Valencene synthase – a biochemical magician and harbinger of transgenic aromas

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ethylene. ISR triggered by volatiles from the strain IN937 is even independent of ethylene signaling pathways. These results are different from previously established PGPRmediated ISR [20] and suggest the presence of an unknown signaling mechanism. Because the bacterial acetoin-pathway leading to the production of 2,3-B is usually triggered by low partial pressures of O2, comparable to conditions in the soil around the root area [19], it is reasonable to assume that at least some root colonizing PGPRs will generate 2,3-B and/or other bioactive volatiles at sufficient concentrations to effect plant responses. As is the case for volatiles released and acting aboveground on leaves, nothing is known about the site and the mechanism of volatile-perception. Interesting future perspectives might be seen in the selection or the genetic modification of PGPR strains to generate enhanced levels and higher release rates of plant growth-promoting volatiles or volatiles that induce systemic resistance. Also the treatment of (aerial parts of) plants with highly active but cheap compounds, such as 2,3-B, for growth-promotion and induction of ISR, might represent a novel and promising strategy in agriculture. References 1 Lynch, J.M. and Whipps, J.M. (1991) Substrate flow in the rhizosphere. In The Rhizosphere and Plant Growth (Keister, D.L. and Cregan, B., eds), pp. 15 – 24, Beltsville Sympos. in Agric. Res 14, Kluwer, Dordrecht, The Netherlands 2 Kloepper, J.W. and Schroth, M.N. (1978) Plant growth-promoting rhizobacteria on radishes. In Proc. of the 4th Int. Conf. on Plant Pathogenic Bacter (Vol. 2), pp. 879 – 882, Station de Pathologie Ve´ge´tale et Phytobacte´riologie, INRA, Angers, France 3 Bashan, Y. and Holguin, G. (1997) Proposal for the division of plant growth-promoting rhizobacteria into two classifications: biocontrolPGPG (plant growth-promoting bacteria) and PGPB. Soil Biol. Biochem. 30, 1225– 1228 4 Persello-Cartieaux, F. et al. (2003) Tales from the underground: molecular plant–rhizobia interactions. Plant Cell Environ. 26, 189–199

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5 Parker, J.E. (2003) Plant recognition of microbial patterns. Trends Plant Sci. 8, 245 – 247 6 Teplitski, M. et al. (2000) Plants secrete substances that mimic bacterial N-acyl homoserine lactone signal activities and affect population density-dependent behaviour in associated bacteria. Mol. Plant –Microbe Interact. 13, 637 – 648 7 Freiberg, C. et al. (1997) Molecular basis of symbiosis between Rhizobium and legumes. Nature 387, 394 – 401 8 Steenhoudt, O. and Vanderleyden, J. (2000) Azospirillum, a free-living nitrogen-fixing bacterium closely associated with grasses: genetic, biochemical and ecological aspects. FEMS Microbiol. Rev. 24, 487 – 506 9 Rodrı´guez, H. and Fraga, R. (1999) Phosphate solubilizing bacteria and their role in plant growth promotion. Biotechnol. Adv. 17, 319 – 339 10 Bloemberg, G.V. and Lugtenberg, B.J.J. (2001) Molecular basis of plant growth promotion and biocontrol by rhizobacteria. Curr. Opin. Plant Biol. 4, 343– 350 11 Walling, L.L. (2001) Induced resistance: from the basic to the applied. Trends Plant Sci. 6, 445– 447 12 Yanni, Y.G. et al. (2001) The beneficial plant growth-promoting association of Rhizobium leguminosarum bv. trifolii with rice roots. Aust. J. Plant Physiol. 28, 845 – 870 13 Walling, L.L. (2000) The myriad of plants responses to herbivores. J. Plant Growth Regul. 19, 195 – 216 14 Holland, M.A. (1997) Occam’s razor applied to hormonology (are cytokinins produced by plants?). Plant Physiol. 115, 865 – 868 15 Shulaev, V. et al. (1997) Airborne signalling by methyl salicylate in plant pathogen resistance. Nature 385, 718 – 721 16 Arimura, G. et al. (2000) Herbivore-induced volatiles from lima bean leaves elicit defense genes in uninfested leaves. Nature 406, 512 – 515 17 Alme´ras, E. et al. (2003) Reactive electrophile species activate defense gene expression in Arabidopsis. Plant J. 34, 205– 216 18 Ryu, C.M. et al. (2003) Bacterial volatiles promote growth in Arabidopsis. Proc. Natl. Acad. Sci. U. S. A. 100, 4927 – 4932 19 Ryu, C.M. et al. (2004) Bacterial volatiles induce systemic resistance in Arabidopsis. Plant Physiol. 134, 1017– 1026 20 Pieterse, C.M.J. et al. (2002) Signaling in rhizobacteria-induced systemic resistance in Arabidopsis thaliana. Plant Biol. 4, 535 – 544 21 Go´mez-Go´mez, L. and Boller, T. (2000) FLS2: an LRR Receptor-like Kinase involved in the perception of the bacterial elicitor flagellin in Arabidopsis. Mol. Cell 5, 1003 – 1011

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Valencene synthase – a biochemical magician and harbinger of transgenic aromas Joe Chappell Plant Physiology/Biochemistry/Molecular Biology Program, Agronomy Department, University of Kentucky, Lexington, KY 40546-0312, USA

Plants have the capacity to produce a wide array of terpene compounds – compounds that provide ecological and physiological benefit to plants. Terpenes also have important practical applications such as flavors, fragrances and medicines. Liat Sharon-Asa and colleagues have recently isolated and characterized a sesquiterpene synthase from Citrus that is providing a new window into the biochemical mechanics of these

Corresponding author: Joe Chappell ([email protected]). www.sciencedirect.com

enzymatic wizards and hints for the future metabolic engineering of terpene biosynthesis. A quick query of GenBank with the search term ‘terpene synthase’ reveals that . 100 such genes have been reported. Many of these are associated with names such as 5-epi-aristolochene synthase or amorpha-4,11-diene synthase – names that might be foreign to many plant biologists, or at least hard to pronounce. However, these names do have meaning to Natural Products chemists and biochemists because they represent the amazing potential

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of plants to generate complex and diverse arrays of chemical compounds – compounds that have stimulated exciting convergences in chemical ecology [1], biochemistry [2], physiology [3], molecular biology [4] and biotechnology research [5]. The recent article by Liat Sharon-Asa et al. [6] extends this research by reporting on the cloning and characterization of yet another terpene synthase gene – a gene that has caught the attention of many scientists in seemingly disparate fields of study for several reasons. New molecular insights from the valencene synthase gene Terpene synthases are classified according to their substrate specificities. Monoterpene synthases catalyze the biosynthesis of linear and cyclic compounds from the 10-carbon substrate geranyl diphosphate (GPP). Sesquiterpene synthases prefer the 15-carbon substrate farnesyl diphosphate (FPP). Diterpene synthases exhibit substrate specificity for the 20-carbon substrate geranylgeranyldiphosphate (GGPP). Perhaps not surprisingly, these enzymes can also be grouped according to their primary amino acid sequence alignments [2]. For instance, sesquiterpene synthases are more closely related to one another than to monoterpene synthases, and angiosperm synthases tend to cluster independently from the gymnosperm synthases. But these simple alignments do not do justice to the chemical richness catalyzed by these enzymes. Figure 1(a) represents a prediction of how FPP might be converted to valencene by valencene synthase (Cstps1), and for comparative purposes to 5-epi-aristolochene by 5-epi-aristolochene synthase (TEAS). Both enzymes are likely to generate a germacrenyl carbocation, a common intermediate for many sesquiterpene synthases. With this

common intermediate, TEAS is thought to create an internal bond forming the bicyclic structure in such a way that the methyl substituent at carbon 7 is positioned up relative to the plane of the bicyclic ring structure. Cstps1 apparently catalyzes internal bond formation in such a way that the same methyl group is oriented behind the plane of the ring structure. But this is not the only distinguishing feature differentiating these two enzymes. A final deprotonation at carbon 6 by Cstps1 results in valencene, whereas deprotonation at carbon 8 by TEAS results in 5-epi-aristolochene. Significant progress has been made during the past decade in detailing the biochemical reactions catalyzed by terpene synthases, but equally exciting has been the recent efforts to reconcile structural features of these enzyme with their catalytic magic. The structure –function relationships of terpene synthases, for example, have been advanced by combinations of crystallographic studies [2,7] and site-directed mutagenesis [8,9]. The isolation of valencene synthase contributes to another emerging trend in the field – comparative analyses. This type of approach assumes that particular structural features of the enzymes contribute to reaction specificity or partial reaction coordinates, and that sequence alignments and molecular modeling should help to tease-out these structural elements. Figure 1(b) represents a comparison of the amino acids known to line the active site of the TEAS enzyme [2] with the corresponding amino acids of Cstps1. Unexpectedly, there appears to be only a single amino acid substitution within the active site residues. This is a significant difference that changes the residue from a relatively bulky, polar charged residue (threonine) to the most benign of amino acids (glycine). However, it is difficult to imagine how this single difference could account H

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Figure 1. (a) Chemical rationalizations for the conversion of farnesyl diphosphate (FPP) to the sesquiterpene reaction products 5-epi-aristolochene and valencene, catalyzed by the tobacco enzyme 5-epi-aristolochene synthase (TEAS) and by the citrus enzyme valencene synthase (Cstps1). (b) Sequence alignment of amino acids lining the active ˚ of a substrate analog co-crystallized site of TEAS with the corresponding positions of Cstps1 reported by Sharon-Asa et al. [6]. The amino acid residues lying within 3 A within the TEAS enzyme, including residues making up the J/K loop (loop between the two a-helices), which clamps down over the active site upon substrate binding, are depicted [2]. The corresponding residues within Cstps1 were initially identified by primary sequence alignment, then visual inspection of the relevant sequences overlaid onto the TEAS 3-dimensional structure. Only 1 residue (highlighted) within the active site of Cstps1 appears to differ from those in TEAS. www.sciencedirect.com

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plastic in plants, and that plants could survive with 50% lower levels or would compensate by increasing biosynthetic rates according to some feedback regulatory mechanism [15]. In the case of engineering terpene metabolism, this would translate into a re-direction of 50% of the carbon normally dedicated to sterol biosynthesis in the cytosol, or to carotenoids in the plastids. In general terms, sterols and carotenoids accumulate to ,2–20 mg/g fresh weight in a tobacco leaf [15]. Accurate, efficient metabolic engineering should therefore result in the accumulation of 1–10 mg of new monoterpene or sesquiterpene product per g fresh weight of mature leaf tissue; levels 100 to 1000 times greater than those reported to date. Sharon-Asa et al. [6] provide two other caveats cautioning against such theoretical expectations. The first arises from their measurements of valencene accumulation by ripening citrus fruits. Valencene accumulates up to a maximum of , 60 mg per g fresh weight of flavedo tissue. This is much lower than the estimated maximum above. Second, although expression of the Cstps1 gene can be induced five- to tenfold by the exogenous application of the growth regulator ethylene, valencene accumulation was not increased much. SharonAsa and colleagues’ [6] explanation for these observations is that valencene is synthesized and accumulates in specific cells making up juice glands or sacs, and that these specialized cells or glands are probably functioning at their maximal capacity. Hence, attempts to engineer valencene metabolism into plants by simple insertion of the valencene synthase gene is not likely to be any more successful than the previous attempts. New approaches will necessarily require additional genes – genes that either up-regulate entire metabolic pathways [16] or enhance novel cellular developmental pathways that can better accommodate metabolic engineering [17].

for such different reaction chemistries (Figure 1). The work by Sharon-Asa et al. [6] has provided new impetus to theoretical comparisons of the valencene synthase gene and also provides an obvious means for testing specific structure – function relationships using conventional sitedirected mutagenesis. Metabolic prospecting Engineering terpene metabolism in plants has turned out to be much more difficult than most scientists imagined. Because monoterpenes, sesquiterpenes and diterpenes appear as relatively simple branch pathways of the mevalonate and methyl erythritol phosphate pathways (Figure 2), significant diversion of carbon to novel terpene accumulation was anticipated by engineering plants with terpene synthases – the branch-point enzymes responsible for diverting carbon flux towards mono-, sesqui- or di-terpene biosynthesis. Several attempts to create plants that accumulate novel sesquiterpenes have been disappointing. When a fungal trichodiene synthase gene was introduced into transgenic tobacco [10] and more recently when an Artemisia annua amorpha-4,11-diene synthase gene was introduced into tobacco [11], only ng quantities of the respective sesquiterpenes were detected per g fresh weight of leaf tissue. Greater, but still rather low levels, of monoterpenes have been observed in plants engineered with monoterpene synthases [5,12– 14], even though monoterpene engineering requires that the monoterpene synthase be properly targeted to the plastid compartment. The authors of these reports have proposed several theories to account for the limited success in the metabolic engineering of terpene metabolism, including limited substrate availability and stringent metabolic regulation. Hypothetically, it would seem reasonable to assume that the pools of sterol and carotenoid are somewhat

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Figure 2. A depiction of the two-terpene biosynthetic pathways known to operate in plant cells and their intracellular locations. Attempts at engineering terpene metabolism have focused on the introduction of terpene synthase genes that potentially divert pathway intermediates [geranyl diphosphate (GPP) and farnesyl diphosphate (FPP)] towards the biosynthesis of novel terpenes (red arrows). The characterization of the Cstps1 gene now provides researchers with an opportunity to engineer plants for highlevel production of valencene, a highly valued flavor and fragrance compound. Adapted from Ref. [18]. Abbreviations: GA, gibberellin; IPP, isopentenyl diphosphate; MEP, methylerythritol phosphate; PGAL, phosphoglyceraldehyde. www.sciencedirect.com

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Acknowledgements Work in my laboratory is support by grants from NSF, NIH and the Kentucky Agricultural Experiment Station.

References 1 Degenhardt, J. et al. (2003) Attracting friends to feast on foes: engineering terpene emission to make crop plants more attractive to herbivore enemies. Curr. Opin. Biotechnol. 14, 169– 176 2 Starks, C.M. et al. (1997) Structural basis for cyclic terpene biosynthesis by tobacco 5-epi-aristolochene synthase. Science 277, 1815 – 1820 3 Dudareva, N. et al. (2003) (E)-beta-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 4 Trapp, S.C. and Croteau, R.B. (2001) Genomic organization of plant terpene synthases and molecular evolutionary implications. Genetics 158, 811 – 832 5 Lu¨ker, J. et al. (2004) Increased and altered fragrance of tobacco plants after metabolic engineering using three monoterpene synthases from lemon. Plant Physiol. 134, 510 – 519 6 Sharon-Asa, L. et al. (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 7 Whittington, D.A. et al. (2002) Bornyl diphosphate synthase: structure and strategy for carbocation manipulation by a terpenoid cyclase. Proc. Natl. Acad. Sci. U. S. A. 99, 15375 – 15380 8 Segura, M.J.R. et al. (2003) Mutagenesis approaches to deduce structure – function relationships in terpene synthases. Nat. Prod. Rep. 20, 304 – 317 9 Seemann, M. et al. (2002) Pentalenene synthase. Analysis of active site

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residues by site-directed mutagenesis. J. Am. Chem. Soc. 124, 7681– 7689 Hohn, T.M. and Ohlrogge, J.B. (1991) Expression of a fungal sesquiterpene cyclase gene in transgenic tobacco. Plant Physiol. 97, 460– 462 Wallaart, T.E. et al. (2001) Amorpha-4,11-diene synthase: cloning and functional expression of a key enzyme in the biosynthetic pathway of the novel antimalarial drug artemisinin. Planta 212, 460 – 465 Lucker, J. et al. (2001) Expression of Clarkia S-linalool synthase in transgenic petunia plants results in the accumulation of S-linalylbeta-D -glucopyranoside. Plant J. 27, 315– 324 Lewinsohn, E. et al. (2001) Enhanced levels of the aroma and flavor compound S-linalool by metabolic engineering of the terpenoid pathway in tomato fruits. Plant Physiol. 127, 1256– 1265 Lavy, M. et al. (2002) Linalool and linalool oxide production in transgenic carnation flowers expressing the Clarkia breweri linalool synthase gene. Mol. Breed. 9, 103– 111 Chappell, J. et al. (1995) Is the reaction catalyzed by 3-hydroxy-3methylglutaryl coenzyme-A reductase a rate-limiting step for isoprenoid biosynthesis in plants. Plant Physiol. 109, 1337– 1343 Lloyd, A.M. et al. (1992) Arabidopsis and Nicotiana anthocyanin production activated by maize regulator-R and regulator-C1. Science 258, 1773– 1775 Payne, T. et al. (1999) Heterologous myb genes distinct from GL1 enhance trichome production when overexpressed in Nicotiana tabacum. Development 126, 671 – 682 Chappell, J. (2002) The genetics and molecular genetics of terpene and sterol origami. Curr. Opin. Plant Biol. 5, 151 – 157

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