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 ofVvCCD1,VvCCD4aandVvCCD4bwas detected in leaf, flower and throughout berry development.VvCCD1expression was constitutive, whereasVvCCD4aexpression was predominant in leaves andVvCCD4bin berries. A transgenic population with a 12-fold range ofVvCCD1expression exhibited a lack of correlation betweenVvCCD1expression and carotenoid substrates and/or apocarotenoid products in leaves, providing proof that thein plantafunction(s) of VvCCD1 in photosynthetically active tissue is distinct from thein vitroactivities 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 ofVvCCD1in a transgenic grapevine population illustrated the divergence between thein vitroenzyme activity and thein plantaactivity 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.

Background: Carotenoids are a heterogeneous group of plant isoprenoids primarily involved in photosynthesis. In plants the cleavage of carotenoids leads to the formation of the phytohormones abscisic acid and strigolactone, and C₁₃-norisoprenoids involved in the characteristic flavour and aroma compounds in flowers and fruits and are of specific importance in the varietal character of grapes and wine. This work extends the previous reports of carotenoid gene expression and photosynthetic pigment analysis by providing an up-to-date pathway analysis and an important framework for the analysis of carotenoid metabolic pathways in grapevine.Results: Comparative genomics was used to identify 42 genes putatively involved in carotenoid biosynthesis/catabolism in grapevine. The genes are distributed on 16 of the 19 chromosomes and have been localised to the physical map of the heterozygous ENTAV115 grapevine sequence. Nine of the genes occur as single copies whereas the rest of the carotenoid metabolic genes have more than one paralogue. The cDNA copies of eleven corresponding genes fromVitis viniferaL. cv. Pinotage were characterised, and four where shown to be functional. Microarrays provided expression profiles of 39 accessions in the metabolic pathway during three berry developmental stages in Sauvignon blanc, whereas an optimised HPLC analysis provided the concentrations of individual carotenoids. This provides evidence of the functioning of the lutein epoxide cycle and the respective genes in grapevine. Similarly, orthologues of genes leading to the formation of strigolactone involved in shoot branching inhibition were identified:CCD7,CCD8andMAX1. Moreover, the isoforms typically have different expression patterns, confirming the complex regulation of the pathway. Of particular interest is the expression pattern of the threeVvNCEDs: Our results support previous findings thatVvNCED3is likely the isoform linked to ABA content in berries.Conclusions: The carotenoid metabolic pathway is 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 a high degree of similarity with other eudicots. Expression and pigment profiling of developing berries provided insights into the most complete grapevine carotenoid pathway representation. This study represents an important reference study for further characterisation of carotenoid biosynthesis and catabolism in grapevine.

In this study, the metabolic and physiological impacts of an altered microclimate on quality-associated primary and secondary metabolites in grape (Vitis vinifera) ‘Sauvignon Blanc’ berries was determined in a high-altitude vineyard. The leaf and lateral shoot removal in the bunch zones altered the microclimate by increasing the exposure of the berries. The physical parameters (berry diameter and weight), primary metabolites (sugars and organic acids), as well as bunch temperature and leaf water potential were predominantly not affected by the treatment. The increased exposure led to higher levels of specific carotenoids and volatile terpenoids in the exposed berries, with earlier berry stages reacting distinctly from the later developmental stages. Plastic/nonplastic metabolite responses could be further classified to identify metabolites that were developmentally controlled and/or responded to the treatment in a predictable fashion (assessed over two consecutive vintages). The study demonstrates that grapevine berries exhibit a degree of plasticity within their secondary metabolites and respond physiologically to the increased exposure by increasing metabolites with potential antioxidant activity. Taken together, the data provide evidence that the underlying physiological responses relate to the maintenance of stress pathways by modulating antioxidant molecules in the berries.

Background and Aims: Carotenoids and chlorophylls perform a number of essential roles in plants making their accurate quantification important to a variety of studies. We aimed to develop an extraction protocol to accurately determine the photosynthetic pigments in grapevine leaf and berry tissue, specifically focusing on limiting the degradation of these pigments. Methods and Results: An extraction protocol for grapevine leaf and berry tissue was systematically optimised by identifying a number of critical parameters. Extracted pigments were analysed using Reversed Phase‐High Performance Liquid Chromatography (RP‐HPLC). Specific parameters that were optimised included avoiding freeze‐drying the material; the volume of acetone and the time required to extract all the pigments from the tissue; the addition of 0.1% (v/v) N‐ethyldiisopropylamine to berry extracts to minimise pigment degradation during the extraction procedure; and avoiding concentration of the extracts that otherwise resulted in differential degradation of pigments. Additionally, the method of extraction and normalisation with an internal standard was adapted and improved for accuracy. The optimised protocol was validated using authentic standards and its utility shown by analysing the pigment content of berries and leaves at different growth stages. Conclusions: A method has been developed that is able to extract and accurately quantify, by means of HPLC profiling, the levels of photosynthetic pigments from grape berries and leaves. The method avoided any degradation of the pigments during the extraction and was applicable to both berries and leaves in different stages of growth and development, indicating its general usefulness to vegetative and reproductive organs, even if their metabolic states are very different. Significance of Study: The divergence of methods used for photosynthetic pigment analysis in plants, each with specific advantages and disadvantages were considered and used to optimise a number of parameters in a single method that proved to be applicable to plant organs in different developmental stages. The method is fast, applicable to vegetative and reproductive grapevine tissues, avoids degradation of pigments and ensures maximum accuracy when quantifying these important pigments.