Cloning and functional characterization of two carotenoid cleavage dioxygenases for ionone biosynthesis in chili pepper (Capsicum annuum L.) fruits

Abstract

Carotenoids are essential nutrients for the human body and contribute to the coloration and aroma traits of crops. Among plant volatile components, β-ionone has profound effects on scent and aroma due to its very low odor thresholds. In plants, β-ionone is produced from β-carotene (β,β-carotene) by carotenoid cleavage dioxygenases (CCDs), especially members of its CCD1 subfamily. In this study, we isolated two CCD1 genes (CaCCD1A and CaCCD1B) from the chili pepper (Capsicum annuum L.). Both genes have their highest expression levels in fruits among different tissues, and their transcript abundances increase during ripening, along with an increasing accumulation of β-ionone. Both CaCCD1A and CaCCD1B were found to cleave β-carotene specifically at the 9–10/9′–10′ site to produce β-ionone. When these enzymes were expressed in bacterial cells genetically engineered to produce oxygenated derivatives of β-carotene, such as zeaxanthin, antheraxanthin, and violaxanthin, β-ionone was still the only cleavage product, suggesting that β-carotene is preferentially cleaved by CaCCD1s instead of being hydroxylated by the bacterial type carotene hydroxylase CrtZ. In bacterial cells accumulating ε-carotene (ε,ε-carotene), the expression of CaCCD1A or CaCCD1B resulted in the production of α-ionone, indicating that both enzymes have cleavage activities regardless of the β- or ε- ring-type at the end of the substrates.