The kinetics of ribulose-1,5-bisphosphate carboxylase/oxygenase in vivo inferred from measurements of photosynthesis in leaves of transgenic tobacco

Abstract

Transgenic tobacco (Nicotiana tabacum L. cv. W38) with an antisense gene directed against the mRNA of the ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) small subunit was used to determine the kinetic properties of Rubisco in vivo. The leaves of these plants contained only 34% as much Rubisco as those of the wild type, but other photosynthetic components were not significantly affected. Consequently, the rate of CO₂ assimilation by the antisense plants was limited by Rubisco activity over a wide range of CO₂ partial pressures. Unlike in the wild-type leaves, where the rate of regeneration of ribulose bisphosphate limited CO₂ assimilation at intercellular partial pressures above 400 ubar, photosynthesis in the leaves of the antisense plants responded hyperbolically to CO₂, allowing the kinetic parameters of Rubisco in vivo to be inferred. We calculated a maximal catalytic turnover rate, k_cat, of 3.5+0.2 mol CO₂·(mol sites)⁻¹·s⁻¹ at 25° C in vivo. By comparison, we measured a value of 2.9 mol CO₂·(mol sites)⁻¹·⁻¹ in vitro with leaf extracts. To estimate the Michaelis-Menten constants for CO₂ and O₂, the rate of CO₂ assimilation was measured at 25° C at different intercellular partial pressures of CO₂ and O₂. These measurements were combined with carbon-isotope analysis (¹³C/¹²C) of CO₂ in the air passing over the leaf to estimate the conductance for transfer of CO₂ from the substomatal cavities to the sites of carboxylation (0.3 mol·m⁻²·s⁻¹·bar⁻¹) and thus the partial pressure of CO₂ at the sites of carboxylation. The calculated Michaelis-Menten constants for CO₂ and O₂ were 259 ±57 μbar (8.6±1.9μM) and 179 mbar (226 μM), respectively, and the effective Michaelis-Menten constant for CO₂ in 200 mbar O₂ was 549 μbar (18.3 μM). From measurements of the photocompensation point (Γ_* = 38.6 ubar) we estimated Rubisco's relative specificity for CO₂, as opposed to O₂ to be 97.5 in vivo. These values were dependent on the size of the estimated CO₂-transfer conductance.