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

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Abstract

It is believed that intracellular carbonic anhydrases (CAs) are essential components of carbon concentrating mechanisms in microalgae. In this study, putative CA-encoding genes were identified in the genome sequences of the marine diatoms Phaeodactylum tricornutum and Thalassiosira pseudonana. Subsequently, the subcellular localizations of the encoded proteins were determined. Nine and thirteen CA sequences were found in the genomes of P. tricornutum and T. pseudonana, respectively. Two of the β-CA genes in P. tricornutum corresponded to ptca1 and ptca2 identified previously. Immunostaining transmission electron microscopy of a PtCA1:YFP fusion expressed in the cells of P. tricornutum clearly showed the localization of PtCA1 within the central part of the pyrenoid structure in the chloroplast. Besides these two β-CA genes, P. tricornutum likely contains five α- and two γ-CA genes, whereas T. pseudonana has three α-, five γ-, four δ-, and one ζ-CA genes. Semi-quantitative reverse transcription PCR performed on mRNA from the two diatoms grown in changing light and CO2 conditions revealed that levels of six putative α- and γ-CA mRNAs in P. tricornutum did not change between cells grown in air-level CO2 and 5% CO2. However, mRNA levels of one putative α-CA gene, CA-VII in P. tricornutum, were reduced in the dark compared to that in the light. In T. pseudonana, mRNA accumulation levels of putative α-CA (CA-1), ζ-CA (CA-3) and δ-CA (CA-7) were analyzed and all levels found to be significantly reduced when cells were grown in 0.16% CO2. Intercellular localizations of eight putative CAs were analyzed by expressing GFP fusion in P. tricornutum and T. pseudonana. In P. tricornutum, CA-I and II localized in the periplastidial compartment, CA-III, VI, VII were found in the chloroplast endoplasmic reticulum, and CA-VIII was localized in the mitochondria. On the other hand, T. pseudonana CA-1 localized in the stroma and CA-3 was found in the periplasm. These results suggest that CAs are constitutively present in the four chloroplastic membrane systems in P. tricornutum and that CO2 responsive CAs occur in the pyrenoid of P. tricornutum, and in the stroma and periplasm of T. pseudonana.

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Abbreviations

Rubisco:

Ribulose-1,5-bisphosphate carboxylase/oxygenase

CA:

Carbonic anhydrase

CER:

Chloroplast endoplasmic reticulum

PPC:

Periplastidial compartment

BLS:

Blob-like structure

CE:

Chloroplast envelope

CCM:

Inorganic carbon concentrating mechanism

TEM:

Transmission electron microscopy

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Acknowledgments

We thank Ms. Nobuko Higashiuchi and Ms. Megumi Fujii for their technical assistance and Ms. Miyabi Inoue for her skilful secretarial assistance. This research was supported by the Ministry of Education, Culture, Sports, Science and Technology of Japan (to Kwansei Gakuin University, Research Center for Environmental Bioscience), and by Steel Industry Foundation for the Advancement of Environmental Protection Technology (to Y. M.), and by funding from the National Science Foundation (NSF-MCB-1024913, NSF OCE-0727997, and NSF OCE-0722374) to AEA.

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  1. Andrew E. Allen

    Present address: J. Craig Venter Institute, 10355 Science Center Drive, San Diego, CA, 92121, USA

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  1. Department of Bioscience, School of Science and Technology, Kwansei Gakuin University, 2-1 Gakuen, Sanda, Hyogo, 669-1337, Japan

    Masaaki Tachibana, Sae Kikutani, Yuri Endo & Yusuke Matsuda

  2. Environmental and Evolutionary Genomics Section, Institut de Biologie de l’Ecole Normale Supérieure (IBENS), CNRS UMR8197 INSERM U1024, Ecole Normale Supérieure, 46 rue d’Ulm, 75005, Paris, France

    Andrew E. Allen & Chris Bowler

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  1. Masaaki Tachibana
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Tachibana, M., Allen, A.E., Kikutani, S. et al. Localization of putative carbonic anhydrases in two marine diatoms, Phaeodactylum tricornutum and Thalassiosira pseudonana . Photosynth Res 109, 205–221 (2011). https://doi.org/10.1007/s11120-011-9634-4

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