MAIN CONCLUSION Molecular function ofRING E3 ligase SbHCI1is involved in ABA-mediated basal heat stress tolerancein sorghum. Global warming generally reduces plant survival, owing to the negative effects of high temperatures on plant development. However, little is known about the role of Really Interesting New Gene (RING) E3 ligase in the heat stress responses of plants. As such, the aim of the present study was to characterize the molecular functions of the Sorghum bicolor ortholog of the Oryza sativa gene for Heat- and Cold-Induced RING finger protein 1 (SbHCI1). Subcellular localization revealed that SbHCI1 was mainly associated with the cytosol and that it moved to the Golgi apparatus under heat stress conditions. The fluorescent signals of SbHCI1 substrate proteins were observed to migrate to the cytoplasm under heat stress conditions. Bimolecular fluorescence complementation (BiFC) and yeast two-hybrid (Y2H) assays revealed that SbHCI1 physically interacted with OsHCI1 ortholog partner proteins in the cytoplasm. Moreover, an in vitro ubiquitination assay revealed that SbHCI1 polyubiquitinated each of the three interacting proteins. The ectopic overexpression of SbHCI1 in Arabidopsis revealed that the protein was capable of inducing abscisic acid (ABA)-hypersensitivity and basal heat stress tolerance. Therefore, SbHCI1 possesses E3 ligase activity and may function as a positive regulator of heat stress responses through the modulation of interacting proteins.

中文翻译：

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高温和脱落酸胁迫下高粱 RING E3 连接酶 SbHCI1 的分子特征

主要结论RING E3连接酶SbHCI1的分子功能参与了ABA介导的高粱基础热胁迫耐受性。由于高温对植物发育的负面影响，全球变暖通常会降低植物的存活率。然而，关于真正有趣的新基因 (RING) E3 连接酶在植物热应激反应中的作用知之甚少。因此，本研究的目的是表征水稻基因的高粱双色直系同源物对热和冷诱导的环指蛋白 1 (SbHCI1) 的分子功能。亚细胞定位显示 SbHCI1 主要与胞质溶胶相关，并且它在热应激条件下移动到高尔基体。观察到 SbHCI1 底物蛋白的荧光信号在热应激条件下迁移到细胞质。双分子荧光互补 (BiFC) 和酵母双杂交 (Y2H) 分析表明 SbHCI1 与细胞质中的 OsHCI1 直向同源蛋白存在物理相互作用。此外，体外泛素化测定显示 SbHCI1 多泛素化三种相互作用蛋白中的每一种。SbHCI1 在拟南芥中的异位过表达表明该蛋白能够诱导脱落酸 (ABA) 超敏反应和基础热应激耐受。因此，SbHCI1 具有 E3 连接酶活性，可通过调节相互作用的蛋白质作为热应激反应的正调节剂。体外泛素化测定表明，SbHCI1 多泛素化了三种相互作用蛋白中的每一种。SbHCI1 在拟南芥中的异位过表达表明该蛋白能够诱导脱落酸 (ABA) 超敏反应和基础热应激耐受。因此，SbHCI1 具有 E3 连接酶活性，可通过调节相互作用的蛋白质作为热应激反应的正调节剂。体外泛素化测定表明，SbHCI1 多泛素化了三种相互作用蛋白中的每一种。SbHCI1 在拟南芥中的异位过表达表明该蛋白能够诱导脱落酸 (ABA) 超敏反应和基础热应激耐受。因此，SbHCI1 具有 E3 连接酶活性，可通过调节相互作用的蛋白质作为热应激反应的正调节剂。