Plant Physiol. (1989) 89, 1351-1357 Received for publication July 1, 1988 0032-0889/0000/1351 /07/$01 .00/0 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 The primary monoterpene accumulated in the glandular tri- oftobacco trichomes are the only leafcells able to incorporate chomes of spearmint (Mentha spicata) is the ketone (-)-carvone radiolabeled acetate into duvane diterpenes; removal ofthese which is formed by cyclization of the C10 isoprenoid intermediate cells severely reduces or eliminates duvane production (22). geranyl pyrophosphate to the olefin (-)-limonene, hydroxylation Thus, it is tempting to speculate that all terpenes found in to (-)-trans-carveol and subsequent dehydrogenation. Selective glandular trichomes are synthesized in situ. However, recent extraction of the contents of the glandular trichomes indicated reports of monoterpene synthesis by undifferentiated cells in that essentially all of the cyclase and hydroxylase activities culture (5, 28), coupled to evidence for monoterpene transport resided in these structures, whereas only about 30% of the in intact plants (7, 31), suggest that the site of synthesis may carveol dehydrogenase was located here with the remainder not necessarily be the same as the site of accumulation. located in the rest of the leaf. This distribution of carveol dehy- drogenase activity was confirmed by histochemical methods. Spearmint (Mentha spicata, Lamiaceae) accumulates large Electrophoretic analysis of the partially purified carveol dehydro- quantities of monoterpenes in glandular trichomes, the major genase from extracts of both the glands and the leaves following constituent of which is (-)-carvone (20). This monocyclic gland removal indicated the presence of a unique carveol dehy- ketone is biosynthesized by a three step pathway (Fig. 1) in drogenase species in the glandular trichomes, suggesting that which the ubiquitous primary intermediate geranyl pyrophos- the other dehydrogenase found throughout the leaf probably phate is cyclized to the olefin (-)-limonene4 (23), which is utilizes carveol only as an adventitious substrate. These results then hydroxylated by a cytochrome P450-dependent mono- demonstrate that carvone biosynthesis takes place exclusively oxygenase to (-)-trans-carveol (F Karp, R Croteau, unpub- in the glandular trichomes in which this natural product accumu- lished data). (-)-trans-Carveol is subsequently dehydrogen- lates. ated to (-)-carvone. This last enzymic step has not been previously demonstrated in spearmint, but has ample prece- dent in monoterpene metabolism in other systems (16, 24). The site of monoterpene biosynthesis in spearmint was studied by investigating the locations of the enzymes catalyz- Many kinds of lipophilic natural products accumulate in ing the three steps in carvone biosynthesis. Procedures for the modified epidermal hairs known as glandular trichomes (17). selective extraction ofenzymes from glandular trichomes were Prominent among these substances are various types of ter- used (12, 18), which allowed comparison of cell-free prepa- penes, including the monoterpenes and sesquiterpenes of the rations from the trichomes with those of whole leaves from essential oils. It has been generally assumed that the terpenes which the glandular trichomes had been removed. Also, since found in glandular trichomes are synthesized there, since the final step in carvone biosynthesis is mediated by a dehy- gland cells display many ultrastructural features indicative of drogenase, histochemical techniques for studying this enzyme active lipid metabolism and secretion (17, 29). However, the type (26, 32) were employed to probe the cellular and sub- difficulties of identifying terpene secretion products micro- cellular location of this activity. scopically have precluded definite proof of this assumption by ultrastructural methods. MATERIALS AND METHODS Direct evidence for the biosynthetic capabilities ofglandular trichomes has come from studies showing that these structures Plant Materials and Reagents can incorporate labeled precursors such as sucrose, acetate, and mevalonate into terpenes (9). In tobacco, glandular tri- Spearmint (Mentha spicata L.) plants were grown from chomes appear to be the sole site for the synthesis ofduvane- stolons under controlled conditions as previously described (1 1). Newly emerged leaves (3-20 mm long) were used in all 'This investigation was supported in part by U.S. Department of experiments. Polystyrene resin (Amberlite XAD4; Rohm and Energy grant (DE-FG06-88ER1 3869) and by Project 0268 from the Haas) was prepared for use by standard procedures (27). All Agricultural Research Center, Washington State University, Pullman, WA 99164. 4Nomenclature used is based on the p-menthane system: (-)- 2 Dedicated to the memory of W. R. Nes, colleague and friend. limonene = 4S-p-mentha- 1(2),8(9)-diene; (-)-trans-carveol = 2S,4R- Present address: Istituto di Botanica Speciale, University ofTurin, p-mentha-l(6),8(9)-dien-2-ol; (-)-carvone = 4R-p-mentha-1(6),8(9)- Italy. dien-2-one. 1351 1 352 GERSHENZON ET AL. Plant Physiol. Vol. 89, 1989 at 195,000g for 2 h. The resulting pellets were resuspended in 0/*. 0 the appropriate buffers for assay. 3 Assay for Geranyl Pyrophosphate: (-)-Limonene Cyclase This activity was measured in a 15 mm sodium/potassium Geranyl (-)-Limonene (-)-trans-Carveol (-)-Carvone phosphate buffer, containing 10% (v/v) glycerol, 5 mM ascor- pyrophosphate bic acid, and 1 mM DTT. One-mL aliquots of the various Figure 1. Pathway for the biosynthesis of (-)-carvone from geranyl extracts were added to Teflon-sealed, screw-capped tubes and pyrophosphate in spearmint. The enzymes involved are geranyl py- rophosphate: (--limonene cyclase (1), (--limonene hydroxylase (2), the reaction initiated by addition of 20 mM MgCl2 and 18 jM and (--trans-carveol dehydrogenase (3). [I-3H]geranyl pyrophosphate (90 Ci/mol) which was synthe- sized and purified by literature procedures (15). As a trap for the volatile olefin products, 1 mL of pentane was carefully other reagents were purchased from Aldrich or Sigma Chem- layered on top of the reaction mixture. Following incubation ical Co. unless otherwise noted. at 30C for 1 h with gentle shaking, the reaction was stopped by vigorous mixing. The limonene generated was extracted Enzyme Extracts from the reaction mixture, and the 3H content determined by chromatographic separation and liquid scintillation counting Extracts ofglandular trichomes were prepared by two meth- essentially as previously described (23). The cyclase activity ods. For the first method, leaves were submerged in prechilled was almost completely restricted (98%) to the 195,000g extraction buffer and gently brushed with a soft-bristle tooth- supernatants. brush (12). The extraction buffer was 100 mm sodium/potas- Assay for (-)-Limonene Hydroxylase sium phosphate (pH 6.5), containing 1 M sucrose, 5 mM MgCl2, 10 mm Na2S205, 50 mm ascorbic acid, 4 mM DTT This activity was determined by incubating 1 mL aliquots (Research Organics), 1 mm EDTA, polyvinylpolypyrrolidone of the preparation in 25 mm sodium/potassium phosphate (1 g/g leaf), and XAD-4 resin (2 g/g leaf). This procedure buffer (pH 7.4), containing 30% (v/v) glycerol and 0.5 mM removed approximately 40 to 60% ofthe glandular trichomes DTT, with 2 mm NADPH and 200 nmol (-)-limonene (op- present on the leaf surface as determined microscopically. tical purity > 80%) for 1 h at 30°C. The reaction was stopped For the second method, extracts were prepared by a mech- by addition of 1 mL diethyl ether followed by vigorous anized procedure in which the leaf surfaces were gently shaking to extract the (-)-trans-carveol formed. After addition abraded with small glass beads (18). Briefly, 5 to 15 g batches of 25 nmol camphor as an internal standard, the ether layer of leaves were extracted using a Bead-Beater cell disrupter was removed and the reaction mixture reextracted twice with (Biospec Products) containing 200 g of0.5 mm diameter glass additional 1 mL portions of ether. The combined ether ex- beads (Biospec Products) and prechilled extraction buffer tracts were decolorized with charcoal, washed with 1 mL of (formulated as described above) added to nearly full volume water, passed through a short column of silica gel (type 60A, of the 10 ounce polycarbonate chamber. Extraction was car- Mallinckrodt) overlaid with anhydrous MgSO4 in a Pasteur ried out in twenty 15 s pulses of operation with the rotor pipet, and concentrated to 200 ,L under vacuum (Savant speed controlled by a rheostat set at 110 V. Between pulses, Speed Vac). the polycarbonate chamber was dismounted and cooled on Reaction products were analyzed by GLC (Hewlett Packard ice for at least 15 s. This procedure resulted in removal of 5890A with 3392A integrator) using a bonded-phase fused- more than 99% of the glandular trichomes as determined silica open-tubular capillary column (30 m x 0.25 mm i.d.) microscopically. coated with a 0.2 ,um film of Superox FA (Ailtech Associates) Leaves recovered from the second gland extraction proce- and operated with H2 (2 mL/min), on-column injection (in- dure were manually homogenized in a Ten-Broeck homoge- jector