ClBZR1L regulates proline metabolism to mediate brassinolide-induced cold tolerance in watermelon (Citrullus lanatus)

Watermelon seedlings often suffer from cold damage caused by the spring cold snaps during early spring cultivation. Brassinolide (BR) significantly improves the cold tolerance of watermelons, but the molecular mechanism remains to be elucidated. This study aimed to elucidate how BR regulates cold tolerance in watermelon seedlings, with a focus on the role of the transcription factor ClBZR1-Like (ClBZR1L) in BR signaling pathway. Physiological and molecular analyses revealed that ClBZR1L is responsive to low-temperature induction and positively modulates cold tolerance. Notably, the overexpression of ClBZR1L increased proline accumulation by approximately 60 % in watermelon seedlings under cold stress, while its silencing reduced proline content to ∼67 % of the control under the combined treatment of BR and low temperature. Further mechanistic investigations via yeast one-hybrid, GUS reporter, and dual luciferase assays confirmed that ClBZR1L directly binds to the promoter of ClP5CS1 (a rate-limiting enzyme gene in proline biosynthesis) and activates its transcription, thereby promoting proline accumulation. These results uncover a novel BR-mediated cold tolerance mechanism in watermelons through the ClBZR1L-ClP5CS1 transcriptional module. The findings offer a mechanistic basis of the development of cold-tolerant watermelon cultivars.