Melatonin plays a crucial role in regulating plant cold tolerance, but the mechanisms underlying signal transduction remain elusive. In this study, we discovered that overexpression of the melatonin biosynthetic gene caffeic acid O-methyltransferase1 (COMT1) enhanced watermelon (Citrullus lanatus) cold tolerance, accompanied by the accumulation of cytosolic free calcium ([Ca2+]cyt), a stimulation of Ca2+ influx, and upregulation of four Ca2+-permeable channel genes (CNGC2/10/17/20). Conversely, knockout of COMT1 exhibited contrasting effects compared to its overexpression. Knocking out the four CNGC genes revealed that only CNGC20 mediates melatonin-induced Ca2+ influx in response to cold stimuli. CNGC20 deletion impeded watermelon callus redifferentiation, prompting us to employ a virus-induced gene silencing strategy to suppress its expression. Silencing CNGC20 compromised COMT1 overexpression-induced [Ca2+]cyt accumulation, Ca2+ influx, and watermelon cold tolerance. Yeast two-hybrid, bimolecular fluorescence complementation, firefly luciferase complementation imaging, and pull-down assays revealed an interaction between CNGC20 and calmodulin7 (CaM7). Overexpressing CaM7 inhibited melatonin-induced [Ca2+]cyt accumulation, Ca2+ influx, and watermelon cold tolerance. Conversely, silencing CaM7 increased [Ca2+]cyt accumulation, Ca2+ influx, and cold tolerance, whereas COMT1 overexpression failed to further enhance these responses in CaM7-silenced plants, indicating the negative regulation role of CaM7 in melatonin-mediated cold responses. Overall, these findings provide insights into the molecular mechanisms underlying melatonin-enhanced plant cold tolerance via Ca2+ signaling, holding potential for breeding/engineering cold-tolerant cucurbit varieties.

中文翻译：

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环核苷酸门控离子通道 20 调控西瓜褪黑激素诱导的钙信号传导和耐寒性


褪黑激素在调节植物耐寒性中起着至关重要的作用，但信号转导的潜在机制仍然难以捉摸。在这项研究中，我们发现褪黑激素生物合成基因咖啡酸 O-甲基转移酶 1 （COMT1） 的过表达增强了西瓜 （Citrullus lanatus） 的耐寒性，伴随着胞质游离钙 （[Ca2+]cyt） 的积累，刺激 Ca2+ 内流，以及上调四个 Ca2+ 通透性通道基因 （CNGC2/10/17/20）。相反，与过表达相比，COMT1 的敲除表现出相反的效果。敲除 4 个 CNGC 基因表明，只有 CNGC20 介导褪黑激素诱导的 Ca2+ 内流以响应寒冷刺激。CNGC20 缺失阻碍了西瓜愈伤组织的再分化，促使我们采用病毒诱导的基因沉默策略来抑制其表达。沉默 CNGC20 损害了 COMT1 过表达诱导的 [Ca2+] cyt 积累、Ca2+ 内流和西瓜耐寒性。酵母双杂交、双分子荧光互补、萤火虫荧光素酶互补成像和沉降测定揭示了 CNGC20 和钙调蛋白 7 （CaM7） 之间的相互作用。过表达 CaM7 抑制褪黑激素诱导的 [Ca2+] cyt 积累、Ca2+ 内流和西瓜耐寒性。相反，沉默 CaM7 增加了 [Ca2+] cyt 积累、Ca2+ 内流和耐寒性，而 COMT1 过表达未能进一步增强 CaM7 沉默植物中的这些反应，表明 CaM7 在褪黑激素介导的冷反应中具有负调节作用。总体而言，这些发现为通过 Ca2+ 信号传导增强褪黑激素增强植物耐寒性的分子机制提供了见解，为培育/工程耐寒葫芦品种具有潜力。