Metabolismof Monoterpenes Cell Cultures officinalis)I · CommonSage(Salvia officinalis)I Biochemical Rationaleforthe Lackof MonoterpeneAccumulation KimberlyL. Falk, JonathanGershenzon,

Plant Physiol. (1990) 93, 1559-15670032-0889/90/93/1 559/09/$01 .00/0

Received for publication January 5, 1990Accepted Aprl 19,1990

Metabolism of Monoterpenes in Cell Cultures ofCommon Sage (Salvia officinalis)I

Biochemical Rationale for the Lack of Monoterpene Accumulation

Kimberly L. Falk, Jonathan Gershenzon, and Rodney Croteau*Institute of Biological Chemistry, and Graduate Program in Plant Physiology, Washington State University,

Pullman, Washington 99164-6340

ABSTRACT

Leaves of common sage (Salvia officinalis) accumulate mono-terpenes in glandular trichomes at levels exceeding 15 milligramsper gram fresh weight at maturity, whereas sage cells in suspen-sion culture did not accumulate detectable levels of monoter-penes (<0.3 nanograms per gram fresh weight) at any stage ofthe growth cycle, even in the presence of a polystyrene resintrap. Monoterpene biosynthesis from [U-14C]sucrose was alsovirtually undetectable in this cell culture system. In vitro assay ofeach of the enzymes required for the sequential conversion ofthe ubiquitous isoprenoid precursor geranyl pyrophosphate to(+)-camphor (a major monoterpene product of sage) in solubleextracts of the cells revealed the presence of activity sufficientto produce (+)-camphor at a readily detectable level (>0.3 micro-grams per gram fresh weight) at the late log phase of growth.Other monoterpene synthetic enzymes were present as well. Invivo measurement of the ability to catabolize (+)-camphor inthese cells indicated that degradative capability exceeded bio-synthetic capacity by at least 1000-fold. Therefore, the lack ofmonoterpene accumulation in undifferentiated sage culturescould be attributed to a low level of biosynthetic activity (relativeto the intact plant) coupled to a pronounced capacity for mono-terpene catabolism.

The accumulation of terpenoid natural products in plantcell cultures has been successfully demonstrated in the casesof diterpenoids and sesquiterpenoids, but rarely in the case ofmonoterpenes. Thus, there are reports of the production ofditerpenoid substances in culture at levels exceeding those ofthe intact plant (42, 43) and the induced accumulation ofsesquiterpene phytoalexins in culture is well documented (12,13, 16), whereas most accounts ofmonoterpene accumulationin cell culture systems (1, 17, 44) describe either very lowlevels of production or compositional patterns that differmarkedly from those of the intact plant.Monoterpenes in intact plants usually accumulate in the

extracellular storage spaces of specialized secretory structures,such as glandular trichomes, resin ducts, or resin cavities (35),

' Research supported in part by U.S. Department of Energy grantDE-FG06-88ER13869 and by Project 0268 from the AgriculturalResearch Center, Washington State University, Pullman, WA 99164.

and considerable evidence indicates that these secretory struc-tures are the primary, if not the exclusive, sites of monoter-pene biosynthesis (21, 41, 49). It might seem then that mon-oterpenes are unlikely to be produced in cell culture systemsin the absence of such organized structures. In fact, undiffer-entiated callus of Mentha piperita (peppermint) showed notrace of monoterpene accumulation (6), whereas M. piperitacallus with adventitious shoots, containing leaflets with glan-dular trichomes, produced significant quantities of monoter-penes (10, 1 1). However, differentiation to the level of glan-dular trichomes is not always a precondition for monoterpeneaccumulation in cell culture (17), indicating that, at leastunder some conditions, monoterpene biosynthesis is possiblein less organized systems.At a fundamental level, the general absence ofmonoterpene

accumulation in undifferentiated cultures could be due to thelack of significant biosynthetic activity or to the presence ofefficient catabolic processes. De novo monoterpene biosyn-thesis, as distinct from monoterpene accumulation (1, 17, 44)or the biotransformation of exogenous monoterpenes (19),has rarely been directly measured (5). Several investigators(2-4, 34) have demonstrated the conversion of labeled mev-alonic acid to more advanced precursors, such as dimethylal-lyl, geranyl, and farnesyl pyrophosphate, in cell-free extractsfrom cultures of diverse essential oil species, but it is notpossible to determine with certainty whether these metabolitesrepresent intermediates in the biosynthesis of monoterpenesor in the formation of higher products such as phytosterols.By contrast, the efficient biotransformation of exogenousmonoterpenes in cell culture ( 19) implies that at least portionsof monoterpene metabolic pathways may be widely presentin these systems. The ability ofplant cell cultures to catabolizeadded monoterpenes (1, 7, 15, 20) suggests that degradativecapability may be critically important in avoiding the toxiceffects of these compounds on the growth and viability ofcells in culture ( 14).

In this paper, we describe the metabolism of (+)-camphorand other monoterpenes in cell cultures of common sage(Salvia officinalis). Camphor metabolism was emphasized inthis study because this bicyclic ketone is one of the majormonoterpenoid products of the intact plant and because thepathway and enzymes of camphor biosynthesis from theubiquitous isoprenoid precursor, geranyl pyrophosphate, are

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well known (Fig. 1) (28, 31 '32). Additionally, the early stepsin the catabolism of camphor, via 1,2-campholide and thecorresponding glucoside-glucose ester (Fig. 1), in S. officinalisleaves have been documented (25, 26). In the present work,the virtual absence of monoterpene accumulation in S. offi-cinalis cell suspension cultures was shown to result from alow level of biosynthetic activity coupled to a pronouncedability to catabolize these compounds.

MATERIALS AND METHODS

Plant Materials, Substrates, and Reagents

Leaves of common sage (Salvia officinalis L.) were surfacesterilized by soaking in 2% aqueous NaOCl containing 0.02%Tween 20 for 10 min followed by rinsing with sterile, distilledwater. Discs (5 mm diameter) cut from the sterilized leaveswere placed on Murashige-Skoog medium (45) containing 0.2mg/L 2,4-D, 1 mg/L kinetin, and 0.8% (w/v) Phytagar (GibcoLaboratories), and the resulting callus was subcultured every4 weeks (since September 1986) onto maintenance mediumcontaining 1.0 mg/L 2,4-D and 1.0 mg/L kinetin, and keptin the dark at 28°C. For the preparation of suspension cul-tures, flasks containing 50 mL maintenance medium withoutagar were each inoculated with 1.5 g of callus tissue, and wereincubated in the dark at 28°C on an orbital shaker (125 rpm).For time-course studies, cultures were initiated with a 10 mLaliquot (approximately 1 g) of first passage suspension cellsharvested at 7 to 10 d (early log phase) and added to freshmedium.The preparation of (+)-[U-'4C]camphor was carried out by

exposing approximately 500 sage plants (28 d old) to 1 mCiof '4CO2 (generated from Na214CO3 at 0.3 Ci/mol) in a sealedacrylic chamber under illumination. After 1 h of exposure,the chamber was flushed with air into a KOH trap. The apicalbuds plus the top leaf pairs were harvested 24 h later, steamdistilled, and the [U-'4C]camphor (- 10 mg at 1.1 mCi/mol)was isolated from the distillate by TLC on silica gel G (hex-anes:ether, 2:1 (v/v)). The sources of (+)-borneol, (+)-cam-phor, (+)-bornyl pyrophosphate, and (+)- 1,2-campholidehave been described (26, 28, 32). [1-3H]Geraniol and [1-3H]geranyl pyrophosphate (100 Ci/mol) were prepared by stand-ard procedures (31). Tritium-labeled monoterpene olefinswere obtained by incubating cell-free extracts from sage leaveswith [1-3H]geranyl pyrophosphate as previously described(37). Radio-GLC analysis of this olefin mixture confirmedthe presence of a-pinene, camphene, p-pinene, myrcene, lim-onene, and sabinene (at a combined specific activity of 100Ci/mol). [U-'4C]Sucrose (671 Ci/mol) was obtained fromNew England Nuclear. All other reagents and biochemicalswere obtained from Aldrich or Sigma Chemical Co. unlessotherwise noted.

Monoterpene Adsorption by Polystyrene Resin in SageCell Culture

To test the efficacy of beaded polystyrene resin (AmberliteXAD-4, Rohm and Haas) as a lipophilic trap for volatilemonoterpenes in suspension culture, flasks containing 40 mLof the maintenance medium and 500 mg of resin (washed

BIOSYNTHESIS

rSo~PP

GeranylPyrophosphate

* CATABOLISM

OPP

BornylPyrophosphate

4

0

Glc

Borneol

* 1 ,2-Campholide

CamphorFigure 1. Pathways for the conversion of geranyl pyrophosphate tocamphor, and for the conversion of camphor to the glucoside-glucoseester of 1,2-campholide.

1 560 FALK ET AL.



MONOTERPENE METABOLISM IN SAGE CELL CULTURE

exhaustively with 95% ethanol and pentane, then air dried)were inoculated with a 10 mL suspension of early log phase(d 7) cells plus either 1.14 uCi of [1-3H]geraniol or 1.03 uCiof sage-derived [3H]monoterpene olefins. Control flasks con-tained 50 mL of the maintenance medium (without cells) and500 mg XAD resin plus either 1.14 uCi of [I -3H]geraniol or1.03 ,uCi of [3H]olefins. After 14 d of incubation, the cultureswere chilled on ice then centrifuged at 200g for 10 min. TheXAD resin, which floated after centrifugation, was separatedfrom the mixture of medium and cells, and washed threetimes with 3 mL aliquots of diethyl ether if the inoculum was[1-3H]geraniol, or twice with pentane (3 mL) and twice withether (3 mL) if the initial inoculum was [3H]olefins. Cellswere filtered from the medium and homogenized in waterwith a Ten-Broeck homogenizer, and the homogenate wasextracted as above, depending on the inoculum. The mediumwas similarly extracted. In flasks without cells, the resin wasseparated from the medium by filtration and extracted asdescribed above. The tritium content of each fraction wasdetermined by scintillation spectrometry.

Accumulation of Monoterpenes in Culture

For analysis of monoterpene accumulation in suspensionculture, eight flasks containing 500 mg XAD resin each wereinoculated with 1 g of early log phase suspension cells andallowed to incubate for 1, 4, 8, 12, 14, 16, 18, or 20 d. On theprescribed day, each culture was harvested, the packed cellvolume of the culture measured after centrifugation at 200g,and the culture frozen before further analysis. After thawing,the resin was separated from the cells as before and washedwith two 3 mL portions of pentane which were passed over ashort column of silica gel (type 60A, Mallinckrodt), overlaidwith anhydrous Na2SO4, to collect the monoterpene olefins.To obtain the oxygenated monoterpenes, the resin was washedtwo more times with 3 mL portions of ether and this extractwas passed over the same silica gel column. The extractscontaining the monoterpene olefins and the oxygenated mon-oterpenes were concentrated to 1 mL, and an internal stand-ard (25 nmol of menthone) was added to each in preparationfor capillary GLC analysis.

Accumulation of Monoterpene Glycosides in Culture

A 50 mL suspension culture in late stationary phase wasused in this experiment. Cells (- 10 g) were separated fromthe medium and homogenized with a Ten-Broeck homoge-nizer in 40 mL methanol containing 0.5 g NaHCO3, 8 mmolglucono-.-lactone to inhibit endogenous glucosidase activity(7), and 100 jg [3-3H]menthol glucoside (27,47) as an internalstandard. After homogenization, the extract was centrifugedat 27,000g for 30 min, and the supernatant combined withthe medium and extracted with pentane:ether (2:1). Theorganic extract was concentrated under vacuum, lyophilizedto near dryness, and then loaded onto a 12 x 100 mm columnof Davisil RP- 18 (Alltech Associates) equilibrated with dis-tilled water. The column was washed with 200 mL of distilledwater, and the glycosides eluted with 200 mL of methanol.The methanol eluate was concentrated to dryness and then

hydrolyzed sequentially with almond ,B-glucosidase (in 100mM acetate-Tris, pH 5.0) and porcine esterase (same buffer,adjusted to pH 8.0) using several portions of fresh enzymeover the course of 3 d to ensure complete hydrolysis (48). Theliberated aglycones were then extracted from the mixture withseveral portions of pentane and the extracts analyzed bycapillary GLC. An aliquot of the extract was also analyzedfor tritium content to determine the recovery of the internalstandard.

Measurement of in Vivo Rate of MonoterpeneBiosynthesis from [U-14C]SucroseTo determine whether monoterpene biosynthesis occurs in

cell culture, this activity was measured in d 13 suspensioncultures to which 0.45 gCi of [U-_4C]sucrose (671 Ci/mol)was added. Since, as described below, a 50 mL suspensionculture could metabolize 0.5 mg (3.2 ,umol) of camphor in 48h, unlabeled camphor was added as a trap according to thefollowing protocol: 2 ,mol (dissolved in a minimum amountof ethanol) was added 1 h prior to [U-'4C]sucrose additionand 6 Mmol were added with the sucrose. Following incuba-tion (12 h), the cultures were steam distilled with 15 mL ofether, using 200 nmol menthol as internal standard, and therecovered camphor was purified by TLC for determination ofradioactivity content.

Preparation and Assay of Monoterpene BiosyntheticActivities in Cell-Free Extracts from Suspension Cultures

Each relevant enzyme activity was measured at 10 periodsin the growth cycle (d 1, 4, 8, 12, 13, 14, 15, 16, 18, and 20).For each time point, a 50 mL culture was harvested and thepacked cell volume determined after centrifugation at 200g.The medium was poured off and the cells were resuspendedin buffer (50 mm Mes-5 mm sodium phosphate [pH 6.5]containing 200 mM sucrose, 10 mm Na2S2O5, 10 mm ascorbicacid, and 5 mm dithiothreitol). After centrifugation at 27,000gfor 15 min, the supernatant was discarded (this fraction wasinactive) and the cells suspended in a minimum amount ofthe above buffer. Cells were homogenized in a Ten-Broeckhomogenizer with 100 mg of polyvinylpolypyrrolidone pergram of cells, and the homogenate slurried with 300 mg ofXAD resin per gram of cells for 10 min on ice. The amountsof polyvinylpolypyrrolidone and XAD used were lower thanthose normally required in extracting monoterpene cyclasesfrom intact plants (24) because cell cultures typically containmuch lower levels of resins, phenolics, and monoterpenesthan the intact plant. After filtration through eight layers ofcheesecloth prewetted with extraction buffer, the filtrate wascentrifuged again at 27,000g for 15 min. and the resultingsupernatant was used as the enzyme source.

Assays were performed as previously described: geranylpyrophosphate:(+)-pinene cyclase and geranyl pyrophos-phate:(-)-pinene cyclase (37, 38); 1,8-cineole cyclase (30);sabinene cyclase (38); (+)-bornyl pyrophosphate cyclase (31);(+)-borneol dehydrogenase (28); and (+)-bornyl pyrophos-phate phosphohydrolase (two enzyme activities that accountfor the sequential hydrolysis of bornyl pyrophosphate to bor-

1561




neol) (32). The typical reaction mixture for the assay ofcyclases (the presumptive rate-limiting enzymes of monoter-pene biosynthesis [22, 33, 39]) contained 40 to 150 Ag proteinin a 1 mL volume with 30 mM MgCl2 and 20 ,AM [1-3H]geranyl pyrophosphate, overlaid with 1 mL pentane in aTeflon-sealed screw-cap vial. The reaction mixture was incu-bated for 90 min at 3O°C and, after chilling in ice, the productswere isolated by solvent extraction and purified by TLC (24).Protein levels were determined by the method of Bradford(9).

Rate of Camphor Catabolism and Assay of Catabolites

A preliminary examination of the ability of cell cultures tocatabolize camphor under various conditions was carried outto aid in the design of an experiment to trace the metabolicfate of the U-'4C-labeled compound. In this examination, 2mg of unlabeled camphor were inoculated aseptically intofour 50 mL cultures (d 13), two of which contained 500 mgXAD resin and two which lacked this terpene adsorbent. Justprior to inoculation with camphor, one culture of each typewas inactivated by autoclaving for 20 min at 121°C and 15psi. All cultures were incubated for 48 h at room temperaturein the dark and the cultures were then chilled on ice and theXAD resin, when present, was separated from the cells andextracted twice with 3 mL of pentane:diethyl ether (2:1 [v/v]).For each culture, the cells were separated from the mediumby filtration and the medium was extracted three times with10 mL of pentane:ether. The cells were homogenized in 10mL of water in a Ten-Broeck homogenizer and then centri-fuged at 27,000g for 10 min. The supernatant was separatedfrom the cell debris and both fractions were extracted twicewith 3 mL portions of pentane:ether. An internal standard(650 nmol menthol) was added to each extract which wasthen concentrated to 1 mL and analyzed by capillary GLC.To examine the pathway of camphor catabolism in sage

cultures, a total of 0.26 ,uCi of [U-'4C]camphor (1.1 mCi/mol) was divided equally among 16 cultures of d 13 cells andthe cultures allowed to incubate for 72 h. The cultures werethen chilled at 4°C for 1 h and the medium filtered from thecells. Half of the medium (-400 mL) was frozen and theother halfwas extracted twice with 400 mL pentane and oncewith 400 mL pentane:ether (2:1 v/v). An aliquot of thecombined organic extract was taken for determination ofradioactivity and, following the addition of carrier standards,camphor and 1,2-campholide were separated by TLC (hex-anes:ether, 1:2 [v/v]) and analyzed by radio-GLC. An aliquotof the remaining aqueous phase was also taken for determi-nation of radioactivity, and a 1 mL aliquot was subjected toacid hydrolysis (2 N HCl, 30°C, 24 h) to give an indication(by the generation of ether-soluble radioactivity) of the pres-ence of monoterpene glycosides in the medium. The remain-ing aqueous phase was retained for the analysis of glycosidesand esters by enzymatic hydrolysis.The collected cells were ruptured using a Bead Beater

(BioSpec Products) with the extraction chamber filled withcold distilled water. Seven 1 min pulses were applied, withthe motor controlled by a rheostat set at 10 V. The resultinghomogenate was placed in a flask with 1 L of CHCl3:MeOH

(2:1, v/v) and kept at 4°C for 1 week. The cellular debris wasthen removed by filtration and the CHCl3:MeOH extractwashed with 500 mL water to afford an aqueous methanolicfraction. An aliquot of this material was taken for determi-nation of radioactivity and the remainder combined with theoriginal medium for the analysis of glycosides and esters byenzymatic hydrolysis as described above. The products liber-ated by hydrolysis were extracted into ether as before andanalyzed by TLC and radio-GLC. A portion of this materialwas also methylated with 14% BF3 in methanol for theanalysis of campholenic acids (as methyl esters) by radio-GLC.The chloroform phase of the cell extract was evaporated to

dryness and the residue saponified in 40 mL of 0.15 N KOHin 15% aqueous methanol on a steam bath for 1 h. Thereaction mixture was cooled on ice and extracted with three50 mL portions of diethyl ether to remove nonsaponifiablelipids (primarily phytosterols). The aqueous phase was acidi-fied (to pH 1.0) and extracted with ether to provide thesaponifiable lipids (fatty acids) which were methylated with14% BF3-MeOH as before. The '4C-content ofthe saponifiableand nonsaponifiable lipids was determined by scintillationspectrometry.

Analytical Procedures

TLC was performed on 1 mm layers of silica gel G. Devel-oped plates were sprayed with a 0.2% ethanolic solution (w/v) of 2,7-dichlorofluorescein and viewed under long-wave UVlight to locate components which were eluted from the gelwith ether. For scintillation spectrometry, 15 mL ofa cocktailconsisting of 0.4% (w/v) Omnifluor (New England Nuclear)dissolved in 30% ethanol in toluene was employed (3H effi-ciency = 40%; 14C efficiency = 96%).

Capillary GLC analyses were performed on a Hewlett-Packard 5890A gas chromatograph with 3392 integrator usingbonded-phase, fused-silica open-tubular columns (30 m x0.25 mm i.d.) coated with either a 0.2 um film of Superox-FA or a 1 ,um film of RSL-150 (Alltech Associates), andoperated using H2 as carrier (2 mL/min) and RD2 (250°C)with on-column or split injection modes. For borneol dehy-drogenase assays, the Superox FA column was programmedfrom 45°C (5 min hold) at 10°C/min to 220°C. For the analysisof monoterpene accumulation, the RSL-150 column wasprogrammed from 70°C (5 min hold) at 10°C/min to 250°C.For the analysis of camphor catabolites, the Superox FAcolumn was programmed from 50°C (5 min hold) at 10°C/min to 220°C.Radio-GLC was performed on a GOW-MAC 550P gas

chromatograph (TCD, He flow rate of 45 mL/min) attachedto a Nuclear Chicago 7357 gas proportional counter. Boththermal conductivity and radioactivity output channels weremonitored with a SICA 7000A chromatogram processor, andthe system was externally calibrated with [3H]toluene or [14C]toluene. For the analysis of 3H-labeled monoterpene olefins,the chromatographic column was 12 feet x 0.125 inch o.d.

2Abbreviations: FID, flame ionization detector; TCD, thermalconductivity detector.

1 562 FALK ET AL.




stainless steel containing 15% Silar lOC on 80/100 meshChromosorb WHP and was programmed from 70°C (15 minhold) at 5°C/min to 1 10°C. For the analysis of 3H-labeledoxygenated monoterpenes, the column used was 12 feet x0.125 inch o.d. stainless steel containing 15% AT-1000 onGas-Chrom Q and was programmed from 1 30°C (5 min hold)at 5°C/min to 180°C. For the analysis of [U-'4C]camphor,1,2-campholide and related catabolites, the column used was12 feet x 0.125 inch o.d. stainless steel containing 15% SE-30 on Chromosorb WHP and was programmed from 90°C(10 min hold) at 3°C/min to 1 30°C.

RESULTS AND DISCUSSION

Monoterpene Accumulation in Suspension Cultures

Monoterpene production was examined in suspension cul-tures of Salvia officinalis that had been generated from callusinitiated from leaf tissue. Preliminary experiments indicatedthat a lipophilic organic phase in the suspension medium,like that employed by Berlin and Witte (8), would be necessaryto trap monoterpenes synthesized by the culture, especiallythe more volatile olefins. After unsuccessful trials with mineraloil, Miglylol (a mixture of triglycerides), and various gaschromatographic stationary phases, it was found that Amber-lite XAD-4, a beaded polystyrene resin, was very efficient attrapping exogenously applied monoterpenes while giving alow background of extractable contaminants when analyzedby gas chromatography, and that this material was onlyslightly inhibitory to cell growth at a concentration of 1% (w/v). The low density resin beads were also easy to separatefrom the cells and medium, since they could be removed byflotation after low speed centrifugation.The recoveries of 3H-labeled monoterpenes (1.95 jig ger-

aniol or 1.40 Ag mixed olefins) added to culture flasks con-taining XAD resin were evaluated after a 14-d incubationperiod. Trials were conducted using both active cultures andflasks containing medium only to evaluate the effect of livingcells on recovery, and in either case the recovery was negligiblein the absence of the resin. Solvent extraction of the resinrecovered 85% of the geraniol added to the culture withoutcells (with negligible levels in the medium), whereas only 15%of this monoterpene was recovered from the resin in theculture with cells (with 10% of the initial radioactivity re-covered in the medium; most of which was not extractable inorganic solvent). This result suggests that the cells played arole in the disappearance of geraniol, possibly by transfor-mation to more volatile substances, or to water-soluble ma-terials. Evidence indicates that plant cells in culture secreteinto the medium high levels of hydrolytic and oxidativeenzymes which are capable of degrading primary and second-ary metabolites (51). The recovery of labeled monoterpeneolefins (a mixture of a-pinene, f-pinene, camphene, myrcene,limonene, and sabinene) from the culture without cells was64% (most was bound to the XAD resin with negligibleamounts remaining in the medium), whereas in the presenceof cells, 58% of the olefins were recovered from the XADresin (with 10% of the initial radioactivity remaining in themedium, most of which was extractable in organic solvent).

The apparent lack of metabolic transformation of the olefinsmay be a consequence ofthe fact that these compounds, beingmore hydrophobic than geraniol, are more favorably parti-tioned into the polystyrene resin, and are therefore less acces-sible to degradative enzymes. In general, the lower recoveryof monoterpene olefins compared to geraniol in the cultureswithout cells may be attributed to the higher volatility of thesecompounds relative to geraniol. For the purpose of evaluatingthe production of monoterpenes in sage cultures, these dataallow prediction that, in the presence of XAD resin, approx-imately 85% of the geraniol (and other oxygenated monoter-penes) and roughly 65% of the monoterpene olefins synthe-sized in the culture can in theory be recovered, in the absenceof cellular catabolism.With the anticipated recoveries as a guide, suspension cul-

tures containing XAD resin were harvested periodicallythroughout a growth cycle of 21 d and examined for thepresence of endogenous monoterpenes by capillary-GLCanalysis of pentane:ether extracts of the resin trap. No meas-urable amounts of monoterpenes were found at any day inthe growth cycle. The use of internal standards showed thatthe limits of detection were 3 ng of monoterpene product per50 mL culture. If cultures were synthesizing monoterpenes ata level comparable to that of leaves on the intact plant,approximately 150 mg of product would be expected toaccumulate per 50 mL culture, given that the monoterpenecontent of sage leaves on a fresh weight basis is usually 1.5%(29) and that stationary phase cultures had a wet weight ofabout 10 g. An examination of glycosidically-bound or ester-ified monoterpenes also failed to detect accumulation atgreater than 35 Ag per culture, which was the limit ofdetectionof this method based on enzymatic hydrolysis.

Monoterpene Biosynthetic Capacity in Culture: Synthesisfrom [U-14C]Sucrose

Despite the negligible recovery of monoterpenes from sagecell cultures, the fact that cells could degrade a significantproportion of added monoterpenes suggested that the biosyn-thesis of these products might take place without net accu-mulation. A culture was therefore supplied with 0.45 ,uCi of[U-'4C]sucrose (700 pmol) during the period of peak mono-terpene biosynthetic activity in early stationary phase (asdetermined by in vitro assay; see below). The production oflabeled camphor was examined since this monoterpene ke-tone is one of the principal products of the intact plant (31).Because of the potential for monoterpene degradation inculture, unlabeled camphor (1.2 mg) was also added in anattempt to trap the labeled biosynthetic product. No XADresin was employed in this experiment. After a 13 h incuba-tion period, which straddled the peak of biosynthetic activity,74% of the unlabeled camphor was recovered, but radio-TLCanalysis showed only 62 pCi (-0. 1 pmol, based on the specificactivity of the starting material) of labeled camphor to bepresent. Although the results of this experiment suggest thatthe level of camphor biosynthetic activity in vivo is extremelylow, a higher rate of camphor biosynthesis might have beenobscured either by dilution ofthe precursor (since it is unlikelythat unlabeled sucrose originally present in the medium had

1 563




been fully depleted) or by very rapid catabolism of the mon-oterpene product (since exogenously applied camphor maynot have fully equilibrated with that generated endogenously).

Monoterpene Biosynthetic Capacity in Culture: In VitroMeasurement of Enzyme Activities

In an attempt to determine if monoterpene biosynthesiswas occurring at a significant rate in cultured cells, the activi-ties of several enzymes of monoterpene biosynthesis weremeasured, including all of those required for the formation ofcamphor from the ubiquitous precursor geranyl pyrophos-phate (28, 31, 32) (Fig. 1). Cell-free extracts were preparedfrom cultures at several points in the growth cycle, and theability to cyclize geranyl pyrophosphate to monoterpene ole-fins, 1,8-cineole, and bornyl pyrophosphate (Fig. 1) was as-sayed. The cyclization of geranyl pyrophosphate representsthe first committed reaction leading to monoterpenes, andthis enzymatic transformation is considered to be a regulatorystep in monoterpene biosynthesis (22, 39). Figure 2A depictsthe growth of sage suspension cultures over a 2 l-d period asmeasured by the packed cell volume ofthe entire culture aftera low speed centrifugation. This overall pattern ofgrowth wasconfirmed by both fresh weight and dry weight measurementsof the cultures made at the same time points. The stationaryphase, which is often found to be the stage during which themost active synthesis of secondary metabolites occurs in cellculture (1, 18, 43, 50), is reached at d 13. The time courses ofenzyme activity are illustrated in Figure 2 for monoterpeneolefin cyclases (38), 1,8-cineole cyclase (30), and bornyl py-rophosphate cyclase (31). Cyclase activity of all types wasvirtually absent throughout most of the cell culture growthcycle, except for a brief period (48 h) near the beginning ofstationary phase at which time these enzymes showed a pro-nounced increase in activity (on either a per culture or per gfresh weight basis). The maximum activity observed for eachof these cyclases was considerably lower than that noted inintact plant tissue. For example, bornyl pyrophosphate cy-clase, which had the highest activity noted for any cyclase insage cultures, registered a peak activity of 13 fmol/s per gfresh weight of cells, approximately 5% of the level noted foryoung, expanding sage leaves (33, 40).Radio-GLC analysis of the products generated by the mon-

oterpene olefin cyclases indicated the presence of ,3-pinene(50%), myrcene (36%), and terpinolene (13%). The appear-ance of a measurable quantity of terpinolene is surprising,considering that this olefin is normally a trace component ofthe olefin mixture produced by the intact plant. Sabinene,camphene, and a-pinene, which are normal components ofsage essential oil (38), were not detected. This distribution ofolefinic products underscores a phenomenon previously ob-served in cell cultures of monoterpene producing species:most cultures do not synthesize the same mixture of mono-terpenes as that found in the intact plant ( 18, 46, 52).

Activities of the subsequent steps in the biosynthesis ofcamphor following formation of bornyl pyrophosphate (Fig.1) were also examined by in vitro assay. Bornyl pyrophosphatephosphohydrolase activity (the summation of two hydrolaseactivities leading to borneol (32]) was consistently higher thancyclase activity throughout the growth curve. This enzyme

activity peaks at a maximum of 140 pmol/s per culture at d13, corresponding to a level of 14 pmol/s per g fresh weight,which is about the same as that observed in extracts of theintact plant (32, 33). Borneol dehydrogenase catalyzes thefinal step of camphor biosynthesis (Fig. 1), and the activity ofthis enzyme peaks at nearly 6 pmol/s per culture at d 13,corresponding to a level of 0.6 pmol/s per g fresh weight,which is about half the level of the intact plant (28, 33). Themaximum activity per culture of the dehydrogenase coincideswith that of the bornyl pyrophosphate cyclase and bornylpyrophosphate hydrolases. Since the dehydrogenase and phos-phohydrolases are present at all stages ofculture development,plots of activity on a per g fresh weight basis exhibit lessvariation than do plots on a per culture basis. Nevertheless, apeak of activity per g was also noted in both cases at d 13(i.e., the period when the cyclases are present).

It is interesting that all of the enzymes of monoterpene

E-20

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_0

a)ENcw

120

90 C_

._..60 0

E

30 N

w

Days

Figure 2. Growth curve and in vitro measurement of the levels ofmonoterpene biosynthetic enzymes in sage suspension cultures.Packed cell volume (0), monoterpene olefin synthase (cyclase) activ-ity (0), and 1,8-cineole cyclase activity (A) are plotted in panel A.Bomyl pyrophosphate cyclase activity x 10° (0), bornyl pyrophos-phate phosphohydrolase activity x 10-3 (U) and borneol dehydro-genase activity x 10-2 (A) are plotted in panel B. The enzyme assaysare described in "Materials and Methods." 1 Unit = fmol/s -culture.

1 564 FALK ET AL.




biosynthesis studied in sage cells exhibit a coordinately regu-lated burst of activity in the culture near the end of thelogarithmic phase of growth and the beginning of stationaryphase. This pattern is frequently observed in enzymologicalstudies of natural product metabolism in cell culture and isbelieved to be a function of the depletion of some essentialnutrient from the culture medium (1, 17, 44). The relativeactivities of the enzymes of camphor biosynthesis measuredare consistent with the cyclization of geranyl pyrophosphatebeing the rate-limiting step of this pathway (33). In cellculture, the maximum cyclase activity is considerably lessthan the activities of the other two enzymes (2% of thedehydrogenase and 0.08% of the phosphohydrolase, com-pared with 40% and 3%, respectively, in the intact plant),suggesting that low cyclase activity may be an importantconstraint on monoterpene biosynthesis in culture.

Monoterpene Catabolic Capacity in Culture: Rate andPathway of Camphor Catabolism

Although the level of monoterpene biosynthesis in culturewas low as judged by in vivo and in vitro measurements,calculation based on the levels of cyclases measured in vitroindicated that about 50 nmol of monoterpenes (22 nmol ofcamphor) would have been produced in a single culture inthe 2 d during which the cyclases were most active. Since thislevel of product would have been easily detected, the lack ofmonoterpene accumulation actually observed might be dueto catabolic processes. To assess the extent of catabolism inculture, a series of camphor feeding experiments were con-ducted. In the first experiment, 2 mg of camphor were ad-ministered to both live cells and heat inactivated cells. Aftera 48 h incubation period, 1.06 mg of the original camphorwas recovered from the flask of inactivated cells, whereas only0.56 mg ofcamphor was recovered from the flask of live cells.These data suggest that the loss of camphor due to volatili-zation is about 50%, and that sage cell cultures are capable ofdegrading about half of the remaining camphor (i.e., about0.5 mg in 48 h).Given the extent of camphor loss to catabolic processes, it

was of interest to examine the pathway of camphor degrada-tion. For this purpose, 0.26 ,OCi of [U-'4C]camphor (0.24mmol) was distributed among 16 cultures at d 13. Afterincubation for 72 h, only 0.01 gCi (-4%) of the initialradioactivity was recovered as camphor and it was determinedthat 0.12 gCi (46%) of the camphor applied was degraded,the remainder having been lost by volatilization. Based onthe rate of camphor loss, it can be estimated that cataboliccapacity exceeds the maximum cyclase activity in sage cul-tures by at least two orders of magnitude.A pathway for the degradation of camphor, via 1,2-cam-

pholide and the corresponding glucoside-glucose ester (Fig.1), has been described in the intact sage plant (25, 26).However, in the present experiment, neither of these twointermediates were detected as metabolites of[U-_4C]camphorin extracts of the medium or the cells, nor were chemicaldegradation products of these metabolites (such as campho-lenic acids [26]) observed. Thus, if 1,2-campholide or itsconjugates are intermediates in the degradation of camphorin culture, they are very rapidly turned over.

The greatest amount of label from exogenous ['4C]camphor(46%) was recovered as water-soluble components of themedium and cells, from which label was not appreciablyliberated by ,B-glucosidase and esterase hydrolysis. No attemptwas made to identify these labeled products, but it is likelythat they represent a wide range of cellular metabolites. Acidhydrolysis of the total water-solubles from medium and cellsreleased 20% of the '4C-label as unidentified ether-solubleconstituents. Small amounts of the total radioactivity appliedto the cells as camphor were recovered in phytosterols (0.3%)and fatty acids (1%).There are significant differences between the pathway of

camphor degradation previously demonstrated in the intactplant and that in cell culture. As mentioned, 1,2-campholidewas not detected in culture, nor was the corresponding glu-coside-glucose ester. Since sucrose in the medium is nearlydepleted at this point of the growth cycle, degradation of theapplied camphor may not necessarily proceed through gly-cosylated intermediates as in the intact plant. The glucoside-glucose ester serves as a phloem transport derivative betweenthe site of monoterpene accumulation in leaves and the siteof catabolism in the roots (25, 26). Transport to a remote sitefor catabolism seems unnecessary in culture, and it appearsthat camphor may be degraded directly (probably via 1,2-campholide to accomplish ring cleavage) to basic metaboliteswithout the intermediacy of glycoconjugates. In the intactplant, the ultimate products of camphor degradation are acyland isoprenoid lipids (25). The lack of significant labeling ofthese compounds in culture likely indicates that these station-ary phase cells are using camphor as a source of energy ratherthan as a carbon source for synthesis of new membraneconstituents.

CONCLUSION

In this investigation, we have shown that undifferentiatedcell suspension cultures of sage exhibit no measurable accu-mulation of either free monoterpenes or conjugated forms. Intheory, the lack of observable accumulation and the lowapparent rate of monoterpene production from [U-'4C]su-crose could be due either to the absence of significant biosyn-thetic activity or to the presence of efficient catabolic proc-esses. Studies with cell-free extracts of cultures indicated thatseveral enzymes of monoterpene biosynthesis are present atactivity levels comparable to those measured in the intactplant, although the cyclases, which are often thought to cata-lyze the rate-limiting step of the pathway (22, 33, 39), arepresent at significantly lower levels than those in the intactplant. Nevertheless, sufficient amounts of enzyme activityappear to be present in culture to produce readily detectablelevels of monoterpenes. The lack of observable accumulationthus indicates that these suspension cultures must readilydegrade monoterpenes, and the efficient degradation of ex-ogenous camphor to water-soluble metabolites was, in fact,demonstrated. Catabolism of exogenous monoterpenes hasbeen shown in cell cultures of a variety of other species (7,15, 20).Undifferentiated cell cultures lack organized structures for

the extracellular storage of monoterpenes, such as resin ductsor the subcuticular space of glandular trichomes. Monoter-

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penes that are secreted into the medium would appear to bemuch more susceptible to enzymatic degradation than thosesequestered in extracellular compartments since, in culture,plant cells typically excrete large amounts of hydrolytic andoxidative enzymes into the medium (51). If cells are unableto store monoterpenes in discrete structures, both extra- andintracellular degradation may, in fact, be critically importantin order to avoid the toxic effects of monoterpenes on growthand viability (14, 36). In intact sage plants, catabolism hasbeen shown to represent a mechanism for the salvage ofcarbon from monoterpene defense compounds in older leaves(23). In culture, catabolism may result from the need todetoxify monoterpenes and provide substrate for cell growth,or could simply be a consequence of the greater accessibilityof monoterpenes to catabolic enzymes in undifferentiatedcells.

ACKNOWLEDGMENTS

We thank Margaret Duffy-Riggle, Henry Fisk, and D. MichaelSatterwhite for technical assistance, Greg Wichelns for raising theplants, and Karen Maertens for typing the manuscript.

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