, Volume 195, Issue 1, pp 88–97 | Cite as

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

  • Susanne von Caemmerer
  • John R. Evans
  • Graham S. Hudson
  • T. John Andrews


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, kcat, 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.

Key words

Gas exchange Nicotiana Photosynthesis (C3Ribulose-1,5-bisphosphate carboxylase/oxygenase kinetics Transgenic tobacco 

Abbreviations and Symbols


CO2-assimilation rate


conductance for CO2 transfer from the substomatal cavities to the sites of carboxylation

Kc, Ko

Michaelis-Menten constants for carboxylation, oxygenation of Rubisco


Vcmax/[active site]


partial pressure of O2 at the site of carboxylation


partial pressure of CO2 at the site of carboxylation


intercellular CO2 partial pressure


‘day’ respiration (non-photorespiratory CO2 evolution)


ribulose 1,5-bisphosphate carboxylase/oxygenase




relative specificity factor for Rubisco


small subunit of Rubisco

Vcmax, Vomax

maximum rates of Rubisco carboxylation, oxygenation


partial pressure of CO2 in the chloroplast at which photorespiratory CO2 evolution equals the rate of carboxylation


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Copyright information

© Springer-Verlag 1994

Authors and Affiliations

  • Susanne von Caemmerer
    • 1
    • 3
  • John R. Evans
    • 1
  • Graham S. Hudson
    • 2
  • T. John Andrews
    • 2
    • 3
  1. 1.Environmental Biology Group, Research School of Biological SciencesAustralian National UniversityCanberra CityAustralia
  2. 2.Cooperative Research Centre for Plant Science, Research School of Biological SciencesAustralian National UniversityCanberra CityAustralia
  3. 3.Molecular Plant Physiology Group, Research School of Biological SciencesAustralian National UniversityCanberra CityAustralia

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