Heterologous expression and molecular and cellular characterization of CaPUB1 encoding a hot pepper U-box E3 ubiquitin ligase homolog
Heterologous expression and molecular and cellular characterization of CaPUB1 encoding a hot pepper U-box E3 ubiquitin ligase homolog
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DOI：
10.1104/pp.106.087965
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发表时间：
2006-12-01
期刊：
影响因子：
7.4
通讯作者：
Kim, Woo Taek
中科院分区：
文献类型：
--
作者：
Cho, Seok Keun;Chung, Hoo Sun;Kim, Woo Taek
U-box基序是在真核生物E3泛素连接酶的多种同工型中发现的一个保守结构域。从缺水辣椒（Capsicum annuum L. cv Pukang）植株中分离出C. annuum推定的U-box蛋白1 (CaPUB1)，该蛋白编码一个在其n端区域含有单个U-box基序的蛋白。体外泛素化和定点诱变实验表明，CaPUB1具有E3泛素连接酶活性，并且U-box基序确实是其酶活性所必需的。RNA凝胶印迹分析显示，CaPUB1 mRNA可被广泛的非生物胁迫（包括干旱、高盐、低温和机械损伤）快速诱导，但不受乙烯、脱落酸或细菌病原体的影响，这表明它在非生物相关防御反应的早期事件中起作用。由于辣椒的转基因工作非常困难，本研究在拟南芥（Arabidopsis thaliana）中过表达CaPUB1，以提供该基因在发育和植物对非生物胁迫反应中的功能的细胞信息。在花椰菜花叶病毒35S启动子的控制下，组成部分表达CaPUB1基因的转基因拟南芥植株的下胚轴和根明显长于野生型，生长速度快于野生型，出现较早的抽苔表型。显微镜分析显示，35STCaPUB1根的小细胞数量增加，导致皮层、内胚层和柱状细胞层排列紊乱，密集分布。此外，过表达capub1的植物对水分胁迫和轻度盐度的敏感性增加。这些结果表明，CaPUB1在拟南芥细胞中发挥功能，从而有效地改变细胞和组织的生长以及对非生物胁迫的反应。比较蛋白质组学分析显示，与野生型相比，35STCaPUB1幼苗中26S蛋白酶体复合物的非atp酶亚基RPN6蛋白水平显著降低。Pull-down和泛素化实验表明，RPN6与CaPUB1相互作用，并在体外以CaPUB1依赖的方式泛素化。虽然CaPUB1的生理功能尚不清楚，但有几种可能性表明它参与了转基因拟南芥幼苗抵抗脱水和高盐度胁迫的生理反应子集。
The U-box motif is a conserved domain found in the diverse isoforms of E3 ubiquitin ligase in eukaryotes. From water-stressed hot pepper (Capsicum annuum L. cv Pukang) plants, we isolated C. annuum putative U-box protein 1 (CaPUB1), which encodes a protein containing a single U-box motif in its N-terminal region. In vitro ubiquitination and site-directed mutagenesis assays revealed that CaPUB1 possessed E3 ubiquitin ligase activity and that the U-box motif was indeed essential for its enzyme activity. RNA gel-blot analysis showed that CaPUB1 mRNA was induced rapidly by a broad spectrum of abiotic stresses, including drought, high salinity, cold temperature, and mechanical wounding, but not in response to ethylene, abscisic acid, or a bacterial pathogen, suggesting its role in the early events in the abiotic-related defense response. Because transgenic work was extremely difficult in hot pepper, in this study we overexpressed CaPUB1 in Arabidopsis (Arabidopsis thaliana) to provide cellular information on the function of this gene in the development and plant responses to abiotic stresses. Transgenic Arabidopsis plants that constitutively expressed the CaPUB1 gene under the control of the cauliflower mosaic virus 35S promoter had markedly longer hypocotyls and roots and grew more rapidly than the wild type, leading to an early bolting phenotype. Microscopic analysis showed that 35STCaPUB1 roots had increased numbers of small-sized cells, resulting in disordered, highly populated cell layers in the cortex, endodermis, and stele. In addition, CaPUB1-overexpressing plants displayed increased sensitivity to water stress and mild salinity. These results indicate that CaPUB1 is functional in Arabidopsis cells, thereby effectively altering cell and tissue growth and also the response to abiotic stresses. Comparative proteomic analysis showed that the level of RPN6 protein, a non-ATPase subunit of the 26S proteasome complex, was significantly reduced in 35STCaPUB1 seedlings as compared to the wild type. Pull-down and ubiquitination assays demonstrated that RPN6 interacted physically with CaPUB1 and was ubiquitinated in a CaPUB1-dependent manner in vitro. Although the physiological function of CaPUB1 is not yet clear, there are several possibilities for its involvement in a subset of physiological responses to counteract dehydration and high-salinity stresses in transgenic Arabidopsis seedlings.