Absence of carbonic anhydrase in chloroplasts affects C3 plant development but not photosynthesis

Abstract

Significance Carbonic anhydrase enzymes located in chloroplast stroma have been hypothesized to facilitate photosynthesis in C3 plants because they catalyze a reaction involving bicarbonate and CO2, 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 CO2 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 CO2 for carbon fixation. Instead, as is also true in microorganisms, carbonic anhydrase is necessary to supply bicarbonate for biosynthetic processes. The enzyme carbonic anhydrase (CA), which catalyzes the interconversion of bicarbonate with carbon dioxide (CO2) and water, has been hypothesized to play a role in C3 photosynthesis. We identified two tobacco stromal CAs, β-CA1 and β-CA5, and produced CRISPR/Cas9 mutants affecting their encoding genes. While single knockout lines Δβ-ca1 and Δβ-ca5 had no striking phenotypic differences compared to wild type (WT) plants, Δβ-ca1ca5 leaves developed abnormally and exhibited large necrotic lesions even when supplied with sucrose. Leaf development of Δβ-ca1ca5 plants normalized at 9,000 ppm CO2. Leaves of Δβ-ca1ca5 mutants and WT that had matured in high CO2 had identical CO2 fixation rates and photosystem II efficiency. Fatty acids, which are formed through reactions with bicarbonate substrates, exhibited abnormal profiles in the chloroplast CA-less mutant. Emerging Δβ-ca1ca5 leaves produce reactive oxygen species in chloroplasts, perhaps due to lower nonphotochemical quenching efficiency compared to WT. Δβ-ca1ca5 seedling germination and development is negatively affected at ambient CO2. Transgenes expressing full-length β-CA1 and β-CA5 proteins complemented the Δβ-ca1ca5 mutation but inactivated (ΔZn-βCA1) and cytoplasm-localized (Δ62-βCA1) forms of β-CA1 did not reverse the growth phenotype. Nevertheless, expression of the inactivated ΔZn-βCA1 protein was able to restore the hypersensitive response to tobacco mosaic virus, while Δβ-ca1 and Δβ-ca1ca5 plants failed to show a hypersensitive response. We conclude that stromal CA plays a role in plant development, likely through providing bicarbonate for biosynthetic reactions, but stromal CA is not needed for maximal rates of photosynthesis in the C3 plant tobacco.