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The carbon concentrating mechanism in Chlamydomonas reinhardtii: finding the missing pieces

Abstract

The photosynthetic, unicellular green alga, Chlamydomonas reinhardtii, lives in environments that often contain low concentrations of CO2 and HCO3 , the utilizable forms of inorganic carbon (Ci). C. reinhardtii possesses a carbon concentrating mechanism (CCM) which can provide suitable amounts of Ci for growth and development. This CCM is induced when the CO2 concentration is at air levels or lower and is comprised of a set of proteins that allow the efficient uptake of Ci into the cell as well as its directed transport to the site where Rubisco fixes CO2 into biomolecules. While several components of the CCM have been identified in recent years, the picture is still far from complete. To further improve our knowledge of the CCM, we undertook a mutagenesis project where an antibiotic resistance cassette was randomly inserted into the C. reinhardtii genome resulting in the generation of 22,000 mutants. The mutant collection was screened using both a published PCR-based approach (Gonzalez-Ballester et al. 2011) and a phenotypic growth screen. The PCR-based screen did not rely on a colony having an altered growth phenotype and was used to identify colonies with disruptions in genes previously identified as being associated with the CCM-related gene. Eleven independent insertional mutations were identified in eight different genes showing the usefulness of this approach in generating mutations in CCM-related genes of interest as well as identifying new CCM components. Further improvements of this method are also discussed.

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Abbreviations

CA:

Carbonic anhydrase

CBB cycle:

Calvin–Benson–Bassham cycle

CCM:

Carbon (dioxide) concentrating mechanism

Rubisco:

Ribulose-1,5-bisphosphate carboxylase/oxygenase

RuBP:

Ribulose-1,5-bisphosphate

References

  • Blanco-Rivero A, Shutova T, Roman MJ, Villarejo A, Martinez F (2012) Phosphorylation controls the localization and activation of the lumenal carbonic anhydrase in Chlamydomonas reinhardtii. PLoS ONE 7(11):e49063. doi:10.1371/journal.pone.0049063

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Borkhsenious ON, Mason CB, Moroney JV (1998) The intracellular localization of ribulose-1,5-bisphosphate Carboxylase/Oxygenase in Chlamydomonas reinhardtii. Plant Physiol 116(4):1585–1591

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Brueggeman AJ, Gangadharaiah DS, Cserhati MF, Casero D, Weeks DP, Ladunga I (2012) Activation of the carbon concentrating mechanism by CO2 deprivation coincides with massive transcriptional restructuring in Chlamydomonas reinhardtii. Plant Cell 24:1860–1875

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Burow MD, Chen ZY, Mouton TM, Moroney JV (1996) Isolation of cDNA clones of genes induced upon transfer of Chlamydomonas reinhardtii cells to low CO2. Plant Mol Biol 31(2):443–448

    Article  CAS  PubMed  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(3):231–241

    Article  CAS  Google Scholar 

  • Dent RM, Haglund CM, Chin BL, Kobayashi MC, Niyogi KK (2005) Functional genomics of eukaryotic photosynthesis using insertional mutagenesis of Chlamydomonas reinhardtii. Plant Physiol 137(2):545–556. doi:10.1104/pp.104.055244

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Depege N, Bellafiore S, Rochaix JD (2003) Role of chloroplast protein kinase Stt7 in LHCII phosphorylation and state transition in Chlamydomonas. Sci STKE 299(5612):1572

    CAS  Google Scholar 

  • Duanmu D, Miller AR, Horken KM, Weeks DP, Spalding MH (2009) Knockdown of limiting-CO2-induced gene HLA3 decreases HCO3 transport and photosynthetic Ci affinity in Chlamydomonas reinhardtii. Proc Natl Acad Sci USA 106(14):5990–5995. doi:10.1073/pnas.0812885106

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Fang W, Si Y, Douglass S, Casero D, Merchant SS, Pellegrini M, Ladunga I, Liu P, Spalding MH (2012) Transcriptome-wide changes in Chlamydomonas reinhardtii gene expression regulated by carbon dioxide and the CO2-concentrating mechanism regulator CIA5/CCM1. Plant Cell 24(5):1876–1893. doi:10.1105/tpc.112.097949

    Article  CAS  PubMed Central  PubMed  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(9):5347–5352. doi:10.1073/pnas.081593498

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Genkov T, Meyer M, Griffiths H, Spreitzer RJ (2010) Functional hybrid rubisco enzymes with plant small subunits and algal large subunits: engineered rbcS cDNA for expression in Chlamydomonas. J Biol Chem 285(26):19833–19841. doi:10.1074/jbc.M110.124230

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Giordano M, Beardall J, Raven JA (2005) CO2 concentrating mechanisms in algae: mechanisms, environmental modulation, and evolution. Ann Rev Plant Biol 56:99–131. doi:10.1146/annurev.arplant.56.032604.144052

    Article  CAS  Google Scholar 

  • Gonzalez-Ballester D, Pootakham W, Mus F, Yang W, Catalanotti C, Magneschi L, de Montaigu A, Higuera JJ, Prior M, Galvan A, Fernandez E, Grossman AR (2011) Reverse genetics in Chlamydomonas: a platform for isolating insertional mutants. Plant Methods 7:24. doi:10.1186/1746-4811-7-24

    CAS  PubMed Central  PubMed  Google Scholar 

  • Gowik U, Westhoff P (2011) The path from C3 to C4 photosynthesis. Plant Physiol 155(1):56–63. doi:10.1104/pp.110.165308

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Hanson DT, Franklin LA, Samuelsson G, Badger MR (2003) The Chlamydomonas reinhardtii cia3 mutant lacking a thylakoid lumen-localized carbonic anhydrase is limited by CO2 supply to rubisco and not photosystem II function in vivo. Plant Physiol 132(4):2267–2275

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Im CS, Grossman AR (2002) Identification and regulation of high light-induced genes in Chlamydomonas reinhardtii. Plant J 30(3):301–313

    Article  CAS  PubMed  Google Scholar 

  • Karlsson J, Clarke AK, Chen ZY, Hugghins SY, Park YI, Husic HD, Moroney JV, Samuelsson G (1998) A novel alpha-type carbonic anhydrase associated with the thylakoid membrane in Chlamydomonas reinhardtii is required for growth at ambient CO2. EMBO J 17(5):1208–1216. doi:10.1093/emboj/17.5.1208

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Ma Y, Pollock SV, Xiao Y, Cunnusamy K, Moroney JV (2011) Identification of a novel gene, CIA6, required for normal pyrenoid formation in Chlamydomonas reinhardtii. Plant Physiol 156(2):884–896. doi:10.1104/pp.111.173922

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Mariscal V, Moulin P, Orsel M, Miller AJ, Fernandez E, Galvan A (2006) Differential regulation of the Chlamydomonas Nar1 gene family by carbon and nitrogen. Protist 157(4):421–433. doi:10.1016/j.protis.2006.06.003

    Article  CAS  PubMed  Google Scholar 

  • Mason CB, Matthews S, Bricker TM, Moroney JV (1991) Simplified procedure for the isolation of intact chloroplasts from Chlamydomonas reinhardtii. Plant Physiol 97(4):1576–1580

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • McKay R, Gibbs SP, Vaughn K (1991) RuBisCo activase is present in the pyrenoid of green algae. Protoplasma 162(1):38–45

    Article  CAS  Google Scholar 

  • Meyer M, Griffiths H (2013) Origins and diversity of eukaryotic CO2-concentrating mechanisms: lessons for the future. J Exp Bot 64(3):769–786. doi:10.1093/jxb/ers390

    Article  CAS  PubMed  Google Scholar 

  • Meyer MT, Genkov T, Skepper JN, Jouhet J, Mitchell MC, Spreitzer RJ, Griffiths H (2012) Rubisco small-subunit alpha-helices control pyrenoid formation in Chlamydomonas. Proc Natl Acad Sci USA 109(47):19474–19479. doi:10.1073/pnas.1210993109

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Miura K, Yamano T, Yoshioka S, Kohinata T, Inoue Y, Taniguchi F, Asamizu E, Nakamura 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(3):1595–1607. doi:10.1104/pp.104.041400

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Morita E, Kuroiwa H, Kuroiwa T, Nozaki H (1997) High localization of ribulose-1, 5-bisphosphate carboxylase/oxygenase in the pyrenoids of Chlamydomonas reinhardtii (Chlorophyta), as revealed by cryofixation and immunogold electron microscopy. J Phycol 33(1):68–72

    Article  CAS  Google Scholar 

  • Moroney JV, Ynalvez RA (2007) Proposed carbon dioxide concentrating mechanism in Chlamydomonas reinhardtii. Eukaryot Cell 6(8):1251–1259

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Moroney JV, Husic HD, Tolbert NE (1985) Effect of carbonic anhydrase inhibitors on inorganic carbon accumulation by Chlamydomonas reinhardtii. Plant Physiol 79(1):177–183

    Article  CAS  PubMed Central  PubMed  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, Kitayama M, Togasaki RK, Tolbert NE (1987) Evidence for inorganic carbon transport by intact chloroplasts of Chlamydomonas reinhardtii. Plant Physiol 83(3):460–463

    Article  CAS  PubMed Central  PubMed  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(3):897–903

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Moroney JV, Ma Y, Frey WD, Fusilier KA, Pham TT, Simms TA, DiMario RJ, Yang J, Mukherjee B (2011) The carbonic anhydrase isoforms of Chlamydomonas reinhardtii: intracellular location, expression, and physiological roles. Photosynth Res 109(1–3):133–149. doi:10.1007/s11120-011-9635-3

    Article  CAS  PubMed  Google Scholar 

  • Moroney JV, Jungnick N, Dimario RJ, Longstreth DJ (2013) Photorespiration and carbon concentrating mechanisms: two adaptations to high O2, low CO2 conditions. Photosynth Res 117(1–3):121–131. doi:10.1007/s11120-013-9865-7

    Article  CAS  PubMed  Google Scholar 

  • Mukherjee B (2013) Investigation of the role of putative inorganic carbon transporters in the carbon dioxide concentrating mechanisms of Chlamydomonas reinhardtii. Dissertation

  • Mukherjee B, Pham TT, Ma Y, Simms TA, Moroney JV (2012) The absence of the periplasmic carbonic anhydrase, CAH1, in the sequenced Chlamydomonas reinhardtii strain CC-503. In: Proceedings of the 15th International Congress on Photosynthesis. Zhejiang University Press, Hangzhou, Beijing, China

  • 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(9):3105–3117. doi:10.1105/tpc.109.071811

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Pollock SV, Colombo SL, Prout DL Jr, Godfrey AC, Moroney JV (2003) Rubisco activase is required for optimal photosynthesis in the green alga Chlamydomonas reinhardtii in a low-CO2 atmosphere. Plant Physiol 133(4):1854–1861. doi:10.1104/pp.103.032078

    Article  CAS  PubMed Central  PubMed  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(1):125–132. doi:10.1007/s11103-004-2650-4

    Article  CAS  PubMed  Google Scholar 

  • Price GD (2011) Inorganic carbon transporters of the cyanobacterial CO2 concentrating mechanism. Photosynth Res 109(1–3):47–57. doi:10.1007/s11120-010-9608-y

    Article  CAS  PubMed  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(52):18228–18233. doi:10.1073/pnas.0405211101

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Price GD, Badger MR, von Caemmerer S (2011) The prospect of using cyanobacterial bicarbonate transporters to improve leaf photosynthesis in C3 crop plants. Plant Physiol 155(1):20–26. doi:10.1104/pp.110.164681

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Price GD, Pengelly JJ, Forster B, Du J, Whitney SM, von Caemmerer S, Badger MR, Howitt SM, Evans JR (2013) The cyanobacterial CCM as a source of genes for improving photosynthetic CO2 fixation in crop species. J Exp Bot 64(3):753–768. doi:10.1093/jxb/ers257

    Article  CAS  PubMed  Google Scholar 

  • Qu Z, Hartzell HC (2008) Bestrophin Cl channels are highly permeable to HCO3 . Am J Physiol Cell Physiol 294(6):C1371–C1377. doi:10.1152/ajpcell.00398.2007

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Ramazanov Z, Mason CB, Geraghty AM, Spalding MH, Moroney JV (1993) The low CO2-inducible 36-kilodalton protein is localized to the chloroplast envelope of Chlamydomonas reinhardtii. Plant Physiol 101(4):1195–1199

    CAS  PubMed Central  PubMed  Google Scholar 

  • Raven JA, Giordano M, Beardall J, Maberly SC (2011) Algal and aquatic plant carbon concentrating mechanisms in relation to environmental change. Photosynth Res 109(1–3):281–296. doi:10.1007/s11120-011-9632-6

    Article  CAS  PubMed  Google Scholar 

  • Raven JA, Giordano M, Beardall J, Maberly SC (2012) Algal evolution in relation to atmospheric CO2: carboxylases, carbon-concentrating mechanisms and carbon oxidation cycles. Philos Trans R Soc Lond Ser B 367(1588):493–507. doi:10.1098/rstb.2011.0212

    Article  CAS  Google Scholar 

  • Rawat M, Henk MC, Lavigne LL, Moroney JV (1996) Chlamydomonas reinhardtii mutants without ribulose-1,5-bisphosphate carboxylase–oxygenase lack a detectable pyrenoid. Planta 198(2):263–270

    Article  CAS  Google Scholar 

  • Shimogawara K, Fujiwara S, Grossman A, Usuda H (1998) High-efficiency transformation of Chlamydomonas reinhardtii by electroporation. Genetics 148(4):1821–1828

    CAS  PubMed Central  PubMed  Google Scholar 

  • Sinetova MA, Kupriyanova EV, Markelova AG, Allakhverdiev SI (2012) Pronina NA (2012) Identification and functional role of the carbonic anhydrase Cah3 in thylakoid membranes of pyrenoid of Chlamydomonas reinhardtii. Biochim BiophysActa 1817(8):1248–1255. doi:10.1016/j.bbabio.2012.02.014

    Article  CAS  Google Scholar 

  • Sizova I, Fuhrmann M, Hegemann P (2001) A Streptomyces rimosus aphVIII gene coding for a new type phosphotransferase provides stable antibiotic resistance to Chlamydomonas reinhardtii. Gene 277(1–2):221–229. doi:10.1016/s0378-1119(01)00616-3

    Article  CAS  PubMed  Google Scholar 

  • Spalding MH, Spreitzer RJ, Ogren WL (1983a) Carbonic anhydrase-deficient mutant of Chlamydomonas reinhardii requires elevated carbon dioxide concentration for photoautotrophic growth. Plant Physiol 73(2):268–272

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Spalding MH, Spreitzer RJ, Ogren WL (1983b) Reduced inorganic carbon transport in a CO2-requiring mutant of Chlamydomonas reinhardii. Plant Physiol 73(2):273–276

    Article  CAS  PubMed Central  PubMed  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(4):1213–1219

    Article  PubMed Central  PubMed  Google Scholar 

  • Van K, Spalding MH (1999) Periplasmic carbonic anhydrase structural gene (Cah1) mutant in Chlamydomonas reinhardtii. Plant Physiol 120(3):757–764

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Villand P, Eriksson M, Samuelsson G (1997) Carbon dioxide and light regulation of promoters controlling the expression of mitochondrial carbonic anhydrase in Chlamydomonas reinhardtii. Biochem J 327(Pt 1):51–57

    CAS  PubMed Central  PubMed  Google Scholar 

  • von Caemmerer S, Quick WP, Furbank RT (2012) The development of C4 rice: current progress and future challenges. Science 336(6089):1671–1672. doi:10.1126/science.1220177

    Article  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(26):10110–10115. doi:10.1073/pnas.0603402103

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Woodger FJ, Badger MR, Price GD (2005) Sensing of inorganic carbon limitation in Synechococcus PCC7942 is correlated with the size of the internal inorganic carbon pool and involves oxygen. Plant Physiol 139(4):1959–1969. doi:10.1104/pp.105.069146

    Article  CAS  PubMed Central  PubMed  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(9):5341–5346. doi:10.1073/pnas.101534498

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Yamano T, Fukuzawa H (2009) Carbon-concentrating mechanism in a green alga, Chlamydomonas reinhardtii, revealed by transcriptome analyses. J Basic Microbiol 49(1):42–51. doi:10.1002/jobm.200800352

    Article  CAS  PubMed  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(9):1453–1468. doi:10.1093/pcp/pcq105

    Article  CAS  PubMed  Google Scholar 

  • Ynalvez RA, Xiao Y, Ward AS, Cunnusamy K, Moroney JV (2008) Identification and characterization of two closely related beta-carbonic anhydrases from Chlamydomonas reinhardtii. Physiol Plant 133(1):15–26. doi:10.1111/j.1399-3054.2007.01043.x

    Article  CAS  PubMed  Google Scholar 

  • Yoshioka S, Taniguchi F, Miura K, Inoue T, Yamano T, Fukuzawa H (2004) The novel Myb transcription factor LCR1 regulates the CO2-responsive gene Cah1, encoding a periplasmic carbonic anhydrase in Chlamydomonas reinhardtii. Plant Cell 16(6):1466–1477. doi:10.1105/tpc.021162

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Zhu XG, Long SP, Ort DR (2008) What is the maximum efficiency with which photosynthesis can convert solar energy into biomass? Curr Opin Biotechnol 19(2):153–159. doi:10.1016/j.copbio.2008.02.004

    Article  CAS  PubMed  Google Scholar 

  • Zhu XG, Long SP, Ort DR (2010) Improving photosynthetic efficiency for greater yield. Ann Rev Plant Biol 61:235–261. doi:10.1146/annurev-arplant-042809-112206

    Article  CAS  Google Scholar 

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Correspondence to James V. Moroney.

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Nadine Jungnick, Yunbing Ma and Bratati Mukherjee contributed equally to this work.

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Jungnick, N., Ma, Y., Mukherjee, B. et al. The carbon concentrating mechanism in Chlamydomonas reinhardtii: finding the missing pieces. Photosynth Res 121, 159–173 (2014). https://doi.org/10.1007/s11120-014-0004-x

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