Band 39, Heft 5

Many herbicides inhibit chloroplast electron transport by interfering with a proteinaceous component of the Q в complex located in the appressed granal membrane. Certain of these herbicides, designated inhibitory uncouplers, also interfere with photophosphorylation and affect other chloroplast-mediated responses, some of which involve components located in the nonappressed granal membrane. The inhibitory uncouplers can be divided into dinoseb (phenolic) types which contain dissociable protons and dicryl (acylanilide) types which are nonionic. The dinoseb types can function as protonophores and shuttle protons across the thylakoid membrane at low concentrations and can alter the integrity of semipermeable membranes at higher concentrations. However, the dicryl types only alter the integrity of the membranes. The inhibitory uncouplers, but not the DCMU-types of electron transport inhibitors: stimulated electron trans- port from DPIPH 2 to methyl viologen; inhibited valifiomycin-induced swelling of intact chloroplasts; increased the permeability of the chloroplast envelope to K + in the absence of an ionophore; prevented energization of the thylakoid membrane by PS I; and increased the permeability of phosphatidyl choline liposomes to protons. Chlorination response patterns obtained with isomers of N-phenyl-2-methylpentanamides in the above reactions, in general, were similar for interference with the Q в complex, i.e., in all assays, dichlorination in the 3,4 or 3,5 positions was associated with maximum inhibitory potency, whereas substitution in an ortho position decreased inhibitory activity. With a series of 1-alkyl-3-(α,α,α-trifluoro-m-tolyl)ureas, maximum inhibition of electron transport was obtained with the butyl derivative, whereas maximum responses for uncoupling and membrane disturbances were obtained with the hexyl or octyl derivatives. Some of the interferences produced by inhibitory uncouplers may result from interactions with the lipoidal components of chloroplast membranes.

The inhibitory activities (pI 50 values) of a series of 3-alkylamino-2-cyanoacrylic acid ester derivatives in relation to photosypthetic electron transport in isolated pea chloroplast suspensions are reported. Assuming such pI 50 values reflect the relative binding affinities for the inhibitor receptor site, the results suggest that several groups in these molecules interact specifically with the binding domain so that a multi-point attachment is formed. Stereochemical factors appear to play a significant role in the interaction.

When DCMU and DCMU-type inhibitor concentrations vary over a large range, different effects may be observed on chlorophyll fluorescence parameters such as fluorescence transients, areas over fluorescence induction curves as well as kinetics of area accumulation. These results would indicate a heterogeneity of PS II electron acceptors, or an inhibitor partitioning behavior dependent on the concentration. Also, a threshold value can be reached at supra-optimal concentrations, beyond which the back reaction is almost completely blocked. Such an approach makes it possible to find out inconsistencies in the results, brought about by dual effects of some inhibitors such as phenol-type herbicides, as previously proposed.

A new spin label nitroxide which is also a herbicide of the urea family has been synthesized. The nitroxide spin label derivative of diuron (ditempuron, DTPU) inhibits electron flow at the site of diuron inhibition of photosystem II (PSII) activity, and it is also reduced by electron flow from one of the first acceptors of PSII. The reduction of the nitroxide group of DTPU is inhibited by diuron or by trypsin treatment of the thylakoids but not by DNP-INT or DBMIB. It is suggested that DTPU binds specifically to and accepts electrons at the Q в site.

In the presence of trypsin and ferricyanide as external electron acceptor, lettuce chloroplasts are resistant to DCMU, showing that the inhibitory site of DCMU is only situated on the acceptor side of photosystem II. However, kinetic properties of the oxygen evolving complex are modified at non-saturating concentrations of DCMU. These changes are interpreted in terms of a model with two distinct charges separation systems on the same center: the auxiliary donor- acceptor system DQ L implicated in the transitions S 1 → S 2 and S 2 → S 3 would be much less affected by DCMU than the main donor-acceptor system YQ H after the first flash.

Diuron (DCMU) induces the photodestruction of pigments, which is the initial herbicidal symptom. As a working hypothesis, it is proposed that this symptom can only be produced when the herbicide dose is sufficiently high to inhibit not only photosystem II electron transport almost completely, but also inhibit (through over oxidation) the natural cyclic electron flow associated with photosystem I as well. Using freshly prepared chloroplasts, studies of DCMU-induced fluorescence changes, and dose responses for inhibition of electron transport, have been compared with a dose response for the photodestruction of pigments in chloroplasts during 24 h illumination. Photodestruction of pigments coincides with the inhibition of cyclic flow.

Transient levels of the fluorescence induction rise were used to quantify partial photosynthesis inhibition by DCMU -type herbicides in whole leaves. Assays in different crop or weed species showed a good accuracy in measurements (generally, variation was lower than 5%). This technique was applied to the problem of varietal selectivity of wheat towards chlortoluron.

The effect of herbicides that inhibit the photosynthetic electron transport at the photosystem II acceptor side has been analyzed in whole plants by using a fluorometric method. The data reported indicate that the apparent variable fluorescence of the induction curve normalized to the control value provides reliable information about the penetration rate and metabolic detoxification of PS II herbicides in whole plants.

The effect of specific proteolytic enzymes on variable fluorescence, p-benzoquinone-mediated oxygen evolution, PS II herbicide (atrazine and bromoxynil) binding, and protein degradation has been analyzed in isolated class II pea chloroplasts. It was found that: 1. Trypsin and a lysine-specific protease effectively reduce the maximum chlorophyll-a fluorescence yield, whereas the initial fluorescence remains almost constant. At the same number of enzymatic activity units both proteases have practically the same effect. 2. Trypsin and a lysine-specific protease inhibit the p-benzoquinone-mediated flash-induced oxygen evolution with trypsin being markedly more effective at the same number of activity units of both enzymes. Unstacked thylakoids exhibit a higher sensitivity to proteolytic degradation by both enzymes. 3. Trypsin and a lysine-specific protease reduce the binding capacity of [ 14 C]atrazine, but enhance that of [ 14 C]bromoxynil (at long incubation times trypsin treatment also impairs bromoxynil binding). At the same specific activity a markedly longer treatment is required for the lysine-specific protease in order to achieve the same degree of modification as with trypsin. 4. Trypsin was found to attack the rapidly-turned-over 32 kDa-protein severely, whereas the lysine-specific protease does not modify this polypeptide. On the other hand, the lysine-specific protease attacks the light harvesting complex II. 5. Under our experimental conditions an arginine-specific protease did not affect chlorophyll-a fluorescence yield, p-benzoquinone-mediated oxygen evolution, herbicide binding and the poly- peptide pattern. Based on these results a mechanism is proposed in which an as yet unidentified polypeptide with exposable lysine residues, as well as the lysine-free “QB-protein” regulate the electron transfer from Q - A to Q B and are involved in herbicide binding.

Many inhibitors prevent the oxidation of the primary electron-accepting quinone (Q A ) by the secondary quinone (Q в ) in photosystem II by displacement of Q в from its binding site. On the other hand, plastoquinone-1 and 6-azido-5-decyl-2,3-dim ethoxy-p-benzoquinone displace herbicides. Binding studies show the herbicide/quinone interaction to be (apparently) competitive. The herbicide binding is influenced differentially by various treatments. In this paper it is shown that the affinity of, for example, bromoxynil is decreased by thylakoid unstacking or by light-or reductant-induced reduction of certain thylakoid components, whereas atrazine affinity remains unchanged. Furthermore, absence of HCO - 3 in the presence of form ate leads to an affinity decrease of bromoxynil and atrazine, but to an increase in i-dinoseb affinity. Other differential photosystem II herbicide effects are known from the literature. Since different and unrelated groups of Q - A oxidation inhibitors have been found, and because of the above-mentioned dissimilarities in binding characteristics for different inhibitor groups, the hypothesis of non-identical, but “overlapping” binding sites for different herbicide groups and the native quinone must be more extensively defined. In this manuscript we evaluate both the competitive herbicide/quinone binding model, and a model in which binding of one ligand alters the protein conformation resulting in a dramatic decrease in the binding affinity of ligands from other chemical groups; in this model ligands from the same or related chemical groups bind competitively. Thus, the latter model proposes that only one herbicide or quinone molecule can be bound with high affinity to the herbicide/quinone binding environment, but it depends on the chemical structure of the ligands whether the binding interaction between two ligands is truly competitive or more indirect (allosteric), mediated through the protein conformation.

The reactivation of the Hill reaction in CO 2 -depleted broken chloroplasts by various concentrations of bicarbonate was measured in the absence and in the presence of photosystem II herbicides. It appears that these herbicides decrease the apparent affinity of the thylakoid membrane for bicarbonate. Different characteristics of bicarbonate binding were observed in chloroplasts of triazine-resistant Amaranthus hybridus compared to the triazine-sensitive biotype. It is concluded that photosystem II herbicides, bicarbonate and formate interact with each other in their binding to the Q в -protein and their interference with photosynthetic electron transport.

Evidence is presented from chlorophyll a fluorescence transient data for two sites of bicarbonate (HCO - 3 *) action in photosystem II. Both the absence of HCO - 3 (HCO - 3 -depleted thylakoids) and a high concentration of HCO - 3 (60 mм HCOj added to non-depleted thylakoids) accelerate the variable chlorophyll a fluorescence rise in the presence of 10 μм diuron (DCMU). In non-HCO - 3 -depleted thylakoids the effect is independent of the order in which HCO - 3 and DCMU are added, whereas in HCO - 3 -depleted thylakoids, the effect is seen only when HCO - 3 is added before DCMU. We propose that the effect seen in HCO - 3 -depleted thylakoids is indirectly due to the binding of HCO - 3 functionally near the site of DCMU binding, which is also where HCO - 3 exerts its major effect on electron transport between the primary quinone Q A and the plastoquinone pool. We suggest that the smaller effect seen in non-HCO - 3 -depleted thylakoids is due to the binding of HCO - 3 at a second, lower affinity site. Binding at this site appears to require light, in con- trast to the higher affinity site, which is inhibited by light. Bathocuproine, an inhibitor of the H 2 O- to-silicomolybdate partial reaction, is synergistic with HCO - 3 in its effect on the variable chlorophyll a fluorescence of non-HCO - 3 -depleted thylakoids, and may bind heterotropically with HCO - 3 . Thus, this second site of HCO - 3 binding appears to be functionally near the bathocuproine binding site.

Chloroplast thylakoid membranes depleted of bicarbonate exhibit a slowed oxidation of the primary quinone acceptor (Q ᴀ ) by the secondary quinone acceptor (Q в ) of photosystem II. The kinetics of these slowed reactions have been followed by using short xenon flashes of light both to excite photosystem II and to probe the redox state of Q ᴀ . Thylakoids incubated with formate but not depleted of bicarbonate showed the same pattern of slowed reaction kinetics of the quinone acceptors as seen in bicarbonate-depleted| thylakoids. This led us to conclude that there was a simple competition between bicarbonate and formate at this site; however, steady-state electron transfer measured with an oxygen electrode showed that although the bicarbonate- depleted thylakoids were indeed inhibited, rates in the formate-incubated thylakoids were only slightly slowed. We suggest that the inhibition seen at the quinone acceptor site of photosystem II depends in a subtle way upon the rate of exchange of bicarbonate and formate at this site.

The effects of formate on the Hill reaction in isolated broken pea chloroplasts were in­vestigated. Addition of formate to chloroplasts has two distinct effects: I. basal electron flow can be stimulated 3-fold; 2. uncoupled electron flow is inhibited. The stimulating effect is due to uncoupling by formate and appears instantaneous. Maximal inhibition by form ate is only observed after prolonged illumination. The inhibitory action of form ate on electron flow can be relieved by bicarbonate *.

Inhibitors of photosystem II reactions from the “diuron-type” and “phenol-type” have been compared regarding their mechanism of action. “Diuron-” as well as “phenol-type” inhibitors act at the acceptor site of photosystem II by displacing the secondary acceptor quinone Q в from its binding site. “Phenol-type” inhibitors additionally interfere with the donor site, which is demonstrated in studies of chlorophyll fluorescence and luminescence. This mechanism of action is shown to be similar but not identical to that reported for hydroxylamine.

In plastoquinone-depleted thylakoids “DCMU-type” herbicides generally have a lower binding affinity as compared to control thylakoids, with the exception of DCMU itself. Contrary, binding properties of phenolic herbicides are not affected by plastoquinone depletion. DCMU in plastoquinone-depleted thylakoids can be displaced from the membrane by various substituted 1,4-benzoquinones, including plastoquinone-1. Labeling patterns by azido-atrazine are compared for thylakoids, plastoquinone-depleted thylakoids, and different photosystem II preparations from spinach and Chlamydomonas. Azido-atrazine tags a 32-34 kDa protein, which seems to be different from another 32-34 kDa protein which is preferentially labeled by a plastoquinone-azide.

Binding of radioactively labelled DCMU, ioxynil and terbutryn to spinach chloroplasts is determined following preillumination by single-turnover, saturating light flashes. With all of the three herbicides binary oscillations of binding are observed. Dark-adapted samples, or those preilluminated by an even number of flashes, bind more inhibitor than samples preilluminated by an odd number of flashes. Binding oscillations depend on inhibitor incubation time and on the seasonal adaptation of the plants. The binding kinetics following a single flash display three phases, the last two of which can be correlated with reoxidation kinetics of the secondary photo- system II acceptor Q - в , as determined by fluorescence measurements. Analysis of the binding at DCMU concentrations up to 10 -7 m free DCMU yields identical binding constants for dark- and flash preilluminated samples, but much less binding sites following one flash. It is concluded that up to 10 -7 m free DCMU, centers with bound do not contribute significantly to total binding. Displacement of Q - в from the binding site is half-saturated at about 10 -6 M DCMU, as monitored via fluorescence induction. The data are considered strong support for the Velthuys ‘inhibitor- Q - в competition model’.

Binding of the phenol-type herbicide ioxynil in intact algal cells is strongly increased under anaerobic conditions. This increase is most pronounced at low concentrations of bound ioxynil (< 3 nmol/mg chlorophyll). The affinity of binding sites is improved, rather than new sites are created. Other phenol-type herbicijdes, as well as uncouplers like carbonylcyanidechlorophenylhydra- zone. also significantly increase ioxynil binding in algae. Binding of DCMU-type herbicides, however, was not affected. These results demonstrate high variability of ioxynil binding in algal cells and are discussed in respect of low affinity binding.

Molecular orbital calculations of the net charge and the π charge distribution in several inhibitors and herbicides of the functionally related group of the diuron and dinoseb type are reported. They confirm the model that urea, aminotriazinone and triazine herbicides all have in common a positive π-charge at a particular atom considered to be essential for binding. Phenol type inhibitors have different charge distribution and a model for their essential features is presented. The calculations support the finding that two different subunits with different binding characteristics are involved in inhibitor and plastoquinone function on the acceptor side of photosystem II. Force-field model building and MO calculations of the charge distribution of a plastoquinone analogue with a butenyl side chain, of two of its semiquinone forms and of the hydroquinone, are reported, as well as their conformation with the lowest energy content and their likely anionic forms.

Triazine herbicides inhibit photosynthesis by blocking electron transport in photosystem II. The target site of the herbicide was identified as a chloroplast thylakoid polypeptide (the Q в protein) of 32,000 daltons. Studies of triazine-resistant weed biotypes suggested that a subtle change in the Q в protein caused the resistance. We have cloned the chloroplast gene (psbA) that codes this protein from herbicide-resistant and herbicide-susceptible biotypes of Solanum nigrum. By DNA sequencing we detected a single base substitution in the psbA gene of the resistant plants, resulting in an amino acid change (serine to glycine for the susceptible to resistance conversion). This mutation is exactly the same one which we have described in a herbicide-resistant biotype of Amaranthus hybridus.

Sequence homology has been found between the L and M subunits of Rhodopseudomonas capsulata reaction centers and the Q в -protein from all species thus far sequenced. The homology between the L subunit and the Q в -protein is contained in the sequence PFHMLG---F----AMHG-LV-S and is believed to have survived three billion years of evolution. A model is presented which associates quinone binding with this highly conserved sequence.

Spinach thylakoids have been photoaffinity labeled with [ 14 C]azidoatrazine in vitro and with specific [ 3 H]amino acids in vivo. The radiolabeled protein has been isolated, cleaved with trypsin, and the resulting fragments have been identified by their characteristic [ 3 H]amino acid incorporation. The peptide pattern has been interpreted using the known gene sequence for the Q B -protein of spinach thylakoids, which is the major protein labeled by both the in vivo and in vitro procedures. When analyzed by gel filtration, over 80% of the 14 C-activity migrated as two peaks: one as undigested protein at the void volume and the other at an apparent MW of 8.35 ± 0.76 kilodaltons. The remaining activity migrated with the included volume as free azidoatrazine. Fragments were analysed for covalent [ 14 C]azidoatrazine binding; the tryptic peptide covalently labeled by [ 14 C]azidoatrazine was found to be the fragment Pro-141 to Arg-225. This peptide is well removed from the site of a single amino acid change at Ser-264 implicated in triazine resistance, but includes a region suggested to be involved in Q в -binding by homology with sequences of two reaction center proteins from Rhodopseudomonas capsulata.

In Bumilleriopsis filiformis, the thylakoid-membrane protein showing the highest turnover in the light has an apparent molecular weight of 35 kDa. Loss of this protein after chlor-amphenicol treatment leads to a corresponding reduction of herbicide-binding sites as well as photosystem-II activity (measured by e.g. H 2 O → silicomolybdate) and cellular photosynthesis. This correlation is quantitated by comparison of the amount of herbicide-binding protein (autoradiography), electron transport, and herbicide binding. An increase of the binding constant after chloramphenicol treatment is evident.

The protein composition of Chlamydomonas reinhardii thylakoids in the molecular weight range of 32-35 kDa was studied. The thylakoids were labelled with 32 P-P i in vivo using P i -starved cell cultures, solubilized with SDS and separated by polyacrylamide gradient gel electrophoresis. The following differentiation of proteins could be accomplished: two proteins were phosphorylated, which were also well stained with Coomassie blue, and one major protein was only detected by the silver staining procedure. The mobility of the latter protein is different in gels with urea, showing an apparently lower molecular weight. In order to investigate whether a functional photosystem II is obligatory for protein phosphorylation, the phosphorylation of thylakoid proteins was studied with a photosystem II deficient mutant. The mutant, which had normal photosystem I activity, but lacked photo- system II activity, could synthesize ATP light dependently; its main labelled protein bands had a molecular weight of 32-35 kDa; it contained the light harvesting protein chlorophyll complex and an unknown protein at 22 kDa. The 32 P incorporation in photosystem II deficient cells was comparable to cells with functional photosystem II units.

Herbicide resistance in Chlamydomonas reinhardii cells was induced by mutagenesis with 5-fluorodeoxyuridine and ethylmethanesulfonate. Four mutant strains were isolated and analyzed for resistance against DCMU-type or phenolic inhibitors of photosynthetic electron transport. The mutants were different in both the extent and the pattern of their resistance: the R/S value, i.e. the ratio of I 50 values of the inhibition of photosynthetic electron transport in isolated resistant and susceptible thylakoids, varied for metribuzin from 10 000 to 36. The mutant MZ-1 was resistant against metribuzin, atrazine and DCMU, whereas the mutant MZ-2 showed resistance mainly against metribuzin and atrazine. The mutant MZ-3 was similar to MZ-1, but showed a lesser extent of resistance against DCMU. The mutant MZ-4 showed resistance against metribuzin, but not against atrazine. These results demonstrate that the resistance against one herbicide of the DCMU-type (metribuzin) must not be accompanied by similar resistance against te other inhibitors. Binding studies with radioactively labeled herbicides, [ 14 C]metribuzin, [ 14 C]atrazine and [ 3 H]DCMU, and isolated thylakoids supported these observations. Phosphorylation of thylakoid membrane proteins was studied with wild-type cells and resistant mutants under in vivo conditions in the light. The 32 P-labeled main proteins bands were in the molecular weight range of 10-14 kDa, 26-29 kDa, 32-35 kDa and 46-48 kDa. The pattern and the extent of incorporation of 32 P were similar for the mutants and the wild-type cells.

The rate of photosynthesis in intact leaves of a triazine-resistant biotype of Amaranthus retroflexus was about 15% lower than that of a triazine-susceptible biotype. Differences in photosynthesis between resistant and susceptible biotypes of Polygonum lapathifolium, Poa annua and Solanum nigrum were not significant, and absent in Chenopodium album and Stellaria media. The results are brought in relation to the appearance of resistant biotypes in the field.

Assay systems have been developed in order to differentiate between the modes of action of certain bleaching herbicides. These include inhibition of chlorophyll or carotenoid biosynthesis, and initiation of pigment degradation. Herbicidal compounds with phytoene desaturase as their primary target site were investigated in a cell-free carotenogenic system from Aphanocapsa. In a comparative study, the structural prerequisites for inhibition of phytoene desaturase were established for both benzophenone analogs and various m-phenoxybenzamides. This inhibitory action of the latter compounds is enhanced by lipophilic groups with certain steric properties.

Among chlorosis-inducing herbicides that interfere with carotenoid synthesis two groups of different potency can be discriminated (group 1: aminotriazole amd haloxidine; group 2 with more extensive photodestructions: pyridazinone herbicides and difunon). After application of herbicides of group 2 colored carotenoids were completely absent and preexisting chlorophyll was degraded by photochemical reactions requiring high light intensity and O 2 , that occurred also at 0°C. In treatments with group 1 herbicides direct photodegradation of chlorophyll was not sufficient to generate the chlorosis. Light-induced interference with constituents of the chloroplast protein synthesis apparatus being more sensitive to photooxidative damage than chlorophyll, appeared to indirectly mediate the chlorosis. In the absence of chloroplast protein synthesis further chlorophyll accumulation is prevented. Photodegradation of chlorophyll in the presence of group 2 herbicides involved the participation of O 2 - radicals and was accompanied by lipid peroxidation. In all herbicide treatments the catalase activity of the leaves was very low. Only in the presence of group 2 herbicides chloroplast enzymes of cytoplasmic origin (e.g. NADP-glyceraldehyde-3-phosphate dehydrogenase) were also inactivated. Rapid inactivation of catalase as well as of NADP-glyceraldehyde-3-phosphate dehydrogenase was induced by exposure of dim-light-grown herbicide-treated leaves to bright light, also at 0°C. In treatments with herbicides of group 2 also other peroxisomal enzymes (e.g. glycolate oxidate, hydroxy-pyruvate reductase) were affected. The elimination of these peroxisomal enzymes also appeared to depend on photooxidative processes of the chloroplast.

Plants grown in the presence of the herbicides assayed synthesized chlorophylls during growth at low fluence rates. Subsequent irradiation with higher fluence rates of red light induced a strong chlorosis with SAN 6706 being a much stronger herbicide than J 852 or amino-triazole. All herbicides assayed also changed the content and composition of chlorophylls, carotenoids and pigment-protein-complexes of the thylakoid membrane and therefore the pigment excitation and chlorophyll fluorescence emission spectra of the plastid. With increasing herbicide toxicity the main characteristic emission bands at 690 and 730 nm disappeared and new emission bands at 715 (J 852) and 700 nm (SAN 6706) appeared. Such “artificial” membranes with a changed pigment composition were very susceptible to light. Presented data may be taken as evidence, that the lack of photoprotective cyclic carotenoids caused by the specific action of a bleaching herbicide is the primary event that may lead to a disturbed formation of the thylakoid membrane and its destruction by light and oxygen.

Cell extracts of the C115 (β-carotene-accumulating) strain of Phycomyces blakesleeanus were incubated with either [2- 14 C]MVA or [1- 14 C]IPP and a range of possible inhibitors of carotenogenesis, including bleaching herbicides, biphenyl compounds and geranylacetone. Several of these compounds were potent inhibitors of β-carotene formation and caused the accumulation of phytoene. No other carotenes were found to accumulate, in vitro.The structures of these inhibitors, compared to that of phytoene, suggest that they affect the enzymic activity of “phytoene dehydrogenase”, possibly by competitive inhibition.

We have studied the effect of three herbicides viz., triazole, pyrazon and metribuzin on total chlorophyll, carotenoids and cell protein in leaves, and PS II and PS I activity in the chloroplasts isolated from control and herbicide treated pea plants. Pyrazon and metribuzin at 10μм concentration affect these parameters resulting in the chlorosis of the leaves.

Diphenyl ethers are the most versatile phytotoxic compounds known so far with respect to their targets in the plant cell. According to their substituents, the following modes of action can be found: (a) inhibition of photosynthetic electron transport effective at two binding sites, (b) peroxidative activity (degradation of cellular constituents), (c) inhibition of carotenogenesis, (d) decrease of ATP-synthase (CF 1 ) activity, (e) inhibition of respiratory electron transport, (f) stress response. This paper presents a condensed overview.

Measurements of ethane and ethylene as indices of in vivo lipid peroxidation and of chemically- induced stress, respectively, were done on mustard seedlings (Sinapis alba), treated with oxyfluorfen[2-chloro-1-(3-ethoxy-4-nitrophenoxy)-4-(trifluoromethyl)benzene]. When seedlings were exposed to low light intensity of 14W/m 2 , only stress-ethylene production but not per- oxidative ethane evolution was significantly higher over 10 days in herbicide-treated seedlings than in the untreated control. Exposure to high light intensity of 390 W/m 2 did not increase stress-ethylene production in oxyfluorfen-treated plants after 24 h. Lipid peroxidation, however, measured as ethane evolution, was substantially higher after 24 h in herbicide-treated plants, exposed to high light intensity, than in the control, and peroxidation was directly related to strong oxidation of antioxidants and to severe membrane damage.

The breakdown of chlorophylls, carotenoids, and linolenic acid together with the formation of malondialdehyde and ethane was followed in isolated pea chloroplast membranes. Breakdown was enhanced by light, oxygen, D 2 O and rose bengal, but retarded by crocetin. The results are discussed in relationship to the role of singlet oxygen in promoting damage in vivo.

Phenyl-t-butylnitrone can be used to trap light-induced radicals with isolated chloroplasts, measurable by ESR technique. The radical signals are dramatically amplified and their lifetime prolonged in the presence of peroxidizing diphenyl ethers. A para-substitution with -NO 2 , - Cl-or - I is necessary, as well as an operating photosynthetic electron transport. The nature of radicals is not yet clear; they may be alkyl or linolenic-acid radicals, but their presence indicates peroxidation. Furthermore, the ESR measurements presented strengthen our hypothesis that peroxidative activity of paraquat and diphenyl ethers are not caused by the same mechanism.

Biogenesis and development of chloroplasts are summarized in the light of new findings; the interactions of different groups of photosynthetic herbicides with chloroplast development are indicated. The effect of two new active ingredients, mevinolin* and sethoxydim **, on chloroplast modification and biogenesis are described. Mevinolin blocks the cytoplasmic HMG-CoA reductase and sterol accumulation and modifies chloroplast differentiation via the formation of a sun-type chloroplast. Sethoxydim inhibits leaf growth in grasses and gramineous crop plants and strongly affects chloroplast biogenesis and differentiation at all stages of development from proplastids via prochloroplasts to young and mature chloroplasts. This applies to thylakoid formation and multiplication, grana formation as well as chloroplast division and chlorophyll and carotenoid accumulation. The chlorophyll fluorescence induction kinetics (Kautsky effect) indicate that photosynthesis is not affected. At higher doses sethoxydim stops all growth responses of maize plants and induces the accumulation of anthocyanins.

The effects of ᴅʟ-phosphinothricin and L-methionine sulfoximine on the enzymes of nitrogen assimilation were studied. Furthermore we investigated the accumulation of ammonia and the photosynthetic activity after the treatment of mustard plants with phosphinothricin. Phosphino-thricin was a specific and very strong inhibitor of glutamine synthetase. Major differences, however, were found between the phosphinothricin affinity of the leaf enzyme and that of the root of mustard plants. The leaf enzyme was 50% inhibited at a concentration of 10 -4 m phosphinothricin (pI 50 = 4), whereas the root enzyme already showed the same effect at a concentration of 2 × 10 -5 m (pI 50 = 4.7). In addition K i values of about 0.03 mм for the leaf enzyme and 0.002 mм for the root enzyme respectively were determined. Phosphinothricin treatment of plants caused an ammonia accumulation in tissues. The accumulation was light dependent. At the beginning of the light period the major sources of ammonia accumulation could be the nitrogen assimilation as well as catabolic processes of nitrogen compounds. A clear contribution of photorespiration was only found when higher concentrations of ammonia were reached. The application of phosphinothricin induced a strong reduction of CO 2 assimilation.

A range of biochemical and physiological changes were monitored in Matricaria inodora following field rate applications of ioxynil and bromoxynil. Bromoxynil showed greater phytotoxicity with decreased reducing sugars, amino acids and proteins occurring within 28 days of treatment, when plant death was apparent. After an initial decline these parameters increased as plants appear to recover from ioxynil treatment. CO 2 fixation was completely inhibited within 4 days in leaves treated with ioxynil and bromoxynil. Ultrastructural effects of both herbicides were similar with chloroplast swelling, decrease in starch grains and thylakoid disruption prior to cellular destruction. In vitro activity of the herbicides on isolated chloroplasts revealed ioxynil to be slightly more inhibitory than bromoxynil toward electron transport and approximately one hundred times more effective as an uncoupler of PMS cyclic photophosphorylation.

Addition of BAS 13-338 (4-chloro-5-dimethylamino-2-phenyl-3(2H)-pyridazinone) to a suspension of chloroplast thylakoids caused an increase in the / level of chlorophyll fluorescence induction without affecting the F 0 level and with a slight decrease in the F max level in a manner similar to the addition of DCMU to a thylakoid suspension. Addition of BAS 13-338 also inhibited the rate of Hill reaction H 2 O → dichlorophenol indophenol with 50% inhibition occurring at about 10 μм BAS 13-338. The inhibition was not reversed by diphenyl carbazide used as an artificial electron donor to photosystem II. These results suggest that the site of inhibition by BAS 13-338 is between Q (next to the primary electron acceptor) and plastoquinone. When the plants were grown in the presence of sublethal dose of BAS 13-338, the following changes were noted in the thylakoids of the treated plants as compared to the thylakoids isolated from the control plants: The F 0 and the normalized variable fluorescence (⊿F/F 0 ) levels increased, chlorophyll a/b ratio decreased, chlorophyll/P700 ratio increased. Furthermore, the rate of photosystem II electron transport both under saturated intensity and the limiting intensity of illumination increased, and the ratio of plastoquinone to Q decreased. These observations have been interpreted as due to an increase in the ratio of photosystem II to photosystem I in plants grown in the presence of BAS 13-338.