The GhWRKY46‐GhGAI Module Mediates Cotton Flowering by Regulating the Expression of Flowering Promotion Factors

Flowering represents a pivotal developmental transition stage in the life cycle of a plant, and the occurrence of flowering at the optimal time is critical for reproductive success. WRKY transcription factors play a vital role in a signaling network that governs a multitude of plant processes. Here, a gene, GhWRKY46, that was differentially expressed in early and late maturing materials was identified via association analysis, and it was specifically expressed in flower buds. Under natural light and temperature conditions, compared to Jin668, the flowering time of OE-GhWRKY46 plants was advanced by approximately 6 days, while the flowering time of CR-GhWRKY46 plants was delayed by approximately 8 days. Transcriptomic data indicated that overexpression or knockout of GhWRKY46 resulted in the activation or repression, respectively, of the photoperiod gene CO-Like and genes related to bud differentiation. Combined RNA-seq and DAP-seq analysis revealed that three genes, namely, GhCOL4, GhCOL2 and GhFPF1-like, may be expressed downstream of GhWRKY46. Dual-luciferase assays and electrophoretic mobility shift assays (EMSAs) demonstrated that GhWRKY46 could directly bind to the W-box and promote the expression of these genes. Similarly, GhFT was also found to be activated by GhWRKY46. Both in vivo and in vitro biochemical analyses demonstrated that GhWRKY46 interacted with GhGAI, and GhGAI could interfere with the transcriptional activation of GhWRKY46, which in turn inhibited the expression of GhCOL4, GhCOL2, GhFPF1-like, and GhFT. In conclusion, this study accurately predicted the GhWRKY46 binding motif, which is important for the construction of regulatory networks of the WRKY family in other crops and introduces a novel regulatory module for the flowering regulation pathway in cotton.