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

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, 13-CA1 and 13-CA5, and produced CRISPR/ Cas9 mutants affecting their encoding genes. While single knockout lines d/3-ca1 and d/3-ca5 had no striking phenotypic differences compared to wild type (WT) plants, d/3-ca1ca5 leaves developed abnormally and exhibited large necrotic lesions even when supplied with sucrose. Leaf development of d/3-ca1ca5 plants normalized at 9,000 ppm CO2. Leaves of d/3-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 d/3-ca1ca5 leaves produce reactive oxygen species in chloroplasts, perhaps due to lower nonphotochemical quenching efficiency compared to WT. d/3-ca1ca5 seedling germination and development is negatively affected at ambient CO2. Transgenes expressing full-length 13-CA1 and 13-CA5 proteins complemented the d/3-ca1ca5 mutation but inactivated (AZn-13CA1) and cytoplasmlocalized (A62-13CA1) forms of 13-CA1 did not reverse the growth phenotype. Nevertheless, expression of the inactivated AZn-13CA1 protein was able to restore the hypersensitive response to tobacco mosaic virus, while d/3-ca1 and d/3-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.

Automated summary

The stromal CAs 13-CA1 and 13-CA5 in tobacco play significant roles in leaf development and CO2 fixation, but they are not necessary for maximal photosynthetic rates in C3 plant tobacco.