Binding site of novel 2-benzylamino-4-methyl-6-trifluoromethyl-1,3,5- triazine herbicides in the D1 protein of Photosystem II

A series of replacement experiments of [¹⁴C]-triazines, [¹⁴C]-atrazine and [7-¹⁴C]-2-benzylamino-4-methyl-6-trifluoromethyl-1,3,5-triazine, bound to thylakoids isolated from wild-type and atrazine-resistant Chenopodium album (lambsquarters) were conducted. Replacement experiments of [¹⁴C]-triazines bound to wild-type Chenopodium thylakoids with non-labeled atrazine and 2-benzylamino-4-methyl-6-trifluoromethyl-1,3,5-triazine were carried out, to elucidate whether benzylamino-1,3,5-triazines use the same binding niche as atrazine. [¹⁴C]-Atrazine and [7-¹⁴C]-2-benzylamino-4-methyl-6-trifluoromethyl-1,3,5-triazine bound to wild-type thylakoids were replaced by non-labeled 2-benzylamino-4-methyl-6-trifluoromethyl-1,3,5-triazine and non-labeled atrazine, respectively. The above two replacements showed mutual competition. To clarify further whether benzylamino-1,3,5-triazines bind at the D1-protein to amino acid residue(s) different from atrazine or not, experiments to replace [7-¹⁴C]-2-benzylamino-4-methyl-6-trifluoromethyl-1,3,5-triazines bound to atrazine-resistant Chenopodium thylakoids by non-labeled atrazine, 2-(4-bromobenzylamino)-4-methyl-6-trifluoromethyl-1,3,5-triazine, DCMU and DNOC were carried out. Although the bound [7-¹⁴C]-2-benzylamino-4-methyl-6-trifluoromethyl-1,3,5-triazine was difficult to be replaced even with high concentrations of atrazine, [¹⁴C]-labeled 1,3,5-triazine was competitively replaced by non-labeled 2-(4-bromobenzylamino)-4-methyl-6-trifluoromethyl-1,3,5-triazine, DCMU or DNOC. Thus, 2-benzylamino-4-methyl-6-trifluoromethyl-1,3,5-triazine herbicides are considered to bind to the same niche at the D1 protein as atrazine, but use amino acid residue(s) different from those involved with atrazine binding. This revised version was published online in August 2006 with corrections to the Cover Date.     

Photosynthetic electron transport inhibition by 2-substituted 4-alkyl-6-benzylamino-1,3,5-triazines with thylakoids from wild-type and atrazine-resistant Chenopodium album

The effect of 2-benzylamino-1,3,5-triazines on photosynthetic electron transport (PET) was measured with thylakoids isolated from atrazine-resistant, wild-type Chenopodium album, and spinach to find novel 1,3,5-triazine herbicides bearing a strong PET inhibition. The PET inhibition assay with Chenopodium (wild-type and resistant), yielded a resistance ratio (R/W = I50 (resistant)/I50 (wild-type)) of 324 for atrazine while for benzylamino-1,3,5-triazine derivatives of diamino-1,3,5-triazines a R/W of 11 to 160 was found. The compounds having a benzylamino group at one of the amino groups in the diamino-1,3,5-triazines have a resistant ratio down to one half to 1/30 of the atrazine value. The average resistance ratio of 21 benzylamino derivatives of monoamino-1,3,5-triazines was found to be about 4.0. The inhibition of 21 benzylamino-1,3,5-triazines assayed with atrazine-resistant Chenopodium thylakoids, indicated by pI50 (R)-values, correlated well with the PET inhibition pI50 (W) of wild-type thylakoids from Chenopodium.     

Low resistance against novel 2-benzylamino-1,3,5-triazine herbicides in atrazine-resistant Chenopodium album plants

The effects of nine novel 2-benzylamino-1,3,5-triazines on photosynthetic reactions were measured in thylakoids isolated from wild-type and atrazine-resistant plants of Chenopodium album. The resistant plants have a mutation of serine for glycine at position 264 of the D1 protein. The measurement of oxygen evolution and chlorophyll a fluorescence induction indicated a 2–4-fold stronger inhibition by the 6-trifluoromethyl analogues of Photosystem II-dependent electron flow than atrazine. Analogues having a 6-methyl-, 6-monofluoromethyl or 6-difluoromethyl substitution were weak inhibitors, indicating that the 6-trifluoro group is very important for strong inhibition. All the nine novel 2-benzylamino-1,3,5-triazines were almost as active in wild-type as in atrazine-resistant thylakoids, indicating that the benzylamino substitution may be important for the lack of resistance in the atrazine-resistant plants. This revised version was published online in June 2006 with corrections to the Cover Date.     

Characterization of the alterations of the chlorophyll a fluorescence induction curve after addition of Photosystem II inhibiting herbicides

The effects of Photosystem II inhibiting herbicides, 3-(3,4-dichlorophenyl)-1,1-dimethylurea (diuron), atrazine and two novel 2-benzylamino-1,3,5-triazine compounds, on photosynthetic oxygen evolution and chlorophyll a fluorescence induction were measured in thylakoids isolated from Chenopodium album (wild type and atrazine-resistant plants) and cyanobacterial intact cells. The resistant plants have a mutation of serine for glycine at position 264 of the D1 protein. Diuron and two members of a novel class of 2-benzylamino-1,3,5-triazine compounds were almost as active in wild-type as in atrazine-resistant thylakoids, indicating that the benzylamino substitution in the novel triazines may be important for the lack of resistance in these atrazine-resistant plants. The inhibition by the herbicides of oxygen evolution in the cyanobacteria was somewhat lower than in the thylakoids of Chenopodium album wild type, probably caused by a slower uptake in the intact cells. The so-called OJIP fluorescence induction curve was measured during a one second light pulse in the absence and in the presence of high concentrations of the four herbicides. In the presence of a herbicide we observed an increase of the initial fluorescence at the origin (Fo′), a higher J level, and a decreased steady state at its P level (Fp). The increase to Fo′ and the decreased leveling Fp are discussed. After dark adaptation about 25% of the reaction centers are in the S₀ state of the oxygen evolving complex with an electron on the secondary electron accepting quinone, Q_B. The addition of a herbicide causes a transfer of the electron on Q_B to the primary quinone acceptor, Q_A, and displacement of Q_B by the herbicide; the reduced Q_A leads to a higher Fo′. The decrease of Fp in the presence of the herbicides is suggested to be caused by inhibition of the photo-electrochemical stimulation of the fluorescence yield. This revised version was published online in August 2006 with corrections to the Cover Date.     

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

Summary The 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 Q_B-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].     

Studies on the light-induced loss of the D1 protein in photosystem-II membrane fragments

PS II membrane fragments produced from higher plant thylakoids by Triton X-100 treatment exhibit strong photoinhibition and concomitant fast degradation of the D1 protein. Involvement of (molecular) oxygen is necessary for degradation of the D1 protein.The herbicides atrazine and diuron, but not ioxynil, partly protect the D1 protein against degradation. Binding of atrazine to the D1 protein is necessary to protect the D1 polypeptide, as shown with PS II membrane fragments from an atrazine-resistant biotype of Chenopodium album which are protected by diuron not by atrazine.Abbreviations atrazine 2-chloro-4-ethylamino-6-isopropylamino-1,3,5-triazine - Chl chlorophyll, diuron - (DCMU) 3-(3,4-dichlorophenyl)-1,1-dimethylurea - DMBQ 2,5-dimethyl-p-benzoquinone - DCIP 2,6-dichlorophenol indophenol - DPC diphenylcarbazide - ioxynil 4-cyano-2,6-diiodophenol - k_b binding constant - Mes 4-morpholinoethanesulfonic acid - P-680 reaction-center chlorophyll a of photosystem-II - PAGE polyacrylamide gel electrophoresis - PS II photosystem-II - Q_A and Q_B primary and secondary quinone electron acceptors - Z electron donor to the photosystem-II reaction center - SDS sodium dodecylsulfate - Tricine N-2-hydroxy-1,1-bis(hydroxymethyl)ethylglycine     

Substituent Effect on Photosynthesis of N2-α-Substituted Benzyl-N4-alkyl-2,4-diamino-6-chloro-s-triazines and Their Phytotoxic Property

The influence of substituents on the activities of a series of N²-α-substituted benzyl-N⁴-alkyl-2,4-diamino-6-chloro-s-triazines as inhibitors of photosystem II (PSII) was examined, and the phytotoxic differences between them and atrazine, as to the photosynthesis in leaf disks, mesophyll cells, intact chloroplasts and broken chloroplasts of spinach, and as to seedling-growth, were discussed. The inhibitory activity of the N²-α,α-dimethylbenzyl-N⁴-ethyl derivative (6), which was comparable on that of atrazine, was lower than those of the N²-α-alkylbenzyl analogues (1 ~5). The N⁴-?-alkyl-N²-α- methylbenzyl derivatives, in spite of the carbon length of the alkyl group, exhibited more potent activity than atrazine, but an a α β substitution of the N⁴-n-alkyl group caused a decrease in the activity with a few exceptions. These data may imply that the space of the binding site on PSII surrounding both the N² and N⁴ amino groups is relatively large. The binding between the receptor site and the N⁴ amino group, however, is easily influenced by a slight structural change in an inhibitor. The herbicidal compounds, N²-α-methylbenzyl-A^⁴-ethyl (1), A^²-α,α-dimethylbenzyl-N⁴-1-methylpropyl (30) and N²-α-methylbenzyl-N⁴,N⁴-diethyl (42) derivatives, exhibited potent inhibitory activity in the seedling growth test under dark/light conditions, whereas atrazine was very poor. The inhibitory activity of compound (1) toward photosynthesis was poor with leaf disks, compared to atrazine, whereas, the order of their activities was the reverse for plant preparations such as abaxial epidermis peeled leaf disks, mesophyll cells, intact chloroplasts and broken chloroplasts. It was indicated that a change in the phytotoxic symptom in the whole plant assay would be correlated to the permeability of the compound through the plant membrane(s).     

Purification and Characterization of a Cryoprotective Protein (Cryoprotectin) from the Leaves of Cold-Acclimated Cabbage

We have purified a protein (cryoprotectin) from the leaves of cold-acclimated cabbage (Brassica oleracea L.) that protects thylakoids from nonacclimated spinach (Spinacia oleracea L.) against freeze-thaw damage. The procedure involves precipitations by heat, ammonium sulfate, and the glycosaminoglycan heparin and column chromatography on Polyamide 6 and a C18 reverse-phase matrix. After reverse-phase chromatography we obtained a single band of an apparent molecular mass of 7 kD when fractions that showed cryoprotective activity were analyzed by sodium dodecyl sulfate gel electrophoresis and silver staining. Gel-filtration experiments confirmed that the active protein is a monomer of 7 kD native molecular mass. This 7-kD protein could be purified only from cold-acclimated cabbage, but not from plants grown under nonacclimating conditions. Using peroxidase-labeled lectins, we show that cryoprotectin is a glycoprotein and that the saccharide moiety contains [alpha]1-3-linked fucose.     

Selective inhibition of photosystem II in spinach by tobacco mosaic virus: an effect of the viral coat protein

Leaves of Spinacia oleracea inoculated with tobacco mosaic virus (TMV) strain PV230 develop mild chlorotic and mosaic symptoms of infection. Thylakoid membranes isolated from these infected leaves showed a reduced Fv/Fm ratio for chlorophyll fluorescence kinetics, at 25 degrees C. The photosystem II (PS II)-mediated electron-transport rate was inhibited 50%, whereas PS I activity was unaffected by virus infection. Protein analysis indicated that TMV coat protein was associated with thylakoids, in particular with the PS II fraction. The results demonstrate that TMV-infected S. oleracea shows inhibition of photosynthetic electron transport through PS II. We propose that the inhibition of photosynthetic activity results from the association of viral coat protein with the PS II complex.     

A multi-biosensor based on immobilized Photosystem II on screen-printed electrodes for the detection of herbicides in river water

A multi-biosensor for detection of herbicides and pollutants was constructed using various photosynthetic preparations as biosensing elements. The photosynthetic thylakoid from Spinacia oleracea L., Senecio vulgaris and its mutant resistant to atrazine were immobilized with (BSA-GA) on the surface of screen-printed sensors composed of a graphite-working electrode and Ag/AgCl reference electrode deposited on a polymeric substrate. The biosensor was composed of four flow cells with independent illumination of 650 nm to activate electron transfer in Photosystem II. The principle of the detection was based on the fact that herbicides selectively block electron transport activity in a concentration-dependent manner and that the four PSII biomediators show differential recognition activity toward herbicides. Changes of the activity were registered amperometrically as rate of photoreduction of the artificial electron acceptor DQ. The setup resulted in a reusable herbicide multibiosensor with a good stability (half-life of 16.7 h for spinach thylakoids) and limit of detection of about 10(-8) M for herbicides recovered in spring in river.     

Mechanical freeze-thaw damage and frost hardening in leaves and isolated thylakoids from spinach. II. Frost hardening reduces solute permeability and increases extensibility of thylakoid membranes

Abstract Thylakoids isolated from cold-acclimated spinach (Spinacia oleracea L.) leaves were more resistant against mechanical freeze-thaw injury measured as plastocyanin release, than thylakoids from non-acclimated leaves. They were more resistant against solute influx during freezing and they were able to re-expand to a larger volume in comparison to non-hardy controls. Likewise, plastocyanin was released from thylakoids of non-acclimated but not of frost-hardy leaves under conditions of mild in situ freezing stress for several days.     

Purification and Characterization of an Inducible s-Triazine Hydrolase from Rhodococcus corallinus NRRL B-15444R

The widespread use and relative persistence of s-triazine compounds such as atrazine and simazine have led to increasing concern about environmental contamination by these compounds. Few microbial isolates capable of transforming substituted s-triazines have been identified. Rhodococcus corallinus NRRL B-15444 has previously been shown to possess a hydrolase activity that is responsible for the dechlorination of the triazine compounds deethylsimazine (6-chloro-N-ethyl-1,3,5-triazine-2,4-diamine) (CEAT) and deethylatrazine (6-chloro-N-isopropyl-1,3,5-triazine-2,4-diamine) (CIAT). The enzyme responsible for this activity was purified and shown to be composed of four identical subunits of 54,000 Da. Kinetic experiments revealed that the purified enzyme is also capable of deaminating the structurally related s-triazine compounds melamine (2,4,6-triamino-1,3,5-triazine) (AAAT) and CAAT (2-chloro-4,6-diamino-1,3,5-triazine), as well as the pyrimidine compounds 2,4,6-triaminopyrimidine (AAAP) and 4-chloro-2,6-diaminopyrimidine (CAAP). The triazine herbicides atrazine and simazine inhibit the hydrolytic activities of the enzyme but are not substrates. Induction experiments demonstrate that triazine hydrolytic activity is inducible and that this activity rises approximately 20-fold during induction.     

Isolation and characterisation of metribuzin-resistant Chlamydomonas reinhardii cells

Chlamydomonas reinhardii cells were treated with 5-fluorodeoxyuridine and ethylmethanesulfonate to induce mutagenesis. The mutant cells were analyzed for resistance against metribuzin (4-amino-6-(t-butyl)-3-methylthio-1,2,4-triazine-5-one). Clones with normal growth were isolated and the mutant cells further characterized. The photosynthetic rates of the mutant cells were about 20% lower than those of wild-type cells. The mutant cells were not only resistant against metribuzin (pI₅₀ lowered from 6.65 to 3.41) but also against bromacil, atrazine, phenisopham and tolerant against 3-(3,4-dichlorophenyl)-1,1-dimethylurea. However, the mutant was more susceptible to phenolic electron-transport inhibitors like bromonitrothymol, ioxynil and i-dinoseb. 2,4-Dinitrophenyl-2′-iodo-3′-methyl-4′-nitro-6′-isopropyl phenyl ether inhibited the wild-type thylakoids more than the mutant. The analysis of the electron transport with artificial electron donors and acceptors showed that only Photosystem II was affected by the mutation and not Photosystem I. Binding experiments with isolated thylakoids of resistant and susceptible cells using [¹⁴C]metribuzin and [³H]-i-dinoseb revealed that metribuzin did not bind specifically to the thylakoids of the mutant cells, but that i-dinoseb did bind to the thylakoids of the mutant, and even better than to the thylakoids of the wild-type cells. Fluorescence studies confirmed these results.     

Trans-4-aminoproline, a phytotoxic metabolite with herbicidal activity produced by Ascochyta caulina

A phytotoxic metabolite, characterized through NMR techniques and synthetic methods as trans-4-aminoproline, was isolated from the culture filtrates of Ascochyta caulina, a promising mycoherbicide for biological control of Chenopodium album. The metabolite, which shows interesting phytotoxic properties, together with ascaulitoxin (recently characterized as N.2-beta-D-glucoside of the unusual bis-amino acid 2,4,7-triamino-5-hydroxyoctandioc acid) and another unidentified compound, compose an active fraction of A. caulina culture filtrates with promising herbicidal properties. When assayed on leaves of host and non host dicots, including wild and cultivated plants, the trans-4-aminoproline showed a wide range of toxicity, with leaves of C. album being the most sensitive. Other interesting aspects were its inefficacy on several monocots, both cultivated and wild, and its lack of antifungal, antibiotic and zootoxic activities. This is the first report on trans-4-aminoproline as naturally occurring compound and phytotoxic metabolite produced by A. caulina.     

Interaction of 2-n-heptyl-4-hydroxyquinoline-N-oxide with photosystem II in chloroplasts and subchloroplast particles

The effects of 2-n-heptyl-4-hydroxyquinoline-N-oxide on electron transport in thylakoids and oxygen-evolving photosystem II particles has been examined. Kinetic fluorescence studies reveal that the site of inhibition for alkyl derivatives of hydroxyquinoline-N-oxide (I50 approximately equal to 2 microM) is located between Q and plastoquinone. Studies with thylakoids isolated from atrazine-resistant pigweed plants indicate that the modification in the Q/B membrane complex that confers increased resistance to inhibition by atrazine also results in decreased sensitivity to inhibition by 2-n-heptyl-4-hydroxyquinoline-N-oxide (resistant/ sensitive ratio = 11). From the results of tetramethylphenylenediamine by-pass experiments, determinations of inhibitor sensitivity in trypsin-treated thylakoids and competitive displacement experiments made with [14C]metribuzin in thylakoids and photosystem II particles, it is suggested that 2-n-heptyl-4-hydroxyquinoline-N-oxide binds in a region of the Q/B complex that is distinct from the 3-(3,4-dichloro)-1,1-dimethyl urea and atrazine binding sites.     

Synthesis and in vitro antitumor activity of 4(3H)-quinazolinone derivatives with dithiocarbamate side chains

A series of 4(3H)-quinazolinone derivatives with dithiocarbamate side chains were synthesized and tested for their in vitro antitumor activity against human myelogenous leukemia K562 cells. Among them, (3,4-dihydro-2-methyl-4-oxoquinazolin-6-yl)methyl 4-(4-fluorophenyl)piperazine-1-carbodithioate 8q exhibited significant inhibitory activity against K562 cells with IC(50) value of 0.5 microM.     

Synthesis and antihypertensive activity of novel 3-benzyl-2-substituted-3H-[1,2,4]triazolo[5,1-b]quinazolin-9-ones

A series of 3-benzyl-2-substituted-3H-[1,2,4]triazolo[5,1-b]quinazolin-9-ones have been synthesized by the cyclocondensation of 3-amino-2-benzylamino-3H-quinazolin-4-one with a variety of one-carbon donors. The starting material 3-amino-2-benzylamino-3H-quinazolin-4-one was synthesized from methyl anthranilate by a novel innovative route. The title compounds were evaluated for their in vivo antihypertensive activity using spontaneously hypertensive rats (SHR). While all the test compounds exhibited significant antihypertensive activity, 3-benzyl-2-methyl-3H-[1,2,4]triazolo[5,1-b] quinazolin-9-one exhibited antihypertensive activity more than the reference standard prazocin.     

Interference by DDT and cyclodiene types of insecticides with chloroplast-associated reactions

The effects of DDT, some of its analogs, and selected cyclodiene insecticides on isolated spinach (Spinacea oleracea L.) thylakoids were identified, characterized, and compared to responses induced by selected herbicides. Except for endrin, the insecticides inhibited light-induced electron transport, altered chlorophyll fluorescence transients, and competitively displaced [14C]atrazine [2-chloro-4-(ethylamino)-6-(isopropylamino)-s-triazine], a known photosystem II inhibitor, from the membranes. The insecticides appeared to act at, or near B, the secondary electron acceptor of photo-system II. Binding of DDT and dieldrin was estimated at 900 and 2200 molecules, respectively, per photosynthetic unit (490 chlorophyll molecules). The insecticides also inhibited valinomycin-induced swelling of the thylakoid membrane. Whereas inhibition of electron transport can be attributed to interaction by the insecticides with a proteinaceous component of the thylakoid membrane, interference with the action of valinomycin may involve interaction with lipoidal constituents of the membrane.     

A new class of 2-(4-cyanophenyl amino)-4-(6-bromo-4-quinolinyloxy)-6-piperazinyl (piperidinyl)-1,3,5-triazine analogues with antimicrobial/antimycobacterial activity

This study presents the synthesis and in vitro pharmacological evaluations of novel 2-(4-cyanophenyl amino)-4-(6-bromo-4-quinolinyloxy)-6-piperazinyl (piperidinyl)-1,3,5-triazines. The title compounds were assayed for their in vitro antimicrobial activity against eight bacteria (Staphylococcus aureus, Bacillus cereus, Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, Salmonella typhi, Proteus vulgaris, Shigella flexneria) and four fungi (Aspergillus niger, Aspergillus fumigatus, Aspergillus clavatus, Candida albicans) using paper disc diffusion and agar streak dilution method as well as against Mycobacterium tuberculosis H37Rv strain using BACTEC MGIT and Lowenstein-Jensen MIC method. The bioassay results indicate that nine compounds namely 5d, 5h, 5n, 5p, 5q, 5r, 5s, 5t and 5u could be considered as possible potential agents with dual antimicrobial and antimycobacterial activities. The structures of the compounds were elucidated with the aid of IR, (1)H NMR, (13)C NMR, (19)F NMR spectroscopy and CHN analysis.     

Quinolones and their N-oxides as inhibitors of photosystem II and the cytochrome b(6)/f-complex

4(1H)-quinolones (2-alkyl- (1), 2-alkyl-3-methyl- (2), 2-methyl-3-alkyl- (3), 1-hydroxy-2-methyl-3-alkyl- (4) and 1-hydroxy-2-alkyl- (5)) with n-alkyl side chains varying from C(5) to C(17) have been synthesized and tested for biological activity in photosystem II and the cytochrome b(6)/f-complex. In photosystem II, quinolones 1 and 2 showed only moderate activity, whereas 3<5<4 (increasing activity) were potent inhibitors. Displacement experiments with [(14)C]atrazine indicated that the quinolones share an identical binding site with other photosystem II commercial herbicides. In the cytochrome b(6)/f-complex, only 3<4 showed enhanced activity. Maximal inhibitory potency was achieved at a carbon chain length of 12-14 A. Further increase of the chain length decreased activity. In a quantitative structure-activity relationship inhibitory activity in photosystem II and the cytochrome b(6)/f-complex could be correlated to the physicochemical parameters lipophilicity pi and/or to STERIMOL L.