Functional characterisation of three members of the Vitis vinifera L. carotenoid cleavage dioxygenase gene family

Abstract

Background: In plants, carotenoids serve as the precursors to C13-norisoprenoids, a group of apocarotenoid compounds with diverse biological functions. Enzymatic cleavage of carotenoids catalysed by members of the carotenoid cleavage dioxygenase (CCD) family has been shown to produce a number of industrially important volatile flavour and aroma apocarotenoids including β-ionone, geranylacetone, pseudoionone, α-ionone and 3-hydroxy-β-ionone in a range of plant species. Apocarotenoids contribute to the floral and fruity attributes of many wine cultivars and are thereby, at least partly, responsible for the "varietal character". Despite their importance in grapes and wine; carotenoid cleavage activity has only been described for VvCCD1 and the mechanism(s) and regulation of carotenoid catabolism remains largely unknown.

Results: Three grapevine-derived CCD-encoding genes have been isolated and shown to be functional with unique substrate cleavage capacities. Our results demonstrate that the VvCCD4a and VvCCD4b catalyse the cleavage of both linear and cyclic carotenoid substrates. The expression of VvCCD1, VvCCD4a and VvCCD4b was detected in leaf, flower and throughout berry development. VvCCD1 expression was constitutive, whereas VvCCD4a expression was predominant in leaves and VvCCD4b in berries. A transgenic population with a 12-fold range of VvCCD1 expression exhibited a lack of correlation between VvCCD1 expression and carotenoid substrates and/or apocarotenoid products in leaves, providing proof that the in planta function(s) of VvCCD1 in photosynthetically active tissue is distinct from the in vitro activities demonstrated. The isolation and functional characterisation of VvCCD4a and VvCCD4b identify two additional CCDs that are functional in grapevine.

Conclusions: Taken together, our results indicate that the three CCDs are under various levels of control that include gene expression (spatial and temporal), substrate specificity and compartmentalisation that act individually and/or co-ordinately to maintain carotenoid and volatile apocarotenoid levels in plants. Altering the expression of VvCCD1 in a transgenic grapevine population illustrated the divergence between the in vitro enzyme activity and the in planta activity of this enzyme, thereby contributing to the efforts to understand how enzymatic degradation of carotenoids involved in photosynthesis occurs. The identification and functional characterisation of VvCCD4a and VvCCD4b suggest that these enzymes are primarily responsible for catalysing the cleavage of plastidial carotenoids.

Figure 1

Molecular phylogenetic analysis of Arabidopsis thaliana and putative Vitis vinifera carotenoid cleavage dioxygenases. A Figtree generated phylogeny based on protein similarity: tree shows the various clades of the CCD family. Proteins indicated with “*” have not been isolated and are based on sequence prediction software. The CCDs are named according to their closest orthologue in Arabidopsis thaliana.

Figure 2

Functionality and substrate specificity of VvCCD1, VvCCD4a and VvCCD4b in a heterologous in vivo bacterial system. CCDs were expressed in Escherichia coli engineered to accumulate specific carotenoids. Volatile apocarotenoids produced after cleavage were determined using GC/MS. Data is represented as the average and standard deviation of three biological repeats (n = 3). pTWIN1 represents the empty vector control. Significant differences between pTWIN1 (control) and VvCCDs are indicated with an asterisk (* = p-value ≤ 0.01). nd = not determined.

Figure 3

Spatial and temporal distribution of VvCCD1, VvCCD4a and VvCCD4b transcripts in distinct grapevine tissues. qRT-PCR analysis of VvCCD1, VvCCD4a and VvCCD4b in leaf, flower and three berry developmental stages. Data are expressed relative to pre-véraison berry stage and normalised to the housekeeping gene, VvEF1a. Relative changes in the total carotenoid and chlorophyll concentrations in the respective tissues are also shown.

Figure 4

qRT-PCR analysis of VvCCD1 expression in the transgenic grapevine population. (A) Expression of the native/endogenous (dark grey square symbol) and transgenic (light grey square symbol) VvCCD1 in lines transformed with the overexpression cassette (CCD1) (n = 3). (B) Expression of VvCCD1 in lines transformed with the silencing cassette (RNAi) (n = 3). Data are expressed relative to the wild-type (WT) expression and normalised to VvGAPDH expression.

Figure 5

The relationship between carotenoids and chlorophylls in the leaves of the grapevine population altered for VvCCD1 expression. RP-HPLC analysis of the VvCCD1-overexpressing (dark grey square symbol), VvCCD1-silenced (RNAi) (light grey triangle symbol) and wild-type (×) grapevine plants used in the study showed positive correlation (R² = 0.90) between the total carotenoid concentration and the total chlorophyll concentration in the leaves (n = 3).

Figure 6

VvCCD-mediated cleavage of members of the carotenoid biosynthetic pathway.The cleavage of tested members of the carotenoid biosynthetic pathway is shown together with the volatile apocarotenoids produced. The 5,6 (5’,6’) and 9,10 (9’,10’) double bond cleavage sites are indicated with a diagonal line (/) through the carotenoid backbone. The circled (light grey circle) double bonds represent an increase in desaturation of the carbon bonds that is required for substrate acceptance.