Marine Biotechnology

, Volume 16, Issue 4, pp 371–384 | Cite as

Rubisco Expression in the Dinoflagellate Symbiodinium sp. Is Influenced by Both Photoperiod and Endosymbiotic Lifestyle

  • Anderson B. Mayfield
  • Yi-Yuong Hsiao
  • Hung-Kai Chen
  • Chii-Shiarng ChenEmail author
Original Article


Although the importance of anthozoan-dinoflagellate (genus Symbiodinium) endosymbioses in the establishment of coral reef ecosystems is evident, little is known about the molecular regulation of photosynthesis in the intra-gastrodermal symbiont communities, particularly with respect to the rate-limiting Calvin cycle enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco). In this study, we analyzed rubisco mRNA (rbcL) and protein (RBCL) concentrations over the diel cycle in both cultured and endosymbiotic Symbiodinium samples. In the former, rbcL expression increased upon illumination and decreased during the dark, a pattern that was upheld under continual dark incubation. A different trend in rbcL expression was observed in endosymbiotic Symbiodinium residing within sea anemone (Aiptasia pulchella) tissues, in which illumination gradually led to decreased rbcL mRNA expression. Unexpectedly, RBCL protein expression did not vary over time within anemone tissues, and in neither cultured nor endosymbiotic samples was a correlation between gene and protein expression documented. It appears, then, that photoperiod, lifestyle, and posttranscriptional regulation are all important drivers of RBCL expression in this ecologically important dinoflagellate.


Cnidarian Dinoflagellate Endosymbiosis Photosynthesis Rubisco Symbiodinium 



ABM was funded by an international research fellowship from the United States National Science Foundation (OCE-0852960) and the Khaled bin Sultan Living Oceans Foundation. NMMBA and National Science Council (NSC 98-2311-B-291-001-MY3 and 101-2311-B-291-002-MY3) provided funds to CSC that were instrumental to the success of the laboratory analyses.

Supplementary material

10126_2014_9558_MOESM1_ESM.jpg (192 kb)
Fig. s1 Primer specificity validation study. (A) Symbiodinium density in Aiptasia pulchella specimens of different symbiotic status (healthy, partially bleached, and fully bleached) was observed with DIC microscopy. (B) The same samples as in (A) were also imaged under a fluorescent microscope. The red fluorescence is from the chlorophyll of the Symbiodinium populations while the green represents autofluorescence of the host. (C) Selected insets in (B) were magnified to better demonstrate Symbiodinium density in hospite. (D) Neither rbcL nor act1 mRNAs were detected in partially and fully bleached specimens after 27 PCR cycles. In partially bleached A. pulchella, only the act1 mRNA could be detected after 40 cycles. (JPEG 191 kb) (1 mb)
Fig. s2 RFLPs. A portion of the Symbiodinium 18s rDNA gene was amplified with PCR, digested with either TaqI or Sau3 AI, and electrophoresed as described in the text. Two clade B Symbiodinium samples from a previous study (Wang et al. 2008) are shown as a reference (A), and four representative samples are shown for each of the following groups: the Symbiodinium cultures (B), anemones of the 12L:12D study (C), and anemones of the primer specificity validation study (PSVS; D). In all panels, lanes 1 and 4, and, when applicable, 7 and 10, represent the undigested, ~1,500 bp PCR product. Lanes 2 and 5, and, when applicable, 8 and 11, represent TaqI-digested amplicons. Lanes 3 and 6, and, when applicable, 9 and 12, represent Sau3 AI-digested amplicons. DNA ladders are shown in each panel. (AI 1025 kb)


  1. Badger MR, Andrews TJ, Whitney SM, Ludwig M, Yellowlees DC, Leggat W, Price GD (1998) The diversity and co-evolution of Rubisco, plastids, pyrenoids and chloroplast-based CCMs in the algae. Can J Bot 76:1052–1071Google Scholar
  2. Bellantuono AJ, Hoegh-Guldberg O, Rodriguez-Lanetty M (2011) Resistance to thermal stress in corals without changes in symbiont composition. Proc R Soc Biol Sci Ser B 279:1100–1107CrossRefGoogle Scholar
  3. Bruyant F, Babin M, Genty B, Prasil O, Behrenfeld MJ, Claustre H, Bricaud A, Garczareck L, Holtzendorf J, Koblizek M, Dousova H, Partensky F (2005) Diel variations in the photosynthetic parameters of Prochlorococcus strain PCC 9511: combined effects of light and cell cycle. Limnol Oceanogr 50:850–863CrossRefGoogle Scholar
  4. Corredor JE, Wawrik B, Paul JH, Tran H, Kerkhof L, López JM, Dieppa A, Cárdenas O (2004) Geochemical rate-RNA in integration study: ribulose-1,5-bisphosphate carboxylase/oxygenase gene transcription and photosynthetic capacity of planktonic photoautotrophs. Appl Environ Microbiol 70:5459–5468PubMedCentralCrossRefPubMedGoogle Scholar
  5. Crawley A, Kline DA, Dunn S, Anthony K, Dove S (2010) The effect of ocean acidification on symbiont photorespiration and productivity in Acropora formosa. Glob Chang Biol 16:851–863CrossRefGoogle Scholar
  6. Davies PS (1993) Endosymbiosis in marine cnidarians. In: John DM, Hawkins SJ, Price JH (eds) Plant-animal interactions in the marine benthos. Clarendon, OxfordGoogle Scholar
  7. Dykens JA, Shick JM (1982) Oxygen production by endo-symbiotic algae controls superoxide dismutase activity in their animal host. Nature 297:579–580CrossRefGoogle Scholar
  8. Fukuda I, Imagawa S, Iwao K, Horiguchi T, Watanabe T (2002) Isolation of actin-encoding cDNAs from symbiotic corals. DNA Res 9:217–223CrossRefPubMedGoogle Scholar
  9. Furla P, Galgani I, Durand I, Allemand D (2000) Sources and mechanisms of inorganic carbon transport for coral calcification and photosynthesis. J Exp Biol 203:3445–3457PubMedGoogle Scholar
  10. Goiran C, Allemand D, Galgani I (1997) Transient Na + stress in symbiotic dinoflagellates after isolation from coral host cells and subsequent immersion in seawater. Mar Biol 129:581–589CrossRefGoogle Scholar
  11. Hobson LA, Morris WJ, Guest KP (1985) Varying photoperiod, ribulose 1,5-bisphosphate carboxylase/oxygenase and CO2 uptake in Thalassiosira fluviatilis (Bacillariophyceae). Plant Physiol 79:833–837PubMedCentralCrossRefPubMedGoogle Scholar
  12. Hollnagel HC, Pinto E, Morse D, Colepicolo P (2010) The oscillation of photosynthetic capacity in Lingulodinium polyedrum is not related to differences in RuBisCo, peridinin or chlorophyll a amounts. Biol Rhythm Res 33:443–458CrossRefGoogle Scholar
  13. Iglesias-Prieto R, Trench RK (1994) Acclimation and adaptation to irradiance in symbiotic dinoflagellates. I. Responses of the photosynthetic unit to changes in photon flux density. Mar Ecol Prog Ser 113:163–175CrossRefGoogle Scholar
  14. Jones RJ, Hoegh-Guldberg O (2001) Diurnal changes in the photochemical efficiency of the symbiotic dinoflagellates (Dinophyceae) of corals: photoprotection, photoinactivation and the relationship to coral bleaching. Plant Cell Environ 24:89–99CrossRefGoogle Scholar
  15. Jordan DB, Ogren WL (1981) Species variation in the specificity of ribulose bisphosphate carboxylase/oxygenase. Nature 291:13–15CrossRefGoogle Scholar
  16. Kreps JA, Kay SA (1997) Coordination of plant metabolism and development by the circadian clock. Plant Cell 9:1235–1244PubMedCentralCrossRefPubMedGoogle Scholar
  17. Kühl M, Cohen Y, Dalsgaard T, Jorgensen BB, Revsbech NP (1995) Microenvironment and photosynthesis of zooxanthellae in scleractinian corals studies with microsensors for O2, pH and light. Mar Ecol Prog Ser 117:159–172CrossRefGoogle Scholar
  18. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685CrossRefPubMedGoogle Scholar
  19. LaJeunesse TC (2002) Diversity and community structure of symbiotic dinoflagellates from Caribbean coral reefs. Mar Biol 141:387–400CrossRefGoogle Scholar
  20. LaJeunesse TC, Smith R, Walther M, Pinzon J, Pettay DT, McGinley M, Aschaffenburg M, Medina-Rosas P, Cupul-Magana AL, Perez AL, Reyes-Bonilla H, Warner ME (2010) Host-symbiont recombination versus natural selection in the response of coral-dinoflagellate symbioses to environmental disturbance. Proc R Soc Biol Sci Ser B 277:2925–2934CrossRefGoogle Scholar
  21. Leggat W, Badger MR, Yellowlees D (1999) Evidence for an inorganic carbon-concentrating mechanism in the symbiotic dinoflagellate Symbiodinium sp. Plant Physiol 121:247–255CrossRefGoogle Scholar
  22. Leggat W, Whitney SM, Yellowlees D (2004) Is coral bleaching due to the instability of the zooxanthellae dark reactions? Symbiosis 37:137–153Google Scholar
  23. Levy O, Achituv Y, Schnider K, Dubinsky Z, Gorbunov M (2004) Diurnal hysteresis in coral photosynthesis. Mar Ecol Prog Ser 268:105–117CrossRefGoogle Scholar
  24. Levy O, Achituv Y, Yacobi YZ, Dubinsky Z, Stambler N (2006) Diel “tuning” of coral metabolism: physiological responses to light cues. J Exp Biol 209:273–283CrossRefPubMedGoogle Scholar
  25. Levy O, Kaniewska P, Alon S, Eisenberg E, Karako-Lampert S, Bay LK, Reef R, Rodriguez-Lanetty M, Miller DJ, Hoegh-Guldberg O (2011) Complex diel cycles of gene expression in coral-algal symbiosis. Science 331:175CrossRefPubMedGoogle Scholar
  26. Lilley RMC, Ralph PJ, Larkum AWD (2010) The determination of activity of the enzyme Rubisco in cell extractions of the dinoflagellate alga Symbiodinium sp. by manganese chemiluminescence and its response to short-term thermal stress of the alga. Plant Cell Environ 33:995–1004CrossRefPubMedGoogle Scholar
  27. Mayfield AB, Gates RD (2007) Osmoregulation in anthozoan-dinoflagellate symbiosis. Comp Biochem Physiol A 147:1–10CrossRefGoogle Scholar
  28. Mayfield SP, Yohn CB, Cohen A, Danon A (1995) Regulation of chloroplast gene expression. Annu Rev Plant Physiol Plant Mol Biol 46:147–166CrossRefGoogle Scholar
  29. Mayfield AB, Hirst MB, Gates RD (2009) Gene expression normalization in a dual-compartment system: a quantitative real-time PCR protocol for symbiotic anthozoans. Mol Ecol Res 9:462–470CrossRefGoogle Scholar
  30. Mayfield AB, Hsiao YY, Fan TY, Chen CS, Gates RD (2010) Evaluating the temporal stability of stress-activated protein kinase and cytoskeleton gene expression in the Pacific corals Pocillopora damicornis and Seriatopora hystrix. J Exp Mar Biol Ecol 395:215–222CrossRefGoogle Scholar
  31. Mayfield AB, Wang LH, Tang PC, Hsiao YY, Fan TY, et al. (2011) Assessing the impacts of experimentally elevated temperature on the biological composition and molecular chaperone gene expression of a reef coral. PLoS One e26529Google Scholar
  32. Mayfield AB, Chan PH, Putnam HP, Chen CS, Fan TY (2012) The effects of a variable temperature regime on the physiology of the reef-building coral Seriatopora hystrix: results from a laboratory-based reciprocal transplant. J Exp Biol 215:4183–4195CrossRefPubMedGoogle Scholar
  33. McClung CR (2001) Circadian rhythms in plants. Annu Rev Plant Physiol Plant Mol Biol 52:139–162CrossRefPubMedGoogle Scholar
  34. Mittag M, Li L, Hastings JW (1998) The mRNA level of circadian regulated Gonyaulax luciferase remains constant over the cycle. Chronobiol Int 15:93–98CrossRefPubMedGoogle Scholar
  35. Morse D, Milos PM, Roux E, Hastings JW (1989) Circadian regulation of bioluminescence in Gonyaulax involves translational control. Proc Natl Acad Sci U S A 86:172–176PubMedCentralCrossRefPubMedGoogle Scholar
  36. Morse D, Salois P, Markovic P, Hastings JW (1995) A nuclear encoded form II Rubisco in dinoflagellates. Science 268:1622–1624CrossRefPubMedGoogle Scholar
  37. Moya A, Tambutte S, Beranger G, Gaume B, Scimeca J, Allemand D, Zoccola D (2008) Cloning and use of a coral 36B4 gene to study the differential expression of coral genes between light and dark conditions. Mar Biotechnol 10:653–663CrossRefPubMedGoogle Scholar
  38. Muscatine L (1990) The role of symbiotic algae in carbon and energy flux in reef corals. In: Dubinsky Z (ed) Ecosystems of the world. Elsevier, AmsterdamGoogle Scholar
  39. Muscatine L, Grossman D, Doino J (1991) Release of symbiotic algae by tropical sea anemones and corals after cold shock. Mar Ecol Prog Ser 77:233–243CrossRefGoogle Scholar
  40. Nassoury N, Fritz L, Morse D (2001) Circadian changes in ribulose-1,5-bisphosphate carboxylase/oxygenase distribution inside individual chloroplasts can account for the rhythm in dinoflagellate carbon fixation. Plant Cell 13:923–934PubMedCentralCrossRefPubMedGoogle Scholar
  41. Paul JH (1996) Carbon cycling: molecular regulation of photosynthetic carbon fixation. Microb Ecol 32:231–245CrossRefPubMedGoogle Scholar
  42. Paul JH, Pichard SL (1998) Phytoplankton activity through the measurement of ribulose bisphosphate carboxylase gene expression (Rubisco). In: Cooksey KE (ed) Molecular approaches to the study of the ocean. Chapman and Hall, LondonGoogle Scholar
  43. Pichard SL, Paul JH (1991) Detection of gene expression in genetically engineered microorganisms and natural phytoplankton populations in the marine environment by mRNA analysis. Appl Environ Microbiol 51:1721–1727Google Scholar
  44. Pichard SL, Paul JH (1993) Gene expression per gene dose: a specific measure of gene expression in aquatic microorganisms. Appl Environ Microbiol 59:451–457PubMedCentralPubMedGoogle Scholar
  45. Pichard SL, Campbell L, Kang JB, Tabita FR, Paul JH (1996) Regulation of ribulose bisphosphate carboxylase expression in natural phytoplankton communities. I. Diel rhythms. Mar Ecol Prog Ser 139:257–265CrossRefGoogle Scholar
  46. Pichard SL, Campbell L, Carder K, Kang JB, Patch J, Tabita FR, Paul JH (1997) Analysis of ribulose bisphosphate carboxylase gene expression in natural phytoplankton communities by group-specific gene probing. Mar Ecol Prog Ser 149:239–253CrossRefGoogle Scholar
  47. Pochon X, Putnam HM, Burki F, Gates RD (2011) Identifying and characterizing alternative molecular markers for the symbiotic and free-living dinoflagellate genus Symbiodinium. PLoS ONE e29816Google Scholar
  48. Porter JW, Muscatine L, Dubinsky Z, Falkowski PG (1984) Primary production and photoadaptation in light-adapted and shade-adapted colonies of the symbiotic coral, Stylophora pistillata. Proc R Soc Biol Sci Ser B 222:161–180CrossRefGoogle Scholar
  49. Rowan R, Powers DA (1991) Molecular genetic identification of symbiotic dinoflagellates (zooxanthellae). Mar Ecol Prog Ser 71:65–73CrossRefGoogle Scholar
  50. Seibt C, Schlichter D (2001) Compatible intracellular ion composition of the host improves carbon assimilation by zooxanthellae in mutualistic symbioses. Naturwissenschaften 88:382–386CrossRefPubMedGoogle Scholar
  51. Sorek M, Levy O (2012) Influence of the quantity and quality of light on photosynthetic periodicity in coral endosymbiotic algae. PLoS ONE e43264Google Scholar
  52. Stat M, Pochon X, Cowie ROM, Gates RD (2009) Specificity in communities of Symbiodinium in corals from Johnston Atoll. Mar Ecol Prog Ser 386:83–96CrossRefGoogle Scholar
  53. Stat M, Bird CE, Pochon X, Chasqui L, Chauka LJ, Concepcion GT, Logan D, Takabayashi M, Toonen RJ, Gates RD (2010) Variation in Symbiodinium ITS2 sequence assemblages among coral colonies. PLoS ONE e15854Google Scholar
  54. Tolosa JM, Schjenken JE, Civiti TD, Clifton VL, Smith R (2007) Column-based method to simultaneously extract DNA, RNA, and proteins from the same sample. Biotechniques 43:799–804CrossRefPubMedGoogle Scholar
  55. van Dolah FM, Lidie K, Morey J, Brunelle S, Ryan J, Monroe E, Haynes B (2007) Microarray analysis of diurnal and circadian regulated genes in the Florida red tide dinoflagellate, Karenia brevis. J Phycol 43:741–752CrossRefGoogle Scholar
  56. Wang LH, Liu YH, Ju YM, Hsiao YY, Fang LS, Chen CS (2008) Cell cycle propagation is driven by light-dark stimulation in a cultured symbiotic dinoflagellate isolated from corals. Coral Reefs 27:823–835CrossRefGoogle Scholar
  57. Whitney SM, Andrews TJ (1998) The CO2/O2 specificity of single-subunit ribulose-bisphosphate carboxylase from the dinoflagellate, Amphidinium carterae. Aust J Plant Physiol 25:131–138CrossRefGoogle Scholar
  58. Whitney SM, Yellowlees D (1995) Preliminary investigations into the structure and activity of ribulose bisphosphate carboxylase from two photosynthetic dinoflagellates. J Phycol 31:138–146CrossRefGoogle Scholar
  59. Whitney SM, Shaw DC, Yellowlees D (1995) Evidence that some dinoflagellates contain a ribulose-1,5-bisphosphate carboxylase/oxygenase related to that of the α-proteobactineria. Proc R Soc Biol Sci Ser B 259:271–275CrossRefGoogle Scholar
  60. Wyman M, Davies JT, Weston K, Crawford DW, Purdie DA (1998) Ribulose-1,5-carboxylase/oxygenase (Rubisco) gene expression and photosynthetic activity in nutrient-enriched mesocosm experiments. Estuar Coast Shelf Sci 46:23–33CrossRefGoogle Scholar
  61. Xu HH, Tabita FR (1996) Ribulose-1,5-bisphosphate carboxylase/oxygenase gene expression and diversity of Lake Erie planktonic microorganisms. Appl Environ Microbiol 62:1913–1921PubMedCentralPubMedGoogle Scholar
  62. Yang YW (2001) Polymorphic symbiosis and phylogenetic analysis of zooxanthellae in the Indo-Pacific scleractinian corals. Master’s thesis. National Sun Yat-Sen University, Kaohsiung, TaiwanGoogle Scholar
  63. Zhang H, Lin S (2003) Complex gene structure of the form II Rubisco in the dinoflagellate Prorocentrum minimum (Dinophyceae). J Phycol 39:1160–1171CrossRefGoogle Scholar
  64. Zhang H, Hou Y, Miranda L, Campbell DA, Sturm NR, Gaasterland T, Lin S (2007) Spliced leader RNA trans-splicing in dinoflagellates. Proc Natl Acad Sci U S A 104:4618–4623PubMedCentralCrossRefPubMedGoogle Scholar
  65. Zinser ER, Lindell D, Johnson ZI, Futschik ME, Steglich C, Coleman ML, Wright MA, Rector T, Steen R, McNulty N, Thompson LR, Chisholm SW (2009) Choreography of the transcriptome, photophysiology, and cell cycle of a minimal photoautotroph, Prochlorococcus. PLoS ONE e5135Google Scholar

Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • Anderson B. Mayfield
    • 1
    • 2
  • Yi-Yuong Hsiao
    • 1
    • 3
  • Hung-Kai Chen
    • 1
    • 3
    • 4
  • Chii-Shiarng Chen
    • 1
    • 3
    • 4
    Email author
  1. 1.Taiwan Coral Research Center (TCRC)National Museum of Marine Biology and AquariumPingtung 944Republic of China
  2. 2.Living Oceans FoundationLandoverUSA
  3. 3.Graduate Institute of Marine BiotechnologyNational Dong-Hwa UniversityPingtungRepublic of China
  4. 4.Department of Marine Biotechnology and ResourcesNational Sun Yat-Sen UniversityKaohsiungRepublic of China

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