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

Abstract

BackgroundIn 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.ResultsThree 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.ConclusionsTaken 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.

A Comprehensive Analysis of Carotenoid Cleavage Dioxygenases Genes in Solanum Lycopersicum

“…As shown in Fig. 5, the degree of conservation was poor in these proteins, which supports the view reported previously for other plants (Auldridge et al 2006a, b;Lashbrooke et al 2013;Vallabhaneni et al 2010;Walter et al 2010).…”

“…It suggested that a duplication event had occurred in the course of evolution. The members of the CCD4 enzymes, as well as those from other plants, often include two copies, namely CCD4a and CCD4b (Huang et al 2009;Lashbrooke et al 2013;Ohmiya et al 2006;Rubio et al 2008;Vallabhaneni et al 2010). The copies of CCD8 are variable in different plant species.…”

Tissue-Specific Apocarotenoid Glycosylation Contributes to Carotenoid Homeostasis in Arabidopsis Leaves

“…However, the transcripts from two grapevine CCD4 genes were recently found to be similarly ripening induced (Lashbrooke et al, 2013). As stated above and despite similar substrate and product preferences of CCD1 and CCD4 in vitro (Vogel et al, 2008;Bruno et al, 2015), results from ccd4-1 and ccd1-1 ccd4-1 mutants are in favor of CCD4 being responsible for AG formation in planta.…”

Development and Use of Biotechnology Tools for Grape Functional Analysis

“…In grapevines, this technology has been recently applied to analyze the function of several genes such as VviAdh [75], VviPIP2 [76], VviCCD1 [77], and VviWRKY33 [78]. Likewise, this method has been proved to be very useful for transgenic complementation [79][80][81], technical studies [82][83][84][85][86][87], promoter analysis [88][89][90][91], subcellular localization analysis [92], and protein production [69,93] in grapevine.…”

“…For instance, the compatibility of A. tumefaciens strains with the plant species represents an important variable to be considered in this kind of assay. Thus, the strains most efficiently used for gene transfer into grapevine are probably C58C1 (pCH32) [69,78,86] and EHA105 [75,77,82,83,85,88,90], which contain extra copies of vir genes that make them hypervirulent [61].…”