The mitogen-activated protein kinase phosphatase PHS1 regulates flowering in Arabidopsis thaliana.

MAIN CONCLUSION: Arabidopsis PHS1, initially known as an actor of cytoskeleton organization, is a positive regulator of flowering in the photoperiodic and autonomous pathways by modulating both CO and FLC mRNA levels. Protein phosphorylation and dephosphorylation is a major type of post-translational modification, controlling many biological processes. In Arabidopsis thaliana, five genes encoding MAPK phosphatases (MKP)-like proteins have been identified. Among them, PROPYZAMIDE HYPERSENSITIVE 1 (PHS1) encoding a dual-specificity protein tyrosine phosphatase (DsPTP) has been shown to be involved in microtubule organization, germination and ABA-regulated stomatal opening. Here, we demonstrate that PHS1 also regulates flowering under long-day and short-day conditions. Using physiological, genetic and molecular approaches, we have shown that the late flowering phenotype of the knock-out phs1-5 mutant is linked to a higher expression of FLOWERING LOCUS C (FLC). In contrast, a decline of both CONSTANS (CO) and FLOWERING LOCUS T (FT) expression is observed in the knock-out phs1-5 mutant, especially at the end of the light period under long-day conditions when the induction of flowering occurs. We show that this partial loss of sensitivity to photoperiodic induction is independent of FLC. Our results thus indicate that PHS1 plays a dual role in flowering, in the photoperiodic and autonomous pathways, by modulating both CO and FLC mRNA levels. Our work reveals a novel actor in the complex network of the flowering regulation.

TL -DNA transformation decreases ABA level.

The endogenous levels of ABA were measured in Agrobacterium rhizogenes A4 Tl -DNA transformed oilseed rape (Brassica napus L. var. oleifera cv. Brutor and cv. Drakkar), cabbage (Brassica oleracea). A4 transformed tobacco (Nicotiana tabacum cv. Xanthi) and their normal counterparts, using high performance liquid chromatography and enzyme-liked immunosorbent assay. Measurements were made on different plant tissues (i. e. floral stem, terminal bud, young leaf, mature leaf, root and root tips) and ABA levels were compared in unstressed and osmotically stressed oilseed rape plants (cv. Brutor). In unstressed Plants. in each of the 5 independent transformation events studied, a significant reduction (about 65% of control) in ABA concentration was observed in all transformed plants. When subjected to an osmotic stress, TL transformed Brutor plants showed a higher ABA accumulation than untransformed plants. The change in ABA content as a consequence of TL -DNA transformation is discussed with regard to phenotype, drought resistance and adaptability.

The intrinsically disordered C-terminal region of Arabidopsis thaliana TCP8 transcription factor acts both as a transactivation and self-assembly domain.

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.

Arabidopsis thaliana lipid phosphate phosphatase 2 is involved in abscisic acid signalling in leaves.

Lipid phosphate phosphatases (LPPs, E.C. 3.1.3.4) catalyse the dephosphorylation of diacylglycerol pyrophosphate (DGPP) and phosphatidic acid (PA), which are secondary messengers in abscisic acid (ABA) signalling. In this study, we investigated the effect of ABA on the expression of AtLPP genes as they encode putative ABA-signalling partners. We observed that AtLPP2 expression was down-regulated by ABA and we performed experiments on Atlpp2-2, an AtLPP2 knockout mutant, to determine whether AtLPP2 was involved in ABA signalling. We observed that Atlpp2-2 plantlets contained about twice as much PA as the wild-type Col-0 and exhibited higher PA kinase (PAK) activity than Col-0 plants. In addition, we showed that ABA stimulated diacylglycerol kinase (DGK) activity independently of AtLPP2 activity but that the ABA-stimulation of PAK activity recorded in Col-0 was dependent on AtLPP2. In order to evaluate the involvement of AtLPP2 activity in guard cell function, we measured the ABA sensitivity of Atlpp2-2 stomata. The inhibition of stomatal opening was less sensitive to ABA in Atlpp2-2 than in Col-0. Watered and water-stressed plants of the two genotypes accumulated ABA to the same extent, thus leading us to consider Atlpp2-2 an ABA-signalling mutant. Taken together our observations show that AtLPP2 is a part of ABA signalling and participate to the regulation of stomatal movements.

Protein tyrosine kinases and protein tyrosine phosphatases are involved in abscisic acid-dependent processes in Arabidopsis seeds and suspension cells.

Protein tyrosine (Tyr) phosphorylation plays a central role in many signaling pathways leading to cell growth and differentiation in animals. Tyr phosphorylated proteins have been detected in higher plants, and the roles of protein Tyr phosphatases and protein Tyr kinases in some physiological responses have been shown. We investigated the involvement of Tyr phosphorylation events in abscisic acid (ABA) signaling using a pharmacological approach. Phenylarsine oxide, a specific inhibitor of protein Tyr phosphatase activity, abolished the ABA-dependent accumulation of RAB18 (responsive to ABA 18) transcripts. Protein Tyr kinase inhibitors like genistein, tyrphostin A23, and erbstatin blocked the RAB18 expression induced by ABA in Arabidopsis (Arabidopsis thaliana). Stomatal closure induced by ABA was also inhibited by phenylarsine oxide and genistein. We studied the changes in the Tyr phosphorylation levels of proteins in Arabidopsis seeds after ABA treatment. Proteins were separated by two-dimensional gel electrophoresis, and those phosphorylated on Tyr residues were detected using an anti-phosphotyrosine antibody by western blot. Changes were detected in the Tyr phosphorylation levels of 19 proteins after ABA treatment. Genistein inhibited the ABA-dependent Tyr phosphorylation of proteins. The 19 proteins were analyzed by matrix-assisted laser-desorption ionization time-of-flight/time-of-flight mass spectrometry. Among the proteins identified were storage proteins like cruciferins, enzymes involved in the mobilization of lipid reserves like aconitase, enolase, aldolase, and a lipoprotein, and enzymes necessary for seedling development like the large subunit of Rubisco. Additionally, the identification of three putative signaling proteins, a peptidyl-prolyl isomerase, an RNA-binding protein, and a small ubiquitin-like modifier-conjugating enzyme, enlightens how Tyr phosphorylation might regulate ABA transduction pathways in plants.

The phs1-3 mutation in a putative dual-specificity protein tyrosine phosphatase gene provokes hypersensitive responses to abscisic acid in Arabidopsis thaliana.

The 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.

Induction of abscisic acid-regulated gene expression by diacylglycerol pyrophosphate involves Ca2+ and anion currents in Arabidopsis suspension cells.

Diacylglycerol pyrophosphate (DGPP) was recently shown to be a possible intermediate in abscisic acid (ABA) signaling. In this study, reverse transcription-PCR of ABA up-regulated genes was used to evaluate the ability of DGPP to trigger gene expression in Arabidopsis (Arabidopsis thaliana) suspension cells. At5g06760, LTI30, RD29A, and RAB18 were stimulated by ABA and also specifically expressed in DGPP-treated cells. Use of the Ca2+ channel blockers fluspirilene and pimozide and the Ca2+ chelator EGTA showed that Ca2+ was required for ABA induction of DGPP formation. In addition, Ca2+ participated in DGPP induction of gene expression via stimulation of anion currents. Hence, a sequence of Ca2+, DGPP, and anion currents, constituting a core of early ABA-signaling events necessary for gene expression, is proposed.

Diacylglycerol pyrophosphate is a second messenger of abscisic acid signaling in Arabidopsis thaliana suspension cells.

In plants, the importance of phospholipid signaling in responses to environmental stresses is becoming well documented. The involvement of phospholipids in abscisic acid (ABA) responses is also established. In a previous study, we demonstrated that the stimulation of phospholipase D (PLD) activity and plasma membrane anion currents by ABA were both required for RAB18 expression in Arabidopsis thaliana suspension cells. In this study, we show that the total lipids extracted from ABA-treated cells mimic ABA in activating plasmalemma anion currents and induction of RAB18 expression. Moreover, ABA evokes within 5 min a transient 1.7-fold increase in phosphatidic acid (PA) followed by a sevenfold increase in diacylglycerol pyrophosphate (DGPP) at 20 min. PA activated plasmalemma anion currents but was incapable of triggering RAB18 expression. By contrast, DGPP mimicked ABA on anion currents and was also able to stimulate RAB18 expression. Here we show the role of DGPP as phospholipid second messenger in ABA signaling.

Plasmalemma abscisic acid perception leads to RAB18 expression via phospholipase D activation in Arabidopsis suspension cells.

Abscisic acid (ABA) plays a key role in the control of stomatal aperture by regulating ion channel activities and water exchanges across the plasma membrane of guard cells. Changes in cytoplasmic calcium content and activation of anion and outward-rectifying K(+) channels are among the earliest cellular responses to ABA in guard cells. In Arabidopsis suspension cells, we have demonstrated that outer plasmalemma perception of ABA triggered similar early events. Furthermore, a Ca(2+) influx and the activation of anion channels are part of the ABA-signaling pathway leading to the specific expression of RAB18. Here, we determine whether phospholipases are involved in ABA-induced RAB18 expression. Phospholipase C is not implicated in this ABA pathway. Using a transphosphatidylation reaction, we show that ABA plasmalemma perception results in a transient stimulation of phospholipase D (PLD) activity, which is necessary for RAB18 expression. Further experiments showed that PLD activation was unlikely to be regulated by heterotrimeric G proteins. We also observed that ABA-dependent stimulation of PLD was necessary for the activation of plasma anion current. However, when ABA activation of plasma anion channels was inhibited, the ABA-dependent activation of PLD was unchanged. Thus, we conclude that in Arabidopsis suspension cells, ABA stimulation of PLD acts upstream from anion channels in the transduction pathway leading to RAB18 expression.