Biochemical Markers and Enzyme Assays for Herbicide Mode of Action and Resistance Studies Franck E
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Weed Science 2015 Special Issue:23–63 Biochemical Markers and Enzyme Assays for Herbicide Mode of Action and Resistance Studies Franck E. Dayan, Daniel K. Owens, Natalia Corniani, Ferdinando Marcos Lima Silva, Susan B. Watson, J’Lynn Howell, and Dale L. Shaner* Key words: Acetolactate synthase, acetyl-CoA carboxylase, cellulose biosynthesis, deoxyxylulose-5- phosphate synthase, dihydropteroate synthase, herbicide resistance, mitosis, mode of action, molecular probe, photosystem I, photosystem II, p-hydroxyphenylpyruvate dioxygenase, phytoene desaturase, protoporphyr- inogen oxidase, serine/threonine protein phosphatases, target site, very long chain fatty acid elongases. Herbicides inhibit biochemical and physiological used successfully in our laboratories. Readers inter- processes or both with lethal consequences. The ested in the specifics of each assay are referred to the target sites of these small molecules are usually literature for other examples that may be more enzymes involved in primary metabolic pathways or suitable for their needs. However, we have taken the proteins carrying out essential physiological func- opportunity to mention key aspects that should be of tions. Herbicides tend to be highly specific for their help in successfully performing each assay. Addition- respective target sites and have served as tools to ally, the effects of herbicides can sometimes be study these physiological and biochemical processes detected before the onset of the visual symptoms by in plants (Dayan et al. 2010b). monitoring the accumulation of intermediates or A few reviews on the modes of action by measuring the integrity of the physiological processes herbicides have been published in recent years they target. These will also be mentioned, where (Duke and Dayan 2011; Fedtke and Duke 2005), appropriate, as a means of rapid identification of but the last comprehensive book on that topic was certain herbicide modes of action (Table 1). nearly 20 yrs old (Devine et al. 1993). Furthermore, The sections in this chapter are organized no compendium of herbicide target-site assays has according to the herbicide classification scheme been available since the publication of the seminal established by the Herbicide Resistance Action book Target Assays for Modern Herbicides and Committee (HRAC) and adopted by the Weed Related Phytotoxic Compounds (Bo¨ger and Sand- Science Society of America as described in the mann 1993), although new modes of action have Herbicide Handbook (Senseman 2007). That classi- been characterized since then (e.g., inhibition of p- fication may need revision because the modes of hydroxyphenylpyruvate dioxygenase, very long fatty action of several herbicides are either new or better acid elongases, cellulose biosynthesis, serine/threo- understood than when the handbook was published. nine protein phosphatases, and deoxyxylulose-5- Those issues will be addressed as they arise in the text. phosphate synthase). The aim of this chapter is to describe assay protocols for all current herbicide General Considerations targets. Because of space constraints, this update will present the most common methods and, whenever Equipment. Unless specifically indicated in the applicable, will describe protocols that have been text, the following instruments were used for these experiments: DOI: 10.1614/WS-D-13-00063.1 1. High-performance liquid chromatography * First, second, fifth, and sixth authors: Plant Physiologist, Plant Physiologist, Support Chemist, and Biological Science (HPLC; Waters Corporation, Milford, MA) Laboratory Technician, Natural Products Utilization Research consisting of a model 600E pump, a model 717 Unit, Agricultural Research Service, U.S. Department of autosampler, a Millennium 2010 controller, a Agriculture, University, MS 38677; third and fourth authors: model 996 photodiode detector, a model 2475 Plant Physiologist and Agronomist, Faculty of Agronomic multi-l–fluorescence detector, and the Empow- Sciences, Sa˜o Paulo State University, Botucatu, Brazil 18610; er software; seventh author: Plant Physiologist, Water Management Research Unit, Agricultural Research Service, U.S. Department of 2. UV3101 PC spectrophotometer (Shimadzu Agriculture, Fort Collins, CO 80526. Corresponding author’s Scientific Instruments, Columbia, MD); E-mail: [email protected] 3. RF-5301 PC spectrofluorophotometer (Shimadzu); Dayan et al.: Biochemical markers and enzyme assays N 23 DownloadedCopyright from https://www.cambridge.org/core © The Authors [2015] This. IP address:is an Open 170.106.40.139 Access , article,on 01 Oct distributed 2021 at 16:49:19 under, subject the termsto the Cambridge of the Creative Core terms Commons of use, available Attribution at https://www.cambridge.org/core/terms license . https://doi.org/10.1614/WS-D-13-00063.1(http://creativecommons.org/licenses/by/4.0/), which permits unrestricted re-use, distribution, and reproduction in any medium, provided the original work is properly cited. https://doi.org/10.1614/WS-D-13-00063.1 Downloaded from 24 N https://www.cambridge.org/core Weed Science 63, Special Issue 2015 Table 1. Summary of the molecular target sites affected by herbicides and their respective Herbicide Resistance Action Committee classification, typical phenotypic responses, metabolic markers, and target assays. Herbicide Target site class Phenotypic response Metabolic marker Target assay . IPaddress: Acetyl-coenzyme A carboxylase A Slow death Crude extract Acetolactate synthasea B Slow death 2-Aminobutyrate Leaf disc Crude extract 170.106.40.139 Photosystem II C1,C2,C3 Chlorosis Chlorophyll fluorescence In planta; isolated chloroplasts Photosystem I D Desiccation Chlorophyll fluorescence In planta; isolated chloroplasts Protoporphyrinogen oxidase E Desiccation Protoporphyrin IX Electrolyte leakage , on Isolated etioplasts, heterologous expression Phytoene desaturase F Bleaching Phytoene Heterologous expression 01 Oct2021 at16:49:19 1 p-Hydroxyphenylpyruvate dioxygenase F2 Bleaching Phytoene Heterologous expression b Deoxyxylulose 5-phosphate synthase F3 Bleaching Chlorophylls Heterologous expression Carotenoids Enolpyruvyl shikimate-3-phosphate synthase G Slow death Shikimate Leaf discs Crude extract Glutamine synthetase H Chlorosis and wilting Ammonia Leaf disc , subjectto theCambridgeCore termsofuse,available at Dihydropteroate synthase I Chlorosis and stunting 4-Aminobenzoate Leaf disc Mitosis K1,K2 Clubbing, short roots Mitotic index Very long chain fatty acid synthase K3 Isolated endoplasmic reticulum membranes Cellulose L In planta cellulose formation Oxidative phosphorylation uncoupler M Desiccation Electrolyte leakage Fatty acid and lipid biosynthesisc N Synthetic auxins O Epinasty Ethylene Auxin transport P Epinasty, antigeotropic response Serine/threonine protein phosphatased Q N/A Crude extract a Also called acetohydroxy acid synthase. b F3 is a complicated classification because it contains compounds with different modes of action. For example, aclonifen is a phytoene desaturase and protoporphyrinogen IX oxidase inhibitor, amitrole is an inhibitor of phytyl synthesis, and clomazone is a deoxyxylulose-5-phosphate synthase inhibitor. c N is not a very accurate classification because it contains compounds targeting very long-chain fatty acids elongases (K3). Such compounds should be moved to that classification. https://www.cambridge.org/core/terms d Q is a proposed new classification for endothall that is now known to inhibit serine/threonine protein phosphatase. 4. Sorvall RC6 Plus centrifuge with SS34 rotor be adjusted on ice instead of at room temperature (Thermo Fisher Scientific Inc, Milford, MA); (RT). Similarly, reactions known to generate 5. Polytron PT-3100 homogenizer (Kinematica significant amount of H+ or OH2 will require Inc., Bohemia, NY); higher buffer concentrations to maintain the pH. 6. Model CU-36L5 incubator (Percival Scientific, Phosphate buffers are very common. The term Inc., Perry, IA); phosphate buffer can describe either sodium phos- 7. Model OS5-FL pulse-modulated fluorometer phate or potassium phosphate buffers. Phosphate (Opti-Science, Hudson, NH); buffers are usually made by mixing different 8. Model DW1 computer-controlled oxygen amounts of monobasic and dibasic stock solutions probe (Hansatech, PP Systems, Amesbury, (see Table 2). For example, a 50-mM sodium- MA) with a fiber-optic light source; phosphate buffer of pH 6.5 is made by mixing 9. Tri-Carb 2100 scintillation counter (PerkinEl- 68.5 ml of 200 mM sodium phosphate monobasic mer, Waltham, MA); with 31.5 ml of 200 mM sodium phosphate dibasic 10. PowerWave XS microplate reader (Bio-Tek and diluting with deionized water to a total volume Instruments, Inc., Winooski, VT); of 400 ml. A similar principle is applied for 11. French press (Thermo Spectronics Instrument potassium phosphate. Alternatively, a number of from Thermo Fisher); Web site tools and online tables can be used to 12. Electrical conductivity meter (model 1056, simplify buffer calculations. Amber Science Inc., Eugene, OR) equipped Although phosphate buffers are useful, their with a model 858 conductivity macro flow cell; metal cations or phosphate components sometimes 13. ThermoMixer-R (Brinkmann Instruments, interfere with enzyme assays, so other buffers, such Inc., Westbury, NY). as Tris or Trizma (tris(hydroxymethyl)methyl- amine, pH 7.5–9.0), HEPES (4-(2-hydroxyethyl)- 1-piperazineethanesulfonic acid, pH 6.8–8.2), and Buffers. Buffers are an important component of MES (2-(N-morpholino)ethanesulfonic acid, pH every