The role of phosphoenolpyruvate carboxylase during C4 photosynthetic isotope exchange and stomatal conductance

Phosphoenolpyruvate carboxylase (PEPC; EC 4.1.1.31) plays a key role during C-4 photosynthesis and is involved in anaplerotic metabolism, pH regulation, and stomatal opening. Heterozygous (Pp) and homozygous (pp) forms of a PEPC-deficient mutant of the C-4 dicot Amaranthus edulis were used to study the effect of reduced PEPC activity on CO2 assimilation rates, stomatal conductance, and Co-13(2) (Delta C-13) and (COO)-O-18(Delta O-18) isotope discrimination during leaf gas exchange. PEPC activity was reduced to 42% and 3% and the rates of CO2 assimilation in air dropped to 78% and 10% of the wild-type values in the Pp and pp mutants, respectively. Stomatal conductance in air (531 mu bar CO2) was similar in the wild-type and Pp mutant but the pp mutant had only 41% of the wild-type steady-state conductance under white light and the stomata opened more slowly in response to increased light or reduced CO2 partial pressure, suggesting that the C-4 PEPC isoform plays an essential role in stomatal opening. There was little difference in Delta C-13 between the Pp mutant (3.0 parts per thousand +/- 0.4 parts per thousand) and wild type (3.3 parts per thousand +/- 0.4 parts per thousand), indicating that leakiness (phi), the ratio of CO2 leak rate out of the bundle sheath to the rate of CO2 supply by the C-4 cycle, a measure of the coordination of C-4 photosynthesis, was not affected by a 60% reduction in PEPC activity. In the pp mutant Delta C-13 was 16 parts per thousand +/- 3.2 parts per thousand, indicative of direct CO2 fixation by Rubisco in the bundle sheath at ambient CO2 partial pressure. Delta O-18 measurements indicated that the extent of isotopic equilibrium between leaf water and the CO2 at the site of oxygen exchange (theta) was low (0.6) in the wild-type and Pp mutant but increased to 0.9 in the pp mutant. We conclude that in vitro carbonic anhydrase activity overestimated theta as compared to values determined from Delta O-18 in wild-type plants.