Plant Physiol, November 2000, Vol. 124, pp. 1335-1348

The Role of Vacuolar Malate-Transport Capacity in Crassulacean Acid Metabolism and Nitrate Nutrition. Higher Malate-Transport Capacity in Ice Plant after Crassulacean Acid Metabolism-Induction and in Tobacco under Nitrate Nutrition¹

Institute of Botany, Darmstadt University of Technology, Schnittspahnstrasse 3-5, D-64287 Darmstadt, Germany

Anion uptake by isolated tonoplast vesicles was recorded indirectly via increased H⁺-transport by H⁺-pumping of the V-ATPase due to dissipation of the electrical component of the electrochemical proton gradient, µ_H+, across the membrane. ATP hydrolysis by the V-ATPase was measured simultaneously after the Palmgren test. Normalizing for ATP-hydrolysis and effects of chloride, which was added to the assays as a stimulating effector of the V-ATPase, a parameter, J_mal^rel, of apparent ATP-dependent malate-stimulated H⁺-transport was worked out as an indirect measure of malate transport capacity. This allowed comparison of various species and physiological conditions. J_mal^rel was high in the obligate crassulacean acid metabolism (CAM) species Kalanchoë daigremontiana Hamet et Perrier, it increased substantially after CAM induction in ice plant (Mesembryanthemum crystallinum), and it was positively correlated with NO₃ nutrition in tobacco (Nicotiana tabacum). For tobacco this was confirmed by measurements of malate transport energized via the V-PPase. In ice plant a new polypeptide of 32-kD apparent molecular mass appeared, and a 33-kD polypeptide showed higher levels after CAM induction under conditions of higher J_mal^rel. It is concluded that tonoplast malate transport capacity plays an important role in physiological regulation in CAM and NO₃ nutrition and that a putative malate transporter must be within the 32- to 33-kD polypeptide fraction of tonoplast proteins.