Changes in carotenoid content and composition and expression of carotenoid biosynthetic genes were analyzed in the flavedo of sweet orange (Citrus sinensis L. Osbeck, cv. Navelate) fruit during development and maturation. Lutein and all-E-violaxanthin were the major carotenoids in chloroplast-containing tissues. During fruit coloration, phytoene, beta-cryptoxanthin, zeaxanthin, and mainly (9Z)-violaxanthin progressively accumulated, and a large proportion of apocarotenoids was also found in the flavedo of full-colored fruits. We have cloned partial and full-length cDNAs corresponding to genes involved in early condensation and desaturase reactions [phytoene synthase (PSY), phytoene desaturase (PDS), and zeta-carotene desaturase (ZDS)], coupled redox reaction (plastid terminal oxidase), cyclizations [beta-lycopene cyclase (beta-LCY) and epsilon-lycopene cyclase (epsilon-LCY)], hydroxylation [beta-carotene hydroxylase (beta-CHX)], and epoxidation [zeaxanthin epoxidase (ZEP)] and analyzed their mRNA accumulation in the flavedo of fruits during development and ripening as compared with those of leaves. Collectively, the results indicated that PDS gene expression correlated with carotenoid content in developing fruit and that up-regulation of PSY and ZDS genes at the onset of fruit coloration would enhance the production of linear carotenes and the flux into the pathway. The shift from the beta,epsilon-branch to the beta,beta-branch of the pathway that originates the changes in carotenoid composition during fruit coloration may be explained by a down-regulation of epsilon-LCY and by the increase of the beta-CHX transcript.

Citrus is the first tree crop in terms of fruit production. The colour of Citrus fruit is one of the main quality attributes, caused by the accumulation of carotenoids and their derivative C30 apocarotenoids, mainly β-citraurin (3-hydroxy-β-apo-8′-carotenal), which provide an attractive orange-reddish tint to the peel of oranges and mandarins. Though carotenoid biosynthesis and its regulation have been extensively studied in Citrus fruits, little is known about the formation of C30 apocarotenoids. The aim of this study was to the identify carotenoid cleavage enzyme(s) [CCD(s)] involved in the peel-specific C30 apocarotenoids. In silico data mining revealed a new family of five CCD4-type genes in Citrus. One gene of this family, CCD4b1, was expressed in reproductive and vegetative tissues of different Citrus species in a pattern correlating with the accumulation of C30 apocarotenoids. Moreover, developmental processes and treatments which alter Citrus fruit peel pigmentation led to changes of β-citraurin content and CCD4b1 transcript levels. These results point to the involvement of CCD4b1 in β-citraurin formation and indicate that the accumulation of this compound is determined by the availability of the presumed precursors zeaxanthin and β-cryptoxanthin. Functional analysis of CCD4b1 by in vitro assays unequivocally demonstrated the asymmetric cleavage activity at the 7′,8′ double bond in zeaxanthin and β-cryptoxanthin, confirming its role in C30 apocarotenoid biosynthesis. Thus, a novel plant carotenoid cleavage activity targeting the 7′,8′ double bond of cyclic C40 carotenoids has been identified. These results suggest that the presented enzyme is responsible for the biosynthesis of C30 apocarotenoids in Citrus which are key pigments in fruit coloration.

The characterization of a novel mutant, named Pinalate, derived from the orange (Citrus sinensis L. Osbeck) Navelate, which produces distinctive yellow fruits instead of the typical bright orange colouration, is reported. The carotenoid content and composition, and ABA content in leaf and flavedo tissue (coloured part of the skin) of fruits at different developmental and maturation stages were analysed. No important differences in leaf carotenoid pattern of both phenotypes were found. However, an unusual accumulation of linear carotenes (phytoene, phytofluene and zeta- carotene) was detected in the flavedo of Pinalate. As fruit maturation progressed, the flavedo of mutant fruit accumulated high amounts of these carotenes and the proportion of cyclic and oxygenated carotenoids was substantially lower than in the parental line. Full-coloured fruit of Pinalate contained about 44% phytoene, 21% phytofluene, 25% zeta-carotene, and 10% of xanthophylls, whereas, in Navelate, 98% of total carotenoids were xanthophylls and apocarotenoids. The ABA content in the flavedo of Pinalate mature fruit was 3-6 times lower than in the corresponding tissue of Navelate, while no differences were found in leaves. Other maturation processes were not affected in Pinalate fruit. Taken together, the results indicate that Pinalate is a fruit-specific alteration defective in zeta-carotene desaturase or in zeta-carotene desaturase-associated factors. Possible mechanisms responsible for the Pinalate phenotype are discussed. Because of the abnormal fruit-specific carotenoid complement and ABA deficiency, Pinalate may constitute an excellent system for the study of carotenogenesis in Citrus and the involvement of ABA in fruit maturation and stress responses.

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