Expression and functional analysis of two lycopene β-cyclases from citrus fruits

Department of Biological and Environmental Sciences, Faculty of Agriculture, Shizuoka University, 836 Ohya, Suruga, Shizuoka, 422-8529, Japan.
Planta (Impact Factor: 3.26). 06/2012; 236(4):1315-25. DOI: 10.1007/s00425-012-1690-2
Source: PubMed


In the present study, two LCYb genes (CitLCYb1 and CitLCYb2) were isolated from Satsuma mandarin (Citrus unshiu Marc.), Valencia orange (Citrus sinensis Osbeck) and Lisbon lemon (Citrus limon Burm.f.) and their functions were analyzed by the color complementation assay in lycopene-accumulating E. coli cells. The results showed that CitLCYb1 and CitLCYb2 shared high identity at the amino acid level among the three citrus varieties. The N-terminal region of the two proteins encoded by CitLCYb1 and CitLCYb2 was predicted to contain a 51-residue chloroplastic transit peptide, which shared low similarity. In Satsuma mandarin, the secondary structures of the CitLCYb1 and CitLCYb2 encoding proteins without the transit peptide were quite similar. Moreover, functional analysis showed that both enzymes of CitLCYb1 and CitLCYb2 participated in the formation of β-carotene, and when they were co-expressed with CitLCYe, α-carotene could be produced from lycopene in E. coli cells. However, although CitLCYb2 could convert lycopene to α-carotene in E. coli cells, its extremely low level of expression indicated that CitLCYb2 did not participate in the formation of α-carotene during the green stage in the flavedo. In addition, the high expression levels of CitLCYb1 and CitLCYb2 during the orange stage played an important role in the accumulation of β,β-xanthophylls in citrus fruits. The results presented in this study might contribute to elucidate the mechanism of carotenoid accumulation in citrus fruits.

Download full-text


Available from: Gang Ma, Sep 29, 2015
  • Source
    • "Our results reinforce and extend the proposal that a reduced expression of the β-LCY2 gene and the altered ratio between the two alleles appears to be a common characteristic of C. paradisi. By contrast, the data suggest that oranges (Alquezar et al. 2009) and mandarins (Zhang et al. 2012) have an enhanced expression of the gene β-LCY2 and also of the allele with higher activity ( "
    [Show abstract] [Hide abstract]
    ABSTRACT: Accumulation of lycopene in citrus fruits is an unusual feature restricted to selected mutants. Grapefruit (Citrus paradisi Macf.) is the Citrus specie with greater number of red-fleshed mutants, but the molecular bases of this alteration are not fully understood. To gain knowledge into the mechanisms implicated in this alteration, we conducted a comparative analysis of carotenoid profile and of the expression of genes related to carotenoid biosynthesis and catabolism in flavedo and pulp of two grapefruit cultivars with marked differences in colouration: the white Marsh and the red Star Ruby. Mature green fruit of Marsh accumulated chloroplastic carotenoids, while mature tissues lacked carot-enoids. However, accumulation of downstream products such as abscisic acid (ABA) and expression of its biosynthetic genes, 9-cis-epoxycarotenoid dioxygenase (NCED1 and NCED2), increased after the onset of colouration. In contrast, red grapefruit accumulated lycopene, phytoene and phytofluene, while ABA content and NCED gene expression were lower than in Marsh, suggesting a blockage in the carotenoid biosynthetic pathway. Expression analysis of three genes of the isoprenoid pathway and nine of the carotenoid biosynthetic pathway revealed virtually no differences in flavedo and pulp between both genotypes, except for the chromoplast-specific lycopene cyclase 2 (β-LCY2) which was lower in the pulp of the red grapefruit. The proportion in the expression of the allele with high (β-LCY2a) and low (β-LCY2b) activity was also similar in the pulp of both geno-types. Therefore, results suggest that reduced expression of β-LCY2 appears to be responsible of lycopene accumulation in the red Star Ruby grapefruit.
    Tree Genetics & Genomes 09/2013; 9(5). DOI:10.1007/s11295-013-0635-7 · 2.45 Impact Factor
  • Source
    • "External and internal coloration of Citrus fruits, as in many other fruits, is due to the accumulation of carotenoids, which provide colours ranging from yellow and orange to the red tint typical of the different species and cultivars (reviewed in Alquézar et al., 2008a; Kato, 2012). accumulation and composition during Citrus fruit maturation is highly regulated by coordinated transcriptional changes in carotenoid biosynthetic genes, and cyclization of lycopene appears to be a key regulatory step in the pathway , redirecting the flow of carotenoids during the transition from chloroplast to chromoplast (Alquézar et al., 2009; Kato, 2012; Zhang et al., 2012). "
    [Show abstract] [Hide abstract]
    ABSTRACT: 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.
    Journal of Experimental Botany 09/2013; 64(14). DOI:10.1093/jxb/ert260 · 5.53 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: The control of gene expression is a crucial regulatory mechanism in carotenoid accumulation of fruits and flowers. We investigated the role of transcriptional regulation of nine genes involved in the carotenoid biosynthesis pathway in three varieties of Cucurbita pepo with evident differences in fruit color. The transcriptional levels of the key genes involved in the carotenoid biosynthesis were higher in flower-, leaf- and fruit skin tissues than flesh tissues. This correlated with higher concentration of carotenoid content in these tissues. The differential expression among the colored and white cultivars detected for some genes, such as LCYe, in combination with other regulatory mechanisms could explain the large differences found in terms of carotenoid content among the three varieties. These results are a first step to elucidate carotenogenesis in C. pepo and demonstrate that in general, regulation of the pathway genes is a critical factor that determines the accumulation of these compounds.
    Journal of Agricultural and Food Chemistry 06/2013; 61(26). DOI:10.1021/jf4004576 · 2.91 Impact Factor
Show more