No 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 ␤-carotene in the fruit, and old-gold (og), a recessive mutation that abolishes ␤-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 ␤-cyclase, an enzyme that converts lycopene to ␤-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 ␤-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.

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.

SummaryThe red colour of tomato (Lycopersicon esculentum) fruits is provided by the carotenoid pigment lycopene whose concentration increases dramatically during the ripening process. A single dominant gene, Del, in the tomato mutant Delta changes the fruit colour to orange as a result of accumulation of δ-carotene at the expense of lycopene. The cDNA for lycopene ε-cyclase (CrtL-e), which converts lycopene to δ-carotene, was cloned from tomato. The primary structure of CRTL-E is 71% identical to the homologous polypeptide from Arabidopsis and 36% identical to the tomato lycopene β-cyclase, CRTL-B. The CrtL-e gene was mapped to a single locus on chromosome 12 of the tomato linkage map. This locus co-segregated with the Del gene. In the wild-type tomato, the transcript level of CrtL-e decreases at the 'breaker' stage of ripening to a nondetectable level in the ripe fruit. In contrast, it increases approximatley 30-fold during fruit ripening in the Delta plants. The Delta mutation does not affect carotenoid composition nor the mRNA level of CrtL-e in leaves and flowers. These results strongly suggest that the mutation Del is an allele of the gene for ε-cyclase. Together with previous data, our results indicate that the primary mechanism that controls lycopene accumulation in tomato fruits is based on the differential regulation of expression of carotenoid biosynthesis genes. During fruit development, the mRNA levels for the lycopene-producing enzymes phytoene synthase (PSY) and phytoene desaturase (PDS) increase, while the mRNA levels of the genes for the lycopene β-and ε-cyclases, which convert lycopene to either β-or δ-carotene, respectively, decline and completely disappear.

SummaryCarotenoids are present in most tissues of higher plants where they play a variety of essential roles. To study the regulation of carotenoid biosynthesis, we have isolated novel mutations in tomato (Solanum lycopersicum) with altered pigmentation of fruit or flowers. Here we describe the isolation and analysis of a tomato mutant, high-pigment 3 (hp3), that accumulates 30% more carotenoids in the mature fruit. Higher concentrations of carotenoids and chlorophyll were also measured in leaves and the pericarp of green fruit. The mutation in hp3 had occurred in the gene for zeaxanthin epoxidase (Zep), which converts zeaxanthin to violaxanthin. Consequently, leaves of the mutant lack violaxanthin and neoxanthin, and flowers contain only minute quantities of these xanthophylls. The concentration in the hp3 mutant of abscisic acid (ABA), which is derived from xanthophylls, is 75% lower than the normal level, making hp3 an ABA-deficient mutant. The plastid compartment size in fruit cells is at least twofold larger in hp3 plants compared with the wild-type. The transcript level in the green fruit of FtsZ, which encodes a tubulin-like protein involved in plastid division, is 60% higher in hp3 than in the wild-type, suggesting that increased plastid division is responsible for this phenomenon. Elevated fruit pigmentation and plastid compartment size were also observed in the ABAdeficient mutants flacca and sitiens. Taken together, these results suggest that ABA deficiency in the tomato mutant hp3 leads to enlargement of the plastid compartment size, probably by increasing plastid division, thus enabling greater biosynthesis and a higher storage capacity of the pigments.