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

Abstract

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 <i>Δβ</i>-<i>ca1</i> and <i>Δβ-ca5</i> had no striking phenotypic differences compared to wild type (WT) plants, <i>Δβ</i>-<i>ca1ca5</i> leaves developed abnormally and exhibited large necrotic lesions even when supplied with sucrose. Leaf development of <i>Δβ</i>-<i>ca1ca5</i> plants normalized at 9,000 ppm CO₂ Leaves of <i>Δβ</i>-<i>ca1ca5</i> 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 <i>Δβ</i>-<i>ca1ca5</i> leaves produce reactive oxygen species in chloroplasts, perhaps due to lower nonphotochemical quenching efficiency compared to WT. <i>Δβ</i>-<i>ca1ca5</i> seedling germination and development is negatively affected at ambient CO₂ Transgenes expressing full-length β-CA1 and β-CA5 proteins complemented the <i>Δβ-ca1ca5</i> 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 <i>Δβ-ca1</i> and <i>Δβ-ca1ca5</i> 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.