Four specific isogenes of the anthocyanin metabolic pathway are systematically co-expressed with the red colour of grape berries
Introduction
The colour of the red wine is essentially due to the release of pigments from the skin of grape berries during the process of wine making. Some red cultivars also accumulate pigments in the flesh of the berry, giving a red colour to the pulp. These cultivars (called “teinturier”) give rise to wines of very dense colour and are most commonly used for blending. Anthocyanins are the predominant pigment in red and black grape berries. Wine colour therefore relies on quantitative and qualitative patterns of anthocyanin accumulation in developing berries that depend on cultivars, maturity of the berries, environmental factors and vineyard management practices (for review see [1]).
Anthocyanin biosynthesis has been characterised in flowers of petunia, snapdragon, and in kernels of maize and the biosynthetic pathway is one of the well-known pathway in plants [2]. Two classes of genes are required for anthocyanin biosynthesis. The structural genes encode the enzymes that directly participate in the formation and storage of anthocyanins and other flavonoids. The regulatory genes regulate the expression of the structural genes, and control the spatial and temporal accumulation of pigments [2], [3], [4]. There is strong divergence of the mechanisms by which the pathway is controlled between organ or plant species [4]. Various mutable alleles for flowers, seeds, and fruits caused by insertion of DNA transposon into genes for anthocyanin pigmentation have been found [5] suggesting that the regulatory genes exert their control on only a subset of biosynthetic genes.
Concerning the anthocyanin biosynthesis pathway in grape berries (Fig. 1), some cDNAs encoding structural genes have been cloned in Vitis vinifera [6]. Using them as probes, Boss et al. [7], [8] showed that only the expression of the gene coding for UFGT was consistently associated with the berry colour, depending on developmental stage and cultivar. Expression analysis of UFGT genes in white cultivars and red-skinned sports revealed that UFGT gene was present, although not expressed in the white cultivars [8], [9]. Moreover, UFGT sequences were identical in white and red-skinned sports including the promotor region suggesting that the phenotypic change from white to red could be the result of a mutation in a regulatory gene controlling the expression of UFGT [10]. Several myb-related genes were isolated from the red berries and delivery of VvmybA1-1, VvmybA1-2 and VvmybA2 to somatic embryos of grape led to the induction of reddish-purple spots and UFGT gene expression in non-coloured embryos [10]. It was shown that the lack of expression of VvmybA1 in white cultivars results from the insertion of a retrotransposon in its promotor region and that this retrotransposon is deleted in red sports [11]. These results provide strong evidence that VvmybA1 is involved in the differences between red and white cultivars.
Recent reports suggest that UFGT may be neither the only gene of the anthocyanin pathway whose expression is associated with colour determination nor the only molecular target of the myb factor. In tomato, the expression of a set of genes involved in anthocyanin biosynthesis is coordinately induced by ANT1 (a myb factor similar to VvMybA1) over-expression that leads to pigmented phenotype [12]. Among the induced transcripts in the ANT1 transgenic tomato, these authors found genes encoding some glucosyl transferases (including UFGT), a chalcone synthase, and a type-I GST, a flavonoid binding protein required for vacuolar transport that is orthologous to the BZ2 gene in maize and AN9 gene in petunia [13]. In apple [14], [15], five genes involved in anthocyanin biosynthesis are coordinately expressed and correlated with anthocyanin concentration. Very similar results were found in Perilla frutescens [16]. In grape, Jeong et al. [17] reported that in the berry skins, CHS2, CHS3, CHI1 and F 3-H2 were, among their gene families, predominantly transcribed during coloration and they suggest a common regulation with UFGT. With the exception of this work, most studies performed to date on grape used non-specific probes and may not have distinguish members among multigenic families. By using both oligo-array and gene-specific RT-PCR, there are now robust methods to specifically assay gene expression in each member of such families.
The present study was performed to identify possible new specific isogenes consistently associated with the determination of colour in grape berries and to question whether entities regulating their expression were the same in different genotypes (red, white, “teinturiers”) and in different tissues (flesh, skin). To capture genes whose expression was best associated with the berry colour, we developed: (i) cDNA SSH libraries obtained by comparing red or white pulp cultivars, (ii) real-time PCR quantification of selected genes obtained from the SSH libraries and (iii) a large scale expression analysis using a 3 K oligo array (isogene specific) and several genotypes × developmental stages × tissue combinations.
Section snippets
Plant material
V. vinifera L. berries from different cultivars, Gamay Noir, Gamay Fréaux and Lacryma Christii were harvested during summers 2002 and 2003 in Domaine de Vassal (ENSA-Montpellier, France). Berries from Pinot Noir and Pinot Blanc were collected during summer 2003 in Domaine du Mont Battois (Beaune, France). Pinot noir and Pinot blanc used were sports of the same Pinot gris cultivar. All samples were collected at the same developmental stage, 1 week after véraison. In 2002, six randomly selected
Anthocyanin profiling in two different cultivars of V. vinifera
The Lacryma berry skins contained about eight-fold more anthocyanins (A520 = 554 g−1 fresh weight) than Gamay berry skins (A520 = 70.4 g−1 fresh weight). The Gamay flesh did not contain any detectable anthocyanins, whereas the Lacryma flesh was strongly coloured (A520 = 93.2 g−1 fresh weight). The berry extracts exhibited anthocyanin profiles (Fig. 2) similar to those previously reported in red skin cultivars [7], [18] with malvidin derivatives as the major form. The other major aglycone such as
A multi step experimental set up including SSH, RT-PCR and oligo array
The aim of this work was to identify best candidate genes involved in the determination of berry colour. For that purpose, we designed a multi-step experimental set up involving SSH library construction, quantitative transcript abundance determination using RT-PCR and large scale isogene profiling using oligo arrays. SSH combines the selectivity of subtractive hybridisation with the sensitivity of PCR [24]. One of its main advantages is to allow the detection of low abundance differentially
Conclusion
Data presented here unambiguously show that a set of co-regulated isogenes, including the previously identified UFGT and three other genes (CHS3, GST, MT) involved in secondary metabolism, systematically correlates with the red colour of the grape berry, regardless if these differences arose from developmental stages, tissues or cultivars. They open the way for further discovery of common regulatory elements.
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2022, Scientia HorticulturaeCitation Excerpt :SlGSTAA was fine-mapping on Chromosome 2, which mutated leading to anthocyanin absent and presented green hypocotyl of tomato seedlings (Zhang et al., 2016a). Similarly, the loss of function of anthocyanin MATEs transporters (AM1, AM3) results in no accumulation of anthocyanin in vesicles (Ageorges et al., 2006; Conn et al., 2003). Therefore, MATEs transporters likely obstruct the conveyance of anthocyanin from the cytoplasm to tonoplast, which makes GST-mediated transportation of anthocyanin from cytoplasm to the central vacuole impossible (Gu et al., 2019).