Roles of diacylglycerol kinases in Arabidopsis thaliana by pharmacological approaches and reverse genetics

Les diacylglycerol kinases catalysent la phosphorylation du diacylglycérol en acide phosphatidique. Nous avons montré que la PLC spécifique des phosphoinositide (PI-PLC) et la diacylglycérol kinase (DGK) régulent négativement l'expression basale de la plupart des gènes DREB2 dans les cellules en suspension d'Arabidopsis thaliana. Les gènes DREB2 codent pour des facteurs de transcription qui se lient aux motifs DRE (Drought Responsive Elements). Ces éléments sont également liés par les facteurs DREB1. Alors que les facteurs DREB2 sont principalement impliqués dans les réponses à la sécheresse et au stress chaud, les DREB1 sont quant à eux induits en réponse au froid. Nous avons également pu montrer que l'inhibition par des agents pharmacologiques des activités PI-PLC ou DGK conduit à l'induction de l'expression basale des gènes DREB1. Cependant, l'induction est beaucoup moins marquée chez les gènes DREB1 que DREB2A, un membre de la famille DREB2. Cela indique que les gènes DREB1 et DREB2, ne sont pas soumis à la même régulation transcriptionnelle et que la signalisation lipidique pourrait en partie expliquer les différences dans la régulation des gènes DREB. Les DGK d'Arabidopsis sont codées par une famille multigénique de 7 gènes. Parmi ces gènes, on retrouve la DGK5 dont les le transcrit peut subir un épissage alternatif, ce qui aboutit à deux transcrits, dont l'un comporte une protéine avec un domaine putatif de liaison à la calmoduline. Le mutant knock-out dgk5.1 à une racine plus courte lorsqu'il est cultivé à 12°C comparé au sauvage. Ce phénotype racinaire est corrélé avec une zone méristématique et des cellules plus petites. La croissance des racines du mutant n'est n'est pas modifiée en présence de la plupart des hormones testées. Pourtant, elle est moins sensible à l'auxine exogène à 12°C par rapport au WT. Le mutant dgk5.1 génère moins de racines secondaires en présence d'auxine exogène que le WT. Le promoteur DR5 n'est pas activé dans le mutant à 12°C par l'IAA exogène dans la zone méristématique, alors qu'il est dans le WT. Nos résultats montrent que le mutant dgk5.1 est altéré dans sa réponse à l'auxine à 12°C, suggérant un rôle de perception/transduction de l’auxine dans les racines courtes.Diacylglycerol kinases catalyse the phosphorylation of diacylglycerol into phosphatidic acid. We show that phosphoinositide dependent-phospholipase C (PI-PLC) and diacylglycerol kinase (DGK) in Arabidopsis thaliana suspension cells negatively regulated the basal expression of most DREB2 genes. DREB2 genes encode transcription factors that bind to Drought Responsive Elements (DRE). Those elements are also bound by DREB1 factors. While DREB2 factors are mostly involved in drought and heat responses, DREB1s are induced in the response to chilling. We show also that the pharmacological inhibition of PI-PLC or DGK leads to the basal induction of DREB1 genes. However, the induction is much less marked for the DREB1 genes than that of DREB2A, a member of the DREB2 family. This illustrates that DREB1 and DREB2 genes, while having the same targets, are not submitted to the same transcription regulation, and that lipid signalling might in part explain these differences in the regulation of the DREB genes. In Arabidopsis, DGKs are encoded by a multigenic family of 7 members. In this thesis, we focus on DGK5. The transcripts can have differential splicing, leading to two mature transcript, one of which leading to a protein with a putative calmodulin binding domain. A dgk5 knocked-out mutant is comparable to the WT, except for shorter root when grown at 12°C. This short root phenotype is correlated with to shorter meristematic zone and smaller cells. The short root phenotype is not altered in presence of most hormones. Yet, the root growth is less sensitive to exogenous auxin at 12°C compared to the WT. Accordingly the mutant produces less secondary roots in presence of exogenous IAA than the WT at 12°C. The DR5 promoter is not activated in the mutant at 12°C by exogenous IAA, in the meristematic zone, while it is in the WT. Our results show that the dgk5.1 mutant is impaired in auxin response at 12°C, suggesting a role of auxin perception /transduction in the short root phenotype

Phosphoglycerolipids are master players in plant hormone signal transduction

International audiencePhosphoglycerolipids are essential structural constituents of membranes and some also have important cell signalling roles. In this review, we focus on phos-phoglycerolipids that are mediators in hormone signal transduction in plants. We first describe the structures of the main signalling phosphoglycerolipids and the metabolic pathways that generate them, namely the phospholi-pase and lipid kinase pathways. In silico analysis of Arabidopsis transcriptome data provides evidence that the genes encoding the enzymes of these pathways are trans-criptionally regulated in responses to hormones, suggesting some link with hormone signal transduction. The involvement of phosphoglycerolipid signalling in the early responses to abscisic acid, salicylic acid and auxins is then detailed. One of the most important signalling lipids in plants is phosphatidic acid. It can activate or inactivate protein kinases and/or protein phosphatases involved in hormone signalling. It can also activate NADPH oxidase leading to the production of reactive oxygen species. We will interrogate the mechanisms that allow the activation/ deactivation of the lipid pathways, in particular the roles of G proteins and calcium. Mediating lipids thus appear as master players of cell signalling, modulating, if not controlling , major transducing steps of hormone signals

DIACYLGLYCEROL KINASE 5 participates in flagellin-induced signaling in Arabidopsis

International audienceAbstract Flagellin perception is a keystone of pattern-triggered immunity in plants. The recognition of this protein by a plasma membrane (PM) receptor complex is the beginning of a signaling cascade that includes protein phosphorylation and the production of reactive oxygen species (ROS). In both Arabidopsis (Arabidopsis thaliana) seedlings and suspension cells, we found that treatment with flg22, a peptide corresponding to the most conserved domain of bacterial flagellin, caused a rapid and transient decrease in the level of phosphatidylinositol (PI) 4,5-bisphosphate along with a parallel increase in phosphatidic acid (PA). In suspension cells, inhibitors of either phosphoinositide-dependent phospholipases C (PLC) or diacylglycerol kinases (DGKs) inhibited flg22-triggered PA production and the oxidative burst. In response to flg22, receptor-like kinase-deficient fls2, bak1, and bik1 mutants (FLAGELLIN SENSITIVE 2, BRASSINOSTEROID INSENSITIVE 1-associated kinase 1, and BOTRYTIS-INDUCED KINASE 1, respectively) produced less PA than wild-type (WT) plants, whereas this response did not differ in NADPH oxidase-deficient rbohD (RESPIRATORY BURST OXIDASE HOMOLOG D) plants. Among the DGK-deficient lines tested, the dgk5.1 mutant produced less PA and less ROS after flg22 treatment compared with WT seedlings. In response to flg22, dgk5.1 plants showed lower callose accumulation and impaired resistance to Pseudomonas syringae pv. tomato DC3000 hrcC-. Transcriptomics revealed that the basal expression of defense-related genes was altered in dgk5.1 seedlings compared with the WT. A GFP-DGK5 fusion protein localized to the PM, where RBOHD and PLC2 (proteins involved in plant immunity) are also located. The role of DGK5 and its enzymatic activity in flagellin signaling and fine-tuning of early immune responses in plant–microbe interactions is discussed

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

International audiencePhosphoinositide-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