Lack of leaf carbonic anhydrase activity eliminates the C₄ carbon-concentrating mechanism requiring direct diffusion of CO₂ into bundle sheath cells

Carbonic anhydrase (CA) performs the first enzymatic step of C₄ photosynthesis by catalysing the reversible hydration of dissolved CO₂ that diffuses into mesophyll cells from intercellular airspaces. This CA-catalysed reaction provides the bicarbonate used by phosphoenolpyruvate carboxylase to generate products that flow into the C₄ carbon-concentrating mechanism (CCM). It was previously demonstrated that the Zea mays ca1ca2 double mutant lost 97% of leaf CA activity, but there was little difference in the growth phenotype under ambient CO₂ partial pressures (pCO₂). We hypothesise that since CAs are among the fastest enzymes, minimal activity from a third CA, CA8, can provide the inorganic carbon needed to drive C₄ photosynthesis. We observed that removing CA8 from the maize ca1ca2 background resulted in plants that had 0.2% of wild-type leaf CA activity. These ca1ca2ca8 plants had reduced photosynthetic parameters and could only survive at elevated pCO₂. Photosynthetic and carbon isotope analysis combined with modelling of photosynthesis and carbon isotope discrimination was used to determine if ca1ca2ca8 plants had a functional C₄ cycle or were relying on direct CO₂ diffusion to ribulose 1,5-bisphosphate carboxylase/oxygenase within bundle sheath cells. The results suggest that leaf CA activity in ca1ca2ca8 plants was not sufficient to sustain the C₄ CCM.