Gibberellin precursor biosynthesis inhibition by EPTC and reversal by R-25788

S-ethyl dipropylthiocarbamate (EPTC) inhibited gibberellic acid (GA) precursor biosynthesis in a cell-free enzyme preparation from unruptured, etiolated sorghum (Sorghum bicolor L. cv. G522 DR) coleoptiles. EPTC, 1 μM, inhibited incorporation of [¹⁴C]mevalonic acid into kaurene 60%, while 10 μM EPTC inhibited ¹⁴C incorporation into kaurene 90%. The precursor of kaurene cyclization (GGPP) increased in ¹⁴C content at both EPTC concentrations. R-25788 reversed the EPTC inhibition of kaurene synthesis. Kaurene oxidation was modified by both EPTC and R-25788. Hypothesized modes of action for EPTC and R-25788 are (a) inhibition of GA synthesis, (b) increased peroxidase activity resulting in increased lignification, (c) increased detoxification by sulfoxidation and carbamoylation, and (d) inhibition of fatty acid synthesis and/or desaturation. These hypotheses are discussed with three of them being incorporated into one working unit which correlates with EPTC and R-25788 symptom phenology. The fourth hypothesis could also fit into this general pattern.     

Diallate inhibition of gibberellin biosynthesis in sorghum coleoptiles

Diallate [S-(2,3-dichloroallyl)diisopropylthiocarbamate] incorporated into sand significantly inhibited sorghum (Sorghum bicolor (L.) Moench cv Funks G 522DR) growth of 14-day-old seed lings. Inhibition was competitively reversed by exogenous giberellic acid (GA₃) (0.1 and 10 ppmw). Diallate inhibited gibberellin (GA) precursor biosynthesis in a cell-free enzyme preparation from unruptured, etiolated sorghum coleoptiles. Diallate (10 μM) inhibited kaurene oxidation 40% with a 2.7 × increase in kauren-ol and a 50% decrease in kaurenoic acid. The GA biosynthesis inhibition correlates with symptom phenology and field use application concentrations. Geranylgeranyl pyrophosphate accumulated 5 × at 0.1 μM diallate concentrations but concomitant kaurene concentration decreases did not occur. At 10 μM diallate, kaurene synthetase was inhibited 33%.     

Inhibition of chitin biosynthesis by buprofezin analogs in relation to their activity controlling Nilaparvata lugens Stål

Buprofezin (Applaud, 2-tert-butylimino-3-isopropyl-5-phenyl-3,4,5,6-tetrahydro-2H-1,3,5-thiadiazin-4-one) strongly inhibited the [³H]chitin synthesis from N-acetyl-d-[1-³H]glucosamine in the brown rice planthopper, Nilaparvata lugens Stål. No inhibition was observed for [³H]-labeled protein biosynthesis from [5-³H]glucose or l-[3,5-³H]tyrosine but [³H]-labeled nucleic acid synthesis from [5-³H]glucose was weakly reduced by buprofezin. The lethal activity of buprofezin analogs related well to their inhibitory potency against chitin biosynthesis in N. lugens nymphs.     

Mode of action of triadimefon in Ustilago avenae

Triadimefon [1-(4-chlorophenoxy)-3,3-dimethyl-(1,2,4-triazol-1-yl)-2-butanone], 1.5–2.0 μ/ml, inhibited the multiplication of sporidia of Ustilago avenae more strongly than it did the increase of dry weight. The treated sporidia appeared swollen, multicellular, and branched. At concentrations of 1.5–100 μg of triadimefon/ml, the oxidation of glucose was not affected. Increase in dry weight and synthesis of protein, RNA, and DNA were inhibited slightly, whereas cell division was acutely arrested. After an incubation period of 9.5 hr, microscopic studies revealed that daughter cells of the treated sporidia also contained one nucleus. In sporidia treated for 6 hr with triadimefon, both the total lipid content and its composition of fatty acids were not appreciably altered. The treated cells, however, differed from control cells by a higher content of free fatty acids. Triadimefon markedly interfered in sterol biosynthesis in Ustilago avenae. Gas chromatographic (glc) analysis and [¹⁴C]acetate incorporation studies indicated that ergosterol biosynthesis was almost completely inhibited by triadimefon; on the other hand, sterol compounds representing precursors of ergosterol (probably 4,4-dimethyl and C-4-methyl sterols) accumulated in treated sporidia. As the results indicate, the inhibition of conversion of immediate sterol precursors to ergosterol may be regarded as the primary target for the action of triadimefon in Ustilago avenae.     

Metolachlor [2-chloro-N-(2-ethyl-6-methylphenyl)-N-(2-methoxy-1-methylethyl)acetamide] inhibition of gibberellin precursor biosynthesis

[2-¹⁴C]Mevalonic acid incorporation into gibberellic acid precursors was measured in cell-free extracts from sorghum [Sorghum bicolor (L.) Moench var. G-522 DR] coleoptiles. ¹⁴C incorporation into ent-kaur-16-ene was inhibited ca. 90% by 10^?7 to 10^?4M metolachlor [2-chloro-N-(2-ethyl-6-methylphenyl)-N-(2-methoxy-1-methylethyl)acetamide]. [¹⁴C]Geranylgeraniol (GG) content increased. [¹⁴C]Farnesol content was not altered and [¹⁴C]geraniol content decreased. Total ¹⁴C incorporation was decreased by metolachlor. In the safener [α-(cyanomethoximino)benacetonitrile]-treated sorghum seed coleoptile cell-free system, total ¹⁴C incorporation increased, [¹⁴C]kaurene and relative kaurence content increased 4× up to 10⁵M metolachlor, and [¹⁴C]farnesol, and [¹⁴C]GG contents increased while relative farnesol and relative GG contents were not influenced by metolachlor. Thus, the inhibition of kaurene synthesis by metolachlor was reversed by the safener. Since the biosynthetic processes are mevalonic acid → geraniol → farnesol → GG → copalylol → kaurene, these data corroborate a proposed gibberellic acid biosynthesis inhibition between GG and kaurene as well as a partial blockage between mevalonic acid and geraniol. Thus, a portion of metolachlor-induced growth inhibitions of sorghum could be explicable on the basis of gibberellic acid biosynthesis inhibitions.     

Physiological responses of corn (Zea mays) to AC 243,997 in combination with valine,leucine, and isoleucine

Various physiological processes were measured in corn after treatment with AC 243,997. Neutral sugar levels in leaves increased 39% over the control 24 hr after application of AC 243,997. Protein synthesis, measured by [¹⁴C]leucine and [¹⁴C]cystine incorporation, and lipid synthesis were not inhibited 24 hr after application of 150 μM of AC 243,997, while respiration and RNA synthesis were inhibited 32 and 15%, respectively. DNA synthesis was severely inhibited (70–90%) by 150 μM of the herbicide 24 hr after application. The inhibition of DNA synthesis by AC 243,997 did not begin until 5 to 7 hr after application. Although protein synthesis rates were apparently unaffected by AC 243,997, the level of the soluble proteins decreased 40% while free amino acid levels increased 32% 24 hr after application of the herbicide. An exogenous supply of valine, leucine, and isoleucine to corn prevented the inhibition of growth and reversed the inhibition of DNA synthesis caused by AC 243,997. All three amino acids at a concentration of 1 mM were needed to provide maximum protection. The results support the hypothesis that AC 243,997 kills plants by interfering with the biosynthesis of valine, leucine, and isoleucine.     

On the antifungal mode of action of tridemorph

Tridemorph (2,6-dimethyl-N-tridecylmorpholine) was active against representative of nearly all taxonomic groups of fungi; gram-positive bacteria were also sensitive although gram-negative were not. Tridemorph, 3–10 μg/ml, inhibited the multiplication of sporidia of Ustilago maydis more strongly than the increase of dry weight. The treated sporidia appeared swollen, multicellular, and sometimes branched. Unsaturated lipophilic compounds like α-tocopherol and trilinolein alleviated the toxicity of tridemorph to Botrytis allii and U. maydis. Protein and RNA syntheses were inhibited slightly. DNA synthesis was rather strongly affected already after 2 hr. Lipid synthesis was first inhibited but later stimulated. At an early stage (2 hr) treated cells differed already from control cells by a higher content of free fatty acids. Tridemorph also inhibited sterol biosynthesis. The antimicrobial spectrum, the characteristic morphology of treated cells of U. maydis, the observations on cross-resistance, the alleviating effect of unsaturated lipophilic compounds, and the alterations in neutral lipid pattern suggest strong similarity of the mode of action of tridemorph with that of the known inhibitors of sterol biosynthesis.     

PCR-based suppression subtractive hybridization analyses of enantioselective gene expression in root tips of wheat treated with optically active urea compounds

The optically active urea compounds R- and S-1-α-methylbenzyl-3-p-tolylurea (R- and S-MBTU) have qualitatively different effects on plant physiology, especially in Gramineae plants. To elucidate the mechanisms by which S-MBTU inhibits root growth of wheat (Triticum aestivum L. var. Chinese Spring), we used PCR-based suppression subtractive hybridization (SSH) and quantitative real-time RT-PCR (qPCR) analyses. Genes related to amino acid biosynthesis, cell cycle and skeleton, protein synthesis, cell wall biosynthesis, glycolysis, signaling, DNA modification, and detoxification were enantioselectively regulated. These expression profiles suggested that the putative mode of action of S-MBTU is disruption of primary amino acid biosynthesis, especially the glutamine synthetase (GS)- and acetohydroxyacid synthase (AHAS)-enzyme systems, and disruption of cell membrane skeleton homeostasis related to a putative α-tubulin 2 (α-TUB2), a putative β-actin (ACT) and β-tubulin 1 (β-TUB1). Other genes specifically affected by S-MBTU may result in disruption of metabolic processes, leading to inhibition of root growth.     

CDAA inhibition of Kaurene oxidation in etiolated sorghum coleoptiles

N,N-Diallyl-2-chloroacetamide (CDAA) (0.25 ppmw; I μM) inhibited growth of 14-day-old sorghum (Sorghum bicolor (L) Moench. cv Funks G 522DR) when the herbicide was incorporated into sand. Kaurene oxidation was inhibited in a cell-free enzyme preparation from 4-day-old unruptured, etiolated coleoptiles. CDAA (1 μM) inhibited incorporation of [¹⁴C]mevalonic acid into kauren-19-oic acid (50%), with resultant increases in concentration of precursors. Thus, inhibition of gibberellin precursor biosynthesis was demonstrated, and this activity would explain many of the morphogenic and biochemical responses of grasses to CDAA.     

Metolachlor influence on growth and terpenoid synthesis

Height and fresh weight of sorghum (Sorghum bicolor L. var GA 522 DR) grown in sand were reduced by metolachlor (2-chloro-N-(2-ethyl-6-methylphenyl)-N-(2-methoxy-1-methylethyl) acetamide) (0, 0.125, 0.25, 0.5, or 1 ppmw) applied preemergence. Significantly different responses were obtained from plants grown at two light intensities (270 and 27 μein/m²/sec). When grown in nutrient solution containing 0, 0.0156, 0.0625, 0.25, 1, 16, or 64 ppmw metolachlor, shoot and root lengths were inhibited by metolachlor; fresh and dry weights of shoot, root, and total plant decreased as metolachlor concentration increased. Carotene content (micrograms per gram fresh weight) in sorghum leaves was decreased by metolachlor. Specific activity of carotene synthesized from [2-¹⁴C] mevalonic acid by carrot (Daucus carota L.) disks was reduced 50% by 10^?5 and 10^?4M metolachlor. Thus, terpenoid biosynthesis is influenced by metolachlor. Gibberellins are terminal products of plant terpenoid biosynthesis, and GA₃ reverses metolachlor inhibition of growth at specific ratios of GA₃ and metolachlor but not at other concentrations. Thus, one effect of metolachlor on plants may be an inhibition of GA synthesis that results in shoot and root growth reductions as metolachlor concentration increases. Other growth responses of plants to metolachlor are unexplained.     

Effects of alachlor on anthocyanin and lignin synthesis in etiolated sorghum (Sorghum bicolor (L.) Moench) mesocotyls

Alachlor, a preemergence herbicide used to control grasses and some broadleaf weeds, was found to inhibit anthocyanin and lignin accumulation in excised sections of mesocotyls from 6 day-old, etiolated sorghum (Sorghum bicolor (L.) Moench) seedlings. Alachlor inhibited anthocyanin and lignin synthesis by 50% at 20 and 28 μM, respectively, with anthocyanin synthesis being inhibited in 1 hr. Other chloroacetanilide herbicides also inhibited anthocyanin synthesis in this system, but all were less active than alachlor. Inhibition of anthocyanin synthesis could not be reversed by compounds from the shikimic acid or phenylpropanoic acid pathways. Anthocyanin synthesis could be restored by removal of alachlor from the incubation solution. Evidence is presented which suggests that alachlor inhibits anthocyanin formation at a point late in the phenylpropanoic acid pathway and not in the shikimic acid pathway.     

Localizing the action of two thiadiazolyl herbicidal derivatives

Enzymatically isolated leaf cells from navy beans (Phaseolus vulgaris L., cv. “Tuscola”) were used to study the effect of buthidazole (3-[5-(1,1-dimethylethyl)-1,3,4-thiadiazol-2-yl]-4-hydroxy-1-methyl-2-imidazolidinone) and tebuthiuron (N-[5-(1,1-dimethylethyl)-1,3,4-thiadiazol-2-yl]-N,N′-dimethylurea) on photosynthesis, protein, ribonucleic acid (RNA), and lipid synthesis. The incorporation of NaH¹⁴CO₃, [¹⁴C]leucine, [¹⁴C]uracil, and [¹⁴C]acetic acid as substrates for the respective metabolic process was measured. Time-course and concentration studies included incubation periods of 30, 60, and 120 min and concentrations of 0.1, 1, 10, and 100 μM of both herbicides. Photosynthesis was very sensitive to both buthidazole and tebuthiuron and was inhibited in 30 min by 0.1 μM concentrations. RNA and lipid syntheses were inhibited 50 and 87%, respectively, by buthidazole and 42 and 64%, respectively, by tebuthiuron after 120 min at 100 μM concentration. Protein synthesis was not affected by any herbicide at any concentration or any exposure time period. The inhibitory effects of buthidazole and tebuthiuron on RNA and lipid syntheses may be involved in the ultimate herbicidal action of these herbicidal chemicals.     

Analogy in the mode of action of fluotrimazole and clotrimazole in Ustilago avenae

Fluotrimazole [BUE 0620; 1-(3-trifluoromethyltriphenyl) 1,2,4-triazole] (20 μg/ml of nutrient solution) and clotrimazole [Bay b 5097; bisphenyl(2-chlorophenyl)-1-imidazolyl methane] (5 μg/ml) did not inhibit dry weight increase and only slightly reduced multiplication of sporidia of Ustilago avenae during the first doubling period (about 4 hr). After 8 hr, both fluotrimazole and clotrimazole more strongly inhibited sporidia multiplication than dry weight increase. As a consequence of treatment with both fungicides the usually single-celled sporidia appear swollen, multicellular, and branched. Both chemicals at a concentration range of 5–100 μg/ml did not affect oxidation of glucose. The effect of fluotrimazole and clotrimazole on protein, DNA, and RNA synthesis was similar to that on dry weight. Following a 6-hr incubation period total lipid synthesis was quantitatively unaffected by both chemicals. As the analysis of major fatty acids of total lipids revealed fluotrimazole substantially induced the synthesis of 20:4 carbon fatty acids, while in clotrimazole-treated sporidia the pattern of fatty acids did not differ from that of control sporidia. Fluotrimazole and clotrimazole produced a higher quantity of free fatty acids in sporidia of U. avenae. Gas-liquid chromatographic analysis of sterol fractions in treated and control sporidia (6 hr) indicated that both fluotrimazole and clotrimazole seriously inhibited ergosterol biosynthesis and concomitantly caused an accumulation of immediate ergosterol precursors which represent C-4-methyl and 4,4-dimethyl sterols. Incorporation of [¹⁴C]acetate for 2 hr into various lipid fractions of sporidia of U. avenae also revealed that radioactivity in C-4-desmethyl sterols in both fluotrimazole- and clotrimazole-treated sporidia was drastically reduced, while the radioactivity of C-4-methyl and 4,4-dimethyl sterols distinctly increased. The data suggest that fluotrimazole and clotrimazole are specific inhibitors of the oxidative demethylation of the C-14-methyl group during ergosterol biosynthesis in U. avenae.     

Antifungal mode of action of triforine

Rapidly growing mycelia of Aspergillus fumigatus treated with 10 μg/ml triforine (N,N′-bis-(1-formamido-2,2,2-trichloroethyl)-piperazine) showed little or no inhibition in dry weight increase prior to 2 h. By 2.5–3 h, triforine inhibited dry weight increase by 85%. The effects of triforine on protein, DNA, and RNA syntheses corresponded to the effect on dry weight increase both in time of onset and magnitude. Neither glucose nor acetate oxidation were inhibited by triforine.Ergosterol synthesis was almost completely inhibited by triforine even in the first hour after treatment. Inhibition of ergosterol synthesis was accompanied by an accumulation of the ergosterol precursors 24-methylenedihydrolanosterol, obtusifoliol, and 14α-methyl-Δ^{8, 24 (28)}-ergostadienol. Mycelia treated with 5 μg/ml of triarimol (α-(2,4-dichlorophenyl)-α-phenyl-5-pyrimidinemethanol) also accumulated the same sterols as well as a fourth sterol believed to be Δ^{5, 7}-ergostadienol.Identification of 4,4-dimethyl-Δ^{8, 24 (28)}-ergostadienol in untreated mycelia indicates that the C-14 methyl group is the first methyl group removed in the biosynthesis of ergosterol by A. fumigatus. The lack of detectable quantities of 4,4-dimethyl-Δ^{8, 24 (28)}-ergostadienol in triforine or triarimol-treated mycelia and the accumulation of C-14 methylated sterols in treated mycelia suggests that both fungicides inhibit sterol C-14 demethylation. The accumulation of Δ^{5, 7}-ergostadienol in triarimol-treated mycelia further implies that triarimol also inhibits the introduction of the sterol C-22(23) double bond.Two strains of Cladosporium cucumerinum tolerant to triforine and triarimol were also tolerant to the fungicide S-1358 (N-3-pyridyl-S-n-butyl-S′-p-t-butylbenzyl imidodithiocarbonate).     

Effect of alachlor on Avena seedlings: Inhibition of growth and interaction with gibberellic acid and indoleacetic acid

The growth of Avena seedlings grown in sand was found to be inhibited by alachlor with the time of onset of inhibition after treatment being a function of herbicide concentration. There was a 12 hr lag period following a subirrigation with 2.5 × 10^?4M alachlor before growth inhibition could be detected. This lag period may be due to uptake and translocation of alachlor from the roots to the site of inhibition or to the exhaustion of certain growth-limiting substance(s) whose biosynthesis is inhibited by alachlor. Additions of gibberellic acid by subirrigation simultaneously with alachlor or after alachlor treatment did not prevent growth inhibition. However, treatment with 10^?3M gibberellic acid 24 hr prior to alachlor treatment overcame the alachlor inhibition. On the other hand, in contrast to gibberellic acid, indoleacetic acid did not prevent inhibition by alachlor.     

Evidence that ethirimol may interfere with adenine metabolism during primary infection of barley powdery mildew

Ethirimol, a hydroxypyrimidine fungicide active against powdery mildews only, inhibited the formation of appressoria during primary infection of barley powdery mildew, Erysiphe graminis f.sp hordei. It also affected other stages of mildew development. Several adenine analogs had similar effects and ethirimol-resistant mildew strains were generally cross-resistant to these. Adenine and adenosine reduced the fungitoxicity of ethirimol. During the formation of appressoria [³H]adenine was incorporated into RNA but [¹⁴C]glycine was not, suggesting that purine biosynthesis did not occur. Ethirimol inhibited this RNA synthesis and it is concluded that the fungicide may interfere with adenine metabolism at some site subsequent to its synthesis.     

Inhibitors of juvenile hormone biosynthesis in corpora allata of the cockroach Periplaneta americana (L.) in vitro

The spontaneous biosynthesis of methyl (2E,6E)-(10R)-10,11-epoxy-3,7,11-trimethyldodeca-2,6-dienoate (juvenile hormone III; JH III) in excised corpora allata of Periplaneta americana was inhibited by a number of synthetic prop-2-ynyl ethers and 1,3-benzodioxole derivatives. One structurally diverse group of compounds inhibited only the final biosynthetic enzyme methyl farnesoate epoxidase (EC.1.14.14.–) at low to moderate concentrations, but at higher concentrations, also inhibited methyl farnesoate (MF) formation, causing an accumulation of MF in the concentration range 10 to 100 μM. For a second, more limited, group of compounds, there was close congruence between the inhibition of JH III biosynthesis and that of total ester (MF plus JH III) biosynthesis over their effective inhibitory concentration ranges. In contrast to the first group, there was no accumulation of MF and these compounds evidently inhibited JH III biosynthesis, at the level of either farnesoic acid esterification by O-methyl transferase (EC.2.1.1.–), or at an earlier step in the biosynthetic pathway that remains to be elucidated.     

The effect of organophosphate insecticides on adrenal corticosterone formation

In vitro and in vivo experiments with Sprague-Dawley rats showed that the three organophosphate insecticides tested (see below) depressed endogenous corticosterone synthesis and blocked corticosteroidogenesis in response to ACTH and cAMP stimulation of a suspension of adrenal cells. Pregnenolone stimulation of adrenal cells was not inhibited at the insecticide concentrations which blocked the ACTH and cAMP stimulation of corticosteroidogenesis. It was concluded that the insecticides act beyond the site of action of ACTH and at or beyond the level of cAMP metabolism and prior to the metabolism of pregnenolone.Insecticides tested were: dichlorvos (O,O-dimethyl-O,2,2-dichlorovinyl phosphate), Dursban (phosphorothioic acid: O,O-diethyl O-3,5,6-trichloro-2-pyridyl ester), and Diazinon (phosphorothioic acid: O,O-diethyl O-(2-isopropyl-6-methyl-4-pyrimidinyl ester).