The UBC27-AIRP3 ubiquitination complex modulates ABA signaling by promoting the degradation of ABI1 in Arabidopsis.
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ABSTRACT:
SUBMITTER: Pan W
PROVIDER: S-EPMC7959499 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The phytohormone abscisic acid (ABA) is an essential part of the plant response to abiotic stressors such as drought. Upon the perception of ABA, pyrabactin resistance (PYR)/PYR1-like (PYL)/regulatory components of ABA receptor (RCAR) proteins interact with co-receptor protein phosphatase type 2Cs to permit activation Snf1-related protein kinase2 (SnRK2) kinases, which switch on ABA signaling by phosphorylating various target proteins. Thus, SnRK2 kinases are central regulators of ABA signaling. However, the mechanisms that regulate SnRK2 degradation remain elusive. Here, we show that SnRK2.3 is degradated by 26S proteasome system and ABA promotes its degradation. We found that SnRK2.3 interacts with AtPP2-B11 directly. AtPP2-B11 is an F-box protein that is part of a SKP1/Cullin/F-box E3 ubiquitin ligase complex that negatively regulates plant responses to ABA by specifically promoting the degradation of SnRK2.3. AtPP2-B11 was induced by ABA, and the knockdown of AtPP2-B11 expression markedly increased the ABA sensitivity of plants during seed germination and postgerminative development. Overexpression of AtPP2-B11 does not affect ABA sensitivity, but inhibits the ABA hypersensitive phenotypes of SnRK2.3 overexpression lines. These results reveal a novel mechanism through which AtPP2-B11 specifically degrades SnRK2.3 to attenuate ABA signaling and the abiotic stress response in Arabidopsis.
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Project description: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.
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Project description:Plants are able to sense and mediate the balance between carbon (C) and nitrogen (N) nutrient availability to optimize metabolism and growth, described as the C/N response. To clarify the C/N signalling mechanism, C/N-insensitive plants were obtained from an Arabidopsis FOX hunting population, which over-expresses full-length cDNAs for individuals. The resulting cni2-D (carbon/nitrogen insensitive 2-dominant) plant was found to overcome the post-germination growth checkpoint and to expand green cotyledons in disrupted high C/low N stress conditions. The CNI2 gene encodes ABI1, a phosphatase type 2C protein, which negatively regulates abscisic acid (ABA) signal transduction. Over-expressors of ABI1 were found to be insensitive to disrupted C/N stress, whereas the loss-of function mutant abi1-2 was hypersensitive, suggesting that ABI1 plays an essential role in the plant C/N response. By contrast, the C/N-dependent growth phenotype observed in wild-type plants was not associated with endogenous ABA content. Accordingly, the ABA-insensitive mutant abi1-1, which could not bind to the ABA-ABA receptor complex, was not insensitive and restored normal sensitivity to high C/low N stress. The canonical ABA signalling mutants abi4 and abi5 were also sensitive to disrupted C/N stress. Further gene expression analysis demonstrated that several genes in the SnRK2s and SnRK1s pathways are transcriptionally affected by high C/low N stress in wild-type plants regardless of the lack of increased endogenous ABA contents, whereas the expression of these genes were significantly suppressed in ABI1 over-expressors. Taken together, these results suggest direct cross-talk between C/N and non-canonical ABA signalling pathways, regulated by ABI1, in plants.
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Project description:Clade A protein phosphatase 2Cs (PP2Cs) are abscisic acid (ABA) co-receptors that block ABA signalling by inhibiting the downstream protein kinases. ABA signalling is activated after PP2Cs are inhibited by ABA-bound PYR/PYL/RCAR ABA receptors (PYLs) in Arabidopsis. However, whether these PP2Cs are regulated by other factors remains unknown. Here, we report that ABI1 (ABA-INSENSITIVE 1) can interact with the U-box E3 ligases PUB12 and PUB13, but is ubiquitinated only when it interacts with ABA receptors in an in vitro assay. A mutant form of ABI1-1 that is unable to interact with PYLs is more stable than the wild-type protein. Both ABI1 degradation and all tested ABA responses are reduced in pub12 pub13 mutants compared with the wild type. Introducing the abi1-3 loss-of-function mutation into pub12 pub13 mutant recovers the ABA-insensitive phenotypes of the pub12 pub13 mutant. We thus uncover an important regulatory mechanism for regulating ABI1 levels by PUB12 and PUB13.
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Project description:Phosphorylation and dephosphorylation events play an important role in the transmission of the ABA signal. Although SnRK2 [sucrose non-fermenting1-related kinase2] protein kinases and group A protein phosphatase type 2C (PP2C)-type phosphatases constitute the core ABA pathway, mitogen-activated protein kinase (MAPK) pathways are also involved in plant response to ABA. However, little is known about the interplay between MAPKs and PP2Cs or SnRK2 in the regulation of ABA pathways. In this study, an effort was made to elucidate the role of MAP kinase kinase kinase18 (MKKK18) in relation to ABA signaling and response. The MKKK18 knockout lines showed more vigorous root growth, decreased abaxial stomatal index and increased stomatal aperture under normal growth conditions, compared with the control wild-type Columbia line. In addition to transcriptional regulation of the MKKK18 promoter by ABA, we demonstrated using in vitro and in vivo kinase assays that the kinase activity of MKKK18 was regulated by ABA. Analysis of the cellular localization of MKKK18 showed that the active kinase was targeted specifically to the nucleus. Notably, we identified abscisic acid insensitive 1 (ABI1) PP2C as a MKKK18-interacting protein, and demonstrated that ABI1 inhibited its activity. Using a cell-free degradation assay, we also established that MKKK18 was unstable and was degraded by the proteasome pathway. The rate of MKKK18 degradation was delayed in the ABI1 knockout line. Overall, we provide evidence that ABI1 regulates the activity and promotes proteasomal degradation of MKKK18.
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Project description:The Met receptor tyrosine kinase is an attractive target for cancer therapy as it promotes invasive tumor growth. SAIT301 is a novel anti-Met antibody, which induces LRIG1-mediated Met degradation and inhibits tumor growth. However, detailed downstream mechanism by which LRIG1 mediates target protein down-regulation is unknown. In the present study, we discovered that SAIT301 induces ubiquitination of LRIG1, which in turn promotes recruitment of Met and LRIG1 complex to the lysosome through its interaction with Hrs, resulting in concomitant degradation of both LRIG1 and Met. We also identified USP8 as a LRIG1-specific deubiquitinating enzyme, reporting the interaction between USP8 and LRIG1 for the first time. SAIT301 triggers degradation of LRIG1 by inhibiting the interaction of LRIG1 and USP8, which regulates ubiquitin modification and stability of LRIG1. In summary, SAIT301 employs ubiquitination of LRIG1 for its highly effective Met degradation. This unique feature of SAIT301 enables it to function as a fully antagonistic antibody without Met activation. We found that USP8 is involved in deubiquitination of LRIG1, influencing the efficiency of Met degradation. The relation of Met, LRIG1 and USP8 strongly supports the potential clinical benefit of a combination treatment of a USP8 inhibitor and a Met inhibitor, such as SAIT301.
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Project description:Differential concentrations of phytohormone trigger distinct outputs, which provides a mechanism for the plasticity of plant development and an adaptation strategy among plants to changing environments. However, the underlying mechanisms of the differential responses remain unclear. Here we report that a high concentration of auxin, distinct from the effect of low auxin concentration, enhances abscisic acid (ABA) responses in Arabidopsis thaliana, which partially relies on TRANS-MEMBERANE KINASE 1 (TMK1), a key regulator in auxin signaling. We show that high auxin and TMK1 play essential and positive roles in ABA signaling through regulating ABA INSENSITIVE 1 and 2 (ABI1/2), two negative regulators of the ABA pathway. TMK1 inhibits the phosphatase activity of ABI2 by direct phosphorylation of threonine 321 (T321), a conserved phosphorylation site in ABI2 proteins, whose phosphorylation status is important for both auxin and ABA responses. This TMK1-dependent auxin signaling in the regulation of ABA responses provides a possible mechanism underlying the high auxin responses in plants and an alternative mechanism involved in the coordination between auxin and ABA signaling.
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