The genes and enzymes of the carotenoid biosynthetic pathway in Vitis vinifera L.

ABSTRACT Background: Carotenoids represent one of a large heterogeneous group of plant isoprenoids primarily involved in photosynthesis. In plants the oxidative and/or enzymatic cleavage of certain carotenoids leads to the formation of the plant hormones abscisic acid and strigolactone, as well as C13-norisoprenoids that play a role in the formation of characteristic flavour and aroma compounds, especially in flowers and fruits and are of specific importance in the varietal character of both grapes and wine. The work presented here aims to provide a baseline for the pathway analysis of carotenoid biosynthetic process in grapevine. Results: Comparative genomics was used to identify 42 genes putatively involved in carotenoid biosynthesis and catabolism in grapevine. The genes were found to be distributed on 16 of the 19 chromosomes and have been localised to the physical map of the heterozygous ENTAV115 grapevine sequence. Only nine of the genes occur as single copies whereas the rest of the carotenoid biosynthetic genes have more than one paralogue. From exon-intron gene analysis, it is clear that exon number is remarkably conserved across species; this phenomenon has been suggested to be important for alternative splicing as a regulatory mechanism. Moreover, the cDNA copies of eleven corresponding genes from Vitis vinifera L. cv. Pinotage were further characterised, including functionality. Microarrays provided expression profiles of the entire pathway in three distinct Sauvignon blanc berry developmental stages, whereas HPLC analysis provided the concentrations of individual carotenoids at the three developmental stages. This provides evidence of the existence and functioning of the taxonomically restricted lutein epoxide (Lx) cycle pigments (Lx and lutein) and their respective genes in grapevine. Similarly, orthologues of genes implicated in the catabolic pathway leading to the formation of the plant hormone strigolactone involved in shoot branching inhibition have been identified (i.e. CCD7, CCD8 and MAX1). Moreover, the different isoforms of the carotenoid biosynthetic genes typically have distinctly different expression patterns, confirming the complex regulation of the pathway. Of particular interest is the expression pattern of the three VvNCEDs: Our results show that VvNCED9 is likely the enzymatic isoform linked to the ABA content in berries. Conclusions: The carotenoid biosynthetic pathway is relatively well characterised, and the genes and enzymes have been studied in a number of plants. The study of the 42 carotenoid pathway genes of grapevine showed that they share high homology with those from eudicots. Expression analysis combined with pigment profiling of developing berries provided new insights into the grapevine carotenoid pathway. Overall, this study represents a sound base and important reference study for further characterisation of carotenoid biosynthesis and catabolism in grapevine and other plant species.