Metabolism of Isolated Leaves: I. Changes in the Protein, Soluble Nitrogenous Compounds, Sugars, and Organic Acids in Tobacco Leaves in Light and Dark

Abstract

Degradation of protein level in detached leaves has long been known and the accumulation of free amino acids and amide at the expense of protein breakdown has been evident from the time of Schulze (1898) followed by Chibnall (6), Mothes (cf. 6), Yemm. (42), Vickery et al (33), Mc Kee (18), Wood et al (41), and Viets et al (38). However the changes in individual amino acids remained uninvestigated because of the lack of proper methods. During recent years paper chromatography has been widely applied as a tool for biochemical investigations and recently an attempt was made to study the amino acid metabolism of leaves after removal from the plant under conditions of light and dark (23, 24). difficulty in detecting some of the overlapping amino acids by the horizontal migration method necessitated examination employing two-dimensional chromatography of amino acids. Since protein metabolism is closely related to other metabolic activities (6, 12) and hence is difficult to interpret without knowledge of the other processes, it was of interest to follow the changes in the other two important fractions, the sugars and the acids, simultaneously occurring in detached leaves. In this way it was hoped to obtain a clear picture of metabolic events operative during the culture of detached leaves. Nicotiana tabacumi L. was selected as the working material, for its metabolic behavior is known through the work of Vickery and his colleagues (33), and it has been found to exhibit characteristic metabolism not only in relation to protein, amino acids, and amides but also with respect to acids. Although the loss o.f malic acid and gain of citric acid has repeatedly been confirmed for Connecticut tobacco leaves cultured in the dark (33, 34, 35, 36), it becomes, however, difficult to explain these results if the normal citric acid cycle (16) is considered operative in these leaves. At the most it could explained by assuming that in detached leaves cultured in the dark the rate of synthesis of citric acid from malic acid exceeded that of the decomposition of citric acid. How darkening of detached leaves would foster these changes is a matter of speculation. Organic acids of the tricarboxylic acid cycle are not normally consumed for meeting the energy requirements of the tissue, they being intermediate oxidation products. Chibnall (6), while calling it an organic acid phase in the metabolism of starving tobacco leaves, strongly felt that such a behavior would most unlikely if the normal Krebs cycle is considered operative. In the latter case the levels of the respective acids should have remained unchanged. Regarding the possibility of an acid cycle participating in Nicotiana leaves to meet its energy requirements, Chibnall (6) himself wrote: The interpretation that I have given to these five sets of starvation experiments is admittedly speculative in that it is based on the hypothesis of an acid cycle in respiration for which there is no ad hoc proof at present and perhaps there never can be (p. 241). This conclusion was, of course, due to lack of the knowledge of pathways of malic-citric conversion involving enzyme systems other than those in the Krebs cycle at that time. Experiments have been conductecl to elaborate the pathway of malic-citric conversion in Nicotiana leaves. success has been mainly due to the finding that the leaves cultured in streptomycin and penicillin solution in dark failed to show the conversion, which ANicotiana tabacumn var. Cheroot leaves carried out when cultured in water in the dark. This observation made it necessary to examine whether the acid metabolism of variety S-20 leaves which (lid not show this conversion, could also altered by culturing on antibiotic solution, and whether the enzymes for malic-citric conversion of the regular citric acid cycle were operative in these leaves. latter has been demonstrated by the simple method of feeding the leaves with acid salts (succinate and citrate).