Abstract

Cold stress is a major environmental factor that detrimentally affects plant growth and development. Melatonin has been shown to confer plant tolerance to cold stress through activating the C-REPEAT BINDING FACTOR (CBF) pathway; however, the underlying modes that enable this function remain obscure. In this study, we investigated the role of H2O2and Ca2+signaling in the melatonin-induced CBF pathway and cold tolerance in watermelon (Citrullus lanatusL.) through pharmacological, physiological, and genetic approaches. According to the results, melatonin induced H2O2accumulation, which was associated with the upregulation ofrespiratory burst oxidase homolog D(ClRBOHD) during the early response to cold stress in watermelon. Besides, melatonin and H2O2induced the accumulation of cytoplasmic free Ca2+([Ca2+]cyt) in response to cold. This was associated with the upregulation ofcyclic nucleotide-gated ion channel 2(ClCNGC2) in watermelon. However, blocking of Ca2+influx channels abolished melatonin- or H2O2-induced CBF pathway and cold tolerance. Ca2+also inducedClRBOHDexpression and H2O2accumulation in early response to cold stress in watermelon. Inhibition of H2O2production in watermelon by RBOH inhibitor or in Arabidopsis byAtRBOHDknockout compromised melatonin-induced [Ca2+]cytaccumulation and melatonin- or Ca2+-induced CBF pathway and cold tolerance. Overall, these findings indicate that melatonin inducesRBOHD-dependent H2O2generation in early response to cold stress. Increased H2O2promotes [Ca2+]cytaccumulation, which in turn induces H2O2accumulation viaRBOHD, forming a reciprocal positive-regulatory loop that mediates melatonin-induced CBF pathway and subsequent cold tolerance.Keywords:melatonin;hydrogen peroxide;calcium signal;CBF-responsive pathway;respiratory burst oxidase homolog D;cold stress