Cloning of tangerine from tomato reveals a carotenoid isomerase essential for the production of beta-carotene and xanthophylls in plants

Abstract

Carotenoid biosynthesis in plants has been described at the molecular level for most of the biochemical steps in the pathway. However, the cis-trans isomerization of carotenoids, which is known to occur in vivo, has remained a mystery since its discovery five decades ago. To elucidate the molecular mechanism of carotenoid isomerization, we have taken a genetic map-based approach to clone the tangerine locus from tomato. Fruit of tangerine are orange and accumulate prolycopene (7Z,9Z,7'Z,9'Z-tetra-cis-lycopene) instead of the all-trans-lycopene, which normally is synthesized in the wild type. Our data indicate that the tangerine gene, designated CRTISO, encodes an authentic carotenoid isomerase that is required during carotenoid desaturation. CRTISO is a redox-type enzyme structurally related to the bacterial-type phytoene desaturase CRTI. Two alleles of tangerine have been investigated. In tangerine(mic), loss of function is attributable to a deletion mutation in CRTISO, and in tangerine(3183), expression of this gene is impaired. CRTISO from tomato is expressed in all green tissues but is upregulated during fruit ripening and in flowers. The function of carotene isomerase in plants presumably is to enable carotenoid biosynthesis to occur in the dark and in nonphotosynthetic tissues.

Figure 1.

The Carotenoid Biosynthesis Pathway in Plants. Enzymes are named according to the designation of their genes. crtI, phytoene desaturase (bacterial type); Lcy-b (CrtL-b), lycopene β-cyclase; CrtL-e, lycopene ɛ-cyclase; CrtR-b, β-ring hydroxylase, CrtR-e, ɛ-ring hydroxylase; CycB, chromoplast-specific lycopene; cyclase; GGPP, geranylgeranyl diphosphate; Nxs, neoxanthin synthase; Pds, phytoene desaturase; Psy, phytoene synthase; Vde, violaxanthin deepoxidase; Zds, ζ-carotene desaturase; Zep, zeaxanthin epoxidase.

Figure 2.

Fruits and Seedlings of Tangerine Mutants. (A) Ripe fruit of the wild type (top) and mutant tangerine^mic (bottom). (B) Immature green fruit of tangerine³¹⁸³ (top) and mutant tangerine^mic (bottom). (C) Light-grown seedlings of the wild type (left) and mutant tangerine^mic (right). (D) Light-grown seedlings of mutant tangerine^mic (left) and the wild type (right).

Figure 3.

Analysis by HPLC of Carotenoids in Tomato Fruit of Wild Type and Two Alleles of tangerine Mutants. Peak 1, all-trans-lycopene; peak 2, di-cis-lycopene; peak 3, prolycopene; peak 4, neurosporene; peak 5, tri-cis-neurosporene; peak 6, ζ-carotene; peak 7, β-carotene; peak 8, phytofluene; peak 9, phytoene. Absorption spectra of peaks are presented in the insets.

Figure 4.

Map-Based Cloning of the tangerine Locus. Fine mapping of chromosome 10 was performed with the restriction fragment length polymorphism markers indicated in the map. The chromosomal segment in the introgression line IL 10-2 that overlaps tangerine is depicted as a bar. Numbers correspond to centimorgans. Numbers in parentheses describe recombination events. Markers were TG408, CT20, CD72, CT57, TG1, and TG241. CEN, centromere.

Figure 5.

Organization of the Genomic Sequences of CRTISO. Closed boxes represent exons. Deletions found in CRTISO of tangerine alleles are indicated. ATG, initiation codon; UGA, stop codon.

Figure 6.

Expression of CRTISO during Tomato Fruit Development. Steady state levels of mRNA of CRTISO, Psy, and Pds were measured by reverse transcriptase–mediated PCR from total RNA isolated from different stages of fruit development in wild-type tomato (cv M82) and mutant tangerine³¹⁸³. PCR products were separated by agarose gel electrophoresis and stained with ethidium bromide. G, mature green fruit; B, breaker stage; R, ripe stage 7 days after breaker; B_×1/3 and B_×3, samples that contained one-third and three times the total RNA, respectively, from breaker-stage fruit.

Figure 7.

Phylogenetic Tree of CRTISO and Related Genes. CRTI, phytoene desaturase from bacteria. Slr1293 is a hypothetical gene from Synechocystis PCC6803. CRTI R. sphaeroides, CRTI Rhodobacter sphaeroides.