Cis-acting elements in the promoter of PtWRKYs.

WRKY is a crucial transcription factor family in plants, participating in a variety of physiological processes and stress responses. In this study, we identified 102 WRKY genes from the poplar genome, randomly distributed on 18 chromosomes and one scaffold, and classified them into three subgroups based on phylogenetic analysis. Members of the same subgroup form similar structures due to their shared relatively conservative domains. All poplar WRKY proteins are hydrophilic, located in the cell nucleus, and form target relationships with a large number of miRNAs, with their promoter containing a large number of stress defense elements. The expansion of the poplar WRKY family mainly occurs through segmental duplication, and they also have abundant cross-species collinearity. Based on RNA-Seq, we identified 83 WRKYs significantly respond to cadmium (Cd) stress. Subsequently, we conducted a study on WRKY95, which was significantly up-regulated in the roots, stems, and leaves under Cd stress. Under cadmium toxicity, the plant height of PsnWRKY95-overexpressing plants increased by 16%−26% compared with the wild type (WT), the root length increased by 12%−27% compared with WT, the peroxidase (POD) activity was 28%−51% higher than that of WT, the chlorophyll content increased by 15%−29% compared with WT, the malondialdehyde (MDA) content decreased by 13%−32% compared with WT, and the electrical conductivity decreased by 9%−20% compared with WT, with the expression levels of POD and HMA1 in the overexpressing plants also being higher than those in WT. Results from yeast experiments demonstrated that PsnWRKY95 can improve Cd tolerance by specifically binding to cadmium (Cd) resistance element G-box, activating the reactive oxygen clearance ability and downstream target gene. This study comprehensively analyzes the basic data of WRKYs, identifying their response to Cd stress and specifically analyzes the stress-resistant function of PsnWRKY95, providing clues for understanding the molecular mechanism of WRKYs resistance to Cd.

WRKY转录因子家族是植物中一类关键的转录因子家族，参与多种生理过程与胁迫应答反应。本研究从杨树基因组中鉴定得到102个WRKY基因，这些基因随机分布于18条染色体与1个基因组支架上，并通过系统发育分析将其划分为3个亚组。同一亚组的成员因共享相对保守的结构域，其蛋白结构具有较高相似性。所有杨树WRKY蛋白均为亲水性蛋白，定位于细胞核内，可与大量miRNA形成靶标调控关系，且其启动子区域含有大量胁迫防御顺式作用元件。杨树WRKY家族的扩张主要通过片段重复事件实现，同时该家族还存在丰富的种间共线性关系。基于RNA测序（RNA-Seq）数据，本研究鉴定出83个WRKY基因可显著响应镉（Cd）胁迫。随后，本研究针对在镉胁迫下根、茎、叶中均显著上调表达的WRKY95展开深入研究。在镉毒性胁迫条件下，过表达PsnWRKY95的植株与野生型（WT）相比，株高提升16%−26%，根长增加12%−27%，过氧化物酶（POD）活性较野生型高出28%−51%，叶绿素含量提升15%−29%，丙二醛（MDA）含量较野生型降低13%−32%，电导率较野生型下降9%−20%；同时过表达植株中POD与HMA1的表达水平也高于野生型。酵母实验结果表明，PsnWRKY95可通过特异性结合镉抗性顺式作用元件G-box，激活活性氧清除能力与下游靶基因，从而提升植物对镉的耐受性。本研究全面解析了杨树WRKY家族的基础数据，明确了其响应镉胁迫的分子模式，并深入剖析了PsnWRKY95的抗逆功能，为解析WRKY家族抵御镉胁迫的分子机制提供了重要线索。