Acquisition and loss of desiccation tolerance in seeds: from experimental model to biological relevance

AbstractMain conclusionBesides being an important model to study desiccation tolerance, the induction of desiccation tolerance in germinated seeds may also play an ecological role in seedling establishment.
 Desiccation tolerance (DT) is the ability of certain organisms to survive extreme water losses without accumulation of lethal damage. This was a key feature in the conquering of dry land and is currently found in all taxa including bacteria, fungi, roundworms and plants. Not surprisingly, studies in various fields have been performed to unravel this intriguing phenomenon. In flowering plants, DT is rare in whole plants (vegetative tissues), yet is common in seeds. In this review, we present our current understanding of the evolution of DT in plants. We focus on the acquisition of DT in seeds and the subsequent loss during and after germination by highlighting and comparing research in two model plants Medicago truncatula and Arabidopsis thaliana. Finally, we discuss the ability of seeds to re-establish DT during post-germination, the possible ecological meaning of this phenomenon, and the hypothesis that DT, in combination with dormancy, optimizes seedling establishment.

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title: A Regulatory Network-Based Approach Dissects Late Maturation Processes Related to the Acquisition of Desiccation Tolerance and Longevity of Medicago truncatula Seeds1[C][W][OPEN]
abstract: A network analysis approach to gene regulation during seed maturation of Medicago truncatula uncovers distinct temporal regulatory programs related to desiccation tolerance, longevity, and pod abscission and the key regulators governing these programs. In seeds, desiccation tolerance (DT) and the ability to survive the dry state for prolonged periods of time (longevity) are two essential traits for seed quality that are consecutively acquired during maturation. Using transcriptomic and metabolomic profiling together with a conditional-dependent network of global transcription interactions, we dissected the maturation events from the end of seed filling to final maturation drying during the last 3 weeks of seed development in Medicago truncatula. The network revealed distinct coexpression modules related to the acquisition of DT, longevity, and pod abscission. The acquisition of DT and dormancy module was associated with abiotic stress response genes, including late embryogenesis abundant (LEA) genes. The longevity module was enriched in genes involved in RNA processing and translation. Concomitantly, LEA polypeptides accumulated, displaying an 18-d delayed accumulation compared with transcripts. During maturation, gulose and stachyose levels increased and correlated with longevity. A seed-specific network identified known and putative transcriptional regulators of DT, including ABSCISIC ACID-INSENSITIVE3 (MtABI3), MtABI4, MtABI5, and APETALA2/ ETHYLENE RESPONSE ELEMENT BINDING PROTEIN (AtAP2/EREBP) transcription factor as major hubs. These transcriptional activators were highly connected to LEA genes. Longevity genes were highly connected to two MtAP2/EREBP and two basic leucine zipper transcription factors. A heat shock factor was found at the transition of DT and longevity modules, connecting to both gene sets. Gain- and loss-of-function approaches of MtABI3 confirmed 80% of its predicted targets, thereby experimentally validating the network. This study captures the coordinated regulation of seed maturation and identifies distinct regulatory networks underlying the preparation for the dry and quiescent states.

title: From Avicennia to Zizania: seed recalcitrance in perspective.
abstract: Considered only in terms of tolerance of, or sensitivity to, desiccation (which is an oversimplification), orthodox seeds are those which tolerate dehydration and are storable in this condition, while highly recalcitrant seeds are damaged by loss of only a small proportion of water and are unstorable for practical purposes. Between these extremes, however, there may be a gradation of the responses to dehydration--and also to other factors--suggesting perhaps that seed behaviour might be best considered as constituting a continuum subtended by extreme orthodoxy and the highest degree of recalcitrance. As the characteristics of seeds of an increasing number of species are elucidated, non-orthodox seed behaviour is emerging as considerably more commonplace--and its basis far more complex--than previously suspected.



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There appears to be little taxonomic relationship among species exhibiting the phenomenon of seed recalcitrance, suggesting that it is a derived trait, with tolerance having been lost a number of times. Although recalcitrant seededness is best represented in the mesic tropics, particularly among rainforest climax species, it does occur in cooler, drier and markedly seasonal habitats. The selective advantages of the trait are considered.

title: Desiccation tolerance in resurrection plants: new insights from transcriptome, proteome and metabolome analysis
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title: Drought, metabolites, and Arabidopsis natural variation: a promising combination for understanding adaptation to water-limited environments.
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title: Desiccation-Tolerant Flowering Plants in Southern Africa
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title: An overview of mechanisms of desiccation tolerance in selected angiosperm resurrection plants
abstract: The vegetative tissues of resurrection plants, like seeds, can tolerate desiccation to 5% relative water content (RWC) for extended periods and yet resume full metabolic activity on re-watering. In this review we will illustrate how this is achieved in a variety of angiosperm resurrection plants, our studies ranging from the ecophysiological to the biochemical level. At the whole plant level, leaf folding and other anatomical changes serve to minimise light and mechanical stress associated with drying and rehydration. The mechanisms of cell wall folding are described for Craterostigma wilmsii and Myrothanmus flabellifolia. Free radicals, radical oxygen species (ROS) usually generated under water-deficit stress by photosynthesis, are minimised by either homoiochlorophylly (e.g. C. wilmsii and M. flabellifolia) or poikilochlorophylly (e.g. Xerophyta sp.). The antioxidant systems of these plants effectively deal with ROS generated by other metabolic processes. In addition to antioxidants common to most plants, resurrection plants also accumulate polyphenols such as 3, 4, 5 tri-O-galloylquinic acid in M. flabellifolia, and seed-associated antioxidants (e.g. 1-cys-peroxiredoxin and metallothionines) as effective ROS scavengers. Sucrose accumulates at low RWC, presumably protecting the sub-cellular milieu against desiccation-induced macromolecular denaturation. _____________________________________________________________________________________________________________

title: A comparison of mechanisms of desiccation tolerance among three angiosperm resurrection plant species
abstract: The mechanisms of protection against mechanical and oxidative stress were identified and compared in the angiosperm resurrection plants Craterostigma wilmsii, Myrothamnus flabellifolius and Xerophyta humilis. Drying-induced ultrastructural changes within mesophyll cells were followed to gain an understanding of the mechanisms of mechanical stabilisation. In all three species, water filled vacuoles present in hydrated cells were replaced by several smaller vacuoles filled with non-aqueous substances. In X. humilis, these occupied a large proportion of the cytoplasm, preventing plasmalemma withdrawal and cell wall collapse. In C. wilmsii, vacuoles were small but extensive cell wall folding occurred to prevent plasmalemma withdrawal. In M. flabellifolius, some degree of vacuolation and wall folding occurred, but neither were sufficient to prevent plasmalemma withdrawal. This membrane was not ruptured, possibly due to membrane repair at plasmodesmata junctions where tearing might have occurred. In addition, the extra-cytoplasmic compartment appeared to contain material (possibly similar to that in vacuoles) which could facilitate stabilisation of dry cells.Photosynthesis and respiration are particularly susceptible to oxidative stress during drying. Photosynthesis ceased at high water contents and it is proposed that a controlled shut down of this metabolism occurred in order to minimise the potential for photo-oxidation. The mechanisms whereby this was achieved varied among the species. In X. humilis, chlorophyll was degraded and thylakoid membranes dismantled during drying. In both C. wilmsii and M. flabellifolius, chlorophyll was retained, but photosynthesis was stopped due to chlorophyll shading from leaf folding and anthocyanin accumulation. Furthermore, in M. flabellifolius thylakoid membranes became unstacked during drying. All species continued respiration during drying to 10% relative water content, which is proposed to be necessary for energy to establish protection mechanisms. Activity of antioxidant enzymes increased during drying and remained high at low water contents in all species, ameliorating free radical damage from both photosynthesis and respiration. The nature and extent of antioxidant upregulation varied among the species. In C. wilmsii, only ascorbate peroxidise activity increased, but in M. flabellifolius and X. humilis ascorbate peroxidise, glutathione reductase and superoxide dismutase activity increased, to various extents, during drying. Anthocyanins accumulated in all species but this was more extensive in the homoiochlorophyllous types, possibly for protection against photo-oxidation.

title: Leaf Shrinkage with Dehydration: Coordination with Hydraulic Vulnerability and Drought Tolerance1[C][W][OPEN]
abstract: Leaf shrinkage is a potential driver of leaf hydraulic vulnerability, especially under mild stress, and carries strong ecological implications. Leaf shrinkage with dehydration has attracted attention for over 100 years, especially as it becomes visibly extreme during drought. However, little has been known of its correlation with physiology. Computer simulations of the leaf hydraulic system showed that a reduction of hydraulic conductance of the mesophyll pathways outside the xylem would cause a strong decline of leaf hydraulic conductance (Kleaf). For 14 diverse species, we tested the hypothesis that shrinkage during dehydration (i.e. in whole leaf, cell and airspace thickness, and leaf area) is associated with reduction in Kleaf at declining leaf water potential (Ψleaf). We tested hypotheses for the linkage of leaf shrinkage with structural and physiological water relations parameters, including modulus of elasticity, osmotic pressure at full turgor, turgor loss point (TLP), and cuticular conductance. Species originating from moist habitats showed substantial shrinkage during dehydration before reaching TLP, in contrast with species originating from dry habitats. Across species, the decline of Kleaf with mild dehydration (i.e. the initial slope of the Kleaf versus Ψleaf curve) correlated with the decline of leaf thickness (the slope of the leaf thickness versus Ψleaf curve), as expected based on predictions from computer simulations. Leaf thickness shrinkage before TLP correlated across species with lower modulus of elasticity and with less negative osmotic pressure at full turgor, as did leaf area shrinkage between full turgor and oven desiccation. These findings point to a role for leaf shrinkage in hydraulic decline during mild dehydration, with potential impacts on drought adaptation for cells and leaves, influencing plant ecological distributions.

title: A Modulating Role for Antioxidants in Desiccation Tolerance1
abstract: Most organisms depend on the availability of water. However, some life-forms, among them plants and fungi, but very few animals, can survive in the desiccated state. Here we discuss biochemical mechanisms that confer tolerance to desiccation in photosynthetic and non-photosynthetic organisms. We first consider damage caused by water removal and point out that free radicals are a major cause of death in intolerant tissue. Free radicals impair metabolism and necessitate protection and repair during desiccation and rehydration, respectively. As a consequence, desiccation tolerance and prolonged longevity in the desiccated state depend on the ability to scavenge free radicals, using antioxidants such as glutathione, ascorbate, tocopherols and free radical-processing enzymes. Some ‘classic’ antioxidants may be absent in lower plants and fungi. On the other hand, lichens and seeds often contain secondary phenolic products with antioxidant properties. The major intracellular antioxidant consistently found in all life forms is glutathione, making it essential to survive desiccation. We finally discuss the role of glutathione to act as a signal that initiates programmed cell death. The failure of the antioxidant system during long-term desiccation appears to trigger programmed cell death, causing ageing and eventual death of the organism. In turn, this suggests that a potent antioxidant machinery is one of the underlying mechanisms of desiccation tolerance.

title: Mechanisms of plant desiccation tolerance.
abstract: Anhydrobiosis ("life without water") is the remarkable ability of certain organisms to survive almost total dehydration. It requires a coordinated series of events during dehydration that are associated with preventing oxidative damage and maintaining the native structure of macromolecules and membranes. The preferential hydration of macromolecules is essential when there is still bulk water present, but replacement by sugars becomes important upon further drying. Recent advances in our understanding of the mechanism of anhydrobiosis include the downregulation of metabolism, dehydration-induced partitioning of amphiphilic compounds into membranes and immobilization of the cytoplasm in a stable multicomponent glassy matrix.

title: An emerging picture of the seed desiccome: confirmed regulators and newcomers identified using transcriptome comparison
abstract: Desiccation tolerance (DT) is the capacity to withstand total loss of cellular water. It is acquired during seed filling and lost just after germination. However, in many species, a germinated seed can regain DT under adverse conditions such as osmotic stress. The genes, proteins and metabolites that are required to establish this DT is referred to as the desiccome. It includes both a range of protective mechanisms and underlying regulatory pathways that remain poorly understood. As a first step toward the identification of the seed desiccome of Medicago truncatula, using updated microarrays we characterized the overlapping transcriptomes associated with acquisition of DT in developing seeds and the re-establishment of DT in germinated seeds using a polyethylene glycol treatment (−1.7 MPa). The resulting list contained 740 and 2829 transcripts whose levels, respectively, increased and decreased with DT. Fourty-eight transcription factors (TF) were identified including MtABI3, MtABI5 and many genes regulating flowering transition and cell identity. A promoter enrichment analysis revealed a strong over-representation of ABRE elements together with light-responsive cis-acting elements. In Mtabi5 Tnt1 insertion mutants, DT could no longer be re-established by an osmotic stress. Transcriptome analysis on Mtabi5 radicles during osmotic stress revealed that 13 and 15% of the up-regulated and down-regulated genes, respectively, are mis-regulated in the mutants and might be putative downstream targets of MtABI5 implicated in the re-establishment of DT. Likewise, transcriptome comparisons of the desiccation sensitive Mtabi3 mutants and hairy roots ectopically expressing MtABI3 revealed that 35 and 23% of the up-regulated and down-regulated genes are acting downstream of MtABI3. Our data suggest that ABI3 and ABI5 have complementary roles in DT. Whether DT evolved by co-opting existing pathways regulating flowering and cellular phase transition and cell identity is discussed.

title: Seed maturation in Arabidopsis thaliana is characterized by nuclear size reduction and increased chromatin condensation
abstract: Most plant species rely on seeds for their dispersal and survival under unfavorable environmental conditions. Seeds are characterized by their low moisture content and significantly reduced metabolic activities. During the maturation phase, seeds accumulate storage reserves and become desiccation-tolerant and dormant. Growth is resumed after release of dormancy and the occurrence of favorable environmental conditions. Here we show that embryonic cotyledon nuclei of Arabidopsis thaliana seeds have a significantly reduced nuclear size, which is established at the beginning of seed maturation. In addition, the chromatin of embryonic cotyledon nuclei from mature seeds is highly condensed. Nuclei regain their size and chromatin condensation level during germination. The reduction in nuclear size is controlled by the seed maturation regulator ABSCISIC ACID-INSENSITIVE 3, and the increase during germination requires two predicted nuclear matrix proteins, LITTLE NUCLEI 1 and LITTLE NUCLEI 2. Our results suggest that the specific properties of nuclei in ripe seeds are an adaptation to desiccation, independent of dormancy. We conclude that the changes in nuclear size and chromatin condensation in seeds are independent, developmentally controlled processes.

title: Desiccation-tolerance in bryophytes: a review
abstract: Desiccation-tolerance (DT), the ability to lose virtually all free intracellular water and then recover normal function upon rehydration, is one of the most remarkable features of bryophytes. The physiology of bryophytes differs in major respects from that of vascular plants by virtue of their smaller size; unlike vascular plants, the leafy shoots of bryophytes equilibrate rapidly with the water potential in their surroundings and tend to be either fully hydrated or desiccated and metabolically inactive. The time required to recover from desiccation increases and degree of recovery decreases with length of desiccation; both also depend upon temperature and intensity of desiccation. Tolerance in at least some species shows phenotypic plasticity. Recovery of respiration, photosynthesis and protein synthesis takes place within minutes or an hour or two; recovery of the cell cycle, food transport and the cytoskeleton may take 24 hours or more. Positive carbon balance is essential to survival of repeated cycles of drying and wetting; significant growth requires continuously wet periods of a few days or more. Male and female gametophytes, and gametophyte and sporophyte, may differ in tolerance. Desiccation-tolerance is essential to dispersal and establishment of spores and vegetative propagules. The mechanisms of DT in bryophytes, including expression of LEA proteins, high content of non-reducing sugars and effective antioxidant and photo-protection, are at least partly constitutive, allowing survival of rapid drying, but changes in gene expression resulting from mRNA sequestration and alterations in translational controls elicited upon rehydration are also important to repair processes following re-wetting. Phylogenetic and ecological considerations suggest that DT is a primitive character of land plants, lost in the course of evolution of the homoiohydric vascular-plant shoot system, but retained in spores, pollen and seeds, and re-evolved in the vegetative tissues of vascular “resurrection plants.” Bryophytes have retained the poikilohydry and DT that are probably the optimal pattern of adaptation at their scale, but modern bryophytes are specialized and diverse, and are removed by the same span of evolutionary time as the flowering plants from their primitive origins.

title: Evolution of Abscisic Acid Synthesis and Signaling Mechanisms
abstract: The plant hormone abscisic acid (ABA) mediates seed dormancy, controls seedling development and triggers tolerance to abiotic stresses, including drought. Core ABA signaling components consist of a recently identified group of ABA receptor proteins of the PYRABACTIN RESISTANCE (PYR)/REGULATORY COMPONENT OF ABA RECEPTOR (RCAR) family that act as negative regulators of members of the PROTEIN PHOSPHATASE 2C (PP2C) family. Inhibition of PP2C activity enables activation of SNF1-RELATED KINASE 2 (SnRK2) protein kinases, which target downstream components, including transcription factors, ion channels and NADPH oxidases. These and other components form a complex ABA signaling network. Here, an in depth analysis of the evolution of components in this ABA signaling network shows that (i) PYR/RCAR ABA receptor and ABF-type transcription factor families arose during land colonization of plants and are not found in algae and other species, (ii) ABA biosynthesis enzymes have evolved to plant- and fungal-specific forms, leading to different ABA synthesis pathways, (iii) existing stress signaling components, including PP2C phosphatases and SnRK kinases, were adapted for novel roles in this plant-specific network to respond to water limitation. In addition, evolutionarily conserved secondary structures in the PYR/RCAR ABA receptor family are visualized.

title: Molecular Basis of the Core Regulatory Network in ABA Responses: Sensing, Signaling and Transport
abstract: ABA is a major phytohormone that regulates a broad range of plant traits and is especially important for adaptation to environmental conditions. Our understanding of the molecular basis of ABA responses in plants improved dramatically in 2009 and 2010, banner years for ABA research. There are three major components; PYR/PYL/ RCAR (an ABA receptor), type 2C protein phosphatase (PP2C; a negative regulator) and SNF1-related protein kinase 2 (SnRK2; a positive regulator), and they offer a double negative regulatory system, [PYR/PYL/RCAR—| PP2C—| SnRK2]. In the absence of ABA, PP2C inactivates SnRK2 by direct dephosphorylation. In response to environmental or developmental cues, ABA promotes the interaction of PYR/PYL/RCAR and PP2C, resulting in PP2C inhibition and SnRK2 activation. This signaling complex can work in both the nucleus and cytosol, as it has been shown that SnRK2 phosphorylates basic-domain leucine zipper (bZIP) transcription factors or membrane proteins. Several structural analyses of PYR/PYL/RCAR have provided the mechanistic basis for this ‘core signaling’ model, by elucidating the mechanism of ABA binding of receptors, or the ‘gate–latch–lock’ mechanism of interaction with PP2C in inhibiting activity. On the other hand, intercellular ABA transport had remained a major issue, as had intracellular ABA signaling. Recently, two plasma membrane-type ABC transporters were identified and shed light on the influx/efflux system of ABA, resolving how ABA is transported from cell to cell in plants. Our knowledge of ABA responses in plants has been greatly expanded from intracellular signaling to intercellular transport of ABA.

title: Land Plants Acquired Active Stomatal Control Early in Their Evolutionary History
abstract: Stomata are pores that regulate plant gas exchange [1]. They evolved more than 400 million years ago [2, 3], but the origin of their active physiological responses to endogenous and environmental cues is unclear [2-6]. Recent research suggests that the stomata of lycophytes and ferns lack pore closure responses to abscisic acid (ABA) and CO(2). This evidence led to the hypothesis that a fundamental transition from passive to active control of plant water balance occurred after the divergence of ferns 360 million years ago [7, 8]. Here we show that stomatal responses of the lycophyte Selaginella [9] to ABA and CO(2) are directly comparable to those of the flowering plant Arabidopsis [10]. Furthermore, we show that the underlying intracellular signaling pathways responsible for stomatal aperture control are similar in both basal and modern vascular plant lineages. Our evidence challenges the hypothesis that acquisition of active stomatal control of plant carbon and water balance represents a critical turning point in land plant evolution [7, 8]. Instead, we suggest that the critical evolutionary development is represented by the innovation of stomata themselves and that physiologically active stomatal control originated at least as far back as the emergence of the lycophytes (circa 420 million years ago) [11].

title: Role of ABA and ABI3 in Desiccation Tolerance
abstract: The hormone pathway that stabilizes seeds may have served more primitive seedless plants in supporting desiccation tolerance. We show in bryophytes that abscisic acid (ABA) pretreatment of moss (Physcomitrella patens) cells confers desiccation tolerance. In angiosperms, both ABA and the transcriptional regulator ABSCISIC ACID INSENSITIVE 3 (ABI3) are required to protect the seed during desiccation. ABA was not able to protect moss cells in stable deletion lines of ABI3 (ΔPpabi3). Hence, moss has the same functional link between ABA, ABI3, and the desiccation tolerance phenotype that is found in angiosperms. Furthermore, we identified 22 genes that were induced during ABA pretreatment in wild-type lines. When their expression was compared with that of ΔPpabi3 during ABA pretreatment and immediately after desiccation, a new target of ABI3 action appears to be in the recovery period.

title: The Re-Establishment of Desiccation Tolerance in Germinated Arabidopsis thaliana Seeds and Its Associated Transcriptome
abstract: The combination of robust physiological models with “omics” studies holds promise for the discovery of genes and pathways linked to how organisms deal with drying. Here we used a transcriptomics approach in combination with an in vivo physiological model of re-establishment of desiccation tolerance (DT) in Arabidopsis thaliana seeds. We show that the incubation of desiccation sensitive (DS) germinated Arabidopsis seeds in a polyethylene glycol (PEG) solution re-induces the mechanisms necessary for expression of DT. Based on a SNP-tile array gene expression profile, our data indicates that the re-establishment of DT, in this system, is related to a programmed reversion from a metabolic active to a quiescent state similar to prior to germination. Our findings show that transcripts of germinated seeds after the PEG-treatment are dominated by those encoding LEA, seed storage and dormancy related proteins. On the other hand, a massive repression of genes belonging to many other classes such as photosynthesis, cell wall modification and energy metabolism occurs in parallel. Furthermore, comparison with a similar system for Medicago truncatula reveals a significant overlap between the two transcriptomes. Such overlap may highlight core mechanisms and key regulators of the trait DT. Taking into account the availability of the many genetic and molecular resources for Arabidopsis, the described system may prove useful for unraveling DT in higher plants.

title: The evolution of vegetative desiccation tolerance in land plants
abstract: Vegetative desiccation tolerance is a widespread but uncommon occurrence in the plant kingdom generally. The majority of vegetative desiccation-tolerant plants are found in the less complex clades that constitute the algae, lichens and bryophytes. However, within the larger and more complex groups of vascular land plants there are some 60 to 70 species of ferns and fern allies, and approximately 60 species of angiosperms that exhibit some degree of vegetative desiccation tolerance. In this report we analyze the evidence for the differing mechanisms of desiccation tolerance in different plants, including differences in cellular protection and cellular repair, and couple this evidence with a phylogenetic framework to generate a working hypothesis as to the evolution of desiccation tolerance in land plants. We hypothesize that the initial evolution of vegetative desiccation tolerance was a crucial step in the colonization of the land by primitive plants from an origin in fresh water. The primitive mechanism of tolerance probably involved constitutive cellular protection coupled with active cellular repair, similar to that described for modern-day desiccation-tolerant bryophytes. As plant species evolved, vegetative desiccation tolerance was lost as increased growth rates, structural and morphological complexity, and mechanisms that conserve water within the plant and maintain efficient carbon fixation were selected for. Genes that had evolved for cellular protection and repair were, in all likelihood, recruited for different but related processes such as response to water stress and the desiccation tolerance of reproductive propagules. We thus hypothesize that the mechanism of desiccation tolerance exhibited in seeds, a developmentally induced cellular protection system, evolved from the primitive form of vegetative desiccation tolerance. Once established in seeds, this system became available for induction in vegetative tissues by environmental cues related to drying. The more recent, modified vegetative desiccation tolerance mechanism in angiosperms evolved from that programmed into seed development as species spread into very arid environments. Most recently, certain desiccation-tolerant monocots evolved the strategy of poikilochlorophylly to survive and compete in marginal habitats with variability in water availability.

title: The evolution of desiccation tolerance in angiosperm plants: a rare yet common phenomenon.
abstract: In a minute proportion of angiosperm species, rehydrating foliage can revive from airdryness or even from equilibration with air of ~0% RH. Such desiccation tolerance is known from vegetative cells of some species of algae and of major groups close to the evolutionary path of the angiosperms. It is also found in the reproductive structures of some algae, moss spores and probably the aerial spores of other terrestrial cryptogamic taxa. The occurrence of desiccation tolerance in the seed plants is overwhelmingly in the aerial reproductive structures; the pollen and seed embryos. Spatially and temporally, pollen and embryos are close ontogenetic derivatives of the angiosperm microspores and megaspores respectively. This suggests that the desiccation tolerance of pollen and embryos derives from the desiccation tolerance of the spores of antecedent taxa and that the basic pollen/embryo mechanism of desiccation tolerance has eventually become expressed also in the vegetative tissue of certain angiosperm species whose drought avoidance is inadequate in micro-habitats that suffer extremely xeric episodes. The protective compounds and processes that contribute to desiccation tolerance in angiosperms are found in the modern groups related to the evolutionary path leading to the angiosperms and are also present in the algae and in the cyanobacteria. The mechanism of desiccation tolerance in the angiosperms thus appears to have its origins in algal ancestors and possibly in the endosymbiotic cyanobacteria-related progenitor of chloroplasts and the bacteria-related progenitor of mitochondria. The mechanism may involve the regulation and timing of the accumulation of protective compounds and of other contributing substances and processes.

title: Evolution, Diversity, and Habitats of Poikilohydrous Vascular Plants
abstract: Water stress is a common environmental constraint in terrestrial ecosystems. Among the various strategies to cope with temporal lack of water, poikilohydry is an important adaptive trait under specific habitat conditions. Desiccation tolerant vascular plants are particularly common among ferns, fern allies, and angiosperms that colonize forest canopies and mainly tropical rock outcrops (e.g., granitic/gneissic inselbergs, ferricretes) where environmental conditions (e.g., high temperatures, lack of water, and soil) are harsh.

title: The Signature of Seeds in Resurrection Plants: A Molecular and Physiological Comparison of Desiccation Tolerance in Seeds and Vegetative Tissues1
abstract: Desiccation-tolerance in vegetative tissues of angiosperms has a polyphyletic origin and could be due to 1) appropriation of the seed-specific program of gene expression that protects orthodox seeds against desiccation, and/or 2) a sustainable version of the abiotic stress response. We tested these hypotheses by comparing molecular and physiological data from the development of orthodox seeds, the response of desiccation-sensitive plants to abiotic stress, and the response of desiccation-tolerant plants to extreme water loss. Analysis of publicly-available gene expression data of 35 LEA proteins and 68 anti-oxidant enzymes in the desiccation-sensitive Arabidopsis thaliana identified 13 LEAs and 4 anti-oxidants exclusively expressed in seeds. Two (a LEA6 and 1-cys-peroxiredoxin) are not expressed in vegetative tissues in A. thaliana, but have orthologues that are specifically activated in desiccating leaves of Xerophyta humilis. A comparison of antioxidant enzyme activity in two desiccation-sensitive species of Eragrostis with the desiccation-tolerant E. nindensis showed equivalent responses upon initial dehydration, but activity was retained at low water content in E. nindensis only. We propose that these antioxidants are housekeeping enzymes and that they are protected from damage in the desiccation-tolerant species. Sucrose is considered an important protectant against desiccation in orthodox seeds, and we show that sucrose accumulates in drying leaves of E. nindensis, but not in the desiccation-sensitive Eragrostis species. The activation of “seed-specific” desiccation protection mechanisms (sucrose accumulation and expression of LEA6 and 1-cys-peroxiredoxin genes) in the vegetative tissues of desiccation-tolerant plants points towards acquisition of desiccation tolerance from seeds.

title: Flow Cytometric Determination of Nuclear Replication Stages in Tomato Seeds during Priming and Germination
abstract: We report relative nuclear DNA contents in tomato (Lycopersicon esculentum)seed and seedling tissues as measured with flow cytometry. We provide information about the replication stage of nuclei in cells of non-imbibed, hydrated and primed seeds and specify the tissue in which DNA replication occurs

title: Seed dormancy and the control of germination.
abstract: Seed dormancy is an innate seed property that defines the environmental conditions in which the seed is able to germinate. It is determined by genetics with a substantial environmental influence which is mediated, at least in part, by the plant hormones abscisic acid and gibberellins. Not only is the dormancy status influenced by the seed maturation environment, it is also continuously changing with time following shedding in a manner determined by the ambient environment. As dormancy is present throughout the higher plants in all major climatic regions, adaptation has resulted in divergent responses to the environment. Through this adaptation, germination is timed to avoid unfavourable weather for subsequent plant establishment and reproductive growth. In this review, we present an integrated view of the evolution, molecular genetics, physiology, biochemistry, ecology and modelling of seed dormancy mechanisms and their control of germination. We argue that adaptation has taken place on a theme rather than via fundamentally different paths and identify similarities underlying the extensive diversity in the dormancy response to the environment that controls germination.

title: The evolution of seed dormancy: environmental cues, evolutionary hubs, and diversification of the seed plants.
abstract: Seed dormancy, by controlling the timing of germination, can strongly affect plant survival. The kind of seed dormancy, therefore, can influence both population and species-level processes such as colonization, adaptation, speciation, and extinction. We used a dataset comprising over 14,000 taxa in 318 families across the seed plants to test hypotheses on the evolution of different kinds of seed dormancy and their association with lineage diversification. We found morphophysiological dormancy to be the most likely ancestral state of seed plants, suggesting that physiologically regulated dormancy in response to environmental cues was present at the origin of seed plants. Additionally, we found that physiological dormancy (PD), once disassociated from morphological dormancy, acted as an 'evolutionary hub' from which other dormancy classes evolved, and that it was associated with higher rates of lineage diversification via higher speciation rates. The environmental sensitivity provided by dormancy in general, and by PD in particular, appears to be a key trait in the diversification of seed plants.

title: Ecological aspects of seed desiccation sensitivity
abstract: The ability of seeds to survive desiccation is an important functional trait and is an integral part of plant regeneration ecology. Despite this, the topic has received relatively little attention from ecologists. In this study, we examine the relationships between seed desiccation tolerance and two important aspects of plant regeneration ecology: habitat and dormancy. This is done by comparative analysis of a data set of 886 tree and shrub species from 93 families. 2 The proportion of species displaying desiccation sensitive seeds declines as the habitat becomes drier, and possibly also cooler, although the latter observation requires cautious interpretation. Desiccation sensitivity is most common in moist, relatively aseasonal vegetation zones, but is infrequent in, though not absent from arid and highly seasonal habitats. 3 The highest frequency of desiccation sensitivity occurs in non-pioneer evergreen rain forest trees, although 48% of the species examined have desiccation tolerant seeds. In contrast, all pioneer taxa within the data set have drying tolerant seeds. 4 Desiccation sensitivity is more frequent in seeds that are non-dormant on shedding ( c. 31%), than dormant ( c. 9%). Highest frequencies of drying tolerance occur in seeds with physical or combinational dormancy, at 99% and 100%, respectively. 5 Although there is an association between non-dormancy and desiccation sensitivity in both tropical and temperate zones, the relationship does not appear to be causal. 6 Wo rking from the hypothesis that seed desiccation sensitivity represents a derived state in extant species, we use the results to investigate and discuss possible ecological trade-offs and associated fitness advantages. These may explain the hypothesized repeated loss of this trait. The frequent association between large seed size and desiccation sensitivity is also considered.

title: The evolution of seeds.
abstract: The evolution of the seed represents a remarkable life-history transition for photosynthetic organisms. Here, we review the recent literature and historical understanding of how and why seeds evolved. Answering the 'how' question involves a detailed understanding of the developmental morphology and anatomy of seeds, as well as the genetic programs that determine seed size. We complement this with a special emphasis on the evolution of dormancy, the characteristic of seeds that allows for long 'distance' time travel. Answering the 'why' question involves proposed hypotheses of how natural selection has operated to favor the seed life-history phenomenon. The recent flurry of research describing the comparative biology of seeds is discussed. The review will be divided into sections dealing with: (1) the development and anatomy of seeds; (2) the endosperm; (3) dormancy; (4) early seed-like structures and the transition to seeds; and (5) the evolution of seed size (mass). In many cases, a special distinction is made between angiosperm and gymnosperm seeds. Finally, we make some recommendations for future research in seed biology.

title: The evolution and biological significance of seeds
abstract: No evolutionary event in the colonization of the land by vascular plants is of greater significance than the appearance of the seed habit. What apparently began as an adaptation enhancing sexual reproduction in the absence of external free water has assumed a far broader importance in terms of survival and dispersal. The essential features of the seed habit are identified in relation to heterospory, megaspore retention, and endosporic gametophyte development and approaches to the seed habit by nonseed plants are reviewed. Seeds first appear in the fossil record of the Late Devonian Period and, in diverse forms and sizes, constitute a significant component of Carboniferous fossil floras. The rise to dominance of plants of gymnospermous affinity in the early Mesozoic and the appearance and rapid adaptive radiation of the angiosperms are discussed in relation to the significance of the seed habit. The structure and development of modern gymnosperm and angiosperm seeds are compared, and the contrasting patter...

title: Implications of the lack of desiccation tolerance in recalcitrant seeds
abstract: A suite of interacting processes and mechanisms enables tolerance of desiccation and storage (conservation) of orthodox seeds in the dry state. While this is a long-term option under optimized conditions, dry orthodox seeds are not immortal, with life spans having been characterized as short, intermediate and long. Factors facilitating desiccation tolerance are metabolic “switch-off” and intracellular dedifferentiation. Recalcitrant seeds lack these mechanisms, contributing significantly to their desiccation sensitivity. Consequently, recalcitrant seeds, which are shed at high water contents, can be stored only in the short-term, under conditions not allowing dehydration. The periods of such hydrated storage are constrained by germination that occurs without the need for extraneous water, and the proliferation of seed-associated fungi. Cryopreservation is viewed as the only option for long-term conservation of the germplasm of recalcitrant-seeded species. This is not easily achieved, as each of the necessary procedures imposes oxidative damage. Intact recalcitrant seeds cannot be cryopreserved, the common practice being to use excised embryos or embryonic axes as explants. Dehydration is a necessary procedure prior to exposure to cryogenic temperatures, but this is associated with metabolism-linked injury mediated by uncontrolled reactive oxygen species generation and failing anti-oxidant systems. While the extent to which this occurs can be curtailed by maximizing drying rate (flash drying) it cannot be completely obviated. Explant cooling for, and rewarming after, cryostorage must necessarily be rapid, to avoid ice crystallization. The ramifications of desiccation sensitivity are discussed, as are problems involved in cryostorage, particularly those accompanying dehydration and damage consequent upon ice crystallization. While desiccation sensitivity is a “fact” of seed recalcitrance, resolutions of the difficulties involved germplasm conservation are possible as discussed.

title: Oleosins prevent oil-body coalescence during seed imbibition as suggested by a low-temperature scanning electron microscope study of desiccation-tolerant and -sensitive oilseeds
abstract: In order to clarify further the physiological role of oleosins in seed development, we characterized the oil-body proteins of several oilseeds exhibiting a range of desiccation sensitivities from the recalcitrant (Theobroma cacao L., Quercus rubra L.), intermediate (Coffea arabica L., Azadirachta indica A. Juss.) and orthodox categories (Sterculia setigera Del., Brassica napus L.). The estimated ratio of putative oleosins to lipid in oil bodies of Q. rubra was less than 5% of the equivalent values for rapeseed oil bodies. No oleosin was detected in T. cacao oil bodies. In A. indica cotyledons, oil bodies contained very low amounts of putative oleosins. Oil bodies both from C. arabica and S. setigera exhibited a similar ratio of putative oleosins to lipid as found in rapeseed. In C. arabica seeds, the central domain of an oleosin was partially sequenced. Using a low temperature field-emission scanning electron microscope, the structural stability of oil bodies was investigated in seeds after drying, storage in cold conditions and rehydration. Despite the absence or relative dearth of oleosins in desiccation-sensitive, recalcitrant oilseeds, oil bodies remained relatively stable after slow or fast drying. In A. indica seeds exposed to a lethal cold storage treatment, no significant change in oil-body sizes was observed. In contrast, during imbibition of artificially dried seeds containing low amounts of putative oleosins, the oil bodies fused to form large droplets, resulting in the loss of cellular integrity. No damage to the oil bodies occurred in imbibed seeds of Q. rubra, C. arabica and S. setigera. Thus the rehydration phase appears to be detrimental to the stability of oil bodies when these are present in large amounts and are lacking oleosins. We therefore suggest that one of the functions of oleosins in oilseed development may be to stabilize oil bodies during seed imbibition prior to germination.

title: Arabidopsis Seed Development and Germination Is Associated with Temporally Distinct Metabolic Switches1[W]
abstract: While the metabolic networks in developing seeds during the period of reserve accumulation have been extensively characterized, much less is known about those present during seed desiccation and subsequent germination. Here we utilized metabolite profiling, in conjunction with selective mRNA and physiological profiling to characterize Arabidopsis (Arabidopsis thaliana) seeds throughout development and germination. Seed maturation was associated with a significant reduction of most sugars, organic acids, and amino acids, suggesting their efficient incorporation into storage reserves. The transition from reserve accumulation to seed desiccation was associated with a major metabolic switch, resulting in the accumulation of distinct sugars, organic acids, nitrogen-rich amino acids, and shikimate-derived metabolites. In contrast, seed vernalization was associated with a decrease in the content of several of the metabolic intermediates accumulated during seed desiccation, implying that these intermediates might support the metabolic reorganization needed for seed germination. Concomitantly, the levels of other metabolites significantly increased during vernalization and were boosted further during germination sensu stricto, implying their importance for germination and seedling establishment. The metabolic switches during seed maturation and germination were also associated with distinct patterns of expression of genes encoding metabolism-associated gene products, as determined by semiquantitative reverse transcription-polymerase chain reaction and analysis of publicly available microarray data. When taken together our results provide a comprehensive picture of the coordinated changes in primary metabolism that underlie seed development and germination in Arabidopsis. They furthermore imply that the metabolic preparation for germination and efficient seedling establishment initiates already during seed desiccation and continues by additional distinct metabolic switches during vernalization and early germination.

title: Seed desiccation: a bridge between maturation and germination.
abstract: The development of orthodox seeds concludes by a desiccation phase. The dry seeds then enter a phase of dormancy, also called the after-ripening phase, and become competent for germination. We discuss physiological processes as well as gene expression and metabolic programs occurring during the desiccation phase in respect to their contribution to the desiccation tolerance, dormancy competence and successful germination of the dry seeds. The transition of developing seeds from the phase of reserve accumulation to desiccation is associated with distinct gene expression and metabolic switches. Interestingly, a significant proportion of the gene expression and metabolic signatures of seed desiccation resemble those characterizing seed germination, implying that the preparation of the seeds for germination begins already during seed desiccation.

title: Temporal profiling of the heat-stable proteome during late maturation of Medicago truncatula seeds identifies a restricted subset of late embryogenesis abundant proteins associated with longevity.
abstract: Developing seeds accumulate late embryogenesis abundant (LEA) proteins, a family of intrinsically disordered and hydrophilic proteins that confer cellular protection upon stress. Many different LEA proteins exist in seeds, but their relative contribution to seed desiccation tolerance or longevity (duration of survival) is not yet investigated. To address this, a reference map of LEA proteins was established by proteomics on a hydrophilic protein fraction from mature Medicago truncatula seeds and identified 35 polypeptides encoded by 16 LEA genes. Spatial and temporal expression profiles of the LEA polypeptides were obtained during the long maturation phase during which desiccation tolerance and longevity are sequentially acquired until pod abscission and final maturation drying occurs. Five LEA polypeptides, representing 6% of the total LEA intensity, accumulated upon acquisition of desiccation tolerance. The gradual 30-fold increase in longevity correlated with the accumulation of four LEA polypeptides, representing 35% of LEA in mature seeds, and with two chaperone-related polypeptides. The majority of LEA polypeptides increased around pod abscission during final maturation drying. The differential accumulation profiles of the LEA polypeptides suggest different roles in seed physiology, with a small subset of LEA and other proteins with chaperone-like functions correlating with desiccation tolerance and longevity.

title: An integrated overview of seed development in Arabidopsis thaliana ecotype WS
abstract: This work is part of a research program aiming at identifying and studying genes involved in Arabidopsis thaliana seed maturation. We focused here on the Wassilewskija ecotype seed development and linked physiological and biochemical data, including protein, oil, soluble sugars, starch and free amino acid measurements, to embryo development, to obtain a complete and thorough reference data set. A. thaliana seed development can be divided into three stages. During early embryogenesis (i.e. morphogenesis), seed weight and lipid content were low whereas important amounts of starch were transiently accumulated. In the second stage, or maturation phase, a rapid increase in seed dry weight was observed and storage oils and proteins were accumulated in large quantities, accounting for approximately 40% of dry matter each at the end of this stage. During the third and last stage (late maturation including acquisition of desiccation tolerance), seed dry weight remained constant while an acute loss of water took place in the seed. Storage compound synthesis ended concomitantly with sucrose, stachyose and raffinose accumulation. This study revealed the occurrence of metabolic activities such as protein synthesis, in the final phase of embryo desiccation. A striking correlation between peaks in hexose to sucrose ratio and transition phases during embryogenesis was observed.

title: The continuing conundrum of the LEA proteins
abstract: Research into late embryogenesis abundant (LEA) proteins has been ongoing for more than 20 years but, although there is a strong association of LEA proteins with abiotic stress tolerance particularly dehydration and cold stress, for most of that time, their function has been entirely obscure. After their initial discovery in plant seeds, three major groups (numbered 1, 2 and 3) of LEA proteins have been described in a range of different plants and plant tissues. Homologues of groups 1 and 3 proteins have also been found in bacteria and in certain invertebrates. In this review, we present some new data, survey the biochemistry, biophysics and bioinformatics of the LEA proteins and highlight several possible functions. These include roles as antioxidants and as membrane and protein stabilisers during water stress, either by direct interaction or by acting as molecular shields. Along with other hydrophilic proteins and compatible solutes, LEA proteins might also serve as “space fillers” to prevent cellular collapse at low water activities. This multifunctional capacity of the LEA proteins is probably attributable in part to their structural plasticity, as they are largely lacking in secondary structure in the fully hydrated state, but can become more folded during water stress and/or through association with membrane surfaces. The challenge now facing researchers investigating these enigmatic proteins is to make sense of the various in vitro defined functions in the living cell: Are the LEA proteins truly multi-talented, or are they still just misunderstood?

title: A review of recalcitrant seed physiology in relation to desiccation-tolerance mechanisms
abstract: A suite of mechanisms or processes that together have been implicated in the acquisition and maintenance of desiccation tolerance in orthodox seeds is discussed in the context of the behaviour of desiccation-sensitive seeds, and where appropriate, parallels are drawn with the situation in vegetative plant tissues that tolerate dehydration. Factors included are: physical characteristics of cells and intracellular constituents; insoluble reserve accumulation; intracellular de-differentiation; metabolic ‘switching off’; presence, and efficient operation, of antioxidant systems; accumulation of putatively protective substances including LEAs, sucrose and other oligosaccharides, as well as amphipathic molecules; the presence and role of oleosins; and the presence and operation of repair systems during rehydration. The variable response to dehydration shown by desiccation-sensitive seeds is considered in terms of the absence or incomplete expression of this suite of mechanisms or processes. Three categories of damage are envisaged: (i) reduction in cell volume which can lead to mechanical damage; (ii) aqueous-based degradative processes, probably consequent upon deranged metabolism at intermediate water contents. This is termed ‘metabolism-induced damage’ and its extent will depend upon the metabolic rate and the rate of dehydration; and (iii) the removal of water intimately associated with macromolecular surfaces leading to denaturation: this is referred to as desiccation damage sensu stricto . The effects of drying rate and the maturity status of seeds are considered in relation to the responses to dehydration, leading to the conclusion that the concept of critical water contents on a species basis is inappropriate. Viewing seed postharvest physiology in terms of a continuum of behaviour is considered to be more realistic than attempting precise categorization. Rapid dehydration of excised embryonic axes (or other explants) from desiccation-sensitive seeds permits retention of viability (in the short term) to water contents approaching the level of non-freezable water. This opens up the possibility of long-term conservation, by cryopreservation techniques, of the genetic resources of species producing non-orthodox seeds.

title: Regulation of the seed to seedling developmental phase transition by the LAFL and VAL transcription factor networks
abstract: In the seed, a fundamental transition between embryo and vegetative phases of plant development is coordinated by the interaction between the AFL and VAL sub‐clades of the plant specific B3 domain transcription factor family. The AFL B3 factors together with LEC1‐type HAP3 transcription factors promote embryo maturation; whereas the VAL B3 factors repress the LEC1/AFL (LAFL) network during seed germination. Recent advances reveal that genes in key developmental programs and hormone signaling pathways are downstream targets of the LAFL network highlighting the central role of the LAFL network in integration of intrinsic developmental and hormonal signals during plant development. The VAL B3 proteins are proposed to mediate repression by recruiting a histone deacetylase complex (HDAC) to LAFL genes that contain the Sph/RY cis‐element recognized by AFL and VAL B3‐DNA‐binding domains. In addition to VAL B3 factors, epigenetic mechanisms are implicated in maintaining repression of LAFL network during vegetative development.

title: Isolation of the Arabidopsis ABI3 gene by positional cloning.
abstract: Arabidopsis abi3 mutants are altered in various aspects of seed development and germination that reflect a decreased responsiveness to the hormone abscisic acid. The ABI3 gene has been isolated by positional cloning. A detailed restriction fragment length polymorphism (RFLP) map of the abi3 region was constructed. An RFLP marker closely linked to the abi3 locus was identified, and by analyzing an overlapping set of cosmid clones containing this marker, the abi3 locus was localized within a 35-kb region. An 11-kb subfragment was then shown to complement the mutant phenotype in transgenic plants, thereby further delimiting the position of the locus. A candidate ABI3 gene was identified within this fragment as being expressed in developing fruits. The primary structure of the encoded protein was deduced from sequence analysis of a corresponding cDNA clone. In the most severe abi3-4 allele, the size of this predicted protein was reduced by 40% due to the presence of a point mutation that introduced a premature stop codon. The predicted ABI3 protein displays discrete regions of high similarity to the maize viviparous-1 protein.

title: Arabidopsis LEAFY COTYLEDON1 Is Sufficient to Induce Embryo Development in Vegetative Cells
abstract: The Arabidopsis LEAFY COTYLEDON1 (LEC1) gene is required for the specification of cotyledon identity and the completion of embryo maturation. We isolated the LEC1 gene and showed that it functions at an early developmental stage to maintain embryonic cell fate. The LEC1 gene encodes a transcription factor homolog, the CCAAT box-binding factor HAP3 subunit. LEC1 RNA accumulates only during seed development in embryo cell types and in endosperm tissue. Ectopic postembryonic expression of the LEC1 gene in vegetative cells induces the expression of embryo-specific genes and initiates formation of embryo-like structures. Our results suggest that LEC1 is an important regulator of embryo development that activates the transcription of genes required for both embryo morphogenesis and cellular differentiation.

title: FUSCA3 encodes a protein with a conserved VP1/AB13-like B3 domain which is of functional importance for the regulation of seed maturation in Arabidopsis thaliana.
abstract: Conditionally lethal mutant alleles of the FUSCA3 (FUS3) gene of Arabidopsis thaliana are specifically defective in the gene expression program responsible for seed maturation. FUS3 was isolated by map-based cloning and expression of the FUS3 cDNA resulted in complementation of the Fus3- phenotype. In the predicted FUS3 gene product, a continuous stretch of more than 100 amino acids shows significant sequence similarity to the B3 domains of the polypeptides encoded by ABI3 (Arabidopsis) and VP1 (maize). FUS3 transcription was detected mainly in siliques and was found to be developmentally regulated during embryogenesis. Transcripts of abnormal sizes were observed in fus3 mutants due to aberrant splicing caused by point mutations at intron termini. Sequence analysis of mutant and wild-type FUS3 alleles, as well as sequencing of fus3 cDNAs, revealed small inframe deletions at two different sites of the coding region. While a deletion between B3 and the C-terminus of the predicted polypeptide was found in conjunction with normal FUS3 function, another deletion located within the conserved B3 domain (as well as truncations therein) were associated with the Fus3- phenotype. It is apparent, therefore, that an intact B3 domain is essential for the regulation of seed maturation by FUS3.

title: LEAFY COTYLEDON2 encodes a B3 domain transcription factor that induces embryo development
abstract: The Arabidopsis LEAFY COTYLEDON2 (LEC2) gene is a central embryonic regulator that serves critical roles both early and late during embryo development. LEC2 is required for the maintenance of suspensor morphology, specification of cotyledon identity, progression through the maturation phase, and suppression of premature germination. We cloned the LEC2 gene on the basis of its chromosomal position and showed that the predicted polypeptide contains a B3 domain, a DNA-binding motif unique to plants that is characteristic of several transcription factors. We showed that LEC2 RNA accumulates primarily during seed development, consistent with our finding that LEC2 shares greatest similarity with the B3 domain transcription factors that act primarily in developing seeds, VIVIPAROUS1/ABA INSENSITIVE3 and FUSCA3. Ectopic, postembryonic expression of LEC2 in transgenic plants induces the formation of somatic embryos and other organ-like structures and often confers embryonic characteristics to seedlings. Together, these results suggest that LEC2 is a transcriptional regulator that establishes a cellular environment sufficient to initiate embryo development.

title: LEA polypeptide profiling of recalcitrant and orthodox legume seeds reveals ABI3-regulated LEA protein abundance linked to desiccation tolerance
abstract: In contrast to orthodox seeds that acquire desiccation tolerance during maturation, recalcitrant seeds are unable to survive drying. These desiccation-sensitive seeds constitute an interesting model for comparative analysis with phylogenetically close species that are desiccation tolerant. Considering the importance of LEA (late embryogenesis abundant) proteins as protective molecules both in drought and in desiccation tolerance, the heat-stable proteome was characterized in cotyledons of the legume Castanospermum australe and it was compared with that of the orthodox model legume Medicago truncatula. RNA sequencing identified transcripts of 16 homologues out of 17 LEA genes for which polypeptides are detected in M. truncatula seeds. It is shown that for 12 LEA genes, polypeptides were either absent or strongly reduced in C. australe cotyledons compared with M. truncatula seeds. Instead, osmotically responsive, non-seed-specific dehydrins accumulated to high levels in the recalcitrant cotyledons compared with orthodox seeds. Next, M. truncatula mutants of the ABSCISIC ACID INSENSITIVE3 (ABI3) gene were characterized.  Mtabi3 seeds were found to be desiccation sensitive when dried below a critical water content of 0.4g H2O g DW–1. Characterization of the LEA proteome of the Mtabi3 seeds revealed a subset of LEA proteins with severely reduced abundance that were also found to be reduced or absent in C. australe cotyledons. Transcripts of these genes were indeed shown to be ABI3 responsive. The results highlight those LEA proteins that are critical to desiccation tolerance and suggest that comparable regulatory pathways responsible for their accumulation are missing in both desiccation-sensitive genotypes, revealing new insights into the mechanistic basis of the recalcitrant trait in seeds.

title: ABI3 controls embryo degreening through Mendel's I locus
abstract: Occurrence of mature green seeds in oil-seed crops such as canola and soybean causes severe losses in revenue. Retention of chlorophyll in seeds can be an undesirable trait as it affects seed maturation, seed oil, and meal quality. We show that the abscisic acid (ABA, plant hormone) dependent transcription factor ABSCISIC ACID INSENSITIVE 3 (ABI3), confers seed degreening by regulating Mendel’s stay-green genes. This study unveils a new role for ABI3 in removal of seed chlorophyll in addition to its functions in embryo maturation and conferring desiccation tolerance. This pathway could be manipulated to tackle the cold-induced green seed problem in oil-seed crops. Chlorophyll (chl) is essential for light capture and is the starting point that provides the energy for photosynthesis and thus plant growth. Obviously, for this reason, retention of the green chlorophyll pigment is considered a desirable crop trait. However, the presence of chlorophyll in mature seeds can be an undesirable trait that can affect seed maturation, seed oil quality, and meal quality. Occurrence of mature green seeds in oil crops such as canola and soybean due to unfavorable weather conditions during seed maturity is known to cause severe losses in revenue. One recently identified candidate that controls the chlorophyll degradation machinery is the stay-green gene, SGR1 that was mapped to Mendel’s I locus responsible for cotyledon color (yellow versus green) in peas. A defect in SGR1 leads to leaf stay-green phenotypes in Arabidopsis and rice, but the role of SGR1 in seed degreening and the signaling machinery that converges on SGR1 have remained elusive. To decipher the gene regulatory network that controls degreening in Arabidopsis, we have used an embryo stay-green mutant to demonstrate that embryo degreening is achieved by the SGR family and that this whole process is regulated by the phytohormone abscisic acid (ABA) through ABSCISIC ACID INSENSITIVE 3 (ABI3); a B3 domain transcription factor that has a highly conserved and essential role in seed maturation, conferring desiccation tolerance. Misexpression of ABI3 was sufficient to rescue cold-induced green seed phenotype in Arabidopsis. This finding reveals a mechanistic role for ABI3 during seed degreening and thus targeting of this pathway could provide a solution to the green seed problem in various oil-seed crops.

title: A mutant of Arabidopsis which is defective in seed development and storage protein accumulation is a new abi3 allele
abstract: In order to investigate the role of the plant hormones gibberellin (GA) and abscisic acid (ABA) in seed development and germination the GA biosynthetic inhibitor, Uniconazol, was used to isolate mutants with abnormal germination profiles. In one of these mutants, the ability to germinate on Uniconazol is due to a mutation in the ABI3 gene. However, unlike the previously reported abi3 mutant, this line displays an array of seed-specific developmental defects. The accumulation of seed reserve proteins is dramatically reduced due to reduced levels of the storage protein mRNA. The embryos remain green throughout development and are desiccation intolerant. However, immature seeds are completely non-dormant and grow normally. These results suggest the ABI3 gene is essential for the synthesis of seed storage proteins and for the protection of the embryo during desiccation.

title: A regulatory role for the ABI3 gene in the establishment of embryo maturation in Arabidopsis thaliana
abstract: The ABI3 gene product of Arabidopsis is essential for correct completion of seed maturation. A severe mutant allele at this locus results in seed that remain green, fail to establish desiccation tolerance, and that germinate at a developmental stage when wild-type seed will not. Moreover, the formation of leaf primordia and xylem differentiation, both characteristic of germinating wild-type seedlings, can be observed in embryos harvested 12 days after flowering. Thus, mature abi3 embryos reach a developmental state that more closely resembles the character of a developing seedling rather than that of a dormant embryo. Previous studies of this gene have resulted in the suggestion that ABI3 is a transducer of abscisic acid induced seed dormancy. Our results demonstrate that the ABI3 gene product can be most accurately described as one of the major regulators of the transition between embryo maturation and early seedling development, rather than simply a transducer of the abscisic acid signal.

title: Natural modifiers of seed longevity in the Arabidopsis mutants abscisic acid insensitive3-5 (abi3-5) and leafy cotyledon1-3 (lec1-3).
abstract: *Seed longevity is an important trait in many crops and is essential for the success of most land plant species. Current knowledge of its molecular regulation is limited. The Arabidopsis mutants abscisic acid insensitive3-5 (abi3-5) and leafy cotyledon1-3 (lec1-3) have impaired seed maturation and quickly lose seed viability. abi3-5 and lec1-3 were used as sensitized genetic backgrounds for the study of seed longevity. We exploited the natural variation of Arabidopsis to create introgression lines from the Seis am Schlern and Shahdara accessions in, respectively, the abi3-5 and lec1-3 backgrounds. These lines carry natural modifiers of the abi3 and lec1 phenotypes. Longevity tests and a proteomic analysis were conducted to describe the seed physiology of each line. The modifier lines showed improved seed longevity. The Shahdara modifiers can partially re-establish the seed developmental programs controlled by LEC1 and restore the accumulation of seed storage proteins that are reduced in abi3-5 and lec1-3. The isolation and characterization of natural modifiers of the seed maturation mutants abi3-5 and lec1-3, and the analysis of their seed proteomes, advance our current understanding of seed longevity.

title: The re-establishment of desiccation tolerance in germinated radicles of Medicago truncatula Gaertn. seeds
abstract: Germinated seeds of Medicago truncatula Gaertn. with a protruded radicle length of 2.7 mm did not survive drying below 0.2 g H2O g–1 dw, as indicated by vital stain assays and the absence of growth resumption after rehydration. The re-establishment of desiccation tolerance was achieved using an osmotic treatment with polyethylene glycol (PEG), combined with a cold treatment. The ability to regain desiccation tolerance after germination was restricted to a period of growth characterized by radicle lengths between 1 and 3 mm. After PEG treatment of germinated seeds with 2.7 mm long radicles at –1.7 MPa at 10°C for 3 d and subsequent drying to 0.04 g H2O g–1 dw, 90% survived and developed into normal seedlings after rehydration. Desiccation tolerance could also be re-established in excised radicles, demonstrating that cotyledons were not essential for this process. Upon PEG incubation, sucrose accumulated rapidly prior to the re-establishment of desiccation tolerance in germinated radicles, regardless of the presence of cotyledons. Induction of MtDHN (a dehydrin) gene expression was correlated with the re-establishment of desiccation tolerance. Furthermore, the PEG-induced expression of MtDHN was repressed when fluridone was added to the PEG solution.

title: Loss of desiccation tolerance during germination in neo-tropical pioneer seeds: implications for seed mortality and germination characteristics†
abstract: Orthodox, desiccation-tolerant seeds lose desiccation tolerance during germination. Here, we quantify the timing of the loss of desiccation tolerance, and explore the implications of this event for seed mortality and the shape of germination progress curves for pioneer tree species. For the nine species studied, all seeds in a seedlot lost desiccation tolerance after the same fixed proportion of their time to germination, and this proportion was fairly constant across the species (0.63–0.70). The loss of desiccation tolerance after a fixed proportion of the time to germination has the implication that the maximum number of seeds in a seedlot that can be killed by a single drying event during germination (Mmax) increases with an increasing time to 50% germination (t50) and an increasing slope of the germination progress curve. Consequently, to prevent the seed population from becoming highly vulnerable to desiccation-induced mortality, species with a greater t50 would be expected to have a shallower germination progress curve. In conclusion, these data suggest that the loss of desiccation tolerance during germination may constitute a significant, but previously unexplored, source of mortality for seeds in seasonal environments with unpredictable rainfall.

title: Disappearance of desiccation tolerance of imbibed crop seeds is not associated with the decline of oligosaccharides
abstract: The relationship between sugar content and loss of desiccation tolerance of hydrated crop seeds (tomato, okra, snow pea, mung bean, and cucumber) was evaluated by imbibing seeds with or without ABA, followed by dehydration and germination. During the process of hydration, but before the seeds lost desiccation tolerance, monosaccharide content increased only slightly, sucrose increased in snow pea, mung bean and cucumber, but maintained its original level in other species and the oligosaccharides declined dramatically. At the time of losing desiccation tolerance, the sucrose content of imbibed seeds was 2-3 times higher than the original level in most species. Positive significant correlation coefficients (r) were found in many, but not all crop seeds between desiccation tolerance and the oligosaccharide mass, or oligo/sucrose ratio. The ratio of oligo/sucrose in intact seeds at the time of losing desiccation tolerance, however, was not a fixed value and varied among species. Oligosaccharides declined significantly in different seed parts of imbibed cucumber seeds while sucrose increased to a higher level in the radicle than in the hypocotyl. Radicles were far more sensitive to desiccation than hypocotyls. The same observation was found for cucumber seeds imbibed in 100 μM ABA, yet desiccation tolerance was largely maintained in hypocotyls and cotyledons. It is concluded that sucrose and oligosaccharides are not the determinants of the loss of desiccation tolerance in hydrated seeds. Imbibed seeds did not show any differences between seed parts in their ability to resynthesize sugars during the process of slow dehydration. Differences in sensitivity to desiccation among seed parts were not due to differences in the initial water content or to the rate of water content increase among seed parts. Physiological regulation of the loss of desiccation tolerance in crop seeds during hydration is discussed.

title: Induction of desiccation tolerance in germinated seeds
abstract: The induction of desiccation tolerance in germinated seeds by incubation in PEG for several days is reported. The induction coincided with an increased sucrose content and the formation of heat-soluble proteins. Germinated seeds can serve as a convenient model system in studies of desiccation tolerance. This finding may have important implications for the agricultural industry.

title: Abscisic acid (ABA) sensitivity regulates desiccation tolerance in germinated Arabidopsis seeds.
abstract: During germination, orthodox seeds lose their desiccation tolerance (DT) and become sensitive to extreme drying. Yet, DT can be rescued, in a well-defined developmental window, by the application of a mild osmotic stress before dehydration. A role for abscisic acid (ABA) has been implicated in this stress response and in DT re-establishment. However, the path from the sensing of an osmotic cue and its signaling to DT re-establishment is still largely unknown. Analyses of DT, ABA sensitivity, ABA content and gene expression were performed in desiccation-sensitive (DS) and desiccation-tolerant Arabidopsis thaliana seeds. Furthermore, loss and re-establishment of DT in germinated Arabidopsis seeds was studied in ABA-deficient and ABA-insensitive mutants. We demonstrate that the developmental window in which DT can be re-established correlates strongly with the window in which ABA sensitivity is still present. Using ABA biosynthesis and signaling mutants, we show that this hormone plays a key role in DT re-establishment. Surprisingly, re-establishment of DT depends on the modulation of ABA sensitivity rather than enhanced ABA content. In addition, the evaluation of several ABA-insensitive mutants, which can still produce normal desiccation-tolerant seeds, but are impaired in the re-establishment of DT, shows that the acquisition of DT during seed development is genetically different from its re-establishment during germination.

title: Stress-associated factors increase after desiccation of germinated seeds of Tabebuia impetiginosa Mart.
abstract: Improved re-establishment of desiccation tolerance was studied in germinated seeds of Tabebuia impetiginosa Mart. by exposing to a polyethylene glycol solution prior to desiccation. The effects of different osmotic potentials and drying rates were studied. In addition, temporary temperature stress and exogenous abscisic acid were applied to evaluate their effect on desiccation tolerance of the protruded radicle. An osmotic potential of −1.7 MPa at 5°C followed by slow drying was most effective in the re-establishment of desiccation tolerance in protruded radicles with a length up to 3 mm. An osmotic potential of −1.4 or −2.0 MPa was less effective. Fast drying completely prevented the re-induction of desiccation tolerance. Cold shock or heat shock prior to osmotic treatment as well as abscisic acid added to the osmotic solution improved desiccation tolerance of protruded radicles. Surprisingly, survival of the germinated seed did not depend on re-establishment of desiccation tolerance in the protruded radicle. Even after the protruded radicle became necrotic and died, the production of adventitious roots from the hypocotyls allowed for survival and the development of high quality seedlings. Thus, T. impetiginosa appeared to be well adapted to the seasonally dry biome in which the species thrives via mechanisms that offer protection against desiccation in the young seedling stage.

title: Metabolic dysfunction and unabated respiration precede the loss of membrane integrity during dehydration of germinating radicles.
abstract: This study shows that dehydration induces imbalanced metabolism before loss of membrane integrity in desiccation-sensitive germinated radicles. Using a photoacoustic detection system, responses of CO(2) emission and fermentation to drying were analyzed non-invasively in desiccation-tolerant and -intolerant radicles of cucumber (Cucumis sativa) and pea (Pisum sativum). Survival after drying and a membrane integrity assay showed that desiccation tolerance was present during early imbibition and lost in germinated radicles. However, tolerance could be re-induced in germinated cucumber radicles by incubation in polyethylene glycol before drying. Tolerant and polyethylene glycol (PEG)-induced tolerant radicles exhibited a much-reduced CO(2) production before dehydration compared with desiccation-sensitive radicles. This difference was maintained during dehydration. In desiccation-sensitive tissues, dehydration induced an increase in the emission of acetaldehyde and ethanol that peaked well before the loss of membrane integrity. Acetaldehyde emission from sensitive radicles was significantly reduced when dehydration occurred in 50% O(2) instead of air. Acetaldehyde/ethanol were not detected in dehydrating tolerant radicles of either species or in polyethylene glycol-induced tolerant cucumber radicles. Thus, a balance between down-regulation of metabolism during drying and O(2) availability appears to be associated with desiccation tolerance. Using Fourier transform infrared spectroscopy, acetaldehyde was found to disturb the phase behavior of phospholipid vesicles, suggesting that the products resulting from imbalanced metabolism in seeds may aggravate membrane damage induced by dehydration.

title: Desiccation and survival in the recalcitrant seeds of Avicennia marina: DNA replication, DNA repair and protein synthesis
abstract: An autoradiographic study was made of leucine and thymidine incorporation into the meristematic root primordia and hypocotyl tips of seeds of the recalcitrant mangrove species, Avicennia marina. The investigations show that although there is a temporary reduction of protein synthesis at shedding, root primordia and surrounding hypocotyl cells of the axis never wholly cease incorporation of [3H]leucine and regain pre-shedding levels of activity within a day. Precursor studies using methyl-[3H]thymidine show that, at shedding, there is a temporary cessation of incorporation into root meristem nuclei that lasts no longer than 48 h and, within a day, pre-shedding levels are regained in the meristem nuclei. Analysis of DNA fragmentation patterns in root tips at the time of shedding, and their ability to repair radiation-induced DNA damage, indicate that DNA repair processes are markedly compromised in these cells if water loss reaches 22%. Protein synthesis and DNA replication are reduced by more than half by a water loss of 18% and 16%, respectively. DNA replication does not fully recover on rehydration after only 8% water loss. DNA fragmentation to nucleosomes indicates a programme of cell death at a water loss of 10%. We suggest that the feature of continuous protein synthesis activity with only a temporary interruption in active cell cycling in A. marina root primordia helps to explain both the rapidity in seedling establishment and the extreme vulnerability to desiccation.

title: Changes in DNA and microtubules during loss and re-establishment of desiccation tolerance in germinating Medicago truncatula seeds.
abstract: Desiccation tolerance (DT) in orthodox seeds is acquired during seed development and lost upon imbibition/germination, purportedly upon the resumption of DNA synthesis in the radicle cells. In the present study, flow cytometric analyses and visualization of microtubules (MTs) in radicle cells of seedlings of Medicago truncatula showed that up to a radicle length of 2 mm, there is neither DNA synthesis nor cell division, which were first detected in radicles with a length of 3 mm. However, DT started to be lost well before the resumption of DNA synthesis, when germinating seeds were dried back. By applying an osmotic treatment with polyethylene glycol (PEG) before dehydration, it was possible to re-establish DT in seedlings with a radicle up to 2 mm long. Dehydration of seedlings with a 2 mm radicle, with or without PEG treatment, caused disassembly of MTs and appearance of tubulin granules. Subsequent pre-humidification led to an almost complete disappearance of both MTs and tubulin granules. Upon rehydration, neither MTs nor tubulin granules were detected in radicle cells of untreated seedlings, while PEG-treated seedlings were able to reconstitute the microtubular cytoskeleton and continue their normal development. Dehydration of untreated seedlings also led to an apoptotic-like DNA fragmentation in radicle cells, while in PEG-treated seedlings DNA integrity was maintained. The results showed that for different cellular components, desiccation-tolerant seedlings may apply distinct strategies to survive dehydration, either by avoidance or further repair of the damages.

title: DNA and desiccation tolerance
abstract: This article reviews mechanisms by which specialized cells of different life forms have overcome the lethal effects of dehydration and considers how the maintenance of genetic information is central to survival As a dynamic and hydrated molecule in vivo, DNA can assume different conformational structures depending upon the water activity, the base sequence and the presence of specific binding proteins The attainment of stable secondary structures that are resistant to degradation in vivo at low water potentials is proposed as a likely accompaniment to desiccation tolerance In addition, chemical modification of bases in DNA, the extent of methylation and conformational changes could determine the expression of different gene sequences as cells pass from desiccation-tolerant to desiccationintolerant states We monitored the integrity of extracted DNA in embryos of seeds and in winddispersed pollen during transition from their desiccation tolerance to desiccation intolerance on hydration and germination. We present evidence to show that the DNA of these two stages is different and that it is the DNA from desiccation-tolerant cells only that retains integrity when the cells are subjected to desiccation regimes We discuss these findings in relation to certain hydration-sensitive DNA structures and to other relevant biological systems

title: Influence of priming-induced nuclear replication activity on storability of pepper ( Capsicum annuum L.) seed
abstract: The effect of two priming treatments in PEG solutions on nuclear replication stages of pepper ( Capsicum annuum L.) embryos were evaluated using flow cytometry. Priming at −1.1 MPa for 10 d induced almost 40% of nuclei in the embryo root tips to enter the synthetic phase of nuclear division, while no DNA synthesis occurred during 6 d at−1.5 MPa and in less than 10% of the nuclei at 10 d. Both priming treatments effectively increased seed performance. A decrease of 2.7 d in mean germination time (MGT) was observed after priming in PEG solution at −1.1 MPa, while the treatment at−1.5 MPa lowered the MGT by 2.1 d. Primed and untreated seeds were subjected to controlled deterioration treatments by exposure to 45°C after equilibration at 75% RH. Both osmotic treatments considerably lowered seed tolerance to adverse storage conditions as compared with untreated seeds. However, seeds in which DNA replication was induced by priming were more sensitive to controlled deterioration than seeds in which priming did not induce nuclei to enter the synthetic phase. This could be a consequence of a higher DNA content, which is a more vulnerable target for mutation inducing factors. Alternatively the increased sensitivity could be a consequence of their more advanced progress in germinative events, making seeds less resistant to deteriorative factors imposed during storage. On the whole, from the present results it appears that the nucleic acid DNA content of the embryo only plays an additive role in influencing seed storability.

title: A plant DNA ligase is an important determinant of seed longevity.
abstract: DNA repair is important for maintaining genome integrity. In plants, DNA damage accumulated in the embryo of seeds is repaired early in imbibition, and is important for germination performance and seed longevity. An essential step in most repair pathways is the DNA ligase-mediated rejoining of single- and double-strand breaks. Eukaryotes possess multiple DNA ligase enzymes, each having distinct roles in cellular metabolism. Here, we report the characterization of DNA LIGASE VI, which is only found in plant species. The primary structure of this ligase shows a unique N-terminal region that contains a β-CASP motif, which is found in a number of repair proteins, including the DNA double-strand break (DSB) repair factor Artemis. Phenotypic analysis revealed a delay in the germination of atlig6 mutants compared with wild-type lines, and this delay becomes markedly exacerbated in the presence of the genotoxin menadione. Arabidopsis atlig6 and atlig6 atlig4 mutants display significant hypersensitivity to controlled seed ageing, resulting in delayed germination and reduced seed viability relative to wild-type lines. In addition, atlig6 and atlig6 atlig4 mutants display increased sensitivity to low-temperature stress, resulting in delayed germination and reduced seedling vigour upon transfer to standard growth conditions. Seeds display a rapid transcriptional DNA DSB response, which is activated in the earliest stages of water imbibition, providing evidence for the accumulation of cytotoxic DSBs in the quiescent seed. These results implicate AtLIG6 and AtLIG4 as major determinants of Arabidopsis seed quality and longevity.

title: The loss of desiccation tolerance during germination: An ultrastructural and biochemical approach
abstract: SummaryRye embryos of high viability and vigour can be imbibed for 1 hour, dehydrated and subsequently rehydrated without harm. However, extension of the imbibition period results in progressive structural damage to cells of both the embryonic root and the coleorhiza. Greatest sensitivity to this treatment is shown by the microtubule assembly system and the plasmalemma which loses its integrity permitting the egress of ribosomes and lipid towards the cell wall. Further stress results in fragmentation of the endoplasmic reticulum, disruption of plastid, mitochondrial and nuclear membranes and the dispersion of the contents of provacuoles. Damage is initiated during the drying of imbibed embryos but it is compounded by subsequent rehydration. Coleorhiza cells, particularly those distal to the root, which normally develop very rapidly during the early hours of germination are most sensitive to desiccation. The onset of sensitivity to desiccation (ca. 3 hours after imbibition) corresponds with a transitory halt in the increasing rate of protein synthesis and with the start of DNA replication. These results are discussed in relation to DNA repair and the “hardening” of seeds to stimulate rapid growth following rehydration.

title: Genetic Analysis of Desiccation Tolerance in Saccharomyces cerevisiae
abstract: Desiccation tolerance, the ability to survive nearly total dehydration, is a rare strategy for survival and reproduction observed in all taxa. However, the mechanism and regulation of this phenomenon are poorly understood. Correlations between desiccation tolerance and potential effectors have been reported in many species, but their physiological significance has not been established in vivo. Although the budding yeast Saccharomyces cerevisiae exhibits extreme desiccation tolerance, its usefulness has been hampered by an inability to reduce tolerance more than a few fold by physiological or genetic perturbations. Here we report that fewer than one in a million yeast cells from low-density logarithmic cultures survive desiccation, while 20–40% of cells from saturated cultures survive. Using this greatly expanded metric, we show that mutants defective in trehalose biosynthesis, hydrophilins, responses to hyperosmolarity, and hypersalinity, reactive oxygen species (ROS) scavenging and DNA damage repair nevertheless retain wild-type levels of desiccation tolerance, suggesting that this trait involves a unique constellation of stress factors. A genome-wide screen for mutants that render stationary cells as sensitive as log phase cells identifies only mutations that block respiration. Respiration as a prerequisite for acquiring desiccation tolerance is corroborated by respiration inhibition and by growth on nonfermentable carbon sources. Suppressors bypassing the respiration requirement for desiccation tolerance reveal at least two pathways, one of which, involving the Mediator transcription complex, is associated with the shift from fermentative to respiratory metabolism. Further study of these regulators and their targets should provide important clues to the sensors and effectors of desiccation tolerance.

title: TOR and RAS pathways regulate desiccation tolerance in Saccharomyces cerevisiae
abstract: Desiccation is thought to impose many stresses. Which of these stresses is responsible for desiccation-induced death and how the stress response is regulated are unknown, however. Here we use Saccharomyces cerevisiae to show that reduction of a 60S biogenesis intermediate via RAS or TOR down-regulation increases desiccation tolerance.

title: Comparative Analysis of the Heat Stable Proteome of Radicles of Medicago truncatula Seeds during Germination Identifies Late Embryogenesis Abundant Proteins Associated with Desiccation Tolerance1[W]
abstract: A proteomic analysis was performed on the heat stable protein fraction of imbibed radicles of Medicago truncatula seeds to investigate whether proteins can be identified that are specifically linked to desiccation tolerance (DT). Radicles were compared before and after emergence (2.8 mm long) in association with the loss of DT, and after reinduction of DT by an osmotic treatment. To separate proteins induced by the osmotic treatment from those linked with DT, the comparison was extended to 5 mm long emerged radicles for which DT could no longer be reinduced, albeit that drought tolerance was increased. The abundance of 15 polypeptides was linked with DT, out of which 11 were identified as late embryogenesis abundant proteins from different groups: MtEm6 (group 1), one isoform of DHN3 (dehydrins), MtPM25 (group 5), and three members of group 3 (MP2, an isoform of PM18, and all the isoforms of SBP65). In silico analysis revealed that their expression is likely seed specific, except for DHN3. Other isoforms of DNH3 and PM18 as well as three isoforms of the dehydrin Budcar5 were associated with drought tolerance. Changes in the abundance of MtEm6 and MtPM25 in imbibed cotyledons during the loss of DT and in developing embryos during the acquisition of DT confirmed the link of these two proteins with DT. Fourier transform infrared spectroscopy revealed that the recombinant MtPM25 and MtEm6 exhibited a certain degree of order in the hydrated state, but that they became more structured by adopting α helices and β sheets during drying. A model is presented in which DT-linked late embryogenesis abundant proteins might exert different protective functions at high and low hydration levels.

title: A postgermination developmental arrest checkpoint is mediated by abscisic acid and requires the ABI5 transcription factor in Arabidopsis
abstract: Seed dormancy is a trait of considerable adaptive significance because it maximizes seedling survival by preventing premature germination under unfavorable conditions. Understanding how seeds break dormancy and initiate growth is also of great agricultural and biotechnological interest. Abscisic acid (ABA) plays primary regulatory roles in the initiation and maintenance of seed dormancy. Here we report that the basic leucine zipper transcription factor ABI5 confers an enhanced response to exogenous ABA during germination, and seedling establishment, as well as subsequent vegetative growth. These responses correlate with total ABI5 levels. We show that ABI5 expression defines a narrow developmental window following germination, during which plants monitor the environmental osmotic status before initiating vegetative growth. ABI5 is necessary to maintain germinated embryos in a quiescent state thereby protecting plants from drought. As expected for a key player in ABA-triggered processes, ABI5 protein accumulation, phosphorylation, stability, and activity are highly regulated by ABA during germination and early seedling growth.

title: The isolation and characterization of abscisic acid-insensitive mutants of Arabidopsis thaliana
abstract: Abscisic acid (ABA) insensitive mutants of Arabidopsis thaliana (L.) Heynh. were isolated by selecting plants which grew well on a medium containing 10 μM ABA. From the progeny of approximately 3500 mutagen-treated seeds, five mutants of at least three different loci were isolated. Three mutants were characterized, moreover, by a reduced seed dormancy and by symptoms of withering, indicating disturbed water relations and, therefore, resembled phenotypically the ABA-deficient mutants we described earlier in this species. Two mutants showed in addition only a reduction of seed dormancy. Compared to wild type, all mutants showed similar or increased levels of endogenous ABA in developing seeds and fruits (siliquae). The role of the different genes involved is discussed in relation to the mechanism of ABA action.

title: The Arabidopsis Abscisic Acid Response Locus ABI4 Encodes an APETALA2 Domain Protein
abstract: Arabidopsis abscisic acid (ABA)–insensitive abi4 mutants have pleiotropic defects in seed development, including decreased sensitivity to ABA inhibition of germination and altered seed-specific gene expression. This phenotype is consistent with a role for ABI4 in regulating seed responses to ABA and/or seed-specific signals. We isolated the ABI4 gene by positional cloning and confirmed its identity by complementation analysis. The predicted protein product shows homology to a plant-specific family of transcriptional regulators characterized by a conserved DNA binding domain, the APETALA2 domain. The single mutant allele identified has a single base pair deletion, resulting in a frameshift that should disrupt the C-terminal half of the protein but leave the presumed DNA binding domain intact. Expression analyses showed that despite the seed-specific nature of the mutant phenotype, ABI4 expression is not seed specific.

title: Mutations at two new Arabidopsis ABA response loci are similar to the abi3 mutations
abstract: Two new Arabidopsis loci involved in ABA response, ABA-insensitive (ABI)4 and ABI5, have been identified by mutation. The abi4 and abi5 mutants were characterized in terms of ABA sensitivity of seed germination, dormancy, seed-specific gene expression and stomatal regulation. Their phenotypes are similar to mutations in ABI3, which is thought to encode a seed-specific transcriptional activator. Analysis of double mutants combining abi4 and abi5 mutations with abi1, abi2 or abi3 mutations also suggests that ABI4 and ABI5 act in the same transduction pathway as ABI3.

title: The Arabidopsis Abscisic Acid Response Gene ABI5 Encodes a Basic Leucine Zipper Transcription Factor
abstract: The Arabidopsis abscisic acid (ABA)–insensitive abi5 mutants have pleiotropic defects in ABA response, including decreased sensitivity to ABA inhibition of germination and altered expression of some ABA-regulated genes. We isolated the ABI5 gene by using a positional cloning approach and found that it encodes a member of the basic leucine zipper transcription factor family. The previously characterized abi5-1 allele encodes a protein that lacks the DNA binding and dimerization domains required for ABI5 function. Analyses of ABI5 expression provide evidence for ABA regulation, cross-regulation by other ABI genes, and possibly autoregulation. Comparison of seed and ABA-inducible vegetative gene expression in wild-type and abi5-1 plants indicates that ABI5 regulates a subset of late embryogenesis–abundant genes during both developmental stages.

title: A null mutation in a bZIP factor confers ABA-insensitivity in Arabidopsis thaliana.
abstract: We have used a modification of the classical ABA-insensitive screen (Koornneef et al. 1984) to isolate novel mutations in the ABA signal transduction pathway of Arabidopsis thaliana. In our screen, mutants were recovered on the basis of their growth-insensitivity to ABA (GIA) rather than germination-insensitivity. Here we present the isolation of the gia1 mutant as well as the identification of the gia1 gene by positional cloning and complementation studies. GIA1 is predicted to code for a bZIP transcription factor with high homology to previously characterized plant bZIP transcription factors (DPBF1, ABFs and TRAB1) known for their ability to bind ABA-responsive DNA elements. Our results provide in vivo evidence that a bZIP factor may indeed be involved in ABA signaling. Since GIA1 turned out to be identical to ABI5, we designated GIA1 as ABI5 in the present paper.

title: Toward the identification and regulation of the Arabidopsis thaliana ABI3 regulon
abstract: The plant-specific, B3 domain-containing transcription factor ABSCISIC ACID INSENSITIVE3 (ABI3) is an essential component of the regulatory network controlling the development and maturation of the Arabidopsis thaliana seed. Genome-wide chromatin immunoprecipitation (ChIP-chip), transcriptome analysis, quantitative reverse transcriptase–polymerase chain reaction and a transient promoter activation assay have been combined to identify a set of 98 ABI3 target genes. Most of these presumptive ABI3 targets require the presence of abscisic acid for their activation and are specifically expressed during seed maturation. ABI3 target promoters are enriched for G-box-like and RY-like elements. The general occurrence of these cis motifs in non-ABI3 target promoters suggests the existence of as yet unidentified regulatory signals, some of which may be associated with epigenetic control. Several members of the ABI3 regulon are also regulated by other transcription factors, including the seed-specific, B3 domain-containing FUS3 and LEC2. The data strengthen and extend the notion that ABI3 is essential for the protection of embryonic structures from desiccation and raise pertinent questions regarding the specificity of promoter recognition.

title: Abscisic acid: emergence of a core signaling network.
abstract: Abscisic acid (ABA) regulates numerous developmental processes and adaptive stress responses in plants. Many ABA signaling components have been identified, but their interconnections and a consensus on the structure of the ABA signaling network have eluded researchers. Recently, several advances have led to the identification of ABA receptors and their three-dimensional structures, and an understanding of how key regulatory phosphatase and kinase activities are controlled by ABA. A new model for ABA action has been proposed and validated, in which the soluble PYR/PYL/RCAR receptors function at the apex of a negative regulatory pathway to directly regulate PP2C phosphatases, which in turn directly regulate SnRK2 kinases. This model unifies many previously defined signaling components and highlights the importance of future work focused on defining the direct targets of SnRK2s and PP2Cs, dissecting the mechanisms of hormone interactions (i.e., cross talk) and defining connections between this new negative regulatory pathway and other factors implicated in ABA signaling.

title: In Vivo Inhibition of Seed Development and Reserve Protein Accumulation in Recombinants of Abscisic Acid Biosynthesis and Responsiveness Mutants in Arabidopsis thaliana.
abstract: In Arabidopsis thaliana, seed development in recombinants of the ABA-deficient aba mutant with the ABA response mutants abi1 or abi3 is compared to wild type and the monogenic parents. Aberrant seed development occurred in the aba,abi3 recombinant and was normal in aba,abi1, abi3 and aba,abi1 seeds. Embryos of the recombinant aba,abi3 seeds maintained the green color until maturity, the seeds kept a high water content, did not form the late abundant 2S and 12S storage proteins, were desiccation intolerant, and often showed viviparous germination. Application of ABA, and particularly of an ABA analog, to the roots of plants during seed development partially alleviated the aberrant phenotype. Seeds of aba,abi3 were normal when they developed on a mother plant heterozygous for Aba. In contrast to seed development, the induction of dormancy was blocked in all monogenic mutants and recombinants. Dormancy was only induced by embryonic ABA; it could not be increased by maternal ABA or ABA applied to the mother plant. It is concluded that endogenous ABA has at least two different effects in developing seeds. The nature of these responses and of the ABA response system is discussed.

title: A mutational analysis of the ABA1 gene of Arabidopsis thaliana highlights the involvement of ABA in vegetative development.
abstract: Much of the literature on the phytohormone abscisic acid (ABA) describes it as a mediator in triggering plant responses to environmental stimuli, as well as a growth inhibitor. ABA-deficient mutants, however, display a stunted phenotype even under well-watered conditions and high relative humidity, which suggests that growth promotion may also be one of the roles of endogenous ABA. Zeaxanthin epoxidase, the product of the ABA1 gene of Arabidopsis thaliana, catalyses the epoxidation of zeaxanthin to antheraxanthin and violaxanthin, generating the epoxycarotenoid precursor of the ABA biosynthetic pathway. This paper gives a description of the molecular and phenotypic characterization of a large series of mutant alleles of the ABA1 gene, which cause different degrees of ABA deficiency, four of them previously isolated (aba1-1, aba1-3, aba1-4, and aba1-6) and the remaining five novel (sañ1-1, sañ1-2, sañ1-3, sañ1-4, and sre3). Molecular analysis of these alleles provides insights into the domains in which they compromise zeaxanthin epoxidase function. The size of the leaves, inflorescences, and flowers of these mutants is reduced, and their rosettes have lower fresh and dry weights than their wild types, as a result of a diminished cell size. Low concentrations of exogenous ABA increase the fresh weight of mutant and wild-type plants, as well as the dry weight of the mutants. The leaves of aba1 mutants are abnormally shaped and fail to develop clearly distinct spongy and palisade mesophyll layers. Taken together, these phenotypic traits indicate, as suggested by previous authors, that ABA acts as a growth promoter during vegetative development. The abnormal shape and internal structure of the leaves of aba1 mutants suggests, in addition, a role for ABA in organogenesis.

title: Seed‐specific immunomodulation of abscisic acid activity induces a developmental switch
abstract: A single‐chain Fv antibody (scFv) gene, which has previously been used to immunomodulate abscisic acid (ABA) activity in transgenic tobacco to create a ‘wilty’ phenotype, was put under control of the seed‐specific USP promoter from Vicia faba and used to transform tobacco. Transformants were phenotypically similar to wild‐type plants apart from their seeds. Anti‐ABA scFv embryo development differed markedly from wild‐type embryo development. Seeds which accumulated similar levels of a scFv that binds to oxazolone, a hapten absent from plants, developed like wild‐type embryos. Anti‐ABA scFv embryos developed green cotyledons containing chloroplasts and accumulated photosynthetic pigments but produced less seed storage protein and oil bodies. Anti‐ABA scFv seeds germinated precociously if removed from seed capsules during development but were incapable of germination after drying. Total ABA levels were higher than in wild‐type seeds but calculated free ABA levels were near‐zero until 21 days after pollination. We show for the first time seed‐specific immunomodulation and the resulting switch from the seed maturation programme to a germination programme. We conclude that the immunomodulation of hormones can alter the development programme of target organs, allowing the study of the directly blocked endogenous molecules and manipulation of the system concerned.

title: Three Arabidopsis SnRK2 protein kinases, SRK2D/SnRK2.2, SRK2E/SnRK2.6/OST1 and SRK2I/SnRK2.3, involved in ABA signaling are essential for the control of seed development and dormancy.
abstract: ABA is an important phytohormone regulating various plant processes, including stress tolerance, seed development and germination. SRK2D/SnRK2.2, SRK2E/SnRK2.6/OST1 and SRK2I/SnRK2.3 are redundant ABA-activated SNF1-related protein kinases 2 (SnRK2s) in Arabidopsis thaliana. We examined the role of these protein kinases in seed development and germination. These SnRK2 proteins were mainly expressed in the nucleus during seed development and germination. The triple mutant (srk2d srk2e srk2i) was sensitive to desiccation and showed severe growth defects during seed development. It exhibited a loss of dormancy and elevated seed ABA content relative to wild-type plants. The severity of these phenotypes was far stronger than that of any single or double SRK2D, SRK2E and SRK2I mutants, including the srk2d srk2i mutant. The triple mutant had greatly reduced phosphorylation activity in in-gel kinase experiments using basic leucine zipper (bZIP) transcription factors including ABI5. Microarray experiments revealed that 48 and 30% of the down-regulated genes in abi5 and abi3 seeds were suppressed in the triple mutant seeds, respectively. Moreover, disruption of the three protein kinases induced global changes in the up-regulation of ABA-repressive gene expression, as well as the down-regulation of ABA-inducible gene expression. These alterations in gene expression result in a loss of dormancy and severe growth defects during seed development. Collectively, these results indicate that SRK2D, SRK2E and SRK2I protein kinases involved in ABA signaling are essential for the control of seed development and dormancy through the extensive control of gene expression.

title: Interaction between sugar and abscisic acid signalling during early seedling development in Arabidopsis
abstract: Sugars regulate important processes and affect the expression of many genes in plants. Characterization of Arabidopsis (Arabidopsis thaliana) mutants with altered sugar sensitivity revealed the function of abscisic acid (ABA) signalling in sugar responses. However, the exact interaction between sugar signalling and ABA is obscure. Therefore ABA deficient plants with constitutive ABI4 expression (aba2-1/35S::ABI4) were generated. Enhanced ABI4 expression did not rescue the glucose insensitive (gin) phenotype of aba2 seedlings indicating that other ABA regulated factors are essential as well. Interestingly, both glucose and ABA treatment of Arabidopsis seeds trigger a post-germination seedling developmental arrest. The glucose-arrested seedlings had a drought tolerant phenotype and showed glucose-induced expression of ABSCISIC ACID INSENSITIVE3 (ABI3), ABI5 and LATE EMBRYOGENESIS ABUNDANT (LEA) genes reminiscent of ABA signalling during early seedling development. ABI3 is a key regulator of the ABA-induced arrest and it is shown here that ABI3 functions in glucose signalling as well. Multiple abi3 alleles have a glucose insensitive (gin) phenotype comparable to that of other known gin mutants. Importantly, glucose-regulated gene expression is disturbed in the abi3 background. Moreover, abi3 was insensitive to sugars during germination and showed sugar insensitive (sis) and sucrose uncoupled (sun) phenotypes. Mutant analysis further identified the ABA response pathway genes ENHANCED RESPONSE TO ABA1 (ERA1) and ABI2 as intermediates in glucose signalling. Hence, three previously unidentified sugar signalling genes have been identified, showing that ABA and glucose signalling overlap to a larger extend than originally thought.

title: The sugar-insensitive1 (sis1) mutant of Arabidopsis is allelic to ctr1.
abstract: Soluble sugar levels affect a diverse array of plant developmental processes. For example, exposure to high levels of glucose or sucrose inhibits early seedling development of Arabidopsis thaliana (L.) Heynh. Media-shift experiments indicate that Arabidopsis seedlings lose their sensitivity to the inhibitory effects of high sugar levels on early development within approximately two days after the start of imbibition. The sugar-insensitive1 (sis1) mutant of Arabidopsis was isolated by screening for plants that are insensitive to the inhibitory effects of high concentrations of sucrose on early seedling development. The sis1 mutant also displays glucose and mannose resistant phenotypes and has an osmo-tolerant phenotype during early seedling development. The sis1 mutant is resistant to the negative effects of paclobutrazol, an inhibitor of gibberellin biosynthesis, on seed germination. Characterization of the sis1 mutant revealed that it is allelic to ctr1, a previously identified mutant with a constitutive response to ethylene.

title: Abscisic acid-induced rearrangement of intracellular structures associated with freezing and desiccation stress tolerance in the liverwort Marchantia polymorpha.
abstract: The plant growth regulator abscisic acid (ABA) is known to be involved in triggering responses to various environmental stresses such as freezing and desiccation in angiosperms, but little is known about its role in basal land plants, especially in liverworts, representing the earliest land plant lineage. We show here that survival rate after freezing and desiccation of Marchantia polymorpha gemmalings was increased by pretreatment with ABA in the presence of increasing concentrations of sucrose. ABA treatment increased accumulation of soluble sugars in gemmalings, and sugar accumulation was further increased by addition of sucrose to the culture medium. ABA treatment of gemmalings also induced accumulation of transcripts for proteins with similarity to late embryogenesis abundant (LEA) proteins, which accumulate in association with acquisition of desiccation tolerance in maturing seeds. Observation by light and electron microscopy indicated that the ABA treatment caused fragmentation of vacuoles with increased cytosolic volume, which was more prominent in the presence of a high concentration of external sucrose. ABA treatment also increased the density of chloroplast distribution and remarkably enlarged their volume. These results demonstrate that ABA induces drastic physiological changes in liverwort cells for stress tolerance, accompanied by accumulation of protectants against dehydration and rearrangement and morphological alterations of cellular organelles.

title: Role of the Arabidopsis Glucose Sensor HXK1 in Nutrient, Light, and Hormonal Signaling
abstract: Glucose modulates many vital processes in photosynthetic plants. Analyses of Arabidopsis glucose insensitive2(gin2) mutants define the physiological functions of a specific hexokinase (HXK1) in the plant glucose-signaling network. HXK1 coordinates intrinsic signals with extrinsic light intensity. HXK1 mutants lacking catalytic activity still support various signaling functions in gene expression, cell proliferation, root and inflorescence growth, and leaf expansion and senescence, thus demonstrating the uncoupling of glucose signaling from glucose metabolism. The gin2 mutants are also insensitive to auxin and hypersensitive to cytokinin. Plants use HXK as a glucose sensor to interrelate nutrient, light, and hormone signaling networks for controlling growth and development in response to the changing environment.

title: Annual changes in dormancy and germination in seeds of Sisymbrium officinale (L.) Scop.
abstract: The effect of environmental conditions on changes in dormancy and germination of seeds of Sisymbrium officinale (L.) Scop, was studied. Seeds were buried in soil in the field or in incubators and at regular intervals, germination of exhumed seeds was tested over a range of conditions. Seeds that were buried in the field showed clear seasonal changes in dormancy. Fluctuations in soil moisture and nitrate content were not required for the changes in dormancy. Temperature seemed to be the only factor regulating these changes. Dormancy was relieved in periods of low temperatures and induced in periods of high temperatures. Fresh seeds and seeds buried for only a few months germinated best at elevated temperatures whereas seeds buried for a longer time germinated best at low-temperatures. Light, nitrate and desiccation stimulated germination of exhumed seeds. As a consequence, these treatments extended germination over a much longer period of the year. Germination of exhumed seeds in Petri dishes at field temperature was reasonably described by a model based on the dual role of temperature, in the regulation of both dormancy and subsequent germination.

title: Are Seasonal Dormancy Patterns in Arabidopsis thaliana Regulated by Changes in Seed Sensitivity to Light, Nitrate and Gibberellin?
abstract: Imbibed seeds of Arabidopsis thaliana (L.) Heynh., passed annually through a pattern of changes in dormancy. Dormancy was broken in summer and re-induced in autumn-winter. A second small germination flush occurred in early spring. The role of sensitivity to light, nitrate and gibberellins (GAs) in regulating annual dormancy patterns and germination was studied with the use of GA-deficient (gal-2) and wild-type seeds. Dark-incubated seeds were exposed to a natural temperature regime for periods up to 18 months and at regular intervals germination capacity of portions of seeds was tested at laboratory conditions. Germination data fitted as logistic dose response curves showed that sensitivity to light varied with the seasons in both genotypes. From interpretation of curve parameters, it is proposed that the observed sensitivity changes involve alterations in the number of receptors, in the binding characteristics of the receptors and/or in the response chain initiated by ligand-receptor interaction. In this response chain GA biosynthesis is stimulated (wild type) and sensitivity to GAs is enhanced (wild type, gal -2). GA sensitivity is also directly influenced by temperature, thus without the interference of light. However, the significance of direct regulation of GA requirement seemed to diminish with prolonged incubation outdoors, whereas reversible changes in light sensitivity remained clear. Therefore, we propose that seasonal dormancy patterns are mainly regulated by changes in sensitivity to light. GA sensitivity contributes to this pattern but is not primarily controlling dormancy. The GA requirement for germination is obvious as gal-2 seeds did not germinate at any time of the year when deprived of applied GAs. However, GA biosynthesis is not required for dormancy control, as a dormancy pattern was also observed in the absence of the capacity to synthesize GAs. Nitrate or sensitivity to nitrate did not contribute to the regulation of dormancy and germination of this species.

title: Dormancy cycling in Arabidopsis seeds is controlled by seasonally distinct hormone-signaling pathways
abstract: Seeds respond to environmental signals, tuning their dormancy cycles to the seasons and thereby determining the optimum time for plant establishment. The molecular regulation of dormancy cycling is unknown, but an extensive range of mechanisms have been identified in laboratory experiments. Using a targeted investigation of gene expression over the dormancy cycle of Arabidopsis seeds in the field, we investigated how these mechanisms are seasonally coordinated. Depth of dormancy and gene expression patterns were correlated with seasonal changes in soil temperature. The results were consistent with abscisic acid (ABA) signaling linked to deep dormancy in winter being repressed in spring concurrent with enhanced DELLA repression of germination as depth of dormancy decreased. Dormancy increased during winter as soil temperature declined and expression of ABA synthesis (NCED6) and gibberellic acid (GA) catabolism (GA2ox2) genes increased. This was linked to an increase in endogenous ABA that plateaus, but dormancy and DOG1 and MFT expression continued to increase. The expression of SNF1-related protein kinases, SnrK 2.1 and 2.4, also increased consistent with enhanced ABA signaling and sensitivity being modulated by seasonal soil temperature. Dormancy then declined in spring and summer. Endogenous ABA decreased along with positive ABA signaling as expression of ABI2, ABI4, and ABA catabolism (CYP707A2) and GA synthesis (GA3ox1) genes increased. However, during the low-dormancy phase in the summer, expression of transcripts for the germination repressors RGA and RGL2 increased. Unlike deep winter dormancy, this represson can be removed on exposure to light, enabling the completion of germination at the correct time of year.

title: Dual Effect of Light on the Gibberellin- and Nitrate-Stimulated Seed Germination of Sisymbrium officinale and Arabidopsis thaliana.
abstract: Red light (R) has a dual effect on the seed germination of the two related species Arabidopsis thaliana and Sisymbrium officinale. The two species provide different means to separate the light-effects. In S. officinale, stimulation of germination by R depends on the stimultaneous presence of nitrate (light-effect I). The effect of both factors is completely blocked by tetcyclacis, an inhibitor of gibberellin (GA)-biosynthesis. Addition of a mixture of gibberellins A(4) and A(7) (GA(4+7)) antagonizes the inhibition. In the absence of nitrate, R shifts germination to lower GA-requirement (light-effect II). In A. thaliana a similar second light-effect is seen on the GA-requirement of GA-deficient ga-1 mutant seeds. R stimulates germination of wild type seeds in water (light-effect I). For both species, light-effect I shows a fluence threshold value of approximately 10(-5) moles per square meter, which is independent of the nitrate concentration. Increasing nitrate concentrations narrow the fluence-range required for maximal germination whereby the product of nitrate concentration and fluence value determines the germination level, indicating a multiplicative interaction between R and nitrate. Fluence-response curves for light-effect II are similar for both species. Germination occurs in the range of 10(-6) to 10(-2) moles per square meter fluence. The maximal level of germination is determined by the level of dark-germination and light-effect II. Increasing GA(4+7) concentrations induce a shift to lower fluence values. It is shown that in the second effect the co-action of R and exogenous GA(4+7) is clearly additive. It is concluded that light-effect I induces a chain of events leading to GA biosynthesis. Light-effect II seems to enhance the sensitivity of the seeds to GAs.

title: LEAF-CANOPY-INDUCED SEED DORMANCY IN A GRASSLAND FLORA
abstract: The germination of 27 species from calcareous grassland was tested in three light treatments in the laboratory: in darkness, under tungsten lamps and under a leaf-canopy. Germination was significantly lower under a leaf-canopy than in the dark in 17 species. The phenomena of leaf-canopy induced dormancy was not associated with the life-span of species, though there was a weak, negative relationship with seed weight amongst 20 perennials in the sample. It is suggested that the co-existence of species at high density in calcareous grasslands may, in part, be explained by differences in germination response to fine-grained variations in the light environment at the soil surface.

title: SOMNUS, a CCCH-Type Zinc Finger Protein in Arabidopsis, Negatively Regulates Light-Dependent Seed Germination Downstream of PIL5[W]
abstract: Light absorbed by seed phytochromes of Arabidopsis thaliana modulates abscisic acid (ABA) and gibberellic acid (GA) signaling pathways at least partly via PHYTOCHROME-INTERACTING FACTOR3-LIKE5 (PIL5), a phytochrome-interacting basic helix-loop-helix transcription factor. Here, we report a new mutant, somnus (som), that germinates in darkness, independently of various light regimens. SOM encodes a nucleus-localized CCCH-type zinc finger protein. The som mutant has lower levels of ABA and elevated levels of GA due to expressional changes in ABA and GA metabolic genes. Unlike PIL5, however, SOM does not regulate the expression of GA-INSENSITIVE and REPRESSOR OF GA1 (RGA/RGA1), two DELLA genes encoding GA negative signaling components. Our in vivo analysis shows that PIL5 activates the expression of SOM by binding directly to its promoter, suggesting that PIL5 regulates ABA and GA metabolic genes partly through SOM. In agreement with these results, we also observed that the reduced germination frequency of a PIL5 overexpression line is rescued by the som mutation and that this rescue is accompanied by expressional changes in ABA and GA metabolic genes. Taken together, our results indicate that SOM is a component in the phytochrome signal transduction pathway that regulates hormone metabolic genes downstream of PIL5 during seed germination.

title: Light activates the degradation of PIL5 protein to promote seed germination through gibberellin in Arabidopsis.
abstract: Angiosperm seeds integrate various environmental signals, such as water availability and light conditions, to make a proper decision to germinate. Once the optimal conditions are sensed, gibberellin (GA) is synthesized, triggering germination. Among environmental signals, light conditions are perceived by phytochromes. However, it is not well understood how phytochromes regulate GA biosynthesis. Here we investigated whether phytochromes regulate GA biosynthesis through PIL5, a phytochrome-interacting bHLH protein, in Arabidopsis. We found that pil5 seed germination was inhibited by paclobutrazol, the ga1 mutation was epistatic to the pil5 mutation, and the inhibitory effect of PIL5 overexpression on seed germination could be rescued by exogenous GA, collectively indicating that PIL5 regulates seed germination negatively through GA. Expression analysis revealed that PIL5 repressed the expression of GA biosynthetic genes (GA3ox1 and GA3ox2), and activated the expression of a GA catabolic gene (GA2ox) in both PHYA- and PHYB-dependent germination assays. Consistent with these gene-expression patterns, the amount of bioactive GA was higher in the pil5 mutant and lower in the PIL5 overexpression line. Lastly, we showed that red and far-red light signals trigger PIL5 protein degradation through the 26S proteasome, thus releasing the inhibition of bioactive GA biosynthesis by PIL5. Taken together, our data indicate that phytochromes promote seed germination by degrading PIL5, which leads to increased GA biosynthesis and decreased GA degradation.

title: Through form to function: root hair development and nutrient uptake.
abstract: Root hairs project from the surface of the root to aid nutrient and water uptake and to anchor the plant in the soil. Their formation involves the precise control of cell fate and localized cell growth. We are now beginning to unravel the complexities of the molecular interactions that underlie this developmental regulation. In addition, after years of speculation, nutrient transport by root hairs has been demonstrated clearly at the physiological and molecular level, with evidence for root hairs being intense sites of H(+)-ATPase activity and involved in the uptake of Ca(2+), K(+), NH(4)(+), NO(3)(-), Mn(2+), Zn(2+), ) and H(2)PO(4)(-).

title: ABI5 acts downstream of ABI3 to execute an ABA-dependent growth arrest during germination.
abstract: The development of a germinating embryo into an autotrophic seedling is arrested under conditions of water deficit. This ABA-mediated developmental checkpoint requires the bZIP transcription factor ABI5. Here, we used abi3-1, which is also unable to execute this checkpoint, to investigate the relative role of ABI3 and ABI5 in this process. In wild-type Arabidopsis plants, ABI3 expression and activity parallel those described for ABI5 following stratification. During this process, transcript levels of late embryogenesis genes such as AtEm1 and AtEm6 are also re-induced, which might be responsible for the acquired osmotic tolerance in germinated embryos whose growth is arrested. ABI5 expression is greatly reduced in abi3-1 mutants, which has low AtEm1 or AtEm6 expression. Cross complementation experiments showed that 35S-ABI5 could complement abi3-1, whereas 35S-ABI3 cannot complement abi5-4. These results indicate that ABI5 acts downstream of ABI3 to reactivate late embryogenesis programmes and to arrest growth of germinating embryos. Although ABI5 is consistently located in the nucleus, chromosomal immunoprecipitation (ChIP) experiments revealed that ABA increases ABI5 occupancy on the AtEm6 promoter.

title: The role of chromatin-remodeling factor PKL in balancing osmotic stress responses during Arabidopsis seed germination.
abstract: Embryogenesis in Arabidopsis results in mature, osmotolerant embryos within dry seeds. Late-embryogenesis programs involve the transcription factors ABI3 and ABI5, which are necessary for osmotolerance. ABI3 and ABI5 are degraded in seeds initiating germination, abolishing their protected state. However, during an early stage of germination, strong osmotic stresses, or ABA exposure maintain ABI3 and ABI5 expression, leading to growth arrest, and osmotolerance. Mild stress stimuli delay ABI3 and ABI5 disappearance, retarding germination but not preventing eventual closure of embryogenesis programs. PICKLE (PKL), a putative chromatin modifier, is necessary to repress ABI3 and ABI5 expression during germination in response to ABA. We show that pkl mutants display persistent high expression of ABI3 and ABI5 upon ABA stimulation. In turn, maintenance of ABI5 expression leads to hypersensitive germination responses to ABA in pkl seeds. We provide evidence that ABI3 and ABI5 are less associated with repressed chromatin in pkl mutants. Our results provide evidence that PKL-dependent repression of embryonic gene expression extends to late-embryogenesis genes and is associated with changes in chromatin. We suggest that effective closure of embryogenesis omostolerance programs during germination prevents excessive plant reactions to stress.

title: The Arabidopsis Histone Deacetylases HDA6 and HDA19 Contribute to the Repression of Embryonic Properties after Germination1[W]
abstract: Histone deacetylase (HDAC) is a chromatin-remodeling factor that contributes to transcriptional repression in eukaryotes. In Arabidopsis (Arabidopsis thaliana), the transcription factors LEAFY COTYLEDON1 (LEC1), FUSCA3 (FUS3), and ABSCISIC ACID INSENSITIVE3 (ABI3) play key roles in embryogenesis. Although the repression of embryogenesis-related genes during germination has been proposed to occur, the role of HDAC in this process has not been elucidated. To address this question, the effects of an HDAC inhibitor and suppression of the Arabidopsis HDAC genes on this process were investigated. Here, we show that treatment of an HDA6 repression line with the HDAC inhibitor trichostatin A resulted in growth arrest and elevated transcription of LEC1, FUS3, and ABI3 during germination. The growth-arrest phenotype of the repression line was suppressed by lec1, fus3, and abi3. An HDA6/HDA19 double-repression line displayed arrested growth after germination and the formation of embryo-like structures on the true leaves of 6-week-old plants even without trichostatin A. The growth-arrest phenotype of this line was rescued by lec1. These results suggest that during germination in Arabidopsis, HDA6 and HDA19 redundantly regulate the repression of embryonic properties directly or indirectly via repression of embryo-specific gene function.

title: HISTONE DEACETYLASE 9 represses seedling traits in Arabidopsis thaliana dry seeds.
abstract: Plant life is characterized by major phase changes. We studied the role of histone deacetylase (HDAC) activity in the transition from seed to seedling in Arabidopsis. Pharmacological inhibition of HDAC stimulated germination of freshly harvested seeds. Subsequent analysis revealed that histone deacetylase 9 (hda9) mutant alleles displayed reduced seed dormancy and faster germination than wild-type plants. Transcriptome meta-analysis comparisons between the hda9 dry seed transcriptome and published datasets demonstrated that transcripts of genes that are induced during imbibition in wild-type prematurely accumulated in hda9-1 dry seeds. This included several genes associated with photosynthesis and photoautotrophic growth such as RuBisCO and RuBisCO activase (RCA). Chromatin immunoprecipitation experiments demonstrated enhanced histone acetylation levels at their loci in young hda9-1 seedlings. Our observations suggest that HDA9 negatively influences germination and is involved in the suppression of seedling traits in dry seeds, probably by transcriptional repression via histone deacetylation. Accordingly, HDA9 transcript is abundant in dry seeds and becomes reduced during imbibition in wild-type seeds. The proposed function of HDA9 is opposite to that of its homologous genes HDA6 and HDA19, which have been reported to repress embryonic properties in germinated seedlings.

title: Triple Loss of Function of Protein Phosphatases Type 2C Leads to Partial Constitutive Response to Endogenous Abscisic Acid1[C][W][OA]
abstract: The phytohormone abscisic acid (ABA) is a key regulator of plant growth and development as well as plant responses to situations of decreased water availability. Protein phosphatases type 2C (PP2Cs) from group A, which includes the ABI1/HAB1 and PP2CA branches, are key negative regulators of ABA signaling. Specifically, HAB1, ABI1, ABI2, and PP2CA have been shown to affect both seed and vegetative responses to ABA. To further understand their contribution to ABA signaling and to unravel possible genetic interactions and functional redundancy among them, we have generated different combinations of double and triple mutants impaired in these PP2Cs. Interestingly, hab1-1pp2ca-1 and abi1-2pp2ca-1 double mutants showed reduced water loss and enhanced resistance to drought stress, which further supports the role of PP2CA in vegetative responses to ABA. Two triple hab1-1abi1-2abi2-2 and hab1-1abi1-2pp2ca-1 mutants were generated, which showed an extreme response to exogenous ABA, impaired growth, and partial constitutive response to endogenous ABA. Thus, transcriptomic analysis revealed a partial up-regulation/down-regulation of a subset of ABA-responsive genes in both triple mutants in the absence of exogenous ABA. Comparison of ABA responses in the different pp2c mutants showed that a progressive increase in ABA sensitivity could be obtained through combined inactivation of these PP2Cs. These results indicate that ABA response is finely tuned by the integrated action of these genes, which is required to prevent a constitutive response to endogenous ABA that might have a deleterious effect on growth and development in the absence of environmental stress.

title: Seed dormancy release in Arabidopsis Cvi by dry after-ripening, low temperature, nitrate and light shows common quantitative patterns of gene expression directed by environmentally specific sensing.
abstract: The depth of seed dormancy can be influenced by a number of different environmental signals, but whether a common mechanism underlies this apparently similar response has yet to be investigated. Full-genome microarrays were used for a global transcript analysis of Arabidopsis thaliana Cape Verde Island accession seeds exposed to dry after-ripening (AR), or low temperature, nitrate and light when imbibed. Germination studies showed that the sensitivity of imbibed seeds to low temperature, nitrate and light was dependent upon the length of time spent AR following harvest. Seeds had an absolute requirement for light to complete dormancy release in all conditions, but this effect required an exposure to a prior dormancy relieving environment. Principal component analyses of the expression patterns observed grouped physiological states in a way that related to the depth of seed dormancy, rather than the type of environmental exposure. Furthermore, opposite changes in transcript abundance of genes in sets associated with dormancy, or dormancy relief through AR, were also related to the depth of dormancy and common to different environments. Besides these common quantitative changes, environment-specific gene expression patterns during dormancy relief are also described. For example, higher transcript abundance for genes linked to the process of nitrate accumulation, and nitrate reduction was associated with dormancy relief. The quantity of GA3ox1 transcripts increased during dormancy relief in all conditions, in particular when dormancy relief was completed by exposure to light. This contrasts with transcripts linked to abscisic acid (ABA) synthesis, which declined. The results are consistent with a role for the ABA/gibberellic acid balance in integrating dormancy-relieving environmental signals.

title: ABA-dependent and ABA-independent signaling in response to osmotic stress in plants.
abstract: Plants have adaptive robustness to osmotic stresses such as drought and high salinity. Numerous genes functioning in stress response and tolerance are induced under osmotic conditions in diverse plants. Various signaling proteins, such as transcription factors, protein kinases and phosphatases, play signal transduction roles during plant adaptation to osmotic stress, with involvement ranging from stress signal perception to stress-responsive gene expression. Recent progress has been made in analyzing the complex cascades of gene expression during osmotic stress response, and especially in identifying specificity and crosstalk in abscisic acid (ABA)-dependent and ABA-independent signaling pathways. In this review, we highlight transcriptional regulation of gene expression governed by two key transcription factors: AREB/ABFs and DREB2A operating respectively in ABA-dependent and ABA-independent signaling pathways.