Band 46, Heft 9-10

A considerable portion of the plant mitochondrial DNA is derived from genome internal duplications. Many of these amplified sequences determine functions of transcription and processing. Among these are promoter regions, sequences defining the 3′ ends of stable mRNAs, potential RNA processing sites and intron domains. Simultaneously, some of these repeated sequences can be active sites of recombination in plant mitochondria. Such duplicat­ed control regions may simplify coordinate expression of different genes.

The behaviour of monomers and trimers of C-phycocyanin and allophycocyanin under high pressure is studied by UV-VIS absorption spectroscopy. It is found that fast variations of pressure (Δp ≥ 500 bar) and/or temperature are accompanied by significant changes in the absorption spectra (intensity and/or spectral shift). The induced differences disappear, however, in part, when the samples are left for several minutes at the final pressure (relaxation effect). The observed spectral variations are different from those connected with a change in state of aggregation and could therefore be due to small modifications of the chromophore-protein arrangement.

Transformed cell suspension and hairy root cultures were established by infecting seedlings of Lupinus polyphyllus and L. hartwegii with various wild type strains of Agrobacterium tume-faciens and A. rhizogenes. Transformation of the cultures was confirmed either by their phyto­hormone autotrophy, detection of opines or southern analysis. Glueosides of genistein and 2′-hydroxygenistein, were found to be the main secondary metabolites in normal and trans­formed suspension cultures as well as in hairy root cultures. Although some of the isoflavone glycosides of the cultures were apparently new constituents of Lupinus, they were afterwards also found in young seedlings.

Some highly productive suspension and hairy root cultures were found among several trans­formed cultures of Lupinus polyphyllus and L. hartwegii. A transformed suspension culture Lupo 30150 and a root culture Luha 15834 containing the highest specific isoflavone glucoside content were characterized and compared with normal phytohormone-dependent lines with respect to product stability as well as to their responsiveness to external triggers, e.g. response to changes in the medium. While phytohormone-dependent suspension cultures lost their ini­tial ability to form increased levels of isoflavonoids on phytohormone-free medium, the trans­formed phytohorm one-independent suspension Lupo 30150 remained a highly productive line, despite the fact that its specific levels decreased to 60% of the initial values during several years in liquid medium. Highest stability of product patterns and levels were noted for the transformed root culture. Phytohorm ones had little effect on growth and isoflavonoid levels in suspension cultures, while they reduced both strongly in root cultures. In the presence of 2,4-D the root culture changed into an aggregated low producing suspension culture from which the root state was recovered on phytohorm one-free medium. As long as the root state was main­ tained, isoflavonoid levels could not be distinctly improved by media variation while specific isoflavonoid levels of suspensions were increased by stress factors such as phosphate depletion. When suspensions were transferred to fresh medium phenylalanine ammonia-lyase was greatly induced within 24 h, while the activity remained nearly unchanged in root cultures.

The very young leaflets of silver lime trees (Tilia tomtentosa), just unfolding from buds, con ­ tained a continuous wax layer without any wax sculptures. The wax on young leaves is quite different in yield and composition than that of mature leaves. After unfolding of leaves a very dynamic biosynthesis of most wax lipids was started. Fifteen days after leaf unfolding the de novo biosynthesis of β-amyrenyl acetate and later on of aldehydes could be detected for the first time. The biosynthesis of wax components in silver lime leaves was finished at the end of June and the wax remained nearly constant in amount and composition during the remaining season. At the same time, when β-amyrenyl acetate was found for the first time, wax sculptures were observed in silver lime leaf waxes coming out of the continuous wax layer, exclusively on the upper leaf side. These wax sculptures increased in quantity in the next weeks and resulted in a crystalloid shape of most solitaire quadrangular rodlets. These crystals were remained all over the season and were formed from β-amyrenyl acetate, the dominating main wax compo­nent {ca. 49% wax).

C-terminal amino acid sequences of two phytochromes sharing less than 40 per cent homol­ogy and two transmitter modules of bacterial sensor proteins that share homologies with phy­tochromes were examined by hydrophobic cluster analysis. Striking coincidences in distribu­tion, size, shape and orientation of hydrophobic and hydrophilic domains of the bacterial transmitter modules and phytochrome sequences were revealed. The results corroborate the view that the folding of the homologous regions of the two groups of proteins is similar. Since the mode of action of phytochrome is not known, the structural coincidences may be indica­tive of how phytochrome transmits signals, although distinct differences should not be over­ looked. Some coinciding structural features in phytochromes and bacterial sensors may tenta­tively be extracted from secondary structure predictions.

CD spectra have been recorded with subunit δ from chloroplast CF 0 CF 1 and with OSCP from mitochondrial MF 0 MF 1 . These subunits are supposed to act similarly at the interface between proton transport through the F 0 -portion and ATP-synthesis in the F 1 -portion of their respective F 0 F 1 -ATPase. Evaluation of the data for both proteins revealed a very high α-helix content of -85% and practically no β-sheets. Despite their low homology on the primary structure level (23% identity) and their different electrostatic properties (pl-values differ by 3 units), spinach δ and porcine OSCP are indistinguishable with respect to their secondary structure as measured by CD. Prediction and analysis of consensual a-helices even in poorly conserved regions indicate α high degree of structural similarity between chloroplast δ and OSCP. In view of the topology and function of δ and OSCP in intact F 0 F 1 these findings are interpreted to indicate the dominance of secondary and tertiary structure over the primary structure in their supposed function between proton flow and ATP-synthesis.

A compilation of 38 sequences for the D1 and 15 sequences for the D 2 reaction center pro­teins of photosystem II is presented. The sequences have been compared and a similarity index that takes into account the degree of conservation and the quality of the changes in each posi­tion has been calculated. The similarity index is used to identify and describe functionally im­portant domains in the D1 / D2 heterodimer. Comparative hydropathy plot are presented for the aminoacid sidechains that constitute the binding domain of the tyrosine radicals, Tyr Z and Tyr D , in photosystem II. The structure around Tyr Z is more hydrophilic than the structure around Tyr D . The hydrophilic residues are clustered in the part of the binding pocket for Tyr Z that is turned towards the lumenal side of the thylakoid membrane. Most prominent is the presence of two conserved carboxylic am inoacids, D1-Asp 170 and D1-Glu 189. Their respec­tive carboxyl-groups come close in space and are proposed to constitute a metal binding site together with D1-Gln 165. The distance between the proposed metal binding site and the cen­ter of the ring of Tyr Z is approximately 7Å . The cavity that constitutes the binding site for Tyr D is composed of residues from the D 2 protein. Its character is more hydrophobic than the Tyr Z site and the environment around Tyr D lacks the cluster of putative metal binding side­-chains.

The inhibition of ATP synthesis, proton uptake and electron transport (basal, phosphory-lating and uncoupled) from water to methylviologen indicates that cacalol and its derivatives act as electron transport inhibitors. Since on one hand photosystem I is not affected and elec­tron transport from DPC to QA is midly affected and on the other hand the electron transport from water to DCIP, and water to silicomolibdate are inhibited, we conclude that the site of inhibition of cacalol is located at the oxygen evolution level. Cacalol derivatives inhibit elec­tron flow between P680 to QA and probably also the Q B site.

N-Acetyl-β-ᴅ-hexosaminidases (EC 3.2.1.52) from Artemia nauplii were isolated and char­acterized. Three different enzymes I, II 1 and II 2 were separated according to their behaviour on anion exchange chromatography and gel filtration columns. Their apparent molecular masses were 83,000 ± 7000, 110,000 ± 10,000 and 56,000 ± 5000 Da with corresponding S-values of 8.6, 11.9 and 7.9. All three enzymes also differ in their apparent pH-optima (5.1, 4.5 and 6.1) and they all bind to concanavalin A. The three enzymes have about the same affinities (app. K m between 0.16 and 0.72 mmol/1) for the three substrates (p-nitrophenyl-N-acetyl-β-ᴅ-glucosamine or p-nitrophenyl-N-acetyl-β-ᴅ-galactosamine and N ,N′-diacetyl-chitobiose) and are therefore N-acetyl-β-ᴅ-hexosaminidases. In contrast, the three enzymes behave quite differently, both in terms of their inhibitor constants and the type of inhibition. The substrates inhibit both enzymes II 1 and II 2 but not enzyme I. On the other hand, N-acetyl-β-ᴅ-galactosamine inhibits enzyme I in a non-competi­tive way but not enzymes II 1 and II 2 . All three enzymes are inhibited by the end product N-acetyl-β-ᴅ-glucosamine, enzyme I in a competitive manner, both enzymes II 1 and II 2 in a non-competitive way. 2-Acetamido-2-deoxy-ᴅ-galactonolactone is a strong inhibitor for en­zyme I (K i = 13 μtmol/l) with much lower affinities towards enzymes II 1 and II 2 (K i = 0.63 and 1.03 mmol/l). All three enzymes are inhibited in a dose-dependent way and completely reversi­ble by α-methyl-mannoside.

Looking for a possible protective effect of heavy water on proteins, cholinesterase and lac­tate dehydrogenase in combination with D 2 O and further protective substances were exposed to a temperature of 60 °C (for 10 min) and about 45 °C (for several days). In combination with glycerine there resulted an additive protective effect; with NaCl and/or albumin being added the individual effects raised to a higher level. In the cold D 2 O protects cholinesterase only against acid denaturation, but in combination with warmth also against basic denaturation.

The concept of using chemical safeners to improve herbicide selectivity became widely known in 1969 with the introduction of naphthalic anhydride (NA) to improve the tolerance of maize to thiocarbamate herbicides. Soon thereafter dichlormid was also developed as a safener for thiocarbamates in maize. At present there are at least eight different chemical safeners that have been developed commercially. Many of these safeners have been most effec­tive for chloroacetamide or thiocarbamate herbicides in monocotyledonous crops such as maize or sorghum. Some are like dichlormid and have been developed as physiologically selec­tive safeners that can be combined in the formulation with the herbicides. Others, like NA , are either less selective or less active in the soil and must be used as dressings on the crop seed. Most of the presently used safeners are for soil active herbicides. The challenge for the future will be to develop safeners that are effective for the newer postemergence herbicides in dicoty­ledonous as well as monocotyledonous crops.

The discovery and commercial success of safeners against thiolcarbamate herbicide injury to corn has stimulated a rapid progress and opened new possibilities for further research and development in the last decade. Compounds with new chemistry, increased efficacy, and a broader selectivity spectrum were synthesized and developed for agricultural use. Structure-activity relationship studies helped to optimize their chemical properties and to understand their biological modes of action. Several examples indicate close similarity between chemical structures possessing herbicidal and safener properties. In some cases this differentiation may be marginal, as shown in crops pretreated with low herbicide doses leading to safening effects. In other examples, however, structural optima for safening and herbicidal efficacy can be clearly differentiated.

Acetals and ketals involving a dichloromethyl group on their central carbon atom were found to be active or highly active as safeners of corn against thiocarbamate and chloroacet-anilide herbicide injury. A mechanism for the biotransformation of these compounds as pro­safeners to the actual dichloroacetic ester safeners is proposed. Similarly, S-and N-analogues of the cyclic acetals (ketals) which show safener activity can also be considered as prosafeners because they can be converted in the biophase by the same mechanism into actual safeners such as dichloroacetic thiolesters or dichloroacetamides sup­porting the bioactivation hypothesis. Characteristic bioactive chemical bond systems in safeners and prosafeners are suggested.

Although detailed examinations have been published on structure-activity relationships of herbicide safeners, only a few data are available on chemical reactivity-safener activity relationships. Chemical reactivity of acetamide type compounds as well as their safener activity against thiocarbamate herbicides change with the number of chlorine substituents in the order: non-chlorinated < monochloro < dichloro. Several compounds of another chemical group, acetals (e.g. MG-191, 2-dichloromethyl-2-methyl-1,3-dioxolane), are also effective safeners for thiocarbamate herbicides. According to our growth room studies, the safener activity of acetals also increases with increasing chlorine content up to two chlorine atoms on the same carbon. A number of differently chlorinated acetals have been synthesized and their acid-catalyzed hydrolysis rate determined in order to establish a relationship between their bioactivity and chemical reactivity. The hydrolysis rate order of acetals containing no, one or two chlorine atoms: non-chlorinated > monochloro > dichloro, is just the opposite than what has been found for acetamides. Thus, safener activity of acetals increases with decreasing chemical reactivity. The opposite reactivity order of acetamides and acetals can be explained by the different mechanisms of their hydrolysis. Dichloroacetals may not be effective safeners in their original structure. In plants, however, they can be biologically activated to active safeners by a transformation other than hydrolysis.

The growing environmental and toxicological concern about the dichloroacetamide type herbicide safeners prompted us to search for alternative safener types. Based on analogy con­siderations, a random screening of structurally unrelated bivalent sulphur compounds was performed, followed by successive structure optimization phases representing both speculative and empirical elements. Dithiocarbamate ester type compounds derived from 1,4-exaza-spiro[4.5]decane revealed highest protecting potency both agains thiolcarbamate and chlor-acetanilide herbicides in corn (Zea mays L.), with the benzylic and allylic esters being the most active.

Herbicide safeners are chemicals used for manipulating the tolerance of large-seeded grass crops to selected soil-applied herbicides. The physiological interactions of herbicides and their respective safeners are characterized by the following facts: a) safeners are most effective when applied prior to or simultaneously with the herbicides whose injury they prevent; b) safeners exhibit a high degree of botanical and chemical specificity protecting only certain grasses against injury caused from specific classes of herbicides; and c) protected grass crops are moderately tolerant to the antagonized herbicides. At the biochemical level, safeners may act either as “bioregulators” regulating the amount of a given herbicide that reaches its target site in an active form or as “antagonists” of herbicidal effects at a similar site of action. A safener-induced enhancement of herbicide detoxication in protected plants is currently viewed as the most apparent mechanism for the action of the currently available safeners. Safeners enhance the conjugation of carbamothioate and chloroacetanilide herbicides with glutathione either by elevating the levels of reduced glutathione (GSH) or by inducing the activity of specific glutathione S̱-transferases (GSTs). A safener-induced enhancement of the activity of other degradative enzymes such as the cytochrome P450-dependent mixed function oxidases or UDP-glucosyl transferases seems to be important for the protective action of safeners against injury from aryloxyphenoxypropionate, imidazolinone, and sulfonylurea herbicides. Metabolic processes related to acetyl-CoA metabolism have been implicated as likely target sites for a competitive antagonism between safeners and chloroacetanilide or carbamothioate herbicides. At the molecular level, the “gene activation” and “gene amplification” theories offer a likely explanation for the action of safeners.

In growth room bioassays, four herbicide safeners {BAS-145138: 1-dichloroacetyl-hexa-hydro-3,3,8a-trimethyl-pyrrolo(1,2a)pyrimidin-6(2H)-one, dichlormid: N,N-diallyl-2,2-dichloroacetamide, flurazole: phenylmethyl 2-chloro-4-(trifluoromethyl)-5-thiazole-carboxylate and MG-191: 2-dichloromethyl-2-methyl-1,3-dioxolane} were compared for their effective­ ness in reducing metazachlor {N-(2,6-dimethylphenyl)-N-(1-pyrazolylmethyl)-chloroacet-amide} injury to maize seedlings. Three of these compounds (BAS-145138, dichlormid and MG-191) and two different compounds (AD-67: N-dichloroacetyl-1-oxa-4-azaspiro-4,5-de-cane and DKA-24: N ,N 2 -diallyl-N 2 -dichloroacetylglycineamide) were also evaluated as safe­ners for acetochlor {N-(2-ethyl-6-methylphenyl)-N-(ethoxymethyl)chloroacetamide} in maize seedlings. Both BAS-145138 and dichlormid were highly effective as safeners for metazachlor and BAS-145138 and DKA-24 were equally good safeners for acetochlor. Metazachlor and acetochlor uptake and metabolism by maize seedlings were enhanced sig­nificantly in response to treatment with every safener examined. Rates of metazachlor and acetochlor uptake and metabolism were greatest in seedlings treated with BAS-145138 -the most effective safener. In the absence of any safener treatment, the non-enzymatic conjugation of the herbicides with glutathione either exceeded (metazachlor) or was similar (acetochlor) to enzymatic conjugation. However, the rate of enzymatic conjugation was generally increased by safener treat­ ment. This was particularly true when the herbicide ( 14 C-labeled) was employed as a conjuga­tion substrate instead of the less specific substrate, CDNB (1-chloro-2,4-dinitrobenzene). A significant linear correlation was observed between safener effectiveness and rates of metazachlor metabolism or glutathione S-transferase activity in maize seedlings. However, a similar correlation could not be established for the five compounds evaluated as safeners for acetochlor.

The influence of MG-191 safener on the uptake, translocation and metabolism of [ 14 C]acetochlor and [ 14 C]EPTC was studied. The amounts of absorbed radioactivity by maize seedlings at 3, 6, 24, and 72 h after applications of [ 14 C]labeled herbicides and [ 14 C]MG-191 were different. Plants treated with [ 14 C]acetochlor took up 30-to 50-fold more radiolabel with­in 72 h than [ 14 C]EPTC-or [ 14 C]MG-191-treated plants. Addition of MG-191 caused only mi­nor changes in the rate of herbicide absorption. EPTC and MG-191 and/or their metabolites moved quickly acropetally and partitioned equally between root and shoot tissues up to 72 h. The amount of acetochlor and/or its labeled metabolites translocated to shoot tissues was less than 10%. MG-191 practically had no influence on herbicide translocation rates. With all chemicals the amounts of water-soluble and unextractable fractions increased while the ratio of hexane-extractable metabolites decreased with time. TLC analyses of both water-and hex­ane-soluble metabolites confirmed the fast metabolism of acetochlor. The acetochlor metabo­lism took place via GSH conjugation and more polar, non-conjugated metabolites compared to parent molecule were detected in hexane-soluble fraction. MG-191 enhanced acetochlor metabolism by decreasing the portion of non-metabolized acetochlor. EPTC metabolism re­sulted in water-soluble metabolites having similar chromatographic properties to those of ace­tochlor. However, there was no safener effect on non-metabolized EPTC content of plants. It appears that MG-191 protects maize against EPTC by enhancing the early rate of conjugation with GSH after initial oxidative metabolism.

The herbicide safener benoxacor (CGA -154281) is effective in protecting corn from meto­lachlor injury. Glutathione-S-transferase (GST) activity of corn seedlings was stimulated by low concentrations of benoxacor as was the formation of a polar metabolite identified as the glutathione (GSH) conjugate of metolachlor. A similar degree of enhancement of metolachlor metabolism was observed in both a metolachlor-tolerant (’Cargill 7567’) and a metolachlor-susceptible (’Northrup-King 9283’) corn line. The total GSH content of shoots of the metolachlor-susceptible corn hybrid was not affected by benoxacor treatment, but an increase was noted for shoots of the tolerant corn hybrid. The two corn hybrids with differential tolerance to metolachlor also differ in their dose response to benoxacor with higher safener concentra­tions failing to induce or inhibit GST activity of the tolerant ’Cargill 7567’ corn line. Stimula­tion of GST activity and a corresponding enhanced rate of metolachlor metabolism can ac­count for the safening effect of benoxacor. These mechanism of action of dichloroacetamide safeners.

Fast protein liquid chromatography (anion exchange) was used to separate glutathione S-transferase isozymes in nontreated etiolated maize shoots and those treated with the herbi­cide safener CGA -1542814-(dichloroacetyl)-3,4-dihydro-3-methyl-2 H-1 ,4-benzoxazine. Non­treated shoots contained isozymes active with the following substrates: trans-cinnamic acid (1 isozyme), atrazine (3 isozymes), 1-chloro-2,4-dinitrobenzene (1 isozyme), metolachlor (2 isozymes) and the sulfoxide derivative of S-ethyl dipropylcarbamothioate (2 isozymes). Pre­treatment of shoots with the safener CGA -154281 (1 μM) had no effect on the activity of the isozymes selective for trans-cinnamic acid and atrazine but increased the activity of the constitutively-expressed isozymes that exhibit activity with 1-chloro-2,4-dinitrobenzene, metola­chlor and the sulfoxide derivative of S-ethyl dipropylcarbamothioate. The safener pretreat­ment also caused the appearance of one new isozyme active with 1-chloro-2,4-dinitrobenzene and one new isozyme active with metolachlor. The results illustrate the complexity of gluta­thione S-transferase activity in etiolated maize shoots, and the selective enhancement of gluta­thione S-transferase isozymes by the safener CGA -154281.

Studies were conducted to determine the biochemical aspects of chloroacetamide injury to maize and the mechanism by which safeners maintain herbicide tolerance, even at reduced temperatures. The objectives of these studies were threefold: one, determine whether gluta­thione (GSH) content varies in maize plants grown at three different temperatures in safener-treated and non-treated plants; two, determine whether glutathione S-transferase (GST) activ­ity varies in plants grown at different temperatures; and three, determine if GSH activity is sensitive to low temperatures in vitro. The herbicide safeners CGA -154281 [4-(dichloroacetyl)-3,4-dihydro-3-methyl-2 H-1 ,4-benzoxazine] and dichlormid [2,2-dichloro-N,N-di-2-propenylacetamide] were used with metolachlor [2-chloro-N-(2-ethyl-6-methylphenyl)-n-(2-methoxy-1-methyl)acetamide] or acetochlor [2-chloro-N-(ethoxymethyl)-N-2-ethyl-6-methylphenyl)-acetamide], respectively, to determine the mechanisms of maize tolerance. CGA -154281 signifi­cantly increased GSH levels in maize seedlings grown at 27 °C compared to non-safened seed­lings, however significant differences were not seen at 17 or 37 °C. Dichlormid increased GSH levels by 1.6-fold at all growth temperatures. Both CGA -154281 and dichlormid increased GST activity significantly at all growth temperatures. The safener-induced GST activity was main­tained at in vitro incubation temperatures of 5 and 15 °C for acetochlor and metolachlor, re­spectively. In contrast, GST activity from non-safened tissue was essentially absent at these temperatures. Therefore, greater GST activity following safener treatment may result in higher levels of herbicide metabolism, even at low temperatures.

The influence of individual or combined treatments of the chloroacetanilide herbicide preti­lachlor and the safener fenclorim on glutathione content and the activity of glutathione reduc­tase (GR, EC 1.6.4.2) and glutathione-S-transferase (GST, EC 2.5.1.18) of rice (Oryza sativa L., var. Lamont) was investigated in a time-course study including 0, 12, 24, 48, and 72 h peri­ods after treatment. Pretilachlor applied alone at 2 μM did not influence the levels of total or reduced glutathione of germinating rice seedlings. Fenclorim applied alone at 20 μM increased significantly the levels of total and reduced glutathione of rice seedlings at 12, 24, and 72 h after treatment. The combination of pretilachlor and fenclorim increased the levels of total and reduced glutathione of rice at all time periods. GR activity extracted from pretilachlor-treated rice seedlings was significantly lower than that extracted from untreated rice seedlings. Pretreatment of rice seedlings with the safener fenclorim increased GR activity at 12 and 24 h, but not at 48 and 72 h after treatment. GR activity extracted from rice seedlings treated with the combination of pretilachlor and fenclorim was significantly higher than that of untreated controls at 12, 24, and 48 h after treatment. Pretreatment of rice seedlings with pretilachlor reduced the activity of GST which catalyzes the conjugation of this herbicide with reduced glutathione at time intervals greater than 24 h. Pretreatment with fenclorim did not have any effect on GST activity of rice seedlings. Pretreatment with the combination of pretilachlor and fenclorim increased GST activity of rice seedlings at the 48 and 72 h periods. These results suggest that a simultaneous application of pretilachlor and fenclorim is critical for the enhance­ment of glutathione and glutathione-related enzyme activity and the protection of rice from pretilachlor injury.

Phytophthora cactorum, Ph. cambivora, Ph. cinnamomi, Ph. citricola, Ph. cryptogea, Ph. drechsleri, Ph. infestans, Ph. megasperma, Ph. parasitica, and Ph. syringae contain GSH-conjugation systems as indicated by the presence of active GST enzyme in addition to GSH. Basal levels of both GSH and GST in the thalli of Phytophthora strains studied did not correlate with either fungal sensitivity to phenylamides (acetochlor, butachlor, metolachlor, dimethachlor, propachlor, ofurace, CGA-29212, metalaxyl, RE-26745, benalaxyl, furalaxyl, LAB-149202) or their acquired resistance to metalaxyl. Thalli of Phytophthora strains from axenic cultures ex­ posed to sublethal concentrations of the above pesticides contained significantly higher levels of both GSH and GST than the untreated controls. This response was independent of the sen­sitivity and tolerance of the strains to phenylamides. When the responses of Phytophthora strains to phenylamide chemicals were compared by means of principal component analysis, four independent components were detected account­ ing for 88% of the total variation. Biological properties (basal and induced levels of GST and GSH, growth intensity, degree of acquired resistance to metalaxyl, sensitivity to propiconazole and to cis-and trans-tridemorph) of the strains contributed differently to this variation. It was concluded that, in contrast to plants, sensitivity or tolerance of Phytophthora species to phenylamide pesticides is not regulated by the efficiency of the GSH-conjugation system. In addition, our data clearly indicate that the acetanilide pesticides have multiple sites of action in the Phytophthora genus.

Levels of non-protein thiols (mostly glutathione, GSH), ascorbic acid (AA), and activities of the enzymes ascorbate peroxidase (AP), glutathione reductase (GR) and GSH S-transferase (GST) were determined in cell-free leaf extracts of acifluorfen-resistant and -sensitive tobacco plants. These parameters were examined also in detached leaves of the above plants exposed to acifluorfen stress. In leaves of untreated plants the AA content was by 40% higher in the resist­ ant biotype as compared to the sensitive ones, but the levels of GSH, AP, GR, and GST did not differ significantly in the two biotypes. However, in the resistant leaves stressed by aci­fluorfen the activity of AP readily increased while in the sensitive leaves it did not change. The levels of GSH and the activities of GR and GST markedly increased in both biotypes after acifluorfen stress, but the induction in the resistant leaves was consistently stronger in each case. The AA contents were increased equally in both biotypes. These parameters were much less affected by paraquat stress. The only significant changes were observed at low concentra­tions of this herbicide (8 x 10 -9 м): when the thiol content and the activity of GST increased in the resistant leaves. Enhanced inducibility of antioxidant systems seems to be involved in resistance of tobacco to acifluorfen stress.

BAS 145138 was moderately effective in protecting corn from injury due to chlorimuron ethyl. BAS 145138 increased the I 50 of chlorimuron ethyl to corn, determined by root-growth in a sandy loam soil, from 6 g chlorimuron ethyl/ha to 25 g/ha. BAS 145138 caused an increase in the rate of chlorimuron ethyl metabolism , but it did not effect the rate of chlorimuron ethyl uptake or the nature of the metabolites produced. Major metabolites included chlorimuron ethyl hydroxylated in the pyrimidine ring (hydroxychlorimuron ethyl), the glucoside of hydroxychlorimuron ethyl, the glutathione conjugate formed by displacement of chlorine, a metabolite characterized as the glutathione conjugate sulfoxide or a hydroxylated glutathione conjugate, four unidentified polar metabolites, and 2-sulfonylamino benzoic acid ethyl ester. Metabolites were characterized by mass spectrometry and hydrolysis. It was concluded that BAS 145138 partially protects corn from chlorimuron ethyl by causing an increase in the rate of herbicide metabolism by hydroxylation, glucoside conjugation and by glutathione conjugation.

Bensulfuron methyl is a sulfonylurea herbicide controlling a broad spectrum of broadleaf and sedge weeds in paddy rice. Safening effects with certain thiolcarbamate herbicides for grass control have been discovered in the process of combination products development. The results of mode of safening studies revealed that the enhancement of metabolic inactivation rate of bensulfuron methyl in plants was the basis of safening. Among these thiolcarbamates, dimepiperate was unique in exhibiting safening action even in direct water-seeded rice. Further studies on dimepiperate effects on root-applied bensulfu­ron methyl indicated that rice root elongation, enhanced metabolism in roots and in shoots, were the key to the safening effects. The translocation rate of bensulfuron methyl, from roots to shoots, on the other hand, was not significantly affected by dimepiperate.

The tolerance of maize to root-applied imazethapyr can be increased by pretreating plants with the potassium salt of naphthalic acid (NAK). This safening effect appears to be the result of NAK stimulating the ability of maize to rapidly metabolize imazethapyr to 5′-hydroxyethyl-imazethapyr, possibly through a mixed function oxidase. The safening effect of NAK can be antagonized by 1-aminobenzotriazole (ABT), a mixed function oxidase inhibitor. The increased rate of hydroxylation of imazethapyr to 5′-hydroxyethyl-imazethapyr immobilizes the herbicide in the root system which decreases the accumulation of herbicide in the meristematic tissue. This decreased accumulation, in turn, lowers the phytotoxicity of imazethapyr on maize.

Naphthalic anhydride is a seed-applied herbicide safener which reduces the toxicity of ima­zethapyr, an imidazolinone herbicide, to maize (Zea mays L.). Protection of maize from ima­zethapyr was dependent on the amount of naphthalic anhydride applied to the seed. Metabo­lism of imazethapyr by maize roots and shoots was increased by exposure of the roots to a solution containing naphthalic anhydride. Increased imazethapyr metabolism due to naph­thalic anhydride treatment of roots was observed within 4 h following safener exposure. Nitro­gen, carbon monoxide and tetcyclacis inhibited imazethapyr metabolism in maize coleoptiles grown from naphthalic anhydride treated seed. This suggests that imazethapyr is metabolized by a cytochrome P-450 monooxygenase. Naphthalic anhydride application to maize seed increased the level of cytochrome P-450 in the seedling shoots. Microsomes isolated from coleoptiles grown from naphthalic anhydride treated seed, but not untreated seed, converted bentazon to hydroxy-bentazon but did not me­ tabolize imazethapyr. Protection of maize from imazethapyr damage by naphthalic anhydride is due to a safener-induced higher rate of imazethapyr metabolism associated with elevated cytochrome P-450 levels. However, this was not demonstrated in vitro for imazethapyr.

The influence of the safener, CGA 185072 (5-chloro-8-quinolinoxy-acetic acid-1-methylhexyl-ester), on the metabolism of the aryloxyphenoxypropanoate herbicide, CGA 184927 (2-propynyl-R-2-[4-(5-chloro-3-fluoro-2-pyridinyloxy)-phenoxy]-propionate), was studied in excised leaves of wheat, barley, and maize. In wheat and barley, CGA 184927 readily under­went ester hydrolysis followed by hydroxylation at the pyridinyl moiety as well as ether cleav­age between the pyridinyl and the phenyl ring. Ether cleavage constituted the minor pathway in both species. All metabolites were subject to glycosyl conjugation. Tetcyclacis strongly in­hibited pyridinyl-ring hydroxylation in wheat. Metabolism by hydroxylation and ether cleav­age was more rapid in wheat than in barley, and was found to be accelerated in the presence of the safener CGA 185072 in both wheat and, to a lesser degree, in barley. Moreover, the safener increased the capacity for O-glycoside formation in wheat as suggested from studies using the ,4C-labelled pyridinyl-ring hydroxylated metabolite as a precursor. In maize, which is highly susceptible to CGA 184927, rapid ester hydrolysis of CGA 184927 and partial conversion of the corresponding carboxylic acid to glycosyl ester conjugate(s) occurred. However, no further transformation of the herbicide was found in maize, both in the absence or presence of CGA 185072. It is concluded that the ability of CGA 185072 to protect wheat from injury by the herbicide, CGA 184927, and to confer partial protection to barley, is related to the ability of the safener to stimulate herbicide metabolism in these crop species.

Metabolism of bentazon and metolachlor by excised shoots and a microsomal fraction iso­lated from the shoots, of 3-day-old, dark-grown, grain sorghum (Sorghum bicolor cv. Funk G 522 DR) seedlings was studied. The effects of seed treatments, on the subsequent metabo­lism of the herbicides, with the safeners naphthalic anhydride, oxabetrinil, and CGA 133205 were compared against surface-sterilization and Captan-treatments. Bentazon was aryl hydroxylated in both in vivo and in vitro studies with the hydroxylated derivative undergoing glycosylation only under in vivo conditions. Both shoots and microsomes isolated from shoots of safener-treated seed showed enhanced metabolism of bentazon relative to the controls. In­ hibition by tetcyclacis, a potent inhibitor of plant cytochrome P-450 monooxygenases, in both the in vivo and in vitro studies, and a requirement for NADPH in the in vitro studies suggested that the formation of hydroxybentazon was mediated by a cytochrome P-450 monooxygenase. Metolachlor was metabolized to polar material and O-desmethylmetolachlor under in vivo conditions. Only the demethylated product was formed in vitro. Shoots isolated from safener-treated seed showed enhanced formation of polar com pounds which were assumed to have arisen from conjugation with glutathione. Tetcyclacis did not affect the formation of the polar components. However, the formation of O-desmethylmetolachlor was depressed in the shoots excised from safener-treated seed under both in vivo and in vitro conditions. Tetcyclacis completely prevented formation of the demethylated metabolite. Hence, formation of this meta­bolite is considered to be P-450 mediated. The differential response obtained with the safeners, i.e., stimulation of aryl hydroxylation of bentazon and depression of metolachlor demethylation, suggests that the reactions are probably catalyzed by different cytochrome P-450 mono­oxygenases.

The monooxygenase suicide inhibitor piperonyl butoxide (PBO) antagonizes the inhibitory activity of the herbicide mefenacet in a root regeneration test system with etiolated cut oat stems. When studying the behaviour of [ 14 C]mefenacet [2-(2-benzothiazolyloxy)-N-methyl-N-phenylacetamide] in cut oat stems that had been pretreated with PBO for 24 h after cutting, an increased rate of uptake and increased concentrations of mefenacet in the tissue were found after PBO as compared to water pretreatment. Increased uptake rates and increased rates of metabolism after PBO pretreatment, supposedly by increased monooxygenation, were also observed with the substrates diuron, 2,4-D and cinnamic acid. Mefenacet induced the same responses as PBO in all systems. The similar actions of mefenacet and PBO are interpreted to suggest that mefenacet might interfere with monooxygenase enzymes. Similar responses, i.e. stimulation of monooxygena­tion of suitable substrates, have been reported for herbicide safeners. An environmentally con­trolled regulatory response system is suggested to be triggered by the different classes of compounds, mefenacet and similar herbicides, monooxygenase inhibitors, and safeners, and to re­spond with increased enzyme activities in the monooxygenase and glutathione detoxification pathways.

S-Ethyl-N,N-dipropylthiocarbamate (EPTC) was oxidized into its corresponding sulfoxide by microsomal fractions from etiolated maize seedlings. This reaction is catalyzed by a hydroperoxide-dependent enzyme, identified as a peroxygenase. The hydroperoxides formed from fatty acids by a lipoxygenase are efficient co-substrates of the EPTC sulfoxidation. The effects of the safener dichlormid on the peroxygenase and lipoxygenase activities were studied in vitro and in vivo. In vitro, the safener is not an inhibitor of these enzymes. Dichlormid seems to act, in vivo, by modulating the amounts of peroxygenase and lipoxygenase in treated plants.

The changes in fatty acid composition of maize leaf lipids caused by EPTC were generally similar to known effects of this herbicide in other plants: decreasing of linolenic acid content and increasing of its precursors, palmitic, stearic, oleic and linoleic acids. However, novel effects were detected in roots where the proportion of minor fatty acid palmitoleic acid was increased from 2.1 to 7.6 and 16.6% by EPTC and EPTC + dichlormid treatments, respective­ly. Simultaneously, the phospholipid content of root lipids was increased by both EPTC as well as EPTC + dichlormid treatments. The possible effects of EPTC and dichlormid on lipid biosynthesis of maize are discussed.

It has been found that various thiocarbamate herbicides, known to alter surface wax and cutin synthesis, inhibit the elongation of fatty acids. We have proposed this as a mode of ac­tion of such compounds. Because it is believed that the sulphoxide metabolites of thiocarba­mates are the active intermediates, we have examined the action of 1-aminobenzotriazole (an inhibitor of sulphoxide formation) on the inhibition of very long-chain fatty acid biosynthesis. In all tissues tested, aminobenzotriazole was able to block the specific inhibitory effect of thio­carbamates on fatty acid elongation. These results add further support to our proposal that fatty acid elongation is a sensitive target site for thiocarbamate herbicides in plants.

The effects of individual or combined treatment of the cyclohexanedione herbicide sethoxy­dim and the safener dichlormid on total lipid synthesis, protein synthesis and acetyl-CoA car­ boxylase (ACCase, EC 6.4.1.12) activity of grain sorghum [Sorghum bicolor (L.) Moench, var. G 623] were investigated. Sethoxydim and dichlormid were tested at concentrations of 0, 5, 50, and 100 μM each. Sethoxydim applied alone at 50 and 100 μm , inhibited the incorporation of [ 14 C]acetate into total lipids of sorghum leaf protoplasts by more than 50% , following a 4 h incubation. Dichlormid antagonized partially the inhibitory effects of sethoxydim on the in­corporation of acetate into total lipids of sorghum protoplasts only when it was used at 100 μM . Sethoxydim applied alone inhibited the incorporation of [ 14 C]leucine into sorghum leaf protoplasts only at 100 μm. Dichlormid was not inhibitory of this process at any concen­tration. The combined effects of sethoxydim and dichlormid on this process were mainly addi­tive indicating no interactions of the two chemicals. Sethoxydim applied alone at 5 and 50 μM inhibited the activity of ACCase extracted from leaf tissues of grain sorghum seedlings by 58 and 90% , respectively. Addition of the safener dichlormid to the assay medium did not inhibit ACCase activity of sorghum leaves even at the high concentration of 50 μM . The combined effects of sethoxydim and dichlormid on the activity of sorghum ACCase were similar to those observed when sethoxydim was used alone. These results indicate that the protection con­ferred by dichlormid on grain sorghum against sethoxydim injury can not be explained on the basis of an antagonistic interaction of the two chemicals on target metabolic processes (lipid synthesis) or target enzymes (ACCase).

This study demonstrates that corn can be partially safened by BAS-145138 against sulfonyl­urea and imidazolinone herbicides. The corn seeds were impregnated with BAS-145138 by soaking the seeds for 6 h in solutions of the safener. BAS-145138 had no effect on corn growth. However, corn shoot and root growth in length, fresh weight, and dry weight was partially protected by BAS-145138 from the phytotoxic effects of the sulfonylurea herbicides thiametu­ron and CGA -136872. Similar protection was given to corn against the phytotoxic activity of the imidazolinone herbicides imazapyr, imazaquin, and imazethapyr. This study suggests that corn could be safened with BAS-145138 against the phytotoxic effects of residual amounts of sulfonylurea and imidazolinone herbicides found in rotations take place.

Zea mays L., cv. Potro, shoots had a higher level of resistance to imazaquin (IQ) and metsul­furon methyl (MSM) than roots. Shoot lengths were increased by pretreating the seeds with commercial 1,8-naphthalic anhydride (NA) at 1% (w/w) or oxabetrinil at 0.2% (w/w). The growth of shoots of safened seeds was unaffected by 400 nᴍ IQ and by 40 nᴍ MSM. The in vivo activity of acetolactate synthase (ALS) extracted from corn shoots and roots was not affected by treatments with IQ or MSM , but pretreatment of seeds with NA or oxabetrinil, prior to germination, caused an increase in the level of extractable ALS from shoots. ALS activity from roots and shoots of NA-pretreated seedlings was increased to a large degree (> 40 %) when the seedlings were germinated on 40 nᴍ MSM , whereas ALS acti­vities from oxabetrinil-pretreated seedlings were enhanced to a lesser degree (≈20%). ALS from unsafened seedlings was inhibited 21% by 400 nᴍ IQ and 70% by 40 nᴍ MSM in vitro, but ALS from roots of seedlings germinated on 400 nᴍ IQ was not inhibited by 400 nᴍ IQ in vitro.

Metolachlor and excess Mn 2+ (acid soil stress) induce alterations in gibberellin precursor biosynthesis that can explain the morphological responses to these physiological stresses. Oxa­betrinil protects sorghum [Sorghum bicolor (L.) Moench] from the influence of metolachlor and excess Mn 2+ . Sorghum cultivar variations in response to excess Mn 2+ are explicable as dif­ferential rates of ent-kaurene biosynthesis between acid soil sensitive and tolerant cultivars. Concentrations of Mn 2+ present in vegetative leaves and reproductive stem tissues were not different. Therefore, cultivar differences in ent-kaurene biosynthesis explain the acid soil toler­ance differences rather than differential Mn 2+ absorption, translocation, and/or compartmentation. Metolachlor and safener responses are found in cellular compartments and tissues that do not match a decreased herbicide concentration through absorption, transport, or degrada­tion as a sole mode of action for safeners.

The effect of clopyralid pretreatment (500 g/ha) on picloram-induced ethylene, ACC (1-aminocyclopropane-1-carboxylic acid), and MACC [1-(malonylamino)-cyclopropane-1-carboxylic acid] was measured in rapeseed plants that were treated with 50 or 100 g/ha of picloram. In contrast to plants that did not receive a clopyralid pretreatment, ethylene biosynthesis was significantly reduced in plants pretreated with clopyralid prior to picloram. Piclo­ram-induced levels of ACC also were significantly reduced in plants receiving pretreatment with clopyralid. In contrast, there was no difference between the levels of MACC in plants that were and were not pretreated with clopyralid. Therefore, the mechanism by which clopyralid pretreatment interferes with picloram-induced synthesis of both ACC and ethylene may be manifested through the blocking of de novo synthesis of ACC synthase normally stimulated by picloram. The lack of significant difference in MACC levels between plants that were and were not pretreated with clopyralid precludes the stimulation of enhanced ACC conversion to MACC as an exclusive mechanism of clopyralid’s antidoting activity. It is likely that the rate of picloram-induced ACC synthesis by plants receiving pretreatment is within their capacity to convert ACC to MACC, thereby limiting the substrate available for conversion to ethylene. In contrast, it appears that the extent of ACC synthesis by plants receiving no pretreatment su­persedes their capacity for conversion to MACC, thereby resulting in greatly enhanced rates of ethylene evolution and subsequent development of injury symptoms.

Plantago altissima L., Plantago lanceolata L., Plantago atrata Hoppe and Plantago argentea Chaix. were examined for iridoids. In all four species aucubin and catalpol were found. Globularin and methyl ester of the desacetylasperulosidic acid were isolated for the first time from Plantaginaceae plants (P. altissima and P. lan­ceolata). Asperuloside was found for the first time in P. altissima as well as dihydroaucubin in P. atrata.

Fully substituted quinones including some naturally occurring oxyquinones acted as inhibitors of yeast gluta­thione reductase (EC 1.6.4.2). They were competitive, mixed or uncompetitive inhibitors for NADPH , possess­ing K i in the range of 1 -200 μᴍ and uncompetitive in­ hibitors for glutathione. Rhein (4,5-dioxy-9,10-anthraquinone-2-carbonic acid) and 9,10-phenanthrenequinone were the most effective inhibitors. It is concluded that certain quinones can bind to the NADP(H)-binding site and to the heteroaromatics binding site at the inter­ face domain (P. A. Karplus, E. F. Pai, and G. E. Schulz, Eur. J. Biochem. 178, 693 -703 (1989)) of the enzyme.