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

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Abstract

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.

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Abbreviations

GPDH:

Glycerol-3-phosphate dehydrogenase

SerB/PSP:

Phosphoserine phosphatase

DHAP:

Dihydroxyacetone phosphate

GPP:

Glycerol-3-phosphate phosphatase

ORF:

Open reading frame

References

  • 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):4135–4144

    PubMed  CAS  Google Scholar 

  • Alonso R, Ramos J (1984) Dual system for potassium transport in Saccharomyces cerevisiae. J Bacteriol 159(3):940–945

    Google Scholar 

  • Avron M (1986) The osmotic components of halotolerant algae. Trends Biochem Sci 11:5–6. doi:10.1016/0968-0004(86)90218-5

    Article  CAS  Google Scholar 

  • Belmans D, Van Laera A (1987) Glycerol cycle enzymes and intermediates during adaptation of Dunaliella teriolecta cells to hyperosmotic stress. Plant Cell Environ 10:185–190

    CAS  Google Scholar 

  • 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

    Article  PubMed  CAS  Google Scholar 

  • 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

    Article  PubMed  CAS  Google Scholar 

  • 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

    Article  PubMed  CAS  Google Scholar 

  • 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

    Article  PubMed  CAS  Google Scholar 

  • 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

    Article  PubMed  CAS  Google Scholar 

  • 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

    Article  PubMed  CAS  Google Scholar 

  • Gee R, Goyal A, Byerrum RU, Tolbert NE (1993) Two isozymes of dihydroxyacetone phosphate reductase from the chloroplasts of Dunaliella tertiolecta. Plant Physiol 103:243–249

    PubMed  CAS  Google Scholar 

  • 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

    Article  PubMed  CAS  Google Scholar 

  • 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

    Article  PubMed  CAS  Google Scholar 

  • 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

    Article  PubMed  CAS  Google Scholar 

  • 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

    Article  PubMed  CAS  Google Scholar 

  • Graham P (2008) Pond scum genomics: the genomes of Chlamydomonas and Ostreococcus. Plant Cell 20:502–507. doi:10.1105/tpc.107.056556

    Article  Google Scholar 

  • 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

    Article  PubMed  CAS  Google Scholar 

  • Haus M, Wegman K (1984) Glycerol-3-phosphate dehydrogenase (EC 1.1.1.8) from Dunaliella teriolecta. I. Purification and kinetic properties. Physiol Plant 60:283–288. doi:10.1111/j.1399-3054.1984.tb06063.x

    Article  CAS  Google Scholar 

  • 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

    Article  PubMed  CAS  Google Scholar 

  • 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

    Article  PubMed  CAS  Google Scholar 

  • 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

    Article  PubMed  CAS  Google Scholar 

  • 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

    Article  PubMed  CAS  Google Scholar 

  • 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

    Article  PubMed  CAS  Google Scholar 

  • 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

    Article  PubMed  CAS  Google Scholar 

  • Oren A (2005) A hundred years of Dunaliella research: 1905–2005. Saline Syst 1:2. doi:10.1186/1746-1448-1-2

    Article  PubMed  Google Scholar 

  • 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

    Article  Google Scholar 

  • 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

    Article  PubMed  CAS  Google Scholar 

  • 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

    Article  PubMed  CAS  Google Scholar 

  • 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

    Article  PubMed  CAS  Google Scholar 

  • 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 145:715–727

    Article  PubMed  CAS  Google Scholar 

  • 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

    Article  CAS  Google Scholar 

  • 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

    Article  PubMed  CAS  Google Scholar 

  • Schramm M (1958) O-phosphoserine phosphatase from baker’s yeast. J Biol Chem 233:1169–1172

    PubMed  CAS  Google Scholar 

  • 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

    Article  PubMed  CAS  Google Scholar 

  • Sussman I, Avron M (1981) Characterization and partial purification of DL-glycerol-phosphatase from Dunaliella salina. Biochim Biophys Acta 661:199–204

    CAS  Google Scholar 

  • 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

    Article  PubMed  CAS  Google Scholar 

  • 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

    Article  PubMed  CAS  Google Scholar 

  • 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)

    CAS  Google Scholar 

Download references

Acknowledgments

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).

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Authors and Affiliations

  1. Shanghai Key Laboratory of Bio-Energy Crops, School of Life Sciences, Shanghai University, 99 Shangda Road, 200444, Shanghai, People’s Republic of China

    Yunxia He, Qianlan Fan, Xiaoliang Sun, Zhengkai Xu & Rentao Song

  2. Institute of Plant Physiology & Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 200032, Shanghai, People’s Republic of China

    Xiangzong Meng & Zhengkai Xu

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  1. Yunxia He
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  2. Xiangzong Meng
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  3. Qianlan Fan
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  4. Xiaoliang Sun
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  5. Zhengkai Xu
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  6. Rentao Song
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Correspondence to Rentao Song.

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Yunxia He and Xiangzong Meng contributed equally to this work.

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He, Y., Meng, X., Fan, Q. et al. Cloning and characterization of two novel chloroplastic glycerol-3-phosphate dehydrogenases from Dunaliella viridis . Plant Mol Biol 71, 193–205 (2009). https://doi.org/10.1007/s11103-009-9517-7

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