Klaus Pfister

The mode of Action of Photosystem II-Specific Inhibitors in Herbicide-Resistant Weed Biotypes

Abstract This report reviews studies which provide evidence defining the mode of action and site of action of photosystem II (PS II) herbicides; the involvement of the secondary electron carrier on the reducing side of PS II (called B) is indicated as the target site for these compounds. These studies of the action of PS II-inhibitors were performed in chloroplasts of various weed species in order to define the mechanism which is responsible for herbicide tolerance at the level of chloroplast membranes in newly discovered triazine-resistant weed biotypes. Many species of triazine-resistant weed biotypes have been collected in North America and Europe. Where data is available, these plants have been found to share the following common features: a) they were discovered in areas where triazine herbicides had been used repeatedly, b) resistance to the triazines is extreme; it is not due to a minor shift in herbicidal response, c) no changes in herbicide uptake, translocation or metabolism - as compared to susceptible biotypes - can be detected, d) resistance is selective for only certain classes of photosynthetic herbicides, and, e) chloroplasts isolated from triazine-resistant weeds display high preferential resistance to the triazines in assays of photosystem II partial reactions. To focus on the mechanism which regulates preferential herbicide activity, we have characterized susceptible and resistant chloroplasts in the presence and absence of herbicides. Properties of the PS II complex of chloroplasts from several different triazine-resistant weed biotypes share the following traits: a) the herbicide binding site (as measured by direct binding of radiolabeled herbicides or by inhibition experiments) is modified such that the affinity for triazines is dramatically reduced. b) alterations in response to many PS II-herbicides occur such that the triazine-resistant chloroplasts are very strongly resistant to all symmetrical triazines, strongly resistant to assymmetrical triazinones, partially resistant to pyridazones and uracils, only slightly resistant to ureas or amides, and increasingly susceptible to nitrophenols, phenols and the herbicide bentazon (all as compared to susceptible chloroplasts), c) there is a change in the reaction kinetics of the electron transport step between the primary and secondary electron acceptors (referred to as Q and B ), and d) in two examples, specific small changes in a membranepolypeptide can be detected in the resistant thylakoids. We suggest that certain amino acids or segments of the apoprotein of B (the bound quinone which acts as the secondary electron carrier) are modified or deleted in these chloroplasts. Such a polypeptide change could affect both the redox poising of the Q-/B reaction pair, and the specific binding of herbicides.

Evidence for a close spatial location of the binding sites for CO2 and for Photosystem II inhibitors

1. CO2-depletion of thylakoid membranes results in a decrease of binding affinity of the Photosystem II (PS II) inhibitor atrazine. The inhibitory efficiency of atrazine, expressed as I50-concentration (50% inhibition) of 2,6-dichlorophenolindophenol reduction, is the same in CO2-depleted as well as in control thylakoids. This shows that CO2-depletion results in a complete inactivation of a part of the total number of electron transport chains. 2. A major site of action of CO2, which had previously been located between the two electron acceptor quinone molecule B (or R) and Photosystem II inhibitor atrazine as suggested by the following observations: (a) CO2-depletion results in a shift of the binding constant (kappa b) of [14C]atrazine to thylakoid membranes indicating a decreased affinity of atrazine to membrane; (b) trypsin treatment, which is known to modify the Photosystem II complex at the level of B, strongly diminishes CO2 stimulation of electron transport reactions in CO2-depleted membranes; and (c) thylakoids from atrazine-resistant plants, which contain a Photosystem II complex modified at the inhibitor binding site, show an altered CO2-stimulation of electron flow. 3. CO2-depletion does not produce structural changes in enzyme complexes involved in Photosystem II function of thylakoid membranes, as shown by freeze-fracture studies using electron microscopy.

Conserved Kinetics at the Reducing Side of Reaction-Center II in Photosynthetic Organisms; Changed Kinetics in Triazine-Resistant Weeds

The decay of chlorophyll variable fluorescence after a “single turnover” flash is generally assumed to represent the reoxidation of the reduced quinone Qa. We have observed that the kinetics of this decay are very similar in a wide variety of species. Comparing 28 different species, we found an average half decay time of 314 ± 46 μsec. No systematic correlations were found between the decay rate and biochemical or physiological specializations such as C2, C4 or CAM. This indicates that structural as well as functional factors controlling photosystem II electron transfer between Qa and Qb are highly conserved. Apparently, the freedom for natural structural variations in this region is very limited. Triazine resistant plants, characterized by an altered amino acid sequence of the D1 protein, have clearly decreased rates of Qa/Qb electron transfer. We found an average half decay time of 946 ± 100 (isec (5 species). However, this three-fold decrease is much less than previously reported. Therefore, if alterations of photosystem II electron transfer efficiency contributes to an often reported reduction of “ecological fitness”, this contribution is smaller than was hitherto assumed.

High-and Low-Affinity Binding of Photosystem II Herbicides to Isolated Thylakoid Membranes and Intact Algal Cells

Abstract High- and low-affinity binding of photosystem II herbicides to isolated thylakoids of Spinacia oleracea and to intact cells of the unicellular green alga Ankistrodesmus braunii were investigated. Complete mutual displacement of bound diuron-type herbicides (e.g. diuron, atrazine, terbutryn) by either diuron- or phenol-type herbicides (e.g. ioxynil, dinoseb) in thylakoids as well as in intact algal cells was found for herbicide concentrations (< 4 nmol bound herbicide/mg Chl) which gave almost saturated high-affinity binding. This demonstrates a high degree of specific binding of these herbicides towards their receptor sites even in intact algal cells. In contrast, phenol-type herbicides are largely unspecifically bound in algal cells. The mechanism of binding of all photosystem II herbicides at the high-affinity (specific) binding site was found to be competitive. Within the group of diuron-type and of phenol-type herbicides as well as between these two groups, graphical and quantitative analysis of the Lineweaver- Burk plot and of the Dixon plot indicated competitive binding. From this a common binding site for both types of herbicides was concluded. The involvement of two different herbicide binding- proteins is discussed. Low-affinity (unspecific) binding was found to be irreversible in contrast to the easily reversible high-affinity binding. Irreversibility was indicated by a lack of displacement. It is proposed that low-affinity binding represents either a partitioning of the herbicides into the lipophilic parts of the membranes or an attachment to distinct receptor sites. Unspecifically bound herbicides might be responsible for several high concentration effects of the photosystem II herbicides, which are described in the literature. Evidences for the possible existence of a second binding site of these herbicides are presented.

Trypsin-Mediated Removal of Herbicide Binding Sites within the Photosystem II Complex

Abstract Trypsin treatment of isolated chloroplast thylakoids resulted in a step-wise modification of surface exposed membrane polypeptides. Early effects of the protease action resulted in a decrease in inhibitory activity of atrazine, diuron, pyrazon, and bromacil, but an initial increase in the activity of bromnitrothymol and dinoseb. Direct measurements of atrazine binding demonstrated that decreased inhibitory activity corresponded to a decreased binding affinity in the treated membranes. Longer term effects of trypsin caused removal of atrazine binding sites and a concomitant block of electron transport chains. The data are consistent with a concept that the traizine receptor protein is a component of the electron transport chain which is successively degraded in two or more steps by protease attack. Polyacrylamide gel electrophoresis of trypsin-treated membranes and sub-membrane fragments derived from these membranes revealed that several polypeptides are membrane surface exposed. The involvement of a 32000 dalton polypeptide in creating the atrazine binding site is discussed.

Comparative Binding of Photosystem II — Herbicides to Isolated Thylakoid Membranes and Intact Green Algae

Abstract The binding of the photosystem II herbicides diuron (DCMU), atrazine (s-triazine), ioxynil and dinoseb (substituted phenols) to isolated spinach thylakoids was saturated in less than 2 min in the dark. In intact cells of the green alga Ankistrodesmus b. it took 10 to 20 min to reach the binding equilibrium. Binding affinity of diuron, atrazine, dinoseb, measured as equilibrium binding constants, was found to be comparable in isolated thylakoids and intact algal cells. For ioxynil, reduced binding affinity was observed in algae. The concentration of binding sites in thylakoids and intact cells was determined to be 300-500 chl/inhibitor binding site, suggesting a 1:1 stoichiometry between bound herbicide and electron transport chains. In intact cells only the phenol herbicides ioxynil and dinoseb showed increased concentrations of binding sites. Strong correlation of herbicide binding and inhibition of electron transport was found for diuron in isolated thylakoids and intact cells. In thylakoids this is valid also for atrazine and dinoseb. For ioxynil a difference between the amount of binding and inhibition was found. This correlation of herbicide binding and inhibition proves that binding specifically occurs at the inhibition site at photosystem H. In addition to the specific binding, for all four herbicides studied, (except for ioxynil in thylakoids) unspecific binding was observed in thylakoids as well as in algae, which was not related to inhibition.

Comparison of Diuron-and Phenol-Type Inhibitors: Additional Inhibitory Action at the Photosystem II Donor Site

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 Qв 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.

The Inhibition of Photosynthetic Light Reactions by Halogenated Naphthoquinones

Abstract Halogenated naphthoquinones act as inhibitors of photosynthetic electron flow. I50 concentra tion for inhibition of methylviologen reduction were found to range between 2 × 10-5 m to 2 × 10-6 M. Comparing their effects on several partial reactions of electron flow, the inhibition site of the naphthoquinones was found to be at the reducing site of PS II. Studies of fluorescence transients in presence of halogenated naphthoquinones give further evidence for a site action similar to that of diuron and different to that of DBMIB. All naphthoquinones act as quenchers of chlorophyll fluorescence with pure chlorophyll a, and with much higher efficiency in green algae and chloroplasts. It is concluded, that the halogenated naphthoquinones act similar to PS II-inhibitors like diuron, but do not share a common binding site at the PS II-complex. Implications of a possible involvement of phylloquinone K 1 in photosynthetic electron transport are discussed. The synthesis of 2-chloro-as well as 2-bromo-3-isopropyl-1,4-naphthoquinone is described.