The KNOX transcription factor ClSP activates ClAPRR2 to regulate dark green stripe formation in watermelon

As a prominent external feature of watermelon, the stripe pattern exhibits remarkable phenotypic diversity, directly impacting commercial value through consumer preference. However, the genetic and molecular mechanisms underlying this important agronomic trait in watermelon remain poorly understood. In this study, we discovered that the total chlorophyll content in dark green stripes (DGS) was significantly higher than that in light green stripes (LGS) or reticular green stripes (RGS). Moreover, the number and size of chloroplasts were significantly increased in the DGS. Genetic analysis identified the KNOX TF ClSP as the most likely candidate for regulating watermelon dark green stripe formation, whose functional disruption substantially impaired chlorophyll biosynthesis and chloroplast development, converting dark green stripes into reticulate stripes. Through transcriptome analysis, we identified approximately 94 differently expressed genes (DEGs) that contain the KNOX TF binding cis-element 'TGAC' in their promoters. Among these genes, the expression pattern of ARABIDOPSIS PSEUDO RESPONSE REGULATOR 2-LIKE (APRR2-like) TF ClAPRR2 closely mirrored that of ClSP, displaying significantly down-regulated transcriptional expression in LGS compared to DGS. Utilizing Y1H, GUS activity, DLR and EMSA assays, we confirmed that ClSP activates the transcriptional activity of ClAPRR2 through promoter binding. Collectively, we propose a potential working model for the ClSP-ClAPRR2 module, which regulates the chlorophyll synthesis and chloroplast development in watermelon fruits, providing new insights into the mechanisms underlying stripe pattern formation.