UNIVERSIDADE ESTADUAL PAULISTA “JÚLIO DE MESQUITA FILHO” FACULDADE DE CIÊNCIAS AGRONÔMICAS CAMPUS DE BOTUCATU MÉTODOS PARA IDENTIFICAÇÃO DE MOLÉCULAS COM ATIVIDADE HERBICIDA COM ÊNFASE NA ROTA DE SÍNTESE DE CAROTENÓIDES NATÁLIA CORNIANI Tese apresentada à Faculdade de Ciências Agronômicas da UNESP – Campus de Botucatu, para obtenção do título de Doutor em Agronomia (Proteção de Plantas) BOTUCATU-SP Dezembro – 2013 UNIVERSIDADE ESTADUAL PAULISTA “JÚLIO DE MESQUITA FILHO” FACULDADE DE CIÊNCIAS AGRONÔMICAS CAMPUS DE BOTUCATU MÉTODOS PARA IDENTIFICAÇÃO DE MOLÉCULAS COM ATIVIDADE HERBICIDA COM ÊNFASE NA ROTA DE SÍNTESE DE CAROTENÓIDES NATÁLIA CORNIANI Orientador: Prof. Dr. Edivaldo Domingues Velini Coorientadores: Profa Dra Ana Catarina Cataneo, Dr. Franck E. Dayan Tese apresentada à Faculdade de Ciências Agronômicas da UNESP – Campus de Botucatu, para obtenção do título de Doutor em Agronomia (Proteção de Plantas) BOTUCATU-SP Dezembro - 2013 III III III “Discovery consists of seeing what everybody else has seen, and thinking what nobody else has thought.” Albert von Szent-Györgyi IV ACKNOWLEDGMENTS Completing a Ph.D. is truly a marathon event, and I would not have been able to complete this journey without the aid of countless people over the past few years. I would like to express my sincere gratitude to my Brazilian advisor, Dr. Edivaldo Domingues Velini, whose expertise added considerably to my graduate experience. His thoughtful advice often served to give me a sense of direction during my Ph.D. studies. I am grateful for his belief in me. A special thanks goes out to my French-American advisor, Dr. Franck E. Dayan, for taking time out of his schedule to advise me. His guidance and fruitful discussion made my Ph.D. experience productive and stimulating. I admire his bright wisdom. I cannot but express my gratitude to Dr. Stephen O. Duke for enabling me to pursue my studies at USDA-NPURU, thereby allowing me to live one of the most amazing experiences of my life. My solemn high esteem to the eminent researcher he is. Appreciation also goes out to Ana Catarina Cataneo, Inês Cechin and Terezinha de Fátima Fumis for all the instances in which their intellectual support and friendship helped me along the way. I would like to thank my friends and lab mates at NUPAM for their support and positive input. I expand my thanks to all the staff for their helpful assistance and kindness. To the staff and students at USDA-NPURU, thank you for welcoming me to your group and for your assistance. My time at Oxford, Mississippi, was made enjoyable in large part due to the many friends that became a part of my life. I am tempted to individually thank all of my friends but fear I might omit someone, instead, I simply and genuinely say: thank you all for your care and trust. I would like to thank and dedicate this work to my parents and sister who are the angels that constantly watch over my head and give me the necessary strength to hold on and persevere. I appreciate the financial support from CAPES and express my gratitude to this agency. I cannot finish without acknowledging how eternally grateful and thankful I am to The Protecting Friend that guides me. Thank you, Dear Lord! V TABLE OF CONTENTS Page LIST OF TABLES .................................................................................................................. VII LIST OF FIGURES ................................................................................................................. VIII 1 RESUMO ............................................................................................................................. 01 2 SUMMARY ......................................................................................................................... 02 3 INTRODUCTION ............................................................................................................... 03 3.1 References ............................................................................................................... 06 4 CHAPTER I: BIOCHEMICAL MARKERS AND ENZYME ASSAYS FOR HERBICIDE MODE OF ACTION AND RESISTANCE STUDIES ................................... 09 Abstract .......................................................................................................................... 09 4.1 Introduction .............................................................................................................. 10 4.2 General considerations ............................................................................................. 12 4.3 Common protocols ................................................................................................... 15 4.4 Acetyl-CoA carboxylase - molecular target of herbicide class A or 1 .................... 19 4.5. Acetolactate Synthase - molecular target of herbicide class B ............................... 22 4.6 Photosystem II - molecular target of herbicide class C1, C2 and C3 ........................ 27 4.7 Photosystem I electron diversion - molecular target of herbicide class D ............... 31 4.8 Protoporphyrinogen oxidase - molecular target of herbicide class E ...................... 31 4.9 Phytoene Desaturase - molecular target of herbicide class F1 ................................ 34 4.10 p-Hydroxyphenylpyruvate Dioxygenase - molecular target of herbicide class F2 38 4.11 Deoxyxylulose-5-Phosphate - molecular target of herbicide class F3 ................... 40 4.12 5-EPSPS - molecular target of herbicide class G ................................................... 41 4.13. Glutamine synthetase assay - molecular target of herbicide class H .................... 45 4.14 7,8-Dihydropteroate synthase - molecular target of herbicide class I .................... 48 4.15 Mitosis - molecular target of herbicide class K1 and K2 ........................................ 52 4.16 Very Long Chain Fatty Acid Elongases - molecular target of herbicide class K3 . 54 4.17 Cellulose biosynthesis - molecular target of herbicide class L .............................. 58 4.18 Oxidative phosphorylation uncoupler - molecular target of herbicide class M ..... 61 4.19 Fatty acid and lipid biosynthesis - molecular target of herbicide class N .............. 62 4.20 Synthetic auxins and auxin transport inhibitors - molecular target of herbicide class O and P .................................................................................................................. 62 VI Page 4.21 Serine/threonine protein phosphatases - molecular target of herbicide class Q..... 62 4.22 Parting comments ................................................................................................... 65 4.23 References ............................................................................................................. 66 5 CHAPTER II: SIMPLE BIOASSAY FOR MEASURING INHIBITORS OF THE 2-C- Methyl-D-erythritol 4-Phosphate (MEP) PATHWAY ............................................................ 74 Abstract .......................................................................................................................... 74 5.1 Introduction .............................................................................................................. 75 5.2 Materials and Methods ............................................................................................. 80 5.2.1 Chemicals .................................................................................................. 80 5.2.2 Plant material ............................................................................................ 84 5.2.3 Bioassays .................................................................................................. 85 5.2.4 Phytoene extraction and determination ..................................................... 85 5.2.5 Statistical analysis ..................................................................................... 85 5.3 Results and Discussion ............................................................................................. 86 5.3.1 Accumulation of phytoene overtime ......................................................... 86 5.3.2 Effect of clomazone and ketoclomazone .................................................. 87 5.3.3 Validity of the bioassay with inhibitors of early steps of the MEP pathway .............................................................................................................. 92 5.3.4 Selectivity of the bioassay ........................................................................ 97 5.3.5 Supplementary studies .............................................................................. 99 5.4 Conclusions .............................................................................................................. 100 5.5 Acknowledgments .................................................................................................... 101 5.6 References ................................................................................................................ 101 VII LIST OF TABLES Page 4.1 Summary of the molecular target sites affected by herbicides, their respective HRAC classification, typical phenotypic responses, metabolic markers and target assays. ............... 11 4.2 Proportion table to obtain 100 ml of 200 mM sodium phosphate buffer at a specific pH1. ......................................................................................................................................... 14 5.1 Chemicals purchased from Sigma-Aldrich (St. Louis, MO 63103). ................................ 80 5.2 Chemicals purchased from Chem-Service (West Chester, PA 19381). ........................... 81 5.3 Effect of BASF experimental compounds on phytoene accumulation in the presence of 200 µM norflurazon. Inhibition of phytoene accumulation by compound treatment was expressed in percentage of inhibition related to maximum accumulation in control assays. All compounds were tested at 100 µM. ................................................................................... 97 5.4 Effect of herbicides with different modes of action on phytoene accumulation in the presence of 200 µM norflurazon. The bioassay was performed in three replications of each treatment. All compounds were tested at 100 µM. .................................................................. 99 VIII LIST OF FIGURES Page 4.1 Left panel shows a time course experiment illustrating the light-independent (�) and light-dependent (�) loss of membrane integrity, compared to solvent control with no inhibitor (�). Arrows represents the 3 time measurements used in the simplified assay shown in panel B. The dotted line represents maximum change in conductivity (obtained after boiling the discs) from Dayan and Watson (2011). ....................................................... 18 4.2 A) Acetyl-CoA carboxylase location and activity in Poaceae. B) Dose-response curve of clodinafop on ACCase activity from wild type (�) and resistant (�) populations of Alopecurus myosuroides (data from Délye et al. 2003). ........................................................ 20 4.3 A) Acetolactate synthase (ALS) catalyzes an important step in the synthesis of branched chain amino acids via two parallel reactions. B) Effect of alanine on acetolactate accumulation in wheat leaf discs. C) Dose-response curve of ALS inhibitor imazamox on ALS activity from wild type (black) and herbicide resistant (white) canola in leaf disc assays. KARI, ketoacid reductoisomerase is inhibited in order to cause 2-acetolactate accumulation. .......................................................................................................................... 24 4.4 A) Diagram of the Z-scheme describing the hill reaction from Dayan et al., 2010. The sites of herbicide interactions are indicated with the arrows. B) Effect of amicarbazone (10 µM) on wild-type (white) and herbicide resistant pigweed (black) photosynthetic electron transport and C) oxygen evolution dose-response curve. ....................................................... 29 4.5 A) Protoporphyrinogen oxidase (PPO) catalyzes the conversion of the colorless protoporphyrinogen IX (Protogen) to the highly fluorescent protoporphyrin IX (Proto). The dotted arrow represents the non-enzymatic step leading to proto accumulation when PPO is inhibited. B) Effect of 10 µM acifluorfen on wild-type (white) and herbicide resistant (black) Amaranthus tuberculatus and C) dose-response curves of acifluorfen on heterologously expressed PPO from wild-type (�) and herbicide resistant (�) Amaranthus tuberculatus. .......................................................................................................................... 33 4.6 A) Phytoene desaturase (PDS) catalyzes the conversion of phytoene to ζ-carotene in the biosynthesis of carotenoids. B) Effect of 12 nM fluridone on phytoene accumulation in wild-type (white) and herbicide resistant (black) Hydrilla verticillata. C) Dose-response curve of fluridone on heterologously expressed PDS from wild-type (�) and herbicide resistant (�) Hydrilla verticillata. ......................................................................................... 35 IX Page 4.7 A) p-Hydroxyphenylpyruvate dioxygenase (HPPD) catalyzes the conversion of p- hydroxyphenylpyruvate (HPP) to homogentisate (HGA). Effect of sulcotrione on B) heterologously expressed Arabidopsis thaliana activity and C) chlorophylls, carotenoids and plastoquinone levels. ........................................................................................................ 37 4.8 A) Deoxyxylulose-5-phosphate synthase (DXS) catalyzes the first step of the MEP pathway leading to IPP synthesis in plastids. ......................................................................... 41 4.9 A) Enoylpyruvyl shikimate-3-phosphate synthase (EPSPS) catalyzes the conversion of shikimate-3-phosphate to enoylpyruvyl shikimate-3-phosphate in the biosynthesis of aromatic amino acids. B) Effect of 100 µM glyphosate on shikimate accumulation in wild- type (white) and herbicide resistant (black) Amaranthus palmeri. C) Dose-response curve of glyphosate on EPSPS activity in crude extracts from wild-type (white) and herbicide resistant (black) Amaranthus palmeri. Notice the much higher activity in the resistant biotype, which is the result of gene amplification. ................................................................. 43 4.10 A) Reaction catalyzed by GS and various detection methods. B) Accumulation of ammonia in sunflower leaf discs after 16 h incubation under light in the presence of increasing amount of glufosinate. ........................................................................................... 47 4.11 A) Dihydropteroate synthase (DHPS) is a key enzyme in the synthesis of folic acid. B) Dose-response curve of asulam on Lemna paucicostata growth (7 days after treatment). C) Effect of 200 µM asulam on DHPS activity as measured by 14C-pABA incorporation in folate in Lemna paucicostata. ................................................................................................. 50 4.12 A) Light micrographs of untreated onion root tips. Cells at various stages of mitosis are indicated in A as p = prophase, m = metaphase, a= anaphase, and t = telophase. Bars represent 100 µm. B) Distribution of phases of mitosis in control (■), podophyllotoxin (≡) and etoposide (□) treated onion root tips. Data is obtained by counting at least 3000 cells per treatment. Cells with chromosomal arrangements deviating from those associated with normal mitotic phases are classified as abnormal. LSD at p = 0.05. Images are from Oliva et al., 2002. ............................................................................................................................. 53 4.13 A) Very long chain fatty acid (VLCFA) elongases catalyze the elongation of acyl- CoA with 18 or more carbons. B) VLCFA profile obtained by incubating leek microsomal X Page fraction with stearoyl-CoA and [14C]-malonyl-CoA. C) Effect of S-metolachlor on VLCFA elongases activity in leek microsomal fraction. ...................................................................... 56 4.14 A) Simplified biochemical pathways involved in cell wall synthesis. The assay consists of incorporating 14C-glucose into cell wall and isolating the cellulose-rich acid- insoluble fraction and quantifying it following biological oxidation and liquid-scintillation counting. B) Typical dose-response curve of dichlobenil on the growth of Lemna paucicostata. C) Effect of dichlobenil on incorporation of 14C-glucose into cellulose-rich acid-insoluble fraction. C= control, T= 10 µM dichlobenil, dotted line marks the maximum inhibition measured. ............................................................................................................... 59 4.15 A) Regulation of protein activity by phosphorylation/dephosphorylation by protein kinases and phosphatases. B) Inhibition of Ser-Thr protein phosphatases by endothall in Lemna paucicostata crude extract. ......................................................................................... 63 5.1 Enzymatic reactions in the MVA in plants and synthesis of the short-chain prenyl diphosphates. Enzymes abbreviations: AACT, acetyl-CoA C-acetyltransferase; HMGS, 3- Hydroxy-3-methylglutaryl-CoA synthase; HMGR, 3-Hydroxy-3-methylglutaryl-CoA reductase; MK, MVA kinase; PMK, Phospho-MVA kinase; MPDC, Diphospho-MVA decarboxylase; IPP isomerase, isopentenyl diphosphate -isomerase; GPP synthase, geranyl diphosphate synthase; FPP synthase, farnesyl diphosphate synthase; GGPP synthase, geranylgeranyl diphosphate synthase. ... ................................................................. 76 5.2 Biosynthesis of carotenoids starts with the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway leading to the formation of IPP, continues with the isoprenoid pathway to obtain GGPP. The first committed step to the synthesis of carotenoids consists of the head to head condensation of 2 GGPP to form phytoene. The steps in bold italics (first and last step shown) denote enzymes inhibited by commercial herbicides .......................................... 77 5.3 Structure of norflurazon, dichlobenil, glufosinate-ammonium, respectively. 5.4 A Structures of experimental diphenyl ether-isoxazole inhibitors of DXS from BASF. .. 83 5.4 B Structures of experimental hydrazone inhibitors of DXS from BASF. ........................ 83 5.4 C Structures of experimental pyrimidine inhibitors of DXR (IspC) from BASF. ........... 83 5.4 D Structures of experimental acylated aminobenzothiazole inhibitors of IspD from BASF. ..................................................................................................................................... 83 5.4 E Structures of experimental azolopyrimidine inhibitors of IspD from BASF. ............... 85 XI Page 5.4 F Structure of experimental isoindoline inhibitor of IspE from BASF. ........................... 85 5.5 Structures of ketoclomazone and analogs 67, 69. ............................................................ 85 5.6 Time-dependent phytoene accumulation in barley (Hordeum vulgare L.) exposed to 200 µM norflurazon. Data represent means of three replications with standard deviation. ... 87 5.7 Dose-response curves showing the effect of the herbicide clomazone (�) with and (�) without phorate on phytoene accumulation induced by 200 µM norflurazon. (A) Green and (B) greening etiolated young barley leaves. Data represent means of three replications with standard deviation. .......................................................................................................... 89 5.8 Dose-response curves of the ketoclomazone a clomazone metabolite) (�) with and (�) without phorate on (A) green and (B) greening etiolated barley leaves. Phytoene was caused to accumulate by the presence of 200 µM norflurazon. Data represent means of three replications with standard deviation. ............................................................................. 91 5.9 Dose-response to the analog 69 on (�) green and (☐) greening etiolated young barley leaves. Data represent means of three replications with standard deviation. ......................... 93 5.10 Dose-response curves of DXR inhibitors (A) FR90098 and (B) fosmidomycin on (�) greening etiolated and ( ) green young barley leaves. Phytoene accumulation was induced in the presence of 200 µM norflurazon. Data represent means of three replications with standard deviation. .................................................................................................................. 95 1 1. RESUMO O manejo de plantas daninhas é um aspecto importante da produção agrícola. A introdução de herbicidas sintéticos, em meados do século 20, tornou o controle de plantas daninhas menos dispendioso e mais eficaz. A introdução de culturas transgênicas resistentes a herbicidas não-seletivos (por exemplo, glifosato e glufosinato) reforçou o estabelecimento dos herbicidas como a principal tecnologia usada para controle em grande escala de plantas daninhas na produção agrícola mundial. No entanto, a pressão de seleção imposta pelos herbicidas levou à evolução generalizada de resistência em populações de plantas daninhas, o que representa uma grande ameaça para a sustentabilidade e rentabilidade dos sistemas de cultivo. Testes confiáveis para a detecção de resistência são pré-requisito para a implementação de estratégias de controle integrado eficazes. Há demanda crescente dos produtores por testes para diagnosticar a resistência de plantas daninhas e aprender a gerenciá-la. Os cientistas desenvolveram protocolos de teste de resistência para inúmeros herbicidas, mas, no Brasil, não há nenhum trabalho compilando essas informações. A evolução da resistência tem também acentuado a necessidade de produtos com novos mecanismos de ação para complementar a falta de atividade dos herbicidas atuais. A rota do metileritritol fosfato (MEP) representa um dos alvos mais promissores para o desenvolvimento de novos herbicidas, bem como para melhorar o valor nutricional de culturas agrícolas. No entanto, há apenas um herbicida comercial, clomazone, alvejando esta rota. Portanto, o primeiro capítulo do presente trabalho consiste em uma compilação de ensaios para medir a atividade de enzimas-alvo e caracterização de resistência de plantas a todos os modos de ação de herbicidas conhecidos. Uma vez que não havia descrito nenhum ensaio in vivo para testar inibidores da rota do MEP, no segundo capítulo é descrito o desenvolvimento de um ensaio com discos foliares rápido, preciso, de baixo custo baseado na medição do fluxo de carbono a partir de gliceraldeído-3-fosfato (GAP) e piruvato a fitoeno, um dos produtos desta via. Além disso, utilizando o ensaio descrito foram caracterizados os efeitos de inibidores conhecidos e experimentais sobre o acúmulo de fitoeno. 2 METHODS FOR IDENTIFYING MOLECULES WITH HERBICIDAL ACTIVITY WITH EMPHASIS ON THE CAROTENOIDS BIOSYNTHESIS PATHWAY. Botucatu, 2013. 109p. Tese (Doutorado em Agronomia/Proteção de Plantas) - Faculdade de Ciências Agronômicas, Universidade Estadual Paulista. Author: Natália Corniani Advisor: Edivaldo Domingues Velini 2. SUMMARY Weed management has always been an important aspect of crop production. The introduction of synthetic herbicides in the mid-20th century has made weed control less expensive and more effective. The introduction of transgenic crops resistant to non-selective synthetic herbicides (e.g., glyphosate and glufosinate) further established the reliance on herbicides as the dominant technology used for large-scale weed control in production agriculture worldwide. However, the selection pressure imposed by herbicides has led to the widespread evolution of herbicide resistance in weed populations, which is a major threat to the sustainability and profitability of cropping systems. Reliable tests for resistance are an essential pre-requisite for the rational implementation of effective integrated control strategies. There is increasing demand from growers to test for weed resistance and learn how to manage it. Scientists have developed resistance-testing protocols for numerous herbicides but, in Brazil, there is no review compiling this information. Evolution of herbicide resistance is also underscoring the need for herbicides with new modes of action to complement those herbicides failing due to resistance. The 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway represents one of the most promising targets to develop new herbicides as well as targets to improve the nutritional value of crop plants. However, there is just one commercial herbicide, clomazone, targeting it. Therefore, in the first chapter of this study there is a compilation of assays to measure the activity of key target enzymes and characterization of plant resistance to all known herbicides mode of action. Since no in vivo assay was available for testing inhibitors of MEP pathway, in the second chapter it is described the development of a rapid, accurate, cheap, readily available leaf disc assay based on the measurement of the carbon flow from glyceraldehyde 3-phosphate (GAP) and pyruvate to phytoene, one of the downstream products of this pathway. Moreover, using the new assay we characterized the effects of well-known and experimental inhibitors on phytoene accumulation. Keywords: MEP pathway, clomazone, herbicides mode of action, resistance, assays. 3 3. INTRODUCTION Global food production needs high productivity with minimal human labour and maximum input-use efficiency (PINGALI; HEISEY, 1999). Weedy plants are the greatest biological constraint to annual crop yields in agricultural systems worldwide (OERKE, 2006). Berca (2004) goes further when he says: “Weeds eat the food of about 1 billion inhabitants”. Significant crop losses due to weeds are simply not acceptable in a world where 2 billion more people will have to be fed in the next 40 years. Herbicides are the key to sustainable crop production and will remain the mainstay for weed control for the foreseeable future (GIANESSI, 2013). The value of the worldwide herbicide market grew by 39% between 2002 and 2011 and is projected to grow by another 11% by 2016 (McDOUGALL, 2013). Despite their many benefits, misuses of herbicides can become a serious problem for the farmer and society. The repetitive use of the same herbicide over many years often results in the selection of herbicide resistant weeds from populations normally well controlled by the same herbicide (PALOU et al., 2008). There are many biological, genetic, herbicide and operational factors driving the dynamics of herbicide resistance evolution in weed species (JASIENIUK et al., 1996). The topic of herbicide resistance is reviewed in several books (POWLES; HOLTUM, 1994; DePRADO et al., 1997; GRESSEL, 2002). Since the first report of resistance to 2,4-D in 1957 in wild carrot (Daucus carota L.) (SWITZER, 1957; WHITEHEAD; SWITZER, 1967), resistance to herbicides has expended to include over 220 species (130 dicots and 90 monocots) involving at least 21 of the 25 known herbicide sites of action (HEAP, 2013). Resistance to herbicides is among the primary concerns in modern agriculture (BURGOS et al., 2013). Although it is a known problem, farmers in many countries detect problems with herbicide efficacy after resistant weed populations are already established in the field (FAO, 2012). Accurate and timely diagnosis is crucial to manage and mitigate resistance successfully (BURGOS, 2013). Herbicide treatments may fail for a variety of reasons and confirmation that herbicide resistance is involved requires reliable tests. In the last decade, the global weed resistance database has expanded significantly (HEAP, 2013), and so has our collective experience in surveying, confirming and evaluating resistance. There is accumulated experience of several assays to characterize resistance to a great variety of herbicides (MOSS, 1995; BECKIE et al., 2000). This necessary information should be compiled as a basis for future work in preventing this problem in other locations, 4 countries and regions. Therefore, herein (Chapter I) we aim to help science practitioners solve questions pertaining to the testing of herbicide resistance based on herbicide bioassays and enzyme target site assays. The protocols are categorized according to herbicide site of action, using the alphabetical classification system adopted by the Herbicide Resistance Action Committee (RETZINGER; MALLORY-SMITH, 1997; SCHMIDT, 1997). We believe that understanding the advantages and limitations of the various assays will be helpful to one choose the appropriate assay protocol and interpret the results properly. A good solution to the emergence of herbicide-resistant weeds would be discover and develop herbicides with new modes of action (MOAs) for which no resistance has evolved. New robust solutions to herbicide-resistant weeds will continually be needed and rewarded in the market place, and only by refocusing our efforts can we provide the new MOAs and weed control technologies that will most certainly be needed in the future (GERWICK, 2004). To date, no new major herbicide mode of action has been introduced in a commercial herbicide active ingredient in the last 20 years (DUKE, 2012). The last novel MOA was introduced in 1980 with p-hydroxyphenyl pyruvate dioxygenase (HPPD, E.C. 1.13.11.27) (MATSUMOTO et al., 2002). All new commercial herbicide products consist of either new premixes or formulations of existing active ingredients, or new active ingredients within existing herbicide classes affecting known target sites (GERWICK, 2010). In spite of major progress in physiology profiling, metabolomics and genomics approaches, the successful identification of a novel MOA is still rarely described (DUKE, 2012). According to Witschel et al. (2013) many enzymatic targets have been suggested to be potentially herbicidal, but only very few have been confirmed so far with herbicidal activity in the greenhouse, and none has been developed to become a commercial herbicide. Duke (2012) mentions that the limitation for obtaining new herbicide modes of action is not due to failures in finding good inhibitors of essential plant enzymes, but rather to the difficulty of finding sensitive, lethal target sites (WITTENBACH; ABELL, 1999). Due to increasing resistance of weeds to almost all available herbicides, especially also to the commercially most important herbicide glyphosate, new MOAs in the herbicide market are of high importance for the future world food production (WELLER et al., 2010). The enzymes of the methylerythritol-4-phosphate (MEP) pathway are especially interesting targets for agrochemicals, as there exist no homologs in 5 mammals, which instead utilize the mevalonate (MVA) pathway to synthesize isopentenyl diphosphate (IPP), and therefore no target related toxicological side-effects of inhibitors of this pathway are expected (WITSCHEL, 2011). Since the MEP pathway is responsible for the biosynthesis of a substantial number of natural compounds of biological and biotechnological importance, this pathway has become an obvious target for the development of potential next generation herbicides (EISENREICH et al., 2004; WITHERS; KEASLING, 2007; CORDOBA et al., 2009). To this end, appropriate test systems are required to ascertain the potential of each new inhibitor as an herbicide (RODRÍGUEZ-CONCEPCIÓN, 2004). Herein (Chapter II) it is described the development of a novel in vivo bioassay by using well-known and experimental inhibitors as tools to perturb MEP pathway flux. The newly developed method proved to be a fast, and reproducible method for identifying inhibitors of MEP enzymes that may be useful to research groups (academia, governmental or industry) seeking to discover new herbicide target sites. 6 REFERENCES1 BECKIE, H.J. et al. Screening for herbicide resistance in weeds. Weed Technology, v.14, p.428-445, 2000. 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CHAPTER I “BIOCHEMICAL MARKERS AND ENZYME ASSAYS FOR HERBICIDE MODE OF ACTION AND RESISTANCE STUDIES” Abstract: This review is an up-to-date compilation of assays to measure the activity of most of the target sites affected by commercial herbicides. The methods described herein are meant to assist anyone interested in measuring the activity of a number of key target enzymes in the biosynthesis of plant macromolecules (e.g., proteins, cell walls and membranes) and the synthesis of their building blocks (e.g., amino acids, sugars and fatty acids) as well physiological processes involving photosynthesis directly (e.g., photosystem I and II) and indirectly (e.g., synthesis of carotenoid, porphyrins, and plastoquinone). Our aim has been to provide practical information to help performs these assays based on our personal experience. These protocols will also be useful for anyone interested in herbicide resistance mechanisms that either involve point mutations, deletions or overexpression that affect the behavior of these proteins and enzymes. Keywords: mode of action, herbicide resistance, target site, molecular probe, acetyl-CoA carboxylase, acetolactate synthase, photosystem I, photosystem II, protoporphyrinogen oxidase, phytoene desaturase, p-hydroxyphenylpyruvate dioxygenase, deoxyxylulose-5- phosphate synthase, dihydropteroate synthase, mitosis, very long chain fatty acid elongases, cellulose biosynthesis, serine/threonine protein phosphatases. 10 4.1 INTRODUCTION Herbicides inhibit biochemical and/or physiological processes with lethal consequences. The target sites of these small molecules are usually enzymes involved in primary metabolic pathways or proteins carrying out essential physiological functions. Herbicides tend to be highly specific for their respective target sites and have served as tools to study these physiological and biochemical processes in plants (DAYAN et al., 2010b). A few reviews on the mode of action of herbicides have been published in recent years (DUKE; DAYAN, 2011; FEDTKE; DUKE, 2005) but the last comprehensive book on this topic is nearly 20 years old (DEVINE et al., 1993). Furthermore, no compendium of herbicide target site assays has been available since the publication of the seminal book entitled ‘Target Assays for Modern Herbicides and Related Phytotoxic Compounds’ (BÖGER; SANDMANN, 1993) although new modes of action have been characterized since then (e.g., inhibition of p-hydroxyphenylpyruvate dioxygenase, very long fatty acid elongases, cellulose biosynthesis, serine/threonine protein phosphatases, and deoxyxylulose-5-phosphate synthase). The aim of this chapter is to describe assay protocols for all current herbicide targets. Due to space constraints, this update will present the most common methods, and whenever applicable, will describe protocols that have been used successfully in our laboratories. Readers interested in the specifics of each assay are referred to the literature for other examples that may be more suitable for their needs. However, we have taken the opportunity to mention key aspects that should be of help in successfully performing each assay. Additionally, the effects of herbicides can sometimes be detected before the onset of the visual symptoms by monitoring the accumulation of intermediates or measuring the integrity of the physiological processes they target. These will also be mentioned as a means of rapid identification of certain herbicide modes of action where appropriate (Table 4.1). 11 Table 4.1 - Summary of the molecular target sites affected by herbicides, their respective Herbicide Resistance Action Committee classification, typical phenotypic responses, metabolic markers and target assays. Target Site Herbicide Class Phenotypic response Metabolic marker Target assay Acetyl-CoA carboxylase A Slow death Crude extract Acetolactate synthase1 B Slow death 2-aminobutyrate Leaf disc, Crude extract Photosystem II C1, C2, C3 Chlorosis Chlorophyll fluorescence In planta, Isolated chloroplasts Photosystem I D Burn-down Chlorophyll fluorescence In planta, Isolated chloroplasts Protoporphyrinogen oxidase E Burn-down Proto Electrolyte leakage, Isolated etioplasts, Heterologous expression Phytoene desaturase F1 Bleaching Phytoene Heterologous expression p-Hydroxyphenylpyruvate dioxygenase F2 Bleaching Phytoene Heterologous expression Deoxyxylulose 5-phosphate synthase2 F3 Bleaching Chlorophylls Carotenoids Chapter II Enolpyruvyl shikimate-3-phosphate synthase G Slow death Shikimate Leaf discs, Crude extract Glutamine synthetase H Chlorosis and wilting Ammonia Leaf disc 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 ER membranes Cellulose L In planta cellulose formation Oxidative phosphorylation uncoupler M Burn-down Electrolyte leakage Fatty Acid and Lipid Biosynthesis3 N Synthetic auxins O Epinasty Ethylene Auxin transport P Epinasty, antigeotropic response Serine/threonine protein phosphatase4 Q n/a Crude extract 1also called Acetohydroxy Acid Synthase (AHAS); 2F3 is a complicated classification because it contains compounds with different modes of action. For example, aclonifen is a PDS and PPO inhibitor, amitrole is an inhibitor of phytyl synthesis, and clomazone is a deoxyxylulose-5-phosphate synthase inhibitor; 3N is not a very accurate classification because it contains compounds targeting VLCFA elongases (K3). Such compounds should be moved to that classification. 4Q is a proposed new classification for endothall that is now known to inhibit serine/threonine protein phosphatase. 12 The sections in this chapter are organized according to the herbicide classification scheme established by the Herbicide Resistance Action Committee (HRAC) and adopted by the Weed Science Society of America (WSSA) as described in the Herbicide Handbook (SENSEMAN, 2007). It has become apparent that this classification is in need of revision since the modes of action of several herbicides are either new or better understood than when the handbook was published. These issues will be addressed as they arise in the text. 4.2 GENERAL CONSIDERATIONS Buffers Buffers are an important component of every assay and readers are encouraged to learn more about how these molecules work. Calbiochem (EMD Biosciences, Inc) has an excellent free downloadable guidebook (MOHAN, 2003). Buffers are aqueous solutions that prevent changes in pH even when a small amount of strong acid or base is added to them. The optimum buffer capacity of a molecule is near its pKa, so this allows for the selection of a buffer to maintain the optimum pH for the purpose of the experiment. However, published protocols generally provide little information regarding buffers, other than the chemical name or acronym and the concentration of the solution. Therefore, the following useful practical information on buffers is provided. Buffer tip #1. Prepare buffers 10X concentrations and stored in 50 ml aliquots in a freezer until use. We use 50 ml BD Falcon tubes (BD Biosciences, San Jose, CA). It is critical to consider under what condition the buffer will be used because the pH of a solution can be affected by temperature or by an enzyme reaction. For example, if an extraction is performed at 4 °C, it is important for the pH of the extraction buffer to be adjusted on ice instead of at room temperature. Similarly, reactions known to generate significant amount of H+ or OH- will require higher buffer concentrations to maintain the pH. Phosphate buffers are very common. The term ‘phosphate buffer’ can describe either sodium phosphate or potassium phosphate buffers. Phosphate buffers are usually made by mixing different amount of monobasic and dibasic stock solutions (see Table 4.2). For example, a 50 mM sodium phosphate buffer of pH 6.5 is made by mixing 13 68.5 ml of 200 mM sodium phosphate monobasic with 31.5 ml of sodium phosphate dibasic and diluting with deionized water to a total volume of 400 ml. A similar principle is applied for potassium phosphate. Alternatively, there are now a number of website tools and online tables that can be used to simplify buffer calculations. While phosphate buffers are very useful, their metal cations or phosphate components sometimes interfere with enzyme assays so other buffers such as TRIS or TRIZMA™ (tris(hydroxymethyl)methylamine, pH 7.5-9.0), HEPES (4-2- hydroxyethyl-1-piperazineethanesulfonic acid, pH 6.8-8.2), MES (2-(N- morpholino)ethanesulfonic acid, pH 5.5-6.7) are often used. Buffer tip #2. Do not use sodium hydroxide to adjust the pH of the buffer if sodium interferes with an assay. Use potassium hydroxide instead; vice versa if potassium is a problem for the assay. Make sure the buffer selected is used within its buffer range (pKa +/- 1 pH unit as a rule of thumb). One should always check methods carefully because it is not unusual to see published protocols using buffers at pH ranges outside their optimum buffer capacity. Buffer tip #3. Prepare Tris-HCl buffer by making up a solution of Tris-free base and adjusting its pH with 1 N HCl. Ethylenediaminetetraacetic acid (EDTA) is often added to buffers because it prevents the deactivation of catalytic sites by removing traces of heavy metals known to interfere with the formation of disulfide bridges. However, EDTA interferes with the activity of enzymes with divalent metal cofactors (e.g., Hg2+ and Ag2+). Again, the judicious use of buffer components requires a good understanding of their functions. Buffer tip #4. Prepare 100 ml of 500 mM EDTA stock by adding 18.6 g of Na2EDTA. 2H2O to 80 ml of deionized water in a beaker on a stir plate and slowly add 1 N NaOH a drop at a time to reach pH 8. When EDTA is solubilized, bring volume to 100 ml and store refrigerated. It remains stable for a long time. Finally, calibrate the pH meter in a range close to the pH of the solution being prepared. The use of color-coded pre-made solutions at specific pHs is very 14 handy. Use the red (pH 4 standard) for solutions below pH 5.5, the yellow (pH 7 standard) for solutions between 5.5 and 8.5, and the blue (pH 10 standard) for solutions above 8.5. Table 4.2 - Proportion table to obtain 100 ml of 200 mM sodium phosphate buffer at a specific pH1. pH NaH2PO4 Na2HPO4 pH NaH2PO4 Na2HPO4 ml ml 5.7 93.5 6.5 6.9 45.0 55.0 5.8 92.0 8.0 7.0 39.0 61.0 5.9 90.0 10.0 7.1 33.0 67.0 6.0 87.7 12.3 7.2 28.0 72.0 6.1 85.0 15.0 7.3 23.0 77.0 6.2 81.5 18.5 7.4 19.0 81.0 6.3 77.5 22.5 7.5 16.0 84.0 6.4 73.5 26.5 7.6 13.0 87.0 6.5 68.5 31.5 7.7 10.5 89.5 6.6 62.5 37.5 7.8 8.5 91.5 6.7 56.5 43.5 7.9 7.0 93.0 6.8 51.0 49.0 8.0 5.3 94.7 1Prepare 500 ml stocks of 200 mM sodium phosphate monobasic monohydrate (NaH2PO4.H2O) = (2.75 g/100 ml water) and 200 mM sodium phosphate dibasic heptahydrate (Na2HPO4.7H2O) = (5.36 g/100 ml water) and mix different ratios to obtain a specific final pH of 200 mM sodium phosphate buffer solution. This solution can then be diluted to lower concentrations with water and the pH will remain the same. Extinction coefficients Beer’s law is commonly encountered in biochemical assays as it establishes the relationship between the absorbance of a compound and its concentration in a solution. A useful rule of thumb is that when expressed in standard units, a 1 mM solution of a compound has an absorbance of �/1000. Beer’s law: � = ��� A = absorbance, unitless; � = molar extinction coefficient, L mol -1 cm-1; C = concentration, mol L-1; and L = cell length, cm. The simple and commonly used working form of the equation is: � = � � In the majority of modern spectrophotometers, the cell length is 1 cm, and therefore does not contribute to the equation. The molar extinction coefficient is an experimentally determined constant of a specific substance at a particular wavelength for which known values are often available from reference books or in the literature. It is 15 common for extinction coefficients to be in units other than molar (mmolar or µmolar) so that final calculated values are in more easily interpretable units. 4.3 COMMON PROTOCOLS Protein quantification The most commonly used total protein assay systems include direct measurement at absorbance 280 nm and colorimetric assays. Absorbance at 280 nm takes advantage of the fact that proteins have absorbance maxima at this wavelength resulting primarily from the presence of aromatic amino acids. However, this method is subject to error due to the presence of many substances that absorb at 280 nm in plant extracts. Protein tip #1. Use quartz cuvettes because plastic cuvettes interfere with readings in the UV range. Colorimetric protein determination methods are based on a reaction between protein and an assay reagent that results in color production directly proportional to amount of protein present in an extract. The amount of protein in the extract is calculated based on a standard curve, often produced using bovine serum albumin. The method of Bradford using Coomassie blue G is one of the most common colorimetric protein assays (BRADFORD, 1976). Protein tip #2. The Bradford system may not be compatible with all buffer components, particularly detergents and imidazole. Those interfering molecules must be removed by gel filtration, dialysis, or some other method before protein concentration can be determined accurately. In some cases, an alternate method or commercially available specialized assays may also be used to overcome this issue. Chlorophyll quantification The method most often used for chlorophyll quantification is that of Arnon (1949) which involves the homogenization of leaf tissues in acetone and centrifugation. However, we prefer the extraction method of Hiscox and Israelstam (1979), because it can be done with a smaller sample size (i.e., 5 mg of tissue) using dimethyl sulfoxide. The chlorophyll extract is stable for several more days than when extracted with acetone, and the analysis can be done using disposable cuvettes, whereas Arnon's method 16 requires the use of glass or quartz cuvettes. In brief, place 5 mg (or more) of leaf tissue in a glass tube with 2 ml of dimethyl sulfoxide. Seal the tube and place it in an oven at 65 °C for 2 h. Chlorophyll tip #1. Parafilm will melt in the oven. Do not use it to seal the tubes. Instead use the heat and chemical resistant Dura Seal film (PGC, Gaithersburg, MD) or tight-fitting culture tube caps. Vortex the samples every 30 min. Transfer the dimethyl sulfoxide to clean tubes and extract the tissue with another 1 ml of dimethyl sulfoxide for an additional 1 h in the oven. Combine the extracts and determine the absorbance of 645 and 663 nm with a spectrophotometer using the equation developed by Arnon (1949). Chlorophyll tip #2. The Arnon equation to quantify chlorophyll is as follows: mg chlorophyll L-1 = A645 x 20.2 + A663 x 8.02 Make sure to dilute the extract if the absorption at 663 nm exceeds 0.7 units because this will be outside the linear range of the equation. Chlorophyll tip #3. Remember that some plants with a thicker cuticle may require longer exposure to dimethyl sulfoxide for complete extraction of chlorophylls. Carotenoid quantification The extraction process for carotenoids is more involved than for chlorophylls. Total carotenoids are extracted from as little as 5 mg of tissue, and total carotenoid concentrations are determined spectrophotometrically according to Sandmann and Böger (1983). Place 5 mg samples of fresh green tissue in 16x100 mm glass tubes containing 3 ml of basic methanol (6% KOH in methanol w/v). All subsequent steps must be performed under dim green light. Homogenize the samples at full speed for 30 seconds, and leave at RT for 15 min. Centrifuge for 5 min at 2,000 g, and transfer the supernatant to clean glass tubes. Extract the carotenoids by partitioning with 3 ml of ether:petrol (1:9 v/v). Mix the tubes vigorously and wait for 5 min. Carotenoid tip #1. Be sure to use petrol (petroleum benzin) with a boiling point range between 80- 110 °C. Petrol with a higher boiling range does not work as well. 17 Add 1.5 ml of saturated sodium chloride to the solution, mix again, and centrifuge at 2,000 g for 5 min. Collect a 2 ml aliquot from the top organic layer, transfer to a clean tube, place the tube on a heat block set on low heat and dry under gentle flow of nitrogen in the hood. Carotenoid tip #2. Disposable cuvettes do not work because acetone reacts with the plastic. Resuspend the carotenoids in 200 µl of acetone and quantify in quartz microcuvettes on a spectrophotometer at 445 nm with an extinction coefficient of ε445 = 2500 (% w/v). Carotenoid tip #3. ε445 = 2500 (% w/v) means that a solution containing 1 g of carotenoid in 100 ml would have an absorbance of 2500 unit at 445 nm. For the purpose of the method described above, µg of carotenoids g-1 FW is obtained with the following equation: μg carotenoids/g FW = ���� � 0.000012 � ! � 10" Where A445 is absorbance of the 3 ml ether:petrol phase obtained at the end of the extraction. Make sure to convert the mg of tissue measured into g FW for the equation. Electrolyte leakage Cultivate plants in a growth chamber with a 16/8 light/dark cycle for 7-10 d until cotyledons are fully expanded. Cucumber seedlings (Cucumis sativus (L.) work well for screening most herbicidal compounds. Place twenty-five 4-mm cotyledon discs on top of 5 ml of bathing medium (2% sucrose/1 mM Mes [2-(N- morpholino)ethanesulfonic acid] buffer that has been adjusted to pH 6.5 at room temperature) in 60x15 mm disposable Petri dishes. Prepare test compounds in 100X stocks and add as 50 µl aliquots to the bathing medium. Expose the control samples to the same volume of solvent (acetone or methanol) in the absence of test compound. Incubate the plates in darkness for 16 h prior to exposure to high light intensity. Leakage tip #1. For optimum detection of mode of action that might be light-dependent, such as the effect of compounds inhibiting protoporphyrinogen oxidase, it is very important to expose the plants to high light intensity without heating the bathing medium as the conductivity meter is very sensitive to temperature change. We expose our discs to 1000 µmol m-2 s1 photosynthetically active radiation (PAR) in an incubator (Model E-30-B, Percival Scientific, Boone, IA). 18 Conductivity of the bathing medium can be measured at many time intervals during the periods of dark and light incubation with a conductivity meter (Figure 4.1, left panel) (DUKE; KENYON, 1993). Alternatively, the experiment can be simplified by making measurements at only three specific time points, namely at the beginning of the dark incubation period, after 16 h (overnight) dark incubation, and after 8 h of light exposure (Figure 4.1, right panel) (DAYAN; WATSON, 2011). Leakage tip #2. Measure maximum conductivity by boiling three samples of each treatment for 20 min. This value serves as an upper limit to gauge how much leakage is observed relative to total possible leakage. Compounds with light-independent modes of action will cause leakage (increase in conductivity) during the period of dark incubation, whereas those with light-dependent modes of action will only cause leakage when the samples are exposed to light. Figure 4.1 - Left panel shows a time course experiment illustrating the light-independent (�) and light-dependent (�) loss of membrane integrity, compared to solvent control with no inhibitor (�). Arrows represents the 3 time measurements used in the simplified assay shown in panel B. The dotted line represents maximum change in conductivity (obtained after boiling the discs) from Dayan and Watson (2011). Time (h) 0 8 16 24 32 40 C o n d u c ti v it y ( ∆ m o h m ) 0.0 0.1 0.2 0.3 0.4 0.5 0.6 A B Start Dark Light 19 4.4 Acetyl-CoA Carboxylase - Molecular Target of Herbicide Class A or 1 Acetyl-CoA carboxylase (ACCase, EC 6.4.1.2) catalyzes the ATP- dependent carboxylation of acetyl-CoA to form malonyl-CoA, the first committed step in fatty acid biosynthesis (Figure 4.2A). There are two forms of ACCase: a prokaryote form consisting of three protein components biotin carboxylase, carboxyltransferase, and biotin carboxylase carrier protein (SASKI; NAGANO, 2004) and a eukaryote form consisting of three functional domains on a single polypeptide ( SASKI; NAGANO, 2004). Most higher plants have the prokaryotic ACCase in the plastids that is responsible for fatty acid biosynthesis and the eukaryotic ACCase in the cytoplasm. However, members of the Poaceae (grasses) only have the eukaryotic form of ACCase in both the plastids and the cytoplasm (SASKI; NAGANO, 2004). The eukaryotic ACCase, but not the prokaryotic ACCase, is inhibited by the cyclohexanediones and the aryloxyphenoxy propionates. Hence, the selectivity of these graminicides is due to the specific inhibition of the eukaryotic form of ACCase. The key component in measuring ACCase activity is the incorporation of 14C from CO2 into a component that is acid- and heat-stable. This enzyme assay can only be conducted in labs that have a license to use radiolabeled material. ACCase tip #1. Radioactive CO2 is released in this assay. The assay should only be conducted in a fume hood or some type of protecting apparatus that will prevent the radioactive carbon from contaminating the immediate atmosphere. Enzyme extraction. The procedure is adapted from Seefeldt et al. (1996) and Yu et al. (2003). Collect 3 g of plant material from the most actively growing shoot tissue, which has the highest level of ACCase. Conduct the protein extraction in a cold chamber at 3 °C, or keep everything on ice during the extraction. Grind the tissue in liquid nitrogen and sterile washed sea sand in a mortar and pestle with 5 ml extraction buffer (100 mM Tris (pH 8), 1 mM EDTA, 10% glycerol, 2 mM L-ascorbic acid, 1 mM PMSF, 20 mM DTT, 0.5% PVP40, 0.5% PVPP). Filter the extract through 4 layers of cheesecloth into a centrifuge tube. Rinse the mortar and pestle with an additional 5 ml of extraction buffer and add to the original extract. Centrifuge the solution at 25,000 g for 15 min and discard the pellet. Add saturated ammonium sulfate drop wise to the supernatant to a final concentration of 66% of the original supernatant volume. Stir the solution for 1 h on ice to allow the protein to precipitate. 20 ACCase tip #2. Saturated ammonium sulfate (100%) is made by adding 761 g of (NH4)2SO4 to 1000 ml dH2O at 25 °C. Adjust pH to 6.8-7.0 with 5 or 10 M NaOH. Centrifuge the solution at 25,000 g for 30 min. Discard the supernatant, and dissolve the pellet in 2.7 ml elution buffer (50 mM Tricine, pH 8.0, 50 mM KCl, and 2.5 mM MgCl2). Pipette the solution onto an equilibrated Sephadex G-25 column (GE Healthcare Bio-Sciences Corp, Piscataway, NJ) and elute with 2.8 ml elution buffer. Samples can be stored in a final 25% glycerol concentration at -80 °C until the assay is conducted. ACCase tip #3. It is critical to extract young tissue to maximize the amount of ACCase isolated. In grasses this will either be the base of the plants, prior to tillering, or the young tillers. Figure 4.2 - A) Acetyl-CoA carboxylase location and activity in Poaceae. B) Dose- response curve of clodinafop on ACCase activity from wild-type (�) and resistant (�) populations of Alopecurus myosuroides (data from Délye et al., 2003). Enzyme assay. Conduct this assay in a fume hood or in a container that will trap CO2. The assay solution consists of 1 ml of 20 mM tricine (pH 8.3), 10 mM KCl, 2 mM MgCl2, 2.5 mM dithiothreitol, 0.1% BSA, 10 mM ATP, 40 mM NaHCO3 which includes 2 kBq of NaH14CO3, and 50 µl of enzyme extract. Incubate the solution at 30 C for 3 min before adding acetyl-CoA at 3 mM final concentration. The blanks include all of the reagents except for acetyl-CoA. Incubate the reaction at 30 °C for 15 min and stop the reaction by adding 20 µl glacial acetic acid. Allow the samples to stand open for 1 21 h to allow the escape of 14CO2. Transfer the assay solution to the surface of a glass fiber filter inside liquid scintillation vials and allow the samples to dry before adding 10 ml scintillation cocktail. Measure the radioactivity in the samples via liquid scintillation spectroscopy. See below for stock solutions and assay procedure. For herbicide sensitivity measurements, include at least 4 different concentrations of the ACCase inhibitor in the assay. A typical curve for inhibition of ACCase is shown in Figure 4.2B (DÉLYE et al., 2003). Stock Solutions 200 mM Tricine Buffer: Dissolve 3.58 g of Tricine in 70 ml of deionized water. Adjust pH with 5 N NaOH to 8.3. Bring volume to 100 ml. Store at room temperature 10% BSA: Dissolve 5 g of molecular biology grade BSA in 50 ml of deionized water. To avoid clumping, dissolve the powder by layering on the surface of the liquid and gently rocking a capped tube until it dissolves. Do not stir. Store in 2 ml aliquots at -20 C. 20 mM MgCl2: Dissolve 0.407 g of MgCl2 into 70 ml of deionized water and bring to 100 ml final volume. Store at room temperature. 0.4 M NaHCO3: Dissolve 5.04 g of NaHCO3 in 100 ml of H2O. Adjust the volume to 150 ml to yield a final concentration of 0.4 M. Filter-sterilize the solution, and store at room temperature. 100 mM KCl: Dissolve 0.746 g of KCl into 70 ml of deionized water and bring to 100 ml final volume. Store at room temperature. NaH14CO3: Make a stock solution with 20 kbq (1,200,000 or 0.5 µCi) per ml in water. Aliquot into 2 ml aliquots and freeze at -20 °C. 1 M dithiothreitol: Dissolve 1.54 g of DTT in 8 ml of water. Adjust the total volume to 10 ml, dispense as 50 µl aliquots into microfuge tubes, and store them in the dark (wrapped in aluminum foil) at -20 °C (indefinitely). Use 1 aliquot per 20 ml of assay buffer. 30 mM acetyl-CoA: Dissolve 0.233 g of acetyl- CoA trilithium salt in 10 ml of water. Prepare fresh for each assay or the stock solution. Stocks can be dispensed into 2 ml aliquots and stored in - 80 °C freezer where they will be stable for 6 months. 100 mM ATP: Dissolve 1.2 g of ATP into 12 ml of water. Adjust pH to 7.0 with 4 N NaOH. Adjust final volume to 20 ml with water. Store in 2 ml aliquots at -20 °C indefinitely. Glacial acetic acid Assay Assay solution Conduct assay in fume hood 2 ml tricine buffer 1. Dispense 0.7 ml of the assay solution into tube. (Assay can be done in scintilation vials.) 2. Add 0.1 ml of a 10X stock solution of ACCase inhibitor. 3. Add 50 µl of enzyme extract to tube. 4. Incubate at 30 °C for 15 min. 5. Initiate reaction by adding 0.2 ml of acetyl-CoA stock solution, except for the blanks to which is added 0.2 ml of water. 6. Incubate at 30 °C for 15 min. 7. Stop reaction by adding 20 µl of glacial acetic acid to each tube. 8. Let tubes stand open for 1 h. 9. Transfer solution to glass fiber filter in scintillation vial and allow to stand for an additional h. 10. Add scintillation cocktail and measure dpm. 2 ml MgCl2 2 ml KCl 50 µl DTT 2 ml ATP 2 ml BSA 2 ml NaHCO3 2 ml NaH14CO3 1.9 ml DI water 22 4.5 Acetolactate Synthase - Molecular Target of Herbicide Class B Acetolactate synthase (ALS, EC 4.1.3.18; also called acetohydroxyacid synthase or AHAS) is an enzyme in the pathway to the three branched chain amino acids leucine, isoleucine, and valine (Figure 4.3A). Plants treated with these inhibitors stop growing, eventually wilting and turning red due to the accumulation of stress-induced anthocyanins. Inhibiting ALS causes accumulation of one of its precursors, 2-ketobutyrate, which is transaminated in situ to 2-aminobutyrate (LOPER et al., 2002). At least part of the phytotoxicity appears to be due to the accumulation of these phytotoxic compounds. Accumulation of 2-aminobutyrate. The effects of ALS inhibitors can be measured by extracting and quantifying the accumulation of 2-aminobutyrate by HPLC. Grind fresh shoots (250 mg) in an ice-cold mortar using liquid nitrogen and homogenize in 900 µl deionized water. Precipitate proteins by adding 50 µl cold trichloroacetic acid to the extract, incubating for 15 min on ice and centrifugation at 45,000 g at 4 °C for 3 min. Prepare the reagent solution by adding 5 µl of undiluted β- mercaptoethanol to 1 ml O-phthaldialdehyde. Derivatize 2-aminobutyrate to a fluorescent form by mixing 100 µl of the reagent solution with a 20 µl aliquot of the supernatant within 2 min prior to analysis. ALS tip #1. O-phthaldialdehyde reagent is used to derivatize primary amines and amino acids. Detection of individual fluorescent derivatives requires reverse-phase HPLC separation. The presence of excess sulfhydryl (in the form of β-mercaptoethanol) is necessary. Analysis is done with a C18 reverse-phase column with the following solvent system. Mobile phase A is 2.7 g sodium acetate trihydrate, 180 ml triethylamine and 3 ml tetrahydrofuran in 1 L of deionized water at pH 7.2. Mobile phase B is 2.7 g sodium acetate in 200 ml deionized water at pH 7.2, 400 ml acetonitrile and 400 ml methanol. Start the gradient at 80% A and 20% B and reach 20% A and 80% B over 20 min. Wash the column with 100% B for 7 min and reequilibrate to the initial conditions for 5 min. The flow rate is 0.5 ml/min. Detect the O-phthaldialdehyde derivatives with a fluorescence spectrophotometer with excitation and emission wavelength set at 340 and 450 nm, respectively. Quantify 2-aminobutyrate based on a calibration curve obtained with technical standard (LI; WANG, 2005). 23 Leaf disc assay. In order to measure ALS activity, it is first necessary to induce acetolactate accumulation (Figure 4.3B). This is achieved by floating leaf discs (50 mg) from young leaves on 4 ml of 25% Murashige and Skoog nutrient medium with 500 µM CPCA (1,1-cyclopropanedicarboxylic acid), an inhibitor of ketol- acid reductoisomerase (KARI), in 60x15 mm disposable Petri dishes (GERWICK et al., 1993) or 96 well microtiter plates. The effect of herbicides targeting ALS can then be evaluated by measuring the reduction in acetolactate levels (Figure 4.3C). Typical dose- response curves range from 0.001 and 1000 µM. ALS tip #2. It is very important to prevent bacterial contamination. Use clean equipment and wash any dirt off the leaves or plants to be tested before sampling. Use very sharp cork borers or cutting equipment. Do not crush leaf tissue during preparation. Handle the plant material as little as possible to prevent cell disruption. If any solutions are cloudy after the incubation period, discard. Bacteria will accumulate very large amounts of acetolactate, but the ALS activity in bacteria is completely resistant to ALS inhibitors. Ampicillin (50 units/ml) can be added to the incubation solution to slow down bacterial growth. ALS tip #3. It is very important to take discs from rapidly growing leaf tissue. For broadleaf species, this is the youngest leaf from which leaf discs can be taken. For seedling grasses, cut segments (5 mm/segment) from the base of the plant, which contain the apical meristem. If grasses are tillering, sampling the tillers is better than using the main stem. 24 Figure 4.3 - A) Acetolactate synthase (ALS) catalyzes an important step in the synthesis of branched chain amino acids via two parallel reactions. B) Effect of alanine on acetolactate accumulation in wheat leaf discs. C) Dose-response curve of ALS inhibitor imazamox on ALS activity from wild-type (black) and herbicide resistant (white) canola in leaf disc assays. KARI, ketoacid reductoisomerase is inhibited in order to cause 2-acetolactate accumulation. Incubate Petri dishes with leaf samples in a growth chamber at 25 °C under fluorescent light (250 µmol m-2 s-1 PAR) for 24 h. Dry leaf discs on paper towels and transfer into 1.5 ml microcentrifuge tubes with 300 µl deionized water and freeze at - 80 °C overnight (the samples are stable until analysis). For analysis, thaw samples and allow acetolactate to diffuse into the solution by shaking the tubes on a thermomixer at 60 °C for 60 min. Centrifuge for 5 min at 20,800 g. Mix a 100 µl aliquot with 10 µl of 6 N H2SO4. Incubate the mixture at 60 °C for 30 min to convert acetolactate to acetoin. 25 Microtiter plates. Fill each well with 100 µl assay buffer. Place 1-3 5-mm diameter leaf discs in each well. To maximize accumulation of acetolactate, 50 mM alanine can be added to the incubation solution (Figure 4.3B). Wrap the plate in plastic wrap to prevent evaporation and incubate under light as described above for 16 to 24 h. Stop the reaction by adding 25 µl of 5% H2SO4 to each well and place the plate at -20 °C, where it can remain until further analysis. After thawing the plate, the leaf discs should be a uniform grey-green, indicating that the acid has completely penetrated the discs. Transfer 75 µl of the extract from each well to another microtiter plate and add 125 µl of the naphthol/creatine reagent described below. Incubate the plate at room temperature for 60 min or at 60 °C for 15 min. If the solutions appear cloudy from precipitate, the plates can be centrifuged and 150 µl of the sample transferred to another plate. Acetoin is quantified by a modified colorimetric assay (WESTERFELD, 1945) by derivatization with α-naphthol to form a red complex. Add 50 µl of 0.5% (w/v) creatine and 50 µl α-naphthol reagents to each well and heat the mixtures at 60 °C for 15 min. After cooling, centrifuge the tubes for 5 min at 20,800 g. Transfer 100 µl to a microtiter plate and record absorbance at 540 nm on a microplate reader (Figure 4.3B, C). ALS tip #4. α-Naphthol reagent (5% w/v) must be made fresh by adding 1 g of α-naphthol to 20 ml of 2.5 N NaOH. α-Naphthol is readily oxidized so the solution should be clear and should not be used if its color is more than faint yellow. Creatine is added to the reaction mixture to enhance the color development. Crude enzyme extraction. It is very important to use young plants and do all the extraction steps on ice. Grind 1 g of young plant tissue in liquid nitrogen and sterile quartz sand using a mortar and pestle. Homogenize the sample after the addition of 3 ml ALS extraction buffer. Stir the homogenate for 2 min, transfer to two 1.5 ml microcentrifuge tubes, and centrifuge for 5 min at 20,800 g at 4 °C. Combine the supernatants and apply 2.5 ml of the extract to a Sephadex G25 column (PD-10, GE Healthcare Biosciences Pittsburgh, PA). This column has to be equilibrated with 25 ml extraction buffer prior to loading the sample. Wash the column with 1 ml of elution buffer and discard the eluant because it contains very little ALS activity. Elute the ALS- 26 containing fraction with an additional 2.5 ml of elution buffer. Total soluble protein is quantified by the method of Bradford (1976). ALS tip #5. If the ALS activity in the extract is very low, it can be enhanced by ammonium sulfate precipitation. It is critical that the extract never freeze and form ice crystals. If the extract forms ice crystals, all the ALS activity is lost. ALS assay. Add 70 µl ALS assay buffer, 20 µl crude enzyme extract and 10 µl herbicide solution to 1.5 ml microcentrifuge tubes. The blank consists of 100 µl ALS assay buffer. Incubate the assays for 1 h at 37 °C and stop the reaction by adding 10 µl of 6 N H2SO4. A positive control estimating the baseline concentration of acetolactate consists of the complete assay performed with 10 µl of 6 N H2SO4 added at the beginning of the reaction. Convert acetolactate to acetoin by heating the acidified samples. Quantify acetoin as described above. ALS tip #6. Relative ALS activity is calculated as follows: #$%&'()$ ��* &+'()(', = �-./012 − (�/56 − 7%&89) (�/.; − (�/56 − 7%&89) � 100 Asample= absorbance of sample at 540 nm, Amax = absorbance of positive control, Amin = absorbance of sample incubated with H2SO4, Ablank = absorbance of assay buffer. Rapid extraction and assay. Another method that can be used to rapidly determine in vitro ALS activity to screen for resistance is to extract the tissue as described above, but the extraction buffer is 2X the ALS assay buffer. In this method, the tissue is first pulverized as described above. Extract the pulverized tissue in 2X assay buffer as described above and filter the extract through cheesecloth into a tube and centrifuge as described above. Use the supernatant directly for the ALS assay. Fill each well in a microtiter plate with 50 µl of either water or 2X the final concentration for the ALS inhibitor. Pipette 50 µl of the supernatant into each well and incubate and analyze as previously described. This assay is very rapid because it eliminates the ammonium sulfate precipitation and the supernatant is used directly in the assay. This assay is suitable for screening for resistance. 27 ALS tip #7. There can be artifacts in the in vitro ALS assay. Certain species, such as legumes, or ageing tissue have an ALS-like activity that is not related to the anabolic form of ALS. This activity produces acetoin directly from pyruvate and is not involved in the biosynthesis of the branched chain amino acids. To determine if this non-ALS assay is present, use two controls. The first control is to immediately stop the reaction by adding acid to the medium right after adding the enzyme preparation. The second control is to stop the reaction with 0.25 N NaOH at the end of the reaction. Using NaOH prevents the conversion of acetolactate to acetoin and one can substract the acetoin produced through the non-ALS enzyme from the total acetoin measured. ALS extraction buffer (100 ml) 100 mM potassium phosphate Start with 80 ml of water, add components, cool on ice, adjust pH to 7.5 and bring to a final volume of 100 ml. 0.5 mM MgCl2 (33.5 µl of 3 M MgCl2) 0.5 mM thiamine pyrophosphate 0.01 mM flavine adenine dinucleotide 1 mM diethyldithiocarbamate 13.5% glycerol (v/v) Elution buffer (200 ml) 100 mM potassium phosphate Start with 160 ml of water, add components, cool on ice, adjust pH to 7.5 and bring to a final volume of 200 ml. 20 mM sodium-pyruvate 0.5 mM MgCl2 (33.5 µl of 3 M MgCl2) ALS assay buffer (100 ml) 25 mM potassium phosphate Start with 80 ml of water, add components, adjust pH to 7.0 at room temperature and bring to a final volume of 100 ml. 0.5 mM MgCl2 (33.5 µl of 3 M MgCl2) 0.5 mM thiamine pyrophosphate 0.02 mM flavine adenine dinucleotide 50 mM sodium pyruvate 4.6 Photosystem II - Molecular Target of Herbicide Class C1, C2 and C3 The paradigm underlying chlorophyll fluorescence analysis is that the light energy absorbed by chlorophylls is used to drive photosynthesis (photochemical energy), and excess energy is released as non-photochemical energy, such as heat and chlorophyll fluorescence (Figure 4.4A). While chlorophyll fluorescence accounts for only 1–2% of the total light absorbed, it is easily measured and has been a powerful tool to investigate plant physiological processes (Figure 4.4B) (DAYAN; ZACCARO, 2012). The 28 light reaction of photosynthesis can also be monitored by measuring the rate of O2 evolution (Figure 4.4C). Chlorophyll fluorescence. Chlorophyll fluorescence can be measured non-destructively on intact plants exposed to herbicides, or on leaf discs floating on 5 ml of bathing medium with known inhibitor concentrations in 60x15 mm disposable Petri plates (Figure 4.4B). With the instrument set on Kinetic Mode, adjust so that the initial Ft (instantaneous fluorescence signal) value in the control samples is approximately 210. Set the instrument detector gain between 75 and 85. Quantum yield is determined by the following light treatment: each cycle consisted of a 0.8 s pulse of saturating light generated with a laser diode actinic source to saturate PSII, followed by a 4 s far-red light pulse used to re-oxidize PSII, and a 10 s delay to allow PSII to regain steady-state conditions. A total of seven cycles are performed for each sample. ETR values are expressed as percents of the ETR average values observed in control treatments. Fluorescence tip #1. The opening of the clip holding the probe at the correct angle over the leaf surface can be made smaller by taping a piece of non-reflective black paper with a smaller hole. The gain may have to be adjusted but this technique works well with leaf discs. Bathing medium (200 ml of 5X) 2% sucrose Start with 160 ml of water, add components and adjust pH to 6.5 at room temperature. 1 mM MES 29 Figure 4.4 - A) Diagram of the Z-scheme describing the hill reaction from Dayan et al., 2010. The sites of herbicide interactions are indicated with the arrows. B) Effect of amicarbazone (10 µM) on wild-type (white) and herbicide resistant pigweed (black) photosynthetic electron transport and C) oxygen evolution dose-response curve. Photosynthetic oxygen evolution. Oxygen evolution can be monitored from intact chloroplasts preparation. Spinach is used routinely in our laboratory because it yields excellent chloroplast preparations, but other plants can be used as well. Homogenize 50 g of tissue and 250 ml of extraction buffer in a blender. Filter the homogenate through one layer of miracloth lined by two layers of cheesecloth and collect it in a cold beaker. Centrifuge the filtrate for 20 min at 6000 g at 4 °C. Resuspend the pellet containing chloroplasts in 0.5 ml of resuspension buffer. Rinse the tubes with 0.5 ml of additional buffer through each tube and combine with the chloroplast extract. This crude 30 chloroplast extract can be used for oxygen evolution, but it contains broken chloroplasts and is contaminated with mitochondria. Oxygen evolution tip #1. Intact chloroplasts can be obtained by centrifugating the crude extract on a sucrose step gradient (30%-52%) 60 min at 80,000 g at 4 °C but this requires an ultracentrifuge and swinging buckets. Transfer the layer containing intact chloroplasts into a pre-cooled tube containing about 3 ml of resuspension buffer. Centrifuge 15 min at 6000 g. Resuspend the pellet in 3 ml of resuspension buffer and collect the clear chloroplasts as a soft pellet after centrifugation for 15 min at 6000 g. O2 evolution can be measured polarographically using an oxygen probe. These experiments are performed under saturating light conditions (2400 µmol m-2 sec-1 photosynthetically active radiation). We use a fiber-optic light with two sources and place one fiber optic over the top of the chamber and the other one perpendicular to the chamber to maximize light exposure. All assays are performed at 30 °C using a recycling water heater. I50 values of inhibition of oxygen evolution can be obtained from dose- response curves (Figure 4.4C). Extraction buffer (200 ml of 5X) 1,650 mM sorbitol Start with 160 ml of water, add components, cool on ice, adjust pH to 7.7 and bring to a final volume of 200 ml. Store at –20 °C in 50 ml aliquots. Dilute 50 ml of stock to 250 ml final volume before use. 50 mM HEPES 25 mM cysteine 5 mM MgCl2 (0.335 ml of 3 M MgCl2) 5 mM EDTA (2.0 ml of 500 mM EDTA) Resuspension buffer (100 ml of 5X) 1,650 mM sorbitol Start with 80 ml of water, add components, cool on ice, adjust pH to 7.7 and bring to a final volume of 100 ml. Store at -20 °C in 25 ml aliquots. Dilute 25 ml of stock to 125 ml final volume before use. 50 mM HEPES 5 mM dithiothreitol 5 mM MgCl2 (0.167 ml of 3 M MgCl2) 5 mM EDTA (1.0 ml of 500 mM EDTA) Oxygen evolution assay buffer (1X) 800 mM sucrose Start with 400 ml of water, add components, bring to 30 °C, adjust pH to 6.2 and bring to a final volume of 500 ml. Maintain at 30 °C in water bath during assay. Store in refrigerator for up to a week. 50 mM MES-NaOH 15 mM CaCl2 1 mM FeCN (potassium ferrocyanide) 31 4.7 Photosystem I electron diversion - Molecular Target of Herbicide Class D Photosystem I (PSI) is on the second part of the Z-scheme of photosynthetic electron transport. It provides reducing the power to reduce nicotinamide adenine dinucleotide phosphate (NADP+) to its reduced form, NADPH, which is required for carbon fixation and other biochemical processes. In daylight, PSI has very strong reducing power, and PSII is generating high levels of O2. Under these conditions, compounds with a redox potential between -300 and -700 mV (e.g., paraquat = -446 mV) can be reduced by receiving electrons from PSI. This results in the production of large quantities of superoxide anion, which, in turn generates other reactive radicals, including the highly toxic hydroxyl radical, ultimately leading to massive lipid peroxidation. In cell membranes, this process quickly causes membrane dysfunction and cell death. There are no specific assays to monitor the mechanism of action of paraquat and other PSI inhibitors. However, the assays described to measure the effect of PSII inhibitors on chlorophyll fluorescence in leaves and on photosynthetic oxygen evolution from isolated chloroplasts can be useful, but PSI inhibitors induce different responses (see “Chlorophyll Fluorescence” in the “Photosystem II” section) (DAYAN; ZACCARO, 2012). The electrolyte leakage experiment described earlier can also provide some information on these types of inhibitors (see “Electrolyte Leakage” in the “Common Protocols section”) (DAYAN; WATSON, 2011). 4.8 Protoporphyrinogen oxidase - Molecular Target of Herbicide Class E Protoporphyrinogen IX oxidase (PPO, EC 1.3.3.4) catalyzes the last biochemical step in common between heme and chlorophyll synthesis (DAYAN; DUKE, 2010). One of the key features of the inhibition of PPO is the rapid accumulation of the highly photodynamic tetrapyrrole protoporphyrin IX (Proto), the product of the reaction (Figure 4.5A) ( DAYAN; DUKE, 2011; MATRINGE; SCALLA, 1988). A key aspect of the mechanism of action of class E inhibitors is the generation of reactive oxygen species in the presence of light. Therefore, the simple electrolyte leakage experiment can easily identify the light-dependent nature of these herbicides (see “Electrolyte Leakage” in the “Common Protocols” section) (DAYAN; WATSON, 2011). Analysis of Proto levels. The effect of PPO inhibitors can be evaluated by measuring the accumulation of Proto in whole plants or in leaf disc (Figure 32 4.5B). A number of methods for the extraction and analysis of Proto from leaf tissues are available. The method presented here has been used successfully with a number of herbicides in our laboratory (DAYAN et al., 1997a; DAYAN et al., 1997b; DAYAN et al., 1997c; LI et al., 2004). PPO tip #1. Prevent light exposure because Proto is highly photodynamic; therefore, all the windows in the laboratory should covered with aluminum foil and several lamps have been mounted with green filters. Homogenize approximately 100 mg of leaf disks in 2 ml of basic methanol (HPLC-grade methanol–0.1 N ammonium hydroxide [9:1 v/v]) at full speed for 15 s and keep in the dark for 10 min. Collect the supernatant after centrifugation at 3,000 g for 3 min. PPO tip #2. Use Corex tubes because regular glass tubes tend to break in the centrifuge. Resuspend the pellet in 1 ml of basic methanol, keep in the dark for 10 min, and collect the supernatant after centrifugation as above. Combine and filter the supernatants through a 0.2-µm nylon syringe filter to remove particles, and store in light- tight glass vials at -20 °C until analysis by HPLC. PPO tip #3. Use scintillation vials covered with aluminum foil, which work well. Proto has a strong absorbance at 400 nm, which is similar to chlorophylls and carotenoids, therefore separation by HPLC is necessary to resolve Proto. Protocols must be adapted for individual HPLC/column systems but as a general principle, a reverse phase column (such as a 4.6 by 250-mm Spherisorb 5-mm ODS-1 preceded by an ODS-5S guard column) [Waters Corporation, Milford, MA]) works well. The solvent system consists of a gradient beginning at 60% HPLC-grade methanol and 40% deionized water. At 10 min, the gradient is 100% methanol to wash the column of lipophilic coextractants; then the solvente system is returned to the original settings after 30 min. PPO tip #4. Proto can be visualized with a photodiode array as a peak with a maximum near 400 nm but the detection limit is not very good. Instead, use a fluorescence detector with excitation and emission wavelength settings at 400 and 630 nm for much greater sensitivity. 33 PPO tip #5. Set the photodiode array detector to scan from 300 to 700 nm and chlorophylls and carotenoids can easily be differentiated according to their respective spectra. Quantify Proto levels in the extracts using a calibration curve obtained with a commercially available Proto standard. Measuring PPO activity. Assaying for PPO activity is a rather delicate process for a number of reasons. Synthesis of the substrate is complicated and dangerous. The assay also requires a spectrofluorophotometer, which is not common in most laboratories. Based on these facts it was decided not to describe the method here. For more information check (DAYAN et al., 2010a; JACOBS; JACOBS, 1987). Figure 4.5 - A) Protoporphyrinogen oxidase (PPO) catalyzes the conversion of the colorless protoporphyrinogen IX (Protogen) to the highly fluorescent protoporphyrin IX (Proto). The dotted arrow represents the non-enzymatic step leading to proto accumulation when PPO is inhibited. B) Effect of 10 µM acifluorfen on wild-type (white) and herbicide resistant (black) Amaranthus tuberculatus and C) dose-response curves of acifluorfen on heterologously expressed PPO from wild-type (�) and herbicide resistant (�) Amaranthus tuberculatus. 34 4.9 Phytoene Desaturase - Molecular Target of Herbicide Class F1. Phytoene desaturase (PDS, EC 1.3.5.5) participates in the carotenoid biosynthesis pathway (DAYAN; DUKE, 2003). Key aspects of this mode of action are the bleaching (reduction of carotenoid and chlorophyll levels) in the newly emerging tissue and the accumulation of phytoene (Figure 4.6A). Please refer to the sections “Chlorophyll Quantification” and “Carotenoid Quantification”. Generally, the bleaching effect of a PDS inhibitor can be distinguished from that of an inhibitor of p-hydroxyphenylpyruvate dioxygenase (HPPD) by applying the herbicides to a leaf and observing where the bleaching occurs. PDS inhibitors are not phloem mobile because they are very lipophilic so bleaching will occur on the new growth within the treated leaf. On the other hand, commercial HPPD inhibitors are less lipophilic and tend to translocate, causing bleaching of the meristematic tissue away from the treated leaf. Phytoene quantification. Phytoene is extracted and quantified by the method of Sprecher et al. (1998), which is very similar to that described for carotenoids analysis. However, more tissue is required and detection is done in petrol because the absorption spectrum of acetone interferes with that of phytoene. Weigh out between 100 to 250 mg fresh shoot tissue and homogenize in 3 ml of basic methanol (6% KOH in methanol w/v) in 16x100 mm glass tubes. Leave the samples at room temperature for 15 min. Centrifuge for 5 min at 2,000 g and transfer the supernatant to clean glass tubes. Extract Phytoene by partitioning with 3 ml of petrol. Vortex the tubes and allow to stand for 5 min. Add a 1.5 ml volume of saturated sodium chloride and vortex the tubes again. Centrifuge at 2,000 g for 5 min. Collect and transfer a 1.25 ml aliquot from the top organic layer of each tube to a 1.5 ml methacrylate semimicro, disposable UV cuvette (Fisherbrand, Thermo Fisher Scientific). Quantify Phytoene on a spectrophotometer at 287 nm with an extinction coefficient of ε287 = 1108 (% w/v) (Figure 4.6B). PDS tip #1. Obtain the micrograms of phytoene per g of FW with the following equation: μg phytoene g-1 FW = �@AB � 0.000027 � ! � 10" Where A287 is the absorbance of the 3 ml petrol solution obtained at the end of the extraction. Make sure to convert the mg of tissue measured into g of FW for the equation. 35 Figure 4.6 - A) Phytoene desaturase (PDS) catalyzes the conversion of phytoene to ζ- carotene in the biosynthesis of carotenoids. B) Effect of 12 nM fluridone on phytoene accumulation in wild-type (white) and herbicide resistant (black) Hydrilla verticillata. C). Dose-response curve of fluridone on heterologously expressed PDS from wild-type (�) and herbicide resistant (�) Hydrilla verticillata. Enzyme assay. There have been many attempts to assay phytoene desaturase activity in leaf crude extracts, but doing so is difficult because the substrate is a hydrocarbon chain with no chromophore and is not readily derivatized. However, it has been done by incorporating 14C-geranylgeranyl-pyrophasphate (14C-GGPP) in isolated thylakoid preparations (SANDMANN, 1993) and in isolated chromoplasts from red bell peppers (Capsicum annuum L.) (CAMARA, 1993). 36 PDS tip #2. Be aware that the problem with this approach is that more than 90% of the 14C-GGPP is incorporated into the phytyl tail of the chlorophyll, and only a small portion ends up in carotenoids. Activity of plant PDS, however, can be expressed heterologously in E. coli using conventional molecular techniques (MICHEL et al., 2004). Pellet cells expressing PDS by centrifugation at 3,000 g for 20 min at 4 °C. Wash the pellets with 0.9% sodium chloride, transfer to 50 ml tubes and centrifuge again. Discard the supernatant and store the pellets at -80 °C. For extraction, thaw the pellets and lyse in 7.5 ml PDS assay buffer using a French press at 140 MPa. Add 75 µl of 100 mM PMSF (in ethanol) to obtain 1 mM protease inhibitor and 75 units of benzonase to degrade DNA and wait 20 min at room temperature. Adding the benzonase is very useful to liquify the thick and sticky consistency of the extract and make the centrifugation step much more effective. Centrifuge the lysate at 16,000 g for 10 min at 4 °C and decant the supernatant into a clean tube on ice. Purify the his-tagged PDS protein on a nickel-activated Hitrap Chelating HP column (GE Healthcare Biosciences, Pittsburgh, PA) according to the manufacturer's protocol and elute with 250 mM imidazole. PDS tip #3. Determine the concentrations of imidazole required to elute the his-tagged protein in preliminary experiments. Desalt by loading a 2.5 ml aliquot of the fraction containing PDS activity on a Sephadex G-25 size exclusion column (GE Healthcare Biosciences, Pittsburgh, PA) which has been equilibrated with assay buffer (200 mM sodium phosphate, pH 7.2). Elute the protein with 3.5 ml of assay buffer, adjust the protein concentration to 100 µg ml-1 and store at -80 °C. Synthesis of phytoene. Crude extracts containing phytoene are produced in E. coli JM101/pACCRT-EB expressing geranylgeranyl pyrophosphate synthase and phytoene synthase enzymes from Erwinia uredovora (EB) (MISAWA et al., 1995). The extract (EB extract) is diluted to 10 mg protein ml-1 and stored at -80 °C. This extract contains ample amount of phytoene to assay PDS activity. 37 PDS assay. Thaw one tube of purified PDS extract and one tube of EB extract. Keep on ice. For each sample, add 100 µl of PDS (10 µg protein) to a microcentrifuge tube with 10 µl of either solvent (acetone or methanol) or 100x herbicide solution, and mix thoroughly. Dose-response curves are obtained by testing technical grade herbicides in concentrations ranging from 1 nM to 1 mM (Figure 4.6C). Incubate the enzyme with the herbicides on ice for 15 min prior to starting the reaction by adding 100 µl of EB crude extract (1 mg total protein). The assay is carried out for 30 min at 30 °C with constant gentle mixing on a thermomixer. Activity drops sharply at incubation times in excess of 30 min. Transfer the reaction mixtures to 12x75 mm disposable glass culture tubes containing 1 ml of basic methanol (6% KOH in methanol w/v). Rinse the microcentrifuge tube with 200 µl of basic methanol and add to the glass tube. Mix the solutions thoroughly. Add 1 ml of ether:petrol (1:9) to each tube, mix and place on ice before adding 500 µl of saturated sodium chloride. Centrifuge at 4 °C and 3,900 g for 10 min. Transfer a 600 µl aliquot of the supernatant to a fresh 12x75 mm disposable glass culture tube and dry in a heat block under a gentle flow of nitrogen. Cool the tube to room temperature, then add 125 µl of acetone. Quantify β-Carotene amounts by spectrophotometry at 425 nm in a quartz cuvette using a mM extinction coefficient of ε425=138. PDS tip #4. Realize that mM ε425 = 138 means that a 1 mM solution of β-carotene has an absorbance of 138 unit at 425 nm. For the PDS method described, obtain the nmoles of β-carotene mg-1 protein h-1 with the following equation: nmol β-carotene mg-1 h-1 = ��@� � 45.29 Assuming that 10 µg protein is assayed for 30 min and β-carotene is quantified in a 125 µl final volume of acetone. Saturated sodium chloride (250 ml) 100 g sodium chloride Add sodium chloride to 250 ml of deionized water and bring to a gentle boil while stirring on a hot plate. Add a very small amount of water gradually until salt is completely dissolved. Cool overnight and store at room temperature. Some salt will crystallize but the solution will be saturated. PDS assay buffer (100 ml) 200 mM sodium phosphate Add 28 ml of 200 mM sodium phosphate monobasic to 72 ml of 200 mM sodium phosphate dibasic to obtain pH 7.2. 38 4.10 p-Hydroxyphenylpyruvate Dioxygenase - Molecular Target of Herbicide Class F2 p-Hydroxyphenylpyruvate dioxygenase (HPPD, EC 1.13.11.27) converts p-hydroxyphenylpyruvate (HPP) to homogentisate (HGA), a step involved in the synthesis of prenyl quinones and tocopherol (Figure 4.7A). Because plastoquinone is an essential cofactor for PDS, the symptoms of HPPD inhibition are the same as that of PDS inhibitors (e.g., reduction in chlorophyll, carotenoids and accumulation of phytoene) (Figure 4.7C). There is no known case, to our knowledge, of naturally evolved resistance due to alteration of the enzyme target. Bioassays. The simple tests measuring chlorophyll and carotenoids levels described at the beginning of this review work well with HPPD inhibitors. As a diagnostic tool, the bleaching pattern of HPPD inhibitors is different from that of the PDS inhibitors because of their differences in phloem mobility. HPPD inhibitors also reduce the cellular levels of plastoquinone, whereas PDS inhibitors do not have that effect. Enzyme assay. HPPD activity is not easily assayed in crude plant extracts, but is easily tested by heterologous expression of the plant gene in E. coli (DAYAN et al., 2007). Conventional molecular techniques are used to express the desired plant HPPD gene in E. coli. Harvest induced cells and wash as described in the phytoene desaturase section. Store cells at -80 °C until use. Resuspend cells in 7.5 ml of extraction buffer and lyse with a French press. Add 6-Aminohexanoic acid and benzamidine to final concentration of 1 mM each, along with 750 unit of benzonase, to the lysed cells. Incubate for 20 min at room temperature. Obtain a cell-free supernatant by centrifugation at 35,000 g for 30 min at 4 °C. HPPD tip #1. Always apply protease inhibitors once the cells have been lysed. These compounds typically have very short half-lives in water and must interact with proteases very rapidly to deactivate them