Identification of carotenoids and candidate genes shaping high pigment chili pepper variety

Pepper (Capsicum spp.) is a major vegetable grown and consumed worldwide. Fruit color is an important exterior quality of fresh and processed peppers. The typical red color of pepper fruit is mainly determined by the content of capsanthin and capsorubin. The biosynthetic pathway of capsanthin and capsorubin has been largely clarified, but the regulators of this pathway remain to be investigated. To decipher the regulatory mechanism of high pigment accumulation in mature red pepper (C. annuum) fruit, carotenoid and transcriptome profiling were performed on two red chili peppers with distinct levels of capsanthin and capsorubin. The total carotenoids of high-pigment genotype (GB23) increased by 168 times during fruit ripening, especially carotenoid compounds such as zeaxanthin, 8-cryptoxanthin and capsanthin. Moreover, the difference in the transcript level of genes encoding phytoene synthase 1 (PSY1), 8-carotene hydroxylase 1 (BCH1), and capsanthin-capsorubin synthase (CCS) was consistent with the difference in carotenoid accumulation between GB23 and GB42 (low-pigment genotype). Further investigation revealed several transcription factors exhibiting similar expression patterns and correlated with capsanthin accumulation. Utilizing weighted gene co-expression network analysis, we identified eight transcription factors, namely bHLH13, Zinc finger protein ZFP3, MADS-RIN, NAC68, PIF3, and WRKY6, as potential regulators of PSY1, BCH1, or CCS genes. Yeast one-hybrid and luciferase reporter assays demonstrated that ZFP3 activates PSY1 and CCS while WRKY6 activates CCS through promoter binding. This comprehensive study sheds light on the regulatory mechanism underlying capsanthin accumulation in chili pepper, offering new insights into this important trait.