Calmodulin1–Calmodulin Binding Transcription Activator (CAM1–CAMTA) negatively regulate the transcription of <i>Fluoride Export Gene 1</i> (<i>FEX1</i>) to mediate fluoride transport in tea (<i>Camellia sinensis</i>)

Fluoride (F) is vital for the growth of tea plants (Camellia sinensis); however, high levels in the leaves can pose a potential threat to human health. Previous studies have shown that F-export genes (FEXs) regulate efflux from the cells in tea plants, but the specific regulatory network remains unclear. In this study, the Calmodulin-Binding Transcription Activator (CAMTA) protein was functionally characterized based on yeast one-hybrid screening assays and previous transcriptomics studies, which showed that it is mainly localized in the nucleus and binds to the G-box (CACGTC) cis-element in the promoter of Fluoride Export Gene 1 (FEX1) as a transcription inhibitor. In addition, yeast-two-hybrid and bimolecular fluorescence complementation assays showed that Calmodulin 1 (CAM1) could interact with CAMTA to form a protein complex that suppressed FEX1 expression. Furthermore, subcellular examination indicated that CAM1 was localized in the nucleus and cytoplasm, suggesting that it might be activated by binding Ca2+ directly in the cytoplasm, subsequently leading to a physical interaction with CAMTA. Finally, silencing of CAMTA and CAM1 in tea plants by using antisense oligodeoxynucleotides increased the expression level of FEX1 and decreased the intracellular concentration of F. Thus, our study identifies a CAM1-CAMTA-FEX1 module that regulates F export in tea leaves, and hence provides new insights for breeding low-F tea varieties.