Herbicide binding in the bacterial photosynthetic reaction center.

The ureas and phenolics are two major classes of herbicides that act on Photosystem II (PSII) and are normally inactive in the photosynthetic reaction centers of purple bacteria. However, the triazine-resistant mutant T4 from Rhodopseudomonas (Rps.) viridis, which has the tyrosine residue at position 222 on the L subunit substituted for phenylalanine (TyrL222Phe), is sensitive to both ureas and phenolics. Since for the first time structural data on urea binding are available, T4 is a particularly interesting model for the herbicide-binding site of PSII.     

A partial reaction in photosystem II: reduction of silicomolybdate prior to the site of dichlorophenyldimethylurea inhibition.

Silicomolybdate functions as an electron acceptor in a Photosystem II water oxidation (measured as O2 evolution) partial reaction that is 3-(3,4-dichlorophenyl)-1, 1-dimethylurea (DCMU) insensitive, that is, reduction os silicomolybdate occurs at or before the level of Q, the primary electron acceptor for Photosystem II. This report characterizes the partial reaction with the principal findings being as follows: 1. Electron transport to silicomolybdate significantly decreased room temperature Photosystem I side of the DCMU had no effect on the fluorescence level, consistent with silicomolybdate accepting electrons at or before Q. In the absence of DCMU, silicomolybdate is also reduced at a site on the Photosystem I side of the DCMU block, prior to or at plastoquinone, since the plastoquinone antagonist dibromothymoquinone (DBMIB) did not affect the electron transport rate. 3. Electron transport from water to silicomolybdate (+ DCMU) is not coupled to ATP formation, nor is there a measurable accumulation of protons within the membrane (measured by amine uptake). Silicomolybdate is not inhibitory to phosphorylation per se since neither cyclic nor post-illumination (XE) phosphorylation were inhibited. 4. Uncouplers stimulated electron transport from water to silicomolybdate in the pH range of 6 to 7, but inhibited at pH values near 8. These data are consistent with the view that when electron flow is through the abbreviated sequence of water to Photosystem II to silicomolybdate (+ DCMU), conditions are not established for the water protons to be deposited within the membrane. Experiments reported elsewhere (Fiaquinta, R.T., Dilley, R.A. and Horton, P.(19741 J. Bioenerg. 6, 167-177) and these data, are consistent with the hypothesis that electron transport between Q and plastoquinone energizes a membrane conformational change that is required to interact with the water oxication system so as to result in the deposition of water protons either within the membrane itself or within the inner oxmotic space.     

Pathways of silicomolybdate photoreduction and the associated photophosphorylation in tobacco chloroplasts

Three sites of silicomolybdate reduction in the electron transport chain of isolated tobacco chloroplasts are described. The relative participation of these sites is greatly influenced by the particular reaction conditions. One site (the only site when the reaction medium contains high concentrations of bovine serum albumin (> 5 mg/ml)) is associated with Photosystem I, since it supports phosphorylation with a P/e₂ value close to 1 and the reaction is totally sensitive to both plastocyanin inhibitors and 3-(3,4-dichlorophenyl)-1,1-dimethylurea. Two other sites of silicomolybdate reduction are associated with Photosystem II. One site is 3-(3,4-dichlorophenyl)-1,1-dimethylurea insensitive and supports phosphorylation when the reaction mixture contains dimethyl sulfoxide and glycerol (protective agents). The P/e₂ value routinely observed is about 0.2. Bovine serum albumin (1–2 mg/ml) can also act as a protective agent, but the efficiency of Photosystem II phosphorylation observed is lower. Silicomolybdate reduction supports virtually no phosphorylation, regardless of the reduction pathway, when the reaction mixture contains no protective agents. This is due to irreversible uncoupling by silicomolybdate itself. The silicomolybdate uncoupling is potentiated by high salt concentrations even in the presence of protective agents. Exposure of chloroplasts to silicomolybdate in the absence of protective agents rapidly inactivates both photosystems.     

Pathways of silicomolybdate photoreduction and associated photophosphorylation in tobacco chloroplasts.

Three sites of silicomolybdate reduction in the electron transport chain of isolated tobacco chloroplasts are described. The relative participation of these sites is greatly influenced by the particular reaction conditions. One site (the only site when the reaction medium contains high concentrations of bovine serum albumin (greater than 5 mg/ml) is associated with Photosystem I, since it supports phosphorylation with a P/e2 value close to 1 and the reaction is totally sensitive to both plastocyanin inhibitors and 3-(3,4-dichlorophenyl)-1,1-dimethylurea. Two other sites of silicomolybdate reduction are associated with Photosystem II. One site is 3-(3,4-dichlorophenyl)-1,1-dimethylurea insensitive and supports phosphorylation when the reaction mixture contains dimethyl sulfoxide and glycerol (protective agents). The P/e2 value routinely observed is about 0.2. Bovine serum albumin (1-2 mg/ml) can also act as a protective agent, but the efficiency of Photosystem II phosphorylation observed is lower. Silicomolybdate reduction supports virtually no phosphorylation, regardless of the reduction pathway, when the reaction mixture contains no protective agents. This is due to irreversible uncoupling by silicomolybdate itself. The silicomolybdate uncoupling is potentiated by high salt concentrations even if the presence of protective agents. Exposure of chloroplasts to silicomolybdate in the absence of protective agents rapidly inactivates both photosystems.     

Dichlorophenylurea-insensitive Reduction of Silicomolybdic Acid by Chloroplast Photosystem II

The photoreduction of silicomolybdate and other heteropoly ions by chloroplasts is insensitive to 3-(3, 4-dichlorophenyl)-1, 1-dimethylurea (DCMU). Both water and diphenylcarbazide can be used as electron source for the reduction. Three different assays for silicomolybdate reduction are described including oxygen evolution, formation of a reduced heteropoly blue silicomolybdate, or an indirect assay for reduced silicomolybdate by redox indicators, such as ferricyanide or cytochrome c. The effects of detergents and tris washing are consistent with silicomolybdate reduction through photosystem II before the DCMU site. The effects of orthophenanthroline and bathophenanthroline indicate chelator-sensitive sites in photosystem II before the site of DCMU action.     

Behaviour of Herbicides in Dicotyledonous Roots Transformed by Agrobacterium rhizogenes: II. TRANSPORT TO REGENERATED SHOOTS

Mugnier, J. 1988. Behaviour of herbicides in dicotyledonousroots transformed by Agrobacterium rhizogenes. II. Transportto regenerated shoots.—J. exp. Bot. 39: 1057–1064. Regenerated plants from roots of Anagallis arvensis transformedby Agrobacterium rhizogenes were propagated in Petri disheson Murashige and Skoog's medium. The roots were exposed to differentherbicides. We report here the relationship between root uptakeand translocation of the herbicides acting upon the shoots.The results show that regenerated Anagallis arvensis plantspropagated in Petri dishes reflected the situation in normalplants in their responses to symplastic herbicides (aminotriazole,glyphosate, 2,4-D) which have strong bleaching or wilting effects.Sucrose seemed to be the critical driving force by which symplasticherbicides were transported from the root towards the shoot.The results applied to a limited range of environmental conditionssince the transport and performance of apoplastic herbicides(S-triazines, triazinones, substituted ureas) was apparentlylimited by the sucrose and moisture conditions in the Petridish. The mode of transport, in phloem versus xylem, of herbicidewithin a transformed root is discussed. Key words: Agrobacterium rhizogenes, herbicides, root organ culture     

Regulation of photosynthetic electron transport by bicarbonate formate and herbicides in isolated broken and intact chloroplasts

In this paper, we have presented a minireview on the interaction of bicarbonate, formate and herbicides with the thylakoid membranes.The regulation of photosynthetic electron transport by bicarbonate, formate and herbicides is described. Bicarbonate, formate, and many herbicides act between the primary quinone electron acceptor Q_A and the plastoquinone pool. Many herbicides like the ureas, triazines and the phenol-type herbicides act, probably, by the displacement of the secondary quinone electron acceptor Q_B from its binding site on a Q_B-binding protein located at the acceptor side of Photosystem II. Formate appears to be an inhibitor of electron transport; this inhibition can be removed by the addition of bicarbonate. There appears to be an interaction of the herbicides with bicarbonate and/or It has been suggested that both the binding of a herbicide and the absence of bicarbonate may cause a conformational alteration of the environment of the Q_B-binding site. The alteration brought about by a herbicide decreases the affinity for another herbicide or for bicarbonate; the change caused by the absence of bicarbonate decreases the affinity for herbicides. Moreover, this change in conformation causes an inhibition of electron transport. A bicarbonate-effect in isolated intact chloroplasts is demonstrated.Paper presented at the FESPP meeting (Strasbourg, 1984)     

Room temperature photooxidation of β-carotene and peripheral chlorophyll in photosystem II reaction centre

Differential kinetic absorption spectra were measured during actinic illumination of photosystem II reaction centres and core complexes in the presence of electron acceptors silicomolybdate and ferricyanide. The spectra of samples with ferricyanide differ from those with both ferricyanide and silicomolybdate. Near-infrared spectra show temporary beta-carotene and peripheral chlorophyll oxidation during room temperature actinic illumination. Peripheral chlorophyll is photooxidized even after decay of beta-carotene oxidation activity and significant reduction of beta-carotene content in both reaction centres and photosystem II core complexes. Besides, new carotenoid cation is observed after about 1 s of actinic illumination in the reaction centres when silicomolybdate is present. Similar result was observed in PSII core complexes. HPLC analyses of illuminated reaction centres reveal several novel carotenoids, whereas no new carotenoid species were observed in HPLC of illuminated core complexes. Our data support the proposal that pigments of inner antenna are a sink of cations originating in the photosystem II reaction centre.     

Mode of action of Photosystem II herbicides studied by thermoluminescence

The mode of action of chemically different herbicides (ureas, pyridazinones, phenylcarbamates, triazines, hydroxyquinolines, hydroxybenzonitriles and dinitrophenols) on photosynthetic electron transport was investigated by measurements of oxygen evolution and thermoluminescence. Depending on the particular herbicide used the thermoluminescence band related to Q (the primary acceptor of Photosystem II) appears at +5, 0 or −14°C. It was shown that these three different peak positions can be ascribed to various redox states of Q, the shifts being due to the binding of herbicides to the chloroplast membrane. Both displacement experiments and additive inhibition of herbicide pairs measured by thermoluminescence and oxygen evolution suggested that the sites of action of these herbicides are on the same protein. However, herbicide treatment of trypsinized chloroplasts showed that there were three different binding sites on the same protein, in agreement with the classification of herbicides into three groups based on thermoluminescence measurements. Our results suggest that the primary and secondary acceptors of Photosystem II (Q and B, respectively) are in close proximity and form a common complex with the herbicide-binding protein within the chloroplast membrane.     

Circular dichroism of the peripheral chlorophylls in photosystem II reaction centers revealed by electrochemical oxidation

Visible absorption spectra and circular dichroism (CD) of the red absorption band of isolated photosystem II reaction centers were measured at room temperature during progressive bleaching by electrochemical oxidation, in comparison with aerobic photochemical destruction, and with anaerobic photooxidation in the presence of the artificial electron acceptor silicomolybdate. Initially, selective bleaching of peripheral chlorophylls absorbing at 672 nm was obtained by electrochemical oxidation at +0.9 V, whereas little selectivity was observed at higher potentials. Illumination in the presence of silicomolybdate did not cause a bleaching but a spectral broadening of the 672-nm band was observed, apparently in response to the oxidation of carotene. The 672-nm absorption band is shown to exhibit a positive CD, which accounts for the 674-nm shoulder in CD spectra at low temperature. The origin of this CD is discussed in view of the observation that all CD disappears with the 680-nm absorption band during aerobic photodestruction.     

Circular dichroism of the peripheral chlorophylls in photosystem II reaction centers revealed by electrochemical oxidation

Visible absorption spectra and circular dichroism (CD) of the red absorption band of isolated photosystem II reaction centers were measured at room temperature during progressive bleaching by electrochemical oxidation, in comparison with aerobic photochemical destruction, and with anaerobic photooxidation in the presence of the artificial electron acceptor silicomolybdate. Initially, selective bleaching of peripheral chlorophylls absorbing at 672 nm was obtained by electrochemical oxidation at +0.9 V, whereas little selectivity was observed at higher potentials. Illumination in the presence of silicomolybdate did not cause a bleaching but a spectral broadening of the 672-nm band was observed, apparently in response to the oxidation of carotene. The 672-nm absorption band is shown to exhibit a positive CD, which accounts for the 674-nm shoulder in CD spectra at low temperature. The origin of this CD is discussed in view of the observation that all CD disappears with the 680-nm absorption band during aerobic photodestruction.     

Identification of the pheophytin-QA-Fe domain of the reducing side of the photosystem II as the Cu(II)-inhibitory binding site.

Oxygen evolution by photosystem II membranes was inhibited by Cu(II) when 2,6-dichlorobenzoquinone or ferricyanide, but not silicomolybdate, was used as electron acceptor. This indicated that Cu(II) affected the reducing side of the photosystem II. The inhibition curves of Cu(II), o-phenanthroline and 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), were compared; the inhibitory patterns of Cu(II) and o-phenanthroline were very similar and different in turn from that of DCMU. Cu(II) did not eliminate or modify the electron paramagnetic resonance signal at g = 8.1 ascribed to the non-heme iron of the photosystem II reaction center, indicating that the inhibition by Cu(II) was not the result of the replacement of the iron by Cu(II). Controlled trypsin digestion of thylakoid membranes inhibited oxygen evolution using 2,6-dichlorobenzoquinone, but had no effect when using ferricyanide or silicomolybdate. Using ferricyanide, oxygen evolution of trypsin-treated thylakoids was insensitive to DCMU but became even more sensitive to Cu(II) and o-phenanthroline than nontreated thylakoids; however, trypsinized thylakoids were insensitive to inhibitors in the presence of silicomolybdate. We conclude that Cu(II) impaired the photosystem II electron transfer before the QB niche, most probably at the pheophytin-QA-Fe domain.     

Evidence for localisation of accumulated chlorophyll cation on the D1-accessory chlorophyll in the reaction centre of Photosystem II

Absorption and circular dichroism spectra of Photosystem II (PS II) reaction centres (RC) were studied and compared with spectra calculated on the basis of point-dipole approximation. Chlorophyll cation was accumulated during a light treatment of PS II RC in the presence of artificial electron acceptor silicomolybdate. Light-induced difference spectra and their calculated counterparts revealed the location of accumulated cation at the accessory chlorophyll of the D1 protein subunit.     

Synthesis and Biological Activity of Urea and Thiourea Derivatives from 2-Aminoheterocyclic Compounds

Thirty-eight N-substituted-N′-(2-thiozolyl and furfuryl)ureas and thioureas were prepared by reaction of 2-aminothiazole and 2-furfurylamine with the appropriate iso(thio)cyanate. All compounds were tested for herbicidal activity and selectivity on seedlings of wheat (a monocotyledonous plant) and cucumber (a dicotyledonous plant). Only one compound (1) out of 14 ureas was characterized by considerable herbicidal activity against the wheat seedlings and two compounds (1 and2)-towards the cucumber seedlings. The phenylurea derivative of 2-aminothiazole (1) was 1.7-fold more and the 3-chlorophenylurea derivative of 2-furfurylamine (23) was equally as active as the standard diuron with respect to selective herbicidal activity. Among 24 thioureas, four compounds (15, 16, 17, and18) to displayed the highest selective herbicidal activity and two other compounds (19 and33) were almost equal to diuron activity. Selective herbicidal ratio (SHR) represents the degree of herbicidal effect of the investigated compounds compared to diuron at both test objects. Four compounds (16,17,18, and23) possessed SHR ≪100 in the wheat seedlings while in the cucumber seedlings they had SHR ≫ 100. Therefore these compounds were substantially more active herbicides to the wheat seedlings as compared to diuron. The cytokinin-like activity of the synthesized compounds was also investigated in terms of betacyanin synthesis and radish cotyledon enlargement. The urea derivatives exhibited mostly high cytokinin-like activity but their activity remained lower than those of kinetin and N-phenyl-N′-(4-pyridyl)urea. The N-(3-fluorophenyl)-N′-(2-thiazolyl)urea (2) possessed the greatest activity at 10 μM while the corresponding compound with 3-chlorophenyl (4) was the most active cytokinin-like substance in the whole concentration range tested. Attention was also given to structure-activity relationships for the screened compounds. In general, the ureas and thioureas containing a 2-thiazole ring were more active than those containing a 2-furfuryl residue. Online publication: 7 April 2005     

Proton translocation and ATP formation coupled to electron transport from H2O to the primary acceptor of photosystem 2.

1. The rate of electron transport from H2O to silicomolybdate in the presence of 3-(3-4-dichlorophenyl)-1,1-dimethylurea (diuron) (which involves the oxygen-evolving enzyme, the photochemistry of photosystem 2 and the primary electron acceptor of photosystem 2) is controlled by internal pH. This is based on the shift of the pH profile of the rate of electron transport upon addition of uncouplers, or by using EDTA-treated chloroplasts. Both stimulation and inhibition of electron transport by addition of uncouplers (depending on external pH) could be observed. These effects are obtained in the diuron-insensitive photoreductions of either silicomolybdate or ferricyanide. These experiments provide strong evidence that a proton translocating site exists in the sequence of the electron transport H2O leads to Q (the primary acceptor of photosystem 2). 2. The photoreduction of silicomolybdate in the presence of diuron causes the formation of delta pH. The value of delta pH depends on the external pH and its maximal value was shown to be 2.4. The calculated internal pH at different external pH values was found to be rather constant, namely between 5.1 -- 5.2. 3. Electron transport from H2O to silicomolybdate (in the presence of diuron) does not support ATP formation. It is suggested that this is due to the fact that the delta pH formed is below the "threshold" delta pH required for the synthesis of ATP. By adding an additional source of energy in the form of a dark diffusion potential created in the presence of K+ and valinomycin, significant amounts of ATP are formed in this system.     

Evidence for alpha-Tocopherol Function in the Electron Transport Chain of Chloroplasts

The effect of three different stable radicals-2,2-diphenyl-1-picrylhydrazyl, 1,3,5-triphenyl-verdazyl, and galvinoxyl-was studied in photosystem II of spinach (Spinacia oleracea) chloroplasts. Inhibition by the three was noted on dimethylbenzoquinone reduction in presence of 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB) and on silicomolybdate reduction in presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) in photosystem II and on the H₂O → methylviologen reaction encompassing both photosystems. Inhibition of all photosystem II reactions except silicomolybdate reduction could be partially restored by α-tocopherol or by 9-ethoxy-α-tocopherone but not by other quinones or radical chasers. On this basis, a functional role for α-tocopherol in the electron transport chain of spinach chloroplasts between the DCMU and DBMIB inhibition sites is postulated.     

Silicomolybdate reduction by isolated pea chloroplasts

Conditions for the optimization of silicomolybdate reduction by isolated pea chloroplasts are described. Maximum rates of reduction are related to time of addition to the chloroplasts and the presence of an oxidizing cofactor, such as ferricyanide. Silicomolybdate or silicomolybdate plus ferricyanide reduction is only partially inhibited by a concentration of CMU which totally abolishes ferricyanide reduction. Evidence for a differing response of the two reduction sites to silicomolbydate is described.     

Metabolism of twelve herbicides by Streptomyces

Experiments were conducted to assess the ability of Streptomyces (strain PS1/5) to metabolize twelve herbicides representing several different classes including: acetanilides, triazines, ureas, uracils, and imidazoles. Incubations in aqueous culture with dextrin as carbon source and either ammonium or Casamino acids as nitrogen source resulted in transformations (>50%) of eight of the herbicides tested: alachlor, metolachlor, atrazine, prometryne, ametryne, linuron, tebuthiuron, and bromacil; the remaining four herbicides (cyanazine, diuron, metribuzin, and imazapyr) were also transformed, but to a lesser extent. In most instances, biotransformations occurred concurrently with growth and results were consistent regardless of the nitrogen source (ammonium vs. Casamino acids). However, in some instances there were differences in rates of biotransformation as a consequence of the nitrogen source (e.g. alachlor, metribuzin), suggesting the selective induction of certain metabolic enzymes; in other instances biotransformations were not associated with growth, suggesting secondary metabolism. An experiment was also conducted to assess the ability of Streptomyces (strain PS1/5) to metabolize atrazine contaminated soil. Inoculation of soil amended with 20 μg/g of atrazine and 5% chitin as carbon source resulted in ca. 78% removal of atrazine within 28 days. These data suggest that Streptomyces species may be potential candidates for soil inoculation to bioremediate herbicide contaminated soils.     

Herbicide-induced changes in charge recombination and redox potential of Q(A) in the T4 mutant of Blastochloris viridis

To gain new insights into the function of photosystem II (PSII) herbicides DCMU (a urea herbicide) and bromoxynil (a phenolic herbicide), we have studied their effects in a better understood system, the bacterial photosynthetic reaction center of the terbutryn-resistant mutant T4 of Blastochloris (Bl.) viridis. This mutant is uniquely sensitive to these herbicides. We have used redox potentiometry and time-resolved absorption spectroscopy in the nanosecond and microsecond time scale. At room temperature the P(+)(*)Q(A)(-)(*) charge recombination in the presence of bromoxynil was faster than in the presence of DCMU. Two phases of P(+)(*)Q(A)(-)(*) recombination were observed. In accordance with the literature, the two phases were attributed to two different populations of reaction centers. Although the herbicides did induce small differences in the activation barriers of the charge recombination reactions, these did not explain the large herbicide-induced differences in the kinetics at ambient temperature. Instead, these were attributed to a change in the relative amplitude of the phases, with the fast:slow ratio being approximately 3:1 with bromoxynil and approximately 1:2 with DCMU at 300 K. Redox titrations of Q(A) were performed with and without herbicides at pH 6.5. The E(m) was shifted by approximately -75 mV by bromoxynil and by approximately +55 mV by DCMU. As the titrations were done over a time range that is assumed to be much longer than that for the transition between the two different populations, the potentials measured are considered to be a weighted average of two potentials for Q(A). The influence of the herbicides can thus be considered to be on the equilibrium of the two reaction center forms. This may also be the case in photosystem II.