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. Summary Statement Zea mays plants containing 0.2% of wild‐type leaf carbonic anhydrase activity had low photosynthesis rates and a 1% CO2‐dependent growth phenotype. The depleted leaf 13C isotopic composition and high CO2 compensation point suggest that these plants had a disrupted C4 carbon‐concentrating mechanism.