THE INDUCED CHEMICAL DEFENSES OF NORWAY SPRUCE [PICEA ABIES (L) KARST.]: ECOLOGICAL FUNCTION AND REGULATION Dissertation zur Erlangung des akademischen Grades doctor rerum naturalium (Dr. rer. nat.) vorgelegt dem Rat der Biologisch-Pharmazeutischen Fakultät der Friedrich-Schiller- Universität Jena von Master of Science Gazmend Zeneli geboren am 13.08.1972 in Kruja (Albanien) Gutachter: 1. Prof. Jonathan Gershenzon (MP Institute for Chemical Ecology- Jena, Germany) 2. Prof. Frank Hellwig (Friedrich-Schiller-Universität- Jena, Germany) 3. Prof. Marco Michelozzi (National Research Council, Firenze- Italy) Tag der mündlichen Prüfung: 19 April 2006 Tag der öffentlichen Verteidigung: 22 Mai 2006 TABLE OF CONTENT TABLE OF CONTENT INTRODUCTION................................................................................................................1 1.1. Plants as organic chemists par excellence...............................................................1 1.2. Keeping the world green: chemical defenses in conifers .......................................2 1.3. Picea abies (L) Karst., ecology, distribution, importance......................................4 1.4. The biology and ecology of bark beetles and white pine weevils ..........................5 1.4.1. “The Dream Team." Bark beetle and blue-stain fungi: Multiple attacks against multiple defenses ...........................................................................................7 1.5. Terpenoids and phenolics.........................................................................................8 1.5.1. Terpenoids: biosynthetic pathways and cellular compartmentation ................8 1.5.2. Phenolic compounds ......................................................................................11 1.6. Mechanism of defense induction and signaling....................................................13 1.7. Methyl jasmonate induces defense responses.......................................................14 1.8. The culture of conifer cells as a system for studying the production of defense compounds ......................................................................................................................16 1.9. Purpose and objectives of the study ......................................................................17 MATERIALS AND METHODS ......................................................................................19 2.1. Effect of methyl jasmonate treatment on defense responses to the pathogen Ceratocystis polonica ......................................................................................................19 2.1.1. Methyl jasmonate treatment and sampling ....................................................19 2.1.2. Measurements of fungal growth, cambium necrosis, and anatomy...............20 2.1.3. Statistical analyses .........................................................................................21 2.2. Effect of methyl jasmonate on defense responses in Picea abies to the bark beetle Ips typographus ....................................................................................................22 2.2.1. Effect of methyl jasmonate treatment on beetle colonization of live trees....22 2.2.2. Effect of methyl jasmonate treatment on beetle colonization, egg laying, and attraction of conspecifics..........................................................................................24 2.2.3. Effect of methyl jasmonate treatment on brood production of Ips typographus..............................................................................................................25 2.2.4. Statistical analyses .........................................................................................26 1 TABLE OF CONTENT 2.3. Effect of jasmonate treatment on defense responses to the white pine weevil (Pissodes strobi), and influence of mechanical wounding and weevil attack on terpene content ...............................................................................................................26 2.3.1. Plant material and treatment...........................................................................26 2.3.2. Preparation of attacked Norway spruce terminal leaders for histological examination..............................................................................................................28 2.3.3. White pine weevil response to treatments......................................................29 2.3.4. Statistical analysis ..........................................................................................29 2.4. Extraction of resin terpenes ...................................................................................29 2.5. Analysis of monoterpenes, sesquiterpenes, and diterpenes.................................30 2.6. Extraction and identification of phenolic compounds .........................................33 2.7. The culture of plant cells ........................................................................................34 2.7.1. Cell line initiation and maintenance...............................................................34 2.7.2. Culture elicitation and metabolic studies measurements ...............................35 RESULTS ...........................................................................................................................38 3.1. Methyl jasmonate, terpenes and resistance to Ceratocystis polonica..................38 3.1.1. Terpene profile of untreated Picea abies wood and bark tissues...................38 3.1.2. Methyl jasmonate increases resin ducts and resin flow .................................39 3.1.3. Methyl jasmonate increases terpene content of Picea abies..........................42 3.1.4. Clonal variation in resin response to methyl jasmonate treatment ................43 3.1.5. Methyl jasmonate increased monoterpenes, sesquiterpenes, and diterpenes, but not phenolics ......................................................................................................45 3.1.6. Methyl jasmonate increases P. abies resistance to C. polonica.....................49 3.2 Methyl jasmonate, terpenes and resistance to Ips typographus ...........................50 3.2.1. Methyl jasmonate increased the number of traumatic resin ducts and the accumulation of terpene resin constituents ..............................................................50 3.2.2. Methyl jasmonate (MJ) reduced Ips typographus colonization of P. abies bark...........................................................................................................................53 3.2.3. Methyl jasmonate reduced Ips typographus aggregation...............................56 3.2.4. Methyl jasmonate reduced Ips typographus reproduction in detached logs..57 2 TABLE OF CONTENT 3.3 Methyl jasmonate, tepenes and resistance to Pissodes strobi, the white pine weevil ...............................................................................................................................58 3.3.1. Effects of jasmonate, weevil feeding and mechanical wounding on terpenoid accumulation ............................................................................................................58 3.3.2. Effect of treatments on formation of traumatic resin ducts............................61 3.3.3. Effects of treatments on white pine weevil performances .............................62 3.4. The use of cell cultures to study defensive terpene formation in spruce............63 3.4.1. Growth of cultured cells.................................................................................63 3.4.2. Monoterpene production in cultured cells......................................................63 3.4.3. Monoterpene biosynthetic activity in cultured cells ......................................66 DISCUSSIONS...................................................................................................................68 4.1. Methyl jasmonate induces resin duct formation..................................................68 4.2. Methyl jasmonate induces increases in terpene content, but only minor changes in terpene composition...................................................................................................70 4.3. Methyl jasmonate did not alter soluble phenolic content....................................72 4.4. Induced terpene defences and resistance to Ceratocystic polonica .....................74 4.5. Induced terpene defences and resistance to bark beetles....................................75 4.6. Induced defenses and resistance to white pine weevils feeding ..........................79 4.7. Induced defense: comparing spruce and herbaceous plants...............................80 4.8. Cost and induced defense .......................................................................................81 4.9. Terpene variability, a rule rather than an exception in Picea abies...................82 4.10. Regulation of terpene formation in Picea abies cell cultures ............................84 SUMMARY ........................................................................................................................87 ZUSAMMENFASUNG .....................................................................................................90 REFERENCES...................................................................................................................94 3 ABBREVIATIONS ANOVA analysis of variance df degree of freedom DMAPP Dimethylallyl diphosphate DMSO Dimethyl sulphoxide DOXP 1-deoxy-D-xylulose