Advances in Botanical Research, Volume 43

Defensive and Sensory Chemical Ecology of Brown Algae

CHARLES D. AMSLER AND VICTORIA A. FAIRHEAD

Department of Biology, The University of Alabama at Birmingham, Birmingham, Alabama 35294‐1170

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . II. Phlorotannins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Chemical Structure and Synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Comparison of Quantification Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Cellular Roles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Putative Ecological Roles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. Spatial and Intra/Interspecific Variability . . . . . . . . . . . . . . . . . . . . . . . . . F. Rates and Cost of Production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G. Current Uncertainty and Future Directions . . . . . . . . . . . . . . . . . . . . . . . III. Nonphlorotannin Antiherbivore Defences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Dictyotales . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Desmarestiales . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Other Orders . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Activated Defences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. Testing Chemical Defence Theories with Brown Algae. . . . . . . . . . . . . . . . . A. Optimal Defence Theory . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Induced Defence Model. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Carbon‐Nutrient Balance and Resource Allocation . . . . . . . . . . . . . . . . D. Tests of Multiple and Other Theories . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. Nonphlorotannin Defences Against Bacteria, Fouling Organisms, and Pathogens. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VI. Volatile Halogenated Organic Compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . VII. Sensory Chemical Ecology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Chemoattraction to Pheromones . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Behaviour and Sensory Capabilities of Spores . . . . . . . . . . . . . . . . . . . . . Advances in Botanical Research, Vol. 43 Incorporating Advances in Plant Pathology Copyright 2006, Elsevier Ltd. All rights reserved.

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0065-2296/06 $35.00 DOI: 10.1016/S0065-2296(05)43001-3

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VIII. Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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ABSTRACT The ecological interactions of brown algae are important as these macroalgae are common and often keystone members in many benthic marine communities. This review highlights their chemical interactions, particularly with potential herbivores, but also with fouling organisms, with potential pathogens, with each other as gametes, and with their microenvironments when they are spores. Phlorotannins, which are phenolic compounds unique to brown algae, have been studied heavily in many of these respects and are highlighted here. This includes recent controversy about their roles as defences against herbivory, as well as new understanding of their roles in primary cellular functions that may, in many instances, be more important than, and which at least have to be considered in concert with, any possible ecological functions. Brown algae have also been useful models for testing theories about the evolution of and ecological constraints on chemical defence. Furthermore, their microscopic motile gametes and spores have the ability to react to their chemical environments behaviourally.

I. INTRODUCTION Chemical ecology can be described as the study of chemically mediated interactions between organisms or between organisms and their environment, and most such interactions can be grouped into three broad categories. One category is chemical communication between organisms, such as brown algal male gamete attraction to pheromones, which is discussed in Section VII.A. A second is organisms sensing and responding to their chemical environments, and in brown algae this is observed in spores, which are able to sense and respond behaviourally to nutrients as described in Section VII.B. However, most studies of the chemical ecology of brown algae fall into the third category, which is chemical defence. Defences can be mounted against predators, pathogens, biofoulers, or competitors. A majority of the research on chemical defences in brown algae to date, and consequently a majority of this review is focused on defences against predators, but we also discuss defences against pathogens and biofoulers in Sections II. D.3 and V. Outside of speculation about potential allelopathic roles of pheromones, as discussed briefly in Section VII.A, we are aware of little work on brown algae examining chemical interactions between competitors, although the report of Ra˚ berg et al. (2005) is a fascinating recent example. The chemicals mediating defensive interactions are usually secondary metabolites (also called natural products) so termed to distinguish them from

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metabolites with primary metabolic roles within the cells of many taxa. However, as discussed in Section II, brown algal phlorotannins are an important exception to this as they have important primary roles in addition to ‘‘secondary’’ functions as chemical defences. Brown algae have been included in numerous recent reviews that have covered various aspects of algal chemical ecology (Amsler, 2001; Amsler and Iken, 2001; Arnold and Targett, 2002; Cronin, 2001; La Barre et al., 2004; Paul and Puglisi, 2004; Paul et al., 2001; Pohnert, 2004; Pohnert and Boland, 2002; Potin et al., 2002; Steinberg and de Nys, 2002; Steinberg et al., 2001, 2002; Targett and Arnold, 2001; Van Alstyne et al., 2001a). Here we have attempted to take a comprehensive approach in reviewing the current state of brown algal chemical ecology, and we believe that this focus on the group is warranted. Brown algae, which comprise the Class Phaeophyceae, are unique in being very far removed phylogenetically from all other eukaryotic macrophytes (i.e., red macroalgae, green macroalgae, bryophytes, and vascular plants, which clade together in modern phylogenetic trees) with the notable exception of the endosymbiotically derived red lineage chloroplasts of brown algae and their heterokont relatives (Baldauf, 2003; Falkowski et al., 2004; Keeling, 2004). A great deal of the conceptual framework of defensive chemical ecology is based on studies of vascular plants (cf. Section IV), and this phylogenetic distinctiveness of the Phaeophyceae makes them ideal tools to test and extend such ideas in trophically analogous but phylogenetically distinct organisms. Brown algae are also very important members of many marine communities ranging from the tropics to near the poles (Lobban and Harrison, 1994) and often dominate these communities in terms of structure and biomass, particularly in temperate and polar waters (e.g., Dayton, 1985a,b, 1990; Schiel and Foster, 1986; Wiencke and Clayton, 2002). Brown algae do occasionally occur in freshwater (Graham and Wilcox, 2000), but we are aware of no studies of their chemical ecology in freshwater systems. Consequently this review focuses entirely on marine and estuarine environments. With respect to defences against predation, we include only chemical forms of defence even though brown algae can also deter herbivory via morphological or otherwise physical mechanisms (e.g., Lewis et al., 1987; Lowell et al., 1991) or via life history adaptations that expose relatively palatable stages to herbivores over a minimal period of time (Lubchenco and Cubit, 1980). Because of the length of this review, we presume that many readers will consult only some parts at any given time and, consequently, we have chosen to include redundancy in two forms in the text. There are a number of published studies that are referred to, but in diVerent contexts, and often

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with diVerent emphasis, both in Section II on phlorotannins and in Section IV on tests of chemical defence theories. Also, particularly because this review is focused on brown algae as a specific taxonomic group, we feel that it is important throughout to be explicit about the ordinal‐level taxonomic relationships between the species discussed. Both similarities and diVerences in the chemical ecology of diVerent brown algal species, as described later, should be considered within a phylogenetic context. To provide such context, we have attempted to exhaustively identify the ordinal‐level classification of each species every time it is discussed, although sometimes using terms such as ‘‘kelp’’ for members of the Laminariales, ‘‘fucoid’’ for members of the Fucales, or ‘‘dictyotalean’’ for members of the Dictyotales. It is hoped that a reader of the entire review or of multiple parts thereof will understand the reasons for this redundancy.

II. PHLOROTANNINS Phlorotannins are polyphenolic polar metabolites with both primary and secondary roles. They occur only in the Phaeophyceae and account for over 10% dry weight in many species or up to 20% in others (Ragan and Glombitza, 1986). Their roles and functions have been the subject of many studies over the last few decades, particularly those roles that relate to antiherbivory and antifouling. Recent reviews have covered several aspects of our current knowledge in relation to phlorotannins and this work will not specifically focus on these areas, but it will still briefly address important areas such as chemical structure and roles at the cellular level. The comprehensive review by Ragan and Glombitza (1986) covered work up to that time, and recent reviews have concentrated on more specific aspects, such as the mediating role phlorotannins play in the interaction between algae and herbivores (Targett and Arnold, 1998, 2001) and on the putative roles of phlorotannins at the cellular level (Schoenwaelder, 2002). Arnold and Targett (2002) provide an overview of marine tannins in general, reviewing work on both vascular and nonvascular taxa, and Paul and Puglisi (2004) include a discussion of phlorotannins in their wide‐ranging review of chemical interactions between marine organisms. A. CHEMICAL STRUCTURE AND SYNTHESIS

1. Structure Phlorotannins are polymers of phloroglucinol (1,3,5‐trihydroxybenzene; Fig. 1) and are classified into six groups on the basis of the chemical structure of the polymer (fucols, phlorethols, fucophlorethols, fuhalols,

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Fig. 1. Chemical structures of phloroglucinol and subunits of the six diVerent structural classes of phlorotannins.

isofuhalos, and eckols; Fig. 1) (Ragan and Glombitza, 1986). They have several properties in common with some vascular plant tannins, although they remain chemically quite diVerent. Properties in common with the condensed tannins of vascular plants include the ability to bind to metal ions and to precipitate protein and carbohydrate from solution (Ragan and

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Glombitza, 1986). The molecular masses of phlorotannins vary between 126 Da and 650 kDa (Targett and Arnold, 2001), but are found most commonly in the 10 to 100 kDa range (Boettcher and Targett, 1993). 2. Location in the cell: soluble and cell wall bound forms The highest concentration of phlorotannins in the cell is found in physodes, which are membrane‐bound vesicles appearing as light‐refractive bodies in the cytoplasm (for reviews, see Ragan and Glombitza, 1986; Schoenwaelder, 2002). Phlorotannins in this form are soluble (Ragan and Glombitza, 1986). Physodes may occur in most tissues of the thallus but commonly occur in the outermost layers (Ragan and Glombitza, 1986; Tugwell and Branch, 1989). Lu¨ der and Clayton (2004) suggest that as much as 90% of the total phlorotannin content of Ecklonia radiata (Order Laminariales) can be found in the epidermal layer. Shibata et al. (2004) reported that the phlorotannin distribution in tissues of three other Ecklonia spp. is similarly concentrated in epidermal layers. Phlorotannins are also found as a constituent of cell walls (Schoenwaelder and Clayton, 1999a), where they are incorporated after release from the physodes (see Section II.C.1). Physodes are able to move to areas of active cell wall formation via interactions with the cytoskeleton (Schoenwaelder and Clayton, 1999b). The cell wall bound phlorotannins are thought to occur in levels an order of magnitude lower than levels of soluble phlorotannins in the cell (Koivikko et al., 2005). 3. Synthesis and metabolic turnover Phlorotannins are generally thought to be synthesised via the acetate–malonate pathway (Herbert, 1989) in a process that may involve a polyketide synthase (PKS) type enzyme complex (Arnold and Targett, 2002). However, Chen et al. (1997) have proposed an alternative hypothesis that they are produced by the shikimic acid pathway, in a manner analogous to vascular plant tannins. Identification of the phlorotannin synthetic pathway is an important goal for future research. This is particularly so if it leads to methodologies to monitor phlorotannin synthesis at the genetic or enzymatic levels, which could potentially help resolve some of the uncertainties in studies of phlorotannins as described later. Arnold and Targett (1998) developed a method using stable isotope (13C) techniques to determine rates of phlorotannin synthesis. In Lobophora variegata (Order Dictyotales) and in Sargassum pteropleuron and Fucus distichus (Order Fucales), phlorotannin synthesis costs represented
1 % of total assimilated carbon when calculated over a range of carbon assimilation rates. Arnold and Targett (1998) emphasised that these investment rates do not include other costs, such as maintaining the metabolic pathways and

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storage structures, which may diVer between species and for diVerent metabolites. They also investigated polymerisation (‘‘aging’’) in Sargassum pteropleuron, observing that
30 kDa polymers formed rapidly in a 5‐h period following the labelling incubation, and that this size class was gradually replaced with 30 kDa polymers from that time on. In a later study these authors used the same methods in field and laboratory experiments that provided evidence for relatively high rates of metabolic turnover in two tropical species (L. variegata and Sargassum hystrix var buxifolium), although the rate for L. variegata (2 days for complete turnover) was considered to be related to exudation under the stressful experimental conditions (Arnold and Targett, 2000). The turnover rate for S. hystrix var. buxifolium was slower at 17 days for a complete removal of 13C from the measurable phlorotannin pool, which was recorded in field experiments considered to involve very low rates of exudation. B. COMPARISON OF QUANTIFICATION METHODS

Several methods provide measures of phlorotannins, either by quantifying the total level of polyphenolics or by specifically measuring levels of tannins (for reviews, see Ragan and Glombitza, 1986; Targett and Arnold, 1998). The chemical behaviour of phlorotannins (as reactive, large, structurally similar, polar metabolites) has led to colorimetric methods becoming accepted as the preferred technique to eVectively quantify the levels of soluble, physode‐bound phlorotannins (Ragan and Glombitza, 1986; Targett and Arnold, 1998). These colorimetric methods, of which there are several, measure the total level of phenolics in the sample, which does not allow diVerentiation of size classes (Stern et al., 1996b), considered an important factor in determining the bioactivity of phlorotannins (see Section II.D.2). Results gained from each procedure will be variously aVected by the exact chemical structure of the phlorotannins in the sample (including size and bond types) and by the choice of reference compound (Ragan and Glombitza, 1986; Stern et al., 1996b; Van Alstyne, 1995). The proliferation of methods means it is sometimes diYcult to make comparisons between studies, but as each method has its advantages in particular instances, it is hard to advocate a single preferred method, and relative comparisons within studies (when results are gained using the same procedure) are still valid. The strong hydrogen‐bonding capacity of phlorotannins means they can be eVectively removed from a sample by treatment with the resin polyvinylpolypyrrolidone (PVPP) (reviewed by Toth and Pavia, 2001). This allows quantification methods to be assessed (e.g., Targett et al., 1995) or to be modified by including PVPP‐treated samples (e.g., Cronin and Hay, 1996a; Peckol

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et al., 1996; Yates and Peckol, 1993) and is useful for creating appropriate controls in experimental design (e.g., Wilkstro¨ m and Pavia, 2004). Extraction of phlorotannins prior to quantification is problematic no matter what assay is preferred. Polyphenolics are prone to oxidation and thus an inert atmosphere and dark, cold conditions are required for any procedure. The ability to oxidise rapidly, in combination with their tendency to precipitate proteins, is likely to result in underestimates of concentrations unless these factors are controlled for (Ragan and Glombitza, 1986). Extraction of phenolics is normally by aqueous alcohol (usually methanol) or aqueous acetone (Ragan and Glombitza, 1986), which minimises extraction of nonphenolic substances (e.g., proteins) that may be inadvertently measured in the quantification assay (Van Alstyne, 1995). Koivikko et al. (2005) compared the eYciency of various solvents for extracting phlorotannins from Fucus vesiculosus, indicating that 70% aqueous acetone was the most eYcient in that case. 1. Folin–Denis A modification of the Folin–Denis procedure (Folin and Denis, 1915) is perhaps the most widely used method of quantifying phlorotannins. During the Folin–Denis assay, polyphenolics are oxidised in reactions linked to the production of stable blue‐coloured molecules (via the reduction of phosphomolybdic and phosphotungstic acids), which allows measurement through spectrophotometry and quantification using a commercially available standard (Ragan and Glombitza, 1986). Although several compounds are known to interfere with the reactions (Ragan and Glombitza, 1986) and the reagent is also reactive with certain nonpolyphenolic compounds, these factors are not considered to aVect results beyond normal error limits (Targett and Arnold, 1998; Targett et al., 1995; but see Appel et al., 2001). 2. Folin–Ciocalteu The Folin–Ciocalteu assay, using a modified reagent (Folin and Ciocalteu, 1927; Waterman and Mole, 1994), represents an improvement of the Folin– Denis assay by reducing levels of precipitates (Van Alstyne, 1995), which allows use with small sample volumes (Stern et al., 1996b). In common with the Folin–Denis assay, the reagent is reactive with compounds other than phenolics, but this represents only a small percentage of the overall measurement (Van Alstyne, 1995). 3. 2,4‐Dimethyloxybenzaldehyde The DMBA assay, another colorimetric assay, depends on the ability of 2,4‐ dimethyloxybenzaldehyde (DMBA) to react specifically with phlorotannins (1,3‐ and 1,3,5‐substituted phenols), but not other phenolics, producing a

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pink‐coloured chromophore (Stern et al., 1996b). This assay has the disadvantage of requiring a standard to be produced for each species (by purification of phlorotannins from it or a very closely related species) in contrast to those aforementioned assays that utilise commercially available phloroglucinol. This is due to the larger variability in reactivity depending on the specific nature of the phlorotannins (Stern et al., 1996b). However, the DMBA assay is less sensitive to interferences from nonphenolic substances (Stern et al., 1996b), and once a standard has been prepared, the simplicity of the assay procedure allows more easily for a sample design with much larger numbers of samples (as long as the samples come from the same, or closely related, species). 4. Cell wall bound Koivikko et al. (2005) published a method for measuring cell wall bound phlorotannins. The method was adapted from vascular plant studies and involves alkaline degradation of the bonds between phenolics and alginic acid in the cell walls. C. CELLULAR ROLES

1. Cell wall structure Phlorotannins have recently been confirmed as being an important component of the brown algal cell wall (Schoenwaelder and Clayton, 1999a) and as being vital for the process of cytokinesis (for a review, see Schoenwaelder, 2002). In zygotes of Acrocarpia paniculata and Hormosira bankisii (Order Fucales), phlorotannins are secreted into cell walls following the fusion and breakup of physode membranes (Schoenwaelder and Clayton, 1998a,b) where they are then thought to complex with alginic acid (Schoenwaelder and Clayton, 1999a). Investigations in the same species also revealed that prior to cell division the physodes form a distinct line across the centre of cell, which occurs before any other cell wall constituents accumulate (Schoenwaelder and Clayton, 1998b). 2. Adhesion to the substrate A secretion of phenolics coincides with the adhesion of zygotes in Acrocarpia paniculata and Fucus gardneri (Order Fucales) where secretion is localised at the point of adhesion (Schoenwaelder and Clayton, 1998a; Vreeland et al., 1998). Secretion of polyphenolics also occurs after fertilisation in Durvillaea potatorum (Order Durvillales; Clayton and Ashburner, 1994). Adhesion of these newly formed zygotes is thought to be via an extracellular glue composed, in part, by phenolics (Vreeland et al., 1998; reviewed in Schoenwaelder, 2002).

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3. Spermatozoid inhibitors During the period in which newly fertilised zygotes remain unprotected from polyspermy (which is potentially deadly), secreted phlorotannins are thought to protect the zygote by impairing sperm motility (Schoenwaelder and Clayton, 1998a; reviewed in Schoenwaelder, 2002). 4. Wound healing That phlorotannins play a role in wound healing has been recognised for over three decades (Fagerberg and Dawes, 1976; Fulcher and McCully, 1971), and recent work has further clarified this role (Lu¨der and Clayton, 2004). Lu¨ der and Clayton (2004) created small wounds with a cork borer in sections of Ecklonia radiata (Order Laminariales) and then followed the healing process for 9 days using microscopy techniques (light, fluorescence, and transmission electron). They found clear evidence of an accumulation of phlorotannins around wound sites, which was evident 1 day after wounding. This became prominent in the nearby medulla after only 3 days and by 9 days dense accumulations were found throughout the medulla of the entire algal section. They observed that new medullary cells produced at the wound site were structurally diVerent and contained several physodes, which increased in number and size over the next few days. Cortical cells were also observed to accumulate physodes. By day 5 the wound surface was composed entirely of cells dense with physodes, which diVerentiated into epidermal cells that remained rich in physodes. They concluded that phlorotannins function in both wound‐sealing (which is consistent with the clotting proposal of Fagerberg and Dawes, 1976) and structural wound‐healing roles (consistent with their role in cell wall formation; see Section II.C.1). In addition, the general accumulation of phlorotannins throughout the medulla was considered to be an antiherbivore response (see Section II.D.1), which may reduce infection (see Section II.D.3). D. PUTATIVE ECOLOGICAL ROLES

1. Antiherbivory: evidence for and against deterrence A multitude of studies have focused on the role of phlorotannins in herbivore defence. These studies have generally utilised feeding experiments that give herbivores the choice between tissues with diVerent phlorotannin contents and artificial foods containing algal extracts or pure compounds, with only the latter method being a test of phlorotannin bioactivity. The concentration and dose of phlorotannins in the oVered food influence greatly the outcome of the algal–herbivore interaction (Pereira and Yoneshigue‐ Valentin, 1999; Targett and Arnold, 1998; Van Alstyne et al., 1999c), and the

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type and nutritional quality of the bioassay food may also be important (Deal et al., 2003; Kubanek et al., 2004). A positive deterrent eVect has not been found consistently and seems to be highly dependent not only on the selection of algae but also on the herbivore species. Even when the same herbivore species is used, the interactions mediated by phlorotannins do not always yield the same results. The numerous studies conducted using Fucus vesiculosus (Order Fucales) demonstrate these points. The gastropod Littorina littorea preferred to graze on F. vesiculosus with a lower phenolic content when oVered the choice between this and higher phenolic content foods (Geiselman and McConnell, 1981; Yates and Peckol, 1993). In contrast, Jormalainen et al. (2004) found a preference of Idotea baltica for phlorotannin (extracted from F. vesiculosus) containing food, an eVect that increased with higher concentrations (10% compared to 5%), which they suggest to be related to host recognition. Also, Hemmi et al. (2004) established that while the I. baltica preferred damaged F. vesiculosus for at least 10 days after clipping, this was not associated with diVerences in phlorotannin levels, as clipping did not induce the production of phlorotannins. Furthermore, crude polar extracts from F. vesiculosus deterred feeding by sea urchins (Arbacia punctulata), but bioassay‐guided fractionation of this extract revealed that deterrence was not due to phlorotannins (Deal et al., 2003). Feeding assays showed that even at concentrations 400% of the natural level (1% dwt), isolated phlorotannins were not an eVective deterrent to feeding by A. punctulata (Deal et al., 2003). Kubanek et al. (2004) also used bioassay‐guided fractionation techniques and found that the deterrent eVect of crude F. vesiculosus extracts was due to defensive compounds other than phlorotannins, but they were not able to isolate the bioactive compounds and did not detect the galactolipid reported by Deal et al. (2003). They tested the eVect of purified phlorotannins (at 3, 6, and 12 natural yield) on the feeding of amphipods (Ampithoe longimana) and sea urchins (A. punctilata), which normally avoid F. vesiculosus, and found no deterrent eVects. The amphipod Ampithoe valida, which is commonly known to eat F. vesiculosus in the field, was deterred only at the 12 concentration (Kubanek et al., 2004). Investigations of other taxa have also produced mixed results. Within the Fucales, Van Alstyne (1988) found that snails (Littorina sitkana) moved away from areas of wounded Fucus gardneri (as F. distichus), which showed phlorotannin accumulation, and preferred to feed on undamaged algae with lower phlorotannin contents. The isopod Idotea granulosa preferred the high phlorotannin content tissue of Ascophyllum nodosum, which may have been due to the higher nitrogen content of that tissue (Pavia et al., 1997). Feeding by the snail Littorina obtusata on Ascophyllum nodosum tissue with a high

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phlorotannin content was decreased (Pavia and Toth, 2000a; Toth and Pavia, 2000b), but feeding by I. granulosa was not aVected by phlorotannin content (Pavia and Toth, 2000a). In a subsequent study the basal stipes of A. nodosum were found to have the highest phlorotannin contents and were consumed less by L. obtusata than other tissue types (Pavia et al., 2002). Phenolics extracted from Sargassum furcatum were deterrent at 2 and 5% of food content to amphipods (Parhyale hawaiensis), but the natural concentration (0.5%) did not deter feeding, consistent with the observed palatability of this species to P. hawaiensis (Pereira and Yoneshigue‐Valentin, 1999). In contrast (Cronin and Hay, 1996a) reported that feeding by amphipods was not influenced by the phlorotannin concentration of Sargassum filipendula. In members of the Laminariales (kelps), Johnson and Mann (1986) reported that the avoidance of the intercalary meristem region of Laminaria longicruris by grazing snails (Lacuna vincta) correlated with a high phenolic content. Abalone (Haliotis rufescens) preferred to feed on phenolic poor species, and phenolics extracted from Dictyoneurum californicum deterred feeding by 90% (Winter and Estes, 1992). However, Wakefield and Murray (1998) concluded that the feeding preferences of Norrisia norrisi (herbivorous snail) were determined more by factors such as habitat and refuge provision or toughness than by phlorotannin content. Feeding preference assays revealed that the snail consistently preferred the high phlorotannin content kelps over other algal species, although comparisons within the kelps did show the least preferred species (Egregia menziesii) was that with the highest phlorotannin content, but this was also the toughest kelp species. In a study of Zonaria angusta (Order Dictyotales), amphipods (Tethygeneia sp. and Hyale rubra) preferred to consume young tissue with a low density of physodes (Poore, 1994). The amphipods preferentially consumed the area directly behind the apical cell row, which has a low density of physodes, in preference to both the single layer of physode‐dense apical meristematic cells and older tissue with moderate densities of physodes (Figs. 2 and 3). A number of studies comparing palatability across orders have also been reported. Steinberg and van Altena (1992) found that some of the Australasian herbivores they tested were deterred from feeding by extracts from some algal species, but in general they found that Australasian marine herbivores were not deterred by phlorotannins. This contrasts to North American herbivores tested (e.g., Tegula funebralis and Tegula brunnea), which were strongly deterred by the presence of phlorotannins (Steinberg, 1984, 1985, 1988; Steinberg and van Altena, 1992). Steinberg et al. (1991) showed that the concentration of phlorotannins (from tropical and temperate species) in foods could not be related to feeding deterrence of tropical herbivorous fish. Also, the herbivorous fish Cebidichthys violaceus was

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Fig. 2. Apical region of Zonaria angustata branches: intact (left) and displaying damage from amphipod feeding (center and right). On the right is a branch in which the apical cell row has been breached by amphipods with the area behind it having been consumed. The center shows a branch in which the dangling apical cell row has broken away. From Poore (1994).

Fig. 3. Distribution of physodes in a longitudinal section of the apical tissues of Zonaria angustata. Physode distribution was compiled from photomicrographs. Physodes are visible as dark‐staining subcellular bodies with high densities in the apical meristem (left) and old growth (right) and in low densities adjacent to the meristem (center). From Poore (1994).

deterred from feeding by the presence of polar extracts of the fucoid Fucus gardneri in its diet, but not by polar extracts from the kelp Macrocystis integrifolia (Ireland and Horn, 1991). Van Alstyne et al. (2001b) examined the feeding preferences of four herbivores (sea urchins and snails) towards tissue that diVered in phlorotannin concentrations (juvenile and adult tissue from six members of the Laminariales and two members of the Fucales). Although they found much variation amongst species and life stages in terms of deterrence level, phlorotannins were apparently not responsible.

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The lack of a consistent feeding response to phlorotannins has led some authors to question whether they can be considered a defensive metabolite (Jormalainen and Honkanen, 2004; Jormalainen et al., 2003) and also highlights the complex role other metabolites play in defence, a fact that is often overlooked but which can confound experimental designs (Deal et al., 2003; Kubanek et al., 2004). 2. EVects on digestion and reproduction Studies of eVects of phlorotannins on the assimilation eYciencies of herbivores, as with those on feeding deterrence, have produced results that are often ambiguous. The specific structure of the phlorotannins occurring in an algal species, in combination with diVerences in the gut environment of herbivores, is thought to be partly responsible for this confusion (see review in Targett and Arnold, 2001). Several features of the gut are important in determining the activity of ingested phlorotannins (such as morphology, pH, enzyme composition, microbial activity) and these factors diVer widely among diVerent herbivores (Targett and Arnold, 2001). The acidity of the gut may be most important in fish, with basic guts correlated with higher assimilation eYciencies (Targett and Arnold, 2001). The presence of surfactants in herbivore gut fluid has also been shown to be involved in ameliorating the eVects of phlorotannins (Tugwell and Branch, 1992). Nevertheless, phlorotannins are considered to aVect food value via their ability to complex with proteins and other macromolecules in the gut, which can aVect assimilation eYciency (Stern et al., 1996a; see review in Targett and Arnold, 2001). Polar extracts (that should include most phlorotannins) of Fucus gardneri (Order Fucales), but not of Macrocystis integrifolia (Order Laminariales), significantly reduced the ability of the fish Cebidichthys violaceus to digest a palatable green alga (Ulva lobata), in particular reducing the assimilation eYciency of nitrogen (Ireland and Horn, 1991). Shell growth of abalone (Haliotis rufescens) was inhibited by the addition of polyphenolics from Dictyoneurum californicum (Order Laminariales) into its diet (Winter and Estes, 1992). In addition, the total carbon and nitrogen assimilations of the herbivorous isopod Idotea baltica decreased with increasing phlorotannin content in an artificial food diet, although as mentioned earlier this herbivore was not deterred from feeding by higher phlorotannin content (Jormalainen and Honkanen, 2004). However, Hemmi and Jormalainen (2004) reported that the body size of female I. baltica was significantly correlated with the phlorotannin content of Fucus vesiculosus (Order Fucales) in their habitat. Also, Toth et al. (2005) found that an increased level of phlorotannins in the diet of Littorina obtusata (fed on Ascophyllum nodosum, Order Fucales) did

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not have an eVect on growth rates, but did reduce significantly the number of viable eggs that the gastropod produced. A high dietary phlorotannin content did not aVect the assimilation eYciencies of three tropical herbivores (fish Sparisoma radians and Sparisoma chrysopterum, which have a basic gut environment, or the crab Mithrax sculptus) (Targett and Arnold, 2001; Targett et al., 1995). Similarly, the growth and conversion eYciencies of the Australasian herbivores Tripneustes gratilla (sea urchin) and Turbo undulata (snail) were not aVected by feeding on a diet high in phlorotannins for several months (Steinberg and van Altena, 1992). Also, a phlorotannin‐rich diet (containing 3 natural concentration of phlorotannins from F. vesiculosus) was correlated with enhanced growth and survivorship of the amphipod Ampithoe valida (Kubanek et al., 2004). In common with vascular plant tannins, molecular size is an important determinant of the eVects of phlorotannins on herbivores. Boettcher and Targett (1993) studied the eVect of diVerent molecular size fractions on the assimilation eYciency of the fish Xiphister mucosus (which has an acidic gut environment; Targett and Arnold, 2001). They utilised force‐feeding bioassays with extracts from various temperate and tropical browns with which X. mucosus cooccurs but is not observed to eat. The high molecular size (>10 kDa) phlorotannins had a significant eVect in reducing the assimilation eYciency (total, organic and protein) of the herbivore. This is the same size class (10–100 kDa) of phlorotannins that was found in the highest concentrations in all but one of the surveyed species. Boettcher and Targett (1993) suggested that diVerences in the molecular size distribution of phlorotannins in diVerent species could be part of the explanation for such wide‐ranging results in deterrence studies. Phlorotannins do, in some cases, aVect the nutritional value of foods, but animals (including marine herbivores) can engage in compensatory feeding to overcome this eVect (Cruz‐Rivera and Hay, 2001). This may mean that some herbivores simply eat more of a particular (phlorotannin containing) species in order to meet nutritional requirements (but see Honkanen et al., 2002). In addition, some herbivores preferred phlorotannin‐containing foods (Jormalainen and Honkanen, 2004; Pavia et al., 1997). These outcomes are not consistent with the fitness benefit that should be associated with a putative defensive metabolite. 3. Antibacterial and antifouling activity The potential antifouling and antibacterial roles of phlorotannins were first proposed in the 1940s and 1960s, respectively, and many subsequent studies have investigated the validity of these roles (reviewed in Ragan and Glombitza, 1986), with results to date also being inconclusive, at least for the

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antifouling role. In addition, note the caveats about the ecological relevance of antifouling or antiepiphytic bacteria bioassays noted in Section V. Fletcher (1975) reported that phlorotannins in the crustose alga Ralfsia spongiocarpa (Order Ectocarpales) were eVective in protecting the thallus from epiphytic fouling. In studies of members of the Fucales, Sieburth and Conover (1965) found that extracts containing Sargassum natans polyphenolics acted as an antibiotic. Phlorotannins extracted from Sargassum tenerrimum inhibit bacterial growth as well as larval settlement of polychaetes (Lau and Qian, 1997) and of barnacles (Lau and Qian, 2000) at ecologically relevant concentrations. Phlorotannins from Fucus spiralis and Ascophyluum nodosum aVected the survival and therefore the habitat choice of the ciliate Voticella marina (Langlois, 1975). A study by Wilkstro¨ m and Pavia (2004) showed that phlorotannins from Fucus vesiculosus inhibit settlement of the fouling barnacle Balanus improvisus at ecologically relevant concentrations (1 mg liter1). The second species they studied was Fucus evanescens, an invasive species, which had quantitatively much lower natural levels of fouling that the native F. vesiculosus. However, in settlement bioassays, Balanus improvisus settled on F. evanescens at higher rates when given a choice between the two species. The authors found that whilst the phlorotannin concentration of the thallus did not diVer between the two species (about 10% dwt in both), the inhibitory eVect of phlorotannins from F. vesiculosus was greater and eVective at lower concentrations than those extracted from F. evanescens. Contradictory results from field experiments (i.e., lower fouling on F. evanescens) and laboratory work (i.e., phlorotannins from F. vesiculosus more eVective antifouling) indicated that the higher eVectiveness of antifouling mechanisms in F. evanescens was not due to settlement inhibition by phlorotannins, but was due to a higher postsettlement mortality (Wilkstro¨ m and Pavia, 2004). The high level of fouling on F. vesiculosus indicates that while phlorotannins from this species can deter larval settlement, other factors are responsible for juvenile mortality. Furthermore, this demonstrates that the settlement inhibition eVect of phlorotannins is not constant even among congeneric species. In another study of F. vesiculosus, Honkanen and Jormalainen (2005) found that the variation in fouling biomass levels, which they observed on diVerent genotypes, was not correlated with tissue phlorotannin content. Lu¨ der and Clayton (2004) suggested that the presence of increased numbers of physodes at wound surfaces in Ecklonia radiata (Order Laminariales) may serve to reduce microbe infection. Molecular size may be an important factor in determining the bioactivity of phlorotannins, including antibiotic roles (Ragan and Glombitza, 1986). By contrast, Jennings and Steinberg (1997) concluded that natural concentrations of phlorotannins at the thallus

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surface of Ecklonia radiata were unlikely to be able to eVectively reduce epiphytism by the green alga Ulva lactuca and found no correlation between epiphyte load and tissue phlorotannin content. Their study of phlorotannin exudation rates in E. radiata found these to be much lower than rates reported in other studies, which mainly utilised stressed algae (Jennings and Steinberg, 1994, 1997). An inhibitory eVect of phlorotannins from E. radiata and Sargassum vestitum (Order Fucales) on the germination of U. lactuca was found, but only at unnaturally high concentrations (>10 mg liter1) (Jennings and Steinberg, 1997). The authors highlighted the importance in these types of studies of measuring or estimating concentrations of phlorotannins where the encounter occurs (i.e. at the boundary layer) and which the larvae or epiphytes are actually likely to encounter so that measuring tissue content is probably not appropriate for studies of epiphytism (Jennings and Steinberg, 1997). 4. Sunscreen Depletion of the ozone layer over the past few decades has led to higher levels of damaging UV‐B radiation reaching shallow water benthic communities (Frederick et al., 1998), which has led to concern over the eVects of UV‐B on benthic organisms (Karentz, 2001; Karentz and Bosch, 2001). Phlorotannins absorb in the UV‐B range of the spectrum (Pavia et al., 1997) and are considered to be one way in which the algal thallus can protect itself from photodestruction caused by UV radiation (reviewed in Schoenwaelder, 2002). A 2‐week exposure to increased UV‐B radiation led to a significant increase in the phlorotannin content of Ascophyllum nodosum (Order Fucales) (Pavia et al., 1997). In a subsequent experiment, Pavia and Brock (2000) found that A. nodosum exposed to ambient UV‐B increase phenolic content after 7 weeks (but not after 2 weeks) in comparison to individuals that had had UV‐B removed from the available spectrum. However, in a field study involving a smaller increase in UV‐B radiation, no increase was found in phlorotannin content in Desmarestia anceps or in Desmarestia menziesii (Order Desmarestiales; V. A. Fairhead, C. D. Amsler, J. B. McClintock, and B. J. Baker, unpublished result). Swanson and Druehl (2002) reported that tissue phlorotannin content increased in Macrocystis integrifolia (Order Laminariales) after exposure to UV‐B radiation (but also UV‐A, which phlorotannins do not absorb well). Phlorotannin exudate from M. integrifolia reduced transmission of UV‐B radiation and also reduced the harmful eVects of UV‐B on developing kelp meiospores (germination rates were higher when water contained exudate) (Swanson and Druehl, 2002). The authors suggested that in regions with high kelp densities the concentration of phlorotannins in the water body

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C. D. AMSLER AND V. A. FAIRHEAD

could alter the spectral characteristics and shield marine organisms from biologically harmful UV‐B (Swanson and Druehl, 2002). However, UV-B did not affect phlorotannins in Fucus gardneri germlings (Order Fucales; Henry and Van Alstyne, 2004). 5. Heavy metal chelation The capacity of phlorotannins to bind to metal ions is well established, which has led to the suggestion that phlorotannins may be able to reduce the toxicity of certain heavy metal pollutants (reviewed in Ragan and Glombitza, 1986; Toth and Pavia, 2000a), but very little work has been done in this area. Ragan et al. (1979) found that phlorotannins are particularly good at chelating divalent metal ions such as copper and lead. The toxicity and anthropogenic release of heavy metals into the oceans means that an ability to detoxify both the cell (e.g., Skipnes et al., 1975; Smith et al., 1986) and the surrounding environment (e.g., Ragan et al., 1980) would be advantageous for the marine system. However, Toth and Pavia (2000a) concluded that probably substances other than phlorotannins (e.g., polysaccharides and/or phytochelatins) are important for the detoxification and resistance to copper accumulation in Ascophyllum nodosum (Order Fucales). Increasing the copper concentration in the surrounding water resulted in accumulation of copper in the algal tissues (without aVecting growth rates), but not in an increase in phlorotannin content. Karez and Periera (1995) reported that a majority of the copper, lead, cadmium, zinc, and chromium in Padina gymnospora (Order Dictyotales) coextracted with phlorotannins and hypothesised that this was because they were chelated by the phenolics. E. SPATIAL AND INTRA/INTERSPECIFIC VARIABILITY

The concentration of phlorotannins has been found to vary from undetectable levels to up to 20% of thallus dry weight, varying within and between species, as well as showing spatial variation on just about every scale (for reviews, see Ragan and Glombitza, 1986; Van Alstyne et al., 2001a). Phlorotannin content has been found to vary within the thallus of an individual (Fairhead et al., 2005a; Pavia et al., 2003; Pfister, 1992; Poore, 1994; Steinberg, 1984; Toth and Pavia, 2002b; Tugwell and Branch, 1989; Tuomi et al., 1989; Van Alstyne et al., 1999a), between individuals in the same population (Fairhead et al., 2005a; Pavia et al., 2003), between diVerent sites ˚ berg, 1996; Pavia et al., 2003; Steinberg, 1989; and populations (Pavia and A Stiger et al., 2004; Targett et al., 1992; Toth and Pavia, 2000a; Van Alstyne et al., 1999a,b), across depths (Connan et al., 2004; Fairhead et al., 2005a; Martinez, 1996), between diVerent species (Fairhead et al., 2005a; Ragan

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and Glombitza, 1986; Steinberg, 1989; Steinberg and van Altena, 1992; Tugwell and Branch, 1989; Van Alstyne et al., 1999a,b; Stiger et al., 2004), between diVerent aged tissue (Pavia et al., 2003; Toth and Pavia, 2002b), across seasons (Connan et al., 2004; Stiger et al., 2004), and across large geographical areas (Steinberg, 1989; Targett et al., 1992; Van Alstyne and Paul, 1990; Van Alstyne et al., 1999b). Because the bioactivity of phlorotannins is often concentration dependant, this high degree of variation in phlorotannin content has important consequences for mediating the interactions between herbivores and brown macroalgae (Targett and Arnold, 2001; Van Alstyne et al., 2001a). Biogeographical patterns in phlorotannin content exist, with Australasian brown algae tending to have, on average, concentrations over five times higher than those from the northeast Pacific (Steinberg, 1989; Steinberg et al., 1995). The ecological and evolutionary consequences of this variation for algal–herbivore interactions are reviewed in Van Alstyne et al. (2001a). There is also evidence for a tropical‐temperate pattern, with tropical brown algae often containing low levels of phlorotannins (<2%) (Pereira and Yoneshigue‐Valentin, 1999; Steinberg, 1986; Van Alstyne and Paul, 1990). This was postulated to be a result of the lack of eVectiveness of phlorotannins against some tropical herbivorous fish (Steinberg, 1986; Steinberg and Paul, 1990). However, not all tropical fish are resistant to phlorotannins (Van Alstyne and Paul, 1990). Also, Pereira and Yoneshigue‐Valentin (1999) demonstrated that although phlorotannins occur in low levels in browns from the tropical south Atlantic (0.5%), sympatric herbivores are deterred by higher concentrations (2–5%), implying that they could be an eVective defence in that system if natural levels were greater. In addition, tropical brown algae do not always have low phlorotannin concentrations. Targett et al. (1992) established that, in contrast to tropical Indo‐Pacific species, many Caribbean species have high phlorotannin levels (e.g., >14%) and that all the species surveyed had concentrations >2% dwt. A later study broadened the geographical range of high phlorotannin concentration phaeophytes to the western tropical Atlantic (Targett et al., 1995). Other studies of members of the Fucales and Laminariales have found within‐species diVerences in the concentration of phenolics across diVerent regions separated by tens to hundreds of kilometres, such as the southern and eastern coasts of Australia and northern New Zealand (Cystophora moniliformis and Ecklonia radiata; Steinberg, 1989), the eastern coast of North America (Fucus vesiculosus; Target et al., 1992) and the western U.S. coastline (a variety of kelps and fucoids, Van Alstyne et al., 1999b). For other species, smaller scales (<1 km) have been more important for detecting patterns of variation than larger geographical scales (Ascophyllum

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˚ berg, 1996). Jormalainen et al. (2003) and Honkanen nodosum; Pavia and A and Jormalainen (2005) both reported a high degree of variation among diVerent populations of F. vesiculosus (which accounted for up to 65% of the total variation). Within‐population and within‐thallus variability of polyphenolic content has also been observed in A. nodosum, as well as interannual variability within populations (Pavia et al., 1999a, 2003). Several instances of interannual variability in phenolic content were also reported by Steinberg (1989) for kelp and fucoid populations. Phenolic content has also been correlated with tissue age and life cycle stages. New tissue in Zonaria angustata (Order Dictyotales) had a significantly lower density of physodes than the older branch tissue (Fig. 3; Poore, 1994). Denton et al. (1990) found that phlorotannin contents diVered between juveniles and adults but that the pattern was species specific for the three species of Fucus (Order Fucales) they investigated. Steinberg (1989) reported that juvenile Ecklonia radiata (Order Laminariales) contained higher levels of phenolics than adult secondary blades. Similarly, Van Alstyne et al. (2001b) examined diVerences in phlorotannin content between juvenile and adult kelps and fucoids and found that juveniles often had higher concentrations, but not always and that in one kelp species the adults displayed higher phenolic levels. By contrast, Pavia et al. (2003) reported that the annual shoots of adult Ascophyllum nodosum (Order Fucales) tend to have a higher phenolic content than juvenile tissue, and Stiger et al. (2004) reported the same pattern in the fucoids Sargassum mangarevense and Turbinaria ornata. The reproductive tissue of the kelps Alaria marginata, Ecklonia maxima, and Macrocystis angustifolia contain higher phenolic contents than vegetative tissue but not those in Alaria nana or Laminaria pallida (Pfister, 1992; Steinberg, 1984; Tugwell and Branch, 1989; Van Alstyne et al., 1999a). By contrast, the phlorotannin concentration in reproductive structures of the fucoids Fucus vesiculosus, Fucus gardneri, and Pelvetia compressea contain lower levels than the vegetative tissue (Tuomi et al., 1989; Van Alstyne et al., 1999a) Variation in phenolic content among meristematic, vegetative, and reproductive tissues was detected in 8 out of 15 kelp species and in 2 of 5 fucoid species surveyed by Van Alstyne et al. (1999a). Meristematic tissue tended to have the highest concentrations (Van Alstyne et al., 1999a). Phlorotannins content also varied significantly throughout the thallus of the kelp Laminaria hyperborean, as well as between individuals and populations (Toth and Pavia, 2002a). The fronds tended to have lower levels than the meristems, and older meristematic tissue tended to have higher concentrations than new meristems, although no consistent diVerences were seen between new and old fronds (Toth and Pavia, 2002a,b). Significant variation in phenolic content was

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detected among stipes, holdfasts, meristems, reproductive, and vegetative tissue in the kelps Alaria marginata, Ecklonia maxima, and Macrocystis angustifolia (Tugwell and Branch, 1989). Similarly, the kelp Ecklonia radiata showed significant within‐thallus variation, with higher content (on a dry weight basis) in the primary blade and stipes compared to secondary blades (Jennings and Steinberg, 1997). The basal stipes of the fucoid Ascophyllum nodosum, considered to have the highest fitness value of the species’ diVerent tissues (Pavia et al., 2002), have the highest phlorotannin concentration and are the least preferred by the gastropod Littorina obtusata (Pavia et al., 2002; Toth et al., 2005). We detected significant within‐thallus and between individual variation in the phlorotannin content of Desmarestia anceps and Desmarestia menziesii (Order Desmarestiales) (Fairhead et al., 2005a). Samples were taken from 13 areas of the thallus of multiple individuals (from various sites and depths), and results showed that whilst within‐thallus variation was significant for each individual, the actual pattern in phenolic content was not consistent across individuals of each species, either overall or grouped by collection site or depth. This spatial variability makes it exceptionally challenging to formulate hypotheses regarding the potential influence of various abiotic (e.g., light) or biotic (e.g., herbivory) factors on phlorotannin production. F. RATES AND COST OF PRODUCTION

1. Costs and trade‐oVs? Several studies have reported a correlation between increased levels of phenolics and decreasing growth rates, which has been taken in some cases as evidence for a trade‐oV between growth and a cost for phlorotannin production. However, most studies did not find this relationship consistently. Decreased growth of Ecklonia radiata (Order Laminariales) was correlated with increased phlorotannin content, but only in spring (Steinberg, 1995). Yates and Peckol (1993) found a correlation between decreased growth of Fucus vesiculosus (Order Fucales) and increased phlorotannin concentration at a site with high nitrogen (where phenolic content was lower overall), but this relationship was not found at their low nitrogen sites. Pfister (1992) found a relationship between lower growth rates and increased phlorotannin content in the fronds of Alaria nana (Order Laminariales), but in the sporophylls (which had the higher phlorotannin content) the relationship between phlorotannin concentration and growth was positive. Growth and phlorotannin content were correlated negatively in annual shoots of Ascophyllum nodosum (Order Fucales), a relationship that was consistent across two populations and over two years of data collection (Pavia et al., 1999b).

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Both Hemmi et al. (2004) and Cronin and Hay (1996a) reported positive relationships between growth and phlorotannin content in Fucus vesiculosus, but Jormalainen et al. (2003) found no evidence of a trade‐oV. In addition, Arnold and Targett (1998) pointed out that the presence of a correlation does not imply causation and that these studies only measured static concentrations, not taking into account the rates of phlorotannin turnover, which have been shown to be relatively high (Arnold and Targett, 2000). Turnover will increase the metabolic cost of maintaining a particular level of defence beyond that accounted for by simply measuring the accumulation of phlorotannins (Arnold and Targett, 2000). Toth et al. (2005) also pointed out that, in addition to which particular algal tissue is under consideration (as these do not all have the same fitness value), cost–benefit relationships must diVer depending on the type and density of the herbivore population. Arnold and Targett (2003) provided a fresh perspective on this issue. They suggested that the cycle of production and the roles of phlorotannins mean that there is in fact no possibility of a trade‐oV between growth and phlorotannin production rates, and they also accounted for the apparent trade‐ oVs reported in previous studies. They proposed a model of production that sees phlorotannins as sequestered in physodes after synthesis (Fig. 4), in which state they are reactive (and therefore measurable) in colorimetric assays. It is in this chemical form that phlorotannins act as secondary metabolites, for example, as a deterrent to herbivores or as antibacterial agents, which is dependant upon their ability to complex with proteins and other macromolecules (see Section II.D.2). These roles are only seen by

Fig. 4. Model proposed by Arnold and Targett (2003) of phlorotannin biosynthesis illustrating the transition of these compounds from reactive chemical defences in physodes to oxidised components of brown algal cell walls (CW). From Arnold and Targett (2003).

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Fig. 5. Diagram explaining the apparent growth defence trade‐oVs in brown algae with variable rates of phlorotannin production as a function of their multiple transitional roles as proposed by Arnold and Targett (2003). (A) In rapidly growing tissues, high rates of physode secretion at cell membranes provide components for cell walls and levels of measurable phlorotannins are low. (B) In slow‐growing tissues, physode decomposition at cell walls slows, production outpaces decomposition, and measured levels of phlorotannins increase. This model predicts a negative correlation between algal growth and phlorotannin content irrespective of any cost for phlorotannin production. From Arnold and Targett (2003).

Arnold and Targett (2003) as transitional, however, and are lost when the phlorotannins are cycled into their primary role of cell wall construction, where they are no longer measurable by colorimetric assays (Fig. 5). The important consequence of this scheme is that it is the requirement for cell wall construction that drives the rates of phlorotannin production and not their secondary role in defence. This means that measurable phlorotannin levels will drop when the requirement for cell wall construction (i.e., growth) is high and will increase in slow‐growing tissue where rates of phlorotannin synthesis outpace rates of decomposition and incorporation into cell walls (Fig. 5). In this way the model allows for apparent trade‐oVs between growth and phlorotannin production without necessarily requiring phlorotannin production to be allocated a cost that competes with growth. If the trade‐ oV with growth processes is eliminated, this requires questions as to the relevance of many ecological defence theories for describing patterns of phlorotannin production (see Section IV). 2. Possible determinants of production or induction Regardless of how well our knowledge of phlorotannin production fits into particular theories concerning the ecology or evolution of chemical defence, there is no doubt that many factors, both biotic and abiotic, can

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be correlated with changes in rates of production. The relative influence of one factor in a particular study may be seen as support for a chosen theory, but overall the picture of phlorotannin production is complex and does not fit easily into any one particular ecological theory (see Section IV). a. Light. Several studies have found correlative evidence for the link between irradiance and phlorotannin production. Current evidence also suggests that UV‐B radiation can induce phlorotannin production, which provides protection for the alga (Pavia and Brock, 2000; Pavia et al., 1997; Swanson and Druehl, 2002; see Section II.D.4). Fucus vesiculosus (Order Fucales) displayed a seasonal pattern in phlorotannin content that was well correlated with irradiance (Peckol et al., 1996). A similar consistent seasonal pattern in phlorotannin concentration has been found in Australian brown algae, with higher concentrations in the spring compared to autumn (Steinberg, 1995; Steinberg and van Altena, 1992). A two‐week reduction in irradiance reduced phlorotannin contents in F. vesiculosus but not in the fucoid Ascophyllum nodosum, and evidence showed that individuals collected from sun‐exposed sites contain higher concentration of phlorotannins than those from more shaded sites (Pavia and Toth, 2000b). We observed that Desmarestia anceps (Order Desmarestiales) had significantly higher phlorotannin concentrations when collected from the upper end of its depth distribution than from the lower part of its distribution, which was not true in Desmarestia menziesii, although that could have been because the depth diVerence was smaller in D. menziesii (Fairhead et al., 2005a). Phlorotannin concentrations in both species increased when they were transplanted from a 15‐m depth to a 5‐m depth regardless of whether they were shielded from UV radiation, suggesting that increased photosynetically active radiation was responsible for the diVerence (V. A. Fairhead, C. D. Amsler, J. B. McClintock, and B. J. Baker, unpublished result). However, neither study was intended to examine the eVects of irradiance alone, so strict irradiance controls were not performed. Consequently, although it is likely that the diVerences observed between phlorotannin levels were because of diVerences in light, this conclusion cannot be definitive. However, other investigations have found no such correlation with irradiance in other species, for example, in the fucoids Sargassum globulariaefolium and Fucus gardneri (as F. distichus) (Steinberg, 1986; Van Alstyne, 1988), or have found the peak concentration to occur in the winter (Ragan and Jensen, 1978). Also, Cronin and Hay (1996a) found no eVect on phlorotannin content of their manipulation of the irradiance environment of the fucoid Sargassum filipendula. If irradiance does play a role in phlorotannin

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production, it is complex and is probably tightly coupled with resource availability and/or growth processes. b. Nutrients. Several studies of Fucus vesiculosus have assessed the impact of nutrients on phlorotannin production, and the results have been inconsistent. Ilvessalo and Tuomi (1989) found an inverse correlation between tissue nitrogen and phlorotannin content in F. vesiculosus (Order Fucales). Yates and Peckol (1993) found that F. vesiculosus collected from low nitrogen sites (with lower tissue‐N content) had higher phenolic content than algae collected from high nitrogen sites (with higher tissue‐N contents) except at certain times of the year when concentrations were low overall. At both sites the phenolic content was inversely correlated to tissue‐N content over the year. However, experimental manipulation of nutrient levels failed to find a consistent reduction in phenolic content in response to nutrient enrichment (which consistently increased tissue‐N). Similarly, Peckol et al. (1996) found inconsistencies between sites (one of which was the same as in the previous study) in their study of the eVects of nitrogen enrichment on the same species. While natural phlorotannin levels were correlated negatively with tissue‐N content, Pavia and Toth (2000b) did not find any eVect of nutrient enrichment on phlorotannin content. Jormalainen et al. (2003) found that nutrient enhancement decreased phlorotannin production in F. vesiculosus, and Hemmi et al. (2004) found a decrease in the phlorotannin content of F. vesiculosus grown in water enriched with N‐P‐K fertiliser, an eVect that became significant between 10 and 38 days after the treatment began (Hemmi and Jormalainen, 2004; Hemmi et al., 2004). Investigations of other species have reported no eVect of nutrient enrichment on phenolic content in the fucoids Sargassum filipendula and Ascophyllum nodosum (Cronin and Hay, 1996a; Pavia and Brock, 2000; Pavia and Toth, 2000b) or the kelp Hedophyllum sessile (Pfister and Van Alstyne, 2003), whereas phlorotannins decreased in embryos of the fucoid Fucus gardneri (Van Alstyne and Pelletreau, 2000) and in the dictyotalean alga Lobophora variegata (Arnold et al., 1995). There was also a negative correlation between phlorotannins and nitrogen levels at their collection sites in the fucoids Turbinaria ornata and Sargassum mangarevense (Stiger et al., 2004). There is probably an influence of nutrients in the phenotypic modification of phlorotannin content, but as with other environmental factors, it is complex and poorly understood. c. Induction by herbivory. Although phlorotannins are probably eVective herbivore deterrents, the eVect is concentration dependant. Low constituent levels may not be enough to deter feeding, but an induction of phlorotannin

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production in response to herbivory will serve to reduce future herbivory, particularly from small, relatively immobile herbivores. Grazing over 2–4 weeks by the snail Littorina obtusata induced the production of phlorotannins in the fucoid Ascophyllum nodosum (Pavia and Brock, 2000; Pavia and Toth, 2000a) and this reduced grazing pressure (i.e., palatability) (Pavia and Toth, 2000a). Pavia and Toth (2000a) also noted that in natural populations of A. nodosum, individuals with more grazing marks had significantly higher phlorotannin levels than individuals with no apparent evidence of grazing. In the same study they demonstrated that the type of herbivore is important because grazing by the isopod Idotea granulosa did not produce an induction response. The authors suggest that these diVerent responses may be due in part to the diVering amounts of damage caused by the two herbivores, as the feeding mechanism of L. obtusata, which is also larger and less motile, may cause greater tissue losses and thus represent a stronger selective pressure. Toth et al. (2005) also reported that the phlorotannin content of A. nodosum increased after 2 weeks of grazing by L. obtusata, more so in the basal shoots than in the apical shoots. In an experiment that utilised snails (Theodoxus fluviatilis) to manipulate levels of fouling organisms on Fucus vesiculosus (Order Fucales), Honkanen and Jormalainen (2005) found that phlorotannin content was higher in algae from a low fouling treatment that included snails. They pointed out that the confounding factor of fouling level makes it diYcult to directly assess the impact of herbivore activity in this case, as a higher fouling level may also aVect phlorotannin production (e.g., by reducing available irradiance); in addition the snails are not known to consume F. vesiculosus and are thought to just feed on the epibiota, removing hyaline hairs (which are involved in nutrient uptake) in the process (Jormalainen et al., 2003). Whether responses to actual herbivory are an adaptive response to grazing or are due to other eVects of grazing (e.g., removal of epibiota or algal biomass, increase in nutrient absorption) that may aVect resource availability remain unclear in many cases (Jormalainen et al., 2003). Jormalainen et al. (2003) addressed this issue in Fucus vesiculosus through manipulations of the epibiota level and of the nutrient environment and also with simulated and actual herbivore presence. Their results suggest that physical contact with the herbivores is important (i.e., mere presence of herbivores but without contact had no eVect), but that this is species specific. Only one of their herbivore species (the snail Theodoxus fluviatilis) induced production of phlorotannins; the presence of another snail (Physa fontinalis) did not result in an increase in phlorotannin content. Neither of these species is thought to actually consume F. vesiculosus and the authors considered the eVect of

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T. fluviatilis to be related to the removal of hyaline hairs, which impacts nutrient assimilation, and not to a defence response. Phlorotannin content in the kelp Laminaria hyperborea decreased in response to grazing by a limpet (Ansates pellucida) and by a gastropod (Lacuna vincta), although this may have been an artefact of the artificial environment (Toth and Pavia, 2002a). In addition, we failed to find any eVect of grazing by two species of amphipods or one species of gastropod on the phlorotannin content of Desmarestia menziesii (Order Desmarestiales; V. A. Fairhead, C. D. Amsler, J. B. McClintock, and B. J. Baker, unpublished result). d. EVect of mechanical wounding. Mechanical wounding has been utilised frequently to test predications about the eVects of natural herbivory. Whilst it would be ideal to closely replicate the actual feeding process (Baldwin, 1990), this is often diYcult in practise and work in this area has been done by clipping, scraping with a blade, or wounding with a hole punch, which should not be assumed to simulate herbivory without testing for its validity (Pavia and Toth, 2000a). Additionally, mechanical wounding does not replicate other parts of the grazing process, such as water‐borne cues (Toth and Pavia, 2000b, see Section II.F.2.e) or, perhaps, specific forms of cell wall degradation (Potin et al., 2002). Nevertheless, several studies utilising diVerent species in both Fucales (Peckol et al., 1996; Van Alstyne, 1988; Yates and Peckol, 1993) and Laminariales (Hammerstrom et al., 1998; Lu¨ der and Clayton, 2004) have found an induction of phlorotannins in response to mechanical wounding within 3 to 5 days after wounding. However, an increase in phlorotannins in response to artificial wounding is not always observed (Hammerstrom et al., 1998; Hemmi et al., 2004; Pavia and Toth, 2000a; Pavia et al., 1997, 2003; Peckol et al., 1996; Steinberg, 1994; Yates and Peckol, 1993). Additionally, in a study of the eVects of mechanical wounding on Desmarestia menziesii, we found no increase in phlorotannin content after mechanical wounding (V. A. Fairhead, C. D. Amsler, J. B. McClintock, and B. J. Baker, unpublished result). Lu¨ der and Clayton (2004) suggested that this discrepancy of results may be partly a consequence of a dilution eVect when the total tissue phlorotannin content is measured, which masks accumulation in the outermost layers of the cell. e. Water‐borne cues. Recent studies have provided evidence for a potential role of water‐borne cues in the induction of phlorotannin production. Ascophyllum nodosum (Order Fucales) significantly increased tissue phlorotannin content when exposed, in a flow‐through system, to water from other aquaria containing snails (Littorina obtusata) grazing on other A. nodosum individuals (Toth and Pavia, 2000b). L. obtusata is a relatively sedentary

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species that feeds slowly so it probably has a restricted feeding range, and feeding on nearby individuals by L. obtusata may provide a reliable cue for upcoming herbivore attacks (Toth and Pavia, 2000b). Water‐borne cues may be particularly useful in an intertidal environment where physical damage is common and as such may not provide an adequate cue for herbivory (Pavia and Toth, 2000a). f. Methyl jasmonate. Arnold et al. (2001) assessed whether the plant growth regulator methyl jasmonate (MeJA), known to be important in vascular plant defence responses, is involved in phlorotannin production. Fucus vesiculosus individuals were incubated with diVering concentrations of airborne MeJA during the low tide period. Phenolic content in samples collected 12 days after incubation showed raised levels in MeJA‐treated individuals at a magnitude similar to that for a simulated grazing (clipping) treatment (Arnold et al., 2001). The authors suggested that further investigations of the role of the octadecanoid pathway are needed, but that MeJA could potentially be used to manipulate phlorotannin levels experimentally (Arnold et al., 2001). G. CURRENT UNCERTAINTY AND FUTURE DIRECTIONS

Perhaps the only conclusive statement one can make regarding phlorotannin distributions is that they are highly variable in space and time. Phenotypic plasticity in response to biotic (e.g., grazing pressure) and abiotic (e.g., nutrient and irradiance levels) factors has been well documented by field surveys and induction experiments, but these responses are by no means consistent, even within one species (e.g., Fucus vesiculosus). In addition, phenotypic responses of brown macroalgae contribute to a large variability of phlorotannin content in response to multiple stimuli, apparently in multiple ways. Genotypic variability may also be contributing to some of the within‐species variability reported (Honkanen and Jormalainen, 2005; Jormalainen and Honkanen, 2004), and the moderate to high rates of phlorotannin turnover within cells that has been reported (Arnold and Targett, 2000) could also influence this variability. This makes attempts to understand the ecological role of phlorotannins (e.g., are they constitutive or induced defences or can they be considered as a group to be herbivore deterrents) very diYcult. Complex interactions between putative production‐controlling factors mean that studies need to incorporate, or at least control for, multiple abiotic and biotic variables in study designs (e.g., Pavia and Brock, 2000; Peckol et al., 1996). Recent advances in our knowledge of physical and biological influences on phlorotannin production should allow

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future work to provide clearer outcomes, and should they be developed, genetic or enzymatic methods to monitor phlorotannin production in response to multiple, interactive stimuli would likely prove invaluable. It is no longer valid to, a priori, equate a feeding deterrent eVect in brown algae with an eVect of phlorotannins or visa versa, even in crude extracts that should be enriched in phlorotannins (e.g., Deal et al., 2003; Hemmi et al., 2004; Kubanek et al., 2004), and we also now know that the type of herbivore (e.g., gut type) influences the outcome of algal–herbivore interactions mediated by phlorotannins (Targett and Arnold, 1998, 2001). Future attempts to understand the ecological roles of phlorotannins need to incorporate these factors. Spatial variability must be studied in combination with assessments of induced defence (e.g., Taylor et al., 2002), as there is good evidence that these responses can aVect concentrations throughout the thallus over time in order to determine intrathallus variation patterns conclusively (Toth et al., 2005). Our current level of knowledge of the influence of environmental factors on the phenotypic variation of phlorotannins is limited, and we are a long way from having the ability to make predictive statements about environmental influences on phenolic content, if indeed these are appropriate. If the primary role of phlorotannins as cell wall constituents is indeed the driving force for phlorotannin production (Arnold and Targett, 2003), then eVorts to understand phlorotannin variability should be focused on the complexities of that process, as the utility for an alga of using phlorotannins as a defensive metabolite would be constrained by it (Pavia et al., 2003). Conversely, there is a large data base from numerous research laboratories correlating phlorotannin levels with defence against herbivores. We would not agree that, based on the few reports where other compounds have been identified as the actual antiherbivore component in crude extracts enriched in phlorotannins, one can assume that phlorotannins are not active as feeding deterrents in other populations or taxa even when past workers did not look for them with exhaustive, organic chemical methodology. However, we also strongly believe that more careful chemical analyses of the bioactive compounds should be performed in the future whenever knowing the specific chemical nature of feeding deterrents is critical in an experimental analysis. Correlations between feeding deterrence and colorimetrically determined phlorotannin levels should be assumed to be nothing more than that, i.e., as correlations, not causes. However, ecologically, such a correlation may be suYcient in many instances. Our own experiences make it clear to us that, although possible, purifying phlorotannins for use in chemical and biological assays without allowing them to become oxidised is far from being a trivial exercise. In addition, the organic chemistry

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technical skills and technology necessary to identify bioactive but nonphlorotannin metabolites within extracts are often not available to many ecologists. So in the end, the specific question being asked should determine the extent to which the involvement of phlorotannins in ecological interactions needs to be correlative vs definitive. The observations that increased levels of phlorotannins often but not always correlate with increased feeding deterrence demonstrate that one cannot assume that there is such a correlation in every species or situation. In our opinion, if the biological relevance of a study involves changes in palatability in addition to phlorotannin levels, one should always couple feeding bioassays on fresh thalli, on ground tissues, and/or on extracts with the phlorotannin content determinations. Unlike the potential technical diYculties presented to ecologists in determining if nonphlorotannin metabolites might be responsible for observed unpalatability, performing feeding bioassays themselves is usually a very practical option. Significantly, in many studies regarding predator–prey interactions in an ecological or evolutionary context, the response of the predator is likely more important than the particular attribute of the prey that caused it.

III. NONPHLOROTANNIN ANTIHERBIVORE DEFENCES A. DICTYOTALES

Dictyotalean algae produce a wide variety of terpenoids, acetogenins, and compounds of mixed biosynthetic origin (terpenoid‐aromatics) that, in contrast to phlorotannins, are commonly bioactive at concentrations of a few percent down to well under 1% algal dw (Hay and Fenical, 1988, 1992; Hay and Steinberg, 1992; Hay et al., 1987b; Paul et al., 2001). The most commonly studied brown algae producing nonphlorotannin secondary metabolites are species in the tropical and warm‐temperate genus Dictyota (Order Dictyotales), with well over 200 known secondary metabolites (Paul et al., 2001). Ecological roles for the bicyclic diterpene alcohol pachydictyol‐A (Fig. 6) have been especially well documented, particularly as antiherbivore defences against fish as well as some sea urchins and amphipods (e.g., Hay and Fenical, 1992; Hay and Steinberg, 1992; Hay et al., 1987a,b, 1988a; Paul, 1992; Pereira et al., 1994), although it is not bioactive against some other herbivores (e.g., Hay and Fenical, 1992; Hay and Steinberg, 1992; Hay et al., 1987a; Paul, 1992; Paul and Van Alstyne, 1988; Pereira et al., 2000a). Dictyota spp. and other members of the Dictyotales also produce a wide

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Fig. 6. Chemical structures of defensive secondary metabolites from Order Dictyotales.

variety of related compounds with known antiherbivore bioactivity such as dictyol B, dictyol B acetate, dictyol E, dictyol H, acutiol A, dolobelladiene 1 (10,18‐diacetoxy‐8‐hydroxy‐2,6‐dolobelladiene), and (6R)‐6‐hydroxydichotoma‐3,14‐diene‐1,17‐dial (Figs. 6 and 7); as with pachydictyol‐A, these have often been shown to be active against some herbivores but not others (e.g., Barbosa et al., 2004; Cronin et al., 1997; Fleury et al., 1994; Hay and Fenical, 1992; Hay et al., 1987a,b, 1988a; Hay and Steinberg, 1992; Pereira

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Fig. 7. Chemical structures of defensive secondary metabolites from Order Dictyotales.

et al., 2000a). Cruz‐Rivera and Hay (2003) demonstrated that at least some of these compounds (a mixture of pachydictyol‐A and dictyol‐E) interact with food quality and were more deterrent to five species of amphipods (but not to one species of isopod) when presented in relatively lower quality

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artificial foods. Although not consistently so, this compound mix also decreased amphipod fitness with the negative eVects greater when the animals were maintained on a lower quality diet (Cruz‐Rivera and Hay, 2003). Based on studies utilising crude extracts that should have contained dictyols, these compounds might also play a role in host choice by amphipods (Pereira et al., 2000b). Vallim et al. (2005) reviewed diterpenes in the Ditcyotaceae from a chemotaxonomic perspective focusing on Dictyota and the most closely related genera in the family. While terpenoids, acetogenins, and related mixed biosynthetic compounds continue to be discovered from the Dictyotales (e.g., Ali et al., 2004; Soto et al., 2003), much recent attention has turned to manipulative or otherwise more detailed investigations into their functions as well as to the identification of other types of compounds with roles in antiherbivore defence of dictyotalean algae. With respect to other classes of bioactive compounds, some of the attention has been focused on sulfated and nonsulfated C11 compounds produced by Dictyopteris spp., which appear to have particularly strong eVects on amphipod herbivory (e.g., Hay et al., 1988b). Amphipods and other mesoherbivores are becoming recognised as potentially having very important roles as macroalgal predators in some communities (e.g., Arrontes, 1999; Brawley, 1992; DuVy and Hay, 2000; Peters, 2003) so defences that appear to be specific to mesoherbivores are of particular interest. Schnitzler et al. (1998) demonstrated that the sulfated compounds 3‐hexyl‐[1,2]dithiepan‐5‐one (a cyclic dithiepanone; Fig. 7) and three related, noncyclic metabolites, including thioacetic acid S‐(3‐oxoundecyl)‐ester (Fig. 7), were strong deterrents of feeding by a sympatric, herbivorous amphipod (Ampithoe longimana) but not a herbivorous sea urchin (Arbacia punctulata). Later, this same group compared palatability to Ampithoe longimana of Dictyopteris membranacea, which produces these sulfated C11 compounds, with D. hoytii, a species that does not produce them as well as with a strain of D. membranacea from culture that had lost the ability to synthesise these compounds (Schnitzler et al., 2001). D. hoytii was strongly preferred over D. membranacea when presented as fresh thallus, dried and ground thallus prepared into artificial foods, or as lipophilic or hydrophilic extracts incorporated into artificial foods. The nonsulfated‐C11‐producing strain of D. membranacea was also preferred over the field‐collected algae that produce the C11 compounds when dried and ground thallus was presented in artificial foods. In the same study, the authors showed that a maintenance diet of artificial foods supplemented with 3‐hexyl‐[1,2]dithiepan‐5‐one and 9‐oxo acid (Fig. 7), a nonsulfated compound likely to be a by‐product of sulfated C11 compound synthesis, both strongly decreased survivorship of amphipods (Schnitzler et al., 2001).

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These sulfated C11 compounds are very similar to a group of compounds released as pheromones by female gametes in a wide variety of brown algae (Amsler and Iken, 2001; Pohnert and Boland, 2002; see Section VII.A). Hay et al. (1998) showed that the pheromone produced by Dictyota, dictyotene (Fig. 9), and six of nine degradation products of dictyotene [most found naturally in Dictyopteris spp.; to our knowledge, the pheromone(s) produced by Dictyopteris spp. female gametes has not been reported] inhibited feeding by the Ampithoe longimana but only 2 of these 10 significantly decreased feeding by Arbacia punctulata. As Hay et al. (1998) discussed, the potential for brown algal pheromones and their degradation products to also function as chemical defences against mesoherbivores is intriguing. Very little information is available on biotic interactions of macroalgal germlings (Amsler et al., 1992) but it is very likely that animals such as micro‐ and mesoherbivores that commonly feed on microalgae would be particularly important predators on recently settled macroalgal propagules. Consequently, assuming some cost to their production, it would be of obvious adaptive benefit to the algae if these compounds and their biosynthetic pathways functioned not only in male gamete attraction, but also in protecting the settled female gametes and, potentially, the resulting zygotes and germlings from predation. These C11 metabolites are not just produced by female gametes but also, as in Dictyopteris, by macroscopic stages of the algae as well as by some microalgae and vascular plants (Boland, 1995; Pohnert and Boland, 2002) so there is no reason to suspect that their production need cease after syngamy (even though in many cases production does appear to cease; Maier and Mu¨ ller, 1986) or that they could not be produced by other life history stages. If they are of adaptive value as antiherbivore defences in settled gametes and zygotes, however, one would predict that they would be equally valuable in defending settled meiospores, asexual spores, and their germlings. We are aware of no such reports of such compounds being produced by these other microscopic life history stages, although this could very likely be because no one has looked for them there. However, it is noteworthy that these pheromones can be quite odoriferous and detectable by human olfaction in cultures of sexually reproducing brown algae (Mu¨ller, 1967, 1989). It would be somewhat surprising if they had gone undetected by phycologists in cultures producing meiospores or asexual spores if they are released at comparable levels to the pheromones from cultures where gametes are being produced. Nevertheless, this is an aspect of brown algal chemical ecology that warrants further attention. Another potentially defensive chemical compound that has recently received attention in the Dictyotales is sulfuric acid in high concentrations. As noted later (Section III.B), this had been documented previously in the

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Desmarestiales, where there is evidence that it does serve a defensive role. Sasaki et al. (1999) reported that three Dictyopteris spp. and Spatoglossum crassum concentrated SO42 with cellular pH levels estimated at 0.5 to 0.9. These authors speculated that the acids might function as antiherbivore defences as in the Desmarestiales, but no ecologically relevant assays were conducted. Sasaki et al. (2004) surveyed 61 brown algal species from 10 orders and extended the known, high acid Dictyotales to 7 of a total 27 species surveyed. Two species from other orders also contained high concentrations of SO42 but the ions were balanced by Ca2þ rather than Hþ so the cells were not highly acidic. It would not be at all surprising if the high pH levels found is these dictyotalean algae had ecologically relevant roles similar to those in the Desmarestiales, but this remains to be determined empirically. Likely as a result of their well‐known terpenoid, acetogenin, and mixed biosynthetic defensive chemistry and their widespread geographic occurrence in tropical and warm temperate seas, members of the Dictyotales have been important models in experimental studies of macroalgal chemical ecology. This includes tests of chemical defence theories as detailed in Section IV, but they have also served as models for other important experimental questions. For example, it has long been thought that grazing pressure is higher in the tropics than at temperate latitudes (e.g., Gaines and Lubchenco, 1982; Vermeij, 1978) and that this has resulted in higher levels of chemical defences in tropical macroalgae (e.g., Fenical, 1980; reviewed by Van Alstyne et al., 2001a). However, this has rarely been experimentally tested. Bolser and Hay (1996) compared the palatability to tropical and temperate sea urchins of congeneric tropical (Bahamas) and temperate (North Carolina) macroalgae from four dictyotalean genera (Dictyopteris, Dictyota, Lobophora, and Padina) and Sargassum (Order Fucales) as well as one red and one green algal genus. When presented as dried thallus powders in artificial foods, the temperate species were, in general, preferred significantly by both tropical and temperate sea urchins with the exception of Dictyota. Assuming that these specific Dictyota spp. are an exception and the tropical vs. temperate pattern is indeed generalisable, one would predict that this would select for increased tolerance to defensive metabolites by tropical herbivores because they are confronted with more heavily defended prey. Cronin et al. (1997) tested that prediction using diterpenoid secondary metabolites from the tropical species Dictyota acutiloba and reported that acutilol A (Fig. 6) and two closely related diterpenoids were indeed more strongly deterrent to temperate than tropical macroherbivores (fish and sea urchins). (See also Section II.E for a discussion of hypotheses about latitudinal variations in phlorotannin concentrations and of tests thereof.)

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Within the North Carolina populations studied by Bolser and Hay (1996), Dictyota spp. from the two regions were equivalent in palatability, but oVshore populations, particularly of D. menstrualis, were usually significantly less palatable than inshore populations, which was reflected in comparisons of palatability between North Carolina and Bahamas species. Using the amphipod Ampithoe longimana as the bioassay animal, Taylor et al. (2003) reported that the increased unpalatability of the North Carolina oVshore populations of D. menstrualis was due primarily to the production of 4
‐hydroxydictyodial A and 18,O‐diohydro‐4
‐hydroxy‐dictyodial A 18‐acetate (Fig. 7) in the oVshore but not inshore populations. The diVerence between populations was not because of diVerences in pactydictyol A or dictyol E, which, as noted earlier, are deterrent to sea urchins and fish but often not to amphipods. Amphipods raised on oVshore populations had decreased growth, reproduction, and survivorship compared to animals raised on inshore algae. These authors also reported that oVshore populations of Sargassum filipendula (Order Fucales) and Spatoglossum schroederi were less palatable to the amphipods than inshore populations. In addition, Pereira et al. (2004) reported that crude extracts of Stypopodium zonale from two widely separated sites in Brazil deterred herbivory by crabs (Pachygrapsus transverses) and sea urchins (Lytechinus variegatus). However, extracts from one site (Bu´ zios) were significantly more deterrent than from the other (Fernando de Noronha), which was correlated with a diVerence in the major defensive metabolites present in the two populations. The major compound produced by the Bu´ zios population was the meroditerpene atomaric acid (Fig. 8), whereas at Fernando de Noronha it was the meroditerpene stypoldione (Fig. 8). Atomaric acid was strongly bioactive against both crabs and sea urchins when presented at the natural concentration found within the Bu´ zios algae, whereas stypoldione was less active as a deterrent for sea urchins and not significantly deterrent to the crabs when presented at the natural concentration from the Fernando de Noronha population. The selective factors that might be responsible for the intraspecific diVerences documented in these studies are unclear, but nevertheless these results are important because they show that there can be marked qualitative diVerences in defensive attributes within individual macroalgal species, even when collected from geographically close but distinct populations. Dictyotalean algae have also been very important models for examination of other aspects of macroalgal–herbivore relationships. For example, although Ampithoe longimana is often used as a bioassay animal for studies of dictyotalean chemical defences along the southeastern United States coast and preferentially feeds on nondefended macroalgae if oVered in the laboratory (e.g., Cruz‐Rivera and Hay, 2003), in nature it actually prefers to live

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Fig. 8. Chemical structures of defensive secondary metabolites from Orders Desmarestiales, Fucales, and Dictyotales.

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and feed on Dictyota menstrualis in this region (DuVy and Hay, 1994). This appears to be an adaptation that reduces predation on the amphipod by omnivorous fish that eat both macroalgae and amphipods but are chemically deterred by D. menstrualis (DuVy and Hay, 1991, 1994). The distribution of A. longimana continues much further north along the U.S. coast than D. menstrualis, and Sotka and Hay (2002) reported that allopatric A. longimana from Connecticut were less resistant to the defensive chemistry from D. menstrualis and also had lower growth rates and fitness than sympatric amphipods when raised on D. menstrualis (and Dictyota ciliolata) in the laboratory. Furthermore, these diVerences were based genetically, as they persisted through two generations in culture. Sotka et al. (2003) expanded upon and collaborated these conclusions by examining this relationship across three sympatric and six allopatric populations of A. longimana. When amphipods from northern and southern populations were crossed experimentally (Sotka, 2003), the hybrid oVspring were intermediate between the parental strains in terms of feeding preference for D. menstrualis, further confirming the genetic basis of resistance to the algal chemical defences. B. DESMARESTIALES

That members of the genus Desmarestia can sequester very high levels of acid within their thalli has been known since at least the 1930s (Kylin, 1931; Miwa, 1931; Wirth and Rigg, 1937), although early workers diVered on the specific acid responsible and it was not until the 1950s that this was resolved to be sulfuric acid across the genus (Eppley and Bovell, 1958; Meuse, 1956). Vacuolar pH within cells is commonly estimated as below 1.0 with H2SO4 comprising 12 to 25% of the thalli by dry weight (Anderson and Velimirov, 1982; Eppley and Bovell, 1958; McClintock et al., 1982; Miwa, 1931; Pelletreau and Muller‐Parker, 2002; Sasaki et al., 1999, 2004). Although previous authors (e.g., Anderson and Velimirov, 1982; Dayton, 1985b) had proposed that the acidity could be a defence against herbivores, it is surprising that an explicit test of this was not published until Pelletreau and Muller‐Parker (2002) demonstrated that sulfuric acid added to artificial foods at levels comparable to those found in Desmarestia munda could explain the unpalatability of thalli of that species to sea urchins. Marine tunicates also sequester sulfuric acid at high concentrations and although its potential defensive role has been somewhat controversial, using techniques similar to Pelletreau and Muller‐Parker (2002), it has been shown that the acid can also serve as an ecologically relevant defence against potential predators of tunicates (McClintock et al., 2004; Pisut and Pawlik, 2002).

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Based on their consensus molecular phylogeny of the Family Desmarestiaceae, Peters et al. (1997) concluded that sequestration of high concentrations of H2SO4 is a derived characteristic in the family and arose only once. However, Desmarestia antarctica, which groups at the base of the family with other southern hemisphere species that are nonacidic, also produces acid (presumably H2SO4), albeit in much lower concentrations than other Desmarestia spp. (pH of cell slurries ¼ 4.5; Moe and Silva, 1989). D. antarctica thalli are very unpalatable to sympatric algal consumers (amphipods, fish, sea stars) and crude extracts (pH neutralised) of D. antarctica are also deterrent to them, but only modestly so (Amsler et al., 2005a). Amsler et al. (2005a) proposed that the strong observed unpalatability of the fresh thalli is likely a combination of deterrent eVects of the modest secondary metabolite deterrence and the weak acidity. Perennial, nonacidic members of the Desmarestiales, particularly Desmarestia anceps, D. menziesii, and Himantothallus grandifolius, dominate shallow water, hard‐bottom communities along the western Antarctic Peninsula, often covering the vast majority of the bottom with biomass levels comparable to temperate kelp forests (Amsler et al., 1995; Brouwer et al., 1995; Neushul, 1965; Wiencke and Clayton, 2002). These nonacidic species are strongly unpalatable as thallus to sympatric sea stars, fish, and amphipods (Amsler et al., 2005a). Although some of this unpalatability could be due to relatively high levels of phlorotannins (Fairhead et al., 2005a), more lipophilic compounds not present in phlorotannin‐enriched extracts were responsible for at least some of the unpalatability in D. menziesii and H. grandifolius as well as in Phaeurus antarcticus (K. Iken, C. D. Amsler, J. B. McClintock, and B. J. Baker, unpublished result; Amsler et al., 2005a). The only specific metabolite identified to date with ecologically relevant bioactivity is menzoquinone (Fig. 8), a hydroquinone with feeding deterrent bioactivity against sea stars (Ankisetty et al., 2004) but not amphipods (C. D. Amsler, unpublished). C. OTHER ORDERS

A majority of chemical defence studies of other brown algae have focused on the potential roles of phlorotannins, as described in Section II.D. Although there are over 280 nonphlorotannin secondary metabolites known from other orders in the Phaeophyceae (Harper et al., 2001), relatively few studies have targeted ecological roles for specific compounds. Deal et al. (2003) reported that the chemical defence of Fucus vesiculosus (Order Fucales) from North Carolina and Maine (USA) against the sea urchin Arbacia punctulata was due to a polar galactolipid (Fig. 8) and one or

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more hydrophilic, nonphenolic compounds rather than to phlorotannins. The latter compound or compounds were unstable and could not be identified. Kubanek et al. (2004) similarly localised the chemically deterrent bioactivity against A. punctulata and two amphipod species in F. vesiculosus from North Carolina and Connecticut to unstable but clearly nonphlorotannin compounds. Sargassum spp. (Order Fucales) are commonly, although not always, unpalatable to sympatric herbivores (e.g., Hay, 1986) and produce significant levels of phlorotannins (e.g., Arnold and Targett, 2000; Stiger et al., 2004), but Cronin and Hay (1996a) reported that the palatability of S. filipendula was not correlated with experimentally manipulated phlorotannin contents. The Antarctic fucoid Cystosphaera jacquinotii is unpalatable to sympatric sea stars, amphipods, and fish (Amsler et al., 2005a) and although phlorotannins could be responsible for all or part of this unpalatability with amphipods and fish, the sea stars were deterred by relatively lipophilic extracts and not by those enriched in phlorotannins (K. Iken, C. D. Amsler, J. B. McClintock, and B. J. Baker, unpublished result; Amsler et al., 2005a). Similarly, Adenocystis utricularis (Order Chordariales) was chemically defended against sympatric Antarctic sea stars and amphipods, but phlorotannin‐enriched extracts were not deterrent (K. Iken, C. D. Amsler, J. B. McClintock, and B. J. Baker, unpublished result; Amsler et al., 2005a). Although potentially challenging technically if chemical instability proves to be common, understanding the potential roles of nonphlorotannin secondary metabolites in chemical defence of brown algae outside the Dictyotales remains an important area for future studies, particularly in the light of recent work (Deal et al., 2003; Kubanek et al., 2004) highlighting the importance of nonphlorotannin defences even in taxa that elaborate high concentrations of phlorotannins. D. ACTIVATED DEFENCES

Activated chemical defences are those that are produced constitutively but maintained in a biologically inactive form until wounding inflicted by a herbivore or other cause cues an enzymatic conversion of this precursor into a bioactive form. Such defences are fundamentally distinct from the induced defences described in Section IV.B because the alga (or other organism) has already made an investment of resources in the defensive compound(s). However, the defensive compounds are stored in a less biologically active form that presumably is less toxic to the producing alga or otherwise less costly to maintain. Activated defences in algae were first identified in green macroalgae (Paul and Van Alstyne, 1992) and have since been identified in a number of other macroalgae and microalgae

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(e.g., Jung et al., 2002; Pohnert, 2002; Steinke et al., 2002; Van Alstyne and Houser, 2003). However, to our knowledge the only report of an attempt to identify them in brown algae is that of Cetrulo and Hay (2000) who surveyed for induced responses in 42 macroalgal species, including 17 brown algal species (11 from Order Dictyotales, 4 Fucales, 2 Scytosiphonales, and 1 Ectocarpales). They reported that extracts from three Dictyota spp. (Order Dictyotales) and Scytosiphon lomentaria (Order Scytosiphonales) that had been severely wounded before extraction were significantly less palatable than extracts of unwounded thalli to sea urchins, fish, or both. These data suggest that defence activation had occurred due to the wounding, although the possibility that the results were caused by loss of some palatable component from the wounded algae cannot be ruled out completely. To our knowledge, compounds that might have been activated have not been identified from these algae, and identification of specific activated defences would, obviously, discount the possibility that the observed changes in palatability were caused by loss of feeding stimulants.

IV. TESTING CHEMICAL DEFENCE THEORIES WITH BROWN ALGAE Almost all chemical defence theories that have been applied to or tested with macroalgae are derived from the terrestrial plant literature. This should not be surprising considering the much greater overall level of research eVort that has been focused on terrestrial organisms. However, the application of these ideas to algae is certainly appropriate considering that macroalgae are analogous to terrestrial plants in being autotrophic and in being unable to behaviourally fight or flee a predator. Consequently, macroalgae provide analogous but phylogenetically distinct models with which to test these ideas about chemical defence and, potentially, to expand upon them in ways not possible with terrestrial organisms. That is particularly true of brown algae because of the much greater phylogenetic separation between them and terrestrial plants compared to red or green macroalgae (Section I); in fact, a majority of studies concerning chemical defence theory in macroalgae have utilised brown algae as models, particularly with respect to the optimal defence theory (ODT) and the induced defence model (IDM). Many of these studies have focused in whole or in part on phlorotannins, which, as noted earlier (Section II.E) display a wide range of variability on multiple scales. For the purposes of this section, in those cases we will follow the original authors in presuming that phlorotannins were indeed the defensive compounds even though in many cases that may not have been

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demonstrated definitively. It would be wise to consider all such studies carefully in the light of currently evolving ideas about the primary and secondary roles of phlorotannins and their eVectiveness as chemical defences (Sections II.E and III.C), particularly where chemical assays were not coupled with feeding bioassays testing palatability of the algal thallus or extracts thereof or where an energetic trade‐oV between allocations to defence versus growth was assumed. All chemical defence theories assume some cost to the production, maintenance, and/or storage of secondary metabolite defences but also assume that such costs are oVset by benefits to the fitness of an organism. Although accurate measurements of total costs of chemical defences can be diYcult, the biosynthesis of secondary metabolites clearly generates significant costs in terrestrial plants (reviewed by Heil, 2002; Purrington, 2000; Stamp, 2003). In brown algae, several authors have quantified costs of phlorotannin production in situ (see Section II.F.1). However, Arnold and Targett (2003) have argued that conclusions about costs from such studies are flawed because they do not consider the primary roles of phlorotannins [but see Koivikko et al. (2005) for evidence that the proportion of phlorotannins devoted to primary roles may be relatively small compared to secondary roles]. With respect to benefits, utilising terrestrial plant models, Mauricio and Rausher (1997), Agrawal (1998), and Baldwin (1998) directly demonstrated cost–benefit relationships and/or increases in fitness with respect to allocation of resources to chemical defence, and similar results continue to be reported in other terrestrial plants (e.g., Glawe et al., 2003). Corresponding studies of the benefits of defence in brown algae have primarily been correlative in linking investment in chemical defences with decreased consumption by or decreased fecundity in herbivores (e.g., Borell et al., 2004; Fairhead et al., 2005b; Toth et al., 2005). An important goal for future research on brown and other macroalgae is the direct demonstration of a fitness benefit to the algae as a consequence of the production of chemical defences. A. OPTIMAL DEFENCE THEORY

The optimal defence theory (Rhoades, 1979) examines within‐organism variations in defensive chemistry in the context of the competing relationship between growth and the production of chemical defences. ODT was developed in the context of the common and often marked diVerences observed in defensive compound allocations to various organs and tissues in terrestrial plants (Denno and McClure, 1983; McKey, 1974, 1979). Assuming that there is some cost to the production of defensive compounds, ODT predicts

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that defences should be directly correlated with risk of attack and inversely correlated with the cost of a particular defence. Furthermore, the theory predicts that within an organism, defences should be diVerentially allocated to those tissues or structures most valuable in terms of fitness and that there should be a correlation between energetic investment and defence in specific tissues. Most tests of ODT predictions in macroalgae have used brown algae as models and most but not all of these studies have provided support for the theory. In a general sense, the portion of a macroalgal thallus most exposed to herbivore attack is the epidermis (or meristoderm as appropriate), as these outer layers are the first thing the predator would encounter. Tugwell and Branch (1989) tested several predictions of ODT in three species of kelps, including the hypothesis that phlorotannins would be most concentrated in the meristoderm, which was indeed the case. The meristoderm layers in Ecklonia maxima and Laminaria pallida had phlorotannin concentrations manyfold higher than cortical and medullary layers, and the concentrations in Macrocystis angustifolia meristoderm layers made up a majority of the total found in the thalli. However, these authors only looked at the distribution of phlorotannins and did not bioassay the thallus layers or extracts thereof to test the hypothesis that the increased phlorotannin concentrations in fact resulted in greater unpalatability. Nevertheless the observation of higher phlorotannin levels in the meristoderm is very consistent with many studies on a wide variety of brown algae that, while not framed in the context of defence theories, have reported higher densities of physodes in epidermal layers (reviewed by Schoenwaelder, 2002; see Section II.A.2). Reproductive tissues are of apparently obvious importance in terms of fitness and, because of relatively dense cytoplasm, might also be expected to often be relatively high in energetic investment. In both senses, the ODT would predict that they have relatively high levels of defence. Kelps (Order Laminariales) are excellent models for testing predictions about resource allocation to reproductive tissues because they typically produce meiospores in relatively large, discrete sori that are often borne on specialised blades (sporophylls). In a seminal paper, Steinberg (1984) demonstrated that sporophylls in Alaria marginata had much higher phlorotannin levels than vegetative blades and were significantly less palatable to herbivorous snails in both laboratory and field assays. Neither tissue nitrogen nor thallus toughness was correlated with the herbivore preference, strongly supporting the hypothesis that the diVerence in palatability was due to chemical defences and in line with ODT predictions. Tugwell and Branch (1989) also examined phlorotannin concentrations in kelp reproductive tissues and reported higher concentrations in sporophylls of Ecklonia maxima and

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Macrocystis angustifolia compared to vegetative tissues but this pattern did not hold for fertile sori in Laminaria pallida, which had very low phlorotannin concentrations. As noted earlier, however, these authors did not assay the eYcacy of the phlorotannins as antipredator defences. Van Alstyne et al. (1999a) measured phlorotannin contents in Alaria marginata from several diVerent sites and reported higher levels in sporophylls from some but not all when compared to vegetative blades (although not compared to the vegetative meristems) but this was not true in A. nana from two sites where there was no significant diVerence in phlorotannin concentrations between vegetative blades and sporophylls. Pfister (1992), however, did report higher phlorotannin levels in sporophylls than in vegetative blades of A. nana from one of the same sites reported on by Van Alstyne et al. (1999a). In both studies, overall phlorotannin levels were relatively low and the extent to which the reported diVerences could be due to intraannual or seasonal variation is unknown. Van Alstyne et al. (1999a) also compared phlorotannin levels between vegetative blades and reproductive tissues (receptacles) in four members of Order Fucales and reported that, in apparent contrast to ODT predictions, they were significantly lower in the receptacles of Fucus gardneri and Pelvetia compressa and not significantly diVerent between these tissues in Fucus spiralis or Pelvetiopsis limitata. Although palatability was not measured, higher phlorotannin contents had been correlated previously with reduced palatability in F. gardneri as (F. distichus, Van Alstyne, 1988). Similarly, Tumoi et al. (1989) reported that phlorotannin levels were lower in receptacles of Fucus vesiculosus than in vegetative tissues (although the receptacles were physically tougher than vegetative tissues). However, this chemical diVerence is only a contradiction if indeed the reproductive tissues are more important in terms of overall fitness. Although as mentioned earlier that makes a great deal of sense intuitively, it may not always be true. As Pavia et al. (2002) have noted, the life history characteristics of the algae need to be considered since, for example, loss of reproductive tissues may be much more detrimental in a short‐lived annual species compared to a long‐ lived perennial. Using the relatively long‐lived fucoid Ascophyllum nodosum as a model, these authors applied demographic elasticity (proportional sensitivity) analysis of population–projection matrices in an attempt to objectively estimate relative fitness values of diVerent components of the thalli. This analysis predicted that fitness values, and therefore by ODT prediction, defences, should be highest in the stipes, intermediate in the annual shoots (which contain the meristems), and lowest in the receptacles. Indeed, both palatability of fresh tissue to the herbivorous snail Littorina obtusata and phlorotannin contents followed these predictions with stipes

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being significantly less palatable and higher in phlorotannins than annual shoots, which were significantly less palatable and higher in phlorotannins than the receptacles. The pattern of fitness value and defensive levels within the Ascophyllum nodosum (Pavia et al., 2002) does make sense intuitively when one considers not only the life history characteristics but also the morphology of the species. Even though meristems and receptacles are obviously important, in an alga like A. nodosum with a single primary axis, damage to that axis (the stipe) that caused or increased the chance of catastrophic breakage would mean the loss of all thallus components above that point, including multiple apical shoots and receptacles. Although based on a subjective rather than objective analysis of fitness value, we have compared within‐ thallus variation chemical and physical defences in two large, nonacidic, perennial members of the Desmarestiales with similar but not identical levels of morphological complexity as a test of ODT predictions (Fairhead et al., 2005b). In terms of thallus construction, although approximately an order of magnitude larger in overall size, Desmarestia anceps is similar to A. nodosum in typically having a single primary axis (stipe) that supports lateral branches containing most of the photosynthetic and reproductive tissues. We reasoned that because breakage of the stipe would result in loss of a large amount of the lateral tissues, on a per unit volume (or biomass) basis, ODT would predict higher levels of defence in the stipes, which indeed was the case with both chemical and physical defences (chemical defences measured as palatability of crude extracts to the sympatric, herbivorous amphipod Gondogeneia antarctica). In contrast, the sympatric and ecologically similar congener Desmarestia menziesii typically has multiple main axes supporting lateral branches, less morphological distinction between main axes and laterals, and fewer orders of branching overall. In D. menziesii there was no diVerence in palatability of crude extracts of main axes and laterals, although the main axes were physically tougher. We also included holdfasts in our analyses, which also diVered morphologically between the species. In D. anceps, the holdfast is composed of multiple, intertwined haptera that are capable of regrowth. Although damage to a holdfast that resulted in an alga becoming dislodged from the substrate is obviously the most extreme form of damage possible for a large macroalga, we reasoned that mesograzer damage to any individual part of the D. anceps holdfast would be less severe in terms of the chances for catastrophic breakage than damage to the primary stipe. This was both because of the multihaptera construction of the holdfast and because small damage to the main stipe, which must withstand much greater physical bending due to wave surge, is more likely to result in catastrophic breakage (cf. Duggins et al., 2001; Koehl and

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Wainwright, 1977; Utter and Denny, 1996) and, based on that assumption, the observations that the holdfast was less chemically defended (although not significantly diVerent in terms of toughness) compared to the primary stipe was consistent with ODT predictions. Conversely, in D. menziesii the holdfast is a single, small discoid structure rather than a mat of individual haptera. Coupled with the smaller diVerence between primary and lateral axes, we predicted that the holdfast tissue would be at least comparable to the main axes in terms of fitness value and indeed they were both significantly tougher than the lateral branches, although all three components were equivalent in terms of chemical defences. It is also of note that although these ODT‐predictable diVerences were evident in crude extract feeding bioassays utilising an important, sympatric herbivore, they were not explained by the marked but much less consistent intrathallus variation in the levels of phlorotannins (Fairhead et al., 2005a). Also based on a subjective rather than objective analysis of fitness value, Taylor et al. (2002) made a very similar prediction about the relative fitness values of thallus components in Sargassum filipendula, reasoning that lower portions of the thallus were most important because they supported the upper, meristematic, and reproductive branches. They reported that these lower portions of the stipes were most unpalatable to a sympatric herbivorous amphipod but that this unpalatability was the result of physical toughness rather than of chemical defences. Several other studies have compared defensive investment in brown algal vegetative tissues in terms of ODT predictions, particularly with respect to meristematic vs. older or more developed sections of the vegetative thallus. Tugwell and Branch (1989) reported that in three kelps, phlorotannin levels in meristems, holdfasts, and stipes were higher than in the blades. Van Alstyne et al. (1999a) reported higher phlorotannin levels in meristems compared to blades in eight kelp species. An additional seven kelp species and four fucoids did not have significant diVerences in phlorotannin levels between the two tissue types, although in the seven kelps, overall phlorotannin levels were usually much lower than in the species displaying a diVerence (Van Alstyne et al., 1999a). As noted previously, neither of these studies directly examined palatability of the specific tissues to herbivores, but Johnson and Mann (1986) did correlate much higher phlorotannin levels in meristems compared to blades in the kelp Laminaria longicuris with palatability to a herbivorous snail. The stipe, holdfast, and midrib were also relatively unpalatable but this appeared to be correlated with physical toughness rather than chemical defence (Johnson and Mann, 1986). Although the chemical nature of deterrence can only be inferred from the presence and concentration of physodes, Poore (1994) documented a

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visually striking example of diVerential levels of defence among meristems, newly formed cells, and older cells in Zonaria angustata (Order Dictyotales). Z. angustata grows from a row of meristemsatic apical cells that are darkly colored because of a high density of physodes (Figs. 2 and 3). Newly formed cells have few physodes and are very lightly colored, and as cells age (correlated with their distance back from the apical cell row), they accumulate physodes (Fig. 3). Palatability of these cell zones to two species of herbivorous amphipods was strongly negatively correlated with the density of physodes and not related to diVerences in physical toughness (Poore, 1994). It is of note that this species also covers its antheridia (male reproductive cells) with a sterile cell layer rich in physodes, which have been suggested to play a role in herbivore deterrence (Phillips and Clayton, 1991). Although the high level of apparent chemical defence in the apical cell row (and over the antheridia) would seem in line with ODT predictions, the fact that the amphipods could rip through the apical cell row and consume the new, palatable cell zone behind it, thereby leading to the loss of entire apical cell rows (Fig. 2), indicates that defending only the apical cells is ineVective in the larger sense, at least with respect to the two amphipod species used in these experiments. Nevertheless, as Poore (1994) discussed, the very great diVerences in palatability of thallus components separated by only a few cells highlight the fact that particularly with small mesograzers, such as amphipods, chemical ecologists may need to consider the distribution and eYcacy of chemical defences over very fine spatial scales within a macroalgal thallus. Cronin and Hay (1996b) tested the ODT prediction that younger, meristemsatic tissues should be more highly defended than older tissues using another member of the Dictyotales, Dictyota ciliolata, and reported that ODT predictions were not met. The younger blade apices were significantly more palatable and less chemically defended than older portions of the thallus. Although some authors have contrasted this with Poore’s (1994) report on Z. angustata because the latter species had a defended meristem (apical cell row) at the branch tips, such a contrast is misleading as it ignores important morphological diVerences between the genera. Even though both are members of the Family Dictyotaceae, Dictyota spp. grow from a single apical cell at the tip of each branch rather than from an apical cell row. In both D. ciliolata and Z. angustata the young cell zone immediately behind the apical cell(s) was significantly more palatable than older tissues farther down the thallus. Cronin and Hay (1996b) speculated that one reason for the diVerence between this and tests in other orders of brown algae supporting predictions of the ODT is that Dictyota spp. lack a known mechanism to transport materials between cells within the thallus (even though closely

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related Dictyopteris menbranacea does have structures morphologically consistent with transport capacity in their midribs; Katsaros and Galatis, 1988). While a lack of translocation capability remains to be proven as an explanation for the diVerences between defences in meristematic and other nonsupportive vegetative tissues in Dictyota spp. (which do not have midribs), it points to an important feature of brown macroalgae that is sometimes (but not always, e.g., Hemmi et al., 2004; Van Alstyne, 1988) overlooked by chemical ecologists when interpreting within‐thallus patterns of chemical defence or in studies of the induction of chemical defence. Although it is commonly recognised that members of the Laminariales have conductive cells (trumpet hyphae) that allow translocation of photosynthate and other substances within the thallus (Graham and Wilcox, 2000; Lobban and Harrison, 1994), other orders of brown algae are likely to have analogous capabilities (Raven, 2003). Members of the Desmarestiales also contain trumpet hyphae (Fritsch, 1945; Raven, 2003) and although members of the Fucales do not have quite such obvious conductive structures, it has been known for over 120 years that there is analogous cytoplasmic continuity between their medullary cells (Hick, 1885; Moss, 1983) and analogous structures may also be present in the Ascoseriales and Scytosiphonales (Raven, 2003). Members of the Fucales are able to translocate inorganic nutrients and photosynthate via filamentous medullary cells within their thalli, although apparently only from older to younger tissues (Diouris and Floc’h, 1984; Floc’h, 1982). Consequently, there is at least a possibility that they could also transport defensive compounds or, with respect to defence induction (Section IV.B), inductive signal molecules within their thalli (cf. Ryan and Pearce, 1998). These features need to be considered more often when both designing and interpreting the results of chemical defence studies, and the potential for transport of defensive compounds and/or inductive signals should be specifically tested more frequently. B. INDUCED DEFENCE MODEL

The induced defence model (Harvell, 1990; Karban and Meyers, 1989) follows from the ODT prediction that defence production should be directly correlated with risk of attack. Assuming that chemical defences are indeed costly to an organism, if predation pressure is variable in space or time one mechanism by which these costs might be reduced is for the organism to produce defences only when under attack by a consumer. Conversely, ODT and IDM would predict constitutive defence production if predation pressure is consistent and heavy. However, temporal and spatial patterns of predation are critical because limiting the production of chemical defences

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to those times when an organism is under attack by a consumer would only be beneficial if the defences could be produced in time to have a significant impact on consumption by the predator. Hay (1996) proposed that inducible chemical defences in macroalgae and other benthic marine organisms would likely be most eVective if produced in response to mesograzers because they cause only partial damage to their prey, at least over short time intervals, and because they often feed on individual macroalgae for long enough periods for a chemical response to be produced. Nevertheless, the critical features of predation are its spatial and temporal scales if induced responses are to be eVective. Not all mesograzers feed on individual prey for long times or are limited to feeding within small spatial ranges (cf. Paul et al., 2001). Also, larger predators such as Antarctic sea stars sometimes prey on an individual organism for very long periods of time (cf. Amsler, 2001; Dayton et al., 1974). As a result, not all small consumers feed in ways that would be predicted by the IDM to select for induced defences, whereas some larger consumers do feed over temporal and spatial scales that would be predicted to select for induced defences in their prey. Induction of defence or ‘‘resistance’’ in brown algae, particularly with the Fucales, has been a relatively active area of research in the past few years and, until very recently (Ceh et al., 2005; Weidner et al., 2004), browns were the only macroalgal group known to have inducible defences against herbivory. Induced chemical defences in macroalgae were first documented in a landmark study by Van Alstyne (1988), which demonstrated that two weeks after simulated grazing (mechanical clipping) of Fucus gardneri (as F. distichus, Order Fucales), clipped thalli were significantly less palatable than controls to herbivorous snails and had significantly higher phlorotannin concentrations in and adjacent to the damaged areas. Within the Fucales, chemical defence induction, detected at least in part via a change in palatability to herbivores, has also been documented in Fucus vesiculosus (Hemmi et al., 2004; Rohde et al., 2004; Yates and Peckol, 1993), Ascophyllum nodosum (Borell et al., 2004; Pavia and Toth, 2000a; Toth and Pavia, 2000b; Toth et al., 2005), Sargassum filipendula (Sotka et al., 2002; Taylor et al., 2002), and Sargassum asperifolium (Ceh et al., 2005). Induction measured as increases in phlorotannins without concurrent measurements of palatability has also been reported (Pavia and Brock, 2000; Peckol et al., 1996). Very nonpolar extracts of Cystoseira myrica that had been subjected to amphipod grazing were significantly less palatable than extracts from untreated controls; however, although there was a trend for the same pattern in intact thalli, the diVerence was not statistically significant (Ceh et al., 2005). Pavia et al. (1997) were unable to detect decreased palatability or increased phlorotannin levels in Ascophyllum nodosum that was clipped

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mechanically in a design similar to that of Van Alstyne (1988). However, Pavia and Toth (2000a) documented an induction of increased phlorotannin levels and a concomitant decrease in palatability when A. nodosum was fed on by the snail Littorina obtusata but not by the isopod Idotea granulosa or when subjected to two diVerent methods of mechanical wounding even though the snails and amphipods removed equivalent algal biomass. This indicates that for at least some macroalgae, induction is not a general response to wounding but rather is cued specifically by specific herbivores (as is also known in terrestrial plants; cf. Karban and Baldwin, 1997). Potin et al. (2002) have hypothesised that the herbivore‐specific signal could be related to specific oligoalginates released during cell wall decomposition and/ or to alginate‐degrading enzymes of the herbivore (cf. Section V). Toth and Pavia (2000b) showed that the inductive signal was a waterborne compound that could induce defence production in ungrazed thalli receiving water from aquaria containing A. nodosum that were being grazed on by L. obtusata (but not by mechanically wounded A nodosum). This result was particularly exciting in the context of the growing terrestrial plant literature on airborne induction of chemical defences between individuals (e.g., Kessler and Baldwin, 2001). [For a more detailed microreview focused on Pavia and Toth (2000a) and Toth and Pavia (2000b), see Amsler (2001).] Particularly interesting in the context of diVusible chemical cues for defence induction identified by Toth and Pavia (2000b), Arnold et al. (2001) reported that a brief exposure to methyl jasmonate, a volatile compound involved with inter‐plant defence induction in terrestrial plants, could significantly stimulate phlorotannin synthesis in Fucus vesiculosus. Neither this compound nor its precursor is known from brown algae, and because the eVects of the increased phlorotannins on palatability to herbivores were not measured, the direct ecological relevance of this observation cannot be assessed. However, methyl jasmonate has been identified in red macroalgae (Bouarab et al., 2004), and these authors speculate that it may have been acquired from the plastid endosymbiont and, hence, potentially may be found in any photosynthetic eukaryote. Rohde et al. (2004) demonstrated that chemical defences (measured as unpalatability) in F. vesiculosus could be induced by direct grazing of either the snail Littorina littorea or the isopod Idotea baltica. Waterborne cues from I. baltica grazing could induce defences in neighboring F. vesiculosus, but this was not true of grazing by L. littorea. Defence levels returned to preinduction levels within 2 weeks. Similar to the work of Pavia and Toth (2000a) and Toth and Pavia (2000b), this demonstrates defence induction by waterborne cues and that the specific herbivore involved significantly aVects the specific algal response. However, there are important diVerences between results and interpretations in the two sets of

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studies. The two macroalgal species are in the same family (Fucaceae) and the snails and isopods are congeners. However, only the snails induced defences in Ascophyllum nodosum while both herbivores did with F. vesiculosus, and snail grazing resulted in waterborne cues with A. nodosum but not F. vesiculosus, even though isopod grazing did result in waterborne cues from F. vesiculosus. Pavia and Toth (2000a) hypothesised that diVerences between responses to the two herbivores by A. nodosum could be due to the snails being much less motile predators than the isopods. They reasoned that the isopods do not remain on an individual alga long enough for an induced response to be eVective. With respect to the responses of F. vesiculosus, Rohde et al. (2004) noted the same diVerences in relative mobility between the herbivores, but suggested that this reduced selection for a waterborne signal for the snails because a single animal remains on an individual alga for relatively long periods of time. The more mobile isopods, however, move more readily between neighboring algae, making an ability to sense and respond to grazed neighbors more adaptive. These are diVerent species at diVerent geographic locations, and these two sets of interpretations need not be mutually exclusive. However, the somewhat reversed reasoning applied in the two sets of circumstances does highlight the fact that we are far from truly understanding the mechanistic or selective relevance of the herbivore‐specific nature of induced defence responses. Pavia’s research group combined their studies of defence induction in Ascophyllum nodosum with their tests of ODT (Pavia et al., 2002; cf. Section IV.A) to extend ODT predictions on within‐thallus variation in constitutive levels of defensive allocation to within‐thallus variation in defence induction. Toth et al. (2005) reported that even though basal stipes, with a higher assigned fitness value, had significantly higher constitutive levels of phlorotannins than lower fitness value apical shoots, and even though both did significantly induce phlorotannin production when grazed by I. obtusata, phlorotannin induction was significantly greater in the more valuable basal stipes. Although feeding deterrence was not measured in this study to correlate with the phlorotannin levels, snails fed basal stipes had lower fecundity than those fed apical shoots, which was significantly more pronounced in snails fed previously grazed algal thalli, indicating that diVerences in within‐thallus defences and their induction in the macroalgae impacted herbivore fitness. Taylor et al. (2002) also combined tests of the ODT and IDM in a comparison of constitutive and induced responses to amphipod herbivory in the fucoid Sargassum filipendula. As noted earlier, the lower stipes, which were judged highest in fitness value, were constitutively defended via physical toughness rather than chemistry. However, in the intermediate valuable upper stipes, which contained the meristems,

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herbivory did induce chemical defence, whereas both upper and lower blades were defended neither physically nor chemically. The most valuable tissues were defended constitutively, the intermediate valuable tissues only when under herbivore attack, and the more readily replaceable tissues not at all, even though some of them are immediately adjacent to the induced upper stipes in the algal thallus. Sotka et al. (2002) confirmed the tissue‐specific nature of the induced response and showed that waterborne cues from neighboring macroalgae did not induce defences in ungrazed thalli. Hemmi et al. (2004) also employed an experimental design that allowed them to test for the spread of an induced response within macroalgal thalli. Mechanical clipping of the fucoid Fucus vesiculosus induced chemical defences measured as decreased palatability to the isopod Idotea baltica, but the induction was very localised and did not spread either laterally or vertically within individual thalli. In addition, the induced responses were relatively short‐lived, lasting more than 10 but less than 38 days. Hemmi et al. (2004) suggested that such a localised and short‐lived response may have little eVect on overall‐thallus defences but might serve to disperse grazing damage within a thallus, thereby decreasing the likelihood of thallus breakage. In these same experiments, nutrient levels were also manipulated and although they did aVect phlorotannin production (see Sections II.F.2.b and IV.C), the phlorotannin levels were not correlated with palatability to I. baltica. Although the experimental design used by these investigators might not have been able to detect the transport of a chemical inducer from younger to older tissues if such a signal was confined to the thallus side on which the wound occurred, it is significant that the design should have detected transport from older tissues towards the apical meristems, the direction in which translocation of photosynthate and ions have been reported in F. vesiculosus (Diouris and Floc’h, 1984; Floc’h, 1982). Borell et al. (2004) confirmed Pavia and Toth’s (2000a) observations on induction of Ascophyllum nodosum (Order Fucales) defences by Littorina obtusata, although they also observed a significant eVect of mechanical clipping on A. nodosum palatability in one of two experimental trials. They utilised this system as a test of the herbivore motility model proposed previously for interactions of terrestrial plants and their insect herbivores (Coleman et al., 1996; Edwards and Wratten, 1987). Consistent with this model, L. obtusata spent more time grazing on the less defended primary and annual shoots compared to the more defended stipes. Feeding damage on thalli with previously induced defences was more diVuse than in uninduced controls, the feeding scars were smaller in previously induced thalli, and total consumption was lower in previously induced thalli.

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Induction of chemical defences has also been examined in Dictyotales, Laminariales, and Desmarestiales. In an intensive study combining field manipulations of amphipod grazing with chemical determinations and feeding bioassays of macroalgae from sites that naturally diVered in grazing pressure in some years but not others, Cronin and Hay (1996c) documented induction of chemical defences in Dictyota menstrualis (Order Dictyotales). Macroalgae from a site that 1 year had higher densities of amphipods than two other sites were less palatable and had higher levels of the defensive compounds dictyol E, pachyodictyol A (Fig. 6), and dictyodial (Fig. 8; cf. Section III.A). In a second year, the amphipod densities at these three sites did not diVer and neither did the palatability nor defensive compound concentrations of the D. menstrualis from them. In a third year, there were diVerences between sites but they were smaller than the first year and although there were small but significantly higher levels of the compounds at the site with higher amphipod densities, there was no significant diVerence in palatability of the macroalgal thalli. Within the individual sites, thalli that had amphipod grazing scars were less palatable and had higher levels of defensive compounds. The induction of these responses was confirmed with manipulative studies placing algae in cages with or without amphipods in the field. In other dictyotalean algae, Peckol and Yates (1997) documented increases in phlorotannin contents after simulated grazing in Lobophora variegata and Padina sanctae‐crucis, although potential changes in palatability were not assayed. Weidner et al. (2004) reported an induction of chemical defences in L. variegata after amphipod grazing measured as decreased palatability in extracts prepared using only a very nonpolar solvent. However, this unpalatability was not observed in intact thallus, indicating that multiple changes had occurred, which on balance did not decrease thallus palatability. Macaya et al. (2005) reported a similar result with Glossophora kunthii (Order Dictyotales), where artificial foods containing nonpolar extracts of algae previously grazed by amphipods were deterrent compared to foods containing extracts from control algae, but there was no diVerence between the palatability of the corresponding fresh thalli. However, they went on to demonstrate that defences eVective in intact thalli were restricted to the apical (meristematic area) and basal (stipe and holdfast) regions of the thalli and suggested that the lack of an induction response in intact thalli was probably because the amphipods simply shifted feeding from the defended apical and basal regions to the undefended tissues in between (Macaya et al., 2005). In addition, these authors reported that the induction response seen in extract containing artificial foods could be elicited both by the mere presence

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of amphipods in water upstream of the thalli or by the presence of amphipods grazing on conspecific algae upstream of the thalli. This indicates that the presence of waterborne cues is involved in stimulating the induced response and suggests that the cues come from the amphipods alone and not from algae in the process of being grazed. In all cases, the diVerence in palatability between extracts of induced and control algae disappeared within 12 days of removing the amphipod grazers (Macaya et al., 2005). Rotha¨ usler et al. (2005) reported similar results using the same amphipod species, but defences were not induced by another amphipod species or by sea urchins (although the experimental design as described apparently did not include autogenic controls; cf. Peterson and Renaud, 1989). In Laminariales, induction of phlorotannins in response to simulated grazing has been reported in Agarum fimbriatum, Hedophyllum sessile, Laminaria complanata, L. groenlandica, and Pleurophycus gardneri (Hammerstrom et al., 1998) and in Ecklonia radiata (Lu¨ der and Clayton, 2004), although palatability changes were not assayed in either study. Toth and Pavia (2002a) tested for phlorotannin induction in Laminaria hyperborea in response to grazing by limpets and snails and observed a decrease in phlorotannins after grazing, which they suggested could have been due to a loss of physode‐rich epidermal layers or to compensatory thallus growth. Macaya et al. (2005) reported no induction of defences in Macrocystis integrifolia in experiments performed in parallel with those described earlier using Glossophora kunthii. Rotha¨ usler et al. (2005) reported decreased palatability of previously grazed Lessonia nigrescens compared to controls in one amphipod species but not in another amphipod or sea urchin species using bioassays apparently lacking autogenic controls. The only study we are aware of looking for defence induction in the Desmarestiales is our own group’s work. We tested for changes in palatability or phlorotannin concentrations in Desmarestia menziesii after grazing by two species of amphipods, a snail species, and mechanical wounding, and we observed no eVect of any of these treatments (V. A. Fairhead, C. D. Amsler, J. B. McClintock, and B. J. Baker, unpublished results). C. CARBON‐NUTRIENT BALANCE AND RESOURCE ALLOCATION

The carbon‐nutrient balance hypothesis (CNBH) predicts that because they have a relative excess of fixed carbon, nitrogen‐limited plants should produce compounds such as phenolics and terpenes that do not contain nitrogen as defensive secondary metabolites and that they should produce more of these compounds as nitrogen limitation increases relative to light (and, therefore, carbon fixation) availability (Bryant et al., 1983). Conversely, the

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CNBH predicts that light‐limited (i.e., carbon‐limited) plants should utilise nitrogen‐containing defences such as alkaloids and cyclic peptides, which are often eVective at much lower concentrations than phenolics or terpenes. The CNBH addresses intraspecific diVerences in defences on ecological timescales, whereas the closely related resource allocation model (RAM) makes similar predictions about defensive compound variation between species over evolutionary timescales (Coley et al., 1985). RAM also predicts that species that evolved in growth‐limiting environments should produce relatively high levels of defensive metabolites because replacing tissue lost to herbivory should be more costly than in more growth‐permissive habitats. A number of investigations of brown macroalgae have tested predictions of the CNBH but almost no studies have attempted to extend RAM predictions to brown or other macroalgae. The most common aspect of CNBH tested with brown macroalgae has been the prediction that nutrient limitation would increase the concentration of phenolics or terpenes (or, conversely, that nutrient enhancement would result in decreased levels). This has usually proven true with phlorotannins, including in studies of the Fucales (Hemmi et al., 2004; Jormalainen et al., 2003; Pavia and Toth, 2000b; Pavia et al., 1999b; Peckol et al., 1996; Tuomi et al., 1989; Van Alstyne and Pelletreau, 2000; Yates and Peckol, 1993), the Laminariales (Hurd et al., 2000), and the Dictyotales (Arnold et al., 1995). However, this has not always been true for phlorotannins in some species of the Dictyotales (Peckol and Yates, 1997), Laminariales (Pfister and Van Alstyne, 2003), or Fucales (Cronin and Hay, 1996a; Pavia and Brock, 2000; Pavia and Toth, 2000b), although increased irradiance did increase phlorotannin concentrations in Sargassum filipendula (Order Fucales; Cronin and Hay, 1996a). The CNBH prediction of a link between nutrients and defences did not hold for the one study testing it with terpenes in the Dictyotales (Cronin and Hay, 1996a) (it also did not predict terpene concentrations in red macroalgae; Puglisi and Paul, 1997). In a unique study, Jormalainen et al. (2003) attributed phlorotannin increases in Fucus vesiculosus in response to grazing by the snail Theodoxus fluviatilis to the removal of hyaline hairs that are likely involved in nutrient uptake, as these were the only parts of the macroalgal thallus consumed. Most of the studies of CNBH predictions of phlorotannin levels did not also examine the influence of them on palatability, so it is particularly significant that two investigations that did incorporate palatability assays reported that increased phlorotannin levels caused by the manipulation of nutrients or light did not result in decreases in palatability (Cronin and Hay, 1996a; Hemmi et al., 2004). Overall, as Cronin (2001) has discussed, the CNBH predicts responses of macroalgae in some instances but not others. In addition, the CNBH has

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recently come under attack in the terrestrial plant literature. Hamilton et al. (2001) reviewed a large number of terrestrial tests of the CNBH and concluded that although it sometimes correctly predicts plant responses to the environment, it is incorrect so often that in their opinion the CNBH is of no use as a predictive model and should be abandoned. This conclusion has been echoed by Koricheva (2002) and Nitao et al. (2002) but refuted by Lerdau and Coley (2002). The number of reports testing CNBH predictions in macroalgae is too low for such a definitive conclusion. However, the mixed results to date in macroalgae, coupled with the much larger terrestrial data base, suggest that the CNBH should be applied with great caution, if at all, as a predictive model for macroalgal responses to the environment. As Cronin (2001) has discussed, the RAM has rarely if ever been tested explicitly with macroalgae, probably because the conditions necessary to do so are usually lacking in marine communities. Based on gross generalisations of extremes in the variation of algal growth rates across taxa and of defensive metabolites across latitudes, Cronin (2001) concluded that the overall evidence from a combination of previous studies (that did not specifically intend to address RAM) was not consistent with RAM predictions. Although that could serve as a useful starting point for directed investigations, the vast generalisations underlying the conclusion should make it nothing more than a starting point. With respect to one aspect of RAM not addressed by Cronin (2001), our research group has been able to test the prediction that carbon‐limited macroalgae should selectively produce nitrogen‐containing secondary metabolites, which are often bioactive at much lower concentrations than nonnitrogenous compounds and, therefore, require a lower total carbon investment. Macroalgae in general produce relatively few nitrogenous secondary metabolites (e.g., Harper et al., 2001; Paul et al., 2001). Several authors have suggested that this is because nitrogen is so commonly growth limiting for macroalgae in the ocean that there has been little selection across broad spatial or temporal scales for an investment of nitrogen in defences (Cronin, 2001; Hay and Fenical, 1988; Hay and Steinberg, 1992). However, one place in the world where this is not true is in coastal waters of Antarctica, where nitrogen and other nutrients rarely if ever limit macroalgal growth and where most macroalgae have very low C:N ratios (Dunton, 2001; Peters et al., 2005; Weykam et al., 1996). We tested the RAM prediction that Antarctic macroalgae should commonly produce nitrogenous secondary metabolites using an extraction and chemical staining protocol specifically targeting such compounds in 24 macroalgal species, including eight ecologically dominant brown algal species (Amsler et al., 2005). Coupling this with standard extractions and a literature search, we found no evidence at all of nitrogenous compounds (Amsler et al., 2005). Although this does not mean that

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nitrogen‐containing defences do not exist in Antarctic macroalgae, it does indicate that, in contradiction to RAM‐based predictions, they are likely no more common than in macroalgae from lower latitudes. D. TESTS OF MULTIPLE AND OTHER THEORIES

Although we have chosen to categorise tests of chemical defence theories in the preceding sections by individual theory, the selective factors presumably driving them in nature certainly do not work in isolation, and a number of authors have examined some of these ideas in combination. Studies combining tests of IDM and CNBH have been particularly common (Hemmi et al., 2004; Jormalainen et al., 2003; Pavia and Brock, 2000; Peckol and Yates, 1997; Peckol et al., 1996; Weidner et al., 2004; Yates and Peckol, 1993). IDM has also been explored in combination with UV radiation stress (cf. Section II.D.4; Macaya et al., 2005; Pavia and Brock, 2000; Pavia et al., 1997). Two theories that make somewhat conflicting predictions are the ODT and the growth‐diVerentiation balance hypothesis (GDBH). GDBH addresses diVerential allocation of defences during growth and subsequent diVerentiation, with the production of defences viewed as part of the process of diVerentiation (Herms and Mattson, 1992). One prediction of the GDBH is that newly produced cells, such as those at and near meristems, should be less defended than older, more diVerentiated cells further from meristems. This is opposite the predictions of ODT, as noted earlier in Section IV.A. Both Cronin and Hay (1996b) and Van Alstyne et al. (1999a) compared and contrasted these predictions (their results are detailed in Section IV.A) with the results of Van Alstyne et al. (1999a) not supporting GDBH in most respects, but the results of Cronin and Hay (1996b) being a better fit with GDBH than ODT. As also noted in Section IV.A, these latter authors suggested that the diVerence could be because they were working with algae from a genus (Dictyota) lacking a known internal translocation mechanism that might be used to transport defensive compounds into young tissues in other macroalgal taxa. There are several other chemical defence theories (cf. Cronin, 2001; Stamp, 2003), but to our knowledge, most have not been tested explicitly using brown algae. One exception is the prediction that stressed organisms would be able to devote fewer resources to defence and, therefore, be more palatable than unstressed individuals (Rhoades, 1985). Renaud et al. (1990) reported that desiccation resulted in the normally unpalatable alga Padina gymnospora (Order Dictyotales) becoming more palatable to sea urchins, although a normally palatable sympatric red alga examined in the same study became less so. Cronin and Hay (1996d) reported that desiccation of Dictyota ciliolata (Dictyotales) also reduced the levels of specific defensive

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metabolites (pachydictol A, dictyol B acetate, and dictyodiol; Figs. 6 and 8) and reduced feeding deterrence in bioassays using sea urchins and amphipods. In addition, UV stress decreased levels of the defensive metabolites, suggesting that palatability would have decreased too, but palatability assays were not performed on the UV‐treated algae (Cronin and Hay, 1996d). UV stress reportedly had no influence on the palatability of either Glossophora kunthii (Order Dictyotales) or Macrocystis integrifolia (Order Laminariales) (Macaya et al., 2005).

V. NONPHLOROTANNIN DEFENCES AGAINST BACTERIA, FOULING ORGANISMS, AND PATHOGENS There have been far fewer ecologically relevant reports of antiepiphytic‐ bacteria or antifouling chemical defences of brown algae (or of other macroalgae) compared to reports of antiherbivore defences, even though there have been many studies looking at the antibacterial or antifouling bioactivity of algal extracts. The reason for this discrepancy is that it is relatively diYcult to demonstrate that observed bioactivities against epiphytic bacteria or fouling organisms in vitro actually have ecological relevance. Many authors, ourselves included, have performed bioassays on extracts or purified compounds from marine organisms at concentrations somehow equivalent to those present in the intact organism, and some studies (e.g., da Gama et al., 2002; Henrikson and Pawlik, 1995) have outplanted gels containing such extracts into the sea to test their eVectiveness in preventing fouling. Those are valuable and important as first steps, but to make a strong case for ecological relevance one must also show that the compounds are present at the surface of the organism or in the boundary layer overlying that surface at bioactive concentrations (Steinberg and de Nys, 2002; Steinberg et al., 2001). Bhadury and Wright (2004) reviewed studies of in vitro antibacterial and antifouling activity in marine algae, including 25 brown algal species in the orders Ectocarpales, Scytosiphonales, Chordariales, Dictyotales, Fucales, Laminariales, and Desmarestiales. Other than to update this by listing several more recent reports on brown algae not included in that review (Amsler et al., 2005b; Freile‐Pelegrin and Morales, 2004; Hellio et al., 2004; Mare´ chal et al., 2004), with the exception of part of the discussion earlier concerning potential eVects of phlorotannins (Section II.D.3), this review concentrates on the few studies where chemical defences are very well supported in an ecological context. The only very clear case of nonphlorotannin brown algal secondary metabolites acting as antifoulants in nature that we are aware of is in

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Dictyota menstrualis (Order Dictyotales). Schmitt et al. (1995) reported that D. menstrualis was noticeably less fouled in nature than other macroalgae and that bryozoan larvae would not settle on D. menstrualis in laboratory bioassays even though they would contact its surface and would settle on several other species of brown and red macroalgae. Extracts of surface rubbings of the algal thallus contained pachydictyol A and dictyol E (Fig. 6; also know to be antiherbivore defences; Section III.A). These surface‐rubbing extracts could prevent fouling when coated onto a settlement substrate, although the rubbings were too small to allow the levels of the compounds to be quantified accurately. Dictyol E was toxic to the larvae when presented in solution, and although pachydictyol A in solution was not toxic, it caused abnormal and delayed development (Schmitt et al., 1995). Neither of the assays with compounds in solution is directly ecologically relevant, but this was the only way to approach the question experimentally, as the larvae would not contact a surface coated with the compounds. Because the compounds were detected at the thallus surface, the assays do demonstrate that there could be adverse consequences to the larvae if they settled on D. menstrualis. Schmitt et al. (1998) extended the examination of the eVects of metabolites in solution on larval development and survival to dictyol B acetate (Fig. 6) and dictyodiol (Fig. 8) and to the larvae of an additional bryozoan species, as well as a hydroid species. It is easier for in vitro assays of defences against potential pathogens to be made ecologically relevant because pathogens do invade the host tissues and often the host cells, so needing to know the actual surface concentrations of defensive compounds is not an impediment. Kubanek et al. (2003) reported that crude extracts of Lobophora variegata (Order Dictyotales) from the Bahamas have particularly strong antifungal bioactivity and identified the cyclic lactone lobophorolide (Fig. 8) as a defensive compound with significant bioactivity at exceptionally low concentrations against both pathogenic and saprophytic sympatric fungi. Although L. variegata from 46 of 51 samples collected at 10 sites in the Bahamas contained measurable, bioactive concentrations of lobophorolide, it was not present in two collections from the Red Sea even though crude extracts of the Red Sea algae did have antifungal bioactivity. This indicates that although L. variegata from diVerent regions may be chemically defended against pathogens, the same specific compounds may not be involved. Although the vast majority of the investigations into the defensive chemical ecology of macroalgae have focused on roles of secondary metabolites, recent work has demonstrated that both red and brown algae employ oxidative burst defences against pathogens, a defensive mechanism that has been recognised in terrestrial plants for a number of years (reviewed

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by Lamb and Dixon, 1997; Mahalingam and FedoroV, 2003; Wojtaszek, 1997). This was first discovered in red algae, where cell wall degradation products elicit a rapid release of H2O2 that is toxic to epiphytic bacteria (Weinberger et al., 1999, 2000, 2001). Ku¨pper et al. (2001) demonstrated an analogous response in Laminaria digitata (Order Laminariales) that produces a rapid oxidative burst of H2O2 and O2– in response to oligomeric degradation products of alginate (a major cell wall component of L. digitata and other brown algae). Ku¨ pper et al. (2001) also reported that H2O2 concentrations in the range released by the algae were toxic to pathogenic, alginate‐degrading bacteria and, using histological techniques, demonstrated that the response was confined primarily to the meristoderm. These authors also began to dissect the underlying physiological mechanisms and showed that at least some of the mechanisms are likely conserved within the oxidative burst responses of algae, terrestrial plants, and animals. Ku¨pper et al. (2002) surveyed 45 species of brown algae from 11 orders for constitutive vs. induced production of H2O2. They reported that oxidative bursts in response to alginate oligomers were common in sporophytes in Laminariales and Desmarestiales but not in their filamentous gametophyte stages. One species (Pylaiella littoralis) from the Ectocarpales had an oxidative burst response but 10 other ectocarpalean species did not. Members of the Fucales had high constitutive production of H2O2 but most did not produce additional H2O2 in response to alginate oligomers. Ku¨ pper et al. (2002) also reported that axenic sporophytes of Macrocystis pyrifera (Order Laminariales) were infected rapidly by pathogenic bacteria when the oxidative burst response was blocked with an NAD(P)H oxidase inhibitor but not in controls and that when non‐axenic M. pyrifera or L. digitata sporophytes were treated with the inhibitor, they were attacked rapidly by their natural bacterial flora. These authors also demonstrated that the oxidative burst response is important in resistance of both M. pyrifera and L. digitata to the pathogenic brown algal endophytes Laminariocolax tomentosoides and Laminariocolax macrocystis, although the response took 7 days to occur and likely involves elicitation of other structural or chemical defences (Ku¨ pper et al., 2002).

VI. VOLATILE HALOGENATED ORGANIC COMPOUNDS An additional response that might be defensive against pathogens, biofoulers, and/or herbivores is the release of volatile halogenated organic compounds

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(VHOCs), such as bromoform, dibromomethane, dibromochloromethane, bromodichloromethane, and chloroiodomethane. That brown algae release these compounds is clear (e.g., Gschwend et al., 1985; Laturnus, 1996, 2001; Laturnus et al., 1996; Manley et al., 1992), but to our knowledge, with the exception of one report on antiepiphyte activity in a coralline red alga (Ohsawa et al., 2001), there is no published evidence of any ecological role for them even though they have been thought to potentially be defensive (e.g., Borchardt et al., 2001; Fenical, 1975; Laturnus et al., 1996) and they have received significant attention because they impact atmospheric processes (e.g., Broadgate et al., 2004; Carpenter et al., 2000; Steinke et al., 2002). It has been suggested that VHOC production is related to the oxidative burst defence mechanism in algae (Abrahamsson et al., 2003; Potin et al., 1999, 2002), and the enzymes responsible for VHOC production (e.g., bromoperoxidases; cf. Colin et al., 2003, 2005) have been shown to be involved in cross‐linking phlorotannins in cell walls (Berglin et al., 2004), which could certainly be involved in responses to damage by grazers or pathogens. A deeper understanding of the ecological and physiological roles of VHOCs in brown and other macroalgae remains an important goal. Relatively high levels of VHOC release have been reported from brown algae in Antarctica (Laturnus, 1995, 2001; Laturnus et al., 1996, 1997). Our research group performed feeding bioassays with bromoform, the most abundant VHOC identified previously from Antarctic macroalgae (Laturnus, 1995; Laturnus et al., 1996) using a common, herbivorous Antarctic amphipod, Gondogeneia antarctica. We observed statistically significant decreases in feeding when bromoform was added to artificial foods at initial concentrations of 3 ppm and above (final concentrations in the foods were likely to have been lower but these were not measured; C. D. Amsler, B. J. Baker, and J. B. McClintock, unpublished result). This suggests that bromoform or other VHOCs could function as feeding deterrents if they were produced in such concentrations. However, we were unable to identify bromoform or a number of other VHOCs using gas chromatography from water samples taken in situ within dense beds of Antarctic brown algae or within pieces of intact or mechanically diced brown algal thalli (B. J. Baker, C. D. Amsler, and J. B. McClintock, unpublished result). The level of detection was orders of magnitude below the concentrations with bioactivity against amphipods (even accounting for loss during artificial food preparation) so we concluded that it was unlikely that VHOCs play a role as defences against amphipod herbivory in this system.

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VII. SENSORY CHEMICAL ECOLOGY Brown macroalgae reproduce primarily via motile spores and gametes, and the chemosensory abilities of some of these motile cells appear to be very important, even though nonmotile spores and female gametes are produced by some groups (Graham and Wilcox, 2000) and at least several species or populations reproduce and are dispersed via vegetative means (e.g., Amsler, 1984; Keum et al., 2003). Usually these motile cells are heterokont. Heterokont cells are biflagellate, possessing an anterior flagellum that bears mastigonemes (stiV hairs) that enable these forward‐projecting flagella to pull the cells through the water, and they have a trailing, smooth posterior flagellum (Cahill et al., 1996; Geller and Mu¨ller, 1981; Graham and Wilcox, 2000). Although the sensory chemical ecology of brown algae has received less research attention than brown algal chemical defences, recognition that sensory abilities are present in male gametes long predates studies of defensive chemical ecology and, more recently, limited attention has been focused on the chemosensory abilities of brown algal spores. A. CHEMOATTRACTION TO PHEROMONES

Motile brown algal male gametes (termed spermatozoids in oogamous species) from a wide variety of taxa locate female gametes via pheromones secreted by the female gametes. This behavioural response has been known since the independent work of Thuret and Pringsheim on Fucus (Order Fucales) in the 1850s and of Berhold and Oltmanns on Ectocarpus (Order Ectocarpales) and Kuckuck on Cutleria (Order Cutleriales) in the 1890s (reviewed by Fritsch, 1945; Maier, 1995; Maier and Mu¨ ller, 1986), although it was not until the 1960s that the specific chemicals responsible for the observed chemoattraction began to be identified (Mu¨ ller, 1967, 1968). At present, 12 specific pheromones (Fig. 9) are known to be released by eggs or settled, previously motile female gametes from over 60 species in 13 orders, with most species releasing more than one of the pheromones in addition to some of their biosynthetic precursors (Maier, 1995; Pohnert and Boland, 2002). The eVective distance at which male gametes could possibly perceive the pheromone signals has been estimated as between less than half a millimeter and one or a few millimeters (Boland et al., 1982, 1983; Maier and Mu¨ ller, 1986; Mu¨ ller, 1981), although that distance is likely to decrease with increasing water motion (Gordon and Brawley, 2004). A majority of the recent attention in this area has focused on the biosynthetic pathways and other biochemical aspects of pheromone production and reception (reviewed by Boland, 1995; Maier, 1995; Pohnert and Boland, 2002), and a

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Fig. 9.

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Chemical structures of brown algal pheromones.

number of partial and comprehensive reviews are available (in particular, Maier, 1995) that describe the chemosensory behaviours, methodology, and evolutionary relationships in great detail (see also Amsler and Iken, 2001; Maier, 1993; Maier and Mu¨ ller, 1986; Mu¨ ller, 1989). Consequently, here we present only a selective overview of pheromone‐related behaviours. In the Laminariales, Desmarestiales, and Sporochnales, pheromones act not only as chemoattractants, but also stimulate the release of male gametes from antheridia (Mu¨ ller et al., 1979, 1982, 1988). In Laminaria digitata (Order Laminariales), the threshold concentration of the pheromone lamoxirene

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Fig. 10. A dark‐field, microflash photomicrography sequence showing mass release and chemotaxis of Laminaria digitata spermatozoa in response to the pheromone lamoxirene. Lamoxirine adsorbed onto a porous glass (Spherosil) particle (*) was placed near a fertile clump of filamentous male gametophytes ( ) at time 0, and the response of spermatozoa at 5, 60, 120, and 180 s thereafter is shown. Small white dots are the dark‐field images of spermatozoa. From Maier (1982).

(Fig. 9), necessary to induce spermatozoid release, is below 1010 M (Maier et al., 1988). Spermatozoid release in response to the pheromone is very rapid (Fig. 10; Maier, 1982) and the gametes swim directly towards the pheromone source (Fig. 10; Maier, 1982; Maier and Mu¨ller, 1990). Because the cells orient their swimming such that they move directly towards the source, this is a true chemotactic behaviour (cf. Amsler and Iken, 2001). In addition to the direct chemotaxis, the spermatozoids exhibit a phobic, near – 1808 turn when the concentration of the lamoxirene is decreasing, resulting in a turn back to the general direction of the source (Maier and Mu¨ ller, 1990).

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In Fucus spiralis (Order Fucales), the spermatozoids appear to rely only on a near 1808 turn, phobic responses to decreasing concentrations of the pheromone, fucoserratene (Fig. 9), in order to remain in the vicinity of a released female gamete (Maier and Mu¨ller, 1986). Spermatozoids of the Australasian fucoid Hormosira banksii also display a phobic response to decreasing concentrations of the pheromone homosirene (Fig. 9), but in addition decrease their frequency of turning as homosirene concentration increases (at high concentrations their swimming pattern is superficially, but not mechanistically, similar to those of the damaged spores illustrated in Fig. 11E) (Maier et al., 1992). Chemoattractive responses such as these that rely on modulation of turning frequency and/or swimming speed are properly referred to as chemokinetic rather than chemotactic responses, with the latter being reserved for behaviours that involve movement directly towards or away from a stimulant source (Amsler and Iken, 2001; Dusenbery, 1992; Fraenkel and Gunn, 1961).

Fig. 11. Swimming patterns of Hincksia irregularis spores tracked by computer‐ assisted motion analysis: (A) straight path type characteristic of a majority of spores within 10–20 minutes after release; (B) search circle path type found in 30–40% of spores during the first 50 min after release; (C) orientation behaviour found in approximately 30% of spores between 40 and 60 min after release; (D) gyration behaviour that precedes settlement, first appearing after approximately 40 min and observed in 34% of spores by 60 min after release; and (E) wobbling behaviour observed occasionally (0–6%) in cells of any age and apparently caused by cell damage. From Iken et al. (2001).

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Perhaps the most thoroughly studied chemoattractive behaviour of brown algal gametes is the chemokinetic attraction of Ectocarpus siliculosus (Order Ectocarpales) to the pheromones ectocarpene (Fig. 9) and, as has been discovered recently (Pohnert and Boland, 2002), preectocarpene (Fig. 9). E. siliculosus is morphologically isogamous but functionally oogamous, as the female gamete settles and begins to secrete pheromones within 30 min of release from the gametangia (Mu¨ ller, 1989). Male gametes respond in four ways (Maier, 1995; Maier and Calenberg, 1994; Mu¨ ller, 1989). They already display a preference to remain close to solid surfaces (a thigmotactic response), which increases in the presence of pheromones. They also decrease their swimming speed (an orthokinetic response) and increase their rate of turning (a klinokinetic response) as pheromone concentrations increase. Mu¨ ller (1978) termed this behaviour ‘‘chemo‐thigmo‐klinokinesis.’’ In addition, the gametes exhibit a phobic response to decreasing pheromone concentrations similar to those described earlier in Laminaria and Fucus (Maier and Calenberg, 1994). The turns made by E. siliculosus gametes are initiated by sharp, sideways beats of their short, rudder‐like posterior flagella (Geller and Mu¨ ller, 1981). The posterior flagella probably play a similar role in other taxa, although they are often longer and probably also have a direct role in locomotion (Maier, 1995). However, a posterior flagellum is not absolutely required in male gametes, as demonstrated by members of the Dictyotales, which are able to respond to the pheromones dictyotene or multifidene (Fig. 9), but which produce only a single anterior, mastagoneme‐bearing flagellum (e.g., Phillips et al., 1990a,b). The observations that female gametes of most species produce multiple pheromones (even though their male gametes may not respond to all of them) and that adult, vegetative tissues sometimes produce pheromones have led to speculation that these pheromones could potentially have allelopathic roles. Mu¨ ller (1981) suggested that pheromones could potentially be used to ‘‘misguide’’ male gametes of a competitor, thereby preventing them from reaching conspecific female gametes and producing viable zygotes. Amsler et al. (1992) noted that observations of diVerential patterns of kelp recruitment following simultaneous spore settlement in nature (Reed, 1990) would, hypothetically at least, be consistent with a hypothesis that early maturing Pterygophora californica female gametophytes stimulate premature release of spermatozoa from Macrocystis pyrifera, thereby limiting the supply of M. pyrifera spermatozoa when M. pyrifera female gametophytes become mature. Boland et al. (1983) suggested six hypothetical scenarios by which a brown algal species might interfere with the pheromone‐mediated communication of a competitor. Their data on pheromone production and relative sensitivity in Ectocarpus siliculosus (Order Ectocarpales) and

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Cutleria multifida (Order Cutleriales) suggested that E. siliculosus should have a competitive advantage in nature. This surprised the authors (Boland et al., 1983), as it has long been presumed that the morphologically simple members of the Ectocarpales represent the most primitive members of the Phaeophyceae and they would have predicted that the more ‘‘advanced’’ species would have the competitive advantage. However, recent evidence has indicated that the Ectocarpales may in fact be among the most derived brown algae (Cho et al., 2004; Draisma et al., 2001) regardless of their simple morphology. Boland et al. (1983) also speculated that pheromones produced by vegetative tissues in Dictyopteris spp. (Order Dictyotales; cf. Section III.A) might interfere with reproduction in competitors or epiphytes, an idea echoed by Maier and Mu¨ ller (1986). B. BEHAVIOUR AND SENSORY CAPABILITIES OF SPORES

Considering the range of chemokinetic and chemotactic responses that brown algal gametes can exhibit in response to pheromones, it should not be surprising that other motile life history phases of brown algae can also sense and respond behaviourally to their chemical microenvironments. However, there has been much less attention focused on the behaviours of these other stages, which include both asexual spores produced by mitosis and spores produced by meiosis as part of a sexual life history (meiospores). Either class of spores can, when motile, be referred to as zoospores. To our knowledge, the only reports of chemosensory behaviours in brown algal zoospores are in members of the Laminariales and Ectocarpales. Chemosensory behaviours have been reported in meiospores from three species of kelps (Order Laminariales): Macrocystis pyrifera and Pterygophora californica from California (Amsler and Neushul, 1989, 1990) and Laminaria japonica from Japan (Fukuhara et al., 2002). Spores of all three species are attracted by nitrate, although M. pyrifera and P. californica are more sensitive to it as they were attracted by 10 M nitrate (Amsler and Neushul, 1989), whereas L. japonica spores did not respond to 11 M nitrate (but did to 200 M, the next highest concentration tested; Fukuhara et al., 2002). Based on manual video observations of P. californica swimming in a nitrate gradient (Amsler and Neushul, 1989), the chemoattractive behaviour in these species has been described as a true chemotactic response. However, a more detailed, quantitative behavioural analysis, such as those conducted by Amsler et al. (1999) and Iken et al. (2001), is needed to confirm this. M. pyrifera spores were not attracted by phosphate, but both P. californica and L. japonica spores were (Amsler and Neushul, 1989; Fukuhara et al., 2002). Again though, the L. japonica spores were much less sensitive than

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P. californica. L. japonica was attracted by 100 M phosphate but not by concentrations of 9.7 M and below, whereas P. californica was significantly attracted by 2 M phosphate (Amsler and Neushul, 1989; Fukuhara et al., 2002). In addition, both M. pyrifera and P. californica were attracted by ammonia (low concentrations) and boron while neither species responded to zinc. Manganese attracted M. pyrifera spores but repelled P. californica spores at the same concentration (18 M). Cobalt attracted M. pyrifera spores but had no aVect with those of P. californica. Glycine, aspartate, and iodine all significantly attracted M. pyrifera but were not tested with P. californica (Amsler and Neushul, 1989). Perhaps the most interesting responses were those of M. pyrifera spores to various concentrations of ammonia and ferrous iron. The spores were attracted by ammonia concentrations at and below 90 M and by 1 M Fe2þ but were repelled by 1000 M ammonia and by 45 M Fe2þ (Amsler and Neushul, 1989). The concentrations of ammonia that attracted the spores are likely to be stimulatory for growth and 1 M Fe2þ is stimulatory for reproduction in the gametophyte generation into which the spores germinate. Conversely, the higher concentrations are likely to be inhibitory for growth and reproduction, respectively (references in Amsler and Neushul, 1989). The gametophyte generation that the spores develop into is an obligate stage of the kelp life history (Graham and Wilcox, 2000). It is also microscopic and so must be able to grow to reproductive maturity in the microenvironment that the spore settles in. Although spores themselves presumably have no need for nutrients (because they do not grow or reproduce until they have germinated), chemotaxis towards nutrients when present in beneficial concentrations and away from them at detrimental concentrations is probably adaptive by increasing the chances that the gametophyte will develop in a permissive microenvironment for growth and gametogenesis. Kelp spores can also respond to nutrients by increasing their rate of settlement. Macrocystis pyrifera spore settlement rates are increased significantly by at least some concentrations of ammonia, nitrate, glycine, phosphate, manganese, boron, and both ferrous and ferric iron (Amsler and Neushul, 1990). Although the responses of Pterygophora californica spores to specific nutrients were not examined, their settlement was increased by a macro‐ and micronutrient mixture (Amsler and Neushul, 1990). Nutrient stimulation of spore settlement is probably like spore chemotaxis to nutrients in making it more likely that a spore will settle in a microenvironment conducive to gametophytic growth and reproduction. However, they appear to be mechanistically distinct. The settlement stimulation response is not present for at least several hours after spores are released even though newly released spores are chemotactic. There are also diVerences in the responses

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of M. pyrifera spores to specific nutrients, for example, to phosphate, which does not attract spores but does increase their settlement rates (Amsler and Neushul, 1989, 1990). Fukuhara et al. (2002) examined the eVects of various nitrate and phosphate concentrations on the number of swimming Laminaria japonica spores as a function of time. This could be, but is not necessarily, an indication of settlement rates. Although sample size, sample variance, or statistical analyses were not indicated in the paper for these data, there was a trend for fewer swimming spores over time in three phosphate concentrations between 3.0 and 9.7 M compared to three concentrations between 0.0 and 1.7 M, suggesting that phosphate may stimulate settlement (Fukuhara et al., 2002). There was no convincing concentration‐dependent pattern for spores swimming in various concentrations of nitrate. In the Ectocarpales, the behaviours of asexually produced spores from Ectocarpus siliculosus and Hincksia irregularis have been examined. E. siliculosus spores diVer from those of kelps in not being attracted to nutrients (either chemotactically or chemokinetically) and in not being stimulated to settle by nutrients (Amsler et al., 1999). However, it was only practical to work with E. siliculosus spores soon after release so the potential for them to develop a settlement stimulation response some hours after release, as is observed in kelp spores, could not be tested. E. siliculosus spores did settle diVerentially to substrates of diVerent surface energy (hydrophobicity or wetabiltiy) with fourfold higher rates of settlement on hydrophobic surfaces compared to either positively or negatively charged hydrophilic surfaces (Amsler et al., 1999). H. irregularis also settles at faster rates on hydrophobic surfaces (Greer and Amsler, 2002, 2004; Greer et al., 2003), as do spores of ulvoid green algae (e.g., Callow and Callow, 1998a,b, 2000; Ista et al., 2004). Hincksia irregularis and other members of the Ectocarpales are morphologically simple as adult thalli, but in contrast to long‐held assumptions, that does not necessarily mean that they are evolutionarily primitive compared to other brown algae with greater morphological complexity (Cho et al., 2004; Draisma et al., 2001). Regardless, studies have shown that the zoospores of H. irregularis are behaviourally complex and capable of integrating multiple chemical and other environmental stimuli in determining when or where to settle and germinate. Iken et al. (2001) documented both quantitative and qualitative changes in spore behaviour over time (Fig. 11) that are consistent with a hypothetical, developmental progression of sensory modes. The ‘‘gyration’’ behaviour (Fig. 11D) that often occurs immediately before final settlement and attachment has been reported previously in macroalgal spores (Fletcher and Callow, 1992), but the preceding progression of behaviours (Fig. 11A–C) and the relative synchrony of their development under tightly controlled laboratory conditions surprised even the study’s authors.

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Quantitative measures of the appearance of an apparently abnormal behaviour (Fig. 11E) have proven useful in identifying secondary metabolites from marine invertebrates with potential antifoulant capabilities against the spores (Greer, 2003; Iken et al., 2003). Furthermore, H. irregularis spores are capable of integrating their sensory responses to soluble chemicals and surface hydrophobicity (Greer et al., 2003), as well as their responses to surface hydrophobicity and gradients of light (Greer and Amsler, 2002, 2004). These are clearly not ‘‘simple’’ propagules of simple algae and, as with kelp spores, one should not be surprised if natural selection has shaped their behaviours. This is an area of brown algal sensory chemical ecology ripe for investigation in the near term, and recent progress on the biochemical interactions and sensory biology of green algal zoospores (e.g., Callow et al., 2002; Ista et al., 2004; Joint et al., 2002; Patel et al., 2003) points to progress that might be expected if the quantitative measures of the responses to chemical and other environmental stimuli that have been used with brown algal spores were integrated with other methodologies that have been pioneered with spores of green algae.

VIII. CONCLUDING REMARKS Although there are several ordinal‐level classification schemes for the Phaeophyceae, a widely used current text (Graham and Wilcox, 2000) includes 14 orders. In preparing this review, one thing that struck us is how often we have discussed members of the Fucales, Laminariales, and Dictyotales and how few times we have mentioned many of the others, particularly in the sections on defensive chemical ecology. In some cases this is because the other orders include relatively few species and/or species that are not particularly common, but in some that is not true (e.g., the Ectocarpales, which is widespread throughout the world’s oceans). What is true of most of the other groups (particularly the Ectocarpales) is that those that are widespread are usually not particularly large, and it should not be surprising that the larger and most common macroalgae in communities have received the most attention. Nevertheless, smaller does not mean less interesting or less important, as illustrated by the increased recent attention on mesoherbivores. Poore’s (1994) report illustrated that the distribution of chemical defences can vary within a distance of one to a few cells within an individual macroalga, and studies of chemical defences in some single‐celled microalgae (e.g., Pohnert et al., 2002; Steinke et al., 2002; Strom et al., 2003; Wichard et al., 2005) show that small algae can elaborate eVective chemical defences. Might it not be possible that diVerences in defensive investment analogous to that

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discussed in Sections II.E or IV.A could also occur in smaller macroalgae such as ectocarpoids? Also, if one scales down the idea of mesoherbivores being important in influencing defensive patterns in some larger macroalgae, might micrograzers be selecting analogous and, perhaps, novel or otherwise unexpected patterns of defence in small macroalgae? Less likely, perhaps, but until someone looks, these are open questions. Regardless, algal– herbivore interactions between filamentous or other small macroalgae and herbivores such as mesograzers could still be important in terms of macroalgal community structure, as Peters (2003) has hypothesised for communities along the western Antarctic Peninsula. The smaller brown algae have proven to be valuable models in studies of sensory chemical ecology, and although their size may impose technical limitations, they deserve experimental attention in studies of defensive chemical ecology. It is hoped that this review has been useful as a summary of how much important work has been done in the subdisciplines comprising brown algal chemical ecology as a whole. We have tried throughout to highlight some of the areas that we feel deserve attention in the coming years. However, those are, of course, only a small subset of the important questions yet to be asked.

ACKNOWLEDGMENTS We are grateful to numerous colleagues who provided preprints or other literature and to those who allowed us to use illustrations from their work. We are particularly grateful to Alistair Poore for sending scans of original illustrations. The manuscript benefited greatly from the comments of Margaret Amsler, James McClintock, and Robert Thacker. We also thank Majdouline LeRoy for preparing chemical structure figures. Manuscript preparation was supported in part by Grants OPP‐0125181 and OPP‐0442769 from the National Science Foundation.

REFERENCES Abrahamsson, K., Choo, K. S., Pedersen, 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. Agrawal, A. A. (1998). Induced responses to herbivory and increased plant performance. Science 279, 1201–1202. Ali, M. S., Pervez, M. K., Ahmed, F. and Saleema, M. (2004). Dichotenol‐A, B and C: The C‐16 oxidised seco‐dolastanes from the marine brown alga Dictyota dichotoma (Huds.) Lamour. Natural Product Research 18, 543–549.

72

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Amsler, C. D. (1984). Culture and field studies of Acinetospora crinita (Carmichael) Sauvageau (Ectocarpaceae, Phaeophyceae) in North Carolina (USA). Phycologia 23, 377–382. Amsler, C. D. and Neushul, M. (1989). Chemotactic eVects of nutrients on spores of the kelps Macrocystis pyrifera and Pterygophora californica. Marine Biology 102, 557–564. Amsler, C. D. and Neushul, M. (1990). Nutrient stimulation of spore settlement in the kelps Pterygophora californica and Macrocystis pyrifera. Marine Biology 107, 297–304. Amsler, C. D., Reed, D. C. and Neushul, M. (1992). The microclimate inhabited by macroalgal propagules. British Phycological Journal 27, 253–270. Amsler, C. D., Rowley, R. J., Laur, D. R., Quetin, L. B. and Ross, R. M. (1995). Vertical distribution of Antarctic Peninsular macroalgae: Cover, biomass, and species composition. Phycologia 34, 424–430. Amsler, C. D., Shelton, K. L., Britton, C. J., Spencer, N. Y. and Greer, G. P. (1999). Nutrients do not influence swimming behaviour or settlement rates of Ectocarpus siliculosus (Phaeophyceae) spores. Journal of Phycology 35, 239–244. Amsler, C. D. (2001). Induced defenses in macroalgae: The herbivore makes a diVerence. Journal of Phycology 37, 353–356. Amsler, C. D. and Iken, K. B. (2001). Chemokinesis and chemotaxis in marine bacteria and algae. In ‘‘Marine Chemical Ecology’’ (J. B. McClintock and B. J. Baker, eds.), pp. 413–430. CRC Press, Boca Raton, FL. Amsler, C. D., Iken, K., McClintock, J. B., Amsler, M. O., Peters, K. J., Hubbard, J. M., Furrow, F. B. and Baker, B. J. (2005a). Comprehensive evaluation of the palatability and chemical defenses of subtidal macroalgae from the Antarctic Peninsula. Marine Ecology Progress Series 294, 141–159. Amsler, C. D., Okogbue, I. N., Landry, D. M., Amsler, M. O., McClintock, J. B. and Baker, B. J. (2005b). Potential chemical defenses against diatom fouling in antarctic macroalgae. Botanica Marina 48, 318–322. Anderson, R. and Velimirov, B. (1982). An experimental investigation of the palatability of kelp bed algae to the sea urchin Parechinus angulosus Leske. PSZNI Marine Ecology 3, 357–373. Ankisetty, S., Nandiraju, S., Win, H., Park, Y. C., Amsler, C. D., McClintock, J. B., Baker, J. A., Diyabalanage, T. K., Pasaribu, A., Singh, M. P., Maiese, W. M., Walsh, R. D., Zaworotko, M. J. and Baker, B. J. (2004). Chemical investigation of predator‐deterred macroalgae from the Antarctic Peninsula. Journal of Natural Products 67, 1295–1302. Appel, H. M., Govenor, H. L., D’Ascenzo, M., Siska, E. and Schultz, J. C. (2001). Limitations of Folin assays of foliar phenolics in ecological studies. Journal of Chemical Ecology 27, 761–778. Arnold, T. M., Tanner, C. E. and Hatch, W. I. (1995). Phenotypic variation in polyphenolic content of the tropical brown alga Lobophora variegata as a function of nitrogen availability. Marine Ecology Progress Series 123, 177–183. Arnold, T. M. and Targett, N. M. (1998). Quantifying in situ rates of phlorotannin synthesis and polymerization in marine brown algae. Journal of Chemical Ecology 24, 577–595. Arnold, T. M. and Targett, N. M. (2000). Evidence for metabolic turnover of polyphenolics in tropical brown algae. Journal of Chemical Ecology 26, 1393–1410. 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.

DEFENSIVE AND SENSORY CHEMICAL ECOLOGY OF BROWN ALGAE

73

Arnold, T. M. and Targett, N. M. (2003). To grow and defend: Lack of tradeoVs for brown algal phlorotannins. Oikos 100, 406–408. 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. Arrontes, J. (1999). On the evolution of interactions between marine mesoherbivores and algae. Botanica Marina 42, 137–155. Baldauf, S. L. (2003). The deep roots of eukaryotes. Science 300, 1703–1706. Baldwin, I. T. (1990). Herbivory simulations in ecological research. Trends in Ecology and Evolution 5, 91–93. Baldwin, I. T. (1998). Jasmonate‐induced responses are costly but benefit plants under attack in native populations. Proceedings of the National Academy of Sciences of the United States of America 95, 8113–8118. Barbosa, J. P., Teixeira, V. L. and Pereira, R. C. (2004). A dolabellane diterpene from the brown alga Dictyota pfaYi as chemical defense against herbivores. Botanica Marina 47, 147–151. 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. Bhadury, P. and Wright, P. C. (2004). Exploitation of marine algae: Biogenic compounds for potential antifouling applications. Planta 219, 561–578. Boettcher, A. A. and Targett, N. M. (1993). Role of polyphenolic molecular size in reduction of assimilation eYciency in Xiphister mucosus. Ecology 74, 891–903. Boland, W. (1995). The chemistry of gamete attraction: Chemical structures, biosynthesis, and (a)biotic degradation of algal pheromones. Proceedings of the National Academy of Sciences of the United States of America 92, 37–43. Boland, W., Marner, F.‐J., Jaenicke, L., Mu¨ ller, D. G. and Folster, E. (1983). Comparative receptor study in gamete chemotaxis of the seaweeds Ectocarpus siliculosus and Cutleria multifida: An approach to interspecific communication of algal gametes. European Journal of Biochemistry 134, 97–103. Boland, W., Terlinden, R., Jaenicke, L. and Mu¨ ller, D. G. (1982). Binding‐mechanism and sensitivity in gamete chemotaxis of the phaeophyte Cutleria multifida. European Journal of Biochemistry 126, 173–179. Bolser, R. C. and Hay, M. E. (1996). Are tropical plants better defended? Palatability and defenses of temperate vs tropical seaweeds. Ecology 77, 2269–2286. 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 oxidised halogen antimicrobials. Applied and Environmental Microbiology 67, 3174–3179. Borell, E. M., Foggo, A. and Coleman, R. A. (2004). Induced resistance in intertidal macroalgae modifies feeding behaviour of herbivorous snails. Oecologia 140, 328–334. 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. Brawley, S. H. (1992). Mesoherbivores. In ‘‘Plant‐Animal Interactions in the Marine Benthos’’ (D. M. John, S. J. Hawkins and J. H. Price, eds.), pp. 235–263. Clarendon Press, Oxford. Broadgate, W. J., Malin, G., Kupper, F. C., Thompson, A. and Liss, P. S. (2004). Isoprene and other non‐methane hydrocarbons from seaweeds: A source of reactive hydrocarbons to the atmosphere. Marine Chemistry 88, 61–73.

74

C. D. AMSLER AND V. A. FAIRHEAD

Brouwer, P. E. M., Geilen, E. F. M., Gremmen, N. J. M. and van Lent, F. (1995). Biomass, cover and zonation pattern of sublittoral macroalgae at Signy Island, South Orkeny Islands, Antarctica. Botanica Marina 38, 259–270. Bryant, J. P., Chapin, F. S., III and Klein, D. R. (1983). Carbon/nutrient balance of boreal plants in relation to vertebrate herbivory. Oikos 40, 357–368. Cahill, D. M., Cope, M. and Hardham, A. R. (1996). Thrust reversal by tubular mastigonemes: Immunological evidence for a role of mastigonemes in forward motion of zoospores of Phytophthora cinnamomi. Protoplasma 194, 18–28. Callow, M. E. and Callow, J. A. (1998a). Attachment of zoospores of the fouling alga Enteromorpha in the presence of zosteric acid. Biofouling 13, 87–95. Callow, M. E. and Callow, J. A. (1998b). Enhanced adhesion and chemoattraction of zoospores of the fouling alga Enteromorpha to some foul‐release silicone elastomers. Biofouling 13, 157–172. Callow, M. E. and Callow, J. A. (2000). Substratum location and zoospore behaviour in the fouling alga Enteromorpha. Biofouling 15, 49–56. Callow, M. E., Jennings, A. R., Brennan, A. B., Seegert, C. E., Gibson, A., Wilson, L., Feinberg, A., Baney, R. and Callow, J. A. (2002). Microtopographic cues for settlement of zoospores of the green fouling alga Enteromorpha. Biofouling 18, 237–245. Carpenter, L. J., Malin, G., Liss, P. S. and Kupper, F. C. (2000). Novel biogenic iodine‐containing trihalomethanes and other short‐lived halocarbons in the coastal east Atlantic. Global Biogeochemical Cycles 14, 1191–1204. Ceh, J., Molis, M., Dzeha, T. M. and Wahl, M. (2005). Induction and reduction of anti‐herbivore defenses in brown and red macroalgae oV the Kenyan coast. Journal of Phycology 41, 726–731. Cetrulo, G. L. and Hay, M. E. (2000). Activated chemical defenses in tropical versus temperate seaweeds. Marine Ecology Progress Series 207, 243–253. Chen, Y., Yan, X. and Fan, X. (1997). A hypothesis on Phaeophyceae polyphenols: Their structural unit and mechanism of the formation (in Chinese with English summary). Oceanologia et Limnologia Sinica 28, 225–232. Cho, G. Y., Lee, S. H. and Boo, S. M. (2004). A new brown algal order, Ishigeales (Phaeophyceae), established on the basis of plastid protein‐coding rbcL, psaA, and psbA region comparisons. Journal of Phycology 40, 921–936. Clayton, M. N. and Ashburner, C. M. (1994). Secretion of phenolic bodies following fertilisation in Durvilaea potatorum (Durvilleales). European Journal of Phycology 29, 1–9. Coleman, R. A., Barker, A. M. and Fenner, M. (1996). Cabbage (Brassica oleracea var Capitata) fails to show wound‐induced defence against a specialist and a generalist herbivore? Oecologia 108, 105–112. Coley, P. D., Bryant, J. B. and Chapin, F. S., III (1985). Resource availability and plant antiherbivore defense. Science 230, 895–899. 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. 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. Connan, S., Goulard, F., Stiger, V., Deslandes, E. and Gall, E. A. (2004). Interspecific and temporal variation in phlorotannin levels in an assemblage of brown algae. Botanica Marina 47, 410–416.

DEFENSIVE AND SENSORY CHEMICAL ECOLOGY OF BROWN ALGAE

75

Cronin, G. (2001). Resource allocation in seaweeds and marine invertebrates. In ‘‘Marine Chemical Ecology’’ (J. B. McClintock and B. J. Baker, eds.), pp. 325–363. CRC Press, Boca Raton, FL. Cronin, G. and Hay, M. E. (1996a). EVects of light and nutrient availability on the growth, secondary chemistry, and resistance to herbivory of two brown seaweeds. Oikos 77, 93–106. Cronin, G. and Hay, M. E. (1996b). Within plant variation in seaweed palatability and chemical defenses: Optimal defense theory versus the growth diVerentiation balance hypothesis. Oecologia 105, 361–368. Cronin, G. and Hay, M. E. (1996c). Induction of seaweed chemical defenses by amphipod grazing. Ecology 77, 2287–2301. Cronin, G. and Hay, M. E. (1996d). Susceptibility to herbivores depends on recent history of both the plant and animal. Ecology 77, 1531–1543. Cronin, G., Paul, V. J., Hay, M. E. and Fenical, W. (1997). Are tropical herbivores more resistant than temperate herbivores to seaweed chemical defenses? Diterpenoid metabolites from Dictyota acutiloba as feeding deterrents for tropical versus temperate fishes and urchins. Journal of Chemical Ecology 23, 289–302. Cruz‐Rivera, E. and Hay, M. E. (2001). Macroalgal traits and the feeding and fitness of an herbivorous amphipod: The roles of selectivity, mixing, and compensation. Marine Ecology Progress Series 218, 249–266. Cruz‐Rivera, E. and Hay, M. E. (2003). Prey nutritional quality interacts with chemical defenses to aVect consumer feeding and fitness. Ecological Monographs 73, 483–506. da Gama, B. A. P., Pereira, R. C., Carvalho, A. G. V., Coutinho, R. and Yoneshigue‐ Valentin, Y. (2002). The eVects of seaweed secondary metabolites on biofouling. Biofouling 18, 13–20. Dayton, P. K. (1985a). Ecology of kelp communities. Annual Review of Ecology and Systematics 16, 215–245. Dayton, P. K. (1985b). The structure and regulation of some South American kelp communities. Ecological Monographs 55, 447–468. Dayton, P. K. (1990). Polar benthos. In ‘‘Polar Oceanography, Part B: Chemistry, Biology, and Geology’’ (W. O. Smith, Jr., ed.), pp. 631–685. Academic Press, New York. Dayton, P. K., Robilliard, G. A., Paine, R. T. and Dayton, L. B. (1974). Biological accommodation in the benthic community at McMurdo Sound, Antarctica. Ecological Monographs 44, 105–128. Deal, M. S., Hay, M. E., Wilson, D. and Fenical, W. (2003). Galactolipids rather than phlorotannins as herbivore deterrents in the brown seaweed Fucus vesiculosus. Oecologia 136, 107–114. Denno, R. F. and McClure, M. S. (1983). ‘‘Variable Plants and Herbivores in Natural and Managed Systems.’’ Academic Press, New York. Denton, A., Chapman, A. R. O. and Markham, J. (1990). Size‐specific concentrations of phlorotannins (anti‐herbivore compounds) in three species of Fucus. Marine Ecology Progress Series 65, 103–104. Diouris, M. and Floc’h, J. Y. (1984). Long‐distance transport of 14C‐labeled assimilates in the Fucales: Directionality, pathway and velocity. Botanica Marina 78, 199–204. Draisma, S. G. A., van Reine, W. F. P., Stam, W. T. and Olsen, J. L. (2001). A reassessment of phylogenetic relationships within the Phaeophyceae based on RUBISCO large subunit and ribosomal DNA sequences. Journal of Phycology 37, 586–603.

76

C. D. AMSLER AND V. A. FAIRHEAD

DuVy, J. E. and Hay, M. E. (1991). Food and shelter as determinants of food choice by an herbivorous marine amphipod. Ecology 72, 1286–1298. DuVy, J. E. and Hay, M. E. (1994). Herbivore resistance to seaweed chemical defense: The roles of mobility and predation risk. Ecology 75, 1304–1319. DuVy, J. E. and Hay, M. E. (2000). Strong impacts of grazing amphipods on the organisation of a benthic community. Ecological Monographs 70, 237–263. Duggins, D., Eckman, J. E., Siddon, C. E. and Klinger, T. (2001). Interactive roles of mesograzers and current flow in survival of kelps. Marine Ecology Progress Series 223, 143–155. Dunton, K. (2001). 15N and 13C measurements of Antarctic Peninsula fauna: Trophic relationships and assimilation of benthic seaweeds. American Zoologist 41, 99–112. Dusenbery, D. B. (1992). ‘‘Sensory Ecology.’’ Freeman, New York. Edwards, P. and Wratten, S. (1987). Ecological significance of wound‐induced changes in plant chemistry.In ‘‘Insects‐Plants. Proceedings of the 6th International Synposium on Insect‐Plant Relationships, Pau 1986’’ (V. Labeyrie, G. Farbes and D. Lachaise, eds.), pp. 213–218. Junk, Dordrecht. Eppley, R. W. and Bovell, C. R. (1958). Sulfuric acid in Desmarestia. Biological Bulletin 115, 101–106. Fagerberg, W. R. and Dawes, C. J. (1976). Studies on Sargassum. I. A light microscope examination of the wound regeneration process in mature stipes of S. filipendula. American Journal of Botany 63, 110–119. Fairhead, V. A., Amsler, C. D., McClintock, J. B. and Baker, B. J. (2005a). Variation in phlorotannin content within two species of brown macroalgae (Desmarestia anceps and D. menziesii ) from the western Antarctic Peninsula. Polar Biology 28, 680–686. Fairhead, V. A., Amsler, C. D., McClintock, J. B. and Baker, B. J. (2005b). Within‐ thallus variation in chemical and physical defenses in two species of ecologically dominant brown macroalgae from the Antarctic Peninsula. Journal of Experimental Marine Biology and Ecology 322, 1–12. Falkowski, P. G., Katz, M. E., Knoll, A. H., Quigg, A., Raven, J. A., Schofield, O. and Taylor, F. J. R. (2004). The evolution of modern eukaryotic phytoplankton. Science 305, 354–360. Fenical, W. (1975). Halogenation in the Rhodophyta: A review. Journal of Phycology 11, 245–259. Fenical, W. (1980). Distribution and taxonomic features of toxin‐producing marine algae. In ‘‘Pacific Seaweed Aquaculture’’ (I. A. Abbott, M. S. Foster and L. F. Eklund, eds.), pp. 144–151. California Sea Grant Program, La Jolla, CA. Fletcher, R. L. (1975). Heteroantagonism observed in mixed algal cultures. Nature 253, 534–535. Fletcher, R. L. and Callow, M. E. (1992). The settlement, attachment, and establishment of marine algal spores. British Phycological Journal 27, 303–329. Fleury, B., Kelecom, A., Pereira, R. C. and Teixeira, V. L. (1994). Polyphenols, terpenes and sterols in Brazilian Dictyotales and Fucales. Botanica Marina 37, 457–462. Floc’h, J. Y. (1982). Uptake of inorganic ions and their long distance transport in Fucales and Laminariales. In ‘‘Synthetic and Degradative Processes in Marine Macrophytes’’ (L. M. Srivastava, ed.), pp. 139–165. Walter de Gruyter, Berlin. Folin, O. and Ciocalteu, V. (1927). On tyrosine and tryptophane determinations in proteins. Journal of Biological Chemistry 73, 627–650.

DEFENSIVE AND SENSORY CHEMICAL ECOLOGY OF BROWN ALGAE

77

Folin, O. and Denis, W. (1915). A colorimetric method for the determination of phenols (and phenol derivatives) in urine. Journal of Biological Chemistry 22, 305–308. Fraenkel, G. S. and Gunn, D. L. (1961). ‘‘The Orientation of Animals, Kineses, Taxes and Compass Reactions.’’ Dover, New York. Frederick, J. E., Qu, Z. and Booth, C. R. (1998). Ultraviolet radiation at sites on the Antarctic coast. Photochemistry and Photobiology 68, 183–190. Freile‐Pelegrin, Y. and Morales, J. L. (2004). Antibacterial activity in marine algae from the coast of Yucatan, Mexico. Botanica Marina 47, 140–146. Fritsch, F. E. (1945).‘‘The Structure and Reproduction of the Algae,’’ Vol. II. Cambridge University Press, Cambridge. Fukuhara, Y., Mizuta, H. and Yasui, H. (2002). Swimming activities of zoospores in Laminaria japonica (Phaeophyceae). Fisheries Science 68, 1173–1181. Fulcher, R. G. and McCully, M. E. (1971). Histological studies on the genus Fucus. V. An autoradiographic and electron microscope study of the early stages of regeneration. Canadian Journal of Botany 49, 161–165. Gaines, S. D. and Lubchenco, J. (1982). A unified approach to marine plant‐ herbivore interactions. II. Biogeography. Annual Review of Ecology and Systematics 13, 111–138. Geiselman, J. and McConnell, O. (1981). Polyphenols in brown algae Fucus vesiculosus and Ascophyllum nodosum: Chemical defenses against the marine herbivorous snail, Littorina littorea. Journal of Chemical Ecology 7, 1115–1133. Geller, A. and Mu¨ ller, D. G. (1981). Analysis of the flagellar beat pattern of male Ectocarpus siliculosus gametes (Phaeophyta) in relation to chemotactic stimulation by female cells. Journal of Experimental Biology 92, 53–66. Glawe, G. A., Zavala, J. A., Kessler, A., Van Dam, N. M. and Baldwin, I. T. (2003). Ecological costs and benefits correlated with trypsin protease inhibitor production in Nicotiana attenuata. Ecology 84, 79–90. Gordon, R. and Brawley, S. H. (2004). EVects of water motion on propagule release from algae with complex life histories. Marine Biology 145, 21–29. Graham, L. E. and Wilcox, L. W. (2000). ‘‘Algae.’’ Prentice Hall, Upper Saddle River, NJ. Greer, S. P. and Amsler, C. D. (2002). Light boundaries and the coupled eVects of surface hydrophobicity and light on spore settlement in the brown alga Hincksia irregularis (Phaeophyceae). Journal of Phycology 38, 116–124. Greer, S. P. (2003). ‘‘Physical and Chemical Factors AVecting the Swimming Behaviour, Settlement, and Survival of Hincksia irregularis (Phaeophyceae) Spores.’’ Ph.D. Dissertation. University of Alabama at Birmingham, Birmingham, Alabama. Greer, S. P. and Amsler, C. D. (2004). Clonal variation in phototaxis and settlement behaviours of Hincksia irregularis (Phaeophyceae) spores. Journal of Phycology 40, 44–53. Greer, S. P., Iken, K. B., McClintock, J. B. and Amsler, C. D. (2003). Individual and coupled eVects of echinoderm extracts and surface hydrophobicity on spore settlement and germination in the brown alga Hincksia irregularis. Biofouling 19, 315–326. Gschwend, P. M., Mac Farlane, J. K. and Newman, K. A. (1985). Volatile halogenated organic compounds released to seawater from temperate marine macroalgae. Science 227, 1033–1035. Hamilton, J., Zangerl, A., De Luca, E. and Berenbaum, M. (2001). The carbon‐ nutrient balance hypothesis: Its rise and fall. Ecology Letters 4, 86–95.

78

C. D. AMSLER AND V. A. FAIRHEAD

Hammerstrom, K., Dethier, M. N. and Duggins, D. O. (1998). Rapid phlorotannin induction and relaxation in five Washington kelps. Marine Ecology Progress Series 165, 293–305. Harper, M. K., Bubni, T. S., Copp, B. R., James, R. D., Lindsay, B. S., Richardson, A. D., Schnabel, P. C., Tasdemir, D., Van Wagoner, R. M., Verbitski, S. M. and Ireland, C. M. (2001). Introduction to the chemical ecology of marine natural products. In ‘‘Marine Chemical Ecology’’ (J. B. McClintock and B. J. Baker, eds.), pp. 3–69. CRC Press, Boca Raton, FL. Harvell, C. D. (1990). The ecology and evolution of inducible defenses. Quarterly Review of Biology 65, 323–340. Hay, M. E. (1986). Associational plant defenses and the maintenance of species diversity: Turning competitors into accomplices. American Naturalist 128, 617–641. Hay, M. E. (1996). Marine chemical ecology: What’s known and what’s next? Journal of Experimental Marine Biology and Ecology 200, 103–134. Hay, M. E., DuVy, J. E., Fenical, W. and Gustafson, K. (1988b). Chemical defense in the seaweed Dictyopteris delicatula: DiVerential eVects against reef fishes and amphipods. Marine Ecology Progress Series 48, 185–192. Hay, M. E., DuVy, J. E., Pfister, C. A. and Fenical, W. (1987a). Chemical defensce against diVerent marine herbivores: Are amphipods insect equivalents? Ecology 68, 1567–1580. Hay, M. E. and Fenical, W. (1988). Marine plant‐herbivore interactions: The ecology of chemical defence. Annual Review of Ecology and Systematics 19, 111–145. Hay, M. E. and Fenical, W. (1992). Chemical mediation of seaweed‐herbivore interactions. In ‘‘Plant‐Animal Interactions in the Marine Benthos’’ (V. M. John, S. J. Hawkins and J. H. Price, eds.), pp. 317–337. Clarendon Press, Oxford, England. Hay, M. E., Fenical, W. and Gustafson, K. (1987b). Chemical defense against diverse coral‐reef herbivores. Ecology 68, 1581–1591. Hay, M. E., Piel, J., Boland, W. and Schnitzler, I. (1998). Seaweed sex pheromones and their degradation products frequently suppress amphipod feeding but rarely suppress sea urchin feeding. Chemoecology 8, 91–98. Hay, M. E., Renaud, P. E. and Fenical, W. (1988a). Large mobile versus small sedentary herbivores and their resistance to seaweed chemical defenses. Oecologia 75, 246–252. Hay, M. E. and Steinberg, P. D. (1992). The chemical ecology of plant‐herbivore interactions in marine versus terrestrial communities. In ‘‘Herbivores: Their Interactions with Secondary Plant Metabolites’’ (G. A. Rosenthal and M. R. Berenbaum, eds.), Vol. II. Academic Press, New York. Heil, M. (2002). Ecological costs of induced resistance. Current Opinion in Plant Biology 5, 345–350. Hellio, C., Marechal, J. P., Veron, B., Bremer, G., Clare, A. S. and Le Gal, Y. (2004). Seasonal variation of antifouling activities of marine algae from the Brittany coast (France). Marine Biotechnology 6, 67–82. Hemmi, A., Honkanen, T. and Jormalainen, V. (2004). Inducible resistance to herbivory in Fucus vesiculosus – duration, spreading and variation with nutrient availability. Marine Ecology Progress Series 273, 109–120. Hemmi, A. and Jormalainen, V. (2004). Geographic covariation of chemical quality of the host alga Fucus vesiculosus with fitness of the herbivorous isopod Idotea baltica. Marine Biology 145, 759–768. Henry, B. E. and Van Alstyne, K. L. (2004). EVects of UV radiation on growth and phlorotannins in Fucus gardneri (Phaeophyceae) juveniles and embryos. Journal of Phycology 40, 527–533.

DEFENSIVE AND SENSORY CHEMICAL ECOLOGY OF BROWN ALGAE

79

Henrikson, A. A. and Pawlik, J. R. (1995). A new antifouling assay method: Results from field experiments using extracts of four marine organisms. Journal of Experimental Marine Biology and Ecology 194, 157–165. Herbert, R. B. (1989). ‘‘The Biosynthesis of Secondary Metabolites.’’ Chapman and Hall, London. Herms, D. A. and Mattson, W. J. (1992). The dilemma of plants: To grow or defend. Quarterly Review of Biology 67, 285–335. Hick, T. (1885). Protoplasmic continuity in the Fucaceae. Journal of Botany London 23, 97–102. Honkanen, T., Jormalainen, V., Hemmi, A., Ma¨ kinen, A. and Heikkila¨ , N. (2002). Feeding and growth of the isopod Idotea baltica on the brown alga Fucus vesiculosus: Roles of inter‐population and within‐plant variation in plant quality. Ecoscience 9, 332–338. Honkanen, T. and Jormalainen, V. (2005). Genotypic variation in tolerance and resistance to fouling in the brown alga Fucus vesiculosus. Oecologia 144, 196–205. Hurd, C. L., Durante, K. M. and Harrison, P. J. (2000). Influence of bryozoan colonisation on the physiology of the kelp Macrocystis integrifolia (Laminariales, Phaeophyta) from nitrogen‐rich and ‐poor sites in Barkley Sound, British Columbia, Canada. Phycologia 39, 435–440. Ilvessalo, H. and Tuomi, J. (1989). Nutrient availability and accumulation of phenolic compounds in the brown alga Fucus vesiculosus. Marine Biology 101, 115–119. Iken, K., Amsler, C. D., Greer, S. P. and McClintock, J. B. (2001). Qualitative and quantitative studies of the swimming behaviour of Hincksia irregularis (Phaeophyceae) spores: Ecological implications and parameters for quantitative swimming assays. Phycologia 40, 359–366. Iken, K., Greer, S. P., Amsler, C. D. and McClintock, J. B. (2003). A new antifouling bioassay monitoring brown algal spore swimming behaviour in the presence of echinoderm extracts. Biofouling 19, 327–334. Ireland, C. D. and Horn, M. H. (1991). EVects of macrophyte secondary chemicals on food choice and digestive eYciency of Cebidichthys violaceus (Girard), and herbivorous fish of temperate marine waters. Journal of Experimental Marine Biology and Ecology 153, 179–194. Ista, L. K., Callow, M. E., Finlay, J. A., Coleman, S. E., Nolasco, A. C., Simons, R. H., Callow, J. A. and Lopez, G. P. (2004). EVect of substratum surface chemistry and surface energy on attachment of marine bacteria and algal spores. Applied and Environmental Microbiology 70, 4151–4157. Jennings, J. G. and Steinberg, P. D. (1994). In situ exudation of phlorotannins by the sublittoral kelp Ecklonia radiata. Marine Biology 121, 349–354. Jennings, J. G. and Steinberg, P. D. (1997). Phlorotannins versus other factors aVecting epiphyte abundance on the kelp Ecklonia radiata. Oecologia 109, 461–473. Johnson, C. R. and Mann, K. H. (1986). The importance of plant defence abilities to the structure of subtidal seaweed communities: The kelp Laminaria longicruris de la Pylaie survives grazing by the snail Lacuna vincta (Montagu) at high population densities. Journal of Experimental Marine Biology and Ecology 97, 231–267. Joint, I., Tait, K., Callow, M. E., Callow, J. A., Milton, D., Williams, P. and Camara, M. (2002). Cell‐to‐cell communication across the prokaryote‐eukaryote boundary. Science 298, 1207. Jormalainen, V. and Honkanen, T. (2004). Variation in natural selection for growth and phlorotannins in the brown alga Fucus vesiculosus. Journal of Evolutionary Biology 17, 807–820.

80

C. D. AMSLER AND V. A. FAIRHEAD

Jormalainen, V., Honkanen, T., Koivikko, R. and Eranen, J. (2003). Induction of phlorotannin production in a brown alga: Defense or resource dynamics? Oikos 103, 640–650. Jung, V., Thibaut, T., Meinesz, A. and Pohnert, G. (2002). Comparison of the wound‐activated transformation of caulerpenyne by invasive and noninvasive Caulerpa species of the Mediterranean. Journal of Chemical Ecology 28, 2091–2105. Karban, R. and Meyers, J. H. (1989). Induced plant responses to herbivory. Annual Review of Ecology and Systematics 20, 331–348. Karban, R. and Baldwin, I. T. (1997). ‘‘Induced Responses to Herbivory.’’ University of Chicago Press. Karentz, D. (2001). Chemical defenses of marine organisms against solar radiational exposure: UV‐absorbing mycosporine‐like amino acids and scytonemin. In ‘‘Marine Chemical Ecology’’ (J. B. McClintock and B. J. Baker, eds.), pp. 481–520. CRC Press, Boca Raton FL. Karentz, D. and Bosch, I. (2001). Influence of ozone‐related increases in ultraviolet radiation on antarctic marine organisms. American Zoologist 41, 3–16. Karez, C. S. and Pereira, R. C. (1995). Metal contents in polyphenolic fractions extracted from the brown alga Padina gymnospora. Botanica Marina 38, 151–155. Katsaros, C. and Galatis, B. (1988). Thallus development in Dictyopteris membanacea (Phaeophyta, Dictyotales). British Phycological Journal 23, 71–88. Keeling, P. J. (2004). Diversity and evolutionary history of plastids and their hosts. American Journal of Botany 91, 1481–1493. Kessler, A. and Baldwin, I. T. (2001). Defensive function of herbivore‐induced plant volatile emissions in nature. Science 291, 2141–2144. Keum, Y. S., Oak, J. H., van Reine, W. F. P. and Lee, I. K. (2003). Comparative morphology and taxonomy of Sphacelaria species with tribuliform propagules (Sphacelariales, Phaeophyceae). Botanica Marina 46, 113–124. Koehl, M. A. R. and Wainwright, S. (1977). Mechanical adaptations of a giant kelp. Limnology and Oceanography 22, 1067–1071. Koivikko, R., Loponen, J., Honkanen, T. and Jormalainen, V. (2005). Contents of soluble, cell‐wall‐bound and exuded phlorotannins in the brown alga Fucus vesiculosus, with implications on their ecological functions. Journal of Chemical Ecology 31, 195–212. Koricheva, J. (2002). The carbon‐nutrient balance hypothesis is dead; long live the carbon‐nutrient balance hypothesis? Oikos 98, 537–539. 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. Kubanek, J., Lester, S. E., Fenical, W. and Hay, M. E. (2004). Ambiguous role of phlorotannins as chemical defenses in the brown alga Fucus vesiculosus. Marine Ecology Progress Series 277, 79–93. 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., Muller, 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.

DEFENSIVE AND SENSORY CHEMICAL ECOLOGY OF BROWN ALGAE

81

¨ pfelsa¨ ure bei einer Braunalge. Hoppe‐ ¨ ber das Vorkommen von A Kylin, H. (1931). U Seyler’s Zeitschrift fur Physiologische Chemie 197, 7–11. La Barre, S. L., Weinberger, F., Kervarec, N. and Potin, P. (2004). Monitoring defensive responses in macroalgae: Limitations and perspectives. Phytochemistry Reviews 3, 371–379. 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. Langlois, G. L. (1975). EVect of algal exudates on substratum selection by motile telotrochs of the marine peritrich ciliate Voticella marina. Journal of Protozoology 22, 115–123. Laturnus, F. (1995). Release of volatile halogenated organic‐compounds by unialgal cultures of polar macroalgae. Chemosphere 31, 3387–3395. Laturnus, F. (1996). Volatile halocarbons released from Arctic macroalgae. Marine Chemistry 55, 359–366. Laturnus, F. (2001). Marine macroalgae in polar regions as natural sources for volatile organohalogens. Environmental Science and Pollution Research 8, 103–108. Laturnus, F., Adams, F. C., Gomez, I. and Mehrtens, G. (1997). Halogenating activities detected in Antarctic macroalgae. Polar Biology 17, 281–284. Laturnus, F., Wiencke, C. and Kloser, H. (1996). Antarctic macroalgae: Sources of volatile halogenated organic compounds. Marine Environmental Research 41, 169–181. Lau, S. C. K. and Qian, P. Y. (1997). Phlorotannins and related compounds as larval settlement inhibitors of the tube‐building polychaete Hydroides elegans. Marine Ecology Progress Series 159, 219–227. Lau, S. C. K. and Qian, P. Y. (2000). Inhibitory eVect of phenolic compounds and marine bacteria on larval settlement of the barnacle Balanus amphitrite amphitrite Darwin. Biofouling 16, 47–58. Lerdau, M. and Coley, P. (2002). Benefits of the carbon‐nutrient balance hypothesis. Oikos 98, 534–536. Lewis, S. M., Norris, J. N. and Searles, R. B. (1987). The regulation of morphological plasticity in tropical reef algae by herbivory. Ecology 68, 636–641. Lobban, C. S. and Harrison, P. J. (1994). ‘‘Seaweed Ecology and Physiology.’’ Cambridge University Press. Lowell, R. B., Markham, J. H. and Mann, K. H. (1991). Herbivore‐like damage induces increased strength and toughness in a seaweed. Proceedings of the Royal Society of London: Biological Sciences 243, 31–38. Lubchenco, J. and Cubit, J. (1980). Heteromorphic life histories of certain marine algae as adaptations to variations in herbivory. Ecology 61, 676–686. Lu¨ der, U. H. and Clayton, M. N. (2004). Induction of phlorotannins in the brown macroalga Ecklonia radiata (Laminariales, Phaeophyta) in response to simulated herbivory: The first microscopic study. Planta 218, 928–937. Macaya, E. C., Rotha¨ usler, E., Thiel, M., Molis, M. and Wahl, M. (2005). Induction of defences and within‐alga variation of palatability in two brown algae from the northern‐central coast of Chile: EVects of mesograzers and UV radiation. Journal of Experimental Marine Biology and Ecology 325, 214–227. Mahalingam, R. and FedoroV, N. (2003). Stress response, cell death and signaling: The many faces of reactive oxygen species. Physiologia Plantarum 119, 56–68. Maier, I. (1982). New aspects of pheromone‐triggered spermatozoid release in Laminaria digitata (Phaeophyta). Protoplasma 113, 137–142.

82

C. D. AMSLER AND V. A. FAIRHEAD

Maier, I. (1993). Gamete orientation and induction of gametogenesis by pheromones in algae and plants. Plant Cell and the Environment 16, 891–907. Maier, I. (1995). Brown algal pheromones. Progress in Phycological Research 11, 51–102. Maier, I. and Calenberg, M. (1994). EVect of extracellular Ca2þ and Ca2þ antagonists on the movement and chemoorientation of male gametes of Ectocarpus siliculosus (Phaeophyceae). Botanica Acta 107, 451–460. Maier, I. and Mu¨ ller, D. G. (1986). Sexual pheromones in algae. Biological Bulletin 170, 145–175. Maier, I. and Mu¨ ller, D. G. (1990). Chemotaxis in Laminaria digitata (Phaeophyceae). I. Analysis of spermatozoid movement. Journal of Experimental Botany 41, 869–876. Maier, I., Mu¨ ller, D. G., Schmid, C., Boland, W. and Jaenicke, L. (1988). Pheromone receptor specificity and threshold concentrations for spermatozoid release in Laminaria digitata. Naturwissenschaften 75, 260–263. Maier, I., Wenden, A. and Clayton, M. N. (1992). The movement of Hormosira banksii (Fucales, Phaeophyta) spermatozoids in response to sexual pheromone. Journal of Experimental Botany 43, 1651–1657. Manley, S. L., Goodwin, K. and North, W. J. (1992). Laboratory production of bromoform, methylene bromide, and methyl iodide by macroalgae and distribution in nearshore southern California waters. Limnology and Oceanography 37, 1652–1659. Mare´ chal, J. P., Culioli, G., Hellio, C., Thomas‐Guyon, H., Callow, M. E., Clare, A. S. and Ortalo‐Magne´ , A. (2004). Seasonal variation in antifouling activity of crude extracts of the brown alga Bifurcaria bifurcata (Cystoseiraceae) against cyprids of Balanus amphitrite and the marine bacteria Cobetia marina and Pseudoalteromonas haloplanktis. Journal of Experimental Marine Biology and Ecology 313, 47–62. Martinez, E. A. (1996). Micropopulation diVerentiation in phenol content and susceptibility to herbivory in the Chilean kelp Lessonia nigrescens (Phaeophyta, Laminariales). Hydrobiologia 327, 205–211. Mauricio, R. and Rausher, M. D. (1997). Experimental manipulation of putative selective agents provides evidence for the role of natural enemies in the evolution of plant defense. Evolution 51, 1435–1444. McClintock, J. B., Amsler, M. O., Amsler, C. D., Southworth, K. J., Petrie, C. and Baker, B. J. (2004). Biochemical composition, energy content and chemical antifeedant and antifoulant defenses of the colonial Antarctic ascidian Distaplia cylindrica. Marine Biology 145, 885–894. McClintock, M., Higenbotham, N., Uribe, E. G. and Cleland, R. E. (1982). Active, irreversible accumulation of extreme levels of hydrogen sulfide in the brown alga, Desmarestia. Plant Physiology 70, 771–774. McKey, D. (1974). Adaptive patterns in alkaloid physiology. American Naturalist 108, 305–320. McKey, D. (1979). The distribution of secondary metabolites within plants. In ‘‘Herbivores: Their Interactions with Secondary Plant Metabolites’’ (G. A. Rosenthal and D. H. Janzen, eds.), pp. 55–133. Academic Press, New York. Meuse, B. J. D. (1956). Free sulfuric acid in the brown alga, Desmarestia. Biochemica et Biophysica Acta 19, 372–374. Miwa, T. (1931). Occurrence of abundant free sulfuric acid in Desmarestia. Proceedings of the 7th Pacific Science Congress, Pacific Science Association 5, 78–80.

DEFENSIVE AND SENSORY CHEMICAL ECOLOGY OF BROWN ALGAE

83

Moe, R. L. and Silva, P. C. (1989). Desmarestia antarctica (Desmarestiales, Phaeophyceae), a new ligulate antarctic species with an endophytic gametophyte. Plant Systematics and Evolution 164, 273–283. Moss, B. L. (1983). Sieve elements in the Fucales. New Phytologist 93, 433–437. Mu¨ ller, D. G. (1967). Ein leichtflu¨ chtiges Gyno‐Gamon der Braunalge Ectocarpus siliculosus. Naturwissenschaften 54, 496–497. Mu¨ ller, D. G. (1968). Versuche zur Charakterisierung eines SexuallockstoVes bei der Braunalge Ectocarpus siliculosus. I. Methoden, Isolierung, und gaschromatographischer Nachweis. Planta 81, 160–168. Mu¨ ller, D. G. (1978). Locomotive responses of male gametes to the species‐specific sex attractant of Ectocarpus siliculosus (Phaeophyta). Archiv fu¨ r Protistenkunde 120, 371–377. Mu¨ ller, D. G. (1981). Sexuality and sexual attraction. In ‘‘The Biology of Seaweeds’’ (C. S. Lobban and M. J. Wynne, eds.) Blackwell Scientific Publications, Oxford. Mu¨ ller, D. G. (1989). The role of pheromones in sexual reproduction of brown algae. In ‘‘Algae as Experimental Systems’’ (A. W. Coleman, L. J. GoV and J. R. Stein‐Taylor, eds.), pp. 201–213. A. R. Liss, New York. Mu¨ ller, D. G., Boland, W., Becker, U. and Wahl, T. (1988). Caudoxirene, the spermatozoid‐releasing and attracting factor in the marine brown alga Perithalia caudata (Phaeophyceae, Sporochnales). Biological Chemistry Hoppe‐Seyler 369, 655–659. Mu¨ ller, D. G., Gassman, G. and Lu¨ ning, K. (1979). Isolation of a spermatozoid‐ releasing and attracting substance from female gametophytes of Laminaria digitata. Nature 279, 430–431. Mu¨ ller, D. G., Peters, A. F., Gassman, G., Boland, W., Marner, F.‐J. and Jaenicke, L. (1982). Identification of a sexual hormone and related substances in the marine brown alga Desmarestia. Naturwissenschaften 69, 290. Neushul, M. (1965). Diving observation of sub‐tidal Antarctic marine vegetation. Botanica Marina 8, 234–243. Nitao, J., Zangerl, A., Berenbaum, M., Hamilton, J. and De Lucia, E. (2002). CNB: Requiescat in pace? Oikos 98, 540–546. Ohsawa, N., Ogata, Y., Okada, N. and Itoh, N. (2001). Physiological function of bromoperoxidase in the red marine alga, Corallina pilulifera: Production of bromoform as an allelochemical and the simultaneous elimination of hydrogen peroxide. Phytochemistry 58, 683–692. Patel, P., Callow, M. E., Joint, I. and Callow, J. A. (2003). Specificity in the settlement‐modifying response of bacterial biofilms towards zoospores of the marine alga Enteromorpha. Environmental Microbiology 5, 338–349. Paul, V. J. (1992). Seaweed chemical defenses on coral reefs. In ‘‘Ecological Roles of Marine Natural Products’’ (V. J. Paul, ed.), pp. 24–50. Comstock Publishing Associates, Ithaca NY. Paul, V. J., Cruz‐Rivera, E. and Thacker, R. W. (2001). Chemical mediation of macroalgal‐herbivore interactions: Ecological and evolutionary perspectives. In ‘‘Marine Chemical Ecology’’ (J. B. McClintock and B. J. Baker, eds.), pp. 227–265. CRC Press, Boca Raton FL. Paul, V. J. and Puglisi, M. P. (2004). Chemical mediation of interactions among marine organisms. Natural Product Reports 21, 189–209. Paul, V. J. and Van Alstyne, K. L. (1988). The use of injested diterpenoids by the ascoglossan opisthobranch Elysia halmedae Macnae as antipredator defenses. Journal of Experimental Marine Biology and Ecology 119, 15–29.

84

C. D. AMSLER AND V. A. FAIRHEAD

Paul, V. J. and Van Alstyne, K. L. (1992). Activation of chemical defenses in the tropical green algae Halimeda spp. Journal of Experimental Marine Biology and Ecology 160, 191–203. ˚ berg, P. (1996). Spatial variation in polyphenolic content of AscoPavia, H. and A phyllum nodosum (Fucales, Phaeophyta). Hydrobiologia 327, 199–203. Pavia, H. and Brock, E. (2000). Extrinsic factors influencing phlorotannin production in the brown alga Ascophyllum nodosum. Marine Ecology Progress Series 193, 285–294. ˚ berg, P. (1999a). Habitat and feeding preferences of Pavia, H., Carr, H. and A crustacean mesoherbivores inhabiting the brown seaweed Ascophyllum nodosum (L.) Le Jol. and its epiphytic macroalgae. Journal of Experimental Marine Biology and Ecology 236, 15–32. ˚ berg, P. (1997). EVects of UV‐B radiation Pavia, H., Cervin, G., Lindgren, A. and A and simulated herbivory on phlorotannins in the brown alga Ascophyllum nodosum. Marine Ecology Progress Series 157, 139–146. Pavia, H. and Toth, G. B. (2000a). Inducible chemical resistance to herbivory in the brown seaweed Ascophyllum nodosum. Ecology 81, 3212–3225. Pavia, H. and Toth, G. B. (2000b). Influence of light and nitrogen on the phlorotannin content of the brown seaweeds Ascophyllum nodosum and Fucus vesiculosus. Hydrobiologia 440, 299–305. ˚ berg, P. (1999b). Trade‐oVs between phlorotannin Pavia, H., Toth, G. B. and A production and annual growth in natural populations of the brown seaweed Ascophyllum nodosum. Journal of Ecology 87, 761–771. ˚ berg, P. (2002). Optimal defense theory: Elasticity Pavia, H., Toth, G. B. and A analysis as a tool to predict intraplant variation in defenses. Ecology 83, 891–897. ˚ berg, P. (2003). Intraspecific variation in Pavia, H., Toth, G. B., Lindgren, A. and A the phlorotannin content of the brown alga Ascophyllum nodosum. Phycologia 42, 378–383. Peckol, P., Krane, J. M. and Yates, J. L. (1996). Interactive eVects of inducible defense and resource availability on phlorotannins in the North Atlantic brown alga Fucus vesiculosus. Marine Ecology Progress Series 138, 209–217. Peckol, P. and Yates, J. L. (1997). Inducible phlorotannin levels in brown algae from backreef sites. Proceedings of the 8th International Coral Reef Symposium 2, 1259–1262. Pelletreau, K. N. and Muller‐Parker, G. (2002). Sulfuric acid in the phaeophyte alga Desmarestia munda deters feeding by the sea urchin Strongylocentrotus droebachiensis. Marine Biology 141, 1–9. Pereira, R. C., Cavalcanti, D. N. and Teixeira, V. L. (2000a). EVects of secondary metabolites from the tropical Brazilian brown alga Dictyota menstrualis on the amphipod Parhyale hawaiensis. Marine Ecology Progress Series 205, 95–100. Pereira, R. C., Donato, R., Teixeira, V. L. and Cavalcanti, D. N. (2000b). Chemotaxis and chemical defenses in seaweed susceptibility to herbivory. Revista Brasileira de Biologia 60, 405–414. Pereira, R. C., Soares, A. R., Teixeira, V. L., Villaca, R. and da Gama, B. A. P. (2004). Variation in chemical defenses against herbivory in southwestern Atlantic Stypopodium zonale (Phaeophyta). Botanica Marina 47, 202–208. Pereira, R. C., Teixeira, V. L. and Kelecom, A. (1994). Chemical defenses against herbivores in marine algae. 1. The brown alga Dictyota dichotoma (Hudson) Lamouroux from Brazil. Anais da Academia Brasileira de Cieˆ ncias 66, 229–235.

DEFENSIVE AND SENSORY CHEMICAL ECOLOGY OF BROWN ALGAE

85

Pereira, R. C. and Yoneshigue‐Valentin, Y. (1999). The role of polyphenols from the tropical brown alga Sargassum furcatum on the feeding by amphipod herbivores. Botanica Marina 42, 441–448. Peters, A. F. (2003). Molecular identification, taxonomy and distribution of brown algal endophytes, with emphasis on species from Antarctica. In ‘‘Proceedings of the 17th International Seaweed Symposium’’ (A. R. O. Chapman, R. J. Anderson, V. Vreeland and I. R. Davison, eds.), pp. 293–302. Oxford University Press, New York. Peters, A. F., van Oppen, M. J. H., Wiencke, C., Stam, W. T. and Olsen, J. L. (1997). Phylogeny and historical ecology of the Desmarestiaceae (Phaeophyceae) support a southern hemisphere origin. Journal of Phycology 33, 294–309. Peters, K. J., Amsler, C. D., Amsler, M. O., McClintock, J. B., Dunbar, R. B. and Baker, B. J. (2005). A comparative analysis of the nutritional and elemental composition of macroalgae from the western Antarctic Peninsula. Phycologia 44, 453–463. Peterson, C. H. and Renaud, P. E. (1989). Analysis of feeding preference experiments. Oecologia 80, 82–86. Pfister, C. A. (1992). Costs of reproduction in an intertidal kelp: Patterns of allocation and life history consequences. Ecology 73, 1586–1596. Pfister, C. A. and Van Alstyne, K. L. (2003). An experimental assessment of the eVects of nutrient enhancement on the intertidal kelp Hedophyllum sessile (Laminariales, Phaeophyceae). Journal of Phycology 39, 285–290. Phillips, J. A. and Clayton, M. N. (1991). Biflagellate spermatozoids in the Dictyotales: The structure of gametes and gametangia in Zonaria angustata (Dictyotales, Phaeophyta). Phycologia 20, 205–214. Phillips, J. A., Clayton, M. N., Maier, I., Boland, W. and Mu¨ ller, D. G. (1990a). Multifidene, the spermatozoid attractant of Zonaria angustata (Dictyotales, Phaeophyta). British Phycological Bulletin 25, 295–298. Phillips, J. A., Clayton, M. N., Maier, I., Boland, W. and Mu¨ ller, D. G. (1990b). Sexual reproduction in Dictyota diemensis (Dictyotales, Phaeophyta). Phycologia 29, 367–379. Pisut, D. and Pawlik, J. (2002). Anti‐predatory chemical defenses of ascidians: Secondary metabolites or inorganic acids? Journal of Experimental Marine Biology and Ecology 270, 203–214. Pohnert, G. (2002). Phospholipase A(2) 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. ‘‘Chemistry of Pheromones and Other Semiochemicals I,’’Vol. 239, pp. 179–219. Pohnert, G. and Boland, W. (2002). The oxylipin chemistry of attraction and defense in brown algae and diatoms. Natural Product Reports 19, 108–122. Pohnert, G., Lumineau, O., CueV, A., Adolph, S., Cordevant, C., Lange, M. and Poulet, S. (2002). Are volatile unsaturated aldehydes from diatoms the main line of chemical defence against copepods? Marine Ecology Progress Series 245, 33–45. Poore, A. G. B. (1994). Selective herbivory by amphipods inhabiting the brown alga Zonaria angustata. Marine Ecology Progress Series 107, 113–123. Potin, P., Bouarab, K., Ku¨ pper, F. 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.

86

C. D. AMSLER AND V. A. FAIRHEAD

Puglisi, M. P. and Paul, V. J. (1997). Intraspecific variation in red alga Portieria hornemannii: Monoterpene concentrations are not influenced by nitrogen or phosphorus enrichment. Marine Biology 128, 161–170. Purrington, C. B. (2000). Costs of resistance. Current Opinions in Plant Biology 3, 305–308. Ra˚ berg, S., Berger‐Jo¨ nsson, R., Bjo¨ rn, A., Grane´ li, E. and Kautsky, L. (2005). EVects of Pilayella littoralis on Fucus vesiculosus recruitment: Implications for community composition. Marine Ecology Progress Series 289, 131–139. Ragan, M. A. and Glombitza, K.‐W. (1986). Phlorotannins, brown algal polyphenols. Progress in Phycological Research 4, 129–241. Ragan, M. A. and Jensen, A. (1978). Quantitative studies on brown algal phenols. II. Seasonal variation in polyphenol content of Ascophyllum nodosum (L.) Le Jol and Fucus vesiculosus (L.). Journal of Experimental Marine Biology and Ecology 34, 245–258. Ragan, M. A., Ragan, C. M. and Jensen, A. (1980). Natural chelators in sea water: Detoxification of Zn2þ by brown algal polyphenols. Journal of Experimental Marine Biology and Ecology 44, 261–267. Ragan, M. A., Smidsrød, O. and Larsen, B. (1979). Chelation of divalent metal ions by brown algal polyphenols. Marine Chemistry 7, 265–271. Raven, J. A. (2003). Long‐distance transport in non‐vascular plants. Plant, Cell and Environment 26, 73–85. Reed, D. C. (1990). The eVects of variable settlement and early competition on patterns of kelp recruitment. Ecology 71, 776–787. Renaud, P. E., Hay, M. E. and Schmitt, T. M. (1990). Interactions of plant stress and herbivory: Intraspecific variation in the susceptibility of a palatable versus unpalatable seaweed to sea urchin grazing. Oecologia 82, 217–226. Rhoades, D. (1979). Evolution of plant chemical defenses against herbivores. In ‘‘Herbivores’’ (G. A. Rosenthal and D. H. Janzen, eds.), pp. 4–54. Academic Press, New York. Rhoades, D. F. (1985). OVensive‐defensive interactions between herbivores and plants: Their relevance in herbivore population dynamics and ecological theory. American Naturalist 125, 205–238. Rohde, S., Molis, M. and Wahl, M. (2004). Regulation of anti‐herbivore defence by Fucus vesiculosus in response to various cues. Journal of Ecology 92, 1011–1018. Rotha¨ usler, E., Macaya, E. C., Molis, M., Wahl, M. and Thiel, M. (2005). Laboratory experiments examining inducible defense show variable responses of temperate brown and red macroalgae. Revista Chilena de Historia Natural (in press). Ryan, C. A. and Pearce, G. (1998). Systemin: A polypeptide signal for plant defensive genes. Annual Review of Cell and Developmental Biology 1998, 1–17. Sasaki, H., Kataoka, H., Kamiya, M. and Kawai, H. (1999). Accumulation of sulfuric acid in Dictyotales (Phaeophyceae): Taxonomic distribution and ion chromatography of cell extracts. Journal of Phycology 35, 732–739. Sasaki, H., Kataoka, H., Murakami, A. and Kawai, H. (2004). Inorganic ion compositions in brown algae, with special reference to sulfuric acid ion accumulations. Hydrobiologia 512, 255–262. Schiel, D. R. and Foster, M. S. (1986). The structure of subtidal algal stands in temperate waters. Oceanography and Marine Biology Annual Review 24, 265–307. Schmitt, T. M., Lindquist, N. and Hay, M. B. (1998). Seaweed secondary metabolites as antifoulants: EVects of Dictyota spp. diterpenes on survivorship,

DEFENSIVE AND SENSORY CHEMICAL ECOLOGY OF BROWN ALGAE

87

settlement, and development of marine invertebrate larvae. Chemoecology 8, 125–131. Schmitt, T. M., Hay, M. E. and Lindquist, N. (1995). Constraints on chemically mediated coevolution: Multiple functions for seaweed secondary metabolites. Ecology 76, 107–123. Schnitzler, I., Boland, W. and Hay, M. E. (1998). Organic sulfur compounds from Dictyopteris spp. deter feeding by an herbivorous amphipod (Ampithoe longimana) but not by an herbivorous sea urchin (Arbacia punctulata). Journal of Chemical Ecology 24, 1715–1732. 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. Schoenwaelder, M. E. A. (2002). The occurrence and cellular significance of physodes in brown algae. Phycologia 41, 125–139. Schoenwaelder, M. E. A. and Clayton, M. N. (1998a). The secretion of phenolic compounds following fertilisation in Acrocarpia paniculata (Fucales, Phaeophyta). Phycologia 37, 40–46. Schoenwaelder, M. E. A. and Clayton, M. N. (1998b). Secretion of phenolic substances into the zygote wall and cell plate in embryos of Hormosira and Acrocarpia (Fucales, Phaeophyceae). Journal of Phycology 34, 969–980. Schoenwaelder, M. E. A. and Clayton, M. N. (1999a). The presence of phenolic compounds in isolated cell walls of brown algae. Phycologia 38, 161–166. Schoenwaelder, M. E. A. and Clayton, M. N. (1999b). The role of the cytoskeleton in brown algal physode movement. European Journal of Phycology 34, 223–229. Shibata, T., Kawaguchi, S., Hama, Y., Inagaki, M., Yamaguchi, K. and Nakamura, T. (2004). Local and chemical distribution of phlorotannins in brown algae. Journal of Applied Phycology 16, 291–296. Sieburth, J. M. and Conover, J. T. (1965). Sargassum tannin, an antibiotic which retards fouling. Nature 208, 52–53. Skipnes, O., Roald, T. and Haug, A. (1975). Uptake of zinc and strontium by brown algae. Physiologia Plantarum 34, 314–320. Smith, K. L., Hann, A. C. and Harwood, J. L. (1986). The subcellular localisation of absorbed copper in Fucus. Physiologia Plantarum 66, 692–698. Sotka, E. E. (2003). Genetic control of feeding preference in the herbivorous amphipod Ampithoe longimana. Marine Ecology Progress Series 256, 305–310. Sotka, E. E. and Hay, M. E. (2002). Geographic variation among herbivore populations in tolerance for a chemically rich seaweed. Ecology 83, 2721–2735. 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. Sotka, E. E., Wares, J. P. and Hay, M. E. (2003). Geographic and genetic variation in feeding preference for chemically defended seaweeds. Evolution 57, 2262–2276. Soto, H., Rovirosa, J. and San‐Martin, A. (2003). A new diterpene from Dictyota crenulata. Zeitschrift Fur Naturforschung Section B‐a Journal of Chemical Sciences 58, 795–798. Stamp, N. (2003). Out of the quagmire of plant defense hypotheses. Quarterly Review of Biology 78, 23–55. Steinberg, P. D. (1984). Algal chemical defense against herbivores: Allocation of phenolic compounds in the kelp Alaria marginata. Science 223, 405–407.

88

C. D. AMSLER AND V. A. FAIRHEAD

Steinberg, P. D. (1985). Feeding preferences of Tegula funebralis and chemical defenses of marine brown algae. Ecological Monographs 55, 333–349. Steinberg, P. D. (1986). Chemical defences and the susceptibility of tropical marine brown algae to herbivores. Oecologia 69, 628–630. Steinberg, P. D. (1988). EVects of quantitative and qualitative variation in phenolic compounds on feeding in three species of marine invertebrate herbivores. Journal of Experimental Marine Biology and Ecology 120, 221–237. Steinberg, P. D. (1989). Biogeographical variation in brown algal polyphenolics and other secondary metabolites: Comparison between temperate Australasia and North America. Oecologia 78, 373–382. Steinberg, P. D. (1994). Lack of short‐term induction of phlorotannins in the Australasian brown algae Ecklonia radiata and Sargassum vestitum. Marine Ecology Progress Series 112, 129–133. Steinberg, P. D. (1995). Seasonal variation in the relationship between growth and phlorotannin production in the kelp Ecklonia radiata. Oecologia 102, 169–173. Steinberg, P. D. and de Nys, R. (2002). Chemical mediation of colonisation of seaweed surfaces. Journal of Phycology 38, 621–629. Steinberg, P. D., de Nys, R. and Kjelleberg, S. (2001). Chemical mediation of surface colonisation. In ‘‘Marine Chemical Ecology’’ (J. B. McClintock and B. J. Baker, eds.), pp. 355–387. CRC Press, Boca Raton, FL. Steinberg, P. D., de Nys, R. and Kjelleberg, S. (2002). Chemical cues for surface colonisation. Journal of Chemical Ecology 28, 1935–1951. Steinberg, P. D., Edyvane, K., de Nys, R., Birdsey, R. and van Altena, I. (1991). Lack of avoidance of phenolic‐rich brown algae by tropical herbivorous fish. Marine Biology 109, 335–343. Steinberg, P. D., Estes, J. A. and Winter, F. C. (1995). Evolutionary consequences of food‐chain length in kelp forest communities. Proceedings of the National Academy of Sciences of the United States of America 92, 8145–8148. Steinberg, P. D. and Paul, V. (1990). Fish feeding and chemical defense of tropical brown algae in Western Australia. Marine Ecology Progress Series 58, 253–259. Steinberg, P. D. and van Altena, I. (1992). Tolerance of marine invertebrate herbivores to brown algal phlorotannins in temperate Australasia. Ecological Monographs 62, 189–222. Steinke, M., Malin, G. and Liss, P. S. (2002). Trophic interactions in the sea: An ecological role for climate relevant volatiles? Journal of Phycology 38, 630–638. Stern, J. L., Hagerman, A. E., Steinberg, P. D. and Mason, P. K. (1996a). Phlorotannin‐protein interactions. Journal of Chemical Ecology 22, 1877–1899. Stern, J. L., Hagerman, A. E., Steinberg, P. D., Winter, F. C. and Estes, J. A. (1996b). A new assay for quantifying brown algal phlorotannins and comparisons to previous methods. Journal of Chemical Ecology 22, 1273–1293. Stiger, V., Deslandes, E. and Payri, C. E. (2004). Phenolic contents of two brown algae, Turbinaria omata and Sargassum mangarevense on Tahiti (French Polynesia): Interspecific, ontogenic and spatio‐temporal variations. Botanica Marina 47, 402–409. Strom, S., Wolfe, G., Holmes, J., Stecher, H., Shimeneck, C., Lambert, S. and Moreno, E. (2003). Chemical defense in the microplankton. I. Feeding and growth rates of heterotrophic protists on the DMS‐producing phytoplankter Emiliania huxleyi. Limnology and Oceanography 48, 217–229. Swanson, A. K. and Druehl, L. D. (2002). Induction, exudation and the UV protective role of kelp phlorotannins. Aquatic Botany 73, 241–253.

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Targett, N. M. and Arnold, T. M. (1998). Predicting the eVects of brown algal phlorotannins on marine herbivores in tropical and temperate oceans. Journal of Phycology 34, 195–205. Targett, N. M. and Arnold, T. M. (2001). EVects of secondary metabolites on digestion in marine herbivores. In ‘‘Marine Chemical Ecology’’ (J. B. McClintock and B. J. Baker, eds.), pp. 391–411. CRC Press, Boca Raton, FL. Targett, N. M., Boettcher, A. A., Targett, T. E. and Vrolijk, N. H. (1995). Tropical marine herbivore assimilation of phenolic‐rich plants. Oecologia 103, 170–179. Targett, N. M., Coen, L. D., Boettcher, A. A. and Tanner, C. E. (1992). Biogeographic comparisons of marine algal polyphenolics: Evidence against a latitudinal trend. Oecologia 89, 464–470. Taylor, R. B., Lindquist, N., Kubanek, J. and Hay, M. E. (2003). Intraspecific variation in palatability and defensive chemistry of brown seaweeds: EVects on herbivore fitness. Oecologia 136, 412–423. Taylor, R. B., Sotka, E. and Hay, M. E. (2002). Tissue‐specific induction of herbivore resistance: Seaweed response to amphipod grazing. Oecologia 132, 68–76. 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. Toth, G. and Pavia, H. (2000a). Lack of phlorotannin induction in the brown seaweed Ascophyllum nodosum in response to increased copper concentrations. Marine Ecology Progress Series 192, 119–126. Toth, G. B. and Pavia, H. (2000b). 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. and Pavia, H. (2001). Removal of dissolved brown algal phlorotannins using insoluble polyvinylpolypyrrolidone (PVPP). Journal of Chemical Ecology 27, 1899–1910. Toth, G. B. and Pavia, H. (2002a). Lack of phlorotannin induction in the kelp Laminaria hyperborea in response to grazing by two gastropod herbivores. Marine Biology 140, 403–409. Toth, G. B. and Pavia, H. (2002b). Intraplant habitat and feeding preference of two gastropod herbivores inhabiting the kelp Laminaria hyperborea. Journal of the Marine Biological Association of the United Kingdom 82, 243–247. Tugwell, S. and Branch, G. M. (1989). DiVerential polyphenolic distribution amoung tissues in the kelps Ecklonia maxima, Laminaria pallida and Macrocystis angustifolia in relation to plant‐defence theory. Journal of Experimental Marine Biology and Ecology 129, 219–230. Tugwell, S. and Branch, G. M. (1992). EVects of herbivore gut surfactants on kelp polyphenol defenses. Ecology 73, 205–215. Tuomi, J., Ilvessalo, H., Sire´ n, S. and Jormalainen, V. (1989). Within‐plant variation in phenolic content and toughness of the brown alga Fucus vesiculosus L. Botanica Marina 32, 505–509. Utter, B. D. and Denny, M. W. (1996). Wave‐induced forces on the giant kelp Macrocystis pyrifera (Agardh): Field test of a computational model. Journal of Experimental Biology 199, 2645–2654. Vallim, M. A., De Paula, J. C., Pereira, R. C. and Teixeira, V. L. (2005). The diterpenes from dictyotacean marine brown algae in the tropical Atlantic American region. Biochemical Systematics and Ecology 33, 1–16.

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Van Alstyne, K. and Paul, V. (1990). The biogeography of polyphenolic compounds in marine macroalgae: Temperate brown algal defenses deter feeding by tropical herbivorous fishes. Oecologia 84, 158–163. Van Alstyne, K. L. (1988). Herbivore grazing increases polyphenolic defenses in the intertidal brown alga Fucus distichus. Ecology 69, 655–663. Van Alstyne, K. L. (1995). Comparison of 3 methods for quantifying brown algal polyphenolic compounds. Journal of Chemical Ecology 21, 45–58. Van Alstyne, K. L., Dethier, M. N. and Duggins, D. O. (2001a). Spatial patterns in macroalgal chemical defenses. In ‘‘Marine Chemical Ecology’’ (J. B. McClintock and B. J. Baker, eds.), pp. 301–324. CRC Press, Boca Raton, FL. Van Alstyne, K. L., Ehlig, J. M. and Whitman, S. L. (1999c). Feeding preferences for juvenile and adult algae depend on algal stage and herbivore species. Marine Ecology Progress Series 180, 179–185. Van Alstyne, K. L. and Houser, L. T. (2003). Dimethylsulfide release during macroinvertebrate grazing and its role as an activated chemical defense. Marine Ecology Progress Series 250, 175–181. Van Alstyne, K. L., McCarthy, J. J., Hustead, C. L. and Duggins, D. O. (1999b). Geographic variation in polyphenolic levels of northeastern Pacific kelps and rockweeds. Marine Biology 133, 371–379. Van Alstyne, K. L., McCarthy, J. J., Hustead, C. L. and Kearns, L. J. (1999a). Phlorotannin allocation among tissues of northeastern Pacific kelps and rockweeds. Journal of Phycology 35, 483–492. Van Alstyne, K. L. and Pelletreau, K. N. (2000). EVects of nutrient enrichment on growth and phlorotannin production in Fucus gardneri embryos. Marine Ecology Progress Series 206, 33–43. Van Alstyne, K. L., Whitman, S. L. and Ehlig, J. M. (2001b). DiVerences in herbivore preferences, phlorotannin production, and nutritional quality between juvenile and adult tissues from marine brown algae. Marine Biology 139, 201–210. Vermeij, G. J. (1978). ‘‘Biogeography and Adaptation.’’ Harvard University Press, Boston, MA. Vreeland, V., Waite, J. H. and Epstein, L. (1998). Polyphenols and oxidases in substratum adhesion by marine algae and mussels. Journal of Phycology 34, 1–8. Wakefield, R. L. and Murray, S. N. (1998). Factors influencing food choice by the seaweed‐eating marine snail Norrisia norrisi (Trochidae). Marine Biology 130, 631–642. Waterman, P. G. and Mole, S. (1994). ‘‘Analysis of Phenolic Plant Metabolites.’’ Blackwell Scientific, Oxford. Weidner, K., Lages, B. G., da Gama, B. A. P., Molis, M., Wahl, M. and Pereira, R. C. (2004). EVect of mesograzers and nutrient levels on induction of defenses in several Brazilian macroalgae. Marine Ecology Progress Series 283, 113–125. 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

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toward Gracilaria conferta (Rhodophyta). Journal of Phycology 37, 418–426. Weykam, G., Go´ mez, I., Wiencke, C., Iken, K. and Klo¨ ser, H. (1996). Photosynthetic characteristics and C:N ratios of macroalgae from King Gorge Island (Antarctica). Journal of Experimental Marine Biology and Ecology 204, 1–22. Wichard, T., Poulet, S. A., Halsband‐Lenk, C., Albaina, A., Harris, R., Liu, D. and Pohnert, G. (2005). Survey of the chemical defence potential of diatoms: Screening of fifty species for 
‐unsaturated aldehydes. Journal of Chemical Ecology 31, 949–958. Wiencke, C. and Clayton, M. (2002). ‘‘Antarctic Seaweeds.’’ ARG Gantner Verlag, KG Ruggell. Wilkstro¨ m, S. A. and Pavia, H. (2004). Chemical settlement inhibition versus post‐ settlement mortality as an explanation for diVerential fouling of two congeneric seaweeds. Oecologia 138, 223–230. Winter, F. C. and Estes, J. A. (1992). Experimental evidence for the eVects of polyphenolic compunds from Dictyoneurum californicum Ruprecht (Phaeophyta:Laminariales) on feeding rate and growth in the red abalone Haliotus rufescens Swainson. Journal of Experimental Marine Biology and Ecology 155, 263–277. Wirth, H. E. and Rigg, G. B. (1937). The acidity of the juice of Desmarestia. American Journal of Botany 24, 68–70. Wojtaszek, P. (1997). Oxidative burst: An early plant response to pathogen infection. Biochemical Journal 322, 681–692. Yates, J. L. and Peckol, P. (1993). EVects of nutrient availability and herbivory on polyphenolics in the seaweed Fucus vesiculosus. Ecology 74, 1757–1766.

FURTHER READING Dethier, M. N., Williams, S. L. and Freeman, A. (2005). Seaweeds under stress: Manipulated stress and herbivory affect critical life-history functions. Ecological Monographs 75, 403–418. Molis, M., Ko¨ ner J., Ko, Y. W., Kim, J.H., and Wahl, M. (in press). Inducible responses in the brown seaweed Ecklonia cave: The role of grazer identity and season. Journal of Ecology.

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

Crop Performance and Improvement, Rothamsted Research Harpenden, Hertfordshire AL5 2JQ, United Kingdom

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . II. Amino Acid Signalling. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. General Amino Acid Control in Yeast . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Plant General Control Nonderepressible‐2 and Evidence of Conservation of Elements of General Amino Acid Control . . . . . . . . III. Carbon Metabolite Sensing and Signalling . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. AMP‐activated Protein Kinase, the Fuel Gauge of Animal Cells . . . B. Regulation of Carbon Metabolism in Yeast . . . . . . . . . . . . . . . . . . . . . . . C. Carbon Metabolite Sensing and Signalling in Plants . . . . . . . . . . . . . . . IV. The Link Between Sugar and Abscisic Acid/Stress Signalling Pathways . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. Prospects. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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ABSTRACT The sensing and signalling mechanisms through which plant cells respond to levels of carbon metabolites and amino acids are reviewed, focusing on the roles of sucrose nonfermenting‐1‐related protein kinase (SnRK1) and general control nonderepressible‐2‐related protein kinase, drawing comparisons with homologous and analogous systems in animals and fungi. Amino acid signalling and general amino acid control Advances in Botanical Research, Vol. 43 Incorporating Advances in Plant Pathology Copyright 2006, Elsevier Ltd. All rights reserved.

0065-2296/06 $35.00 DOI: 10.1016/S0065-2296(05)43002-5

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in yeast are described, and evidence for the presence of a similar system in plants is discussed. Carbon metabolite signalling in animal, fungal, and plant systems is described. The roles of SnRK1 in sugar sensing and the regulation of carbohydrate metabolism, starch accumulation, sterol biosynthesis, the cell cycle, and development are described. The link between sugar and abscisic acid signalling is discussed.

I. INTRODUCTION A few hours after we eat a meal we start to feel hungry and the gap between eating and wanting to eat again will shorten if we use up energy by exercising or if our bodies have a high demand for nutrients for growth. We regard this as a whole organism eVect, but nutrient availability also has profound eVects at the cellular level. Cells sense and respond to the availability of nutrients, in other words their metabolic status, in several ways. They modify their processes for nutrient uptake, they mobilise or build up intracellular reserves, and they regulate the demand imposed by biosynthetic pathways. One of the great fascinations of this subject is that the regulatory mechanisms that maintain and respond to cellular metabolic status are ancient and must have emerged in primitive eukaryotes before the divergence of plants, animals, and fungi. While these mechanisms have continued to evolve in all three kingdoms, they have been conserved suYciently to be instantly recognisable from higher plants to fungi and humans. Hence, the regulatory pathway that is activated in human skeletal muscle cells to enable them to meet the energy demands of exercise has the same central players as that which enables yeast cells to start making the enzymes required to use alternative carbon sources in the absence of glucose and that which enables the cells in a growing potato tuber to make the enzymes required for the breakdown of sucrose arriving from the leaves. These regulatory processes begin with the sensing of metabolite levels. Indeed, the interest of plant scientists in this field stemmed from the realisation that metabolites themselves initiate signals that have profound eVects on enzyme activity and gene expression, thereby influencing the flux of compounds through metabolic pathways. In this way, metabolite sensing and signalling aVects and is aVected by processes such as photosynthesis, the cell cycle, biotic and abiotic stresses from the environment, nutrient availability, and the partitioning of resources within the plant (Fig. 1). It is, therefore, a crucial factor in determining the survival and productivity of plants and their adaptation to extreme environments. The rapid advances in genomics, transcriptomics, proteomics, and metabolomics mean that there is now a great opportunity to make progress in the elucidation of the sensing and signalling mechanisms that maintain the

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Fig. 1. Schematic showing how metabolic status aVects and is aVected by a range of cellular processes.

Fig. 2. The partitioning of assimilated carbon and nitrogen arriving at a sink organ such as a seed or tuber. The partitioning of assimilate is an important determinant of both yield and quality.

metabolic status of plant cells and regulate and integrate metabolic pathways. This is essential for the development of rational strategies for the modification of metabolism and resource partitioning (Fig. 2). However, the target of a

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broad understanding of these mechanisms remains a considerable way oV. They include the activation and deactivation of enzymes by phosphorylation, the thioredoxin system, the control of transcription, translation, post‐ translational modification and protein turnover, as well as the interactions shown in Fig. 1. This review deals with the sensing and signalling mechanisms through which plant cells respond to levels of carbon metabolites and amino acids, focussing in particular on the roles of sucrose nonfermenting‐1 (SNF1)‐ related protein kinase (SnRK1) and general control nonderepressible‐2 (GCN2)‐related protein kinase. It covers the metabolites that are sensed and what is known about the genes, transporters, metabolic enzymes, transcription factors, and hormones that are involved. Throughout, comparisons are drawn with homologous and analogous systems in animals and fungi.

II. AMINO ACID SIGNALLING A. GENERAL AMINO ACID CONTROL IN YEAST

Amino acid synthesis and protein production in budding yeast (Saccharomyces cerevisiae) are regulated in a wonderfully elegant manner in response to amino acid levels. Amino acid starvation causes a general reduction in protein synthesis and initiates changes in expression of a huge number of genes in a process known as general amino acid control (Hinnebusch, 1992). Fundamental to general amino acid control is the protein kinase GCN2 (Wek et al., 1989). The substrate for GCN2 is the  subunit of eukaryotic translation initiation factor‐2 (eIF‐2), which is phosphorylated by GCN2 at serine‐51 (Samuel, 1993) (Fig. 3). eIF‐2 is a trimeric factor (subunits , , and ) that can bind either GDP or GTP. However, only when bound to GTP is it able to carry out its physiological function of binding Met‐tRNA to the ribosome and transferring it to the 40S ribosomal subunit. Following attachment of the [eIF‐2.GTP.Met‐tRNA] complex to the 40S subunit, the GTP is hydrolyzed to GDP and Pi and eIF‐2 is released as an inactive [eIF‐2. GDP] complex. Phosphorylation of eIF‐2 inhibits the recycling of bound GDP to GTP, decreasing the rate of protein synthesis. GCN2‐mediated phosphorylation of eIF‐2 under conditions of amino acid deprivation increases the expression of amino acid biosynthesis genes through the action of a transcriptional activator, GCN4 (Hinnebusch, 1997) (Fig. 4). Upregulation of GCN4 occurs at the translational level, with translation initiating from a downstream initiation codon that is not used

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Fig. 3. The mechanism by which GCN2 inhibits protein synthesis in yeast. GCN2 is activated in response to low amino acid levels and phosphorylates the  subunit of eIF‐2. Phosphorylation of eIF‐2 inhibits the recycling of eIF2‐bound GDP to GTP, preventing eIF2 from binding Met‐tRNAi (a special tRNA used for initiation) to the ribosome and transferring it to the 40S ribosomal subunit, thereby decreasing the rate of protein synthesis. Figure kindly provided by Yuhua Zhang.

Fig. 4. A decrease in the binding of Met‐tRNAi to eIF2 causes an increase in the translation of the transcription factor, GCN4, which activates the expression of a large number of genes, including many that encode enzymes involved in amino acid biosynthesis. The free amino acid pool is therefore replenished.

under normal conditions (Hinnebusch, 1992, 1994). Microarray analysis identified 539 yeast genes that are induced by amino acid starvation through the action of GCN4 (Natarajan, 2001). These included genes in

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every amino acid biosynthetic pathway except cysteine, as well as genes encoding amino acid precursors, vitamin biosynthetic enzymes, peroxisomal components, mitochondrial carrier proteins, autophagy proteins, and many genes encoding protein kinases and transcription factors. GCN2 is believed to be activated through interaction with uncharged tRNA (Wek et al., 1989). The region of the protein responsible for this is a domain of approximately 400 amino acid residues that shows significant sequence similarity with histidyl‐tRNA synthetases (Wek et al., 1989). The presence of adjacent kinase catalytic and His‐tRNA synthetase‐like domains is indicative of a GCN2‐type protein kinase. Note that a second mechanism that is distinct from this amino acid response but acts through GCN2 to induce the synthesis of GCN4 is initiated by glucose limitation (Yang et al., 2000). Induction of GCN4 during carbon starvation is believed to enhance the storage of amino acids in the vacuoles, thereby facilitating re‐entry into exponential growth when glucose becomes available. Homologues of GCN2 have been identified in Drosophila melanogaster (Santoyo et al., 1997) and Neurospora crassa (Sattlegger et al., 1998), and there are two other eIF‐2 kinases that have similar catalytic domains to GCN2 but do not contain a histidyl‐tRNA synthetase‐like domain and respond to diVerent stimuli. These are the heme‐regulated inhibitor (HRI), which has been cloned from rabbit and rat (Chen et al., 1991; Mellor et al., 1994a), and the double‐stranded RNA‐dependent kinase (PKR), which has been cloned from human (Meurs et al., 1990). HRI is activated in response to heme deficiency (Chen et al., 1991; Mellor et al., 1994a) and PKR to the presence of double‐stranded RNAs after virus infection (Icely et al., 1991; Mellor et al., 1994b; Meurs et al., 1990). B. PLANT GENERAL CONTROL NONDEREPRESSIBLE‐2 AND EVIDENCE OF CONSERVATION OF ELEMENTS OF GENERAL AMINO ACID CONTROL

The phosphorylation site at serine‐51 is conserved in eIF‐2 homologues from a wide range of eukaryotes, including wheat, and yeast GCN2 will phosphorylate wheat eIF‐2 at this position (Chang et al., 1999, 2000). Furthermore, a GCN2 homologue from Arabidopsis was identified in 2003 (Zhang et al., 2003). This gene, AtGCN2, which is present as a single copy on chromosome 3, encodes a protein of 140 kDa showing approximately 30% identity overall with yeast GCN2, rising to 44% in the catalytic domain. Expression of AtGCN2 in yeast gcn2 mutants complements the mutation, enabling growth in the presence of sulfometuron methyl, an inhibitor of branched chain amino acid biosynthesis, and 3‐aminotriazole, an inhibitor of histidine biosynthesis. Screening of EST and genomic databases with the

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AtGCN2 sequence reveals the presence of similar genes and transcripts in rice, wheat, barley, potato, soybean, sugar beet, sugarcane, Medicago, cotton, poplar, onion, lotus, and Zinnia. However, perhaps surprisingly, there is no obvious candidate for a GCN4 homologue in Arabidopsis that is identifiable on the basis of sequence similarity with yeast GCN4. There is a database entry for a ‘‘GCN4‐complementing’’ gene called GCP1 (accession AJ130878), but this entry dates to the late 1990s and to the author’s knowledge data supporting the claim have never been published. The link between amino acid signalling, nitrogen use eYciency, crop yield, and quality makes the elucidation of amino acid signalling pathways an important target for plant scientists. There is evidence of co‐ordinated regulation of genes encoding enzymes of amino acid biosynthesis in plants. For example, blocking histidine biosynthesis in Arabidopsis with a specific inhibitor, IRL 1803, has been shown to increase the expression of eight genes involved in the synthesis of aromatic amino acids, histidine, lysine, and purines (Guyer et al., 1995). Genes encoding tryptophan biosynthesis pathway enzymes have also been shown to be induced by amino acid starvation (caused by, for example, glyphosate treatment) in Arabidopsis (Zhao et al., 1998). The identification of AtGCN2 supports the hypothesis that at least some elements of general amino acid control are present in plants, but clearly much has to be done to elucidate the whole signalling pathway. It will also be important to determine how amino acid signalling links with the broader signalling network that responds to nitrate availability. Amino acid signalling has the potential to aVect many metabolic and developmental processes. Just one possible example is the regulation of storage protein (prolamin) gene expression in cereal seeds (reviewed by Shewry et al., 2003). The expression of prolamin genes responds sensitively to the availability of nitrogen in the grain (DuVus and Cochrane, 1992; Giese and Hopp, 1984) and many contain a conserved sequence, approximately 30 bp long and positioned around 300 bp upstream of the transcription start site (Forde et al. 1985) (Fig. 5). This sequence contains two conserved motifs, TGTAAAGT and G(A/G)TGAGTCAT; the former has been called the endosperm motif (Hammond‐Kosack et al., 1993) (E motif in Fig. 5) and the latter the GCN4‐like motif (GLM), nitrogen element, or N motif (Hammond‐Kosack et al., 1993; Mu¨ ller and Knudsen, 1993). The central part of the N motif is similar to the binding site of GCN4, the sequence of which is TGACTC, and this element acts as a negative element at low nitrogen levels and a positive element when nitrogen levels are adequate (Mu¨ller and Knudsen, 1993). Two transcription factors, ESBF‐II (Hammond‐Kosack et al., 1993) and SPA (Albani et al., 1997), have been shown to bind this

Fig. 5. Schematic showing the regulatory elements in the promoters of families of wheat storage protein genes. The N motif is similar to the binding site of the yeast transcription factor, GCN4.

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motif, although neither is closely related to GCN4 in terms of amino acid sequence.

III. CARBON METABOLITE SENSING AND SIGNALLING A. AMP‐ACTIVATED PROTEIN KINASE, THE FUEL GAUGE OF ANIMAL CELLS

AMP‐activated protein kinase (AMPK) was one of the first protein kinases to be studied biochemically. It was purified originally as separate activities that caused time‐ and ATP‐dependent inactivations of HMG‐CoA reductase (Beg et al., 1973) or acetyl‐CoA carboxylase (Carlson and Kim, 1973). Later it became clear that these activities were functions of the same protein kinase. The primary function of AMPK is the conservation of ATP and it achieves this function through the phosphorylation and inactivation of regulatory enzymes of ATP‐consuming pathways, such as acetyl‐CoA carboxylase (fatty acid synthesis) (Davies et al., 1990, 1992) and HMG‐CoA reductase (sterol/isoprenoid synthesis) (Clarke and Hardie, 1990; Gillespie and Hardie, 1992). It also regulates gene expression, inhibiting the glucose activation of genes involved in glucose and lipid metabolism in liver cells, including genes encoding pyruvate kinase and fatty acid synthase (Leclerc et al., 1998; Woods et al., 2000). Activation of AMPK has been demonstrated in response to a variety of stresses in mammalian cells, including exercise in skeletal muscle (Winder and Hardie, 1996), interruption of the blood supply in heart muscle (Kudo et al., 1995), treatment of cells with deoxyglucose or high levels of fructose (Moore et al., 1991; Sato et al., 1993), heat shock (Corton et al., 1994), arsenate, and other inhibitors of oxidative metabolism (Corton et al., 1994; Witters et al., 1991). AMPK is attracting increasing interest because it has been implicated in insulin regulation, type 2 diabetes mellitus, and obesity. Mutations in AMPK are associated with a severe heart defect (hypertrophy and arrhythmia). As its name suggests, AMPK is activated allosterically by 50 ‐adenosine monophosphate (AMP) (Carling et al., 1987, 1989). It is also activated by phosphorylation by an upstream protein kinase that itself is activated by AMP (Hawley et al., 1996). AMP binding acts in two other ways to increase AMPK activity: it makes AMP a better substrate for the upstream protein kinase and a worse substrate for protein phosphatase‐2C (Carling et al., 1989; Corton et al., 1995; Davies et al., 1995; Hawley et al., 1995). Activation of AMPK by AMP is antagonised by high concentrations of ATP.

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Fig. 6. Pathways for activation of AMP‐activated protein kinase (AMPK), the ‘‘fuel gauge’’ of mammalian cells, and its targets.

This means that AMPK can act as a fuel gauge for animal cells (Hardie and Carling, 1997) (Fig. 6). Levels of ATP, AMP, and ADP are held in equilibrium by the enzyme adenylate kinase, which catalyses the interconversion of ATP and AMP to two molecules of ADP. Continuous production of ATP when a cell is well nourished leads to a high ATP:ADP ratio, pushing this reaction in the direction of ADP and depleting the pool of AMP. Conversely, when ATP is not being supplied in suYcient amounts by glycolysis, the TCA cycle, and respiration, the ATP:ADP ratio falls and AMP levels rise. AMPK comprises three subunits, a catalytic  subunit and accessory and subunits, in a heterotrimeric complex (Davies et al., 1994; Mitchelhill et al., 1994) (Fig. 7). Amino acid and DNA sequencing of the three subunits (Carling et al., 1994; Gao et al., 1995, 1996; Mitchelhill et al., 1994; Woods et al., 1996) revealed that AMPK was the animal homologue of sucrose nonfermenting‐1 (SNF1) of budding yeast (Saccharomyces cerevisiae) and SNF1‐related protein kinase‐1 (SnRK1) of plants. More recently, a protein kinase upstream of AMPK has been identified as LKB1, a tumour repressor that had not been linked previously to metabolic regulation (Woods et al., 2003). In humans, mutations in LKB1 cause Peutz–Jeghers syndrome, suVerers of which are prone to develop rare

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Fig. 7. Components of SnRK1 (plants), AMPK (mammals), and SNF1 (yeast). Plant subunits are labelled in black, animal subunits in mid‐grey, and yeast subunits in light grey. Note that plants have several proteins with some similarity with the AMPK and SNF4 subunits; this subunit dissociates from the catalytic subunit when glucose availability is adequate, allowing the regulatory domain of the catalytic subunit to fold over the catalytic domain, causing inactivation.

cancers. This discovery caused great excitement because it suggested a link between cancer and diabetes, and might explain why exercise provides some protection against both. AMPK phosphorylates a synthetic peptide (His Met Arg Ser Ala Met Ser Gly Leu His Leu Val Lys Arg Arg) known as the SAMS peptide based on the sequence around the primary phosphorylation site for AMPK on rat acetyl‐CoA carboxylase. This has enabled a relatively simple assay for AMPK activity to be developed that measures the rate of phosphorylation of the SAMS peptide using radiolabelled ATP as a phosphate donor (Davies et al., 1989). B. REGULATION OF CARBON METABOLISM IN YEAST

1. SNF1 and glucose repression In yeast, the expression of genes and processes involved in aerobic metabolism or the metabolism of carbon sources other than glucose is prevented if glucose is present above a concentration of about 0.2% (w/v) (Dickinson,

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1999). This process of glucose repression is the over‐riding mechanism for regulating carbon metabolism. It aVects the utilisation of alternative carbon sources, gluconeogenesis, the synthesis of dozens of enzymes, respiration, and the biogenesis of mitochondria and peroxisomes even if other carbon sources are provided. It ensures that glucose is always consumed first, regardless of what other carbon sources are available, and that the glucose is consumed entirely by fermentation to ethanol. Aerobic metabolism commences only when glucose levels are low. A critical component of this fundamental signalling mechanism in budding yeast is a protein kinase called sucrose nonfermenting‐1 (SNF1) (Fig. 8). SNF1, a 72‐kDa protein with an N‐terminal protein serine/threonine kinase domain followed by a C‐terminal regulatory domain (Celenza and Carlson, 1986), was given its name because a snf1 mutant was first identified in a screen for mutants that were unable to switch on the invertase gene SUC2 in response to glucose deprivation. It is now known to be required for growth on other fermentable carbon sources such as galactose and maltose and on nonfermentable carbon sources such as glycerol and ethanol (reviewed by Dickinson, 1999). As well as being required for the derepression of essentially all glucose‐repressed genes, SNF1 aVects sporulation, cell cycle control, glycogen accumulation, peroxisome biogenesis, and regulation of enzyme activity. A transcription factor that is almost certainly a substrate for SNF1 is the transcriptional repressor MIG1. MIG1 contains several potential SNF1 phosphorylation sites, and mutation of these results in ¨ stling and Ronne, 1998). The constitutive repression of transcription (O cellular location of MIG1 is determined by glucose: the addition of glucose causes it to move to the nucleus and the removal of glucose causes it to return to the cytosol (DeVit et al., 1997). Like AMPK, to which it is now known to be homologous, the SNF1 protein kinase actually comprises three subunits (Fig. 7), of which the catalytic subunit is encoded by the SNF1 gene (Celenza and Carlson, 1986). The second subunit in the complex is a protein called SNF4, a regulatory subunit that is required for full activity (Celenza et al., 1989) and which is homologous to the subunit of AMPK. The interaction between SNF1 and SNF4 appears to be regulated by glucose, and in conditions of glucose deprivation, SNF4 counteracts autoinhibition by SNF1 (Jiang and Carlson, 1996). The third subunit, homologous to the subunit of AMPK, is one from a class of proteins that comprises SIP1, SIP2, and GAL83, three related proteins that are interchangeable and may target the complex to diVerent substrates (Yang et al., 1994). SIP1, SIP2, and GAL83 contain two conserved domains: the ASC domain (association with SNF1 complex) (Jiang and Carlson, 1997; Yang et al., 1994) and the KIS domain

Fig. 8. Possible pathways for activation of sucrose nonfermenting‐1 (SNF1) of yeast, and its targets. Carbon ‘‘flow’’ from glucose to ethanol or CO2 is indicated with black arrows; signalling interactions are shown with grey arrows; and dotted lines are used where the mechanism for an interaction is not known.

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(kinase interacting sequence) (Jiang and Carlson, 1997). They appear to form ‘‘scaVold’’ subunits on which the SNF1 and SNF4 proteins assemble (Jiang and Carlson, 1997). Direct interaction between SNF1 and SNF4 proteins only occurs under derepressing (low glucose) conditions (Jiang and Carlson, 1996) (Fig. 7), and this interaction is necessary for the protein kinase activity of SNF1 to be expressed (Celenza and Carlson, 1989; Woods et al., 1994). Using the SAMS peptide as a substrate, activity of the SNF1 complex has been shown to increase dramatically within 5 min of glucose removal (Wilson et al., 1996; Woods et al., 1994). As with the activation of AMPK, this is due to phosphorylation. However, even now, the intracellular signal(s) responsible for triggering this activation remains unknown. Under conditions where SNF1 is activated there are large increases in AMP and decreases in ATP (Wilson et al., 1996) and, by analogy with mammalian AMPK, these nucleotides would be good candidates to be the signals. However, unlike AMPK, no direct eVects of AMP or ATP have been demonstrated on SNF1 in vitro. Three protein kinases that lie upstream of SNF1 (ELM1, PAK1, and TOS3) have now been identified (Hong et al., 2003; Sutherland et al., 2003). It has been proposed that glucose is sensed in yeast by the enzyme hexokinase (Fig. 8). This enzyme has a metabolic function in catalysing the conversion of glucose to glucose‐6‐phosphate (the first stage in glycolysis), but it is found not only in the cytosol where it performs its catalytic function, but also in the nucleus (Herrero et al., 1998; Randez‐Gil et al., 1998). Furthermore, mutations in HXK2, which encodes the major hexokinase PII isoform in S. cerevisiae, cause partially constitutive expression of glucose‐repressed genes (Entian, 1980). However, further work has shown a good correlation between the overall residual hexokinase catalytic activity of diVerent mutants and their ability to exhibit glucose repression (Ma et al., 1989; Rose et al., 1991). This suggests that hexokinase PII has a role in producing the signal molecule, but does not sense or initiate a signal. Despite this, hexokinase is now widely regarded as a ubiquitous glucose sensor in eukaryotes (Rolland et al., 2001). The catalytic activity of hexokinase has been shown to be aVected by components of the trehalose biosynthetic pathway. This pathway involves two enzymes, trehalose phosphate synthase (TPS), which catalyses the formation of trehalose‐6‐phosphate (T6P) from glucose‐6‐phosphate and UDP‐glucose, and trehalose phosphate phosphatase (TPP), which catalyses the dephosphorylation of T6P to trehalose; trehalose is cleaved by another enzyme, trehalase. Both TPS and T6P have an inhibitory eVect on hexokinase, thereby regulating the flux of carbon into glycolysis (Thevelein

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and Hohmann, 1995). This is crucial because it ensures that cellular ATP levels are not depleted through the buildup of glycolytic intermediates (Bonini et al., 2000; Noubhani et al., 2000). This finding has stimulated great interest in the role of T6P as a sensed metabolite in both yeast and plant systems. 2. Glucose induction Whatever the role of glucose in the repression/derepression signalling pathway, it is definitely sensed to initiate a signal in a diVerent pathway, that of glucose induction (Fig. 9). S. cerevisiae has 17 hexose transporters encoded by HXT genes that are expressed diVerentially, depending on the concentration of glucose that is available; high‐aYnity, low‐capacity transporters are expressed when the glucose concentration is low, whereas low‐aYnity,

Fig. 9. Glucose induction in yeast. Glucose is sensed by low‐ and high‐aYnity glucose sensors, SNF3 and RGT2, located in the membrane, and a signal is generated that results in the inactivation of RGT1, a transcriptional repressor of low‐ and high‐ aYnity glucose transporter (HXT) gene promoters. High‐aYnity glucose transporter gene promoters also bind the repressor MIG1, which is active only at high glucose concentrations. When no glucose is present, RGT1 represses all of the HXT genes and none are expressed; when glucose is present at low concentrations, neither RGT1 nor MIG1 is active so all of the HXT genes are expressed; when glucose is present at high concentrations, MIG1 represses the genes encoding high‐aYnity transporters and only the low‐aYnity transporters are expressed.

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high‐capacity transporters are expressed when the glucose concentration is high (Boles and Hollenberg, 1997; Kruckeberg, 1996; Reifenberger et al., 1997). This process ensures that the cell possesses an optimal complement of glucose transporters whatever the medium glucose concentration. Low‐ and high‐aYnity glucose sensors, SNF3 and RGT2, are located in the membrane. Binding of glucose to the sensors causes a signal to be generated that results in inactivation of a protein called RGT1, a transcriptional repressor of HXT gene promoters. The promoters of genes of high‐aYnity glucose transporters also bind the repressor MIG1, which is active at high glucose concentrations (Ozcan and Johnston, 1996). When no glucose is present, none of the HXT genes is expressed because of the action of RGT1. At low concentrations, neither RGT1 nor MIG1 is active so HXT genes are expressed, whereas at high concentrations, only low‐aYnity transporters are expressed because of the action of MIG1. C. CARBON METABOLITE SENSING AND SIGNALLING IN PLANTS

1. SnRK1 A plant homologue of SNF1 and, as was discovered later, of AMPK was first described in 1991 (Alderson et al., 1991). It encodes a 58‐kDa protein showing 48% amino acid sequence identity with SNF1 and AMPK, rising to 62–64% amino acid sequence identity in the kinase catalytic domain. Homologous genes have since been identified and characterised in many plant species and given the name SNF1‐related protein kinase‐1 (SnRK1) (Halford and Hardie, 1998). They are present in small‐ to medium‐sized gene families, comprising, for example, three members in Arabidopsis and 10–20 in barley. The SnRK1 gene family of cereals can be subdivided further into two groups, SnRK1a and SnRK1b, on the basis of amino acid sequence similarity and expression patterns (Halford and Hardie, 1998; Hannappel et al., 1995). A SNF4/AMPK homologue called AtSNF4 has been cloned from Arabidopsis by partial complementation of a snf4 mutant (Kleinow et al., 2000), whereas genes related to the SIP1/SIP2/GAL83/AMPK family have been cloned from Arabidopsis (AKIN 1 and AKIN 2) and potato (StubGAL83) (Bouly et al., 1999; Lakatos et al., 1999). AKIN 1 and AKIN 2 interact with SnRK1 in the two‐hybrid system but also with the yeast SNF1 and SNF4 proteins (Bouly et al., 1999). Potato StubGAL83 was isolated by screening a yeast two‐hybrid cDNA library with a potato SnRK1 cDNA (Lakatos et al., 1999). A maize homologue of AtSNF4 was given the name AKIN because it was found to contain an N‐terminal KIS domain fused with a C‐terminal domain similar to SNF4, AMPK , and AtSNF4 (Lumbreras et al., 2001).

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Re‐analysis of the Arabidopsis AtSNF4 gene shows that it too encodes a protein with this N‐terminal KIS domain. The reason for this domain fusion is not clear. Two other families of plant proteins show similarity with SNF4. These are the PV42 family, which includes PV42 from bean (Phaseolus vulgaris) and AKIN from Arabidopsis (Abe et al., 1995; Bouly et al., 1999), and the SnIP1 family (Slocombe et al., 2002). These show 20–25% amino acid sequence identity with SNF4 and interact with SnRK1 in two‐hybrid assays and in vitro. However, they do not complement the snf4 mutation in yeast and are unique to plants. Both PV42 and SnIP1 will align with SNF4 and AMPK , but show little sequence similarity with each other apart from a short, hydrophobic motif called the SnIP motif (Slocombe et al., 2002). The PV42 and SnIP1 families are unique to plants. 2. SnRK2 and SnRK3 The situation in plants is complicated further by the presence of two other subfamilies of protein kinases, SnRK2 and SnRK3, which contain catalytic domains with sequences that place them clearly within the SNF1 family. The relationship of these protein kinases with other members of the SNF1 family and other protein kinase families is represented in the dendrograms in Figs. 10 and 11A. SnRK2 and SnRK3 have 42–45% amino acid sequence identity with SnRK1, SNF1, and AMPK in the catalytic domain, but no similarity in the regulatory domain (Fig. 11B) and they are unique to plants.

Fig. 10. Dendrogram showing the relationships of subfamilies within the SNF1 family and between the SNF1 family and other families of protein kinases. The SNF1 family includes not only SNF1 (yeast) itself, SnRK1 (plants), and AMPK (mammals), but also two other subfamilies of protein kinases that are unique to plants, SnRK2 and SnRK3, and the cell cycle control proteins, NIM1 (fission yeast) and NIK1 (budding yeast).

Fig. 11. (A) Dendrogram showing the divergence of SNF1‐related protein kinases in plants into three subfamilies: SnRK1, SnRK2, and SnRK3. (B) Schematic showing the structures of SnRK1, SnRK2, and SnRK3 compared with SNF1 and AMPK. Note that there is considerable variation in size within the SnRK3 family. Figures within the diagrams give the amino acid sequence identity with SnRK1 in the catalytic and (where significant) regulatory domains.

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The SnRK2 subfamily includes PKABA1 from wheat, which is involved in mediating ABA‐induced changes in gene expression (Anderberg and Walker‐ Simmons, 1992; Go´ mez‐Cadenas et al., 1999), while the SnRK3 family includes SOS2, an Arabidopsis protein kinase involved in conferring salt tolerance (Halfter et al., 2000; Liu et al., 2000). Importantly, there is no evidence of functional redundancy between these subfamilies of protein kinases. There is some evidence that SnRK3 activity is calcium dependent, unlike that of SnRK1 and SnRK2, although it is not clear how important this regulation is (reviewed by Harper et al., 2004). The completion of the Arabidopsis genome sequencing project means that the full complement of the Arabidopsis SnRK gene family can now be identified. It comprises three SnRK1 genes, two of which (referred to previously as AKIN10 and AKIN11) are active while one (previously known as AKIN30) is silent (Deveraj Jhurreea, unpublished data), nine SnRK2 genes, and 29 SnRK3 genes (Halford et al., 2003a; Hrabak et al., 2003). It is an interesting evolutionary question as to why these subfamilies of SNF1‐ related protein kinase genes are absent from animals and fungi. One interpretation of the dendrogram shown in Fig. 10 is that these gene families were present in primitive eukaryotes, diverging from what would become SNF1, AMPK, and SnRK1 before the separation of plants, animals, and fungi. They were then lost in animals and fungi. However, I propose that SnRK2 and SnRK3 arose from duplication of the SnRK1 gene during plant evolution. The duplication event resulted in accelerated evolution and divergence of the SnRK2 and SnRK3 gene families, explaining their position on the dendrogram. Given the little that we do know about the function of SnRK2s and SnRK3s, one can hypothesise that their emergence allowed plants to develop networks that link environmental stress and metabolite signalling. Exactly when the SnRK2 and SnRK3 gene families diverged from SnRK1 is not yet clear. Two SnRK1 homologues have been identified in the moss, Physcomitrella (Thelander et al., 2004). As yet there is no report of the presence of SnRK2 or SnRK3 genes in this species, but a Chlamydomonas protein kinase called Sac3 involved in the response to sulphur limitation is a member of the SnRK2 family (Davies et al., 1999). 3. Substrates of SnRK1 a. Peptides. Once SnRK1 had been shown to be homologous to AMPK, it was possible to confirm that SnRK1 and a plant protein kinase related to AMPK that had been studied at the biochemical level in Grahame Hardie’s laboratory in Dundee were one and the same (Ball et al., 1995; Barker et al., 1996). Like AMPK, this protein kinase, which had hitherto been known as HMG‐CoA reductase kinase, had been shown to phosphorylate the SAMS

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peptide (MacKintosh et al., 1992). What is more, a purification procedure had already been developed, using cauliflower florets as a source (Ball et al., 1994). A recognition motif for SnRK1 has been established using variant peptide substrates (Weekes et al., 1993). It comprises the phosphorylated serine (SnRK1 will phosphorylate threonine but phosphorylates serine much more eYciently), hydrophobic residues at positions
5 and þ4 relative to the serine, and at least one basic residue, preferably at
3 but tolerated at
4 (Fig. 12). The AMARA peptide (Fig. 12) in which the minimal recognition motif is retained but other residues are alanine apart from the basic C terminus, which is not essential for phosphorylation, appears to be a better substrate than the SAMS peptide (Dale et al., 1995). More recently, basic residues at positions
6 and þ5 have been shown to enhance activity, and a proline residue at position
4 has been found to favour phosphorylation by SnRK1 relative to CDPKs (Huang and Huber, 2001). It is notable that whenever SAMS or AMARA peptide kinase activity has been purified, SnRK1 has accounted for most of it. A minor SAMS peptide kinase activity has been tentatively assigned to SnRK2 but has not been characterised in detail (Ball et al., 1994; Barker et al., 1996; Crawford et al., 2001; Sugden et al., 1999b). Plants expressing antisense SnRK1 sequences retain some AMARA and SAMS peptide kinase activities (Purcell et al., 1998), but it is not clear whether this is residual SnRK1 activity, the activity of SnRK2 and/or SnRK3, or nonspecific background. Mutants of Physcomitrella that lack SnRK1 have almost no AMARA peptide kinase activity at all (Thelander et al., 2004). Intriguingly, the SnIP motif found in PV42 and SnIP1 contains the sequence Hyd‐Xxx‐Bas‐Xxx‐Xxx‐Xxx‐Xxx‐Xxx‐Xxx‐Hyd, where Hyd represents a hydrophobic residue and Bas a basic residue. This resembles the SnRK1 recognition sequence without the target serine residue; such sites act as pseudo‐substrates in the regulatory subunits of the cAMP‐dependent kinase PKA of mammals (Taylor et al., 1990).

Fig. 12. Consensus sequence for phosphorylation by SnRK1 (top row). Required residues are shown in bold. ‘‘Bas’’ indicates a basic residue and ‘‘Hyd’’ a hydrophobic residue. Residues shown in brackets are not required but may enhance phosphorylation. SnRK1 will tolerate the basic residue being at
4 with respect to the serine instead of
3. The middle and bottom rows give the sequences of the AMARA and SAMS peptides, both of which are good substrates for SnRK1.

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b. HMG‐CoA reductase. The demonstration that HMG‐CoA reductase kinase and SnRK1 were one and the same established HMG‐CoA reductase as a substrate for SnRK1. The protein kinase had been shown to phosphorylate and inactivate a bacterially‐expressed Arabidopsis HMG‐CoA reductase in vitro (Ball et al., 1994); the phosphorylation site is Ser‐577. HMG‐CoA reductase catalyses the NADH‐dependent reduction of 3‐ hydroxy‐3‐methylglutaryl‐coenzyme A (HMG‐CoA) to mevalonic acid. In animals and yeast, this is the first committed step in the only pathway for the production of isopentenyl diphosphate, a five‐carbon unit from which all isoprenoids are made. Indeed, it is the overall rate‐limiting step for the isoprenoid biosynthetic pathway. Mevalonate synthesis via HMG‐CoA reductase is also a key step in isoprenoid biosynthesis in plants, although plants possess a second, plastidic pathway for IPP synthesis (Eisenreich et al., 1996). In particular, the HMG‐CoA reductase pathway appears to be the route through which phytosterols are synthesised. Plants contain multiple HMG‐CoA reductases; Arabidopsis, for example, has two: HMG1 and HMG2 (Enjuto et al., 1994). Both HMG1 and HMG2 contain a SnRK1 target site, indeed the site (Met Lys Tyr Asn Arg Ser Ser Arg Asp Ile) is conserved in all of the plant HMG‐CoA reductases characterised so far, with the first of the serine residues being the target for phosphorylation (Halford and Hardie, 1998). Note that it is a suboptimal site in that the basic residue is at
4 with respect to the target serine rather than at
3. The regulation of sterol biosynthesis in plants is of interest to biotechnologists because of the eYcacy of dietary phytosterols in reducing blood cholesterol in humans; with optimised dietary intake they can lower human serum total‐ and low‐density lipoprotein (LDL) or ‘‘bad’’ cholesterol by 10% (Katan et al., 2003). The predominant naturally‐occurring phytosterols, which include ‐sitosterol, campesterol, and stigmasterol, are related structurally to cholesterol (Fig. 13). It has been suggested that they competitively inhibit the uptake of cholesterol from the small intestine (Westrate and Meijer, 1998). Alternatively, they may lead to an increase in the expression of the sterol transporter (Ostlund, 2004; Plat and Mensink, 2002), leading to increased excretion of sterols, including cholesterol, back to the gut lumen. The significance of HMG‐CoA reductase regulation by SnRK1 in the control of phytosterol biosynthesis has been demonstrated by producing transgenic tobacco plants expressing modified HMG‐CoA reductases lacking a SnRK1 target site (Hey et al., in press). Levels of seed sterols were up to 2.5‐fold higher in plants expressing the transgene than in wild type. However, other regulatory mechanisms aVect the activity of HMG‐CoA reductase in plants. One of these must involve the N‐terminal,

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Fig. 13. Diagram showing similarity in the structure of cholesterol and the predominant, naturally‐occurring phytosterols.

membrane‐spanning domain because seed phytosterol levels in tobacco plants expressing an N‐terminally truncated HMG‐CoA reductase lacking this domain were enhanced by up to 3.2‐fold (Harker et al., 2003). c. Sucrose phosphate synthase and nitrate reductase. Two other important enzymes, sucrose phosphate synthase (SPS) and nitrate reductase (NR), have been shown to be substrates for SnRK1. SPS is phosphorylated

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at Ser‐158 and NR at Ser‐543 (Bachmann et al., 1996b; Douglas et al., 1995; Su et al., 1996). In both cases phosphorylation results in inactivation of the enzyme, although the inactivation of NR also requires the binding of a 14‐3‐3 protein to the phosphorylation site (Bachmann et al., 1996a; Moorhead et al., 1996). SPS catalyzes the first step in the synthesis of sucrose, and the fact that SnRK1 potentially regulates SPS activity makes SnRK1 a possible key regulator of source to sink carbon flux and acclimation to carbon supply. Increased leaf SPS activity, for example, would result in more sucrose synthesis, and more sucrose would be available for export to sinks such as seeds and tubers. SnRK1 could be involved in the inactivation of SPS to reduce the export of sucrose from the leaf in response to environmental conditions that limit carbon supply. Phosphorylation at Ser‐158 causes an inactivation at subsaturating concentrations of the substrate, glucose‐6‐phosphate, and high concentrations of the inhibitor, phosphate (McMichael et al., 1993). Nitrate reductase catalyses the first step in the assimilation of nitrogen from nitrate into organic compounds such as amino acids. Although SnRK1 is only one of several protein kinases that phosphorylate nitrate reductase, this potential regulatory mechanism is particularly important because it could be a link between carbon and nitrogen signalling. d. Small heat shock protein (HSP). SnRK1 has been shown to interact with and phosphorylate a small heat shock protein from barley (Slocombe et al., 2004). Although the small HSP is a less eVective substrate than HMG‐ CoA reductase, it is phosphorylated at a specific phosphorylation site towards the N‐terminal end. This site does not conform to the consensus sequence for a SnRK1 target site, which probably explains why the small HSP is a relatively poor substrate. Some other small heat shock proteins are known to be phosphorylated. For example, mammalian small HSPs are substrates for MAPK‐activated protein kinase‐2 (Freshney et al., 1994; Rouse et al., 1994) and maize mitochondrial small heat shock protein HSP22 is phosphorylated significantly at the N terminus in vivo, although the protein kinase responsible has not been identified (Lund et al., 2001). At present, it is not known if N‐terminal phosphorylation of small HSPs by SnRKI leads to a change in function. 4. Redox regulation of ADP‐glucose pyrophosphorylase (AGPase) ADP‐glucose pyrophosphorylase is a plastidic enzyme that catalyses a key step in starch biosynthesis, the conversion of glucose‐1‐phosphate to ADP‐ glucose. Its activity is regulated to a great extent by post‐translational redox modulation; AGPase is a heterotetramer of two small and two large

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subunits, and the regulatory mechanism involves formation of a cysteine bridge between the two small subunits (Fu et al., 1998). Redox regulation of AGPase plays an important role in the control of starch synthesis in response to carbon supply in potato tubers (Tiessen et al., 2002). In tuber discs cut from plants and incubated in the absence of sugars, redox activation of AGPase decreases, but this can be prevented by feeding with either sucrose or glucose (Tiessen et al., 2003). Fascinatingly, palatinose, a nonmetabolisable analogue of sucrose, mimics the sucrose eVect, indicating that sucrose does not have to be metabolised to initiate a signal. Furthermore, sucrose‐ induced redox activation of AGPase does not occur in discs cut from tubers with reduced SnRK1 activity, whereas glucose‐induced activation does. This shows that sucrose‐ and glucose‐induced redox activation of AGPase involve diVerent signalling pathways and puts SnRK1 clearly in the sucrose sensing and signalling pathway but not, in this case, in the glucose sensing and signalling pathway. It also begs the question of how the signal is passed from the cytosol to the plastid, as there is no evidence of SnRK1 being present in plastids. 5. Involvement of SnRK1 in the regulation of gene expression The regulation of gene expression in response to sugars was first demonstrated by Jen Sheen in 1990. She used photosynthetic gene promoter/reporter gene fusions to show that seven maize photosynthetic genes were repressed by glucose or sucrose in a maize protoplast system. The expression of the ribulose 1,5‐bisphosphate carboxylase/oxygenase small subunit (RbcS) gene was shown to be reduced by glucose in cell suspensions of Chenopodium rubrum (Krapp et al., 1993); in the same system, chlorophyll a/b binding protein (Cab) and thylakoid ATPase delta subunit gene expression was reduced by glucose, whereas Cab gene expression was shown to be reduced by sucrose in oilseed rape cell cultures (Harter et al., 1993; Krapp et al., 1993). The demonstration that expression of photosynthetic genes is under feedback regulation by sugars was a landmark discovery (reviewed by Sheen, 1994). The list goes on: genes encoding isocitrate lyase and malate synthase, the two key enzymes involved in the glyoxylate cycle, were shown to be repressed by glucose (Graham et al., 1994). Similarly, the expression of genes encoding ‐amylase was shown to be induced by sugar starvation in rice suspension cultures and repressed by feeding with sucrose, fructose, or glucose (Yu et al., 1991). Conversely, the expression of a gene encoding ‐ amylase was found to be induced by sugars in rosette leaves of Arabidopsis (Mita et al., 1995). Nowadays, of course, broad pictures of eVects on gene expression can be gained by performing microarray experiments, and in 2004 Price and

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co‐workers showed that 444 Arabidopsis genes are upregulated by glucose, including those involved in biotic and abiotic responses, carbohydrate metabolism, N metabolism, lipid metabolism, inositol metabolism, secondary metabolism, nucleic acid related activities, protein synthesis and degradation, transport, signal transduction (e.g., protein kinases and phosphatases, and transcription factors), hormone synthesis, and cell growth or structure. A similar number (534) are downregulated. Section III.C.4 described the eVects of sucrose and glucose feeding on AGPase activity, noting that sucrose and glucose signalling involves diVerent signalling pathways. Experiments on the eVects of these two sugars on gene expression had already indicated that this was the case. For example, sucrose synthase activity increases in response to sucrose but is not aVected by glucose (Salanoubat and Belliard, 1989; Sowokinos and Varns, 1992). Fu and Park (1995) showed that this eVect is mediated at the level of gene expression; they described two diVerentially‐expressed potato sucrose synthase genes, one of which (SUS4) is expressed only in tubers under normal conditions but can be induced in detached leaves in response to incubation with high concentrations of sucrose. The other (SUS3) is expressed predominantly in stems and roots and is not induced by sucrose treatment. Neither gene responds to glucose. Sucrose synthase activity and the onset of starch biosynthesis have also been shown to coincide with an increase in sucrose levels and a decrease in hexose levels in the developing cotyledons of Vicia faba (Weber et al., 1996). Because sucrose synthase catalyses the reversible conversion of sucrose and UDP to UDP‐glucose and fructose, this finding is perhaps not surprising. Nevertheless, the discovery that sucrose can be sensed independently of glucose is an important one. It should be remembered when considering this that the eVects of sucrose and glucose depend in part on the presence in the tissue that is being studied of enzymes such as invertase, sucrose synthase, and sucrose phosphate synthase that interconvert them. If sucrose is cleaved to hexoses prior to entry into a cell, then it is unlikely to initiate a diVerent signal to glucose. Other examples of specifically sucrose‐ induced gene expression include a patatin gene in potato (Grierson et al., 1994) (patatin is the major storage protein in potato tubers) and a nitrate reductase gene in Arabidopsis (Cheng et al., 1992). So what role does SnRK1 play in sucrose or glucose regulation of gene expression? Such a role is suggested strongly by the ability of SnRK1 to complement mutations in SNF1 in yeast. One of the functions of SNF1 is the transcriptional regulation of genes encoding enzymes of carbohydrate metabolism. SnRK1 will perform this role in yeast snf1 mutants to the extent that the yeast can utilise sucrose and nonfermentable carbon sources such as

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ethanol and glycerol (Alderson et al., 1991; Muranaka et al., 1994). It should be emphasised that this results from the derepression of gene expression, not some other function of SNF1. Confirmation of a role for SnRK1 in regulating gene expression in plants was obtained in 1998 using potato plants expressing an antisense SnRK1 gene specifically in the tubers or leaves (Purcell et al., 1998). The expression of sucrose synthase was much lower in tubers expressing the antisense SnRK1 gene than in wild‐type and could not be induced by incubation of excised leaves expressing the antisense SnRK1 gene, again in contrast to wild‐type. Further investigations have shown that the expression of genes encoding ADP‐glucose pyrophosphorylase, ‐amylase, and sucrose phosphate synthase decrease as a result of antisense inhibition of SnRK1 gene expression in potato tubers, whereas both sucrose synthase and ADP‐ glucose pyrophosphorylase gene expression increases if SnRK1 is overexpressed (McKibbin et al., unpublished data). An ‐amylase gene promoter has also been shown to be repressed by antisense SnRK1 in wheat embryos (Laurie et al., 2003). SnRK1 therefore has the potential to control starch biosynthesis and mobilisation at multiple control points, at the levels of both gene expression and post‐translational modulation of enzyme activity, and in response to the levels of sucrose (sucrose synthase, AGPase) or glucose (‐amylase) (Fig. 14). Clearly, subtle regulation of SnRK1 itself and its downstream targets must determine whether starch is accumulated or mobilised. For example, antisense inhibition of SnRK1 may indicate that SnRK1 is required for the expression of a particular gene, but the presence or absence of transcription factors will determine whether SnRK1 regulates the expression of that gene in a specific tissue, cell type, or developmental phase. 6. SnRK1 is implicated in cell cycle control Antisense inhibition of SnRK1 induces a large mitotic cell size in potato cell cultures (Francis and Halford, unpublished data), suggesting that SnRK1 could cross‐talk with components of cell cycle signalling. In yeast cells, however, overexpression of SnRK1 causes a reduction in cell volume to one‐third of normal (Dickinson et al., 1999). There has been evidence of a link between metabolite signalling and the cell cycle in yeast for some time: snf1 mutants fail to arrest in the G1 phase of the cell cycle under conditions of nutrient deprivation (Thompson‐Jaeger et al., 1991) and have a shorter G1 period than the wild‐type (Aon and Cortassa, 1999). Furthermore, SNF1 is related to NIK1 of Saccharomyces cerevisiae and its homologue, NIM1, in Saccharomyces pombe (Fig. 10). NIK1 interacts with the CDC28 complex and is a negative regulator of

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Fig. 14. Points of influence and modes of action for SnRK1 in the starch biosynthetic pathway. Carbon ‘‘flow’’ through the pathway is shown with black arrows; signalling interactions are indicated with grey arrows.

SWE1, a protein kinase involved in the calcium‐dependent control of mitosis (Tanaka and Nojima, 1996). ELM1, one of the protein kinases upstream of SNF1, has also been implicated in the regulation of SWE1. ELM1 mutants have a prolonged mitotic delay, fail to regulate polar bud growth during mitosis, and are defective in cytokinesis (Sreenivasan and Kellogg, 1999). A role for SnRK1 in meristems was suggested by Pien et al. (2001) based on the finding that genes encoding SnRK1, sucrose synthase, and AGPase showed an asymmetric pattern of expression within tomato leaf meristems. A possible role for SnRK1 in meristems could be to link metabolic status to cell cycle control. 7. Phenotypic eVects of manipulating SnRK1 activity Manipulation of SnRK1 activity can have dramatic eVects on plant development, the most extreme of which is to prevent plant growth altogether. For example, eVorts to produce transgenic potato plants expressing antisense SnRK1 under the control of a constitutive promoter (CaMV35S) failed (Purcell et al., 1998). This was despite multiple attempts being made and the fact that successful transformation experiments were being performed alongside using the same antisense sequence but tissue‐specific promoters, suggesting that the failure resulted from a lethal eVect of the transgene during tissue culture.

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Transgenic Arabidopsis plants have now been produced expressing antisense and RNAi SnRK1 sequences under the control of a CaMV35S promoter (unpublished data). These were produced by the floral dip method so no tissue culture was involved, perhaps explaining why these transformation experiments were successful while those with potato were not. A ‘‘lethal’’ eVect has also been observed with expression of an antisense SnRK1 sequence in barley pollen (Zhang et al., 2001). Analysis of pollen grains carrying an antisense SnRK1 transgene showed that they had arrested at the binucleate stage of development, were small, pear shaped, contained little or no starch, and were nonfunctional. Carbohydrate metabolism plays an important role in pollen grain development, and pollen infertility is often associated with a lack of starch accumulation, brought about by mutation (Joppa et al., 1966) or physiological stress (Graham, 1975; Ito, 1978; Saini et al., 1984). Starch acts as a major energy source for development, germination, and tube growth (Baker and Baker, 1979; Clement et al., 1994; Franchi et al., 1996; Pacini and Franchi, 1988). It is possible that inhibition of starch accumulation caused the arrest in pollen development. A less severe phenotype, delayed sprouting, has been observed in potato cv. Desiree tubers expressing an antisense SnRK1 sequence (Halford et al., 2003b). The transgenic tubers do not sprout at all if kept at 4 8C, even after long‐term storage (Fig. 15), although they will sprout when potted up and transferred to a glasshouse. However, recent experiments with cv. Prairie tubers expressing an antisense SnRK1 sequence have failed to replicate this result (unpublished data), indicating that the genetic background plays an important role in the appearance of this phenotype. SnRK1 activity has also been manipulated in the moss, Physcomitrella patens (Thelander et al., 2004). The advantage of using this species is that

Fig. 15. Tubers from wild‐type potato cv. Desiree and transgenic potato cv. Desiree expressing an antisense SnRK1 gene.

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knockout mutants can be produced. Physcomitrella patens contains two SnRK1 genes, and the double knockout mutant shows a complex phenotype of abnormal development, premature senescence, hypersensitivity to auxin, hyposensitivity to cytokinin, and a requirement for continuous light. The requirement for continuous light may again indicate a perturbation in starch accumulation and breakdown, as this has been shown to be required for Arabidopsis to grow normally in a day/night cycle (Caspar et al., 1985, 1991; Lin et al., 1988). 8. Summary of ‘‘downstream’’ eVects of SnRK1 Like its fungal and animal counterparts, SNF1 and AMPK, SnRK1 can control aspects of carbon metabolism at multiple points, potentially aVecting sterol biosynthesis, the accumulation and breakdown of sucrose and starch (with implications for sucrose transport, storage organ development, and carbon partitioning), and the assimilation of nitrogen into organic compounds. It is also implicated in cross talk with cell cycle control mechanisms and in the control of development. Its ‘‘downstream’’ eVects are summarised in Fig. 16. 9. Upstream factors: activation of SnRK1 It has been something of a frustration that the metabolite that is sensed at the ‘‘top’’ of the SnRK1 signal transduction pathway has remained elusive. Feeding experiments with diVerent sugars and plant tissues have failed to give convincing, repeatable results, although glucose‐6‐phosphate has been shown to inhibit SnRK1 in vitro (Toroser et al., 2000). However, it is beginning to be teased out, and the candidates 50 ‐AMP and trehalose‐6‐ phosphate are stimulating considerable interest. Section III.A described the role of 50 ‐AMP as a signalling molecule in animal cells. This metabolite, which accumulates in animal cells as a result of nutrient and other stresses, activates AMPK allosterically. No such activation occurs with SnRK1 (or SNF1), but 50 ‐AMP does modulate the phosphorylation state of SnRK1 and therefore its activity. SnRK1 is regulated in vitro by phosphorylation on a threonine residue within the so‐called T‐loop (Sugden et al., 1999a); dephosphorylation and inactivation have been found to be inhibited by low concentrations of 50 ‐AMP (Sugden et al., 1999a). The implication of this is that 50 ‐AMP could activate the protein kinase upstream of SnRK1 (Fig. 17). Attempts to identify and purify this protein kinase in order to test this hypothesis have proved unsuccessful. However, this work has been given a new impetus by the identification of analogous protein kinases in humans (LKB1) and yeast (ELM1, PAK1, and TOS3). Although a clear homologue

Fig. 16. Targets and modes of action for SnRK1.

Fig. 17. Possible pathways for activation of SNF1‐related protein kinase‐1 (SnRK1). Carbon ‘‘flow’’ from glucose to CO2 is indicated with black arrows; signalling interactions are shown with grey arrows; and dotted lines are used where the mechanism for an interaction is not known.

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of these protein kinases does not exist in plants, there are a number of hitherto uncharacterised candidates from Arabidopsis (unpublished data). There is no doubt that post‐translational modulation of SnRK1 activity is important; experiments in potato have shown that, for example, activity in minitubers is over 40 times higher than in mature tubers while the levels of transcript do not diVer (Man et al., 1997). As described in Section III.B. T6P has an inhibitory eVect on hexokinase in yeast, thereby regulating flux into glycolysis. While it may not perform this exact function in plants, evidence has shown it to be an essential component of metabolic signalling (Eastmond et al., 2002; Pellny et al., 2004; Schluepman et al., 2003, 2004). Furthermore, increasing evidence shows that SnRK1 interacts with and possibly regulates the trehalose biosynthetic pathway. For example, several Arabidopsis trehalose phosphate synthases contain a SnRK1 target site, and expression of one of the two SnRK1 genes in Arabidopsis correlates with T6P content (Schleupmann et al., 2004). Trehalose feeding has been shown to induce the expression of AGPase and starch synthesis in Arabidopsis (Wingler et al., 2000), further evidence of a possible link between trehalose metabolism and SnRK1. Glucose itself is attractive as a signalling molecule because it is universal to all organisms as a source of energy and carbon skeletons. Yeast does sense glucose levels through the membrane‐located sensors SNF3 and RGT2 (Section III.B, Fig. 9), but these initiate signals only through the glucose induction pathway. The metabolite that is sensed to initiate a signal through SNF1 and the more important glucose repression pathway remains elusive. If glucose is a signalling molecule, then one possible mechanism through which it could be sensed is its interaction with hexokinase. In plants, hexokinase has been associated particularly with photosynthetic metabolism as a mechanism that links the carbohydrate status of leaves to photosynthetic gene expression as a means of feedback control (Jang et al., 1997; reviewed by Jang and Sheen, 1997). Proving that hexokinase is a glucose sensor and signalling molecule is diYcult because of its important metabolic role (Halford et al., 1999). Any attempt to alter hexokinase activity in transgenic or mutant plants or with inhibitors may cause changes in other potentially important signalling molecules downstream of hexokinase, such as glucose‐6‐phosphate and 50 ‐AMP. However, Moore and co‐workers (2003) used targeted mutagenesis of one of the Arabidopsis hexokinase genes, HXK1, to produce enzymes that were catalytically inactive; glucose binding still occurred but glucose‐6‐ phosphate was not formed. In transgenic plants the two catalytically inactive hexokinases mediated glucose‐dependent developmental arrest and repression of chlorophyll accumulation without altering catalytic activity

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or glucose‐6‐phosphate and fructose‐6‐phosphate content. This separation of catalytic and signalling functions is good evidence of a signalling role for hexokinase. Nevertheless, frustratingly given that the hexokinase signalling hypothesis was proposed in the mid‐1990s, there is still no convincing hypothesis of how hexokinase generates a signal or what signalling pathway transduces that signal. Hexokinase signalling appears to aVect gene expression, cell proliferation, root and inflorescence growth, leaf expansion, and senescence. Some of these eVects may be mediated through cross‐talk with light and hormone signalling. For example, there is good evidence for interaction and antagonism between hexokinase signalling, light, and cytokinin in the regulation of leaf senescence (Hwang and Sheen, 2001; Wingler et al., 1998). Evidence also shows that glucose signalling interacts with auxin signalling to promote or inhibit growth, depending on tissue or glucose concentration (Moore et al., 2003). Thus, many of the growth‐promoting and hormonal‐like properties of sugars may be explained in part through interactions between hexokinase and hormone signalling pathways. Hexokinase sensing and signalling cannot be the whole story as far as glucose sensing goes in plants is concerned because hexose levels in the vacuole or apoplast of tobacco plants have been shown to be sensed (Heineke et al., 1994; Herbers et al., 1996) and hexokinase is a cytosolic enzyme. It is also notable that the pathway for hexose phosphate production from sucrose via sucrose synthase (the predominant pathway in storage organs) does not involve hexokinase or the production of glucose at all (Fig. 17). Other candidates for glucose sensors are sugar transport proteins (STPs) (Fig. 17). Arabidopsis has 14 STP genes (reviewed by Sherson et al., 2003), the best characterised of which, AtSTP1, encodes a protein that transports galactose, xylose, and mannose, as well as glucose, and responds sensitively to extracellular sugar availability. The glucose to be transported is produced by the hydrolysis of incoming sucrose to glucose and fructose by cell wall invertases, of which Arabidopsis has six. The expression of diVerent invertases and STPs may enable the plant to change its hexose utilisation in response to diVerent environmental conditions; it has also been suggested that STPs have a role in hexose sensing and signal initiation (Sherson et al., 2003). Note that in some tissues and cell types sucrose is imported into the cell (Fig. 17), and the mechanism for fructose import into the cell is still unknown. Whether SnRK1 is involved in hexose sensing and signalling is not clear. Its involvement in regulating ‐amylase (‐AMY2) gene expression in cultured wheat endosperms (Laurie et al., 2003) suggests that it is. The ‐AMY2 gene is glucose‐repressible and starvation‐inducible, making it similar to glucose‐repressed, SNF1‐controlled genes in yeast. SNF1 activity

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does respond rapidly and sensitively to glucose levels (Wilson et al., 1996), although the sensing mechanism is not known. However, this has never been demonstrated unambiguously for SnRK1. SnRK1 certainly appears to be required for the transduction of signals that are initiated by sucrose. The evidence for this was described earlier; the involvement of SnRK1 in regulating sucrose synthase gene expression and AGPase activity in response to sucrose is quite clear. The fact that independent sensing of sucrose has evolved in plants is not surprising, as sucrose is found in all plants, is the end product of photosynthesis, the major transported sugar and the starting point for energy metabolism, and the synthesis of structural components of cells. Nevertheless, the involvement of SnRK1 is interesting because it does not perform a similar function in animals or fungi. It is not known what lies upstream of SnRK1 in the sucrose signalling pathway. However, sucrose transport in plants is mediated by proton‐ coupled sucrose transporters (SUT) (Lalonde et al., 1999, 2003). These SUT proteins are related structurally to the HXT transporters and related glucose sensors of yeast (Section III.B). The SUT transporters have diVerent aYnities for sucrose, with SUT1 being the high‐aYnity transporter and SUT4 the low‐aYnity transporter, while SUT2 appears to be a combined transporter and sensor (Barker et al., 2000; Weise et al., 2000). All three proteins have been localised in sieve elements (Reinders et al., 2002).

IV. THE LINK BETWEEN SUGAR AND ABSCISIC ACID/STRESS SIGNALLING PATHWAYS In the last few years there has been a burgeoning interest in understanding the genetics of cross‐talk between hormone and metabolic pathways, particularly ABA and sugar signalling (Brocard‐GiVord et al., 2003). This was stimulated in part by the results of screens carried out to identify Arabidopsis mutants that are impaired in their response to sugar (sugar response mutants). Several of the mutants identified in these screens turned out to be ABA related, leading to the hypotheses that sugar signalling is mediated directly by ABA (Arenas‐Huertero et al., 2000; Smeekens, 2000), that ABA modulates sugar signalling by priming tissues to respond to sugars (Rook et al., 2001), or that although essentially separate, ABA and sugar signalling converge and cross‐talk through specific factors (Halford and Paul, 2003). Such factors would be expected to be involved in the control of developmental events such as germination and seedling establishment that are sensitive to both ABA and sugars. One of the ABA‐related mutants implicated in sugar signalling is abi5 (ABA‐insensitive 5). Like all of the abi mutants, abi5 was isolated because of the ability of mutant seeds to germinate in the presence of normally

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inhibitory concentrations of ABA (Finkelstein, 1994). The ABI5 gene was found to encode a basic leucine zipper (bZIP) transcription factor that regulates expression of a subset of late embryogenesis‐abundant (LEA) genes (Finkelstein and Lynch, 2000; Lopez‐Molina and Chua, 2000). These LEA genes include AtEM1 and AtEM6. ABI5 is expressed most strongly in seeds but also in vegetative and floral tissues, where it is induced by ABA and stress treatments (Brocard et al., 2002). The ABI5 protein interacts with another transcription factor, ABI3, in two‐hybrid experiments (Nakamura et al., 2001). The two transcription factors have also been shown to act synergistically with one another in the activation of AtEM6, phaseolin, and barley HVA1 and HVA22 gene promoters in rice protoplast transient expression experiments (Gampala et al., 2002). Significantly, their action in these experiments was inhibited by overexpression of aba1–1, a dominant‐negative allele of protein phosphatase ABI1, suggesting that phosphorylation plays a part in their regulation. ABI3 encodes a B3‐binding domain transcription factor and is homologous to the maize gene VP1. Both ABI3 and VP1 have attracted interest because of the significance of VP1/ABI3 in controlling the transition between seed development and germination (Kurup et al., 2000; McKibbin et al., 2002). The product of another ABI gene, ABI4, is believed to be involved with ABI3 and ABI5 in a regulatory network (Brocard et al., 2002), although the nature of the interaction is not known. ABI4 encodes an Apetela2‐type transcription factor (Finkelstein et al., 1998). Overexpression of ABI3, 4, and 5 causes hypersensitivity to sugar as well as ABA, as measured by the ability to germinate in the presence of sugar/ ABA (Brocard et al., 2002). Mutations in ABI4 have been identified in a number of sugar response mutants: impaired sucrose induction‐3 (isi3), sucrose uncoupled‐6 (sun6), sucrose insensitive‐5 (sis5), and glucose insensitive‐6 (gin6). The abi5 mutant is also glucose and mannose insensitive (Arenas‐Huertero et al., 2000; Laby et al., 2000).

V. PROSPECTS Protein phosphorylation is one of the major mechanisms by which cells react to extracellular signals, regulate cellular processes, and respond to nutritional and environmental stimuli. The characterisation and functional analysis of protein kinases therefore represents an important target for plant scientists. SnRK1 and the genes that encode it have been characterised from the molecular level through to the identification of substrates and

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downstream eVects on sucrose synthase, ‐amylase, sucrose phosphate synthase, and ADP‐glucose pyrophosphorylase gene expression. These studies have placed SnRK1 firmly in the sugar sensing signal transduction pathway and at the heart of the control of carbon partitioning. Somewhat disappointingly, SnRK1 remains one of only a few plant protein kinases to have been characterised at the molecular and biochemical level. While less mature, studies on GCN2 in plants suggest that it plays a role in controlling amino acid biosynthesis in response to amino acid availability. A key question is to what extent is a general amino acid control system like that of yeast operating in plant cells. A great opportunity and challenge of the coming years is to exploit the rapidly expanding genomic data and the new ‘‘omics’’ technologies and bioinformatics to increase the pace of discovery in the elucidation of the signalling networks in which SnRK1 and GCN2 operate. The establishment of a consensus sequence for the target phosphorylation site in SnRK1 substrates makes the use of bioinformatics particularly appealing. However, the degeneracy within the target site and the tolerance of apparently any residue at some positions adjacent to the target serine (Fig. 12) make searches for proteins containing the site less than trivial, and specific search tools will have to be developed. Proteomic/phosphoproteomic analyses are based on the identification of proteins that become more or less abundant or are phosphorylated to a greater or lesser degree in response to a particular treatment. The proteins are identified by matrix assisted laser desorption ionisation time of flight (MALDI‐ToF) mass spectrometry (MS) and/or liquid chromatography‐ MS/MS. It should enable components of protein kinase cascades and their target enzymes and transcription factors to be identified much more rapidly than has been possible before. It may even be possible to identify the conduits between pathways that link them into networks. The feasibility of such an approach has been demonstrated in an analysis of the Arabidopsis plasma membrane (Nu¨ hse et al., 2004). This identified receptor‐like and other protein kinases, putative signalling proteins, transporters, ATPases, nuclear proteins, proteins involved in lipid metabolism, traYcking and cellolose synthesis, and others that are phosphorylated.

ACKNOWLEDGMENT Rothamsted Research receives grant‐aided support from the Biotechnology and Biological Sciences Research Council (BBSRC) of the United Kingdom.

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REFERENCES Abe, H., Kamiya, Y. and Sakurai, A. A. (1995). cDNA clone encoding yeast SNF4‐ like protein from Phaseolus vulgaris. Plant Physiology 110, 336. Albani, D., Hammond‐Kosack, M. C. U., Smith, C., Conlan, S., Colot, V., Holdsworth, M. and Bevan, M. W. (1997). The wheat transcriptional activator SPA: A seed‐specific bZIP protein that recognises the GCN4‐like motif in the bifactorial endosperm box of prolamin genes. The Plant Cell 9, 171–184. Alderson, A., Sabelli, P. A., Dickinson, J. R., Cole, D., Richardson, M., Kreis, M., Shewry, P. R. and Halford, N. G. (1991). Complementation of snf1, a mutation aVecting global regulation of carbon metabolism in yeast, by a plant protein kinase cDNA. Proceedings of the National Academy of Sciences USA 88, 8602–8605. Anderberg, R. J. and Walker‐Simmons, M. K. (1992). Isolation of a wheat cDNA clone for an abscisic acid‐inducible transcript with homology to protein kinases. Proceedings of the National Academy of Sciences USA 89, 10183–10187. Aon, M. G. and Cortassa, S. (1999). Quantitation of the eVects of disruption of catabolite (de)repression genes on the cell cycle behaviour of Saccharomyces cerevisiae. Current Microbiology 38, 57–60. Arenas‐Huertero, F., Arroyo, A., Zhou, L., Sheen, J. and Leon, P. (2000). Analysis of Arabidopsis glucose insensitive mutants, gin5 and gin6, reveals a central role of the plant hormone ABA in the regulation of plant vegetative development by sugar. Genes and Development 14, 2085–2096. Bachmann, M., Huber, J. L., Liao, P. C., Gage, D. A. and Huber, S. C. (1996a). The inhibitor protein of phosphorylated nitrate reductase from spinach (Spinaea oleracea) leaves is a 14‐3‐3 protein. FEBS Letters 387, 127–131. Bachmann, M., Shiraishi, N., Campbell, W. H., Yoo, B. C., Harmon, A. C. and Huber, S. C. (1996b). Identification of Ser‐543 as the major regulatory phosphorylation site in spinach leaf nitrate reductase. Plant Cell 8, 505–517. Baker, H. G. and Baker, I. (1979). Starch in angiosperm pollen grains and its evolutionary significance. American Journal of Botany 66, 591–600. Ball, K. L., Barker, J. H. A., Halford, N. G. and Hardie, D. G. (1995). Immunological evidence that HMG‐CoA reductase kinase‐A is the cauliflower homologue of the RKIN1 subfamily of plant protein kinases. FEBS Letters 377, 189–192. Ball, K. L., Dale, S., Weekes, J. and Hardie, D. G. (1994). Biochemical characterisation of two forms of 3‐hydroxy‐3‐methyl‐glutaryl CoA reductase kinase from cauliflower (Brassica oleracea). European Journal of Biochemistry 219, 743–750. Barker, J. H. A., Slocombe, S. P., Ball, K. L., Hardie, D. G., Shewry, P. R. and Halford, N. G. (1996). Evidence that barley 3‐hydroxy‐3‐methylglutaryl‐ Coenzyme A reductase kinase is a member of the sucrose nonfermenting‐1‐ related protein kinase family. Plant Physiology 112, 1141–1149. Barker, L., Ku¨ hn, C., Weise, A., Schulz, B., Gebhardt, C., Hirner, B., Hellmann, H., Schulze, W., Ward, J. M. and Frommer, W. B. (2000). SUT2, a putative sucrose sensor in sieve elements. Plant Cell 12, 1153–1164. Beg, Z. H., Allmann, D. W. and Gibson, D. M. (1973). Modulation of 3‐hydroxy‐3‐ methylglutaryl coenzyme A reductase activity with cAMP and with protein fractions of rat liver cytosol. Biochemical and Biophysical Research Communications 54, 1362–1369.

130

N. G. HALFORD

Boles, E. and Hollenberg, C. P. (1997). The molecular genetics of hexose transport in yeasts. FEMS Microbiology Reviews 21, 85–111. Bonini, B. M., Van Vaeck, C., Larsson, C., Gustafsson, L., Ma, P., Winderickx, J., Van Dijck, P. and Thevelein, J. M. (2000). Expression of Escherichia coli otsA in a Saccharomyces tps1 mutant restores growth and fermentation with glucose and control of glucose influx into glycolysis. Biochemical Journal 350, 261–268. Bouly, J. P., Gissot, L., Lessard, P., Kreis, M. and Thomas, M. (1999). Arabidopsis thaliana homologues of the yeast SIP and SNF4 proteins interact with AKIN1, a SNF1‐like protein kinase. Plant Journal 18, 541–550. Brocard, I. M., Lynch, T. J. and Finkelstein, R. R. (2002). Regulation and role of the Arabidopsis abscisic acid‐insensitive 5 gene in abscisic acid, sugar, and stress response. Plant Physiology 129, 1533–1543. Brocard‐GiVord, I. M., Lynch, T. J. and Finkelstein, R. R. (2003). Regulatory networks in seeds integrating developmental, abscisic acid, sugar, and light signaling. Plant Physiology 131, 78–92. Carling, D., Aguan, K., Woods, A., Verhoeven, A. J. M., Beri, R. K., Brennan, C. H., Sidebottom, C., Davison, M. D. and Scott, J. (1994). Mammalian AMP‐activated protein kinase is homologous to yeast and plant protein kinases involved in the regulation of carbon metabolism. Journal of Biological Chemistry 269, 11442–11448. Carling, D., Clarke, P. R., Zammit, V. A. and Hardie, D. G. (1989). Purification and characterisation of the AMP‐activated protein kinase: Co‐purification of acetyl‐CoA carboxylase and 3‐hydroxy‐3‐methylglutaryl CoA reductase kinase activity. European Journal of Biochemistry 186, 129–136. Carling, D., Zammit, V. A. and Hardie, D. G. (1987). A common bicyclic protein kinase cascade inactivates the regulatory enzymes of fatty acid and cholesterol biosynthesis. FEBS Letters 223, 217–222. Carlson, C. A. and Kim, K. H. (1973). Regulation of hepatic acetyl coenzyme A carboxylase by phosphorylation and dephosphorylation. Journal of Biological Chemistry 248, 378–380. Caspar, T., Huber, S. C. and Somerville, C. (1985). Alterations in growth, photosynthesis, and respiration in a starchless mutant of Arabidopsis thaliana (L.) deficient in chloroplast phosphoglucomutase activity. Plant Physiology 79, 11–17. Caspar, T., Lin, T. P., Kakefuda, G., Benbow, L., Preiss, J. and Somerville, C. R. (1991). Mutants of Arabidopsis with altered regulation of starch degradation. Plant Physiology 95, 1181–1188. Celenza, J. L. and Carlson, M. (1986). A yeast gene that is essential for release from glucose repression encodes a protein kinase. Science 233, 1175–1180. Celenza, J. L. and Carlson, M. (1989). Mutational analysis of the Saccharomyces cerevisiae SNF1 protein kinase and evidence for functional interaction with the SNF4 protein. Molecular and Cellular Biology 9, 5034–5044. Celenza, J. L., Eng, F. J. and Carlson, M. (1989). Molecular analysis of the Snf4 gene of Saccharomyces cerevisiae: Evidence for the physical association of the Snf4 protein with the Snf1 protein kinase. Molecular and Cellular Biology 9, 5045–5054. Chang, L.‐Y., Yang, W. Y., Browning, K. and Roth, D. (1999). Specific in vitro phosphorylation of plant eIF2 by eukaryotic eIF2 kinases. Plant Molecular Biology 41, 363–370. Chang, L.‐Y., Yang, W. Y. and Roth, D. (2000). Functional complementation by wheat eIF2 in the yeast GCN2‐mediated pathway. Biochemical and Biophysical Research Communications 279, 468–474.

REGULATION OF CARBON AND AMINO ACID METABOLISM

131

Chen, J.‐J., Throop, M. S., Gehrke, L., Kuo, I., Pal, J. K., Brodsky, M. and London, I. M. (1991). Cloning of the cDNA of the heme‐regulated eukaryotic initiation factor‐2 alpha (eIF2) kinase of rabbit reticulocytes: Homology to yeast GCN2 protein kinase and human double‐stranded RNA‐dependent eIF2 kinase. Proceedings of the National Academy of Sciences USA 88, 7729–7733. Cheng, C.‐L., Acedo, G. N., Cristinsin, M. and Conkling, M. A. (1992). Sucrose mimics the light induction of Arabidopsis nitrate reductase gene transcription. Proceedings of the National Academy of Sciences USA 89, 1861–1864. Clarke, P. R. and Hardie, D. G. (1990). Regulation of HMG‐CoA reductase: Identification of the site phosphorylated by the AMP‐activated protein kinase in vitro and in intact rat liver. EMBO Journal 9, 2439–2446. Clement, C., Chavant, L., Burrus, M. and Audran, J. C. (1994). Anther starch variations in Lilium during pollen development. Sexual Plant Reproduction 7, 347–356. Corton, J. M., Gillespie, J. G. and Hardie, D. G. (1994). Role of the AMP‐activated protein kinase in the cellular stress response. Current Biology 4, 315–324. Corton, J. M., Gillespie, J. G., Hawley, S. A. and Hardie, D. G. (1995). 5‐Aminoimidazole‐4‐carboxamide ribonucleoside: A specific method for activating AMP‐activated protein kinase in intact cells. European Journal of Biochemistry 229, 558–565. Crawford, R. M., Halford, N. G. and Hardie, D. G. (2001). Cloning of DNA encoding a catalytic subunit of SNF1‐related protein kinase‐1 (SnRK1‐1) and immunological analysis of multiple forms of the kinase in spinach leaf. Plant Molecular Biology 45, 731–741. Dale, S., Wilson, W. A., Edelman, A. M. and Hardie, D. G. (1995). Similar substrate recognition motifs for mammalian AMP‐activated protein kinase, higher plant HMG‐CoA reductase kinase‐A, yeast SNF1, and mammalian calmodulin‐dependent protein kinase I. FEBS Letters 361, 191–195. Davies, J. P., Yildiz, F. H. and Grossman, A. R. (1999). Sac3, an Snf1‐like serine/ threonine kinase that positively and negatively regulates the responses of Chlamydomonas to sulfur limitation. Plant Cell 11, 1179–1190. Davies, S. P., Carling, D. and Hardie, D. G. (1989). Tissue distribution of the AMP‐ activated protein kinase, and lack of activation by cyclic AMP‐dependent protein kinase, studies using a specific and sensitive peptide assay. European Journal of Biochemistry 186, 123–128. Davies, S. P., Carling, D., Munday, M. R. and Hardie, D. G. (1992). Diurnal rhythm of phosphorylation of rat‐liver acetyl‐CoA carboxylase by the AMP‐activated protein kinase, demonstrated using freeze‐clamping: EVects of high‐ fat diets. European Journal of Biochemistry 203, 615–623. Davies, S. P., Hawley, S. A., Woods, A., Carling, D., Haystead, T. A. J. and Hardie, D. G. (1994). Purification of the AMP‐activated protein kinase on ATP‐g‐ Sepharose and analysis of its subunit structure. European Journal of Biochemistry 223, 351–357. Davies, S. P., Helps, N. R., Cohen, P. T. W. and Hardie, D. G. (1995). 50 ‐AMP inhibits dephosphorylation, as well as promoting phosphorylation, of the AMP‐activated protein kinase: Studies using bacterially expressed human protein phosphatase‐2C and native bovine protein phosphatase‐2A(C). FEBS Letters 377, 421–425. Davies, S. P., Sim, A. T. R. and Hardie, D. G. (1990). Location and function of three sites phosphorylated on rat acetyl‐CoA carboxylase by the AMP‐activated protein kinase. European Journal of Biochemistry 187, 183–190.

132

N. G. HALFORD

DeVit, M. J., Waddle, J. A. and Johnston, M. (1997). Regulated nuclear translocation of the Mig1 repressor. Molecular Biology of the Cell 8, 1603–1618. Dickinson, J. R. (1999). Carbon metabolism. In ‘‘The Metabolism and Molecular Physiology of Saccharomyces cerevisiae’’ (J. R. Dickinson and M. Schweizer, eds.), pp. 23–55. Taylor and Francis, London. Dickinson, J. R., Cole, D. and Halford, N. G. (1999). A cell cycle role for a plant sucrose nonfermenting‐1‐related protein kinase (SnRK1) is indicated by expression in yeast. Plant Growth Regulation 28, 169–174. Douglas, P., Morrice, N. and MacKintosh, C. (1995). Identification of a regulatory phosphorylation site in the hinge 1 region of nitrate reductase from spinach (Spinacea oleracea) leaves. FEBS Letters 377, 113–117. DuVus, C. M. and Cochrane, M. P. (1992). Grain structure and composition. In ‘‘Barley: Genetics, Biochemistry, Molecular Biology and Biotechnology’’ (P. R. Shewry, ed.), pp. 291–317. CAB International, Wallingford. Eastmond, P. J., van Dijken, A. J., Spielman, M., Kerr, A., Tissier, A. F., Dickinson, H. G., Jones, J. D., Smeekens, S. C. and Graham, I. A. (2002). Trehalose‐6‐ phosphate 1, which catalyses the first step in trehalose synthesis, is essential for Arabidopsis embryo development. Plant Journal 29, 225–235. Eisenreich, W., Menhard, B., Hylands, P. J., Zenk, M. H. and Bacher, A. (1996). Studies on the biosynthesis of taxol: The taxane carbon skeleton is not of mevalonoid origin. Proceedings of the National Academy of Sciences USA 93, 6431–6436. Enjuto, M., Balcells, L., Campos, N., Caelles, C., Arro, M. and Boronat, A. (1994). Arabidopsis thaliana contains two diVerentially expressed 3‐hydroxy‐3‐ methylglutaryl‐CoA reductase genes, which encode microsomal forms of the enzyme. Proceedings of the National Academy of Sciences USA 91, 927–931. Entian, K. D. (1980). Genetic and biochemical evidence for hexokinase PII as a key enzyme involved in carbon catabolite repression in yeast. Molecular and General Genetics 178, 633–637. Finkelstein, R. R. (1994). Mutations at two new Arabidopsis ABA response loci are similar to the abi3 mutations. Plant Journal 5, 765–771. Finkelstein, R. R. and Lynch, T. J. (2000). The Arabidopsis abscisic acid response gene ABI5 encodes a basic leucine zipper transcription factor. Plant Cell 12, 599–609. Finkelstein, R. R., Wang, M. L., Lynch, T. J., Rao, S. and Goodman, H. M. (1998). The Arabidopsis abscisic acid response locus ABI4 encodes an APETALA 2 domain protein. Plant Cell 10, 1043–1054. Forde, B. G., Heyworth, A., Pywell, J. and Kreis, M. (1985). Nucleotide sequence of a B1 hordein gene and the identification of possible upstream regulatory elements in endosperm storage protein genes from barley, wheat and maize. Nucleic Acids Research 13, 7327–7339. Franchi, G. G., Bellani, L., Nepi, M. and Pacini, E. (1996). Types of carbohydrate reserves in pollen: Localisation, systematic distribution and ecophysiological significance. Flora 191, 143–159. Freshney, N. W., Rawlinson, L., Guesdon, F., Jones, E., Cowley, S., Hsuan, J. and Saklatvala, J. (1994). Interleukin‐1 activates a novel protein cascade that results in the phosphorylation of hsp27. Cell 78, 1039–1049. Fu, H. and Park, W. D. (1995). Sink‐ and vascular‐associated sucrose synthase functions are encoded by diVerent gene classes in potato. Plant Cell 7, 1369–1385. Fu, Y. B., Ballicora, M. A., Leykam, J. F. and Preiss, J. (1998). Mechanism of reductive activation of potato tuber ADP‐glucose pyrophosphorylase. Journal of Biological Chemistry 273, 25045–25052.

REGULATION OF CARBON AND AMINO ACID METABOLISM

133

Gampala, S. S. L., Finkelstein, R. R., Sun, S. S. M. and Rock, C. D. (2002). ABI5 interacts with abscisic acid signaling eVectors in rice protoplasts. Journal of Biological Chemistry 277, 1689–1694. Gao, G., Fernandez, S., Stapleton, D., Auster, A. S., Widmer, J., Dyck, J. R. B., Kemp, B. E. and Witters, L. A. (1996). Non‐catalytic ‐ and ‐subunit isoforms of the 50 ‐AMP‐activated protein kinase. Journal of Biological Chemistry 271, 8675–8681. Gao, G., Widmer, J., Stapleton, D., The, T., Cox, T., Kemp, B. E. and Witters, L. A. (1995). Catalytic subunits of the porcine and rat 50 ‐AMP‐activated protein kinase are members of the SNF1 protein kinase family. Biochimica et Biophysica Acta 1266, 73–82. Giese, H. and Hopp, E. (1984). Influence of nitrogen nutrition on the amount of hordein, protein Z and ‐amylase messenger RNA in developing endosperms of barley. Carlsberg Research Communications 49, 365–383. Gillespie, J. G. and Hardie, D. G. (1992). Phosphorylation and inactivation of HMG‐CoA reductase at the AMP‐activated protein kinase site in respons to fructose treatment of isolated rat hepatocytes. FEBS Letters 306, 59–62. Go´ mez‐Cadenas, A., Verhey, S. D., Holappa, L. D., Shen, Q., Ho, T.‐H. D. and Walker‐Simmons, M. K. (1999). An abscisic acid‐induced protein kinase, PKABA1, mediates abscisic acid‐suppressed gene expression in barley aleurone layers. Proceedings of the National Academy of Sciences USA 96, 1767–1772. Graham, I. A., Denby, K. J. and Leaver, C. J. (1994). Carbon catabolite repression regulates glyoxylate cycle gene expression in cucumber. Plant Cell 6, 761–772. Graham, R. D. (1975). Male sterility in wheat plants deficient in copper. Nature 254, 514–515. Grierson, C., Du, J. S., Zabala, M. D., Beggs, K., Smith, C., Holdsworth, M. and Bevan, M (1994). Separate cis sequences and trans factors direct metabolic and developmental regulation of a potato tuber storage protein gene. Plant Journal 5, 815–826. Guyer, D., Patton, D. and Ward, E. (1995). Evidence for cross‐pathway regulation of metabolic gene expression in plants. Proceedings of the National Academy of Sciences USA 92, 4997–5000. Halford, N. G. and Hardie, D. G. (1998). SNF1‐related protein kinases: Global regulators of carbon metabolism in plants? Plant Molecular Biology 37, 735–748. Halford, N. G., Hey, S., Jhurreea, D., Laurie, S., McKibbin, R. S., Paul, M. J. and Zhang, Y. (2003a). Metabolic signalling and carbon partitioning: Role of Snf1‐related (SnRK1) protein kinase. Journal of Experimental Botany 54, 467–475. Halford, N. G., Hey, S., Jhurreea, D., Laurie, S., McKibbin, R. S., Zhang, Y. and Paul, M. (2003b). Dissection and manipulation of metabolic signalling pathways. Annals of Applied Biology 142, 25–31. Halford, N. G. and Paul, M. J. (2003). Carbon metabolite sensing and signalling. Plant Biotechnology Journal 1, 381–398. Halford, N. G., Purcell, P. C. and Hardie, D. G. (1999). Is hexokinase really a sugar sensor in plants? Trends in Plant Science 4, 117–120. Halfter, U., Ishitani, M. and Shu, J.‐K. (2000). The Arabidopsis SOS2 protein kinase physically interacts with and is activated by the calcium‐binding protein, SOS3. Proceedings of the National Academy of Sciences USA 97, 3535–3740.

134

N. G. HALFORD

Hammond‐Kosack, M. C. U., Holdsworth, M. J. and Bevan, M. W. (1993). In vivo footprinting of a low molecular weight glutenin gene (LMWG‐1D1) in wheat endosperm. EMBO Journal 12, 545–554. Hannappel, U., Vicente‐Carbajosa, J., Barker, J. H. A., Shewry, P. R. and Halford, N. G. (1995). DiVerential expression of two barley SNF1‐related protein kinase genes. Plant Molecular Biology 27, 1235–1240. Hardie, D. G. and Carling, D. (1997). The AMP‐activated protein kinase: Fuel gauge of the mammalian cell? European Journal of Biochemistry 246, 259–273. Harker, M., Holmberg, M., Clayton, J. C., Gibbard, C. L., Wallace, A. D., Rawlins, S., Hellyer, S. A., Lanot, A. and SaVord, R. (2003). Enhancement of seed phytosterol levels by expression of an N‐terminal truncated Hevea brasiliensis (rubber tree) 3‐hydroxy‐3‐methylglutaryl‐CoA reductase. Plant Biotechnology Journal 1, 113–121. Harper, J. F., Breton, G. and Harmon, A. (2004). Decoding Ca2þ signals through plant protein kinases. Annual Review of Plant Biology 55, 263–288. Harter, K., Talke‐Messerer, C., Barz, W. and Scha¨ fer, E. (1993). Light and sucrose‐ dependent gene expression in photomixotrophic cell suspension cultures and protoplasts of rape. Plant Journal 4, 507–516. Hawley, S. A., Davison, M., Woods, A., Davies, S. P., Beri, R. K., Carling, D. and Hardie, D. G. (1996). Characterisation of the AMP‐activated protein kinase kinase from rat liver, and identification of threonine‐172 as the major site at which it phosphorylates and activates AMP‐activated protein kinase. Journal of Biological Chemistry 271, 27879–27887. Hawley, S. A., Selbert, M. A., Goldstein, E. G., Edelman, A. M., Carling, D. and Hardie, D. G. (1995). 50 ‐AMP activates the AMP‐activated protein kinase cascade, and Ca2þ/calmodulin the calmodulin‐dependent protein kinase I cascade, via three independent mechanisms. Journal of Biological Chemistry 270, 27186–27191. Heineke, D., Wildenberger, K., Sonnewald, U., Willmitzer, L. and Heldt, H. W. (1994). Accumulation of hexoses in leaf vacuoles: Studies with transgenic tobacco plants expressing yeast‐derived invertase in the cytosol, vacuole or cytoplasm. Planta 194, 29–33. Herbers, K., Meuwly, P., Frommer, W. B., Metraux, J. P. and Sonnewald, U. (1996). Systemic acquired resistance mediated by the ectopic expression of invertase: Possible hexose sensing in the secretory pathway. Plant Cell 8, 793–803. Herrero, P., Martinez‐Campa, C. and Moreno, F. (1998). The hexokinase 2 protein participates in regulatory DNA‐protein complexes necessary for glucose repression of the SUC2 gene in Saccharomyces cerevisiae. FEBS Letters 434, 71–76. Hey, S. J., Powers, S. J., Beale, M. H., Hawkins, N. D., Ward, J. L. and Halford, N. G. (in press). Enhanced seed phytosterol accumulation through expression of a modified HMG‐CoA reductase. Plant Biotechnology Journal. Hinnebusch, A. G. (1992). General and pathway‐specific regulatory mechanisms controlling the synthesis of amino acid biosynthetic enzymes in Saccharomyces cerevisiae. In ‘‘Molecular and Cellular Biology of the Yeast Saccharomyces’’ (E. W. Jones, J. R. Pringle and J. B. Broach, eds.), Vol. 2, pp. 319–414. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Hinnebusch, A. G. (1994). Translational control of GCN4: An in vivo barometer of initiation factor activity. Trends in Biochemical Sciences 19, 409–414. Hinnebusch, A. G. (1997). Translational regulation of yeast GCN4: A window on factors that control initiator‐tRNA binding to the ribosome. Journal of Biological Chemistry 272, 21661–21664.

REGULATION OF CARBON AND AMINO ACID METABOLISM

135

Hong, S. P., Leiper, F. C., Woods, A., Carling, D. and Carlson, M. (2003). Activation of yeast Snf1 and mammalian AMP‐activated protein kinase by upstream kinases. Proceedings of the National Academy of Sciences USA 100, 8839–8843. Hrabak, E. M., Chan, C. W. M., Gribskov, M., Harper, J. F., Choi, J. H., Halford, N., Kudla, J., Luan, S., Nimmo, H. G., Sussman, M. R., Thomas, M., Walker‐Simmons, K., Zhu, J.‐K. and Harmon, A. C. (2003). The Arabidopsis CDPK‐SnRK superfamily of protein kinases. Plant Physiology 132, 666–680. Huang, J. Z. and Huber, S. C. (2001). Phosphorylation of synthetic peptides by a CDPK and plant SNF1‐related protein kinase: Influence of proline and basic amino acid residues at selected positions. Plant and Cell Physiology 42, 1079–1087. Hwang, I. and Sheen, J. (2001). Two‐component circuitry in Arabidopsis signal transduction. Nature 413, 383–389. Icely, P. L., Gros, P., Bergeron, J. J. M., Devault, A., Afar, D. E. H. and Bell, J. C. (1991). TIK, a novel serine threonine kinase, is recognised by antibodies directed against phosphotyrosine. Journal of Biological Chemistry 266, 16073–16077. Ito, N. (1978). Male sterility caused by cooling treatment at the young microspore stage in rice plants. XVI. Changes in carbohydrates, nitrogenous and phosphorous compounds in rice anthers after cooling treatment. Japanese Journal of Crop Science 17, 318–323. Jang, J. C. and Sheen, J. (1997). Sugar sensing in higher plants. Trends in Plant Science 2, 208–214. Jang, J.‐C., Leon, P., Zhou, L. and Sheen, J. (1997). Hexokinase as a sugar sensor in higher plants. Plant Cell 9, 5–19. Jiang, R. and Carlson, M. (1996). Glucose regulates protein interactions within the yeast Snf1 protein kinase complex. Genes and Development 10, 3105–3115. Jiang, R. and Carlson, M. (1997). The Snf1 protein kinase and its activating subunit, Snf4, interact with distinct domains of the Sip1/Sip2/Gal83 component in the kinase complex. Molecular and Cellular Biology 17, 2099–2106. Joppa, L. R., McNeal, F. H. and Welsh, J. R. (1966). Pollen and anther development in cytoplasmic male sterile wheat (Triticum aestivum L.). Crop Science 6, 296–335. Katan, M. B., Grundy, S. M., Jones, P., Law, M., Miettinen, T. and Paoletti, R. (2003). EYcacy and safety of plant stanols and sterols in the management of blood cholesterol levels. Mayo Clinic Proceedings 78, 965–978. Kleinow, T., Bhalerao, R., Breuer, F., Umeda, M., Salchert, K. and Koncz, C. (2000). Functional identification of an Arabidopsis SNF4 orthologue by screening for heterologous multicopy suppressors of snf4 deficiency in yeast. Plant Journal 23, 115–122. Krapp, A., Hofmann, B., Scha¨ fer, C. and Stitt, M. (1993). Regulation of the expression of rbcS and other photosynthetic genes by carbohydrates: A mechanism for the ‘sink’ regulation of photosynthesis? Plant Journal 3, 817–828. Kruckeberg, A. L. (1996). The hexose transporter family of Saccharomyces cerevisiae. Archives of Microbiology 166, 283–292. Kudo, N., Barr, A. J., Barr, R. L., Desai, S. and Lopaschuk, G. D. (1995). High rates of fatty acid oxidation during reperfusion of ischemic hearts are associated with a decrease in malonyl‐CoA levels due to an increase in 50 ‐AMP‐ activated protein kinase inhibition of acetyl‐CoA carboxylase. Journal of Biological Chemistry 270, 17513–17520.

136

N. G. HALFORD

Kurup, S., Jones, H. D. and Holdsworth, M. J. (2000). Interactions of the developmental regulator ABI3 with proteins identified from developing Arabidopsis seeds. Plant Journal 21, 143–155. Laby, R. J., Kincaid, M. S., Kim, D. G. and Gibson, S. I. (2000). The Arabidopsis sugar‐insensitive mutants sis4 and sis5 are defective in abscisic acid synthesis and response. Plant Journal 23, 587–596. Lakatos, L., Klein, M., Hofgen, R. and Banfalvi, Z. (1999). Potato StubSNF1 interacts with StubGAL83: A plant protein kinase complex with yeast and mammalian counterparts. Plant Journal 17, 569–574. Lalonde, S., Boles, E., Hellmann, H., Barker, L., Patrick, J. W., Frommer, W. B. and Ward, J. M. (1999). The dual function of plant sugar carriers in transport and in nutrient sensing. Plant Cell 11, 707–726. Lalonde, S., Tegeder, M., Throne‐Holst, M., Frommer, W. B. and Patrick, J. W. (2003). Phloem loading and unloading of amino acids and sugars. Plant Cell and Environment 26, 37–56. Laurie, S., McKibbin, R. S. and Halford, N. G. (2003). Antisense SNF1‐related (SnRK1) protein kinase gene represses transient activity of an ‐amylase (‐Amy2) gene promoter in cultured wheat embryos. Journal of Experimental Botany 54, 739–747. Leclerc, I., Mar Alba`, M., Kleinow, T., Koncz, C. and Page`s, M. (1998). The 50 ‐AMP‐activated protein kinase inhibits the transcriptional stimulation by glucose in liver cells, acting through the glucose response complex. FEBS Letters 431, 180–184. Lin, T.‐P., Caspar, T., Somerville, C. and Preiss, J. (1988). Isolation and characterisation of a starchless mutant of Arabidopsis thaliana (L.) Heynh lacking ADPglucose pyrophosphatase activity. Plant Physiology 86, 1131–1135. Liu, J., Ishitani, M., Halfter, U., Kim, C.‐S. and Shu, J.‐K. (2000). The Arabidopsis thaliana SOS2 gene encodes a protein kinase that is required for salt tolerance. Proceedings of the National Academy of Sciences USA 97, 3730–3734. Lopez‐Molina, L. and Chua, N. H. (2000). A null mutation in a bZIP factor confers ABA‐insensitivity in Arabidopsis thaliana. Plant and Cell Physiology 41, 541–547. Lumbreras, V., Mar Alba`, M., Kleinow, T., Koncz, C. and Page`s, M. (2001). Domain fusion between SNF1‐related kinase subunits during plant evolution. EMBO Reports 2, 55–60. Lund, A. A., Rhoads, D. M., Lund, A. L., Cerny, R. and Elthon, T. E. (2001). In vivo modifications of the maize mitochondrial small heat stress protein, HSP22. Journal of Biological Chemistry 276, 29924–29929. Ma, H., Bloom, L. M., Walsh, C. T. and Botstein, D. (1989). The residual enzymatic phosphorylation activity of hexokinase II mutants is correlated with glucose repression in Saccharomyces cerevisiae. Molecular and Cellular Biology 9, 5643–5649. MacKintosh, R. W., Davies, S. P., Clarke, P. R., Weekes, J., Gillespie, J. G., Gibb, B. J. and Hardie, D. G. (1992). Evidence for a protein kinase cascade in higher plants: 3‐hydroxy‐3‐methylglutaryl‐CoA reductase kinase. European Journal of Biochemistry 209, 923–931. Man, A. L., Purcell, P. C., Hannappel, U. and Halford, N. G. (1997). Potato SNF1‐ related protein kinase: Molecular cloning, expression analysis and peptide kinase activity measurements. Plant Molecular Biology 34, 31–43. McKibbin, R. S., Wilkinson, M. D., Bailey, P. C., Flintham, J. E., Andrew, L. M., Lazzeri, P. A., Gale, M. D., Lenton, J. R. and Holdsworth, M. J. (2002).

REGULATION OF CARBON AND AMINO ACID METABOLISM

137

Transcripts of Vp‐1 homeologues are misspliced in modern wheat and ancestral species. Proceedings of the National Academy of Sciences USA 99, 10203–10208. McMichael, R. W., Jr., Klein, R. R., Salvucci, M. E. and Huber, S. C. (1993). Identification of the major regulatory phosphorylation site in sucrose phosphate synthase. Archives of Biochemistry and Biophysics 307, 248–252. Mellor, H., Flowers, K. M., Kimball, S. R. and JeVerson, L. S. (1994a). Cloning and characterisation of cDNA encoding rat hemin‐sensitive initiation factor‐2 alpha (eIF2) kinase: Evidence for multi‐tissue expression. Journal of Biological Chemistry 269, 10201–10204. Mellor, H., Flowers, K. M., Kimball, S. R. and JeVerson, L. S. (1994b). Cloning and characterisation of a cDNA encoding rat PKR, the double‐stranded RNA‐ dependent eukaryotic initiation factor‐II kinase. Biochimica et Biophysica Acta 1219, 693–696. Meurs, E., Chong, K., Galabru, J., Thomas, N. S. B., Kerr, I. M., Williams, B. R. G. and Hovanessian, A. G. (1990). Molecular cloning and characterisation of the human double stranded RNA‐activated protein kinase induced by interferon. Cell 62, 379–390. Mita, S., Suzuki‐Fujii, K. and Nakamura, K. (1995). Sugar‐inducible expression of a gene for ‐amylase in Arabidopsis. Plant Physiology 107, 895–904. Mitchelhill, K. I., Stapleton, D., Gao, G., House, C., Michell, B., Katsis, F., Witters, L. A. and Kemp, B. E. (1994). Mammalian AMP‐activated protein kinase shares structural and functional homology with the catalytic domain of yeast Snf1 protein kinase. Journal of Biological Chemistry 269, 2361–2364. Moore, B., Zhou, L., Rolland, F., Hall, Q., Cheng, W.‐H., Liu, Y.‐X., Hwang, I., Jones, T. and Sheen, J. (2003). Role of the Arabidopsis glucose sensor HXK1 in nutrient, light and hormonal signalling. Science 300, 332–336. Moore, F., Weekes, J. and Hardie, D. G. (1991). AMP triggers phosphorylation as well as direct allosteric activation of rat liver AMP‐activated protein kinase: A sensitive mechanism to protect the cell against ATP depletion. European Journal of Biochemistry 199, 691–697. Moorhead, G., Douglas, P., Morrice, N., Scarabel, M., Aitken, A. and MacKintosh, C. (1996). Phosphorylated nitrate reductase from spinach leaves is inhibited by 14‐3‐3 proteins and activated by fusicoccin. Current Biology 6, 1104–1113. Mu¨ ller, M. and Knudsen, S. (1993). The nitrogen response of a barley C‐hordein promoter is controlled by positive and negative regulation of the GCN4 and endosperm box. Plant Journal 4, 343–355. Muranaka, T., Banno, H. and Machida, Y. (1994). Characterisation of the tobacco protein kinase NPK5, a homologue of Saccharomyces cerevisiae SNF1 that constitutively activates expression of the glucose‐repressible SUC2 gene for a secreted invertase of S. cerevisiae. Molecular and Cellular Biology 14, 2958–2965. Nakamura, S., Lynch, T. J. and Finkelstein, R. R. (2001). Physical interactions between ABA response loci of Arabidopsis. Plant Journal 26, 627–635. Natarajan, K., Meyer, M. R., Jackson, B. M., Slade, D., Roberts, C., Hinnebusch, A. G. and Marton, M. J. (2001). Transcriptional profiling shows that Gcn4p is a master regulator of gene expression during amino acid starvation in yeast. Molecular and Cellular Biology 21, 4347–4368. Noubhani, A., Bunoust, O., Rigoulet, M. and Thevelein, J. M. (2000). Reconstitution of ethanolic fermentation in permeabilised spheroplasts of wild type and trehalose 6‐phosphate synthase mutants of the yeast Saccharomyces cerevisiae. European Journal of Biochemistry 267, 4566–4576.

138

N. G. HALFORD

Nu¨ hse, T. S., Stensballe, A., Jensen, O. N. and Peck, S. C. (2004). Phosphoproteomics of the Arabidopsis plasma membrane and a new phosphorylation site database. Plant Cell 16, 2394–2405. ¨ stling, J. and Ronne, H. (1998). Negative control of the Mig1p repressor by Snf1p‐ O dependent phosphorylation in the absence of glucose. European Journal of Biochemistry 252, 162–168. Ostlund, R. E., Jr. (2004). Phytosterols and cholesterol metabolism. Current Opinion in Lipidology 15, 37–41. Ozcan, S. and Johnston, M. (1996). Two diVerent repressors collaborate to restrict expression of yeast glucose transporter genes HXT2 and HXT4 to low levels of glucose. Molecular and Cellular Biology 16, 5536–5545. Pacini, E. and Franchi, G. G. (1988). Amylogenesis and Amylolysis during pollen grain development. In ‘‘Sexual Reproduction in Higher Plants’’ (M. Cresti, P. Gori and E. Pacini, eds.), pp. 181–186. Springer, Berlin. Pellny, T. K., Ghannoum, O., Conroy, J. P., Schluepmann, H., Smeekens, S., Andralojc, J., Krause, K.‐P., Goddijn, O. and Paul, M. J. (2004). Genetic modification of photosynthesis with E.coli genes for trehalose synthesis. Plant Biotechnology Journal 2, 71–82. Pien, S., Wyrzykowska, J. and Fleming, A. J. (2001). Novel marker genes for early leaf development indicate spatial regulation of carbohydrate metabolism within the apical meristem. Plant Journal 25, 663–674. Plat, J. and Mensink, R. P. (2002). Increased intestinal ABCA1 expression contributes to the decrease in cholesterol absorption after plant stanol consumption. FASEB Journal 16, 1248–1253. Price, J., Laxmi, A., St Martin, S. K. and Jang, J. C. (2004). Global transcription profiling reveals multiple sugar signal transduction mechanisms in Arabidopsis. Plant Cell 16, 2128–2150. Purcell, P. C., Smith, A. M. and Halford, N. G. (1998). Antisense expression of a sucrose nonfermenting‐1‐related protein kinase sequence in potato results in decreased expression of sucrose synthase in tubers and loss of sucrose‐ inducibility of sucrose synthase transcripts in leaves. Plant Journal 14, 195–202. Randez‐Gil, F., Herrero, P., Sanz, P., Prieto, J. A. and Moreno, F. (1998). Hexokinase PII has a double cytosolic‐nuclear localisation in Saccharomyces cerevisiae. FEBS Letters 425, 475–478. Reifenberger, E., Boles, E. and Ciriacy, M. (1997). Kinetic characterisation of individual hexose transporters of Saccharomyces cerevisiae and their relation to the triggering mechanisms of glucose repression. European Journal of Biochemistry 245, 324–333. Reinders, A., Schulze, W., Ku¨ hn, C., Barker, L., Schulz, A., Ward, J. M. and Frommer, W. B. (2002). Protein‐protein interactions between sucrose transporters of diVerent aYnities co‐localised in the same enucleate sieve element. Plant Cell 14, 1567–1577. Rolland, F., Winderickx, J. and Thevelein, J. M. (2001). Glucose‐sensing mechanisms in eukaryotic cells. Trends in Biochemical Sciences 26, 310–317. Rook, F., Corke, F., Card, R., Munz, G., Smith, C. and Bevan, M. W. (2001). Impaired sucrose‐induction mutants reveal the modulation of sugar‐induced starch biosynthetic gene expression by abscisic acid signalling. Plant Journal 26, 421–433. Rose, M., Albig, W. and Entian, K. D. (1991). Glucose repression in Saccharomyces cerevisiae is directly associated with hexose phosphorylation by hexokinase PI and PII. European Journal of Biochemistry 199, 511–518.

REGULATION OF CARBON AND AMINO ACID METABOLISM

139

Rouse, J., Cohen, P., Trigon, S., Morange, M., Alonso‐Llamazares, A., Zamanillo, D., Hunt, T. and Nebreda, A. R. (1994). A novel kinase cascade triggered by stress and heat shock that stimulates MAPKAP kinase‐2 and phosphorylation of the small heat shock protein. Cell 78, 1027–1037. Saini, H. S., Sedgley, M. and Aspinall, D. (1984). Developmental anatomy in wheat of male sterility induced by heat stress, water deficit or abscisic acid. Australian Journal of Plant Physiology 11, 243–253. Salanoubat, M. and Belliard, G. (1989). The steady‐state level of potato sucrose synthase mRNA is dependant on wounding, anaerobiosis and sucrose concentration. Gene 84, 181–185. Samuel, C. E. (1993). The eIF2 protein kinases, regulators of translation in eukaryotes from yeasts to humans. Journal of Biological Chemistry 268, 7603–7608. Santoyo, J., Alcalde, J., Mendez, R., Pulido, D. and de Haro, C. (1997). Cloning and characterisation of a cDNA encoding a protein synthesis initiation factor‐ 2 alpha (eIF2) kinase from Drosophila melanogaster: Homology to yeast GCN2 protein kinase. Journal of Biological Chemistry 272, 12544–12550. Sato, R., Goldstein, J. L. and Brown, M. S. (1993). Replacement of Serine‐871 of hamster 3‐hydroxy‐3‐methylglutaryl CoA reductase prevents phosphorylation by AMP‐activated protein kinase and blocks inhibition of sterol synthesis induced by ATP depletion. Proceedings of the National Academy of Sciences USA 90, 9261–9265. Sattlegger, E., Hinnebusch, A. G. and Barthelmess, I. B. (1998). cpc‐3, the Neurospora crassa homologue of yeast GCN2, encodes a polypeptide with juxtaposed eIF2 kinase and histidyl‐tRNA synthetase‐related domains required for general amino acid control. Journal of Biological Chemistry 273, 20404–20416. Schluepman, H., Pellny, T., van Dijken, A., Smeekens, S. and Paul, M. J. (2003). Trehalose 6‐phosphate is indispensable for carbohydrate utilisation and growth in Arabidopsis thaliana. Proceedings of National Academy of Sciences USA 100, 6849–6854. Schluepman, H., van Dijken, A., Aghdasi, M., Wobbes, B., Paul, M. and Smeekens, S. (2004). Trehalose‐mediated growth inhibition of Arabidopsis seedlings is due to trehalose‐6‐phosphate accumulation. Plant Physiology 135, 879–890. Sheen, J. (1990). Metabolic repression of transcription in higher plants. Plant Cell 2, 1027–1038. Sheen, J. (1994). Feedback control of gene expression. Photosynthesis Research 39, 427–438. Sherson, S. M., Alford, H. L., Forbes, S. M., Wallace, G. and Smith, S. M. (2003). Roles of cell‐wall invertases and monosaccharide transporters in the growth and development of Arabidopsis. Journal of Experimental Botany 54, 525–531. Shewry, P. R., Halford, N. G. and Lafiandra, D. (2003). The genetics of wheat gluten proteins. Advances in Genetics 49, 111–184. Slocombe, S. P., Beaudoin, F., Donaghy, P. G., Hardie, D. G., Dickinson, J. R. and Halford, N. G. (2004). SNF1‐related protein kinase (SnRK1) phosphorylates class I heat shock protein. Plant Physiology and Biochemistry 42, 111–116. Slocombe, S. P., Laurie, S., Bertini, L., Beaudoin, F., Dickinson, J. R. and Halford, N. G. (2002). Molecular cloning of SnIP1, a novel protein that interacts with SNF1‐related protein kinase (SnRK1). Plant Molecular Biology 49, 31–44.

140

N. G. HALFORD

Smeekens, S. (2000). Sugar‐induced signal transduction in plants. Annual Review of Plant Physiology and Plant Molecular Biology 51, 49–81. Sowokinos, J. R. and Varns, J. L. (1992). Induction of sucrose synthase in potato tissue culture: EVect of carbon source and metabolic regulators on sink strength. Journal of Plant Physiology 139, 672–679. Sreenivasan, A. and Kellogg, D. (1999). The Elm1 kinase functions in a mitotic signaling network in budding yeast. Molecular and Cellular Biology 19, 7983–7994. Su, W., Huber, S. C. and Crawford, N. M. (1996). Identification in vitro of a post‐ translational regulatory site in the hinge 1 region of Arabidopsis nitrate reductase. Plant Cell 8, 519–527. Sugden, C., Crawford, R. M., Halford, N. G. and Hardie, D. G. (1999a). Regulation of spinach SNF1‐related (SnRK1) kinases by protein kinases and phosphatases is associated with phosphorylation of the T loop and is regulated by 50 ‐AMP. Plant Journal 19, 433–439. Sugden, C., Donaghy, P., Halford, N. G. and Hardie, D. G. (1999b). Two SNF1‐ related protein kinases from spinach leaf phosphorylate and inactivate 3‐hydroxy‐3‐methylglutaryl‐coenzyme A reductase, nitrate reductase and sucrose phosphate synthase in vitro. Plant Physiology 120, 257–274. Sutherland, C. M., Hawley, S. A., McCartney, R. R., Leech, A., Stark, M. J., Schmidt, M. C. and Hardie, D. G. (2003). Elm1p is one of three upstream kinases for the Saccharomyces cerevisiae SNF1 complex. Current Biology 13, 1299–1305. Tanaka, S. and Nojima, H. (1996). Nik1, a Nim1‐like protein kinase of S. cerevisiae, interacts with the Cdc28 complex and regulates cell cycle progression. Genes to Cells 1, 905–921. Taylor, S. S., Buechler, J. A. and Yonemoto, W. (1990). cAMP‐dependent protein kinase: Framework for a diverse family of regulatory enzymes. Annual Review of Biochemistry 59, 971–1005. Thelander, M., Olssen, T. and Ronne, H. (2004). Snf1‐related protein kinase 1 is needed for growth in a normal day‐night life cycle. EMBO Journal 23, 1900–1910. Thevelein, J. and Hohmann, S. (1995). Trehalose synthase: Guard to the gate of glycolysis in yeast? Trends in Biochemical Sciences 20, 3–10. Thompson‐Jaeger, S., Francois, J., Gaughran, J. P. and Tatchell, K. (1991). Deletion of SNF1 aVects the nutrient response of yeast and resembles mutations which activate the adenylate cyclase pathway. Genetics 12, 697–706. Tiessen, A., Hendriks, J. H. M., Stitt, M., Branscheid, A., Gibon, Y., Farre, E. M. and Geigenberger, P. (2002). Starch synthesis in potato tubers is regulated by post‐translational redox modification of ADP‐glucose pyrophosphorylase: A novel regulatory mechanism linking starch synthesis to the sucrose supply. Plant Cell 14, 2191–2213. Tiessen, A., Prescha, K., Branscheid, A., Palacios, N., McKibbin, R., Halford, N. G. and Geigenberger, P. (2003). Evidence that SNF1‐related kinase and hexokinase are involved in separate sugar‐signalling pathways modulating post‐ translational redox activation of ADP‐glucose pyrophosphorylase in potato tubers. Plant Journal 35, 490–500. Toroser, D., Plaut, Z. and Huber, S. C. (2000). Regulation of a plant SNF1‐ related protein kinase by glucose‐6‐phosphate. Plant Physiology 123, 403–411. Weber, H., Buchner, P., Borisjuk, L. and Wobus, U. (1996). Sucrose metabolism during cotyledon development of Vicia faba L. is controlled by the concerted action of both sucrose phosphate synthase and sucrose synthase: Expres-

REGULATION OF CARBON AND AMINO ACID METABOLISM

141

sion patterns, metabolic regulation and implications for seed development. Plant Journal 9, 841–850. Weekes, J., Ball, K. L., Caudwell, F. B. and Hardie, D. G. (1993). Specificity determinants for the AMP‐activated protein kinase and its plant homologue analyzed using synthetic peptides. FEBS Letters 334, 335–339. Weise, A., Barker, L., Ku¨ hn, C., Lalonde, S., Buschmann, H., Frommer, W. B. and Ward, J. M. (2000). A new subfamily of sucrose transporters, SUT4, with low aYnity/high capacity localised in enucleate sieve elements of plants. Plant Cell 12, 1345–1355. Wek, R. C., Jackson, B. M. and Hinnebusch, A. G. (1989). Juxtaposition of domains homologous to protein kinases and histidyl transfer RNA synthetases in GCN2 protein suggests a mechanism for coupling GCN4 expression to amino acid availability. Proceedings of the National Academy of Sciences USA 86, 4579–4583. Westrate, J. A. and Meijer, G. W. (1998). Plant phytosterol‐enriched margarines and reduction of plasma total‐ and LDL‐cholesterol concentrations in normocholesterolemic and mildly hypercholesterolemic subjects. European Journal of Clinical Nutrition 52, 334–343. Wilson, W. A., Hawley, S. A. and Hardie, D. G. (1996). The mechanism of glucose repression/derepression in yeast: SNF1 protein kinase is activated by phosphorylation under derepressing conditions, and this correlates with a high AMP:ATP ratio. Current Biology 6, 1426–1434. Winder, W. W. and Hardie, D. G. (1996). Inactivation of acetyl‐CoA carboxylase and activation of AMP‐activated protein kinase in muscle during exercise. American Journal of Physiology 270, E299–E304. Wingler, A., Fritzius, T., Wiemken, A., Boller, T. and Aeschbacher, R. A. (2000). Trehalose induces the ADP‐glucose pyrophosphorylase gene, ApL3, and starch synthesis in Arabidopsis. Plant Physiology 124, 105–114. Wingler, A., von Schaewen, A., Leegood, R. C., Lea, P. J. and Quick, W. P. (1998). Regulation of leaf senescence by cytokinin, sugars and light: EVects on NADH‐dependent hdroxypyruvate reductase. Plant Physiology 116, 329–335. Witters, L.A, Nordlund, A. C. and Marshall, L. (1991). Regulation of intracellular acetyl‐CoA carboxylase by ATP depletors mimics the action of the 50 ‐ AMP‐activated protein kinase. Biochemical and Biophysical Research Communications 181, 1486–1492. Woods, A., Azzout‐Marniche, D., Foretz, M., Stein, S. C., Lemarchand, P., Ferre, P., Foufelle, F. and Carling, D. (2000). Characterisation of the role of AMP‐activated protein kinase in the regulation of glucose‐activated gene expression using constitutively active and dominant negative forms of the kinase. Molecular and Cellular Biology 20, 6704–6711. Woods, A., Cheung, P. C. F., Smith, F. C., Davison, M. D., Scott, J., Beri, R. K. and Carling, D. (1996). Characterisation of AMP‐activated protein kinase and subunits: Assembly of the heterotrimeric complex in vitro. Journal of Biological Chemistry 271, 10282–10290. Woods, A., Johnstone, S. R., Dickerson, K., Leiper, F. C., Fryer, L. G., Neumann, D., Schlattner, U., Wallimann, T., Carlson, M. and Carling, D. (2003). LKB1 is the upstream kinase in the AMP‐activated protein kinase cascade. Current Biology 13, 2004–2008. Woods, A., Munday, M. R., Scott, J., Yang, X., Carlson, M. and Carling, D. (1994). Yeast SNF1 is functionally related to mammalian AMP‐activated protein kinase and regulates acetyl‐CoA carboxylase in vivo. Journal of Biological Chemistry 269, 19509–19515.

142

N. G. HALFORD

Yang, R. J., Wek, S. A. and Wek, R. C. (2000). Glucose limitation induces GCN4 translation by activation of gcn2 protein kinase. Molecular and Cellular Biology 20, 2706–2717. Yang, X., Jiang, R. and Carlson, M. (1994). A family of proteins containing a conserved domain that mediates interaction with the yeast Snf1 protein kinase complex. EMBO Journal 13, 5878–5886. Yu, S.‐M., Kuo, Y.‐H., Sheu, G., Sheu, Y.‐J. and Liu, L.‐F. (1991). Metabolic derepression of ‐amylase gene expression in suspension‐cultured cells of rice. Journal of Biological Chemistry 266, 21131–21137. Zhang, Y., Shewry, P. R., Jones, H., Barcelo, P., Lazzeri, P. A. and Halford, N. G. (2001). Expression of antisense SnRK1 protein kinase sequence causes abnormal pollen development and male sterility in transgenic barley. Plant Journal 28, 431–442. Zhang, Y., Dickinson, J. R., Paul, M. J. and Halford, N. G. (2003). Molecular cloning of an Arabidopsis homologue of GCN2, a protein kinase involved in co‐ordinated response to amino acid starvation. Planta 217, 668–675. Zhao, J., Williams, C. C. and Last, R. L. (1998). Induction of Arabidopsis tryptophan pathway enzymes and camalexin by amino acid starvation, oxidative stress, and an abiotic elicitor. Plant Cell 10, 359–370.

Opportunities for the Control of Brassicaceous Weeds of Cropping Systems Using Mycoherbicides

AARON MAXWELL AND JOHN K. SCOTT

Cooperative Research Centre for Australian Weed Management and The Commonwealth Scientific and Industrial Research Organisation (CSIRO) Entomology, PO Wembley, WA 6913, Australia

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . II. Pathogens of Wild Radish . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Biotrophic Pathogens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Necrotrophic Pathogens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Host Specificity. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . III. Strategies to Increase Virulence or Specificity . . . . . . . . . . . . . . . . . . . . . . . . . A. Multiple Pathogens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Chemical Synergists . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Phytotoxic Metabolites . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Gene Manipulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. Epidemiology with Respect to Cropping Conditions . . . . . . . . . . . . . . . . . . . V. Inoculum Production and Formulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Industrial Scale Inoculum Production. . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Formulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VI. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Advances in Botanical Research, Vol. 43 Incorporating Advances in Plant Pathology Copyright 2006, Elsevier Ltd. All rights reserved.

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0065-2296/06 $35.00 DOI: 10.1016/S0065-2296(05)43003-7

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ABSTRACT Mycoherbicides oVer another option in controlling weeds, especially in light of increasing problems with herbicide resistance. Wild radish is a weed of cropping systems for which pathogens found in Australia include a suite of species with diVering host range that could be developed into mycoherbicides useful against weed species from the Brassicaceae. Potentially useful fungal pathogens found in Australia are Alternaria alternata, A. brassicae, A. brassicicola, A. japonica, Fusarium oxysporum, and Hyaloperonospora parasitica. Mycoherbicide candidates are considered in terms of their host specificity, virulence, epidemiological properties, industrial scale inoculum production, and suitability for liquid or solid formulation. Another critical issue is how to avoid nontarget damage in crops of the Brassicaceae such as canola. Hyaloperonospora parasitica has potentially the most specific pathovars for the control of wild radish, but is not well suited for mycoherbicide production. Whereas Alternaria japonica is less host specific than H. parasitica, it is probably the most suited for development as a mycoherbicide. This review identifies opportunities to overcome some of the limitations associated with delivering reliable bioherbicide technology through improved identification of pathogens associated with Brassicaceae, a better understanding of their epidemiology, and possible modifications to improve their mycoherbicide potential.

I. INTRODUCTION The number and extent of herbicide‐resistant weeds are increasing worldwide, with populations of some weeds resistant to several diVerent herbicide groups. One approach to this problem is to develop plant pathogens into mycoherbicides against either a narrow or a broad spectrum of weed species. Although mycoherbicides have been used successfully in horticultural and aquatic situations where moisture is not limiting, their development has largely failed in cropping situations where moisture levels are low or unpredictable. Recent advances in biotechnology and plant pathology oVer ways to overcome some of the problems that have plagued the commercialisation of mycoherbicide candidates. This review explores how the some of these advances could be applied to select, develop, and improve mycoherbicide candidates. We use as a model the wild radish (Raphanus raphanistrum), which is one of Australia’s and the world’s most problematic herbicide‐resistant weeds. Weeds from the family Brassicaceae are a major problem in cropping and horticultural situations worldwide. In southern Australian cropping regions, important brassicaceous weeds include Brassica tournefortii Gouan, Capsella bursapastoris (L.) Medikus, Diplotaxis tenuifolia (L.) DC, Raphanus raphanistrum L., Rapistrum rugosum (L.) All., Sinapis arvensis L., Sisymbrium oYcinale (L.) Scop., Sisymbrium orientale L., and Sisymbrium thellungii O.Schulz (Heap, 2005; Jones et al., 2000; Lemerle et al., 2001). Weeds of cropping systems, of which brassicaceous weeds are an important component, are estimated to cost Australian agriculture at least $1.4 billion annually (Sinden et al., 2004).

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Raphanus raphanistrum is the most important brassicaceous weed species in Australia because of the cost of control and the development of multiple herbicide‐resistant populations. Some populations in southern Australia are resistant across four diVerent modes of action (Walsh et al., 2004): Photosystem II inhibitors such as atrazine; acetolactate synthase (ALS) inhibitors such as chlorsulfuron; auxin analogues such as 2,4‐D; and phyotoene desaturase (PDS) inhibitors such as diflufenican. One response to this problem is to use biological control, either the classical approach of introducing exotic biological control agents or the development of biopesticides from pathogens already present in Australia. At the species level, all known pathogens considered for classical biological control of R. raphanistrum are already recorded on brassicaceous crop or weed species in Australia (Scott and Jourdan, unpublished; Scott et al., 2002). However, recent developments in molecular taxonomic studies have detected cryptic pathogens of the Brassicaceae (Mendes‐Pereira et al., 2003), and it is likely more cryptic or otherwise unknown host‐specific pathogens will emerge, possibly in the Mediterranean regions that are the origins of wild radish. At present the classical biological control option does not seem feasible for wild radish in Australia. The other main biological control method is the use of bioherbicides, for which indigenous or locally established pathogens can be used. It appears that there are no mycoherbicides being developed for weeds in this family, although a diverse range of at least 18 non‐brassicaceous species is being targeted worldwide (Charudattan and Dinoor, 2000). This review focuses on the potential of developing pathogens found in Australia as mycoherbicides against wild radish.

II. PATHOGENS OF WILD RADISH According to published and unpublished herbarium records, 96 fungal species are recorded on brassicaceous hosts in Australia. Table I shows the 26 species found on Raphanus in Australia, recorded elsewhere on radish species (Williams, 1993) and present on other brassicaceous plants in Australia (consequently are potentially found on wild radish), and those species also associated with the crop canola. Although bacterial (Imaizumi et al., 1997) and viral (Charudattan et al., 2003) pathogens have been tested for the biological control of weeds, these are not considered in this review. In the following sections on biotrophic and necrotrophic pathogens, the species in Table I are considered and reduced to a short list (Table II) for further analysis on the priority for further research.

TABLE I A Comparison of Known Fungal Pathogens of Raphanus Recorded on Raphanus spp. and Brassica napus in Southern Australia Based on Cook and Dube´ (1989), Khangura et al. (1999), Kharbanda et al. (2001), Sampson and Walker (1982), Shivas (1989), Simmonds (1966) and Unpublished Herbarium Records from the Qld Herbarium, NSW Herbarium, Australian Pest and Disease Database (APDD) and the Western Australian Department of Agriculture Records (DAR) Host rangeb

Radish hostc

Canola hostc

Other hostsc

NA Oidium sp. NA NA

PV W PV N

þ
þ þ

þ þ þ þ

þ þ þ þ

NA NA NA

N W W

þ




þ þ


þ þ

Sclerotium rolfsii Sacc. Alternaria alternata (Fr.) Keissler Alternaria brassicae (Berk.) Sacc. Alternaria brassicicola (Schw.) Wiltshire Alternaria japonica Yoshii Alternaria longissima Deighton and MacGarvie Botryotrichum piluliferum Saccardo and Marchal

W PV N N N N



þ




þ
þ þ
þ

þ þ þ þ þ


U




þ

þ

Pathogen (teleomorph)a Obligate biotrophs Albugo candida (Pers. ex. Chev.) Kuntze Erysiphe polygoni DC. Hyaloperonospora parasitica (Pers. ex Fr.) Fr. Plasmodiophora brassicae Wor. Facultative biotrophs Aphanomyces raphani Kendrick Phytophthora megasperma Drechsler Pythium acanthicum Drechsler Necrotrophs and hemibiotrophs Athelia rolfsii (Curzi) Tu and Kimbrough Unknown Unknown Unknown Unknown Unknown Chaetomium piluliferum J. Daniels

Pathogen (anamorph)

Unknown Glomerella cingulata (Stonem.) Spauld. and Schrenk Unknown Unknown Leptosphaeria maculans (Desmaz.) Ces. and De Not. Macrophomina phaeseolina (Tassi) Goid. Mycosphaerella brassicicola (Duby) Lindau in Engl. and Prantl Mycosphaerella capsellae Inman and Sivansen Unknown Unknown Thanatephorus cucumeris (A.B. Frank) Donk Unknown a

Colletotrichum coccodes (Wallr.) Hughes Colletotrichum gloeosporoides (Penz.) Penz. and Sacc Fusarium avenaceum (Corda: Fr.) Sacc. Fusarium oxysporum Schlecht. Emend Snyder and Hansen Plenodomus lingam (Tode ex Fr.) Ho¨ nel, formerly Phoma lingam (Tode: Fr.) Desmaz. Rhizoctonia bataticola (Tassi) E.J. Butler NA Pseudocercosporella capsellae (Ellis and Everh.) Deighton Sclerotinia minor Jagger Sclerotinia sclerotiorum (Lib.) de Bary Rhizoctonia solani Ku¨ hn (esp. zymogram group 5) Verticillium dahliae Kleb.

W W

þ







þ

W PV





þ



þ

N

þ

þ

þ

U N





þ


þ þ

N

þ

þ

þ

W W W



þ

þ þ þ

þ þ þ

W

þ




þ

Ninety‐six fungi are recorded from other brassicaceous hosts in Australia. Pathovar (PV) indicates that this species contains pathovars that may be specific to Raphanus, narrow host range (N) indicates that this species is recorded only on the Brassicaceae and may be specialised on hosts within this family, wide host range (W) indicates that this species is recorded across many genera, including those outside the Brassicaceae, uncertain host range (U) indicates that the host range is unclear from the published literature. c þ, recorded on this host. Other hosts refer to other Brassicaceae. b

TABLE II Matrix Indicating Criteria for Mycoherbicide Suitability Against Fungal Pathogens of Raphanus

Pathogen

Likely virulencea

Alternaria alternata Alternaria brassicae Alternaria brassicicola Alternaria japonica Fusarium oxysporum Leptosphaeria maculans Mycosphaerella capsellae

L‐H M M H M M M

Industrial inoculum production in similar taxa Solid Solid Solid Solid

and biphasic and biphasic and biphasic and biphasic Liquid Nil Nil

Formulation suitabilityb

Epidemiological traits suited to Australian cropping systems

Priority for future research

WOW WOW WOW WOW Solid; alginate U U

Yes Mediteranean and temperate Warmer northern zone Yes U Yes U

Low High High High High Low Low

a Virulence is based on the proportion of articles published since 1994 referring to pathogenicity on Raphanus species and from reports on the impact of each pathogen species on R. raphanistrum or where unavailable on R. sativus; Low (L), Moderate (M), High (H). b WOW, water, oil water; U, unknown.

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A. BIOTROPHIC PATHOGENS

Obligate biotrophic pathogens such as the four listed in Table I are usually not appropriate for development as mycoherbicides because they cannot be cultured artificially. It may be possible to grow Hyaloperonospora parasitica (¼Peronospora parasitica) or another of the obligate biotrophic species on a brassicaceous hosts and then distribute infected straw on farms in an augmentative control strategy, for example, to speed up the reinfection process where there is low survival of a useful pathogen over summer. H. parasitica is the most frequently recorded biotrophic disease on Raphanus and it has host‐specific races (Dickinson and Greenhalgh, 1977; Greenhalgh and Dickinson, 1975; Satou and Fukumoto, 1996; Silue´ et al., 1996) and possibly distinct species specialising on Raphanus (Choi et al., 2003). Therefore, H. parasitica is the most promising candidate for the biological control of wild radish using this strategy. Facultative biotrophs such as Aphanomyces raphani can be grown on artificial media, but their requirements for sporulation may be more stringent than for necrotrophic fungi (Singh and Pavgi, 1977). The mycoherbicide DeVine is a formulation of the facultative biotroph Phytophthora palmivora for the control of strangler vine in citrus orchards and hence this group of pathogens shows potential for mycoherbicide development. Of the three facultative biotroph species considered in Table I, A. raphani is the only one that may be suYciently host specific to consider for mycoherbicide development. This pathogen infects plant roots primarily via the production of motile zoospores that require free soil moisture. Therefore the biology of this species may not be ideal for mycoherbicide development against wild radish in Australia where the cropping areas have highly seasonal rainfall and free soil moisture is short‐lived. It may be possible to overcome this limitation through appropriate formulation and targeting the timing of application to coincide with predicted rainfall events early in the season when radish seedlings are emerging.

B. NECROTROPHIC PATHOGENS

The necrotrophic and hemibiotrophic pathogens recorded on brassicaceous hosts (Table I) are generally more suited than biotrophic species for development as mycoherbicides because they can be mass produced more readily. Seven promising candidates were identified from Table I on the basis of evidence of host specialisation within the Brassicaceae and then compared against their fit to criteria desirable in a mycoherbicide (Table II). Although not all of these species are recorded on wild radish in Australia, they are

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recorded from cultivated radish or wild radish elsewhere. All of these fungal species are present in Australia, and isolates obtained could be screened for their eYcacy in causing disease on the desired host weed. Colletotrichum higginsianum is an additional species of interest that has not been recorded in Australia. However, it is possible that the recording of Colletotrichum gloeosporoides is actually C. higginsianum because the former is morphologically plastic and has been confused with C. higginsianum (Sutton, 1980). This is likely because C. higginsianum is a species that specialises on brassicaceous hosts. If further research clarifies this situation, then C. higginsianum would be added to the list of species in Table II. In order to develop one of these necrotrophic or hemibiotrophic pathogens into a mycoherbicide, the following principles need to be addressed. First, the pathogen needs to be suitably host specific and virulent on the target weed; second, it must be suitable for industrial scale inoculum production; third, the conditions that are required to produce a disease epidemic need to be well understood; and finally, the formulation of the product needs to be optimised to maximise disease development, be robust in transport and long‐term storage conditions, and be compatible with spray technology on the farm. The pathogens identified as potential candidates for mycoherbicide development against brassicaceous weeds are considered in light of these requirements. C. HOST SPECIFICITY

Based on molecular and hybridisation evidence, wild radish is very closely related to cultivated radish (Snow et al., 2001) and is closely related to brassica crops. Raphanus is estimated to have diverged from the B. rapa/B. oleracea lineage more recently than B. nigra diverged from the B. rapa/B. oleracea lineage (Yang et al., 2002) (Fig. 1). Thus host specificity is the most important element to consider when selecting pathogens for further study. Significantly, although Raphanus is a monophyletic genus, Brassica is polyphyletic and some species such as B. oxyrhina are more closely related to Raphanus than to most other Brassica species (Warwick and Sauder, 2005). Therefore, host range testing needs to be based on the recent molecular phylogeny of the Brassicaceae rather than relying on current taxonomic generic placement of taxa. Fusarium oxysporum has a broad host range and attacks both monocot and dicot hosts. However, within this species there are host‐specific pathovars. In some cases these pathovars may have diVerent chromosome numbers, be phylogenetically distinct, and could represent actual species, for example, F. oxysporum f. sp conglutinans VCG 0101 and F. oxysporum

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Fig. 1. Phylogenetic tree for Brassica rapa (Br), Brassica oleracea (Bo), Brassica nigra (Bn), Raphanus sativus (Rs) and Lepidium virginicum (Lv) reconstructed from nucleotide substitution (k) data of four intergenic spacers in the trnT–trnF and trnD– trnT fragments. When more than one sequence is available for a particular OTU, the values of K are the mean of all possible pairwise comparisons, as described in Wolfe et al. (1987). Reprinted from Yang et al. (2002) with permission from Elsevier.

f. sp. raphani VCG 0102 (Momol and Kistler, 1992). There are three forma speciales associated with Brassicaceae hosts, and Fusarium oxysporum f. sp. raphani is the most specialised to attack Raphanus (Bosland and Williams, 1987). This pathovar has been characterised by nuclear DNA markers that correspond to virulence on Raphanus hosts (Momol and Kistler, 1992). In a molecular study, Baayen et al. (2000) determined that both monophyletic and non‐monophyletic forma speciales occur in F. oxysporum and speculated that this pattern could be explained by the horizontal gene transfer amongst or between Fusarium species, conferring virulence on a particular host. Alternatively, the pattern of phylogenetically unrelated forma speciales of the same host could be due to the independent evolution of pathogenicity genes against a given host in diVerent lineages of F. oxysporum. Although there is some evidence for the monophyletic origins for three forma speciales of F. oxysporum specialising on diVerent Brassicaceae hosts (Kistler et al., 1987; Kistler et al., 1991; Momol and Kistler, 1992), the molecular phylogeny of these taxa remains unresolved and they may represent distinct species. Although F. oxysporum is recorded on Brassicaceae hosts in Australia, F. oxysporum f. sp. raphani, which attacks Raphanus, has not and its presence would need to be ascertained before this pathogen could be considered for local development as a bioherbicide. Leptosphaeria maculans is pathogenic on wild radish and other brassicaceous species. There is evidence of some degree of host specialisation within the L. maculans species complex (Chen and Seguin‐Swartz, 1999; Johnson and Lewis, 1994), and recent phylogenetic evidence suggests that there are at

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least two species within this complex, which includes the recently described L. biglobosa (Mendes‐Pereira et al., 2003). Chen and Seguin‐Swartz (1999) found variability amongst R. raphanistrum individuals in terms of their susceptibility to L. maculans, which suggests that selection for resistance to this pathogen could develop across wild radish populations following repeated exposure to the proposed mycoherbicide. In addition to this limitation, mycoherbicides based on the sexual (Leptosphaeria) or asexual (Plenodomus/Phoma) stages of this genus have not been developed and therefore industrial scale production of propagules may not be cost eVective. Finally, L. maculans is a major pathogen of canola (Howlett, 2004; Williams, 1992) and any mycoherbicide based on this pathogen will need to be of significant benefit to oVset any risks to farming activities. Rhizoctonia solani is pathogenic on a broad range of hosts, but zymogram groups within this species complex are diVerentially pathogenic according to host. In a study of R. solani from crops and weeds in Australia, isolates from ZG5 (AG2‐1) resulted in 100% inhibition of R. raphanistrum seedling emergence compared to 72 and 64% inhibition of B. napus and Triticum aestivum seedling emergence, respectively (Khangura et al., 1999). Therefore, although this pathovar shows some degree of specialisation, it still causes considerable damage to two of the most important crops in southern Australia. The R. solani groups are therefore unlikely to be of any value as a potential mycoherbicide against wild radish. Furthermore, Rhizoctonia does not produce spores in culture and industrial scale propagule production of this pathogen is likely to be diYcult. There are four species of Alternaria that show some degree of host specialisation on brassicaceous species: A. alternata, A. brassicae, A. brassicicola, and A. japonica. The first of these, A. alternata, forms a complex that contains many host‐specific pathovars on non‐brassicaceous taxa, many of which appear to be determined by the production of host‐specific toxins (HSTs) (Hatta et al., 2002; Kusaba and Tsuke, 1994). However, there is no published information on the existence of isolates uniquely pathogenic on Raphanus or that produce HSTs specific for either Brassica or Raphanus species. Of the three remaining Alternaria species; A. japonica is the most frequently cited on Raphanus and is reported as being most specialised for that genus (Rotem, 1994), A. brassicicola appears to be most common on B. oleraceae, and A. brassicae is associated most frequently with oilseed rape B. campestris (Humpherson‐Jones, 1992; Humpherson‐Jones and Phelps, 1989; Sharma and Kolte, 1994). According to a study of pre‐ and post‐ penetration events across eight brassicaceous hosts (not including Raphanus species), A. brassicicola was the least specialised, A. brassicae was most

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specialised for infection of B. nigra, and A. japonica was most specialised for infection of Sinapis alba. In another study, the species most susceptible to A. brassicae was B. nigra followed in order of least to greatest resistance by B. juncea, B. campestris, B. napus, B. oleracea, and B. carinata (Bansal et al., 1990). None of the necrotrophic species discussed, with the possible exception of F. oxysporum f. sp. raphani, are uniquely pathogenic on Raphanus and hence all pose a risk to canola in terms of mycoherbicide development. Of the necrotophic genera discussed, Alternaria and Fusarium are the most promising for mycoherbicide development in terms of their potential for industrial scale inoculum production. For this reason the following discussion on virulence, inoculum production formulation, and epidemiology focuses on these two genera.

III. STRATEGIES TO INCREASE VIRULENCE OR SPECIFICITY A. MULTIPLE PATHOGENS

Plant disease may be more destructive when combinations of pathogens are involved (Jeger, 2001). Synergistic interactions between pathogens used in weed biological control have been recorded for Cirsium arvense (Guske et al., 2004) in Germany and Xanthium occidentale in Australia (Morin et al., 1993a,b). The potential for synergistic pathogen interactions is an area that requires more exploration and in particular the possibility of applying biotrophic and necrotrophic pathogens simultaneously. Plant defense response to these types of pathogens is upregulated according to two diVerent biochemical pathways: salicylic acid (SA) and jasmonic acid/ethylene (JA), respectively (Tierens et al., 2002). Therefore, a simultaneous assault by the two types of pathogen may render the host more susceptible to disease. Pathogen combinations that could be considered for wild radish include A. japonica and H. parasitica or A. japonica and Aphanomyces raphani. Unless advances in tissue culture or biotechnology enable H. parasitica to be grown in vitro, this pathogen would need to be applied as an infected radish straw‐based formulation. The Alternaria could be sprayed as a foliar mycoherbicide after the radish has emerged early in the cropping phase, but it would be more cost eVective if it were applied as a solid formulation along with radish straw. Similarly, Alternaria and Aphanomyces would be applied simultaneously in a solid formulation such as ‘‘stabileze’’ (Quimby et al., 1999) early in the cropping season to target emerging wild radish seedlings.

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Some chemical herbicides make plants more susceptible to disease either via interference with the physical barriers of the plant to attack or to the biochemical defense pathways of the host. The coupling of chemical growth regulators or herbicides with pathogens has resulted in synergistically higher rates of weed control in many situations (Grant et al., 1990; Hoagland, 1996, 2001; Schnick and Boland, 2004; Tierens et al., 2002). A number of chemical compounds and fungal taxa have been tested that may also be suitable for use against brassica weeds. Probably the earliest recorded herbicide–pathogen interaction against host plants include glyphosate with the pathogens Phytophthora megasperma and Alternaria cassiae against soybean (Keen et al., 1982) and Senna obtusifolia (Cassia obtusifolia) (Sharon et al., 1992), respectively. The application of glyphosate inhibits the enzyme 5‐enolpyruvylshikimate 3‐phosphate synthase, which is involved in the production of plant defense compounds such as phytoalexins. The application of sublethal doses of glyphosate to S. obtusifolia suppressed the accumulation of phytoalexins in response to Alternaria cassiae inoculation and led to a fivefold reduction in the amount of conidia required to kill the plants (Sharon et al., 1992). At the same time there was no increase in susceptibility of the closely related non‐target crop plant Glycine max. A similar interaction with glyphosate has been recorded for the fungal taxa Colletotrichum (Le´ ger et al., 2001) and Fusarium avenaceum (Oleskevich et al., 1998). This suggests that pathovars of Alternaria alternata, A. brassicae, A. brassicicola, A. japonica, and Fusarium oxysporum could be tested for synergistic interactions with glyphosate in controlling brassicaceous weeds such as R. raphanistrum. Watson and Ahn (2001) also noted that chemicals such as thidiazuron (a plant growth regulator) and basagram (a herbicide) have been applied with Colletotrichum coccodes to control Abutilon theophrastii. These chemicals may decrease the capacity of the plant to outgrow the disease. Bromoximal þ MCPA, Metribuzin, Diclofop, Imazethapyr, and Sethoxydim have been used in combination with Colletotrichum gloeosporioides f. sp. malvae to control Malva pusilla (Grant et al., 1990). Some of these chemicals inhibited spore germination and the chemical and biological agents had to be applied separately in order to generate a positive interaction for weed control. Hoagland (2001) lists several other herbicides for which synergistic interactions with four pathogens have been patented and all of these pathogens belong in the genera Alternaria, Colletotrichum, and Fusarium. Significantly, none of these interactions led to an increase in the host range of the pathogens tested (Hoagland, 1996).

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C. PHYTOTOXIC METABOLITES

There is a long list of phytotoxic metabolites that are derived from fungal pathogens such as brefeldin A (Vurro et al., 1998), cercosporin (Assante et al., 1977), colletotrichin (Duke et al., 1992), destruxins (Buchwaldt and Jenson, 1991), fumonisins, and AAL toxin (Abbas et al., 1995; Gelderblom et al., 1988). These could potentially be developed into chemical herbicides or applied in conjunction with a pathogen of wild radish in order to increase disease expression synergistically. Vurro et al. (1998) found that some of these phytotoxic compounds, such as brefeldin A from Alternaria, suppress plant defense enzymes at very low concentrations, even below those required for phytotoxic symptoms such as tissue necrosis. Most of these phytotoxic compounds have a broader host range than the pathogen from which they are derived (Hoagland, 2001) and therefore the use of these metabolites would need to be accompanied by the breeding of resistance in crop plants. For example, destruxins, which are produced by Alternaria brassicae (Buchwaldt and Jenson, 1991), cause necrosis to a much greater range of plants than A. brassicae from which it was isolated. There are at least 36 of these compounds that are diVerentially toxic to various plant and insect species (Pedras et al., 2002). Destruxin B is most phytotoxic to Brassica species and its toxicity decreases according to increasing phylogenetic distance from this genus (Buchwaldt and Green, 1992). There may be other destruxins in this class of compounds that are more toxic to Raphanus and the identification of such compounds could be achieved by screening isolates of A. brassicae and A. japonica from radish hosts. Some of the other pathogens listed in Table I, such as Alternaria japonica, could be tested for the production of phytotoxic compounds against brassica weeds. These metabolites could be developed into chemical herbicides, for example, as occurred with glufosinate and bialaphos, which were developed from a herbicidal strain of a streptomycete. Bialophos is unique in terms of herbicides in that it is produced via a fermentation process. Glufosinate is the synthetic ammonium salt of phosphinothricin (PPT), and resistance to this herbicide has been conferred on over 20 crop plants (Hoagland, 2001), including Brassica napus (Stringam et al., 2003), via the insertion of either bar or pat genes from Streptomyces hygroscopicus or S. viridochromosgenes, respectively. The identification of new compounds from fungal pathogens could lead to similar developments for controlling brassica weeds in a broad range of crops, including those in the Brassicaceae. Another possibility is to introduce genes for phytotoxic compounds into a minor pathogen to create hypervirulence.

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Amsellem et al. (2002) and Gressel (2001) have proposed coupling virulence genes with fail‐safe mechanisms to prevent spread and transgene introgression into crop pathogens in order to improve a mycoherbicide (Fig. 2). Strategies to prevent hypervirulent pathogen spread include creating asporogenous mutants that require an exogenous chemical or defined media to sporulate or that are only capable of infecting via artificial spores. Introgression of transgenes to other fungi would be restricted by flanking the virulence genes with mitigator genes such as antisporulation or antimelanin genes that would reduce the fitness of a fungus that received the gene. Amsellem et al. (2002) demonstrated that adding Fusarium virulence genes to the fungus Colletotrichum coccodes increased its pathogenicity to the weed Abutilon theophrasti without changing the pathogen host range. Genes coding for plant toxins could also be introduced into host‐specific pathovars of brassicaceous weeds. Pathogens (hypervirulent or otherwise) could also be

Fig. 2. Fail‐safe mechanisms for hypervirulent biocontrol agents. Dual fail‐safe mechanisms to prevent (step 1) spread of biocontrol agents and (step 2) their introgression into other organisms. a, chlamydospores; b, microconidia; c, macroconidia; d, ascus with ascospores; e, sclerotia; f, asporogenic mycelia. Reprinted from Gressel (2001) with permission from Elsevier.

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modified genetically to reduce their persistence and dissemination by knocking out genes associated with sporulation or modifying with mating type genes. These traits are especially important for targeting brassicaceous weeds in order to reduce the impact on closely related, nontarget brassicaceous crops such as canola. Ideally, the candidate gene product should be specifically phytotoxic for wild radish and be under the control of one or a few closely linked genes. Destruxins are one group of phytotoxic metabolites that may have this potential. Because a destruxin gene has been cloned from M. anisopliae (Bailey et al., 1996), it should be possible to clone destruxin B and other destruxin genes phytotoxic to Raphanus hosts. This will facilitate the genetic transformation of a candidate mycoherbicide or at least the selection of virulent isolates. Genes from A. brassicae coding for destruxins could be inserted into another host‐specific pathogen such as F. oxysporum f. sp. raphani or a weak pathogen such as Colletotrichum higginsianum. The gene would be introduced under the control of a promoter that is induced during infection or under the control of a chemically induced promoter such as the ethanol‐ induced alcA gene promoter. In the latter scenario, the mycoherbicide and ethanol could be applied together to induce the gene. This would limit the virulence of this pathogen to the initial infection of the weed and reduce the risk to subsequent crops of a related species such as canola to which the organism may also be a weak pathogen. The alcA gene from Aspergillus could be suited to this role because it is a widely used system for the induction of heterologous genes in a range of organisms, including other fungi as well as plants and animals. Nikolaev et al. (2002) have successfully transferred a heterologous gene construct under the control of the alc promoter into a fungal species that lacks the inducible alcA gene. They transferred the inducible and strongly expressed alcA gene encoding alcohol dehydrogenase I from Aspergillus nidulans together with the activator gene alcR in the industrial fungus Aspergillus niger. Induced expression of the alcA gene was similar in the transformed A. niger to the donor A. nidulans, as monitored by alcA transcription, alcohol dehydrogenase activity, and heterologous expression of the reporter enzyme, ‐glucuronidase (Fig. 3). It has also been shown that the expression of genes under the control of the alcA promoter is dose dependent and reversible. Using alcR alcA:Gus reporter constructs, Roslan et al. (2001) found that transgene expression in Arabidopsis reached a peak five days after ethanol induction and by 11 days after induction expression had fallen to levels only marginally above the noninduced state. Therefore the production of a phytotoxin under the control of this inducible promoter

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Fig. 3. ‐Glucuronidase (GUS) activity of A. niger and A. nidulans transformants containing gpd:alcR and palcA:uidA constructs. (A) Kinetics of GUS activity of A. niger transformant containing gpd:alcR and palcA:uidA genes grown under various growth conditions: noninduced conditions (NI, ); induced by 50 mM EMK ( ); induced by 1% ethanol (Et,
); and repressed by 1% glucose in the presence of 1% ethanol (EtG, ). GUS activity was followed up to 12 h and expressed in units per milligram as described in Section II. (B) Kinetics of ‐glucuronidase activity in A. niger and A. nidulans transformants containing gpd:alcR and alcA:uidA genes grown in the presence of 0.1% yeast extract and induced with 1% ethanol up to 12 h. Transformants in A. niger ( ) and A. nidulans ( ) are shown. GUS activity was expressed in units per milligram as described in Section II. (Reprinted from Nikolaev et al., 2002 with permission from Elsevier.)



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could be limited to the time of application after which the production of the phytotoxin would fall and the organism would remain relatively benign to subsequent brassica crop plants. Ideally, a mycoherbicide based on high expression of a phytotoxic metabolite should be developed in conjunction with crop plants that are resistant to the phytotoxin either through host selection or the introduction of genes

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for disabling the phytotoxin. A potential source of resistance to destruxin B is Sinapis alba, which is resistant to A. brassicae partly because it detoxifies destruxin B rapidly via sequential hydroxylation and glucosylation processes (Pedras et al., 2000, 2001). The breakdown product of this process, hydroxydestruxin B, also induced defense‐related phytoalexins in the resistant species S. alba but not in the susceptible species B. napus. Sharma et al. (2002) evaluated 38 Brassicaceae species for resistance to A. brassicae and identified 8 highly resistant species that could prove useful sources of resistance for introgression to crop species. Resistance genes against Alternaria pathogens have been introduced into Brassica crop plants from diverse sources, including fungi (Mora and Earle, 2001) and unrelated plant species (Kanrar et al., 2002). Potentially F. oxysporum is a good candidate pathogen for gene manipulation because protocols for genetic transformation have been developed for this species (Momol and Kistler, 1992). However, the fact that this species is also a pathogen that kills immune compromised mammals, including humans (Ortoneda et al., 2004), may make it an especially contentious pathogen to modify genetically. The safest strategy would be to utilise knowledge gained via techniques such as targeted gene knockout and genomics research (Gold et al., 2001) to further understand pathogenesis and then select or modify virulent isolates of A. brassicae or A. japonica in conjunction with breeding resistance in susceptible crop species such as canola. Other fungal metabolites with phytotoxic activity include cercosporin, fumonisins, and the related AAL toxins. However, these are also mammalian toxins and therefore not likely to be developed into herbicides in their own right. It may be possible to insert genes for these compounds into host‐specific pathogens of brassicaceous weeds in a similar way to that suggested for destruxins. Mycotoxins are thought to be short‐lived in the environment (Hoagland, 2001) and some, such as fumonisin B, are excreted rapidly when ingested by non‐human primates (Shephard and Snijman, 1999). Mycotoxins have been associated with an increasing number of human health problems (Hoagland, 2001). Therefore the potential detrimental aVects of using a mycotoxin‐producing bioherbicide would need to be studied carefully in order to identify risks to human health and to determine the safety of their use. The risks associated with genetically manipulated organisms are managed through the appropriate legislation, and the requirements may change from one nation to another. In Australia, these risks are managed through the regulatory framework of the Gene Technology Act 2000 and the Gene Technology Regulations that came into eVect on June 21, 2001. Any genetically modified mycoherbicides against brassicaceous weeds would be subject to this process.

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IV. EPIDEMIOLOGY WITH RESPECT TO CROPPING CONDITIONS Mycoherbicide applications aim to maximise initial infection rates and rely on disease development to kill plants at the early seedling stage. The initial establishment of disease is critical and for this reason formulations are designed to increase infection rates by maintaining moisture levels at the infection surface. However, the success of a mycoherbicide may also depend on subsequent polycyclic disease dynamics, which include variables identified by Van der Plank (1963, 1984), such as the length of the latent period, the number of infection cycles in a season, host resistance, and external (environmental) factors. If the plant is able to outgrow the initial infection, then the continuous production of spores and the reinfection of growing points on the plant are likely to be important in the eventual success of the disease epidemic. All three Alternaria species specialising on Brassicaceae will infect the host at all developmental stages and may cause seedling stunting or death (Valkonen and Koponen, 1990). Disease development associated with A. brassicae and A. brassicicola increases with host age and can impact severely on pod development, leading to reduced seed set and seed viability (Humpherson‐ Jones, 1992). Sporulation in all three Alternaria species is rapid (12–48 h in vitro) (Humpherson‐Jones and Phelps, 1989), and the latent phase of infection is brief (Verma and Saharan, 1994). Because these species cause disease on all host stages and result in polycyclic epidemics, they are good candidates for mycoherbicide development against brassicaceous weeds. There is an interaction between temperature and humidity for the induction of sporulation in all three species. At 208C a minimum of 87 and 83% relative humidity is required for sporulation of A. brassicae and A. brassicicola, respectively (Humpherson‐Jones and Phelps, 1989). In vivo, the optimal sporulation temperature for A. brassicae is lower (18–248C) than for A. brassicicola (20–308C) (Humpherson‐Jones and Phelps, 1989). Although all three species are able to germinate and infect their hosts relatively quickly, provided that free moisture is available, A. brassicae is most rapid and infection occurs within 6 h at optimal temperatures compared to 18 h for A. brassicicola (Humpherson‐Jones, 1992). Free moisture as rain, dew, or 100% humidity for a minimum of 9–18 h at optimal temperatures between about 20 and 308C is required for infection by all three species. Alternaria brassicae has the lowest optimal temperature range for germination (21–288C) and infection (19–218C) of the three species (Degenhardt et al., 1982). The optimal temperature for germination is 28–318C in A. brassicicola and 23–318C for A. japonica, and the optimal

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temperature for infection is 25–278C for A. brassicicola and 17–298C for A. japonica (Degenhardt et al., 1982). The temperature requirements of A. brassicae and A. japonica are within the range typical of the early part of the cropping season in temperate or Mediterranean climates. In Australia, temperatures in the Western Australian cropping region (April–May) are likely to range from 8 to 278C (Anonymous, 2005a). Cropping regions of southwest Australia are characterised by a Mediterranean type of climate with cool wet winters and hot dry summers (Love, 2005). The cropping zone of south‐eastern Australia is characterised by a temperate climate, with spring rainfall and temperatures within the range required for Alternaria infection during the early part of autumn (Anonymous, 2005b). Cropping is usually restricted to winter unless irrigation is used. The temperature requirements for A. brassicicola are higher and therefore a mycoherbicide based on this species is likely to suit a warmer subtropical climate. The cropping zone of northern New South Wales and southern Queensland is characterised by a tropical to subtropical climate. Alternaria japonica is less well studied, but appears to have a greater range of climatic suitability, is more specialised on Raphanus, and therefore appears to be the best candidate overall. An advantage in the suitability of an organism for mycoherbicide development would be a capacity to germinate under conditions of repeated short moisture periods. This is because extended periods of wetness in Australian cropping areas are not reliable. However, Bailey (2004) found that A. cirsinoxia was able to infect and cause disease on Canada thistle (Cirsium arvense) under intermittent leaf wetness and drying, although viability and disease expression was less than that recorded for continuous wetness of 24–48 h, and a similar pattern may be true for Alternaria species attacking Raphanus. Another epidemiological trait of Alternaria species is their capacity to survive in plant debris or soil to provide inoculum for disease outbreaks in subsequent seasons (Rotem, 1994). Alternaria brassicae and A. brassicicola remain viable for a long period of time as spores on seed coat or as mycelium in seed as well as in infected plant debris for up to 12 weeks or more (Humpherson‐Jones, 1989). This could present problems if these pathogens were developed as a mycoherbicide because they may cause disease in susceptible brassica crops planted in subsequent seasons. This could be managed through selecting strains that are non‐pathogenic to canola, developing non‐sporulating mutants, breeding resistant canola varieties, and by leaving a break period between the use of the mycoherbicide and the

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planting of canola. A break period has been identified as the solution to this type of issue for the biological control fungus Sclerotinium sclerotorium in pastures (Bourdoˆ t et al., 2000; De Jong et al., 2002).

V. INOCULUM PRODUCTION AND FORMULATION A. INDUSTRIAL SCALE INOCULUM PRODUCTION

Because inoculum is of fundamental importance in disease initiation and development, maximising inoculum production through industrial scale fermentation techniques and through appropriate formulation is of prime consideration in developing a mycoherbicide. Propagule production may be achieved in either a liquid or solid‐state fermentation process. Liquid fermentation is preferred because it is the most cost‐eVective technology for growing fungi, but solid state fermentation is often more conducive to fungal spore production (Lomer et al., 2001). Purifying spore preparations from submerged fermentation systems involves centrifugation or filtration techniques that are considered easier and result in a less bulky formulation than techniques for recovering spores from solid‐state or biphasic systems (Auld and Morin, 1995). However, a cyclone air stream technology for the separation of conidia from solid‐state systems has been developed that is viable commercially (Lomer et al., 2001). Of the species listed in Table II, industrial scale inoculum production has previously only been considered for Alternaria and Fusarium genera. In general, Alternaria has been considered as a bioherbicide in other systems (Bailey, 2004; Green and Bailey, 2000; Green et al., 2001; Walker and Boyette, 1986). This genus usually does not produce conidia in liquid culture but will do so in solid‐state fermentation (Daigle et al., 1998; Masangkay et al., 2000). In contrast, Fusarium conidia can be produced in liquid culture (Amsellem, 1999; Quimby et al., 2001). The method of conidia production may aVect the characteristics of the conidia produced with respect to long‐ term viability and spore surface characteristics such as hydrophobicity, as discussed later. For some biopesticide fungi, certain spore types will be produced in liquid and others only on a solid substrate at the interface between substrate and air (Lomer et al., 2001). These various types of conidia are characterised by diVerent cell surface properties, which have implications for infection and for mycoherbicide formulation. Many Ascomycete and Basidiomycete fungal conidia attach to hydrophobic leaf surfaces by virtue of conidia wall

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proteins known as hydrophobins (Wo¨ sten, 2001). Hydrophobin membranes enable spores to attach rapidly and strongly to hydrophobic leaf surfaces and anchor appressoria that penetrate the leaf surface. In other fungal species, spore attachment is facilitated by the production of mucilage, the composition of which may include glycoproteins, lipids, and polysaccharides (Hamer et al., 1988; Sugui et al., 1998; Tucker and Talbot, 2001). This mucilage may be water soluble and assist in the dispersal of spores in rain splash. Spore surface properties may determine the type of mycoherbicide formulation that is most suitable. Hydrophobic conidia are suited to an oil‐based formulation or they can be applied in a water‐based formulation if an emulsifying agent is added (Feng et al., 1994). Fungi such as Colletotrichum, which form hydrophilic mucilaginous matrices around their spores, may be suited to a water‐based formulation without the need to add emulsifying agents. This may in part explain why Auld et al. (1990) were able to obtain successful infection of Xanthium spinosum in field inoculations with a simple liquid formulation of Colletotrichum orbiculare. Extremely hydrophobic spores are likely to clump in water‐based formulation and hence result in uneven dispersal on the target plant and variable levels of infection. B. FORMULATION

A range of solid formulations have been developed, which have been reviewed comprehensively by Auld et al. (2003). Fusarium species have been deployed successfully as mycoherbicides in solid formulations. These include the use of F. oxysporum in a Pesta formulation for the control of sunflower broomrape (Figs. 4 and 5) (Shabanna et al., 2003) and the ‘‘stabileze’’ process of Quimby et al. (1999). Shabanna et al. (2003) compared 19 Pesta formulations, using two types of fungal spores and eight adjuvants, in terms of their shelf‐life and for eYcacy under greenhouse conditions. Amending Pesta with yeast extract reduced the loss of viability of microconidia and chlamydospores during production of the formulations. Viability was greatest when granules were stored at 38C (Figs. 4 and 5). The five most eVective formulations of Pesta caused reduction in Orobanche biomass by 67–80%. For the control of wild radish, one of these formulations could be adopted for the soil‐borne pathogens F. oxysporum f. sp. raphanus or Aphanomyces raphani. This approach would be suitable because Fusarium species have already been applied in these formulations to target other weeds (Amsellem et al., 1999; Shabanna et al., 2003) and because F. oxysporum f. sp. raphanus

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Fig. 4. Shelf life (viable colony‐forming units, cfu) of Fusarium oxysporum f. sp. orthocerus–Pesta microconidia (PM) 1, 5 and 7 at various storage conditions (water activities, aw and temperature levels). Data points are means with standard error bars. Reprinted from Shabana et al. (2003) with permission from Elsevier.

is primarily a root pathogen able to germinate and infect the plant under moist soil conditions (Toyota and Kimura, 1992). Liquid formulations are likely to be better than solid formulations against wild radish or other weeds in dry cropping situations because the

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Fig. 5. Shelf life (viable colony forming units, cfu) of Fusarium oxysporum f. sp. orthocerus –Pesta chlamydospores (PC) 14, 15 and 18 at various storage conditions (water activities, aw and temperature levels). Data points are means with standard error bars. Reprinted from Shabana et al. (2003) with permission from Elsevier.

former are more likely to work under low or unpredictable rainfall events. Mycoherbicides in liquid form are applied as a spray in a similar manner to conventional herbicides and are formulated in order to reduce dew dependency for germination and infection of plant shoots (Auld et al., 2003).

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Of the pathogens listed, the most suited to this approach are the Alternaria species because they are foliar pathogens and are resistant to ultraviolet and other harsh conditions (Rotem, 1994). Auld et al. (2003) reviewed bioherbicide formulations and indicated that water‐in‐oil‐in‐water (WOW) emulsions were better than many other liquid formulations for reducing dew dependence in the field. In the past Alternaria conidia have been produced in solid or biphasic systems (Babu et al., 2004; ´ vila et al., 2000; Masangkay et al., 2000), but not in Daigle et al., 1998; De A submerged fermentation, indicating that their conidia probably only form at the interface between the substrate and the air. Therefore Alternaria spores are probably hydrophobic and would suit a simple, invert, or WOW emulsion, which, as well as facilitating an even distribution of spores, will also reduce dew dependence. Alternaria species have been applied as spores in oil emulsions (Babu et al., 2003; Connick et al., 1991; Shabana et al., 1997) and as spores or mycelia in an alginate gel formulated in either an aqueous or an oil‐based emulsion (Shabana et al., 1995, 1997).

VI. CONCLUSION In terms of host specificity, the biotrophic pathogens A. candida, A. raphani, and H. parasitica f. sp. raphani show the most promise to selectively control wild radish in canola cropping areas (Table II). However, these species are limited by the likely diYculty and cost of producing inoculum on an industrial scale. It may be possible to overcome these diYculties through innovative culture, tissue culture, or biotechnological solutions. First, the ideal candidate species from amongst these would need to be identified by investigating their molecular taxonomy, species boundaries, and host range on Brassica species. Second, the capacity for each of these species to infect under low moisture cropping climates would need to be investigated in terms of dew and temperature requirements and other epidemiological features. If a candidate was identified that could create the disease epidemic required without impacting on canola, then the challenge of developing cost‐ eVective industrial scale inoculum production could be investigated, possibly through tissue culture techniques or gene manipulation of the pathogen to grow on artificial media. Alternaria brassicae, A. brassicicola, A. japonica, and Fusarium oxysporum are the necrotrophic pathogens with the most potential for mycoherbicide development (Table II). The greatest diYculty with these taxa is their potential to attack canola. This could be overcome by identifying whether the pathogenicity of these organisms was due to the production of phytotoxic

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metabolites and then breeding (either by conventional or molecular techniques) host crops that are able to detoxify the compound. The first priority in the necrotroph‐based strategy would be to determine whether there are highly virulent strains of any of these pathogens on wild radish in Australia. Second, the basis of that virulence would need to be determined, and if it were largely due to the production of host‐specific toxins, then such compounds would need to be characterised. Concurrently the virulence of these isolates on canola should be assessed and if they are not pathogenic on canola then further host range testing could be pursued with the view to developing the mycoherbicide along conventional lines. If the HST is responsible for pathogenicity, then canola hosts could be screened for resistance and genes responsible for their detoxification identified (either from canola or from the pathogen). These genes could then be selected for or inserted into canola hosts to enhance resistance. The possibility of synthesising or otherwise fermenting and isolating the phytotoxic compound along the lines of bialophos/glufosinate could be researched. The final product could be a pure mycoherbicide, a combination of the phytotoxic compound and some host‐specific pathogen, or the phytotoxic compound applied alone.

ACKNOWLEDGMENTS We thank the CRC for Australian Weed Management and CSIRO Entomology for their support during this work. Nuccia Eyres from the Agriculture Department of Western Australia assisted by searching the APDD and CCDB databases for pathogen records, and Louise Morin also provided a list of pathogen records from NSW and Qld herbaria. We thank Gavin Ash, Bruce Auld, and Bernie Dell for their comments on drafts of the manuscript.

REFERENCES Abbas, H. K., Tanaka, T. and Duke, S. O. (1995). Pathogenicity of Alternaria alternata and Fusarium moniliforme and phytotoxicity of AAL‐toxin, and fumonisn B1 on tomato varieties. Journal of Phytopathology 143, 329–334. Amsellem, Z., Zidack, N. K., Quimby, P. C. and Gressel, J. (1999). Long‐term dry preservation of viable mycelia of two mycoherbicidal organisms. Crop Protection 18, 643–649. Amsellem, Z., Cohen, B. A. and Gressel, J. (2002). Engineering hypervirulence in a mycoherbicidal fungus for eYcient weed control. Nature Biotechnology 20, 1035–1039. Anonymous (2005a). Australian Bureau of Meteorology: http://www.bom.gov.au/ climate/map/climate_avgs/a12a.shtml. Web site consulted 25 June 2005.

168

A. MAXWELL AND J. K. SCOTT

Anonymous (2005b). World Meteorological Organisation: http://www.worldweather. org/185/m185.htm.Web site consulted 25 June 2005. Assante, G., Locci, R., Camarda, L., Merlini, L. and Nasini, G. (1977). Screening of the genus Cercospora for secondary metabolites. Phytochemistry 16, 243–247. Auld, B. A., Hetherington, S. D. and Smith, H. E. (2003). Advances in bioherbicide formulation. Weed Biology and Management 3, 61–67. Auld, B. A. and Morin, L. (1995). Constraints in the development of bioherbicides. Weed Technology 9, 638–652. Auld, B. A., Say, M. M., Rindings, H. I. and Andrews, J. (1990). Field application of Colletotrichum orbiculare to control Xanthium spinosum. Agriculture Ecosystems and Environment 32, 315–323. Baayen, R. P., O’Donnell, K., Bonants, P. J. M., Cigelnik, E., Kroon, L. P. N. M., Roebroeck, E. J. A. and Waalwijk, C. (2000). Gene genealogies and AFLP analyses in the Fusarium oxysporum complex identify monophyletic and nonmonophyletic formae speciales causing wilt and rot disease. Phytopathology 90, 891–900. Babu, R. M., Sajeena, A. and Seetharaman, K. (2003). Bioassay of the potentiality of Alternaria alternata (Fr.) keissler as a bioherbicide to control waterhyacinth and other aquatic weeds. Crop Protection 22, 1005–1013. Babu, R. M., Sajeena, A. and Seetharaman, K. (2004). Solid substrate for production of Alternaria alternata conidia: A potential mycoherbicide for the control of Eichhornia crassipes (water hyacinth). Weed Research 44, 298–304. Bailey, A. M., Kershaw, M. J., Hunt, B. A., Paterson, I. C., Charnley, A. K., Reynolds, S. E. and Clarkson, J. M. (1996). Cloning and sequence analysis of an intron‐containing domain from a peptide synthetase‐encoding gene of the entomopathogenic fungus Metarhizium anisopliae. Gene 173, 195–197. Bailey, K. L. (2004). Microbial weed control: An oV‐beat application of plant pathology. Canadian Journal of Plant Pathology 26, 239–244. Bansal, V. K., Se´ guin‐Swartz, G., Rakow, G. F. W. and Petrie, G. A. (1990). Reaction of Brassica species to infection by Alternaria brassicae. Canadian Journal of Plant Science 70, 1159–1162. Bosland, P. W. and Williams, P. H. (1987). An evaluation of Fusarium oxysporum from crucifers based on pathogenicity, isozyme polymorphism, vegetative compatibility and geographic origin. Canadian Journal of Botany 65, 2067–2073. Bourdoˆ t, G. W., Saville, D. J., Hurrell, G. A., Harvey, I. C. and De Jong, M. D. (2000). Risk analysis of Sclerotinia sclerotiorum for biological control of Cirsium arvense in pasture: Sclerotium survival. Biocontrol Science and Technology 10, 411–425. Buchwaldt, L. and Green, H. (1992). Phytotoxicity of destruxin B and its possible role in the pathogenesis of Alternaria brassicae. Plant Pathology 41, 55–63. Buchwaldt, L. and Jensen, J. S. (1991). HPLC purification of destruxins produced by Alternaria brassicae in culture and leaves of Brassica napus. Phytochemistry 30, 2311–2316. Charudattan, R. and Dinoor, A. (2000). Biological control of weeds using plant pathogens: Accomplishments and limitations. Crop Protection 19, 691–695. Charudattan, R., Elliot, M., DeValerio, J. T., Hiebert, E. and Pettersen, M. E. (2003). Tobacco mild green mosaic virus: A virus‐based bioherbicide. In ‘‘Proceedings of the XI International Symposium on Biological Control of Weeds’’ (J. M. Cullen, D. T. Briese, D. J. Kriticos, W. M. Lonsdale, L. Morin and J. K. Scott, eds.), p. 63. CSIRO Entomology, Canberra, Australia.

OPPORTUNITIES FOR THE CONTROL OF BRASSICACEOUS WEEDS

169

Chen, C. Y. and Se´ guin‐Swartz, G. (1999). Reaction of wild crucifers to Leptosphaeria maculans, the causal agent of blackleg of crucifers. Canadian Journal of Plant Pathology 21, 361–367. Choi, Y.‐J., Hong, S.‐B. and Shin, H.‐D. (2003). Diversity of the Hyaloperonospora parasitica complex from core brassicaceous hosts based on ITS rDNA sequences. Mycological Research 107, 1314–1322. Connick, W. J., Daigle, D. J. and Quimby, P. C. (1991). An improved invert emulsion with high water‐retention for mycoherbicide delivery. Weed Technology 5, 442–444. Cook, R. P. and Dube´ , A. J. (1989). ‘‘Host‐Pathogen Index of Plant Diseases in South Australia’’ Field Crops Pathology Group, South Australian Department of Agriculture. Daigle, D. J., Connick, W. J., Boyette, C. D., Jackson, M. A. and Dorner, J. W. (1998). Solid‐state fermentation plus extrusion to make biopesticide granules. Biotechnology Techniques 12, 715–719. ´ vila, Z. R., De Mello, S. C. M., Ribeiro, Z. M. D. and Fontes, E. M. G. (2000). De A Production of Alternaria cassiae inoculum. Pesquisa Agropecua´ ria Brasileira 35, 533–541. Degenhardt, K. J., Petrie, G. A. and Morrall, R. A. A. (1982). EVects of temperature on spore germination and infection of rapeseed by Alternaria brassicae, A. brassicicola and A. raphani. Canadian Journal of Plant Pathology 4, 115–118. De Jong, M. D., Bourdoˆ t, G. W., Powell, J. and Goudriaan, J. (2002). A model of the escape of Sclerotinia sclerotiorum ascospores from pasture. Ecological Modelling 150, 83–105. Dickinson, C. H. and Greenhalgh, J. R. (1977). Host range and taxonomy of Peronospora on crucifers. Transactions of the British Mycological Society 69, 111–116. Duke, S. O., Gohbara, M., Paul, R. N. and Duke, M. V. (1992). Colletotrichin causes rapid membrane damage to plant cells. Journal of Phytopathology 134, 289–305. Feng, M. G., Poprawski, T. J. and Khachatourians, G. G. (1994). Production, formulation and application of the entomopathogenic fungus Beauveria bassiana for insect control: Current status. Biocontrol Science and Technology 4, 3–34. Gelderblom, W. C. A., Jaskiewicz, M. K., Marasas, W. F. O., Thiel, P. G., Horak, R. M., Vleggaar, R. and Kriek, N. P. J. (1988). Fumonisins‐novel mycotoxins with cancer‐promoting activity produced by Fusarium moniliforme. Applied and Environmental Microbiology 54, 1806–1811. Gold, S. E., Garcia‐Pedrajas, M. D. and Martinez‐Espinoza, A. D. (2001). New (and used) approaches to the study of fungal pathogenicity. Annual Review of Phytopathology 39, 337–365. Grant, N. T., Prusinkiewicz, E., Mortensen, K. and Makowski, R. M. D. (1990). Herbicide interactions with Colletotrichum gloeosporioides f. sp. malvae a bioherbicide for round‐leaved mallow (Malva pusilla) control. Weed Technology 4, 716–723. Green, S. and Bailey, K. L. (2000). EVects of leaf maturity, infection site and application rate of Alternaria cirsinoxia conidia on infection of Canada thistle (Cirsium arvense). Biological Control 19, 167–174. Green, S., Mortensen, K. and Bailey, K. L. (2001). Host range, temperature response, survival and overwintering of Alternaria cirsinoxia. Biological Control 20, 57–64.

170

A. MAXWELL AND J. K. SCOTT

Greenhalgh, J. R. and Dickinson, C. H. (1975). DiVerential reactions of three crucifers to infection by Peronospora parasitica (Pers. ex Fr.) Fr. Phytopathologische Zeitschrift 84, 131–141. Gressel, J. (2001). Potential failsafe mechanisms against the spread and introgression of transgenic hypervirulent biocontrol fungi. Trends in Biotechnology 19, 149–154. Guske, S., Schulz, B. and Boyle, C. (2004). Biocontrol options for Cirsium arvense with indigenous fungal pathogens. Weed Research 44, 107–116. Hamer, J. E., Howard, R. J., Chumley, F. G. and Valent, B. (1988). A mechanism for surface attachment in spores of a plant pathogenic fungus. Science 239, 288–290. Hatta, R., Ito, K., Hosaki, Y., Tanaka, T., Tanaka, A., Yamamoto, M., Akimitsu, K. and Tsuge, T. (2002). A conditionally dispensable chromosome controls host‐specific pathogenicity in the fungal plant pathogen Alternaria alternata. Genetics 161, 59–70. Heap, I. (2005). The international survey of herbicide resistant weeds. Available: www.weedscience.com. (Web site consulted 4 February 2005). Hoagland, R. E. (1996). Chemical interactions with bioherbicides to improve eYcacy. Weed Technology 10, 651–674. Hoagland, R. E. (2001). Microbial allelochemicals and pathogens as bioherbicidal agents. Weed Technology 15, 835–857. Howlett, B. J. (2004). Current knowledge of the interaction between Brassica napus and Leptosphaeria maculans. Canadian Journal of Plant Pathology 26, 245–252. Humpherson‐Jones, F. M. (1989). Survival of Alternaria brassicae and Alternaria brassicicola on crop debris of oilseed rape and cabbage. Annals of Applied Biology 115, 45–50. Humpherson‐Jones, F. M. (1992). Epidemiology and control of dark leaf spot of brassicas. In ‘‘Alternaria Biology, Plant Diseases and Metabolites’’ (J. Chelkowski and A. Visconti, eds.), pp. 267–288. Elsevier, Amsterdam. Humpherson‐Jones, F. M. and Phelps, K. (1989). Climatic factors influencing spore production in Alternaria brassicae and Alternaria brassicicola. Annals of Applied Biology 114, 449–458. Imaizumi, S., Nishino, T., Miyabe, K., Fujimori, T. and Yamada, M. (1997). Biological control of annual bluegrass (Poa annua L.) with a Japanese isolate of Xanthomonas campestris pv. Poae (JT‐P482). Biological Control 8, 7–14. Jeger, M. J. (2001). Biotic interactions and plant‐pathogen associations. In ‘‘Biotic Interactions in Plant‐Pathogen Associations’’ (M. J. Jeger and N. J. Spence, eds.), pp. 1–14. CABI Press, Wallingford. Johnson, R. D. and Lewis, B. G. (1994). Variation in host range, systemic infection and epidemiology of Leptosphaeria maculans. Plant Pathology 43, 269–277. Jones, R., Alemseged, Y., Medd, R. and Vere, D. (2000). The distribution, density and economic impact of weeds in the Australian annual winter cropping system. CRC Weed Management Systems, Australia Technical Series 4, 1–53. Kanrar, S., Venkateswari, J. C., Kirti, P. B. and Chopra, V. L. (2002). Transgenic expression of hevein, the rubber tree lectin, in Indian mustard confers protection against Alternaria brassicae. Plant Science 162, 441–448. Keen, N. T., Holliday, M. J. and Yoshikawa, M. (1982). EVects of glyphosate on glyceollin production and the expression of resistance to Phytophthora megasperma f. sp. glycinea in soybean. Phytopathology 72, 1467–1470.

OPPORTUNITIES FOR THE CONTROL OF BRASSICACEOUS WEEDS

171

Khangura, R. K., Barbetti, M. J. and Sweetingham, M. W. (1999). Characterisation and pathogenicity of Rhizoctonia species on canola. Plant Disease 83, 714–721. Kharbanda, P. D., Fitt, B. D. L., Lange, R. M., West, J. S., Lamey, A. H. and Phillips, D. V. (2001). Diseases of Rapeseed ¼ Canola (B. napus L. and Brassica rapa L.) http://www.apsnet.org/online/common/names/rapeseed. asp (Consulted 10 May 2004). Kistler, H. C., Bosland, P. W. and Benny, U. (1987). Relatedness of strains of Fusarium osysporum from crucifers measured by examination of mitochondrial and ribosomal DNA. Phytopathoglogy 77, 1289–1293. Kistler, H. C., Momol, E. A. and Benny, U. (1991). Repetitive genomic sequences for determining relatedness among strains of Fusarium osysporum. Phytopathoglogy 81, 331–336. Kusaba, M. and Tsuke, T. (1994). Nuclear ribosomal DNA variation and pathogenic specialisation in Alternaria fungi known to produce host‐specific toxins. Applied and Environmental Microbiology 60, 3055–3062. Le´ ger, C., Hallett, S. G. and Watson, A. K. (2001). Performance of Colletotrichum dematium for the control of fireweed (Epilobium angustifolium) improved with formulation. Weed Technology 15, 437–446. Lemerle, D., Blackshaw, R. E., Smith, A. B., Potter, T. D. and Marcroft, S. J. (2001). Comparative survey of weeds surviving in triazine‐tolerant and conventional canola crops in south‐eastern Australia. Plant Protection Quarterly 16, 37–40. Lomer, C. J., Bateman, R. P., Johnson, D. L., Langewald, J. and Thomas, M. (2001). Biological control of locusts and grasshoppers. Annual Review of Entomology 46, 667–702. Love, G. (2005). Impacts of climate variability on regional Australia. Proceedings of the OUTLOOK 2005 conference. National Convention Centre, Canberra, 1–2 March 2005, Accessed from http://www.abareconomics.com/outlook/ PDF/loveg.pdf. Masangkay, R. F., Paulitz, T. C., Hallett, S. G. and Watson, A. K. (2000). Solid substrate production of Alternaria alternata f. sp. sphenocleae conidia. Biocontrol Science and Technology 10, 399–409. Mendes‐Pereira, E., Balesdent, M‐H., Brun, H. and Rouxel, T. (2003). Molecular phylogeny of the Leptosphaeria maculans‐L. biglobosa species complex. Mycological Research 107, 1287–1304. Momol, E. A. and Kistler, H. C. (1992). Mitochondrial plasmids do not determine host range in crucifer‐infecting strains of Fusarium oxysporum. Plant Pathology 41, 103–112. Mora, A. A. and Earle, E. D. (2001). Resistance to Alternaria brassicicola in transgenic broccoli expressing a Trichoderma harzianum endochitinase gene. Molecular Breeding 8, 1–9. Morin, L., Auld, B. A. and Brown, J. F. (1993a). Interaction between Puccinia xanthii and facultative parasitic fungi on Xanthium occidentale. Biological Control 3, 288–295. Morin, L., Auld, B. A. and Brown, J. F. (1993b). Synergy between Puccinia xanthii and Colletotrichum orbiculare on Xanthium occidentale. Biological Control 3, 296–310. Nikolaev, I., Mathieu, M., van de Vondervoort, P. J. I., Visser, J. and Felenbok, B. (2002). Heterologous expression of the Aspergillus nidulans alcR–alcA system in Aspergillus niger. Fungal Genetics and Biology 37, 89–97. Oleskevich, C., Shamoun, S. F., Vesonder, R. F. and Punja, Z. K. (1998). Evaluation of Fusarium avenaceum and other fungi for potential as biological control

172

A. MAXWELL AND J. K. SCOTT

agents of invasive Rubus species in British Columbia. Canadian Journal of Plant Pathology 20, 12–18. Ortoneda, M., Guarro, J., Madrid, M. P., Caracuel, Z., Roncero, M. I. G., Mayayo, E. and Di Pietro, A. (2004). Fusarium oxysporum as a multihost model for the genetic dissection of fungal virulence in plants and mammals. Infection and Immunity 72, 1760–1766. Pedras, M. S. C., Biesenthal, C. J. and Zaharia, I. L. (2000). Comparison of the phytotoxic activity of the phytotoxin destruxin B and four natural analogs. Plant Science 156, 185–192. Pedras, M. S. C., Zaharia, I. L., Gai, Y., Zhou, Y. and Ward, D. E. (2001). In planta sequential hydroxylation and glycosylation of a fungal phytotoxin: Avoiding cell death and overcoming the fungal invader. Proceedings of the National Academy of Sciences USA 98, 747–752. Pedras, M. S. C., Zaharia, I. L. and Ward, D. E. (2002). The destruxins: Synthesis, biosynthesis, biotransformation and biological activity. Phytochemistry 59, 579–596. Quimby, P. C., Mercadier, G., Meikle, W., Vega, F., Fargues, J. and Zidack, N. (2001). Enhancing biological control through superior formulations: A worthy goal but still a work in progress. In ‘‘Enhancing Biocontrol Agents and Handling Risks’’ (M. Vurro, J. Gressel, T. Butt, G. E. Harman, A. Pilgeram, R. J. St. Le´ ger and D. L. Nuss, eds.), pp. 86–95. IOS Press, Amsterdam. Quimby, P. C., Zidack, N. K., Boyette, C. D. and Grey, W. E. (1999). A simple method for stabilising and granulating fungi. Biocontrol Science and Technology 9, 5–8. Roslan, H. A., Salter, M. G., Wood, C. D., White, M. R. H., Croft, K. P., Robson, F., Coupland, G., Doonan, J., Laufs, P., Tomsett, A. B. and Caddick, M. X. (2001). Characterisation of the ethanol‐inducible alc gene‐expression system in Arabidopsis thaliana. The Plant Journal 28, 225–235. Rotem, J. (1994). ‘‘The Genus Alternaria Pathology, Epidemiology and Pathogenicity.’’ APS Press, St. Paul, MN. Sampson, P. J. and Walker, J. (1982). ‘‘An Annotated List of Plant Diseases in Tasmania.’’ Tasmanian Department of Agriculture, Hobart, Australia. Satou, M. and Fukumoto, F. (1996). The host range of downy mildew, Peronospora parasitica, from Brassica campestris, chinese cabbage and turnip crops and Raphanus sativus, japanese radish crop. Annals of the Phytopathology Society of Japan 62, 402–407. Schnick, P. J. and Boland, G. J. (2004). 2,4‐D and Phoma herbarum to control dandelion (Taraxacum oYcinale). Weed Science 52, 808–814. Scott, J. K., Vitou, J. and Jourdan, M. (2002). Review of the potential for biological control of wild radish (Raphanus raphanistrum) based on surveys in the Mediterranean region. In ‘‘Proceedings of the 13th Australian Weeds Conference’’ (H. SpaVord Jacob, J. Dodd and J. H. Moore, eds.), pp. 377–380. Plant Protection Society of Western Australia, Perth. Shabana, Y. M., Baka, Z. A. M. and Abdel‐Fattah, G. M. (1997). Alternaria eichhorniae, a biological control agent for waterhyacinth: Mycoherbicidal formulation and physiological and ultrastructural host responses. European Journal of Plant Pathology 103, 99–111. Shabana, Y. M., Charudattan, R. and Elwakil, M. A. (1995). Evaluation of Alternaria eichhorniae as a bioherbicide for waterhyacinth (Eichhornia crassipes) in greenhouse trials. Biological Control 5, 136–144. Shabana, Y. M., Mu¨ ller‐Sto¨ ver, D. and Sauerborn, J. (2003). Granular Pesta formulation of Fusarium oxysporum f. sp. orthoceras for biological control of

OPPORTUNITIES FOR THE CONTROL OF BRASSICACEOUS WEEDS

173

sunflower broomrape: EYcacy and shelf‐life. Biological Control 26, 189–201. Sharma, S. R. and Kolte, S. J. (1994). EVect of soil‐applied NPK fertilisers on severity of black spot disease (Alternaria brassicae) and yield of oilseed rape. Plant and Soil 167, 313–320. Sharma, G., Kumar, V. D., Haque, A., Bhat, S. R., Prakash, S. and Chopra, V. L. (2002). Brassica coenospecies: A rich reservoir for genetic resistance to leaf spot caused by Alternaria brassicae. Euphytica 125, 411–417. Sharon, A., Amsellem, Z. and Gressel, J. (1992). Glyphosate suppression of an elicited defense response; increased susceptibility of Cassia obtusifolia to a mycoherbicide. Plant Physiology 98, 654–659. Shephard, G. S. and Snijman, P. W. (1999). Elimination and excretion of a single dose of the mycotoxin fumonisin B2 in a non‐human primate. Food and Chemical Toxicology 37, 111–116. Shivas, R. G. (1989). Fungal and bacterial diseases in Western Australia. Journal of the Royal Society of Western Australia 72, 1–62. Silue´ , D., Nashaat, N. I. and Tirilly, Y. (1996). DiVerential responses of Brassica oleracea and B. rapa accessions to seven isolates of Peronospora parasitica at the cotyledon stage. Plant Disease 80, 142–144. Simmonds, J. H. (1966). ‘‘Host Index of Plant Diseases in Queensland.’’ Queensland Department of Primary Industries, Brisbane, Australia. Sinden, J., Jones, R., Hester, S., Odom, D., Kalisch, C., James, R. and Cacho, O. (2004). The economic impact of weeds in Australia. CRC for Australian Weed Management Technical Series 8, 1–55. Singh, S. L. and Pavgi, M. S. (1977). Aphanomyces root rot of cauliflower. Mycopathologia 61, 167–172. Snow, A. A., Uthus, K. L. and Culley, T. M. (2001). Fitness of hybrids between weedy and cultivated radish: Implications for weed evolution. Ecological Applications 11, 934–943. Stringam, G. R., Ripley, V. L., Love, H. K. and Mitchell, A. (2003). Transgenic herbicide tolerant canola: The Canadian experience. Crop Science 43, 1590–1593. Sugui, J. A., Leite, B. and Nicholson, R. L. (1998). Partial characterisation of the extracellular matrix released onto hydrophobic surfaces by conidia and conidial germlings of Colletotrichum graminicola. Physiological and Molecular Plant Pathology 52, 411–425. Sutton, B. C. (1980). ‘‘The Coelomycetes, Fungi Imperfecti with Pycnidia, Acervuli and Stromata.’’ CABI Publishing, Wallingford, UK. Tierens, K. F. M.‐J., Thomma, B. P. H. J., Bari, R. P., Garmier, M., Eggermont, K., Brouwer, M., Penninckx, I. A. M. A., Broekaert, W. F. and Cammue, B. P. A. (2002). Esa1, an Arabidopsis mutant with enhanced susceptibility to a range of necrotrophic fungal pathogens, shows a distorted induction of defense responses by reactive oxygen generating compounds. The Plant Journal 29, 131–140. Toyota, K. and Kimura, M. (1992). Growth of Fusarium oxysporum f. sp. raphani in the host rhizosphere. Japanese Journal of Soil Science and Plant Nutrition 63, 566–570. Tucker, S. L. and Talbot, N. J. (2001). Surface attachment and pre‐penetration stage development by plant pathogenic fungi. Annual Review of Phytopathology 39, 385–417. Valkonen, J. P. T. and Koponen, H. (1990). The seed‐borne fungi of Chinese cabbage (Brassica pekinensis), their pathogenicity and control. Plant Pathology 39, 510–516.

174

A. MAXWELL AND J. K. SCOTT

Van der Plank, J. E. (1963). ‘‘Plant Diseases: Epidemics and Control.’’ Academic Press, New York. Van der Plank, J. E. (1984). ‘‘Disease Resistance in Plants.’’ 2nd Ed., Academic Press, Orlando, FL. Verma, P. R. and Saharan, G. S. (1994). ‘‘Monograph on Alternaria Diseases of Crucifers.’’ Research Branch Agriculture and Agri‐Food Canada, Saskatoon, Canada. Vurro, M., Evidente, A., Andolfi, A., Zonno, M. C., Giordano, F. and Motta, A. (1998). Brefeldin A and , ‐dehydrocurvularin, two phytotoxins from Alternaria zinniae, a biocontrol agent of Xanthium occidentale. Plant Science 138, 67–79. Walker, H. L. and Boyette, C. D. (1986). Influence of sequential dew periods on biocontrol of sicklepod (Cassia obtusifolia) by Alternaria cassiae. Plant Disease 70, 962–963. Walsh, M. J., Powles, S. B., Beard, B. R., Parkin, B. T. and Porter, S. A. (2004). Multiple herbicide resistance across four modes of action in wild radish (Raphanus raphanistrum). Weed Science 52, 287–306. Warwick, S. I. and Sauder, C. A. (2005). Phylogeny of tribe brassiceae (Brassicaceae) based on chloroplast restriction site polymorphisms and nuclear ribosomal internal transcribed spacer and chloroplast trnL intron sequences. Canadian Journal of Botany 83, 467–483. Watson, A. K. and Ahn, B.‐S. (2001). Better control of weeds with enhanced bioherbicides. In ‘‘Enhancing Biocontrol Agents and Handling Risks’’ (M. Vurro, J. Gressel, T. Butt, G. E. Harman, A. Pilgeram, R. J. St. Le´ ger and D. L. Nuss, eds.), pp. 106–113. IOS Press, Amsterdam. Williams, P. H. (1992). Biology of Leptosphaeria maculans. Canadian Journal of Plant Pathology 14, 30–35. Williams, P. H. (1993). Diseases of Crucifers (Brassica and Raphanus spp.) http:// www.apsnet.org/online/common/names/crucifer.asp. (Web site consulted 10 May 2004). Wolfe, K. H., Li, W.‐H. and Sharp, P. M. (1987). Rates of nucleotide substitution vary greatly among plant mitochondrial, chloroplast and nuclear DNAs. Proceedings of the National Academy of Science USA 84, 9054–9058. Wo¨ sten, H. A. B. (2001). Hydrophobins: Multipurpose proteins. Annual Review of Microbiology 55, 625–646. Yang, Y.‐W., Tai, P.‐Y., Chen, Y. and Li, W.‐H. (2002). A study of the phylogeny of Brassica napus, B. nigra, Raphanus sativus and their related genera using noncoding regions of chloroplast DNA. Molecular Phylogenetics and Evolution 23, 268–275.

Stress Resistance and Disease Resistance in Seaweeds: The Role of Reactive Oxygen Metabolism

MATTHEW J. DRING

Queen’s University Marine Laboratory, Portaferry, Co. Down, BT22 1PF Northern Ireland, United Kingdom

I. II. III. IV.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Environmental Stress on Seaweeds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Reactive Oxygen Metabolism (ROM) in Vascular Plants and Seaweeds . . .. . Interactions Between ROM and Environmental Stress Factors . . . . . . . . . A. Freezing, Desiccation and Osmotic Stress . . . . . . . . . . . . . . . . . . . . . . . . . B. High Light and Ultraviolet Stress . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Heavy Metal Stress . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Mechanical Stress . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. Responses of Seaweeds to Infection: Interaction with ROM. . . . . . . . . . . . VI. Summary and Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

176 177 179 183 185 188 190 192 193 197 201

ABSTRACT It has become clear over the last 15–20 years that the immediate eVect of a wide range of environmental stresses, and of infection, on vascular plants is to increase the formation of reactive oxygen species (ROS) and to impose oxidative stress on the cells. Since 1994, suYcient examples of similar responses in a broad range of marine macroalgae have been described to show that reactive oxygen metabolism also underlies the mechanisms by which seaweeds respond (and become resistant) to stress and infection. Desiccation, freezing, low temperatures, high light, ultraviolet radiation, and heavy metals all tend to result in a gradual and continued buildup of ROS because photosynthesis is inhibited and excess energy results in the formation of singlet oxygen. The response to other stresses (infection or oligosaccharides which Advances in Botanical Research, Vol. 43 Incorporating Advances in Plant Pathology Copyright 2006, Elsevier Ltd. All rights reserved.

0065-2296/06 $35.00 DOI: 10.1016/S0065-2296(05)43004-9

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signal that infection is occurring, mechanical stress, hyperosmotic shock) is quite diVerent—a more rapid and intense, but short‐lived production of ROS, described as an ‘‘oxidative burst’’—which is attributed to activation of NADPH oxidases in the plasma membrane. Seaweed species that are able to survive such stresses or resist infection have the capacity to remove the ROS through a high cellular content of antioxidant compounds, or a high activity of antioxidant enzymes.

I. INTRODUCTION Seaweeds that grow in the subtidal zone live in relatively constant and stress‐ free environments, subjected to only small, slow changes in temperature, salinity, and supply of oxygen, inorganic carbon, and other nutrients. The most obvious stress occurs towards the bottom of the photic zone, where irradiance is limiting, or in regions of low nutrient availability, but changes occur slowly so that physiological processes have time to adjust. In the intertidal zone, however, the situation is very diVerent. The disadvantages of terrestrial habitats (unreliable water supply, variable temperatures, exposure to ultraviolet radiation) are combined with the disadvantages of aquatic habitats (variable irradiance and nutrient supply) and, to these, are added the mechanical stresses imposed by the waves. To survive in some intertidal sites, therefore, seaweeds may need to be able to tolerate rapid and frequent changes in temperature and desiccation stress and in salinity and irradiance (both photosynthetically active and ultra‐violet radiation), as well as the abrasion, pressure, and drag associated with wave action. Most work on environmental stresses in seaweeds has described the responses of physiological processes to stress and little attention has been paid to the cellular mechanisms by which plants limit the damage that has been (or may be) caused by stress. This is unfortunate because such approaches are more likely to indicate how the stress is detected, the mechanism of the response, and the way in which plants may become tolerant to a stress (cf. Davison and Pearson, 1996). Consequently, it has been diYcult to generalise about typical responses to a wide range of stresses or to identify the signals by which stresses are perceived. As Davison and Pearson (1996) pointed out, however, there were signs in the mid‐1990s that the production of reactive oxygen species (ROS) was associated with more than one type of stress and that reactive oxygen metabolism (ROM) could play a central role. These signs have strengthened in the last 10 years, and more evidence has accumulated, but the techniques that are needed for exploring ROM have been restricted to relatively few laboratories and there has been no review since 1996 that has highlighted the potential importance of ROM in seaweed ecophysiology. In this period, however, a new field of seaweed research has

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arisen, stimulated by the growing importance of the aquaculture of seaweeds for commercial applications—the study of the susceptibility of seaweeds to disease (‘‘phycopathology’’; Bouarab et al., 2001a,b; Potin et al., 1999)—and this has revealed that ROM is also involved in the responses of several seaweeds to infection by potentially pathogenic organisms and in the ability of such species to resist infection. This work has indicated the parallels between resistance to environmental stress and resistance to disease and has strengthened the importance of ROM to the ecology and exploitation of seaweeds. The aims of this review are as follows. 1. To assemble evidence for a central and integrating role of ROM in the responses of seaweeds to a range of apparently discrete environmental stresses and in the ability of seaweed species to resist and survive such stresses. 2. To demonstrate the similarities and parallels between the responses of seaweeds to environmental stresses and those to infection by potential pathogens (or to chemicals that appear to act as signals of such infection), and in their resistance to both types of stress. The treatment will concentrate on physiological responses and physiological evidence (including some of the basic methods for detecting ROS) rather than on the detailed biochemistry of ROM and on the ecological and aquacultural implications of ROM for seaweeds in order to encourage seaweed physiologists and ecologists to become more familiar with the basic concepts of ROM and to seek further evidence of its importance from a wider range of species.

II. ENVIRONMENTAL STRESS ON SEAWEEDS This section summarises the range of environmental stresses that act upon seaweeds in their natural habitats and discusses recent examples of work exploring the physiological responses to such stresses. It remains true that, as Davison and Pearson (1996) pointed out, most current research into environmental stresses in seaweeds describes the responses of individual physiological processes to stress and fails to investigate the cellular mechanisms by which seaweeds detect diVerent stresses and limit the damage that they cause. The stress that has received most attention in recent years, for obvious reasons, is ultra‐violet (UV) radiation, but it is diYcult to distinguish between the eVects of UV and the eVects of high irradiances of photosynthetically

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active radiation (PAR) with which it is always associated in natural environments (Dring et al., 2001). Nevertheless, it is clear that both UV and high light result in damage to the photosynthetic apparatus, although this has been deduced most frequently in recent years by measuring chlorophyll fluorescence using pulse amplitude moderated (PAM) fluorometers instead of measuring oxygen or carbon exchange. The immediate damage caused by both types of radiation has been linked to D1 protein (e.g., Bouchard et al., 2005) and cyclobutane pyrimidine dimers (van de Poll et al., 2002), whereas the longer term response leading to resistance is associated with the production of mycosporine amino acids (MAAs; Franklin et al., 1999; Krabs et al., 2004). There is increasing recent evidence (reviewed later) that the immediate responses of seaweeds to high irradiances of PAR and/or UV radiation are associated with some aspect of ROM. Such stresses are experienced by seaweed species at the upper limits of their depth distribution in upper sublittoral or intertidal zones, but the lower depth limit for the distribution of many species may be determined by low irradiance, which can be regarded as a distinct type of stress. This is not an acute stress and does not cause direct damage, except through long‐term reduction of energy and carbon supply. The typical responses, either acclimative or adaptive (a distinction that is not explored here), involve changes in the concentration and/or composition of photosynthetic pigments (Raven et al., 2000). Similarly, the low availability of CO2 may impose a stress that is chronic rather than acute, although such stress is rarely encountered in the sea because of the high concentration of bicarbonate, and seaweeds display a range of CO2‐ concentrating mechanisms that enable them to maintain adequate supplies of inorganic carbon (Giordano et al., 2005). The main stresses experienced by intertidal seaweeds are, of course, those associated with emersion, which include desiccation, high temperature, low temperature (especially freezing), and either increased (through evaporation) or decreased (through freshwater runoV) salinity. The impact of all of these stresses tends to be similar because they all result, directly or indirectly, in osmotic challenges to the cells of seaweeds (Pearson and Davison, 1994), and the responses or adaptations may involve the production of osmolytes (Edwards et al., 1988; Kirst, 1990). However, the first indications that ROM might be involved in the environmental responses of seaweeds came from studies of the responses of Fucus spp. to desiccation, freezing, and low temperatures (see later). High temperatures may exert an eVect on metabolic activity that is independent of their contribution towards osmotic stress, and responses have been observed involving the formation of heat stress proteins (Lewis et al., 2001; Li and Brawley, 2004). These responses do not appear to involve ROM.

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Deficiencies in the supply of essential nutrients (especially N, P) are rarely encountered in seaweeds in their natural habitats, although evidence shows that species growing higher on the shore have stronger nutrient uptake mechanisms than species lower on the shore or in the sublittoral, and this is thought to be a response to the limited time available for nutrient uptake because of the longer periods of tidal emersion (Hurd and Dring, 1990; Phillips and Hurd, 2004). However, the damaging eVects of emersion are exerted more rapidly on the process of photosynthesis than on nutrient uptake so that seaweeds are unlikely to respond acutely or specifically to nutrient stress in the sea. Heavy metals, however, are known to exert a damaging eVect on several aspects of seaweed development (Nielsen et al., 2003) and may cause a rapid inhibition of photosynthesis. Again, ROM appears to be involved (see later). Wave action imposes a severe mechanical stress on seaweeds, particularly in the intertidal zone, and several examples of apparent morphological responses to the severity of wave action (or the degree of shelter) have been described. Although wave action is an acute stress, no acute response is possible—apart from breakage of the frond and loss of photosynthetic tissue. Nevertheless, in transplant experiments, individual plants can be seen to have acclimated to a changed wave environment (e.g., Alaria; Sundene, 1962), which suggests that plants can detect and respond to signals indicating the severity of wave action, which probably involves some form of mechanical stress. It is of interest, therefore, that ROM has been implicated in the responses of two quite diVerent seaweeds to mechanical damage (Eucheuma, Collen and Pedersen, 1994; Dasycladus, Ross et al., 2005).

III. REACTIVE OXYGEN METABOLISM (ROM) IN VASCULAR PLANTS AND SEAWEEDS The term ‘‘reactive oxygen metabolism’’ is used in this review to refer to the full range of biochemical reactions involving what are commonly called ‘‘reactive oxygen species.’’ ROS are forms of oxygen, sometimes combined with hydrogen, which, as the name suggests, are more reactive than the relatively stable molecule O2, which is common in the atmosphere. In fact, this stable form of oxygen is strictly a ‘‘triplet oxygen’’ molecule [3O2], in which the two unpaired electrons have the same spin quantum number, and this imposes a restriction on the rate at which the molecule reacts with the many oxidisable substrates in natural environments on Earth. In photosynthetic organisms under light‐saturated conditions, triplet oxygen may be

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photosensitised by an excited triplet chlorophyll molecule to generate ‘‘singlet oxygen’’ [1O2], a form of the dioxygen molecule in which the spin restriction is removed and, consequently, reactivity is increased greatly. Singlet oxygen can react with pigments, proteins, and lipids and is regarded as the main cause of photoinhibition of photosynthesis in plants, including the degradation of D1 protein (Krieger‐Liszkay, 2005). Another of the reactive oxygen species is the so‐called ‘‘superoxide radical’’ [
O2], whose extra reactivity is due to the fact that it is both a radical (i.e., has unpaired electrons in its outermost shell) and an anion. Superoxide can be formed directly from singlet oxygen or by reduction of triplet oxygen (Fig. 1), usually as a result of leakage of electrons from respiratory or photosynthetic electron transport pathways (Alscher et al., 1997). At physiological pH, the superoxide radical does not react rapidly with cellular components (Wojtaszek, 1999) and ‘‘disproportionates’’ in solution into another ROS (hydrogen peroxide, H2O2) and triplet oxygen: 3 2
O 2 þ 2H ! H2 O2 þ O2


ð1Þ

This reaction may occur spontaneously, but is enhanced greatly by the activity of superoxide dismutase (SOD), which is found in many compartments of the cell, including the cytosol, chloroplasts, and mitochondria, as well as extracellularly. Several distinct forms of SOD have been found in photosynthetic organisms, incorporating diVerent metals as cofactors (Alscher et al., 2002; Asada, 1999). The thylakoid membranes and cytosol of vascular plants and some algae contain a CuZnSOD, whereas the forms of SOD found in the stroma of the chloroplasts contain iron and those in the mitochondria and peroxisomes contain manganese (Fig. 2). It is the activity of these various forms of SODs in plants that is thought to provide the major protection against the damaging eVects of superoxide (Pinto et al., 2003). Scavenging of superoxide by SODs results in the production of H2O2, which, although less damaging than the superoxide radical, is far from

Fig. 1. Formation of diVerent reactive oxygen species (ROS) from molecular (triplet) oxygen (adapted from Wojtaszek, 1999).

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Fig. 2. Localisation of diVerent forms of superoxide dismutase (SODs) in plant cells (redrawn from Alscher et al., 2002).

harmless. Partly because H2O2 is less reactive and more stable than other ROS, it may diVuse away from its site of formation, into other cells or, especially for aquatic plants, into the outside environment. Significant concentrations can build up in the medium surrounding cultures of both microalgae (e.g., up to 0.9 M by the raphidophyte, Heterosigma akashiwo; Twiner et al., 2001) and macroalgae (e.g., up to 4.0 M by the green seaweed Ulva rigida; Collen and Pedersen, 1996). H2O2 may also participate in a number of enzymatic reactions within plant and algal cells, which are described later. However, reaction between the superoxide radical and H2O2 (the Haber–Weiss reaction) results in the most reactive of the ROS, the hydroxyl radical [
OH]:  3
O 2 þ H2 O2 !
OH þ OH þ O2


ð2Þ

This reaction is not thought normally to generate significant quantities of
OH in plant cells, but the presence of transition metals, such as Fe3þ or Cu2þ, enhances the rate substantially (Pinto et al., 2003). Another mechanism by which transition metals may promote the formation of
OH is the Fenton reaction, in which reduced forms of metal ions (e.g., of Fe, Cu, or Cr)

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are first oxidised by H2O2 and subsequently reduced by reaction with superoxide (Pinto et al., 2003; Wojtaszek, 1999): H2 O2 þ Fe2þ ðor Cuþ Þ !
OH þ OH þ Fe3þ ðor Cu2þ Þ

ð3Þ

2þ þ 3þ 2þ 3
O 2 þ Fe ðor Cu Þ ! Fe ðor Cu Þ þ O2

ð4Þ

Because of its relative stability, hydrogen peroxide is the most readily detected ROS in and around plants and algae, and there is an extensive literature on its reactions and interactions with a variety of biochemicals. The primary reaction of H2O2 is its breakdown into water and triplet oxygen. This may occur spontaneously but, in most cells, is catalysed by catalase: H2 O2 þ H2 O2 ! 2H2 O þ 3 O2


ð5Þ

Another mechanism by which H2O2 can be detoxified is through its reaction with ascorbate, which is mediated by ascorbate peroxidase (APX; see Alscher et al., 1997). This results in the oxidation of ascorbate in the cell or organelle, but ascorbate is regenerated by the oxidation of glutathione (GSH – a tripeptide: glutamate þ cysteine þ glycine), which is regenerated in turn by glutathione reductase (GR) using reduced NADP (Fig. 3). Catalase, the various forms of SOD, and the enzymes involved in this ascorbate/glutathione pathway (APX, DHAR, GR) are referred to frequently as ‘‘antioxidant enzymes,’’ as they are collectively involved in the removal of ROS from various compartments of plant cells and in the reduction of oxidative stress. The activity of such enzymes has been detected in a wide range of seaweed species, including Fucus spp. (Collen and Davison, 1999b, 2001), the green algae Ulva rigida (Collen and Pedersen, 1996), Acrosiphonia penicilliformis, Monostroma arcticum, Chaetomorpha linum, Chaetomorpha melagonium (Aguilera et al., 2002b), and the red algae Devaleraea ramentacea,

Fig. 3. Reactions of the ascorbate‐glutathione pathway (also known as Halliwell–Asada pathway; adapted from Wojtaszek, 1999).

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Palmaria palmata (Aguilera et al., 2002b), Polysiphonia arctica (Dummermuth et al., 2003), Kappaphycus alvarezii (Barros et al., 2003), Chondrus crispus, and Mastocarpus stellatus (Collen and Davison, 1999c; Lohrmann et al., 2004). Although the strategy of most plants seems to be to rid themselves of H2O2 as quickly as possible, H2O2 appears to have a beneficial role in some plants and algae through its interaction with various peroxidase enzymes to produce secondary metabolites of considerable importance. These include lignin in woody plants (Wojtaszek, 1999) and a range of halocarbons in algae. Haloperoxidases have been detected in representatives of all three major groups of seaweeds (Table I), in which they catalyse the production of mainly bromocarbons and iodocarbons from H2O2, bromide, or iodide and an organic substrate. Many of the products are volatile and may escape into the atmosphere (Carpenter et al., 2000) so that there is considerable current interest in establishing the relative contributions that seaweeds and human activities may make towards ozone depletion through brominated hydrocarbons in the atmosphere (Carpenter and Liss, 2000; Goodwin et al., 1997). The products of haloperoxidase activity in seaweeds may also be part of defence mechanisms against microorganisms (McConnell and Fenical, 1977), herbivores (Gschwend et al., 1985), or larval settlement (Ohsawa et al., 2001; Ohshiro et al., 1999) and are thought to assist in the uptake of bromide and iodide in Laminaria spp. (Ku¨ pper et al., 1998). However, it is diYcult to establish a function for all of the many and varied halocarbons produced by seaweeds, and several investigators of these haloperoxidases have concluded that their main function is to detoxify H2O2 (Collen et al., 1994; Manley and Barbero, 2001; Pedersen et al., 1996). Indeed, Manley and Barbero (2001) considered that the internal bromoperoxidase of Ulva lactuca may be the major H2O2‐removing enzyme because of its relatively low Km for H2O2. Peroxidases seem, therefore, to play an important role in the ROM of seaweeds.

IV. INTERACTIONS BETWEEN ROM AND ENVIRONMENTAL STRESS FACTORS The relationship between ROM and environmental stress in vascular plants was first established in the late 1980s (Foyer et al., 1997), and attempts to look for similar relationships in the algae were made soon afterwards, in the mid‐ to late 1990s—first for the freshwater dinoflagellate Peridinium gatunense (Butow et al., 1994, 1997) and then for the first seaweeds—in the response of the cultivated seaweed Eucheuma playcladum to mechanical

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TABLE I Haloperoxidase Enzymes and Their Products in Seaweeds Species Chlorophyta Ulva lactuca U. rigida U. lens Cladophora glomerata Enteromorpha spp. Phaeophyceae Macrocystis pyrifera Laminaria digitata L. digitata Laminaria spp. Ascophyllum nodosum A. nodosum Fucus vesiculosus

Enzymesa

Reference

BrPO

CHBr3

BrPO BrPO

CHBr3 CH2Br2, CHBr3 CHCl3, CHBr3, CH2I2 CHCl3, CHBr3, CH2I2

Manley and Barbero, 2001 Pedersen et al., 1996 Ohshiro et al., 1999 Abrahamsson et al., 2003 Abrahamsson et al., 2003

CH2Br2, CHBr3

Goodwin et al., 1997

I‐uptake

Ku¨ pper et al., 1998

HPO IPO, BrPO IPO, BrPO HPO

Colin et al., 2003 Almeida et al., 2001 Vilter et al., 1983

V‐HPO CHCl3, CHBr3, CH2I2

Ecklonia stolonifera

V‐BPO

Saccorhiza polyschides

V‐IPO

Rhodophyta Meristiella gelidium Laurencia sp. Eucheuma denticulatum E. denticulatum Corallina pilulifera

V‐BPO

C. C. C. C.

V‐BPO V‐BPO V‐BPO V‐BPO

pilulifera pilulifera oYcinalis oYcinalis

Products

Weyand et al., 1999 Abrahamsson et al., 2003 Hara and Sakurai, 1998 Almeida et al., 1998

Various I, Br, and Cl hydrocarbons CHBr3

Collen et al., 1994 Pedersen et al., 1996 Pedersen et al., 1996 Sundstrom et al., 1996

Various I, Br, and Cl hydrocarbons

Mtolera et al., 1996

CHBr3

Shimonishi et al., 1998 Ohsawa et al., 2001 Ohshiro et al., 2002 Carter et al., 2002 Isupov et al., 2000

a

BrPO, bromoperoxidase; HPO, haloperoxidase; IPO, iodoperoxidase; V‐BPO, vanadium peroxidases.

stress (Collen and Pedersen, 1994) and in the resistance of diVerent species of Fucus to the stresses of the intertidal environment (Collen and Davison, 1999a,b).

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STRESS AND DISEASE RESISTANCE IN SEAWEEDS A. FREEZING, DESICCATION AND OSMOTIC STRESS

The first study of ROM in seaweeds of the intertidal zone (Collen and Davison, 1999a,b) examined the responses of Fucus spiralis, F. evanescens, and F. distichus, which are typically found in this sequence from the top to the bottom of the intertidal on rocky shores of the northwest Atlantic to single exposures to either freezing (6 h in darkness at 16 to 18 8C) or desiccation (6 h in darkness at 4 8C after drying in low humidity air to 40% of fresh weight) treatments. The physiological responses of the fronds exposed to these treatments were measured by PAM fluorometry (Fv:Fm) and the production of reactive oxygen was estimated by following the oxidation of an intracellular dye, 20 ,70 ‐dichlorohydrofluorescein diacetate (DCFH‐DA) to the fluorescent 20 ,70 ‐dichlorofluorescein (DCF; Collen and Davison, 1997) and also by measuring the release of H2O2 into the medium. The Fv:Fm values obtained immediately after the freezing treatment indicated that only F. distichus had been aVected adversely by the treatment, and the production of DCF was also increased by freezing only in F. distichus (Collen and Davison, 1999a). However, freezing resulted in a significant increase in H2O2 production only in F. spiralis. The desiccation treatment produced a better diVerentiation among the three species in terms of DCF formation, as it increased sevenfold in F. distichus and threefold in F. evanscens, but was not aVected significantly in F. spiralis (Table II). These indications that freezing and desiccation treatments caused the production of ROS in the thalli of low‐shore species but not in high‐shore species suggested that resistance to such stresses might be associated with the ability of species to reduce the oxidative stress produced by the treatments. This could be achieved by possessing relatively high concentrations of antioxidant compounds (tocopherol, ‐carotene, ascorbate, glutathione) or high activities of the antioxidant enzymes (catalase, SOD, APX, GR) described in

TABLE II Production of Reactive Oxygen (Measured Using Dichlorohydrofluorescein, DCF) by Three Species of Fucus after Desiccation Treatment (6 h in Darkness at 40% Water Content) and 2 h Recovery in Seawater (Collen and Davison, 1999a)a Rate of DCF production (nmol g FW1 h1) Species Fucus spiralis F. evanescens F. distichus

Control 0.094a 0.115a 0.157ab

Values with diVerent subscript letters are significantly diVerent at p ¼ 0.05.

a

Desiccated 0.157ab 0.384b 1.11c

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the previous section, and these concentrations and activities were measured in thalli of the same three Fucus species by Collen and Davison (1999b). The concentrations of the antioxidant compounds did not correlate clearly with the positions of the three species on the shore (or with their response to the stress treatments), but F. distichus exhibited lower activities of catalase, SOD, and APX than the other two species. There were fewer diVerences in antioxidants or enzymes to account for the diVerent responses of F. spiralis and F. evanescens to stress, but the activities of APX and SOD in F. spiralis were considerably higher on a chlorophyll basis, and Collen and Davison (1999b) suggested that this may indicate higher concentrations in the chloroplasts and, therefore, more eYcient scavenging of ROS produced in the light when photosynthesis is inhibited by the stresses imposed. Similar indications of diVerences in antioxidant metabolism with vertical position on the shore, but this time within a single species, have been obtained for the New Zealand red alga Stictosiphonia arbuscula (Burritt et al., 2002). Thalli of this species from the top and bottom of its zone on the shore were maintained at diVerent relative humidities for 12, 24, or 48 h, and their antioxidant metabolism and the leakage of amino acids through their membranes were examined after rehydration. Specimens from low on the shore lost more amino acids through the plasma membrane and produced more H2O2 than high‐shore specimens; these diVerences seemed to be related to the greater activity of APX and GR in thalli from the high shore rather than to higher concentrations of antioxidants. These results support the conclusions of Collen and Davison (1999a,b) about the importance of ROM in enabling seaweeds to survive desiccation stress and are particularly valuable in that they show that intraspecific diVerences occur in a red alga from the southern hemisphere, in addition to interspecific diVerences among brown algae from the northern hemisphere. Studies of three other intertidal species (two red and one brown) from the northwest Atlantic have provided further evidence for the connection between antioxidants and the positions that species occupy in the intertidal zonation and have shown that there may be seasonal variations in the concentrations of antioxidants and the activities of antioxidant enzymes. The morphologically similar pair of red algae, Chondrus crispus and Mastocarpus stellatus, occur together throughout much of their tidal range, but M. stellatus can reach greater tidal heights and was assumed, therefore, to be more tolerant of environmental stress. Although the response of these species to stress treatments was not tested, the rate at which M. stellatus scavenged H2O2 (i.e., reduced the concentration of added H2O2 in seawater) and the resistance of its photosynthetic apparatus (measured as Fv:Fm) to the addition of H2O2 or Rose Bengal (a stain that increases the formation of

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singlet oxygen; Foote, 1967) was greater than for C. crispus (Collen and Davison, 1999c). The concentration of ascorbate and the activity of catalase were also higher in M. stellatus, but C. crispus had more glutathione, and the diVerences in other antioxidants and enzyme activities were less clear cut. A more recent study of the same two species (Lohrmann et al., 2004) showed that there are substantial seasonal variations in the antioxidant concentrations and enzyme activities—both tend to be higher in the winter months than in the summer—which may explain some of the inconsistencies in the earlier data. The most interesting point to emerge from these two studies is that both species appear to acclimate to low temperatures in the winter by increasing their capacity for scavenging ROS, which is thought to be because photosynthesis is more likely to be light saturated at low temperatures so that ROS production will be higher. The activities of catalase, SOD, and APX also declined between February and August in Fucus vesiculosus from the same region (Collen and Davison, 2001). Unfortunately, this study was continued only to the following October and, by this time, recovery was apparent only in APX. The activity of GR did not change over this period, and neither did the concentration of glutathione, whereas ascorbate was three times higher in August and October than in February. After 2 weeks of growth at 0 8C, with or without a daily freezing treatment, or at 20 8C, the content of ascorbate and glutathione had also not changed in F. vesiculosus, but the activity of SOD had increased markedly in the freezing treatment, and that of both APX and GR had decreased in the 20 8C treatment (Collen and Davison, 2001). There is good evidence, therefore, for phenotypic acclimation of the antioxidant enzymes in brown and red algae in response to low temperature and/or freezing treatments, which are consistent with the hypothesis that thalli develop cellular mechanisms to reduce oxidative stress under conditions that are likely to increase such stresses. However, the detailed responses of individual species show considerable variation, and no one antioxidant or enzyme can be used as a marker of the degree of stress being experienced by seaweeds or of the degree of resistance of thalli to intertidal stresses. The physiological responses of the photosynthetic apparatus of Fucus distichus to freezing stress were found to be very similar to those resulting from osmotic dehydration (Pearson and Davison, 1994) and also to the eVects of desiccation stress during emersion on the shore. This suggests that another form of environmental stress that seaweeds may experience—hyperosmotic stress as a result of evaporation of seawater during emersion—will give rise to similar physiological responses. There has been evidence for some time that the ability of vascular plants to tolerate high salinity is associated with ROM (e.g., Gossett et al., 1994; Herna´ ndez et al., 1995; Kalir and

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PoljakoV‐Mayber, 1981) but, although antioxidant responses to hypersaline stress have been recorded in Dunaliella (Jahnke and White, 2003), none of the many studies of the eVects of salinity changes on seaweeds have considered how ROM might be aVected. A recent and welcome exception to this, which (because of its technical sophistication) has much broader implications than simply confirming that seaweeds respond to increased salinity by generating ROS, is a study of Fucus embryos by Coelho et al. (2002). Two‐celled embryos of Fucus serratus were incubated for 20 min in chloromethyl‐20 ,70 ‐dichlorohydrofluorescein diacetate (CM‐DCFH‐DA) and then subjected to a hyperosmotic shock by transferring them to seawater containing 2 M sorbitol. The fluorescence that developed as the CM‐DCFH‐DA was oxidised to DCF by superoxide or H2O2 was monitored by confocal laser microscopy (as described by Rijstenbil et al., 2000) and was detectable in the plasma membrane of the rhizoid apex within 12 s of the start of the osmotic shock treatment. It then spread to the subapical regions within 40–120 s, where it continued to increase for up to 20 min. Simultaneous monitoring of the concentration and distribution of calcium ions within the embryos showed a sharp transient increase in Ca2þ within 10–12 s in the same location as the initial ROS production, which was then propagated as a wave from the apex through the rest of the cell. The production of ROS in the subapical regions was shown to be located exclusively in the mitochondria and was considered to be a separate and secondary response to the osmotic shock, which was initiated as the Ca2þ wave spread through the cell (Fig. 4). This exciting and far‐reaching study suggests that the primary response to osmotic shock (perhaps initiated by the withdrawal of the plasma membrane from the cell wall as a result of dehydration) is the production of ROS outside the plasma membrane, which provides the signal for an increase in Ca2þ channel activity (Fig. 4). The influx of Ca2þ is propagated as a wave through the cytoplasm and is, in turn, a signal for more widespread production of ROS by the mitochondria. It is of interest that ROS production was clearly not associated with the chloroplast in this system, so that, unlike the responses to other intertidal stresses discussed so far, the ROS did not originate from any activity in the photosynthetic apparatus and was not a result of inhibition of photosynthesis. B. HIGH LIGHT AND ULTRAVIOLET STRESS

The photosynthetic apparatus is certainly involved, however, in the responses of seaweeds to high irradiances of PAR and/or UV radiation. High light and UV stress has been studied extensively in seaweeds, but

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Fig. 4. Proposed signalling pathway during hyperosmotic stress in Fucus embryos. Osmotic change is sensed by an unidentified osmosensor (1), which induces ROS production within 10 s (2). H2O2 is produced outside the plasma membrane and may be involved in cell wall strengthening. H2O2 also diVuses into the cell, leading to localised peripheral increase. External H2O2 production also increases Ca2þ channel activity (3). Downstream events include Ca2þ wave propagation during the subsequent 10 to 60 s (4), followed by a Ca2þ increase in the mitochondria (5) and mitochondrial ROS production (6). T‐bars, inhibiting eVects: Br2BAPTA, dibromoglycine, N1N1‐(1,2‐ethanediylbis(oxy‐2,1‐phenylene))bis(N‐car‐boxymethyl))‐ tetrapotassium salt (a Ca2þ chelator); U73122, phospholipase C inhibitor; FCCP, carbon‐ylcyanide p‐trifluoromethoxyphenyl hydrazone (Coelho et al., 2002).

mainly by looking at eVects on pigments, photosynthetic rates, and chlorophyll fluorescence (see review by Franklin and Forster, 1997). The first studies of ROM in seaweeds in relation to these factors were in the mid‐ 1990s and involved Eucheuma denticulatum and Ulva rigida, both of which were shown to generate H2O2 when exposed to high irradiances of visible light (600 and 800 mol m2 s1, respectively; Mtolera et al., 1995; Collen and Pedersen, 1996). A further study of E. denticulatum showed that this species released a considerable range of volatile halocarbons in high irradiances, which was attributed to the production of H2O2 followed by its reaction with haloperoxidases (Table I; Mtolera et al., 1996). In both of these studies of E. denticulatum, high pH was found to increase the production of H2O2 and volatile halocarbons, and it was suggested that the primary cause of ROS production might have been limitations in the supply of inorganic carbon (especially as Eucheuma is dependent on dissolved CO2), but the end result of photoinhibition of photosynthesis by high light or limitation by low

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CO2 is the same: absorption of excess photons by chlorophyll and increased probability of generating singlet oxygen and/or other ROS. Several more recent studies of the influence of high irradiances on a range of seaweed species have compared activities of the antioxidative enzymes in diVerent species, in a single species in diVerent seasons, or exposed to diVerent irradiances or UV treatments (Aguilera et al., 2002a,b; Bischof et al., 2002; Choo et al., 2004; Davison et al., 2000; Rossa et al., 2002). All of this work is broadly consistent with the hypothesis that both UV radiation and high irradiances of visible radiation result in the production of ROS within seaweed thalli and that the ability to withstand these stresses is associated with the capacity of the cells to scavenge the ROS produced through their content of antioxidants or the activity of the same range of antioxidative enzymes that has already been described (i.e., catalase, SOD, APX, GR). Attempts to distinguish between the eVects of UV and of high irradiances of PAR have been no more successful than with physiological responses (e.g., Bischof et al., 2002), and it seems safest to conclude that both wave bands may stimulate ROS production. For the purposes of this review, the major points are that the absorption of more photons than the photosynthetic apparatus can process will increase the likelihood of ROS formation and that species or individuals with a greater ability to reduce the eVects of these ROS will be better able to recover when the stress is relieved. As a postscript to this summary of the interactions between ROM and high light/UV stress, it may be instructive to examine a recent study of the responses of phytoplankton to similar types of stress (Sunda et al., 2002). The basic findings were that environmental factors that result in oxidative stress, including solar UV radiation, CO2 limitation, and Fe limitation, tend to increase the cellular content of dimethylsulphoniopropionate (DMSP) and its breakdown products (e.g., dimethylsulphide, acrylate, dimethylsulphoxide) and that all of these products are as eVective as antioxidants as ascorbate and glutathione. DMSP received a lot of attention in the 1980s as an osmoregulator in seaweeds (Edwards et al., 1988; Kirst, 1990; Reed, 1983), but this function of the metabolite has started to be questioned (Van Alstyne et al., 2003). If DMSP and its relatives can join the list of antioxidants formed by seaweeds, it should be possible to reexamine earlier studies of the responses of seaweeds to salinity changes in the light of our modern understanding of ROM. C. HEAVY METAL STRESS

Several transition metals participate directly in ROM in plants because they act as cofactors in enzymes (e.g., SOD) or catalyse the Haber–Weiss and Fenton reactions (see Section III). However, these roles do not account for

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the observations that heavy metals increase oxidative stress in plant cells (Pinto et al., 2003). This eVect is more probably due to their disruption of photosynthetic electron transport chains, leading to the formation of the superoxide radical and singlet oxygen (Asada and Takahashi, 1987). In any case, the toxic eVects of heavy metals seem to be related to the formation of ROS; again, the ability of species to resist heavy metals stress is a function of their capacity for scavenging ROS. The relationship between ROM and heavy metal stress in algae has been reviewed recently (Pinto et al., 2003), and this section is restricted to the few examples of work involving seaweed species. Two of these relate to the eVects of copper on the green alga Enteromorpha (Ratkevicius et al., 2003; Rijstenbil et al., 1998). The glutathione redox ratio (GSH:[GSH þ 0.5GSSG]) decreased with the copper content of the thalli in E. prolifera from the Scheldt estuary in Holland (Rijstenbil et al., 1998) from a 1:1 balance between GSH and GSSG to about 0.2. This suggests that APX is actively oxidising ascorbate to DHA and that the balance between GSH and GSSG is shifted towards GSSG as ascorbate is regenerated (see Fig. 3). Glutathione ratios in specimens of E. prolifera collected from the Scheldt estuary and of E. linza from the Thermaikos Gulf (Greece) also decreased with the copper content of the thalli so that the ratio seemed to act as an indicator of oxidative stress–if not specifically of copper stress–in these species. A more detailed study of ROM and copper stress in Enteromorpha compressa was conducted in regions of northern Chile that were seriously polluted by wastes from copper mines (Ratkevicius et al., 2003). The activity of APX was almost undetectable in sites unaVected by the mine discharge, but increased almost 40 times in polluted sites. DHA levels also increased strongly in polluted sites, as did the ratio between DHA and ascorbate, but the total glutathione content (i.e., GSH þ GSSG) decreased by over 200 times compared with thalli from unpolluted shores and was so low that the glutathione ratio in Cu‐stressed algae was unmeasurable. Results from these two studies provide clear evidence that high copper concentrations induce oxidative stress in Enteromorpha and that the ascorbate/glutathione pathway is used to scavenge the ROS produced, but the exact balance between diVerent chemicals in the pathway appears to vary from species to species within the genus. In this respect, the situation seems to be similar to that observed in the responses of red and brown seaweeds to intertidal stresses. Recent work on the short‐term eVects of copper and cadmium on the red seaweed Gracilaria tenuistipitata provides another example of heavy metals inducing oxidative stress and increasing the activity of antioxidative enzymes (Collen et al., 2003). This species appeared to be more sensitive to copper than to cadmium, as copper stimulated increases in the activity of

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catalase, SOD, and APX, whereas cadmium aVected catalase only, but both metals increased the cellular content of two common antioxidants, ‐carotene and lutein. D. MECHANICAL STRESS

One of the earliest reports of increased ROM in a seaweed species was described as a response to ‘‘mechanical stress’’ (Collen and Pedersen, 1994), which is of considerable interest because it seems to suggest that another environmental stress to which seaweeds are regularly exposed– wave action–may be mediated by ROM. However, the mechanical stress that resulted in a short (10 min) burst of vigorous H2O2 production in the tropical carageenophyte Eucheuma platycladum was to break the thalli into 2‐cm pieces (Collen and Pedersen, 1994) so that the ROS production may have been from broken cells rather than from living cells subjected to sublethal mechanical stress analogous to wave action. In addition, there was some doubt about whether the method used to detect ROS, luminol‐ dependent chemiluminescence, is a reliable indicator of biological ROS production, as the luminol radical can spontaneously reduce oxygen to superoxide (Fridovich, 1997). Consequently, the case for ROM being stimulated by mechanical stress and/or wave action had to be regarded as unproven, and deserving further investigation, for a long time after the initial report. There is now evidence, however, from a very diVerent macroalga, the green coencytic alga Dasycladus vermicularis, that broken cells may generate an oxidative burst (Ross et al., 2005) that has some similarities to that observed by Collen and Pedersen (1994). Injuring the giant cells of D. vermicularis resulted in a wound plug in which a browning reaction was observed after 35–45 min. Browning occurred earlier in the presence of 1 mM H2O2 and was delayed by catalase and completely prevented by 1 mM ascorbate. These results suggested that oxidative reactions were involved, and incubating injured algae with DCDF‐DA revealed a sharp increase in fluorescence (due to DCF formation) after 40 min at the site of the injury. Quantifying the release of H2O2 showed a slow increase for 30 min after the injury, followed by a rapid rise for the next 10 min, with a plateau being reached after 90 min. Peroxidase activity could be detected histochemically in the wound plug for the first time 35–45 min after the injury (Ross et al., 2005). Although the production of ROS in response to injury in Dasycladus appears to be considerably slower than in Eucheuma, evidence that both species produce an ‘‘oxidative burst’’ suggests that these responses are analogous to the responses of plant and algal cells to infection by pathogens, which is the subject of the next section of this review.

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V. RESPONSES OF SEAWEEDS TO INFECTION: INTERACTION WITH ROM An ‘‘oxidative burst’’ (i.e., the sudden but short‐lived release of ROS) is now known to be a common response of vascular plant cells to challenges with pathogens, including fungal zoospores, viruses, and bacteria (Lamb and Dixon, 1997; Wojtaszek, 1997). It was soon recognised that such a response must involve chemical recognition of the pathogen by the host plant, and much of the detailed investigation of the phenomenon has involved challenging suspension‐cultured cells with extracts (e.g., cell wall fragments) of the pathogens, or specific ‘‘elicitor’’ chemicals, which induce the oxidative burst response. Whereas some of these elicitors are derived from the pathogen (e.g., chitosans), others appear to be derived from the host and are frequently oligosaccharides resulting from degradation by the pathogen of the host cell wall (e.g., oligogalacturonides; see Wojtaszek, 1997). Evidence shows that three quite distinct groups of seaweeds may also respond, apparently defensively, with oxidative bursts when ‘‘challenged’’ with oligosaccharide elicitors, and, interestingly, the elicitors so far described include all three groups of cell wall polysaccharides that are unique to seaweeds: agars, carrageenans, and alginates. Only one of these seaweed systems is clearly associated with infection by a specific pathogen (Chondrus crispus and its green algal endophyte Acrochaete operculata; Bouarab et al., 1999). The other two show responses to appropriate elicitors (Gracilaria conferta to oligoagars, Weinberger et al., 1999; Laminaria digitata to oligoalginates, Ku¨ pper et al., 2001) that are assumed to be defensive, but no specific pathogen has been identified that causes the host to produce the elicitors. The most intensively studied system is the association between Chondrus crispus and Acrochaete operculata (Bouarab et al., 1999, 2001a,b; Weinberger et al., 2002, 2005). In common with other members of the Gigartinales, Chondrus crispus has an isomorphic life history, in which the sporophytes and gametophytes are morphologically indistinguishable. However, the two life history phases can be separated by examining the carrageenans they contain because sporophytes produce ‐carrageenans, whereas gametophytes produce ‐ and ‐carrageenans. The two phases can also be separated by their susceptibility to infection by A. operculata: sporophytes are completely invaded by the filaments of the endophyte, leading to fragmentation of the thalli and death, whereas infection of the gametophyte is restricted to the epidermis and cortex and causes little damage. The link between carrageenan composition and susceptibility of the two phases is complex, but has been unravelled successfully by Bouarab et al. (1999).

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Fig. 5. Sulphated oligosaccharide signalling in the Chondrus crispus–Acrochaete operculata host–pathogen association (from Bouarab et al., 1999). (A) Tetrasporophyte of C. crispus releases ‐carrageenan oligosaccharides from the extracellular matrix during infection, which signal to pathogen to control host defense reactions. ‐Carrageenan fragments enhance the synthesis of specific polypetides in A. operculata, including a peroxidase isoform that quenches ROS (labelled ‘‘AOS,’’ active oxygen species) released by the host. This results in stimulated virulence of the pathogen. (B) Gametophyte of C. crispus releases ‐carrageenan oligosaccharides during infection, which do not induce production of ROS in pathogen and consequently hinder the expression of genes involved with virulence. Furthermore, pathogen recognition by the host is enhanced, triggering an oxidative burst, which prevents further invasion. ROS are detrimental to the pathogen and may participate in oxidative cross‐linking of the host cell walls and induce gene‐regulated defense responses. Altogether, pathogen virulence is inhibited. Filled hexagons represent recognition signals from A. operculata (L‐asparagine; Weinberger et al., 2002); half‐square‐ended bars, putative receptors for ‐carrageenan oligosaccharides;

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Unusually in plant–pathogen interactions, the oligosaccharides produced by the host appear to control the infectivity of the pathogen rather than the defensive responses of the host. Consequently, oligocarrageenans do not induce an oxidative burst in either the gametophytes or the sporophytes of Chondrus, but such bursts are induced by cell‐free extracts of Acrochaete, although the intensity of the burst is 10 times greater in the gametophyte than in the sporophyte. However, ‐ and, to a lesser extent, ‐carrageenans elicit an oxidative burst from filaments of Acrochaete within 5 min of application, and also stimulate the synthesis, over the next 4 to 8 days of new proteins, including peroxidases that quench the oxidative burst produced in the host and so facilitate the spread of the infection. As a result, even gametophytes succumb to infection from Acrochaete that has been cultured for some days with ‐carrageenan. Bouarab et al. (1999) suggested, therefore, that ‐carrageenan released from the sporophyte of Chondrus in the initial stages of infection stimulates the production of ROS by Acrochaete, which acts as a signal for the activation of specific pathogenicity genes (Fig. 5). These result in the synthesis of a number of proteins, including peroxidases, that scavenge the ROS produced by the host and, in this way, inhibit activation of the defensive mechanism of the host. Infection of the gametophyte, however, liberates ‐carrageenan, which inhibits ROS production in the pathogen and prevents the induction of peroxidase synthesis. Consequently, the oxidative burst in the host, aided by a specific signal from Acrochaete (now known to be L‐asparagine; Weinberger et al., 2002) recognised only by the gametophyte, is able to activate the defensive system and further invasion is prevented (Fig. 5). Some more details of this defensive system have been elucidated recently (Weinberger et al., 2005). The L‐asparagine released by Acrochaete acts as a substrate for the production of H2O2 by an apoplastic L‐amino acid oxidase, and the concentrations of H2O2 that result are suYcient to reduce the settlement of Acrochaete zoospores on the surface of the host. In the agarophyte Gracilaria conferta, oligosaccharides derived from agarose with between two and eight disaccharide units (i.e., 4 to 16 sugar residues) stimulated an increased consumption of oxygen that peaked 3 min after their addition and decreased gradually over the next 20 min (Weinberger et al., 1999). This oxygen consumption was correlated with the release of H2O2 into the medium (detected again by DCF formation) and in half‐circle‐ended bars, putative receptors for ‐carrageenan oligosaccharides; R and R, putative receptors for signals from A. operculata; scissors, carrageenolytic activity; T‐bars, inhibiting eVects. Broken lines and italicised captions refer to inhibited processes or pathways.

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the rate of bromination of phenol red, both of which processes were independent of light. These responses were also associated with the elimination of an agar‐degrading bacterial flora that had been inoculated onto the surface of the thalli (Flavobacterium‐Cytophaga 1D; Weinberger and Friedlander, 2000; Weinberger et al., 2001) and were, therefore, considered to be equivalent to the defensive oxidative burst of vascular plants (and of Acrochaete and Chondrus) in response to the first products of cell wall degradation. The bacterial flora could be inhibited either directly by the ROS produced in the oxidative burst or by toxic halocarbons resulting from haloperoxidase activity. Elicitation of the response was restricted to oligosaccharides derived from agar, as a wide range of other oligo‐, di‐, and monosaccharides was completely ineVective. Oligoagars containing six to eight disaccharide units proved to be most eVective, possibly because molecules of this size retain enough of their three‐dimensional structure to be recognised, although an ecological advantage might be that, by reacting to relatively large molecules, the alga can respond to the earliest stages of infection (Weinberger et al., 2001). Most species of Gracilaria and Gracilariopsis that were tested responded similarly, although to a lesser extent than G. conferta, but 18 other seaweeds from outside these genera, including other agarophytes, such as Gelidium spp., showed no increase in oxygen uptake after the addition of neoagarohexaose (with three disaccharide units). If light and oligoagars were present, the tips of the thalli became bleached after 10 to 16 h, but this response required higher concentrations of oligoagars, was inhibited by catalase, and was considered to be a nonspecific response to the extra oxidative stress imposed by light. Remarkably similar responses have been reported in sporophytes of Laminaria digitata to treatment with short lengths of alginate chains (oligoalginates; Ku¨ pper et al., 2001). Again, oxygen consumption increased rapidly within 2–3 min of the application of the elicitors, reaching a peak up to eight times the basal rate of dark respiration. This enhanced oxygen consumption was correlated with a burst of H2O2 production, which resulted in peak concentrations in the external medium after 5–10 min followed by a gradual decline to zero after 30–40 min. Both the increased oxygen consumption and the H2O2 burst were dependent on the presence of guluronic acid residues in the oligoalginate elicitor. The molecules used contained 15–25 sugar acid residues, and those that contained guluronic acid alone (oligoguluronates) had the greatest eVect; those with a mixture of guluronic and mannuronic acid had a more limited eVect, whereas oligomannuronates (with mannuronic acid alone) had no eVect. Using DCDF‐DA and confocal laser microscopy, the site of the oxidative burst was shown to be the outer, cortical region of

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the thallus, and the most active types of sporophyte tissue were the young blades 40–80 cm from the stipe. Older blade tissue and the meristem were much less reactive, and filamentous gametophytes of the same species showed no response. As with the comparable phenomenon in Gracilaria, the biological significance of the oxidative burst elicited in Laminaria is thought to be its inhibition of bacterial pathogens and epiphytes, and concentrations of H2O2 similar to those produced at the surface of the thalli were shown to kill alginate‐degrading bacteria (e.g., Pseudoalteromonas elyakovii) and to inhibit the growth of an epiphytic strain ‘‘Ldm2’’ isolated from decayed L. digitata in the field. Ku¨pper et al. (2002) have also shown that alginate‐induced oxidative bursts increase the resistance of L. digitata to infection by the brown algal endophyte Laminariocolax tomentosoides. One important diVerence between the production of ROS in response to pathogens or elicitors and that in response to most environmental stresses is that the former are independent of photosynthesis (e.g., the oxidative burst of Gracilaria occurs in darkness, see earlier discussion). This must mean that the origin of the ROS cannot be via the photosynthetic apparatus, either by the photosensitisation of triplet oxygen by excited chlorophyll molecules or by the leakage of electrons from the photosynthetic electron transport pathway. However, the oxidative bursts in all these systems (and also that in the osmotic stress response of Fucus embyos, which is also independent of photosynthesis; Coelho et al., 2002) can be completely inhibited by diphenylene iodonium (DPI). This is an irreversible inhibitor of flavin‐containing enzymes, including the superoxide‐generating NAD(P)H oxidases of mammalian neutrophils (O’Donnell et al., 1993) and of higher plants (Auh and Murphy, 1995; Pugin et al., 1997), and it seems probable that NADPH oxidases or similar flavoenzymes (e.g., the L‐amino acid oxidase of Chondrus) are the source of the ROS in these light‐independent oxidative bursts of seaweeds.

VI. SUMMARY AND CONCLUSIONS It has become clear since the mid‐1980s that the immediate eVect of a wide range of environmental stresses, and of infection, on vascular plants is to increase the formation of ROS and to impose oxidative stress on the cells (Alscher et al., 2002; Wojtaszek, 1999). It is important to emphasise that the production of ROS occurs continuously in plant and algal cells as a result of the leakage of electrons from respiratory or photosynthetic electron transport pathways (Alscher et al., 1997), but, under unstressed conditions, the

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rates of production and of scavenging are in balance and large concentrations of ROS do not accumulate. This low‐level production of ROS is illustrated nicely by the faint fluorescence of DCF around the main axis of an uninjured thallus of Dasycladus (Ross et al., 2005). It is the imposition of stress, whether by environmental factors or pathogen attack (or the chemical elicitors that signal such attack), that causes a major increase in the rate of production of ROS and results in their accumulation within or around the plant. The ability of species to survive the environmental stress or resist the attack is related to the capacity of the cells to scavenge the ROS, and this capacity is a function of the cellular concentration of antioxidant compounds or the activity of antioxidant enzymes. This review has shown that these generalisations about vascular plants also hold true for some seaweeds from all of the major evolutionary lineages, but the distribution of diVerent types of response among the algal groups is extremely patchy (Table III). Although the responses of ROM in Fucus spp. to desiccation, freezing, low temperatures, and salinity have been well studied, it would be valuable to extend these types of investigation to other genera of brown algae, which are known to be subject to similar stresses. Similarly, ROM in red and green algae has rarely been studied in relation to these types of intertidal stress, and there is considerable scope to broaden the range of examples in which ROM appears to be involved. The lack of studies of ROM in relation to salinity is particularly conspicuous, and a reopening of this field might be especially valuable in view of the recent indications that DMSP can act as an antioxidant in phytoplankton (Sunda et al., 2002), as it has long been considered to be an important osmolyte in marine macroalgae (Kirst, 1990). Brown algae feature less frequently than reds and greens in studies of ROM in relation to high light and UV stress, even though the eVects of such stresses on the growth and photosynthesis of brown algae have received much attention (e.g., Dring et al., 2001; Gevaert et al., 2002; Harker et al., 1999; Kuhlenkamp et al., 2001; Schoenwaelder et al., 2003). There is a need for photoinhibition studies in brown algae to take note of recent developments. Although the mechanisms for scavenging ROS seem to involve a similar range of antioxidant compounds and enzymes in all seaweeds (and, indeed, vascular plants), regardless of the type of stress that has triggered the ROS production, it is diYcult, if not impossible, to generalise about the precise biochemical pathways that are used. Some stress‐tolerant species contain high amounts of some antioxidants and not others; other species seem to build up high activities of certain enzymes, whereas other enzymes remain relatively inactive. It seems likely that, because there are so many possible routes by which ROS can be scavenged (see, e.g., Wojtaszek, 1999), the exact

TABLE III Summary of Responses to Environmental Stress or Infection that Involve Reactive Oxygen Metabolism among Seaweed Species from DiVerent Groups Seaweed species Type of stress

Brown algae

Red algae

Green algae

Desiccation

Fucus spp.

Stictosiphonia arbuscula

No examples

Freezing Low temperature

Fucus spp. F. vesiculosus

No examples Chondrus crispus, Mastocarpus stellatus

No examples No examples

Salinity (hyper‐ osmotic shock) High light/UV

F. serratus

No examples

No examples

Dictyota dichotoma

Eucheuma denticulatum Palmaria palmata Coccotylus truncatus Phycodrys rubens

Ulva spp. Monostroma arcticum Acrosiphonia penicilliformis

Heavy metals

No examples

Gracilaria tenuistipitata

Enteromorpha spp.

Mechanical

No examples

Eucheuma platycladum

Dasycladus vermicularis

Infection (or oligo‐ saccharide elicitors)

Laminaria digitata

Chondrus crispus Gracilaria spp.

Acrochaete operculata

References Collen and Davison, 1999a; Burritt et al., 2002 Collen and Davison, 1999a Collen and Davison, 2001; Collen and Davison, 1999c; Lohrmann et al., 2004 Coelho et al., 2002 Davison et al., 2000; Mtolera et al., 1995, 1996; Collen and Pedersen, 1996; Bischof et al., 2002; Aguilera et al., 2002a; Aguilera et al., 2002b Collen et al., 2003; Rijstenbil et al., 1998; Ratkevicius et al., 2003 Collen and Pedersen, 1994; Ross et al., 2005 Ku¨ pper et al., 2001; Bouarab et al., 1999; Weinberger et al., 1999, 2001

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pathway followed on any one occasion depends on the species, the state of acclimation of the individual, and the microenvironment at the time. It may not be useful, therefore, to seek a common indicator or monitor of oxidative stress among the compounds or enzymes that have been measured so frequently. What is of greater interest is the source of, and the precise trigger for, the production of ROS. In principle, there seem to be two common sources for the increase in ROS that results from the imposition of stress or infection: the photosynthetic apparatus (either excited chlorophyll molecules or the photosynthetic electron transport chain) or flavin‐containing NADPH oxidases located in the plasma membrane. As we have seen, many of the environmental stresses to which seaweeds are exposed result in a reduction of the rate of photosynthesis, which means that more photons are absorbed than can be utilised. Under these conditions, excited chlorophyll molecules may photosensitise triplet oxygen to form singlet oxygen, which, in turn, may cause damage to the D1 protein that results in longer term photoinhibition. This seems to be the most plausible explanation of the eVects of stress imposed by desiccation, freezing, low temperatures, high light, UV radiation, and heavy metals, all of which tend to result in a gradual and continued buildup of ROS, but more positive confirmation of this explanation in a number of seaweed examples for each type of stress would be valuable. The pattern of ROS production in response to other stresses (infection or its elicitors, mechanical stress, hyperosmotic shock), however, is quite diVerent—a more rapid and intense, but short‐lived production, resulting in an ‘‘oxidative burst’’—and has sometimes been shown to occur in the dark. As discussed earlier, this type of response is completely inhibited by DPI, which indicates that NADPH oxidases are involved. The outstanding question, therefore, is: what triggers the sudden burst of activity of such oxidases? The recent identification of L‐asparagine as the elicitor and substrate for the oxidative burst of gametophytes of Gracilaria in response to infection by Acrochaete (Weinberger et al., 2005) provides an answer for this specific example, but cannot be generalised to others. The suggestion that the trigger for the response of Fucus embryos to hyperosmotic shock may be the separation of the plasma membrane from the cell wall (Coelho et al., 2002) is also intriguing, but, again, is unlikely to lead to a broad generalisation. The search goes on, therefore, and, as in so many other examples, will need to be concentrated into an ever shorter time period (less than 10 s from the start of the stress) and an ever smaller space (less than 10 m).

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REFERENCES Abrahamsson, K., Choo, K. S., Pedersen, 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. Aguilera, J., Bischof, K., Karsten, U., Hanelt, D. and Wiencke, C. (2002a). Seasonal variation in ecophysiological pattern in macroalgae from an Arctic fjord. 2. Pigment accumulation and biochemical defence systems against high light stress. Marine Biology 140, 1087–1095. Aguilera, J., Dummermuth, A., Karsten, U., Schriek, R. and Wiencke, C. (2002b). Enzymatic defences against photooxidative stress induced by ultraviolet radiation in Arctic marine macroalgae. Polar Biology 25, 432–441. Almeida, M., Filipe, S., Humanes, M., Maia, M. F., Melo, R., Severino, N., da Silva, J. A. L., da Silva, J. J. R. F. and Wever, R. (2001). Vanadium haloperoxidases from brown algae of the Laminariaceae family. Phytochemistry 57, 633–642. Alscher, R. G., Donahue, J. L. and Cramer, C. L. (1997). Reactive oxygen species and antioxidants: Relationships in green cells. Physiologia Plantarum 100, 224–233. Alscher, R. G., Erturk, N. and Heath, L. S. (2002). Role of superoxide dismutases (SODs) in controlling oxidative stress in plants. Journal of Experimental Botany 53, 1331–1341. Asada, K. (1999). The water‐water cycle in chloroplasts: Scavenging of active oxygens and dissipation of excess photons. Annual Review of Plant Physiology and Plant Molecular Biology 50, 601–639. Asada, K. and Takahashi, M. (1987). Production and scavenging of active oxygen in photosynthesis. In ‘‘Photoinhibition’’ (D. J. Kyle, C. Osmond and C. J. Arntzen, eds.), pp. 227–297. Elsevier, New York. Auh, C.‐K. and Murphy, T. M. (1995). Plasma membrane redox enzyme is involved in the synthesis of O2 and H2O2 by Phytophthora elicitor‐stimulated rose cells. Plant Physiology 107, 1241–1247. Barros, M. P., Granbom, M., Colepicolo, P. and Pedersen, M. (2003). Temporal mismatch between induction of superoxide dismutase and ascorbate peroxidase correlates with high H2O2 concentration in seawater from clofibrate‐treated red algae Kappaphycus alvarezii. Archives of Biochemistry and Biophysics 420, 161–168. Bischof, K., Hanelt, D., Aguilera, J., Karsten, U., Voegele, B., Sawall, T. and Wiencke, C. (2002). Seasonal variation in ecophysiological patterns in macroalgae from an Arctic fjord. 1. Sensitivity of photosynthesis to ultraviolet radiation. Marine Biology 140, 1097–1106. Bouarab, K., Kloareg, B., Potin, P. and Correa, J. (2001a). Ecological and biochemical aspects in algal infectious diseases. Cahiers de Biologie Marine 42, 91–100. 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., 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.

202

M. J. DRING

Bouchard, J. N., Campbell, D. A. and Roy, S. (2005). EVects of UV‐B radiation on the D1 protein repair cycle of natural phytoplankton communities from three latitudes (Canada, Brazil, and Argentina). Journal of Phycology 41, 273–286. Burritt, D. J., Larkindale, J. and Hurd, C. L. (2002). Antioxidant metabolism in the intertidal red seaweed Stictosiphonia arbuscula following desiccation. Planta 215, 829–838. Butow, B., Wynne, D. and Tel‐Or, E. (1994). Response of catalase activity to environmental stress in the freshwater dinoflagellate Peridinium gatunense. Journal of Phycology 30, 17–22. Butow, B., Wynne, D. and Tel‐Or, E. (1997). Antioxidative protection of Peridinium gatunense in Lake Kinneret: Seasonal and daily variation. Journal of Phycology 33, 780–786. Carpenter, L. J. and Liss, P. S. (2000). On temperate sources of bromoform and other reactive organic bromine gases. Journal of Geophysical Research‐ Atmospheres 105, 20539–20547. Carpenter, L. J., Malin, G., Liss, P. S. and Ku¨ pper, F. C. (2000). Novel biogenic iodine‐containing trihalomethanes and other short‐lived halocarbons in the coastal east Atlantic. Global Biogeochemical Cycles 14, 1191–1204. Carter, J. N., Beatty, K. E., Simpson, M. T. and Butler, A. (2002). Reactivity of recombinant and mutant vanadium bromoperoxidase from the red alga Corallina oYcinalis. Journal of Inorganic Biochemistry 91, 59–69. Choo, K. S., Snoeijs, P. and Pedersen, M. (2004). Oxidative stress tolerance in the filamentous green algae Cladophora glomerata and Enteromorpha ahlneriana. Journal of Experimental Marine Biology and Ecology 298, 111–123. Coelho, S. M., Taylor, A. R., Ryan, K. P., Sousa‐Pinto, I., Brown, M. T. and Brownlee, C. (2002). Spatiotemporal patterning of reactive oxygen production and Ca2þ wave propagation in Fucus rhizoid cells. Plant Cell 14, 2369–2381. 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. Collen, J. and Davison, I. R. (1997). In vivo measurement of active oxygen production in the brown alga Fucus evanescens using 20 ,70 ‐dichlorohydrofluorescein diacetate. Journal of Phycology 33, 643–648. Collen, J. and Davison, I. R. (1999a). Reactive oxygen production and damage in intertidal Fucus spp. (Phaeophyceae). Journal of Phycology 35, 54–61. Collen, J. and Davison, I. R. (1999b). Reactive oxygen metabolism in intertidal Fucus spp. (Phaeophyceae). Journal of Phycology 35, 62–69. Collen, J. and Davison, I. R. (1999c). Stress tolerance and reactive oxygen metabolism in the intertidal red seaweeds Mastocarpus stellatus and Chondrus crispus. Plant, Cell and Environment 22, 1143–1151. Collen, J. and Davison, I. R. (2001). Seasonality and thermal acclimation of reactive oxygen metabolism in Fucus vesiculosus (Phaeophyceae). Journal of Phycology 37, 474–481. Collen, J., Ekdahl, A., Abrahamsson, K. and Pedersen, M. (1994). The involvement of hydrogen peroxide in the production of volatile halogenated compounds by Meristiella gelidium. Phytochemistry 36, 1197–1202. Collen, J. and Pedersen, M. (1994). A stress‐induced oxidative burst in Eucheuma platycladum (Rhodophyta). Physiologia Plantarum 92, 417–422.

STRESS AND DISEASE RESISTANCE IN SEAWEEDS

203

Collen, J. and Pedersen, M. (1996). Production, scavenging and toxicity of hydrogen peroxide in the green seaweed Ulva rigida. European Journal of Phycology 31, 265–271. Collen, J., Pinto, E., Pedersen, M. and Colepicolo, P. (2003). Induction of oxidative stress in the red macroalga Gracilaria tenuistipitata by pollutant metals. Archives of Environmental Contamination and Toxicology 45, 337–342. Davison, I. R., Collen, J. and Fegley, J. C. (2000). Reactive oxygen metabolism in the tropical brown alga Dictyota dichotoma. Journal of Phycology 36 (Suppl.), 16. Davison, I. R. and Pearson, G. A. (1996). Stress tolerance in intertidal seaweeds. Journal of Phycology 32, 197–211. Dring, M. J., Wagner, A. and Luning, K. (2001). Contribution of the UV component of natural sunlight to photoinhibition of photosynthesis in six species of subtidal brown and red seaweeds. Plant Cell and Environment 24, 1153–1164. Dummermuth, A. L., Karsten, U., Fisch, K. M., Koenig, G. M. and Wiencke, C. (2003). Responses of marine macroalgae to hydrogen‐peroxide stress. Journal of Experimental Marine Biology and Ecology 289, 103–121. Edwards, D. M., Reed, R. H., Chudeck, J. A., Foster, R. and Stewart, W. D. P. (1988). Osmoacclimation in Enteromorpha intestinalis: Long‐term eVects of osmotic stress on organic solute concentrations. Marine Biology 98, 467–476. Foote, C. S. (1967). Photosensitised oxidation and singlet oxygen consequences in biological systems. In ‘‘Free Radicals in Biology’’ (W. A. Prior, ed.), 2nd Edn., pp. 85–113. Academic Press, New York. Foyer, H. F., Lopez‐Delgado, H., Dat, J. F. and Scott, I. M. (1997). Hydrogen peroxide‐ and glutathione‐associated mechanisms of acclimatory stress tolerance and signalling. Physiologia Plantarum 100, 241–254. Franklin, L. A. and Forster, R. M. (1997). The changing irradiance environment: Consequences for marine macrophyte physiology, productivity and ecology. European Journal of Phycology 32, 207–232. Franklin, L. A., Yakovleva, I., Karsten, U. and Luning, K. (1999). Synthesis of mycosporine‐like amino acids in Chondrus crispus (Florideophyceae) and the consequences for sensitivity to ultraviolet B radiation. Journal of Phycology 35, 682–693. Fridovich, I. (1997). Superoxide anion radical (O2), superoxide dismutases, and related matters. Journal of Biological Chemistry 272, 18515–18517. Gevaert, F., Creach, A., Davoult, D., Holl, A. C., Seuront, L. and Lemoine, Y. (2002). Photo‐inhibition and seasonal photosynthetic performance of the seaweed Laminaria saccharina during a simulated tidal cycle: Chlorophyll fluorescence measurements and pigment analysis. Plant Cell and Environment 25, 859–872. Giordano, M., Beardall, J. and Raven, J. A. (2005). CO2 concentrating mechanisms in algae: Mechanisms, environmental modulation and evolution. Annual Review of Plant Biology 56, 99–131. Goodwin, K. D., North, W. J. and Lidstrom, M. E. (1997). Production of bromoform and dibromomethane by giant kelp: Factors aVecting release and comparison to anthropogenic bromine sources. Limnology and Oceanography 42, 1725–1734. Gossett, D. R., Millhollon, E. P. and Lucas, M. C. (1994). Antioxidant responses to NaCl stress in salt‐tolerant and salt‐sensitive cultivars of cotton. Crop Science 34, 706–714.

204

M. J. DRING

Gschwend, P. M., Macfarlane, J. K. and Newman, K. A. (1985). Volatile halogenated organic‐compounds released to seawater from temperate marine macroalgae. Science 227, 1033–1035. Hara, I. and Sakurai, T. (1998). Isolation and characterisation of vanadium bromoperoxidase from a marine macroalga, Ecklonia stolonifera. Journal of Inorganic Biochemistry 72, 23–28. Harker, M., BerkaloV, C., Lemoine, Y., Britton, G., Young, A. J., Duval, J. C., Rmiki, N. E. and Rousseau, B. (1999). EVects of high light and desiccation on the operation of the xanthophyll cycle in two marine brown algae. European Journal of Phycology 34, 35–42. Herna´ ndez, J. A., Olmos, E., Corpas, F. J., Sevilla, F. and del Rio, L. A. (1995). Salt induced oxidative stress in chloroplasts of pea plants. Plant Science 105, 151–167. Hurd, C. L. and Dring, M. J. (1990). Phosphate uptake by intertidal algae in relation to zonation and season. Marine Biology 107, 281–289. Isupov, M. N., Dalby, A. R., Brindley, A. A., Izumi, Y., Tanabe, T., Murshudov, G. N. and Littlechild, J. A. (2000). Crystal structure of dodecameric vanadium‐dependent bromoperoxidase from the red algae Corallina oYcinalis. Journal of Molecular Biology 299, 1035–1049. Jahnke, L. S. and White, A. L. (2003). Long‐term hyposaline and hypersaline stresses produce distinct antioxidant responses in the marine alga Dunaliella tertiolecta. Journal of Plant Physiology 160, 1193–1202. Kalir, A. and PoljakoV‐Mayber, A. (1981). Changes in activity of malate dehydrogenase, catalase, peroxidase and superoxide dismutase in leaves of Halimone portulacoides (L.) Allen exposed to high sodium chloride concentrations. Annals of Botany 47, 75–85. Kirst, G. O. (1990). Salinity tolerance of eukaryotic marine algae. Annual Review of Plant Physiology and Plant Molecular Biology 41, 21–53. Krabs, G., Watanabe, M. and Wiencke, C. (2004). A monochromatic action spectrum for the photoinduction of the UV‐absorbing mycosporine‐like amino acid shinorine in the red alga Chondrus crispus. Photochemistry and Photobiology 79, 515–519. Krieger‐Liszkay, A. (2005). Singlet oxygen production in photosynthesis. Journal of Experimental Botany 56, 337–346. Kuhlenkamp, R., Franklin, L. A. and Luning, K. (2001). EVect of solar ultraviolet radiation on growth in the marine macroalga Dictyota dichotoma (Phaeophyceae) at Helgoland and its ecological consequences. Helgoland Marine Research 55, 77–86. 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, 2057–2080. Ku¨ pper, F. C., Schweigert, N., Gall, E. A., Legendre, J. M., Vilter, H. and Kloareg, B. (1998). Iodine uptake in Laminariales involves extracellular, haloperoxidase‐mediated oxidation of iodide. Planta 207, 163–171. 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.

STRESS AND DISEASE RESISTANCE IN SEAWEEDS

205

Lewis, S., Donkin, M. E. and Depledge, M. H. (2001). Hsp70 expression in Enteromorpha intestinalis (Chlorophyta) exposed to environmental stressors. Aquatic Toxicology 51, 277–291. Li, R. and Brawley, S. H. (2004). Improved survival under heat stress in intertidal embryos (Fucus spp.) simultaneously exposed to hypersalinity and the eVect of parental thermal history. Marine Biology 144, 205–213. Lohrmann, N. L., Logan, B. A. and Johnson, A. S. (2004). Seasonal acclimatisation of antioxidants and photosynthesis in Chondrus crispus and Mastocarpus stellatus, two co‐occurring red algae with diVering stress tolerances. Biological Bulletin, Marine Biological Laboratory, Woods Hole 207, 225–232. Manley, S. L. and Barbero, P. E. (2001). Physiological constraints on bromoform (CHBr3) production by Ulva lactuca (Chlorophyta). Limnology and Oceanography 46, 1392–1399. McConnell, O. and Fenical, W. (1977). Halogen chemistry of red alga Asparagopsis. Phytochemistry 16, 367–374. Mtolera, M. S. P., Collen, J., Pedersen, M., Ekdahl, A., Abrahamsson, K. and Semesi, A. K. (1996). Stress‐induced production of volatile halogenated organic compounds in Eucheuma denticulatum (Rhodophyta) caused by elevated pH and high light intensities. European Journal of Phycology 31, 89–95. Mtolera, M. S. P., Collen, J., Pedersen, M. and Semesi, A. K. (1995). Destructive hydrogen peroxide production in Eucheuma denticulatum (Rhodophyta) during stress caused by elevated pH, high light intensities and competition with other species. European Journal of Phycology 30, 289–297. Nielsen, H. D., Brown, M. T. and Brownlee, C. (2003). Cellular responses of developing Fucus serratus embryos exposed to elevated concentrations of Cu2þ. Plant Cell and Environment 26, 1737–1747. O’Donnell, V., Tew, D. G., Jones, O. T. G. and England, P. J. (1993). Studies on the inhibitory mechanism of iodonium compounds with special reference to neutrophil NADPH oxidase. Biochemical Journal 290, 41–49. Ohsawa, N., Ogata, Y., Okada, N. and Itoh, N. (2001). Physiological function of bromoperoxidase in the red marine alga, Corallina pilulifera: Production of bromoform as an allelochemical and the simultaneous elimination of hydrogen peroxide. Phytochemistry 58, 683–692. Ohshiro, T., Nakano, S., Takahashi, Y., Suzuki, M. and Izumi, Y. (1999). Occurrence of bromoperoxidase in the marine green macroalga, Ulvella lens, and emission of volatile brominated methane by the enzyme. Phytochemistry 52, 1211–1215. Pearson, G. A. and Davison, I. R. (1994). Freezing stress and osmotic dehydration in Fucus distichus (Phaeophyta): Evidence for physiological similarity. Journal of Phycology 30, 257–267. Pedersen, M., Collen, J., Abrahamsson, K. and Ekdahl, A. (1996). Production of halocarbons from seaweeds: An oxidative stress reaction? Scientia Marina (Barcelona) 60, 257–263. Phillips, J. C. and Hurd, C. L. (2004). Kinetics of nitrate, ammonium, and urea uptake by four intertidal seaweeds from New Zealand. Journal of Phycology 40, 534–545. Pinto, E., Sigaud‐Kutner, T. C. S., Leitao, M. A. S., Okamoto, O. K., Morse, D. and Colepicolo, P. (2003). Heavy metal‐induced oxidative stress in algae. Journal of Phycology 39, 1008–1018.

206

M. J. DRING

Potin, P., Bouarab, K., Ku¨ pper, F. and Kloareg, B. (1999). Oligosaccharide recognition signals and defence reactions in marine plant‐microbe interactions. Current Opinion in Microbiology 2, 276–283. Pugin, A., Frachisse, J. M., Tavernier, E., Bligny, R., Gout, E., Douce, R. and Guern, J. (1997). Early events induced by the elicitor cryptogein in tobacco cells: Involvement of a plasma membrane NADPH oxidase and activation of glycolysis and the pentose phosphate pathway. Plant Cell 9, 2077–2091. Ratkevicius, N., Correa, J. A. and Moenne, A. (2003). Copper accumulation, synthesis of ascorbate and activation of ascorbate peroxidase in Enteromorpha compressa (L.) Grev. (Chlorophyta) from heavy metal‐enriched environments in northern Chile. Plant Cell and Environment 26, 1599–1608. Raven, J. A., Kubler, J. E. and Beardall, J. (2000). Put out the light, and then put out the light. Journal of the Marine Biological Association of the United Kingdom 80, 1–25. Reed, R. H. (1983). Measurement and osmotic significance of dimethylsulphoniopropionate in marine macroalgae. Marine Biology Letters 4, 173–181. Rijstenbil, J. W., Coelho, S. M. and Eijsackers, M. (2000). A method for the assessment of light‐induced oxidative stress in embryos of fucoid algae via confocal laser scan microscopy. Marine Biology 137, 763–774. Rijstenbil, J. W., Haritonidis, S., Malea, P., Seferlis, M. and Wijnholds, J. A. (1998). Thiol pools and glutathione redox ratios as possible indicators of copper toxicity in the green macroalgae Enteromorpha spp. from the Scheldt Estuary (SW Netherlands, Belgium) and Thermaikos Gulf (Greece, N Aegean Sea). Hydrobiologia 385, 171–181. Ross, C., Ku¨ pper, F. C., Vreeland, V., Waite, J. H. and Jacobs, R. S. (2005). Evidence of a latent oxidative burst in relation to wound repair in the giant unicellular chlorophyte Dasycladus vermicularis. Journal of Phycology 41, 531–541. Rossa, M. M., de Oliveira, M. C., Okamoto, O. K., Lopes, P. F. and Colepicolo, P. (2002). EVect of visible light on superoxide dismutase (SOD) activity in the red alga Gracilariopsis tenuifrons (Gracilariales, Rhodophyta). Journal of Applied Phycology 14, 151–157. Schoenwaelder, M. E. A., Wiencke, C., Clayton, M. N. and Glombitza, K. W. (2003). The eVect of elevated UV radiation on Fucus spp. (Fucales, Phaeophyta) zygote and embryo development. Plant Biology 5, 366–377. Shimonishi, M., Kuwamoto, S., Inoue, H., Wever, R., Ohshiro, T., Izumi, Y. and Tanabe, T. (1998). Cloning and expression of the gene for a vanadium‐ dependent bromoperoxidase from a marine macroalga, Corallina pilulifera. FEBS Letters 428, 105–110. Sunda, W., Kieber, D. J., Kiene, R. P. and Huntsman, S. (2002). An antioxidant function for DMSP and DMS in marine algae. Nature 418, 317–320. Sundene, O. (1962). The implications of transplant and culture experiments on the growth and distribution of Alaria esculenta. Nytt Magasin for Botanikk 9, 155–174. Sundstrom, J., Collen, J., Abrahamsson, K. and Pedersen, M. (1996). Halocarbon production and in vivo brominating activity of Eucheuma denticulatum. Phytochemistry 42, 1527–1530. Twiner, M. J., Dixon, S. J. and Trick, C. G. (2001). Toxic eVects of Heterosigma akashiwo do not appear to be mediated by hydrogen peroxide. Limnology and Oceanography 46, 1400–1405.

STRESS AND DISEASE RESISTANCE IN SEAWEEDS

207

Van Alstyne, K. L., Pelletreau, K. N. and Rosario, K. (2003). The eVects of salinity on dimethylsulfoniopropionate production in the green alga Ulva fenestrata Postels et Ruprecht (Chlorophyta). Botanica Marina 46, 350–356. van de Poll, W. H., Hanelt, D., Hoyer, K., Buma, A. G. J. and Breeman, A. M. (2002). Ultraviolet‐B‐induced cyclobutane‐pyrimidine dimer formation and repair in Arctic marine macrophytes. Photochemistry and Photobiology 76, 493–500. Vilter, H., Glombitza, K. and Grawe, A. (1983). Peroxidases from Phaeophyceae. I. Extraction and detection of the peroxidases. Botanica Marina 26, 331–340. 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., Pohnert, G., Berndt, M. L., Bouarab, K., Kloareg, B. and Potin, P. (2005). 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., 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., 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. Weyand, M., Hecht, H. J., Kiess, M., Liaud, M. F., Vilter, H. and Schomburg, D. (1999). X‐ray structure determination of a vanadium‐dependent haloperoxidase from Ascophyllum nodosum at 2.0 angstrom resolution. Journal of Molecular Biology 293, 595–611. Wojtaszek, P. (1997). Oxidative burst: An early plant response to pathogen infection. Biochemical Journal 322, 681–692.

Nutrient Sensing and Signalling in Plants: Potassium and Phosphorus

ANNA AMTMANN,* JOHN P. HAMMOND,{ PATRICK ARMENGAUD* AND PHILIP J. WHITE{

*Plant Science Group, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow G12 8QQ, United Kingdom { Warwick Horticulture Research International, University of Warwick, Wellesbourne, Warwick CV35 9EF, United Kingdom

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . II. Potassium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Potassium Nutrition of Plants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Transcriptional Responses to K . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. K Perception and Cellular Signalling Events . . . . . . . . . . . . . . . . . . . . . . D. Systemic Signalling of Plant K Status. . . . . . . . . . . . . . . . . . . . . . . . . . . . . III. Phosphorus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Phosphorus Nutrition of Plants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Transcriptional Responses to P. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Phosphorus Perception and Cellular Signalling Events . . . . . . . . . . . . . D. Systemic Signalling of Plant P Status . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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ABSTRACT Potassium and phosphorus are important macronutrients for crops but are often deficient in the field. Very little is known about how plants sense fluctuations in K and P and how information about K and P availability is integrated at the whole Advances in Botanical Research, Vol. 43 Incorporating Advances in Plant Pathology Copyright 2006, Elsevier Ltd. All rights reserved.

0065-2296/06 $35.00 DOI: 10.1016/S0065-2296(05)43005-0

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plant level into physiological and metabolic adaptations. This chapter reviews recent advances in discovering molecular responses of plants to K and P deficiency by microarray experiments. These studies provide us not only with a comprehensive picture of adaptive mechanisms, but also with a large number of transcriptional markers that can be used to identify upstream components of K and P signalling pathways. On the basis of the available information we discuss putative receptors and signals involved in the sensing and integration of K and P status both at the cellular and at the whole plant level. These involve membrane potential, voltage‐dependent ion channels, intracellular Ca and pH, and transcription factors, as well as hormones and metabolites for systemic signalling. Genetic screens of reporter lines for transcriptional markers and metabolome analysis of K- and P-deficient plants are likely to further advance our knowledge in this area in the near future.

I. INTRODUCTION Management of mineral nutrients is one of the biggest challenges that plants face during their life span. In contrast to animals, which can move or migrate to open up new food resources, plants are confined to a particular patch of soil and the minerals that are available from it. In a natural ecosystem, endogenous plant species and microbes are adapted to the nutrient diet on oVer and they form communities to optimally exploit and recreate resources. By contrast, in agricultural systems, high‐density monoculture crops deplete soil minerals quickly and therefore rely on external supplies for most of their major nutrients, particularly nitrogen (N), potassium (K), and phosphorus (P). A balanced supply of mineral nutrients is crucial for both quantity and quality of the crop, but is rarely achieved in the field. Compound fertilisers, containing N, P, and K, have a long tradition in Europe. In developing countries, the introduction of high‐yield crop varieties has been accompanied by a steep increase in N fertilisation, whereas application of P and K fertilisers has often been neglected (Gething, 1993; Laegreid et al., 1999). In many areas this has led to the depletion of arable land for these two nutrients. Even in Europe K and P can be limiting due to leaching or soil structure and chemistry (Syers, 1998). Furthermore, if uptake rates are high, depletion zones can form in the plant rhizosphere (Marschner, 1995). An insuYcient supply of K and P leads to ineYcient usage of nitrate, which can leak into the groundwater and cause environmental problems (Laegreid et al., 1999). To achieve an optimal relationship between the costs and the benefits of fertiliser usage, it is not only important to monitor and improve arable soils continuously, but also to fully understand the role of individual nutrients for plant growth and development, the interaction between diVerent nutrients, and the way plants respond to impaired nutrient balance in the soil. The study of mineral nutrition has a long tradition in the plant sciences. Pioneering work was carried out during the 19th and early 20th century by

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Saussure, Sachs, Knop, Lipman, and others to determine requirements for individual nutrients, resulting in a list of macro‐ and micronutrients essential for plants (Arnon and Stout, 1939; Epstein 1965, 1972). During the second half of the 20th century the scientific focus of research into plant nutrition moved towards studying pathways for nutrient transport (e.g., Epstein, 1973; Epstein et al., 1963), and 50 years of experimental research employing isotope fluxes, electrophysiology, and molecular biology have resulted in a vast, although still incomplete, body of data on ion channels and transporters that mediate the uptake of mineral nutrients and their redistribution within cellular compartments and plant tissues (see compilations by Blatt, 2004; Williams, 2000). In parallel, biochemical analysis has provided us with knowledge of how mineral nutrients are assimilated and metabolised (reviewed by Crawford et al., 2000; Kochian, 2000; Plaxton and Carswell, 1999). More recently, plant scientists have started to investigate how plants sense nutrients and integrate information on the availability of diVerent nutrients. Much of this research has focussed on carbon and nitrogen (Filleur et al., 2005; Moore et al., 2003; Rolland and Sheen, 2005; Zhang and Forde, 1998), whereas less is known about the perception and signalling of other nutrients. In this context it is important to remember that not only the supply of mineral nutrients, but also the plant’s demand for them, fluctuate diurnally and over a plant’s life span and depend on other environmental factors. This review focuses on plant responses to varying supplies of potassium (K) and phosphorus (P). Both nutrients are required in significant amounts for plant growth and development. Whereas P is an important component of many structural macromolecules, metabolites, and signalling molecules (Plaxton and Carswell, 1999; Vance et al., 2003), K is not assimilated into organic matter. Nevertheless, K also plays an important role in metabolism as it functions as a cofactor of many enzymes and is required for charge balancing and transport of metabolites (Marschner, 1995; Wyn Jones and Pollard, 1983). Two types of acclimatory responses can be distinguished when plants encounter nutrient shortage. The first response is linked to nutrient uptake and homeostasis. Under deficiency plants increase their capacity and affinity for nutrient uptake and activate transport processes that assist the remobilisation and redistribution of stored nutrients to support growing and metabolically active tissues, often at the cost of older and less crucial cell types. Nutrient homeostasis involves both transcriptional and posttranslational regulation of a diverse set of ion transporters in diVerent membranes and diVerent cell types (Amtmann et al., 2004; Rausch and Bucher, 2002; Smith et al., 2000b; Tester and Leigh, 2001). Although many of these

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transporters have been characterised with respect to their expression pattern, membrane localisation, biophysical properties, and regulation, very little is known about how their function and regulation are integrated at the cellular or whole plant level. The second response involves the reprogramming of plant growth, development, and metabolism with the aim of using limited resources optimally. Research into the signal pathways underlying these responses and their causal relationships is in its infancy. In order to respond to fluctuating nutrient supply, plants must sense changes in the soil environment, generate signals both at the cellular and at the systemic level, perceive and integrate these signals at specific sites of action, and translate them into a concatenated whole plant response. Many responses to K and P deficiency have been characterised in detail and several sites of action have been identified. There is also some evidence as to which signalling pathways may be involved in nutrient signalling, but there is a general lack of knowledge about primary receptors of nutrient stress. In most cases it is not known which stimulus is perceived: the nutrient itself, a physicochemical parameter linked to nutrient concentration (e.g., ionic strength, membrane potential, pH), or a change in specific metabolites? Cellular ion homeostasis aims to maintain constant levels of K and P in the root cytoplasm, and it is therefore generally assumed that the site of perception resides at the soil/root interface, i.e., in the plasma membrane of root epidermal and cortical cells. However, even this notion is questionable, as current methods to measure cytoplasmic K and P concentrations in individual cells are not sensitive enough to resolve small fluctuations of these ions in the cytoplasm. Constant improvements in in vivo nuclear magnetic resonance and imaging techniques are promising and there is hope that we will soon be able to obtain a dynamic picture of nutrient and metabolite concentrations in tissues, individual cell types, and even cellular compartments. The study of signalling pathways usually takes a reverse approach. Rather than starting the search for molecular components of nutrient sensing at the level of receptors and primary signals, the experiments commence with a well‐defined response and work backwards by identifying molecular processes that are necessary to obtain this response. Physiological and metabolic responses have only limited value for such an experimental design as they usually represent the end point of a complex interplay between diVerent signalling pathways or reflect secondary responses to defective biochemical processes. Current knowledge suggests that most plant responses manifest themselves through changes in gene expression. The relative ease with which to measure transcript levels of a large number of genes has contributed to much of the recent progress in the identification of signalling pathways.

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These experiments are often combined with a forward genetics approach, where a mutagenised population of transgenic plants carrying a promoter– reporter construct for a nutrient‐regulated gene is screened for mutant lines that lack the transcriptional response or have a constitutive response. Although individual genes that are regulated by nutrient stress have been identified and used in signalling studies, the development of full genome microarrays (transcriptomics) has allowed us to identify a large number of transcriptional markers for nutrient stresses, many of which will be an invaluable help for future studies into nutrient sensing. In particular, in the case of K, very few genes that respond to varying K supply had been identified prior to microarray technology. Much of this review is therefore dedicated to the transcriptional responses to K and P stress discovered in recent microarray studies. The review then proceeds by summarising current knowledge on perception and signalling events located upstream of the observed responses. Although transcripts are the most commonly used markers for stress signalling in plants, it should be emphasised that other responses can also reveal useful information on plant nutrient sensing. For example, the characterisation of biophysical properties of ion channels has deepened our understanding of signalling pathways, linking external K concentrations with K homeostasis. Another area of potential progress in our understanding of nutrient signalling is metabolism. Primary metabolites of nitrogen assimilation and carbon fixation have emerged as potential signals for the downstream integration of carbon–nitrogen metabolism in response to an external imbalance between nitrogen availability and photosynthesis (Coruzzi and Bush, 2001; Stitt and Fernie, 2003). Similarly, we can assume that some metabolites might play important roles as systemic signals for K and P deficiency. Advanced high‐resolution technology for measuring a large number of metabolites in individual tissue samples (metabolomics) will facilitate the identification of potential metabolic signals involved in plant responses to fluctuating P and K supply.

II. POTASSIUM A. POTASSIUM NUTRITION OF PLANTS

Potassium is the most abundant inorganic cation in plants, comprising up to 10% of a plant’s dry weight (Leigh and Jones, 1984). Potassium is an important macronutrient for plants, which carries out vital functions in metabolism, growth, and stress adaptation. These functions can be classified into those that rely on high and relatively stable concentrations of K in

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certain cellular compartments or tissues and those that rely on K movement among diVerent compartments, cells, and tissues. The first class of functions includes enzyme activation, stabilisation of protein synthesis, neutralisation of negative charges on proteins, and maintenance of cytoplasmic pH homeostasis (Marschner, 1995). The optimal K concentration for enzyme activation and protein synthesis is of the order 100 mM (Wyn Jones and Pollard, 1983). Thus for optimal metabolic activity, cells rely on controlled potassium concentrations of around 100 mM in metabolically active compartments, i.e., the cytoplasm, the nucleus, the stroma of chloroplasts, and the matrix of mitochondria. Other roles of potassium are linked to its high mobility. This is particularly evident where K movement is the driving force for osmotic changes as, for example, in stomatal movement, light‐driven and seismonastic movements of organs, or phloem transport. In other cases, K movement provides a charge‐balancing counterflux, essential for sustaining the movement of other ions. Thus energy production through Hþ‐ATPases relies on overall Hþ/K exchange (Tester and Blatt, 1989; Wu et al., 1991), and transport of sugars, amino acids, and nitrate is accompanied by K fluxes (Marschner, 1995). The directed movement of potassium is also required for growth. Accumulation of potassium in plant vacuoles creates the necessary osmotic potential for cell extension. Rapid cell extension relies on high mobility of the used osmoticum and therefore only few other inorganic ions (e.g., Naþ) can replace K in this role (Reckmann et al., 1990). Once cell growth has come to a halt, maintenance of osmotic potentials can be carried out by less mobile molecules such as sugars, and K ions can partly be recovered from vacuoles (Marschner, 1995; PoVenroth et al., 1992). Plants have mechanisms to accumulate potassium from very low external concentrations, and because of its high mobility, potassium can be redistributed quickly between diVerent compartments and tissues under fluctuating external potassium conditions. Plants therefore grow well over a wide range of external K supplies (approximately 10 M to 10 mM). As for all nutrients, critical concentrations for starvation or toxicity depend on other environmental factors. In particular, stress conditions that are linked directly to potassium availability, such as drought and salinity, narrow the window for potassium suYciency. Due to the vital role that potassium plays in plant growth and metabolism, potassium‐deficient plants show a very general phenotype, which is characterised by reduced growth, especially of aerial parts. A halt in lateral root growth has been described for K‐starved Arabidopsis thaliana plants grown on agar plates (Armengaud et al., 2004). Physiological symptoms of potassium deficiency include reduced photosynthesis and impaired regulation of transpiration. A study on rice varieties that diVer in their K use

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eYciency showed that K‐eYcient genotypes had a greater relative tillering and grain‐filling rate in low K compared to K‐ineYcient genotypes (Yang et al., 2004). These parameters were correlated with leaf K concentrations, net photosynthetic rate, stomatal conductance, and Rubisco activity. Although these findings can be explained easily with known roles of K in stomatal function and enzyme activation, it is diYcult to determine which K‐dependent process is the most crucial one in creating deficiency symptoms. For example, a lack of K will impede the establishment of Hþ gradients, inhibit the activity of photosynthetic enzymes, and disturb source‐sink transport of sugars; all these factors have an impact on photosynthetic rates. For many plants, a decrease in chlorophyll levels under K deficiency has been reported. Interestingly, when K‐starved red beet plants were supplied with nontoxic levels of Na (which can replace K in its osmotic role), chlorophyll levels were still lower than in K‐suYcient plants, but no diVerence in photosynthetic rates was observed (Subbarao et al., 2000). Inhibition of phloem transport in K‐starved plants has been suggested as the reason for low starch content in storage organs such as potato tubers (Marschner, 1995). However, a study in alfalfa roots found that other sugars accumulate in roots during K deficiency, and the authors suggest that reduced starch synthesis in these plants is a result of direct K dependency of the starch synthase enzyme rather than a lack of available sugars (Volenec et al., 1996). K deficiency also has an impact on nitrogen metabolism. Low levels of total N were measured in K‐starved alfalfa roots, together with low concentrations of soluble protein and vegetative storage proteins (Volenec et al., 1996). This could be due to K dependency of N‐metabolising enzymes or to K dependency of nitrate and amino acid transport. Surprisingly, interactions between N and K, although well established in the field, have not yet been studied at the molecular level. In contrast to well‐described K deficiency symptoms, acclimation responses of K‐starved plants are less well documented. An increase in root surface area to increase the nutrient uptake potential, which is a typical response to P deficiency, has not been reported for K‐starved Arabidopsis plants. However, a recent study involving a number of crop species showed an increase in root surface areas under low K, which was solely due to an increase in root hair length (Høgh‐Jensen and Pedersen, 2003). Root hair growth was correlated positively with K accumulation and was more pronounced in rape and cereals (rye, ryegrass, and barley) than in legumes (lucerne, red clover, and pea). The question whether this response is lacking in Arabidopsis or whether it can only be observed in specific conditions is important, as root hair mutants could provide a useful means to identify K receptors and signalling pathways.

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An increase of the diamine putrescine is a well‐established feature of K‐ deficient plants (Boucherau et al., 1999; Richards and Coleman, 1952). The exact role of putrescine in K stress acclimation remains to be elucidated, but it has been suggested that polyamines act as metabolic buVers by binding to charged macromolecules during stress (Altman and Levin, 1993). Polyamines also regulate ion channels and, therefore, could be involved in K homeostasis. A clear distinction between detrimental stress symptoms and stress acclimation responses is diYcult and will only be achieved through the identification and characterisation of mutants. B. TRANSCRIPTIONAL RESPONSES TO K

1. Responses linked to ion transport Several microarray studies have been carried out with the aim to identify transcriptional targets of varying external K supply. Maathuis et al. (2003) employed a custom‐designed Arabidopsis membrane transporter microarray (AMT chip) carrying 50‐mer probes for some 1000 known and putative membrane transporter genes to identify transcriptional responses of Arabidopsis roots during plant acclimation to low K, low Ca, and increased NaCl. In contrast to Ca and salt stress, which generated diVerential expression in a large number of genes, K starvation led to only a small number of genes showing diVerential expression between starved and resupplied plants. Even more striking was the lack of transcript changes for genes encoding known and putative K transporters. Because the study was carried out on mature plants, which had been grown in K‐suYcient medium for several weeks prior to the experiments (lasting 3 to 96 h), the lack of transcriptional response could be explained by the fact that the plants had accumulated suYcient K to complete their life cycle, without the need for further uptake. Nevertheless, K redistribution between diVerent tissues must have taken place in these plants as a small but significant reduction in total shoot K levels was observed. It can be concluded that regulation of K transporters for tissue K homeostasis occurs mostly at the post-translational level, a notion that is supported by other studies that observed transcriptional regulation of K channels by several plant hormones and stresses but not in response to K starvation (Pilot et al., 2003). The only known K channel that shows a significant transcriptional response to K starvation and resupply is SKOR1, an outward rectifying K channel, which is localised in the xylem parenchyma and thought to be involved in the delivery of K to the shoot (Gaymard et al., 1998; Pilot et al., 2003). The observed downregulation of SKOR1 during K starvation might be a means of retaining K in the roots under these conditions (Maathuis et al., 2003). Amongst the other

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transporters, vacuolar proton pumps and aquaporins were transcriptionally regulated by K stress, but they also responded to Ca and NaCl stress, indicating a general role in ion homeostasis. A downregulation of both the V‐type ATPase and the pyrophosphates in response to decreasing supply of K and Ca suggests that the proton gradient across the tonoplast is an important parameter in regulating ion uptake and release from the vacuole during fluctuating K and Ca supply, thus supporting intracellular homeostasis of these ions. K starvation also induced the downregulation of several genes encoding water channels (aquaporins). This response also occurred during Ca starvation, and because neither of the two treatments created a change in external water potential, it is likely that the observed regulation of aquaporins does not reflect an osmotic adaptation. The authors discussed the possibility that a change in transmembrane water permeability might change the ratio of apoplastic to symplastic ion flow, which could be important for root‐shoot allocation of K and Ca (Maathuis et al., 2003). A weak transcriptional response to K starvation in mature Arabidopsis plants was also reported by Gierth et al. (2005), who used AVymetrix full genome arrays to monitor transcript levels in roots after plant exposure to low K for 6 h to 7 days. By contrast, a study by Armengaud et al. (2004) with spotted full genome arrays based on the Qiagen probe set identified a large number of K‐regulated genes. Several diVerences between this and the other studies might explain the discrepancy. First, Armengaud and colleagues (2004) used much younger plants for their experiments. Not only did these plants require much higher rates of K uptake for extensive growth, but they were also grown from germination in low K conditions and therefore had no opportunity to build up significant K stores. Second, the identification of K‐regulated genes by Armengaud et al. (2004) was not based solely on starvation treatments but included resupply of K after long‐term starvation. This experimental design allowed the scientists to monitor rapid responses of K‐depleted plants to external K. Third, data analysis in the Armengaud study employed a rank‐based procedure (Breitling et al., 2004), which is better suited to identify subtle but significant changes in transcript levels than fold‐change cutoVs applied in the other studies. Nevertheless, Armengaud et al. (2004) also found a general lack of transcriptional responses of K transporters to external K. Only HAK5, a member of the KUP/HAK family of K transporters, was consistently found to be upregulated in roots of K‐starved plants both on microarrays (Armengaud et al., 2004; Gierth et al., 2005; Hampton et al., 2004) and in real‐time polymerase chain reaction analysis (Ahn et al., 2004). Not only does this point to an important role of HAK5 in high‐aYnity K uptake (Gierth et al., 2005), but it also identifies HAK5 as a useful marker to study K sensing at the root

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level (Shin et al., 2004). KUP3, another member of the KUP/HAK gene family, had previously reported to be induced by K starvation in roots of Arabidopsis seedlings (Kim et al., 1998), but this response was not detected in other studies (Gierth et al., 2005; Maathuis et al., 2003). By contrast, Hampton et al. (2004) reported downregulation of KUP3 in shoots of K‐starved plants. They also found K‐dependent expression of several genes encoding putative glutamate receptors (GLR1.2, GLR 1.3) and cyclic‐ nucleotide gated channels (CNGC1, CNGC13). It appears that although a few K channels and transporters respond transcriptionally to changes in the external K supply, their response is dependent on the exact growth conditions and the plant developmental stage. Interestingly, homologues of KUP3 and HAK5 were also found to be downregulated in tomato roots after the addition of nitrate to the growth medium (Wang et al., 2000). Conversely, Armengaud et al. (2004) reported that a group of three genes encoding NRT2‐type nitrate transporters was downregulated during K starvation and upregulated quickly upon K resupply. Interestingly, this response was accompanied by the transcriptional regulation of several putative amino acid and peptide transporters, as well as several genes encoding N assimilatory enzymes. A similar interplay is found for ammonium and K. On the one hand, ammonium is known to aVect K uptake (Santa‐Maria et al., 2000; Spalding et al., 1999). On the other hand, Maathuis et al. (2003) found that ammonium transporters of the AMT family were diVerentially expressed in roots of K‐deplete and K‐replete plants. These findings provide a starting point for molecular studies into K–N interactions and suggest that N metabolites might be involved in K stress signalling. Two putative Ca transporters are regulated by external K: ACA1, a root plasma membrane Ca pump, and CAX3, a putative vacuolar cation/H antiporter (Armengaud et al., 2004). Regulation of these transporters links K stress to Ca homeostasis and could be indicative of a role for Ca in replacing K as an osmoticum. Several Ca‐regulated genes were diVerentially expressed in K‐treated Arabidopsis seedlings such as calmodulins and Ca‐dependent protein kinases, which points to the possibility that Ca signalling is involved in K sensing (see later). 2. Responses in nontransporter genes In shoots of Arabidopsis seedlings, a large number of genes related to the plant hormone jasmonic acid (JA) respond to K starvation and K resupply (Armengaud et al., 2004). These include enzymes involved in JA biosynthesis, such as lipoxygenase, allene oxide synthase, and allene oxide cyclase, as well as downstream targets of JA, such as vegetative storage proteins (VSPs),

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and proteins involved in wounding and pathogen defense (e.g., plant defensins, PDFs and polygalacturonase-inhibiting proteins, PGIPs). Interestingly, very few genes related to salicylic acid, another hormone involved in pathogen defence, were found to be regulated by K. Studies in our laboratory (P. Armengaud and A. Amtmann, unpublished results) confirmed not only an increase of JA as well as other oxylipins during K stress, but also that many of the transcriptional responses to K were absent in the JA signalling mutant coi1. Thus, JA appears to take a prominent role in mediating leaf responses to K stress. Whether these responses lead to stress acclimation by assisting the plant in nutrient storage and remobilisation or have a role in protecting K‐starved plants against increased pathogen attack remains to be elucidated. Another important class of K‐regulated genes identified by Armengaud et al. (2004) comprises genes encoding cell wall proteins. Many cell wall proteins, such as extensins and xyloglucan glucosyltransferases, were upregulated quickly upon K resupply. This indicates a rapid adjustment of cell wall properties prior to the reinstatement of K‐driven cell extension and growth. Signal pathways involved in this response can now be studied in more detail using the identified K‐regulated cell wall genes as molecular markers. Cell wall localised processes could also play a role in nutrient stress perception. For example, several genes for cell wall arabinogalactan proteins (AGPs) responded to changes in external K supply. As for animal cells, an involvement of AGPs in signal transduction has been proposed for plants (Schultz et al., 1998). Peroxidases also featured strongly among K‐regulated transcripts (Armengaud et al., 2004; Shin and Schachtman, 2004). One reason for this might be their involvement in growth‐related cell wall responses (e.g., oxidative cross‐linkage of cell wall components). Another reason for transcriptional regulation of peroxidases during K stress might be related to their function in ROS detoxification. In fact, H2O2 was identified as an important signal in K stress perception (Shin and Schachtman, 2004; see later). In addition to the K‐dependent functional gene classes described earlier, many individual K‐regulated genes were found in the microarray studies cited earlier. These included several transcription factors of diverse gene families (e.g., B3, WRKY, bZIP, scarecrow‐like, C3H4‐RING finger, AP2) and various protein kinases and phosphatases (serine/threonine PK, CIPK, PP2C). Furthermore, K stress aVected the expression of many crucial enzymes of the primary metabolism, such as enzymes for N, S, and P assimilation (e.g., glutamate dehydrogenase, ATP sulfurylase, asparagine synthase, 6P‐gluconolactonase), pyruvate synthesis (PEP carboxylase, PEP carboxykinase, malic enzyme), and sugar metabolism (glucose‐6‐phophate‐dehydrogenase, starch

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synthase), as well as secondary metabolism (e.g., members of the FAD‐linked oxidoreductase family). These changes reflect both known and unknown metabolic disturbances in K‐starved plants and also suggest that extensive metabolic reprogramming is underway in K‐starved plants. Figure 1 shows an attempt to summarise the physiological, metabolic, and signalling events reflected in microarray data. This diagram [an updated version of a similar figure in Armengaud et al. (2004)] includes new findings from JA signalling mutants and metabolic profiling. For example, K‐starvation induction of the arginine decarboxylase gene (ADC2), although reported to be JA dependent (Perez‐Amador et al., 2002), was still found in the JA signalling mutant coi1 (P. Armengaud and A. Amtmann, unpublished results), and polyamine production is therefore no longer placed downstream of JA. Furthermore, biochemical studies suggest that the regulation of NRT2 nitrate transporter genes during K stress is downstream of a K eVect on N assimilation rather than vice versa (see later). It should be pointed out that although microarray data provide a useful framework to identify the molecular processes underlying plant responses to K stress, they do not reveal the reason for the observed change in transcript. The question whether a transcript is regulated because certain upstream processes are disturbed or because it is part of an acclimation response can only be answered by detailed studies at the single gene level. C. K PERCEPTION AND CELLULAR SIGNALLING EVENTS

1. Membrane potential and cytoplasmic Ca The first point of contact between the plant and its soil environment is the root apoplast. It is generally assumed that the root apoplast reflects the soil K concentration, but depending on the availability of exchangeable K in the soil and plant K uptake rates, K gradients between the site of uptake (root cell plasma membrane) and the bulk soil solution might develop when K supply is low. Conversely, in conditions of high K supply, K might accumulate outside the Casparian strip or in the symplast of the xylem parenchyma if transport to the shoot is slow. Apoplastic K concentration and membrane voltage of root cells are the most likely primary stimuli during K stress (see later), whereas cytoplasmic K concentrations appear to remain constant under varying external K (Walker et al., 1996). The two stimuli are connected, as the electrical properties of the plasma membrane are strongly dependent on external K. Potassium channels together with the proton ATPase dominate the electric conductance of the plasma membrane and, therefore, their relative conductance determines the membrane potential, which in turn determines the driving force for K movement. Voltage‐dependent K

Fig. 1. Overview of K stress responses reflected in gene expression data. Putative components of K deficiency and adaptive responses are shown in boxes. Connecting lines are based on K‐responsive genes identified by Armengaud et al. (2004) (shown in italics) and other published information (see Armengaud et al., 2004). Solid arrows indicate stimulation; dashed lines indicate inhibition. Known K deficiency symptoms are shown in white boxes. Putative components of signaling events are indicated in dark grey. Lighter grey shading marks diVerent JA‐dependent processes potentially leading to adaptive nutrient management and defense responses. For further discussion, see text.

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channels are the best established ‘‘K receptors’’ as they respond to both voltage and external K with changes in their gating (Blatt, 1988; Blatt and Gradmann, 1997; Maathuis and Sanders, 1997; Schroeder et al., 1994). In the simplest model of a membrane‐delimited regulatory loop, K channels act both as receptors and as eVectors. For example, some inward rectifying channels shift their activation potential with the K equilibrium potential (EK), thus preventing K loss under low external Kþ (Maathuis et al., 1997). There is also evidence that proton ATPase activity is stimulated directly by external K (Briskin and Poole, 1983; Hall and Williams, 1991). This leads to a more complex but still a plasma membrane‐delimited model of a regulatory loop, which involves the plasma membrane proton pump as a second receptor (Amtmann et al., 1999). Circumstantial evidence suggests that external pH could be an important parameter in this model: (1) Measurements of apoplastic pH after application of fusicoccin (Amtmann et al., 1999; Felle, 1998) showed that the cell wall pH is indeed clamped by proton pump activity. (2) Apoplastic pH regulates root K channels with a Km that is in the physiological range of cell wall pH (Amtmann et al., 1999; Zimmermann et al., 1998). Figure 2 shows a summary of receptors (membrane transporters) and system parameters (K concentration, voltage, and pH) in this model. The final output of such a loop, in terms of transmembrane K flux in response to a change in external K, is diYcult to predict, as

Fig. 2. Model of early signalling events in response to Kþ stress. The simplest scenario (1) involves voltage‐dependent plasma membrane K channels and the membrane potential ( ). An extended regulatory loop (2) includes the plasma membrane Hþ‐ATPase and external pH. A further extension (3) considers the eVect of membrane potential changes on plasma membrane Ca2þ channels, a change in cytoplasmic Ca2þ concentration, and the eVect of the latter on vacuolar K channels as well as gene expression.

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many of the caused changes are counteractive. On the one hand, a negative shift in EK in low external K concentrations will hyperpolarise the membrane and lead to channel activation. On the other hand, inactivation of the pump in low K will lead to membrane depolarisation, thus decreasing channel open probability, and this eVect will be exacerbated by an increase in external pH. Exact quantification of the eVect of a change in external K on K fluxes requires knowledge of the membrane potential, apoplastic pH, and the regulatory profiles of K channels and pumps in the specific membrane and cell type. Although membrane‐delimited regulatory systems can contribute to cellular K homeostasis, they are not suYcient to explain many of the observed downstream eVects of changes in K supply (e.g., changes in gene expression). A simple extension of the aforementioned model considers processes that will respond to changes in the membrane potential. The best‐known example for such a process is the opening of voltage‐dependent Ca channels, which will cause Ca influx into the cell, raising the cytoplasmic Ca concentration (Grabov and Blatt, 1997; Trewavas, 2000). A K clamp of the membrane voltage can be used to induce intracellular Ca waves experimentally. Thus, Allen et al. (2001) showed that they could exploit the voltage dependence of hyperpolarisation‐activated Ca channels in guard cells and create controlled oscillations of cytoplasmic Ca by repeatedly changing between a depolarising buVer (100 mM KCl) and a hyperpolarising buVer (0.1 mM KCl). Because root cells contain both depolarisation‐ and hyperpolarisation‐ activated Ca channels (Miedema et al., 2001; Thion et al., 1998), a more complex Ca signature would be expected here in response to changes in external K. Furthermore, K fluctuations in nature will be much slower than the protocol used by Allen and colleagues (2001) and therefore changes in plasma membrane Ca fluxes might be counteracted easily by intracellular Ca homeostasis. Cytoplasmic Ca signals occur in response to many environmental stimuli (Scrase‐Field and Knight, 2003), but have not yet been reported in relation to K stress. It is important to note that although membrane depolarisation occurs in response to many diVerent ions (e.g., increase of external Naþ, ammonium, phosphate) membrane hyperpolarisation to very negative membrane voltages can only be achieved by decreasing external K. The large proportion of Ca‐regulated proteins (e.g., calmodulins, protein kinases) among K‐regulated transcripts strongly suggests some involvement of intracellular Ca in the plant’s response to K stress, but it remains to be elucidated whether their role is in Ca signalling or Ca homeostasis. Cytoplasmic Ca could link the external K stimulus not only to post-translational and post-transcriptional responses via Ca‐dependent kinases, phosphatases, and transcription factors, but also to an adjustment of tonoplast K fluxes

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for cellular K homeostasis (Fig. 2). Indeed, K‐permeable vacuolar channels (SV and VK) are activated by Ca and would release K into the cytoplasm in response to a rise of cytoplasmic Ca levels (Allen and Sanders, 1996, 1997). 2. Reactive oxygen species Reactive oxygen species such as H2O2 have been shown to be key regulators in a range of physiological processes, such as stomatal closure, hypersensitive response to pathogens, root gravitropism, and root cell elongation (Foreman et al., 2003; Laloi et al., 2004). It has been observed that K‐deficient plants produce H2O2 and that this response is required for some transcriptional responses to K starvation (Shin and Schachtman, 2004). Thus, inhibition of ROS production suppresses the transcriptional response of two peroxidases, HAK5 and the transcription factor WRKY9 to K deprivation. The transcription of these genes was no longer induced when diphenylene iodonium (DPI), an inhibitor of NADPH oxidase, was applied. Their transcriptional response to low K was absent in the Arabidopsis rdh2 mutant, which is mutated in one of the catalytic subunits of the NADPH oxidase. Application of H2O2 to K‐replete plants was in some but not all cases suYcient to induce gene expression (Shin and Schachtman, 2004). Interestingly, the addition of H2O2 to a growth medium containing millimolar K activated a high‐aYnity K uptake component, which was otherwise only apparent in growth medium containing micromolar external K. Because H2O2 application was not suYcient to induce HAK5 expression, this component is unlikely to be carried by HAK5. If ROS are an important signal in plant acclimation to low K, the question arises as to how this signal is created, which are downstream signalling events, and what is its physiological role. ROS are produced either in the apoplast by cell wall peroxidases and amine oxidases or as a by‐product of metabolic pathways localised in diVerent cellular compartments. Although the source of ROS production during K stress has not been identified, the observed transcriptional regulation of several cell wall peroxidases might be indicative for an apoplastic oxidative burst similar to the one caused by pathogens. The fact that many disease‐related genes were present in microarray data also points to the possibility that K stress shares ROS signalling pathways with defense responses. Downstream signalling events of ROS production in Arabidopsis include the MAPK cascade as well as redox‐state‐dependent kinases, phosphatases, and transcription factors (Laloi et al., 2004). ROS also activate Ca channels, a response that is essential for root hair growth (Foreman et al., 2003). Which, if any, of these events are also involved in responses to K stress remains to be studied. The fact that the ROS synthesis mutant rdh2 is characterised by short root hairs points to a role of ROS in increasing root

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hair length to enhance K uptake. As mentioned earlier, a root hair response to K deprivation was observed in several crop species (Høgh‐Jensen and Pedersen, 2003) but has not been reported for Arabidopsis. There is some evidence that the occurrence of a root hair response in Arabidopsis depends on ammonium being present in the growth medium (R. Gaxiola, personal communication). D. SYSTEMIC SIGNALLING OF PLANT K STATUS

1. Hormonal signals The notion of long‐distance signals to communicate K deficiency seems unnecessary, as no molecule moves faster around the plant than K itself. However, similar to the cellular situation where maintenance of constant K levels in the cytoplasm requires signals to communicate the external K concentration to intracellular compartments (e.g., vacuole, nucleus), eYcient K homeostasis at the tissue level requires systemic signals between K‐deplete and K‐replete tissues. Another reason why K is unlikely to report on plant K status is that K concentrations are generally in the millimolar range and, therefore, large absolute changes are required to achieve significant relative changes. This means that K sensors are either extremely sensitive or that the plant risks considerable depletion of K before any responses occur. Short‐ term K resupply experiments performed by Armengaud et al. (2004) on K‐ starved Arabidopsis seedlings demonstrated that within 6 h of K resupply many shoot genes responded, although no significant change of overall shoot K content was recorded at this stage. However, this does not exclude the possibility that selective shoot tissues already experienced a considerable change in K concentration. A detailed analysis of the dynamic and spatial pattern of tissue K concentrations in response to a change in external K is required to understand the nature of K communication at the whole plant level. Thus, certain cell types that experience depletion or resupply of K earlier than others could send signals to other tissues that are still suYcient/ depleted. In theory, two types of systemic signalling can be distinguished. The first type involves a sensor that records the apoplastic K concentration and translates it into a signal that moves between diVerent cellular compartments, cells, or tissues. The sensory machinery, which is likely to include membrane potential, H2O2 and Ca (see earlier discussion), can be located in root cells that are in direct contact with the soil‐apoplast continuum or in leaf cells that are particularly well or particularly poorly supplied with K (e.g., xylem parenchyma and epidermis respectively, Karley et al., 2000). Abscisic acid (as free ABA or ABA conjugates, Sauter et al., 2005) is a typical root/ shoot signal for osmotic stress (Shinozaki and Yamaguchi‐Shinozaki, 2000),

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but available microarray data do not indicate the occurrence of an ABA signal during K stress, i.e., known ABA reporter genes such as COR78 and KIN2 were not aVected by a change in K supply (Armengaud et al., 2004). Microarray analysis did, however, suggest changes in auxin (Armengaud et al., 2004) and ethylene (Shin and Schachtman, 2004) in response to K stress. Significant ethylene production by Arabidopsis seedlings was detected within 6 h after K removal. Further studies are required to prove an involvement of hormonal long‐distance signals in plant K stress signalling and to identify upstream and downstream events. 2. Metabolic signals A second type of systemic signal could originate from the breakdown of cellular homeostasis and subsequent K depletion in specific cell types or tissues. It has been shown that under salt stress, cytoplasmic K levels in leaf epidermal cells can be as low as 15 mM, whereas cytoplasmic K levels in mesophyll cells are maintained around 70 mM (Cuin et al., 2003). This finding not only confirms that plants prioritise tissue allocation of K, it also shows that in nonprioritised tissues, cytoplasmic K levels can drop to values that are considerably lower than the K optimum for protein stability and enzyme activity (Wyn Jones and Pollard, 1983). Resulting changes in protein and metabolite levels could inform K‐replete tissues of the whole plant K status. Most enzymes require K (50–150 mM) for optimal function in vitro. The activity of some 60 enzymes has been shown to depend on K (Wyn Jones and Pollard, 1983). Many of these enzymes are involved in sugar and nitrogen metabolism (e.g., starch synthase, asparaginase). Pyruvate kinase (PK) was one of the first enzymes for which K dependency was discovered (Kachmar and Boyer, 1953), and the kinetic and structural properties related to K activation have since been studied in detail. Although K‐binding sites in PK are well conserved among eukaryotic enzymes, they are absent in some bacterial PKs that do not require K for activation. Replacement of a Glu117 residue in close vicinity to the K‐binding site by lysine renders the rabbit muscle PK K independent (Laughlin and Reed, 1997), thus providing evidence that genetic engineering could be used as a means to decrease K sensitivity of this enzyme. The K dependency of eukaryotic PK activity is very steep in the physiological range of cellular K concentrations (Km ¼ 14 mM, Laughlin and Reed, 1997). In plants, PK activity has been used as an indicator for cation balance in cucumber leaves (Ruiz et al., 1999). The microarray study by Armengaud et al. (2004) showed that several enzymes in the PK pathway are regulated during K stress (Fig. 3); in particular, upregulation of two genes for malic enzyme, which catalyses an alternative pathway for pyruvate synthesis, was observed. This is likely to be a direct response to decreased pyruvate levels

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in K‐depleted tissue. Because PK is a central regulator of C/N metabolism (Smith et al., 2000a), many of the observed changes in transcripts and metabolite levels under K deficiency might lie downstream of a decrease in PK activity. Similar to PK, any K‐dependent enzyme could theoretically function as a ‘‘K sensor’’ with its products and substrates acting as signals. However, it is important to remember that such a ‘‘signal pathway’’ would require a change of K in the cytoplasm or other metabolically active cellular compartment. Only dynamic and spatial fine mapping of K levels, transcripts, and metabolites in diVerent tissues during K stress will answer the question of whether the observed downstream responses are limited to K‐depleted cells or whether K‐ dependent enzymes are involved in systemic signalling between K‐deplete and K‐replete tissues. A recent analysis of amino acid levels showed that K‐starved Arabidopsis plants have increased levels of glutamine and decreased levels of glutamate (P. Armengaud and A. Amtmann, unpublished results). This finding suggests that some enzymes required for N assimilation are K dependent. Both amino acids are highly mobile between cells and tissues and are likely to act as signals for membrane transporters. Glutamine has been shown to repress the nitrate transporter AtNRT2.1 transcriptionally (Nazoa et al., 2003), and it is likely that the downregulation of NRT2 genes under K deficiency observed by Armengaud et al. (2004) is a consequence of increased glutamine levels. Glutamate might directly regulate ion channels of the putative glutamate receptor family. This large gene family in Arabidopsis (20 members) is poorly characterised in plants (Davenport 2002; Kang and Turano, 2003), but animal glutamate receptors transport a range of cations, including K, Na, and Ca. The possibility that glutamate aVects cation currents was supported by the observation that glutamate applied to root cells causes a large membrane depolarisation and a change of cytoplasmic Ca (Dennison and Spalding, 2001). Therefore, glutamate might play a role both as a cellular signal in K‐depleted cells and as a long‐distance signal informing K‐replete tissues of whole plant K status.

III. PHOSPHORUS A. PHOSPHORUS NUTRITION OF PLANTS

Phosphorus constitutes 0.1 to 1.4% of the dry matter of a typical plant (Broadley et al., 2004). It is an essential element, which is present primarily in small metabolites, nucleic acids, and phospholipids (Marschner, 1995). However, P is also required for diverse homeostatic and signal transduction

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cascades, as the activities of many proteins are regulated by phosphorylation/dephosphorylation reactions. Plant roots acquire P from the soil solution as phosphate (Pi, H2PO4 ). The concentration of Pi in the soil solution is low (2 to 10 M), which limits its diVusion to the root system and may result in Pi depletion in the rhizosphere of a rapidly growing plant. Thus, although soil P concentrations are often high, little Pi may be available for uptake by plants. For this reason, plants have evolved many strategies to increase their ability to acquire P from the soil and to cope with periods of low Pi availability (Hammond et al., 2004a; Raghothama, 2005; Vance et al., 2003). Plants that lack P increase the eVective surface area of their root systems by increasing carbohydrate partitioning to the root, accelerating the

Fig. 3. Hypothetical signalling cascades regulating transcription during P starvation. Several cell autonomous and systemic signalling cascades may regulate transcription in P‐starved plants. (1) Some immediate transcriptional changes in roots respond indiscriminately to changes in the ionic environment. These include the upregulation of genes involved in plant defence responses. (2) Some immediate transcriptional changes respond directly to Pi in the rhizosphere and result in an increase in the density and elongation of root hairs. The production of ethylene is implicated in this process. (3) Some immediate transcriptional changes in roots respond specifically to Pi withdrawal. These include the (weak) upregulation of PHR1, which encodes a MYB‐CC transcription factor whose activity might be regulated posttranscriptionally. The PHR1 transcriptional cascade upregulates genes encoding other transcription factors, riboregulators, protein kinases, Pi transporters, RNases, phosphatases, and metabolic enzymes through the P1BS cis element. (4) Reduced P transport to the shoot results rapidly in a decrease in shoot P concentration. This inhibits shoot growth before root growth is aVected and leads indirectly to an increase in the root:shoot ratio. (5) Reduced shoot Pi supply aVects [Pi]cyt and primary metabolism, both through allosteric interactions and transcriptional changes, leading to an increase in the concentrations of starch, sucrose, and organic acids in leaves. (7) Increased leaf sucrose concentrations result in (a) an increase in sucrose transport to the root, through the parallel upregulation of sucrose transporters, (b) a reduction of photosynthesis, through downregulation of many photosystem subunits and small subunits of RuBisCo, and (c) the production of anthocyanins through a TTG1‐TT8/EGL3‐PAP1/PAP2‐dependent cascade. (7) A decrease in shoot P upregulates the expression of genes encoding enzymes involved in the synthesis of sulpholipids and galactolipids, possibly through the P1BS cis element. (8) An increase in sucrose transported in the phloem to the root (a) promotes root growth and results in an increase in the root:shoot ratio and (b) acts as a systemic signal to modulate the expression of the AtPHR1 transcriptional cascade upregulating genes encoding riboregulators, Pi transporters, RNases, phosphatases, and metabolic enzymes. It also regulates the expression of other transport proteins. (9) An increased translocation of carbohydrates and organic acids in the phloem stimulates the release of organic acids from the root. (10) A reduction in cytokinin and altered auxin transport within the root initiates and promotes lateral root elongation. The location of lateral root elongation is determined by P availability in the rhizosphere, which is perceived by the root tip.

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initiation and growth of lateral (Chevalier et al., 2003; Forde and Lorenzo, 2001; Lynch and Brown, 2001; Malamy, 2005) or cluster roots (Dinkelaker et al., 1995), and by increasing the number and length of root hairs [Fig. 3, processes (2), (8), and (10); Bates and Lynch 1996; Forde and Lorenzo, 2001; He et al., 2005; Jungk, 2001]. They also form more associations with mycorrhizal fungi (Karandashov and Bucher, 2005). They increase the phosphate influx capacity of their root cells severalfold (Lee, 1993; Rausch and Bucher, 2002; Smith et al., 2003) and they secrete protons, enzymes, and organic acids into the rhizosphere [Fig. 3, processes (3), (8), and (9)] to release Pi from organic and inorganic sources in the soil (Brinch‐Pedersen et al., 2002; Dinkelaker et al., 1995; Lo´ pez‐Bucio et al., 2000; Miller et al., 2001; Narang et al., 2000). In parallel, diverse metabolic changes occur in P‐starved plants, enabling them to utilise P more eYciently. It is noteworthy that small metabolites, nucleic acids, and phospholipids contribute about equally to leaf P content. Thus, metabolism adjusts to P starvation by employing reactions that do not require Pi or adenylates (Hammond et al., 2004a; Plaxton and Carswell, 1999; Vance et al., 2003), by inducing intracellular phosphatases and nucleases that remobilise P from cellular metabolites and nucleic acids (Bariola et al., 1994; Brinch‐Pedersen et al., 2002; del Pozo et al., 1999), and by replacing phospholipids in thylakoid and extraplastidic membranes by galactolipids and sulpholipids [Fig. 3, process (7); Andersson et al., 2003; Essigmann et al., 1998; Ha¨ rtel et al., 2000; Yu et al., 2002]. Through these responses, plants acclimate to periods of P starvation. However, one unfortunate consequence of P starvation is the accumulation of sucrose and starch in leaves [Fig. 3, processes (5) and (6)]. The accumulation of sucrose reduces the expression of many genes involved in photosynthesis and eventually leads to a decline in photosynthetic performance [Fig. 3, process (6); Martin et al., 2002; Paul and Pellny, 2003; Rook and Bevan, 2003]. The characteristic accumulation of anthocyanins in response to P deficiency is thought to protect nucleic acids from UV damage and chloroplasts from the photoinhibitory damage caused by P‐limited photosynthesis [Fig. 3, process (6); Hoch et al., 2001]. In order to respond appropriately to P starvation, a plant must first perceive a lack of P and then generate signalling cascades to initiate and coordinate its acclimatory responses (Franco‐Zorrilla et al., 2004; Hammond et al., 2004a; Ticconi and Abel, 2004; Vance et al., 2003). It is assumed that these acclimatory responses result from changes in gene expression, and significant progress towards understanding the control of transcriptional responses to P starvation has been achieved through the phenotypic characterisation of mutants and the judicious application of molecular biology and transcriptional profiling technologies.

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B. TRANSCRIPTIONAL RESPONSES TO P

The expression of thousands of genes is altered when plants have insuYcient P for growth (Al‐Ghazi et al., 2003; Hammond et al., 2003, 2004a,b, 2005; Misson et al., 2005; Uhde‐Stone et al., 2003; Wang et al., 2002; Wasaki et al., 2003; Wu et al., 2003). These phosphate starvation‐responsive (PSR) genes have been grouped into ‘‘early’’ genes that respond rapidly (within hours after P withdrawal), transiently, and often nonspecifically to P withdrawal and ‘‘late’’ genes that alter the morphology, physiology or metabolism of plants upon prolonged P starvation (Hammond et al., 2003, 2004a,b). The early transcriptional responses to P withdrawal include increased expression of genes encoding general stress‐related proteins, such as chitinases and peroxidases, that respond indiscriminately to many biotic and abiotic challenges (Hammond et al., 2003, 2004b; Misson et al., 2005; Wang et al., 2002). However, they also include altered expression of genes encoding various transcription factors (e.g., zinc finger proteins, HD‐ZIP, WRKY transcription factors, MYB‐CC transcription factors, bHLH DNA‐ binding proteins; Hammond et al., 2003, 2004b; Rubio et al., 2001; Todd et al., 2004; Wang et al., 2002; Wu et al., 2003), riboregulators (e.g., At4/IPS family; Burleigh and Harrison, 1999; Hou et al., 2005; Liu et al., 1997; Martı´n et al., 2000; Mu¨ ller et al., 2004; Shin et al., 2004; Wasaki et al., 2003), and other components of intracellular signalling cascades, such as protein kinases and protein phosphatases, that respond more specifically to P withdrawal (Hammond et al., 2003, 2004b; Wang et al., 2002; Wu et al., 2003). The late transcriptional responses to P withdrawal are primarily acclimatory responses. In general, they improve the acquisition of P from the soil and/or promote the eYcient use of P within the plant (Hammond et al., 2004a; Vance et al., 2003). In roots, the expression of genes encoding members of the Pht1 Pi transporter family (Al‐Ghazi et al., 2003; Rausch and Bucher, 2002; Smith et al., 2003; Wasaki et al., 2003), intracellular and apoplastic RNases (Ko¨ ck et al., 1995; Mu¨ ller et al., 2004; Wasaki et al., 2003), and phosphatases (Baldwin et al., 2001; Berger et al., 1995; del Pozo et al., 1999; Haran et al., 2000; Miller et al., 2001; Mu¨ ller et al., 2004; Petters et al., 2002; Stenzel et al., 2003; Wasaki et al., 2003) are increased gradually during P starvation. Genes encoding enzymes of the glycolytic pathway are upregulated, leading to the production of organic acids, as are genes encoding putative plasma membrane organic acid channel proteins (Uhde‐ Stone et al., 2003; Wasaki et al., 2003). Genes encoding proteins regulating or regulated by the activity of plant hormones (Al‐Ghazi et al., 2003; Hammond et al., 2004a; Misson et al., 2005; Uhde‐Stone et al., 2003; Wu

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et al., 2003) are also aVected by prolonged P starvation. Changes include the increased expression of auxin responsive genes, such as AIR1, AIR3, AIR9, AIR12, HRGP, and LRP1, that control lateral root development (Al‐Ghazi et al., 2003; Casson and Lindsey, 2003; Uhde‐Stone et al., 2003), the expression of several genes involved in ethylene biosynthesis (ACC oxidase, methioninesynthase and S‐adenosyl methionine synthetase) and signalling (EREB2), which could mediate the transcription of ethylene‐responsive genes (Rietz et al., 2004; Uhde‐Stone et al., 2003; Wu et al., 2003), and the expression of genes encoding cytokinin oxidases, which may be involved in the breakdown of cytokinin in roots, thereby releasing the negative control cytokinins have on root development (Hammond et al., 2004a; Uhde‐Stone et al., 2003). Interestingly, root systems of diVerent plant species, and diVerent genotypes of a particular species, vary considerably in the extent of their morphological change, and their sensitivity, to reduced P supply (Chevalier et al., 2003; Hodge, 2004; Lynch and Brown, 2001; Narang et al., 2000; Robinson, 1994). This may allow the genes influencing such traits to be identified through quantitative genetic analyses (Lynch and Brown, 2001; Vreugdenhil et al., 2005). In shoots, the expression of genes encoding proteins that have an impact on both primary and secondary metabolism is influenced by P starvation. Genes encoding many photosystem subunits and small subunits of RuBisCo are downregulated, and genes encoding PEP carboxylases, sucrose synthases, fructose‐1,6‐bisphosphatases, UDP‐glucose pyrophosphorylases, and sucrose transporters are upregulated in P‐deficient plants (Ciereszko et al., 2001; Hammond et al., 2004a; Paul and Pellny, 2003; Plaxton and Carswell, 1999; Pen˜ aloza et al., 2005; Toyota et al., 2003; Uhde‐Stone et al., 2003; Wu et al., 2003). Several of these changes reflect the necessity to bypass ATP‐ and Pi‐dependent enzymes when P is scarce and the changing metabolism required to generate energy and carbon skeletons during P deficiency (Plaxton and Carswell, 1999). Genes encoding enzymes involved in anthocyanin biosynthesis and genes encoding enzymes that remobilise P from cellular molecules, such as phosphatases, nucleases, and phosphodiesterases, are also upregulated in leaves of P‐deficient plants (Hammond et al., 2003, 2004a,b; Uhde‐Stone et al., 2003; Vance et al., 2003; Wu et al., 2003). In addition, the expression of genes encoding enzymes that increase the proportion of galactolipids and sulpholipids in thylakoid and extraplastidic membranes are also increased in P‐deficient plants (Essigmann et al., 1998; Hammond et al., 2003; Ha¨ rtel et al., 2000; Misson et al., 2005; Wasaki et al., 2003; Yu et al., 2002), which enables photosynthesis to be maintained despite a reduction in phospholipid content. It is thought that many of the late transcriptional changes are induced by increasing leaf sugar concentrations,

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as 22% of genes responding to P deficiency are also diVerentially regulated in shoots of an Arabidopsis mutant (pho3 ¼ suc2; Lloyd and Zakhleniuk, 2004) with elevated leaf sugar concentrations (J. P. Hammond, unpublished results). Interestingly, genes encoding protein kinases and phosphatases were significantly more represented amongst these genes, suggesting that they may be involved in fine‐tuning enzyme activities. It has been observed that PSR genes can be clustered into coregulated groups (regulons) that exhibit specific temporal and spatial expression patterns in response to unique sets of environmental and/or developmental stimuli in addition to P withdrawal. Thus, it has been suggested that P withdrawal elicits several diVerent regulatory cascades and that each subset of PSR genes might share a common regulatory cascade (Franco‐Zorrilla et al., 2004; Hammond et al., 2003, 2004a,b; Vance et al., 2003). The next section presents the temporal sequence, tissue specificity, and orchestration of transcriptional responses to increasing P deficiency in plants. C. PHOSPHORUS PERCEPTION AND CELLULAR SIGNALLING EVENTS

1. Membrane potential and cytoplasmic Ca When the ionic environment of a plant cell changes, the electrochemical gradients for ion movement across its plasma membrane also change. Thus, one of the immediate consequences of drastic changes in the ionic composition of the rhizosphere is altered ionic flux across the plasma membrane of root cells, which can cause changes in membrane potential (Mimura, 1999). Whether a change in the flux of a particular nutrient will aVect the membrane potential depends on the contribution of the related fluxes to the overall membrane conductance. Whereas K is a major determinant of the membrane potential, P fluxes under normal conditions are relatively small and will not make a large contribution to the membrane conductance. However, after prolonged starvation, resupply of P to the medium has been shown to cause fast and transient depolarisations, which increased with the length of prestarvation and were followed by long‐term hyperpolarisation (Dunlop and Gardiner, 1993). Several conclusions can be drawn from these findings: (1) Phosphate transport is coupled to the movement of a net positive charge (reflecting PO43 /nHþ cotransport), (2) P fluxes increase with increasing P deficiency (possibly due to transcriptional upregulation of high‐aYnity P transporters), and (3) long‐term P starvation results in decreased pump activity (possibly due to a rise in cytoplasmic pH or depletion of ATP stores). Thus removal of P is unlikely to have drastic eVects on the membrane potential, but long‐term starvation and resupply will have a strong impact on the membrane potential, which can be linked to a number

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of downstream responses as described earlier for K. Whether a cytosolic Ca2þ signal is caused by changes in external P remains to be determined, but such perturbations in cell membrane potential have the potential to alter Ca2þ influx and, thereby, cytosolic Ca2þ concentrations ([Ca2þ]cyt; White 2000; White and Broadley, 2003). This may account for some of the similarities between the ‘‘early’’ nonspecific transcriptional changes in response to P withdrawal and other nutritional, abiotic, and biotic challenges (Hammond et al., 2003). Pathogenesis‐related genes in particular are characteristically triggered by Ca2þ signals (White and Broadley, 2003). Nevertheless, in addition to the initial, nonspecific changes in gene expression, P‐specific responses are also initiated. 2. Transcription factors and promoter elements The transcriptional response of some genes in root cells to P withdrawal appears to be cell autonomous. The expression of these genes changes only a few hours after P withdrawal and, therefore, is unlikely to be regulated by a systemic signal (Hammond et al., 2003; Wang et al., 2002; Wu et al., 2003). They may also occur in roots of defoliated plants. During this period there is little change in cytoplasmic Pi concentration [Pi]cyt, which is maintained in the range 2 to 10 mM until P deficiency becomes severe (Lee et al., 1990; Mimura, 1999; Schachtman et al., 1998). Thus, any immediate changes in gene transcription observed in roots following P withdrawal are unlikely to be initiated by changes in [Pi]cyt. However, it is possible that they are initiated in response to changes in vacuolar or apoplastic Pi concentration or to changes in metabolism. The expression of several transcription factors changes rapidly in response to P stress. These include members of the WRKY, WD40, bHLH, MYB, HD‐ZIP, zinc finger, and At4/IPS families (Burleigh and Harrison, 1999; Hammond et al., 2003, 2004b; Hou et al., 2005; Liu et al., 1997; Martı´n et al., 2000; Misson et al., 2005; Mu¨ ller et al., 2004; Rubio et al., 2001; Shin et al., 2004; Todd et al., 2004; Wang et al., 2002; Wasaki et al., 2003; Wu et al., 2003). The MYB transcription factors are early components of the transcriptional cascades initiated specifically by P withdrawal. Members of the MYB‐CC subfamily appear to be essential to these transcriptional cascades (Rubio et al., 2001; Todd et al., 2004). These proteins contain a MYB DNA‐binding domain, a second domain predicted to form a coiled coil (CC) usually involved in protein–protein interactions, and a glutamate‐ rich C terminus found in transcriptional activators. There are at least 15 genes encoding members of the MYB‐CC subfamily in Arabidopsis (Fig. 4). These include AtPHR1 (At4g28610), AtPHR2 (At1g79430), AtNSR1 (At3g04030), and AtNSR2 (At5g18240). A suggested subfamily

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nomenclature of ‘‘MCC’’ (MYB‐CC) proteins is proposed here (Fig. 2). Transcription of several of these genes, including AtPHR1, AtPHR2, and AtMCC6, respond to P starvation (Hammond et al., 2005; Rubio et al., 2001; Todd et al., 2004; Wu et al., 2003). The AtPHR1, AtPHR2, and AtMCC6 proteins appear to be positive regulators of P starvation responses. The Arabidopsis phr1 mutant shows less of an increase in shoot/root ratio, reduced accumulation of anthocyanins, and lower expression of Pi‐responsive genes under P starvation than wild‐type plants (Rubio et al., 2001). The Arabidopsis AtPHR1 signalling pathway has been elucidated and appears to have been conserved during the evolution of plants. The AtPHR1 protein recognises the DNA sequence (GnATATnC), termed the P1BS element, which is present in the promoters of many ‘‘late’’ PSR genes (Franco‐Zorrilla et al., 2004; Hammond et al., 2004a; Hou et al., 2005; Misson et al., 2005; Rubio et al., 2001). In Arabidopsis, these include genes encoding transcription factors, protein kinases, Pi transporters, RNases, phosphatases, metabolic enzymes, and enzymes involved in the synthesis of sulpholipids and galactolipids (Franco‐Zorrilla et al., 2004; Rubio et al., 2001). The transcription of many of these genes is repressed in P‐starved plants by the application of phosphonate/phosphite (H2PO3 or HPO32 ), a nonmetabolised analog of Pi, implicating a Pi‐sensing mechanism in these transcriptional cascades (Ticconi et al., 2001, 2004; Varadarajan et al., 2002). Mutations in either PHR1 or the P1BS cis element reduce the expression of genes whose promoters contain the P1BS sequence during P starvation (Franco‐Zorrilla et al., 2004; Rubio et al., 2001; Schu¨ nmann et al., 2004). It is noteworthy that some of these genes encode components of biochemical signal transduction cascades and transcription factors, which may become the next amplification or selectivity step in the signalling cascade in response to P withdrawal. Curiously, however, the P1BS element is present in the promoters of 15 to 20% of Arabidopsis genes and is not preferentially contained within promoters of P‐regulated genes (Hammond et al., 2003). This suggests that the activity of AtPHR1 could be regulated post-transcriptionally in response to P withdrawal or that more than one cis element is required to eVect P‐specific transcriptional responses. It is noteworthy that promoters harbouring the P1BS element often also contain PHO‐like sequences, CACGTd and/or CdhGTGG (d ¼ G,T or A, h ¼ C,T or A), resembling a functional cis element present in the promoters of genes in the yeast PHO operon (Hammond et al., 2003, 2004a,b; Liu et al., 1997, 2005; Schu¨ nmann et al., 2004; Vance et al., 2003). However, although the PHO‐like sequence CACGTd was found to be present significantly more frequently in PSR genes

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(Hammond et al., 2003), there is no direct evidence to confirm its role in transcriptional regulation during P starvation (Schu¨ nmann et al., 2004). However, evidence shows that transcriptional repressors also regulate PSR genes, as mobility shift assays with promoter fragments from PSR genes detect DNA‐binding proteins in nuclear extracts from P‐replete plants that are absent in P‐starved plants (Mukatira et al., 2001). 3. Signals involved in root morphological adaptations Some changes in gene expression respond to the rhizosphere P concentration. These changes occur irrespective of whether another part of the root system is supplied with P. One physiological process responding directly to low rhizosphere P concentrations is the development of transfer cells with high Hþ‐ATPase activities in the rhizodermis of tomato roots (Schikora and Schmidt, 2002). Another physiological process responding to local Pi availability is the proliferation of root hairs in places with low rhizosphere P concentration (Bates and Lynch, 1996). Significantly, root hair development is unaVected in the Arabidopsis phr1 mutant (Rubio et al., 2001). It is thought that interactions between increasing concentrations of auxin and ethylene increase the abundance and length of root hairs. Auxin is thought to increase trichoblast cell file number, and ethylene is important for root hair formation (He et al., 2005; Zhang et al., 2003b). The expression of several genes involved in ethylene biosynthesis (ACC oxidase, methioninesynthase and S‐adenosyl methionine synthetase) is increased in plants lacking P (Uhde‐Stone et al., 2003; Wu et al., 2003). In Arabidopsis, AtTTG1 (a WD40 protein), in tandem with AtTTG2 (a WRKY transcription factor), is implicated in root hair initiation. The current model is that AtTTG1 interacts with specific bHLH and MYB proteins to form heteromeric complexes that control gene expression through the consensus bHLH‐binding site CAnnTG, which also resembles the PHO‐like binding site (Hammond et al., 2003). In the root epidermis, AtTTG1 forms a ternary complex with the bHLHs AtGL3 or AtEGL3 and the MYB AtWER to maintain the atrichoblast state, or AtCPC, to allow hair development (Bernhardt et al., 2003; Casson and Lindsey, 2003; Montiel et al., 2004; Zhang et al., 2003a). Fig. 4. (A) Phylogenetic tree of Arabidopsis MYB‐like transcription factors homologous to AtPHR1 that contain both a SHAQKYF myb‐like DNA‐binding domain and a domain predicted to form a coiled coil (CC) structure implicated in protein–protein interactions. Protein alignments were performed using the AlignX program in Vector NTI 9.0 (Invitrogen, Paisley, UK). A suggested subfamily nomenclature of ‘‘MCC’’ (MYB‐CC) proteins is proposed. (B) The predicted amino acid sequences of the conserved SHAQKYF myb‐like DNA‐binding domain and CC domains present in the 15 Arabidopsis MYB‐CC proteins. Conserved amino acid residues are marked with an asterisk.

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Although the expression of AtTTG1 appears to be unaVected by P starvation, the expression of several key bHLH and MYB proteins is influenced by P starvation (Wu et al., 2003). The elongation of root hairs relies on the activity of hyperpolarisation‐activated Ca2þ channels (HACCs), which elevate [Ca2þ]cyt at their apex (Foreman et al., 2003; White, 1998). This enables cell expansion to proceed through the fusion of membrane vesicles with the apical plasma membrane. The activity of HACCs is increased by reactive oxygen species (Foreman et al., 2003). Thus, root hairs might be longer where rhizosphere Pi concentrations are low because Ca2þ influx through HACCs is increased or prolonged in these regions, not only because rhizosphere Ca2þ concentrations are likely to be greater, but also because Pi deficiency induces oxidative stress (Hammond et al., 2003; Jungk 2001). It is also important to recognise that lateral roots form preferentially in patches of the soil with high Pi availability (Drew, 1975; Hodge, 2004; Robinson, 1994). This phenomenon, which is more intense in P‐starved plants, suggests that lateral rooting might be regulated by (at least) two P sensors. The first, which responds to plant P concentration, predisposes a root system to lateral root formation, and the second, which responds to rhizosphere Pi concentration, determines where lateral roots are formed. This is analogous to the proliferation of cluster roots in patches of high Pi availability only when the plant has a low P status (Dinkelaker et al., 1995). Evidence suggests that plant P status is perceived in the shoot and systemic signals predispose the root system to lateral root formation. By contrast, the root tip appears to sense Pi availability in the rhizosphere, as the proliferation of lateral roots in a P‐rich patch only occurs when the root tip grows through the patch (Drew, 1975; Forde and Lorenzo, 2001; Hodge, 2004; Linkohr et al., 2002; Robinson, 1994). D. SYSTEMIC SIGNALLING OF PLANT P STATUS

1. Shoot responses When P is unavailable to plant roots, Pi transport to the xylem is reduced immediately and the xylem Pi concentration declines (Jeschke et al., 1997; Mimura, 1999). This often leads to a decrease in shoot P concentration, and a reduction in shoot growth before root growth is aVected (Clarkson and Scattergood, 1982; Cogliatti and Clarkson, 1983). This results in an increase in the plant root:shoot ratio. Thus, P acts as its own root/shoot signal. The proteins responsible for loading Pi into the xylem are unknown. However, some insight into the regulation of P transport to the shoot has been obtained from the Arabidopsis pho1 mutant. This mutant exhibits reduced

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P transport to the shoot, and it has been suggested that PHO1, which is expressed in the root stele and upregulated weakly upon P starvation, encodes a membrane protein that functions in P sensing (Hamburger et al., 2002). Ten homologs of PHO1 in Arabidopsis may be involved in coordinating diverse P starvation responses in plants. All contain an SPX domain, which is implicated in P sensing in yeast and G‐protein interactions (Wang et al., 2004). The transcription of many genes responds rapidly to P starvation in the shoot. It has been argued that changes in the expression of these genes are unlikely to be initiated by changes in [Pi]cyt because [Pi]cyt might be maintained until P deficiency becomes severe. However, because the [Pi]cyt of photosynthetic cells decreases rapidly when leaves are illuminated (Mimura, 1999), it is not inconceivable that P starvation might aVect [Pi]cyt dynamics in these cells. Such changes in [Pi]cyt may result in immediate (kinetic) changes in metabolism as a consequence of perturbed metabolite concentrations (Plaxton and Carswell, 1999). In particular, it has been suggested that reduced [Pi]cyt might promote both starch and sucrose biosynthesis, either by releasing enzymes from allosteric inhibition or through transcriptional regulation of genes encoding key enzymes (Mu¨ ller et al., 2004; Plaxton and Carswell, 1999). These changes in cell biochemistry are unlikely to reflect simply a reduced C requirement for growth during P starvation because carbohydrate metabolism returns to unstressed levels after supplying P for 2 days to P‐starved plants that have not grown significantly (Mu¨ ller et al., 2004). One of the first physiological manifestations of P starvation in shoots is the accumulation of sucrose. This may result in (1) an increase in the amount of sucrose transported to the root, (2) a reduction of photosynthesis, and (3) the production of anthocyanins (Plaxton and Carswell, 1999; Ticconi and Abel, 2004). Many genes are coregulated in shoots by increased sucrose and P starvation. These include genes encoding many photosystem subunits and small subunits of RuBisCo, which are downregulated, and enzymes involved in the production of sucrose and starch, anthocyanin biosynthesis, and sucrose transporters, which are upregulated by both increased sucrose and P starvation (Ciereszko et al., 2001; Franco‐Zorrilla et al., 2005; Lloyd and Zakhleniuk, 2004; Toyota et al., 2003). It is possible, therefore, that the regulation of some genes responding to P starvation could be aVected indirectly through changes in leaf sucrose concentration. However, because many of these responses are repressed by phosphonate in P‐starved plants (Carswell et al., 1996, 1997), a Pi‐sensing mechanism is also implicated. An increase in the transcription of sucrose transporters, in combination with an increase in leaf sucrose concentration, could account for the increase in carbohydrate transport to the root and the consequent increase in the

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root:shoot ratio observed during P starvation. The reduction of photosynthesis in P‐deficient plants is probably a direct consequence of the accumulation of sugars, as many of the genes impacting on photosynthesis are repressed by sucrose (Martin et al., 2002; Paul and Pellny, 2003; Rook and Bevan, 2003), although P deficiency may partly mitigate the sugar repression of photosynthetic genes (Hurry et al., 2000). Similarly, the production of anthocyanins during P starvation could be initiated by an increase in sucrose concentration. An interaction among WD40, bHLH, and MYB proteins is thought to control flavonoid metabolism in many plants (Irani et al., 2003). In Arabidopsis, AtTTG1 and AtTTG2 are implicated in the control of anthocyanin biosynthesis in vegetative tissue (Johnson et al., 2002; Shirley et al., 1995; Walker et al., 1999). The current model is that AtTTG1 forms a ternary complex with the bHLHs AtTT8 or AtEGL3 and the MYB proteins AtPAP1 (AtMYB75) or AtPAP2 (AtMYB90) to control flavonoid biosynthesis in leaves (Baudry et al., 2004; Zhang et al., 2003a). As in roots, the expression of AtTTG1 appears to be unaVected by P starvation (Hammond et al., 2003, 2004b). However, the expression of AtPAP1 and AtPAP2 is upregulated not only by P starvation, and in the P‐deficient shoots of the pho1 mutant (J. P. Hammond, http://aVymetrix.arabidopsis. info/narrays/experimentpage.pl?experimentid¼102), but also by high sucrose concentrations (Lloyd and Zakhleniuk, 2004). 2. Shoot–root signals The transcription of many genes in roots reflects the shoot, not the root, P concentration. This is clearly observed when Pi is available to only a portion of the root system and gene expression is monitored both in roots supplied with Pi and those that are not. Evidence from such experiments suggests that systemic signals regulate the transcription of many genes involved in acclimation to P starvation in plant roots. Several systemic signals have been proposed that may initiate genetic responses to P starvation. These include reduced Pi translocation in the phloem (Drew and Saker, 1984; Jeschke et al., 1997; Mimura, 1999), increased sucrose translocation in the phloem (Liu et al., 2005), reduced cytokinin, and/or increased auxin translocation to the root (Hammond et al., 2004a; Hou et al., 2005; Martı´n et al., 2000; Nacry et al., 2005). Although the activity of the primary transcriptional regulator implicated in responses to P starvation, AtPHR1, is thought to respond rapidly to immediate environmental signals (Rubio et al., 2001), the expression of many genes that may be regulated through AtPHR1 appears to be also regulated by systemic signals. For example, members of the At4/IPS gene family are

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downregulated systemically when Pi is available to only a portion of the root system (Burleigh and Harrison, 1999; Hou et al., 2005; Liu et al., 1997). This is consistent with the elevated expression of At4 in roots of the Arabidopsis pho1 mutant, despite their high P status (Burleigh and Harrison, 1999). These observations suggest that a systemic signal controlled by shoot P status represses these genetic responses to P withdrawal. Another key physiological response to P starvation that may be regulated by both AtPHR1 and a systemic signal is Pi uptake by root cells. The ability of roots to take up Pi is correlated with their expression of genes encoding high‐aYnity, plasma membrane Pi transporters of the Pht1 subfamily (Dong et al., 1999; Shin et al., 2004; Smith et al., 2003). Transcripts from many members of the Pht1 subfamily increase rapidly in roots of P‐starved plants (Karandashov and Bucher, 2005; Karthikeyan et al., 2002; Mu¨ ller et al., 2004; Rausch and Bucher, 2002; Schu¨ nmann et al., 2004; Shin et al., 2004; Smith et al., 2003). When only a portion of the root system is supplied with Pi or when Pi is supplied by mycorrhizal associations, the transcription of Pht1 genes in root cells, and their Pi uptake, is not determined by their P concentration but by the P status of the shoot (Chiou et al., 2001; Drew and Saker, 1984; Rausch et al., 2001; Smith et al., 2003). This observation originally led researchers to postulate that Pi uptake was determined by Pi loading into the xylem and that this might be regulated by shoot P status (or shoot P demand) through the retranslocation of Pi in the phloem, which decreases during P starvation (Drew and Saker, 1984; Jeschke et al., 1997; Mimura, 1999). This interpretation is consistent with the increased expression of genes encoding Pi transporters and Pi uptake by roots of Zn‐deficient plants (Huang et al., 2000; Marschner and Cakmak, 1986) and the Arabidopsis pho2 mutant (Dong et al., 1998), which accumulate excessive Pi in the shoot and retranslocate less Pi from the shoot to the root. However, evidence shows that the expression of Pi transporters in Arabidopsis and lupin roots is upregulated during the light period and in response to the addition of sucrose to the nutrient solution bathing the roots (Lejay et al., 2003; Liu et al., 2005; Raghothama, 2005). This suggests that an increase in sucrose transport from the shoot to the root in response to shoot P starvation could regulate the expression of these genes. It might also explain why, when growth is limited by nitrogen or sulphur, Pi transporters fail to be induced by Pi deprivation (Smith et al., 1999). Curiously, upon resupplying Pi to roots, although their Pi uptake decreases rapidly, Pi may accumulate to toxic levels in leaves, suggesting a flaw in this regulatory mechanism (Adalsteinsson et al., 1994; Clarkson and Scattergood 1982; Cogliatti and Clarkson, 1983; Green et al., 1973).

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3. Metabolic signals Interestingly, a systemic sucrose signal has been implicated in the regulation of other genes responding to P starvation in roots. These include genes encoding transport proteins, such as AtAMT1.1, AtNrt1.1, LeNrt2, LeIRT1, and LaMATE (Al‐Ghazi et al., 2003; Lejay et al., 2003; Liu et al., 2005; Wang et al., 2002; Wu et al., 2003), and phosphatases, such as AtVSP, AtACP5, AtPAP12, and LaSAP1 (Baldwin et al., 2001; Berger et al., 1995; Li et al., 2002; Liu et al., 2005). Indeed, the Arabidopsis pho3 (¼suc2) mutant, which lacks a sucrose transporter that loads the phloem, does not secrete acid phosphatase in response to P starvation (Lloyd and Zakhleniuk, 2004). Interestingly, the promoters of many of these genes contain both the AtPHR1‐binding motif (GnATATnC) and the PHO‐like sequence (CACGTd, where d ¼ G/T) that is overrepresented in genes responding to P starvation and apparently functions as an enhancer for plant genes responding to sugars and biotic and abiotic stress (Hammond et al., 2004a,b; Liu et al., 2005). It has been speculated that proteins from the bZIP class of transcription factors might bind to the latter (Hammond et al., 2004a). Interestingly, sucrose represses the translation of several bZIP transcription factors through a highly conserved, translated, upstream open reading frame encoding a peptide of 25 to 42 amino acids in their 50 ‐untranslated region (Wiese et al., 2004). Five Arabidopsis bZIP genes harbour this sequence: AtbZIP1, AtbZIP2, AtbZIP11, AtbZIP44, and AtbZIP53. Interestingly, the expression of all these genes is aVected by P starvation and/or carbohydrate accumulation (J.P. Hammond, http://aVymetrix.arabidopsis.info/narrays/ experimentpage.pl?experimentid¼102; Lloyd and Zakhleniuk, 2004; Price et al., 2004; Wu et al., 2003). Promoter dissection of the soybean VspB gene identified two contiguous domains that mediated sucrose induction and phosphate inhibition (Tang et al., 2001). Intriguingly, the domain mediating phosphate inhibition contains a sequence (CATTAATTAG) that may bind HD‐ZIP proteins as transcriptional repressors and is found in the promoters of other genes responding to both sucrose accumulation and P starvation (Tang et al., 2001; Toyota et al., 2003). However, sucrose is not the only signal that has an impact on the expression of genes encoding At4/IPS riboregulators, Pi transporters, and acid phosphatases. The expression of all these genes decreases rapidly (within 30 min) in roots and shoots of P‐starved plants when resupplied with Pi. This apparently occurs before a change in carbohydrate concentration can be detected, but is consistent with an increase in tissue Pi concentration (Mu¨ ller et al., 2004). The repression of expression of genes encoding At4/IPS riboregulators, Pi transporters, and acid phosphatases in P‐starved plants by phosphonate has implicated a Pi sensor in their regulation (Ticconi et al., 2001, 2004; Varadarajan et al.,

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2002). However, because phosphonate can be translocated rapidly throughout the plant, the location of the Pi sensor is unknown (Guest and Grant, 1991). The AtPDR2 gene has also been implicated in this transcriptional cascade, as (1) changes in the expression of these genes are more sensitive to P starvation, and greater, in the pdr2 mutant and (2) pdr2 plants grown in the presence of phosphonate have a wild‐type phenotype (Ticconi et al., 2004). Nevertheless, it would be interesting to determine whether sucrose and starch concentrations accumulate in leaves of phosphonate‐treated, P‐starved plants. 4. Hormonal signals Cytokinin has been implicated in the control of transcriptional responses to P starvation and has also been suggested as a systemic signal. Cytokinin concentrations are lower in roots of P‐starved plants, and the exogenous application of cytokinins suppresses the expression of At4, AtIPS1, AtPT1, and AtACP5 in roots of P‐starved Arabidopsis (Martı´n et al., 2000) and OsIPS1 and OsIPS2 in roots of P‐starved rice (Hou et al., 2005). Because the AtCRE1 receptor protein has been implicated in regulating these responses in Arabidopsis, it is noteworthy that the expression of AtCRE1 is induced by cytokinins and downregulated by P starvation (Franco‐Zorrilla et al., 2002). However, cytokinin is unlikely to be the systemic signal itself because the addition of exogenous cytokinins to a portion of the root system cannot repress the transcription of PSR genes systemically (Franco‐Zorrilla et al., 2004). Exogenous cytokinins also repress lateral rooting in P‐starved plants (Franco‐Zorilla et al., 2002), and the phenotype of the Arabidopsis pho2 mutant, which has a longer primary root and fewer lateral roots than wild‐ type plants, indicates the influence of shoot P status on this trait (Chen et al., 2000; Williamson et al., 2001). Plants lacking P are also more sensitive to an auxin‐induced increase in lateral root number and density (Lo´ pez‐Bucio et al., 2002). Thus, it is thought that a decrease in cytokinin concentration releases a negative control on root development, initiation of lateral roots is promoted by increasing auxin concentrations, and interactions between increasing auxin and ethylene concentrations promote lateral root elongation in P‐starved plants (Franco‐Zorilla et al., 2004; Hammond et al., 2004a; Lo´ pez‐Bucio et al., 2005; Lynch and Brown, 2001; Malamy, 2005; Nacry et al., 2005; Ticconi and Abel, 2004; Vance et al., 2003). Further, it has been suggested that a reduction of primary root growth in P‐starved plants may initiate the development of lateral roots by altering auxin transport from the shoot through the root (Ticconi and Abel, 2004). It is clear that the expression of genes encoding proteins regulating or regulated by the activity of

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plant hormones changes appropriately in plants during P starvation (Al‐Ghazi et al., 2003; Hammond et al., 2004a; Rietz et al., 2004; Uhde‐ Stone et al., 2003; Wu et al., 2003), but little is known of their systemic regulation. However, sucrose does not appear to be the systemic signal because growing Arabidopsis plants on media containing sucrose had no eVect on the morphological responses of their root system to P starvation (Williamson et al., 2001) and when Arabidopsis plants were grown on media containing high sucrose:N ratios, lateral root initiation was inhibited almost completely (Malamy, 2005). In addition to changes in the concentrations of Pi, sucrose, and plant hormones in the phloem sap during P starvation, changes in the concentrations of other solutes are also apparent (Jeschke et al., 1997). To compensate for the decreased Pi concentration of the phloem sap, the concentrations of inorganic anions, such as chloride, amino acids, such as glutamate, and organic acids, such as malate, succinate, shikimate, and oxalate, increase in phloem sap during P starvation (Jeschke et al., 1997). The phloem sap also includes submillimolar concentrations of several organic P metabolites, such as nucleotides and hexose phosphates, whose response to P starvation is unknown. These solutes may act as systemic signals and/or contribute to acclimatory responses to P starvation. Interestingly, the presence of glutamate in the rhizosphere of the root tip inhibits primary root growth, but this eVect is not observed when glutamate is added to other parts of the root system (Filleur et al., 2005). In some situations the exudation of organic acids into the rhizosphere parallels the production of organic acids in the shoot. Because the exudation of organic acids by roots is of fundamental importance for releasing Pi from organic and inorganic sources in the soil, it is noteworthy that this may be controlled systemically through the regulation of shoot metabolism.

IV. CONCLUSIONS The ability to perceive and integrate information on nutrient availability in the soil is a fundamental prerequisite for plant survival. Enhancing our knowledge of the molecular processes involved in plant nutrient stress responses is an important contribution plant scientists can make to the development of sustainable agricultural practices. We have shown in this review that the combination of a wide range of experimental techniques has led to an impressive body of data, which provides a good foundation for future progress in this field. Transcriptional profiling of K‐ and P‐starved plants has created a wealth of novel information on plant molecular

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responses to nutrient stress and has provided us with a large number of potential molecular markers for K and P sensing in plants. Forward and reverse genetics studies are now required to identify signalling elements that lie upstream of individual marker genes and to characterise the physiological function of individual K‐ and P‐regulated genes in stress acclimation. In addition to research at the single gene level, future progress can be expected from further ‘‘omics’’ studies. One methodology that is likely to deliver exciting new insights into nutrient signalling is metabolic profiling, which not only facilitates the discovery of putative metabolite signals, but can also help us pin down primary enzymatic targets of K and P stress. To obtain a truly comprehensive picture of the signalling events and acclimation processes occurring under K and P deficiency, fine mapping of K and P levels, together with transcripts, proteins, and metabolites, in tissues and cellular compartments, as well as an increased dynamic resolution during stress, are required. Correlation of all these system parameters in time and space should allow us to outline a first systemic model of their complex causal relationships. Targeted manipulation of key elements of such a model can then be used to test the predicted links and refine the model.

ACKNOWLEDGMENTS Work by PJW and JPH was supported by the Department for Environment, Food and Rural AVairs, UK (Projects HH3501SFV and HH3504SPO). Research in the laboratory of AA and PA is supported by the Biotechnology and Biological Sciences Research Council.

REFERENCES Adalsteinsson, S., Schjørring, J. K. and Jense´ n, P. (1994). Regulation of phosphate influx in winter wheat: Root‐shoot phosphorus interactions. Journal of Plant Physiology 143, 681–686. Ahn, S. J., Shin, R. and Schachtman, D. P. (2004). Expression of KT/KUP genes in Arabidopsis and the role of root hairs in Kþ uptake. Plant Physiology 134, 1135–1145. Al‐Ghazi, Y., Muller, B., Pinloche, S., Tranbarger, T. G., Nacry, P., Rossignol, M., Tardieu, F. and Doumas, P. (2003). Temporal responses of Arabidopsis root architecture to phosphate starvation: Evidence for the involvement of auxin signalling. Plant, Cell and Environment 26, 1053–1066. Allen, G. J. and Sanders, D. (1996). Control of ionic currents in guard cell vacuoles by cytosolic and luminal calcium. Plant Journal 10, 1055–1069. Allen, G. J. and Sanders, D. (1997). Vacuolar ion channels of higher plants. Advances in Botanical Research 25, 217–252.

246

A. AMTMANN ET AL.

Allen, G. J., Chu, S. P., Harrington, C. L., Schumacher, K., HoVmann, T., Tang, Y. Y., Grill, E. and Schroeder, J. I. (2001). A defined range of guard cell calcium oscillation parameters encodes stomatal movements. Nature 411, 1053–1057. Altman, A. and Levin, N. (1993). Interactions of polyamines and nitrogen nutrition in plants. Physiologia Plantarum 89, 653–658. Amtmann, A., Jelitto, T. C. and Sanders, D. (1999). Kþ‐Selective inward‐rectifying channels and apoplastic pH in barley roots. Plant Physiology 120, 331–338. Amtmann, A., Armengaud, P. and Volkov, V. (2004). Potassium nutrition and salt stress. In ‘‘Membrane Transport in Plants’’ (M. R. Blatt, ed.), pp. 293–339. Blackwell, Oxford, UK. Andersson, M. X., Stridh, M. H., Larsson, K. E., Liljenberg, C. and Sandelius, A. S. (2003). Phosphate‐deficient oat replaces a major portion of the plasma membrane phospholipids with the galactolipid digalactosyldiacylglycerol. FEBS Letters 537, 128–132. Armengaud, P., Breitling, R. and Amtmann, A. (2004). The potassium‐dependent transcriptome of Arabidopsis reveals a prominent role of jasmonic acid in nutrient signaling. Plant Physiology 136, 2556–2576. Arnon, D. I. and Stout, P. R. (1939). The essentiality of certain elements in minute quantity for plants with special reference to copper. Plant Physiology 14, 371–375. Baldwin, J. C., Karthikeyan, A. S. and Raghothama, K. G. (2001). LEPS2, a phosphorus starvation‐induced novel acid phosphatase from tomato. Plant Physiology 125, 728–737. Bariola, P. A., Howard, C. J., Taylor, C. B., Verburg, M. T., Jaglan, V. D. and Green, P. J. (1994). The Arabidopsis ribonuclease gene RNS1 is tightly controlled in response to phosphate limitation. Plant Journal 6, 673–685. Bates, T. R. and Lynch, J. P. (1996). Stimulation of root hair elongation in Arabidopsis thaliana by low phosphorus availability. Plant, Cell and Environment 19, 529–538. Baudry, A., Heim, M. A., Dubreucq, B., Caboche, M., Weisshaar, B. and Lepiniec, L. (2004). TT2, TT8 and TTG1 synergistically specify the expression of BANYULS and proanthocyanidin biosynthesis in Arabidopsis thaliana. Plant Journal 39, 366–380. 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. Berger, S., Bell, E., Sadka, A. and Mullet, J. E. (1995). Arabidopsis thaliana Atvsp is homologous to soybean VspA and VspB, genes encoding vegetative storage protein acid phosphatases, and is regulated similarly by methyl jasmonate, wounding, sugars, light and phosphate. Plant Molecular Biology 27, 933–942. Blatt, M. R. (ed.) (2004). ‘‘Membrane Transport in Plants.’’ Blackwell, Oxford, UK. Blatt, M. R. (1988). Potassium‐dependent, bipolar gating of Kþ channels in guard cells. Journal Membrane Biology 102, 235–246. Blatt, M. R. and Gradmann, D. (1997). Kþ sensitive gating of the Kþ outward rectifier in Vicia guard cells. Journal Membrane Biology 158, 241–256. Boucherau, A., Aziz, A, Larher, F. and Martin‐Tanguy, J. (1999). Polyamines and environmental challenges: Recent development. Plant Science 140, 103–125. Breitling, R., Armengaud, P., Amtmann, A. and Herzyk, P. (2004). Rank products: A simple, yet powerful, new method to detect diVerentially regulated genes in replicated microarray experiments. FEBS Letters 573, 83–92.

NUTRIENT SENSING AND SIGNALLING IN PLANTS

247

Broadley, M. R., Bowen, H. C., Cotterill, H. L., Hammond, J. P., Meacham, M. C., Mead, A. and White, P. J. (2004). Phylogenetic variation in the shoot mineral concentration of angiosperms. Journal of Experimental Botany 55, 321–336. Brinch‐Pedersen, H., Sorensen, L. D. and Holm, P. B. (2002). Engineering crop plants: Getting a handle on phosphate. Trends in Plant Sciences 7, 118–125. Briskin, D. P. and Poole, R. J. (1983). Plasma membrane ATPase of red beet forms as phosphorylated intermediate. Plant Physiology 78, 507–512. Burleigh, S. H. and Harrison, M. J. (1999). The down‐regulation of Mt4‐like genes by phosphate fertilisation occurs systemically and involves phosphate translocation to the shoots. Plant Physiology 119, 241–248. Carswell, C., Grant, B. R., Theodorou, M. E., Harris, J., Niere, J. O. and Plaxton, W. C. (1996). The fungicide phosphonate disrupts the phosphate‐starvation response in Brassica nigra seedlings. Plant Physiology 110, 105–110. Carswell, M. C., Grant, B. R. and Plaxton, W. C. (1997). Disruption of the phosphate‐starvation response of oilseed rape suspension cells by the fungicide phosphonate. Planta 203, 67–74. Casson, S. A. and Lindsey, K. (2003). Genes and signalling in root development. New Phytologist 158, 11–38. Chen, D. L., Delatorre, C. A., Bakker, A. and Abel, S. (2000). Conditional identification of phosphate‐starvation‐response mutants in Arabidopsis thaliana. Planta 211, 13–22. Chevalier, F., Pata, M., Nacry, P., Doumas, P. and Rossignol, M. (2003). EVects of phosphate availability on the root system architecture: Large scale analysis of the natural variation between Arabidopsis accessions. Plant, Cell and Environment 26, 1839–1850. Chiou, T.‐J., Liu, H. and Harrison, M. J. (2001). The spatial expression patterns of a phosphate transporter (MtPT1) from Medicago truncatula indicate a role in phosphate transport at the root/soil interface. Plant Journal 25, 281–293. Ciereszko, I., Johansson, H., Hurry, V. and Kleczkowski, L. A. (2001). Phosphate status aVects the gene expression, protein content and enzymatic activity of UDP‐glucose pyrophosphorylase in wild‐type and pho mutants of Arabidopsis. Planta 212, 598–605. Clarkson, D. T. and Scattergood, C. B. (1982). Growth and phosphate transport in barley and tomato plants during the development of, and recovery from, phosphate stress. Journal of Experimental Botany 33, 865–875. Cogliatti, D. H. and Clarkson, D. T. (1983). Physiological changes in, and phosphate uptake by potato plants during development of, and recovery from phosphate deficiency. Physiologia Plantarum 58, 287–294. Coruzzi, G. and Bush, D. R. (2001). Nitrogen and carbon nutrient and metabolite signaling in plants. Plant Physiology 125, 61–64. Crawford, N. M., Kahn, M. L., Leustek, T. and Long, S. R. (2000). Nitrogen and sulfur. In ‘‘Biochemistry and Molecular Biology of Plants’’ (B. B. Buchanan, W. Gruissem and R. L. Jones, eds.), pp. 786–849. ASPP, Rockville. Cuin, T. A., Miller, A. J., Laurie, S. A. and Leigh, R. A. (2003). Potassium activities in cell compartments of salt‐grown barley leaves. Journal of Experimental Botany 54, 657–661. Davenport, R. (2002). Glutamate receptors in plants. Ann Bot (London) 90, 549–557. del Pozo, J. C., Allona, I., Rubio, V., Leyva, A., de la Pen˜ a, A., Aragoncillo, C. and Paz‐Ares, J. (1999). A type 5 acid phosphatase gene from Arabidopsis thaliana is induced by phosphate starvation and by some other types of phosphate mobilising/oxidative stress conditions. Plant Journal 19, 579–589.

248

A. AMTMANN ET AL.

Dennison, K. L. and Spalding, E. P. (2001). Glutamate‐gated calcium fluxes in Arabidopsis. Plant Physiology 124, 1511–1514. Dinkelaker, B., Hengeler, C. and Marschner, H. (1995). Distribution and function of proteoid roots and other root clusters. Botanical Acta 108, 183–200. Dong, B., Rengel, Z. and Delhaize, E. (1998). Uptake and translocation of phosphate by pho2 mutant and wild‐type seedlings of Arabidopsis thaliana. Planta 205, 251–256. Dong, B., Ryan, P. R., Rengel, Z. and Delhaize, E. (1999). Phosphate uptake in Arabidopsis thaliana: Dependence of uptake on the expression of transporter genes and internal phosphate concentrations. Plant, Cell and Environment 22, 1455–1461. Drew, M. C. (1975). Comparison of the eVects of a localised supply of phosphate, nitrate ammonium and potassium on the growth of the seminal root system, and the shoot, in barley. New Phytologist 75, 479–490. Drew, M. C. and Saker, L. R. (1984). Uptake and long‐distance transport of phosphate, potassium and chloride in relation to internal ion concentrations in barley: Evidence of non‐allosteric regulation. Planta 160, 500–507. Dunlop, J. and Gardiner, S. (1993). Phosphate uptake, proton extrusion and membrane electropotentials of phosphorus‐deficient. Trifolium repens L. Journal of Experimental Botany 44, 1801–1808. Epstein, E. (1965). Mineral metabolism. In ‘‘Plant Biochemistry’’ (J. Bonner and J. E. Varner, eds.), pp. 438–466. Academic Press, London. Epstein, E. (1972). ‘‘Mineral Nutrition of Plants: Principles and Perspectives.’’ Wiley, New York. Epstein, E. (1973). Mechanisms of ion transport through plant cell membranes. Critical Reviews in Cytology 34, 123–168. Epstein, I., Rains, D. W. and Elzam, O. E. (1963). Resolution of dual mechanisms of potassium absorption by barley roots. Proceedings of the National Academy of Sciences of the USA 49, 684–692. Essigmann, B., Gu¨ ler, S., Narang, R. A., Linke, D. and Benning, C. (1998). Phosphate availability aVects the thylakoid lipid composition and the expression of SQD1, a gene required for sulfolipid biosynthesis in Arabidopsis thaliana. Proceedings of the National Academy of Sciences of the USA 95, 1950–1955. Felle, H. H. (1998). The apoplastic pH of the Zea mays root cortex as measured with pH‐sensitive microelectrodes: Aspects of regulation. Journal of Experimental Botany 49, 987–995. Filleur, S., Walch‐Liu, P., Gan, Y. and Forde, B. G. (2005). Nitrate and glutamate sensing by plant roots. Biochemical Society Transactions 33, 283–286. Forde, B. and Lorenzo, H. (2001). The nutritional control of root development. Plant and Soil 232, 51–68. 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. Franco‐Zorrilla, J. M., Gonza´ lez, E., Bustos, R., Linhares, F., Leyva, A. and Paz‐ Ares, J. (2004). The transcriptional control of plant responses to phosphate limitation. Journal of Experimental Botany 55, 285–293. Franco‐Zorrilla, J. M., Martin, A. C., Leyva, A. and Paz‐Ares, J. (2005). Interactions between phosphate‐starvation, sugar, and cytokinin signaling in Arabidopsis and the roles of cytokinin receptors CRE1/AHK4 and AHK3. Plant Physiology 138, 847–857.

NUTRIENT SENSING AND SIGNALLING IN PLANTS

249

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. Plant Journal 32, 353–360. Gaymard, F., Pilot, G., Lacombe, B., Bouchez, D., Bruneau, D., Boucherez, J., Michaux‐Ferriere, N., Thibaud, J. B. and Sentenac, H. (1998). Identification and disruption of a plant shaker‐like outward channel involved in Kþ release into the xylem sap. Cell 94, 647–655. Gething, P. A. (1993). ‘‘The Potassium‐Nitrogen Partnership.’’ IPI Research Topics 13, Bern, Switzerland. Gierth, M., Ma¨ ser, P. and Schroeder, J. I. (2005). The potassium transporter AtHAK5 functions in Kþ deprivation‐induced high‐aYnity Kþ uptake and AKT1 Kþ channel contribution to Kþ uptake kinetics in Arabidopsis roots. Plant Physiology 137, 1105–1114. Grabov, A. and Blatt, M. R. (1997). Parallel control of the inward‐rectifier Kþ channel by cytosolic free Ca2þ and pH in Vicia guard cells. Planta 201, 84–95. Green, D. G., Ferguson, W. S. and Warder, F. G. (1973). Accumulation of toxic levels of phosphorus in the leaves of phosphorus‐deficient barley. Canadian Journal of Plant Science 53, 241–246. Guest, D. and Grant, B. (1991). The complex action of phosphonates as antifungal agents. Biological Reviews 66, 159–187. Hall, J. L. and Williams, L. E. (1991). Properties and functions of proton pumps in higher plants. Pestic. Science 32, 339–351. Hamburger, D., Rezzonico, E., MacDonald‐Comber Pete´ tot, J., Somerville, C. and Poirier, Y. (2002). Identification and characterisation of the Arabidopsis PHO1 gene involved in phosphate loading to the xylem. Plant Cell 14, 889–902. Hammond, J. P., Bennett, M. J., Bowen, H. C., Broadley, M. R., Eastwood, D. C., May, S. T., Rahn, C., Swarup, R., Woolaway, K. E. and White, P. J. (2003). Changes in gene expression in Arabidopsis shoots during phosphate starvation and the potential for developing smart plants. Plant Physiology 132, 578–596. Hammond, J. P., Broadley, M. R., Craigon, D. J., Higgins, J., Emmerson, Z., Townsend, H., White, P. J. and May, S. T. (2005). Using genomic DNA‐ based probe‐selection to improve the sensitivity of high‐density oligonucleotide arrays when applied to heterologous species. Plant Methods, In Press. Hammond, J. P., Broadley, M. R. and White, P. J. (2004a). Genetic responses to phosphorus deficiency. Annals of Botany 94, 323–332. Hammond, J. P., White, P. J. and Broadley, M. R. (2004b). Diagnosing phosphorus deficiency in plants. Aspects of Applied Biology 72, 89–98. Hampton, C. R., Bowen, H. C., Broadley, M. R., Hammond, J. P., Mead, A., Payne, K. A., Pritchard, J. and White, P. J. (2004). Cesium toxicity in Arabidopsis. Plant Physiology 136, 3824–3837. Haran, S., Logendra, S., Seskar, M., Bratanova, M. and Raskin, I. (2000). Characterisation of Arabidopsis acid phosphatase promoter and regulation of acid phosphatase expression. Plant Physiology 124, 615–626. Ha¨ rtel, H., Do¨ rmann, P. and Benning, C. (2000). DGD1‐independent biosynthesis of extraplastidic galactolipids after phosphate deprivation in Arabidopsis. Proceedings of the National Academy of Sciences of the USA 97, 10649–10654. He, Z., Ma, Z., Brown, K. M. and Lynch, J. P. (2005). Assessment of inequality of root hair density in Arabidopsis thaliana using the Gini coeYcient: A close look at the eVect of phosphorus and its interaction with ethylene. Annals of Botany 95, 287–293.

250

A. AMTMANN ET AL.

Hoch, W. A., Zeldin, E. L. and McCown, B. H. (2001). Physiological significance of anthocyanins during autumnal leaf senescence. Tree Physiology 21, 1–8. Hodge, A. (2004). The plastic plant: Root responses to heterogeneous supplies of nutrients. New Phytologist 162, 9–24. Høgh‐Jensen, H. and Pedersen, M. B. (2003). Morphological plasticity by crop plants and their potassium use eYciency. Journal of Plant Nutrition 26, 969–984. Hou, X. L., Wu, P., Jiao, F. C., Jia, Q. J., Chen, H. M., Yu, J., Song, X. W. and Yi, K. K. (2005). Regulation of the expression of OsIPS1 and OsIPS2 in rice via systemic and local Pi signalling and hormones. Plant, Cell and Environment 28, 353–364. Huang, C., Barker, S. J., Langridge, P., Smith, F. W. and Graham, R. D. (2000). Zinc deficiency up‐regulates expression of high‐aYnity phosphate transporter genes in both phosphate‐suYcient and ‐deficient barley roots. Plant Physiology 124, 415–422. ˚ ., Furbank, R. and Stitt, M. (2000). The role of inorganic Hurry, V., Strand, A phosphate in the development of freezing tolerance and the acclimatisation of photosynthesis to low temperature is revealed by the pho mutants of Arabidopsis thaliana. Plant Journal 24, 383–396. Irani, N. G., Hernandez, J. M. and Grotewold, E. (2003). Regulation of anthocyanin pigmentation. Recent Advances in Phytochemistry 37, 59–78. Jeschke, W. D., Kirkby, E. A., Peuke, A. D., Pate, J. S. and Hartung, W. (1997). EVects of P deficiency on assimilation and transport of nitrate and phosphate in intact plants of castor bean (Ricinus communis L.). Journal of Experimental Botany 48, 75–91. Johnson, C. S., Kolevski, B. and Smyth, D. R. (2002). TRANSPARENT TESTA GLABRA2, a trichome and seed coat development gene of Arabidopsis, encodes a WRKY transcription factor. Plant Cell 14, 1359–1375. Jungk, A. (2001). Root hairs and the acquisition of plant nutrients from soil. Journal of Plant Nutrition and Soil Science 164, 121–129. Kachmar, J. F. and Boyer, P. D. (1953). Kinetic analysis of enzyme reactions. II. The potassium activation and calcium inhibition of pyruvic phophoferase. Journal of Biological Chemistry 200, 669–683. Kang, J. and Turano, F. J. (2003). The putative glutamate receptor 1.1 (AtGRL1.1) functions as a regulator of carbon and nitrogen metabolism in Arabidopsis thaliana. Proceedings of the National Academy of Sciences of the USA 27, 6872–6877. Karandashov, V. and Bucher, M. (2005). Symbiotic phosphate transport in arbuscular mycorrhizas. Trends in Plant Science 10, 22–29. Karley, A. J., Leigh, R. A. and Sanders, D. (2000). Where do all the ions go? The cellular basis of diVerential ion accumulation in leaf cells. Trends Plant Science 5, 465–470. Karthikeyan, A. S., Varandarajan, D. K., Mukatira, U. T., D’Urzo, M. P., Damsz, B. and Raghothama, K. G. (2002). Regulated expression of Arabidopsis phosphate transporters. Plant Physiology 130, 221–233. Kim, E. J., Kwak, J. M., Uozumi, N. and Schroeder, J. I. (1998). AtKUPI: An Arabidopsis gene encoding high‐affinity potassium transport activity. Plant Cell 10, 51–62. Ko¨ ck, M., Lo¨ Zer, A., Abel, S. and Glund, K. (1995). cDNA structure and regulatory properties of a family of starvation‐induced ribonucleases from tomato. Plant Molecular Biology 27, 477–485. Kochian, L. V. (2000). Molecular physiology of mineral nutritient acquisition, transport and utilisation. In ‘‘Biochemistry and Molecular Biology of Plants’’

NUTRIENT SENSING AND SIGNALLING IN PLANTS

251

(B. B. Buchanan, W. Gruissem and R. L. Jones, eds.), pp. 786–849. ASPP, Rockville. Laegreid, M., Bockman, O. C. and Kaarstad, O. (1999). ‘‘Agriculture, Fertilisers and the Environement.’’ CABI, Oxon, UK. Laloi, C., Apel, K. and Danon, A. (2004). Reactive oxygen signalling: The latest news. Current Opinion in Plant Biology 7, 323–328. Laughlin, L. T. and Reed, G. H. (1997). The monovalent cation requirement of rabbit muscle pyruvate kinase is eliminated by substitution of lysine for glutamate 117. Archives of Biochemistry and Biophysics 348, 262–267. Lee, R. B. (1993). Control of net uptake of nutrients by regulation of influx in barley plants recovering from nutrient deficiency. Annals of Botany 72, 223–230. Lee, R. B., RatcliVe, R. G. and Southon, T. E. (1990). 31P NMR measurements of the cytoplasmic and vacuolar Pi content of mature maize roots: Relationships with phosphorus status and phosphate fluxes. Journal of Experimental Botany 41, 1063–1078. Leigh, R. A. and Jones, R. G. W. (1984). A hypothesis relating critical potassium concentrations for growth to the distribution and functions of this ion in the plant‐cell. New Phytology 97, 1–13. Lejay, L., Gansel, X. Z., Cerezo, M., Tillard, P., Mu¨ ller, C., Krapp, A., von Wire´ n, N., Daniel‐Vedele, F. and Gojon, A. (2003). Regulation of root ion transporters by photosynthesis: Functional importance and relation to hexokinase. Plant Cell 15, 2218–2232. Linkohr, B. I., Williamson, L. C., Fitter, A. H. and Leyser, H. M. O. (2002). Nitrate and phosphate availability and distribution eVects on root system architecture of Arabidopsis. Plant Journal 29, 751–760. Li, D., Zhu, H., Liu, K., Liu, X., Leggewie, G., Udvardi, M. and Wang, D. (2002). Purple acid phosphatases of Arabidopsis thaliana: Comparative analysis and diVerential regulation by phosphate deprivation. Journal of Biological Chemistry 277, 27772–27781. Liu, C., Muchhal, U. S. and Raghothama, K. G. (1997). DiVerential expression of TPSI1, a phosphate starvation‐induced gene in tomato. Plant Molecular Biology 33, 867–874. Liu, J., Samac, D. A., Bucciarelli, B., Allan, D. L. and Vance, C. P. (2005). Signaling of phosphorus deficiency‐induced gene expression in white lupin requires sugar and phloem transport. Plant Journal 41, 257–268. Lloyd, J. C. and Zakhleniuk, O. V. (2004). Responses of primary and secondary metabolism to sugar accumulation revealed by microarray expression analysis of the Arabidopsis mutant, pho3. Journal of Experimental Botany 55, 1221–1230. Lo´ pez‐Bucio, J., Herna´ ndez‐Abreu, E., Sa´ nchez‐Caldero´ n, 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. Lo´ pez‐Bucio, J., Herna´ ndez‐Abreu, E., Sa´ nchez‐Caldero´ n, L., Pe´ rez‐Torres, A., Rampey, R. A., Bartel, B. and Herrerra‐Estrella, L. (2005). An auxin transport independent pathway is involved in phosphate stress‐induced root architectural alterations in Arabidopsis: Identification of BIG as a mediator of auxin pericycle cell activation. Plant Physiology 137, 681–691. Lo´ pez‐Bucio, J., Nieto‐Jacobo, M. F., Ramı´rez‐Rodrı´guez, V. and Herrera‐Estrella, L. (2000). Organic acid metabolism in plants: From adaptive physiology to transgenic varieties for cultivation in extreme soils. Plant Science 160, 1–13.

252

A. AMTMANN ET AL.

Lynch, J. P. and Brown, K. M. (2001). Topsoil foraging: An architectural adaptation of plants to low phosphorus availability. Plant and Soil 237, 225–237. Maathuis, F. and Sanders, D. (1997). Regulation of Kþ absorption in plant root cells by external Kþ: Interplay of diVerent Kþ transporters. Journal of Experimental Botany 48, 451–458. Maathuis, F. J., Ichida, A. M., Sanders, D. and Schroeder, J. I. (1997). Roles of higher plant Kþ channels. Plant Physiology 114, 1141–1149. Maathuis, F. J., Filatov, V., Herzyk, P., G, C. K., K, B. A., Chen, S., Green, B. J., Li, Y., Madagan, K. L., Sanchez‐Fernandez, R., Forde, B. G., Palmgren, M. G., Rea, P. A., Williams, L. E., Sanders, D. and Amtmann, A. (2003). Transcriptome analysis of root transporters reveals participation of multiple gene families in the response to cation stress. Plant J. 35, 675–692. Malamy, J. E. (2005). Intrinsic and environmental response pathways that regulate root system architecture. Plant Cell and Environment 28, 67–77. Marschner, H. (1995). ‘‘Mineral Nutrition of Higher Plants,’’ 2nd Edn. Academic Press, London. Marschner, H. and Cakmak, I. (1986). Mechanism of phosphorus‐induced zinc deficiency in cotton. II. Evidence for impaired shoot control of phosphorus uptake and translocation under zinc deficiency. Physiologia Plantarum 68, 491–496. Martı´n, A. C., del Pozo, J. C., Iglesias, J., Rubio, V., Solano, R., de la Pen˜ a, A., Leyva, A. and Paz‐Ares, J. (2000). Influence of cytokinins on the expression of phosphate starvation responsive genes in Arabidopsis. Plant Journal 24, 559–567. Martin, T., Oswald, O. and Graham, I. A. (2002). Arabidopsis seedling growth, storage lipid mobilisation, and photosynthetic gene expression are regulated by carbon:nitrogen availability. Plant Physiology 128, 472–481. Miedema, H., Bothwell, J. H. F., Brownlee, C. and Davies, J. M. (2001). Calcium uptake by plant cells: Channels and pumps acting in concert. Trends in Plant Science 6, 514–519. Miller, S. S., Liu, J., Allan, D. L., Menzhuber, C. J., Fedorova, M. and Vance, C. P. (2001). Molecular control of acid phosphatase secretion into the rhizosphere of proteoid roots from phosphorus‐stressed white lupin. Plant Physiology 127, 594–606. Mimura, T. (1999). Regulation of phosphate transport and homeostasis in plant cells. International Review of Cytology 191, 149–200. Misson, J., Raghothama, K. G., Jain, A., Jouhet, J., Block, M. A., Bligny, R., Ortet, P., CreV, A., Somerville, S., Rolland, N., Doumas, P., Nacry, P., Herrerra‐ Estrella, L., Nussaume, L. and Thibaud, M.‐C. (2005). A genome‐wide transcriptional analysis using Arabidopsis thaliana AVymetrix gene chips determined plant responses to phosphate deprivation. Proceedings of the National Academy of Sciences of the USA 102, 11934–11939. Montiel, G., Gantet, P., Jay‐Allemand, C. and Breton, C. (2004). Transcription factor networks. Pathways to the knowledge of root development. Plant Physiology 136, 3478–3485. Moore, B. L., Zhou, L., Rolland, F., Hall, Q., Cheng, W.‐H., Liu, Y.‐X., Jones, T. L. and Sheen, J. (2003). Role of the Arabidopsis glucose sensor HXK1 in nutrient, light and hormonal signaling. Science 300, 332–336. Mu¨ ller, R., Nilsson, L., Krintel, C. and Nielsen, T. H. (2004). Gene expression during recovery from phosphate starvation in roots and shoots of Arabidopsis thaliana. Physiologia Plantarum 122, 233–243.

NUTRIENT SENSING AND SIGNALLING IN PLANTS

253

Mukatira, U. T., Liu, C., Varadarajan, D. K. and Raghothama, K. G. (2001). Negative regulation of phosphate starvation‐induced genes. Plant Physiology 127, 1854–1862. 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. Narang, R. A., Breune, A. and Altman, T. (2000). Analysis of phosphate acquisition eYciency in diVerent Arabidopsis accessions. Plant Physiology 124, 1786–1799. Nazoa, P., Vidmar, J. J., Tranbarger, T. J., Mouline, K., Damiani, I., Tillard, P., Zhuo, D., Glass, A. D. and Touraine, B. (2003). Regulation of the nitrate transporter gene AtNRT2.1 in Arabidopsis thaliana: Responses to nitrate, amino acids and developmental stage. Plant Molecular Biology 52, 689–703. Paul, M. J. and Pellny, T. K. (2003). Carbon metabolite feedback regulation of leaf photosynthesis and development. Journal of Experimental Botany 54, 539–547. Pen˜ aloza, E., Mun˜ oz, G., Salvo‐Garrido, H., Silva, H. and Cocuera, L. J. (2005). Phosphate deficiency regulates phosphoenolpyruvate carboxylase expression in proteoid root clusters of white lupin. Journal of Experimental Botany 56, 145–153. Perez‐Amador, M. A., Leon, J., Green, P. J. and Carbonell, J. (2002). Induction of the arginine decarboxylase ADC2 gene provides evidence for the involvement of polyamines in the wound response in Arabidopsis. Plant Physiology 130, 1454–1463. Petters, J., Go¨ bel, C., Scheel, D. and Rosahl, S. (2002). A pathogen‐responsive cDNA from potato encodes a protein with homology to a phosphate starvation‐induced phosphatase. Plant Cell Physiology 43, 1049–1053. Pilot, G., Gaymard, F., Mouline, K., Cherel, I. and Sentenac, H. (2003). Regulated expression of Arabidopsis shaker Kþ channel genes involved in Kþ uptake and distribution in the plant. Plant Molecular Biology 51, 773–787. Plaxton, W. C. and Carswell, M. C. (1999). Metabolic aspects of the phosphate starvation response in plants. In ‘‘Plant Responses to Environmental Stress: From Phytohormones to Genome Reorganization’’ (H. R. Lerner, ed.), pp. 350–372. Dekker, New York. PoVenroth, M., Green, D. B. and Tallman, G. (1992). Sugar concentration in guard cells of Vicia faba illuminated with red or blue light. Plant Physiology 98, 1460–1471. Price, J., Laxmi, A., St. Martin, S. K. and Jang, J. C. (2004). Global transcription profiling reveals multiple sugar signal transduction mechanisms in Arabidopsis. Plant Cell 16, 2128–2150. Raghothama, K. G. (2005). Phosphorus. In ‘‘Plant Nutritional Genomics’’ (M. R. Broadley and P. J. White, eds.), pp. 112–126. Blackwell, Oxford, UK. Rausch, C. and Bucher, M. (2002). Molecular mechanisms of phosphate transport in plants. Planta 216, 23–37. Rausch, C., Daram, P., Brunner, S., Jansa, J., Laloi, M., Leggewie, G., Amrhein, N. and Bucher, M. (2001). A phosphate transporter expressed in arbuscle‐ containing cells in potato. Nature 414, 462–470. Reckmann, U., Scheibe, R. and Raschke, K. (1990). Rubisco activity in guard cells compared with the solute requirement for stomatal opening. Plant Physiology 92, 246–253. Richards, F. J. and Coleman, E. G. (1952). Occurrence of putrescine in potassium deficient barley. Nature 170, 460–461.

254

A. AMTMANN ET AL.

Rietz, S., Holk, A. and Scherer, G. F. E. (2004). Expression of the patatin‐related phospholipase A gene AtPLA IIA in Arabidopsis thaliana is up‐regulated by salicylic acid, wounding, ethylene, and iron and phosphate deficiency. Planta 219, 743–753. Robinson, D. (1994). The response of plants to non‐uniform supplies of nutrients. New Phytologist 127, 635–674. Rolland, F. and Sheen, J. (2005). Sugar sensing and and signalling networks in plants. Biochemical Society Transactions 33, 269–271. Rook, F. and Bevan, M. W. (2003). Genetic approaches to understanding sugar‐ response pathways. Journal of Experimental Botany 54, 495–501. Rubio, V., Linhares, F., Solano, R., Martı´n, A. C., Iglesias, J., Leyva, A. and Paz‐ Ares, J. (2001). A conserved MYB transcription factor involved in phosphate starvation signalling both in vascular plants and in unicellular algae. Genes and Development 15, 2122–2133. Ruiz, J. M., Moreno, D. A. and Romero, L. (1999). Pyruvate kinase activity as an indicator of the level of K, Mg, and Ca in leaves and fruits of cucumber: The role of potassium fertilisation. Journal of Agricultural Food Chemistry 47, 845–849. Santa‐Maria, G. E., Danna, C. H. and Czibener, C. (2000). High‐aYnity potassium transport in barley roots: Ammonium‐sensitive and ‐insensitive pathways. Plant Physiology 123, 297–306. Sauter, A., Dietz, K. J. and Hartung, W. (2005). A possible stress physiological role of abscisic acid conjugates in root‐to‐shoot signalling. Plant, Cell and Environment 25, 223–228. Schachtman, D. P., Reid, R. J. and Ayling, S. M. (1998). Phosphorus uptake by plants: From soil to cell. Plant Physiology 116, 447–453. Schu¨ nmann, P. H. D., Richardson, A. E., Vickers, C. E. and Delhaize, E. (2004). Promoter analysis of the barley Pht1;1 phosphate transporter gene identifies regions controlling root expression and responsiveness to phosphate deprivation. Plant Physiology 136, 4205–4214. Schikora, A. and Schmidt, W. (2002). Formation of transfer cells and Hþ‐ATPase expression in tomato roots under P and Fe deficiency. Planta 215, 304–311. Schroeder, J. I., Ward, J. M. and Gassmann, W. (1994). Perspectives on the physiology and structure of inward‐rectifying Kþ channels in higher plants: Biophysical implications for Kþ uptake. Annual Review of Biophysics and Biomolecular Structure 23, 441–471. Schultz, C., Gilson, P., Oxley, D., Youl, J. and Bacic, A. (1998). GPI‐anchors on arabinogalactan‐proteins: Implications for signalling in plants. Trends Plant Science 3, 426–431. Scrase‐Field, A. M. G. and Knight, M. R. (2003). Calcium: Just a chemical switch? Current Opinion in Plant Biology 6, 500–506. Shin, R. and Schachtman, D. P. (2004). Hydrogen peroxide mediates plant root cell response to nutrient deprivation. Proceedings of the National Academy of Sciences of the USA 101, 8827–8832. Shin, H., Shin, H.‐S., Dewbre, G. R. and Harrison, M. J. (2004). Phosphate transport in Arabidopsis: Pht1;1 and Pht1;4 play a major role in phosphate acquisition from both low‐ and high‐phosphate environments. Plant Journal 39, 629–642. Shinozaki, K. and Yamaguchi‐Shinozaki, K. (2000). Molecular responses to dehydration and low temperature: DiVerences and cross‐talk between two stress signaling pathways. Current Opinion in Plant Biology 3, 217–223. Shirley, B. W., Kubasek, W. L., Storz, G., Bruggemann, E., Koornneef, M., Ausubel, F. M. and Goodman, H. M. (1995). Analysis of Arabidopsis mutants deficient in flavonoid biosynthesis. Plant Journal 8, 659–671.

NUTRIENT SENSING AND SIGNALLING IN PLANTS

255

Smith, C. R., Knowles, V. L. and Plaxton, W. C. (2000a). Purification and characterisation of cytosolic pyruvate kinase from Brassica napus (rapeseed) suspension cell cultures. European Journal of Biochemistry 267, 4477–4485. Smith, F. W., Cybinski, D. H. and Rae, A. L. (1999). Regulation of expression of genes encoding phosphate transporters in barley roots. In ‘‘Plant Nutrition: Molecular Biology and Genetics’’ (G. Gissel‐Nielsen and A. Jensen, eds.), pp. 145–150. Kluwer Academic, The Netherlands. Smith, F. W., Mudge, S. R., Rae, A. L. and Glassop, D. (2003). Phosphate transport in plants. Plant and Soil 248, 71–83. Smith, F. W., Rae, A. L. and Hawksford, M. J. (2000b). Molecular mechanisms of phosphate and sulphate transport in plants. Biochimica and Biophysica Acta 1465, 236–245. Spalding, E. P., Hirsch, R. E., Lewis, D. R., Qi, Z., Sussman, M. R. and Lewis, B. D. (1999). Potassium uptake supporting plant growth in the absence of AKT1 channel activity: Inhibition by ammonium and stimulation by sodium. Journal of Generic Physiology 113, 909–918. Stenzel, I., Ziethe, K., Schurath, J., Hertel, S. C., Bosse, D. and Ko¨ ck, M. (2003). DiVerential expression of the LePS2 phosphatase gene family in response to phosphate availability, pathogen infection and during development. Physiologia Plantarum 118, 138–146. Stitt, M. and Fernie, A. R. (2003). From measurements of metabolites to metabolomics: An ‘on the fly’ perspective illustrated by recent studies of carbon‐ nitrogen interactions. Current Opinion in Biotechnology 14, 136–144. Subbarao, G. V., Wheeler, R. M., Stutte, G. W. and Levine, L. H. (2000). Low potassium enhances sodium uptake in red‐beet under moderate saline conditions. Journal of Plant Nutrition 23, 1449–1470. Syers, J. K. (1998). ‘‘Soil and Plant Potassium in Agriculture.’’ The Fertiliser Society, York, UK. Tang, Z., Sadka, A., Morrishige, D. T. and Mullet, J. E. (2001). Homeodomain leucine zipper proteins bind to the phosphate response domain of the soybean VspB tripartite promoter. Plant Physiology 125, 797–809. Tester, M. and Blatt, M. R. (1989). Direct measurement of Kþ channels in thylakoid membranes by incorporation of vesicles into planar lipid bilayers. Plant Physiology 91, 249–252. Tester, M. and Leigh, R. A. (2001). Partitioning of nutrient transport processes in roots. Journal of Experimental Botany 52, 445–457. Thion, L., Mazars, C., Nacry, P., Bouchez, D., Moreau, M., Ranjeva, R. and Thuleau, P. (1998). Plasma membrane depolarisation‐activated calcium channels, stimulated by microtubule‐depolymerizing drugs in wild‐type Arabidopsis thaliana protoplasts, display constitutively large activities and a longer half‐life in ton 2 mutant cells aVected in the organisation of cortical microtubules. Plant Journal 13, 603–610. Ticconi, C. A. and Abel, S. (2004). Short on phosphate: Plant surveillance and countermeasures. Trends in Plant Science 9, 548–555. Ticconi, C. A., Delatorre, C. A. and Abel, S. (2001). Attenuation of phosphate starvation responses by phosphite in Arabidopsis. Plant Physiology 127, 963–972. Ticconi, C. A., Delatorre, C. A., Lahner, B., Salt, D. E. and Abel, S. (2004). Arabidopsis pdr2 reveals a phosphate‐sensitive checkpoint in root development. Plant Journal 34, 801–814. Todd, C. D., Zeng, P., Rodriguez Huete, A. M., Hoyos, M. E. and Polacco, J. C. (2004). Transcripts of MYB‐like genes respond to phosphorus and nitrogen deprivation in Arabidopsis. Planta 219, 1003–1009.

256

A. AMTMANN ET AL.

Toyota, K., Koizumi, N. and Sato, F. (2003). Transcriptional activation of phosphoenolpyruvate carboxylase by phosphorus deficiency in tobacco. Journal of Experimental Botany 54, 961–969. Trewavas, A. (2000). Signal perception and transduction. In ‘‘Biochemistry and Molecular Biology of Plants’’ (B. B. Buchanan, W. Gruissem and R. L. Jones, eds.), pp. 786–849. ASPP, Rockville. Uhde‐Stone, C., Zinn, K. E., Ramirez‐Ya´ n˜ ez, M., Li, A., Vance, C. P. and Allan, D. L. (2003). Nylon filter arrays reveal diVerential gene expression in proteoid roots of white lupin in response to phosphorous deficiency. Plant Physiology 131, 1064–1079. Vance, C. P., Uhde‐Stone, C. and Allan, D. L. (2003). Phosphorus acquisition and use: Critical adaptations by plants for securing a nonrenewable resource. New Phytologist 157, 423–447. Varadarajan, D. K., Karthikeyan, A. S., Matilda, P. D. and Raghothama, K. G. (2002). Phosphite, an analog of phosphate, suppresses the coordinated expression of genes under phosphate starvation. Plant Physiology 129, 1232–1240. Volenec, J. J., Joern, B. C., Rong, L. and Cunningham, S. M. (1996). EVects of potassium nutrition on carbohydrate and protein metabolism in alfalfa roots. In ‘‘Frontiers in Potassium Nutrition’’ (D. M. Oosterhuis and G. A. Berkowitz, eds.), pp. 77–82. Potash & Phosphate Institute, Georgia. Vreugdenhil, D., Aarts, M. G. M. and Koornneef, M. (2005). Exploring natural genetic variation to improve plant nutrient content. In ‘‘Plant Nutritional Genomics’’ (M. R. Broadley and P. J. White, eds.), pp. 201–219. Blackwell, Oxford, UK. Walker, A. R., Davisin, P. A., Bolognesi‐Winfield, A. C., James, C. M., Srinivasan, N., Blundell, T. L., Esch, J. J., Marks, M. D. and Gray, J. C. (1999). The TRANSPARENT TESTA GLABRA1 locus, which regulates trichome diVerentiation and anthocyanin biosynthesis in Arabidopsis, encodes a WD40 repeat protein. Plant Cell 11, 1337–1349. Walker, D. J., Leigh, R. A. and Miller, A. J. (1996). Potassium homeostasis in vacuolate plant cells. Proceedings of the National Academy of Sciences of the USA 93, 10510–10514. Wang, R., Guegler, K., LaBrie, S. T. and Crawford, N. M. (2000). Genomic analysis of a nutrient response in Arabidopsis reveals diverse expression patterns and novel metabolic and potential regulatory genes induced by nitrate. Plant Cell 12, 1491–1509. Wang, Y., Ribot, C., Rezzonico, E. and Poirier, Y. (2004). Structure and expression profile of the Arabidopsis PHO1 gene family indicates a broad role in inorganic phosphate homeostasis. Plant Physiology 135, 400–411. Wang, Y.‐H., Garvin, D. F. and Kochian, L. V. (2002). Rapid induction of regulatory and transporter genes in response to phosphorus, potassium, and iron deficiencies in tomato roots: Evidence for cross talk and root/rhizosphere‐ mediated signals. Plant Physiology 130, 1361–1370. Wasaki, J., Yonetani, R., Kuroda, S., Shinano, T., Yazaki, J., Fujii, F., Shimbo, K., Yamamoto, K., Sakata, K., Sasaki, T., Kishimoto, N., Kikuchi, S., Yamagishi, M. and Osaki, M. (2003). Transcriptomic analysis of metabolic changes by phosphorus stress in rice plant roots. Plant, Cell and Environment 26, 1515–1523. White, P. J. (1998). Calcium channels in the plasma membrane of root cells. Annals of Botany 81, 173–183. White, P. J. (2000). Calcium channels in higher plants. Biochimica et Biophysica Acta 1465, 171–189.

NUTRIENT SENSING AND SIGNALLING IN PLANTS

257

White, P. J. and Broadley, M. R. (2003). Calcium in plants. Annals of Botany 92, 487–511. Wiese, A., Elzinga, N., Wobbes, B. and Smeekens, S. (2004). A conserved upstream open reading frame mediates sucrose‐induced repression of translation. Plant Cell 16, 1717–1729. Williams, L. E. (ed.) (2000). Plant membrane transporters: Structure function and regulation. Biochimica and Biophysica Acta 1465. Williamson, L. C., Ribrioux, S. P. C. P., Fitter, A. H. and Leyser, H. M. O. (2001). Phosphate availability regulates root system architecture in Arabidopsis. Plant Physiology 126, 875–882. Wu, P., Ma, L., Hou, X., Wang, M., Wu, Y., Liu, F. and Deng, X. W. (2003). Phosphate starvation triggers distinct alterations of genome expression in Arabidopsis roots and leaves. Plant Physiology 132, 1260–1271. Wu, W., Peters, J. and Berkowitz, G. A. (1991). Surface charge‐mediated eVects of Mg2þ and Kþ flux across the chloroplast envelope are associated with regulation of stroma pH and photosynthesis. Plant Physiology 97, 580–587. Wyn Jones, R. J. and Pollard, A. (1983). Proteins, enzymes and inorganic ions. In ‘‘Encyclopedia of Plant physiology’’ (A. Lauchli and A. Pirson, eds.), pp. 528–562. Springer, Berlin. Yang, X. E., Liu, J. X., Wang, W. M., Ye, Z. Q. and Luo, A. C. (2004). Potassium internal use eYciency relative to growth vigor, potassium distribution, and carbohydrate allocation in rice genotypes. Journal of Plant Nutrition 27, 837–852. Yu, B., Xu, C. and Benning, C. (2002). Arabidopsis disrupted in SQD2 encoding sulfolipid synthase is impaired in phosphate‐limited growth. Proceedings of the National Academy of Sciences of the USA 99, 5732–5737. Zhang, F., Gonzalez, A., Zhao, M., Payne, T. and Lloyd, A. (2003a). A network of redundant bHLH proteins functions in all TTG1‐dependent pathways of Arabidopsis. Development 130, 4859–4869. Zhang, H. and Forde, B. G. (1998). An Arabidopsis MADS box gene that controls nutrient‐induced changes in root architecture. Science 16, 279(5349), 407–409. Zhang, Y.‐J., Lynch, J. P. and Brown, K. M. (2003b). Ethylene and phosphorus availability have interacting yet distinct eVects on root hair development. Journal of Experimental Botany 54, 2351–2361. Zimmermann, S., Talke, I., Ehrhardt, T., Nast, G. and Muller‐Rober, B. (1998). Characterization of SKT1, an inwardly rectifying potassium channel from potato, by heterologous expression in insect cells. Plant Physiology 116, 879–890.

Advances in

BOTANICAL RESEARCH Incorporating Advances in Plant Pathology

Editor-in-Chief J. A. CALLOW

School of Biosciences, The University of Birmingham, Birmingham, United Kingdom

Editorial Board J. S. HESLOP-HARRISON M. KREIS R. A. LEIGH P. R. SHEWRY D. SOLTIS

University of Leicester, United Kingdom Universite de Paris-Sud, Orsay, France University of Cambridge, United Kingdom IACR-Long Ashton Research Station, United Kingdom University of Florida at Gainesville, USA

CONTRIBUTORS TO VOLUME 43

CHARLES D. AMSLER Department of Biology, The University of Alabama at Birmingham, Birmingham, Alabama 35294–1170 ANNA AMTMANN Plant Science Group, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow G12 8QQ, United Kingdom PATRICK ARMENGAUD Plant Science Group, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow G12 8QQ, United Kingdom MATTHEW J. DRING Queen’s University Marine Laboratory, Portaferry, Co. Down, BT22 1PF Northern Ireland, United Kingdom VICTORIA A. FAIRHEAD Department of Biology, The University of Alabama at Birmingham, Birmingham, Alabama 35294–1170 NIGEL G. HALFORD Crop Performance and Improvement, Rothamsted Research, Harpenden, Hertfordshire AL5 2JQ, United Kingdom JOHN P. HAMMOND Warwick Horticulture Research International, University of Warwick, Wellesbourne, Warwick CV35 9EF, United Kingdom AARON MAXWELL Cooperative Research Centre for Australian Weed Management and The Commonwealth Scientific and Industrial Research Organisation (CSIRO) Entomology, PO Wembley, WA 6913, Australia JOHN K. SCOTT Cooperative Research Centre for Australian Weed Management and The Commonwealth Scientific and Industrial Research Organisation (CSIRO) Entomology, PO Wembley, WA 6913, Australia PHILIP J. WHITE Warwick Horticulture Research International, University of Warwick, Wellesbourne, Warwick CV35 9EF, United Kingdom

CONTENTS OF VOLUMES 33–42

Contents of Volume 33 Foliar Endophytes and Their Interactions with Host Plants, with Specific Reference to the Gymnospermae W.-M. KRIEL, W. J. SWART and P. W. CROUS Plants in Search of Sunlight D. KOLLER The Mechanics of Root Anchorage A. R. ENNOS Molecular Genetics of Sulphate Assimilation M. J. HAWKESFORD and J. L. WRAY Pathogenicity, Host-Specificity, and Population Biology of Tapesia spp., Causal Agents of Eyespot Disease of Cereals J. A. LUCAS, P. S. DYER and T. D. MURRAY

Contents of Volume 34 BIOTECHNOLOGY OF CEREALS Edited by Peter Shewry Cereal Genomics K. J. EDWARDS and D. STEVENSON Exploiting Cereal Genetic Resources R. J. HENRY

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CONTENTS OF VOLUMES 33–42

Transformation and Gene Expression P. BARCELO, S. RASCO-GAUNT, C. THORPE and P. A. LAZZERI Opportunities for the Manipulation of Development of Temperate Cereals J. R. LENTON Manipulating Cereal Endosperm Structure, Development and Composition to Improve End Use Properties P. R. SHEWRY and M. MORELL Resistance to Abiotic Freezing Stress in Cereals M. A. DUNN, G. O’BRIEN, A. P. C. BROWN, S. VURAL and M. A. HUGHES Genetics and Genomics of the Rice Blast Fungus Magnaporthe grisea: Developing an Experimental Model for Understanding Fungal Diseases of Cereals N. J. TALBOT and A. J. FOSTER Impact of Biotechnology on the Production of Improved Cereal Varieties R. G. SOLOMON and R. APPELS Overview and Prospects P. R. SHEWRY, P. A. LAZZERI and K. J. EDWARDS

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

CONTENTS OF VOLUMES 33–42

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

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

xi

xii

CONTENTS OF VOLUMES 33–42

Nematodes S. A. MacFARLANE, R. NEILSON and D. J. F. BROWN Other Vectors R. T. PLUMB

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

CONTENTS OF VOLUMES 33–42

xiii

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

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

xiv

CONTENTS OF VOLUMES 33–42

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 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

CONTENTS OF VOLUMES 33–42

xv

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

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

AUTHOR INDEX

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

A Aarts, M. G. M., 232, 255 Abbas, H. K., 155, 167 Abdel-Fattah, G. M., 166, 172 Abe, H., 109, 129 Abel, S., 230, 231, 235, 239, 242, 243, 247, 250, 255 ˚ berg, P., 11, 12, 15, 17, 18, 19, 20, 21, 24, A 27, 29, 44, 45, 49, 51, 55, 57, 83, 84 Abrahamsson, K., 61, 71, 183, 184, 189, 199, 201, 202, 205, 206 Acedo, G. N., 117, 131 Adalsteinsson, S., 241, 245 Adams, F. C., 61, 81 Adas, F., 50, 73 Adolph, S., 3, 70, 85 Aeschbacher, R. A., 124, 141 Afar, D. E. H., 98, 135 Aghdasi, M., 124, 142 Agrawal, A. A., 42, 71 Aguan, K., 102, 130 Aguilera, J., 182, 183, 190, 199, 201, 207 Ahmed, F., 33, 71 Ahn, B.-S., 154, 174 Ahn, S. J., 217, 245 Aitken, A., 115, 137 Akimitsu, K., 152, 170 Albaina, A., 70, 90 Albani, D., 99, 129 Albig, W., 106, 138 Alcalde, J., 98, 139 Alderson, A., 108, 118, 129 Alemseged, Y., 144, 170 Alford, H. L., 125, 139 Al-Ghazi, Y., 231, 232, 242, 244, 245 Ali, M. S., 33, 71 Allain, E. J., 61, 73 Allan, D. L., 211, 229, 230, 231, 232, 233, 235, 237, 240, 241, 242, 243, 244, 251, 252, 255 Allen, G. J., 223, 224, 245, 246 Allmann, D. W., 101, 129 Allona, I., 230, 231, 247 Almeida, M., 184, 201 Alonso-Llamazares, A., 115, 138 Alscher, R. G., 180, 181, 182, 197, 201 Altman, A., 216, 246

Altman, T., 230, 232, 252 Amrhein, N., 241, 253 Amsellem, Z., 154, 156, 162, 163, 167, 173 Amsler, C. D., 3, 18, 21, 34, 38, 39, 40, 42, 45, 46, 49, 50, 56, 59, 62, 63, 64, 65, 66, 67, 68, 69, 70, 72, 76, 77, 79, 82, 85 Amsler, M. O., 38, 39, 40, 56, 59, 72, 82, 85 Amtmann, A., 209, 211, 214, 217, 218, 219, 220, 221, 222, 225, 226, 227, 246 Anderberg, R. J., 111, 129 Anderson, R., 38, 72 Andersson, M. X., 230, 246 Andolfi, A., 155, 174 Andralojc, J., 124, 138 Andrew, L. M., 127, 136 Andrews, J., 163, 168 Ankisetty, S., 39, 72 Anonymous, 161, 167 Aon, M. G., 118, 129 Apel, K., 224, 250 Appel, H. M., 8, 72 Aragoncillo, C., 230, 231, 247 Arenas-Huertero, F., 126, 127, 129 Armengaud, P., 209, 211, 214, 217, 218, 219, 220, 221, 225, 226, 227, 246 Arnold, T. M., 3, 4, 6, 7, 8, 10, 14, 15, 19, 22, 23, 25, 28, 29, 40, 42, 50, 55, 72, 73, 88 Arnon, D. I., 211, 246 Arro, M., 113, 132 Arrontes, J., 33, 73 Arroyo, A., 126, 127, 129 Asada, K., 180, 191, 201 Ashburner, C. M., 9, 74 Aspinall, D., 120, 138 Assante, G., 155, 168 Audran, J. C., 120, 131 Auh, C.-K., 197, 201 Auld, B. A., 153, 162, 163, 165, 166, 168, 171 Auster, A. S., 102, 133 Ausubel, F. M., 240, 254 Ayling, S. M., 234, 257 Aziz, A., 216, 246 Azmi, A., 240, 243, 252 Azzout-Marniche, D., 101, 141

260

AUTHOR INDEX

B Baayen, R. P., 151, 168 Babu, R. M., 166, 168 Bacher, A., 113, 132 Bachmann, M., 115, 129 Bacic, A., 219, 254 Bailey, A. M., 157, 168 Bailey, K. L., 161, 162, 168, 169 Bailey, P. C., 127, 136 Baka, Z. A. M., 166, 172 Baker, B. J., 18, 21, 38, 39, 40, 42, 45, 46, 56, 59, 72, 76, 82, 85 Baker, H. G., 120, 129 Baker, I., 120, 129 Baker, J. A., 39, 72 Bakker, A., 243, 247 Balcells, L., 113, 132 Baldauf, S. L., 3, 73 Baldwin, I. T., 27, 42, 50, 73, 77, 80 Baldwin, J. C., 231, 242, 246 Balesdent, M.-H., 145, 152, 171 Ball, K. L., 111, 112, 113, 129, 140 Ballicora, M. A., 142 Baney, R., 70, 74 Banfalvi, Z., 108, 136 Banno, H., 118, 137 Bansal, V. K., 153, 168 Barbero, P. E., 183, 184, 205 Barbetti, M. J., 146, 152, 171 Barbosa, J. P., 31, 73 Barcelo, P., 120, 141 Bari, R. P., 153, 154, 173 Bariola, P. A., 230, 246 Barker, A. M., 52, 74 Barker, J. H. A., 108, 111, 112, 129, 134 Barker, L., 126, 129, 136, 138, 140 Barker, S. J., 241, 250 Barr, A. J., 101, 135 Barr, R. L., 101, 135 Barros, M. P., 183, 201 Bartel, B., 243, 251 Barthelmess, I. B., 98, 139 Barz, W., 116, 134 Bateman, R. P., 162, 171 Bates, T. R., 230, 237, 246 Baudry, A., 240, 246 Beard, B. R., 145, 174 Beardall, J., 178, 203, 206 Beatty, K. E., 184, 202 Beaudoin, F., 109, 115, 139 Becker, U., 63, 83 Beg, Z. H., 101, 129 Beggs, K., 117, 133 Bell, E., 231, 242, 246 Bell, J. C., 98, 135 Bellani, L., 120, 132 Belliard, G., 117, 139 Benbow, L., 121, 130

Bennett, M. J., 231, 232, 233, 234, 235, 237, 238, 240, 249, 257 Benning, C., 230, 232, 248, 249, 256 Benny, U., 151, 171 Berenbaum, M., 56, 77, 83 Berger, S., 231, 242, 246 Berger-Jo¨ nsson, R., 2, 85 Bergeron, J. J. M., 98, 135 Berglin, M., 61, 73 Beri, R. K., 101, 102, 130, 134, 141 BerkaloV, C., 198, 204 Berkowitz, G. A., 214, 256 Berndt, M. L., 193, 195, 200, 207 Bernhardt, C., 237, 246 Bertini, L., 109, 139 Bevan, M. W., 99, 117, 126, 129, 133, 138, 230, 240, 253 Bhadury, P., 58, 73 Bhalerao, R., 108, 135 Bhat, S. R., 159, 173 Biesenthal, C. J., 159, 172 Birdsey, R., 12, 88 Bischof, K., 190, 199, 201, 207 Bjo¨ rn, A., 2, 85 Blackshaw, R. E., 144, 171 Blatt, M. R., 211, 214, 222, 223, 246, 254 Blevins, D. G., 257 Bligny, R., 197, 205, 231, 232, 234, 235, 252 Block, M. A., 231, 232, 234, 235, 252 Bloom, L. M., 106, 136 Blundell, T. L., 240, 255 Bockman, O. C., 210, 250 Boettcher, A. A., 6, 7, 8, 15, 18, 19, 73, 88 Boland, G. J., 154, 172 Boland, W., 3, 33, 34, 47, 62, 63, 64, 66, 67, 73, 78, 82, 83, 85, 86 Boles, E., 108, 126, 130, 136, 138 Boller, T., 124, 141 Bolognesi-Winfield, A. C., 240, 255 Bolser, R. C., 35, 36, 73 Bonants, P. J. M., 151, 168 Bonini, B. M., 107, 130 Boo, S. M., 67, 69, 74 Booth, C. R., 17, 77 Borchardt, S. A., 61, 73 Borisjuk, L., 117, 140 Boronat, A., 113, 132 Borrell, E. M., 42, 49, 52, 73 Bosch, I., 17, 80 Bosland, P. W., 151, 168, 171 Bosse, D., 231, 254 Bothwell, J. H. F., 223, 224, 238, 248, 252 Botstein, D., 106, 136 Bouarab, K., 27, 50, 61, 73, 85, 177, 193, 194, 195, 199, 200, 201, 205, 207 Bouchard, J. N., 178, 201 Boucherau, A., 216, 246 Boucherez, J., 216, 248 Bouchez, D., 216, 223, 248, 257

AUTHOR INDEX Bouly, J. P., 108, 109, 130 Bourdo˚ t, G. W., 162, 168, 169 Bovell, C. R., 38, 76 Bowen, H. C., 217, 218, 227, 231, 232, 233, 234, 235, 237, 238, 240, 247, 249, 257 Boyer, P. D., 226, 250 Boyette, C. D., 153, 162, 163, 166, 169, 172, 174 Boyle, C., 153, 170 Branch, G. M., 6, 14, 18, 20, 21, 43, 46, 89 Branscheid, A., 116, 140 Bratanova, M., 231, 249 Brawley, S. H., 33, 62, 73, 77, 178, 204 Breeman, A. M., 178, 207 Breitling, R., 214, 217, 218, 219, 220, 221, 225, 226, 227, 246 Bremer, G., 58, 78 Brennan, A. B., 70, 74 Brennan, C. H., 102, 130 Breton, C., 237, 252 Breton, G., 111, 134 Breuer, F., 108, 135 Breune, A., 230, 232, 252 Brinch-Pedersen, H., 230, 247 Brindley, A. A., 184, 204 Briskin, D. P., 222, 247 Britton, C. J., 67, 69, 72 Britton, G., 198, 204 Broadgate, W. J., 61, 73 Broadley, M. R., 217, 218, 227, 229, 230, 231, 232, 233, 234, 235, 237, 238, 240, 242, 243, 244, 247, 249, 256 Brocard, I. M., 127, 130 Brocard-GiVord, I. M., 126, 130 Brock, E., 17, 24, 25, 26, 28, 49, 55, 57, 83 Brodsky, M., 98, 131 Broekaert, W. F., 153, 154, 173 Brouwer, M., 153, 154, 173 Brouwer, P. E. M., 39, 73 Brown, J. F., 153, 171 Brown, K. M., 230, 232, 237, 243, 249, 251, 256 Brown, M. S., 101, 139 Brown, M. T., 179, 188, 189, 197, 199, 200, 202, 205 Browning, K., 98, 130 Brownlee, C., 179, 188, 189, 197, 199, 200, 202, 205, 223, 224, 238, 248, 252 Bruggemann, E., 240, 254 Brun, H., 145, 152, 171 Bruneau, D., 216, 248 Brunner, S., 241, 253 Bryant, J. B., 55, 74 Bryant, J. P., 54, 74 Bubni, T. S., 39, 56, 78 Bucciarelli, B., 235, 240, 241, 242, 251 Bucher, M., 211, 230, 231, 241, 250, 253 Buchner, P., 117, 140

261

Buchwaldt, L., 155, 168 Buechler, J. A., 112, 140 Buma, A. G. J., 178, 207 Bunoust, O., 107, 137 Burleigh, S. H., 231, 234, 241, 247 Burritt, D. J., 186, 199, 202 Burrus, M., 120, 131 Buschmann, H., 126, 140 Bush, D. R., 213, 247 Bustos, R., 230, 233, 235, 243, 248 Butler, A., 184, 202 Butow, B., 183, 202 C Caboche, M., 240, 246 Cacho, O., 144, 173 Caddick, M. X., 157, 172 Caelles, C., 113, 132 Cahill, D. M., 62, 74 Cakmak, I., 241, 251 Calenberg, M., 66, 81 Callow, J. A., 69, 70, 74, 79, 83 Callow, M. E., 58, 69, 70, 74, 76, 79, 82, 83 Camara, M., 70, 79 Camarda, L., 155, 168 Cammue, B. P. A., 153, 154, 173 Campbell, D. A., 178, 201 Campbell, W. H., 115, 129 Campos, N., 113, 132 Canivenc, G., 240, 243, 252 Caracuel, Z., 159, 172 Card, R., 126, 138 Carling, D., 101, 102, 103, 106, 130, 131, 134, 141 Carlson, C. A., 101, 130 Carlson, M., 102, 104, 106, 130, 134, 135, 141 Carpenter, L. J., 61, 74, 183, 202 Carr, H., 20, 83 Carswell, C., 239, 247 Carswell, M. C., 211, 230, 232, 239, 253 Carter, J. N., 184, 202 Carvalho, A. G. V., 58, 75 Caspar, T., 121, 130, 136 Casson, S. A., 232, 237, 247 Caudwell, F. B., 112, 140 Cavalcanti, D. N., 30, 31, 33, 84 Ceh, J., 49, 74 Celenza, J. L., 104, 106, 130 Cerezo, M., 241, 242, 251 Cerny, R., 115, 136 Cervin, G., 11, 15, 17, 24, 27, 49, 57, 84 Cetrulo, G. L., 41, 74 Chan, C. W. M., 111, 134 Chang, L.-Y., 98, 130 Chapin, F. S., III, 54, 55, 74 Chapman, A. R. O., 20, 75 Charnley, A. K., 157, 168

262

AUTHOR INDEX

Charudattan, R., 145, 166, 168, 172 Chavant, L., 120, 131 Chen, C. Y., 151, 152, 169 Chen, D. L., 243, 247 Chen, H. M., 231, 234, 235, 240, 241, 243, 249 Chen, J.-J., 98, 131 Chen, Y., 6, 74, 150, 151, 174 Cheng, C.-L., 117, 131 Cheng, W.-H., 124, 125, 137, 211, 252 Cherel, I., 216, 253 Cheung, P. C. F., 102, 141 Chevalier, F., 230, 232, 247 Chiou, T.-J., 241, 247 Cho, G. Y., 67, 69, 74 Choi, J. H., 111, 134 Choi, Y.-J., 149, 169 Chong, K., 98, 137 Choo, K. S., 61, 71, 184, 190, 201, 202 Chopra, V. L., 159, 170, 173 Chu, S. P., 223, 246 Chua, N. H., 127, 136 Chudeck, J. A., 178, 190, 203 Chumley, F.G., 163, 170 Ciereszko, I., 232, 239, 247 Cigelnik, E., 151, 168 Ciocalteu, V., 8, 76 Ciriacy, M., 108, 138 Clare, A. S., 58, 78, 82 Clarke, P. R., 101, 112, 130, 131, 136 Clarkson, D. T., 238, 241, 247, 256 Clarkson, J. M., 157, 168 Clayton, J. C., 114, 134 Clayton, M., 3, 39, 90 Clayton, M. N., 6, 9, 10, 16, 27, 47, 54, 65, 66, 74, 81, 82, 85, 87, 198, 206 Cleland, R. E., 38, 82 Clement, C., 120, 131 Cochrane, M. P., 99, 132 Cocuera, L. J., 232, 252 Coelho, S. M., 188, 189, 197, 199, 200, 202, 206 Coen, L. D., 18, 19, 88 Cogliatti, D. H., 238, 241, 247 Cohen, B. A., 156, 167 Cohen, P., 101, 115, 131, 138 Cole, D., 103, 108, 118, 129, 132 Coleman, E. G., 216, 253 Coleman, R. A., 42, 49, 52, 73, 74 Coleman, S. E., 69, 70, 79 Colepicolo, P., 180, 181, 182, 183, 190, 191, 199, 201, 202, 205, 206 Coley, P. D., 55, 56, 74, 81 Colin, C., 61, 74, 184, 202 Collen, J., 181, 182, 183, 184, 185, 186, 187, 189, 190, 191, 192, 199, 202, 203, 205, 206 Collins, D. O., 59, 80 Colot, V., 129

Conkling, M. A., 117, 131 Conlan, S., 99, 129 Connan, S., 18, 19, 74 Connick, W. J., 162, 166, 169 Conover, J. T., 16, 87 Conroy, J. P., 124, 138 Cook, R. P., 146, 169 Cope, M., 62, 74 Copp, B. R., 39, 56, 78 Cordevant, C., 3, 70, 85 Corke, F., 126, 138 Corpas, F. J., 187, 204 Correa, J., 177, 191, 193, 194, 195, 199, 201, 205 Cortassa, S., 118, 129 Corton, J. M., 101, 131 Coruzzi, G., 213, 247 Costa, S., 224, 238, 248 Cotterill, H. L., 227, 247 Coupland, G., 157, 172 Coutinho, R., 58, 75 Cowley, S., 115, 132 Cox, T., 102, 133 Craigon, D. J., 231, 235, 249 Cramer, C. L., 180, 182, 197, 201 Crawford, N. M., 115, 139, 211, 247 Crawford, R. M., 112, 121, 131, 139 Creach, A., 198, 203 CreV, A., 231, 232, 234, 235, 252 Cristinsin, M., 117, 131 Croft, K. P., 157, 172 Cronin, G., 3, 7, 12, 22, 24, 25, 31, 35, 40, 47, 53, 55, 56, 57, 58, 74, 75 Cruz-Rivera, E., 3, 15, 30, 31, 33, 36, 49, 56, 75, 83 Cubit, J., 3, 81 CueV, A., 3, 70, 85 Cuin, T. A., 226, 257 Culioli, G., 58, 82 Culley, T. M., 150, 173 Cunningham, S. M., 215, 255 Cybinski, D. H., 241, 254 Czibener, C., 218, 253 D da Gama, B. A. P., 36, 49, 53, 57, 58, 75, 84, 90 Daigle, D. J., 162, 166, 169 Dalby, A. R., 184, 204 Dale, S., 112, 113, 129, 131 Damiani, I., 227, 252 Damsz, B., 241, 250 Daniel-Vedele, F., 241, 242, 251 Danna, C. H., 218, 253 Danon, A., 224, 250 Daram, P., 241, 253 D’Ascenzo, M., 8, 72 da Silva, J. A. L., 184, 201

AUTHOR INDEX da Silva, J. J. R. F., 184, 201 Dasycladus, 179 Dat, J. F., 183, 203 Davenport, R., 227, 247 Davies, J. M., 223, 224, 238, 248, 252 Davies, J. P., 111, 131 Davies, S. P., 101, 102, 103, 112, 131, 134, 136 Davisin, P. A., 240, 255 Davison, I. R., 176, 177, 178, 182, 183, 184, 185, 186, 187, 190, 199, 202, 203, 205 Davison, M., 101, 134 Davison, M. D., 102, 130, 141 Davoult, D., 198, 203 Dawes, C. J., 10, 76 Dayton, L. B., 49, 75 Dayton, P. K., 3, 38, 49, 75 Deal, M. S., 11, 14, 29, 39, 40, 75 ´ vila, Z. R., 166, 169 De A Degenhardt, K. J., 160, 161, 169 de Haro, C., 98, 139 De Jong, M. D., 162, 168, 169 Delage, L., 61, 73, 74, 184, 202 de la Pen˜ a, A., 230, 231, 234, 240, 243, 247, 252 Delatorre, C. A., 235, 242, 243, 247, 255 Delhaize, E., 235, 237, 241, 248, 254 del Pozo, J. C., 230, 231, 234, 240, 243, 247, 252 del Rio, L. A., 187, 204 De Lucia, E., 56, 77, 83 De Mello, S. C. M., 166, 169 Demidchik, V., 224, 238, 248 Denby, K. J., 116, 133 Deng, X. W., 231, 232, 234, 235, 237, 238, 242, 244, 256 Denis, W., 8, 76 Dennison, K. L., 227, 247 Denno, R. F., 42, 75 Denny, M. W., 46, 89 Denton, A., 20, 75 de Nys, R., 3, 12, 58, 88 de Oliveira, M. C., 190, 206 De Paula, J. C., 33, 89 Depledge, M. H., 178, 204 Desai, S., 101, 135 Deslandes, E., 18, 19, 20, 25, 40, 74, 88 Dethier, M. N., 3, 18, 19, 27, 35, 54, 77, 89 DeValerio, J. T., 145, 168 Devault, A., 98, 135 DeVit, M. J., 104, 132 Dewbre, G. R., 218, 231, 241, 254 Dickerson, K., 102, 141 Dickinson, C. H., 149, 169, 170 Dickinson, H. G., 124, 132 Dickinson, J. R., 98, 103, 104, 108, 109, 115, 118, 129, 132, 139, 141 Dietz, K. J., 225, 253

263

Dinkelaker, B., 230, 238, 247 Dinoor, A., 145, 168 Diouris, M., 48, 52, 75 Di Pietro, A., 159, 172 Dixon, R. A., 60, 80, 193, 204 Dixon, S. J., 181, 206 Diyabalange, T. K., 39, 72 Dolan, L., 224, 238, 248 Donaghy, P., 112, 115, 139, 140 Donahue, J. L., 180, 182, 197, 201 Donato, R., 33, 84 Dong, B., 241, 248 Donkin, M. E., 178, 204 Doonan, J., 157, 172 Do¨ rmann, P., 230, 232, 249 Dorner, J. W., 162, 166, 169 Douce, R., 197, 205 Douglas, P., 115, 132, 137 Doumas, P., 230, 231, 232, 234, 235, 240, 242, 243, 244, 245, 247, 252 Draisma, S. G. A., 67, 69, 75 Drew, M. C., 238, 240, 241, 248 Dring, M. J., 175, 178, 179, 198, 203, 204 Druehl, L. D., 17, 18, 24, 88 Du, J. S., 117, 133 Dube´ , A. J., 146, 169 Dubreucq, B., 240, 246 DuVus, C. M., 99, 132 DuVy, J. E., 30, 31, 33, 38, 75, 78 Duggins, D. O., 3, 18, 19, 27, 35, 45, 54, 76, 77, 89, 90 Duke, M. V., 155, 169 Duke, S. O., 155, 167, 169 Dummermuth, A., 182, 183, 190, 199, 201, 203 Dunbar, R. B., 59, 85 Dunlop, 233 Dunton, K., 56, 76 Durante, K. M., 55, 79 D’Urzo, M. P., 241, 250 Dusenbery, D. B., 65, 76 Duval, J. C., 198, 204 Dyck, J. R. B., 102, 133 Dzeha, T. M., 49, 74 E Earle, E. D., 159, 171 Eastmond, P. J., 124, 132 Eastwood, D. C., 231, 232, 233, 234, 235, 237, 238, 240, 249 Eckman, J. E., 45, 76 Edelman, A. M., 101, 112, 131, 134 Edwards, D. M., 178, 190, 203 Edwards, P., 52, 76 Edyvane, K., 12, 88 Eggermont, K., 153, 154, 173 Ehlig, J. M., 10, 13, 20, 89, 90 Ehrhardt, T., 222, 256

264

AUTHOR INDEX

Eijsackers, M., 188, 206 Eisenreich, W., 113, 132 Ekdahl, A., 183, 184, 189, 199, 202, 205 Elliot, M., 145, 168 Elthon, T. E., 115, 136 Elwakil, M. A., 166, 172 Elwing, H., 61, 73 Elzam, O. E., 211, 248 Elzinga, N., 242, 256 Emmerson, Z., 231, 235, 249 Eng, F. J., 104, 130 England, P. J., 197, 205 Enjuto, M., 113, 132 Entian, K. D., 106, 132, 138 Eppley, R. W., 38, 76 Epstein, E., 211, 248 Epstein, I., 211, 248 Epstein, L., 9, 90 Eranen, J., 14, 19, 22, 25, 26, 55, 57, 79 Erturk, N., 180, 181, 197, 201 Esch, J. J., 240, 255 Essigmann, B., 230, 232, 248 Estes, J. A., 7, 8, 9, 12, 14, 19, 88, 91 Eucheuma, 179 Evidente, A., 155, 174 F Fagerberg, W. R., 10, 76 Fairhead, V. A., 18, 21, 39, 42, 45, 46, 76 Falkowski, P. G., 3, 76 Fan, X., 6, 74 Fargues, J., 162, 172 Farre, E. M., 116, 140 FedoroV, N., 60, 81 Fedorova, M., 230, 231, 252 Fegley, J. C., 190, 199, 203 Feinberg, A., 70, 74 Felenbok, B., 157, 158, 171 Felle, H. H., 222, 248 Feng, M. G., 163, 169 Fenical, W., 11, 14, 15, 29, 30, 31, 33, 35, 39, 40, 56, 59, 61, 75, 76, 78, 80, 183, 205 Fenner, M., 52, 74 Ferguson, W. S., 241, 249 Fernandez, S., 102, 133 Fernie, A. R., 213, 254 Ferrari, K. E., 28, 50, 73 Ferre, P., 101, 141 Filipe, S., 184, 201 Filleur, S., 211, 244, 248 Finkelstein, R. R., 126, 127, 130, 132, 137 Finlay, J. A., 69, 70, 79 Fisch, K. M., 183, 203 Fitt, B. D., 146, 171 Fitter, A. H., 238, 243, 244, 251, 256 Fleming, A. J., 119, 138 Fletcher, R. L., 16, 69, 76

Fleury, B., 31, 76 Flintham, J. E., 127, 136 Floc’h, J. Y., 48, 52, 75, 76 Flowers, K. M., 98, 137 Foggo, A., 42, 49, 52, 73 Folin, O., 8, 76 Folster, E., 62, 66, 67, 73 Fontes, E. M. G., 166, 169 Foote, C. S., 187, 203 Forbes, S. M., 125, 139 Forde, B. G., 99, 132, 211, 230, 238, 244, 248, 256 Foreman, J., 224, 238, 248 Foretz, M., 101, 141 Forster, R. M., 189, 203 Foster, M. S., 3, 86 Foster, R., 178, 190, 203 Foufelle, F., 101, 141 Foyer, H. F., 183, 203 Frachisse, J. M., 197, 205 Fraenkel, G. S., 65, 77 Franchi, G. G., 120, 132, 138 Francois, J., 118, 140 Franco-Zorrilla, J. M., 230, 233, 235, 239, 243, 248 Franklin, L. A., 178, 189, 198, 203, 204 Frederick, J. E., 17, 77 Freile-Pelegrin, Y., 58, 77 Freshney, N. W., 115, 132 Fridovich, I., 192, 203 Friedlander, M., 60, 90, 193, 195, 196, 199, 206, 207 Fritsch, F. E., 48, 62, 77 Fritzius, T., 124, 141 Frommer, W. B., 125, 126, 129, 134, 136, 138, 140 Fryer, L. G., 102, 141 Fu, H., 116, 117, 132 Fu, Y. B., 142 Fujii, F., 231, 232, 234, 255 Fujimori, T., 145, 170 Fukuhara, Y., 67, 68, 69, 77 Fukumoto, F., 149, 172 Fulcher, R. G., 10, 77 Furbank, R., 240, 250 Furrow, F. B., 39, 40, 56, 72 G Gage, D. A., 115, 129 Gai, Y., 159, 172 Gaines, S. D., 35, 77 Galabru, J., 98, 137 Galatis, B., 48, 80 Gale, M. D., 127, 136 Gall, E. A., 18, 19, 74, 183, 184, 204 Gampala, S. S. L., 127, 132 Gan, Y., 211, 244, 248 Gansel, X. Z., 241, 242, 251

AUTHOR INDEX Gantet, P., 237, 252 Gao, G., 102, 133, 137 Gaquerel, E., 50, 73 Garcia-Pedrajas, M. D., 159, 169 Gardiner, 233 Garmier, M., 153, 154, 173 Garvin, D. F., 231, 234, 242, 255 Gassman, G., 63, 83 Gassmann, W., 222, 254 Gaughran, J. P., 118, 140 Gaymard, F., 216, 248, 253 Gebhardt, C., 126, 129 Gehrke, L., 98, 131 Geigenberger, P., 116, 140 Geilen, E. F. M., 39, 73 Geiselman, J., 11, 77 Gelderblom, W. C. A., 155, 169 Geller, A., 62, 66, 77 Gething, P. A., 210, 248 Gevaert, F., 198, 203 Ghannoum, O., 124, 138 Gibb, B. J., 112, 136 Gibbard, C. L., 114, 134 Gibon, Y., 116, 140 Gibson, A., 70, 74 Gibson, D. M., 101, 129 Gibson, S. I., 127, 135 Gierth, M., 217, 218, 249 Giese, H., 99, 133 Gillespie, J. G., 101, 112, 131, 133, 136 Gilson, P., 219, 254 Giordano, F., 155, 174 Giordano, M., 178, 203 Gissot, L., 108, 109, 130 Glass, A. D., 227, 252 Glassop, D., 230, 231, 241, 254 Glawe, G. A., 42, 77 Glombitza, K.-W., 4, 5, 6, 7, 8, 15, 16, 18, 85, 184, 198, 206 Glund, K., 231, 250 Go¨ bel, C., 231, 253 Goddijn, O., 124, 138 Gohbara, M., 155, 169 Gojon, A., 241, 242, 251 Gold, S. E., 159, 169 Goldstein, E. G., 101, 134 Goldstein, J. L., 101, 139 Gomez, I., 56, 61, 81, 90 Go´ mez-Cadenas, A., 111, 133 Gonzalez, A., 237, 240, 246, 256 Gonza´ lez, E., 230, 233, 235, 243, 248 Goodman, H. M., 127, 132, 240, 254 Goodwin, K., 61, 82, 183, 184, 203 Gordon, R., 62, 77 Gossett, D. R., 187, 203 Goudriaan, J., 162, 169 Goulard, F., 18, 19, 74 Gout, E., 197, 205 Govenor, H. L., 8, 72

265

Grabov, 223 Gradmann, D., 222, 246 Graham, I. A., 116, 124, 132, 133, 230, 240, 252 Graham, L. E., 3, 48, 62, 68, 70, 77 Graham, N. S., 257 Graham, R. D., 120, 133, 241, 250 Granbom, M., 183, 201 Grane´ li, E., 2, 85 Grant, B. R., 239, 243, 247, 249 Grant, N. T., 154, 169 Grawe, A., 184, 206 Gray, J. C., 240, 255 Green, D. B., 214, 253 Green, D. G., 241, 249 Green, H., 155, 168 Green, P. J., 230, 246 Green, S., 162, 169 Greenhalgh, J. R., 149, 169, 170 Greer, G. P., 67, 69, 72 Greer, S. P., 65, 67, 69, 70, 77, 79 Gremmen, N. J. M., 39, 73 Gressel, J., 154, 156, 162, 163, 167, 170, 173 Grey, W. E., 153, 163, 172 Gribskov, M., 111, 134 Grierson, C., 117, 133 Grill, E., 223, 246 Gros, P., 98, 135 Grossman, A. R., 111, 131 Grotewold, E., 240, 250 Grundy, S. M., 113, 135 Gschwend, P. M., 61, 77, 183, 203 Guarro, J., 159, 172 Guern, J., 60, 80, 193, 196, 197, 199, 204, 205 Guesdon, F., 115, 132 Guest, D., 243, 249 Gu¨ ler, S., 230, 232, 248 Gunn, D. L., 65, 77 Guske, S., 153, 170 Gustafson, K., 30, 31, 33, 78 Gustafsson, L., 107, 130 Guyer, D., 99, 133 H Hagerman, A. E., 7, 8, 9, 14, 88 Halford, N. G., 93, 98, 99, 103, 108, 109, 111, 112, 113, 115, 116, 118, 119, 120, 121, 124, 125, 126, 129, 131, 132, 133, 134, 136, 138, 139, 140, 141 Halfter, U., 111, 133, 136 Hall, J. L., 222, 249 Hall, Q., 124, 125, 137, 211, 252 Hallett, S. G., 154, 162, 166, 171 Halsband-Lenk, C., 70, 90 Hama, Y., 6, 87 Hamburger, D., 239, 249

266

AUTHOR INDEX

Hamer, J. E., 163, 170 Hamilton, J., 56, 77, 83 Hammerstrom, K., 27, 54, 77 Hammond, J. P., 209, 217, 218, 227, 229, 230, 231, 232, 233, 234, 235, 237, 238, 240, 242, 243, 244, 247, 249 Hammond-Kosack, M. C. U., 99, 129, 133 Hampton, C. R., 217, 218, 249 Hanelt, D., 178, 190, 199, 201, 207 Hannappel, U., 108, 124, 134, 136 Haque, A., 159, 173 Hara, I., 184, 203 Haran, S., 231, 249 Hardham, A. R., 62, 74 Hardie, D. G., 101, 102, 103, 106, 108, 111, 112, 113, 115, 121, 124, 125, 129, 130, 131, 133, 134, 136, 137, 139, 140, 141 Haritonidis, S., 191, 199, 206 Harker, M., 114, 134, 198, 204 Harmon, A. C., 111, 115, 129, 134, 135 Harper, J. F., 111, 134 Harper, M. K., 39, 56, 78 Harrington, C. L., 223, 246 Harris, J., 239, 247 Harris, R., 70, 90 Harrison, M. J., 218, 231, 234, 241, 247, 254 Harrison, P. J., 3, 48, 55, 79, 81 Ha¨ rtel, H., 230, 232, 249 Harter, K., 116, 134 Hartung, W., 225, 238, 240, 241, 244, 250, 253 Harvell, C. D., 48, 78 Harvey, I. C., 162, 168 Harwood, J. L., 18, 87 Hatch, W. I., 25, 28, 50, 55, 72, 73 Hatta, R., 152, 170 Haug, A., 18, 87 Hawksford, M. J., 257 Hawley, S. A., 101, 102, 106, 125, 131, 134, 140, 141 Hay, M. E., 7, 11, 12, 14, 15, 22, 24, 25, 29, 30, 31, 33, 34, 35, 36, 38, 39, 40, 41, 46, 47, 49, 51, 52, 53, 55, 56, 57, 58, 59, 73, 74, 75, 78, 80, 86, 87, 89 Haystead, T. A. J., 102, 131 He, Z., 230, 237, 249 Heap, I., 144, 170 Heath, L. S., 180, 181, 197, 201 Hecht, H. J., 184, 207 Heikkila¨ , N., 15, 79 Heil, M., 42, 78 Heim, M. A., 240, 246 Heineke, D., 125, 134 Heldt, H. W., 125, 134 Hellio, C., 58, 78, 82 Hellmann, H., 126, 129, 136 Hellyer, S. A., 114, 134 Helps, N. R., 101, 131

Hemmi, A., 11, 14, 15, 22, 25, 27, 29, 48, 49, 52, 55, 57, 78, 79 Hendriks, J. H. M., 116, 140 Hengeler, C., 230, 238, 247 Henrikson, A. A., 58, 78 Henry, B. E., 91, 91 Herbers, K., 125, 134 Herbert, R. B., 6, 78 Herms, D. A., 57, 79 Herna´ ndez, J. A., 187, 204 Hernandez, J. M., 240, 250 Herna´ ndez-Abreu, E., 243, 251 Herrero, P., 106, 134, 138 Herrerra-Estrella, L., 230, 231, 232, 234, 235, 243, 251, 252 Hertel, S. C., 231, 254 Herzyk, P., 217, 246 Hester, S., 144, 173 Hetherington, S. D., 163, 165, 166, 168 Hey, S., 111, 120, 133 Heyworth, A., 99, 132 Hick, T., 48, 79 Hiebert, E., 145, 168 Higenbotham, N., 38, 82 Higgins, J., 231, 235, 249 Hinnebusch, A. G., 96, 97, 98, 134, 137, 139, 140 Hirner, B., 126, 129 Hirsch, R. E., 218, 254 Ho, T.-H. D., 111, 133 Hoagland, R. E., 154, 155, 159, 170 Hoch, W. A., 230, 249 Hodge, A., 232, 238, 249 HoVmann, T., 223, 246 Hofgen, R., 108, 136 Hofmann, B., 116, 135 Høgh-Jensen, H., 215, 225, 249 Hohmann, S ., 106, 140 Holappa, L. D., 111, 133 Holdsworth, M., 99, 117, 127, 129, 133, 135, 136 Holk, A., 232, 244, 253 Holl, A. C., 198, 203 Hollenberg, C. P., 108, 130 Holliday, M. J., 154, 170 Holm, P. B., 230, 247 Holmberg, M., 114, 134 Holmes, J., 70, 88 Hong, S.-B., 149, 169 Hong, S. P., 106, 134 Honkanen, T., 6, 8, 9, 11, 14, 15, 16, 19, 22, 25, 26, 27, 28, 29, 42, 48, 49, 52, 55, 57, 78, 79, 80 Hopp, E., 99, 133 Hopp, H. G., 207 Hoppe, H. G., 60, 90, 193, 195, 196, 199, 206 Horak, R. M., 155, 169 Horn, M. H., 13, 14, 79

AUTHOR INDEX Hosaki, Y., 152, 170 Hou, X. L., 231, 232, 234, 235, 237, 238, 240, 241, 242, 243, 244, 249, 256 House, C., 102, 137 Houser, L. T., 41, 90 Hovanessian, A. G., 98, 137 Howard, C. J., 230, 246 Howard, R. J., 163, 170 Howlett, B. J., 152, 170 Hoyer, K., 178, 207 Hoyos, M. E., 231, 234, 235, 255 Hrabak, E. M., 111, 134 Hsuan, J., 115, 132 Huang, C., 241, 250 Huang, J. Z., 112, 135 Hubbard, J. M., 39, 40, 56, 72 Huber, J. L., 115, 129 Huber, S. C., 112, 115, 121, 129, 130, 135, 136, 139, 140 Humanes, M., 184, 201 Humpherson-Jones, F. M., 152, 160, 161, 170 Hunt, B. A., 157, 168 Hunt, T., 115, 138 Huntsman, S., 190, 198, 206 Hurd, C. L., 55, 79, 179, 186, 199, 202, 204, 205 Hurrell, G. A., 162, 168 Hurry, V., 232, 239, 240, 247, 250 Hustead, C. L., 18, 19, 20, 44, 46, 57, 90 Hwang, I., 124, 125, 135, 137 Hylands, P. J., 113, 132 I Icely, P. L., 98, 135 Ichida, A. M., 222, 251 Iglesias, J., 231, 234, 235, 237, 240, 243, 252, 253 Iken, K. B., 3, 34, 39, 40, 56, 63, 64, 65, 67, 69, 70, 72, 77, 79, 90 Ilvessalo, H., 18, 20, 25, 44, 55, 89, 91 Imaizumi, S., 145, 170 Inagaki, M., 6, 87 Inoue, H., 184, 206 Irani, N. G., 240, 250 Ireland, C. D., 13, 14, 39, 56, 78, 79 Ishitani, M., 111, 133, 136 Ista, L. K., 69, 70, 79 Isupov, M. N., 184, 204 Ito, K., 152, 170 Ito, N., 120, 135 Itoh, N., 61, 83, 183, 184, 205 Izumi, Y., 183, 184, 204, 205, 206 J Jackson, B. M., 96, 97, 98, 137, 140 Jackson, M. A., 162, 166, 169 Jacobs, R. S., 192, 198, 199, 206

267

Jaenicke, L., 62, 63, 64, 66, 67, 73, 82, 83 Jaglan, V. D., 230, 246 Jahnke, L. S., 188, 204 Jain, A., 231, 232, 234, 235, 252 James, C. M., 240, 255 James, R., 144, 173 James, R. D., 39, 56, 78 Jang, J. C., 116, 124, 135, 138, 242, 253 Jansa, J., 241, 253 Jaskiewicz, M. K., 155, 169 Jay-Allemand, C., 237, 252 JeVerson, L. S., 98, 137 Jeger, M. J., 153, 170 Jelitto, T. C., 222, 246, 257 Jennings, A. R., 70, 74 Jennings, J. G., 16, 17, 21, 79 Jensen, A., 18, 24, 85, 86 Jensen, J. S., 155, 168 Jensen, O. N., 128, 137 Jensen, P., 241, 245 Jensen, P. R., 59, 80 Jeschke, W. D., 238, 240, 241, 244, 250 Jhurreea, D., 111, 120, 133 Jia, Q. J., 231, 234, 235, 240, 241, 243, 249 Jiang, R., 104, 106, 135, 141 Jiao, F. C., 231, 234, 235, 240, 241, 243, 249 Joern, B. C., 215, 255 Johansson, G., 61, 71, 184, 201 Johansson, H., 232, 239, 247 Johnson, A. S., 183, 187, 199, 204 Johnson, C. R., 12, 46, 79 Johnson, C. S., 240, 250 Johnson, D. L., 162, 171 Johnson, R. D., 151, 170 Johnston, M., 104, 108, 132, 138 Johnstone, S. R., 102, 141 Joint, I., 70, 79, 83 Jones, E., 115, 132 Jones, H., 120, 141 Jones, H. D., 127, 135 Jones, J. D., 124, 132 Jones, J. D. G., 224, 238, 248 Jones, O. T. G., 197, 205 Jones, P., 113, 135 Jones, R., 144, 173 Jones, R. G. W., 213, 251 Jones, T., 124, 125, 137 Jones, T. L., 211, 252 Joppa, L. R., 120, 135 Jormalainen, V., 6, 8, 9, 11, 14, 15, 16, 18, 19, 20, 22, 25, 26, 27, 28, 29, 42, 44, 48, 49, 52, 55, 57, 78, 79, 80, 89 Jouhet, J., 231, 232, 234, 235, 252 Jourdan, M., 145, 172 Jung, V., 41, 79 Jungk, A., 230, 238, 250

268

AUTHOR INDEX

K Kaarstad, O., 210, 250 Kachmar, J. F., 226, 250 Kahn, M. L., 211, 247 Kakefuda, G., 121, 130 Kalir, A., 187, 204 Kalisch, C., 144, 173 Kamiya, M., 35, 38, 86 Kamiya, Y., 109, 129 Kang, J., 227, 250 Kanrar, S., 159, 170 Karandashov, V., 230, 241, 250 Karban, R., 48, 50, 80 Karentz, D., 17, 80 Karez, C. S., 18, 80 Karley, A. J., 225, 250 Karsten, U., 178, 182, 183, 190, 199, 201, 203, 207 Karthikeyan, A. S., 231, 235, 241, 242, 246, 250, 255 Kashman, Y., 60, 90, 196, 199, 207 Katan, M. B., 113, 135 Kataoka, H., 35, 38, 86 Katsaros, C., 48, 80 Katsis, F., 102, 137 Katz, M. E., 3, 76 Kautsky, L., 2, 85 Kawaguchi, S., 6, 87 Kawai, H., 35, 38, 86 Kearns, L. J., 18, 20, 44, 46, 57, 90 Keeling, P. J., 3, 80 Keen, N. T., 154, 170 Keifer, P. A., 59, 80 Kelecom, A., 30, 31, 76, 84 Kellogg, D., 119, 139 Kelly, R. F., 61, 73 Kemp, B. E., 102, 133, 137 Kerr, A., 124, 132 Kerr, I. M., 98, 137 Kershaw, M. J., 157, 168 Kervarec, N., 3, 80 Kessler, A., 42, 50, 77, 80 Keum, Y. S., 62, 80 Khachatourians, G. G., 163, 169 Khangura, R. K., 146, 152, 171 Kharbanda, P. D., 146, 171 Kieber, D. J., 190, 198, 206 Kiene, R. P., 190, 198, 206 Kiess, M., 184, 207 Kikuchi, S., 231, 232, 234, 255 Kim, 218 Kim, C.-S., 111, 136 Kim, D. G., 127, 135 Kim, K. H., 101, 130 Kimball, S. R., 98, 137 Kimura, M., 164, 173 Kincaid, M. S., 127, 135 Kirkby, E. A., 238, 240, 241, 244, 250 Kirst, G. O., 178, 190, 198, 204

Kirti, P. B., 159, 170 Kishimoto, N., 231, 232, 234, 255 Kistler, H. C., 151, 159, 171 Kjelleberg, S., 3, 58, 88 Kleczkowski, L. A., 232, 239, 247 Klein, D. R., 54, 74 Klein, M., 108, 136 Klein, R. R., 115, 136 Kleinow, T., 101, 108, 135, 136 Klinger, T., 45, 76 Kloareg, B., 27, 50, 60, 61, 73, 74, 80, 85, 90, 177, 183, 184, 193, 194, 195, 196, 197, 199, 200, 201, 202, 204, 205, 207 Klo¨ ser, H., 56, 61, 81, 90 Knight, M. R., 223, 254 Knoll, A. H., 3, 76 Knowles, V. L., 211, 227, 254 Knudsen, S., 99, 137 Kochian, L. V., 211, 231, 234, 242, 250, 255 Ko¨ ck, M., 231, 250, 254 Koehl, M. A. R., 45, 80 Koenig, G. M., 183, 203 Koivikko, R., 6, 8, 9, 14, 19, 22, 25, 26, 42, 55, 57, 79, 80 Koizumi, N., 232, 239, 242, 255 Kolevski, B., 240, 250 Kolte, S. J., 152, 173 Koncz, C., 101, 108, 135, 136 Koornneef, M., 232, 240, 254, 255 Koponen, H., 160, 173 Koricheva, J., 56, 80 Krabs, G., 178, 204 Krane, J. M., 53, 55, 57, 84 Krapp, A., 116, 135, 241, 242, 251 Krause, K.-P., 124, 138 Kreis, M., 99, 108, 109, 118, 129, 130, 132 Krieger-Liszkay, A., 178, 204 Kriek, N. P. J., 155, 169 Krintel, C., 231, 234, 239, 241, 242, 252 Kroon, L. P. N. M., 151, 168 Kruckeberg, A. L., 108, 135 Kubanek, J., 11, 14, 15, 29, 36, 40, 59, 80, 89 Kubasek, W. L., 240, 254 Kubler, J. E., 178, 206 Kudla, J., 111, 134 Kudo, N., 101, 135 Kuepper, F., 205 Kuhlenkamp, R., 198, 204 Ku¨ hn, C., 126, 129, 138, 140 Kumar, V. D., 159, 173 Kuo, I., 98, 131 Kuo, Y.-H., 116, 141 Kupper, F. C., 206 Ku¨ pper, F. C., 60, 61, 73, 74, 80, 85, 183, 184, 192, 193, 196, 197, 198, 199, 201, 204 Kuroda, S., 231, 232, 234, 255 Kurup, S., 127, 135

AUTHOR INDEX Kusaba, M., 152, 171 Kuwamoto, S., 184, 206 Kylin, H., 38, 80 L La Barre, S. L., 3, 80 Laby, R. J., 127, 135 Lacombe, B., 216, 248 Laegreid, M., 210, 250 Lafiandra, D., 99, 139 Lages, B. G., 49, 53, 57, 90 Lahner, B., 235, 242, 243, 255 Lakatos, L., 108, 136 Laloi, C., 224, 250 Laloi, M., 241, 253 Lalonde, S., 126, 136, 140 Lamb, C., 60, 80, 193, 204 Lambert, S., 70, 88 Lamey, A. H., 146, 171 Lange, M., 3, 70, 85 Lange, R. M., 146, 171 Langewald, J., 162, 171 Langhamer, O., 14, 21, 22, 26, 29, 42, 49, 51, 89 Langlois, G. L., 16, 81 Langridge, P., 241, 250 Lanot, A., 114, 134 Larher, F., 216, 246 Larkindale, J., 186, 199, 202 Larsen, B., 18, 86 Larsson, C., 107, 130 Larsson, K. E., 230, 246 Last, R. L., 99, 142 Laturnus, F., 61, 81 Lau, S. C. K., 16, 81 Lauer, M. J., 257 Laufs, P., 157, 172 Laughlin, L. T., 226, 250 Laur, D. R., 39, 72 Laurie, S., 109, 111, 118, 120, 125, 133, 136, 139, 226, 257 Law, M., 113, 135 Laxmi, A., 116, 138, 242, 253 Lazzeri, P. A., 120, 127, 136, 141 Lea, P. J., 125, 141 Leaver, C. J., 116, 133 Leblanc, C., 61, 74, 184, 202 Leclerc, I., 101, 136 Lee, I. K., 62, 80 Lee, M. M., 237, 246 Lee, R. B., 230, 234, 250 Lee, S. H., 67, 69, 74 Leech, A., 106, 140 Leegood, R. C., 125, 141 Le Gal, Y., 58, 78 Legendre, J. M., 183, 184, 204 Le´ ger, C., 154, 171 Leggewie, G., 241, 242, 251, 253

269

Leigh, R. A., 211, 213, 220, 225, 226, 250, 251, 257 Leiper, F. C., 102, 106, 134, 141 Leitao, M. A. S., 180, 181, 182, 191, 205 Leite, B., 163, 173 Leize-Wagner, E., 61, 74, 184, 202 Lejay, L., 241, 242, 251 Lemarchand, P., 101, 141 Lemerle, D., 144, 171 Lemoine, Y., 198, 203, 204 Lenton, J. R., 127, 136 Leon, J., 220, 252 Leon, P., 124, 126, 127, 129, 135 Lepiniec, L., 240, 246 Lerdau, M., 56, 81 Lessard, P., 108, 109, 130 Lester, S. E., 11, 14, 15, 29, 40, 80 Leustek, T., 211, 247 Levin, N., 216, 246 Levine, L. H., 215, 257 Lewis, B. D., 218, 254 Lewis, B. G., 151, 170 Lewis, D. R., 218, 254 Lewis, S., 178, 204 Lewis, S. M., 3, 81 Leykam, J. F., 142 Leyser, H. M. O., 238, 243, 244, 251, 256 Leyva, A., 230, 231, 233, 234, 235, 237, 239, 240, 243, 247, 248, 252, 253 Li, A., 231, 232, 237, 244, 255 Li, D., 242, 251 Li, R., 178, 204 Li, W.-H., 150, 151, 174 Liao, P. C., 115, 129 Liaud, M. F., 184, 207 Lidstrom, M. E., 183, 184, 203 Liljenberg, C., 230, 246 Lin, T.-P., 121, 130, 136 Lindgren, A., 11, 15, 17, 18, 19, 20, 24, 27, 29, 49, 57, 84 Lindquist, N., 36, 59, 86, 89 Lindsay, B. S., 39, 56, 78 Lindsey, K., 232, 237, 247 Linhares, F., 230, 231, 233, 234, 235, 237, 240, 243, 248, 253 Linke, D., 230, 232, 248 Linkohr, B. I., 238, 251 Linstead, P., 224, 238, 248 Liss, P. S., 41, 61, 70, 73, 74, 88, 183, 202 Littlechild, J. A., 184, 204 Liu, C., 231, 234, 235, 241, 251 Liu, D., 70, 90 Liu, F., 231, 232, 234, 235, 237, 238, 242, 244, 256 Liu, H., 241, 247 Liu, J., 111, 136, 230, 231, 235, 240, 241, 242, 251, 252 Liu, J. X., 215, 256 Liu, K., 242, 251

270

AUTHOR INDEX

Liu, L.-F., 116, 141 Liu, X., 242, 251 Liu, Y.-X., 124, 125, 137, 211, 252 Lloyd, A., 237, 240, 246, 256 Lloyd, J. C., 233, 239, 240, 242, 251 Lobban, C. S., 3, 48, 81 Locci, R., 155, 168 Lo¨ Zer, A., 231, 250 Logan, B. A., 183, 187, 199, 204 Logendra, S., 231, 249 Lohrmann, N. L., 183, 187, 199, 204 Lomer, C. J., 162, 171 London, I. M., 98, 131 Long, S. R., 211, 247 Lopaschuk, G. D., 101, 135 Lopes, P. F., 190, 206 Lopez, G. P., 69, 70, 79 Lo´ pez-Bucio, J., 230, 243, 251 Lopez-Delgado, H., 183, 203 Lopez-Molina, L., 127, 136 Loponen, J., 6, 8, 9, 42, 80 Lorenzo, H., 230, 238, 248 Love, G., 161, 171 Love, H. K., 155, 173 Lowell, R. B., 81 Luan, S., 111, 134 Lubchenco, J., 3, 35, 77, 81 Lucas, M. C., 187, 203 Lu¨ der, U. H., 6, 10, 16, 27, 54, 81 Lumbreras, V., 108, 136 Lumineau, O., 3, 70, 85 Lund, A. A., 115, 136 Lund, A. L., 115, 136 Lu¨ ning, K., 63, 83, 178, 198, 203, 204 Luo, A. C., 215, 256 Lynch, J. P., 230, 232, 237, 243, 246, 249, 251, 256 Lynch, T. J., 126, 127, 130, 132, 137 M Ma, H., 106, 136 Ma, L., 231, 232, 234, 235, 237, 238, 242, 244, 256 Ma, P., 107, 130 Ma, Z., 230, 237, 249 Maathuis, F., 216, 217, 218, 222, 251 Maathuis, F. J., 257 Macaya, E. C., 53, 54, 57, 58, 81, 86 MacDonald-Comber Pete´ tot, J., 239, 249 MacFarlane, J. K., 61, 77, 183, 203 Machida, Y., 118, 137 MacKintosh, C., 115, 132, 137 MacKintosh, R. W., 112, 136 Madrid, M. P., 159, 172 Mahalingam, R., 60, 81 Maia, M. F., 184, 201 Maier, I., 34, 47, 62, 63, 64, 65, 66, 67, 81, 82, 85

Maiese, W. M., 39, 72 Ma¨ kinen, A., 15, 79 Makowski, R. M. D., 154, 169 Malamy, J. E., 230, 243, 244, 251 Malea, P., 191, 199, 206 Malin, G., 41, 61, 70, 73, 74, 88, 183, 202 Man, A. L., 124, 136 Manley, S. L., 61, 82, 183, 184, 205 Mann, K. H., 3, 12, 46, 79, 81 Mar Alba`, M., 101, 108, 136 Marasas, W. F. O., 155, 169 Marcroft, S. J., 144, 171 Marechal, J. P., 78 Mare´ chal, J. P., 58, 82 Markham, J. H., 3, 20, 75, 81 Marks, M. D., 240, 255 Marner, F.-J., 62, 63, 66, 67, 73, 83 Marschner, H., 210, 211, 214, 215, 227, 230, 238, 241, 247, 251 Marshall, L., 101, 141 Martı´n, A. C., 231, 234, 235, 237, 239, 240, 243, 248, 252, 253 Martin, T., 230, 240, 252 Martinez, E. A., 18, 82 Martinez-Campa, C., 106, 134 Martinez-Espinoza, A. D., 159, 169 Martin-Tanguy, J., 216, 246 Marton, M. J., 97, 137 Masangkay, R. F., 162, 166, 171 Ma¨ ser, P., 217, 218, 249 Mason, P. K., 14, 88 Mathieu, M., 157, 158, 171 Matilda, P. D., 235, 242, 255 Mattson, W. J., 57, 79 Mauricio, R., 42, 82 Maxwell, A, 143 May, S. T., 231, 232, 233, 234, 235, 237, 238, 240, 249, 257 Mayayo, E., 159, 172 Mazars, C., 223, 257 McCarthy, J. J., 18, 19, 20, 44, 46, 57, 90 McCartney, R. R., 106, 140 McClintock, J. B., 18, 21, 38, 39, 40, 42, 45, 46, 56, 59, 65, 67, 69, 70, 72, 76, 77, 79, 82, 85 McClintock, M., 38, 82 McClure, M. S., 42, 75 McConnell, O., 11, 77, 183, 205 McCown, B. H., 230, 249 McCoy, W. F., 61, 73 McCully, M. E., 10, 77 McKey, D., 42, 82 McKibbin, R. S., 111, 116, 118, 120, 125, 127, 133, 136, 140 McMichael, R. W., Jr., 115, 136 McNeal, F. H., 120, 135 Meacham, M. C., 227, 247 Mead, A., 217, 218, 227, 247, 249 Medd, R., 144, 170

AUTHOR INDEX Mehrtens, G., 61, 81 Meijer, G. W., 113, 140 Meikle, W., 162, 172 Meinesz, A., 41, 79 Mellor, H., 98, 137 Melo, R., 184, 201 Mendes-Pereira, E., 145, 152, 171 Mendez, R., 98, 139 Menhard, B., 113, 132 Mensink, R. P., 113, 138 Menzhuber, C. J., 230, 231, 252 Mercadier, G., 162, 172 Merlini, L., 155, 168 Metraux, J. P., 125, 134 Meurs, E., 98, 137 Meuse, B. J. D., 38, 82 Meuwly, P., 125, 134 Meyer, M. R., 97, 137 Meyers, J. H., 48, 80 Michaux-Ferriere, N., 216, 248 Michel, G., 61, 74 Michell, B., 102, 137 Michels, J. J., 61, 73 Miedema, H., 223, 224, 238, 248, 252 Miettinen, T., 113, 135 Miller, A. J., 220, 226, 257 Miller, S. S., 230, 231, 252 Millhollon, E. P., 187, 203 Milton, D., 70, 79 Mimura, T., 233, 234, 238, 239, 240, 241, 252 Misson, J., 231, 232, 234, 235, 252 Mita, S., 116, 137 Mitchelhill, K. I., 102, 137 Mitchell, A., 155, 173 Miwa, T., 38, 82 Miyabe, K., 145, 170 Mizuta, H., 67, 68, 69, 77 Moe, R. L., 39, 82 Moenne, A., 191, 199, 205 Mole, S., 8, 90 Molis, M., 49, 50, 51, 53, 54, 57, 58, 74, 81, 86, 90 Momol, E. A., 151, 159, 171 Montiel, G., 237, 252 Moore, B., 124, 125, 137, 211, 252 Moore, F., 101, 137 Moorhead, G., 115, 137 Mora, A. A., 159, 171 Morales, J. L., 58, 77 Morange, M., 115, 138 Moreau, M., 223, 257 Moreno, D. A., 226, 253 Moreno, E., 70, 88 Moreno, F., 106, 134, 138 Morin, L., 153, 162, 168, 171 Morrall, R. A. A., 160, 161, 169 Morrice, N., 115, 132, 137 Morrishige, D. T., 242, 254

271

Morse, D., 180, 181, 182, 191, 205 Mortensen, K., 154, 162, 169 Moss, B. L., 48, 82 Motta, A., 155, 174 Mouline, K., 216, 227, 252, 253 Mtolera, M. S., 184, 189, 199, 205 Muchhal, U. S., 231, 234, 235, 241, 251 Mudge, S. R., 230, 231, 241, 254 Mueller, D. G., 197, 204 Mukatira, U. T., 237, 241, 250 Muller, B., 231, 232, 240, 242, 243, 244, 245, 252 Mu¨ ller, C., 241, 242, 251 Mu¨ ller, D. G., 34, 47, 60, 62, 63, 64, 65, 66, 67, 73, 77, 80, 81, 82, 83, 85 Mu¨ ller, M., 99, 137 Mu¨ ller, R., 231, 234, 239, 241, 242, 252 Muller-Parker, G., 38, 84 Muller-Rober, B., 222, 256 Mu¨ ller-Sto¨ ver, D., 163, 164, 165, 172 Mullet, J. E., 231, 242, 246, 254 Munday, M. R., 101, 106, 131, 141 Mun˜ oz, G., 232, 252 Munz, G., 126, 138 Murakami, A., 35, 38, 86 Muranaka, T., 118, 137 Murphy, T. M., 197, 201 Murray, S. N., 12, 90 Murshudov, G. N., 184, 204 Mylona, P., 224, 238, 248 N Nacry, P., 223, 230, 231, 232, 234, 235, 240, 242, 243, 244, 245, 247, 252, 257 Nakamura, K., 116, 137 Nakamura, S., 127, 137 Nakamura, T., 6, 87 Nakano, S., 183, 184, 205 Nandiraju, S., 39, 72 Narang, R. A., 230, 232, 248, 252 Nashaat, N. I., 149, 173 Nasini, G., 155, 168 Nast, G., 222, 256 Natarajan, K., 97, 137 Nazoa, P., 227, 252 Nebreda, A. R., 115, 138 Nepi, M., 120, 132 Neumann, D., 102, 141 Neushul, M., 34, 39, 66, 67, 68, 69, 72, 83 Newman, K. A., 61, 77, 183, 203 Nicholson, R. L., 163, 173 Nielsen, H. D., 179, 205 Nielsen, T. H., 231, 234, 239, 241, 242, 252 Niere, J. O., 239, 247 Nieto-Jacobo, M. F., 230, 243, 251 Nikolaev, I., 157, 158, 171 Nilsson, L., 231, 234, 239, 241, 242, 252 Nimmo, H. G., 111, 134

272

AUTHOR INDEX

Nishino, T., 145, 170 Nitao, J., 56, 83 Nojima, H., 119, 140 Nolasco, A. C., 69, 70, 79 Nordlund, A. C., 101, 141 Norris, J. N., 3, 81 North, W. J., 61, 82, 183, 184, 203 Noubhani, A., 107, 137 Nu¨ hse, T. S., 128, 137 Nussaume, L., 231, 232, 234, 235, 252 O Oak, J. H., 62, 80 Odom, D., 144, 173 O’Donnell, K., 151, 168 O’Donnell, V., 197, 205 Ogata, Y., 61, 83, 183, 184, 205 Ohsawa, N., 61, 83, 183, 184, 205 Ohshiro, T., 183, 184, 205, 206 Okada, N., 61, 83, 183, 184, 205 Okamoto, O. K., 180, 181, 182, 190, 191, 205, 206 Oleskevich, C., 154, 171 Olmos, E., 187, 204 Olsen, J. L., 67, 69, 75 Olssen, T., 111, 112, 120, 140 Ortalo-Magne´ , A., 58, 82 Ortet, P., 231, 232, 234, 235, 252 Ortoneda, M., 159, 172 Osaki, M., 231, 232, 234, 255 ¨ stling, J., 104, 137 O Ostlund, R. E., Jr., 113, 138 Oswald, O., 230, 240, 252 Oxley, D., 219, 254 Ozcan, S., 108, 138 P Pacini, E., 120, 132, 138 Page`s, M., 101, 108, 136 Paine, R. T., 49, 75 Pal, J. K., 98, 131 Palacios, N., 116, 140 Paoletti, R., 113, 135 Park, W. D., 116, 117, 132 Park, Y. C., 39, 72 Parkin, B. T., 145, 174 Pasaribu, A., 39, 72 Pata, M., 230, 232, 247 Pate, J. S., 238, 240, 241, 244, 250 Patel, P., 70, 83 Paterson, I. C., 157, 168 Patrick, J. W., 126, 136 Patton, D., 99, 133 Paul, M., 120, 126 Paul, M. J., 98, 111, 124, 133, 138, 141, 142, 230, 232, 240, 252 Paul, R. N., 155, 169

Paul, V. J., 3, 4, 19, 30, 31, 35, 40, 49, 55, 56, 75, 83, 85, 88, 89 Paulitz, T. C., 162, 166, 171 Pavgi, M. S., 149, 173 Pavia, H., 7, 8, 11, 12, 14, 15, 16, 17, 18, 19, 20, 21, 22, 24, 25, 26, 27, 28, 29, 42, 44, 45, 49, 50, 51, 52, 54, 55, 57, 83, 84, 89, 90 Pawlik, J. R., 38, 58, 78, 85 Payne, K. A., 217, 218, 249 Payne, T., 237, 240, 256 Payri, C. E., 18, 19, 20, 25, 40, 88 Paz-Ares, J., 230, 231, 233, 234, 235, 237, 239, 240, 243, 247, 248, 252, 253 Pearce, G., 48, 86 Pearson, G. A., 176, 177, 178, 187, 203, 205 Peck, S. C., 128, 137 Peckol, P., 7, 8, 11, 21, 24, 25, 27, 28, 49, 53, 55, 57, 84, 91 Pedersen, M., 61, 71, 181, 182, 183, 184, 189, 190, 191, 192, 199, 201, 202, 205, 206, 215, 225, 249 Pedras, M. S. C., 155, 159, 172 Pelletreau, K. N., 25, 38, 55, 84, 90, 190, 206 Pellny, T. K., 124, 138, 142, 230, 232, 240, 252 Pen˜ aloza, E., 232, 252 Penninckx, I. A. M. A., 153, 154, 173 Pereira, R. C., 10, 12, 18, 19, 30, 31, 33, 36, 49, 53, 57, 58, 73, 75, 76, 80, 84, 89, 90 Perez-Amador, M. A., 220, 252 Pe´ rez-Torres, A., 243, 251 Pervez, M. K., 33, 71 Peters, A. F., 33, 39, 60, 63, 71, 80, 83, 84, 197, 204 Peters, J., 214, 256 Peters, K. J., 39, 40, 56, 59, 72, 85 Peterson, C. H., 54, 85 Petrie, C., 38, 82 Petrie, G. A., 153, 160, 161, 168, 169 Petters, J., 231, 253 Pettersen, M. E., 145, 168 Peuke, A. D., 238, 240, 241, 244, 250 Pfister, C. A., 18, 20, 21, 25, 30, 31, 44, 55, 78, 85 Phelps, K., 152, 160, 170 Phillips, D. V., 146, 171 Phillips, J. A., 47, 66, 85 Phillips, J. C., 179, 205 Piel, J., 34, 78 Pien, S., 119, 138 Pilot, G., 216, 248, 253 Pinloche, S., 231, 232, 242, 244, 245 Pinto, E., 180, 181, 182, 191, 199, 202, 205 Pisut, D., 38, 85 Plat, J., 113, 138 Plaut, Z., 121, 140

AUTHOR INDEX Plaxton, W. C., 211, 227, 230, 232, 239, 247, 253, 254 PoVenroth, M., 214, 253 Pohnert, G., 3, 27, 33, 34, 41, 50, 61, 62, 66, 70, 79, 85, 86, 90, 193, 194, 195, 200, 207 Poirier, Y., 239, 249, 255 Polacco, J. C., 231, 234, 235, 255 PoljakoV-Mayber, A., 187, 204 Pollard, A., 211, 214, 226, 256 Poole, R. J., 222, 247 Poore, A. G. B., 12, 13, 18, 20, 46, 47, 70, 85 Poprawski, T. J., 163, 169 Porter, S. A., 145, 174 Potin, P., 3, 27, 50, 60, 61, 73, 74, 80, 85, 177, 184, 193, 194, 195, 196, 197, 199, 200, 201, 202, 204, 205, 207 Potter, T. D., 144, 171 Poulet, S., 3, 70, 85 Poulet, S. A., 90 Powell, J., 162, 169 Powles, S. B., 145, 174 Prakash, S., 159, 173 Preiss, J., 121, 130, 136, 142 Prescha, K., 116, 140 Price, J., 116, 138, 242, 253 Prieto, J. A., 106, 138 Pritchard, J., 217, 218, 249 Prusinkiewicz, E., 154, 169 Pugin, A., 197, 205 Puglisi, M. P., 3, 4, 55, 83, 85 Pulido, D., 98, 139 Punja, Z. K., 154, 171 Purcell, P. C., 112, 118, 119, 124, 133, 136, 138 Purrington, C. B., 42, 85 Pywell, J., 99, 132 Q Qi, Z., 218, 254 Qian, P. Y., 16, 81 Qu, Z., 17, 77 Quetin, L. B., 39, 72 Quick, W. P., 125, 141 Quigg, A., 3, 76 Quimby, P. C., 153, 162, 163, 166, 167, 169, 172 R Ra˚ berg, S., 2, 85 Rae, A. L., 230, 231, 241, 254, 257 Ragan, C. M., 18, 86 Ragan, M. A., 4, 5, 6, 7, 8, 15, 16, 18, 24, 85, 86 Raghothama, K. G., 229, 231, 232, 234, 235, 241, 242, 246, 250, 251, 252, 253, 255

273

Rahn, C., 231, 232, 233, 234, 235, 237, 238, 240, 249 Rains, D. W., 211, 248 Rakow, G. F. W., 153, 168 Ramı´rez-Rodriguez, V., 230, 251 Ramirez-Ya´ n˜ ez, M., 231, 232, 237, 244, 255 Ramı´rz, M. E., 39, 84 Rampey, R. A., 243, 251 Randez-Gil, F., 106, 138 Ranjeva, R., 223, 257 Rao, S., 127, 132 Raschke, K., 214, 253 Raskin, I., 231, 249 RatcliVe, R. G., 234, 250 Ratkevicius, N., 191, 199, 205 Rausch, C., 211, 230, 231, 241, 253 Rausher, M. D., 42, 82 Raven, J. A., 3, 48, 76, 86, 178, 203, 206 Rawlins, S., 114, 134 Rawlinson, L., 115, 132 Reckmann, U., 214, 253 Reed, D. C., 34, 66, 72, 86 Reed, G. H., 226, 250 Reed, R. H., 178, 190, 203, 206 Reid, R. J., 234, 257 Reifenberger, E., 108, 138 Reinders, A., 126, 138 Renaud, P. E., 30, 54, 57, 78, 85, 86 Rengel, Z., 241, 248 Reynolds, S. E., 157, 168 Rezzonico, E., 239, 249, 255 Rhoades, D. F., 42, 57, 86 Rhoads, D. M., 115, 136 Ribeiro, Z. M. D., 166, 169 Ribot, C., 239, 255 Ribrioux, S. P. C., 243, 244, 256 Richard, C., 60, 90, 196, 199, 207 Richards, F. J., 216, 253 Richardson, A. D., 39, 56, 78 Richardson, A. E., 235, 237, 241, 254 Richardson, M., 108, 118, 129 Rietz, S., 232, 244, 253 Rigg, G. B., 38, 91 Rigoulet, M., 107, 137 Rijstenbil, J. W., 188, 191, 199, 206 Rindings, H. I., 163, 168 Ripley, V. L., 155, 173 Rmiki, N. E., 198, 204 Roald, T., 18, 87 Roberts, C., 97, 137 Robilliard, G. A., 49, 75 Robinson, D., 232, 238, 253 Robson, F., 157, 172 Rock, C. D., 127, 132 Rodriguez Huete, A. M., 231, 234, 235, 255 Roebroeck, E. J. A., 151, 168 Rohde, S., 49, 50, 51, 86 Rolland, F., 106, 124, 125, 137, 138, 211, 252, 253

274

AUTHOR INDEX

Rolland, N., 231, 232, 234, 235, 252 Romero, L., 226, 253 Roncero, M. I. G., 159, 172 Rong, L., 215, 255 Ronne, H., 104, 111, 112, 120, 137, 140 Rook, F., 126, 138, 230, 240, 253 Rosahl, S., 231, 253 Rosario, K., 190, 206 Rose, M., 106, 138 Roslan, H. A., 157, 172 Ross, C., 192, 198, 199, 206 Ross, R. M., 39, 72 Rossa, M. M., 190, 206 Rossignol, M., 230, 231, 232, 240, 242, 243, 244, 245, 247, 252 Rotem, J., 152, 161, 166, 172 Roth, D., 98, 130 Rotha¨ usler, E., 53, 54, 57, 58, 81, 86 Rouse, J., 115, 138 Rousseau, B., 198, 204 Rouxel, T., 145, 152, 171 Rovirosa, J., 33, 87 Rowley, R. J., 39, 72 Roy, S., 178, 201 Rubio, V., 230, 231, 234, 235, 237, 240, 243, 247, 248, 252, 253 Ruiz, J. M., 226, 253 Ryan, C. A., 48, 86 Ryan, K. P., 188, 189, 197, 199, 200, 202 Ryan, P. R., 241, 248 S Sabelli, P. A., 108, 118, 129 Sadka, A., 231, 242, 246, 254 SaVord, R., 114, 134 Saharan, G. S., 160, 174 Saini, H. S., 120, 138 Sajeena, A., 166, 168 Sakata, K., 231, 232, 234, 255 Saker, L. R., 240, 241, 248 Saklatvala, J., 115, 132 Sakurai, A. A., 109, 129 Sakurai, T., 184, 203 Salanoubat, M., 117, 139 Salau¨ n, J.-P., 27, 50, 61, 73, 85 Salchert, K., 108, 135 Saleema, M., 33, 71 Salt, D. E., 235, 242, 243, 255 Salter, M. G., 157, 172 Salvo-Garrido, H., 232, 252 Salvucci, M. E., 115, 136 Samac, D. A., 235, 240, 241, 242, 251 Sampson, P. J., 146, 172 Samuel, C. E., 96, 139 Sa´ nchez-Caldero´ n, L., 243, 251 Sandelius, A. S., 230, 246 Sanders, D., 222, 224, 225, 245, 246, 250, 251, 257

Sanderson, J., 256 San-Martin, A., 33, 87 Santa-Maria, G. E., 218, 253 Santoyo, J., 98, 139 Sanz, P., 106, 138 Sasaki, H., 35, 38, 86 Sasaki, T., 231, 232, 234, 255 Sato, F., 232, 239, 242, 255 Sato, R., 101, 139 Satou, M., 149, 172 Sattlegger, E., 98, 139 Sauder, C. A., 150, 174 Sauerborn, J., 163, 164, 165, 172 Sauter, A., 225, 253 Saville, D. J., 162, 168 Sawall, T., 190, 199, 207 Say, M. M., 163, 168 Scarabel, M., 115, 137 Scattergood, C. B., 238, 241, 247, 256 Schachtman, D. P., 217, 219, 224, 226, 234, 245, 254, 257 Scha¨ fer, C., 116, 135 Scha¨ fer, E., 116, 134 Scheel, D., 231, 253 Scheibe, R., 214, 253 Scherer, G. F. E., 232, 244, 253 Schiefelbeim, J., 237, 246 Schiel, D. R., 3, 86 Schikora, A., 237, 254 Schjør˚ ring, J. K., 241, 245 Schlattner, U., 102, 141 Schluepmann, H., 124, 138, 142 Schmid, C., 64, 82 Schmidt, M. C., 106, 140 Schmidt, W., 237, 254 Schmitt, T. M., 57, 59, 86 Schnabel, P. C., 39, 56, 78 Schnick, P. J., 154, 172 Schnitzler, I., 33, 34, 78, 86 Schoenwaelder, M. E. A., 4, 6, 9, 10, 17, 43, 86, 87, 198, 206 Schofield, O., 3, 76 Schomburg, D., 184, 207 Schriek, R., 182, 183, 190, 199, 201 Schroeder, J. I., 217, 218, 222, 223, 246, 249, 251, 254 Schultz, C., 219, 254 Schultz, J. C., 8, 72 Schulz, A., 126, 138 Schulz, B., 126, 129, 153, 170 Schulze, W., 126, 129, 138 Schumacher, K., 223, 246 Schu¨ nmann, P. H. D., 235, 237, 241, 254 Schurath, J., 231, 254 Schweigert, N., 183, 184, 204 Scott, I. M., 183, 203 Scott, J., 102, 106, 130, 141, 143, 145, 172 Scrase-Field, A. M. G., 223, 254 Searles, R. B., 3, 81

AUTHOR INDEX Sedgley, M., 120, 138 Seegert, C. E., 70, 74 Seetharaman, K., 166, 168 Seferlis, M., 191, 199, 206 Se´ guin-Swartz, G., 151, 152, 153, 168, 169 Selbert, M. A., 101, 134 Semesi, A. K., 184, 189, 199, 205 Sentenac, H., 216, 248, 253 Seskar, M., 231, 249 Seuront, L., 198, 203 Severino, N., 184, 201 Sevilla, F., 187, 204 Shabana, Y. M., 163, 164, 165, 166, 172 Shamoun, S. F., 154, 171 Sharma, S. R., 152, 159, 173 Sharon, A., 154, 173 Sharp, P. M., 151, 174 Sheen, J., 116, 124, 125, 126, 127, 129, 135, 137, 139, 211, 252, 253 Shelton, K. L., 67, 69, 72 Shen, Q., 111, 133 Shephard, G. S., 159, 173 Sherson, S. M., 125, 139 Sheu, G., 116, 141 Sheu, Y.-J., 116, 141 Shewry, P. R., 99, 108, 111, 112, 118, 120, 129, 134, 139, 141 Shibata, T., 6, 87 Shimbo, K., 231, 232, 234, 255 Shimeneck, C., 70, 88 Shimonishi, M., 184, 206 Shin, H., 218, 231, 241, 254 Shin, H.-D., 149, 169 Shin, H.-S., 218, 231, 241, 254 Shin, R., 217, 219, 224, 226, 234, 245, 254 Shinano, T., 231, 232, 234, 255 Shinozaki, K., 225, 257 Shiraishi, N., 115, 129 Shirley, B. W., 240, 254 Shivas, R. G., 146, 173 Shu, J.-K., 111, 133, 136 Siddon, C. E., 45, 76 Sidebottom, C., 102, 130 Sieburth, J. M., 16, 87 Sierzputowska-Gracz, H., 257 Sigaud-Kutner, T. C. S., 180, 181, 182, 191, 205 Silue´ , D., 149, 173 Silva, H., 232, 252 Silva, P. C., 39, 82 Sim, A. T. R., 101, 131 Simmonds, J. H., 146, 173 Simons, R. H., 69, 70, 79 Simpson, J., 243, 251 Simpson, M. T., 184, 202 Sinden, J., 144, 173 Singh, M. P., 39, 72 Singh, S. L., 149, 173 Sire´ n, S., 18, 20, 44, 55, 89

275

Siska, E., 8, 72 Skipnes, O., 18, 87 Slade, D., 97, 137 Slocombe, S. P., 109, 111, 112, 115, 129, 139 Smeekens, S., 124, 126, 132, 138, 139, 142, 242, 256 Smidsrød, O., 18, 86 Smith, A. B., 144, 171 Smith, A. M., 112, 118, 119, 138 Smith, C., 99, 117, 126, 129, 133, 138 Smith, C. R., 211, 227, 254 Smith, F. C., 102, 141 Smith, F. W., 230, 231, 241, 250, 254, 257 Smith, H. E., 163, 165, 166, 168 Smith, K. L. C., 18, 87 Smith, S. M., 125, 139 Smyth, D. R., 240, 250 Snijman, P. W., 159, 173 Snoeijs, P., 61, 71, 184, 190, 201, 202 Snow, A. A., 150, 173 Soares, A. R., 36, 84 Solano, R., 231, 234, 235, 237, 240, 243, 248, 252, 253 Somerville, C., 121, 130, 136, 239, 249 Somerville, S., 231, 232, 234, 235, 252 Song, X. W., 231, 234, 235, 240, 241, 243, 249 Sonnewald, U., 125, 134 Sorensen, L. D., 230, 247 Sotka, E. E., 29, 38, 46, 49, 51, 52, 87, 89 Soto, H., 33, 87 Sousa-Pinto, I., 188, 189, 197, 199, 200, 202 Southon, T. E., 234, 250 Southworth, K. J., 38, 82 Sowokinos, J. R., 117, 139 Spalding, E. P., 218, 227, 247, 254 Spencer, N. Y., 67, 69, 72 Spielman, M., 124, 132 Sreenivasan, A., 119, 139 Srinivasan, N., 240, 255 Stam, W. T., 67, 69, 75 Stamp, N., 42, 57, 87 Stapleton, D., 102, 133, 137 Stark, M. J., 106, 140 Stearns, G. W., 61, 73 Stecher, H., 70, 88 Stein, S. C., 101, 141 Steinberg, P. D., 3, 7, 8, 9, 12, 14, 15, 16, 17, 18, 19, 20, 21, 24, 27, 30, 31, 43, 56, 58, 78, 79, 87, 88 Steinke, M., 41, 61, 70, 88 Stensballe, A., 128, 137 Stenzel, I., 231, 254 Stern, J. L., 7, 8, 9, 14, 88 Stewart, W. D. P., 178, 190, 203 Stiger, V., 18, 19, 20, 25, 40, 74, 88 Stitt, M., 116, 135, 140, 213, 240, 250, 254 St Martin, S. K., 116, 138, 242, 253 Storz, G., 240, 254

276

AUTHOR INDEX

Stout, P. R., 211, 246 ˚ ., 240, 250 Strand, A Stridh, M. H., 230, 246 Stringam, G. R., 155, 173 Strom, S., 70, 88 Stutte, G. E., 215, 257 Su, W., 115, 139 Subbarao, G. V., 215, 257 Sugden, C., 112, 121, 139, 140 Sugui, J. A., 163, 173 Sullards, M. C., 59, 80 Sun, G., 231, 235, 249 Sun, S. S. M., 127, 132 Sunda, W., 190, 198, 206 Sundene, O., 179, 206 Sundstrom, J., 184, 206 Sussman, M. R., 111, 134, 218, 254 Sutherland, C. M., 106, 140 Sutton, B. C., 150, 173 Suzuki, M., 183, 184, 205 Suzuki-Fujii, K., 116, 137 Swanson, A. K., 17, 18, 24, 88 Swarup, R., 231, 232, 233, 234, 235, 237, 238, 240, 249 Sweetingham, M. W., 146, 152, 171 Syers, J. K., 210, 254 T Tai, P.-Y., 150, 151, 174 Tait, K., 70, 79 Takahashi, M., 191, 201 Takahashi, Y., 183, 184, 205 Talbot, N. J., 163, 173 Talke, I., 222, 256 Talke-Messerer, C., 116, 134 Tallman, G., 214, 253 Tanabe, T., 184, 204, 206 Tanaka, A., 152, 170 Tanaka, S., 119, 140 Tanaka, T., 152, 155, 167, 170 Tang, Y. Y., 223, 246 Tang, Z., 242, 254 Tanner, C. E., 18, 19, 25, 28, 50, 55, 72, 73, 88 Tardieu, F., 231, 232, 242, 244, 245 Targett, N. M., 3, 4, 6, 7, 8, 10, 14, 15, 18, 19, 22, 23, 28, 29, 40, 42, 50, 72, 73, 88 Targett, T. E., 7, 8, 15, 19, 88 Tasdemir, D., 39, 56, 78 Tatchell, K., 118, 140 Tavernier, E., 197, 205 Taylor, A. R., 188, 189, 197, 199, 200, 202 Taylor, C. B., 230, 246 Taylor, F. J. R., 3, 76 Taylor, R. B., 29, 36, 46, 49, 51, 52, 87, 89 Taylor, S. S., 112, 140 Tegeder, M., 126, 136

Teixeira, V. L., 30, 31, 33, 36, 73, 76, 84, 89 Tel-Or, E., 183, 202 Terlinden, R., 62, 73 Tester, M., 211, 214, 254, 257 Tew, D. G., 197, 205 Thacker, R. W., 3, 30, 49, 56, 83 The, T., 102, 133 Thelander, M., 111, 112, 120, 140 Theodorou, M. E., 239, 247 Thevelein, J. M., 106, 107, 130, 137, 138, 140 Thibaud, J. B., 216, 248 Thibaud, M.-C., 231, 232, 234, 235, 252 Thibaut, T., 41, 79 Thiel, M., 53, 54, 57, 58, 81, 86 Thiel, P. G., 155, 169 Thion, L., 223, 257 Thomas, M., 108, 109, 111, 130, 134, 162, 171 Thomas, N. S. B., 98, 137 Thomas-Guyon, H., 58, 82 Thomma, B. P. H. J., 153, 154, 173 Thompson, A., 61, 73 Thompson-Jaeger, S., 118, 140 Throne-Holst, M., 126, 136 Throop, M. S., 98, 131 Thuleau, P., 223, 257 Ticconi, C. A., 230, 235, 239, 242, 243, 255 Tierens, K. F. M.-J., 153, 154, 173 Tiessen, A., 116, 140 Tillard, P., 227, 241, 242, 251, 252 Tirilly, Y., 149, 173 Tissier, A. F., 124, 132 Todd, C. D., 231, 234, 235, 255 Tomsett, A. B., 157, 172 Toroser, D., 121, 140 Torres, M. A., 224, 238, 248 Toth, G. B., 7, 12, 14, 18, 19, 20, 21, 22, 24, 25, 26, 27, 28, 29, 42, 44, 45, 49, 50, 51, 52, 54, 55, 84, 89 Touraine, B., 227, 252 Townsend, H., 231, 235, 249 Toyota, K., 164, 173, 232, 239, 242, 255 Tranbarger, T. J., 227, 231, 232, 242, 244, 245, 252 Trewavas, A., 223, 255 Trick, C. G., 181, 206 Trigon, S., 115, 138 Trimmer, P., 257 Tsuge, T., 152, 170 Tsuke, T., 152, 171 Tucker, S. L., 163, 173 Tugwell, S., 6, 14, 18, 20, 21, 43, 46, 89 Tuomi, J., 18, 20, 25, 44, 55, 89, 91 Turano, F. J., 227, 250 Twiner, M. J., 181, 206

AUTHOR INDEX U Udvardi, M., 242, 251 Uhde-Stone, C., 211, 229, 230, 231, 232, 233, 235, 237, 243, 244, 255 Umeda, M., 108, 135 Uribe, E. G., 38, 82 Uthus, K. L., 150, 173 Utter, B. D., 46, 89 V Valent, B., 163, 170 Valkonen, J. P. T., 160, 173 Vallim, M. A., 33, 89 Van Alstyne, K. L., 3, 7, 8, 10, 11, 13, 18, 19, 20, 24, 25, 27, 30, 35, 40, 41, 44, 46, 48, 49, 50, 55, 57, 83, 85, 89, 90, 91, 91, 190, 206 van Altena, I., 12, 15, 18, 24, 88 Vance, C. P., 211, 229, 230, 231, 232, 233, 235, 237, 240, 241, 242, 243, 244, 251, 252, 255 Van Dam, N. M., 42, 77 van de Poll, W. H., 178, 207 Van der Plank, J. E., 160, 174 van de Vondervoort, P. J. I., 157, 158, 171 Van Dijck, P., 107, 130 van Dijken, A. J., 124, 132, 142 Van Dorsselaer, A., 61, 74, 184, 202 van Lent, F., 39, 73 Van Onckelen, H., 240, 243, 252 van Reine, W. F. P., 62, 67, 69, 75, 80 Van Vaeck, C., 107, 130 Van Wagoner, R. M., 39, 56, 78 Varadarajan, D. K., 235, 241, 242, 250, 255 Varns, J. L., 117, 139 Vega, F., 162, 172 Velimirov, B., 38, 72 Venkateswari, J. C., 159, 170 Verbitski, S. M., 39, 56, 78 Verburg, M. T., 230, 246 Vere, D., 144, 170 Verhey, S. D., 111, 133 Verhoeven, A. J. M., 102, 130 Verma, P. R., 160, 174 Vermeij, G. J., 35, 90 Veron, B., 58, 78 Vesonder, R. F., 154, 171 Vicente-Carbajosa, J., 108, 134 Vickers, C. E., 235, 237, 241, 254 Vidmar, J. J., 227, 252 Villaca, R., 36, 84 Vilter, H., 61, 73, 183, 184, 204, 206, 207 Visser, J., 157, 158, 171 Vitou, J., 145, 172 Vleggaar, R., 155, 169 Voegele, B., 190, 199, 207 Volenec, J. J., 215, 255 Volkov, V., 211, 246

277

von Schaewen, A., 125, 141 von Wire´ n, N., 241, 242, 251 Vreeland, V., 9, 90, 192, 198, 199, 206 Vreugdenhil, D., 232, 255 Vrolijk, N. H., 7, 8, 15, 19, 88 Vurro, M., 155, 174 W Waalwijk, C., 151, 168 Waddle, J. A., 104, 132 Wagner, A., 178, 198, 203 Wagner, E., 61, 74, 184, 202 Wahl, M., 49, 50, 51, 53, 54, 57, 58, 74, 81, 86, 90 Wahl, T., 63, 83 Wainwright, S., 45, 80 Waite, J. H., 9, 90, 192, 198, 199, 206 Wakefield, R. L., 12, 90 Walch-Liu, P., 211, 244, 248 Walker, A. R., 240, 255 Walker, D. J., 220, 257 Walker, H. L., 162, 174 Walker, J., 146, 172 Walker-Simmons, K., 111, 135 Walker-Simmons, M. K., 111, 129, 133 Wallace, A. D., 114, 134 Wallace, G., 125, 139 Wallimann, T., 102, 141 Walsh, C. T., 106, 136 Walsh, M. J., 145, 174 Walsh, R. D., 39, 72 Wang, 218 Wang, D., 242, 251 Wang, M. L., 127, 132, 231, 232, 234, 235, 237, 238, 242, 244, 256 Wang, W., 215, 256 Wang, Y., 239, 255 Wang, Y.-H., 231, 234, 242, 255 Ward, D. E., 155, 159, 172 Ward, E., 99, 133 Ward, J. M., 126, 129, 136, 138, 140, 222, 254 Warder, F. G., 241, 249 Wares, J. P., 38, 87 Warwick, S. I., 150, 174 Wasaki, J., 231, 232, 234, 255 Watanabe, M., 178, 204 Waterman, P. G., 8, 90 Watson, A. K., 154, 162, 166, 171, 174 Weber, H., 117, 140 Weekes, J., 101, 112, 113, 129, 136, 137, 140 Weidner, K., 49, 53, 57, 90 Weinberger, F., 3, 60, 80, 90, 177, 193, 194, 195, 196, 199, 200, 201, 206, 207 Weise, A., 126, 129, 140 Weisshaar, B., 240, 246 Wek, R. C., 96, 98, 140, 141 Wek, S. A., 98, 141

278

AUTHOR INDEX

Welsh, J. R., 120, 135 Wenden, A., 65, 82 West, J. S., 146, 171 Westrate, J. A., 113, 140 Wever, R., 184, 201, 206 Weyand, M., 184, 207 Weykam, G., 56, 90 Weyland, M., 207 Wheeler, R. M., 215, 257 White, A. L., 188, 204 White, M. R. H., 157, 172 White, P. J., 209, 217, 218, 227, 229, 230, 231, 232, 233, 234, 235, 237, 238, 240, 242, 243, 244, 247, 249, 256, 257 Whitman, S. L., 10, 13, 20, 89, 90 Wichard, T., 70, 90 Widmer, J., 102, 133 Wiemken, A., 124, 141 Wiencke, C., 3, 39, 56, 61, 81, 90, 178, 182, 183, 190, 198, 199, 201, 203, 204, 206, 207 Wiese, A., 242, 256 Wijnholds, J. A., 191, 199, 206 Wilcox, L. W., 3, 48, 62, 68, 70, 77 Wildenberger, K., 125, 134 Wilkinson, M. D., 127, 136 Wilkstro¨ m, S. A., 8, 16, 90 Williams, B. R. G., 98, 137 Williams, C. C., 99, 142 Williams, L. E., 211, 222, 249, 256 Williams, P., 70, 79 Williams, P. H., 145, 151, 152, 168, 174 Williamson, L. C., 238, 243, 244, 251, 256 Willmitzer, L., 125, 134 Wilson, D., 11, 14, 29, 39, 40, 75 Wilson, L., 70, 74 Wilson, W. A., 106, 112, 125, 131, 141 Win, H., 39, 72 Winder, W. W., 101, 141 Winderickx, J., 106, 107, 130, 138 Wingler, A., 124, 125, 141 Winter, F. C., 7, 8, 9, 12, 14, 19, 88, 91 Wirth, H. E., 38, 91 Witters, L. A., 101, 102, 133, 137, 141 Wobbes, B., 124, 142, 242, 256 Wobus, U., 117, 140 Wojtaszek, P., 60, 91, 180, 182, 183, 193, 197, 198, 207 Wolfe, G., 70, 88 Wolfe, K. H., 151, 174 Wood, C. D., 157, 172 Woods, A., 101, 102, 106, 130, 131, 134, 141 Woolaway, K. E., 231, 232, 233, 234, 235, 237, 238, 240, 249 Wo¨ sten, H. A. B., 163, 174 Wratten, S., 52, 76 Wright, P. C., 58, 73

Wu, P., 231, 232, 234, 235, 237, 238, 240, 241, 242, 243, 244, 249, 256 Wu, W., 214, 256 Wu, Y., 231, 232, 234, 235, 237, 238, 242, 244, 256 Wyn Jones, R. J., 211, 214, 226, 256 Wynne, D., 183, 202 Wyrzykowska, J., 119, 138 X Xu, C., 230, 232, 256 Y Yakovleva, I., 178, 203 Yamada, M., 145, 170 Yamagishi, M., 231, 232, 234, 255 Yamaguchi, K., 6, 87 Yamaguchi-Shinozaki, K., 225, 257 Yamamoto, K., 231, 232, 234, 255 Yamamoto, M., 152, 170 Yan, X., 6, 74 Yang, R. J., 98, 141 Yang, W. Y., 98, 130 Yang, X., 104, 106, 141 Yang, X. E., 215, 256 Yang, Y.-W., 150, 151, 174 Yasui, H., 67, 68, 69, 77 Yates, J. L., 7, 8, 11, 21, 24, 25, 27, 28, 49, 53, 55, 57, 84, 91 Yazaki, J., 231, 232, 234, 255 Ye, Z. Q., 215, 256 Yi, K. K., 231, 234, 235, 240, 241, 243, 249 Yildiz, F. H., 111, 131 Yonemoto, W., 112, 140 Yoneshigue-Valentin, Y., 10, 12, 19, 58, 75, 84 Yonetani, R., 231, 232, 234, 255 Yoo, B. C., 115, 129 Yoshikawa, M., 154, 170 Youl, J., 219, 254 Young, A. J., 198, 204 Yu, B., 230, 232, 256 Yu, J., 231, 234, 235, 240, 241, 243, 249 Yu, S.-M., 116, 141 Z Zabala, M. D., 117, 133 Zaharia, I. L., 155, 159, 172 Zakhleniuk, O. V., 233, 239, 240, 242, 251 Zamanillo, D., 115, 138 Zammit, V. A., 101, 130 Zangerl, A., 56, 77, 83 Zavala, J. A., 42, 77 Zaworotko, M. J., 39, 72 Zeldin, E. L., 230, 249 Zeng, P., 231, 234, 235, 255

AUTHOR INDEX Zenk, M. H., 113, 132 Zhang, F., 237, 240, 246, 256 Zhang, H., 211, 256 Zhang, Y., 98, 111, 120, 133, 141 Zhang, Y.-J., 237, 256 Zhao, J., 99, 142 Zhao, M., 237, 240, 256 Zhou, L., 124, 125, 126, 127, 129, 135, 137, 211, 252

Zhou, Y., 159, 172 Zhu, H., 242, 251 Zhu, J.-K., 111, 135 Zhuo, D., 227, 252 Zidack, N. K., 153, 162, 163, 167, 172 Ziethe, K., 231, 254 Zimmermann, S., 222, 256 Zinn, K. E., 231, 232, 237, 244, 255 Zonno, M. C., 155, 174

279

SUBJECT INDEX

A AAL toxins, 155, 159 ABA, 225 sugar signalling and, 126–7 ABI4, 127 ABI5 protein, 127 Abscisic acid, sugar and, 126–7 Abutilon theophrastii, 154, 156–7 ACA1, 218 ACC oxidase, 232 Acclimation responses, to potassium deficiency, 215 Acetate-malonate pathway, 6 Acetogenins, 33 Acrocarpia paniculata, 9 Acrochaete operculata, 193, 200 sulphated oligosaccharide signalling in, 194 Acrosiphona penicilliformis, 182 ADC2. See Arginine decarboxylase Adenocystis utricularis, 40 50 -adenosine monophosphate (AMP), 101 as signalling molecule, 121 ADP-glucose pyrophosphorylase (AGPase), 115–16 glucose and, 117 sucrose and, 117 AVymetrix full genome arrays, 217 Agarum fimbriatum, 54 AGPase. See ADP-glucose pyrophosphorylase AGPs. See Arabinogalactin proteins AIR1, 232 AIR3, 232 AIR9, 232 AIR12, 232 AKIN10, 111 AKIN11, 111 AKIN , 108 Alaria marginata, 20, 43 Alaria nana, 20 Alternaria alternata, 152, 160 Alternaria brassicae, 152, 157, 160, 166 Alternaria brassicola, 152, 160, 166 Alternaria japonica, 152, 153, 155, 161, 166 Alternaria species, 162 in plant debris, 161–2 properties of, 160 temperature and, 160–1 AMARA peptide, 112 Amino acid signalling, 96–101

GCN2 and, 98–101 in yeast, 96–8 AMP. See 50 -adenosine monophosphate AMP-activated protein kinase (AMPK) activation of, 101 in carbon metabolite signalling, 101–3 components of, 103 function of, 101 pathways for activation of, 102 subunits of, 102 Ampithoe longimana, 33, 34, 36 Ampithoe valida, 11, 15 AMPK. See AMP-activated protein kinase AMT. See Arabidopsis membrane transporter Ansates pellucida, 27 Antibacterial activity, phlorotannins in, 15–17 Antifouling activity, phlorotannins in, 15–17 Antiherbivore defenses activated, 40–1 of desmarestiales, 38–9 of dictyotales, 30–8 for various orders, 39–40 Antiherbivory roles, of phlorotannins, 10–14 Antioxidants, 186 Aphanomyces candida, 166 Aphanomyces raphani, 149, 153, 163, 166 APX, 187, 190 Arabidopsis phylogenetic tree of, 236 reductases of, 113 transgenic, 120 Arabidopsis membrane transporter (AMT), 216 Arabidopsis thaliana, 214 Arabinogalactin proteins (AGPs), 219 Arbacia punctulata, 11, 33, 34, 39–40 Arginine decarboxylase (ADC2), 220 Ascophyllum nodosum, 11, 16, 18, 21, 24, 27, 45, 49–50 defense induction in, 51–2 Ascorbate-glutathione pathway, reactions of, 182 Aspergillus nidulans, 157 GUS activity of, 158 Aspergillus niger, 157 GUS activity of, 158 AtACP5, 243 AtAMT1.1, 242 AtGCN2, 98–9 identification of, 99

282

SUBJECT INDEX

AtNSR1, 234 AtNSR2, 234 AtPAP1, 240 AtPAP2, 240 AtPDR2, 243 AtPHR1 transcriptional cascade, 228, 234, 235, 241 AtSNF4, 108 AtSTP1, 125 AtTTG1, 237, 240 AtTTG2, 237, 240 AtWER, 237 B Bacteria, defenses against, 58–60 Balanus improvisus, 16 bHLH, 234, 240 Biocontrol agents, hypervirulent, 156 Biopesticide fungi, 126 Biosynthesis, phlorotannin, 22 Biotrophic pathogens, of wild radish, 149 Brassica napus, 155 Brassica nigra, phylogenetic tree for, 151 Brassica oleracea, phylogenetic tree for, 151 Brassica rapa, 150 phylogenetic tree for, 151 Brassica tournefortii, 144 Brassicaceae, problems caused by, 144–5 Brefeldin A, 155 Brown algae bacteria defenses of, 58–60 chemical defense theories and, 41–58 chemical ecology of, 3 chemical studies of, 2 defense induction in, 49 feeding deterrents eVects in, 29 fouling organisms and, 58–60 in freshwater systems, 3 growth defense trade-oVs in, 23 pathogens and, 58–60 pheromone chemical structure of, 63 VHOC release in, 61 bZIP genes, 242 C CACGTd sequence, 235 Calcium (Ca) phosphorous and, 233–4 potassium and, 220–4 CaMV35S, 119 Capsella bursapastoris, 144 Carbon, arriving at sink organs, 95 Carbon metabolites, 101–26 glucose induction and, 107–8 regulation of, 103–8 glucose repression, 103–7 SNF1, 103–7

SnRK1 and, 108–9 SnRK2 and, 109–11 SnRK3 and, 109–11 Carbon-nutrient balance hypothesis (CNBH), 54–7 Catalase, 182, 187 CAX3, 218 CDC28 complex, 118 CdhGTGG sequence, 235 Cebidichthys violaceus, 12, 14 Cell cycle control, SnRK1 in, 118–19 Cell wall bound forms, 6, 9 structure, 9 Cell wall proteins, encoding of, 219 Cellular ion homeostasis, 212 Cellular processes, metabolic status and, 95 Cellular signalling events phosphorous and, 233–4 potassium and, 220–5 Cercosporin, 155 Chaetomorpha linum, 182, 186 Chaetomorpha melagonium, 182 Chelation, heavy metal, 18 Chemical communication, 2 Chemical defense theories brown algae and, 41–58 CNBH, 54–7 induced defense, 48–54 optimal defense, 42–8 Chemical ecology of brown algae, 3 defined, 2 macroalgal, 35 sensory, 62–70 chemoattraction to pheromones, 62–7 spore capabilities, 67–70 Chemoattraction, to pheromones, 62–7 Chemokinetic responses, 65 of Ectocarpus, 66 Chemotactic responses, 65 Chemotaxis, of Laminaria digitata, 64 Cholesterol, 113 structure of, 114 Chondrus crispus, 183, 186–7, 193 sulphated oligosaccharide signalling in, 194 CNBH. See Carbon-nutrient balance hypothesis Colletotrichin, 155 Colletotrichum coccodes, 154, 156 Colletotrichum gloeosporoides, 154 Colletotrichum glucosporoides, 150 Colletotrichum higginsianum, 150, 157 Colletotrichum orbiculare, 163 Colorimetric assays, 23 Conidia, 163 hydrophobic, 163 COR78, 226

SUBJECT INDEX Crop yield, 99 Cropping conditions, epidemiology and, 160–2 Cutleria, 62 Cutleria multifida, 67 Cyclic-nucleotide gated channels, 218 Cytokinin, 232, 243 D D1 protein, 178 Dasycladus vermicularis, 192, 198 DCDF-DA, 192, 196–7 Defense induction in Ascophyllum nodosum, 51 in brown algae, 49–50 cues for, 50 Delayed sprouting, 120 Desiccation, 178, 185–8 Desmarestia anceps, 17, 24, 39, 45 Desmarestia antarctica, 39 Desmarestia menziesii, 17, 21, 39, 45 Desmarestia munda, 38 Desmarestiales antiherbivore defenses of, 38–9 chemical structures of secondary metabolites, 37 Destruxins, 155, 157 Devaleraea ramentacea, 183 Dictyol E, 59 Dictyoneurum californicum, 12, 14 Dictyopteris membranacea, 33 Dictyota acutiloba, 35 Dictyota ciliolata, 47, 57 Dictyota menstrualis, 36, 53, 59 Dictyotales, 4 antiherbivore defenses of, 30–8 biosynthetic compounds in, 33 chemical structures of secondary metabolites, 37 defensive secondary metabolites from, 31 DiVusible chemical cues, 50 Digestion, phlorotannins in, 14–15 2,4-Dimethyloxybenzaldehyde (DMBA) assay, 8–9 Diplotaxis tenuifolia, 144 DMBA. See 2,4-Dimethyloxybenzaldehyde assay DMSP, 190 Drosophila melanogaster, 98 Durvillaea potatorum, 9 E Ecklonia maxima, 20, 43 Ecklonia radiata, 10, 16–17, 20, 21 Ectocarpus, 62 chemokinetic responses of, 66

283

Ectocarpus siliculosus, 66 spores of, 69 eIF-2 binding of, 97 release of, 96 ELM1, 121 Emersion, 178 Enteromorpha compressa, 191 Enteromorpha prolifera, 191 Environmental stress factors desiccation, 185–8 freezing, 185–8 heavy metal, 190–2 high light, 188–90 mechanical, 192 osmotic stress, 185–8 ROM and, 183–91 ultraviolet, 188–90 EREB2, 232 ESBF-II, 99 Ethylene biosynthesis, 232 Eucheuma denticulatum, 189 Eucheuma playcladum, 183, 192 Extensins, 219 F Feeding deterrents, 29 Flavobacterium-Cytophaga, 196 Folin-Denis procedure, 8 Fouling organisms, defenses against, 58–60 Freezing, 185–8 Freshwater systems, brown algae in, 3 Fucales, 4 chemical structures of secondary metabolites, 37 phlorotannins in, 44, 55 Fucus distichus, 6 freezing stress on, 187–8 Fucus embryos, 200 hyperosmotic stress in, 189 Fucus evanescens, 16, 185 Fucus gardneri, 9, 11, 13, 14, 20, 24, 44 Fucus serratus, 188 Fucus spiralis, 16, 44, 65, 185 Fucus vesiculosus, 8, 11, 14, 20, 21, 25, 26, 28, 44, 50, 51, 187 Fumonisins, 155 Fusarium oxysporum, 150, 153, 157, 159, 162, 163, 166 shelf life of, 164, 165 Fusicoccin, 222 G GAL83, 104 Galactolipids, 230 Gametophytes, 193 GCN4-like motif (GLM), 99

284

SUBJECT INDEX

GCP1, 99 GDBH. See Growth-diVerentiation balance hypothesis GDP, binding, 96 Gene expression hexokinase in, 125 SnRK1 in, 116–18 Gene Technology Act 2000, 159 Gene Technology Regulations, 159 General control nonderepressible-2 (GCN2) related protein kinase, 96 activation of, 98 general amino acid control and, 98–101 homologues of, 98 protein synthesis inhibition and, 97 GLM. See GCN4-like motif Glossophora kunthii, 53, 58 Glu117, 226 Glucose, as signalling molecule, 124 Glucose induction in carbon metabolites, 107–8 in yeast, 107 Glucose repression, regulation of, 103–7 b-Glucuronidase (GUS) in A. nidulans, 158 in A. niger, 158 Glutamate, 227 Glutamate receptors, 218 Glutamine, 227 Glycolysis, T6P and, 124 Gondogeneia antarctica, 45, 61 Gracilaria conferta, 195–6, 196–7, 200 Gracilaria tenuistipitata, 191–2 Growth-diVerentiation balance hypothesis (GDBH), 57 GTP, binding, 96 GUS. See b-Glucuronidase H H2O2, 181, 183, 219 HACCs. See Hyperpolarisation-activated Ca2þ channels HAK5, 217, 224 Haloperoxidases, 183 in seaweeds, 184 HD-ZIP, 231, 234, 242 Heavy metal stress, 190–2 Hedophyllum sessum, 54 Herbivores, 29. See also Antiherbivore defenses Herbivory induction, phlorotannin production and, 25–7 Heterosigma akashiwo, 181 Hexokinase catalytic activity of, 106–7 in gene expression, 125 as glucose sensor, 124 Himantothallus grandifolius, 39

Hincksia irregularis spores of, 69–70 swimming patterns of, 65 HMG-CoA reductase, 113–14 Hormonal signals phosphorous and, 243–4 of potassium status, 225–6 Hormosira bankisii, 9, 65 HRGP, 232 Hyaloperonaspora parasitica, 149, 166 Hyperpolarisation-activated Ca2þ channels (HACCs), 238 I IDM. See Induced defense model Idotea baltica, 14, 50 Idotea granulosa, 11, 12 Induced defense model (IDM), 41, 48–54 Induction, by herbivory, 25–7 Infection responses, of seaweeds, 193–7 Inoculum production formulation, 163–6 industrial scale, 162–3 Intertidal zonation, 186 Ion transport, potassium and, 216–18 Irradiation, 57 Isocitrate lyase, 116 J JA. See Jasmonic acid Jasmonic acid (JA), 218 K Kappaphycus alvarezii, 183 Kelp spores, 68–9 KIN2, 226 KUP3, 218 L Lactone lobophloride, 59 Laminaria, 12, 20 Laminaria digitata, 60, 63, 193, 196 chemotaxis of, 64 Laminaria hyperborea, 54 Laminaria japonica, spores of, 67–8 Laminaria longicuris, 46 Laminaria pallida, 43 Laminariales, 4 Laminariocolax macrocystis, 60 Laminariocolax tomentosoides, 60 Lamoxirene, 64 LDL. See Low-density lipoprotein Lepidium virginicum, phylogenetic tree for, 151 Leptosphaeria maculans, 151–2

SUBJECT INDEX Lessonia negriscens, 54 Light, phlorotannin production and, 24–5 Light stress, 188–90 Lignin, 183 Littorina littorea, 11 Littorina obtusata, 11, 14, 26, 44, 50 LKB1, 102–3 Lobophora variegata, 6, 25, 59 Low-density lipoprotein (LDL), 113 LRP1, 232 Lytechinus variegatus, 36 M Macroalgae, RAM testing on, 56 Macroalgal chemical ecology, 35 Macrocystis angustifolia, 43, 44, 60 Macrocystis integrifolia, 13, 14, 17, 21, 54 Macrocystis pyrifera, 60, 66 spores of, 67–8 Malate, 244 Malate synthase, 116 MALDI-ToF mass spectrometry, 128 Malva pusilla, 154 MAPK cascade, 224 Mastocarpus stellatus, 183, 186–7 Mechanical stress, 192 Mechanical wounding, eVects of, 27 Membrane potential phosphorous and, 233–4 potassium and, 220–4 Meristems, 47 Metabolic signals phosphorous and, 242–3 of potassium, 226–7 Metabolic status, cellular processes and, 95 Methioninesynthase, 232 Methyl jasmonate, 28 MIG1, 104, 108 Mineral nutrients, management of, 210 Mithrax sculptus, 15 Monostroma arcticum, 182, 186 MYB-CC, 228, 231, 234, 235, 240 Mycoherbicides cropping conditions and, 160–2 inoculum production and, 162–6 for Raphanus pathogens, 148 uses for, 144 Mycotoxins, 159 N NADPH oxidases, 197 Necrotrophic pathogens, of wild radish, 149–50 Neurospora crassa, 98 NIK1, 118 NIM1, 118 Nitrate reductase (NR), 114–15

285

Nitrogen (N), 210 arriving at sink organs, 95 use eYciency of, 99 Nontransporter genes, potassium and, 218–20 Norrisia norrisi, 12 NR. See Nitrate reductase NRT2, 227 Nutrients fluctuating supply of, 212 phlorotannin production and, 25 Nutrition phosphorous in, 227–30 potassium in, 213–16 O ODT. See Optimal defense theory Oligocarrageenans, 195 Optimal defense theory (ODT), 41 brown algae and, 42–8 OsIPS1, 243 Osmotic stress, 185–8 in Fucus embryos, 189 Osmoticum, 214 Oxalate, 244 P P1BS, 228, 235 Pachydictyol-A, 30, 59 Pachygraphsus transverses, 36 Padina gymnospora, 57 PAK1, 121 Palmaria palmata, 183 PAR, 190 seaweed and, 177–8 Pathogens, defenses against, 58–60 Pelvetia, 20 Peridinium gatunense, 183 Phaeophyceae, 3, 4 Phaeurus antarcticus, 39 Phenolics, 19, 55 Pheromones, 34 chemical structure of brown algal, 63 chemoattraction to, 62–7 Phloroglucinol, 4 chemical structures of, 5 Phlorotannins biosynthesis model, 22 cellular roles of, 9–10 in cell wall structure, 9 in spermatozoid inhibitors, 10 in substrate adhesion, 9–10 in wound healing, 10 chemical structure of, 4–6 defined, 4 ecological roles of, 10–18 antibacterial, 15–17

286

SUBJECT INDEX

Phlorotannins (cont.)

antifouling, 15–17 antiherbivory, 10–14 in digestion, 14–15 heavy metal chelation, 18 in reproduction, 14–15 sunscreen, 17–18 extraction of, 8 in Fucales, 44, 55 future directions of, 28–30 growth defense trade-oVs and, 23 hydrogen-bonding capacity of, 7 intraspecific variability of, 18–21 location of, 6 metabolic turnover of, 6–7 production costs of, 21–8 induction by herbivory, 25–7 light and, 24–5 mechanical wounding and, 27 methyl jasmonate in, 28 nutrients and, 25 water-borne cues and, 27–8 quantification methods for, 7–9 cell wall bounds, 9 DMBA assay for, 8–9 Folin-Ciocalteu assay, 8 Folin-Denis procedure, 8 spatial variability of, 18–21 structural classes of, 5 synthesis of, 6–7 PHO1, 239 PHO-like sequences, 235 Phosphate-starvation response (PSR) genes, 231 Phospholipids, 230 transcriptional responses to, 231–3 Phosphorous (P), 210 calcium and, 233–4 cellular signalling events and, 233–8 deficiency, 212 hormonal signals and, 243–4 membrane potential and, 233–4 metabolic signals and, 242–3 nutrition, 227–30 in plants, 211 promoter elements and, 234–7 root morphological adaptations and, 237–8 shoot responses, 238–40 shoot-root signals and, 240–1 signalling cascades regulating transcription during, depletion, 228 signalling pathways, 212–13 systemic signalling of plant status, 238–44 transcriptional elements and, 234–7 Phosphorylation protein, 127–8 by SnRK1, 112

Photosynthesis, 230 PHR1 transcriptional cascade, 228 Pht1 Pi transporters, 231 Physa fontinalis, 26 Physcomitrella patens, 120–1 Physodes, 46 Phytophthora megasperma, 154 Phytophthora palmivora, 149 Pi, 241 PKABA1, 111 PKS. See Polyketide synthase Polyketide synthase (PKS), 6 Polysiphonia arctica, 183 Polyvinylpolypyrrolidone (PVPP), 7 Potassium (K), 210 calcium and, 220–4 cellular signalling events and, 220–5 deficiency, 212, 215 hormonal signals of, 225–6 ion transport and, 216–18 membrane potential and, 220–4 metabolic signals, 226–7 nontransporter genes and, 218–20 nutrition, 213–16 in plants, 211 roles of, 214 ROS and, 224–5 signalling pathways, 212–13 stress responses, 221 systemic signalling of, 225–7 transcriptional responses to, 216–20 Potato tubers, 120 Prolamin, 99 Promoter elements, phosphorous and, 234–7 Protein phosphorylation, 127–8 Proton pumps, 222 PSR. See Phosphate-starvation response Pterogophora californica, 66 spores of, 67–8 PV42, 112–13 PVPP. See Polyvinylpolypyrrolidone Pylaiella littoralis, 60 Pyruvate kinase (PK), 226–7 Pyruvate synthesis, 219 Q Qiagen probes, 217 R Ralfsia spongiocarpa, 16 RAM. See Resource allocation model Raphanus raphanistrum, 144 Raphanus raphanistrum, 144, 145 fungal pathogens of, 146–7 mycoherbicides for, 148 Raphanus sativus, phylogenetic tree for, 151

SUBJECT INDEX Rapistrum rugosum, 144 Reactive oxygen, production of, 185 Reactive oxygen metabolism (ROM), 186 defined, 179 environmental stress factors and, 183–91, 199 production of, 176–7 in seaweeds, 179–83 seaweeds interaction with, 193–7 Reactive oxygen species (ROS), 190 defined, 179 formation of, 180 potassium and, 224–5 production of, 176–7 scavenging, 198–9 triggers for, 200 Regulatory elements, in wheat storage protein genes, 100 Regulatory processes, 94 Reproduction, phlorotannins in, 14–15 Resource allocation model (RAM), 55–6 testing, 56 RGT1, 108 RGT2, 108 Rhizoctonia napus, 152 Rhizoctonia solani, 152 ROM. See Reactive oxygen metabolism Root morphological adaptations, phosphorous and, 237–8 ROS. See Reactive oxygen species RuBisCo, 232, 239 S S-adenosyl methionine synthase, 232 SAMS peptide, 103 SNF1 activity and, 106 Sargassum filipendula, 12, 24, 36, 40, 46, 55 Sargassum furcatum, 12 Sargassum globulariaefolium, 24 Sargassum mangarevense, 25 Sargassum natans, 16 Sargassum pteropleuron, 6, 7 Sargassum tenerrimum, 16 Sargassum vestitum, 17 Scytosiphon lomentaria, 41 Seaweeds environmental stresses in, 176–7, 177–9 desiccation, 185–8 freezing, 185–8 heavy metal, 190–2 high light, 188–90 mechanical, 192 osmotic stress, 185–8 ultraviolet, 188–90 haloperoxidases in, 184 infection responses and, 193–7 properties of, 176–7

287

ROM in, 179–83, 199 ROM interaction with, 193–7 Secondary metabolites, 2 chemical structures of, 37 from Dictyotales, 31 Senna obtusifolia, 154 Shikimate, 244 Shoot responses, phosphorous in, 238–40 Shoot-root signals, phosphorous and, 240–1 Sinapis alba, 159 Sinapis arvensis, 144 Sinapsis alba, 153 Sink organs carbon arriving at, 95 nitrogen arriving at, 95 SIP1, 104 SIP2, 104 Sisymbrium oYcinale, 144 Sisymbrium orientale, 144 Sisymbrium thellungii, 144 SKOR1, 216 Small heat shock protein (HSP), 115 SNF1. See Sucrose nonfermenting-1 SNF1-related protein kinase (SnRK1), 96 AGPase and, 115–16 in carbon metabolite sensing, 108–9 in cell cycle control, 118–19 components of, 103 downstream eVects of, 121 in gene expression, 116–18 HMG-CoA reductase and, 113–14 NR and, 114–15 pathways for, 123 peptides, 111–12 phenotypic eVects of manipulating, 119–21 phosphorylation by, 112 recognition motif for, 112 small HSP and, 115 SPS and, 114–15 targets for, 122 upstream factors, 121–6 SnIP1, 112–13 SnRK1. See SNF1-related protein kinase SnRK2, in carbon metabolite sensing, 109–11 SnRK3, in carbon metabolite sensing, 109–11 SOD. See Superoxide dismutase SPA, 99 Sparisoma chrysopterum, 15 Sparisoma radians, 15 Spatoglossum crassum, 35 Spermatozoid inhibitors, 10 Spermatozoid release, 64 Spores, behavior and sensory capabilities of, 67–70

288

SUBJECT INDEX

Sporophytes, 193 SPS. See Sucrose phosphate synthase Sterol biosynthesis, 113 Stictosiphonia arbuscula, 186 Streptomyces hygroscopicus, 155 Streptomyces viridochromosgenes, 155 Stress potassium and responses to, 221, 222 signalling, 213 sugar and, 126–7 Stypopodium zonale, 36 Substrates, adhesion to, 9–10 Succinate, 244 Sucrose, 230 Sucrose nonfermenting-1 (SNF1), 96 activation pathways for, 105 components of, 103 divergence of, 110 regulation of, 103–7 subfamilies of, 109 subunits of, 104 Sucrose phosphate synthase (SPS), 114–15 Sucrose synthase, 117 Sucrose transporters (SUT), 126 Sugar abscisic acid/stress signalling pathways and, 126–7 metabolism, 219–20 Sulfuric acid, 34–5, 38 Sulphated oligosaccharide signalling, 194 Sulpholipids, 230 Sunscreen, phlorotannins and, 17–18 Superoxide dismutase (SOD), 180, 187 localisation of, 181 Superoxide radicals, 180 SUT. See Sucrose transporters SWE1, 119 T T6P. See Trehalose-6-phosphate Terpenes, 55 Terpenoids, 33 Thalli, 53–4 Theodoxus fluviatilis, 26–7, 55 TOS3, 121 TPP. See Trehalose phosphate phosphatase TPS. See Trehalose phosphate synthase Transcription factors, phosphorous and, 234–7 Transcriptional responses, to potassium, 216–20 Trehalose phosphate phosphatase (TPP), 106–7 Trehalose phosphate synthase (TPS), 106 Trehalose-6-phosphate (T6P), 106 in glycolysis, 124 Tripneustes gratilla, 15 Triticum aestivum, 152

Turbinaria ornata, 20, 25 Turbo undulata, 15 U Ulva lactuca, 17, 183 Ulva rigida, 181, 189 UV radiation, seaweed and, 177–8 UV stress, 188–90 UV-B radiation, 17 V Vascular plant tannins, 5 Vegetative storage proteins (VSPs), 219 VHOCs. See Volatile halogenated organic compounds Volatile halogenated organic compounds (VHOCs), 60–1 Voticella marina, 16 VSPs. See Vegetative storage proteins W Water-borne cues, 27–8 WD40, 234, 240 Weeds, herbicide-resistant, 144 Wild radish chemical synergists, 154 gene manipulation of, 156–9 pathogens of, 145–53 biotrophic, 149 host specificity, 150–3 multiple, 153 necrotrophic, 149–50 phytotoxic metabolites of, 155 Wound healing, phlorotannins in, 10 WRKY transcription factors, 231, 234 X Xanthium occidentale, 153 Xanthium spinosum, 163 Xiphister mucosus, 15 Xyloglucan glucosyltransferases, 219 Y Yeast amino acid control in, 96–8 glucose induction in, 107 Z Zinc finger, 234 Zonaria angusta, 12, 20, 47 Zonaria angustata apical region of, 13 physode distribution in, 13

CONTENTS

CONTRIBUTORS TO VOLUME 43 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

vii

CONTENTS OF VOLUMES 33–42 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

ix

Defensive and Sensory Chemical Ecology of Brown Algae CHARLES D. AMSLER AND VICTORIA A. FAIRHEAD I. II. III. IV. V.

Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Phlorotannins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Nonphlorotannin Antiherbivore Defences. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Testing Chemical Defence Theories with Brown Algae. . . . . . . . . . . . . . . . . . . . . . . Nonphlorotannin Defences Against Bacteria, Fouling Organisms, and Pathogens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VI. Volatile Halogenated Organic Compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VII. Sensory Chemical Ecology. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VIII. Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

2 4 30 41 58 60 62 70 71

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 I. II. III. IV. V.

Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Amino Acid Signalling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Carbon Metabolite Sensing and Signalling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The Link Between Sugar and Abscisic Acid/Stress Signalling Pathways . . . . . . . . Prospects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

94 96 101 126 127 129

Opportunities for the Control of Brassicaceous Weeds of Cropping Systems Using Mycoherbicides AARON MAXWELL AND JOHN K. SCOTT I. Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . II. Pathogens of Wild Radish . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

144 145

vi

CONTENTS

III. IV. V. VI.

Strategies to Increase Virulence or Specificity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Epidemiology with Respect to Cropping Conditions . . . . . . . . . . . . . . . . . . . . . . . . . Inoculum Production and Formulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

153 160 162 166 167

Stress Resistance and Disease Resistance in Seaweeds: The Role of Reactive Oxygen Metabolism MATTHEW J. DRING I. II. III. IV. V. VI.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Environmental Stress on Seaweeds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Reactive Oxygen Metabolism (ROM) in Vascular Plants and Seaweeds . . . . . . . . . Interactions Between ROM and Environmental Stress Factors . . . . . . . . . . . . . . . . Responses of Seaweeds to Infection: Interaction with ROM. . . . . . . . . . . . . . . . . . . Summary and Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

176 177 179 183 193 197 201

Nutrient Sensing and Signalling in Plants: Potassium and Phosphorus ANNA AMTMANN, JOHN P. HAMMOND, PATRICK ARMENGAUD AND PHILIP J. WHITE I. II. III. IV.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Potassium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Phosphorus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

210 213 227 244 245

AUTHOR INDEX . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

259

SUBJECT INDEX . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

281