Anthocyanin production in selected cell lines of grape (Vitis vinifera L.)

Summary Anthocyanin production of two lines ofVitis vinifera cell cultures, i.e., 5.4 and 13.1, which were obtained from the same starting material after 20 and 37 mo. of clonal selection, respectively, was investigated. Cell suspension cultures of lines 5.4 and 13.1 maintained an anthocyanin content of 0.44 ± 0.15 and 1.02 ± 0.31 mg·g⁻¹ fresh weight during 50 and 32 weekly maintenance subcultures, respectively. Under anthocyanin-promoting culture conditions, both lines showed an enhancement of their anthocyanin level by approximately fourfold. While line 5.4 accumulated peonidin 3-glucoside and cyanidin 3-glucoside in decreasing order, line 13.1 accumulated primarily peonidin 3-p-coumaroylglucoside with lesser amounts of malvidin monoglucoside. Results show that while the anthocyanin content was improved during the course of repeated selections, the anthocyanin composition was modified markedly favoring the accumulation of more metabolically-advanced anthocyanins.     

An NMR microscopic study of grape (Vitis vinifera L.)

Summary Mature healthy grape berries and berries wound-inoculated with the fungusBotrytis cinerea were examined by¹H NMR microimaging using 2D and 3D spin echo and gradient echo procedures. These NMR images were compared with representations obtained by conventional histology, where possible using the same specimens. 3D imaging datasets from excised seeds were reconstructed by surface rendering and maximum intensity projection to allow interpretation of their internal structure. T₂-weighted spin echo images revealed the major features of the pericarp, septum and loculi of whole berries. T₁-weighted images were less discriminatory of parenchyma tissues in the fruit but revealed the endosperm in seeds as a chemically shifted feature. A non-invasive study by T₁-weighted spin echo NMR imaging of infection byB. cinerea over a 6-day period showed that the disease spread throughout the exocarp but failed to spread in the mesocarp, a result confirmed by histological examination of the same specimen. Surface rendering of 3D datasets of excised seeds revealed the two ruminations of the endosperm and the distal location of the chalaza. The position of the embryonic axis was revealed in T₂-weighted maximum intensity projections. This noninvasive study revealed the need to apply a range of imaging techniques and parameters to visualise the structural features of the different parts of the grape berry.Abbrevations BF bright field - FDA fluorescein diacetate - FI field inhomogeneity - FOV field of view - NMR nuclear magnetic resonance - RF radiofrequency - T₁ spin-lattice relaxation time - T₂ spin-spin relaxation time - TE echo time - TMS tetramethylsilane - TR repeat time     

Recovery of bioactive compounds from white grape (Vitis vinifera L.) stems as potential antimicrobial agents for human health

The grape is a matrix rich in bioactive compounds and its production generates large quantities of by-products, such as grape stems, which, to date, present low commercial value. However, there is a growing interest in the application of this material as a source of phenolic compounds. Therefore, the present study aims at assessing the phytochemical profile of (poly)phenolic extracts of white Portuguese grape stem varieties produced in the Região Demarcada do Douro (Portugal). The antioxidant activity determined by several assays, as well as the antimicrobial activity using the disc diffusion method against human gastrointestinal pathogenic bacteria of the hydromethanolic extracts, were evaluated. This work presents very positive results as the rich composition in phenolic compounds (94.71–123.09 mg GA⁻¹ and 0.02–73.79 mg g⁻¹ for the total phenol content and for individual phenolics, respectively) presented by grape stems can explain the high antioxidant (0.37–1.17 mmol Trolox g⁻¹) and antimicrobial activities against, essentially, Gram-positive bacteria, and in some cases with higher efficacy than commercial antibiotics. Thus, demonstrating that this wine by-product should deserve greater attention from the pharmaceutical industries due to its excellent biological properties and characteristics not yet applied.     

Cellular expansion and gene expression in the developing grape (Vitis vinifera L.)

Summary. Expression profiles of genes involved in cell wall metabolism and water transport were compared with changes in grape (Vitis vinifera L.) berry growth, basic chemical composition, and the shape, size, and wall thickness of cells within tissues of the berry pericarp. Expression of cell wall-modifying and aquaporin genes in berry pericarp tissues generally followed a bimodal expression profile with high levels of expression coinciding with the two periods of rapid berry growth, stages I and III, and low levels of expression corresponding to the slow-growth period, stage II. Cellular expansion was observed throughout all tissues during stage I, and only mesocarp cellular expansion was observed during stage III. Expansion of only exocarp cells was evident during transition between stages II and III. Cell wall-modifying and aquaporin gene expression profiles followed similar trends in exocarp and mesocarp tissues throughout berry development, with the exception of the up-regulation of pectin methylesterase, pectate lyase, two aquaporin genes (AQ1 and AQ2), and two expansin genes (EXP3 and EXPL) during stage II, which was delayed in the exocarp tissue compared with mesocarp tissue. Exocarp endo-(1→3)-β-glucanase and expansin-like gene expression was concurrent with increases in epidermal and hypodermal cell wall thickness. These results indicate a potential role of the grape berry skin in modulating grape berry growth. Correspondence: P. Bowen, Pacific Agri-Food Research Centre, 4200 Highway 97, Summerland, BC V0H 1Z0, Canada     

Accumulation of peonidin 3-glucoside enhanced by osmotic stress in grape (Vitis vinifera L.) cell suspension

Cell cultures of grapes, Vitis vinifera L. cv Gamay Fréaux were grown under different conditions of external osmotic potential induced by an increase of sucrose concentration or by the addition of mannitol to the culture medium. Addition of 82 mM mannitol or increasing sucrose concentration to 132 mM had similar effects on repressing growth. Cyanidin 3-glucoside, peonidin 3-glucoside and peonidin 3-p-coumaroylglucoside are three main anthocyanins of Vitis cells. Increasing osmotic potential from –0.43 MPa to –0.8 MPa in the medium resulted in a significant intracellular accumulation of anthocyanin especially peonidin 3-glucoside in the pigmented cells. High osmotic potential appears to stimulate the methylation of anthocyanins. Osmotic potential is an important culture factor and may be useful in the controlling of anthocyanin production and composition.     

Factors affecting isolation of protoplasts from leaves of grape (Vitis vinifera)

A method of isolating grape mesophyll protoplasts was developed to facilitate the eventual use of genetic engineering techniques in this species. The effects of several factors influencing protoplast isolation could be evaluated quickly by using leaf disks 1 cm in diameter and known volumes of maceration and wash media. The best yields of mesophyll protoplasts were obtained using medium sized leaves of grapevines kept in the dark for 24 hours prior to maceration in 1% Cellulysin, 0.5% Macerase, 0.7 M mannitol, 5 ppm 2, 4 D, 0.1 ppm BAP, 1/10 strength Murashige and Skoog medium, and incubated at 22°C in cool-white fluorescent light (70–100 E m^-2 s^-1) for 24 hours. Over 30×10⁶ protoplasts per cm² of leaf were produced using these conditions. This method of screening factors affecting protoplast isolation could be applicable to other species.     

Action of UV and visible radiation on chlorophyll fluorescence from dark-adapted grape leaves (Vitis vinifera L.)

Grapevine plants (Vitis vinifera L. cv. Silvaner) were cultivated under shaded conditions in the absence of UV radiation in a greenhouse, and subsequently placed outdoors under filters transmitting natural radiation, or screening out the UV-B (280 to 315 nm), or screening out the UV-A (315 to 400 nm) and the UV-B spectral range. All conditions decreased maximum chlorophyll fluorescence (F_M) and increased minimum chlorophyll fluorescence (F₀) from dark-adapted leaves; however, with increasing UV, F_M quenching was stimulated but increases in F₀ were reduced. The F_V/F_M ratio (where F_V=F_M-F₀) was clearly reduced by visible radiation (VIS): UV-B caused a moderate extra-reduction in F_V/F_M. Exposure of leaves (V. vinifera L. cv. Bacchus) to UV or VIS lamps quenched the F_M to similar extents; further, UV-B doses comparable to the field, quenched F₀. A model was developed to describe how natural radiation intensities affect PS II and thereby change leaf fluorescence. Fitting theory to experiment was successful when the same F_M yield for UV- and VIS-inactivated PS II was assumed, and for lower F₀ yields of UV- than for VIS-inactivated PS II. It is deduced, that natural UV can produce inactivated PS II exhibiting relatively high F_V/F_M. The presence of UV-inactivated PS II is difficult to detect by measuring F_V/F_M in leaves. Hence, relative concentrations of intact PS II during outdoor exposure were derived from F_M. These concentrations, but not F_V/F_M, correlated reasonably well with CO₂ gas exchange measurements. Consequently, PS II inhibition by natural UV could be a main factor for UV inhibition of photosynthesis.This revised version was published online in October 2005 with corrections to the Cover Date.     

Phenology of flowering and starch accumulation in grape (Vitis vinifera L.) cuttings and vines

BACKGROUND AND AIMS: A reliable protocol for flowering and fruiting in cuttings was developed with the aim of (a) studying inflorescence and flower development in grapevine cuttings and field plants, and (b) assisting haploid plant production. METHODS: Inflorescence and flower development was studied in 'Gewurztraminer' (GW) and 'Pinot Noir' (PN) grape vines and cuttings grown in a glasshouse, along with variations in starch in the flowers. As there is a strong relationship between flower development and starch, the starch content of reproductive structures was estimated. KEY RESULTS: Inflorescence and flower development were similar in the vines and cuttings with consistent differences between the two cultivars. Indeed, the ontogenesis of male and female organs is not synchronous in GW and PN, with both female and male meiosis occurring earlier in PN than in GW. Moreover, changes of starch reserves were similar in the two plant types. CONCLUSIONS: Cuttings have a similar reproductive physiology to vines, and can be used to study grape physiology and to develop haploid plants.     

Regeneration of plants from embryogenic callus-derived protoplasts of Garganega and Sangiovese grapevine (Vitis vinifera L.) cultivars

Plant Cell, Tissue and Organ Culture (PCTOC) - Tissue culture systems have contributed to the massive propagation of bamboos. However, micro-propagated plants show a low percentage of rooting and...     

Transformation of grape (Vitis vinifera L.) zygotic-derived somatic embryos and regeneration of transgenic plants

Summary Transgenic grape plants were regenerated from somatic embryos derived from immature zygotic embryos of seedless grape (Vitis vinifera L.) selections. Somatic embryos were bombarded twice with 1 m gold particles using the Biolistic PDS-1000/He device (Bio-Rad Laboratories) and then exposed to Agrobacterium tumefaciens strain C58/Z707 containing the binary plasmid pGA482GG or pCGN7314. Following cocultivation, secondary embryos were allowed to proliferate on Emershad/Ramming proliferation (ERP) medium for 6 weeks before selection on ERP medium containing 20–40 g/ml kanamycin (kan). Transgenic embryos were identified after 3–5 months under selection and allowed to germinate and develop into rooted plants on Woody Plant Medium containing 1 M 6-benzylaminopurine (BAP), 1.5% sucrose, 0.3% activated charcoal and 0.75% agar. Integration of the foreign genes into these grapevines was verified by growth in the presence of kan, positive GUS and PCR assays, and Southern analysis.     

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.     

Influence of constitutive phenolic compounds on the response of grapevine (Vitis vinifera L.) leaves to infection by Plasmopara viticola

Flavonols and hydroxycinnamic acids are known to contribute to plant resistance against pathogens, but there are few reports on the implication of flavonols in the resistance of grapevine against Plasmopara viticola, and none on the involvement of hydroxycinnamic acids. In order to analyze the effect of flavonols on P. viticola infection, variable amounts of flavonols were induced by different light conditions in otherwise phenologically identical leaves. Differences in content of leaf hydroxycinnamic acids were induced at the same time. A non-invasive monitoring of flavonols and hydroxycinnamic acids was performed with Dualex leaf-clip optical sensors. Whatever the light condition, there were no significant changes in flavonol or in hydroxycinnamic acid contents for control and inoculated leaves during the development of P. viticola until 6 days after inoculation. The violet-blue autofluorescence of stilbenes, the main phytoalexins of grapevine that accumulate in inoculated leaves, was used as an indicator of infection by P. viticola. The implication of leaf constitutive flavonols and hydroxycinnamic acids in the defence of Vitis vinifera against P. viticola could be investigated in vivo thanks to this indicator. The increase in stilbene violet-blue autofluorescence started earlier for leaves with low flavonol content than for leaves with higher content, suggesting that constitutive flavonols are able to slow down the infection by P. viticola. On the contrary, constitutive hydroxycinnamic acids did not seem to play a role in defence against P. viticola. The non-destructive nature of the methods used alleviates the major problem of destructive experiments: the large variability in leaf phenolic contents.     

葡萄胚胎发育与败育过程中胚珠的多胺含量变化

花后40~60d期间,种子败育型无核葡萄胚珠的3种多胺含量明显低于有核葡萄,据此认为无核葡萄胚珠中多胺含量急剧下降和较低的多胺水平可能是导致葡萄胚胎败育的重要原因。(Spd Spm)/Put和Spm/PAs比值变化与葡萄胚胎发育密切相关,较低的比值不利于胚胎的正常分化。     

一种测定葡萄木质部压力梯度的新方法

通过同时测定葡萄木质部导管液流平均流量和流速,应用变形的哈根-泊萧叶方程,研究了测定葡萄木质部压力梯度的新方法。结果表明,在直径分别为7mm的葡萄茎段和4mm的葡萄叶柄上,用此新方法测得的木质部压力梯度与实际加在其上的压力梯度吻合的很好,说明此新方法可用于连续、实时测定葡萄木质部压力梯度。用此新方法在离体葡萄枝上测定发现,随光照强度增强,其木质部压力梯度成线性正相关增加。     

Stabilization of erythrocytes against oxidative and hypotonic stress by tannins isolated from sumac leaves (Rhus typhina L.) and grape seeds (Vitis vinifera L.)

Erythrocytes are constantly exposed to ROS due to their function in the organism. High tension of oxygen, presence of hemoglobin iron and high concentration of polyunsaturated fatty acids in membrane make erythrocytes especially susceptible to oxidative stress. A comparison of the antioxidant activities of polyphenol-rich plant extracts containing hydrolysable tannins from sumac leaves (Rhus typhina L.) and condensed tannins from grape seeds (Vitis vinifera L.) showed that at the 5-50 μg/ml concentration range they reduced to the same extent hemolysis and glutathione, lipid and hemoglobin oxidation induced by erythrocyte treatment with 400 μM ONOO(-) or 1 mM HClO. However, extract (condensed tannins) from grape seeds in comparison with extract (hydrolysable tannins) from sumac leaves stabilized erythrocytes in hypotonic NaCl solutions weakly. Our data indicate that both hydrolysable and condensed tannins significantly decrease the fluidity of the surface of erythrocyte membranes but the effect of hydrolysable ones was more profound. In conclusion, our results indicate that extracts from sumac leaves (hydrolysable tannins) and grape seeds (condensed tannins) are very effective protectors against oxidative damage in erythrocytes.     

Direct shoot organogenesis and plant regeneration from leaves of grape (Vitis spp.)

Adventitious shoots developed from in vitro-grown leaves of Vitis vinifera cultivars Cabernet Sauvignon, French Colombard, Grenache, Thompson Seedless (syn. Sultana) and White Riesling, V. rupestris cv. St. George (syn. du Lot) and V. vinifera × rupestris cv. Ganzin 1. Leaf explants less than 15 mm long were excised from nodal cultures and cultured on Murashige and Skoog or Nitsch and Nitsch-based regeneration media with 0, 1, 2 or 4 mgl^-1 6-benzylaminopurine (BAP). Adventitious shoots developed within 4 weeks at the petiolar stub and occasionally from wounded lamina tissues. Shoot organogenesis occurred only on media containing BAP and at a higher frequency with 2 mgl^-1 than with 1 or 4 mgl^-1. On media containing 2 mgl^-1 BAP, 47, 67, 60, and 42%, respectively, of leaf explants of Cabernet Sauvignon, French Colombard, Thompson Seedless, and White Riesling produced adventitious shoots compared to 14, 14, and 29%, respectively, for Grenache, St. George, and Ganzin 1. Solid culture medium was superior to liquid medium and transfer frequency on solid medium did not affect the regeneration frequency. Further shoot growth was promoted by the transfer of regenerating tissues to fresh regeneration medium. More than 80% of explants initially producing adventitious buds exhibited further shoot growth, developing an average of more than 6 shoots each. Shoots rooted easily and the resulting plants appeared morphologically identical to parent vines.     

Phenolics and antioxidant activity in different cultivars/clones of Vitis vinifera L. seeds over two years

Grape seeds (GS) are one of the most important by-products of the winery and grape juice industry. GS contain a vast array of health-giving metabolites, including several polyphenols known for their remarkable antioxidant activity. In this study, it has been evaluated the nutraceutical potential of seeds from 15 grapevine cultivars/clones in terms of total phenols, monomeric, dimer, polymeric, gallate esters flavan 3-ols composition and in vitro antiradical activity. Considerable quali-quantitative differences in polyphenol content and AA had been observed among GS from different cultivars/clones collected in 2013 and 2014. In most of the investigated cultivars/clones, the better values of TP, FL and AA were recorded in GS collected in 2014. The polymeric procyanidins were significantly higher in 2014 in all the cultivars, while the gallate esters were significantly higher in 2013. These differences can be attributed to different climatic conditions in 2013 and 2014. In conclusion, the data here reported help to identify the best source of GS for the recovery of bioactive polyphenols and to valorize these waste materials.     

Isolation and characterization of cell walls from the mesocarp of mature grape berries (Vitis vinifera)

Cell walls have been isolated from the mesocarp of mature grape (Vitis vinifera L.) berries. Tissue homogenates were suspended in 80% (v/v) ethanol to minimise the loss of water-soluble wall components and wet-sieved on nylon mesh to remove cytoplasmic material. The cell wall fragments retained on the sieve were subsequently treated with buffered phenol at pH 7.0, to inactivate any wall-bound enzymes and to dislodge small amounts of cytoplasmic proteins that adhered to the walls. Finally, the wall preparation was washed with chloroform/methanol (1:1, v/v) to remove lipids and dried by solvent exchange. Scanning electron microscopy showed that the wall preparation was essentially free of vascular tissue and adventitious protein of cytoplasmic origin. Compositional analysis showed that the walls consisted of approximately 90% by weight of polysaccharide and less than 10% protein. The protein component of the walls was shown to be rich in arginine and hydroxyproline residues. Cellulose and polygalacturonans were the major constituents, and each accounted for 30–40% by weight of the polysaccharide component of the walls. Substantial varietal differences were observed in the relative abundance of these two polysaccharides. Xyloglucans constituted approximately 10% of the polysaccharide fraction and the remainder was made up of smaller amounts of mannans, heteroxylans, arabinans and galactans. Received: 26 November 1996 / Accepted: 30 January 1997     

Accumulation of anthocyanins enhanced by a high osmotic potential in grape (Vitis vinifera L.) cell suspensions

A cell suspension of grape, Vitis vinifera L. cv Gamay Fréaux, was grown under different conditions of water stress (high external osmotic potential) induced by an increase of sucrose concentration or by the addition of mannitol to the culture medium. Best growth (cell density) was achieved in the low osmotic potential medium. Increasing the osmotic potential of the medium from –0.5 MPa to –0.9 MPa medium resulted in a significant increase in accumulation of anthocyanins in pigmented cells. Regulation of the osmotic potential of culture medium may be useful in controlling anthocyanin production.