Carotenoid pigments in plants fulfill indispensable functions in photosynthesis. Carotenoids that accumulate as secondary metabolites in chromoplasts provide distinct coloration to flowers and fruits. In this work we investigated the genetic mechanisms that regulate accumulation of carotenoids as secondary metabolites during ripening of tomato fruits. We analyzed two mutations that affect fruit pigmentation in tomato (Lycopersicon esculentum): Beta (B), a single dominant gene that increases beta-carotene in the fruit, and old-gold (og), a recessive mutation that abolishes beta-carotene and increases lycopene. Using a map-based cloning approach we cloned the genes B and og. Molecular analysis revealed that B encodes a novel type of lycopene beta-cyclase, an enzyme that converts lycopene to beta-carotene. The amino acid sequence of B is similar to capsanthin-capsorubin synthase, an enzyme that produces red xanthophylls in fruits of pepper (Capsicum annum). Our results prove that beta-carotene is synthesized de novo during tomato fruit development by the B lycopene cyclase. In wild-type tomatoes B is expressed at low levels during the breaker stage of ripening, whereas in the Beta mutant its transcription is dramatically increased. Null mutations in the gene B are responsible for the phenotype in og, indicating that og is an allele of B. These results confirm that developmentally regulated transcription is the major mechanism that governs lycopene accumulation in ripening fruits. The cloned B genes can be used in various genetic manipulations toward altering pigmentation and enhancing nutritional value of plant foods.

Neoxanthin, a precursor of the plant hormone abscisic acid, is an allenic xanthophyll recognized as the last product of carotenoid synthesis in green plants. A cDNA for neoxanthin synthase (NSY) was isolated from tomato using a molecular approach based on the mechanistic and structural similarities of NSY to two other closely related carotenogenic enzymes, lycopene cyclase (LCY) and capsanthin-capsorubin synthase (CCS). The identified tomato NSY cDNA (T.NSY) encodes a 56-kDa plastid-targeted protein that when expressed in Escherichia coli, catalyzes the conversion of violaxanthin to neoxanthin. In tobacco leaves that transiently express T.NSY, an increase in neoxanthin content with a concomitant decrease in violaxanthin is observed. NSY is structurally similar to LCY and CCS. However, in Cyanobacteria, the generally accepted progenitor of plastids, both CCS and NSY are absent while LCY is present. LCY catalyzes a simplified version of the reaction catalyzed by NSY and CCS suggesting that these two enzymes were remodeled from LCY during higher plant evolution to create new forms of oxidized carotenoids.