Positive Interaction between H2O2 and Ca2+ Mediates Melatonin-Induced CBF Pathway and Cold Tolerance in Watermelon (Citrullus lanatus L.)

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 H₂O₂ and Ca²⁺ signaling in the melatonin-induced CBF pathway and cold tolerance in watermelon (<i>Citrullus lanatus</i> L.) through pharmacological, physiological, and genetic approaches. According to the results, melatonin induced H₂O₂ accumulation, which was associated with the upregulation of <i>respiratory burst oxidase homolog D</i> (<i>ClRBOHD</i>) 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 <i>cyclic nucleotide-gated ion channel 2</i> (<i>ClCNGC2</i>) in watermelon. However, blocking of Ca²⁺ influx channels abolished melatonin- or H₂O₂-induced CBF pathway and cold tolerance. Ca²⁺ also induced <i>ClRBOHD</i> 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 <i>AtRBOHD</i> knockout compromised melatonin-induced [Ca²⁺]_cyt accumulation and melatonin- or Ca²⁺-induced CBF pathway and cold tolerance. Overall, these findings indicate that melatonin induces <i>RBOHD</i>-dependent H₂O₂ generation in early response to cold stress. Increased H₂O₂ promotes [Ca²⁺]_cyt accumulation, which in turn induces H₂O₂ accumulation via <i>RBOHD</i>, forming a reciprocal positive-regulatory loop that mediates melatonin-induced CBF pathway and subsequent cold tolerance.