Localization and characterization θ carbonic anhydrases in Thalassiosira pseudonana.

Carbonic anhydrase (CA) is a crucial component for the operation of CO2-concentrating mechanisms (CCMs) in the majority of aquatic photoautotrophs that maintain the global primary production. In the genome of the centric marine diatom, Thalassiosira pseudonana, there are four putative gene sequences that encode θ-type CA, which was a type of CA recently identified in marine diatoms and green algae. In the present study, specific subcellular locations of four θCAs, TpθCA1, TpθCA2, TpθCA3, and TpθCA4 were determined by expressing GFP-fused proteins of these TpθCAs in T. pseudonana. As a result, C-terminal GFP fusion proteins of TpθCA1, TpθCA2, and TpθCA3 were all localized in the chloroplast; TpθCA2 was at the central chloroplast area, and the other two TpθCAs were throughout the chloroplast. Immunogold-labeling transmission electron microscopy was further performed for the transformants expressing TpθCA1:GFP and TpθCA2:GFP with anti-GFP-monoclonal antibody. TpθCA1:GFP was localized in the free stroma area, including the peripheral pyrenoid area. TpθCA2:GFP was clearly located as a lined distribution at the central part of the pyrenoid structure, which was most likely the pyrenoid-penetrating thylakoid. Considering the presence of the sequence encoding the N-terminal thylakoid-targeting domain in the TpθCA2 gene, this localization was likely the lumen of the pyrenoid-penetrating thylakoid. On the other hand, TpθCA4:GFP was localized in the cytoplasm. Transcript analysis of these TpθCAs revealed that TpθCA2 and TpθCA3 were upregulated in atmospheric CO2 (0.04% CO2, LC) levels, while TpθCA1 and TpθCA4 were highly induced under 1% CO2 (HC) condition. The genome-editing knockout (KO) of TpθCA1, by CRISPR/Cas9 nickase, gave a silent phenotype in T. pseudonana under LC-HC conditions, which was in sharp agreement with the case of the previously reported TpθCA3 KO. In sharp contrast, TpθCA2 KO is so far unsuccessful, suggesting a housekeeping role of TpθCA2. The silent phenotype of KO strains of stromal CAs suggests that TpαCA1, TpθCA1, and TpθCA3 may have functional redundancy, but different transcript regulations in response to CO2 of these stromal CAs suggest in part their independent roles.

Nawaly H ，Tanaka A ，Toyoshima Y ，Tsuji Y ，Matsuda Y ... -  《-》

Localization of putative carbonic anhydrases in two marine diatoms, Phaeodactylum tricornutum and Thalassiosira pseudonana.

Tachibana M ，Allen AE ，Kikutani S ，Endo Y ，Bowler C ，Matsuda Y ... -  《-》

Localization of putative carbonic anhydrases in the marine diatom, Thalassiosira pseudonana.

Samukawa M ，Shen C ，Hopkinson BM ，Matsuda Y ... -  《-》

Thylakoid luminal θ-carbonic anhydrase critical for growth and photosynthesis in the marine diatom Phaeodactylum tricornutum.

Kikutani S ，Nakajima K ，Nagasato C ，Tsuji Y ，Miyatake A ，Matsuda Y ... -  《-》

Molecular aspects of the biophysical CO2-concentrating mechanism and its regulation in marine diatoms.

Diatoms operate a CO2-concentrating mechanism (CCM) that drives upwards of 20% of annual global primary production. Recent progress in CCM research in the marine pennate diatom Phaeodactylum tricornutum revealed that this diatom directly takes up HCO3- from seawater through low-CO2-inducible plasma membrane HCO3- transporters, which belong to the solute carrier (SLC) 4 family. Apart from this, studies of carbonic anhydrases (CAs) in diatoms have revealed considerable diversity in classes and localization among species. This strongly suggests that the CA systems, which control permeability and flux of dissolved inorganic carbon (DIC) by catalysing reversible CO2 hydration, have evolved from diverse origins. Of particular interest is the occurrence of low-CO2-inducible external CAs in the centric marine diatom Thalassiosira pseudonana, offering a strategy of CA-catalysed initial CO2 entry via passive diffusion, contrasting with active DIC transport in P. tricornutum. Molecular mechanisms to transport DIC across chloroplast envelopes are likely also through specific HCO3- transporters, although details have yet to be elucidated. Furthermore, recent discovery of a luminal θ-CA in the diatom thylakoid implied a common strategy in the mechanism to supply CO2 to RubisCO in the pyrenoid, which is conserved among green algae and some heterokontophytes. These results strongly suggest an occurrence of convergent coevolution between the pyrenoid and thylakoid membrane in aquatic photosynthesis.

Tsuji Y ，Nakajima K ，Matsuda Y 《-》