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First published online November 12, 2004; 10.1104/pp.104.050229 Plant Physiology 136:4215-4227 (2004) © 2004 American Society of Plant Biologists Organization of Monoterpene Biosynthesis in Mentha. Immunocytochemical Localizations of Geranyl Diphosphate Synthase, Limonene-6-Hydroxylase, Isopiperitenol Dehydrogenase, and Pulegone Reductase1Institute of Biological Chemistry, Washington State University, Pullman, Washington 99164
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.
Monoterpenes are a large and diverse class of volatile C10 isoprenoids that are the major constituents of many plant essential oils and resins. These natural products play important chemoecological roles in the interactions of plants with their environments. Some monoterpenes have been implicated as allelopathic agents, and they often directly, or indirectly, protect plants from herbivores and pathogens (Pickett, 1991  ; Harborne, 1991; Langenheim, 1994; Wise and Croteau, 1999; Hallahan, 2000). As important constituents of floral scents, monoterpenes also function to attract pollinators (Dudareva and Pichersky, 2000). Some plants release volatile monoterpenes and sesquiterpenes in response to herbivore damage that function to attract predatory insects that in turn feed on, or parasitize, the herbivorous insects (Langenheim, 1994; Degenhardt et al., 2003). In conifer species, mechanical wounding, insect attack, or applications of methyl jasmonate can induce resin secretion and differentiation of traumatic resin ducts within wounded tissues, producing a protective barrier of resin at the site of wounding (Steele et al., 1995; Trapp and Croteau, 2001; Franceschi et al., 2002; Martin et al., 2002). Many plant species constitutively produce large quantities of terpenoid-rich resins and essential oils within specialized glandular tissues, such as glandular trichomes, secretory cavities, and secretory ducts (Fahn, 1979, 2000). These natural stores of plant terpenoids probably serve as deterrents to herbivorous insects, but they also provide commercially important sources of flavorings, fragrances, resins, and pharmaceuticals (Langenheim, 1994; Wise and Croteau, 1999). The glandular cells of these secretory tissues are of interest for their remarkable ability to rapidly generate substantial amounts of specific terpenoid products.
Peppermint (Mentha x piperita) has been employed as a model system for the study of monoterpene biosynthesis (Wise and Croteau, 1999
We previously described the immunocytochemical localization of (–)-4S-limonene synthase (the committed step of the pathway; Fig. 2) to the stroma of peppermint glandular trichome leucoplasts (Turner et al., 1999 ). Here we present the subcellular localizations of four additional enzymes that mediate major steps of p-menthane monoterpene biosynthesis (Fig. 2): geranyl diphosphate synthase (GPPS, the prenyltransferase that condenses IPP and DMAPP to produce the C10 precursor of (–)-limonene), spearmint (Mentha spicata) limonene-6-hydroxylase (L6OH, for the regiospecific hydroxylation of limonene; in peppermint, the related limonene-3-hydroxylase produces isopiperitenol), isopiperitenol dehydrogenase (IPD, for the oxidation of isopiperitenol to isopiperitenone), and pulegone reductase (PR, catalyzing the reduction of the  4,8 double bond to produce menthone on route to menthol). With the exception of GPPS, these are the first localizations for these enzymes in plants, and the first localizations of any steps in a monoterpene biosynthetic pathway later than the early step of ring closure catalyzed by monoterpene synthases. Monoterpene biosynthesis is known to initiate in plastids (Eisenreich et al., 1997; Turner et al., 1999; Tholl et al., 2004), but in addition, we localized enzymes to endoplasmic reticulum, mitochondria, and the cytosol. We combine these observations with data relating to the monoterpene content of peppermint gland cells, the secretion rate, and monoterpene solubility to present a new model of monoterpene intracellular translocation and secretion.
Specificity of Affinity-Purified Antibodies Western-blot analysis employing crude gland cell extracts of peppermint and spearmint indicated that after affinity purification the polyclonal antibodies to GPPS, L6OH, IPD, and PR used in this study labeled single protein bands corresponding to the correct Mrs for their target enzymes (Fig. 3).
GPPS Localization
The monoterpene biosynthetic pathway in peppermint (Fig. 2) initiates with the formation of the C5 terpenoid precursors IPP and DMAPP derived from pyruvate and glyceraldehyde-3-phosphate by the plastidial nonmevalonate, methylerythritol phosphate (MEP) pathway (Eisenreich et al., 1997
Presumably, the subcellular distribution of GPPS is an important factor affecting substrate allocation for the production of monoterpenes. The preprotein sequences of both the large and small subunits of Mentha GPPS contain probable N-terminal transit peptides (Burke et al., 1999
Within the leucoplasts, the GPPS-SSU label appeared to be frequently, but not consistently, associated with tubular plastid membranes. This was most apparent with chemically fixed glands for which staining more clearly contrasted tubular thylakoid membranes from stroma (Fig. 4A). With freeze-substituted plastids, the contrast between the tubules and the densely staining stroma was usually poor, often making the tubules especially difficult to distinguish. Figure 4B illustrates a small lobe of a freeze-substituted plastid within which the tubules are especially apparent.
GPPS-LSU was also localized to the leucoplasts of secretory phase glandular cells of the peltate glandular trichomes (Fig. 5A), but strong labeling also occurred within chloroplasts of mesophyll chlorenchyma cells adjacent to thylakoid membranes (Fig. 5B) and within the leucoplasts of small capitate glandular trichome secretory cells (Fig. 5D). The labeling in chloroplasts and leucoplasts of capitate trichomes probably represents cross-labeling of another plastidial short-chain prenyltransferase, most likely GGPPS that shares 65% to 75% sequence identity with GPPS-LSU. The lack of labeling of capitate gland plastids by the GPPS-SSU antibody (Fig. 5E), and also by anti-limonene synthase antiserum (Turner et al., 1999
In Mentha, the majority of geranyl diphosphate produced by GPPS is cyclized by (–)-limonene synthase to form (–)-(4S)-limonene (Fig. 2; Colby et al., 1993
Immunocytochemical localization of L6OH in spearmint is shown in Figure 6. Labeling occurred in the cap cells of secretory phase peltate glandular trichomes (Fig. 6, A and B) but was absent from all other cell types and tissues. Labeling was consistent with the association with SER (Fig. 6A). Preservation of spearmint glandular cells by conventional tissue fixation was poor, but it was adequate to determine the subcellular location of the enzyme. Intense labeling occurred in basal regions of the glandular cells near leucoplasts, as well as along the cell periphery, but very little labeling occurred adjacent to vacuoles in distal regions of the cells (Fig. 6B). Our immunolocalizations of L6OH are entirely consistent with predictions based on sequence analysis. PSORT (Nakai and Horton, 1999
IPD Localization
In peppermint, (–)-trans-isopiperitenol is oxidized by (–)-trans-IPD to produce the ketone (–)-isopiperitenone (Fig. 2), while a nearly identical enzyme functions in spearmint to oxidize the C6 hydroxyl of (–)-trans-carveol to (–)-carvone, the major constituent of spearmint oil. Peppermint IPD is an operationally soluble 27-kD, pyridine nucelotide-dependent, short-chain dehydrogenase with a pH optimum of 10.5 and a strong preference for NAD as cofactor (K. Ringer, E. Davis, and R. Croteau, unpublished data; Kjonaas et al., 1985 Affinity-purified anti-IPD strongly labeled mitochondria of secretory stage glandular cap cells of peppermint peltate glandular trichomes (Fig. 7, A and B), whereas very little labeling was seen in presecretory stage gland cells. Mitochondria of other cell types were unlabeled, including those of the stalk cells (not shown) and adjacent mesophyll parenchyma (Fig. 7D). A similar pattern was seen with spearmint glands with labeling of secretory-stage gland cell mitochondria (Fig. 7C). Presumably, this labeling represents carveol dehydrogenase (CD) because the amino acid sequences of spearmint CD and peppermint IPD share 99% identity (K. Ringer, E. Davis, and R. Croteau, unpublished data).
Following the oxidation of (–)-trans-isopiperitenol by IPD, the endocyclic-double bond of (–)-isopiperitenone is reduced by (–)-isopiperitenone reductase to yield (+)-cis-isopulegone. An isomerase then mediates the shift of the 8,9 double bond of (+)-cis-isopulegone to the 4,8 position to produce (+)-pulegone (Fig. 2).
(+)-PR is an operationally soluble 38-kD protein and a member of the medium-chain dehydrogenase/reductase superfamily, which catalyses the NADPH-dependent reduction of the
We present the tissue and subcellular distributions of four essential enzymes of monoterpene biosynthesis in Mentha, peppermint GPPS, spearmint L6OH, peppermint IPD, and peppermint PR. The localizations of L6OH, IPD, and PR represent the first for any monoterpene biosynthetic enzymes downstream of the plastidial monoterpene cyclase, limonene synthase, and indicate the diversity of organelles involved in the biosynthetic process. At the tissue level, with the exception of GPPS-LSU, labeling for each of these enzymes was found only within glandular cap cells of secretory stage peltate glandular trichomes. This observation is consistent with the findings of McConkey et al. (2000) and Gershenzon et al. (2000) that monoterpene biosynthesis in peppermint is transcriptionally regulated, and that peak abundances of the relevant transcripts, enzymes, and activities and are associated with young leaves containing the highest surface densities of secretory stage peltate glands. The chloroplast labeling by the anti-GPPS-LSU antibody probably represents cross-reactivity with another plastidial prenyltransferase, most likely GGPPS with which GPPS-LSU shares greater than 70% identity (Burke et al., 1999). Since abundant anti-GPPS-SSU labeling occurred only in peltate glandular trichomes, functional GPPS heteromers must be restricted to the glandular cells. Although the plastids of the small capitate glandular trichomes were intensely stained by the anti-GPPS-LSU antibodies, they were not labeled by antibodies against GPPS-SSU or limonene synthase (Turner et al., 1999), indicating that the capitate glands do not produce significant amounts of monoterpenes.
Our immunocytochemical localizations demonstrated a high degree of compartmentalization within the glandular cells, with GPPS localized to gland cell leucoplasts, spearmint limonene-6-hydroxylase associated with SER membranes, IPD found within the mitochondrial matrix, and the apparently soluble PR occurring as a cytoplasmic enzyme. The plastid localization of GPPS was expected because the preproteins of both GPPS subunits contain predicted N-terminal plastid targeting transit peptides (Burke et al., 1999
Previous studies have provided substantial evidence that monoterpene biosynthesis is initiated within gland cell plastids but little evidence for the involvement of other organelles. Thus, a correlation was found between the presence of leucoplasts in glandular cells and the presence of monoterpenes in the gland secretions (Cheniclet and Carde, 1985
Bouvier et al. (2000)
The subcellular compartmentalization of monoterpene biosynthesis in diverse locales within peppermint glandular cells presents questions concerning the coordinated intracellular movement of monoterpene metabolites between organelles during production and secretion. In speculating about this process, enzyme localization in the context of intracellular transport, possible oil secretion mechanisms, and the known monoterpene content of peppermint gland cells must be considered. The rate of oil secretion is moderately rapid, in that the glandular cells of a peltate glandular trichome secrete approximately twice their cellular volume when filling the subcuticular oil storage space in roughly 20 to 30 h (Turner et al., 2000a
The C3-oxygenated monoterpenes, such as menthol, have some water solubility (Weidenhamer et al., 1993
Published aqueous solubility estimates for monooxygenated monoterpenes vary widely (Fichan et al., 1999 ), but with the exception of limonene, water solubility of the monoterpene metabolites appears to be similar to, or greater than, our estimated values for their concentrations in gland cells (Weidenhamer et al., 1993; Fichan et al., 1999). Therefore, simple diffusion could play an important role in subcellular monoterpene translocation.
The water solubility of limonene is very low (<0.15 mM) compared to the solubilities of its C3-oxygenated derivatives (>1–5 mM; Weidenhamer et al., 1993
Hydroxylation of monoterpene olefins greatly increases their water solubility (Weidenhamer et al., 1993
PR was localized to the cytosol, and preliminary immunocytochemical results indicate that menthone reductase is also cytosolic (G. Turner and R. Croteau, unpublished data); thus, the final steps of monoterpene biosynthesis in peppermint occur in the cytosol. Within peppermint glandular cells, most lipid-like deposits are associated with ER membranes. These deposits are especially abundant at ER of the peripheral cytoplasm, suggesting a directional movement of lipids (terpenoids) toward the secretory plasma membrane (Turner et al., 2000a
Plant Materials, Secretory Cell Extracts, and Antibody Production
Peppermint (Mentha x piperita L. cv Black Mitcham) plants were grown with a 16-h photoperiod in a controlled environment chamber as previously described (Gershenzon et al., 2000
Production of antibodies to recombinant L6OH (Lupien et al., 1999
Partially purified recombinant PR obtained with Source 30Q anion-exchange chromatography, as described above, was further purified for use in affinity purification of polyclonal anti-PR antibodies. Source 30Q fractions demonstrating ample PR activity were combined and loaded onto a phenyl sepharose FPLC column (Amersham Biosciences) in a loading buffer containing final concentrations of 50 mM sodium phosphate buffer, 1 mM dithiothreitol, 10% (v/v) glycerol, and 1.5 M (NH4)2SO4. After washing with 10 mL of the loading buffer, proteins were eluted with a decreasing ammonium sulfate gradient [1.5–0 M (NH4)2SO4], and fractions were assayed for PR activity (Ringer et al., 2003 The cDNA encoding IPD was also subcloned into the pBAD-TOPO vector (Invitrogen, Carlsbad, CA) and expressed in E. coli One Shot TOP10 cells (Invitrogen) to generate a fusion protein bearing a C-terminal His6 tag. One-liter cultures were grown to an optical density of A600 = 0.5, induced with 0.1% Ara, and then grown overnight at 17°C. Soluble extracts were prepared as before, and the recombinant proteins were purified using a Ni-NTA agarose column (Qiagen USA, Valencia, CA) as per the manufacturer's instructions. The His6-tagged recombinant protein (purity >95%) was then used for affinity purification of anti-IPD antibodies.
All crude antisera were affinity purified before use for immunocytochemistry. The total IgG fractions from antisera and preimmune sera were first isolated by FPLC using HiTrap Protein A affinity columns (Amersham Biosciences). Approximately 3 mL of serum was diluted with 7 mL of 20 mM sodium phosphate buffer, pH 7.5, then filtered and loaded onto a 1-mL protein A column. After rinsing with 10 mL of 20 mM phosphate buffer, the bound IgG was eluted with 2 mL of citric acid-sodium phosphate buffer, pH 3.5, and immediately neutralized with 120 µL of 1 M Tris-HCl, pH 9. The resulting antisera IgG fraction typically contained approximately 1 mg protein/mL. The IgG fraction was then affinity purified against the corresponding target protein. For this purpose, approximately 300 µg of the purified recombinant target enzyme was covalently linked to 0.5 mL (109 beads) of tosyl-activated M-280 magnetic dynabeads (Dynal A.S., Oslo) as per the manufacturer's instructions. Magnetic fields from small neodynium magnets were used to isolate the coated beads during antibody purification. Coated dynabeads were first suspended in a blocking solution of 3% bovine serum albumin (BSA) in Tris-buffered saline containing Tween 20 (TBST; 10 mM Tris-HCl, pH 7.5, 250 mM NaCl, and 0.3% Tween 20) for 2 to 4 h at 4°C, and then incubated overnight at 4°C in a solution containing 300 µL of protein A purified antisera IgG and 1.2 mL of the BSA-TBST blocking solution. The beads were then rinsed three times for 10 min each with TBST and finally with H2O before eluting the bound IgG with 300 µL of Gly-HCl buffer, pH 2.5, for 45 s. The IgG solution was immediately neutralized with 30 µL of 1 M Tris-HCl, pH 8.5. The affinity-purified IgG solution was then centrifuged at 10,000g for 5 min to remove any remaining protein-coated dynabeads.
Immunoblots were performed to test the specificity of antibodies toward the target enzymes (Fig. 3). Secretory cell protein extracts were separated using one-dimensional SDS-PAGE on 10% to 12% gels and then electroblotted to nitrocellulose according to instructions for the Bio-Rad (Hercules, CA) Mini-Protean Gel Electrophoresis and Mini-Transblot systems. The nitrocellulose blots were immersed for 2 to 4 h (room temp) or overnight (4°C) in a blocking solution containing either 5% nonfat dry milk in TBST, or 3% BSA in TBST. Blots were rinsed with TBST and then transferred to half strength blocking solution (2.5% dry milk or 1.5% BSA) containing a dilute solution of the purified primary antibody. Blots were incubated in primary antibody solutions for 4 h (room temp) or overnight (4°C). The blots were then rinsed three times (10 min each) in TBST and then transferred to a half strength blocking solution containing a 1:5,000 dilution of goat anti-rabbit antibodies conjugated to alkaline phosphatase. Blots were incubated with the secondary antibodies for 1 to 2 h (room temperature), then rinsed well with TBST and water prior to two 10-min rinses in alkaline phosphatase reaction buffer (0.1 M Tris-HCl, pH 9.5, with 0.1 M NaCl, and 5 mM MgCl2). Antibody-labeled proteins were stained for several minutes in alkaline phosphatase reaction buffer containing 0.5 mg nitro-blue tetrazolium/mL and 0.17 mg 5-bromo-4-chloro-3-indolyl phosphate/mL to form dark deposits on the immunolabeled proteins. The reaction was stopped by washing blots with 20 mM Tris-HCl, pH 7.5, containing 0.5 mM EDTA.
Rapid freezing, freeze-substitution, and embedment in LR White resin (Ted Pella, Redding, CA) of high-pressure frozen, freeze-substituted peppermint leaf tissues were performed in the Electron Microscopy Laboratory at the University of California, Berkeley. Small leaf discs, approximately 1 mm in diameter, from young peppermint leaves were rapidly frozen in a Balzers (BAL TEC AG, Balzers, Liechtenstein) model HPM 010 high pressure freezer and then freeze-substituted at –90°C for 72 h in a Leica (Wetzler, Germany) AFS automatic freeze substitution device. The freeze-substitution fluid consisted of anhydrous acetone containing 0.25% uranyl acetate, 0.1% glutaraldehyde, and 0.01% OsO4. After warming slowly to room temperature, the acetone mixture was exchanged for ethanol, and the ethanol was then exchanged for LR White resin in a short, graded series of steps. Infiltration with LR White resin was allowed to proceed overnight at room temp. The resin was then polymerized in a Pelco model 3440 research microwave oven (Ted Pella) at full power for 45 min. Spearmint and some peppermint specimens were fixed by immersing 1-mm leaf discs overnight in a chilled (4°C) fixative solution containing 0.5% (v/v) glutaraldehyde, 2% (v/v) paraformaldehyde, and 50 mM PIPES buffer, pH 7.3. These specimens were then dehydrated in a graded ethanol series and infiltrated with LR White resin. After infiltration, the resin was allowed to polymerize overnight at 50°C in a conventional oven. Sectioning was accomplished using a Diatome (Diatome U.S., Hatfield, PA) diamond knife and a Leica Ultracut R ultramicrotome at the Washington State University Electron Microscopy Center. Silver sections were collected on uncoated 300-mesh nickel grids.
Thin sections on nickel grids were incubated for 1 to 2 h at room temp in a TBST blocking solution containing 1% (w/v) IgG-free BSA (Jackson ImmunoResearch Laboratories, West Grove, PA) and 1% (v/v) normal donkey serum. After blocking, specimens were transferred to a primary antibody solution containing TBST-BSA (0.5% IgG-free BSA) and either affinity-purified anti-serum IgG, or the equivalent concentration of the appropriate protein A purified preimmune IgG. The concentrations of the IgG solutions were determined by Bradford protein assays (Bio-Rad) using an IgG standard curve. Typically, each grid was incubated for 4 h room temp in 30 µL of solution containing 5 to 15 µg IgG/mL. After incubation with the primary antibody, the sections were rinsed a minimum of three times for 10 min with TBST, then transferred to a TBST blocking solution containing 2.5% to 5% colloidal gold conjugated donkey anti-rabbit secondary antibodies (Jackson ImmunoResearch). Sections were incubated for 1 to 2 h, rinsed with TBST, and followed by rinses with distilled water. After immuno-labeling, the immunogold-labeled sections were counter-stained for 12 min with a uranyl acetate-KMnO4 solution consisting of 3 parts 2% aqueous uranyl acetate, and 1 part 1% KMnO4, mixed and filtered immediately prior to staining (Franceschi et al., 1994
Protein sequences were examined for possible organelle targeting motifs with the following prediction programs: TargetP (Emanuelsson et al., 2000
Since both the dimensions of the glandular cells and the volume of the SCS are known (Turner et al., 2000a Upon request, all novel materials described in this publication will be made available in a timely manner for noncommercial research purposes, subject to the requisite permission from any third-party owners of all or parts of the material. Obtaining any permissions will be the responsibility of the requestor.
We thank Kent McDonald and the staff of the Electron Microscope Laboratory at the University of California, Berkeley, for assistance with freeze-substituted samples. We thank the staff of the Electron Microscopy Center at Washington State University for technical support, John Rogers for helpful discussions, Yujia Wu, Stefan Jennewein, Ed Davis, Kerry Ringer, and Charles Burke for helpful discussions and technical assistance, and Julianna Gothard for growing the plants. Received August 2, 2004; returned for revision September 28, 2004; accepted September 29, 2004.
1 This work was supported by the U.S. Department of Energy, the Mint Industry Research Council, and the Washington State University Agricultural Research Center (Project 0268).  Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.104.050229. * Corresponding author; e-mail croteau{at}wsu.edu; fax 1–509–335–7643.
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ABSTRACT
RESULTS
ski et al., 2001
-D-thiogalactopyranoside and grown overnight at 15°C. The cells were then pelleted by centrifugation at 5,000 rpm, resuspended in chilled 50 mM MOPSO buffer, pH 7.5, that contained 1 mM benzamidine, and sonicated three times for 15 s at 1/4 power with a Virsonic sonicator (VirTis). The resulting lysate was centrifuged at 18,000g for 30 min, then at 195,000g for 90 min at 4°C, to afford the soluble enzyme fraction that was separated by chromatography on Source 30Q anion-exchange resin (Amersham Biosciences, Piscataway, NJ) with a salt gradient (0–1 M NaCl) in 50 mM MOPSO buffer, pH 7.5. The target enzymes, located by activity assay (Ringer et al., 2003
-helices in prokaryotic membrane proteins: the dense alignment surface method. Protein Eng 10: 673–676