A Rapeseed WRKY Transcription Factor Phosphorylated by CPK Modulates Cell Death and Leaf Senescence by Regulating the Expression of ROS and SA-Synthesis-Related Genes

Abstract

Salicylic acid (SA) and reactive oxygen species (ROS) are two well-defined inducers of leaf senescence. Here, we identified a novel WRKY transcription factor gene <i>WSR1</i> (<i>WRKY regulating SA and ROS 1</i>) in <i>Brassica napus</i> (rapeseed) in promoting SA and ROS production, which eventually led to leaf senescence thereafter. Its expression increased in senescing leaves. Ca²⁺-dependent protein kinase (CPK) 5 and -6 interacted with and phosphorylated BnaWSR1. Overexpression of phosphomimic <i>BnaWSR1</i> (<i>BnaWSR1ca</i>) in rapeseed protoplasts elicited ROS production and cell death while its ectopic expression in <i>Arabidopsis</i> enhanced SA and ROS levels and, hence, accelerated leaf senescence. Furthermore, BnaWSR1ca activated the expression of <i>Isochorismate Synthase 1</i> (<i>ICS1</i>), <i>Respiratory Burst Oxidase Homologue</i> (<i>Rboh</i>) <i>D</i>, and <i>Senescence-Associated Gene 14</i> (<i>SAG14</i>). Electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation-quantitative PCR (ChIP-qPCR) assays demonstrated that BnaWSR1ca directly bound to promoter regions of <i>ICS1</i>, <i>RbohD</i>, and <i>SAG14.</i> These data have identified a CPK-WSR1 module that integrates SA and ROS to control cell death and leaf senescence.