René Scalla

Protoporphyrinogen oxidase inhibition by three peroxidizing herbicides: Oxadiazon, LS 82-556 and M&B 39279

Three chemically unrelated peroxidizing molecules, namely oxadiazon [5‐(t‐butyl)‐3‐(2,4‐dichloro‐5‐isopropoxyphenyl)‐1,3,4‐oxadiazol‐2‐one], LS 82‐556 [(S)3‐N‐(methylbenzyl)carbamoyl‐5‐propionyl‐2,6‐lutidine] and M&B 39279 [5‐amino‐4‐cyano‐1‐(2,6‐dichloro‐4‐trifluoromethylphenyl)pyrazol], are potent inhibitors of plant, yeast and mouse protoporphyrinogen oxidase.

Effects of acifluorfen-methyl on cucumber cotyledons: Porphyrin accumulation☆

Abstract The nitrodiphenyl ether herbicide acifluorfen-methyl and the pyridine derivative LS 82-556 induce porphyrin accumulation in green cucumber cotyledons. When experiments are done with intact plants absorbing the herbicide through the roots, that accumulation is light-dependent. 3-(3,4-Dichlorophenyl)-1,1-dimethylurea (DCMU) which prevents cellular damages under these conditions (M. Matringe and R. Scalla, Pestic. Biochem. Physiol. 26 , 150 (1986), also inhibits porphyrin accumulation. In contrast, when detached cotyledons are cut into pieces and floated on herbicide solutions, porphyrins accumulate in the dark. Accordingly, DCMU does not inhibit porphyrin accumulation or protect the tissues against herbicidal effects. Furthermore, 4,6-dioxoheptanoic acid, and inhibitor of tetrapyrrole biosynthesis, prevents both porphyrin accumulation and phytotoxic effects. The same results can be obtained with norflurazon, an inhibitor of carotenoid biosynthesis; in this case, inhibition of porphyrin biosynthesis is probably a secondary consequence of abnormal development of chloroplasts. These results indicate that the herbicidal activity of diphenyl ether-type herbicides probably results from their ability to interfere with the metabolism of tetrapyrroles.

Hydroxylation and N-demethylation of chlorotoluron by wheat microsomal enzymes.

Abstract In vitro metabolism of the herbicide chlorotoluron was studied in various subcellular fractions from suspension-cultured cells of wheat ( Triticum aestivum ) treated with cyometrinil. The highest rates of degradation of herbicide were found in microsomal fractions exhibiting a NADPH-cytochrome P -450 (cytochrome c ) reductase activity. Membrane fractions supported the ring-methyl hydroxylation and the first N -demethylation of chlorotoluron in the presence of molecular oxygen and NADPH. Cell treatment by cyometrinil increased the rates of these two reactions, the activity of lauric acid in-chain hydroxylase and the levels of cytochromes P -450. Ring-methyl hydroxylation and N -demethylation of chlorotoluron were drastically reduced by para -chloromercuribenzoate and by compounds draining electrons from NADPH-cytochrome P -450 (cytochrome c ) reductase. Ring-methyl hydroxylase was also strongly inhibited by CO, with partial reversion by light. Combination of these data allow to postulate that ring-methyl hydroxylase of wheat belongs to the family of cytochrome P -450 monooxygenases. In contrast, the N -monodemethylated metabolite could also be formed in the presence of cumene hydroperoxide, and the N -demethylase activity was not affected by CO. These distinctive properties still raise some questions about the nature of the monooxygenase(s) involved in the first N -demethylation of chlorotoluron in wheat cells.

Interactions of various agrochemicals with cytochrome P-450-dependent monooxygenases of wheat cells

Abstract Levels of cytochrome P-450s and rates of monooxygenase activities were studied in microsomes prepared from wheat cell suspension cultures. Cytochrome contents and enzymatic activities, namely, the enzymatic systems responsible for chlorotoluron ring-methyl hydroxylation and N-demethylation, lauric acid hydroxylase, and cinnamic acid 4-hydroxylase, were enhanced after pretreatment of cells with 2,4-D, procloraz, mecoprop, chlorotoluron, and oxime ether safeners. Effects of various agrochemicals were also determined on chlorotoluron ring-methyl hydroxylase and N-demethylase, following their direct addition to microsomal preparations. Plant growth regulators and fungicides, as well as piperonyl butoxide decreased both activities, tetcyclacis, and procloraz being markedly inhibitory. Naphthalic anhydride, oxime ether safeners, dichlormid, and tridiphane had only weak effects. The substrate specificities of chlorotoluron ring-methyl hydroxylase and N-demethylase were also investigated using structural analogues of the herbicide. Diuron was the strongest inhibitor among the tested phenylureas. Other herbicides that can be metabolized by wheat affected both activities to different extents. However, diclofop enhanced only chlorotoluron N-demethylase.

Effect of the cytochrome P-450 inactivator 1-aminobenzotriazole on the metabolism of chlortoluron andisoproturon in wheat☆

Abstract Roots of young wheat plants ( Triticum aestivum cv Clement) were treated with [ 14 C]chlortoluron or [ 14 C]isoproturon alone or mixed with 1-aminobenzotriazole (ABT), a mechanism-based inactivator of cytochrome P -450 monooxygenases. Radioactivity extracted from shoots slightly decreased during periods of metabolism, this decrease being reduced by ABT in the case of isoproturon. ABT strongly inhibited the metabolism of both herbicides. Accumulation of metabolites was generally depressed in the presence of ABT; however, levels of the free N -monodemethylated derivatives were little or not affected. It is concluded that ABT is a synergist of chlortoluron and isoproturon in wheat because it inhibits the enzymes responsible for the ring-alkyl hydroxylation of chlortoluron and putatively of isoproturon, as well as the second N -demethylation of isoproturon. The possibility that those enzymes belong to the class of cytochrome P -450 monooxygenases is discussed.

The double-stranded RNA associated with the ‘447’ cytoplasmic male sterility in Vicia faba is packaged together with its replicase in cytoplasmic membranous vesicles

The 447 male sterility trait in Vicia faba is strictly correlated with the presence of well-defined membranous vesicles or ‘cytoplasmic spherical bodies’ not found in fertile isogenic maintainer plants, and by the occurrence of a discrete high molecular weight double-stranded RNA. We have purified these cytoplasmic membranous vesicles and find that they contain the dsRNA together with an RNA-dependent RNA polymerase whose activity depends upon the presence of Mg2+, requires the four-nucleoside triphosphates and is unaffected by inhibitors of cellular transcriptases, e.g. α-amanitin and Actinomycin D. The dsRNA can be labelled in vitro by incubating the cytoplasmic vesicles with radioactive NTPs, and the RNA synthesized in vitro is also in a double-stranded form as judged by its resistance to RNase digestion at high salt and its behaviour upon CF-11 chromatography. Treatment of the vesicles with a non-ionic detergent releases the dsRNA in the form of a complex with the RNA-dependent RNA polymerase. The enzyme can still carry out the specific synthesis of dsRNA in these solubilized complexes. The cytoplasmic vesicles therefore isolate this vertically transmitted, self-replicating dsRNA from the cellular milieu: the possible mode of action and relevance of this novel genetic element to the 447 cytoplasmic male sterility trait are discussed.

Competitive interaction of three peroxidizing herbicides with the binding of [3H]acifluorfen to corn etioplast membranes

The specific binding of the herbicide acifluorfen 5‐[2‐chloro‐4‐(trifluoromethyl)phenoxy]‐2‐nitrobenzoic acid to corn etioplast membranes is competitively inhibited by protoporphyrinogen IX, the substrate of protoporphyrinogen oxidase. Three other peroxidizing molecules, oxadiazon [5‐ter‐butyl‐3‐(2,4‐dichloro‐5‐isopropoxyphenyl)‐1,3,4‐oxadiazol‐2‐one], LS 82556 [(S)3‐N‐(methylbenzyl)carbamoyl‐5‐propionyl‐2,6‐lutidine], and M&B 39279 [5‐amino‐4‐cyano‐1‐(2,6‐dichloro‐4‐trifluoromethylphenyl)pyrazol], also compete with acifluorfen for its binding site. The four herbicides thus bind to the same site, or to closely located sites, on the enzyme protoporphyrinogen oxidase.

An in vivo study of a nonstructural polypeptide synthesized upon TMV infection and its identification with a polypeptide synthesized in vitro from TMV RNA

Abstract Proteins from tobacco leaves infected with tobacco mosaic virus (TMV) were labeled in vivo with radioactive leucine, and were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A large virus-induced polypeptide was found in every subcellular fraction of the leaf homogenate, and provided that membranous fractions were defatted before analysis, it had a nearly identical migration rate in all fractions with an apparent MW of about 130,000. In nondefatted preparations, electrophoretic migration was retarded. The rate of in vivo incorporation of [ 3 H]leucine into this protein was maximal 3 or 4 days after inoculation, and was not inhibited by a mixture of actinomycin D and chloramphenicol. The 30,000 g -sedimentable fraction of the homogenates contained most of the virus-induced polypeptide per unit weight of protein. To determine whether or not the 130,000-MW polypeptides isolated from two distinct fractions of the leaf homogenate were identical, they were labeled in vivo and purified from both the 30,000 g -sedimentable, and from the soluble, nonsedimentable fractions and compared by cyanogen bromide-peptide analysis on polyacrylamide gels. Comparisons were also made to the 130,000-MW polypeptide synthesized in vitro in a messenger-dependent reticulocyte lysate system, using TMV RNA as messenger. Close similarity was found between hydrolysis products, suggesting that the sedimentable and the soluble in vivo , and the in vitro polypeptides are identical molecular species.

Phytotoxicity and metabolism of chlortoluron in two wheat varieties

Abstract Varietal susceptibility of winter wheat to chlortoluron, 1-(3-chloro-4-methylphenyl)-3,3 dimethylurea, has been studied in two varieties, Corin (susceptible) and Clement (tolerant). After a 24-hr root absorption of the herbicide, phytotoxicity was estimated from growth measurements. When administered at 12 to 96 μM concentrations, the herbicide reduced the growth of both varieties. A significant selective effect was found at 96 μM. Measurements of chlorophyll fluorescence-induction kinetics allowed to discriminate between the two varieties treated with 12 to 48 μM chlortoluron. The metabolism of chlortoluron was studied following absorption of 24 μM solutions. Both varieties produced the same pattern of metabolites but the tolerant variety degraded the herbicide and the phytotoxic mono-N-demethylated metabolite at a slightly higher rate. An unexpected result was that the more susceptible variety possessed a very significant ability to metabolize chlortoluron. In conclusion, it appears that further studies are necessary before deciding whether the differences in susceptibility of the two varieties can be explained by the only metabolic factor.

RNA containing intracellular particles in cytoplasmic male sterile faba bean (Vicia faba L.)

Abstract The ribosomal fraction of pistil and another tissues of fertile and sterile plants was analyzed by centrifugation in sucrose gradients. By comparison with fertile tissues, analyses of sterile material revealed an additional peak. Electron microscope observations showed that it was correlated to the presence of cytoplasmic spherical bodies (CSB) of approx. 62 nm in diameter. These CSB, also observed in ultrathin sections of ovules, are made up of a circular unit membrane enclosing an electron dense zone. Their UV spectrum indicates the presence of a nucleic acid which, according to the results of electrophoretic analysis and of nuclease treatments, is probably single-stranded RNA. This RNA resists heat treatment and has an apparent molecular weight much higher than 2 × 10 6 . We found a strict correlation between the presence of these CSB and the genetic factor of cytoplasmic male sterility (CMS), since CSB were detected in male sterile genotypes but not in maintainers, a restored hybrid or a revertant.