SlERF.F2 integrates carotenoid metabolism and chloroplast development via dual transcriptional repression in tomato

Tomato (Solanum lycopersicum) is popular among consumers worldwide for its abundant nutritional value, and carotenoids are important metabolites in tomato with significant nutritional and physiological significance. Previous studies have demonstrated that AP2/ethylene response factor (ERF) transcription factors are key regulators involved in carotenoid biosynthesis. However, most studies have focused on ERF activators that positively regulate carotenoid biosynthesis, while the mechanisms by which Ethylene-responsive element binding factor-associated Amphiphilic Repression (EAR) motif-containing ERF repressors modulate this process remain largely unidentified. Here, we identified a AP2/ERF transcription factor SlERF.F2, which harbors EAR motifs and is co-expressed with key enzyme genes in the carotenoid biosynthesis. Although SlERF.F2 contains the EAR motif typically characterizing negative regulatory transcription factors, the LC-MS carotenoids profiling analysis revealed that SlERF.F2 overexpression significantly increased total carotenoid content in fruit, whereas SlERF.F2 knockout reduced the carotenoids level. Biochemical and molecular assays revealed that SlERF.F2-mediated transcriptional repression of the carotenoid metabolism β-carotene hydroxylase gene (SlBCH1) and the green shoulder formation suppressor gene (SlBEL2), which is dependent on the presence of its C-terminal EAR motif. SlERF.F2 suppresses the expression of the SlBCH1 and SlBEL2 genes, thereby reducing β-carotene degradation and promoting green shoulder formation in tomatoes, respectively. Through this negative-negative regulatory mechanism, SlERF.F2 achieves carotenoid accumulation in tomatoes. Furthermore, we found that SlERF.F2 interacts with the corepressor SlTPL2 via the C-terminal EAR motif, synergistically inhibiting the activity of the SlBCH1 gene. Collectively, our study unveils a dual-function regulatory module where SlERF.F2 integrates carotenoid metabolism and chloroplast development, offering a strategic target for enhancing tomato fruit quality.