Skip to main content
SpringerLink
Log in

Expression of Bacterial l-aspartate-α-decarboxylase in Tobacco Increases β-Alanine and Pantothenate Levels and Improves Thermotolerance

  • Published:
Plant Molecular Biology Aims and scope Submit manuscript

Abstract

l-Aspartate-α-decarboxylase catalyzes the decarboxylation of l-aspartate to generate β-alanine and carbon dioxide. This is an unusual pyruvoyl-dependent enzyme unique to prokaryotes that undergoes limited self-processing. The Escherichia coli panD gene encoding l-aspartate-α-decarboxylase was expressed under a constitutive promoter in transgenic tobacco. Transgene expression was verified by assays based on RNA blots, immunoblots and enzyme activity in vitro. The panD lines had increased levels of leaf β-alanine (1.2- to 4-fold), pantothenate (3.2- to 4.1-fold) and total free amino acids (up to 3.7-fold) compared to wild-type and vector controls. Growth of homozygous lines expressing E. coli l-aspartate-α-decarboxylase was less affected than that of the control lines when the plants were stressed for 1 week at 35 °C. When transferred from 35 to 30 °C for 3 weeks, the PanD transgenic lines recovered significantly (P ≤ 0.001) better than the controls: PanD lines had on an average 54% and 84% greater fresh and dry weights respectively, compared to the controls. Homozygous lines expressing E. coli l-aspartate-α-decarboxylase had significantly greater thermotolerance (P ≤ 0.05) during germination. At 42 °C, 95% of two T3 PanD transgenic line seeds germinated after 12 days compared to 73% for the wild-type seeds. Our results indicated that E. colil-aspartate-α-decarboxylase was correctly processed and active in the transgenic eukaryotic host and its expression resulted in increased thermotolerance in tobacco.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  • A. Albert V. Dhanaraj U. Genschel G. Khan M.K. Ramjee R. Pulido B.L. Sibanda F. Delft M. Witty T.L. Blundel A.G. Smith C. Abell (1998) ArticleTitleCrystal structure of aspartate decarboxylase at 2.2 Å resolution provides evidence for an ester in protein self-processing Nat. Struct. Biol. 5 289–293 Occurrence Handle10.1038/nsb0498-289 Occurrence Handle1:CAS:528:DyaK1cXitlantbo%3D Occurrence Handle9546220

    Article  CAS  PubMed  Google Scholar 

  • R. Ameziane K. Bernhard D. Lightfoot (2000) ArticleTitleExpression of the bacterial gdhA gene encoding a NADPH glutamate dehydrogenase in tobacco affects plant growth and development Plant Soil 221 47–57 Occurrence Handle10.1023/A:1004794000267 Occurrence Handle1:CAS:528:DC%2BD3cXlslyns74%3D

    Article  CAS  Google Scholar 

  • G. An P.R. Ebert A. Mitra S.B. Ha (1988) Binary vectors S.B. Gelvin R.A. Schilperoort (Eds) Plant Molecular Biology Manual, vol. A3 Kluwer Dordrecht 1–19

    Google Scholar 

  • N. Bouche H. Fromm (2004) ArticleTitleGABA in plants: just a metabolite? Trends Plant Sci. 9 110–115 Occurrence Handle10.1016/j.tplants.2004.01.006 Occurrence Handle1:CAS:528:DC%2BD2cXhslCjs7Y%3D Occurrence Handle15003233

    Article  CAS  PubMed  Google Scholar 

  • S. Chichkova J. Arellano C.P. Vance G. Hernandez (2001) ArticleTitleTransgenic tobacco plants that overexpress alfalfa NADH-glutamate synthase have higher carbon and nitrogen content J. Exp. Bot. 52 2079–2087 Occurrence Handle1:CAS:528:DC%2BD3MXotFSqsLc%3D Occurrence Handle11604446

    CAS  PubMed  Google Scholar 

  • S. Chopra H. Pai A. Ranganathan (2002) ArticleTitleExpression, purification, and biochemical characterization of Mycobacterium tuberculosis aspartate decarboxylase PanD Protein Expr. Purif. 25 533–540 Occurrence Handle1:CAS:528:DC%2BD38XmtFWiurw%3D Occurrence Handle12182836

    CAS  PubMed  Google Scholar 

  • J.E. Cronan (1980) ArticleTitleβ-Alanine synthesis in Escherichia coli J. Bact. 141 1291–1297 Occurrence Handle1:CAS:528:DyaL3cXhvFCju7o%3D Occurrence Handle6767707

    CAS  PubMed  Google Scholar 

  • N. Dusch A. Puhler J. Kalinowski (1999) ArticleTitleExpression of the Corynebacterium glutamicum panD gene encoding l-aspartate-α-decarboxylase leads to pantothenate overproduction in Escherichia coli Appl. Environ. Microbiol. 65 1530–1539 Occurrence Handle1:CAS:528:DyaK1MXitlart70%3D Occurrence Handle10103247

    CAS  PubMed  Google Scholar 

  • W.M. Fouad (2004) Metabolic engineering for β-alanine overproduction and stress tolerance in plants: expression of Escherichia coli L-aspartate-α-decarboxylase in transgenic tobacco University of Florida Gainesville, FL 99

    Google Scholar 

  • S.I. Fuentes D.J. Allen A. Ortiz-Lopez G. Hernandez (2001) ArticleTitleOver-expression of cytosolic glutamine synthetase increases photosynthesis and growth at low nitrogen concentrations J. Exp. Bot. 52 1071–1081 Occurrence Handle10.1093/jexbot/52.358.1071 Occurrence Handle1:CAS:528:DC%2BD3MXltVeisrs%3D Occurrence Handle11432923

    Article  CAS  PubMed  Google Scholar 

  • J. Fu R. Sampalo F.M. Gallardo Canovas E.G. Kirby (2003) ArticleTitleAssembly of a cytosolic pine glutamine synthetase holoenzyme in the leaf of transgenic poplar leads to an enhanced vegetative growth of young plants Plant Cell Environ. 26 411–418 Occurrence Handle10.1046/j.1365-3040.2003.00972.x Occurrence Handle1:CAS:528:DC%2BD3sXjtVyltL0%3D

    Article  CAS  Google Scholar 

  • G. Galili R. Hoefgen (2002) ArticleTitleMetabolic engineering of amino acids and storage proteins in plants Metab. Eng. 4 3–11 Occurrence Handle10.1006/mben.2001.0203 Occurrence Handle1:CAS:528:DC%2BD38XlsVSrtw%3D%3D Occurrence Handle11800569

    Article  CAS  PubMed  Google Scholar 

  • F. Kaplan J. Kopka D.W. Haskell W. Zhao K.C. Schiller N. Gatzke D.Y. Sung C.L. Guy (2004) ArticleTitleExploring the temperature-stress metabolome of Arabidopsis Plant Physiol. 136 4159–4168 Occurrence Handle10.1104/pp.104.052142 Occurrence Handle1:CAS:528:DC%2BD2MXjtVakug%3D%3D Occurrence Handle15557093

    Article  CAS  PubMed  Google Scholar 

  • S. Klapheck H. Zopes H.G. Levels L. Bergmann (1988) ArticleTitleProperties and localization of the homoglutathione synthetase from Phaseolus coccineus leaves Physiol. Plant 74 733–739 Occurrence Handle1:CAS:528:DyaL1MXptFSlsA%3D%3D

    CAS  Google Scholar 

  • S.P. Long E.A. Ainsworth A. Rogers D.R. Ort (2004) ArticleTitleRising atmospheric carbon dioxide: plants FACE the future Annu. Rev. Plant Biol. 55 591–628 Occurrence Handle10.1146/annurev.arplant.55.031903.141610 Occurrence Handle1:CAS:528:DC%2BD2cXlvFeisb8%3D Occurrence Handle15377233

    Article  CAS  PubMed  Google Scholar 

  • A.D. Hanson D.A. Gage (1991) ArticleTitleIdentification and determination by fast atom bombardment mass spectrometry of the compatible solute choline-o-sulfate in Limonium species and other halophytes Aust. J. Plant Physiol. 18 317–327 Occurrence Handle1:CAS:528:DyaK38XisFaisLo%3D

    CAS  Google Scholar 

  • R.R. Mayer J.H. Cherry D. Rhodes (1990) ArticleTitleEffects of heat shock on amino acid metabolism of cowpea cells Plant Physiol. 94 796–810 Occurrence Handle1:CAS:528:DyaK3cXmt1yjtr4%3D

    CAS  Google Scholar 

  • A.D. Mehta N.W. Seidler (2005) ArticleTitleβ-Alanine suppresses heat inactivation of lactate dehydrogenase J. Enzyme Inhib. Med. Chem. 20 199–203 Occurrence Handle10.1080/14756360400020538 Occurrence Handle1:CAS:528:DC%2BD2MXjvVOksbc%3D Occurrence Handle15968825

    Article  CAS  PubMed  Google Scholar 

  • W.K. Merkel B.P. Nichols (1996) ArticleTitleCharacterization and sequence of the Escherichia coli panBCD gene cluster FEMS Microbiol. Lett. 143 247–252 Occurrence Handle1:CAS:528:DyaK28XlvVant7k%3D Occurrence Handle8837478

    CAS  PubMed  Google Scholar 

  • A. Migge E. Carrayol B. Hirel T.W. Becker (2000) ArticleTitleLeaf-specific overexpression of plastidic glutamine synthetase stimulates the growth of transgenic tobacco seedlings Planta 210 252–260 Occurrence Handle10.1007/PL00008132 Occurrence Handle1:CAS:528:DC%2BD3cXisVajug%3D%3D Occurrence Handle10664131

    Article  CAS  PubMed  Google Scholar 

  • J.F. Moran I. Iturbe-Ormaetxe M.A. Matamoros M.C. Rubio M.R. Clemente I.J. Brewin M. Becana (2000) ArticleTitleGlutathione and homoglutathione synthetases of legume nodules: cloning, expression, and subcellular localization Plant Physiol. 124 1381–1392 Occurrence Handle10.1104/pp.124.3.1381 Occurrence Handle1:CAS:528:DC%2BD3cXotlWqsL0%3D Occurrence Handle11080313

    Article  CAS  PubMed  Google Scholar 

  • I.C. Oliveira T. Brears T.J. Knight A. Clark G.M. Coruzzi (2002) ArticleTitleOverexpression of cytosolic glutamine synthetase. Relation to nitrogen, light, and photorespiration Plant Physiol. 129 1170–1180 Occurrence Handle10.1104/pp.020013 Occurrence Handle1:CAS:528:DC%2BD38XlsFajsLg%3D Occurrence Handle12114571

    Article  CAS  PubMed  Google Scholar 

  • G.L. Peterson (1977) ArticleTitleA simplification of the protein assay method of Lowry et al., which is more generally applicable Anal. Biochem. 83 709–716 Occurrence Handle10.1016/0003-2697(77)90043-4

    Article  Google Scholar 

  • S.B. Raman B. Rathinasabapathi (2003) ArticleTitleβ-Alanine N-methyltransferase of Limonium latifolium. cDNA cloning and functional expression of a novel N-methyltransferase implicated in the synthesis of the osmoprotectant β-alanine betaine Plant Physiol. 132 1642–1651 Occurrence Handle10.1104/pp.103.020453 Occurrence Handle1:CAS:528:DC%2BD3sXlsFGhu7c%3D Occurrence Handle12857843

    Article  CAS  PubMed  Google Scholar 

  • S.B. Raman B. Rathinasabapathi (2004) ArticleTitlePantothenate synthesis in plants Plant Sci. 167 961–968 Occurrence Handle10.1016/j.plantsci.2004.06.019 Occurrence Handle1:CAS:528:DC%2BD2cXmsVGisrk%3D

    Article  CAS  Google Scholar 

  • M.K. Ramjee U. Genschel C. Abell A.G. Smith (1997) ArticleTitleEscherichia coli l-aspartate-α-decarboxylase: preprotein processing and observation of reaction intermediates by electrospray mass spectrometry Biochem. J. 323 661–669 Occurrence Handle1:CAS:528:DyaK2sXjtFGlsLs%3D Occurrence Handle9169598

    CAS  PubMed  Google Scholar 

  • B. Rathinasabapathi K.F. McCue D.A. Gage A.D. Hanson (1994) ArticleTitleMetabolic engineering of glycine betaine synthesis: plant betaine aldehyde dehydrogenases lacking typical transit peptides are targeted to tobacco chloroplasts where they confer betaine aldehyde resistance Planta 193 155–162 Occurrence Handle10.1007/BF00192524 Occurrence Handle1:CAS:528:DyaK2cXisVamu7o%3D Occurrence Handle7764986

    Article  CAS  PubMed  Google Scholar 

  • B. Rathinasabapathi C.A. Sigua J. Ho D.A. Gage (2000) ArticleTitleOsmoprotectant β-alanine betaine synthesis in the Plumbaginaceae: S-adenosyl-l-methionine dependent N-methylation of β-alanine to its betaine is via N-methyl and N, N-dimethyl β-alanines Physiol. Plant. 109 225–231 Occurrence Handle10.1034/j.1399-3054.2000.100302.x Occurrence Handle1:CAS:528:DC%2BD3cXkvFCrurY%3D

    Article  CAS  Google Scholar 

  • B. Rathinasabapathi W.M. Fouad C.A. Sigua (2001) ArticleTitle β-Alanine betaine synthesis in the Plumbaginaceae: purification and characterization of a trifunctional, S-adenosyl-l-methionine-dependent N-methyltransferase from Limonium latifolium leaves Plant Physiol. 126 1241–1249 Occurrence Handle10.1104/pp.126.3.1241 Occurrence Handle1:CAS:528:DC%2BD3MXlsFentb0%3D Occurrence Handle11457974

    Article  CAS  PubMed  Google Scholar 

  • D. Rhodes P.J. Rich D.G. Brunk (1989) ArticleTitleAmino acid metabolism of Lemna minor L. IV. 15N-Labeling kinetics of the amine and amino groups of glutamine and asparagines Plant Physiol. 89 1161–1171 Occurrence Handle1:CAS:528:DyaL1MXksFChsL0%3D

    CAS  Google Scholar 

  • L. Rizhsky H. Liang J. Shuman V. Shulaev S. Davletova R. Mittler (2004) ArticleTitleWhen defense pathways collide. The response of Arabidopsis to a combination of drought and heat stress Plant Physiol. 134 1683–1696 Occurrence Handle10.1104/pp.103.033431 Occurrence Handle1:CAS:528:DC%2BD2cXjsFKmsbs%3D Occurrence Handle15047901

    Article  CAS  PubMed  Google Scholar 

  • D. Rontein W.I. Wu D.R. Voelker A.D. Hanson (2003) ArticleTitleMitochondrial phosphatidylserine decarboxylase from higher plants. Functional complementation in yeast, localization in plants, and overexpression in Arabidopsis Plant Physiol. 132 1678–1687 Occurrence Handle10.1104/pp.103.023242 Occurrence Handle1:CAS:528:DC%2BD3sXlsFGgsrw%3D Occurrence Handle12857846

    Article  CAS  PubMed  Google Scholar 

  • Sambrook, J., Fritsch, E.F. and Maniatis, T. 1989. Molecular Cloning: A Laboratory Manual, 2 edn. Cold Spring Harbor Laboratory Press

  • InstitutionalAuthorNameSAS Institute (2002) SAS/Graph User’s Guide, Version 8.01 SAS Institute Cary, NC

    Google Scholar 

  • R.A. Sherwood (2001) Amino acid measurement in body fluids using PITC derivatives C. Cooper N. Packer K. Williams (Eds) Amino Acid Analysis Protocols. Methods in Molecular Biology NumberInSeriesvol. 159 Humana Press Inc Totowa, NJ 169–175

    Google Scholar 

  • M. Stitt A. Krapp (1999) ArticleTitleThe interaction between elevated carbon dioxide and nitrogen nutrition: the physiological and molecular background Plant Cell Physiol. 22 1365–3040

    Google Scholar 

  • D.R. Taub J.R. Seemann J.S. Coleman (2000) ArticleTitleGrowth in elevated CO2 protects photosynthesis against high-temperature damage Plant Cell Environ. 23 649–656 Occurrence Handle10.1046/j.1365-3040.2000.00574.x Occurrence Handle1:CAS:528:DC%2BD3cXltFWit7g%3D

    Article  CAS  Google Scholar 

  • W.H. White P.L. Gunyuzlu J.H. Toyn (2001) ArticleTitleSaccharomyces cerevisiae is capable of de nova pantothenic acid biosynthesis involving a novel pathway of β-alanine production from spermine J. Biol. Chem. 276 10794–10800 Occurrence Handle1:CAS:528:DC%2BD3MXjvFCqtb0%3D Occurrence Handle11154694

    CAS  PubMed  Google Scholar 

  • J.M. Williamson G.M. Brown (1979) ArticleTitlePurification and properties of l-aspartate-α-decarboxylase, an enzyme that catalyzes the formation of β-alanine in Escherichia coli J. Biol. Chem. 254 8074–8082 Occurrence Handle1:CAS:528:DyaE1MXlsFegtbs%3D Occurrence Handle381298

    CAS  PubMed  Google Scholar 

  • Y. Wong D.E. Taylor (1993) ArticleTitleA rapid CTAB DNA isolation technique useful for RAPD fingerprinting and other PCR applications BioTechniques 14 748–750

    Google Scholar 

  • S.D. Wullschleger T.J. Tschaplinski R.J. Norby (2002) ArticleTitlePlant water relations at elevated CO2-implications for water-limited environments Plant Cell Environ. 25 319–331 Occurrence Handle10.1046/j.1365-3040.2002.00796.x Occurrence Handle11841673

    Article  PubMed  Google Scholar 

  • B.W. Wyse W.O. Song J.H. Walsh R.G. Hansen (1985) Pantothenic acid J. Augustin B.P. Klein D. Becker P.B. Venugopal (Eds) Methods in Vitamin Assay Wiley–Interscience Publication New York 399–416

    Google Scholar 

  • H. Xiong B.A. Stanley B.L. Tekwani A.E. Pegg (1997) ArticleTitleProcessing of mammalian and plant S-adenosylmethionine decarboxylase proenzymes J. Biol. Chem. 272 28342–28348 Occurrence Handle1:CAS:528:DyaK2sXntlCru74%3D Occurrence Handle9353291

    CAS  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Plant Molecular and Cellular Biology Program, Horticultural Sciences Department, University of Florida, Hull Road, 1301 Fifield Hall, P.O. Box 110 690, Gainesville, FL, 32611-0690, USA

    Walid M. Fouad & Bala Rathinasabapathi

Authors
  1. Walid M. Fouad
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Bala Rathinasabapathi
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Bala Rathinasabapathi.

Additional information

This is Florida Agricultural Experiment Station journal series number R-10355. W.M.F. was supported by the Egypt Development Training fellowship and by the UF College of Agriculture and Life Sciences assistantship.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Fouad, W.M., Rathinasabapathi, B. Expression of Bacterial l-aspartate-α-decarboxylase in Tobacco Increases β-Alanine and Pantothenate Levels and Improves Thermotolerance. Plant Mol Biol 60, 495–505 (2006). https://doi.org/10.1007/s11103-005-4844-9

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11103-005-4844-9

Keywords

Search

Navigation