, Volume 256, Issue 1, pp 117–130 | Cite as

Coordination between photorespiration and carbon concentrating mechanism in Chlamydomonas reinhardtii: transcript and protein changes during light-dark diurnal cycles and mixotrophy conditions

  • S. Tirumani
  • K.M. Gothandam
  • Basuthkar J RaoEmail author
Original Article


Carbon concentrating mechanism (CCM) and photorespiration (PR) are interlinked and co-regulated in Chlamydomonas reinhardtii, but conditions where co-regulation alters are not sufficiently explored. Here, we uncover that PR gene transcripts, like CCM transcripts, are induced even in the dark when both processes are not active. Such diurnal cycles show that transcript levels peak in the middle of 12 h day, decline by early part of 12-h dark followed by their onset again at mid-dark. Interestingly, the onset in the mid-dark phase is sensitive to high CO2, implying that the active carbon sensing mechanism operates even in the dark. The rhythmic alterations of both CCM and PR transcript levels are unlinked to circadian clock: the “free-running state” reveals no discernible rhythmicity in transcript changes. Only continuous light leads to high transcript levels but no detectable transcripts were observed in continuous dark. Asynchronous continuous light cultures, upon shifting to low from high CO2 exhibit only transient induction of PR transcripts/proteins while CCM transcript induction is stable, indicating the loss of co-regulation between PR and CCM gene transcription. Lastly, we also describe that both CCM and PR transcripts/proteins are induced in low CO2 even in mixotrophic cultures, but only in high light, the same being attenuated in high CO2, implying that high light is a mandatory “trigger” for CCM and PR induction in low CO2 mixotrophy. Our study provides comprehensive analyses of conditions where CCM and PR were differently regulated, setting a paradigm for a detailed mechanistic probing of these responses.


Carbon concentrating mechanism Circadian clock Transcriptional regulation Light-dark cycles Mixotrophy Photorespiration 



Alanine:α-ketoglutarate aminotransf erase


Carbonic anhydrase 3


Carbon concentrating mechanism


Chloroplast carrier protein 1


Photosystem II protein D1


Glycine decarboxylase H protein


Glycolate dehydrogenase


Hydroxypyruvate reductase


Low CO2 inducible membrane protein


Low CO2 inducible membrane protein


Light harvesting complex stress-related chlorophyll a/b binding protein


Phosphoglycolate phosphatase




Ribulose-1,5-bisphosphate carboxylase-oxygenase


Serine:glyoxylate aminotransferase

TAP medium

Tris acetate phosphate medium

TP medium

Tris phosphate medium



This work was supported by J.C. Bose Fellowship Grant, DST (10X-217) and Department of Atomic Energy Grant, Government of India (12P0123) to Prof. Basuthkar Jagadeeshwar Rao. We also thank Soumajit Saha for his help in microscopy imaging and valuable inputs throughout the project.

Authors’ contribution

BJ and TS conceived and designed the research. TS conducted the experiments. BJ contributed new reagents or analytical tools. BJ, TS, and KM analyzed the data. BJ wrote the manuscript. All authors read and approved the manuscript.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

709_2018_1283_Fig8_ESM.png (655 kb)

(PNG 654 kb)

709_2018_1283_MOESM1_ESM.tif (4.8 mb)
High resolution image (TIF 4874 kb)


  1. Ashworth J, Coesel S, Lee A, Armbrust EV, Orellana MV, Baliga NS (2013) Genome-wide diel growth state transitions in the diatom Thalassiosira pseudonana. Proc Natl Acad Sci U S A 110:7518–7523. CrossRefGoogle Scholar
  2. Atkinson N, Feike D, Mackinder LCM, Meyer MT, Griffiths H, Jonikas MC, Smith AM, Mc Cormick AJ (2016) Introducing an algal carbon concentrating mechanism into higher plants: location and incorporation of key components. Plant Biotechnol J 14:1302–1315. CrossRefGoogle Scholar
  3. Badger MR, John Andrews T, Whitney S, Ludwig M, Yellowlees DC, Leggat W, Dean Price G (1998) The diversity and coevolution of Rubisco, plastids, pyrenoids, and chloroplast-based CO 2 -concentrating mechanisms in algae 1. 76:1052–1071. doi:
  4. Bauwe H, Hagemann M, Fernie AR (2010) Photorespiration: players, partners and origin. Trends Plant Sci 15:330–336. CrossRefGoogle Scholar
  5. Bauwe H, Hagemann M, Kern R, Timm S (2012) Photorespiration has a dual origin and manifold links to central metabolism. Curr Opin Plant Biol 15:269–275. CrossRefGoogle Scholar
  6. Becker B (2013) Snow ball earth and the split of Streptophyta and Chlorophyta. Trends Plant Sci 18:180–183. CrossRefGoogle Scholar
  7. Beezley BB, Gruber PJ, Frederick SE (1976) Cytochemical localization of Glycolate dehydrogenase in mitochondria of Chlamydomonas. Plant Physiol 58:315–319. CrossRefGoogle Scholar
  8. Bernstein E (1960) Synchronous division in Chlamydomonas moewusii. Science 131:1528–1529. CrossRefGoogle Scholar
  9. Blackwell RD, Murray AJS, Lea PJ, Kendall AC, Hall NP, Turner JC, Wallsgrove RM (1988) The value of mutants unable to carry out photorespiration. Photosynth Res 16:155–176. CrossRefGoogle Scholar
  10. Blasing OE (2005) Sugars and circadian regulation make major contributions to the global regulation of diurnal gene expression in Arabidopsis. the Plant Cell Online 17:3257–3281. CrossRefGoogle Scholar
  11. Buchanan BB (1980) Role of light in the regulation of chloroplastic enzyms. Annu Rev Plant Physiol 31:341–374. CrossRefGoogle Scholar
  12. Chaudhari VR, Vyawahare A, Bhattacharjee SK, Rao BJ (2015) Enhanced excision repair and lack of PSII activity contribute to higher UV survival of Chlamydomonas reinhardtii cells in dark. Plant Physiol Biochem 88:60–69. CrossRefGoogle Scholar
  13. Chen ZY, Burow MD, Mason CB, Moroney JV (1996) A low-CO2-inducible gene encoding an alanine: alpha-ketoglutarate aminotransferase in Chlamydomonas reinhardtii. Plant Physiol 112(2):677–684CrossRefGoogle Scholar
  14. Cross FR, Umen JG (2015) The Chlamydomonas cell cycle. Plant J 82:370–392. CrossRefGoogle Scholar
  15. Duanmu D, Miller AR, Horken KM, Weeks DP, Spalding MH (2009) Knockdown of limiting- CO2 –induced gene HLA3 decreases HCO 3- transport and photosynthetic Ci affinity in Chlamydomonas reinhardtii. doi:
  16. Eisenhut M, Ruth W, Haimovich M, Bauwe H, Kaplan A, Hagemann M (2008) The photorespiratory glycolate metabolism is essential for cyanobacteria and might have been conveyed endosymbiontically to plants. Proc Natl Acad Sci U S A 105:17199–17204. CrossRefGoogle Scholar
  17. Espinoza C, Degenkolbe T, Caldana C, Zuther E, Leisse A, Willmitzer L, Hincha DK, Hannah MA (2010) Interaction with diurnal and circadian regulation results in dynamic metabolic and transcriptional changes during cold acclimation in arabidopsis. PLoS One 5:e14101. CrossRefGoogle Scholar
  18. 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 24:1876–1893. CrossRefGoogle Scholar
  19. Fernie AR, Bauwe H, Eisenhut M, Florian A, Hanson DT, Hagemann M, Keech O, Mielewczik M, Nikoloski Z, Peterhänsel C, Roje S, Sage R, Timm S, von Cammerer S, Weber APM, Westhoff P (2013) Perspectives on plant photorespiratory metabolism. Plant Biol 15:748–753. CrossRefGoogle Scholar
  20. Foyer CH, Bloom AJ, Queval G, Noctor G (2009) Photorespiratory metabolism: genes, mutants, energetics, and redox signaling. Annu Rev Plant Biol 60:455–484. CrossRefGoogle Scholar
  21. Foyer CH, Neukermans J, Queval G, Noctor G, Harbinson J (2012) Photosynthetic control of electron transport and the regulation of gene expression. J Exp Bot 63:1637–1661. CrossRefGoogle Scholar
  22. Fujiwara S, Ishida N, Tsuzuki M (1996) Circadian expression of the carbonic anhydrase gene, Cah1, in Chlamydomonas reinhardtii. Plant Mol Biol 32:745–749. CrossRefGoogle Scholar
  23. 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 U S A 98:5347–5352. CrossRefGoogle Scholar
  24. Gao H, Wang Y, Fei X, Wright DA, Spalding MH (2015) Expression activation and functional analysis of HLA3, a putative inorganic carbon transporter in Chlamydomonas reinhardtii. 1–11. doi:
  25. Geiger DR, Servaites JC (1994) Diurnal regulation of photosynthetic carbon metabolism in C3 plants. Ann Rev Plant Physiol Plant Mol Biol 45:235–256. CrossRefGoogle Scholar
  26. Goldschmidt M, Rochaix JD (2006) EMBO Practical Course: Molecular Genetics of Chlamydomonas Google Scholar
  27. Gorman SD, Levine RP, Gorman DS, Levine RP (1965) Cytochrome f and plastocyanin: their sequence in the photosynthetic electron transport chain of Chlamydomonas reinhardi. Proc Natl Acad Sci 54:1665–1669. CrossRefGoogle Scholar
  28. Govindjee DS (2011) Adventures with cyanobacteria: a personal perspective. Front Plant Sci 2:28. CrossRefGoogle Scholar
  29. Harmer SL, Hogenesch JB, Straume M, Chang HS, Han B, Zhu T, Wang X, Kreps JA, Kay SA (2000) Orchestrated transcription of key pathways in Arabidopsis by the circadian clock. Science (New York, NY) 290:2110–2113. CrossRefGoogle Scholar
  30. Howell SH, Walker LL (1977) Transcription of the nuclear and chloroplast genomes during the vegetative cell cycle in Chlamydomonas reinhardi. Dev Biol 56:11–23. CrossRefGoogle Scholar
  31. Johnson CH, Stewart PL, Egli M (2011) The Cyanobacterial circadian system: from biophysics to bioevolution. Annu Rev Biophys 40:143–167. CrossRefGoogle Scholar
  32. Kasting JF (1987) Theoretical constraints on oxygen and carbon dioxide concentrations in the Precambrian atmosphere. Precambrian Res 34:205–229. CrossRefGoogle Scholar
  33. Kinmonth-Schultz HA, Golembeski GS, Imaizumi T (2013) Circadian clock-regulated physiological outputs: dynamic responses in nature. Semin Cell Dev Biol 24:407–413. CrossRefGoogle Scholar
  34. Kozaki A, Takeba G (1996) Photorespiration protects C3 plants from photooxidation. Nature 384:557–560. CrossRefGoogle Scholar
  35. Matsuo T, Ishiura M (2010) New insights into the circadian clock in chlamydomonas. Int Rev Cell Mol Biol 280:281–314. CrossRefGoogle Scholar
  36. McClung CR, Hsu M, Painter JE, Gagne JM, Karlsberg SD, Salomé PA (2000) Integrated temporal regulation of the photorespiratory pathway. Circadian regulation of two Arabidopsis genes encoding serine hydroxymethyltransferase. Plant Physiol 123:381–392. CrossRefGoogle Scholar
  37. Mitchell MC, Meyer MT, Griffiths H (2014) Dynamics of carbon-concentrating mechanism induction and protein relocalization during the dark-to-light transition in synchronized Chlamydomonas reinhardtii. Plant Physiol 166:1073–1082. CrossRefGoogle Scholar
  38. Mittag M, Kiaulehn S, Johnson CH (2005) Update on the circadian clock in Chlamydomonas reinhardtii. Plant Physiol 137:399–409. CrossRefGoogle Scholar
  39. Miura K, Yamano T, Yoshioka S, Kohinata T, Inoue Y (2004) Expression Profiling-Based Identification of CO 2 -Responsive Genes Regulated by CCM1 Controlling a Carbon-Concentrating Mechanism in Chlamydomonas reinhardtii 1. 135:1595–1607. doi:
  40. Monnier A, Liverani S, Bouvet R, Jesson B, Smith JQ, Mosser J, Corellou F, Bouget F-Y (2010) Orchestrated transcription of biological processes in the marine picoeukaryote Ostreococcus exposed to light/dark cycles. BMC Genomics 11:192. CrossRefGoogle Scholar
  41. Moroney JV, Wilson BJ, Tolbert NE (1986) Glycolate metabolism and excretion by Chlamydomonas reinhardtii. Plant Physiol 82:821–826. CrossRefGoogle Scholar
  42. 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. CrossRefGoogle Scholar
  43. Moroney JV, Jungnick N, DiMario RJ, Longstreth DJ (2013) Photorespiration and carbon concentrating mechanisms: two adaptations to high O2, low CO2 conditions. Photosynth Res 117:121–131. CrossRefGoogle Scholar
  44. Nakamura Y, Kanakagiri S, Van K, He W, Spalding MH (2005) Disruption of the glycolate dehydrogenase gene in the high-CO 2 -requiring mutant HCR89 of Chlamydomonas reinhardtii. Can J Bot 83:820–833. CrossRefGoogle Scholar
  45. Nelson B, Tolbert NE (1970) Glycolate Dehydrogenase in Green Algae. Arch Biochem Biophys 141:102–110. CrossRefGoogle Scholar
  46. Nisbet EG, Grassineau NV, Howe CJ, Abell PI, Regelous M, Nisbet RER (2007) The age of Rubisco: the evolution of oxygenic photosynthesis. Geobiology 5:311–335. CrossRefGoogle Scholar
  47. Polukhina I, Fristedt R, Dinc E, Cardol P, Croce R (2016) Carbon supply and Photoacclimation cross talk in the green alga Chlamydomonas reinhardtii. Plant Physiol 172:1494–1505. CrossRefGoogle Scholar
  48. Queval G, Foyer CH (2012) Redox regulation of photosynthetic gene expression. Philos Trans R Soc Lond Ser B Biol Sci 367:3475–3485. CrossRefGoogle Scholar
  49. 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 109:937–944CrossRefGoogle Scholar
  50. Reddy AB, Rey G (2014) Metabolic and nontranscriptional circadian clocks: eukaryotes. Annu Rev Biochem 83:165–189. CrossRefGoogle Scholar
  51. Ruts T, Matsubara S, Wiese-Klinkenberg A, Walter A (2012) Diel patterns of leaf and root growth: endogenous rhythmicity or environmental response? J Exp Bot 63:3339–3351. CrossRefGoogle Scholar
  52. Sage RF (2004) The evolution of C 4 photosynthesis. New Phytol 161:341–370. CrossRefGoogle Scholar
  53. Sage RF, Sage TL, Kocacinar F (2012) Photorespiration and the evolution of C 4 photosynthesis. Annu Rev Plant Biol 63:19–47. CrossRefGoogle Scholar
  54. Sanders MA, Salisbury JL (1995) Immunofluorescence microscopy of cilia and flagella. Meth Cell Biol 47:163–169. CrossRefGoogle Scholar
  55. Somerville CR, Ogren WL (1982) Genetic modification of photorespiration. Trends Biochem Sci 7:171–174. CrossRefGoogle Scholar
  56. Soon Im C, Grossman AR (2002) Identification and regulation of high light-induced genes in Chlamydomonas reinhardtii. Plant J 30:301–313. CrossRefGoogle Scholar
  57. Spalding MH (1989) Photosynthesis and photorespiration in freshwater green algae. Aquat Bot 34:181–209. CrossRefGoogle Scholar
  58. Suzuki K, Marek LF, Spalding MH (1990) A Photorespiratory mutant of Chlamydomonas reinhardtii. Plant Physiol 93:231–237. CrossRefGoogle Scholar
  59. Thines B, Harmon FG, Li J, Chua N-H (2010) Four easy pieces: mechanisms underlying circadian regulation of growth and development this review comes from a themed issue on growth and development edited. Curr Opin Plant Biol 14:31–37. CrossRefGoogle Scholar
  60. Timm S, Florian A, Arrivault S, Stitt M, Fernie AR, Bauwe H (2012) Glycine decarboxylase controls photosynthesis and plant growth. FEBS Lett 586:3692–3697. CrossRefGoogle Scholar
  61. Timm S, Florian A, Wittmiß M, Jahnke K, Hagemann M, Fernie AR, Bauwe H (2013) Serine acts as a metabolic signal for the transcriptional control of photorespiration-related genes in Arabidopsis. Plant Physiol 162:379–389. CrossRefGoogle Scholar
  62. Tirumani S, Kokkanti M, Chaudhari V, Shukla M, Rao BJ (2014) Regulation of CCM genes in Chlamydomonas reinhardtii during conditions of light-dark cycles in synchronous cultures. Plant Mol Biol 85:277–286. CrossRefGoogle Scholar
  63. Tolbert NE (1997) The C 2 oxidative photosynthetic carbon cycle. Annu Rev Plant Physiol Plant Mol Biol 48:1–25. CrossRefGoogle Scholar
  64. Tolbert NE, Harrison M, Selph N (1983) Aminooxyacetate stimulation of glycolate formation and excretion by chlamydomonas. Plant Physiol 72:1075–1083CrossRefGoogle Scholar
  65. 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 17:325–329. CrossRefGoogle Scholar
  66. Tural B, Moroney JV (2005) Regulation of the expression of photorespiratory genes in Chlamydomonas reinhardtii. Can J Bot 83:810–819. CrossRefGoogle Scholar
  67. Wingler A, Lea PJ, Quick WP, Leegood RC (2000) Photorespiration: metabolic pathways and their role in stress protection. Philos Trans R Soc Lond Ser B Biol Sci 355:1517–1529. CrossRefGoogle Scholar
  68. Xiang Y, Zhang J, Weeks DP (2001) The Cia5 gene controls formation of the carbon concentrating mechanism in Chlamydomonas reinhardtii. Proc Natl Acad Sci U S A 98:5341–5346. CrossRefGoogle Scholar
  69. Xie X, Huang A, Gu W, Zang Z, Pan G, Gao S, He L, Zhang B, Niu J, Lin A, Wang G (2016) Photorespiration participates in the assimilation of acetate in Chlorella sorokiniana under high light. New Phytol 209:987–998. CrossRefGoogle Scholar
  70. Yamano T, Miura K, Fukuzawa H (2008) Expression analysis of genes associated with the induction of the carbonconcentrating mechanism in Chlamydomonas reinhardtii. Plant Physiol 147:340–354. CrossRefGoogle Scholar
  71. Yamano T, Sato E, Iguchi H, Fukuda Y, Fukuzawa H, Buchanan BB (2015) Characterization of cooperative bicarbonate uptake into chloroplast stroma in the green alga Chlamydomonas reinhardtii. doi:
  72. Zelitch I, Schultes NP, Peterson RB, Brown P, Brutnell TP (2009) High glycolate oxidase activity is required for survival of maize in normal air. Plant Physiol 149:195–204. CrossRefGoogle Scholar
  73. Zhong HH, Young JC, Pease EA., Hangarter RP, McClung CR (1994) Interactions between Light and the Circadian Clock in the Regulation of CAT2 Expression in Arabidopsis Plant physiology 104:889–898Google Scholar

Copyright information

© Springer-Verlag GmbH Austria, part of Springer Nature 2018

Authors and Affiliations

  • S. Tirumani
    • 1
    • 2
  • K.M. Gothandam
    • 2
  • Basuthkar J Rao
    • 1
    • 3
    Email author
  1. 1.B-202, Department of Biological SciencesTata Institute of Fundamental ResearchMumbaiIndia
  2. 2.School of Bio Sciences and TechnologyVIT UniversityVelloreIndia
  3. 3.Indian Institute of Science Education and ResearchTirupatiIndia

Personalised recommendations