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Plant Molecular Biology

, Volume 85, Issue 4–5, pp 429–441 | Cite as

Transgenic Arabidopsis expressing osmolyte glycine betaine synthesizing enzymes from halophilic methanogen promote tolerance to drought and salt stress

  • Shu-Jung Lai
  • Mei-Chin Lai
  • Ren-Jye Lee
  • Yu-Hsuan Chen
  • Hungchen Emilie Yen
Article

Abstract

Glycine betaine (betaine) has the highest cellular osmoprotective efficiency which does not accumulate in most glycophytes. The biosynthetic pathway for betaine in higher plants is derived from the oxidation of low-accumulating metabolite choline that limiting the ability of most plants to produce betaine. Halophilic methanoarchaeon Methanohalophilus portucalensis FDF1T is a model anaerobic methanogen to study the acclimation of water-deficit stresses which de novo synthesize betaine by the stepwise methylation of glycine, catalyzed by glycine sarcosine N-methyltransferase (GSMT) and sarcosine dimethylglycine N-methyltransferase. In this report, genes encoding these betaine biosynthesizing enzymes, Mpgsmt and Mpsdmt, were introduced into Arabidopsis. The homozygous Mpgsmt (G), Mpsdmt (S), and their cross, Mpgsmt and Mpsdmt (G × S) plants showed increased accumulation of betaine. Water loss from detached leaves was slower in G, S, and G × S lines than wild-type (WT). Pot-grown transgenic plants showed better growth than WT after 9 days of withholding water or irrigating with 300 mM NaCl. G, S, G × S lines also maintained higher relative water content and photosystem II activity than WT under salt stress. This suggests heterologously expressed Mpgsmt and Mpsdmt could enhance tolerance to drought and salt stress in Arabidopsis. We also found a twofold increase in quaternary ammonium compounds in salt-stressed leaves of G lines, presumably due to the activation of GSMT activity by high salinity. This study demonstrates that introducing stress-activated enzymes is a way of avoiding the divergence of primary metabolites under normal growing conditions, while also providing protection in stressful environments.

Keywords

Halophilic methanoarchaea Glycine betaine Glycine sarcosine N-methyltransferase (GSMT) Sarcosine dimethylglycine N-methyltransferase (SDMT) Salt tolerance Transgenic Arabidopsis 

Abbreviations

GSMT

Glycine sarcosine N-methyltransferase

LC/MS

Liquid chromatograph–mass spectrometry

QAC

Quaternary ammonium compound

RWC

Relative water content

RWL

Rate of water loss

SDMT

Sarcosine dimethylglycine N-methyltransferase

Notes

Acknowledgments

This work has been supported by Grants NSC 100-2321-B-005-005-MY3 from the National Science Council, Taiwan. We are grateful to the Instrument Center, NCHU, Taiwan for assistance with LC/MS analyses.

Supplementary material

11103_2014_195_MOESM1_ESM.pdf (332 kb)
Supplementary material 1 (PDF 332 kb)

References

  1. Ahmad R, Kim MD, Back KH, Kim HS, Lee HS, Kwon SY, Murata N, Chung WI, Kwak SS (2008) Stress-induced expression of choline oxidase in potato plant chloroplasts confers enhanced tolerance to oxidative, salt, and drought stresses. Plant Cell Rep 27:687–698PubMedCrossRefGoogle Scholar
  2. Allakhverdiev SI, Hayashi H, Nishiyama Y, Ivanov AG, Aliev JA, Klimov VV, Murata N, Carpentier R (2003) Glycine betaine protects the D1/D2/Cytb559 complex of photosystem II against photo-induced and heat-induced inactivation. J Plant Physiol 160:41–49PubMedCrossRefGoogle Scholar
  3. Auton M, Rösgen J, Sinev M, Holthauzen LMF, Bolen DW (2011) Osmolyte effects on protein stability and solubility: a balancing act between backbone and side-chains. Biophys Chem 159:90–99PubMedCentralPubMedCrossRefGoogle Scholar
  4. Ben Hassine A, Ghanem ME, Bouzid S, Lutts S (2008) An inland and a coastal population of the Mediterranean xerohalophyte species Atriplex halimus L. differ in their ability to accumulate proline and glycine betaine in response to salinity and water stress. J Exp Bot 59:1315–1326PubMedCrossRefGoogle Scholar
  5. Boyer JS (1982) Plant productivity and environment. Science 218:443–448PubMedCrossRefGoogle Scholar
  6. Chen THH, Murata N (2008) Glycine betaine: an effective protectant against abiotic stress in plants. Trends Plant Sci 13:499–505PubMedCrossRefGoogle Scholar
  7. Chen THH, Murata N (2011) Glycine betaine protects plants against abiotic stress: mechanisms and biotechnological applications. Plant, Cell Environ 34:1–20CrossRefGoogle Scholar
  8. Chen S-Y, Lai M-C, Lai S-J, Lee Y-C (2009) Characterization of osmolyte betaine synthesizing sarcosine dimethylglycine N-methyltransferase from Methanohalophilus portucalensis. Arch Microbiol 191:735–743PubMedCrossRefGoogle Scholar
  9. Clough SJ, Bent AF (1998) Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16:735–743PubMedCrossRefGoogle Scholar
  10. Diamant S, Eliahu N, Rosenthal D, Goloubinoff P (2001) Chemical chaperones regulate molecular chaperones in vitro and in cells under combined salt and heat stresses. J Biol Chem 276:39586–39591PubMedCrossRefGoogle Scholar
  11. Einset J, Nielsen E, Connolly EL, Bones A, Sparstad T, Winge P, Zhu JK (2007) Membrane-trafficking RabA4c involved in the effect of glycine betaine on recovery from chilling stress in Arabidopsis. Physiol Plant 130:511–518CrossRefGoogle Scholar
  12. Einset J, Winge P, Bones AM, Connolly EL (2008) The FRO2 ferric reductase is required for glycine betaine’s effect on chilling tolerance in Arabidopsis roots. Physiol Plant 134:334–341PubMedCrossRefGoogle Scholar
  13. Flowers TJ, Colmer TD (2008) Salinity tolerance in halophytes. New Phytol 179:945–963PubMedCrossRefGoogle Scholar
  14. Giraud E, Ho LH, Clifton R, Carroll A, Estavillo G, Tan YF, Howell KA, Ivanova A, Pogson BJ, Millar AH, Whelan J (2008) The absence of ALTERNATIVE OXIDASE1a in Arabidopsis results in acute sensitivity to combined light and drought stress. Plant Physiol 147:595–610PubMedCentralPubMedCrossRefGoogle Scholar
  15. Goel D, Singh AK, Yadav V, Babbar SB, Murata N, Bansal KC (2011) Transformation of tomato with a bacterial codA gene enhances tolerance to salt and water stresses. J Plant Physiol 168:1286–1294PubMedCrossRefGoogle Scholar
  16. Grieve CM, Grattan SR (1983) Rapid assay for determination of water soluble quaternary ammonium compounds. Plant Soil 70:303–307CrossRefGoogle Scholar
  17. Hanson AD, May AM, Grumet R, Bode J, Jamieson GC, Rhodes D (1985) Betaine synthesis in chenopods: localization in chloroplasts. Proc Natl Acad Sci USA 82:3678–3682PubMedCentralPubMedCrossRefGoogle Scholar
  18. Harb A, Krishnan A, Ambavaram MM, Pereira A (2010) Molecular and physiological analysis of drought stress in Arabidopsis reveals early responses leading to acclimation in plant growth. Plant Physiol 154:1254–1271PubMedCentralPubMedCrossRefGoogle Scholar
  19. Hasegawa PM, Bressan RA, Zhu JK, Bohnert HJ (2000) Plant cellular and molecular responses to high salinity. Annu Rev Plant Physiol Plant Mol Biol 51:463–499PubMedCrossRefGoogle Scholar
  20. Huang J, Hirji R, Adam L, Rozwadowski KL, Hammerlindl JK, Keller WA, Selvaraj G (2000) Genetic engineering of glycine betaine production toward enhancing stress tolerance in plants: metabolic limitations. Plant Physiol 122:747–756PubMedCentralPubMedCrossRefGoogle Scholar
  21. Kathuria H, Giri J, Nataraja KN, Murata N, Udayakumar M, Tyagi AK (2009) Glycinebetaine-induced water-stress tolerance in codA-expressing transgenic indica rice is associated with up-regulation of several stress responsive genes. Plant Biotechnol J 7:512–526PubMedCrossRefGoogle Scholar
  22. Kempf B, Bremer E (1998) Uptake and synthesis of compatible solutes as microbial stress responses to high-osmolality environment. Arch Microbiol 170:319–330PubMedCrossRefGoogle Scholar
  23. Kimura Y, Kawasaki S, Yoshimoto H, Takegawa K (2010) Glycine betaine biosynthesized from glycine provides an osmolyte for cell growth and spore germination during osmotic stress in Myxococcus xanthus. J Bacteriol 192:1467–1470PubMedCentralPubMedCrossRefGoogle Scholar
  24. Krall JP, Edwards GE (1992) Relationship between photosystem II activity and CO2 fixation in leaves. Physiol Plant 86:180–187CrossRefGoogle Scholar
  25. Lai S-J, Lai M-C (2011) Characterization and regulation of the osmolyte betaine synthesizing enzymes GSMT and SDMT from halophilic methanogen Methanohalophilus portucalensis. PLoS ONE 6:e25090PubMedCentralPubMedCrossRefGoogle Scholar
  26. Lai M-C, Ciulla R, Roberts MF, Sowers KR, Gunsalus RP (1995) Extraction and detection of compatible intracellular solutes. In: Sowers KR, Schreier HJ (eds) Archaea, a laboratory manual, methanogens protocols. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, pp 349–368Google Scholar
  27. Lai M-C, Yang D-R, Chuang M-J (1999) Regulatory factors associated with synthesis of the osmolyte glycine betaine in the halophilic methanoarchaeon Methanohalophilus portucalensis. Appl Environ Microbiol 65:828–833PubMedCentralPubMedGoogle Scholar
  28. Lai M-C, Wang C–C, Chuang M-J, Wu Y-C, Lee Y-C (2006) Effects of substrate and potassium on the betaine-synthesizing enzyme glycine sarcosine dimethylglycine N-methyltransferase from a halophilic methanoarchaeon Methanohalophilus portucalensis. Res Microbiol 157:948–955PubMedCrossRefGoogle Scholar
  29. Lu W-D, Chi Z-M, Su C-D (2006) Identification of glycine betaine as compatible solute in Synechococcus sp. WH8102 and characterization of its N-methyltransferase genes involved in betaine synthesis. Arch Microbiol 186:495–506PubMedCrossRefGoogle Scholar
  30. Ma QQ, Wang W, Li YH, Li DQ, Zou Q (2006) Alleviation of photoinhibition in drought-stressed wheat (Triticum aestivum) by foliar-applied glycine betaine. J Plant Physiol 163:165–175PubMedCrossRefGoogle Scholar
  31. Naleway JJ (1992) Histochemical, spectrophotometric, and fluorometric GUS substrates. In: Gallagher SR (ed) GUS protocols: using the GUS gene as a reporter of gene expression. Academic Press, San Diego, pp 61–76Google Scholar
  32. Nyyssölä A, Kerovuo J, Kaukinen P, von Weymarn N, Reinikainen T (2000) Extreme halophiles synthesize betaine from glycine by methylation. J Biol Chem 275:22196–22201PubMedCrossRefGoogle Scholar
  33. Nyyssölä A, Reinikainen T, Leisola M (2001) Characterization of glycine sarcosine N-methyltransferase and sarcosine dimethylglycine N-methyltransferase. Appl Environ Microbiol 67:2044–2050PubMedCentralPubMedCrossRefGoogle Scholar
  34. Park EJ, Jeknić Z, Chen TH, Murata N (2007) The codA transgene for glycinebetaine synthesis increases the size of flowers and fruits in tomato. Plant Biotechnol J 5:422–430PubMedCrossRefGoogle Scholar
  35. Quan R, Shang M, Zhang H, Zhao Y, Zhang J (2004) Engineering of enhanced glycine betaine synthesis improves drought tolerance in maize. Plant Biotechnol J 2:477–486PubMedCrossRefGoogle Scholar
  36. Rhodes D, Hanson AD (1993) Quaternary ammonium and tertiary sulfonium compounds in higher-plants. Annu Rev Plant Physiol Plant Mol Biol 44:357–384CrossRefGoogle Scholar
  37. Roberts MF (2000) Osmoadaptation and osmoregulation in archaea. Front Biosci 5:796–812CrossRefGoogle Scholar
  38. Roberts MF, Lai M-C, Gunsalus RP (1992) Biosynthetic pathways of the osmolytes N-acetyl-β-lysine, β-glutamine, and glycine betaine in Methanohalophilus strain FDF1 suggested by nuclear magnetic resonance analyses. J Bacteriol 174:6688–6693PubMedCentralPubMedGoogle Scholar
  39. Roesser M, Muller V (2001) Osmoadaptation in bacteria and archaea: common principles and differences. Environ Microbiol 3:743–754PubMedCrossRefGoogle Scholar
  40. Rontein D, Basset G, Hanson AD (2002) Metabolic engineering of osmoprotectant accumulation in plants. Metab Eng 4:49–56PubMedCrossRefGoogle Scholar
  41. Singh M, Sharma NK, Prasad SB, Yadav SS, Narayan G, Rai AK (2013) The freshwater cyanobacterium Anabaena doliolum transformed with ApGSMT-DMT exhibited enhanced salt tolerance and protection to nitrogenase activity, but became halophilic. Microbiology 159:641–648PubMedCrossRefGoogle Scholar
  42. Sleator RD, Hill C (2001) Bacterial osmoadaptation: the role of osmolytes in bacterial stress and virulence. FEMS Microbiol Rev 26:49–71CrossRefGoogle Scholar
  43. Smart RE, Bingham GE (1974) Rapid estimates of relative water content. Plant Physiol 53:258–260PubMedCentralPubMedCrossRefGoogle Scholar
  44. Sulpice R, Tsukaya H, Nonaka H, Mustardy L, Chen THH, Murata N (2003) Enhanced formation of flowers in salt-stressed Arabidopsis after genetic engineering of the synthesis of glycinebetaine. Plant J 36:165–176PubMedCrossRefGoogle Scholar
  45. Waditee R, Tanaka Y, Aoki K, Hibino T, Jikuya H, Takano J, Takabe T (2003) Isolation and functional characterization of N-methyltransferase that catalyze betaine synthesis from glycine in a halotolerant photosynthetic organism Aphanothece halophytica. J Biol Chem 278:4932–4942PubMedCrossRefGoogle Scholar
  46. Waditee R, Bhuiyan NH, Rai V, Aoli K, Tanaka Y, Hibino T, Suzuki S, Jagendorf AT, Takabe T, Takabe T (2005) Gene for direct methylation of glycine provides high levels of glycine betaine and abiotic-stress tolerance in Synechococcus and Arabidopsis. Proc Natl Acad Sci USA 102:1318–1323PubMedCentralPubMedCrossRefGoogle Scholar
  47. Waditee-Sirisattha R, Singh M, Kageyama H, Sittipol D, Rai AK, Takabe T (2012) Anabaena sp. PCC7120 transformed with glycine methylation genes from Aphanothece halophytica synthesized glycine betaine showing increased tolerance to salt. Arch Microbiol 194:909–914PubMedCrossRefGoogle Scholar
  48. Weigel D, Glazebrook J (2002) Arabidopsis: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring HarborGoogle Scholar
  49. Wingler A, Lea PJ, Quick WP, Leegood RC (2000) Photorespiration: metabolic pathways and their role in stress protection. Philos Trans R Soc Lond B Biol Sci 55:1517–1529CrossRefGoogle Scholar
  50. Wyn Jones RG, Storey R (1981) Betaines. In: Paleg LG, Aspinall D (eds) The physiology and biochemistry of drought resistance in plants. Academic Press, New York, pp 171–204Google Scholar
  51. Yang J, Yen HE (2002) Early salt stress effects on the changes in chemical composition in leaves of ice plant and Arabidopsis. A Fourier transform infrared spectroscopy study. Plant Physiol 130:1032–1042PubMedCentralPubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2014

Authors and Affiliations

  1. 1.Department of Life SciencesNational Chung Hsing UniversityTaichungTaiwan
  2. 2.Instrument Center and Department of ChemistryNational Chung Hsing UniversityTaichungTaiwan

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