Monoterpene Biosynthesis in the GlandularTrichomes of - NCBI

Plant Physiol. (1989) 89, 1351-1357 0032-0889/0000/1351 /07/$01 .00/0

Received for publication July 1, 1988 and in revised form December 2, 1988

Biochemical and Histochemical Localization of Monoterpene Biosynthesis in the Glandular Trichomes of Spearmint (Mentha spicata)" 2 Jonathan Gershenzon, Massimo Maffei3, and Rodney Croteau* Institute of Biological Chemistry, Washington State University, Pullman, Washington 99164-6340 ABSTRACT

type diterpenes found in the glandular exudate. The head cells of tobacco trichomes are the only leaf cells able to incorporate radiolabeled acetate into duvane diterpenes; removal of these cells severely reduces or eliminates duvane production (22). Thus, it is tempting to speculate that all terpenes found in glandular trichomes are synthesized in situ. However, recent reports of monoterpene synthesis by undifferentiated cells in culture (5, 28), coupled to evidence for monoterpene transport in intact plants (7, 31), suggest that the site of synthesis may not necessarily be the same as the site of accumulation. Spearmint (Mentha spicata, Lamiaceae) accumulates large quantities of monoterpenes in glandular trichomes, the major constituent of which is (-)-carvone (20). This monocyclic ketone is biosynthesized by a three step pathway (Fig. 1) in which the ubiquitous primary intermediate geranyl pyrophosphate is cyclized to the olefin (-)-limonene4 (23), which is then hydroxylated by a cytochrome P450-dependent monooxygenase to (-)-trans-carveol (F Karp, R Croteau, unpublished data). (-)-trans-Carveol is subsequently dehydrogenated to (-)-carvone. This last enzymic step has not been previously demonstrated in spearmint, but has ample precedent in monoterpene metabolism in other systems (16, 24). The site of monoterpene biosynthesis in spearmint was studied by investigating the locations of the enzymes catalyzing the three steps in carvone biosynthesis. Procedures for the selective extraction of enzymes from glandular trichomes were used (12, 18), which allowed comparison of cell-free preparations from the trichomes with those of whole leaves from which the glandular trichomes had been removed. Also, since the final step in carvone biosynthesis is mediated by a dehydrogenase, histochemical techniques for studying this enzyme type (26, 32) were employed to probe the cellular and subcellular location of this activity.

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.

Many kinds of lipophilic natural products accumulate in modified epidermal hairs known as glandular trichomes (17). Prominent among these substances are various types of terpenes, including the monoterpenes and sesquiterpenes of the essential oils. It has been generally assumed that the terpenes found in glandular trichomes are synthesized there, since gland cells display many ultrastructural features indicative of active lipid metabolism and secretion (17, 29). However, the difficulties of identifying terpene secretion products microscopically have precluded definite proof of this assumption by ultrastructural methods. Direct evidence for the biosynthetic capabilities of glandular trichomes has come from studies showing that these structures can incorporate labeled precursors such as sucrose, acetate, and mevalonate into terpenes (9). In tobacco, glandular trichomes appear to be the sole site for the synthesis of duvane-

MATERIALS AND METHODS Plant Materials and Reagents

Spearmint (Mentha spicata L.) plants were grown from stolons under controlled conditions as previously described (1 1). Newly emerged leaves (3-20 mm long) were used in all experiments. Polystyrene resin (Amberlite XAD4; Rohm and Haas) was prepared for use by standard procedures (27). All

'This investigation was supported in part by U.S. Department of Energy grant (DE-FG06-88ER1 3869) and by Project 0268 from the Agricultural Research Center, Washington State University, Pullman, WA 99164. 2 Dedicated to the memory of W. R. Nes, colleague and friend. Present address: Istituto di Botanica Speciale, University of Turin, Italy.

4Nomenclature used is based on the p-menthane system: (-)limonene = 4S-p-mentha- 1(2),8(9)-diene; (-)-trans-carveol = 2S,4Rp-mentha-l(6),8(9)-dien-2-ol; (-)-carvone = 4R-p-mentha-1(6),8(9)dien-2-one. 1351

1 352

GERSHENZON ET AL.

0/*.

at 195,000g for 2 h. The resulting pellets were resuspended in the appropriate buffers for assay.

0 3

Geranyl

pyrophosphate

(-)-Limonene

(-)-trans-Carveol

Plant Physiol. Vol. 89, 1989

(-)-Carvone

Figure 1. Pathway for the biosynthesis of (-)-carvone from geranyl pyrophosphate in spearmint. The enzymes involved are geranyl pyrophosphate: (--limonene cyclase (1), (--limonene hydroxylase (2), and (--trans-carveol dehydrogenase (3).

other reagents were purchased from Aldrich or Sigma Chemical Co. unless otherwise noted. Enzyme Extracts

Extracts of glandular trichomes were prepared by two methods. For the first method, leaves were submerged in prechilled extraction buffer and gently brushed with a soft-bristle toothbrush (12). The extraction buffer was 100 mm sodium/potassium phosphate (pH 6.5), containing 1 M sucrose, 5 mM MgCl2, 10 mm Na2S205, 50 mm ascorbic acid, 4 mM DTT (Research Organics), 1 mm EDTA, polyvinylpolypyrrolidone (1 g/g leaf), and XAD-4 resin (2 g/g leaf). This procedure removed approximately 40 to 60% of the glandular trichomes present on the leaf surface as determined microscopically. For the second method, extracts were prepared by a mechanized procedure in which the leaf surfaces were gently abraded with small glass beads (18). Briefly, 5 to 15 g batches of leaves were extracted using a Bead-Beater cell disrupter (Biospec Products) containing 200 g of 0.5 mm diameter glass beads (Biospec Products) and prechilled extraction buffer (formulated as described above) added to nearly full volume of the 10 ounce polycarbonate chamber. Extraction was carried out in twenty 15 s pulses of operation with the rotor speed controlled by a rheostat set at 110 V. Between pulses, the polycarbonate chamber was dismounted and cooled on ice for at least 15 s. This procedure resulted in removal of more than 99% of the glandular trichomes as determined microscopically. Leaves recovered from the second gland extraction procedure were manually homogenized in a Ten-Broeck homogenizer to obtain extracts of whole leaves from which the glandular trichomes had been removed. Homogenization was carried out in the same extraction buffer, without XAD-4, but with additional polyvinylpolypyrrolidone (0.5 g/g leaf). Following homogenization, the extract was slurried with XAD-4 (1 g/g leaf) for 5 min. All extracts were filtered by passage through eight layers of premoistened cheesecloth and then four layers of 80 ,um nylon mesh (Small Parts, Inc.). Filtered extracts were centrifuged at 195,000g for 2 h. The supernatants obtained were divided into several portions, dialyzed to specific assay conditions as noted below, and then concentrated by ultrafiltration (Amicon YM-30) to a small volume suitable for assay. The 195,000g pellets were resuspended in extraction buffer, manually homogenized, stirred with XAD (1 g/g leaf) to ensure complete removal of endogenous monoterpenes, filtered through cheesecloth and nylon mesh, and centrifuged again

Assay for Geranyl Pyrophosphate: (-)-Limonene Cyclase This activity was measured in a 15 mm sodium/potassium phosphate buffer, containing 10% (v/v) glycerol, 5 mM ascorbic acid, and 1 mM DTT. One-mL aliquots of the various extracts were added to Teflon-sealed, screw-capped tubes and the reaction initiated by addition of 20 mM MgCl2 and 18 jM [I -3H]geranyl pyrophosphate (90 Ci/mol) which was synthesized and purified by literature procedures (15). As a trap for the volatile olefin products, 1 mL of pentane was carefully layered on top of the reaction mixture. Following incubation at 30C for 1 h with gentle shaking, the reaction was stopped by vigorous mixing. The limonene generated was extracted from the reaction mixture, and the 3H content determined by chromatographic separation and liquid scintillation counting essentially as previously described (23). The cyclase activity was almost completely restricted (98%) to the 195,000g supernatants. Assay for (-)-Limonene Hydroxylase This activity was determined by incubating 1 mL aliquots of the preparation in 25 mm sodium/potassium phosphate buffer (pH 7.4), containing 30% (v/v) glycerol and 0.5 mM DTT, with 2 mm NADPH and 200 nmol (-)-limonene (optical purity > 80%) for 1 h at 30°C. The reaction was stopped by addition of 1 mL diethyl ether followed by vigorous shaking to extract the (-)-trans-carveol formed. After addition of 25 nmol camphor as an internal standard, the ether layer was removed and the reaction mixture reextracted twice with additional 1 mL portions of ether. The combined ether extracts were decolorized with charcoal, washed with 1 mL of water, passed through a short column of silica gel (type 60A, Mallinckrodt) overlaid with anhydrous MgSO4 in a Pasteur pipet, and concentrated to 200 ,L under vacuum (Savant Speed Vac). Reaction products were analyzed by GLC (Hewlett Packard 5890A with 3392A integrator) using a bonded-phase fusedsilica open-tubular capillary column (30 m x 0.25 mm i.d.) coated with a 0.2 ,um film of Superox FA (Ailtech Associates) and operated with H2 (2 mL/min), on-column injection (injector temperature ambient), temperature programming (45° for 5 min, then 10°/min to 2200 with 15 min hold), and flame ionization detector (detector temperature 230C). (-)-transCarveol was identified by comparison of retention time and mass spectrum with an authentic standard and was quantitated by comparison of detector response to that of the internal standard. Control assays extracted immediately after substrate addition (i.e. zero time) were used to determine the background of endogenous volatile substances in each extract. The small amounts of trans-carveol usually found in these control assays were subtracted from those produced in fullterm assays to determine enzymic production. Limonene hydroxylase activity was found only in the 195,000g pellet, and was not evident in boiled preparations of this type. Assay for (-)-trans-Carveol Dehydrogenase One-mL aliquots of the extract in 100 mm sodium/potassium phosphate buffer (pH 8.0), containing 10% (v/v) glycerol

LOCALIZATION OF MONOTERPENE BIOSYNTHESIS IN SPEARMINT TRICHOMES

and 1 mM DTT, were incubated in the presence of 1 mM NADP and 200 nmol (-)-carveol (62% trans, 38% cis, SCM Specialty Chemicals) for 1 h at 30°C. Reaction products were extracted with ether and analyzed by GLC as described above for hydroxylase assays. Only the (-)-trans-carveol isomer was enzymically oxidized to (-)-carvone. Carveol dehydrogenase activity was principally confined (about 90%) to the 195,000g

supernatants. Partial Purification of (-)-trans-Carveol Dehydrogenase

Carveol dehydrogenase preparations from glandular trichome extracts and whole-leaf (minus trichomes) extracts were purified separately for electrophoretic comparison. After extraction, filtration and centrifugation as detailed above, the supernatants were dialyzed against 100 mm sodium/potassium phosphate buffer (pH 7.0), containing 10% (v/v) glycerol, 2 mm ascorbic acid, 1 mM DTT, and 1 mm EDTA, and then concentrated by ultrafiltration (Amicon YM-30) to volumes of approximately 10 mL. Powdered (NH4)2SO4 was added until 25% saturation was achieved and the precipitated protein was removed by centrifugation at 27,000g for 10 min. The supernatants were then brought to 60% saturation with (NH4)2S04 and centrifuged again. The resulting pellets contained over 97% of the carveol dehydrogenase activity originally present in the extracts. These preparations were suspended in a minimum volume of 100 mm phosphate buffer (pH 8.0), containing 10% (v/v) glycerol, 1 mm ascorbic acid, 0.5 mM DTT, and 0.5 mM EDTA, and applied to a 2.5 x 110 cm column of Sephacryl S-200 (Pharmacia) equilibrated with the same buffer. The column was pumped at a flow rate of 25 mL/h, and 5.3 mL fractions were collected and assayed for carveol dehydrogenase activity, which eluted at approximately 1.55 void volumes. Fractions containing the dehydrogenase were combined, concentrated by ultrafiltration and, after a buffer change by which 1% (w/v) sorbitol was substituted for the 10% (v/v) glycerol, the preparation was lyophilized and stored under N2 at -25°C. As a result of these steps, the total purification of carveol dehydrogenase was 30- to 40fold and recovery was 60 to 80%. Separation of Dehydrogenase Activities by PAGE A comparison between carveol dehydrogenase activities located in the glandular trichomes and those located in the rest of the leaf was made by nondenaturing discontinuous polyacrylamide gel electrophoresis of the corresponding partially purified preparations. Vertical slab gels (16 cm x 18 cm x 1 mm) employing the discontinuous system of Laemmli (25) were cast with a separating gel of 7.5% acrylamide. Prior to loading the sample, the slab gel was subjected to electrophoresis at 40 mA constant current to remove the residual persulfate used in polymerization (21). Lyophilized preparations of carveol dehydrogenase, partially purified as described above, were dissolved in a buffer, formulated to preserve catalytic activity, that contained 50 mM diazabicyclooctane (pH 9.0), 25% (w/v) sucrose, 6 mm 3-mercaptopropionic acid, 5 mm ascorbic acid, 1 mm DTT, and 20 mM NaCl. Aliquots containing from 50 to 200 ,ug protein were loaded into 1 cmwide wells. Two gels at a time were run in a Hofer SE 600 vertical slab gel apparatus with a 4°C cooling bath at a constant

1 353

current of 40 mA for 3 h (approximately 1000 V/h). Standard electrode buffers (25 mm Tris, 192 mM glycine) according to Laemmli (25) were used, but the cathode buffer also contained 1 mm sodium thioglycolate, 3 mM 3-mercaptopropionic acid, and 1 mM EDTA to aid in persulfate removal (4). Gel slices were stained for carveol dehydrogenase activity by incubation in 100 mm phosphate buffer (pH 8.0), containing 0.4 mm nitroblue tetrazolium, 0.065 mM phenazine methosulfate, 0.75 mM NAD, and saturating levels of (-)-carveol. Incubation was carried out for approximately 2 h in the dark in a sealed glass bottle that had been purged with N2. Stained slices were scanned at 633 nm with an LKB 2202 Ultroscan laser densitometer. Control slices assayed without (-)-carveol, or from runs without enzyme, showed no detectable bands. Protein concentrations were estimated using the BioRad protein assay based upon the dye-binding technique of Bradford (3) with BSA as the standard. Histochemical Localization of (-)-trans-Carveol Dehydrogenase Immature leaves (5-10 mm) collected before the plants flowered were cut in half and incubated immediately with the reaction mixture described below in Teflon-sealed screwcapped tubes covered with aluminum foil. Incubation was carried out for 45 min at 30°C with gentle agitation. The composition of the reaction mixture, based on previous studies of dehydrogenase histochemistry (26, 32) and extensive preliminary trials, was 20 mm sodium cacodylate (pH 7.5), 3 mM MgCl2, 1% (w/v) sucrose, 2% (w/v) polyvinylpyrrolidone (Mr 360,000), 0.01% (v/v) Tween 20, 0.6 mm nitroblue tetrazolium, 1 mm phenazine methosulfate, 0.5 mM NADP, and 0.5 mM (-)-carveol. Reaction conditions were optimized in a series of experiments by varying concentrations of nitroblue tetrazolium, phenazine methosulfate, NADP, (-)-carveol, and incubation time. Tween 20 was added to enhance tissue penetration of the hydrophobic substrate, while sucrose and polyvinylpyrrolidone reduced browning of the tissue. A brief (10 s) dip of the leaves in pentane prior to incubation significantly enhanced the apparent reaction rate, presumably by removing cuticle wax and thereby assisting substrate uptake. Direct comparisons were made between leaves incubated with the above reaction mixture and controls incubated without carveol. To distinguish carveol dehydrogenase from alcohol (ethanol) dehydrogenase activity, experiments were also undertaken with 0.5 mm ethanol as a substrate in place of carveol, and by including in the reaction mixture 15 mm pyrazole, an inhibitor of alcohol dehydrogenase. After incubation, the leaves were rinsed with 20 mm sodium cacodylate (pH 7.0) for 20 min and then fixed with 3% (v/v) glutaraldehyde in 20 mm sodium cacodylate buffer for 1 h. Fixation prior to incubation greatly reduced the rate of the dehydrogenase reaction. After washing the fixed tissue with sodium cacodylate for 30 min, the leaves were postfixed with 1% (W/V) OS04 in 10 mm sodium cacodylate (pH 7.0) for 90 min at room temperature, then washed with distilled water for 45 min, dehydrated in a graded ethanol series ending with 100% propylene oxide, and embedded in Spurr's low viscosity resin (30). Silver-gold thin sections were obtained with a Reichert OM U2 ultramicrotome. Sections were stained with lead citrate, uranyl acetate, and lead citrate again, in sequence

GERSHENZON ET AL.

1 354

4

-,4

cu"

*

5

a

.

SC

t.".,. i I:,

"f;

-t i.,

1. : ./. :., g I'AL

_ek



(14) and viewed with a Hitachi H-300 transmission electron microscope.

RESULTS Localization of Carvone Biosynthetic Capability by CellFree Assays To determine whether (-)-carvone biosynthesis takes place specifically in the glandular trichomes of spearmint, the activities ofthe enzymes associated with the relevant pathway were measured in cell-free extracts ofthe glandular trichomes. Two types of procedures were employed to extract selectively the glandular contents. In the first method, leaves were submerged in extraction buffer and then gently brushed with a toothbrush to obtain a glandular trichome extract containing the material from only 40 to 60% of the glandular trichomes present on the leaf surface, but with essentially no contamination from underlying tissue (12). All three enzymes involved in the synthesis of carvone from the ubiquitous isoprenoid intermediate geranyl pyrophosphate (i.e. the cyclase, hydroxylase, and dehydrogenase) were readily detected in this extract indicating that the glandular trichomes were capable of carvone biosynthesis. To determine the biosynthetic capability of the trichome extract relative to that of the rest of the leaf, a second experiment was performed using a more efficient procedure for gland removal. Leaf surfaces were abraded with glass beads (18) to remove over 99% of all the glandular trichomes (Figs. 2 and 3) without extensive damage to the underlying leaf tissue. Using this method, an extract of the contents of virtually all of the glandular trichomes on the leaf was obtained. The remaining leaf tissue was extracted by homogenization to give a preparation representing the leaf free of gland contents. Comparison of these two extracts showed that two of the three enzymes of the carvone pathway, geranyl pyrophosphate: (-)-limonene cyclase and (-)-limonene hydroxylase, were almost completely restricted to the glandular trichomes (Table I). The traces of these two activities in the remainder of the leaf probably result from incomplete removal of glandular trichomes from this preparation. The third enzyme, (-)trans-carveol dehydroganase, was found in substantial amounts in both the glandular trichomes and the rest of the leaf (Table I).

1355

Histochemical Localization of Carveol Dehydrogenase To confirm that carveol dehydrogenase activity is present throughout the cells of the leaf, an adaptation of a commonly used method for histochemical localization of dehydrogenases was employed. This method is based on the reduction of tetrazolium salts to insoluble formazans by NADPH, with phenazine methosulfate as an intermediate electron carrier (26, 32). Leaf sections were incubated with nitroblue tetrazolium, phenazine methosulfate, NADP, and the substrate (-)trans-carveol, and then fixed and prepared for transmission electron microscopy. The presence of carveol dehydrogenase was indicated by small dark granules of formazan precipitate. Formazan granules were found in cells of all tissues of the leaf, including the epidermis, the palisade layer, the spongy parenchyma, the vascular bundles, and both types of glandular trichomes, peltate and capitate (Fig. 4). Granules in the glandular trichomes were associated with the smooth endoplasmic reticulum (Fig. 6), while granules in the cells of the rest of the leaf were often associated with smooth endoplasmic reticulum, and also with other membrane systems (Fig. 7). Controls incubated without carveol showed only sporadic reaction zones in some mesophyll cells, with no consistent subcellular location (Fig. 5). To examine the possibility that the putative carveol dehydrogenase activity was due to alcohol (ethanol) dehydrogenase, leaf sections were incubated with ethanol as a substrate under otherwise identical conditions. The reaction attributed to alcohol dehydrogenase activity was observed only in mesophyll cells and was localized near the plasmalemma (Fig. 8), a pattern clearly distinguishable from that produced by carveol dehydrogenase. Pyrazole, an inhibitor of alcohol dehydrogenase, also inhibited the reaction of carveol dehydrogenase and was thus not useful as a diagnostic tool. Therefore, the summation of both histochemical and in vitro results were consistent, and indicated that carveol dehydrogenase activity is present in glandular trichomes and in the rest of the leaf.

Electrophoretic Comparison of Carveol Dehydrogenase Activities from Glandular Trichomes and the Remaining Leaf The oxidation of carveol is the final step in carvone biosynthesis and it therefore seems unlikely that the alcohol would

Figures 2 and 3. Scanning electron micrographs (x90) of spearmint leaf surfaces before (Fig. 2) and after (Fig. 3) selective removal of the glandular trichomes with glass beads (PT, peltate glandular trichomes). More than 99% of these structures were removed by this method. Arrows in Figure 3 show former locations of glandular trichomes. Samples for scanning electron microscopy were air-dried ovemight at room temperature and gold-coated. Specimens were viewed with an ETEC Autoscan U-1 at 30 kV. Figures 4-8. Histochemical localization of carveol dehydrogenase in sections of young spearmint leaves. Leaves were incubated with the reaction mixtures described in "Materials and Methods," then fixed and prepared for transmission electron microscopy. The presence of carveol dehydrogenase is indicated by a precipitate of formazan granules. Figure 4. Peltate glandular trichome and adjacent mesophyll following incubation with carveol showing a precipitate in all tissues (CU, cuticle; SS, subcuticular space; SC, secretory cell; ST, stalk cell; BC, basal cell; E, epidermis; M, mesophyll) (x1370). Figure 5. Control section incubated without carveol, showing only sporadic precipitate in the mesophyll (x1370). Figure 6. Section of a secretory cell illustrating the subcellular location (arrows) of carveol dehydrogenase in smooth endoplasmic reticulum (SER) (x68,400). Figure 7. Mesophyll cell showing precipitate associated with smooth endoplasmic reticulum and other membrane systems (CW, cell wall)

(x 1 3,700).

Figure 8. Subcellular localization of alcohol (ethanol) dehydrogenase (arrows) near the plasmalemma of a mesophyll cell indicating that carveol dehydrogenase and alcohol dehydrogenase are not equivalent (x13,700).

1356

GERSHENZON ET AL.

Table I. Localization of Enzymes of Carvone Biosynthesis in Spearmint Extracts Glandular trichomes were first removed from the leaves by abrasion with glass beads, and the remaining leaves were then homogenized. The preparation and assay of each enzyme is described in "Materials and Methods." Activity Enzyme

Glandular

Glandular Remaining trchomes

trichomes

leaf

Geranyl pyrophosphate:

nkat/kg tissue 5220 25.0

99.5

(--limonene cyclase (-)-trans-Carveol dehydro-

150 8080

96.4 30.9

(-)Limonene hydroxylase

5.6

18,100

%

genase

A


turing PAGE, stained to reveal carveol dehydrogenase activ-

ity, displayed significant differences between the two preparations. Only a single staining band was present in the trichome-free leaf extracts, whereas two bands of activity were present in extracts of the glandular trichomes (Fig. 9). The slower moving and less intense of these two bands had the same mobility as the single band in the trichome-free leaf extract. A plausible interpretation is that the dehydrogenase unique to the glandular trichome extract is the enzyme responsible for carveol oxidation in the intact plant (such highly selective monoterpenol dehydrogenases have been previously reported in other plants [8, 16]), whereas the enzyme present in both extracts represents a dehydrogenase activity found in all tissues which, while capable of oxidizing carveol under in vitro conditions, is probably not associated with monoterpene biosynthesis in vivo. Thus, the enzymes responsible for the entire pathway of carvone biosynthesis in spearmint appear to be restricted to the glandular trichomes.

cv,

co

C.) %us

a) CO

coU1)

B

0)

0 a L-

ID

-c:a) m 0

I

I

U1) L..

0o 40

50

60

70

Mobility (mm) Figure 9. PAGE of partially purified carveol dehydrogenase preparations from glandular trichomes (A) and from the remainder of the leaf following gland removal (B). Gels were run, stained, and scanned as described under "Materials and Methods." The glandular trichome extract contains two species of carveol dehydrogenase activity, whereas the extract of the remainder of the leaf contains only the slower migrating species.

be transported out of the glandular trichomes for conversion to the ketone and then transported back into the trichomes for storage. Rather, it seems more probable that carveol oxidation would occur within the trichomes, perhaps catalyzed by a unique species of dehydrogenase. To evaluate this possibility, the carveol dehydrogenase activities from both the glandular trichome extract and the remaining leaf (free of trichomes) extract were separately purified by combination of (NH4)2S04 precipitation and gel filtration. The activities from both extracts coeluted on gel filtration. However nondena-

DISCUSSION For many years, glandular trichomes have been regarded as the primary site of monoterpene synthesis in mints and related essential oil plants (1, 9, 17). The present results demonstrate that these structures are in fact the sole sites of monoterpene biosynthesis in the leaves of spearmint. In conjunction with the work of Keene and Wagner on the diterpenes oftobacco (22), it can now be suggested that all terpenes accumulated in, or exuded from, glandular trichomes are probably synthesized in these highly specialized structures. Since monoterpene alcohols are known to be transported as glycosides (7, 31) (i.e. in mature peppermint (+)-neomethylf3-D-glucoside is transported from leaves to rhizomes and catabolized at this site [10]), it is conceivable that the monoterpenes of spearmint are first synthesized elsewhere in the leaf and then transported to the glandular trichomes. However, this possibility appears to be eliminated by the present results. Additionally, it is extremely unlikely that monoterpenes are biosynthesized elsewhere in the plant since stem and root extracts are devoid of limonene cyclase activity (J Gershenzon, R Croteau, unpublished data). Because monoterpene synthesis is restricted to the glandular trichomes, it is evident that the timing of gland ontogeny will directly influence the concentration of monoterpenes in a leaf and how this varies throughout leaf development. Typically, most glandular trichomes are initiated very early in leaf development and begin to accumulate monoterpenes before the leaves are 5 mm long (1). Therefore, monoterpene concentrations (per gram tissue) are relatively high in young leaves and decline with further development (13). Ultrastructural studies have concluded that terpene synthesis within the glandular trichome occurs principally in the secretory cells rather than the stalk cell or the basal cell (see Fig. 4) (17, 29); the present results support this generalization. Carveol dehydrogenase activity, as indicated by the presence of formazan granules, was always much more prominent in secretory cells than in other cell types for both peltate and capitate trichomes. At the subcellular level, carveol dehydrogenase activity in spearmint secretory cells was associated with smooth endoplasmic reticulum. This result is somewhat surprising in light ofthe observation that the carveol dehydro-


genase was operationally soluble (>90% ofthe activity resided in the 195,000g supernatant) and suggests that the enzyme is released from membranes during tissue extraction (2). However, the subcellular localization of carveol dehydrogenase must be viewed with some caution since, in the histochemical procedures used in this study, NADPH and phenazine methosulfate are freely diffusible and so may produce artefacts (26). Smooth endoplasmic reticulum has been frequently observed in cells assumed to produce terpene secretions ( 17, 19, 29). Yet, the lack of suitable methods for identifying terpenoids under the electron microscope has prevented any ultrastructural demonstration of the direct involvement of smooth endoplasmic reticulum in terpene biogenesis. Recently, however, the synthesis of sesquiterpene olefins in Citrofortunella mitis fruits was shown to be associated with a membrane fraction containing endoplasmic reticulum (2). Another type of organelle often linked with monoterpene synthesis is the leucoplast, a plastid of complex shape without thylakoids (6). Plastids of this description were present in the secretory cells of spearmint glandular trichomes. However, no histochemical evidence was found for the association of carveol dehydrogenase activity with these organelles. Leucoplasts in spearmint glands might serve as compartments for some of the earlier steps of monoterpene synthesis. Detailed evaluation of the localization of monoterpene biogenesis at the subcellular level must await the development of immunochemically based techniques.

10. Croteau R, Martinkus C (1979) Metabolism of monoterpenes: Demonstration of (+)-neomenthyl-fl-D-glucoside as a major 11.

12.

13. 14.

15. 16.

17. 18.

19. 20.

ACKNOWLEDGMENTS

21.

We thank Greg Wichelns for raising the plants, Nancy Madsen for typing the manuscript, and the staff of the Electron Microscopy Center, Washington State University, for assistance.

22.

23.

LITERATURE CITED 1. Amelunxen F (1965) Elektronenmikroskopische Untersuchungen an den Druisenschuppen von Mentha piperita L. Planta Med

13: 457-473 2. Belingheri L, Pauly G, Gleizes M, Marpeau A (1988) Isolation by an aqueous two-polymer phase system and identification of endomembranes from Citrofortunella mitis fruits for sesquiterpene hydrocarbon synthesis. J Plant Physiol 132: 80-85 3. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248-254 4. Brewer JM (1967) Artifact produced in disc electrophoresis by ammonium persulfate. Science 156: 256-257 5. Charlwood BV, Charlwood KA (1983) The biosynthesis of monoand sesquiterpenes in tissue culture. Biochem Soc Trans 11: 592-593 6. Cheniclet C, Carde J-P (1985) Presence of leucoplasts in secretory cells and of monoterpenes in the essential oil: a correlative study. Isr J Bot 34: 219-238 7. Croteau R (1987) Biosynthesis and catabolism of monoterpenoids. Chem Rev 87: 929-954 8. Croteau R, Felton M (1980) Substrate specificity ofmonoterpenol dehydrogenases from Foeniculum vulgare and Tanacetum vulgare. Phytochemistry 19: 1343-1347 9. Croteau R, Johnson MA (1984) Biosynthesis of terpenoids in glandular trichomes. In E Rodriguez, PL Healy, I Mehta, eds, Biology and Chemistry of Plant Trichomes. Plenum, New York, pp 133-185

1357

24.

25. 26. 27.

28. 29. 30. 31. 32.

metabolite of (-)-menthone in peppermint (Mentha piperita). Plant Physiol 64: 169-175 Croteau R, Venkatachalam KV (1986) Metabolism of monoterpenes: Demonstration that (+)-cis-isopulegone, not piperitenone, is the key intermediate in the conversion of (-)-isopiperitenone to (+)-pulegone in peppermint (Mentha piperita). Arch Biochem Biophys 249: 306-315 Croteau R, Winters JN (1982) Demonstration of the intercellular compartmentation of l-menthone metabolism in peppermint (Mentha piperita). Plant Physiol 69: 975-977 Croteau R, Felton M, Karp F, Kjonaas R (1981) Relationship of camphor biosynthesis to leaf development in sage (Salvia officinalis). Plant Physiol 67: 820-824 Daddow LYM (1983) A double lead stain method for enhancing contrast of ultrathin sections in electron microscopy: a modified multiple staining technique. J Microsc 129: 147-153 Davisson VJ, Woodside AB, Poulter CD (1985) Synthesis of allylic and homoallylic isoprenoid pyrophosphates. Methods Enzymol 110: 130-144 Dehal SS, Croteau R (1987) Metabolism of monoterpenes: specificity of the dehydrogenases responsible for the biosynthesis of camphor, 3-thujone and 3-isothujone. Arch Biochem Biophys 258: 287-291 Fahn A (1979) Secretory Tissues in Plants. Academic Press, New York Gershenzon J, Duffy MA, Karp F, Croteau R (1987) Mechanized techniques for the selective extraction of enzymes from plant epidermal glands. Anal Biochem 163: 159-164 Gleizes M, Carde J-P, Pauly G, Bernard-Dagan C (1980) In vivo formation of sesquiterpene hydrocarbons in the endoplasmic reticulum of pine. Plant Sci Lett 20: 79-90 Guenther E (1974) The Essential Oils, Vol III (reprinted). Krieger, Huntington, NY, p 681 Heeb MJ, Gabriel 0 (1984) Enzyme localization in gels. Methods Enzymol 104: 416-439 Keene CK, Wagner GJ (1985) Direct demonstration of duvatrienediol biosynthesis in glandular heads of tobacco trichomes. Plant Physiol 79: 1026-1032 Kjonaas R, Croteau R (1983) Demonstration that limonene is the first cyclic intermediate in the biosynthesis of oxygenated p-menthane monoterpenes in Mentha piperita and other Mentha species. Arch Biochem Biophys 220: 79-89 KJonaas R, Venkatachalam KV, Croteau R (1985) Metabolism of monoterpenes: Oxidation of isopiperitenol to isopiperitenone, and subsequent isomerization to piperitenone by soluble enzyme preparations from peppermint (Mentha piperita) leaves. Arch Biochem Biophys 238: 49-60 Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680685 Lewis PR (1977) Other cytochemical methods for enzymes. In AM Glauert, ed, Practical Methods in Electron Microscopy, Vol 5. North-Holland, Amsterdam, pp 225-287 Loomis WD, Lile JD, Sandstrom RP, Burbott AJ (1979) Adsorbent polystyrene as an aid in plant enzyme isolation. Phytochemistry 18: 1049-1054 Nabeta K, Ohnishi Y, Hirose T, Sugisawa H (1983) Monoterpene biosynthesis by callus tissues and suspension cells from Perilla species. Phytochemistry 22: 423-425 Schnepf E (1974) Gland cells. In AW Robards, ed, Dynamic Aspects of Plant Ultrastructure. McGraw-Hill, London, pp 331-357 Spurr AR (1969) A low-viscosity epoxy resin embedding medium for electron microscopy. J Ultrastruct Res 26: 31-43 Stahl-Biskup E (1987) Monoterpene glycosides, state-of-the-art. Flavour Fragr J 2: 75-82 Van Noorden CJF (1984) Histochemistry and Cytochemistry of Glucose-6-Phosphate Dehydrogenase. Gustav Fischer, Stuttgart