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 H₂O₂ and Ca²⁺ 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 H₂O₂ 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 H₂O₂ induced the accumulation of cytoplasmic free Ca²⁺ ([Ca²⁺]_cyt) in response to cold. This was associated with the upregulation of cyclic 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 induced ClRBOHD expression and H₂O₂ accumulation in early response to cold stress in watermelon. Inhibition of H₂O₂ production in watermelon by RBOH inhibitor or in Arabidopsis by AtRBOHD knockout compromised melatonin-induced [Ca²⁺]_cyt accumulation and melatonin- or Ca²⁺-induced CBF pathway and cold tolerance. Overall, these findings indicate that melatonin induces RBOHD-dependent H₂O₂ generation in early response to cold stress. Increased H₂O₂ promotes [Ca²⁺]_cyt accumulation, which in turn induces H₂O₂ accumulation via RBOHD, forming a reciprocal positive-regulatory loop that mediates melatonin-induced CBF pathway and subsequent cold tolerance. [ABSTRACT FROM AUTHOR]