Durham E-Theses Breeding phaseolus vulgaris (common bean) for resistance to the major pest bruchids Zabrotes subfasciatus and Acanthoscelides obtectus. Biochemical bases for seed resistance in wild lines Minney, Benjamin Hugo How to cite: Minney, Benjamin Hugo (1990) Breeding phaseolus vulgaris (common bean) for resistance to the major pest bruchids Zabrotes subfasciatus and Acanthoscelides obtectus. Biochemical bases for seed resistance in wild lines, Durham theses, Durham University. Available at Durham E-Theses Online: http://etheses.dur.ac.uk/5995/ Use policy The full-text may be used and/or reproduced, and given to third parties in any format or medium, without prior permission or charge, for personal research or study, educational, or not-for-prot purposes provided that: • a full bibliographic reference is made to the original source • a link is made to the metadata record in Durham E-Theses • the full-text is not changed in any way The full-text must not be sold in any format or medium without the formal permission of the copyright holders. Please consult the full Durham E-Theses policy for further details. Academic Support Oce, Durham University, University Oce, Old Elvet, Durham DH1 3HP e-mail: [email protected] Tel: +44 0191 334 6107 http://etheses.dur.ac.uk 2 Breeding Phaseolus vulgaris (common bean) for resistance to the major pest bruchids Zabrotes subfasciatus and Acanthoscelides obtectus. Biochemical bases for seed resistance in wild lines by Benjamin Hugo Minney Submitted for the degree of: Doctorate of Philosophy to: Department of Biological Sciences Durham University The copyright of this thesis rests with the author. 1990 No quotation from it should be pubhshed without his prior written consent and information derived from it should be acknowledged. page 1.2 1 8 AUG 1992 Abstract: Phaseolus vulgaris (common bean) is an important source of protein for human and animal consumption. Economic losses post-harvest in storage are primarily due to the bruchid beetles Acanthoscelides obtectus (Bean Weevil) and Zabrotes subfasciams (Mexican Bean Weevil). Wild-lines exhibiting resistance to these two species have been found and the mechanisms of resistance to each species are investigated. The mechanisms of resistance to the two species are found to be multiple, and different for each species. In the case of Z. subfasciatus, the presence of a novel storage protein and absence of the conventional storage protein constitutes the primary mechanism. The novel protein ("arcelin") is antimetabolic when included in artificial diets. In vitro digestibility studies indicate that it is indigestible to Z. subfasciatus larval gut proteases, and since arcelin constitutes the major protein of the seed the larvae starve. Arcelin has a similar amino- acid sequence to PHA. Also present is WBAI, a highly specific inhibitor of larval amylase of the two bruchid pests Z. subfasciatus and Callosobruchus maculatus, whilst having virtually no inhibitory activity on the mammalian amylase, nor on other insect, bacterial nor fungal enzymes. WBAI is similar in gross structure to the conventional amylase inhibitor obtainable from RKB (commercial P. vulgaris), and both are also immunologically similar to PHA. Both of these mechanisms are suitable for incorporation into commercial seed, and the former has already been tested, using meal from F2 seeds, in feeding trials using rats, confirming absence of mammalian toxicity. Resistance to A. obtectus damage is accompanied by reduced starch content, and high content of an acidic polysaccharide (whose structure has not been elucidated). No protein cause for resistance was found. Inheritance of resistance to A. obtectus is recessive. Since the factor responsible for resistance is not a primary gene product and is expressed recessively, this factor is unsuitable for incorporation into breeding lines to be used for developing commercial cultivars. page 1.3 Dedication Close on four years of work is dedicated to Dr Angharad Gatehouse, always a friend and mentor. I dedicate this volume to Ali and Emma. Acknowledgements I would like to thank almost everyone in the department for assistance of one kind or another at one time or another; and also to the staff of Microcomputing Ltd for help whilst writing up. Special thanks are due to Dr John Gatehouse my supervisor, Ms Julia Bryden and Ms Michelle Atterby for technical assistance, and Mr John Gilroy for extensive advice. Dr P. Dobie and Dr C. Haines, and the team at ODNRI Chatham, provided much assistance, and Professor D. Boulter allowed the use of Depanment facilides. The work was funded by a grant from ODA. Declaration The work in this thesis is all original except where acknowledged. Technical work was provided by J. Bryden and M. Atterby; bioassays on artificial diets were performed at ODNRI (Slough or Chatham), and I am indepted to Drs J and A Gatehouse for guidance. The copyright of this thesis rests with the author. No quotation from it should be published without my prior written consent and information derived from it should be acknowledged. page 1.4 Abbreviations 28k Protein subunits of 28kDa relative molecular mass 35k Protein subunits of 35kDa relative molecular mass Arc Arcelin (similar amino-acid sequence to Phaseolus lectin) b.p. years before present CBAI Cultivated Bean Amylase Inhibitor (inhibitor of animal amylase found in cultivated Phaseolus vulgaris) Chi^ A statistical test comparing frequencies observed with frequencies expected for a null hypothesis HO Da Dalton (a measure of protein molecular size) df degrees of freedom of a set of data et al. et alias, and other workers F,, Fj, F3 generations of hybrid cross Gll Globulin protein fraction II, Phaseolus globulin HO Null hypothesis (useful for Chi^ tests) ibidem in the same place Relative molecular mass P probability level for a particular null hypothesis PHA Phaseolus HemAgglutinin {Phaseolus lectin) r, r^ correlation coefficient between two variables WBAI Wild Bean Amylase Inhibitor (inhibitor of bruchid amylase found in wild Phaseolus vulgaris) page 1.5 Table of Contents Introduction 1 Beans and Beetles 116 1.1 ORIGINS OF THE COMMON BEAN PHASEOLUS VULGARIS I.I6 1.2 BRUCHIDAE 1.17 1.2.a Lift History of Zabrotes subfasciatus Boheman 1.18 1.2.b Life History of Acanthoscelides obtectus (Say) 1.20 1.3 SPECIFICITY I-22 1.4 ECONOMIC IMPORTANCE I.24 Introduction 2 Physical barriers 129 2.1 ENVIRONMENT I-30 2.2 TESTA I.30 2.2.a Testa Hardness 1-31 2.2.b Deterrence and Recognition 1-32 2.3 SEED SIZE I-33 Introduction 3 & 4 Biochemical Basis of Resistance 134 Introduction 3 Non-Protein Antimetabolites 136 3.1 ALKALOIDS I-36 3.2 NON-PROTEIN AMINO-ACIDS I-37 3.3 TERPENOIDS I-38 3.4 POLYPHENOLS I-39 3.5 CYANOGENS I-39 3.6 POLYSACCHARIDES I-40 3.7 FLAVONES AND ISOFLAVONES 1-41 Introduction 4 Protein Antimetabolites ^ ^2 page 1.6 4.1 LECTINS I-42 4.2 ARCELIN 1.44 4.3 CARBOHYDRASE INHIBITORS 1.45 4.4 PROTEASE INHIBITORS I.46 4.5 NUTRITIONAL UTILISATION 1.47 Introduction 5 Incorporating Resistance into Commercial Lines 1.49 5.1 CROSS BREEDING I SO 5.2 GENETIC ENGINEERING I.52 5.3 OVERCOMING RESISTANCE 1.53 5.4 MULTICOMPONENT DEFENSIVE MECHANISMS 1.54 Introduction 6 Conclusions and Introduction to Experimental Work 1.55 6.1 AIMS OF THE PROJECT I.56 6.2 THE EXPERIMENTAL WORK 1.57 Materials and Methods 7 Insect Bioassays and Artificial Diets MM.58 7.1 INSECT BIO ASSAY, SEEDS OF ACCESSIONS MM.58 7.2 ARTIFICIAL DIETS MM.58 7.3 INSECT BIOASSAY, ARTIFICIAL DIETS. MM.59 7.3.a Numeric Analysis MM.60 7.3.b Chi-Squared Test MM.60 7.3.C Analysis of Variance MM.61 7.3.d Paired t-test MM.61 Materials and Methods 8 Purification of Proteins MM.62 8.1 GENERAL METHODS MM.62 8.1.a Ion Exchange MM.62 8.1.b Gel Filtration MM.62 8.1.C Ammonium sulphate precipitation MM.63 8.2 EXTRACTION OF ALBUMIN AND GLOBULIN PROTEINS MM.63 page 1.7 8.3 ARCELIN 4 MM.64 8.4 a-AMYLASE INHIBITOR MM.65 8.5 PPTE MM.66 8.5.a Method 1 MM.66 8.5.b Method 2 MM.67 Materials and Methods 9 Protein Characterisation MM.69 9.1 HEMAGGLUTINATION MM.69 9.2 PREPARATION OF ANTIBODIES MM.TO 9.3 POLY-ACRYLAMIDE GEL ELECTROPHORESIS MM.72 9.3.a Basic SDS-PAGE MM.72 9.3.b Preparation of Samples MM.72 9.3.C Recipes for Different Densities of Acrylamide MM.74 9.3.d Visualising Protei is MM.77 9.3.e Visualising Glyco Groups on Proteins MM.77 9.4 SIZING AND QUANTITATION OF SUBUNITS ON SDS- PAGE MM.78 9.5 WESTERN BLOTTING MM.80 9.6 ELECTROBLOTTING TO PVDF MEMBRANE PRIOR TO SEQUENCING MM.83 9.7 N-TERMINAL ANALYSIS MM.84 9.8 QUANTITATION OF THE LEVEL OF GLYCOSYLATION MM.85 9.9 ENZYMATIC DEGLYCOSYLATION OF GLYCOPROTEINS MM.86 9.10 CHEMICAL DEGLYCOSYLATION MM.87 Materials and Methods 10 Carbohydrate Analysis MM.89 10.1 PHENOL-SULPHURIC ACID ASSAY. GENERAL TEST FOR CARBOHYDRATE MM.89 10.2 CARBAZOLE ASSAY FOR URONIC ACIDS MM.90 10.3 L-CYSTEINE/SULPHURIC ACID ASSAY FOR HEXOSES MM.91 10.4 STARCH DETERMINATION BY UV METHOD MM.92 Materials and Methods 11 page 1.8 Enzyme and Inhibitor Characterisation MM.94 11.1 PREPARATION OF LARVAL GUT EXTRACT MM.95 11.2 PROTEASE ACTIVITY MM.96 11.3 a-AMYLASE INHIBITION MM.98 11.3.a Alternative Amylase Assay MM.104 Results 12 Zabrotes subfasciatus Resistance R105 12.1 BIOASSAY OF ACCESSIONS R.ios 12.2 ANALYSIS OF COMPONENTS R.IO7 12.3 EFFECT OF SEED FRACTIONS ON LARVAL DEVELOPMENT R.iii 12.4 SEPARATION OF ALBUMIN PROTEINS AND SOLUBLE CARBOHYDRATES Rin 12.5 DISCUSSION R.120 Results 13 Mechanism of Arcelin Resistance R 123 13.1 IN VITRO DIGESTION OF PURE PROTEINS R.123 13.2 DIGESTION OF TOTAL BEAN MEALS R.129 13.2.a Arcelin 4 in G12953 R-129 13.2.b Other arcelin-containing lines R.129 13.3 INCLUSION OF ARCELIN 3 IN ARTIFICIAL DIETS R.I30 13.4 OVERCOMING EXHIBITED RESISTANCE OF G 12953 BY PHASEOLIN SUPPLEMENTATION R-133 13.5 INCLUSION OF ARCELIN IN MAMMALIAN DIETS R.I37 13.6 CONCLUSIONS TO MECHANISM OF ARCELIN RESISTANCE R140 Results 14 Characterisation of Arcelin 4 R 143 14.1 MOLECULAR SIZE R i43 14.2 PHYTOHEMAGGLUTINATION ACTIVITY R.147 14.3 N-TERMINAL ANALYSIS AND SEQUENCING OF ARCELIN 4 R.150 page 1.9 14.4 ANTIBODIES AND SCREENING F2 GENERATION SEEDS R.ISI 14.5 COMPARING ARCELIN TYPES R.155 14.6 USAGE OF ARCELIN DuRD^^G GERMINATION R.157 14.7 C.