Pesticide Biochemistry and Physiology 101 (2011) 182–190

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Pesticide Biochemistry and Physiology

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Plant cell membrane as a marker for light-dependent and light-independent mechanisms of action ⇑ Franck E. Dayan , Susan B. Watson

USDA-ARS, Natural Products Utilization Research Unit, P.O. Box 8048, University, MS 38677, USA article info abstract

Article history: Plant cells possess a number of membrane bound organelles that play important roles in compartmen- Received 18 June 2011 talizing a large number of biochemical pathways and physiological functions that have potentially harm- Accepted 7 September 2011 ful intermediates or by-products. The plasma membrane is particularly important as it holds the entire Available online 22 September 2011 cellular structure whole and is at the interface between the cell and its environment. Consequently, breaches in the integrity of the lipid bilayer, often via reactive oxygen species (ROS)-induced stress mem- Keywords: brane peroxidation, result in uncontrolled electrolyte leakage and in cell death. A simple 3-step bioassay Plasma membrane was developed to identify compounds that cause electrolyte leakage and to differentiate light-dependent Mode of action mechanisms of action from those that work in darkness. representative of all known modes of Herbicide Mechanism of action action (as well as several natural phytotoxins) were selected to survey their effects on membrane integ- Light-dependent rity of cucumber cotyledon discs. The most active compounds were those that are known to generate ROS Light-independent such as the electron diverters and uncouplers ( and dinoterb) and those that either were photo- Electrolyte leakage dynamic (cercosporin) or caused the accumulation of photodynamic products (acifluorfen-methyl and Conductivity ). Other active compounds targeted lipids (diclofop-methyl, triclosan and pelargonic acid) Peroxidation or formed pores in the plasma membrane (syringomycin). Herbicides that inhibit amino acid, protein, nucleotide, cell wall or microtubule synthesis did not have any effect. Therefore, it was concluded that the plant plasma membrane is a good biomarker to help identify certain herbicide modes of action and their dependence on light for bioactivity. Published by Elsevier Inc.

1. Introduction photosynthesis. The high level of physiological activity occurring within plastids is reflected by the presence of at least 700 different Plant cells, as do all eukaryotic cells, have a plasma membrane proteins. Similarly, mitochondria play a central role in eukaryotic enclosing the cytoplasm and a number of membrane-bound sub- cells by providing ATP by the process of oxidative phosphorylation. cellular organelles where vital biochemical and physiological func- Mitochondria are also involved in many other cellular functions tions are compartmentalized. In addition to providing a physical including numerous catabolic or anabolic reactions and apoptotic barrier between the cell and its environment, the plasma mem- cell death [2]. brane also contains a large number of proteins that perform impor- The nuclear genetic material is compartmentalized within a tant functions (e.g., regulation of ion and metabolite transport and double-membrane envelope punctuated by pores formed by supra- cell wall biosynthesis) and participate in responses to biotic and molecular protein structures (nuclear pores complex) [3]. The nu- abiotic stresses. cleus stores genes on chromosomes that encode proteins and Two of the organelles, the plastids and mitochondria, have fairly regulatory factors, as well as participating in many signaling re- complex membrane systems consisting of outer and inner enve- sponses and cellular activities. lopes that provide a spatial separation from the rest of the cellular Most of the volume of the plant cell is occupied by the tonoplast physiological functions and enclose many important biochemical enclosed vacuole. Plants store various secondary metabolites and pathways. The two membrane system surrounding plastids is the potentially toxic by-products that would otherwise be harmful to location of important processes such as the synthesis of glyceroli- the cells [4]. The tonoplast holds numerous proteins (i.e., pumps, pids, pigments (chlorophylls, carotenoids), and prenylquinones carriers and ion channels) that support the carry-out the transfer (plastoquinone and a-tocopherol) [1]. Plastids also enclose of these compounds in the vacuole. thylakoid membranes organized to conduct the light reaction of Higher plants also possess several different sorts of peroxi- somes (i.e., glyoxysomes, leaf peroxisomes, and unspecialized ⇑ Corresponding author. Fax: +1 662 915 1035. peroxisomes). These organelles contain enzymes of the fatty-acid E-mail addresses: [email protected], [email protected] (F.E. Dayan). b-oxidation cycle, the glyoxylate cycle, the photorespiration

0048-3575/$ - see front matter Published by Elsevier Inc. doi:10.1016/j.pestbp.2011.09.004 F.E. Dayan, S.B. Watson / Pesticide Biochemistry and Physiology 101 (2011) 182–190 183

0 0 0 0 pathway and the H2O2-scavenging pathway [5]. Other endomem- 6,11-dione; AAL-toxin A; Hypericin, 4,5,7,4 ,5 ,7 -hexahydroxy-2,2 - branes present within plant cells include the endoplasmic reticu- dimethylnaphthodianthrone; Syringomycin; Acifluorfen-methyl, 5- lum and golgi body which are central to the synthesis, sorting, [2-chloro-4-(trifluoromethyl)phenoxy]-2-nitro-benzoic acid meth storage and packaging of protein and lipid reserves. In addition yl ester; Norflurazone, 4-chloro-5-(methylamino)-2-[3-(trifluoro- to the plasma membrane and organelles, lipids also protect plants methyl)phenyl]-3(2H)-pyridazinone; , 2-amino-4-(hydr from dehydration and biotic attacks by sealing the aerial plant or- oxymethylphosphinyl)butanoic acid were purchased from Sigma– gans within a layer of waxes deposited over the epidermal cell Aldrich (St. Louis, MO 63103). walls [6,7]. Fluridone, 1-methyl-3-phenyl-5-[3-(trifluoromethyl)phenyl]- In view of the complex organization and important functions of 4(1H)-pyridinone was a gift from SePRO Inc. (Carmel, IN 46032); plasma and endo-membranes, any xenobiotic that destabilizes the Cinmethylin, (1R,2S,4S)-rel-1-methyl-4-(1-methylethyl)-2-[(2-met integrity of lipid bilayers, either directly or indirectly, has cata- hylphenyl)methoxy]-7-oxabicyclo[2.2.1]heptanes was a gift from strophic consequences leading to cellular death. Biological or Dupont de Nemours (Newark, DE 19711); Dehydrozaluzanin C, chemical processes frequently generate reactive oxygen species (3aS,6aR,9aR,9bS)-octahydro-3,6,9-tris(methylene)-azuleno[4,5-b] (ROS) as by-products of normal cellular metabolism, but cells are furan-2,8(3H,4H)-dione was kindly provided by Dr. J.C.G. Galindo equipped with antioxidants and ROS-scavenging enzyme cycles (University of Cadiz, Spain). that, under normal circumstances, quench their potentially harm- ful effects. However, biotic and abiotic stresses can induce high 2.2. Electrolyte leakage levels of ROS that can overwhelm the natural plant protective mechanisms, often resulting in lipid peroxidation of plant mem- Cucumber seedlings (Cucumis sativus (L.) var. straight eight) branes [8]. While the composition of membranes modulates their were grown in a growth chamber with a 16/8 light/dark cycle for sensitivity to ROS [9], all membranes are susceptible to peroxida- 10 days. Twenty-five 4-mm cotyledon discs were placed on a 2% tion which makes them important biomarkers for tissue damage sucrose/1 mM 2-(N-morpholino)ethanesulfonic acid buffer (MES, [10,11]. pH 6.5) containing 100 lM of each of the compounds tested [12] The purpose of this survey is to use a simple three-step assay to in 60 15 mm Petri plates. Each plate contained 5 mL of buffer. test selected herbicides representative of all the known herbicide Control tissues were exposed to the same amount of acetone as mechanisms of action and determine their effect on membrane treated tissues but without the test compounds. The final concen- integrity. A number of natural phytotoxins are also included in this tration of acetone in the dishes was 1% (v/v). Plates were incubated survey. Any activity detected on membrane stability is discussed in in darkness for 16 h prior to exposure to high light intensity the context of the compounds respective mechanisms of action and (1000 lmol m2 s1) photosynthetically active radiation (PAR) in their potential requirement for light. an incubator (Model E-30-B, Percival Scientific, Boone, IA 50036). Measurements were made using an electrical conductivity meter (Model 1056, Amber Science, Eugene, OR 97402) equipped with a 2. Materials and methods model 858 Conductivity Macro Flow cell at the beginning of the dark incubation period, another measurement was made after 2.1. Chemicals 16 h (overnight), at which time the samples were placed in the light and a final measurement was made after 8 h of light exposure. Diclofop-methyl, 2-[4-(2,4-dichlorophenoxy)phenoxy]-propaic Each experiment consisted of three replicates. Maximum conduc- acid methyl ester; , 2-chloro-N-(2,6-diethylphenyl)-N-(me tivity was measured by boiling three samples of each treatment thoxymethyl)-acetamide; Sulfentrazone, N-[2,4-dichloro-5-[4-(difl for 20 min. uoromethyl)-4,5-dihydro-3-methyl-5-oxo-1H-1,2,4-triazol-1-yl]- phenyl]-methanesulfonamide; , 2-[(2-chlorophe nyl)me thyl]-4,4-dimethyl-3-isoxazolidinone; , 6-chloro-N2-ethyl- 3. Results and discussion N4-(1-methylethyl)-1,3,5-triazine-2,4-diamine; , 1H-2,1, 3-benzothiadiazin-4(3H)-one, 3-(1-methylethyl)-2,2-dioxide; Para Herbicides have unique affinities for their respective molecular quat, 1,10-dimethyl-4,40-bipyridinium; Dinoterb, 2-(1,1-dimethyl- target site within important plant biochemical pathways and/or ethyl)-4,6-dinitro-phenol; , 2-[4,5-dihydro-4-methyl-4- physiological processes [13,14]. Most often, these stresses eventu- (1-methylethyl)-5-oxo-1H-imidazol-2-yl]-3-pyridinecarboxylic ally generate ROS that can lead to membrane peroxidation result- acid; Metsulfuron, 2-[[[[(4-methoxy-6-methyl-1,3,5-triazin-2-yl) ing in the uncontrolled release of the cell’s electrolytes. This can be amino]carbonyl]amino]sulfonyl]-benzoic acid; , N-(pho monitored by measuring the electrical conductivity of a medium sphonomethyl)glycine; , N-[(4-aminophenyl)sulfonyl]-car- on which plant samples are exposed to various inhibitors [12,15]. bamic acid methyl ester; , 7-oxabicyclo[2.2.1]heptane- Time-course studies monitoring the conductivity of a bathing med- 2,3-dicarboxylic acid; 2,4-D, 2-(2,4-dichlorophenoxy)-acetic acid; ium during an initial period of dark incubation and a subsequent , 3,7-dichloro-8-quinolinecarboxylic acid were pur- period of light exposure can discern compounds that have light- chased from ChemService (West Chester, PA 19381). dependent mechanisms of action from those that do not Carbetamide, (2R)-N-ethyl-2-[[(phenylamino)carbonyl]oxyl]pro (Fig. 1A). A simplified three-step assay was developed to provide panamide; EPTC, N,N-dipropyl-carbamothioic acid S-ethyl ester; a rapid on–off determination of membrane destabilization and Isoxaflutole, (5-cyclopropyl-4-isoxazolyl)[2-(methylsulfonyl)-4- the requirement for light in the mechanism of action (Fig. 1B). (trifluoromethyl)phenyl]-methanone; Triclosan, 5-chloro-2-(2,4- Since plasma membrane and subcellular membranes are essen- dichlorophenoxy)-phenol; Pelargonic acid, nonanoic acid; Diuron, tial for compartmentalizing and supporting cellular physiological N0-(3,4-dichlorophenyl)-N,N-dimethyl-; , N-(1- processes, a loss of membrane stability negatively affects biochem- ethylpropyl)-3,4-dimethyl-2,6-dinitrobenzenamine; Oryzalin, 4- ical functions and cell viability. Therefore, membrane integrity (dipropylamino)-3,5-dinitro-benzenesulfonamide; Dichlobenil, 2, should be a good marker to monitor biotic and abiotic stresses 6-dichlorobenzonitrile; Isoxaben, N-[3-(1-ethyl-1-methylpropyl)- on plants [16], including stresses induced by herbicides. 5-isoxazolyl]-2,6-dimethoxy-benzamide; MSMA, monosodi um Herbicide representatives of all the known modes of action, as methylarsonate; Cercosporin, (13bR)-5,12-dihydroxy-8,9-bis[(2R)- well as selected phytotoxins were tested on cucumber cotyledons 2-hydroxypropyl]-7,10-dimethoxy-perylo[1,12-def]-1,3-dioxepin- discs using this simple three-step assay. 184 F.E. Dayan, S.B. Watson / Pesticide Biochemistry and Physiology 101 (2011) 182–190

ABTable 1 List of compounds tested targeting electron transport. Start 0.6 Dark Light Dark WSSA classification Chemical Light Photosystem II inhibitors 0.5 C1 Atrazine C2 Bentazon C3 Diuron 0.4 Photosystem I inhibitor 0.3 D Paraquat Oxidative phosphorylation uncouplers 0.2 M Dinoterb Z Monosodium methylarsonate Conductivity (mohm) 0.1

0.0 Start 0 8 16 24 32 40 AB Dark Light Time (h) 0.6 ) Fig. 1. (A) Time course illustrating the light-dependent and light-independent lost of membrane integrity. Twenty-five leaf or cotyledon discs of young green tissues mohm Δ are floating on 5 mL of buffer (5 mM MES with 1% sucrose), with or without test 0.4 compounds in 6 well plates. The discs are incubated in the dark overnight (16 h) and then exposed to high light intensity for another 8 h (24 h after start of experiment). Arrows represents the three time measurements used in this survey to differentiate compounds that cause light-dependent loss of membrane integrity 0.2 from those that cause light-independent effects. s = Solvent control; h = light- Conductivity ( Conductivity dependent electrolyte leakage; 5 = light-independent electrolyte leakage. (B) Conductivity measured in the bathing medium from discs treated with compound A (light-independent mechanism of action) or compound B (light-dependent 0.0 mechanism of action). The dotted line represents maximum change in conductivity (obtained after boiling the discs). Atrazine Bentazon Diuron Paraquat Dinoterb MSMA

Fig. 2. Electrolyte leakage induced by herbicides targeting electron transport. Data 3.1. Herbicides targeting electron transport represents means of three replications with standard deviation. The dotted line represents maximum leakage obtained by boiling the cotyledon discs. Most physiological electron transports involve potentially reac- tive and harmful intermediates that can generate ROS. For this reason, these processes are universally confined to the microenvi- inhibitors. Bipyridilium herbicides are dication molecules that be- ronments provided by lipid bilayers. The light reaction of photo- come highly reactive free radicals upon acceptance of electrons synthesis is compartmentalized within the thylakoid membranes from PSI. These unstable free radicals undergo autoxidation back of the chloroplasts and catalyzes the light driven splitting of water, to the parent ion. The subsequent generation of ROS from the bipy- which releases O2 and provides the electrons required to assimilate ridilium cycle overwhelms the plant’s natural quenching mecha- CO2 into chemical energy. nisms [17]. The rapid cycling between the native herbicide ion and its reactive radical, and the high flow rate of electrons in PSI, 3.1.1. Inhibition of photosystem II accounts for the rapid desiccation of foliar tissues treated with Photosynthesis uses light energy to photoenergize electrons such inhibitors [17]. Accordingly, paraquat (Table 1) caused a rapid and inevitably generates highly reactive intermediates that can light-dependent loss of membrane integrity (Fig. 2). cause oxidative damage to the photosynthetic apparatus. These intermediates are normally quenched by several antioxidative sys- 3.1.3. Uncouplers tems [8]. However, herbicides can overwhelm these protective The herbicide dinoterb (Table 1) is a synthetic phenol that is no mechanisms by interrupting the electron flow by interacting with longer used. Dinoterb was one of the most active herbicides tested the plastoquinone binding site on photosystem II (PSII). Interest- in inducing electrolyte leakage. The effect was most pronounced in ingly, none of the PSII inhibitors tested (e.g., atrazine, diuron and the presence of light (Fig. 2), though the onset of toxicity occurred bentazon, Table 1) caused any notable electrolyte leakage (Fig. 2), during the period of dark incubation. The strong effect of dinoterb though diuron has been reported to induce small levels of leakage may be due to the fact that it has a multifaceted mechanism of ac- in Lemna [12]. This may be due to the fact that Lemna is generally tion involving the inhibition of photosynthesis, uncoupling oxida- very sensitive to herbicides and the assay was terminated after 8 h tive phosphorylations and interfering with respiratory oxygen of light exposure. consumption [18]. Monosodium methanearsonate (MSMA, Table 1) is an herbicide 3.1.2. Diversion of electron from photosystem I that has an unknown mechanism of action, but the rapid dessica- Under normal circumstances, energized electrons are trans- tion of the foliage of plants treated with this herbicide suggests ferred from PSII to plastocyanin via plastoquinone and ultimately that it causes cell membrane destruction [19]. Early studies on arrive at the four redox factors in the core protein complex of pho- MSMA suggested that it may act as an uncoupler of mitochondrial tosystem I (PSI). Unlike PSII inhibitors that block the electron oxidative phopshorylation [20]. The mode of action of MSMA may transfer to the natural electron acceptor in the photosynthetic also involve inhibition of photosynthesis and respiration [21] as electron transport chain, bipyridylium herbicides are themselves well as forming complexes with sulfhydryl-containing enzymes electron acceptors on PSI. This gives them a more intimate partic- [22]. MSMA had very little effect on electrolyte conductivity when ipation in the development of phytotoxic symptoms than PSII tested at the standard 100 lM concentration of this assay (Fig. 2). F.E. Dayan, S.B. Watson / Pesticide Biochemistry and Physiology 101 (2011) 182–190 185

This concentration is too low to exert any effect since MSMA is a Start high rate use herbicide whose foliar application requires up to 50 Dark times higher concentrations to achieve satisfactory weed control 0.6 Light [20]. Our data is consistent with a previous study comparing the ROS-dependent electrolyte leakage induced by acifluorfen, para- quat and MSMA in cocklebur and cotton [23]. In that study, both 0.4 acifluorfen and paraquat caused electrolyte leakage at that concen- tration, whereas MSMA was not active. A ten-fold higher concen- tration of MSMA did eventually lead to a modest level of leakage after 48 h of incubation. 0.2

3.2. Herbicides targeting pigment biosynthesis 0.0

3.2.1. Carotenoid synthesis FluridoneNorflurazone Sulcotrione Clomazone Sulfentrazone Carotenoids play a number of important roles in plants, the -methyl most critical one is to protect the photosynthetic apparatus from Fig. 3. Electrolyte leakage induced by herbicides targeting pigment synthesis. Data photodegradation under high light intensity by quenching the ex- represents means of three replications with standard deviation. The dotted line cess energy released. A successful mechanism of herbicide action is represents maximum leakage obtained by boiling the cotyledon discs. inhibition of the carotenoid biosynthesis pathway, decreasing the concentration of coloured carotenoid and causing the photody- 3.2.1.3. Inhibition of the non-mevalonate pathway. The importance namic destruction of chlorophyll molecules, resulting in a whiten- of carotenoids in plant survival is evident in the fact that it is the ing (photobleaching) of the green tissues [24]. target site for an additional herbicide, clomazone, with an entirely different mechanism of action. This herbicide inhibits the methyl- 3.2.1.1. Inhibition of phytoene desaturase (PDS). The first known tar- erythritol phosphate (MEP) isoprenoid pathway [29] which re- get site in carotenoid biosynthesis is the enzyme PDS which cata- duces the levels of plastid-synthesized isoprenoids (e.g., carote- lyzes the rate-limiting conversion of phytoene into phytofluene noids and phytol) while the production of cytosol-synthesized [25]. Since this herbicide target site does not affect the levels of secondary terpenoids is not affected. Clomazone is the only com- preexisting pools of carotenoids, none of the PDS inhibitors tested mercial herbicide to affect the MEP pathway, although it does so (fluridone and norflurazon, Table 2) induced loss of membrane indirectly. Clomazone (Table 2), a proherbicide, is converted into integrity in the assay used in this paper (Fig. 3). However, a similar the active metabolite 5-ketoclomazone in planta. This metabolite concentration of norflurazon elicited some electrolyte leakage in a is a potent inhibitor of the first step in the plastid-specific, MEP ter- previous study [12]. penoid pathway, 1-deoxy-D-xylulose-5-phosphate synthase [30,31]. Clomazone caused a small amount of electrolyte leakage (Fig. 3). While inhibition of carotenoid synthesis may not be the 3.2.1.2. Inhibition of p-hydroxyphenylpyruvate dioxygenase cause of leakage, it is possible that the effect of clomazone on phy- (HPPD). Carotenoid synthesis can also be inhibited indirectly by tyl biosynthesis may be involved. targeting HPPD. HPPD catalyzes the formation of homogentisic acid, which is a key precursor of eight different tocochromanols 3.2.2. Chlorophyll synthesis (tocopherols and tocotrienols) and prenyl quinones. The latter pre- 3.2.2.1. Inhibition of protoporphyrinogen oxidase (PPO). The primary nylquinone is a required cofactor for PDS [26]. Therefore, inhibition herbicide target in chlorophyll biosynthesis is PPO. This enzyme of HPPD indirectly reduces PDS activity by reducing the pool of catalyzes the last step heme and chlorophyll synthesis have in available plastoquinone [27,28]. In plants, the enzyme is the target common, and inhibitors of this enzyme cause rapid light-depen- site of b-triketone herbicides (e.g., sulcotrione and isoxaflutole, Ta- dent desiccation of foliage. The symptoms observed on the foliage ble 2). While isoxaflutole did not cause any leakage (data not of the treated plants include leaf cupping, crinkling, bronzing, and shown), the b-triketone herbicide sulcotrione caused a small in- necrosis due to loss of membrane integrity [32]. The peroxidative crease in conductivity, especially in the presence of light (Fig. 3). damage to the plasma membrane is due to the unregulated cyto- The cause of this small disturbance of membrane integrity is not solic accumulation of the highly photodynamic protoporphyrin IX known. intermediate, which is responsible for the herbicidal action of these inhibitors [33]. Differential susceptibility to PPO inhibitors is due in large part to differences in protoporphyrin IX accumula- Table 2 tion [34] and antioxidative systems [35]. Additionally, some plants List of herbicides tested targeting pigment synthesis. may be protected from such inhibitors by rapidly degrading the WSSA classification Chemical accumulating protoporphyrin IX [36]. Consistent with their mode Phytoene desaturase inhibitors of action, both acifluorfen-methyl and sulfentrazone (Table 2) F1 Fluridone caused dramatic loss of membrane integrity upon exposure of F1 Norflurazone the samples to light (Fig. 3). p-Hydroxyphenylpyruvate dioxygenase inhibitors F2 Isoxaflutole 3.3. Herbicides targeting amino acid, protein or nucleotide F2 Sulcotrione biosynthesis 1-Deoxy-D-xylulose phosphate synthase inhibitor F3 Clomazone 3.3.1. Amino acid synthesis Protoporphyrinogen oxidase inhibitors 3.3.1.1. Inhibition of acetolactate synthase (ALS). ALS (also known as E Acifluorfen-methyl E Sulfentrazone acetohydroxy acid synthase, AHAS) catalyzes the first step in the synthesis of the branched-chain amino acids (, leucine, and 186 F.E. Dayan, S.B. Watson / Pesticide Biochemistry and Physiology 101 (2011) 182–190 isoleucine). The reaction involves the synthesis of either 2-aceto- lactate from two molecules of pyruvate or 2-aceto-2-hydroxybuty- Start rate from a molecule each of pyruvate and 2-ketobutyrate. ALS Dark Light belongs to a superfamily of thiamin diphosphate-dependent en- 0.6 zymes that are capable of catalyzing a variety of reactions, includ- ing both the oxidative and nonoxidative decarboxylation of 2- ketoacids. This cofactor is bound by a divalent metal ion such as 0.4 Mg2, which coordinates to the diphosphate group of the thiamin diphosphate and to two highly conserved residues in these pro- teins [37]. ALS inhibitors were introduced in the 1980s and have 0.2 become valuable tools for weed management in agronomic pro- duction systems around the world. Unfortunately, they also repre- sent the group of herbicides with the greatest number of weed 0.0 biotypes identified with resistance. Imazapyr and metsulfuron (Ta- ble 3) were tested as representative of the herbicides inhibiting Asulam ALS. While these herbicides are known to be active at very low Imazapyr Glyphosate Glufosinate Cinmethylin Endothall concentrations [19], they did not induce any electrolyte leakage Fig. 4. Electrolyte leakage induced by herbicides targeting amino acid, protein or at the high concentration tested (Fig. 4). nucleotide synthesis. Data represents means of three replications with standard deviation. The dotted line represents maximum leakage obtained by boiling the cotyledon discs. 3.3.1.2. Inhibition of 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS). EPSPS is one of the key enzymes on the shikimate path- way responsible for the biosynthesis of the essential aromatic ami- (Fig. 4), although prolonged exposure is likely to result in a greater no acids, phenylalanine, tryptophan and tyrosine. Glyphosate effect due to ammonia toxicity. (Table 3), the only commercial inhibitor of EPSPS, is currently the most popular herbicide in the world because of the broad spectrum 3.3.2. Protein synthesis of weed control achieved when applied on genetically engineered 3.3.2.1. Inhibition of tyrosine aminotransferase. Tyrosine amino- glyphosate-resistant crops. This systemic herbicide is not very po- transferase catalyzes the conversion of tyrosine to 4-hydroxyphen- tent, requiring nearly 400 lM (Four times the concentration used ylpyruvate. Cinmethylin (Table 3) is a structural analog to the in this study) to inhibit the growth of the usually sensitive aquatic natural product 1,4-cineole that was developed in the early model plant Lemna paucicostata [38]. Accordingly, glyphosate did 1980’s to control important grass weeds such as green foxtail (Se- not lead to any detectable loss of membrane integrity in our study taria viridis) and barnyardgrass (Echinochloa crus-galli) and sup- (Fig. 4). press the growth of several broadleaf weeds [42]. The mode of action of cinmethylin eluded scientists for many years in spite of several research efforts. It was recently reported that the mecha- 3.3.1.3. Inhibition of glutamine synthetase (GS). GS catalyzes the key nism of cinmethylin involves inhibition of plant tyrosine amino- step in glutamine synthesis and plays a key role in ammonia transferase [43]. Unlike other inhibitors of amino acid synthesis, assimilation in plants. It catalyzes the condensation of glutamate cinmethylin caused a moderate level of electrolyte leakage (Fig. 4). and ammonia to form glutamine which turns out to be one of the rare instances where inorganic nitrogen is incorporated into 3.3.2.2. Inhibition of protein phosphatases. Protein phosphatases cat- organic forms. Glufosinate (Table 3) is the only commercial inhib- alyze a multitude of reactions crucial to the regulation of nearly itor of GS, though a number of other molecules are known to share every cellular process such as gene transcription and translation, this molecular target site [39,40]. The inhibition of this process re- metabolism, protein–protein interactions, protein activity, and sults in the accumulation of ammonia in plant cells to toxic levels. apoptosis. Endothall is a relatively old herbicide that was intro- The potent inhibition of GS by glufosinate is associated with its duced in the 1950’s (Table 3) whose mode of action has remained ability to mimic the phosphinic acid of glutamic acid, the natural elusive [19]. While early work reported general reductions of substrate of this enzyme [41]. During the relatively short duration mRNA and protein synthesis, these observations were the result of our assays, glufosinate induced very little electrolyte leakage of a general reduction in the overall physiological and biochemical processes of plants exposed to endothall [44,45]. Endothall is now Table 3 known to be a strong inhibitor of plant serine/threonine protein List of compounds tested targeting amino acid, protein or phosphatases that control many cellular processes [46]. Endothall nucleotide synthesis. has also been reported to cause membrane dysfunction [47] and WSSA classification Chemical similar results were observed in this study (Fig. 4). Acetolactate synthase inhibitors B Imazapyr 3.3.3. Inhibition of dihydropteroate synthetase B Metsulfuron Dihydropteroate synthetase is involved in the synthesis of tet- EPSP Synthase inhibitor rahydrofolate and other folic acids [48]. Folic acids are necessary G Glyphosate for the catalysis of key metabolic functions, such as the synthesis Glutamine synthetase inhibitor of methionine, pantothenate, purines, and thymidylate. Asulam H Glufosinate (Table 3) is an inhibitor of this enzyme, and is a systemic herbicide Tyrosine aminotransferase inhibitor but is not a very potent molecule. The aquatic model plant L. pauc- Z Cinmethylin icostata is normally sensitive to most xenobiotics, but to reduce its Protein phosphatase inhibitor growth by 50% requires a concentration of nearly 500 lM asulam, NC Endothall which is five times higher than the concentration used in this study Dihydropteroate synthetase inhibitor [38]. This may account for the lack of activity measured in our as- I Asulam say (Fig. 4). F.E. Dayan, S.B. Watson / Pesticide Biochemistry and Physiology 101 (2011) 182–190 187

3.4. Herbicides targeting lipid and wax biosynthesis Start Dark 3.4.1. Inhibition of acetyl-CoA carboxylase (ACCase) 0.6 Light

ACCase is a biotin-dependent enzyme that catalyzes the irre- ) versible carboxylation of acetyl-CoA to produce malonyl-CoA mohm Δ through its two catalytic activities, biotin carboxylase and carb- 0.4 oxyltransferase. The most important function of ACC is to provide the malonyl-CoA substrate for the biosynthesis of fatty acids. The diphenyl ether diclofop-methyl is an inhibitor of ACCase (Table 4). Although this herbicide is known to be grass selective because it 0.2 targets the multifunctional eukaryotic form of ACCase found in ( Conductivity the grass chloroplast [49], the concentration (100 lM) used in this study was sufficiently high to induce electrolyte leakage (Fig. 5). 0.0 While the primary mechanism of action of dichlofop-methyl in- volves inhibition of ACCase, it has been observed in previous stud- EPTC Alachlor Carbetamide Triclosan ies that this herbicide also destabilizes membranes and alters their Diclofop-methyl Pelargonicacid electrophysiological potentials [50,51]. Fig. 5. Electrolyte leakage induced by herbicides targeting lipid biosynthesis and waxes. Data represents means of three replications with standard deviation. The dotted line represents maximum leakage obtained by boiling the cotyledon discs. 3.4.2. Inhibition of enoyl (acyl carrier protein) reductase (ENR) ENR catalyzes an important step in fatty acid elongation, and disruption of this step has deleterious effect on very-long-chain the plasma membrane VLCFA content reaches critical levels to ob- fatty acids [52]. While no commercial herbicide targets ENR, the di- tain significant disruption of the membrane integrity. phenyl ether triclosan (Table 4) is a potent inhibitor of this enzyme [53] and induced a very strong light-independent electrolyte leak- 3.4.4. Wax removal age in plants (Fig. 5). It has been suggested that this very lipophilic Lastly, the natural product derived pelargonic acid (nonanoic inhibitor may also have direct membranotropic effects perturbing acid, Table 4) is a non selective postemergence herbicide that in- the bilayer assembly, leading to rapid loss membrane integrity jures both monocotyledonous and dicotyledonous plant weed [54]. and crop species. Its mechanism of action involves the stripping of cuticular waxes which results in rapid dessication of the foliage [57]. Consequently, intercalation of perlargonic acid within the li- 3.4.3. Inhibition of very-long chain fatty acid synthase (VLCFA pid bilayer induces light-independent membranotropic destabili- synthase) zation as well as a light-dependent membrane peroxidation from Very-long-chain fatty acids (VLCFAs) are formed by a micro- radicals derived from photosensitized chlorophyll displaced from somal elongase system in the endoplasmic reticulum. Chloroaceta- the thylakoid membranes [58]. Pelargonic acid may also cause mides are irreversible tight-binding inhibitors of the condensing the degradation of linolenic acid in the thylakoid membranes. This VLCFA synthase. While the plant contains in total about 1% VLCFAs, herbicide did not have any effect when tested at 100 lM, but plasma membranes are particularly rich (22%) in saturated VLCFAs, caused a dramatic disruption of membrane activity when tested primarily in the form of phospholipids. Treatment of intact seed- at 0.1% (v/v) (Fig. 5), which is a more realistic concentration for this lings with chloroacetamides markedly reduced the VLCFA content natural short-chain fatty acid herbicide [19]. in the plasma membrane and membranes with reduced VLCFA content are less stable and lose their functions, eventually leading 3.5. Herbicides with other mechanisms of action to death of the plant [55]. A number of herbicides target very-long-chain fatty acid syn- 3.5.1. Inhibition of microtubule assembly thase [19,56]. Of these, carbetamide, alachlor and EPTC (Table 4) Microtubules are formed by the self-assembly of heterodimeric were tested. While carbetamide and EPTC did not have any effect, tubulin units. Dinitroaniline herbicides (e.g., pendimethalin and alachlor did cause a small amount of light-independent membrane oryzalin, Table 5) bind to a-tubulin and prevent the polymeriza- disruption over the short time of this assay (Fig. 5). This is probably tion of free tubulin subunits into microtubule [19]. The subsequent due to the fact that inhibition of VLCFA synthesis is relatively slow loss of spindle apparatus stops the movement of the chromosomes acting, requiring long term exposure before significant decreases in to the poles during mitosis, and the cell cycle is arrested in a pro- metaphase configuration. While pendimethalin had no effect on cellular leakage, oryzalin causes some loss of membrane integrity Table 4 List of herbicides tested targeting lipid biosynthesis and waxes. which was more pronounced upon light exposure (Fig. 6).

WSSA classification Chemical 3.5.2. Inhibition of cellulose biosynthesis Acetyl-CoA carboxylase inhibitor Cellulose deposition on the outer surface of the plasma mem- A Diclofop-methyl brane is directed by the microtubule cytoskeleton, therefore there Lipid biosynthesis inhibitor is some overlap between the effects of herbicides targeting these N EPTC two processes [59,60]. For example, isoxaben (Table 5) interferes Very-long-chain fatty acid elongase inhibitors with the cellulose synthase (CESA) complex without affecting cor- K2 Carbetamide tical microtubule depolymerization, but still affects the skeletal K3 Alachlor microtubule organization [61]. Isoxaben stops the activity of the Enoyl CoA reductase inhibitor CESA complex by interfering with an early step in the incorpora- NC Triclosan tion of sugars into the cellulosic cell wall components [59,62].In Cuticular wax removal spite of its close association with membrane localized processes, Z Pelargonic acid isoxaben did not disrupt the bilayers integrity (Fig. 6). Dichlobenil 188 F.E. Dayan, S.B. Watson / Pesticide Biochemistry and Physiology 101 (2011) 182–190

Table 5 3.6. Selected natural phytotoxins List of compounds tested with other modes of action.

WSSA classification Chemical 3.6.1. Inhibition of HPPD Microtubule inhibitors The natural b-triketone herbicide leptospermone (Table 6), an K1 Pendimethalin inhibitor of HPPD [65], has the same mechanism of action as the K1 Oryzalin herbicides sulcotrione and isoxaflutole (Table 2). It caused electro- Cellulose biosynthesis inhibitors lyte leakage in the dark and became more pronounced in the sam- L Dichlobenil ples exposed to light (Fig. 7) and the effect was greater than with L Isoxaben sulcotrione (see Fig. 3). The cause of this loss of membrane integ- Synthetic rity is not known, but it suggests that the natural product may O 2,4-D have a secondary target site. O Quinclorac

3.6.2. Inhibition of ceramide synthase AAL toxin (Table 6) was included in this survey tested because (Table 5), on the other hand, inhibits a later step in cellulose bio- its disruption of sphingolipid synthesis causes the accumulation synthesis. While its precise mechanism of action is not well under- of phytosphingosine and sphinganine which render the plasma stood, this herbicide binds to MAP20, a protein known to be membrane porous [66]. However, this toxin did not have the ex- involved in cell wall formation [63]. Interestingly, dichlobenil pected effect at the concentration used in this assay (Fig. 7). While caused a small amount of electrolyte leakage similar to that of some species (i.e., black nightshade and Lemna) respond to concen- the microtubule inhibitor oryzalin (Fig. 6). trations of AAL toxin as low as 5 lM, cucumber requires at least 400 lM to exhibit a phytotoxic response [67]. 3.5.3. Synthetic auxins Auxins are an important class of phytohormones that influence 3.6.3. Inhibition of protein phosphatase and aspartate virtually every aspect of plant growth and development. Of these, aminotransferase indole-3-acetic acid is thought to act as a ‘master hormone’ in Cantharidin (Table 6) is a natural terpenoid analog of the herbi- the complex network of interactions with other phytohormones. cide endothall (Table 4) produced by the blister beetle (Epicauta Auxins generally regulate cell division and elongation and develop- spp.) and the Spanish fly (Lytta vesicatoria) as a protection mecha- mental processes [64]. Synthetic ‘superauxins’ (e.g., 2,4-D, Table 5) nism. This toxin inhibits plant serine/threonine protein phospha- have been used in agriculture for many years. These compounds tases [68] and its ability to destabilize membranes was stronger induce the same type of plant responses as natural endogenous than that observed with endothall (compare Figs. 4 and 7). auxins, but have a longer and more intense effect on plants, partic- Cornexistin (Table 6) is a microbial product isolated from the ularly on dicot weeds. In spite of its central role in regulating plant fungus Paecilomyces variotii SANK 21086 that has postemergence growth, 2,4-D did not affect the membrane stability in the cucum- herbicidal activity on several important weeds [69]. While this tox- ber cotyledon assay (Fig. 6). in did not have any detectable effect in our assay (data not shown), Quinclorac (Table 5) is a relatively new and highly selective it has been reported to disrupt plasma membrane and tonoplast herbicide that differs from traditional herbicides of this class integrity. Cornexistin appears to be a proherbicide that is con- in its ability to selectively control grasses by inducing leaf chloro- verted in vivo into a bioactive metabolite that inhibits aspartate sis, wilting and necrosis of the foliage. Its mechanism of action in- aminotransferase (AAT). volves the stimulation of ACC synthase activity in the root tissue, leading to an accumulation of ACC in shoots, where it is converted 3.6.4. Photodynamic natural products to high levels of ethylene and cyanide by the activity of ACC oxi- Cercosporin (Table 6) is a highly photodynamic toxin produced dase. This accumulation of cyanide is the toxic agent which causes by members of the fungal genus Cercospora. Its mechanism of ac- cell and plant death. The lack of effect by quinclorac in our assay tion is similar to that obtained with the bright red dye rose bengal, may be due to the fact that the generation of cyanide occurs specif- involving the production of both singlet oxygen and superoxide in ically in monocots (Fig. 6). the presence of light [70,71]. As such, it induced a strong loss of

Table 6 Start List of natural phytotoxins tested. Dark Light WSSA classification Chemical 0.6

) p-Hydroxyphenylpyruvate dioxygenase inhibitors F2 Leptospermone

mohm

Δ Ceramide synthase inhibitor 0.4 NC AAL toxin AAT and Protein phosphatase inhibitor NC Cantharidin 0.2 NC Cornexistin

Conductivity ( Conductivity Photodynamic natural product NC Cercosporin NC Hypericin 0.0 Enoyl-CoA reductase NC Cyperin Oryzalin Isoxaben 2,4-D Pore forming Pendimethalin Dichlobenil Quinclorac NC Syringomycin Fig. 6. Electrolyte leakage induced by herbicides with other modes of action. Data Unknown mode of action represents means of three replications with standard deviation. The dotted line NC Dehydrozalulanine C represents maximum leakage obtained by boiling the cotyledon discs. F.E. Dayan, S.B. Watson / Pesticide Biochemistry and Physiology 101 (2011) 182–190 189

Start product) or light-independent mechanisms (e.g., dinoterb). Com- Dark pounds targeting various steps of lipid synthesis also destabilized Light 0.6 membranes primarily via light-independent processes (inhibitors

) of ACCase, enoyl-ACP reductase and very-long-chain fatty acid syn- thase). Other compounds either had little or no effect. This survey mohm

Δ also identified weaknesses that must be considered if one was to 0.4 design a high throughput assay to screen for herbicides that affect membrane stability. Slow acting herbicides may not be identified within the time allocated by the current experimental design, 0.2 and the target species selected may be naturally more tolerant to

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