Abstract

Cold stress significantly limits the growth and productivity of plants. WRKY transcription factors play crucial roles in response to a variety of abiotic stimuli by modulating abscisic acid (ABA) signaling. Nevertheless, their specific role in regulating cold tolerance in Chrysanthemum indicum var. aromaticum ( Ci. aromaticum ) remains to be further explored. In this study, we demonstrated that CiWRKY33 , a member of the WRKY-I group, positively regulates cold tolerance in Ci. aromaticum . Expression of CiWRKY33 was induced by ABA treatment and cold stress, and the protein was primarily localized in the nucleus. By facilitating the accumulation of ABA and antioxidant capability under cold stress, CiWRKY33 overexpression in tobacco dramatically improved cold tolerance. Meanwhile, transient silencing of expression in Ci. aromaticum reduced cold tolerance. CiWRKY33 directly binds to the promoter of CiNCED3 and activates its transcription, and this activation was strengthened through interaction with CiWRKY12. These findings reveal that the CiWRKY33-CiWRKY12- CiNCED3 module enhances cold tolerance by promoting ABA biosynthesis. This mechanism also provides a crucial molecular basis for genetically engineering plants to improve cold resistance.