Complex Regulation by Apetala2 Domain-Containing Transcription Factors Revealed through Analysis of the Stress-Responsive TdCor410b 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下可被诱导数倍。对TdCor410b启动子的缺失分析显示，存在一个具有功能的C重复（C-repeat, CRT）元件。通过对小麦和大麦cDNA文库开展酵母单杂交（yeast one-hybrid）筛选，共鉴定出7种可结合该CRT元件的转录因子（Transcription Factors, TFs）；其中仅1种属于DREB类转录因子，剩余6种编码的乙烯响应因子（Ethylene Response Factors, ERFs）分属3个独立的亚家族。对这些转录因子的结合选择性分析表明，全部7种因子均可结合CRT元件（GCCGAC）；而6种ERFs中有3种可同时结合CRT元件与乙烯响应性GCC盒（GCC-box, GCCGCC）。TaERF4亚家族成员可特异性结合CRT元件，既不结合GCC盒，也不识别DRE元件（ACCGAC）。分子建模与定点诱变研究发现，TaERF4类蛋白的APETALA2（Apetala2, AP2）结构域中存在一个保守于单子叶植物的单一位点Pro42，该位点是决定该亚家族DNA识别特异性的关键残基。我们推测，DREB与ERF蛋白均可通过单一关键CRT元件调控Cor410b基因的表达；其中TaERF4亚家族成员是Cor410b基因表达的特异性正向调控因子。