Photosynthetic carbon fixation is often limited by CO2 availability, which led to the evolution of CO2 concentrating mechanisms (CCMs). Some diatoms possess CCMs that employ biochemical fixation of bicarbonate, similar to C4 plants, but whether biochemical CCMs are commonly found in diatoms is a subject of debate. In the diatom Phaeodactylum tricornutum, phosphoenolpyruvate carboxylase (PEPC) is present in two isoforms, PEPC1 in the plastids and PEPC2 in the mitochondria. We used real-time quantitative polymerase chain reaction, Western blots, and enzymatic assays to examine PEPC expression and PEPC activity, under low and high concentrations of dissolved inorganic carbon (DIC). We generated and analyzed individual knockout cell lines of PEPC1 and PEPC2, as well as a PEPC1/2 double-knockout strain. While we could not detect an altered phenotype in the PEPC1 knockout strains at ambient, low or high DIC concentrations, PEPC2 and the double-knockout strains grown under ambient air or lower DIC availability conditions showed reduced growth and photosynthetic affinity for DIC while behaving similarly to wild-type (WT) cells at high DIC concentrations. These mutants furthermore exhibited significantly lower 13 C/12 C ratios compared to the WT. Our data imply that in P. tricornutum at least parts of the CCM rely on biochemical bicarbonate fixation catalyzed by the mitochondrial PEPC2.

Department of Bioscience School of Biological and Environmental Sciences 1 Gakuen Uegahara Sanda Hyogo 669 1330 Japan

Fachbereich Biologie Universität Konstanz 78457 Konstanz Germany

Institute of Parasitology Biology Centre Czech Academy of Sciences 370 05 České Budějovice Czech Republic

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Andersson I, Backlund A. 2008. Structure and function of Rubisco. Plant Physiology Biochemistry 46: 275-291.

Armbrust EV, Berges JA, Bowler C, Green BR, Martinez D, Putnam NH, Zhou S, Allen AE, Apt KE, Bechner M et al. 2004. The genome of the diatom Thalassiosira pseudonana: ecology, evolution, and metabolism. Science 306: 79-86.

Ascencio J, Bowes G. 1983. Phosphoenolpyruvate carboxylase in Hydrilla plants with varying CO2 compensation points. Photosynthesis Research 4: 151-170.

Attwood PV, Cleland W. 1986. Decarboxylation of oxalacetate by pyruvate carboxylase. Biochemistry 25: 8191-8196.

Badger MR, Andrews TJ, Whitney S, Ludwig M, Yellowlees DC, Leggat W, Price GD. 1998. The diversity and coevolution of Rubisco, plastids, pyrenoids, and chloroplast-based CO2-concentrating mechanisms in algae. Canadian Journal of Botany 76: 1052-1071.

Bailleul B, Berne N, Murik O, Petroutsos D, Prihoda J, Tanaka A, Villanova V, Bligny R, Flori S, Falconet D et al. 2015. Energetic coupling between plastids and mitochondria drives CO2 assimilation in diatoms. Nature 524: 366-369.

Beardall J, Mukerji D, Glover H, Morris I. 1976. The path of carbon in photosynthesis by marine phytoplankton. Journal of Phycology 12: 409-417.

Beardall J, Raven JA. 2020. Acquisition of inorganic carbon by microalgae and cyanobacteria. In: Wang Q, ed. Microbial photosynthesis. Singapore City, Singapore: Springer, 151-168.

Besnard G, Pinçon G, D'Hont A, Hoarau J-Y, Cadet F, Offmann B. 2003. Characterisation of the phosphoenolpyruvate carboxylase gene family in sugarcane (Saccharum spp.). Theoretical and Applied Genetics 107: 470-478.

Birmingham BC, Colman B. 1979. Measurement of carbon dioxide compensation points of freshwater algae. Plant Physiology 64: 892-895.

Bowes G. 2010. Single-cell C4 photosynthesis in aquatic plants. In: Raghavendra A, Sage R, eds. C4 photosynthesis and related CO2 concentrating mechanisms, In: Advances in photosynthesis and respiration. Dordrecht, the Netherlands: Springer, 63-80.

Bowler C, Allen AE, Badger JH, Grimwood J, Jabbari K, Kuo A, Maheswari U, Martens C, Maumus F, Otillar RP et al. 2008. The Phaeodactylum genome reveals the evolutionary history of diatom genomes. Nature 456: 239-244.

Bradford MM. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 72: 248-254.

Burkhardt S, Amoroso G, Riebesell U, Sültemeyer D. 2001. CO2 and HCO3− uptake in marine diatoms acclimated to different CO2 concentrations. Limnology and Oceanography 46: 1378-1391.

Chang KS, Jeon H, Seo S, Lee Y, Jin E. 2014. Improvement of the phosphoenolpyruvate carboxylase activity of Phaeodactylum tricornutum PEPCase 1 through protein engineering. Enzyme and Microbial Technology 60: 64-71.

Clement R, Dimnet L, Maberly SC, Gontero B. 2016. The nature of the CO2-concentrating mechanisms in a marine diatom, Thalassiosira pseudonana. New Phytologist 209: 1417-1427.

Clement R, Jensen E, Prioretti L, Maberly SC, Gontero B. 2017. Diversity of CO2-concentrating mechanisms and responses to CO2 concentration in marine and freshwater diatoms. Journal of Experimental Botany 68: 3925-3935.

Colman B, Rotatore C. 1995. Photosynthetic inorganic carbon uptake and accumulation in two marine diatoms. Plant, Cell & Environment 18: 919-924.

Dong L-Y, Masuda T, Kawamura T, Hata S, Izui K. 1998. Cloning, expression, and characterization of a root-form phosphoenolpyruvate carboxylase from Zea mays: comparison with the C4-form enzyme. Plant and Cell Physiology 39: 865-873.

Doyle EL, Booher NJ, Standage DS, Voytas DF, Brendel VP, VanDyk JK, Bogdanove AJ. 2012. TAL Effector Nucleotide Targeter (TALE-NT) 2.0: tools for TAL effector design and target prediction. Nucleic Acids Research 40: W117-W122.

Edwards GE, Franceschi VR, Voznesenskaya EV. 2004. Single-cell C4 photosynthesis versus the dual-cell (Kranz) paradigm. Annual Review of Plant Biology 55: 173-196.

Ewe D. 2015. Living well with a scrambled metabolism: CO2 fixation and carbohydrate pathways in the diatom Phaeodactylum tricornutum. PhD thesis, Konstanz University, Konstanz, Germany.

Ewe D, Tachibana M, Kikutani S, Gruber A, Bártulos CR, Konert G, Kaplan A, Matsuda Y, Kroth PG. 2018. The intracellular distribution of inorganic carbon fixing enzymes does not support the presence of a C4 pathway in the diatom Phaeodactylum tricornutum. Photosynthesis Research 137: 263-280.

Falkowski P, Raven J. 1997. An introduction to photosynthesis in aquatic systems. In: Aquatic photosynthesis. Malden, MA, USA: Blackwell Science, 1-32.

Flori S, Jouneau PH, Bailleul B, Gallet B, Estrozi LF, Moriscot C, Bastien O, Eicke S, Schober A, Bártulos CR et al. 2017. Plastid thylakoid architecture optimizes photosynthesis in diatoms. Nature Communications 20: 15885.

Giordano M, Beardall J, Raven JA. 2005. CO2 concentrating mechanisms in algae: mechanisms, environmental modulation, and evolution. Annual Review of Plant Biology 56: 99-131.

Gowik U, Engelmann S, Bläsing O, Raghavendra AS, Westhoff P. 2006. Evolution of C4 phosphoenolpyruvate carboxylase in the genus Alternanthera: gene families and the enzymatic characteristics of the C4 isozyme and its orthologues in C3 and C3/C4 Alternantheras. Planta 223: 359-368.

Graven H, Allison CE, Etheridge DM, Hammer S, Keeling RF, Levin I, Meijer HAJ, Rubino M, Tans PP, Trudinger CM et al. 2017. Compiled records of carbon isotopes in atmospheric CO2 for historical simulations in CMIP6. Geoscientific Model Development 10: 4405-4417.

Gruber A, Kroth PG. 2017. Intracellular metabolic pathway distribution in diatoms and tools for genome-enabled experimental diatom research. Philosophical Transactions of the Royal Society B 372: 20160402.

Guillard RR. 1975. Culture of phytoplankton for feeding marine invertebrates. In: Smith ML, Chanley MH, eds. Culture of marine invertebrate animals. New York, NY, USA: Plenum Press, 29-60.

Guillard RR, Ryther JH. 1962. Studies of marine planktonic diatoms: I. Cyclotella Nana Hustedt, and Detonula Confervacea (CLEVE) Gran. Canadian Journal of Microbiology 8: 229-239.

Gutierrez M, Gracen V, Edwards G. 1974. Biochemical and cytological relationships in C4 plants. Planta 119: 279-300.

Haimovich-Dayan M, Garfinkel N, Ewe D, Marcus Y, Gruber A, Wagner H, Kroth PG, Kaplan A. 2013. The role of C4 metabolism in the marine diatom Phaeodactylum tricornutum. New Phytologist 197: 177-185.

Hennon GM, Hernández Limón MD, Haley ST, Juhl AR, Dyhrman ST. 2017. Diverse CO2-induced responses in physiology and gene expression among eukaryotic phytoplankton. Frontiers in Microbiology 8: 2547.

Hill AV. 1910. The possible effects of the aggregation of the molecules of haemoglobin on its dissociation curves. Journal of Physiology 40: 4-7.

Hopkinson BM, Dupont CL, Allen AE, Morel FM. 2011. Efficiency of the CO2-concentrating mechanism of diatoms. Proceedings of the National Academy of Sciences, USA 108: 3830-3837.

Hopkinson BM, Dupont CL, Matsuda Y. 2016. The physiology and genetics of CO2 concentrating mechanisms in model diatoms. Current Opinion in Plant Biology 31: 51-57.

Hopkinson BM, Meile C, Shen C. 2013. Quantification of extracellular carbonic anhydrase activity in two marine diatoms and investigation of its role. Plant Physiology 162: 1142-1152.

Huang W, Haferkamp I, Lepetit B, Molchanova M, Hou S, Jeblick W, Río Bártulos C, Kroth PG. 2018. Reduced vacuolar β-1,3-glucan synthesis affects carbohydrate metabolism as well as plastid homeostasis and structure in Phaeodactylum tricornutum. Proceedings of the National Academy of Sciences, USA 115: 4791-4796.

Jeffrey SW, Humphrey GF. 1975. New spectrophotometric equations for determining chlorophylls a, b, c1 and c2 in higher plants, algae and natural phytoplankton. Biochemie und Physiologie der Pflanzen 167: 191-194.

Jenkins C. 1987. 3, 3-Dichloro-2-dihydroxyphosphinoylmethyl-2-propenoate, a new, specific inhibitor of phosphoenolpyruvate carboxylase. Biochemistry International 14: 219-226.

Jensen EL, Clement R, Kosta A, Maberly SC, Gontero B. 2019. A new widespread subclass of carbonic anhydrase in marine phytoplankton. The ISME Journal 13: 2094-2106.

Kaplan A, Reinhold L. 1999. CO2 concentrating mechanisms in photosynthetic microorganisms. Annual Review of Plant Biology 50: 539-570.

Karlusich JJP, Bowler C, Biswas H. 2021. Carbon dioxide concentration mechanisms in natural populations of marine diatoms: insights from Tara oceans. Frontiers in Plant Science 12: 659.

Kikutani S, Nakajima K, Nagasato C, Tsuji Y, Miyatake A, Matsuda Y. 2016. Thylakoid luminal θ-carbonic anhydrase critical for growth and photosynthesis in the marine diatom Phaeodactylum tricornutum. Proceedings of the National Academy of Sciences, USA 113: 9828-9833.

Kroth P. 2002. Protein transport into secondary plastids and the evolution of primary and secondary plastids. International Review of Cytology 221: 191-255.

Kroth PG, Chiovitti A, Gruber A, Martin-Jezequel V, Mock T, Parker MS, Stanley MS, Kaplan A, Caron L, Weber T et al. 2008. A model for carbohydrate metabolism in the diatom Phaeodactylum tricornutum deduced from comparative whole genome analysis. PLoS ONE 3: e1426.

Kustka AB, Milligan AJ, Zheng H, New AM, Gates C, Bidle KD, Reinfelder JR. 2014. Low CO2 results in a rearrangement of carbon metabolism to support C4 photosynthetic carbon assimilation in Thalassiosira pseudonana. New Phytologist 204: 507-520.

Lara MV, Casati P, Andreo CS. 2002. CO2-concentrating mechanisms in Egeria densa, a submersed aquatic plant. Physiologia Plantarum 115: 487-495.

Lara MV, Chuong SD, Akhani H, Andreo CS, Edwards GE. 2006. Species having C4 single-cell-type photosynthesis in the Chenopodiaceae family evolved a photosynthetic phosphoenolpyruvate carboxylase like that of Kranz-type C4 species. Plant Physiology 142: 673-684.

Launay H, Huang W, Maberly SC, Gontero B. 2020. Regulation of carbon metabolism by environmental conditions: a perspective from diatoms and other chromalveolates. Frontiers in Plant Science 11: 1033.

Lee RE, Kugrens P. 1998. Hypothesis: the ecological advantage of chloroplast ER - the ability to outcompete at low dissolved CO2 concentrations. Protist 149: 341-345.

Lepetit B, Sturm S, Rogato A, Gruber A, Sachse M, Falciatore A, Kroth PG, Lavaud J. 2013. High light acclimation in the secondary plastids containing diatom Phaeodactylum tricornutum is triggered by the redox state of the plastoquinone pool. Plant Physiology 161: 853-865.

Losh JL, Young JN, Morel FMM. 2013. Rubisco is a small fraction of total protein in marine phytoplankton. New Phytologist 198: 52-58.

Maberly SC, Ball LA, Raven JA, Sültemeyer D. 2009. Inorganic carbon acquisition by chrysophytes. Journal of Phycology 45: 1052-1061.

Magnin NC, Cooley BA, Reiskind JB, Bowes G. 1997. Regulation and localization of key enzymes during the induction of Kranz-less, C4-type photosynthesis in Hydrilla verticillata. Plant Physiology 115: 1681-1689.

Matsuda Y, Hara T, Colman B. 2001. Regulation of the induction of bicarbonate uptake by dissolved CO2 in the marine diatom, Phaeodactylum tricornutum. Plant, Cell & Environment 24: 611-620.

Matsuda Y, Hopkinson BM, Nakajima K, Dupont CL, Tsuji Y. 2017. Mechanisms of carbon dioxide acquisition and CO2 sensing in marine diatoms: a gateway to carbon metabolism. Philosophical Transactions of the Royal Society B: Biological Sciences 372: 20160403.

McGinn PJ, Morel FM. 2008. Expression and inhibition of the carboxylating and decarboxylating enzymes in the photosynthetic C4 pathway of marine diatoms. Plant Physiology 146: 300-309.

Michaelis L, Menten ML. 1913. Die kinetik der invertinwirkung. Biochemische Zeitschrift 49: 333-369.

Morel F, Reinfelder J, Roberts S, Chamberlain C, Lee J, Yee D. 1994. Zinc and carbon co-limitation of marine phytoplankton. Nature 369: 740-742.

Morel FM, Cox EH, Kraepiel AM, Lane TW, Milligan AJ, Schaperdoth I, Reinfelder JR, Tortell PD. 2002. Acquisition of inorganic carbon by the marine diatom Thalassiosira weissflogii. Functional Plant Biology 29: 301-308.

Nakajima K, Tanaka A, Matsuda Y. 2013. SLC4 family transporters in a marine diatom directly pump bicarbonate from seawater. Proceedings of the National Academy of Sciences, USA 110: 1767-1772.

Nelson DM, Tréguer P, Brzezinski MA, Leynaert A, Quéguiner B. 1995. Production and dissolution of biogenic silica in the ocean: revised global estimates, comparison with regional data and relationship to biogenic sedimentation. Global Biogeochemical Cycles 9: 359-372.

O'Leary MH. 1981. Carbon isotope fractionation in plants. Phytochemistry 20: 553-567.

Prihoda J, Tanaka A, de Paula WB, Allen JF, Tirichine L, Bowler C. 2012. Chloroplast-mitochondria cross-talk in diatoms. Journal of Experimental Botany 63: 1543-1557.

Rao SK, Reiskind JB, Bowes G. 2008. Kinetic analyses of recombinant isoforms of phosphoenolpyruvate carboxylase from Hydrilla verticillata leaves and the impact of substituting a C4-signature serine. Plant Science 174: 475-483.

Reinfelder JR. 2010. Carbon concentrating mechanisms in eukaryotic marine phytoplankton. Annual Review of Marine Science 3: 291-315.

Reinfelder JR, Kraepiel AM, Morel FM. 2000. Unicellular C4 photosynthesis in a marine diatom. Nature 407: 996-999.

Reinfelder JR, Milligan AJ, Morel FM. 2004. The role of the C4 pathway in carbon accumulation and fixation in a marine diatom. Plant Physiology 135: 2106-2111.

Reiskind JB, Bowes G. 1991. The role of phosphoenolpyruvate carboxykinase in a marine macroalga with C4-like photosynthetic characteristics. Proceedings of the National Academy of Sciences, USA 88: 2883-2887.

Riebesell U, Wolf-Gladrow D, Smetacek V. 1993. Carbon dioxide limitation of marine phytoplankton growth rates. Nature 361: 249-251.

Roberts K, Granum E, Leegood RC, Raven JA. 2007. C3 and C4 pathways of photosynthetic carbon assimilation in marine diatoms are under genetic, not environmental, control. Plant Physiology 145: 230-235.

Rotatore C, Colman B, Kuzma M. 1995. The active uptake of carbon dioxide by the marine diatoms Phaeodactylum tricornutum and Cyclotella sp. Plant, Cell & Environment 18: 913-918.

Sage RF. 2004. The evolution of C4 photosynthesis. New Phytologist 161: 341-370.

Sage RF, Sage TL, Kocacinar F. 2012. Photorespiration and the evolution of C4 photosynthesis. Annual Review of Plant Biology 63: 19-47.

Salvucci ME, Bowes G. 1981. Induction of reduced photorespiratory activity in submersed and amphibious aquatic macrophytes. Plant Physiology 67: 335-340.

Sanjana NE, Cong L, Zhou Y, Cunniff MM, Feng G, Zhang F. 2012. A transcription activator-like effector toolbox for genome engineering. Nature Protocols 7: 171-192.

Serif M, Lepetit B, Weißert K, Kroth PG, Rio Bartulos CR. 2017. A fast and reliable strategy to generate TALEN-mediated gene knockouts in the diatom Phaeodactylum tricornutum. Algal Research 23: 186-195.

Shen C, Dupont CL, Hopkinson BM. 2017. The diversity of carbon dioxide-concentrating mechanisms in marine diatoms as inferred from their genetic content. Journal of Experimental Botany 68: 3937-3948.

Smith SR, Abbriano RM, Hildebrand M. 2012. Comparative analysis of diatom genomes reveals substantial differences in the organization of carbon partitioning pathways. Algal Research 1: 2-16.

Svensson P, Biasing OE, Westhoff P. 1997. Evolution of the enzymatic characteristics of C4 phosphoenol pyruvate carboxylase: a comparison of the orthologous PPCA phosphoenol pyruvate carboxylases of Flaveria trinervia (C4) and Flaveria pringlei (C3). European Journal of Biochemistry 246: 452-460.

Tanaka R, Kikutani S, Mahardika A, Matsuda Y. 2014. Localization of enzymes relating to C4 organic acid metabolisms in the marine diatom, Thalassiosira pseudonana. Photosynthesis Research 121: 251-263.

Taylor L, Nunes-Nesi A, Parsley K, Leiss A, Leach G, Coates S, Wingler A, Fernie AR, Hibberd JM. 2010. Cytosolic pyruvate,orthophosphate dikinase functions in nitrogen remobilization during leaf senescence and limits individual seed growth and nitrogen content. The Plant Journal 62: 641-652.

Tsuji Y, Kusi-Appiah G, Kozai N, Fukuda Y, Yamano T, Fukuzawa H. 2021. Characterization of a CO2-concentrating mechanism with low sodium dependency in the centric diatom Chaetoceros gracilis. Marine Biotechnology 23: 456-462.

Tsuji Y, Mahardika A, Matsuda Y. 2017. Evolutionarily distinct strategies for the acquisition of inorganic carbon from seawater in marine diatoms. Journal of Experimental Botany 68: 3949-3958.

Voznesenskaya EV, Franceschi VR, Kiirats O, Artyusheva EG, Freitag H, Edwards GE. 2002. Proof of C4 photosynthesis without Kranz anatomy in Bienertia cycloptera (Chenopodiaceae). The Plant Journal 31: 649-662.

Whitney SM, Sharwood RE, Orr D, White SJ, Alonso H, Galmés J. 2011. Isoleucine 309 acts as a C4 catalytic switch that increases ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) carboxylation rate in Flaveria. Proceedings of the National Academy of Sciences, USA 108: 14688-14693.

Young JN, Heureux AMC, Sharwood RE, Rickaby REM, Morel FMM, Whitney SM. 2016. Large variation in the Rubisco kinetics of diatoms reveals diversity among their carbon-concentrating mechanisms. Journal of Experimental Botany 67: 3445-3456.

Young JN, Hopkinson BM. 2017. The potential for co-evolution of CO2-concentrating mechanisms and rubisco in diatoms. Journal of Experimental Botany 68: 3751-3762.

Yu G, Kroth PG, Gruber A. 2017. Controlled supply of CO2 to batch cultures of the diatom Phaeodactylum tricornutum. Endocytobiosis and Cell Research 28: 62-66.

Zaslavskaia LA, Lippmeier JC, Kroth PG, Grossman AR, Apt KE. 2000. Transformation of the diatom Phaeodactylum tricornutum (Bacillariophyceae) with a variety of selectable marker and reporter genes. Journal of Phycology 36: 379-386.

Zhang Y, Yin L, Jiang HS, Li W, Gontero B, Maberly SC. 2014. Biochemical and biophysical CO2 concentrating mechanisms in two species of freshwater macrophyte within the genus Ottelia (Hydrocharitaceae). Photosynthesis Research 121: 285-297.

Zhao S, Fernald RD. 2005. Comprehensive algorithm for quantitative real-time polymerase chain reaction. Journal of Computational Biology 12: 1047-1064.

ASAFind 2.0: multi-class protein targeting prediction for diatoms and algae with complex plastids