Molecular Characterization of Carbonic Anhydrase Genes in and Their Potential Roles in Symbiotic Nitrogen Fixation

Carbonic anhydrase (CA) plays a vital role in photosynthetic tissues of higher plants, whereas its non-photosynthetic role in the symbiotic root nodule was rarely characterized. In this study, 13 CA genes were identified in the model legume by comparison with Arabidopsis CA genes. Using qPCR and promoter-reporter fusion methods, three previously identified nodule-enhanced CA genes (, , and ) have been further characterized, which exhibit different spatiotemporal expression patterns during nodule development. was expressed in the central infection zone of the mature nodule, including both infected and uninfected cells. was restricted to the vascular bundle of the root and nodule. As for , it was expressed in most cell types of nodule primordia but only in peripheral cortical cells and uninfected cells of the mature nodule. Using CRISPR/Cas9 technology, the knockout of or both and its homolog, , did not result in abnormal symbiotic phenotype compared with the wild-type plants, suggesting that LjβCA1 or LjαCA1/2 are not essential for the nitrogen fixation under normal symbiotic conditions. Nevertheless, the nodule-enhanced expression patterns and the diverse distributions in different types of cells imply their potential functions during root nodule symbiosis, such as CO fixation, N assimilation, and pH regulation, which await further investigations.