The Cotton GhWRKY91 Transcription Factor Mediates Leaf Senescence and Responses to Drought Stress in Transgenic Arabidopsis thaliana

Lijiao Gu,Qiang Ma,Shuxun Yu,Hantao Wang,Congcong Wang,Chi Zhang,Hengling Wei

doi:10.3389/fpls.2019.01352

Abstract

WRKY transcription factors (TFs) play essential roles in the plant response to leaf senescence and abiotic stress. However, the WRKY TFs involved in leaf senescence and stress tolerance in cotton (Gossypium hirsutum L.) are still largely unknown. In this study, a WRKY gene, GhWRKY91, was isolated and thoroughly characterized. Transcriptional activity assays showed that GhWRKY91 could activate transcription in yeast. The expression pattern of GhWRKY91 during leaf senescence, and in response to abscisic acid (ABA) and drought stress was evaluated. β-Glucuronidase (GUS) activity driven by the GhWRKY91 promoter in transgenic Arabidopsis was reduced upon exposure to ABA and drought treatments. Constitutive expression of GhWRKY91 in Arabidopsis delayed natural leaf senescence. GhWRKY91 transgenic plants exhibited increased drought tolerance and presented delayed drought-induced leaf senescence, as accompanied by reinforced expression of stress-related genes and attenuated expression of senescence-associated genes (SAGs). Yeast one-hybrid (Y1H) assays and electrophoretic mobility shift assays (EMSAs) revealed that GhWRKY91 directly targets GhWRKY17, a gene associated with ABA signals and reactive oxygen species (ROS) production. A transient dual-luciferase reporter assay demonstrated that GhWRKY91 activated the expression of GhWRKY17. Our results suggest that GhWRKY91 might negatively regulate natural and stress-induced leaf senescence and provide a foundation for further functional studies on leaf senescence and the stress response in cotton.

Highlights

The WRKY gene family is a large family of plant-specific transcription factors (TFs) that play important roles in various processes, such as stem elongation (Zhang et al, 2011), pathogen resistance (Pan and Jiang, 2014), leaf senescence (Xie et al, 2014), panicle development (Xiang et al, 2017), trichome and seed coat development (Johnson et al, 2002), pollen development (Guan et al, 2014), fruit ripening (Jiang et al, 2017), and biotic and abiotic stress responses
A multiple sequence alignment revealed that the GhWRKY91 protein includes one WRKY domain that comprises approximately 60 amino acids with a highly conserved amino acid sequence (WRKYGQK) and a C2H2 putative zinc finger motif, indicating that GhWRKY91 belongs to the group II family (Figure 1B)
Growing evidence has shown that WRKY TFs are widely involved in plant development, leaf senescence and various abiotic/biotic stress responses (Lim et al, 2007; Yu et al, 2012; Xiao et al, 2013; Pan and Jiang, 2014)

Summary

Introduction

The WRKY gene family is a large family of plant-specific transcription factors (TFs) that play important roles in various processes, such as stem elongation (Zhang et al, 2011), pathogen resistance (Pan and Jiang, 2014), leaf senescence (Xie et al, 2014), panicle development (Xiang et al, 2017), trichome and seed coat development (Johnson et al, 2002), pollen development (Guan et al, 2014), fruit ripening (Jiang et al, 2017), and biotic and abiotic stress responses (Pan and Jiang, Abbreviations: ABA, abscisic acid; EMSA, electrophoretic mobility shift assay; GUS, β-glucuronidase; qRT-PCR, quantitative real-time PCR; ROS, reactive oxygen species; SAGs, senescence-associated genes; TFs, transcription factors; and WT, wild-type.The Function of GhWRKY91 Gene2014). Genes expression significantly increases during leaf senescence include those that encode TFs such NACs (NAM, ATAF1/2 and CUC2) and WRKYs (Balazadeh et al, 2008; Guo et al, 2010; Lin et al, 2015; Wu et al, 2016). AtWRKY22 has been shown to promote and delay leaf senescence in overexpression plants and in T-DNA insertion mutants under dark conditions, respectively (Zhou et al, 2011). Atwrky mutants exhibit a jasmonic acid (JA)-induced early leaf senescence phenotype in which auxin functions as an antagonist (Jiang et al, 2014). OsWRKY23 has been shown to be involved in dark-induced leaf senescence in transgenic Arabidopsis plants (Jing et al, 2009). Two cotton WRKY TFs, GhWRKY27 (Gu et al, 2019) and GhWRKY42 (Gu et al, 2018b), were recently identified to promote leaf senescence in transgenic Arabidopsis plants, suggesting that WRKY TFs have important roles during leaf senescence in cotton

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