DISTRIBUTION OF TARTARIC ACID IN THE LEAVES OF CERTAIN ANGIOSPERMS

STAFFORD, HELEN A. (Reed Coll., Portland, Oregon.) Distribution of tartaric acid in the leaves of certain angiosperms. Amer. Jour. Bot. 46(5): 347-352. 1959.-An optically active isomer of tartaric acid has been definitely identified and quantitatively analyzed in the leaves of 9 species of angiosperms, and trace amounts have been tentatively identified in about 22 others out of 49 species examined. In species where identification was positive, the quantity of tartrate varied from 5 to 200 gmoles/g. fresh wt. of leaf tissue; identification was based on paper chromatography and on the metavanadate colorimetric test. The 9 species include Vitis vinifera, V. labruscana, V. californica, Parthenocissus tricuspidata, P. quinquefolia, Pelargonium hortorum, Bauhinia malabarica, Phaseolus vulgaris, and Coleus blumei. Optical rotation data of the isolated acid indicate that the tartrate in Pelargonium and Parthenocissus quinquefolia is the (+) -isomer similar to that reported for Vitis vinifera, but the opposite to that reported for Bauhinia reticulata. The relationship of tartrate to other organic acids is discussed. WHILE MALIC ACID is generally the dominant organic acid that accumulates in the cells of higher plants, occasionally tartaric, citric, or oxalic acid is found in as great or even greater amounts (Pierce and Appelman, 1943). Although tartaric acid has been reported to be present in numerous species (Buchs, 1957), the large majority of older identifications are questionable, and actual quantitative data have been lacking except in a few isolated instances (Franzen and Helwert, 1923; Thimann and Bonner, 1950; Vickery and Palmer, 1954). Furthermore, since the work of Pasteur (1848) on the isomers of tartrate, the optical activity of the tartrate isolated from plants rarely has been checked. One exception is the confirmation of the presence of the (+) -isomer in wine made from the fruits of Vitis vinifera, and the first report of large quantities of the opposite or (-)-isomer in both fruits and leaves of Bauhinia reticulata (Rabate and Gourevitch, 1938). So far, the mesoisomer never has been identified in any plant tissue, although fungi appear to possess enzymes capable of converting epoxysuccinate to mesotartrate (Martin and Foster, 1955). Since some of the isomers of tartrate are active in cell-free enzyme systems isolated from higher plants (Stafford, 1957), the present investigation to restudy the problem of the distribution of tartrate was undertaken, based on more rigid criteria to establish its presence. Leaves, rather than fruits, were analyzed because they are more suitable for research on cell-free enzymes in plants with low pH values, and because there is some indication that the major site of the origin of tartrate is in the leaf (Amerine, 1956). Identification of tartaric acid was considered positive only if all 3 of the following criteria were 1 Received for publication October 27, 1958. This investigation was supported by a grant from the National Science Foundation (NSF-G4651). The author is indebted to Miss Maryanna Pearson for her assistance throughout this work. met: (1) the identification by paper chromatography of an acid spot at the expected Rf value; (2) the detection of an orange spot at the same Rf value after spraying with a solution of sodium metavanadate; (3) the isolation of sufficient amounts for a quantitative colorimetric analysis based on the metavanadate method. This test is relatively specific for either the (+) or (-)-isomer but will not distinguish between them and will not detect mesotartrate. MATERIALS AND METHODS. Leaves were obtained either from greenhouse or native sources. In a few cases, as indicated, air-dried specimens were used and the values for dry wt. were converted to fresh wt. by arbitrarily assuming that the dry wt. was 15% of the fresh wt. Fresh samples were either extracted immediately upon picking or were first frozen for storage. Rootstocks of grape plants were supplied by Dr. C. J. Alley of the Department of Viticulture at the University of California, Davis, California, while seeds of Bauhinia malabarica were made available by Dr. R. B. Ledin of the University of Florida Sub-tropical Experiment Station. About 3 g. fresh wt. of leaves were used as the starting material for extraction. The leaves were ground in a small Waring blendor in hot 50% ethyl alcohol, and Dowex 50 (H+) was added to decationize the sample. After centrifugation, the supernatant and washings of the residue were evaporated to dryness under reduced pressure at 50?C. Only rarely can the crude extract be used directly in the quantitative analysis based on the metavanadate test; generally a partial purification is necessary. The residue was taken up in about 7 ml. of water and suitable aliquots (0.4-3.0 ml.) were applied as a 30-cm.-long streak to filter paper and were chromatographed in an ether-formic acid-water mixture (5:2:1 v/v). A small strip 0.5 cm. wide was cut from the center of the paper and sprayed with a plh indicator (0.1%o bromcresol green) to detect the position of the acids. Afterwards, a second