LC–MS-Based Profiling Provides New Insights into Apocarotenoid Biosynthesis and Modifications in Citrus Fruits

Apocarotenoids contribute to fruit color and aroma, which are critical quality and marketability attributes. Previously, we reported that the red peels of citrus fruits, which are characterized by higher expression levels of a carotenoid cleavage dioxygenase 4 b ( CitCCD 4 b ) gene, accumulate higher levels of β-citraurin and β-citraurinene than yellow peels. Here, we identified and quantified 12 apocarotenoids, either volatile or nonvolatile, in citrus peel using liquid chromatography-mass spectrometry (LC-MS). Our results show that red peels contain also dramatically higher amounts of β-apo-8'-carotenal, crocetin dialdehyde known from saffron, β-citraurol, β-cyclocitral, and 3-OH-β-cyclocitral and up to about 17-fold higher levels of 3-OH-β-cyclocitral glucoside (picrocrocin isomer). The content of these apocarotenoids was also significantly increased in differen CitCCD 4 b -overexpressing transgenic callus lines, compared with corresponding controls. Transient expression of CitCCD 4 b in Nicotiana benthamiana leaves resulted in a striking increase in the 3-OH-β-cyclocitral level and the accumulation of picrocrocin. Thus, our work reinforces the specific function of CitCCD4b in producing C 10 apocarotenoid volatiles and C 30 pigments in citrus peel and uncovers its involvement in the biosynthesis of picrocrocin, C 20 dialdehyde, and C 30 alcohol apocarotenoids, suggesting the potential of this enzyme in metabolic engineering of apocarotenoids and their derivatives.