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.

Non-invasive micro-test technology (NMT) reveals dynamic ionic/molecular concentration gradients by measuring fluxes of ions and small molecules in liquid media in 1D, 2D or 3D fashions with sensitivity up to pico-(10-12) or femto-(10-15) moles per cm 2 per second. NMT has been applied to study metabolism, signal transduction, genes and/or proteins physiological functions related to transmembrane ionic/molecular activities with live samples under normal conditions or stress. Data on ion and/or molecule homeostasis (IMH) by NMT in biomedical sciences, plant and crop sciences, environmental sciences, marine and space biology as well as traditional Chinese medicine are reviewed.

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.

Plants employ sophisticated volatile-mediated signaling mechanisms to defend against herbivore attacks; however, the molecular pathways underlying these processes remain poorly understood. Herein, we unveil the molecular mechanism by which the herbivore-induced volatile (E)-4,8-dimethyl-1,3,7-nonatriene (DMNT) activates jasmonic acid (JA) biosynthesis in tea plants (Camellia sinensis). We demonstrate that DMNT triggers early signaling events in tea plants, characterized by a rapid influx of Ca2+ in mesophyll cells, which subsequently initiates a signaling cascade involving the CsCAMTA3 (calmodulin-binding transcription activator, CAMTA). Using luciferase reporter assays, yeast 1-hybrid analysis, electrophoretic mobility shift assays, and antisense oligonucleotide silencing, we demonstrated that CsCAMTA3 directly binds to the CG-box element in the promoter region of the key transcription factor CsWRKY70 and activates its expression. Subsequently, CsWRKY70 binds to the W-box motif in the CsLOX3 (lipoxygenase, LOX) promoter, driving the expression of the JA biosynthetic gene and promoting the accumulation of JA and jasmonoyl-isoleucine (JA-Ile). Silencing CsCAMTA3 or CsWRKY70 significantly inhibits JA biosynthesis and reduces tea plant resistance to tea geometrid (Ectropis obliqua). Furthermore, the suppression of both CsCAMTA3 and CsWRKY70 significantly diminished the ability of tea plants to synthesize JA and JA-Ile in response to DMNT signal induction. Our findings reveal a Ca2+-CAMTA3-WRKY70-LOX3 signaling cascade induced by DMNT, broadening the theoretical framework of plant–plant communication.