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Sugar accumulation in grape berries. Cloning of two putative vacuolar invertase cDNAs and their expression in grapevine tissues.

During grape berry (Vitis vinifera L.) ripening, sucrose transported from the leaves is accumulated in the berry vacuoles as glucose and fructose. To study the involvement of invertase in grape berry ripening, we have cloned two cDNAs (GIN1 and GIN2) from berries. The cDNAs encode translation products that are 62% identical to each other and both appear to be vacuolar forms of invertase. Both genes are expressed in a variety of tissues, including berries, leaves, roots, seeds, and flowers, but the two genes have distinct patterns of expression. In grape berries, hexose accumulation began 8 weeks postflowering and continued until the fruit was ripe at 16 weeks. Invertase activity increased from flowering, was maximal 8 weeks postflowering, and remained constant on a per berry basis throughout ripening. Expression of GIN1 and GIN2 in berries, which was high early in berry development, declined greatly at the commencement of hexose accumulation. The results suggest that although vacuolar invertases are involved in hexose accumulation in grape berries, the expression of the genes and the synthesis of the enzymes precedes the onset of hexose accumulation by some weeks, so other mechanisms must be involved in regulating this process.

Amino Acid Sequence↗

A class IV chitinase is highly expressed in grape berries during ripening.

Chitinase activity increased markedly at the onset of ripening in grape (Vitis vinifera L.) berries and continued to increase throughout the sugar accumulation phase of berry development. In contrast, beta-1,3-glucanase activity was not detected in grape berries at any stage of development. Two closely related chitinase cDNAs (VvChi4A and VvChi4B) were cloned from grapes. Sequence and Southern analysis indicate that these two clones may represent alleles of the same gene. The predicted proteins are acidic and have a signal peptide followed by a cysteine-rich, chitin-binding domain and a catalytic region. An analysis of their sequences indicates that they are class IV chitinase. The deduced protein sequence of VvChi4A has a high level of identity with the 32- and 28-kD chitinases present as haze proteins in wine. Expression of VvChi4 was high in berries and low in flowers but was not detected in leaves, roots, or seeds. No expression was detected in berries 2 to 8 weeks postflowering, but expression was high 12 to 16 weeks postflowering, which coincided with sugar accumulation and an increase in chitinase activity. Constitutive expression of VvChi4 appears to be fruit-specific and induced at high levels in grapes during ripening.

Amino Acid Sequence↗

Coordinate accumulation of antifungal proteins and hexoses constitutes a developmentally controlled defense response during fruit ripening in grape.

During ripening of grape (Vitis labruscana L. cv Concord) berries, abundance of several proteins increased, coordinately with hexoses, to the extent that these became the predominant proteins in the ovary. These proteins have been identified by N-terminal amino acid-sequence analysis and/or function to be a thaumatin-like protein (grape osmotin), a lipid-transfer protein, and a basic and an acidic chitinase. The basic chitinase and grape osmotin exhibited activities against the principal grape fungal pathogens Guignardia bidwellii and Botrytis cinerea based on in vitro growth assays. The growth-inhibiting activity of the antifungal proteins was substantial at levels comparable to those that accumulate in the ripening fruit, and these activities were enhanced by as much as 70% in the presence of 1 m glucose, a physiological hexose concentration in berries. The simultaneous accumulation of the antifungal proteins and sugars during berry ripening was correlated with the characteristic development of pathogen resistance that occurs in fruits during ripening. Taken together, accumulation of these proteins, in combination with sugars, appears to constitute a novel, developmentally regulated defense mechanism against phytopathogens in the maturing fruit.

Amino Acid Sequence↗

Differential screening indicates a dramatic change in mRNA profiles during grape berry ripening. Cloning and characterization of cDNAs encoding putative cell wall and stress response proteins.

We used differential screening to isolate ripening-associated cDNAs from a Shiraz grape (Vitis vinifera L.) berry cDNA library. A rapid increase in the mRNA levels of a number of cDNAs not present in unripe fruit occurred in grape berries at the onset of ripening. The putative translation products of some of these clones had homologs in other species that are involved in cell wall structure. These included four proline-rich proteins, a small protein that is similar to the non-catalytic, N-terminal domain of some pectin methylesterases, and two other glutamate-rich proteins. The remainder of the clones encoded putative stress response proteins. These included two thaumatin-like proteins, a metallothionein, a transcription factor, a cytochrome P450 enzyme, and proteins induced by water, sugar, and/or cold stress in other species. Many of the homologs of the grape cDNAs thought to be involved in cell wall structure or stress-related responses also accumulate in a developmental manner in other plants. This may indicate that the grape mRNAs accumulate in response to stresses such as the storage of high concentrations of sugars and rapid cell expansion, or they may accumulate as part of the ripening developmental program.

Amino Acid Sequence↗

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.

Journal Article↗

A grape ASR protein involved in sugar and abscisic acid signaling.

The function of ASR (ABA [abscisic acid]-, stress-, and ripening-induced) proteins remains unknown. A grape ASR, VvMSA, was isolated by means of a yeast one-hybrid approach using as a target the proximal promoter of a grape putative monosaccharide transporter (VvHT1). This promoter contains two sugar boxes, and its activity is induced by sucrose and glucose. VvMSA and VvHT1 share similar patterns of expression during the ripening of grape. Both genes are inducible by sucrose in grape berry cell culture, and sugar induction of VvMSA is enhanced strongly by ABA. These data suggest that VvMSA is involved in a common transduction pathway of sugar and ABA signaling. Gel-shift assays demonstrate a specific binding of VvMSA to the 160-bp fragment of the VvHT1 promoter and more precisely to two sugar-responsive elements present in this target. The positive regulation of VvHT1 promoter activity by VvMSA also is shown in planta by coexpression experiments. The nuclear localization of the yellow fluorescent protein-VvMSA fusion protein and the functionality of the VvMSA nuclear localization signal are demonstrated. Thus, a biological function is ascribed to an ASR protein. VvMSA acts as part of a transcription-regulating complex involved in sugar and ABA signaling.

Abscisic Acid↗

Bioconversion of grape must into modulated gluconic acid production by Aspergillus niger ORS-4.410.

AIMS: Analysis of regulators for modulated gluconic acid production under surface fermentation (SF) condition using grape must as the cheap carbohydrate source, by mutant Aspergillus niger ORS-4.410. Replacement of conventional fermentation condition by solid-state surface fermentation (SSF) for semi-continuous production of gluconic acid by pseudo-immobilization of A. niger ORS-4.410. METHODS AND RESULTS: Grape must after rectification was utilized for gluconic acid production in batch fermentation in SF and SSF processes using mutant strain of A. niger ORS-4.410. Use of rectified grape must led to the improved levels of gluconic acid production (80-85 g l(-1)) in the fermentation medium containing 0.075% (NH4)2HPO4; 0.1% KH2PO4 and 0.015% MgSO4.7H2O at an initial pH 6.6 (+/-0.1) under surface fermentation. Gluconic acid production was modulated by incorporating the 2% soybean oil, 2% starch and 1% H2O2 in fermentation medium at continuously high aeration rate (2.0 l min(-1)). Interestingly, 95.8% yield of gluconic acid was obtained when A. niger ORS-4.410 was pseudo-immobilized on cellulose fibres (bagasse) under SSF. Four consecutive fermentation cycles were achieved with a conversion rate of 0.752-0.804 g g(-1) of substrate into gluconic acid under SSF. CONCLUSIONS: Use of additives modulated the gluconic acid production under SF condition. Semi-continuous production of gluconic acid was achieved with pseudo-immobilized mycelia of A. niger ORS-4.410 having a promising yield (95.8%) under SSF condition. SIGNIFICANCE AND IMPACT OF THE STUDY: The bioconversion of grape must into modulated gluconic acid production under SSF conditions can further be employed in fermentation industries by replacing the conventional carbohydrate sources and expensive, energy consuming fermentation processes.

Aspergillus niger↗

Isolation of an ethanol-tolerant endospore-forming Gram-negative Brevibacillus sp. as a covert contaminant in grape tissue cultures.

AIMS: To characterize the alcohol-surviving bacterial isolate ARBG1 from in vitro grapes (Vitis vinifera). METHODS AND RESULTS: Two bacterial strains that survived in covert form in grape cultures were isolated from the spent alcohol used for disinfecting the tools of which one (ARBG2) was characterized earlier. The present study describes characterization of the second isolate, ARBG1. Nutrient agar (NA)-derived colonies of ARBG1 displayed consistently Gram-negative staining rods (2-4x0.5-0.6 micro) substantiated by KOH mucoid thread test. Older cultures (3-7 days) showed emergence of Gram-negative staining, oblong, phase-refractile cells with ellipsoidal spores. The growth and sporulation were modified by growth medium and incubation temperature with the optimum around 37 degrees C. Identification attempts involving microscopic, biochemical, Biolog or fatty acid profiling approaches brought in mixed and inconclusive results. PCR amplification of 16S rDNA was not successful with the standard primers 27F and 1492R but with 27F and a modified primer ARBG1-RP1. The identity of the isolate was established as Brevibacillus sp. based on partial 16S rDNA sequence data from eight single colonies with Gram-positive Brevibacillus choshinensis as the closest match (99.5%). Spotting tests on NA employing spore suspension in aqueous ethanol (0%, 25%, 50%, 60%, 70%, 80% or 90%, v/v) indicated unhindered bacterial-survival in alcohol for 1 month, and that at 2 or 4 months revealed 90% ethanol as more sporicidal than lower levels, corroborated by plating results. Grape microcuttings inoculated with ARBG1 showed substantial general colonization of shoots, roots and medium but low endophytic colonization. CONCLUSIONS: The rare type of spore-producing consistently Gram-negative bacterial isolate ARBG1 was identified as Brevibacillus sp. based on 16S rDNA sequence similarity. The alcohol-defying organism was nonpathogenic and survived in covert form in grape cultures. Aqueous 90% ethanol appeared more sporicidal than lower levels. SIGNIFICANCE AND IMPACT OF THE STUDY: Characterization of an unusual endospore-forming Gram-negative bacterium, observation that some bacteria may fall outside the purview of standard 16S rDNA primers, elucidation of the threats of covert bacteria in plant tissue cultures and alcohol-mediated lateral transmission of spore formers, and the revelation that 70-80% ethanol may not be the most effective bactericidal concentration for all bacteria.

Base Sequence↗

Three grape CBF/DREB1 genes respond to low temperature, drought and abscisic acid.

The C-repeat (CRT)-binding factor/dehydration-responsive element (DRE) binding protein 1 (CBF/ DREB1) transcription factors control an important pathway for increased freezing and drought tolerance in plants. Three CBF/DREB1-like genes, CBF 1-3, were isolated from both freezing-tolerant wild grape (Vitis riparia) and freezing-sensitive cultivated grape (Vitis vinifera). The deduced proteins in V. riparia are 63-70% identical to each other and 96-98% identical to the corresponding proteins in V. vinifera. All Vitis CBF proteins are 42-51% identical to AtCBF1 and contain CBF-specific amino acid motifs, supporting their identification as CBF proteins. Grape CBF sequences are unique in that they contain 20-29 additional amino acids and three serine stretches. Agro-infiltration experiments revealed that VrCBF1b localizes to the nucleus. VrCBF1a, VrCBF1b and VvCBF1 activated a green fluorescent protein (GFP) or glucuronidase (GUS) reporter gene behind CRT-containing promoters. Expression of the endogenous CBF genes was low at ambient temperature and enhanced upon low temperature (4 degrees C) treatment, first for CBF1, followed by CBF2, and about 2 d later by CBF3. No obvious significant difference was observed between V. riparia and V. vinifera genes. The expression levels of all three CBF genes were higher in young tissues than in older tissues. CBF1, 2 and 3 transcripts also accumulated in response to drought and exogenous abscisic acid (ABA) treatment, indicating that grape contains unique CBF genes.

Abscisic Acid↗

Pythium periplocum, an aggressive mycoparasite of Botrytis cinerea causing the gray mould disease of grape-vine.

Pythium periplocum Dreschler has been found to be an aggressive mycoparasite of Botrytis cinerea, the causal agent of the gray mould disease of the grape-vine. When grown together, the former enters the latter's mycelium, branches freely within, coagulates its cytoplasm and finally tears its hyphae apart, bringing about widespread destruction of the grape-vine pathogen. Extensive coiling around the host, as reported in the case of other mycoparasites belonging to the genus Pythium, has not been observed here. The infected mycelium of B. cinerea fails to infect the grape-vine and does not induce the characteristic gray mould symptoms. Since P. periplocum is not a grape-vine parasite, it could be useful for the biological control of B. cinerea. A brief account of this mycoparasitism is discussed in this article.

Botrytis↗

Grape seed extract prevents gentamicin-induced nephrotoxicity and genotoxicity in bone marrow cells of mice.

The protection conferred by grape seed extract against gentamicin-induced nephrotoxicity and bone marrow chromosomal aberrations have been evaluated in adult Swiss albino mice. The activity of reduced glutathione peroxidase (GSH peroxidase), the levels of glutathione (GSH) and lipid peroxidation as malondialdehyde (MDA) in the kidneys homogenates, serum urea and creatinine were measured, and in addition the changes in kidney histology and bone marrow chromosomes were investigated. Gentamicin (80 mg/kg b.wt. intraperitoneally for 2 weeks) induced kidney damage as indicated from a pronounced changes in kidney histology, a significant increase in serum urea and creatinine and MDA content in the kidney homogenate. While the activity of the antioxidant enzyme GSH peroxidase and the level of GSH were significantly decreased. Gentamicin induced genotoxicity indicated by increased the number of aberrant cells and different types of structural chromosomal aberrations (fragment, deletion and ring chromosome) and showed no effect on mitotic activity of the cell. Pretreatment with grape seed extract (7 days) and simultaneously (14 days) with gentamicin significantly protected the kidney tissue by ameliorating its antioxidant activity. Moreover, grape seed extract significantly protected bone marrow chromosomes from gentamicin induced genotoxicity by reducing the total number of aberrant cells, and different types of structural chromosomal aberrations. It could be concluded that grape seed extract acts as a potent antioxidant prevented kidney damage and genotoxicity of bone marrow cells.

Animals↗

Reduction of myocardial ischemia reperfusion injury with regular consumption of grapes.

Recently several polyphenolic antioxidants derived from grape seeds and skins have been implicated in cardioprotection. This study was undertaken to determine if the grapes were equally cardioprotective. Sprague Dawley male rats were given (orally) standardized grape extract (SGE) for a period of three weeks. Time-matched control experiments were performed by feeding the animals 45 microg/100 of glucose plus 45 microg/100 g fructose per day for three weeks. After 30 days, rats were sacrificed, hearts excised and perfused via working-mode. Hearts were made ischemic for 30 min followed by two hours of reperfusion. At 100 mg/kg and at 200 mg/kg, SGE provided significant cardioprotection as evidenced by improved post-ischemic ventricular recovery and reduced amount of myocardial infarction. No cardioprotection was apparent when rats were given grape samples at a dose of 50 mg/100 g/day. In vitro studies demonstrated that the SGE could directly scavenge superoxide and hydroxyl radicals which are formed in the ischemic reperfused myocardium. The results demonstrate that the heats of the rats fed SGE reduced myocardial ischemia reperfusion injury by functioning as in vivo antioxidant.

Animals↗

Study of Spanish grape mycobiota and ochratoxin A production by Isolates of Aspergillus tubingensis and other members of Aspergillus section Nigri.

The native mycobiota of five grape varieties grown in Spain has been studied. Four (Bobal, Tempranillo, Garnacha, and Monastrell) were red varieties and one (Moscatel) was white. The main fungal genera isolated were Alternaria, Cladosporium, and Aspergillus. The isolation frequency of Aspergillus spp. section Nigri in contaminated samples was 82%. Ochratoxin A (OTA) production was assessed using yeast extract-sucrose broth supplemented with 5% bee pollen. Cultures of 205 isolates from this section showed that 74.2% of Aspergillus carbonarius and 14.3% of Aspergillus tubingensis isolates produced OTA at levels ranging from 1.2 to 3,530 ng/ml and from 46.4 to 111.5 ng/ml, respectively. No Aspergillus niger isolate had the ability to produce this toxin under the conditions assayed. Identification of the A. niger aggregate isolates was based on PCR amplification of 5.8S rRNA genes and its two intergenic spacers, internal transcribed spacer 1 (ITS1) and ITS2, followed by digestion with restriction endonuclease RsaI of the PCR products. The restriction patterns were compared with those from strains of A. niger CECT 2807 and A. tubingensis CECT 20393, held at the Spanish Collection of Type Cultures. DNA sequencing of the ITS1-5.8S rRNA gene-ITS2 region of the OTA-producing isolates of A. tubingensis matched 99 to 100% with the nucleotide sequence of strain A. tubingensis CBS 643.92. OTA determination was accomplished by liquid chromatography with fluorescence detection. OTA confirmation was carried out by liquid chromatography coupled to ion trap mass spectrometry. The results showed that there are significant differences with regard to the isolation frequency of ochratoxinogenic fungi in the different grape varieties. These differences were uncorrelated to berry color. The ability of A. tubingensis to produce OTA and the influence of grape variety on the occurrence of OTA-producing fungi in grapes are described in this report for the first time.

Aspergillus↗

Control of Byssochlamys and Related Heat-resistant Fungi in Grape Products.

Heat-resistant strains of Byssochlamys fulva, B. nivea, and other heat-resistant fungi were isolated from vineyard soil, grapes, grape-processing lines, and waste pomace. They are known to remain in grape juice occasionally and to grow in grape juice products. Ascospores of these fungi have a D value (decimal reduction time) of about 10 min at 190 F (88 C), but in the presence of 90 muliters of SO(2) per liter (normally added to the juice) the D value was cut in half. Filtration through a commercial diatomaceous filter aid (also a common processing step) entrapped all but about 0.001% of experimentally added spores. Thus, heat in the presence of SO(2) and filtration together can reduce the population of these spores by several orders of magnitude. Growth was also prevented by benzoate or sorbate in low concentrations. Oxygen must be reduced to extremely low levels before lack of oxygen limits growth.

Journal Article↗

Reduced FVC among California grape workers.

Pulmonary disease due to organic and inorganic dust inhalation represents a significant health problem in the agricultural community. In response to concern over the potential adverse health effects of agricultural exposures, and to address the question of whether the specific crop worked was associated with decreased lung function, we conducted a cross-sectional survey of pulmonary function in a population of 238 grape workers, 355 tomato workers, and 166 citrus workers in California's Central Valley. In addition, 92 workers (66 males and 26 females) performed spirometry before and after an 8-h work shift. Multiple regression analysis of pulmonary function in all 759 workers revealed that age, height, and smoking-adjusted mean preshift FVC was lower in grape workers than in citrus workers in each of three sex and age groups studied: decreased 314 ml for males greater than or equal to 25 yr of age p less than 0.05, 82 ml for males less than 25 yr, and 83 ml for females greater than or equal to 20 yr. Mean adjusted preshift FVC was also lower in grape workers than tomato workers (decreased 249 ml in males greater than or equal to 25 yr [p less than 0.05], 51 ml for males less than 25 yr, and 46 ml for females greater than or equal to 20 yr). Adjusted mean FEV1 was not different by crop, but FEV1/FVC and forced-expiratory flow from 25 to 75% of the FVC (FEF25-75) were significantly elevated in grape workers of the three age and sex groups compared with citrus or tomato workers.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗

Inhibitory effects of green tea and grape juice on the phenol sulfotransferase activity of mouse intestines and human colon carcinoma cell line, Caco-2.

Tea and fruit juices are beverages consumed daily all over the world. The present study reports the inhibitory effects of these beverages on the activity of mammalian intestinal phenol sulfotransferases (P-STs). Green tea strongly inhibited the E. coli-expressed mouse intestinal P-ST activity in vitro. (-)-Epigallocatechin gallate (EGCG) was found to be the most potent inhibitor among the catechins tested (IC50=0.93 microM). (-)EGCG also inhibited the P-ST activity of the human colon carcinoma cell line, Caco-2. Kinetic analysis showed that the inhibition was competitive. Among fruit juices examined (apple, grape, grapefruit and orange), grape juice exhibited the most potent inhibitory action on the P-ST activity of mouse intestines and human colon carcinoma cells. The inhibitory activity of grape juice was located mainly in the skin and seeds. Flavonols, such as quercetin and kaempferol, inhibited the P-ST activity at low concentrations. These observations suggest the possible inhibition of P-ST activity in human intestines by green tea or grape juice.

Animals↗

S-Adenosyl-L-methionine-dependent O-methylation of 2-hydroxy-3-alkylpyrazine in wine grapes: a putative final step of methoxypyrazine biosynthesis.

The final step of 2-methoxy-3-alkylpyrazine (MP) biosynthesis has been presumed to involve O-methylation of 2-hydroxy-3-alkylpyarzine (HP), although this reaction has never been demonstrated in organisms. We detected 2-hydroxy-3-isobutylpyrazine (IBHP) and 2-hydroxy-3-isopropylpyrazine (IPHP) in unripe grapes, and S-adenosyl-L-methionine-dependent O-methyltransferase (OMT) activity toward HP in crude extracts from young shoots and unripe grapes that accumulate MP at different levels. The levels of HP in the berries and stems were estimated by using 2-hydroxy-3-sec-butylpyrazine as an internal standard. The OMT activity observed in the crude extracts from young shoot and berries was extremely low, but no MP-decomposing activity was detected in the solutions. The levels of HP and OMT activity were closely related with the level of MP in the grapes. These results indicate that the predicted final step of MP biosynthesis exists in wine grapes.

Chromatography, High Pressure Liquid↗

Seasonal movement and distribution of the grape mealybug (Homoptera: Pseudococcidae): developing a sample program for San Joaquin Valley vineyards.

The grape mealybug, Pseudococcus maritimus (Ehrhorn), is an important pest of table grapes in California's San Joaquin Valley. The mealybug causes direct damage by infesting grape bunches, resulting in very low economic injury levels. To develop a sampling program to help growers predict damage and make control decisions, we destructively sampled six entire grapevines each month to determine mealybug abundance and within-vine distribution. These absolute counts were then used to evaluate several relative sampling methods: sticky tape barriers on canes, excised spur samples, standard-sized pieces of bark, timed counts, and nondestructive counts on spurs. At midseason we sampled additional vines to correlate mealybug numbers with economic damage at harvest. Finally, mealybug life stages and natural enemies were recorded throughout the study. Timed 5-min counts show the strongest correlation with total mealybug numbers, and a simple count of mealybugs on three spurs per vine at midseason is the best predictor of economic damage. Mealybugs completed approximately equals 2.5 generations in 1998. Comparison to data on mealybug development suggests that exceptionally long growing seasons could exacerbate infestations by allowing the completion of a third generation. No mealybugs were found on bunches before early August, when second-generation crawlers moved out of the bark. Grape bunches that touched old wood had significantly higher damage and mealybug densities. The majority of mealybugs were always found in protected locations (under the bark of the trunk, spurs or canes), indicating the need for chemical or biological controls that can penetrate these refugia.

Animals↗