Purification and Characterization of a O-Methyltransferase Capable of Methylating 2-Hydroxy-3-alkylpyrazine from Vitis vinifera L. (cv. Cabernet Sauvignon)

An S-adenosyl-L-methionine-dependent O-methyl-transferase capable of methylating 2-hydroxy-3-alkyl-pyrazine (HP) was purified 7,300-fold to apparent homogeneity with an 8.2% overall recovery from Vitis vinifera L. (cv. Cabernet Sauvignon) through a purification procedure including column chromatography on DEAE-Sepharose FF, Ether-5PW, hydroxyapatite, G2000SWXL, and DEAE-5PW. The relative molecular mass of the native enzyme estimated on gel permeation chromatography was 85 kDa, and the subunit molecular mass was estimated to be 41 kDa on SDS-polyacrylamide gel electrophoresis. The enzyme also methylates caffeic acid. The V_max for IBHP and caffeic acid were 0.73 and 175 pkatals/mg, respectively, and the respective K_m for IBHP and caffeic acid were 0.30 and 0.032 mM. The optimum pH for IBHP (8.5) was different from that for caffeic acid (7.5).     

Proteomic analysis reveals differences between Vitis vinifera L. cv. Chardonnay and cv. Cabernet Sauvignon and their responses to water deficit and salinity

The impact of water deficit and salt stress on two important wine grape cultivars, Chardonnay and Cabernet Sauvignon, was investigated. Plants were exposed to increasing salinity and water deficit stress over a 16 d time period. Measurements of stem water potentials, and shoot and leaf lengths indicated that Chardonnay was more tolerant to these stresses than Cabernet Sauvignon. Shoot tips were harvested every 8 d for proteomic analysis using a trichloroacetic acid/acetone extraction protocol and two-dimensional gel electrophoresis. Proteins were stained with Coomassie Brilliant Blue, quantified, and then 191 unique proteins were identified using matrix-assisted laser desorption ionization time of flight/time of flight mass spectrometry. Peptide sequences were matched against both the NCBI nr and TIGR Vitis expressed sequence tag (EST) databases that had been implemented with all public Vitis sequences. Approximately 44% of the protein isoforms could be identified. Analysis of variance indicated that varietal difference was the main source of protein expression variation (40%). In stressed plants, reduction of the amount of proteins involved with photosynthesis, protein synthesis, and protein destination was correlated with the inhibition of shoot elongation. Many of the proteins up-regulated in Chardonnay were of unclassified or of unknown function, whereas proteins specifically up-regulated in Cabernet Sauvignon were involved in protein metabolism.     

Potassium nutrition of Cabernet Sauvignon grapevines (Vitis vinifera L.) as affected by shoot trimming

As potassium (K) requirement of grapevine (Vitis viniferaL.) berries is high and phloem translocation from mature leaves to developing organs is well established, it was posited that shoot trimming, a widely applied technique which alters the source-sink balance and the mature-to-immature foliage ratio of a canopy, may influence K deficiency. Six-year-old Cabernet Sauvignon grapevines were grown in 0.045 m³ pots (one plant per pot) filled with a soil sampled from a vineyard previously displaying K deficiency symptoms. Two levels of K supply (K₀, no K added; K_1, 25 g K per pot added in five splits from bloom to post veraison) were tested on vines that for each level were left (a) untrimmed and trimmed at ten main leaves with (b) or without (c) maintenance of lateral shoots in a split-plot design. Potassium concentration of leaf blades, berries and canes, vegetative growth and leaf gas exchange were recorded throughout the season; yield, grape quality and must characteristics were determined at fruit maturity. While adding potassium to the soil resulted in higher K concentration in blades of both main and lateral shoots and berries at harvest, trimming with removal of lateral shoots resulted in lower blade K concentration of the main leaves at harvest and more severe K deficiency symptoms regardless of K soil availability. Berry K concentration and the fraction of whole-plant K partitioned to clusters were not significantly affected by trimming. Gas-exchange, vegetative growth, yield and grape quality were not affected by the seasonal K fertilization, whereas the latter was impaired when trimming with excision of lateral shoots was applied. The results indicate that if shoot trimming is not followed by an adequate regrowth of secondary shoots an excessive depletion of potassium from the retained old basal leaves occurs during fruit maturity and increases the risk of leaf K deficiency, particularly in the K₀ treatment.     

高温胁迫下不同光强对‘赤霞珠’葡萄PSII活性及恢复的影响

本文研究了高温与不同光强结合处理对‘赤霞珠’葡萄叶片PSII活性及恢复的影响。结果表明,高温黑暗处理（40℃,0μmaol·m-2．s-1）导致叶片PSII最大光化学效率（Fv/Fm）、反应中心吸收的光能用于电子传递的量子产额（ψEo）与单位反应中心光能的传递（ETo/RC）降低明显,且无恢复趋势,K点相对荧光（Vk）、单位反应中心光能的吸收（ABS／RC）与捕获（TRo／RC）显著升高。高温弱光处理（40℃,200μmol·m-2．s-1）后的叶片PSII活性明显恢复,ETo／RC降低明显,TRo/RC无显著变化。高温强光（40℃,1600μmol·m-2．S-1）处理导致单位面积有活性反应中心数量（RC/CSm）抑制程度最大,恢复程度较低。实验结果说明,高温处理下黑暗对葡萄PSII功能活性及恢复均会造成抑制,而弱光可以显著缓解高温对葡萄叶片的胁迫作用,并促进PSII的恢复,强光导致胁迫下的PSII功能抑制最明显。     

Purification and Characterization of Geranyl Diphosphate Synthase from Vitis vinifera L. cv Muscat de Frontignan Cell Cultures

A geranyl diphosphate synthase (EC 2.5.1.1), which catalyzes the formation of geranyl diphosphate from dimethylallyl diphosphate and isopentenyl diphosphate, was isolated from Vitis vinifera L. cv Muscat de Frontignan cell cultures. Purification of the enzyme was achieved successively by ammonium sulfate precipitation and chromatography on DEAE-Sephacel, hydroxylapatite, Mono Q, Phenyl Superose, Superose 12, and preparative nondenaturing polyacrylamide gels. The enzyme formed only geranyl diphosphate as a product. In all cases, neither neryl diphosphate, the cis isomer, nor farnesyl diphosphate was detected. The enzyme showed a native molecular mass of 68 [plus or minus] 5 kD as determined by gel permeation. On sodium dodecyl sulfate polyacrylamide gels, geranyl diphosphate synthase purified to electrophoretic homogeneity migrated with a molecular mass of 66 [plus or minus] 2 kD. Michaelis constants for isopentenyl diphosphate and dimethylallyl diphosphate were 8.5 and 56.8 [mu]M, respectively. The enzyme required Mn2+ and Mg2+ as cofactors and its activity was enhanced by Triton X-100. Inorganic pyrophosphate, aminophenylethyl diphosphate, and geranyl diphosphate had inhibitory effects on the enzyme.     

Changes in protein abundance during powdery mildew infection of leaf tissues of Cabernet Sauvignon grapevine (Vitis vinifera L.)

A comparative analysis of differentially expressed proteins in a susceptible grapevine (Vitis vinifera ‘Cabernet Sauvignon’) during the infection of Erysiphe necator, the causal pathogen of grapevine powdery mildew (PM), was conducted using iTRAQ. The quantitative labeling analysis revealed 63 proteins that significantly changed in abundance at 24, 36, 48, and 72 h post inoculation with powdery mildew conidiospores. The functional classification of the PM‐responsive proteins showed that they are involved in photosynthesis, metabolism, disease/defense, protein destination, and protein synthesis. A number of the proteins induced in grapevine in response to E. necator are associated with the plant defense response, suggesting that PM‐susceptible Cabernet Sauvignon is able to initiate a basal defense but unable to restrict fungal growth or slow down disease progression.     

砧木对河北昌黎产区赤霞珠葡萄生长和果实品质的影响

以7个砧木嫁接的赤霞珠和自根为材料,研究不同砧木对河北昌黎产区赤霞珠葡萄生长、结果和品质的影响.结果表明:5C和5BB显著提高赤霞珠主干粗度,5C显著提高赤霞珠主梢粗度;188-08、5BB和5C显著提高赤霞珠可溶性固形物含量,188-08和5BB显著提高还原糖含量;101-14M和3309C显著降低可滴定酸含量,5C和101-14M显著提高果皮多酚和花青素含量,101-14M显著提高果皮单宁含量;3309C、110R、5C和101-14M显著提高赤霞珠单株产量.采用模糊评价法综合评价7个砧木对赤霞珠生长、品质和产量等因子的影响,在河北昌黎地区独特的气候和环境条件下,7种砧木嫁接赤霞珠的综合评价均优于自根,适宜砧木顺序依次为5C、101-14M、3309C、5BB、188-08、110R、SO4.     

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

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Vessel-diameter distribution in roots of grapevines (Vitis vinifera L. cv. Shiraz)

The distribution frequency patterns of diameter of xylem vessels and percentage of total predicted axial conductances were studied in 190-day and 212-day-old main roots of grapevine (Vitis vinifera L. cv. Shiraz) grown under well-watered and stressed conditions. The protoxylem were the first to mature and were responsible for most of the theoretical conductance in root segments between the tip and 2.5 cm from the tip. Some large xylem vessels retained cross walls and protoplasm up to 22.5 cm from the tip. Statistical tests using the Kolmogorov-Smirnov two sample test showed that the pattern of distribution frequency of xylem vessels classified in different diameter classes varied with distance from the root tip. The distribution frequency of xylem vessels was similar in both well-watered and stressed plants from the tip up to 15 cm from the tip. At distances further from the tip the distribution frequency of xylem vessels of well-watered plants was significantly different from that of stressed plants, with the former having more larger vessels than the latter. The pattern of vessel distribution frequency was different from that of percent total axial conductance (K_h) predicted with fewer large vessels carrying most of the axial flow.     

Surprising structural lability of a cysteine-S-conjugate precursor of 4-methyl-4-sulfanylpentan-2-one, a varietal aroma in wine of Vitis vinifera L. cv. Sauvignon blanc

4-Methyl-4-sulfanylpentan-2-one (1; 4MSP) provides a characteristic aroma compound of wines made from Vitis vinifera L. cv. Sauvignon blanc. 4MSP has a strong box-tree odor with a very low perception threshold and is derived from the cysteinylated precursor S-(1,1-dimethyl-3-oxobutyl)cysteine (4; P-4MSP). P-4MSP is transformed into 4MSP during alcoholic fermentation and is an excellent marker of varietal aroma potential. An improved synthesis of P-4MSP as well as of its deuterium-labeled analogue [D(6)]-P-4MSP is described. Several analytical methods (NMR, IR, LSI-MS, GC/MS, ESI-MS(n)) were combined to elucidate spontaneous reversible structural changes of P-4MSP at different pH values. At low pH, P-4MSP has a linear keto form. The keto-enol tautomerism was observed at neutral pH. At pH 8, the formation of N-substituted intramolecular hemiaminal was characterized by ESI-MS and ESI-MS(n) experiments. The hemiaminal loses H(2)O at high pH to produce a cycloimine, which is easily opened by acid hydrolysis. The keto-enol tautomerism explained the incorporation of only six D-atoms during the preparation of the P-4MSP deuterated standard even if [D(10)]mesityl oxide was used. Derivatization conditions for GC/MS analysis strongly affected the ratio of the monosilylated intramolecular cyclic form and the disilylated linear form of P-4MSP. The structural changes of P-4MSP may have a considerable impact on the development of methods of measuring varietal aroma potential.     

Ultrastructure and germination of Vitis vinifera cv. Loureiro pollen

The cultivar Loureiro of Vitis vinifera is one of the most economically important, recommended in almost the totality of the Regi?o Demarcada dos Vinhos Verdes. In vineyards, the grape productivity of this cultivar is normal while in others it is extremely low. The aim of this work was to study the morphology and germination of Vitis vinifera cv. Loureiro pollen with high and low productivity. The pollen grain was examined under light, transmission and scanning electron microscopy. Typically V. vinifera pollen present three furrows but in the cultivar Loureiro we found tricolporated and acolporated (without furrows or pores) pollen grains. Both pollen types present generative and vegetative cells with the usual aspect and a dense cytoplasm rich in organelles. In the acolporated pollen a continuous exine layer and an irregular intine layer were observed. Differences were found in the starch accumulation, since only in tricolporated pollen abundant plastids filled with numerous starch granules were observed. To determine the causes of the low productivity of this cultivar we tested pollen viability by the fluorochromatic reaction and pollen germinability by in vitro assays. We observed that the acolporated pollen grain is viable, but no germination was recorded.     

Fruit ripening in Vitis vinifera: light intensity before and not during ripening determines the concentration of 2-methoxy-3-isobutylpyrazine in Cabernet Sauvignon berries

The roles of light and temperature in the accumulation of the vegetal impact compound 2-methoxy-3-isobutylpyrazine (MIBP) in grape (Vitis vinifera L.) berries were determined. Individual clusters were exposed to various light intensities using neutral density shade cloth before ripening, during ripening or throughout the season in three growing seasons. A recently developed method using headspace solid-phase microextraction combined with GC-MS in the selected ion-monitoring mode was employed to measure MIBP in berries. Berry MIBP concentration increased subsequent to berry set, reached a maximum prior to onset of ripening, and then decreased thereafter until harvest. Complete shading of clusters increased the concentration of MIBP more than 100% compared to unshaded controls in 2 out of 3 years. Light increasingly inhibited MIBP concentrations up to 25-50% of ambient light intensities (1500 μmol photons m(-2) s(-1) ). However, only changes in light intensity before ripening had any effect on MIBP accumulation or final MIBP concentration. Analyses of weather data showed that the 1 year in which shading was ineffective was unusually warm, warm early in the season, and had more hot days and higher early season degree days than the other 2 years. In controlled environment experiments, warm growth conditions reduced MIBP concentrations in fruit about as much as light exposure reduced MIBP concentrations in the field experiments. The results indicate that both light and temperature significantly affect MIBP in harvested fruit, but that the light environment during ripening does not significantly affect MIBP concentrations in the berries at harvest.     

Free radical scavenging of grape pomace extracts from Cabernet sauvingnon (Vitis vinifera)

Pressed grape pomace obtained from the wine production of Cabernet sauvignon (Vitis vinifera) vintage was dried until 9.8% moisture content, ground and submitted to extraction of soluble components from different extraction techniques. Low pressure extractions were performed with ethanol maceration followed by fractionation with n-hexane, dichloromethane, butanol and ethyl acetate. These solvents were furthermore applied for soxhlet extraction. Supercritical fluid extraction (SFE) was also performed to obtain grape pomace extracts by using pure CO(2) and CO(2) with ethanol as co-solvent in concentrations of 10, 15 and 20%w/w. The operating condition used in high pressure extractions was 150bar and 40 degrees C. The antioxidant activity of the grape pomace extracts was determined considering the free radical scavenging assay using 1,1-Diphenyl-2-picrylhydrazyl (DPPH) and was correlated with the total phenol content determined according to the Folin-Ciocalteu method. The results obtained in DPPH tests indicate the highest antioxidant activity of 96.6+/-0.3%AA, with an IC(50) value of 13+/-1, for the extracts obtained with ethyl acetate in solid-liquid extraction. The highest yield values were achieved in soxhlet extraction with ethanol (13.2%w/w) and with butanol (12.2%w/w), and also by SFE with 15% ethanol (9.2%w/w). The lipophilic composition of grape pomace extracts was evaluated by gas chromatography-mass spectrometry with the identification of components like linoleic acid and ethyl linoleate, with important therapeutic activities.     

Photorespiration and photoprotection of grapevine (<Emphasis Type="Italic">Vitis vinifera</Emphasis> L. cv. Cabernet Sauvignon) under water stress

In order to investigate the photoprotective function of photorespiration in grapevine under water stress, potted grapevines (Vitis vinifera L. cv. Cabernet Sauvignon) were randomly divided into three uniform groups for well-watered [watered every morning to keep the relative water content (RWC) of soil over 70 %], water-stress adapted (drought-adapted at 30 % relative soil water content for 30 days), and water stress without adaptation treatment (water-stressed to 30 % relative soil water content for 3 days). Net assimilation rate (A _N), stomatal conductance (g _s), substomatal CO₂ concentration (C _i), transpiration rate (E), actual photochemical efficiency of PSII (Φ_PSII), and maximum photochemical efficiency of PSII (F_v/F_m) were recorded by combining measurements of gas exchange and chlorophyll fluorescence. Gross photorespiration (P_r), photosynthetic electron partitioning (J_C/J_T), photochemical quenching coefficient (qP), and non-photochemical quenching (NPQ) were also calculated. The ratio of net assimilation rate to transpiration rate (A _N/E) was used as an indicator of water use efficiency (WUE). A _N, apparent P_r, Φ_PSII, F_v/F_m, qp, and g _s decreased, NPQ increased, and gross P_r sustained at a high level under water stress. This suggests that both photorespiration and energy dissipation play important roles in protecting photosynthetic apparatus against photoinhibition. C _i in water-stressed plants without adaptation treatment increased, which indicates the leaves suffered a non-stomatal limitation, while the water-stress adaped plants only suffered a stomatal limitation indicated by low C _i.     

Three-year field response of drip-irrigated grapevine (Vitis vinifera L., cv. Tempranillo) to soil salinity

The aim of this work was to evaluate the effects of Fe deficiency on the activity of several metabolic enzymes (PEPC, PK, PFK, G6PDH and G3PDH), along with the function of the antioxidant enzymes (SK, SDH and PAL) in two lines of Medicago ciliaris, TN11.11 and TN8.7. Plants were grown in a greenhouse under controlled conditions. After germination and pre-treatment, plants were transferred for hydroponic culture. Three treatments were used: 30 μM Fe (+Fe), 0 μM Fe (?Fe) and 30 μM Fe + 10 mM NaHCO₃ (+Bic.). Our results showed that all the enzymatic activities increased in extracts of Fe-deficient roots when compared to the control. The above increases in the activity were particularly evident for the bicarbonate-treated roots of TN11.11. PEPC activity was increased by 277% in TN11.11 plants with the addition of bicarbonate to the nutrient solution. Our results indicate also that, in the two lines of Medic, the activity of SK, SDH and PAL in leaves and roots were increased under Fe deficiency (either direct or induced by bicarbonate), to a greater extent in TN11.11 plants. Furthermore, a considerable increase in lipid peroxidation of roots and leaves of Fe-deficient plants was observed in TN8.7 when compared to TN11.11 plants. Our data suggest that the TN11.11 line is more effective in overcoming Fe deficiency than TN8.7. The tolerance of TN11.11 to Fe deficiency is related to its ability to modulate the carbohydrate metabolism and to increase secondary metabolism pathways.     

Purification and characterization of a novel O-methyltransferase from Flammulina velutipes

An enzyme catalyzing the methylation of phenolic hydroxyl groups in polyphenols was identified from mycelial cultures of edible mushrooms to synthesize O-methylated polyphenols. Enzyme activity was measured to assess whether methyl groups were introduced into (?)-epigallocatechin-3-O-gallate (EGCG) using SAM as a methyl donor, and (?)-epigallocatechin-3-O-(3-O-methyl)-gallate (EGCG3″Me), (?)-epigallocatechin-3-O-(4-O-methyl)-gallate (EGCG4″Me), and (?)-epigallocatechin-3-O-(3,5-O-dimethyl)-gallate (EGCG3″,5″diMe) peaks were detected using crude enzyme preparations from mycelial cultures of Flammulina velutipes. The enzyme was purified using chromatographic and two-dimensional electrophoresis. The purified enzyme was subsequently analyzed on the basis of the partial amino acid sequence using LC–MS/MS. Partial amino acid sequencing identified the 17 and 12 amino acid sequences, VLEVGTLGGYSTTWLAR and TGGIIIVDNVVR. In database searches, these sequences showed high identity with O-methyltransferases from other mushroom species and completely matched 11 of 17 and 9 of 12 amino acids from five other mushroom O-methyltransferases.     

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.     

葡萄实生树阶段转变过程中的内源多胺含量变化

用高效液相色谱法测定二年生巨峰葡萄自然实生树不同节位叶片、芽、韧皮部中内源多胺含量的结果表明,随着节位的升高,在3种组织中的腐胺(Put)和亚精胺(Spd)含量增加,21～25节腐胺增加幅度最大,自21节起亚精胺含量陡增;21～25节叶片中的精胺(Spm)含量出现高峰.说明21节左右可能是童性消失进入生殖生长期的临界点.