Molecular Biology Reports

, Volume 38, Issue 5, pp 3375–3382 | Cite as

Molecular cloning and characterization of a vacuolar H+-pyrophosphatase from Dunaliella viridis



The halotolerant alga Dunaliella adapts to exceptionally high salinity and possesses efficient mechanisms for regulating intracellular Na+. In plants, sequestration of Na+ into the vacuole is driven by the electrochemical H+ gradient generated by H+ pumps, and this Na+ sequestration is one mechanism that confers salt tolerance to plants. To investigate the role of vacuolar H+ pumps in the salt tolerance of Dunaliella, we isolated the cDNA of the vacuolar proton-translocating inorganic pyrophosphatase (V–H+-PPase) from Dunaliella viridis. The DvVP cDNA is 2,984 bp in length, codes for a polypeptide of 762 amino acids and has 15 transmembrane domains. The DvVP protein is highly similar to V–H+-PPases from other green algae and higher plant species, in terms of its amino acid sequence and its transmembrane model. A phylogenetic analysis of V–H+-PPases revealed the close relationship of Dunaliella to green algal species of Charophyceae and land plants. The heterologous expression of DvVP in the yeast mutant G19 (Δena1-4) suppressed Na+ hypersensitivity, and a GFP-fusion of DvVP localized to the vacuole membranes in yeast, indicating that DvVP encodes a functional V–H+-PPase. A northern blot analysis showed a decrease in the transcript abundance of DvVP at higher salinity in D. viridis cells, which is in contrast to the salt-induced upregulation of V–H+-PPase in some plants, suggesting that the expression of DvVP under salt stress may be regulated by different mechanisms in Dunaliella. This study not only enriched our knowledge about the biological functions of V–H+-PPases in different organisms but also improved our understanding of the molecular mechanism of salt tolerance in Dunaliella.


Dunaliella viridis H+-pyrophosphatase Cloning Functional characterization Expression analysis Salt tolerance 





Vacuolar H+-pyrophosphatase


Open reading frame


Untranslated region


Expressed sequence tag



We are grateful to Dr. Alonso Rodriguez-Navarro (Universidad Politécnica de Madrid, Spain) for providing the yeast G19 mutant, Dr. Erin K. O’Shea (University of California, San Francisco) for providing the plasmid EB0666 and Dr. Anu Saloheimo (VTT Biotechnology, Finland) for providing the pAJ401 vector. This work was supported by National Natural Sciences Foundation of China (30871278, 30970242), Ministry of Agriculture of China (2008ZX08003-001, 2008ZX08003-005) and the Research Foundation from Shanghai Municipal Education Commission (09DZ2271800).


  1. 1.
    Oren A (2005) A Hundred years of Dunaliella research: 1905–2005. Saline Systems 1:2. doi: 10.1186/1746-1448-1-2 PubMedCrossRefGoogle Scholar
  2. 2.
    Apse MP, Aharon GS, Snedden WA, Blumwald E (1999) Salt tolerance conferred by overexpression of a vacuolar Na+/H+ antiport in Arabidopsis. Science 285:1256–1258. doi: 10.1126/science.285.5431.1256 PubMedCrossRefGoogle Scholar
  3. 3.
    Zhu JK (2003) Regulation of ion homeostasis under salt stress. Curr Opin Plant Biol 6:441–445. doi: 10.1016/S1369-5266(03)00085-2 PubMedCrossRefGoogle Scholar
  4. 4.
    Gimmler H (2000) Primary sodium plasma membrane ATPase in salt-tolerant algae: facts and fictions. J Exp Bot 51:1171–1178PubMedCrossRefGoogle Scholar
  5. 5.
    Goyal A (2007) Osmoregulation in Dunaliella, part II: photosynthesis and starch contribute carbon for glycerol synthesis during a salt stress in Dunaliella tertiolecta. Plant Physiol Biochem 45:705–710. doi: 10.1016/j.plaphy.2007.05.009 PubMedCrossRefGoogle Scholar
  6. 6.
    Popova LG, Shumkova GA, Andreev IM, Balnokin YV (2005) Functional identification of electrogenic Na+-translocating ATPase in the plasma membrane of the halotolerant microalga Dunaliella maritima. FEBS Letters 579:5002–5006. doi: 10.1016/j.febslet.2005.07.087 PubMedCrossRefGoogle Scholar
  7. 7.
    Katz A, Pick U (2001) Plasma membrane electron transport coupled to Na+ extrusion in the halotolerant alga Dunaliella. Biochim Biophys Acta 1504:423–431. doi: 10.1016/S0005-2728(01)00157-8 PubMedCrossRefGoogle Scholar
  8. 8.
    Rea PA, Poole RJ (1993) Vacuolar H+-translocating pyrophosphatases. Annu Rev Plant Physiol, Plant Mol Biol 44:157–180. doi: 10.1146/annurev.pp.44.060193.001105 CrossRefGoogle Scholar
  9. 9.
    Maeshima M (2000) Vacuolar H+-pyrophosphatase. Biochim Biophys Acta 1465:37–51PubMedCrossRefGoogle Scholar
  10. 10.
    Taiz L (1992) The plant vacuole. J Exp Bot 172:113–122Google Scholar
  11. 11.
    Fukuda A, Chiba K, Maeda M, Nakamura A, Maeshima M, Tanaka Y (2004) Effect of salt and osmotic stresses on the expression of genes for the vacuolar H+-pyrophosphatase, H+-ATPase subunit A, and Na+/H+ antiporter from barley. J Exp Bot 55:585–594. doi: 10.1093/jxb/erh070 PubMedCrossRefGoogle Scholar
  12. 12.
    Gao F, Gao Q, Duan X, Yue G, Yang A, Zhang J (2006) Cloning of an H+-PPase gene from Thellungiella halophila and its heterologous expression to improve tobacco salt tolerance. J Exp Bot 57:3259–3270. doi: 10.1093/jxb/erl090 PubMedCrossRefGoogle Scholar
  13. 13.
    Guo S, Yin H, Zhang X, Zhao F, Li P, Chen S, Zhao Y, Zhang H (2006) Molecular cloning and characterization of a vacuolar H+-pyrophosphatase gene, SsVP, from the halophyte Suaeda salsa and its overexpression increases salt and drought tolerance of Arabidopsis. Plant Mol Biol 60:41–50. doi: 10.1007/s11103-005-2417-6 PubMedCrossRefGoogle Scholar
  14. 14.
    Gaxiola RA, Li JS, Undurraga S, Dang LM, Allen GJ, Alper SL, Fink GR (2001) Drought- and salt-tolerant plants result from overexpression of the AVP1 H+-pump. Proc Natl Acad Sci USA 98:11444–11449. doi: 10.1073/pnas.191389398 PubMedCrossRefGoogle Scholar
  15. 15.
    Park S, Li J, Pittman JK, Berkowitz GA, Yang H, Undurraga S, Morris J, Hirschi KD, Gaxiola RA (2005) Up-regulation of a H+-pyrophosphatase (H+-P Pase) as a strategy to engineer drought-resistant crop plants. Proc Natl Acad Sci USA 102:18830–18835. doi: 10.1073/pnas.0509512102 PubMedCrossRefGoogle Scholar
  16. 16.
    Wang W, Xu Z, Song R (2006) Identification of two Dunaliella sp based on nuclear ITS rDNA sequences. J Shanghai University (Nat Sci Edn) 12:84–88Google Scholar
  17. 17.
    Sun Y, Yang Z, Gao X, Li Q, Zhang Q, Xu Z (2005) Expression of foreign gene in Dunaliella by electrophoration. Mol Biotechnol 30:185–192. doi: 10.1385/MB:30:3:185 PubMedCrossRefGoogle Scholar
  18. 18.
    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 25:4876–4882PubMedCrossRefGoogle Scholar
  19. 19.
    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
  20. 20.
    Quintero FJ, Garciadeblas B, Rodríguez-Navarro A (1996) The SAL1 gene of Arabidopsis, encoding an enzyme with 3′(2′), 5′-bisphosphate nucleotidase and inositol 1-phosphatase activities, increases salt tolerance in yeast. Plant Cell 8:529–537PubMedCrossRefGoogle Scholar
  21. 21.
    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 13:219–228PubMedCrossRefGoogle Scholar
  22. 22.
    Gietz D, St Jean A, Woods RA, Schiest RH (1992) Improved method for high efficiency transformation of intact yeast cells. Nucl Acid Res 20:1425CrossRefGoogle Scholar
  23. 23.
    Alonso R, Ramos J (1984) Dual system for potassium transport in Saccharomyces cerevisiae. J Bacteriol 159:940–945Google Scholar
  24. 24.
    Lau WT, Howson RW, Malkus P, Schekman R, O’Shea EK (2000) Pho86p, an endoplasmic reticulum (ER) resident protein in Saccharomyces cerevisiae, is required for ER exit of the high-affinity phosphate transporter Pho84p. Proc Natl Acad Sci USA 97:1107–1112PubMedCrossRefGoogle Scholar
  25. 25.
    Vida TA, Emr SD (1995) A new vital stain for visualizing vacuolarmembrane dynamics and endocytosis in yeast. J Cell Biol 128:779–792PubMedCrossRefGoogle Scholar
  26. 26.
    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 423:36–42. doi: 10.1016/j.gene.2008.06.029 PubMedCrossRefGoogle Scholar
  27. 27.
    Li Q, Gao X, Sun Y, Zhang Q, Song R, Xu Z (2006) Isolation and characterization of a sodium- dependent phosphate transporter gene in Dunaliella viridis. Biochem Biophys Res Commun 340:95–104. doi: 10.1016/j.bbrc.2005.11.144 PubMedCrossRefGoogle Scholar
  28. 28.
    Gaxiola RA, Palmgren MG, Schumacher K (2007) Plant proton pumps. FEBS Letters 581:2204–2214. doi: 10.1016/j.febslet.2007.03.050 PubMedCrossRefGoogle Scholar
  29. 29.
    Drozdowicz YM, Rea PA (2001) Vacuolar H+ pyrophosphatases: from the evolutionary backwaters into the mainstream. Trends Plant Sci 6:206–211. doi: 10.1016/S1360-1385(01)01923-9 PubMedCrossRefGoogle Scholar
  30. 30.
    Zhen RG, Kim EJ, Rea PA (1997) Acidic residues necessary for pyrophosphate-energized pumping and inhibition of the vacuolar H + -pyrophosphatase by N, N′-dicyclohexylcarbodiimide. J Biol Chem 272:22340–22348. doi: 10.1074/jbc.272.35.22340 PubMedCrossRefGoogle Scholar
  31. 31.
    Gaxiola RA, Rao R, Sherman A, Grisafi P, Alper S, Fink GR (1999) The Arabidopsis thaliana proton transporters, AtNhx1 and Avp1, can function in cation detoxification in yeast. Proc Natl Acad Sci USA 96:1480–1485PubMedCrossRefGoogle Scholar
  32. 32.
    Ballesteros E, Donaire JP, Belver A (1996) Effects of salt stress on H+-ATPase and H+-PPase activities of tonoplast-enriched vesicles isolated from sunflower roots. Physiol Plant 97:259–568CrossRefGoogle Scholar
  33. 33.
    Brini F, Gaxiola RA, Berkowitz GA, Masmoudi K (2005) Cloning and characterization of a wheat vacuolar cation/proton antiporter and pyrophosphatase proton pump. Plant Physiol Biochem 43:347–354. doi: 10.1016/j.plaphy.2005.02.010 PubMedGoogle Scholar
  34. 34.
    Otoch MLO, Sobreira ACM, de-aragao MEF, Orellano EG, Lima MGS, de-Melo DF (2001) Salt modulation of vacuolar H+-ATPase and H+-phrophosphatase activities in Vigna unguiculata. J Plant Physiol 158:545–551. doi: 10.1078/0176-1617-00310 CrossRefGoogle Scholar
  35. 35.
    Parks GE, Dietrich MA, Schumaker KS (2002) Increased vacuolar Na+/H+ exchange activity in Salicornia bigelovii Torr. in response to NaCl. J Exp Bot 53:1055–1065PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2010

Authors and Affiliations

  1. 1.Shanghai Key Laboratory of Bio-energy CropsSchool of Life Sciences, Shanghai UniversityShanghaiChina
  2. 2.Institute of Plant Physiology and EcologyShanghai Institutes for Biological Sciences, Chinese Academy of SciencesShanghaiChina

Personalised recommendations