The role of the glucosinolate-myrosinase system for the interaction of Brassicaceae with the turnip sawfly Athalia rosae (L.) Dissertation zur Erlangung des naturwissenschaftlichen Doktorgrades der Julius-Maximilians-Universit¨at W¨urzburg vorgelegt von Nora Verena Travers-Martin, geb. Martin aus Kassel W¨urzburg 2007 Eingereichtam: ................................................. Mitglieder der Promotionskommission Vorsitzender: ................................................. Erstgutachterin: Prof. Dr. Caroline M¨uller, Universit¨at Bielefeld Zweitgutachter: Prof. Dr. Martin J. M¨uller, Universit¨at W¨urzburg TagdesPromotionskolloquiums: ................................................. Promotionsurkunde ausgeh¨andigtam: ................................................. Contents 1 Introduction 11 1.1Theglucosinolate-myrosinasesystem...................... 11 1.2Herbivoresonglucosinolate-containingplants................. 14 1.3Variationinplantdefence............................ 16 1.4Studysystemandscopeofwork........................ 18 1.5Hypothesesandquestions............................ 21 2 Revised determination of soluble and insoluble myrosinase activities in plant extracts 23 2.1Introduction.................................... 25 2.2Materialsandmethods.............................. 28 2.2.1 Plantmaterialandprocessingofsamples............... 28 2.2.2 Myrosinaseassay............................. 28 2.2.3 Glucosinolate-retardingpropertiesofSephadexA-25......... 30 2.2.4 Substrate specificity of soluble and insoluble myrosinases ...... 30 2.2.5 Effectsofascorbicacidonglucosedetermination........... 31 2.3Results....................................... 31 2.3.1 Freeze-drying effects on myrosinase activity concentrations . 31 2.3.2 Glucosinolate-retardingpropertiesofSephadexA-25......... 32 2.3.3 Activity and substrate specificity of soluble and insoluble myrosinases 33 2.3.4 Effectsofascorbicacidonglucosedetermination........... 34 2.4Discussion..................................... 36 2.4.1 Samplepreparation........................... 36 2.4.2 Assayconditions............................. 37 2.4.3 Detection................................. 38 2.4.4 Conclusions................................ 40 3 Induction of plant responses by a sequestering insect: Relationship of glucosinolate concentration and myrosinase activity 43 3.1Introduction.................................... 46 3.2Materialsandmethods.............................. 48 3.2.1 Planting and induction experiment . ................ 48 3.2.2 Analysisofglucosinolates........................ 49 3.2.3 Analysisofmyrosinaseactivity..................... 50 3.2.4 Analysesofprimarymetabolitesandwatercontent.......... 51 3.2.5 Statisticalanalyses............................ 51 3.3Results....................................... 52 3.3.1 Glucosinolatelevels........................... 52 3.3.2 Myrosinaseactivity............................ 55 3.3.3 Correlationsbetweenglucosinolateandmyrosinaselevels...... 56 3.3.4 Primarymetabolitesandwatercontent................ 57 3.4Discussion..................................... 58 3.4.1 Conclusions................................ 61 4 Specificity of plant responses in Sinapis alba L. and their effects on a specialist herbivore 63 4.1Introduction.................................... 65 4.2Methodsandmaterials.............................. 67 4.2.1 Planting and induction experiment . ................ 67 4.2.2 Analysisofglucosinolates........................ 67 4.2.3 Analysisofmyrosinaseactivity..................... 68 4.2.4 Analysesofsolubleproteinandwatercontents............ 69 4.2.5 Behavioural experiments with Athalia rosae .............. 69 4.2.6 Plantvascularconnectivity....................... 70 4.2.7 Statisticalanalyses............................ 70 4.3Results....................................... 71 4.3.1 Damagepatterns............................. 71 4.3.2 Glucosinolateconcentrationsandmyrosinaseactivities........ 73 4.3.3 Solubleproteinandwatercontent................... 76 4.3.4 Behavioural experiments with Athalia rosae .............. 77 4.3.5 Plantvascularconnectivity....................... 78 4.4Discussion..................................... 79 4.4.1 Specificity according to mode of induction . ...... 79 4.4.2 Specificityaccordingtoplantcultivar................. 80 4.4.3 Consequencesforinteraction...................... 80 4.4.4 Conclusions................................ 82 5 Matching plant defence syndromes with preference and performance of a specialist herbivore 83 5.1Introduction.................................... 85 5.2Materialsandmethods.............................. 87 5.2.1 Plantandinsectmaterial........................ 87 5.2.2 Leafchemistry.............................. 88 5.2.3 Leafmorphologyandwatercontent.................. 91 5.2.4 Insectperformanceparameters..................... 91 5.2.5 Insectpreferencebioassays....................... 92 5.2.6 Statisticalanalyses............................ 92 5.3Results....................................... 95 5.3.1 Plantdefencesyndromes......................... 95 5.3.2 Insectperformanceandpreference...................101 5.3.3 Multipleregressionanalysesonplantandinsecttraits........111 5.4Discussion.....................................114 5.4.1 Plantdefencesyndromes.........................114 5.4.2 Insectperformanceandpreference...................115 5.4.3 Matchingofplantandinsectclusters..................117 5.4.4 Conclusions................................118 6 Discussion 121 6.1Variationwithinplantindividualsandshort-termeffects...........121 6.2Variationbetweenplantspeciesandlong-termeffects.............130 6.3Comparisonbetweenshort-termandlong-termeffects............134 6.4Perspectivesandfutureprospects........................137 A Growth of organs after induction treatments 139 B Syndrome supplementary material 145 Bibliography 155 List of Figures 179 List of Tables 181 Summary 183 Zusammenfassung 187 10 11 Chapter 1 Introduction Vascular plants are in general immobile and tied to their immediate environment. In contrast to mobile animals, changes within the habitat need to be coped with directly at the site. Plants therefore require plastic mechanisms of defence against abiotic and biotic stresses (Rosenthal and Berenbaum, 1991). Unpalatability towards insects, one major group of herbivores, is achieved via morphologic characters, e.g. trichomes or leaf toughness, or via defensive secondary metabolites. The chemical nature of these bioactive compounds varies greatly: from alkaloids, over terpenoids and cyanogenic glycosides to glucosinolates (Rosenthal and Berenbaum, 1992). Furthermore, plants can apply pheno- logical or environmental escape mechanisms, e.g. high regrowth capacity, simultaneous leaf expansion, or occurrence in habitats with low densities of herbivores. In this study the interaction of Brassicaceae with the turnip sawfly Athalia rosae (L.) (Hy- menoptera: Tenthredinidae) is examined with emphasis on the glucosinolate-myrosinase system. The larvae of this herbivorous sawfly are specialised to feed on glucosinolate- containing crucifers. Short-term impacts of larval feeding on the physiology of white mustard (Sinapis alba L.), the responses’ specificity and their potential defence effects are investigated. Furthermore, the defensive properties of seven plant species are evalu- ated with regard to nutrition, mechanical and chemical defence and their individual and combined importance for the long-term performance and preference of A. rosae. 1.1 The glucosinolate-myrosinase system The binary glucosinolate-myrosinase system is a very prominent plant defence mechanism (Louda and Mole, 1991). Glucosinolates, once known as mustard oil glucosides, are amino 12 acid derived compounds which are hydrolysed upon tissue damage by the enzymes my- rosinases (Figure 1.1). The resulting breakdown products, called mustard oils, are mostly toxic (Wittstock et al., 2003). The system is primarily found in the plant order Brassi- cales and especially within the family Brassicaceae (Rodman, 1991). Within this clade a monophyletic origin is assumed including 15 plant families, e.g. Capparaceae, Caricaceae, and Tropaeolaceaee. However, the genus Drypetes (Euphorbiaceae) contains species which produce glucosinolates and thus this particular defence mechanism was at least developed twice during the evolution of terrestrial plants (Rodman et al., 1998). A: glucosinolate classes S Glu S Glu S Glu N - N - N - O SO 3 HO O SO 3 N O SO 3 H Aliphatic glucosinolates, Aromatic glucosinolates, Indolic glucosinolates, e.g. 2-propenylgls. e.g. p-hydroxybenzylgls. e.g. indol-3-ylmethylgls. B: enzymatic mechanism R S Glu Glucosinolates 1 N - O SO 3 Myrosinases Primary reaction products D-Glucose 2 R SH Thiohydroximate-O-sulphonates 3 N - O SO 3 Secondary reaction products Hydrogensulphate 4 5 Epithionitriles Oxazolidine-2-thiones Thiocyanates Nitriles Isothiocyanates Figure 1.1: The glucosinolate-myrosinase system (modified from Wittstock and Halkier, 2002). A - Examples of aliphatic, aromatic and indolic glucosinolate structures; B - enzymatic mechanism: upon tissue damage glucosinolates and myrosinases interact to form the primary reaction products D-glucose and thiohydroximate-O-sulphonates. The aglucone is unstable and rearranges to yield hydrogensulphate and one or more compounds of a variety of further decomposition products called mustard oils. Numbers in black circles refer to metabolites for possible detection of activity (Chapter 2). Abbreviations - gls: glucosinolate, R: modified side chain, Glu: D-glucose.