Volume 48, Issue 3-4

Otto Warburg, the father of cellular bioenergetics, seems to have been the first investigator to report on inhibition of a plant biochemical reaction by a progenitor of a selective herbicide. The year was 1920 and the compound was phenylurethane (ethyl N-phenylcarbam ate or EPC). Warburg found that it strongly inhibited photosynthesis in Chlorella. EPC did not develop into a commercial herbicide, but the isopropyl derivatives (propham and chlorpropham) which were introduced in the late 1940s became selective herbicides. The phenylureas (monuron and diuron) were introduced in the early 1950s and shortly thereafter, interference with the Hill reaction by both phenylureas and phenylcarbamates was reported. During the latter part of the 1950s, into the 1960s, and even now, additional herbicidal chemistry was and is being announced that interferes with the Hill reaction. Duysens, in 1963, identified the site of action of diuron, i.e., on the acceptor side of PS II. Corwin Hansch, in 1966 introduced the SAR or QSAR concept in which inhibitory action of Hill inhibitors was related to various chemical and physical parameters. Because of differential responses to partial, thylakoid-associated reactions, the Hill inhibitors were subsequently divided into two groups: pure electron transport inhibitors (phenylureas, s-triazines, triazinones, and uracils) and inhibitory uncouplers (acylanilides, dinitrophenols, benzimidazoles, dinitroanilines, and benzonitriles). The inhibitory uncouplers (dinoseb-types), unlike the diuron-types, uncoupled photophosphorylation by interacting with the coupling factor complexes in both chloroplasts and intact mitochondria. Additionally, the bi-pyridyliums were shown to be reduced by PS I, hence, diverted electrons from the native acceptor. Field observations of triazine resistance were reported in 1970 and resistance was subse­ quently demonstrated at the thylakoid level. Application of the techniques of genetic engineering and biotechnology resulted in identification of the 32 kD a herbicide-binding protein and determination of its amino acid sequence. Crystallization and X-ray examination of the photosynthetic reaction center from Rhodopseudomonas by Michel et al. in the mid-1980s provided new models to account for interactions of herbicides with the D -1 protein. During the 1980s, herbicides were identified that interfered with biochemical machinery in chloroplasts that is not involved in electron transport and light harvesting: inhibition of lipid biosynthesis by aryloxyphenoxypropionates and cyclohexanediones, aromatic amino acid bio­ synthesis by glyphosate, branched chain amino acid biosynthesis by sulfonylureas and imidazolinones, carotenoid biosynthesis by pyridazinones, and porphyrin biosynthesis by diphenylethers and oxadiazoles. The current status of research in most, if not all, of the above areas was reported through oral and poster presentations at this Omiya Symposium.

Representatives of two structural types of cyanoacrylate PS II inhibitors have been studied in respect to their pI 50 values and qualitative rate of binding with wild type and S 264 G mutant thylakoids isolated from Brassica napus. Both are potent inhibitors of photosynthetic electron transport and both show a large discrimination between wild type and mutant thylakoids under equilibrium conditions. However one, an N-methylanilino cyanoacrylate, has an initial rapid reaction with both wild type and mutant thylakoids, but continues to react slowly with the wild type species until equilibrium is reached, while the other, a benzylamino cyanoacry­ late, equilibrates rapidly with both species as does the classical PS II inhibitor, atrazine. These differences in kinetic behaviour have been interpreted in terms of different H-bond interactions with the serine-264 hydroxyl group. It is suggested that the slow binding reaction is due to the N -methylanilino compound interacting as an H-bond acceptor with the serine-264 hydroxyl hydrogen thus disrupting an intramolecular ser-264-his-252 H-bond within the D 1 peptide. Rapid equilibration on the other hand, has been attributed to the benzylamino derivative acting as an H-bond donor to the serine-264 hydroxyl oxygen and strengthening the 264 -252 H -bond by conjugation. It is proposed that atrazine and other classical PS II inhibitors act in this way and that this may explain their ability to inhibit trypsin degradation of the D 1 peptide, if the 264-252 intramolecular H-bond plays an important role in stabilizing the peptide conform ation. It is also speculated that photodegradation may be related to the ability of Q B - to act as an H-bond acceptor and disrupt the 264-252 H-bond.

Many 3-N-methylanilino cyanoacrylate derivatives are potent inhibitors of photosynthetic electron transport at the PS II level in a thylakoid system isolated from Pisum sativum. These inhibitors are somewhat unusual in that they take more than 15 min to equilibrate with the binding site in contrast to most classical PS II inhibitors which tend to equilibrate rapidly i.e. within the mixing time of seconds. The effects of mono-and disubstituents in the aryl nucleus on inhibitory activity were studied. The nature and position of the substituent had no detectable influence on equilibration rate but a pronounced influence on inhibitory activity as measured by equilibrium pI 50 values. Substituent position enhanced inhibitory activity in the order 3′ > 2′ ⋙ 4′ . As regards the nature of the substituent, size appeared to be the most important parameter -the smaller the substituent the greater the activity. Such influences were in contrast to those in analogous 3-anilino-and 3-benzylamino-2-cyano-acrylate series where the comparable order of substituent position effect was 3′ and 4′ ⋙ 2′ and where substituent hydro-phobicity rather than size was the most important factor. Disubstituted fluoro derivatives have given interesting insights into the preferred orientation of these inhibitors at the binding site with the 2′-and 5′-positions interacting positively, the 4′-position, negatively, and the 3′-position neutrally.

For thirty years the study of Quantitative Structure Activity Relationships (QSAR) has been an active area of research aimed at developing an understanding of the interactions be­ tween inhibitors of photosynthetic electron transport and the herbicide binding site in the Photosystem II (PS II) reaction centre. Many QSAR studies of PS II inhibitors with diverse chemical structures have emphasized the hydrophobic nature of the binding domain, with lipophilicity being the dominant determinant of Hill inhibition activity. The cyanoacrylate classes of PS II inhibitors also show a diversity of active structures and considerable variation in inhibition potency with minor alterations to structure. QSAR analysis and examination of chirality in cyanoacrylate inhibitors has also shown the importance of steric factors in determining activity. Different modes of binding for different classes of cyanoacrylates have been identified; a classical urea-type relationship between activity and hydrophobicity and another type of interaction in which the lipophilicity or electronic nature of phenyl substituents plays little part and the size of the substituents is of primary importance. Because size and shape are parameters of great importance in determining the topography of a binding site, QSAR studies of flexible PS II inhibitors such as cyanoacrylates will continue to be important in elucidating the intricacies of inhibitor/PS II interactions.

The orientation of acridones, xanthones, 1,4-benzo-and naphthoquinones within the photosystem II Q B herbicide-binding niche was studied by means of mild trypsination and by estimation of pI 50 -values in Chlamydomonas reinhardtii D 1 mutants (Val 219 > lie, Ala 251 > Val, Phe 255 > Tyr, Ser 264 > Ala, Asn 266 > Thr, and Leu 275 > Phe). As judged from the R/S-values (ratios of I 50 -values resistant versus susceptible type) close to 1 in all mutants, the acridones and xanthones do not have strong interactions with the parent amino acids. Contrary, the quinones exhibit extreme low R/S-values down to 0.003 (for 2,5-dibromo-3-methyl-6-isopropyl-1,4-benzoquinone; DBMIB) in the Ser 264 mutant. This extreme negative cross resistance or supersensitivity indicates that the quinones do not form a hydrogen bond to the serine hydroxyl group.

A range of 4-hydroxypyridines were synthesized and their activity as PET inhibitors were investigated with regard to their structural resemblance to plastoquinone in photosynthetic electron transport (PET). The activity of these compounds was markedly enhanced upon modifying their structures: introduction of halogens into both the 3-and 5-positions of the pyridine ring and additional substitution at the α-position of the side chain at 6-position were effective among others in enhancing the activity. Insertion of a phenyl ring into the side chain at 6-position of the pyridine ring also increased the activity. Substituents on the phenyl ring greatly affected the activity: when substituted with an appropriate functional group, the compounds became 10-to 100-fold more active. The mode of action of both halogenated and non-halogenated 4-hydroxypyridines were investigated by means of thermoluminescence measurements and cross resistance examination against atrazine-resistant thylakoids having mutation in D 1 protein. It was inferred that upon halogenation, 4-hydroxypyridines changed their mode of action from plastoquinone-pool inhibitors to phenol-type inhibitors.

Esters of 3-nitrophloroglucinecarboxylic acid were found to be active photosystem II (PS II) inhibitors, as were the amides, indicating that, in their structures, the substituted phloroglucinol nuclei themselves plays a major roles in PS II inhibition. Among the phenylureidoalkyl esters tested, 3,4-dichlorophenylurea compound having two amino hydrogen atoms showed high activities. Optimal activity was associated with compounds in which the oxygen atom and the urea structure in the ester side chain were connected via an ethylene group.

Through the studies on structure-activity relationships of 5-acyl-3-(1-aminoalkylidene)-4-hydroxy-2 H-pyran-2,6(3 H)-dione derivatives in photosystem II (PS II) inhibition, overall lipophilicity of the molecule was found to be a major determinant for the activity. In the substituted N -benzyl derivatives, not only the lipophilicity but also the electronic and steric characters of the substituents greatly affected the activity. Their mode of PS II inhibition seemed to be similar to that of DCMU , whereas pyran-enamine derivatives needed to be highly lipophilic to block the electron transport in thylakoid membranes, which in turn diminished the permeability through biomembranes.

Interactions of halogenated benzoquinones with the acceptor side of Photosystem (PS) II were studied by measuring fluorescence induction curves and flash-induced absorbance changes in PS II particles isolated from Synechococcus vulcanus. Following results were obtained: 1) Addition of some halogenated benzoquinones prior to 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) in the dark increased the area above the fluorescence induction curve (work integral) by a factor of two. 2) Based on the ability to increase the fluorescence work integral, halogenated benzoquinones could be divided into two groups. 3) 2,6-Dichlorobenzo-quinone (2,6-DCBQ), trichlorobenzoquinone (TCBQ) and tetrahalogenated benzoquinones except tetrafluorobenzoquinone (fluoranil) (group A) increased the work integral, but 2,5-DCBQ and fluoranil (group B) did not. 4) Rapid reoxidation of Q A - was observed in the presence of quinones which belong to group A. These results were interpreted in terms of dark oxidation of by quinones belonging to group A. Possible mechanisms of oxidation of Q 400 by these quinones in the dark are discussed.

We have recently shown that binding affinities of benzoquinones can be estimated by two methods in photosystem (PS) II particles (K. Satoh et al., Biochim. Biophys. Acta 1102, 45-52 (1992)). Using these methods we calculated the binding affinity of thymoquinone (2-methyl-5-isopropyl-p-benzoquinone) to the Q B site and studied how the quinone accepts electrons in oxygen-evolving PS II particles isolated from the thermophilic cyanobacteria, Synechococcus elongatus and S. vulcanus. The results are as follows: (1) The binding constant of thymoqui­ none to the Q B site determined by several methods was around 0.33 mᴍ . (2) At low thymoquinone concentrations the quinone was supposed to accept electrons via Q B -plastoquinone, whereas at high concentrations the quinone seemed to bind to the Q B site and accept an electron directly from Q - A . Lower rates of photoreduction of the quinone at high concentrations were attributed to a slower turnover rate of the quinone at the Q B site than that of endogenous plastoquinone. (3) A model for the function of plastoquinone at the Q B site, which can explain all the results, was presented. According to this model, the plastoquinone molecule at the Q B site is not replaced by another plastoquinone molecule. Instead, it transfers electrons to pool plastoquinone molecules by turning over its head group but remaining its long side chain bound to the PS II complexes.

Previous studies of structure/activity relationships of photosystem II inhibitors, including comparisons of their inhibitory power in herbicide-resistant and susceptible chloroplasts, have led to predict the role of hydrogen bonding, associated to hydrophobicity, in the binding onto the Q B site. The crystallographic structures of bacterial reaction centers now allow these bonds to be identified. In order to be able to understand the binding of various herbicides and the effects of resistance mutations within the Q B site, a reliable estimation of hydrogen bonding strengths is needed. We show here, by calculating interactions with model compounds, controlled by physicochemical measurements, that the hydrogen bonding properties of the C = X nucleophilic moiety present in most PS II inhibitors are different for triazines as compared to urea or amide derivatives. Semiempirical methods (AM 1) fail to reproduce the energies of hy­drogen bonds between a triazine ring nitrogen and a phenolic proton. An empirical method (SIBFA), designed to reproduce interaction energies, has been adapted with the aim of calculating the binding energies of various herbicides with models of the Q B site.

The D 2 protein contains an extended loop (the D-de loop) between helices D and de at the reducing side of photosystem II (PS II). Characterization of D 2 mutants of the cyanobacterium Synechocystis sp. PCC 6803 has indicated that the length and amino acid com position of the D-de loop are not critical for basic PS II functions, although most of the residues in that region are conserved phylogenetically. Here we show using herbicide binding and electron-flow inhibition measurements that drastic modifications in the D-de loop of the D 2 protein modify the interaction of some PS II-directed herbicides with their binding niche. The stability of (semi-)reduced Q B in its binding pocket is altered in at least two of the mutants, as indicated by a shifted peak temperature of the thermoluminescence signal originating from charge recombination involving Q B . These results suggest a close functional association between the D-de loop of the D 2 protein and the Q B /herbicide-binding environment, which is viewed as being coordinated mostly by residues of the D 1 protein. This represents one of the first examples of modification of the Q B / herbicide-binding domain by mutations in the D 2 protein.

The prefered binding orientations for the herbicide DCMU within the Q B -binding site of the D 1 protein model from a photosystem II reaction centre have been determined. Calculation of the intermolecular energy between the herbicide and the binding site has been instrumental in obtaining optimum positions reinforced by experimental results from mutation studies and herbicide binding to analogous bacterial reaction centres. We have shown that two binding sites are possible, one involving a hydrogen bond to and the other to the Ser 264 residue. In both cases, which are more important for the stabilization of the interactions.

Various herbicides inhibit photosynthesis by displacing the second electron acceptor Q B from its binding site at the D 1 protein. Different amino acid substitutions within this binding site have been found to reduce herbicide affinities, thereby conferring herbicide resistance. In Synechocystis PCC 6714 we have selected 7 single mutants and 6 double mutants resistant to various herbicides due to amino acid substitutions at different positions in the Q B pocket. Characterization of these mutants consists in molecular determination of the mutations in the psbAl genes and in transformation of Synechocystis PCC 6803 by the cloned mutated genes to analyze the role of the mutations in the mutant phenotypes. Comparison with equivalent Chlamydomonas mutants is presented. These studies allow us to specify the interactions of several amino acid residues with herbicides and Q B and with each other. Furthermore some Synechocystis mutants present additional characteristics such as an increased sensitivity to photoinhibition, or resistance to formate or modification of the oscillatory pattern of oxygen evolution. Among the 6 point-mutations giving herbicide resistance, only those located at the limit of the loop and the parallel helix produced additional effects on photosystem II function.

A series of 20 triazines (derivatives of 2-alkylamino-4-benzylam ino-6-chloro-1,3,5-triazines) and 37 triazinones (derivatives of 3-alkyl-4-amino-6-phenyl-1,2,4-triazin-5-ones) is tested for inhibitory potency in photosynthetic electron flow through photosystem II of wild type Chlamydomonas reinhardtii and of five mutants with aminoacid substitutions in the D 1 protein at valine 219, alanine 251, phenylalanine 255, serine 264, and leucine 275. The data are used for computer modelling of the possible location of the compounds within a three dimensional model of the Q B -binding niche of the D 1 protein.

Photosynthesis inhibition data were determined with thylakoids of the green alga Chlamydomonas reinhardtii on twenty-two 2-bromo-4-nitro-6-alkyl -and 2,4-dinitro-6-alkyl-phenols. Wild type and five different mutants of the algae were employed with defined changes in the psbA gene and a single amino acid substitution in the herbicide binding protein D 1 of photo­ system II. Their response to the various inhibitors was quite different from that of the wild type. Tolerance as well as enhanced inhibitory activity was found in the mutants, with tolerance in all phenols in the val219 ile and of some in the phe255 tyr mutant and supersensitivity of most phenols in the ala251 val, ser264 ala and leu275 phe mutant. This implies that the phenols are bound specifically to the D 1 protein. Structure-activity studies revealed parabolic relationships with all descriptors increasing with the alkyl chain length for wild type and most mutant inhibition values. Molecular modelling chains in different phenols in the Q B binding niche same direction.

The absence of the extrinsic Mn-stabilizing 33 kDa protein in the IC 2 mutant of Synechocystis PCC 6803 disturbs the redox cycling of the water splitting system and retards the formation of its higher S-states (I. Vass, K. Cook, S. Deak, S. R. Mayes, and J. Barber, Biochim. Biophys. Acta 1102, 195-201 (1992)). We have performed analyses of the flashinduced oxygen exchange in the mutated cyanobacterium to clarify further the role of the 33 kDa protein. Under aerobic conditions, both the wild type and IC2 mutant show a relatively slow signal of oxygen rise on the first flash which is increased about twice by the addition of 10 μᴍ DCMU and significantly diminished by lowering the oxygen concentration in the medium. According to action spectra measurements, this mode of apparent oxygen release is mediated by PS I and can be attributed to a light induced inhibition of respiratory activity. In contrast to the wild type, having the usual oxygen evolution flash pattern with a periodicity of four, the IC2 mutant shows a binary oscillation pattern of flash-induced respiratory oxygen exchange at a flash frequency 10 Hz, being dampened with DCMU or by a lower flash frequency (< 1 Hz). Oxygen evolution due to water splitting is clearly seen in the IC2 mutant when background far-red illumination is applied to saturate the signal due to respiratory inhibition, but a quadruple oscillatory component of flash-induced oxygen evolution appears only in the presence of artificial electron acceptors under partial aerobic conditions. The mutant possesses a higher PS I/PS II ratio compared to the wild type, as judged from both the flashinduced yields and quantum efficiencies of the steady-state rates of the oxygen exchange reactions. Estimates of antenna sizes indicate about a 20% decrease of optical cross-section at 675 nm of the PS II unit in IC2 mutants in comparison with the wild type. It is suggested that the absence of the 33 kDa protein leads to a modification of the PS II assembly and because of the slowing down of the S-state cycle, the rate of cyclic electron flow around PS II is enhanced. It seems that the absence of the 33 kDa protein in Synechocystis 6803 also disturbs energy transfer between adjacent PS II core complexes and may also alter their association with the phycobilisomes.

Studies on thermodynamics and kinetics of electron transfer from Q A- to Q B (Q B - ) were performed by monitoring laser flash induced changes of the relative fluorescence emission as a function of temperature (220 K < T < 310 K) in isolated thylakoids and PS II membrane fragments. In addition, effects of bivalent metal ions on PS II were investigated by measuring conventional fluorescence induction curves, oxygen evolution, manganese content and atrazine binding mostly in PS II membrane fragments. It was found: a) the normalized level of the fluorescence remaining 10 s after the actinic flash (F t /F 0 ) steeply increases at temperatures below -10 to - 20 °C, b) the fast phase of the transient fluorescence change becomes markedly retarded with decreasing temperatures, c) among different cations (Cu 2+ , Zn 2+ , Cd 2+ , Ni 2+ , Co 2+ ) only Cu 2+ exhibits marked effects in the concentration range below 100 μᴍ and d) Cu 2+ decreases the normalized variable fluorescence, inhibits oxygen evolution and diminishes the affinity to atrazine binding without affecting the number of binding sites. The content of about four manganeses per functionally competent oxygen evolving complex is not changed by [Cu 2+ ] < 70 μᴍ. Based on these findings it is concluded: i) a temperature dependent equilibrium between an inactive (I) and active (A) state of Q A - reoxidation by Q B (Q B - ) is characterized by standard enthalpies ΔH° of 95 kJ mol -1 and 60 kJ mol -1 and standard entropies ΔS° of 370 kJ K -1 mol -1 and 240 kJ K -1 mol -1 in isolated thylakoids and PS II membrane fragments, respectively, ii) the activation energies of Q A - reoxidation by plastoquinone bound to the Q B site are about 30 kJ mol -1 (thylakoids) and 40 kJ mol -1 (PS II membrane fragments) in 220 K < T < 300 K, and iii) Cu 2+ causes at least a two-fold effect on PS II by modifying the atrazine binding affinity at lower concentrations ( ~ 5 μᴍ) and interference with the redox active tyrosine Y z at slightly higher concentration ( ~ 10 μᴍ) leading to blockage of oxygen evolution.

In recent papers the heterogeneous nature of photosystem (PS) II core phosphorylation has been revealed (Giardi et al., BBRC 176, 1298 -1305 (1991); Plant Physiol. 100, 1948 -1954 (1992)). In this paper the action of endogenous and exogenous phosphatases both on the distribution of phosphorylated PS II core populations and on herbicide-binding activity in photosystem II preparations from Spinacia oleracea L. has been investigated. The results indicate that these phosphatases modify the photosystem II core phosphorylation heterogeneity at a different level. Dark incubation causes a partial dephosphorylation of D 1 and D 2 proteins by endogenous phosphatase(s) and changes the relative distribution of phosphorylated photosystem II core populations, while the action of the alkaline phosphatase leads to extensive dephosphorylation and to the detachment of PsbH protein from the photosystem II core. Dephosphorylation by the two alternative methods results in a differential modification of herbicide-binding activity. It is suggested that photosystem II heterogeneity with respect to the herbicide action, observed in vivo, could be a consequence of PS II core phosphorylation heterogeneity.

Light-induced degradation of the D 1 protein of photosystem II (PS II) was determined by radioactive pulse-chase labelling experiments in intact leaves of Schefflera polybotrya. PS II photochemical efficiency was monitored by measuring chlorophyll fluorescence. A significant and consistent decline in the F v / F m ratio was taken to indicate photoinhibition. The formation and degradation of a modified form of the D 1 protein, D 1*, was different under photoinhibi-tory or non-photoinhibitory light conditions. At photoinhibitory irradiance greater amounts of D 1 * were formed relative to D 1, and the degradation of D 1* was slower when compared with non-photoinhibitory irradiance. The formation and degradation of D 1* were therefore shown to be at least partly light intensity dependent. Higher light intensities appeared to slow D 1* degradation, which suggests a modification in PS II turnover properties.

In this paper the current status of the so-called bicarbonate effect is presented. Several chemicals (such as formate, azide, nitrite and nitric oxide) are known to inhibit the two-electron gate of photosystem II (PS II). A remerkable slowing down of Q A - reoxidation and an increase in equilibrium [Q A - ] have been observed after the second or the subsequent, but not the first, flash when thylakoid membranes are treated with formate, etc. And, significantly, these effects are totally and uniquely reversed upon bicarbonate addition. The current hypothesis is that bicarbonate functions as a proton shuttle that stabilizes the binding niche of Q B - and stimulates platoquinol formation. This bicarbonate effect must involve both the D 1 and D 2 proteins since various herbicide-resistant D 1 mutants (e.g., D 1 -S264A , D 1 -L275F), as well as some D 2 mutants (e.g., D 2 -R251S, D 2 -R 233Q) have been found to be differentially sensitive to formate. The D 2-arginine (233, 251) effects are specific since D 2 -R 139H mutant and an­ other mutant in which an extra arginine was inserted, between F 223 and E 224 , behaves like the wild type. Data in the literature suggest that the bicarbonate binding must also involve Fe in the PS II Q A -Fe -Q B complex. In contrast, the Q A -Fe -Q B complex and the two-electron gate of both green and purple photosynthetic bacteria, including the M -E 234 G , Q and V mutants, are insensitive to bicarbonate-reversible inhibitors. We will also address the question of the nature of the active species involved and the possible role of bicarbonate in vivo.

We have investigated the electron transfer kinetics for reduction of plastoquinone by photo­ system II in six mutant strains of Chlamydomonas reinhardtii by following the decay of the high fluorescence state after flash activation, and compared the separate reactions of the two-electron gate with those of a wild type strain. By analysis of the electron transfer kinetics, and separate measurement of the equilibrium constant for stabilization of the bound semiquinone after one flash, we have been able to deconvolute the contributions of rate constants and equilibrium constants for plastoquinone binding and electron transfer to the overall process. Two mutations, S 264 A and A 251 V, led to a marked slowing of kinetics for reduction of plastoquinone to the bound semiquinone. In S 264 A , the second electron transfer was also slower, but was normal in A 251 V. In mutant G 256 D , the electron transfer kinetics were normal after the first flash, but slowed after the second. In mutants L 257 F , V 219 I, and F 255 Y , the electron transfer kinetics after both flashes were similar to those in wild type. We discuss the results in terms of a model which provides a description of the mechanism of the two-electron gate in terms of measured kinetic and equilibrium constants, and we give values for these parameters in all strains tested.

The psbL gene is a member of the psbEFLJ gene cluster in the cyanobacterium Synechocystis sp. PCC 6803 and higher plants. psbL, a 4.5 kDa protein encoded by this gene, is a component of the photosystem II complex. The amino acid sequence of this protein indicates that it has a single membrane-spanning a-helical domain. We have used a targeted mutagenesis technique to delete the coding region of the psbL gene in Synechocystis 6803. The resultant mutant strain T345 did not show any PSII-mediated oxygen evolution activity and, as a result, could not grow under photoautotrophic conditions. However, it had normal PSI activity. The chlorophyll to phycobilin ratio in the T345 cells was significantly lower than that in the wild type cells. Fluorescence emission spectra (77 K) of the mutant cells showed the absence of a 695 nm band that usually originates from the PSII complex. Binding assays with radioactive diuron demonstrated that the mutant cells did not have any herbicide binding activity. However, immunostaining experiments showed that both the D 1 (the herbicide binding protein) and the D 2 proteins of the PSII reaction center were present at > 25 % of their normal levels in the thylakoid membranes of the T345 mutant cells. Our data indicate that the PsbL protein is essential for the normal functioning of PSII.

Resistance to s-triazine-herbicides in weeds is the most widespread and extensively studied of all intraspecific herbicide-resistance. It is of interest that the resistant biotype appears in some limited genera such as Amaranthus spp. and Chenopodium spp. much more frequently than in many other significant weeds. We examined the response of cultured Amaranthus retroflexus cells to atrazine in comparison with those of several other plant species to understand what causes this differentially inter-specific response. Atrazine scarcely inhibited the cell growth of either atrazine-resistant and susceptible-Amaranthus cells. Tobacco cells, however, could not grow as cultured Amaranthus cells in high concentrations of atrazine even under heterotrophic culture conditions. Atrazine-resistant tobacco cells were also sensitive to high concentrations of atrazine. The inhibition of cell growth by this secondary effect of atrazine was also observed in cultured wheat and rice cells. Atrazine-sensitive Chenopodium cells are relatively more resistant to high concentrations of atrazine. The importance of potential tolerance to the secondary effects of atrazine is discussed with respect to the frequent occurrence of triazine- resistant biotypes in limited plant species.

Triazine-resistant and susceptible Chenopodium album plants were grown at low and at high light irradiances. At the lower light irradiance the dry matter production of the resistant and the susceptible plants were almost similar. At the higher irradiance the resistant biotype had a significantly lower production. Fluorescence studies showed that the photochemical yield and the photosystem II electron transport rate were lower in the resistant biotype. It could be demonstrated in intact leaves that the lower productivity of the resistant biotype is caused by a higher sensitivity to photoinhibition. However, when studying effects of photoinhibition on electron flow and photophosphorylation in isolated thylakoids of the two biotypes, no significant differences between resistant and susceptible plant materials were observed. It is suggest­ ed that the difference between resistant and susceptible biotypes connected with processes protective against photoinhibition in intact leaves, are lost during the isolation of thylakoids.

Since the first discovery of s-triazine-resistance (R ) in higher plants, the altered D -1 protein product of the psbA gene has been regarded as less photosynthetically efficient in those R biotypes of a species. Decreases in electron transport function in the chloroplast have been believed to be the cause of decreased carbon assimilation rates and plant productivity in many reports. W hat is less clear in the literature is whether this change in D -1 structure and electron transport function directly modifies whole-leaf photosynthesis and plant productivity or only indirectly influences these functions. The dynamic nature of these responses have led several to conclude that the primary effect of R is complex, involves more than one aspect of photosynthesis, and can be mitigated by other processes in the system. Electron transport limitations are only one possible regulatory point in the photosynthetic pathway leading from light-harvesting and the photolysis of water, through ribulose bisphosphate carboxylase/oxygenase, to sucrose biosynthesis and utilization. Herein we discuss this complex issue, arguing that D -1 function can’t be evaluated in isolation from the leaf, the organism, and possibly from the community response. Carbon assimilation in R and the susceptible wild type (S) is a function of several interacting factors. These include the pleiotropic effects resulting from the psbA mutation (the dynamic reorganization of the R chloroplast) interacting with other regulatory components of photosynthesis, microenvironmental conditions, and time (including ontogeny and time of day). Previous work in our laboratory indicated a consistant, differential, pattern of Chi a fluorescence, carbon assimilation, leaf temperature, and stomatal function between S and R Brassica napus over the course of a diurnal light period and with ontogeny, i.e. R is a chronomutant. Dekker and Sharkey have shown that the primary limitation to photosynthesis changes with changes in leaf temperature, and that electron transport limitations in R may be significant only at higher temperatures. The recognized R plants interact with the environment in a different way than does S. Under environmental conditions highly favorable to plant growth, S often has an advantage over R. Under certain less favorable conditions to plant growth, stressful conditions, R can be at an advantage over S. These conditions may have been cool (or hot), low light conditions interacting with other biochemical and diurnal plant factors early and late in the photoperiod, as well as more complex physiological conditions late in the plant’s development. It can be envisioned that there were environmental conditions in the absence of s-triazine-herbicides in which R had an adaptive advantage over the more numerous S individuals in a population of a species. Under certain conditions R might have exploited a photosynthetic niche under-utilized by S.

The mechanism of selective herbicidal action of sethoxydim was investigated by using cultured root tips of corn and pea. Sethoxydim inhibited the growth of susceptible corn root at concentrations of 3 nᴍ and above, but did not affect that of pea at 0.1 mᴍ. Meristematic cells in cultured roots were arrested at the G 1 and G 2 stages of the cell division cycle by sucrose starvation, and resumed growth and cell division (proliferation) upon sucrose addition. Corn root growth was not inhibited by sethoxydim even at 0.01 mᴍ when the roots were arrested by sucrose starvation. Corn roots that resumed growth upon sucrose provision were more sensitive to sethoxydim than those kept in constant growth condition. Better absorption of [ 14 C]sethoxydim into the meristematic region of corn roots was observed when cells were in the proliferative condition, which was not observed when cells were arrested by sucrose starvation. No stronger uptake of the herbicide was observed into pea meristems in either growth condition. In the cell cycle study, stronger absorption of [ 14 C]sethoxydim into the corn root meristem took place at a certain limited period prior to the S (DNA synthesis) stage. The physiological effects and the better absorption of sethoxydim clearly depended on cell cycle progression of the corn root meristem, while fatty acid synthesis, as well as its inhibition by sethoxydim, was neither associated with cell cycle progression nor with stronger absorption of the herbicide. The selective herbicidal action of sethoxydim may be caused by interference with selected stages (G 1 and/or the transition step between G 1 and S stages) of the cell division cycle, where affinity sites for the herbicide might be expressed.

In higher plants the biosynthetic machinery of de novo fatty acid biosynthesis, measured as [ 14 C]acetate incorporation into fatty acids, is predominantly located in plastids. A key enzyme in this pathway is the biotin-containing acetyl-CoA carboxylase (ACC , EC 6.4.1.2) which catalyzes the ATP-dependent carboxylation of acetyl-CoA to malonyl-CoA. The ACC from Poaceae is very efficiently blocked by two herbicide classes, the cyclohexane-1,3-diones (e.g. sethoxydim, cycloxydim) and the aryloxyphenoxy-propionic acids (e.g. diclofop, fluazifop). It is shown that within the Poaceae not only different species but also different varieties exist which exhibit an altered sensitivity and tolerance towards both herbicide classes, which points to a mutation of the target enzyme ACC. In purifying the ACC we extended our research to the possible presence of other biotin-containing plant enzymes. In protein preparations from maize, oat, barley, pea and lentil we were able to demonstrate the carboxylation of acetyl-CoA, propionyl-CoA and methylcrotonyl-CoA. The two herbicide classes not only block the ACC, but also the activity of the propionyl-CoA carboxylase (PCC ), whereas the methylcrotonyl- CoA carboxylase (MCC ), a distinct biotin-containing enzyme from mitochondria, is not affected. MCC may play a role in isoprenoid catabolism. Whether PCC is a separate plastid enzyme or only a side activity of ACC is under current investigation. The efficiency of the graminicides in sensitive Poaceae is then not only determined by the inhibition of ACC, malonyl-CoA and fatty acid biosynthesis, but also by the exclusion of the PCC-catalyzed metabolic pathways of the plant cell.

Twenty-eight 1-ethyl-and 1-propyl-5 -substituted imidazoles were synthesized an d their bleaching activity was evaluated by using the lettuce seedling test. 5-Phenyl-1-propylim idazole (1) caused distinct chlorosis, while none of the c ompounds with various alkenyl groups at the 5-position o the imidazole ring show ed this bleaching activity. Introduction of a bromo, chloro, fluoro, methyl or trifluoromethyl groupa t the 4-position on the benzene ring of com­pound 1 increased the activity in comparison with that of compound 1. 5-[4-(3-Chlorobenzyl-oxy)phenyl]-l-ethylimidazole (15) an d l-ethyl-5-[4-(3-methylbenzyloxy)phenyl]imidazole (21), analogs with a large substituent at the 4 -position on the benzene ring, also caused pronounced chlorosis in the lettuce seedlings. Both compounds 1 and 15 a t 30 ppm decreased total c arot­enoid content in the lettuce seedlings to less than 40% of that in the control, and the reduction of total carotenoid content correlated well with treatment dose.

Recent work on the molecular biology of carotenogenic genes and enzymes showed that there are two different and unrelated types of phytoene desaturase. One can be found in bacteria and fungi whereas the other is present in cyanobacteria, algae, and higher plants. Studies with herbicidal inhibitors showed that only the latter type of enzyme is affected by the bleaching herbicides norflurazon, fluridone, and flurtam one. I 50 values have been determined for inhibition of phytoene desaturase in either solubilized membranes of the cyanobacterium Synechococcus, membrane preparation of E. coli in which the corresponding gene from Synechococcus was expressed or purified and reactivated phytoene desaturase from this E. coli transformant. The values differ by two orders of magnitude. The importance of the lipid environment of the integral membrane protein phytoene desaturase for its interaction with herbicides has been discussed. With the purified phytoene desaturase it could also be shown that the pyrimidine derivative J852 which is an inhibitor of ζ-carotene desaturase also inhibits phytoene desaturase with an I 50 value which is about 2700 times higher than for inhibition of phytoene desaturation.

The bleaching herbicide flurtamone shows a concentration-dependent inhibition of phytoene desaturase activity in a cell-free assay. Structure-activity studies were carried out with 26 derivatives. For this purpose, I 50 values for inhibition of formation of colored carotenoids in cultures of Synechococcus and with some selected compounds in vitro inhibition data were measured. For meta-substituted 4-phenyl derivatives a quantitative structure-activity correlation was calculated. Regression analysis show ed a significant contribution of lipophilicity π and steric parameter B 2 of the substituents to inhibitory activity of these derivatives. In addition, the stericrequirement of 4-phenyl substituents was further revealed by the substitution pattern at different positions. In general, mono substituents at the meta position were most favorable and only the very small fluoro group was allowed as a second substituent. When the ligands at position 2 of the furanone heterocycle were modified, phenyl or para-fluorophenyl groups were most advantageous. Among the available compounds with different alkyl substitutions of the 5-amino group, the ethyl derivative show ed the highest inhibitory activity whereas the one with the branched isopropyl group w as quite inactive.

Protoporphyrinogen oxidase (Protox) inhibitors and other compounds which block or stimulate the porphyrin pathway can cause sufficient levels of porphyrins to accumulate in plant tissues for severe photo dynamic damage to occur. The gross symptomology for all of these por­phyrinogenic herbicides is similar. Porphyrin accumulation induced by three porphyrinogenic herbicides acifluorfen (AF), δ-aminolevulinic acid (ALA), and 2,2′-dipyridyl (DY) was determined by in vivo spectrophotometry and HPLC methods. The averaged in vivo difference spec­tra between untreated and AF-treated (30 μᴍ for 20 h in darkness) yellow cucumber cotyledon discs approximated the absorption spectra of protoporphyrin IX (Proto IX). There was also an enhanced peak near 503 nm. Treatment of cotyledon discs with ALA alone generated a difference spectrum of protochlorophyllide (PChlide) in combination with Mg -Proto IX or Mg-Proto IX monomethyl ester (Mg-Proto IX ME). With ALA and AF in combination , the PChlide and Mg-Proto IX portions of the difference spectrum were reduced and the Proto IX peak and peak near 503 nm were increased. DY treatment yielded a difference spectrum with peaks approximating those of Proto IX and Mg-Proto IX ME , along with a peak near 503 nm . The presence of all porphyrins detected by in vivo spectrophotometry except for the 503 nm peak was confirmed with HPLC . Proto IX monomethyl ester was found by HPLC to be espe­cially elevated in treatments with AF. The in vivo 503 nm peak and in vitro studies with Protox-containing barley etioplast preparations suggest that p rototetrahydroporphyrin IX (an oxida­tion state intermediate between protoporphyrinogen IX and Proto IX) may accumulate under some conditions. These data demonstrate that rapid in vivo spectrophotometric studies can provide much of the qualitative results of HPLC studies and can confirm that in vitro results correspond with the in vivo situation.

Protoporphyrinogen oxidase, the last enzyme of the common tetrapyrrole biosynthetic pathway, is inhibited by several peroxidizing compounds resulting in accumulation of photodynamic tetrapyrroles, mainly protoporphyrin IX. The inhibition characteristics of two chemi­cally unrelated compounds were studied using membrane bound protoporphyrinogen oxidase from corn etioplasts. As shown by Lineweaver-Burk-analysis, the inhibition of enzyme activity by the diphenyl ether oxyfluorfen and the cyclic imide MCI 15 are competitive with respect to the substrate. The competitive interaction of protoporphyrinogen and the two chemically un­related inhibitors indicate a relative specificity of the binding site. The reversibility of oxyfluorfen inhibition was evaluated by dilution experiments and was shown to be independent of the presence of DTT. The analysis of structure-activity-relationship on protoporphyrinogen oxidase inhibition was investigated with para-substituted derivatives of phenyl-3,4,5,6-tetrahydro-phthalimides. The results obtained by QSAR -calculation yielded a good correlation of the inhibitory activity determined by the lipophilicity of the para-substituent. These data point to one binding region of the inhibitors within a lipophilic environment associated with the active center of the enzyme.

The crystal structure of 3-(4-chloro-5-cyclopentyloxy-2-fluorophenyl)-5-isopropylideneoxazolidine- 2,4-dione (BW -4), chlorophthalim and oxyfluorfen were determined by X -ray analysis. The molecular size of these compounds was almost the same, but the angle between the oxazolidine and the phenyl ring (BW -4), the imide and phenyl ring (chlorophthalim ) and between the two phenyl rings (oxyfluorfen) differed. The phytotoxic activities, determined by growth inhibition of Scenedesmus cells, loss of chlorophyll, and peroxidative destruction of photosynthetic pigments, were different among these compounds. The angle and length of BW-3, BW-4 and oxyfluorfen showing best phytotoxic activity were found approximately 65 -95 ° and 11 - 13 Å, respectively. These preliminary findings indicate that both angle and length of the molecule have some bearing on peroxidative activity.

A group of highly active N -phenylimide photobleaching herbicides have been synthesized. These N -phenylimide herbicides as well as diphenyl ether herbicides induce protoporphyrin IX accumulation and inhibit protoporphyrinogen oxidase activity at extremely low concentrations in higher plants. The binding of a 14 C -labeled N -phenylimide herbicide S-23121 [N-[4-chloro- 2-fluoro-5-[(1-m ethyl-2-propynyl)oxy]phenyl]-3,4,5,6-tetrahydrophthalimide] to the solubilized plastid fractions of greening corn seedlings is competed by the diphenyl ether herbicide acifluorfen-ethyl, but not by diuron, an inhibitor of photosynthetic electron transport. These results indicate a similar mode of action for both N -phenylimide and diphenyl ether herbicides. In order to investigate the mechanism of photobleaching herbicides at the molecular level, a strain of Chlamydomonas reinhardtii RS-3 resistant to N -phenylimide S-23142 [N -(4-chloro- 2-fluoro-5-propargyloxyphenyl)-3,4,5,6-tetrahydrophthalimide] was isolated by mutagenesis with N -m ethyl-N′-nitro-N -nitrosoguanidine. The 90% inhibition concentration of N -phenylimide S-23142 for growth of RS-3 was 100 times higher than that for wild type. Maximum accumulation of protoporphyrin IX was reached at 0.03 μᴍ of S-23142 for the wild type and 3 μᴍ for RS-3. RS-3 was resistant to oxadiazon, oxyfluorfen and acifluorfen-ethyl which had been shown to have the same mechanism of action as N -phenylimide herbicides, but not to paraquat, diuron or fluridone. Genetic analysis of RS-3 strain showed that the resistance results from a dominant mutation ( rs-3) in the nuclear genome. The magnesium protoporphyrin IX synthesizing activity from 5-am inolevulinic acid in chloroplast fragments isolated from RS-3 was less sensitive to S-23142 than that from wild type (CC-407). Protoporphyrinogen oxidase activity in Percoll™ -purified chloroplasts from RS-3 was also less sensitive to S-23142 than that from wild type. These results indicate that the resistance of RS-3 is specific for photobleaching herbicides, and that the mutation is related to protoporphyrinogen oxidase, the primary site of the photobleaching herbicide action.

Common structural features were investigated for “light-dependent herbicides” (LDH s, also called peroxidizing or photobleaching herbicides). Quantum chemical calculations of 143 herbicidal compounds revealed that LUMO levels of LDH s were similar and strikingly low. Using the LUMO position as an anchor, presumably known structure-activity relationships could be explained. Overall molecular similarity between oxyfluorfen and chlorophthalim was examined by molecular field fitting. The result supported LUMO position correspondence.

Effects of S-23142 on protoporphyrin IX (Proto IX) biosynthesis in chloroplasts isolated from Spinacia oleracea L. were examined using reverse phase HPLC with fluorescence monitoring. The synthesis of Proto IX was inhibited to a level of 50% by 10 -9 ᴍ of S-23142 in this system. The effects of S-23142 was also tested in chloroplasts isolated from two types of photomixotrophic tobacco cells, wild type and S-23142 tolerant cells. The biosynthesis of both the wild type cells and YZI-1 S cells were inhibited at 50% by 10 -9 ᴍ and 10 -7 ᴍ of S-23142, respectively. It is, therefore evident that the mutation in the tolerant cell is associated with the Protox. To investigate the localization of Proto IX biosynthesis and the target site of S-23142, spinach chloroplasts were osmotically broken and separated into stroma and membrane (thylakoid and envelope) fractions. A very active Proto IX synthesis from ALA was found in the stromal fraction, while no activity of Proto IX synthesis was observed in the membrane fraction. These results suggest that most Proto IX synthetic activity and a target-site of S-23142 exist in the stromal fraction.

Several acrylic acid derivatives incorporating elements of sulfonylurea structure around a central vinylogous core were synthesized and found to be moderate inhibitors of the enzyme, acetohydroxyacid synthase (AHAS). Some com pounds showed post-emergence herbicidal activity against mustard. Biological activity was found to be markedly influenced by the nature of the 2-arylcarbamoyl group. When this group contained a phenyl ring the compounds were inactive. A 4,6-dim ethoxypyrimidin-2-ylcarbamoyl function resulted in the best activity but this activity was highly dependent on the nature of the 4,6-substituents on the pyrimidine ring, with 4,6-dim ethylpyrimidines showing very little activity. The structures of the most active compound, ethyl 3-(2-chlorophenyl)sulfonylamino-3-m ethylthio-2-[(4,6-dimethoxypyrimidin- 2-yl)carbam oyl]acrylate, and an inactive analogue, 4,6-dimethylpyrimidin-2-yl-3-(2-chlorophenyl) sulfonylamino-3-methylthio-2-cyanoacrylate, were determined by X -ray crystallography. The resulting structures showed several conformational differences which may play an important role in inhibitor binding to AHAS and the resultant herbicidal activity.

Nitronate compounds resembling the aci-carboxylate reaction intermediate in the reaction catalyzed by isopropylmalate isomerase were investigated as enzyme inhibitors and potential herbicides. The nitronic acids of 1-hydroxy-2-nitrocyclopentane-1-carboxylic acid, nitroisopropylmalate and of 1-hydroxy -2-nitrocyclohexane-1-carboxylic acid were all potent inhibitors of isopropylmalate isomerase. The nitroalkane/nitronate pK a values of these were 7.3, 9.5 and 11.1, respectively. The perturbed pK a values of the first and last compound were und erstandable on the basis of the opposing effects of 5- and 6-membered ring strain on the ionization of the nitro group. As the only compound significantly in the nitronate form at physiological pH , it was not surprising that 1-hydroxy-2-nitrocyclopentane-1-carboxylic acid was the best herbicide. Its specific mode of action was confirmed by the fact that 1-hydroxy-2-nitrocyclopentane- 1-carboxylic acid inhibited the growth of carrot cells in liquid culture and was specifically reversed by leucine.

Glufosinate (phosphinothricin) irreversibly blocks the glutamine synthetase which subsequently gives rise to an accumulation of ammonium and to a strong decrease in some amino acids, especially glutamine and glutamate. Under atmospheric conditions (400 ppm CO 2 , 21% O 2 ) glufosinate causes a rapid inhibition of photosynthesis, too. H ow ever, under non-photo respiratory conditions (1000 ppm CO 2 , 2% O 2 ) only a slight inhibition of photosynthesis occurs with glufosinate. Since under both conditions an accumulation of ammonium occurs, it is concluded that inhibition of photosynthesis is not induced by the higher concentrations of ammonium. The results rather suggest that the absence of amino donors in the glycolate pathway leads to a break-down of the transamination reaction of glyoxylate to glycine. This causes an inhibition of photorespiration and as a further consequence the inhibition of photosynthesis. There are two hypotheses for explaining this phenomenon. One of them supposes that the blockade in the glycolate pathway produces a lack of Calvin cycle intermediates which subsequently is the cause of the inhibition of photo synthesis. The other one suggests a direct inhibition of the ribulose-1,5-bisphosphate carboxylase by the accumulation of glyoxylate and P-glycolate. After treatment with different intermediates of the Calvin cycle and photorespiration to gether with glufosinate no decrease in the inhibition of photosynthesis could be measured. This suggests that the inhibition of photosynthesis is not induced by a depletion of intermediates of the Calvin cycle. Tests on the effect of glyoxylate and P-glycolate on ribulosebisphosphate carboxylase activity showed that in crude leaves extracts the enzyme activity can only be inhibited by high concentrations of these substances. However, in intact spinach chloroplasts the enzyme activity can be blocked by using much lower concentrations of glyoxylate. This may indicate that the ribulosebisphosphate carboxylase activase is affected by this metabolite and that this may be the reason for an inhibition of photosynthesis after treatment with glufosinate.

Paraquat(methylviologen), a widely used nonspecific herbicide, is ptoreduced in the primary photochemical reaction of photosystemI. Using two types of the photosystem I reaction center preparations, i.e. one with FeS A /FeS B and the other without, the immediate electron donor to methylviologen was determined to be FeS x (P 430 ) rather than FeS A /FeS B .

The soybean cultivars “ Kwangkyo” and “ Hood ” are differentially sensitive to the bipyridylium herbicide paraquat (1,1′-dimethyl-4 ,4′-bipyridiniumion). This was confirmed by visible injury observations, measurements of desiccation levels and chlorophyll content, and tracings of chlorophyll fluorescence induction of fully expanded first trifoliate leaves of these two cultivars after exposure to a wide range of paraquat concentrations. The margin of this intraspecific differential tolerance to paraquat was narrow an d the ratio of the paraquat concentrations causing 50% injury to the tolerant Kwangkyo and to the susceptible Hood soybean (approximate tolerance factor) was found to be 10. Paraquat at 1 μᴍ or higher inhibited rapidly the CO 2 fixation capacity of leaf mesophyll cells, isolated enzymatically from both cultivars. Thus, the tolerance of Kwangkyo soybean to paraquat does not appear to result from any differences at the site of paraquat action in chloroplast membranes. At early time periods (30 min to 2 h) after treatment with 100 μᴍ of paraquat, chlorophyll fluorescence induction was completely suppressed in first trifoliate leaves of Hood, but not in those of Kwangkyo soybean. At longer time periods (≧ 3 h), paraquat suppressed chlorophyll fluorescence induction similarly in leaves of both soybean cultivars. These results suggest that reduced mobility or a delayed release of paraquat in the mesophyll cells of Kwangkyo may be involved in the observed tolerance of this soybean cultivar to this herbicide.

The effect of p araquaton hydroxyl radical production , glutathione and ascorbate content, and glutathione reductase and superoxide dismutase activities was assayed in the tolerant “Kwangkyo” and the susceptible “ Hood ” cultivars of soybean [Glycine max (L.) Merr.]. Seedlings of both cultivars at the fully expanded first trifoliate leaf stage were treated with paraquat at 0, 1, 10, 100, 500, and 1000 μᴍ. The in vivo generation of hydroxyl radicals was estimated by measuring methanesulfonic acid (MSA ) produced in soybean leaves treated with dimethylsulfoxide (DMSO ) and paraquat. The levels of paraquat-induced production of hydroxyl radicals were similar in Kwangkyo and Hood soybean. Ascorbate levels in the two cultivars were comparable and were reduced by paraquat treatment. Total glutathione levels were similar in both cultivars, but Kwangkyo had more glutathione in the reduced form than Hood. Treatment with low concentrations of paraquat enhanced oxidized glutathione levels in Hood. At high concentrations, paraquat decreased the levels of total and reduced glutathione in both cultivars. The endogenous activities of glutathione reductase and superoxide dismutase were similar in Kwangkyo and Hood soybean. Treatment with selected concentrations of paraquat enhanced the activity of these enzymes in trifoliates of the tolerant Kwangkyo. Overall, the results of this study do not suggest a strong involvement of components of the ascorbate-glutathione cycle in the observed tolerance of Kwangkyo soybean to the herbicide paraquat.

Involvement of active oxygen species in the accumulation of early light-inducible protein (ELIP) in mature pea plants was investigated under light stress conditions. Here we provide evidence that hydroxyl radicals and singlet oxygen interfere with ELIP expression at both , transcriptional and posttranslational levels. Addition of free radical scavengers can alleviate the detrimental action of the radicals almost completely at the level of transcription but only a partial protection was observed at the level of proteins. The data suggest that the damage of ELIP occurs during its insertion into thylakoid membranes close to the free radical sources.

Five photosynthesis inhibiting herbicides were compared for their mobility in soil columns used as the penetration model. The vertical movement of these compounds in soil was analyzed by a newly developed technique in which herbicide distribution was determ ined by the intensity of chlorophyll fluorescence in Chlorella cells suspended in aqueous extracts of soil samples from various depths. With the aid of a fluorescence microplate scanner, measurement of chlorophyll fluorescence was performed with great efficiency. Photosynthesis inhibiting activity of tested herbicides was determined beforehand by the same method and served in calculating herbicide concentrations in sample extracts. Among tested compounds, hexazinone showed the greatest mobility and diuron the smallest, suggesting water solubility is one of the factors which govern the soil mobility of herbicides.

The low -temperature fluorescence excitation analysis of different protochlorophyllide (PChlide) forms has been extended to the UV part of the spectrum . A new band at about 360 nm was detected in excitation spectra of active PChlide forms bound to isolated protochlorophyllide holochrome. This band is very similar to the absorbance of NADPH in this region and its intensity depends on the redox state of the surrounding medium. On illumination at low temperature the intensity of the band decreases considerably with out any corresponding changes in the redox state of “ free” NADPH in the surrounding medium. A new intermediate state exhibiting a mixed excitation spectrum between PChlide and chlorophyllide (Chlide) was detected in the course of PChlide photoconversion.