Catalytic activities and chloroplast import of carotenogenic enzymes from citrus

Citrus fruits are a rich source of carotenoids. cDNAs for carotenoid biosynthetic enzymes have been identified from their flavedos and juice sacs, and were used to examine expression patterns of carotenogenic genes during fruit development by several groups. However, functions of most of the gene products have not been verified yet. in this report, we examined catalytic activities of two carotenogenic enzymes from navel orange (Citrus sinensis Osbeck), phytoene desaturase (CitPds) and lycopene-beta-cyclase (CitLcyb), and one enzyme from Satsuma mandarin (Citrus unshiu Marcow), lycopene-F-cyclase (CitLcye). We also conducted in vitro import assay of the three proteins along with two other carotenogenic enzyme from navel orange, phytoene synthase (CitPsy) and carotenoid beta-ring hydroxylase (CitChyb), using isolated chloroplasts, and confirmed their plastid localization and the presence of transit peptides that were cleaved upon import. Furthermore, we examined their suborganellar localization. CitPsy was found to be peripherally associated with the membrane, while CitPds was mainly recovered in the soluble fraction. By contrast, CitLcyb and CitLcye were targeted both to the soluble and to the membrane compartments, although the latter showed a stronger association to the membrane than the former. Finally, CitChyb was exclusively inserted into the chloroplast internal membranes. These data should help us better understand the mechanism of carotenoid biosynthesis.