Vitis vinifera root and leaf metabolic composition during fruit maturation: implications of defoliation

Grapevine (Vitis vinifera) roots and leaves represent major carbohydrate and nitrogen (N) sources, either as recent assimilates, or mobilized from labile or storage pools. This study examined the response of root and leaf primary metabolism following defoliation treatments applied to fruiting vines during ripening. The objective was to link alterations in root and leaf metabolism to carbohydrate and N source functioning under conditions of increased fruit sink demand. Potted grapevine leaf area was adjusted near the start of véraison to 25 primary leaves per vine compared to 100 leaves for the control. An additional group of vines were completely defoliated. Fruit sugar and N content development was assessed, and root and leaf starch and N concentrations determined. An untargeted GC/MS approach was undertaken to evaluate root and leaf primary metabolite concentrations. Partial and full defoliation increased root carbohydrate source contribution towards berry sugar accumulation, evident through starch remobilization. Furthermore, root myo‐inositol metabolism played a distinct role during carbohydrate remobilization. Full defoliation induced shikimate pathway derived aromatic amino acid accumulation in roots, while arginine accumulated after full and partial defoliation. Likewise, various leaf amino acids accumulated after partial defoliation. These results suggest elevated root and leaf amino N source activity when leaf N availability is restricted during fruit ripening. Overall, this study provides novel information regarding the impact of leaf source restriction, on metabolic compositions of major carbohydrate and N sources during berry maturation. These results enhance the understanding of source organ carbon and N metabolism during fruit maturation.     

Localization of stilbene synthase in Vitis vinifera L. during berry development

The localization of stilbene synthase (STS) (EC 2.3.1.95) in grape berry (Vitis vinifera L.) was investigated during fruit development. The berries were collected at 2, 4, 7, 11, and 15 weeks postflowering from the cultivar Nebbiolo during the 2005 and 2006 growing seasons. High-performance liquid chromatography analysis showed that berries accumulated cis- and trans-isomers of resveratrol mainly in the exocarp throughout fruit development. Immunodetection of STS protein was performed on berry extracts and sections with an antibody specifically developed against recombinant grape STS1. In agreement with resveratrol presence, STS was found in berry exocarp tissues during all stages of fruit development. The labeled epidermal cells were few and were randomly distributed, whereas nearly all the outer hypodermis cells were STS-positive. The STS signal decreased gradually from exocarp to mesocarp, where the protein was detected only occasionally. At the subcellular level, STS was found predominantly within vesicles (of varying size), along the plasma membrane and in the cell wall, suggesting protein secretion in the apoplast compartment. Despite the differences in fruit size and structure, the STS localization was the same before and after veraison, the relatively short developmental period during which the firm green berries begin to soften and change color. Nevertheless, the amount of protein detected in both exocarp and mesocarp decreased significantly in ripe berries, in agreement with the lower resveratrol content measured in the same tissues. The location of STS in exocarp cell wall is consistent with its role in synthesizing defense compounds and supports the hypothesis that a differential localization of phenylpropanoid biosynthetic machinery regulates the deposition of specific secondary products at different action sites within cells.     

Ultrastructure of the cuticle during growth of the grape berry (Vitis vinifera L.)

The outer surface morphology and the ultrastructure of grape berries during growth were examined by electron microscopy. The cuticle began to form before anthesis as highly organized and tightly appressed cuticular ridges. During the period of rapid expansion, the cuticular material spread out over the grape berry. At the same time, an outer wax layer of about 0.5 μm was indentified. As growth proceeded, the cuticular material flattened out and eventually disappeared. At the final stage of growth, the berry had a smooth, continuous and homogeneous cuticle with a thickness of 3 μm.     

Transporters expressed during grape berry (Vitis vinifera L.) development are associated with an increase in berry size and berry potassium accumulation

Potassium accumulation is essential for grapevine (Vitis vinifera L.) growth and development, but excessive levels in berries at harvest may reduce wine quality particularly for red wines. In addition to decreasing the free acid levels, potassium also combines with tartaric acid to form largely insoluble potassium bitartrate. This precipitates during winemaking and storage, resulting in an increase in wine pH that is associated with negative impacts on wine colour, flavour, and microbiological stability. For these reasons, a better understanding of potassium transport and accumulation within the vine and berries is important for producing fruit with improved winemaking characteristics. Here two genes encoding KUP/KT/HAK-type potassium transporters that are expressed in grape berries are described. Their function as potassium transporters was demonstrated by complementation of an Escherichia coli mutant. The two transporters are expressed most highly in the berry skin during the first phase of berry development (pre-veraison), with similar patterns in two grapevine varieties. The timing and location of expression of these transporters are consistent with an involvement in potassium accumulation in grape berries.     

A grape berry (Vitis vinifera L.) cation/proton antiporter is associated with berry ripening

We have cloned and characterized VvNHX1, a gene encoding a vacuolar cation/H(+) antiporter from Vitis vinifera cv. Cabernet Sauvignon. VvNHX1 belongs to the vacuolar NHX protein family and showed high similarity to other known vacuolar antiporters. The expression of VvNHX1 partially complements the salt- and hygromycin-sensitive phenotypes of an ena1-4 nhx1 yeast strain. Immunoblots of vacuoles of yeast expressing a VvNHX1, together with the expression of a VvNHX1-GFP (green fluorescent protein) chimera demonstrated that VvNHX1 localized to the vacuoles. VvNHX1 displayed low affinity K(+)/H(+) and Na(+)/H(+) exchange activities (12.8 and 40.2 mM, respectively). The high levels of expression of VvNHX1 during the véraison and post-véraison stages would indicate that the increase in vacuolar K(+) accumulation, mediated by VvNHX1, is needed for vacuolar expansion. This process, together with the rapid accumulation of reducing sugars, would drive water uptake to the berry and the concomitant berry size increase, typical of the post-véraison stage of growth.     

cDNA microarray analysis of developing grape (Vitis vinifera cv. Shiraz) berry skin

Microarray analysis of Vitis vinifera cv. Shiraz developing berries has revealed the expression patterns of several categories of genes. Microarray slides were constructed from 4,608 PCR-amplified cDNA clones derived from a ripening grape berry cDNA library. The mRNA expression levels of the genes represented by these cDNAs were measured in flowers, week 2 post-flowering whole berries, week 5, week 8, week 10 (véraison, green berries), week 12 and week 13 berry skin. In addition, a comparison of RNA expression in pigmented and unpigmented berry skin at véraison (week 10) was undertaken. Image and statistical analysis revealed four sets of genes with distinctive and similar expression profiles over the course of berry development. The first set was composed of genes which had maximum RNA expression in flowers, followed by a steady decrease in expression. The most prominent group within this set were genes which have a role in photosynthesis. The second set of cDNAs was dominated by genes involved in flavonoid biosynthesis and had a peak of expression week 2 post-flowering. The data indicate co-ordinate regulation of flavonoid biosynthetic genes which code for the enzymes 4-coumarate-CoA ligase, chalcone synthase, chalcone isomerase, flavonone hydroxylase, anthocyanidin reductase and cytochrome b5. The third set of cDNAs exhibited maximum expression week 5 post-flowering, midway between flowering and véraison, a period of rapid berry growth. This set of cDNAs is dominated by genes which code for structural cell wall proteins. The fourth set of genes was dramatically up-regulated at véraison and remained up-regulated until 13 weeks post-flowering. This set of genes was composed of a diverse range of genes, a reflection of the complexity of ripening, most with no known function.     

Structure and dynamics of reconstituted cuticular waxes of grape berry cuticle (Vitis vinifera L.)

Cuticular waxes from grape berry cuticle of Vitis vinifera L. have been investigated by X-ray diffraction, differential scanning calorimetry and gas chromatography. The waxes were mainly composed of n-alcohols and n-fatty acids and a considerable amount (about 30%) of the cyclic terpenoid oleanolic acid. The physical techniques used showed that the waxes have a high degree of molecular order in spite of the presence of the cyclic component. Molecular dynamics of the reconstituted waxes were measured to characterize the transport properties of the cuticular waxes that form the transport-limiting barrier of plant cuticles. For this purpose, the diffusion coefficient of labelled cholesterol, imitating the terpenoic acid, was measured. The value obtained was around 10^-21 m² s^-1 indicating a low mobility of the cyclic part of the reconstituted waxes. Temperature dependence of the diffusion coefficient was studied in the range of 5-45C. Arrhenius plot analysis yielded a high activation energy, 196.4 kJ mol^-1, of the diffusion process. This indicates dense molecular packing of reconstituted cuticular waxes.     

The genes and enzymes of the carotenoid metabolic pathway in Vitis vinifera L

ABSTRACT: BACKGROUND: Carotenoids are a heterogeneous group of plant isoprenoids primarily involved inphotosynthesis. In plants the cleavage of carotenoids leads to the formation of thephytohormones abscisic acid and strigolactone, and C13-norisoprenoids involved in thecharacteristic flavour and aroma compounds in flowers and fruits and are of specificimportance in the varietal character of grapes and wine. This work extends the previousreports of carotenoid gene expression and photosynthetic pigment analysis by providing anup-to-date pathway analysis and an important framework for the analysis of carotenoidmetabolic pathways in grapevine. RESULTS: Comparative genomics was used to identify 42 genes putatively involved in carotenoidbiosynthesis/catabolism in grapevine. The genes are distributed on 16 of the 19 chromosomesand have been localised to the physical map of the heterozygous ENTAV115 grapevinesequence. Nine of the genes occur as single copies whereas the rest of the carotenoidbiosynthetic genes have more than one paralogue. The cDNA copies of eleven correspondinggenes from Vitis vinifera L. cv. Pinotage were characterised, and four where shown to befunctional. Microarrays provided expression profiles of 39 accessions in the metabolicpathway during three berry developmental stages in Sauvignon blanc, whereas an optimisedHPLC analysis provided the concentrations of individual carotenoids. This provides evidenceof the functioning of the lutein epoxide cycle and their respective genes in grapevine.Similarly, orthologues of genes leading to the formation of strigolactone involved in shootbranching inhibition were identified: CCD7, CCD8 and MAX1. Moreover, the isoformstypically have different expression patterns, confirming the complex regulation of thepathway. Of particular interest is the expression pattern of the three VvNCEDs: Our resultssupport previous findings that VvNCED3 is likely the isoform linked to ABA content inberries. CONCLUSIONS: The carotenoid biosynthetic pathway is well characterised, and the genes and enzymes havebeen studied in a number of plants. The study of the 42 carotenoid pathway genes ofgrapevine showed that they share a high degree of similarity with other eudicots. Expressionand pigment profiling of developing berries provided insights into the most completegrapevine carotenoid pathway representation. This study represents an important referencestudy for further characterisation of carotenoid biosynthesis and catabolism in grapevine.     

A carotenoid cleavage dioxygenase from Vitis vinifera L.: functional characterization and expression during grape berry development in relation to C13-norisoprenoid accumulation

A potential Carotenoid Cleavage Dioxygenase (CCD) gene was identified among a Vitis vinifera L. EST collection and a full-length cDNA (VvCCD1) was isolated. Recombinant expression of VvCCD1 confirmed that the gene encoded a functional CCD. Experimental evidence was obtained that VvCCD1 cleaves zeaxanthin symmetrically yielding 3-hydroxy-beta-ionone, a C(13)-norisoprenoidic compound, and a C(14)-dialdehyde. Expression of the gene was studied by real-time PCR at different developmental stages of grape berries from Muscat of Alexandria and Shiraz cultivars. A significant induction of the gene expression approaching véraison was observed in both cultivars. In parallel, the C(13)-norisoprenoid level increased from véraison to maturity in both cultivars.     

Genetic mapping of grapevine ( Vitis vinifera L.) applied to the detection of QTLs for seedlessness and berry weight

Parental and consensus genetic maps of Vitis vinifera L. (2n = 38) were constructed using a F₁ progeny of 139 individuals from a cross between two partially seedless genotypes. The consensus map contained 301 markers [250 amplification fragment length polymorphisms (AFLPs), 44 simple sequence repeats (SSRs), three isozymes, two random amplified polymorphic DNAs (RAPDs), one sequence-characterized amplified region (SCAR), and one phenotypic marker, berry color] mapped onto 20 linkage groups, and covered 1,002 cM. The maternal map consisted of 157 markers covering 767 cM (22 groups). The paternal map consisted of 144 markers covering 816 cM (23 groups). Differences in recombination rates between these maps and another unpublished map are discussed. The major gene for berry color was mapped on both the paternal and consensus maps. Quantitative trait loci (QTLs) for several quantitative subtraits of seedlessness in 3 successive years were searched for, based on parental maps: berry weight, seed number, seed total fresh and dry weights, seed percent dry matter, and seed mean fresh and dry weights. QTLs with large effects (R² up to 51%) were detected for all traits and years at the same location on one linkage group, with some evidence for the existence of a second linked major QTL for some of them. For these major QTLs, differences in relative parental effects were observed between traits. Three QTLs with small effects (R² from 6% to 11%) were also found on three other linkage groups, for berry weight and seed number in a single year, and for seed dry matter in 2 different years.     

Genetic transformation of Vitis vinifera via organogenesis

Background  

Efficient transformation and regeneration methods are a priority for successful application of genetic engineering to vegetative propagated plants such as grape. The current methods for the production of transgenic grape plants are based on Agrobacterium-mediated transformation followed by regeneration from embryogenic callus. However, grape embryogenic calli are laborious to establish and the phenotype of the regenerated plants can be altered.     

Berry morphology and composition in irrigated and non-irrigated grapevine (Vitis vinifera L.)

The present study was carried out in a 5-year-old vineyard (Vitis vinifera L., cv. Aglianico) located in Southern Italy. Half of the plants (IRR) were fully irrigated, whereas the other half were not irrigated (NIRR). In both of the treatments, plant water status, gas exchange, photosynthetic efficiency and productive performance were determined. The arid conditions resulted in significant decreases in stem water potential in NIRR (minimum values of -1.34 and -1.52 MPa in IRR and NIRR, respectively). The values of yield per plant, cluster weight and total berry weight were significantly higher in IRR. Grape berries were separated into four weight classes, and morphometric and microscopic analyses were carried out to measure and calculate berry skin characteristics. Irrigation determined a marked shift toward heavier (+23% in the class ≥ 1.25 g) and bigger (336.35 mm3 vs 299.15 mm3) berries, and induced significant changes in other morphometric berry parameters. No differences among berry weight classes and irrigation treatments were observed for berry skin thickness. In all of the berry weight classes, total anthocyanins extracted from berry skins were significantly higher in NIRR than in IRR (12301.53 and 9585.52 mg kg?1 fresh berry skin, respectively), and appeared to be positively related to berry weight, whereas total flavonols were not significantly different between the two treatments. Qualitative changes in the levels of single anthocyanin and flavonol compounds were detected between IRR and NIRR. In addition, iron, copper and zinc, whose high concentration can negatively affect wine quality, were significantly higher in the IRR treatment. The results highlighted that the absence of irrigation did not determine decreases in grape quality. Such data can be of primary importance in environments where water availability is by far the most important limiting factor for plant growth.     

Sugars and Organic Acids of Vitis vinifera

Glucose, fructose, galactose, sucrose, maltose, melibiose, raffinose, and stachyose were identified in the leaves, bark, roots, and berries of Vitis vinifera L. var. Thompson Seedless. In addition to these sugars, verbascose and manninotriose were found in the leaves and bark.

Malic, tartaric, citric, isocitric, ascorbic, cis-aconitic, oxalic, glycolic, glyoxylic, succinic, lactic, glutaric, fumaric, pyrrolidone carboxylic, α-ketoglutaric, pyruvic, oxaloacetic, galacturonic, glucuronic, shikimic, quinic, chlorogenic, and caffeic acids were identified in the leaves, bark, roots, and berries.

Glucose, fructose, sucrose, malate, tartrate, and citrate were determined quantitatively in the leaf, petiole, xylem, bark, tendril, bud, puduncle pedicel, berry, lateral roots, and main roots at 4 separate physiological stages of growth. In addition, changes in the concentrations of fructose, glucose, malate, and tartrate in leaves were measured during a 36-day period starting from budburst.

     

Phases of Berry Growth in Vitis vinifera

The course of berry growth in Vitis vinifera has been interpreteddifferently by various authors. Divisions into two, three orfour phases have been postulated, though, in the latter cases,without any objective criteria for their delimitation. To clarifythis point, investigations were carried out with the cultivarsBacchus and Madeleine. Fresh and dry wt curves showed a double sigmoid course and threetransition points could be clearly defined. The central transitionpoint, occurring around 42 d after anthesis, may be definedas the change-over from the first to the second growth phase.Both the course of the fresh weight curve when plotted logarithmicallyand the relative growth rates argue against there being a phaseof slow growth at the beginning of the first growth phase. Indeed,the relative increase in fresh weight is maximal at the beginningof the first growth phase. The delimitation of a separate phase of little or no growthin the region of the transition from the first to the secondgrowth phase is not supported. The definitions of such phaseboundaries is arbitrary. The smaller the number of seeds perberry, the earlier is the first growth phase ended and the secondgrowth phase, including veraison, begun. During the first growth phase there is high cell division activity.The average area of an epidermal cell reached its minimum at8–11 d after anthesis, with a simultaneous strong increasein cell number. The maximal number of epidermal cells was attainedtowards the end of the first growth phase. The growth of the embryo showed no relation to the double-sigmoidalgrowth of the pericarp. Final embryo size was reached at 70–75d after anthesis. Seed d. wt, on the other hand, showed a biphasicincrease. The results presented here support the division of berry growthof V. vinifera into just two phases. Vitis vinifera L., grape vine, berry growth, growth phases     

Vitis vinifera in the Iberian Peninsula: A review

The origin of the Eurasian grapevine, Vitis vinifera L. and the different steps in the development of viticulture from the wild grapevine to the modern varietal situation in the Iberian Peninsula are reviewed here. The ancestors of Vitis vinifera subs. sylvestris, the only representative of the Vitis genus in natural Eurasian and North African ecosystems, appeared in the late Miocene. In the Iberian Peninsula, human action has significantly reduced the habitat of this species, whose oldest palaeobotanical remains go back to the late Pleistocene. The introduction of viticulture in Iberia is connected with the relationships between the indigenous population and the trading colonies founded by Phoenicians and Greeks. In this way, there is evidence of wine production and consumption, even as a ritual and prestige item, going back to the third millennium BP. Chloroplast genome studies carried out in wild and cultivated plants indicate that a secondary domestication process occurred in the Iberian Peninsula and this is reflected in the “A” chlorotype, which contrasts with the predominance of the “B” and “C” chlorotypes in wild and domesticated vines in the Transcaucasian region, where domesticated grapes at sites of the Shulaveri Culture (Georgia) are evidence of early viticulture around 8000 BP.     

Molecular linkage maps of Vitis vinifera L. and Vitis riparia Mchx

Two linkage maps for grape (Vitis spp.) have been developed based on 81 F(1) plants derived from an interspecific cross between the wine cultivar Moscato bianco (Vitis vinifera L.) and a Vitis riparia Mchx. accession, a donor of pathogen resistance traits. The double pseudotest-cross mapping strategy was applied using three types of molecular markers. The efficiency of SSRs to anchor homologous linkage groups from different Vitis maps and the usefulness of AFLPs in saturating molecular linkage maps were evaluated. Moreover, the SSCP technique was developed based on sequence information in public databases concerning genes involved in flavonoid and stilbene biosynthesis. For the maternal genetic map a total of 338 markers were assembled in 20 linkage groups covering 1,639 cM, whereas 429 loci defined the 19 linkage groups of the paternal map which covers 1,518 cM. The identification of 14 linkage groups common to both maps was possible based on 21 SSR and 19 AFLP loci. The position of SSR loci in the maps presented here was consistent with other published mapping experiments in Vitis.