Plant Molecular Biology

, Volume 71, Issue 1–2, pp 193–205 | Cite as

Cloning and characterization of two novel chloroplastic glycerol-3-phosphate dehydrogenases from Dunaliella viridis

  • Yunxia He
  • Xiangzong Meng
  • Qianlan Fan
  • Xiaoliang Sun
  • Zhengkai Xu
  • Rentao Song


Dunaliella, a unicellular green alga, has the unusual ability to survive dramatic osmotic stress by accumulating high concentrations of intracellular glycerol as a compatible solute. The chloroplastic glycerol-3-phosphate dehydrogenase (GPDH) has been considered to be the key enzyme that produces glycerol for osmoregulation in Dunaliella. In this study, we cloned the two most prominent GPDH cDNAs (DvGPDH1 and DvGPDH2) from Dunaliella viridis, which encode two polypeptides of 695 and 701 amino acids, respectively. Unlike higher plant GPDHs, both proteins contained extra phosphoserine phosphatase (SerB) domains at their N-termini in addition to C-terminal GPDH domains. Such bi-domain GPDHs represent a novel type of GPDH and are found exclusively in the chlorophyte lineage. Transient expression of EGFP fusion proteins in tobacco leaf cells demonstrated that both DvGPDH1 and DvGPDH2 are localized in the chloroplast. Overexpression of DvGPDH1 or DvGPDH2 could complement a yeast GPDH mutant (gpd1Δ), but not a yeast SerB mutant (ser2Δ). In vitro assays with purified DvGPDH1 and DvGPDH2 also showed apparent GPDH activity for both, but no SerB activity was detected. Surprisingly, unlike chloroplastic GPDHs from plants, DvGPDH1 and DvGPDH2 could utilize both NADH and NADPH as coenzymes and exhibited significantly higher GPDH activities when NADH was used as the coenzyme. Q-PCR analysis revealed that both genes exhibited transient transcriptional induction of gene expression upon hypersalinity shock, followed by a negative feedback of gene expression. These results shed light on the regulation of glycerol synthesis during salt stress in Dunaliella.


Dunaliella Glycerol-3-phosphate dehydrogenase Functional characterization Gene expression Glycerol synthesis 



Glycerol-3-phosphate dehydrogenase


Phosphoserine phosphatase


Dihydroxyacetone phosphate


Glycerol-3-phosphate phosphatase


Open reading frame



We are grateful to Dr. Bernard A. Prior (Stellenbosch University, South Africa) for providing the gpd1Δ mutant, Dr. Lennart Adler (Göteborg University, Sweden) for providing the gpp1Δgpp2Δ mutant and Dr. Anu Saloheimo (VTT Biotechnology, Finland) for providing the pAJ401 plasmid. This work was supported by National Natural Sciences Foundation of China (30871278, 30471119, 30470857), Ministry of Education of China (NCET-06-0435), Ministry of Agriculture of China (2008ZX08003-005), Fok Ying Tung Education Foundation (101024) and the Research Foundation from Shanghai Municipal Education Commission (228495).

Supplementary material

11103_2009_9517_MOESM1_ESM.doc (54 kb)
(DOC 52 kb)
11103_2009_9517_MOESM2_ESM.tif (125 kb)
(TIFF 125 kb)
11103_2009_9517_MOESM3_ESM.tif (136 kb)
(TIFF 135 kb)
11103_2009_9517_MOESM4_ESM.tif (559 kb)
(TIFF 558 kb)
11103_2009_9517_MOESM5_ESM.tif (167 kb)
(TIFF 166 kb)
11103_2009_9517_MOESM6_ESM.tif (102 kb)
(TIFF 101 kb)


  1. Albertyn J, Hohmann S, Thevelein JM, Prior BA (1994) GPD1, which encodes glycerol-3-phosphate dehydrogenase, is essential for growth under osmotic stress in Saccharomyces cerevisiae, and its expression is regulated by the high-osmolarity glycerol response pathway. Mol Cell Biol 14(6):4135–4144PubMedGoogle Scholar
  2. Alonso R, Ramos J (1984) Dual system for potassium transport in Saccharomyces cerevisiae. J Bacteriol 159(3):940–945Google Scholar
  3. Avron M (1986) The osmotic components of halotolerant algae. Trends Biochem Sci 11:5–6. doi: 10.1016/0968-0004(86)90218-5 CrossRefGoogle Scholar
  4. Belmans D, Van Laera A (1987) Glycerol cycle enzymes and intermediates during adaptation of Dunaliella teriolecta cells to hyperosmotic stress. Plant Cell Environ 10:185–190Google Scholar
  5. Ben-Amotz A, Avron M (1973) The role of glycerol in the osmotic regulation of the halophilic alga D. parva. Plant Physiol 51:875–878. doi: 10.1104/pp.51.5.875 PubMedCrossRefGoogle Scholar
  6. Ben-Amotz A, Avron M (1974) Isolation, characterization and partial purification of a reduced nicotinamide adenine dinucleotide phosphate-dependent dihydroxyacetone reductase from the halophilic alga D. parva. Plant Physiol 53:628. doi: 10.1104/pp.53.4.628 PubMedCrossRefGoogle Scholar
  7. Collet J, Stroobant V, Pirard M, Delpierre G, Schaftingen EV (1998) A new class of phosphotransferases phosphorylated on an aspartate residue in an amino-terminal DXDX(T/V) motif. J Biol Chem 273(23):14107–14112. doi: 10.1074/jbc.273.23.14107 PubMedCrossRefGoogle Scholar
  8. Gee R, Byerrum RU, Gerber DW, Tolbert NE (1988a) Dihydroxyacetone phosphate reductase in plants. Plant Physiol 86:98–103. doi: 10.1104/pp.86.1.98 PubMedCrossRefGoogle Scholar
  9. Gee R, Goyal A, Gerber DW, Byerrum RU, Tolbert NE (1988b) Isolation of dihydroxyacetone phosphate reductase from Dunaliella chloroplasts and comparison with isozymes from spinach leaves. Plant Physiol 88:896–903. doi: 10.1104/pp.88.3.896 PubMedCrossRefGoogle Scholar
  10. Gee R, Goyal A, Byerrum RU, Tolbert NE (1989) Two isozymes of dihydroxyacetone phosphate reductase in Dunaliella. Plant Physiol 91:345–351. doi: 10.1104/pp.91.1.345 PubMedCrossRefGoogle Scholar
  11. Gee R, Goyal A, Byerrum RU, Tolbert NE (1993) Two isozymes of dihydroxyacetone phosphate reductase from the chloroplasts of Dunaliella tertiolecta. Plant Physiol 103:243–249PubMedGoogle Scholar
  12. Ghoshal D, Mach D, Agarwal M, Goyal A (2002) Osmoregulatory isoform of dihydroxyacetone phosphate reductase from Dunaliella tertiolecta: purification and characterization. Protein Expr Purif 24:404–411. doi: 10.1006/prep.2001.1588 PubMedCrossRefGoogle Scholar
  13. Gietz D, St Jean A, Woods RA, Schiest RH (1992) Improved method for high efficiency transformation of intact yeast cells. Nucleic Acids Res 20(6):1425. doi: 10.1093/nar/20.6.1425 PubMedCrossRefGoogle Scholar
  14. Goyal A (2007a) Osmoregulation in Dunaliella, part I: effects of osmotic stress on photosynthesis, dark respiration and glycerol metabolism in Dunaliella tertiolecta and its salt-sensitive mutant (HL 25/8). Plant Physiol Biochem 45(9):696–704. doi: 10.1016/j.plaphy.2007.05.008 PubMedCrossRefGoogle Scholar
  15. Goyal A (2007b) Osmoregulation in Dunaliella, part II: photosynthesis and starch contribute carbon for glycerol synthesis during a salt stress in Dunaliella tertiolecta. Plant Physiol Biochem 45(9):705–710. doi: 10.1016/j.plaphy.2007.05.009 PubMedCrossRefGoogle Scholar
  16. Graham P (2008) Pond scum genomics: the genomes of Chlamydomonas and Ostreococcus. Plant Cell 20:502–507. doi: 10.1105/tpc.107.056556 CrossRefGoogle Scholar
  17. Guan Z, Meng X, Sun Z, Xu Z, Song R (2008) Characterization of duplicated Dunaliella viridis SPT1 genes provides insights into early gene divergence after duplication. Gene 423(1):36–42. doi: 10.1016/j.gene.2008.06.029 PubMedCrossRefGoogle Scholar
  18. Haus M, Wegman K (1984) Glycerol-3-phosphate dehydrogenase (EC from Dunaliella teriolecta. I. Purification and kinetic properties. Physiol Plant 60:283–288. doi: 10.1111/j.1399-3054.1984.tb06063.x CrossRefGoogle Scholar
  19. He Q, Qiao D, Bai L, Zhang Q, Yang W, Li Q, Cao Y (2007) Cloning and characterization of a plastidic glycerol 3-phosphate dehydrogenase cDNA from Dunaliella salina. J Plant Physiol 164:214–220. doi: 10.1016/j.jplph.2006.04.004 PubMedCrossRefGoogle Scholar
  20. Hohmann S (2002) Osmotic stress signaling and osmoadaptation in yeast. Microbiol Mol Biol Rev 66(2):300–372. doi: 10.1128/MMBR.66.2.300-372.2002 PubMedCrossRefGoogle Scholar
  21. Husic HD, Tolbert NE (1986) Effect of osmotic stress on carbon metabolism in Chlamydomonas reinhardtii. Plant Physiol 82:594–596. doi: 10.1104/pp.82.2.594 PubMedCrossRefGoogle Scholar
  22. Kozak M (1991) An analysis of vertebrate mRNA sequences: intimations of translational control. J Cell Biol 115:887–903. doi: 10.1083/jcb.115.4.887 PubMedCrossRefGoogle Scholar
  23. Liska AJ, Shevchenko A, Pick U, Katz A (2004) Elevated photosynthesis and redox energy production contribute to salinity tolerance in Dunaliella as revealed by homology-based proteomic analysis. Plant Physiol 136:2806–2817. doi: 10.1104/pp.104.039438 PubMedCrossRefGoogle Scholar
  24. Merchant SS et al (2007) The Chlamydomonas genome reveals the evolution of key animal and plant functions. Science 318:245–251. doi: 10.1126/science.1143609 PubMedCrossRefGoogle Scholar
  25. Oren A (2005) A hundred years of Dunaliella research: 1905–2005. Saline Syst 1:2. doi: 10.1186/1746-1448-1-2 PubMedCrossRefGoogle Scholar
  26. Osami M, Yamato Y, Keiji N, Takayuki F, Takayuki S, Syunsuke H, Yoshiki N, Tsuneyoshi K (2008) Genome analysis and its significance in four unicellular algae, Cyanidioshyzon merolae, Ostreococcus tauri, Chlamydomonas reinhardtii, and Thalassiosira pseudonana. J Plant Res 121:3–17. doi: 10.1007/s10265-007-0133-9 CrossRefGoogle Scholar
  27. Ou X, Ji C, Han X, Zhao X, Li X, Mao Y, LL W, Bartlam M, Rao Z (2006) Crystal structures of human glycerol 3-phosphate dehydrogenase 1 (GPD1). J Mol Biol 357:858–869. doi: 10.1016/j.jmb.2005.12.074 PubMedCrossRefGoogle Scholar
  28. Pahlman A, Granath K, Ansell R, Hohmann S, Adler L (2001) The yeast glycerol 3-phosphatases Gpp1p and Gpp2p are required for glycerol biosynthesis and differentially involved in the cellular responses to osmotic, anaerobic, and oxidative stress. J Biol Chem 276:3555–3563. doi: 10.1074/jbc.M007164200 PubMedCrossRefGoogle Scholar
  29. Remize F, Barnavon L, Dequin S (2001) Glycerol export and glycerol-3-phosphate dehydrogenase, but not glycerol phosphatase, are rate limiting for glycerol production in Saccharomyces cerevisiae. Metab Eng 3:301–312. doi: 10.1006/mben.2001.0197 PubMedCrossRefGoogle Scholar
  30. Rep M, Albertyn J, Thevelein JM, Prior BA, Hohmann S (1999) Different signalling pathways contribute to the control of GPD1 gene expression by osmotic stress in Saccharomyces cerevisiae. Microbiology 145:715–727PubMedCrossRefGoogle Scholar
  31. Sadka A, Lers A, Zamir A, Avron M (1989) A critical examination of the role of the de novo protein sythesis in the osmotic adaptation of the halotolerant alga Dunaliella. FEBS Lett 244:93–98. doi: 10.1016/0014-5793(89)81170-6 CrossRefGoogle Scholar
  32. Saloheimo A, Henrissat B, Hoffrén A, Teleman O, Penttilä M (1994) A novel, small endoglucanase gene, egl5, from Trichoderma reesei isolated by expression in yeast. Mol Microbiol 13(2):219–228. doi: 10.1111/j.1365-2958.1994.tb00417.x PubMedCrossRefGoogle Scholar
  33. Schramm M (1958) O-phosphoserine phosphatase from baker’s yeast. J Biol Chem 233:1169–1172PubMedGoogle Scholar
  34. Sun Y, Yang Z, Gao X, Li Q, Zhang Q, Xu Z (2005) Expression of foreign gene in Dunaliella by electrophoration. Mol Biotechnol 30(3):185–192. doi: 10.1385/MB:30:3:185 PubMedCrossRefGoogle Scholar
  35. Sussman I, Avron M (1981) Characterization and partial purification of DL-glycerol-phosphatase from Dunaliella salina. Biochim Biophys Acta 661:199–204Google Scholar
  36. Tamura K, Dudley J, Nei M, Kumar S (2007) MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol Biol Evol 24:1596–1599. doi: 10.1093/molbev/msm092 PubMedCrossRefGoogle Scholar
  37. Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG (1997) The ClustalX windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 25:4876–4882. doi: 10.1093/nar/25.24.4876 PubMedCrossRefGoogle Scholar
  38. Wang W, Xu ZK, Song RT (2006) Identification of two Dunaliella sp. based on nuclear ITS rDNA sequences. J Shanghai Univ 12(1):84–88 (Natural Science Edition)Google Scholar

Copyright information

© Springer Science+Business Media B.V. 2009

Authors and Affiliations

  • Yunxia He
    • 1
  • Xiangzong Meng
    • 2
  • Qianlan Fan
    • 1
  • Xiaoliang Sun
    • 1
  • Zhengkai Xu
    • 1
    • 2
  • Rentao Song
    • 1
  1. 1.Shanghai Key Laboratory of Bio-Energy Crops, School of Life SciencesShanghai UniversityShanghaiPeople’s Republic of China
  2. 2.Institute of Plant Physiology & Ecology, Shanghai Institutes for Biological SciencesChinese Academy of SciencesShanghaiPeople’s Republic of China

Personalised recommendations