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


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.


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



Carbonic anhydrases


Carbon dioxide


CO2-concentrating mechanism


Inorganic carbon


Dissolved inorganic carbon




6 h of incubation in light (mid-light)


12 h of incubation in light (complete-light)


6 h of incubation in dark (mid-dark)


12 h of incubation in dark (complete-dark)


Quantitative real time PCR


Reverse transcription PCR



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)


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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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