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Plant Molecular Biology

, Volume 85, Issue 3, pp 277–286 | Cite as

Regulation of CCM genes in Chlamydomonas reinhardtii during conditions of light–dark cycles in synchronous cultures

  • Srikanth Tirumani
  • Mallikarjuna Kokkanti
  • Vishal Chaudhari
  • Manish Shukla
  • Basuthkar J. Rao
Article

Abstract

We have investigated transcript level changes of CO2-concentrating mechanism (CCM) genes during light–dark (12 h:12 h) cycles in synchronized Chlamydomonas reinhardtii at air-level CO2. CCM gene transcript levels vary at various times of light–dark cycles, even at same air-level CO2. Transcripts of inorganic carbon transporter genes (HLA3, LCI1, CCP1, CCP2 and LCIA) and mitochondrial carbonic anhydrase genes (CAH4 and CAH5) are up regulated in light, following which their levels decline in dark. Contrastingly, transcripts of chloroplast carbonic anhydrases namely CAH6, CAH3 and LCIB are up regulated in dark. CAH3 and LCIB transcript levels reached maximum by the end of dark, followed by high expression into early light period. In contrast, CAH6 transcript level stayed high in dark, followed by high level even in light. Moreover, the up regulation of transcripts in dark was undone by high CO2, suggesting that the dark induced CCM transcripts were regulated by CO2 even in dark when CCM is absent. Thus while the CAH3 transcript level modulations appear not to positively correlate with that of CCM, the protein regulation matched with CCM status: in spite of high transcript levels in dark, CAH3 protein reached peak level only in light and localized entirely to pyrenoid, a site functionally relevant for CCM. Moreover, in dark, CAH3 protein level not only reduced but also the protein localized as a diffused pattern in chloroplast. We propose that transcription of most CCM genes, followed by protein level changes including their intracellular localization of a subset is subject to light–dark cycles.

Keywords

Air-level CO2 Bicarbonate transporter Carbonic anhydrase CO2-concentrating mechanism Light–dark cycles Transcriptional regulation 

Abbreviations

CAs

Carbonic anhydrases

CO2

Carbon dioxide

CCM

CO2-concentrating mechanism

Ci

Inorganic carbon

DIC

Dissolved inorganic carbon

L:D

Light:dark

ML

6 h of incubation in light (mid-light)

CL

12 h of incubation in light (complete-light)

MD

6 h of incubation in dark (mid-dark)

CD

12 h of incubation in dark (complete-dark)

qRT-PCR

Quantitative real time PCR

RT-PCR

Reverse transcription PCR

Notes

Acknowledgments

This work would not been possible without the help and cooperation from BJ lab people whom we profusely thank. We want to thank Prof. J.V. Moroney for sharing CAH4/5 antibody preparation. A special acknowledgement is due to Dr Ullas Kolthur and Upasana Roy for their help in qPCR work. MK is thankful to M Vijayalakshmi (HOD), Botany and Microbiology Department, Acharya Nagarjuna University for sanctioning leave to carry out this work. MK is also thankful to Prof. G Dr PCO Reddy (YV University, Kadapa) for useful discussions and help. BJR acknowledges JC Bose fellowship grant (DST). Department of Atomic Energy (Government of India) grant to Tata Institute of Fundamental Research (TIFR), Mumbai.

Supplementary material

11103_2014_183_MOESM1_ESM.tif (1.1 mb)
Supplementary Fig. 1: Brightfield imaging of synchronized cells from ML, CL, MD & CD. (Scale-bar: 50 μm) (TIFF 1176 kb)
11103_2014_183_MOESM2_ESM.tif (15 mb)
Supplementary Fig. 2: Confocal image stacks from left to right of anti-CAH3-Ab immunofluorescence analyses of ML and MD cell population. Autofluorescence image stacks of chloroplast cups and brightfield image of the same population provide the reference orientation of cells (TIFF 15332 kb)
11103_2014_183_MOESM3_ESM.tif (14.5 mb)
Supplementary Fig. 3: Confocal image stacks from left to right of anti-CAH4/5-Ab immunofluorescence analyses of ML and MD cell population. Autofluorescence image stacks of chloroplast cups and brightfield image of the same population provide the reference orientation of cells (TIFF 14828 kb)
11103_2014_183_MOESM4_ESM.tif (3.2 mb)
Supplementary Fig. 4: CCM components of C. reinhardtii are depicted in a schematic model (Modified from Spalding 2009; Wang et al. 2005). Ci flow is from left (outside of cell) to right (thylakoid lumen/pyrenoid). White boxes depict genes whose transcripts are up regulated in light (LCI1, HLA3, LCIA, CCP1, CCP2, CAH4 and CAH5) and those in black with partial white boxes depict gene transcripts up regulated in dark and light (CAH3, CAH6 and LCIB). Genes in grey boxes are not under study here (TIFF 3298 kb)
11103_2014_183_MOESM5_ESM.docx (23 kb)
Supplementary material 5 (DOCX 22 kb)

References

  1. Bernstein E (1960) Synchronous division in Chlamydomonas moewusii. Science 131:1528–1529PubMedCrossRefGoogle Scholar
  2. Blanco-Rivero A, Shutova T, Roman MJ, Villarejo A, Martinez F. (2012) Phosphorylation controls the localization and activation of the luminal carbonic anhydrase in Chlamydomonas reinhardtii. Plos One 7(11):e49063Google Scholar
  3. Borkhsenious ON, Mason CB, Moroney JV (1998) The intracellular localization of ribulose-1,5-bisphosphate carboxylase/oxygenase in Chlamydomonas reinhardtii. Plant Physiol 116:1585–1591PubMedCentralPubMedCrossRefGoogle Scholar
  4. 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 106:5990–5995PubMedCentralPubMedCrossRefGoogle Scholar
  5. 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 149:929–937PubMedCentralPubMedCrossRefGoogle Scholar
  6. Ehara T, Osafune T, Hase E (1995) Behavior of mitochondria in synchronized cells of Chlamydomonas reinhardtii (Chlorophyta). J Cell Sci 108(Pt 2):499–507PubMedGoogle Scholar
  7. Eriksson M, Villand P, Gardestrom P, Samuelsson G (1998) Induction and regulation of expression of a low-CO2-induced mitochondrial carbonic anhydrase in Chlamydomonas reinhardtii. Plant Physiol 116:637–641PubMedCentralPubMedCrossRefGoogle Scholar
  8. 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 24:1876–1893PubMedCentralPubMedCrossRefGoogle Scholar
  9. Giordano M, Norici A, Forssen M, Eriksson M, Raven JA (2003) An anaplerotic role for mitochondrial carbonic anhydrase in Chlamydomonas reinhardtii. Plant Physiol 132:2126–2134PubMedCentralPubMedCrossRefGoogle Scholar
  10. Gorman DS, Levine RP (1965) Cytochrome f and plastocyanin: their sequence in the photosynthetic electron transport chain of Chlamydomonas reinhardtii. Proc Natl Acad Sci USA. 54(6):1665–1669Google Scholar
  11. Im CS, Zhang Z, Shrager J, Chang CW, Grossman AR (2003) Analysis of light and CO(2) regulation in Chlamydomonas reinhardtii using genome-wide approaches. Photosynth Res 75:111–125PubMedCrossRefGoogle Scholar
  12. Karlsson J, Hiltonen T, Husic HD, Ramazanov Z, Samuelsson G (1995) Intracellular carbonic anhydrase of Chlamydomonas reinhardtii. Plant Physiol 109:533–539PubMedCentralPubMedCrossRefGoogle Scholar
  13. 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 17:1208–1216PubMedCentralPubMedCrossRefGoogle Scholar
  14. 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 156:884–896PubMedCentralPubMedCrossRefGoogle Scholar
  15. Mitra M, Lato SM, Ynalvez RA, Xiao Y, Moroney JV (2004) Identification of a new chloroplast carbonic anhydrase in Chlamydomonas reinhardtii. Plant Physiol 135:173–182PubMedCentralPubMedCrossRefGoogle Scholar
  16. 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 109:133–149PubMedCrossRefGoogle Scholar
  17. Ohnishi N, Mukherjee B, Tsujikawa T, Yanase M, Nakano H, Moroney JV, Fukuzawa H (2010) Expression of a low CO(2)-inducible protein, LCI1, increases inorganic carbon uptake in the green alga Chlamydomonas reinhardtii. Plant Cell 22:3105–3117PubMedCentralPubMedCrossRefGoogle Scholar
  18. Palmqvist K, Yu JW, Badger MR (1994) Carbonic anhydrase activity and inorganic carbon fluxes in low-and high-Ci cells of Chlamydomonas reinhardtü and Scenedesmus obliquus. Physiologia Plant 90:537–547CrossRefGoogle Scholar
  19. Rawat M, Moroney JV (1995) The regulation of carbonic anhydrase and ribulose-1,5-bisphosphate carboxylase/oxygenase activase by light and CO2 in Chlamydomonas reinhardtii. Plant Physiol 109:937–944PubMedCentralPubMedGoogle Scholar
  20. Riano-Pachon DM, Correa LG, Trejos-Espinosa R, Mueller-Roeber B (2008) Green transcription factors: a Chlamydomonas overview. Genetics 179:31–39PubMedCentralPubMedCrossRefGoogle Scholar
  21. Shukla M, Minda R, Singh H, Tirumani S, Chary KV, Rao BJ (2012) UVI31 + is a DNA endonuclease that dynamically localizes to chloroplast pyrenoids in C. reinhardtii. PLoS ONE 7:e51913PubMedCentralPubMedCrossRefGoogle Scholar
  22. Spalding MH (2008) Microalgal carbon-dioxide-concentrating mechanisms: Chlamydomonas inorganic carbon transporters. J Exp Bot 59:1463–1473PubMedCrossRefGoogle Scholar
  23. Spalding MH (2009) The CO2-concentrating mechanism and carbon assimilation, vol 2. Springer, New YorkGoogle Scholar
  24. Spalding MH, Van K, Wang Y, Nakamura Y (2002) Acclimation of Chlamydomonas to changing carbon availability. Funct Plant Biol 29:221–230CrossRefGoogle Scholar
  25. Sultemeyer DF, Miller AG, Espie GS, Fock HP, Canvin DT (1989) Active CO(2) transport by the green alga Chlamydomonas reinhardtii. Plant Physiol 89:1213–1219PubMedCentralPubMedCrossRefGoogle Scholar
  26. Tripathi U, Sarada R, Ravishankar GA (2001) A culture method for microalgal forms using two-tier vessel providing carbon-dioxide environment: studies on growth and carotenoid production. World J Microbiol Biotechnol 17:325–329CrossRefGoogle Scholar
  27. Van K, Spalding MH (1999) Periplasmic carbonic anhydrase structural gene (Cah1) mutant in Chlamydomonas reinhardtii. Plant Physiol 120:757–764PubMedCentralPubMedCrossRefGoogle Scholar
  28. 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 83:796–809CrossRefGoogle Scholar
  29. Wang B, Liu CQ, Wu Y (2005) Effect of heavy metals on the activity of external carbonic anhydrase of microalga Chlamydomonas reinhardtii and microalgae from karst lakes. Bull Environ Contam Toxicol 74:227–233PubMedCrossRefGoogle Scholar
  30. 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 98:5341–5346PubMedCentralPubMedCrossRefGoogle Scholar
  31. Yamano T, Fukuzawa H (2009) Carbon-concentrating mechanism in a green alga, Chlamydomonas reinhardtii, revealed by transcriptome analyses. J Basic Microbiol 49:42–51PubMedCrossRefGoogle Scholar
  32. Yamano T, Miura K, Fukuzawa H (2008) Expression analysis of genes associated with the induction of the carbon-concentrating mechanism in Chlamydomonas reinhardtii. Plant Physiol 147:340–354PubMedCentralPubMedCrossRefGoogle Scholar
  33. 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 51:1453–1468PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2014

Authors and Affiliations

  • Srikanth Tirumani
    • 1
    • 2
  • Mallikarjuna Kokkanti
    • 2
  • Vishal Chaudhari
    • 1
  • Manish Shukla
    • 1
  • Basuthkar J. Rao
    • 1
  1. 1.B-202, Department of Biological SciencesTata Institute of Fundamental ResearchMumbaiIndia
  2. 2.Department of Botany and MicrobiologyAcharya Nagarjuna UniversityGunturIndia

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