The Metabolism of the Organic Acids of Tobacco Leaves * XVIII. EFFECT OF CULTURE OF EXCISED LEAVES IN SOLUTIONS OF POTASSIUM L,-ISOCITRATE HUBERT

Among the unsolved problems of the biochemistry of organic acids in green leaf tissue is the position of isocitric acid in the metabolic system. This acid accumulates in substantial amounts in the leaves of plants of the botanical family Crassulaceae, and has been found in moderate amounts in a number of other species, notably the fruit of the blackberry (Rubus fructicosus), where it was first discovered in nature (I), and the leaves of the foxglove (Digitalis purpurea) (2). Nevertheless, inasmuch as isocitric acid is a member of the tricarboxylic acid cycle, there is little doubt that its distribution, at least in trace amounts, is far wider than these and a few other observations would suggest. Indirect evidence for this is the identification of isocitric dehydrogenase in such plant tissues as cucumber and pea seeds (3), Avenu coleoptile (4), and parsley root (Petroselinum hortense) (5), and in the leaves of chickweed (Xtellaria media), elder (Sambucus nigra) (6)) and tomato (Lycopersicon esculentum) (7). The accumulation of isocitric acid in high concentrations in leaves of crassulacean plants, and to a moderate extent also in a few species in other botanical families, suggests, however, that this substance fulfils some function in them which is quite different from what may be regarded as its usual function as a member of the tricarboxylic acid cycle. Leaves of Bryophyllum calycinum normally contain from 12 to 16 g of isocitric acid per kg of fresh weight. Extensive study of the acids of these leaves during exposure to diurnal alternation of light and dark, or to extended periods of light or of darkness, has shown that isocitric acid remains essentially unaltered in amount throughout the treatment. Notwithstanding this, the malic acid present may decrease from 14 to 2 g per kg during a lo-hour period of illumination and return almost to the initial high level during the following night (8). Even on exposure of the leaves of this plant to light for 64 hours, the isocitric acid in one experiment remained unchanged (9), although malic acid rapidly diminished to a low level. In another similar test, isocitric acid diminished by about 9% of the amount initially present during exposure to light for 74 hours (10). Citric acid undergoes diurnal variation in amount in Bryophyllum leaves in a manner essentially parallel with that of malic acid, although to a much smaller extent with respect to the quantity involved (10). It would appear that the isocitric acid stored in such remarkable amounts’in the leaves of crassulacean plants is used up in the