Cold Stress-Induced (Z)-3-Hexenol and Thymol Enhance Cold Tolerance of Tea Plants by Activating Ca²⁺ Signalling

Volatile organic compounds (VOCs) released under cold stress have emerged as important mediators of stress tolerance. However, the specific functional VOCs and the mechanisms through which they confer cold tolerance remain largely unknown. In this study, we established a Fluo-8-based calcium detection system in tea (Camellia sinensis) protoplasts to investigate the interplay between cold-induced VOCs, calcium signalling, and cold tolerance. We identified (Z)-3-hexenol and thymol as key VOCs that significantly enhance tea plant cold tolerance by activating cytosolic calcium signalling. These VOCs upregulated cold-responsive genes (CsICE1, CsCBF1, and CsCBF2), enhanced antioxidant enzyme activities (SOD and POD), and improved photosynthetic efficiency under cold stress. Furthermore, we revealed that CsCDPK4, a calcium-dependent protein kinase, acts as a key mediator of (Z)-3-hexenol and thymol-induced calcium signalling. Silencing CsCDPK4 abolished the beneficial effects of (Z)-3-hexenol and thymol, underscoring its critical role in translating calcium signals into physiological adaptations. Our findings provide a mechanistic framework linking VOC perception, calcium signalling, and cold stress tolerance, offering novel strategies for enhancing crop resilience against abiotic stress in the face of climate challenges.