The Transcription Factor VvbHLH053 Regulates the Expression of Copper Homeostasis-Associated Genes VvCTr5/6 and VvFRO4 and Confers Root Development in Grapevine

Chlormequat chloride (CCC) has been demonstrated to inhibit plant growth and strengthen seedlings. The present study demonstrated that the root growth of <i>Thompson seedless</i> grapevine seedlings was significantly enhanced by the application of CCC treatment. Nevertheless, the precise mechanism by which CCC regulates plant root growth remains to be elucidated. Consequently, an RNA-sequencing (RNA-Seq) analysis was conducted on grapevine roots subjected to CCC treatment and those undergoing natural growth. A total of 819 differentially expressed genes were identified. Subsequently, Gene Ontology (GO) functional enrichment and weighted gene co-expression network analysis (WGCNA) identified the Copper (Cu) homeostasis-associated genes, <i>VvCTr4/5/6/8</i> and <i>VvFRO4</i>, which play a pivotal role in mediating the effect of CCC. To further elucidate the transcription factor regulating these Cu homeostasis-associated genes, the key transcription factor VvbHLH053 was identified based on the PlantTFDB database, WGCNA results, and expression patterns under CCC treatment. Furthermore, multiple bHLH binding sites were identified on the promoters of <i>VvCTr4/5/6</i> and <i>VvFRO4</i>. The GUS activity analysis and dual-luciferase assay demonstrated that VvbHLH053 can directly regulate the expression of <i>VvCTr5/6</i> and <i>VvFRO4</i>. These findings reveal the feedback mechanism of grapevine root growth mediated by CCC and establish a direct functional relationship between CCC, VvbHLH053, and Cu homeostasis-associated genes that regulate root growth.