The Arabidopsis ABA-Activated Kinase OST1 Phosphorylates the bZIP Transcription Factor ABF3 and Creates a 14-3-3 Binding Site Involved in Its Turnover

indicates that members of the Snf1-Related Kinases 2 family (SnRK2) are essential in mediating various stress-adaptive responses. Recent reports have indeed shown that one particular member, OPEN STOMATA (OST)1, whose kinase activity is stimulated by the stress hormone abscisic acid (ABA), is a direct target of negative regulation by the core ABA co-receptor complex composed of PYR/PYL/RCAR and clade A Protein Phosphatase 2C (PP2C) proteins. and that phospho-T451 is important for stabilization of ABF3. on T451 to create a 14-3-3 binding motif. In a wider physiological context, we propose that the long term responses to ABA that require sustained gene expression is, in part, mediated by the stabilization of ABFs driven by ABA-activated SnRK2s

Mécanismes de régulation des réponses au stress hydrique par la protéine kinase OPEN STOMATA1

En réponse à la sécheresse, la phytohormone acide abscissique (ABA) induit la fermeture des stomates, afin de limiter les pertes en eau, et une modification de l expression de gènes dont les produits participent au maintien de l intégrité cellulaire. Les kinases SnRK2 régulent la signalisation de l ABA. Nous avons défini le motif optimal de phosphorylation par la kinase SnRK2.6/OST1, jouant un rôle clé dans les stomates, et avons utilisé ces données pour prédire les cibles putatives d OST1 in silico. Le facteur de transcription ABF3 apparaît comme un substrat physiologique d OST1 dans les stomates. OST1 phosphoryle ABF3 in vitro sur plusieurs sites LXRXXS/T. L un d eux correspond à la T451, située dans un motif conservé, qui une fois phosphorylée, crée un motif de liaison aux protéines 14-3-3. De plus, la T451 est indispensable à la phosphorylation et à la stabilisation d ABF3 in vivo en réponse à l ABA. Ces données suggèrent que la phosphorylation des ABF par les SnRK2 et la liaison des 14-3-3 qui en résulte, protègeraient ces facteurs de la dégradation, permettant l expression de gènes ABA-dépendants. Dans une approche ciblée, nous avons identifié le motif de phosphorylation d OST1 dans la NADPH oxydase AtrbohF impliquée dans la fermeture stomatique. OST1 interagit avec, et phosphoryle, AtrbohF. Ceci suggère une régulation de l activité NADPH oxydase par OST1. Nos résultats étayent donc le rôle central d OST1 dans les voies de l ABA, au niveau de l expression génique et de la fermeture stomatique. De plus, l approche prédictive permettra d identifier de nouvelles cibles des SnRK2 et de contribuer à une meilleure compréhension de la signalisation de l ABA.In response to drought, the plant hormone abscisic acid (ABA) promotes stomatal closure to prevent water loss by transpiration, and regulates the expression of many genes that participate to the dehydration tolerance. ABA-activated SnRK2 kinases are regulators of this signaling pathway. We defined the substrate preferences of the SnRK2 kinase OST1, a key component of ABA signaling in guard cells, and used these data to predict putative substrates in silico. Our results designate the transcription factor ABF3 as genuine OST1 substrate in guard cells. In vitro, OST1 phosphorylates ABF3 on multiple sites including the one corresponding to T451, which once phosphorylated, creates a binding site for 14-3-3 proteins. Furthermore, T451 is important for both ABF3 phosphorylation and stabilization in response to ABA in vivo. These findings suggest that the phosphorylation of ABF transcription factors by ABA-activated SnRK2 kinases and the subsequent binding of 14-3-3 protect these factors from degradation, leading to ABA-responsive genes expression. In a targeted approach, we identified putative OST1 phosphorylation sites in the NADPH oxidase AtrbohF which plays a role in stomatal closure. OST1 interacts with and phosphorylates AtrbohF, suggesting a regulation of NADPH oxidase activity by OST1 in guard cell. Together, our results imply that OST1 regulates ABA signaling in guard cell through the phosphorylation and the regulation of several proteins involved in the regulation of gene expression and in stomatal closure. Furthermore, the predictive strategy could help the identification of new SnRK2 substrates and therefore contribute to a better understanding of ABA signaling.ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF

Protein Phosphatases 2C Regulate the Activation of the Snf1-Related Kinase OST1 by Abscisic Acid in Arabidopsis[W]

The plant hormone abscisic acid (ABA) orchestrates plant adaptive responses to a variety of stresses, including drought. This signaling pathway is regulated by reversible protein phosphorylation, and genetic evidence demonstrated that several related protein phosphatases 2C (PP2Cs) are negative regulators of this pathway in Arabidopsis thaliana. Here, we developed a protein phosphatase profiling strategy to define the substrate preferences of the HAB1 PP2C implicated in ABA signaling and used these data to screen for putative substrates. Interestingly, this analysis designated the activation loop of the ABA activated kinase OST1, related to Snf1 and AMPK kinases, as a putative HAB1 substrate. We experimentally demonstrated that HAB1 dephosphorylates and deactivates OST1 in vitro. Furthermore, HAB1 and the related PP2Cs ABI1 and ABI2 interact with OST1 in vivo, and mutations in the corresponding genes strongly affect OST1 activation by ABA. Our results provide evidence that PP2Cs are directly implicated in the ABA-dependent activation of OST1 and further suggest that the activation mechanism of AMPK/Snf1-related kinases through the inhibition of regulating PP2Cs is conserved from plants to human