Abstract
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.
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Data availability
All data generated or analyzed during this study are included in this published article and its supplementary information files. The cDNA sequences that presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article.
Abbreviations
- CA:
-
Carbonic anhydrase
- CCM:
-
CO2-concentrating mechanism
- CER:
-
Chloroplast endoplasmic reticulum
- DIC:
-
Dissolved inorganic carbon
- FCP8:
-
Fucoxanthin chlorophyll a/c binding protein 8
- Rubisco:
-
Ribulose-1,5-bisphosphate carboxylase/oxygenase
- TEM:
-
Transmission electron microscopy
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Acknowledgements
We are grateful to Prof. Luke C. M. Mackinder and Dr. Onyou Nam of the University of York who have critically read the manuscript. We wish to thank Mrs Nobuko Higashiuchi and Mrs Eri Nakayama of Kwansei Gakuin University for their technical assistance.
Funding
This work was supported by JSPS Kakenhi Kiban Research (A) (grant no. 19H01153 to YM), by JSPS Kakenhi Innovative Areas (grant no. 16H06557 to YM), by JST CREST ‘Cell dynamics’ (grant no. JPMJCR20E1 to YM), by Grant-in-Aid for Young Scientists (B) (grant no. 17K15326 to Y.T.).
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HN and YM: conceptualization; HN and YM methodology; HN and YT: investigation and formal analysis, with supervision YT, YM; AT: performed the immuno-labeling experiment; YM: funding acquisition; HN, AT, YT, and YM: writing—original draft preparation; HN, AT, YT, YT, and YM: writing—review and editing; all authors agreed to the final contents of the paper.
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Nawaly, H., Tanaka, A., Toyoshima, Y. et al. Localization and characterization θ carbonic anhydrases in Thalassiosira pseudonana. Photosynth Res 156, 217–229 (2023). https://doi.org/10.1007/s11120-023-01007-z
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DOI: https://doi.org/10.1007/s11120-023-01007-z