Comparative Effects of Substituted Pyrimidines on Growth and Gibbereilin Biosynthesis

The fungicide a-(2,4dlhloropheyl)-a-phenyl5-pyrne methyl alcohol (triarimol) and four other structural analos of this substance, in which one or more of the substituents were varied, were tested for their comparative effects on growth and gibbereilin biosynthesis in the fimgus GibbereJluiuwro Each of the five analogs tested was capable of inhibiting growth as measured by dry weight In 5-day-old cultures. Three of them la-(2-chlorophenyl)-a44-chlor nyl)-5-pyre methyl alcohol, fenarimol; a-(2-chlorophenyl)-a-(4-fluorophenyl)-5-pyrmine methyl alcohol, nuarimol and triarimoll were effective at appreciably lower concent:stions than the other two la-(4-chlorophenyl)-a-(l-methylethyl)-5-pyridine methyl alcohol, experimental compound EL 509; and a-cyclopropyl-a-(4methoxyphenyl)-5-pyrimldine methyl alcohol, ancymidoll. All five substances also inhibited gibberellin production as measured by gibbereflin content of fungus fitrates. The relative effectiveness of the compounds as inhibitors of growth and gibberellin production were similar. These analogs were also shown to inhibit eat-kaurene oxidation by microsomal preparations from fungal mycelia. Thus, the site of inhibition of gibberellin biosynthesis may be the same for the fungus as the one affected by this group of substances in hgher plant tissues. The structure-activity relatshs between the analogs are opposite to those observed in higher plant tissues. The fungicides fenarimol, nuarmol, and triarimol, which were most effective in inhibiting growth and gibberellin biosynthesis in the fungus, were much less effective than EL 509 and ancymidol in inhibiting growth and gibberellin biosynthesis in higher plants. These results indicate that the ent-kaurene oxidase systems from the two sources have somewhat different molecular characterstics, and thus, interact differently with this group of substances. Ancymidol3 is a potent inhibitor of growth and GA4 biosyn' Supported by Grant PCM 76-19279 from the National Science Foundation and by Grant GM 07065 from the National Institutes of Health. 'To whom reprint requests should be addressed. 3Trivial names: Ancymidol (also referred to in the literature as experimental compound EL 531 or the commercial preparation A-REST) is the trivial name for a-cyclopropyl-a-(4-methoxyphenyl)-5-pyrimidine methyl alcohol; triarimol (experimental compound EL 273) is the trivial name for a-(2,4-dichlorophenyl)-a-phenyl-5-pyrimidine methyl alcohol; ent-kaurene and ent-kaurenoic acid refer to ent-kaur-16-ene and ent-kaur-16-en19-oic acid, respectively. Other potential inhibitors used in this study include: fernarimol (EL 222), a-(2-chlorophenyl)-a (4-chlorophenyl)-5pyrimidine methyl alcohol; nuarimol (EL 228), a-(2-chlorophenyl)-a-(4fluorophenyl)-5-pyrimidine methyl alcohol; and EL 509, a-(4-chlorophenyl)-a-(l-methylethyl)-5-pyrimidine methyl alcohol. The structures of all five inhibitors are provided in the accompanying paper (I 1). 'Abbreviation: GA, gibberellin. thesis in higher plants (9-11, 17, 22, 24). It has been shown previously to be ineffective at similar concentrations on the fungus, Gibberellafujikuroi (10, 17). Shive and Sisler (22) compared the effects of ancymidol and triarimol on growth and GA contents of beans and G. fujikuroi. They reported that these compounds do inhibit GA biosynthesis in the fungus, although triarimol, an experimental fungicide, is more effective than ancymidol. Ali et al. (1) also reported on the fungitoxic activity of ancymidol. In the accompanying paper (11), we have compared the effects of a number of ancymidol analogs, including triarimol, on the growth of pea seedlings and the rate of kaurene oxidation in cell-free enzyme extracts of Marah macrocarpus liquid endosperm. The results indicate that the analogs with fungicidal activity do inhibit ent-kaurene oxidation, but only at relatively high concentrations. Because of the seeming high degree of specificity of ancymidol for three mixed-function oxygenase reactions ofthe biosynthetic pathway for GAs in the enzyme system from M. macrocarpus (10), the apparent differences in the structure-activity relationships of these compounds in higher plants and G. fujikuroi (11, 22), and the apparent lack of growth inhibitory activity for the fungus by other GA biosynthesis inhibitors (8, 12, 16, 18), we have compared the effects of five structural analogs, including ancymidol and triarimol, on growth, ent-kaurene oxidation, and GA biosynthesis in the fungus G. fujikuroi. MATERIALS AND METHODS Fungus Culture. The stock culture of G. fujikuroi used in these studies was a single-cell isolate from the M 419 strain ofAmerican Type Culture Collection No. 917 maintained on potato dextrose agar. The liquid culture medium used for determination of effects of inhibitors on the mycelial growth of G. fujikuroi was as described previously (10). Suspensions (1 ml) of 3-d-old cultures (100 ml) of fungus (exponential growth phase) were transferred to fresh media containing appropriate concentrations of potential inhibitors. These flasks were then incubated for 5 d on a shaker under conditions previously described (10). Determination of Growth. After 5 d incubation, the mycelia were harvested by vacuum filtration on a Buchner funnel. The culture flasks were rinsed twice with distilled H20, and the mycelia were also washed gently. The culture filtrates were stored at 3°C for extraction for GAs. The collected mycelia were oven dried on preweighed filter papers for 72 h, and dry weights were determined repeatedly until constant. GA Extractions. Ten percent of the total volume of each filtrate was withdrawn. The pH of these filtrates was adjusted to 2.5 with dilute HCI. Filtrates were extracted twice with equal volumes of ethyl acetate. Organic extracts were combined and dried over anhydrous sodium sulfate, decanted, and evaporated to dryness under vacuum on a rotary evaporator at 40°C. The dried residue 712 www.plantphysiol.org on November 11, 2017 Published by Downloaded from Copyright © 1982 American Society of Plant Biologists. All rights reserved. PYRIMIDINE EFFECTS: GIBBERELLA FUJIKUROI was then taken up in 1 ml of acetone and stored under refrigeration. The acetone extract was applied to thin layers (0.25 mm) of silica gel 60. The TLC plates were developed to 15 cm with H20, (7) and the upper 5 cm of silica gel were removed. The GAs in this portion of the gel were eluted with 10 ml of acetone, and the acetone was evaporated. The extract was taken up in Tris buffer, adjusted to pH 8, and extracted three times with 2 ml benzene to remove nonacidic organic contaminants. The aqueous phases were adjusted to pH 2.5 with HC1 and diluted with ethanol to a final concentration of 25% in 0.05% aqueous Tween-20. These preparations were refrigerated for later analysis by the dwarf pea bioassay and GC-MS. Procedure for GA Bioassays. Pisum sativum (Progress No. 9) seeds were planted in sterile vermiculite in plastic pots (15 cm in diameter) 10 to 15 d prior to treatment. These plants were grown in a greenhouse under 16-h photoperiods. The light intensity was supplemented with and day length extended by Gro-Lux fluorescent lamps yielding approximately 800 ft-c. The temperature ranged from 20 to 25°C. Pots were irrigated alternately with water and half-strength Hoagland solution. Pots were thinned to contain 10 uniform seedlings, and each of the 10 plants per pot received a 10 plI application of experimental organic extract on the shoot tip after initial height measurements had been recorded. An additional set of bioassay plants was treated in the same way with a 1:9 dilution of each sample. This was done to insure responses within the linear range of the bioassay and to provide confiming data. Two sets of plants were also used for standard curve determinations. One set was treated with samples of GA3 ranging in concentration from 0.01 to 100 ,ug/,ul. The other set was treated with the same concentrations of GA3 which had been previously extracted from buffer and chromatographed under the same conditions as the fungus extracts. GC-MS and Single Ion Current Monitoring of GAs. Samples prepared for bioassay were also utilized for GC-MS. Aliquots were re-extracted into ethyl acetate at pH 2.5, taken to dryness, dissolved in ether, treated with diazomethane (generated from Nmethyl-nitroso-p-toluene sulfonamide), followed by treatment in closed containers at 60°C for 30 min with bis-(trimethylsilyl)trifluoroacetamide plus 1% trimethylchlorosilane. These derivatized samples were injected into a computerized Varian MAT CH7 combined gas chromatograh-mass spectrometer fitted with a 4ft x Y8-inch column of 7% OV 210 coated on HP Chromsorb W. Chromatography was done isothermal at 230°C with injection and detection chambers at 290°C. Helium was the carrier gas at 30 cc min-'. MS (10 scans min-) was at 70 ev. The identity of GA3 was confirmed by comparison of mass spectra and retention times with an authentic standard and with published mass spectra (4). GA3 content was monitored by single ion current monitoring at m/e = 504. Incorporation of 12-54CiMevalonic Acid into GA3. Aqueous [214C]mevalonic acid (0.5 ,umol; 10.9 mCi/mmol) was Millipore filtered and added aseptically to growing cultures (50 ml medium buffered at pH 7.5 with 0.05 M potassium phosphate) of G.fujikuroi 44 h prior to harvest. The culture filtrates were extracted, first with an equal volume of benzene/acetone (3:1), and then acidified to pH 2.5 and extracted for GAs as described above. The organic extracts were combined, dried over sodium sulfate and chromatographed on thin layers of silica gel to 15 cm in hexane followed by redevelopment to 10 cm with benzene/ethyl acetate/NH40H (90:9.5:0.5). This system separates many of the intermediates in the GA biosynthetic pathway from ent-kaurene through ent-kaurenoic acid while leaving more polar acids at the origin (10). The gel at the origin (0.5 cm) was then removed, extracted with acidic ethyl acetate, and rechromatographed on a new TLC plate with isopropyl ether:acetone:acetic acid (90:30:1.0) with authentic nonradioactive GA3 as a marker. The gel corresponding to the position of GA3 in an ancymidol-treated sample was extracted, combined with 100 mg GA3, and recrystallized to constant specific radioactivity from methanol. Cell-Free Ent-Kaurene Oxidation. Microsomal preparations from mycelia of actively growing G. fujikuroi were prepared and assayed as described previously (10) for ent-kaurene oxidase activity in the presence and absence of all five inhibitors. The ['4C]entkaurene used as substrate was prepared biosynthetically from ['4C]mevalonic acid (13.65 mCi/mmol) in cell-free extracts of M. macrocarpus endosperm (10). Radioactivity Determinations. Radioactive products separated by TLC were located on the plate by means of a Packard radiochromatogram scanner (model 7201) with a 2-mm window. Gel segments were scraped from the TLC plate and counted in a Packard liquid scintillation spectrometer. Source and Purity of Reagents. N-methyl-N-nitroso-p-toluene sulfonamide, NADPH, flavin adenine dinucleotide, and GA3 (Grade III) were purchased from Sigma. Regisil RC-2(bis-(trimethylsilyl)-trifluoroacetamide + 1% trimethylchlorosilane) was purchased from Regis Chemical, Morton Grove, IL. The potential inhibitors were all gifts from Eli Lilly and Co. They are all presumably racemic mixtures. Ancymidol and EL 509 are experimental plant growth regulators, while fenarimol, nuarimol, and triarimol are experimental fungicides. All other chemicals were reagent grade, and all solvents used in derivatizations were redistilled before use.