Horticultural Reviews, Volume 27

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HORTICULTURAL REVIEWS Volume 27

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Horticultural Reviews is sponsored by: American Society for Horticultural Science

Editorial Board, Volume 27 Nigel H. Banks Frederick T. Davies Susan Lurie

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HORTICULTURAL REVIEWS Volume 27

edited by

Jules Janick Purdue University

NEW YORK

John Wiley & Sons, Inc. / CHICHESTER / WEINHEIM / BRISBANE / SINGAPORE / TORONTO

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Copyright © 2001 by John Wiley & Sons, Inc. All rights reserved. No part of this publication may be reproduced, stored in a retrieval system or transmitted in any form or by any means, electronic, mechanical, photocopying, recording, scanning or otherwise, except as permitted under Sections 107 or 108 of the 1976 United States Copyright Act, without either the prior written permission of the Publisher, or authorization through payment of the appropriate per-copy fee to the Copyright Clearance Center, 222 Rosewood Drive, Danvers, MA 01923, (978) 750-8400, fax (978) 750-4744. Requests to the Publisher for permission should be addressed to the Permissions Department, John Wiley & Sons, Inc., 605 Third Avenue, New York, NY 10158-0012, (212) 850-6011, fax (212) 850-6008, E-Mail: PERMREQ @ WILEY.COM. This publication is designed to provide accurate and authoritative information in regard to the subject matter covered. It is sold with the understanding that the publisher is not engaged in rendering professional services. If professional advice or other expert assistance is required, the services of a competent professional person should be sought. This title is also available in print as ISBN 0-471-38790-8. Some content that appears in the print version of this book may not be available in this electronic edition. For more information about Wiley products, visit our web site at www.Wiley.com

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Contents

Contributors

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Dedication: John V. Possingham

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Keith Boardman

1. The Molecular Biology of Flowering

1

Steve van Nocker I. II. III. IV. V. VI. VII.

Introduction Arabidopsis as a Model for Flowering-Time Studies Floral Inductive Pathways Role of Carbohydrates Control of Meristem Identity Competency Conclusion and Perspectives Literature Cited

2. Floral Homeotic Gene Regulation

1 2 3 20 21 25 28 30

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Robert G. Franks and Zhongchi Liu I. II. III. IV. V.

Introduction Conservation of the ABC Functions in Angiosperms Positive Regulators of Floral Organ Identity Genes Negative Regulators of Floral Organ Identity Genes Summary Literature Cited

42 51 56 63 69 71 v

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CONTENTS

3. Lingonberry: Botany and Horticulture

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Inger Hjalmarsson and Rodomiro Ortiz I. II. III. IV. V. VI.

Introduction History Botany Management of Natural Stands Horticulture Summary and Future Prospects Literature Cited

4. Caper Bush: Botany and Horticulture

80 81 87 93 99 111 114

125

Gabriel O. Sozzi I. II. III. IV. V. VI. VII. VIII.

Introduction Botany Ecophysiology Horticulture Postharvest Technology Composition and Utilization International Trade Concluding Remarks Literature Cited

126 132 137 140 156 159 170 172 173

5. Water Relations and Irrigation Scheduling in Grapevine

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M. H. Behboudian and Zora Singh I. II. III. IV. V. VI.

Introduction Phenology Aspects of Water Relations Irrigation of Vineyards Quality Attributes for Wine, Dried, Table, and Juice Grapes Future Prospects Literature Cited

6. Physiology and Biochemistry of Superficial Scald of Apples and Pears

190 191 193 207 215 218 219

227

Morris Ingle I. II.

Introduction Scald Symptoms and Cell Changes

228 228

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CONTENTS

III. IV. V. VI.

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Biochemistry of Scald Physiology of Scald A Model of Scald Development Prospects Literature Cited

7. Health Functional Phytochemicals of Fruit

229 245 253 259 262

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Wilhelmina Kalt I. II. III. IV. V. VI.

Introduction Citrus Grapes and Wine Vaccinium Other Fruits Conclusions Literature Cited

8. Producing Sods over Plastic in Soilless Media

270 282 291 298 303 307 308

317

Henry F. Decker I. II. III. IV. V. VI. VII. VIII. IX. X. XI.

Introduction Producing Sods in Soilless Media Development of the Concept Producing Mature Sods over Plastic Producing Sods for Golf Greens Solving the Problem of a Stable Continuum Subsequent Proposals in the Genre Manufacturing Sods New Machinery Future Potential Summary Literature Cited

318 319 321 327 330 331 333 338 340 342 345 346

Subject Index

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Cumulative Subject Index

355

Cumulative Contributor Index

377

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Contributors M. H. Behboudian, Institute of Natural Resources, College of Sciences, Massey University, Private Bag 11222, Palmerston North, New Zealand Keith Boardman, 6 Somers Crescent, Canberra ACT 2603, Australia Henry F. Decker, Buckeye Bluegrass Farms, Inc., Box 176, Ostrander, OH 43061 Robert G. Franks, Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, MD 20742 Inger Hjalmarsson, The Nordic Gene Bank, Smedjevägen 3, PO Box 41, S-230 53 Alnarp, Sweden Morris Ingle, Division of Plant and Soil Sciences, 1090 AG SCI BD, PO Box 6108, West Virginia University, Morgantown, WV 26506 Wilhelmina Kalt, Agriculture and Agri-Food Canada, Atlantic Food and Horticulture Research Centre, Kentville, Nova Scotia B4N 1J5, Canada Zhongchi Liu, Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, MD 20742 Steve van Nocker, Department of Horticulture, Michigan State University, 390 Plant and Soil Science Building, East Lansing, MI 48824 Rodomiro Ortiz, IITA c/o Lambourn & Co., Carolyn House, 26 Dingwall Road, Croydon CR9 3EE, UK Zora Singh, Department of Horticulture, Muresk Institute of Agriculture, Curtin University of Technology, GPO Box U 1987, Perth, WA 6845, Australia Gabriel O. Sozzi, Departamento de Biología Aplicada y Alimentos, Facultad de Agronomía, Universidad de Buenos Aires, Avda. San Martín 4453, C 1417 DSE Buenos Aires, Argentina

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John V. Possingham

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Dedication: John V. Possingham This volume is dedicated to Dr. John Possingham, an outstanding scientist and administrator, in recognition of his outstanding contribution to Australian horticulture and for his international efforts in horticultural science and viticulture. John was born in 1929 and grew up on a small horticultural property in rural South Australia. He studied agriculture and plant physiology at the Universities of Adelaide in Australia and at Oxford in England. He initially worked in the field of plant nutrition at the Laboratories of the Commonwealth Scientific and Industrial Research Organisation (CSIRO) in Canberra establishing that manganese was essential for the photochemical reactions of higher plant chloroplasts. In 1962 he accepted responsibility for CSIRO’s program of research in horticulture that was mainly concerned with grapevines and centered at Merbein Victoria. He subsequently established horticultural laboratories at Adelaide, Darwin, and Brisbane that, together with an existing postharvest horticulture group at Sydney, enabled CSIRO’s Division of Horticulture to carry out a wide-ranging program of basic and applied horticultural research concentrating mainly on wine and raisin grape vines and on a range of selected tropical and subtropical fruit crops. At Merbein, Dr. Possingham established a major grapevine improvement program that covered virus-tested introductions from overseas and the breeding of new cultivars suited to the warm irrigated conditions of inland Australia. This program also included the development of grapevine rootstocks that were tolerant of Australian plant parasitic nematodes. New cultivars released from the grape breeding program and adopted by the viticultural industries include ‘Tarrango’ for light red wine; ‘Carina’, a black seedless raisin; and the table grape ‘Marroo Seedless’. A number of other potential cultivars are being evaluated by industry. Dr. Possingham also obtained major government funding and established a vineyard mechanization program based on importing prototype grape harvesting machines from the University of California, Davis and Cornell University. Vine training and management systems were developed for use in mechanically harvested vineyards. These included minimal pruning, which is now used extensively in warm to hot grape growing regions in both Australia and California. xi

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DEDICATION: JOHN V. POSSINGHAM

The work of the Division of Horticulture assisted the recent rapid expansion of the Australian wine industry and enabled it to become a major player in world markets. It provided the industry with high-quality vine planting material and developed training systems that enabled Australian wine-grape vineyards to become highly mechanized. Together with other members of the Division he was awarded both a CSIRO Medal for his contributions to vine improvement and an Ian McLennan Award from Industry for his foresight in developing a program of work on vineyard mechanization for Australia. Dr. Possingham was involved with other scientists in many aspects of the Division’s program including the minimal pruning of grapevines and the evaluation of introduced grape cultivars. He contributed to the understanding of how the waxy layers of raisin grapes control water loss and the elucidation of a major role of endotrophic mycorrhizae in the uptake of phosphorus by grapevines. Research concerned with grapevine physiology, biotechnology, and molecular biology was developed at the Division’s laboratory located on the Waite Institute Campus. Studies on grapevines included the features of and factors affecting flowering, fruit set, photosynthesis, response to salinity, and root/shoot hormonal interactions. Biotechnology investigations were aimed at better methods for grapevine propagation and systems for virus elimination. Recent work has been concerned with methods for the DNA finger-printing of grapevines and techniques for their genetic modification. Some genetically modified grapevines are currently undergoing field trials. Throughout his career, Dr. Possingham has maintained a personal research program concerned with factors involved with the division of higher plant chloroplasts. This work included both structural and biochemical studies and provided support for the long-held hypothesis that the plastids of higher plants arise from the division of pre-existing plastids and cannot be formed de novo. He showed that chloroplasts of spinach are highly polyploid and that division can be temporally separated from the synthesis of chloroplast located DNA. Using barley mutants he demonstrated that the polymerase used for c-DNA replication is nuclear coded and a component in the complex control exerted by the nucleus over plastic division. Dr. Possingham has contributed to international horticulture via a number of FAO/United Nations Development Program (UNDP) missions, but more importantly through his work with the International Society for Horticultural Science (ISHS). He established the Viticulture Section within ISHS and has been a Council member for Australia for a number of years. He is in his second term as a member of the ISHS Board and is currently the Society’s Vice President. In Australia he was directly

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involved in setting up the Australian Society for Horticultural Science and was its second President. He is a fellow of the Australian Institute of Agricultural Science and Technology, the Australian Academy of Technological Science and Engineering, the Russian Academy of Agricultural Sciences, and holds a DSc from the University of Oxford. In his “retirement” he grows wine grapes and makes wine on highly mechanized vineyards near Adelaide. John is known throughout Australia and the world for his generosity and warmth, for his dedication to high standards, and for his love of good fellowship and fine wines. Keith Boardman Formerly Chairman and Chief Executive CSIRO, Canberra, Australia

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1 The Molecular Biology of Flowering Steve van Nocker Department of Horticulture Michigan State University East Lansing, MI 48824

I. INTRODUCTION II. ARABIDOPSIS AS A MODEL FOR FLOWERING-TIME STUDIES III. FLORAL INDUCTIVE PATHWAYS A. Photoperiodic Induction 1. Light Effects on Flowering in Arabidopsis 2. The Endogenous Clock 3. Entrainment of the Clock by Light 4. Other Photoperiodic Pathway Genes B. Non-Photoperiodic Induction: The Autonomous Pathway C. Vernalization D. Induction by Gibberellins IV. ROLE OF CARBOHYDRATES V. CONTROL OF MERISTEM IDENTITY A. Meristem Identity Genes B. Integration of Flowering Pathways and Activation of Meristem Identity Genes VI. COMPETENCY VII. CONCLUSION AND PERSPECTIVES LITERATURE CITED

I. INTRODUCTION Of the myriads of developmental processes that define plant form and function, flowering is of exceptional interest to horticulturalists. The vast majority of horticulturally important crops are in some way dependent upon flowering, whether the flower is the primary goal of production, or is simply required for a crop to be produced. Much effort is currently being put into regulating the timing of flowering. In floriculture crops, the interest is in abbreviating or extending the vegetative Horticultural Reviews, Volume 27, Edited by Jules Janick ISBN 0-471-38790-8 © 2001 John Wiley & Sons, Inc. 1

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phase in order to create an aesthetically pleasing balance between leaves and flowers, or to conveniently induce or repress flowering to take advantage of market potential. In ornamental foliage plants, and agronomically important plants that are grown for their leaf tissues (e.g., lettuce, spinach, and other greens), it is highly desirable to suppress flowering as long as possible. Also, in woody plants, there is a great deal of interest in finding means to abbreviate the vegetative phase, which in some species can last ten or more years and is probably the single most limiting factor for germplasm improvement through traditional breeding techniques. Most efforts at controlling flowering time have involved manipulation of environmental conditions or the application of synthetic growth regulators. However, these approaches can increase production costs and labor requirements. In addition, the use of many traditionally utilized chemical compounds is becoming restricted. Alternative approaches to manipulate flowering—including biotechnology—will require a better understanding of the associated molecular mechanisms. The physiology and phenomenology of the developmental transition from vegetative growth to reproductive growth—flowering—has been studied for many years, but only in approximately the last 10 years have the molecular mechanisms begun to be addressed. Flowering is ultimately determined by genes that govern the identity of the meristem, promoting or repressing floral fate versus shoot fate. When and how these genes are activated, in response to environmental cues and/or developmental progression, is a fascinating question. As might be expected from the incredible diversity of flowering strategies employed in nature, it is now becoming apparent that flowering at the molecular level involves an extraordinarily complex web of interactive pathways. Here we review the current knowledge about the genetics and molecular biology of flowering in Arabidopsis thaliana, the only plant in which these aspects of flowering have been extensively studied.

II. ARABIDOPSIS AS A MODEL FOR FLOWERING-TIME STUDIES Arabidopsis thaliana is an herbaceous weed of the mustard family with a natural distribution throughout the Northern Hemisphere (Meyerowitz 1989; Meinke et al. 1998). In addition to its many qualities that make it a superior model for plant biology in general (i.e., small size, rapid life cycle, and well-characterized genome), Arabidopsis is especially attractive as a subject for flowering-time studies because the timing of flow-

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ering can be strongly influenced by environmental conditions (e.g., light and temperature), thus permitting the molecular analyses of the associated input pathways. In this species, at least fifty genes have been identified that act directly or indirectly either to promote or to repress flowering (Levy and Dean 1998). Many of these genes have been identified through a traditional genetic approach. Delayed flowering can result from loss of function of genes that presumably act to promote flowering, whereas accelerated flowering can result from loss of function of flowering-repressor genes. Although several repressor genes have been identified, most mutagenic approaches have targeted genes that act to promote flowering (Redei 1962; Koornneef et al. 1991). This is in part because the genotypes commonly used in the laboratory flower soon after germination in photoperiodically inductive conditions (long-day photoperiods), and mutants that flower even earlier, are difficult to discern in large populations. Screens designed to find early-flowering mutants among late-flowering genetic backgrounds, or employing photoperiodically noninductive conditions, should result in the identification of additional repressor genes.

III. FLORAL INDUCTIVE PATHWAYS An interesting finding coming from genetic analyses is that no single mutation completely eliminates flowering. This was an early indication that flowering is promoted by at least two pathways that can operate in a parallel, or partially redundant, manner. That such redundancy should have evolved makes sense, given the crucial importance of flowering in maintaining the species. The promotive genes identified through genetic analyses have traditionally been assigned into distinct groups based on the sensitivity of the mutant phenotype to environmental conditions, and these groups have formerly been considered to define the pathways (Martinez-Zapater et al. 1994; Coupland 1995). Mutations in a subset of flowering-time genes predominately affect the photoperiodic control of flowering, such that the flowering habit of the corresponding mutant tends toward day-neutrality. Mutations in another subset of floweringtime genes result in delayed flowering without a significant loss of photoperiodic sensitivity—i.e., these mutants flower later than wild-type plants under both photoperiodically inductive and noninductive conditions. Because mechanisms for sensing daylength are evidently intact in the latter mutants, the corresponding genes are supposed to function in an environmentally “autonomous” pathway that acts in parallel with the “photoperiodic” pathway to eventually initiate flowering, even

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under unfavorable conditions (Martinez-Zapater et al. 1994; Coupland 1995; Amasino 1996). Another characteristic of mutants in the autonomous pathway is that they exhibit a significant vernalization response—i.e., the late-flowering phenotype can be fully “rescued” by a long-term cold treatment given to the imbibed seed or young plant. In contrast, cold is largely ineffective to accelerate flowering of the photoperiodic pathway mutants (Martinez-Zapater et al. 1994). Koornneef et al. (1991, 1998a) used double-mutant analysis to examine the epistatic relationships between the commonly studied floweringpromoting genes in an attempt to better define such pathways. The rationale for this type of approach is as follows: if two genes operate in a more-or-less linear pathway, then loss of both genes’ function should confer a phenotype that is similar to that of the single mutant (i.e., the double mutant should flower no later than either single mutant). However, if genes operate in parallel pathways, a significant enhancement of the late flowering might be conferred by combining the mutant alleles. A caveat to this type of genetic approach is that it is only valid when using complete loss-of-function alleles, as enhancement of the phenotype should be expected when partially functional alleles operating in the same pathway are combined. In general, the results of these experiments were inconsistent with the simple assignment of flowering-time genes to independent pathways. This suggests that there is significant interaction (“crosstalk”) between pathways. Another finding from these studies was that flowering was not prevented even when combining mutations in genes considered to act in the photoperiodic and autonomous pathway. Thus, the redundancy of flowering pathways is more extensive than was previously thought. A. Photoperiodic Induction 1. Light Effects on Flowering in Arabidopsis. As in many other plants, both light quality (wavelength) and photoperiod strongly influence flowering time in Arabidopsis. In general, flowering in this species is delayed by red light and accelerated by blue light (Brown and Klein 1971; Eskins 1992). The molecular biology of the major photoreceptors in plants, the red/blue-sensitive phytochromes and green/blue/UV-A-sensitive cryptochromes, has been extensively reviewed and will not be discussed here (Barnes et al. 1997; Cashmore 1998; Whitelam and Devlin 1998; Ahmad 1999; Cashmore et al. 1999; Deng and Quail 1999; Briggs and Huala 1999). Mutations that abrogate synthesis of the phytochrome chromophore and therefore result in an absence of functional phytochrome, or mutations that specifically result in loss of the major light-stable phy-

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tochrome, PHYB, confer early flowering, suggesting that the negative effect of red light is mediated by PHYB. Mutants lacking function of the CRYPTOCHROME1 (CRY1) gene exhibit delayed flowering that is evident in both long and short days (King and Bagnall 1996; Coupland 1997). This phenotype is especially striking when plants are grown under blue light (Bagnall et al. 1996), suggesting that CRY1 mediates blue-light promotion of flowering. In contrast, mutants lacking function of CRY2 (allelic to the previously described flowering time gene FHA) exhibit a much-reduced photoperiodic response, flowering much later than wild type in long days and slightly earlier than wild type in short days (Koornneef et al. 1991; Guo et al. 1998). In addition, constitutive expression of the CRY2 gene in transgenic plants accelerates flowering in short days, but not long days. Unlike in cry1 mutants, flowering in plants lacking CRY2 is accelerated by blue light (Guo et al. 1998). Given the delay in flowering in white light conferred by loss of CRY2 activity, one interpretation of this data is that CRY2 normally acts not as a direct positive regulator under blue, but as a negative regulator of the repression of flowering imposed by PHYB (Guo et al. 1998). 2. The Endogenous Clock. In plants, as in other organisms, one or more molecular mechanisms sustain oscillations with periods of approximately 24 h. The circadian rhythms generated by these endogenous “clocks” allow plants to anticipate daily variations in environmental conditions and thereby optimize their responses to them. One example is the family of LHC genes encoding light-harvesting chlorophyll a/b-binding (CAB) proteins, which are upregulated in a diurnal manner before the expected onset of illumination (Piechulla 1988; Nagy et al. 1988). A large body of physiological evidence implicates the clock in mediating the effects of photoperiod on flowering. Evidence is also accumulating that light quality as well influences flowering time by virtue of its effects on the clock. Thus, the clock has a central and very important role in flowering. How might a self-sustaining oscillatory mechanism in plants be composed at the molecular level? Some clues come from research on the Drosophila (fruit fly) clock mechanism that controls eclosion (emergence from the pupae) and locomotor activity. This clock is essentially comprised of an oscillatory mechanism set up through the interactions between two proteins, TIMELESS (TIM) and PERIOD (PER). Transcription of both the PER and TIM genes increases during the subjective day, from a minimum rate near the onset of illumination (referred to as Zeitgeiber time 0, or Zt0) and reaching a maximum rate at approximately Zt12 (Hardin et al. 1992). Maximal accumulation of PER and

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TIM mRNA is offset 2 to 4 hours, whereas maximal accumulation of the proteins is offset another 2 to 4 hours (So and Rosbash 1997). Thus, PER and TIM protein levels reach a maximum at Zt16-20, a point during which transcription of the genes is rapidly decreasing. In fact, transcriptional repression of the PER and TIM genes is a direct result of the increase in protein levels. Heterodimerization between PER and TIM allow the proteins to gain entrance into the nucleus, where they block the transcription of their own genes by the CLOCK and BMAL1 transcription factors (Vosshall et al. 1994; Gekakis et al. 1995; Darlington et al. 1998). The inhibition of transcription by PER/TIM allows the circadian cycle to begin anew. Constant turnover of the mRNAs and proteins is necessary for the oscillations to continue. The TIM protein is thought to be destabilized through phosphorylation by the product of the DOUBLETIME gene, which is structurally related to the kinase domain of human casein kinase Iε (Kloss et al. 1998; Price et al. 1998). In addition, in the absence of TIM, PER protein fails to accumulate, suggesting that TIM functions directly or indirectly to stabilize PER (Price et al. 1995). Although great progress has been made in understanding the basics of this Drosophila clock mechanism, how the clock operates in plants is mostly unknown. PER, TIM, and other components of the fly clock were discovered through traditional genetic analysis. Arabidopsis displays numerous visible phenotypes that cycle in a circadian manner [e.g., movements of cotyledons and primary leaves (Engelmann et al. 1992), alterations in the rate of hypocotyl elongation (Dowson-Day and Millar 1999), and changes in stomatal aperture (Somers et al. 1998b)], but in all cases these phenotypes are subtle and thus not useful for mutant screening. Millar et al. (1995) generated a synthetic circadian phenotype by expressing the firefly luciferase gene under the control of an LHC gene promoter in transgenic plants. Screens using this LHC:LUC genetic background yielded numerous mutants. The best-characterized, designated toc1-1, exhibits a slightly shorter period length of LHC mRNA expression in both constant light and constant darkness (Millar et al. 1995; Somers et al. 1998b). In addition, the mRNA expression of members of at least one other circadian-cycling nuclear gene family, GRP7/8 (see below), is altered in a similar manner (Kreps and Simon 1997). Although toc1-1 plants were originally reported to be phenotypically indistinguishable from wild-type plants, more careful observations revealed that toc1-1 plants were disrupted in multiple circadian cycling phenotypes. In addition, toc1-1 plants exhibited aberrant floral initiation, flowering earlier than wild-type plants under short photoperiods and later than wild-type plants under long photoperiods (Somers et al. 1998b). These findings suggest that the multiple circadian processes

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and the timing of flowering are controlled either by a single clock, or by multiple related clocks sharing the TOC1 component. The TOC1 gene was recently cloned and found to encode a protein with homology to the receiver domain of response regulators from two-component signal transduction systems (Strayer et al. 2000). A dominant mutation in the LATE ELONGATED HYPOCOTYL (LHY) gene leads to loss of rhythmic mRNA expression of clock-regulated genes and defects in multiple clock-influenced phenotypes, including flowering time and circadian leaf movements (Schaffer et al. 1998). In wild-type plants, LHY mRNA levels oscillate in a circadian manner, whereas LHY mRNA is expressed at a constitutive high level in the lhy mutant (Schaffer et al. 1998). In addition, in transgenic plants containing a singly copy of a lhy mutant allele, cycling of the endogenous wildtype LHY mRNA is suppressed. These findings indicate that LHY is part of a feedback circuit that regulates its own mRNA expression. The LHY gene product is a member of a large family of proteins structurally related to the vertebrate proto-oncogenic transcription factor c-Myb (Martin and Paz-Ares 1997). In Arabidopsis, this family also includes the product of the CCA1 (CIRCADIAN CLOCK ASSOCIATED 1) gene, originally identified as a factor that bound a LHC promoter element essential for its regulation by light and the clock (Wang et al. 1997). Like LHY mRNA, CCA1 mRNA and protein also cycle with a circadian rhythm (Wang and Tobin 1998). Constitutive expression of CCA1 mRNA under control of the strong, viral CaMV 35S promoter (35S:CCA1) in transgenic Arabidopsis, like constitutive expression of LHY mRNA from the mutant lhy allele, leads to the disruption of the circadian mRNA expression patterns of various clock-regulated genes, including LHY, and such plants exhibit delayed flowering in long-day conditions (Wang and Tobin 1998). These findings suggest that both LHY and CCA1 are potential key components of a central clock mechanism. That the clock defect conferred by loss of CCA1 function is apparent even in the presence of LHY activity indicates that, despite their structural similarities and similar effects by constitutive expression, LHY and CCA1 do not have strictly redundant roles (Green and Tobin 1999). Like that of TIM, the activity of the CCA1 and LHY proteins may be negatively regulated by phosphorylation. Both proteins are substrates for the protein kinase CK2 in vitro (Sugano et al. 1998, 1999). Constitutive expression of CKB3 mRNA, encoding a regulatory subunit of CK2, in Arabidopsis mimics the effects of loss of CCA1 function, substantially shortening the rhythm periods of multiple clock-regulated genes. However, in contrast to the delay of flowering conferred by loss of CCA1, flowering is accelerated in 35S:CKB3 plants (Sugano et al. 1999).

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GIGANTEA (GI) is another member of the set of genes involved in the promotion of flowering by long days. In plants grown in a regular lightdark photoperiod, mRNA levels for GI oscillate in a diurnal pattern, and studies in which plants were kept in constant light or darkness indicate that GI is under clock control (Fowler et al. 1999; Park et al. 1999). In 35S:CCA1 or 35S:LHY plants, this rhythmic expression pattern is disrupted, indicating that GI is regulated by these genes (Fowler et al. 1999). However, disruption of GI function also affects expression of CCA1 and LHY mRNA. In some gi mutant backgrounds, the amplitude of CCA1 and LHY oscillations are diminished and periodicity becomes less obvious (Fowler et al. 1999; Park et al. 1999). The finding that GI may act both upstream and downstream of the clock genes CCA1 and LHY suggests that GI is intimately associated with the clock. Mutants lacking GI function also exhibit reduced seedling deetiolation under red light, suggesting that GI could be involved in PHYB signaling (Huq et al. 2000). How GI might carry out its function has not been determined. GI was recently cloned and encodes a large protein that is predicted by computer modeling to contain transmembrane domains (Fowler et al. 1999; Park et al. 1999). However, more recent evidence indicates that the GI protein is localized to the nucleus (Huq et al. 2000). The GI transcript was detected in both very young seedlings and mature plants, and is apparently not restricted to any specific tissue type (Fowler et al. 1999). Open reading frames have been identified from both rice and maize that would encode proteins with significant amino acid sequence identity to the GI protein (Fowler et al. 1999; J. Liu and S. van Nocker, unpublished data). Because structural homology is often associated with functional homology, it is possible that these monocot GI orthologs are also involved in flowering. The current efforts to better characterize the rice genome, and determine gene function in maize through reversed genetics approaches, will allow this idea to be tested (Goff 1999; Martienssen 1998; Walbot, 2000). Other potential clock genes include members of the GRP7/8 (also called CCR1/2) family. These genes encode small proteins containing an interesting bipartite structure (van Nocker and Vierstra 1993; Carpenter et al. 1994). The amino-terminal domain contains a specific RNA-binding consensus sequence termed the RRM motif (found also in the flowering-time genes FCA and FPA; below), whereas the carboxyl-terminal region is greatly enriched in glycine residues, a configuration seen in many plant cell wall proteins (Showalter 1993; Cassab 1998). These genes are expressed to high levels in meristematic tissues, and, in addition to being regulated in a circadian pattern, are upregulated by lowered temperatures

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(Heintzen et al. 1994; Kreps and Simon 1997). The protein products of these genes also oscillate with a circadian period, and are localized to the nucleus (Heintzen et al. 1994). Constitutive expression of the GRP7 gene in transgenic Arabidopsis suppresses the circadian oscillations of mRNAs for both the endogenous GRP7 gene and for GRP8, suggesting that the respective proteins are involved in a mutual, autoregulatory feedback loop. This effect of GRP7 on the oscillations of its own transcript is not mediated entirely through its promoter, suggesting that at least some regulation occurs at the posttranscriptional level (Staiger and Apel 1999). As previously mentioned, GRP7/8 expression is affected by impairment of TOC1 function. Interestingly, however, unlike in toc1 mutants, the rhythmic expression patterns of specific clock-regulated genes were not affected in 35S:GRP7 plants (Heintzen et al. 1997). This suggests both that the GRP7/8 clock acts downstream from TOC1, and that the output of the AtGRP7/8 oscillator is limited. The function of the so-called AtGRP7/8 “slave” oscillator is not known, as phenotypic abnormalities associated with constitutive expression of GRP7 have not been reported. In light of the intimate relationship between circadian rhythms and flowering, it is reasonable to hypothesize that these genes are somehow involved in the regulation of flowering time. On the other hand, genes encoding small proteins exhibiting the RRM motif/glycine-rich bipartite structure have also been identified in mammals, amphibians, ascidians, and cyanobacteria (Nishiyama et al. 1997; Danno et al. 1997; Uochi and Asashima 1998; Tanaka et al. 2000; Maruyama et al. 1999). At least a subset of these genes cycle in a diurnal manner and/or are inducible by lowered temperatures (Nishiyama et al. 1997, 1998; Sato and Maruyama 1997; Maruyama et al. 1999; Danno et al. 1997). Thus, these RRM-GRP proteins may carry out a function that is conserved among kingdoms. 3. Entrainment of the Clock by Light. A notable feature of clocks in all organisms yet studied is that the innate period is somewhat longer or shorter than 24 h. In order to cycle with a precise daily rhythm, the clock must be entrained, or synchronized, each day. In Drosophila, light serves to entrain the clock by initiating the phosphorylation and rapid degradation of the TIM protein (Hunter-Ensor et al. 1996; Myers et al. 1996; Zeng et al. 1996). This results in a phase delay in the evening, when TIM is being continually resynthesized, and a phase advance in the morning, when TIM is not effectively replaced. The light signals are perceived by cryptochrome, a protein that is similar in amino acid sequence to the CRY proteins in plants. Upon illumination, CRY undergoes a photochemical change that allows a physical interaction with the TIM protein and presumably initiates the degradation process (Ceriani et al. 1999).

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As in other organisms, under constant illumination, the period of the clock can be modulated by light in an intensity-dependent manner (Somers et al. 1998a,b). Although this phenomenon may not be physiologically relevant, it has proven useful for determining the potential role of specific genes in the light entrainment of the clock. Millar et al. (1995) found that both red and blue wavelengths were effective in shortening the period of LHC:LUC expression, suggesting the involvement of phytochromes and potentially also cryptochromes. By examining the period length of LHC:LUC expression in phyA, phyB, cry1, or cry2 mutant plants, Somers et al. (1998a) concluded that red light signals are transmitted to the clock by phytochromes A and B, with PHYA acting under low intensities and PHYB acting under high intensities, whereas blue light inputs are provided by PHYA and cryptochromes. Interestingly, loss of CRY2 shortened the period under low-fluence blue light, but had little effect under the higher fluences where photoperiodic timing of flowering is affected. Unlike in Drosophila, no direct interactions between photoreceptors and clock components have been demonstrated in plants, and clock function may depend on a mechanism to transmit information from photoreceptors to the clock. The EARLY FLOWERING 3 (ELF3) gene may play such a role. Plants lacking ELF3 activity display phenotypes that mimic phyB mutations (i.e., increased hypocotyl elongation in red light and petiole length). However, mutations in ELF3 and PHYB have additive effects when combined, suggesting that ELF3 is not simply a component of a PHYB signal transduction pathway (Reed et al. 2000). Unlike mutations in PHYB, which alter the periodicity of the clock (above), mutations in ELF3 abolish rhythmicity, and do so in a light-dependent manner (Hicks et al. 1996). The CCA1 gene is rapidly and transiently upregulated in response to light, specifically red light (Wang et al. 1997), suggesting that CCA1 could also be involved in transmitting signals to the clock from phytochrome. Whether or not the phosphorylation of CCA1 or LHY is associated with their turnover, as is the case with TIM, has not been reported. However, as in the Drosophila clock, protein degradation is an important process of the plant clock mechanism. Two homologous genes have been identified in Arabidopsis that might play a role in the turnover of clock components. These genes, FKF1 and ZEITLUPE (ZTL), encode proteins containing an F-box motif (Nelson et al. 2000; Somers et al. 2000). Where studied in other organisms, F-box-containing proteins act in the recognition of degradation substrates for the ubiquitin proteolytic pathway (Patton et al. 1998; Kornitzer and Ciechanover 2000). FKF1 transcripts oscillate in a circadian manner, whereas ZTL mRNA expression

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is apparently not under clock control. However, mutations in both genes confer a similar phenotype, with flowering delayed primarily under photoinductive conditions. In addition to the F-box, the FKF1/ZTL proteins contain a segment similar to the flavin-binding domain in the bluelight receptor NPH1 involved in phototropism. At least ztl mutants exhibit a period-lengthening phenotype that is strongly light-dependent, and at least the FKF1 promoter is selectively activated under white or blue light. Taken together, these observations indicate that these proteins may function as light-dependent clock regulators (Nelson et al. 2000; Somers et al. 2000). 4. Other Photoperiodic Pathway Genes. CONSTANS (CO), another of the promotive photoperiod pathway genes, was one of the first of the flowering-time genes to be cloned, and thus has been the most extensively studied (Putterill et al. 1995). The CO protein contains zincfinger-type DNA-binding domains common to the GATA1 family of transcription factors, and thus likely acts as a component of the transcriptional apparatus. Known mutations in CO are semidominant. Where a mutation results in complete loss of function of the gene, semidominance is an indicator that the respective gene product is limiting for the respective process (i.e., that relative levels of the gene product are important). Consistent with this, increasing CO activity, either constitutively through adding extra copies of the gene in transgenic plants (Putterill et al. 1995), or transiently by activating the protein in an inducible system (Simon et al. 1996), is sufficient to trigger flowering. In addition, consistent with its role in promoting flowering under photoinductive conditions, the mRNA levels of CO are elevated in long-day grown Arabidopsis plants relative to short-day grown plants (Putterill et al. 1995), and this regulation is accomplished at least in part by transcriptional upregulation of the gene (Suarez-Lopez et al. 1998). CO was found to be expressed in both leaf and stem tissue, but the very low abundance of the mRNA complicated a more thorough analysis of spatial expression patterns (Putterill et al. 1995). Its likely function as a transcription factor and regulation at the transcriptional level suggests that CO is an intermediate in a cascade of transcriptional events. What could be upstream regulators and downstream targets of CO? CRY2 and PHYB likely act upstream of CO as indirect positive and negative regulators, respectively, because mutations in CRY2 decrease CO mRNA expression (Guo et al. 1998), and the early-flowering seen in phyB mutants in short days is alleviated in a genetic background compromised for CO activity (Putterill et al. 1995). Onouchi et al. (1998) clarified the relationship between CO and several other photoperiodic

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pathway genes by examining the effect of 35S:CO expression in photoperiodic pathway-mutant backgrounds. The premise of this experiment was that if CO acted in a genetic pathway downstream from GENE X, then removal of GENE X function should have no effect on the phenotype conferred by constitutive expression of CO (i.e., constitutive expression of CO would be epistatic to loss of GENE X function). In contrast, if CO acted upstream of GENE X, then adding CO activity should make no difference to the phenotype conferred by loss of GENE X function. In this case, the 35S:CO transgene was completely epistatic to gi and lhy, suggesting that CO acts downstream from these two genes. In contrast, 35S:CO had only a small effect in genetic backgrounds in which expression of the FT or FWA flowering-time genes was disrupted (see below), suggesting that these two genes function downstream from CO. Extending this experimental approach, Onouchi et al. (2000) discovered a target of CO by searching for mutations that suppressed the earlyflowering phenotype conferred by constitutive CO expression. This gene, designated SUPPRESSOR OF CONSTITUTIVE EXPRESSION OF CO1 (SOC1), encodes a protein containing a domain designated the MADS box. This motif is present in other proteins known to bind DNA as homo- or heterodimeric complexes (Trobner et al. 1992; Riechmann and Meyerowitz 1997). SOC1 is expressed in the shoot and inflorescence apical meristem as well as the leaf primordia in response to inductive photoperiods (Samach et al. 2000). Consistent with the results of Onouchi et al. (1998), Samach et al. (2000) identified the FT gene (see below) as a very early downstream target of CO activity. In the approach used here, the CO coding sequence was translationally fused to the ligand-binding domain of the rat glucocorticoid receptor. This CO-GR fusion protein was expressed constitutively in transgenic plants, and could be directed to the nucleus and thus “activated” by application of the synthetic glucocorticoid hormone dexamethasone. In this case, FT transcript accumulation was seen within two hours of dexamethasone application. This experimental approach also resulted in the identification of two other early downstream targets of CO, AtP5CS2 involved in proline biosynthesis, and ACS10, encoding a potential 1-aminocyclopropane-1-carboxylic acid (ACC) synthase involved in the production of ethylene. AtP5CS2 is apparently essential for the elongation of the internodes that occurs upon flowering in Arabidopsis (bolting), as reduction in AtP5CS2 expression in transgenic plants eliminated this response (Nanjo et al. 1999). Although ethylene plays an obvious role in flowering in other species (e.g., Bromeliads), the precise role of ethylene in flowering in Arabidopsis is not known (Bernier et al. 1981). Mutants insensitive to ethylene exhibit slightly

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delayed flowering, but the molecular mechanism of this effect has not been explored (Guzman and Ecker 1990). CO exists as a member of a gene “family,” or group of genes that encode structurally related proteins (Ledger et al. 1996). It is possible that these CO-like (COL) genes also have a role in flowering, however Putterill et al. (1997) have noted that at least one of these genes, COL1, does not seem to be expressed at higher levels in inductive photoperiods, and to date has not been identified as important in flowering by traditional genetic analyses. In other species studied [i.e., apple (Hoon et al. 1999) and Brassica napus (Robert et al. 1998)], families of CO homologs also exist. It has been suggested that the function of CO in apple could be different from that in Arabidopsis based on the apparent abundance of mRNAs of two of the apple genes in the developing flower and fruit (Hoon et al. 1999), but such studies are complicated by the ambiguity of the evolutionary relationships between the identified apple genes and the Arabidopsis CO and COL genes, and the fact that the spatial pattern of CO expression in Arabidopsis has not been fully established. In Brassica napus, CO homologs are found at genomic positions corresponding to quantitative trait loci (QTL) affecting flowering time, and at least one of the Brassica CO homologs is functionally homologous to its Arabidopsis counterpart, because it is able to complement the flowering-time defect conferred by the co-2 mutation when expressed in transgenic Arabidopsis (Robert et al. 1998). A gene encoding a protein closely related to CO has recently been cloned from the short-day plant Pharbitis nil through an assay designed to identify genes that are upregulated in response to inductive photoperiods (i.e., short days; J. Liu and H. Kende, pers. commun). This is an important finding because the fact that CO is upregulated in both species in response to inductive photoperiods, even though the plants are of opposite flowering habits, suggests that the molecular mechanisms that are distinct between plants of varying photoperiodic responses lie genetically upstream of CO. In maize, ancestrally a short-day plant, the INDETERMINATE1 (ID1) gene promotes flowering in response to inductive photoperiods (Singleton 1946; Galinat and Naylor 1951). This is the only gene cloned to date that has unequivocally been demonstrated to be involved in flowering time in species other than Arabidopsis. ID1 encodes a protein containing zinc-finger motifs, suggesting that it binds DNA. It is expressed predominately in the leaf and influences flowering in a noncell-autonomous manner, and thus possibly regulates the production of a transmissible signal (Colasanti et al. 1998). There is no strong structural homology between ID1 and any of the Arabidopsis flowering-time genes that have been cloned to date, and the family of ID1-like genes that

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do exist in Arabidopsis (Colasanti et al. 1998) have not yet been reported to be involved in flowering. This could indicate a significant divergence in flowering mechanisms between Arabidopsis and maize. B. Non-photoperiodic Induction: The Autonomous Pathway As mentioned previously, loss of function of photoperiodic pathway genes does not prevent flowering but merely delays it, suggesting that at least one redundant pathway exists. Numerous flowering-time genes have been identified that are presumed to work outside of the photoperiodic control of flowering in the so-called autonomous pathway. LUMINIDEPENDENS (LD) was one of the first flowering-time genes identified (Redei 1962), and one of the first plant genes cloned through T-DNA mutagenesis (Lee et al. 1994a). In this technique, segments of DNA of known sequence are transferred into a plant by Agrobacterium, where they integrate into the genome at random locations. Interruption of a gene by the T-DNA often results in loss of gene function, and the corresponding gene sequence can be easily cloned by simple molecular techniques (Azpiroz-Leehan and Feldman 1997). The LD gene encodes a large protein containing two interesting structural features. First, a homeodomain—a nucleic acid-binding motif found in developmentally important proteins from yeast, plants, and animals—is found near the amino terminus. The homeodomain in LD is highly homologous to that found within the Drosophila Distal-less protein, which functions as a developmental switch to initiate limb formation (Cohen 1990). It also closely resembles the homeodomain found in Mata1, a yeast protein that acts as one component of a heterodimeric factor that represses expression of haploid-specific genes (Johnson and Herskowitz 1985). The other interesting structural feature is an acidic carboxyl-terminal region enriched in glutamine residues and containing short, homopolymeric glutamine stretches. These structural features are common to the activation domains of known transcriptional activators such as Drosophila Antennapedia and herpes virus VP16 (Gerber et al. 1994; Triezenberg 1995). Thus, it is possible that LD acts as a transcriptional regulator. Consistent with this proposed role, the LD protein contains nuclear localization signals and is localized to the nucleus. LD is expressed ubiquitously throughout the plant, with a concentration of mRNA expression in proliferating tissues, including the shoot, root, and floral apices (Aukerman et al. 1999). The function of LD may have diverged through evolution. An orthologous gene has been characterized from maize (van Nocker et al. 2000). The maize LD gene is highly homologous to its Arabidopsis counterpart,

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containing both the homeodomain and the potential transcriptional activator region, and exhibits an analogous mRNA expression pattern in the maize plant. However, when expressed in transgenic Arabidopsis containing an ld mutation, it does not complement the flowering-time defect, but instead causes developmental abnormalities associated with the shoot and floral meristems (van Nocker et al. 2000). What function this gene has been recruited for in maize is not presently known, but should be revealed by analyses of transposon-tagged lines. Although the activity of most plant genes studied to date seems to be controlled predominately at the transcriptional level, recent evidence suggests that posttranscriptional control may be an important factor in the regulation of flowering. The FCA gene was cloned and found to encode a large protein containing RRM motifs thought to mediate binding to RNA (Macknight et al. 1997). In support of the structural suggestion of function, the FCA protein binds to RNA in vitro, with a preference for G- and U-rich sequences (Macknight et al. 1997). The FCA gene produces multiple transcripts as a result of alternative splicing and transcriptional termination. Only one of these, which is a minority of FCA transcripts, would encode the presumed, full-length protein, and splicing to produce the full-length “active” FCA mRNA is likely to be regulated, as high-level expression of the genomic FCA sequence in transgenic plants resulted in only a minor increase in the amount of active mRNA (Macknight et al. 1997). Interestingly, it appears that FCA is able to promote flowering in a cell non-autonomous manner, because flowering is not delayed in periclinal chimaeras that express FCA only in the epidermal cell layers (Furner et al. 1996). In addition to the RNA-binding motifs, the presumed active form of the FCA protein contains a region designated the WW motif that contains two closely spaced tryptophan residues (Bork and Sudol 1994). This is potentially an essential component of the FCA protein, as it is excluded from the protein encoded by the strong fca-1 allele (Macknight et al. 1997). In other systems, WW motifs mediate interactions with protein partners containing proline-rich regions (Kay et al. 2000). Proteins or RNAs that interact with FCA have not been identified. One possibility is the protein encoded by FY. Mutations in FY do not further enhance the late flowering conferred by loss of FCA function (Koornneef et al. 1998a), suggesting that the two gene products operate in close proximity. In contrast, mutations in two other autonomous pathway genes, FPA and FVE, greatly enhance the lateness of fca (and fy) mutants. This suggests some redundancy in the mechanism of the autonomous pathway. However, as previously cautioned, this type of genetic analysis is contingent on mutations creating a complete loss of function, and even

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in cases where the gene has been cloned, this is difficult to demonstrate. FPA was recently cloned and, like FCA, encodes a protein containing RRM-type RNA-binding motifs (R. Amasino, pers. commun.). The cloning of FY, FVE, and another autonomous pathway gene, FLD, have not yet been reported. Semidominant mutations in the SHORT VEGETATIVE PHASE (SVP) gene confer photoperiod-sensitive early flowering (Hartmann et al. 2000). SVP encodes a MADS-box transcription factor that is expressed in the apical meristem during the vegetative phase, but apparently not in the inflorescence apical meristem. This expression pattern is consistent with its role as a repressor of flowering. SVP mRNA is also expressed in the early floral meristem, suggesting a role in flower development. However, loss of SVP function confers no gross floral defects, indicating that a function at this stage is redundant or minor (Hartmann et al. 2000). The genetic relationships between SVP and other autonomous pathway genes have not been characterized. C. Vernalization In many plants, flowering can be accelerated or induced by exposure to a long period of near-freezing temperatures. This is a commonly employed reproductive strategy that allows for flowering and seed production in the environmentally favorably period following natural winter. This phenomenon, termed vernalization, has been studied for decades at the physiological level but only recently at the molecular level. The lack of molecular work addressing vernalization is partly due to the fact that in Arabidopsis thaliana, the commonly utilized laboratory strains flower soon after germination, and extended cold treatments do little to further abbreviate the vegetative phase (Koornneef et al. 1998b). However, most natural ecotypes of Arabidopsis behave as winter annuals, flowering extremely late in the absence of cold, but very early when exposed to cold for extended periods. The flowering habit among natural ecotypes is largely determined by allelic variation at two loci, designated FRIGIDA (FRI) and FLOWERING LOCUS C (FLC); (Lee et al. 1993; Koornneef et al. 1998b). “Early” alleles at either loci behave similarly to presumed null alleles created by induced mutation, suggesting that natural early alleles have lost function (Michaels and Amasino 1999). FLC is expressed predominately in the vegetative apex and roots, but is absent from the inflorescence apex. Expression of FLC mRNA is apparently not significantly decreased as the plant proceeds through the vegetative phase, suggesting that repression of flowering by FLC can be overcome by developmental progression (Sheldon et al.

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1999). FLC encodes a MADS-box-containing protein (Michaels and Amasino 1999; Sheldon et al. 1999). Because other MADS-box proteins are known to work as heterodimers (Trobner et al. 1992), it is possible that FLC has a DNA-binding partner. One possibility is SVP, as both genes are expressed in the shoot apical meristem specifically during the vegetative phase. However, unlike those of FLC, mRNA levels of SVP are not diminished after extended cold treatments (Hartmann et al. 2000). The activity of FLC is semidominant, and transgenic plants containing extra copies of the FLC genomic sequence never flower without cold, acting in essence as biennials (Michaels and Amasino 1999; Sheldon et al. 1999). Importantly, these findings suggest that the difference in flowering habit between winter-annual plants and biennial plants could be quantitative rather than qualitative. The cloning of FRI has recently been reported; this gene encodes a protein that does not exhibit significant sequence identity to any other protein of known function (Johanson et al. 2000). In Arabidopsis, a genotype conferring the winter-annual habit can also be synthesized by impairing the function of the promotive autonomous pathway genes (Koornneef et al. 1991), and, like repression of flowering imposed by FRI, the block to flowering resulting from the loss of autonomous-pathway gene function is also dependent on FLC activity (Lee et al. 1994b; Koornneef et al. 1994; Sanda and Amasino 1996a,b). These data suggest that the flowering-repressive activity of FLC is both positively regulated by FRI and negatively regulated by autonomous pathway genes. Consistent with this idea, FLC mRNA expression is increased both in genotypes containing late FRI alleles, and in autonomous-pathway gene mutants (Michaels and Amasino 1999; Sheldon et al. 1999). FLC mRNA expression is decreased after extended cold exposures (Michaels and Amasino 1999), suggesting that vernalization involves molecular events “upstream” from FLC. The winterannual habit conferred by loss of autonomous-pathway gene function is not dependent on FRI, indicating that neither the activity of FRI nor the autonomous pathway genes is necessary for the vernalization response. Thus, although these genes set up a requirement for cold for flowering, they are unlikely to be directly involved in the associated cold signal transduction. Specific components involved in transmitting the signal from the cold stimulus to FLC expression have not yet been identified. Using a genetic approach, Chandler et al. (1996) identified at least two loci, designated VRN1 and VRN2, that could play such a role. These mutants were isolated in an fca mutant background based on a lack of vernalization response. FLC mRNA levels are only partially decreased after

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cold treatment in these mutants, consistent with the idea that VRN1 and VRN2 function upstream to regulate FLC expression (Sheldon et al. 2000). Ishitani et al. (1998) identified a recessive mutation, hos1-1, that constitutively activated gene expression from a cold-responsive promoter. hos1-1 plants exhibited accelerated flowering, suggesting that HOS1 might normally act as a negative regulator of vernalization. However, these results were difficult to interpret because hos1-1 conferred pleiotropic effects on growth, and the specific genetic background utilized (C24) is normally early flowering due to an “early” FLC allele (Sanda and Amasino 1996a). The well characterized cold-regulated (COR) genes involved in the process of acclimation probably have little or no role in vernalization, as freezing tolerance is not affected in the vrn mutants (Chandler et al. 1996), and constitutive expression of members of the CBF family of transcriptional activators upregulates COR gene expression in a winter annual line in the absence of cold, but has no effect on flowering time (J. Liu and S. van Nocker, manuscript submitted). Some characteristics of vernalization, including the requirement for cell division for the vernalized state to be attained and the stability of the vernalized state through mitosis, suggests an epigenetic mechanism (Wellensiek 1964). One possibility is the covalent modification of DNA through cytosine methylation. Evidence for the involvement of DNA methylation in the vernalization response has been presented by Burn et al. (1993) and Brock and Davidson (1994), who found that the promotion of flowering by extended cold in Arabidopsis and wheat, respectively, could be partially substituted for by exposure of plants to the ribonucleotide analog 5-azacytidine (5-azaC). Treatment with this compound results in demethylation of DNA. In the study by Burn et al. (1993), flowering was reportedly accelerated only in genotypes that are known to exhibit a strong vernalization response. Thus, the partial substitution for cold treatment conferred by 5-azaC apparently acted specifically upon the vernalization pathway. It was hypothesized that extended cold results in the selective demethylation and transcriptional activation of floral-promotive genes (Finnegan 1998). A further possible link between DNA methylation and vernalization was hypothesized by Finnegan et al. (1996), who reported that antisense expression of the METHYLTRANSFERASE1 (MET1) gene in transgenic Arabidopsis resulted in both decreased genomic DNA methylation levels and early flowering. This early flowering was apparently associated with decreased FLC mRNA abundance (Sheldon et al. 1999), again suggesting specificity for the vernalization pathway. In contrast to these results, Ronemus et al. (1996) found that MET1 antisense expression

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conferred highly pleiotropic effects, including slightly delayed flowering, in transgenic Arabidopsis. The apparent contradictions between these two reports could reflect differences in environmental conditions or genetic backgrounds used. However, neither group utilized a genetic background that exhibits a strong vernalization response. Other aspects of these results should also be interpreted with caution. Goto and Hamada (1988) and Chandler and Dean (1994) demonstrated that growth of plants on the nucleotide analog 5-bromodeoxyuridine, which does not result in reduced DNA methylation, could also accelerate flowering. D. Induction by Gibberellins The influence of GAs on flowering in many plants is well known (Lang 1965; Zeevaart 1983). In long-day rosette plants such as Arabidopsis, GAs generally have an inductive effect, and this is especially striking in Arabidopsis where flowering is delayed by growth in short days, or in winter-annual genotypes grown in the absence of cold. Consistent with this, flowering is delayed in the ga1 mutant that is defective in GA biosynthesis, and in the gai mutant, which is insensitive to GAs (Koornneef and van der Veen 1980; Koornneef et al. 1985). In addition, plants carrying mutations in the SPINDLY (SPY) gene, which exhibit a constitutive GA response, flower early (Jacobsen and Olszewki 1993). Exogenously applied GA is able to promote flowering in all late mutants studied, and mutations in GA biosynthesis or perception are interactive with all flowering-promotive genes studied, especially those grouped into the photoperiodic pathway (Putterill et al. 1995; Simpson et al. 1999; our independent observations). Consistent with the strongly interactive effect with photoperiod pathway genes, ga mutant plants are apparently unable to flower when grown in short days, and gai plants flower extremely late under such conditions (Wilson et al. 1992). Thus, it appears that the production of GAs represents an additional pathway to flowering that operates in parallel with the photoperiodic pathway, and, to some extent, the autonomous pathway as well. The role of GAs in vernalization is unclear. Although GAs are able to promote flowering in winter-annual genotypes (thereby bypassing the requirement for cold), winter-annual genotypes containing the ga or gai mutations still exhibit a normal vernalization response (R. Amasino, pers. commun.; Chandler et al. 2000). This would suggest that GAs are not necessary for vernalization. However, as GA production is not completely eliminated in the ga mutant (J.A.D. Zeevaart, pers. commun.), such results should be interpreted with caution. In addition, although GAI has an obvious role in GA signal transduction during vegetative

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growth, other yet unidentified GA-signaling components could be involved in the flowering response.

IV. ROLE OF CARBOHYDRATES Carbohydrates have long been known to play a key role in flowering (Bernier et al. 1993). The concentration of sucrose, the major translocated sugar in most plants, increases dramatically in phloem exudates upon photoinduction in both short-day and long-day plants, even when the photoinductive treatment does not result in a net increase in photosynthesis (Bodson and Outlaw 1985; Houssa et al. 1991; Corbesier et al. 1998). One of the earliest biochemically detectable changes in the shoot meristem upon photoinduction is the accumulation of sucrose (Bodson and Outlaw 1985) and labeling experiments suggest that this sucrose originates not from increased photosynthesis, but from mobilization of sugars from reserve carbohydrates such as starch in the leaves and stem (Bodson et al. 1977). Arabidopsis will flower in complete darkness if the aerial portion of the plant is supplied with sucrose or glucose (Redei et al. 1974; Goto 1982; Araki and Komeda 1993). Under such conditions, the lateflowering phenotype conferred by mutations in GI, CO, FCA, FPA, and FVE was complemented or nearly complemented. In contrast, flowering was not promoted by these conditions in plants carrying mutations in FWA or FT (Araki and Komeda 1993; Roldan et al. 1999). These surprising results suggest that the fundamental mechanism of both the photoperiodic and autonomous pathways could be the delivery of sugars to the shoot apex! Sucrose is synthesized in the cytosol from the products of photosynthesis or starch degradation, transported to and loaded into the phloem, translocated throughout the plant, unloaded from the phloem, and then transported from cell to cell. This complicated routing provides many opportunities for control of sugar transport, and thus it is likely that many genes are involved. Interestingly, mutations in the GI gene are pleiotropic in that mutants accumulate excess levels of starch in the leaves and stem (Eimert et al. 1995). At least one other Arabidopsis mutant that was originally identified as a starch accumulator, carbohydrate accumulation mutant1 (cam1), was found to flower late relative to wild-type plants, especially when grown under continuous light (Eimert et al. 1995). High starch content per se does not seem to be the direct cause of the flowering-time defect, because the flowering-time defect conferred by gi and cam1 was not rescued in genetic backgrounds where starch synthesis was dis-

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rupted (Eimert et al. 1995). Other mutants that lack starch, ADP-glucose pyrophosphorylase1 (adg1) and phosphoglucomutase1 (pgm1), and at least one other mutant that accumulates starch, starch-in-excess1 (sex1), also exhibit delayed flowering, but only under photoperiods of less than 16 h (Lin et al. 1988; Caspar et al. 1985; Caspar et al. 1991; Corbesier et al. 1998). The observation that both the overabundance of starch, and lack of starch, can affect flowering in a similar manner further suggests that flowering is not directly affected by starch content. In fact, the lack or excess of starch in the pgm1 and sex1 mutants, respectively, seems to disrupt carbohydrate metabolism in a similar manner, as in both mutants soluble sugars (including sucrose) accumulate to abnormally high levels (Caspar et al. 1985; Caspar et al. 1991). Thus, it seems probable that the predominant effector of flowering in these mutants is the levels of sugars. Arabidopsis plants grown at low temperature also accumulate soluble sugars, and this may be related to the delayed flowering seen under these conditions.

V. CONTROL OF MERISTEM IDENTITY The shoot and flower are, in spite of their radical difference in morphology, essentially analogous structures produced by the meristem. The fate of meristems—to generate flowers rather than shoots—is governed by a group of meristem identity genes, which are activated during the transition to flowering. This group of genes in turn controls expression both of the floral organ identity genes, which control the development of the floral organs, and cadastral genes, which regulate the boundaries of expression of the organ identity genes. The molecular biology of flower development is beyond the scope of this review, and the reader is referred to recent excellent discussions on this topic (Bowman 1997; Sessions et al. 1998). A. Meristem Identity Genes As with genes influencing the timing of flowering, genes involved in influencing meristem identity have been identified by screening for mutants in which meristem identity is altered. Such screens have identified genes that both positively and negatively regulate the shoot-toflower transition. In Arabidopsis plants homozygous for the recessive terminal flower 1 (tfl1) mutation, the normally indeterminate inflorescence terminates in a single flower, and lateral shoots develop as solitary flowers (Shannon and Meeks-Wagner 1991; Alvarez et al. 1992).

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Thus, a presumed function of TFL1 is to keep the inflorescence meristem in an indeterminate state. Plants lacking TFL1 activity also flower slightly early, suggesting that TFL1 functions during the vegetative phase as a repressor of the shoot-to-inflorescence transition (Shannon and Meeks-Wagner 1991; Schultz and Haughn 1993). TFL encodes a member of a small protein family exhibiting limited homology to mammalian Raf kinase inhibitor protein (RKIP). RKIP is a membrane-associated protein that regulates Raf-1 kinase, which is intimately involved in signal transduction cascades controlling cell proliferation and differentiation in mammals (Ferrell 1996). The amino-terminus of RKIP is cleaved off to form a small peptide hormone, leading to the speculation that TFL may in a similar manner be the progenitor of a small signaling peptide involved in flowering (Bradley et al. 1997). That intercellular signaling should be involved in flowering is expected, as the meristem must function as a unit to organize flower primordia even though it is composed of clonally unrelated cells (see below). Constitutive expression of the TFL1 gene in transgenic Arabidopsis confers a phenotype that is essentially opposite to that seen in tfl1 mutants—such plants exhibit delayed flowering, and produce secondary inflorescences that are not subtended by cauline leaves (Ratcliffe et al. 1998). Because Arabidopsis flowers are not normally found in association with leaves (bracts), such structures can be interpreted as a conversion of flowers to inflorescence shoots. Conversion of flowers to shoots is also seen in plants carrying loss-offunction mutations in a group of genes best typified by LEAFY (LFY). In plants carrying strong lfy alleles, early-arising (basal) flowers are completely transformed into shoots, whereas those that develop in more apical positions exhibit partial floral character. In plants carrying very weak lfy alleles, secondary shoots subtended by cauline leaves develop at the first few positions normally occupied by flowers (Schultz and Haughn 1991). That flowers eventually do develop even in the absence of LFY activity indicates that other genes function in a partially redundant manner to promote the inflorescence-to-floral switch. One of these is APETALA1 (AP1). Loss of AP1 function phenocopies very weak lfy alleles with respect to inflorescence structure, but dramatically enhances the phenotype of lfy plants, such that in lfy/ap1 double mutants, even the most apical nodes produce structures with strong shoot characteristics (Bowman et al. 1993; Huala and Sussex 1992; Weigel et al. 1992). Strong ap1 alleles also confer a striking floral phenotype—sepals found in the outer whorl of the flower exhibit leaf-like characteristics, and often subtend secondary flowers (Irish and Sussex 1990). This phenotype can be interpreted as a partial reversion of the flower into a shoot, further implicat-

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ing AP1 in meristem identity (Mandel et al. 1992). Strong constitutive expression of both LFY and AP1 in transgenic plants results in premature transformation of the shoot into a flower, mimicking loss of TFL function (Weigel and Nilsson 1995; Mandel and Yanofsky 1995). Incredibly, Arabidopsis LFY is able to accomplish this even in a divergent tree species, aspen, suggesting a high degree of conservation of meristem identity function during evolution (Weigel and Nilsson 1995). That loss-of-function mutations in genes such as LFY and AP1 exhibit additive phenotypic effects when combined is evidence that at least two pathways are normally involved in establishing the floral meristem (Shannon and Meeks-Wagner 1993). The genetic evidence indicating that these pathways are partially redundant is reinforced by experiments showing that the shoot-to-flower conversion conferred by 35S:AP1 is not dependent on LFY, and that mutations in AP1 cannot fully suppress this effect in 35S:LFY plants (Mandel and Yanofsky 1995; Weigel and Nilsson 1995). However, these two pathways are strongly interactive. In primordia destined to become flowers, LFY mRNA expression precedes that of AP1, and AP1 upregulation is delayed in lfy plants (Simon et al. 1996; Hempel et al. 1997; Liljegren et al. 1999). In addition, ectopic activation of LFY activity results in premature AP1 expression (Parcy et al. 1998). These findings suggest that LFY acts as a positive regulator of AP1. Conversely, LFY is expressed prematurely in primordia of 35S:AP1 plants, suggesting a reciprocal positive regulation between the two genes (Liljegren et al. 1999). The protein encoded by LFY does not resemble any other known protein (Weigel et al. 1992), but numerous lines of evidence indicate that it acts as a transcription factor. LFY protein is localized to the nucleus and is able to mediate transcriptional activation in yeast when fused to a suitable activation domain (Parcy et al. 1998). Consistent with the genetic evidence that LFY positively regulates AP1 expression, the LFY protein binds to a potential AP1 promoter element in vitro (Parcy et al. 1998). Moreover, Wagner et al. (1999) demonstrated through the use of an inducible LFY-GR fusion protein that LFY is able to rapidly activate AP1 expression even in the presence of cycloheximide, suggesting a direct interaction. Another gene with a promotive role in floral identity is CAULIFLOWER (CAL). The function of this gene is apparently completely redundant with that of AP1, such that in an otherwise wild-type genetic background, plants lacking CAL activity appear normal. However, in plants lacking both CAL and AP1 activity, meristems never develop determinate floral character and continue to proliferate, and inflorescences develop into structures that resemble tiny versions of the garden

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vegetable for which the gene is named (Bowman et al. 1993). The phenotypic similarity between Arabidopsis cal/ap1 double mutants and cauliflower led Kempin et al. (1995) to investigate functional conservation of CAL genes in the two species. In both Brassica oleracea, and its cauliflower derivative (Brassica oleracea, var. botrytis) CAL is expressed in a spatial and temporal pattern similar to that seen with CAL in Arabidopsis. However, the gene from var. botrytis encodes a protein that is significantly truncated and is probably nonfunctional (Kempin et al. 1995), suggesting a molecular explanation for what is probably the most popularly recognized natural inflorescence variation. Both AP1 and CAL encode proteins containing a MADS-box domain, consistent with roles as transcription factors (Mandel et al. 1992; Kempin et al. 1995). The essentially opposite phenotypes conferred by loss of TFL and LFY/AP1/CAL function indicates that these genes operate antagonistically, and molecular studies support this conclusion. Expression of both TFL and LFY mRNAs is maintained at a low level in the shoot apex during the vegetative phase, and is upregulated upon the transition to flowering. However, their expression is spatially separated, with TFL mRNA present in the center of the meristem, and LFY mRNA present only in emerging primordia (Bradley et al. 1997). In plants lacking LFY, AP1, or CAL activity, TFL expression extends into the lateral primordia (Ratcliffe et al. 1999), whereas in plants constitutively expressing LFY, TFL expression is greatly decreased (Ratcliffe et al. 1999; Liljegren et al. 1999). Like AP1 and CAL1, at least two other genes are known to have redundant roles in promoting floral identity. Mutations in APETALA2 (AP2), for example, do not confer strong flower-to-shoot conversion, but instead enhance both lfy and ap1 phenotypes (Shannon and Meeks-Wagner 1993). Plants carrying mutations in the UNIDENTIFIED FLORAL ORGANS (UFO) gene resemble weak lfy mutants in that basal inflorescence nodes exhibit some shoot identity (Levin and Meyerowitz 1995; Wilkinson and Haughn 1995). In addition, in short-day-grown ufo plants, the transition to an inflorescence meristem is incomplete, and reversion to a vegetative meristem can occur (Wilkinson and Haughn 1995). Like the aforementioned FKF1 and ZTL, UFO encodes an F-boxcontaining protein, implicating this factor in the selective elimination of other, possibly regulatory proteins (Ingram et al. 1995). The AGAMOUS gene, better known for its role in governing floral organ identity in the inner whorls, also functions to promote floral meristem identity by regulating meristem determinacy. The normally determinate floral meristem becomes indeterminate in ag mutant plants, and under short-day conditions can even completely revert to an inflorescence meristem (Yanofsky et al. 1990; Mizukami and Ma 1995, 1997).

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B. Integration of Flowering Pathways and Activation of Meristem Identity Genes Genes affecting the timing of flowering are obvious candidates for direct regulators of meristem identity genes, and recent genetic and molecular studies have demonstrated numerous interactions between the two classes of genes. Simon et al. (1996) utilized the inducible CO-GR expression system described above to examine the response of the LFY, AP1, and TFL1 genes to increased CO activity. Within 24 h of CO:GR activation in plants grown in inductive photoperiods, LFY and TFL1 mRNAs accumulated to detectable levels, and detectable AP1 mRNA upregulation occurred after ~72 h. The delayed expression of AP1 relative to that of LFY is in accordance with genetic evidence that LFY acts upstream of AP1 (above). These kinetics were similar to those seen upon the transfer of short-day-grown plants to inductive photoperiods (Simon et al. 1996). In contrast, when CO-GR was activated in short-day-grown plants, LFY and TFL1 were again activated within 24 h, but the delay in the upregulation of expression of AP1 was extended to ~120 h. The authors concluded that since inductive photoperiods were more effective than CO to activate AP1, an additional, unknown mechanism operating in inductive photoperiods is involved in the upregulation of AP1 (Simon et al. 1996). In fact, the flowering time genes FT and FWA (see below) may play a role that is redundant with that of LFY in activating AP1. Evidence for this was presented by Ruiz-Garcia et al. (1997), who showed that the phenotype conferred by a strong allele of lfy is greatly enhanced in an ft or fwa mutant background. It is known that GAs promote flowering at least in part through upregulation of LFY, because in the ga1 mutant, LFY promoter activity is reduced, and its upregulation in response to inductive photoperiods is delayed relative to wild-type plants. In addition, 35S:LFY expression can partially complement the flowering defect conferred by ga1 in short day conditions (Blazquez et al. 1998). Recently, Blazquez and Weigel (2000) demonstrated that distinct cis elements in the LFY promoter mediate the induction of LFY in response to GAs or inductive photoperiods. Thus, it appears that LFY represents an integration point of at least the photoperiod pathway and the GA pathway.

VI. COMPETENCY A multitude of physiological studies has indicated that flowering is dependent not only on the ability of the leaf to produce the floral stimulus, but also on the ability of the shoot apical meristem to respond to

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it (Lang 1965). The shoot apical meristem is a group of specialized cells found within the apex at the growing tip of shoots. In Arabidopsis, as in other plants, the meristem displays typical tunica-corpus organization, recognizable as early as the torpedo stage of embryogenesis (Long et al. 1996). The tunica layers (L1 and L2) are propogated through anticlinal cell divisions, and thus the L1, L2, and L3 tend to be clonally unrelated. The meristem can display an additional level of organization that is superimposed on the tunica-corpus structure. This consists of radially symmetric “zones” that are often distinguished by mitotic activity and cell size and density (Vaughan 1952; Steeves and Sussex 1989). The central initiation zone, at the summit of the meristem, is characterized by a group of large cells with prominent vacuoles that apparently divide very slowly. Flanking the central initiation zone is a ring of smaller, more densely cytoplasmic, proliferative cells termed the peripheral zone. In the peripheral zone, groups of cells are recruited into leaf or flower primordia where they may soon assume specialized roles. Immediately subtending the central initiation zone is a group of proliferative cells referred to as the rib, or file, meristem that produces the internal tissues of the plant stem. When given inductive photoperiods, the commonly studied annual genotypes of Arabidopsis flower soon after germination. Before the transition to flowering in such young plants, the apical meristem is small and without easily recognized cytological zonation. However, when the vegetative phase is extended (i.e., through growth in short-day photoperiods, or in winter-annual genotypes lacking cold treatment) the zonal pattern described above becomes more apparent (Vaughan 1952; Besnard-Wibaut 1981). The appearance of well-defined zonal organization in Arabidopsis has been correlated with the ability of the plant to exhibit a significant flowering response to applied GAs or long days (Besnard-Wibaut 1981), suggesting that appropriate meristem structure could constitute the morphological basis of competency. The molecular determinants of competency remain unknown. It is important to note that although transgenic Arabidopsis expressing LFY in a constitutive manner flower very early, they still progress through a short vegetative phase (Weigel and Nilsson 1995). That LFY is insufficient to force early-arising primordia into a floral fate suggests that genes controlling meristem competence act genetically downstream, or in a separate pathway, from LFY. Two candidates are FWA and FT. Mutations in these genes cause delayed flowering, and are epistatic to a 35S:LFY transgene [i.e., constitutive expression of LFY is unable to rescue that late-flowering phenotype conferred by fwa and ft mutations (Nilsson et al. 1998)].

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All known mutations in FWA are dominant. Dominance of mutations often indicates that the mutant gene product has gained activity. This could happen if the gene product were increased in abundance, or if it usually existed in the “off” position and were turned “on” by the mutation in a manner similar to the action of an upstream signaling molecule. Indeed, FWA mRNA levels were found to be increased in fwa mutants relative to wild-type plants, suggesting that the dominance of the mutation indeed results from increased FWA activity. Interestingly, it appears that the known mutant alleles of fwa result from epimutation, a class of mutation that does not disrupt the DNA sequence. In the case of fwa mutations, constitutive expression of the gene is associated with a reduction in methylation of DNA residues found in the promoter region of the gene (W. Soppe, pers. commun.). The FWA gene was recently cloned and found to encode a transcription-factor-like protein (M. Koornneef and W. Soppe, pers. commun,). Because a gain in function of FWA results in later flowering, the function of the wild-type FWA gene is likely to repress flowering. Alleles of FT conferring late flowering are recessive and likely result from decreased function, suggesting that FWA acts in a manner opposite that of FT. The Arabidopsis FT gene was recently cloned by activation tagging (Kobayashi et al. 1999; Kardailsky et al. 1999). In this approach, random plant genes are transcriptionally activated by the nearby insertion of T-DNAs containing strong enhancer sequences, and function of the activated gene is surmised based on the resulting phenotype. Activation tagging has become a powerful technique for the identification of genes whose products are normally limiting in a pathway affecting a given phenotype (Lindsey et al. 1998). The product of the FT gene is structurally related to that of TFL, suggesting that like TFL, the FT protein may function in cell-to-cell signaling (Kardailsky et al. 1999). FT mRNA is expressed throughout the aerial tissues of the plant, and is not localized to any specific domain within the shoot apex (Kobayashi et al. 1999; Kardailsky et al. 1999). This expression becomes evident only near the floral transition, and, consistent with being regulated by CO (see above), this upregulation of expression is delayed in the co mutant and in shortday conditions. However, upregulation of expression still occurs in plants lacking CO activity, indicating that FT is also regulated by another mechanism. As might be expected from a gene controlling meristem competence, constitutive expression of FT in transgenic plants results in a nearly complete elimination of the vegetative phase, as such plants form a flower after only ~2 leaves, which are embryonic in origin. Other genes that have been postulated to play a role in competence include EMBRYONIC FLOWER (EMF) 1 and 2. Plants carrying mutant

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alleles of these genes produce a modified flower upon germination (Sung et al. 1992; Bai and Sung 1995; Yang et al. 1995; Chen et al. 1997). Plants carrying strong emf1 alleles develop no leaves at all, indicating that the vegetative phase has been completely bypassed. Mutations in EMF2 confer a milder but similar phenotype, with a few small leaves produced on a modified inflorescence. The very early flowering associated with loss of EMF function suggests that the EMF genes normally act as strong repressors of reproductive development. It is possible that the two genes operate in distinct genetic pathways, as combining strong emf1 and emf2 alleles leads to severe developmental defects that could be considered an additive phenotype (Yang et al. 1995). Although cloning of the EMF genes has not been reported, some indication of their mode of action comes from examining interactions of emf mutations with other mutations affecting the timing of flowering. Mutations in GI or CO, for example, have no effect on emf1 or emf2 phenotype; that CO and GI are not required for the expression of the emf mutant phenotype suggests that CO and GI act as upstream regulators of the EMF genes. However, mutations in other flowering-time genes, including FCA, LD, FVE, FY, FHA, FPA, FWA, and FT resulted in a partial rescue of the early flowering emf phenotype, with mutations in FWA and FT having the greatest effect (Huang and Yang 1998; Page et al. 1999). These results suggest that the EMF genes, rather than having a general repressive effect on flowering, act specifically as downstream negative regulators of the photoperiodic pathway. Finally, it is interesting to note that constitutive expression of LFY was not sufficient to fully rescue the flowering defect conferred by the ga mutation in short days (Blazquez et al. 1998). This suggests that in addition to playing a role in LFY activation, gibberellins play a role in promoting competency. VII. CONCLUSION AND PERSPECTIVES Research into the molecular mechanisms of flowering is now entering its second decade. Many flowering genes have been identified in the model plant Arabidopsis thaliana, and most of these have now been cloned. In addition, the functions of most of the known flowering-time genes have been assigned within one of the multiple parallel pathways that promote flowering in this plant (Fig. 1.1). However, the research accomplished to date should be considered merely as a foundation for future work, as many aspects of flowering in this plant remain unexplored. For example, in many cases, the relationships and interactions among the genes in these pathways have been surmised based solely on

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florigen

COLD vernalization

molecular events at the shoot apex

sugars

VRN1 VRN2 LD, FCA, FPA, FVE, FLD

FT FWA FLC SOC1

FRI

AP1 LFY

GA molecular events at the leaf ATP5CS2

endogenous clock

LIGHT

TOC1, LHY, CCA1, ELF3, FKF1, ZTL, GI

phytochromes cryptochromes

starch

internode elongation

CO

sugars

ACS10

florigen ethylene production

Fig. 1.1. Schematic diagram of molecular pathways leading to flowering presumably operating in the leaf and in the shoot apex. Assignment of gene function to the leaf or apex is based predominantly on the reported mRNA expression pattern of the genes. Relationships among genes depicted here are based on genetic and/or molecular evidence. Arrows indicate a generally positive regulation, whereas lines with blocks indicate a repressive regulation. Not all of the genes referred to in the text are shown. The relationship between the CO gene, the hypothetical substance florigen, and genes acting downstream of florigen is especially speculative.

genetic evidence, and need to be confirmed with molecular and biochemical data. Some more general questions remain to be answered as well. For example, in spite of the wealth of physiological data suggesting that flowering is the result of a transmissible signaling substance (“florigen”), produced in the leaves and acting at the shoot apex, the molecular identity of this signal is still unknown. The possibility that flowering can be manipulated in species other than Arabidopsis through molecular methods is largely dependent on the degree to which flowering-time mechanisms have been conserved through evolution. In order to prove their utility in solving horticultural

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problems, the models proposed to describe floral initiation based on genetic and molecular studies in Arabidopsis will likely need to be evaluated in plants with dissimilar flowering habits. With very few exceptions (see above), this is an area of research that has remained largely unexplored. Probably the most attractive opportunity in this regard is maize, which has traditionally been the most popular monocot model for developmental studies, and has diverged significantly from Arabidopsis in terms of genetics, physiology, and anatomy. Current efforts underway to discover maize gene function by high-throughput, reversed-genetics approaches should greatly simplify this task (Martienssen 1998; Walbot, 2000). The apparent conservation of function of meristem identity genes among species offers some indication that the function of flowering-time genes may be similarly conserved. As the intriguingly complex pathways that constitute flowering become more completely characterized in Arabidopsis, this area holds much promise for future research.

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2 Floral Homeotic Gene Regulation Robert G. Franks and Zhongchi Liu* Dept. of Cell Biology and Molecular Genetics University of Maryland College Park, MD 20742

I. INTRODUCTION A. The ABC Model B. The MADS-domain Multi-gene Family C. The A Class Genes: AP2 and AP1 D. The B Class Genes: AP3 and PI E. The C Class Genes: AG, HUA1 and HUA2 F. Novel Class Genes: SEP1, SEP2, and SEP3 II. CONSERVATION OF THE ABC FUNCTIONS IN ANGIOSPERMS A. Dicotyledonous Species B. Monocotyledonous Species III. POSITIVE REGULATORS OF FLORAL ORGAN IDENTITY GENES A. Meristem Identity Genes: LFY and AP1 B. LFY, a Direct Activator of AP1 C. LFY, a Direct Activator of AG D. Two Phases of Regulation: Initiation and Maintenance of B Gene Expression E. UFO, a Coregulator of B Gene Expression IV. NEGATIVE REGULATORS OF FLORAL ORGAN IDENTITY GENES A. Temporal and Spatial Regulators of AG B. Repression of AP1 Expression in Floral Whorls 3–4 C. Restriction of B Gene Expression to Floral Whorls 2–3 V. SUMMARY LITERATURE CITED

*We thank Beth Krizek for photographs used for Figures 2.2E and 2.2F. Our research on Arabidopsis flower development is supported by the U.S. Department of Agriculture 9835304-6714 (Z.L.), U.S. Department of Energy 02-00ER20281 (Z.L.), and a NIH postdoctoral fellowship GM20426-01 (R.G.F.).

Horticultural Reviews, Volume 27, Edited by Jules Janick ISBN 0-471-38790-8 © 2001 John Wiley & Sons, Inc. 41

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I. INTRODUCTION In the past decade, a major milestone in plant developmental biology has been the elucidation of the molecular genetic basis underlying floral organ specification. An elegant ABC model explains how three classes of genes (A, B, C classes) direct the development of four types of floral organs (Coen and Meyerowitz 1991; Weigel and Meyerowitz 1994). This model enables one to design and engineer the structure of flowers in a predictable manner by altering the expression of these ABC class floral homeotic genes (Jack et al. 1994; Krizek and Meyerowitz 1996a). Although the function of the ABC genes has been studied extensively, only more recently has the regulatory mechanism of their expression been elucidated. Both positive and negative regulators of ABC gene expression have been identified and, in some cases, significant progress has been made toward understanding the molecular mechanisms underlying the regulation of their expression. This review will focus on the regulation of ABC gene expression in Arabidopsis but will highlight similarities and differences among the ABC genes found in other plant species. For simplicity and uniformity, standard Arabidopsis nomenclature is used throughout this review even if a gene is from a different species. Specifically, uppercase letters identify wild-type genes or gene products and lowercase letters identify mutant genes or mutants. Additionally, the names of genes and mutants are shown in italic type, while the names of proteins are not italicized. For example, AGAMOUS (AG) refers to the wild type gene, agamous (ag) refers to the mutation or mutant, and AGAMOUS (AG) refers to the protein. A. The ABC Model The structure of the Arabidopsis flower is typical of many angiosperm flowers (Fig. 2.1A; Fig. 2.2A). It is made up of four types of floral organs arranged in four concentric circles or whorls. The sterile perianth organs, sepals and petals, comprise the outer two whorls while the reproductive organs, stamens and carpels, make up the inner two whorls. Despite dramatic variation in the number, color, and shape of floral organs in different species this arrangement of sepal, petal, stamen, and carpel, from the outermost whorl to the innermost whorl, is fixed in the majority of the angiosperm species. This cross-species similarity suggests that the molecular genetic systems responsible for patterning of floral organs are similar in the majority of flowering plants.

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A: A diagram of an Arabidopsis flower

B: Wild-type Whorls: 1

Whorls Sepal (Se)

1

Petal (Pe)

2

Se

2

3

4

Pe

Sta

Ca

A

Stamen (Sta) 3 Carpel (Ca)

B

4

C C: Class A mutants Whorls: 1

Ca

A B C

-

2 Sta

D: Class B mutants

3

4

1

2

Sta

Ca

Se

Se

A B C

3 Ca

E: Class C mutants

4 Ca

1

2

3

Se

Pe

Pe

4 Se

A

-

B C

-

Fig. 2.1. The ABC model. (A) A diagram of an Arabidopsis flower showing four sepals in whorl 1, four petals in whorl 2, six stamens in whorl 3, and two fused carpels in whorl 4. (B) In wild-type, the domains of the A, B, and C activities are indicated by the filled boxes. A class activity is only present in whorls 1–2; B class activity is in whorls 2–3; and C class activity is in whorls 3–4. (C) In A class mutants, such as ap2 or ap1, the A activity is absent. This results in the expansion of the C activity into all four whorls and the homeotic transformation of first whorl sepals to carpels and second whorl petals to stamens. (D) In B class mutants such as ap3 or pi, the B activity is absent. As a result, second whorl organs develop into sepals while third whorl organs develop into carpels. (E) In C class mutants, such as ag, the C activity is absent. This results in the expansion of the A activity into all four whorls and the homeotic transformation of third whorl organs into petals and forth whorl organs into sepals and another flower.

The analysis of mutations that alter the pattern of organ identity within the flower has generated a greater understanding of the molecular genetics of floral pattern specification. Floral homeotic mutations result in the substitution or replacement of one organ type by another organ type. (Fig. 2.2B–D; Bateson 1894; Acquaah et al. 1992). The isolation of floral homeotic mutations provided the initial clues to the genetic basis of floral patterning (Meyerowitz et al. 1989). It was the analyses of these floral homeotic mutants and the genetic interactions among them in Arabidopsis thaliana and Antirrhinum majus that led to

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Fig. 2.2. Microscopic photography of Arabidopsis flowers. (A) A wild-type flower with four sepals in whorl 1, four petals in whorl 2, six stamens in whorl 3, and two fused carpels in whorl 4. (B) A strong class A floral homeotic mutant, ap2-2. Two whorl 1 organs are carpel-like (arrow). The other two whorl 1 organs are still sepal-like. All whorl 2 organs are absent, and the whorl 3 consists of a single stamen. The whorl 4 carpels are similar to wild-type. (C) A class B floral homeotic mutant, pi-1. Organs in the outer two whorls are all sepals (arrowhead indicates a whorl 2 sepal). Organs in the inner two whorls are all carpels (arrow indicates a whorl 3 carpel). (D) A class C homeotic mutant, ag-1. Whorl 1 consists of four sepals; whorl 2 consists of four petals; whorl 3, however, is converted into six petals; and whorl 4 is a new flower with similar sepal, petal, petal arrangement (not shown in this picture). (E) A flower from a transgenic plant that ectopically expresses both B and C class genes: AP3, PI, and AG (Krizek and Meyerowitz 1996a). (F) A flower from a transgenic plant that ectopically expresses AP3 and PI and, at the same time, carries the ag-3 mutation (Krizek and Meyerowitz 1996a). Photos of (E) and (F) are gifts of Beth Krizek.

the establishment of the ABC model (Bowman et al. 1991b; Coen and Meyerowitz 1991; Weigel and Meyerowitz 1994). The ABC model places floral homeotic mutants into one of the three classes: A, B, or C and thus defines three classes of gene activities (Fig. 2.1B). In the outermost (first) whorl, the activity of the A class genes specifies the development of sepals. In whorl 2, where both A and B class genes are active, petal development is specified. In whorl 3, B and

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C activities together specify stamen identity, and in the innermost (fourth) whorl, C activity alone specifies carpel development. Thus, the A and B activities are predicted to overlap in whorl 2, while B and C activities overlap in whorl 3. In contrast, the A and C activities are predicted to be present in their respective domains without overlap. To account for this, the model predicts that the A and C activities are antagonistic to each other. The A activity in the first two whorls inhibits expression of C genes and vice versa. This tenant of the model is supported by the altered organ identity displayed in the A class homeotic mutants in which the pattern of organs in whorls one through four is carpel, stamen, stamen, carpel, respectively. This is consistent with the postulate that the C activity has spread to all four whorls (Fig. 2.1C). In C class mutants, the A activity is proposed to spread throughout all four whorls (Fig. 2.1E). Since the primary function of the ABC genes is to specify floral organ identity, the ABC class floral homeotic genes are also termed the “organ identity genes.” Molecular genetic analyses of homeotic mutations in the fruit fly Drosophila melanogaster indicated that homeotic genes encode master regulatory proteins. These transcriptional regulators control developmental programs that cooperate to generate a particular organ type (Gehring and Hiromi 1986). The molecular isolation of members of the ABC class floral homeotic genes indicates that these genes also function as master regulators of organ specific developmental programs (Weigel and Meyerowitz 1994). The ABC genes all encode DNA-binding transcription factors. Furthermore, in situ examination of their RNA expression patterns have largely supported the basic tenets of the ABC model. Their expression domains largely coincide with the domains of their activity as predicted by the ABC model. For example, the A class gene APETALA1 (AP1) is expressed in the first two whorls, while the C class gene AGAMOUS (AG) is expressed in the third and fourth whorls (Drews et al. 1991; Mandel et al. 1992b). Furthermore, the expression of the C class gene AG is expanded throughout all four whorls in the floral meristems of some A class mutants (Drews et al. 1991). Hence, the spatially restricted function of the ABC genes is largely regulated at the RNA level. There are, however, exceptions. For example, the A class gene APETALA2 (AP2) is expressed in all floral whorls although its activity is limited to whorls 1–2 (Jofuku et al. 1994). In this case, AP2 activity is likely regulated by post-transcriptional mechanisms. Clearly, one of the next major challenges in the field of flower pattern formation is to elucidate how the spatially and temporally restricted ABC activities are regulated. Currently, most investigations in this area of research are conducted in Arabidopsis, which will be the focus of this review.

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B. The MADS-domain Multi-gene Family With the exception of AP2, all other ABC genes encode a highly conserved 56 amino acid domain called the MADS-domain and thus are members of a multi-gene family (Riechmann and Meyerowitz 1997a). The name of the MADS-domain was derived from the four founding members: MCM1, yeast; AG, Arabidopsis; DEFICIENS (DEF), Antirrhinum; and SRF, human. The basic N-terminal half of the MADS domain is essential for DNA binding and the C-terminal half of the MADS domain is required for dimerization (Riechmann et al. 1996b). In the majority of plant MADS domain-containing proteins, a second conserved domain, the K box, was identified because of its similarity to the coiled-coil domain of keratin (Ma et al. 1991). The distinctive feature of the K box is the disposition of hydrophobic residues with a spacing that permits the formation of amphipathic α-helices (Ma et al. 1991; Pnueli et al. 1991). Between the MADS domain and the K box is a less strictly conserved linker (L) region. Amino acids in the L region and the K box have been shown to be important for the partner specificity in dimer formation (Riechmann et al. 1996b). MADS-domain proteins function as dimers and bind to a core consensus site CC(A/T)6GG, which is known as the CArG-box (SchwarzSommer et al. 1992; Wynne and Treisman 1992; Huang et al. 1993; Shiraishi et al. 1993). However, different MADS-domain family members can possess related but distinct DNA-binding specificity (Nurrish and Treisman 1995). Nevertheless, functional specificity (i.e. distinct organ identity activity) of the MADS-domain proteins is independent of their DNA-binding specificity. For example, hybrid genes were generated by swapping the amino terminal half of the MADS domain of the Arabidopsis proteins AP1, AP3, PI, and AG with the corresponding portion of human MEF2A or SRF proteins. Such hybrid proteins, having acquired the in vitro binding specificity of MEF2A or SRF, are able to perform the specific functions of the corresponding Arabidopsis genes in transgenic plants (Riechmann and Meyerowitz 1997b; Krizek and Meyerowitz 1996b). Thus, interactions between these MADS proteins with additional cofactors are probably crucial for the specific organ identity functions. Although AP3 and PI can each dimerize with AG or AP1 in vitro, these complexes do not bind to CArG boxes (Riechmann et al. 1996b). Only AP3/PI heterodimers can bind to the CArG boxes. This partner specificity of AP3 and PI suggests that the combinatorial mode of action between A and B genes in whorl 2 and between B and C in whorl 3 is not achieved through direct interactions between A and B proteins in

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whorl 2 or interaction between B and C proteins in whorl 3 (Riechmann et al. 1996a). For example, petal specific gene expression in whorl 2 does not appear to result from the activity of AP1/PI or AP1/AP3 heterodimers. Rather petal identity in whorl 2 is specified by the interaction of genes regulated by AP1 homodimers and genes regulated by AP3/PI heterodimers. C. The A Class Genes: AP2 and AP1 A class genes are defined as those required to specify sepal and petal identity. Interestingly, all A class genes appear to have other functions in addition to organ identity specification. AP2 has at least two functions: the specification of sepal and petal identity and the repression of AG RNA expression in whorls 1–2 (Bowman et al. 1991a; Drews et al. 1991). In strong loss-of-function ap2 mutants, when both functions of AP2 are defective, AG expression is extended to whorls 1–2, causing carpelloid structures in whorl 1 and staminoid petals or loss of petals in whorl 2 (Fig. 2.1C; Fig. 2.2B). Further, AG was found to be expressed precociously at earlier stages and at elevated levels, which may be responsible for the loss of floral organs in strong ap2 mutants (Fig. 2.2B). Unlike other ABC genes, AP2 is unique in that it encodes a novel, putative nuclear protein with two 68 amino acid repeat sequences, dubbed the AP2 domain. The AP2 domain has been predicted to perform functions of protein-protein dimerization and DNA-binding (Jofuku et al. 1994). AP2 is a member of a multi-gene family (Riechmann and Meyerowitz 1998). Studies with other family members such as the ethyleneresponsive element binding proteins (EREBPs) demonstrated that the AP2-domain recognizes and binds to DNA specifically in an 11-bp sequence (TAAGAGCCGCC), the GCC box (Ohme-Takagi and Shinshi 1995). Hence, AP2-domain containing proteins define a novel class of plant transcription factors. AP2 mRNA is detected in all floral whorls as well as in vegetative tissues, indicating that the spatially restricted activity of AP2 in whorls 1–2 must depend on post-transcriptional regulation. AP1 is another A function floral organ identity gene. Unlike AP2, AP1 does not negatively regulate AG expression in whorls 1–2 (GustafsonBrown et al. 1994). In ap1 mutants, whorl 1 organs are bract/leaf-like; whorl 2 organs are usually absent; whorls 3–4 are usually normal (Irish and Sussex 1990). Thus, AP1 activity is required for the development of sepals and petals. Unique to ap1 mutants is the formation of secondary flowers in the axils of first whorl bract/leaf-like organs, suggesting that ap1 mutant flowers partially adopt the fate of inflorescence shoots (Irish and Sussex 1990). This second defect of ap1 in flower/shoot

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transition will be discussed in a later section of this review. AP1 encodes a MADS box protein and AP1 mRNA is initially expressed throughout the floral meristem and later becomes restricted to whorls 1–2 (Mandel et al. 1992b). The spatially-restricted expression of AP1 at later stages is consistent with its role as an A class gene (Bowman et al. 1993; Mandel et al. 1992b; Gustafson-Brown et al. 1994). D. The B Class Genes: AP3 and PI APETALA3 (AP3) and PISTILLATA (PI) are the Arabidopsis B class genes. Mutations in either AP3 or PI cause similar homeotic transformations in whorls 2–3 such that second whorl organs develop as sepals and third whorl organs develop as carpels (Fig. 2.1D; Fig. 2.2C; Bowman et al. 1989; Hill and Lord 1989). AP3 and PI both encode MADS domain proteins that have been shown to bind DNA only as AP3/PI heterodimers (Jack et al. 1992; Goto and Meyerowitz 1994; Riechmann et al. 1996a; Riechmann et al. 1996b; Hill et al. 1998; Tilly et al. 1998). Obligatory heterodimer formation explains why both AP3 and PI are required to specify petal and stamen identity. Ectopic expression studies involve artificially expressing a gene in a new spatial or temporal domain, in which the gene is normally not expressed. The 35S promoter from the cauliflower mosaic virus (CaMV) is a constitutive plant promoter that is frequently used to drive the expression of genes in all tissues and at all developmental stages. If AP3 and PI together are sufficient to confer the B class activity, then ectopically expressing AP3 and PI in all four floral whorls would result in a flower of petals in the outer two whorls and stamens in the inner two whorls. Transgenic plants that constitutively and simultaneously express both AP3 and PI under the direction of the 35S promoter develop flowers that have petals in whorls 1–2 and stamens in whorls 3–4 (Jack et al. 1994; Krizek and Meyerowitz 1996a). Ectopic expression of both B class and C class genes led to the production of Arabidopsis flowers with stamens in all four whorls (Fig. 2.2E; Krizek and Meyerowitz 1996a). Thus, AP3 and PI together are both necessary and sufficient for the B activity within the context of a flower. AP3 and PI initially are not expressed in identical domains. AP3 mRNA is detected in whorls 2–3 plus in a small number of cells at the base of the first whorl (Weigel and Meyerowitz 1993; Tilly et al. 1998), while PI RNA is detected in whorls 2–4 (Goto and Meyerowitz 1994). At later stages of flower development, the expression of both genes is restricted to petals and stamens. Maintenance of this later expression in petals and stamens requires the functional activity of both AP3 and PI,

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suggesting a system of autoregulation (Jack et al. 1992; Goto and Meyerowitz 1994). Presumably, auto- and cross-regulation of AP3 and PI are responsible for the similar expression domains of AP3 and PI in later stages of flower development. E. The C Class Genes: AG, HUA1, and HUA2 AG was the first C class gene identified and isolated (Bowman et al. 1989; Bowman et al. 1991b; Yanofsky et al. 1990). AG plays a key role in specifying stamen and carpel identity. In ag loss-of-function mutants (Fig. 2.1E; Fig. 2.2D), the A activity is expanded into whorls 3–4, where stamens are replaced by petals, and carpels are replaced by a new flower. This results in a floral pattern of sepal, petal, petal, (sepal, petal, petal)n. The generation of flowers within a flower reveals a second role of AG: to maintain the determinacy of the floral meristem. AG encodes a MADSdomain containing protein (Yanofsky et al. 1990). As predicted by the ABC model, AG mRNA is detected in the inner two whorls during early floral stages. AG mRNA continues to be expressed in stamens and carpels during later stages and eventually becomes restricted to specific cell types within the stamens and carpels (Bowman et al. 1991a; Drews et al. 1991). Ectopic expression of AG under the 35S promoter in transgenic Arabidopsis or tobacco plants causes homeotic conversion from sepals into carpels and from petals into stamens (Mizukami and Ma 1992; Mandel et al. 1992a). Thus, AG appears not only necessary but also sufficient to specify stamen and carpel identity within the context of a flower. In summary, AG has at least three functions: repressing A class gene activity in whorls 3–4, specifying stamen and carpel organ identity, and maintaining the determinacy of floral meristems. For many years, it was thought that AG was the only C class gene because AG alone appeared sufficient to specify C activity, and several mutageneses only yielded additional ag alleles without identifying mutations in other genes that confer similar phenotypes. However, genetic redundancy and/or embryonic or seedling lethality may have prevented the isolation of mutations in additional C class genes. Chen and Meyerowitz (1999) searched for new C class genes by looking for mutations that enhanced the phenotype of the weak ag-4 allele. Flowers of ag-4 plants can still make stamens in the third whorl because of residual activity of the mutant ag gene product (Sieburth et al. 1995; Chen and Meyerowitz 1999). Mutations that enhance the ag-4 phenotype are expected to convert the third whorl stamens into petals, as is seen in the stronger ag alleles. Chen and Meyerowitz (1999) successfully

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isolated two new C class genes, HUA1 and HUA2 (Hua means “flower” in Chinese). Either one of the hua1 or hua2 mutations weakly enhances ag-4, while hua1 hua2 double mutants strongly enhance the ag-4 phenotype. Both hua1 and hua2 single mutant flowers are phenotypically normal. Flowers of the hua1 hua2 double mutants show a weak stamen and carpel phenotype. In the first few flowers, lateral stamens are petaloid, and the gynoecia are enlarged toward their tips and constricted along their sides. The lack of phenotype in hua1 and hua2 single mutants explains why they were only recovered in genetic screens when AG activity is compromised. Genetic analyses indicated that HUA1 and HUA2 share a redundant role with AG in all aspects of AG function: repression of A class gene expression, stamen and carpel identity specification, and regulation of floral determinacy. HUA1 and HUA2 act in parallel or together with AG in the specification of C activity because AG mRNA expression is not affected in the hua1 or hua2 mutants. Similarly, HUA2 expression is not altered in ag mutants. Since HUA2 encodes a novel protein that contains multiple nuclear localization signals and additional motifs, it is likely that HUA2 is a transcription factor and acts as a cofactor of the AG gene. F. Novel Class Genes: SEP1, SEP2, and SEP3 Recently, another class of floral homeotic mutants has been described (Pelaz et al. 2000). This new class is encoded by three genes: SEPALLATA1 (SEP1), SEP2, and SEP3. All three genes are MADS-box containing genes and were isolated based on their sequence similarity to AG. In fact, SEP1, SEP2, and SEP3 were previously named AGAMOUS-LIKE2 (AGL2), AGL4, and AGL9, respectively (Ma et al. 1991; Mandel and Yanofsky 1998). A reverse genetic approach was used to identify mutations in SEP1, SEP2, and SEP3. Reverse genetics refers to a variety of techniques that can be used to generate mutations in a particular gene whose sequence is known. Since we have now entered an era of genomics, the large number of DNA sequences available in Arabidopsis, rice, tomato, and a few other plant species allow the discovery of many genes and gene families. However, in a majority of the cases, the function of these genes remains unknown. Thus, reverse genetic approaches are now more frequently employed and are crucial to illuminate gene function. These approaches are labeled “reverse” because they lead from gene sequence to mutation in the opposite direction of the typical “forward” genetic approach that leads from mutation to gene sequence.

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In the reverse genetic approach employed by Pelaz et al. (2000), polymerase chain reaction (PCR) amplification was used to identify transposable element insertions within the SEP gene of interest. After generating individual mutations in each of the SEP genes, phenotypic analyses indicated that mutations in any one of the SEP genes alone cause only subtle phenotypes. However, sep1 sep2 sep3 triple mutants displayed a striking phenotype in which all floral organs in the first three whorls were sepals or sepal-like organs. The fourth whorl was converted into a new flower that repeats this same floral pattern (Pelaz et al. 2000). The phenotype displayed by the sep1 sep2 sep3 triple mutant is very similar to “bc” double mutants (such as pi ag or ap3 ag), suggesting that SEP1, SEP2, and SEP3 are required for B and C class gene expression or for their activity. Because of redundancy among SEP1, SEP2, and SEP3, removing one of these SEP genes by mutations normally would not reveal such a requirement. SEP1, SEP2, and SEP3 are all expressed just prior to the expression of the B and C class genes and are expressed throughout whorls 2–4 (SEP1 and SEP2 are also expressed in whorl 1 in young flowers) (Pelaz et al. 2000). The initial patterns of B and C class gene expression are not altered in the sep1 sep2 sep3 triple mutants, suggesting that SEP1, SEP2, and SEP3 are not required for the initiation of B or C class gene expression. Thus, it has been suggested that SEP1, SEP2, and SEP3 regulate B and C class genes post-transcriptionally. One possible mechanism for this post-transcriptional regulation is by a direct interaction between the SEP1, SEP2, and SEP3 gene products and the B and C class gene products. Yeast two-hybrid assays have revealed a number of interactions between SEP proteins and B and C gene products (Fan et al. 1997).

II. CONSERVATION OF THE ABC FUNCTIONS IN ANGIOSPERMS The ABC genes provide an excellent opportunity to understand the evolutionary conservation and divergence of floral development in angiosperms. Studies carried out in both dicot and monocot plants suggest that the basic genetic mechanisms that determine floral organ identity are conserved across angiosperms. For most of the ABC genes, cognate homologs (orthologs) can be identified in diverse angiosperm species. For some of these homologs, functional analogy to corresponding Arabidopsis genes has been confirmed (see Table 2.1) by studying loss-of-function mutants, transgenic plants ectopically expressing these

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Table 2.1. ABC genes in different plants. Only those genes are included that exhibit functional homology with corresponding Arabidopsis genes except ZAP1 and ZMM2 in Maize and BLIND in Petunia. Class

Arabidopsis

Antirrhinum

Petunia

Maize

Rice

A

APETALA1 APETALA2 APETALA3 PISTILLATA AGAMOUS

SQUAMOSA — DEFICIENS GLOBOSA PLENA

— BLINDy GREEN PETAL FBP1, PMADS2 pMADS3, FBP6

ZAPz — SILKY1 — ZAG1, ZMM2z

— — OsMADS16 OsMADS4 OsMADS3

SEP1, 2, 3

—

FBP2

—

LHS1

B C Novel (BC) z

Only sequence and expression data are available (Mena et al., 1995). Only genetic data are available (de Vlaming et al., 1984; Tsuchimoto et al., 1993).

y

homologs, transgenic plants expressing antisense genes to corresponding homologs, or functional complementation of Arabidopsis mutants with homologs isolated from different species. One major theme from these studies (mostly on the B and C class genes) is that organ identity genes in different species differ both in the number of genes involved (due to gene duplication) and in the distribution of functional roles among these duplicated genes. Because dicots and monocots are on separate branches of the angiosperm phylogenetic tree, the conservation of the ABC model in dicot and monocot species suggests that the ABC model represents an ancient regulatory network that in all likelihood is generally applicable to most angiosperms. In this review, we will focus on studies where functional data on ABC gene homologs are available. For a more extensive review of angiosperm flower development, see Irish and Kramer (1998). For reviews on grass species, see Schmidt and Ambrose (1998) and Ma and dePamphilis (2000). A. Dicotyledonous Species Like Arabidopsis, Antirrhinum majus is a dicot plant that has been extensively studied and has contributed greatly to the establishment of the ABC model. Although Arabidopsis (Brassicaceae) and Antirrhinum (Scrophulariaceae) are widely divergent dicot species and belong to different subclasses, the phenotype of the corresponding Antirrhinum mutants (Carpenter and Coen 1990; Schwarz-Sommer et al. 1990) are quite similar to Arabidopsis mutants (Coen and Meyerowitz 1991). DEF and GLO, orthologs of AP3 and PI, respectively, are both required for B function in Antirrhinum (Sommer et al. 1990; Trobner et al. 1992). How-

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ever, the expression domains of these orthologs are switched. In Arabidopsis, PI is expressed early in whorls 2–4, while AP3 is expressed in whorls 1–3. In Antirrhinum, DEF is expressed early in whorls 2–4, while GLO is expressed largely in whorls 2–3 (Schwarz-Sommer et al. 1992; Trobner et al. 1992). Despite the difference in expression, AP3 and DEF are functionally conserved as demonstrated by cross species complementation (Irish and Yamamoto 1995; Samach et al. 1997). Similar expression studies in several other species showed that the DEF expression pattern represents an ancestral condition, while the AP3 expression pattern represents an exception rather than the rule (Irish and Kramer 1998). Based on sequence similarity, there are two petunia PI-like genes, FBP1 and pMADS2 (Angenent et al. 1992, 1994; Kush et al. 1993). Consistent with it being a B function gene, FBP1 was shown to be required for petal and stamen development in co-suppression experiments (Angenent et al. 1992, 1994). However, pMADS2 appears to encode a redundant function, since loss of pMADS2 function has no phenotypic effect (van der Krol et al. 1993). The petunia AP3 homolog, pMADS1 is also called GREEN PETAL (GP), because mutations in this gene cause a homeotic conversion from petals to green sepals but do not affect stamen development (van der Krol et al. 1993). Furthermore, ectopic expression of pMADS1/GP under the control of the 35S promoter in wild-type plants resulted in partial conversion of sepals into petaloid organs, but had no effect on stamen identity (Halfter et al. 1994). The fact that pMADS1/GP is not required for stamen development suggests that petunia may have another, as yet unidentified, AP3 homolog specifying the B function in whorl 3. Thus, petunia differs from Arabidopsis and Antirrhinum both in an increased number of B class genes and in the limitation of B class gene activity to only one whorl. AG orthologs from various species are more conserved than are the B class genes, both with respect to DNA sequence and gene function. In Antirrhinum, PLENA (PLE) is considered the functional ortholog of AG (Table 2.1), as both the expression pattern and the function of PLE are similar to AG in Arabidopsis (Bradley et al. 1993). Petunia has two AGlike genes, pMADS3 and FBP6, both of which appear to have expression patterns consistent with a reproductive function (Tsuchimoto et al. 1993; Angenent et al. 1995; Kater et al. 1998). Ectopic expression of pMADS3 either via 35S promoter in transgenic plants or via gain-offunction alleles led to the homeotic conversion of whorl 1 and whorl 2 generating organs with carpelloid and staminoid features, respectively. However, ectopic expression of FBP6 did not lead to homeotic transformations (Kater et al. 1998).

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The Antirrhinum ortholog of the A class gene AP1 is SQUAMOSA (SQUA). SQUA, like AP1, plays a role both in meristem identity and organ identity; however, its role in organ identity determination is rather limited, as flowers of squa mutants are often nearly wild type (Huijser et al. 1992). The Antirrhinum mutant ovulata exhibits a phenotype similar to that of ap2. However, ovulata is a gain-of-function allele of the C class gene PLE that is caused by a transposon insertion into the second intron of PLE and may have disrupted binding sites for negative regulators (Bradley et al. 1993). Two genes STYLOSA (STY) and FISTULATA (FIS) together control the restriction of the C gene PLE to the inner whorls of the flower. Genetic and expression studies, however, indicated that the effect of STY and FIS on PLE expression is indirect and that STY and FIS are more general regulators of flower development (Motte et al. 1998). In petunia, the blind (bl) mutants exhibit a phenotype similar to A class mutants, which includes homeotic conversion from sepals to carpelloid sepals and petals into antheroid structures (de Vlaming et al. 1984; Angenent et al. 1992; Tsuchimoto et al. 1993). In addition, the petunia C class gene pMADS3 was ectopically expressed in bl mutants (Tsuchimoto et al. 1993), indicating that BL, like AP2, is a negative regulator of C class genes. However, the molecular nature of BL is unknown. Orthologs of Arabidopsis AP2 have not been identified in Antirrhinum, petunia, or other species (Table 2.1). The tomato TM5 gene and its petunia homolog FBP2 are expressed in the inner three whorls, all of which are defective when TM5 or FBP2 are inactivated by antisense constructs or co-suppression (Angenent et al. 1994; Pneuli et al. 1994). Although the phenotypes in tomato and petunia are slightly different, in both cases, petals are transformed into sepals or leaf-like organs, and additional whorls of organs or new flowers can develop in the center of the flower. This phenotype resembles the Arabidopsis sep1 sep2 sep3 triple mutants (Pelaz et al. 2000). Sequence analyses indicate that indeed SEP1, SEP2, and SEP3 are most closely related to TM5 and FBP2 (Purugganan et al. 1995). The presence of three redundant SEP genes in Arabidopsis suggests that tomato and petunia may also possess multiple genes for the same function. The antisense and co-suppression approaches used to knock out TM5 and FBP2 might have simultaneously abolished the activity of other redundant genes of TM5 or FBP2 due to their high levels of sequence similarity. B. Monocotyledonous Species Maize (Zea mays) is a monocot grass species that has been extensively characterized resulting in the development of a host of useful molecular genetic tools. In particular, transposon tagging and other reverse

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genetic approaches are available. A number of maize MADS box genes have been isolated and their functions have been assayed by reverse genetic, as well as forward genetic, approaches. Grass flowers have stamens and carpels, but lack obvious sepals and petals. Instead, grass-specific organs known as glumes, lemma, palea, and lodicules surround the stamens and carpels. Recent studies of a maize mutant silky1 provides compelling developmental evidence for recognizing lodicules as modified petals, and possibly, palea and lemma as modified sepals (Ambrose et al. 2000). SILKY1 encodes an AP3-like gene in maize. Homeotic transformations of stamens to pistils and lodicules to organs resembling lemma/palea are exhibited in silky1 mutants. In situ hybridization indicates SILKY1 is localized to lodicule and stamen primordia. Thus genetic, morphological, sequence, and expression data all support that SILKY1 is a B function gene (Ambrose et al. 2000; Ma and dePamphilis 2000). The maize C class gene ZAG1 was identified first by sequence homology to AG (Schmidt et al. 1993). Subsequently, reverse genetic approaches created a putative null allele of zag1 (Mena et al. 1995). While the ABC model would predict a loss of both reproductive organ development and floral meristem determinacy, only the later phenotype was evident, with supernumerary carpels being reiterated within the zag1 florets. Although ZAG1 is expressed in both stamens and carpels, the zag1 mutation does not affect stamen development. One possible explanation is the existence of partially redundant C class genes in maize. Indeed, ZMM2, a gene closely related to ZAG1, has been isolated (Mena et al. 1995; Schmidt and Ambrose 1998). The expression pattern, sequence, and map position of ZMM2 all suggest that it is a duplicate gene with activities that are non-identical, but partially overlapping with those of ZAG1. Consistent with the genetic analyses, ZAG1 is more highly expressed in carpels and ZMM2 more highly expressed in stamens. Thus, it is highly likely that in maize the AG-like activity is shared by two genes. Several MADS box genes that play important roles in controlling flower development in rice have also been studied. Using antisense experiments, Kang et al. (1998) demonstrated that the rice MADS box genes OsMADS3 (Oryza sativa MADS box gene 3) and OsMADS4 are putative orthologs of AG and PI respectively. OsMADS16 gene has been proposed as a homolog of AP3 based on its amino acid sequence similarity to AP3, its expression pattern, and its interaction with OsMADS4 in yeast (Moon et al. 1999). The leafy hull sterile 1 (lhs1) mutations display mutant phenotypes similar to transgenic plants expressing a dominant-negative mutant form of OsMADS1, suggesting that the lhs1 mutation may be defective in the OsMADS1 gene (Kinoshita et al. 1976; Jeon et al. 2000). The fact that

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wild-type OsMADS1 can rescue lhs1 mutants further indicates that OsMADS1 and LHS1 encode the same gene (Jeon et al. 2000). Strong lhs mutants exhibit leafy palea and lemma, and partial transformation from lodicules to leafy palea and lemma. Additional carpels and a new flower may be generated. This mutant phenotype and the sequence similarity between LHS1 (OsMADS1) and TM5 and FBP2 suggests that LHS1 may encode a functional homolog of TM5, FBP2, and the Arabidopsis SEP genes. However, the phenotype of lhs also suggests that LHS1 has an additional role during the development of palea and lemma during late stages of flower development. In this respect LHS is similar to AP1.

III. POSITIVE REGULATORS OF FLORAL ORGAN IDENTITY GENES A. Meristem Identity Genes: LFY and AP1 At the beginning stages of Arabidopsis development, the primary shoot produces leaves with axillary second order shoots. Later, at the transition to reproductive phase, the primary shoot switches to producing flowers. Two genes LEAFY (LFY) and AP1 are necessary and sufficient for this developmental switch (Irish and Sussex 1990; Huala and Sussex 1992; Weigel et al. 1992; Bowman et al. 1993; Mandel and Yanofsky 1995; Weigel and Nilsson 1995). Loss-of-function mutations in these two genes cause the conversion (to varying degrees) from flowers to second order shoots. Conversely, constitutive expression of either LFY or AP1 cause the conversion from shoots to flowers. Thus LFY and AP1 are referred to as “meristem identity genes.” Specifically, in lfy mutants, flowers are replaced by leaves and second order shoots. In ap1 mutants, (leaf-like) bracts develop in the first whorl and secondary flowers develop in the axils of first whorl floral organs. Most strikingly, ap1 enhances the defects of lfy. In lfy ap1 double mutants, leaf-like organs arise in a spiral fashion (a feature of shoot) rather than whorled fashion (a feature of flowers), and all flowers are replaced by shoot-like structures. Thus, LFY and AP1 have partially redundant roles in floral meristem identity specification, with LFY playing a more prominent role than AP1. For more specific reviews regarding floral inductive pathways leading to the activation of LFY and AP1, see Yanofsky (1995), Koornneef (1997), Weigel (1997 and 1998), Ma (1998), and van Nocker (2001). Although the LFY protein does not share sequence homology with other known families of DNA-binding proteins, LFY can bind DNA in a sequence specific manner and stimulate transcription in yeast cells

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when fused to a heterologous activation domain (Weigel et al. 1992; Parcy et al. 1998). Thus, LFY may activate the floral program by transcriptional activation of target genes involved in flower development. This hypothesis is consistent with LFY RNA and protein expression that precedes the transcriptional activation of ABC genes. Specifically, LFY mRNA is expressed in early floral meristem, and is transiently expressed in sepal, petal, stamen, and carpel primordia (Weigel et al. 1992). The LFY protein is expressed in a similar pattern and localizes to the nucleus, consistent with a role in transcriptional activation (Parcy et al. 1998). To further understand the regulatory relationship between meristem identity genes and ABC genes, Weigel and Meyerowitz (1993) examined ABC gene expression in lfy and ap1 single and double mutants. They found that LFY and AP1 are positive regulators of ABC gene transcription. In lfy mutants, early AG expression is delayed, and the initial expression domain is smaller than in wild-type flowers. The expression of AG mRNA in ap1 mutants is relatively normal, however, AG RNA entirely fails to accumulate in the center of ap1 lfy double mutant flowers. Therefore, AG expression is more strongly affected in the ap1 lfy double mutants than in either single mutant. Similarly, both the amount and the domain of expression of AP3 and PI are severely reduced in strong lfy mutants. The function of AP1 in activating AP3 and PI only becomes obvious when LFY activity is reduced or eliminated as shown in lfy ap1 double mutants. Like B and C class genes, the expression of AP1 is delayed and reduced in lfy mutants (Ruiz-Garcia et al. 1997; Liljegren et al. 1999). Since AP2 RNA is detected in a variety of non-floral tissues, including leaves and stems, AP2 transcription is likely regulated independently of the meristem identity genes (Jofuku et al. 1994). B. LFY, a Direct Activator of AP1 The dual roles of AP1 as a meristem identity gene and an A class organ identity gene correlate well with its two phases of expression. AP1 is initially expressed in the entire floral meristem and later becomes restricted to the first two whorls (Bowman et al. 1993; Mandel et al. 1992b; Gustafson-Brown et al. 1994). This expression pattern correlates well with an early function of AP1 in meristem identity specification and a later function of AP1 in sepal and petal identity specification. LFY is an obvious candidate activator of AP1 expression as AP1 RNA expression in the floral meristem is initiated soon after LFY is first detected and AP1 RNA expression is delayed and reduced in lfy mutants (Ruiz-Garcia et al. 1997; Liljegren et al. 1999).

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Evidence that LFY can directly activate AP1 comes from experiments in which constitutive and ectopic expression of LFY under the 35S promoter led to precocious expression of AP1 in young seedlings (Parcy et al. 1998). Thus the role of LFY in activating AP1 can be separated from its role in floral meristem specification. Consistent with the idea that AP1 expression is directly regulated by LFY, in vitro DNA-binding assays showed that a high affinity binding site for LFY is present in a minimal AP1 promoter. Additionally, LFY, upon fusion to a strong transcriptional activation domain, can activate the expression of a reporter gene in yeast, which is under the control of an AP1 promoter (Parcy et al. 1998). Using a post-translational inducible system, Wagner et al. (1999) demonstrated that AP1 is an immediate target of LFY. Specifically, a steroid hormone-inducible LFY switch was constructed. This construct (35S::LFY-GR) uses the CaMV-35S promoter to express the LFY coding sequence that has been fused to a glucocorticoid receptor (GR) hormone binding domain. In the absence of the steroid hormone dexamethasone, the LFY-GR fusion protein is held in the cytoplasm and is non-functional. In the presence of dexamethasone, the LFY-GR fusion protein moves to the nucleus and is able to perform its function as a transcriptional activator. As the translocation of the LFY-GR protein into the nucleus does not depend on protein synthesis, a direct effect of LFY on its target gene transcription can be evaluated in the presence of cyclohexamide (a protein synthesis inhibitor). The LFY-GR protein was able to rescue defects of AP1 expression at early stages even in the presence of cyclohexamide, indicating that LFY directly activates AP1 at early stages. However, the ability of LFY-GR to rescue defects of AP1 expression during later stages is dependent on protein synthesis. Thus, the two phases of AP1 expression appear to be controlled by different regulatory mechanisms. While LFY directly activates AP1 expression during early stages of flower development, the effect of LFY on AP1 expression is indirect at later stages. C. LFY, a Direct Activator of AG Despite the knowledge that LFY and AP1 are required for AG expression, it has been difficult to determine if the effect of LFY and AP1 on AG expression is direct or indirect. For example, a failure to activate AG might simply result from the fact that the shoot structures formed in lfy or lfy ap1 mutants never acquire any floral identity. Fortunately, a gainof-function LFY protein LFY:VP16 is able to separate the role of LFY in meristem identity specification from the role of LFY in regulating AG

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(Parcy et al. 1998). Specifically, LFY:VP16 is a fusion protein between LFY and a strong transcriptional activation domain from the Herpes Simplex Virus (HSV) VP16 protein, generating a form of LFY that is constitutively active with respect to transcriptional activation. The LFY:VP16 fusion protein was expressed in developing plants from the endogenous LFY promoter, and thus the expression pattern of LFY:VP16 mimics the expression of the wild-type LFY protein. Transgenic plants harboring the LFY:VP16 construct initiate floral meristem formation normally; however, the floral organs display homeotic transformations, where sepals are transformed into carpels in whorl 1 and petals are transformed into stamens in whorl 2. In situ hybridization showed that these LFY:VP16 plants exhibit both ectopic and precocious AG RNA expression. When the LFY:VP16 plants were crossed to the strong loss-of-function ag-1 mutant, the floral organs in whorls 1–2 were largely restored to normal sepals and petals. Hence, ectopic and precocious AG expression is chiefly responsible for the abnormal floral organs formed in these LFY:VP16 plants. When the LFY: VP16 was ectopically expressed in developing seedlings under the control of the 35S promoter, seedlings were growth-arrested and AG was ectopically expressed (Parcy et al. 1998). The induction of ectopic AG expression in pre-flowering seedlings by LFY:VP16 further indicates that LFY:VP16 is able to activate AG in non-floral tissues. Thus, the effect of LFY:VP16 on AG is rather direct and does not require proper floral meristem formation. However, a 35S::LFY construct that drives the ectopic expression of the wild-type LFY protein is not sufficient to generate pre-flowering seedling expression of AG; rather the presence of the strong VP16 activation domain is required (Parcy et al. 1998). Two models were proposed to explain this observation. The first model postulates that there is a repressor that is present in whorls 1–2 of the floral meristem and in the vegetative tissues that normally prevents LFY from activating AG expression. In this model the enhanced transcriptional activity of the LFY:VP16 protein overcomes this repressor activity. Alternatively, in wild-type plants, the LFY protein is assisted by the action of a co-activator only expressed in the center of the floral meristem. Hence, AG is only activated by LFY in the center of a wild-type flower. According to this model, the strong transcriptional activity of LFY:VP16 can activate AG transcription independently of any co-activators. Analysis of the AG cis-regulatory sequences revealed that sequences within the second AG intron are necessary and sufficient for the wildtype AG expression pattern (Sieburth and Meyerowitz 1997; Busch et al. 1999). At least two redundant enhancers within the intronic sequences

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mediate the expression of AG (Busch et al. 1999). These enhancers are LFY responsive, as they respond appropriately to expression of the LFY:VP16 construct or to the loss of LFY activity in the lfy mutant. Using deletion analysis, one functional enhancer was found within a 230 base pair (bp) fragment of the intron. Within this fragment, two LFY binding sequences, that are 31 bp apart, were detected by gel shift analysis. These sequences are similar to each other and to previously defined LFY binding sites in the AP1 promoter (Parcy et al. 1998). Alterations to the binding site sequence that prevent the binding of LFY in vitro also cause the enhancer fragments to lose their responsiveness to both wild type LFY as well as LFY:VP16 proteins in vivo (Busch et al. 1999). These results suggest that LFY-dependent stimulation of AG expression requires the direct binding of LFY to the enhancer sequences of AG. In addition, two CArG boxes have been identified within the 3′ activation cis-element where a LFY binding site is also located (Deyholos and Sieburth 2000). These CArG boxes could serve as binding sites for MADS-domain proteins such as AP1. D. Two Phases of Regulation: Initiation and Maintenance of B Gene Expression The regulation of AP3 and PI can be divided into two phases: the early initiation phase and the later maintenance phase. These two different phases are regulated by distinct sets of genes. The early phase of AP3 and PI expression are positively regulated by meristem identity genes LFY and AP1 (Weigel and Meyerowitz 1993; Goto and Meyerowitz 1994). AP3 and PI expression is significantly reduced in strong lfy mutants, whose flowers lack petals and stamens. Hence, LFY is required to initiate AP3 and PI expression. Although, ap1 mutations alone have little effect on B class gene expression, lfy ap1 double mutants display no detectable AP3 and PI expression (Weigel and Meyerowitz 1993), suggesting that AP1 does play a role in B class gene activation. Activation of AP3 by LFY apparently relies on a mechanism different from those for the activation of AG and AP1. AP1 and AG can be activated in ectopic tissues in response to the ectopic expression of wild type LFY (in the case of AP1) or LFY:VP16 (in the case of AG). However, ectopic and constitutive expression of LFY by 35S::LFY or LFY::VP16 failed to activate AP3 ectopically. Apparently, B class genes require additional factors for their activation. Results from several experiments (see below) indicate that the activation of the B class genes requires the UNUSUAL FLORAL ORGANS (UFO) gene. When LFY and UFO are both constitutively expressed in seedlings (35S::LFY and 35S::UFO), they

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can induce AP3 expression in seedlings (Parcy et al. 1998). Thus, activation of AP3, like that of AP1 and AG, is not dependent on the formation of floral meristems. Similarly, flower-independent activation of PI is observed in seedlings where both LFY and UFO are expressed constitutively (Honma and Goto 2000). LFY-GR experiments that directly test the effect of LFY and UFO on AP3 or PI expression in vivo in the presence of protein synthesis inhibitors will be required to determine if LFY and UFO are immediate upstream activators of AP3 and PI. The maintenance (late) phase of AP3 and PI expression is dependent on the activity of AP3 and PI. The autoregulatory role of AP3 and PI is observed in ap3 and pi mutants where both AP3 and PI late phase expression is reduced while the early phase expression is not affected (Jack et al. 1994; Goto and Meyerowitz 1994). Three CArG boxes were identified between –90 to –180 of the AP3 promoter (Hill et al. 1998; Tilly et al. 1998). These same elements are necessary for maintaining AP3 expression in petals and stamens (Hill et al. 1998). AP3/PI heterodimers can bind to CArG box 1 and 3 in vitro in a sequence specific manner (Hill et al. 1998; Tilly et al. 1998). In addition, AP3-GR, which is activated by steroid hormone, can induce AP3 expression in the absence of de novo protein synthesis (Honma and Goto 2000). Thus, direct interaction between AP3/PI and the CArG boxes in AP3 promoter is responsible for maintaining late phase AP3 transcription. Interestingly, the promoter or intron sequences of PI do not contain any CArG box. This raised the possibility that AP3/PI heterodimers may not directly bind to PI cis-elements. Indeed, electrophoretic mobility shift assays (EMSA) failed to detect binding of AP3/PI to the proximal promoter element of PI. Using a similar AP3-GR system, it was found that the ability of AP3/PI heterodimer to activate PI transcription requires de novo protein synthesis (Honma and Goto 2000). Thus, in contrast to the direct autoregulation of AP3 by AP3/PI, autoregulation of PI transcription by AP3/PI is indirect and requires de novo synthesis of additional factor(s). Using a promoter fusion to the uidA reporter gene encoding bglucuronidase (GUS), the cis-regulatory region of AP3 and PI promoters have been dissected. Minimal promoters of about 727 bp for AP3 (Hill et al. 1998) and 498 bp for PI (Honma and Goto 2000) were identified that can direct the wild-type pattern of respective gene expression. Hence, these fragments contain all necessary cis-regulatory elements. Within 727 bp of the AP3 minimal promoter, multiple cis-acting elements were identified that control temporal and spatial subsets of the AP3 expression. Two elements for initiating early stage AP3 expression were identified; one is located proximally and the other is located

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distally within the promoter of AP3. The three CArG boxes described earlier exist within the proximal early element (Hill et al. 1998). In addition, a petal-specific element and two stamen-elements were identified that direct AP3 expression during maintenance phase (Hill et al. 1998; Tilly et al. 1998). Interestingly, genomic sequences of the AP3 and PI promoters do not show any sequence similarity (Honma and Goto 2000). Within the 498 bp PI promoter, multiple, discrete cis-regulatory elements were identified. The elements located in the distal region of the PI promoter are generally involved in the early initiation phase of PI expression while the element more proximal to the PI promoter is involved in the maintenance phase regulation. Thus, while cis-elements required for initiation and maintenance are located in distinct, separable regions of the PI promoter, the cis-elements required for initiation and maintenance of AP3 expression overlap. E. UFO, a Coregulator of B Gene Expression As described earlier, UFO activity is essential for B gene expression and ufo mutants exhibit floral organ identity defects that are similar to partial loss-of-function lfy and to B class mutants (Wilkinson and Haughn 1995; Levin and Meyerowitz 1995). In ufo mutants, AP3 and PI RNA level is reduced (Wilkinson and Haughn 1995; Levin and Meyerowitz 1995; Hill et al. 1998). Constitutive expression of UFO under the control of 35S promoter results in precocious and ectopic activation of AP3 and PI in flowers, confirming a positive regulatory role of UFO for B gene expression (Lee et al. 1997; Honma and Goto 2000). Consistent with its role as an upstream activator, UFO RNA accumulates in the floral meristem before the onset of AP3 expression. UFO is expressed initially throughout the entire floral meristem, but later is restricted to whorls 2–3 (Ingram et al. 1995; Lee et al. 1997). Thus, the presence of both LFY and UFO in whorls 2–3 might be necessary to initiate B gene expression. What is the relationship between LFY and UFO in activating AP3? Clearly, the simple hierarchical models of UFO acting downstream of LFY or LFY acting downstream of UFO are inconsistent with the following observations. First, LFY and UFO expression is activated independently (Levin and Meyerowitz 1995; Lee et al. 1997). Second, constitutive expression of LFY fails to rescue ufo mutants and, conversely, constitutive expression of UFO fails to rescue lfy mutants (Weigel and Nilsson 1995; Lee et al. 1997). In other words, the ability of the 35S::UFO construct to activate B class gene expression is dependent on wild-type LFY activity (Lee et al. 1997) and vice versa. Based on these

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data, a model has been proposed in which UFO and LFY are coregulators of B genes. LFY is responsible for general floral-specific activation of B gene transcription and UFO confers regional specificity to whorls 2–3 (Lee at al. 1997). The UFO gene encodes a protein with a F-box motif (Ingram et al. 1995). F-box proteins are part of the heteromeric ubiquitin ligase complex known as SCF (Skp1, Cdc53/cullin, F-box), which plays a key role in the degradation of a variety of regulatory proteins (Patton et al. 1998). The F-box protein in each SCF complex probably acts as a receptor to recruit specific protein targets for degradation. UFO has been shown to interact in vitro with Arabidopsis Skp1-like proteins ASK1 and ASK2 (Samach et al. 1999), supporting the idea that UFO may be a component of the SCF complex. In addition, ask1 mutants exhibit mosaic organs in their flowers similar to those seen in ufo mutants (Zhao et al. 1999), suggesting a role of Skp1-like proteins in B class gene regulation. Thus, LFY and UFO might act by distinct mechanisms to coordinately regulate B class gene expression. For example, LFY binds to AP3 promoter and activates AP3 transcription, whereas UFO might act as a member of the heteromeric ubiquitin ligase complex that specifically removes repressors of AP3 transcription via ubiquitin-mediated protein degradation.

IV. NEGATIVE REGULATORS OF FLORAL ORGAN IDENTITY GENES It has become evident that combined activities of both positive regulators, such as LFY, and negative regulators are necessary to specify the proper temporal and spatial domains of ABC gene expression. A number of negative regulators of the ABC genes in Arabidopsis have been identified that play crucial roles in delimiting ABC gene expression to specific domains. Many of these negative regulators have been identified through genetic screens for floral mutants that exhibit partial or complete homeotic transformation from one floral organ type to another. Based on the ABC model, one can interpret the mutant phenotype by predicting an ectopic expression of a particular class or classes of the ABC genes. The predictions can be easily tested with in situ hybridization experiments to examine the specific A, B, or C gene expression in the newly isolated mutant background. Once confirmed, these newly isolated mutations define negative regulators of the ABC genes. Despite the identification and molecular cloning of a large number of the negative regulators of the C class gene AG, the mechanism of negative regulation is still not well understood.

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Work to date suggests that a number of negative regulators work in a coordinated fashion to initiate and maintain AG repression. A. Temporal and Spatial Regulators of AG 1. APETALA2 (AP2). AP2 is both an A class organ identity gene and a repressor of AG, specifically in whorls 1–2. The DNA binding motifs found in AP2 suggests that AP2 could directly interact with DNA and AP2-domain containing proteins have been shown to bind to DNA elements termed GCC boxes (Ohme-Takagi and Shinshi 1995). However, the absence of any GCC box in the cis-regulatory elements of AG (Deyholos and Sieburth 2000) indicates that AP2 may mediate negative regulation indirectly, via other intermediate steps. Thus far, no direct DNA binding of AP2 to the AG cis-elements has been reported. Bomblies et al. (1999) examined the cis-regulatory sequences of AG that mediate the repressive action of AP2. They also tested if the repressive effect of AP2 on AG depends on the activity of LFY. Sequences within the second intron of AG that have been shown to be required for activation of AG expression by LFY (Busch et al. 1999) are also required for the repression of AG expression by AP2 (Sieburth and Meyerowitz 1997; Bomblies et al. 1999). The two independent LFY responsive enhancers identified within the large intron (Busch et al. 1999) are also involved in mediating the repressive effect of AP2 (Bomblies et al. 1999). However, additional experiments suggest that AP2 may regulate AG expression through both LFY-dependent and LFY-independent mechanisms. It is possible that the LFY-independent mechanism might be mediated by AP1 (Deyholos and Sieburth 2000). 2. LEUNIG (LUG). Since AP2 is expressed throughout the developing floral meristem, the spatially restricted activity of AP2 in repressing AG in the first two whorls must depend on additional levels of regulation (Jofuku et al. 1994). This spatially restricted activity of AP2 was initially thought to be conferred by the presence of other co-regulators that are only present in the first two whorls. One candidate is LUG, which was identified in a screen for enhancers of a weak ap2 allele (Liu and Meyerowitz 1995). Plants with mutations in the LUG gene exhibit homeotic transformations similar to, but less severe than, ap2 mutants (Komaki et al. 1988; Liu and Meyerowitz 1995). These lug mutants also display ectopic and precocious expression of AG RNA, suggesting that LUG is required for proper repression of AG. Furthermore, lug ap2 double mutants exhibit more severe homeotic transformations than either single mutants. In situ hybridization experiments indicated that the

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mechanism of this enhancement is through the increased ectopic expression of AG. The enhancement of defects seen in the lug ap2 double mutants suggests that LUG and AP2 share partially redundant functions. In addition, dominant interactions were observed between strong ap2 alleles and lug mutations, suggesting either that these two gene products might interact directly or that an activity composed of both LUG and AP2 is required above a threshold level for proper AG repression. LUG was recently cloned (Conner and Liu 2000) and found to encode a nuclear protein that has an overall domain structure similar to a class of functionally related transcriptional co-repressors including Tup1p of yeast and Groucho of Drosophila (Hartley et al. 1988; Williams and Trumbly 1990). A common structure shared by all members of this class includes Q-rich regions near the N-terminus of the protein and 6–7 WD repeats at the C-terminus. The WD repeats [named so because the repeat often ends with the amino acids tryptophan (W) and aspartic acid (D)] have been shown to mediate protein-protein interactions and are found in proteins with a wide variety of biochemical functions (Neer et al. 1994; Smith et al. 1999). The mechanism of this class of transcriptional co-repressors has been extensively studied in yeast and Drosophila. Several mechanisms were implicated such as interfering with the interaction between activators and the general transcriptional machinery (quenching); interacting with the general transcriptional machinery (direct repression) or by affecting chromatin organization. The Tup1p protein, although it cannot bind to DNA on its own, can interact with a variety of DNA binding transcription factors and mediate transcriptional repression through any of the above mechanisms. In particular, it was shown that Tup1p can organize repressive chromatin structure through direct interaction with the N-terminal region of histones H3 and H4 (Edmondson et al. 1996). LUG may function similarly by interacting with AP2 or other unidentified DNA-binding transcription factors to bring about transcriptional repression of AG. In situ hybridization showed that LUG mRNA is ubiquitously expressed in all floral whorls (Conner and Liu 2000). Hence, like AP2, additional factors or post-transcriptional modifications are needed to limit the activity of LUG to whorls 1–2. Recently, two additional genetic enhancers of lug were identified, SEUSS (SEU) and LARSON (LSN) (R. Franks, X. Bao and Z. Liu, unpublished data). The seu mutants display a phenotype that is very similar to, albeit weaker than, lug mutants. Furthermore, seu lug double mutants display an enhanced phenotype that is characterized by strong precocious and ectopic expression of AG and enhanced homeotic transformations of floral organs, particularly in the first two whorls. lsn mutant

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does not have an obvious phenotype on its own but shows strong enhancement of lug in floral organ identity specification. Both SEU and LSN, together with LUG and AP2, are candidates for members of a transcriptional repression system that regulates AG expression. 3. AINTEGUMENTA (ANT). Mutations in ANT cause narrower floral organ shape and a decrease in floral organ number (Elliott et al. 1996; Klucher et al. 1996). Furthermore, ant mutants are female sterile due to failure of integument initiation and megasporogenesis. Although ant single mutants rarely show homeotic transformation of organ identity, ant dramatically enhances the organ identity defects of ap2 and lug (Elliott et al. 1996; Krizek et al. 2000; Liu et al. 2000). This enhancement correlates with an increased ectopic AG expression in ant ap2 and ant lug double mutants. Hence, ANT is likely another redundant repressor of AG. ANT encodes a member of the AP2 family of DNA-binding transcriptional regulators (Elliott et al. 1996; Klucher et al. 1996); the sequence similarity between ANT and AP2 may underlie their functional redundancy in AG repression. 4. CURLY LEAF (CLF). The clf mutants are characterized by narrow and curled rosette and cauline leaves as well as short stem internodes (Goodrich et al. 1997). The clf flowers display narrow petals, and partial homeotic transformations in whorls 1–2. These phenotypes resemble those reported for plants in which the AG gene was ectopically expressed (Mizukami and Ma 1992). RNA gel blot and in situ analysis indicated that AG was ectopically expressed in leaves and in developing petals of clf mutants at later stages of flower development. Double mutant analyses with ag-3 indicate that the ag mutation is epistatic to clf and thus the clf phenotype in leaves and flowers was dependent upon AG activity. Hence, CLF is another repressor of AG expression with primary roles in leaves, stems and, to a lesser extent, flowers. CLF encodes a protein with extensive sequence similarity to the product of a Drosophila polycomb group gene, ENHANCER OF ZESTE (Goodrich et al. 1997). Drosophila polycomb group genes appear to form multimeric complexes that interact with DNA to bring about heritable maintenance of transcriptional states and there is indirect evidence that they do so by modifying chromatin structure (Carrington and Jones 1996). Like the polycomb group genes, CLF appears to be required for the maintenance of repression and not for the initiation of repression of AG. This interpretation is consistent with the observation that ectopic AG expression was only detected at later stages of flower development. CLF is expressed in 8-day-old seedlings throughout the apical meristem,

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leaf primordia, and leaves (Goodrich et al. 1997). It is also expressed in the infloresence meristem and in all four whorls of the flower. The fact that AG and CLF RNA are co-expressed suggests that CLF RNA expression is not sufficient to repress AG expression. 5. STERILE APETALA (SAP). Flowers of sap mutants resemble those of ap2 or lug mutants with carpelloid sepals and loss of petals (Byzova et al. 1999). In sap mutants, AG RNA is not only detected ectopically in floral whorls 1 and 2, but also in inflorescence meristems. SAP is thus another negative regulator of AG expression. Consistent with a role in AG repression, mutations in SAP enhance the organ identity defects of ap2. To test the regulatory relationship between SAP and AP2, AP2 RNA expression was examined in sap mutants and vice versa. AP2 RNA expression was unaltered in sap mutants and SAP RNA expression was not altered in ap2 mutants. Thus, SAP and AP2 do not appear to regulate each other at the transcriptional level. Like other negative regulators of AG, SAP appears to possess additional functions as revealed by defects of sap mutants in female gametophyte development and by defects of sap ag double mutants in meristem identity determination. SAP encodes a protein with serine-rich and glycine-rich domains that are often found in eukaryotic transcriptional regulators. 6. FILAMENTOUS FLOWER (FIL). The effect of the fil mutation is complex; genetic analyses of fil mutants indicated that FIL is required for the maintenance and growth of inflorescence meristems, floral meristems, and floral organs (Sawa et al. 1999a; Chen et al. 1999). More relevant to this review are the findings that AG is ectopically expressed in fil mutants in floral whorls 1 and 2 and that homeotic transformations in whorls 1–2 are enhanced in ap2 fil and lug fil double mutants (Chen et al. 1999). These data suggest that FIL is yet another member of the AG negative regulators. FIL encodes a nuclear protein that contains a zinc finger and an HMG box-like domain, suggesting a role in transcriptional regulation (Sawa et al. 1999b). Unexpectedly, FIL RNA expression is restricted to the abaxial side of the developing leaves and floral organs, and 35S::FIL plants display an abaxialization of leaves (Sawa et al. 1999b). Thus, FIL controls the identity of the abaxial side of lateral organs. B. Repression of AP1 Expression in Floral Whorls 3–4 In wild type, AP1 is initially expressed in the entire floral primordia, but at later stages AP1 RNA is restricted to whorls 1–2 (Mandel et al. 1992b; Gustafson-Brown et al. 1994). The inhibition of AP1 expression in

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whorls 3–4 results from negative regulation by AG that becomes expressed in whorls 3 and 4 at this stage. In ag loss-of-function mutants, AP1 RNA is expanded to all four whorls (Gustafson-Brown et al. 1994). In the ap2 or lug single mutants, when AG expression is expanded to all four whorls, AP1 RNA is absent from all four whorls (Gustafson-Brown et al. 1994; Liu and Meyerowitz 1995), suggesting that the ectopic AG in whorls 1–2 represses AP1 transcription. However, the mechanism by which AG brings about repression of AP1 transcription is presently unclear. The C class genes HUA1 and HUA2 also participate in the negative regulation of AP1 (Chen and Meyerowitz 1999). In hua1 hua2 double mutants, ectopic AP1 expression was seen in late stages in carpel walls and occasionally in stamens. HUA1 and HUA2 may act in parallel with AG to repress the expression of AP1 in whorls 3–4, possibly as transcriptional co-regulators. C. Restriction of B Gene Expression to Floral Whorls 2–3 1. SUPERMAN (SUP). The sup mutants exhibit supernumerary stamens interior to the third whorl stamens at the expense of carpels (Schultz et al. 1991; Bowman et al. 1992). In situ hybridization experiments indicate that the B class genes AP3 and PI are ectopically expressed in whorl 4 in sup mutants. Additionally, ap3 sup and pi sup double mutants exhibit phenotypes similar to the ap3 or pi single mutants. From these molecular and genetic experiments, SUP was originally thought to function as a negative regulator of B class genes in whorl 4. SUP encodes a nuclear protein with a single zinc finger and a putative basic leucine zipper motif, suggesting a role in transcriptional regulation (Sakai et al. 1995). However, in situ hybridization revealed that SUP is expressed in whorl 3, not in whorl 4. Further, SUP RNA expression is, in fact, dependent on AP3 (Sakai et al. 1995). Initial AP3 expression precedes SUP RNA expression, and ectopic AP3 expression under 35S promoter causes ectopic SUP expression. In addition, SUP RNA is much reduced or absent in ap3 mutant flowers, while the onset of AP3 expression in the sup mutant is normal. These new findings do not support the earlier hypothesis that SUP represses B gene expression in whorl 4. Two alternative models were proposed (Sakai et al. 1995); in one SUP functions to prevent the spread of AP3 activity from whorl 3 to whorl 4, while in the second model SUP functions to limit the extent of cell proliferation in whorl 3. In either model, SUP acts to maintain a boundary between whorls 3 and 4.

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2. LEUNIG (LUG) and CURLY LEAF (CLF). In addition to their roles as negative regulators of AG, LUG and CLF also negatively regulate B class gene expression. Patches of ectopic AP3 and PI expression detected in whorl 1 organs of lug mutant flowers suggest that LUG represses B class gene expression in whorl 1 (Liu and Meyerowitz 1995). Similarly, ectopic AP3 expression was detected in leaves of clf mutants, suggesting a role of CLF in repressing AP3 expression in leaves (Goodrich et al. 1997). Whether these effects reflect a direct action of LUG or CLF on B class genes is unknown. These studies, however, suggest that the regulation of B genes employs at least two mechanisms: a region-specific coactivator such as UFO and negative regulators such as LUG.

V. SUMMARY The initial activation of the ABC genes in a flower specific fashion is dependent upon meristem identity genes such as LFY and AP1. Later, ABC gene expression is spatially refined by a combination of other positive regulators, such as UFO, and negative regulators, such as AP2, LUG, ANT, CLF, and SAP. The combined activity of both positive and negative regulators insures proper spatial and temporal expression of the ABC genes and thus the stereotypical structure of a given flower. Now that many of the key regulatory molecules have been identified and isolated, the challenge for the future is to further clarify the molecular mechanisms underlying ABC gene regulation. Clearly a variety of mechanisms are employed in this process. Evidence to date suggests that both transcriptional and post-transcriptional mechanisms are employed and that generating the proper ABC expression domains likely requires targeted degradation of specific repressors, autoregulatory enforcement mechanisms, and recruitment of transcriptional co-repressors. The identification of an increasingly large number of genes involved in ABC gene regulation suggests that the mechanism of ABC gene regulation is rather complex and many questions remained unanswered. For example, what is the molecular or biochemical basis underlying the genetic enhancement or dominant interaction among these mutants? Do these ABC regulators physically interact directly? Do they regulate each other’s expression? Future experiments involving immunoprecipitation assays and/or yeast two-hybrid assays will allow us to test physical interactions among these ABC regulators. Examination of the expression of these genes by RNA in situ hybridization and immunolocalization in different mutant backgrounds will illuminate the regulatory

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relationship among these genes. For genes that encode DNA-binding domains, gel-shift assays may help to identify their target ABC genes. Further deletion analyses of the promoters of the target ABC genes and testing these deletion constructs using gel-shift and transgenic reporter assays will help to locate sequence motifs that are crucial for ABC gene expression. Similarly, site-directed mutagenesis and domain-swamping —for example, between AP2 and ANT—may help to assess their DNAbinding and transcriptional activation potential and target-site specificity. Thus far, many of the expression studies have only looked at RNA expression. It is now necessary to examine the protein expression of these genes to determine if translation of the RNA molecules is spatially or temporally regulated. Alternatively, regulation of the subcellular localization of regulatory proteins may explain the whorl-specific gene activity. These studies will lead to a more complete picture of the molecular hierarchy responsible for ABC gene regulation. This review has focused on the genetic basis of ABC gene regulation and has treated floral organ identity specification as independent from environmental events. As we further clarify the molecular mechanisms of floral organ identity, we may be able to better predict the effects of environmental influences, such as the effect of photoperiod, hormone, and temperature on floral development. Understanding the interaction between the genetic programs and environmental factors will be critical for controlling traits of agricultural varieties in the field. As our understanding of the genetic mechanisms of organ identity specification grows, so does our ability to engineer new floral variants. One can envision a multitude of applications in the areas of horticulture and agriculture. For example, to engineer environmentally friendly ornamental types (such as pollen-free or fruit-free cultivars), or to facilitate outcrossing and simplify breeding programs, novel methods of generating male-sterile plants will be highly desirable. By specifically repressing the B or C class genes in whorl 3 through transgenic techniques, one may create flowers whose stamens are converted into carpels or petals. Alternatively, our knowledge of organ identity genes could be used to increase the visual diversity of floral variants. Replacing reproductive structures with petals leads to showier flowers, as is seen in a number of presently existing “double flowers” (Acquaah et al. 1992) and in Arabidopsis transgenic plants (Fig. 2.2F). New chimeric organs that result from a partial homeotic transformation may have useful or visually interesting properties: stamenoid petals that are more tubular or sepalloid petals that are more resistant to wilting and thus increase the life of the cut flower. Increasing the proportion of a given organ within the flower may enhance the yield of certain floral-derived products. Yields

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of saffron, derived from stigmatic tissue of the crocus flower, might be improved if ectopic expression of C class genes is employed to generate additional carpels. As we continue to illustrate the “blueprints” of floral development, we hope to enable “agricultural architects” of the future to rationally design floral types to better meet societal needs. In addition to the technical hurdles that lie ahead, we also face the multi-disciplinary challenge of managing these new variants such that environmental and socioeconomic effects are carefully considered.

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3 Lingonberry: Botany and Horticulture Inger Hjalmarsson The Nordic Gene Bank, Smedjevägen 3, P.O. Box 41, S-230 53 Alnarp, Sweden Rodomiro Ortiz The Royal Veterinary and Agricultural University, Department of Agricultural Sciences, 40 Thorvaldsensvej, DK-1871 Frederiksberg C, Denmark

I. INTRODUCTION II. HISTORY A. Early B. 18th and 19th Centuries C. 20th Century D. Horticultural Research in the Northern Hemisphere 1. Nordic Region 2. Other Sites in the Northern Hemisphere III. BOTANY A. Taxonomy and Geographic Distribution B. Morphology 1. Vegetative 2. Reproductive C. Ecology D. Other Vaccinium Species in Scandinavia 1. Bilberry 2. Bog Bilberry 3. Small-fruited European Cranberry 4. Dwarf Cranberry IV. MANAGEMENT OF NATURAL STANDS A. Photosynthesis B. Biomass Production C. Seed Ecology and Regeneration D. Berry Production E. Effects of Forestry Management F. Experiments in Natural Habitats Horticultural Reviews, Volume 27, Edited by Jules Janick ISBN 0-471-38790-8 © 2001 John Wiley & Sons, Inc. 79

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V. HORTICULTURE A. Propagation 1. Plants Collected Directly from the Wild 2. Shoot Cuttings and Rhizomes 3. Seedling Plants B. Frost Protection C. Culture 1. Soil 2. Nutrients 3. Mycorrhiza 4. Mulching 5. Irrigation D. Plant Pathology 1. Weed Control 2. Diseases E. Crop Improvement 1. Early Studies of Ecotypes 2. Breeding at Balsgård 3. Description of Swedish Cultivars 4. Description of North American Cultivars VI. SUMMARY AND FUTURE PROSPECTS LITERATURE CITED

I. INTRODUCTION Lingonberry (Vaccinium vitis-idaea L., Ericaceae) is a perennial, evergreen dwarf shrub that is indigenous to Scandinavia, where the peasized, bright-red fruit is picked from wild stands. Lingonberry is known as puolukka in Finland, as tyttebær in Norway and Denmark, and as lingon in Sweden. Lingonberry jam, with or without sugar, may be eaten with porridge, potatoes, bread, pancakes, cow and reindeer milk, herring, black blood pudding, meatballs, and steak among other foods. The berries have also been used for soups and beverage. Retzius (1806) recommended lingonberry drinks for fever patients. Furthermore, lingonberry has been used as an anti-scorbutic (Nyman 1868), and because of its richness in glycosides (Bandzatiene 1999), as a diarrhea medication (Stodola and Volak 1986). Folk medicine recommends that lingonberry tea, derived from leaves, be used against rheumatism (Henriksson 1923b) and as a remedy for urinary tract infections (Nielsen 1978). Recent reports suggest that lingonberry may have anticancer attributes due to high anthocyanin content (Bomser et al. 1996). Lingonberry jam is a traditional delicacy and, although considered a luxury today, it was once one of the few staples available to poor people. While no longer necessary for survival, berry picking has become a recreational activity for many and jam making has moved from homes

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to processing plants. Industry requires a continuous supply of the berries and shortages of labor have led to the start of domestication of the crop. Lingonberry has also been grown as an ornamental plant or shrub in Scandinavian gardens and landscapes since the 17th century (Adelswärd 1994; Balwoll and Weisaeth 1994; Lundqvist 1995) and is an important component of Christmas decorations. Since the 1960s, cultural studies have been carried out in Sweden, Finland, and Norway but published information is scattered. This review article focuses on Nordic lingonberry and includes botany, management of natural stands, cultural practices, and breeding. Information about lingonberry research in Germany, the former Soviet Union, Poland, and North America has also been included.

II. HISTORY A. Early There is little documented information about berry utilization in Scandinavia before Linnaeus. Remnants of lingonberry wine in Danish graves from the Bronze Age are the first proof of its use in the home (Brøndegaard 1987). The Icelandic law books (Grágrás) of the 13th century stipulated that berry-picking on other people’s land must be limited to what can be eaten on the spot, thereby indicating the importance of lingonberry as human food (Armfelt Hansell 1969). There are a few other published reports of wild lingonberry in the Middle Ages (Eriksson et al. 1979). The Italian diplomat Magalotti (1674) wrote the first thorough description of wild lingonberry in Sweden after his journey through the country in the 17th century. By that time lingonberry was also mentioned in Nordic gardening books. In 1651, André Mollet, the French gardener working for Queen Christina, published Le Jardin de Plaisir and suggested the use of lingonberry for parterres de broderie (hedge gardens) instead of the less adapted box model (Adelswärd 1994; Lundqvist 1995). The first Norwegian gardening book, written in 1694 by Christian Gartner, recommended planting lingonberry for culinary and medical purposes (Balwoll and Weisaeth 1994). B. 18th and 19th Centuries Botanists such as Linnaeus (1748) and Retzius (1806) described lingonberry during the 18th and 19th centuries. The economist Fischerström (1779) also discussed lingonberry in his dictionary about Swedish

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households and natural science. Furthermore, lingonberry recipes were published in the cookbooks of the time. In addition, lingonberry was included in crofters’ (tenants’) contracts and mentioned in local horticultural records. For example, Waern (1834) reported on the cultivation of white lingonberry, Vaccinium Vitis idaea fructu albo, in the garden of Baldersnäs (Sweden). Lingonberry was also considered in ethnology studies by Hyltén-Cavallius (1868) and in diet investigations by Keyland (1919) and Grøn (1942). Although lingonberry has been considered among the most important of the fruit jellies (Retzius 1806), no evidence exists about its importance in the diet of earlier times. Eriksson et al. (1979) indicated that berry harvest was most valuable to poor people, especially in years of bad crops when lingonberry could mean survival. Crofters’ contracts in the 19th century often stated that the crofter’s family should pick a certain quantity of berries for further delivery to the estate (Armfelt Hansell 1969). Women and children did most of this work and they also picked the family supply. The berries were originally picked by hand, but Fischerström (1779) described the earliest picking tool. The use of these tools became more widespread and, at the beginning of the 20th century, a debate started about whether this practice was harmful. However, experiments at the Royal Swedish Academy of Agriculture concluded that the tools were harmless to the plants (Sylvén 1918). Johansson (1983), von Zabeltizt (1989), and Dale et al. (1994) have described the development of other lingonberry harvesting aids. Throughout the history of this region, lingonberry has been important as a supplier of energy and vitamins. Lingonberry fruit differs from most of the other wild berry species owing to its long-term storage potential. Consequently, berries are kept from one year to the next without sugar, a product that was rare in most Scandinavian homes until the 19th century (Kuuse 1982). Traditionally, berries are placed in jars and preserved by pouring clear water over them to produce a dish known as “water-lingon” in Swedish cuisine (Retzius 1806). The storage ability of the berries depends primarily on their benzoic acid content, with up to 65 mg benzoic acid per 100 g of berries (Karlsson and Malmberg 1974). Lingonberry fruit also contains large amounts of aroma compounds and anthocyanins (Anjou and von Sydow 1967; Andersen 1985), negligible amounts of proteins, and only small amounts of minerals, although lingonberry fruit provides 4 mg vitamin C, 0.02 mg carotene, and 67 kcal of energy per 100 g (Statens Livsmedelsverk 1978). According to Fuchs and Wretling (1991), lingonberry fruit has 7 g sugar/100 g and 24 g titrable acids/L fruit juice.

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Lingonberry has a long history of commerce in the Nordic countries. Linnaeus (1732) described how farmers in northern Sweden sent their berries to the markets in Stockholm. The moor farmers of Jylland (Denmark) traded lingonberry with Copenhagen in the 1800s (Brøndegaard 1987). The first reference to export of lingonberry from Norway was dated 1835 (Valset 1976; Graff 1991). However, the “days of glory” for lingonberry began at the turn of the century, in conjunction with the development of the railway. In 1902, a record quantity of 20 million kg of lingonberries was exported from Sweden (Wikmark 1907). The Swedish berries were mainly sold to Germany, where they competed with berries from Norway and Finland. C. 20th Century Taking care of wild lingonberries captured people’s interest in Finland, Norway, and Sweden at the beginning of the 20th century. For example, Norwegian committees were established to make berry picking more efficient (Valset 1976; Graff 1991), whereas Swedes started an industry with dried lingonberry and blueberry (Lind 1916). According to official statistics, the exports remained high throughout the 1930s (Eriksson et al. 1979; Graff 1991). Finnish exports during this decade varied between 2.4 and 6.7 million kg yearly (Anon 1983). The demand for lingonberry continued to be high during World War II, when people were encouraged by their governments to create contingency stocks in case there were shortages of other fruits. Lingonberry exports never surpassed earlier levels after World War II (Eriksson et al. 1979). But commercial lingonberry harvest has been reported from Alaska, Nova Scotia, and Newfoundland in North America (Holloway 1984). The commercial lingonberry harvest in Finland from 1977 to 1985 was estimated to vary between 1.7 and 10.2 million kg annually, of which 35 to 80% was exported (Hiirsalmi and Lehmushovi 1993). Swedish exports during the same period fluctuated between 1.5 and 4.2 million kg (Holmberg 1987). In 1985 the highest economic return (85 million Swedish Krone) was realized. Finland and Sweden import some wild berries but remain net exporters of lingonberries. In Norway, however, more fruit are imported than exported. The Norwegian food industry uses about 1 million kg of lingonberry yearly and an additional 0.2 to 0.4 million kg are sold on the fresh market. The majority of these berries are imported (Nes 1994). Foreign trade in wild lingonberry remains important in Scandinavia (Statistics Sweden 1994). Per capita consumption of wild small fruits (mainly lingonberry and blueberry) in Sweden was estimated to be 0.6 kg in 1990 (Statens Jordbruksverk 1994).

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D. Horticultural Research in the Northern Hemisphere 1. Nordic Region. Lingonberry is a healthy component of the Scandinavian diet and is considered an exotic fruit by many people outside the region, which suggests that this crop will always have a market. However, the question persists whether the crop should be picked solely from native stands or through commercial production, or both. Interest in commercial harvest from wild stands has decreased but the industry demands a reliable annual supply. Sweden. AB Bjäre Industrier, the Swedish producer of lingonberry jam, initiated the first lingonberry plantings at Vången (Skepparslöv) in 1962 and another 5 ha were planted at the Ottarp farm (Ryssby) from 1966 to 1968 (Teär 1972). Additional small plantations were started later in both middle and northern Sweden, and supplementary experiments were carried out in the Department of Pomology at the former Agricultural College of Sweden in Alnarp (Fernqvist 1977). A Nordic symposium about domestication of wild berries was held in 1974 at Karlstad (Fernqvist 1974). Most of this early research was published in Swedish, but English abstracts were printed for the ISHS International Symposium on Vaccinium Culture (Fernqvist 1977; Hjalmarsson 1993). Since the late 1970s, most lingonberry research has been carried out at the Department of Horticultural Breeding in the Swedish University of Agricultural Sciences (SLU) in Balsgård. Pilot plantations are run parallel to the plant breeding experiments and provide continuous experience on practical cultivation. The first plantations were planted with micropropagated plants of ‘Sussi’ and ‘Sanna’. Plant spacing was 40 cm in a zig-zag pattern that aimed to create a dense carpet of lingonberry vegetation (Eckerbom 1990). Currently a row system with plant spacing of 25–30 cm × 80 × 100 cm is recommended (Nilsson and Rumpunen 1997). Finland. The first cultivation experiments began in 1968 at the Institute of Horticulture in Piikkiö (southwest Finland). The broad research program involved experiments in natural habitats, field studies on plant material, soil, liming, fertilization, and shading among others. Research results were reported in Finnish journals, and review articles were also written (Lehmushovi 1977b; Hiirslami 1989). Current research is carried out by the Department of Plant Production of the University of Helsinki and focuses on weed control (Saario 1998). Norway. The first commercial lingonberry field in Norway was planted in the mid-1960s. In 1974 public research began at Kise Research Station, where the experiments focused on plant material, plant establish-

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ment, water requirement, and weed control (Nes 1994). In 1983, a group of farmers in southeastern Norway started a lingonberry project; however, it did not lead to any extensive production (Vestrheim et al. 1994). Seven years later, a private farmer imported Dutch and German cultivars for field planting. At the same time, the Agricultural University of Norway (NLH) started cultivar trials, which are still going on (Vestrheim et al. 1994). 2. Other Sites in the Northern Hemisphere Eastern Europe. Former Soviet researchers started their research on lingonberry domestication in the mid 1960s (Paal 1992) owing to a rising demand and low productivity of wild lingonberry. Propagation methods, seed germination, factors influencing root development in cuttings, mulching, phenology, somatic embryogenesis, and micropropagation were included in their research agenda (Butkus et al. 1989; Labokas and Budriuniene 1989; Audrina 1996; Banner 1996, 1998; Bandzaitiene 1998; Kutas 1998; Kutas and Sidorovitch 1998). In Poland, mulching was also investigated (Pliszka and Scibisz 1985). Plant growth was enhanced in mulched plots but it did not improve fruit yield. Testing foreign lingonberry germplasm to identify new high-yielding cultivars has also been an important activity in Eastern Europe. A high second harvest was reported for Dutch and German cultivars in Byelorussia (Pavlovsky and Ruban 1998). However, this second harvest of foreign cultivars was low in Latvia (Audrina 1996) and Russia (Tiak and Cherkasov 1998). Four promising selections were made from the local material in Latvia (Audrina 1996) and two Russian cultivars (‘Kostromskaya Rozovaya’ and ‘Kostromichka’) were released in the mid1990s (Tiak and Cherkasov 1998). It seems that seedlings from northern regions have earlier growth and faster development than those from southern sites (Reier and Paal 1998). Plant breeding through chromosome doubling (CD) with colchicine was unsuccessful in Byelorussia. The CD-derived plants did not improve fruit yield because of few flowers and pollen sterility (Morozov 1998). Wild tetraploid lingonberry accessions collected in Magadan (Russia) were crossed with other Vaccinium species, but the hybrid seeds did not always germinate. Germany. Lingonberry research was started in 1973 by Prof. G. Liebster at the Institute of Fruit in Weinhenstephan (Liebster 1977, 1984; Müller 1982). Morphology and physiology research led to an efficient method for vegetative propagation of the Dutch cultivar ‘Koralle’ and the German

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cultivars (‘Erntedank,’ ‘Erntekrone,’ and ‘Erntesegen’). The German cultivars are selections from wild lingonberry stands (Zillmer 1985). Further investigations at Wilhelm Dierking Beerenobst included soils for lingonberry cultivation, mulching, and fertilizers (Dierking and Krüger 1984; Dierking 1985). The University of Hannover started research on lingonberry nutrition in the 1980s to determine the correlation between yield and growth with nutrient levels using the leaf analysis (Krüger and Naumann 1984a,b,c; Krüger 1985). There were some attempts by German scientists to obtain hybrids between lingonberry and cranberry (Vaccinium macrocarpon Ait.) (Christ 1977), as well as in vitro propagation of lingonberry clones (Gebhardt and Friedrich 1986). Tissue culture has been confirmed as a means for rapid mass propagation of lingonberry (Riechers and Bünemann 1989) and to facilitate fruit picking, a harvest machine was developed by von Zabelitz (1989). Nowadays, about 35 ha are grown commercially in Germany (Dierking and Dierking 1993). North America. Wild lingonberry fruit are collected commercially in Alaska, Nova Scotia, and Newfoundland (Holloway 1984; Hendrickson 1997). Since 1965 researchers at Fairbanks (Alaska) have been working in lingonberry improvement. Early investigations included assessment of substrates for lingonberry cultivation, influence of light intensity on growth, gibberellic acid effect on fruit set, chilling temperature requirements, factors affecting rooting of stem cuttings, and seed propagation (Hall and Bell 1970; Holloway et al. 1982a,b; Holloway et al. 1982; Holloway et al. 1983; Holloway 1985). In Newfoundland, lingonberry researchers are studying crop establishment, maturity dates, pests affecting the crop, and the best cultural practices to enhance earliness and fruit yield (Penney et al. 1997). Researchers working in the Department of Horticulture at the University of Wisconsin–Madison have investigated physiology (photoperiod response) and cultural practices (weed control, humus application) needed to introduce lingonberry as a new fruit crop in the northern United States (Stang et al. 1993a,b, 1994; Stang 1994). They have established small-scale demonstration plots and determined fruit processing requirements. Cuddy (1998) has reviewed advances in lingonberry cultivation at Wisconsin. North American scientists have tested cultivars and selections from the wild (Penney et al. 1977; Estabrooks 1997). In the mid-1990s, the University of Wisconsin–Madison released two lingonberry cultivars for commercial production (Stang et al. 1994), ‘Splendor’ and ‘Regal’, both

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selections from Finnish exotic wild germplasm. Vaccinium reticulatum has been crossed with lingonberry because this diploid evergreen wild species, native to Hawaii, has a large fruit size among other interesting characteristics (Zeldin and McCown 1997). Some North American researchers (Hosier et al. 1985; Serres et al. 1994) have investigated micropropagation methods including basal media, plant growth regulators, and culture conditions. III. BOTANY A. Taxonomy and Geographic Distribution The genus Vaccinium includes approximately 400 species (Galletta and Ballington 1996) dispersed from the arctic to subtropical regions and mountainous tropics (Hutchinson 1969). Five Vaccinium species occur in Scandinavia: V. vitis idaea (2n = 24), V. myrtillus L. (2n = 24), V. uliginosum L. (2n = 24, 48, 72), V. oxycoccus L. (2n = 24, 48, 72), and V. microcarpum (Turcz.) Hook. (2n = 24) (Hylander 1955) (Plate 3.1A). The chromosome numbers in brackets are those reported by Luby et al. (1990). The rare hybrid Vaccinium × intermedia Ruthe was described by Ritchii (1955a) as an intermediate between V. vitis-idaea L. and V. myrtillus L. Although this hybrid has been seldom reported in the Nordic Region, it has been seen sometimes in Sweden (Scania and Stockholm) and in two locations at Jylland, Denmark (Lagerberg 1948). Triploid forms of Vaccinium vitis-idaea have been reported in Sweden (Ising 1950) and in Finland (Ahokas 1971). Hultén and Fries (1986) have mapped the distribution of the circumpolar lingonberry (Fig. 3.1), and Hultén (1971) provided details about the abundance of lingonberry in the forests of Finland, Norway, and Sweden and its sparse occurrence on the calcareous soils of southern Sweden and the Danish islands. Lingonberry is also common on the moors of Jylland (Brøndegaard 1987; Hultén 1971) and grows at 1800 m above sea level in Jotunheimen, Norway (Hultén 1958; Lagerberg 1948). Lingonberry seems to be a European crop (Plate 3.1B), although the Pacific Northwest in the United States has observed a significant planting of lingonberry in recent years (Finn 1999). Vaccinium vitis-idaea is the only species in the section Vitis-idaea (Moench) Koch (Galetta and Ballington 1996). Hultén (1949) divided lingonberry into two subspecies: subsp. vitis-idaea L. and subsp. minus (G. Lodd.) Hultén. Both subspecies are found in the arctic mountains of Norway. Subspecies vitis-idaea predominates in Eurasia, while subsp.

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Fig. 3.1. Lingonberry in the Northern Hemisphere (From: Hultén and Fries 1986, with kind authorization from Koeltz Scientific Books).

minus prevails in the mountains of North America. Apparently, the two subspecies hybridize in Scandinavia (Hultén and Fries 1986). B. Morphology 1. Vegetative. Lingonberry is as an evergreen, small shrub with subterranean rhizomes, and aerial shoots varying from 5 to 30 cm tall (Hultén 1958) (Fig. 3.2). The subsp. minus is shorter and has smaller leaves and berries than subsp. vitis-idaea. The leaves of subsp. vitis-idaea possess conspicuous venation, while the venation is inconspicuous in subsp. minus (Hultén 1949). The leathery leaves of lingonberry are ovate with a thick, glossy upper surface and a pale, glandular lower surface. These characteristics allow the plant to survive the cold and windy winters of the North without desiccation. The lingonberry plants typically have leaves ranging in length from 4 to 29 mm and width from 2 to 16 mm (Teär 1972).

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Lingonberry plants collected from the Nordic forest.

2. Reproductive. Floral initiation in Vaccinium species starts in late spring or early summer (Bell and Burchill 1955; Eck 1966b). Flowering occurs for one month between May and June in the southern part of Scandinavia, and four weeks later in Lapland (Hultén 1971) (Fig 3.3). In these northern latitudes, secondary flowering on current year shoots is rare, as noted in middle Europe by Hegi (1927) and Ritchii (1955b). At Wisconsin, flower initiation occurs at 8 to 12 h day length and a minimum of 8 weeks seems to be needed for maximum flower induction (Stang et al. 1993a). It is possible to distinguish between floral and vegetative buds of lingonberry in August at northern latitudes. The vegetative buds are larger (2–3 mm) and wider (1 mm) and they have a tendency to bend downward. The inflorescence is a slightly pendulous raceme of 4 to 6 flowers (Teär 1972). The lingonberry flowers (Plate 3.1C) are white to pinkishred, 4 to 6 mm in length, urceolate, and possess 4 to 5 petals (Knuth 1899). These flowers are hermaphroditic and epigynous with 4 to 5 locules per ovary and 15 to 20 ovules per carpel. The flower has 8 stamens that open by pores (also known as porandrous). The form of their sepals, hairiness of their filaments, and length of their style (Hultén

Fig. 3.3.

Lingonberry plants in blossom.

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1949) differentiate the flowers of the two subspecies. The dark red, globular berry of the subsp. vitis-idaea ripens in September and has, on an average, a diameter of 6 to 8 mm (Teär 1972). The style is closely surrounded by anthers; the stigma and anthers mature nearly simultaneously but the anthers may mature later. The stigma rises outside the corolla, which suggests that wind might be important for pollination (Hagerup 1954). However, most authors (Haslerud 1974; Lehmushovi 1977a) consider entomogamy essential for fruit set. Bumblebees (Bombus terrestis L. and B. pratorum L.) were the principal pollinating species at Ottarp (Eriksson 1975). However, some years the bumblebee workers were not ready until the last half of the flowering period (Ängeby 1978). Nectaries, hidden beneath the stamens, produce a large amount of nectar that attracts insects. Cross-pollination is effected as the insect passes the stigma, depositing pollen, and in the process of getting to the nectaries, pollen from the anthers is transferred to the insect. The anthers (Plate 3.1D) are touched afterwards, thus releasing pollen grains to the surrounding environment. Pollen grains of lingonberry are arranged in tetrads, and the rate of germination exceeds 80% (Eriksson 1975; Lehmushovi 1977a). Pollen tubes required five days to grow through the style. Different botanical varieties of lingonberry are mentioned in the literature. Among them, variety ovata J. Henriksson, exhibiting oblong-ovate berries, occurs in the county of Dalsland (Sweden) and in northern Norway (Henriksson 1923a; Jørstad 1960). Swedish botanists have also noted lingonberry stands with white berries (Nyman 1868; Lagerberg 1948). Temperatures between 15 and 20°C enhance pollination, while temperatures above 25°C lower fruit set (Eriksson 1975; Hjalmarsson 1997). Fruit set was greatest following artificial cross-pollination (64%) and open-pollination (58%), whereas fruit set after self-pollination and in isolated flowers were only 28% and 2%, respectively, in wild stands. Seed set ranged from 10.9 per berry in open-pollinated plants to 4.1 in isolated plants. There were 8.6 seeds per berry after cross-pollination, while 3.8 seeds were obtained after self-pollination. Similar results were obtained with the cultivars ‘Sanna’ and ‘Sussi’ (Hjalmarsson 1997). C. Ecology Teär (1972) was the first Nordic scientist to thoroughly investigate vegetative and reproductive growth of wild lingonberry. His research was aimed at obtaining information about plant ecology to facilitate a predictable process of domestication. Since then botanists and foresters

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have further investigated native lingonberry populations together with other common understory species in the forests of northern Scandinavia. The effect of light on the annual growth differed according to species (Kellomäki 1977). In lingonberry the relationship between the amount of photosynthesis and productivity was nonlinear. Maximum growth was reached at low photosynthetic light flux. Bilberry and lingonberry were abundant after the closure of the canopy at the expense of more light-dependent species. Kellomäki argued that lingonberry possesses phenotypic and reproductive plasticity, which is common among vascular plants in ground cover communities. Branching occurred on one-year-old shoots in young ramets as well as from buds on older shoots after rejuvenation in deciduous bilberry and evergreen lingonberry in habitats of northern Finland (Tolvanen 1995). Lingonberry has a predominantly monopodial growth habit, but shoot growth stopped in terminal inflorescences after a few growing seasons. This indicates a sympodial branching system, i.e., older ramets grew more horizontally than younger ramets. Terminal buds mainly developed into vegetative shoots in the northern Finnish forest understory, whereas a great number of lateral buds were activated in open habitats. This, however, did not lead to any change in the total number of new shoots. Instead, flower production was greatest in open habitats. Differences in growth habits between the two sites indicated high morphological plasticity, allowing the species to respond rapidly to changing environments. D. Other Vaccinium Species in Scandinavia Berry crops have always been important components of human diets, although some of the berry species have remained economically important only locally. Lingonberry has been considered among the major new berry crops (Finn 1999), but other Vaccinium species indicated below have been locally harvested and may become economically important new crops in the Nordic region. Most of their fruit continues to be harvested from wild stands, although owing to their attributes some of these species are attracting the attention of the industry either for processing (e.g., for juices or jams) or for pharmaceutical purposes. As expected in this process, lingonberry and other Vaccinium species are being domesticated to shift the harvest from wild or native stands to their commercial cultivation in farmers’ fields, which will ensure a stable fruit supply for the industry (Finn 1999). Hence, potential markets for these new crops are playing an important role in the process of domestication of lingonberry and other Nordic Vaccinium species.

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1. Bilberry (V. myrtillus). This species is a perennial low bush whose leaves fall off in autumn (Mossberg et al. 1992). The angular twigs remain green during the winter. The leaves are not leathery as with lingonberry, but are thin and pointed with denting edges. Bilberry flowers between May and June, and the flowers grow one by one. The flower is almost round and pale green with a reddish tone. Berries ripen in July to August and most commonly they are blue and covered with dew. However, they may also be black and shiny and are called “shoemaker’s berry.” Bilberry is common in most of Scandinavia, is harvested from the wild and eaten fresh, as jam, cream, or soup. Bilberries, which have a long history in European folk medicine (Morazzoni and Bombardelli 1996), are still used as a medical treatment against diarrhea. 2. Bog Bilberry (V. uliginosum). This small bush (10–75 cm tall), which flowers between May and June in Scandinavia, stretches across circumboreal Northern Hemisphere regions (Finn 1999). It may be seen as a common under shrub, especially where heath-lands are turning to swamp and along lakeside forest. The berries are blue, oval, covered with dew and harvested from the wild plants (Mossberg et al. 1992). Some accessions have been crossed with V. corymbosum to improve winter hardiness and obtain early harvest in highbush blueberry. The cultivar ‘Aron’ was developed following this breeding approach (Hiirsalmi 1989; Hiirsalmi and Lehmushovi 1993). The fruit juice has no color (opposite to bilberry), and the taste is often described as flat and stale. 3. Small-fruited European Cranberry (V. oxycoccus). This species is similar to the American cranberry, but is much smaller (4–8 cm tall). The leaves (6-8 mm long) are evergreen (Mossberg et al. 1992). The plant flowers between June and July, and each flower cluster has 2 to 4 flowers. The round-shaped berry diameters are 8 to 10 mm, and the berry ripens late. Hence, berry picking is preferred after the first winter frost. This small-fruited European cranberry is common in Scandinavia, although not in the very northern mountain area. The berries are used for jam or alcoholic beverage. 4. Dwarf Cranberry (V. microcarpum). This Vaccinium species is even smaller than the small-fruited European cranberry and extends to the very north of Scandinavia (Mossberg et al. 1992). The leaves are 3 to 8 mm long, and the plant flowers between June and July. The flowers grow one by one or two together, and the berries (5–6 mm) are more oval than those of the small-fruited European cranberry.

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IV. MANAGEMENT OF NATURAL STANDS A. Photosynthesis Seasonal carbon dioxide assimilation per unit leaf mass was three times higher in the deciduous bog bilberry (V. uliginosum) than in evergreen lingonberry in sub-arctic environments (Karlsson 1982). However, lingonberry was light saturated for a longer period, i.e., 80 to 86% of the time in July and 60 to 70% in early August. Lingonberry leaves are largest on the mid-position of each yearly shoot segment, and have axes with uniform and horizontal angles. Old lingonberry leaves are important for a rapid CO2 assimilation in the spring (Karlsson 1982). Old leaves need two weeks to build up to full photosynthetic capacity. Similarly, lingonberry plants are able to extend their growing season late in autumn. Increased leaf age, however, affects maximal photosynthetic capacity. Leaves will only retain 2/3 of their original capacity during the second growing season, and in the following years an additional 10% will be lost annually (Karlsson 1982). Hence, lingonberry leaves need four growing seasons to assimilate the same amount of carbon dioxide as bog bilberry leaves assimilate during one season. Current year shoot growth in lingonberry was mainly supported during early summer by photosynthetic products of older leaves (Karlsson 1982). Photosynthesis in bog bilberry and lingonberry was similar. In response to drought, lingonberry has a great ability to survive in dry environments. The two species respond differently to light utilization and water economy, and occupy distinct niches in natural subarctic environments. B. Biomass Production There were, on average, 101 to 231 m rhizomes per m2 in a large number of selected lingonberry plots in natural habitats in Sweden (Teär 1972). Annual production was calculated at 13 to 55 new rhizomes per m2. New rhizomes typically grew 15 to 20 cm, and never had a growth rate exceeding 40 cm. Rhizomes had buds, which may develop new shoots under or above ground. A total of 150 dormant buds were found per m of rhizome. New plants were usually developed in groups near where the rhizomes originated, and only about 15% of these originated from buds at the terminal ends. Every year 10 to 25% of the old plants were replaced by new plants through rejuvenation. The average plant age was 4 years, but plants as old as 11 years were found. The total amount of dry biomass varied between 153 and 579 g per m2 (Teär 1972). About

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half the biomass was underground and rhizomes accounted for 10 to 20%. The above ground biomass was equally divided between shoots and leaves, with the latter constituting 78% of the annual production of biomass. A lingonberry plant had on average 3.3 shoots, and on average produced 1.7 new shoots per plant every year, but more than half the plants had only one new shoot. Most of the shoots were produced from vegetative one-year-old shoots. Large plants produced more shoots than small plants, though the ratio (new shoots/total number of shoots) was lower. Furthermore, flowering shoots were shorter than vegetative shoots, and produced new shoots from stems that flowered late in the growing season. On average there were 8 to 9 leaves per shoot. Flowering shoots had more leaves than those that were strictly vegetative. At the beginning of the summer, current-year leaves accounted for 70% of the total leaf number (Teär 1972). Leaves growing on shoot tips were more pointed than others growing below the shoot tips. The number of plants with flower buds increased with the age of the plants. Plants with 5 to 6 older shoots had mostly new flowering shoots (2.8–3.1). However, the share of new fertile shoots within a plant decreased as it grew in size. Most fertile shoots were in clear felled areas, while the opposite occurred in forests with Norway spruce. On average, there was one flower bud per flowering shoot. The number of flowers per cluster varied from 4.1 to 5.9, while there were, on average, 3 to 4 berries and a maximum of 14 berries per cluster. Increased shoot growth and higher levels of nitrogen, phosphorous, and potassium were noted after irrigation and fertilization, while the photosynthetic rate was the same in controls and treatments (Karlsson 1985). These changes were more conspicuous in lingonberry than in bog bilberry. Lingonberry had fewer old leaves per shoot after the treatment. These results suggested that lingonberry, as previously reported in other evergreen species, has decreased leaf longevity when nutrient levels are increased in the substrate. C. Seed Ecology and Regeneration Fruit set was lower after self-pollination than after open-pollination (Fröborg 1996). A few lingonberry seeds were recorded in the seed bank of a sub-arctic pine-birch forest in Lapland, Finland (Vieno et al. 1993). Vaccinium species lack developed seed banks even though they are known to have high seed production (Eriksson and Fröborg 1996). The work of Eriksson and Fröborg focused on “windows of opportunity,” i.e., spatially and temporally unpredictable conditions in which seedling

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recruitment was possible. Lingonberry seedlings favor moist soil with high organic content. Likewise, sudden optimal conditions at a specific micro-site may lead to establishment of new seedlings within stands of adults. Extensive production of well dispersed seeds but no extended dormancy will take advantage of such favorable situations. Furthermore, recruitment through “windows of opportunity” could explain unexpected high genetic variation within populations of persistent clonal plants. Regeneration of understory species after fire was investigated in Northern Sweden (Schimmel 1989, Schimmel and Granström 1996). To survive or escape a forest fire, plant material must either be able to withstand high temperatures or be deeply buried. Rhizomes of lingonberry were killed after 10 minutes at 55° to 59°C. The main part of the subterranean runners grew in the middle or a little below the middle of the humus layer known as mor, which occurs by decomposition in the superficial soil layers instead of its surface. A small proportion of rhizomes in the mineral soil occurred in thick humus layer. The total bud bank ranged from 470 to 950 shoots per m2. Slightly burned plots produced more sprouts than clipped plots that weren’t burned. Sprouting from rhizomes in the upper horizon increased after burning. Improved nutrient status in the soil could explain this phenomenon. If fires were limited to the moss layer, pre-fire coverages were reached within 2 to 4 years. The most severe fires, however, eliminated lingonberry as well as other Vaccinium species. Tolvanen et al. (1995) mapped the recovery of lingonberry after removal of annual branches or ramets in a boreal forest. Recovery proceeded unexpectedly high even after the most severe treatment (100% removal of ramets). When whole ramets were removed the percentage of new growth emerging from basal buds increased. About 42 to 112% of the above ground biomass and 60 to 70% of the coverage were regained after three growing seasons. D. Berry Production The National Forestry Survey did the first research project regarding wild berry production in Swedish forests (Eriksson et al. 1979; Kardell 1980). Production of bilberry, raspberry, and lingonberry were analyzed on 44,000 sites during three years (1974 to 1977), and berries were counted and weighed. Lingonberry occurred on 1.2 million ha out of 23.5 million ha of productive Swedish forests. The occurrence of lingonberry varied little with the density of the forest. Coverage was about 5% on clear-cut areas and in young forests, while about 7% were

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recorded for old established forests. In northern Sweden, lingonberry was more common than bilberry and raspberry. In this region, coverage was highest (8–9%) in stands of Scots pine, while it was 4% for Norwegian spruce. Productivity was relatively high on clear-cut areas, in young and very old forests, whereas productivity was low on 80 to 90% of the forests. Most of the berries (70–80%) were produced in the forests of northern and central Sweden. Between 70 and 80% of the total production was considered available for harvest. The National Forest Survey for 1978 through 1980 investigated the occurrence of cloudberry, small-fruited cranberry, and lingonberry (Kardell and Carlsson 1982; Kardell 1986). The inventories involved both forests and bogs. The latter covered 5.1 million ha with average lingonberry coverage of 1.1%. The results indicated that lingonberry preferred mineral soil, where the coverage increased from south to north, while the opposite occurred in bogs. A negative relationship was reported between lingonberry coverage and site elevation. The total mean berry production during this investigation was 209 million kg yearly. Only 4% of this yield was from bogs. The yield as a whole was widely distributed over the country. The highest yields (13.8 and 17.3 kg /ha) were noted from forests and bogs in central Sweden. Graff (1991) calculated the total Norwegian lingonberry production, which varied between 44 and 115 million kg per year. Raatikainen (1988) and Raatikainen et al. (1984) noted that variation in berry production depended on different factors, such as tree-canopy density and lingonberry coverage in Finland. The total annual production was estimated at 180 to 200 million kg, with an average yield of 8 kg/ha in the forests. About 80% of these berries were considered harvestable. However, lingonberry yield varies from one year to another due to night frost in June (Kardell and Carlsson 1982). About 50% of the flower buds, flowers, and green fruits are killed at temperatures below –1.5°, –3.1°, or –3.5°C, respectively (Teär 1972). There was also an association between snow cover and yield. Lingonberry buds were unable to withstand minimum temperatures, which varied between –25° and –32°C in January (Raatikainen and Vänninen 1988). After the exceptionally cold winter of 1985, highest yields were harvested from plants that had overwintered below a thick snow cover. The annual production of lingonberry in the Nordic countries can be estimated at 500 million kg, of which 80% is considered available for harvest. However, only 2 to 11% of the total production is collected. Through a mail survey, Hultman (1983) found that 38% of the Swedish population picked 15 L of lingonberry per person annually. People in northern Sweden gathered more berries than those in the south. The per-

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centage of berries picked was 28% in the south and 7% in the north, i.e., 11% of the production was harvested in Sweden. Only 3 to 5% of the total lingonberry production was picked in Norway (Graff 1991), and 2 to 7% of lingonberry production was harvested in Finland (Saastamoinen 1981). During the 1970s the average picker gathered 14 L per year (Hultman 1983). Saastamoinen and Lohiniva (1989) studied lingonberry picking in the Rovaniemi region of Lapland. About 70% of the households participated in the harvest. The total quantity of wild berries collected was 2 to 4 times larger than the national average. This is considered typical for rural communities in northern, eastern, and central Finland. Approximately 86% of the families in five communities in central Finland picked lingonberry and 9 to 44% of the total production in this area was gathered (Rossi et al. 1984). E. Effects of Forestry Management As lingonberry picking is a popular family pastime, there is an apprehension that modern forestry could threaten berry production. Consequently, the section of Environmental Forestry of the Swedish University of Agricultural Sciences (SLU) started a series of experiments at 27 testing sites across Sweden in 1976 with the aim of studying the relationship between silviculture and the development of grand cover vegetation. The original status of the sites was mapped during the first year. Different treatments that included thinning, clear cutting, fertilization, and soil disruption were carried out. Results from these experiments were reported after 5, 10, and 15 years (Kardell and Eriksson 1983, 1990, 1995). Lingonberry biomass was reduced by 15 to 20% directly after thinning of the overstory trees. Recovery was slow over time but thinning was positive for vegetative lingonberry development and, after 15 years, the coverage was slightly higher than in the control plots. Additionally, thinning resulted in a 3- to 4-fold yield increase. Clear cutting also led to decreased lingonberry biomass in the beginning, but after 15 years the plants had completely regained their positions. Lingonberry thus has good competing capacity on clear cuts and takes advantage of extra light. Clear cuts may be exposed to spring frosts, but in general these are good areas for berry production. On average a 4-fold yield gain was recorded. Development after fertilization with 150 kg N/ha was negative, as lingonberry coverage and yield decreased by 12 and 45%, respectively, following fertilization. Soil disruption caused a 20% biomass reduction after clear cutting and reduced berry production over 14 years by 39%.

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During a six-year period, removal of branches in clear-cut areas doubled yield, while stump removal had the opposite effect (Kardell and Wärne 1981; Kardell 1992). Pinus contorta forests allow less light penetration and lingonberry yields were 46% lower in those forests than those growing with P. silvestris (Kardell and Eriksson 1989, 1990). Kardell and Eriksson (1990) predicted that lingonberry production in Sweden could decrease by 10%, owing to soil fertility, mechanical soil disruption, and the widespread introduction of Pinus contorta. In contrast, minimal use of fertilizers and machinery, thinning, and clear cutting followed by dead branch removal appears to benefit lingonberry growth and production. Flavor of berries harvested from plants grown in fertilized plots was judged by a test panel to be slightly inferior to those plants grown in unfertilized plots (Kardell et al. 1981; Åkerstrand et al. 1988). The berries from fertilized plots had higher N levels during the year of treatment. Furthermore, molds and yeast were common in the samples from fertilized plots, resulting in reduced storage life. However, the effects of fertilization were considered small in comparison with those associated with environment and post-harvest handling. F. Experiments in Natural Habitats A prerequisite for successful field planting is the knowledge of the conditions prevailing in natural habitats (Teär 1972; Lehmushovi 1977a). This point was considered for ecological research by Teär as well as by Finnish researchers, who focused their investigations on wild stands from 1968 to 1976. Soil characteristics, temperature, light, and moisture were investigated to elucidate the factors promoting vegetative and reproductive development. The effects of fertilizer applications were also studied. One of the most important factors influencing yield is the weather during flowering. Frost, severe drought, or abundant rain may lead to 60 to 100% loss of buds, flowers, and unripe berries (Lehmushovi 1977a). In the years with favorable weather, this loss ranged from 30 to 60%. Crosspollination by insects and good light conditions are also essential for fruit set. The results of fertilizer trials in natural populations in forests are contradictory. A 2- to 3-fold gain in fruit yield after fertilization has been reported (Lehmushovi and Hiirslami 1972; Lehmushovi 1977a), but if the natural habitat included competing grasses and broad-leaved herbs, there was no benefit. Teär (1972) found a lower percentage of flowering shoots in fertilized plots than in unfertilized plots. The fertilizers did not

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affect the number of flower buds per shoot; however, their use resulted in slight increase in the production of rhizomes, new shoots, and leaves.

V. HORTICULTURE A. Propagation 1. Plants Collected Directly from the Wild. The cheapest way to propagate lingonberry is by digging up plants from natural habitats. Plants may be dug early in the spring, before growth starts, or late in the autumn when it has ceased. If homogeneous clumps of lingonberry were chosen and subdivided before planting, 73% survived when there were four plants per division, while only 18% survived when there were 15 plants per division (Teär 1972). Material collected from native stands should be planted with only the shoots above ground, because the lingonberry plants develop roots in the uppermost soil layer. These plants were susceptible to drought and frost heaving due to their fine, fibrous, and shallow root system. Plant survival ranged from 30 to 90% (Öster 1974). Plants could be established more reliably if the plants were first grown in humid peat nursery beds. After three growing seasons, plants that were first grown in nursery beds had better coverage (80%) than those directly planted from the forest (50%), though these coverages were 80% and 90%, respectively, the following year (I. Hjalmarsson, unpublished). After 3 to 5 years with an initial density of 80 to 100 plants per m2, an even cover was achieved with 400 to 600 plants per m2 (Teär 1972). Plant materials from sunny locations were more difficult to establish than those from shaded locations (Öster 1974). Selected plants had stronger sympodial growth, shorter shoots, greater number of leaves, twice as many flower buds per flowering shoot, and 3 to 4 times more shoots per plant than those from native stands (Teär 1972). In a 5- to 6-year-old lingonberry field, more than 1000 flower buds per m2 were observed, while there were only 300 to 500 flower buds per m2 in the forest (Lehmushovi 1975). The major drawbacks with wild plantlets are that they have a poor rate of establishment, are heterogeneous, and are difficult to handle. 2. Shoot Cuttings and Rhizomes. Propagation by shoot cuttings has been successful (Öster 1974; Lehmushovi 1975). The best results were obtained with mature spring and autumn shoots, while soft wood shoots taken during the summer were the most difficult to root (I. Hjalmarsson, unpublished). The shoots required about eight weeks for rooting and the

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most favorable substrate was peat. An average rooting percentage of 85% was achieved when mist and peat beds were used (Lehmushovi 1975). Pre-treatments with auxins also promoted rooting and almost 100% rooting was obtained if cuttings were over-wintered in non-heated greenhouses. The shoots were taken from September to October, placed in moistened peat, and wrapped with transparent plastic. By March or April the plants were ready to be transplanted in the field. The best shoot cuttings of ‘Sanna’ were collected in spring and late summer (Gustavsson 1998). However, cuttings from the current year growth were more uniform than mature shoots. Thus, softwood cuttings taken in late summer were recommended. Rooting was more successful in outdoor plastic tunnels than in the greenhouse for ‘Sanna’ (Gustavsson 1998). Plants from cuttings generally grow well in the field and tend to crop early. However, their rhizomes develop slowly. Plants raised from micro propagation produce rhizomes more easily. Rhizomes can propagate lingonberry, therefore shallowly planting 5 to 10 cm rhizome pieces in boxes with moist peat has been recommended (Öster 1974). One or two shoots normally develop at the terminal end, while roots are produced basically. Best results were achieved during spring and late summer, when 60 to 80% of the material was rooted. Similar results were reported by Lehmushovi (1975), who, nevertheless, concluded that rhizomes were difficult to procure and use for propagation. Lack of strong rhizomes with well-developed buds may result in weak plants. Also, rhizomes are sensitive to drought and therefore impossible to plant directly in the field without first growing in a nursery. 3. Seedling Plants. Lingonberry is easily propagated by seeds. The berry contains many seeds that can be cleaned by pulverizing the berry with water in a blender. Well-developed seeds will sink to the bottom and the rest of the mix is then decanted. Seeds germinate well directly after harvest but can also be cold-stored. About 72% seed germination (in dry seeds that were kept in a refrigerator for six months) has been observed after two weeks in damp sand (Lehmushovi 1975). Results from experiments with whole berries, pulverized berries, or direct seed sowing in the field at Ottarp were not encouraging. The acids in the fruit flesh inhibited seed germination (Karlsson and Malmberg 1974), and seedling plants were very susceptible to drought immediately after sprouting. The most preferred sowing appears to be in boxes with fertilizer-free peat and a top layer of sand during winter (Hjalmarsson 1993). The seeds are not covered because light is necessary

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3. LINGONBERRY: BOTANY AND HORTICULTURE

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for germination, which begins in about three weeks at 20° to 25°C. Seedlings are watered once a week with a complete nutrient solution, given additional light, and then transplanted as soon as they can be handled. After hardening-off at the beginning of July, they are transplanted to the fields. In greenhouse experiments, the number of leaves increased from 5 to 23 after three months, and shoot length from 1 to 7 cm (Hjalmarsson 1977). The development of leaves and shoots was simultaneous. However, the burst of leaves at the terminal end occurred in flushes and 3 to 4 leaves were born at a time, with 10 days between each flush. When the plants had reached 4 cm in height (11 leaves), the first lateral shoots became visible just above the cotyledons. Seedlings grown outdoors produced dense stands; after two years, for each m2 there were 2755 current year shoots, 330 flowering shoots, 430 flower clusters, and 28 m of rhizomes, which accounted for 9.4% of the total biomass dry weight (Hjalmarsson 1977). Lingonberry seedlings also develop rhizomes when they are very young. In addition, seedlings offer a quick way of producing a large quantity of plants, but fruit production could be delayed by 2 to 3 years due to juvenility and seedlings are genetically diverse. B. Frost