Absence of carbonic anhydrase in chloroplasts affects C 3 plant development but not photosynthesis

Significance Carbonic anhydrase enzymes located in chloroplast stroma have been hypothesized to facilitate photosynthesis in C 3 plants because they catalyze a reaction involving bicarbonate and CO 2 , a substrate of the carbon-fixing enzyme RuBisCO. To test this possibility, tobacco mutants completely lacking chloroplast stromal carbonic anhydrase activity were produced by CRISPR/Cas9 mutagenesis. The plants displayed normal photosystem II activity and CO 2 assimilation but also abnormal development and increased reactive oxygen species and stromal pH. We conclude that chloroplast carbonic anhydrase does not play a direct role in providing CO 2 for carbon fixation. Instead, as is also true in microorganisms, carbonic anhydrase is necessary to supply bicarbonate for biosynthetic processes.