Jaime A . Teixeira da Silva Floriculture , Ornamental and Plant Biotechnology

S: VOLUME I Structure, Metabolism, Development, Physiology and Genetics Part 1 Flower growth, development, patterning, senescence and death Page 1. Genetics of floral development and patterning. MP Running, USA ...................................................................................... 1-11 Flowers are valued both for their beauty and their economic importance, as flower parts form the major source of food for both humans and animals. Flowers also present an intriguing model for biological pattern formation: a small group of flower meristem cells gives rise to several different organ types in stereotypical positions in rapid succession. The use of molecular genetics approaches in the study of flower development has led to a series of insights into the biological mechanisms of generating flower patterns. This review focuses on the current knowledge of the genetic basis of flower patterning in Arabidopsis thaliana. Flower patterning requires several steps: the specification of lateral meristems as flowers instead of shoots, the establishment of proper floral organ identity, and the initiation of organs in correct number and positions. Floral meristem identity genes integrate environmental cues to establish a flower program, including the activation of flower homeotic genes, which specify sepal, petal, stamen, and carpel formation. Organ initiation occurs largely independently of organ identity and requires proper control of flower meristem size, proper regulation of cell division orientation, and spacing mechanisms that remain poorly defined. Future challenges include identifying additional genes involved in establishing the flower pattern, along with identifying more genes that act downstream of patterning genes to confer the final size and shape of flower organs across diverse species. 2. The utility of snapdragon (Antirrhinum majus) to study flower development and coloration. N Efremova, R Castillo, C Navarro, Germany ............................................................................................................. 12-24 The flower of the common ornamental snapdragon (Antirrhinum majus L.) and its mutant forms have been subject of study since the middle of the 19 century when its advantages as an experimental system were first recognised. In recent years, Antirrhinum has been one of the plant organisms contributing to the understanding of basic biological processes such as flower initiation and formation. Antirrhinum genetics combined with molecular approaches have enabled the cloning of numerous genes with a role in different aspects of flower development like the control of floral-organ identity, floral symmetry, inflorescence architecture and colouration. Newly developed research tools now allow studies of gene expression profiles during different stages of development and genes can be analysed in their genomic context. Comparison with Arabidopsis reveals conservation and divergence of gene functions leading to the formation of a basic floral structure. Uncovering peculiarities in these processes in different species demonstrates the usefulness of using different model systems to explain natural variability. 3. Molecular mechanisms of hormone functions in flowering. H Yu, T Dhavale, Singapore, S Yang, China ........................... 25-32 Flowering in plants involves several sequential developmental programs from floral transition to floral organ development. In most species, the shoot apical meristems initially generate primordia that develop into vegetative organs, and are reprogrammed to give rise to floral primordia during the transition to flowering. These floral primordia can differentiate into flowers with different whorls of floral organs during flower development. Although striking progress has been made in understanding of flowering mechanisms in recent years, there are still many unanswered fundamental questions. The function of plant hormones (phytohormones) in flowering process is one of these puzzles. The phytohormones, including abscisic acid, auxin, cytokinin, ethylene, gibberellin acid (GA), brassinosteroid, and jasmonic acid, are small molecules affecting a wide range of developmental programs in plants. Some of these phytohormones, such as GA, show significant impact on the control of flowering time and floral organ development. In the last several years, molecular genetic studies in the genetically facile model plant Arabidopsis thaliana have provided significant insights into the molecular basis of various roles of phytohormones in plant development. Here we summarize the recent advances in our understanding of molecular mechanisms of hormone functions in flowering, with the emphasis on the GA, cytokinin, and auxin pathways. 4. Florogenesis in flower bulbs: classical and molecular approaches. MA Flaishman, R Kamenetsky, Israel ....................... 33-43 Flower bulbs belong to numerous botanical taxa and show remarkable diversity with regard to morphology, developmental biology, genetic control and response to the environment. In this chapter we review the current data on morpho-physiological and biochemical aspects of the transition of bulbous plants from the vegetative to the generative phase, the development of reproductive organs from initiation to anthesis, and regulation of these processes by internal and external factors. We also discuss the factors involved in florogenesis with special emphasis on the prospects and future investigations of biochemical and molecular mechanisms of florogenesis in flower bulbs. Elucidating developmental mechanisms in these species may greatly contribute to the regulation of their florogenesis, as well as to the understanding of developmental processes in other higher plants. Molecular characterization of genes involved in flower morphology will help in developing novel floral architectures in ornamental bulbs by classical breeding or by genetic manipulations using transformation systems. In addition, knowledge of bulb periodicity is essential for the control of flowering, while introducing modifications in this periodicity constitutes the basis of the techniques used to promote or delay flowering. 5. The differentiation of perianth morphologies in monocotyledonous plants. A Kanno, Japan .......................................... 44-50 The morphological transition of the first whorl of flowers from petaloid tepals into sepals occurred frequently during the diversification of angiosperms. The Arabidopsis class B genes APETALA3 (AP3) and PISTILLATA (PI), and the Antirrhinum class B genes DEFICIENS (DEF) and GLOBOSA (GLO), are required for petal development in whorl 2, and their homologs have been isolated and characterized from various plants. A recent study of tulip flowers indicates that the morphology of petaloid tepals in whorls 1 and 2 is consistent with the expansion of class B gene expression. To clarify the evolutionary transition between sepals and petals in monocotyledonous plants, we isolated and characterized the class B genes from Agapanthus, Muscari and Tricyrtis, whose perianths consist of petaloid tepals in two whorls, and from Tradescantia, Commelina and Habenaria, whose perianths are differentiated into sepals and petals. Gene expression was examined by Northern hybridization and RT-PCR using dissected floral organs, or by in situ hybridization, and revealed the expansion of class B gene expression in whorl 1 in Agapanthus, Muscari and Tricyrtis, and a lack of DEF(AP3)-like gene expression in whorl 1 in Commelina, Tradescantia and Habenaria species. These results suggest that the DEF (AP3)-like gene expression pattern may be correlated with a morphological transition from petaloid tepals into sepals in the first whorl in monocot flowers. 6. A proteomics approach to the study of distyly in Turnera species. D Khosravi, Canada/USA, KWM Siu, JS Shore, Canada .............................................................................................. 51-60 Distylous species of Turnera are strongly self-incompatible, therefore they provide an excellent system for investigations of the proteins involved in distyly. Distyly is a genetic polymorphism in which there are two self-incompatible but cross-compatible morphs. The longversus short-styled morphs have a reciprocal arrangement of reproductive organs. Little is known of the molecular basis of distyly in any species. Previous molecular investigations of Turnera species identified two proteins that are specific to the transmitting tissue of short styles: a polygalacturonase and an α-dioxygenase. Here we continue to search for proteins that might be involved in distyly, since we have not yet identified the genes that occur at the S-locus and no reliable candidate proteins from pollen have been identified to date. Comparative investigations were undertaken to detect and identify morph-specific proteins from styles and pollen using a proteomics approach. Using 2-D PAGE, and mass spectrometry, two new proteins were identified that are specific to short styles: a putative cysteine protease and a putative β-expansin. Neither the cysteine protease nor the β-expansin was detected in mutants or in self-compatible homostylous species of Turnera. They are absent in short styles sampled one day prior to anthesis. We speculate upon the function of these two proteins in the selfincompatibility system of short-styled plants. We also discovered a number of proteins unique to anthers of the longor short-styled morph. We have not yet identified these proteins, but this remains an important aspect of our future work. 7. Mutations affecting corolla symmetry in sunflower. M Fambrini, D Bertini, G Cionini, V Michelotti, C Pugliesi, Italy ........... 61-70 The inflorescence of sunflower (Helianthus annuus L.) is heterogamous with zygomorphic ray flowers located in the outermost whorl of the head and actinomorphic disk flowers arrayed in arcs radiating from the center of the head. The ray flowers are sterile with large flat ovaries with no ovules; disk flowers are hermaphrodite, carrying both male and female organs. Two mutants with altered corolla symmetry are known in sunflower: the first, named Chrysanthemoides (Chry) is characterized by a shift from the polysymmetric corolla of disk flowers into a monosymmetric-like corolla; the second, named tubular ray flower (turf), shows a shift from the zygomorphic corolla of ray flowers into a nearly actinomorphic tubular-like corolla. We report in detail the morphological floral features of Chry and turf, demonstrating that both mutations also affect the development of stamens and carpels. Most disk flowers found in the peripheral whorls of Chry heads showed drastic reduction in stamen length, as well as the absence of ovules, and developed an unbranched style. In contrast, tubular-like ray flowers of turf achieved the ability to differentiate both fertile stamens and ovules. Homeotic transformations were also identified in the tubular-like ray flowers of turf, affecting both filaments and anthers that displayed petaloid-like traits. Notably, the turf mutation was not completely stable, occasionally reverting to nearly “wild-type” phenotype. Our results point to a primary role for TURF and CHRY in the programming of the corolla symmetry and suggest a key interaction of both genes with floral organ identity genes. 8. From the wild to the market: breeding of floral size and architecture in ornamentals. J Weiss, E Cano-Vicente, M Egea-Cortines, Spain ........................................................................................................ 71-75 The enormous number of ornamental plants present on the market today is the result of a continuous effort by private companies and individuals to obtain new plants that are attractive and have a long life. In spite of the obvious diversity caused both by choosing different plants and looking for new flowers, the fact is that floral development is a very well conserved developmental program. In this review we will give a perspective of three aspects that have been fundamental for the current status of ornamentals as commodities. First we will describe the general floral program, changes that have happened between some ornamentals and their wild relatives, and finally we will give a short description of some of the methods that are used to obtain new varieties. 9. Male gametophyte development and function. D Honys, D Reňák, Czech Republic, D Twell, UK ............................................ 76-87 Male gametophyte development in higher plants is a complex process that requires the coordinated participation of various cell and tissue types and their associated specific gene expression patterns. The male gametophytic life cycle can be divided into a developmental phase leading to the formation of mature pollen grains, and a functional or progamic phase, beginning with the impact of the grains on the stigma surface and ending at double fertilisation. Pollen ontogeny is also an excellent model in which to dissect the cellular networks that control cell growth, polarity, cellular differentiation and cell signaling. Here we present an overview of important cellular processes in male gametophyte development and recent advances in genetics and genomic approaches that are advancing the field significantly. Genetic approaches have identified a growing number of gametophytic mutants affecting discrete steps during the developmental or progamic phases that are now beginning to uncover some of the key molecular processes involved. With recent technological advances pollen transcriptomic studies now provide the first genome-wide view of male reproductive cell development in Arabidopsis These studies reveal at least two successive global gene expression programs and the identity of a large number of male gametophyte-specific genes and putative transcriptional regulators. Transcriptome analysis has revealed a striking overrepresentation of cell wall metabolism, cytoskeleton and signaling genes in preparation for the progamic phase. This quantum leap in gene-centered knowledge highlights the functional specialization of this pathway and offers many new opportunities for the dissection of cellular processes that control male reproductive success. 10. The role of polyamines in relation to flowering senescence. N Bagni, A Tassoni, Italy ............................................................ 88-95 Cut carnation (var. Reiko) and gerbera (var. Lisa) flowers were held under standard environmental conditions and treated with different polyamine (PA) concentrations, either by spraying directly on the petals or by supplying them in the vase water. In carnation, the greatest delay of senescence was evidenced with 10 mM spermidine (spd) in the watering solution (10 mM spd-V), while no significant effect was obtained by spraying the flowers. PA levels (free, PCA-soluble and PCA-insoluble conjugated) were determined in carnation control and 10 mM spd-V treated flowers. Starting from day 3, spd was absorbed from the watering solution and accumulated in the petals both in the free and PCA-soluble fractions. Putrescine (put) levels also increased with 10 mM spd-V treatment. Total anthocyanin, DNA, protein content and protease activity were determined in control and 10 mM spd-V supplied carnations. Spd was shown to stabilise and retard the degradation of DNA. In gerbera, the best results were obtained with 0.1 mM spd spray and with 10 mM spd supplied in the watering solution. Both these treatments prevented the bending of the stem. Free and PCA-soluble conjugated PAs were determined in control, 0.1 mM spd and 10 mM spd-V gerbera petals. In contrast to carnation, only 0.1 mM spd spray induced an increase in endogenous free and PCA-soluble spd and put content, while endogenous PA levels did not vary in 10 mM spd-V flowers. No evident difference in total DNA degradation was detected in control and treated gerberas. 11. Abscisic acid and cut flower senescence. A Ferrante, P Vernieri, Italy ................................................................................... 96-100 The keeping quality of cut flowers, during the postharvest stages, is very important in the global market, where floricultural items must be extremely competitive. Postharvest physiological analyses of senescence point to plant hormones playing important roles in the promotion or inhibition of flower and leaf senescence. Among them, ethylene and abscisic acid (ABA) are the most important. Ethylene has been extensively studied and much information is available for modulating senescence in ethylene-sensitive flowers. In contrast, the role of ABA during cut flower senescence has been wrongly assigned secondary importance. In this chapter, the effect of ABA on leaf and flower senescence has been briefly reviewed in select species, with a brief discussion of ABA and gene expression. 12. Genetic control of floral abscission. RB Aalen, MA Butenko, G-E Stenvik, NM Tandstad, Norway, SE Patterson,USA ................................................................... 101-108 Abscission is a process whereby plants shed their organs such as leaves, flowers or floral organs after having served their function. Arabidopsis thaliana is an ideal model for investigating the process of floral organ abscission since it is not obscured by other processes like senescence. Abscission is a program of cell separation that takes place between layers of small cells differentiated into an abscission zone at the base of the organ to be shed. Investigation of Arabidopsis mutants has shown that many of the growth regulators including ethylene, auxin, methyl jasmonate and gibberellic acid affect floral organ abscission. For example, mutations in ethylene receptor genes and other genes in the ethylene response signal transduction pathway lead to delayed floral organ abscission. However, observations on the delayed in abscission (dab) mutants and the inflorescence deficient in abscission (ida) mutant indicate that ethylene independent pathways regulate abscission, as these mutants have normal sensitivity to ethylene. Other mutants with delayed abscission have been identified; however, many have additional phenotypes not associated with abscission. Thus, we have selected to present an in depth discussion on ida. IDA encodes a small putative peptide ligand, and genes and transcripts encoding IDA-like (IDL) proteins have been identified in Arabidopsis and other dicot and monocot plant species. Promoters of IDL Arabidopsis genes can direct reporter gene expression in the floral organ abscission zone, at the base of the floral pedicel and in the columella cells of the root cap, suggesting involvement in abscission or abscission-like processes, like root cap sloughing. Overexpression of IDA leads to shedding of organs that normally are not abscised in Arabidopsis, e.g. the pedicel, indicating that ectopic expression of IDA can activate preformed abscission cells. Preliminary experiments indicate that the ida phenotype can be recreated by RNA interference. Scientists’ knowledge from studies on IDA and other genes regulating floral abscission may be utilized to modify floral crops, and we will present these applications. In summary, these recent insights leave the community poised to modify abscission in many ornamentals. 13. Molecular basis of ethylene signal transduction and control of abscission of flowers in Delphinium. S Abe, S Kuroda, Y Hirose, E Davies, Japan ............................................................................................................................. 109-123 Florets of plants belonging to the genus Delphinium are known to undergo severe senescence a few days after harvesting the cut flowers, as well as in response to environmental cues such as rainfall and pollination. An ethylene sensing mechanism is thought to be involved. Recently, the molecular basis of the ethylene signal transduction pathway has begun to be uncovered. Ethylene Response Sensor (ERS)type ethylene receptors are likely to be involved in the senescence of florets. ERS1 for the ethylene sensor gene and CTR1 for its downstream factor have been discovered in Delphinium and are likely to be involved in the abscission of florets. This chapter describes the structure, expression, and possible functions in abscission of florets in Delphinium. We postulate that floret abscission in Delphinium is caused by elevated levels of the ethylene receptor, ERS1, and that these will be influenced by endogenous ethylene generated after cutting the stem. This ethylene signal transduction pathway is crucial in the shelf life of cut flowers and in garden longevity of intact flowers against environmental cues, and poses a way to enhance them by either conventional methods or modern biotechnology such as transgenic plants. 14. Ornamental cut flowers: physiology in practice. JA Teixeira da Silva, Japan ....................................................................... 124-140 Ornamental plants occupy an important part of society, culture, religion, and economy. Cut flowers from several ornamentals bear both a “butter and blade” symbolism, but independent of the viewpoint, there is no doubt that the quality of longevity is of great importance. In this review, the current status of postharvest technology applied to cut flowers and foliage is discussed. Basic considerations of the physical, biochemical and genetic mechanisms underlying some of the processes central to cut flower and foliage deterioration, such as abscission, senescence and programmed cell death, and some of their cross-talk mechanisms have been included. Moreover, through examples, solutions to increasing longevity through improvement of cultural practices and sanitation and through genetic engineering are covered, providing practical solutions to the global cut flower market. Long live the flower! 15. Programmed Cell Death in plants and flowers. GE Drury, P Gallois, UK ............................................................................... 141-156 Programmed Cell Death is a process common to all multicellular organisms so far studied. Over the last thirty years it has been very well characterised in mammalian systems, as well as in organisms such as the nematode, insects and amphibians. Cell death in plants remained poorly understood in comparison until research in recent years has led to Programmed Cell Death (PCD) becoming a more eminent research area. Plant PCD occurs at many stages during development, and can occur in response to environmental conditions. Some of the better characterised occurrences of PCD include xylem formation, senescence and the hypersensitive response to pathogens and studying the occurrence of such processes will continue to bettering our understanding of a biologically imperative and prevalent process. Research in plant PCD has been heavily influenced by findings in mammalian PCD, but has yet to fully elucidate the events that culminate in PCD. The extent that PCD is conserved between the two systems is likely to be limited, and although evidence exists which implies there are similarities, the emergence of plant specific mediators has seen a shift in research focus. 16. Lace plant: a novel system for studying developmental programmed cell death. AHLAN Gunawardena, C Navachandrabala, Canada, M Kane, USA, NG Dengler, Canada ................................................... 157-162 The submerged aquatic plant, lace plant (Aponogeton madagascariensis, Aponogetonaceae) has a highly unusual leaf form, in which holes perforate the leaf blade in a regular lattice-like pattern. These perforations are formed by programmed cell death (PCD) shortly after leaf emergence from the apical region of the corm. Because PCD occurs in predictable locations in relation to the leaf vein pattern and at a known stage of leaf expansion, and because the thin, aquatic leaves are tractable to live-imaging microscopy, lace plant is an attractive model system for studying developmentally-regulated PCD in plants. One limitation of carrying out developmental and physiological analyses is the unknown effects of associated micro-organisms found in aquarium culture. Therefore, a primary objective of this study has been to establish and maintain axenic cultures of this species. We first characterized the formation of perforations through PCD in axenic cultures and found that the PCD process in culture did not differ significantly from that previously described. Since ethylene is an important component of PCD signaling pathways during many forms of plant PCD and also functions in growth regulation of other submerged aquatics, we also carried out experiments using 0.05 mM AgNO3 as an inhibitor of ethylene perception and found a significant reduction in the number of perforations formed per leaf under treatment conditions compared to control plants. While preliminary, these experiments indicate that axenically cultured lace plant has considerable potential as a model system for the study of developmentally-regulated PCD in plants. 17. In vitro culture of the fern Platycerium bifurcatum as a tool for developmental and physiological studies. M Camloh, Slovenia ...................................................................................................................................................................... 163-170 The staghorn fern, Platycerium bifurcatum is an epiphytic homosporous fern. Different phases of its life cycle – spores, gametophytes and various parts of sporophytes – constitute useful systems for developmental and physiological studies. The morphological simplicity and simple culture requirements of the gametophyte enable various aspects of growth and development to be observed. Its great morphogenetic potential when cultured in vitro enables study of morphogenesis, including adventitious bud development and apospory. Direct adventitious bud development occurs on the whole surface of the excised juvenile leaf without use of any growth regulators. Morphogenesis was also obtained on excised bud scales. Apospory, the formation of gametophytes from sporophytic tissue without meiosis or sporulation, was induced on wounded juvenile leaves regularly and at a high frequency. In this article we provide an overview of the research on P. bifurcatum using in vitro techniques. Spore germination, gametophyte development and morphogenesis are discussed with respect to its potential use in plant development research, as well as to point out some similarities between ferns and seed plants. 18. Zinnia elegans is an excellent model for xylogenesis: in vitro and in planta. E Pesquet, Sweden, A Jauneau, D Goffner, France ................................................................................................................... 171-178 Vascular tissues play an essential role in plant growth, allowing for transport of raw sap via the xylem from roots to leaves and elaborated sap by the phloem from source to sink. The formation of xylem, or xylogenesis, is an integral part of the growth of each organ and allows vascular continuity in the organism, interconnecting organs and establishing irrigation of the entire plant. Xylem is a complex tissue comprised of several cell types (parenchyma, conducting cells and fibers), each formed by specific developmental processes and each with distinct morphological characteristics. One interesting cell type is the conducting cell, called the tracheary element (TE), which, in order to be functional, must undergo secondary cell wall formation and programmed cell death (PCD) processes. This results in the formation of an interconnected series of strengthened hollow tubes suitable for raw sap conduction. One of the major difficulties in studying TE formation in planta is that very few cells actually differentiate at a given moment and a given time. In order to study TE formation, an in vitro TE differentiation system has been established from isolated mesophyll cells of an ornamental asteraceae, Zinnia elegans. This system is characterized by a high rate of differentiation and the semi-synchronous nature of the morphological changes that occur. In order to compare TE formation in vitro and in planta, we provide herein i) a detailed anatomical description of the vascular system of Zinnia elegans, ii) a morphological comparison of TE differentiation in vitro vs. in planta and iii) gene expression localization both in planta and in vitro. The morphological and gene expression data provided in this chapter allow us to integrate in vitro-derived information in the context of the whole plant. Part 2 Cellular mechanisms 19. The nuclear and chromosomal architecture of plant cells: development and dynamics. S Fujimoto, S Matsunaga, K Fukui, Japan ................................................................................................................................. 179-184 In eukaryotic nuclei, genomic DNA is compacted as a result of interaction with various proteins. A hierarchical model is most commonly used to elucidate DNA organization in the nucleus. First, DNA is wound around core histones to form a nucleosome then interaction between linker DNA and histone H1 generates a heterogeneously folded 30-nm-long fiber structure. This fiber structure is organized into a 5-200 kb-long loop, which attaches to the nuclear matrix or scaffold, and during mitosis, forms the higher structure necessary for chromosome formation. This review is concerned with the physical properties of plant nuclei and features of proteins related to plant nuclear and chromosomal architecture. Some chromatin proteins are conserved among eukaryotes; however, many proteins related to nuclear structure are not. Therefore, identification of plant-specific proteins using visual screening such as random GFP fusion might contribute to our understanding of the specific characters of plant nuclei. 20. Mechanisms of cytokinesis in flowering plants: new pieces for an old puzzle. JM Seguí-Simarro, Spain, LA Staehelin, USA ............................................................................................................................. 185-196 Cell division is the most distinctive stage of the cell cycle of eukaryotic cells due to the major changes in cell architecture that accompany the cell dividing process. Progress in plant cell division research during the past 130 years has been driven to a significant extent by improvements in light and electron microscope techniques. Most recently, improvements in cryofixation and freeze-substitution specimen preparation methods together with the use of dual-axis electron tomography techniques for visualizing the cryofixed samples has led to new structural and functional insights into the complex process of cell plate assembly as well of the associated changes in the phragmoplast cytoskeletal system. The main purpose of this chapter is to summarize the contribution of 3-D electron microscopy to the understanding of the mechanisms underlying cytokinesis in flowering plants. This information also provides an improved framework for interpreting light microscope observations of living cells, and for formulating new molecular hypotheses of plant cell cytokinesis. 21. Plant chimeras. J Wegner, France .............................................................................................................................................. 197-202 This review chapter covers the origin of chimeras, explains the conditions for their existence and the reasons for their changes. The most common forms of chimeras and their features are described, and commonly used terms are explained. Furthermore, analytical methods are provided allowing the identification of a given plant as a chimera and to determine its structure. Finally, the impact chimeras may have on plant breeding is described, as are ways of making use of them when creating new varieties. 22. Plant peptide hormones, from defense to pollen self-incompatibility, cell fate and development: small peptides as signaling molecules in plants. DS Moura, MC Silva-Filho, Brazil ............................................................. 203-209 The first peptide hormone isolated from plants was an 18 amino acid peptide called systemin. Systemins are found in several Solanaceae species and are involved in the signaling transduction pathway responsible for the local and systemic production of defense proteins against herbivorous insects. Since the discovery of systemin, several other peptides involved in a diverse range of physiological processes have been identified in plants. Phytosulfokines (PSKs) are 5-amino-acid sulfated peptides initially isolated from suspension culture cells of asparagus and later from several other species. PSKs are mitogenic factors that have been implicated in cell proliferation and differentiation although their role in vivo is still unclear. The self-incompatibility (SI) system found in brassica is controlled by the S-locus that contains among others, a gene that codes for the S-locus cysteine rich protein (SP11/SCR). The SP11/SCRs are peptides of 47-60 amino acids in length that after interacting with their receptors on the stigmatic papilla cell membrane trigger the SI response through a signaling pathway that involves an S-locus glycoprotein, an arm-repeat protein and a recently identified membrane-anchored protein kinase (MLPK). The Arabidopsis mutant clv3 is defective in a gene encoding a 79 extracellular signaling peptide named CLAVATA3 (CLV3). The CLV3 signaling pathway determines cell fate in the shoot apical meristem. RALF, a 5kDa secreted plant peptide, has been isolated from several plant species and cDNAs coding for the RALF precursor have been found in all plant tissues throughout the plant kingdom. RALF exogenously applied to germinating seeds of tomato and Arabidopsis was able to inhibit root growth and the peptide is though to have a role in a basic cellular process. A review of the recent findings in this developing field in plant science highlighting the role and structure of the peptide ligands and the signaling pathways that are initialized by them will be presented herein. 23. Mitochondrial somatic genetics and homeotic effects on flower morphology. RJ Rose, JT Fitter, DW McCurdy, MB Sheahan, Australia ........................................................................................................ 210-215 Homeotic floral structure, notably carpelloidy (stamen feminization), caused by alteration of the mitochondrial genome is a morphological manifestation of cytoplasmic male sterility (CMS). Mitochondria, which are usually maternally inherited are characterized by a recombinationally active genome and by being a dynamic organelle, undergoing fusion and fission. It is these latter phenomena that contribute to mitochondrial genomic changes that in turn lead to carpelloidy. There are three ways homeotic changes due to mitochondria can be induced: by mutation, by alloplasmic effects as a result of sexual hybridization and backcrossing, and by somatic hybridization or cybridization. All three ways are influenced by the multipartite nature of the mitochondrial genome, the recombination propensity of the mitochondrial genome and the fusion and fission of mitochondria to create novel, chimaeric genes and operons that produce “male sterilizing factors”. All three ways are also influenced by nuclear-cytoplasmic relationships, both in affecting the mitochondrial genome through nuclear restorer genes and in responding to the consequences of the mitochondrial genomic effects which influence nuclear homeotic genes. This interplay within the mitochondrial population and between mitochondria and nucleus probably acts to prevent deleterious mutations, but with crosses between species or wider somatic hybridization the effect is to produce an adult that cannot produce pollen, thereby acting as a speciation barrier. As a consequence, however, fascinating floral morphologies are produced. 24. Genome structure and gene expression in polyploids. SC Pessino, LG Martelotto, Argentina ........................................... 216-223 A raise in the number of total genomic complements in the nucleus or polyploidization is a common event in the evolutionary history of most angiosperms. New phenotypes often arise with polyploid formation and determine their success in nature or their further selection for use in agriculture. Enormous progress has been achieved in the last few years regarding the understanding of the molecular mechanisms concerned with polyploidization. It was shown that while in a number of species it appears to involve extensive alterations of the progenitor genomes, full additivity associated to genetic and epigenetic stasis is observed in others. Genomic changes are usually followed by a refined gene expression re-patterning which have most probably an adaptive significance. It could involve dosage-regulation, altered regulatory interactions and both rapid and long-term genetic and epigenetic changes. The objective of this chapter is to summarize the principal mechanisms leading to genome alterations and novel forms of gene expression in polyploid plants and briefly analyze their evolutionary significance. 25. Sex chromosomes in plants. R Hobza, B Vyskot, Czech Republic .......................................................................................... 224-235 A majority of plant species are cosexuals forming male (stamens) and female (pistils) sexual organs in each flower. About 5% of species are strictly dioecious and form unisexual flowers, either male or female, on different individuals. A high number of plant species represent intermediate stages, i.e., different forms of flowers are present on one individual (e.g. monoecy) or sexually different individuals occur in populations of plants (e.g. gynoecy). It is well documented that these sexual forms also represent intermediate steps in the evolution of sexuality. Similarly as in animals, there are two basic mechanisms of sex determination in plants: genetic and environmental (hormonal). Among the dioecious species, only a few of them have evolved heteromorphic sex chromosomes, especially white campion (Silene latifolia) and common sorrel (Rumex acetosa). In these two classical species, different sex chromosome-based mechanisms have been described: white campion has the male dominant chromosome Y (the mammalian type of sex determination), while in sorrel the sexuality is controlled by a ratio between the number of X chromosomes and the number of autosomal sets (the drosophila system). Recent molecular analyses show that the plant sex chromosomes are evolutionarily much younger compared with the sex chromosomes in animal species. This fact makes them the optimum models to study early stages of sex chromosome evolution. 26. A wonder plant-microbe interaction between white campion and anther smut. W Uchida, Y Kazama, S Matsunaga, S Kawano, Japan ............................................................................................................. 236-242 Infection with the smut fungus Microbotryum violaceum induces the development of male organs in female flowers of the dioecious plant Silene latifolia. The development of stamen primordia is prematurely arrested in healthy females, whereas in infected females it is derepressed, resulting in smutted anthers filled with fungal teliospores instead of pollen grains. Light-microscope analyses revealed that the derepressed stamens of infected females formed anther wall layers similar to those of healthy males. At this stage, the floral homeotic Bfunction gene, SLM2, was expressed both in the stamen primordia of healthy males and in the derepressed primordia of infected females, but not in the arrested primordia of healthy females. SLM2 expression was induced in female S. latifolia by fungal infection, independently of the presence of the Y chromosome. To investigate the host-parasite interactions, electron-microscopic analysis was performed following fixation by a high-pressure freezing method. Parasitic fungal hyphae were observed throughout all organs in the young floral buds of infected plants. No morphological change in the fungus was found before or after the derepression of stamen primordia in infected females. The fungi were often observed adjacent to dying sporogenous cells of infected female anthers. We found an increasing number of dead and dying sporogenous cells, among which the fungus formed initial teliospores. The fungus accelerates cell death in the anther and utilizes constituents of dead host cells to form mature teliospores. 27. Integrating knowledge of transcription factors and cell engineering for modelling Catharanthus roseus: prospects and bottlenecks. S Hedhili, France, PK Pati, India, P Gantet, France ..................................................................... 243-252 In Catharanthus roseus, studies on flavonoid as well as terpenoid indole alkaloids biosynthetic pathway holds immense potential looking at its ornamental and important medicinal properties. In general, studies on flavonoid pathway provide us the basis to realise that transcription factors are more promising and efficient molecular tools to engineer plant secondary metabolite pathways. Modelling plants with the knowledge of transcription factors not only helps in coordinated regulation of gene expression and cellular differentiation involved in the biosynthesis of a metabolite but also provides tremendous flexibility in bypassing the requirement to characterize different biosynthetic pathways at the biochemical or molecular level. The present chapter discusses the concept of transcription factor and regulation of key genes of terpenoid indole alkaloid (TIA) biosynthesis pathway in C. roseus. The need for functional interactions and determination of an efficient combination between transcription factors, genes and promoter sequences is also highlighted. For effective manipulation of C. roseus integration of the knowledge of transcription factors and efficient transformation/regeneration protocols is highly essential. The chapter highlights the various approaches, current status and major bottlenecks of genetic manipulation in C. roseus. Development of a new strategy to overcome the existing blockade in modelling C. roseus will greatly facilitate in improved production of TIAs and thus could be a boom to the pharmaceutical industry. 28. Glimpse into mechanisms of signal transduction in plant cells: role of non-receptor protein tyrosine kinases. A Dash, India ................................................................................................................................................................................. 253-255 Two orders of green alga (Cladophorales and Charales) were investigated for the presence of protein tyrosine kinase activity. Proteins of 70 and 85 kD were found to be tyrosine phosphorylated in Cladophora fracta, with an additional phosphorylated band evident at the 120 kD region in Chara vulgaris, suggestive of the presence of putative tyrosine kinase activity in these algal species. A 70 kD protein was immunoprecipitated from both species using a polyclonal antibody against non-receptor protein tyrosine kinase Syk. These observations supported the presence of Syk-like kinase in the green algal species, which could have an important role in algal physiology. Parallels to signal transduction mechanisms in higher plants are drawn. Part 3 Light, vernalization, clocks, rhythms and movements 29. Plant photoreceptors and the photoperiodic induction of flowering. BL Montgomery, USA ............................................... 256-262 Plant photoreceptors perceive light and transmit the information to downstream components that regulate the growth and development of flowering plants throughout the plant life cycle. Three major classes of photoreceptors are active in flowering plants, including the red/far-red absorbing phytochromes, blue-light-absorbing cryptochromes and phototropins, and UV-B absorbing photoreceptors. The phytochromes and blue light photoreceptors have defined impacts on the photoperiodic induction of flowering in higher plants. This chapter will review the effects of phytochromes and blue light receptors, and the downstream components that they control, on the photoperiodic induction of flowering. 30. Thermoperiodic control of shoot elongation and involvment of gibberellins. J-Anders Stavang, R Moe, JE Olsen, Norway ............................................................................................................................ 263-267 Stem elongation in many species is affected by the relationship between day (DT) and night temperature (NT). When plants are grown at the same average diurnal temperature, stem elongation increases with an increase in difference (DIF) between DT and NT, from a negative to a positive value. A negative DIF treatment (low DT and high NT) is a tool to produce compact flower plants and vegetable seedlings with short internodes without a delay in production time in commercial plant production in greenhouses. Also, in a number of flowering plant species, a short temperature drop of 2-4 hours in the beginning of the light period is efficient and commonly used to reduce stem elongation. Exogenous gibberellins (GA) stimulate stem elongation under negative DIF in a number of species. Also, negative DIF treatments and short diurnal temperature drops significantly reduce the level of active GAs as compared to a high DT/low NT regime or constant temperature. Using pea as a model system, we have shown that transcriptional control of a GA-deactivation gene, GA2-oxidase2 (PsGA2ox2) is a key component in mediating thermoperiodic regulation of GA1 levels and stem elongation. Inactivation of GA1 by 2-oxidation apparently results in the reduced GA1 levels and reduced stem elongation under negative DIF compared to zero and positive DIF. Also a temperature drop of 2 h in light increased the expression of PsGA2ox2, while a temperature drop in darkness had no effect on expression of GA-deactivation genes, but instead slightly stimulated expression of three GA-biosynthesis genes in pea, NA, GA20-oxidase1 and GA3-oxidase1. The observed expression patterns contribute to explain why stem elongation is more restricted by temperature drops in light than in darkness and suggest that GA-metabolism is involved in plant acclimation to low temperature both in light and darkness. 31. The rhythms of circumnutation in higher plants. A Charzewska, Poland ............................................................................... 268-275 Circumnutation is a rhythmic, rotary movement of elongating plant organs, such as stems, tendrils and roots. The tip of a plant organ describes ellipses, or circles, or pendulum-like movements that can alternate between the clockwise and counterclockwise direction. Circumnutation is caused by a turgor wave rotating around the stem that drives the helical, acidic growth of the stem. Although the basic rhythm of the movement is ultradian, circumnutation is also found to be modulated in a circadian and infradian way. Circadian modulation is brought about by the plant biological clock that drives circumnutation rhythms even in the absence of environmental cues, at least for several days. The role of the clock is also visible in the phenomenon of anticipation of light/dark cycles by rotating plants. Infradian modulation of circumnutation acquires a regular rhythm of the movement activity every several days. It is hypothesized to be correlated with stem modular growth. Circumnutation is a universal and unique oscillation in higher plants. The present chapter contains a review of recent studies executed on sunflower, Arabidopsis, rice and bean plants. The movement physiology based on recent studies is also discussed. 32. The use of photoperiodic lighting in floriculture in Mediterranean conditions: Gypsophila paniculata. J Lopez, A Gonzalez, Spain ......................................................................................................................................................... 276-281 Gypsophila paniculata is used as a cut flower. Its beautiful inflorescence (panicle) is formed by many white or pink coloured, small flowers. This is a species native to the Mediterranean, which means that it is suited to intermediate environmental conditions for its natural growth, and although, being a long day plant, it needs a great number of light hours to bloom. These plants show excellent vegetative and floral development in non-natural growth condition cycles as long as their thermic and lighting needs are covered. It can be grown continuously with 2 to 3 cycles of flowering per year and can last 2 to 3 years. It needs sandy or porous soil in order to regulate its moisture uptake, while excess water can seriously harm it. These conditions can be provided by localised irrigation. Its rapid growth with resulting numerous, small secondary stems mean that it needs a guide, which is usually provided by several meshes at different heights. Vegetative development out of season is improved by the application of growth regulators e.g. gibberellins. Pests and diseases can be controlled. Stems are harvested when 30 or 40% of the flowers have started to open, although similar good quality results can be obtained by applying artificial opening techniques when only 5% of the flowers are open. Correct handling will ensure the longest possible vase life. Some varieties show different characteristics e.g. flower size, light needs, yield, duration of cultivation cycle and flowering phase, among others. Part 4 Colour and scents 33. Flavonoid compounds in flowers: genetics and biochemistry. O Yu, M Matsuno, S Subramanian, USA ............................ 282-292 Flavonoids are one of the main determinants of flower colors. Flavonoid biosynthesis is one of the most extensively studied natural product metabolism. Flavonoid compounds are produced by the phenylpropanoid pathway, a major secondary pathway that exists in all higher plants. Most enzymes involved in this pathway and their corresponding genes have been characterized; some proteins have been crystallized and structurally resolved. This chapter aims to provide a basic understanding of flavonoid biosynthesis and its regulation in flowers. The chapter has been organized into three sections: the first outlines key enzymatic reactions that lead to the production of anthocyanin and related copigments; the second summarizes major regulating factors that affect the biochemistry and genetics of the flavonoid compounds in flowers; and the last section describes examples of genetic engineering attempts that produced novel flower colors in many plant species by modifying the flavonoid biosynthesis pathway. 34. Model plants and functional genomic approaches applied to the characterization of genes involved in floral scent biosynthesis. G Scalliet, Switzerland, S Baudino, JM Cock, P Hugueney, France .......................................................................................... 293-299 Flower scents have been subjected to extensive chemical characterization for many years, providing detailed analyses of the complex mixture of volatiles emitted by flowers. However, the past ten years have seen a rapid acceleration of progress in flower scent research, with the characterization of numerous genes involved in scent biosynthesis. This review focuses on the evolution of flower scent research, from the early biochemical to the present genomic approaches, illustrating how the use of different plant models, combined with functional genomic approaches, contributed to the present knowledge in this field. 35. Molecular breeding of flower color. K-Y To, C-K Wang, Taiwan ............................................................................................... 300-310 Flower color contributes mainly to the market value of an ornamental plant, and coloration of flowers is determined by three classes of pigments: flavonoids, carotenoids and betalains. Flavonoids and carotenoids are widespread; however, betalains can be found only in plants of several genera in the order Caryophyllales, which belongs to one small group of angiosperms. Among these pigments, flavonoids (mainly anthocyanins) are the most common flower pigments contributing to a range of colors from yellow to orange to red to purple. During the past few decades, flavonoid biosynthetic pathway leading to anthocyanin production has been well established in various plant species, and genetic engineering of flavonoid/anthocyanin biosynthesis has been used to produce cultivars with novel pigmentation in flowers. Here we summarize the current status of molecular approaches in breeding flower coloration, and describe our study and prospective regarding flower color modification. 36. Causes of flower colour patterns with a focus on chimerical patterns. K Olbricht, S Plaschil, F Pohlheim, Germany ..... 311-319 Colour patterns in flowers are of ornamental value and are important for histological studies. Similar flower patterns may have different causes. Bicoloured flowers may result from genetically inherited patterns, instable alleles of floral genes, infections, or chimeral dispositions. A transgenic Petunia Juss. serves as an example of an epigenetic chimera. Bicoloured cultivars of Angelonia Humb. et Bonpl., Pelargonium L ́Herit. ex Ait., Petunia, and Saintpaulia H. Wendl. and other species are examined in terms of their flower colour pattern. The investigation of the causes of flower patterns is based on inheritance analysis (self-pollination), double marking of the different apex layers, grafting and somatic segregation (spontaneous and by in vitro callus culture). In the case of chimerally determined flower patterns, conclusions about the participation of apex layers in the histogenesis of flower tissues can additionally be drawn by apex layer double marking. 37. Isolation and analysis methods of volatile compounds from flowers and leaves. V Radulescu, E Oprea, S Chiliment, Romania ........................................................................................................................... 320-325 The paper has an introductory part, which presents the basic principles for different methods of isolation and determination of volatile compounds from plants. In the experimental part, a comparison is made between the chemical composition of different plant products (essential oil, infusion and tincture), using as research material flowers and leaves of Salvia officinalis. The essential oil from leaves and flowers of S. officinalis was obtained through hydro-distillation in a Neo-Clevenger apparatus. The volatile compounds from infusion and tincture were extracted using solid phase extraction (C18-SPE) and liquid-liquid extraction with hexane and dichloromethane. The volatile oil diluted with dichloromethane and the infusion and tincture extracts were analysed by gas chromatography coupled with mass spectrometry. The results were also compared with those obtained through headspace at 50°C and 80°C. The chemical composition of volatile oils extracted from leaves and flowers are very similar. The only significant difference is a higher contribution of monoterpenic hydrocarbons in flowers (8.59%) compared to leaves (4.54%). The analysis of volatile compounds from S. officinalis leaves shows that through infusion and maceration most of the monoterpenic hydrocarbons are lost. In headspace analysis, the amount of volatile compounds at an equilibrium temperature of 80°C is more than five times higher than at 50°C (the average ratio is 5.24). Part 5 Metabolism, metabolites and hormones 38. Targeted transcriptomics to elucidate the regulation of benzenoid synthesis in Petunia hybrida. JC Verdonk, MA Haring, AJ van Tunen, RC Schuurink, The Netherlands ............................................................................... 326-338 Flowers attract specific pollinators by a combination of color, shape and fragrance, which is referred to as a pollination syndrome. Generally, the fragrance emission by the flowers is at a maximal level when the pollinator is active, and consists of volatile molecules derived from different biochemical pathways. Studies regarding the fragrance of flowers have focused on scent composition rather than on the enzymes involved in its production and regulation. In Petunia hybrida cv. Mitchell, the fragrance of the flowers consists almost exclusively of benzenoids, which are mainly emitted during the evening and night. Microarrays were used to identify cDNAs whose expression increased with the production of floral benzenoids in the evening. We found genes from the shikimate pathway and the post-shikimate pathway that ultimately provides precursors for phenylpropanoid and benzenoid synthesis to be upregulated in the afternoon, prior to benzenoid production. Additionally, we identified an R2R3-like MYB transcription factor, ODORANT1, which regulates the expression of two genes in the shikimate pathway, and two post-shikimate pathway genes chorismate mutase (CM) and phenylalanine ammonia lyase (PAL). Moreover, the expression of several genes that have a function in the biosynthesis of S-Adenosyl methionine (SAM) correlated with scent emission. SAM is the methyl donor in the reaction that forms methyl benzoate, the most abundant compound in the headspace of petunia Mitchell. These results illustrate the power of using targeted metabolomics and transcriptomics to unravel the biosynthesis of secondary metabolites. 39. Plastid isoprenoids: biogenesis and molecular regulation. F Bouvier, JC Isner, Switzerland, MS Alexis, France, O Dogbo, Ivory Coast, B Camara, France ............................................ 339-358 Plastid isoprenoids comprise a wide array of compounds (e.g. carotenoids, chlorophylls, tocopherols, phylloquinone, plastoquinone and diverse bioactive low molecular weight derivatives). Their biosynthesis starts either with isopentenyl diphosphate or dimethylallyl diphosphate and involves multiple enzymic transformations. Through a combination of biochemical and molecular approaches our knowledge about the genes and the enzymes governing their synthesis has significantly advanced. This review will discuss specific examples of this progress. 40. Lipid biosynthesis in Spermatophyta. M Iriti, F Faoro, Italy .................................................................................................... 359-372 Lipids are a heterogeneous group of compounds essential for all living organisms. Lipid bilayers provide a spatial separation between the extracellular medium and cytoplasm, as well as subcellular compartmentalization. Besides, lipids are a predominant form of chemical energy storage in seeds and some of them, namely octadecanoid (jasmonates) and eicosanoid (prostanoids) oxylipins act as mediators entailed in defence and immunity, via phospholipase/lipoxygenase (LOX)/cyclooxygenase (COX) pathway. Other classes of lipids, include, isoprenoids (consisting of pigments, hormones, vitamins and essential oils), phytosterols and polyunsatured fatty acids (PUFA), such as omega-3, that enhance functional properties of foodstuffs, protecting against chronic disease. In this chapter, lipid biosynthesis will be examined, despite its complexity, emphasising on the parallelism/diversity between plants and animals, with the aim of offering a comprehensive description, beneficial for any biotechnological approach. 41. The potential of carbohydrates in plant growth regulation. D Lišková, P Capek, K Kollárová, L Slováková, A Kákošová, Slovakia ................................................................................... 373-378 Attention was devoted to enzymes assumed to be involved in the elongation/cessation of growth after treatment with galactoglucomannan oligosaccharides (GGMOs). Endoglycanases, endo-(1-4)-β-D-glucanase, endo-(1-3),(1-4)-β-D-glucanase, endo-(1-4)-β-D-mannanase, as well as XET, showed remarkable activity changes after GGMOs treatment indicating their role in cell wall remodeling and cell wall milieu stabilization during cessation of elongation growth. Studies on peroxidase – the enzyme participating in cell wall tightening – showed that elongation growth inhibited by GGMOs may be associated with the process of cell wall rigidification catalysed by peroxidase. Previously obtained results on glycosidases are also discussed. 42. Cytokinins and plant phenolics: a cooperative metabolism. J Frébortová, O Novák, P Galuszka, I Frébort, Czech Republic ................................................................................ 379-384 Cytokinin dehydrogenase (CKX) is a key enzyme in regulation of the levels of active cytokinins in plants. We have previously reported that CKX can use products of oxidation of plant phenolics by tyrosinase and peroxidase as acceptors of electrons from oxidized cytokinin and that the phloem sap of maize seedlings contains unknown phenolic precursor of this acceptor. In the present work we investigated a set of substituted phenolic acids for their ability to serve as precursors of electron acceptors in the CKX reaction and analyzed the phloem sap of maize seedlings for the presence of carboxylic phenolic acids using HPLC with a Q-TOF detector. Only p-coumaric, caffeic and chlorogenic acids were converted to effective electron acceptors in the presence of mushroom tyrosinase. Ferulic acid showed weak activity and analogous compounds derived from 4-hydroxybenzoic acid were not active. Compounds effective in in vitro CKX assay were identified in the phloem sap. 43. Flower ovens and solar furnaces. I Lamprecht, Germany, CM Romero, L Blanco, Colombia, JA Teixeira da Silva, Japan ......................................................... 385-404 Struggle for life means struggle for energy, in animals and in plants. Because of their sessile existence plants depend strongly on photosynthesis, sometimes even on exploiting other plants. Metabolism is in general much lower in plants than in animals, but in special periods the demand may increase significantly. Two means of coping with such situations that are mainly connected with inflorescence, pollination and/or seed production are discussed in this chapter. The first deals with thermogenic plants, which use their own energy sources to increase the metabolic turnover rates and to heat up blossoms (“metabolic climax”, “flare-up”). Temperature increases of up to 35 K and for some hours, days or even weeks are observed. The mass specific rates are so high that they become equal to those of the most active animals like honeybee queens or hovering hummingbirds. A few thermogenic species even regulate the heat production to keep their elevated temperatures constant. Thus, there are a number of parallels to phenomena well known from homoeothermic animals and their brown fat tissue in hibernators or young mammals. The second means uses the direct sun light to achieve the necessary energy. Blossoms and leaves orient in a favourite angle with the incident beams to obtain a maximum quantum flux per area (phototropism). Specialists among them even follow the course of the sun by blossoms and/or leaves during the day (“solar tracking”, “heliotropism”). And some have bowl shaped flowers concentrating the radiation to their centres. 44. Osmoregulation versus osmoprotection: re-evaluating the role of compatible solutes. S Shabala, TA Cuin, Australia ............................................................................................................................... 405-416 Growth and production of ornamentals is severely limited by dehydration stress imposed on plants by salinity, drought or temperature extremes. The ability of plants to accumulate so-called “compatible solutes” (small water-soluble molecules that may be accumulated in cells at high concentrations, without affecting metabolic reactions in the cytosol or major organelles), is frequently cited as a key mechanism for increasing dehydration tolerance. This review summarises recent progress in our understanding of the physiological roles of compatible solutes in plant adaptive responses to the environment and investigates prospects for using molecular tools to genetically engineer crop and ornamental species with greater dehydration stress tolerance. Despite the central dogma in stress physiology that the adaptive role of compatible solutes is in mediating osmotic adjustment, their concentrations in the cell are frequently far too low for conventional osmotic adjustment. Osmoprotective and/or regulatory roles are more likely. Such roles include PSII protection and repair, membrane and enzyme protection, ROS scavenging, energy sinks and redox potential buffering, the use of osmolytes as a source of N and C during the recovery from stress, signalling, and control over ion transport and partitioning. Major problems hindering the use of transgenic approach to improve plant dehydration tolerance are discussed, and prospects for future research are highlighted. It is concluded that combining transgenic approaches with in planta physiological measurements of plant adaptive responses at various levels of plant structural organization will help us to fully appreciate the complex role of osmolytes in cellular metabolism and make substantial progress in improving plant dehydration tolerance under field conditions.