Lack of leaf carbonic anhydrase activity eliminates the C-4 carbon-concentrating mechanism requiring direct diffusion of CO2 into bundle sheath cells

Carbonic anhydrase (CA) performs the first enzymatic step of C-4 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 C-4 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 (pCO(2)). 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 C-4 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 pCO(2). Photosynthetic and carbon isotope analysis combined with modelling of photosynthesis and carbon isotope discrimination was used to determine if ca1ca2ca8 plants had a functional C-4 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 C-4 CCM.