Vacuolar Contents of Fruit Subepidermal Cells from Vitis Species

Abstract

termination of phenolic compounds, sugars, acids, and cations, the skins were removed manually from the grapes, rinsed briefly with distilled H20, frozen in N2, and freeze-dried. To prevent oxidation of phenolic compounds, vacuum was released with N2, and samples were stored in N2 atmosphere., Preparation of Vacuoles. Grape berries (1 kg) were rinsed briefly in 70%o aqueous ethanol and dried on paper towels, and the skins were manually removed by gentle squeezing and cut in half. Batches of about 30 g of skins were rinsed with 100 ml buffer (17 mm citric acid, 70 mim Na2HPO4, and 1.2 M sorbitol at pH 6.0) for 30 min in 1,000-ml beakers on a reciprocal shaker (100 rpm). The buffer was replaced with a Cellulysin (1.5%; Calbiochem) and Macerase (1%; Calbiochem) solution in the same volume as the above buffer. The grape skins were digested for 7 h at room temperature, the incubation medium decanted, and the protoplasts sedimented at 100g in a centrifuge. The sediment was resuspended in the rinsing buffer from above (1,500 ml) and filtered through a glass wool layer in a Buchner funnel under mild suction (aspirator). This treatment produced vacuoles from protoplasts. The vacuoles were sedimented at 100g, resuspended in 225 ml rinsing buffer, and sedimented by gravity twice. The number of vacuoles in a preparation was determined in a well slide using 5 ,Al aliquot and 20 ul. buffer.Disruption of Vacuoles. The vacuole preparations were transferred into 40-ml centrifuge tubes and the vacuoles sedimented at 50g. The supernatant buffer was decanted, the volume made up to 40 ml with distilled H20, and the preparation homogenized by sonication. Microscopic examination of this preparation showed that all vacuoles had lysed. The homogenate was centrifuged at 180,000g for 2 h to remove membranous material. The supernatant (37 ml) was decanted, frozen in N2, and stored at -40 C.Identification of Anthocyanins. The anthocyanins in DeChaunac grapes have been previously identified (28).Determination of Total Anthocyanin Content in Grape Skins.Freeze-dried grape skin powder (1.0 g) was extracted with 0.1% aqueous HCI (100 ml), the absorbance of this solution measured at 520 nm, and the anthocyanin concentration determined using E = 33,000 1-mol-1cm-' (27).Determination of the Total Anthocyanin Content in Vacuole Preparations. One ml of the vacuole lysate was used for the determination, as described above.Quantitative Determination of the Individual Anthocyanins. The individual anthocyanin content of grape skins and vacuole preparations was determined by quantitative adaptation ofa previously described HPLC method (28). Only pigments present in significant amounts were quantitated. For the establishment of anthocyanin standard curves, the following pigment stock solutions were pre-

Maintenance Carbon Cycle in Crassulacean Acid Metabolism Plant Leaves

“…The changes in intensity of peak 5 ( Fig. 1) (8,11,12,22,26,28). Thus, it is possible that sugars may be stored temporarily in the vacuole during the day.…”

A Rapid Determination Method for Anthocyanin Profiling in Grape Genetic Resources

“…1). No complex formation between anthocyanins and metal ions or flavonoids is involved in the color expression of grapes (Cheynier and Rigaud, 1986;Kitamura et al, 2005a;Moskowitz and Hrazdina, 1981). The total level of anthocyanins increased throughout ripening, whereas composition of the individual anthocyanins did not vary greatly (Boss et al, 1996a;Shiraishi and Watanabe, 1994a).…”

The Vacuolar Localization of Grapevine Peroxidase Isoenzymes Capable of Oxidizing 4-Hydroxystilbenes

“…In this case, an inhibition of 50% was achieved for a cyanin concentration of 50 |JM. These results suggest that the functionality of the 4-HS-oxidizing peroxidase system in anthocyani(di)n-containing vacuoles is doubtful, even more when the anthocyani(di)n concentration found in vacuoles of subepidermal grape-vine cells is in the order of 1 -1 0 m M [28].…”

“…This is sup ported by the fact that resveratrol production [30,31], peroxidase [32], and anthocyani(di)ns [28] are mainly located in the epidermal and the subepider mal cell layers which constitute the berry skin.…”

“…Thus, the particular expres sion o f the phenolic metabolism which exists in the whole fruit is conserved in the in vitro culture as re gards the various forms of anthocyani(di)ns which accumulated [24,25]. In this context, the localiza tion of 4-HS oxidizing isoperoxidases in vacuoles raises the question o f their functionality, since anthocyani(di)ns are mainly located in vacuoles (Table II), and these com pounds are long-known strong antioxidant agents [26,27], Besides, the pH of the grape-vine vacuolar sap is strongly acid (around 3.0) [28], and this may give rise to a non functionality of the 4-HS-oxidizing system. For these reasons, the 4-HS-oxidizing activity of grape-vine peroxidase was analyzed as regards pH and anthocyani(di)n concentration.…”