Structural basis of cyanobacterial photosystem II Inhibition by the herbicide terbutryn

Herbicides that target photosystem II (PSII) compete with the native electron acceptor plastoquinone for binding at the Q_B site in the D1 subunit and thus block the electron transfer from Q_A to Q_B. Here, we present the first crystal structure of PSII with a bound herbicide at a resolution of 3.2 Å. The crystallized PSII core complexes were isolated from the thermophilic cyanobacterium Thermosynechococcus elongatus. The used herbicide terbutryn is found to bind via at least two hydrogen bonds to the Q_B site similar to photosynthetic reaction centers in anoxygenic purple bacteria. Herbicide binding to PSII is also discussed regarding the influence on the redox potential of Q_A, which is known to affect photoinhibition. We further identified a second and novel chloride position close to the water-oxidizing complex and in the vicinity of the chloride ion reported earlier (Guskov, A., Kern, J., Gabdulkhakov, A., Broser, M., Zouni, A., and Saenger, W. (2009) Nat. Struct. Mol. Biol. 16, 334–342). This discovery is discussed in the context of proton transfer to the lumen.     

Mode of action of a herbicide : johnsongrass and methanearsonic Acid

Johnsongrass (Sorghum halepense (L.) Pers.) is sensitive to methanearsonate, foliar application resulting in a topkill. Investigation of the pattern of photosynthesis by radioautography revealed an accumulation of malate in methanearsonate-treated leaves. Accumulation of malate was attributed to an inhibition of NADP⁺-malic enzyme which was found to be sensitive to sulfhydryl group reagents including arsenosomethane, CH₃AsO. Methanearsonate was found to act as an oxidant in the Hill reaction using spinach chloroplasts, the photoproduct being a sulfhydryl group reagent.

These results suggest that methanearsonate inhibits CO₂ release from malate in bundle sheath cells, depriving the plant of its source of carbon for sucrose production. The mechanism of inhibition of enzymes sensitive to sulfhydryl group reagents by arsenosomethane is addressed.

     

抗草甘膦转基因大豆对非靶标节肢动物群落多样性的影响

在田间自然条件下,用直接观察法和吸虫器研究了抗草甘膦转基因大豆对豆田节肢动物群落的影响.结果表明,种植抗除草剂转基因大豆的豆田节肢动物群落和非转基因亲本大豆豆田节肢动物群落的物种数,优势集中性指数、多样性指数相似程度较高,它们之间差异不显著,说明抗除草剂转基因大豆对节肢动物群落多样性无明显影响.     

Photosynthesis in rice under a salt stress

In four cultivars of Oryza sativa L., a gradual decrease in the activity of photosystems 1 and 2 as well as in chlorophyll (Chl) fluorescence transients and emission at 688 nm was observed with an increase in NaCl concentration. This decrease was more pronounced in salt-sensitive cultivars as compared to the tolerant ones. A drastic decrease in net photosynthetic rate was found in both cultivar types.     

Photosynthesis in rice under a salt stress

In four cultivars of Oryza sativa L., a gradual decrease in the activity of photosystems 1 and 2 as well as in chlorophyll (Chl) fluorescence transients and emission at 688 nm was observed with an increase in NaCl concentration. This decrease was more pronounced in salt-sensitive cultivars as compared to the tolerant ones. A drastic decrease in net photosynthetic rate was found in both cultivar types. This revised version was published online in June 2006 with corrections to the Cover Date.     

Photosynthesis at ambient and elevated humidity over a growing season in soybean

Daytime rates of net photosynthesis of upper canopy leaflets of soybeans were compared on 17 days for leaflets exposed to air at the ambient humidity and at a higher humidity. Leaflets at the higher humidity had higher rates of net photosynthesis on 16 of the 17 days. The daily total of net photosynthesis of leaflets at the higher humidity was on average 1.32 times that for leaflets at ambient humidity. A strong limitation of net photosynthesis by ambient humidity was found throughout the growing season.     

Inhibition of photosystem II of nitrogen-fixing blue-green alga Nostoc linckia by the rice-field herbicide benthiocarb

Effects of rice-field herbicide benthiocarb (S(4-chlorobenzyl)-N,N-diethyl thiolcarbamate) was studied on the nitrogen-fixing blue-green alga Nostoc linckia. The herbicide caused inhibition of growth and heterocyst formation, an increase in intensity of photoacoustic signals, and a four-fold reduction in oxygen evolution, but did not affect dark O2-uptake. The inhibition of growth and heterocyst formation was relieved by 500 micrograms/ml glucose. A Het-Nif- mutant of Nostoc muscorum failed to show an increase in reversion, frequency after treatment with 10 micrograms/ml benthiocarb for 1 hr.     

Inhibitors of photosystem II and the topology of the herbicide and QB binding polypeptide in the thylakoid membrane

The folding through the thylakoid membrane of the D-1 herbicide binding polypeptide and of the homologous D-2 subunit of photosystem II is predicted from comparison of amino acid sequences and hydropathy index plots with the folding of the subunits L and M of a bacterial photosystem. As the functional amino acids involved in Q and Fe binding in the bacterial photosystem of R. viridis, as indicated by the X-ray structure, are conserved in the homologous D-1 and D-2 subunits of photosystem II, a detailed topology of the binding niche of Q_B and of herbicides on photosystem II is proposed. The model is supported by the observed amino acid changes in herbicide tolerant plants and algae. These changes are all in the binding domain on the matrix side of the D-1 polypeptide, and turn out to be of functional significance in the Q_B binding.New inhibitors of Q_B function are described. Their chemical structure, i.e. pyridones, quinolones, chromones and benzodiones, contains the features of the phenolic type herbicides. Their essential elements, -charges at particular atoms, QSAR and steric requirements for optimal inhibitory potency are discussed and compared with the classical herbicides of the urea/triazine type.     

Redox reactions on the reducing side of photosystem II in chloroplasts with altered herbicide binding properties

Measurements of chlorophyll fluorescence have been used to monitor electron transfer from Q (the primary electron acceptor of photosystem II) to B (the bound quinone which serves as the secondary acceptor) in chloroplasts isolated from atrazine-susceptible and atrazine-resistant pigweed chloroplasts. The Q^? → B electron transfer was at least 10-fold slower in the plastids from resistant plants. Binary oscillations in the rate of Q^? decay after a series of flashes were of opposite phase in the two types. The data are interpreted to indicate that the apoprotein of B is altered in the photosytem II complex of the two types of plants—this is correlated to altered binding affinity of herbicides to this component and may be related to altered redox properties of the bound quinone cofactor.     

Photosynthesis, photorespiration and productivity of wheat and soybean genotypes

The results of the numerous measurements obtained during the last 40 years on gas exchange rate, photosynthetic carbon metabolism by exposition in ¹⁴CO₂ and activities of primary carbon fixation enzyme, ribulose‐1,5‐bisphosphate carboxylase/oxygenase (RuBPC/O), in various wheat and soybean genotypes grown over a wide area in the field and contrasting in photosynthetic traits and productivity are presented in this article. It was established that high productive wheat genotypes (7–9 t ha^?1) with the optimal architectonics possess higher rate of CO₂ assimilation during the leaf ontogenesis. Along with the high rate of photosynthesis, high values of photorespiration are characteristic for the high productive genotypes. Genotypes with moderate (4–5 t ha^?1) and low (3 t ha^?1) grain yield are characterized by relatively low rates of both CO₂ assimilation and photorespiration. A value of photorespiration constitutes 28–35% of photosynthetic rate in contrasting genotypes. The activities of RuBPC and RuBPO were changing in a similar way in the course of the flag leaf and ear elements development. High productive genotypes are also characterized by a higher rate of biosynthesis and total value of glycine–serine and a higher photosynthetic rate. Therefore, contrary to conception arisen during many years on the wastefulness of photorespiration, taking into account the versatile investigations on different aspects of photorespiration, it was proved that photorespiration is one of the evolutionarily developed vital metabolic processes in plants and the attempts to reduce this process with the purpose of increasing the crop productivity are inconsistent.     

Amino acid biosynthetic enzymes as targets of herbicide action

Several structurally-unrelated herbicides act by blocking amino acid biosynthesis. Since amino acid metabolism is similar in plants and well-studied, manipulatable microbial systems, the opportunity exists for a particularly productive interaction between microbial and plant molecular biology. Such a symbiosis may lead to new methods for the identification and design of crop protection chemicals.     

A reassessment of the use of herbicide binding to measure photosystem II reaction centres in plant thylakoids

The binding of the herbicide atrazine to thylakoid membranes is often used to quantify Photosystem II reaction centres. Two atrazine binding sites, with high and low affinities, have been observed on the D1 and D2 polypeptides of Photosystem II, respectively (McCarthy S., Jursinic P. and Stemler A. (1988) Plant Physiol. 86S:46). We have observed that the accessibility of the low-affinity binding sites is variable, being limited in freshly isolated thylakoids or in fresh frozen-thawed thylakoids, but increasing during storage of the membranes on ice. In contrast, the accessibility of the high-affinity binding sites, which are titratable at low concentrations (< 500 nM) of herbicide, is much less variable, although the dissociation constant is greatly influenced by ethanol. We conclude that to quantify Photosystem II reaction centres by atrazine binding, it is sufficient and more reliable to assay only the high-affinity binding sites.     

Development and use of domain-specific antibodies in a characterization of the large subunits of soybean photosystem 1.

The molecular architecture of the soybean photosystem 1 reaction center complex was examined using a combination of surface labeling and immunological methodology on isolated thylakoid membranes. Synthetic peptides (12 to 14 amino acids in length) were prepared which correspond to the N-terminal regions of the 83 and 82.4 kDa subunits of photosystem 1 (the PsaA and PsaB proteins, respectively). Similarly, a synthetic peptide was prepared corresponding to the C-terminal region of the PsaB subunit. These peptides were conjugated to a carrier protein, and were used for the production of polyclonal antibodies in rabbits. The resulting sera could distinguish between the PsaA and PsaB photosystem 1 subunits by Western blot analysis, and could identify appropriate size classes of cyanogen bromide cleavage fragments as predicted from the primary sequences of these two subunits. When soybean thylakoid membranes were surface-labeled with N-hydroxysuccinimidobiotin, several subunits of the complete photosystem 1 lipid/protein complex incorporated label. These included the light harvesting chlorophyll proteins of photosystem 1, and peptides thought to aid in the docking of ferredoxin to the complex during photosynthetic electron transport. However, the PsaA and PsaB subunits showed very little biotinylation. When these subunits were examined for the domains to which biotin did attach, most of the observed label was associated with the N-terminal domain of the PsaA subunit, as identified using a domain-specific polyclonal antisera.     

Mutations in the D1 subunit of photosystem II distinguish between quinone and herbicide binding sites.

The structure-activity relationships of the plastoquinone QB binding domain in the D1 subunit of photosystem II (PSII) were investigated by characterization of mutations introduced in the D1 protein. Eight novel point mutations in the gene psbA, which encodes D1, were generated in the cyanobacterium Synechocystis PCC6803 by site-specific mutagenesis in vitro. The effects of the resulting modifications in D1 on electron transfer in PSII and on herbicide binding were analyzed. The results extend the structural analogies between the secondary quinone binding site in D1 and in subunit L of the photosynthetic reaction center in purple bacteria. The involvement of Phe255, Ser264, and Leu271 of D1 in plastoquinone binding and electron transfer in PSII was established. An indirect effect of Tyr254 on the binding of QB was demonstrated. Changes in binding of herbicides and QB to D1 as a result of the mutations revealed specific interactions between amino acid residues in D1 and the plastoquinone and distinguished between the binding sites of QB and herbicides.     

Photosynthesis involvement in the mechanism of action of diphenyl ether herbicides

Photosynthesis is not required for the toxicity of diphenyl ether herbicides, nor are chloroplast thylakoids the primary site of diphenyl ether herbicide activity. Isolated spinach (Spinacia oleracea L.) chloroplast fragments produced malonyl dialdehyde, indicating lipid peroxidation, when paraquat (1,1′-dimethyl-4,4′-bipyridinium ion) or diuron [3-(3,4-dichlorophenyl)-1,1-dimethylurea] were added to the medium, but no malonyl dialdehyde was produced when chloroplast fragments were treated with the methyl ester of acifluorfen (methyl 5-[2-chloro-4-(trifluoromethyl)phenoxy]-2-nitrobenzoic acid), oxyfluorfen [2-chloro-1-(3-ethoxy-4-nitrophenoxy)-4-(trifluoromethyl)benzene], or MC15608 (methyl 5-[2-chloro-4-(trifluoromethyl)phenoxy]-2-chlorobenzoate). In most cases the toxicity of acifluorfen-methyl, oxyfluorfen, or MC15608 to the unicellular green alga Chlamydomonas eugametos (Moewus) did not decrease after simultaneous treatment with diuron. However, diuron significantly reduced cell death after paraquat treatment at all but the highest paraquat concentration tested (0.1 millimolar). These data indicate electron transport of photosynthesis is not serving the same function for diphenyl ether herbicides as for paraquat. Additional evidence for differential action of paraquat was obtained from the superoxide scavenger copper penicillamine (copper complex of 2-amino-3-mercapto-3-methylbutanoic acid). Copper penicillamine eliminated paraquat toxicity in cucumber (Cucumis sativus L.) cotyledons but did not reduce diphenyl ether herbicide toxicity.     

Photoinhibition of photosystem II under environmental stress

Inhibition of the activity of photosystem II (PSII) under strong light is referred to as photoinhibition. This phenomenon is due to an imbalance between the rate of photodamage to PSII and the rate of the repair of damaged PSII. In the “classical” scheme for the mechanism of photoinhibition, strong light induces the production of reactive oxygen species (ROS), which directly inactivate the photochemical reaction center of PSII. By contrast, in a new scheme, we propose that photodamage is initiated by the direct effect of light on the oxygen-evolving complex and that ROS inhibit the repair of photodamaged PSII by suppressing primarily the synthesis of proteins de novo. The activity of PSII is restricted by a variety of environmental stresses. The effects of environmental stress on damage to and repair of PSII can be examined separately and it appears that environmental stresses, with the exception of strong light, act primarily by inhibiting the repair of PSII. Studies have demonstrated that repair-inhibitory stresses include CO₂ limitation, moderate heat, high concentrations of NaCl, and low temperature, each of which suppresses the synthesis of proteins de novo, which is required for the repair of PSII. We postulate that most types of environmental stress inhibit the fixation of CO₂ with the resultant generation of ROS, which, in turn, inhibit protein synthesis.     

Photoinhibition of photosystem II under environmental stress

Inhibition of the activity of photosystem II (PSII) under strong light is referred to as photoinhibition. This phenomenon is due to an imbalance between the rate of photodamage to PSII and the rate of the repair of damaged PSII. In the "classical" scheme for the mechanism of photoinhibition, strong light induces the production of reactive oxygen species (ROS), which directly inactivate the photochemical reaction center of PSII. By contrast, in a new scheme, we propose that photodamage is initiated by the direct effect of light on the oxygen-evolving complex and that ROS inhibit the repair of photodamaged PSII by suppressing primarily the synthesis of proteins de novo. The activity of PSII is restricted by a variety of environmental stresses. The effects of environmental stress on damage to and repair of PSII can be examined separately and it appears that environmental stresses, with the exception of strong light, act primarily by inhibiting the repair of PSII. Studies have demonstrated that repair-inhibitory stresses include CO(2) limitation, moderate heat, high concentrations of NaCl, and low temperature, each of which suppresses the synthesis of proteins de novo, which is required for the repair of PSII. We postulate that most types of environmental stress inhibit the fixation of CO(2) with the resultant generation of ROS, which, in turn, inhibit protein synthesis.     

Inhibition of photosystem II (PSII) electron transport as a convenient endpoint to assess stress of the herbicide linuron on freshwater plants

Effects of the herbicide linuron on photosynthesis of the freshwater macrophytes Elodea nuttallii (Planchon) St. John, Myriophyllum spicatum L., Potamogeton crispus L., Ranunculus circinatus Sibth., Ceratophyllum demersum L. and Chara globularis (Thuill.), and of the alga Scenedesmus acutus Meyen, were assessed by measuring the efficiency of photosystem II electron flow using chlorophyll fluorescence. In a series of single-species laboratory tests several plant species were exposed to linuron at concentrations ranging from 0 to 1000 μg l−1. It was found that the primary effect of linuron, inhibition of photosystem II electron flow, occurred with a half-lifetime of about 0.1 to 1.9 h after addition of linuron to the growth medium. The direct effect of the herbicide on photosynthesis appeared to be reversible. Complete recovery from the inhibition occurred with a half-lifetime of 0.5 to 1.8 h after transfer of linuron treated plants to linuron free medium. The EC50,24h of the inhibition of photosystem II electron transport by linuron was about 9–13 μg l−1 for most of the macrophytes tested. For S. acutus the EC50,72h for inhibition of photosystem II electron flow was about 17 μg l−1 for the free suspension, and 22 μg l−1 for cells encapsulated in alginate beads. In a long-term indoor microcosm experiment, the photosystem II electron flow of the macrophytes E. nuttallii, C. demersum and the alga Spirogyra sp. was determined during 4 weeks of chronic exposure to linuron. The EC50,4weeks for the long-term exposure was 8.3, 8.7 and 25.1 μg l−1 for E. nuttallii, C. demersum and Spirogyra, respectively. These results are very similar to those calculated for the acute effects. The relative biomass increase of E. nuttallii in the microcosms was determined during 3 weeks of chronic exposure and was related to the efficiency of photosystem II electron transport as assessed in the different treatments. It is concluded that effects of the photosynthesis inhibiting herbicide on aquatic macrophytes, algae and algae encapsulated in alginate beads can be conveniently evaluated by measuring photosystem II electron transport by means of chlorophyll fluorescence. This method can be used as a rapid and non-destructive technique in aquatic ecological research. This revised version was published online in August 2006 with corrections to the Cover Date.