Skip to main content
SpringerLink
Log in

Functional Characterization of a Melon Alcohol Acyl-transferase Gene Family Involved in the Biosynthesis of Ester Volatiles. Identification of the Crucial Role of a Threonine Residue for Enzyme Activity*

  • Published:
Plant Molecular Biology Aims and scope Submit manuscript

Abstract

Volatile esters, a major class of compounds contributing to the aroma of many fruit, are synthesized by alcohol acyl-transferases (AAT). We demonstrate here that, in Charentais melon (Cucumis melo var. cantalupensis), AAT are encoded by a gene family of at least four members with amino acid identity ranging from 84% (Cm-AAT1/Cm-AAT2) and 58% (Cm-AAT1/Cm-AAT3) to only 22% (Cm-AAT1/Cm-AAT4). All encoded proteins, except Cm-AAT2, were enzymatically active upon expression in yeast and show differential substrate preferences. Cm-AAT1 protein produces a wide range of short and long-chain acyl esters but has strong preference for the formation of E-2-hexenyl acetate and hexyl hexanoate. Cm-AAT3 also accepts a wide range of substrates but with very strong preference for producing benzyl acetate. Cm-AAT4 is almost exclusively devoted to the formation of acetates, with strong preference for cinnamoyl acetate. Site directed mutagenesis demonstrated that the failure of Cm-AAT2 to produce volatile esters is related to the presence of a 268-alanine residue instead of threonine as in all active AAT proteins. Mutating 268-A into 268-T of Cm-AAT2 restored enzyme activity, while mutating 268-T into 268-A abolished activity of Cm-AAT1. Activities of all three proteins measured with the prefered substrates sharply increase during fruit ripening. The expression of all Cm-AAT genes is up-regulated during ripening and inhibited in antisense ACC oxidase melons and in fruit treated with the ethylene antagonist 1-methylcyclopropene (1-MCP), indicating a positive regulation by ethylene. The data presented in this work suggest that the multiplicity of AAT genes accounts for the great diversity of esters formed in melon.

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. Aggelis I. John Z. Karvouny D. Grierson (1997) ArticleTitleCharacterization of two cDNA clones for mRNAs expressed during ripening of melon (Cucumis melo L.) fruits Plant Mol. Biol. 33 313–322 Occurrence Handle10.1023/A:1005701730598 Occurrence Handle9037149

    Article  PubMed  Google Scholar 

  • A. Aharoni L.C.P. Keizer H.J. Bouwmeester Z. Sun M. Alvarez-Huerta H.A. Verhoeven J. Blass A.M.M.L. Houwelingen ParticleVan R.C.H. Vos ParticleDe H. Voet ParticleVan der R.C. Jansen M. Guis J. Mol R.W. Davis M. Schena A.J. Tunen ParticleVan A.P. O’Connell (2000) ArticleTitleIdentification of the SAAT gene involved in strawberry flavor biogenesis by use of DNA microarrays Plant Cell 12 647–661 Occurrence Handle10.1105/tpc.12.5.647 Occurrence Handle10810141

    Article  PubMed  Google Scholar 

  • S.F. Altschul T.L. Madden A.A. Schaffer J. Zhang Z. Zhang W. Miller D.J. Lipman (1997) ArticleTitleGapped BLAST and PSI-BLAST: a new generation of protein database search programs Nucleic Acids Res. 25 3389–3402 Occurrence Handle10.1093/nar/25.17.3389 Occurrence Handle9254694

    Article  PubMed  Google Scholar 

  • C. Aubert N. Bourger (2004) ArticleTitleInvestigation of volatiles in Charentais Cantaloupe melons (Cucumis melo var Cantalupensis) Characterization of aroma constituents in some cultivars J. Agric. Food Chem. 52 4522–4528 Occurrence Handle10.1021/jf049777s Occurrence Handle15237961

    Article  PubMed  Google Scholar 

  • R. Ayub M. Guis M. Ben Amor L. Gillot J.P. Roustan A. Latché M. Bouzayen J.C. Pech (1996) ArticleTitleExpression of ACC oxidase antisense gene inhibits ripening of cantaloupe melon fruits Nat. Biotech. 14 862–866 Occurrence Handle10.1038/nbt0796-862

    Article  Google Scholar 

  • A.-D. Bauchot D.S. Mottram A.T. Dodson P. John (1998) ArticleTitleEffect of aminocyclopropane-1-carboxylic acid oxidase antisense gene on the formation of volatile esters in Cantaloupe Charentais melon (cv. Védrantais) J. Agric. Food Chem. 46 4787–4792 Occurrence Handle10.1021/jf980692z

    Article  Google Scholar 

  • R.M. Beaudry (1999) ArticleTitleEffect of O2 and CO2 partial pressure on selected phenomena affecting fruit and vegetable quality Postharv. Biol. Technol. 15 293–303 Occurrence Handle10.1016/S0925-5214(98)00092-1

    Article  Google Scholar 

  • J.C. Beaulieu C. Grimm (2001) ArticleTitleIdentification of volatile compounds in cantaloupe at various developmental stages using solid phase microextraction J. Agric. Food Chem. 49 1345–1352 Occurrence Handle10.1021/jf0005768 Occurrence Handle11312862

    Article  PubMed  Google Scholar 

  • J. Beekwilder M. Alavarez-Huerta E. Neef F.W.A. Verstappen H.J. Bouwmeester A. Aharoni (2004) ArticleTitleFunctional characterization of enzymes forming volatile esters from strawberry and banana Plant Physiol. 135 1865–1878 Occurrence Handle10.1104/pp.104.042580 Occurrence Handle15326278

    Article  PubMed  Google Scholar 

  • M.M. Bradford (1976) ArticleTitleA rapid and sensitive method for the quantitation of microgram quantities of protein using the principle of protein-dye binding Anal. Biochem. 72 248–254 Occurrence Handle942051

    PubMed  Google Scholar 

  • P.K. Boss C. Davies S.P. Robinson (1996) ArticleTitleAnalysis of the expression os anthocyanin pathway genes in developing Vitis vinifera cv. Shiraz grape berries and the implications for pathway regulation Plant Physiol. 111 1059–1066 Occurrence Handle12226348

    PubMed  Google Scholar 

  • A.M. Dandekar G. Teo B.G. Defilippi S. Uratsu A.J. Passey A.A. Kader J.R. Stow R.J. Colgan D.J. Jazmes (2004) ArticleTitleEffect of down-regulation of ethylene biosynthesis on fruit flavor complex in apple fruit Transgenic Res. 13 373–384 Occurrence Handle10.1023/B:TRAG.0000040037.90435.45 Occurrence Handle15517996

    Article  PubMed  Google Scholar 

  • J. D’Auria F. Chen E. Pichersky (2002) ArticleTitleCharacterization of an acyltransferase capable of synthesizing benzylbenzoate and other volatile esters in flowers and damaged leaves of Clarkia breweri Plant Physiol. 130 466–476 Occurrence Handle10.1104/pp.006460 Occurrence Handle12226525

    Article  PubMed  Google Scholar 

  • B.G. Defilippi A.M. Dandekar A.A. Kader (2004) ArticleTitleImpact of suppression of ethylene action or biosynthesis on flavor metabolites in apple (Malus domestica Borkh) fruits J. Agr. Food Chem. 52 5694–5701

    Google Scholar 

  • B.G. Defilippi A.A. Kader A.M. Dandekar (2005) ArticleTitleApple aroma: alcohol acyltransferase, a rate limiting step for ester biosynthesis, is regulated by ethylene Plant Sci. 168 1199–1210 Occurrence Handle10.1016/j.plantsci.2004.12.018

    Article  Google Scholar 

  • N. Dudareva J.C. D’Auria K.H. Nam R.A. Raguso E. Pichersky (1998) ArticleTitleAcetyl-CoA: benzylalcohol acetyltransferase – an enzyme involved in floral scent production in Clarkia breweri Plant J. 14 297–304 Occurrence Handle10.1046/j.1365-313X.1998.00121.x Occurrence Handle9628024

    Article  PubMed  Google Scholar 

  • N. Dudareva E. Pichersky (2000) ArticleTitleBiochemical and molecular genetic aspects of floral scents Plant Physiol. 122 627–633 Occurrence Handle10.1104/pp.122.3.627 Occurrence Handle10712525

    Article  PubMed  Google Scholar 

  • N. Dudareva E. Pichersky J. Gershenzon (2004) ArticleTitleBiochemistry of plant volatiles Plant Physiol. 135 1893–1902 Occurrence Handle10.1104/pp.104.049981 Occurrence Handle15326281

    Article  PubMed  Google Scholar 

  • S. Elss C. Preston C. Hertzig F. Heckel E. Richling P. Schreier (2005) ArticleTitleAroma profiles of pineapple fruit (Ananas comosus L., Merr.) and pineapple products Lebensm-Wissu-Technol. 38 263–274

    Google Scholar 

  • X. Fan J. Mattheis D. Buchanan (1998) ArticleTitleContinuous requirement of ethylene for apple fruit volatile synthesis J. Agric. Food Chem. 46 1959–1963 Occurrence Handle10.1021/jf970877q

    Article  Google Scholar 

  • F. Flores F. El Yahyaoui G. Billerbeck ParticleDe F. Romojaro A. Latché M. Bouzayen J.C. Pech C. Ambid (2002) ArticleTitleRole of ethylene in the biosynthetic pathway of aliphatic ester aroma volatiles in Charentais Cantaloupe melons J. Exp. Bot. 53 201–206 Occurrence Handle10.1093/jexbot/53.367.201 Occurrence Handle11807123

    Article  PubMed  Google Scholar 

  • M. Guis R. Botondi M. Ben Amor R. Ayub M. Bouzayen J.C. Pech A Latché (1997) ArticleTitleRipening-associated biochemical traits of cantaloupe charentais melons expressing an antisense ACC oxidase transgene J. Amer. Soc. Hort. Sci. 122 748–751

    Google Scholar 

  • F. Jeanmougin J.D. Thompson M. Gouy D.G. Higgins T.J. Gibson (1998) ArticleTitleMultiple sequence alignment with Clustal X Trends Biochem. Sci. 23 403–405 Occurrence Handle10.1016/S0968-0004(98)01285-7 Occurrence Handle9810230

    Article  PubMed  Google Scholar 

  • M. Harada Y. Ueda T. Iwata (1985) ArticleTitlePurification and some properties of alcohol acyltransferase from banana fruit Plant Cell Physiol. 26 1067–1074

    Google Scholar 

  • V. Homatidou S. Karvouni V. Dourtouglou C. Poulos (1992) ArticleTitleDetermination of total volatile components of Cucumis melo L. Variety Cantaloupensis J. Agric. Food Chem. 40 1385–1388 Occurrence Handle10.1021/jf00020a019

    Article  Google Scholar 

  • S. Lurie C. Pre-Aymard U. Ravid O. Larkov E. Fallik (2002) ArticleTitleEffect of 1-methylcyclopropene on volatile emission and aroma in cv Anna apples J. Agr. Food Chem. 50 4251–4256 Occurrence Handle10.1021/jf0200873

    Article  Google Scholar 

  • X. Ma J. Koepke S. Panjikar G. Fritzch J. Stöckigt (2005) ArticleTitleCrystal structure of vinorine synthase, the first representative of the BAHD superfamily J. Biol. Chem. 280 13576–13583 Occurrence Handle10.1074/jbc.M414508200 Occurrence Handle15665331

    Article  PubMed  Google Scholar 

  • J.P. Mattheis J.K. Fellman (1999) ArticleTitlePre-harvest factors influencing flavor of fresh fruit and vegetables Postharv. Biol. Technol. 15 227–232 Occurrence Handle10.1016/S0925-5214(98)00087-8

    Article  Google Scholar 

  • R. Medina-Suarez K. Manning J. Fletcher J. Aked C.R. Bird G. Seymour (1997) ArticleTitleGene expression in the pulp of ripening bananas. Two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis of in vitro translation products and cDNA cloning of 25 different ripening-related mRNAs Plant Physiol. 115 453–461 Occurrence Handle10.1104/pp.115.2.453 Occurrence Handle9342865

    Article  PubMed  Google Scholar 

  • N.A. Mir R. Perez P. Schwallier R. Beaudry (1999) ArticleTitleRelationship between ethylene response manipulation and volatile production in Jonagold variety apples J. Agric. Food Chem. 47 2653–2659 Occurrence Handle10.1021/jf981150k Occurrence Handle10552540

    Article  PubMed  Google Scholar 

  • Y.W. Nam L. Tichit M. Leperlier B. Cuerq I. Marty J.M. Lelièvre (1999) ArticleTitleIsolation and characterization of mRNAs differentially expressed during ripening of wild strawberry (Fragaria vesca L.) fruits Plant Mol. Biol. 39 629–636 Occurrence Handle10.1023/A:1006179928312 Occurrence Handle10092188

    Article  PubMed  Google Scholar 

  • Nicholas , K.B. and Nicholas H.B.J. 1997. GENEDOC: a tool for editing and annotating multiple sequence alignments, distributed by the authors

  • R.D.M. Page (1996) ArticleTitleTREE VIEW: an application to display phylogenic trees on personal computers Comput. Appl. Biosci. 12 357–358 Occurrence Handle8902363

    PubMed  Google Scholar 

  • N.M.M. Paillard (1990) The flavour of apples, pears and quinces I.D. Morton A.J. McLeod (Eds) Food Flavours, PartC. The Flavour of Fruit Elsevier Amsterdam, The Netherlands 1–41

    Google Scholar 

  • A.G. Perez C. Sanz R. Olías J.J. Ríos J.M. Olías (1996) ArticleTitleEvolution of strawberry alcohol acyltransferase activity during fruit development and storage J. Agric. Food Chem. 44 3286–3290 Occurrence Handle10.1021/jf960040f

    Article  Google Scholar 

  • E. Pichersky D.R. Gang (2000) ArticleTitleGenetics and biochemistry of secondary metabolites in plants: an evolutionary perspective Trends Plant Sci. 5 439–445 Occurrence Handle10.1016/S1360-1385(00)01741-6 Occurrence Handle11044721

    Article  PubMed  Google Scholar 

  • B. St Pierre P. Laflamme A.M. Alarco V. Luca ParticleDe (1998) ArticleTitleThe terminal O-acetyltransferase involved in vindoline biosynthesis defines a new class of proteins responsible for co-enzyme A-dependent acyl transfer Plant J. 14 703–713 Occurrence Handle10.1046/j.1365-313x.1998.00174.x Occurrence Handle9681034

    Article  PubMed  Google Scholar 

  • B. St Pierre V. De Luca (2000) Evolution of acyltranferase genes: origin and diversification of the BAHD superfamily of acyltransferases involved in secondary metabolism R.I. John R. Romeo L. Varin V. Luca ParticleDe (Eds) Recent Advances in Phytochemistry. Evolution of Metabolic Pathways Elsevier Science Publishing Oxford 285–315

    Google Scholar 

  • C. Sanz J.M. Olias A.G. Perez (1997) Aroma biochemistry of fruit and vegetables F.A. Tomas-Barberan R.J. Robins (Eds) Phytochemistry of Fruit and Vegetables Oxford Science Pub Oxford, UK 313–329

    Google Scholar 

  • M. Shalit N. Katzir Y. Tadmor O. Larkov Y. Burger F. Schalechet E. Lastochkin U. Ravid O. Amar M. Edelstein Z. Karchi E. Lewinsohn (2001) ArticleTitleAcetyl-CoA: alcohol acetyl transferase activity and aroma formation in ripening melon fruits J. Agric. Food Chem. 49 794–799 Occurrence Handle10.1021/jf001075p Occurrence Handle11262031

    Article  PubMed  Google Scholar 

  • M. Shalit I. Guterman H. Volpin E. Bar T. Tamari N. Menda Z. Adam D. Zamir A. Vainstein D. Weiss E. Pichersky E. Lewinsohn (2003) ArticleTitleVolatile ester formation in roses. Identification of an acetyl-coenzyme A geraniol/citronellol° acetyl transferase in developing rose petals Plant Physiol. 131 1868–1876 Occurrence Handle10.1104/pp.102.018572 Occurrence Handle12692346

    Article  PubMed  Google Scholar 

  • H. Shiota (1993) ArticleTitleNew esteric components in the volatiles of banana fruit (Musa sapientum L.) J. Agric. Food Chem. 41 2056–2062 Occurrence Handle10.1021/jf00035a046

    Article  Google Scholar 

  • J. Song F. Bangerth (1996) ArticleTitleThe effect of harvest date on aroma compound production from ‘Golden Delicious’ apple fruit and relationship to respiration and ethylene production Postharv. Biol. Technol. 8 259–269 Occurrence Handle10.1016/0925-5214(96)00020-8

    Article  Google Scholar 

  • Y. Wang S.G. Wyllie D.N. Leach (1996) ArticleTitleChemical changes during the development and ripening of the fruit of Cucumis melo (cv Makdimon) J. Agric. Food Chem. 44 210–216l Occurrence Handle10.1021/jf9503568

    Article  Google Scholar 

  • S.G. Wyllie J.K. Fellmann (2000) ArticleTitleFormation of volatile branched chain esters in bananas (Musa sapientum L.) J. Agric. Food Chem. 48 3493–3496 Occurrence Handle10.1021/jf0001841 Occurrence Handle10956138

    Article  PubMed  Google Scholar 

  • F. Yahyaoui C. Wongs-Aree A. Latché R. Hackett D. Grierson J.C. Pech (2002) ArticleTitleMolecular and biochemical characteristics of a gene encoding an alcohol acyl-transferase involved in the generation of aroma volatile esters during melon ripening Eur. J. Biochem. 269 2359–2366 Occurrence Handle10.1046/j.1432-1033.2002.02892.x Occurrence Handle11985619

    Article  PubMed  Google Scholar 

  • H. Yoshimoto D. Fujiwara T. Momma K. Tanaka H. Sone N. Nagasawa Y. Tamai (1999) ArticleTitleIsolation and characterization of the ATF2 gene encoding alcohol acyltransferase II in the bottom fermenting yeast Saccharomyces pastorius Yeast 15 409–417 Occurrence Handle10.1002/(SICI)1097-0061(19990330)15:5<409::AID-YEA366>3.3.CO;2-H Occurrence Handle10219999

    Article  PubMed  Google Scholar 

  • I. Zabetakis M.A. Holden (1997) ArticleTitleStrawberry flavour: analysis and biosynthesis J. Sci. Food Agric. 74 421–434 Occurrence Handle10.1002/(SICI)1097-0010(199708)74:4<421::AID-JSFA817>3.3.CO;2-Y

    Article  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. UMR 990 INRA/INPT-ENSAT “Génomique et Biotechnologie des Fruits”, Av. de l’Agrobiopole, BP 32607, F-31326, Castanet-Tolosan Cedex, France

    Islam El-Sharkawy, Daniel Manríquez, Francisco B. Flores, Farid Regad, Mondher Bouzayen, Alain Latché & Jean-Claude Pech

Authors
  1. Islam El-Sharkawy
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Daniel Manríquez
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Francisco B. Flores
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Farid Regad
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Mondher Bouzayen
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Alain Latché
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Jean-Claude Pech
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Jean-Claude Pech.

Additional information

*Accession numbers Cm-AAT1 (CAA94432), Cm-AAT2 (AAL77060), Cm-AAT3 (AAW51125), and Cm-AAT4 (AAW51126).

Islam El-Sharkawy, Daniel Manriquez, Francisco B. Flores: These authors contributed equally to the work

Rights and permissions

Reprints and permissions

About this article

Cite this article

El-Sharkawy, I., Manríquez, D., Flores, F.B. et al. Functional Characterization of a Melon Alcohol Acyl-transferase Gene Family Involved in the Biosynthesis of Ester Volatiles. Identification of the Crucial Role of a Threonine Residue for Enzyme Activity*. Plant Mol Biol 59, 345–362 (2005). https://doi.org/10.1007/s11103-005-8884-y

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11103-005-8884-y

Keywords

Search

Navigation