reinhardtii. The EMBO Journal 19, 2139–2151. Moseley, J. L., Allinger, T., Herzog, S., Hoerth, P., Wehinger, E., Merchant, S. and Hippler, M. (2002a). Adaptation to Fe‐deficiency requires remodeling of the photosynthetic apparatus. The EMBO Journal 21, 6709–6720. Moseley, J. L., Page, M. D., Pergam, N., Eriksson, M., Quinn, J., Soto, J., Theg, S., Hippler, M. and Merchant, S. (2002b). Reciprocal expression of two di‐iron enzymes aVecting photosystem I and light‐harvesting complex accumulation. The Plant Cell 14, 673–688. Murata, Y., Ma, J. F., Yamaji, N., Ueno, D., Nomoto, K. and Iwashita, T. (2006). A specific transporter for iron(III)‐phytosiderophore in barley roots. The Plant Journal 46, 563–572. Nakanishi, H., Yamaguchi, H., Sasakuma, T., Nishizawa, N. K. and Mori, S. (2000). Two dioxygenase genes, Ids3 and Ids2, from Hordeum vulgare are involved in the biosynthesis of mugineic acid family phytosiderophores. Plant Molecular Biology 44, 199–207. Naumann, B., Stauber, E. J., Busch, A., Sommer, F. and Hippler, M. (2005). N‐terminal processing of Lhca3 is a key step in remodeling of the photosystem I‐light‐harvesting complex under iron deficiency in Chlamydomonas reinhardtii. The Journal of Biological Chemistry 280, 20431–20441. Negishi, T., Nakanishi, H., Yazaki, J., Kishimoto, N., Fujii, F., Shimbo, K., Yamamoto, K., Sakata, K., Sasaki, T., Kikuchi, S., Mori, S. and Nishizawa, N. K. (2002). cDNA microarray analysis of gene expression during Fe‐deficiency stress in barley suggests that polar transport of vesicles is implicated in phytosiderophore secretion in Fe‐deficient barley roots. The Plant Journal 30, 83–94. Neilands, J. B., Konopka, K., Schwyn, B., Coy, M., Francis, R. T., Paw, H. and Bagg, A. (1987). Comparative biochemistry of microbial iron assimilation. In ‘‘Iron Transport in Animals, Plants and Microorganisms’’. (G. Winkelmann, D. van der Helm and J. B. Neilands, eds.), pp. 3–34. VCH Chemie, Weinheim, Germany. Nishio, J. N., Taylor, S. E. and Terry, N. (1985). Changes in thylakoid galactolipids and proteins during iron nutrition‐mediated chloroplast development. Plant Physiology 77, 705–711. Ogo, Y., Itai, R. N., Nakanishi, H., Inoue, H., Kobayashi, T., Suzuki, M., Takahashi, M., Mori, S. and Nishizawa, N. K. (2006). Isolation and characterization of IRO2, a novel iron‐regulated bHLH transcription factor in graminaceous plants. Journal of Experimental Botany 57, 2867–2878. Ouerdane, L., Mari, S., Czernic, P., Lebrun, M. and Lobinski, R. (2006). Speciation of non‐covalent nickel species in plant tissue extracts by electrospray Q‐TOFMS/MS after their isolation by 2D size exclusion hydrophilic interaction LC (SEC‐HILIC) monitored by ICP‐MS. Journal of Analytical Atomic Spectrometry 21, 676–683. Pascal, N. and Dorne, A. J. (1994). Inhibition of fatty acid desaturation in sycamore cells deprived of iron. FEBS Letters 353, 95–98. Pascal, N., Block, M. A., Pallett, K. E., Joyard, J. and Douce, R. (1995). Inhibition of carotenoids biosynthesis in sycamore cells deprived of iron. Plant Physiology and Biochemistry 33, 97–104.

176

J.‐F. BRIAT

Pedler, J. F., Parker, D. R. and Crowley, D. E. (2000). Zinc deficiency‐induced phytosiderophore release by the Triticaceae is not consistently expressed in solution culture. Planta 211, 120–126. Pilon‐Smits, E. A., Garifullina, G. F., Abdel‐Ghany, S., Kato, S., Mihara, H., Hale, K. L., Burkhead, J. L., Esaki, N., Kurihara, T. and Pilon, M. (2002). Characterization of a NifS‐like chloroplast protein from Arabidopsis. Implications for its role in sulfur and selenium metabolism. Plant Physiology 130, 1309–1318. Pushnik, J. C., Miller, G. W. and Manwaring, J. H. (1984). The role of iron in higher plant chlorophyll biosynthesis, maintenance and chloroplast biogenesis. Journal of Plant Nutrition 7, 733–758. Qu, L. Q., Yoshihara, T., Ooyama, A., Goto, F. and Takaiwa, F. (2005). Iron accumulation does not parallel the high expression level of ferritin in transgenic rice seeds. Planta 222, 225–233. Raven, J. A., Evans, M. C. W. and Korb, R. (1999). The role of trace metals in photosynthetic electron transport in O2‐evolving organisms. Photosynthesis Research 60, 111–149. Roberts, L. A., Pierson, A. J., Panaviene, Z. and Walker, E. L. (2004). Yellow stripe1. Expanded roles for the maize iron‐phytosiderophore transporter. Plant Physiology 135, 112–120. Robinson, N. J., Procter, C. M., Connolly, E. L. and Guerinot, M. L. (1999). A ferric‐ chelate reductase for iron uptake from soils. Nature 397, 694–697. Rogers, E. E. and Guerinot, M. L. (2002). FRD3, a member of the multidrug and toxin eZux family, controls iron deficiency responses in Arabidopsis. The Plant Cell 14, 1787–1799. Rogers, E. E., Eide, D. J. and Guerinot, M. L. (2000). Altered selectivity in an Arabidopsis metal transporter. Proceedings of the National Academy of Sciences USA 97, 12356–12360. Rroco, E., Kosegarten, H., Harizaj, F., Imani, J. and Mengel, K. (2003). The importance of soil microbial activity for supply of iron to sorghum and rape. European Journal of Agronomy 19, 487–493. Sakaguchi, T., Nishizawa, N. K., Nakanishi, H., Yoshimura, E. and Mori, S. (1999). The role of potassium in the secretion of mugineic acids family phytosiderophores from iron‐deficient barley roots. Plant and Soil 215, 221–227. Schaaf, G., Ludewig, U., Erenoglu, B. E., Mori, S., Kitahara, T. and von Wiren, N. (2004). ZmYS1 functions as a proton‐coupled symporter for phytosiderophore‐ and nicotianamine‐chelated metals. The Journal of Biological Chemistry 279, 9091–9096. Schaaf, G., Honsbein, A., Meda, A. R., Kirchner, S., Wipf, D. and von Wiren, N. (2006). AtIREG2 encodes a tonoplast transport protein involved in iron‐ dependent nickel detoxification in Arabidopsis thaliana roots. The Journal of Biological Chemistry 281, 25532–25540. Schmidke, I., Kruger, C., Frommichen, R., Scholz, G. and Stephan, U. W. (1999). Phloem loading and transport characteristics of iron in interaction with plant‐endogenous ligands in castor bean seedlings. Physiologia Plantarum 106, 82–89. Sharma, A., Johri, B. N., Sharma, A. K. and Glick, B. R. (2003). Plant growth‐ promoting bacterium Pseudomonas sp. strain GRP3 influences iron acquisition in mungbean (Vigna radiata L. Wilzeck). Soil Biology and Biochemistry 35, 887–894. Sharma, S. and Sanwal, G. G. (1992). EVect of iron deficiency on the photosynthetic system of maize. Plant Physiology 140, 527–530.

IRON DYNAMICS IN PLANTS

177

Shikanai, T., Mu¨ller‐Moule´, P., Munekage, Y., Niyogi, K. K. and Pilon, M. (2003). PAA1, a P‐type ATPase of Arabidopsis, functions in copper transport in chloroplasts. The Plant Cell 15, 1333–1346. Shingles, R., North, M. and McCarty, R. E. (2002). Ferrous ion transport across chloroplast inner envelope membranes. Plant Physiology 128, 1022–1030. Shojima, S., Nishizawa, N. K., Fushiya, S., Nozoe, S., Irifune, T. and Mori, S. (1990). Biosynthesis of phytosiderophores: In vitro biosynthesis of 20 ‐deoxymugineic acid from l‐methionine and nicotianamine. Plant Physiology 93, 1497–1503. Siebner‐Freibach, H., Hadar, Y. and Chen, Y. (2003). Siderophores sorbed on Ca‐ montmorillonite as an iron source for plants. Plant and Soil 251, 115–124. Singh, D. P., Cornah, J. E., Hadingham, S. and Smith, A. G. (2002). Expression analysis of the two ferrochelatase genes in Arabidopsis in diVerent tissues and under stress conditions reveals their diVerent roles in haem biosynthesis. Plant Molecular Biology 50, 773–788. Smith, A. G., Santana, M. A., Wallace‐Cook, A. D. M., Roper, J. M. and Labbe‐ Bois, R. (1994). Isolation of a cDNA encoding chloroplast ferrochelatase from Arabidopsis thaliana by functional complementation of a yeast mutant. The Journal of Biological Chemistry 269, 13405–13413. Smith, B. N. (1984). Iron in higher plants: Storage and metabolic role. Journal of Plant Nutrition 7, 759–766. Spiller, S. C. and Terry, N. (1980). Limiting factors in photosynthesis. II. Iron stress diminishes photochemical capacity by reducing the number of photosynthetic units. Plant Physiology 65, 121–125. Spiller, S. C., Castelfranco, A. M. and Castelfranco, P. A. (1982). EVects of iron and oxygen on chlorophyll biosynthesis. Plant Physiology 69, 107–111. Spiller, S. C., Kaufman, L. S., Thompson, W. F. and Briggs, W. R. (1987). Specific mRNA and rRNA levels in greening pea leaves during recovery from iron deficiency. Plant Physiology 84, 409–414. Stacey, M. G., Osawa, H., Patel, A., Gassmann, W. and Stacey, G. (2006). Expression analyses of Arabidopsis oligopeptide transporters during seed germination, vegetative growth and reproduction. Planta 223, 291–305. Stephan, U. W. and Scholz, G. (1993). Nicotianamine: Mediator of transport of iron and heavy metals in the phloem. Physiologia Plantarum 88, 522–529. Stephan, U. W., Schmidke, I. and Pich, A. (1994). Phloem translocation of Fe, Cu, Mn and Zn in Ricinus seedlings in relation to the concentration of nicotianamine, an endogenous chelator of divalent metal ions, in diVerent seedling parts. Plant and Soil 165, 181–188. Stevenson, F. J. (1994). ‘‘Humus Chemistry: Genesis, Composition, Reactions’’. Wiley J. & Sons, New York, USA. Stocking, C. R. (1975). Iron deficiency aVects the structure and the physiology of maize chloroplasts. Plant Physiology 55, 626–631. Suzuki, T., Masuda, T., Singh, D. P., Tan, F.‐C., Tsuchiya, T., Shimada, H., Ohta, H., Smith, A. G. and Takamiya, K. (2002). Two types of ferrochelatase in photosynthetic and non‐photosynthetic tissues of cucumber; their diVerence in phylogeny, gene expression and localization. The Journal of Biological Chemistry 277, 4731–4737. Suzuki, M., Takahashi1, M.,Tsukamoto,T., Watanabe, S., Matsuhashi, S., Yazaki, J., Kishimoto, N., Kikuchi, S., Nakanishi, H., Mori, S. and Nishizawa, N. K. (2006). Biosynthesis and secretion of mugineic acid family phytosiderophores in zinc‐deficient barley. The Plant Journal 48, 85–97.

178

J.‐F. BRIAT

Takagi, S. (1976). Naturally occuring iron‐chelating compounds in oat‐ and rice‐root washings. I. Activity measurement and preliminary characterization. Soil Science and Plant Nutrition 22, 423–433. Takahashi, Y. and Tokumoto, U. (2002). A third bacterial system for the assembly of iron‐sulfur clusters with homologs in archaea and plastids. The Journal of Biological Chemistry 277, 28380–28383. Takahashi, Y., Mitsui, A., Hase, T. and Matsubara, H. (1986). Formation of the iron‐ sulfur cluster of ferredoxin in isolated chloroplasts. Proceedings of the National Academy of Sciences USA 83, 2434–2437. Takahashi, Y., Mitsui, A. and Matsubara, H. (1991a). Formation of the Fe‐S cluster of ferredoxin in lysed spinach chloroplasts. Plant Physiology 95, 97–103. Takahashi, Y., Mitsui, A., Fujiota, Y. and Matsubara, H. (1991b). Roles of ATP and NADPH in formation of the Fe‐S cluster of spinach ferredoxin. Plant Physiology 95, 104–110. Takahashi, M., Yamaguchi, H., Nakanishi, H., Shioiri, T., Nishizawa, N. K. and Mori, S. (1999). Cloning two genes for nicotianamine aminotransferase, a critical enzyme in iron acquisition (Strategy II) in graminaceous plants. Plant Physiology 121, 947–956. Takahashi, M., Terada, Y., Nakai, I., Nakanishi, H., Yoshimura, E., Mori, S. and Nishizawa, N. K. (2003). Role of nicotianamine in the intracellular delivery of metals and plant reproductive development. The Plant Cell 15, 1263–1280. Tang, C., Robson, A. D. and Dilworth, M. J. (1990). The role of iron in nodulation and nitrogen fixation in Lupinus auguistifolius L. The New Phytologist 115, 61–67. Terry, N. (1980). Limiting factors in photosynthesis. I. Use of iron stress to control photochemical capacity in vivo. Plant Physiology 65, 114–120. Terry, N. and Zayed, A. M. (1995). Physiology and biochemistry of leaves under iron deficiency. In ‘‘Iron Nutrition in Soil and Plants’’ (J. Abadia, ed.), pp. 283–294. Kluwer Academic Publishers, The Netherlands. Theil, E. C. and Briat, J. F. (2004). ‘‘Plant Ferritin and Non‐Heme Iron Nutrition in Humans.’’ HarvestPlus Technical Monograph No. 1. International Food Policy Research Institute and International Center for Tropical Agriculture (CIAT), Washington, D.C. and Cali, 17 pp. Thomine, S., Wang, R., Ward, J. M., Crawford, N. M. and Schroeder, J. I. (2000). Cadmium and iron transport by members of a plant metal transporter family in Arabidopsis with homology to Nramp genes. Proceedings of the National Academy of Sciences USA 97, 4991–4996. TiYn, L. O. and Chaney, R. L. (1973). Translocation of iron from soybean cotyledons. Plant Physiology 52, 393–396. Tottey, S., Block, M. A., Allen, M., Westergren, T., Albrieux, C., Scheller, H. V., Merchant, S. and Jensen, P. E. (2003). Arabidopsis CHL27, located in both envelope and thylakoid membranes, is required for the synthesis of protochlorophyllide. Proceedings of the National Academy of Sciences USA 100, 16119–16124. Touraine, B., Boutin, J. P., Marion‐Poll, A., Briat, J. F., Peltier, G. and Lobre´aux, S. (2004). Nfu2: A scaVold protein required for [4Fe‐4S] and ferredoxin iron‐ sulphur cluster assembly in Arabidopsis chloroplasts. The Plant Journal 40, 101–111. Val, J., Monge, E., Heras, L. and Abadia, J. (1987). Changes in photosynthetic pigments composition in higher plants as aVected by iron nutrition status. Journal of Plant Nutrition 10, 995–1001.

IRON DYNAMICS IN PLANTS

179

Van Wuytswinkel, O., Vansuyt, G., Grignon, N., Fourcroy, P. and Briat, J. F. (1999). Iron homeostasis alteration by ferritin overexpression in transgenic tobacco. The Plant Journal 17, 93–98. Vansuyt, G., Mench, M. and Briat, J. F. (2000). Soil‐dependent variability of leaf iron accumulation in transgenic tobacco overexpressing ferritin. Plant Physiology and Biochemistry 38, 499–506. Vansuyt, G., Robin, A., Briat, J. F., Curie, C. and Lemanceau, P. (2007). Iron acquisition from Fe‐pyoverdine by Arabidopsis thaliana. Molecular Plant Microbe Interactions 20, 441–447. Vert, G., Grotz, N., Dedaldechamp, F., Gaymard, F., Guerinot, M. L., Briat, J. F. and Curie, C. (2002). IRT1, an Arabidopsis transporter essential for iron uptake from the soil and for plant growth. The Plant Cell 14, 1223–1233. Vert, G., Briat, J. F. and Curie, C. (2003). Dual regulation of the Arabidopsis high‐ aYnity root iron uptake system by local and long‐distance signals. Plant Physiology 132, 796–804. Von Wiren, N., Mori, S., Marschner, H. and Romheld, V. (1994). Iron ineYciency in maize mutant ys1 (Zea mays L. cv Yellow‐Stripe) is caused by a defect in uptake of iron phytosiderophores. Plant Physiology 106, 71–77. Von Wiren, N., Marschner, H. and Ro¨mheld, V. (1996). Roots of iron‐eYcient maize also absorb phytosiderophore‐chelated zinc. Plant Physiology 111, 1119–1125. Vos, J. W., Safadi, F., Reddy, A. S. and Hepler, P. K. (2000). The kinesin‐like calmodulin binding protein is diVerentially involved in cell division. The Plant Cell 12, 979–990. Walter, A., Ro¨hmeld, V., Marschner, H. and Crowley, D. E. (1994a). Iron nutrition of cucumber and maize: EVect of Pseudomonas putida YC3 and its siderophore. Soil Biology and Biochemistry 26, 1023–1031. Walter, A., Ro¨hmeld, V., Marschner, H. and Mori, S. (1994b). Is the release of phytosiderophores in zinc‐deficient wheat plants a response to impaired iron utilization? Physiologia Plantarum 92, 493–500. Wang, Y., Brown, H. N., Crowley, D. E. and Szaniszlo, P. J. (1993). Evidence for direct utilization of a siderophore, ferrioxamine B, in axenically grown cucumber. Plant Cell and Environment 16, 579–585. Waters, B. M., Blevins, D. G. and Eide, D. J. (2002). Characterization of FRO1, a pea ferric‐chelate reductase involved in root iron acquisition. Plant Physiology 129, 85–94. Waters, B. M., Chu, H. H., DiDonato, R. J., Roberts, L. A., Eisley, R. B., Lahner, B., Salt, D. E. and Walker, E. L. (2006). Mutations in Arabidopsis yellow stripe‐ like1 and yellow stripe‐like3 reveal their roles in metal ion homeostasis and loading of metal ions in seeds. Plant Physiology 141, 1446–1458. Welch, R. M. (1995). Micronutrient nutrition of plants. Critical Review in Plant Sciences 14, 49–82. Winder, T. L. and Nishio, J. N. (1995). Early iron deficiency stress response in leaves of sugar beet. Plant Physiology 108, 1487–1494. Wittenberg, J. B., Wittenberg, B. A., Day, D. A., Udvardi, M. K. and Appleby, C. A. (1996). Siderophore‐bound iron in the peribacteroid space of soybean root nodules. Plant and Soil 178, 161–169. Xu, X. M. and Møller, S. G. (2004). AtNAP7 is a plastidic SufC‐like ATP‐binding cassette/ATPase essential for Arabidopsis embryogenesis. Proceedings of the National Academy of Sciences USA 101, 9143–9148. Xu, X. M. and Møller, S. G. (2006). AtSufE is an essential activator of plastidic and mitochondrial desulfurases in Arabidopsis. The EMBO Journal 25, 900–909.

180

J.‐F. BRIAT

Xu, X. M., Adams, S., Chua, N. H. and Møller, S. G. (2005). AtNAP1 represents an atypical SufB protein in Arabidopsis plastids. The Journal of Biological Chemistry 280, 6648–6654. Yabe, T. and Nakai, M. (2006). Arabidopsis AtIscA‐I is aVected by deficiency of Fe–S cluster biosynthetic scaVold AtCnfU‐V. Biochemical and Biophysical Research Communications 340, 1047–1052. Yabe, T., Morimoto, K., Kikuchi, S., Nishio, K., Terashima, I. and Nakai, M. (2004). The Arabidopsis chloroplastic NifU‐like protein CnfU, which can act as an iron‐sulfur cluster scaVold protein, is required for biogenesis of ferredoxin and photosystem I. The Plant Cell 16, 993–1007. Ye, H., Garifullina, G. F., Abdel‐Ghany, S., Zhang, L., Pilon‐Smits, E. A. H. and Pilon, M. (2005). AtCpNifS is required for iron–sulfur cluster formation in ferredoxin in vitro. Planta 220, 602–608. Ye, H., Abdel‐Ghany, S., Anderson, T. D., Pilon‐Smits, E. A. H. and Pilon, M. (2006). CpSufE activates the cysteine desulfurase CpNifS for chloroplastic Fe‐S cluster formation. The Journal of Biological Chemistry 281, 8958–8969. Yehuda, Z., Shenker, M., Ro¨hmeld, V., Marschner, H., Hadar, Y. and Chen, Y. (1996). The role of ligand exchange in the uptake of iron from microbial siderophores by graminaceous plants. Plant Physiology 112, 1273–1280. Yi, Y. and Guerinot, M. L. (1996). Genetic evidence that induction of root Fe(III) chelate reductase activity is necessary for iron uptake under iron deficiency. The Plant Journal 10, 835–844. Yoshida, K., Kitahara, S., Ito, D. and Kondo, T. (2006). Ferric ions involved in the flower color development of the Himalayan blue poppy, Meconopsis grandis. Phytochemistry 67, 992–998. Yuan, Y. X., Zhang, J., Wang, D. W. and Ling, H. Q. (2005). AtbHLH29 of Arabidopsis thaliana is a functional ortholog of tomato FER involved in controlling iron acquisition in strategy I plants. Cell Research 15, 613–621. Zancani, M., Peresson, C., Biroccio, A., Federici, G., Urbani, A., Murgia, I., Soave, C., Micali, F., Vianello, A. and Macri, F. (2004). Evidence for the presence of ferritin in plant mitochondria. European Journal of Biochemistry 271, 3657–3664.

Plants and Arbuscular Mycorrhizal Fungi: Cues and Communication in the Early Steps of Symbiotic Interactions

VIVIENNE GIANINAZZI‐PEARSON,* NATHALIE SE´JALON‐DELMAS,{ ANDREA GENRE,{ SYLVAIN JEANDROZ*,} AND PAOLA BONFANTE{

*UMR INRA 1088/CNRS 5184/Universite´ de Bourgogne Plant‐Microbe‐Environment, INRA, CMSE, BP 86510, Dijon Cedex 21065, France { UMR 5546, Equipe de Mycologie Ve´ge´tale, Poˆle de Biotechnologie Ve´ge´tales, Chemin de Borde‐Rouge, BP 42617, Castanet‐Tolosan 31326, France { Dipartimento di Biologia Vegetale, Universita` di Torino, I.P.P.‐C.N.R., Viale Mattioli 25, Torino 10125, Italy } UMR INRA/UHP 1136 Interactions Arbres‐Microorganismes, Universite´ H. Poincare´ Nancy I, Faculte´ des Sciences, BP 239, Vandœuvre Le`s Nancy 54506, France

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . II. Rhizosphere Signaling in Symbiotic Interactions . . . . . . . . . . . . . . . . . . . . . . . . . . A. Plant Signals in the Presymbiotic Stage: The Case of Flavonoids ..... B. Nonflavonoid Rhizosphere Signals ......................................... C. Identification of the Hyphal Branching Factor: The Strigolactone Story ...................................................... D. Fungal Signaling to Host Roots: Myc Factors ........................... III. Plant Genetic Programs: Mycorrhiza‐Defective Mutants . . . . . . . . . . . . . . . . . IV. Molecular Cross Talk and Signaling Pathways . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. Plant Cell Responses to Fungal Colonization: Tissue and Cell Specificity .... VI. Interface Biogenesis: New Facts/New Hypotheses. . . . . . . . . . . . . . . . . . . . . . . . . Advances in Botanical Research, Vol. 46 Incorporating Advances in Plant Pathology Copyright 2008, Elsevier Ltd. All rights reserved.

182 184 186 188 190 192 193 197 201 204

0065-2296/08 $35.00 DOI: 10.1016/S0065-2296(07)46005-0

182

V. GIANINAZZI‐PEARSON ET AL.

VII. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 206 Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 207 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 208

ABSTRACT The ubiquitous nature of arbuscular mycorrhiza (AM) pleads for common molecular and genetic determinants across diVerent plant taxa. The cellular processes determining compatibility in early interactions prior to and during cell contact between arbuscular mycorrhizal fungi and plant roots are starting to be unraveled. The root epidermis is an active checkpoint where signal exchanges and control over root colonization occur. Root‐secreted flavonoids, flavonols, and strigolactones can act as rhizosphere signals in stimulating presymbiotic fungal growth, although their mechanism of action on the fungal cell is as yet unknown. Likewise, fungal signals (Myc factors) activate early plant responses with induction of genes related to signal transduction pathways and biogenesis of a prepenetration apparatus designed to accommodate intracellular fungal growth from appressoria into epidermal cells. Evidence from genetical, transcriptional, and physiological studies points to the implication of calcium as a secondary messenger in signaling pathways leading to early host cell responses. Future challenges for research are to decipher the complexity of symbiosis signaling and to provide new insights into the specificity of the molecular dialogue between AM symbionts.

I. INTRODUCTION Mycorrhizal associations are the root symbioses of the large majority of terrestrial plants, and the beneficial fungi involved represent an important component in the ecology and biology of most soils (Smith and Read, 1997). In the case of arbuscular mycorrhiza (AM), benefits to the plant are multiple but their symbiotic nature is principally characterized by bilateral exchanges where the photosynthetic host receives mineral nutrients and the fungus acquires carbohydrates. Mycorrhizal research has entered the mainstream of biology, thanks mainly to DNA technologies and genomics, which are providing new tools to discover symbiont communication, development, and diversity, and to reveal the contribution of symbiotic partners to the functioning of mycorrhizal associations. Several features of AM associations are considered as landmarks, unanimously accepted by the scientific community. First of all, land plants and AM fungi share a long coevolutionary history, which originated more than 450 million years ago (Remy et al., 1994) and which has ensured the widespread distribution of AM fungi. Reciprocal nutrient exchange within a functional symbiosis requires extensive physical contact between the partners, which results from profound readjustments in the plant and fungal tissues during root colonization (Harrison, 1999). Being asexual, obligately biotrophic, multinucleate, and unculturable microbes,

CUES AND COMMUNICATION IN ARBUSCULAR MYCORRHIZA

183

AM fungi have been considered intractable organisms from a taxonomic point of view for many years. On the basis of ribosomal gene sequences, they have been moved to a new phylum, the Glomeromycota (Schu¨ßler et al., 2001), and their hierarchical position has been confirmed with the remodeling of the entire phylogenetic tree of fungi (James et al., 2006). More than 150 species have been described so far within the Glomeromycota, but their number and genetic features are a dynamic field of research, as witnessed by debates (reviewed by Pawlowska, 2005). In order to set the scene and provide the background for subsequent discussion, the main aspects of root colonization by the symbiotic fungi are outlined first. Glomeromycota are highly dependent on their hosts for fitness and survival, and hyphae germinating from their large asexual spores can only grow for a few days in the absence of a plant. On recognition of the host plant, these presymbiotic hyphae diVerentiate hyphopodia‐like appressoria on the root epidermis, which in turn form hyphal pegs that cross the root epidermis and initiate infection units to colonize the root cortex. Interactions between the model legume Lotus japonicus and the AM fungus Gigaspora margarita oVer a good example of the diVerent types of root colonization patterns (Arum, Paris) that can occur (Dickson, 2004). The swollen appressorium developing at the root surface gives rise to an intercellular hypha, which usually separates two adjacent epidermal cells to reach their base. Here, it penetrates the radial wall of the epidermal cell and develops into the cell lumen to form the first interface compartment between symbiont cells (Bonfante et al., 2000). This series of events is a crucial step in the interaction, where reciprocal recognition, localized diVerentiation of the appressorium, cell wall breaching, and intracellular accommodation of the fungal symbiont in a novel apoplastic compartment represent the result of complementary, coordinated strategies in both partners, which provide the beneficial fungus access to internal root tissues without causing damage to the plant. Once the fungus has overcome this epidermal checkpoint and reached the inner root layers, it spreads through the parenchymal cortex by hyphal coils and/or intercellular hyphae and eventually forms intracellular, highly branched structures called arbuscules (Bonfante, 1984). These intracellular fungal structures are surrounded by a newly built apoplastic space, which is bordered by a specialized plant membrane (Gianinazzi‐Pearson et al., 2000; Harrison et al., 2002). The construction of this interface compartment, which regulates reciprocal nutrient exchanges between symbiont cells, results from an intense reorganization of plant cell contents and activity, ranging from specific gene activation (Gianinazzi‐Pearson and Brechenmacher, 2004) to localized cell wall and membrane deposition (Balestrini and Bonfante, 2005), cytoskeleton remodeling (Genre and Bonfante, 1997, 1998), organelle mobilization

184

V. GIANINAZZI‐PEARSON ET AL.

(Lohse et al., 2005), and phosphate transport (Karandashov and Bucher, 2005; Liu et al., 1998). Arbuscules are ephemeral structures with a life span of a few days (Toth and Miller, 1984). When host cell reorganization reaches its peak, arbuscule tips, then branches collapse, and finally the host cell contents gradually adopt an aspect similar to that prior to colonization (Jacquelinet‐ Jeanmougin et al., 1987). Interest in this ancient and widespread plant symbiosis has expanded exponentially over the last few years both from a fundamental viewpoint of interorganism interactions and for their potential exploitation in the development of sustainable plant production systems (Science Citation Index, Web of Science, http://scientific.thomson.com/products/wos/). The ubiquitous nature of AM associations, together with the constant structural and functional features characterizing the root–fungal relationships, pleads for common molecular and genetic determinants across diVerent plant taxa. Consequently, recent developments have been toward research that is based on model AM host–fungal combinations (Gianinazzi‐Pearson et al., 2007; Parniske, 2004), which may appear restrictive at first sight but provide a strategy for putting cell programs into a more general context with broader relevance. There are several reviews covering diVerent aspects of the AM symbiosis, including molecular and cell interactions (see citations in Gianinazzi‐Pearson et al., 2007 and Reinhardt, 2007). The purpose of this chapter is not to repeat earlier reviews, but rather to pinpoint events that drive early interactions between AM fungi and plants and propose future lines of research to unravel their role as prerequisites to reciprocal compatibility between the actors of the symbiosis.

II. RHIZOSPHERE SIGNALING IN SYMBIOTIC INTERACTIONS The rhizosphere is generally considered to be a narrow zone of soil where roots stimulate or inhibit microbial populations and activities in their vicinity. Roots may change the physical and chemical properties of the soil through mucilage production and the excretion of water‐soluble compounds in root exudates. Low‐molecular weight compounds, such as amino acids, organic acids, sugars, hormones, enzymes, secondary metabolites like phenolics and terpenoids, comprise the majority of root exudates (Walker et al., 2003). A large body of knowledge suggests that root exudates act as messengers that communicate and initiate biological and physiological interactions between roots and microorganisms, which in turn will produce signals essential for root colonization. Many studies have shown that root exudates from host

CUES AND COMMUNICATION IN ARBUSCULAR MYCORRHIZA

185

plants stimulate spore germination, hyphal branching, and growth of AM fungi (Be´card et al., 2004), and mathematical models of the colonization process by Glomus mosseae indicate that the portion of the root where exudation is greatest is most likely to become colonized (Schwab et al., 1991). The phosphate status of host plants can aVect the metabolism of compounds that accelerate AM fungal growth (Elias and Safir, 1987; Nagahashi and Douds, 1996). The application of phosphate to plants can decrease root membrane permeability, and with it alter the production or composition of root exudates (Amijee et al., 1989; Graham, 1982; Graham et al., 1981; Nagahashi and Douds, 2000; Ratnayake et al., 1978; Schwab et al., 1983; Tawaraya et al., 1994, 1996). In addition, phosphate deficiency aVects the exudation of flavonoids and other unidentified compounds (Akiyama et al., 2002; Franken and Gna¨dinger, 1994; Nair et al., 1991; Rossiter and Beck, 1996; Siqueira et al., 1991; Tawaraya et al., 1998; Xie et al., 1995). AM symbiosis establishment can also alter root exudates so that they no longer stimulate fungal growth or even inhibit further colonization (Pinior et al., 1999; Vierheilig et al., 1998b), indicating that in addition to being involved in presymbiotic stages, root exudates also play an important role in regulating root colonization by a negative feedback mechanism allowing the plant to control excessive colonization. The root signal that systemically inhibits further colonization is still unknown. While flavonoids stimulating root colonization can increase under low‐phosphate conditions, flavonoids inhibitory to AM fungi can accumulate under high plant phosphate status following mycorrhiza establishment (Guenoune et al., 2001; Larose et al., 2002). Cytokinin and salicylic acid have also been proposed to be involved in the autoregulation. Cytokinin levels are altered in AM colonized roots (Allen et al., 1980; Shaul‐Keinan et al., 2002), and reduced salicylic acid levels in Nah G tobacco plants increase root mycorrhization (Herrera‐Medina et al., 2003). The role of root exudates in the inability of certain plant species to form mycorrhiza is less clear. Nonmycorrhizal plants arose late in evolution (
100 million years), and their loss of compatibility with AM fungi may be polyphyletic (Brundrett, 2002). Distinction between host and nonhost plants may occur as soon as the presymbiotic stage but the mechanisms by which nonhost exudates influence AM fungal development remain an enigma. It has been suggested that they may simply lack compounds required to stimulate hyphal branching, growth, and/or chemotaxis (Bue´e et al., 2000; Gemma and Koske, 1988; Glenn et al., 1985, 1988), or that they may release inhibitory compounds (Gadkar et al., 2003; Nagahashi and Douds, 2000). Appressorium development is not elicited on nonhost roots, and this lack of appressoria formation is always associated with the absence of diVerential hyphal morphogenesis during the presymbiotic stage (Giovannetti et al., 1994).

186

V. GIANINAZZI‐PEARSON ET AL.

Intergeneric grafts between mycorrhizal pea and nonmycorrhizal Lupinus have provided evidence for a shoot‐produced inhibitor of the symbiosis in Lupinus (Gianinazzi‐Pearson and Gianinazzi, 1992). Lupinus presents high concentrations of alcaloids in leaves, which are translocated throughout the whole plant. However, a role of these compounds in the nonmycorrhizal status of Lupinus seems unlikely as they do not inhibit hyphal growth, suggesting that Lupinus probably also lacks stimulatory compounds (Vierheilig et al., 1995). Glucosinolate that may be metabolized into isothiocyanate, an antifungal compound, has been extracted from roots of nonmycorrhizal Brassica and shown to inhibit spore germination of AM fungi (Schreiner and Koide, 1993; Vierheilig and Ocampo, 1990), but no inhibitory compounds have been found to be representative of other nonmycorrhizal families (Barker et al., 1998a; Schreiner and Koide, 1993). A. PLANT SIGNALS IN THE PRESYMBIOTIC STAGE: THE CASE OF FLAVONOIDS

Evidence that root and fungal signals must be involved in the AM symbiosis came from early observations but the identity of their molecular nature is still at its beginning. Spores of AM fungi can germinate spontaneously in the absence of a host (asymbiotic stage) (Bianciotto et al., 1995; Douds and Schenck, 1991; Gianinazzi‐Pearson et al., 1989; Giovannetti et al., 1993; Mosse, 1959). However, further directional growth and intense branching of the germ tube (presymbiotic stage), which favor fungal contact with the root (appressoria formation) and the establishment of symbiosis, require the presence of root compounds. In the absence of a host plant, spore germination is arrested before complete depletion of carbon resources (Bago et al., 1999; Be´card and Piche´, 1989a). The branching response, resulting in ‘‘fan‐like structures’’ (Powell, 1976) later called ‘‘arbuscule‐like structures’’ (Mosse, 1988), is stimulated by root exudates of host but not nonhost plant species (Be´card and Piche´, 1990; Gianinazzi‐Pearson et al., 1989), showing that an AM fungus senses its host plant and is able to discriminate from nonhost plants. Using mycorrhiza‐defective pea mutants, Balaji et al. (1995) proposed that factors inducing hyphal branching are diVerent from those promoting root penetration and/or colonization. Indeed, the pea mutants were not aVected in stimulatory compounds but rather in root signals inducing appressorium development and root colonization. At present, root factors that aVect spore germination, presymbiotic hyphal proliferation, and appressorium diVerentiation have not been reported. The major influence of root exudates on AM fungi is to stimulate hyphal growth and branching rather than spore germination. Phenolics, including flavonoids, have been proposed as the root molecules that could be involved

187

CUES AND COMMUNICATION IN ARBUSCULAR MYCORRHIZA

in the stimulation of presymbiotic fungal growth. Flavonoids, which are lipophilic to water‐soluble molecules derived from the phenylpropanoid pathway, play an active role in the regulation of symbiotic and pathogenic interactions (Peters and Verma, 1990). They are widely present in the plant kingdom and more than 4000 thousand have been identified in vascular plants (Harborne, 1988). Their eVects on AM fungi can be (1) stimulation of fungal spore germination, (2) promotion of hyphal growth, and (3) to favor root colonization. A number of isolated flavonoids can activate spore germination of Gigaspora or Glomus species in the micromolar range (Fig. 1) (Gianinazzi‐Pearson et al., 1989; Graham, 1982; Kape et al., 1992; Siqueira et al., 1982; Tsai and Phillips, 1991). The smaller spores of Glomus appear more dependent on an external stimulus for germination than species with large spores (e.g., Gigaspora). Flavonoids or flavonols naturally released from host roots enhance hyphal growth (Be´card et al., 1992; Bel‐ Rhlid et al., 1993; Poulin et al., 1993; Tsai and Phillips, 1991) and promote AM colonization (Nair et al., 1991). AM fungal responses to flavonoids are not uniform and they can diVer between fungi, flavonoid concentration,

B

A

C

OH

OH

OH O

HO

O

HO

OH

OH OH

OH

O

HO

O

HO

HO

O

I

H O

OCH3

O

OH O

OH O

G OH

OH

OCH3

OH

OH

F

OH

OH O

OH O

O

E

D

HO

OH O

HO

OH

O

OH OH

OCH3 O

HO O

HO

OH OH

O OH

O

Fig. 1. Structure of flavonoid‐related molecules known to influence AM fungal spore germination and/or hyphal growth. A, B, C, and D are flavonol structures: A, quercetin; B, kaempferol; C, apigenin; and D, luteolin. E, F, and G are flavanones: E, hesperitin; F, naringenin; and G, biochanin A. G, H, and I are isoflavonoids: G, biochanin A; H, formononetin; and I, myricetin.

188

V. GIANINAZZI‐PEARSON ET AL.

and type of molecule. For example, quercetin, apigenin, and two flavanones (hesperitin and naringenin) all stimulate hyphal growth of G. margarita but the isoflavones biochanin A and formononetin are inhibitory (Be´card et al., 1992; Chabot et al., 1992). Be´card et al. (1992) proposed a possible structure– activity relationship and hypothesized that a hydroxyl group in position 3 is essential to confer activity on hyphal growth of the flavonols quercetin and kaempferol (Fig. 1). However, this could not explain the activity on spore germination of luteolin and apigenin, which do not possess a hydroxyl group in position 3 (Fig. 1). Vierheilig et al. (1998a) suggested that AM fungi might have genus‐, or even species‐, specific requirements during symbiosis, and Larose et al. (2002) proposed that flavonoid patterns of accumulation may be diVerentially modulated depending on the fungal genera. Flavonoid profiles in root exudates diVer considerably between legumes (Phillips, 2000), and this variability is supposed to enable symbiotic Rhizobia to distinguish their hosts from nonlegumes (Mithofer, 2002). This flavonoid‐based host specificity in the nodulation symbiosis may be reminiscent of a preexisting flavonoid‐ fungal specificity in the AM symbiotic program. There is as yet no clue as to the physiological basis of the promoting eVects of flavonoids on AM fungal development. Interestingly, they are known for their estrogenic activity in vertebrates. Poulin et al. (1997) reported that flavonoid activity on AM fungal growth could be blocked by pure antiestrogens and that ‐estradiol stimulates hyphal growth of Glomus intraradices, suggesting a potential estrogen‐like binding site in this fungus. However, the central role of flavonoids in AM interactions was questioned when Be´card et al. (1995) showed that maize mutants deficient in chalcone synthase, an enzyme in the flavonoid synthesis pathway, induced a same hyphal branching activity in AM fungi as compared to wild‐type plants and developed comparable levels of mycorrhization. In addition, carrot roots transformed with Ri T‐DNA used for in vitro culture of AM fungi do not produce flavonoids. Finally, significant amounts of quercetin, kaempferol, and myricetin have been detected in Arabidopsis thaliana, a nonmycorrhizal plant (Burbulis et al., 1996). Taken together, these data suggest that flavonoids are not essential compounds for mycorrhization and that other chemicals may stimulate AM fungal growth, perhaps synergistically with flavonoids.

B. NONFLAVONOID RHIZOSPHERE SIGNALS

Volatiles have been reported as growth stimulants of AM fungi (Saint‐John et al., 1983) but usually in synergy with root exudates. Be´card and Piche´ (1989b) identified the active volatile compound as CO2, assuming that CO2

CUES AND COMMUNICATION IN ARBUSCULAR MYCORRHIZA

189

serves as carbon source through an active CO2 dark fixation (Bago et al., 1999). However, CO2 and root exudates act synergistically to induce germ tube elongation and each alone has little or no eVect. In contrast, the positive eVect of quercetin is independent of CO2 (Chabot et al., 1992). Consequently, the CO2 eVect on AM fungi may only be visible with other compounds present in root exudates. As mentioned above, AM fungi exhibit a specific pattern of profuse branching in the vicinity of host plant roots (Giovannetti et al., 1993, 1996; Tawaraya et al., 1996). The identity of the branching factor exuded from host roots has been the object of considerable research. Using dialysis membranes and a sandwich system, Giovannetti et al. (1996) determined that the branching factor from Ocimum basilicum had a molecular weight lower than 500 Da. The development of an in vitro bioassay for hyphal branching in germinating spores of the genus Gigaspora has facilitated chemical analysis of the active compound released from roots. Nagahashi and Douds (1999) showed that dilution of crude root exudates reduces branching, while increasing root exudate concentrations induces ‘‘bushier, 3‐D type branching,’’ and finally at higher doses the ‘‘arbuscule‐like structures.’’ The ‘‘bushier branching’’ is normally observed when a hyphal tip grows close to a host root surface while the ‘‘arbuscule‐like structures’’ develop on external hyphae growing in limited nutrient conditions (Be´card and Fortin, 1988; Mosse, 1988; Mosse and Hepper, 1975). The physiological significance of these latter structures has not been determined, but they could mimic the branched absorbing structures (BAS) formed by G. intraradices (Bago et al., 1998), which are supposed to be involved in nutrient uptake by the fungus. A fraction of carrot root exudates prepurified by Bue´e et al. (2000) proved to be active in inducing branching of all AM fungal species tested, contrasting with flavonoids, which display stimulatory, inhibitory, or neutral activity, depending on the AM fungal species. The branching activity was induced also by root exudates of the chalcone synthase maize mutants, excluding once again the hypothesis of flavonoids as the active molecules. The active fraction of root exudates was called ‘‘branching factor,’’ and was supposed to contain several molecules. The branching factor was characterized as being lipophilic and of small molecular weight, in agreement with Giovannetti et al. (1996). It is active in stimulating hyphal branching at very low concentrations and is absent from nonhost plants like A. thaliana or sugar beet (Bue´e et al., 2000). Detailed investigations of the physiological eVects of this ‘‘branching factor’’ have shown that it is accompanied by increased mitotic activity in hyphae (Bue´e et al., 2000) and eVects at the mitochondrial level (Tamasloukht et al., 2003). One to four hours after addition of the branching factor to germinating spores of Gigaspora rosea, biogenesis of mitochondria

190

V. GIANINAZZI‐PEARSON ET AL.

increases and they change in shape and orientation. This cellular response is accompanied by very early induction of several genes related to mitochondrial activity and by an increase in oxygen consumption. These eVects precede the first morphological responses observed 6–24 h after stimulation. The increase in mitochondrial activity can be associated with the increase in phosphate uptake and plasma membrane ATPase activity reported by Lei et al. (1991). Increased transcription in presence of host root exudates has also been reported for a gene encoding a copper–zinc superoxide dismutase (SOD) from G. margarita (Lanfranco et al., 2005). Since this enzyme plays a role in the detoxification of stress‐related molecules, it may be linked to the action of root exudates on mitochondria and on the subsequent increased production of activated oxygen species. Jolicoeur et al. (1998) demonstrated that the cytosolic pH of hyphae was more alkaline when G. rosea spores were germinating in the vicinity of host roots, which could explain observations that transmembrane electric potential becomes more negative after addition of plant root extracts to G. margarita spores (Ayling et al., 2000). These authors concluded to a direct eVect of root compounds on the membrane of G. margarita, rather than an eVect mediated through modified gene expression. There is, as yet, no evidence for the involvement of host root exudates in the regulation of genes involved in AM fungal biotrophy (Tre´panier et al., 2005). In addition, even if the respiratory response to carrot root exudates is conserved in Gigaspora and Glomus (Tamasloukht et al., 2003), there is no clue as to putative diVerential eVects of branching factors on AM fungal species or eventual host specificities. To date, the mechanisms by which root exudates enhance hyphal branching in AM fungi remain obscure but a scenario has been proposed by Be´card et al. (2004) where the ensemble of metabolic changes induced by root factors enables an AM fungus to exploit its own growth potential and so ensure the developmental switch from the asymbiotic stage of spore germination to presymbiotic hyphal branching. C. IDENTIFICATION OF THE HYPHAL BRANCHING FACTOR: THE STRIGOLACTONE STORY

A breakthrough in deciphering plant–AM fungal signaling events was the identification of the L. japonicus branching factor as 5‐deoxy strigol, belonging to the strigolactone family (Akiyama et al., 2005) (Fig. 2). These authors demonstrated activation of branching in G. margarita by a natural strigolactone purified from L. japonicus, as well as by the chemical analogue GR24 (Fig. 2). Strigolactones were previously isolated as seed germination stimulants of the root parasitic weeds Striga and Orobanche (Bouwmeester et al., 2003), which devastate important food crops (maize, sorghum, millet, rice)

191

CUES AND COMMUNICATION IN ARBUSCULAR MYCORRHIZA

A

B O C A

C

O

O C A

B O

O D

O O C

B

A O

O

O D

O

B H

O

O

O D

O

Fig. 2. Structure of strigolactones that stimulate the hyphal branching response in AM fungi. (A) sorgolactone, (B) GR24 (chemical analogue), and (C) 5‐deoxy strigol.

and legumes. It now turns out that strigolactones are detected as host‐ derived signals both by beneficial fungal symbionts and by parasitic weeds. Strigolactones are active on parasitic weed germination and AM fungal spore germination at very low concentrations (pico to nanomolar). An active component in root exudates of sorghum has also been identified as a sorgolactone (Fig. 2), which stimulates not only Gigaspora branching but also G. intraradices spore germination rates (Besserer et al., 2006). Moreover, strigolactones act on the AM fungus in a way similar to the branching factor from carrot roots, with a rapid increase in respiration rate and in mitochondria biogenesis, morphology, and motility (Besserer et al., 2006). Purification of strigolactones and related compounds has been hampered until now due to their very low concentration in root exudates. This is consistent with a role as a signal molecule and explains why they have been described in few plants since their first discovery (Cook et al., 1966). The chemical lifetime of strigolactones under natural soil conditions may be very short, enabling these chemicals to convey positional information about the roots of living host plants. The presence of strigolactones in both monocots like sorghum and dicots like Lotus suggests a widespread occurrence among angiosperms (Akiyama and Hayashi, 2006; Akiyama et al., 2005; Besserer et al., 2006), which is consistent with the large host spectrum of AM fungi. They have been described by many authors as sesquiterpene lactones, but very little is known about their metabolic pathway or regulation, apart from the involvement of the carotenoid pathway in their biosynthesis (Matusova et al., 2005). The production of parasitic weed stimulants in nonmycorrhizal plant families is largely unreported. The only exception is A. thaliana, for which there are indications of their synthesis but at lower concentrations than in mycorrhizal plants like carrot or tobacco (Goldwasser and Yoder, 2001; Westwood, 2000). However, other classes of active molecules may not be ruled out in AM interactions. Neither Akiyama et al. (2005) nor Besserer et al. (2006) could reproduce the root exudate‐induced branching pattern of

192

V. GIANINAZZI‐PEARSON ET AL.

‘‘arbuscule‐like structures’’ on hyphae using GR24, even at millimolar concentrations. This suggests that strigolactones may act synergistically with other molecules, from the same chemical family or not. The role of strigolactones in fungal appressoria formation has not yet been investigated. In parasitic weeds, after stimulation of seed germination by strigolactones, haustoria development is triggered by other molecules like xenognosin A, phenolics, quinones, and anthocyanidins (Chang and Lynn, 1986; Lynn et al., 1981). It may be hypothesized that flavonoids, which play a role in AM interactions (see above), could also act on appressorium formation and/ or root colonization subsequent to presymbiotic stimulation of hyphal growth and branching by strigolactones. D. FUNGAL SIGNALING TO HOST ROOTS: MYC FACTORS

Microbial symbionts must communicate their presence to host plants (Long, 1996) and plants need to distinguish friends from foes. In the same way that legume root flavonoids may activate nodulation genes in Rhizobia, host root compounds may activate mycorrhization genes in AM fungi. Larose et al. (2002) reported that alfalfa root flavonoid content increased in the presence of mycelium and spores of G. intraradices, before any physical contact between the symbionts, pointing toward the existence of signals derived from the fungus and sensed by the host plant. Hyphae (Gigaspora, Glomus) growing in the vicinity of host–roots but separated by a membrane release a diVusible signal (or signals) of less than 3.5 kDa that induce MtENOD11 gene expression in Medicago truncatula. This expression is synchronous with the induction of hyphal branching, and does not occur with dead spores or fungal pathogens (Kosuta et al., 2003). M. truncatula mutants defective for both nodulation and mycorhization (dmi/Mtsym, Section 3) respond to the AM fungi with the same induction of MtENOD11, whereas they are totally blocked for the Nod factor response (Catoira et al., 2000), suggesting the existence of a signal transduction pathway in presymbiotic AM interactions that is independent of the Nod factor transduction pathway. This conclusion is reinforced by the fact that G. mosseae and Sinorhizobium meliloti activate diVerent sets of signal transduction‐related M. truncatula genes during early interactions with host roots (Sanchez et al., 2005; Weidmann et al., 2004). Furthermore, molecules released by AM spores can induce a transient and rapid calcium response in soybean cells (see Section 3) even after such cells have been challenged with a Nod factor, indicating that the receptors involved in the rhizobial and fungal signal perception are diVerent (Navazio et al., 2007). DiVusible AM fungal signals can also induce lateral root formation in M. truncatula (Ola´h et al., 2005), while root pathogens

CUES AND COMMUNICATION IN ARBUSCULAR MYCORRHIZA

193

do not have the same eVect. Lateral root formation induced by the fungal signal(s) requires the symbiosis‐related M. truncatula genes DMI1 and DMI2, but not DMI3. Consequently, either the diVusible fungal compound(s) promoting lateral root formation should diVer from the compound(s) inducing MtENOD11 gene expression (Kosuta et al., 2003), or the same compound(s) can activate two diVerent pathways. These diVerent observations, coupled with the chitinous nature of the Nod factor backbone, have led to the speculation that AM fungi (which are much more ancient than Rhizobia) could produce a related ‘‘Myc’’ factor with a role in mycorrhizal signaling similar to that of Nod factors in the nodulation symbiosis. The identification of several plant genes, which are activated during both nodulation and AM interactions in legumes, as well as the discovery of symbiosis‐defective mutants (Section 3), has led to the conclusion that some common mechanisms may regulate root responses to signals from Rhizobia and AM fungi. The next challenge is to identify Myc factors and to describe the Myc factor‐related signaling pathway. Because of the genetic and genomic knowledge accumulated on legumes, this plant family represents a major resource for such investigations.

III. PLANT GENETIC PROGRAMS: MYCORRHIZA‐DEFECTIVE MUTANTS Genetic analyses of interactions between roots and AM fungi have known important advances with the identification of plant mutants (obtained by EMS, gamma irradiation, fast neutron or transposon‐tagging mutagenesis) that are impaired in the development of the symbiosis. These have so far provided evidence for the existence of genes that control root compatibility with the symbiotic fungi in M. truncatula, Pisum sativum, L. japonicus, Vicia faba, Phaseolus vulgaris, Melilotus alba, Zea mays, and Lycopersicon esculentum (Barker et al., 1998b; Borisov et al., 2004; David‐Schwartz et al., 2001, 2003; Duc et al., 1989; Kistner et al., 2005; Lum et al., 2002; Morandi et al., 2005; Oldroyd and Downie, 2004; Paszkowski et al., 2006). Isolation of corresponding genes from mutant backgrounds has been instrumental in identifying some of the plant gene functions essential to the first steps of symbiosis establishment. The cellular and molecular characterization of root interactions with fungal symbionts in mycorrhiza‐defective mutants point to a role of symbiosis‐related plant genes in pathways for specifically sensing and responding to AM fungal signals, and give clues as to how biologically active root or fungal factors may regulate cell functions linked to a successful symbiosis.

194

V. GIANINAZZI‐PEARSON ET AL.

Few plant mutants have been reported so far that are aVected in the presymbiotic phase of AM interactions prior to fungal contact with the root surface (Table I). Absence of appressoria formation on roots of a maize mutant (nope1) indicates a defect in early plant–fungal communication (Paszkowski et al., 2006), and two nonallelic tomato mutants (pmi1, pmi2) aVect precontact events by reducing spore germination and hyphal growth prior to appressoria formation (David‐Schwartz et al., 2001, 2003). Root exudates alone from one tomato mutant (pmi1) were suYcient to impair hyphal growth, suggesting that the corresponding gene may somehow control the release of inhibitory compounds by the host plant (Gadkar et al., 2003). Other observations that plead for a role of symbiosis‐related plant genes in controlling inhibitory root responses to AM fungi are, on the one hand, that AM fungal gene expression can be significantly downregulated in appressoria formed on roots of the dmi3/Mtsym13 mycorrhiza‐resistant mutant of M. truncatula (P. Seddas, M. C. Arias, D. van Tuinen, and V. Gianinazzi‐Pearson, unpublished data) and, on the other, that AM fungal appressoria elicit defence‐related wall reactions and gene expression in pea mutated for PsSYM9 (DMI3/MtSYM13 homologue) (Gollotte et al., 1993; Ruiz‐Lozano et al., 1999). At the same time, none of the mycorrhiza‐ defective plant mutants tested so far show an alteration in their phenotype vis‐a`‐vis either root or aerial pathogens (Catoira et al., 2000; Gao et al., 2006; Gianinazzi‐Pearson et al., 1994; Mellersh and Parniske, 2006), indicating diVerences in the genetic determinants controlling early processes in symbiotic and pathogenic interactions, even though there exists some overlap in host transcriptional responses at later stages (Gu¨imil et al., 2005). Most of the early stage mutants that have been described support appressoria formation by AM fungi but are aVected in the process of root penetration (Table I). By characterizing cytological and molecular events in mycorrhiza‐defective mutants, it has been possible to start to position the plant processes that are regulated by the corresponding symbiosis‐related genes. In the M. truncatula and pea mutants dmi2/Mtsym2 and Pssym9, AM fungal development is blocked at the junction of epidermal cells where the appressoria induce unusual thickening of subtending walls (Calantzis et al., 2001; Gollotte et al., 1993). The colonization process is also blocked at the surface of the epidermis in the double L. japonicus mutants pollux‐13 har1‐1 and ccamk‐5 har1‐1 (Murray et al., 2006) while inactivation of LjSYM15 aVects the separation of adjacent epidermal cells below appressoria that is required for the passage of hyphae into underlying root layers, and mutations in LjSYM2 (LjSYMRK), LjSYM3 (LjNUP33), and LjSYM4 (LjCASTOR) aVect the intracellular passage of hyphae through neighboring epidermal or hypodermal cells (Bonfante et al., 2000; Demchenko et al., 2004;

TABLE I Plant Mutants AVected in Early Interactions with AM Fungi

Z. mays

L. esculentum

L. japonicus

P. sativum

Mutated gene identity

M. truncatula

Mutant phenotypea


Reference(s)

nope1

–

–

–

–

–

App

–

pmi1, pmi2

–

–

–

–

– –

rmc –

– pollux

– –

– dmi1

– ion channel

Sp/Hyred, Appþ, Pen
Appþ, Penþ/
Appþ, Penþ/


– –

– –

Pssym8 Pssym19

– dmi2/Mtsym2

– receptor‐like kinase

Appþ, Pen
Appþ, Penþ/


–

–

– SymRK‐2 Ljsym2 –

Pssym9

dmi3/Mtdmi13

Appþ, Pen


– –

– –

– –

– –

Appþ, Penþ/
Appþ, Pen


–

–

–

–

–

Appþ, Penþ/


Kistner et al., 2005

–

–

Ljsym3 castor‐2 Ljsym4‐2 castor‐4 Ljsym4‐4 Ljsym15‐2

calcium/calmodulin‐ dependent kinase – ion channel

Paszkowski et al., 2006 David‐Schwartz et al., 2001, 2003 Gao et al., 2001 Kistner et al., 2005; Morandi et al., 2005 Albrecht et al., 1998 Kistner et al., 2005; Morandi et al., 2005 Catoira et al., 2000; Morandi et al., 2005 Kistner et al., 2005 Kistner et al., 2005

–

–

Appþ, Pen


Kistner et al., 2005

–

–

–

–

Appþ, Pen


Murray et al., 2006

–

–

–

–

–

Appþ, Pen


Murray et al., 2006

–

–

pollux‐13 har1‐1 ccamk‐5 har1‐1 castor‐23 har1‐1

calcium/calmodulin‐ dependent kinase –

–

–

–

Appþ, Pen


Murray et al., 2006

a App
: no appressorium formation; Appþ, Pen
: appressoria formed, no root penetration; Sp/Hyred: reduced spore germination and hyphal growth prior to appressorium formation; Appþ, Penþ/
: appressoria formed but root penetration and/or appressorium formation dependent on culture conditions and/or AM fungal isolate.

196

V. GIANINAZZI‐PEARSON ET AL.

Wegel et al., 1998). Inactivation of symbiosis‐related plant genes in L. japonicus also has profound eVects on fungal morphology, inducing extensive branching, swollen or deformed appressoria/hyphopodia on mutant roots (Kistner et al., 2005; Murray et al., 2006). However, detailed observations indicate somatic instability of the mutant phenotype in many cases, with fungal invasion of root tissues occurring as a delayed or rare event depending on plant age, growth conditions, or the AM fungal species involved (Demchenko et al., 2004; Gao et al., 2001; Kistner et al., 2005; Morandi et al., 2005; Paszkowski et al., 2006). Present exceptions appear to be the dmi3/Mtsym13 mutant of M. truncatula and the castor‐2, Ljsym15‐2, pollux‐ 13 har1‐1, ccamk‐5 har1‐1, and castor‐23 har1‐1 mutants of L. japonicus (Table I) (Kistner et al., 2005; Morandi et al., 2005; Murray et al., 2006). The phenotypic modifications in fungal–root interactions in M. truncatula and L. japonicus mutants are accompanied by changes in the molecular dialogue between the symbionts. Inactivation of the symbiosis‐related plant genes alters the perception of fungal signals by roots, including gene responses that are part of signal‐transduction pathways, and abolishes AM‐induced transcriptional activity or protein synthesis (Amiour et al., 2006; Kistner et al., 2005; Sanchez et al., 2005; Weidmann et al., 2004). Legumes are the principle model species for genetic studies of plant determinants regulating early steps in AM formation. This has been largely spurred by the fact that legume mutants impaired in AM are also aVected in nodulation, indicating partially shared genetic programs and setting the scene for a common signal transduction pathway in the two root symbioses (see also Section 2). Early indications that AM fungi and Nod factors share steps in a signal transduction pathway came from the observation that mutation of the pea gene PsSYM8 interferes with ENOD5 and ENOD12A responses to both symbiotic stimuli (Albrecht et al., 1998). Since then, symbiosis‐related gene homologues have been isolated from mutant backgrounds of P. sativum, M. truncatula, Medicago sativa, and L. japonicus. Four sets of genes that are essential for root penetration by AM fungi have been characterized and they encode a leucine‐rich repeat receptor‐like kinase (PsSYM19/MtDMI2/LjSYMRK/MSNORK ) (Endre et al., 2002; Stracke et al., 2002), plastid ion channels (MtDMI1/LjCASTOR/LjPOLLUX) (Ane´ et al., 2004; Imaizumi‐Anraku et al., 2005), a nuclear localized calcium‐ and calmodulin‐binding protein kinase (PsSYM9/MtDMI3/LotusCCamK) (Le´vy et al., 2004; Mitra et al., 2004), and a nucleoporin (LotusNUP33) (Kanamori et al., 2006). These diVerent proteins are also needed for early steps in the Nod factor signaling pathway of the nodulation symbiosis where they participate in intracellular calcium responses. They have been positioned in a model signal transduction pathway where the leucine‐rich repeat

CUES AND COMMUNICATION IN ARBUSCULAR MYCORRHIZA

197

receptor‐like kinase and plastid ion channel proteins, necessary for Nod factor induction of calcium spiking, are upstream of the calcium‐ and calmodulin‐binding protein kinase, which is assumed to be involved in deciphering the calcium signal (Oldroyd and Downie, 2006). Their participation in the AM symbiosis suggests that calcium should function as a second messenger also in mycorrhizal signaling. The use of soybean cell cultures stably expressing the Ca2þ bioluminescent indicator aequorin has recently provided direct evidence of intracellular Ca2þ changes in response to the culture medium of spores of G. margarita germinating in the absence of the plant partner (Navazio et al., 2007). Rapid and transient elevations in cytosolic free Ca2þ were recorded, indicating that diVusible molecules released by the mycorrhizal fungus are perceived by host plant cells through a Ca2þ‐mediated signaling.

IV. MOLECULAR CROSS TALK AND SIGNALING PATHWAYS Novel avenues of research into gene pathways or networks driving the molecular scenario in AM development have opened up with the advent of transcriptomic and proteomic technologies adapted to the analyses of such complex biological systems. Nevertheless, there is still a paucity of information about molecular responses in roots prior to and during initial contact with AM fungi, mainly due to diYculties in synchronizing developmental events in order to dissect gene expression patterns and correlate them with these early stages of the symbiosis. Time‐course transcript profiling of genes activated in functional AM in M. truncatula has identified a number of plant genes that are already induced in response to appressoria and before intraradical mycelium develops (Brechenmacher et al., 2004; Massoumou et al., 2007). Several belong to protein‐encoding gene families that have been implicated in defence strategies against pests or pathogens (glutathione‐S‐ transferase, PR 10 protein, putative wound‐induced protein, germin‐like protein, serine protease, defensin, Kunitz‐type trypsin inhibitor, subtilisin inhibitor). Specific signal molecules produced by pathogens (elicitors) trigger defence reactions in plant tissues. It has been suggested that wall components of AM fungi may also act as elicitors since extracts from extraradical mycelium of G. intraradices can induce phytoalexin synthesis in soybean cotyledons (Lambais, 2000). Enhanced gene expression, protein synthesis, and/or enzyme activities involved in the synthesis of flavonoid compounds have also been reported during the early stages of AM development (Amiour et al., 2006; Bonanomi et al., 2001; Volpin et al., 1994), and increased expression

198

V. GIANINAZZI‐PEARSON ET AL.

of a disease‐related pea gene (PI206) is directly proportional to the number of appressoria developing on roots (Ruiz‐Lozano et al., 1999). Peroxidase, chitinase, catalase, and 1–3 glucanase activities also increase during early AM plant–fungal interactions then decline strongly as root colonization develops (Blilou et al., 2000; David et al., 1998; Lambais and Medhy, 1993; Spanu and Bonfante‐Fasolo, 1988; Spanu et al., 1989; Vierheilig et al., 1994; Volpin et al., 1994). The fact that plant hydrolases, which can attack fungal cell walls (chitinase, 1–3 glucanase), do not prevent root colonization may be explained by a low aYnity or inaccessibility of these enzymes to chitin or 1–3 glucan of the fungal cell wall (Gianinazzi‐Pearson et al., 1996). In conclusion, the amplitude and kinetics of defence responses to AM development diVer markedly from what is typical of plant invasion by pathogenic microorganisms (Gianinazzi‐Pearson et al., 1996) and mechanisms responsible for the AM‐related control have yet to be elucidated. The possible role of symbiosis‐related plant genes in controlling inhibitory root responses has already been evoked. Alternatively, elicitor degradation or the ability of AM fungi to suppress plant defence responses during symbiotic interactions could explain the balance between induction and suppression of plant defence reactions during development of the AM symbiosis. Other plant genes that are upregulated by appressorium formation have been identified in pea and M. truncatula and several have predicted functions in signal perception, transduction, transcription, and/or translation. It has been speculated that a Clp serine protease gene in pea, which is transiently activated during the appressorium stage of fungal–root interactions, may have multiple functions in the control of key regulatory proteins within a signal transduction pathway in the symbiosis (Roussel et al., 2001). Transcripts of another pea gene, PsENOD12A, accumulate when an AM fungus forms appressoria on pea roots and as the epidermis is penetrated (Albrecht et al., 1998). The function of PsENOD12 is unclear but nucleotide sequences indicate that it may encode cell wall proteins induced by AM fungi, in which case it could contribute to assembling the interface compartment initiated prior to cell penetration (see Section 5). Interestingly, expansins and extensins also appear to be involved in initial cell‐to‐cell contact (Weidmann et al., 2004). This class of extracellular proteins may also be operating during epidermal penetration in construction of the interface surrounding the penetrating hypha (Section 5) and/or be involved in producing signaling molecules from the plant cell wall. By targeting gene expression in root systems of M. truncatula where appressorium formation by G. mosseae was synchronized, 21 genes associated with putative signal transduction events have been identified among plant genes activated during this early event in mycorrhiza development (Sanchez et al., 2005; Weidmann et al., 2004).

CUES AND COMMUNICATION IN ARBUSCULAR MYCORRHIZA

199

These M. truncatula genes encode such proteins as a receptor kinase, calcium lipid‐binding protein, casein kinase, 14.3.3 protein that can interact with calcium‐dependent kinases (Camoni et al., 1998), and annexin that belongs to a family of calcium‐binding proteins (Morgan and Pilar Fernandez, 1997). Proteins that change conformation or catalytic activity on binding calcium allow the cellular perception and transduction of the signal generated by intracellular calcium oscillations (Oldroyd and Downie, 2006). The fact that AM fungal molecules can induce a calcium response in soybean cell cultures (see Section 3) highlights the implication of such a calcium‐modulated signaling pathway in early host cell responses to AM fungi. Nitric oxide (NO) is another well‐known signal molecule in animal systems and it has been identified as a key signaling molecule in plants, where it has diverse functions in a broad spectrum of pathophysiological and developmental processes (Wendehenne et al., 2004). It has been hypothesized that NO may also be involved in AM symbiosis (Vieweg et al., 2005; Weidmann et al., 2004). Nitrate reductase (NR) is a central enzyme in nitrogen assimilation in plants (reducing nitrate to nitrite) that can also catalyze the reduction of nitrite, in high concentrations, to NO (Yamasaki and Sakihama, 2000). Plant NR gene expression and protein activity are reduced or not aVected in functional AM (Hildebrandt et al., 2002; Kaldorf et al., 1998; M. Massoumou, S. Jeandroz, and V. Gianinazzi‐Pearson, unpublished data). However, M. truncatula NR gene expression is, in contrast, enhanced in response to appressorium formation by G. mosseae or G. intraradices (Weidmann et al., 2004; Fig. 3) where it seems more likely to be related to NO metabolism

J5 I

6d NI

TRV25 I

10 d NI

T

GSNO

NR NIR Mtgapdh1

Fig. 3. Plant gene activation related to nitric oxide (NO) metabolism in roots of wild‐type (J5) and myc‐mutant (TRV25) Medicago truncatula. Transcript profiling by RT‐PCR of nitrate reductase (NR) and nitrite reductase (NIR) gene expression in roots inoculated (I) or not (NI) by Glomus intraradices BEG141, and after 4 h treatment or not (T) with an NO donor (GSNO). At 6 days (6 d) after inoculation, 3.5 appressoria/cm root developed on wild‐type (J5) plants; 7 appressoria/cm root developed on dmi3/Mtsym3 mutant roots (TRV25) 10 days (10 d) after inoculation. Mtgapdh1 is a constitutively expressed plant gene encoding a glyceraldehyde phosphate dehydrogenase. (M. Massoumou and S. Jeandroz, unpublished data).

200

V. GIANINAZZI‐PEARSON ET AL.

rather than nitrate assimilation. Nitrite reductase (NiR) is likewise suspected to participate in NO production but through another enzymatic pathway (Sto¨hr and Stremlau, 2006), and recent results have shown that NiR gene expression is also stimulated in response to appressorium formation by G. intraradices on M. truncatula roots (Fig. 3). To test the hypothesis that M. truncatula NR and NiR may be related to NO metabolism, noninoculated roots were treated with NO donors (NONOate and GSNO). Monitoring of NR and NiR gene expression showed that, as in the case of appressorium formation, GSNO treatment activates both plant genes (Fig. 1). The mobile nature of NO, and its chemical reactivity with various cell targets, makes it a potentially important molecule in cell responses. The downstream eVects of NO may be induced directly by its interactions with, for example, ion channel proteins or proteins that regulate gene expression, or indirectly by interactions with signaling proteins such as protein kinases or with secondary messenger‐generative enzymes (Neill et al., 2003). In this context, GSNO treatment also increases transcript levels of a M. truncatula MAPK gene (M. Massoumou, S. Jeandroz, and V. Gianinazzi‐Pearson, unpublished data), which is activated by appressorium formation (Weidmann et al., 2004). Finally, plant NR and NiR transcript accumulation is not aVected when appressoria develop on roots of the mycorrhiza‐deficient dmi3/Mtsym13 mutant of M. truncatula (Fig. 1), indicating that NR and NiR gene expression may be regulated in vivo by a complex signal transduction pathway. NO appears to act through cGMP and cADPR to activate intracellular Ca2þ‐ permeable channels, and also plays a role in elevating free cytosolic Ca2þ (Wendehenne et al., 2004). This is particularly relevant in relation to the fact that DMI3 encodes a Ca2þ and calmodulin‐dependent protein kinase in M. truncatula. NO production during AM interactions has to be verified in situ, using fluorescent specific probes like DAF‐2A, and its role in the symbiosis elucidated. Interestingly, NO production occurs in functional nodules in Rhizobium interactions where it appears to be unrelated to defence or cell death activation (Baudouin et al., 2006). Considerably less is known about fungal processes involved in plant recognition and signal transduction prior to root penetration, or those active in regulating hyphal growth arrest and cytoplasm retraction that occurs after spore germination in the absence of a host root. A gene of G. mosseae encoding a putative hedgehog protein with GTPase activity (GmGIN1) is mainly expressed during spore germination prior to contact with the plant and completely shut down during symbiosis (Requena et al., 2002). Such a protein could be involved in the signaling cascade controlling growth arrest and further programmed cell death of hyphae in the absence of a signal from the host plant. An analysis of transcriptome modifications in germinated

CUES AND COMMUNICATION IN ARBUSCULAR MYCORRHIZA

201

sporocarps of G. mosseae, triggered in synchrony with appressorium formation on a host root, identified 27 upregulated genes putatively encoding proteins with functions in signaling, transduction, general cell metabolism, defence/stress responses, or of unknown function (Breuninger and Requena, 2004). Some proteins have a potential role in Ca2þ‐based signaling pathways (calmodulin, leucine zipper protein, Ca2þ‐induced Ras inactivator, Ca2þ‐ATPase) that may be an indication of Ca2þ as a second messenger in the fungal perception of a plant signal leading to appressorium formation. Calcium‐ and calmodulin‐dependent signaling during appressorium formation has been demonstrated in pathogenic fungi.

V. PLANT CELL RESPONSES TO FUNGAL COLONIZATION: TISSUE AND CELL SPECIFICITY Morphological observations dating back to the seventies carefully demonstrated that an AM fungus is always surrounded by a membrane of host origin, enclosed in a newly built apoplastic compartment, when it develops within root cells (Scannerini and Bonfante, 1983). The development of in situ protocols (immunolabeling and in situ hybridization), new technological platforms (confocal microscopy and in vivo imaging), the availability of transformed plants with GUS or GFP marker genes and of mutants impaired in their colonization capabilities, as well as the improvement of in vitro mycorrhization systems, have all contributed to the achievement of new insights into the understanding of plant cell responses to root colonization. Liverwort thalli and fern gametophytes are examples of AM‐like associations that do not take place in root organs (Bonfante, 1984), demonstrating that not only the root organ architecture, but even the diploid stage, is not a strict prerequisite to successful colonization by symbiotic fungi. These observations may also have some relevance from an evolutionary point of view. First land plants known to host AM‐like fungi, like Aglaophyton, did not possess true roots (Remy et al., 1994) and the colonized tissues were derived from a vegetative meristem. Strictly speaking, adventitious roots have the same origin and a large number of experiments have demonstrated that these roots are highly susceptible to AM colonization. Therefore, it can be concluded that the first symbiotic niche of AM fungi was epigeous tissues and that even today they do not always require a root for the colonization of plant tissues. Interestingly, rhizobial colonization of stem tissues has been reported in the literature (Goormachtig et al., 2004), although this is limited to tropical legumes and often let aside by mainstream research on nodulation. This suggests that both so‐called root symbioses can be considered as

202

V. GIANINAZZI‐PEARSON ET AL.

present forms of previous interactions where organ specificity was not a rule. On the other hand, it is known that leaf pathogens like Magnaporthe may colonize roots (Sesma and Osbourn, 2004) with a developmental program largely overlapping their better known behavior above ground. Altogether, fungi interacting with plant tissues may be more versatile than generally considered with regards to the host organs they target. In contrast, multicellular tissue organization seems to be a mandatory requirement for fungal colonization: isolated or cultured cells are never colonized, even when put under strong fungal pressure, as in laboratory experiments (Fig. 4A and B). For example, when soybean cells are cocultured with germinating spores of G. margarita, a dense hyphal web develops to surround the individual cells, which often become attached to the hyphae. However, specific adhesion structures such as appressoria are never observed (B. Baldan, L. Navazio, and P. Mariani, unpublished data), suggesting that signaling events triggered by appressorium induction are inoperative in such cell cultures. That said, not all cell types in an organized tissue can be colonized: only epidermal and cortical cells apparently represent a convenient niche for AM fungi. Root meristems and diVerentiating tissues are never colonized, neither are the endodermis, the vascular tissues, or specialized cortical cells, such as idioblasts or those containing raphides or accumulating phenols. To our knowledge, evidence‐based explanations for such a selective colonization pattern have not yet been proposed. Existence of a plant control over AM fungal colonization is obvious from cellular and genetic evidence. It can be hypothesized that plant accommodation

Fig. 4. Gigaspora margarita–Glycine max interactions. (A) Dark field microscopy image showing contacts among hyphae and cultured soybean cells. No appressoria or cell penetration events are present. Bar ¼ 15 mm. (B. Baldan, unpublished data). (B) Brightfield image showing cotton blue‐stained arbuscules in the cortex of a colonized root. Bar ¼ 30 mm.

CUES AND COMMUNICATION IN ARBUSCULAR MYCORRHIZA

203

responses are only programmed in epidermal and cortical tissues, although the mechanisms involved remain obscure. Root responses to AM fungi can therefore be described on a spatial and temporal scale, depending on the cell type involved (epidermis, cortex) and the corresponding step in root colonization. While investigations of expression profiles for AM‐regulated plant genes have demonstrated profound diVerences with respect to the timescale (Liu et al., 2003), the notion that epidermal and cortical cells may be programmed diVerently is a relatively new concept (Genre and Bonfante, 2005). AM colonization is normally the result of multiple, isolated infection points scattered on the root surface, the development of which is not usually synchronized. In addition, it is diYcult to observe the colonization process in vivo without inactivating the symbiosis and destroying tissue vitality. It is only with the achievement of in vitro mycorrhization of root organ cultures (Chabaud et al., 2002) that critical advances have been possible toward the direct observation of the living interaction. This has also opened the way to studying plant responses even before fungal contact, highlighting the presence of a molecular dialogue between the partners, until then completely unexplored (Kosuta et al., 2003). The root epidermis is the first barrier to all soil microorganisms during their colonization process and it is therefore likely to be the site of recognition mechanisms as well as accommodation/defence responses (Parniske, 2004). As already mentioned, mycorrhizal mutant plants have pointed to the importance of epidermal cells in early steps in the establishment of the AM symbiosis and, as for the Nod factor response in nodule interactions (Oldroyd and Downie, 2006), the epidermis appears to be the site of the earliest plant responses to the still evanescent Myc factor of AM fungi (see Section 2). Rather than being a passive barrier, epidermal cells are an active checkpoint where signal exchanges and a strong control over root colonization occur (Demchenko et al., 2004; Novero et al., 2002). Direct evidence for this has come from the recent description of prepenetration responses designing, a few hours before cell penetration, the track that the AM fungus will subsequently follow in the host cell. In creating an intracellular niche to host another organism within living cells, the AM interaction resembles all other cases of endosymbioses, where the guest organisms are confined into specialized membrane‐bordered spaces. The impact of AM fungi on root cell contents has promoted investigations of the role of the host cytoskeleton as a key structure permitting root cell colonization (Takemoto and Hardham, 2004). While increases in the complexity of microtubule (Blancaflor et al., 2001; Bonfante et al., 1996; Genre and Bonfante, 1997) and actin microfilament (Genre and Bonfante, 1998) organization are striking in arbuscule‐colonized cortical cells, evidence for

204

V. GIANINAZZI‐PEARSON ET AL.

the pivotal role of the cytoskeleton also in fungal accommodation during early stages of root colonization was first provided by studies of the L. japonicus Ljsym4‐2 mutant. When an AM fungus achieves epidermal cell penetration of mutant roots, the absence of a correct cytoskeletal response leads to plant cell death and colonization arrest (Genre and Bonfante, 2002). Taken as a whole, host cell reorganization in response to the symbiotic fungus can be explained by the need for preserving cell integrity, on the one hand, and for optimizing reciprocal compatibility, on the other. In particular, the repositioning of cytoskeleton, as well as endoplasmic reticulum (ER) and Golgi bodies mediating localized membrane proliferation and cell wall deposition, is most likely involved in the construction between symbiont cells of the novel interface compartment.

VI. INTERFACE BIOGENESIS: NEW FACTS/NEW HYPOTHESES The above‐mentioned technological advances have made live cell imaging possible, especially of the epidermal tissue, which is most easily accessible to direct microscopic observation. An important advance in understanding the mechanisms of interface construction in AM interactions has come from the combined application of several up‐to‐date techniques. The first description of in vivo epidermal cell responses to fungal contact was possible through in vivo imaging of GFP‐labeled cell components in mycorrhizal root organ cultures using confocal microscopy (Genre et al., 2005). The main discovery from this research stands in the observation of a novel, ephemeral apparatus, the prepenetration apparatus (PPA), which is assembled from the first moment of surface contact between an AM appressorium and the root, and which is supposed to be responsible for the assembly of the interface compartment prior to cell penetration. The PPA is organized in the epidermal cell cytoplasm below the appressorium as soon as this fungal structure develops at the root surface and remains visible for a few hours, until fungal penetration occurs (Fig. 5). This short life span, together with the possible diYculties in preserving such a membrane structure, accounts for the lack of earlier observations of the PPA. In detail, appressorium contact with the outer epidermal cell triggers the repositioning of the plant nucleus in vicinity of the contact site (Fig. 5A and B). This repositioning, which occurs in a couple of hours, is accompanied by the assembly of localized patches of ER, reorganization of cytoskeleton, and the subsequent appearance of a polarized array of microfilaments radiating from the contact area. After this first step, the nucleus starts to migrate anew,

CUES AND COMMUNICATION IN ARBUSCULAR MYCORRHIZA

A

B

C

205

D

Fig. 5. Scheme of the prepenetration apparatus (PPA) development in a root epidermal cell of Medicago truncatula. Upon contact with the appressorium of an arbuscular mycorrhizal fungus (A), the plant nucleus repositions underneath the contact site (B). A second nuclear migration toward the inner tangential wall coordinates the assembly of a transcellular column of cytoplasm, containing a central membranous thread (C). Finally, a penetration hypha crosses the cell wall and grows within the new‐built interface compartment as the PPA disassembles (D).

toward the cell wall that faces the cortex. This movement is accomplished in 2–4 h and corresponds to complete development of the PPA. It results in a column of cytoplasm being assembled between the nucleus and the contact site, containing a very high density of cytoskeletal fibers and ER cisternae. When nuclear migration terminates, a fine thread of plasma membrane has been laid down in the middle of the PPA to form an apoplastic tunnel (Fig. 5C). Only then fungal penetration occurs and, interestingly, the hypha grows exactly along the route traced by the PPA (Fig. 5D), which then starts to disassemble about 6–8 h after appressorium contact. These observations unambiguously demonstrate an active control by the plant cell over the infection process: fungal development is arrested at the stage of appressorium until the apoplastic tunnel—the future interface—is completed. In addition, intracellular fungal growth is confined to the new compartment, indicating that the epidermal cell has traced in advance a route that the hyphal tip will follow in order to access the root inner tissues. The homology between the PPA and the Rhizobium‐induced infection thread in nodule symbioses is a visible sign of the evolutionary relationship between the two symbioses. The existence of the PPA also opens several new questions about AM interactions (Smith et al., 2006), especially concerning the specificity of such a response to AM fungi or any root‐penetrating microorganism, or the nature of the local signal(s) triggering cell polarization and PPA orientation. In an attempt to address these questions, experiments where GFP‐transformed roots are challenged with diVerent fungi or by mere physical stimulations using a micromanipulator have been designed (A. Genre and coworkers, unpublished data). Initial observations from these experiments suggest that nuclear repositioning can be triggered by physical contact, while

206

V. GIANINAZZI‐PEARSON ET AL.

the assembly of ER patches only occurs in the presence of a living fungus. Furthermore, a completely assembled PPA has only been observed in the presence of AM fungi. Altogether, these observations suggest the existence of a specific fungal signal that orientates plant responses, shifting them toward fungal accommodation and, as a first requirement, interface assembly. As a counterpart of the plant strigolactones, perceived by AM fungi as signals of the presence of a potential host (see Section 2), such fungal signals or Myc factors are supposed to be the main actors of the signaling dialogue that precedes and accompanies root colonization. The molecular programs that regulate early signal exchange and transduction between AM symbionts is of uttermost importance for the mycorrhizal scientific community and one of the key questions in deciphering communication in the symbiosis.

VII. CONCLUSIONS Many of the compounds found in root exudates display both antifungal and antibacterial activities. Thus, it is likely that several secreted compounds act synergistically. This may be true also for stimulatory compounds. Flavonoids have been shown to either stimulate or inhibit AM establishment, depending of the stage of the symbiosis considered and the nutrient status of the plant. Strigolactones have recently been described as new rhizosphere signals involved in early interactions between AM symbionts. The role of these new molecules in the presymbiotic stage is quite well described now, even if the mechanism of action on the fungal cell is still not elucidated. However, research is still in its infancy and their potential role in other stages of the symbiosis is not known. Likewise, the combined eVects with flavonoids or other unknown active molecules are not yet described. Their role as essential molecules for AM symbiosis establishment will be assessed only when plant mutants unable to produce strigolactones will be available. The AM symbiosis is considered nonspecific, but some diVerences in functional compatibility are becoming apparent. Multiple stimulatory molecules capable of eliciting diVerential growth responses in AM fungi may provide the mechanisms by which plants favor interactions with a given symbiont. Root exudate profiles from model plants would be necessary to describe these molecules. The low concentration of active molecules, which hampered characterization of the branching factor for some years, is no longer a problem, thanks to improved isolation protocols and mass spectrometry. The coming years should see an impetus in this new field of plant research.

CUES AND COMMUNICATION IN ARBUSCULAR MYCORRHIZA

207

The process of compatibility between plants and AM fungi requires a complex cascade of events, some of which are starting to be deciphered. Pathways underlying the early cell processes leading to successful establishment of an AM symbiosis must require activation of specific signal transduction‐related events in both partners. Although the structure of AM fungal signals has yet to be identified, it is already evident that some steps are shared in legume perception of symbiotic Nod and Myc factors. Convergence of genetical, transcriptional, and physiological evidence points to calcium as a secondary messenger in AM interactions and to the involvement of a calcium‐modulated signaling pathway in early host cell responses to AM fungi. The implication of plant NO in the initial molecular dialogue between AM symbionts is also an emerging possibility. A challenge for future research will be to unravel the complexity of such symbiosis signaling and to provide new insights into the specificity of the molecular dialogue of host roots with AM fungi (Harrison, 2005). The discovery of new symbiosis‐defective plant mutants and further exploitation of the genomic and cDNA resources available for model legumes like M. truncatula and L. japonicus (www.noble.org/MedicagoHandbook/; www.lotusjaponicus.org/) will be instrumental for the identification of the host genes involved. The molecular forces governing the role played by the fungus in the development of the AM symbiosis are more diYcult to define. While it is well acknowledged that plant SYM genes control the accommodation process leading to root colonization, fungal genes that could be involved in such a process are completely unknown. Similarly, the morphogenetic mechanisms determining appressorium diVerentiation, intraradical, and intracellular growth are at least as obscure. Advances are hampered by the limited amount of sequence information presently accessible in public databases for AM fungi (e.g., ESTs in NCBI ¼ 10,000 for Glomus and 1100 for Gigaspora). However, breakthroughs are expected within the near future from the ongoing international eVort to sequence the first genome of an AM fungus, G. intraradices, and to expand on the number of available EST sequences (http://darwin.nmsu.edu/~fungi/).

ACKNOWLEDGMENTS Contributions to this chapter were partly funded by MIUR projects (FIRB and PRIN), CEVIOBEM as well as by CNR grants (P.B.), and by the Conseil Re´gional de Bourgogne (V.G.‐P. and S.J.). We thank M. Massoumou (INRA), P. Seddas (INRA), and B. Baldan (CNR) for access to unpublished data, and D. van Tuinen for useful discussion. We apologize to all those researchers whose work could not be included due to page limitations.

208

V. GIANINAZZI‐PEARSON ET AL.

REFERENCES Akiyama, K. and Hayashi, H. (2006). Strigolactones: Chemical signals for fungal symbionts and parasitic weeds in plant roots. Annals of Botany 97, 925–931. Akiyama, K., Matsuoka, H. and Hayashi, H. (2002). Isolation and identification of a phosphate deficiency‐induced C‐glycosyl flavonoid that stimulates arbuscular mycorrhiza formation in melon roots. Molecular Plant‐Microbe Interactions 15, 334–340. Akiyama, K., Matsuzaki, K. and Hayashi, H. (2005). Plant sesquiterpenes induce hyphal branching in arbuscular mycorrhizal fungi. Nature 435, 824–827. Albrecht, C., Geurts, R., Lapeyrie, F. and Bisseling, T. (1998). Endomycorrhizae and rhizobial Nod factors both require SYM 8 to induce the expression of the early nodulin genes PsENOD5 and PsENOD12A. The Plant Journal 15, 605–614. Allen, M. F., Moore, T. S. and Christen, M. (1980). Phytohormone changes in Bouteloua gracilis infected by vesicular‐arbuscular mycorrhizae. I. Cytokinin increases in host plants. Canadian Journal of Botany 58, 371–374. Amijee, F., Tinker, P. B. and Stribley, D. P. (1989). The development of endomycorrhizal root systems. VII. A detailed study of eVects of soil phosphorus on colonization. The New Phytologist 111, 435–446. Amiour, N., Recorbet, G., Robert, F., Gianinazzi, S. and Dumas‐Gaudot, E. (2006). Mutations in DMI3 and SUNN modify the appressorium‐responsive root proteome in arbuscular mycorrhiza. Molecular Plant‐Microbe Interactions 19, 988–997. Ane´, J.‐M., Kiss, G. B., Riely, B. K., Penmetsa, R. V., Oldroyd, G. E. D., Ayax, C., Le´vy, J., Debelle´, F., Baek, J.‐M., Kalo, P., Rosenberg, C., Roe, B. A., et al. (2004). Medicago truncatula DMI1 required for bacterial and fungal symbioses in legumes. Science 303, 1364–1367. Ayling, S. M., Smith, S. E. and Smith, F. A. (2000). Transmembrane electric potential diVerence of germ tubes of arbsucular mycorrhizal fungi responds to external stimuli. The New Phytologist 147, 631–639. Bago, B., Azcon‐Aguilar, C., Goulet, A. and Piche´, Y. (1998). Branched absorbing structures (BAS): A feature of the extraradical mycelium of symbiotic arbuscular mycorrhizal fungi. The New Phytologist 139, 375–388. Bago, B., PfeVer, P. E., Douds, D. D., Brouillette, J., Be´card, G. and Shachar‐Hill, Y. (1999). Carbon metabolism in spores of the arbuscular mycorrhizal fungus Glomus intraradices as revealed by NMR spectroscopy. Plant Physiology 121, 263–272. Balaji, B., Poulin, M.‐J., Vierheilg, H. and Piche´, Y. (1995). Responses of an arbuscular mycorrhizal fungus, Gigaspora margarita, to exudates and volatiles from the Ri T‐DNA transformed roots of non mycorrhizal and mycorrhizal mutants of Pisum sativum L. Sparkle. Experimental Mycology 19, 275–283. Balestrini, R. and Bonfante, P. (2005). The interface compartment in arbuscular mycorrhizae: A special type of cell wall? Plant Biosystems 139, 8–15. Barker, S. J., Tagu, D. and Delp, G. (1998a). Regulation of root and fungal morphogenesis in mycorrhizal symbioses. Plant Physiology 116, 1201–1207. Barker, S. J., Stummer, B., Gao, L., Dispain, I., O’Connor, P. J. and Smith, S. E. (1998b). A mutant of Lycopersicon esculentum Mill. with highly reduced VA mycorrhizal colonization: Isolation and preliminary characterisation. The Plant Journal 15, 791–797.

CUES AND COMMUNICATION IN ARBUSCULAR MYCORRHIZA

209

Baudouin, E., Pieuchot, L., Engler, G., Pauly, N. and Puppo, A. (2006). Nitric oxide is formed in Medicago truncatula‐Sinorhizobium meliloti functional nodules. Molecular Plant‐Microbe Interactions 19, 970–975. Be´card, G. and Fortin, J. A. (1988). Early events of vesicular‐arbuscular mycorrhiza formation on Ri T‐DNA transformed roots. The New Phytologist 108, 211–218. Be´card, G. and Piche´, Y. (1989a). New aspects on the acquisition of biotrophic status by a vesicular‐arbuscular mycorrhizal fungus, Gigaspora margarita. The New Phytologist 112, 77–83. Be´card, G. and Piche´, Y. (1989b). Fungal growth stimulation by CO2 and root exudates in vesicular arbuscular‐mycorrhizal symbiosis. Applied and Environmental Microbiology 55, 2320–2325. Be´card, G. and Piche´, Y. (1990). Physiological factors determining vesicular‐ arbuscular mycorrhizal formation in host and non‐host Ri T‐DNA transformed roots. Canadian Journal of Botany 68, 1260–1264. Be´card, G., Douds, D. D. and PfeVer, P. E. (1992). Extensive hyphal growth of vesicular‐arbuscular mycorrhizal fungi in the presence of CO2 and flavonols. Applied and Environmental Microbiology 58, 821–825. Be´card, G., Taylor, L. P., Douds, D. D., PfeVer, P. E. and Donner, L. (1995). Flavonoids are not necessary plant signal compounds in arbuscular mycorrhizal symbiosis. Molecular Plant‐Microbe Interactions 8, 252–258. Be´card, G., Kosuta, S., Tamasloukht, M., Se´jalon‐Delmas, N. and Roux, C. (2004). Partner communication in the arbuscular mycorrhizal interaction. Canadian Journal of Botany 82, 1186–1197. Bel‐Rhlid, R., Chabot, S., Piche´, Y. and Cheˆnevert, R. (1993). Isolation and identification of flavonoids from Ri T‐DNA transformed roots (Daucus carota) and their significance in vesicular‐arbuscular mycorrhiza. Phytochemistry 33, 1369–1371. Besserer, A., Puech‐Page`s, V., Kiefer, P., Gomez‐Roldan, V., Jauneau, A., Roy, S., Portais, J. C., Roux, C., Be´card, G. and Se´jalon‐Delmas, N. (2006). Strigolactones stimulate arbuscular mycorrhizal fungi by activating mitochondria. PLoS Biology 4, e226. Bianciotto, V., Barbiero, G. and Bonfante, P. (1995). Analysis of the cell cycle in an arbuscular mycorrhizal fungus by flow cytometry and bromodeoxyuridine labelling. Protoplasma 188, 161–169. Blancaflor, E. B., Zhao, L. and Harrison, M. J. (2001). Microtubule organisation in root cells of Medicago truncatula during development of an arbuscular mycorrhizal symbiosis with Glomus versiforme. Protoplasma 217, 154–165. Blilou, I., Bueno, P., Ocampo, J. A. and Garcia‐Garrido, J. M. (2000). Induction of catalase and ascorbate peroxidase activities in tobacco roots inoculated with the arbuscular mycorrhizal Glomus mosseae. Mycological Research 104, 722–725. Bonanomi, A., Oetiker, J. H., Guggenheim, R., Boller, T., Wiemken, A. and Vo¨geli‐ Lange, R. (2001). Arbuscular mycorrhiza in mini‐mycorrhizotrons: First contact of Medicago truncatula roots with Glomus intraradices induces chalcone synthase. The New Phytologist 150, 573–582. Bonfante, P. (1984). Anatomy and morphology of VA mycorrhizae. In ‘‘VA Mycorrhizae’’ (C. L. Powell and D. J. Bagyaraj, eds.), pp. 5–33. CRC Press, Boca Raton, FL. Bonfante, P., Bergero, R., Uribe, X., Romera, C., Rigau, J. and Puigdome´nech, P. (1996). Transcriptional activation of a maize alpha‐tubulin gene in mycorrhizal maize and transgenic tobacco plants. The Plant Journal 9, 737–743.

210

V. GIANINAZZI‐PEARSON ET AL.

Bonfante, P., Genre, A., Faccio, A., Martini, I., Schauser, L., Stougaard, J., Webb, J. and Parniske, M. (2000). The Lotus japonicus LjSym4 gene is required for the successful symbiotic infection of root epidermal cells. Molecular Plant‐ Microbe Interactions 13, 1109–1120. Borisov, A. Y., Voroshilova, V. A., Zhukov, V. A., Zhernakov, A. I., Danilova, T. N., Shtark, O. Y., Naumkina, T. S., Tsyganov, V. E., Madsen, L. H., Sanjuan, J., Olivares, J. Priefer, U. B., et al. (2004). Pea (Pisum sativum) regulatory genes controlling development of nitrogen‐fixing nodules and arbuscular mycorrhiza. In ‘‘Biology of Plant‐Microbe Interactions’’ (I. Tikhonovich, B. Lugtenberg and N. Provorov, eds.), Vol. 4, pp. 502–505. IS‐MPMI, St. Paul, MN. Bouwmeester, H., Matusova, J. R., Zongkui, S. and Beale, M. (2003). Secondary metabolite signalling in host‐parasitic plant interactions. Current Opinion in Plant Biology 6, 358–364. Brechenmacher, L., Weidmann, S., van Tuinen, D., Chatagnier, O., Gianinazzi, S., Franken, P. and Gianinazzi‐Pearson, V. (2004). Expression profiling of up‐ regulated plant and fungal genes in early and late stages of Medicago truncatula‐Glomus mosseae interactions. Mycorrhiza 14, 253–262. Breuninger, M. and Requena, N. (2004). Recognition events in AM symbiosis: Analysis of fungal gene expression at the early appressorium stage. Fungal Genetics and Biology 41, 794–804. Brundrett, M. C. (2002). Coevolution of roots and mycorrhizas of land plants. The New Phytologist 154, 275–304. Bue´e, M., Rossignol, M., Jauneau, A., Ranjeva, R. and Be´card, G. (2000). The pre‐ symbiotic growth of arbuscular mycorrhizal fungi is induced by a branching factor partially purified from plant root exudates. Molecular Plant‐Microbe Interactions 13, 693–698. Burbulis, I. E., Iacobucci, M. and Shirley, B. W. (1996). A null mutation in the first enzyme of flavonoid biosynthesis does not aVect male fertility in Arabidopsis. The Plant Cell 8, 1013–1025. Calantzis, C., Morandi, D., Arnould, C. and Gianinazzi‐Pearson, V. (2001). Cellular interactions between G. mosseae and a Myc‐ dmi2 mutant in Medicago truncatula. Symbiosis 30, 97–108. Camoni, L., Harper, J. F. and Palmgren, M. G. (1998). 14‐3‐3 proteins activated a plant calcium‐dependent protein kinase (CDPK). FEBS Letters 430, 381–384. Catoira, R., Galera, R., de Billy, F., Penmetsa, V., Journet, E.‐P., Mailler, F., Rosenberg, C., Cook, D., Gough, C. and De´narie´, J. (2000). Four genes of Medicago truncatula controlling components of a Nod factor transduction pathway. The Plant Cell 12, 1647–1665. Chabaud, M., Venard, C., Defaux‐Petras, A., Be´card, G. and Barker, D. G. (2002). Targeted inoculation of Medicago truncatula in vitro root cultures reveals MtENOD11 expression during early stages of infection by arbuscular mycorrhizal fungi. The New Phytologist 156, 265–273. Chang, M. and Lynn, D. G. (1986). The haustoria and the chemistry of host recognition in parasitic angiosperms. Journal of Chemical Ecology 12, 561–579. Cook, C. E., Whichard, L. P., Turner, B., Wall, M. E. and Egley, G. H. (1966). Germination of witchweed (Striga lutea Lour.): Isolation and properties of a potent stimulant. Science 154, 1189–1190. David, R., Itzhaki, H., Ginzberg, I., Gafni, Y., Galili, G. and Kapulnik, Y. (1998). Suppression of tobacco basic chitinase gene expression in response to colonisation by the arbuscular mycorrhizal fungus Glomus intraradices. Molecular Plant‐Microbe Interactions 11, 489–497.

CUES AND COMMUNICATION IN ARBUSCULAR MYCORRHIZA

211

David‐Schwartz, R., Badani, H., Smadar, W., Levy, A. A., Galili, G. and Kapulnik, Y. (2001). Identification of a novel genetically controlled step in mycorrhizal colonization: Plant resistance to infection by fungal spores but not extra‐radical hyphae. The Plant Journal 27, 561–569. David‐Schwartz, R., Gadkar, V., Wininger, S., Bendov, R., Galili, G., Levy, A. A. and Kapulnik, Y. (2003). Isolation of a premycorrhizal infection (pmi2) mutant of tomato, resistant to arbuscular mycorrhizal colonization. Molecular Plant‐Microbe Interactions 16, 382–388. Demchenko, K., Winzer, T., Stougaard, J., Parniske, M. and Pawlowski, K. (2004). Distinct roles of Lotus japonicus SYMRK and SYM15 in root colonisation and arbuscule formation. The New Phytologist 163, 381–392. Dickson, S. (2004). The Arum‐Paris continuum of mycorrhizal symbioses. The New Phytologist 163, 187–200. Douds, D. D. and Schenck, N. C. (1991). Relationship of colonization and sporulation by VA mycorrhizal fungi to plant nutrient and carbohydrate contents. The New Phytologist 116, 621–627. Duc, G., Trouvelot, A., Gianinazzi‐Pearson, V. and Gianinazzi, S. (1989). First report of non‐mycorrhizal plant mutants (Myc
) obtained in pea (Pisum sativum L.) and fababean (Vicia faba L.). Plant Science 60, 215–222. Elias, K. S. and Safir, G. R. (1987). Hyphal elongation of Glomus fasciculatus in response to root exudates. Applied and Environmental Microbiology 53, 1928–1933. Endre, G., Kereszt, A., Kevei, Z., Mihacea, S., Kalo, P. and Kiss, G. B. (2002). A receptor kinase gene regulating symbiotic nodule development. Nature 417, 962–966. Franken, P. and Gna¨dinger, F. (1994). Analysis of parsley arbuscular endomycorrhiza: Infection development and mRNA levels of defense related‐genes. Molecular Plant‐Microbe Interactions 7, 612–620. Gadkar, V. R., David‐Schwartz, R., Nagahashi, G., Douds, D. D., Wininger, S. and Kapulnik, Y. (2003). Root exudate of pmi tomato mutant M161 reduces AM fungal proliferation in vitro. FEMS Microbiology Letters 223, 193–198. Gao, L.‐L., Delp, G. and Smith, S. E. (2001). Colonization patterns in a mycorrhiza‐ defective mutant tomato vary with diVerent arbuscular‐mycorrhizal fungi. The New Phytologist 151, 477–491. Gao, L.‐L., Smith, F. A. and Smith, S. E. (2006). The rmc locus does not aVect plant interactions or defence‐related gene expression when tomato (Solanum lycopersicon) is infected with the root fungal parasite, Rhizoctonia. Functional Plant Biology 33, 289–296. Gemma, J. N. and Koske, R. E. (1988). Seasonal variation in spore abundance and dormancy of Gigaspora gigantea and in mycorrhizal inoculum potential of a dune soil. Mycologia 80, 211–216. Genre, A. and Bonfante, P. (1997). A mycorrhizal fungus changes microtubule orientation in tobacco root cells. Protoplasma 199, 30–38. Genre, A. and Bonfante, P. (1998). Actin versus tubulin configuration in arbuscule‐ containing cells from mycorrhizal tobacco roots. The New Phytologist 140, 745–752. Genre, A. and Bonfante, P. (2002). Epidermal cells of a symbiosis‐defective mutant of Lotus japonicus show altered cytoskeleton organisation in the presence of a mycorrhizal fungus. Protoplasma 219, 43–50. Genre, A. and Bonfante, P. (2005). Building a mycorrhizal cell: How to reach compatibility between plants and arbuscular mycorrhizal fungi. Journal of Plant Interactions 1, 3–13.

212

V. GIANINAZZI‐PEARSON ET AL.

Genre, A., Chabaud, M., Timmers, T., Bonfante, P. and Barker, D. (2005). Arbuscular mycorrhizal fungi elicit a novel intracellular apparatus in Medicago truncatula root epidermal cells before infection. The Plant Cell 17, 3489–3499. Gianinazzi‐Pearson, V. and Brechenmacher, L. (2004). Functional genomics of arbuscular mycorrhiza: Decoding the symbiotic cell programme. Canadian Journal of Botany 82, 1228–1234. Gianinazzi‐Pearson, V. and Gianinazzi, S. (1992). Influence of intergeneric grafts between host and non‐host legumes on formation of vesicular‐arbuscular mycorrhiza. The New Phytologist 120, 505–508. Gianinazzi‐Pearson, V., Branzanti, B. and Gianinazzi, S. (1989). In vitro enhancement of spore germination and hyphal growth of a vesicular‐arbuscular mycorrhizal fungus by host root exudates and plant flavonoids. Symbiosis 7, 243–255. Gianinazzi‐Pearson, V., Gollotte, A., Dumas‐Gaudot, E., Franken, P. and Gianinazzi, S. (1994). Gene expression and molecular modifications associated with plant responses to infection by arbuscular mycorrhizal fungi. In ‘‘Advances in Molecular Genetics of Plant‐Microbe Interactions’’ (D. M. Downie and A. E. Osbourn, eds.), Vol. 3, pp. 179–186. Kluwer Academic Publishers, Boston. Gianinazzi‐Pearson, V., Dumas‐Gaudot, E., Gollotte, A., Tahiri‐Alaoui, A. and Gianinazzi, S. (1996). Cellular and molecular defence‐related root responses to invasion by arbuscular mycorrhizal fungi. The New Phytologist 133, 45–57. Gianinazzi‐Pearson, V., Arnould, C., Oufattole, M., Arango, M. and Gianinazzi, S. (2000). DiVerential activation of Hþ‐ATPase genes by an arbuscular mycorrhizal fungus in root cells of transgenic tobacco. Planta 211, 609–613. Gianinazzi‐Pearson, V., Maldonado‐Mendoza, I., Lopez‐Meyer, M., Weidmann, S. and Harrison, M. J. (2007). Arbuscular mycorrhiza. In ‘‘The Medicago Handbook’’ (U. Mathesius, E. P. Journet and L. W. Sumner, eds.), ISBN 0-9754303-1-9. http://www.noble.org/MedicagoHandbook/ Giovannetti, M., Sbrana, C., Citernesi, A. S. and Logi, C. (1993). DiVerential hyphal morphogenesis in arbuscular mycorrhizal fungi during pre‐infection stages. The New Phytologist 125, 587–593. Giovannetti, M., Sbrana, C. and Logi, C. (1994). Early processes involved in host recognition by arbuscular mycorrhizal fungi. The New Phytologist 127, 703–709. Giovannetti, M., Sbrana, C., Citernesi, A. S. and Avio, L. (1996). Analysis of factors involved in fungal recognition responses to host‐derived signals by arbuscular mycorrhizal fungi. The New Phytologist 133, 65–71. Glenn, M. G., Chew, F. S. and Williams, P. H. (1985). Hyphal penetration of Brassica (Cruciferae) roots by a vesicular‐arbuscular mycorrhizal fungus. The New Phytologist 99, 403–472. Glenn, M. G., Chew, F. S. and Williams, P. H. (1988). Influence of glucosynolate content of Brassica (Cruciferae) roots on the growth of vesicular‐arbuscular mycorrhizal fungi. The New Phytologist 110, 217–225. Goldwasser, Y. and Yoder, J. I. (2001). DiVerential induction of Orobanche seed germination by Arabidopsis thaliana. Plant Science 160, 951–959. Gollotte, A., Gianinazzi‐Pearson, V., Giovannetti, M., Sbrana, C., Avio, L. and Gianinazzi, S. (1993). Cellular localization and cytochemical probing of resistance reactions to arbuscular mycorrhizal fungi in a ‘locus A’ myc
mutant of Pisum sativum (L.). Planta 191, 112–122.

CUES AND COMMUNICATION IN ARBUSCULAR MYCORRHIZA

213

Goormachtig, S., Capoen, W. and Holsters, M. (2004). Rhizobium infection: Lessons from the versatile nodulation behaviour of water‐tolerant legumes. Trends in Plant Science 9, 518–522. Graham, J. H. (1982). EVect of citrus root exudates on germination of chlamydospores of the vesicular‐arbuscular mycorrhiza fungus Glomus epigeum. Mycologia 74, 831–835. Graham, J. H., Leonard, R. T. and Menge, J. A. (1981). Membrane mediated decrease in root exudation responsible for phosphorus inhibition of vesicular‐arbuscular mycorrhiza formation. Plant Physiology 68, 548–552. Guenoune, D., Galili, S., Phillips, D. A., Volpin, H., Chet, I., Okon, Y. and Kapulnik, Y. (2001). The defense response elicited by the pathogen Rhizoctonia solani is suppressed by colonization of the AM‐fungus Glomus intraradices. Plant Science 160, 925–932. Gu¨imil, S., Chang, H.‐S., Zhu, T., Sesma, A., Osbourn, A., Roux, C., Ioannidis, V., Oakeley, E. J., Docquier, M., Descombes, P., Briggs, S. P. and Paszkowski, U. (2005). Comparative transcriptomics of rice reveals an ancient pattern of response to microbial colonization. Proceedings of the National Academy of Sciences USA 102, 8066–8070. Harborne, J. B. (1988). ‘‘Flavonoids: Advances in Research,’’ Vol. 2. Chapman and Hall, London, UK. Harrison, M. J. (1999). Molecular and cellular aspects of the arbuscular mycorrhizal symbiosis. Annual Review of Plant Physiology 50, 361–389. Harrison, M. J. (2005). Signaling in the arbuscular mycorrhizal symbiosis. Annual Review of Microbiology 59, 19–42. Harrison, M. J., Dewbre, G. R. and Liu, J. Y. (2002). A phosphate transporter from Medicago truncatula involved in the acquisiton of phosphate released by arbuscular mycorrhizal fungi. The Plant Cell 14, 2413–2429. Herrera‐Medina, M. J., Gagnon, H., Piche´, Y., Juan, A., Ocampo, J. A., Garcia Garrido, J. M. and Vierheilig, H. (2003). Root colonization by arbuscular mycorrhizal fungi is aVected by the salicylic acid content of the plant. Plant Science 164, 993–998. Hildebrandt, U., Schmelzer, E. and Bothe, H. (2002). Expression of nitrate transporter genes in tomato colonized by an arbuscular mycorrhizal fungus. Physiologia Plantarum 115, 125–136. Imaizumi‐Anraku, H., Takeda, N., Charpentier, M., Perry, J., Miwa, H., Umehara, Y., Kouchi, H., Murakami, Y., Mulder, L., Vickers, K., Pike, J. Downie, A. J., et al. (2005). Plastid proteins crucial for symbiotic fungal and bacterial entry into plant roots. Nature 433, 527–531. Jacquelinet‐Jeanmougin, J., Gianinazzi‐Pearson, V. and Gianinazzi, S. (1987). Endomycorrhizas in the Gentianaceae. II. Ultrastructural aspects of symbiont relationships in Gentiana lutea L. Symbiosis 3, 269–286. James, T., KauV, F., Schoch, C., Matheny, P., Hofstetter, V., Cox, C., Celio, G., Gueidan, C., Fraker, E., Miadlikowska, J., Lumbsch, H. T. Rauhut, A., et al. (2006). Reconstructing the early evolution of fungi using a six‐gene phylogeny. Nature 443, 818–822. Jolicoeur, M., Germette, S., Gaudette, M., Perrier, M. and Be´card, G. (1998). Intracellular pH in arbuscular mycorrhizal fungi. Plant Physiology 116, 1279–1288. Kaldorf, M., Schmelzer, E. and Bothe, H. (1998). Expression of maize and fungal nitrate reductase genes in arbuscular mycorrhiza. Molecular Plant‐Microbe Interactions 11, 439–448. Kanamori, N., Madsen, L. H., Radutoiu, S., Frantescu, M., Quistgaard, E. M., Miwa, H., Downie, J. A., James, E. K., Felle, H. H., Haaning, L. L.,

214

V. GIANINAZZI‐PEARSON ET AL.

Jensen, T. H., Sato, S., et al. (2006). A nucleoporin is required for induction of Ca2þ spiking in legume nodule development and essential for rhizobial and fungal symbiosis. Proceedings of the National Academy of Sciences USA 103, 359–364. Kape, R., Wex, K., Parniske, M., Go¨rge, E., Wetzel, A. and Werner, D. (1992). Legume root metabolites and VA mycorrhizal fungus. Journal of Plant Physiology 141, 45–60. Karandashov, V. and Bucher, M. (2005). Symbiotic phosphate transport in arbuscular mycorrhizas. Trends in Plant Science 10, 22–29. Kistner, C., Winzer, T., Pitzschke, A., Mulder, L., Sato, S., Kaneto, T., Tabata, S., Sandal, N., Stougaard, J., Webb, K. J., Szczyglowski, K. and Parniske, M. (2005). Seven Lotus japonicus genes required for transcriptional reprogramming of the root during fungal and bacterial symbiosis. The Plant Cell 17, 2217–2229. Kosuta, S., Chabaud, M., Lougnon, G., Gough, C., De´narie, J., Barker, D. G. and Be´card, G. (2003). A diVusible factor from arbuscular mycorrhizal fungi induces symbiosis‐specific MtENOD11 expression in roots of Medicago truncatula. Plant Physiology 131, 1–11. Lambais, M. R. (2000). Regulation of plant defence‐related genes in arbuscular mycorrhizae. In ‘‘Current Advances in Mycorrhizae Research’’ (G. K. Podila and D. D. Douds, eds.), pp. 45–59. The American Phytopathological Society, MN. Lambais, M. R. and Medhy, M. C. (1993). Suppression of endochitinase, ‐1.3‐ endoglucanase, and chalcone isomerase expression in bean vesicular‐ arbuscular mycorrhizal roots under diVerent soil phosphate conditions. Molecular Plant‐Microbe Interactions 6, 75–83. Lanfranco, L., Novero, M. and Bonfante, P. (2005). The mycorrhizal fungus Gigaspora margarita possesses a CuZn superoxide dismutase that is up‐regulated during symbiosis with legume hosts. Plant Physiology 137, 1319–1330. Larose, G., Cheˆnevert, R., Moutoglis, P., Gagne, S., Piche´, Y. and Vierheilig, H. (2002). Flavonoid levels in roots of Medicago sativa are modulated by the development stage of the symbiosis and the root colonizing arbuscular‐ mycorrhizal fungus. Journal of Plant Physiology 159, 1329–1339. Lei, J., Be´card, G., Catford, J. G. and Piche´, Y. (1991). Root factors stimulate P uptake and plasmalemma ATPase activity in vesicular‐arbuscular mycorrhizal fungus, Gigaspora margarita. The New Phytologist 118, 289–294. Le´vy, J., Bres, C., Geurts, R., Chalhoub, B., Kulikova, O., Duc, G., Journet, E.‐P., Ane´, J.‐M., Lauber, E., Bisseling, T., De´narie´, J., Rosenberg, C., et al. (2004). A putative Ca2þ and calmodulin‐dependent protein kinase required for bacterial and fungal symbioses. Science 303, 1361–1364. Liu, H., Trieu, A. T., Blaylock, L. A. and Harrison, M. J. (1998). Cloning and characterization of two phosphate transporters from Medicago truncatula roots: Regulation in response to phosphate and to colonization by arbuscular mycorrhizal (AM) fungi. Molecular Plant‐Microbe Interactions 11, 14–22. Liu, J., Blaylock, L., Endre, G., Cho, J., Town, C. D., VandenBosch, K. and Harrison, M. J. (2003). Transcript profiling coupled with spatial expression analyses reveals genes involved in distinct developmental stages of the arbuscular mycorrhizal symbiosis. The Plant Cell 15, 2106–2123. Lohse, S., Schliemann, W., Ammer, C., Kopka, J., Stracke, D. and Fester, T. (2005). Organization and metabolism of plastids and mitochondria in arbuscular mycorrhizal roots of Medicago truncatula. Plant Physiology 139, 329–340. Long, S. R. (1996). Rhizobium symbiosis: Nod factors in perspective. The Plant Cell 8, 1885–1898.

CUES AND COMMUNICATION IN ARBUSCULAR MYCORRHIZA

215

Lum, M. R., Li, Y., LaRue, T. A., David‐Schwartz, R., Kapulnik, Y. and Hirsch, A. M. (2002). Investigation of four classes of non‐nodulating white sweet clover (Melilotus alba annua Desr.) mutants and their responses to arbuscular‐ mycorrhizal fungi. Integrative and Comparative Biology 42, 295–303. Lynn, D. G., SteVens, J. C., Kamat, V. S., Graden, D. W., Shabanowitz, J. and Riopel, J. L. (1981). Isolation and characterization of the first host recognition substance for parasitic angiosperms. Journal of the American Chemical Society 103, 1868–1870. Massoumou, M., van Tuinen, D., Chatagnier, O., Arnould, C., Brechenmacher, L., Sanchez, L., Selim, S., Gianinazzi, S. and Gianinazzi‐Pearson, V. (2007). Medicago truncatula gene responses specific to arbuscular mycorrhiza interactions with diVerent species and genera of Glomeromycota. Mycorrhiza 17, 223–234. Matusova, R., Rani, K., Verstappen, F. W., Franssen, M. C., Beale, M. H. and Bouwmeester, H. J. (2005). The Strigolactone germination stimulants of the plant‐parasitic Striga and Orobanche spp. are derived from the carotenoid pathway. Plant Physiology 139, 920–934. Mellersh, D. and Parniske, M. (2006). Common symbiosis genes of Lotus japonicus are not required for intracellular accommodation of the rust fungus Uromyces loti. The New Phytologist 170, 641–644. Mithofer, A. (2002). Suppression of plant defence in rhizobia‐legume symbiosis. Trends in Plant Science 7, 440–444. Mitra, R. M., Gleason, C. A., Edwards, A., Hadfield, J., Downie, J. A., Oldroyd, G. E. D. and Long, S. R. (2004). A Ca2þ/calmodulin‐dependent protein kinase required for symbiotic nodule development: Gene identification by transcript‐based cloning. Proceedings of the National Academy of Sciences USA 101, 4701–4705. Morandi, D., Prado, E., Sagan, M. and Duc, G. (2005). Characterisation of new symbiotic Medicago truncatula (Gaertn.) mutants, and phenotypic or genotypic complementary information on previously described mutants. Mycorrhiza 15, 283–289. Morgan, R. and Pilar Fernandez, M. (1997). Distinct annexin subfamilies in plants and protists diverged prior to animal annexins from a common ancestor. Journal of Molecular Evolution 44, 178–188. Mosse, B. (1959). The regular germination of resting spores and some observations on the growth requirements of Endogone sp. causing vesicular‐arbuscular mycorrhiza. Transactions of the British Mycological Society 42, 273–286. Mosse, B. (1988). Some studies relating to ‘‘independent’’ growth of vesicular‐ arbuscular endophytes. Canadian Journal of Botany 66, 2533–2540. Mosse, B. and Hepper, C. M. (1975). Vesicular‐arbuscular mycorrhizal infections in root organ cultures. Physiology and Plant Pathology 5, 215–223. Murray, J., Geil, J., Wagg, C., Karas, B., Szczyglowski, K. and Peterson, R. L. (2006). Genetic suppressors of Lotus japonicus har1‐1 hypernodulation show altered interactions with Glomus intraradices. Functional Plant Biology 33, 749–755. Nagahashi, G. and Douds, D. D. (1996). Phosphorus amendment inhibits hyphal branching of the VAM fungus Gigaspora margarita directly and indirectly through its eVects on root exudation. Mycorrhiza 6, 403–408. Nagahashi, G. and Douds, D. D. (1999). Rapid and sensitive bioassay to study signals between root exudates and arbuscular mycorrhizal fungi. Biotechnology Techniques 13, 893–897. Nagahashi, G. and Douds, D. D. (2000). Partial separation of root exudate components and their eVects upon the growth of germinated spores of AM fungi. Mycological Research 104, 1453–1464.

216

V. GIANINAZZI‐PEARSON ET AL.

Nair, M. G., Safir, G. R. and Siqueira, J. O. (1991). Isolation and identification of vesicular‐arbuscular mycorrhiza stimulatory compounds from clover (Trifolium repens) roots. Applied and Environmental Microbiology 57, 434–439. Navazio, N., Moscatiello, R., Genre, A., Novero, M., Baldan, B., Bonfante, P. and Mariani, P. (2007). A diVusible signal from arbuscular mycorrhizal fungi elicits a transient cytosolic calcium elevation in host plant cells. Plant Physiology, 144, 673–681. Neill, S. J., Desikan, R. and Hancock, J. T. (2003). Nitric oxide signalling in plants. The New Phytologist 159, 11–35. Novero, M., Faccio, A., Genre, A., Stougaard, J., Webb, K. J., Mulder, L., Parniske, M. and Bonfante, P. (2002). Dual requirement of the LjSym4 gene for mycorrhizal development in epidermal and cortical cells of Lotus japonicus roots. The New Phytologist 154, 741–749. Ola´h, B., Brie`re, C., Be´card, G., De´narie´, J. and Gough, C. (2005). Nod factors and a diVusible factor from arbuscular mycorrhizal fungi stimulate lateral root formation in Medicago truncatula via the DMI1/DMI2 signalling pathway. The Plant Journal 44, 195–207. Oldroyd, G. E. D. and Downie, J. A. (2004). Calcium, kinases and nodulation signalling in legumes. Nature Reviews 5, 566–576. Oldroyd, G. E. D. and Downie, J. A. (2006). Nuclear calcium changes at the core of symbiosis signalling. Current Opinion in Plant Biology 9, 351–357. Parniske, M. (2004). Molecular genetics of the arbuscular mycorrhizal symbiosis. Current Opinion in Plant Biology 7, 414–421. Paszkowski, U., Jakovleva, L. and Boller, T. (2006). Maize mutants aVected at distinct stages of the arbuscular mycorrhizal symbiosis. The Plant Journal 47, 165–173. Pawlowska, T. E. (2005). Genetic processes in arbuscular mycorrhizal fungi. FEMS Microbiology Letters 251, 185–192. Peters, N. K. and Verma, D. P. (1990). Phenolic compounds as regulators of gene expression in plant‐microbe relations. Molecular Plant‐Microbe Interactions 3, 4–8. Phillips, D. A. (2000). Biosynthesis and release of rhizobial nodulation gene inducers by legumes. In ‘‘Prokaryotic Nitrogen Fixation: A Model System for the Analysis of a Biological Process’’ (E. W. Triplett, ed.), pp. 349–364. Horizon Scientific, Wyndham, UK. Pinior, A., Wyss, U., Piche´, Y. and Vierheilig, H. (1999). Plants colonized by AM fungi regulate further root colonization by AM fungi through altered root exudation. Canadian Journal of Botany 77, 891–897. Poulin, M. J., Bel‐Rhlid, R., Piche´, Y. and Cheˆnevert, R. (1993). Flavonoids released by carrot (Daucus carota) seedlings stimulate hyphal development of vesicular‐arbuscular mycorrhizal fungi in the presence of CO2 enrichment. Journal of Chemical Ecology 19, 2317–2327. Poulin, M.‐J., Simard, J., Catford, J.‐G., Labrie, F. and Piche´, Y. (1997). Response of symbiotic endomycorrhizal fungi to estrogens and anti‐estrogens. Molecular Plant‐Microbe Interactions 10, 481–487. Powell, C. L. (1976). Development of mycorrhizal infections from Endogone spores and infected root segments. Transactions of the British Mycological Society 66, 439–445. Ratnayake, M., Leonard, R. T. and Menge, J. A. (1978). Root exudation in relation to supply of phosphorus and its possible relevance to mycorrhizal formation. The New Phytologist 81, 543–552. Reinhardt, D. (2007). Programming good relations—development of the arbuscular symbiosis. Current Opinion in Plant Biology 10, 1–8.

CUES AND COMMUNICATION IN ARBUSCULAR MYCORRHIZA

217

Remy, W., Taylor, T. N., Hass, H. and Kerp, H. (1994). Four hundred‐million‐year‐ old vesicular arbuscular mycorrhizae. Proceedings of the National Academy of Sciences USA 91, 11841–11843. Requena, N., Mann, P., Hampp, R. and Franken, P. (2002). Early developmentally regulated genes in the arbuscular mycorrhizal fungus Glomus mosseae: Identification of GmGIN1, a novel gene with homology to the C‐terminus of metazoan hedgehog proteins. Plant and Soil 244, 129–139. Rossiter, R. C. and Beck, A. B. (1996). Physiological and ecological studies on the oestrogenic isoflavones in subterranean clover. Australian Journal of Agriculture Research 17, 447–456. Roussel, H., van Tuinen, D., Franken, P., Gianinazzi, S. and Gianinazzi‐Pearson, V. (2001). Signalling between arbuscular mycorrhizal fungi and plants: Identification of a gene expressed during early interactions by diVerential RNA display analysis. Plant and Soil 232, 13–19. Ruiz‐Lozano, J. M., Roussel, H., Gianinazzi, S. and Gianinazzi‐Pearson, V. (1999). Defence genes are diVerentially induced by a mycorrhizal fungus and Rhizobium in wild type and symbiosis‐defective pea genotypes. Molecular Plant‐Microbe Interactions 12, 976–984. Saint‐John, T. V., Hays, R. I. and Reid, C. P. (1983). Influence of a volatile compound on formation of vesicular‐arbuscular mycorrhizas. Transactions of the British Mycological Society 81, 153–154. Sanchez, L., Weidmann, S., Arnould, C., Bernard, A. R., Gianinazzi, S. and Gianinazzi‐Pearson, V. (2005). P. fluorescens and G. mosseae trigger DMI3‐dependent activation of genes related to a signal transduction pathway in roots of Medicago truncatula. Plant Physiology 139, 1065–1077. Scannerini, S. and Bonfante, P. (1983). Comparative ultra structural analysis of mycorrhizal associations. Canadian Journal of Botany 61, 917–923. Schreiner, R. P. and Koide, R. T. (1993). Mustards, mustard oils and mycorrhizas. The New Phytologist 123, 107–113. Schwab, S. M., Menge, J. A. and Leonhard, R. T. (1983). Quantitative and qualitative eVects of phosphorus on extracts and exudates of sudan grass in relation to vesicular arbuscular mycorrhiza formation. Plant Physiology 73, 761–765. Schwab, S. M., Menge, J. A. and Tinker, P. B. (1991). Regulation of nutrient transfer between host and fungus in vesicular‐arbuscular mycorrhizas. The New Phytologist 117, 387–398. Schu¨ßler, A., Schwarzott, D. and Walker, C. (2001). A new fungal phylum, the Glomeromycota: Phylogeny and evolution. Mycological Research 105, 1413–1421. Sesma, A. and Osbourn, A. E. (2004). The rice leaf blast pathogen undergoes developmental processes typical of root‐infecting fungi. Nature 431, 582–586. Shaul‐Keinan, O., Gadkar, V., Gintzberg, I., Gru¨nzweig, J. M., Chet, I., Elad, Y., Wininger, S., Belausov, E., Eshed, Y., Atzmon, N., Ben‐Tal, Y. and Kapulnik, Y. (2002). Hormone concentrations in tobacco roots change during arbuscular colonization with Glomus intraradices. The New Phytologist 154, 501–507. Siqueira, J. O., Hubbel, D. H. and Schenck, N. C. (1982). Spore germination and germ tube growth of a vesicular‐mycorrhizal fungus in vitro. Mycologia 74, 952–959. Siqueira, J. O., Safir, G. R. and Nair, M. G. (1991). Significance of phenolic compounds in plant soil microbial systems. Critical Reviews in Plant Science 10, 63–121. Smith, S. E. and Read, D. J. (1997). ‘‘Mycorrhiza Symbiosis,’’ 2nd Edn. Academic Press, San Diego, CA.

218

V. GIANINAZZI‐PEARSON ET AL.

Smith, S. E., Barker, S. J. and Zhu, Y.‐G. (2006). Fast moves in arbuscular mycorrhizal symbiotic signalling. Trends in Plant Science 11, 369–371. Spanu, A. U. and Bonfante‐Fasolo, P. (1988). Cell‐wall‐bound peroxidase activity in roots of mycorrhizal Allium porrum. The New Phytologist 109, 119–124. Spanu, P., Boller, T., Ludwig, A., Wiemken, A., Faccio, A. and Bonfante‐Fasolo, P. (1989). Chitinase in roots of mycorrhizal Allium porrum: Regulation and localization. Planta 177, 447–455. Stracke, S., Kistner, C., Yoshida, S., Mulder, L., Sato, S., Kaneko, T., Tabata, S., Sandal, N., Stougaard, J., Sczyblowski, K. and Parniske, M. (2002). A plant receptor‐like kinase required for both bacterial and fungal symbiosis. Nature 417, 959–962. Sto¨hr, C. and Stremlau, S. (2006). Formation and possible roles of nitric oxide in plant roots. Journal of Experimental Botany 57, 463–470. Takemoto, D. and Hardham, A. R. (2004). The cytoskeleton as a regulator and target of biotic interactions in plants. Plant Physiology 136, 3864–3876. Tamasloukht, M. B., Sejalon‐Delmas, N., Kluver, A., Roux, C., Be´card, G. and Franken, P. (2003). Root factors induce mitochondrial‐related gene expression and fungal respiration during the developmental switch from asymbiosis to presymbiosis in the arbuscular mycorrhizal fungus Gigaspora rosea. Plant Physiology 131, 1468–1478. Tawaraya, K., Sasai, K. and Wagatsuma, T. (1994). EVect of phosphorus application on the content of amino acids and reducing sugars in the rhizosphere and VA mycorrhizal infection of white clover. Soil Science and Plant Nutrition 40, 539–543. Tawaraya, K., Watanabe, S. and Yoshida, E. (1996). EVect of onion (Allium cepa) root exudates on the hyphal growth of Gigaspora margarita. Mycorrhiza 6, 57–59. Tawaraya, K., Hashimoto, K. and Wagatsuma, T. (1998). EVect of root exudate fractions from P‐deficient and P‐suYcient onion on root colonization by the arbuscular mycorrhizal fungus Gigaspora margarita. Mycorrhiza 8, 67–70. Toth, R. and Miller, R. M. (1984). Dynamics of arbuscule development and degeneration in a Zea mays mycorrhiza. American Journal of Botany 71, 449–460. Tre´panier, M., Be´card, G., Moutoglis, P., Willemot, C., Gagne´, S., Avis, T. J. and Rioux, J. A. (2005). Dependence of arbuscular mycorrhizal fungi on their plant host for palmitic acid synthesis. Applied and Environmental Microbiology 71, 5341–5347. Tsai, S. M. and Phillips, D. A. (1991). Flavonoids released naturally from alfalfa promote development of symbiotic Glomus spores in vitro. Applied and Environmental Microbiology 57, 1485–1488. Vierheilig, H. and Ocampo, J. A. (1990). EVect of isothiocyanates on germination of spores of Glomus mosseae. Soil Biology and Biochemistry 22, 1161–1162. Vierheilig, H., Alt, M., Mohr, U., Boller, T. and Wiemken, A. (1994). Ethylene biosynthesis and activities of chitinase and ‐1,3‐glucanase in the roots of host and non‐host plants of vesicular‐arbuscular mycorrhizal fungi after inoculation with Glomus mosseae. Journal of Plant Physiology 143, 337–343. Vierheilig, H., Alt, M., Ma¨der, P., Boller, T. and Wiemken, A. (1995). Spreading of Glomus mosseae, a vesicular‐arbuscular mycorrhizal fungus, across the rhizosphere of host and non‐host plants. Soil Biology and Biochemistry 27, 1113–1115. Vierheilig, H., Bago, B., Albrecht, C., Poulin, M.‐P. and Piche´, Y. (1998a). Flavonoids and arbuscular‐mycorrhizal fungi. In ‘‘Flavonoids in the Living System’’ (J. A. Manthey and B. S. Buslig, eds.), pp. 9–33. Plenum Press, New York.

CUES AND COMMUNICATION IN ARBUSCULAR MYCORRHIZA

219

Vierheilig, H., Alt‐Hug, M., Engel‐Streitwolf, R., Ma¨der, P. and Wiemken, A. (1998b). Studies on attractional eVect of root exudates on hyphal growth of an arbuscular mycorrhizal fungus in soil compartment‐membrane system. Plant and Soil 203, 133–144. Vieweg, M. F., Hohnjec, N. and Ku¨ster, H. (2005). Two genes encoding truncated hemoglobins are regulated during root nodule and arbuscular mycorrhiza symbioses of Medicago truncatula. Planta 220, 757–766. Volpin, H., Elkind, Y., Okon, Y. and Kapulnik, Y. (1994). A vesicular arbuscular mycorrhizal fungus (Glomus intraradix) induces a defense response in alfalfa roots. Plant Physiology 104, 683–689. Walker, T. S., Bais, H. P., Grotewold, E. and Vivenco, J. M. (2003). Root exudation and rhizosphere biology. Plant Physiology 132, 44–51. Wegel, E., Schauser, L., Sandal, J., Stougaard, J. and Parniske, M. (1998). Mycorrhiza mutants of Lotus japonicus define genetically independent steps during symbiotic infection. Molecular Plant‐Microbe Interactions 11, 933–936. Weidmann, S., Sanchez, L., Descombin, J., Chatagnier, O., Gianinazzi, S. and Gianinazzi‐Pearson, V. (2004). Fungal elicitation of signal transduction‐ related plant genes precedes mycorrhiza establishment and requires the dmi3 gene in Medicago truncatula. Molecular Plant‐Microbe Interactions 17, 1385–1393. Wendehenne, D., Durner, J. and Klessig, D. F. (2004). Nitric oxide: A new player in plant signalling and defence responses. Current Opinion in Plant Biology 7, 449–455. Westwood, J. H. (2000). Characterization of the Orobanche‐Arabidopsis system for studying parasite‐host interactions. Weed Science 48, 742–748. Xie, Z. P., Staehelin, C., Vierheilig, H., Wiemken, A., Jabbouri, S., Broughton, W. J., Vo¨geli‐Lange, R. and Boller, T. (1995). Rhizobial nodulation factors stimulate colonization of nodulating and nonnodulating soybeans. Plant Physiology 108, 1519–1525. Yamasaki, H. and Sakihama, Y. (2000). Simultaneous production of nitric oxide and peroxynitrite by plant nitrate reductase: In vitro evidence for the NR‐dependent formation of active nitrogen species. FEBS Letters 468, 89–92.

Dynamic Defense of Marine Macroalgae Against Pathogens: From Early Activated to Gene‐Regulated Responses

AUDREY COSSE, CATHERINE LEBLANC AND PHILIPPE POTIN

Centre National de la Recherche Scientifique, Universite´ Pierre et Marie Curie‐Paris6, Laboratoire International Associe´ «Dispersal and Adaptation in Marine Species», Unite´ Mixte de Recherche 7139 ‘‘Marine Plants and Biomolecules,’’ Station Biologique, BP 74, F29682 RoscoV Cedex, France

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . II. Dynamic Defense of Marine Algae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Pathogen Perception in Higher Eukaryotes ............................... B. Recognition of the Attacker(s) in Seaweeds ............................... III. The Oxidative Burst Machinery in Marine Algae . . . . . . . . . . . . . . . . . . . . . . . . . A. Oxidative Burst in Higher Plants: Definition, Effects, and Involved Enzymes ........................................................ B. DPI‐Sensitive Oxidative Burst in Algae–Pathogen Interactions ....... C. NADPH Oxidase‐Like Genes in Algae .................................... D. Other Sources of ROS in Algae–Pathogen Interactions ................. IV. The Defensive Role of Halogenation in Marine Algae . . . . . . . . . . . . . . . . . . . . A. Halogen Metabolism and Halogenating Enzymes in Algae ............ B. A Direct Role in Cell Protection ............................................ C. Chemical Defense Molecules ................................................ V. Oxylipins as Mediators of Defense Responses in Marine Algae. . . . . . . . . . . A. Lipase‐Activated Release and Lipoxygenase‐Mediated Transformation of FFAS .................................................... B. Oxylipin Pathways in Marine Algae ........................................ C. Biological and Ecological Functions of Oxylipins........................ VI. Transcriptional Responses in Marine Algae: Mining Defense Genes . . . . . A. Genomic and Transcriptomic Data in Marine Algae .................... B. Mining Defense‐Related Gene in Brown Algae........................... C. Mining Defense‐Related Gene in Red Algae.............................. Advances in Botanical Research, Vol. 46 Incorporating Advances in Plant Pathology Copyright 2008, Elsevier Ltd. All rights reserved.

222 224 224 226 228 228 229 230 232 233 233 235 236 238 239 240 242 245 245 247 250

0065-2296/08 $35.00 DOI: 10.1016/S0065-2296(07)46006-2

222

A. COSSE ET AL.

VII. Conclusions and Prospects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 252 Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 254 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 254

ABSTRACT In striking contrast with the knowledge on biotic interactions in terrestrial crop or wild plants, defense induction and regulation have hardly been studied in marine benthic algae (seaweeds), which are nonvascular, multicellular, photosynthetic ‘‘marine plants.’’ This chapter highlights recent progresses which have been made during the last decade in the understanding of activated and induced‐defense mechanisms in seaweeds. It appears that marine plants resemble terrestrial plants and animals in their basic mechanisms for pathogen recognition and signaling, including the oxidative burst machinery, the generation of halogen oxidants, and the peroxidation of fatty acids. Sharing a number of common traits of the innate immunity responses with higher eukaryotes, brown and red seaweeds provide ideal models to test the hypothesis that these essential cell functions have arisen in the sea. The recent development of important genomic resources in marine algae, including whole‐genome projects and large expressed sequence tags (ESTs) libraries will reinforce this research field.

I. INTRODUCTION Macrophytic marine algae are nonvascular, multicellular, and photosynthetic eukaryotes. These ‘‘marine plants,’’ commonly known as seaweeds, dominate the rocky intertidal or submerged reef‐like habitats in coastal regions of ocean temperate and cold waters, as well as in tropical coral reefs. Unlike most microscopic algae (microalgae), seaweeds, such as kelps and rockweeds, are mostly sessile organisms attached to the bottom (benthic), produce high biomass, and determine the structure of the ecosystem (i.e., kelp forests). Most phylogenetic studies support an ancient origin of photosynthetic eukaryotes (algae and plants) with the primary endosymbiosis that gave rise to the first alga having occurred after the split of the Plantae (i.e., Rhodophyta—red algae, the Glaucophyta, and the Viridiplantae—green algae plus land plants) from the opisthokonts sometime before about 1.5 billions years ago (Yoon et al., 2004). The remaining algae obtained their plastid through secondary or tertiary endosymbiosis. In the first case, a nonphotosynthetic protist engulfed a red or a green alga (Gibbs, 1993) resulting in a ‘‘secondary’’ plastid, whereas in the second case, an alga containing a secondary plastid was engulfed (Yoon et al., 2005). Phaeophyceae (brown algae) belong to the same lineage as unicellular Bacillariophyceae (diatoms) and oomycetes, stemming from a secondary endosymbiosis between a plastid‐less protist and an ancestral unicellular red alga (Baldauf, 2003). It results that marine algae define a vast assemblage of autotrophic organisms, including lineages of unicellular

MARINE MACROALGAE AGAINST PATHOGENS

223

microscopic algae and three lineages comprising multicellular benthic macroalgae (green, red, and brown seaweeds). Benthic macroalgae are confronted by a continuous challenge with microorganisms and eukaryotic propagules and also with grazers. Wild populations of marine algae are often plagued by various pathogens including bacteria (Sawabe et al., 1998, 2000), oomycetes (Ku¨pper and Mu¨ller, 1999; Sparrow, 1969), plasmodiophoraleans (Maier et al., 2000), viruses (Mu¨ller et al., 1998), filamentous algae, and pathogenic endophytes (Correa et al., 1988; Ellertsdottir and Peters, 1997). Among the pathologies recently recorded in marine macroalgae, several studies show marine bacteria as causative agents. For example, this is the case in coralline lethal orange disease, a severe infection aVecting various coral‐reef‐building coralline algae (Littler and Littler, 1995). Pseudoalteromonas bacteriolytica was identified as the marine bacterium that is the causative agent of red spot disease of the maricultured algal species Laminaria japonica (Sawabe et al., 2000). Harvell and colleagues proposed that the frequency of disease among marine macroorganisms has increased in recent decades and may continue to increase, because of climate change and human activities that stress hosts, introduce pathogens to new areas, and provide microbes with favorable conditions for growth (Harvell et al., 1999, 2002). Such diseases may be highly destructive, as reported for the kombu (Ishikawa and Saga, 1989) and nori (Fujita et al., 1972) aquaculture fields. These colonizers cause disease, degradation, inhibition of photosynthesis, increase in hydrodynamic drag, and many other detrimental eVects. Consequently, seaweeds have evolved a variety of defensive mechanisms against being colonized. Seaweeds depend on their chemical repertoire to influence interactions with other organisms and with the environment (Paul et al., 2006b). A part of these chemicals may provide constitutive barriers against grazers or parasites. Seaweeds constitutively produce a plethora of secondary metabolites, many of which can act as antimicrobial compounds during defense against microorganisms (de Nys and Steinberg, 2002; Kubanek et al., 2003) or grazers (Paul and Puglisi, 2004). However, considering the fundamental question of the investment of physiological resources in defense structures or metabolites (Amsler and Fairhead, 2006), it is obvious that marine algae have also developed activated and induced‐defense mechanisms. However, in striking contrast with the knowledge on host–pest interactions in terrestrial crop or wild plants, defense induction and regulation have hardly been studied in marine algae. The last decade was marked by significant progress in the understanding of signaling events and biochemical responses involved in activated and induced‐ defense mechanisms of marine algae toward pathogen attack. In this chapter, we focus on basic mechanisms for pathogen recognition and signaling,

224

A. COSSE ET AL.

including the oxidative burst machinery, the generation of halogen oxidants, and the peroxidation of fatty acids (Fig. 1). We also refer to work about responses of phytoplankton to grazing (Pohnert, 2005) and to wound‐ activated responses in both micro‐ and macroalgae (Pohnert, 2004), which share common transduction and defense pathways with responses to pathogens in macroalgae. One important objective of the chapter is also to highlight recent developments in the field of marine genomics which will allow apply powerful transcriptomic approaches in the field of algal pathology for deciphering defense metabolic pathways and the mode of action of some defensive or hormone‐like chemicals. For further information on ecological aspects, the reader is referred to other reviews about algal pathology (Andrews, 1976, 1977; Bouarab et al., 2001a; Correa and Sanchez, 1996) and chemical ecology (Amsler and Fairhead, 2006; Ianora et al., 2006; Paul and Puglisi, 2004; Paul et al., 2006b).

II. DYNAMIC DEFENSE OF MARINE ALGAE A. PATHOGEN PERCEPTION IN HIGHER EUKARYOTES

In plant–pathogen interactions, a key diVerence between resistance and susceptibility to the infection is the timely recognition of the invading pathogen, which allows a rapid and eVective activation of the host defense mechanisms. Resistant plants are capable of rapidly deploying a wide variety of defense responses that prevent pathogen ingress whereas susceptible plants exhibit much slower and weaker responses that fail to restrict pathogen growth and spreading. In both host‐specific gene for gene interactions and nonhost resistance, cell–cell recognition between host and pathogen involves signals referred to as elicitors. A growing body of evidence indicates that a major common feature of innate immunity in animals and higher plants is the capability to recognize invariant microbe‐ or pathogen‐associated molecular patterns (MAMPs or PAMPs) that are characteristic of microorganisms but that are not found in potential hosts (Medzhitov and Janeway, 2002; Nu¨rnberger et al., 2004). MAMPs include cell wall components of microorganisms such as peptidoglycans, lipoteichoic acid (LTA) of Gram‐positive bacteria, and lipopolysaccharides (LPS) of Gram‐negative bacteria. LPS have been recognized as elicitors of innate immunity in plants (Silipo et al., 2005). The induction of an oxidative burst (Gerber et al., 2004; Meyer et al., 2001) and the activation of phosphorylation cascades (Gerber and Dubery, 2004) by LPS from bacterial plant pathogens have been shown in terrestrial plants. It was

Sea water

Exogenous elicitor Pathogen Herbivore

Toxicity for pathogen

VHOCs Halogen Metabolism HPO

Endogenous elicitor Cell wall degrading Cell wall

Receptor (?)

K+ channel

O2

NADPH oxidase

Plasma membrane

SOD

O2−

H2O2

Phospholipase

NADPH

NADP+

Cytoplasm

Accumulation of phenolic compounds

Galactolipase Ions fluxes Free fatty acids

H2O2

Protein kinases Phosphatases

Catalase

Oxylipin pathway

Glutation peroxidase cycle

O2

H2O PAL Phenylpropanoid pathway

Cross-linking cell wall

Halide fluxes

Ascorbate/ Glutathione cycle Shikimate pathway

X−

Lipid peroxidation

Ca2+ channel Oxidative Pentose Phosphate pathway

X+

LOX

MAPK

H2O2

Redox State Altered

Activation of defense genes Nucleus

Fig. 1. Representative scheme of defense reactions induced in seaweed/pathogen interactions. Recognition of pathogen‐derived or endogenous elicitors by algal cells triggers the production of signaling molecules, which either activate defense‐specific biochemical pathways or directly aVect cell metabolism. SOD, superoxide dismutase; LOX, lipoxygenase; MAPK, mitogen activated protein kinase; PAL, phenylalanine ammonia lyase; HPO, haloperoxidase; VHOCs, volatile halogenated organic compounds.

226

A. COSSE ET AL.

demonstrated that one of the most prominent features of animal innate immunity, LPS‐mediated NO production, is apparent in higher plants in response to a variety of LPS from animal or plant pathogens and from rhizobacteria (Zeidler et al., 2004). In terrestrial plants, an important class of elicitors of nonhost resistance is constituted by oligosaccharins, that is, polysaccharide fragments with signaling activities (Coˆte´ and Hahn, 1994; Coˆte´ et al., 1998; Darvill et al., 1992; John et al., 1997; Ryan and Farmer, 1991), but also glycoproteins and glycopeptides (Ebel and Cosio, 1994). The relevance of elicitors such as oligosaccharins as in vivo factors in defense systems is supported by their possible natural occurrence during plant–microbe interactions (Fritig et al., 1998). In contrast, in marine plant–microbe interactions, a few pathosystems provided cues to understand the mechanisms of pathogen recognition (Bouarab et al., 2001b; Potin et al., 1999, 2002). B. RECOGNITION OF THE ATTACKER(S) IN SEAWEEDS

The first few examples of attack recognition through the perception of endogenous (i.e., released by the host) oligosaccharide elicitors were simultaneously reported in the brown alga Laminaria digitata and the red alga Gracilaria conferta. In L. digitata, it was shown that oligosaccharides derived from alginate, the main component of its cell wall, elicit a strong, rapid, and transient oxidative burst. A clear structure–activity relationship determines the capacity of alginate oligosaccharides to elicit an oxidative burst in L. digitata (Ku¨pper et al., 2001). A strong response is elicited only by the homo‐ oligomeric fragments of poly‐
‐1,4‐L‐guluronic acid, a structural conformation similar to that of the poly‐
‐1,4‐L‐galacturonic acid blocks of pectins, which allows intermolecular associations in the presence of calcium ions. In G. conferta, bacterial degradation products of its main cell wall polysaccharides, namely agarose, trigger an oxidative burst and can induce necrosis similar to the symptoms of the white tip bacterial disease reported in pond cultures of this alga (Weinberger et al., 1999). The oligoagars that were most active in eliciting the oxidative response in G. conferta were the dodeca‐ to hexadeca‐saccharides (Weinberger et al., 2001). This recognition mechanism appears to be as finely tuned as that recognizing glucan or pectin elicitors in higher plants: only 0.01–0.5 mM concentrations of the optimally sized agaro‐oligosaccharides were necessary to induce half‐maximal reactions. As agar oligomers readily adopt a helicoidal conformation, and as it takes just three disaccharidic repeating units to complete the pitch of the agarose helix, it is likely that oligoagars are

MARINE MACROALGAE AGAINST PATHOGENS

227

perceived by G. conferta in their ordered single‐ or double‐helix conformations rather than as random coils. In this aspect of specific elicitor generation and recognition, a clear relation of structure–activity was observed showing a common feature with pathogen perception in higher plants (Fig. 1). Still, it is not clear so far whether recognition of bacteria relies solely on endogenous elicitors such as oligoguluronates and oligoagars, released from the algal cell wall during bacterial attack, or whether algae can also recognize exogenous elicitors, like MAMPs. Whilst there is a certain body of knowledge on exogenous elicitors in higher plants (Wojtaszek, 1997), there is only one report of a bacterial elicitor of the marine red alga G. conferta (Weinberger and Friedlander, 2000) mentioning low molecular weight (700–1500 Da) peptide elicitors, without their exact structure being known. Emission of H2O2 was described in L. digitata following the application of LPS from various sources such as pathogenic bacteria of mammals and marine bacteria (Ku¨pper et al., 2006). This study is the first report of an oxidative burst in the context of LPS perception in any algal lineage. LPS from Salmonella arbortus equi, which induced severe inflammatory responses, including oxidative burst in mammalian hosts, was proved to be the most eYcient in triggering an oxidative burst in L. digitata. Although the oxidative burst is considerably delayed, it is in similar range to that observed after elicitation by oligoguluronates and is also sensitive to diphenylene diodonium (DPI). The destructive association established between the carrageenophytic red alga Chondrus crispus and its green algal endophytic pathogen Acrochaete operculata provides another example of attack recognition through the perception of pathogen elicitors. The green alga A. operculata is a primary invasive organism of C. crispus, which features an isomorphic life history. When zoospores settle and germinate, the vegetative filaments of the parasite completely invade the host diploid sporophytic fronds. In contrast, haploid gametophytic fronds are not infected beyond the epidermis and outer cortex (Bouarab et al., 1999; Correa and McLachlan, 1992, 1994; Correa et al., 1988). The life cycle phases of C. crispus diVer by the degree of sulfation of their cell wall carrageenans. However, in contrast to G. conferta or L. digitata, C. crispus does not recognize carrageenan oligosaccharides as defense elicitors, but cell‐free extracts from the pathogen A. operculata elicit an oxidative burst in C. crispus gametophytes (Bouarab et al., 1999). Interestingly, C. crispus gametophytes synthesize ultraviolet (UV)‐absorbing compounds around the sites of A. operculata zoospore penetration, whereas this response is absent in the sensitive generation (Bouarab et al., 2004). These findings are reminiscent of the deposition of phenolic compounds in higher plant–pathogen interactions (McLusky et al., 1999). Although the exact

228

A. COSSE ET AL.

structure of these UV‐absorbing compounds remains to be elucidated, their aromatic nature suggests that the perception of the pathogen induces the phenylpropanoid metabolism. Indeed, A. operculata extracts activated two key enzymes of this pathway, shikimate dehydrogenase and phenylalanine ammonia‐lyase (Bouarab et al., unpublished data). During the last decade, a few experimental studies have shown that brown seaweeds may also specifically recognize attackers, such as grazers, as pointed by the specific induction of defensive terpenes, such as pachydictol A, dictyol B acetate, and dictyodiol in Dictyota (Cronin and Hay, 1996) and of phlorotannins in Fucales, which function as defense compounds is more controversial (Arnold and Targett, 2002; Pavia and Toth, 2000; Sotka et al., 2002; Taylor et al., 2002; Toth and Pavia, 2000; Toth et al., 2005). Therefore, inducible defense mechanisms can be an important strategy to cope with some herbivores (Ianora et al., 2006). However, in comparison to terrestrial plant–herbivore interactions, signal perception and spreading is poorly understood (Ianora et al., 2006; Toth and Pavia, 2000). The search for the chemical nature of herbivore cues and water‐borne signals has not been successful up to now.

III. THE OXIDATIVE BURST MACHINERY IN MARINE ALGAE A. OXIDATIVE BURST IN HIGHER PLANTS: DEFINITION, EFFECTS, AND INVOLVED ENZYMES

In green terrestrial plants, the production of reactive oxygen species (ROS) via consumption of oxygen in a so‐called oxidative burst is one of the earliest responses following pathogen recognition. Apoplastic generation of superoxide (O
2 ) or its dismutation product hydrogen peroxide (H2O2) has been documented following recognition of variety of pathogens. Avirulent pathogens, recognized via the action of disease resistance R gene products in plant immune system, elicit a biphasic ROS accumulation with low‐amplitude, transient first phase, followed by a sustained phase of much higher magnitude that correlates with disease resistance (Lamb and Dixon, 1997). Virulent pathogens that avoid host recognition induce only the transient low amplitude first phase of this response, suggesting a role for ROS in the establishment of the defenses. In line with this conclusion, elicitors of defense response also trigger an oxidative burst (Torres et al., 2006). ROS may have direct cytotoxic eVects on pathogen (Mellersh et al., 2002). In plants cells, ROS can directly cause strengthening of host cell wall via cross‐ linking of glycoproteins and also lipid peroxidation (Lamb and Dixon, 1997).

MARINE MACROALGAE AGAINST PATHOGENS

229

It is also evident that ROS are important signals inducing other defense lines and mediating defense gene activation (Hancock et al., 2001; Levine et al., 1994; Neill et al., 2002). Thus, they orchestrate a variety of secondary responses, like synthesis of pathogenesis‐related protein and phytoalexins. In most plant–pathogen interaction, a DPI‐sensitive, membrane located NADPH oxidase represents the enzymatic source of ROS (Torres and Dangl, 2005). The NADPH oxidase, also known as the respiratory burst oxidase (RBO), was initially described in mammalian phagocytes as a multicomponent complex mediating microbial killing (Lambeth, 2004). Gp91phox is the enzymatic subunit of this oxidase. It is responsive for electron transfer to molecular oxygen to generate O
2 . Since the late 1990, a number of homologues of gp91phox, named respiratory burst oxidase homologues (rboh), have been discovered in higher plants (Bolwell, 1999). Several reports demonstrate that the members of rboh family mediate the production of ROS during defense responses (Torres and Dangl, 2005). Moreover, unlike mammals, higher plants seem to possess a unique protein required for NADPH oxidase activity. Like in mammals, in higher plants such as Solanaceae in which several NADPH oxidases have been isolated, each isoform may have distinct implication in generation of defense reactions and probably diVerent functions (Torres and Dangl, 2005). As shown using virus‐ induced gene silencing, rboh homologues encoded by genes constitutively expressed contribute to phase I of the oxidative burst, whereas rboh homologue genes which are specifically induced by elicitors provide de novo synthesized proteins required for phase II of the burst (Yoshioka et al., 2001).

B. DPI‐SENSITIVE OXIDATIVE BURST IN ALGAE–PATHOGEN INTERACTIONS

In marine macroalgae, an oxidative burst, DPI sensitive, has also been reported upon the perception of pathogen and algal cell wall‐derived elicitors (Bouarab et al., 1999; Ku¨pper et al., 2001; Weinberger and Friedlander, 2000). In L. digitata, the oligoguluronate‐induced H2O2 emission was shown to control the growth of epiphytic, potentially pathogenic bacteria (Ku¨pper et al., 2001, 2002). This elicitation also increases the resistance of the alga to infection by its brown algal endophyte Laminariocolax tomentosoides (Ku¨pper et al., 2002). Similar observations are made in the red alga G. conferta, where the oligoagar‐elicited oxidative burst results in the elimination of the bacterial epiflora (Weinberger and Friedlander, 2000). The occurrence of an oxidative burst associated with the perception of cell‐free extracts of its specific pathogen, the green algal endophyte A. operculata, was shown to play a crucial role in the resistance of C. crispus (Bouarab et al., 1999).

230

A. COSSE ET AL.

Thus, these studies suggest that the oxidative burst is involved in natural and induced immunity of algae (Fig. 1). In the diVerent studies, where a DPI‐sensitive, presumably membrane bound NADPH oxidase has been suggested being involved in H2O2 production, some common steps of the signal transduction conducting to the enzyme activation have been shown. In L. digitata (Ku¨pper et al., 2001), C. crispus (Bouarab, 2000), and G. conferta (Weinberger et al., 2005a,b), several lines of pharmacological evidence suggest that the activation of NADPH‐oxidase involved requires the opening of ion channels (Kþ, Naþ, Hþ, and halides), calcium translocation, the activation of phospholipases, and the involvement of protein kinases. In these cases, the oxidative burst is associated with a refractory state. After a first elicitation with oligosaccharides, subsequent challenges with the same elicitor remain ineVective during several hours. These observations are correlated with pharmacological analysis, indicating involvement of phosphorylation events. Moreover, they suggest the existence of a specific receptor for oligosaccharides. No report has yet been published concerning the occurrence of an oxidative burst in response to herbivore attacks in algae, whereas it is known that early wound responses in plants involved the production of ROS, as in pathogen responses (Orozco‐Cardenas and Ryan, 1999; Orozco‐Cardenas et al., 2001). However, it has been shown that mechanical injuries induced ROS production in the red alga Euchema platycladum (Colle´n et al., 1994) and in the green alga Dasycladus vermicularis (Ross et al., 2005a). This coenocytic green alga is able to form rapid wound plugs to prevent cytoplasmic loss, upon injury (Ross et al., 2005b). In D. vermicularis, the DPI inhibition of ROS production suggests that the second phase of wound repair is based on the activation of a putative NADPH‐oxidase enzyme, 35 min after injury, leading to a micromolar‐level production of H2O2. This latent oxidative burst is proposed to be involved, through catalysis by peroxidases, in oxidative cross‐linking of phenolic cell wall components, during plug browning and hardening (Ross et al., 2005a). C. NADPH OXIDASE‐LIKE GENES IN ALGAE

A gene encoding a homologue of RBO gp91phox, named Ccrboh, present in a single copy, has been isolated from C. crispus (Herve´ et al., 2006). During infection of C. crispus gametophyte by A. operculata zoospores, Ccrboh is induced and maintained at high level during at least 24 h, whereas it is expressed at a very basal low level in standard conditions. Induction of Ccrboh mRNA accumulation occurred when germinating zoospores started to penetrate through the host cell wall. Such enhancement taking place

231

MARINE MACROALGAE AGAINST PATHOGENS

during the first hours of pathogen attack may reveal a role of Ccrboh in early defense response. Added to Ccrboh gene identified, search performed in available genome and expressed sequence tags (ESTs) algal databases permitted to identify sequences showing common features of NADPH oxidases in other algae such as the red unicellular Cyanidioschyzon merolae, the red macroalga Porphyra yezoensis, and the two diatoms Phaeodactylum tricornutum and Thalassiosira pseudonana. Any sequence showing similarity to rboh genes has been found in L. digitata (Herve´ et al., 2006). It is interesting to note that the length of diatoms amino acid sequence was approximately the same size as that of the human gp91phox, whereas the red algal RBO were at least 200 amino acids longer. These amino acids are inserted between the two NADPH‐binding sites forming four additional transmembrane domains compared to the architecture of gp91phox subunits in NADPH oxidases in other organisms. All the rboh homologs identified present some features common to NADPH oxidases, contributing to the catalytic activity, such as the FAD and NADPH binding sites, but no calcium binding EF‐hand motif was found as an extension of the N‐terminal end of algal enzymes (Fig. 2). Indeed, the phylogenetic analysis revealed that NADPH oxidases homologues in red algae and diatoms constitute an independent cluster, which emerged early in evolution from a common ancestor of the ferric

gp91phox in neutrophils

Membrane Cytoplasm

rboh in red algae

Membrane Cytoplasm

Fig. 2. Schematic representation of human gp91phox and red algae homologues. Indicated are the orientation of the protein in the membrane with the transmembrane domains (barrels), the putative position of the two histidine residues (H), the FAD and the NADPH binding sites. Adapted from Herve´ et al. (2006).

232

A. COSSE ET AL.

reductase and NADPH oxidases (Herve´ et al., 2006). These results unvalidate the hypothesis of Lalucque and Silar which postulated that NADPH oxidase constitute the redox signaling system that allows the gaining of multicellularity in the course of evolution (Lalucque and Silar, 2003). D. OTHER SOURCES OF ROS IN ALGAE–PATHOGEN INTERACTIONS

Several other sources have been suggested for the production of ROS in higher plants, including apoplastic peroxidases (Martinez et al., 1998), as well as various oxidases such as oxalate oxidase (Zhang et al., 1995), amine oxidase (Rea et al., 2002), carbohydrate oxidase (Custers et al., 2004), or other oxidases (Thordal‐Christensen et al., 1997). There is also some evidence of the occurrence of other sources of ROS in marine algae. 1. Gracilaria conferta versus Gracilaria chilensis: Two diVerent sources of ROS production Contrarily to the expectation of a conserved mechanism of elicitor perception in related species, G. chilensis markedly departs from G. conferta in its response to incubation with agar oligosaccharides (Weinberger et al., 2005b). While this latter species recognizes agar oligosaccharides as a primary elicitor, which triggers the activation of an NADPH oxidase followed by a potent oxidative burst, agar oligosaccharides are the substrate of an agar oxidase in G. chilensis, only leading to a moderated release of H2O2. Using an electron microscopy technique to detect ROS, it was shown that the agar oxidase enzyme is apoplastic whereas the NAPDH oxidase is localized at the plasma membrane (Weinberger et al., 2005b). In contrast with G. conferta, in G. chilensis, a repeated supply of agar oligosaccharides results in production of H2O2. A refractory state was not observed, suggesting that oligoagar played a role as a substrate for the production of ROS rather than as a signal. Like in G. conferta, pharmacological experiments and microscopic observations were carried out in G. chilensis. The release of H2O2 was sensitive to some inhibitors of flavoenzymes and salicylhydroxamic acid (SHAM), which are known to interfere with the action mode of various metalloenzymes. KCN and NaN3, two well‐ known inhibitors of heme and copper enzymes, also inhibited the response. Indeed, there are several evidences that an enzymatic oxidation of oligoagar occurred generating aldehydes accumulation in the medium. Microscopic observations confirmed that an oxidase is present in the cell wall of G. chilensis. This enzyme presumably catalyzes the transfer of electron from oligoagar to molecular oxygen, thereby generating H2O2 and oxidized oligoagar. Only oligoagar larger than disaccharide proved to be suitable

MARINE MACROALGAE AGAINST PATHOGENS

233

substrates for oligoagar oxidase, and only agarase can generate these saccharides. Up to now, cell macerating microorganisms are the only known source of agarase. It is therefore reasonable to suspect a role of oligoagar oxidase, which is constitutively expressed, in a latent defense of G. chilensis against agar hydrolysis (Weinberger et al., 2005b). 2. Enzymatic sources of ROS in C. crispus challenged with pathogen extracts Some aspects of biochemical bases of C. crispus immunity and pathogen virulence have been highlighted above. The sulfated polysaccharides from the cell wall matrix of C. crispus were shown to control endophyte penetration, with ‐carrageenans from the sensitive sporophytic generation increasing and ‐carrageenans from the resistant gametophytes reducing the endophyte virulence (Bouarab et al., 1999). In response to the perception of ‐carrageenan oligosaccharides, A. operculata secretes a nitrogen storage compound, L‐asparagine, which in return induces H2O2 emission by the red alga (Weinberger et al., 2002). It has been reported that secreted L‐asparagine is the substrate of an L‐amino acid oxidase in the host (Weinberger et al., 2005a). The settlement of A. operculata zoospores on thallus sporophyte can be drastically reduced by the addition of L‐asparagine and this eVect was prevented by simultaneous addition of catalase. In the gametophyte, the half of bacterial epiflora is eliminated by addition of L‐asparagine. In this view, it is presumed that L‐asparagine does not act as a specific signal for a receptor in C. crispus and that L‐amino acid oxidase from the host is involved in the control of the early stages of the infection by A. operculata (Weinberger et al., 2005a).

IV. THE DEFENSIVE ROLE OF HALOGENATION IN MARINE ALGAE A. HALOGEN METABOLISM AND HALOGENATING ENZYMES IN ALGAE

A rapid response, likely to constitute a chemical defense specific to the marine environment, is the emission of volatile halogenated organic compounds (VHOCs). An increased production of such iodinated, brominated, or chlorinated carbon skeletons is associated with oxidative stress from various origins (Mtolera et al., 1996). Marine organisms and especially seaweeds are known for a long time to concentrate halides from their environment. Hence, the element iodine has been discovered in the ashes of brown algal kelps by Courtois in the beginning of the 19th century (Courtois, 1813). In L. digitata, the iodine content

234

A. COSSE ET AL.

can reach 4.7% of dry weight in young plantlets, corresponding to a concentration factor of up to 106 (Ku¨pper et al., 1998), but it shows some variations depending on the algal tissue, age, season, and location (Ar Gall et al., 2004). Concerning bromine, red algae belonging to the Ceramiales, are the highest accumulators with up to 3.7% of dry weight (Saenko et al., 1978). Up to now, it is diYcult to draw a general feature about halide contents, especially in relation with phylogenetic data. Because of the importance of iodine in human health, studies have been focused on iodine contents in commercial seaweeds (Teas et al., 2004), and showed that this element is mainly present as iodide (Hou et al., 1997; Shah et al., 2005). Iodine accumulators also present high level of bromine (Saenko et al., 1978), suggesting common uptake pathway and retention species. Nevertheless, the central question still remains the biological function of halides in algae. In marine macroalgae, a particular class of peroxidases, the vanadium‐ dependent haloperoxidases (vHPOs), are likely to play a central role both in the iodine and bromine uptake and in the production of iodinated, brominated, or chlorinated low molecular weight carbon skeletons, referred to as VHOCs (Butler and Carter‐Franklin, 2004; Leblanc et al., 2006). Haloperoxidases catalyze the oxidation of halides in the presence of H2O2 to produce a diVusible halogen intermediate Xþ (XOH, X2, X
3 ) which can halogenate various organic substrates. They are named according to the most electronegative halide that they can oxidize, that is, chloroperoxidases (CPOs) can catalyze the oxidation of chloride as well as of bromide and iodide, bromoperoxidases (BPOs) react with bromide and iodide, whereas iodoperoxidases (IPOs) are specific of iodide. In red and brown macroalgae, vHPO activities have been widely detected and most of them have been identified as BPOs, whereas nonheme haloperoxidases have been identified in the green lineage (Vilter, 1995). L. digitata featured two distinct vHPO gene families, one coding for BPO activities, and another coding for IPOs (Colin et al., 2003, 2005; Vilter, 1995). These latter enzymes could be involved in the highly eYcient mechanism of iodine accumulation in kelps (Colin et al., 2003; Ku¨pper et al., 1998). In microalgae, halogenation of organic compounds is known to involve halide methyl transferases (Manley, 2002; Moore et al., 1996) and no vHPO has been yet identified on genomic data obtained from diatoms (Scala et al., 2002). A novel type of enzyme hydroperoxide halolyase, which generates halogenated aldehydes, has been described in the marine diatom Stephanopyxis turris (Wichard and Pohnert, 2006). Several putative vHPO homologues and dehalogenase enzymes have also been identified in EST databases of C. crispus and Ectocarpus siliculosus (Colle´n et al., 2006; Cock, personal communication), highlighting the importance of halogen metabolism in red and brown algae.

235

MARINE MACROALGAE AGAINST PATHOGENS

Chemical defense

Volatile halocarbons

I−, Br−

I2

Sea water Cell wall

Org. XOH,

X3−,

X2

ROS scavenging

Cell wall strengthening by oxidative crosslinking

vHPO

I−, Br−

H2O2 Plasma membrane

NADPH oxidase

Anion channel? Cytoplasm

Fig. 3. Schematic view of putative biological roles of vanadium‐dependent haloperoxidases (vHPOs) and of halogen metabolism in defense responses of marine macroalgae. Org, organic compound.

One way to establish the biological function of halides is to study the biochemical specificity, the regulation and the subcellular localization of the enzymes, directly involved in halogenation processes in seaweeds. In brown algal kelps, vHPOs form large multigenic families (Colin et al., 2003, 2005), which some members could have evolved toward specialized defense functions. Based on the recent progress about biochemical and biological functions of vHPOs and about the remobilization of halide pool under stress, we have schematized the putative central functions of vHPO and halides in a context of cellular defense, that is, oxidative detoxification, cell wall strengthening and chemical defense, through halocarbon production (Fig. 3). B. A DIRECT ROLE IN CELL PROTECTION

In the marine environment, the production of VHOCs has been extensively investigated and is directly correlated with phytoplankton blooms or with the abundance of macroalgae along the coast. The last 10 years have seen an accumulation of qualitative and quantitative data on this biogenic production, because these compounds and in particular the iodinated forms have a potential significant impact on ozone destruction and on the formation of clouds over the remote ocean (McFiggans et al., 2004; O’Dowd et al., 2002). Laboratory experiments have also explored the physiological basis of the production of halogenated compounds by seaweeds, with the aim of quantifying and predicting the real impact of this biogenic source on global chemistry of atmosphere. Abiotic stresses such as high light

236

A. COSSE ET AL.

(Mtolera et al., 1996), UV exposure (Laturnus et al., 2004), temperature changes (Abrahamsson et al., 2003) have been shown to increase both the levels of ROS and VHOCs produced by the algae. As reviewed above, the perception of pathogen attacks is followed by the rapid and intense production of ROS in the surrounding of the cell, which has to protect itself by setting up antioxidative strategies. In L. digitata elicited by oligoguluronates, an increase in the emission of iodine‐containing halocarbons and molecular iodine I2 was monitored (Malin et al., 2001; Palmer et al., 2005). Similarly, our research group has shown an upregulation of VHOCs production upon defense elicitation in the two red algae Hydropuntia sp. (formerly G. conferta) (Weinberger et al., personal communication) and C. crispus (Bouarab et al., personal communication). In a context of defense oxidative burst, the vHPO could then take part in the scavenging of H2O2 through VHOCs production (Fig. 1). Another putative role of vHPOs is related to the oxidative cross‐linking of alginates and polyphenols, leading to algal adhesion and/or cell wall strengthening (Vreeland et al., 1998). In vitro cross‐linked polyphenols have been obtained using purified vHPO in the presence of H2O2, bromide, or iodide (Berglin et al., 2004; Bitton et al., 2006). During early stages of defense responses, both halide and H2O2 are released outside the cell membrane. Indeed in the presence of exogenously added H2O2, an increase in radioactive iodine eZux was observed in L. digitata (Ku¨pper et al., 1998), and a net iodine eZux was also induced by oligoguluronate elicitation in the same brown alga (Fievet et al., unpublished results). As some isoforms of vBPO enzymes were reported to be extracellular in Fucales (Krenn et al., 1989) and in giant kelps (Butler et al., 1990; Jordan and Vilter, 1991), we proposed that cell wall localized vHPO should catalyze the oxidative cross‐linking of alginates and polyphenols, with the increased concentrations of H2O2 and halides, leading to cell wall strengthening and mechanical protection against pathogen or herbivore, as also observed in land plant defense responses (Brisson et al., 1994).

C. CHEMICAL DEFENSE MOLECULES

The correlation between stress‐activated oxidative metabolism and increase in the emission of VHOC has first conducted some authors to propose that halogenated compounds should only be secondary waste products of ROS detoxification processes (Pederse´n et al., 1996). Their specific selection as defense compounds in algae has been controversial for a long time, whereas a number of these compounds were known to have potent antibiotic activities (Manley, 2002; Wever et al., 1991).

MARINE MACROALGAE AGAINST PATHOGENS

237

In 2001, a first laboratory study has demonstrated that bromoform produced by the red algae Corallina pilulifera and Lithophyllum yessoense inhibited the growth of epiphytic diatoms on the algal surface, and that this anti‐proliferating eVect was dependent on the activities of vBPO enzymes (Ohsawa et al., 2001). In the red alga Asparagopsis armata, bromoform and dibromoacetic acid are produced in high quantity and stored with other brominated compounds in specialized gland cells. When released at the surface of the thallus, they displayed antibiotic activity against epiphytic bacteria (Paul et al., 2006a). In the same alga, brominated compounds have also a role in feeding deterrence of mesograzers, because more intensive consumption of algae were observed, when brominating metabolism was switched oV by bromide starvation of algal cultures (Paul et al., 2006a). In C. crispus gametophytes, three VHOCs, upregulated after defense elicitation, were toxic against algal spores and germlings of A. operculata (Bouarab et al., personal communication). In red algae, VHOCs seem then to have an important physiological role in activated defense responses, acting as biocidal or repelling substances against microorganisms and herbivores (Paul et al., 2006a), but also preventing the settlement of epiphytic (Ohsawa et al., 2001) or pathogenic algae (Bouarab et al., personal communication). Concerning brown algal species, no experimental demonstration is yet available about the defensive functions of VHOCs. Iodovolatilization, that is, molecular iodine emission, has been correlated with oxidative stresses in L. digitata (Palmer et al., 2005). I2 is formed chemically with I
and H2O2 but a rapid formation can be catalyzed by vHPOs. The strong antiseptic activities of iodine species are known for a long time, and aqueous or alcoholic iodine solutions are traditionally used for medical applications. Chemical studies of iodine speciation in aqueous solution have shown that one of the most active forms was I2 for disinfection (Gottardi, 1999). In iodine‐concentrating brown algae, the oxidative burst, the concomitant iodine eZux, and the presence of extracellular vHPOs are also hypothesized to take part in a very eYcient early defense response, by eliminating pathogen using molecular iodine. Algal halogenated compounds are known to interfere with bacterial signaling system and to induce the dispersal of bacterial biofilms. A haloperoxidase system from L. digitata, producing hypohalous acid, can deactivate acylated homoserine lactones (AHLs), the cell‐to‐cell signaling molecules that are involved in quorum sensing, swarming, luminescence, and biofilms formation and dispersal in Gram‐negative bacteria (Borchardt et al., 2001). The red alga Delisea pulchra from the southeastern coast of Australia produces a range of halogenated enones and furanones, some of which have specific eVects on colonization phenotypes of marine bacteria at concentrations found on the surface of the alga (Maximilien et al., 1998).

238

A. COSSE ET AL.

Brominated furanones resemble AHLs structurally (Givskov et al., 1996) and show inhibitory activity at ecologically realistic concentrations in AHL bioassays. It was shown that they can control bacterial colonization of surfaces by specifically interfering with AHL‐mediated gene expression at the level of the LuxR protein (Manefield et al., 2001). This may illustrate a natural defense mechanism to prevent biofouling on the surface of this marine alga, and the evolution of specialized halogenation functions, in relation with signaling processes. Another domain, which remains to be explored, is the putative role of these halogenated compounds in distance signaling between algae.

V. OXYLIPINS AS MEDIATORS OF DEFENSE RESPONSES IN MARINE ALGAE As mentioned in a review about lipids and lipid metabolism in marine algae (Guschina and Harwood, 2006), there is a growing body of evidence for the involvement of oxylipin pathways in the defense mechanisms of seaweeds against pathogens and in the chemical defense of diatoms against grazing. In both metazoans and green plant lineages, enzymatic and nonenzymatic oxygenation of fatty acids results in the generation of a wide variety of oxygenated derivatives (oxylipins), which play a role either in cell signaling or as toxins (Weber, 2002). The biosynthesis of these oxylipin messengers is preceded by an early and rapid lipase‐like activation reflected by a release of free fatty acids (FFAs) which are subjected to an oxidative cascade involving lipoxygenases (LOXs), cyclooxygenase‐like and cytochromes P450 (CYPs) enzymes (Fig. 4). Members of the eicosanoid family of lipid mediators have been studied extensively regarding their biosynthesis from eicosanoids, that are, hydroperoxides derived from C20 polyunsaturated fatty acids (PUFAs), and their function in the regulation of cell diVerentiation, immune responses, and homeostasis in animal systems (Funk, 2001). In contrast, terrestrial plants, which have lost the capability to synthesize arachidonic acid, use derivatives of C18 (octadecanoids) and C16 (hexadecanoids) fatty acids as developmental or defense hormones (Weber, 2002). The biosynthesis of these oxylipins involves LOXs, which are enzymes that catalyze the oxygenation of PUFAs into hydroperoxy derivatives, rapidly converted to a broad range of oxygenated derivatives and to other secondary products (Funk, 2001; Howe and Schilmiller, 2002). In terrestrial higher plants, this oxidative cascade leads to the synthesis of the cyclopentenone jasmonic acid (JA), a key defense hormone described in a variety of crops or cell cultures (Heitz et al., 1997; Howe, 2005; Ishiguro et al., 2001; Seo et al., 2001; Turner et al., 2002). In animals, an alternative pathway involving cyclooxygenases also

239

MARINE MACROALGAE AGAINST PATHOGENS

Lipase activation Poly-Unsaturated Fatty Acids (PUFA)

Membrane Lipids

R

C18 C16

R⬘

Phospholipases Galactolipases

C20

Activation of oxylipin synthesis LOX OCTADECANOIDS EICOSANOIDS

R

C20:4

R⬘ OOH AOC AOS Jasmonates

COX?

PUFA Hydroperoxides

LOX

RED

Ketols Hydroxides

EAS/ PEROX Epoxides/ Hydroxides

HPL Prostaglandins

Aldehydes O

COOMe

COOH

O HO

OH

Fig. 4. Schematic view of conserved oxylipin pathways that were dissected in marine algae. Insert shows that like in animals, an alternative pathway from C20:4, which may also involve cyclooxygenases in seaweeds, leads to the synthesis of important hormones with a cyclopentenone structure, known as prostaglandins. AOS, allene oxide synthase; AOC, allene oxide cyclase; HPL, hydroperoxide lyase; LOX, lipoxygenase; EAS, epoxy‐alcohol synthase; PEROX, peroxygenase; RED, reductase; COX, cyclooxygenase.

leads to the synthesis of important hormones with a cyclopentenone structure, known as prostaglandins (Funk, 2001). Another pathway derived from phospholipase‐released arachidonic acid, involving 5‐LOX, generates other potent eicosanoid lipid mediators referred to as leukotrienes (Funk, 2001). Marine algae are known to contain oxylipins of the eicosanoid family, including prostaglandins and leukotrienes, as well as octadecanoids (Gerwick et al., 1999; Guschina and Harwood, 2006). However, the function of these pathways has hardly been investigated (Potin et al., 2002). A. LIPASE‐ACTIVATED RELEASE AND LIPOXYGENASE‐MEDIATED TRANSFORMATION OF FFAS

In both micro‐ and macroalgae, the activation of oxylipin‐based chemical defense is initiated by lipase‐like enzymes (Bouarab et al., 2004; Ku¨pper et al., 2006), phospholipase A2 (Pohnert, 2002), and/or galactolipases

240

A. COSSE ET AL.

(Cutignano et al., 2006; Lion et al., 2006), that can be activated by signaling (Bouarab et al., 2004; Ku¨pper et al., 2006) or act immediately after rupture of cell compartments during wounding (Cutignano et al., 2006; Lion et al., 2006; Pohnert, 2002). In marine diatoms, it was shown that chloroplast‐derived glycolipids are the main substrates for the biosynthetic pathway that produces antiproliferative polyunsaturated aldehydes in broken cells of Thalassiosira rotula (Cutignano et al., 2006). This process, which is associated with the formation of FFAs and lyso compounds from polar lipids but not triglycerides, is largely dependent on glycolipid hydrolytic activity, in addition to phospholipase A2 as previously suggested (Pohnert, 2002). The wound response of the multicellular red alga G. chilensis also involves the release of FFA as well as the hydroxylated eicosanoids, 8R‐hydroxy eicosatetraenoic acid (8‐HETE) and 7S,8R‐dihydroxy eicosatetraenoic acid (7,8‐di‐HETE). While the release of free arachidonic acid and subsequent formation of 8‐HETE is controlled by phospholipase A, 7,8‐di‐HETE production is independent of this lipase. This dihydroxylated fatty acid might be directly released from galactolipids that contained 8‐HETE or 7,8‐di‐HETE (Lion et al., 2006). In C. crispus also, challenging gametophytic thalli with A. operculata cell‐free extracts dramatically change their FFA composition. Significant levels of arachidonic acid (C20:4), linolenic acid (C18:3), and stearidonic acid (C18:4) accumulate (Bouarab et al., 2004). Challenging the sporophytes of the brown alga L. digitata with LPS from various sources also results in the activation of a rapid release of FFAs (Ku¨pper et al., 2006) with a concomitant accumulation of oxidized derivatives of linolenic (C18:2) and eicosapentaenoic acid (C20:5). Other strong inducers of the oxidative burst in Laminaria such as oligoguluronates could not induce the release of FFAs nor oxylipin production. These results suggest that diVerent signaling pathways are involved in the induction of the oxidative burst and oxylipin production. B. OXYLIPIN PATHWAYS IN MARINE ALGAE

On the basis of the structure of the oxylipins characterized so far, red and brown marine algae probably contain 5R‐, 8R‐, 9S‐, 12S‐, and 15S‐LOXs that act on eicosanoic (C20) PUFAs, as well as !3‐, !6‐, !9‐, and !10‐LOXs that act on octadecanoic PUFAs (C18) (Gerwick et al., 1999). The characterization of four new oxylipins in Rhodymenia pertusa strongly supports the occurrence of a functional 5R‐LOX in this red alga (Jiang et al., 2000). They are probably generated through a ‘‘leukotriene A‐type intermediate,’’ making R. pertusa a member of the relatively small club of nonmammalian organisms that synthesize leukotriene‐A‐like substances (Gerwick et al., 1999).

MARINE MACROALGAE AGAINST PATHOGENS

241

Other red algae mainly produce oxylipins by the pathways initiated by 8‐, 9‐, and 12‐LOXs (Gerwick et al., 1999). At the molecular level, a cDNA encoding a putative 12‐LOX was identified in the gametophyte of Porphyra purpurea (Liu and Reith, 1994) while a polyenoic fatty acid isomerase, which converts arachidonic acid into a conjugated triene, was purified and cloned from Ptilota filicina (Zheng et al., 2002). Following the oxidative burst in the red alga C. crispus, various lipid hydroperoxides are produced, which were identified by a combination of liquid/gas chromatography and mass spectrometry (Bouarab et al., 2004). Two LOX isoforms, which were specific for the metabolism of linoleic acid, were upregulated following the oxidative burst in C. crispus, and LOX inhibitors abolished the natural resistance of C. crispus gametophytes (Bouarab et al., 2004). Some marine algae can form volatile aldehydes such as n‐hexanal, hexenals, and nonenals. In higher plants, C6 aldehydes, n‐hexanal (2), (Z)‐3‐ and (E)‐2‐hexenal, are ubiquitously found. In general, they are formed from C18 fatty acids, such as linoleic or linolenic acid, through the synthesis of 13‐hydroperoxides by LOX, followed by their stereospecific cleavage by 13‐hydroperoxide lyase (HPL). This enzymatic pathway is a branch of a highly divergent biosynthetic pathway, called either the (phyto)oxylipin or octadecanoid pathway (Ble´e, 1998). Some plants, such as pear, cucumber, or melon can form both C9 and C6 aldehydes (Noordermeer et al., 2001). Some marine algae can also form C6 and C9 aldehydes, but their precise biosynthetic pathway is less understood. In the brown algae, Laminaria spp., C9 aldehydes such as (E)‐2‐nonenal could be found in relatively large quantities, followed by C6 aldehydes, such as n‐hexanal, (Z)‐3‐ and (E)‐2‐hexenal. Recently, it was shown that L. angustata forms the aldehydes enzymatically. The algae form C9 aldehydes almost exclusively from C20 fatty acid, such as arachidonic acid, through the formation of 12(S)HPETE, while C6 aldehydes are derived from either C18 or C20 fatty acids, through formation of 13(S)‐hydroperoxyoctadecadienoic acid or 15(S)HPETE (Boonprab et al., 2003). The HPL that accounts for the formation of C9 aldehydes from 12(S) HPETE, seems to be the one highly specific to hydroperoxides of C20 fatty acids, because partially purified HPL, which is responsible for C6 aldehyde formation, shows little activity to hydroperoxides of C20 fatty acids (Boonprab et al., 2003). By surveying various species of marine algae including Phaeophyta, Rhodophyta, and Chlorophyta, it was shown that almost all the marine algae have HPL activity. Thus, a wide distribution of the enzyme is expected. It has been shown that the diatom T. rotula can form short‐chain aldehydes having C10 structures, such as (E, Z)‐2,4‐decadienal and (E, Z, Z)‐2,4,7‐decatrienal, which inhibit egg cleavage of copepods

242

A. COSSE ET AL.

(Miralto et al., 1999). They were shown to be involved in an indirect chemical defense in diatoms (Pohnert, 2002, 2005). A novel class of oxylipins based on C16 PUFAs in diatoms has been described (d’Ippolito et al., 2006) providing new compounds as candidates to participate in signaling and/or allelopathy mechanisms. C10 aldehydes are proposed to be formed from arachidonic acid through synthesis of 11‐hydroperoxyeicosatetraenoic acid catalyzed by LOX followed by cleavage by HPL (Pohnert, 2000; Pohnert and Boland, 1996). It was also established from the work on the structure and biosynthesis of brown algal pheromones that the biosynthesis of C11 hydrocarbons in both diatoms and brown algae relies on eicosanoid precursors (Pohnert and Boland, 2002). From the work conducted on diatoms, it was inferred that brown algae use a LOX pathway, HPL coupled to generate 9‐, 12‐, or 15‐hydroxyeicosatetraenoic acid (HETE). In the brown alga L. digitata, it was shown that the early events of the perception of LPS include the accumulation of oxylipins such as 13‐hydroxyoctadecatrienoic acid (13‐HOTrE) and 15‐hydroxyeicosapen-taenoic acid (15‐HEPE) (Ku¨pper et al., 2006). It is interesting to note that oligoguluronates did not induce such a release of FFA and synthesis of hydroxyl derivatives of C18 and C20. From this observation, it can be concluded that in L. digitata, the oxylipin pathway activated in response to LPS challenging is independent of the oxidative burst. So, this study highlights that some features of defense mechanisms are conserved among the eukaryotes, from mammals and plant to algae, including recognition of MAMPs, oxidative burst, and oxylipin signaling. C. BIOLOGICAL AND ECOLOGICAL FUNCTIONS OF OXYLIPINS

Altogether, marine algae are well documented to contain a variety of oxylipins of pharmacological interest, but until very recently nearly nothing was known of their biological functions in the algae (Gerwick et al., 1999). In diatoms, in addition with their indirect defense function against copepod grazing (Pohnert, 2005), it has been shown that the aldehydes can induce a stress surveillance system in intact cells distant from wounded cells (Vardi et al., 2006). Exposure to wounded diatom‐derived reactive aldehydes trigger intracellular calcium transients and the generation of NO by calcium‐ dependent NO synthase‐like activity, which determines cell fate (i.e., immune resistance vs cell death). Like in diatoms, results point at the importance of the oxylipin pathways in the regulation of seaweed induced defenses (Bouarab et al., 2004; Lion et al., 2006). The volatile methyl ester of the plant defense hormone jasmonate (MeJA) was shown to trigger the accumulation of phlorotannins in the

MARINE MACROALGAE AGAINST PATHOGENS

243

common rockweed Fucus vesiculosus when exposed at low tide. The timing and magnitude of this increase are similar to those induced by herbivores in brown algal fucoids suggesting that analogs of cyclopentenone structures may play a role in the development of antiherbivore responses in Fucus tissues, including those responses involving interplant communication (Arnold et al., 2001). It was suggested that oxylipins produced by marine macroalgae may play important detrimental roles in herbivore fitness (Bouarab et al., 2004). In addition, the by‐products of the biogenesis of fatty acid‐derived C8 and C11 hydrocarbons which compose the sexual pheromones of marine heterokont algae and sulfurylated C11 compounds were shown to play also an important role as chemical defenses against herbivores. Studies on the brown alga Dictyopteris spp. have shown that 9‐oxo nonadienoic acid deters amphidod grazers (Schnitzler et al., 2001). In brown algal kelps, it was also shown that Laminaria spp. synthesize polyunsaturated aldehydes of similar structure and using closely related biosynthetic pathways than diatoms (Boonprab et al., 2003); however, their biological functions have not yet been tested in kelps. In response to wounding, the red alga G. chilensis releases FFAs as well as hydroxylated eicosanoids and this liberation of oxylipins was shown to be part of the defense of G. chilensis against epiphytism (Lion et al., 2006). Given the importance of the impact of mechanical wounding on the induction of defense responses during insect feeding in higher plants (Mitho¨fer et al., 2005), such a response to grazers in seaweeds clearly need some additional careful investigations. In red algae, challenging C. crispus sporophytes, the generation of which is naturally invaded by the green endophyte A. operculata, with hydroperoxieicosatetraenoic acid (HPETE), hydroperoxioctadecadienoic acid (HPODE), or MeJA induced a transient resistance to A. operculata infection, confirming the dual nature of the oxylipin defense pathway in this alga (Bouarab et al., 2004). To our knowledge, no conclusive evidence exists that MeJA is an endogenous compound in C. crispus; however, MeJA has been detected in the incubation medium of cell‐free extracts of this alga with linolenic acid (Bouarab et al., 2004). MeJA is also taken up by C. crispus and causes a dose‐dependent production of oxylipins (Gaquerel et al., 2007). The presence of MeJA has been reported in another florideophyte red alga, Gelidium latifolium (Krupina and Dathe, 1991). In addition, the enzymes involved in the metabolization of linolenic acid leading to the synthesis of jasmonate have been identified in two other florideophyte red algae, Lithothamnion coralloides and Gracilariopsis sp. (Hamberg and Gardner, 1992; Hamberg and Gerwick, 1993). It was inferred that MeJA has a potentially important function in the regulation of the biotic defense in the red macroalga C. crispus. MeJA

244

A. COSSE ET AL.

activated the oxidative metabolism of C20 and C18 PUFAs and generated hydroperoxides and cyclopentenones, such as prostaglandins and oxygenated fatty acids (Bouarab et al., 2004; Gaquerel et al., 2007). Addition of MeJA to C. crispus also induced increased activities of enzymes potentially involved in defense reactions, such as shikimate dehydrogenase, phenylalanine ammonium lyase, and an enzyme that hydroxylates PUFAs (Bouarab et al., 2004; Gaquerel et al., 2007). The transcription of defense‐related genes was also upregulated after MeJA addition, that is, glutathione S‐transferase (GST) and NADPH oxidase, a possible key protein in defense mechanisms in red algae (Gaquerel et al., 2007; Herve´ et al., 2006). Transcription analysis of the C. crispus NADPH oxidase gene Ccrboh revealed that it is overexpressed after incubation in the presence of MeJA and hydroxyperoxides derivatives of arachidonic acid. The main eVects were observed in the presence of MeJA, derivatives from linoleic acid, and 12‐HpETE, derived from arachidonic acid (Herve´ et al., 2006). Such results show that NADPH oxidase‐homologue gene expression is related to both eicosanoid and octadecanoid signaling pathways, a striking feature, which has not been observed so far in other eukaryotic lineages. The upregulation of defense‐related proteins by MeJA is followed by an induced resistance in C. crispus toward the pathogenic green algal endophyte A. operculata (Bouarab et al., 2004). Altogether, this indicates that MeJA, or a related compound, has a physiological role in C. crispus and other advanced red algae and one of the functions is as a stress hormone. In conclusion, red and brown algae are likely to use both animal‐like (eicosanoid) and higher plant‐like (octadecanoid) oxylipins in the regulation of defense metabolism toward protection against pathogens and grazers or in response to elicitors of defense responses. This duality of the oxylipin metabolism is also found in mammalian cells, where there is emerging evidence that, besides the C20:4 derivatives, the oxygenation products from C18:2 can act as defense compounds (Ishizaki et al., 1995a,b). An hypothetical scenario to account for the presence of both the C20 and C18 PUFAs oxygenation cascades in these phylogenetically distant lineages as well as in other eukaryotic branches was proposed by Bouarab et al. (2004). It assumed that these two categories of lipid derivatives preexisted before the radiation of extant eukaryotes. Within the past few years, we have gained many new insights into the roles of fatty acid‐based molecules as potent signals in seaweeds as a result of the use of sensitive biochemical methods for the identification of algal oxylipins and of the development of powerful methods to monitor gene expression. Other functions such as antimicrobial properties should also be investigated, as illustrated in higher plants (Prost et al., 2005). Preliminary results of

MARINE MACROALGAE AGAINST PATHOGENS

245

signaling studies demonstrate the presence of multiple signaling molecules in a biochemical cascade, a situation similar to prostaglandin signaling in mammals (Funk, 2001) or jasmonate cascade in higher plants (Howe, 2005). It is evident that, apart from jasmonates, there are many other fatty acid‐ derived signals in algae. Following the initial reports of their activity, we must now begin to dissect these signaling pathways genetically. The knowledge of the genome of the filamentous brown alga Ectocarpus should be of enormous help to identify candidate biosynthetic genes and to help identifying mutants. Furthermore, the availability of DNA microarrays will be an important tool for investigating the signaling function and pathways of individual fatty acid‐derived regulators. Although the need for genetic tools favors Ectocarpus as a model alga for research in fatty acid signaling, we should not overlook other algal species and phyla because these might synthesize and perceive diVerent fatty acid‐derived signals.

VI. TRANSCRIPTIONAL RESPONSES IN MARINE ALGAE: MINING DEFENSE GENES A. GENOMIC AND TRANSCRIPTOMIC DATA IN MARINE ALGAE

The last decade, we have observed an explosion of genomic information used not only to establish the gene content of organisms, but also to characterize environmental and developmental processes that modulate gene expression, and to understand how gene content and expression patterns may explain the ecological niche of living organisms. There are few algae for which the nuclear genome has been sequenced. Complete or nearly complete sequence of the genomes of the red alga C. merolae (http://merolae.biol.s.u‐tokyo.ac.jp/; Matsuzaki et al., 2004), the diatom T. pseudonana (http://genome.jgi‐psf.org/thaps1/thaps1.home.html; Armbrust et al., 2004), and the picoeukaryote green alga Ostreococcus tauri (http://genome.jgi‐psf.org/Ostta4/Ostta4.home.html; Derelle et al., 2006)) have been made publicly available. Other genomes either sequenced and not totally released or in the process of being sequenced include the diatom P. tricornutum (http://genome.jgi‐psf.org/Phatr2/Phatr2.home.html; Maheswari et al., 2005; Scala et al., 2002), the green alga Chlamydomonas reinhardtii (http://genome. jgi‐psf.org/Chlre3/Chlre3.home.html), and the brown alga E. siliculosus (http:// www.cns.fr/externe/Francais/Projets/Projet_KY/KY.html). In the absence of complete genome information, the exploitation of general or dedicated EST cDNA libraries is a promising strategy for identifying genes. Among algal species, ESTs from the red algae P. yezoensis

246

A. COSSE ET AL.

(Nikaido et al., 2000), C. crispus (Colle´n et al., 2006), and Gracilaria gracilis (Lluisma and Ragan, 1997), from the brown alga L. digitata (Cre´pineau et al., 2000; Roeder et al., 2005), from the diatom P. tricornutum (Scala et al., 2002), and from the green alga Ulva linza (Stanley et al., 2005) provide the most important resources, available in the public EST database of the National Center for Biotechnology Information (NCBI) (http://www.ncbi. nlm.nih.gov/dbEST/). Some other ESTs libraries unreleased, or only partially, have been generated from diVerent macroalgal species (Fucus serratus, F. vesisulosus, Laminaria saccharina, E. siliculosus, U. linza) and diatoms (Fragilariopsis cylindrus). These projects are currently being organized into a database through the Marine Genomics Europe Network of Excellence. Up to now, the molecular bases of algal innate immunity have been barely explored and very few studies have been focused on molecular responses after biotic stress. However, the global increase of algal EST data has allowed screening putative candidates for defense or pathogen‐related genes, by searching similarities with genes already described in other organisms. Despite of the relatively low number of algal sequences in databases, these ‘‘in silico’’ approaches constitute a preliminary step toward understanding the transcriptional changes that occur during biotic interactions in marine algae. Here, we draw up a nonexhaustive inventory of putative defense genes, tagged by homology search. P. yezoensis is one of the algae, which has the most abundant ESTs data. To identify gene candidates related to the morphological and physiological diVerence between the leafy gametophyte and the filamentous sporophyte generations of P. yezoensis, large‐scale expressed sequence tag analysis was conducted in the two life cycle phases. A total of 10,265 EST sequences were generated from the sporophyte (Asamizu et al., 2003), in addition to the 10,154 ESTs from the gametophyte (Nikaido et al., 2000). Thus, 4496 putative diVerent cDNAs were obtained. Only 22% of them are represented in both generations and only 32% were given a putative biochemical function. The putative defense genes underlined by these works are connected to active oxygen metabolism, detoxification process, shikimate and phenylpropanoid pathways, and contain several heat shock protein (HSP) families. The complete list of P. yezoensis ESTs is available at http://www.kazusa.or.jp/en/ plant/porphyra/EST/. In the brown algal phylum, E. siliculosus genome sequencing is in progress and nearly 26,000 ESTs have yet been obtained from the two life cycle phases (Mark Cock, personal communication). These data are not yet released in public databases. A private access to primary data allows us to indicate that like in P. yezoensis, cDNA from active oxygen metabolism, detoxification process, and shikimate and phenylpropanoid pathways are identified.

MARINE MACROALGAE AGAINST PATHOGENS

247

Among the published data in brown algae, ESTs from L. digitata are the most abundant, with about 3000 sequences (Roeder et al., 2005). They highlight essentially cDNA involved in detoxification process and HSP families. B. MINING DEFENSE‐RELATED GENE IN BROWN ALGAE

1. Specific response to cope with oxidative stress in brown algae From the ESTs available in brown macroalgae, a singular aspect of oxygen metabolism can be noticed in this phylum. Surprisingly, in L. digitata, L. saccharina, F. serratus, and F. vesiculosus, no gene coding for classical antioxidant enzymes has been found in libraries constructed with RNA extracted from algae incubated under stress conditions. In particular, in a L. digitata protoplast library (Roeder et al., 2005), no ESTs coding for well‐ conserved key enzymes of ROS scavenging, such as catalase, superoxide dismutase, glutathione peroxidase or ascorbate peroxidase, which are typically strongly expressed by cells under oxidative stress conditions (Desikan et al., 2001; Menezes‐Benavente et al., 2004; Mittler, 2002) were identified. These proteins are usually well conserved at the amino acid level among eukaryotes, and if present, their transcripts would probably have been identified. The only well‐represented EST among the diVerent brown algal libraries, coding for a classical antioxidant enzyme, codes for a methionine sulfoxide reductase (MSR). It has been proposed that cellular response to oxidative stress involves two major components, the methionine residues in proteins, which act as endogenous scavengers of oxidants, and MSR, which subsequently reduces methionine sulfoxides (oxidized methionine) back to methionine (Levine et al., 1996). The apparent MSR over‐representation seems to indicate that protein compartment plays a key role in antioxidant processes. Related to this antioxidant aspect, it is remarkable to note that brown and red algae feature a variety of haloperoxidase isoforms that have iodinating or brominating activity. Several genes have been characterized, representing multigenic families in L. digitata (Colin et al., 2003). We have mentioned above that these enzymes catalyze the oxidation of halides to hypohalous acids in the presence of H2O2. They are responsive of VHOCs emission, associated with oxidative response to various stress. A previously unidentified gene subfamily of BPO, named vBPO‐II, was identified in L. digitata protoplast ESTs library. This vBPO‐II is markedly diVerent from the constitutive isoform, vBPO‐I. It has been confirmed by Northern blot experiments that BPO‐II genes are not expressed in sporophytes under normal conditions but are highly induced after protoplasts isolation (Roeder et al., 2005). These results suggest that BPO‐II gene is specifically induced in protoplast cells,

248

A. COSSE ET AL.

probably in response to the severe oxidative stress. Taking into account the absence of ESTs coding for classical antioxidant enzymes in libraries and considering the high level of haloperoxidases transcripts, it will be of special interest to test the hypothesis that algae may use the diversity of haloperoxidases isoforms, to dedicate specific members to ROS scavenging. Genes coding for haloperoxidases may represent the first typically marine defense‐ related gene. The development of functional approaches to invalidate or overexpress these candidate genes represents a major challenge for this field of research. 2. DiVerential expression methods toward identification of defense‐related genes in brown algae The potential of ESTs libraries as an initial strategy to mine putative defense genes candidates has been illustrated in some recent studies in brown macroalgae. ESTs catalogs have been obtained to compare transcripts in the macroscopic sporophyte of L. digitata with those of the microscopic gametophyte (Cre´pineau et al., 2000). Of the 905 ESTs sequenced, about 44% were given putative functions. However, one limitation of this approach, which analyzed ESTs from algae growing under normal conditions, was that most of the ESTs identified correspond to housekeeping genes, especially those encoding ribosomal proteins. Therefore, libraries from protoplasts were considered as a favorable alternative experimental tool to enrich the repertoire of genes involved in stress responses. As a consequence, another cDNA library dedicated to transcript analysis in L. digitata protoplast has been investigated (Roeder et al., 2005). Protoplasts are naked, isolated, and severely stressed cells. They may be thought of as having been subjected to an ultimate form of tissue wounding. The loss of the cell wall, physical support, and communication with other cells, is a deeply disturbing experience for a vegetative cell and this can be regarded as a combination of mechanical and osmotic stress. Indeed, in L. digitata, protoplast generation was shown to result in a severe oxidative stress and up to 120 mM H2O2 was found in the digestion medium (Benet et al., 1997). Moreover, protoplasts are prepared with the use of cell wall degrading enzymes, involving long incubations in the presence of eliciting cell wall fragments, such as oligoguluronates, known to elicit an oxidative burst in L. digitata sporophytes (Ku¨pper et al., 2001, 2002). The use of protoplasts cDNA libraries would favor the identification of genes important in oxidative stress and defense responses. The sequencing of this L. digitata protoplast cDNA library generated 1985 ESTs (Roeder et al., 2005). Forty‐five percent of them showed significant

MARINE MACROALGAE AGAINST PATHOGENS

249

amino acid sequence similarity to sequences in the GeneBank database. More than 23% of the identified ESTs belonged to the ‘‘stress defense’’ class, which was three times larger than in sporophyte database. It is, however, noteworthy that the most highly expressed sequences are related to a small number of genes, HSP70 and HSP90, vBPO, GST and thioredoxin, which thus appear to have a key role in stress response of L. digitata. The HSPs aid in the folding of damaged and newly formed proteins as well as in preventing protein aggregations (Wang et al., 2004). The high number of HSP coding ESTs in the protoplast library could reveal the necessity for the cell to protect protein from oxidation damage and to prevent cell death. Also induced in the protoplast, several putative chloroplastic thioredoxin and thioredoxin reductases are components of the thioredoxin system, which is essential for the redox regulation (Arner and Holmgren, 2000). Usually, thioredoxins are induced during the oxidation stress response in plants (Laloi et al., 2004; Meyer et al., 2005) and also bacteria, yeast, and mammals (Mu¨ller, 1991; Ritz et al., 2000). Finally, as already mentioned and as pointed by Roeder and colleagues (2005), haloperoxidases are likely to play a major role in coping with oxidative stress, in replacement of classical antioxidant enzymes. Another major original trait highlighted by protoplast transcripts analysis is related to the way the brown alga L. digitata copes with a severe oxidative stress. It is suggested that a second line of defense against ROS is probably provided by a particular class of GSTs. Some diVerent GSTs showing significant similarity to the sigma class were strongly expressed in L. digitata protoplasts. The role of GSTs in oxidative stress and in defense has been well documented. These proteins are generally considered as detoxifying enzymes (Hayes and McLellan, 1999; Marrs, 1996). So far, sigma‐type GSTs have been found only in mammals. They have been shown to detoxify exogenous toxic products, to metabolize lipid peroxidation products, and to display a prostaglandin synthase activity in mammals (Hayes and McLellan, 1999). This is the first report of such a sigma GST in a photosynthetic organism (Roeder et al., 2005). In addition, ESTs coding for 6‐phospho‐gluconate‐dehydrogenase (6PGDH) are well represented in the protoplast library (Roeder et al., 2005). This is the second limiting enzyme of the pentose phosphate pathway. Pentose phosphate pathway represents the major source of reducing power (NADPH) for protecting cell from oxidative stress (Kruger and von Schaewen, 2003). We assume that the pentose phosphate pathway is involved in defense response of L. digitata due to its ability to produce NADPH that can be used to maintain the redox equilibrium of the cell. From this protoplast approach, we have enriched in stress‐related genes the transcriptomic data available in L. digitata. To progress toward the

250

A. COSSE ET AL.

identification of genes specifically induced in host–pathogen interaction, we constructed a suppression subtractive hybridization (SSH) library of sporophyte challenge or not with the oligoguluronate elicitor (A. C. et al., unpublished results). Defense gene candidate issued from these two approaches, protoplast EST library and SSH library, have been used to design a dedicated stress macroarray and to monitor their expression after elicitation by oligoguluronates. This study provides the first analysis of transcriptomic changes that occur in defense context in marine brown algae. It confirms that protoplast tool and EST library are useful strategies to discover molecular markers of defense responses. C. MINING DEFENSE‐RELATED GENE IN RED ALGAE

1. Strategies of identification of defense‐related genes ESTs libraries from protoplasts (2002 ESTs) and thallus (2052 ESTs) of C. crispus were also released (Colle´n et al., 2006). Among the 2291 nonredundant sequences identified, 50% could not be assigned a putative function. This observation reflects the specificity of the metabolism of the red algal lineage and the scarcity of relevant information in the protein databases. The fraction of stress‐related ESTs was five times higher in the protoplast library (18%) than in the thallus library (3.5%). The similar projects carried out on two nonrelated brown and red macroalgae allow highlighting some interesting common features. Both C. crispus and L. digitata protoplasts cDNA libraries feature an increased EST number of stress‐related genes, genes important for cell organization and conserved hypothetical proteins, and a decreased EST number of genes involved in protein synthesis. The induction of genes such as HSP, GST, and BPO may reflect that cells attempt to reduce oxidative stress damages. The particularity of halogen metabolism is again underlined, as indicated by another group of ESTs found in C. crispus, encoding for haloalkane dehalogenase. In bacteria, this haloalkane dehalogenase cleave carbon–halogen bonds (Janssen et al., 1994). In addition to the general action of the numerous GST, haloalkane dehalogenase could be dedicated to the detoxification of the halocarbons. Contrasting with these common points, some particular diVerences can be noted. Genes relevant to reactive oxygen metabolism and coding for well‐ known antioxidant enzymes, like NADPH oxidase, ascorbate, and several other peroxidases, catalase, MSR, and Mn‐SOD have been identified in the red alga and not in L. digitata. Nevertheless, if such antioxidant activities are known to be important for abiotic stress tolerance in C. crispus (Colle´n and Davison, 1999), their genes were not statistically overexpressed in the protoplast library. Another transcript, coding for an L‐amino acid oxidase, is

MARINE MACROALGAE AGAINST PATHOGENS

251

potentially a defense‐related gene since the oxidation of L‐asparagine was shown to be important in the recognition and defense of C. crispus against its green algal pathogen A. operculata (Weinberger et al., 2002, 2005a). Moreover, C. crispus presents the originality to feature many animal‐related genes, like some genes with similarities to ovoperoxidases, eosinophil peroxidases, and some cytochrome P450 enzymes. The firsts are possibly involved in ROS scavenging and the second possibly involved in secondary metabolism or FFA oxidation cascades in this red alga (Bouarab et al., 2004). The presence of these animal‐related genes and some traits of metabolism implied as a consequence are in concordance with the study of Nozaki et al. (2003), which puts red algae as the most basal eukaryotic photosynthetic group. 2. Development of microarrays in red algae As indicated by these two examples, ESTs libraries and protoplast approaches are interesting tools to provide a large repertoire of stress gene sequences, which can be used to investigate responses to ecological and physiological situations in algae. The sequence data generated by the ESTs libraries from C. crispus have been used to construct a cDNA microarray and analyze genes expression in biotic defense. The work of Colle´n et al. (2006) is the first report to our knowledge of a real transcriptomic study of defense mechanism in marine algae. This medium‐scale expression profiling approach to identify genes regulated by MeJA clearly shows changes in gene expression in C. crispus. Roles for jasmonates in biotic defense mechanisms, that is, as a response to grazing‐induced wounds, and in response to various abiotic stresses are well documented in higher plants (Howe, 2005). It is also known that MeJA has a potentially important function in regulation of the biotic defense in the red alga C. crispus (Bouarab et al., 2004; Gaquerel et al., 2007) since MeJa activated the oxidative metabolism of C20 and C18 PUFAs and induced the production of hydroperoxides and cyclopentenones, such as prostaglandins and oxygenated fatty acids (Bouarab et al., 2004; Gaquerel et al., 2007). The transcription of defense‐related genes was also upregulated after MeJA addition, that is, GST and NADPH oxidase (Gaquerel et al., 2007; Herve´ et al., 2006). This upregulation is followed by an induced resistance in C. crispus to the pathogenic green algal endophyte A. operculata (Bouarab et al., 2004). The changes in the transcriptome were monitored using cDNA microarrays with 1920 diVerent cDNA representing 1295 unique genes. The analysis showed that 6% of genes studied showed a significant response to the addition of MeJA at some time points and 4‐ to 11‐fold changes in expression were detected for 1% of them. The predominant pattern is an increased expression of stress genes with a simultaneous downregulation of genes involved in energy conversion and general metabolism. Potential stress

252

A. COSSE ET AL.

genes that are the most upregulated include GST, HSP, peroxidase, xenobiotic reductase, and drug resistance protein. The active oxygen metabolism is certainly involved in response to MeJA treatment, as shown by the potential increased expression of NADPH oxidase, peroxiredoxin, catalase, ascorbate peroxidase, Mn‐SOD, and MSR. Interestingly, it was found that the gene of DHAP synthase, which is the first enzyme of the shikimate pathway, is overexpressed. These findings are reminiscent of transcriptional analyses carried out in Arabidopsis thaliana (Taki et al., 2005; von Rad et al., 2005). This similarity indicates that the red macroalgae share some classical defense response with higher plants, like the response to MeJA, even though the oxylipin metabolism in C. crispus has unusual features, representing an intermediate between metazoan and higher plant metabolism (Bouarab et al., 2004). The lack of knowledge concerning defense‐related genes in marine algae is essentially due to the weak quantity of genomic and transcriptomic information presently available. Extensive ESTs libraries from P. yezoensis and E. siliculosus have already permitted to identify numerous genes, involved in the principal plant defense pathways and also in some innate immunity responses of metazoans. These genomic resources provide a start point to design new strategies using large‐scale or dedicated DNA arrays to further dissect defense‐related transcriptomic changes. For instance, if the genomic background is now available in marine macroalgae, there is only little experimental evidence of the transcriptional responses involved in defense mechanisms.

VII. CONCLUSIONS AND PROSPECTS The past decade has witnessed a substantial growth in our understanding of active defense pathways against pathogens in marine algae. One major challenge in studies of algal defense mechanisms is to understand the adaptation and role of typically animal, bacterial‐, or plant‐like properties and metabolism in the context of the marine environment and life‐history strategy of micro‐ and macroalgae. In this context, as testified by the evolution of the oxidative burst machinery, the generation of halogen oxidants, and the oxidative metabolism of fatty acids, marine algae provide ideal models to test some hypothesis about the evolution of these key mechanisms of innate immunity during the crown diversification of eukaryotes (Baldauf, 2003) and to show that these essential cell functions have arisen in the sea. This chapter underlines that studying such a vast assemblage of evolutionary lineages of autotrophic organisms is a diYcult task and suVers from the small size of the scientific community in this field. Some important signaling

MARINE MACROALGAE AGAINST PATHOGENS

253

pathways remained nearly unexplored. For example, reactive nitrogen species (RNS), which are key regulators of immune responses in metazoans and higher plants, were hardly detected in seaweeds. As mentioned earlier, diatoms produce NO in response to some aldehydes (Vardi et al., 2006). Only recently, the green coenocytic alga D. vermicularis has also been shown to produce NO in response to wounding. Pharmacological approaches have suggested that the signaling pathways leading to ROS and RNS productions share similarities with defense and wounding signal transduction in higher plant systems (Ross et al., 2006). However, in contrast with higher plants which display a burst of NO production in responses to bacterial LPS, attempts to detect it in response to LPS treatment in L. digitata remained unsuccessful (Ku¨pper et al., 2006). Further studies are required to explore these essential signal transduction pathways including other key aspects of the regulation of defense responses such as phosphorylation events, redox control, and transcriptional eVectors. In contrast to terrestrial plants, seaweeds, as nonvascular aquatic organisms, do not appear to have developed systemic responses (Potin et al., 2002). A fascinating challenge that remains, however, is to identify molecules that are involved both in intraplant signaling and in communication between conspecifics. The growing field of oxylipin research in marine algae (Bouarab et al., 2004; Pohnert, 2005) has provided the first candidates, such as MeJA or endogenous hydroxy fatty acids that induces important transcriptional changes (Colle´n et al., 2006; Herve´ et al., 2006). Other candidates might be synthesized by the metabolism of volatile hydrocarbons. To better understand the complex interactions between seaweeds and pathogens, the field must continue to unravel the relative contributions of diVerent defense pathways and also the possible avoidance or suppression of algal defenses. There is a pressing need to develop more powerful general models for marine algae that are well adapted to the application of genomic and genetic approaches to extend the description of the important characteristics of these lineages to the molecular level. An extensive survey of potential model organisms among the brown algae (Peters et al., 2004) has concluded to develop the filamentous alga E. siliculosus as a model species. In this small filamentous alga, genetic approaches are amenable to address key issues in the innate immunity and defense metabolism and possibly signaling, through large‐scale UV mutagenesis, mutant screens relevant in the context of defense responses, and a combination of genetics, analytical chemistry, biochemistry, and transcriptomic approaches to further characterize mutant phenotypes. Given this new context in the field of seaweed research, it is clearly a very exciting time to study biotic interactions of marine algae, and many new insights in the mechanisms of defense are expected in the coming years.

254

A. COSSE ET AL.

ACKNOWLEDGMENTS We are grateful to C. Herve´ who provided an illustration from her work and to numerous colleagues for valuable discussion. M. Cock is thanked for having provided access to the E. siliculosus EST database. Manuscript preparation was supported in part by CNRS, the Conseil Re´gional de Bretagne (Programme de Recherche d’Inte´reˆt Re´gional financed by (PRIR‐N8 560408; A3CBL9) and the Institut Franc¸ais de la Biodiversite´ (Programme Biodiversite´ et changement Global). A.C. was supported by a fellowship from the Conseil Re´gional de Bretagne. Part of A.C. Ph.D. work was performed in the frame of ‘‘Marine Genomics Europe’’ NoE 7 (EC contract N8 GOCE‐ CT‐2004‐505403).

REFERENCES Abrahamsson, K., Choo, K. S., Pederse´n, M., Johansson, G. and Snoeijs, P. (2003). EVects of temperature on the production of hydrogen peroxide and volatile halocarbons by brackish‐water algae. Phytochemistry 64, 725–734. Amsler, C. D. and Fairhead, V. A. (2006). Defensive and sensory chemical ecology of brown algae. Advances in Botanical Research 43, 1–91. Andrews, J. H. (1976). The pathology of marine algae. Biological Reviews 51, 211–253. Andrews, J. H. (1977). Observations on the pathology of seaweeds in the Pacific Northwest. Canadian Journal of Botany 55, 1019–1027. Ar Gall, E., Ku¨pper, F. C. and Kloareg, B. (2004). A survey of iodine contents in Laminaria digitata. Botanica Marina 47, 30–37. Armbrust, E. V., Berges, J. A., Bowler, C., Green, B. R., Martinez, D., Putnam, N. H., Zhou, S., Allen, A. E., Apt, K. E., Bechner, M., Brzezinski, M. A., Chaal, B. K., et al. (2004). The genome of the diatom Thalassiosira pseudonana: Ecology, evolution, and metabolism. Science 306, 79–86. Arner, E. S. J. and Holmgren, A. (2000). Physiological functions of thioredoxin and thioredoxin reductase. European Journal of Biochemistry 267, 6102–6109. Arnold, T. M. and Targett, N. M. (2002). Marine tannins: The importance of a mechanistic framework for predicting ecological roles. Journal of Chemical Ecology 28, 1919–1934. Arnold, T. M., Targett, N. M., Tanner, C. E., Hatch, W. I. and Ferrari, K. E. (2001). Evidence for methyl jasmonate‐induced phlorotannin production in Fucus vesiculosus (Phaeophyceae). Journal of Phycology 37, 1026–1029. Asamizu, E., Nakajima, M., Kitade, Y., Saga, N., Nakamura, Y. and Tabata, S. (2003). Comparison of RNA expression profiles between the two generations of Porphyra yezoensis (Rhodophyta), based on Expressed Sequence Tag frequency analysis. Journal of Phycology 39, 923–930. Baldauf, S. L. (2003). The deep roots of eukaryotes. Science 300, 1703–1706. Benet, H., Ar Gall, E., Asensi, A. and Kloareg, B. (1997). Protoplast regeneration from gametophytes and sporophytes of some species in the order Laminariales (Phaeophyceae). Protoplasma 199, 39–48. Berglin, M., Delage, L., Potin, P., Vilter, H. and Elwing, H. (2004). Enzymatic Cross‐ linking of a phenolic polymer extracted from the marine alga Fucus serratus. Biomacromolecules 5, 2376–2383.

MARINE MACROALGAE AGAINST PATHOGENS

255

Bitton, R., Ben‐Yehuda, M., Davidovich, M., Balazs, Y., Potin, P., Delage, L., Colin, C. and Bianco‐Peled, H. (2006). Structure of algal‐born phenolic polymeric adhesives. Macromolecular Bioscience 6, 737–746. Ble´e, E. (1998). Phytooxylipins and plant defense reactions. Progress in Lipid Research 37, 33–72. Bolwell, G. P. (1999). Role of active oxygen species and NO in plant defence responses. Current Opinion in Plant Biology 2, 287–294. Boonprab, K., Matsui, K., Akakabe, Y., Yotsukura, N. and Kajiwara, T. (2003). Hydroperoxy‐arachidonic acid mediated n‐hexanal and (Z)‐3‐ and (E)‐2‐ nonenal formation in Laminaria angustata. Phytochemistry 63, 669–678. Borchardt, S. A., Allain, E. J., Michels, J. J., Stearns, G. W., Kelly, R. F. and McCoy, W. F. (2001). Reaction of acylated homoserine lactone bacterial signaling molecules with oxidized halogen antimicrobials. Applied and Environmental Microbiology 67, 3174–3179. Bouarab, K. (2000). Bases biochimiques et cellulaires des interactions plante‐ pathoge`ne dans le syste`me Chondrus crispus—Acrochaete operculata Doctorat Oce´anologie Biologique et Environnement Marin, Universite´ de Paris VI, Paris. Bouarab, K., Potin, P., Correa, J. and Kloareg, B. (1999). Sulfated oligosaccharides mediate the interaction between a marine red alga and its green algal pathogenic endophyte. Plant Cell 11, 1635–1650. Bouarab, K., Kloareg, B., Potin, P. and Correa, J. A. (2001a). Ecological and biochemical aspects in algal infectious diseases. Cahier de Biologie Marine 42, 91–100. Bouarab, K., Potin, P., Weinberger, F., Correa, J. and Kloareg, B. (2001b). The Chondrus crispus‐Acrochaete operculata host‐pathogen association, a novel model in glycobiology and applied phycopathology. Journal of Applied Phycology 13, 185–193. Bouarab, K., Adas, F., Gaquerel, E., Kloareg, B., Salaun, J. P. and Potin, P. (2004). The innate immunity of a marine red alga involves oxylipins from both the eicosanoid and octadecanoid pathways. Plant Physiology 135, 1838–1848. Brisson, L. F., Tenhaken, R. and Lamb, C. (1994). Function of oxidative cross‐ linking of cell wall structural proteins in plant disease resistance. Plant Cell 6, 1703–1712. Butler, A. and Carter‐Franklin, J. N. (2004). The role of vanadium bromoperoxidase in the biosynthesis of halogenated marine natural products. Natural Product Reports 21, 180–188. Butler, A., Soedjak, H. S., Polne‐Fuller, M., Gibor, A., Boyen, C. and Kloareg, B. (1990). Studies of vanadium‐bromoperoxidase using surface and cortical protoplasts of Macrocystis pyrifera (Phaeophyta). Journal of Phycology 26, 589–592. Colin, C., Leblanc, C., Wagner, E., Delage, L., Leize‐Wagner, E., van Dorsselaer, A., Kloareg, B. and Potin, P. (2003). The brown algal kelp Laminaria digitata features distinct bromoperoxidase and iodoperoxidase activities. Journal of Biological Chemistry 278, 23545–23552. Colin, C., Leblanc, C., Michel, G., Wagner, E., Leize‐Wagner, E., van Dorsselaer, A. and Potin, P. (2005). Vanadium‐dependent iodoperoxidases in Laminaria digitata, a novel biochemical function diverging from brown algal bromoperoxidases. Journal of Biological Inorganic Chemistry 10, 156–166. Colle´n, J. and Davison, I. R. (1999). Stress tolerance and reactive oxygen metabolism in the intertidal red seaweeds Mastocarpus stellatus and Chondrus crispus. Plant, Cell and Environment 22, 1143–1151.

256

A. COSSE ET AL.

Colle´n, J., Pederse´n, M. and Bornman, C. H. (1994). A stress‐induced oxidative burst in Eucheuma platycladum (Rhodophyta). Physiologia Plantarum 92, 417–422. Colle´n, J., Herve´, C., Guisle‐Marsollier, I., Le´ger, J. J. and Boyen, C. (2006). Expression profiling of Chondrus crispus (Rhodophyta) after exposure to methyl jasmonate. Journal of Experimental Botany 57, 3869–3881. Correa, J. A. and McLachlan, J. L. (1992). Endophytic algae of Chondrus crispus (Rhodophyta). IV. Detrimental eVects on the host following infections by Acrochaete operculata and A. heteroclada. Marine Ecology Progress Series 81, 73–87. Correa, J. A. and McLachlan, J. L. (1994). Endophytic algae of Chondrus crispus (Rhodophyta). V. Fine structure of the infection by Acrochaete operculata. European Journal of Phycology 29, 33–47. Correa, J. A. and Sanchez, P. A. (1996). Ecological aspects of algal infectious diseases. Hydrobiologia 327, 89–95. Correa, J. A., Nielsen, R. and Grund, D. W. (1988). Endophytic algae of Chondrus crispus Stackhouse. II. Acrochaete heteroclada sp. nov., Acrochaete operculata sp. nov., and Phaeophila dendroides (Crouan frat.) Batters (Chlorophyta). Journal of Phycology 24, 528–539. Coˆte´, F. and Hahn, M. G. (1994). Oligosaccharins: Structures and signal transduction. Plant Molecular Biology 26, 1379–1411. Coˆte´, F., Ham, K. S., Hahn, M. G. and Bergmann, C. W. (1998). Oligosaccharide elicitors in host‐pathogen interactions. Generation, perception, and signal transduction. In ‘‘Subcellular Biochemistry, Plant Microbe Interactions’’ (B. B. Biswas and H. K. Das, eds.), Vol. 29, pp. 385–432. Plenum Press, New York. Courtois, M. B. (1813). Sur un nouvel acide forme´ avec la substance de´couverte par M. Courtois. Annales de Chimie et de Physique 88, 311–318. Cre´pineau, F., Roscoe, T., Kaas, R., Kloareg, B. and Boyen, C. (2000). Characterisation of complementary DNAs from the expressed sequence tag analysis of life cycle stages of Laminaria digitata (Phaeophyceae). Plant Molecular Biology 43, 503–513. Cronin, G. and Hay, M. E. (1996). Induction of seaweed chemical defenses by amphipod grazing. Ecology 77, 2287–2301. Custers, J. H. H. V., Harrison, S. J., Sela‐Buurlage, M. B., van Deventer, E., Lageweg, W., Howe, P. W., van der Meijs, P. J., Ponstein, A. S., Simons, B. H., Melchers, L. S. and Stuiver, M. H. (2004). Isolation and characterisation of a class of carbohydrate oxidases from higher plants, with a role in active defence. The Plant Journal 39, 147–160. Cutignano, A., d’Ippolito, G., Romano, G., Lamari, N., Cimino, G., Febbraio, F., Nucci, R. and Fontana, A. (2006). Chloroplastic glycolipids fuel Aldehyde biosynthesis in the marine diatom Thalassiosira rotula. Chembiochem: A European Journal of Chemical Biology 7, 450–456. Darvill, A., Augur, C., Bergmann, C., Carlson, R. W., Cheong, J. J., Eberhard, S., Hahn, M. G., Lo, M. V., Marfa, V., Meyer, B., Mohnen, D., O’Neil, M. A., et al. (1992). Oligosaccharins‐oligosaccharides that regulate growth, development and defense responses in plants. Glycobiology 2, 181–198. de Nys, R. and Steinberg, P. D. (2002). Linking marine biology and biotechnology. Current Opinion in Biotechnology 13, 244–248. Derelle, E., Ferraz, C., Rombauts, S., Rouze, P., Worden, A. Z., Robbens, S., Partensky, F., Degroeve, S., Echeynie, S., Cooke, R., Saeys, Y., Wuyts, J., et al. (2006). Genome analysis of the smallest free‐living eukaryote

MARINE MACROALGAE AGAINST PATHOGENS

257

Ostreococcus tauri unveils many unique features. Proceedings of the National Academy of Sciences of the United States of America 103, 11647–11652. Desikan, R., A. H.‐Mackerness, S., Hancock, J. T. and Neill, S. J. (2001). Regulation of the Arabidopsis transcriptome by oxidative stress. Plant Physiology 127, 159–172. d’Ippolito, G., Cutignano, A., Tucci, S., Romano, G., Cimino, G. and Fontana, A. (2006). Biosynthetic intermediates and stereochemical aspects of aldehyde biosynthesis in the marine diatom Thalassiosira rotula. Phytochemistry 67, 314–322. Ebel, J. and Cosio, E. G. (1994). Elicitors of plant defense responses. International Review of Cytology 148, 1–36. Ellertsdottir, E. and Peters, A. F. (1997). High prevalence of infection by endophytic brown algae in populations of Laminaria spp. (Phaeophyceae). Marine Ecology Progress Series 146, 135–143. Fritig, B., Heitz, T. and Legrand, M. (1998). Antimicrobial proteins in induced plant defense. Current Opinion in Immunology 10, 16–22. Fujita, Y., Zenitani, B., Nakao, Y. and Matsubara, T. (1972). Bacteriological studies on diseases of cultured laver.II. Bacteria associated with diseased laver. Bulletin of the Japanese Society of Scientific Fisheries 38, 565–569. Funk, C. D. (2001). Prostaglandins and leukotrienes: Advances in eicosanoid biology. Science 294, 1871–1875. Gaquerel, E., Herve, C., Labriere, C., Boyen, C., Potin, P. and Salaun, J. P. (2007). Evidence for oxylipin synthesis and induction of a new polyunsaturated fatty acid hydroxylase activity in Chondrus crispus in response to methyljasmonate. Biochimica Biophysica Acta 1771, 565–575. Gerber, I. B. and Dubery, I. A. (2004). Protein phosphorylation in Nicotiana tabacum cells in response to perception of lipopolysaccharides from Burkholderia cepacia. Phytochemistry 65, 2957–2966. Gerber, I. B., Zeidler, D., Durner, J. and Dubery, I. A. (2004). Early perception responses of Nicotiana tabacum cells in response to lipopolysaccharides from Burkholderia cepacia. Planta 218, 647–657. Gerwick, W. H., Roberts, M. A., Vulpanovici, A. and Ballantine, D. L. (1999). Biogenesis and biological function of marine algal oxylipins. Lipoxygenases and Their Metabolites 447, 211–218. Gibbs, S. P. (1993). The evolution of the algal chloroplast. In ‘‘Origins of Plastids. Symbiogenesis, Prochlorophytes and the Origin of Chloroplasts’’ (R. A. Lewin, ed.), pp. 107–121. Chapman and Hall, New York. Givskov, M., de Nys, R., Manefield, M., Gram, L., Maximilien, R., Eberl, L., Molin, S., Steinberg, P. D. and Kjelleberg, S. (1996). Eukaryotic interference with homoserine lactone‐mediated prokaryotic signalling. Journal of Bacteriology 178, 6618–6622. Gottardi, W. (1999). Iodine and disinfection: Theoretical study on mode of action, eYciency, stability and analytical aspects in the aqueous system. Archiv der Pharmazie and Pharmaceutical and Medicinal Chemistry 332, 151–157. Guschina, I. A. and Harwood, J. L. (2006). Lipids and lipid metabolism in eukaryotic algae. Progress in Lipid Research 45, 160–186. Hamberg, M. and Gardner, H. W. (1992). Oxylipin pathway to jasmonates: Biochemistry and biological significance. Biochimica et Biophysica Acta (BBA)— Lipids and Lipid Metabolism 1165, 1–18. Hamberg, M. and Gerwick, W. H. (1993). Biosynthesis of vicinal dihydroxy fatty‐ acids in the red alga Gracilariopsis lemaneiformis—Identification of a sodium‐dependent 12‐lipoxygenase and a hydroperoxide isomerase. Archives of Biochemistry and Biophysics 305, 115–122.

258

A. COSSE ET AL.

Hancock, J. T., Desikan, R. and Neill, S. J. (2001). Role of reactive oxygen species in cell signalling pathways. Biochemical Society Transactions 29, 345–350. Harvell, C. D., Kim, K., Burkholder, J. M., Colwell, R. R., Epstein, P. R., Grimes, D. J., Hofmann, E. E., Lipp, E. K., Osterhaus, A., Overstreet, R. M., Porter, J. W., Smith, G. W., et al. (1999). Review: Marine ecology—Emerging marine diseases—Climate links and anthropogenic factors. Science 285, 1505–1510. Harvell, C. D., Mitchell, C. E., Ward, J. R., Altizer, S., Dobson, A. P., Ostfeld, R. S. and Samuel, M. D. (2002). Ecology—Climate warming and disease risks for terrestrial and marine biota. Science 296, 2158–2162. Hayes, J. and McLellan, L. (1999). Glutathione and glutathione‐dependent enzymes represent a co‐ordinately regulated defence against oxidative stress. Free Radical Research 31, 273–300. Heitz, T., Bergey, D. R. and Ryan, C. A. (1997). A gene encoding a chloroplast‐ targeted lipoxygenase in tomato leaves is transiently induced by wounding, systemin, and methyl jasmonate. Plant Physiology 114, 1085–1093. Herve´, C., Tonon, T., Colle´n, J., Corre, E. and Boyen, C. (2006). NADPH oxidases in eukaryotes: Red algae provide new hints! Current Genetics 49, 190–204. Hou, X., Chai, C., Qian, Q., Yan, X. and Fan, X. (1997). Determination of chemical species in iodine in some seaweeds. The Science of The Total Environment 204, 215–221. Howe, G. A. (2005). Jasmonate: An oxylipin signal with many roles in plants. Vitamins and Hormones 72, 431–456. Howe, G. A. and Schilmiller, A. L. (2002). Oxylipin metabolism in response to stress. Current Opinion in Plant Biology 5, 230–236. Ianora, A., Boersma, M., Casotti, R., Fontana, A., Harder, J., HoVmann, F., Pavia, H., Potin, P., Poulet, S. A. and Toth, G. (2006). New trends in marine chemical ecology. Estuaries and Coasts 29, 531–551. Ishiguro, S., Kawai‐Oda, A., Ueda, J., Nishida, I. and Okada, K. (2001). The defective in anther dehiscence 1gene encodes a novel phospholipase A1 catalyzing the initial step of jasmonic acid biosynthesis, which synchronizes pollen maturation, anther dehiscence, and flower opening in Arabidopsis. The Plant Cell 13, 2191–2209. Ishikawa, Y. and Saga, N. (1989). The diseases of economically valuable seaweeds and pathology in Japan. In ‘‘Current Topics in Marine Biotechnology’’ (S. Miyachi, I. Karube and Y. Ishda, eds.), pp. 215–218. Fuji Technology Press Ltd, Tokyo. Ishizaki, T., Shigemori, K., Nakai, T., Miyabo, S., Ozawa, T., Chang, S. W. and Voelkel, N. F. (1995a). Leukotoxin, 9,10‐epoxy‐12‐octadecenoate causes edematous lung injury via activation of vascular nitric‐oxide synthase. American Journal of Physiology‐Lung Cellular and Molecular Physiology 13, L65–L70. Ishizaki, T., Shigemori, K., Yamamura, Y., Matsukawa, S., Nakai, T., Miyabo, S., Hayakawa, M., Ozawa, T. and Voelkel, N. F. (1995b). Increased nitric‐ oxide biosynthesis in leukotoxin, 9,10‐epoxy‐12‐octadecenoate injured lung. Biochemical and Biophysical Research Communications 210, 133–137. Janssen, D. B., Pries, F. and Van der Ploeg, J. R. (1994). Genetics and biochemistry of dehalogenating enzymes. Annual Review of Microbiology 48, 163–191. Jiang, Z. D., Ketchum, S. O. and Gerwick, W. H. (2000). 5‐Lipoxygenase‐derived oxylipins from the red alga Rhodymenia pertusa. Phytochemistry 53, 129–133. John, M., Rohrig, H., Schmidt, J., Walden, R. and Schell, J. (1997). Cell signalling by oligosaccharides. Trends in Plant Science 2, 111–115.

MARINE MACROALGAE AGAINST PATHOGENS

259

Jordan, P. and Vilter, H. (1991). Extraction of proteins from material rich in anionic mucilages: Partition and fractionation of vanadate‐dependant bromoperoxidases from the brown algae Laminaria digitata and L. saccharina in aqueous polymer two‐phase systems. Biochimica Biophysica Acta 1073, 98–106. Krenn, B. E., Tromp, M. G. M. and Wever, R. (1989). The brown alga Ascophyllum nodosum contains two diVerent vanadium bromoperoxidases. Journal of Biological Chemistry 32, 19287–19292. Kruger, N. J. and von Schaewen, A. (2003). The oxidative pentose phosphate pathway: Structure and organisation. Current Opinion in Plant Biology 6, 236–246. Krupina, M. V. and Dathe, W. (1991). Occurrence of jasmonic acid in the red alga Gelidium latifolium. Zeitschrift fu¨r Naturforschung 46, 1127–1259. Kubanek, J., Jensen, P. R., Keifer, P. A., Sullards, M. C., Collins, D. O. and Fenical, W. (2003). Seaweed resistance to microbial attack: A targeted chemical defense against marine fungi. Proceedings of the National Academy of Sciences of the United States of America 100, 6916–6921. Ku¨pper, F. C. and Mu¨ller, D. G. (1999). Massive occurrence of the heterokont and fungal parasites Anisolpidium, Eurychasma and Chytridium in Pylaiella littoralis (Ectocarpales, Phaeophyceae). Nova Hedwigia 69, 381–389. Ku¨pper, F. C., Schweigert, N., Ar Gall, E., Legendre, J.‐M., Vilter, H. and Kloareg, B. (1998). Iodine uptake in Laminariales involves extracellular, haloperoxidase‐mediated oxidation of iodide. Planta 207, 163–171. Ku¨pper, F. C., Kloareg, B., Guern, J. and Potin, P. (2001). Oligoguluronates elicit an oxidative burst in the brown algal kelp Laminaria digitata. Plant Physiology 125, 278–291. Ku¨pper, F. C., Mu¨ller, D. G., Peters, A. F., Kloareg, B. and Potin, P. (2002). Oligoalginate recognition and oxidative burst play a key role in natural and induced resistance of sporophytes of laminariales. Journal of Chemical Ecology 28, 2057–2081. Ku¨pper, F. C., Gaquerel, E., Boneberg, E.‐M., Morath, S., Salau¨n, J.‐P. and Potin, P. (2006). Early events in the perception of lipopolysaccharides in the brown alga Laminaria digitata include an oxidative burst and activation of fatty acid oxidation cascades. Journal of Experimental Botany 57, 1991–1999. Laloi, C., Mestres‐Ortega, D., Marco, Y., Meyer, Y. and Reichheld, J. P. (2004). The Arabidopsis cytosolic thioredoxin h5 gene induction by oxidative stress and its W‐box‐mediated response to pathogen elicitor. Plant Physiology 134, 1006–1016. Lalucque, H. and Silar, P. (2003). NADPH oxidase: An enzyme for multicellularity? Trends in Microbiology 11, 9–12. Lamb, C. and Dixon, R. A. (1997). The oxidative burst in plant disease resistance. Annual Review of Plant Physiology and Plant Molecular Biology 48, 251–275. Lambeth, J. D. (2004). Nox enzymes and the biology of reactive oxygen. Nature Reviews Immunology 4, 181–189. ¨ berg, G. (2004). Ultraviolet radiation Laturnus, F., Svensson, T., Wiencke, C. and O aVects emission of ozone‐depleting substances by marine macroalgae: Results from a laboratory incubation study. Environmental Science & Technology 38, 6605–6609. Leblanc, C., Colin, C., Cosse, A., Delage, L., La Barre, S., Morin, P., Fie´vet, B., Voiseux, C., Ambroise, Y., Verhaeghe, E., Amouroux, D., Donard, O., et al. (2006). Iodine transfers in the coastal marine environment: The key role of brown algae and of their vanadium‐dependent haloperoxidases. Biochimie 88, 1773–1785.

260

A. COSSE ET AL.

Levine, A., Tenhaken, R., Dixon, R. A. and Lamb, C. (1994). H2O2 from the oxidative burst orchestrates the plant hypersensitive disease resistance response. Cell 79, 583–593. Levine, R. L., Mosoni, L., Berlett, B. S. and Stadtman, E. R. (1996). Methionine residues as endogenous antioxidants in proteins. Proceedings of the National Academy of Sciences of the United States of America 93, 15036–15040. Lion, U., Wiesemeier, T., Weinberger, F., Beltran, J., Flores, V., Faugeron, S., Correa, J. and Pohnert, G. (2006). Phospholipases and galactolipases trigger oxylipin‐mediated wound‐activated defence in the red alga Gracilaria chilensis against epiphytes. Chembiochem 7, 457–462. Littler, M. M. and Littler, D. S. (1995). Impact of clod pathogen on pacific coral reefs. Science 267, 1356–1360. Liu, Q. Y. and Reith, M. E. (1994). Isolation of a gametophyte‐specific cDNA encoding a lipoxygenase from the red alga Porphyra purpurea. Molecular Marine Biology and Biotechnology 3, 206–209. Lluisma, A. O. and Ragan, M. A. (1997). Expressed sequence tags (ESTs) from the marine red alga Gracilaria gracilis. Journal of Applied Phycology 9, 287–293. Maheswari, U., Montsant, A., Goll, J., Krishnasamy, S., Rajyashri, K. R., Patell, V. M. and Bowler, C. (2005). The Diatom EST Database. Nucleic Acids Research 33, D344–D347. Maier, I., Parodi, E., Westermeier, R. and Mu¨ller, D. G. (2000). Maullinia ectocarpii gen. et sp nov (Plasmodiophorea), an intracellular parasite in Ectocarpus siliculosus (Ectocarpales, Phaeophyceae) and other filamentous brown algae. Protist 151, 225–238. Malin, G., Ku¨pper, F. C., Carpenter, L. J., Baker, A., Broadgate, W., Kloareg, B. and Liss, P. S. (2001). Trace gas production by seaweeds: Defense, oxidative stress, signalling and atmospheric significance. Journal of Phycology 37, 32. Manefield, M., Welch, M., Givskov, M., Salmond, G. P. and Kjelleberg, S. (2001). Halogenated furanones from the red alga, Delisea pulchra, inhibit carbapenem antibiotic synthesis and exoenzyme virulence factor production in the phytopathogen Erwinia carotovora. Federation of European Microbiological Societies Microbiology Letters 205, 131–138. Manley, S. L. (2002). Phytogenesis of halomethanes: A product of selection or a metabolic accident? Biogeochemistry 60, 163–180. Marrs, K. A. (1996). The functions and regulation of glutathione S‐transferase in plants. Annual Review of Plant Physiology and Plant Molecular Biology 47, 127–158. Martinez, C., Montillet, J. L., Bresson, E., Agnel, J. P., Dai, G. H., Daniel, J. F., Geiger, J. P. and Nicole, M. (1998). Apoplastic peroxidase generates superoxide anions in cells of cotton cotyledons undergoing the hypersensitive reaction to Xanthomonas campestris pv. malvacearum race 18. Molecular Plant‐Microbe Interactions 11, 1038–1047. Matsuzaki, M., Misumi, O., Shin, I. T., Maruyama, S., Takahara, M., Miyagishima, S. Y., Mori, T., Nishida, K., Yagisawa, F., Yoshida, Y., Nishimura, Y., Nakao, S., et al. (2004). Genome sequence of the ultrasmall unicellular red alga Cyanidioschyzon merolae 10D. Nature 428, 653–657. Maximilien, R., de Nys, R., Holmstro¨m, C., Gram, L., Givskov, M., Crass, K., Kjelleberg, S. and Steinberg, P. D. (1998). Chemical mediation of bacterial surface colonisation by secondary metabolites from the red alga Delisea pulchra. Aquatic Microbial Ecology 15, 233–246. McFiggans, G., Coe, H., Burgess, R., Allan, J., Cubison, M., Alfarra, M. R., Saunders, R., Saiz‐Lopez, A., Plane, J. M. C., Wevill, D., Carpenter, L. J., Rickard, A. R., et al. (2004). Direct evidence for coastal iodine particles

MARINE MACROALGAE AGAINST PATHOGENS

261

from Laminaria macroalgae—Linkage to emissions of molecular iodine. Atmospheric Chemistry and Physics 4, 701–713. McLusky, S. R., Bennett, M. H., Beale, M. H., Lewis, M. J., Gaskin, P. and Mansfield, J. W. (1999). Cell wall alterations and localized accumulation of feruloyl‐30 ‐methoxytyramine in onion epidermis at sites of attempted penetration by Botrytis allii are associated with actin polarisation, peroxidase activity and suppression of flavonoid biosynthesis. The Plant Journal 17, 523–534. Medzhitov, R. and Janeway, C. A. J. (2002). Decoding the patterns of self and nonself by the innate immune system. Science 296, 298–300. Mellersh, D. G., Foulds, I. V., Higgins, V. J. and Heath, M. C. (2002). H2O2 plays diVerent roles in determining penetration failure in three diverse plant‐ fungal interactions. The Plant Journal 29, 257–268. Menezes‐Benavente, L., Teixeira, F. K., Alvim Kamei, C. L. and Margis‐Pinheiro, M. (2004). Salt stress induces altered expression of genes encoding antioxidant enzymes in seedlings of a Brazilian indica rice (Oryza sativa L.). Plant Science 166, 323–331. Meyer, A., Pu¨hler, A. and Niehaus, K. (2001). The lipopolysaccharides of the phytopathogen Xanthomonas campestris pv. campestris induce an oxidative burst reaction in cell cultures of Nicotiana tabacum. Planta 213, 214–222. Meyer, Y., Reichheld, J. P. and Vignols, F. (2005). Thioredoxins in Arabidopsis and other plants. Photosynthesis Research 86, 419–433. Miralto, A., Barone, G., Romano, G., Poulet, S. A., Ianora, A., Russo, L., Buttino, I., Mazzarella, G., Laabir, M., Cabrini, M. and Giacobbe, M. G. (1999). The indidious eVect of diatoms on copepod reproduction. Nature 402, 173–176. Mitho¨fer, A., Wanner, G. and Boland, W. (2005). EVects of feeding Spodoptera littoralis on lima bean leaves. II. Continuous mechanical wounding resembling insect feeding is suYcient to elicit herbivory‐related volatile emission. Plant Physiology 137, 1160–1168. Mittler, R. (2002). Oxidative stress, antioxidants and stress tolerance. Trends in Plant Science 7, 405–410. Moore, R. M., Webb, M., Tokarczyk, R. and Wever, R. (1996). Bromoperoxidase and iodoperoxidase enzymes and production of halogenated methanes in marine diatom cultures. Journal of Geophysical Research 101, 20899–20908. Mtolera, M., Colle´n, J., Pederse´n, M., Ekdahl, A., Abrahamsson, K. and Semesi, A. (1996). Stress induced production of volatile halogenated organic compounds in Eucheuma denticulatum (Rhodophyta) caused by elevated pH and high light intensity. European Journal of Phycology 31, 89–95. Mu¨ller, E. G. (1991). Thioredoxin deficiency in yeast prolongs S phase and shortens the G1 interval of the cell cycle. Journal of Biological Chemistry 266, 9194–9202. Mu¨ller, D. G., Kapp, M. and Knippers, R. (1998). Viruses in marine brown algae. Advances in Virus Research 50, 49–67. Neill, S. J., Desikan, R., Clarke, A., Hurst, R. D. and Hancock, J. T. (2002). Hydrogen peroxide and nitric oxide as signalling molecules in plants. Journal of Experimental Botany 53, 1237–1247. Nikaido, I., Asamizu, E., Nakajima, M., Nakamura, Y., Saga, N. and Tabata, S. (2000). Generation of 10,154 expressed sequence tags from a leafy gametophyte of a marine red alga, Porphyra yezoensis. DNA Research 7, 223–227. Noordermeer, M. A., Veldink, G. A. and Vliegenthart, J. F. G. (2001). Fatty acid hydroperoxide lyase: A plant cytochrome P450 enzyme involved in wound healing and pest resistance. Chembiochem 2, 494–504.

262

A. COSSE ET AL.

Nozaki, H., Matsuzaki, M., Takahara, M., Misumi, O., Kuroiwa, H., Hasegawa, M., Shini, T., Kohara, Y., Ogasawara, N. and Kuroiwa, T. (2003). The phylogenetic position of red algae revealed by multiple nuclear genes from mitochondria‐containing eukaryotes and an alternative hypothesis on the origin of plastids. Journal of Molecular Evolution 56, 485–497. Nu¨rnberger, T., Brunner, F., Kemmerling, B. T. and Piater, L. (2004). Innate immunity in plants and animals: Striking similarities and obvious diVerences. Immunological Reviews 198, 249–266. O’Dowd, C. D., Jimenez, J. L., Bahreini, R., Flagan, R. C., Seinfeld, J. H., Ha¨meri, K., Pirjola, L., Kulmala, M., Jennings, G. S. and HoVmann, T. (2002). Marine aerosol formation from biogenic iodine emissions. Nature 417, 632–636. Ohsawa, N., Ogata, Y., Okada, N. and Itoh, N. (2001). Physiological function of bromoperoxidase in the red marine alga, Corallina pilulifera: Production of bromoformas an allelochemical and the simultaneous elimination of hydrogen peroxide. Phytochemistry 58, 683–692. Orozco‐Cardenas, M. L. and Ryan, C. A. (1999). Hydrogen peroxide is generated systemically in plant leaves by wounding and systemin via the octadecanoid pathway. Proceedings of the National Academy of Sciences of the United States of America 96, 6553–6557. Orozco‐Cardenas, M. L., Narvaez‐Vasquez, J. and Ryan, C. A. (2001). Hydrogen peroxide acts as a second messenger for the induction of defense genes in tomato plants in response to wounding, systemin, and methyl jasmonate. The Plant Cell 13, 179–191. Palmer, C. J., Anders, T. L., Carpenter, L. J., Ku¨pper, F. C. and McFiggans, G. (2005). Iodine and halocarbon response of Laminaria digitata to oxidative stress and links to atmospheric new particle production. Environmental Chemistry 2, 282–290. Paul, V. J. and Puglisi, M. P. (2004). Chemical mediation of interactions among marine organisms. Natural Product Reports 21, 189–209. Paul, N. A., de Nys, R. and Steinberg, P. D. (2006a). Seaweed‐herbivore interactions at a small scale: Direct tests of feeding deterrence by filamentous algae. Marine Ecology Progress Series 323, 1–9. Paul, V. J., Puglisi, M. P. and Ritson‐Williams, R. (2006b). Marine chemical ecology. Natural Product Reports 23, 153–180. Pavia, H. and Toth, G. B. (2000). Inducible chemical resistance in the brown seaweed Ascophylum nodosum. Ecology 81, 3212–3225. Pederse´n, M., Colle´n, J., Abrahamsson, K. and Ekdahl, A. (1996). Production of halocarbons from seaweeds: An oxidative stress reaction? Scientia Marina 60, 257–263. Peters, A. F., Marie, D., Scornet, D., Kloareg, B. and Cock, M. J. (2004). Proposal of Ectocarpus siliculosus (Ectocarpales, Phaeophyceae) as a model organism for brown algal genetics and genomics. Journal of Phycology 40, 1079–1088. Pohnert, G. (2000). Wound‐activated chemical defense in unicellular planktonic algae. Angewandte Chemie— International Edition 39, 4352–4354. Pohnert, G. (2002). Phospholipase A2 activity triggers the wound‐activated chemical defense in the diatom Thalassiosira rotula. Plant Physiology 129, 103–111. Pohnert, G. (2004). Chemical defense strategies of marine organisms. Topics in Current Chemistry 239, 179–219. Pohnert, G. (2005). Diatom/copepod interactions in plankton: The indirect chemical defense of unicellular algae. Chembiochem 6, 946–959. Pohnert, G. and Boland, W. (1996). Biosynthesis of the algal pheromone hormosirene by the fresh‐water diatom Gomphonema parvulum (Bacillariophyceae). Tetrahedron 52, 10073–10082.

MARINE MACROALGAE AGAINST PATHOGENS

263

Pohnert, G. and Boland, W. (2002). The oxylipin chemistry of attraction and defense in brown algae and diatoms. Natural Product Reports 19, 108–122. Potin, P., Bouarab, K., Ku¨pper, F. C. and Kloareg, B. (1999). Oligosaccharide recognition signals and defence reactions in marine plant‐microbe interactions. Current Opinion in Microbiology 2, 276–283. Potin, P., Bouarab, K., Salau¨n, J.‐P., Pohnert, G. and Kloareg, B. (2002). Biotic interactions of marine algae. Current Opinion in Plant Biology 5, 308–317. Prost, I., Dhondt, S., Rothe, G., Vicente, J., Rodriguez, M. J., Kift, N., Carbonne, F., GriYths, G., Esquerre‐Tugaye, M. T., Rosahl, S., Castresana, C., Hamberg, M., et al. (2005). Evaluation of the antimicrobial activities of plant oxylipins supports their involvement in defense against pathogens. Plant Physiology 139, 1902–1913. Rea, G., Metoui, O., Infantino, A., Federico, R. and Angelini, R. (2002). Copper amine oxidase expression in defense responses to wounding and Ascochyta rabiei Invasion. Plant Physiology 128, 865–875. Ritz, D., Patel, H., Doan, B., Zheng, M., Aslund, F., Storz, G. and Beckwith, J. (2000). Thioredoxin 2 is involved in the oxidative stress response in Escherichia coli. Journal of Biological Chemistry 275, 2505–2512. Roeder, V., Colle´n, J., Rousvoal, S., Corre, E., Leblanc, C. and Boyen, C. (2005). Identification of stress gene transcripts in Laminaria digitata (Phaeophyceae) protoplast cultures by Expressed Sequence Tag analysis. Journal of Phycology 41, 1227–1235. Ross, C., Ku¨pper, F. C., Vreeland, V., Herbert Waite, J. and Jacobs, R. S. (2005a). Evidence of a latent oxidative burst in relation to wound repair in the giant unicellular chlorophyte Dasycladus vermicrularis. Journal of Phycology 41, 531–541. Ross, C., Vreeland, V., Herbert Waite, J. and Jacobs, R. S. (2005b). Rapid assembly of a wound plug: Stage one of a two‐stage wound repair mechanism in the giant unicellular chlorophyte Dasycladus vermicularis (Chlorophyceae). Journal of Phycology 41, 46–54. Ross, C., Ku¨pper, F. C. and Jacobs, R. S. (2006). Involvement of reactive oxygen species and reactive nitrogen species in the wound response of Dasycladus vermicularis. Chemistry & Biology 13, 353–364. Ryan, C. A. and Farmer, E. E. (1991). Oligosaccharide signals in plants: A current assessment. Annual Review of Plant Physiology and Plant Molecular Biology 42, 651–674. Saenko, G. N., Kravtsova, Y. Y., Ivanenko, V. V. and Sheludko, S. I. (1978). Concentration of iodine and bromine by plants in the Seas of Japan and Okhotsk. Marine Biology 47, 243–250. Sawabe, T., Makino, H., Tatsumi, M., Nakano, K., Tajima, K., Iqbal, M. M., Yumoto, I., Ezura, Y. and Christen, R. (1998). Pseudoalteromonas bacteriolytica sp. nov., a marine bacterium that is the causative agent of red spot disease of Laminaria japonica. International Journal of Systematic Bacteriology 48, 769–774. Sawabe, T., Tanaka, R., Iqbal, M. M., Tajima, K., Ezura, Y., Ivanova, E. P. and Christen, R. (2000). Assignment of Alteromonas elyakovii KMM 162(T) and five strains isolated from spot‐wounded fronds of Laminaria japonica to Pseudoalteromonas elyakovii comb. nov and the extended description of the species. International Journal of Systematic and Evolutionary Microbiology 50, 265–271. Scala, S., Carels, N., Falciatore, A., Chiusano, M. L. and Bowler, C. (2002). Genome properties of the diatom Phaeodactylum tricornutum. Plant Physiology 129, 993–1002.

264

A. COSSE ET AL.

Schnitzler, I., Pohnert, G., Hay, M. and Boland, W. (2001). Chemical defense of brown algae (Dictyopteris spp.) against the herbivorous amphipod Ampithoe longimana. Oecologia 126, 515–521. Seo, H. S., Song, J. T., Cheong, J.‐J., Lee, Y.‐H., Lee, Y.‐W., Hwang, I., Lee, J. S. and Choi, Y. D. (2001). Jasmonic acid carboxyl methyltransferase: A key enzyme for jasmonate‐regulated plant responses. Proceedings of the National Academy of Sciences of the United States of America 98, 4788–4793. Shah, M., Wuilloud, R. G., Kannamkumaratha, S. S. and Caruso, J. A. (2005). Iodine speciation studies in commercially available seaweed by coupling diVerent chromatographic techniques with UV and ICP‐MS detection. Journal of Analytical Atomic Spectrometry 20, 176–182. Silipo, A., Molinaro, A., Sturiale, L., Dow, J. M., Erbs, G., Lanzetta, R., Newman, M. A. and Parrilli, M. (2005). The elicitation of plant innate immunity by lipooligosaccharide of Xanthomonas campestris. Journal of Biological Chemistry 280, 33660–33668. Sotka, E. E., Taylor, R. B. and Hay, M. E. (2002). Tissue‐specific induction of resistance to herbivores in a brown seaweed: The importance of direct grazing versus waterborne signals from grazed neighbors. Journal of Experimental Marine Biology and Ecology 277, 1–12. Sparrow, F. K. J. (1969). Zoosporic marine fungi from the Pacific Northwest (U.S.A.). Archive Mikrobiology 66, 129–146. Stanley, M. S., Perry, R. M. and Callow, J. A. (2005). Analysis of Expressed Sequence Tags from the green alga Ulva linza (Chlorophyta). Journal of Phycology 41, 1219–1226. Taki, N., Sasaki‐Sekimoto, Y., Obayashi, T., Kikuta, A., Kobayashi, K., Ainai, T., Yagi, K., Sakurai, N., Suzuki, H., Masuda, T., Takamiya, K.‐I., Shibata, D., et al. (2005). 12‐Oxo‐phytodienoic acid triggers expression of a distinct set of genes and plays a role in wound‐induced gene expression in Arabidopsis. Plant Physiology 139, 1268–1283. Taylor, R. B., Sotka, E. E. and Hay, M. E. (2002). Tissue‐specific induction of herbivore resistance: Seaweed response to amphipod grazing. Oecologia 132, 68–76. Teas, J., Pino, S., Critchley, A. and Braverman, L. E. (2004). Variability of iodine content in common commercially available edible seaweeds. Thyroid 14, 836–841. Thordal‐Christensen, H., Zhang, Z., Wei, Y. and Collinge, D. B. (1997). Subcellular localization of H2O2 in plants. H2O2 accumulation in papillae and hypersensitive response during the barley‐powdery mildew interaction. The Plant Journal 11, 1187–1194. Torres, M. A. and Dangl, J. L. (2005). Functions of the respiratory burst oxidase in biotic interactions, abiotic stress and development. Current Opinion in Plant Biology 8, 397–403. Torres, M. A., Jones, J. D. and Dangl, J. L. (2006). Reactive oxygen species signaling in response to pathogens. Plant Physiology 141, 373–378. Toth, G. B. and Pavia, H. (2000). Water‐borne cues induce chemical defense in a marine alga Ascophyllum nodosum. Proceedings of the National Academy of Sciences of the United States of America 97, 14418–14420. Toth, G. B., Langhamer, O. and Pavia, H. (2005). Inducible and constitutive defenses of valuable seaweed tissues: Consequences for herbivore fitness. Ecology 86, 612–618. Turner, J. G., Ellis, C. and Devoto, A. (2002). The Jasmonate signal pathway. The Plant Cell 14, S153–164.

MARINE MACROALGAE AGAINST PATHOGENS

265

Vardi, A., Formiggini, F., Casotti, R., De Martino, A., Ribalet, F., Miralto, A. and Bowler, C. (2006). A stress surveillance system based on calcium and nitric oxide in marine diatoms. PLoS Biology 4, 411–419. Vilter, H. (1995). Vanadium‐dependent haloperoxidases. In ‘‘Metal ions in Biological Systems’’ (H. Sigel and A. Sigel, eds.), Vol. 31, pp. 325–362. Marcel Dekker, Inc., New York, Basel, Hong Kong. von Rad, U., Mueller, M. J. and Durner, J. (2005). Evaluation of natural and synthetic stimulants of plant immunity by microarray technology. New Phytologist 165, 191–202. Vreeland, V., Ng, K. L. and Epstein, L. (1998). cDNA sequence and active recombinant vanadium bromoperoxidase from Fucus embryos. Molecular Biology of the Cell 9, 1043. Wang, W., Vinocur, B., Shoseyov, O. and Altman, A. (2004). Role of plant heat‐ shock proteins and molecular chaperones in the abiotic stress response. Trends in Plant Science 9, 244–252. Weber, H. (2002). Fatty acid‐derived signals in plants. Trends in Plant Science 7, 217–224. Weinberger, F. and Friedlander, M. (2000). Response of Gracilaria conferta (Rhodophyta) to oligoagars results in defense against agar‐degrading epiphytes. Journal of Phycology 36, 1079–1086. Weinberger, F., Friedlander, M. and Hoppe, H.‐G. (1999). Oligoagars elicit a physiological response in Gracilaria conferta (Rhodophyta). Journal of Phycology 35, 747–755. Weinberger, F., Richard, C., Kloareg, B., Kashman, Y., Hoppe, H.‐G. and Friedlander, M. (2001). Structure‐activity relationships of oligoagar elicitors toward Gracilaria conferta (Rhodophyta). Journal of Phycology 37, 418–426. Weinberger, F., Pohnert, G., Kloareg, B. and Potin, P. (2002). A signal released by an endophytic attacker acts as a substrate for a rapid defensive reaction of the red alga Chondrus crispus. ChemBioChem 3, 1260–1263. Weinberger, F., Pohnert, G., Berndt, M. L., Bouarab, K., Kloareg, B. and Potin, P. (2005a). Apoplastic oxidation of L‐asparagine is involved in the control of the green algal endophyte Acrochaete operculata Correa & Nielsen by the red seaweed Chondrus crispus Stackhouse. Journal of Experimental Botany 56, 1317–1326. Weinberger, F., Leonardi, P., Miravalles, A., Correa, J. A., Lion, U., Kloareg, B. and Potin, P. (2005b). Dissection of two distinct defense‐related responses to agar oligosaccharides in Gracilaria chilensis (Rhodophyta) and Gracilaria conferta (Rhodophyta). Journal of Phycology 41, 863–873. Wever, R., Tromp, M. G. M., Krenn, B. E., Marjani, A. and Van Tol, M. (1991). Brominating activity of the seaweed Ascophyllum nodosum: Impact on the biosphere. Environmental Science and Technology 25, 446–449. Wichard, T. and Pohnert, G. (2006). Formation of halogenated medium chain hydrocarbons by a lipoxygenase/hydroperoxide halolyase‐mediated transformation in planktonic microalgae. Journal of American Chemical Society 128, 7114–7115. Wojtaszek, P. (1997). Oxidative burst: An early plant response to pathogen infection. Biochemical Journal 322, 681–692. Yoon, H. S., Hackett, J. D., Ciniglia, C., Pinto, G. and Bhattacharya, D. (2004). A molecular timeline for the origin of photosynthetic eukaryotes. Molecular Biology and Evolution 21, 809–818.

266

A. COSSE ET AL.

Yoon, H. S., Hackett, J. D., Van Dolah, F. M., Nosenko, T., Lidie, L. and Bhattacharya, D. (2005). Tertiary endosymbiosis driven genome evolution in dinoflagellate algae. Molecular Biology and Evolution 22, 1299–1308. Yoshioka, H., Sugie, K., Park, H. J., Maeda, H., Tsuda, N., Kawakita, K. and Doke, N. (2001). Induction of plant gp91phox homolog by fungal cell wall, arachidonic acid, and salicylic acid in potato. Molecular Plant‐Microbe Interactions 14, 725–736. Zeidler, D., Zahringer, U., Gerber, I., Dubery, I., Hartung, T., Bors, W., Hutzler, P. and Durner, J. (2004). Innate immunity in Arabidopsis thaliana: Lipopolysaccharides activate nitric oxide synthase (NOS) and induce defense genes. Proceedings of the National Academy of Sciences of the United States of America 101, 15811–15816. Zhang, Z., Collinge, D. B. and Thordal‐Christensen, H. (1995). Germin‐like oxalate oxidase, a H2O2‐producing enzyme, accumulates in barley attacked by the powdery mildew fungus. The Plant Journal 8, 139–145. Zheng, W., Wise, M. L., Wyrick, A., Metz, J. G., Yuan, L. and Gerwick, W. H. (2002). Polyenoic fatty acid isomerase from the marine alga Ptilota filicina: Protein characterization and functional expression of the cloned cDNA. Archives of Biochemistry and Biophysics 401, 11–20.

AUTHOR INDEX

Numbers in bold refer to pages on which full references are listed.

A Aasamaa, K., 112, 123 Abadia, A., 158, 168 Abdel-Ghany, S. E., 161, 168, 176, 180 Abel, S., 44, 61 Abrahamsson, K., 236, 254, 261, 262 Achard, P., 51, 61 Adak, S., 19, 20, 24 Agre, P., 76, 77, 79, 123, 125, 128, 129, 131, 132, 136 Aida, M., 40, 62 Akiyama, K., 185, 190, 191, 208 Albrecht, C., 195, 196, 198, 208, 218 Alexander, M., 19, 32 Alexandersson, E., 93, 107, 118, 123, 133 Allen, M. F., 185, 208 Alleva, K., 88, 96, 123 Aloni, E., 51, 62 Aloni, R., 51, 62 Alvarez, M. I., 10, 24 Ameglio, T., 115, 124 Amijee, F., 185, 208 Amiour, N., 196, 197, 208 Amodeo, G., 88, 123, 124, 127 Amsler, C. D., 223, 224, 254 Andaluz, S., 159, 168 Anderson, I. C., 18, 19, 24 Andrews, J. H., 224, 254 Ane´, J.-M., 196, 208, 214 Anthony, T. L., 84, 124 Ar Gall, E., 234, 254, 259 Araujo, W. L., 20, 24 Arias, M. C., 194 Armbrust, E. V., 245, 254 Arner, E. S. J., 249, 254 Arnold, T. M., 228, 243, 254 Aroca, R., 104, 105, 124 Arosio, P., 141, 172 Arrese-Igor, C., 54, 62 Arrivault, S., 157, 168 Arruebarrena Di Palma, A., 19 Arulanantham, A. R., 159, 169 Asami, T., 28, 55, 62, 66, 72 Asamizu, E., 246, 254, 261 Axtell, M. J., 56, 62 Ayling, S. M., 190, 208

Azad, A. K., 89, 119, 124 Azaizeh, H., 95, 102, 124 B Bago, B., 186, 189, 208, 218 Bais, H. P., 13, 24, 219 Balaji, B., 186, 208 Baldan, B., 202, 216 Baldauf, S. L., 222, 252, 254 Balestrini, R., 183, 208 Balk, J., 161, 169 Baluska, F., 10, 24, 31 Bar-Ness, E., 146, 169 Barassi, C. A., 11, 19, 24, 25, 26, 69 Barbieri, P., 13, 15, 24 Barea, J. M., 12, 24 Bari, R., 52, 57, 62 Barker, S. J., 186, 193, 208, 218 Barroso, J. B., 16, 24 Bartel, D. P., 57, 62, 67, 68, 71, 73 Bashan, Y., 10, 11, 12, 13, 18, 25, 29 Bashir, K., 147, 169 Bastarrachea, F., 12, 33 Bates, T. R., 52, 62 Baudouin, E., 21, 25, 200, 209 Bauer, P., 151, 169, 172, 174 Baulcombe, D. C., 56, 66 Beaudoin, N., 50, 62 Be´card, G., 185, 186, 187, 188, 189, 190, 208, 209, 210, 214, 216, 218 Beck, A. B., 185, 217 Beeckman, T., 41, 62, 63, 64 Bel-Rhlid, R., 187, 209, 217 Beligni, M. V., 14, 25 Benet, H., 248, 254 Benfey, P. N., 6, 30, 38, 39, 40, 42, 62, 64, 65, 66, 67, 68, 70, 74, 124 Benkova, E., 40, 43, 62, 65, 66 Bennett, M. J., 43, 62, 63, 64, 72 Bereczky, Z., 142, 149, 151 Berger, F., 41, 62 Berglin, M., 236, 254 Berleth, T., 44, 62, 65 Bernhardt, C., 25, 41, 62 Bethke, P. C., 15, 25 Beuron, F., 80, 124

268

AUTHOR INDEX

Bewley, J. D., 120, 124 Bhalla, P. L., 40, 71 Bianciotto, V., 186, 209 Biela, A., 83, 124, 131 Bienert, G. P., 83, 124, 133 Bird, L. E., 20, 25 Birnbaum, K., 93, 94, 124 Bitton, R., 236, 255 Black, M., 120, 124 Blancaflor, E. B., 203, 209 Ble´e, E., 241, 255 Blilou, I., 43, 44, 62, 67, 198, 209 Bliss, F. A., 54, 71 Bloemberg, G. V., 13, 25 Bloom, A. J., 13, 25, 104, 124 Boddey, R. M., 18, 25 Boerjan, W., 43, 51, 62 Bohlool, B., 54, 72 Boland, W., 242, 261, 262, 263, 264 Bollman, K. M., 57, 63 Bolwell, G. P., 229, 255 Bonanomi, A., 197, 209 Bonfante, P., 66, 183, 194, 201, 203, 204, 208, 209, 210, 212, 214, 216, 217 Bonfante-Fasolo, P., 198, 218 Boonprab, K., 241, 243, 255 Borchardt, S. A., 237, 255 Borisov, A. Y., 193, 210 Boron, W. F., 83, 125, 131, 171 Borsani, O., 56, 58, 63 Bothe, H., 13, 18, 25, 30, 31, 33, 213, Bots, M., 117, 118, 119, 124 Bouarab, K., 224, 226, 227, 228, 229, 230, 233, 236, 237, 239, 240, 241, 242, 243, 244, 251, 252, 253, 255, 263, 265 Bouche, N., 56, 63 Boursiac, Y., 87, 94, 95, 100, 102, 125, 135 Bouwmeester, H., 190, 210 Boyer, J. S., 121, 134 Brachmann, A., 60, 63 Brady, S. M., 50, 63 Bray, E. A., 60, 63 Brechenmacher, L., 72, 183, 197, 210, 212, 215 Breuninger, 201, 210 Brewin, N. J., 46, 63 Briat, J. F., 138, 139, 142, 143, 144, 149, 155, 156, 158, 163, 165, 166, 169, 170, 173, 174, 178, 179 Bright, L. J., 47, 55, 63 Brill, W. J., 54, 64 Brisson, L. F., 236, 255 Brown, M. E., 12, 24, 25 Bruggemann, W., 153, 169 Brumbarova, T., 151, 169 Brundrett, M. C., 185, 210

Bucher, M., 184, 214 Bue´e, M., 185, 189, 210 Bughio, N., 155, 169 Burbulis, I. E., 188, 210 Burton, J. W., 150, 167, 169 Busi, M. V., 163, 169 Butler, A., 234, 236, 255 C Caetano-Anolle’s, G., 47, 63 Cakmak, I., 157, 169 Calantzis, C., 194, 210 Camoni, L., 199, 210 Carroll, B. J., 54, 63, 64 Carter-Franklin, J. N., 234, 255 Carvajal, M., 97, 102, 103, 104, 125, 127, 130 Casanovas, E. M., 11, 25 Casimiro, I., 43, 63, 64, 68 Castignetti, D., 19, 25 Cataldo, D. A., 153, 170 Catoira, R., 192, 194, 195, 210 Celenza, J. L., Jr., 43, 63 Cellier, M., 142, 170 Chabaud, M., 203, 210, 212, 214 Chabot, S., 187, 188, 189, 209 Chakrabarti, N., 85, 125 Chaney, R. L., 141, 178 Chang, M., 192, 210 Chaumont, F., 77, 124, 125, 126, 127, 131 Chen, J. G., 50, 51, 63 Chen, O. S., 163, 170 Chen, X., 56, 63 Chen, Y. H., 155, 170 Chen, Y., 19, 25, 26, 169, 172, 177, 180 Chilley, P. M., 45, 63 Chiou, T. J., 57, 63, 65 Choi, D. W., 20, 26 Chory, J., 44, 70 Chow, K.-S., 160, 170 Chrispeels, M. J., 77, 79, 93, 109, 113, 114, 124, 125, 127, 128, 130, 131, 133, 135 Clark, F. E., 18, 19, 31 Clarkson, D. T., 6, 26, 104, 125, 127, 129 Cline, G. R., 146, 170 Cochard, H., 111, 114, 115, 124, 125 Cock, M. J., 246, 254, 262 Cohen, M. F., 19, 20, 26 Colangelo, E. P., 139, 151, 170 Colin, C., 234, 235, 247, 255, 259 Colle´n, J., 230, 234, 246, 250, 251, 253, 255, 256, 258, 261, 262, 263 Combier, J. P., 59, 63 Conn, V. M., 20, 26 Connolly, E. L., 152, 157, 170, 176 Cook, C. E., 191, 210 Cook, D. R., 47, 70, 73, 211

AUTHOR INDEX Cooney, R. V., 15, 26 Cooper, G. J., 83, 125 Cornah, J. E., 160, 170, 177 Correa, J. A., 223, 224, 227, 255, 256, 265 Correa-Aragunde, N., 5, 6, 7, 10, 13, 21, 22, 26, 29 Cosgrove, D. J., 121, 125 Cosio, E. G., 226, 257 Costa, S., 27, 38, 41, 64, 65 Coˆte´, F., 226, 256 Courtois, M. B., 233, 256 Crane, B. R., 20, 24, 28, 32 Crawford, N. M., 5, 26, 178 Cre´pineau, F., 246, 248, 256 Crespi, M., 37, 63, 65, 69 Creus, C. M., 10, 11, 12, 14, 15, 17, 19, 21, 22, 24, 26, 69 Cronin, G., 228, 256 Crowley, D. E., 146, 170, 176, 179 Cueto, M., 21, 26 Curie, C., 142, 147, 148, 164, 170, 173, 179 Cutignano, A., 240, 256, 257 Cutruzzola´, F., 16, 17, 26 D D’Ippolito, G., 242, 256, 257 Dan, H., 53, 54, 64 Dangl, J. L., 229, 264 Daniels, M. J., 79, 82, 86, 125 Darvill, A., 226, 256 Dathe, W., 243, 259 David, R., 198, 210 David-Schwartz, R., 193, 194, 195, 211, 215 Davis, T. D., 3, 28 Davison, I. R., 250, 255 Day, D. A., 47, 64, 172, 173, 175, 179 Day, J. M., 11, 27 Dazzo, F. B., 54, 64, 74 De Groot, B. L., 85, 126 De Klerk, G. J., 4, 27 De Nys, R., 223, 256, 257, 260, 262 De Smet, I., 42, 43, 50, 64, 72, 73 Deak, K. I., 5, 26, 49, 50, 64, 65 Dean, R. M., 82, 83, 126, 132 Del Papa, M. F., 54, 64 Dell’Amico, J., 103, 126 Demchenko, K., 194, 196, 203, 211 Derelle, E., 245, 256 Desikan, R., 216, 247, 257, 258, 261 Dhanoa, P. K., 5 Dharmasiri, N., 30, 44, 64, 70 Di Laurenzio, L., 40, 64, 71 Dickson, S., 183, 211 DiDonato, R. J., 41, 42, 64, 179 DiDonato, R. J., Jr., 154, 170 Ding, X., 109, 112, 126 Dix, D. R., 146, 171

269

Dixit, R., 118, 119, 126, 128 Dixon, R. A., 228, 259, 260 Dobbelaere, S., 10, 12, 13, 15, 27, 32 Do¨bereiner, J., 11, 18, 25, 27, 30 Dolan, L., 7, 9, 27, 31, 32, 38, 62, 64, 65 Dorne, A. J., 159, 175 Douds, D. D., 185, 186, 189, 208, 209, 211, 214, 216 Downie, J. A., 46, 70, 193, 197, 199, 203, 214, 215, 216, 219 Drakakaki, G., 165, 166, 171 Drew, M. C., 103, 126 Dubery, I. A., 224, 257 Dubrovsky, J. G., 41, 64 Duc, G., 67, 193, 211, 214, 215 Durrett, T. P., 153, 171 Duzan, H. M., 55, 64 E Ebel, J., 226, 257 Eckhardt, U., 149, 171 Eide, D., 148, 171 Elias, K. S., 185, 211 Ellertsdottir, E., 223, 257 Elorza, A., 140, 171 Endre, G., 196, 211, 215 Everard, J. D., 103, 126 F Fahlgren, N., 57, 65 Fairhead, V. A., 223, 224, 254 Fallik, E., 12, 27 Farmer, E. E., 226, 263 Farquhar, M. L., 6, 31 Feldman, L., 40, 65 Felle, H. H., 8, 27, 213 Feng, H., 154, 171 Fennell, A., 104, 126 Ferreira, F. J., 45, 65, 71, 72 Fetter, K., 82, 90, 91, 126 Fievet, 236 Fischer, M., 77, 128 Fitz Gerald, J. N., 42, 65 Flexas, J., 116, 117, 126 Forde, B. G., 5, 33, 53, 66, 73, 74, 130 Foreman, J., 8, 27, 48, 65 Fortin, J. A., 189, 209 Fotiadis, D., 79, 126, 129 Franco, C. M. M., 20, 26 Franco-Zorrilla, J. M., 52, 65 Frangne, N., 108, 126 Franken, P., 185, 210, 211, 212, 217, 218 Fraysse, L. C., 108, 126 Fricke, W., 121, 126 Friedlander, M., 227, 229, 265

270

AUTHOR INDEX

Friml, J., 43, 62, 65 Fritig, B., 226, 257 Frugier, F., 55, 63, 65 Fu, D., 79, 84, 85, 126 Fu, X., 51, 65 Fujii, H., 57, 65 Fujita, Y., 223, 257 Fujiyoshi, Y., 85, 90, 126, 131 Fukaki, H., 42, 44, 65, 66, 70, 73, 74 Fukuhara, T., 121, 126 Funk, C. D., 238, 239, 245, 257 G Gadkar, V. R., 185, 194, 211 Gage, D. J., 38, 65 Gallagher, K. L., 41, 65 Galli, E., 13, 15, 24 Galvez, S., 37, 64 Galway, M. E., 7, 27 Gan, Y., 53, 66, 73 Gao, L.-L., 194, 195, 196, 211 Gao, Y. P., 121, 127 Gaquerel, E., 243, 244, 251, 255, 257, 259 Garcı´a de Salamone, I. E., 12, 27 Garcı´a-Mata, C., 9, 27, 29 Gardner, H. W., 243, 257 Gechev, T. S., 48, 66 Gemma, J. N., 185, 212 Genre, A., 66, 183, 203, 204, 210, 211, 212, 216 Gerbeau, P., 80, 83, 88, 122, 127, 131, 134 Gerber, I. B., 224, 257 Gerwick, W. H., 239, 240, 241, 242, 243, 257, 258, 266 Geurts, R., 46, 66, 208, 214 Ghassemian, M., 50, 66 Gianinazzi, S., 186, 208, 210, 211, 212, 213, 215, 217, 219 Gianinazzi-Pearson, V., 38, 66, 183, 186, 187, 194, 198, 210, 211, 212, 213, 215, 217, 219 Gibbs, S. P., 222, 257 Gilroy, S., 7, 24, 27, 33 Giovannetti, M., 185, 186, 189, 212, 213 Givskov, M., 238, 257, 260 Glenn, M. G., 185, 212, 213 Glick, B. R., 11, 27, 176 Gna¨dinger, F., 185, 211 Goda, H., 44, 66 Goldwasser, Y., 191, 212 Gollotte, A., 194, 212 Goormachtig, S., 201, 213 Goto, F., 165, 166, 171, 176 Gottardi, W., 237, 257 Gout, E., 103, 127, 134 Graham, J. H., 38, 66, 185, 187, 213 Graham, N., 38, 63

Green, L. S., 151, 171 GresshoV, P. M., 47, 54, 63, 64, 74, 210 Gros, P., 142, 170 Grotz, N., 155, 170, 171, 179 Grusak, M. A., 149, 154, 166, 171, 174 Guckert, A., 12, 27 Guenoune, D., 185, 213 Guenther, J. F., 89, 103, 127, 135 Guerinot, M. L., 138, 139, 148, 151, 153, 155, 170, 171, 172, 173, 176, 179, 180 Gu¨imil, S., 194, 213 Gunshin, H., 142, 171 Guo, H. S., 57, 66 Gupta, K. J., 15, 28 Guschina, I. A., 238, 239, 257 H Hachez, C., 93, 94, 127 Hahn, M. G., 64, 226, 256 Haissig, B. E., 3, 28 Hakman, I., 120, 132 Hamberg, M., 243, 257, 263 Hamilton, A. J., 56, 66 Hamman, T., 44, 66 Han, K. H., 51, 66 Hanba, Y. T., 117, 127 Hancock, J. T., 229, 257, 258, 261 Harari, A., 12, 13, 28 Harberd, N. P., 51, 61, 65 Harborne, J. B., 187, 213 Hardham, A. R., 203, 218 Harrison, M. J., 38, 66, 182, 183, 209, 212, 213, 214 Harrison, P. M., 141, 172 Hartmann, A., 14, 16, 22, 28 Harvell, C. D., 223, 258 Harwood, J. L., 238, 239, 257 Hasegawa, P. M., 55, 66, 69 Hay, M. E., 228, 256, 264 Hayashi, H., 191, 208 Hayes, J., 249, 258 He, Y., 10, 33 Heitz, T., 238, 257, 258 Helariutta, Y., 40, 42, 64, 66, 74 Hendriks, G., 90, 127 Henzler, T., 83, 87, 103, 104, 125, 127 Hepler, P. K., 8, 27, 179 Hepper, C. M., 189, 215 Herrera, M., 83, 127 Herrera-Medina, M. J., 185, 213 Herve´, C., 230, 231, 232, 244, 251, 253, 254, 256, 257, 258 Higuchi, M., 45, 66 Hildebrandt, U., 199, 213 Hinsinger, P., 145, 172 Hippler, M., 159, 172, 175 Hirsch, A. M., 37, 46, 66, 215

AUTHOR INDEX Hoarau, J., 104, 127, 129 Hocking, P. J., 166, 172 Hofer, R. M., 7, 28 Ho¨fte, H., 120, 127 Holbrook, N. M., 110, 111, 115, 124, 128, 132 Holguin, G., 11, 25 Hollocher, T. C., 19, 25 Holmgren, A., 249, 254 Hong, I., 20, 28 Ho¨rdt, W., 146, 172 Horgan, J. M., 53, 67 Hose, E., 101, 128 Hou, X., 234, 258 Howe, G. A., 238, 245, 251, 258 Hoyos, M. E., 5, 73 Huang, A. X., 5, 31 Hukin, D., 123, 128 Huynen, M. A., 163, 172 Hyde, B. B., 141, 142, 172 I Ianora, A., 224, 228, 258, 261 Ikeda, S., 119, 128 Imaizumi-Anraku, H., 72, 196, 213 Ishibashi, K., 85, 128 Ishiguro, S., 238, 258 Ishikawa, F., 77, 128, 133 Ishikawa, Y., 223, 258 Ishizaki, T., 244, 258 J Jacobsen, S. E., 56, 66 Jacquelinet-Jeanmougin, J., 184, 213 Jagnow, G., 19, 29 Jahn, T. P., 83, 124 Jain, D. K., 12, 28, 30 Jakob, C., 83, 135 Jakoby, M., 151, 172 James, T., 183, 213 Janeway, C. A. J., 224, 261 Jang, J. Y., 101, 128 Janssen, D. B., 250, 258 Jasid, S., 16, 28 Javot, H., 81, 93, 96, 97, 99, 105, 128, 131, 134 Jeandroz, S., 199, 200 Jetten, M. S. M., 17, 18, 28 Jiang, Z. D., 240, 258 Johansson, I., 77, 82, 89, 128, 129, 133 John, M., 226, 258 Johnson, K. D., 120, 128 Jolicoeur, M., 190, 214 Joly, R. J., 96, 97, 130 Jones, D. L., 8, 28

271

Jones, D., 7, 27 Jones-Rhoades, M. W., 57, 67, 71 Jordan, P., 236, 259 Jost, R., 54, 67 Jung, J. S., 79, 128 Ju¨rgens, G., 44, 66 Jurkevitch, E., 146, 172 K Kaiser, B. N., 142, 150, 172 KaldenhoV, R., 77, 99, 108, 112, 124, 126, 128, 129, 131, 133, 135 Kaldorf, M., 199, 214 Kamaluddin, M., 103, 129 Kanamori, N., 196, 214 Kapazoglou, A., 161, 172 Kape, R., 187, 214 Kapulnik, Y., 12, 13, 27, 28, 210, 211, 213, 215, 217, 219 Karandashov, V., 184, 214 Karlsson, M., 80, 82, 123, 128, 129, 133, 134 Karmoker, J. L., 104, 129 Katsuhara, M., 99, 127, 129, 130 Kepinski, S., 44, 67 Kers, J. A., 20, 28 Kidner, C. A., 56, 67 Kieber, J. J., 9, 28, 45, 65, 69, 71, 72 Kim, S. A., 143, 155, 167, 172 Kim, S. Y., 8, 28 King, J. J., 51, 67 Kispal, G., 157, 172, 173 Kistner, C., 193, 195, 196, 214, 218 Klebl, F., 83, 129 Kloepper, J. W., 10, 28, 31 Kobayashi, T., 152, 172, 175 Koide, R. T., 186, 217 Koike, S., 154, 166, 172 Kopriva, S., 53, 67 Koske, R. E., 185, 211 Kosuta, S., 192, 193, 203, 209, 214 Kramer, P. J., 103, 132 Krenn, B. E., 236, 259, 265 Krishnan, P., 120, 129 Krueger, C., 154, 172 Kruger, N. J., 249, 259 Krupina, M. V., 243, 259 Krusell, L., 47, 67 Kubanek, J., 223, 259 Kundu, B. S., 13, 28 Ku¨pper, F. C., 223, 226, 227, 229, 230, 234, 236, 239, 240, 242, 248, 253, 254, 259, 260, 262, 263 Kurihara, Y., 56, 67 Kuroha, T., 4, 28 Kushnir, S., 156, 173 Kutz, A., 5, 29, 53, 67

272

AUTHOR INDEX

L Labandera-Gonza´lez, C. A., 10, 30 Lagos-Quintana, M., 55, 67 Laize´, V., 80, 129 Laloi, C., 66, 249, 259 Lalucque, H., 232, 259 Lamattina, L., 4, 5, 9, 14, 15, 17, 19, 21, 22, 25, 26, 27, 29, 30, 69 Lamb, C., 228, 255, 259, 260 Lambais, M. R., 197, 198, 214 Lambeth, J. D., 229, 259 Lambrecht, M., 13, 29 Lamotte, O., 9, 29 Lanfranco, L., 190, 214 Lang, E., 19, 29 Lanquar, V., 142, 156, 173 Lanter, D. J., 54, 67 Lanteri, M. L., 4, 9, 22, 29, 30, 69 Larose, G., 185, 188, 192, 214 LaRue, T. A., 37, 46, 67, 215 Lassalles, J. P., 96, 131, 132 Laturnus, F., 236, 259 Laulhere, J. P., 142, 149, 166, 173 Layer, G., 163, 173 Le Jean, M., 164, 166, 170, 173 Leblanc, C., 234, 255, 259, 263 Lee, R. C., 55, 67 Lee, S. H., 104, 105, 129 Lei, J., 190, 214 Leitch, V., 90, 129 Lemanceau, P., 145, 173, 179 Leon, S., 162, 173 Lescure, A. M., 142, 173 Leshem, Y. Y., 9, 29 Levanony, H., 12, 25, 29 Levesque, M. P., 40, 67 Levier, K., 150, 173 Levine, A., 229, 260 Levine, R. L., 247, 260 Le´vy, J., 196, 208, 214 Leyser, O., 43, 44, 67, 72 Lezhneva, L., 161, 173 Li, H. M., 161, 173 Li, L., 149, 151, 155, 172, 173 Li, M., 52, 67 Lian, H. L., 100, 101, 129 Liang, Y., 55, 63, 68 Lill, R., 157, 172, 173 Lindermayr, C., 9, 29 Lindsay, W. L., 138, 145, 174 Ling, H. Q., 149, 151, 170, 171, 173, 174, 180 Linkohr, B. I., 5, 29, 52, 67 Lion, U., 240, 242, 243, 260, 265 Little, D. Y., 53, 68 Littler, D. S., 223, 260 Littler, M. M., 223, 260 Liu, J., 63, 203, 214 Liu, L. H., 83, 129 Liu, Q. Y., 241, 260

Liu, Y., 13, 29, 31 Ljung, K., 43, 68, 72, 73 Lluisma, A. O., 246, 260 Lobre´aux, S., 141, 142, 143, 161, 166, 169, 173, 174, 178 Lohse, S., 184, 215 Loiseau, L., 161, 174 Lombardo, M. C., 7, 10, 13, 21, 22, 29, 69 Long, S. R., 71, 192, 214, 215 Lopez, F., 103, 104, 129 Lopez-Bucio, J., 48, 52, 53, 68 Lopez-Millan, A. F., 150, 168, 174 Loque, D., 83, 129 Lott, J. N. A., 143, 174 Loudet, O., 42, 68 Louet, E., 145, 174 Lu, C., 59, 68 Lucena, C., 150, 174 Lugtenberg, B. J. J., 13, 25 Lum, M. R., 193, 215 Lundberg, J. O. N., 18, 33 Luu, D. T., 77, 125, 129, 134 Lynch, J. P., 52, 62, 68, 69 Lynch, J., 37, 68 Lynn, D. G., 192, 210, 215 M Ma, J. F., 77, 83, 86, 105, 122, 130, 146, 174, 175 Ma, S., 123, 130 Ma, Z, 52, 69 Maathuis, F. J., 102, 130 Maggio, A., 96, 97, 130 Maheswari, U., 245, 260 Maier, I., 223, 260 Malamy, J. E., 5, 6, 29, 30, 37, 42, 48, 49, 50, 53, 64, 65, 68, 70, 74 Malin, G., 236, 260 Mallory, A. C., 51, 56, 57, 68, 73 Mallory, T. E., 42, 68 Manefield, M., 238, 257, 260 Manley, S. L., 234, 236, 260 Manz, B., 120, 130 Marchant, A., 43, 62, 68 Marentes, E., 149, 166, 174 Mariani, P., 202, 216 Marin-Olivier, M., 118, 119, 130 Markhart, A., 94, 104, 126 Marques, C., 51, 69 Marrs, K. A., 249, 260 Marschner, H., 138, 148, 169, 174, 179, 180 Martienssen, R. A., 57, 67 Martin, A. C., 52, 65, 69, 71 Martinez, C., 232, 260 Martinez-Ballesta, M. C., 102, 103, 130 Martre, P., 90, 91, 96, 99, 100, 101, 105, 109, 110, 113, 115, 130, 131

AUTHOR INDEX Maruyama-Nakashita, A., 53, 69 Masalha, J., 145, 174 Mason, M. G., 45, 69, 71, 72 Massoumou, M., 197, 199, 200, 215 Mathesius, U., 46, 69 Matsuzaki, M., 245, 260, 262 Matthews, M. A., 104, 132 Matusova, R., 191, 215 Maurel, C., 77, 82, 83, 89, 93, 96, 97, 120, 122, 123, 125, 127, 128, 130, 133, 134, 135 Maximilien, R., 237, 257, 260 McEntyre, E., 120, 131 McFiggans, G., 235, 260, 262 McKie, A. T., 153, 174 McLachlan, J. L., 227, 256 McLellan, L., 249, 258 McLusky, S. R., 227, 261 Medhy, M. C., 198 Medzhitov, R., 224, 261 Melkonian, J., 104, 105, 131 Mellersh, D. G., 228, 261 Mellersh, D., 194, 215 Menezes-Benavente, L., 247, 261 Merchan, F., 55, 69 Meyer, A., 224, 261 Meyer, C., 16, 30 Meyer, Y., 249, 261 Midha, S., 19, 20, 30 Miller, C. R., 51, 69 Miller, G.W., 176, 184 Miller, R. M., 38, 66 Miralto, A., 242, 261, 265 Mitho¨fer, A., 188, 215, 243, 261 Mitra, R. M., 196, 215 Mittler, R., 48, 69, 247, 261 Miura, K., 52, 69 Miyawaki, K., 45, 66, 69 Molina-Favero, C., 15, 17, 19, 20, 22, 38, 69 Møller, S. G., 161, 162, 163, 179, 180 Monge, E., 158, 174, 178 Moore, C. M., 20, 30 Moore, R. M., 234, 261 Morandi, D., 193, 195, 196, 210, 215 Moreau, S., 150, 172, 174, 175 Morgan, R., 199, 215 Morillon, R., 96, 109, 113, 114, 130, 131, 132, 133 Moseley, J. L., 159, 175 Moseley, J., 160, 175 Moshelion, M., 81, 125, 126, 127, 131 Mosse, B., 186, 189, 215, 216 Mouchel, C. F., 37, 44, 45, 70 Mtolera, M., 233, 236, 261 Mulder, L., 21, 30, 213, 214, 216, 218 Mullen, R.T., 5

273

Mu¨ller, D. G., 223, 249, 259, 260, 261 Murata, K., 78, 79, 84, 85, 131 Murata, Y., 148, 175 Murray, J., 194, 195, 196, 216 N Nacry, P., 52, 70, 71 Nag, R., 50, 70 Nagahashi, G., 185, 189, 211, 216 Nair, M. G., 185, 187, 216, 218 Nakai, M., 161, 180 Nakanishi, H., 152, 169, 172, 175, 176, 177, 178 Nakano, M., 20, 30 Nakhoul, N. L., 83, 131 Nardini, A., 110, 111, 112, 114, 115, 125, 131, 135 Naumann, B., 159, 175 Navarro, L., 21, 30, 59, 70 Navazio, L., 202, 216 Navazio, N., 192, 197, 216 Nawy, T., 40, 70 Negishi, T., 147, 175 Neilands, J. B., 145, 175 Neill, S. J., 200, 216, 229, 257, 258, 261 Nejsum, L. N., 89, 131 Nemhauser, J. L., 44, 70 Neuer, G., 18, 30 Niemietz, C. M., 77, 82, 83, 87, 96, 101, 123, 131, 135 Nikaido, I., 246, 261 Nikiforova, V. J., 53, 54, 70 Nishimura, C., 45, 70 Nishimura, R., 47, 70 Nishio, J. N., 158, 159, 175, 179 Noordermeer, M. A., 241, 261 North, G. B., 100, 130, 131 Novero, M., 203, 214, 216 Nozaki, H., 251, 262 Nu¨rnberger, T., 224, 262 O O’Brien, M., 118, 119, 131 O’Dowd, C.D., 235, 262 Ocampo, J. A., 186, 209, 213, 218 Ogo, Y., 152, 175 Ohashi, Y., 8, 30, 52, 70 Ohsawa, N., 237, 262 Ohshima, Y., 96, 109, 132 Okon, Y., 10, 11, 27, 28, 29, 30, 31, 32, 213, 219 Okushima, Y., 44, 65, 70 Ola´h, B., 192, 216 Oldroyd, G. E. D., 46, 70, 193, 197, 199, 203, 208, 215, 216

274

AUTHOR INDEX

Oliviusson, P., 120, 132 Ono, K., 116, 134 Orozco-Cardenas, M. L., 230, 262 Osbourn, A. E., 202, 217 Otvos, K., 6, 30 Ouerdane, L., 153, 175 Overbeck, H. J., 19, 33 P Pagnussat, G. C., 4, 5, 7, 13, 21, 22, 29, 30 Palmer, C. J., 236, 237, 262 Papen, H., 19, 30 Parniske, M., 184, 194, 203, 210, 211, 214, 215, 216, 218, 219 Parsons, L. R., 103, 132 Pascal, N., 158, 159, 169, 175 Pasquinelli, A. E., 55, 70, 71 Paszkowski, U., 193, 194, 195, 196, 213, 216 Pate, J. S., 166, 172 Patrikin, D. G., 12, 30 Patriquin, D. G., 12, 28 Paul, E. A., 18, 19, 31 Paul, V. J., 223, 224, 237, 262 Pavia, H., 228, 258, 262, 264 Pawlowska, T. E., 183, 216 Pederse´n, M., 236, 254, 256, 261, 262 Pedler, J. F., 157, 176 Pedrosa, F. O., 18, 27 Peng, G., 11, 31 Penmetsa, R. V., 47, 70, 71, 73, 208 Penteado-Stephan, M., 16, 31, 33 Peragine, A., 56, 71 Pereira, P. A., 54, 70 Peters, A. F., 223, 253, 257, 259, 262 Peters, N. K., 187, 215 Peterson, C. A., 92, 93, 134 Peterson, R. L., 6, 31, 215 Peyrano, G., 102, 132 Pezeshgi, S., 166, 171 Phillips, D. A., 187, 188, 208, 213, 216, 218 Piche´, Y., 186, 188, 208, 209, 213, 214, 216, 217, 219 Pilar Fernandez, M., 199, 215 Pilon-Smits, E. A., 162, 176 Pinior, A., 185, 216 Pitts, R. J., 7, 31 Pohnert, G., 224, 234, 239, 240, 242, 253, 260, 262, 263, 264, 265 Polacco, J., 4, 21, 29 Polacco, J. C., 6, 29, 73 Potin, P., 226, 239, 253, 254, 255, 257, 258, 259, 263, 265 Poulin, M.-J., 187, 188, 208, 217 Powell, C. L., 186, 217 Prasad, G. V. R., 80, 83, 132 Preston, G. M., 77, 82, 128, 132 Prinsen, E., 12, 22, 31 Procino, G., 89, 132

Prost, I., 244, 263 Puglisi, M. P., 223, 224, 262 Puntarulo, S., 15, 17, 21, 22, 26, 28, 30 Pushnik, J. C., 158, 176 Q Qiu, Y. L., 38, 73 Qu, L. Q., 166, 176 Quigley, F., 77, 132 R Radin, J. W., 104, 132 Ragan, M. A., 246, 260 Raghothama, K. G., 52, 69, 71 Rajagopalan, R., 59, 62, 71, 73 Ramahaleo, T., 81, 109, 132 Rashotte, A. M., 45, 71 Ratnayake, M., 185, 217 Raven, J. A., 161, 176 Rea, G., 232, 263 Read, D. J., 182, 218 Reed, R. C., 43, 71 Reinhardt, D., 184, 217 Reinhart, B. J., 55, 70, 71 Reith, M. E., 241, 260 Remans, T., 53, 71, 73 Remington, D. L., 43, 71 Remy, W., 182, 201, 217 Rennenberg, H., 30, 53, 67 Requena, 200, 201, 210, 217 Rhoades, M. W., 57, 71 Ribaudo, C. M., 12, 31 Riefler, M., 45, 71 Ritz, D., 249, 263 Rivers, R. L., 83, 126, 132 Roberts, D. M., 85, 86, 126, 127, 132, 135 Roberts, E., 12, 31 Roberts, J. L., 12, 31 Roberts, L. A., 147, 158, 170, 176, 179 Robinson, N. J., 148, 153, 176 Roeder, V., 246, 247, 248, 249, 263 Rogers, E. E., 148, 151, 153, 171, 176 Rosazza, J. P. N., 19, 20, 25, 26, 31 Ross, C., 230, 253, 263 Rossiter, R. C., 185, 217 Roussel, H., 198, 217 Roux, C., 97, 209, 213, 218 Rroco, E., 145, 176 Rubio, V., 52, 65, 69, 71 Ruiz-Lozano, J. M., 194, 198, 217 Ryan, C. A., 53, 68, 226, 230, 258, 262, 263 Ryan, E., 8, 31 Ryu, C. M., 11, 31 S Sack, L., 109, 110, 111, 112, 114, 132, 135 Saenko, G. N., 234, 263

AUTHOR INDEX Safir, G. R., 185, 211, 218 Saga, N., 223, 254, 258, 261 Saint-John, T. V., 188, 217 Saito, C., 102, 133 Sakaguchi, T., 147, 176 Sakihama, Y., 199, 219 Sakr, S., 108, 115, 125, 133 Sakurai, J., 77, 133 Salleo, S., 115, 131, 135 Samaj, J., 10, 24, 31 Sanchez, L., 192, 196, 198, 215, 217, 219 Sanchez, P. A., 224, 256 Sanders, O. I., 83, 133 Santa-Catarina, C., 5 Santoni, V., 90, 93, 94, 127, 128, 131, 133 Sanwal, G. G., 159, 176 Sarda, X., 108, 133 Sari, M. A., 19, 20, 31 Sarig, S., 10, 27, 31 Savage, D. F., 85, 133 Sawabe, T., 223, 263 Scala, S., 234, 245, 246, 263 Scannerini, S., 201, 217 Schaaf, G., 147, 153, 158, 164, 176 Schenck, N. C., 186, 211, 218 Scheres, B., 38, 39, 40, 45, 62, 67, 71 Schiefelbein, J. W., 8, 27, 31 Schilmiller, A. L., 238, 258 Schmidke, I., 154, 176, 177 Schnitzler, I., 243, 264 Scholz, G., 154, 174, 176, 177 Schreiner, R. P., 186, 217 Schu¨ßler, A., 183, 217 Schuurmans, J. A., 121, 133 Schwab, A. P., 138, 145, 174 Schwab, S. M., 185, 217 Seddas, P., 194, 207 Segal Floh, E. I., 6 Seo, H. S., 238, 264 Sesma, A., 202, 213, 217 Shah, M., 234, 264 Shangguan, Z. P., 100, 104, 133 Sharma, A. K., 146, 176 Sharma, S., 159, 176 Shaul-Keinan, O., 185, 217 Shaw, P., 41, 64 She, X. P., 5, 31 Shikanai, T., 158, 177 Shin, R., 48, 71 Shingles, R., 155, 177 Shojima, S., 146, 177 Sieberer, T., 43, 72 Siebner-Freibach, H., 146, 177 Siefritz, F., 95, 99, 101, 105, 123, 133, 135 Signora, L., 53, 64, 72 Silar, P., 232, 259 Silipo, A., 224, 264 Simontacchi, M., 15, 17, 20, 22, 26, 28, 30

275

Singh, D. P., 160, 170, 177 Singh, M. B., 40, 72 Singleton, P. W., 54, 55, 72 Siqueira, J. O., 185, 187, 216, 218 Sjo¨vall-Larsen, S., 89, 123, 133 Smith, A. G., 160, 170, 177 Smith, B. N., 158, 177 Smith, F. A., 205, 211, 218 Smith, S. E., 182, 208, 211, 218 Sober, A., 112, 123 Son, J. K., 20, 31 Sorin, C., 38, 51, 57, 72 Sotka, E. E., 228, 264 Souter, M. A., 45, 72 Spanu, P., 198, 218 Sparrow, F. K. J., 223, 264 Spiller, S. C., 158, 159, 160, 177 Stacey, G., 37, 46, 72, 177 Stacey, M. G., 165, 177 Stanley, M. S., 246, 264 Steenhoudt, O., 11, 13, 16, 17, 18, 19, 20, 31, 32 SteVens, B., 3, 32 Steinberg, P. D., 223, 256, 257, 260, 262 Stepanova, A. N., 45, 72 Stephan, U. W., 154, 172, 173, 176, 177 Steudle, E., 81, 83, 87, 91, 92, 93, 95, 100, 102, 108, 109, 124, 125, 127, 128, 133, 134, 135, 136 Stevenson, F. J., 145, 177 Stimart, D. P., 51, 67 Stocking, C. R., 158, 159, 177 Sto¨hr, C., 5, 15, 16, 22, 30, 32, 200, 218 Stoimenova, M., 16, 28, 32 Stracke, S., 196, 218 Stremlau, S., 5, 22, 32, 200, 218 Stuehr, D. J., 20, 24, 32 Sudhamsu, J., 20, 32 Sui, H., 78, 79, 84, 86, 134 Sun, M. H., 108, 134 Sunkar, R., 57, 58, 63, 72 Suzuki, A., 55, 72 Suzuki, M., 158, 175, 177 Suzuki, T., 160, 177 Swarup, R., 43, 64, 72 T Tajkhorshid, E., 85, 125, 134, 136 Takagi, S., 145, 146, 178 Takahashi, F., 3, 32 Takahashi, M., 146, 164, 169, 172, 175, 178 Takahashi, Y., 161, 178 Takano, J., 77, 83, 86, 106, 134 Takemoto, D., 203, 218 Taki, N., 252, 264 Tamasloukht, M. B., 189, 190, 218 Tang, A. C., 121, 134

276

AUTHOR INDEX

Tang, C., 150, 178 Tanimoto, M., 9, 32 Tansengco, M. I., 47, 72 Targett, N. M., 228, 254 Tawaraya, K., 185, 189, 218 Taylor, R. B., 228, 264 Teale, W. D., 57, 72 Teas, J., 234, 264 Temmei, Y., 90, 91, 134 Terashima, I., 116, 127, 134 Terry, N., 158, 159, 169, 175, 177, 178 Terskikh, V. V., 120, 134 Theil, E. C., 149, 169, 178 Thomine, S., 142, 173, 178 Thordal-Christensen, H., 232, 264, 266 Tien, T. M., 12, 32 TiYn, L. O., 141, 178 Timmers, A. C., 46, 72 To, J. P., 45, 72 Todd, C. D., 5, 53, 72 Tokumoto, U., 161, 178 Tomos, A. D., 108, 134 Tornroth-Horsefield, S., 79, 85, 86, 88, 89, 90, 91, 122, 134 Torres, M. A., 228, 229, 264 Tosques, I. E., 17, 32 Toth, G. B., 184, 228, 262, 264 Tottey, S., 158, 178 Touraine, B., 161, 173, 178 Tournaire-Roux, C., 82, 88, 97, 98, 103, 122, 134, 135 Tre´panier, M., 190, 218 Tsai, S. M., 187, 218 Tsuda, M., 114, 134 Tuberosa, R., 37, 73 Turner, J. G., 238, 264 Tyerman, S. D., 77, 82, 83, 87, 96, 97, 102, 123, 131, 134, 135, 136 Tyree, M. T., 110, 114, 115, 125, 133, 134, 135 U Uehlein, N., 83, 116, 124, 131, 135 Ullrich, W. R., 15, 32 Ulmasov, T., 44, 73 V Val, J., 158, 178 Van der Weele, C. M., 5, 32, 49, 73 Van Tuinen, D., 194, 210, 215, 217 Van Wuytswinkel, O., 165, 169, 179 Vande Broek, A., 11, 12, 29, 31, 32 Vander Willigen, C., 120, 121, 123, 135 Vanderleyden, J., 11, 13, 29, 31, 32 Vanneste, S., 44, 73 Vansuyt, G., 146, 166, 179 Vardi, A., 242, 253, 265

Vartanian, N., 50, 73 Vaucheret, H., 56, 63, 68, 71 Vazquez, F., 56, 73 Veereshlingam, H., 47, 73 Vera-Estrella, R., 90, 135 Verbavatz, J. M., 79, 135 Verkman, A. S., 80, 135 Verma, D. P., 187, 216 Vernoux, T., 42, 73 Verstraete, W., 19, 32 Vert, G., 148, 150, 151, 157, 165, 179 Veselova, T. V., 120, 121, 135 Vierheilig, H., 185, 186, 188, 198, 208, 213, 214, 218, 219 Vieweg, M. F., 199, 219 Villalba, S., 79, 136 Vilter, H., 234, 236, 254, 259, 265 Voinnet, O., 56, 70, 73 Volpin, H., 197, 198, 213, 219 Von Rad, U., 252, 265 Von Schaewen, A., 249, 259 Von Wiren, N., 146, 147, 157, 169, 176, 179 Vos, J. W., 147, 179 Voßwinkel, R., 16, 33 Vreeland, V., 236, 263, 265 W Walch-Liu, P., 53, 73 Walker, T. S., 184, 219 Wallace, I. S., 85, 86, 127, 135 Walter, A., 146, 157, 179 Wan, X., 88, 135 Wan, X. C., 97, 135 Wang, B., 38, 73 Wang, J. W., 57, 74 Wang, W., 249, 265 Wang, Y., 146, 179 Wareing, P. F., 53, 67 Watanabe, Y., 56, 67 Waters, B. M., 149, 154, 164, 165, 166, 167, 174, 179 Weber, H., 238, 265 Wegel, E., 196, 219 Weidmann, S., 192, 196, 198, 199, 200, 210, 212, 217, 219 Weig, A., 77, 135 Weig, A. R., 83, 135 Weinberger, F., 226, 227, 229, 230, 232, 233, 236, 251, 255, 260, 265 Weitzberg, E., 18, 33 Welch, R. M., 157, 179 Wendehenne, D., 199, 200, 219 Werner, T., 45, 74 Werner-Felmayer, G., 19, 33 Westgate, E., 108, 135 Westwood, J. H., 191, 219 Wever, R., 236, 259, 261, 265 White, P., 8, 33

AUTHOR INDEX Wichard, T., 234, 265 Wightman, B., 55, 74 Williams, L., 57, 74 Wilmoth, J. C., 44, 74 Winder, T. L., 159, 179 Winicov, I., 55, 74 Wittenberg, J. B., 150, 179 Witzel, K. P., 19, 33 Wojtaszek, P., 227, 265 Wopereis, J., 47, 54, 74 Wrage, N., 16, 18, 19, 33 Wymer, C. L., 8, 33 Wysocka-Diller, J. W., 42, 74 X Xia, Y., 11, 33 Xie, C. H., 11, 16, 33 Xie, Q., 57, 66, 74 Xie, Z. P., 185, 219 Xiong, L., 50, 74 Xu, X. M., 161, 162, 179, 180 Y Yabe, T., 161, 180 Yamasaki, H., 19, 20, 26, 199, 219 Yang, L., 57, 74 Yasui, M., 84, 136 Ye, H., 162, 168, 180 Ye, Q., 87, 127, 129, 136 Ye, R. W., 16, 17, 30, 33 Yeager, M., 82, 125 Yehuda, Z., 146, 180

Yi, Y., 138, 148, 171, 180 Yoder, J. I., 191, 212 Yokota, A., 11, 16, 33 Yoon, H. S., 222, 265, 266 Yoshida, K., 164, 180 Yoshioka, H., 229, 266 Yu, X., 84, 105, 129, 136 Yuan, Y. X., 151, 180 Z Zamudio, M., 12, 33 Zancani, M., 156, 164, 180 Zardoya, R., 79, 136 Zayed, A. M., 158, 178 Zeidler, D., 226, 257, 266 Zemojtel, T., 5, 16, 33 Zhang, B., 56, 74 Zhang, H., 5, 33, 53, 63, 64, 72, 74 Zhang, W. H., 97, 136 Zhang, Z., 232, 264, 266 Zheng, W., 241, 266 Zhu, C., 9, 33 Zhu, F., 87, 136 Zhu, J. K., 57, 63, 65, 72 Zimmer, W., 14, 16, 18, 22, 28, 31, 33 Zimmermann, H. M., 100, 136 Zimmermann, U., 108, 129, 134, 136 Zumft, W. G., 16, 17, 33 Zwiazek, J. J., 97, 103, 129, 135 Zwieniecki, M. A., 113, 115, 124, 128, 132, 136

277

SUBJECT INDEX

A Abscisic acid (ABA) role in plant water status, 101 in water stress condition, 45, 50 Abscisic acid insensitive 3 mutant (abi3), 50 Acer saccharum, 111 Acid loading treatment, in roots water transportation, 97–8 Acrochaete operculata, 227–9, 237, 244 Acylated homoserine lactones (AHLs), 237 Adiantum lunulatum, 111 ADP-ribosylation factor 1 (ARF1), 147 Adventitious root (AR), 3–5, 7, 9, 13–14, 20, 22–3 formation and environmental conditions, 51 Aerobic denitrification, 17 Agave deserti, 100 Aglaophyton, 201 alf3/4 mutants, aberrant lateral root formation 3/4 mutants, 42–3 Allorhizic root system, in Arabidopsis, 38 Aquaporins (AQP), 76–9, 83–7, 89–90 cell hydrostatic pressure in water channel regulation, 87–8 expression in flowers, 118–20 in leaves, 107–8 in seeds, 120–2 functional characterization methods, 80–2 homologues, of A. thaliana and E.coli, relationship, 77–8 phosphorylation and methylation, 89–90 reproductive organs and, 117–22 reverse genetics role, 98–100 role, in leaves and roots, 93, 106–17 structural motifs, 84–6 temperature eVect on leaf water transport and, 112–13 tetrameric channels, 78–9 Aquaporins gating, molecular mechanisms heterotetramer formation, regulation, 90–1 mercury blocking, 86–7 osmotic and hydrostatic pressure regulation, 87–8 oxygen species regulation, 87 post-translational modifications, 89–90 protons and calcium regulation, 88

Arabidopsis, 5, 77–8, 80, 83–4, 86, 88, 90, 96–7, 101–3, 106–7, 109, 112–15, 118, 120 aquaporin expression, 99 auxins and NO regulation, 7–8 epidermal cell layer in, 38 mercury eVects on seeds germination, 121 mutants, 148 mutation, 42 plant adaptation to phosphorus deficiency, 52 root mass spectrometry analysis, 94 root water transport inhibition, 98 Arabidopsis NOS1 gene, 5 Arabidopsis PIP2;1, structural model, 84 Arabidopsis thaliana, 77, 140, 142–3, 188 elongation and diVerentiation zone, 38 root system, 38–42 root tissues organization, 41 Arbuscular mycorrhiza (AM), 182 Arbuscule-colonized cortical cells, 203 Ar/R constriction in aquaporins, 85 Arthrobacter sp., 19 Asparagopsis armata, 237 AtFRO2/6, ferric-chelate reductase genes, 154–5 AtIREG2–green fluorescent protein (GFP), 153 AtIRT1/2, iron transporter genes, 155 AtMTP3 gene, 157 AtNOS1. See Arabidopsis NOS1 gene AtNramp3/4, 142–3 atnrt2.1–1 mutant in root architecture, 53 AtOPT3 gene, 165 Atrichoblasts cells, 7, 41 AtYSLs gene, 148, 164–5 AUX/IAA genes, 43–4 Auxin-response elements (AREs), 44 Auxin-responsive factor ARF17, 51 Auxin role adventitious root formation, 3, 51 phophorus deficient conditions, 52 RAM formation, 40 root architecture, 42–4 Auxin signaling F-box gene 1, 2, and 3 (tir1/afb1–3), 40 Azospirillum, 3, 10, 12–16, 18 beneficial eVects, 11 NO and auxin synthesis, 22–3

280

SUBJECT INDEX

Azospirillum, (cont. )

role, at cellular level NO sources and PGPR, 15–21 root growth initiation, 13–15 Azospirillum amazonense, 16 Azospirillum brasilense, 12–13 Azospirillum irakense, 16 Azospirillum lipoferum, 13 Azospirillum oryzae, 16 B Bacillus spp., 16 Bacterial NOS role, 19–20 Bacteroids, in leguminous plant roots, 46 BDL/IAA12 protein, 44 Benthic macroalgae, 223 Beta vulgaris, 88 Biological nitrogen fixation (BNF), 11 Bodenlos (bdl) gene, 40 Boron transport, in plants, 106 Branched absorbing structures (BAS), 189 Brassica napus, 108, 121 Brassica oleracea, 118–19 Brassinosteroid (BR) role, in plants development, 44 Brevis radix protein (BRX), 44 Bromoperoxidases (BPOs), 234 Brown algae, mining defense-related gene in expression methods, defense-related genes identification, 248–50 specific response to cope with oxidative stress in, 247–8 C Ca2þ-dependent protein kinases, 4 Caenorhabditis elegans, 55 Caþion, role in root hair cell, 8–9 Calcium-binding proteins, 199 Calmodulin, diurnally regulated transcription of, 147 CAM. See Crassulacean acid metabolism 2-(4-carboxyphenyl)-4,4,5,5tetramethylimidazoline-1-oxyl3-oxide, 5, 14–15 Ccrboh gene, 231 Ccrboh mRNA, germinating zoospores, 230 CC1 transporter, of yeast, 155 CDK. See Cyclin-dependent kinase CDPKs. See Ca2þ-dependent protein kinases Cell level water transportation, in roots, 96 Cell pressure probe techniques, 81 cGMP, guanylate cyclase-catalyzed synthesis, 4 Chamydomonas reinhardtii, 159

Chara corallina, 87 CHIP28 protein, 76 Chlamydomonas reinhardtii, 245 Chloronerva, 149 p-chlorophenoxy isobutyric acid, 15 Chlorosis, 138, 146, 158 Chondrus crispus, 227, 229–30, 244 CK receptors, root growth and, 45 Confocal microscopy, 204 Constitutive photomorphogenesis and dwarf (CPD), 44–5 Constitutive triple response gene, 9 Corallina pilulifera, 237 Cordia alliodora, 114 Cortical cells (CCs), 41 Cortical microtubules randomization, in pericycle cells, 10 CO2 transportation in aquaporins, 83–4 in leaves, 116–17 CPTIO. See 2-(4-carboxyphenyl)-4,4,5,5tetramethylimidazoline-1-oxyl3-oxide Crassulacean acid metabolism, 108–9 CRE1/HK4 gene, 45 C-terminal reductase domain, 20 CTR1 gene. See Constitutive triple response gene Cucumis sativus, 4 Cyanidioschyzon merolae, 231, 245 CycB1;1 marker gene, 42 Cyclic guanosine 30 ,50 -monophosphate (cGMP), 20 Cyclin-dependent kinase, 6 Cys residue, in human AQP1, 86 Cytokinin oxidase (CKX), 45 Cytokinin response 1/histidin kinase 4 (CRE1/AHK4), 45 Cytokinins (CKs) role phosphorus deficient condition, 52 root development, 45 D Dasycladus vermicularis, 230 Delisea pulchra, 237 Denitrification, 16 Nap and Nar role, 17 NO production, 18 Deoxymugineic acid synthase (DMAS), 146 Dicer-like 1(DCL1), 55 Dictyopteris spp., 243 dig3 mutant, 50 Dissimilative nitrite reductase, 17 Distal root apex, in A. thaliana, 38 Divalent cation transporter 1 (DCT1), 142 Divalent metal ion transporter 1 (DMT1), 142 DwMYB2 transgenic plants, 155

SUBJECT INDEX E Ectocarpus siliculosus, 234, 245–6 Endoplasmic reticulum (ER), 204 Endosymbioses, 203 Escherichia coli, 17 Ethylene role in cell walls, 9 phophorus deficient condition, 52 root growth, 45 in root primordia, 3 eto1-1, 9 Etylene requirement, in JA’s function, 9 Euchema platycladum, 230 Evaporative flux method, 109–10 Expressed sequence tags (ESTs), 231 F FC genes, in Arabidopsis, 160 Fe(III)-chelate reductase protein, 149 Fe(III)-MA transport system, 147 Fe–NA OsYSL2 transporter, rice seeds, 166 fer, mutants, 149, 151 Ferritins, 141–2 Ferrochelatase (FC), 160 Ferrous iron transporter IRT1, 157 Fe-S biogenesis, 161–2 iron donors for, 163–4 Fe–S scaVold protein NFU2, 161 fet3fet4 yeast mutant strain, 147 Fet4p transporter, 146 Flavonoids, plant signals in presymbiotic stage and, 186–8 Frataxin, 163 frd3, mutant of Arabidopsis, 153 Free fatty acids (FFAs), 238 FR01 gene in pea, 149 FRO and ZIP (IRT) family, for tomato genes and cDNAs, 149 FRO2 gene, 148 FRU/FIT1, transcription factor, 153 Fucus vesiculosus, 243 Fungal-root interactions, 198 Fungal spore germination, 187 Fungal symbionts, 193 G Gas exchange measurements, in CO2 transportation, 117 Gelidium latifolium, 243 GFP. See Green fluorescent protein Gibberellin (GA) role, in osmotic stress conditions, 40, 50–1 Gigaspora, germination of, 187 Gigaspora margarita, 183 Glomeromycota, 183 Glomus intraradices, 188, 199–200

281

GmDMT1 transporter, 150 Gracilaria conferta, 226–7, 229–30 Gracilariopsis sp., 243 Graptopetalum paraguayens, 109 Green fluorescent protein, 90, 102 GTP-binding protein (GTPase), 147 H har1 gene, 47 H4B. See 6R-tetrahydrobiopterin Heme, 160. See also Iron iron donors for, 163–4 Heterologous systems, water measurement, 81–2 Heterotrophic nitrification, 18–19 High pressure flow meter, 95, 110, 114 Hormonal signaling genes, 40 root growth regulation, 42–5 ‘‘Hourglassfold’’ model, 79 HPFM. See High pressure flow meter Human gp91phox, 231 HvYS1 mRNA, epidermal root cells, 148 13-Hydroperoxide lyase (HPL), 241 Hydropuntia sp., 236 Hypernodulating mutants, legume root systems and, 47 I IAA14 protein, 42 Intergeneric grafts, 186 Iron biofortification, 165 circulation, long distance, 153–5 dynamics, 168 intracellular compartmentation in plant cell, 156 mobilization after germination metabolic-induced dynamics, 140–1 molecular aspects of seed iron remobilization, 141–3 seed iron mobilization, 141 photosynthesis and heme and Fe–S cluster biosynthesis, 160–4 impairment, iron deficiency and, 158–60 and reproduction in plants iron during flowering, 164–5 iron unloading and storage in seeds, 165–7 uptake by roots in grasses, 146–8 in nongrasses, 148–50 regulation of expression of plant high aynity iron transport systems, 150–2

282

SUBJECT INDEX

Iron (cont. )

uptake from rhizosphere and distribution, 144 Iron acquisition and circulation, in vegetative growth iron uptake by roots, 146–53 iron/zinc uptake and compartmentation in plants, 157–8 long distance iron circulation, in plant, 153–5 plant/soil/microorganisms interactions, in rhizosphere, 143–6 subcellular compartmentation of iron, 155–7 Iron-sulfur cluster (ISC), 161 Iron transport protein (ITP), 154 IRT cDNA(LeIRT1 and LeIRT2), transporter in tomato, 149 IRT1 gene, 157 IRT1 promoter-GUS fusion, anther filament, 165 Isopentenyltransferases (IPTs), 45 J Jasmonic acid (JA), 9, 238 Juglans regia, 110 K Kalanchoe daigremontiana, 109 L Laminaria digitata, 226, 230, 233, 236–7 Laminaria japonica, 223 Laminariocolax tomentosoides, 229 Latd gene, 47 Late embryogenesis abundant (LEA), 154 Lateral root development, 5–6, 10, 13–15 Lateral roots (LRs), 3, 12, 37 formation, auxin and NO role, 5–6 and legume nodule primordia, 46 nitrogen concentration, 52–3 organogenesis, 41–2 osmotic stress, 49–50 primordium initiation, 41–2 Leaf hydraulic conductance (Kleaf), 109–14 physiological regulation cold embolism repair, 115–16 light role, 114 transpiration demand, 113–14 water eVect, 115 Leaves aquaporin expression, 107–8 aquaporin function in water transportation, 112–13 CO2 transportation, 116–17 Kleaf physiological functions, 113–16

water transport in, 106–10 water transport pathways, 110–11 LeFRO1 gene, 149 Leghemoglobin, 150 Legume mutants role, in nodule formation, 47 Legume nodules organogenesis, 46 Legume roots and environmental factors, 54–5 systems, supernodulating mutants role, 46–7 Leguminous plant and rhizobial interaction, 46 Light eVects, root water transport and, 103–4 Lipopolysaccharides (LPS), 224 Lipoteichoic acid (LTA), 224 Lipoxygenases (LOXs), 238 Lithophyllum yessoense, 237 Lithothamnion coralloides, 243 Lotus, 191 Lotus japonicus, 54, 85, 103, 183, 204 LRD. See Lateral root development lsi1, molecular characterization, 105–6 Lsi1 role in Xenopus oocytes, 83 Lupinus, 186 Lupinus albus, 21 M Macaranga triloba, 111 Macroalgal species, 246 Macrophytic marine algae, 222 Magnaporthe, 201 Maize YS1 gene, 147 Major intrinsic protein (MIPs), 76 molecules transportation by, 83 primary sequence analyses, 79 sequence analysis of, 77 Mammalian Nramp2, 142 MAPK. See Mitogen-activated protein kinase Marine algae dynamic defense of pathogen perception in higher eukaryotes, 224–6 recognition of attacker(s) in seaweeds, 226–8 halogenation, defensive role in chemical defense molecules, 236–8 direct role in cell protection, 235–6 halogen metabolism and halogenating enzymes, 233–5 mining defense genes and transcriptional responses in brown algae, 247–50 genomic and transcriptomic data in, 245–7 red algae, 250–2

SUBJECT INDEX oxidative burst machinery in DPI-sensitive in algae-pathogen interactions, 229–30 in higher plants, 228–9 NADPH oxidase-like genes in algae, 230–2 ROS in algae–pathogen interactions, sources, 232–3 oxylipins, defense responses mediators in, 238–9 biological and ecological functions of, 242–5 lipase activated release and lipoxygenase-mediated transformation of FFAs, 239–40 oxylipin pathways, 240–2 Mass spectrometry analysis, of Arabidopsis roots, 94 Medicago truncatula, 21, 47, 55, 150, 192, 199, 200 MeJA. See Methyl jasmonate Membrane-bound nitrate reductase, 17 Mercury inhibition for water transport in roots, 96–7 leaf water transport and, 112 Mesembryanthemum crystallinum, 121 Methyl jasmonate, 9 Miconia argentea, 114 Microarray assessment of Quiescent center functions, 40 Microbe or pathogen-associated molecular patterns (MAMPS or PAMPs), 224 Microbial symbionts, 192 MicroRNAs (miRNAs), 21, 44 role in plant stress response, 56–7 MIR399, phosphorus homeostasis and, 57–8 MIR160, root growth and, 57 Mitogen-activated protein kinase, 4, 22 Mobilization of sulfur (SUF), 161 Monopteros (mp) gene, 40 MP/ARF5 protein, 44 Mugineic acids (MAs), 146 Multicellular tissue organization, 202 Mycorrhiza-defective mutants, 193–4 Mycorrhizal root organ, 204 N naat tobacco plants, 164 NADPH oxidases, 8, 229 NA-Fe transport, 164 Nap. See Periplasmic nitrate reductase Nar. See Membrane-bound nitrate reductase Nas. See Soluble assimilatory nitrate reductase

283

N-hydroxy-L-arginine, 19 Nicotianamine amino transferase (NAAT), 146 Nicotianamine (NA), 146 Nicotiana, NO induces, 9 NIPs. See Nodulin 26-like intrinsic proteins Nir. See Dissimilative nitrite reductase Nitrate concentration, in root growth, 52–3 Nitrate reductase (NR), 199 Nitric oxide (NO), 2–10, 13–21 and ethylene in plant maturation and senescence, 9 LR formation, 6 production by Azospirillum brasilense, 13–14 in RHF, 7–9 role in root formation, 4 scavenger, 5 as signaling molecules, 199 Nitric oxide reductase, 16–17 Nitric oxide synthase, 5, 16, 19–21 Nitrification, definition, 18 Nitrite reductase (NiR), 200 Nitrogenase, 150 Nitrogen fixation (NIF), 161 Nitrogen-fixing nodules, in leguminous plant, 46 Nitrous oxide reductase, 16–17 Nocardia, 19–20 NOD26. See Nodulin-26 Nod factor signaling pathway, 196, 203 Nodulation symbiosis, 188 Nodulin-26, 77 Nodulin 26-like intrinsic proteins, 77 NOHA. See N-hydroxy-L-arginine Noncoding RNAs, role in root growth and development, 55–9 Nongrasses, iron uptake by roots A. thaliana, 148 legumes, 149–50 tomato, 149 Nor. See Nitric oxide reductase NOS. See Nitric oxide synthase Nos. See Nitrous oxide reductase NOSoxy. See N-terminal oxygenase domain NOSred. See C-terminal reductase domain nramp3/4, 142 Nramp proteins, 142 N-terminal oxygenase domain, 20 Nutrient movement, in plants, 105–6 O Ocimum basilicum, 189 Oligopeptides transporter (OPT), 165 Opuntia acanthocarpa, 97, 100 Organ level water transportation, in roots acid loading, 97–8 mercury inhibition, 96–7

284

SUBJECT INDEX

Organ level water transportation, in roots (cont. )

reverse genetics, 98–100 9-, 12-, or 15-hydroxyeicosatetraenoic acid (HETE), 242 OsYSL rice genes, 154 Oxylipin-based chemical defense activation, 239 Oxylipin pathways, marine algae, 239 P Paracoccus denitrificans, 17 Parasitic weed stimulants production, 191 Parenchymal cortex, 183 Pathogenic microorganisms, 198 PCIB. See p-chlorophenoxy isobutyric acid PEG. See Poly ethylene glycol Pericycle founder cells, in LR development, 41 Periplasmic nitrate reductase, 17–18, 20 PGP4 gene, 44 PGPR. See Plant growth-promoting rhizobacteria Phaeodactylum tricornutum, 231, 245 Phaseolus vulgaris, 89 Phosphate starvation response1 (PHR1), P signaling pathway and, 52 Phosphorus deficiency, root growth and, 52 Phosphorylation, of SoPIP2;1, 89–90 Photosynthetic apparatus, 158 Phytoalexins, 229 Phytohormone production in plant growth, 12 PIN genes, role in auxin distribution, 40, 43–4 Plant adaptation and nutrient deficiencies, 51–4 Plant-AM fungal signaling events identification, 190 Plant aquaporin gating properties, 86–91 Plant-fungal communication/ interactions, 194, 198 Plant growth-promoting rhizobacteria, 3, 10–13, 15–23, 38 NO production and functions and, 20 and plant interaction, 15, 22 root development and, 10–21 Plant high aVnity iron transport systems, expression regulation iron uptake in grasses, 152 through phloem, 154 through xylem, 153–4 IRT/FRO system in nongrasses plants, 150–2 long distance iron translocation, 154–5 Plant protoplasts and vacuoles, swelling and shrinking, 81

Plant root architecture and abiotic stresses, 47–8 functions, 36–7 Plant/soil/microorganisms interactions, in rhizosphere, 143–6 Plant symbiosis, 184 Plant tissues, colonization of, 201 PLS gene, 45 Polyethylene glycol, 100 Polyunsaturated fatty acids (PUFAs), 238 Populus tremula, 112 Porphyra purpurea, 241 Porphyra yezoensis, 231, 246 Potential iron transporters, AtIREG1, AtFRD3 and Nramp1, 155 Prepenetration apparatus (PPA), 204 Pressure probe measurements, 81, 94–7, 108 Presymbiotic hyphal branching, 190 Promoter- -glucuronidase fusions, 106 Proteoliposomes, 82–3 Pseudoalteromonas bacteriolytica, 223 Pseudomonas, 146 Pseudomonas spp., 16–17 PsFRO1 gene, 154 P. syringae, 58 Ptilota filicina, 241 Pyrroline-5-carboxylate dehydrogenase (P5CDH), 58 Q Quantitative trait loci (QTL), 37 Quercus rubra, 110–11 Quiescent center (QC), 39 R Ralstonia eutropha, 17 Reactive oxygen species (ROS), 8, 48, 228 in algae–pathogen interactions enzymatic sources, in C. crispus, 233 Gracilaria conferta vs. Gracilaria chilensis, 232–3 Receptor for activated C kinase 1A (RACK1A), 51 Red algae, mining defense-related gene in identification of, 250–1 microarrays in, 251–2 Red spot disease, 223 Reproductive organs, aquaporins role in flowers, 118–20 in seeds, 120–2 Respiratory burst oxidase homologues (rboh), 229 Reverse genetics, role in aquaporins, 98–100 RGS1 protein, 50 Rhizobia, 59, 192 and leguminous plant interaction, 46 Rhizosphere, 143

SUBJECT INDEX iron deficiency induces acidification of, 148 iron uptake from, 144 plant–microorganism interactions in, 144 plant/soil/microorganisms interactions in, 143 Rhodobacter sphaeroides, 17 Rhodococcus spp., 20 Rhodymenia pertusa, 240 Ribosomal gene sequences, 183 Ribulose bisphosphate carboxylase, role in CO2 transportation, 116 Ricinus communis, 154 RNA-induced silencing complex (RISC), 55–6 Root apical meristem (RAM), 37–8 functions and formation, 39–40 Root colonization mathematical models of, 185 signals essential for, 184 Root-colonizing bacteria, 3 Root exudates, role of, 185 Root ferric chelate reductase FRO2, 157 Root hair formation (RHF) in arabidopsis, interaction between ethylene and JAs, 9–10 features, 6–10, 13–14 Root meristemless 1 mutant (rml1), 42 Root primordium development, in Arabidopsis, 41–2 Root(s) bacterial IAA and, 12–13 definition and role, 3, 10, 91 growth and development eVects of PGPR, 10–21 NO, adventitious root formation and, 3–5 NO in lateral root development, 5–6 regulation, endogenous signals, 42–5 root hair formation features, 6–10 small noncoding RNAs and, 55–9 and soil conditions, 42–7 hair diVerentiation, positive regulator, 9 organ architecture, 201 pathogens, 192 pressure estimation, 95 water uptake, 92–3 Root system architecture in A. thaliana, 38–42 legume root formation and abiotic stress conditions, 54–5 LR development role, 41 water excess and nutrient availability, 51–4 water stress and adaptation, 47–51 Root water, aquaporins expression, 93–4 precept of root water uptake, 91–3

285

Root water transport chilling-induced inhibition, 104–5 hypoxic eVect on, 103 measurements high pressure flow meter, 95 pressure chambers, 94–5 root pressure, 95 nutrient eVect on, 104 salinity stress eVect on, 101–3 stimuli eVects hypoxia, 103 light, 103–4 nutrient status, 104 salinity stress, 101–3 temperature and, 104–5 water stress, 100–1 Root zones, in A. thaliana, 38 S Saccharomyces cerevisiae, 146 Salmonella arbortus equi, 227 Sap flow determination, 95 Scarecrow (SCR) transcription factor, 40–1 scr mutants, 40 SDH2-1/2/3, 140–1 Seaweed/pathogen interactions, defense reactions induced in, 225 Seed germination stimulation, 192 Seed iron remobilization after germination, molecular aspects of ferritins, plastid iron storage proteins, 141–2 natural resistance associated macrophage protein (nramp), 142–3 vacuole iron transporter (VIT1), 142–3 Sequence analysis, of MIPs, 77 Short root (SHR) transcription factor, 40–1 shr mutants, 40 Silicon transport in plants, 105 Sinorhizobium meliloti, 21, 192 Small basic intrinsic proteins (SIPs), 77 Small interfering RNAs (siRNAs), plant stress response and, 56 Soil conditions and leguminous plant, 46 Soil-plant-atmosphere continuum, water flows, 91 Soils ferromagnesium silicate forms in, 145 iron in, 144–5 plants and microbes on iron availability in, 145–6 Solanum chacoense, 118–19 Solitary root-1 (slr-1), 42 Soluble assimilatory nitrate reductase, 16 SpM7918 IAA producer, 13 Spore germination, 186–7 Stephanopyxis turris, 234 Stomatal conductance (Gs), 106, 109, 117

286

SUBJECT INDEX

Stomatal conductance (Gs), (cont. )

water transportation in leaf and, 106–7 Stopped flow spectrophotometry, 88 Stopped-flow techniques, 80 Strigolactones, role of, 192 Sulfur concentration and root growth, 53–4 Sulphate transporter 1;2 (SULTR1;2), 53 Superoxide dismutase (SOD), 190 Suppression subtractive hybridization (SSH), 250 Symbiosis-related plant genes inactivation, 196 Symbiotic fungi, root colonization by, 183 T Tag bacteria, green fluorescent protein interactions, 13 Temperature, eVect on root water transport, 104–5 6R-tetrahydrobiopterin, 20 Thalassiosira pseudonana, 231, 245 Thalassiosira rotula, 240 Thylakoid density, 158 Thylakoid sugar beet proteome analysis, 159

-tonoplast intrinsic proteins ( -TIP), 77 Tradescantia virginiana, 108 Trans-acting siRNAs (tasiRNAs), 44 Transcription factors genes, 40 Transport inhibitor resistant 1 (tir1), 40 Trichoblasts cells, 7 Trichoblasts formation and epidermal cells, 41 Trifolium repens, 55 Trp pathways increase the IAA levels, 12 V Vacuum pump method, 109 Vanadium-dependent haloperoxidases (vHPOs), 234 Video-microscopy role, 81

vit1-1, 143 Volatile halogenated organic compounds (VHOCs), 233 W Water excess and adventitious root formation, 51 stress and plant root architecture, 47–8 stress eVect on root water transport, 100–1 Water transportation in leaves aquaporin role, 112–13 HPFM method of, 110 measurement techniques, 108–10 pathways, 110–11 in roots cell level, 96 organ level, 96–100 Water uptake rate by roots, equation, 91–2 Western blot analyses, of aquaporins in flowers, 118 X Xenopus, 80–1, 83, 88–9, 106, 116 oocytes, transport Fe(II)–NA, 154 Xenopus laevis, 81 Y Y-shaped deformation of root hair, 12 YSLs genes, 164, 166–7 Z Zea mays, 108 ZIP gene family, 150 Zn-PS complexes, 157–8

CONTRIBUTORS TO VOLUME 46

CARLOS ALBERTO BARASSI Unidad Integrada Balcarce, INTA‐ Facultad de Cs. Agrarias, Universidad Nacional de Mar del Plata, CC 276, 7620 Balcarce, Argentina PAOLA BONFANTE Dipartimento di Biologia Vegetale, Universita` di Torino, I.P.P.‐C.N.R., Viale Mattioli 25, Torino 10125, Italy JEAN‐FRANC ¸ OIS BRIAT Biochimie et Physiologie Mole´culaire des Plantes, Centre National de la Recherche Scientifique (UMR 5004), Institut National de la Recherche Agronomique, Universite´ Montpellier 2, E´cole Nationale Supe´rieure d’Agronomie, 2 Place Viala, F‐34060 Montpellier Cedex 2, France NATALIA CORREA‐ARAGUNDE Instituto de Investigaciones Biolo´gicas, Universidad Nacional de Mar del Plata, CC 1245, 7600 Mar del Plata, Argentina AUDREY COSSE Centre National de la Recherche Scientifique, Universite´ Pierre et Marie Curie‐Paris6, Laboratoire International Associe´ «Dispersal and Adaptation in Marine Species», Unite´ Mixte de Recherche 7139 ‘‘Marine Plants and Biomolecules,’’ Station Biologique, BP 74, F29682 RoscoV Cedex, France MARTIN CRESPI Institut des Sciences du Ve´ge´tal, Centre National de la Recherche Scientifique, 91198 Gif sur Yvette, France ´ NICA CREUS Unidad Integrada Balcarce, INTA‐Facultad CECILIA MO de Cs. Agrarias, Universidad Nacional de Mar del Plata, CC 276, 7620 Balcarce, Argentina FLORIAN FRUGIER Institut des Sciences du Ve´ge´tal, Centre National de la Recherche Scientifique, 91198 Gif sur Yvette, France ANDREA GENRE Dipartimento di Biologia Vegetale, Universita` di Torino, I.P.P.‐C.N.R., Viale Mattioli 25, Torino 10125, Italy VIVIENNE GIANINAZZI‐PEARSON UMR INRA 1088/CNRS 5184/ Universite´ de Bourgogne Plant‐Microbe‐Environment, INRA, CMSE, BP 86510, Dijon Cedex 21065, France SILVINA GONZALEZ‐RIZZO Institut des Sciences du Ve´ge´tal, Centre National de la Recherche Scientifique, 91198 Gif sur Yvette, France CAROLINE HARTMANN Universite´ Paris VII‐Denis Diderot, 2 place Jussieu, 75251 Paris Cedex 5, France and Institut des Sciences du Ve´ge´tal, Centre National de la Recherche Scientifique, 91198 Gif sur Yvette, France SYLVAIN JEANDROZ UMR INRA/UHP 1136 Interactions Arbres‐ Microorganismes, Universite´ H. Poincare´ Nancy I, Faculte´ des

x

CONTRIBUTORS

Sciences, BP 239, Vandœuvre Le`s Nancy 54506, France and UMR INRA 1088/CNRS 5184/Universite´ de Bourgogne Plant‐Microbe‐ Environment, INRA, CMSE, BP 86510, Dijon Cedex 21065, France MARIANA JOVANOVIC Institut des Sciences du Ve´ge´tal, Centre National de la Recherche Scientifique, 91198 Gif sur Yvette, France LORENZO LAMATTINA Instituto de Investigaciones Biolo´ gicas, Universidad Nacional de Mar del Plata, CC 1245, 7600 Mar del Plata, Argentina MARI´A LUCIANA LANTERI Instituto de Investigaciones Biolo´gicas, Universidad Nacional de Mar del Plata, CC 1245, 7600 Mar del Plata, Argentina PHILIPPE LAPORTE Institut des Sciences du Ve´ge´tal, Centre National de la Recherche Scientifique, 91198 Gif sur Yvette, France CATHERINE LEBLANC Centre National de la Recherche Scientifique, Universite´ Pierre et Marie Curie‐Paris6, Laboratoire International Associe´ «Dispersal and Adaptation in Marine Species», Unite´ Mixte de Recherche 7139 ‘‘Marine Plants and Biomolecules,’’ Station Biologique, BP 74, F29682 RoscoV Cedex, France ´ RIE LEFEBVRE Universite´ Paris VII‐Denis Diderot, 2 place VALE Jussieu, 75251 Paris Cedex 5, France and Institut des Sciences du Ve´ge´tal, Centre National de la Recherche Scientifique, 91198 Gif sur Yvette, France CHRISTINE LELANDAIS‐BRIE`RE Universite´ Paris VII‐Denis Diderot, 2 place Jussieu, 75251 Paris Cedex 5, France and Institut des Sciences du Ve´ge´tal, Centre National de la Recherche Scientifique, 91198 Gif sur Yvette, France MARI´A CRISTINA LOMBARDO Departamento de Biologı´a, Universidad Nacional de Mar del Plata, CC 1245, 7600 Mar del Plata, Argentina and Instituto de Investigaciones Biolo´gicas, Universidad Nacional de Mar del Plata, CC 1245, 7600 Mar del Plata, Argentina CHRISTOPHE MAUREL Biochimie et Physiologie Mole´culaire des Plantes, SupAgro/CNRS/INRA/UM2 UMR 5004, 2 Place Viala, F‐34060 Montpellier Cedex 1, France CELESTE MOLINA‐FAVERO Unidad Integrada Balcarce, INTA‐Facultad de Cs. Agrarias, Universidad Nacional de Mar del Plata, CC 276, 7620 Balcarce, Argentina OLIVIER POSTAIRE, Biochimie et Physiologie Mole´culaire des Plantes, SupAgro/CNRS/INRA/UM2 UMR 5004, 2 Place Viala, F‐34060 Montpellier Cedex 1, France PHILIPPE POTIN Centre National de la Recherche Scientifique, Universite´ Pierre et Marie Curie‐Paris6, Laboratoire International Associe´ «Dispersal and Adaptation in Marine Species», Unite´ Mixte de Recherche 7139 ‘‘Marine Plants and Biomolecules,’’ Station Biologique, BP 74, F29682 RoscoV Cedex, France

CONTRIBUTORS

xi

NATHALIE SE´JALON‐DELMAS UMR 5546, Equipe de Mycologie Ve´ge´tale, Poˆle de Biotechnologie Ve´ge´tales, Chemin de Borde‐Rouge, BP 42617, Castanet‐Tolosan 31326, France LIONEL VERDOUCQ Biochimie et Physiologie Mole´culaire des Plantes, SupAgro/CNRS/INRA/UM2 UMR 5004, 2 Place Viala, F‐34060 Montpellier Cedex 1, France

CONTENTS OF VOLUMES 35–45 Series Editor (Volumes 35–44) J.A. CALLOW School of Biosciences, University of Birmingham, Birmingham, United Kingdom

Contents of Volume 35 Recent Advances in the Cell Biology of Chlorophyll Catabolism H. THOMAS, H. OUGHAM and S. HORTENSTEINER The Microspore: A Haploid Multipurpose Cell A. TOURAEV, M. PFOSSER and E. HEBERLE-BORS The Seed Oleosins: Structure Properties and Biological Role J. NAPIER, F. BEAUDOIN, A. TATHAM and P. SHEWRY Compartmentation of Proteins in the Protein Storage Vacuole: A Compound Organelle in Plant Cells L. JIANG and J. ROGERS Intraspecific Variation in Seaweeds: The Application of New Tools and Approaches C. MAGGS and R. WATTIER Glucosinolates and Their Degradation Products R. F. MITHEN

xiv

CONTENTS OF VOLUMES 35–45

Contents of Volume 36 PLANT VIRUS VECTOR INTERACTIONS Edited by R. Plumb Aphids: Non-Persistent Transmission T. P. PIRONE and K. L. PERRY Persistent Transmission of Luteoviruses by Aphids B. REAVY and M. A. MAYO Fungi M. J. ADAMS Whitefly Transmission of Plant Viruses J. K. BROWN and H. CZOSNEK Beetles R. C. GERGERICH Thrips as Vectors of Tospoviruses D. E. ULLMAN, R. MEIDEROS, L. R. CAMPBELL, A. E. WHITFIELD, J. L. SHERWOOD and T. L. GERMAN Virus Transmission by Leafhoppers, Planthoppers and Treehoppers (Auchenorrhyncha, Homoptera) E. AMMAR and L. R. NAULT Nematodes S. A. MacFARLANE, R. NEILSON and D. J. F. BROWN Other Vectors R. T. PLUMB

CONTENTS OF VOLUMES 35–45

Contents of Volume 37 ANTHOCYANINS IN LEAVES Edited by K. S. Gould and D. W. Lee Anthocyanins in Leaves and Other Vegetative Organs: An Introduction D. W. LEE and K. S. GOULD Le Rouge et le Noir: Are Anthocyanins Plant Melanins? G. S. TIMMINS, N. M. HOLBROOK and T. S. FEILD Anthocyanins in Leaves: History, Phylogeny and Development D. W. LEE The Final Steps in Anthocyanin Formation: A Story of Modification and Sequestration C. S. WINEFIELD Molecular Genetics and Control of Anthocyanin Expression B. WINKEL-SHIRLEY Differential Expression and Functional Significance of Anthocyanins in Relation to Phasic Development in Hedera helix L. W. P. HACKETT Do Anthocyanins Function as Osmoregulators in Leaf Tissues? L. CHALKER-SCOTT The Role of Anthocyanins for Photosynthesis of Alaskan Arctic Evergreens During Snowmelt S. F. OBERBAUER and G. STARR Anthocyanins in Autumn Leaf Senescence D. W. LEE A Unified Explanation for Anthocyanins in Leaves? K. S. GOULD, S. O. NEILL and T. C. VOGELMANN

xv

xvi

CONTENTS OF VOLUMES 35–45

Contents of Volume 38 An Epidemiological Framework for Disease Management C. A. GILLIGAN Golgi-independent Trafficking of Macromolecules to the Plant Vacuole D. C. BASSHAM Phosphoenolpyruvate Carboxykinase: Structure, Function and Regulation R. P. WALKER and Z.-H. CHEN Developmental Genetics of the Angiosperm Leaf C. A. KIDNER, M. C. P. TIMMERMANS, M. E. BYRNE and R. A. MARTIENSSEN A Model for the Evolution and Genesis of the Pseudotetraploid Arabidopsis thaliana Genome Y. HENRY, A. CHAMPION, I. GY, A. PICAUD, A. LECHARNY and M. KREIS

Contents of Volume 39 Cumulative Subject Index Volumes 1–38

Contents of Volume 40 Starch Synthesis in Cereal Grains K. TOMLINSON and K. DENYER The Hyperaccumulation of Metals by Plants M. R. MACNAIR Plant Chromatin — Learning from Similarities and Differences J. BRZESKI, J. DYCZKOWSKI, S. KACZANOWSKI, P. ZIELENKIEWICZ and A. JERZMANOWSKI

CONTENTS OF VOLUMES 35–45

xvii

The Interface Between the Cell Cycle and Programmed Cell Death in Higher Plants: From Division unto Death D. FRANCIS The Importance of Extracellular Carbohydrate Production by Marine Epipelic Diatoms G. J. C. UNDERWOOD and D. M. PATERSON Fungal Pathogens of Insects: Cuticle Degrading Enzymes and Toxins A. K. CHARNLEY

Contents of Volume 41 Multiple Responses of Rhizobia to Flavonoids During Legume Root Infection JAMES E. COOPER Investigating and Manipulating Lignin Biosynthesis in the Postgenomic Era CLAIRE HALPIN Application of Thermal Imaging and Infrared Sensing in Plant Physiology and Ecophysiology HAMLYN G. JONES Sequences and Phylogenies of Plant Pararetroviruses, Viruses, and Transposable Elements CELIA HANSEN and J. S. HESLOP-HARRISON

Role of Plasmodesmata Regulation in Plant Development ARNAUD COMPLAINVILLE and MARTIN CRESPI

xviii

CONTENTS OF VOLUMES 35–45

Contents of Volume 42 Chemical Manipulation of Antioxidant Defences in Plants ROBERT EDWARDS, MELISSA BRAZIER-HICKS, DAVID P. DIXON and IAN CUMMINS The Impact of Molecular Data in Fungal Systematics P. D. BRIDGE, B. M. SPOONER and P. J. ROBERTS Cytoskeletal Regulation of the Plane of Cell Division: An Essential Component of Plant Development and Reproduction HILARY J. ROGERS Nitrogen and Carbon Metabolism in Plastids: Evolution, Integration, and Coordination with Reactions in the Cytosol ALYSON K. TOBIN and CAROLINE G. BOWSHER

Contents of Volume 43 Defensive and Sensory Chemical Ecology of Brown Algae CHARLES D. AMSLER and VICTORIA A. FAIRHEAD Regulation of Carbon and Amino Acid Metabolism: Roles of Sucrose Nonfermenting-1-Related Protein Kinase-1 and General Control Nonderepressible-2-Related Protein Kinase NIGEL G. HALFORD Opportunities for the Control of Brassicaceous Weeds of Cropping Systems Using Mycoherbicides AARON MAXWELL and JOHN K. SCOTT Stress Resistance and Disease Resistance in Seaweeds: The Role of Reactive Oxygen Metabolism MATTHEW J. DRING Nutrient Sensing and Signalling in Plants: Potassium and Phosphorus ANNA AMTMANN, JOHN P. HAMMOND, PATRICK ARMENGAUD and PHILIP J. WHITE

CONTENTS OF VOLUMES 35–45

xix

Contents of Volume 44 Angiosperm Floral Evolution: Morphological Developmental Framework PETER K. ENDRESS Recent Developments Regarding the Evolutionary Origin of Flowers MICHAEL W. FROHLICH Duplication, Diversification, and Comparative Genetics of Angiosperm MADS-Box Genes VIVIAN F. IRISH Beyond the ABC-Model: Regulation of Floral Homeotic Genes LAURA M. ZAHN, BAOMIN FENG and HONG MA Missing Links: DNA-Binding and Target Gene Specificity of Floral Homeotic Proteins RAINER MELZER, KERSTIN KAUFMANN ¨ NTER THEIßEN and GU Genetics of Floral Development in Petunia ANNEKE RIJPKEMA, TOM GERATS and MICHIEL VANDENBUSSCHE Flower Development: The Antirrhinum Perspective BRENDAN DAVIES, MARIA CARTOLANO and ZSUZSANNA SCHWARZ-SOMMER Floral Developmental Genetics of Gerbera (Asteraceae) TEEMU H. TEERI, MIKA KOTILAINEN, ANNE UIMARI, SATU RUOKOLAINEN, YAN PENG NG, URSULA MALM, ¨ NEN, SUVI BROHOLM, ROOSA LAITINEN, ¨ LLA EIJA PO PAULA ELOMAA and VICTOR A. ALBERT Gene Duplication and Floral Developmental Genetics of Basal Eudicots ELENA M. KRAMER and ELIZABETH A. ZIMMER

xx

CONTENTS OF VOLUMES 35–45

Genetics of Grass Flower Development CLINTON J. WHIPPLE and ROBERT J. SCHMIDT Developmental Gene Evolution and the Origin of Grass Inflorescence Diversity SIMON T. MALCOMBER, JILL C. PRESTON, RENATA REINHEIMER, JESSIE KOSSUTH and ELIZABETH A. KELLOGG Expression of Floral Regulators in Basal Angiosperms and the Origin and Evolution of ABC-Function PAMELA S. SOLTIS, DOUGLAS E. SOLTIS, SANGTAE KIM, ANDRE CHANDERBALI and MATYAS BUZGO The Molecular Evolutionary Ecology of Plant Development: Flowering Time in Arabidopsis thaliana KATHLEEN ENGELMANN and MICHAEL PURUGGANAN A Genomics Approach to the Study of Ancient Polyploidy and Floral Developmental Genetics JAMES H. LEEBENS-MACK, KERR WALL, JILL DUARTE, ZHENGUI ZHENG, DAVID OPPENHEIMER and CLAUDE DEPAMPHILIS Series Editors (Volume 45– ) JEAN-CLAUDE KADER Laboratoire Physiologie Cellulaire et Mole´culaire des Plantes, CNRS, Universite´ de Paris, Paris, France MICHEL DELSENY Laboratoire Ge´nome et De´veloppement des Plantes, CNRS IRD UP, Universite´ de Perpignan, Perpignan, France

Contents of Volume 45 RAPESEED BREEDING History, Origin and Evolution S. K. GUPTA and ADITYA PRATAP

CONTENTS OF VOLUMES 35–45

xxi

Breeding Methods B. RAI, S. K. GUPTA and ADITYA PRATAP The Chronicles of Oil and Meal Quality Improvement in Oilseed Rape ABHA AGNIHOTRI, DEEPAK PREM and KADAMBARI GUPTA Development and Practical Use of DNA Markers KATARZYNA MIKOLAJCZYK Self-Incompatibility RYO FUJIMOTO and TAKESHI NISHIO Fingerprinting of Oilseed Rape Cultivars ´ ˇ URN and JANA Z ˇ ALUDOVA VLADISLAV C Haploid and Doubled Haploid Technology L. XU, U. NAJEEB, G. X. TANG, H. H. GU, G. Q. ZHANG, Y. HE and W. J. ZHOU Breeding for Apetalous Rape: Inheritance and Yield Physiology LIXI JIANG Breeding Herbicide-Tolerant Oilseed Rape Cultivars PETER B. E. MCVETTY and CARLA D. ZELMER Breeding for Blackleg Resistance: The Biology and Epidemiology W. G. DILANTHA FERNANDO, YU CHEN and KAVEH GHANBARNIA Development of Alloplasmic Rape MICHAL STARZYCKI, ELIGIA STARZYCKI and JAN PSZCZOLA Honeybees and Rapeseed: A Pollinator–Plant Interaction D. P. ABROL

xxii

CONTENTS OF VOLUMES 35–45

Genetic Variation and Metabolism of Glucosinolates NATALIA BELLOSTAS, ANNE DORTHE SØRENSEN, JENS CHRISTIAN SØRENSEN and HILMER SØRENSEN Mutagenesis: Generation and Evaluation of Induced Mutations SANJAY J. JAMBHULKAR Rapeseed Biotechnology VINITHA CARDOZA and C. NEAL STEWART, JR. Oilseed Rape: Co-existence and Gene Flow from Wild Species RIKKE BAGGER JØRGENSEN Evaluation, Maintenance, and Conservation of Germplasm RANBIR SINGH and S. K. SHARMA Oil Technology ¨ US BERTRAND MATTHA