Cultures of Common Sage (Salvia Officinalis)'

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Plant Physiol. (1993) 101: 1231-1237

lnduction and Characterization of a Cytochrome P-450-Dependent Camphor Hydroxylase in Tissue Cultures of Common Sage (Salvia officinalis)’

Christoph Funk and Rodney Croteau* lnstitute of Biological Chemistry and Department of Biochemistry and Biophysics, Washington State University, Downloaded from https://academic.oup.com/plphys/article/101/4/1231/6066014 by guest on 02 October 2021 Pullman, Washington 991 64-6340

weight) was observed in these undifferentiated cells, even (+)-Camphor, a major monoterpene of the essential oil of com- when provisions wer; made for trapping this volatile, hydro- mon sage (Salvia officinalis), is catabolized in senescent tissue, and phobic product (Falk et al., 1990). This lack of accumulation the pathway for the breakdown of this bicyclic ketone has been was attributed to the high rate of camphor catabolic activity previously elucidated in sage cell-suspension cultures. In the initial of suspension cultures (Falk et al., 1990), a process that may step of catabolism, camphor is oxidized to 6-exo-hydroxycamphor, mimic the metabolic turnover of camphor produced in the and the corresponding NADPH- and O,-dependent hydroxylase oil glands of the intact plant (Croteau et al., 1984, 1987). activity was demonstrated in microsomal preparations of sage cells. Severa1 well-established inhibitors of cytochrome P-450-dependent Altematively, many monoterpenes are toxic to plant suspen- reactions, including cytochrome c, clotrimazole, and CO, inhibited sion cultures (Brown et al., 1987) and the metabolism of the hydroxylation of camphor, and CO-dependent inhibition was camphor by sage cells might thus represent such a detoxifi- partially reversed by blue light. Upon treatment of sage suspension cation system (Benveniste et al., 1982; Hendry, 1986). Similar cultures with 30 mM MnCI2, camphor-6-hydroxylase activity was detoxification systems are known in animal tissues in which induced up to 7-fold. A polypeptide with estimated molecular mass inducible, microsomal Cyt P-450-dependent monooxygen- of 58 kD from sage microsomal membranes exhibited antigenic ases of broad specificity are involved in xenobiotic metabo- cross-reactivity in western blot experiments with two heterologous lism (Walker-Griffin et al., 1979), including the metabolism polyclonal antibodies raised against cytochrome P-450 camphor- of dietary monoterpenes (Waller, 1969; Karp and Croteau, 5-exo-hydroxylase from Pseudomonas putida and cytochrome P- 1988). 450 limonene-6s-hydroxylase from spearmint (Mentha spicata). Dot blotting indicated that the concentration of this polypeptide Feeding experiments with cultured sage cells have dem- increased with camphor hydroxylase activity in microsomes of onstrated that camphor is transformed, in sequence, to 6-exo- Mn’+-induced sage cells. These results suggest that camphor-6- hydroxycamphor (3), 6-oxocamphor (4), a-campholonic acid exo-hydroxylase from sage is a microsomal cytochrome P-450 (5), and 2-hydroxycampholonic acid (6) (Fig. 1) (Funk et al., monooxygenase that may share common properties and epitopes 1992). The hydroxylation of camphor is, seemingly, the first with bacterial and other plant monoterpene hydroxylases. step in the metabolism of this monoterpene ketone by micro- organisms and animals as well (Waller, 1969). Indeed, based on the substrate and the nature of the reaction catalyzed, the The biosynthetic capacity of plant cell cultures to produce presumptive sage hydroxylase would appear to resemble Cyt various monoterpenoid, sesquiterpenoid, and diterpenoid P-~~OCAM,the camphor-5-exo-hydroxylase isolated from substances has been demonstrated; however, the accumula- Pseudomonas putida upon which much of our current knowl- tion of monoterpenes in cultured cell systems is only rarely edge of Cyt P-450 monooxygenases is based (Sligar and observed (Banthorpe et al., 1986; Charlwood et al., 1989). Murray, 1986). Studies with undifferentiated suspension cultures of common In this paper, we describe the properties of camphor-6- sage (Salvia officinalis) showed that the enzymes directly em-hydroxylase from sage. The results indicate that this responsible for the conversion of the ubiquitous isoprenoid enzyme is an inducible, microsomal Cyt P-450 monooxygen- precursor, geranyl pyrophosphate (Fig. 1, structure l)’, to (+)- ase that shares many characteristics with other monoterpene camphor (Z), a major monoterpene of the intact plant, were hydroxylases, and that it may have at least one epitope in readily detected during the late logarithmic phase of growth. common with the bacterial Cyt P-450~~~and the Cyt P-450 limonene-6-hydroxylase from spearmint. Yet, no significant accumulation of camphor (C0.3 ng/g fresh MATERIALS AND METHODS This investigation was supported in part by U.S. Department of Suspension Cultures and Reagents Energy Grant DE-FG06-91ER13869,by Project 0268 from the Wash- ington State University Agricultura1 Research Center, and by a Swiss Suspension cultures of common sage (Salvia officinalis), National Science Foundation Postdoctoral Fellowship (to C.F.). initiated from leaves, were maintained in Murashige and * In the text, boldface arabic numerals refer to the structures shown in Figure 1. Abbreviations: FAD, flavin adenine dinucleotide; FMN, flavin * Corresponding author; fax 1-509-335-7643. mononucleotide. .*1131 1232 Funk anid Croteau Plant Physiol. Vol. 101, 1993

(195,OOOg pellets) was performed in 10-mL screw-cap culture tubes containing a total volume of 1 mL of 50 m~ sodium phosphate buffer (pH 7.0) with 1 mM EDTA, 1 mM DTT, 1 mM NADPH, and 10% (v/v) glycerol. The reaction was ppo21 ---O+ 2 3 initiated by addition of (+)-[3Hz]camphor(70 nmol, 7.4 Ci/ mol) and incubated at 32OC for 90 min. The reaction mixture was then chilled on ice, 5 pg of 6-exo-hydroxycamphor was added as carrier, and the products were extracted with diethyl ether (2 X 1 mL). This extract was dried by passage through a short column of anhydrous MgS04, the eluate was concen-

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HOO HOOC1 separated by TLC (0.2-mm silica gel sheets, Kodak) using 1 ether:pentane (1:1, v/v) as developing solvent. After visuali- 4 5 6 zation by exposure to I2 vapor, the zone containing 6-hydroxycamphor (RF = 0.2) was excised, and the radioactivity Figure 1. Pathway for (+)-camphormetabolism in sage. The struc- contained therein was detennined by liquid scintillation spec- tures are geranyl pyrophosphate (I), (+)-camphor (2), 6-exo-hy- trometry (in 10 mL of cocktail consisting of 0.4% [w/v] droxycamphor (3), 6-oxocamphor (4), a-campholonic acid (S), and 2-hydroxycampholonic acid (6).PPO is the pyrophosphate moiety. Omnifluor [New England Nuclear] dissolved in 30% ethanol in toluene) (3H efficiency = 40%). When inhibitors of the camphor hydroxylase were tested, Skoog medium (Murashige and Skoog, 1962), supplemented they were added to the reaction mixture 5 min prior to the with 3% Suc as well as 2,4-D and kinetin (1 mg/L each) as addition of substrate and subsequent incubation. For testing described earlier (Funk et al., 1992). For induction experi- inhibition with CO, the reaction mixtures were saturated (by ments, aliquots of suspension cultures in early stationary or bubbling for 5 min) with gas mixtures containing 10,50, and late linear growth were taken by means of a sterile pipette 90% CO with 10% O2and the remainder N2 before addition and treated with the different inducers. Stock solutions of of substrate and incubation in the dark. Light reversal of CO inducers, in water or 30% aqueous ethanol, were sterilized inhibition was evaluated by incubation of treated samples by filtration (0.2 Mm), and control cultures were treated with and controls under light of 450 nm maximal output (Karp et the corresponding amounts of these sterilized solvents. al., 1987). (+)-[3-3H2]Camphor (7.4 Ci/mol) was prepared as de- CO-difference spectra were recorded as described by Es- scribed previously and 6-exo-hydroxycamphor was isolated tabrook and Wemngloer (1978) and the amount of Cyt P- from a camphor-treated sage suspension culture (Funk et al., 450 was calculated by assuming an extinction coefficient of 1992). Acrylamide and bisacrylamide were obtained from 91 m~-'(Omura and Sato, 1964). I'rotein concentration was Hoefer Scientific (San Francisco, CA), polyvinylidene diflu- estimated by the method of Bradford (1976), utilizing the onde (Immobilon-P) membranes were from the Millipore dye-binding reagent and bovine 7-globulin standard from Corp. (Bedford, MA), and, unless otherwise noted, a11 other Bio-Rad Laboratories. reagents for SDS-PAGE, western blotting, and immunostaining were obtained from Bio-Rad Laboratories or Sigma Chem- Determination of Cinnamate-4-Hydroxylase Activity and ical Co. Other reagents and biochemicals were from Sigma Total Phenolics or Aldrich Chemical Co. For the estimation of the cinnamate-4-hydroxylase activity, a spectrophotometric assay described by Lamb and Rubery lsolation and Assay of Camphor-6-exo-Hydroxylase (1975) was used. Total phenolic concentration was estimated For the isolation of (+)-camphor-6-exo-hydroxylase, tissue using Folin-Ciocalteau reagent as described by Koumba- cultures were harvested in late logarithmic growth phase (7- Koumba and Macheix (1982). 9 d after subcultivation). During a11 isolation steps, the tem- perature was maintained at O to 4OC. Cells (50 g) were SDS-PAGE and Western lmmunoblotting extracted in phosphate buffer (200 mL, 100 mM NaP04, pH 7.0, 1 mM EDTA, 1 mM DTT) by means of a glass Ten-Broeck SDS-PAGE was performed in a Hoefer-Scientific gel elec- ' homogenizer. After addition of polyvinylpolypyrrolidone trophoresis apparatus (SE 600), utilizing 1.5-mm-thick dis- (2%, w/v) and stimng on ice for 10 min, large particulate continuous gels containing 10% total acrylamide (2.7% bis- matter was removed by centrifugation (3000g, 15 min). A acrylamide) and 0.1% SDS (Laemmli, 1970). Proteins (20- further centrifugation step (lO,OOOg, 15 min) yielded the 100 Pg/lane) were diluted in sample loading buffer, heated crude enzyme preparation from which the light membranes on a steam bath for severa1 minutes, and then subjected to were obtained by ultracentrifugation (195,00Og, 90 min). The electrophoresis at 45 V for 18 h. Separated polypeptides were pellet containing the microsomes was either resuspended in electroblotted onto polyvinylidene difluoride membranes phosphate assay buffer (see below) or immediately frozen with a Hoefer TE-52 Transphor apparatus using Tris-Gly under argon and stored at -7OOC. buffer containing 15% methanol (Towbin et al., 1979). Blot- The assay for (+)-camphor-6-hydroxylase activity in crude ted proteins were visualized either by staining with Coo- extracts (10,OOOg supematants) and microsomal preparations massie .brilliant blue or by immunostaining. Molecular mass Cyt P-450 Camphor Hydroxylase 1233 markers (Diversified Biotech) were used to estimate the mo- enzyme system, as are other plant and animal Cyt I'-450 lecular mass of peptides in western blot experiments. systems (West, 1980; Mihaliak et al., 1993). Protein blots were blocked with 3% nonfat dry milk and During subcellular fractionation, a considerable part of the incubated with primary antibody (1:lOOO dilution) or preim- camphor hydroxylase activity observed in the crude extracts mune controls. Polyclonal antiserum, raised in rabbits against was lost and could not be accounted for by reassembly of Pseudomonas putida camphor 5-exo-hydroxylase, was ob- fractions. This loss of activity could result from the afore- tained from Oxygene (Dallas, TX) (as anti-Cyt P-450 CIAl mentioned degradation of the Cyt or by separation of the immunoglobulin G fraction). Polyclonal antibodies were various components required for hydroxylase activity (i.e. raised in rabbits against the solubilized, anion-exchange chro- Cyt P-450 reductase, prosthetic groups, etc.). The addition of matography- and SDS-PAGE-purified limonene-6-hydrox- a small amount of the soluble enzyme fraction (up to 20% of total assay volume) or flavins (FAD and FMN to 2.5 ~LM)to ylase from spearmint (Mentha spicata) (Karp et al., 1990) Downloaded from https://academic.oup.com/plphys/article/101/4/1231/6066014 by guest on 02 October 2021 using the subcutaneous implanted-ball technique (Ried et al., the resuspended microsomes did enhance the camphor-6- 1992). Alkaline phosphatase-conjugated goat anti-rabbit im- hydroxylase activity of this fraction by roughly 3-fold (to munoglobulin G (1:3000 dilution, Pierce) was used as the approximately 4 pkat/mg protein). Such stimulation of Cyt secondary antibody with 5-bromo-4-chloro-3-indolyl phos- P-450 catalysis has been reported previously for severa1 phate as substrate (Garfin and Bers, 1989). systems (West, 1980; Karp et al., 1987, 1990).

RESULTS AND DISCUSSION Product ldentification and Reaction Parameters

Demonstration and Localization of Incubation of microsomal preparations of sage cell-suspen- Camphor-6-exo-Hydroxylase Activity sion cultures with (+)-[3-3H2]camphorand appropriate cofac- tors gave rise to only one radioactive product, coincident with The first step in the catabolism of (+)-camphor in undif- authentic 6-hydroxycamphor, by radio-GLC analysis. This ferentiated sage cells is the transformation of this monoter- biosynthetic product was confirmed as the 6-exo epimer pene ketone to 6-exo-hydroxycamphor (Funk et al., 1992). (>96%) by capillary GLC-MS. These analytical methods have The reaction resembles that catalyzed by the well-known Cyt been described previously in the context of the in vivo P-45OCAMfrom Pseudomonas (Sligar and Murray, 1986), and metabolism of (+)-camphor (Funk et al., 1992). the responsible enzyme is one of the growing number of The microsomal camphor-6-exo-hydroxylase system re- plant Cyt P-450-dependent monooxygenases (Donaldson quired molecular oxygen and a reduced pyridine nucleotide and Luster, 1991). Whereas camphor-6-hydroxylase func- (Table I). Incubations without a pyridine nucleotide revealed tions in the catabolism of a natural product, other monoter- no detectable hydroxylation, whereas simply flushing the pene hydroxylases isolated from higher plants are involved head space of the reaction mixture with argon reduced the in biosynthetic pathways (Karp et al., 1987, 1990; Karp and hydroxylation rate by over 75%. NADPH could be replaced Croteau, 1988, 1993) that are expressed in highly differen- with NADH, but the latter supported the reaction at a lower tiated glandular tissues (Gershenzon et al., 1987, 1989, 1991). rate (21%), probably due to less efficient redox coupling via From in vivo feeding experiments with unlabeled substrate, the capacity for (+)-camphor catabolism in sage cell-suspension cultures was estimated to be 10 nmol h-' g-' fresh Table I. Reaction conditions and inhibition of camphor hydroxylase weight in early stationaryllate linear growth. However, optimum camphor-6-hydroxylase activity measured in crude The (+)-camphor-6-exo-hydroxylase from suspension cultures of common sage (S. officinalis) was assayed at a microsomal protein cell-free extracts (10,OOOg supernatants with [+]-[3-3H2]cam- concentration of 1.5 mg/mL and at a (+)-camphorconcentration of phor as substrate) was only 10 to 20% of the in vivo flux (i.e. 70 p~.A relative rate of 100% = 2.5 pkat/mL, and the SD was in all 1-2 nmol h-' g-' fresh weight). The poor extractability and cases within 16% of the mean of the triplicate determinations. stability of higher plant P-450 Cyts are well known (Donald- Conditions Relative Rate son and Luster, 1991; Mihaliak et al., 1993), and in the present instance the loss of activity was most likely due to % inefficient isolation of this membranous enzyme system as 1 mM NADPH + 0, 1O0 well as to degradation; species absorbing at 420 nm in the 1 mM NADPH + Ar 23 0, O CO-difference spectra were observed in the soluble protein 1 mM NADH + 02 21 fraction as well as in the growth medium itself (Yu and 1 mM NADPH + O2+ 2.5 p~ each FAD + FMN 240 Gunsalus, 1974; McMurry and Groves, 1986). Complete system (1 mM NADPH + 02)plus Differential centrifugation experiments indicated that 10% co + 10% 02 66 roughly 90% of the camphor hydroxylase activity was local- 50% CO + 1O% O2 44 ized in the light membrane fraction (195,0008 pellet). Only a 90% co + 10% o, 27 small portion (approximately 10%) of the total hydroxylase 50% CO + 10% O2 + 450 nm of light , 89 activity was found in the heavy membrane fraction (lO,OOOg), 90% CO + 10% O2 + 450 nm of light 49 most likely due to aggregation of light membranes with other 50 pM Cytc 5 organelles, and no detectable hydroxylase activity was found 20 p~ Clotrimazole 29 in the soluble protein fraction (195,OOOg supernatant). These 50 p~ Clotrimazole 17 results indicate that camphor-6-hydroxylase is a microsomal 1 rnM ,LI-Mercaptoethanol 73 1234 Fun Crotead kan u Plant Physiol. Vol. 101, 1993

0.008 -1.5 « Lod 0.006

0.004

0.002

0.000 400 450 500 Q WAVELENGTH (nm)

0.0 Downloaded from https://academic.oup.com/plphys/article/101/4/1231/6066014 by guest on 02 October 2021 30 50 Figure 2. CO-difference spectrum of microsomal proteins from a 20 40 sage cell-suspension culture (60 protein/mLg 0M ) that were reduced MnCI2 CONCENTRATION (mM)

with Na2S2O4. The maximum absorption of the CO adduct at 450 nm (minus the average 470- to 500-nm background) was used to Figure 3. Dose dependence of the induction of camphor-6-hy-

estimat t P-45Cy e 0e contenth pmol/mg8 2 t a t f proteino . droxylase (C6H) activit MnCIn yi 2-treated sage cell-suspension cultures. Cultured cell afted 7 s( r transfer) were treated with increasing

concentration f MnCIso incubated 2an additionan d a an r d, fo h 8 1 l the cell fres hd microsoma weighan ) (T t l camphor hydroxylase NADH:Cy rathe5 b t r than NADPH:Cyt P-450 reductasee Th . flavin moiety of the reductase component of plant Cyt P-450 activit ) wery(• e determined microsomae Th . l protein contens wa t system knows s i especiall e b o nt y labile durin isolatioe gth n unchanged ove course 18-e th r th hf e o experiment . of these membranous proteins (Madyastha and Coscia, 1978). As indicated previouslyn i N , inclusioFM d an botf D no hFA the incubation mixture enhanced hydroxylation activity sev- eralfold (Tabl. eI) The pH optimum for camphor hydroxylation was determined in several buffers to be about 7.0, with activity reduced hala uni t H a p fabou o tt abov% 60 t belor eo maximume wth . Under optimum conditions, linear with respec proteio t t n Pre- anti- anti- Coomassie immune (P-450 ) (P-450 ) blue concentration and time, an apparent Km value for (+)-cam- CAM LH phos estimatewa r 4 JI3 M t basea d n doublo d e reciprocal M S C M C Mw plot f initiao s l hydroxylation velocity versus substrate concentration.

Inhibition of Camphor-6-Hydroxylase Several classical inhibitors of hydroxylation reactions are often used to examine the involvement of a Cyt P-450

Tabl . eEffecII f potentialo t inducers camphorn o hydroxylation Camphor-6-exo-hydroxylase activit 7-d-oln yi d suspension cultures of common sage (5. officinalis) exposed to different inducers for 18 h was measured after gel filtration of the enzyme extracts to remove residual inducer. For description of the assay system, see Table I. Inducer Concentration Relative Activity

Figur . Wester4 e n blot analysi f cultureo s d sage cell proteins. None 100 Cultured cells were harvested in late logarithmic growth and ex- (+)-Camphor 1 mM 36 tractede 10,000th d an g, supernatant, 195,000g supernatantd an , (+)-Fenchone 1 ITIM 34 195,000g pellet were prepare differentiay db l centrifugation. Fifty (— )-Menthone 1 mM 57 micrograms of the crude protein fraction (C), the soluble proteins MgCI 30 mM 113 2 e microsomath d (S)an , l protein ) wer(M s e% resolve10 a n o d MnCI 30 mM 694 2 discontinuous SDS-polyacrylamide gel, transferred to a polyvinyli- HgCI 10 MM 213 2 dene difluoride membrane probed an , d wit preimmune hth e con- FeCI 50 MM 125 3 trol, polyclonal antibodies raised against camphor-5-exo-hydroxyl- MnCI + FeCI 25 mM + 10 MM 44 2 3 ase fro . put/dmP a [anti-(P-450 AM)d polyclonaan L l antibodies 2,4-D 20 MM 63 C raised against limonene-6-hydroxylase from M. sp/cata [anti-(P- Ethanol 0.2% 94 450 )]. Proteins were stained with Coomassie brilliant blue, and DMSO 1% 63 LH molecular mass markers (Mw) are indicated in kD. Cyt P-450 Camphor Hydroxylase 1235

o> hydroxylase activity was almost completely inhibited (Table E I).

0.3 - Spectral Evidence It was not possible to demonstrate the presence of Cyt P- 45 n crud0i e extract CO-differency sb e spectra becausf o e interfering pigments. However, microsomal preparations- re , X CO o duced with sodium dithionite, yielded useful CO-difference spectra (Fig. 2) from which a total Cyt P-450 content of 28 CL 0.0 0.0 pmol/mg protein (15 pmol/g fresh weight) was estimated. in 0 10 20 30 40 50 60 70 Downloaded from https://academic.oup.com/plphys/article/101/4/1231/6066014 by guest on 02 October 2021

INCUBATION TIME (h) Inductio f Camphor-6-exo-Hydroxylasno e Many Cyt P-450 monooxygenases involved in catabolic transformations in animals, microbes, and plants are induced by treatment with the corresponding oxygenase substrate 1 c t- in (Parke, 1957; Benveniste et al., 1982; Sligar and Murray, 2 -^ I < 1986). Cyt P-450 monooxygenase systems in plants have also Q_ been induced by treatment with growth hormones, heavy 2 7 6 3 4 2 7 1 2 1 7 5 3. 0 metals, and other xenobiotics (Reichhart et al., 1980; Adele INCUBATION TIME (h) et al., 1981). The influence of several potential inducers on camphor Figur . Time5 e cours f inductioeo f (+)-camphor-6-hydroxylasno e hydroxylase activity in sage suspension cultures following 18 (C6H) following treatment with 30 mM MnCI2. (+)-Camphor-6- h of exposure is summarized in Table II. Camphor and other hydroxylas totad ean l activit) phenolicinducen i O ) , A y(• , d(A s (closed symbols) and noninduced (open symbols) cells are plotted monoterpene ketones did not enhance the rate of camphor in A. Dot blots of total microsomal protein probed with polyclonal hydroxylatio n extractni f treateso d cells. However f celli , s antibodies raised against limonene-6-hydroxylase fro . spicatmM a were treated with metal ions the activity was induced, re-

[anti-(P-450 provideLHe )]ar Tota. B dn i l microsomal protein content 7-folo t p sultinu d n increasa n gi camphor-6-hydroxylasn ei e 2 1 did not change significantly over the 72-h time course. The slight activity after Mn " " (MnCl2) treatment. Very similar results offse hydroxylasn i t e e th activit o t blod e yan tdu intensite b y yma have been reported for the induction of cinnamate-4-hydrox- high background and apparent accumulation of inactive protein. ylase activit Jerusalen yi m artichoke slices M exposem 5 2 o dt

monooxygenase, among which the photoreversible inhibition anti-(P-450LH) by CO is the most definitive (West, 1980; Mihaliak et al., 1993). Increasing (frolevelO C mf so 10-90% with constant CM C+ S+ M+ Mw O2 at 10%) revealed increasing inhibition of camphor hydroxylation (Tabl Thes. eI) e inhibition rates, ranging fro4 m3 inhibitio% to73 n relativ e correspondinth o et g controle ar , typica f thoso l e observed with other plant-derived monoterpene hydroxylases (Karp et al., 1987,1990; Karp and Croteau, 1993). Partial light-reversa inhibitioO C f o l n (Tabls wa ) eI 96 demonstrated by incubation of reaction mixtures under blue light (45 maximum 0n m output). N-Substituted imidazoles have been use inhibi- o dt P t Cy t 5 5 450 terpene hydroxylases (Karp et al., 1990), and, in the present instance, clotrimazol shows vere eb wa yo nt effective 36 in inhibiting camphor hydroxylase activity by nearly 90% at 29 a concentration of 50 pM. Inhibition of camphor hydroxylatio |8-mercaptoethanonby l (Tablalshas o eI) bee n shown

for the bacterial P-450CAM system (Yu and Gunsalus, 1974). NADPH-Cyt P-450 reductase, a key component of the microsomal monooxygenase system, conduct transfee sth f ro Figure 6. Western blot analysis of the crude (C) and microsomal electrono tw s from NADP t P-450Cy Ho t . This flavoprotein + is also able to transfer electrons to Cyt c and may be assayed (M) proteins from untreated sage cells and of the crude (C ), soluble (S+), and microsomal (M+) proteins from suspension cells treated by this means (Donaldson and Luster, 1991). Because Cyt c with 30 mM MnCI2 for 18 h. The blots were probed with polyclonal serves as an alternate electron acceptor, this Cyt can be used antibodies raised against limonene-6-hydroxylase from M. spicata to inhibit Cyt P-450-dependent monooxygenases (West, [anti-(P-450LH)L and the molecular mass markers (Mw) are indicated 1980) and, in the presence of 50 /UM Cyt c, camphor-6- inkD. 1236 Funk and Croteau Plant Physiol. Vol. 101, 1993

Mn2+(Reichhart et al., 1980). The optimal concentration for cross-reacted essentially with only the 58-kD polypeptide in hydroxylase induction in sage cell suspensions (18 h of ex- microsomal preparations from both Mn2+-treated and un- posure) was determined to be about 30 mM MnCh (Fig. 3); treated cells (Fig. 6), the results are consistent with the at this concentration, there was no observable lysis or adverse assumption that the 58-kD protein does represent the cam- effect on cell fresh weight or protein content. phor-6-hydroxylase and that the induction of this activity is accompanied by a coordinate increase in enzyme protein. Antigenic Cross-Reactivity of Sage Proteins with However, the possibility cannot be eliminated that other Heterologous Polyclonal Antibodies Mn2+-inducible Cyt I’-450 species at 58 kD are recognized by the antibodies and thus contribute to the observed time- Very little information is available on the immunological course of protein accumulation and may obscure the identity relatedness of bacterial, animal, and plant Cyt P-450 enzymes of the camphor hydroxylase. Downloaded from https://academic.oup.com/plphys/article/101/4/1231/6066014 by guest on 02 October 2021 that catalyze similar reactions. Antibodies raised against bac- In general properties, the camphor-6-hydroxylase resem- teria1 Cyt P-450 species have been shown to cross-react with bles other monoterpene hydroxylases (Karp et al., 1987,1990; severa1 microsomal, wound-inducible proteins from pea epi- Karp and Croteau, 1993; Mihaliak et al., 1993) and fulfills cotyls (Stewart and Schuler, 1989) and an anti-(Cyt P-450) the established criteria for a Cyt P-450 monooxygenase monoclonal antibody raised against rat Cyt P-450 isoform c (West, 1980). The induction by Mn2+is reminiscent of other recognizes a bean microsomal protein tentatively identified plant P-450 species involved in defense responses (Reichhart as cinnamate hydroxylase (Bolwell and Dixon, 1986); rabbit et al., 1980) and detoxification reactions (Adele et al., 1981; anti-rat Cyt P-450 also inhibits the P-450-dependent hydrox- Mougin et al., 1990). Cinnamate-4-hydroxylase is induced ylation of the monoterpene (+)-sabinene in S. officinalis mi- by Mn2+ in some species (Reichhart et al., 1980), and, al- crosomes (Karp and Croteau, 1987). though cinnamate hydroxylase was readily observed in sage To evaluate the possible immunological similarity between cell microsomal preparations (approximately 3 pkat/mg pro- the sage camphor hydroxylase and other Cyt P-450 mono- tein), this activity was unaltered by treatment of the suspen- terpene hydroxylase proteins, different cell fractions were sion cultures with Mn2+; total phenolic content was also subjected to SDS-PAGE and the separated peptides were unaltered by Mn2+ treatment (Fig. 5A). The connection be- transferred onto polyvinylidene difluoride membranes. These tween Mn2+ induction of camphor-6-hydroxylase and the blots were then probed with two different -heterologous apparent role of this enzyme in monoterpene catabolism in polyclonal antibodies, one raised in rabbits against the cam- sage is at present unclear phor-5-hydroxylase from P. putida (anti-[P-450~~~]),the other raised in rabbits against the limonene-6-hydroxylase ACKNOWLEDGMENTS from spearmint (M. spicata) (anti-[P-45OLH]),No polypeptides We thank Charles Mihaliak for preparing the anti-(limonene hy- in crude extracts of sage cells (containing both soluble and droxylase) polyclonal antibodies and for assistance with the westem membranous proteins) cross-reacted with preimmune con- blots, A1 Koepp for preparing the substrate, and Joyce Tamura-Brown trols (Fig. 4). However, two polypeptides in these extracts for typing the manuscript. (one at 56 kD, the other at 58 kD) showed strong crossreactivity with both polyclonal antibodies. The analysis of Received November 16, 1992; accepted January 11, 1993. microsomal and soluble proteins separately showed that the Copyright Clearance Center: 0032-0889/93/101/1231/07. 58-kD polypeptide was localized in the microsomes, as was the Cyt P-450 camphor hydroxylase activity, whereas the 56- LITERATURE ClTED kD polypeptide was localized in the soluble fraction, which Adele P, Reichhart D, Salaün JP, Benveniste I, Durst F (1981) contained negligible hydroxylase activity. The soluble frac- Induction of cytochrome P-450 by 2,4-dichlorophenoxyacetic acid tion showed a strong 420-nm absorbance in the CO-differ- in higher plant tissue. Plant Sci Lett 22 39-46 ence spectrum, suggestive of degraded Cyt P-450 (Yu and Banthorpe DV, Branch SA, Njar VCO, Osborne MG, Watson DG Gunsalus, 1974). Other plant Cyt P-450 proteins show similar (1986) Ability of plant callus cultures to synthesize and accumulate lower terpenoids. Phytochemistry 25 629-636 molecular masses, such as cinnamate hydroxylase from Je- Benveniste I, Gabriac B, Fonne R, Reichhart D, Salaün JP, Simon rusalem artichoke at 56 kD (Gabriac et al., 1985), a Cyt P- A, Durst F (1982) Higher plant cytochrome P-450: microsomal 450 purified from avocado at 47 kD (O’Keefe and Leto, 1989), electron transfer and xenobiotic oxidation. ln E Hietanen, M Lai- and a Cyt P-450 purified from tulip bulbs at 52.5 kD (Higashi tinen, O Hanninen, eds, Cytochrome P-450, Biochemistry, Bio- physics and Environmental Implications. Elsevier Biomedical et al., 1985). Press, Amsterdam, pp 201-208 The induction of camphor-6-exo-hydroxylase activity in Bolwell GP, Dixon RA (1986) Membrane-bound hydroxylases in Mn2+-treatedsage cells offered the possibility of examining elicitor-treated bean cells. Rapid induction of the synthesis of prolyl the time-course coordination of catalysis and the level of the hydroxylase and a putative cytochrome P-450. Eur J Biochem 159 58-kD microsomal polypeptide presumed to represent the 163-1 69 Bradford MM (1976) A rapid and sensitive method for the quanti- Cyt P-450 hydroxylase. The specific activity of the camphor- tation of protein utilizing the principle of protein-dye binding. 6-exo-hydroxylase of Mn2+-treated sage suspension cells Ana1 Biochem 72 248-254 reached maximum (7-fold) about 12 h after exposure (Fig. Brown JT, Hegarty PK, Charlwood BV (1987) The toxicity of 5A) and the level of the microsomal protein recognized by monoterpenes to plant cell cultures. Plant Sci 48: 195-201 Charlwood BV, Charlwood KA (1991) Terpenoid production in anti-(Cyt I‘-450 limonene hydroxylase) followed closely plant cell cultures. ln JB Harborne, FA Tomas-Barberan, eds, (maximum at about 17 h) in dot-blot experiments (Fig. 5B). Ecological Chemistry and Biochemistry of Plant Terpenoids. Clar- Because the anti-(limonene hydroxylase) polyclonal antibody endon Press, Oxford, pp 95-132 Cyt P-450 Camphor Hydroxylase 1237

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