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.

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.

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.

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.

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.

The Significance of Hydrogen Sulfide for Arabidopsis Seed Germination.

Hydrogen sulfide (H2S) recently emerged as an important gaseous signaling molecule in plants. In this study, we investigated the possible functions of H2S in regulating Arabidopsis seed germination. NaHS treatments delayed seed germination in a dose-dependent manner and were ineffective in releasing seed dormancy. Interestingly, endogenous H2S content was enhanced in germinating seeds. This increase was correlated with higher activity of three enzymes (L-cysteine desulfhydrase, D-cysteine desulfhydrase, and ß-cyanoalanine synthase) known as sources of H2S in plants. The H2S scavenger hypotaurine and the D/L cysteine desulfhydrase inhibitor propargylglycine significantly delayed seed germination. We analyzed the germinative capacity of des1 seeds mutated in Arabidopsis cytosolic L-cysteine desulfhydrase. Although the mutant seeds do not exhibit germination-evoked H2S formation, they retained similar germination capacity as the wild-type seeds. In addition, des1 seeds responded similarly to temperature and were as sensitive to ABA as wild type seeds. Taken together, these data suggest that, although its metabolism is stimulated upon seed imbibition, H2S plays, if any, a marginal role in regulating Arabidopsis seed germination under standard conditions.

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.

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.

Crosstalk between reactive oxygen species and hormonal signalling pathways regulates grain dormancy in barley.

Seed dormancy, defined as the inability to germinate under favourable conditions, is controlled by abscisic acid (ABA) and gibberellins (GAs). Phytohormone signalling interacts with reactive oxygen species (ROS) signalling regarding diverse aspects of plant physiology and is assumed to be important in dormancy alleviation. Using dormant barley grains that do not germinate at 30 °C in darkness, we analysed ROS content and ROS-processing systems, ABA content and metabolism, GA-responsive genes and genes involved in GA metabolism in response to hydrogen peroxide (H2O2) treatment. During after-ripening, the ROS content in the embryo was not affected, while the antioxidant glutathione (GSH) was gradually converted to glutathione disulphide (GSSG). ABA treatment up-regulated catalase activity through transcriptional activation of HvCAT2. Exogenous H2O2 partially alleviated dormancy although it was associated with a small increase in embryonic ABA content related to a slight induction of HvNCED transcripts. H2O2 treatment did not affect ABA sensitivity but up-regulated the expression of HvExpA11 (GA-induced gene), inhibited the expression of HvGA2ox3 involved in GA catabolism and enhanced the expression of HvGA20ox1 implicated in GA synthesis. In barley, H2O2 could be implicated in dormancy alleviation through activation of GA signalling and synthesis rather than repression of ABA signalling.

Release of sunflower seed dormancy by cyanide: cross-talk with ethylene signalling pathway.

Freshly harvested sunflower (Helianthus annuus L.) seeds are considered to be dormant because they fail to germinate at relatively low temperatures (10 degrees C). This dormancy results mainly from an embryo dormancy and disappears during dry storage. Although endogenous ethylene is known to be involved in sunflower seed alleviation of dormancy, little attention had been paid to the possible role of cyanide, which is produced by the conversion of 1-aminocyclopropane 1-carboxylic acid to ethylene, in this process. The aims of this work were to investigate whether exogenous cyanide could improve the germination of dormant sunflower seeds and to elucidate its putative mechanisms of action. Naked dormant seeds became able to germinate at 10 degrees C when they were incubated in the presence of 1 mM gaseous cyanide. Other respiratory inhibitors showed that this effect did not result from an activation of the pentose phosphate pathway or the cyanide-insensitive pathway. Cyanide stimulated germination of dormant seeds in the presence of inhibitors of ethylene biosynthesis, but its improving effect required functional ethylene receptors. It did not significantly affect ethylene production and the expression of genes involved in ethylene biosynthesis or in the first steps of ethylene signalling pathway. However, the expression of the transcription factor Ethylene Response Factor 1 (ERF1) was markedly stimulated in the presence of gaseous cyanide. It is proposed that the mode of action of cyanide in sunflower seed dormancy alleviation does not involve ethylene production and that ERF1 is a common component of the ethylene and cyanide signalling pathways.