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PubMed1974

Rapid estimates of relative water content.

Relative water content may be accurately estimated using the ratio of tissue fresh weight to tissue turgid weight, termed here relative tissue weight. That relative water content and relative tissue weight are linearly related is demonstrated algebraically. The mean value of r(2) for grapevine (Vitis vinifera L. cv. Shiraz) leaf tissue over eight separate sampling occasions was 0.993. Similarly high values were obtained for maize (Zea mays cv. Cornell M-3) (0.998) and apple (Malus sylvestris cv. Northern Spy) (0.997) using a range of leaf ages. The proposal by Downey and Miller (1971. Rapid measurements of relative turgidity in maize (Zea mays L.). New Phytol. 70: 555-560) that relative water content in maize may be estimated from water uptake was also investigated for grapevine leaves; this was found to be a less reliable estimate than that obtained with relative tissue weight. With either method, there is a need for calibration, although this could be achieved for relative tissue weight at least with only a few subsamples.

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PubMed1975

Stereoisomeric Characterization of Tartaric Acid Produced during l-Ascorbic Acid Metabolism in Plants.

Labeled tartaric acids from Pelargonium crispum apices which had been fed l-ascorbic acid-6-(14)C and Vitis labrusca and Parthenocissus inserta tissues which had been fed l-ascorbic acid-1-(14)C were examined by chemical means to determine chiral configuration. In each instance, label was associated with (+)-tartaric acid.Similar experiments with labeled tartaric acid from P. crispum which had been labeled with d-glucose-1-(14)C or -6-(14)C led to the same result. No evidence was obtained for formation of labeled meso-tartaric acid in experiments described above. The recent suggestion of H. Ruffner and D. Rast (Z. Pflanzenphysiol. 73: 45-55, 1974) that conversion of l-ascorbic acid to tartaric acid in plants is a nonenzymatic process is re-examined in the light of present findings.

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PubMed1975

The influence of temperature on malic Acid metabolism in grape berries: I. Enzyme responses.

Phosphoenolpyruvate (PEP) carboxylase activity in immature ;Carignane' grape berries (Vitis vinifera L.) had a temperature optimum of about 38 C, whereas malic enzyme activity rose with increasing temperature between 10 and 46 C. In vitro temperature inactivation rates for the PEP carboxylase were markedly greater than for the malic enzyme activity. From the simultaneous action of malic acid-producing enzymes (PEP carboxylase and malic dehydrogenase) and malic acid-degradating enzyme (malic enzyme) systems at different temperatures, the greatest tendency for malic acid accumulation in immature grape berries was at 20 to 25 C. Time-course measurements of enzymic activity from heated, intact berries revealed greater in vivo temperature stability for the malic enzyme activity than for the PEP carboxylase activity.

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PubMed1976

Changes in anthocyanin and phenolics content of grapevine leaf and fruit tissues treated with sucrose, nitrate, and abscisic Acid.

Sucrose (0.04 to 0.12 m) induces accumulation of both total phenolics and anthocyanin in leaf discs of grapevine (Vitis vinifera L.) incubated in intermittent light. Abscisic acid (20 muM) and 2-chloroethyl phosphonic acid (60 mum) act synergistically with the sucrose to enhance its induction of both total phenolics and anthocyanin. The magnitude of this interaction depends on leaf age. Nitrate (30 mm) inhibits sucrose induction of phenolics and anthocyanin. Levels of total phenolics and anthocyanin changed independently.

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PubMed1977

Factors affecting the extraction of intact ribonucleic Acid from plant tissues containing interfering phenolic compounds.

Using conventional methods it is impossible to extract RNA as uncomplexed intact molecules from the leaves of grapevines (Vitis vinifera L.) and from a number of woody perennial species that contain high levels of reactive phenolic compounds. A procedure involving the use of high concentrations of the chaotropic agent sodium perchlorate prevents the binding of phenolic compounds to RNA during extraction. Analyses of the phenolics present in plant tissues used in these experiments indicate that there is a poor correlation between the total phenolic content and the complexing of RNA. However, qualitative analyses suggest that proanthocyanidins are involved in the tanning of RNA during conventional extractions.

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PubMed1978

Biosynthesis of (+)-Tartaric Acid from l-[4-C]Ascorbic Acid in Grape and Geranium.

The metabolic fate of l-[4-(14)C]ascorbic acid has been examined in the grape (Vitis labrusca L.) and lemon geranium (Pelargonium crispum L. L'Hér. cv. Prince Rupert) under conditions comparable to data from l-[1-(14)C]ascorbic acid and l-[6-(14)C]ascorbic acid experiments. In detached grape leaves and immature berries, l-[4-(14)C]ascorbic acid and l-[1-(14)C]ascorbic acid were equivalent precursors to carboxyl labeled (+)-tartaric acid. In geranium apices, l-[4-(14)C]ascorbic acid yielded internal labeled (+)-tartaric acid while l-[6-(14)C]ascorbic acid gave an equivalent conversion to carboxyl labeled (+)-tartaric acid. These findings clearly show that two distinct processes for the synthesis of (+)-tartaric acid from l-ascorbic acid exist in plants identified as (+)-tartaric acid accumulators. In grape leaves and immature berries, (+)-tartaric acid synthesis proceeds via preservation of a four-carbon fragment derived from carbons 1 through 4 of l-ascorbic acid while carbons 3 through 6 yield (+)-tartaric acid in geranium apices.

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PubMed1978

Conversion of labeled substrates to sugars, cell wall polysaccharides, and tartaric Acid in grape berries.

[U-(14)C]Sucrose, myo-[U-(14)C]inositol, [6-(14)C]- and [U-(14)C]glucuronate, UDP-[U-(14)C]glucuronate, [U-(14)C]gluconate, and l-[1-(14)C]ascorbic acid were fed into grape berries, Vitis labrusca L. cv. Delaware, at intervals throughout the ripening process and incorporation of (14)C into several metabolites was studied.[U-(14)C]Sucrose was the most effective precursor of cellulose in young grape berries and of glucose and fructose in mature berries. On the other hand, UDP-[U-(14)C]glucuronate was the best precursor of pectic substance, followed by [(14)C]glucuronate and myo-[U-(14)C]inositol. l-[1-(14)C]Ascorbic acid was the most effective precursor of tartaric acid. In young berries, [U-(14)C]sucrose and [U-(14)C]gluconate also produced labeled tartaric acid, the latter a somewhat better precursor in the 3 weeks following flowering. The remaining test compounds were only poor sources of (14)C for tartaric acid although all three, glucuronate, UDP-glucuronate, and myo-inositol, were utilized by the grape berry for pectin biosynthesis.These results strongly indicate that tartaric acid is synthesized by a C-1 oxidation mechanism of hexose in young grape berries.

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PubMed1983

Determination of C distribution in photosynthetic serine and phosphoglycerate from grape leaves.

Serine and phosphoglyceric acid are the classical marker intermediates of photorespiration and reductive carbon assimilation in C(3) plants. The present paper introduces a new and fast method for the determination of (14)C distribution in these compounds by selective elimination of C-3 (NaIO(4)) or C-1 (ninhydrin/ceric sulfate). Reproducibility of the procedure was found to be better than +/-1% upon degradation of [U-(14)C]serine and [U-(14)C]glycerate standards.Determination of labeling patterns in (dephosphorylated) phosphoglycerate and serine, extracted from grape leaf discs (Vitis vinifera L.) after administration of (14)CO(2) for 1 minute showed that (14)C distribution in serine changes considerably with changing assimilatory conditions. Interpretations of data basing on unusual labeling patterns of serine must therefore be accepted with caution.

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PubMed1984

Synthesis of l-(+)-Tartaric Acid from l-Ascorbic Acid via 5-Keto-d-Gluconic Acid in Grapes.

5-Keto-l-idionic acid ( identical with5-keto-d-gluconic acid, d-xylo-5-hexulosonic acid) was found as a metabolic product of l-ascorbic acid in slices of immature grapes, Vitis labrusca L. cv ;Delaware'. Specifically labeled compounds, recognized as metabolic products of l-ascorbic acid in grapes, were fed to young grape tissues to investigate the metabolic pathway from l-ascorbic acid to l-(+)-tartaric acid.Label from dehydro-l-[1-(14)C]ascorbic acid, 2-keto-l-[1-(14)C]idonic acid (l-xylo-2-hexulosonic acid), l-[1-(14)C]idonic acid, or 5-keto-l-[1-(14)C] idonic acid was incorporated into l-(+)-tartaric acid in high yields as it was in the l-[1-(14)C]ascorbic acid experiment. In a double label experiment involving a mixture of l-[1-(14)C]idonic acid and l-[2-(3)H]idonic acid, the (3)H/(14)C ratios of 5-keto-l-idonic acid and l-(+)-tartaric acid synthesized in young grape leaves were almost the same as the value of the l-idonic acid fed. Label from 5-keto-l-[6-(14)C]idonic acid was incorporated into sugars and insoluble residue in the same way as l-[6-(14)C]ascorbic acid was metabolized in grapes.These results provide strong evidence that in grapes l-(+)-tartaric acid is synthesized from the C(4) fragment that corresponds to the C1 to C4 group of the 5-keto-l-idonic acid derived from l-ascorbic acid via 2-keto-l-idonic acid and l-idonic acid.

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PubMed1986

Presence and identification of polyamines in xylem and Phloem exudates of plants.

Polyamines were identified by high performance liquid chromatography (benzoylation) and by thin layer chromatography (dansylation) in xylem exudates from stems of sunflowers (Helianthus annuus [L.]), mung bean (Vigna radiata [L.] Wilczek), grapevine (Vitis vinifera [L.] cv Grenache), and orange (Citrus sinensis [L.] Osbeck, cv Valencia), as well as in phloem sap (using elution into EDTA) of sunflower and mung bean plants. Putrescine was the major polyamine detected, ranging in concentrations of 150 to 9200 picomoles per milliliter exudate, whereas only trace amounts of spermine were detected. High amounts of putrescine and spermidine were found in EDTA eluates (possibly phloem sap) as compared with elution into water. Concentrations of putrescine and spermidine in xylem exudates were related to the physiological conditions of the plants prior to exudate collection. More putrescine was found in exudates of older than in younger sunflower plants, and salt stress applied to sunflower plants resulted in a higher concentration of putrescine and spermidine in the exudate. The presence and abundance of putrescine and spermidine in xylem and phloem exudates indicate that polyamines may be translocated in plants. This long-distance translocation further supports the hypothesis that polyamines have a regulatory role in plant growth and response to stress.

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PubMed1989

Net CO(2) Assimilation and Carbohydrate Partitioning of Grapevine Leaves in Response to Trunk Girdling and Gibberellic Acid Application.

Leaf net CO(2) assimilation rate (A), stomatal conductance (g(s)), carboxylation efficiency, and foliar nonstructural carbohydrates were measured on mature, field-grown Vitis vinifera L. (cv Thompson Seedless) vines that had been trunk girdled, sprayed with gibberellic acid, or both, shortly after anthesis. Girdling reduced A, g(s), and carboxylation efficiency when measured 2 weeks after imposition of the treatments. Diurnal measurements indicated that A of girdled vines was less than that of control vines between 1000 and 1800 hours. Gibberellic acid mitigated the depressing effect of girdling on g(s) during the same diurnal measurements. The concentrations of foliar carbohydrates were greatest for the girdled vines, followed by the combination treatment and were lowest for the control vines. Foliar carbohydrates were greater for girdled vines 4 weeks after the treatments were imposed, however, by this time there was no significant difference in A between the control and girdled vines. Two and 4 weeks after the experiment was initiated root carbohydrate concentrations were less for the girdled vines when compared to the control vines. The data indicate that the reduction in A of girdled grapevines is not associated with the accumulation of leaf nonstructural carbohydrates following the girdling treatment.

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PubMed1991

Characterization of heat injury in grapes using h nuclear magnetic resonance methods : changes in transverse relaxation times.

Transverse relaxation times (T(2)) of tissue water ((1)H) in leaves and suspension cultured cells of grape hybrids (Vitis spp. cv ;Venus' and ;Veeblanc') were measured by nuclear magnetic resonance at various temperatures. The tissue water was characterized by two T(2) time constants. A sharp decrease in T(2) for the major fraction of tissue water was observed in association with heat injury, as measured by electrolyte leakage and triphenyltetrazolium chloride reduction in both leaves and suspension cultured cells. The changes in T(2) as a result of heat injury were irreversible, as indicated by a temperature dependent hysteresis of T(2). Studies using a paramagnetic probe (Mn(+2)) indicated that the plasma membrane was irreversibly damaged at the killing temperature, resulting in a loss of cell compartmentalization. Tissue water in heat-killed samples was characterized by only a single T(2).

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PubMed1991

Plant NAD(H)-Glutamate Dehydrogenase Consists of Two Subunit Polypeptides and Their Participation in the Seven Isoenzymes Occurs in an Ordered Ratio.

The structure and function of NAD(H)-glutamate dehydrogenase in plants was studied by using grapevine (Vitis vinifera L. cv Sultanina) callus grown under different nitrogen sources. The enzyme consists of two subunit-polypeptides, alpha and beta, with similar antigenic properties but with different molecular mass and charge. The two polypeptides have molecular masses of 43.0 and 42.5 kilodaltons, respectively. The holoenzyme is hexameric and is resolved into seven isoenzymes by native gel electrophoresis. Two-dimensional native/SDS-PAGE revealed that the 1 and 7 isoenzymes are homohexamers and the isoenzymes 2 through 6 are hybrids of the two polypeptides following an ordered ratio. The total quantity of alpha- and beta-polypeptides and the isoenzymic pattern was altered by the exogenous nitrogen source. The sample derived from callus grown on nitrate or glutamic acid contained a slightly greater amount of beta-polypeptide and of the more cathodal isoenzymes, whereas alpha-polypeptide and the more anodal isoenzymes predominated in callus grown in the presence of either ammonium or glutamine. The anabolic reaction was correlated with the alpha- and the catabolic reaction with the beta-polypeptide; this could suggest that each isoenzyme exhibits anabolic and catabolic function of different magnitude. The isoenzymic patterns did not obey the expected binomial distribution proportions.

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PubMed1991

Vacuolar Release of 1-(Malonylamino)cyclopropane-1-Carboxylic Acid, the Conjugated Form of the Ethylene Precursor.

The mechanisms underlying the vacuolar retention or release of 1-(malonylamino)cyclopropane-1-carboxylic acid (MACC), the conjugated form of the ethylene precursor, has been studied in grape (Vitis vinifera) cells grown in vitro using the technique of compartmental analysis of radioisotope elution. Following its accumulation in the vacuole, M[2,3-(14)C]ACC could be released from cells when the vacuolar pH was artificially lowered by external buffers from its initial value of 6.2 to below the critical pH of 5.5. Successive release and retention of vacuolar MACC could be achieved by switching the vacuolar pH from values lower and higher than 5.5. The rate constant of efflux was highly correlated with the vacuolar pH. In plant tissues having low vacuolar pH under natural conditions, e.g. apple fruits (pH 4.2) and mung bean hypocotyls (pH 5.3), an efflux of M[2,3-(14)C]ACC also occurred. Its rate constant closely corresponded to the theorical values derived from the correlation established for grape cells. Evidence is presented that the efflux proceeded by passive lipophilic membrane diffusion only when MACC was in the protonated form. In contrast to other organic anions like malic acid, the mono and diionic species could not permeate the tonoplast, thus indicating the strict dependence of MACC retention upon the ionic status of the molecule and the absence of carrier-mediated efflux.

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PubMed1992

Hysteresis and cooperative behavior of a latent plant polyphenoloxidase.

Appearance of a lag period dependent on pH in the expression of the catecholase activity of a polyphenoloxidase extracted in a latent state from Airen grape (Vitis vinifera L.) berries, is revealed, suggesting the hysteretic nature of the enzyme. The lag time was independent of enzyme concentration, indicating that slow pH-induced conformational changes in the protein must occur during assay. Results obtained by varying substrate concentration show that the system presents hyperbolic or cooperative kinetics depending on the pH of the assay.

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PubMed1992

Aberrant processing of polyphenol oxidase in a variegated grapevine mutant.

Bruce's Sport is a mutant grapevine (Vitis vinifera L.) with green and white variegated fruit derived from the Sultana variety. The white regions of tissue have decreased polyphenol oxidase (PPO) activity resulting in a reduced capacity for browning. Active PPO from Sultana grapes was purified and had an apparent molecular weight of 40,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Western blots indicated that mature Sultana grapes contained a single 40-kilodalton PPO, and young Sultana berries also had small quantities of a 60-kilodalton protein. Bruce's Sport grapes had much less of the 40-kilodalton PPO and greater amounts of the 60-kilodalton band. Protease digestion of Bruce's Sport extracts decreased the proportion of the 60-kilodalton protein and increased the 40-kilodalton band. A cDNA clone of grape PPO was used to probe a northern blot of Sultana and Bruce's Sport RNA and hybridized to a 2.2-kilobase transcript in both grapevines. The level of PPO mRNA was high in the early stages of berry development but then declined. The results suggest that in grapevine the active 40-kilodalton form of PPO is synthesized as a precursor protein of at least 60 kilodaltons, and normal processing is interrupted in Bruce's Sport resulting in the accumulation of the 60-kilodalton inactive preform of PPO.

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PubMed2006

Abiotic stress generates ROS that signal expression of anionic glutamate dehydrogenases to form glutamate for proline synthesis in tobacco and grapevine.

Glutamate dehydrogenase (GDH) may be a stress-responsive enzyme, as GDH exhibits considerable thermal stability, and de novo synthesis of the alpha-GDH subunit is induced by exogenous ammonium and senescence. NaCl treatment induces reactive oxygen species (ROS), intracellular ammonia, expression of tobacco (Nicotiana tabacum cv Xanthi) gdh-NAD;A1 encoding the alpha-subunit of GDH, increase in immunoreactive alpha-polypeptide, assembly of the anionic isoenzymes, and in vitro GDH aminating activity in tissues from hypergeous plant organs. In vivo aminating GDH activity was confirmed by gas chromatorgraphy-mass spectrometry monitoring of (15)N-Glu, (15)N-Gln, and (15)N-Pro in the presence of methionine sulfoximine and amino oxyacetic acid, inhibitors of Gln synthetase and transaminases, respectively. Along with upregulation of alpha-GDH by NaCl, isocitrate dehydrogenase genes, which provide 2-oxoglutarate, are also induced. Treatment with menadione also elicits a severalfold increase in ROS and immunoreactive alpha-polypeptide and GDH activity. This suggests that ROS participate in the signaling pathway for GDH expression and protease activation, which contribute to intracellular hyperammonia. Ammonium ions also mimic the effects of salinity in induction of gdh-NAD;A1 expression. These results, confirmed in tobacco and grape (Vitis vinifera cv Sultanina) tissues, support the hypothesis that the salinity-generated ROS signal induces alpha-GDH subunit expression, and the anionic iso-GDHs assimilate ammonia, acting as antistress enzymes in ammonia detoxification and production of Glu for Pro synthesis.

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PubMed2004

Processes contributing to photoprotection of grapevine leaves illuminated at low temperature.

Photoinactivation of photosystem II (PSII) and energy dissipation at low leaf temperatures were investigated in leaves of glasshouse-grown grapevine (Vitis vinifera L. cv. Riesling). At low temperatures (< 15 degrees C), photosynthetic rates of CO(2) assimilation were reduced. However, despite a significant increase in the amount of light excessive to that required by photosynthesis, grapevine leaves maintained high intrinsic quantum efficiencies of PSII (F(v)/F(m)) and were highly resistant to photoinactivation compared to other species. Non-photochemical energy dissipation involving xanthophylls and fast D1 repair were the main protective processes reducing the 'gross' rate of photoinactivation and the 'net' rate of photoinactivation, respectively. We developed an improved method of energy dissipation analysis that revealed up to 75% of absorbed light is dissipated thermally via pH- and xanthophyll-mediated non-photochemical quenching at low temperatures (5-15 degrees C) and moderate (800 micro mol quanta m(-2) s(-1)) light. Up to 20% of the energy flux contributing to electron transport was dissipated via photorespiration when taking into account temperature-dependent mesophyll conductance; however, this flux used in photorespiration was only a relatively small amount of the total absorbed light energy. Photoreduction of O(2) at photosystem I (PSI) and subsequent superoxide detoxification (water-water cycle) was more sensitive to inhibition by low temperature than photorespiration. Therefore the water-water cycle represents a negligibly small energy sink below 15 degrees C, irrespective of mesophyll conductance.

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