Cheng Mei-Chun, Kuo Wen-Chieh, Wang Yi-Ming, Chen Hsing-Yu, Lin Tsan-Piao
Institute of Plant Biology, National Taiwan University, 1 Roosevelt Road, Section 4, Taipei, 10617, Taiwan.
New Phytol. 2017 Feb;213(3):1156-1167. doi: 10.1111/nph.14272. Epub 2016 Oct 27.
Ethylene Response Factor 1 (ERF1) plays a crucial role in biotic and abiotic stress responses. Previous studies have shown that ERF1 regulates stress-responsive gene expression by binding to different cis-acting elements in response to various stress signals. ERF1 was also reported to be unstable in the dark, and it regulates hypocotyl elongation. Here, we elucidated the mechanism underlying degradation of ERF1. Yeast two-hybrid screening showed that UBIQUITIN-CONJUGATING ENZYME 18 (UBC18) interacted with ERF1. The interaction between ERF1 and UBC18 was verified using pull-down assays and coimmunoprecipitation analyses. We then compared the ERF1 protein abundance in the UBC18 mutant and overexpression plants. Based on the results of protein degradation and in vivo ubiquitination assays, we proposed that UBC18 mediates ERF1 ubiquitination and degradation. ERF1 was more stable in UBC18 mutants and less stable in UBC18 overexpression lines compared with that in wild-type plants. ERF1 was degraded by the 26S proteasome system via regulation of UBC18 and promotes dark-repression of downstream genes and proline accumulation. UBC18 negatively regulated drought and salt stress responses by altering the abundance of ERF1 and the expression of genes downstream of ERF1.
乙烯反应因子1（ERF1）在生物和非生物胁迫反应中起关键作用。先前的研究表明，ERF1通过响应各种胁迫信号与不同的顺式作用元件结合来调节胁迫响应基因的表达。据报道，ERF1在黑暗中不稳定，并且它调节下胚轴伸长。在这里，我们阐明了ERF1降解的潜在机制。酵母双杂交筛选表明，泛素结合酶18（UBC18）与ERF1相互作用。使用下拉试验和免疫共沉淀分析验证了ERF1与UBC18之间的相互作用。然后，我们比较了UBC18突变体和过表达植物中ERF1蛋白的丰度。基于蛋白质降解和体内泛素化试验的结果，我们提出UBC18介导ERF1的泛素化和降解。与野生型植物相比，ERF1在UBC18突变体中更稳定，而在UBC18过表达系中更不稳定。ERF1通过UBC18的调节被26S蛋白酶体系统降解，并促进下游基因的黑暗抑制和脯氨酸积累。UBC18通过改变ERF1的丰度和ERF1下游基因的表达来负向调节干旱和盐胁迫反应。
