Complex Regulation by Apetala2 Domain-Containing Transcription Factors Revealed through Analysis of the Stress-Responsive <em>TdCor410b</em> Promoter from Durum Wheat

Expression of the wheat dehydrin gene Cor410b is induced several fold above its non-stressed levels upon exposure to stresses such as cold, drought and wounding. Deletion analysis of the TdCor410b promoter revealed a single functional C-repeat (CRT) element. Seven transcription factors (TFs) were shown to bind to this CRT element using yeast one-hybrid screens of wheat and barley cDNA libraries, of which only one belonged to the DREB class of TFs. The remaining six encoded ethylene response factors (ERFs) belong to three separate subfamilies. Analysis of binding selectivity of these TFs indicated that all seven could bind to the CRT element (GCCGAC), and that three of the six ERFs could bind both to the CRT element and the ethylene-responsive GCC-box (GCCGCC). The TaERF4 subfamily members specifically bound the CRT element, and did not bind either the GCC-box or DRE element (ACCGAC). Molecular modeling and site-directed mutagenesis identified a single residue Pro42 in the Apetala2 (AP2) domain of TaERF4-like proteins that is conserved in monocotyledonous plants and is responsible for the recognition selectivity of this subfamily. We suggest that both DREB and ERF proteins regulate expression of the Cor410b gene through a single, critical CRT element. Members of the TaERF4 subfamily are specific, positive regulators of Cor410b gene expression.小麦脱水素基因Cor410b的表达，在遭受低温、干旱及机械损伤等胁迫时，其转录水平较非胁迫状态可被诱导数倍上调。对TdCor410b启动子的缺失分析显示，其仅含有一个具有功能活性的C重复（CRT）元件。通过对小麦和大麦互补DNA（cDNA）文库开展酵母单杂交筛选，共鉴定得到7种可结合该CRT元件的转录因子（TFs），其中仅1种隶属于脱水应答元件结合蛋白（DREB）家族。剩余6种均为乙烯应答因子（ERFs），分属于3个不同的亚家族。对这些转录因子的结合选择性分析结果表明，7种转录因子均可结合CRT元件（核心序列GCCGAC）；6种ERFs中有3种可同时结合CRT元件与乙烯应答GCC盒（核心序列GCCGCC）。TaERF4亚家族成员仅特异性结合CRT元件，既不识别GCC盒，也不结合脱水应答元件（DRE，核心序列ACCGAC）。分子建模与定点突变实验证实，TaERF4类蛋白的APETALA2（AP2）结构域中存在一个保守残基Pro42，该位点在单子叶植物中高度保守，是决定该亚家族转录因子识别特异性的关键位点。本研究提出，DREB蛋白与ERF蛋白均可通过单一关键CRT元件调控Cor410b基因的表达；TaERF4亚家族成员是Cor410b基因表达的特异性正向调控因子。