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.

中文翻译：

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H2O2 和 Ca2+ 之间的正相互作用介导褪黑激素诱导的 CBF 途径和西瓜 (Citrullus lanatus L.) 的耐寒性

冷胁迫是对植物生长发育产生不利影响的主要环境因素。褪黑激素已被证明通过激活 C-REPEAT 结合因子 (CBF) 途径赋予植物对冷胁迫的耐受性；然而，启用此功能的底层模式仍然模糊不清。在这项研究中，我们通过药理学、生理学和遗传学方法研究了 H ₂ O ₂和 Ca ²⁺信号在褪黑激素诱导的 CBF 通路和西瓜 ( Citrullus lanatus L.)耐冷性中的作用。结果表明，褪黑激素诱导H ₂ O ₂积累，这与呼吸爆发氧化酶同系物D的上调有关（ClRBOHD）在西瓜对冷应激的早期反应期间。此外，褪黑激素和H ₂ O ₂诱导细胞质游离Ca ²⁺ ([Ca ²⁺ ] _cyt )的积累以响应寒冷。这与西瓜中环核苷酸门控离子通道 2 ( ClCNGC2 )的上调有关。然而，Ca ²⁺流入通道的阻断消除了褪黑激素或H ₂ O ₂诱导的CBF途径和冷耐受性。Ca ²⁺也诱导ClRBOHD表达和 H ₂ O ₂西瓜对冷胁迫的早期反应中的积累。通过 RBOH 抑制剂抑制西瓜中的 H ₂ O ₂产生或通过AtRBOHD敲除抑制拟南芥中的 H ₂ O ₂生成，从而损害了褪黑激素诱导的 [Ca ²⁺ ]_细胞积累和褪黑激素或 Ca ²⁺诱导的 CBF 途径和耐寒性。总的来说，这些发现表明褪黑激素在对冷应激的早期反应中诱导依赖于RBOHD 的H ₂ O ₂生成。增加的 H ₂ O ₂促进 [Ca ²⁺ ]_细胞色素积累，进而诱导 H ₂ O₂通过RBOHD积累，形成一个相互的正调节循环，介导褪黑激素诱导的 CBF 途径和随后的耐寒性。