Vitis Vinifera Cell Suspension Cultures Research Articles

Melatonin and phenolics biosynthesis-related genes in Vitis vinifera cell suspension cultures are regulated by temperature and copper stress

Temperature and copper stress are two common abiotic stresses in viticulture. To better understand biosynthesis of melatonin and phenolics in response to these two abiotic stresses, cell suspension cultures of Vitis vinifera were treated with high temperature (HT, 38 °C), low temperature (LT, 16 °C) and 100 μM CuSO4 to investigate whether genes associated with the biosynthesis of melatonin (TDC, SNAT and ASMT) and phenolics (PAL, C4H, CHS, CHI and STS) were involved in response to these two abiotic stresses, as well as the effects of these two stresses on total phenolics, total flavonoids contents and oxidative stress. The expression patterns indicated that TDC, SNAT, ASMT, PAL and C4H were significantly upregulated in response to HT, while CHS and CHI first increased and then decreased, and STS was significantly downregulated. SNAT was significantly upregulated in response to LT, while TDC, ASMT, C4H and CHS first increased and then decreased, and PAL, CHI and STS were significantly downregulated. Copper treatment significantly induced the expression of TDC, SNAT and ASMT, and ASMT was the most significantly induced. C4H, CHI and STS were significantly upregulated in response to copper treatment, while PAL first increased and then decreased. Both HT and LT treatment significantly enhanced the contents of total phenolics and total flavonoids. The malondialdehyde (MDA) content of the cells treated with HT significantly increased. The treatment with CuSO4 significantly enhanced the contents of MDA and H2O2 in the cells. The results suggest that melatonin and phenolics biosynthesis-related genes were involved in HT, LT and copper stress response. The same gene showed different response patterns to the different stresses. HT and copper stress may cause oxidative stress. Whether the biosynthesis of melatonin and phenolics in grapes was also involved in response to temperature and copper stress will provide inspiration for the practice of stress-resistant viticulture

Effect of Salicylic Acid on the Gene Transcript and Protein Accumulation of Flavonoid Biosynthesis-related Enzymes in Vitis vinifera Cell Suspension Cultures

Salicylic acid (SA) as a plant signal molecule plays an important regulatory role in stimulating the accumulation of plants’ defensive compounds. Numerous studies have shown its regulating effects on the biosynthesis of flavonoids. However, the controlling mechanism needs to be made clear further. Here, Vitis vinifera L. ‘Cabernet Sauvignon’ cell suspension cultures were treated with SA and its synthesis inhibitor paclobutrazol (Pac) to investigate their effects on flavonoid biosynthesis. The accumulation of gene transcripts and proteins of flavonoid biosynthesis pathway enzymes, including chalcone synthase (VvCHS), chalcone isomerase (VvCHI), dihydroflavonol 4-reductase (VvDFR), and anthocyanidin synthase (VvANS), were detected using real-time polymerase chain reaction (RT-PCR), quantitative polymerase chain reaction (qPCR), and western-blotting techniques. The results showed that treatment with exogenous SA at the proper concentration enhanced the production of flavonoids such as anthocyanins and proanthocyanidins (PAs) inside the suspension-cultured cells. The induction of anthocyanins and PAs was found to be time course-dependent. The mRNA and protein accumulation of VvCHS, VvCHI, VvDFR, and VvANS in the treatment system were enhanced too, and the changes were observed concomitantly. Treatment of Pac inhibited the induction of the accumulation of gene transcripts and proteins in accordance with the decreased accumulation of flavonoids. These results suggest that exogenous SA could induce both transcript and protein accumulation of flavonoid biosynthesis-related enzymes and in turn enhance the accumulation of flavonoid compounds such as anthocyanins and PAs in suspension-cultured grape cells.

The effect of methyl jasmonate and light irradiation treatments on the stilbenoid biosynthetic pathway in Vitis vinifera cell suspension cultures

Grape stilbenes are a well-known family of plant polyphenolics that have been confirmed to have many biological activities in relation to health benefits. In the present study, we investigated the effect of methyl jasmonate (MeJA) elicitor at four different concentrations (25, 50, 100 and 200 μM) in combination or not with high-level light irradiation (10,000 LUX) on a cell line obtained from the pulp of Vitis vinifera cv. Shahani. Our results showed that the stilbene synthesis pathway is inhibited by high-light conditions. A concentration of 50 μM MeJA was optimum for efficient production and high accumulation of total phenolics and total flavonoids as well as total stilbenoids. Furthermore, we showed that there is a significant negative correlation between the production of these metabolites and cell growth. These data provide valuable information for the future scale-up of cell cultures for the production of these very high value compounds in bioreactor system.

Combined elicitation of chitosan and ultraviolet C enhanced stilbene production and expression of chitinase and β-1,3-glucanase in Vitis vinifera cell suspension cultures

Cell suspension cultures of Vitis vinifera were treated with chitosan and ultraviolet C (UV-C) irradiation, alone or in combination, to investigate the relationship between the production of stilbene and the transcript profiles of genes encoding the enzymes involved in stilbene synthesis. Treatment at the proper concentration or dosage enhanced both stilbene production inside the cells and trans-resveratrol accumulation in the culture medium without loss of biomass, and the combined treatment was the most efficient. Total intracellular stilbene content was markedly increased by the joint use of chitosan and UV-C, reaching the maximum at 60 h (1945.91 ± 70.73 μg g−1 DW; 207.23 % higher than in untreated cells) after elicitation. When cell cultures were supplemented with both elicitors, we observed a synergistic effect on the accumulation of trans-resveratrol release into the culture medium with the maximal concentration of 3.05 ± 0.17 mg l−1 at 60 h. Total phenolics and total flavonoids contents were also highly increased after elicitations. The expression levels of genes associated with stilbene biosynthesis were significantly up-regulated in response to the two elicitors, and the combined elicitation showed a synergistic effect on the expression of stilbene synthase. Furthermore, the expression levels and enzyme activities of two pathogenesis-related proteins, chitinase and β-1,3-glucanase, increased after treatment. The results suggest that the combined treatment of chitosan and UV-C irradiation can significantly enhance the production of stilbene in V. vinifera cell cultures and may induce defense response by increasing the expression and activity levels of chitinase and β-1,3-glucanase.

Oligochitosan and sodium alginate enhance stilbene production and induce defense responses in Vitis vinifera cell suspension cultures

Vitis vinifera L. cv. Cabernet Sauvignon cell suspension cultures were treated with oligochitosan (OCS) and sodium alginate (NaAlg) at various concentrations for different exposure times to investigate the effects on stilbene biosynthesis. In this study, adding optimal concentrations of OCS (100 mg l−1) and NaAlg (100 mg l−1) significantly enhanced both stilbene production inside the cells and trans-resveratrol accumulation in the culture medium without loss of biomass. OCS and NaAlg markedly increased total intracellular stilbene production (1212.05 ± 36.83 and 990.74 ± 24.24 μg g−1 DW; 96.32 and 60.48 % higher than in untreated cells, respectively), and trans-resveratrol release into the culture medium was induced to the maximum of 3.26 ± 0.14 and 2.76 ± 0.12 mg l−1, respectively. Total phenolics and total flavonoids contents were also highly increased after the application of the two elicitors. The expression levels of genes involved in stilbene and flavonoid biosynthesis were significantly up-regulated in the presence of OCS and NaAlg. Further, the expression levels and enzyme activities of two pathogenesis-related proteins, chitinase and β-1,3-glucanase, were induced after the addition of elicitors. The results suggest that OCS and NaAlg can significantly enhance the production of stilbene in V. vinifera cell cultures and may induce the defense responses by the up-regulation of chitinase and β-1,3-glucanase at transcript and enzyme activity levels.

Erratum to: Biosynthesis of phenolic compounds in Vitis vinifera cell suspension cultures: Study on Hydroxycinnamoyl CoA: ligase

In cell suspensions cultures from grape berry pulp (Vitis vinifera cv. Gamay fréaux)hydroxycinnamoyl CoA ligase (CoAL) displayed maximum activity (100 %) forp-coumaric acid and then, in decreasing order, for ferulic acid (81.3 %) and caffeic acid (60.4 %). No activity was detected with sinapic and cinnamic acids. The changes in CoAL activity during the growth cycle of the culture displayed two peaks : the highest (6 h after subculturing) was linked with a strong increase in protein caused by dilution ; the second was weaker and occurred on the 7th day of culture.Grape cell suspension accumulated mainly peonidin (Pn) and cyanidin (Cy) glucosides (Pn 3-glucoside, Cy 3-glucoside, Pn 3-acetylglucoside, Pn 3-caffeylglucoside, Pn 3-p-coumarylglucoside, and Cy 3-p-coumarylglucoside). Maximum accumulation of anthocyanins was associated with the exponential growth phase of the culture and might be the result of the substantial increase in CoAL activity resulting from the effect of dilution. The second enzyme activity peak was probably oriented towards the acylation of anthocyanins since the percentage of acylated forms increased with time after subculturing.

Enhanced Resveratrol Production in Vitis vinifera Cell Suspension Cultures by Heavy Metals Without Loss of Cell Viability

The effects of heavy metal ions (Co(2+), Ag(+), Cd(2+)) on cell viability and secondary metabolite production, particularly anthocyanins and phenolic acids in Vitis vinifera cell suspension cultures, were investigated. Of these, Co at all three used concentrations (5.0, 25, and 50μM), Ag, and Cd at low concentration (5.0μM) were most effective to stimulate the phenolic acid production, increasing the 3-O-glucosyl-resveratrol up to 1.6-fold of the control level (250.5 versus 152.4μmol/g), 4h after the treatments. Meanwhile, the elicitors at effective concentrations did not suppress cell growth, while the cell viability maintained. In contrast, Ag and Cd at high concentrations (25 and 50μM) remarkably reduced the cell viability, decreasing the cell viability up to about 15% of the control level, 24h after the treatments. The heavy metal ions did not affect the anthocyanin production. These observations show how, in a single system, different groups of secondary products can show distinct differences in their responses to potential elicitors. The 1,1-diphenyl-2-picrylhydrazyl radical scavenging activity, peroxidase activity, medium pH value, and conductivity were only slightly elevated by the heavy metal ions. The results suggest that some of the secondary metabolites production was stimulated by the used elicitors, but there was not a stress response of the cells.

Characterization of anthocyanic vacuolar inclusions in Vitis vinifera L. cell suspension cultures

Anthocyanic vacuolar inclusions (AVIs) are intra-vacuolar structures capable of concentrating anthocyanins and are present in over 50 of the highest anthocyanin-accumulating plant species. Presence of AVIs alters pigment intensity, total anthocyanin levels, pigment hue and causes bathochromic shifts in a spatio-temporal manner within various flowers, vegetables and fruits. A year-long study on Vitis vinifera cell suspension cultures found a strong correlation between AVI prevalence and anthocyanin content, but not the number of pigmented cells, growth rate or stilbene content. Furthermore, enhancement of the prevalence of AVIs and anthocyanins was achieved by treatment of V. vinifera cell suspension cultures with sucrose, jasmonic acid and white light. A unique autofluorescence of anthocyanins was used to demonstrate microscopically that AVIs proceed from the cytosol across the tonoplast and were able to coalesce intravacuolarly, with fewer, larger AVIs predominating as cells mature. Purification and characterisation of these bodies were performed, showing that they were dense, highly organic structures, with a lipid component indicative of membrane-encasement. These purified AVIs were also shown to comprise long-chain tannins and possessed an increased affinity for binding acylated anthocyanins, though no unique protein component was detected.

Metabolomic profiling of Vitis vinifera cell suspension culture elicited with silver nitrate by 1H NMR spectrometry and principal components analysis

The metabolomic profiling of Vitis vinifera cell suspension cultures with and without silver nitrate was performed by 1H NMR (nuclear magnetic resonance) spectrometry and principal components analysis (PCA), to assess the efficacy of this method for the characterization and monitoring of plant cell lines. The PCA of the 1H NMR spectra of the aqueous fractions allowed a clear discrimination of V. vinifera cell suspension culture samples with and without silver nitrate treatment by the first three principal components (PC1, PC2, and PC3), which cumulatively accounted for 95.9% of the variation in all variables. In particular, the score plots by the combining PC1 versus PC2 and PC2 versus PC3 facilitated an excellent separation of samples. In addition, the major peaks in 1H NMR spectra contributing to the discrimination were assigned to lactate, alanine, acetic acid, choline, fructose, α-glucose, and sucrose. This method based on metabolomic analysis allows the efficient monitoring and the differentiation of normal cell suspension system from elicited systems without any prepurification steps.

Galloylated catechins and stilbene diglucosides in Vitis vinifera cell suspension cultures

Suspension cultures of Vitis vinifera were found to produce catechins and stilbenes. When cells were grown in a medium inducing polyphenol synthesis, (−)-epicatechin-3- O-gallate, dimeric procyanidin B-2 3′- O-gallate and two resveratrol diglucosides were isolated, together with a new natural compound that was identified as cis-resveratrol-3,4′- O- β-diglucoside by spectroscopical methods.

Carbon-14 biolabelling of wine polyphenols in Vitis vinifera cell suspension cultures

14C- l-phenylalanine is incorporated into a range of polyphenolic compounds when fed to grape cell cultures. Optimisation of several parameters such as the quantity of precursor applied and the duration of metabolism led to incorporation yields of 15% and to specific activities of 875 μCi g −1 in stilbenes. Purification of the products by several chromatographic steps is reported. Both trans- and cis-resveratrols were easily obtained by enzymatic hydrolysis of their corresponding glucosides, with specific activity of 1200–1400 μCi g −1. The specific radioactivity obtained for all the compounds is suitable for in vivo feeding trials to trace their metabolic fate when consumed by animals and for in vitro activity mechanism studies. Indeed, these polyphenols seem to be implicated in the health benefits associated with regular and moderate wine consumption but little is known about their pharmacokinetics and cellular uptake.

Production of 13C-labelled anthocyanins by Vitis vinifera cell suspension cultures.

The use of plant cell cultures for producing isotopically (13C) labelled phenolic substances is reported. Vitis vinifera cells synthesize high levels of anthocyanins when they are cultured in a polyphenol synthesis-inducing medium. Three major anthocyanin monoglucosides found in red wine were identified in grape cells: cyanidin-3-O-beta-glucoside, peonidin-3-O-beta-glucoside, and malvidin-3-O-beta-glucoside. Kinetic study of the intracellular level of phenylalanine and its metabolites showed that it is preferable to add this precursor to grape cell suspensions after the 5th day of culture, i.e. at the beginning of the exponential growth phase. After adding phenylalanine to the culture medium, its uptake was complete and the accumulation of anthocyanins in grape cells was stimulated. Incorporation of [1-13C]-phenylalanine into anthocyanins was measured by means of 13C satellites in the proton NMR spectrum. The maximal rate of 13C enrichment anthocyanins obtained with this technique reached 65%. The production of 13C labelled phenolic compounds was undertaken in order to investigate their absorption and metabolism in humans.

Regulation of polyphenol production in Vitis vinifera cell suspension cultures by sugars.

Sucrose was found to modulate polyphenol accumulation in Vitis vinifera cell cultures. The production of anthocyanins increased 12-fold after addition of 0.15 M sucrose, while that of stilbenes was only slightly affected. Sucrose did not play a physical role because metabolic sugars were required for the induction of polyphenol accumulation. Indeed, the polyols, mannitol and sorbitol, had no effect on this accumulation. We established a model system to investigate the mechanism of sucrose regulation of polyphenol production without inhibition of grape cell growth. After addition of sucrose to the culture medium, the major sugars accumulated in grape cells were glucose and fructose, reaching 40% of the dry weight. The increase in the level of these hexoses closely coincided with the increase in anthocyanin accumulation in grape cells.

Characterization and expression of caffeoyl-coenzyme A 3-O-methyltransferase proposed for the induced resistance response of Vitis vinifera L.

Cell-suspension cultures of Vitis vinifera L. cv Pinot Noir accumulated resveratrol upon fungal elicitation, and the activity of S-adenosyl-L-methionine:trans-caffeoyl-coenzyme A 3-O-methyl-transferase (CCoAOMT), yielding feruloyl-CoA, increased to a transient maximum at 12 to 15 h. CCoAOMT cDNA was cloned from the elicited cells and was shown to encode a polypeptide highly homologous to CCoAOMTs from cells of Petroselinum species or Zinnia species. The expression of the cDNA in Escherichia coli revealed that grapevine CCoAOMT methylates both caffeoyl- and 5-hydroxyferuloyl-coenzyme A and is probably involved in phenolic esterification and lignification. Commercial plant activators induce the disease-resistance response of test plants and are considered to mimic the action of salicylic acid. Among these chemicals, 2,6-dichloroisonicotinic acid and benzo(1,2,3)-thiadiazole-7-carbothioic acid S-methyl ester provoke systemic acquired resistance (SAR) and were also shown to induce the expression of class III chitinase in grapevine. The SAR response is classified by an unchanged phenotype of tissues, but the mechanistic basis is unknown. Treatment of the cultured V. vinifera cells with either fungal elicitor or low concentrations of salicylic acid and 2,6-dichloroisonicotinic acid, respectively, raised the CCoAOMT or stilbene synthase transcript abundance, suggesting that grapevine is capable of the SAR response, whereas benzo(1,2,3)-thiadiazole-7-carbothioic acid S-methyl ester was ineffective. The data imply for the first time (to our knowledge) that the expression of phenyl-propanoid genes in grapevine is induced by SAR activators without phenotypic consequences and suggest a role for CCoAOMT and stilbene synthase in the disease-resistance response leading beyond the level of pathogenesis-related proteins as markers of the SAR.

Characterization and activities of S-adenosyl- l-methionine:cyanidin 3-glucoside 3′- O-methyltransferase in relation to anthocyanin accumulation in Vitis vinifera cell suspension cultures

A cell suspension of Vitis vinifera cv Gamay Fréaux was grown in a maintenance and an anthocyanin-promoting medium (APM). Time-course changes in anthocyanin accumulation and S-adenosyl- l-methionine:cyanidin 3-glucoside 3′- O-methyltransferase (CGMT) activity were examined throughout the growth cycle. An increase in anthocyanin accumulation mainly due to peonidin 3-glucoside and peonidin 3- p-coumaroylglucoside i.e. 3′-methylated derivatives of cyanidin 3-glucoside (Cy3G), of cells grown in APM was observed. The higher activity of CGMT in cells grown in the APM might be responsible for the enhanced formation of 3′-methylated anthocyanins. CGMT isolated from cell suspension cultures has been purified approx. 35-fold with 3.4% enzyme activity recovery using anion exchange, chromatofocusing and gel filtration liquid chromatography. Mg 2+ ions strongly enhanced the reaction rate at a concentration of 5 mM. Optimal pH of CGMT ranged from 7.75 to 9.75. Michaelis-Menten kinetics were observed at pH 8.75 with respect to the substrates Cy3G and S-adenosyl- l-methionine with K m values of 199 and 18 μM respectively. S-adenosyl- l-homocysteine showed a non-competitive inhibition with an approx. K i value of 50 μM. Relative to Cy3G, the activity of CGMT was 13% with cyanidin. No activity was observed with delphinidin or cyanidin 3- p-coumaroylglucoside.

Condensed tannin and anthocyanin production in Vitis vinifera cell suspension cultures

Suspension cultures of Vitis vinifera were cultured in different media in order to establish a model system for promoting high levels of phenolic substances identical with those found in wine. These media were: a low sucrose maintenance medium (MM) and four high sucrose media (differing mainly in sucrose and mineral contents) which were shown to induce secondary metabolism. In MM medium, polyphenol accumulation in the cells was low, and concentrations of 0.1 mg/gfw for condensed tannins and 0.3 mg/gfw for anthocyanins were reached within two weeks of cultivation. Values of 1.4 and 6.4 mg/gfw, respectively, were obtained with a low nitrate and high sucrose medium (HM1), but cell proliferation was reduced. To obtain a maximal production of polyphenols, we investigated the most effective conditions for cell growth and polyphenol production (a high mineral and high sucrose medium, IM1; inoculum dilution of 1.25:10). Under these conditions, the cells produced mainly anthocyanins (1100 mg/l), proanthocyanidins (300 mg/l) and catechins (25 mg/l).

Condensed tannin and anthocyanin production in Vitis vinifera cell suspension cultures

Condensed tannin and anthocyanin production in Vitis vinifera cell suspension cultures

Growth kinetics of vitis vinifera cell suspension cultures: I. Shake flask cultures

Vitis vinifera cell suspension cultures carried out in shake flasks were closely examined for biomass growth and cell division in relation to carbohydrate, NH(4), NO(3)PO(4), and dissolved oxygen (DO)consumption. After inoculation, the oxygen uptake rate of the cultures measured on-tine was observed to increase continuously to a maximum value of 3.8 mmol O(2)L(-1)h(-1) at day 7 when cell division ceased and dissolved oxygen reached its lowest level of 17% air saturation. During this first phase of growth, the specific oxygen uptake rate remained constant at approximately 0.6 mmol 02 O(2) g(-1) dw h(-1)or approximately 2.2 mumol O(2), (10(6) cells)(-1) h(-1) whereas dry biomass concentration increased exponentially from 1.5 to 6.0 g dw L(-1). Thereafter, dry biomass concentration increased linearly to approximately 14 g dw L(-1) at day 14 following nitrate and carbohydrate uptake. During this second phase of growth, the biomass wet-to-dry weight ratio was found to increase in an inverse relationship with the estimated osmotic pressure of the culture medium. This corresponded to inflection points in the dry and wet biomass concentration and packed cell volume curves. Furthermore, growth and nutrient uptake results suggest that extracellular ammonium or phosphate ion availability may limit cell division. These findings indicate that cell division and biomass production of plant cell cultures may not always be completely associated, which suggests important new avenues to improve their productivity. (c) 1995 John Wiley & Sons, Inc.