Vitis Amurensis Research Articles

Somatic embryos cultures of Vitis amurensis Rupr. in air-lift bioreactors for the production of biomass and resveratrol

Resveratrol are the most important bioactive compounds found in Vitis amurensis. In this study, a somatic embryo induction system for V. amurensis was established in air-lift bioreactors for the production of biomass and resveratrol. The somatic embryos biomass growth was low on solid medium (69.60 g L−1) compared to in liquid medium in bioreactor (329.45 g L−1). Bioreactor cultures were found to be superior compared with solid medium culture not only in terms of biomass but also resveratrol productivity. Various culture parameters, including culture method, inoculum density, carbon source, and organic compounds were optimized. An inoculum density of 20 g L−1 embryogenic calli was found suitable for the accumulation of biomass and resveratrol production, whereas 10 g L−1 embryogenic calli increased the amount of resveratrol per fresh weight in somatic embryos. For bioreactor culturing, sucrose was an optimum carbon source and 500 mg L–1 casein hydrolysate acid was conducive to the biomass and resveratrol production. This result indicates that an efficient protocol for the large-scale production of resveratrol can be achieved by bioreactor culturing of V. amurensis somatic embryos and can be used as a source of medicinal raw materials.

Stilbene accumulation and expression of stilbene biosynthesis pathway genes in wild grapevine Vitis amurensis Rupr.

We detected and quantified six stilbenes ( cis -piceid, t -piceid, t -ε-viniferin, cis -ε-viniferin, t -resveratrol, and t -δ-viniferin) in the leaves, petioles, berry skins, and seeds of wild-growing Vitis amurensis . The highest content of stilbenes and expression of stilbene biosynthesis genes were in the probes collected in the autumn and after ultraviolet elicitation. Stilbenes, including the best-studied stilbene resveratrol, are known to display valuable bioactivities and protect plants against various pathogens. There is a lack of studies on stilbene quantities and spectrum combined with an analysis of the stilbene biosynthesis pathway gene expression in Vitaceae species, despite grapevine is an important source of stilbenes. This study presents an analysis of stilbene spectrum, stilbene content, and expression of stilbene biosynthesis genes both in natural conditions and after ultraviolet (UV-C) elicitation in the leaves, petioles, berry skins, and seeds of wild-growing Vitis amurensis, a highly stress-tolerant plant species. Using HPLC analysis, we detected six main stilbenes: cis-piceid (up to 0.257mg/g of dry weight (DW) of plant material), t-piceid (up to 0.055mg/g DW), t-ε-viniferin (up to 0.122mg/g DW), cis-ε-viniferin (up to 0.031mg/g DW), t-resveratrol (from 0.004 to 0.121mg/g DW), and t-δ-viniferin (up to 0.019mg/g DW). The stilbenes were actively synthesized in the leaves (total stilbenes 0.39mg/g DW) and berry skins (total stilbenes 0.249mg/g DW) of V. amurensis collected in the autumn. qRT-PCR revealed that the stilbene synthase (STS), resveratrol O-glucosyltransferase (Glu1), and polyphenol oxidase (PPO1) genes were actively expressed in the analyzed tissues. The resveratrol methyltransferase (Romt1) gene, which is known to catalyze biosynthesis of pterostilbene, was also expressed, but no pterostilbene has been detected in V. amurensis. The content of all detected stilbenes and expression of stilbene biosynthesis genes increased after UV-C treatment, except for Romt1. The data are important for understanding the stilbene biosynthesis in grapevine.

The complete chloroplast genome sequence of Chinese wild grape Vitis amurensis (Vitaceae: Vitis L.)

Vitis amurensis is a species of wild grape with high ecological, medicinal, and economic values. Here, the complete chloroplast genome of this plant was assembled from whole-genome high-throughput sequencing data. The circular double-stranded DNA molecule is 160,953 bp in size, including a pair of inverted repeats (26,354 bp each) separated by large (89,187 bp) and small (19,058 bp) single-copy regions. The chloroplast genome contains 133 genes, including 88 protein-coding genes (80 PCG species), 37 transfer RNA genes (29 tRNA species) and eight ribosomal RNA genes (four rRNA species), 20 of which are duplicated, including eight protein-coding, eight tRNA, and four rRNA genes. The base composition is asymmetric (30.93 % A, 19.07 % C, 18.33 % G, 31.67 % T) with an overall A + T content of 62.60 %. A phylogenetic analysis based on complete chloroplast genome sequences showed that V. amurensis is closely related to V. vinifera, V. aestivalis, and V. rotundifolia.

Vam3, a Compound Derived from Vitis amurensis Rupr., Attenuated Colitis-Related Tumorigenesis by Inhibiting NF-κB Signaling Pathway.

Background: Chronic inflammation is one of the important mediators of colitis-related colon cancer (CRC). Abundant mast cells (MCs) were observed in the tumor microenvironment and mediators released upon MC activation play an important role in the process of chronic inflammation. Previously, we found that activation of intestine mucosal MCs recruited and modulated the inflammatory CD11b+Gr1+ cells to promote the CRC development. In the current study we investigated the effects of Vam3, a resveratrol dimer with potent anti-inflammatory effects, on CRC development.Methods: RBL-2H3 cells, a basophilic leukemia cell line, were pretreated with 2.5 or 5 µM Vam3 and then stimulated with dinitrophenol-conjugated bovine serum albumin (DNP-BSA) plus lipopolysaccharide (LPS). The MC degranulation was determined by measuring β-hexosaminidase release. Generation of TNF-α and IL-6 in RBL-2H3 cells or in peritoneal macrophages was determined by ELISA and real-time qPCR. NF-κB p65 and phospho-NF-κB p65 expression was determined by Western blotting. NF-κB activity in RAW264.7 cells was determined by luciferase reporter assay. CRC was induced in C57BL/6 mice by intraperitoneal injection of azoxymethane (AOM), followed by oral exposure to dextran sodium sulfate (DSS). Vam3 at 50 mg/kg, or disodium cromoglycate (DSCG, MC stabilizer) at 100 mg/kg, or vehicle were administrated to the mice 4 weeks after DSS withdrawal. Levels of TNF-α, IL-6, and mouse MC protease-1 were determined by ELISA. Infiltration of CD11b+Gr1+ cells was determined by flow cytometry analysis. One-way ANOVA was used to compare difference between groups.Results: Pretreatment with Vam3 significantly inhibited RBL-2H3 cell degranulation and inflammatory cytokine production from RBL-2H3 cells and from peritoneal macrophages. After Vam3 treatment, NF-κB activity in RAW264.7 cells, and expressions of phospho-NF-κB p65 in RBL-2H3 cells and in peritoneal macrophages were significantly down-regulated. In the AOM plus DSS-induced CRC murine model, the Vam3 and DSCG-treated mice had less tumor numbers than those treated with vehicle. Expression of phospho-NF-κB p65, production of inflammatory cytokines, and infiltration of MCs and CD11b+Gr1+ cells were attenuated in the Vam3-treated mice.Conclusion: Vam3 treatment could attenuate the CRC development. This effect may be due to its inhibition on NF-κB signaling pathway in MCs and macrophages of the inflamed intestines.

Overexpression of pathogen-induced grapevine TIR-NB-LRR gene VaRGA1 enhances disease resistance and drought and salt tolerance in Nicotiana benthamiana.

The NBS-LRR proteins encoded by the majority of R genes represent important intracellular receptors that directly or indirectly recognize pathogen effector proteins, which subsequently activate plant defense responses. In this study, a novel Plasmopara viticola-induced TIR-NBS-LRR gene, named VaRGA1, was cloned from leaf tissues of a highly downy mildew-resistant Vitis amurensis "Shuanghong" grapevine. The fluorescence signal of the VaRGA1-GFP fusion protein was clearly partitioned to the cytoplasm and nucleus. The expression of the VaRGA1 gene was strongly induced during early stages of infection by P. viticola, and was also significantly upregulated after drought and salt treatments. Accordingly, grapevine leaves transiently expressing the VaRGA1 gene manifested increased resistance to P. viticola, and the overexpression of the VaRGA1 gene in Nicotiana benthamiana conferred enhanced resistance to Phytophthora parasitica through the activation of salicylic acid (SA) signaling and phenylpropanoid pathways and could also increase tolerance to drought and salt stresses at the germination and vegetable growth stages. These findings indicate that the grapevine VaRGA1 gene may function as the immune and non-immune receptors against biotic and abiotic stresses and that there may be signaling overlap between biotic and abiotic responses.

Characterisation of seed oils from different grape cultivars grown in China.

To explore the potential of the large amount of grape pomace in wineries of China, oils of three Eurasiangrape cultivars (Chardonnay, Merlot and Carbernet Sauvignon) and two Chinese traditional grape cultivars (Vitis amurensis and Vitis davidii), were characterised. The results showed seed oil properties differ for various grape varities. Grape seed oils were demonstrated to be good sources of polyunsaturated fatty acid (PUFA) (63.88-77.12%), sterols (227.99-338.83mg/100g oil) and tocotrienols (320.08-679.24mg/kg oil). Seed oil of V. amurensis exhibited the highest values of polyunsaturated fatty acid, total tocotrienols, total tocols and DPPH· scavenging capacity. Seed oil of Carbernet Sauvignon had the highest contents of squalene, total sterols, total tocopherols and total phenolics. Principal component analysis five grape cultivars differentiated on the basis of bioactive components content and antioxidant properties.

Striking changes in volatile profiles at sub-zero temperatures during over-ripening of ‘Beibinghong’ grapes in Northeastern China

The evolution of free and glycosidically bound volatile compounds in ‘Beibinghong’ (Vitis vinifera×Vitis amurensis) grape berries throughout on-vine over-ripening and freezing processes was studied in two vintages. The aroma profiles of ‘Beibinghong’ icewine berries were characterized by C6 compounds, higher alcohols and terpenoids in free fractions and carbonyl compounds, higher alcohols, C6 alcohols and terpenoids in bound fractions. With regard to free volatile compounds, there was a decrease in the concentration of C6 compounds, terpenols and norisoprenoids and an increase of terpene oxides during over-ripening process. A striking alteration of volatile profile occurred at sub-zero temperatures, particularly for the free fractions such as C6 alcohols, higher alcohols and oxidative terpene derivatives. These changes were attributed to a series of reactions (biotransformation, oxidation and anaerobic metabolism) induced by water loss and especially freeze-thaw cycles. PCA revealed temperature and rainfall affected the accumulation of volatile compounds during over-ripening processes.

Antioxidant and Hepatoprotective Effects of Procyanidins from Wild Grape (Vitis amurensis) Seeds in Ethanol-Induced Cells and Rats.

In the present study, we characterized the antioxidant and hepatoprotective mechanisms underlying of wild grape seed procyanidins (WGP) against oxidative stress damage in ethanol-treated HepG2 cell and Sprague-Dawley (SD)-rat models. In HepG2 cells, WGP not only diminished the ethanol (EtOH, 100 mM)-induced reactive oxygen species (ROS) formation and cytochrome P450 2E1 (CYP2E1) expression, but also renovated both the activity and expression of antioxidant enzymes including catalase, superoxide dismutase, and glutathione peroxidase. Additionally, to investigate the hepatoprotective effect of WGP, rats were orally administered 10 or 50 mg/kg WGP once daily for seven days prior to the single oral administration of EtOH (6 g/kg). The results show that WGP administration decreased the EtOH-induced augment of the levels of serum aspartate transaminase and alanine transaminase as well as serum alcohol and acetaldehyde. WGP treatment upregulated the activities and protein levels of hepatic alcohol dehydrogenase, aldehyde dehydrogenase, and antioxidant enzymes but downregulated the protein expression level of liver CYP2E1 in EtOH-treated rats. Moreover, the decreased phosphorylation levels of mitogen activated protein kinases (MAPKs) by ethanol were induced in both HepG2 cell and rat models. Overall, pretreatment of WGP displayed the protective activity against EtOH-mediated toxicity through the regulation of antioxidant enzymes and alcohol metabolism systems via MAPKs pathways.

Phenolic and Chromatic Properties of Beibinghong Red Ice Wine during and after Vinification.

The phenolic and chromatic characteristics of a special red ice wine made from a Vitis amurensis × V. vinifera hybrid cultivar Beibinghong were studied. Results from two different vintages (2013 and 2014) showed that during vinification, the phenolic acid content increased, while the level of flavonoids (flavonols, flavan-3-ols, and anthocyanins) reduced by a variable extent. The color intensity and red % decreased together with a decrease in anthocyanin content. This was accompanied by an increase in hue as well as yellow %. The final phenolic content was found to be between 119.54 and 180.93 mg/L, with anthocyanins as the predominant phenolic group (92.06%–93.03%), of which 3,5-O-diglucosidic anthocyanins made up 53.55%–79.04%. Phenolic acids were the primary non-anthocyanin phenolics at about 6.64%–7.5%. The phenolic contents and color parameters of Beibinghong dry red wine and several V. vinifera dry red wines of superior color quality were also used in an attempt to clarify the relationship between phenolics and color in the Beibinghong red ice wine. By using Pearson correlation analysis and principal component analysis (PCA), it was found that 3,5-O-diglucosidic anthocyanins and protocatechuic acid were the only characteristic phenolics that differentiated Beibinghong wines from the other selected red wines from more traditional varieties. They were also the main phenolics to be positively correlated with the hue and yellow % of the wine at the early stages leading into maturation. Their presence might, therefore, explain the relatively high hue and yellow % of Beibinghong ice wine.

Breeding for cold-resistant, seedless grapes from Chinese wild Vitis amurensis using embryo rescue

ABSTRACTHybridisation was carried out between stenospermocarpic seedless grape cultivars (female parents) and Chinese wild Vitis amurensis or its hybrids with cold-resistance alleles (male parents). The aim of this paper was: (1) to evaluate the effects of the addition of the plant growth regulator indole-3-acetic acid (IAA) to the embryo development medium MM3 + 500 mg L−1 casein hydrolysate + 1 mmol L−1 serine; (2) to obtain a batch of seedless hybrid grape offspring by embryo rescue; and (3) to screen suitable genotypes for embryo rescue. The results indicated that adding 1.75 mg L−1 IAA promotes embryo development, germination and plantlet formation in the combinations of ‘Ruby Seedless’ × ‘00-1-5’ and ‘Beauty Seedless’ × ‘Beichun’. However, it inhibits the combinations ‘Perlette’ × ‘00-1-5’ and ‘Neptune’ × ‘00-1-5’. ‘Crimson Seedless’, ‘Ruby Seedless’ and ‘Flame Seedless’ were more suitable as female parents than ‘Neptune’, ‘Perlette’, ‘Beauty Seedless’, ‘Heshen Seedless’, ‘Jupiter’ and ‘Su-67’. The Hybrid ‘00-1-5’ performed best as the male parent for cold-resistant seedless grape embryo rescue. A total of 267 new hybrid progenies derived from 11 cross combinations were obtained by embryo rescue. GLSP1 and SCF27, two special primers of the molecular marker linked to seedless gene in grape, were employed to screen the progenies of five cross combinations for possible seedlessness. Five of 22 strains obtained from two cross combinations had the marker GLSP1-569; 43 of 89 strains obtained from three cross combinations had the marker SCF27-2000. These marked strains were preliminarily identified as being seedless.

Expression of Vitis amurensis NAC26 in Arabidopsis enhances drought tolerance by modulating jasmonic acid synthesis.

The growth and fruit quality of grapevines are widely affected by abnormal climatic conditions such as water deficits, but many of the precise mechanisms by which grapevines respond to drought stress are still largely unknown. Here, we report that VaNAC26, a member of the NAC transcription factor family, was upregulated dramatically during cold, drought and salinity treatments in Vitis amurensis, a cold and drought-hardy wild Vitis species. Heterologous overexpression of VaNAC26 enhanced drought and salt tolerance in transgenic Arabidopsis. Higher activities of antioxidant enzymes and lower concentrations of H2O2 and O2 (-) were found in VaNAC26-OE lines than in wild type plants under drought stress. These results indicated that scavenging by reactive oxygen species (ROS) was enhanced by VaNAC26 in transgenic lines. Microarray-based transcriptome analysis revealed that genes related to jasmonic acid (JA) synthesis and signaling were upregulated in VaNAC26-OE lines under both normal and drought conditions. VaNAC26 showed a specific binding ability on the NAC recognition sequence (NACRS) motif, which broadly exists in the promoter regions of upregulated genes in transgenic lines. Endogenous JA content significantly increased in the VaNAC26-OE lines 2 and 3. Our data suggest that VaNAC26 responds to abiotic stresses and may enhance drought tolerance by transcriptional regulation of JA synthesis in Arabidopsis.

Protective effect of an ethanol extract mixture of Vitis amurensis, Aralia cordata, and Glycyrrhizae radix against ischemia-induced brain injury in rats and excitotoxicity in cultured neurons

Vitis amurensis, Aralia cordata, and Glycyrrhizae radix have been widely used as oriental medicinal plants in Korea, China and Japan and found to possess anti-oxidative and anti-inflammatory activities. A previous study demonstrated a protection of an ethanol extract (SSB) of a mixture of three medicinal plants of Vitis amurensis, Aralia cordata, and Glycyrrhizae radix against β amyloid protein-induced memory impairment. The current study was conducted to investigate the neuroprotective effect of SSB against ischemiainduced brain injury. Transient focal cerebral ischemia was induced by 2 hr middle cerebral artery occlusion followed by 24 hr reperfusion (MCAO/reperfusion) in rats. Oral administration of SSB (5, 10 and 25 mg/kg) 30 min before and 1 h after MCAO, and 1 h after reperfusion reduced MCAO/reperfusion-induced brain infarct and edema formation. SSB also inhibited development of behavioral disabilities in MCAO/reperfusion-treated rats. Exposure of cultured cortical neurons to 500 μM glutamate for 12 hr resulted in neuronal cell death. SSB (1-10 μg/mL) inhibited glutamateinduced neuronal death, elevation of intracellular calcium concentration ([Ca2+]i), and generation of reactive oxygen species (ROS). These results suggest that the neuroprotective effect of SSB against ischemia-induced brain damage might be associated with its anti-excitotoxic activity and that SSB may have a therapeutic role for prevention of neurodegeneration in stroke.

Influence of overexpression of the true and false alternative transcripts of calcium-dependent protein kinase CPK9 and CPK3a genes on the growth, stress tolerance, and resveratrol content in Vitis amurensis cell cultures

Calcium-dependent protein kinases (CDPKs or CPKs) are important Ca2+ sensors in plants implicated in plant abiotic and biotic stress responses, e.g. in drought-induced stomata closure or pathogen-induced stilbene production. We previously found that the VaCPK9 gene of Vitis amurensis was subjected to unusual post-transcriptional processing mediated by canonical splice sites leading to production of three mRNA isoforms that lacked important catalytic kinase subdomains. We also detected numerous unusual VaCPK9 and VaCPK3a cDNAs possessing short direct repeated sequences instead of canonical splice sites and suggested that these cDNAs were false alternative transcripts generated by reverse transcriptase template switching in vitro. The present study has been attempted to determine if the full-length and spliced CDPKs play a role in the grapevine growth regulation, stilbene biosynthesis, and abiotic stress adaptation. We generated transgenic V. amurensis callus cultures overexpressing different true and false VaCPK9 and VaCPK3a transcripts under control of the CaMV 35S promoter. Overexpression of the full-length VaCPK9 and VaCPK3a transcripts slightly increased biomass accumulation but did not affect resistance to abiotic stresses and biosynthesis of resveratrol (3,5,4′-trihydroxy-trans-stilbene) in the calli. Overexpression of the true alternative VaCPK9SF1, VaCPK9SF2, and VaCPK9SF3 transcripts considerably improved growth of the transgenic calli, while overexpression of the false alternative VaCPK3aSF1, VaCPK3aSF2, and VaCPK3aSF3 transcripts did not considerably influence it. The results show that the VaCPK9 and VaCPK3a genes function in growth regulation of V. amurensis but not in stilbene biosynthesis and abiotic stress tolerance. Alternative splicing of VaCPK9 may serve to reinforce the positive effect of VaCPK9 expression on biomass accumulation.

Stilbene accumulation in cell cultures of Vitis amurensis Rupr. overexpressing VaSTS1, VaSTS2, and VaSTS7 genes

Stilbenes (resveratrol, piceid, viniferins, etc.) are acting as phytoalexins and possess valuable biologically active properties. To improve stilbene production in grapevine, the effect of VaSTS1, VaSTS2, and VaSTS7 gene overexpression on stilbene production in callus cell cultures of wild grapevine Vitis amurensis Rupr. were accessed. Overexpression of VaSTS1 increased the total content of stilbenes by 1.2–2.1 times up to 0.36 mg per g of the dry weight (mg g−1 dry wt) or 0.036 % of dry wt, while overexpression of VaSTS2 and VaSTS7 increased the content of stilbenes by 1.5–3.5 and 2.5–2.8 times (up to 0.48 and 0.60 mg g−1 dry wt), respectively. P-coumaric acid (CA), a precursor of stilbenes, induced stilbene content and production in all cell lines with the highest positive effect on stilbene accumulation being observed in the STS2- and STS7-transgenic cell lines. After the CA feeding, the stilbene content increased up to 1.09–1.16 mg g−1 dry wt in the STS2- and one STS7-transgenic cell lines and only up to 0.35 and 0.40 mg g−1 dry wt in the control and STS1-transgenic cell lines, respectively. Thus, overexpression of VaSTS1, VaSTS2, and VaSTS7 genes had a positive effect on stilbene accumulation in the cells of V. amurensis cultivated in vitro. T-resveratrol and t-resveratrol glycosides were the prevalent stilbenes synthesized in the STS-transgenic calli before and after CA feeding. The results obtained are innovative for plant biotechnology, since stilbene production in plant cell cultures overexpressing a STS gene has not been studied until now.

Overexpression of Muscadinia rotundifolia CBF2 gene enhances biotic and abiotic stress tolerance in Arabidopsis.

C-repeat-binding factor dehydration-responsive element-binding factor 1C (CBF2/DREB1C) gene encodes a small family of transcriptional activator that has been described as playing an important role in freezing tolerance and cold acclimation of plants. We here report that CBF2 gene also plays an important role in the early response to the pathogen infection of grapevine downy mildew disease. The expression level of CBF2 increased dramatically and reached a peak at 7h after infection in immune grapevine Muscadinia rotundifolia 'Noble', which was much faster than moderate resistant Vitis amurensis 'PI1288' and susceptible Vitis vinifera 'Cabernet Sauvignon'. Muscadinia rotundifolia MrCBF2 exhibited amino acid domains characteristic of Vitis CBF2 proteins with unique features including rich serine repeats and slight differences in NLS, DSAWRL, and AP2 domains. The MrCBF2 gene was introduced to Arabidopsis 'COL0' which are susceptible to downy mildew pathogen. The transgenic lines showed an increased resistance to downy mildew disease and more accumulation of SA as well as higher expression of pathogenesis-related (PR) genes (AtPR1, AtPR4, and AtPR5) as a consequence of MrCBF2 overexpression. Besides, constitutive expression of MrCBF2 enhanced phytohormone abscisic acid (ABA)-independent drought tolerance of transgenic plants. Freezing tolerance of transgenic lines was also enhanced accompanied with an increase in the expression of the cold-regulated genes AtCOR, AtCOR15A, AtKIN1, AtRD29A, and AtSuSy. In addition, the development of MrCBF2-overexpressing plants was seen to be altered and resulted in growth retardation, dwarfism, late flowering, and prone rosette leaves, which may be because of an increase in the gene expression of partial DELLA proteins and DDF1.

An Extended Leaf Disc Test for Virulence Assessment in Plasmopara Viticola and Detection of Downy Mildew Resistance in Vitis

Viticulture is continuously suffering significant economic loss from downy mildew epidemics. Although the infection biology of the pathogen Plasmopara viticola is well understood, little is known regarding the population diversity of this oomycete and on the mechanisms responsible for compatible or incompatible reactions with different host genotypes. The discrimination of strains with different virulence is a fundamental step for the assessment of pathotypes in local populations and could help to develop measures for preventing economic loss. We here describe an extended and slightly modified bioassay for the assessment of virulence by means of sporulation intensity. Simultaneously, the necrotic reaction of host genotypes is considered and allows identification of different resistance strategies. Comparison of infections symptoms of 12 host genotypes after inoculation with five different single sporangium strains of Plasmopara viticola showed that: 1. resistance diversity is high in some Vitis vinifera. Cultivars; 2. Vitis riparia showed the strongest resistance amongst the four tested North American species; 3. Asian Vitaceae harbor fully resistant as well as highly susceptible genotypes. In addition, microscopic investigation of inoculated leaf discs from Vitis amurensis and Ampelopsis japonica unraveled, that despite lack of sporulation, a mycelium may grow to a certain point in resistant hosts. The necrotic reaction in V. amurensis indicates a different mechanism of resistance when compared to A. japonica, where no necrosis was found.

GLUCOSE-FRUCTOSE INDEX IN THE GRAPES

Results summarize literature and experimental data on the content of glucose and fructose of different varieties in grapes belonging to different botanical species of Vitis. The ratio of glucose and fructose indicator can be used for fermentation control and prevention of under fermentation in the production of dry wines, as well as an identification parameter to assess the authenticity of grape juice and concentrated must. The object of the study were grapes of red and white winemaking European and autochthonous varieties, belonging to Vitis, as well as varieties of new selection (Aligote, Albilio, Verdelho, Sersial, Rkatsiteli, White Muscat, Cabernet-Sauvignon, Bastardo of Magarach, Kephesiya, Ekim kara, Golubok). Sugar content in grape samples was in the range of 180-260 g/l. Total hexoses were determined by HPLC method according to a modified methodology developed by the Department of Chemistry and Biochemistry of Wine of "FSBSI "Magarach ". It was established that the value range of the glucose-fructose index in the grapes cultivated in different viniviticultural regions of the world makes 0.74-1.19. It has been revealed that the glucose-fructose index decreases with the ripening of berries. Low index values are characteristic for the grape that ripens at high temperatures and was cultivated in regions with hot climate. High index values are characteristic of table grapes and winemaking grape varieties of the species Vitis labrusca, Vitis amurensis and interspecific hybrids. Within the botanical species we can identify varieties that tend to accumulate higher volumes of either glucose or fructose. These patterns are equally characteristic of white and red grape varieties. The analytical analyzes of the Crimean winemaking grape varieties resulted in the establishment of the glucose-fructose index for the first time, varying within the range of 0.9-1.06.

Overexpression of VaPAT1, a GRAS transcription factor from Vitis amurensis, confers abiotic stress tolerance in Arabidopsis.

VaPAT1 functions as a stress-inducible GRAS gene and enhanced cold, drought and salt tolerance in transgenic Arabidopsis via modulation of the expression of a series of stress-related genes. The plant-specific GRAS transcription factor family regulates diverse processes involved in plant growth, development and stress responses. In this study, VaPAT1, a GRAS gene from Vitis amurensis was isolated and functionally characterized. Sequence alignment and phylogenetic analysis showed that VaPAT1 has a high sequence identity to CmsGRAS and OsCIGR1, which belong to PAT1 branch of GRAS family and function in stress resistance. The transcription of VaPAT1 was markedly induced by stress-related phytohormone abscisic acid (ABA) and various abiotic stress treatments such as cold, drought and high salinity, however, it was repressed by exogenous gibberellic acid (GA) application. Overexpression of VaPAT1 increased the cold, drought and high salinity tolerance in transgenic Arabidopsis. When compared with wild type (WT) seedlings, the VaPAT1-overexpression lines accumulated higher levels of proline and soluble sugar under these stress treatments. Moreover, stress-related genes such as AtSIZ1, AtCBF1, AtATR1/MYB34, AtMYC2, AtCOR15A, AtRD29A and AtRD29B showed higher expression levels in VaPAT1 transgenic lines than in WT Arabidopsis under normal growth conditions. Together, our results indicated that VaPAT1 functions as a positive transcriptional regulator involved in grapevine abiotic stress responses.

Grape selection for resistance to biotic and abiotic environmental factors

Most of the viticultural regions of the USSR are located under conditions of limiting biotic and abiotic factors, with frosts, drought, fungal diseases, phylloxera, mites, grape berry moths and some others being of primary importance. The main breeding organizations have been creating for more than 40 years new table and wine cultivars with complex resistance according to long-term programs. These cultivars are own-rooted and capable of wintering in outdoor culture with a limited amount of spray treatments, if any. In crossing, Amur grape and its hybrids, cultivars Seibel and Seyve Villard and some others are used as donors of resistance. Using biophysical and cytoembryological methods, gametes are treated with physical and chemical mutagenic factors in order to increase the variability range of F(1) seedlings, aiming at higher efficiency of selection. The process of selection is accelerated if seedlings are grown hydroponically. Analysis of the F(1) hybrid population determines the nature of the inheritance of valuable agricultural characters and the selection of pairs. The in vitro method is used when seedlings are grown from non-vital seeds, callus embryoids and in accelerated propagation of valuable genotypes providing virus and bacteria elimination. More than 50 cultivars with complex resistance have been bred during 35 years. More than 10 of them have been recommended for culture (Moldova, Lyana, Vostorg, Sukholimanski biely, Pervenets Magaracha, and others), while the remainder are being tested in different viticultural regions of the Soviet Union.

VaCPK21, a calcium-dependent protein kinase gene of wild grapevine Vitis amurensis Rupr., is involved in grape response to salt stress

Ca2+-dependent protein kinases (CDPKs) are known to play important roles in signal transduction in response to various abiotic stresses that limit crop yields. Vitis amurensis is a wild grape species that possesses a high adaptive potential and displays a high resistance to environmental stresses, such as low temperatures or microbial pathogens. The roles of most grape CDPKs in the adaptation to various stresses have not yet been uncovered. A recent CDPK gene expression analysis revealed that expression of VaCPK21 gene was significantly up-regulated under salt stress and, to a less degree, under high mannitol and high temperature stresses in V. amurensis plant cuttings. In this study, the effects of overexpressing the VaCPK21 gene in callus cell lines of V. amurensis and transgenic plants of A. thaliana on their responses to abiotic stresses were investigated. The VaCPK21-overexpressing cell cultures of V. amurensis, soil-grown plants of A. thaliana, and seedlings of A. thaliana exhibited higher tolerance to salt stress in comparison with the controls transformed with the “empty” vector. Heat, cold, and drought stress resistance of the transgenic V. amurensis calli and A. thaliana plants was comparable to that of the controls. Under salt stress conditions, the stress-related genes AtCOR15, AtCOR47, AtCAT1, AtCSD1, AtNHX1, AtKIN1, AtRD26, and AtRD29B demonstrated strong up-regulation in the VaCPK21-transgenic but not in the control Arabidopsis plants. Together, the results indicate that VaCPK21 may act as a positive regulator involved in the grapevine response to salt stress.