Malate-Dependent Proton Transport in Tonoplast Vesicles Isolated from Orchid Leaves Correlates with the Expression of Crassulacean Acid Metabolism

The relationship between the expression of crassulacean acid metabolism (CAM) and the electrophoretic transport of malate 2− into isolated tonoplast vesicles was investigated for leaves of three orchid species. The thick-leaved orchid species Epidendrum ibaguense exhibited a diel rhythm in both cell-sap titratable acidity and cell-sap malate concentration, characteristic of CAM, whereas the thin-leaved C3 species Coelogyne cristata and Coelogyne fimbriata did not. Tonoplast vesicles were prepared from leaf mesophyll homogenates of these three species by a combination of differential and sucrose density gradient centrifugation and were used to study the effects of anions on ATP- and PPi-dependent H+ transport. In tonoplast vesicles from C. cristata and C. fimbriata, chloride stimulated H+ transport (measured as quinacrine-fluorescence quenching) more effectively than malate. But in tonoplast vesicles from E. ibaguense, malate was as effective as chloride in stimulating H+ transport, reflecting the greater fluxes of malate across the tonoplast in this species. A number of other 1,4-dicarboxylates stimulated H+ transport in tonoplast vesicles from E. ibaguense, their order of effectiveness being fumarate > oxalacetate ≥ malate ≥ 2-mercaptosuccinate ≥ succinate > tartrate. The stimulation of H+ transport was not stereospecific, but required the 1,4-dicarboxylate to adopt the trans-carboxyl geometric configuration. Neither shorter-chain (oxalate, malonate) nor longer-chain (glutarate, adipate) dicarboxylates supported H+ transport. The specificity of organic-acid anion transport at the tonoplast of E. ibaguense was similar to that of Kalanchoë daigremontiana and thus appears to be a characteristic of the malate-influx system in CAM plants. © 1992, Gustav Fischer Verlag, Stuttgart. All rights reserved.