Regulation of partitioned sterol biosynthesis in Saccharomyces cerevisiae.

Using yeast strains with null mutations in structural genes which encode delta-aminolevulinic acid synthetase (HEM1), isozymes of 3-hydroxy-3-methylglutaryl coenzyme A (HMG1 and HMG2), squalene epoxidase (ERG1), and fatty acid delta 9-desaturase (OLE1), we were able to determine the effect of hemes, sterols, and unsaturated fatty acids on both sterol production and the specific activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) in Saccharomyces cerevisiae. We found that the HMGR isozymes direct essentially equal amounts of carbon to the biosynthesis of sterols under heme-competent conditions, despite a huge disparity (57-fold) in the specific activities of the reductases. Our results demonstrate that palmitoleic acid (16:1) acts as a rate-limiting positive regulator and that ergosterol acts as a potent inhibitor of sterol production in strains which possess only the HMGR1 isozyme (HMG1 hmg2). In strains which contain only the HMGR2 isozyme (hmg1 HMG2), sterol production was inhibited by oleic acid (18:1) and to a lesser degree by ergosterol. The specific activities of the two reductases (HMGR1 and HMGR2) were found to be differentially regulated by hemes but not by ergosterol, palmitoleic acid, or oleic acid. The disparate effects of unsaturated fatty acids and sterols on these strains lead us to consider the possibility of separate, compartmentalized isoprenoid pathways in S. cerevisiae.     

Hmg-coA reductase gene family in cotton (Gossypium hirsutum L.): unique structural features and differential expression of hmg2 potentially associated with synthesis of specific isoprenoids in developing embryos.

As a first step towards understanding the biosynthesis of isoprenoids that accumulate in specialized pigment glands of cotton at the molecular level, two full-length genes (hmg1 and hmg2) were characterized encoding hmg-coA reductase (HMGR; EC 1.1.1.34), the enzyme that catalyzes the formation of a key isoprenoid precursor. Cotton hmgr genes exhibited features typical of other plant genes, however, hmg2 encodes the largest of all plant HMGR enzymes described to date. HMG2 contains several novel features that may represent functional specialization of this particular HMGR isoform. Such features include a unique 42 amino acid sequence located in the region separating the N-terminal domain and C-terminal catalytic domain, as well as an N-terminal hydrophobic domain that is not found in HMG1 or other HMGR enzymes. DNA blot analysis revealed that hmg1 and hmg2 belong to small subfamilies that probably include homeologous loci in allotetraploid cotton (Gossypium hirsutum L.). Ribonuclease protection assays revealed that hmg1 and hmg2 are differentially expressed in a developmentally- and spatially-modulated manner during morphogenesis of specialized terpenoid-containing pigment glands in embryos. Induced expression of hmg2 coincided with a possible commitment to sesquiterpenoid biosynthesis in developing embryos, although other developmental processes also requiring HMGR cannot be excluded.     

Expression of the Hevea brasiliensis (H.B.K.) Mull. Arg. 3-Hydroxy-3-Methylglutaryl-Coenzyme A Reductase 1 in Tobacco Results in Sterol Overproduction

A genomic fragment encoding one (HMGR1) of the three 3-hydroxy-3-methylglutaryl coenzyme A reductases (HMGRs) from Hevea brasiliensis (H.B.K.) Mull. Arg. (M.-L. Chye, C.-T. Tan, N.-H. Chua [1992] Plant Mol Biol 19: 473-484) was introduced into Nicotiana tabacum L. cv xanthi via Agrobacterium transformation to study the influence of the hmg1 gene product on plant isoprenoid biosynthesis. Transgenic plants were morphologically indistinguishable from control wild-type plants and displayed the same developmental pattern. Transgenic lines showed an increase in the level of total sterols up to 6-fold, probably because of an increased expression level of hmg1 mRNA and a corresponding increased enzymatic activity for HMGR, when compared with the level of total sterols from control lines not expressing the hmg1 transgene. In addition to the pathway end products, campesterol, sitosterol, and stigmasterol, some biosynthetic intermediates such as cycloartenol also accumulated in transgenic tissues. Most of the overproduced sterols were detected as steryl-esters and were likely to be stored in cytoplasmic lipid bodies. These data strongly support the conclusion that plant HMGR is a key limiting enzyme in phytosterol biosynthesis.     

Three genes encode 3-hydroxy-3-methylglutaryl-coenzyme A reductase in Hevea brasiliensis: hmg1 and hmg3 are differentially expressed

The enzyme 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR) catalyses an important step in isoprenoid biosynthesis in plants. In Hevea brasiliensis, HMGR is encoded by a small gene family comprised of three members, hmg1, hmg2 and hmg3. We have previously described hmg1 and hmg2 (Plant Mol Biol 16: 567–577, 1991). Here we report the isolation and characterization of hmg3 genomic and cDNA clones. In comparison to hmg1 which is more highly expressed in laticifers than in leaves, the level of hmg3 mRNA level is equally abundant in laticifers and leaves. In situ hybridization experiments showed that the expression of hmg3 is not cell-type specific while hmg1 is expressed predominantly in the laticifers. Primer-extension experiments using laticifer RNA showed that hmg1 is induced by ethylene while hmg3 expression remains constitutive. The hmg3 promoter, like the promoters of most house-keeping genes, lacks a TATA box. Our results suggest that hmg1 is likely to encode the enzyme involved in rubber biosynthesis while hmg3 is possibly involved in isoprenoid biosynthesis of a housekeeping nature.     

Mitochondrial localization of the mevalonate pathway enzyme 3-Hydroxy-3-methyl-glutaryl-CoA reductase in the Trypanosomatidae

3-Hydroxy-3-methyl-glutaryl-CoA reductase (HMGR) is a key enzyme in the sterol biosynthesis pathway, but its subcellular distribution in the Trypanosomatidae family is somewhat controversial. Trypanosoma cruzi and Leishmania HMGRs are closely related in their catalytic domains to bacterial and eukaryotic enzymes described but lack an amino-terminal domain responsible for the attachment to the endoplasmic reticulum. In the present study, digitonin-titration experiments together with immunoelectron microscopy were used to establish the intracellular localization of HMGR in these pathogens. Results obtained with wild-type cells and transfectants overexpressing the enzyme established that HMGR in both T. cruzi and Leishmania major is localized primarily in the mitochondrion and that elimination of the mitochondrial targeting sequence in Leishmania leads to protein accumulation in the cytosolic compartment. Furthermore, T. cruzi HMGR is efficiently targeted to the mitochondrion in yeast cells. Thus, when the gene encoding T. cruzi HMGR was expressed in a hmg1 hmg2 mutant of Saccharomyces cerevisiae, the mevalonate auxotrophy of mutant cells was relieved, and immunoelectron analysis showed that the parasite enzyme exhibits a mitochondrial localization, suggesting a conservation between the targeting signals of both organisms.     

Co-expression of N-terminal truncated 3-hydroxy-3-methylglutaryl CoA reductase and C24-sterol methyltransferase type 1 in transgenic tobacco enhances carbon flux towards end-product sterols

The enzymes 3-hydroxy-3-methylglutaryl CoA reductase (HMGR) and C24-sterol methyltransferase type 1 (SMT1) have been proposed to be key steps regulating carbon flux through the sterol biosynthesis pathway. To further examine this hypothesis, we co-expressed the catalytic domain of Hevea brasiliensis HMGR (tHMGR) and Nicotiana tabacum SMT1 in tobacco, under control of both constitutive and seed-specific promoters, resulting in increased accumulation of total sterol in seed tissue by 2.5- and 2.1-fold, respectively. This enhancement is greater than when tHMGR and SMT1 were expressed singularly where, for example, seed-specific expression enhanced total sterols by 1.6-fold. Significantly, the relative level of 4-desmethyl sterols (end-product sterols) was higher in seed co-expressing tHMGR and SMT1 from seed-specific promoters (79% of total sterols) than when co-expressed from constitutive promoters (59% of total sterols) and similar to wild-type seed (80% of total sterols). These results demonstrate that HMGR and SMT1 work in concert to control carbon flux into end-product sterols and that the sterol composition can be controlled by the temporal activity of the promoters driving transgene expression. In addition, constitutive expression of the transgenes resulted in elevated accumulation of substrates for C4-demethylation reactions, which indicates that one or several enzymes catalysing such reactions limit carbon flow to end-product sterols, at least in a physiological situation when the carbon flow is upregulated.     

Glucose controls phosphoregulation of hydroxymethylglutaryl coenzyme A reductase through the protein phosphatase 2A-related phosphatase protein, Ppe1, and Insig in fission yeast

HMG-CoA reductase (HMGR) catalyzes a rate-limiting step in sterol biosynthesis and is a key control point in the feedback inhibition that regulates this pathway. Through the action of the membrane protein Insig, HMGR synthesis and degradation are regulated to maintain sterol homeostasis. The fission yeast Schizosaccharomyces pombe encodes homologs of HMGR and Insig called hmg1(+) and ins1(+), respectively. In contrast to the mammalian system, Ins1 regulates Hmg1 by a nondegradative mechanism involving phosphorylation of the Hmg1 active site. Here, we investigate the role of the Ins1-Hmg1 system in coupling glucose sensing to regulation of sterol biosynthesis. We show that Ins1-dependent Hmg1 phosphorylation is strongly induced in response to glucose withdrawal and that HMGR activity is correspondingly reduced. We also find that inability to activate Hmg1 phosphorylation under nutrient limiting conditions results in overaccumulation of sterol pathway intermediates. Furthermore, we show that regulation of Hmg1 phosphorylation requires the protein phosphatase 2A-related phosphatase Ppe1 and its regulator Sds23. These results describe a mechanism by which cells tune the rate of sterol synthesis to match nutrient availability.     

HMG-GoA reductase and terpenoid phytoalexins: Molecular specialization within a complex pathway

Terpenoid phytoalexins and other defense compounds play an important role in disease resistance in a variety of plant families but have been most widely studied in solanaceous species. The rate-limiting step in terpenoid phytoalexin production is mediated by 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR), which catalyzes mevalonic acid synthesis. HMGRs are involved in the biosynthesis of a broad array of terpenoid compounds, and distinct isoforms of HMGR may be critical in directing the flux of pathway intermediates into specific end products. Plant HMGRs are encoded by a small gene family, and genomic or cDNA sequences encoding HMGR have been isolated from several plant species. In tomato, four genes encode HMGR; these genes are differentially activated during development and stress responses. One gene, hmg 2 , is activated in response to wounding and a variety of pathogenic agents suggesting a role in sesquiterpene phytoalexin biosynthesis. In contrast, expression patterns of tomato hmg l suggest a role in sterol biosynthesis and cell growth. Other plant species show an analogous separation of specific HMGR isoforms involved in growth and/or housekeeping function and inducible isoforms associated with biosynthesis of phytoalexins or other specialized "natural products". We are applying a variety of cell and molecular techniques to address whether subcellular localization and/or differential expression of these isoforms are key factors in determining end product accumulation during development and defense.     

Ubiquitin is conjugated by membrane ubiquitin ligase to three sites, including the N terminus, in transmembrane region of mammalian 3-hydroxy-3-methylglutaryl coenzyme A reductase: implications for sterol-regulated enzyme degradation

The stability of the endoplasmic reticulum (ER) glycoprotein 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR), the key enzyme in cholesterol biosynthesis, is negatively regulated by sterols. HMGR is anchored in the ER via its N-terminal region, which spans the membrane eight times and contains a sterol-sensing domain. We have previously established that degradation of mammalian HMGR is mediated by the ubiquitin-proteasome system (Ravid, T., Doolman, R., Avner, R., Harats, D., and Roitelman, J. (2000) J. Biol. Chem. 275, 35840-35847). Here we expressed in HEK-293 cells an HA-tagged-truncated version of HMGR that encompasses all eight transmembrane spans (350 N-terminal residues). Similar to endogenous HMGR, degradation of this HMG(350)-3HA protein was accelerated by sterols, validating it as a model to study HMGR turnover. The degradation of HMG(240)-3HA, which lacks the last two transmembrane spans yet retains an intact sterol-sensing domain, was no longer accelerated by sterols. Using HMG(350)-3HA, we demonstrate that transmembrane region of HMGR is ubiquitinated in a sterol-regulated fashion. Through site-directed Lys --> Arg mutagenesis, we pinpoint Lys(248) and Lys(89) as the internal lysines for ubiquitin attachment, with Lys(248) serving as the major acceptor site for polyubiquitination. Moreover, the data indicate that the N terminus is also ubiquitinated. The degradation rates of the Lys --> Arg mutants correlates with their level of ubiquitination. Notably, lysine-less HMG(350)-3HA is degraded faster than wild-type protein, suggesting that lysines other than Lys(89) and Lys(248) attenuate ubiquitination at the latter residues. The ATP-dependent ubiquitination of HMGR in isolated microsomes requires E1 as the sole cytosolic protein, indicating that ER-bound E2 and E3 enzymes catalyze this modification. Polyubiquitination of HMGR is correlated with its extraction from the ER membrane, a process likely to be assisted by cytosolic p97/VCP/Cdc48p-Ufd1-Npl4 complex, as only ubiquitinated HMGR pulls down p97.     

Structural and functional conservation between yeast and human 3-hydroxy-3-methylglutaryl coenzyme A reductases, the rate-limiting enzyme of sterol biosynthesis.

The pathway of sterol biosynthesis is highly conserved in all eucaryotic cells. We demonstrated structural and functional conservation of the rate-limiting enzyme of the mammalian pathway, 3-hydroxy-3-methyl-glutaryl coenzyme A reductase (HMG-CoA reductase), between the yeast Saccharomyces cerevisiae and humans. The amino acid sequence of the two yeast HMG-CoA reductase isozymes was deduced from DNA sequence analysis of the HMG1 and HMG2 genes. Extensive sequence similarity existed between the region of the mammalian enzyme encoding the active site and the corresponding region of the two yeast isozymes. Moreover, each of the yeast isozymes, like the mammalian enzyme, contained seven potential membrane-spanning domains in the NH2-terminal region of the protein. Expression of cDNA clones encoding either hamster or human HMG-CoA reductase rescued the viability of hmg1 hmg2 yeast cells lacking this enzyme. Thus, mammalian HMG-CoA reductase can provide sufficient catalytic function to replace both yeast isozymes in vivo. The availability of yeast cells whose growth depends on human HMG-CoA reductase may provide a microbial screen to identify new drugs that can modulate cholesterol biosynthesis.     

Arabidopsis thaliana squalene epoxidase 1 is essential for root and seed development

Squalene epoxidase converts squalene into oxidosqualene, the precursor of all known angiosperm cyclic triterpenoids, which include membrane sterols, brassinosteroid phytohormones, and non-steroidal triterpenoids. In this work, we have identified six putative Arabidopsis squalene epoxidase (SQE) enzymes and used heterologous expression in yeast to demonstrate that three of these enzymes, SQE1, SQE2, and SQE3, can epoxidize squalene. We isolated and characterized Arabidopsis sqe1 mutants and discovered severe developmental defects, including reduced root and hypocotyl elongation. Adult sqe1-3 and sqe1-4 plants have diminished stature and produce inviable seeds. The sqe1-3 mutant accumulates squalene, consistent with a block in the triterpenoid biosynthetic pathway. Therefore, SQE1 function is necessary for normal plant development, and the five SQE-like genes remaining in this mutant are not fully redundant with SQE1.     

Genetic evidence for the role of isopentenyl diphosphate isomerases in the mevalonate pathway and plant development in Arabidopsis

Isopentenyl/dimethylallyl diphosphate isomerase (IPI) catalyzes the interconversion of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), which are the universal C(5) units of isoprenoids. In plants, IPP and DMAPP are synthesized via the cytosolic mevalonate (MVA) and plastidic methylerythritol phosphate (MEP) pathways, respectively. However, the role of IPI in each pathway and in plant development is unknown due to a lack of genetic studies using IPI-defective mutants. Here, we show that the atipi1atipi2 double mutant, which is defective in two Arabidopsis IPI isozymes, exhibits dwarfism and male sterility under long-day conditions and decreased pigmentation under continuous light, whereas the atipi1 and atipi2 single mutants are phenotypically normal. We also show that the sterol and ubiquinone levels in the double mutant are <50% of those in wild-type plants, and that the male-sterile phenotype is chemically complemented by squalene, a sterol precursor. In vivo isotope labeling experiments using the atipi1atipi2 double mutant revealed a decrease in the incorporation of MVA (in its lactone form) into sterols, with no decrease in the incorporation of MEP pathway intermediates into tocopherol. These results demonstrate a critical role for IPI in isoprenoid biosynthesis via the MVA pathway, and they imply that IPI is essential for the maintenance of appropriate levels of IPP and DMAPP in different subcellular compartments in plants.     

Regulation of tomato HMG1 during cell proliferation and growth

The enzyme 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR, EC 1.1.1.34) is encoded by a small multigene family in tomato (Lycopersiconesculentum Mill.) and catalyzes the synthesis of mevalonic acid (MVA), a committed step in the biosynthesis of sterols and isoprenoids. A chimeric HMG1::GUS reporter gene fusion was used to analyze the regulation of HMG1 gene expression in detail. HMG1 promoter 5′ deletion mutants established the boundary of a fully inducible promoter. In HMG1::GUS transgenic tomato plants, histochemical staining with 5-bromo-3-indolyl-glucuronide demonstrated that HMG1 was primarily expressed in shoot and root meristems, and in young tomato fruit. This result was confirmed by both HMG1 in-situ hybridization and RNA gel blot analysis. Tomato suspension cell experiments showed that steady-state HMG1 mRNA accumulated during lag and exponential growth phases, but not during the stationary phase. Transient expression of the HMG1::GUS in tissue culture cells treated with mevinolin indicated that HMG1 expression was subject to feedback regulation by a biosynthetic product derived from MVA. These results suggest that a primary, although not exclusive, role of HMG1 is to supply the MVA demand associated with cell division and growth. Received: 26 October 1998 / Accepted: 16 December 1998     

Involvement of de Novo Protein Synthesis,Protein Kinase,Extracellular Ca2+, and Lipoxygenase in Arachidonic Acid Induction of 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase Genes and Isoprenoid Accumulation in Potato (Solanum tuberosum L.)

A series of inhibitors were tested to determine the participation of de novo protein synthesis, protein kinase activity, extracellular Ca2+, and lipoxygenase activity in arachidonic acid elicitation of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) gene expression and sesquiterpene phytoalexin biosynthesis in potato (Solanum tuberosum L. cv Kennebec). Gene-specific probes were used to discriminate effects on the expression of two HMGR genes (hmg1 and hmg2) that respond differentially in tuber tissue following wounding or elicitor treatment. Inhibition of protein synthesis with cycloheximide completely blocked arachidonate-induced hypersensitive necrosis and browning, including HMGR gene induction and phytoalexin accumulation. This suggests that proteins necessary for coupling arachidonic acid reception to HMGR mRNA accumulation are either rapidly turned over or not present constitutively and are induced following elicitor treatment. Staurosporin, a potent inhibitor of protein kinases, and ethyleneglycol-bis([beta]-aminoethyl ether)-N,N[prime]-tetraacetic acid, a Ca2+ chelator, inhibited arachidonate-induction of hmg2 gene expression and phytoalexin accumulation but did not inhibit the wound-induced expression of hmg1. However, staurosporin inhibited arachidonate's suppression of hmg1 gene expression. Eicosatetraynoic acid, a lipoxygenase inhibitor that suppresses elicitor-induced phytoalexin accumulation, also inhibited arachidonate's suppression of hmg1 and induction of hmg2. The results indicate that arachidonate's suppression of hmg1 and activation of hmg2 depend on a common intermediate or set of intermediates whose generation is sensitive to the inhibitors tested.     

A link between sterol biosynthesis, the cell wall, and cellulose in Arabidopsis

A crucial role for sterols in plant growth and development is underscored by the identification of three Arabidopsis sterol biosynthesis mutants that exhibit embryonic defects: fackel (fk), hydra1 (hyd1), and sterol methyltransferase 1/cephalopod (smt1/cph). We have taken a dual approach of sterol profiling and ultrastructural analysis to investigate the primary defects underlying the mutant phenotypes. Comprehensive gas chromatography GC-MS analysis of hyd1 in comparison to fk reveals an abnormal accumulation of unique sterol intermediates in each case. Sterol profiling of the fk hyd1 double mutant provides genetic evidence that FK C-14 reductase acts upstream of HYD1 C-8,7 isomerase. Despite distinct differences in sterol profiles, fk and hyd1 as well as smt1/cph share ultrastructural features such as incomplete cell walls and aberrant cell wall thickenings in embryonic and post-embryonic tissues. The common defects are coupled with ectopic callose and lignin deposits. We show that all three mutants exhibit a deficiency in cellulose, but are not reduced in pectin and sugars of the cell wall and cytosol. The sterol biosynthesis inhibitors 15-azasterol and fenpropimorph also cause cell wall gaps in dividing root cells and a reduction in bulk cellulose, corroborating that the cell wall abnormalities are due to altered sterol composition. Our results demonstrate that sterols are crucial for cellulose synthesis in the building of the plant cell wall.     

A Genetic and Pharmacological Analysis of Isoprenoid Pathway by LC-MS/MS in Fission Yeast

Currently, statins are the only drugs acting on the mammalian isoprenoid pathway. The mammalian genes in this pathway are not easily amenable to genetic manipulation. Thus, it is difficult to study the effects of the inhibition of various enzymes on the intermediate and final products in the isoprenoid pathway. In fission yeast, antifungal compounds such as azoles and terbinafine are available as inhibitors of the pathway in addition to statins, and various isoprenoid pathway mutants are also available. Here in these mutants, treated with statins or antifungals, we quantified the final and intermediate products of the fission yeast isoprenoid pathway using liquid chromatography-mass spectrometry/mass spectrometry. In hmg1-1, a mutant of the gene encoding 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR), ergosterol (a final sterol product), and squalene (an intermediate pathway product), were decreased to approximately 80% and 10%, respectively, compared with that of wild-type cells. Consistently in wild-type cells, pravastatin, an HMGR inhibitor decreased ergosterol and squalene, and the effect was more pronounced on squalene. In hmg1-1 mutant and in wild-type cells treated with pravastatin, the decrease in the levels of farnesyl pyrophosphate and geranylgeranyl pyrophosphate respectively was larger than that of ergosterol but was smaller than that of squalene. In Δerg6 or Δsts1 cells, mutants of the genes involved in the last step of the pathway, ergosterol was not detected, and the changes of intermediate product levels were distinct from that of hmg1-1 mutant. Notably, in wild-type cells miconazole and terbinafine only slightly decreased ergosterol level. Altogether, these studies suggest that the pleiotropic phenotypes caused by the hmg1-1 mutation and pravastatin might be due to decreased levels of isoprenoid pyrophosphates or other isoprenoid pathway intermediate products rather than due to a decreased ergosterol level.     

Is the Reaction Catalyzed by 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase a Rate-Limiting Step for Isoprenoid Biosynthesis in Plants?

3-Hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) catalyzes the irreversible conversion of 3-hydroxy-3-methylglutaryl coenzyme A to mevalonate and is considered a key regulatory step controlling isoprenoid metabolism in mammals and fungi. The rate-limiting nature of this enzyme for isoprenoid biosynthesis in plants remains controversial. To investigate whether HMGR activity could be limiting in plants, we introduced a constitutively expressing hamster HMGR gene into tabacco (Nicotiana tabaccum L.) plants to obtain unregulated HMGR activity. The impact of the resulting enzyme activity on the biosynthesis and accumulation of particular isoprenoids was evaluated. Expression of the hamster HMGR gene led to a 3- to 6-fold increase in the total HMGR enzyme activity. Total sterol accumulation was consequently increased 3- to 10-fold, whereas end-product sterols such as sitosterol, campesterol, and stigmasterol were increased only 2-fold. The level of cycloartenol, a sterol biosynthetic intermediate, was increased more than 100-fold. Although the synthesis of total sterols appears to be limited normally by HMGR activity, these results indicate that the activity of one or more later enzyme(s) in the pathway must also be involved in determining the relative accumulation of end-product sterols. The levels of other isoprenoids such as carotenoids, phytol chain of chlorophyll, and sesquiterpene phytoalexins were relatively unaltered in the transgenic plants. It appears from these results that compartmentation, channeling, or other rate-determining enzymes operate to control the accumulation of these other isoprenoid end products.