Common Sage (Salvia officinalis)I - Metabolism of Monoterpenes in Cell Cultures of - Plant Physiology
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Plant Physiol. (1990) 93, 1559-1567 Received for publication January 5, 1990
0032-0889/90/93/1 559/09/$01 .00/0 Accepted Aprl 19,1990
Metabolism of Monoterpenes in Cell Cultures of
Common 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 and considerable evidence indicates that these secretory struc-
Leaves of common sage (Salvia officinalis) accumulate mono- tures are the primary, if not the exclusive, sites of monoter-
terpenes in glandular trichomes at levels exceeding 15 milligrams pene biosynthesis (21, 41, 49). It might seem then that mon-
per gram fresh weight at maturity, whereas sage cells in suspen- oterpenes are unlikely to be produced in cell culture systems
sion culture did not accumulate detectable levels of monoter- in the absence of such organized structures. In fact, undiffer-
penes (0.3 micro- under some conditions, monoterpene biosynthesis is possible
grams per gram fresh weight) at the late log phase of growth. in less organized systems.
Other monoterpene synthetic enzymes were present as well. In At a fundamental level, the general absence of monoterpene
vivo measurement of the ability to catabolize (+)-camphor in accumulation in undifferentiated cultures could be due to the
these cells indicated that degradative capability exceeded bio- lack of significant biosynthetic activity or to the presence of
synthetic capacity by at least 1000-fold. Therefore, the lack of efficient catabolic processes. De novo monoterpene biosyn-
monoterpene accumulation in undifferentiated sage cultures thesis, as distinct from monoterpene accumulation (1, 17, 44)
could be attributed to a low level of biosynthetic activity (relative or the biotransformation of exogenous monoterpenes (19),
to the intact plant) coupled to a pronounced capacity for mono-
terpene catabolism. 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 extracts
from cultures of diverse essential oil species, but it is not
possible to determine with certainty whether these metabolites
The accumulation of terpenoid natural products in plant represent intermediates in the biosynthesis of monoterpenes
cell cultures has been successfully demonstrated in the cases or in the formation of higher products such as phytosterols.
of diterpenoids and sesquiterpenoids, but rarely in the case of By contrast, the efficient biotransformation of exogenous
monoterpenes. Thus, there are reports of the production of monoterpenes in cell culture ( 19) implies that at least portions
diterpenoid substances in culture at levels exceeding those of of monoterpene metabolic pathways may be widely present
the intact plant (42, 43) and the induced accumulation of in these systems. The ability of plant cell cultures to catabolize
sesquiterpene phytoalexins in culture is well documented (12, added monoterpenes (1, 7, 15, 20) suggests that degradative
13, 16), whereas most accounts of monoterpene accumulation capability may be critically important in avoiding the toxic
in cell culture systems (1, 17, 44) describe either very low effects of these compounds on the growth and viability of
levels of production or compositional patterns that differ cells in culture ( 14).
markedly from those of the intact plant. In this paper, we describe the metabolism of (+)-camphor
Monoterpenes in intact plants usually accumulate in the and other monoterpenes in cell cultures of common sage
extracellular storage spaces of specialized secretory structures, (Salvia officinalis). Camphor metabolism was emphasized in
such as glandular trichomes, resin ducts, or resin cavities (35), this study because this bicyclic ketone is one of the major
' Research supported in part by U.S. Department of Energy grant monoterpenoid products of the intact plant and because the
DE-FG06-88ER13869 and by Project 0268 from the Agricultural pathway and enzymes of camphor biosynthesis from the
Research Center, Washington State University, Pullman, WA 99164. ubiquitous isoprenoid precursor, geranyl pyrophosphate, are
1559
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Copyright (c) 2020 American Society of Plant Biologists. All rights reserved.1 560 FALK ET AL. Plant Physiol. Vol. 93, 1990
well known (Fig. 1) (28, 31 '32). Additionally, the early steps
in the catabolism of camphor, via 1,2-campholide and the
corresponding glucoside-glucose ester (Fig. 1), in S. officinalis
leaves have been documented (25, 26). In the present work, BIOSYNTHESIS
the virtual absence of monoterpene accumulation in S. offi-
cinalis cell suspension cultures was shown to result from a
low level of biosynthetic activity coupled to a pronounced
ability to catabolize these compounds. rSo~PP
MATERIALS AND METHODS
Plant Materials, Substrates, and Reagents
Leaves of common sage (Salvia officinalis L.) were surface
sterilized by soaking in 2% aqueous NaOCl containing 0.02%
Tween 20 for 10 min followed by rinsing with sterile, distilled Geranyl
water. Discs (5 mm diameter) cut from the sterilized leaves Pyrophosphate
were placed on Murashige-Skoog medium (45) containing 0.2
mg/L 2,4-D, 1 mg/L kinetin, and 0.8% (w/v) Phytagar (Gibco
Laboratories), and the resulting callus was subcultured every
4 weeks (since September 1986) onto maintenance medium
containing 1.0 mg/L 2,4-D and 1.0 mg/L kinetin, and kept
* CATABOLISM
in the dark at 28°C. For the preparation of suspension cul- OPP
tures, flasks containing 50 mL maintenance medium without
agar were each inoculated with 1.5 g of callus tissue, and were
incubated in the dark at 28°C on an orbital shaker (125 rpm).
For time-course studies, cultures were initiated with a 10 mL
aliquot (approximately 1 g) of first passage suspension cells Bornyl
harvested at 7 to 10 d (early log phase) and added to fresh Pyrophosphate 0
medium.
The preparation of (+)-[U-'4C]camphor was carried out by
exposing approximately 500 sage plants (28 d old) to 1 mCi
of '4CO2 (generated from Na214CO3 at 0.3 Ci/mol) in a sealed
acrylic chamber under illumination. After 1 h of exposure, Glc
the chamber was flushed with air into a KOH trap. The apical
buds plus the top leaf pairs were harvested 24 h later, steam
distilled, and the [U-'4C]camphor (- 10 mg at 1.1 mCi/mol)
was isolated from the distillate by TLC on silica gel G (hex-
4
anes:ether, 2:1 (v/v)). The sources of (+)-borneol, (+)-cam-
phor, (+)-bornyl pyrophosphate, and (+)- 1,2-campholide Borneol
have 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 olefins
were obtained by incubating cell-free extracts from sage leaves
with [1-3H]geranyl pyrophosphate as previously described
(37). Radio-GLC analysis of this olefin mixture confirmed
the presence of a-pinene, camphene, p-pinene, myrcene, lim-
* 1 ,2-Campholide
onene, and sabinene (at a combined specific activity of 100
Ci/mol). [U-'4C]Sucrose (671 Ci/mol) was obtained from
New England Nuclear. All other reagents and biochemicals
were obtained from Aldrich or Sigma Chemical Co. unless
otherwise noted.
Monoterpene Adsorption by Polystyrene Resin in Sage Camphor
Cell Culture Figure 1. Pathways for the conversion of geranyl pyrophosphate to
camphor, and for the conversion of camphor to the glucoside-glucose
To test the efficacy of beaded polystyrene resin (Amberlite ester of 1,2-campholide.
XAD-4, Rohm and Haas) as a lipophilic trap for volatile
monoterpenes in suspension culture, flasks containing 40 mL
of the maintenance medium and 500 mg of resin (washed
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Copyright (c) 2020 American Society of Plant Biologists. All rights reserved.MONOTERPENE METABOLISM IN SAGE CELL CULTURE 1561
exhaustively with 95% ethanol and pentane, then air dried) hydrolyzed sequentially with almond ,B-glucosidase (in 100
were inoculated with a 10 mL suspension of early log phase mM acetate-Tris, pH 5.0) and porcine esterase (same buffer,
(d 7) cells plus either 1.14 uCi of [1-3H]geraniol or 1.03 uCi adjusted to pH 8.0) using several portions of fresh enzyme
of sage-derived [3H]monoterpene olefins. Control flasks con- over the course of 3 d to ensure complete hydrolysis (48). The
tained 50 mL of the maintenance medium (without cells) and liberated aglycones were then extracted from the mixture with
500 mg XAD resin plus either 1.14 uCi of [I -3H]geraniol or several portions of pentane and the extracts analyzed by
1.03 ,uCi of [3H]olefins. After 14 d of incubation, the cultures capillary GLC. An aliquot of the extract was also analyzed
were chilled on ice then centrifuged at 200g for 10 min. The for tritium content to determine the recovery of the internal
XAD resin, which floated after centrifugation, was separated standard.
from the mixture of medium and cells, and washed three
times with 3 mL aliquots of diethyl ether if the inoculum was Measurement of in Vivo Rate of Monoterpene
[1-3H]geraniol, or twice with pentane (3 mL) and twice with Biosynthesis from [U-14C]Sucrose
ether (3 mL) if the initial inoculum was [3H]olefins. Cells
were filtered from the medium and homogenized in water To determine whether monoterpene biosynthesis occurs in
with a Ten-Broeck homogenizer, and the homogenate was cell culture, this activity was measured in d 13 suspension
extracted as above, depending on the inoculum. The medium cultures to which 0.45 gCi of [U-_4C]sucrose (671 Ci/mol)
was similarly extracted. In flasks without cells, the resin was was added. Since, as described below, a 50 mL suspension
separated from the medium by filtration and extracted as culture could metabolize 0.5 mg (3.2 ,umol) of camphor in 48
described above. The tritium content of each fraction was h, unlabeled camphor was added as a trap according to the
determined by scintillation spectrometry. following protocol: 2 ,mol (dissolved in a minimum amount
of ethanol) was added 1 h prior to [U-'4C]sucrose addition
Accumulation of Monoterpenes in Culture and 6 Mmol were added with the sucrose. Following incuba-
tion (12 h), the cultures were steam distilled with 15 mL of
For analysis of monoterpene accumulation in suspension ether, using 200 nmol menthol as internal standard, and the
culture, eight flasks containing 500 mg XAD resin each were recovered camphor was purified by TLC for determination of
inoculated with 1 g of early log phase suspension cells and radioactivity content.
allowed to incubate for 1, 4, 8, 12, 14, 16, 18, or 20 d. On the
prescribed day, each culture was harvested, the packed cell Preparation and Assay of Monoterpene Biosynthetic
volume of the culture measured after centrifugation at 200g, Activities in Cell-Free Extracts from Suspension Cultures
and the culture frozen before further analysis. After thawing,
the resin was separated from the cells as before and washed Each relevant enzyme activity was measured at 10 periods
with two 3 mL portions of pentane which were passed over a in the growth cycle (d 1, 4, 8, 12, 13, 14, 15, 16, 18, and 20).
short column of silica gel (type 60A, Mallinckrodt), overlaid For each time point, a 50 mL culture was harvested and the
with anhydrous Na2SO4, to collect the monoterpene olefins. packed cell volume determined after centrifugation at 200g.
To obtain the oxygenated monoterpenes, the resin was washed The medium was poured off and the cells were resuspended
two more times with 3 mL portions of ether and this extract in buffer (50 mm Mes-5 mm sodium phosphate [pH 6.5]
was passed over the same silica gel column. The extracts containing 200 mM sucrose, 10 mm Na2S2O5, 10 mm ascorbic
containing the monoterpene olefins and the oxygenated mon- acid, and 5 mm dithiothreitol). After centrifugation at 27,000g
oterpenes were concentrated to 1 mL, and an internal stand- for 15 min, the supernatant was discarded (this fraction was
ard (25 nmol of menthone) was added to each in preparation inactive) and the cells suspended in a minimum amount of
for capillary GLC analysis. the above buffer. Cells were homogenized in a Ten-Broeck
homogenizer with 100 mg of polyvinylpolypyrrolidone per
Accumulation of Monoterpene Glycosides in Culture gram of cells, and the homogenate slurried with 300 mg of
XAD resin per gram of cells for 10 min on ice. The amounts
A 50 mL suspension culture in late stationary phase was of polyvinylpolypyrrolidone and XAD used were lower than
used in this experiment. Cells (- 10 g) were separated from those normally required in extracting monoterpene cyclases
the medium and homogenized with a Ten-Broeck homoge- from intact plants (24) because cell cultures typically contain
nizer in 40 mL methanol containing 0.5 g NaHCO3, 8 mmol much lower levels of resins, phenolics, and monoterpenes
glucono-.-lactone to inhibit endogenous glucosidase activity than the intact plant. After filtration through eight layers of
(7), and 100 jg [3-3H]menthol glucoside (27,47) as an internal cheesecloth prewetted with extraction buffer, the filtrate was
standard. After homogenization, the extract was centrifuged centrifuged again at 27,000g for 15 min. and the resulting
at 27,000g for 30 min, and the supernatant combined with supernatant was used as the enzyme source.
the medium and extracted with pentane:ether (2:1). The Assays were performed as previously described: geranyl
organic extract was concentrated under vacuum, lyophilized pyrophosphate:(+)-pinene cyclase and geranyl pyrophos-
to near dryness, and then loaded onto a 12 x 100 mm column phate:(-)-pinene cyclase (37, 38); 1,8-cineole cyclase (30);
of Davisil RP- 18 (Alltech Associates) equilibrated with dis- sabinene cyclase (38); (+)-bornyl pyrophosphate cyclase (31);
tilled water. The column was washed with 200 mL of distilled (+)-borneol dehydrogenase (28); and (+)-bornyl pyrophos-
water, and the glycosides eluted with 200 mL of methanol. phate phosphohydrolase (two enzyme activities that account
The methanol eluate was concentrated to dryness and then for the sequential hydrolysis of bornyl pyrophosphate to bor-
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Copyright (c) 2020 American Society of Plant Biologists. All rights reserved.1 562 FALK ET AL. Plant Physiol. Vol. 93, 1990
neol) (32). The typical reaction mixture for the assay of (2:1, v/v) and kept at 4°C for 1 week. The cellular debris was
cyclases (the presumptive rate-limiting enzymes of monoter- then removed by filtration and the CHCl3:MeOH extract
pene biosynthesis [22, 33, 39]) contained 40 to 150 Ag protein washed with 500 mL water to afford an aqueous methanolic
in a 1 mL volume with 30 mM MgCl2 and 20 ,AM [1-3H] fraction. An aliquot of this material was taken for determi-
geranyl pyrophosphate, overlaid with 1 mL pentane in a nation of radioactivity and the remainder combined with the
Teflon-sealed screw-cap vial. The reaction mixture was incu- original medium for the analysis of glycosides and esters by
bated for 90 min at 3O°C and, after chilling in ice, the products enzymatic hydrolysis as described above. The products liber-
were isolated by solvent extraction and purified by TLC (24). ated by hydrolysis were extracted into ether as before and
Protein levels were determined by the method of Bradford analyzed by TLC and radio-GLC. A portion of this material
(9). was also methylated with 14% BF3 in methanol for the
analysis of campholenic acids (as methyl esters) by radio-
Rate of Camphor Catabolism and Assay of Catabolites GLC.
A preliminary examination of the ability of cell cultures to The chloroform phase of the cell extract was evaporated to
catabolize camphor under various conditions was carried out dryness and the residue saponified in 40 mL of 0.15 N KOH
to aid in the design of an experiment to trace the metabolic in 15% aqueous methanol on a steam bath for 1 h. The
fate of the U-'4C-labeled compound. In this examination, 2 reaction mixture was cooled on ice and extracted with three
mg of unlabeled camphor were inoculated aseptically into 50 mL portions of diethyl ether to remove nonsaponifiable
four 50 mL cultures (d 13), two of which contained 500 mg lipids (primarily phytosterols). The aqueous phase was acidi-
XAD resin and two which lacked this terpene adsorbent. Just fied (to pH 1.0) and extracted with ether to provide the
prior to inoculation with camphor, one culture of each type saponifiable lipids (fatty acids) which were methylated with
was inactivated by autoclaving for 20 min at 121 °C and 15 14% BF3-MeOH as before. The '4C-content ofthe saponifiable
psi. All cultures were incubated for 48 h at room temperature and nonsaponifiable lipids was determined by scintillation
in the dark and the cultures were then chilled on ice and the spectrometry.
XAD resin, when present, was separated from the cells and
extracted twice with 3 mL of pentane:diethyl ether (2:1 [v/v]). Analytical Procedures
For each culture, the cells were separated from the medium TLC was performed on 1 mm layers of silica gel G. Devel-
by filtration and the medium was extracted three times with oped plates were sprayed with a 0.2% ethanolic solution (w/
10 mL of pentane:ether. The cells were homogenized in 10 v) of 2,7-dichlorofluorescein and viewed under long-wave UV
mL of water in a Ten-Broeck homogenizer and then centri- light to locate components which were eluted from the gel
fuged at 27,000g for 10 min. The supernatant was separated with ether. For scintillation spectrometry, 15 mL of a cocktail
from the cell debris and both fractions were extracted twice consisting of 0.4% (w/v) Omnifluor (New England Nuclear)
with 3 mL portions of pentane:ether. An internal standard dissolved in 30% ethanol in toluene was employed (3H effi-
(650 nmol menthol) was added to each extract which was ciency = 40%; 14C efficiency = 96%).
then concentrated to 1 mL and analyzed by capillary GLC. Capillary GLC analyses were performed on a Hewlett-
To examine the pathway of camphor catabolism in sage Packard 5890A gas chromatograph with 3392 integrator using
cultures, a total of 0.26 ,uCi of [U-'4C]camphor (1.1 mCi/ bonded-phase, fused-silica open-tubular columns (30 m x
mol) was divided equally among 16 cultures of d 13 cells and 0.25 mm i.d.) coated with either a 0.2 um film of Superox-
the cultures allowed to incubate for 72 h. The cultures were FA or a 1 ,um film of RSL- 150 (Alltech Associates), and
then chilled at 4°C for 1 h and the medium filtered from the operated using H2 as carrier (2 mL/min) and RD2 (250°C)
cells. Half of the medium (-400 mL) was frozen and the with on-column or split injection modes. For borneol dehy-
other half was extracted twice with 400 mL pentane and once drogenase assays, the Superox FA column was programmed
with 400 mL pentane:ether (2:1 v/v). An aliquot of the from 45°C (5 min hold) at 10°C/min to 220°C. For the analysis
combined organic extract was taken for determination of of monoterpene accumulation, the RSL-150 column was
radioactivity and, following the addition of carrier standards, programmed from 70°C (5 min hold) at 10°C/min to 250°C.
camphor and 1,2-campholide were separated by TLC (hex- For the analysis of camphor catabolites, the Superox FA
anes:ether, 1:2 [v/v]) and analyzed by radio-GLC. An aliquot column was programmed from 50°C (5 min hold) at 10°C/
of the remaining aqueous phase was also taken for determi- min to 220°C.
nation of radioactivity, and a 1 mL aliquot was subjected to Radio-GLC was performed on a GOW-MAC 550P gas
acid hydrolysis (2 N HCl, 30°C, 24 h) to give an indication chromatograph (TCD, He flow rate of 45 mL/min) attached
(by the generation of ether-soluble radioactivity) of the pres- to a Nuclear Chicago 7357 gas proportional counter. Both
ence of monoterpene glycosides in the medium. The remain- thermal conductivity and radioactivity output channels were
ing aqueous phase was retained for the analysis of glycosides monitored with a SICA 7000A chromatogram processor, and
and esters by enzymatic hydrolysis. the system was externally calibrated with [3H]toluene or [14C]
The collected cells were ruptured using a Bead Beater toluene. For the analysis of 3H-labeled monoterpene olefins,
(BioSpec Products) with the extraction chamber filled with the chromatographic column was 12 feet x 0.125 inch o.d.
cold distilled water. Seven 1 min pulses were applied, with
the motor controlled by a rheostat set at 10 V. The resulting 2Abbreviations: FID, flame ionization detector; TCD, thermal
homogenate was placed in a flask with 1 L of CHCl3:MeOH conductivity detector.
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Copyright (c) 2020 American Society of Plant Biologists. All rights reserved.MONOTERPENE METABOLISM IN SAGE CELL CULTURE 1 563
stainless steel containing 15% Silar lOC on 80/100 mesh The apparent lack of metabolic transformation of the olefins
Chromosorb WHP and was programmed from 70°C (15 min may be a consequence of the fact that these compounds, being
hold) at 5°C/min to 1 10°C. For the analysis of 3H-labeled more hydrophobic than geraniol, are more favorably parti-
oxygenated monoterpenes, the column used was 12 feet x tioned into the polystyrene resin, and are therefore less acces-
0.125 inch o.d. stainless steel containing 15% AT-1000 on sible to degradative enzymes. In general, the lower recovery
Gas-Chrom Q and was programmed from 1 30°C (5 min hold) of monoterpene olefins compared to geraniol in the cultures
at 5°C/min to 180°C. For the analysis of [U-'4C]camphor, without cells may be attributed to the higher volatility of these
1,2-campholide and related catabolites, the column used was compounds relative to geraniol. For the purpose of evaluating
12 feet x 0.125 inch o.d. stainless steel containing 15% SE- the production of monoterpenes in sage cultures, these data
30 on Chromosorb WHP and was programmed from 90°C allow prediction that, in the presence of XAD resin, approx-
(10 min hold) at 3°C/min to 1 30°C. imately 85% of the geraniol (and other oxygenated monoter-
penes) and roughly 65% of the monoterpene olefins synthe-
RESULTS AND DISCUSSION sized in the culture can in theory be recovered, in the absence
of cellular catabolism.
Monoterpene Accumulation in Suspension Cultures With the anticipated recoveries as a guide, suspension cul-
Monoterpene production was examined in suspension cul- tures containing XAD resin were harvested periodically
tures of Salvia officinalis that had been generated from callus throughout a growth cycle of 21 d and examined for the
initiated from leaf tissue. Preliminary experiments indicated presence of endogenous monoterpenes by capillary-GLC
that a lipophilic organic phase in the suspension medium, analysis of pentane:ether extracts of the resin trap. No meas-
like that employed by Berlin and Witte (8), would be necessary urable amounts of monoterpenes were found at any day in
to trap monoterpenes synthesized by the culture, especially the growth cycle. The use of internal standards showed that
the more volatile olefins. After unsuccessful trials with mineral the limits of detection were 3 ng of monoterpene product per
oil, Miglylol (a mixture of triglycerides), and various gas 50 mL culture. If cultures were synthesizing monoterpenes at
chromatographic stationary phases, it was found that Amber- a level comparable to that of leaves on the intact plant,
lite XAD-4, a beaded polystyrene resin, was very efficient at approximately 150 mg of product would be expected to
trapping exogenously applied monoterpenes while giving a accumulate per 50 mL culture, given that the monoterpene
low background of extractable contaminants when analyzed content of sage leaves on a fresh weight basis is usually 1.5%
by gas chromatography, and that this material was only (29) and that stationary phase cultures had a wet weight of
slightly inhibitory to cell growth at a concentration of 1% (w/ about 10 g. An examination of glycosidically-bound or ester-
v). The low density resin beads were also easy to separate ified monoterpenes also failed to detect accumulation at
from the cells and medium, since they could be removed by greater than 35 Ag per culture, which was the limit of detection
flotation after low speed centrifugation. of this method based on enzymatic hydrolysis.
The recoveries of 3H-labeled monoterpenes (1.95 jig ger-
aniol or 1.40 Ag mixed olefins) added to culture flasks con- Monoterpene Biosynthetic Capacity in Culture: Synthesis
taining XAD resin were evaluated after a 14-d incubation from [U-14C]Sucrose
period. Trials were conducted using both active cultures and Despite the negligible recovery of monoterpenes from sage
flasks containing medium only to evaluate the effect of living cell cultures, the fact that cells could degrade a significant
cells on recovery, and in either case the recovery was negligible proportion of added monoterpenes suggested that the biosyn-
in the absence of the resin. Solvent extraction of the resin thesis of these products might take place without net accu-
recovered 85% of the geraniol added to the culture without mulation. A culture was therefore supplied with 0.45 ,uCi of
cells (with negligible levels in the medium), whereas only 15% [U-'4C]sucrose (700 pmol) during the period of peak mono-
of this monoterpene was recovered from the resin in the terpene biosynthetic activity in early stationary phase (as
culture with cells (with 10% of the initial radioactivity re- determined by in vitro assay; see below). The production of
covered in the medium; most of which was not extractable in labeled camphor was examined since this monoterpene ke-
organic solvent). This result suggests that the cells played a tone is one of the principal products of the intact plant (31).
role in the disappearance of geraniol, possibly by transfor- Because of the potential for monoterpene degradation in
mation to more volatile substances, or to water-soluble ma- culture, unlabeled camphor (1.2 mg) was also added in an
terials. Evidence indicates that plant cells in culture secrete attempt to trap the labeled biosynthetic product. No XAD
into the medium high levels of hydrolytic and oxidative resin was employed in this experiment. After a 13 h incuba-
enzymes which are capable of degrading primary and second- tion period, which straddled the peak of biosynthetic activity,
ary metabolites (51). The recovery of labeled monoterpene 74% of the unlabeled camphor was recovered, but radio-TLC
olefins (a mixture of a-pinene, f-pinene, camphene, myrcene, analysis showed only 62 pCi (-0. 1 pmol, based on the specific
limonene, and sabinene) from the culture without cells was activity of the starting material) of labeled camphor to be
64% (most was bound to the XAD resin with negligible present. Although the results of this experiment suggest that
amounts remaining in the medium), whereas in the presence the level of camphor biosynthetic activity in vivo is extremely
of cells, 58% of the olefins were recovered from the XAD low, a higher rate of camphor biosynthesis might have been
resin (with 10% of the initial radioactivity remaining in the obscured either by dilution of the precursor (since it is unlikely
medium, most of which was extractable in organic solvent). that unlabeled sucrose originally present in the medium had
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Copyright (c) 2020 American Society of Plant Biologists. All rights reserved.1 564 FALK ET AL. Plant Physiol. Vol. 93, 1990
been fully depleted) or by very rapid catabolism of the mon- activity peaks at a maximum of 140 pmol/s per culture at d
oterpene product (since exogenously applied camphor may 13, corresponding to a level of 14 pmol/s per g fresh weight,
not have fully equilibrated with that generated endogenously). which is about the same as that observed in extracts of the
intact plant (32, 33). Borneol dehydrogenase catalyzes the
Monoterpene Biosynthetic Capacity in Culture: In Vitro final step of camphor biosynthesis (Fig. 1), and the activity of
Measurement of Enzyme Activities this 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,
In an attempt to determine if monoterpene biosynthesis which is about half the level of the intact plant (28, 33). The
was occurring at a significant rate in cultured cells, the activi- maximum activity per culture of the dehydrogenase coincides
ties of several enzymes of monoterpene biosynthesis were with that of the bornyl pyrophosphate cyclase and bornyl
measured, including all of those required for the formation of pyrophosphate hydrolases. Since the dehydrogenase and phos-
camphor from the ubiquitous precursor geranyl pyrophos- phohydrolases are present at all stages of culture development,
phate (28, 31, 32) (Fig. 1). Cell-free extracts were prepared plots of activity on a per g fresh weight basis exhibit less
from cultures at several points in the growth cycle, and the variation than do plots on a per culture basis. Nevertheless, a
ability to cyclize geranyl pyrophosphate to monoterpene ole- peak of activity per g was also noted in both cases at d 13
fins, 1,8-cineole, and bornyl pyrophosphate (Fig. 1) was as- (i.e., the period when the cyclases are present).
sayed. The cyclization of geranyl pyrophosphate represents It is interesting that all of the enzymes of monoterpene
the first committed reaction leading to monoterpenes, and
this enzymatic transformation is considered to be a regulatory
step in monoterpene biosynthesis (22, 39). Figure 2A depicts
the growth of sage suspension cultures over a 2 l-d period as
measured by the packed cell volume of the entire culture after 120
a low speed centrifugation. This overall pattern of growth was
confirmed by both fresh weight and dry weight measurements
of the cultures made at the same time points. The stationary E
-20
phase, which is often found to be the stage during which the E 90 C
_
most active synthesis of secondary metabolites occurs in cell 0
culture (1, 18, 43, 50), is reached at d 13. The time courses of ._..
enzyme activity are illustrated in Figure 2 for monoterpene 60 0
olefin cyclases (38), 1,8-cineole cyclase (30), and bornyl py- 0
toa) 10
E
rophosphate cyclase (31). Cyclase activity of all types was
virtually absent throughout most of the cell culture growth 0C 30
N
cycle, except for a brief period (48 h) near the beginning of w
stationary phase at which time these enzymes showed a pro-
nounced increase in activity (on either a per culture or per g
fresh weight basis). The maximum activity observed for each
of these cyclases was considerably lower than that noted in
intact plant tissue. For example, bornyl pyrophosphate cy-
clase, which had the highest activity noted for any cyclase in
sage cultures, registered a peak activity of 13 fmol/s per g
fresh weight of cells, approximately 5% of the level noted for CO
%-
young, expanding sage leaves (33, 40).
Radio-GLC analysis of the products generated by the mon-
oterpene olefin cyclases indicated the presence of ,3-pinene _0
(50%), myrcene (36%), and terpinolene (13%). The appear-
ance of a measurable quantity of terpinolene is surprising, a)
E
considering that this olefin is normally a trace component of N
c
the olefin mixture produced by the intact plant. Sabinene, w
camphene, and a-pinene, which are normal components of
sage essential oil (38), were not detected. This distribution of
olefinic products underscores a phenomenon previously ob-
served in cell cultures of monoterpene producing species: Days
most cultures do not synthesize the same mixture of mono-
terpenes as that found in the intact plant ( 18, 46, 52). Figure 2. Growth curve and in vitro measurement of the levels of
Activities of the subsequent steps in the biosynthesis of monoterpene biosynthetic enzymes in sage suspension cultures.
Packed cell volume (0), monoterpene olefin synthase (cyclase) activ-
camphor following formation of bornyl pyrophosphate (Fig. ity (0), and 1,8-cineole cyclase activity (A) are plotted in panel A.
1) were also examined by in vitro assay. Bornyl pyrophosphate Bomyl pyrophosphate cyclase activity x 10° (0), bornyl pyrophos-
phosphohydrolase activity (the summation of two hydrolase phate phosphohydrolase activity x 10-3 (U) and borneol dehydro-
activities leading to borneol (32]) was consistently higher than genase activity x 10-2 (A) are plotted in panel B. The enzyme assays
cyclase activity throughout the growth curve. This enzyme are described in "Materials and Methods." 1 Unit = fmol/s -culture.
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Copyright (c) 2020 American Society of Plant Biologists. All rights reserved.MONOTERPENE METABOLISM IN SAGE CELL CULTURE 1 565
biosynthesis studied in sage cells exhibit a coordinately regu- The greatest amount of label from exogenous ['4C]camphor
lated burst of activity in the culture near the end of the (46%) was recovered as water-soluble components of the
logarithmic phase of growth and the beginning of stationary medium and cells, from which label was not appreciably
phase. This pattern is frequently observed in enzymological liberated by ,B-glucosidase and esterase hydrolysis. No attempt
studies of natural product metabolism in cell culture and is was made to identify these labeled products, but it is likely
believed to be a function of the depletion of some essential that they represent a wide range of cellular metabolites. Acid
nutrient from the culture medium (1, 17, 44). The relative hydrolysis of the total water-solubles from medium and cells
activities of the enzymes of camphor biosynthesis measured released 20% of the '4C-label as unidentified ether-soluble
are consistent with the cyclization of geranyl pyrophosphate constituents. Small amounts of the total radioactivity applied
being the rate-limiting step of this pathway (33). In cell to the cells as camphor were recovered in phytosterols (0.3%)
culture, the maximum cyclase activity is considerably less and fatty acids (1%).
than the activities of the other two enzymes (2% of the There are significant differences between the pathway of
dehydrogenase and 0.08% of the phosphohydrolase, com- camphor degradation previously demonstrated in the intact
pared with 40% and 3%, respectively, in the intact plant), plant and that in cell culture. As mentioned, 1,2-campholide
suggesting that low cyclase activity may be an important was not detected in culture, nor was the corresponding glu-
constraint on monoterpene biosynthesis in culture. coside-glucose ester. Since sucrose in the medium is nearly
depleted at this point of the growth cycle, degradation of the
Monoterpene Catabolic Capacity in Culture: Rate and applied camphor may not necessarily proceed through gly-
Pathway of Camphor Catabolism cosylated intermediates as in the intact plant. The glucoside-
glucose ester serves as a phloem transport derivative between
Although the level of monoterpene biosynthesis in culture the site of monoterpene accumulation in leaves and the site
was low as judged by in vivo and in vitro measurements, of catabolism in the roots (25, 26). Transport to a remote site
calculation based on the levels of cyclases measured in vitro for catabolism seems unnecessary in culture, and it appears
indicated that about 50 nmol of monoterpenes (22 nmol of that camphor may be degraded directly (probably via 1,2-
camphor) would have been produced in a single culture in campholide to accomplish ring cleavage) to basic metabolites
the 2 d during which the cyclases were most active. Since this without the intermediacy of glycoconjugates. In the intact
level of product would have been easily detected, the lack of plant, the ultimate products of camphor degradation are acyl
monoterpene accumulation actually observed might be due and isoprenoid lipids (25). The lack of significant labeling of
to catabolic processes. To assess the extent of catabolism in these compounds in culture likely indicates that these station-
culture, a series of camphor feeding experiments were con- ary phase cells are using camphor as a source of energy rather
ducted. In the first experiment, 2 mg of camphor were ad- than as a carbon source for synthesis of new membrane
ministered to both live cells and heat inactivated cells. After constituents.
a 48 h incubation period, 1.06 mg of the original camphor
was recovered from the flask of inactivated cells, whereas only CONCLUSION
0.56 mg of camphor was recovered from the flask of live cells.
These data suggest that the loss of camphor due to volatili- In this investigation, we have shown that undifferentiated
zation is about 50%, and that sage cell cultures are capable of cell suspension cultures of sage exhibit no measurable accu-
degrading about half of the remaining camphor (i.e., about mulation of either free monoterpenes or conjugated forms. In
0.5 mg in 48 h). theory, the lack of observable accumulation and the low
Given the extent of camphor loss to catabolic processes, it apparent rate of monoterpene production from [U-'4C]su-
was of interest to examine the pathway of camphor degrada- crose could be due either to the absence of significant biosyn-
tion. For this purpose, 0.26 ,OCi of [U-'4C]camphor (0.24 thetic activity or to the presence of efficient catabolic proc-
mmol) was distributed among 16 cultures at d 13. After esses. Studies with cell-free extracts of cultures indicated that
incubation for 72 h, only 0.01 gCi (-4%) of the initial several enzymes of monoterpene biosynthesis are present at
radioactivity was recovered as camphor and it was determined activity levels comparable to those measured in the intact
that 0.12 gCi (46%) of the camphor applied was degraded, plant, although the cyclases, which are often thought to cata-
the remainder having been lost by volatilization. Based on lyze the rate-limiting step of the pathway (22, 33, 39), are
the rate of camphor loss, it can be estimated that catabolic present at significantly lower levels than those in the intact
capacity exceeds the maximum cyclase activity in sage cul- plant. Nevertheless, sufficient amounts of enzyme activity
tures by at least two orders of magnitude. appear to be present in culture to produce readily detectable
A pathway for the degradation of camphor, via 1,2-cam- levels of monoterpenes. The lack of observable accumulation
pholide and the corresponding glucoside-glucose ester (Fig. thus indicates that these suspension cultures must readily
1), has been described in the intact sage plant (25, 26). degrade monoterpenes, and the efficient degradation of ex-
However, in the present experiment, neither of these two ogenous camphor to water-soluble metabolites was, in fact,
intermediates were detected as metabolites of [U-_4C]camphor demonstrated. Catabolism of exogenous monoterpenes has
in extracts of the medium or the cells, nor were chemical been shown in cell cultures of a variety of other species (7,
degradation products of these metabolites (such as campho- 15, 20).
lenic acids [26]) observed. Thus, if 1,2-campholide or its Undifferentiated cell cultures lack organized structures for
conjugates are intermediates in the degradation of camphor the extracellular storage of monoterpenes, such as resin ducts
in culture, they are very rapidly turned over. or the subcuticular space of glandular trichomes. Monoter-
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Copyright (c) 2020 American Society of Plant Biologists. All rights reserved.1 566 FALK ET AL. Plant Physiol. Vol. 93, 1990
penes that are secreted into the medium would appear to be 14. Brown JT, Hegarty PK, Charlwood BV (1987) The toxicity of
much more susceptible to enzymatic degradation than those monoterpenes to plant cell cultures. Plant Sci 48: 195-201
15. Carriere F, Gil G, Tapie P, Chagvardieff P (1989) Biotransfor-
sequestered in extracellular compartments since, in culture, mation of geraniol by photoautotrophic, photomixotrophic
plant cells typically excrete large amounts of hydrolytic and and heterotrophic plant cell suspensions. Phytochemistry 28:
oxidative enzymes into the medium (51). If cells are unable 1087-1090
to store monoterpenes in discrete structures, both extra- and 16. Chappell J, Nable R, Fleming P, Anderson RA, Burton HR
intracellular degradation may, in fact, be critically important (1987) Accumulation of capsidiol in tobacco cell cultures
treated with fungal elicitor. Phytochemistry 26: 2259-2260
in order to avoid the toxic effects of monoterpenes on growth 17. Charlwood BV, Brown JT, Moustou C, Morris GS, Charlwood
and viability (14, 36). In intact sage plants, catabolism has KA (1988) The accumulation of isoprenoid flavour compounds
been shown to represent a mechanism for the salvage of in plant cell cultures. In P Schreier, ed, Bioflavour '87: Analysis,
carbon from monoterpene defense compounds in older leaves Biochemistry, Biotechnology. Walter de Gruyter, Berlin, pp
303-314
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detoxify monoterpenes and provide substrate for cell growth, and sesquiterpenes in tissue culture. Biochem Soc Trans 11:
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ACKNOWLEDGMENTS sity Press, Cambridge, pp 15-34
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We thank Margaret Duffy-Riggle, Henry Fisk, and D. Michael by a Vitis vinifera cell suspension employing a two-phase
Satterwhite for technical assistance, Greg Wichelns for raising the system. Plant Cell Rep 6: 427-430
plants, and Karen Maertens for typing the manuscript. 21. Croteau R (1977) Site of monoterpene biosynthesis in Majorana
hortensis leaves. Plant Physiol 59: 519-520
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