The ClWRKY53-ClBCM/ClSGR module controls plant senescence via chlorophyll degradation in watermelon

Senescence is a complex biological process coordinately regulated by multiple genes at the molecular level. Deciphering its regulatory mechanisms holds significant potential for enhancing crop yield and stress resistance. However, the study on identification of senescence-related genes in watermelon has been limited by low genetic diversity. In this study, we identified an early-senescence watermelon inbred line, WM103, which displayed a pale green phenotype at the seedling stage that transitions to yellow at maturity. Genetic analysis indicated the early-senescence phenotype was controlled by a single recessive gene. Combined by BSA-seq and linkage analysis in a large F2 population, we identified Cla97C10G186360 as the candidate gene, which encoded a BALANCE OF CHLOROPHYLL METABOLISM (ClBCM) protein. Further functional validation through virus-induced gene silencing (VIGS) and CRISPR/Cas9-mediated knockout confirmed that the down-regulation and loss-of-function of ClBCM can accelerate senescence. RNA-seq analysis revealed that the ClBCM was involved in chlorophyll metabolism pathway and these chlorophyll degradation related genes were significantly up-regulated in WM103. Molecular interaction assays revealed a direct physical interaction between ClBCM and ClSGR. Furthermore, we found WRKY family transcription factors were significantly enriched in differentially expressed genes. In vivo and in vitro experiments showed ClWRKY53 directly bound to the ClBCM promoter and suppressed its transcription, thereby promoting chlorophyll degradation and senescence. These findings provide novel insights into the molecular regulation of senescence in watermelon and establish a theoretical framework for genetic improvement of fruit yield and stress tolerance in cucurbit crops.