ADVANCES IN EXPERIMEN TAL MEDICINE AND BIOLOGY . Volume 464 FUE LL ee aa s0]| (OH OVA 21110.) RS IAIE (0 F Paul Kolodziejezyk, John Re Whitaker, Agustin Lopez Munguia, EUORGl NaN me] osCHEMICALS VIA HIGHER PLANT BIOENGINEERING Edited by Fereidoon Shahidi Memorial University of Newfoundland St, John, Newfoundland, Canada Paul Kolodziejezyk POS Pilot Plant Corporation Saskatoon, Saskatchewan, Canada John R. Whitaker University of California Davis, California Agustin Lopez Munguia Institute of Biotechnology-UNAM Cuernavaca, Morelos, Mexico and Glenn Fuller USDA/ARS Albany, California Kluwer Academic/Plenum Publishers New York, Boston, Dordrecht, London, MoscowBased on proceedings of a symposium on Chemicals via Higher Plant Bioengineering, held at the Sth North American Chemical Congress, November I1-15, 1997, in Cancun, Mexico ISBN 0-306-46117-X ©1999 Kluwer Academic/Plenum Publishers, New York 233 Spring Street, New York, N.Y. 10013 0987654321 ACL. record for this book is available from the Library of Congress Al rights reserved No part of this book may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, microfilming, recording, or otherwise, without written permission from the Publisher Printed in the United States of AmericaBIOCHEMICAL AND MOLECULAR TOOLS FOR THE PRODUCTION OF USEFUL TERPENE PRODUCTS FROM PEPPER (C4PSICUM ANNUUM) Edmundo Lozoya-Gloria Genetic Engineering Department CINVESTAV-IPN / Irapuato Unit P.O. Box 629, C.P. 36500 Irapuato, Gto., México ABSTRACT Among other natural products such as colorants and flavorants, natural fungicides like the pepper phytoalexin capsidiol, and the related biochemical pathways, may be used for practical approaches. Key enzymes such as 3-hydroxy-3-methylglutaryl Coenzyme A: reductase, the farnesyl pyrophosphate synthase and and farnesyl pyrophosphate cyclases are known and some related genes have been isolated. However, specific enzymes for im- portant and final modifications as methylation and others, are still to be studied. Construc- tion of chimeric enzymes allowed already the synthesis of different products and the possibilities of designing new enzymes by gene manipulation to produce unknown and useful chemicals are open. INTRODUCTION Pepper (Capsicum annuum) fruits have been traditionally used for the production of natural color (carotenoids) and pungent flavor (capsaicinoids), either directly as the fruit itself or after processing (Govindarajan, 1986). However, there are many other pepper products (Table 1) with interesting biological effects and most of them have been identi- fied in fruits. Other pepper tissues may also produce chemicals according to the role these products play in the plant physiology (Rhodes, 1994), Chemicals via Higher Plant Bioengineering, edited by Shahidi et al. Kluwer Academic / Plenum Publishers, New York, 1999. 63E, Lozoya-Gloria ans NM Nn ‘ordanopeuodique ‘nynue ‘oruapanue ‘s1uaBoursreanue uesarsenuE ye ‘yso8peue ‘oruo8i9qTe SOMO “aangoioidoreday ‘ondodeuouua ‘aqoydiyonsg ‘o1ydoneo Kwhue ‘oLnunssoowoynue ‘snunseBnUE Iwdr>esTHOgE any ‘aurer9q Tr rr SORT SPT TORE UTR a ar ‘sanuoxaud-souro ‘onneisordnue ‘oneseownue ‘oequouswUe ‘ousmOyna|NUE “KuoeUUTEyUUE “ueZaTFeNUE amy pise-o1uajouty-0 PUTA TUPI ay TTD a ir prrc280808 ‘samtuisuenosnau “squor0 Kur ‘odour “tueroenuoooKur ‘onotut‘aatsua}odsy eos “on o stpmaypenue snaue‘naampaueadogyquegoe ose re TENSOPONT Sarr TesTUrsgy (.deddag [J9¢,, aeaseunjog) “] wnuue umnoisdes Ut sontANoe peorTOjOIg s1OKp pure S|EOHUOYD “T IGRI,E. Lozoya-Gloria ‘ qa\ 247 e oxgeyteAe axe saouasojas oy190dg ‘s}8u~/n08'wH8-sxe-uns/:dny ‘ous GOAN ayng “y sours pur roquinig-wonsyoog "Ww uaydars TRION - SUV - VaSN - asequieg ooauIayoOrKYd saseqeied [eotueioqoutpe pue peoMYyDOIKYd "9IAIES YamRAsaY [eMYNoUaY Woy pardepy ia ‘oproiquoy ‘aprorSuny ‘anamrp ‘snmsouqoeg ‘oprouaiseq ‘2arssmnue ‘ondosnue ‘oneutpsEAUe ‘OIZu2|feNUE “SHTBSOTEIST"SaTFIOTOTCOTECAT ‘oprorBuny ‘onrowondo ‘oandssezitoo “ounynue ‘s1waBoreunadsnu ‘Asoreuumelsuanue ‘oquaBoxpuENTe Kody ‘aanuaaaud-re0ueo “TuE|NUINS-SNO ‘sOWMAUE neUASENUE ‘Orso yeUR ‘OHUDYDOTDTE “apiouny ‘soxey ‘aprouatong ‘weprxonue ‘aandaorounue ‘axndaorsounwe‘sruaBeynuunue“o|saBjeue“a.uadLaq|e any uaaukut (pamunuo2) “1 998,Biochemical and Molecular Tools for the Production of Useful Terpene Products or Among physiological processes, one of the most important shared by all plants, is the defense response against potentially damaging biotic and abiotic agents. A plant de- fense response is recognized by the presence of dark or black spots on the surface of in- fected leaves or stems. These spots are formed by dead plant cells and several plant Products to avoid the spreading of infecting microorganisms. This is the hypersensitive re- sponse (HR) triggered by avirulent (non-pathogenic) organisms. If microorganisms over- come the plant defense mechanisms and escape HR, the infection process spreads causing disease and death of the whole plant (Dixon and Harrison, 1990). Different signals recognized somehow by Capsicum annuum after pathogen’s attack or similar stimuli, elicit the corresponding HR responses. For example, some lipopolysac- charides from enteric bacteria and Xanthomonas campestris prevent the pepper HR re- sponse elicited by avirulent X campestris strains (Newman et al., 1997). Also, the coat protein but not the RNA from tobamovirus is required for the elicitation of the Capsicum L2 gene-mediated resistance (de la Cruz et al., 1997). Other proteins related with defense responses have been identified in pepper as a 34 kDa beta-1,3-glucanase with antifungal activity against Phytophthora capsici. This enzyme acted synergistically with a basic pep- per 32 kDa chitinase to inhibit hyphal growth of F oxysporum and P. capsici (Kim and Hwang, 1997). The gene encoding a pepper endo-beta-1,4,glucanase was isolated al- though it was related to fruit ripening and enhanced transcription rate was achieved by ethylene-treatment of immature green fruits (Harpster et al., 1997). A fruit specific defen- sin-type protein J1 with antifungal properties and the respective gene, were isolated and characterized. The mature J1 protein contained eight cysteines forming four intramolecu- lar disulfide bridges. This protein was not detected in healthy green fruits but it was strongly accumulated in wounded green fruits and during ripening (Meyer et al., 1996). However, although these proteins are directly related with plant defense responses, there is also the elicited production of chemicals with antibiotic or deterrent effect on plant pathogens which plays a main role in this response. These chemicals called phy- toalexins, are absent or present at only very low amounts in healthy tissues (Darvill and Albersheim, 1984). The most well known pepper phytoalexin is the bicyclic sesquiterpene capsidiol. It has a recognized fungicide effect but it was also reported an inhibitory effect of capsidiol, on the contraction induced by histamine, acetylcholine and other agonists in isolated guinea-pig ileum and trachea and, on prostaglandin synthesis in vitro (Stoessl et al., 1972; Nasiri et al., 1993). The use of capsidiol as a natural fungicide instead of polluting agro. chemicals is attractive. Also, the molecular mechanism involved in the elicitation of cap- sidiol production after precise stimuli recognition may be manipulated to control the expression of other genes. Finally, the specificity of the enzymes involved in the bio- chemical reactions related to the capsidiol pathway may be modified to synthesize other useful products. SESQUITERPENE BIOSYNTHETIC PATHWAY AND KEY ENZYMES Terpenes are part of quite different plant natural substances and constitute the vola- tiles and essential oils as well as the natural rubber. They are among the most useful and widespread products in nature. The biological effects of some terpenes are known not only in plants but also for insects and animals. Terpenes are classified according to the number of isoprene units (one isoprene = five carbons). Monoterpenes contain two isoprene units,68 E. Lozoya-Gloria sesquiterpenes contain three isoprene units. Diterpenes, triterpene and te:raterpene con- tains four, six and eight isoprene units, respectively. Terpenoids are the corresponding de- rivatives including different groups like alcohols, aldehydes or ketones (Banthorpe and Charlwood, 1980; Chappell, 1995). 3-Hydroxy-3-methylglutaryl Coenzyme A : reductase (HMGR) The synthesis of mevalonic acid (MVA) from acetyl Coenzyme A is the first key step of sesquiterpene biosynthetic pathway (Fig. 1). This step is catalyzed by the 3-hydroxy-3- methylglutaryl Coenzyme A : reductase (HMGR) enzyme encoded by plant gene families and differentially expressed according to the recognized stimuli (developmental stage, pathogen’s attack or other stress conditions) (Weissenborn et al., 1995). HMGR isoenzymes have been located in mitochondria, chloroplast and microsomes membranes (Brooker and Russell, 1975; Wong et al., 1982), Genomic and CDNA-HMGR clones from several plant species has been reported (Learned and Fink, 1989; Narita and Gruissem 1989; Ferrer et al., 1990; Bach et al., 1991; Chye et al., 1991; Stermer et al., 1991; Choi et al., 1992; Genschik et al., 1992; Maldonado-Mendoza et al., 1992; Park et al., 1992; Aoyagi et al., 1993; Enjuto et al., 1994; Joost et al., 1995). Plant HMGR en- zymes are 60-65 kDa average size. In spite of their demonstrated membrane location, the amino acid sequences at the amino terminus is highly divergent between plant species and none of these shows any signal or transit peptide for targeting to the mitochondria or plas- tid. On the other hand, the carboxy terminus contains the active site and the transmem- brane hydrophobic domain and comparison of amino acid sequences at this region shows a high similarity of more than 90% (Cane, 1981; Campos and Boronat, 1995; Denbow et al., 1996). Genes encoding for specific HMGR isoforms are related to the sesquiterpenic phy- toalexin production and accumulation in Solanaceae (tobacco, cotton, potato and tomato) (Stermer et al., 1991; Choi et al., 1992; Chappell et al., 1995; Joost et al., 1995; Weissen- born et al., 1995). HMGR isoenzymes are also regulated at the protein level as shown by transgenic tobacco plants holding foreign HMGR genes (Chappell et al., 1995; Weissen- born etal., 1995). Farnesyl Pyrophosphate Synthase (FPS) After mevalonate is produced, it is converted to isopentenyl pyrophosphate (IPP) by mevalonate kinases and mevalonate 5-diphosphate decarboxylase enzymes. The IPP isom- erase enzyme catalyze the production of the IPP isomer, the dimethylally! pyrophosphate (DMAPP). The condensation of DMAPP with IPP produces geranyl pyrophosphate (GPP), which is the precursor of all monoterpenes. Subsequent addition of IPP molecules gives rise to pyrophosphate intermediates such as farnesyl pyrophosphate (FPP), 2 common pre- cursor of all sesquiterpenes and sterols as well as geranylgeranyl pyrophosphate (GGPP), the precursor of carotenoids and gibberellins. The farnesyl pyrophosphate synthase (FPS), a prenyltransferase enzyme, is responsible for the production of FPP (Chappell, 1995). Prenyltransferases are homodimers of 70-80 kDa and contain a number of con- served regions. The pyrophosphate moiety of the IPP and one ion metal (Mg”* or Mn**) are bound to two aspartate-rich domains involved in the catalytic reaction (Chen et al., 1994; Chappell, 1995), FPS-DNA sequences have been cloned from A. thaliana cDNA (Delourme et al., 1994; Cunillera et al., 1996), Lupinus albus (Attucci et al., 1995), Cap- sicum annuum (Hugueney et al., 1996), Hevea brasiliensis (Adiwilaga and Kush, 1996)Biochemical and Molecular Tools for the Production of Useful Terpene Products o Monoterpenes ‘Sterols, t ss LN, GPP fe Fes we Aor hydrox. ava copetoy 5- epiaristolochene cooH * v hava itite Shame aq Se 2NADP 6 to vetispiradiene| HMEcoa ~S.COA COOH Se wmulene ake Arey ot i Figure 1. Sesquiterpene biosynthetic pathway. Biosynthesis initiates with acetylk-Coenzyme A (Acetyl-CoA), in- termediates and final products are shown: acetoacetyl-Coenzyme A (Acetoacetyl-CoA), 3-hydroxy-3-methylglu- taryl Coenzyme A (HMG-CoA), mevalonic acid (MVA), isopentenyl pyrophosphate (IPP), dimenthylallyl pyrophosphate (DMAPP), geranyl pyrophosphate (GPP), farnesyl pyrophosphate (FPP). Enzymes ate key steps are: 3-hydroxy-3-methyl glutaryl Coenzyme A: reductase (HMGR), famesyl pyrophosphate synthase (FPS), squalene synthase (SS), 5-epi-aristolochene syntahse (EAS), 5-epi-aristolochene hydroxylases (5-EA hydrox.), vetispiradiene synthase (VS), caninene synthase (CAD). Condensation of two molecules is indicated (2x) for ace- tyl-Coa and FPP.70 E. Lozoya-Gloria and Artemissia annua (Matsushita et al., 1996). The C. annuum-FPS gene was expressed in E. coli (Hugueney et al., 1996). This enzyme provides the substrate for sesquiterpenes and other isoprenoid path- ways yielding main end products involved in growth and development of plants such as phytosterols, dolicols, ubiquinones and abscissic acid, among others. Channeling of FPS substrates and products may be directed toward specific pathways as sterols, cyclic ses- quiterpenes or GGPP to produce longer terpenes, After cellulase treatment mimicking fun- gal attack, the pepper FPS was synthesized de novo to increase the FPP pool required for the capsidiol production by the farnesyl pyrophosphate cyclase (FPC) enzyme activity (Hugueney et al., 1996). Farnesyl Pyrophosphate Cyclases (FPC) or Sesquiterpene Cyclases After FPP production at least three enzymes are participating in the same branching step, the squalene synthase (SS), the farnesyl pyrophosphate cyclases (FPC) and the far. nesyl protein transferases. The SS enzyme activity initiates the sterols biosynthesis, and the farnesyl protein transferases are related to primary metabolism. The FPCs or sesquiter- pene cyclase enzymes initiates secondary metabolism where sesquiterpenic phytoalexins, essential oils and other final products are included (Chappell, 1995). A large variety of cy- clic sesquiterpenes are synthezised by plants suggesting a broad stereospecificity of the enzymes involved ia their biosynthesis (McCaskill and Croteau, 1995; Hugueney et al., 1996). These enzymes are often unstable, hard to isolate and produced as mixtures. Most of the necessary substrates are not commercially available and must be chemically synthe- sized, sometimes at high cost. In spite of the broad stereospecificity, the catalytic activity of sesquiterpene cyclases is a common feature present in all terpene cyclases. The cyclic molecule results after join- ing a carbon atom at the diphosphate moiety of the FPP molecule to another carbon atom double bond-associated on the same molecule. The selection of the carbon atom double bond-associated is specific for each cyclase activity in order to yield particular sesquiter- penes (Croteau, 1992). Sesquiterpene cyclase has been purified from Salvia officinalis synthesizing the ses- quiterpenic essential oils, humulene and caryophyllene (Dehal and Croteau, 1988). The to- bacco 5-epi-aristolochene synthase (tEAS) synthesizing the precursor of capsidiol was elicitor-inducible and purified from cellulase-treated suspension cell cultures (Vogeli and Chappell, 1990). The patchoulol synthase was purified from Pogostemon cablin (patch- ouli) and was responsible for the (-)-patchoulol production, another essential oil (Munck and Croteau, 1990). From these enzymes only the tobacco EAS is related to plant defense responses and although cell cultures have been proved as a quite convenient system, it is not very similar to waat fungal pathogens do at the beginning of the infection process. The plant roots are the first tissues directly affected by different factors like fertiliz~ ers, pesticides, a complex mixture of microorganisms and are also essentials for the water and nutrient uptake. This means that the adaptability and defense mechanisms of roots should be very efficient in order to protect the rest of the plant. However, phytoalexin pro- duction in roots has not been studied as deep as in other plant organs despite of the impor- tance of these tissues. ‘We studied the capsidiol production of in vitro pepper root’s cultures. After elicita- tion with cellulase treatment, capsidiol was the only chemical produced and excreted by roots to the culture media, in contrast to pepper fruits or tobacco cell cultures which re- lease other substances as detected by thin layer chromatography (TLC). The carbon supplyBiochemical and Molecular Tools for the Production of Useful Terpene Products n (e.g. sucrose) has a significant effect on the phytoalexin root’s production. Pepper cap- sidiol is also synthesized from 5-epi-aristolochene and the presence of the inducible pEAS- enzyme correlated with the capsidiol production as shown by immunodetection assays with monoclonal antibodies rose against the pure tEAS. The maximum pEAS enzyme ac- tivity was found between 6 to 8 hours after elicitation, this assay was carried out just after addition of 0.1 to 1.0 % of insoluble polyvinylpolypirrolidone (PVPP) to eliminate the phenolic compounds formed by grinding of the root tissue (Chavez-Moctezuma and Lo- zoya-Gloria, 1996). The finding of capsidiol excretion from roots to the surrounding environment could be helpful to find out a practical procedure to stimulate the phytoalexin production in field or greenhouse conditions. This fact may avoid or decrease the use of polluting agrochemi- cals but ensuring the plant protection against pathogen’s attack. The inducible pEAS was partially purified from cellulase-treated pepper fruits by a procedure involving filtration of cell homogenates through a column of polyvinylpolypyr- rolidone and a combination of ion exchange and hydrophobic interaction chromatogra- phies. This method yielded a final enzyme recovery of 5% and a 91-fold enrichment over the starting material. The pEAS was a 60 kDa polypeptide and catalyzed the conversion of FPP to a single product which comigrated on argentation-TLC plates with that formed by tobacco EAS. The pepper enzyme required Mg” for activity and other cations such as Mn** and Ca”* failed to activate it. A K,, value of 6.2 iM for FPP was calculated. The effect of related isoprenoids on the cyclase activity was determined. While squalene showed no effect, farnesol and capsidiol were competitive (K,, 20 uM) and non- competitive inhibitors, respectively. The competitive inhibition of EAS by farnesol, sug- gested that early interaction of the substrate FPP with the enzyme catalytic pocket may not involve the pyrophosphate residue. These results were consistent with the observed failure of nucleotides triphosphates to affect EAS activity. The mechanism of EAS inhibition by capsidiol was analyzed and a non-competitive type of inhibition would be consistent with our observations. The physiological implications of EAS inhibition by capsidiol are diffi- cult to predict but considering that capsidiol produced by pepper root cultures was ex- creted to the extracellular medium, the enzyme and inhibitor mixing is most probably an unlikely event. Furthermore, capsidiol is rapidly degraded when added to unelicited chili pepper cells (Stoessl et al., 1977). These findings suggest that EAS inhibition by capsidiol may merely reflect an in vitro effect with no implications for in vivo regulation (Cano- Camacho et al., 1997). Two nearly identical elicitor-inducible tobacco genomic genes (gEAS3 and gEAS4) were isolated and a full size tEAS-cDNA was expressed in E. coli to produce a soluble and active tEAS enzyme (Facchini and Chappell, 1992; Back et al., 1994). One genomic gene (gVS1) from Hyoscyamus muticus coding for the inducible vetispiradieno synthase was isolated and one complete cVS1-cDNA was expressed in E. coli (Back and Chappell, 1995). The (+)-d-cadinene synthase (CAD) enzyme catalyzes the production of the phy- toalexin gossypol. Three CAD-cDNAs (pXC1, pXC14 and cad1-A) from cell suspension cultures from G. arboreum were isolated. The pXC1 enzyme expressed in E. coli, cata- lyzed the synthesis of (+)-d-cadinene, an intermediate in the biosynthesis of gossypol and lacinilene C phytoalexins in cotton (Chen et al., 1995; Chen et al., 1996). Comparison of cDNA sequences of sesquiterpene cyclases, shows an aspartate-rich consensus domain (DDXXD) as well as the cysteine and histidine residues as part of the catalytic site (Chappell, 1995). Domain swapping with exchange of specific DNA regions between cDNAs of tEAS and VS was attempted to verify the above hypothesis between the tEAS and VS cDNAs. The constructs were expressed in E. coli and the reaction prod-n E. Lozoya-Gloria ucts of the coded chimeric enzymes were identified. The exon 4 of the tEAS gene was specific for the reaction product of the tobacco enzyme (5-epi-aristolochene) and the exon 6 of the VS gene for the Hyoscyamus enzyme product (vetispiradiene). The combination of these two domains in the surrounding nucleotide sequence of tobacco resulted in a new enzyme able to synthesize the two different reaction products. A possible explanation is that many terpene cyclases yield multiple reaction products. The putative organization of two separated and different domains into the chimeric peptide, could produce a new activ- ity and new products (Back and Chappell, 1996). Interestingly the multifunctional quimeric enzyme exhibited both a tighter binding of FPP and a faster conversion of FPP to products than either of its wild-type parents (Mathis et al., 1997). The crystal structure of tEAS alone and complexed with FPP analogs was analyzed. The enzymic mechanism for the bicyclic product, S-epi-aristolochene, was proposed as well as the basis to understand the stereochemical selectivity displayed by other cyclases (Starks et al., 1997). Specific Enzymes of Final Steps After the specific cyclization of the particular sesquiterpenes, other modifications occurs as methylation, hydroxylation, demethylation, isomerization, hydration and reduc- tion reactions. The enzymes catalyzing the !- and 3-hydroxylation reaction of the 5-epi- aristolochene produced by pepper were studied. This reaction is NADPH dependent, requires molecular O, (Whitehead et al., 1989) and was suppressed by inhibitors of cyto- chrome P-450 (Hoshino et al., 1994). A 5-epi-aristolochene 3-hydroxylase was partially purified and located in microsomes of elicited green pepper fruits (Hoshino et al., 1995). CONCLUSIONS Pepper (Capsicum annum) is one of the major crops in the third world countries and used worldwide mainly because of the desirable flavor and color of its fruits. A large number of chemicals and corresponding biological activities have been identified in pep- per fruits (Table 1). However, other tissues like roots are also producers of useful chemi- cals. Here we were focused only in some terpenes but other biosynthetical pathways producing phenylpropanoids, alkaloids and other secondary metabolites are functioning at the same time. Many different terpenes need to be synthesized in quite different tissues, during sev- eral developmental stages of the plant in addition to those terpenes needed to respond to a variety of stimuli from the surrounding biosystem. This could be understood considering many specific isoprenoid pathways. Some of these pathways would be producing terpenes constitutively, some could be triggered for the fast synthesis of specific isoprenoids and some others will be producing tiny amounts of highly active terpenes. Isoprenoid-related isoenzymes and the existence of gene families for key enzymes such as HMGR, FPS and FPC may explain the functioning of specific pathways. The right genes must be expressed from the respective promoters in fine concert to end with the right chemical for the required function and at the right time (Yin et al., 1997). These genes and others to be identified opens up many possibilities for manipulation of the pro- duction of specific and new useful terpenes for human beings. The gene manipulation di- rected to produce chimeric or new enzymes (Back and Chappell, 1996; Zook et al., 1996) would allow the production of new molecules for medicine, flavors, fragrances, food addi- tives, essences, natural pesticides and others.Biochemical and Molecular Tools for the Production of Useful Terpene Products B Also, there is much more work to be done in order to understand how the produced chemicals function by themselves. In animals, farnesylation of HMGR enzymes has been reported as being the responsible step for an increased enzyme degradation (Correll et al., 1994). In plants, farnesol and other isoprenoids are also involved in prenylation of some proteins (Shipton et al., 1995). Brassinosteroids are related to the signal transduction mechanism of plant development (Li and Chory, 1997; Li et al., 1997). Other more com- plex relations have been found where natural products from insects and plants interact, such as volicitin, a normal compound found in the oral secrertion of the beet armyworm caterpillar, which elicits plant volatiles attracting natural enemies of the caterpillar (Turlings et al., 1995; Alborn et al., 1997). Finally, besides the genetic analysis, much remains to be done to understand the bio- chemistry, and spatial regulation of the enzymes and proteins related to isoprenes and other plant natural products (Heintze et al., 1990; Stachelin, 1997). REFERENCES Adiwilaga, K.; Kush, A. Cloning and characterization of cDNA encoding farnesyl diphosphate synthase from rub- ber tree (Hevea brasiliensis). Plant Mol. Biol. 1996, 30, 935-946. 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