13C Nuclear Magnetic Resonance Studies of Crassulacean Acid Metabolism in Intact Leaves of Kalanchoë tubiflora

13C nuclear magnetic resonance spectroscopy of intact leaves of Kalanchoë tubiflora was used to observe Crassulacean acid metabolism in vivo. 13C signals from C-4 of malate were observed after overnight exposure of leaves to 13CO2. Illumination of the labeled leaves resulted in a gradual decrease in the malate signals. After a period of darkness in normal air, 13C signals were detected in all four carbons of malate in the previously labeled leaves. The 13C nuclear magnetic resonance spectrum of malate in solution was pH dependent, which allowed an estimation of the vacuolar pH from the whole leaf spectrum. The pH was 4.0 following a 14-hour dark period, but rose to greater than 6.0 after 6 hours of illumination.