Grape Cell Cultures Research Articles

Characterization of a multifunctional caffeoyl-CoA O-methyltransferase activated in grape berries upon drought stress

Drought stress affects anthocyanin accumulation and modification in vegetative and reproductive plant tissues. Anthocyanins are the most abundant flavonoids in grape (Vitis vinifera L.) coloured berry genotypes and are essential markers of grape winemaking quality. They are mostly mono- and di-methylated, such modifications increase their stability and improve berry quality for winemaking. Anthocyanin methylation in grape berries is induced by drought stress. A few caffeoyl-CoA O-methyltransferases (CCoAOMTs) active on anthocyanins have been described in grape. However, no drought-activated O-methyltransferases have been described in grape berries yet.In this study, we characterized VvCCoAOMT, a grapevine gene known to induce methylation of CoA esters in cultured grape cells. Transcript accumulation of VvCCoAOMT was detected in berry skins, and increased during berry ripening on the plant, and in cultured berries treated with ABA, concomitantly with accumulation of methylated anthocyanins, suggesting that anthocyanins may be substrates of this enzyme. Contrary as previously observed in cell cultures, biotic stress (Botrytis cinerea inoculation) did not affect VvCCoAOMT gene expression in leaves or berries, while drought stress increased VvCCoAOMT transcript in berries. The recombinant VvCCoAOMT protein showed in vitro methylating activity on cyanidin 3-O-glucoside. We conclude that VvCCoAOMT is a multifunctional O-methyltransferase that may contribute to anthocyanin methylation activity in grape berries, in particular under drought stress conditions.

Grape apoplasmic β-1,3-glucanase confers fungal disease resistance in Arabidopsis

To identify apoplasmic proteins in grape cells, we investigated proteins secreted into grape cell culture medium and detected β-1,3-glucanase VvGHF17 in the medium. VvGHF17 is expressed constitutively in grape leaves and berry pulp and skin. Experiments using VvGHF17:GFP fusion protein demonstrated that VvGHF17 is localized at the apoplasmic space of cells, suggesting that VvGHF17 is apoplasmic β-1,3-glucanase. To understand how VvGHF17 protects grapevine against phytopathogens, we created Arabidopsis transgenic plants expressing VvGHF17. VvGHF17-expressing Arabidopsis plants exhibited disease resistance to both Botrytis cinerea and Colletotrichum higginsianum, but not to Pseudomonas syringae pv. tomato DC3000. The inhibitory effect of VvGHF17 to the infection by the fungi was promoted when VvGHF17 protein was expressed in large amounts in the plants. In conclusion, apoplasmic VvGHF17 may be a candidate for the transgene to create transgenic grapevines that are robust to multiple fungal attacks.

VaCPK20, a calcium-dependent protein kinase gene of wild grapevine Vitis amurensis Rupr., mediates cold and drought stress tolerance

Abiotic stresses, such as drought, salinity, cold and heat, are major environmental factors that limit crop productivity. Vitis amurensis Rupr. is a wild grapevine species displaying a high level of abiotic and biotic stress resistance. Protein kinases, including Ca2+-dependent protein kinases (CDPKs), are known to mediate plant acclimation to various environmental changes. However, the functions of most grape CDPKs have not been clarified. A recent CDPK gene expression analysis revealed that 10 CDPK genes of V. amurensis were up-regulated under different abiotic stress treatments. The expression of the VaCPK20 gene was significantly up-regulated under low and high temperature stress in V. amurensis. In the current study, the effects of overexpressing the VaCPK20 gene in callus cell lines of V. amurensis and transgenic plants of A. thaliana on their responses to abiotic stresses were investigated. Transgenic Arabidopsis overexpressing the VaCPK20 gene showed higher tolerance to freezing and drought stresses, and transgenic grape cell cultures overexpressing the VaCPK20 gene showed higher resistance to cold stress in comparison with the controls transformed by the “empty” vector. Heat and salt stress resistance of the transgenic V. amurensis calli and A. thaliana was comparable to that of the wild type and vector controls. Overexpression of the VaCPK20 gene increased the expression of stress-responsive genes, such as COR47, NHX1, KIN1, or ABF3, in the transgenic Arabidopsis plants under non-stress conditions, after freezing, and under drought stress. The results imply that VaCPK20 may act as a regulatory factor involved in cold and drought stress response pathways.

Quantitative proteomic analysis of cabernet sauvignon grape cells exposed to thermal stresses reveals alterations in sugar and phenylpropanoid metabolism.

Grapes (Vitis vinifera) are a valuable fruit crop and wine production is a major industry. Global warming and expanded range of cultivation will expose grapes to more temperature stresses in future. Our study investigated protein level responses to abiotic stresses, with particular reference to proteomic changes induced by the impact of four different temperature stress regimes, including both hot and cold temperatures, on cultured grape cells. Cabernet Sauvignon cell suspension cultures grown at 26°C were subjected to 14 h of exposure to 34 and 42°C for heat stress, and 18 and 10°C for cold stress. Cells from the five temperatures were harvested in biological triplicates and label-free quantitative shotgun proteomic analysis was performed. A total of 2042 non-redundant proteins were identified from the five temperature points. Fifty-five proteins were only detected in extreme heat stress conditions (42°C) and 53 proteins were only detected at extreme cold stress conditions (10°C). Gene Ontology (GO) annotations of differentially expressed proteins provided insights into the metabolic pathways that are involved in temperature stress in grape cells. Sugar metabolism displayed switching between alternative and classical pathways during temperature stresses. Additionally, nine proteins involved in the phenylpropanoid pathway were greatly increased in abundance at extreme cold stress, and were thus found to be cold-responsive proteins. All MS data have been deposited in the ProteomeXchange with identifier PXD000977 (http://proteomecentral.proteomexchange.org/dataset/PXD000977).

Regulation of resveratrol production in Vitis amurensis cell cultures by calcium-dependent protein kinases.

Resveratrol is a naturally occurring plant stilbene that exhibits a wide range of valuable biological and pharmacological properties. Although the beneficial effects of trans-resveratrol to human health and plant protection against fungal pathogens are well-established, little is known about the molecular mechanisms regulating stilbene biosynthesis in plant cells. It has been recently shown that overexpression of the calcium-dependent protein kinase VaCPK20 gene considerably increased resveratrol accumulation in cell cultures of Vitis amurensis. It is possible that calcium-dependent protein kinases (CDPKs) play an important role in the regulation of resveratrol biosynthesis. In the present work, we investigated the effects of overexpression of other members of the CDPK multigene family (VaCPK9, VaCPK13, VaCPK21, and VaCPK29) on resveratrol accumulation and growth parameters of grape cell cultures. The obtained data show that overexpression of VaCPK29 increased resveratrol content 1.6-2.4-fold and fresh biomass accumulation 1.1-1.4-fold in the four independently transformed cell lines of V. amurensis compared with that in the empty vector-transformed calli. However, overexpression of the VaCPK9, VaCPK13, and VaCPK21 genes did not considerably affect resveratrol content and fresh/dry biomass accumulation in the independently transformed cell lines of V. amurensis. VaCPK29-transformed calli were capable of producing between 1.02 and 1.39 mg/l of resveratrol, while the control calli produced 0.48 to 0.79 mg/l of resveratrol. The data indicate that the VaCPK9, VaCPK13, and VaCPK21 genes are not involved in the regulation of stilbene biosynthesis in grape cells, while the VaCPK29 and VaCPK20 genes are implicated in resveratrol biosynthesis as positive regulators.

Enhancement of phenolics, resveratrol and antioxidant activity by nitrogen enrichment in cell suspension culture of Vitis vinifera.

Ammonium nitrate (NH4NO3), an important nitrogen source (34% N), has been used as an elicitor to stimulate plant growth and development as well as induce secondary metabolites under controlled conditions. In the present paper, we investigated the enhancement of cell biomass, total phenolics, resveratrol levels, and antioxidant activity of Vitis vinifera cv. Pok Dum by nitrogen enrichment (MS medium supplemented with NH4NO3 at 0, 500, 1,000, 5,000 and 10,000 mg/L). The highest accumulations of biomass, phenolics and resveratrol contents were observed at 8.8-fold (86.6 g DW/L), 15.9-fold (71.91 mg GAE/g DW) and 5.6-fold (277.89 µg/g DW) by the 14th day, in the medium supplemented with 500 mg/L NH4NO3. Moreover, the antioxidant activities of cultured grape cells estimated by the DPPH● and ABTS●+ assay were positively correlated with phenolics and resveratrol, and the maximum activity was also observed in cultured cells with 500 mg/L NH4NO3 at 176.11 and 267.79 mmol TE/100 g DW, respectively.

Effects of chitosan on the protein profile of grape cell culture subcellular fractions

Grapevine is a large source of healthy polyphenols for human diet, and red table-grapes and wines are the main source of stilbenes. These compounds are important both in the plant defence system and for human health. In the present study, Vitis vinifera cv. Barbera cell cultures were treated with 50 μg/mL chitosan and proteomic analyses on soluble and membrane subcellular fractions were performed against suitable controls. Three soluble stilbene synthase protein spots, four stilbene synthase spots in the microsomal fraction and four spots of membrane ATPase subunits were identified, the accumulation of which was modulated in response to chitosan treatment. Present proteomic and immunolocalisation data seem to provide evidence supporting the hypothesis that a stilbene biosynthetic multi-enzyme complex is associated with the intracellular membrane. In addition, proteomic analyses showed a general decrease in the accumulation of proteins belonging to different primary metabolism pathways, both in the soluble and membrane fractions. In particular, energy, sugar and amino acid metabolisms were down-regulated as a consequence of chitosan and acetic acid treatments. These metabolic modifications could lead to a consistent change in the profile and amount of metabolites stored in grape berries, with consequent effects on taste, flavour, organoleptic and nutraceutical properties of derived food products.

Optimisation of phenolic compound biosynthesis in grape (Bogazkere Cv .) callus culture

The objective of this study was to improve metabolite accumulation of grape cells by manipulating culture and environmental conditions. Response surface methodology (RSM) was used to design and conduct the study with the independent variables being: percent depletion of KH 2 PO 4 or NH4NO3 and light intensity. Wounded grape leaves (Bogazkere cv .) were used for the initiation of callus formation. Grape calli were placed on the modified Murashige and Skoog (MS) media (from 0 to 75% KH 2 PO 4 or NH 4 NO 3 depleted) under various light regimes (from 30 to 120 μmol m -2 s -1 ), and incubated in a controlled environment for three weeks to stimulate secondary metabolite biosynthesis. The anthocyanin - total phenolic (TP) contents of the calli were determined using chemical analyses and the antioxidant capacity of the cultures were determined by using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay. Higher KH2PO4 depletion and 75 μmol m -2 s -1 light intensity favored the biosynthesis of anthocyanins and the other phenolic compounds and resulted in elevated antioxidant capacity in the cultures. However, an increase in KH 2 PO 4 or NH 4 NO 3 depletion resulted in a significant decrease in cell weight. Similar to that, increase of light intensity was correlated with decrease of cell weight. Further elevation of light intensity and KH 2 PO 4 -NH4NO3 depletion was fatal to grape cells and resulted in formation of non-growing waterybrownish cultures. Individual phenolic compounds found in the grape cell cultures were identified using the gas chromatography-flame ionization detector (GC-FID) system. Catechin and epicatechin were identified as the most abundant simple phenolic compounds present in the cultures. Keywords : Antioxidant capacity, callus, grape, phenolic compounds, response surface methodology. African Journal of Biotechnology Vol. 12(25), pp. 3922-3933

Хромосомная вариабельность клеток виноградаVitis amurensisRupr., трансформированных растительным онкогеномrolB

The numbers of chromosomes and nucleoli in cultured cells of Vitis amurensis transformed with the rolB oncogene from A. rhizogenes have been studied. In general, the integration of the rolB gene in grape DNA mostly caused the elevation of the level of the chromosome variability, as well as higher numbers of nucleoli in the cultured cells. The possible influence of the observed chromosomal modifications on the productivity parameters of the grape cell cultures is discussed.

Copper Transport and Compartmentation in Grape Cells

Copper-based fungicides have been widely used against several grapevine (Vitis vinifera L.) diseases since the late 1800s when the Bordeaux mixture was developed, but their intensive use has raised phytotoxicity concerns. In this study, physiological, biochemical and molecular approaches were combined to investigate the impacts of copper in grape cells and how it is transported and compartmented intracellularly. Copper reduced the growth and viability of grape cells (CSB, Cabernet Sauvignon Berry) in a dose-dependent manner above 100 µM and was accumulated in specific metal ion sinks. The copper-sensitive probe Phen Green SK was used to characterize copper transport across the plasma membrane of CSB cells. The transport system (K(m) = 583 µM; V(max) = 177 × 10(-6) %ΔF min(-1) protoplast(-1)) was regulated by copper availability in the culture medium, stimulated by Ca(2+) and inhibited by Zn(2+). The pH-sensitive fluorescent probe ACMA (9-amino-6-chloro-2-methoxyacridine) was used to evaluate the involvement of proton-dependent copper transport across the tonoplast. Cu(2+) compartmentation in the vacuole was dependent on the transmembrane pH gradient generated by both V-H(+)-ATPase and V-H(+)-pyrophosphatase (PPase). High copper levels in the growth medium did not affect the activity of V-H(+)-PPase but decreased the magnitude of the H(+) gradient generated by V-H(+)-ATPase. Expression studies of VvCTr genes showed that VvCTr1 and VvCTr8 were distinctly affected by CuSO(4) availability in grape cell cultures and that both genes were highly expressed in the green stage of grape berries.

Influence of calcium influx induced by the calcium ionophore, A23187, on resveratrol content and the expression of CDPK and STS genes in the cell cultures of Vitis amurensis

The present study examines the effect of cal- cium influx induced by the calcium ionophore (CI) on the biosynthesis of resveratrol and the expression of stilbene synthase (STS) and calcium-dependent protein kinase (CDPK) genes in cell cultures of Vitis amurensis, which have different levels of resveratrol production. The present study utilized the control cell culture V2 of V. amurensis, which contains no more than 0.02 % dry weight (DW) of resveratrol, in addition to rolB transgenic cell cultures VB1 and VB2, which have increased resveratrol contents (0.1-0.8 % DW). Treatment with the CI at a 1 lM con- centration significantly increased STS gene expression (6 of 10 analyzed STS genes) and resveratrol production in the control V2 cell culture by fourfold; however, use of the CI at 10 lM significantly decreased resveratrol production by 2-4 fold in all cell cultures tested. In the control V2 grape cell culture, treatment with the CI increased expression of all of the CDPK genes except VaCDPK1a and VaCDPK3a. In the rolB transgenic VB2 grape cell culture treated with the CI, we detected alterations in expression of several CDPK genes, but these changes in gene expression were not significant. Our results indicated that treatment with 1 lM of the CI increased resveratrol content and production in control grape cells by selectively increasing the expres- sion of STS genes. Conversely, the CI treatment did not significantly increase resveratrol content and production, or the expression of CDPK or STS genes in the rolB transgenic cells. Likely, untreated VB2 cells have increased concen- trations of cytoplasmic calcium, and therefore, treatment with the CI did not significantly change CDPK expression. These results suggest that the rolB gene has an important role in the regulation of calcium-dependent transduction pathways in transformed cells.

Complementary Approaches To Gauge the Bioavailability and Distribution of Ingested Berry Polyphenolics

Two different strategies for investigating the likely fate, after ingestion, of natural, bioactive berry constituents (anthocyanins and other non-nutritive flavonoids) are compared. A model of the human gastrointestinal tract (TIM-1) that mimicked the biological environment from the point of swallowing and ingestion through the duodenum, jejunum, and ileum (but not the colon) was used to monitor the stability and bioaccessibility of anthocyanins from both maqui berry and wild blueberry. TIM-1 revealed that most anthocyanins were bioaccessible between the second and third hours after intake. Alternatively, biolabeled anthocyanins and other flavonoids generated in vitro from berry and grape cell cultures were administered to in vivo (rodent) models, allowing measurement and tracking of the absorption and transport of berry constituents and clearance through the urinary tract and colon. The advantages and limitations of the alternative strategies are considered.

Stimulation of anthocyanin synthesis in grape (Vitis vinifera) cell cultures by pulsed electric fields and ethephon

Anthocyanin from grape cell cultures can be used as a natural alternative to synthetic dyes; particularly due to their reported health-promoting properties. In this study, production of anthocyanin in cell suspension culture of Vitis vinifera was evaluated following treatment with either ethephon and/or pulsed electric fields (PEF). Overall, total production of anthocyanin increased in treated cells compared to untreated cells. Treatment of cell suspension with PEF at day 14 of culture resulted in 1.7-fold increase (1.42 mg/g DW) in anthocyanin content when compared to control cells; while, treatment with ethephon resulted in 2.3-fold increase (1.99 mg/g DW) in anthocyanin content. When cells were treated with both ethephon and PEF, 2.5-fold increase in anthocyanin content (2.2 mg/g DW) was observed. These findings demonstrate that PEF induces a defense response in plant cells, and it may also alter the dielectric properties of cells and/or cell membranes, and would serve as a viable elicitor of secondary metabolites in plant cell cultures.

Regulation of resveratrol biosynthesis in grape cell cultures by salicylic acid and rol genes of Agrobacterium rhizogenes

Regulation of resveratrol biosynthesis in grape cell cultures by salicylic acid and rol genes of Agrobacterium rhizogenes

Increasing sucrose concentrations promote phenylpropanoid biosynthesis in grapevine cell cultures

Vitis vinifera cell suspensions are a suitable model system to study the metabolic regulation of a large range of high valuable polyphenols that are important in understanding the physiology of the plant and for nutraceutical, pharmaceutical and medical purposes. Increasing sucrose concentrations were found to promote cell growth and phenylpropanoid biosynthesis in grape cell cultures obtained from cv. Barbera immature berries. This led to an intracellular accumulation and/or release into the media of specific polyphenol families (in particular, anthocyanins, catechins and stilbenes). This effect was partially correlated with a sucrose modulation of the transcription of some key biosynthetic enzymes, such as phenylalanine ammonia lyase, chalcone synthase, chalcone-flavanone isomerase and stilbene synthase. Total catechin amounts, both endogenous and released in the media, were increased in proportion to the sugar concentration, as were anthocyanin and stilbene production. Sugar treatment notably improved the endogenous accumulation and release in the culture media of resveratroloside, a resveratrol mono-glycoside, which is the most abundant stilbene found in grape cultures, especially in cv. Barbera. We hypothesize that high sucrose concentrations (exceeding those naturally-occurring in ripe berries) could play a role in plant defense via the induction of secondary metabolites, such as stilbenes.

Stimulation of anthocyanin synthesis in grape (Vitis vinifera) suspension cultures by different enzymes

The use of coloring as additives in foods and drinks is a significant factor for food manufactures and customers alike in determining the acceptability of processed foods. The accumulation of colors and pigments in cell cultures can facilitate by direct selection of high producing cell lines. Anthocyanins are compounds that provide some of the coloring pigment of plants, flowers and fruits. Anthocyanin from grape cell cultures can be used as a natural alternative to synthetic dyes, particularly because of their various health-promoting properties. The present study was concentrated on the production of anthocyanin in suspension culture of Vitis vinifera by exposing them to different enzymes that can act as elicitors. In order to enhance the productivity of anthocyanin from grape (Vitis vinifera) cell cultures, different enzymes such as Indanyl isoleucine, Linolenoyl Glutamine, Malonyl coenzyme A and saliva were applied to the cell cultures. The results indicate that the treatment with Indanyl isoleucine and saliva can give higher concentration of anthocyanin than control ones in the cell culture during 12 days. This shows the effect of enzymes as elicitors which can affect the anthocyanin synthesis in the grape cells. Keywords: anthocyanin, plant cell culture, elicitation, enzymes.

ABA Initiates Anthocyanin Production in Grape Cell Cultures

Abscisic acid (ABA) has a well-known positive impact on grape ripening, especially color development, but its role in the initiation of anthocyanin synthesis remains unclear. To elucidate this point, ABA treatment was applied to a simple Vitisvinifera model, consisting of Cabernet Sauvignon cell suspensions that do not spontaneously produce anthocyanins under laboratory conditions. Endogenous ABA levels, the expression of some genes in the upstream part of the anthocyanin pathway, and anthocyanin content were determined. Exogenous ABA treatment sharply increased cell ABA content and induced both structural and regulatory genes involved in anthocyanin production. These changes were promptly detected, as early as 6 h after ABA treatment, whereas anthocyanin production was observed only after 4 days in culture. These results demonstrate that ABA promotes anthocyanin synthesis in grape cell culture.

Effect of Elicitors and Precursors on the Synthesis of Anthocyanin in Grape Vitis vinifera Cell Cultures

Problem statement: Nowadays, plant cell suspension cultures and immobilized cells are being utilized for the higher yield and quality of the products than extraction of whole plants. Anthocyanins are compounds found in plants that have powerful antioxidant properties. They also provide some of the coloring or pigment of plants, flowers and fruits. Anthocyanin from grape cell cultures can be used as a natural alternative to synthetic dyes, particularly in light of their various health-promoting properties. To enchance the production of anthocyanin in grape (Vitis vinifera) cell culture, the effects of elicitation and precursor feeding on the colorant production were investigated. Approach: In this study, salicylic acid and ethephon were used as elicitors and, phenylalanine and skhikimic acids were used as precursors to improve the productivity of useful metabolites for archiving high concentration in Vitis vinifera suspension cultures. When the cells were elicitated with 50 µL/25 mL suspension salyclic acid, the anthocyanin concentration was increased to 0.03 µg mg-1 in 18 days culture as compared to that in the unelicitated cells. Results: As for precursors, both of the shikimic acid and phenylalanine could promote the synthesis of anthocyanin in the grape cell cultures. After 18 days of the treatment with shikimic acid, it had shown that the anthocyanin concentration in the treatment was 0.05 µg mg-1 more than the control in the cell cultures. Conclusion: In the cell cultures with phenylalanine showed that anthocyanin synthesis was 0.03 µg mg-1 higher than that of without phenylalanine.

FLAVOUR BIOSYNTHESIS PATHWAYS IN GRAPE CELL CULTURES: SESQUITERPENES BIOSYNTHESIS

FLAVOUR BIOSYNTHESIS PATHWAYS IN GRAPE CELL CULTURES: SESQUITERPENES BIOSYNTHESIS

Induction of secondary metabolism in grape cell cultures by jasmonates.

The use of a genetic approach to study the biosynthetic pathways leading to the production of secondary metabolites in grapes is difficult given the long generation times and difficulty in transforming this species. In the present study, GC/MS and microarray experiments were used to identify compounds produced in grape cell cultures in response to jasmonates and to examine the expression of genes from pathways that produce these secondary metabolites. Methyl jasmonate (MeJA) and jasmonic acid (JA) treatments resulted in the production of at least 25 compounds with sesquiterpene-like mass spectra in the cell cultures. A significantly greater amount of proanthocyanidins was produced in the MeJA-treated cell cultures compared with controls and stilbene biosynthesis was induced in both MeJA- and JA-treated cells. Salicylic acid (SA) suppressed the MeJA-associated increase in sesquiterpenes and proanthocyanidins, but SA did not suppress the stilbene production induced by MeJA treatment. The mechanism by which jasmonates induced secondary metabolite production in cultured grape cells varied depending on the pathway. The increased production of proanthocyanidins and stilbenes was associated with the induction of all of the genes in associated biosynthesis pathways, including those involved in the production of phenylalanine, whereas increased sesquiterpene synthesis was linked to the induction of certain genes from relevant biosynthesis pathways.