The phytohormone methyl jasmonate (MeJA) enhances plant cold stress tolerance, but the underlying mechanisms remain elusive. Here, we discovered that MeJA induces a transient Ca2+ influx and elevated cytoplasmic free Ca2+ ([Ca2+]cyt) levels during the watermelon (Citrullus lanatus) response to cold stress. Conversely, silencing jasmonic acid carboxyl methyltransferase (ClJMT), encoding an enzyme that methylates JA to MeJA, led to contrasting effects compared with MeJA application. Upon cold exposure, MeJA rapidly and continuously upregulated 2 Ca2+-permeable channel genes, namely cyclic nucleotide-gated ion channel (ClCNGC) 2 and ClCNGC20. Silencing ClCNGC2 or ClCNGC20 attenuated MeJA-induced Ca2+ influx, [Ca2+]cyt accumulation, C-REPEAT BINDING FACTOR (CBF) pathway activation, and watermelon cold tolerance. Accordingly, ClCNGC2 or ClCNGC20 overexpression increased Ca2+ influx, [Ca2+]cyt levels, and expression of the CBF regulon and improved freezing tolerance in transgenic Arabidopsis thaliana plants. Multiple assays showed that ClCNGC2 and ClCNGC20 do not directly interact. Interestingly, silencing ClCNGC2 or ClCNGC20 abolished MeJA-induced upregulation of ClCNGC20 or ClCNGC2, respectively, in watermelon response to cold, demonstrating their reciprocal activation at the transcriptional level. Collectively, these findings suggest a mutual dependence between ClCNGC2 and ClCNGC20 in mediating MeJA-induced Ca2+ influx followed by [Ca2+]cyt elevation, subsequently activating the CBF pathway and enhancing cold tolerance in plants. This study provides insights into the molecular mechanisms underlying MeJA-mediated plant cold tolerance, holding potential for the breeding or engineering of cold-resistant cucurbit varieties.

Nitric oxide (NO) is a pivotal gaseous signaling molecule that plays a critical role in regulating plant tolerance to cold stress; however, the underlying mechanisms of signal transduction remain poorly elucidated. In this study, knockout of nitrate reductase 1 (ClNR1), a crucial gene for NO biosynthesis, led to reduced cold tolerance in watermelon (Citrullus lanatus), accompanied by downregulation of cycle nucleotide-gated channel (ClCNGC) 20, a key Ca2+-permeable channel gene, decreased Ca2+ influx, and upregulation of calmodulin (ClCaM) 2/5/7. Conversely, application of the NO donor sodium nitroprusside (SNP) exhibited contrasting effects compared with NR1 knockout. Silencing ClCNGC20 counteracted SNP-induced Ca2+ influx, downregulation of ClCaM 2/5/7, and cold tolerance. Silencing ClCaM2/5/7 alleviated the inhibition on C-REPEAT-BINDING FACTOR (ClCBF) expression and cold tolerance induced by ClNR1 knockout or ClCNGC20 silencing. Multiple experimental approaches revealed the interactions between ClCaM2/5/7 and voltage-dependent anion channel (ClVDAC) 1 proteins. Overexpression of ClVDAC1 hindered the induction of ClCBF expression and cold tolerance triggered by SNP or CaCl2, whereas ClVDAC1 silencing mitigated the inhibition on ClCBF expression and cold tolerance caused by ClNR1 knockout or ClCNGC20 silencing. Additionally, cold stress rapidly triggered Ca2+ influx, which stimulated NO production. These findings suggest that Ca2+ influx promotes NO generation, which leads to further Ca2+ influx via upregulating ClCNGC20, forming a positive feedback loop that enhances cold tolerance. Furthermore, ClCaM2/5/7 interacts with ClVDAC1 to negatively regulate the NO- and Ca2+ signaling-mediated C-repeat-binding factor pathway and subsequent cold tolerance.