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.

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.

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.

The Arabidopsis DREB2 genetic pathway is constitutively repressed by basal phosphoinositide-dependent phospholipase C coupled to diacylglycerol kinase.

Phosphoinositide-dependent phospholipases C (PI-PLCs) are activated in response to various stimuli. They utilize substrates provided by type III-Phosphatidylinositol-4 kinases (PI4KIII) to produce inositol triphosphate and diacylglycerol (DAG) that is phosphorylated into phosphatidic acid (PA) by DAG-kinases (DGKs). The roles of PI4KIIIs, PI-PLCs, and DGKs in basal signaling are poorly understood. We investigated the control of gene expression by basal PI-PLC pathway in Arabidopsis thaliana suspension cells. A transcriptome-wide analysis allowed the identification of genes whose expression was altered by edelfosine, 30 µM wortmannin, or R59022, inhibitors of PI-PLCs, PI4KIIIs, and DGKs, respectively. We found that a gene responsive to one of these molecules is more likely to be similarly regulated by the other two inhibitors. The common action of these agents is to inhibit PA formation, showing that basal PI-PLCs act, in part, on gene expression through their coupling to DGKs. Amongst the genes up-regulated in presence of the inhibitors, were some DREB2 genes, in suspension cells and in seedlings. The DREB2 genes encode transcription factors with major roles in responses to environmental stresses, including dehydration. They bind to C-repeat motifs, known as Drought-Responsive Elements that are indeed enriched in the promoters of genes up-regulated by PI-PLC pathway inhibitors. PA can also be produced by phospholipases D (PLDs). We show that the DREB2 genes that are up-regulated by PI-PLC inhibitors are positively or negatively regulated, or indifferent, to PLD basal activity. Our data show that the DREB2 genetic pathway is constitutively repressed in resting conditions and that DGK coupled to PI-PLC is active in this process, in suspension cells and seedlings. We discuss how this basal negative regulation of DREB2 genes is compatible with their stress-triggered positive regulation.

Phosphatidylinositol synthase activity from plant membrane fractions and from E. coli-expressed recombinant peptides.

Phosphatidylinositol (PtdIns) synthase is a lipid-synthesizing enzyme responsible for the synthesis of the phospholipid, PtdIns. Its enzymatic properties have been studied in in vitro assays using either membrane-enriched fractions or the purified protein in reconstituted lipid vesicles as a source of enzyme. More recently the specificities in terms of substrate preferences have also been studied using the recombinant protein expressed in Escherichia coli. This chapter deals with the purification of membranes as a source of PtdIns synthase before focusing on the in vitro assays of the enzymatic activities of the protein and, briefly, on the analysis of the product.

Assessment of mitochondria as a compartment for phosphatidylinositol synthesis in Solanum tuberosum.

The outer mitochondrial membrane is particularly rich in phosphatidylinositol (PtdIns), a phospholipid found in different amounts in all eukaryotic membranes, but not synthesized in situ by all. PtdIns is therefore subjected to traffic from the synthesizing membranes to the non-synthesizing ones. The contribution of mitochondria to the cell PtdIns pool has never been the focus of a specific study in plants, whereas in yeast, the presence of the enzyme responsible for synthesis, PtdIns synthase (PIS, cytidine 5'-diphospho-1,2-diacyl-sn-glycerol:myo-inositol 3-phosphatidyltransferase, EC 2.7.8.11), has clearly been demonstrated in mitochondria. As these organelles have now been shown to be responsible for the synthesis of several lipids, the present work aimed at evaluating mitochondria as a compartment for the synthesis of PtdIns in plants. The sub-cellular localization of PIS was studied in Solanum tuberosum L. by membrane fractionation, enzymatic analysis and by confocal microscopy in living cells. In potato, beside the endoplasmic reticulum, the activity of PIS was found to be tightly associated to mitochondria. Using a fluorescent reporter fusion, the enzyme was also found to be associated to these organelles. The enzyme was not present at the plasma membrane. A comparison of the localization in other cell systems suggests that the mitochondrial localization could be regulated.

A new method for accurately measuring Delta(1)-pyrroline-5-carboxylate synthetase activity.

Proline is a key factor in plant adaptation to environmental stresses. The Delta(1)-pyrroline-5-carboxylate synthetase catalyzes the first committed step and the rate-limiting step for proline biosynthesis in both plants and mammals. This enzyme catalyzes the reduction of glutamate to pyrroline-5-carboxylate in two sequential steps including the phosphorylation and the reduction of its precursor. Several methods were established to assay P5CS activity but however none of them are fully reliable. Therefore, we developed a new simple and reliable assay which is based on the quantification of Pi. This assay allowed us to determine the optimal pH, the apparent K(m) and V(m) of P5CS with regard to ATP and glutamate.

Changes in electron transport pathways in endoplasmic reticulum of rapeseed in response to cold.

We studied changes induced by cold on electron transfer pathways (linked to NADH or NADPH oxidation) in endoplasmic reticulum of rapeseed hypocotyls (Brassica napus L.) from a freezing-sensitive variety (ISL) and freezing-tolerant variety (Tradition). Plantlets were grown at 22 degrees C then submitted to a cold shock of 13 or 35 days at 4 degrees C. We measured the content in NADH, NADPH, NAD and NADP of the hypocotyls and the redox power was estimated by the reduced versus oxidized nucleotide ratio. The contents in cytochromes b (5) and P-450, electron acceptors of NADH and NADPH respectively, were determined by differential spectrophotometry. Finally, activity of both NADH-cytochrome b (5) reductase (E.C.1.6.2.2) and NADPH cytochrome P-450 reductase (E.C.1.6.2.4) was determined by reduction of exogenous cytochrome c. Results show that during cold shock, along with an increase of linolenic acid content, there was a general activation of the NADPH pathway which was observed more quickly in Tradition plantlets than in ISL ones. Due to transfer of electrons that can occur between NADPH reductase and cytochrome b (5), this could favor fatty acid desaturation in Tradition, explaining why linolenic acid accumulation was more pronounced in this variety. Besides, more cytochrome P-450 accumulated in ISL that could compete for electrons needed by the FAD3 desaturase, resulting in a relative slower enrichment in 18:3 fatty acid in these plantlets.