BrRAV8 negatively modulates thermotolerance through suppressing cellulose biosynthesis in flowering Chinese cabbage (Brassica campestris L. ssp. chinensis var. utilis Tsen et Lee)

Abstract

High temperature stress severely impairs the growth and development of flowering Chinese cabbage. RAV transcription factors are well-characterized regulators of plant responses to salt and drought stresses, and cellulose plays a fundamental role in stress adaptation. However, the molecular mechanism by which RAVs regulate thermotolerance through mediating cellulose biosynthesis in flowering Chinese cabbage remains to be elucidated. Herein, we systematically investigated 14 RAV transcription factors and 36 cellulose biosynthesis-related genes in flowering Chinese cabbage seedlings (21 days after sowing) under high temperature stress (42 °C). Notably, BrRAV8, BrCESA7, and BrCSLB3.2 exhibited significant induction under heat stress. Transcriptional activation assays demonstrated that BrRAV8 lacked transcriptional activation capacity and likely functioned as a transcriptional repressor. BrRAV8 overexpression significantly downregulated BrCESA7 and BrCSLB3.2 expression, reducing cellulose content and increasing reactive oxygen species (ROS) accumulation, thereby leading to decreased thermotolerance. Conversely, BrRAV8 silencing produced the opposite effects. Additionally, silencing BrCESA7 or BrCSLB3.2 markedly compromised thermotolerance, accompanied by elevated ROS level and diminished cellulose accumulation. Through yeast one-hybrid, dual luciferase, and electrophoretic mobility shift assays, we confirmed that BrRAV8 directly bound to the promoters of BrCESA7 and BrCSLB3.2 to transcriptionally repress their expression. Collectively, our study uncovered a previously unrecognized regulatory module in which BrRAV8 inhibited the cellulose biosynthesis by suppressing the upregulation of BrCESA7 and BrCSLB3.2 to attenuate heat tolerance. This work not only significantly expands our understanding of the molecular regulatory network underlying heat stress response, but also identifies critical genetic resources for developing heat-resistant cultivars, ultimately contributing to improved heat tolerance and productivity in heat-stress cultivation of flowering Chinese cabbage.