Browsing by Author "Young, Philip R."

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    Field-grown grapevine berries use carotenoids and the associated xanthophyll cycles to acclimate to UV exposure differentially in high and low light (shade) conditions
    (Frontiers Media, 2016) Joubert, Chandre; Young, Philip R.; Eyeghe-Bickong, Hans A.; Vivier, Melane A.
    Light quantity and quality modulate grapevine development and influence berry metabolic processes. Here we studied light as an information signal for developing and ripening grape berries. A Vitis vinifera Sauvignon Blanc field experiment was used to identify the impacts of UVB on core metabolic processes in the berries under both high light (HL) and low light (LL) microclimates. The primary objective was therefore to identify UVB-specific responses on berry processes and metabolites and distinguish them from those responses elicited by variations in light incidence. Canopy manipulation at the bunch zone via early leaf removal, combined with UVB-excluding acrylic sheets installed over the bunch zones resulted in four bunch microclimates: (1) HL (control); (2) LL (control); (3) HL with UVB attenuation and (4) LL with UVB attenuation. Metabolite profiles of three berry developmental stages showed predictable changes to known UV-responsive compound classes in a typical UV acclimation (versus UV damage) response. Interestingly, the berries employed carotenoids and the associated xanthophyll cycles to acclimate to UV exposure and the berry responses differed between HL and LL conditions, particularly in the developmental stages where berries are still photosynthetically active. The developmental stage of the berries was an important factor to consider in interpreting the data. The green berries responded to the different exposure and/or UVB attenuation signals with metabolites that indicate that the berries actively managed its metabolism in relation to the exposure levels, displaying metabolic plasticity in the photosynthesis-related metabolites. Core processes such as photosynthesis, photo-inhibition and acclimation were maintained by differentially modulating metabolites under the four treatments. Ripe berries also responded metabolically to the light quality and quantity, but mostly formed compounds (volatiles and polyphenols) that have direct antioxidant and/or “sunscreening” abilities. The data presented for the green berries and those for the ripe berries conform to what is known for UVB and/or light stress in young, active leaves and older, senescing tissues respectively and provide scope for further evaluation of the sink/source status of fruits in relation to photosignalling and/or stress management.
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    Functional characterisation of three members of the Vitis vinifera L. carotenoid cleavage dioxygenase gene family
    (BioMed Central, 2013-10) Lashbrooke, Justin G.; Young, Philip R.; Dockrall, Samantha J.; Vasanth, Krishnan; Viviers, Melane A.
    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.
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    The genes and enzymes of the carotenoid metabolic pathway in Vitis vinifera L
    (BioMed Central, 2012-06) Young, Philip R.; Lashbrooke, Justin G.; Alexandersson, Erik; Jacobson, Dan; Moser, Claudio; Velasco, Riccardo; Vivier, Melané A.
    Abstract 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 from Vitis vinifera L. 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, CCD8 and MAX1. Moreover, the isoforms typically have different expression patterns, confirming the complex regulation of the pathway. Of particular interest is the expression pattern of the three VvNCEDs: Our results support previous findings that VvNCED3 is 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.
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    Seeing the forest through the (phylogenetic) trees : functional characterisation of grapevine terpene synthase (VviTPS) paralogues and orthologues
    (MDPI, 2021-07-26) Smit, Samuel J.; Vivier, Melane A.; Young, Philip R.; Nussbaumer, Thomas; Seetharam, Arum
    Gene families involved in specialised metabolism play a key role in a myriad of ecophysiological and biochemical functions. The Vitis vinifera sesquiterpene synthases represent the largest subfamily of grapevine terpene synthase (VviTPS) genes and are important volatile metabolites for wine flavour and aroma, as well as ecophysiological interactions. The functional characterisation of VviTPS genes is complicated by a reliance on a single reference genome that greatly underrepresents this large gene family, exacerbated by extensive duplications and paralogy. The recent release of multiple phased diploid grapevine genomes, as well as extensive whole-genome resequencing efforts, provide a wealth of new sequence information that can be utilised to overcome the limitations of the reference genome. A large cluster of sesquiterpene synthases, localised to chromosome 18, was explored by means of comparative sequence analyses using the publicly available grapevine reference genome, three PacBio phased diploid genomes and whole-genome resequencing data from multiple genotypes. Two genes, VviTPS04 and -10, were identified as putative paralogues and/or allelic variants. Subsequent gene isolation from multiple grapevine genotypes and characterisation by means of a heterologous in planta expression and volatile analysis resulted in the identification of genotype-specific structural variations and polymorphisms that impact the gene function. These results present novel insight into how grapevine domestication likely shaped the VviTPS landscape to result in genotype-specific functions.
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    The transcriptional responses and metabolic consequences of acclimation to elevated light exposure in grapevine berries
    (Frontiers Media, 2017) Du Plessis, Kari; Young, Philip R.; Eyeghe-Bickong, Hans A.; Vivier, Melane A.
    An increasing number of field studies that focus on grapevine berry development and ripening implement systems biology approaches; the results are highlighting not only the intricacies of the developmental programming/reprogramming that occurs, but also the complexity of how profoundly the microclimate influences the metabolism of the berry throughout the different stages of development. In a previous study we confirmed that a leaf removal treatment to Sauvignon Blanc grapes, grown in a highly characterized vineyard, primarily affected the level of light exposure to the berries throughout their development. A full transcriptomic analysis of berries from this model vineyard details the underlying molecular responses of the berries in reaction to the exposure and show how the berries acclimated to the imposing light stress. Gene expression involved in the protection of the photosynthetic machinery through rapid protein-turnover and the expression of photoprotective flavonoid compounds were most significantly affected in green berries. Overall, the transcriptome analysis showed that the berries implemented multiple stress-mitigation strategies in parallel and metabolite analysis was used to support the main findings. Combining the transcriptome data and amino acid profiling provided evidence that amino acid catabolism probably contributed to the mitigation of a likely energetic deficit created by the upregulation of (energetically) costly stress defense mechanisms. Furthermore, the rapid turnover of essential proteins involved in the maintenance of primary metabolism and growth in the photosynthetically active grapes appeared to provide precursors for the production of protective secondary metabolites such as apocarotenoids and flavonols in the ripening stages of the berries. Taken together, these results confirmed that the green grape berries responded to light stress much like other vegetative organs and were able to acclimate to the increased exposure, managing their metabolism and energy requirements to sustain the developmental cycle toward ripening. The typical metabolic consequences of leaf removal on grape berries can therefore now be linked to increased light exposure through mechanisms of photoprotection in green berries that leads toward acclimation responses that remain intact until ripening.