Regiospecific cytochrome P450 limonene hydroxylases from mint (Mentha) species: cDNA isolation, characterization, and functional expression of (-)-4S-limonene-3-hydroxylase and (-)-4S-limonene-6-hydroxylase.

The oxygenation pattern of the cyclic monoterpenoids of commercial mint (Mentha) species is determined by regiospecific cytochrome P450-catalyzed hydroxylation of the common olefinic precursor (-)-4S-limonene. In peppermint (Mentha x piperita), C3-allylic hydroxylation leads to (-)-trans-isopiperitenol, whereas in spearmint, C6-allylic hydroxylation leads to (-)-trans-carveol. The microsomal limonene-6-hydroxylase was purified from the oil glands of spearmint, and amino acid sequences from the homogeneous enzyme were used to design PCR primers with which a 500-bp amplicon was prepared. This nondegenerate probe was employed to screen a spearmint oil gland cDNA library from which the corresponding full-length cDNA was isolated and subsequently confirmed as the C6-hydroxylase by functional expression using the baculovirus-Spodoptera system. The probe was also utilized to isolate two closely related full-length cDNA species from a peppermint oil gland cDNA library which were confirmed as the limonene-3-hydroxylase by functional expression as before. Deduced sequence analysis of these regiospecific cytochrome P450 monooxygenases indicates that both enzymes bear a typical amino-terminal membrane anchor, consistent with the microsomal location of the native forms, exhibit calculated molecular weights of 56,149 (spearmint) and about 56,560 (peppermint), and are very similar in primary sequence (70% identity and 85% similarity). The availability of these regiochemically distinct, yet very closely related, recombinant hydroxylases and their corresponding genes provides a unique model system for understanding structure-function relationships in cytochrome P450 substrate binding and catalysis, and a means for transgenic manipulation of monoterpene biosynthetic pathways in plants.     

Organization of monoterpene biosynthesis in Mentha. Immunocytochemical localizations of geranyl diphosphate synthase, limonene-6-hydroxylase, isopiperitenol dehydrogenase, and pulegone reductase

We present immunocytochemical localizations of four enzymes involved in p-menthane monoterpene biosynthesis in mint: the large and small subunits of peppermint (Mentha x piperita) geranyl diphosphate synthase, spearmint (Mentha spicata) (-)-(4S)-limonene-6-hydroxylase, peppermint (-)-trans-isopiperitenol dehydrogenase, and peppermint (+)-pulegone reductase. All were localized to the secretory cells of peltate glandular trichomes with abundant labeling corresponding to the secretory phase of gland development. Immunogold labeling of geranyl diphosphate synthase occurred within secretory cell leucoplasts, (-)-4S-limonene-6-hydroxylase labeling was associated with gland cell endoplasmic reticulum, (-)-trans-isopiperitenol dehydrogenase labeling was restricted to secretory cell mitochondria, while (+)-pulegone reductase labeling occurred only in secretory cell cytoplasm. We discuss this pathway compartmentalization in relation to possible mechanisms for the intracellular movement of monoterpene metabolites, and for monoterpene secretion into the extracellular essential oil storage cavity.     

Functional expression of regiospecific cytochrome P450 limonene hydroxylases from mint (Mentha spp.) in Escherichia coli and saccharomyces cerevisiae

The oxygenation pattern of the essential oil monoterpenes of commercial mint (Mentha) species is determined by regiospecific cytochrome P450-catalyzed hydroxylation of the common olefinic precursor (-)-4S-limonene. In spearmint (M. spicata), C6-allylic hydroxylation leads to (-)-trans-carveol and thence (-)-carvone, whereas in peppermint (M. x piperita), C3-allylic hydroxylation leads to (-)-trans-isopiperitenol and ultimately (-)-menthol. cDNAs encoding the C6-hydroxylase and C3-hydroxylase from spearmint and peppermint, respectively, were isolated by a combination of reverse genetic and homology-based cloning methods (S. Lupien, F. Karp, M. Wildung, and R. Croteau, Arch. Biochem. Biophys. 368, 181-192, 1999). Although both hydroxylase genes were confirmed by functional expression using the baculovirus-Spodoptera system, too little protein was available by this approach to permit detailed study of the structure-function relationships of these catalysts, especially the substrate binding determinants that underlie the regiochemistry and stereochemistry of the reactions. Therefore, heterologous overexpression systems based on Escherichia coli and Saccharomyces cerevisiae were developed to produce several N-terminally modified versions of the recombinant hydroxylases. Ancillary methods for the solubilization, purification, and reconstitution (with recombinant spearmint cytochrome P450 reductase) of the limonene hydroxylases were also devised, with which substrate binding behavior and precise regiochemistry and stereochemistry of product formation were determined.     

Monoterpene biosynthesis: specificity of the hydroxylations of (-)-limonene by enzyme preparations from peppermint (Mentha piperita), spearmint (Mentha spicata), and perilla (Perilla frutescens) leaves

Microsomal preparations from the epidermal oil glands of Mentha piperita, Mentha spicata, and Perilla frutescens leaves catalyze the NADPH- and O2-dependent allylic hydroxylation of the monoterpene olefin (-)-limonene at C-3, C-6, and C-7, respectively, to produce the corresponding alcohols, (-)-trans-isopiperitenol, (-)-trans-carveol, and (-)-perillyl alcohol. These transformations are the key steps in the biosynthesis of oxygenated monoterpenes in the respective species, and the responsible enzyme systems meet most of the established criteria for cytochrome P450-dependent mixed function oxygenases. The reactions catalyzed are completely regiospecific and, while exhibiting only a modest degree of enantioselectivity, are highly specific for limonene as substrate. Of numerous monoterpene olefins tested, including several positional isomers of limonene, only the 8,9-dihydro analog served as an alternate substrate for ring (C-3 and C-6) hydroxylation, but not side chain (C-7) hydroxylation. In addition to the regiospecificity of the allylic hydroxylation, these enzymes are also readily distinguishable based on differential inhibition by substituted imidazoles.     

Monoterpene double-bond reductases of the (-)-menthol biosynthetic pathway: isolation and characterization of cDNAs encoding (-)-isopiperitenone reductase and (+)-pulegone reductase of peppermint

Random sequencing of a peppermint essential oil gland secretory cell cDNA library revealed a large number of clones that specified redox-type enzymes. Full-length acquisitions of each type were screened by functional expression in Escherichia coli using a newly developed in situ assay. cDNA clones encoding the monoterpene double-bond reductases (-)-isopiperitenone reductase and (+)-pulegone reductase were isolated, representing two central steps in the biosynthesis of (-)-menthol, the principal component of peppermint essential oil, and the first reductase genes of terpenoid metabolism to be described. The (-)-isopiperitenone reductase cDNA has an open reading frame of 942 nucleotides that encodes a 314 residue protein with a calculated molecular weight of 34,409. The recombinant reductase has an optimum pH of 5.5, and K(m) values of 1.0 and 2.2 microM for (-)-isopiperitenone and NADPH, respectively, with k(cat) of 1.3s(-1) for the formation of the product (+)-cis-isopulegone. The (+)-pulegone reductase cDNA has an open reading frame of 1026 nucleotides and encodes a 342 residue protein with a calculated molecular weight of 37,914. This recombinant reductase catalyzes the reduction of the 4(8)-double bond of (+)-pulegone to produce both (-)-menthone and (+)-isomenthone in a 55:45 ratio, has an optimum pH of 5.0, and K(m) values of 2.3 and 6.9 microM for (+)-pulegone and NADPH, respectively, with k(cat) of 1.8s(-1). Deduced sequence comparison revealed that these two highly substrate specific double-bond reductases show less than 12% identity. (-)-Isopiperitenone reductase is a member of the short-chain dehydrogenase/reductase superfamily and (+)-pulegone reductase is a member of the medium-chain dehydrogenase/reductase superfamily, implying very different evolutionary origins in spite of the similarity in substrates utilized and reactions catalyzed.     

Metabolic engineering of monoterpene biosynthesis: two-step production of (+)-trans-isopiperitenol by tobacco

Monoterpenoid biosynthesis in tobacco was modified by introducing two subsequent enzymatic activities targeted to different cell compartments. A limonene-3-hydroxylase (lim3h) cDNA was isolated from Mentha spicata L. 'Crispa'. This cDNA was used to re-transform a transgenic Nicotiana tabacum'Petit Havana' SR1 (tobacco) line expressing three Citrus limon L. Burm. f. (lemon) monoterpene synthases producing (+)-limonene, gamma-terpinene and (-)-beta-pinene as their main products. The targeting sequences of these synthases indicate that they are probably localized in the plastids, whereas the sequence information of the P450 hydroxylase indicates targeting to the endoplasmatic reticulum. Despite the different location of the enzymes, the introduced P450 hydroxylase proved to be functional in the transgenic plants as it hydroxylated (+)-limonene, resulting in the emission of (+)-trans-isopiperitenol. Some further modifications of the (+)-trans-isopiperitenol were also detected, resulting in the additional emission of 1,3,8-p-menthatriene, 1,5,8-p-menthatriene, p-cymene and isopiperitenone.     

Purification of 4S-limonene synthase, a monoterpene cyclase from the glandular trichomes of peppermint (Mentha x piperita) and spearmint (Mentha spicata).

The p-menthane monoterpenes of the Mentha species are biosynthesized from geranyl pyrophosphate via the monocyclic olefin 4S-limonene. A monoterpene cyclase was isolated from both Mentha x piperita (peppermint) and Mentha spicata (spearmint) that catalyzes the cyclization of geranyl pyrophosphate to 4S-limonene. This enzyme, 4S-limonene synthase, was purified to apparent homogeneity by dye ligand, anion exchange, and hydrophobic interaction chromatography. Since the monoterpenes of Mentha are synthesized and secreted in modified epidermal hairs called glandular trichomes, an extract of isolated glandular trichome cells was used as the source of this enzyme. A combination of gel permeation chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that purified 4S-limonene synthase had a native molecular weight of 56,000 and was monomeric. The principal product of the enzyme was enantiomerically pure (-)-4S-limonene, and a catalytic constant of 0.3/s was determined. The basic properties of 4S-limonene synthase from both M. x piperita and M. spicata are identical and, in general, are similar to those of other monoterpene, sesquiterpene, and diterpene cyclases isolated from microorganisms and higher plants.     

Limonene Synthase, the Enzyme Responsible for Monoterpene Biosynthesis in Peppermint, Is Localized to Leucoplasts of Oil Gland Secretory Cells

Circumstantial evidence based on ultrastructural correlation, specific labeling, and subcellular fractionation studies indicates that at least the early steps of monoterpene biosynthesis occur in plastids. (4S)-Limonene synthase, which is responsible for the first dedicated step of monoterpene biosynthesis in mint species, appears to be translated as a preprotein bearing a long plastidial transit peptide. Immunogold labeling using polyclonal antibodies raised to the native enzyme demonstrated the specific localization of limonene synthase to the leucoplasts of peppermint (Mentha × piperita) oil gland secretory cells during the period of essential oil production. Labeling was shown to be absent from all other plastid types examined, including the basal and stalk cell plastids of the secretory phase glandular trichomes. Furthermore, in vitro translation of the preprotein and import experiments with isolated pea chloroplasts were consistent in demonstrating import of the nascent protein to the plastid stroma and proteolytic processing to the mature enzyme at this site. These experiments confirm that the leucoplastidome of the oil gland secretory cells is the exclusive location of limonene synthase, and almost certainly the preceding steps of monoterpene biosynthesis, in peppermint leaves. However, succeeding steps of monoterpene metabolism in mint appear to occur outside the leucoplasts of oil gland cells.     

Regulation of monoterpene accumulation in leaves of peppermint

Plants synthesize numerous classes of natural products that accumulate during development and are thought to function as constitutive defenses against herbivores and pathogens. However, little information is available about how the levels of such defenses are regulated. We measured the accumulation of monoterpenes, a model group of constitutive defenses, in peppermint (Mentha x piperita L.) leaves and investigated several physiological processes that could regulate their accumulation: the rate of biosynthesis, the rate of metabolic loss, and the rate of volatilization. Monoterpene accumulation was found to be restricted to leaves of 12 to 20 d of age, the period of maximal leaf expansion. The rate of monoterpene biosynthesis determined by (14)CO(2) incorporation was closely correlated with monoterpene accumulation, as determined by gas chromatographic analysis, and appeared to be the principal factor controlling the monoterpene level of peppermint leaves. No significant catabolic losses of monoterpenes were detected throughout leaf development, and monoterpene volatilization was found to occur at a very low rate, which, on a monthly basis, represented less than 1% of the total pool of stored monoterpenes. The composition of volatilized monoterpenes differed significantly from that of the total plant monoterpene pool, suggesting that these volatilized products may arise from a separate secretory system. With the demonstration that the rate of biosynthesis is the chief process that determines monoterpene accumulation in peppermint, efforts to improve production in this species can now focus on the genes, enzymes, and cell differentiation processes that regulate monoterpene biosynthesis.     

Molecular evaluation of a spearmint mutant altered in the expression of limonene hydroxylases that direct essential oil monoterpene biosynthesis

Gamma irradiation of Scotch spearmint created a mutant line, 643-10-74, which has an altered essential oil reminiscent of peppermint because the monoterpene metabolites in the oil glands of the mutant are predominantly oxygenated at the C3 position of the p-menthane ring instead of the C6 position normally found in spearmint. The limonene hydroxylase genes responsible for directing the regiochemistry of oxygenation were cloned from Scotch spearmint and mutant 643 and expressed in Escherichia coli. The limonene bydroxylase from the wild-type parent hydroxylated the C6 position while the enzyme from the mutant oxygenated the C3 position. Comparison of the amino acid sequences with other limonene hydroxylases showed that the mutant enzyme was more closely related to the peppermint limonene-3-hydroxylases than to the spearmint limonene-6-hydroxylases. Because of the sequence differences between the Scotch spearmint and mutant 643 limonene hydroxylases, it is most likely that the mutation did not occur within the structural gene for limonene hydroxylase but rather at a regulatory site within the genome that controls the expression of one or the other regiospecific variants.     

A candidate cDNA clone for (−)-limonene-7-hydroxylase from Perilla frutescens

Cytochrome P450 mono-oxygenases from peppermint, spearmint and perilla (all members of the family Lamiaceae) mediate the regiospecific hydroxylation of the parent olefin (−)-limonene to produce essential oil components oxygenated at C3, C6 and C7, respectively. Cloning, expression and mutagenesis of cDNAs encoding the peppermint limonene-3-hydroxylase and the spearmint limonene-6-hydroxylase have allowed the identification of a single amino acid residue which determines the regiospecificity of oxygenation by these two enzymes. A hybridization strategy provided a cytochrome P450 limonene hydroxylase cDNA from perilla with which to further evaluate the structural determinants of regiospecificity for oxygenation of the common substrate (−)-limonene. The perilla cDNA was a partial clone of 1550 bp (lacking the N-terminal membrane insertion domain), and shared 66% identity with the peppermint 3-hydroxylase and spearmint 6-hydroxylase at the amino acid level. The perilla cytochrome P450 was expressed in Escherichia coli as a chimeric protein fused with the N-terminal membrane insertion domain of the limonene-3-hydroxylase. The kinetically competent recombinant protein was characterized and shown to produce a mixture of C3-, C6- and C7-hydroxylated limonene derivatives with a distribution of 33%, 14% and 53%, respectively.     

Metabolism of Monoterpenes: Conversion of l-Menthone to l-Menthol and d-Neomenthol by Stereospecific Dehydrogenases from Peppermint (Mentha piperita) Leaves

The monoterpene ketone l-menthone is specifically converted to l-menthol and l-menthyl acetate and to d-neomenthol and d-neomenthyl-beta-d-glucoside in mature peppermint (Mentha piperita L. cv. Black Mitcham) leaves. The selectivity of product formation results from compartmentation of the menthol dehydrogenase with the acetyl transferase and that of the neomenthol dehydrogenase with the glucosyl transferase. Soluble enzyme preparations, but not particulate preparations, from mature peppermint leaves catalyzed the NADPH-dependent reduction of l-menthone to both epimeric alcohols, and the two dehydrogenases responsible for these stereospecific transformations were resolved by affinity chromatography on Mātrex Gel Red A. Both enzymes have a molecular weight of approximately 35,000, possess a K(m) for NADPH of about 2 x 10(-5)m, are very sensitive to inhibition by thiol-directed reagents, and are not readily reversible. The menthol dehydrogenase showed a pH optimum at 7.5, exhibited a K(m) for l-menthone of about 2.5 x 10(-4)m, and also reduced d-isomenthone to d-neoisomenthol. The neomenthol dehydrogenase showed a pH optimum at 7.6, exhibited a K(m) for l-menthone of about 2.2 x 10(-5)m, and also reduced d-isomenthone to d-isomenthol. These stereochemically distinct, but otherwise similar, enzymes are of key importance in determining the metabolic fate of menthone in peppermint, and they are probably typical of the class of dehydrogenases thought to be responsible for the metabolism of monoterpene ketones during plant development.     

Inhibition by the essential oils of peppermint and spearmint of the growth of pathogenic bacteria

The effects of the, essential oils of peppermint (Mentha piperita L.), spearmint Mentha spicata L.) and Japanese mint (Mentha, arvensis L.), of four major constituents of the esssential oil of peppermint, and of three major constituents of the essential oil of spearmint, on the proliferation of Helicobacter pylori, Salmonella enteritidis, Escherichia coli O157:H7, methicillin-resistant Staphylococcus aureus (MRSA) and methicillin sensitive Staphylococccus aureus (MSSA) were examined. The essential oils and the various constituents inhibited the proliferation of each strain in liquid culture in a dose-dependent manner. In addition, they exhibited bactericidal activity in phosphate-buffered saline. The antibacterial activities varied among the bacterial species tested but were almost the same against antibiotic-resistant and antibiotic-sensitive strains of Helicobacter pylori and S. aureus. Thus, the essential oils and their constituents may be useful as potential antibacterial agents for inhibition of the growth of pathogens.     

Characterization and mechanism of (4S)-limonene synthase, a monoterpene cyclase from the glandular trichomes of peppermint (Mentha x piperita).

(4S)-Limonene synthase, a monoterpene cyclase isolated from the secretory cells of the glandular trichomes of Mentha x piperita (peppermint), catalyzes the cyclization of geranyl pyrophosphate to (4S)-limonene, a key intermediate in the biosynthesis of p-menthane monoterpenes in Mentha species. The enzyme synthesizes principally (-)-(4S)-limonene (greater than 94% of the total products), plus several other monoterpene olefins. The general properties of (4S)-limonene synthase resemble those of other monoterpene cyclases. The enzyme shows a pH optimum near 6.7, an isoelectric point of 4.35, and requires a divalent metal ion for catalysis, either Mg2+ or Mn2+, with Mn2+ preferred. The Km value measured for geranyl pyrophosphate was 1.8 microM. The activity of (4S)-limonene synthase was inhibited by sodium phosphate, sodium pyrophosphate, and reagents directed against the amino acids cysteine, methionine, and histidine. In the presence of Mn2+, geranyl pyrophosphate protected against cysteine-directed inhibition, suggesting that at least one cysteine residue is located at or near the active site. Experiments with alternate substrates and substrate analogs confirmed many elements of the proposed reaction mechanism, including the binding of geranyl pyrophosphate in the form of a complex with the divalent metal ion, the preliminary isomerization of geranyl pyrophosphate to linalyl pyrophosphate (a bound intermediate capable of cyclization), and the participation of a series of carbocation:pyrophosphate anion pairs in the reaction sequence.     

Characterization of a novel laccase produced by the wood-rotting fungus Phellinus ribis

(+)-Menthofuran is an undesirable monoterpenoid component of peppermint (Mentha x piperita) essential oil that is derived from the alpha,beta-unsaturated ketone (+)-pulegone. Microsomal preparations, from the oil gland secretory cells of a high (+)-menthofuran-producing chemotype of Mentha pulegium, transform (+)-pulegone to (+)-menthofuran in the presence of NADPH and molecular oxygen, implying that menthofuran is synthesized by a mechanism analogous to that of mammalian liver cytochrome P450s involving the hydroxylation of the syn-methyl group of (+)-pulegone, spontaneous intramolecular cyclization to the hemiketal, and dehydration to the furan. An abundant cytochrome P450 clone from a peppermint oil gland cell cDNA library was functionally expressed in Saccharomyces cerevisiae and Escherichia coli and shown to encode the (+)-menthofuran synthase (i.e., (+)-pulegone-9-hydroxylase). The full-length cDNA contains 1479 nucleotides, and encodes a protein of 493 amino acid residues of molecular weight 55,360, which bears all of the anticipated primary structural elements of a cytochrome P450 and most closely resembles (35% identity) a cytochrome P450 monoterpene hydroxylase, (+)-limonene-3-hydroxylase, from the same source. The availability of this gene permits transgenic manipulation of peppermint to improve the quality of the derived essential oil.     

Isoprenoid biosynthesis. Metabolite profiling of peppermint oil gland secretory cells and application to herbicide target analysis

Two independent pathways operate in plants for the synthesis of isopentenyl diphosphate and dimethylallyl diphosphate, the central intermediates in the biosynthesis of all isoprenoids. The mevalonate pathway is present in the cytosol, whereas the recently discovered mevalonate-independent pathway is localized to plastids. We have used isolated peppermint (Mentha piperita) oil gland secretory cells as an experimental model system to study the effects of the herbicides fosmidomycin, phosphonothrixin, methyl viologen, benzyl viologen, clomazone, 2-(dimethylamino)ethyl diphosphate, alendronate, and pamidronate on the pools of metabolites related to monoterpene biosynthesis via the mevalonate-independent pathway. A newly developed isolation protocol for polar metabolites together with an improved separation and detection method based on liquid chromatography-mass spectrometry have allowed assessment of the enzyme targets for a number of these herbicides.     

Agrobacterium-mediated transformation of commercial mints

Commercial peppermint (P) (Mentha × piperita L. ev. Black Mitcham), native spearmint (NS) (M. spicata L.) and Scotch spearmint (SS) (M. × gracillis Sole cv Baker) petioles and orange mint (OM) (M. citrata Ehrh.) leaf disks were cocultivated with a number of Agrobacterium tumefaciens strains. P, SS and OM initiated tumor-like callus tissue on growth regulator-free MS medium after cocultivation with strain A281, a hypervirulent agropine strain containing Ti plasmid pTiBo542. Callus did not initiate from explants cocultivated with strain C58, a virulent nopaline strain; with A 136, a plasmidless strain, or from uninoculated controls. A281-derived callus was maintained on growth regulator-free medium in the absence of antibiotics for up to two years with no bacterial outgrowth. No shoots regenerated from any of the tumors on regeneration medium. Five of seven OM callus lines assayed gave a positive signal for agropine. DNA extracted from OM tumor tissue hybridized to a DNA probe specific to the T-DNA region of pTi plasmid. Genomic Southern analysis of DNA from tumors of P and SS indicated that one to a few copies of the T-DNA integrated into the mint chromosomes. PCR amplification of genomic DNA with primers specific for one of the T-DNA encoded genes yielded fragments that, when analyzed by restriction enzyme mapping and on Southern blots, corresponded to the cytokinin biosynthesis gene ipt. These results demonstrate transformation of three species of mint and the potential for using A. tumefaciens to transfer economically important genes into commercial mint cultivars.Abbreviations BA benzyladenine - CW coconut water - Cef cefotaxime - P peppermint - SS scotch spearmint - NS native spearmint - OM orange mint - BM basal medium - MS Murashige and Skoog (1962) - PAR photosynthetically active radiation - CTAB hexadecylatrimethylammonium bromide - ipt isopentenyl transferase Received for publication 1994. Published as Miscellaneous Paper No. 1482 of the Delaware Agricultural Experiment Station. Contribution No. 317 of the Department of Plant and Soil Sciences. Mention of trade names in this publication does not imply endorsement by the Delaware Agricultural Experiment Station of products named, nor criticism of similar ones not named.     

Biosynthesis of the Monoterpenes Limonene and Carvone in the Fruit of Caraway : I. Demonstration of Enzyme Activities and Their Changes with Development

The biosynthesis of the monoterpenes limonene and carvone in the fruit of caraway (Carum carvi L.) proceeds from geranyl diphosphate via a three-step pathway. First, geranyl diphosphate is cyclized to (+)-limonene by a monoterpene synthase. Second, this intermediate is stored in the essential oil ducts without further metabolism or is converted by limonene-6-hydroxylase to (+)-trans-carveol. Third, (+)-trans-carveol is oxidized by a dehydrogenase to (+)-carvone. To investigate the regulation of monoterpene formation in caraway, we measured the time course of limonene and carvone accumulation during fruit development and compared it with monoterpene biosynthesis from [U-¹⁴C]Suc and the changes in the activities of the three enzymes. The activities of the enzymes explain the profiles of monoterpene accumulation quite well, with limonene-6-hydroxylase playing a pivotal role in controlling the nature of the end product. In the youngest stages, when limonene-6-hydroxylase is undetectable, only limonene was accumulating in appreciable levels. The appearance of limonene-6-hydroxylase correlates closely with the onset of carvone accumulation. At later stages of fruit development, the activities of all three enzymes declined to low levels. Although this correlates closely with a decrease in monoterpene accumulation, the latter may also be the result of competition with other pathways for substrate.     

Biochemical and Histochemical Localization of Monoterpene Biosynthesis in the Glandular Trichomes of Spearmint (Mentha spicata)

The primary monoterpene accumulated in the glandular trichomes of spearmint (Mentha spicata) is the ketone (−)-carvone which is formed by cyclization of the C₁₀ isoprenoid intermediate geranyl pyrophosphate to the olefin (−)-limonene, hydroxylation to (−)-trans-carveol and subsequent dehydrogenation. Selective extraction of the contents of the glandular trichomes indicated that essentially all of the cyclase and hydroxylase activities resided in these structures, whereas only about 30% of the carveol dehydrogenase was located here with the remainder located in the rest of the leaf. This distribution of carveol dehydrogenase activity was confirmed by histochemical methods. Electrophoretic analysis of the partially purified carveol dehydrogenase from extracts of both the glands and the leaves following gland removal indicated the presence of a unique carveol dehydrogenase species in the glandular trichomes, suggesting that the other dehydrogenase found throughout the leaf probably utilizes carveol only as an adventitious substrate. These results demonstrate that carvone biosynthesis takes place exclusively in the glandular trichomes in which this natural product accumulates.