A comparative study on the regulation of C3 and C4 carboxylation processes in the constitutive crassulacean acid metabolism (CAM) plant Kalanchoe daigremontiana and the C3-CAM intermediate Clusia minor

A comparison of carbon metabolism in the constitutive crassulacean acid metabolism (CAM) plant Kalanchoe daigremontiana Hamet et Perr. and the C3-CAM intermediate Clusia minor L. was undertaken under controlled environmental conditions where plants experience gradual changes in light intensity, temperature and humidity at the start and end of the photoperiod. The magnitude of CAM activity was manipulated by maintaining plants in ambient air or by enclosing leaves overnight in an atmosphere of N2 to suppress C4 carboxylation. Measurements of diel changes in carbon-isotope discrimination and organic acid content were used to quantify the activities of C3 and C4 carboxylases in vivo and to indicate the extent to which the activities of phosphoenolpyruvate carboxylase (PEPCase), ribulose-1,5-bisphosphate carboxylase-oxygenase (Rubisco) and decarboxylation processes overlap at the start and end of the photoperiod. These measurements in vivo were compared with measurements in vitro of changes in the diet sensitivity of PEPCase to malate inhibition. The results demonstrate fundamental differences in the down-regulation of PEPCase during the day in the two species. While PEPCase is inactivated within the first 30 min of the photoperiod in K. daigremontiana, the enzyme is active for 4 h at the start and 3 h at the end of the photoperiod in C. minor. Enclosing leaves in N2 overnight resulted in a two- to threefold increase in PEPCase-mediated CO2 uptake during Phase II of CAM in both species. However, futile cycling of CO2 between malate synthesis and decarboxylation does not occur during Phase II in either species. In terms of overall carbon balance, C4 carboxylation accounted for approximately 20% of net daytime assimilation in both species under control conditions, increasing to 30-34% after a night in N2. Although N2-treated leaves of K. daigremontiana took up 25% more CO2 than control leaves during the day this was insufficient to compensate for the loss of CO2 taken up by CAM the previous night. In contrast, in N2- treated leaves of C. minor, the twofold increase in daytime PEPCase activity and the increase in net CO2 uptake by Rubisco during Phase III compensated for the inhibition of C4 carboxylation at night in terms of diel carbon balance.