PHYTOENE SYNTHASE FROM NARCISSUS-PSEUDONARCISSUS - FUNCTIONAL EXPRESSION, GALACTOLIPID REQUIREMENT, TOPOLOGICAL DISTRIBUTION IN CHROMOPLASTS AND INDUCTION DURING FLOWERING

A cDNA coding for the carotenoid biosynthetic enzyme phytoene synthase was cloned from a Narcissus pseudonarcissus flower cDNA library, and the corresponding protein was overexpressed in insect cells using the baculovirus lipofection system. The full-length overexpressed enzyme e xhibited very reduced catalytic activity compared with an overexpresse d N-truncated form, with its transit sequence removed by site-directed mutagenesis. The shortened form readily bound quantitatively to lipid bilayers. Although it was active with liposomes prepared from plastid lipids, with phospholipid liposomes it was not, even though associati on took place. In this latter case, free galactose was capable of subs tituting for galactolipids, resulting in enzymatic activity. It is con cluded that galactolipids are involved in catalytic activity, but do n ot serve as a membrane anchor. Antibodies raised against the recombina nt enzyme made it possible to distinguish between a membrane-bound and a soluble, protein-complexed inactive form of phytoene synthase, pres ent in the chromoplast stroma. These findings and data on phytoene syn thase mRNA and protein expression presented here are discussed in term s of a possible regulatory role in color formation during chromoplast (flower) development.