An alternative pathway to beta -carotene formation in plant chromoplasts discovered by map-based cloning of beta and old-gold color mutations in tomato

Abstract

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.

Figure 1

The carotenoid biosynthesis pathway in tomato. CrtL-b, lycopene β-cyclase; CrtL-e, lycopene ɛ-cyclase; CrtR-b, β-ring hydroxylase, CrtR-e, ɛ-ring hydroxylase; GGDP, geranylgeranyl diphosphate; Ggps, geranylgeranyl diphosphate synthase; Pds, phytoene desaturase; Psy, phytoene synthase; Vde, violaxanthin deepoxidase; Zds, ζ-carotene desaturase; Zep, zeaxanthin epoxidase.

Figure 2

Fruits and flowers of wild type (wt) and mutants Beta, old-gold (og), and old-gold crimson (og^c). Ex-B, transgenic expression of B in wt plants; anti-B, transgenic wt expressing of antisense of B.

Figure 3

Positional cloning of the gene B. Fine mapping of chromosome 6 was done with RFLP markers indicated above the map (centromere position appears as a circle). Chromosomal segments in IL 6–3 and IL 6–2 that overlap B are depicted as black bars. The genetic map, based on the number of recombinants between CT193 and TG578, was derived from scoring a population of 1,335 F₂ plants obtained from a cross between Beta and the wild-type M-82. The relative positions of YAC 310, YAC 271, and YAC 153 are given below the map as empty bars.

Figure 4

Expression of B, CrtL-b, and Pds in fruits and flowers of tomato. The relative amount of mRNA of B, CrtL-b, and Pds was measured concomitantly by RT-PCR from total RNA isolated from different stages of fruit ripening and flower development of wild-type (WT) L. esculentum (M82) and from the mutant Beta. PCR-amplified DNA fragments were separated by PAGE and autoradiographed. (A) WT fruits. (B) Beta fruits. (C) Flowers of wild type and B. Fruit ripening stages: G, mature green; B, breaker; BE, early breaker; BL, late breaker; O, orange; P, pink; R, red; Rx3 and Rx1/3 are samples that contained three times or one-third the total RNA from red fruit. L, leaf. Flowering stages: Fy, preanthesis flower; Fo, postanthesis flower.

Figure 5

Organization of the genomic sequences upstream to the promoters of B and b.

Figure 6

Frame-shift mutations in the B gene from old-gold (og) and old-gold-crimson (og^c). WT, wild type.