Plants are sessile and unable to avoid environmental stresses, such as drought, high temperature, and high salinity, which often limit the overall plant growth. Plants have evolved many complex mechanisms to survive these abiotic stresses via post-translational modifications. Recent evidence suggests that ubiquitination plays a crucial role in regulating abiotic stress responses in plants by regulating their substrate proteins. Here, we reported the molecular function of a RING finger E3 ligase, Oryza sativa Drought, Heat and Salt-induced RING finger protein 1 (OsDHSRP1), involved in regulating plant abiotic stress tolerance via the Ub/26S proteasome system. The OsDHSRP1 gene transcripts were highly expressed under various abiotic stresses such as NaCl, drought, and heat and the phytohormone abscisic acid (ABA). In addition, in vitro ubiquitination assays demonstrated that the OsDHSRP1 protein possesses a RING-H2 type domain that confers ligase functionality. The results of yeast two-hybrid (Y2H), in vitro pull-down, and bimolecular fluorescence complementation assays support that OsDHSRP1 is able to regulate two substrates, O. sativa glyoxalase (OsGLYI-11.2) and O. sativa abiotic stress-induced cysteine proteinase 1 (OsACP1). We further confirmed that these two substrate proteins were ubiquitinated by OsDHSRP1 E3 ligase and caused protein degradation via the Ub/26S proteasome system. The Arabidopsis plants overexpressing OsDHSRP1 exhibited hypersensitivity to drought, heat, and NaCl stress and a decrease in their germination rates and root lengths compared to the control plants because the degradation of the OsGLYI-11.2 protein maintained lower glyoxalase levels, which increased the methylglyoxal amount in transgenic Arabidopsis plants. However, the OsDHSRP1-overexpressing plants showed no significant difference when treated with ABA. Our finding supports the hypothesis that the OsDHSRP1 E3 ligase acts as a negative regulator, and the degradation of its substrate proteins via ubiquitination plays important roles in regulating various abiotic stress responses via an ABA-independent pathway.

中文翻译：

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水稻干旱，热和盐诱导的无名指蛋白1（OsDHSRP1）负调节非生物胁迫响应基因的表达。

植物是无柄的，无法避免环境压力，例如干旱，高温和高盐度，这通常会限制整个植物的生长。植物已经进化出许多复杂的机制来通过翻译后修饰来抵抗这些非生物胁迫。最近的证据表明，泛素化通过调节其底物蛋白在调节植物的非生物胁迫响应中起着至关重要的作用。在这里，我们报道了RING手指E3连接酶，水稻干旱，热和盐诱导的RING手指蛋白1（OsDHSRP1）的分子功能，该分子通过Ub / 26S蛋白酶体系统参与调节植物的非生物胁迫耐受性。OsDHSRP1基因转录物在各种非生物胁迫（例如NaCl，干旱和高温以及植物激素脱落酸（ABA））下高表达。此外，体外泛素化试验表明，OsDHSRP1蛋白具有赋予连接酶功能的RING-H2型结构域。酵母双杂交（Y2H），体外下拉和双分子荧光互补测定的结果支持OsDHSRP1能够调控O. sativa乙二醛乙二醛酶（OsGLYI-11.2）和O. sativa非生物胁迫引起的半胱氨酸蛋白酶1（OsACP1）。我们进一步证实，这两种底物蛋白被OsDHSRP1 E3连接酶泛素化，并通过Ub / 26S蛋白酶体系统引起蛋白降解。与对照植物相比，过表达OsDHSRP1的拟南芥植物对干旱，高温和NaCl胁迫表现出超敏性，并且发芽率和根长降低，这是因为OsGLYI-11.2蛋白的降解保持了较低的乙二醛酶水平，这增加了转基因拟南芥植物中的甲基乙二醛含量。但是，用ABA处理时，过表达OsDHSRP1的植物无明显差异。我们的发现支持以下假设：OsDHSRP1 E3连接酶充当负调节剂，其底物蛋白通过泛素化的降解在通过ABA独立途径调节各种非生物胁迫响应中起着重要作用。