Characterization of Promoter Activation of ZmCOLD1 and Interaction between ZmCOLD1-10A and ZmCT2 in Maize under Cold Stress

As an important thermophilous crop, maize is sensitive to low temperature stress. A GPCR-like G protein, ZmCOLD1, has been confirmed that is related to cold tolerance in maize. However, the regulatory mechanism of ZmCOLD1 under cold stress is unclear. In this study, we employed transgenic Arabidopsis plants, maize protoplasts, and Nicotiana benthamiana systems to characterize the structure and activation of ZmCOLD1-10A promoter and its protein interaction. Analysis revealed that the promoter region contains multiple cis-acting elements, including cold-responsive elements (DRE core, MYB, and MYB-like sequences), ABA-responsive elements (ABRE, ABRE3a/ABRE4, and motif IIb), and other special elements. Promoter activity assays demonstrated that the ZmCOLD1-10A promoter is markedly induced under cold stress, dark treatment, and ABA treatment. Functional studies in transgenic Arabidopsis showed that overexpression of ZmCOLD1-10A significantly enhanced cold tolerance, with substantial reduction in electrolyte leakage compared to wild-type plants. Using luciferase reporter assays, we identified that the core interaction site between ZmCOLD1 and ZmCT2 involves the HL domain of ZmCOLD1-10A and the C-terminal of ZmCT2. These findings establish a foundation for further investigation into G proteins and GTG protein-mediated cold stress signaling in maize.