Wright State University CORE Scholar Browse all Theses and Dissertations Theses and Dissertations 2010 Constitutive and Jasmonate-Inducible Defenses in Phloem of Two North American and Two Asian Ash Species Grown in a Common Garden Qin Wang Wright State University Follow this and additional works at: https://corescholar.libraries.wright.edu/etd_all Part of the Biology Commons Repository Citation Wang, Qin, "Constitutive and Jasmonate-Inducible Defenses in Phloem of Two North American and Two Asian Ash Species Grown in a Common Garden" (2010). Browse all Theses and Dissertations. 986. https://corescholar.libraries.wright.edu/etd_all/986 This Thesis is brought to you for free and open access by the Theses and Dissertations at CORE Scholar. It has been accepted for inclusion in Browse all Theses and Dissertations by an authorized administrator of CORE Scholar. For more information, please contact [email protected]. CONSTITUTIVE AND JASMONATE-INDUCIBLE DEFENSES IN PHLOEMOF TWO NORTH AMERICAN AND TWO ASIAN ASH SPECIES GROWN IN A COMMON GARDEN A Thesissubmittedin partial fulfillment of the requirements forthe degree of Master of Science By QIN WANG B.S.,ShenYangAgriculture University,2007 2010 Wright State University WRIGHT STATEUNIVERSITY SCHOOL OF GRADUATESTUDIES March14,2010 I HEREBY RECOMMEND THAT THETHESIS PREPARED UNDER MY SUPERVISION BY QIN WANG ENTITLED Constitutive andJasmonate-inducible defensesinphloem of twoNorthAmericanand twoAsianashspeciesgrowninthe commongarden. BE ACCEPTED IN PARTIAL FULFILLMENTOF THE REQUIRMENTS FOR THEDEGREEOF Master ofScience. DonCipollini,Ph.D. ThesisDirector DavidGoldstein,Ph.D. Department Chair Committee onFinal Examination DonCipollini,Ph.D. Thomas Rooney,Ph.D. JohnO.Stireman,Ph.D. JohnA.Bantle,Ph.D. Vice President forResearchand Graduate StudiesandInterim Deanof Graduate Studies Abstract Wang,Qin.M.S.Department of Biological Sciences,Wright State University, 2009.Constitutive andjasmonate-inducible defensesinphloem oftwoNorth American andtwoAsianashspeciesgrownin the common garden. EmeraldAshBorer(EAB) is more damagingtoNorthAmericanashes thanAsian ashes.Variationinthe resistance ofashspecies tofeedingbylarvae of EAB maybe relatedtovariationinlevelsofchemical defensesinthe phloem. I comparedconstitutive andmethyl jasmonate (MeJA)-inducible levelsof several chemical defenses inthe phloem of youngManchurian,Chinese,white,andgreenashes.Manchurianashisknowntobe highlyresistant toattack byEAB inthe field,while white and greenashes are both susceptible.The hypothesesof this experiment were that: (1) Manchurian(Fraxinus mandshurica)andChinese ash(Fraxinus chinensis) trees’ phloem or bark contains concentrationsof unique secondarycompounds andhigher level of defense protein activities thanwhite andgreenash.(2) MeJA treatment will induce increasesincontent of protein,Peroxidase (POD),polyphenol oxidase (PPO) content andmost of the secondary compounds inall species.Phloem extractsof Manchurianash showedhigher total soluble proteincontent andsignificantlyfaster browningthanwhite and greenash,but lower peroxidase activityandpolyphenoloxidase activity.Activitiesof PPO,POD,phenolic andlignincontent,andthe rate of browningreactions were not foundtoexhibit differences betweenMeJA treatedanduntreatedgroups.However,our HPLC results revealedManchurian ash containednine unique phenolic compounds: Homovanillic Alcohol,Esculetin,Esculin Related Cmpd,Esculin,Fraxin,Fraxidinhexoside, Pinoresinol dihexoside,Calceolarioside A,Calceolarioside B.We alsofoundobvious increases inspecific compounds(of the 20compounds we reanalyzedinthe four ash species) inGreenash,Chinese ashandManchurianashinthe MeJA treatedgroup.Also, Canonical Discriminant Function Analysisoffive commoncompounds(Tyrosol Hexoside,Mandshurin, Pinoresinol compound,Syringaresinol andOleuropein) revealed that Manchurianashwasdifferent from the otherthree ashes, but closer toGreenashand Chinese ash.Greenash andChinese ashwere similar toeachother.White ashwasvery iii different from all otherthree ashes.Inthis study, the highlyresistant Manchurianash wasdistinguishable from the highlysusceptible white ashbyhavinghigher total soluble proteincontent anda significantlyfaster browning.The susceptible greenashwas distinguishable from Manchurianash byhavinglower total proteinlevelsanda lower degree of browning. The phloem chemistryof greenandChinese ashwaslargelysimilar inthis study,indicatingthat theymaybe similar intheirresistance toEAB,andWhite ashshowedhadlevelsof peroxidase activity,polyphenoloxidase activity,content of phenolic andlowerrate of browningreactionthan the other three species.From the result of Discriminate Function Analysis,White ashalsoshowedmaximal separationfrom the others.Since White ashisas susceptible asGreen ashinNorthAmerica,White ashmay containdifferent compoundsor enzymes thanGreenthat maybe attractive to EAB. iv T A B L E O F C O N T E N T S INTRODUCTION……………………………………………………………………....1 THE INVASION OF EMERALD ASH BORER TO GENUS FRAXINUS ..…...1 OBJECTIVES………………………………………...……………………………3 HYPOTHESES………………………………………………………..……….....3 BACKGROUND OF EMERALD ASH BORER………..…………..…………….3 CONTROL METHODS...…………………………..……………………..……….4 A. PHYSICAL CONTROL…………………………………..……………..…4 B.BIOLOGICAL CONTROL……………………………………..……….....4 C.CHEMICAL CONTROL…………………………………..………….....…5 RESISTANCEIN ASIAN ASH SPECIES……………………..………………...6 METHYL JASMONATEINDUCED PLANT DEFENSES ……..……………..8 MATERIALS AND METHOD ………………………..……………..……………...…10 PLANTSPECIES AND GROWTH ………….…………………….………....…10 TREATMENTS-MeJA……..………………….…………………………..……..10 MEASUREMENTS……..…………………………………………………..…...11 DEFENSE PROTEIN EXTRACTION AND ANALYSIS………..………….....11 ASH PHENOLIC AND LIGNIN EXTRACTION…………...………….............13 IDENTIFICATION OFPHENOLIC SECONDARY COMPOUNDS…...….......14 STATISTICALANALYSIS…………………………………...……...………....15 RESULTS………………………………………………………………………………16 PEROXIDASE ACTIVITY………………...……………………………….....16 POLYPHENOLOXIDASE ACTIVITY ……………………………..………..16 v SOLUBLE PHENOLIC CONCENTRATIONS……………………..………...17 TOTAL AMOUNT OFLIGNIN……………………..…………………….….17 TRYPSIN INHIBITOR ACTIVITY………………………..………………….17 PROTEIN ACTIVITY……………………..………………………………..…17 RATE OF BROWNING REACTION…………………………..……………..18 HPLC………..…………….…………………………………………….….….18 RESULTSOF THE CANONICAL DISCRIMINANT ANALYSIS……..……18 DISCUSSION…………………….…………………………………………………....20 LITERATURE CITED…………………………………………………………………26 vi L I S T O F F I G U R E S FIGURE 1. CONSTITUTIVEAND MEJA –INDUCIBLE PEROXIDASE ACTIVITIES IN PHLOE M EXTRACTSOF MANSHURIAN,CHINESE,WHITE AND GREEN ASH……………………………………………………...………………37 FIGURE 2. CONSTITUTIVEAND MEJA –INDUCIBLE POLYPHENOLOXIDASEACTIVITIES IN PHLOE M EXTRACTSOF MANSHURIAN, CHINESE,WHITE AND GREEN ASH ……………………………………………………………………………….…....38 FIGURE 3. CONSTITUTIVEAND MEJA –INDUCIBLE SOLUBLE PHENOLIC CONCENTRATION IN PHLOEM EXTRACTS OF MANSHURIAN,CHINESE,WHITE AND GREEN ASH ………………………………………………………………...…...39 FIGURE 4. CONSTITUTIVEAND MEJA –INDUCIBLE TOTALLIGNIN IN PHLOEM EXTRACTS OF MANSHURIAN,CHINESE,WHITE AND GREEN ASH ……………………….…………………………………………………….……....39 FIGURE 5. CONSTITUTIVEAND MEJA –INDUCIBLE TRYPSIN INHIBITOR ACTIVITY IN PHLOEM EXTRACTS OF MANSHURIAN,CHINESE,WHITE AND GREEN ASH ………………………………………………………………………..….40 FIGURE 6. CONSTITUTIVEAND MEJA –INDUCIBLE SOLUBLE PROTEIN CONTENT IN PHLOEM EXTRACTS OF MANSHURIAN,CHINESE,WHITE AND GREEN ASH ………………………………………………………………………...…41 FIGURE 7. CONSTITUTIVEAND MEJA –INDUCIBLE RATE OF BROWNING REACTION IN PHLOEM EXTRACTSOF MANSHURIAN,CHINESE,WHITE AND vii GREEN ASH ……………………………..………………………………………….…41 FIGURE 8. COMPARISION OF HPLC PROFILES AT280 NM OF CONSTITUTIVE PHLOEMEXTRACTS (100%METHANOL)OF TWO MANCHURIAN ASH GROUPS: TREATED AND CONTROL GROUPS OVERLAY ……………………….………….42 FIGURE 9. COMPARISION OF HPLC PROFILES AT280 NM OF CONSTITUTIVEPHLOEMEXTRACTS (100% METHANOL)OF TWO CHINESE ASH GROUPS: TREATED AND CONTROL GROUPS OVELAY……………….….42 FIGURE 10. COMPARISION OF HPLC PROFILES AT280 NM OF CONSTITUTIVE PHLOEMEXTRACTS (100%METHANOL)OF TWO WHITE ASH GROUPS: TREATED AND CONTROL GROUPS OVERLAY. …………………………..….….43 FIGURE 11. COMPARISION OF HPLC PROFILES AT280 NM OF CONSTITUTIVE PHLOEMEXTRACTS (100%METHANOL)OF TWO GREEN ASH GROUPS: TREATED AND CONTROL GROUPS OVERLAY …………………………...,,……43 FIGURE 12. RESULT OF THE DISCRIMINANT FUNCTION ANALYSIS OF FIVE COMMON COMPOUNDS (TYROSOL HEXOSIDE,MANDSHURIN,PINORESINOL, SYRINGARESINOL AND OLEUROPEIN)ALL FOUR SPECIES SHARE……...…44 FIGURE 13. MAXIMUM LIKEHOOD TREE OF A REDUCED SET OF THE ITS DATA.……………….……………………………………………………………....…45 viii L I S T O F T A B L E S TABLE 1. RESULTOF A ONE WAY ANOVA SEVERAL DEFENSE RELATED CHEMICAL TRAITS IN FOUR ASH SPECIES…………………………………….…32 TABLE 2. LISTOF PHENOLIC COMPOUNDS THAT WEREEXAMED IN ASH PHLOEMSAMPLES BY HPLC ……………………………………………………….33 TABLE 3. MEAN PEAK AREAS (±SE) OF SELECTED INDIVIDUALPHENOLIC COMPOUNDS IN WHITE,GREEN,CHINESE AND MANCHUEIAN ASH IN CONTROL AND MEJA TREAED GROUPS ………………………. ……..……….…34 TABLE 4. CANONICALLOADING OF THEINDEPENDED VARIABLESON FIVE COMMON COMPOUNDS AMONG 4ASH SPECIES……………………………….36 - ix ACKNOWLEDGEMENTS I wouldlike toextendmysincere gratitude to mysupervisor,DonCipollini for his instructive advice,encouragement,andsupport from the initial tothe final level enabled me todevelop anunderstanding ofthe subjectandcompletionofthis twoyearthesis. I am heartily thankful tomycommittee members Thomas RooneyandJohnO Stiremanfor their assistance andadvice.I wouldalsolike to thankStephanie Enright and DeahLeiurance forassisting with statistical analysis. Finally,I am indebtedtoall