The grape ubiquitin ligase VpRH2 is a negative regulator in response to ABA treatment.

MAIN CONCLUSION: Ubiquitin ligase VpRH2 is a negative regulator in the grape ABA pathway by inhibiting ABL1, PYR1 and GRP2A expressions, and its promoter is inhibited by ABA treatment. In higher plants, ubiquitin ligases play key roles in various cellular processes. As in our previous study (Wang et al. in J Exp Bot 68:1669-1687, 2017), grape RING-H2-type ubiquitin ligase gene VpRH2 and its promoter was induced by powdery mildew and showed resistance to the disease. Diverse small-molecule hormones, like salicylic acid (SA), methyl jasmonate (MeJA) or abscisic acid (ABA), play pivotal roles in plant resistance. Here we found that VpRH2 expression could be induced by SA and MeJA treatment, but inhibited by ABA treatment. The promoter of VpRH2 revealed a similar variation trend under exogenous hormone treatments as the gene expression by GUS activity assay. By a series of deletion fragments, the promoter fragment of VpRH2-P656 to VpRH2-P513 was necessary in response to MeJA treatment, and the inhibition of ABA treatment to the VpRH2 promoter was independent of the ABRE motif. Over-expression of VpRH2 in Arabidopsis thaliana plants displayed ABA-insensitive phenotypes at the germination stage compared to wild type plants. In VpRH2 over-expressing Vitis vinifera cv. Thompson Seedless plants after ABA treatments, the expression of the ABA pathway related genes ABL1 and PYR1 showed a suppresive trend. Moreover, VpGRP2A (an VpRH2-interacting protein) also showed a suppresive trend in response to ABA treatment in VpRH2-overexpressing plants. Our results demonstrate that VpRH2 is a negative regulator in the grape ABA signal pathway by inhibiting ABL1, PYR1 and GRP2A expressions, and its promoter was also inhibited by ABA treatment.

Optimization of Catechin and Proanthocyanidin Recovery from Grape Seeds Using Microwave-Assisted Extraction.

Grape seed extract (GSE) is a rich source of condensed flavonoid tannins, also called proanthocyanidins (PACs). The high molecular weight of polymeric PAC limits their biological activity due to poor bioavailability. The present study was undertaken to explore the potential applicability of microwave-assisted extraction (MAE) to convert GSE-PAC into monomeric catechins. A central composite design (CCD) was used to optimize the processing conditions for the MAE. The maximum total yield of monomeric catechins (catechin, epicatechin, and epicatechin gallate) and PAC were 8.2 mg/g dry weight (DW) and 56.4 mg catechin equivalence (CE)/g DW, respectively. The optimized MAE condition was 94% ethanol, 170 °C temperature, and a duration of 55 min. Compared to the results for PACs extracted via conventional extraction (Con) (94% ethanol; shaking at 25 °C for 55 min), MAE yielded 3.9-fold more monomeric catechins and 5.5-fold more PACs. The MAE showed higher antioxidant capacity and α-glucosidase inhibitory activity than that of the conventional extract, suggesting the potential use of the MAE products of grape seeds as a functional food ingredient and nutraceutical.

Evolvement of nutraceutical onion plants engineered for resveratrol biosynthetic pathway.

KEY MESSAGE: Genetically engineered onion expressing codon-optimized VvSTS1 gene accumulated stilbenes and extended life span in yeast and can serve as potential nutraceutical. Resveratrol (RV) is a natural polyphenolic compound found in certain plant species including grapes. RV is well known for its nutraceutical properties and to assuage several disease conditions. Onion is the second most consumed vegetable worldwide and contains large quantities of precursor molecules, malonyl-CoA and para-coumaroyl-CoA that are needed for RV biosynthesis. The present study reports the development of nutraceutical onion by engineering RV biosynthetic pathway. A codon-optimized grapevine synthetic stilbene synthase gene (VvSTS1) was synthesized using native grapevine sequence. Six-week-old healthy yellowish compact nodular calli were co-cultivated with Agrobacterium tumefaciens harbouring pCAMBIA1300-hpt II-CaMV35S-VvSTS1-nos. PCR analysis revealed the presence of VvSTS1 and hpt II genes in putative transgenics. Southern blot analysis confirmed the integration of VvSTS1 gene and independent nature of transformants. LC-ESI-HRMS analysis revealed the accumulation of variable quantities of RV (24.98-50.18 µg/g FW) and its glycosylated form polydatin (33.6-67.15 µg/g FW) in both leaves and bulbs, respectively, indicating the successful engineering of RV biosynthetic pathway into onion. The transgenic onion bulb extracts extended the life span in haploid yeast. The transgenic onion accumulating RV and polydatin, developed for the first of its kind, may serve as a potential nutraceutical resource.

Alternating temperature in postharvest cooling treatment of 'Fiano' and 'Falanghina' grapes affects cell wall enzyme rate, berry softening and polyphenols.

BACKGROUND: In wine production, temperature control is important and cooling techniques are frequently used to cool down grape must. Grape compound extraction is facilitated by enzymatic cell wall disassembly. Accordingly, our hypothesis is that the use of an alternating temperature could favor enzyme activity and thus berry compound extraction. RESULTS: White wine grapes (cvs 'Falanghina' and 'Fiano') were kept at 4 °C or at an alternating temperature (4 and 20 °C with a 6-h interval) for 24 h. During the cooling treatment, berry skin resistance, berry firmness and native enzymes from the cell wall were analyzed; sugars, total acidity, malic acid and polyphenols were also measured. In 'Falanghina', α-d-glucosidase, ß-d-galactosidase and pectinmethylesterase increased in grape berry kept at an alternating temperature, and the berry firmness decreased and the polyphenol content was greater than in the other sample. In 'Fiano', polygalacturonase and pectinmethylesterase were responsable for berry softening and, overall, with an alternating temperature, the activity was higher and, consequently, sugars and polyphenol contents were higher. CONCLUSION: Instead of just cooling wine grape, a 24-h cycle of low and high temperature on wine grape after harvest could be used to increase cell wall enzyme activity and, consequently, decrease the berry firmness, favouring an increase in sugars and polyphenol contents. © 2018 Society of Chemical Industry.

Structural and Functional Analysis of UGT92G6 Suggests an Evolutionary Link Between Mono- and Disaccharide Glycoside-Forming Transferases.

Glycosylation mediated by UDP-dependent glycosyltransferase (UGT) is one of the most common reactions for the biosynthesis of small molecule glycosides. As glycosides have various biological roles, we characterized UGT genes from grapevine (Vitis vinifera). In silico analysis of VvUGT genes that were highly expressed in leaves identified UGT92G6 which showed sequence similarity to both monosaccharide and disaccharide glucoside-forming transferases. The recombinant UGT92G6 glucosylated phenolics, among them caffeic acid, carvacrol, eugenol and raspberry ketone, and also accepted geranyl glucoside and citronellyl glucoside. Thus, UGT92G6 formed mono- and diglucosides in vitro from distinct compounds. The enzyme specificity constant Vmax/Km ratios indicated that UGT92G6 exhibited the highest specificity towards caffeic acid, producing almost equal amounts of the 3- and 4-O-glucoside. Transient overexpression of UGT92G6 in Nicotiana benthamiana leaves confirmed the production of caffeoyl glucoside; however, the level of geranyl diglucoside was not elevated upon overexpression of UGT92G6, even after co-expression of genes encoding geraniol synthase and geraniol UGT to provide sufficient precursor. Comparative sequence and 3-D structure analysis identified a sequence motif characteristic for monoglucoside-forming UGTs in UGT92G6, suggesting an evolutionary link between mono- and disaccharide glycoside UGTs. Thus, UGT92G6 functions as a mono- and diglucosyltransferase in vitro, but acts as a caffeoyl glucoside UGT in N. benthamiana.

Changes in sugar content and related enzyme activities in table grape (Vitis vinifera L.) in response to foliar selenium fertilizer.

BACKGROUND: Spraying selenium (Se) fertilizer is an effective method for Se-enriched fruit production. Sugar content in fruit is the major factor determining berry quality. However, changes in sugar metabolism in response to Se fertilizer are unclear. Hence, this study was conducted to identify the effects of Se fertilizer on sugar metabolism and related enzyme activities of grape berries. Additionally, production of leaves with and without Se fertilizer was also investigated. RESULTS: Acid invertase (AI) activity, total soluble sugar and Se content in berries, and photosynthetic rate in leaves produced under Se fertilizer treatments were higher than that of control. Glucose and fructose were the primary sugars in berries, with a trace of sucrose. In both berries and leaves, neutral invertase activity was lower than AI, there was no significant difference in neutral invertase, sucrose synthase and sucrose phosphate synthase between Se fertilizer-treated and control. In berries, AI showed a significant positive correlation with glucose and fructose; also Se content was significantly correlated with sugar content. CONCLUSION: AI played an important role in the process of sugar accumulation in berries; high AI activity in berries and photosynthetic rate in leaves could explain the mechanism by which Se fertilizer affected sugar accumulation in berries. © 2017 Society of Chemical Industry.

Stabilization by multipoint covalent attachment of a biocatalyst with polygalacturonase activity used for juice clarification.

Derivatized-agarose supports are suitable for enzyme immobilization by different methods, taking advantage of different physical, chemical and biological conditions of the protein and the support. In this study, agarose particles were modified with MANAE, PEI and glyoxyl groups and evaluated to stabilize polygalacturonase from Streptomyces halstedii ATCC 10897. A new immobilized biocatalyst was developed using glyoxyl-agarose as support; it exhibited high performance in degrading polygalacturonic acid and releasing oligogalacturonides. Maximal enzyme activity was detected at 5h of reaction using 0.05g/mL of immobilized biocatalyst, which released 3mg/mL of reducing sugars and allowed the highest product yield conversion and increased stability. These results are very favorable for pectin degradation with reusability up to 18 successive reactions (90h) and application in juice clarification. Plum (4.7°Bx) and grape (10.6°Bx) juices were successfully clarified, increasing reducing sugars content and markedly decreasing turbidity and viscosity.

Mass spectrometric and enzymatic evidence confirm the existence of anthocyanidin 3,5-O-diglucosides in cabernet sauvignon (Vitis vinifera L.) grape berries.

It has been widely accepted that anthocyanidin 3,5-O-diglucosides do not exist in Vitis vinifera L. Cabernet Sauvignon (CS) berries. However, our anthocyanin analyses using HPLC-ESI-MS/MS detected the existence of a low level of anthocyanidin 3,5-O-diglucosides in the Cabernet Sauvignon grape berries grown in China. The authenticity of these samples was confirmed with microsatellite markers. The existence of anthocyanidin 3,5-O-diglucoside was further verified by the enzymatic evidence for the first time. Four putative 5-O-glucosyltransferase (5GT) genes were isolated from the Cabernet Sauvignon berries. The enzymatic analysis showed that a recombinant protein (designated as Vv5GT3) glucosylated the 3-O- and 5-O-positions of anthocyanidins and flavonols. A phylogenetic analysis revealed that this bifunctional enzyme belongs to the 5GT subfamily of UDP-glycosyltransferases. This finding brought a new understanding of the anthocyanins' profile and their biosynthesis in V. vinifera and would be helpful for further investigations of the mechanism of accumulation of anthocyanidin diglucosides in Cabernet Sauvignon berries in China's wine-producing regions.

Biosynthetic origin of E-resveratrol accumulation in grape canes during postharvest storage.

Grape canes are vineyard waste products containing valuable phytochemicals of medicine and agriculture interest. Grape canes storage is critical for the accumulation of these bioactive compounds. In the present study, we investigated the changes in stilbenoid phytochemical composition during grape cane storage and the influence of the temperature on final concentrations. A strong increase in the concentration of the monomer E-resveratrol (approximately 40-fold) was observed during the first 6 weeks of storage at 20 °C in eight different grape varieties without any change in oligomer concentrations. The E-resveratrol accumulation was temperature-dependent with an optimal range at 15-20 °C. A 2 h heat-shock treatment aiming at protein denaturation inhibited E-resveratrol accumulation. The constitutive expression of key genes involved in the stilbene precursor biosynthesis along with an induction of stilbene synthase (STS) expression during the first weeks of storage contribute to a de novo biosynthesis of E-resveratrol in pruned wood grapes.

Measurement of polyphenol oxidase activity using optical waveguide lightmode spectroscopy-based immunosensor.

Polyphenol oxidase (PPO) is an important quality index during food processing involving heat-treatment and sensitive determination of PPO activity has been a critical concern in the food industry. In this study, a new measurement of PPO activity exploiting an optical waveguide lightmode spectroscopy-based immunosensor is presented using a polyclonal anti-PPO antibody that was immobilized in situ to the surface of a 3-aminopropyltriethoxysilane-treated optical grating coupler activated with glutaraldehyde. When analysed with a purified PPO fraction from potato tubers, a linear relationship was found between PPO activities of 0.0005607-560.7U/mL and the sensor responses obtained. The sensor was applicable to measurement of PPO activity in real samples that were prepared from potato tubers, grapes and Kimchi cabbage, and the analytical results were compared with those obtained by a conventional colorimetric assay measuring PPO activity. When tested for long-term stability, the sensor was reusable up to 10th day after preparation.

The evolutionary history and diverse physiological roles of the grapevine calcium-dependent protein kinase gene family.

Calcium-dependent protein kinases (CDPKs) are molecular switches that bind Ca(2+), ATP, and protein substrates, acting as sensor relays and responders that convert Ca(2+) signals, created by developmental processes and environmental stresses, into phosphorylation events. The precise functions of the CDPKs in grapevine (Vitis vinifera) are largely unknown. We therefore investigated the phylogenetic relationships and expression profiles of the 17 CDPK genes identified in the 12x grapevine genome sequence, resolving them into four subfamilies based on phylogenetic tree topology and gene structures. The origins of the CDPKs during grapevine evolution were characterized, involving 13 expansion events. Transcriptomic analysis using 54 tissues and developmental stages revealed three types of CDPK gene expression profiles: constitutive (housekeeping CDPKs), partitioned functions, and prevalent in pollen/stamen. We identified two duplicated CDPK genes that had evolved from housekeeping to pollen-prevalent functions and whose origin correlated with that of seed plants, suggesting neofunctionalization with an important role in pollen development and also potential value in the breeding of seedless varieties. We also found that CDPKs were involved in three abiotic stress signaling pathways and could therefore be used to investigate the crosstalk between stress responses.

Structure and expression profiling of a novel calcium-dependent protein kinase gene, CDPK3a, in leaves, stems, grapes, and cell cultures of wild-growing grapevine Vitis amurensis Rupr.

KEY MESSAGE : VaCDPK3a is actively expressed in leaves, stems, inflorescences, and berries of Vitis amurensis and may act as a positive growth regulator, but is not involved in the regulation of resveratrol biosynthesis. Calcium-dependent protein kinases (CDPKs) are known to play important roles in plant development and defense against biotic and abiotic stresses. It has previously been shown that CDPK3a is the predominant CDPK transcript in cell cultures of wild-growing grapevine Vitis amurensis Rupr., which is known to possess high resistance against environmental stresses and to produce resveratrol, a polyphenol with valuable pharmacological effects. In this study, we aimed to define the full cDNA sequence of VaCDPK3a and analyze its organ-specific expression, responses to plant hormones, temperature stress and exogenous NaCl, and the effects of VaCDPK3a overexpression on biomass accumulation and resveratrol content in V. amurensis calli. VaCDPK3a was actively expressed in all analyzed V. amurensis organs and tissues and was not transcriptionally regulated by salt and temperature stresses. The highest VaCDPK3a expression was detected in young leaves and the lowest in stems. A reduction in the VaCDPK3a expression correlated with a lower rate of biomass accumulation and higher resveratrol content in calli of V. amurensis under different growth conditions. Overexpression of the VaCDPK3a gene in the V. amurensis calli significantly increased cell growth for a short period of time but did not have an effect on resveratrol production. Further subculturing of the transformed calli resulted in cell death and a decrease in expression of the endogenous VaCDPK3a. The data suggest that while VaCDPK3a acts as a positive regulator of V. amurensis cell growth, it is not involved in the signaling pathway regulating resveratrol biosynthesis and resistance to salt and temperature stresses.

Identification of adducts between an odoriferous volatile thiol and oxidized grape phenolic compounds: kinetic study of adduct formation under chemical and enzymatic oxidation conditions.

HPLC-MS and (1)H, (13)C, and 2D NMR analyses were used to identify new addition products between 3-sulfanylhexan-1-ol (3SH) and o-quinones derived from (+)-catechin, (-)-epicatechin, and caftaric acid. The kinetics of formation of these adducts were monitored in a wine model solution and in a must-like medium by HPLC-UV-MS with the aim of understanding the chemical mechanism involved in reactions between volatile thiols and o-quinones. One o-quinone-caftaric acid/3SH adduct, three o-quinone-(+)-catechin/3SH adducts, and three o-quinone-(-)-epicatechin/3SH adducts were characterized. Caftaric acid was oxidized faster than (-)-epicatechin and (+)-catechin when these phenolic compounds were incubated in a one-component mixture with polyphenoloxidase (PPO) in the presence of 3SH. Consequently, o-quinone-caftaric acid formed adducts with 3SH more rapidly than o-quinone-(+)-catechin and o-quinone-(-)-epicatechin in the absence of other nucleophilic species. Furthermore, o-quinone-(-)-epicatechin reacted faster than o-quinone-(+)-catechin with 3SH. Sulfur dioxide decreased the yield of adduct formation to a significant extent. Under chemical oxidation conditions, the rates and yields of adduct formation were lower than those observed in the presence of PPO, and o-quinone-caftaric acid was slightly less reactive with 3SH, compared to oxidized flavan-3-ols. The identification of o-quinone-caftaric acid/3SH and o-quinone-(+)-catechin/3SH adducts in a must matrix suggests that the proposed reaction mechanism is responsible for 3SH loss in dry wines during their vinification and aging process.

Isolation and characterization of latent and active polyphenoloxidase in BRS Clara (CNPUV 154-147 × Centennial seedless) and BRS Morena (Marroo seedless × Centennial seedless) seedless table grapes.

The seedless grapes BRS Clara and BRS Morena, developed in Brazil, are currently growing in popularity due to their premium texture and taste. However, there are no reports on the polyphenoloxidase (PPO) from these cultivars. In this paper, active and latent PPO from BRS Clara and BRS Morena seedless grapes were extracted using the non-ionic detergents Triton-X-100 (active) and Triton-X-114 (latent), and their catecholase activities were characterized. The PPO extracted using Triton-X-110 exhibited maximum activities at pH 6.0 and at 25 °C. Above 30 °C, a gradual decline in activities was noted, with complete inactivation at 60 °C. The PPO from grapes extracted with Triton-X-114 was activated with 0.2% of the ionic detergent sodium dodecyl sulfate (SDS), and exhibited maximum activities at pH 5.5 and at 30 °C. It was stable until the temperature reached 60 °C.

Over-expression of a grape stilbene synthase gene in tomato induces parthenocarpy and causes abnormal pollen development.

A novel strategy to induce parthenocarpy in tomato fruits by the induction of resveratrol biosynthesis in flower tissues was exploited. Two transgenic tomato lines were considered: a higher resveratrol-producing (35SS) line, constitutively expressing a grape stilbene synthase cDNA, and a lower resveratrol-producing (LoxS) line, expressing stilbene synthase under a fruit-specific promoter. The expression of the stilbene synthase gene affected flavonoid metabolism in a different manner in the transgenic lines, and in one of these, the 35SS line, resulted in complete male sterility. Resveratrol was synthesised either in 35SS or LoxS tomato flowers, at an even higher extent (about 8-10 times) in the former line. We further investigated whether stilbene synthase expression may have resulted in impaired naringenin accumulation during flower development. In the 35SS flowers, naringenin was significantly impaired by about 50%, probably due to metabolic competition. Conversely, the amount of glycosylated flavonols increased in transgenic flowers, thereby excluding the diminished production of flavonols as a reason for parthenocarpy in tomato. We further investigated whether resveratrol synthesis may have resulted changes to pollen structure. Microscopic observations revealed the presence of few and abnormal flake-like pollen grains in 35SS flowers with no germination capability. Finally, the analysis of coumaric and ferulic acids, the precursors of lignin and sporopollenin biosynthesis, revealed significant depletion of these compounds, therefore suggesting an impairment in structural compounds as a reason for pollen ablation. These overall outcomes, to the best of our knowledge, reveal for the first time the major role displayed by resveratrol synthesis on parthenocarpy in tomato fruits.

Generation and characterization of anti-chitinase monoclonal antibodies.

Class IV chitinase, an allergenic protein of Vitis vinifera (grape), was purified by anion exchange chromatography and used for immunization of Balb/c mice. Monoclonal antibodies (MAbs) were raised by hybridoma technology using Sp2/0 myeloma cells. Finally after three limiting dilutions, six stable clones were generated. Antibody isotyping showed that IgG(2a), IgG(2b), and IgM were produced by one, two, and three of the clones, respectively. All of the MAbs had kappa light chain. The affinities were in the range of 3 × 10(8) to 1.2 × 10(9) M(-1). The MAbs were specific for grape chitinase as confirmed by Western blotting. In conclusion, we successfully produced several MAbs against grape class IV chitinase, which could be used for assessment of this allergen in different grape cultivars.

Pectin-derived oligosaccharides increase color and anthocyanin content in Flame Seedless grapes.

BACKGROUND: Grapes grown in warm weather do not develop a desirable red color and require the use of products to enhance berry color. Pectin-derived oligosaccharides (PDOs) have been shown to have a role in various responses including plant defense, growth and development. In this work a mixture of PDOs with 3-20 degrees of polymerization was applied to Vitis vinifera cv. Flame Seedless grapes under field conditions and compared to the effects of ethephon (an ethylene-releasing compound). The effect of treatments on grape color, anthocyanin content and phenylalanine ammonia lyase (PAL) mRNA levels was evaluated. RESULTS: PDOs treatment increased berry color measured by the Color Index of Red Grapes (CIRG) and anthocyanin content, compared to ethephon and untreated berries (control); 1.5, 1 and 0.5 mg mL⁻¹ PDOs increased berry color by 30%, 27% and 26%, respectively, when compared to control berries. Levels of PAL mRNA accumulating in berries treated with PDOs were elevated within the first 24 h of treatment. CONCLUSIONS: PDOs enhanced the color and anthocyanin content of Flame Seedless grape berries possibly due by the induction of PAL mRNA expression. The results demonstrated that PDOs can be used to improve fruit quality aspects such as berry skin color.

Water deficit increases stilbene metabolism in Cabernet Sauvignon berries.

The impact of water deficit on stilbene biosynthesis in wine grape (Vitis vinifera) berries was investigated. Water deficit increased the accumulation of trans-piceid (the glycosylated form of resveratrol) by 5-fold in Cabernet Sauvignon berries but not in Chardonnay. Similarly, water deficit significantly increased the transcript abundance of genes involved in the biosynthesis of stilbene precursors in Cabernet Sauvignon. Increased expression of stilbene synthase, but not that of resveratrol-O-glycosyltransferase, resulted in increased trans-piceid concentrations. In contrast, the transcript abundance of the same genes declined in Chardonnay in response to water deficit. Twelve single nucleotide polymorphisms (SNPs) were identified in the promoters of stilbene synthase genes of Cabernet Sauvignon, Chardonnay, and Pinot Noir. These polymorphisms resulted in eight changes within the predicted cis regulatory elements in Cabernet Sauvignon and Chardonnay. These results suggest that cultivar-specific molecular mechanisms might exist that control resveratrol biosynthesis in grapes.

Chemical and biochemical change of healthy phenolic fractions in winegrape by means of postharvest dehydration.

Clusters of Aleatico winegrape were picked at 18 degrees Brix and placed at 10, 20, or 30 degrees C, 45% relative humidity (RH) and 1.5 m/s of air flow to dehydrate the berries up to 40% of loss of initial fresh weight. Sampling was done at 0, 10, 20, 30, and 40% weight loss (wl). Selected polyphenols and sugar content (expressed as SSC = soluble solids content) both measured on dry weight basis, polyphenol oxidase (PPO), and phenylpropanoid pathway gene expression were analyzed. Phenolic acids increased significantly at 20% wl at 20 degrees C, while at 10 degrees C the increase was lower. Stilbenes (trans-resveratrol and trans-piceid) and catechins rose more than double to 100 mg/kg and more than 3-fold to 135 mg/kg at 20 degrees C and 10% wl. At 10 degrees C the increase of these compounds was less, but higher than initial values. At 30 degrees C, except for a significant rise at 10% wl for catechins and stilbenes, all the rest of the compounds diminished. Anthocyanins increased at 10 and 20 degrees C, but decreased at 30 degrees C. PPO rapidly increased at 20 and 30 degrees C at 10% wl and then declined, while at 10 degrees C the activity lasted longer. Relative gene expression of phenylalanine ammonia lyase (PAL), stilbene synthase (STS), chalcone isomerase (CHI), dihydroflavonol reductase (DFR) were upregulated at 10 degrees C more than at 20 degrees C, at 20% wl, while at 30 degrees C the gene expression was downregulated.

One step purification of the grape vacuolar invertase.

Invertase is a major protein of grape juice and wine. Accordingly, in order to study the biochemical and structural characteristics of this protein and for a better understanding of its physico-chemical properties, large amounts of the pure protein are needed. A simple method for the purification of the grape vacuolar invertase in a preparative-scale is described in this work. The grape protein was isolated and purified from must by ultrafiltration and anion exchange chromatography. The identification and purity determination of the grape invertase fraction were assessed by SDS-PAGE, and were then confirmed using nanoLC-chip-MS/MS analysis. The laboratory fractionation procedure presented in this work generated large quantities of pure grape vacuolar invertase from must.