The metabolic profiles of Eugenia astringens and E. uniflora (Myrtaceae) sensitive seeds affect desiccation.

Myrtaceae species are abundant in tropical Atlantic rainforests, but 41% of the 5500 species of this family are of extreme conservation concern. Eugenia astringens and E. uniflora are native Brazilian Myrtaceae species that occur in the same habitats and produce desiccation-sensitive (DS) seeds. We hypothesized that their seed desiccation-sensitivity degree is associated with specific metabolic signatures. To test it, we analyzed the germination and metabolic profiles of fresh and desiccated seeds. The water content (WC) at which at least half of the seeds survived desiccation was lower in E. astringens (0.17 g H2 O g-1 DW) than in E. uniflora (0.41 g H2 O g-1 DW). We identified 103 annotated metabolites from 3261 peaks in both species, which differed in their relative contents between E. astringens and E. uniflora seeds. The main differences in seed metabolic profiles include several protective molecules in the group of carbohydrates and organic acids and amino acid contents. The relative contents of monosaccharides and disaccharides, malic and quinic acids, amino acids and saturated fatty acids may have taken part in the distinct DS behaviour of E. astringens and E. uniflora seeds. Our study provides evidence of the relationship between desiccation sensitivity, seed viability and metabolic profile of tropical seeds by comparing two closely related Eugenia species with different DS degrees.

Impact of climate perturbations on seeds and seed quality for global agriculture.

In agriculture, seeds are the most basic and vital input on which croplands productivity depends. These implies a good starting material, good production lines and good storage options. High-quality seed lots must be free of pests and pathogens and contain a required degree of genetic purity. Seeds need also to be stored in good condition between harvest and later sowing, to insure later on the field a good plant density and higher crop yield. In general, these parameters are already widely accepted and considered in many countries where advanced technologies evaluate them. However, the more and more frequently devastating climate changes observed around the world has put seed quality under threat, and current seeds may not be adapted to hazardous and unpredictable conditions. Climate-related factors such as temperature and water availability directly affect seed development and later germination. For these reasons, investigating seed quality in response to climate changes is a step to propose new crop varieties and practices that will bring solutions for our future.

In-Depth Proteomic Analysis of the Secondary Dormancy Induction by Hypoxia or High Temperature in Barley Grains.

In barley, incubation of primary dormant (D1) grains on water under conditions that do not allow germination, i.e. 30°C in air and 15°C or 30°C in 5% O2, induces a secondary dormancy (D2) expressed as a loss of the ability to germinate at 15°C in air. The aim of this study was to compare the proteome of barley embryos isolated from D1 grains and D2 ones after induction of D2 at 30°C or in hypoxia at 15°C or 30°C. Total soluble proteins were analyzed by 2DE gel-based proteomics, allowing the selection of 130 differentially accumulated proteins (DAPs) among 1,575 detected spots. According to the protein abundance profiles, the DAPs were grouped into six abundance-based similarity clusters. Induction of D2 is mainly characterized by a down-accumulation of proteins belonging to cluster 3 (storage proteins, proteases, alpha-amylase inhibitors and histone deacetylase HD2) and an up-accumulation of proteins belonging to cluster 4 (1-Cys peroxiredoxin, lipoxygenase2 and caleosin). The correlation-based network analysis for each cluster highlighted central protein hub. In addition, most of genes encoding DAPs display high co-expression degree with 19 transcription factors. Finally, this work points out that similar molecular events accompany the modulation of dormancy cycling by both temperature and oxygen, including post-translational, transcriptional and epigenetic regulation.

Intracellular reactive oxygen species trafficking participates in seed dormancy alleviation in Arabidopsis seeds.

Reactive oxygen species (ROS) release seed dormancy through an unknown mechanism. We used different seed dormancy-breaking treatments to decipher the dynamics and localization of ROS production during seed germination. We studied the involvement of ROS in the breaking of Arabidopsis seed dormancy by cold stratification, gibberellic acid (GA3 ) and light. We characterized the effects of these treatments on abscisic acid and gibberellins biosynthesis and signalling pathways. ROS, mitochondrial redox status and peroxisomes were visualized and/or quantified during seed imbibition. Finally, we performed a cytogenetic characterization of the nuclei from the embryonic axes during seed germination. We show that mitochondria participate in the early ROS production during seed imbibition and that a possible involvement of peroxisomes in later stages should still be analysed. At the time of radicle protrusion, ROS accumulated within the nucleus, which correlated with nuclear expansion and chromatin decompaction. Taken together, our results provide evidence of the role of ROS trafficking between organelles and of the nuclear redox status in the regulation of seed germination by dormancy.

Oxidative signalling in seed germination and early seedling growth: an emerging role for ROS trafficking and inter-organelle communication.

Underground early development of higher plants includes two distinct developmental processes, seed germination and then skotomorphogenesis, a mechanism which favours elongation of the hypocotyl and helps the seedling to find light. Interestingly, both processes, which are regulated by plant hormones, have been shown to depend on reactive oxygen species metabolism and to be related to mitochondrial retrograde signalling. Here we review the recent outcomes in this field of research and highlight the emerging role of ROS communication between organelles and cell compartments. We point out the role of mitochondria as an environmental and developmental sensor organelle that regulates ROS homeostasis and downstream events and we propose future directions of research that should help better understanding the roles of ROS in germination and seedling emergence.

Physiological and Environmental Regulation of Seed Germination: From Signaling Events to Molecular Responses.

A timely and efficient seed germination is critical for plantlets' establishment and robustness as well as plant development and plant performance in both natural ecosystems and agrosystems [...].

Label-Free Quantitative Proteomics Reveal the Involvement of PRT6 in Arabidopsis thaliana Seed Responsiveness to Ethylene.

In Arabidopsis thaliana, the breaking of seed dormancy in wild type (Col-0) by ethylene at 100 µL L-1 required at least 30 h application. A mutant of the proteolytic N-degron pathway, lacking the E3 ligase PROTEOLYSIS 6 (PRT6), was investigated for its role in ethylene-triggered changes in proteomes during seed germination. Label-free quantitative proteomics was carried out on dormant wild type Col-0 and prt6 seeds treated with (+) or without (-) ethylene. After 16 h, 1737 proteins were identified, but none was significantly different in protein levels in response to ethylene. After longer ethylene treatment (30 h), 2552 proteins were identified, and 619 Differentially Expressed Proteins (DEPs) had significant differences in protein abundances between ethylene treatments and genotypes. In Col, 587 DEPs were enriched for those involved in signal perception and transduction, reserve mobilization and new material generation, which potentially contributed to seed germination. DEPs up-regulated by ethylene in Col included S-adenosylmethionine synthase 1, methionine adenosyltransferase 3 and ACC oxidase involved in ethylene synthesis and of Pyrabactin Resistance1 acting as an ABA receptor, while DEPs down-regulated by ethylene in Col included aldehyde oxidase 4 involved in ABA synthesis. In contrast, in prt6 seeds, ethylene did not result in strong proteomic changes with only 30 DEPs. Taken together, the present work demonstrates that the proteolytic N-degron pathway is essential for ethylene-mediated reprogramming of seed proteomes during germination.

Dynamics of Protein Phosphorylation during Arabidopsis Seed Germination.

Seed germination is critical for early plantlet development and is tightly controlled by environmental factors. Nevertheless, the signaling networks underlying germination control remain elusive. In this study, the remodeling of Arabidopsis seed phosphoproteome during imbibition was investigated using stable isotope dimethyl labeling and nanoLC-MS/MS analysis. Freshly harvested seeds were imbibed under dark or constant light to restrict or promote germination, respectively. For each light regime, phosphoproteins were extracted and identified from dry and imbibed (6 h, 16 h, and 24 h) seeds. A large repertoire of 10,244 phosphopeptides from 2546 phosphoproteins, including 110 protein kinases and key regulators of seed germination such as Delay Of Germination 1 (DOG1), was established. Most phosphoproteins were only identified in dry seeds. Early imbibition led to a similar massive downregulation in dormant and non-dormant seeds. After 24 h, 411 phosphoproteins were specifically identified in non-dormant seeds. Gene ontology analyses revealed their involvement in RNA and protein metabolism, transport, and signaling. In addition, 489 phosphopeptides were quantified, and 234 exhibited up or downregulation during imbibition. Interaction networks and motif analyses revealed their association with potential signaling modules involved in germination control. Our study provides evidence of a major role of phosphosignaling in the regulation of Arabidopsis seed germination.

Retrograde signalling from the mitochondria to the nucleus translates the positive effect of ethylene on dormancy breaking of Arabidopsis thaliana seeds.

Ethylene and reactive oxygen species (ROS) regulate seed dormancy alleviation, but the molecular basis of their action and crosstalk remains largely unknown. Here we studied the mechanism of Arabidopsis seed dormancy release by ethylene using cell imaging, and genetic and transcriptomics approaches, in order to tackle its possible interaction with ROS homeostasis. We found that the effect of ethylene on seed germination required ROS production by the mitochondrial electron transport chain. Seed response to ethylene involved a mitochondrial retrograde response (MRR) through nuclear ROS production and upregulation of the MRR components AOX1a and ANAC013, but also required the activation of the ethylene canonical pathway. Together our data allowed deciphering of the mode of action of ethylene on seed germination and the associated dynamics of ROS production. Our findings highlight the occurrence of retrograde signalling in seed germination.

Handing off iron to the next generation: how does it get into seeds and what for?

To ensure the success of the new generation in annual species, the mother plant transfers a large proportion of the nutrients it has accumulated during its vegetative life to the next generation through its seeds. Iron (Fe) is required in large amounts to provide the energy and redox power to sustain seedling growth. However, free Fe is highly toxic as it leads to the generation of reactive oxygen species. Fe must, therefore, be tightly bound to chelating molecules to allow seed survival for long periods of time without oxidative damage. Nevertheless, when conditions are favorable, the seed's Fe stores have to be readily remobilized to achieve the transition toward active photosynthesis before the seedling becomes able to take up Fe from the environment. This is likely critical for the vigor of the young plant. Seeds constitute an important dietary source of Fe, which is essential for human health. Understanding the mechanisms of Fe storage in seeds is a key to improve their Fe content and availability in order to fight Fe deficiency. Seed longevity, germination efficiency and seedling vigor are also important traits that may be affected by the chemical form under which Fe is stored. In this review, we summarize the current knowledge on seed Fe loading during development, long-term storage and remobilization upon germination. We highlight how this knowledge may help seed Fe biofortification and discuss how Fe storage may affect the seed quality and germination efficiency.

Microtubule self-organisation during seed germination in Arabidopsis.

BACKGROUND: Upon water uptake and release of seed dormancy, embryonic plant cells expand, while being mechanically constrained by the seed coat. Cortical microtubules (CMTs) are key players of cell elongation in plants: their anisotropic orientation channels the axis of cell elongation through the guidance of oriented deposition of load-bearing cellulose microfibrils in the cell wall. Interestingly, CMTs align with tensile stress, and consistently, they reorient upon compressive stress in growing hypocotyls. How CMTs first organise in germinating embryos is unknown, and their relation with mechanical stress has not been investigated at such an early developing stage. RESULTS: Here, we analysed CMT dynamics in dormant and non-dormant Arabidopsis seeds by microscopy of fluorescently tagged microtubule markers at different developmental time points and in response to abscisic acid and gibberellins. We found that CMTs first appear as very few thick bundles in dormant seeds. Consistently, analysis of available transcriptome and translatome datasets show that limiting amounts of tubulin and microtubule regulators initially hinder microtubule self-organisation. Seeds imbibed in the presence of gibberellic acid or abscisic acid displayed altered microtubule organisation and transcriptional regulation. Upon the release of dormancy, CMTs then self-organise into multiple parallel transverse arrays. Such behaviour matches the tensile stress patterns in such mechanically constrained embryos. This suggests that, as CMTs first self-organise, they also align with shape-derived tensile stress patterns. CONCLUSIONS: Our results provide a scenario in which dormancy release in the embryo triggers microtubule self-organisation and alignment with tensile stress prior to germination and anisotropic growth.

A New Role for Plastid Thioredoxins in Seed Physiology in Relation to Hormone Regulation.

In Arabidopsis seeds, ROS have been shown to be enabling actors of cellular signaling pathways promoting germination, but their accumulation under stress conditions or during aging leads to a decrease in the ability to germinate. Previous biochemical work revealed that a specific class of plastid thioredoxins (Trxs), the y-type Trxs, can fulfill antioxidant functions. Among the ten plastidial Trx isoforms identified in Arabidopsis, Trx y1 mRNA is the most abundant in dry seeds. We hypothesized that Trx y1 and Trx y2 would play an important role in seed physiology as antioxidants. Using reverse genetics, we found important changes in the corresponding Arabidopsis mutant seeds. They display remarkable traits such as increased longevity and higher and faster germination in conditions of reduced water availability or oxidative stress. These phenotypes suggest that Trxs y do not play an antioxidant role in seeds, as further evidenced by no changes in global ROS contents and protein redox status found in the corresponding mutant seeds. Instead, we provide evidence that marker genes of ABA and GAs pathways are perturbed in mutant seeds, together with their sensitivity to specific hormone inhibitors. Altogether, our results suggest that Trxs y function in Arabidopsis seeds is not linked to their previously identified antioxidant roles and reveal a new role for plastid Trxs linked to hormone regulation.

The Histone Chaperone HIRA Is a Positive Regulator of Seed Germination.

Histone chaperones regulate the flow and dynamics of histone variants and ensure their assembly into nucleosomal structures, thereby contributing to the repertoire of histone variants in specialized cells or tissues. To date, not much is known on the distribution of histone variants and their modifications in the dry seed embryo. Here, we bring evidence that genes encoding the replacement histone variant H3.3 are expressed in Arabidopsis dry seeds and that embryo chromatin is characterized by a low H3.1/H3.3 ratio. Loss of HISTONE REGULATOR A (HIRA), a histone chaperone responsible for H3.3 deposition, reduces cellular H3 levels and increases chromatin accessibility in dry seeds. These molecular differences are accompanied by increased seed dormancy in hira-1 mutant seeds. The loss of HIRA negatively affects seed germination even in the absence of HISTONE MONOUBIQUITINATION 1 or TRANSCRIPTION ELONGATION FACTOR II S, known to be required for seed dormancy. Finally, hira-1 mutant seeds show lower germination efficiency when aged under controlled deterioration conditions or when facing unfavorable environmental conditions such as high salinity. Altogether, our results reveal a dependency of dry seed chromatin organization on the replication-independent histone deposition pathway and show that HIRA contributes to modulating seed dormancy and vigor.

Role of ethylene and proteolytic N-degron pathway in the regulation of Arabidopsis seed dormancy.

Primary dormant seeds of Arabidopsis thaliana did not germinate in darkness at temperature higher than 10-15°C. Ethylene improved the germination of dormant wild-type (Col-0) seeds at 25°C in darkness but seeds of the mutant affected in the proteolytic N-degron pathway, proteolysis6 (prt6), were insensitive to ethylene suggesting that PRT6 was involved in dormancy release by ethylene. The substrates of the N-degron pathway, the Ethylene Response Factors from group VII (HRE1, HRE2, RAP2.2, RAP2.3, and RAP2.12), were identified to be involved in this insensitivity with an increased germination in prt6 rap2.2 rap2.3 rap2.12 rather than in prt6 hre1 hre2, which also indicated that the three RAPs acted downstream of PRT6, while the two HREs acted upstream of PRT6. Ethylene reduced the expression of the three RAPs in Col-0 seeds but they were maintained or induced by ethylene in prt6 seeds. The promoting effect of ethylene was associated with a down-regulation of dormancy-related genes in gibberellins (GAs) and abscisic acid (ABA) signaling, such as RGA, RGL2, and ABI5, and with a strong decrease in ABA/GA4 ratio in the presence of ethylene. In contrast, we show that the insensitivity of prt6 seeds to ethylene was mainly related to GA signaling disturbance.

Regulatory actors and alternative routes for Arabidopsis seed germination are revealed using a pathway-based analysis of transcriptomic datasets.

Regulation of seed germination by dormancy relies on a complex network of transcriptional and post-transcriptional modifications during seed imbibition that controls seed adaptive responses to environmental cues. High-throughput technologies have brought significant progress in the understanding of this phenomenon and have led to identify major regulators of seed germination, mostly by studying the behaviour of highly differentially expressed genes. However, the actual models of transcriptome analysis cannot catch additive effects of small variations of gene expression in individual signalling or metabolic pathways, which are also likely to control germination. Therefore, the comprehension of the molecular mechanism regulating germination is still incomplete and to gain knowledge about this process we have developed a pathway-based analysis of transcriptomic Arabidopsis datasets, to identify regulatory actors of seed germination. The method allowed quantifying the level of deregulation of a wide range of pathways in dormant versus non-dormant seeds. Clustering pathway deregulation scores of germinating and dormant seed samples permitted the identification of mechanisms involved in seed germination such as RNA transport or vitamin B6 metabolism, for example. Using this method, which was validated by metabolomics analysis, we also demonstrated that Col and Cvi seeds follow different metabolic routes for completing germination, demonstrating the genetic plasticity of this process. We finally provided an extensive basis of analysed transcriptomic datasets that will allow further identification of mechanisms controlling seed germination.

The MPK8-TCP14 pathway promotes seed germination in Arabidopsis.

The accurate control of dormancy release and germination is critical for successful plantlet establishment. Investigations in cereals hypothesized a crucial role for specific MAP kinase (MPK) pathways in promoting dormancy release, although the identity of the MPK involved and the downstream events remain unclear. In this work, we characterized mutants for Arabidopsis thaliana MAP kinase 8 (MPK8). Mpk8 seeds presented a deeper dormancy than wild-type (WT) at harvest that was less efficiently alleviated by after-ripening and gibberellic acid treatment. We identified Teosinte Branched1/Cycloidea/Proliferating cell factor 14 (TCP14), a transcription factor regulating germination, as a partner of MPK8. Mpk8 tcp14 double-mutant seeds presented a deeper dormancy at harvest than WT and mpk8, but similar to that of tcp14 seeds. MPK8 interacted with TCP14 in the nucleus in vivo and phosphorylated TCP14 in vitro. Furthermore, MPK8 enhanced TCP14 transcriptional activity when co-expressed in tobacco leaves. Nevertheless, the stimulation of TCP14 transcriptional activity by MPK8 could occur independently of TCP14 phosphorylation. The comparison of WT, mpk8 and tcp14 transcriptomes evidenced that whereas no effect was observed in dry seeds, mpk8 and tcp14 mutants presented dramatic transcriptomic alterations after imbibition with a sustained expression of genes related to seed maturation. Moreover, both mutants exhibited repression of genes involved in cell wall remodeling and cell cycle G1/S transition. As a whole, this study unraveled a role for MPK8 in promoting seed germination, and suggested that its interaction with TCP14 was critical for regulating key processes required for germination completion.

A multiscale approach reveals regulatory players of water stress responses in seeds during germination.

Seed germination is regulated by environmental factors, particularly water availability. Water deficits at the time of sowing impair the establishment of crop plants. Transcriptome and proteome profiling was used to document the responses of sunflower (Helianthus annuus) seeds to moderate water stress during germination in two hybrids that are nominally classed as drought sensitive and drought tolerant. Differences in the water stress-dependent accumulation reactive oxygen species and antioxidant enzymes activities were observed between the hybrids. A pathway-based analysis of the hybrid transcriptomes demonstrated that the water stress-dependent responses of seed metabolism were similar to those of the plant, with a decreased abundance of transcripts encoding proteins associated with metabolism and cell expansion. Moreover, germination under water stress conditions was associated with increased levels of transcripts encoding heat shock proteins. Exposure of germinating seeds to water stress specifically affected the abundance of a small number of proteins, including heat shock proteins. Taken together, these data not only identify factors that are likely to play a key role in drought tolerance during seed germination, but they also demonstrate the importance of the female parent in the transmission of water stress tolerance.

The signalling role of ROS in the regulation of seed germination and dormancy.

Reactive oxygen species (ROS) are versatile compounds which can have toxic or signalling effects in a wide range living organisms, including seeds. They have been reported to play a pivotal role in the regulation of seed germination and dormancy but their mechanisms of action are still far from being fully understood. In this review, we sum-up the major findings that have been carried out this last decade in this field of research and which altogether shed a new light on the signalling roles of ROS in seed physiology. ROS participate in dormancy release during seed dry storage through the direct oxidation of a subset of biomolecules. During seed imbibition, the controlled generation of ROS is involved in the perception and transduction of environmental conditions that control germination. When these conditions are permissive for germination, ROS levels are maintained at a level which triggers cellular events associated with germination, such as hormone signalling. Here we propose that the spatiotemporal regulation of ROS production acts in concert with hormone signalling to regulate the cellular events involved in cell expansion associated with germination.

5' to 3' mRNA Decay Contributes to the Regulation of Arabidopsis Seed Germination by Dormancy.

The regulation of plant gene expression, necessary for development and adaptive responses, relies not only on RNA transcription but also on messenger RNA (mRNA) fate. To understand whether seed germination relies on the degradation of specific subsets of mRNA, we investigated whether the 5' to 3' RNA decay machinery participated in the regulation of this process. Arabidopsis (Arabidopsis thaliana) seeds of exoribonuclease4 (xrn4) and varicose (vcs) mutants displayed distinct dormancy phenotypes. Transcriptome analysis of xrn4-5 and vcs-8 mutant seeds allowed us to identify genes that are likely to play a role in the control of germination. Study of 5' untranslated region features of these transcripts revealed that specific motifs, secondary energy, and GC content could play a role in their degradation by XRN4 and VCS, and Gene Ontology clustering revealed novel actors of seed dormancy and germination. Several specific transcripts identified as being putative targets of XRN4 and VCS in seeds (PECTIN LYASE-LIKE, ASPARTYL PROTEASE, DWD-HYPERSENSITIVE-TO-ABA3, and YELLOW STRIPE-LIKE5) were further studied by reverse genetics, and their functional roles in the germination process were confirmed by mutant analysis. These findings suggest that completion of germination and its regulation by dormancy also depend on the degradation of specific subsets of mRNA.

Awake1, an ABC-Type Transporter, Reveals an Essential Role for Suberin in the Control of Seed Dormancy.

The mother plant plays an important dynamic role in the control of dormancy of her progeny seed in response to environmental signals. In order to further understand the mechanisms by which this dormancy control takes place in Arabidopsis (Arabidopsis thaliana), we conducted a forward genetic screen to isolate mutants that fail to enter dormancy in response to variation in temperature during seed set. We show that, for the first of these mutants, designated awake1, the maternal allele is required for entry into strongly dormant states and that awake1 mutants show seed phenotypes shown previously to be associated with the loss of suberin in the seed. We identify awake1 as an allele of ABCG20, an ATP-binding cassette transporter-encoding gene required for the transport of fatty acids during suberin deposition, and show that further suberin-deficient mutants have seed dormancy defects. Seed coat suberin composition is affected by temperature during seed maturation, but this response appears to be independent of ABCG20. We conclude that seed coat suberin is essential for seed dormancy imposition by low temperature and that the exclusion of oxygen and water from the seed by the suberin and tannin layers is important for dormancy imposition.