Chi Bao Do

Effects of low nitrate and high sugar concentrations on anthocyanin content and composition of grape (Vitis vinifera L.) cell suspension

In pigmented cells of Vitis vinifera suspension cultures, best accumulation of anthocyanins was obtained when nitrate concentration was reduced from 25 mM to 6.25 mM and when sucrose concentration was increased from 88 mM to 132 mM. Under such conditions growth was greatly decreased. However, cell viability was maintained. The increases in anthocyanins in pigmented cells were due largely to increases in peonidin — glucoside. The high sucrose and the low nitrate concentrations can be one of the important culture factors in controlling of anthocyanin production by cell cultures.

Accumulation of anthocyanins enhanced by a high osmotic potential in grape (Vitis vinifera L.) cell suspensions.

A cell suspension of grape, Vitis vinifera L. cv Gamay Fréaux, was grown under different conditions of water stress (high external osmotic potential) induced by an increase of sucrose concentration or by the addition of mannitol to the culture medium. Best growth (cell density) was achieved in the low osmotic potential medium. Increasing the osmotic potential of the medium from −0.5 MPa to −0.9 MPa medium resulted in a significant increase in accumulation of anthocyanins in pigmented cells. Regulation of the osmotic potential of culture medium may be useful in controlling anthocyanin production.

Accumulation of peonidin 3-glucoside enhanced by osmotic stress in grape (Vitis vinifera L.) cell suspension

Cell cultures of grapes, Vitis vinifera L. cv Gamay Fréaux were grown under different conditions of external osmotic potential induced by an increase of sucrose concentration or by the addition of mannitol to the culture medium. Addition of 82 mM mannitol or increasing sucrose concentration to 132 mM had similar effects on repressing growth. Cyanidin 3-glucoside, peonidin 3-glucoside and peonidin 3-p-coumaroylglucoside are three main anthocyanins of Vitis cells. Increasing osmotic potential from −0.43 MPa to −0.8 MPa in the medium resulted in a significant intracellular accumulation of anthocyanin especially peonidin 3-glucoside in the pigmented cells. High osmotic potential appears to stimulate the methylation of anthocyanins. Osmotic potential is an important culture factor and may be useful in the controlling of anthocyanin production and composition.

Effects of high ammonium concentrations on growth and anthocyanin formation in grape (Vitis vinifera L.) cell suspension cultured in a production medium

Cultivating Vitis vinifera cell suspensions in a production medium which is characterized by high sucrose and low nitrate concentrations (132 mM and 6.25 mM respectively) repressed growth but enhanced the intracellular accumulation of anthocyanins, especially peonidin 3-glucoside. Increasing the ammonium concentration of the production medium from 2 to 8–16 mM increased growth and decreased the accumulation of anthocyanins and peonidin 3-glucoside specifically. Instead, peonidin 3-p-coumaroylglucoside accumulated. At 24 mM ammonium concentration, growth was inhibited and accumulation of peonidin 3-p-coumaroylglucoside was significant (p<0.05) and represented 42% of total anthocyanins after 12 days of culture compared with 19% in the production medium with 2 mM ammonium.

Isolation and characterization of a UDP-glucose: cyanidin 3-O-glucosyltransferase from grape cell suspension cultures (Vitis vinifera L.)

Abstract An enzyme catalyzing the transfer of the glucosyl moiety of UDP-glucose to the 3-hydroxyl group of cyanidin has been isolated from a Vitis vinifera cell suspension culture. The enzyme was purified 75-fold by ion-exchange, chromatofocusing and gel filtration liquid chromatography with a recovery of 3.8%. The enzyme had a pH optimum at 8.0. The glucosyltransferase showed the highest activity with cyanidin as acceptor but also utilized delphinidin to a significant degree. Pelargonidin, peonidin and malvidin were glucosylated but at a lower rate. An apparent K m value of 1.2 mM for UDP-glucose was determined. At an equal concentration of UDP-glucose the apparent K m values of the enzyme for the acceptors were 18 μM for cyanidin (3′, 4′ OH) and 28 μM for delphinidin (3′, 4′, 5′ OH). The purified enzyme had an isoelectric point of 4.55 and a molecular weight of 56 kDa. Substrate affinity of the enzyme indicates that it is the first step of two anthocyanin branches which begin with cyanidin 3-glucoside and delphinidin 3-glucoside. Also, the lower activity of the enzyme with 3′- and 5′- O -methylated derivatives of cyanidin and delphinidin would explain in part why methylation occurs after the glucosylation step.

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

Abstract A cell suspension of Vitis vinifera cv Gamay Freaux 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.

Anthocyanin production in selected cell lines of grape (Vitis vinifera L.)

SummaryAnthocyanin production of two lines ofVitis vinifera cell cultures, i.e., 5.4 and 13.1, which were obtained from the same starting material after 20 and 37 mo. of clonal selection, respectively, was investigated. Cell suspension cultures of lines 5.4 and 13.1 maintained an anthocyanin content of 0.44 ± 0.15 and 1.02 ± 0.31 mg·g−1 fresh weight during 50 and 32 weekly maintenance subcultures, respectively. Under anthocyanin-promoting culture conditions, both lines showed an enhancement of their anthocyanin level by approximately fourfold. While line 5.4 accumulated peonidin 3-glucoside and cyanidin 3-glucoside in decreasing order, line 13.1 accumulated primarily peonidin 3-p-coumaroylglucoside with lesser amounts of malvidin monoglucoside. Results show that while the anthocyanin content was improved during the course of repeated selections, the anthocyanin composition was modified markedly favoring the accumulation of more metabolically-advanced anthocyanins.

Anthocyanin release from grape (Vitisvinifera L.) cell suspension

SummaryIn order to develop a protocol for the release of intravacuolarly-stored anthocyanins from grapevine (Vitis vinifera L.) cell suspension with preserved viability, the effects of extracellular pH modifications, of solid adsorbents in the culture medium (two-phase system) and of chemical permeabilization were investigated. The solid adsorbent Amberlite IR-120 and chemical permeabilization were efficient in enabling the release of up to 70–80% of vacuolar anthocyanins. However, the release was always at the expense of a proportionate loss in cell viability. Results demonstrate the irreversible nature of anthocyanin sequestration.