A self-amplifying NO-H₂S loop mediates melatonin-induced CBF-responsive pathway and cold tolerance in watermelon

Melatonin is a pivotal bioactive molecule that enhances plant cold stress tolerance, but the precise mechanisms remain enigmatic. Here, we have discovered that overexpressing melatonin biosynthetic gene ClCOMT1 or applying exogenous melatonin activates the C-repeat binding factor (CBF)-responsive pathway and enhances watermelon cold tolerance. This enhancement is accompanied by elevated levels of nitric oxide (NO) and hydrogen sulfide (H₂S), along with upregulation of nitrate reductase 1 (ClNR1) and L-cysteine desulfhydrase (ClLCD) genes involved in NO and H₂S generation respectively. Conversely, knockout of ClCOMT1 exhibits contrasting effects compared to its overexpression. Furthermore, application of sodium nitroprusside (SNP, a NO donor) and NaHS (a H₂S donor) promotes the accumulation of H₂S and NO, respectively, activating the CBF pathway and enhancing cold tolerance. However, knockout of ClNR1 or ClLCD abolished melatonin-induced H₂S or NO production respectively and abrogated melatonin-induced CBF pathway and cold tolerance. Conversely, supplementation with SNP and NaHS restored the diminished cold response caused by ClCOMT1 deletion. Additionally, deletion of either ClNR1 or ClLCD eliminated NaHS- or SNP-induced cold response, respectively. Overall, these findings suggest a reciprocal positive-regulatory loop between ClNR1-mediated NO and ClLCD-mediated H₂S, which plays a crucial role in mediating the melatonin-induced enhancement of cold tolerance.