Re-establishment Of Desiccation Tolerance Research Articles

The re-establishment of desiccation tolerance in germinated tomato (Solanum lycopersicum) seeds

Abstract Desiccation tolerance (DT) of seeds, one of the plant's environmental adaptation mechanisms, allows them to survive as seeds in a quiescent state under extremely water-deficient conditions during the plant's life cycle, followed by seed germination and seedling establishment under favourable water conditions. The seed-DT is lost after radicle emergence; however, there is a developmental period called the ‘DT window’ during which the germinating seeds can re-induce DT following a cue from their ambient low water potential (i.e. mild osmotic stress). The DT re-inducibility within the DT window has been used as a model biosystem for understanding molecular mechanisms that activate/supress DT in a number of plant species. However, the characteristics of the DT window for species particularly important to the agroindustry are still largely fragmented. Here, physiological analyses were performed, aiming to elucidate the properties of the DT window in tomato, a model species for Solanaceae, holding a key strategic position for the seed industry and commercial use around the world. We revealed that (i) the DT window of tomato seeds is closed when the developing radicle reaches about 4 mm after germination, (ii) the most effective ambient water potential to re-induce DT into seeds is about −1.5 MPa and (iii) there is organ specificity of DT re-induction with hypocotyls, showing a longer DT window than cotyledons and roots in post-germination seeds.

Role of reactive oxygen species and antioxidative enzymes in the loss and re-establishment of desiccation tolerance in germinated pea seeds

This study highlights the regulatory function of ROS and its detoxifying enzymes during the loss and re-establishment of desiccation tolerance (DT) in PEG pretreated and untreated germinated pea (Pisum sativum L.) seeds with 2 mm long radicles. Dehydration of untreated pea seedlings to initial water content (IWC; 0.11 gH2O g−1 DM) and below IWC (0.06 gH2O g−1 DM) reduced the DT to 60 and 20 %, respectively. Significant amelioration in DT was observed in PEG pretreated seedlings by ∼100 % and 70 % following dehydration at IWC and below IWC, respectively. Rendering DT in the PEG pretreated seedlings was negatively associated with an extensive reduction in superoxide (∼2.3 folds) and H2O2 (∼1.8 folds) levels compared to the untreated seedlings. Enhanced expression, levels (quantitative) and number (isoenzyme) of superoxide dismutase (SOD), catalase (CAT), and ascorbate peroxidase (APX) in the PEG pretreated seedlings negatively correlated with the lowering ROS levels and its associated oxidative damage. It is concluded that the re-induction of increased antioxidative capacity is a key factor in the re-establishment of DT in PEG-pretreated pea seedlings. Additionally, PEG pretreatment resulted in 50 % disappearance of superoxide in the sub-apical zone (SZ) of the radicle compared to the radicle of untreated seedlings dehydrated below IWC. Further, in situ detection of superoxide accumulation in the apical zone (AZ) was NADPH-oxidase dependent but in the SZ it was independent of NADPH-oxidase. Our results showed that the osmotic stress induced the expression of antioxidative enzymes in germinated pea seeds and made them desiccation tolerant against oxidative stress caused by desiccation/drought.

Mechanistic insights derived from re-establishment of desiccation tolerance in germinating xerophytic seeds: Caragana korshinskii as an example.

Germplasm conservation strongly depends on the desiccation tolerance (DT) of seeds. Xerophytic seeds have strong desiccation resistance, which makes them excellent models to study DT. Although some experimental strategies have been applied previously, most methods are difficult to apply to xerophytic seeds. In this review, we attempted to synthesize current strategies for the study of seed DT and provide an in-depth look at Caragana korshinskii as an example. First, we analyze congenital advantages of xerophytes in the study of seed DT. Second, we summarize several strategies used to study DT and illustrate a suitable strategy for xerophytic species. Then, based on our previous studies work with C. korshinskii, a feasible technical strategy for DT re-establishment is provided and we provide illustrate some special molecular mechanisms seen in xerophytic seeds. Finally, several steps to unveil the DT mechanism of xerophytic seeds are suggested, and three scientific questions that the field should consider are listed. We hope to optimize and utilize this strategy for more xerophytic species to more systematically decipher the physiological and molecular processes of seed DT and provide more candidate genes for molecular breeding.

Proteomic Analysis of Desiccation Tolerance and Its Re-Establishment in Different Embryo Axis Tissues of Germinated Pea Seeds.

The model of loss and re-establishment of desiccation tolerance (DT) in germinated seeds has been well developed to explore the mechanisms associated with DT, but little attention has been paid to the tissue variation in this model. Herein, we investigated DT in different embryo axis tissues of germinated pea seeds and its re-establishment by poly(ethylene glycol) (PEG) treatment and then employed an iTRAQ-based proteomic method to explore the underlying mechanisms. DT varied among the four embryo axis parts of germinated seeds: epicotyl > hypocotyl-E (hypocotyl part attached to the epicotyl) > hypocotyl-R (hypocotyl part attached to the radicle) > radicle. Meanwhile, PEG treatment of germinated seeds resulted in a differential extent of DT re-establishment in these tissues. Proteins involved in detoxification and stress response were enriched in desiccation-tolerant hypocotyls-E and epicotyls of germinated seeds, respectively. Upon rehydration, proteome change during dehydration was recovered in the hypocotyls-E but not in the radicles. PEG treatment of germinated seeds led to numerous changes in proteins, in abundance in desiccation-sensitive radicles and hypocotyls-R, of which many accumulated in the hypocotyls-E and epicotyls before the treatment. We hypothesized that accumulation of groups 1 and 5 LEA proteins and proteins related to detoxification, ABA, ethylene, and calcium signaling contributed mainly to the variation of DT in different tissues and its re-establishment.

Functional characterization of an unobtrusive protein, CkMT4, in re-establishing desiccation tolerance in germinating seeds

Desiccation tolerance (DT) is gradually lost during seed germination, while it can be re-established by pre-treatment with polyethylene glycol (PEG) and/or abscisic acid (ABA). Increasing knowledge is available on several stress-related proteins in DT re-establishment in herb seeds, but limited information exists on novel proteins in wood seeds. This study aimed to investigate the role of metallothionein CkMT4, a protein species with the highest fold increase in abundance in Caragana korshinskii seeds on PEG treatment. The fluctuation in mRNA levels of CkMT4 during seed development was consistent with the changes in DT, and the expression of CkMT4 could be up-regulated by ABA. Besides metal-binding capacity, CkMT4 might supply Cu2+/Zn2+ to superoxide dismutase (SOD) under high redox potential provided by PEG treatment for excess reactive oxygen species (ROS) scavenging. The overexpression of CkMT4 in yeast results in enhanced oxidation resistance. Experimentally, this study demonstrated the overexpression of CkMT4 in Arabidopsis seeds benefited the re-establishment of DT and enhanced the activity of SOD. On the whole, these findings suggested that CkMT4 facilitated the re-establishment of DT in C. korshinskii seeds mainly through diminishing excess ROS, which put the mechanism underlying the re-establishment of DT in xerophytic wood seeds into a new perspective.

In situ DNA fragmentation during the re-establishment of desiccation tolerance in germinated seeds of Cedrela fissilis Vell.

Abstract: Dehydration is a necessary procedure prior to exposing seeds to long term storage, but this is associated with metabolism-linked injury mediated by cell injury. In order to assess cellular alterations during re-establishment of desiccation tolerance (DT) in C. fissilis germinated seeds and their relation to DNA damage, we verified the occurrence of DNA fragmentation through the TUNEL test and its evidence through the cytological analyses. To re-establish DT, germinated seeds were incubated for 72 h in polyethylene glycol (PEG, -2.04 MPa) before dehydration in silica gel (at 10% moisture content) followed by rehydration. The moisture content changes during the reestablishment of the desiccation tolerance was accomplished. (DT)TdT-dUPT terminal nick-end labeling (TUNEL) was used to assess rates of cell death. TUNEL staining was performed using Click-iT-TUNEL Alexa Flour imaging assay. The TUNEL test showed a consistent DNA fragmentation in the 2 and 5 mm long radicles. Moreover, nuclear and chromosomal alterations were observed in the 5 mm meristematic root cell cycle, contributing to the identification of diagnostic markers of cell death.

Gene expression of late embryogenesis abundant proteins, small heat shock proteins and peroxiredoxin and oxidation of lipid and protein during loss and re-establishment of desiccation tolerance in Pisum sativum seeds

The aim of the present work was to investigate the mechanism of loss and re-establishment of desiccation tolerance (DT) in germinated Pisum sativum seeds (seedlings). Dehydration of pea seedlings of 2 mm radicle length was accompanied by concomitant loss of DT from 100 to 60 and 20% when dehydrated to initial water content (IWC) and below IWC, respectively. Desiccation of seedlings was associated with accumulation of malondialdehyde (MDA) and carbonyls (oxidized product of lipid and protein, respectively) with increased solute leakage and decreased seedling viability. Also, the transcripts of all the protective proteins like late embryogenesis abundant proteins (LEA; PsDHN2, PsDHN3 and PsSBP65), small heat shock proteins (sHSP; PsHSP17.7, PsHSP18.1, PsHSP18.2 and PsHSP22.7) and peroxiredoxin (Prx; 2-Cys Prx) increased in seedlings desiccated to IWC but massive loss (5- to 9-folds) was discernible when desiccated below IWC. The PEG pre-treatment re-established the DT from 20 to 70% in seedlings dehydrated below IWC. The re-induction of DT in PEG-treated seedlings was accompanied with enhanced seedling viability and gene expression (5- to 10-folds) of all the protective proteins (LEA, sHSP and 2-Cys Prx) with substantial reduction in the levels of MDA, carbonyls and electrolyte leakage. The role of genes encoding the protective proteins, MDA, carbonyls, TTC reduction and solute leakage was discussed during loss and re-establishment of DT in pea seedlings.

ITRAQ-based quantitative proteomic analysis reveals pathways associated with re-establishing desiccation tolerance in germinating seeds of Caragana korshinskii Kom.

Mature seeds of Caragana korshinskii Kom. lose desiccation tolerance (DT) during germination. However, DT can be re-induced by polyethylene glycol (PEG) or abscisic acid (ABA), and this provides a novel system to analyse the mechanism of DT. Although the global gene expression during the re-establishment of DT treated by PEG or ABA has been characterized, the changes in protein species abundance are still unknown. Here, we used an iTRAQ-based quantitative proteomics approach to perform a comparative analysis of protein abundances in germinated seeds of PEG-treated, ABA-treated and untreated radicles in response to the re-establishment of DT. A total of 3009 proteins were quantified; compared with the untreated samples, 274 and 261 differentially abundant protein species were found in PEG- and ABA-treated samples, respectively. The observed protein abundance patterns showed a clear overlap in seeds with ABA-and PEG-induced re-establishment of DT. During the re-establishment of DT, the expression of auxin-repressed protein and metallothionien-like protein was induced, and the content of proline increased significantly; however, proteins related to cell proliferation were found to be expressed at lower levels, and the glycolytic pathway was partially inhibited. These results provide promising insights into the molecular mechanisms underlying the re-establishment of DT in germinated seeds. Biological significanceThe major challenge for seed storage is the complexity of the tolerance to desiccation. Re-establishment of desiccation tolerance (DT) in germinating seed as a model was used for DT researching. Although several researches have been focusing on the transcriptome of DT re-establishment, little proteomic information remained to be available. In the present study, the iTRAQ-based quantitative proteomic analysis was employed to identify the differentially accumulated protein species from radicles of germinating C. korshinskii seedling after ABA-, PEG- treatment or not. The data obtained not only provides basic information about proteome, but also characterize the quantitative changes that occur in the abundance of proteins species during DT re-establishment. These results are helpful understanding the mechanism of DT re-establishment induced by PEG or ABA.

Modulating role of ROS in re-establishing desiccation tolerance in germinating seeds of Caragana korshinskii Kom.

In close agreement with visible germination, orthodox seeds lose desiccation tolerance (DT). This trait can be regained under osmotic stress, but the mechanisms are poorly understood. In this study, germinating seeds of Caragana korshinskii Kom. were investigated, focusing on the potential modulating roles of reactive oxygen species (ROS) in the re-establishment of DT. Germinating seeds with 2 mm long radicles can be rendered tolerant to desiccation by incubation in a polyethylene glycol (PEG) solution (-1.7 MPa). Upon PEG incubation, ROS accumulation was detected in the radicles tip by nitroblue tetrazolium chloride staining and further confirmed by confocal microscopy. The PEG-induced re-establishment of DT was repressed when ROS scavengers were added to the PEG solution. Moreover, ROS act downstream of abscisic acid (ABA) to modulate PEG-mediated re-establishment of DT and serve as a new inducer to re-establish DT. Transcriptomic analysis revealed that re-establishment of DT by ROS involves the up-regulation of key genes in the phenylpropanoid-flavonoid pathway, and total flavonoid content and key enzyme activity increased after ROS treatment. Furthermore, DT was repressed by an inhibitor of phenylalanine ammonia lyase. Our data suggest that ROS play a key role in the re-establishment of DT by regulating stress-related genes and the phenylpropanoid-flavonoid pathway.

<b>Loss and re-establishment of desiccation tolerance in the germinated seeds of <em>Sesbania virgata</em> (Cav.) (Pers.)

This research aimed to investigate the cellular alterations during the loss and re-establishment of desiccation tolerance (DT) in germinated Sesbania virgata seeds. The loss of DT was characterized in germinated seeds with increasing radicle lengths (1, 2, 3, 4 and 5 mm) when subjected to dehydration in silica gel, followed by rehydration. To re-establish DT, the germinated seeds were incubated for 72h in polyethylene glycol (PEG, -2.04 MPa) with or without ABA (100 μM) before dehydration in silica gel. Cell viability was assessed by seedling survival, and DNA integrity was evaluated by gel electrophoresis. Seeds with 1 mm radicle length survived dehydration to the original moisture content (MC) of the dry seed (approximately 10%). PEG treatment was able to re-establish DT, at least partially, with 2, 3 and 4 mm but not in 5 mm radicle lengths. Germinated seeds treated with PEG+ABA performed better than those treated only with PEG, and DT was re-established even in germinated seeds with a 5 mm radicle length. Among the PEG-treated germinated seeds dehydrated to 10% MC, DNA integrity was maintained only in those with a 1 mm radicle length.

A gene co-expression network predicts functional genes controlling the re-establishment of desiccation tolerance in germinated Arabidopsis thaliana seeds.

Main conclusion During re-establishment of desiccation tolerance (DT), early events promote initial protection and growth arrest, while late events promote stress adaptation and contribute to survival in the dry state. Mature seeds of Arabidopsis thaliana are desiccation tolerant, but they lose desiccation tolerance (DT) while progressing to germination. Yet, there is a small developmental window during which DT can be rescued by treatment with abscisic acid (ABA). To gain temporal resolution and identify relevant genes in this process, data from a time series of microarrays were used to build a gene co-expression network. The network has two regions, namely early response (ER) and late response (LR). Genes in the ER region are related to biological processes, such as dormancy, acquisition of DT and drought, amplification of signals, growth arrest and induction of protection mechanisms (such as LEA proteins). Genes in the LR region lead to inhibition of photosynthesis and primary metabolism, promote adaptation to stress conditions and contribute to seed longevity. Phenotyping of 12 hubs in relation to re-establishment of DT with T-DNA insertion lines indicated a significant increase in the ability to re-establish DT compared with the wild-type in the lines cbsx4, at3g53040 and at4g25580, suggesting the operation of redundant and compensatory mechanisms. Moreover, we show that re-establishment of DT by polyethylene glycol and ABA occurs through partially overlapping mechanisms. Our data confirm that co-expression network analysis is a valid approach to examine data from time series of transcriptome analysis, as it provides promising insights into biologically relevant relations that help to generate new information about the roles of certain genes for DT.Electronic supplementary materialThe online version of this article (doi:10.1007/s00425-015-2283-7) contains supplementary material, which is available to authorized users.

Re-induction of desiccation tolerance after germination of Cedrela fissilis Vell. seeds.

This work aimed to characterize the re-induction of desiccation tolerance (DT) in germinated seeds, using polyethylene glycol (PEG 8000). Cell changes were investigated through cytological assays (cell viability and transmission electronic microscopy) as well as DNA integrity during loss and re-establishment of DT. The loss of DT was characterized by drying germinated seeds with different radicle lengths (1, 2, 3, 4 and 5 mm) in silica gel, decreasing the moisture content to ten percentage points intervals, followed by pre-humidification (100% RH / 24 h) and rehydration. To re-induce DT, germinated seeds were treated for 72 h with PEG (-2.04 MPa) and PEG (-2.04 MPa) + ABA (100 µM) before dehydration. Germinated seeds did not tolerate desiccation to 10% moisture content, irrespectively of the radicle length. However, when incubated in PEG, those with 1 and 2 mm long radicle attained 71% and 29% survival, respectively. The PEG+ABA treatment was efficient to re-establish DT in seeds with 1 mm long radicles (100% survival). The ultrastructural assays of the cells of germinated seeds with 2 and 5 mm length confirmed the obtained physiological results. Germinated seeds of C. fissilis constitute a useful tool for desiccation tolerance investigations.

Abscisic acid (ABA) sensitivity regulates desiccation tolerance in germinated Arabidopsis seeds

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.

An emerging picture of the seed desiccome: confirmed regulators and newcomers identified using transcriptome comparison.

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.

The Re-Establishment of Desiccation Tolerance in Germinated Arabidopsis thaliana Seeds and Its Associated Transcriptome

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.

Stress-associated factors increase after desiccation of germinated seeds of Tabebuia impetiginosa Mart.

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.

Transcriptome profiling uncovers metabolic and regulatory processes occurring during the transition from desiccation‐sensitive to desiccation‐tolerant stages in Medicago truncatula seeds

To investigate regulatory processes and protective mechanisms leading to desiccation tolerance (DT) in seeds, 16086-element microarrays were used to monitor changes in the transcriptome of desiccation-sensitive 3-mm-long radicles of Medicago truncatula seeds at different time points during incubation in a polyethylene glycol (PEG) solution at -1.7 MPa, resulting in a gradual re-establishment of DT. Gene profiling was also performed on embryos before and after the acquisition of DT during maturation. More than 1300 genes were differentially expressed during the PEG incubation. A large number of genes involved in C metabolism are expressed during the re-establishment of DT. Quantification of C reserves confirms that lipids, starch and oligosaccharides were mobilised, coinciding with the production of sucrose during the early osmotic adjustment. Several clusters of gene profiles were identified with different time-scales. Genes expressed early during the PEG incubation belonged to classes involved in early stress and adaptation responses. Interestingly, several regulatory genes typically expressed during abiotic/drought stresses were also upregulated during maturation, arguing for the partial overlap of ABA-dependent and -independent regulatory pathways involved in both drought and DT. At later time points, in parallel to the re-establishment of DT, upregulated genes are comparable with those involved in late seed maturation. Concomitantly, a massive repression of genes belonging to numerous classes occurred, including cell cycle, biogenesis, primary and energy metabolism. The re-establishment of DT in the germinated radicles appears to concur with a partial return to the quiescent state prior to germination.

NMR metabolite profiling analysis reveals changes in phospholipid metabolism associated with the re‐establishment of desiccation tolerance upon osmotic stress in germinated radicles of cucumber

The adaptation of metabolism is thought to play a role in the acquisition of desiccation tolerance (DT). However, the importance of such a role and whether specific regulatory pathways exist remain to be assessed. Using in vitro 31P and 13C nuclear magnetic resonance (NMR) spectroscopy and biochemical assays, we analysed metabolite profiles of perchloric extracts from germinating radicles of cucumber to identify changes in carbon and phosphate metabolism associated with DT. Emerged radicles measuring 2 mm long can be rendered tolerant to desiccation by incubation in a polyethylene glycol (PEG) solution with a water potential of 1.5 MPa. However, in 4-mm-long emerged radicles, this treatment was ineffective. This manipulable system enabled the discrimination of changes in metabolites associated with DT from those associated with the response to osmotic stress. Independent of radicle length, the PEG treatment resulted in an increase in sucrose (Suc) content, whereas glucose (Glc), fructose (Fru) and the hexose phosphate pool, as well as phosphoenolpyruvate decreased three- to fourfold. In addition, three derivatives arising early during phospholipid catabolism (glycerylphosphorylcholine, glycerylphosphorylethanolamine and glycerylphosphorylinositol) appeared in the PEG-treated radicles. Interestingly, phospholipid degradation was much more pronounced in osmotically challenged radicles that remain sensitive to drying. This was proved by the appearance of catabolites, such as phosphocholine and phosphoethanolamine, solely in 4 mm PEG-treated radicles. Furthermore, glycerol-3-phosphate and its derivative 3-phosphoglycerate increased significantly. Our data suggest that the metabolic response leading to the re-establishment of DT is not entirely identical to that of an osmotic response. It is inferred that membrane remodelling and/or increased phospholipid catabolism is an adaptive response common to osmotic adjustment and DT but is controlled differently in tolerant and sensitive radicles.

Changes in DNA and microtubules during loss and re-establishment of desiccation tolerance in germinating Medicago truncatula seeds

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.

The re-establishment of desiccation tolerance in germinated radicles ofMedicago truncatulaGaertn. seeds

Germinated seeds ofMedicago truncatulaGaertn. with a protruded radicle length of 2.7 mm did not survive drying below 0.2 g H2O g–1dw, 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–1dw, 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 ofMtDHN(a dehydrin) gene expression was correlated with the re-establishment of desiccation tolerance. Furthermore, the PEG-induced expression ofMtDHNwas repressed when fluridone was added to the PEG solution.