The Seed and the Metabolism Regulation.

The seed represents a critical stage in the life cycle of flowering plants. It corresponds to a dry structure carrying the plant embryo in dormant or quiescent state. Orthodox seeds possess a very low water content, preventing biochemical reactions, especially respiration. If the desiccation of living organisms leads to a loss of homeostasis, structure, and metabolism, the seeds go through it successfully thanks to their structure, cellular organization, and growth regulation. Seeds set up a certain number of sophisticated molecules to protect valuable macromolecules or organelles from dehydration/rehydration cycles. Moreover, dormancy takes place in a coordinated process with environmental cues in order to ensure embryo development at the most appropriate conditions for the establishment of the new plant. Moreover, repair processes are programmed to be ready to operate to maximize germination success and seed longevity. This review focuses on the physiology of the seed as related to hydration forces, respiration, and biochemical reactions in the transition from thermodynamically undefined dry state to self-sustained living system. Such processes are of importance for basic knowledge of the regulation of metabolism of living organisms, but also for the control of germination in the context of climate change due to global warming.

Biphasic activation of survival and death pathways in Arabidopsis thaliana cultured cells by sorbitol-induced hyperosmotic stress.

Hyperosmotic stresses represent some of the most serious abiotic factors that adversely affect plants growth, development and fitness. Despite their central role, the early cellular events that lead to plant adaptive responses remain largely unknown. In this study, using Arabidopsis thaliana cultured cells we analyzed early cellular responses to sorbitol-induced hyperosmotic stress. We observed biphasic and dual responses of A. thaliana cultured cells to sorbitol-induced hyperosmotic stress. A first set of events, namely singlet oxygen (1O2) production and cell hyperpolarization due to a decrease in anion channel activity could participate to signaling and osmotic adjustment allowing cell adaptation and survival. A second set of events, namely superoxide anion (O2-) production by RBOHD-NADPH-oxidases and SLAC1 anion channel activation could participate in programmed cell death (PCD) of a part of the cell population. This set of events raises the question of how a survival pathway and a death pathway could be induced by the same hyperosmotic condition and what could be the meaning of the induction of two different behaviors in response to hyperosmotic stress.

A Correlative Study of Sunflower Seed Vigor Components as Related to Genetic Background.

Seed vigor is an important trait that determines seed performance in the field, which corresponds to seed germination rate and seedling establishment. Previous works brought helpful equations to calculate several parameters allowing vigor characterization. In this work we used base water potential (Ψb), base temperature (Tb) and seed lot (Ki) constants to characterize the vigor of 44 sunflower seed lots. Contrasting responses to water or temperature stress and storage potential were recorded within this population, the most interesting being the opposite responses between Ψb and Ki. The genotypes that were resistant to water stress presented low ability for storage and vice versa. Furthermore, Ψb and Ki presented narrow ranges while Tb showed important variability within the 44 genotypes. The analysis of the whole dataset showed that these constants are not correlated to each other or to the seed size, suggesting that genetic background is the most important determining factor in seed performance. Consequently, vigor characterization of genotypes is needed in the crop selection process in order to optimize agricultural productivity.

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.

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.

Re-localization of hormone effectors is associated with dormancy alleviation by temperature and after-ripening in sunflower seeds.

Temperature is the primary factor that affects seed dormancy and germination. However, the molecular mechanism that underlies its effect on dormancy alleviation remained largely unknown. In this study, we investigate hormone involvement in temperature induced germination as compared to that caused by after-ripening. Dormant (D) sunflower seeds cannot germinate at 10 °C but fully germinate at 20 °C. After-ripened seeds become non-dormant (ND), i.e. able to germinate at 10 °C. Pharmacological experiments showed the importance of abscisic acid (ABA), gibberellins (GAs) and ethylene in temperature- and after-ripening-induced germination of sunflower seeds. Hormone quantification showed that after-ripening is mediated by a decline in both ABA content and sensitivity while ABA content is increased in D seeds treated at 10 or 20 °C, suggesting that ABA decrease is not a prerequisite for temperature induced dormancy alleviation. GAs and ethylene contents were in accordance with germination potential of the three conditions (GA1 was higher in D 20 °C and ND 10 °C than in D 10 °C). Transcripts analysis showed that the major change concerns ABA and GAs metabolism genes, while ABA signalling gene expression was significantly unchanged. Moreover, another level of hormonal regulation at the subcellular localization has been revealed by immunocytolocalization study. Indeed, ABA, protein Abscisic acid-Insensitive 5 (ABI5), involved in ABA-regulated gene expression and DELLA protein RGL2, a repressor of the gibberellins signalling pathway, localized mainly in the nucleus in non-germinating seeds while they localized in the cytosol in germinating seeds. Furthermore, ACC-oxidase (ACO) protein, the key ethylene biosynthesis enzyme, was detected in the meristem only in germinating seeds. Our results reveal the importance of hormone actors trafficking in the cell and their regulation in specialized tissue such as the meristem in dormancy alleviation and germination.

Activation of plasma membrane H+-ATPases participates in dormancy alleviation in sunflower seeds.

Using various inhibitors and scavengers we took advantage of the size of sunflower (Helianthus annuus) seeds to investigate in vivo the effects of hormones, namely abscisic acid (ABA) and ethylene (ET), and reactive oxygen species (ROS) on the polarization of dormant (D) and non-dormant (ND) embryonic seed cells using microelectrodes. Our data show that D and ND seed cells present different polarization likely due to the regulation of plasma membrane (PM) H+-ATPase activity. The data obtained after addition of hormones or ROS scavengers further suggest that ABA dependent inhibition of PM H+-ATPases could participate in dormancy maintenance and that ET-and ROS-dependent PM H+-ATPase stimulation could participate in dormancy release in sunflower seeds.

Revisiting the Role of Ethylene and N-End Rule Pathway on Chilling-Induced Dormancy Release in Arabidopsis Seeds.

Dormant Arabidopsis (Arabidopsis thaliana) seeds do not germinate easily at temperatures higher than 10⁻15 °C. Using mutants affected in ethylene signaling (etr1, ein2 and ein4) and in the N-end-rule pathway of the proteolysis (prt6 and ate1-ate2) we have investigated the effects of cold and ethylene on dormancy alleviation. Ethylene (10⁻100 ppm) and 2⁻4 days chilling (4 °C) strongly stimulate the germination of wild type (Col-0) seeds at 25 °C. Two to four days of chilling promote the germination at 25 °C of all the mutants suggesting that release of dormancy by cold did not require ethylene and did not require the N-end-rule pathway. One mutant (etr1) that did not respond to ethylene did not respond to GA3 either. Mutants affected in the N-end rule (prt6 and ate1-ate2) did not respond to ethylene indicating that also this pathway is required for dormancy alleviation by ethylene; they germinated after chilling and in the presence of GA3. Cold can activate the ethylene signaling pathway since it induced an accumulation of ETR1, EINI4, and EIN2 transcripts, the expression of which was not affected by ethylene and GA3. Both cold followed by 10 h at 25 °C and ethylene downregulated the expression of PRT6, ATE1, ATE2, and of ABI5 involved in ABA signaling as compared to dormant seeds incubated at 25 °C. In opposite, the expression of RGA, GAI, and RGL2 encoding three DELLAs was induced at 4 °C but downregulated in the presence of ethylene.

One Way to Achieve Germination: Common Molecular Mechanism Induced by Ethylene and After-Ripening in Sunflower Seeds.

Dormancy is an adaptive trait that blocks seed germination until the environmental conditions become favorable for subsequent vegetative plant growth. Seed dormancy is defined as the inability to germinate in favorable conditions. Dormancy is alleviated during after-ripening, a dry storage period, during which dormant (D) seeds unable to germinate become non-dormant (ND), able to germinate in a wide range of environmental conditions. The treatment of dormant seeds with ethylene (D/ET) promotes seed germination, and abscisic acid (ABA) treatment reduces non-dormant (ND/ABA) seed germination in sunflowers (Helianthus annuus). Metabolomic and transcriptomic studies have been performed during imbibition to compare germinating seeds (ND and D/ET) and low-germinating seeds (D and ND/ABA). A PCA analysis of the metabolites content showed that imbibition did not trigger a significant change during the first hours (3 and 15 h). The metabolic changes associated with germination capacity occurred at 24 h and were related to hexoses, as their content was higher in ND and D/ET and was reduced by ABA treatment. At the transcriptional level, a large number of genes were altered oppositely in germinating, compared to the low-germinating seeds. The metabolomic and transcriptomic results were integrated in the interpretation of the processes involved in germination. Our results show that ethylene treatment triggers molecular changes comparable to that of after-ripening treatment, concerning sugar metabolism and ABA signaling inhibition.

Integrating proteomics and enzymatic profiling to decipher seed metabolism affected by temperature in seed dormancy and germination.

Temperature is an important environmental factor affecting seed dormancy and germination. The mechanism by which temperature induces germination in dormant seeds is however still unclear. Proteomic study has been performed in dormant sunflower seeds during imbibition at permissive and non-permissive temperatures for germination, 20 and 10 °C, respectively. Proteome analysis showed an increase of proteins belonging to metabolism and energy from the first hours of imbibition followed by a decrease of proteins involved in protein metabolism and seed storage in germinating compared to non-germinating seeds. Proteomic study was completed by polysome and proteasome activity assessment and enzymatic profiling on several altered proteins involved in metabolism and energy. Results showed that 20 °C treatment induced the activation of both protein synthesis and degradation processes, the latter being related to proteasome activity during the germination sensu stricto, and to other degradation processes such as proteases during the post-germination. Interestingly, enzymatic profiles showed that TCA cycle and glycolysis were more active in non-germinating seeds in the phase I of the germination sensu stricto. This result suggests the regulation of central metabolism activity in germinating seeds. The control of energy production during imbibition seems to be involved in molecular networks controlling seed dormancy and germination.

Determination of Protein Carbonylation and Proteasome Activity in Seeds.

Reactive oxygen species (ROS) have been shown to be toxic but also function as signaling molecules in a process called redox signaling. In seeds, ROS are produced at different developmental stages including dormancy release and germination. Main targets of oxidation events by ROS in cell are lipids, nucleic acids, and proteins. Protein oxidation has various effects on their function, stability, location, and degradation. Carbonylation represents an irreversible and unrepairable modification that can lead to protein degradation through the action of the 20S proteasome. Here, we present techniques which allow the quantification of protein carbonyls in complex protein samples after derivatization by 2,4-dinitrophenylhydrazine (DNPH) and the determination proteasome activity by an activity-based protein profiling (ABPP) using the probe MV151. These techniques, routinely easy to handle, allow the rapid assessment of protein carbonyls and proteasome activity in seeds in various physiological conditions where ROS may act as signaling or toxic elements.

Deciphering the dual effect of lipopolysaccharides from plant pathogenic Pectobacterium.

Lipopolysaccharides (LPS) are a component of the outer cell surface of almost all Gram-negative bacteria and play an essential role for bacterial growth and survival. Lipopolysaccharides represent typical microbe-associated molecular pattern (MAMP) molecules and have been reported to induce defense-related responses, including the expression of defense genes and the suppression of the hypersensitive response in plants. However, depending on their origin and the challenged plant, LPS were shown to have complex and different roles. In this study we showed that LPS from plant pathogens Pectobacterium atrosepticum and Pectobacterium carotovorum subsp. carotovorum induce common and different responses in A. thaliana cells when compared to those induced by LPS from non-phytopathogens Escherichia coli and Pseudomonas aeruginosa. Among common responses to both types of LPS are the transcription of defense genes and their ability to limit of cell death induced by Pectobacterium carotovorum subsp carotovorum. However, the differential kinetics and amplitude in reactive oxygen species (ROS) generation seemed to regulate defense gene transcription and be determinant to induce programmed cell death in response to LPS from the plant pathogenic Pectobacterium. These data suggest that different signaling pathways could be activated by LPS in A. thaliana cells.

Reactive oxygen species, abscisic acid and ethylene interact to regulate sunflower seed germination.

Sunflower (Helianthus annuus L.) seed dormancy is regulated by reactive oxygen species (ROS) and can be alleviated by incubating dormant embryos in the presence of methylviologen (MV), a ROS-generating compound. Ethylene alleviates sunflower seed dormancy whereas abscisic acid (ABA) represses germination. The purposes of this study were to identify the molecular basis of ROS effect on seed germination and to investigate their possible relationship with hormone signalling pathways. Ethylene treatment provoked ROS generation in embryonic axis whereas ABA had no effect on their production. The beneficial effect of ethylene on germination was lowered in the presence of antioxidant compounds, and MV suppressed the inhibitory effect of ABA. MV treatment did not alter significantly ethylene nor ABA production during seed imbibition. Microarray analysis showed that MV treatment triggered differential expression of 120 probe sets (59 more abundant and 61 less abundant genes), and most of the identified transcripts were related to cell signalling components. Many transcripts less represented in MV-treated seeds were involved in ABA signalling, thus suggesting an interaction between ROS and ABA signalling pathways at the transcriptional level. Altogether, these results shed new light on the crosstalk between ROS and plant hormones in seed germination.

Ethylene, a key factor in the regulation of seed dormancy.

Ethylene is an important component of the gaseous environment, and regulates numerous plant developmental processes including seed germination and seedling establishment. Dormancy, the inability to germinate in apparently favorable conditions, has been demonstrated to be regulated by the hormonal balance between abscisic acid (ABA) and gibberellins (GAs). Ethylene plays a key role in dormancy release in numerous species, the effective concentrations allowing the germination of dormant seeds ranging between 0.1 and 200 µL L(-1). Studies using inhibitors of ethylene biosynthesis or of ethylene action and analysis of mutant lines altered in genes involved in the ethylene signaling pathway (etr1, ein2, ain1, etr1, and erf1) demonstrate the involvement of ethylene in the regulation of germination and dormancy. Ethylene counteracts ABA effects through a regulation of ABA metabolism and signaling pathways. Moreover, ethylene insensitive mutants in Arabidopsis are more sensitive to ABA and the seeds are more dormant. Numerous data also show an interaction between ABA, GAs and ethylene metabolism and signaling pathways. It has been increasingly demonstrated that reactive oxygen species (ROS) may play a significant role in the regulation of seed germination interacting with hormonal signaling pathways. In the present review the responsiveness of seeds to ethylene will be described, and the key role of ethylene in the regulation of seed dormancy via a crosstalk between hormones and other signals will be discussed.

Could FaRP-Like Peptides Participate in Regulation of Hyperosmotic Stress Responses in Plants?

The ability to respond to hyperosmotic stress is one of the numerous conserved cellular processes that most of the organisms have to face during their life. In metazoans, some peptides belonging to the FMRFamide-like peptide (FLP) family were shown to participate in osmoregulation via regulation of ion channels; this is, a well-known response to hyperosmotic stress in plants. Thus, we explored whether FLPs exist and regulate osmotic stress in plants. First, we demonstrated the response of Arabidopsis thaliana cultured cells to a metazoan FLP (FLRF). We found that A. thaliana express genes that display typical FLP repeated sequences, which end in RF and are surrounded by K or R, which is typical of cleavage sites and suggests bioactivity; however, the terminal G, allowing an amidation process in metazoan, seems to be replaced by W. Using synthetic peptides, we showed that amidation appears unnecessary to bioactivity in A. thaliana, and we provide evidence that these putative FLPs could be involved in physiological processes related to hyperosmotic stress responses in plants, urging further studies on this topic.

Translatome profiling in dormant and nondormant sunflower (Helianthus annuus) seeds highlights post-transcriptional regulation of germination.

Seed dormancy, which blocks germination in apparently favourable conditions, is a key regulatory control point of plant population establishment. As germination requires de novo translation, its regulation by dormancy is likely to be related to the association of individual transcripts to polysomes. Here, the polysome-associated mRNAs, that is, the translatome, were fractionated and characterized with microarrays in dormant and nondormant sunflower (Helianthus annuus) embryos during their imbibition at 10°C, a temperature preventing germination of dormant embryos. Profiling of mRNAs in polysomal complexes revealed that the translatome differs between germinating and nongerminating embryos. Association of transcripts with polysomes reached a maximum after 15 h of imbibition; at this time-point 194 polysome-associated transcripts were specifically found in nondormant embryos and 47 in dormant embryos only. The proteins corresponding to the polysomal mRNAs in nondormant embryos appeared to be very pertinent for germination and were involved mainly in transport, regulation of transcription or cell wall modifications. This work demonstrates that seed germination results from a timely regulated and selective recruitment of mRNAs to polysomes, thus opening novel fields of investigation for the understanding of this developmental process.

Is gene transcription involved in seed dry after-ripening?

Orthodox seeds are living organisms that survive anhydrobiosis and may display dormancy, an inability to germinate at harvest. Seed germination potential can be acquired during a prolonged period of dry storage called after-ripening. The aim of this work was to determine if gene transcription is an underlying regulatory mechanism for dormancy alleviation during after-ripening. To identify changes in gene transcription strictly associated with the acquisition of germination potential but not with storage, we used seed storage at low relative humidity that maintains dormancy as control. Transcriptome profiling was performed using DNA microarray to compare change in gene transcript abundance between dormant (D), after-ripened non-dormant (ND) and after-ripened dormant seeds (control, C). Quantitative real-time polymerase chain reaction (qPCR) was used to confirm gene expression. Comparison between D and ND showed the differential expression of 115 probesets at cut-off values of two-fold change (p<0.05). Comparisons between both D and C with ND in transcript abundance showed that only 13 transcripts, among 115, could be specific to dormancy alleviation. qPCR confirms the expression pattern of these transcripts but without significant variation between conditions. Here we show that sunflower seed dormancy alleviation in the dry state is not related to regulated changes in gene expression.

Role of protein and mRNA oxidation in seed dormancy and germination.

Reactive oxygen species (ROS) are key players in the regulation of seed germination and dormancy. Although their regulated accumulation is a prerequisite for germination, the cellular basis of their action remains unknown, but very challenging to elucidate due to the lack of specificity of these compounds that can potentially react with all biomolecules. Among these, nucleic acids and proteins are very prone to oxidative damage. RNA is highly sensitive to oxidation because of its single-stranded structure and the absence of a repair system. Oxidation of mRNAs induces their decay through processing bodies or results in the synthesis of aberrant proteins through altered translation. Depending on the oxidized amino acid, ROS damage of proteins can be irreversible (i.e., carbonylation) thus triggering the degradation of the oxidized proteins by the cytosolic 20S proteasome or can be reversed through the action of thioredoxins, peroxiredoxins, or glutaredoxins (cysteine oxidation) or by methionine sulfoxide reductase (methionine oxidation). Seed dormancy alleviation in the dry state, referred to as after-ripening, requires both selective mRNA oxidation and protein carbonylation. Similarly, seed imbibition of non-dormant seeds is associated with targeted oxidation of a subset of proteins. Altogether, these specific features testify that such oxidative modifications play important role in commitment of the cellular functioning toward germination completion.

Post-transcriptional regulation of GORK channels by superoxide anion contributes to increases in outward-rectifying K⁺ currents.

· Ion fluxes are ubiquitous processes in the plant and animal kingdoms, controlled by fine-tuned regulations of ion channel activity. Yet the mechanism that cells employ to achieve the modification of ion homeostasis at the molecular level still remains unclear. This is especially true when it comes to the mechanisms that lead to cell death. · In this study, Arabidopsis thaliana cells were exposed to ozone (O3). Ion flux variations were analyzed by electrophysiological measurements and their transcriptional regulation by RT-PCR. Reactive oxygen species (ROS) generation was quantified by luminescence techniques and caspase-like activities were investigated by laser confocal microscopy. · We highlighted the delayed activation of K(+) outward-rectifying currents after an O3 -induced oxidative stress leading to programmed cell death (PCD). Caspase-like activities are detected under O3 exposure and could be decreased by K(+) channel blocker. Molecular experiments revealed that the sustained activation of K(+) outward current could be the result of an unexpected O2 ·â» post-transcriptional regulation of the guard cell outward-rectifying K(+) (GORK) channels. · This consists of a likely new mode of regulating the processing of the GORK mRNA, in a ROS-dependent manner, to allow sustained K(+) effluxes during PCD. These data provide new mechanistic insights into K(+) channel regulation during an oxidative stress response.