Abstract
\Lycopene beta-cyclases (LCYBs) are key enzymes functioning at the branch point of the carotenoid biosynthesis pathway and determining the lycopene content in citrus fruit. There are two types of LCYBs, each of which possesses two different alleles (LCYb1a, LCYb1b, LCYb2a, and LCYb2b) in most citrus species. In this study, we performed detailed analyses of enzyme activities and transcript levels of all LCYB alleles in 'Anliu' and 'Hong Anliu' sweet orange (Citrus sinensis). The results showed that low transcript level of the chromoplast-specific CsLCYb2 gene and preferential expression of the inefficient CsLCYb2b allele may be involved in the massive lycopene accumulation in 'Hong Anliu' sweet orange. We further isolated the LCYb2 genes from other citrus species and discovered extensive allelic sequence variations highly associated with the evolutionary relationship among species. To investigate the site(s) determining the differences in enzyme activities, we detailedly analyzed the sequence variations and performed site-directed mutations to verify two amino acids at positions 72 and 359 critically determining enzyme activities of LCYb2 alleles. To understand the regulatory mechanisms of gene expression, we isolated two LCYb2 promoters from sweet orange. Bioinformatics analysis of promoter sequences revealed many common and exclusive cis-elements. Functional characterization of promoter activities by transient expression assay in tomato showed that both promoters were active in chromoplast-rich fruits. In summary, this study not only reinforces the important roles of the LCYb2 genes in lycopene accumulation in citrus but also provides new information about allelic sequence variations, critical amino acids, and promoter characteristics of the LCYb2 genes.