Clade A Type 2C protein phosphatases (PP2CAs) negatively regulate abscisic acid (ABA) signaling and have diverse functions in plant development and in response to various stresses. In this study, we showed that overexpression of the rice ABA receptor OsPYL/RCAR3 reduces the growth retardation observed in plants exposed to osmotic stress. By contrast, overexpression of the OsPYL/RCAR3-interacting protein OsPP2C09 rendered plant growth more sensitive to osmotic stress. We tested whether OsPP2CAs activate an ABA-independent signaling cascade by transfecting rice protoplasts with luciferase reporters containing the drought-responsive element (DRE) or ABA-responsive element (ABRE). We observed that OsPP2CAs activated gene expression via the cis-acting drought-responsive element. In agreement with this observation, transcriptome analysis of plants overexpressing OsPP2C09 indicated that OsPP2C09 induces the expression of genes whose promoters contain DREs. Further analysis showed that OsPP2C09 interacts with DRE-binding (DREB) transcription factors and activates reporters containing DRE. We conclude that, through activating DRE-containing promoters, OsPP2C09 positively regulates the drought response regulon and activates an ABA-independent signaling pathway.
Overexpression of abscisic acid (ABA) receptors has been reported to enhance drought tolerance, but also to cause stunted growth and decreased crop yield. Here, we constructed transgenic rice for all monomeric ABA receptors and observed that only transgenic rice over-expressing OsPYL/RCAR7 showed similar phenotype with wild type, without total yield loss when grown under normal growth condition in a paddy field. Even though transgenic rice over-expressing OsPYL/RCAR7 showed neither an ABA-sensitivity nor an osmotic stress tolerance in plate assay, it showed drought tolerance. We investigated the ABA-dependent interaction with OsPP2CAs and ABA signaling induction by OsPYL/RCAR7. In yeast two hybrid assay, OsPYL/RCAR7 required critically higher ABA concentrations to interact with OsPP2CAs than other ABA receptors, and co-immunoprecipitation assay showed strong interaction under ABA treatment. When ABA-responsive signaling activity was monitored using a transient expression system in rice protoplasts, OsPYL/RCAR7 had the lowest ABA-responsive signaling activity as compared with other ABA receptors. OsPYL/RCAR7 also showed weak suppression of phosphatase activity as compared with other ABA receptors in vitro. Transcriptome analysis of transgenic rice over-expressing OsPYL/RCAR7 suggested that only a few genes were induced similar to control under without exogenous ABA, but a large number of genes was induced under ABA treatment compared with control. We conclude that OsPYL/RCAR7 is a novel functional ABA receptor that has low ABA signaling activity and exhibits high ABA dependence. These results lay the foundation for a new strategy to improve drought stress tolerance without compromising crop growth.
The stress hormone abscisic acid (ABA) helps plants to survive under abiotic stresses; however, its use as an agrochemical is limited by its chemical instability and expense. Here, we report the development of an in vivo screening system to isolate chemicals able to induce ABA signalling responses in rice (Oryza sativa) protoplasts. This system consists of an ABA-hypersensitive synthetic promoter containing ABRE and DRE motifs driving a luciferase reporter gene. After efficiently transfecting rice protoplasts with this construct, we screened chemicals library with a similar molecular weight and chemical structure to ABA and identified one chemical, S7, that induced ABA signalling by mediating interactions between the group I and II OsPYL receptors and certain OsPP2CAs in a yeast two-hybrid assay. In an in vitro pulldown assay, S7 was found to mediate a weak interaction between OsPYL5/8 and various OsPP2CAs. S7 treatments did not affect seedling growth or seed germination, but could reduce water loss. Rice seedlings treated with S7 exhibited transcriptome profiles that partially overlapped those treated with ABA. Taken together, we concluded that S7 is a new partial ABA agonist, which has potential use in future dissections of ABA signalling and as an agrochemical.One of the biggest challenges in agriculture is how to improve the productivity of crops under worsening environmental conditions, such as droughts and high temperatures caused by global climate change 1,2 . The plant stress hormone abscisic acid (ABA) and its functional mechanisms have therefore been intensively investigated in an effort to develop new ways of enhancing crop adaptations to stress. ABA is a sesquiterpenoid known to play central roles in adaptive responses to abiotic stresses such as drought, cold, and salt 3,4 . When plants experience abiotic stress conditions, their endogenous ABA levels increase to activate the ABA signalling pathways that regulate adaptive programmes, thereby enabling their survival in the adverse conditions 5 .The earliest ABA signalling event is triggered by ABA-dependent interactions among three different types of proteins: the ABA receptors named pyrabactin resistance/PYR like/regulatory component (PYR/PYL/RCAR (PYL)) proteins, the clade A phosphatase type 2Cs (PP2CAs), and the subfamily 2 members of SNF1-related protein kinases (SnRK2s). ABA is initially recognized by the ABA receptor PYL. Conformational changes in PYL upon ABA binding enables PYL-ABA to interact with the PP2CAs, thereby form a PYL-ABA-PP2CA complex 6-8 . The formation of this complex releases the SnRK2s from PP2CA-SnRK2 complexes, enabling them to phosphorylate downstream factors such as the ABRE-binding transcription factors or SLOWLY ACTIVATED ANION CHANNEL 1 (SLAC1), which in turn initiate ABA-mediated transcriptional regulation 9-11 .Although ABA signalling regulates many important abiotic stress response pathways, its potential as an agrochemical to improve stress tolerance in plants is limited by its chemical instability, rapid catabolism,...
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