A transgenic approach to understanding the influence of carbonic anhydrase on C¹⁸OO discrimination during C₄ photosynthesis

The oxygen isotope composition of atmospheric CO₂ is an important signal that helps distinguish between ecosystem photosynthetic and respiratory processes. In C₄ plants the carbonic anhydrase (CA)-catalyzed interconversion of CO₂ and bicarbonate (HCO ₃^-) is an essential first reaction for C₄ photosynthesis but also plays an important role in the CO₂-H ₂O exchange of oxygen as it enhances the rate of isotopic equilibrium between CO₂ and water. The C₄ dicot Flaveria bidentis containing genetically reduced levels of leaf CA (CA_leaf) has been used to test whether changing leaf CA activity influences online measurements of C¹⁸OO discrimination (Δ¹⁸O) and the proportion of CO₂ in isotopic equilibrium with leaf water at the site of oxygen exchange (θ). The Δ¹⁸O in wild-type F. bidentis, which contains high levels of CA relative to the rates of net CO₂ assimilation, was less than predicted by models of Δ¹⁸O. Additionally, Δ¹⁸O was sensitive to small decreases in CA _leaf. However, reduced CA activity in F. bidentis had little effect on net CO₂ assimilation, transpiration rates (E), and stomatal conductance (g_s) until CA levels were less than 20% of wild type. The values of θ determined from measurements of Δ¹⁸O and the ¹⁸O isotopic composition of leaf water at the site of evaporation (δ_e) were low in the wild-type-F. bidentis and decreased in transgenic plants with reduced levels of CA activity. Measured values of θ were always significantly lower than the values of θ predicted from in vitro CA activity and gas exchange. The data presented here indicates that CA content in a C₄ leaf may not represent the CA activity associated with the CO₂-H₂O oxygen exchange and therefore may not be a good predictor of θ during C₄ photosynthesis. Furthermore, uncertainties in the isotopic composition of water at the site of exchange may also limit the ability to accurately predict θ in C₄ plants.