Plant Cell Membrane As a Marker for Light-Dependent and Light-Independent Herbicide Mechanisms of Action ⇑ Franck E
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Pesticide Biochemistry and Physiology 101 (2011) 182–190 Contents lists available at SciVerse ScienceDirect Pesticide Biochemistry and Physiology journal homepage: www.elsevier.com/locate/pest Plant cell membrane as a marker for light-dependent and light-independent herbicide 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. Herbicides 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 (paraquat and dinoterb) and those that either were photo- Electrolyte leakage dynamic (cercosporin) or caused the accumulation of photodynamic products (acifluorfen-methyl and Conductivity sulfentrazone). 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; Glufosinate, 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 Â 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 mÀ2 sÀ1) 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; Alachlor, 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; Clomazone, 2-[(2-chlorophe nyl)me thyl]-4,4-dimethyl-3-isoxazolidinone;