Natural variation in ClTFL1 contributes to reduced branching in watermelon by attenuating the ClTFL1-ClGRF5 interaction

Optimal branching architecture in watermelon improves yields and reduces labor costs. The fewer-branching (fb) mutant is a natural watermelon mutant exhibiting reduced branching, with ClTFL1 proposed as a candidate gene for this trait. However, direct evidence and the underlying regulatory mechanism remain unclear. Here, we show that the fb mutant phenotype co-segregates with a nonsynonymous SNP in the coding region of ClTFL1. Knockout of ClTFL1 in a normal-branching watermelon background resulted in reduced branching, loss of tendrils, and terminal flower formation. In contrast, overexpression of ClTFL1 enhanced branching and delayed flowering. Moreover, overexpression of ClTFL1 in the fb mutant rescued the reduced branching phenotype, restoring it to a normal branching architecture. Biochemical analyses revealed that ClTFL1 interacts with ClGRFs predominantly in the cytoplasm and associates with ClFD in the nucleus. ClTFL1 WT associates with ClFD through ClGRF5, which acts as a molecular bridge to support the formation of a ternary regulatory complex. This complex represses the auxin early-response gene ClGH3 and the floral meristem identity gene ClAP1 and is associated with enhanced branching and delayed flowering. By contrast, the natural A165E substitution in ClTFL1 weakened its interaction with ClGRF5 and was associated with reduced repression of ClAP1 and ClGH3. Additionally, ClTFL1 antagonized ClFT-mediated activation of the ClAP1 and ClGH3 promoters. Collectively, our findings provide a key genetic target and novel insights into the optimization of watermelon plant architecture.