Participation of inorganic orthophosphate in regulation of the ribulose-1,5-bisphosphate carboxylase activity in response to changes in the photosynthetic source-sink balance

Sink-limited conditions, defined as treatment with continuous illumination, cause a reduction in the rate of photosynthetic fixation of CO2 in single-rooted leaves of soybean (Glycine max. Merr. cv. Turunoko). We suggested previously that this reduction is due to a deactivation of ribulose-1,5-bisphosphate carboxylase (RuBPcase, E.C. 4.1.1.39) that is caused by a decrease in the level of Pi in the leaves [Sawada et al. (1989) Plant Cell Physiol. 30: 691, Sawada et al. (1990) Plant Cell Physiol. 31: 697]. In the present study, the mechanism of regulation of RuBPcase activity by Pi was examined. The activity of RuBPcase in the sink-limited leaves, exposed for 6 or 7 d to continuous illumination to alter the source/sink balance, was enhanced with increasing concentrations of Pi, in a CO2-free preincubation medium in the presence of 5 mM MgCl2 The maximum value [6.3 μmole CO2 (mg Chl)−1 min−1] was obtained at approximately 5 mM Pi after a 5 min incubation, being 3 times of the activity without the preincubation. The activity of a crude preparation of RuBPcase that had been deactivated by removal of CO2 and Mg2+ ions by the gel filtration was 5.2–9.3 nmole CO2 (mg protein)−1 min−1 and was also enhanced by Pi plus Mg2+ ions. The maximum value [147–151 nmole CO2 (mg protein)−1 min−1] was attained at 5 mM Pi after a 5 min incubation. The cycle of activation and inactivation of deactivated crude RuBPcase was perfectly reversible by addition of Pi to the enzyme and removal of Pi from the enzyme. Levels of free Pi and of esterified phosphate in the sink-limited leaves were 69% and 31% of the total phosphate, respectively. By contrast, in the control leaves, these values were 87% and 13%, respectively. These results support our previously stated hypothesis and indicate an important role for free Pi in the regulation of RuBPcase activity, in particular in sink-limited plants.