A Tripartite Amplification Loop Involving WRKY75, Salicylic Acid and ROS Accelerates Leaf Senescence in Arabidopsis

Leaf senescence is a highly coordinated and complicated process with the integration of numerous internal and environmental signals. Salicylic acid (SA) and reactive oxygen species (ROS) are two well-defined inducers of leaf senescence, whose contents progressively and inter-dependently increase during leaf senescence via a yet unknown mechanism. Here, we have characterized a newly identified positive regulator of leaf senescence, WRKY75, and demonstrated that knock-down or knock-out of WRKY75 delays, while over-expression of WRKY75 accelerates age-dependent leaf senescence. The WRKY75 transcription is induced by age, SA, H2O2, as well as multiple plant hormones. Meanwhile, WRKY75 is able to promote SA production by inducing the transcription of SA INDUCTION-DEFICIENT 2 (SID2), and suppress H2O2 scavenging partly by repressing the transcription of CATALASE 2 (CAT2). Genetic analysis reveals that the SID2 mutation or an increase of catalase activity rescues the precocious leaf senescence phenotype evoked by WRKY75 over-expression. Based on these results, we propose a “tripartite amplification loop” model in which WRKY75, SA and ROS undergo a gradual but self-sustained rise driven by three interlinked positive feedback regulations. This tripartite amplification loop provides a molecular framework connecting the upstream signals, such as age, ethylene, JA and ABA, to the downstream regulatory network executed by those SA-responsive and H2O2-responsive transcription factors during leaf senescence. Overall design: We performed transcriptome profiles analysis of 28-day-old Col-0 and WRKY75RNAi leaves by RNA-seq. The third and fourth leaves of Col-0 and WRKY75RNAi plants were collected and ground into powder in liquid nitrogen. Total RNA was extracted using the RNeasy Plant Mini kit (QIAGEN). RNA-sequencing and differential gene expression analysis was performed by the BIOPIC institution at Peking University.叶片衰老是一个高度协调且复杂的生物学过程，整合了众多内源信号与环境信号。水杨酸（Salicylic acid, SA）与活性氧（Reactive oxygen species, ROS）是两类已被充分证实的叶片衰老诱导因子，二者的含量在叶片衰老进程中逐步升高且相互依赖，但其具体调控机制至今尚未明确。本研究鉴定得到一个全新的叶片衰老正向调控因子WRKY75，并证实：敲低或敲除WRKY75可延缓叶片衰老，而过表达WRKY75则会加速年龄依赖性叶片衰老。WRKY75的转录可被年龄、SA、过氧化氢（Hydrogen peroxide, H₂O₂）以及多种植物激素诱导。同时，WRKY75可通过诱导水杨酸诱导缺陷2（SA INDUCTION-DEFICIENT 2, SID2）的转录促进SA的合成，并通过抑制过氧化氢酶2（CATALASE 2, CAT2）的转录部分抑制H₂O₂的清除。遗传分析显示，SID2突变或过氧化氢酶活性升高可挽救WRKY75过表达所诱导的早衰表型。基于上述结果，本研究提出了"三方放大环路"模型：WRKY75、SA与ROS通过三项相互关联的正向反馈调控，实现逐步且自我维持的含量升高。该三方放大环路构建了一套分子框架，可将上游信号（如年龄、乙烯、茉莉酸（JA）与脱落酸（ABA））与叶片衰老过程中由SA响应型及H₂O₂响应型转录因子所执行的下游调控网络联系起来。 实验设计：本研究通过RNA测序（RNA-seq）对28日龄的哥伦比亚野生型拟南芥（Col-0）与WRKY75 RNA干扰株系（WRKY75RNAi）的叶片开展转录组分析。采集Col-0与WRKY75RNAi植株的第三、第四片叶片，在液氮中研磨成粉末，使用RNeasy植物微量提取试剂盒（QIAGEN）提取总RNA。RNA测序与差异基因表达分析由北京大学BIOPIC研究机构完成。