Donald E. Moreland

Simazine: Degradation by Corn Seedlings.

The herbicide 2-chloro-4,6-bis(ethylamino)-s-triazine (simazine) is converted to 2-hydroxy-4,6-bis(ethylamino)-s-triazine (hydroxysimazine) in vivo by corn seedlings and in vitro by corn extracts. Hydroxysimazine is considered to be a detoxified form of the herbicide. In vitro conversion was produced by reacting simazine with either a cyclic hydroxamate (2,4-dihydroxy-3-keto-7-methoxy-1,4-benzoxazine) or its glucoside. The latter compounds may mediate at least some of the in vivo conversion in corn.

Inhibition of photosynthesis and respiration by substituted 2,6-dinitroaniline herbicides: I. Effects on chloroplast and mitochondrial activities☆

Abstract Effects of 12 substituted 2,6-dinitroaniline herbicides [ N -butyl- N -ethyl-α,α,α-trifluoro-2,6-dinitro- p -toluidine (benefin), 2,6-dinitro- N,N -dipropylcumidine (isopropalin), 4-(methylsulfonyl)-2,6-dinitro- N,N -dipropylaniline (nitralin), 3,5-dinitro- N 4 ,N 4 -dipropylsulfanilamide (ozyzalin), α,α,α-trifluoro-2,6-dinitro- N,N -dipropyl- p -toluidine (trifluralin), and seven experimental derivatives] were measured on electron transport and phosphorylation in isolated spinach ( Spinacia oleracea L.) chloroplasts and mung bean ( Phaseolus aureus Roxb.) mitochondria. In chloroplasts, the 2,6-dinitroanilines inhibited both photoreduction and coupled photophosphorylation with water as the electron donor and with ferricyanide as oxidant, and cyclic photophosphorylation with phenazine methosulfate as the electron mediator under an argon gas phase. In mitochondria, all of the 2,6-dinitroanilines except nitralin markedly inhibited phosphorylating electron transport (state 3 respiration) with malate as substrate, and most were strong inhibitors of NADH and succinate oxidation. Some also inhibited nonphosphorylating (state 4 respiration) electron transport. Interference on the energy-generating pathway was suggested by a limited stimulation of state 4 respiration measured either as a steady-state or transitional condition. However, the strong interference imposed on electron transport prevented an accurate evaluation of their effect on the energy-generating pathway. Inhibition of oxidative and photophosphorylation by the 2,6-dinitroanilines in isolated mitochondria and chloroplasts suggests that if the compounds should partition into the organelles in vivo , photosynthesis and respiration would be inhibited. Because of the dependence of biochemical and physiological processes on oxidative and photophosphorylation for the energy (ATP) needed to drive the reactions, interference with ATP production could be one of the major mechanisms through which phytotoxicity is expressed by the 2,6-dinitroanilines.

Selective inhibition of cytochrome P450 isozymes by the herbicide synergist tridiphane

Abstract Tridiphane [2-(3,5-dichlorophenyl)-2-(2,2,2-trichloroethyl)oxirane] cross-reacts as a synergist to both herbicides and insecticides. As reported herein, tridiphane inhibited the monooxygenase activity of one or more mouse hepatic cytochrome P450 isozymes. With hepatic microsomes isolated from mice pretreated with phenobarbitol (PB) as well as with purified P450 enzymes, tridiphane efficiently inhibited demethylase and deethylase activities catalyzed by PB-induced P450 enzymes, i.e., I 50 values of approximately 4.0 μ M . However, tridiphane was a weak inhibitor of enzyme activities catalyzed by hepatic microsomes isolated from 3-methylcholanthrene (3-MC)-pretreated mice. Type I binding spectra were obtained with hepatic microsomes isolated from uninduced mice ( K s = 12.6 μM ) and PB-induced mice ( K s = 1.0 μM ), and with a purified enzyme isolated from PB-induced mice ( K s = 0.12 μM ). However, tridiphane did not form difference spectra with microsomes isolated from 3-MC-treated mice or with a purified P450 enzyme isolated from 3-MC-treated mice. Inhibition of p -nitroanisole O -demethylase suggested that tridiphane acted as a competitive inhibitor of enzyme activity and produced a K m of 440 μ M with a K i between 0.72 and 1.04 μ M .

Effects of herbicides on the light-activated, magnesium-dependent ATPase of isolated spinach (Spinacia oleracea L.) chloroplasts☆

Abstract Isolated spinach ( Spinacia oleracea L.) chloroplasts contain a Mg +2 -dependent ATPase that is activated by light in the presence of dithiothreitol (DTT) and phenazine methosulfate (PMS). Effects of 11 herbicides, known to affect photophosphorylation in isolated chloroplasts, were measured on ATPase activity when added prior to illumination, on the postillumination dark activity of the ATPase, and on the light-induced synthesis of ATP mediated by DTT and PMS. When added prior to illumination, activity of the ATPase was stimulated by low, and inhibited by high, molar concentrations of chlorpropham, dicryl, dinoseb, ioxynil, oryzalin, perfluidone, propanil, and 4,6,7-trichloro-2-(trifluoromethyl)benzimidazole (TCTFB). The light activation of the ATPase was not affected by diuron, bromacil, or atrazine. Perfluidone, dinoseb, ioxynil, and TCTFB stimulated, whereas chlorpropham, dicryl, oryzalin, propanil, atrazine, bromacil, and diuron had no effect on postillumination hydrolytic activity. The light-induced synthesis of ATP mediated by DTT and PMS was inhibited strongly by chlorpropham, dicryl, dinoseb, ioxynil, oryzalin, perfluidone, propanil, and TCTFB. Because the ATPase reactions are considered to represent the reversal of the terminal reactions of photophosphorylation, inhibition of these reactions implies that the compounds tested, except for diuron, atrazine, and bromacil, have a site of action on the ATP-generating pathway that is separate from the site involved in the inhibition of the Hill reaction.

Inhibition of photosynthesis and respiration by substituted 2,6-dinitroaniline herbicides: II. Effects on responses in excised plant tissues and treated seedlings☆

Abstract Effects of 12 substituted 2,6-dinitroaniline herbicides were measured on photosynthesis and respiration in excised tissue, and tissue excised from seedlings treated with α,α,α-trifluoro-2,6-dinitro-N,N-dipropyl-p-toluidine (trifluralin). The compounds partially inhibited photosynthesis (measured as oxygen evolution) of strips cut from spinach (Spinacia oleracea L.) leaf discs. Only 3,5-dinitro-N4,N4-dipropyl-sulfanilamide (oryzalin) strongly inhibited respiration (measured as oxygen uptake) of excised mung bean (Phaseolus aureus Roxb.) root tips and hypocotyls after a short treatment time. However, root tips excised from corn (Zea mays L.) seedlings germinated in contact with one representative 2,6-dinitroaniline (trifluralin) showed suppressed oxygen uptake. In addition, phosphorylation was completely uncoupled from oxidation in mitochondria isolated from primary roots of corn seedlings after germination of the seed in contact with trifluralin for 96 hr. Interference with photosynthesis and respiration both in vivo and in vitro by the 2,6-dinitroanilines has been demonstrated. Deviations in growth and metabolism produced by these herbicides can be explained partially by interference with the oxidative and photoproduction of ATP, if recognition is extended to the role played by ATP energy in maintaining cellular and nuclear activities. However, the importance of interference with ATP production to the phytotoxic action of the 2,6-dinitroanilines remains to be established.

Comparative effects of dichlobenil and its phenolic alteration products on photo- and oxidative phosphorylation☆

Abstract Effects of dichlobenil (2,6-dichlorobenzonitrile) and its phenolic degradation products (2,6-dichloro-3-hydroxybenzonitrile and 2,6-dichloro-4-hydroxybenzonitrile) were compared on electron transport and phosphorylation in isolated spinach ( Spinacia oleracea L.) chloroplasts and mung bean ( Phaseolus aureus Roxb.) mitochondria. In chloroplasts, the hydroxylated derivatives inhibited both photoreduction and coupled photophosphorylation with water as the electron donor and with ferricyanide as oxidant, and cyclic photophosphorylation with phenazine methosulfate as the electron mediator under an argon gas phase. In mitochondria, the phenolic derivatives acted as uncouplers of oxidative phosphorylation as evidenced by the stimulation of ADP-limited respiration, circumvention of oligomycin-inhibited non-ADP-limited respiration, and the induction of ATPase activity. Treatment of excised mung bean hypocotyls by the phenolic derivatives also resulted in a very rapid and drastic lowering of ATP levels. In all assays, only limited, if any, interference was expressed by dichlobenil even at relatively high molar concentrations. Inhibition of oxidative and photophosphorylation by the phenolic degradation products, but not by dichlobenil, suggests that if there is a delay between the formation of the hydroxylated compounds and their conjugation, photosynthesis and respiration will be inhibited. Because biochemical and physiological processes depend on oxidative and photophosphorylation for the energy (ATP) needed to drive the reactions, interference with ATP production could be one of the major mechanisms through which phytotoxicity is expressed by the phenolic degradation compounds of the herbicide, if they should accumulate in the free from. Species selectivity may be related to the rate of formation of the phenolic products in different plants and the rapidity of conjugate formation.

Interference by flavone and flavonols with chloroplast-mediated electron transport and phosphorylation

Abstract The effects of flavone and seven flavonols on the light-induced electron transport and phosphorylation of isolated spinach ( Spinacia oleracea L.) chloroplasts were investigated. With the exception of flavonol (3-hydroxyflavone), all of the compounds interacted with components of both the ATP-generating and electron transport pathways. Flavonol only interacted with the phosphorylation pathway. Interference with the phosphorylation pathway was evidenced by the greater sensitivity of the phosphorylation reaction than coupled whole-chain electron transport, inhibition of cyclic phosphorylation, inhibition of the light-activated Mg 2+ -ATPase, and inhibition of the heat-activated Ca 2+ -ATPase associated with CF 1 . The overall decreasing order of effectiveness for inhibition of cyclic phosphorylation was: galangin > quercetin = kaempferol = myricetin = flavonol > fisetin > flavone > morin. On the electron transport pathway, all of the compounds, except flavonol, interacted with the Q B -protein complex as, evidenced by inhibition of uncoupled electron transport, alteration of chlorophyll fluorescence transients, and competitive displacement of previously bound radiolabeled atrazine [2-chloro-4-(ethylamino)-6-(isopropylamino)- s -triazine]. The decreasing order of effectiveness for inhibition of uncoupled electron transport was: fisetin > quercetir = galangin > kaempferol > flavone > morin.

2H-1,4-benzoxazin-3(4H)-one, an intermediate in the biosynthesis of cyclic hydroxamic acids in maize

Abstract 2H-1,4-Benzoxazin-3(4H)-one has been identified as an intermediate in the biosynthesis of cyclic hydoxamic acids in maize by showing that the deuterium labelled compound is incorporated into DIMBOA with high retention of deuterium and high efficiency, by trapping radioactivity from [ 14 C] anthranilic acid in a pool of unlabelled benzoxazinone administered to maize shoots, and by showing that benzoxazinone is oxidized to DIBOA by maize microsomes in an NADPH- and oxygen-dependent reaction.

Effects of dibromothymoquinone on mung bean mitochondrial electron transfer and membrane fluidity.

The effects of the quinone analog dibromothymoquinone on electron transfer in isolated mung bean mitochondria are described. Both the main, cyanide-sensitive and the alternate, cyanide-insensitive pathways are inhibited by dibromothymoquinone but in markedly different fashions. Half-maximal inhibition appeared at 40 microM and 20 microM dibromothymoquinone for the cyanide-sensitive and alternate pathways, respectively. With succinate as the electron donor, dibromothymoquinone inhibited the alternate pathway at a single site; showing a mixed, non-competitive type inhibition. On the succinate, cyanide-sensitive pathway dibromothymoquinone showed two sites of inhibition and neither coincides with the site of inhibition associated with the alternate pathway. With malate as the electron donor, two sites of inhibition by dibromothymoquinone were observed regardless of the pathway measured. Dibromothymoquinone also inhibited the rate of valinomycin-induced swelling of isolated mung bean mitochondria. Steady-state kinetics showed the inhibition to be non-competitive with respect to valinomycin. Additionally dibromothymoquinone was observed to increase the fluorescence polarization associated with the hydrophobic probe 1,6-diphenylhexatriene. The results indicated that dibromothymoquinone decreased the fluidity of the inner mitochondrial membrane and suggested that the inhibition of mitochondrial electron transfer by dibromothymoquinone may be associated with this decrease in membrane fluidity. The relationship of the multisite nature of the inhibition of electron transfer by dibromothymoquinone and the possible role of mobile electron carriers such as ubiquinone on the main and alternate respiratory pathways of higher plants is discussed.

Interference by luteolin, quercetin, and taxifolin with chloroplast-mediated electron transport and phosphorylation

SummaryThe effects of luteolin, quercetin, and taxifolin on light induced phosphorylation and electron transport in isolated, greenhouse-grown, spinach (Spinacia oleracea L.) thylakoids were investigated. Luteolin and quercetin interacted with components associated with both the ATP-generating pathway and the electron-transport pathway. However, the action of taxifolin involved only the phosphorylation pathway. Interference with the phosphorylation pathway was evidenced by the greater sensitivity of phosphorylation than oxygen uptake in coupled whole-chain electron transport, inhibition of the light-activated Mg2+-ATPase, and inhibition of the Ca2+-ATPase associated with CF1. The following order of decreasing inhibitory effectiveness was exhibited: luteolin > quercetin >>> taxifolin. On the electron-transport pathway, luteolin and quercetin interfered with the activity of the QB-protein complex as evidenced by inhibition of the partial reaction with diphenylcarbazide as the electron donor and 2,6-dichlorophenolindophenol as electron acceptor; alteration of the chlorophyll fluorescence transients; and competitive displacement of radiolabeled atrazine [2-chloro-4-(ethylamino)-6-(isopropylamino)-s-triazine].