The Role of Phosphoenolpyruvate Carboxylase during C4 Photosynthetic Isotope Exchange and Stomatal Conductance

Abstract

Abstract 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 CO2 at the site of oxygen exchange (θ) 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 θ as compared to values determined from Δ18O in wild-type plants.