Control of Flowering in the Grapevine (Vitis vinifera L.): Formation of Inflorescences in Vitro by Isolated Tendrils

Photosynthesis, primary productivity, N content, and N₂ fixation were determined as a function of applied NH₄⁺ in peas (Pisum sativum L. cv. Alaska) which were inoculated or not inoculated with Rhizobium leguminosarum. Cabon dioxide exchange rate (CER) increased 10-fold, total N content 7-fold, and total dry weight 3-fold in 26-day-old uninoculated plants as applied NH₄⁺ was increased from 0 to 16 millimolar. In inoculated plants of the same age CER and dry weight were maximal at 2 millimolar NH₄⁺, and total N content increased between 0 and 2 millimolar NH₄⁺ but did not change significantly with higher NH₄⁺ applications. Per cent N content of uninoculated plants was significantly lower than that of inoculated plants except at the highest NH₄⁺ concentration (16 millimolar). Symbiotic N₂ fixation by inoculated plants was maximal in peas grown with 2 millimolar NH₄⁺; and apparent relative efficiency of N₂ fixation, calculated from C₂H₂ reduction and H₂ evolution, was maximal in the 2 to 4 millimolar NH₄⁺ concentration range. The capacity to fix N₂ through the Rhizobium-legume symbiosis significantly enhanced the rate and efficiency of photosynthesis and plant N content when NH₄⁺ concentration in the nutrient solution was below 8 millimolar. Above 8 millimolar NH₄⁺ concentration uninoculated plants had greater CER, N content, and dry weight.     

Glucose Transport in Vitis vinifera L. Protoplasts

Transport of glucose and its analogues was studied in grapevine(Viris vinfera L. cv. Soultanina) leaf protoplasts. The transportsystem was hexose specific and the stereospecificity was closelyrelated to carbon-1 of the glucose molecule. Glucose structuralanalogues were not metabolized beyond the stage of phosphorylationand differences between these compounds and glucose were observedin their transport rates and in their specificity for the carrier.Concentration-dependent uptake of labelled glucose by grapevineprotoplasts was linear for concentrations higher than 1?5 molm^–3 at lower concentrations a saturating pattern was observed.The carrier was driven by the proton motive force and the substrateentered the cell probably in an unchanged form. Efflux studieswere not useful as an indication of the rate of metabolism orassimilation of transported compounds in grapevine protoplasts. Key words: Sugar transport, protoplasts, grapevine     

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.     

Morphogenetic Effects of Roots and of Some Synthetic Cytokinins in Vitis vinifera L.

In woody cuttings of the grape vine, inflorescences of fertilebuds usually fail to develop and atrophy soon after bud burst.Results presented here indicate that this effect is relatedto absence of roots at planting. Inflorescences were retainedin pre-rooted cuttings which were propagated by holding thetemperature of the medium at 25° C and the air temperatureat 4° C. Similar effects on inflorescence growth were obtainedby applying synthetic cytokinins to the bases of unrooted cuttingsin solution cultures, and by applications to emergent inflorescences.Promotion of inflorescence growth was found with 6-benzyl-aminopurine(BAP) and 6-(benzylamino)-9-(2-tetrahydropyranyl)-9H purine(‘SD 8₃₃₉’). Stimulation of inflorescence growthby BAP was accompanied by reduction in vegetative growth, andby development of red pigments in inflorescences and leaves.Results of cincturing experiments indicate that BAP is transportedacropetally in the xylem of vines. Effects of roots, and effectsof synthetic cytokinins, are discussed in relation to recentdiscoveries of endogenous cytokinins in the ascending sap ofvines.     

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     

Effect of Root Temperature on Cytokinin Activity in Root Exudate of Vitis vinifera L

Root exudates of plants of Vitis vinifera L. cv. Thompson Seedless, grown in nutrient cultures with root temperatures maintained at either 20° or 30° and with shoots at a common air temperature, were assayed for cytokinin activity. After chromatography of freeze-dried sap on paper with n-butanol/acetic acid/water (4:1:1). activity was detected with a soybean callus assay. For both root temperatures, major activity appeared between R_F 0.6 and R_F 0.8, at about the same concentration in each case. The major difference between the 2 samples was the presence of activity at R_F 0.1 to 0.2 in the 20° sample and its absence in the 30° sample.

The higher root temperature resulted in increased shoot and root elongation, increased dry matter accumulation by both shoots and roots, and also altered the morphological appearance of the roots.

     

Linkage disequilibrium in cultivated grapevine, Vitis vinifera L

We present here the first study of linkage disequilibrium (LD) in cultivated grapevine, Vitis vinifera L. subsp. vinifera (sativa), an outcrossing highly heterozygous perennial species. Our goal was to characterize the amount and pattern of LD at the scale of a few centiMorgans (cM) between 38 microsatellite loci located on five linkage groups, in order to assess its origin and potential applications. We used a core collection of 141 cultivars representing the diversity of the cultivated compartment. LD was evaluated with both independence tests and multilocus r ² , both on raw genotypic and reconstructed haplotypic data. Significant genotypic LD was found only within linkage groups, extending up to 16.8 cM. It appeared not to be influenced by the weak structure of the sample and seemed to be mainly of haplotypic origin. Significant haplotypic LD was found over 30 cM. Both genotypic and haplotypic r ² values declined to around 0.1 within 5–10 cM, suggesting a rather narrow genetic base of the cultivated compartment and limited recombination since domestication events. These first results open up a few application opportunities for association mapping of QTLs and marker assisted selection. Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

The Relationship Between the Effects of CCC on Root Growth and Cytokinin Levels in the Bleeding Sap of Vitis vinifera L.

The growth retardant (2-chloroethyl)trimethylammonium chloride(CCC) induced swollen root tips on seedlings of grape vines(Vilis vinifera L.) as well as on plants grown from cuttings.In both cases CCC had to be applied to the growth medium forthe response to be expressed; spraying the shoots reduced stemgrowth without inducing swollen roots. N-dimethylarminosuccinamicacid (B995) was ineffective in causing these root symptoms ongrapes, nor did root swellings appear on five other speciestreated with CCC. On the other hand, kinetin resulted in graperoots which in some respects resembled those treated with CCC. Two regions of cytokinin activity were detected on chromatogramsof bleeding sap from grape-vines grown in serated nutrientculture solutions compared with only one region in sap fromplants grown in soil or other solid media. Activity of a regionof high mobility was increased by CCC applications to the culturesolutions; effects of CCC on a region of low mobility, whichmay be a bound derivative of the other, were variable. Boththe concentration and absolute amounts of cytokinin activityin the sap were increased by CCC, indicating that this retardantprobably affected cytokinin synthesis by the root tip. The effectof CCC on cytokinin levels in the sap diminished during thebleeding period. The results are interpreted to indicate that in grapes, CCCacts directly on the root meristem to increase cytokinin production.Swelling of the tips is probably a consequence of the elevatedcytokinin levels in this region.     

Mechanism of Arginine Transport in Vitis vinifera L. Protoplasts

Protoplasts were isolated from leaves of in vitro grown axenicshoots of grapevine (Vitis vinifera L. cv. Soultanina) and usedto study the characteristics of arginine transport. Uptake waslinear up to at least 60 min and the rate did not differ significantlybetween light and dark assaying conditions whereas incubationin darkness for 24 h caused a 70% reduction in uptake rate,which was probably not due to an energy dependent factor. Kineticsanalysis revealed a biphasic uptake curve. The high affinitycomponent had a K_m, of 2.2 mol m^–3. Optimum pH value was5.5. Two carrier systems, one for basic and neutral and onefor acidic amino acids were identified. Use of inhibitors revealedthat those associated with ATP metabolism inhibited arginineuptake; more specifically, the proton motive force appearedto be the predominant energy source. Metabolic products of labelledarginine were consistent with the operation of the Krebs-Henseleitcycle. Key words: Grapevine protoplast, grapevine tissue culture, arginine transport     

Infection of Vitis vinifera (cv Chardonnay) Inflorescences by Colletotrichum acutatum and Greeneria uvicola

Detached Vitis vinifera (cv Chardonnay) inflorescences were inoculated with spore suspensions of either Colletotrichum acutatum or Greeneria uvicola at 25°C, and a combination of light microscopy, scanning electron microscopy and plating out of inoculated flowers on to dichloran rose bengal chloramphenicol agar was used to investigate the time frame for infection. Colletotrichum acutatum infection commenced within 2 h of inoculation, while infection by G. uvicola commenced between 12 and 18 h postinoculation. All parts of the flowers were infected by both fungi.     

Genetic structure and differentiation in cultivated grape,Vitis vinifera L

222 cultivated (Vitis vinifera) and 22 wild (V. vinifera ssp. sylvestris) grape accessions were analysed for genetic diversity and differentiation at eight microsatellite loci. A total of 94 alleles were detected, with extensive polymorphism among the accessions. Multivariate relationships among accessions revealed 16 genetic groups structured into three clusters, supporting the classical eco-geographic grouping of grape cultivars: occidentalis, pontica and orientalis. French cultivars appeared to be distinct and showed close affinity to the wild progenitor, ssp. sylvestris from south-western France (Pyrenees) and Tunisia, probably reflecting the origin and domestication history of many of the old wine cultivars from France. There was appreciable level of differentiation between table and wine grape cultivars, and the Muscat types were somewhat distinct within the wine grapes. Contingency chi2 analysis indicated significant heterogeneity in allele frequencies among groups at all loci. The observed heterozygosities for different groups ranged from 0.625 to 0.9 with an overall average of 0.771. Genetic relationships among groups suggested hierarchical differentiation within cultivated grape. The gene diversity analysis indicated narrow divergence among groups and that most variation was found within groups (approximately 85%). Partitioning of diversity suggested that the remaining variation is somewhat structured hierarchically at different levels of differentiation. The overall organization of genetic diversity suggests that the germplasm of cultivated grape represents a single complex gene pool and that its structure is determined by strong artificial selection and a vegetative mode of reproduction.     

Restriction fragment length polymorphism and molecular taxonomy in Vitis vinifera L.

Forty-six accessions of grapevine (V. vinifera L.) were compared by restriction fragment length polmorphism (RFLP) analysis, and 111 informative or unique restriction fragments were found that revealed an important level of polymorphism. RFLP patterns were compared in two ways: by calculating electrophoretic similarity degree values further analyzed by principal component analysis and by studying the distribution of rare restriction fragments. Six taxonomic groups could be defined, which partially confirmed relationships derived from ampelographical data. Our data support the existence of ecogeographical groups.     

Cloning and characterization of Vine-1, a LTR-retrotransposon-like element in Vitis vinifera L., and other Vitis species.

We report the organization of a grapevine chimeric gene Adhr-Vine-1, composed by an Adhr gene, into which a retroelement, Vine-1, was inserted. Sequence analysis revealed that Adhr is a member of the Adh multigene family, but does not correspond to any other grapevine Adh described to date. Vine-1, albeit defective, is the most complete LTR (long terminal repeat)-retrotransposon-like element described in Vitis vinifera L. It is 2392 bp long, with two almost identical LTRs (287 bp) in the same orientation, and flanked by direct repeats of a 5 bp host DNA. This element presents other features, characteristic of retroviruses and retrotransposons including inverted repeats, a primer binding site, and a polypurine tract. It has a single open reading frame (ORF) of 581 amino acids, potentially encoding for a gag protein and parts of the protease and integrase proteins. Vine-1 is most likely related to the copia-like type family, but with no significant similarity to any previously described plant retrotransposon or inserted element, nor to any eukaryotic element described to date. Vine-1 element has been found in Adhr at the same location in different V. vinifera cultivars, but not in some other analyzed Vitis species. These data suggest that Vine-1 insertion in Adhr is specific to V. vinifera, and has occurred after the Adh isogene separation, but prior to cultivar development. Sequences related to Vine-1 were revealed in multiple copies in the V. vinifera genome and, to a lesser extent, in other analyzed Vitis species. The polymorphism observed prompts us to question the role played by transposition in the evolution of the Vitis genus.     

Anthocyanin and flavonol profiles of Vitis vinifera L. cv Rufete grapes

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Induction of somatic embryogenesis and plantlets in tendrils of Vitis vinifera L.

Somatic embryogenesis was observed in callus initiated from tendril explants of Vitis vinifera L. cvs. Thompson, Sonaka and Tas-e-Ganesh on Emershad and Ramming medium supplemented with 1 μm 6-benzylaminopurine. Low-frequency conversion to shoots was obtained in the third and fourth subculture on the same medium. Emerging shoots subsequently formed complete plantlets on liquid rooting medium containing 1 μm indole-3-acetic acid. The possible use of tendrils as a novel explant for somatic embryogenesis in grape is discussed. Received: 3 March 1997 / Revision received: 21 May 1997 / Accepted: 25 June 1997     

Effect of CCC on the growth of roots of Vitis vinifera L.

Summary When plants of Vitis vinifera L. were grown in solution cultures containing (2-chloroethyl)trimethylammonium chloride (CCC), roots became shorter and thicker than those of untreated control plants. The effect of CCC on vine roots could be counteracted by gibberellic acid simultaneously applied either to the solution cultures or to aerial organs of the plant.     

Regulation of Inflorescence Growth in Cuttings of the Grape Vine (Vitis vinifera L.)

In woody cuttings of the grape vine inflorescences of fertilebuds usually atrophy soon after bud burst. Inflorescences areretained if leaves are removed from the elongating shoot, butremoval of apices and roots does not affect inflorescence growthif leaves are present. With defoliated cuttings, inflorescencegrowth is stimulated by removal of apices but depressed by removalof roots. Applications of indole acetic acid to the petiolestumps of defoliated cuttings did not replace the effect ofleaves and induce atrophy of inflorescences. Inflorescence growthin defoliated cuttings was inhibited by 2,4-dichlorophenoxyaceticacid (2,4-D), but concentrations which depressed inflorescencegrowth produced toxicity symptoms. Applications of 2,3,5-tri-iodobenzoicacid to petioles and leaf laminae were without effect on inflorescencegrowth. Treatment of inflorescences with the cytokinin 6-(benzylamino)-9-(2-tetrahydropyranyl)-9H-purine(SD 8₃₃₉) promoted inflorescence growth in both the presenceand the absence of leaves. Inhibitory effects of 2,4-D on inflorescencegrowth were partially reversible by application of SD 8₃₃₉.Response of inflorescences to gibberellic acid required eitherremoval of leaves or addition of SD 8₃₃₉.     

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.