Abstract
Increased production of terpenes and many other aroma-related volatiles occurs with the onset of ripening in apple (Malus domestica Borkh.) fruit. The gaseous plant hormone ethylene plays a key role in the induction of volatile synthesis, but the mechanism is not yet understood. Using a degenerate primer based on a short conserved sequence shared by several sesquiterpene synthases, reverse transcription–polymerase chain reaction with RNA isolated from peel tissue of ‘Law Rome’ apples yielded an approx. 800-bp gene fragment. This was used to screen a cDNA library generated from the peel tissue mRNA. A full-length terpene synthase (TS) cDNA 1,931 nucleotides long was isolated. The 1,728-bp open reading frame encodes a protein 576 amino acids long with a molecular mass of 66 kDa. Sequence analysis of the apple TS showed it to be most similar to several monoterpene synthases. Oddly, the TS includes an RR(X8)W motif near the N-terminus that is common among monoterpene synthases but it lacks the plastid transit peptide sequence typically associated with genes of that group. Expression of the apple TS gene in Escherichia coli gave myc-epitope-tagged and untagged proteins estimated at approx. 68 and approx. 66 kDa, respectively. In assays of sesquiterpene synthase activity, with farnesyl diphosphate as substrate, the untagged bacterially expressed TS gene product synthesized (E,E)-α-farnesene almost exclusively. In monoterpene synthase assays, with geranyl diphosphate as substrate, the untagged apple TS produced only (E)-β-ocimene, albeit at much reduced levels. Addition of a C-terminal myc tag appeared to completely prevent production of soluble protein under all of the expression conditions tested. This is the first report of an (E,E)-α-farnesene synthase gene (AFS1; GenBank accession number AY182241) from a flowering plant. RNA gel blots showed that AFS1 transcript increased about 4-fold in peel tissue of apple fruit during the first 4 weeks of storage at 0.5°C. In contrast, when fruit were treated at harvest with 1-methylcyclopropene, a blocker of ethylene action, AFS1 mRNA declined sharply over the initial 4 weeks of cold storage, and fell to nearly undetectable levels by 8 weeks.
Similar content being viewed by others
Abbreviations
- AA :
-
Amino acid
- DPA :
-
Diphenylamine
- EI :
-
Electron impact
- FDP :
-
Farnesyl diphosphate
- FS :
-
(E,E)-α-Farnesene synthase
- GC–MS :
-
Gas chromatography–mass spectrometry
- GDP :
-
Geranyl diphosphate
- IPTG :
-
Isopropyl β-d-1-thiogalactopyranoside
- 1-MCP :
-
1-Methylcyclopropene
- ORF :
-
Open reading frame
- RACE :
-
Rapid amplification of cDNA ends
- RT–PCR :
-
Reverse transcription–polymerase chain reaction
- TS :
-
Terpene synthase
- UTR :
-
Untranslated region
References
Anet EFLJ (1970) Synthesis of (E,Z)-α-, (Z,Z)-α-, and (Z)-β-farnesene. Aust J Chem 23:2101–2108
Anet EFLJ, Coggiola IM (1974) Superficial scald, a functional disorder of stored apples. X—Control of α-farnesene autoxidation. J Sci Food Agric 25:293–298
Bengtsson M, Bäckman A-C, Liblikas I, Ramirez MI, Borg-Karlson A-K, Ansebo L, Anderson P, Löfqvist J, Witzgall P (2001) Plant odor analysis of apple: antennal response of codling moth females to apple volatiles during phenological development. J Agric Food Chem 49:3736–3741
Bohlmann J, Steele CL, Croteau R (1997) Monoterpene synthases from grand fir (Abies grandis): cDNA isolation, characterization, and functional expression of myrcene synthase, (−)-(4S)-limonene synthase, and (−)-(1S,5S)-pinene synthase. J Biol Chem 272:21784–21792
Brown DS, Buchanan JR, Hicks JR (1966) Volatiles from apple fruits as related to variety, maturity and ripeness. Proc Am Soc Hort Sci 88:99–108
Croteau R, Cane DE (1985) Monoterpene and sesquiterpene cyclases. Methods Enzymol 110:383–405
Cunningham DG, Acree TE, Barnard J, Butts R, Braell P (1986) Analysis of apple volatiles. Food Chem 19:137–147
Du Y, Poppy GM, Powell W, Pickett JA, Wadhams LJ, Woodcock CM (1998) Identification of semiochemicals released during aphid feeding that attract parasitoid Aphidius ervi. J Chem Ecol 24:1355–1368
Du Z, Bramlage WJ (1994) Roles of ethylene in the development of superficial scald in ‘Cortland’ apples. J Am Soc Hort Sci 119:516–523
Dudareva N, Martin D, Kish CM, Kolosova N, Gorenstein N, Faldt J, Miller B, Bohlmann J (2003) (E)-β-Ocimene and myrcene synthase genes of floral scent biosynthesis in snapdragon: function and expression of three terpene synthase genes of a new terpene synthase subfamily. Plant Cell 15:1227–1241
Emanuelsson O, Nielsen H, von Heijne G (1999) ChloroP, a neural network-based method for predicting chloroplast transit peptides and their cleavage sites. Protein Sci 8:978–984
Fan X, Mattheis JP, Blankenship S (1999) Development of apple superficial scald, soft core, core flush, and greasiness is reduced by MCP. J Agric Food Chem 47:3063–3068
Gong Y, Tian MS (1998) Inhibitory effect of diazocyclopentadiene on the development of superficial scald in Granny Smith apple. Plant Growth Regul 26:117–121
Hartmann M-A, Wentzinger L, Hemmerlin A, Bach TJ (2000) Metabolism of farnesyl diphosphate in tobacco BY-2 cells treated with squalestatin. Biochem Soc Trans 28:794–796
Huelin FE, Coggiola IM (1968) Superficial scald, a functional disorder of stored apples. IV—effect of variety, maturity, oiled wraps and diphenylamine on the concentration of α-farnesene in the fruit. J Sci Food Agric 19:297–301
Huelin FE, Coggiola IM (1970) Superficial scald, a functional disorder of stored apples. VII—effect of applied α-farnesene, temperature, and diphenylamine on scald and the concentration and oxidation of α-farnesene in the fruit. J Sci Food Agric 21:584–589
Huelin FE, Murray KE (1966) α-Farnesene in the natural coating of apples. Nature 210:1260–1261
Ingle M, D’Souza MC (1989) Physiology and control of superficial scald of apples: a review. HortScience 24:28–31
Ju Z, Curry E (2000) Lovastatin inhibits α-farnesene synthesis without affecting ethylene production during fruit ripening in ‘Golden Supreme’ apples. J Am Soc Hort Sci 125:105–110
Landolt PJ, Brumley JA, Smithhisler CL, Biddick LL, Hofstetter RW (2000) Apple fruit infested with codling moth are more attractive to neonate codling moth larvae and possess increased amounts of E,E-alpha farnesene. J Chem Ecol 26:1685–1699
Mattheis JP, Fellman JK, Chen PM, Patterson ME (1991) Changes in headspace volatiles during physiological development of Bisbee Delicious apple fruit. J Agric Food Chem 39:1902–1906
Meigh DF, Filmer AAE (1969) Natural skin coating of the apple and its influence on scald in storage. III. α-farnesene. J Sci Food Agric 20:139–143
Mir N, Beaudry R (1999) Effect of superficial scald suppression by diphenylamine application on volatile evolution by stored Cortland apple fruit. J Agric Food Chem 47:7–11
Mir N, Perez R, Beaudry RM (1999) A poststorage burst of 6-methyl-5-hepten-2-one (MHO) may be related to superficial scald development in Cortland apples. J Am Soc Hort Sci 124:173–176
Ohloff G, Seibl J, Kováts E (1964) Die α-Verbindungen acyclischer Monoterpene. Liebigs Ann Chem 675:83–101
Pechous SW, Whitaker BD (2002) Cloning and bacterial expression of a 3-hydroxy-3-methylglutaryl-CoA reductase cDNA (HMG1) from peel tissue of apple fruit. J Plant Physiol 159:907–916
Phillips MA, Wildung MR, Williams DC, Hyatt DC, Croteau R (2003) cDNA isolation, functional expression, and characterization of (+)-α-pinene synthase and (−)-α-pinene synthase from loblolly pine (Pinus taeda): stereocontrol in pinene biosynthesis. Arch Biochem Biophys 411:267–276
Romani R, Ku L (1966) Direct gas chromatographic analysis of volatiles produced by ripening fruit. J Food Sci 31:558–562
Rowan DD, Allen JM, Fielder S, Spicer JA, Brimble MA (1995) Identification of conjugated triene oxidation products of α-farnesene in apple skin. J Agric Food Chem 43:2040–2045
Rowan DD, Hunt MB, Fielder S, Norris J, Sherburn MS (2001) Conjugated triene oxidation products of α-farnesene induce symptoms of superficial scald on stored apples. J Agric Food Chem 49:2780–2787
Rupasinghe HPV, Paliyath G, Murr DP (1998) Biosynthesis of α-farnesene and its relation to superficial scald development in ‘Delicious’ apples. J Am Soc Hort Sci 123:882–886
Rupasinghe HPV, Murr DP, Paliyath G, Skog L (2000a) Inhibitory effect of 1-MCP on ripening and superficial scald development in ‘McIntosh’ and ‘Delicious’ apples. J Hort Sci Biotechnol 75:271–276
Rupasinghe HPV, Paliyath G, Murr DP (2000b) Sesquiterpene α-farnesene synthase: partial purification, characterization, and activity in relation to superficial scald development in apples. J Am Soc Hort Sci 125:111–119
Rupasinghe HPV, Almquist KC, Paliyath G, Murr DP (2001) Cloning of hmg1 and hmg2 cDNAs encoding 3-hydroxy-3-methylglutaryl coenzyme A reductase and their expression and activity in relation to α-farnesene synthesis in apple. Plant Physiol Biochem 39:933–947
Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning; a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory Press, Plainview, NY
Scutareanu P, Drukker B, Bruin J, Posthumus MA, Sabelis MW (1997) Volatiles from Psylla-infested pear trees and their possible involvement in attraction of anthocorid predators. J Chem Ecol 23:2241–2260
Sharon-Asa L, Shalit M, Frydman A, Bar E, Holland D, Or E, Lavi U, Lewinsohn E, Eyal Y (2003) Citrus fruit flavor and aroma biosynthesis: isolation, functional characterization, and developmental regulation of Cstps1, a key gene in the production of the sesquiterpene aroma compound valencene. Plant J 36:664–674
Smith DL, Starrett DA, Gross KC (1998) A gene coding for tomato fruit β-galactosidase II is expressed during fruit ripening−cloning, characterization, and expression pattern. Plant Physiol 117:417–423
Sutherland ORW, Hutchins RFN (1972) α-Farnesene, a natural attractant for codling moth larvae. Nature 239:170
Warburg O, Christian W (1942) Isolierung and Kristallisation des Gärungsferments Enolase. Biochem Z 310:384–424
Watkins CB, Barden CL, Bramlage WJ (1993) Relationships among α-farnesene, conjugated trienes and ethylene production with superficial scald development of apples. Acta Hort 343:155–160
Watkins CB, Nock JF, Whitaker BD (2000) Responses of early, mid, and late season apple cultivars to postharvest application of 1-methylcyclopropene (1-MCP) under air and controlled atmosphere storage conditions Postharvest Biol Technol 19:17–32
Wearing C, Hutchins RFN (1973) α-Farnesene, a naturally occurring oviposition stimulant for the codling moth Lespeyresia pomonella. J Insect Physiol 19:1251–1256
Whitaker BD (2000) DPA treatment alters α-farnesene metabolism in peel of ‘Empire’ apples stored in air or 1.5% O2 atmosphere. Postharvest Biol Technol 18:91–97
Whitaker BD, Saftner RA (2000) Temperature-dependent autoxidation of conjugated trienols from apple peel yields 6-methyl-5-hepten-2-one, a volatile implicated in induction of scald. J Agric Food Chem 48:2040–2043
Whitaker BD, Solomos T (1997) Scald prevention and reduction of α-farnesene synthesis and oxidation in ‘Granny Smith’ and ‘Empire’ apples. Proc 7th Intl Contr Atm Res Conf 2:91–97
Whitaker BD, Solomos T, Harrison DJ (1997) Quantification of α-farnesene and its conjugated trienol oxidation products from apple peel by C18-HPLC with UV detection. J Agric Food Chem 45:760–765
Whitaker BD, Solomos T, Harrison DJ (1998) Synthesis and oxidation of α-farnesene during high and low O2 storage of apple cultivars differing in scald susceptibility. Acta Hort 464:165–171
Acknowledgments
We thank Karen Green for her invaluable technical assistance and David Smith for his expert guidance and advice. Thanks also to David DeVilbiss in the Chemicals Affecting Insect Behaviour Laboratory at Beltsville for performing the GC–MS analyses. This work was funded in part by a research grant from the Washington Tree Fruit Research Commission.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Pechous, S.W., Whitaker, B.D. Cloning and functional expression of an (E,E)-α-farnesene synthase cDNA from peel tissue of apple fruit. Planta 219, 84–94 (2004). https://doi.org/10.1007/s00425-003-1191-4
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00425-003-1191-4