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 C₄ 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 C₄ dicot Amaranthus edulis were used to study the effect of reduced PEPC activity on CO₂ assimilation rates, stomatal conductance, and ¹³CO₂ (Δ¹³C) and C¹⁸OO (Δ¹⁸O) 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 μbar 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 C₄ PEPC isoform plays an essential role in stomatal opening. There was little difference in Δ¹³C between the Pp mutant (3.0‰ ± 0.4‰) and wild type (3.3‰ ± 0.4‰), indicating that leakiness (φ), the ratio of CO₂ leak rate out of the bundle sheath to the rate of CO₂ supply by the C₄ cycle, a measure of the coordination of C₄ photosynthesis, was not affected by a 60% reduction in PEPC activity. In the pp mutant Δ¹³C was 16‰ ± 3.2‰, indicative of direct CO₂ fixation by Rubisco in the bundle sheath at ambient CO₂ partial pressure. Δ¹⁸O measurements indicated that the extent of isotopic equilibrium between leaf water and the CO ₂ at the site of oxygen exchange (φ) 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 φ as compared to values determined from Δ¹⁸O in wild-type plants.