Effects of reduced carbonic anhydrase activity on CO₂ assimilation rates in Setaria viridis: A transgenic analysis

In C₄ species, the major β-carbonic anhydrase (β-CA) localized in the mesophyll cytosol catalyses the hydration of CO₂ to HCO₃ ^-, which phosphoenolpyruvate carboxylase uses in the first step of C₄ photosynthesis. To address the role of CA in C₄ photosynthesis, we generated transgenic Setaria viridis depleted in β-CA. Independent lines were identified with as little as 13% of wild-type CA. No photosynthetic defect was observed in the transformed lines at ambient CO₂ partial pressure (pCO₂). At low pCO₂, a strong correlation between CO₂ assimilation rates and CA hydration rates was observed. C¹⁸O¹⁶O isotope discrimination was used to estimate the mesophyll conductance to CO₂ diffusion from the intercellular air space to the mesophyll cytosol (gm) in control plants, which allowed us to calculate CA activities in the mesophyll cytosol (Cm). This revealed a strong relationship between the initial slope of the response of the CO₂ assimilation rate to cytosolic pCO₂ (AC_m) and cytosolic CA activity. However, the relationship between the initial slope of the response of CO₂ assimilation to intercellular pCO₂ (ACi) and cytosolic CA activity was curvilinear. This indicated that in S. viridis, mesophyll conductance may be a contributing limiting factor alongside CA activity to CO₂ assimilation rates at low pCO₂.