Diurnal changes in carbon dioxide exchange rate (CER), stomatal conductance (g(s)), photosynthetic enzyme activities and metabolite levels relating to the C4 pathway and the Calvin cycle in sugarcane (Saccharum sp. cv. NiF4) leaves were characterized during a natural 24 h day-night cycle. The activities of phosphoenolpyruvate carboxylase (PEPcase), NADP-malic enzyme (NADP-ME), pyruvate, P1 dikinase (PPDK), ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) and chloroplast fructose-1,6-bisphosphatase (FBPase) all exhibited distinct diurnal changes. The levels of C4 metabolites, malate, phosphoenolpyruvate (PEP) and pyruvate, showed large diurnal fluctuations, but the oxaloacetate levels were extremely low and exhibited no significant changes in the day-night cycle. Diurnal changes in CER and g(s) paralleled the changes in radiation of sunlight. The results in this study suggest that at midday when CER is in steady-state and saturated by full sunlight, CER is limited by PPDK activity
under lower radiation of sunlight, i.e., before CER is saturated in the morning and during the period when CER is decreasing in the afternoon, CER is possibly limited by PEPcase activity or g(s).