Positive Interaction between H(2)O(2) and Ca(2+) Mediates Melatonin-Induced CBF Pathway and Cold Tolerance in Watermelon (Citrullus lanatus L.)

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 H(2)O(2) and Ca(2+) signaling in the melatonin-induced CBF pathway and cold tolerance in watermelon (Citrullus lanatus L.) through pharmacological, physiological, and genetic approaches. According to the results, melatonin induced H(2)O(2) accumulation, which was associated with the upregulation of respiratory burst oxidase homolog D (ClRBOHD) during the early response to cold stress in watermelon. Besides, melatonin and H(2)O(2) induced the accumulation of cytoplasmic free Ca(2+) ([Ca(2+)](cyt)) in response to cold. This was associated with the upregulation of cyclic nucleotide-gated ion channel 2 (ClCNGC2) in watermelon. However, blocking of Ca(2+) influx channels abolished melatonin- or H(2)O(2)-induced CBF pathway and cold tolerance. Ca(2+) also induced ClRBOHD expression and H(2)O(2) accumulation in early response to cold stress in watermelon. Inhibition of H(2)O(2) production in watermelon by RBOH inhibitor or in Arabidopsis by AtRBOHD knockout compromised melatonin-induced [Ca(2+)](cyt) accumulation and melatonin- or Ca(2+)-induced CBF pathway and cold tolerance. Overall, these findings indicate that melatonin induces RBOHD-dependent H(2)O(2) generation in early response to cold stress. Increased H(2)O(2) promotes [Ca(2+)](cyt) accumulation, which in turn induces H(2)O(2) accumulation via RBOHD, forming a reciprocal positive-regulatory loop that mediates melatonin-induced CBF pathway and subsequent cold tolerance.