Abscisic acid (ABA) is a plant hormone that mediates abiotic stress tolerance and regulates growth and development. ABA binds to members of the PYL/RCAR ABA receptor family that initiate signal transduction inhibiting type 2C protein phosphatases. Although crosstalk between ABA and the hormone Jasmonic Acid (JA) has been shown, the molecular entities that mediate this interaction have yet to be fully elucidated. We report a link between ABA and JA signaling through a direct interaction of the ABA receptor PYL6 (RCAR9) with the basic helix-loop-helix transcription factor MYC2. PYL6 and MYC2 interact in yeast two hybrid assays and the interaction is enhanced in the presence of ABA. PYL6 and MYC2 interact in planta based on bimolecular fluorescence complementation and co-immunoprecipitation of the proteins. Furthermore, PYL6 was able to modify transcription driven by MYC2 using JAZ6 and JAZ8 DNA promoter elements in yeast one hybrid assays. Finally, pyl6 T-DNA mutant plants show an increased sensitivity to the addition of JA along with ABA in cotyledon expansion experiments. Overall, the present study identifies a direct mechanism for transcriptional modulation mediated by an ABA receptor different from the core ABA signaling pathway, and a putative mechanistic link connecting ABA and JA signaling pathways.

Adverse environmental (abiotic) stressors, such as prolonged periods of drought, limit production in modern agriculture. The phytohormone abscisic acid (ABA) is a key hormone produced under abiotic stress conditions and is a regulator of diverse plant stress resistance and developmental processes. Understanding ABA-mediated regulation of plant stress responses and plant development is key to improving abiotic stress resistance of modern crop varieties.

The phytohormone abscisic acid (ABA) plays crucial roles in plant development and plant responses to environmental stresses. Although ABA receptors and a minimal set of core molecular components have recently been discovered, understanding of the ABA signaling pathway is still far from complete. In this work, we characterized the function of ROP11, a member of the plant-specific ROP small GTPases family, in the ABA signaling process. ROP11 is preferentially expressed in guard cells in all plant organs with stomata. Expression of a constitutively active ROP11 (CA-ROP11) suppresses ABA-mediated responses, whereas reduced expression of ROP11 or expression of its dominant-negative form (DN-ROP11) causes the opposite phenotypes. The affected ABA-mediated responses by ROP11 include seed germination, seedling growth, stomatal closure, induction of ABA-responsive genes, as well as plant response to drought stress. Furthermore, we showed that ROP11 and its closest-related family member, ROP10, act in parallel in mediating these responses. ABA treatment does not affect ROP11 transcription and protein abundance; however, it causes the accumulation of CA-ROP11 in the nucleus. These results demonstrated that ROP11 is a negative regulator of multiple ABA responses in Arabidopsis.

The phytohormone abscisic acid (ABA) is critical to plant development and stress responses. Abiotic stress triggers an ABA signal transduction cascade, which is comprised of the core components PYL/RCAR ABA receptors, PP2C-type protein phosphatases, and protein kinases. Small GTPases of the ROP/RAC family act as negative regulators of ABA signal transduction. However, the mechanisms by which ABA controls the behavior of ROP/RACs have remained unclear. Here, we show that an Arabidopsis guanine nucleotide exchange factor protein RopGEF1 is rapidly sequestered to intracellular particles in response to ABA. GFP-RopGEF1 is sequestered via the endosome-prevacuolar compartment pathway and is degraded. RopGEF1 directly interacts with several clade A PP2C protein phosphatases, including ABI1. Interestingly, RopGEF1 undergoes constitutive degradation in pp2c quadruple abi1/abi2/hab1/pp2ca mutant plants, revealing that active PP2C protein phosphatases protect and stabilize RopGEF1 from ABA-mediated degradation. Interestingly, ABA-mediated degradation of RopGEF1 also plays an important role in ABA-mediated inhibition of lateral root growth. The presented findings point to a PP2C-RopGEF-ROP/RAC control loop model that is proposed to aid in shutting off ABA signal transduction, to counteract leaky ABA signal transduction caused by “monomeric” PYL/RCAR ABA receptors in the absence of stress, and facilitate signaling in response to ABA.

The phytohormone abscisic acid (ABA) regulates many key processes in plants, such as seed germination, seedling growth, and abiotic stress tolerance. In recent years, a minimal set of core components of a major ABA signaling pathway has been discovered. These components include a RCAR/PYR/PYL family of ABA receptors, a group of PP2C phosphatases, and three SnRK2 kinases. However, how the interactions between the receptors and their targets are regulated by other proteins remains largely unknown. In a companion paper published in this issue, we showed that ROP11, a member of the plant-specific Rho-like small GTPase family, negatively regulates multiple ABA responses in Arabidopsis. The current work demonstrated that the constitutively active ROP11 (CA-ROP11) can modulate the RCAR1/PYL9-mediated ABA signaling pathway based on reconstitution assays in Arabidopsis thaliana protoplasts. Furthermore, using luciferase complementation imaging, yeast two-hybrid assays, co-immunoprecipitation assays in Nicotiana benthamiana and bimolecular fluorescence complementation assays, we demonstrated that CA-ROP11 directly interacts with ABI1, a signaling component downstream of RCAR1/PYL9. Finally, we provided biochemical evidence that CA-ROP11 protects ABI1 phosphatase activity from inhibition by RCAR1/PYL9 and thus negatively regulates ABA signaling in plant cells. A model of how ROP11 acts to negatively regulate ABA signaling is presented.

Edited by Ulf-Ingo FlüggeKeywords: ROPGEF1 ROPGEF4 ROP11 GTPase Regulator ABA response Stomatal closure a b s t r a c t ROPs constitute a family of plant-specific, RHO-like small GTPases that serve as molecular switches in a wide range of signaling pathways. The activities of ROPs are regulated by guanine nucleotide exchange factors (GEFs). ROP11, a member of the ROP GTPase family in Arabidopsis, is a negative regulator of multiple ABA responses. In this study, we show that ROPGEF1 and ROPGEF4 interact with ROP11 on plasma membranes in guard cells. Furthermore, our analyses of ROPGEF1/4 knockout mutants and overexpressing lines suggested that ROPGEF1 and ROPGEF4 are specific regulators of ROP11 function in ABA-mediated stomatal closure. Structured summary of protein interactions:ROPGEF9 and Rop11 physically interact by protein complementation assay (View interaction) ROPGEF9 physically interacts with Rop11 by two hybrid (View interaction) ROPGEF1 and Rop11 physically interact by bimolecular fluorescence complementation (View interaction) ROPGEF4 physically interacts with Rop11 by two hybrid (View interaction) ROPGEF2 and Rop11 physically interact by protein complementation assay (View interaction) Rop11 physically interacts with ROPGEF1 by anti tag coimmunoprecipitation (View interaction) ROPGEF1 and Rop11 physically interact by protein complementation assay (View interaction) ROPGEF4 and Rop11 physically interact by protein complementation assay (View interaction) ROPGEF8 physically interacts with Rop11 by two hybrid (View interaction) ROPGEF8 and Rop11 physically interact by protein complementation assay (View interaction) ROPGEF2 physically interacts with Rop11 by two hybrid (View interaction) Rop11 physically interacts with ROPGEF4 by anti tag coimmunoprecipitation (View interaction) ROPGEF4 and Rop11 physically interact by bimolecular fluorescence complementation (View interaction) ROPGEF1 physically interacts with Rop11 by two hybrid (View interaction)

Abscisic acid (ABA) plays essential roles in plant development and responses to environmental stress. ABA induces subcellular translocation and degradation of the guanine nucleotide exchange factor RopGEF1, thus facilitating ABA core signal transduction. However, the underlying mechanisms for ABA-triggered RopGEF1 trafficking/degradation remain unknown. Studies have revealed that RopGEFs associate with receptor-like kinases to convey developmental signals to small ROP GTPases. However, how the activities of RopGEFs are modulated is not well understood. Type 2C protein phosphatases stabilize the RopGEF1 protein, indicating that phosphorylation may trigger RopGEF1 trafficking and degradation. We have screened inhibitors followed by several protein kinase mutants and find that quadruple-mutant plants in the calcium-dependent protein kinases (CPKs) disrupt ABA-induced trafficking and degradation of RopGEF1. Moreover, partially impairs ABA inhibition of cotyledon emergence. Several CPKs interact with RopGEF1. CPK4 binds to and phosphorylates RopGEF1 and promotes the degradation of RopGEF1. CPK-mediated phosphorylation of RopGEF1 at specific N-terminal serine residues causes the degradation of RopGEF1 and mutation of these sites also compromises the RopGEF1 overexpression phenotype in root hair development in Our findings establish the physiological and molecular functions and relevance of CPKs in regulation of RopGEF1 and illuminate physiological roles of a CPK-GEF-ROP module in ABA signaling and plant development. We further discuss that CPK-dependent RopGEF degradation during abiotic stress could provide a mechanism for down-regulation of RopGEF-dependent growth responses.

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