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The intracellular distribution of inorganic carbon fixing enzymes does not support the presence of a C4 pathway in the diatom Phaeodactylum tricornutum

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Abstract

Diatoms are unicellular algae and important primary producers. The process of carbon fixation in diatoms is very efficient even though the availability of dissolved CO2 in sea water is very low. The operation of a carbon concentrating mechanism (CCM) also makes the more abundant bicarbonate accessible for photosynthetic carbon fixation. Diatoms possess carbonic anhydrases as well as metabolic enzymes potentially involved in C4 pathways; however, the question as to whether a C4 pathway plays a general role in diatoms is not yet solved. While genome analyses indicate that the diatom Phaeodactylum tricornutum possesses all the enzymes required to operate a C4 pathway, silencing of the pyruvate orthophosphate dikinase (PPDK) in a genetically transformed cell line does not lead to reduced photosynthetic carbon fixation. In this study, we have determined the intracellular location of all enzymes potentially involved in C4-like carbon fixing pathways in P. tricornutum by expression of the respective proteins fused to green fluorescent protein (GFP), followed by fluorescence microscopy. Furthermore, we compared the results to known pathways and locations of enzymes in higher plants performing C3 or C4 photosynthesis. This approach revealed that the intracellular distribution of the investigated enzymes is quite different from the one observed in higher plants. In particular, the apparent lack of a plastidic decarboxylase in P. tricornutum indicates that this diatom does not perform a C4-like CCM.

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Acknowledgements

We are grateful to D. Ballert (Universität Konstanz) for the genetic transformation of P. tricornutum and the cultivation of the transformed cell lines; to the Bioimaging Center at the University of Konstanz for providing the LSM 510 META; to Dr. Katsura Izui for providing the DCDP and to Dr. Pavel Hrouzek (Centre Algatech) for providing the MitoTracker. This study was supported by the German-Israel Foundation for Scientific Research and Development (GIF; Project No. G-1011-199.12/2008 to A.K. and P.G.K.); the University of Konstanz, the Grant-in-Aid for Scientific Research B (Grant No. 24310015 to Y. M.); the Grant-in-Aid for Scientific Research on Innovative Areas (Grant No. 16H06557 to Y. M.); the Japan Society for the Promotion of Science (JSPS; Grant No. 26870750 to S. K.) and by a 2 years Postdoctoral Researcher Fellowship (Mzdová podpora postdoktorandů) from the Academy of Sciences of the Czech Republic (to D.E.).

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Authors and Affiliations

  1. Fachbereich Biologie, Universität Konstanz, 78457, Konstanz, Germany

    Daniela Ewe, Ansgar Gruber, Carolina Río Bártulos & Peter G. Kroth

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

    Masaaki Tachibana, Sae Kikutani & Yusuke Matsuda

  3. Department of Plant and Environmental Sciences, Edmond J. Safra Campus–Givat Ram, Hebrew University of Jerusalem, 91904, Jerusalem, Israel

    Aaron Kaplan

  4. Centre Algatech, Institute of Microbiology of the Czech Academy of Sciences, Třeboň, Czech Republic

    Daniela Ewe & Grzegorz Konert

  5. Lion Corporation Pharmaceutical Laboratories No.1, Odawara, Kanagawa, 256-0811, Japan

    Masaaki Tachibana

  6. Tech Manage Corp., Tokyo, 160-0023, Japan

    Sae Kikutani

  7. Biology Centre, Institute of Parasitology, Czech Academy of Sciences, České Budějovice, Czech Republic

    Ansgar Gruber

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  1. Daniela Ewe
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Correspondence to Daniela Ewe.

Electronic supplementary material

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11120_2018_500_MOESM1_ESM.tif

Online Resource Figure 5—Additional images of proteins which subcellular localization has been experimentally proven in a second and independent laboratory. Presented are fluorescence microscopy images showing transformed P. tricornutum cells with expressed protein::GFP fusion proteins of PEPCK, PPDK and ME1. Bright field, chlorophyll auto-fluorescence (red), GFP fluorescence (green) and a merged image, showing an overlap of the chlorophyll auto- and GFP fluorescence, are shown from left to right. Scale bars of each image represent 5 µm. “full” or “pre” suffixes after the enzyme’s name indicate whether a full length or pre-sequence was used for plasmid construction. PEPCK and ME1 are located in the mitochondrion and PPDK is located in the cytosol. P. tricornutum strain UTEX 642 was used to generate the PPDKfull and strain UTEX 646 was used to generate the PEPCKpre and ME1pre transformed cell lines. Abbreviations: PEPCK: phosphoenolpyruvate carboxykinase; PPDK: pyruvate phosphate dikinase; ME: malic enzyme. (TIF 37800 KB)

Online Resource Table IV (DOCX 17 KB)

11120_2018_500_MOESM3_ESM.xlsx

Online Resource Table V—A list of enzymes including protein IDs and localizations that are mentioned in this study. “Target P” (Emanuelsson et al. 2000) shows the highest probability for the predicted subcellular localization. The chosen organism groups are “Non-plant” for P. tricornutum and “Plant” for A. thaliana. The output style is: cTP - chloroplast transit peptide; mTP - mitochondrial transit peptide; SP - secretory pathway, other - cytosol, “cleavage site” indicates the sequence motif surrounding the signal peptide cleavage site predicted by SignalP 3.0 NN (Bendtsen et al. 2004) (*indicates manual predictions of the putative cleavage site motif), “localization experiment” indicates the results of experimental localizations cited in “reference/comments”. (XLSX 37 KB)

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Ewe, D., Tachibana, M., Kikutani, S. et al. The intracellular distribution of inorganic carbon fixing enzymes does not support the presence of a C4 pathway in the diatom Phaeodactylum tricornutum. Photosynth Res 137, 263–280 (2018). https://doi.org/10.1007/s11120-018-0500-5

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