Expression and functional analysis of two lycopene β-cyclases from citrus fruits.
ABSTRACT 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.
Postharvest Biology and Technology 01/2015; 99:99–104. DOI:10.1016/j.postharvbio.2014.08.002 · 2.63 Impact Factor
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ABSTRACT: External colour of Citrus fruits is one of the most important quality traits and a decisive factor for consumer acceptance. Pigmentation of fruit peel is highly diverse among the different species and cultivars of the genus Citrus, ranging from the green of limes to the yellow of lemons, orange in mandarins and sweet oranges, and pink in red grapefruits. Colouration of the peel is due to the presence of two main pigments: chlorophylls which provide green colour, and carotenoids, which are responsible for the characteristic colouration of mature fruits of most species and cultivars. Anthocyanins are a third group of pigments, providing a red to purple tint, in a specific group, blood oranges, and mainly restricted to the flesh. Chlorophylls and carotenoids are isoprenoid-derived pigments, synthesized and accumulated in plastids and, therefore, changes in these compounds during natural ripening are driven by the transformation of chloroplasts into chromoplasts. Most of the structural genes involved in chlorophylls and carotenoids metabolism have been characterized in Citrus, concluding that content and composition of these pigments are mostly genetically determined, and highly regulated at the transcriptional level. However, other mechanisms such as post-transcriptional regulation, the formation of specific suborganellar structures or stabilizing-complexes may also operate. Environmental factors, such as light and temperature, are known to play critical influence in the development of colouration and that biochemical and molecular bases of their action are being elucidated. Moreover, nutritional status (mainly nitrogen and sugars) is a key determinant of the rate and intensity of peel colouration. The consensus hypothesis establishes that peel colouration is governed by environmental and nutritional factors acting throughout the action of different hormonal signals. In this review we summarize content and composition of main pigments in the peel of fruits of relevant Citrus species and varieties. A comprehensive overview of metabolic pathways implicated in the metabolism of the main pigments, with emphasis on the key regulatory steps, gene expression and their regulation during fruit ripening and in response to environmental, nutritional and hormonal signals is critically revised and discussed.Scientia Horticulturae 09/2013; 163. DOI:10.1016/j.scienta.2013.08.014 · 1.50 Impact Factor
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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; DOI:10.1007/s11295-013-0635-7 · 2.44 Impact Factor