A natural variant of NON-RIPENING promotes fruit ripening in watermelon

The regulation of non-climacteric fruit ripening by the transcription factor NON-RIPENING (NOR) is poorly understood. Here, we identified that the NOR homolog in the non-climacteric fruit watermelon (Citrullus lanatus) was located within the selective sweep and sweetness quantitative trait locus that was selected during domestication from landraces to cultivars. ClNOR knockout substantially delayed fruit ripening, and the fruits of the knockout plants had lower abscisic acid (ABA) levels, lighter colored flesh, and were less sweet compared to wild type. Transcriptome analysis and DNA affinity purification sequencing revealed that ClNOR targeted the Basic Leucine Zipper gene ClbZIP1, which links ClNOR to genes that do not have a ClNOR-binding motif in their promoters, such as the ABA biosynthesis gene, 9-cis-epoxycarotenoid dioxygenase ClNCED1 and the chromoplast phosphate transporter gene ClPHT4;2. The double mutant Clnor Clbzip1 exhibited delayed fruit ripening, lower ABA level, and lighter colored flesh. Its delayed ripening phenotype was stronger than that of the Clbzip1 single mutant. Additionally, the ClNORT,T haplotype in cultivated watermelon resulted in higher ClbZIP1 expression, but ClNORC,T from landraces and ClNORC,G from ancestral watermelon did not. Heterologous ClNORT,T expression rescued the delayed ripening phenotype of the Slnor knockout in tomato (Solanum lycopersicum). This natural variant (564T/C) of ClNOR promoted fruit ripening by enhancing target genes transcription. Overall, these findings will help elucidate the evolutionary mechanisms of nonclimacteric fruit ripening.