CsCIPK20 Improves Tea Plant Cold Tolerance by Modulating Ascorbic Acid Synthesis Through Attenuation of CsCSN5-CsVTC1 Interaction

Low temperature is a limiting environmental factor for tea plant growth and development. CBL-interacting protein kinases (CIPKs) are important components of the calcium pathway and involved in plant development and stress responses. Herein, we report the function and regulatory mechanisms of a low-temperature-inducible gene, CsCIPK20, in tea plants. The overexpression of CsCIPK20 in Arabidopsis and its transient knockdown in tea plants confirmed its positive role in cold resistance. Notably, the ascorbic acid (AsA) levels increased in the overexpression lines and decreased in the CsCIPK20 knockdown tea plants under freezing stress. Transcriptomic analysis revealed that genes involved in flavonoid metabolism, glutathione metabolism, and AsA biosynthesis were significantly regulated by CsCIPK20. Furthermore, we found that CsCSN5, a key component of the COP9 signalosome, interacted with CsCIPK20 to mediate CsCIPK20 degradation. CsCSN5 interacted with CsVTC1, a key enzyme in AsA biosynthesis, and mediated CsVTC1 degradation. Knockdown of CsVTC1 in tea plants enhanced sensitivity to low temperature. Moreover, we demonstrated that CsCIPK20 competed with CsVTC1 to bind to CsCSN5, which protected CsVTC1 from degradation mediated by CsCSN5 and contributed to AsA accumulation. Overall, our findings uncovered a mechanistic framework through which the CsCIPK20-CsCSN5-CsVTC1 module mediated AsA accumulation and low-temperature resistance in tea plants.