SummaryBlue-light-dependent photomorphogenesis in Arabidopsis is regulated principally by the cryptochrome flavintype photoreceptors, which control hypocotyl growth inhibition, cotyledon and leaf expansion, and the expression of light-regulated genes. Interestingly the plant hormone cytokinin induces similar responses when added exogenously to germinating seedlings, suggesting a link between cryptochrome and cytokinin signalling pathways. In this work we explore the relationship between cryptochrome and cytokinin signalling pathways in the promotion of photomorphogenesis. The effect of exogenously added cytokinins on hypocotyl growth inhibition occurs in the dark, and is largely independent and additive to that of cryptochromes in blue light, via distinct signalling pathways. By contrast, cytokinin-dependent stimulation of anthocyanin accumulation occurs only in light, and interacts with the signalling pathway downstream of cryptochrome 1 (CRY1) 2 at the level of transcript accumulation of anthocyanin biosynthetic genes. Mutants in elongated hypocotyl 5 (hy5) 3 , a downstream intermediate in the CRY1 signalling pathway, show a reduced induction of anthocyanin accumulation in blue light by cytokinins, similar to that observed for cryptochrome (cry1) mutants. Furthermore cytokinins are shown to increase levels of HY5 protein accumulation, suggesting that cytokinins may function by reducing HY5 degradation by COP1 (constitutively photomorphogenic 1). As both cryptochrome and cytokinin signalling pathways increase HY5 protein levels, and as HY5 binds to the promoters of anthocyanin biosynthetic enzymes to stimulate gene expression, it is concluded that the regulation of HY5 protein stability represents a point of convergence between cryptochrome and cytokinin signalling pathways.

Summary Many plants accumulate proline, a compatible osmolyte, in response to various environmental stresses such as water deficit and salinity. In some stress responses, plants generate hydrogen peroxide (H2O2) that mediates numerous physiological and biochemical processes. The aim was to study the relationship between stress‐induced proline accumulation and H2O2 production. Using pharmacological and reverse genetic approaches in Arabidopsis thaliana, we investigated the role of NADPH oxidases, Respiratory burst oxidase homologues (Rboh), in the induction of proline accumulation was investigated in response to stress induced by either 200 mM NaCl or 400 mM mannitol. Stress from NaCl or mannitol resulted in a transient increase in H2O2 content accompanied by accumulation of proline. Dimethylthiourea, a scavenger of H2O2, and diphenylene iodonium (DPI), an inhibitor of H2O2 production by NADPH oxidase, were found to significantly inhibit proline accumulation in these stress conditions. DPI also reduced the expression level of Δ1‐pyrroline‐5‐carboxylate synthetase, the key enzyme involved in the biosynthesis of proline. Similarly, less proline accumulated in knockout mutants lacking either AtRbohD or AtRbohF than in wild‐type plants in response to the same stresses. Our data demonstrate that AtRbohs (A. thaliana Rbohs) contribute to H2O2 production in response to NaCl or mannitol stress to increase proline accumulation in this plant.

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Oxidation of proline in response to dark-induced senescence in Arabidopsis leaves allows this amino acid to be used as an alternative respiratory substrate and contributes to the production of glutamate and energy that can be used for nutrient remobilization.

SummaryThe plant hormone abscisic acid (ABA) controls numerous physiological traits: dormancy and germination of seeds, senescence and resistance to abiotic stresses. In order to get more insight into the role of protein tyrosine phosphatase (PTP) in ABA signalling, we obtained eight homozygous T-DNA insertion lines in Arabidopsis thaliana PTP genes. One mutant, named phs1-3, exhibited a strong ABA-induced inhibition of germination as only 26% of its seeds germinated after 3 days instead of 92% for the Columbia (Col-0) line. Genetic and molecular analyses of phs1-3 showed that it bears a unique T-DNA insertion in the promoter of the gene and that the mutation is recessive. PHS1 expression in the mutant is about half that of the Col-0 line. The upregulation of two ABA-induced genes (At5g06760, RAB18) and the downregulation of two ABA-repressed genes (AtCLC-A, ACL) are enhanced in the phs1-3 mutant compared with the wild-type. The 'in planta' aperture of phs1-3 stomata is reduced and the inhibition of the light-induced opening of stomata by ABA is stronger in phs1-3 leaves than in Col-0 leaves. Finally, PHS1 expression is upregulated in the presence of ABA in both phs1-3 and Col-0 but more intensively in the mutant. Thus, phs1-3 is hypersensitive to ABA. Taken together, these results show that PHS1, which encodes a dual-specificity PTP, is a negative regulator of ABA signalling.

TCPs are plant specific transcription factors with non-canonical basic helix-loop-helix domains. While Arabidopsis thaliana has 24 TCPs involved in cell proliferation and differentiation, their mode of action has not been fully elucidated. Using bioinformatic tools, we demonstrate that TCP transcription factors belong to the intrinsically disordered proteins (IDP) family and that disorder is higher in class I TCPs than in class II TCPs. In particular, using bioinformatic and biochemical approaches, we have characterized TCP8, a class I TCP. TCP8 exhibits three intrinsically disordered regions (IDR) made of more than 50 consecutive residues, in which phosphorylable Ser residues are mainly clustered. Phosphorylation of Ser-211 that belongs to the central IDR was confirmed by mass spectrometry. Yeast two-hybrid assays also showed that the C-terminal IDR corresponds to a transactivation domain. Moreover, biochemical experiments demonstrated that TCP8 tends to oligomerize in dimers, trimers and higher-order multimers. Bimolecular fluorescence complementation (BiFC) experiments carried out on a truncated form of TCP8 lacking the C-terminal IDR indicated that it is effectively required for the pronounced self-assembly of TCP8. These data were reinforced by the prediction of a coiled coil domain in this IDR. The C-terminal IDR acts thus as an oligomerization domain and also a transactivation domain. Moreover, many Molecular Recognition Features (MoRFs) were predicted, indicating that TCP8 could interact with several partners to fulfill a fine regulation of transcription in response to various stimuli.

BackgroundPhospholipases D (PLD) are major components of signalling pathways in plant responses to some stresses and hormones. The product of PLD activity is phosphatidic acid (PA). PAs with different acyl chains do not have the same protein targets, so to understand the signalling role of PLD it is essential to analyze the composition of its PA products in the presence and absence of an elicitor.Methodology/Principal findingsPotential PLD substrates and products were studied in Arabidopsis thaliana suspension cells treated with or without the hormone salicylic acid (SA). As PA can be produced by enzymes other than PLD, we analyzed phosphatidylbutanol (PBut), which is specifically produced by PLD in the presence of n-butanol. The acyl chain compositions of PBut and the major glycerophospholipids were determined by multiple reaction monitoring (MRM) mass spectrometry. PBut profiles of untreated cells or cells treated with SA show an over-representation of 160/18∶2- and 16∶0/18∶3-species compared to those of phosphatidylcholine and phosphatidylethanolamine either from bulk lipid extracts or from purified membrane fractions. When microsomal PLDs were used in in vitro assays, the resulting PBut profile matched exactly that of the substrate provided. Therefore there is a mismatch between the acyl chain compositions of putative substrates and the in vivo products of PLDs that is unlikely to reflect any selectivity of PLDs for the acyl chains of substrates.ConclusionsMRM mass spectrometry is a reliable technique to analyze PLD products. Our results suggest that PLD action in response to SA is not due to the production of a stress-specific molecular species, but that the level of PLD products per se is important. The over-representation of 160/18∶2- and 16∶0/18∶3-species in PLD products when compared to putative substrates might be related to a regulatory role of the heterogeneous distribution of glycerophospholipids in membrane sub-domains.

Indole acetic acid (IAA). abscisic acid (ABA), and zeatin plus zeatin riboside (Z + ZR) were determined daily in cuttings of Lycopersicon esculentum Mill. cv. Craigella (C) and the Craigella Lateral Suppressor (CLS) mutant during the first 5 days of the root‐forming process. A solid‐phase enzyme immunoassay using specific anti‐hormone antibodies was used following a one‐step HPLC purification procedure. The hormone measurements were made in cuttings divided into 4 parts. The main variations occurred in the terminal bud and in the basal part of the hypocotyl of the two tomato varieties, i.e. significant IAA and ABA increases during the first 2 days followed by a more or less fast return to the initial values at day 4 or 5. This is probably due to the ablation of the root system. Z + ZR levels dramatically decreased in the basal part of the Craigella hypocotyl 1 day after cutting. Contrary to CLS, C hypocotyls recovered the initial high levels when roots regenerated (day 5). This is probably linked to the greater ability of roots to produce cytokinins in C plants than in CLS ones. The first step of root formation (reactivation of the pericyclic cells and formation of root primordia) corresponded to a high IAA/Z + ZR ratio in the root‐forming tissue, whereas the second step (elongation of young roots) was characterized by a low ratio with low levels of hormones.