Investigating the Regulatory Targets of the WRKY75 Transcription Factor during Age-Associated Leaf Senescence in Arabidopsis thaliana

Abstract

Leaf senescence is the final stage of leaf development when leaf tissues degrade as a strategy to reallocate nutrients from older leaves to newly developing structures (i.e. new roots, leaves, and inflorescences). This degradative process is largely controlled by differential gene expression. WRKY75 expression is highly expressed at the start of senescence and continuously increases throughout the entire process. Here we investigate the role of WRKY75 as a positive regulator of leaf senescence by attempting to identify its direct targets in developmentally senescing leaves. Here we nominated nine candidate WRKY75 target genes during senescence based on RNA-seq analysis. These genes showed significantly lower expression in the wrky75-2 allele compared to WT leaf six harvested from 38-day-old plants. Two biological replicates of real-time qPCR for the nine genes of interest revealed no significant difference in expression in wrky75-2 allele compared to WT. Furthermore, a promoter analysis of the nine WRKY75 target genes identified several putative W-boxes, however a chromatin immunoprecipitation using a WRKY75-HA tagged line, followed by quantitative PCR (ChIP-qPCR) demonstrated amplification of our negative controls negating the validity of WRKY75 binding to the interrogated W-boxes. Our study suggests the wkry75-2 allele is too weak to identify significant differentially expressed genes and advocate for the use of improved CRISPR-Cas9 mutants that have been recently published. Thus, further studies using one of the more robust wrky75-Cas9 mutant lines is necessary for proper identification of direct targets during leaf senescence.