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Insertional suppressors of Chlamydomonas reinhardtii that restore growth of air-dier lcib mutants in low CO2

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Abstract

Chlamydomonas reinhardtii and other microalgae show adaptive changes to limiting CO2 conditions by induction of CO2-concentrating mechanisms. The limiting-CO2-inducible gene, LCIB, encodes a soluble plastid protein and is proposed to play a role in trapping CO2 released by CAH3 (thylakoid lumen carbonic anhydrase) catalyzed dehydration of accumulated Ci, especially in low CO2 (L-CO2; ~0.04% CO2) conditions. To gain further insight into the mechanisms of Ci uptake and accumulation in L-CO2 acclimated C. reinhardtii, we performed an insertional mutagenesis screen to isolate extragenic suppressors that restore the growth of lcib mutants (pmp1 and ad1) in L-CO2. Four independent suppressors are described here and classified by their photosynthetic affinities for Ci and expression patterns of known limiting-CO2-inducible transcripts. Genetic analysis of the four suppressors identified two allelic, dominant suppressors (su4 and su5), and two recessive suppressors (su1 and su8). Consistent with the suppression phenotype, both the relative affinities of photosynthetic O2 evolution and internal Ci accumulation in all four suppressors were substantially increased relative to pmp1/ad1 in L-CO2 acclimated cells. The relative affinities of pmp-su1 and ad-su8 for Ci were nearly the same as wild type, but that of pmp-su4/su5 was intermediate between pmp-su1 and pmp1. Also, the interactions between lcib mutations and each of the three suppressors varied over the range of CO2 acclimation states. Our results suggest complex contributions of LCIB-dependent and independent active Ci uptake/accumulation systems in various CO2 acclimation states and therefore provide new clues about the roles played by LCIB in limiting Ci acclimation.

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References

  • Amoroso G, Sültemeyer DF, Thyssen C, Fock HP (1998) Uptake of HCO3 − and CO2 in cells and chloroplasts from the microalgae Chlamydomonas reinhardtii and Dunaliella tertiolecta. Plant Physiol 116:193–201

    Article  CAS  Google Scholar 

  • Badger MR, Kaplan A, Berry JA (1980) Internal inorganic carbon pool of Chlamydomonas reinhardtii: evidence for a carbon dioxide concentrating mechanism. Plant Physiol 66:407–413

    Article  PubMed  CAS  Google Scholar 

  • Chomczynski P, Sacchi N (2006) The single-step method of RNA isolation by acid guanidinium thiocyanate–phenol–chloroform extraction: twenty-something years on. Nat Protol 1:581–585

    Article  CAS  Google Scholar 

  • Colombo SL, Pollock SV, Eger KA, Godfrey AC, Adams JE, Mason CB, Moroney JV (2002) Use of the bleomycin resistance gene to generate tagged insertional mutants of Chlamydomonas reinhardtii that require elevated CO2 for optimal growth. Funct Plant Biol 29:231–241

    Article  CAS  Google Scholar 

  • Duanmu D, Miller AR, Horken KM, Weeks DP, Spalding MH (2009a) Knockdown of limiting-CO2-induced gene HLA3 decreases HCO3 − transport and photosynthetic Ci affinity in Chlamydomonas reinhardtii. Proc Natl Acad Sci USA 106:5990–5995

    Article  PubMed  CAS  Google Scholar 

  • Duanmu D, Wang Y, Spalding MH (2009b) Thylakoid lumen carbonic anhydrase (CAH3) mutation suppresses air-dier phenotype of LCIB mutant in Chlamydomonas reinhardtii. Plant Physiol 149:929–937

    Article  PubMed  CAS  Google Scholar 

  • Fukuzawa H, Miura K, Ishizaki K, Kucho KI, Saito T, Kohinata T, Ohyama K (2001) Ccm1, a regulatory gene controlling the induction of a carbon-concentrating mechanism in Chlamydomonas reinhardtii by sensing CO2 availability. Proc Natl Acad Sci USA 98:5347–5352

    Article  PubMed  CAS  Google Scholar 

  • Funke RP, Kovar JL, Weeks DP (1997) Intracellular carbonic anhydrase is essential to photosynthesis in Chlamydomonas reinhardtii at atmospheric levels of CO2. Plant Physiol 114:237–244

    Article  PubMed  CAS  Google Scholar 

  • Giordano M, Beardall J, Raven JA (2005) CO2 concentrating mechanisms in algae: mechanisms, environmental modulation, and evolution. Annu Rev Plant Biol 56:99–131

    Article  PubMed  CAS  Google Scholar 

  • Harris EH (1989) The Chlamydomonas source book: a comprehensive guide to biology and laboratory use. Academic Press Inc, San Diego, pp 419–446

    Google Scholar 

  • Karlsson J, Clarke AK, Chen ZY, Hugghins SY, Park Y-II, Husic HD, Moroney JV, Samuelsson G (1998) A novel α-type carbonic anhydrase associated with the thylakoid membrane in Chlamydomonas reinhardtii is required for growth at ambient CO2. EMBO J 17:1208–1216

    Article  PubMed  CAS  Google Scholar 

  • Kindle KL (1990) High-frequency nuclear transformation of Chlamydomonas reinhardtii. Proc Natl Acad Sci USA 87:1228–1232

    Article  PubMed  CAS  Google Scholar 

  • Long SP, Ainsworth EA, Rogers A, Ort DR (2004) Rising atmospheric carbon dioxide: plants face the future. Annu Rev Plant Biol 55:591–628

    Article  PubMed  CAS  Google Scholar 

  • Mariscal V, Moulin P, Orsel M, Miller AJ, Fernández E, Galván A (2006) Differential regulation of the Chlamydomonas Nar1 gene family by carbon and nitrogen. Protist 157:421–433

    Article  PubMed  CAS  Google Scholar 

  • Miura K, Yamano T, Yoshioka S, Kohinata T, Inoue Y, Taniguchi F, Asamizu E, Nakaura Y, Tabata S, Yamato KT, Ohyama K, Fukuzawa H (2004) Expression profiling-based identification of CO2-responsive genes regulated by CCM1 controlling a carbon-concentrating mechanism in Chlamydomonas reinhardtii. Plant Physiol 135:1595–1607

    Article  PubMed  CAS  Google Scholar 

  • Moroney JV, Ynalvez RA (2007) Proposed carbon dioxide concentrating mechanism in Chlamydomonas reinhardtii. Eukaryot Cell 6:1251–1259

    Article  PubMed  CAS  Google Scholar 

  • Moroney JV, Tolbert NE, Sears BB (1986) Complementation analysis of the inorganic carbon concentrating mechanism of Chlamydomonas reinhardtii. Mol Gen Genet 204:199–203

    Article  CAS  Google Scholar 

  • Moroney JV, Husic HD, Tolbert NE, Kitayama M, Manuel LJ, Togasaki RK (1989) Isolation and characterization of a mutant of Chlamydomonas reinhardtii deficient in the CO2 concentrating mechanism. Plant Physiol 89:897–903

    Article  PubMed  CAS  Google Scholar 

  • Nakamura Y, Kanakagiri S, Van K, He W, Spalding MH (2005) Disruption of the glycolate dehydrogenase gene in the high-CO2-requiring mutant HCR89 of Chlamydomonas reinhardtii. Can J Bot 83:820–833

    Article  CAS  Google Scholar 

  • Ohnishi N, Mukherjee B, Tsujikawa T, Yanase M, Nakano H, Moroney JV, Fukuzawa H (2010) Expression of a low CO2-inducible protein, LCI1, increases inorganic carbon uptake in the green alga Chlamydomonas reinhardtii. Plant Cell 22:3105–3117

    Article  PubMed  CAS  Google Scholar 

  • Palmqvist K, Sjoberg S, Samuelsson G (1988) Induction of inorganic carbon accumulation in the unicellular green algae Scenedesmus obliquus and Chlamydomonas reinhardtii. Plant Physiol 87:437–442

    Article  PubMed  CAS  Google Scholar 

  • Palmqvist K, Yu J-W, Badger MR (1994) Carbonic anhydrase activity and inorganic carbon fluxes in low and high Ci of Chlamydomonas reinhardtii and Scenedesmus obliquus. Physiol Plant 90:537–547

    Article  CAS  Google Scholar 

  • Pollock SV, Prout DL, Godfrey AC, Lemaire SD, Moroney JV (2004) The Chlamydomonas reinhardtii proteins Ccp1 and Ccp2 are required for long-term growth, but are not necessary for efficient photosynthesis, in a low-CO2 environment. Plant Mol Biol 56:125–132

    Article  PubMed  CAS  Google Scholar 

  • Price GD, Maeda SI, Omata T, Badger MR (2002) Modes of active inorganic carbon uptake in the cyanobacterium, Synechococcus sp. PCC7942. Funct Plant Biol 29:131–149

    Article  CAS  Google Scholar 

  • Price GD, Woodger FJ, Badger MR, Howitt SM, Tucker L (2004) Identification of a SulP-type bicarbonate transporter in marine cyanobacteria. Proc Natl Acad Sci USA 101:18228–18233

    Article  PubMed  CAS  Google Scholar 

  • Rymarquis LA, Handley JM, Thomas M, Stern DB (2005) Beyond complementation. Map-based cloning in Chlamydomonas reinhardtii. Plant Physiol 137:557–566

    Article  PubMed  CAS  Google Scholar 

  • Sizova I, Fuhrmann M, Hegemann P (2001) A Streptomyces rimosus aphVIII gene encoding for a new type phosphotransferase provides stable antibiotic resistance to Chlamydomonas reinhardtii. Gene 277:221–229

    Article  PubMed  CAS  Google Scholar 

  • Spalding MH (2008) Microalgal carbon-dioxide-concentrating mechanisms: Chlamydomonas inorganic carbon transporters. J Exp Bot 59:1463–1473

    Article  PubMed  CAS  Google Scholar 

  • Spalding MH, Spreitzer RJ, Ogren WL (1983a) Carbonic anhydrase-deficient mutant of Chlamydomonas reinhardtii requires elevated carbon dioxide concentration for photoautotrophic growth. Plant Physiol 73:268–272

    Article  PubMed  CAS  Google Scholar 

  • Spalding MH, Spreitzer RJ, Ogren WL (1983b) Reduced inorganic carbon transport in a CO2-requiring mutant of Chlamydomonas reinhardtii. Plant Physiol 73:273–276

    Article  PubMed  CAS  Google Scholar 

  • Spalding MH, Van K, Wang Y, Nakamura Y (2002) Acclimation of Chlamydomonas to changing carbon availability. Funct Plant Biol 29:221–230

    Article  CAS  Google Scholar 

  • Sültemeyer DF, Klöck G, Kreuzberg K, Fock HP (1988) Photosynthesis and apparent affinity for dissolved inorganic carbon by cells and chloroplasts of Chlamydomonas reinhardtii grown at high and low CO2 concentrations. Planta 176:256–260

    Article  Google Scholar 

  • Sültemeyer DF, Miller AG, Espie GS, Fock HP, Canvin DT (1989) Active CO2 transport by the green alga Chlamydomonas reinhardtii. Plant Physiol 89:1213–1219

    Article  PubMed  Google Scholar 

  • Sültemeyer DF, Fock HP, Canvin DT (1991) Active uptake of inorganic carbon by Chlamydomonas reinhardtii: evidence for simultaneous transport of HCO3 − and CO2 and characterization of active CO2 transport. Can J Bot 69:995–1002

    Article  Google Scholar 

  • Suzuki K (1995) Phosphoglycolate phosphatase-deficient mutants of Chlamydomonas reinhardtii capable of growth under air. Plant Cell Physiol 36:95–100

    CAS  Google Scholar 

  • Thyssen C, Hermes M, Sültemeyer D (2003) Isolation and characterization of Chlamydomonas reinhardtii mutants with an impaired CO2-concentrating mechanism. Planta 217:102–112

    PubMed  CAS  Google Scholar 

  • Van K, Spalding MH (1999) Periplasmic carbonic anhydrase structural gene (Cah1) mutant in Chlamydomonas reinhardtii. Plant Physiol 120:757–764

    Article  PubMed  CAS  Google Scholar 

  • Van K, Wang Y, Nakamura Y, Spalding MH (2001) Insertional mutants of Chlamydomonas reinhardtii that require elevated CO2 for survival. Plant Physiol 127:607–614

    Article  PubMed  CAS  Google Scholar 

  • Vance P, Spalding MH (2005) Growth, photosynthesis and gene expression in Chlamydomonas over a range of CO2 concentrations and CO2/O2 ratios: CO2 regulates multiple acclimation states. Can J Bot 83:796–809

    Article  CAS  Google Scholar 

  • Wang Y, Spalding MH (2006) An inorganic carbon transport system responsible for acclimation specific to air levels of CO2 in Chlamydomonas reinhardtii. Proc Natl Acad Sci USA 103:10110–10115

    Article  PubMed  CAS  Google Scholar 

  • Wang Y, Spalding MH (2007) CO2 concentrating mechanisms in eukaryotic microalgae. Funct Plant Sci Biotechnol 1:120–128

    Google Scholar 

  • Wirtz W, Stitt M, Heldt HW (1980) Enzymic determination of metabolites in the subcellular compartments of Spinach protoplasts. Plant Physiol 66:187–193

    Article  PubMed  CAS  Google Scholar 

  • Xiang Y, Zhang J, Weeks DP (2001) The cia5 gene controls formation of the carbon concentrating mechanism in Chlamydomonas reinhardtii. Proc Natl Acad Sci USA 98:5341–5346

    Article  PubMed  CAS  Google Scholar 

  • Yamano T, Tsujikawa T, Hatano K, Ozawa S, Takahashi Y, Fukuzawa H (2010) Light and low-CO2-dependent LCIB-LCIC complex localization in the chloroplast supports the carbon-concentrating mechanism in Chlamydomonas reinhardtii. Plant Cell Physiol 51:1453–1468

    Article  PubMed  CAS  Google Scholar 

  • Zamora I, Feldman JL, Marshall WF (2004) PCR-based assay for mating type and diploidy in Chlamydomonas. BioTechniques 37:534–536

    PubMed  CAS  Google Scholar 

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Acknowledgments

This research was supported by the National Research Initiative Competitive Grant no. 2007-35318-18433 from the U.S. Department of Agriculture (to M.H.S.), as well as by the College of Agriculture and Life Sciences and the College of Liberal Arts and Sciences at Iowa State University.

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Authors and Affiliations

  1. Department of Genetics, Development and Cell Biology, Iowa State University, Ames, IA, 50011, USA

    Martin H. Spalding

  2. Department of Molecular and Cellular Biology, University of California, Davis, CA, 95616, USA

    Deqiang Duanmu

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  1. Deqiang Duanmu
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  2. Martin H. Spalding
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Correspondence to Martin H. Spalding.

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Duanmu, D., Spalding, M.H. Insertional suppressors of Chlamydomonas reinhardtii that restore growth of air-dier lcib mutants in low CO2 . Photosynth Res 109, 123–132 (2011). https://doi.org/10.1007/s11120-011-9642-4

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  • DOI: https://doi.org/10.1007/s11120-011-9642-4

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