Plant Physiol. (1985) 79, 1098-11060032-0889/85/79/1098/09/$01.00/0

Stereochemical Aspects of the Biosynthesis of the Side Chain of91,19-Cyclopropyl Sterols in Maize Seedlings Treated withTridemorphl

Received for publication April 12, 1985 and in revised form August 20, 1985

MICHELE BLADOCHA2 AND PIERRE BENVENISTE*Laboratoire de Biochimie Vegetale, ERA No. 487 du Centre National de la Recherche Scientifique, 28, rueGoethe, 67083, Strasbourg Cedex, France

ABSTRACT

9j,19-Cyclopropyl sterols such as 24-methyl pollinastanol accumulatedramatically in maize (Zea mays L. var LG 11) seedlings treated withTridemorph (2,6-dimethyl-N-tridecyl-morpholine), a systemic fungicide(M. Bladocha, P. Benveniste, Plant Physiol 1983 41: 756-762). Incontrast to the situation in control plants where 24-ethyl sterols predom-inate largely, 24-methyl sterols were more than 98% of total cyclopropylsterols. In addition, 24-methyl cyclopropyl sterols were a mixture of (24-R)- and (24-S)-24-methyl epimers and are similar in that respect to the24-methyl cholesterol of control plants. The presence of two epimers atC-24 has been previously explained by the operation of two routes (M.Zakelj, L. J. Goad, Phytochemistry 1983 22: 1931-1936). One mayproceed via A2428)- and A2`')-sterols to produce the (24-R)-24-methylepimer. The other route may involve reduction of either a A2^2-)_, a A23-,or a A'-sterol intermediate to give the (24-S)-24-methyl epimer. Suchintermediates have been searched for in excised Zea mays axes grownaseptically in the presence of Tridemorph and either 15-'4Cimevalonicacid, or [Me-`4C1-L-methionine. Whereas A2'28)- and A24(25)-cyclopropylsterols were found in relatively large amounts, only traces of radioactivitywere associated with A'-sterols. Gas chromatography/mass spectrome-try analysis of the sterols from axes grown in the presence of [Me-2H31L-methionine showed that A24(2g)-cyclopropyl sterols contained only two2H atoms at C-28 as expected and that the 24-methyl pollinastanolfraction contained species with two 2H atoms and no species with three2H atoms. These results indicate that both (24-R)- and (24-S)-epimersoriginate from a common A28) precursor. After incubation of the axiswith 15-'4C,(4-R)-43H,mevalonic acid, the 24-methyl pollinastanol hada 3H:'4C atomic ratio of 4:6 which is consistent with the intermediacy ofa A2(5)-sterol. All these data are in accordance with a pathway whereA24(28)-cyclopropyl sterols are isomerized to give A24(25)-cyclopropyl ster-ols which in turn would be reduced nonregiospecifically to yield both (24-R)- and (24-S)-24-methyl pollinastanols. A plausible'mechanism for thereduction step is discussed.

9Al, 19-Cyclopropyl sterols accumulate dramatically in suspen-sion cultures of plant cells (9, 27) and in maize seedlings (2, 3)

' Supported by the Centre National de la Recherche Scientifique,Equipe de Recherches Associee 487, and grants ATP No. 3998 and ATPNo. 3882.

2Present address, Department of Chemistry, Stanford University,Stanford, CA 94305.

grown in the presence of Tridemorph3 or Fenpropimorph, twosystemic fungicides (19, 20). These results suggested that thesechemicals interfere with the cycloeucalenol-obtusifoliol isomer-ase in plant cells. This was demonstrated recently by showingthat Tridemorph is a strong inhibitor of the cycloeucalenol-obtusifoliol isomerase in vitro (21). A noteworthy feature ofcyclopropylsterols from treated plants was that they consistedessentially of 24-methyl sterols (98% of total cyclopropyl sterols(3). This is in contrast with the situation for A5-sterols from thecontrol where 24-ethyl sterols predominate (more than 70% oftotal A5-sterols). It has been frequentely shown that in mosthigher plants 24-ethyl cholesterol is predominantly the (24-R)-24-ethyl epimer4, sitosterol (Ja) (Fig. 1). This contrasts with 24-methyl cholesterol which is a mixture of (24-R)- and (24-S)-24-methyl epimers: campesterol (2a) and dihydrobrassicasterol (Sa)in the same material (16-18).The cooccurrence of these two sterols in several plants raises

questions regarding their biosynthetic origins. It has been sug-gested (13) that the (24-R)-24-methyl-cholesterol (2a) is producedby a route requiring isomerization of a 24-methylene interme-diate (3a) to the 24-methyl-A24(25) sterol (4a), which is thenreduced stereospecifically to 2a. The isolation of ergosta-5,24-dien-3j#-ol (4a) from Withania somnifera (12) provides somesupport for such a route. It has been suggested (5) that a possibleprecursor of the (24-S)-24-methyl-cholesterol (5a) might be cy-

'Abbreviations and chemical nomenclature: Tridemorph, 2,6-di-methyl-N-tridecyl-morpholine; COI, cycloeucalenol-obtusifoliol isomer-ase; Fenpropimorph, 4-(3-[4-tert-butylphenylJ-2-methyl)propyl-2,6-di-methyl morpholine; cycloartenol (lSb), 4,4,14a-trimethyl-9f3,19-cyclo-5a-cholest-24-en-3f#-ol; 24-methylene cycloartanol (3b), 4,4,14a-trime-thyl-9ft, 19-cyclo-5a-ergost-24(28)-en-313-ol; cyclolaudenol (6b), 4,4,14a-trimethyl-9,,19-cyclo-5a-ergost-25(27)-en-3,-ol; cyclosadol (7b), 4,4,14a-trimethyl-913,19-cyclo-5a-ergost-E-23-en-3B-ol; cycloeucalenol (3c),4,14a-dimethyl-9#, 1 9-cyclo-5a-ergost-24(28)-en-3B-ol; obtusifoliol,4,14a-dimethyl-5a-ergosta-8,24(28)-dien-3j8-ol; 31-nor cyclolaudenol(6c), 4,14a-dimethyl-9B, l9-cyclo-5a-ergost-25(27)-en-3B-ol; 31-nor cy-clobranol (4c), 4,14a-dimethyl-9,,19-cyclo-5a-ergost-24(25)-en-3fB-ol;24-dihydrocycloeucalenol (2c and Sc), 4,14a,(24-R)-, and 4,14a,(24-S)-trimethyl-9#, 19-cyclo-5a-cholestan-31-ol, respectively; 24-methylenepollinastanol (3d), 14a-methyl-96, 19-cyclo-5a-ergost-24(28)-en-3fi-ol;(244)-24-methyl pollinastanol (2d and 5d) 14a,(24-R)-, and 14a,(24-S)-dimethyl-9#,19-cyclo-5a-cholestan-31-ol respectively; cyclofontumienol(8c), 4a,14a-dimethyl-9jd,19-cyclo-5a-stigmast-Z-24(28)-en-3j8-ol; 24-ethyl pollinastanol (ld), 4a,14a-dimethyl-9#, 19-cyclo-5a-stigmastane-3,3-ol; stigmasterol (JOa), stigmasta-5,E-22-dien-3(B-ol. Structures aregiven in Figure 1.4We have adopted in this paper the use of the 24-R- and 24S-

nomenclature in place of the 24a- and 24,B-convention.1098 www.plantphysiol.orgon January 7, 2020 - Published by Downloaded from

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BIOSYNTHESIS OF STEROLS IN TRIDEMORPH TREATED MAIZE

Ado Met,46T

OH

OHIR

11

0

R13

-tTNH>~~

OH

OHR

12

^*,~0R

14

T

R a

HO

a

R = R2 = CH3

R= H, R2 = CH3

b

c

R1=R2 Z H d

FIG. 1. Speculative biosynthetic routes leading to (24-R)-24-methyl and (24-S)-24-methyl sterols. Adapted from Zakelj and Goad (30). (U), '4C-Atom originating from C-5 of mevalonic acid.

clolaudenol (6b), a A25-sterol which has also the 24-S-methylconfiguration (8). Some experimental support for these twobiosynthetic routes has been given by McKean and Nes (13),and by recent experiments using [2-'4C, (4-R)-4-3H1)]mevalonicacid (30) which show that the (24-R)- and (24-S)-methyl-choles-terol mixture had a 3H:'4C atomic ratio of 2.82:5, compatiblewith the use oftwo routes, one proceeding via A24(28>- and A24(25)_sterols to produce the (24-R)-methyl-cholesterol with a 3H:'4Catomic ratio of 2:5, the other route possibly involving reductionof either a A24(28)_ or a A25-sterol intermediate to give the (24-S)-methyl-cholesterol with a 3H:'4C atomic ratio of 3:5.A third possibility has been opened following the isolation of

A23-sterols (7a,b) from Zea mays (10, 24) and by the suggestionthat they may be also the precursors of the (24-S)-methyl sterols.Definitive demonstration of the biosynthesis of (24-R)- and (24-S)-methyl-cholesterol raises some difficulties since the mixture

ofthe two epimers constitute generally less than 30% ofthe totalsterols in higher plants. We have taken advantage ofthe fact thatin maize seedlings treated with Tridemorph, 24-methyl-9#, 19-cyclopropyl sterols represented more than 98% of total cyclopro-pyl sterols and were also a mixture of (24-R)- and (24-S)-24-methyl epimers to carry out experiments aimed at increasing ourknowledge of the biosynthetic mechanism of both (24-R)- and(24-S)-methyl-9#, 19-cyclopropyl sterols. The present work allowsa plausible biosynthetic pathway to be proposed for the synthesisofboth (24-R)- (2d) and (24-S)-24-methyl pollinastanol (5d), themajor cyclopropyl sterols found in maize seedlings treated withTridemorph.

MATERIALS AND METHODSPlant Material. Maize (Zea mays L. var LG 11) caryopses

were germinated and grown in moist vermiculite in the light at

T

Ado Met

Ado Met

T4r

R9

T

RI

1099

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BLADOCHA AND BENVENISTE

25°C for various periods of time. Tridemorph (5 mg) was dis-solved in water (1 L) and the caryopses were soaked for 8 h inthe solution; then the caryopses were germinated and grown invermiculite in the same way as the controls except that thevermiculite was continuously soaked with the Tridemorph so-lution (1 L/d- 100 seedlings) in place of pure water.

Culture of Maize Axis. Maize caryopses were sterilized with a3% solution of NaOCl containing 2% (w/v) of Triton X-100 for20 min, then they were germinated aseptically at 25C for 48 hin the presence of 65 gM Tridemorph. The axes were excisedfrom the caryopses under aseptic conditions and grown in lightat 26°C in a liquid synthetic medium (7, 1 1) supplemented withTridemorph and various labeled or unlabeled biosynthetic pre-cursors. Under these conditions, a plant developed and could begrown for more than a week.

Authentic Materials and Labeled Precursors. A pure (>98%)sample of Tridemorph was kindly provided by Dr. H. Pommer(Badische Anilin und Soda Fabrik Agricultural Research Station,Limburgerhof, Federal Republic of Germany). Fenpropi-morph:4-(3-(4-tert-butylphenyl)-2-methyl)propyl-2,6-dimethyl-morpholine was a kind gift of Dr. H. Pommer. [5-'4CJMevalonicacid (50 mCi/mmol) and [Me-'4C]-L-methionine (56 mCi/mmol), were purchased from the Commissariata l'Energie Ato-mique (CEA), Gif-sur-Yvette, France. [(4-R)-4-3HJ]Mevalonicacid (1 Ci/mmol) was purchased from Amersham, England. [Me-2H3]-L-Methionine was purchased from the CEA. We warmlythank the following scientists for gifts of the compounds: 24-methylene cycloartanol (Dr. T. Itoh, Tokyo University, Japan),cyclolaudenol (Professor D. Arigoni, Zurich, Switzerland), 31-norcyclolaudenol (Dr. A. S. Narula, Duke University, Durham,NC), cycloeucalenol (Dr. P. Schmitt, our laboratory), (24-R)-24-Dihydrocycloeucalenol (Dr. A. H. Conner, Madison, WI), and25-dihydrocyclolaudenol (Dr. M. Mihailovic, Zurich, Switzer-land).

Incorporation Experiments. (a) [Me-'4C]-L-Methionine: themaize axes (25-30) were grown in the presence of the labeledmethionine (30,uCi, 6,gCi/jsmol), unlabeled mevalonic acid (5mM), and Tridemorph (651AM) in a total volume of medium of10 ml. After 3 d of growth, the plantlets were harvested, frozen,and lyophilized. (b). [Me-2H3]-L-Methionine: the maize axes weregrown as described in (a), but in the presence of the deuteratedL-methionine (2 mM) in place of the radioactive methionine.After 6 d of growth, the plantlets were harvested. In someexperiments, a slight necrosis of the leaves was noticed, whichwas possibly caused by the high amountof L-methionine used.(c) [(4-R)-4-3H,,5-'4C]Mevalonic acid: the maize axes were grown

as described in (b), but with unlabeled L-methionine (2 mM) inplace ofthe deuterated species and a mixture of[5-'4C]mevalonicacid (10,uCi, 33,Ci/4mol) and [(4-R)4_3HJ]mevalonic acid (50

,uCi, 333 gCi/gmol). After 6 d of growth, the plantlets wereharvested.

Analytical Procedure. Sterols were extracted and analyzedfrom the lyophilizedmaterial as described previously (2, 3, 27).Only modifications to this procedure will be detailed here. Thesemodifications concern GC which was performed with a CarloErba GC model 4160 equipped with a flame ionization detectorand a WCOT glass capillary column (25 m x 0.25 mm i.d.),coated withOV,, "on column" injection, H2 2 ml/min. Thetemperature program used included a fast rise from60° to 230°C(20°C x min-'), then a slow rise from 23°C to 280°C(2°C xmin-'). Argentation TLC using washed CHC13 was performedon the mixture of the acetates ofla, 2a,Sa and of stigmasterol(JOa) as described previously. In these conditions, JOa could beeasily separated from the mixture ofla, 2a, andSa. To separatethese latter sterols, HPLC was used as detailed below.

Epoxidations. The acetates of 24-methylpollinastanolpurifiedas described previously (3, 27) were dissolved in a saturated

solution (0.5 ml) ofp-nitro-perbenzoic acid in diethyl ether ('40mg/ml). In these conditions, contaminating 5'- or A8-sterylacetates which were otherwise difficult to eliminate, were epoxi-dized, but not the cyclopropyl steryl acetates which do notcontain a double bond. After conventional work-up, the epoxidesof the A5- or A8-steryl acetates were unambiguously separatedfrom the 24-methyl pollinastanyl acetate by TLC using cyclo-hexane-ethyl acetate (85:15, v/v) as the developing solvent. Afterthis step, the recovered cyclopropyl steryl acetate gave a singlepeak in capillary column GC.

Selective Cleavage of Methylidene Groups (29). To the radio-active material, cochromatographing with 24-methylene-cycloar-tanyl acetate (3b-acetate), was added cold 3b-acetate (2 mg) andcyclolaudenyl acetate (6b-acetate) (2 mg). The mixture of 4,4-dimethyl steryl acetates (4 mg) was dissolved in a 10% solutionofOSO4 in pyridine (0.1 ml). The solution was kept at roomtemperature overnight before addition of sodium metabisulfite(20 mg) in water (0.2 ml). The mixture was stirred for 2 h, dilutedwith water (2 ml), and extracted (3x) with hexane. The extractwas washed with H20, dried, and evaporated. The residue wassubmitted to TLC on silica gel (CHC13:CH30H, 92:8, v/v) togive 3,3-acetoxy-24-methylcycloartan-24,28-diol (Jlb) (RF 0.58)and 3,B-acetoxy-24-methylcycloartan-25,26-diol (12b) (RF 0.45).Compound Jlb was dissolved in 1,4-dioxan (0.1 ml) and NaIO4(2 mg), and H20 (1 ml) were added. The mixture was stirredovernight, diluted with H20, and extracted with hexane, and theextract was washed and taken to dryness. The product waspurified by TLC on silica gel (CHC13.CH30H, 97:3, v/v) to give24-oxo-cycloartanyl acetate (13b), (1 mg); PMR spectrum wasin full agreement with the literature (15): PMR (CDC13): 6 0.355(lH,d, H-19 endo), 0.568 (lH,d,H-19 ecto), 1.090 (6H,d,J=7Hz, H-26 and H-27), 2.061 (3H,s,-O-C-CH3), 2,455 (lH,m,H-25), 2.625 (2H,m,H-23), 4.555 (lH,m,H-3a). Compound 12bwas treated in a similar manner to give 24-methyl-25-oxo-26-norcycloartanyl acetate (14b) (0.8 mg); PMR spectrum was infull agreement with the literature (15): PMR (CDC13): 6 0.354(lH,d,H-19 endo), 0.564 (lH,d,H-19 ecto), 1.090 (3H,d,J=7 Hz,H-28), 2.061 (3H,s,-O-C-CH3), 2.135 (3H,s,H-27),2.453 (lH,m,H-24, 4.555(lH,m, H-3a).An identical procedure was employed in the case of the labeled

fraction cochromatographing with cycloeucalenyl acetate (3c-acetate). To this fraction was added unlabeled 3c-acetate (2 mg)and 31-nor cyclolaudenyl acetate (6c-acetate) (0.5 mg). Themixture of 4a-methyl steryl acetates (1.5 mg) was treated asdescribed above and yielded3,-acetoxy-cycloeucalan-24,28-diol(Ilc) (RF 0.58) and 3,3-acetoxy-31-nor-24-methylcycloartan-25,26-diol (12c) (RF 0.45). Finally,Jlc and12c yielded the 24-oxo-(13c) and 25-oxo- (14c) derivatives.HPLC. Analyses have been performed on a Waters apparatus

equipped with an injector model U6K, a pump 6000 A. Thedetection of steryl acetates was done at 214 nm with a spectro-photometer Waters model 480. Quantitative measurements weredone with the help of an integrator Spectra Physics SP 4270. Thecolumn used was a gBondapak C18, the solvent of elution was amixture of CH30H-H20 (97:3, v/v) at a flow of1 ml-min-'.The HPLC technique was applied: (a) on the mixture ofcholes-teryl-, 24-methyl cholesteryl-, and 24-ethyl cholesteryl acetatesresulting from a A5-steryl acetate mixture which had been pre-viously submitted to analytical argentation TLC (see above) inorder to eliminate steryl acetates with a double bond in the lateralchain (e.g. isofucosteryl- or stigmasteryl acetates) (3). In this caseseparation of 24-methyl- and 24-ethyl cholesteryl acetates (reten-tion time, 29.70 and 34.05 min, respectively) was easily obtained;(b) on the mixture of 24-methyl- and 24-ethyl pollinastanylacetates resulting from a steryl acetate mixture which had beensubmitted before to analytical argentation TLC (see above) inorder to eliminate steryl acetates with a double bond in the lateral

1100 Plant Physiol. Vol. 79, 1985

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BIOSYNTHESIS OF STEROLS IN TRIDEMORPH TREATED MAIZE

chain (e.g. 24-methylene pollinastanyl acetate) and then to epox-idation (see above) in order to eliminate contaminating A5- andA8-sterols. In this case, 24-methyl- and 24-ethyl pollinastanylacetates (retention times, 29.8 and 33.5 min, respectively) wereeasily separated. Each operation yielded about 0.5 mg of theformer and 10 gg of the latter. Aftrer repetitive injections, pure24-methyl pollinastanyl acetate (40 mg) and 24-ethyl pollinas-tanyl acetate (0.5 mg) were obtained and were submitted to PMRanalysis.GC-MS of 24-128-2H2jMethylene (Methyl) Sterols. GC-MS

was carried out at an ionizing energy of 70 eV. The separationwere carried out on a quartz capillary column (25 m x 0.25 mmi.d.) coated with SE-30. Determination of the corrected relativeintensities of the peaks corresponding to the various deuteratedspecies (M + 1, M + 2, etc....) corresponding to a component(M) was performed taking into account the natural abundanceof the '3C isotope in the samples.

Quantification of the (24-R)-24-Methyl- and (24-S)-MethylEpimers by PMR Spectrometry. PMR was carried out in CDC13solution on a Brucker 200 MHz and a Varian 300 MHz. Todetermine the configurations at C-24 from the PMR spectra, weused the following authentic compounds: (24-R)-24-dihydrocy-cloeucalenol (2c) (4) and (24-R)- and (24-S)-24-methyl cycloar-tanol (2b and Sb, respectively) (14). The two last epimers havebeen chemically synthesized by M. Mihailovic (Zurich, Switzer-land). With the help of these compounds, the configuration atC-24 of the 24-methyl cyclopropyl sterols from maize seedlingscould be determined without any ambiguity. Details relative toPMR spectra and their interpretation will be given elsewhere.

RESULTS

Sterol Composition of Roots of Z. mays Treated with Tride-morph. The results of the analysis of the sterols of the roots fromZ. mays treated with Tridemorph (17 AM) for 3 weeks are givenin Table I. The dramatic accumulation of 9,B1,9-cyclopropylsterols (90% of total sterols) has been previously commentedupon (3). The following features are noteworthy in the contextof the present work: (a) 24-methyl pollinastanol (40% of totalsterols) is the major sterol of treated plants; (b) 31-nor cyclo-branol, a A2,125) cyclopropyl sterol presenting a great biosyntheticinterest (see "Discussion"), has been identified in relative largeamounts; (c) sterols with a 10 C side chain (e.g. sitosterol) whichpredominate in the control (18), are only minor components intreated plants; by contrast sterols with a 9 C side chain (e.g. 24-methyl pollinastanol, 24-dihydrocycloeucalenol) represent morethan 90% of total cyclopropyl sterols in treated plants; (d) notrace of cyclopropyl sterols possessing a A22 double bond weredetected in treated plants whereas the A22 double bond is presentin the sterols (e.g. stigmasterol) of control plants.24-Methyl Pollinastanol and 24-Dihydro Cycloeucalenol, are

a Mixture of Epimers at C-24. The PMR spectrum of the 24-methyl pollinastanol from Z. mays treated with Tridemorphclearly showed it to be a mixture of the (24-R)- and (24-S)-isomers (Table II). From the intensities of the doublets for theC-21, C-27, and C-28 methyl protons it was estimated that themixture contained 20% of the (24-R)-epimer (2d) and 80% ofthe (24-S)-epimer (Sd). The 24-dihydro cycloeucalenol wasshown to be also a mixture of the (24-R)- and (24-S)-isomers. Inthis case, the mixture contained 45% of the (24-R)-epimer (2c)and 55% of the (24-S)-epimer (Sc) (Table I). Similar results havebeen obtained in the case of the 24-methyl cyclopropyl sterolsfrom leaves of Triticum sativum treated with Fenpropimorph(M.F. Costet, unpublished data). The 200 MHz PMR spectrumof the 24-ethyl-cholesterol from roots of control maize wasidentical to that of authentic sitosterol (la) thus demonstratingthat it consisted predominantly of the (24-R)-isomer, sitosterol(Ja), in agreement with previous results (23, 30). However, the

Table I. Sterolsfrom Roots of Control and 17 1M Tridemorph-TreatedMaize Seedlings

% of Total Sterols

Control Treated

24-Methyl-cholesterol (2a, 5a)24-Ethyl-cholesterol (Ja)Stigmasterol (lOa)

24-Methyl-pollinastanol (2d, 5d)24-Methylene-pollinastanol (3d)24-Ethyl-pollinastanol

Cycloeucalenol (3c)24-Dihydrocycloeucalenol (2c, Sc)31-nor cyclobranol (4c)Cyclofontumienol (8c)

Cycloartenol (15b)24-Methylene cycloartanol (3b)

Total A5-sterols5'-Sterols with a 9 C side chainA5-Sterols with a 10 C side chair.

Total 9,1, 19-cyclopropyl sterolsCyclopropyl sterols with a 9 C side

chainCyclopropyl sterols with a 10 C side

chain

24 (45, 55)222 (100, O)b46

0

0

0

0

0

0

3

4

43 (20, 80)C4

l

18

13 (45, 55)5

l

1 2Traces I

8962575

2 90

95

3a,b,c,d Percentage of (24-R)- and of (24-S)-epimers in 24-methyl-cho-

lesterol (a), 24-ethyl-cholesterol (b), 24-methyl-pollinastanol (c), and 24-dihydrocycloeucalenol (d).

PMR spectrum of the 24-methyl cholesteryl acetate from rootsof control maize clearly showed it to be a mixture of the (24-R)-and (24-S)-isomers (23). It was estimated that the mixture con-tained 40 to 50% of the (24-R)-epimer (2a) and 50 to 60% of the(24-S)-epimer (5a) in agreement with previous determinationsperformed on maize coleoptiles (25). Zakelj and Goad (30)reported that Z. mays seedlings contain a mixture of 20 to 30%of 2a and 70 to80% of Sa. Therefore, it appears from this studythat 24-methyl pollinastanol which is the major sterol of treatedplants (40% of total sterols) consisted of a mixture of epimers atC-24 in which the (24-S)-epimer Sd largely predominates,whereas 24-ethyl cholesterol, a major sterol of the control ispredominantly the (24-R)-epimer la. This striking differencesuggested that Tridemorph could affect at least indirectly thestereochemistry of some steps involved in the C-24 alkylationprocess. According to the Liverpool group, the presence of (24-R)- and (24-S)-24-methyl sterol epimers could be explained bythe operation of two routes (6, 30), one proceeding via A24128)and A24'25'-sterols to produce the (24-R)-24-methyl sterols suchas 2a, the other route involving reduction of either a A24(28) or aA25-sterol intermediate to give the (24-S)-24-methyl sterols suchas 5a (Fig. 1). A plausible A25-sterol intermediate could becyclolaudenol (6b) since it possesses the appropriate stereochem-istry at C-24, is widely found in the plant kingdom, and is derivedfrom the C-24-methylation reaction in a way shown in Figure 1.Since 93, 19-cyclopropyl sterols accumulate considerably in Z.mays treated with Tridemorph, cyclolaudenol (6b), if formed,should be easily detectable as well as its derivative 31-nor cyclo-laudenol (6c). Therefore, we have looked for the presence ofthese two compounds in our material.

Presence of Cyclolaudenol and 31-nor Cyclolaudenol. Whenthe 4,4-dimethyl steryl acetate fraction from Z. mays shoots issubmitted to analytical argentation TLC, three products are

1 101

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BLADOCHA AND BENVENISTE

Table II. PMR (300 MHz) Chemical Shifts (a) ofthe Proton Signals of(24-R)- and (24-S)-24-Methyl-9p,19-Cyclopropyl Sterols and of(24-t)-24-Ethyl Pollinastanol Isolatedfrom Z. mays Seedlings Treated with Tridemorph

C-19C-18a C-21(S) C-21(R) C-26 C-27(R) C-27(S) C-28 C-30 C-32 C-29 C-3Ha

Endo Exo

2Cb 0.894 0.140 0.391 0.856 0.861 0.809 0.782 0.970 0.981 3.207s d d d d d d s d m

J=4C J=4 J=6.5 J=6.8 J=6.5 J=7 J=6.5 J,= 10J2=55bd 0.868 0.859 0.787 0.783

d d d dJ=6.4 J=6.7 J=7 J=6.5

2c + 5c 0.894 0.139 0.392 0.870 0.856 0.860 0.809 0.787 0.784 0.970 0.982 3.218s d d d d d d d d s d dt

J=3.5 J=3.5 J=6.5 J=6.8 J=6.8 J=6.5 J=6.5 J=6.5 J=6.5 J,= 10J2=52d + Sd 0.891 0.066 0.433 0.869 -' 0.858 0.808 0.785 0.781 0.959 3.688

s d d d d d d d s mJ=3.5 J=3.5 J=6.5 J=7 J=6.5 J=6.5 J=6.5

Id 0.895 0.074 0.445 0.872 0.842 0.820 0.961 0.852 4.795s d d d d d s t m

J=4 J=4 J=6.5 J=6 J=6.5 J=7a Protons carried by the carbon atoms numbered according to the official nomenclature for sterols. The letters R and S mean that the protons

belong to a sterol with the (24-R) or (24-S) configuration. b 2c, (24-R)-24-dihydrocycloeucalenol from Pseudotsuga menziesii (4); 5b, synthetic(24-S)-24-dihydrocyclolaudenol; 2c and Sc, (24-R)- and (24-S)-24-dihydrocycloeucalenols from Z. mays; 2d and Sd, (24-R)- and (24-S)-24-methylpollinastanols from Z. mays; Id, (24-t)-24-ethyl pollinastanyl acetate from Z. mays. c Coupling constants in Hz. d Data from Dr. Mihailovic(14). '-, Not resolved.

detected in order of polarity cyclosadyl acetate (7b-acetate),cycloartenyl acetate (S5b-acetate), and a mixture of 24-methyl-ene-cycloartanyl- (3b) and cyclolaudenyl- (6b) acetates (15, 30).The acetate of 7b was not detectable by GC in our material. Theacetate of6b was difficult to detect since it has a behavior similarto the acetate of 3b as well in argentation TLC as in GC.Therefore, labeling experiments and chemical derivatizationwere necessary. Z. mays axes were grown in sterile conditions inthe presence of [5-'4C]mevalonic acid or [Me-'4C]L-methionine.

15-'4CIMevalonic Experiments. The incubations were per-formed for 3 d, in order to obtain sufficient radioactivity insterols. The 4,4-dimethyl sterylacetate fraction was submitted toargentation TLC as described previously (27); to the recoveredlabeled 24-methylene cycloartanyl (3b)-acetate, cold 3b-acetate(2 mg) and cyclolaudenyl (6b) acetate (2 mg) were added, themixture was submitted to Os04 oxidation and NaIO4 cleavageas described in the experimental section. This procedure allowsthe separation of two ketones (13b) andl4b); 13b and 14b were

unambiguously identified by PMR. As shown in Table III, 13bcontained 95% ofthe radioactivity initially present in the fractioncomigrating with 3b- and 6b-acetates, whereas only 3% of thisradioactivity was associated to 14b. The 4a-methyl steryl acetatefraction was treated in the same way, it was first submitted toargentation TLC. To the labeled cycloeucalenyl (3c)-acetate frac-tion, cold 3c-acetate (2 mg) and 6b-acetate (2 mg) were added.6b-Acetate was used to protect any labeled 6c-acetate presentsince it was shown that 6b- and 6c-acetates and their derivativeshad the same RF in TLC. The results showed that the 24-oxo(13c) derivative of 3c-acetate contained more than 95% of theinitial radioactivity whereas negligible radioactivity was associ-ated to the 25-oxo (14c) derivative of 6c-acetate. This providedevidence that 31-nor cyclolaudenyl (3c)-acetate is barely detect-able in our material.

IMe-'4C1L-Methionine Incorporations. The use of this precur-sor allowed the specific labeling of the C-28 carbon atom of24-methyl sterols. Therefore, the selective cleavage of the meth-

Table III. Attempts to Detect Cyclolaudenol (6b) and 31-nor Cyclolaudenol (6c)following incorporation of[S-'4C]Mevalonic Acid and [Me-'4C1-L-Methionine in Z mays Axes Cultured Aseptically

The 24-methylene cycloartanyl acetate and the cycloeucalenyl acetate containing firtions were submittedsuccessively to Os04 oxidation then to periodate cleavage to yield ketones 13b, 14b, and 13c, 14c, respectively,as described in the experimental section.

[5-'4C]Mevalonic Acid [Me-'4C]-L-Methioninedpm

24-Methylene cycloartanyl (3b)-acetate con-taining fraction 12,850 14,000

24-oxo-Cycloartanyl (13b)-acetate 12,200 (95%r 024-methyl-25-oxo-26-norcycloartanyl (14b)-

acetate 380 (3%)- 200 (1.5%rCycloeucalenyl (3c)-acetate containing frac-

tion 20,20024-oxo-31-nor cycloartanyl (13c)-acetate 19,200 (95%)b ND24-methyl-25-oxo-26,31-bisnorcycloartanyl

(14c)-acetate 140 (<1%)P NDa Per cent of the radioactivity initially present in the fraction comigrating with 3b-acetate. bPer cent of

the radioactivity initially present in the fraction comigrating with 3c-acetate.

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BIOSYNTHESIS OF STEROLS IN TRIDEMORPH TREATED MAIZE

ylidene group would lead to complete disappearance of theradioactivity in the case of 3b-acetate but to retention of theradioactivity in the case of 6b-acetate. Thus, the use of this labelwould be a very sensitive indicator of the formation in oursystems of A25 sterols such as 6c. As shown in Table III, all theradioactivity initially present in the fraction comigrating with3b-acetate has been eliminated after methylidene group cleavagesince 13b is completely devoid of radioactivity. In the sameconditions 14b contained 1.5% of the initial radioactivity, con-firming that 6b is formed in small amounts in our system. Inconclusion, the use of radioactive precursors has given evidencethat 24-methylene cycloartanol is the major product of themethylation reaction operating in vivo and that A25-sterols suchas cyclolaudenol (6b) which could be also expected as a methyl-ation product is formed in small amounts, in agreement withresults obtained previously by Zakelj and Goad (15, 30).

[Me-2H31-L-Methionine Incorporations and MS Analysis ofthe Sterols. The results reported above provide evidence thatsmall amounts of a A25 sterol such as 6b are detectable in oursystem. Although slightly radioactive, 6b could however be met-abolically highly active. To find out if6b could play a role in 24-methyl cyclopropyl sterol biosynthesis, we performed experi-ments with [Me-2H3J-L-methionine. From Figure 1, as discussedpreviously by Lenton et al. (1 1), it follows that the involvementof A25-sterols such as 6b (or A23-sterols such as 7b) would lead to24-methyl sterol species with 3 2H atoms at C-28, whereas theuse of the A24128) A24"25) sterol--24-methyl sterol pathwaywould lead to species with 2 2H atoms at C-28. The incorporationwas performed on Z. mays axes grown aseptically in vitro for 6d. The results of the [Me-'4C]-L-methionine incorporationsshowed that the radioactivity associated with 3b-acetate is totallyeliminated after selective cleavage of the 24-methylidene group.This demonstrated that the label associated with the methylgroup of the AdoMet was not randomized in all C atoms of thesterol molecule but specifically associated with the C-28 position.Therefore, [Me-2H3]-L-methionine could be used as a precursorwith little risk of randomization of the label in other H atoms ofthe sterols. Acetylation ofthese sterols and analytical argentationTLC (cyclohexane-toluene, 8:2, v/v) permitted the isolation of:(a) cycloartenyl (15b)-acetate and 24-methylene cycloartanyl(3b)-acetate; (b) cycloeucalenyl acetate (3c); (c) 24-methylenepollinastanyl (3d)-acetate; (d) 24-methyl pollinastanyl (2d, Sd)-acetates; and (e) 24-methyl cholesteryl (2a, Sa)-acetates and 24-ethyl cholesteryl (la)-acetate. These were submitted to GC-MSas described elsewhere (12). The results are summarized in TableIV. The acetate of 15b showed a major peak at m/z 408 corre-sponding to an unlabeled component with a mass spectrumwhich confirmed its identity. The absence of label in JSb is inagreement with the fact that 15b is not methylated at C-24, andconfirms that the label of [Me-2H+-L-methionine is not random-ized in the sterol molecule. The acetate of 3b showed twocomponents; one was unlabeled 3b-acetate (M+ at m/z 482)

while the other showed M+ at m/z 484. Other ions in the mass

spectrum of3b at m/z 467, 422,407, and 300 were accompaniedby ions 2 mass units higher thus showing that the second com-ponent was 24-[28-2H2]methylenecycloartanyl acetate whichcomprised (Table IV) 25% of the mixture. No component wasdetectable at M+ + 3 mass units. These results agree with theexpected mechanism of3b biosynthesis (Fig. 1) (5, 6). The acetateof3c showed two components; one was unlabeled 3c-acetate (M+at m/z 468) while the other showed M+ at m/z 470. Other ionsin the mass spectrum of 3c at m/z 453, 408, 393, and 300 wereaccompanied by ions 2 mass units higher thus showing that thesecond component was [28-2H2]cycloeucalenyl acetate whichcomprised about 25% of the mixture (Table V). No componentwas detectable at M+ + 3 mass units. The acetate of 3d showedtwo components; one was 3d-acetate (M+ at m/z 454) while theother showed M+ at m/z 456. Other ions in the mass spectrumof 3d at m/z 439, 394, 379, and 300 were accompanied by ions2 mass units higher thus proving that the second component was24-[28-2H2]methylene pollinastanyl acetate which comprised25% ofthe mixture (Table IV). These results showed that no lossof 2H did occur during the 3b -. 3c -- 3d transformation. Themixture of acetates of 2d and 5d showed also two components;one corresponded to the acetates ofthe two epimers at C-24 (2d,Sd) (M+ at m/z 456), while the other showed M+ at m/z 458.Other ions in the mass spectrum of the acetates of 2d and Sd atm/z 441, 396, 381, and 302 were accompanied by ions 2 massunits higher thus revealing that the second component was 24-[28-2H2]methyl pollinastanyl acetate which comprised 18% ofthe mixture (two experiments) (Table IV). No component wasdetectable at M+ + 3 mass units. This indicates that during thebiosynthesis of both 2d and 5d, loss of one deuterium atompresent in the initial [Me-2H3]AdoMet does occur, suggestingthat both 2d and 5d should be formed from a 24-methylenecyclopropyl sterol such as 3b, 3c, or 3d and thus excluding theinvolvement of A25_ or A23-sterols such as 6b, 6c, or 7b. Finally,the mixture of the two epimers at C-24 (2a- and 5a-acetates) of24-methyl cholesterol showed essentially a major peak at m/z382 corresponding to an unlabeled component. No componentswere detectable at M+ + 2 or M+ + 3 mass units. An identicalresult was observed in the case of 24-ethyl-cholesterol (Ja) andof stigmasterol (JOa). This shows that AY-sterols were not labeledby [Me-2H3]-L-methionine in conditions where cyclopropyl ster-ols contained more than 20% ofM + 2 species.

15-14C,(4-R)_4-3HI]Mevalonic Acid Incorporation into the9i,19-Cyclopropyl Sterols of Z. mays Axis Grown in Vitro. Thedata given above are not in agreement with the involvement ofA25_ or A23-sterols such as 6b, 6c, or 71 during the biosynthesisof 24-methyl cyclopropyl sterols (2d or 5d); they suggest morelikely that these sterols should be formed from 24-methylenecyclopropyl sterols such as 3b, 3c, or 3d. The following questionmay be raised: are 24-methylene cyclopropyl sterols directlyhydrogenated to give either 2d or 5d or both compounds (6) or

Table IV. Distribution ofLabeled Species among one Defined Sterol Moleculefollowing Incorporation of[Me-2H31 -L-Methionine in Z. mays Axes Grown in Sterile Conditions

Mo M+ I M+2 M+3

Cycloartenyl (15b)-acetate Iooa 0 0 024-Methylene cycloartanyl (3b)-acetate 62 13 25 0Cycloeucalenyl (3c)-acetate 64 5 30 1

72 4 24 124-Methylene pollinastanyl (3d)-acetate 67 7 26 024-methyl pollinastanyl (2d + 5d)-acetateb 73 8 19 0

82 1 17 024-methyl cholesteryl (2a + 5a)-acetateb 95 5 0 0

a Relative per cent of the unlabeled and labeled species corresponding to a given steryl acetate. b Mixtureof the two (24-R)- and (24-S)-24-methyl epimers (nonseparated in 25 m capillary column).

1103

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BLADOCHA AND BENVENISTE

Table V. Incorporation of[5-'4C,(4-R)-4-3Hu]Mevalonic Acid into the 9f1,19-Cyclopropyl-Sterols of Z. maysRootsfrom Axes Grown in Vitro in the Presence of65igM Tridemorph

Observed Corrected TheoreticalSterol 3H:'4C ratio 3H:'4C atomic ratioa 3H:'4C atomic ratio

Cycloartenyl (15b)acetate 3.58 6:6 6:64.01 6:6

24-Methylene cycloartanyl (3b)-acetate 3.60 6.06:6 6:64.01 6.00:6

Cycloartane-24-en-3-one 2.99 5.01:6 5:63.31 4.96:6

Cycloeucalenyl (3c)-acetate 3.05 5.12:6 5:63.43 5.13:6

Cycloeucal-24(28)-en-3-one 2.99 5.01:6 5:631-nor cyclobranyl (4c)-acetate 2.67 3.99:6 4:624-Methylene pollinastanyl (3d)-acetate 2.96 4.97:6 5:6

3.31 4.95:624-methyl pollinastanyl (2d + 5d)b-acetate 2.40 4.02:6 4:6 for (24-R)-epimer

2.73 4.08:6

aThe 3H:'4C atomic ratios were calculated from the experimental 3H:'4C value obtained for 15b-ace-tate. b Mixture of the two (24-R)- and (24-S)-24-methyl epimers (20%:80%, respectively).

are they first isomerized into A24(25"-sterols which in turn arehydrogenated to give 24-methyl cyclopropyl sterols as it has beensuggested for (24-R)-24-methyl sterol biosynthesis (30)? To testthese possibilities, incorporations of [5-'4C,(4-R)-4-3H]mevalonicacid as conducted on Z. mays axes grown in vitro in sterileconditions for 6 d. The labeled sterols produced were extractedand separated as described in the experimental section. Allpurified compounds gave only one symmetrical peak after GCon capillary columns. The results of the 'H:'4C ratio determina-tions are given in the Tabel V. These 3H:'4C atomic ratios werecalculated from the experimental 'H:'4C values obtained forcycloartenyl (15b) acetate in which the labeling pattern had beenestablished previously (22). Since incubations were conductedfor very long times (6 d), it was necessary to check the absenceof randomization of the labels and of nonsymmetrical labelingof sterols as discussed previously (1). This was done by selectiveoxidation of 15b to the cycloartane-24-en-3-one. Calculation ofthe 3H:'4C atomic ratio showed that the oxidation step led to lossof one T atom in full agreement with the expected biosyntheticlocation of the label indicated in Figure 1. Calculation of the3H:'4C atomic ratio in 3b acetate did not show any loss of Tatom in agreement with the mechanism postulated for the C-methylation of plant sterols (6, 18). The value found for 3c-acetate is slightly higher than that expected (5.00:6) consideringthe mechanism of the C-4-demethylation step (6), however oxi-dation of 3c to the corresponding ketone led to the theoreticalvalue. The value found for 31-norcyclobranyl (4c)-acetate isconsistent with the assumption that A2"'25)-sterols such as 4cwould derive from A24'28)-sterols by an isomerization ofthe A24(28)_double bond, a step involving loss of the T atom which isexpected to be located at C-25 (Fig. 1) (6, 18). The value foundfor 24-methylene pollinastanyl (3d)-acetate is consistent withthose found for the others 24-methylene cyclopropyl sterols suchas 3b and 3c. The value found for 24-methyl pollinastanyl acetatewhich is a mixture of the two (24-R)- and (24-S)-24-methylepimers would imply that a tritium atom is lost during reductionof 3d to both 2d and Sd. This result is consistent with theinvolvement of A24t(2S)-sterols such as 4c in the biosynthesis ofboth 2d and Sd.

DISCUSSIONThe incubations of Z. mays axes grown in vitro in the presence

ofTridemorph and various 2H-, 3H-, '4C-labeled precursors haveled to the following results.

A25-Cyclopropyl Sterols are Present in Small Amounts

Whereas &24(25)-Cyclopropyl Sterols are Present in Large Quan-tities in Z. mays Roots. The first part of this work was devotedto the the search of A25-cyclopropyl sterols such as cyclolaudenol(6b) and 3 1-norcyclolaudenol (6c). These compounds were notdetectable significantly by GC under conditions which allowedtheir separation from the A24(28)-sterols (3b, 3c). The use oflabeledprecursors [5-'4C]mevalonic acid or [Me-'4C]-L-methionine ofhigh specific activity showed that after chromatographic separa-tion and formation of specific chemical derivatives, low radio-activity was found to be associated with the derivatives (14b,14c) of carrier 6b and 6c. By contrast, 31-nor cyclobranol (4c), aA24'25'-sterol, is easily detected by GC and strongly labeled when[5-'4C]mevalonic acid is used as precursor, 4c accumulates dra-matically and becomes a major cyclopropyl sterol when Triticumvulgare is treated by Fenpropimorph (M. Costet, unpublisheddata). However, other A24(25)-cyclopropyl-sterols such as cyclob-ranol (4b) or 30,3 1-bisnor cyclobranol (4d) were not detected inour material. The identification of a A24(25)-cyclopropyl sterol inaddition to A24(28)-cyclopropyl and to 24-methyl cyclopropylsterols and the fact that they are labeled when radioactive pre-cursors of sterols are used, is consistent with the hypothesis that4c is involved in the formation ofa (24-R)-24-methyl cyclopropylsterol (2c or 2d) as suggested previously in the case of (24-R)-24-methyl cholesterol (2a) (30) (Fig. 1). Although present in smallamounts, A25-sterols such as 6b or 6c would have an importantmetabolic role if their turnover was high; they could be involved,for example, in the biosynthesis of (24-S)-24-methyl cyclopropylsterols such as Sc or Sd as suggested previously in the case of thebiosynthesis of (24-S)-24-methyl cholesterol (Sa) (30) (Fig. 1). Itshould be noted at this stage that the (24-S)-epimer of 24-methylcyclopropyl sterols predominates (70% of total 24-methyl cyclo-propyl sterols) in our materials, while A25-sterols (6b, 6c) arepresent in very low amounts with respect to A24(28)-sterols (3b, c,d) which occupy an equivalent position in the biosyntheticschemes of Figure 1.

24-Methyl-Cyclopropyl Sterols Biosynthesized from [Me 2H31-L-Methionine Contain only Species with Two Deuterium Atomsat C-28 in Addition to Unlabeled Species. No species with three2H atoms at C-28 were detectable. As explained in "Results,"such data mean that A24,28)-cyclopropyl sterols (3b, 3c, 3d) wouldplay an obligatory role in 24-methyl cyclopropyl sterol (2d, Sd)biosynthesis but would not totally exclude A25-(6b) or &23-cyclo-propyl sterols (7b) as intermediates since isomerization of A24(28)_sterols to A23_ or A25-sterols and vice versa may be considered.However, direct biogenetic relationship between A25_ or A23_

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BIOSYNTHESIS OF STEROLS IN TRIDEMORPH TREATED MAIZE

cyclopropyl sterols and 24-methyl cyclopropyl sterols withoutpassage through A24'28)-cyclopropyl sterols seem to be excludedby our results. A remarkable feature ofthe [Me2H3]-L-methionineincorporation experiments is the absence of any label detectablein A5-sterols. This suggests strongly that no synthesis of A5-sterolsoccurs in Z. mays axes grown in vitro in the presence of theTridemorph during the time (6 d) of incorporation of labeledmethionine.

24-Methyl Pollinastanol Biosynthesized from 15-'4C,(4-R)4-3H1jMevalonic Acid Contains one Tritium Atom Less than 24-Methylene Pollinastanol. This result provides evidence that the25-3H atom present in 3b, c, d is removed during conversion toboth (24-R)- and (24-S)-24-methyl pollinastanols (2d and 5d,respectively). Such a result suggests the involvement of A2'425)_cyclopropyl sterols such as 4b, c, d during the biosynthesis ofboth 2d and Sd. Such a route had been already postulated toexplain the biosynthesis ofthe (24-R)-24-methyl-cholesterol (30),but the passage through A25(27) or A23-sterols was suggested byZakelj and Goad (30) to explain the biosynthesis of the (24-S)-24-methyl-cholesterol on the basis of the observed 3H:'4C atomicratios. These authors have rationalized the observed ratios onthe basis of the proportions of (24-R)-24-methyl and (24-S)-24-methyl epimers (2a and Sa, respectively) shown to be present inZ. mays and the proposed routes of production indicated inFigure 1; 2a comprises about 20 to 30% of the mixture and

26 ~~p22 27

24 25

R1 dj T

3d

Si

Rj

would have a 3H:'4C ratio of 2:6 if produced via a A21425)-sterol,whereas Sa accounts for 70 to 80% of the mixture and wouldhave a 3H:'4C ratio of 3:6 if its biosynthesis involves a A25-sterolintermediate or a A23-sterol precursor (30). Thus, the predicted3H:'4C atomic ratio (2.70-2.80:6) for the 24-methyl-cholesterol(2a and 5a) was in agreement with the observed value of 2.82:6.If such a rationalization was applied in our case, the predicted3H:'4C atomic ratio for the 24-methyl-pollinastanol (2d and Sd)would be in the order of 4.80:6 since 24-methyl-pollinastanolcontained 20% of the (24-R)-epimer (2d) and 80% of the (24-S)-epimer (5d) which is in complete disagreement with the observedvalue of 4:6. To explain our results we propose the simplebiosynthetic scheme depicted in Figure 2. According to thisscheme, a unique route is sufficient to lead to both (24-R)- and(24-S)-epimers of 24-methylpollinastanol. This route operatesvia A24(28)- and A24(25)-sterols to give the 24-methyl cyclopropylsterols. To explain the formation of the two epimers at C-24, wepostulate that the enzymic hydrogenation of the A2'"25)-cyclopro-pyl sterol, which is expected to be regio- and stereospecific whenthe substrate is 9a and would lead to (24-R)-24-ethyl-cholesterol(la) (6), would no longer be regiospecific when the substrate is4d. We suggest more precisely that the reaction would first consistof an addition of a H+ on the Re face of the A2125) leading to athree center carbocation and that an attack ofH- from NAD(P)Hwould then occur either at C-24 or at C-25 leading to the

T

Si

R 1-t R

H + Re4d

H Re FIG. 2. Biosynthetic pathway leading to (24-R)- and(24-S)-24-methylpollinastanol (2d and 5d). Comparisonwith the pathway proposed (30) for (24-R)-24-ethylcholesterol (Ja) biosynthesis. In 3d and 8a, C-26 (pro-R methyl group at C-25) has been shown to originatefrom C-2 of mevalonic acid and C-27 (pro-S methylgroup) from C-6 (28). RI, steroid skeleton.

'2627

5d (24-S) 2d (24-R)

1105

11 01 If

RI I f

I1. II1-

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BLADOCHA AND BENVENISTE

formation of (24-S)-24-methyl-pollinastanol (Sd) and to (24-R)-24-methyl-pollinastanol (2d), respectively. To explain the differ-ences of regiospecificity observed when the substrate is a 24-methyl A2"<25)-sterol such as 4d in place of a 24-ethyl A24(25)-sterolsuch as 9a, one can suggest that steric hindrance caused by thebulky ethyl group at C-24 would favor the attack of the H- fromNAD(P)H at C-25 whereas no such constraints would exist inthe case of the 24-methyl A24(25>-sterol, allowing attack of the H-at both C-24 and C-25. Such a mechanism is in agreement withthe identification of 3 1-norcyclobranol (4c) and the results of theincorporation of Me-2H3]-L-methionine and of [5-'4C,(4-R)-4-3Hilmevalonic acid; however, the demonstration would be com-plete if it could be shown that 24-ethyl-pollinastanol (Id) con-tained only the (24-R)-24-ethyl epimer, as shown already in thecase of the 24-ethyl-cholesterol (la) (23, 30). Unfortunately, Idwas present in too-small amounts (1% of total sterols) to allowunambiguous determination of the chirality at C-24. Attemptswill be made to prepare more ofId and to use PMR at higherfield (400 MHz) to obtain a better resolution. An interestingconsequence of the pathway proposed in Figure 2 is that, ifcarbon 26 was labeled, the resulting configuration at C-25 of thetwo epimers at C-24 (2d andSd) would be opposite. Experimentsusing incorporation of'3CH313C02H as described previously (28)and '3C NMR spectroscopy to determine the chirality at C-25 of2d and5d are in progress to check the above considerations.

Finally, important information which derives from this studyis that drugs like Tridemorph or Fenpropimorph, acting on aspecific target such as the COI, not only lead to an accumulationof sterol intermediates (e.g.9,B, 19-cyclopropyl sterols), but alsoto changes in the configuration of the C-24 alkyl (methyl orethyl) substituents. The present study suggests that Tridemorphor Fenpropimorph would affect indirectly the stereochemistry ofthe A24(5) bond hydrogenation involved in the last step of theC-24 alkylation process. The explanation for this probably rests onthe existence of strong structural differences between 9#,B19-cyclopropyl- and A5-sterols which are the substrates of the A2'tt25)hydrogenasee in control and Tridemorph-treated plants, respec-

tively. Of course the considerations, developed above and themodel depicted in Figure 2 are only relevant to the biosynthesisofcyclopropyl sterols in treated plants and would not obligatorilyapply to the biosynthesis of the (24-R)- and (24-S)-24-methyl-A5-sterols in control plants. Therefore, our results do not contra-dict the biosynthetic scheme previously proposed (30) but pointout that although Tridemorph acts directly on a simple targetenzyme of sterol biosynthesis (21), it leads to indirect effects onseveral enzymic steps of this biosynthesis.

Acknowledgments-We warmly thank Dr. Mihailovic (ETH, Zurich) for havingperforming the 300 MHz PMR spectra. We are undebted to Dr. G. Teller for theGC-MS determinations and to Dr. B. Fritig for discussions and advice.

LfTERATURE CITED

1. ARMAREGO WLF,LJ GOAD,TW GOODWIN 1973 Biosynthesis of a-spinasterolfrom [2-"C, (4-R)-4-'H,jmevalonic acid by Spinacia oleracea and Medicagosativa. Phytochemistry 12: 2181-2187

2. BENVENISTE P, M BLADOCHA, MFCOSTET, A EHRHARDT 1984 Use of inhibitorsof sterol biosynthesis to study plasmalemma structure and function. In ABoudet, G Alibert, G Marigo, P Lea, eds, Journal of the Annual Proceedingsof the Phytochemical Society of Europe. Oxford University Press, Oxford,pp 283-300

3. BLADOCHA M, P BENVENISTE 1983 Manipulation by Tridemorph a systemicfungicide, of the sterol composition of maize leaves and roots. Plant Physiol71: 756-762

4. CONNER AH, DO FOSTER 1981 Triterpenes from Douglas fir sapwood. Phyto-chemistry 20: 2543-2546

5. GOAD LJ, TW GOODWIN 1972 The biosynthesis of plant sterols. In L Rhein-hold, Y Liwschitz, eds, Progress in Phytochemistry, Vol 3. Interscience,London, pp 113-198

6. GOODWIN TW 1981 Biosynthesis of plant sterols and other triterpenoids. InJW Porter, SL Spurgeon, eds, Biosynthesis of Isoprenoid Compounds, vol 1.John Wiley & Sons, New York, pp 443-480

7. GREEN CE, RL PHILLIPS 1975 Plant regeneration from tissue cultures of maize.Crop Sci 15:417-421

8. HENRY JA, DS IRVINE, FS SPRING 1955 Triterpenoids. Part XXXIV. Theconstitution of cyclolaudenol. J Chem Soc 1607-1615

9. HOSOKAWA G, GW PATTERSON, WR LUSBY 1984 Effect of Triarimol, Tride-morph and Triparanol on sterol biosynthesis in carrot, tobacco and soybeansuspension cultures. Lipids 19:449456

10. ITOH T, N SHIMIzu, T. TAMURA, T MATSUMOTO 1981 24-Methyl-E-23-dehy-drolophenol, a new sterol and two other 24-methyl-E-A23-sterols in Zea maysgerm oil. Phytochemistry 20:1353-1356

11. LENTON JR, LJ GOAD, TW GOODWIN 1975 Sitosterol biosynthesis in Hordeumvulgare. Phytochemistry 14: 1523-1528

12. LOCKLEY WJS, DP ROBERTS, H REES, TW GOODWIN 1974 24-Methylcholesta-5,24(25-dien-3#-ol: a new sterol from Withania somnifera. Tetrahedron Lett3773-3776

13. McKEAN ML, WR NES 1977 Evidence for separate intermediate in thebiosynthesis of 24a- and 24,-alkyl sterols in tracheophytes. Phytochemistry16: 683-686

14. MIHAILOVIC M 1984 Biosynthesis of phytosterols in Trebouxia spp: stericcourse of the C-alkylation step. Dissertation Eidgenossische TechnischeHochschule 7535, Zurich

15. MIsso NLA, LJ GOAD 1983 The synthesis of 24-methylene cycloartanol,cyclosadol and cyclolaudenol by a cell free preparation from Zea maysshoots. Phytochemistry 22: 2473-2479

16. NES WR, K KREVITZ, S BEHZADAN 1976 Configuration at C-24 of 24-methyland 24-ethyl cholesterol. Tracheophytes Lipids1 1:1 18-126

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1106 Plant Physiol. Vol. 79, 1985

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