Isolation and Characterization of Two Germacrene A Synthase cDNA Clones from Chicory1 Harro J. Bouwmeester*, Jan Kodde, Francel W.A. Verstappen, Iris G. Altug, Jan-Willem de Kraker, and T. Eelco Wallaart2 Plant Research International, Business Unit Cell Cybernetics, P.O. Box 16, 6700 AA Wageningen, The Netherlands (H.J.B., J.K., F.W.A.V.); Department of Organic Chemistry, Hamburg University, D–20146 Hamburg, Germany (I.G.A.); Department of Organic Chemistry, Wageningen Agricultural University, Dreijenplein 8, 6703 HB Wageningen, The Netherlands (J.-W.d.K.); and University Centre for Pharmacy, Department of Pharmaceutical Biology, University of Groningen, Antonius Deusinglaan 1, 9713 AV Groningen, The Netherlands (T.E.W.) Chicory (Cichorium intybus) sesquiterpene lactones were recently shown to be derived from a common sesquiterpene intermediate, (ϩ)-germacrene A. Germacrene A is of interest because of its key role in sesquiterpene lactone biosynthesis and because it is an enzyme-bound intermediate in the biosynthesis of a number of phytoalexins. Using polymerase chain reaction with degenerate primers, we have isolated two sesquiterpene synthases from chicory that exhibited 72% amino acid identity. Heterologous expression of the genes in Escherichia coli has shown that they both catalyze exclusively the formation of (ϩ)-germacrene A, making this the first report, to our knowledge, on the isolation of (ϩ)-germacrene A synthase (GAS)-encoding genes. Northern analysis demonstrated that both genes were expressed in all chicory tissues tested albeit at varying levels. Protein isolation and partial purification from chicory heads demonstrated the presence of two GAS proteins. On MonoQ, these proteins co-eluted with the two heterologously produced proteins. The Km value, pH optimum, and MonoQ elution volume of one of the proteins produced in E. coli were similar to the values reported for the GAS protein that was recently purified from chicory roots. Finally, the two deduced amino acid sequences were modeled, and the resulting protein models were compared with the crystal structure of tobacco (Nicotiana tabacum)5-epi-aristolochene synthase, which forms germacrene A as an enzyme-bound intermediate en route to 5-epi-aristolochene. The possible involvement of a number of amino acids in sesquiterpene synthase product specificity is discussed. The chicory (Cichorium intybus) plant contains bit- al., 1990). Several of these sesquiterpene lactones ter sesquiterpene lactones, such as lactucin, 8-deoxy- such as tenulin (from Helenium amarum), helenalin lactucin, and lactupicrin, in most of its organs e.g. (from sneezeweed, Helenium autumnale), and parthe- (tap) roots, leaves, and stems and also in the etiolated nin (from Parthenium histerophorus) have been de- heads, which are eaten as a vegetable in some parts of scribed as having anti-feedant activity on herbivo- the world (Rees and Harborne, 1985; Beek et al., 1990; rous insects and vertebrate herbivores (Picman, Price et al., 1990). These sesquiterpene lactones were 1986). In addition, many sesquiterpene lactones were shown to have significant anti-feedant activity (Rees shown to possess pharmacological activities. For ex- and Harborne, 1985). In addition, Monde et al. (1990) ample, parthenolide from feverfew (Tanacetum demonstrated the induction of an anti-fungal gua- parthenium) has an anti-migraine effect (Hewlett et ianolide sesquiterpene lactone in chicory upon infec- al., 1996). Finally, anti-fungal, anti-bacterial, anti- tion with Pseudomonas cichorii. Other composite plant protozoan, schistomicidal, and molluscicidal activi- species such as lettuce (Lactuca salva and Lactuca sa- ties have been reported for many sesquiterpene lac- tiva), radicchio (Cichorium intybus), endive (Cichorium tones (Picman, 1986). endiva), and artichoke (Cynara scolymus) have been de Kraker et al. (1998, 2001, 2002) showed that the sesquiterpene lactones in chicory and probably also demonstrated to contain similar sesquiterpene lac- in a large number of other plant species originate tones as bitter constituents (Herrmann, 1978; Price et from a common germacrane precursor, (ϩ)- germacrene A. The biosynthesis of this sesquiterpene 1 This work was supported in part by Nunhems Zaden BV and olefin from the ubiquitous sesquiterpene precursor the R&D Subsidy for Technological Co-operation (project BTS farnesyl diphosphate (FDP) is catalyzed by a (ϩ)- 97102; to H.J.B., F.W.A.V., and J.K.). germacrene A synthase (GAS; Fig. 1). In a number of * Corresponding author; e-mail [email protected] additional steps, the germacrene A precursor is oxi- ur.nl; fax 0031–317–418094. 2 Present address: GenoClipp Biotechnology B.V., Meditech dized into germacrene A carboxylic acid (de Kraker Center, L.J. Zielstraweg 1, 9713 GX, Groningen, The Netherlands. et al., 2001) that is further oxidized to produce the Article, publication date, and citation information can be found lactone ring (de Kraker et al., 2002). This is then at www.plantphysiol.org/cgi/doi/10.1104/pp.001024. further functionalized and/or cyclized to the respec- 134 Plant PhysiologyDownloaded, May 2002, from Vol. on 129,January pp. 134–144,13, 2020 www.plantphysiol.org- Published by www.plantphysiol.org © 2002 American Society of Plant Biologists Copyright © 2002 American Society of Plant Biologists. All rights reserved. Isolation of Two Germacrene A Synthase cDNAs from Chicory the expected length of about 550 bp were obtained. Sequencing of both fragments revealed homology to known sesquiterpene synthases present in public da- tabases. We subsequently used both fragments as probes for cDNA library screening. This resulted in the isolation of two different, full-length cDNAs CiGASsh and CiGASlo containing a putative open reading frame of 1,674 (558 amino acids; hence, sh for short) and 1,749 bp (583 amino acids; hence, lo for long; Fig. 2). CiGASsh encodes a protein of 64.4 kD with a calculated pI of 4.89. CiGASlo encodes a pro- tein of 67.1 kD with a calculated pI of 5.19. The two sequences exhibited 72% identity on the deduced Figure 1. Biosynthetic pathway of sesquiterpene lactones in chicory. amino acid level. Both genes exhibited highest ho- Solid arrows indicate enzymatic steps previously demonstrated (de mology with the (ϩ)-␦-cadinene synthases from Gos- Kraker et al., 1998, 2001, 2002). 1, GAS; 2, germacrene A hydrox- sypium arboreum (among others Q39760, Q39761, and ylase, 3, germacrene A alcohol dehydrogenase(s); 4, costunolide O49853) and cotton (Gossypium hirsutum; P93665), the synthase; 5, further modifications. Broken arrows indicate postulated potato (Solanum tuberosum) vetispiradiene synthase further steps (de Kraker et al., 2002). (AAD02223), and the tobacco (Nicotiana tabacum; T03714) and pepper (AJ005588) 5-epi-aristolochene synthases. tive guaianolide, eudesmanolide, and germacranol- The catalytic activity of the two encoded proteins ide sesquiterpene lactones (Fig. 1; de Kraker et al., was examined using an enzyme assay on a cell-free 2002). The work by de Kraker et al. on the biosyn- extract of Escherichia coli BL 21 (DE3) harboring the thesis of sesquiterpene lactones was carried out using two different cDNAs in the pET 11d vector. Radio- gas liquid chromatography (radio-GLC) showed that chicory taproots and, so far, little is known about the 3 activity of the GAS in other plant organs or about its both extracts catalyzed the conversion of [ H]FDP to genetic regulation. a radiolabeled product co-eluting with germacrene A In addition to being an intermediate in sesquiter- (Fig. 3). A cell-free extract of E. coli BL 21 (DE3) pene lactone biosynthesis, germacrene A is in itself harboring an empty vector did not produce any apo- an important compound. For a long time, its detec- lar radiolabeled products. GC-mass spectroscopy tion in some systems escaped attention because of its (GC-MS) analysis showed that retention times (not rather high sensitivity to temperature and acidic con- shown) and mass spectra (Fig. 3) of the major peak ditions (de Kraker et al., 1998). However, (Ϫ)- were identical to those of an authentic standard of germacrene A has been identified as the alarm pher- germacrene A, thus, confirming that both cDNAs omone in spotted alfalfa (Medicago sativa) aphids encode a GAS. (Nishino et al., 1977). An unidentified enantiomer of Finally, the possibility was checked that the two germacrene A has been identified as an important enzymes catalyze the formation of two different constituent of spider mite induced volatiles in sweet enantiomers of germacrene A. This was done by pepper (Capsicum annuum; C. van de Boom, T.A. van GC-MS analysis using an enantioselective column in Beek, and M. Dicke, unpublished data). Germacrene combination with the principle of (stereoselective) heat-induced rearrangement of germacrene A to A has also been demonstrated to be an (enzyme- ␤ bound) intermediate in the biosynthesis of 5-epi- -elemene (de Kraker et al., 1998). At an injection aristolochene and vetispiradiene, which are the ses- port temperature of 150°C, germacrene A was the major product of both the short and the long pro- quiterpene precursors of phytoalexins such as ␣ ␤ capsidiol and debneyol (Whitehead et al., 1989). Be- tein. Small amounts of -selinene, -selinene, and cause of the importance of germacrene A both as an selina-4,11-diene, which are proton-induced rear- intermediate and as end product in many plant- rangement products (i.e. they are not produced en- organism interactions, we decided to clone and char- zymatically)