Plant Physiol. (1983) 71, 756-762 0032-0889/83/71/0756/07/$00.50/0 Manipulation by Tridemorph, a Systemic Fungicide, of the Sterol Composition of Maize Leaves and Roots' Received for publication June 16, 1982 and in revised form December 1, 1982 MICHELE BLADOCHA AND PIERRE BENVENISTE Laboratoire de Biochimie Vegetale, Institut de Botanique, 67083-Strasbourg Cedex, France ABSTRACT work deals with the action of Tridemorph, a systemic fungicide (19, 20), on sterol biosynthesis in Zea mays plants. Tridemorph The roots of maize (Zea mays L. var LG11) seedlings were watered with has been shown to block the CO2 (26) in suspension cultures of a solution of Tridemorph (2,6-dhmethyl-N-tridecyl-morphollne), a systemic Rubus fruticosus cells, resulting in an accumulation of 9i,B19- fungicide, for 3 to 4 weeks from the onset of germination. Very few A5- cyclopropyl sterols. The results reported here show that, when sterols, the major sterols ofthe control, were detected in the treated plnts, Tridemorph is given to maize plants, 9,8,19-cyclopropyl sterols and 9,8,19-cyckpropyl sterols accumulated dramatically in both roots and accumulate dramatically in roots and leaves, and the usual A5- leaves. A5-sterols were also found when low concentrations of the drug sterols (stigmasterol and sitosterol) are lost accordingly. were used. The time course of the accumulation of the new sterols has been studied in plants treated with various concentrations (1-20 milligrams per liter) of Tridemorph. We found that: (a) cycloeucalenol-obtusifoliol MATERIALS AND METHODS isomerase, an enzyme opening the cyclopropane ring ofcyclopropyl sterols, was strongly Inibited by the drug; and (b) the drug dhfused readily from Plant Material. Maize (Zea mays L. var LG 11) caryopses were the roots to the whole plant and reached Its enzymic targets in most of the germinated and grown in moist vermiculite in the light at 25°C leaf cells. The data obtained offer an opportunity to evaluate the physio- for 3 weeks. Tridemorph was dissolved in water and the caryopses logical and biochemical consequence of the almost complete replacement were soaked for 8 h in the solution; then the caryopses were of A5-sterols by cyclopropyl sterols in higher plant cells. germinated and grown in vermiculite in the same way as the controls except that the vermiculite was continuously soaked with the Tridemorph solution (1 l/d- 100 seedlings) in place of pure water. Plants were measured after various times of growth, and the mean height and standard deviation of the mean for each treatment were calculated. The maximum SD for any treatment was 5.4 cm. The role of sterols in the membranes of higher plants is still Authentic Materials. Tridemorph, a fungicide discovered at the poorly understood (7). In particular, the significance of the pres- Agricultural Experimental Station ofthe B.A.S.F. (Limburgerhof, ence of a bulky ethyl group at position 24 of the lateral chain in West Germany), was kindly provided by Dr. P. Leroux (Institut most plant sterols is not known. To find out more about this National de la Recherche Agronomique, Versailles, France). Cy- subject, we have modified the sterol composition of higher plant cloeucalenol was extracted from tallow wood (Eucalyptus mycro- membranes using inhibitors ofplant sterol biosynthesis, and have corys) kindly provided by Prof. R.A. Massy Westropp (Adelaide, studied the result of such modification on the structure and Australia). Cycloartenol was kindly supplied by Dr. A.S. Narula function of plant membranes. This strategy was applied first to a (Canberra, Australia). model system: suspension cultures of bramble cells. With this Analytical Procedure. The roots and leaves (about 1.5 g dry material it was possible, by using suitable drugs, to replace almost weight) of maize plants were harvested after various times of completely the normally 9resent sterols (mostly sitosterol, a A5- growth, frozen, and lyophilized; the lyophilized tissues were sterol) with others (e.g. A -sterols, A,'4-sterols, and 9f,B19-cyclo- ground in an ultra-Turrax homogenizer in the presence of dichlo- propyl sterols) (27). The logical next step was to study the effects romethane:methanol (2:1, v/v). After evaporation of the solvent, of drugs interfering with sterol biosynthesis on more complex organisms such as whole plants. There are few published reports 2Abbreviations and chemical nomenclature: COI, cycloeucalenol-ob- of such studies: some years ago it was shown that some plant- tusifoliol isomerase; Tridemorph, 2,6-dimethyl-N-tridecyl-morpholine; growth retardants, such as tris(2-diethylaminoethyl)-phosphate RRT, relative retention time; PMR, proton magnetic resonance; cycloeu- trihydrochloride (SKF-7797), interfere with sterol biosynthesis in calenol, 4a.14a-dimethyl-91,19-cyclo-5a-ergost-24 (28)-en-3,8-ol; obtusi- Pharbitis nil (2). Later, it was noted that drugs (AMO 1618, CCC) foliol, 4a,14a-dimethyl-5a-ergosta-8,24(28)-dien-3,8-ol; cyclofontumienol, which were considered to inhibit gibberellin biosynthesis also 4a,14a-dimethyl-9,B,19-cyclo-5a-stigmast-Z-24(28)-en-3,B-ol; 31-nor cy- acted on plant sterol biosynthesis (8). SKF 7997 was shown to clobranol, 4a,14a-dimethyl-9,I,19-cyclo-5a-ergost-24(28)-en-3fi-ol; 24- inhibit sterol biosynthesis in tobacco (22). More recently, Triari- methylene po11inastanol, 14a-methyl-9,B,19-cyclo-5a-ergost-24(28)-en-3,B- mol (a fungicide) and Ancymidol (a plant-growth retardant) were ol; 24-methyl pollinastanol, 14a-methyl-9fl,19-cyclo-5a-ergostane-318-ol; reported to retard the growth of Phaseolus vulgaris seedlings but 24-ethyl pollinastanol, 14a-methyl-9/8,19-cyclo-5a-stigmastane-3,8-ol; 24- not to noticeably affect the qualitative and quantitative distribu- ethylidene pouinastanol,14a-methyl-9B,19-cyclo-5a-stigmast-Z-24(28)-en- tion of the main sterols present (28), even though these drugs 3,8-ol; cycloartenol, 4,4,14a-trimethyl-9f.,19-cyclo-5a-cholest-24-en-3,8-ol; inhibit ergosterol biosynthesis in Ustilago maydis (21). The present 24-methylene cycloartanol, 4,4,14a-trimethyl-9fl,19-cyclo-5a-ergost- 24(28)-en-3fl-ol; 24-methylene lophenol, 4a-methyl-5a-ergosta-7,24-dien- ' Supported by the Centre National de la Recherche Scientifique, 3,B-ol; 24-ethylidene lophenol, 4a-methyl-5a-stigmasta-7,Z-24(28)-dien- Equipe de Recherche Associee 487, and Grant ATP No. 3998. 3fi-ol; isofucosterol, stigmasta-5,Z-24(28)-dien-3,B-ol. 756 CYCLOPROPYL STEROL ACCUMULATION IN MAIZE 757 the residue was saponified using KOH (6%, w/v) in methanol. coated with SE 30. 31-Nor cyclobranyl acetate: MS m/e (relative The unsaponifiable matter was extracted three times with hexane, intensity): 468 (M+) (14), 425 (M+-43) (2), 408 (M+-60) (100), 393 and these extracts were pooled and dried. After evaporation of (M+-60-15) (94), 283 (M+-lateral chain-60) (22), 324 (a) (11), 300 the solvent, the residue was chromatographed on Merck HF 254 (b) (9), 241 (c) (9). a represented a McLafferty fragmentation, b plates (0.2 mm) with dichloromethane as the solvent (two runs). was a fragment characteristic of the cyclopropane ring, and c The bands of 4,4-dimethyl sterols, 4a-methyl sterols, and 4-des- derived from the cleavage of the D cycle (26). methyl sterols were scraped off and each type was eluted. The PMR Spectrometry. PMR spectrometry was carried out in three classes of compound were acetylated at room temperature CDC13 solution on a Brucker 200 MHz spectrometer. The data for 14 h using a mixture of pyridine (20 pl), acetic anhydride (50 (Table I) gave considerable information about the chemical struc- pi), and toluene (50 ,ul). Excess reagents were evaporated under ture of the new compounds, as follows: reduced pressure and the crude acetates purified by TLC using (24@)-24-Methyl-polftnastanol. A careful examination of the dichloromethane. Each of three classes of acetates was analyzed PMR spectrum (Table I) showed that this compound was a by GLC with a Carlo Erba GC model 4160 equipped with a flame mixture of the (24R)- and (24S)-24-methyl epimers. This conclu- ionization detector and a glass capillary column (15 m x 0.25 mm) sion was obtained by comparison of our PMR data with data from coated with OV-73. The temperature program used included a published reports dealing with the PMR of 24-methyl sterols (24) fast rise from 60 to 220°C (30°C x min-'), then a slow rise from and, more precisely, of synthetic mixtures of (24R)- and (24S)-24- 220 to 2800C (30C x mini). An internal standard of cholesterol methyl sterols (4). was used. The total amount of sterols present in each class was 24-Dihydro CycloeucalenoL This compound was also a mixture quantified using a Varian CDS 11 integrator. To find the RRT, of the (24R) and (24S)-24-methyl epimers. The identification was the products were also analyzed using a GC fitted with two FID achieved by comparison of our PMR and MS data with those and two glass columns (1.50 m x 3 mm) packed with either 1% already published for this compound (5). SE-30 or 1% OV-17. The RRT (cholesterol, RRT = 1.0) have CyclofontumienoL The major features of this spectrum were the been given elsewhere (26) except for 31-Nor cyclobranyl acetate, presence of a signal resonating at 81.591, characteristic of the C- for which the RRT (OV-17) = 2.57. Analytical argentation TLC, 28 vinylic methyl; the presence of a quartet (8 5.122) that corre- using cyclohexane-toluene (7:3, v/v) as the developing solvent, sponded to the C-28 vinylic proton; and the presence of a typical with 15 h of migration, was performed on each class of steryl septet that corresponded to the C-25 proton and whose chemical acetate, and the resulting bands were analyzed by GLC.