The Transcription Factors <scp>NFYA1</scp> and <scp>GBF3</scp> Jointly Regulate <i>CHS2</i> to Promote Tangeretin Accumulation and Cold Tolerance in <i>Citrus</i>

Tangeretin has been known as a polymethoxylated flavone conferring both phytoprotection and nutraceutical value, but cryoprotective roles and molecular regulation of tangeretin under abiotic stresses remain largely unexplored. In this study, we demonstrated that cold treatment led to greater accumulation of tangeretin and upregulation of Chalcone Synthase 2 (CiCHS2) in Ichang papeda (Citrus ichangensis), a cold-hardy citrus species, relative to a cold-sensitive genotype. CiCHS2, localised in the endoplasmic reticulum, was shown to function in cold tolerance by modulating tangeretin synthesis. We revealed that the transcription factors CiNFYA1 and CiGBF3 act as transcriptional activators of CiCHS2 by interacting with the CCAAT and G-box (CACGTG) elements, respectively, in the gene promoter. Furthermore, CiNFYA1 could regulate CiGBF3 by binding to the gene promoter. In addition, CiNFYA1 physically interacted with CiGBF3, and the resulting protein complex further promoted transactivation of CiCHS2. Moreover, CiNFYA1 and CiGBF3 were demonstrated to play a positive role in the modulation of cold tolerance by regulating CHS2-mediated tangeretin accumulation. Taken together, our findings unravel a hierarchical regulatory network wherein CiNFYA1-CiGBF3 cooperatively activated CiCHS2-dependent tangeretin biosynthesis in response to cold stress. These results advance our understanding of the molecular regulation of tangeretin accumulation under cold stress and provide valuable targets for engineering cold-tolerant crops through metabolic engineering.