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August 8-12, 2009 Towson University

Towson, Maryland

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MEETING AGENDA

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Phytochemical Society of North America

49th Annual Meeting

and

Symposia

Biologically Active

Phytochemicals

August 8 – 12, 2009

On the Campus of

Towson University

Towson, Maryland

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Conference and Symposia Organizers

JamesA.Saunders,TowsonUniversity,ChairOrganizingCommittee

BenjaminMatthews,USDA

NadimAlkharouf,TowsonUniversity

JedFahey,JohnsHopkinsUniversity

MarkA.Bernards,UniversityofWesternOntario

ErinYoung,TowsonUniversity

TimMartin,TowsonUniversity

ChristinaEngstrom,TowsonUniversity

TissaThomas,TowsonUniversity

ChristopherSaunders,TowsonUniversity

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August 8, 2009 Saturday

PSNA Executive Committee meeting 3:00 - 5:00 PM RM 360 Smith Hall Registration and Arrival 2:00 – 8:00 PM Chesapeake Rooms Stud Student Union Key Note Presentation James Duke 6:00 – 7:00 PM The Herbal Village Chesapeake Rooms, Student Union Spices and Culinary Herbs in Folk Medicine and Phytomedicine Reception 7:00 – 8:30 PM Chesapeake Rooms Student Union

August 9, 2009 Sunday

Session I: Moderator James A. Saunders, Towson University Chao Lu Dean Graduate School, Towson University Opening Remarks 8:30 – 9:00 AM Lecture Room 326 Smith Hall Jed Fahey 9:00 – 9:45 AM Lecture Room 326 Johns Hopkins University Smith Hall The action of glucosinolates / isothiocyanates on Helicobacter pylori

A. Douglas Kinghorn 9:45 – 10:30 AM Lecture Room 326 Ohio State University Smith Hall Discovery of Potential Anticancer Agents from Tropical Plants

Morning Break 10:30 – 11:00 AM EloyRodriguez 11:00– 11:20 AM Lecture Room 326 CornellUniversity Smith Hall The Biochemical Evolution of Phytomedicines ZhonghuaWang,HongHan,ReinhardJetter 11:20 – 11:40 AM Lecture Room 326 UniversityofBritishColumbia Smith Hall Isolation and Characterization of the Kalanchoe daigremontiana Cyclases forming the Cuticular Triterpenoids Glutinol and Friedelin

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CedricB.Baker 11:40-12:00 PM Lecture Room 326 MercerUniversity‐CollegeofPharmacyandHealthSciences Smith HallBioactive Phytochemicals in Medical Foods for Phytotherapy Lunch 12:00 – 1:05 PM Chesapeake Rooms Student Union

Session II: Moderator Cecilia McIntosh, East Tennessee State University

Jinhui Dou 1:10 - 1:55 PM Lecture Room 326 Food and Drug Administration Smith Hall Botanical Drug Development in the U.S. Poster Introductions (2 minute rounds) 1:55 – 3:15 PM Lecture Room 326 Smith Hall Poster Session and refreshments 3:15 – 5:00 PM Chesapeake Rooms Student Union

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August 10, 2009 Monday

Session III: Moderator Roland Roberts, Towson University

Jeffrey Weidenhamer 8:45 – 9:30 AM Lecture Room 326 Ashland University Smith Hall Analytical Strategies for Ecologically Active Phytochemicals Vonnie Shields 9:30 – 10:15 AM Lecture Room 326 Towson University Smith Hall The Effects of Alkaloids on the Feeding Behavior and Neurophysiology of Insects

Morning Break 10:15 – 10:45 AM

Neish Speaker Gale G. Bozzo 10:45 – 11:15 AM Lecture Room 326 UniversityofGuelph Smith Hall Salvage of Folate Catabolites in Plants: Biochemical Characterization of p-Aminobenzoylglutamate Hydrolase Neish Speaker Syed G.A. Moinuddin, Dhrubojyoti D. Laskar, Chanyoung Ki, Laurence B. Davin, Norman G. Lewis 11:15 – 11:45 AM Lecture Room 326 Washington State University Smith Hall COMT (CAFFEIC ACID O-METHYL TRANSFERASE) Mutations and Effects on Both Lignin Primary Structure and Deconstruction Lunch 11:50 – 1:00 PM Chesapeake Rooms Student Union

Session IV: Moderator Tim Dwyer, Stevenson University

Xing Cong Li 1:10 – 1:55 PM Lecture Room 326 University of Mississippi Smith Hall Drug Discovery of Natural Products for Pharmacognosy Cristobal L. Miranda and Jan F. Stevens 1:55 – 2:15 PM Lecture Room 326 Oregon State University Smith Hall

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Xanthohumol and Related Prenylated Flavonoids from Hops Inhibit Production of Inflammatory Mediators in Activated Monocytes and Macrophages Daniel K. Owens and Cecilia A. McIntosh 2:15 – 2:35 PM Lecture Room 326 East Tennessee State University Smith HallIdentification, Biochemical Characterization, and Structure Function Analysis of a Flavonol 3-O-Glucosyltransferase from Citrus Paradisi Ming-Zhu Shi, Matthew Gromlich, Li-Li Zhou, De-Yu Xie North Carolina State University 2:35 – 2:55 PM Lecture Room 326 Smith Hall Red Callus Culture of Arabidopsis as a Tool for Analyzing the Regulation Mechanism Controlling the Function of Pap1 Transcription Factor Poster Session and Refreshments 3:00 – 4:00 PM Chesapeake Rooms Student Union PSNA Business meeting, 4:00 – 5:00 PM Chesapeake Rooms Student Union

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August 11, 2009 Tuesday

Session V: Moderator John Thor Arnason, University of Ottawa

Bill J. Baker 8:45 – 9:30 AM Lecture Room 326 University of South Florida Smith Hall Antarctic Red Marine Algae Produces Influenza Inhibitory Chemistry Michael C. Tims 9:30 – 10:15 AM Lecture Room 326 National Institutes Standards & Technology Smith Hall A Little Biology Goes a Long Way: Evaluation of Extraction Methods for Gallic Acid, Catechins and Xanthines in Green Tea

Morning Break 10:15 – 10:45 AM

Ralph L. Reed and Jan F. Stevens 10:45 – 11:05 AM Lecture Room 326 Oregon State University Smith Hall Glucosinolate Degradation Products in Fermented Meadowfoam Seed Meal and Their Herbicidal Activities Sungbeom Lee, Marco Herde, Christiane Gatz, and Dorothea Tholl

Virginia Tech 11:05 – 11:25 AM Lecture Room 326 Smith Hall UnravelingTheBiosynthesisOfVolatilesInPlantDefense:ASingleCYTP450EnzymeIs Responsible For the Conversion of Geranyllinalool to the Insect­inducedHomoterpeneTMTTinARABIDOPSISTHALIANA Jim Tokuhisa, Alice Mweetwa, Norma Manrique Constanza, Danielle Hunter, Idit Ginzberg and Richard Veilleux 11:25 – 11:45 AM Lecture Room 326 Virginia Tech Smith Hall TheSteroidalGlycoalkaloidsofSolanumChacoense LydiaMeador,PatrickT.Walker,Ming­ZhuShi,andDe­YuXieNorthCarolinaStateUniversity 11:45 – 12:05 PM Lecture Room 326 Characterization of PAP1-Transgenic Cell Suspension Culture through Growth, Anthocyanins, and Gene Expression Lunch 12:05 – 1:15 PM Chesapeake Rooms Student Union

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Session VI: Moderator, David Gang, University of Arizona

Neish Speaker De­YuXie NorthCarolinaStateUniversity 1:20 – 1:50 PM Lecture Room 326 Smith Hall Red cell culture of the PAP1transgene: a functional tool to understanding the regulatory mechanism of MYB75 controlling the biosyntheses of both anthocyanins and proanthocyanidins Kevin Spelman 1:50 – 2:10 PM Lecture Room 326 Muséum national d'Histoire naturelle Smith Hall Spilanthol, undeca-2E-ene-8, 10-diynoic acid isobutylamide and extracts from Spilanthes acmella demonstrate anti-plasmodial activity M.S.C.Pedras,S.Hossain,Z.Minic,andQ.­A.ZhengUniversityofSaskatchewan 2:10 – 2:30 PM Lecture Room 326Plant Defenses and Pathogen Attack, Tricking Plant Pathogens Smith Hall Phanikanth V. Turlapati, Laurence B. Davin, Norman G. Lewis, Washington State University 2:30 – 2:50 PM Lecture Room 326 Smith Hall Tellimagrandin II Biosynthesis in Tellima grandiflora: C–C coupling STEP Young Investigators Program 3:00 – 4:30 PM Chesapeake Rooms Student Union Banquet 6:00 – 7:30 PM Chesapeake Rooms Student Union Dinner Presentation Norman Farnsworth 7:30 – 8:30 PM Chesapeake Rooms University of Illinois at Chicago Student Union

YSONGOCAMRAHP and the Search for Bioactive Chemicals

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August 12, 2009 Wednesday

Session VI: Moderator Norman Lewis, Washington State University

Sangeeta Dhaubhadel 9:00 – 9:45 AM Lecture Room 326 Agriculture and Agri-Food Canada Smith Hall Regulation of Isoflavonoid Synthesis in Soybean Seeds

Mark Bernards 9:45 – 10:30 AM Lecture Room 326 University of Western Ontario Smith Hall The Ecological Role(s) of Ginsenosides Lili Zhou, Craig Yencho, DE-Yu Xie 10:30 – 10:50 AM Lecture Room 326

North Carolina State University Smith Hall HPLC/MS Profiling of Anthocyanin and Carotenes of Sweet potato Genotypes with Variable Flesh Colors Sung-Jin Kim, Daniel G. Vassão, Laurence B. Davin, Norman G. Lewis; Washington State University 10:50 – 11:10 AM Lecture Room 326 Formation of Allyl/Propenyl Phenols in Liquid Media: Substrate Versatility of Monolignol Acyltranserases and Allyl/Propenyl Phenol Synthases Masaomi Yamamura, Shiro Suzuki, Takefumi Hattori, Toshiaki Umezawa Kyoto University 11:10 – 11:30 AM Lecture Room 326 Smith Hall The Subunit Composition Of Hinokiresinol Synthase Controls Enantiometirc Composition Of Hinokiresinol Formation William F. Reynolds UniversityofToronto 11:30 – 11:50 AM Lecture Room 326 Smith Hall The Sensitivity of HMBC Spectra used for Natural Product Structure Determination by NMR can Depend Dramatically on Parameter Choices

RAPEditorialMeetings12:00‐2:00PM SmithHall360

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INVITEDPRESENTATIONSKeyNotePresentation SPICESANDCULINARYHERBSINFOLKMEDICINEANDPHYTOMEDICINEJamesDukeTheHerbalGarden,GreenPharmacyGarden, 8210MurphyRoad,Fulton,Maryland20759,JimDuke@comcast.netIn this, the70thyear for the spiceOldBay,nowundernearbyMcCormickSpiceCo., JimDukeinhis80thyear,discussesthemedicinalpotentialofthespicesinOldBay.NaturallyJimstartsoutwithBay itself,Laurusnobilis, since thisyear2009,bay,alias laurel, is theHerbSociety’s'herboftheyear.Bayhasrecentlybeenshowntoberichintheantimalarialphytochemical luteolin. Celery seed, which spared Dukemany bout's of gout, is rich inapigenin,cayenneinCOX‐2‐Icapsaicin;mustardinchemopreventiveisothiocyanates,blackpepper in piperine, clove in eugenol, allspice and the antinociceptivemyrcene, ginger ingingerols and shogoals, nutmeg in sabinene, cardamom in the anticholinesterase 1,8‐cineole and cinnamon in spasmolytic cinnamic acid. Then Duke will go into proven orpotential medicinal biologically active phytochemicals in dozens of other Duke believesthat, under current technologies, most spices have around 5000 identifiablephytochemicals, almost if not all biologically active andwithmany pleiomeric activities.AndDukepredictsthat'llapproach50,000perspicespecies.Dukeistryingtoseekorderinthis nearly chaotic complexity. His phytochemical database at the USDA, fromwhich heretiredin1995,isstillavailablegratisathttp://www.ars‐grin.gov/duke.Youcanfinddataon200spicesinthisdatabase.Dukehascloseto100culinaryherbsandspicesgrowinginhisGreenPharmacyGarden.Seehttp://www.greenpharmacy.comTHEACTIONOFGLUCOSINOLATES/ISOTHIOCYANATESONHELICOBACTERPYLORI JedWFahey,KKStephenson,KLWade,PTalalay,MYamamoto,AYanakaBloombergSchoolofPublicHealth­CenterforHumanNutrition,Dept.ofPharmacologyandMolecularSciences,JohnsHopkinsUniversity,725N.WolfeStreet,Baltimore,Maryland21205jfahey@jhmi.edu Three-day-old broccoli sprouts, a widely-available human food, suppress infections with Helicobacter pylori, a bacterium that is the cause of the most common infection found in humans worldwide. Helicobacter infections are now recognized as the major causes of persistent gastritis and peptic ulcers, and infected people (about half of the world’s population) are at 3- to 6-times the risk of stomach cancer as non-infected persons. Although eradication of the infectious agent is the treatment of choice for symptomatic individuals, the need for eradication

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(compared to reducing severity of infection), is currently a matter of scientific debate. In addition, resistance to multiple antibiotics has developed in the many Helicobacter strains. We have previously reported upon the potent in-vitro activity of sulforaphane from broccoli sprouts against H. pylori1,2. We now provide evidence for the beneficial effects of broccoli sprouts in both a mouse model of the H. pylori infection and in a clinical intervention on a Helicobacter-infected Japanese population3. We report studies on mice infected with a human strain of Helicobacter and maintained on a high salt diet to aggravate the severity of gastritis. One group of mice received a broccoli sprout puree, rich in a sulforaphane precursor, in the drinking water for 8 weeks, and was compared with a control group receiving no sprouts. The broccoli sprout-treated Helicobacter-infected mice showed reduced gastric colonization with H. pylori, considerably reduced gastric inflammation and atrophy, lower levels of two inflammatory signaling molecules, and decreased oxidative DNA damage, in comparison to infected control mice. The human trial involved 48 Helicobacter-infected Japanese men and women (age range 23 -73) of whom 25 received fresh broccoli sprouts (70 grams daily for 8 weeks) and 23 consumed an equivalent quantity of fresh alfalfa sprouts during the same period. The severity of Helicobacter infection was assessed at the time of enrollment, after 4 and 8 weeks of treatment, and 8 weeks after termination of intervention. The urea breath test (a test routinely used by physicians to assess whether their patients are colonized by H. pylori bacteria), and the levels of stool antigens which signal the presence of Helicobacter infection, served as biomarkers of the severity of the Helicobacter infections. In addition measurement of pepsinogens in the serum served as biomarkers of severity of inflammation of the gastric mucosa. All the values were significantly lowered during the treatment period in subjects consuming broccoli sprouts, but not in the alfalfa (placebo) recipients. These values returned to pretreatment levels 8 weeks after broccoli sprout feeding was discontinued. These animal and human studies raise the probability that broccoli sprouts can enhance protection of the mucosa lining the stomach against H. pylori-induced oxidative stress, improve the course of the infection in human beings, and reduce the risk of stomach cancer. Since stomach cancer is the second most common and the second most deadly cancer worldwide, these findings may have highly important public health implications.

1 Fahey JW, X Haristoy, PM Dolan, et al. (2002) Proc. Natl. Acad. Sci. USA 99: 7610-7615. 2 Haristoy X, JW Fahey, I Scholtus, & A Lozniewski. (2005) Planta Medica 71: 326-330. 3 Yanaka A, JW Fahey, A Fukumoto, et al. (2009) Cancer Prevention Research 2(4): 353-360. DISCOVERYOFPOTENTIALANTICANCERAGENTSFROMTROPICALPLANTSDouglasKinghorn,LiPan,YulinRen,Ah­ReumHan,andYeDeng,DivisionofMedicinalChemistryandPharmacognosy,CollegeofPharmacy,TheOhioStateUniversity,Columbus,OH43210­1297,E­mailkinghorn.4@osu.edu.

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Natural products from plants and microbes have played an important role in cancerchemotherapyforseveraldecades(1).Inareviewhighlightingoncologydrugdevelopmentover a recent 25‐year period it was concluded that a majority of the anticancer drugsapproved in North America, Europe, and Japan are either natural products or theirderivatives or synthetic molecules based on natural product pharmacophores (2).Currently,ourgroupiscollaboratinginamultidisciplinaryandmulti‐institutionalresearchprojectunderthe“programproject”mechanism,inwhichnewnaturalproductanticancerdrugleadsareobtainedfromadiversegroupoforganisms,constitutedbytropicalplants,aquatic cyanobacteria, and filamentous fungi. Crude extracts are subjected to a panel ofcell‐basedandtarget‐basedbioassays,withselectedleadsthensubjectedtoactivity‐guidedfractionation. The overall organizational plan for this project will be described, as wellprogress made in the isolation and structural determination of bioactive secondarymetabolitesfromseveraltropicalplants,includingGarcinialaterifloraBlume(Clusiaceae),Garcinia mangostana L. (Clusiaceae), and Hyptis brevipes Piot. (Lamiaceae). ThroughcollaborativeworkwithcolleaguesinTheOhioStateMedicalCenter,ithasbeenfoundthatsilvestrol, a 1H‐cyclopenta[b]benzofuran derivative from Aglaia foveolata Pannell(Meliaceae), has selective inhibitory effects for B‐cell leukemias (4). In an attempt todiscovernewminoranalogsof silvestrol,a large‐scale recollectionof thestembarkofA.foveolatahasbeeninvestigatedphytochemicallyandbiologically.(FundingbygrantsU19CA52956andP01CA125066fromNCI/NIHisgratefullyacknowledged).References1. Cragg,G.M.;Kingston,D.G. I.;Newman,D. J.,Eds.AnticancerAgents fromNatural

Products;CRCPress/Taylor&Francis:BocaRaton,FL,2005.2. Newman,D.J.;Cragg,G.M.J.Nat.Prod.2007,70,461‐477.3. Kinghorn, A. D.; Carcache‐Blanco, E. J.; Chai, H.‐B.; Orjala, J.; Farnsworth, N. R.;

Soejarto,D.D.;Oberlies,N.H.;Wani,M.C.;Kroll,D.J.; Pearce,C.J.;Swanson,S.M.;Kramer,R.A.;Rose,W.C.;Emanuel,S.;Vite,G.D.;Jarjoura,J.;Cope,F.O.PureAppl.Chem.2009,81,1051‐1063.

4. Lucas,D.M.;Edwards,R.B.;Lozanski,G.;West,D.A.;Shin,J.D.;Vargo,M.A.;Davis,M.E.;Rozewski,D.M.; Johnson,A. J.; Su,B.‐N.;Goettl, V.M.; Lin,T. S.; Lehman,A;Zhang,X.;Jarjoura,D.;Newman,D.J.;Byrd,J.C.;Kinghorn,A.D.;Grever,M.R.Blood2009,113,4656‐4666.

DevelopingNewBotanicalDrugsfromMedicinalPlants–AFDABotanicalReviewer’sPerspectiveJinhuiDou,Ph.D.,BotanicalReviewTeam,OfficeofDrugEvaluationI(HFD­101),CDER,FoodandDrugAdministration,SilverSpring,MDIntheUnitedStates,botanicalsthatareknowntotheconsumersas“traditional,alternative&complementarymedicines(TACM)”or“herbalmedicines,naturalhealthproducts”areusuallyfallenintooneofthetwomajorcategoriesofFDAregulatedproducts,i.e.,dietarysupplementsanddrugs.

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Therearesignificantdifferencesbetweentheregulatoryapproachesforbotanicaldrugsanddietarysupplements,andassociatedrequirementsonquality,safetyandefficacyfortheirmarketing.Fordietarysupplements,nopre‐marketingapprovalbytheFDAisrequired,however,dietarysupplementsarenotpermittedtobeardiseaseclaims(onlystructure/functionclaimsarepossible).Fordrugproducts,pre‐marketingapprovalbytheFDAisrequired.Besides,drugproductsaresubjecttorisk/benefitevaluationsthroughnon‐clinicalstudies,andmoreimportantly,well‐designedclinicaltrials.Notsurprisingly,withtensofthousandofdietarysupplementproducts(manywithoneormorebotanicals),onlyahandfulofbotanicaldrugsaremarketedintheU.S.SincethepublicationoftheGuidance,therehasbeengrowinginterestinbotanicaldrugdevelopmentjudgedbytheincreasingnumbersofbotanicalINDsandpre‐INDconsultations,acumulativetotalofover400andgrowing.FewofthebotanicalINDswithphase1/2clinicaltrialshavetodateadvancedintolate‐phaseclinicaltrials.Sofar,theVeregen®NDAremainstheonlyonesubmittedandsubsequentlyapproved.Althoughthereasonsforthisarenodoubtdifferentindifferentcases,howtoproducemultiplebatchesofqualifieddrugsubstances(orproducts)thatmeetthehighqualitystandardsremainsasoneofthebiggestchallenges.AdiscussionofthechallengesfacingnewbotanicaldrugdevelopmentcouldshedlightontheseeminglylowpercentageofbotanicalINDsenteringlate‐phaseclinicaltrials.We,ofcause,hopethiswillchange,andthatmoremedicalplantderivedmixtures(suchasactivefractions)alongwithpurifiedcompoundswillbefurtherdevelopedasnewdrugswithmoresuccess.ANALYTICALSTRATEGIESFORECOLOGICALLYACTIVEPHYTOCHEMICALSJeffreyD.WeidenhamerDepartmentofChemistry,AshlandUniversity,Ashland,OH44805USAjweiden@ashland.eduPlantsproduceawidevarietyofhighlyphytotoxicchemicals,someofwhichhaveactivitiescomparable to synthetic herbicides. The possibility that plants exert direct chemical, orallelopathic, effects on neighboring plants as well as on soil microflora has attractedconsiderableresearchinterest. Thesuccessofcertaininvasiveplantshasbeenattributedto phytotoxic root exudates. Otherwork suggests that in conifer forests,monoterpenesreleased from pine roots and litter inhibit nitrogen mineralization and nitrification.However,thereisagenerallackofinformationaboutthespatialandtemporaldynamicsofallelochemicals in the soil that is a barrier to testing hypotheses of allelopathic effects.Whensoilsbeneathsuspectedallelopathicplantsareanalyzed,concentrationsaretypicallylow,andthishasbeencitedasevidencethatthesecompoundsdonotplayasignificantroleinplant‐plantinteractions. However,staticconcentrationsintheenvironmentreflectthecurrent balance of input vs. output rates for a compound. Because plant roots competewith both microorganisms and other processes that remove allelochemicals from soilsolution, flux rates are likely to be a key component of toxicity. Polydimethylsiloxane

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(PDMS) sorbents, which are widely used in analytical techniques such as solid phasemicroextraction (SPME) and stir bar sorptive extraction (SBSE), are being applied tomeasure the dynamics of thiophenes produced by marigold roots in soil. Variousapproaches, including PDMS‐coated wires and PDMS sheets, have been tested thus far.Resultsshowthatthedistributionofmarigoldallelochemicalsintherootzoneisspatiallyheterogeneous and dynamic over time. Given the high potency of these thiophenes inbioassays,theamountsrecoveredcanreadilybeconceivedtobebiologicallyactive.Thesetechniquesappeartobebroadlyapplicable to theanalysisof lipophilicrootexudates. Inaddition,SPMEhasbeenusedtosuccessfullymonitortheuptakeofsoil‐applied1,8‐cineoleby target plants. Our results indicate that PDMS sorbents are a useful tool for studyingecologicallyactivephytochemicals.THEEFFECTSOFALKALOIDSONTHEFEEDINGBEHAVIORANDNEUROPHYSIOLOGYOFINSECTS

VonnieD.C.Shields,TimothyL.Martin,KatelynF.Beattie,NicoleS.Arnold,andKristenP.SmithDepartmentofBiologicalSciences,TowsonUniversity,8000YorkRoad,Towson,MD21252VShields@towson.eduAlkaloidsrepresentoneofthelargestchemicallyheterogenousgroupsofphytochemicals.They occur in 20‐30%of higher plants and have approximately 12,000 known chemicalstructures.Theyarefoundmostcommonlyindicotyledonousangiospermfamilies,suchasRubiaceae, Apocynaceae, and Solanaceae. Plants containing alkaloids are generallyconsideredtobefeedingdeterrentsformanyinsectsandareimportantininfluencingfoodselection. Our research is aimed at understanding the role of phytochemicals for insectbehaviorandphysiology.Here,wefocusontheeffectofalkaloidsontheinsectlarvaltastesystem.Lepidopteranlarvae,suchasthegypsymoth,possesstwopairsofsensoryorgansontheirmouthparts,thelateralandmedialstyloconicsensilla,whicharethoughttoplayanimportant role in host‐plant selection including thedetection of phytochemicals, such asalkaloids.Thesestyloconicsensillaeachbearfivebipolarneurons,fourchemosensoryandone mechanosensory. They are considered to be the main sensory organs involved infeeding. In one experimental approach, we conducted feeding behavioral bioassays ongypsy moth larvae using a series of alkaloids to determine their potency as feedingdeterrents. The results of this study provided innovative insights into the variability ofalkaloidsaseffectivefeedingdeterrentswhentheywereappliedtonaturalversusartificialsubstrates. When we conducted dose‐response experiments, we observed increasingfeeding deterrent responses for all the alkaloids tested and found that the alkaloidsexhibiteddifferentdeterrencythresholdconcentrations.Inanotherseriesofexperiments,we used a single cell electrophysiological tip‐recording method to characterize thetemporal firing patterns and sensitivities of the receptor cells housed within eachstyloconicsensillumofgypsymothlarvaeinresponsetostimulationwithalkaloids. Thismethodallowedustoevaluatehowthegustatorysensoryinputwasencodedaspatternsofnerveimpulsesbyreceptorcellsandsenttoprocessingcentersinthebrainoftheinsect.

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Wefoundthatonereceptorcell(i.e.,deterrentcell)inbothsensillarespondedrobustlytoselected alkaloids and exhibited varying sensitivity, which correlated well with ourbehavioral results. The results of our study have the potential to suggest deterrentalkaloids as possible novel candidates in designing suitable new strategies for cropprotectionfrominsectpests.ThisresearchwassupportedbyNIHgrant1R15DC007609‐01toV.D.S.DRUGDISCOVERYOFNATURALPRODUCTSFORPHARMACOGNOSY IKHLASA.KHANNationalCenterforNaturalProductsResearchandDepartmentofPharmacognosy,SchoolofPharmacy,TheUniversityofMississippi,MS38677,ikhan@olemiss.eduNatural products offer a vast and virtually unlimited source of new agents for both thepharmaceutical and agrochemical industries. The National Center for Natural ProductsResearch(NCNPR)wascreatedtobringtogetheranallianceofacademia,government,andthe pharmaceutical and agrochemical industries to integrate research, development, andcommercializationofpotentiallyusefulnaturalproducts.Development inscienceandtechnologyhas impactedeveryaspectofour lives.Themostprecious thing among them is health.We all agree that everyone should have access tohealth coverage and access tomedicine but in reality its becoming difficult even inwelldevelopedcountriesduetohighcostofit.Themodelofmodernmedicineisnotaddressingthe currentneedand theneedof the future.Eightypercentof theworldpopulation stillreliesontraditionalmedicine.Thesametimenaturalproductsarebecomingmorepopularaspeoplearebecomingmorehealthconscious.Naturalproductscanbedevelopedasadrugorasaherbalmedicine.Themain issue formakingherbal preparation aswell respectedmedicine is standardization.Herbalproductstudiescannotbeconsideredscientificallyvalid if theproduct testedwasnot authenticated and characterized in order to ensure reproducibility in themanufacturingoftheproductinquestion.Recent developments in natural products chemistry atNCNPR related to drug discoveryandherbalproductswillbepresented.A PROTEIN WITH STRONG ANTI‐INFLUENZA ACTIVITY FROM THE ANTARCTICREDMARINEALGAGIGARTINASKOTTSBERGII

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J.AlanMaschek1,CindyBucher2,AlbertovanOlphen2andBillJBaker.11UniversityofSouthFlorida,DepartmentofChemistry,2UniversityofSouthFlorida,DepartmentofGlobalHealth,4202E.FowlerAve,Tampa,FL,33620,bjbaker@cas.usf.edu

With an estimated 3 to 5 million infections and as many as 500,000 deaths from thecomplicationsof influenza infectionseachyear, there liesacriticalneedto identifynoveldrugclassesandstructureswhichcanbeexploitedforantiviraldevelopment.Abioassay‐guidedfractionationofextractsfromtheredmarinealgaeGigartinaskottsbergii,collectednearAnvers Island,Antarctica, significantly inhibited the reproductionof influenza virusA/Wyoming/3/2003 (H3N2) inMDCK cells in vitro with an IC50 value of 4 μg/mL. Thevirus‐inhibitoryeffectwasselective,dose‐dependent,strain‐specificandthevirusinducedcytopathogeniceffect (CPE)wasreducedatnon‐toxicconcentrationsof theextract.SDS‐Gel electrophoresis and sequencing of the active fraction reveals homologywith lectins.Insight intothemechanismofactionviahemagglutinationandplaqueassaysuggestscellprotection by interference of viral docking. Our research suggests that activity fromcurrent over‐the‐counter anti‐influenza vitamin supplements from the same genus andspeciesdoesnotarisefromthepreviouslyreportedantiviralsulfatedpolysaccharides.

ALITTLEBIOLOGYGOESALONGWAY:EVALUATIONOFEXTRACTIONMETHODSFORGALLICACID,CATECHINSANDXANTHINESINGREENTEAMichaelC.TimsandLaneC.SanderNationalInstitutesStandards&Technology,100BureauDriveMS8392,Gaithersburg,MD,20899,michael.tims@nist.gov Greentea(Camelliasinensis),apopularbeverageenjoyedworldwide,hasbeenreportedtocontaincompoundsusedintreatmentofcancer,genitalwarts,cardiovasculardisease,andasanantimicrobial.Accuratemeasurementanddetectionofthesecompoundsisnecessaryforassuringgoodmanufacturingpracticesandintheconductofclinicaltrials.AspartofacollaborativeeffortbetweentheNationalInstituteofStandardsandTechnology(NIST)andtheNational Institutes ofHealth (NIH)Office ofDietary Supplements analyticalmethodshavebeendeveloped for thedeterminationof catechins, gallic acid, theanineandmethylxanthinesinasuiteofgreenteacontainingstandardreferencematerials(SRMs).Themetabolicpoolofcatechinsingreentealeaftissuecanbealteredbybothbioticfactorsandtealeafprocessing.Traditionalgreentealeafextractionmethodsunderestimated‘true’analyte levels in green tea leaves, particularly levels of epigallocatechin gallate (EGCG).Understanding the biology and chemistry of catechins, plant primary cell walls and theantimicrobial properties of catechin galloyl esters helped guide analytical methoddevelopment.Enzymeassistedextractionusingof0.1%EDTAcombinedwithasecondarypressurizedfluidextractionincreasedtherecoveryofcatechinsandlimitedinterferenceof

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biopolymer fragments to optimize extraction efficiency. This talk describes efforts todevelop quantitative extraction procedures for use in the certification of green teacontainingSRMscurrentlyunderdevelopment.YSONGOCAMRAHPANDTHESEARCHFORBIOACTIVECHEMICALSNormanR.FarnsworthDepartmentofMedicinalChemistryandPharmacognosy,UniversityofIllinoisatChicago,833S.WoodStreet(M/C781),Chicago,IL60612­7231norman@uic.eduAnoverviewofthetaxonomicdistributionofphytochemicalsandclassesofbiologicalactivityforplantspecieswillbepresented.Acornucopiaofdata,rangingfromclinicaltrialstoinvitroanalysisofbotanicalextractsandpurecompounds,toreportsontheethnomedicaluseofplants,willbeconsidered,comparedandcontrasted.Tidbitsandanecdotes,designedtoedify,aiddigestion,andkeeptheaudienceawake,willbeoffered.REGULATIONOFISOFLAVONOIDSYNTHESISINSOYBEANSEEDSSangeetaDhaubhadelSouthernCropProtectionandFoodResearchCenter,AgricultureandAgri­FoodCanada,1391SandfordSt,London,Ontario,N5V4T3Canada,sangeeta.dhaubhadel@agr.gc.ca

Isoflavonoids are a diverse group of secondary metabolites that accumulate in soybeanseedsduringdevelopment.Theyplayimportantrolesintheinteractionbetweenplantsandtheenvironment.Severalclinicalstudieshavesuggestedaroleforisoflavonoidsinhumanhealthandnutrition.Theamountofisoflavonoidspresentinsoybeanseedsisinfluencedbybothgeneticandenvironmentalfactorsthatarenotfullyunderstood.Ourstudiessuggestthattotalisoflavonoidaccumulationinsoybeanseedisaresultofdenovosynthesiswithinthe seed as well as transport from maternal tissues. We have conducted a global geneexpressionanalysisofdevelopingsoybeanseedsintwocultivarsthatdifferinthelevelofisoflavonoid accumulation. The results established that the differential expression ofchalcone synthase (CHS)7 and CHS8 genes determines the differences in the levels ofisoflavonoid accumulation in soybean seed. A search for factor (s) that regulate theexpression of the key isoflavonoid biosynthetic genes has identified a MYB family of

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transcription factor that recognizes and binds with specific sequence within the CHS8promoterandregulatestheCHS8geneexpression.ThedirectevidenceoftheinvolvementoftheMYBfactorinisoflavonoidbiosynthesiswasconfirmedbyRNAisilencing.

THEECOLOGICALROLE(S)OFGINSENOSIDESMarkA.Bernards1,DimitreIvanov1,AndreeaNeculai1,LinaYousef2andRobertNicol31DepartmentofBiologyandtheBiotron,TheUniversityofWesternOntario,London,ON,Canada,N6A5B7,2SchoolofEnvironmentandNaturalResources,TheOhioStateUniversity,Columbus,OH43210,3UniversityofGuelphRidgetownCampus,Ridgetown,ON,N0P2C0bernards@uwo.ca.Ginsenosides are triterpenoid saponins produced by Panax spp (ginseng), and arepurported to possess medicinal properties. However, we wanted to know why ginsengmakesthesecompounds,sincetheyaccumulateupto6‐7%ofthedryweightofroots. Inourinitialexperimentsweestablishedthat,likeothersaponins,ginsenosidesarefungitoxic(albeit only mildly) to some soil borne microorganisms in vitro; however, we alsodemonstratedthatthegrowthofothermicroorganisms(especiallyaggressivepathogensofginsengplants)wasenhancedinthepresenceofginsenosides.Further,wenotedthattheprofile of ginsenosides recovered from the medium of pathogens such as Pythiumirregularewassignificantlyaltered.Thatis,whenP.irregularewasculturedinthepresenceof a purified (>90%) ginsenoside mixture, nearly all of the 20(S)‐protopanaxadiolginsenosides(Rb1,Rb2,Rc,Rd,and toa limitedextentG‐XVII)weremetabolized into theminorginsenosideF2,atleasthalfofwhichappearedtobeinternalizedbytheorganism.Nometabolism of the 20(S)‐protopanaxatriol ginsenosides (Rg1 and Re) was evident. Themetabolismofginsenosideswasdependentonextracellularglycosidasessecretedintotheculturemediumbythepathogen.Tofurtherexploretheapparentselectivemetabolismof20(S)‐protopanaxadiolginsenosidesbyextracellularenzymesofP. irregulare,wepurifiedandpartially sequenced three glycosidase enzymes, anddemonstrated them to be acidicproteins (pI of 4.5‐5.0), consisting of an apparent high molecular weight (~160 kDa)homodimerof78kDasubunits,withβ(1→6)activity,andtwomonomericenzymesof61and 57 kDa, with β(1→2) activity. Importantly, the appearance of these specificglycosidases in the culture medium of P. irregulare is dependent on the presence ofginsenosides in the culturemedium, suggesting that their expression is triggered by thepresenceofthesecompounds.Wespeculatethattheroleoftheseextracellularglycosidasesis likely to help Pythium find its host, and/or obtain nutrients/growth factors from itsenvironment.NEISHSPEAKERS

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SALVAGEOFFOLATECATABOLITESINPLANTS:BIOCHEMICALCHARACTERIZATIONOFp‐AMINOBENZOYLGLUTAMATEHYDROLASEGaleG.BozzoDepartmentofPlantAgriculture,UniversityofGuelph,Guelph,Ontario,Canada Folatesareessentialcofactorsforone‐carbontransferreactionsinmostorganisms,butaremade only by plants andmicrobes. Folates are comprised of a pterin, p‐aminobenzoate(pABA), and glutamate moieties. Although the enzymatic steps governing folatebiosynthesisarewelldescribedinplants,verylittleisknownaboutfolatecatabolismandsalvage. Folates are quite susceptible to oxidative breakdown, yielding a pterin and p‐aminobenzoylglutamate(pABAGlu)oritspolyglutamates.In plants, folate degradation is on the order of 10% per day. Salvage of pABAGlu issuggested by the following: (i) pABAGlu does accumulate relative to folate pools; (ii)ArabidopsisandtomatotissuesreadilyconvertedsuppliedpABAGlutothefolatesynthesisprecursor, pABA, and glutamate; and (iii) in vitro studies confirmed the presence ofpABAGluhydrolase(PGH)activityinplants.BiochemicalcharacterizationofPGHactivitysuggeststhepresenceofseveralisoformsinbothArabidopsisandpea.ApartiallypurifiedArabidopsis root PGH hydrolyzes pABAGlu, as well as folate. This activity was stronglyinhibited by ametal chelator and stimulated byMn2+, pointing to ametalloenzyme. TheArabidopsisgenomewassearchedforproteinssimilartoPseudomonascarboxypeptidaseG,which contains zinc and is the only enzyme yet confirmed to attack pABAGlu. The solesignificantmatcheswere auxin conjugate hydrolase familymembers and theAt4g17830protein. None was found to have significant PGH activity, suggesting that this activityresides in hitherto unrecognized enzymes. The finding that Arabidopsis has folate‐hydrolyzingactivitypointstoanenzymaticcomponentoffolatedegradationinplants.COMT (CAFFEIC ACIDO‐METHYL TRANSFERASE) MUTATION AND EFFECTS ONBOTHLIGNINPRIMARYSTRUCTUREANDDECONSTRUCTIONSyed G.A. Moinuddin, Dhrubojyoti D. Laskar, Chanyoung Ki, Laurence B. Davin,Norman G. Lewis; Institute of Biological Chemistry, Washington State University,Pullman,WA99164­6340,syed@wsu.edu

“CaffeicacidO‐methyltransferase”(COMT)wasoriginallyviewedasbeingabispecificOMTbased on in vitro assays and transgenicmanipulations, i.e.whereby itwas thoughtthatitwasabletoO‐methylatecaffeicacidand5‐hydroxyferulicacidmoietiesinvivointhemonolignolpathway.

Atanassova et al., 1995, however, demonstrated unambiguously that “COMT” onlycatalyzes the regiospecific methylation of 5‐hydroxy‐G (5‐OH‐G) units (G = guaiacyl)substrates in angiosperms, this resulting in formationof the so‐calledS (syringyl) lignin.These studies also established, however, that down‐regulation of “COMT” in transgenictobacco linesresulted innearabolitionof thesyringyl (S)component in lignins,whereas

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there was apparently little to no alteration in the G levels of the lignin formed, i.e.suggesting that “COMT”was a 5‐hydroxyguaiacylO‐methyl transferase. We now reportthat the lignin primary structure in Arabidopsis COMT mutated lines has benzodioxanesubstructures in its ligninwhoseoverall8‐O‐4′ inter‐unit linkagefrequency isapparentlybeing conserved relative towild type. These subunits are,however, readily cleavableas5OH‐G/G and 5OH‐G/5OH‐G/Gmoieties in the lignin. Specifically, plant cell wall lignindeconstruction,andidentificationofthereleasedsubunitsbyNMRspectroscopicandwetchemical analyses, together with confirmation by chemical syntheses, have providedfurther evidence for lignin macromolecular assembly being conserved i.e. in a highlyprogrammed(templateguided)manner.Inthiscase,formationof5‐OH‐Gunitsresultsinthesemoietiesbeing8‐O‐4′linkedtotheligninprimarystructureasdepicted

CleavablesubunitsinCOMT‐derivedlignin

SupportedbyDOE(DE‐FG02‐97ER20259)andtheDOEBioEnergyScienceCenter.REDCELLCULTUREOFTHEPAP1TRANSGENE:AFUNCTIONALTOOLTOUNDERSTANDINGTHEREGULATORYMECHANISMOFMYB75CONTROLLINGTHEBIOSYNTHESESOFBOTHANTHOCYANINSANDPROANTHOCYANIDINSDe­YuXieDepartmentofPlantBiology,NorthCarolinaStateUniversity,Raleigh,NC27695,emailaddress:dxie@ncsu.eduPAP1 is a plant MYB R2R3 transcription factor. Arabidopsis pap1­D (production ofanthocyanin pigment 1­Dominant) is a dominant mutant generated by T‐DNA activationtagging(Borevitzetal.,2000).Theinsertionof4x35SenhancersequencesadjacenttothestartofthePAP1generesultsintheoverexpressionofthistranscriptionfactor,whichleadsto the enhanced accumulation of anthocyanins in most of the organs. Several previousreportsshowedthattheoverexpressionofPAP1/MYB75couldenhanceoractivateseveralpathwaygenesofboththeanthocyaninsandproanthocyanidins.Arecentecologicalstudyshowed that the anthocyanin formation in Arabidopsis could be independent of thetranscription of the PAP1. Calliwere induced from leaves ofpap1­Dmutants and PAP1‐transgenictobaccoinourlaboratory.InductionofcelldifferentiationledtoobtainmentofseveraldifferentPAP1‐transgenic cell linesof tobacco, including redandwhite cell lines,

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whichtranscribedasimilarlevelofthePAP1transgene,butthewhitecellseitherdidnotproduceanthocyaninsorproducedaonlytracelevelofanthocyanins.Inthisreport,IwillpresentanddiscusstheuseoftheredcellcultureasafunctionaltoolforasystemsstudyofregulatorymechanismofMYB75.CONTRIBUTEDPRESENTATIONSTHEBIOCHEMICALEVOLUTIONOFPHYTOMEDICINESEloyRodriguez,ChemicalBiology­EcologyandMedicalEthnobotanyLaboratory,CornellUniversity,Ithaca,NY14853Structurallycomplexbio‐medicinalmoleculesinplantsareprimarilytheresultofmillionsofyearsofco‐evolutionaryinteractionsbetweenplantsandherbivores.Theseherbivoresor predators, include insects, herbivorous animals, fungi, bacteria and viruses, with alatitudinal gradient in bioactivity evident from the tropics (more complex and bioactivemoleculesversusplantsof the cold temperate zones). It is therefore,not surprising, thatsome natural poisons are of medicinal value, since they inhibit primary processes,enzymes,kinases,proteases, formproteinadductsand induceapoptosis in insectsand inhumancancercells.Inthispresentation,theevolution,chemistryandantitumoractivitiesof novel bark spiro‐lignans from tropical plants are presented, followed by a briefdiscussion on how plant medicinal use was acquired by the early apes(zoopharmacognosy)andlearnedbytheearlyhominids.ISOLATIONANDCHARACTERIZATIONOFTHEKALANCHOEDAIGREMONTIANACYCLASESFORMINGTHECUTICULARTRITERPENOIDSGLUTINOLANDFRIEDELINZhonghuaWang1,HongHan1,ReinhardJetter1,2

1DeptofBotany,UnivofBritishColumbia,VancouverV6T1Z4,Canada2DeptofChemistry,UnivofBritishColumbia,VancouverV6T1Z1,CanadaPentacyclic triterpenoids are a large group of secondary metabolites found in manydifferentplantspecies,eitherasglycosideconjugatesorasaglycones.Thefirstcommittedstep in triterpenoid biosynthesis is the cyclization of 2,3‐oxidosqualene into variousisomericproductsC30H50O.Thetransformationproceedsviaepoxideprotonation,multipleringformationreactions,1,2‐methyland1,2‐hydrideshiftsandafinaldeprotonationstep,all catalyzed by a single enzyme. Different multifunctional triterpenol synthases and/orcombinationsofseveralproduct‐specificenzymesaccountforthediversityoftriterpenoidsfoundineachplantspecies.Onemajordifferencebetweenalltheseenzymesishowmany1,2‐shiftstheyfacilitatebeforereleasingthefinalproducts.All thetriterpenoidsynthasesdescribedtodatecatalyzereactions involvingonlya few1,2‐shifts,andrelatively little is

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knownabouttherearrangementmechanismsbeyondthatpoint.Therefore,thegoalofthisproject was to identify and characterize triterpenoid synthases catalyzing a maximumnumberof1,2‐shiftsleadingtotheformationofglutinolandfriedelin.TwonoveltriterpenoidsynthasegeneswereisolatedfromKalanchoedaigremontianaandheterologous expression in yeast, followed by GC‐FID and GC‐MS analysis, showed thattheycodeforafriedelinsynthaseandaglutinolsynthase.Sequencecomparisonsbetweenbothenzymes, togetherwith three‐dimensional structuremodeling,wereused topredictamino acids involved in determining product specificity. To test these predictions,chimeragenesisandsite‐directedmutagenesisexperimentswerecarriedout.Theproductprofilesofthealteredenzymesweresignificantlychanged,mostnotablyinthepercentagesof glutinol and friedelin. The implications of these results for the mechanisms oftriterpenoidcyclizationwillbediscussed.Our findings also have implications for the biological functions of plant triterpenoids.Friedelin and glutinol accumulate to high concentrations in the cuticular wax of K.daigremontiana,where they likely play an important role in protecting the plant againstbiotic and/or abiotic stress. As a result, understanding the genetics and biochemistry oftriterpenoids will also provide us further insight into their physiological and ecologicalroles.BIOACTIVEPHYTOCHEMICALSINMEDICALFOODSFORPHYTOTHERAPY CedricB.Baker,Pharm.D.,AdjunctClinicalAssistantProfessor MercerUniversity­CollegeofPharmacyandHealthSciences,Atlanta,Ga.30341Email:baker691@umn.edu Medicalfoodsarefindingtheirwayontopharmacyshelvesasprescriptiononlyitems.Thearea of food as medicine is amorphous and the termmedical food conveys this uniqueconfusion.Medical foodsare inrealitynot foods.Medical foodsareusuallysome formofdietarysupplementcomposedofbioactivephytochemicals,singularorcomposite,ofGRASstatus in anon‐foodmatrix.Theobjectiveof thispaper is to explore themajor issuesofbioactive phytochemicals inmedical foods that are used for phytotherapy in the UnitedStates.Thesemajorissuesare:regulation,safety,efficacy,synergy,andpharmacovigilance.TheregulatoryissueisproblematicformedicalandpharmacypracticeduetothefactthattheFDAdoesnotrequiresafetyorefficacyevaluationformedicalfoods.TheFDAclassifiesmedical foods not as drugs, but as foods. Type 2 medical foods have prescription onlystatus.TherearenoevaluationstandardsformedicalfoodsintheU.S.andthisfactmakestheissueextremelydifficultforhealthcareprofessionals.Thisisaquagmire.Theissuesofsafetyandefficacyclearlysufferduetothisregulatoryquagmire.Becauseofthissingularregulatory environment formedical foods some companies use this loop hole tomarketproducts that after review by experts have been found to be of unproven efficacy.

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Flavocoxid is an example. Complicating the clinical picture is the fact that somephytochemicalswithbioactivitycanbesourcedfromnon‐plantnaturalsourcessuchasCo‐enzymeQ‐10.ItisworthmentioningthatCo‐Q‐10canexistasamedicalfood(ubiquinol)oradietarysupplement(ubiquinone).Phytotherapyhasvastpotential intheemergingareaof integrative therapeutics. This promising area is due in part to the unique aspect ofbioactivephytochemicalsinmulti‐constituentproductscommonlyexhibitingsynergy.Thisuniqueareaofphytochemicalsynergyinmulti‐constituentnaturalproductsisinformedbythe pleiomorphic expression of physiological and pharmacological effects of individualphytochemicalsandphyto‐combinationsinfoodandnon‐foodmatricies.Thecurrentissueof pharmacovigilance is of great importance to modern U.S. public health. This area iscertainlytopicaltophytotherapyfromthestandpointofphytochemical‐druginteractions.Blackpepperextractisaconstitiuentinatleastonemedicalfood.Thisproductcontainsaspecificformulationof(98%)piperineat5mgs.Blackpepperextracthasthepotentialforthe inhibition of several P‐450(CYP) isoforms and p‐glycoprotien substrates. Levels ofevidence evaluation shows that there are many possible clinically important druginteractionswithpiperine,andat leastoneprobable interactionofpiperinewith lithium.Thisareaofpharmacovigilanceneedsurgentattentioninmedicalandpharmacyeducationand practice. The regulatory issues that currently plague medical foods will resolve asbioactivephytochemicalsundergoconclusiveclinicaltrialsforsafetyandefficacy.Bioactivephytochemicals(boththosewith‐inthefoodsmatrixandthosewith‐inanon‐foodmatrix)will become first‐line treatment options in the emerging paradigm of integrative phyto‐pharmacotherapyasU.S.medicalandpharmacypracticeevolve.TheFDAishardatworktryingtofindwaystodealwiththequagmireoftheregulatorystatusoffunctionalfoods,dietarysupplements, andmedical foods. It isadynamicandexciting time tobeactive inbioactivephytochemicals.CHARACTERIZATIONOFPAP1‐TRANSGENICCELLSUSPENSIONCULTURETHROUGHGROWTH,ANTHOCYANINS,ANDGENEEXPRESSIONLydiaMeador1,2,PatrickT.Walker1,Ming­ZhuShi1,andDe­YuXie1*1:DepartmentofPlantBiology,NorthCarolinaStateUniversity,Raleigh,NC276952:REU­NSFstudentfromBotanyDepartment,OklahomaStateUniversity,Stillwater,OK74077*:correspondent(dxie@ncsu.edu)Anthocyaninsareplant flavonoidpigmentsprovidingpink/red/blue/purplecoloration inmost plants. They attract both pollinators and seed dispersers. In addition, theirantioxidative activity protects plants from radiation‐induced damage on cells.Furthermore, theirpresence in foodprovidesstrongantioxidativeusesbenefitinghumanhealth. PAP1 is one of the R2R3‐MYB transcription factors directly regulating thebiosynthesisofanthocyanins.WehaveestablishedPAP1‐transgeniccallusandsuspensioncell cultures of tobacco. In this study, we characterize the growth of cells, anthocyaninformation, andgeneexpression in suspension‐cultured transgenic redcells (6R)ofPAP1during a25‐day cultureperiod.Transgenicwhite cells (6W)ofPAP1andwild‐type cells(P3)wereusedascontrols.Sampleswerecollectedfromsixtimepointsatdays0,5,10,15,20, and 25. Growth curves for the change in fresh weight were measured for thesesuspensionculturecells.Thebiomassincreaseofthe6Rcellsfollowedasigmoid‐liketrend.

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The biomass increase of the P3 cells followed a logarithmic trend. The fresh weightincreaseoftheP3cellswasfasterthanthatofthe6Rcells.Adynamictrendofanthocyaninlevelswereobservedinthe6Rcells.TheP3cellsdidnotproduceanthocyanins.Inaddition,wewill present and discuss the anthocyanin structures and profiles and the expressionlevelsofthePAP1transgeneanditstargetedpathwaygenes(e.g.PAL,CHS,DFR,andANS).ThisresearchisfundedUSDA‐NRI(proposalnumber2006‐1334). XANTHOHUMOLANDRELATEDPRENYLATEDFLAVONOIDSFROMHOPSINHIBITPRODUCTIONOFINFLAMMATORYMEDIATORSINACTIVATEDMONOCYTESANDMACROPHAGESCristobalL.MirandaandJanF.Stevens,LinusPaulingInstituteandtheDepartmentofPharmaceuticalSciences,OregonStateUniversity,Corvallis,Oregon97331e­mail:fred.stevens@oregonstate.eduXanthohumol is a prenylated chalcone‐type flavonoid found in hops (Humulus lupulus),beer, and in several dietary supplements currently on themarket. Our objective of thisstudywastodeterminetheanti‐inflammatoryactivitiesofxanthohumol,isoxanthohumol,and 17 related flavonoids as well as to determine structure‐activity relationships. Theisoflavone,genistein,andtheflavonol,quercetin,werealsoincludedinthestudy.Theanti‐inflammatory activities of the 21 flavonoids were measured by their ability to inhibitlipopolysaccharide(LPS)‐inducednitricoxide(NO)productioninmousemacrophageRAW264.7andtoinhibitLPS‐inducedcytokineproductioninhumanmonocyticTHP‐1cells.8‐Geranylnaringenin and xanthohumols B and C were most active as inhibitors of LPS‐inducedNOproduction inRAW264.7cellsat20µM. InTHP‐1cells,8‐geranylnaringeninwasthemostactiveinhibitorofLPS‐inducedproductionofMCP‐1andIL‐6followedby6‐geranylnaringenin and isoxanthohumol. Addition of prenyl groups to naringenin andchalconaringenin aswell as substitutionof prenyl groupswith geranyl groups showed aprofoundincreaseofinhibitoryeffectsonMCP‐1andIL‐6productioninTHP‐1cells,whichcannot be explained by the resulting increase in lipophilicity. The type of flavonoid(chalcone, dihydrochalcone, flavanone, isoflavone, or flavonol) appeared tohave aminorimpactoninhibitoryactivity.IDENTIFICATION,BIOCHEMICALCHARACTERIZATION,ANDSTRUCTUREFUNCTIONANALYSISOFAFLAVONOL3‐O‐GLUCOSYLTRANSFERASEFROMCITRUSPARADISIDanielK.Owens1,3andCeciliaA.McIntosh1,21DepartmentofBiologicalSciences,EastTennesseeStateUniversity,JohnsonCity,TN,376142SchoolofGraduateStudies,EastTennesseeStateUniversity,JohnsonCity,TN,376143owens1@etsu.edu

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Glucosylation is a predominant flavonoid modification reaction affecting the solubility,stability,andsubsequentbioavailabilityofthesecompounds.Incitrus,flavonoidglycosidesaffect taste characteristics making the associated glucosyltransferases particularlyinteresting targets for biotechnology applications. In this work, a grapefruit (Citrusparadisi var. Duncan) glucosyltransferase gene (GQ141630) was identified, cloned, andintroducedintothepETrecombinantproteinexpressionsystemutilizingprimersdesignedagainst a predicted flavonol/anthocyanidin glucosyltransferase gene (AY519364) frombloodorange(Citrussinensisvar.tarocco).TheencodedCitrusparadisiproteinis51.2kDawithapredictedpIof6.27andis96%identicaltotheCitrussinensishomologue.Anumberofcompoundsfromvariousflavonoidsubclassesweretested,andtheenzymeglucosylatedonly the flavonol aglycones quercetin (Kmapp=67 µM; Vmax=20.45 pKat/µg), kaempferol(Kmapp=12µM;Vmax=11.63pKat/µg),andmyricetin(Kmapp=33µM;Vmax=12.21pKat/µg)anddidnotglucosylatetheanthocyanidinaglycone,cyanidin.Glucosylationoccurredatthe3 hydroxyl position as confirmed by HPLC and TLC analyses with certified referencecompounds.TheoptimumpHwas7.5withapronouncedbuffereffectnotedforreactionsperformedinTris‐HClbuffer.TheenzymewasinhibitedbyCu2+,Fe2+,andZn2+aswellasUDP(Kiapp=69.5µM),whichisaproductofthereaction.Treatmentoftheenzymewithavariety of amino acid modifying compounds suggests that cysteine, histidine, arginine,tryptophan, and tyrosine residues are important for activity. The thoroughcharacterization of this Citrus paradisi flavonol 3‐O‐glucosyltransferase adds to thegrowing base of glucosyltransferase knowledge, and will be used for continuedinvestigationsintostructurefunctionrelationships.PLANTDEFENSESANDPATHOGENATTACK,TRICKINGPLANTPATHOGENSM.S.C.Pedras,S.Hossain,Z.Minic,andQ.­A.ZhengDepartmentofChemistry,UniversityofSaskatchewan,Saskatoon,SK,S7N5C9,CanadaThe “arms race” between plants and their attackers whether microbes or pests, is anoutcome of co‐evolution that continues to cause enormous crop losses. To devisesustainable methods to prevent and deter plant pathogens, the molecular interactionbetween crucifers and their pathogenic fungi is under intense investigation. Cruciferousplants(e.g.canola,mustard,cauliflower,cabbage, turnip,etc.)producecomplexblendsofmetabolites with diverse ecological roles such as self‐protection against microbialpathogens,pestsandothersortsofstress,whiletheirfungalpathogensproducephytotoxicmetabolites that facilitate plant invasion. Phytoalexins (induced) and phytoanticipins(constitutive) of crucifers have a verywide range of structures and biological activities.Fromour results, it is apparent thatmanyof thesenaturalproducts involved in cruciferdefense reactions (both constitutive and induced metabolites) are detoxified by theirfungal pathogens. Our screening results using synthetic and natural compound librarieshaveshownthatthesefungaldetoxificationscanbeinhibited.Takingasleadstructuresafewplantnaturaldefenses,anewgenerationofinhibitorsoffungalenzymesweredesigned

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andshowntobespecificagainstparticularpathogens.Themostrecentmetabolicstudiesandchallengesofthisstrategytotreatplantfungaldiseaseswillbepresented.TELLIMAGRANDINIIBIOSYNTHESIS INTELLIMAGRANDIFLORA: C–CCOUPLINGSTEPPhanikanthV.Turlapati,LaurenceB.Davin,NormanG.Lewis;InstituteofBiologicalChemistry,WashingtonStateUniversity,Pullman,WA99164­6340,phani60@mail.wsu.edu

Ellagitannins are an important class of hydrolysable tannins distributed across a wide range of plant species, and to date at least 500 ellagitannins have been structurally characterized. Besides their intrinsic antioxidant properties, some are bioactive (including those with antitumour, antiviral and anticancer activities). However, the biosynthetic pathway leading to formation of this structurally diverse group is poorly understood, at least in terms of monomeric radical-radical coupling. In this context, some of the major biosynthetic routes in ellagitannins are considered to result from oxidative C–C coupling of galloyl groups and oxidative C–O coupling leading to their oligomerization. Yet, very few studies have investigated the biochemical aspects of these coupling mechanisms. In Tellima grandiflora, putative laccases were initially reported as involved in oxidative C–C intramolecular coupling leading to formation of tellimagrandin II (monomer) and C–O intramolecular coupling to form cornusiin E (dimer). In our studies, however, no evidence to support laccase involvement in either biochemical pathway could be made. The present study was thus aimed to understand clearly the enzyme(s) involved in the oxidative coupling transformation(s) leading to tellimagrandin II. From our ongoing studies, an enzyme involved in C–C coupling leading to formation of tellimagrandin II from 1,2,3,4,6-penta-O-galloyl-β-D-glucopyranose as a substrate was detected and localized to isolated chloroplasts, with the latter being purified by percoll gradients. Similar results were also obtained using purified leaf protoplasts for in vitro reactions. Since no plant laccases have yet been reported as localized to chloroplasts, the existence of another class of oxidases is most likely. We propose polyphenol oxidases as the preferred candidates because of their localization to chloroplasts. Efforts are underway to obtain pure chloroplasts in bulk so that the enzyme(s) can be isolated to homogeneity. The results from the present study partially address the previous enigma of enzymatic oxidative transformations to this broad class of hydrolysable tannins. These findings herein thus place the coupling enzyme in the same subcellular compartments as for the substrate and products.

Supported by NSF (MCB 0417291) and DOE (DE-FG02-97ER20259). REDCALLUSCULTUREOFARABIDOPSISASATOOLFORANALYZINGTHEREGULATIONMECHANSIMCONTROLLINGTHEFUNCTIONOFPAP1TRANSCRIPTIONFACTORMing­ZhuShi,MatthewGromlich,Li­LiZhou,De­YuXie*DepartmentofPlantBiology,NorthCarolinaStateUniversity,Raleigh,NC27695*emailaddress:dxie@ncsu.edu

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PAP1 is a plant MYB R2R3 transcription factor. Arabidopsis pap1­D (production ofanthocyanin pigment 1­Dominant) is a dominant mutant generated by T‐DNA activationtagging(Borevitzetal.,2000).Theinsertionof4x35SenhancersequencesadjacenttothestartofthePAP1generesultsintheoverexpressionofthistranscriptionfactor,whichleadsto theenhancedaccumulationofanthocyanins inmostof theorgans. Calliwere inducedusingpap1­Dleavesasexplants.Severalanthocyanin‐producingcalluslineswereobtainedaftermultipletimesofsubcultureandselection.Asacontrol,wild‐type(WT)callusculturewas also established in the same condition. Seven anthocyanins in pap1 callus wereidentified by liquid chromatography mass spectrometry (HPLC‐MS) analysis. HydrolysisandLC‐MSanalysisshowedthatcyanidinformedthepredominantcorestructureoftheseanthocyanins.RT‐PCR result confirmed that theaccumulationof anthocyanins in the redcalluswereresultfromtheoverexpressionofthePAP1gene.Theexpressionlevelsoftheanthocyanin pathway genes, especially late pathway genes (eg. DFR and ANS), weredramaticallyincreasedintheredcalliincomparisonwiththeWTcalli.This red calli culture and the biosynthesis of anthocyanins provided a useful tool tounderstandtheregulationmechanismofPAP1inArabidopsiscellsandthemechanismsofhow other factors control the activity of this transcription factor. We investigated theeffects of different nitrogen sources and their concentrations on the biosynthesis ofanthocyanins.WetestedthreecombinationsofNH4NO3andKNO3concentrationsintheMSmedium:20mMNH4NO3and18.8mMKNO3,10mMNH4NO3and9.4mMKNO3,0NH4NO3and 9.4 mM KNO3. Higher concentration of NH4NO3 and KNO3 used in the MS mediuminhibitedthebiosynthesisofanthocyaninsandchangedtheprofilesofanthocyanins.Semi‐quantitative RT‐PCR analysis showed that higher concentrations of these two nitrogennutrientsdownregulatedthetranscriptionallevelsofPAP1and4analyzedpathwaygenes(PAL,CHS,DFR,andANS).TheeffectsofthePAP1overexpressiononglobaltranscriptomewillbediscussedinthispresentation.SPILANTHOL,UNDECA‐2E‐ENE‐8,10‐DIYNOICACIDISOBUTYLAMIDEANDEXTRACTSFROMSPILANTHESACMELLADEMONSTRATEANTI‐PLASMODIALACTIVITY

KevinSpelman,Biologiefonctionnelledesprotozoaires,DéveloppementetDiversitéMoléculaire,Muséumnationald'Histoirenaturelle,phytochemks@gmail.com

GalenicextractsfromSpilanthesspp.areactivelyusedtotreatmalariainAfricaandIndia.Yet there is thus farnodataonactiveconstituentsormosteffectiveextractionmethods.Spilanthol (deca‐2E,6Z,8E‐trienoic acid isobutylamide) the predominant alkylamide inSpilanthes spp., and undeca‐2E‐ene‐8,10‐diynoic acid isobutylamide, a lesser occurringalkylamide, were found to have IC50s at 15 μg/mL and 40 μg/mL, respectively, onPlasmodiumfalciparumstrainPFBand6μg/mLand18μg/mLonthechloroquineresistantP.falciparumK1strain.FurtherinvestigationsshowedthatspilantholandthewaterextractofS. acmella reduced theparasitemia53%and59% inmice infectedwithP. yoelii yoelli12XNLat5μg/mLand50μg/mL,respectively.The95%ethanolextractofS.acmellawas,

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unexpectedly,lesseffective(36%reductioninparasitemia)at50μg/mL.Thisprovidesthefirst evidence supporting S. acmella against malaria and provides the first evidencedemonstratingactiveconstituentsinS.acmellaagainstP.falciparum.GLUCOSINOLATE DEGRADATION PRODUCTS IN FERMENTED MEADOWFOAMSEEDMEALANDTHEIRHERBICIDALACTIVITIESRalph L. Reed and Jan F. Stevens, Department of Pharmaceutical Sciences, OregonStateUniversity,Corvallis,Oregon97331,e­mail:fred.stevens@oregonstate.edu

Meadowfoam (Limnanthes alba) is an oilseed crop in western Oregon. The seed mealcontains 2‐4% of the glucosinolate, glucolimnanthin, and minor amounts of otherglucosinolates.Wehavedevelopedamethodfortheconversionofseedmealglucosinolatesinto its degradation products by fermenting enzyme‐inactive seed meal with smallquantitiesof enzyme‐activemeadowfoamseeds.Glucolimnanthinwas converted into thecorresponding isothiocyanate,nitrile,andthioamide.Thethioamidedegradationproduct,2‐(3‐methoxyphenyl)‐ethanethioamide,was identified by isolation andHPLC‐UV and LC‐MS/MS comparison with a synthetic standard. This chemical has not previously beenreported as a naturally occurring compound. We were able to increase the yield of 3‐methoxybenzylcyanide(nitrile)and the thioamidedegradationproductbyco‐incubationof seed meal with enzyme‐active seeds and FeSO4 (10 mM), at the expense of thecorresponding isothiocyanate. In collaboration with Dr. Machado (Columbia BasinAgricultural Research Center, Pendleton, Oregon, OSU), the herbicidal activity ofglucolimnanthin and the isothiocyanate, nitrile, acetamide, and thioamide degradationproducts was determined in a coleoptile emergence assay using downy brome (Bromustectorum)seedsplantedonalayerofsoil.Theorderofherbicidalpotencywasfoundtobe:glucolimnanthin (least potent) < isothiocyanate < acetamide < nitrile < thioamide (mostpotent). Fermentation ofmeadowfoam seedmeal by treatmentwith activeL. alba seedssignificantly increased the herbicidal potency compared to no treatment or shamtreatment.Theherbicidalactivitywasfurtherenhancedbytreatmentofseedmeal inthepresenceofFeSO4.Theherbicidalpotencyofthetreated(fermented)mealscorrelatedwellwiththecontentoftheisothiocyanate,nitrileandthioamidedegradationproducts.Acknowledgments–ThisresearchisfundedbyUSDAgrantCSREES2005­34407­15670andbyNaturalPlantProducts,Inc.,Salem,Oregon.UNRAVELING THE BIOSYNTHESIS OF VOLATILES IN PLANT DEFENSE: A SINGLECYTP450ENZYMEISRESPONSIBLEFORTHECONVERSIONOFGERANYLLINALOOLTOTHEINSECT‐INDUCEDHOMOTERPENETMTTinARABIDOPSISTHALIANASungbeomLee1,MarcoHerde2,ChristianeGatz3,andDorotheaTholl11Department of Biological Sciences, Latham Hall, Virginia Tech, Blacksburg, VA 24060sungbeom@vt.edu;tholl@vt.edu

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2DepartmentofBiochemistryandMolecularBiology,MichiganStateUniversitymherde@msu.edu(currentaddress)3Albrecht‐von‐Haller‐Institut for Plant Sciences, Untere Karspuele 1A, Georg‐August‐Universität,Göttingen,GermanyVolatile secondary metabolites such as homoterpenes released from vegetative planttissues have significant functions in plant defense by directly warding‐off herbivorousinsects or attracting natural enemies of herbivores. This intriguing defense system hasdrawnincreasingattentiontothemetabolicengineeringofvolatilesasatoolindevelopingalternative pest controls. The C16‐homoterpene 4,8,12‐trimethyltrideca‐1,3,7,11‐tetraene(TMTT) is emitted from aerial parts ofmany plants including crops such asmaize, limabean,tomatoandalfalfaaswellasthemodelplantArabidopsisthaliana.TMTTemissionisinducedupon insectandmiteattackand functions inattractingparasitesorpredatorsofthese pests. Besides insect feeding, emission of TMTT can also be induced by fungalelicitorsandbacterialpathogeninfection.WeareinterestedinidentifyingthekeyenzymaticstepsintheTMTTmetabolicpathway.Herewedemonstrate for the first timethatCYP82G1(At3g25180),aCytP450enzymeofthe Arabidopsis CYP82 family, is responsible for the single‐step conversion of the C20‐precursor geranyllinalool to TMTT.We describe that expression of theCYP82G1 gene iscoordinated with that of geranyllinalool synthase in Arabidopsis leaves upon treatmentwith the fungal elicitor alamethicin, particularly in a COI‐1 dependent manner. TMTTemission isabsent inCYP82G1 loss‐of‐functionmutantsunderelicitor‐inducedconditionsbutcanberestoredbycomplementationwiththeCYP82G1geneexpressedundercontrolof theconstitutiveCaMV35Spromoter.Notably, invitroenzymeassayswithrecombinantCYP82G1 and in vivo substrate­feeding assays in yeast cells over‐expressing CYP82G1revealabroadersubstratespecificityoftheenzymebyconvertingnotonlygeranyllinaloolbutalso itsC15‐analognerolidol to the respectiveC11‐homoterpeneDMNT.HistochemicalCYP82G1 promoter‐GUS assays confirm inducible expression of this gene in leaves uponinsectfeedingandshowthatconstitutiveexpressionislimitedtostemsandinflorescences.THESTEROIDALGLYCOALKALOIDSOFSOLANUMCHACOENSEJimTokuhisa,AliceMweetwa,NormaManriqueConstanza,DanielleHunter,IditGinzberg2andRichardVeilleuxDepartmentofHorticulture,VirginiaTech;2AgriculturalResearchOrganization,VolcaniCenter,Israel;email:tokuhisa@vt.eduSteroidal glycoalkaloids are a class of plant natural products that are produced mostabundantlybyspeciesoftheSolanoideaesubfamilyoftheSolanaceae.Theyareglycosidesof nitrogen‐containing steroidal derivatives. These compounds are recognized as plantdefensecompoundsthatareeffectiveagainstherbivoreandfungalattack.TheSolanoideaeincludes the two most important vegetable crops produced world‐wide‐‐potato andtomato.ThewildprogenitorsofcultivatedSolanumcancontaininexcessof20mgofSGAs

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per 100 grams fresh weight, which are levels that are considered unsafe for humanconsumption because of the toxic cellular properties of SGAs as anticholinesterases andmembrane disruptors. Prehistoric breeding practices initiated, and contemporarybreedingprogramshavemaintainedlevelsofSGAsinnewcultivarsthatarelowenoughforsafe consumption of potatoes but high enough to contribute to the familiar and safeorganolepticpropertiesofunripegreentomatoesandpotatoskins.Despitetheimportanceof SGAs as natural pesticides and as potential human toxins, we have a limitedunderstandingofhowtheyaremade.To provide a molecular genetic basis for future Solanum breeding, we are usingbiochemical, molecular, and genetic approaches to identify the enzymes comprising thenovel biosynthetic pathway of steroidal glycoalkaloids with a particular interest in theenzymesassociatedwithsteroidalalkaloidformation.Wehaveisolatedandcharacterizedagenecodingforsqualenesynthasefromawildpotatospecies.Wearedevelopingahighthroughput protocol to extract and purify SGAs in cultivated andwild Solanum and areoptimizingseparationtechnologiesforHPLCandLC‐MStoquantifyandidentifySGAs. Inaddition,wearedevelopingmethodsfortransientgeneexpressioninSolanumtoscreenforgenesinvolvedinSGAbiosynthesis.To understand SGA formation in S. chacoense, we have determined SGA content andcomposition in various tissues during a developmental time course. This wild potatospecies produces leptines, SGAs that are highly toxic to the Colorado potato beetleLeptinotarsa decimlineata, and has SGA levels that are at least 2‐fold higher than themaximum target levels defined by breeders. The possibility that these two traits can bereconciled through introgression of S. chacoense with cultivated potato has drivenconsiderable interest in this species. The plant also produces the two major SGAsassociated with cultivated potato, a‐chaconine and a‐solanine, and additionallydehydrocommersonine. OurresultsindicateastructuraldiversityofSGAsinS.chacoensethat isgeneratedbycombinationsof threedistinctglycosidesand the formationof threemajoraglycones,leadingtotheaccumulationofsevendistinctSGAstructuresoccurringinatissue‐specificmanner.THE SUBUNIT COMPOSITION OF HINOKIRESINOL SYNTHASE CONTROLSENANTIOMERICCOMPOSITIONOFHINOKIRESINOLFORMATIONMasaomiYamamuraa,ShiroSuzukib,TakefumiHattoria,ToshiakiUmezawaa,baInstituteforSustainableHumanosphere,bInstituteofSustainabilityScience,KyotoUniversity,Uji,Kyoto611­0011,Japan(tumezawa@rish.kyoto­u.ac.jp). Norlignansareaclassofphenylpropanoidswithdiphenylpentanecarbonskeletons(C6‐C5‐C6)thatarefoundmainlyinconifersandmonocotyledons.Anorlignan,hinokiresinol,hasanEorZdoublebondinitsmolecule.Bothhinokiresinolshaveantifungalactivityandcanbeproducedinresponsetostress,suchasfungalinfectionandsapwooddrying,suggestingthattheyaresynthesizedinvivoforplantprotection.

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Previously, our findings have indicated that (Z)‐hinokiresinol was synthesized from adimericphenylpropanoidester,4‐coumaryl4‐coumarate,byanenzymepreparationfromelicitor‐treated A. officinalis cells. In addition, (E)‐hinokiresinol was formed from 4‐coumaryl4‐coumaratebyanenzymepreparedfromCryptomeriajaponica(Japanesecedar)cells.Then,wepurified (Z)‐hinokiresonol synthase (HRS) fromA. officinalis, and isolatedcDNAs encoding the enzyme. The enzyme was found to be composed of two subunits(HRSα and HRSβ). When each recombinant subunit was incubated individually with 4‐coumaryl4‐coumarate,only(E)‐hinokiresonolwasformed.Ontheotherhand,incubatingthesamesubatratewithamixtureofthetwosubunitsgaveriseto(Z)‐hinokiresinolthatisthe geometric isomer accumulated in A. officinalis. Thus, the subunit composition cancontrol the geometric isomerism of the product,which is quite interesting from organicchemicalaspects.

Interestingly, in addition to the cis­trans regulation, the enantiomeric composition ofhinokiresinol was found to be controled by the subunit composition of HRS. Thus, (Z)‐hinokiresinolformedbythemixtureofHRSαandHRSβwasopticallypure(+)‐enantiomer.It is noteworthy that the naturally occurring (Z)‐hinokiresinol in A. officinalis is alsoopticallypure (+)‐enantiomer. In contrast, (E)‐hinokiresinol formed following incubationwith HRSα or HRSβ was not optically pure [(‐) > (+), 20.6 or 9.0% enantiomer excess,respectively].Therefore,itwasdemonstratedthatthesubunitcompositionofHRScontrolsnotonlygeometricselectivitybutalsoenantiomericselectivityinhinokiresinolformation.

HPLC/MS PROFILING OF ANTHOCYANIN AND CAROTENES OF SWEET POTATOGENOTYPESWITHVARIABLEFLESHCOLORSLiliZhou*1,CraigYencho2,DE­YuXie1.1DepartmentofPlantBiology,NorthCarolinaState University, Raleigh, NC 27695. 2 Department of Horticulture, North CarolinaStateUniversity,Raleigh,NC27695.Email‐lzhou3@ncsu.edu.Sweetpotato isoneof topnutritionalcropsworldwide.A largenumberofvarietieshavebeencreatedforvalue‐improvedproducts,e.g.yellowandpurplestoragerootswithhighantioxidativeeffects.Inthispresentation,wereportanapplicationofHPLC/MSanalysisforevaluation of anthocyanins and carotenoids of 20 sweet potato clones (varieties) withvariable flesh colors including white, yellow, orange, and purple. Spectrophotometericanalysisat530nmshoweddramaticvariationoftotalanthocyaninlevelsfrom0.43mg/gdwto1.36mg/gdwamong8purplefleshedgenotypes.Thegenotypepur04‐123producesthe highest level of total anthocyanins (1.36 mg/g dw). Yellow‐purple flesh pur04‐051,yellowfleshgenotypes,andwhitefleshgenotypeL258,Won‐MI,andSuwon122producerelative low levels of anthocyanins. Every purple sweet potato has a unique HPLCchromatographyprofile.Morethan18anthocyaninsand4carotenoidswereidentifiedviaLC‐ESI‐MS analysis. In addition, new anthocyanins identified from this research will bediscussed.Themethoddevelopedhereforanalysisofanthocyaninsandcarotenoidscanbe

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usednotonlyfornutritionalevaluationoffreshsweetpotatorootsbutalsoasanimportanttoolforselectionofsweetpotatovarieties.FORMATION OF ALLYL/PROPENYL PHENOLS IN LIQUID MEDIA: SUBSTRATEVERSATILITY OF MONOLIGNOL ACYLTRANSFERASES AND ALLYL/PROPENYLPHENOLSYNTHASESSung­Jin Kim, Daniel G. Vassão, Laurence B. Davin, Norman G. Lewis; Institute ofBiological Chemistry, Washington State University, Pullman, WA 99164­6340,davin@wsu.edu Monomeric allyl/propenyl phenols are important phenylpropanoid constituents ofmanyplant aromas and flavours, e.g. in spices and herbs, and some find traditionalmedicinaluses as well, for example the anesthetic/antimicrobial eugenol from clove oil. Suchcompoundsare thought toplayapartwithin thedefensivearsenalofplants,although insome cases they also serve as floral pollinator attractants. We are now pursuingopportunities for themassive production of these compounds for their potential use aspetrochemical substitutes; i.e. allyl/propenyl phenols are typically liquids of highcombustion energies, and are chemically related to styrenes, forming high‐molecularweightpolymersunderappropriatereactionconditions.As a first step towards the metabolic engineering of these biosynthetic pathways intoselectedplant species,wehavedemonstrated the capacity of geneticallymodifiedE. coliliquidcultures toproduceallyl/propenylphenols fromthecorrespondingmonolignols inacceptable yields. As part of this effort, we have isolated and characterizedmonolignolacyltransferases and allyl/propenylphenol synthases from Larrea tridentata (creosotebush)andPiperregnellii(pariparoba).Here,wepresentourresultsregardingthebacterialproductionofallyl/propenylphenols,and discuss the optimization of this bacterial system as a stepping stone towardsengineering this pathway into tobacco and, ultimately, poplar. All the enzymeswehaveexamined here display high substrate versatility, being able to efficiently process all themonolignols and monolignol acetates tested in vitro. Additionally, we will discuss thephylogeneticrelationshipsbetweentheseproteinsandotherphenylpropanoidreductases.

SupportedbyNSF(MCB0417291)andUSDA(68‐3A75‐7‐612).

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THESENSITIVITYOFHMBCSPECTRAUSEDFORNATURALPRODUCTSTRUCTUREDETERMINATIONBYNMRCANDEPENDDRAMATICALLYONPARAMETERCHOICESWilliamF.Reynolds,DepartmentofChemistry,UniversityofToronto,Toronto,ON,CanadaM5S3H6wreynold@chem.utoronto.caTwodimensionalHMBCNMRspectraareofcriticalimportancefordeterminingstructuresofunknownnaturalproductsbecausetheyallowonetotietogethermolecularfragmentsintocompleteskeletalstructures.HoweverHMBCspectraarealsotheleastsensitiveofthesetof2Dspectrausedforstructuredetermination.Wedemonstratethat,byappropriatechoiceofacquisitionandprocessingparameters,onecangetclosetoaten‐foldsensitivityincreaseforthesespectracomparedtoresultsusingparametersfromawidelyusedtextwhichsuggestsparametersforawiderangeofNMRexperiments.POSTERPRESENTATIONSPOSTER1BIOASSAY‐DIRECTEDISOLATIONOFHYPOTENSIVEALKALOIDFROMHOLARRHENAPUBESCENSKAftab1,SBUsmani2,SBegum2andBSSiddiqui21DepartmentofPharmacology&Therapeutics,PeshawarMedicalCollege,Peshawar.2H.E.J.ResearchInstituteofChemistry,UniversityofKarachi,Karachi,Pakistan.aftabk@cyber.net.pk Holarrhenapubescens belongs to the familyApocynacea, commonlyknownas “kurchi” ishighly reputed in traditional medicine as a remedy for amoebic dysentery and otherintestinalailment.Bioassay‐directedfractionation[1]oftheethanolicextractofHolarrhenapubescensresultedintheisolationofsteroidalalkaloidsi.e.HolamideandPubscinine.Holamideshowedathreeprotondoubletat1.45(J=6.56Hz)andtwoABdoublesat3.17and3.00eachforonproton(J=12.06Hz)inthe1HNMRspectrumsuggestedthatitbelongsto conanine series of alkaloid (A class of compoundwith the steroid nucleus and a fivemembers heterocyclic ringwith nitrogen). In contrast Pubscinine showed onemethyl at1.28 while the doublet is missing a three proton singlet was observed at 2.28 due to avinylicmethylindicatedadoublebondinthe18,20–epiminoringoftheconanineseriesofalkaloids.

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In anaesthetized rats, the Holamide and Pubscinine caused a fall in blood pressure in adose‐dependent manner. Pretreatment of animals Atropine completely abolished thehypotensive response of Acetycholine; whereas hypotensive effect of Holamide andPubscininewerenotmodifiedbyAtropine[1].SimilarlyAcetylcholineproducedcontractileeffect in guinea‐pig ileum,whichwas antagonized by atropine, however both (HolamideandPubscinine)failedtoproducedanystimulantresponseonguinea‐pigileum.Thesedataindicate that the steroidal alkaloids i.e. Holamide and Pubscinine from Holarrhenapubescens mediated hypotensive response through a mechanism different to that ofAcetycholine.1. Aftab,K.,Shaheen,F.,Mohammad,F.V.,Noorwala,M.,Ahmed,V.U.Phyto‐pharmacologyofsaponinsfromSymphytumofficinale,AdvExpMedBiol1996;404:429‐442.POSTER2ACOMPARATIVESTUDYOFTHEINVITROCYTOPROTECTIVEACTIVITIESOFCANADIANANDSIBERIANPOPULATIONSOFRHODIOLAROSEAFidaAhmed,SteffanyA.L.BennettandJohnT.ArnasonNeuralRegenerationLaboratoryandOttawaInstituteofSystemsBiology,DepartmentofBiochemistry,Microbiology,andImmunology,andCentreforAdvancedResearchinEnvironmentalGenomics(CAREG),DepartmentofBiology,UniversityofOttawa,30MarieCurie,Gendron283,K1N6N5,fahme074@uottawa.caRhodiolaroseaL.(Arcticroot,roserootorgoldenroot)isawidelyknownmedicinalplantusedinthecircumpolarregionsofEurasiaandrecentlydiscoveredintheNunavikregionofQuebec,Canada.This shrubhasbeenused traditionally to stimulate thenervous system,decreasedepression,andenhance longevity,workperformanceandresistancetovariousemotional, physical and mental stressors. The phytochemical and pharmacologicalpropertiesoftheCanadianpopulationshaveyettobestudied,particularlywithrespecttotheir neuroprotective activities. Here, we describe a preliminary comparative study ofCanadian(Nunavik)andSiberian(Eurasian)populationsofR. roseawithrespect to theircytoprotective activity against neurodegenerative endoplasmic reticulum (ER) stresspathways, which are prevalent in the cellular pathology of many diseases, includingdiabetes and Alzheimer and Parkinson’s disease. Using rat adrenal pheochromocytomacells (PC12‐AC) as the cell model of interest and challenging themwith two known ERstressors, thapsigarginandthecalciumionophoreA23187,weshowthatcrudeethanolicextractsofthetwodifferentpopulationscontainspecificcytoprotectiveactivity,andactviadifferent mechanisms to improve cell survival compared to controls. The subsequentidentification and purification of active compounds using bioassay‐guided isolationtechniqueswill enable identificationofpotentialplant‐derivedERstress inhibitors for invivotestinginanimalmodelsofhumandisease.

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POSTER3ARTEMISININ BIOSYNTHESIS PROFILES IN ARTEMISIA ANNUA GROWING INGROWTHCHAMBERFatimaAlejos­Gonzalez, Li­LiZhou,CaroleH. Saravitz, JanetL. Shurtleff, andDe­YuXie*DeptofPlantBiologyNorthCarolinaStateUniversityRaleigh,NC,27695,*emailaddress:dxie@ncsu.eduMalariaisoneofthetopthreedevastatingdiseasesintheworldcausingthelossofpeople’slife.TheparasiteresponsibleforthevastmajorityoffatalmalariainfectionsisPlasmodiumfalciparum,whichcankillpatients inonlya fewhours. Thefirstnaturalproductusedtotreat malaria was quinine. However, Plasmodium falciparum has developed resistanceagainst this first antimalarial drug and also,more recently, to other related antimalarialdrugs (chloroquine, mefloquine, primaquine, etc.). Artemisia annua L. (Qing hao) is anindigenousmedicinal plant fromChina. In1972 from the leavesofA. annua, a groupofChinesechemistsisolatedthenaturalproductartemisinin,whichisanovelsesquiterpenelactonehighlyactiveagainstP. falciparum.Thecontentofartemisinin inA.annua isverylow,intherangeof0.01‐0.8%ofdryweightofleavesdependentuponthegrowthregions.Certain progresses have beenmade in understanding the biosynthesis of artemisinin bytheseresearchers;however,commercialproductionneedsnumerousadditionaleffortsdueto low yield. Themain barrier to increasing the production of artemisinin is due to theunknownmechanismof thebiosynthesis inplants. Inorder toovercome thebarrier,wehave optimized a growth condition to investigate the biosynthesis of artemisinin. In thisstudy,weshowagrowthcondition toeffectively reduce thedaysofgrowthrequired forflower development. This growth condition allows themedicinal plant to be grown in agrowthchamberlikeanArabidopsisthalianaplant.Theinflorescencedevelopmentbeginsfromthe15thnodeofmostofplantswhentheyareapproximately60‐day’sold.Seedsarematureforcollectionwhentheplantsarenearly80‐day’sold.HPLC‐MSanalysiswasusedto comparatively profile artemisinin and its precursors. The content of artemisinin fromthe1stleafto17thleafisgraduallyincreasedfromatraceleveltonearly2%indryweight.Thelevelsofartemisininintheinflorescencesdevelopedfromthe15th,16th,and17thnodesarenearly2%.Thesepreliminarydatashowthatthebiosynthesisofartemisininisstronglyplant development‐dependent. In addition, from plants grown in growth chambers, wehave already established a self‐pollinated population for studying the biosynthesis ofartemisinin and metabolic engineering of the antimalarial drug. The biosynthesis ofartemisinin in theplantswillbediscussed inourpresentation.Thisproject is fundedbyNorthCarolinaBiotechnologyCenter.POSTER4

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ANALYSISOFPOTATO(Solanumtuberosum)CYP98AGENES

StephanieAndersonandMarkA.BernardsDepartmentofBiologyandtheBiotronTheUniversityofWesternOntarioLondon,Ontario,Canada,N6A5B7sander74@uwo.caThe process of suberization has important implications in thewound‐healing process inplants. Its formationhasbeenshown to inhibitpathogen invasionand topreventwaterloss. Suberin is a complex biopolymer comprised of a poly(aliphatic) domain (SPAD)similar to that of cutin and a poly(phenolic) domain (SPPD) similar to that of lignin.Formationof the SPADandSPPDmustbe coordinately regulatedbyplants andmustbedepositedinaspecificmanner.DuetothesimilaritiesoftheSPPDtolignin,itisimportantto identify a specific enzymatic step or reaction within general phenylpropanoidmetabolismthatactsexclusivelywithinsuberizationinordertostudytheregulationofthesuberizationprocess.Theconversionofp‐coumaroyltyraminetocaffeoyltyramineappearsto be specific to suberization and could fulfill the role of amarker forwound‐induciblesuberization in potato. This unique step is catalyzed by p‐coumaroyltyramine 3‐hydroxylase(CYP98A),acytochromeP450thatusesNADPH‐reductaseandoxygentoaidin the 3‐hydroxylation of substrates involved in phenylpropanoid metabolism (e.g., p‐coumaroyltyramine, p‐coumaroylshikimate, p‐coumaroylquinate, and 4‐coumaroyl‐4’‐hydroxyphenyllacticacid).Thegoalofthisresearchisto identifyputativepotatoCYP98Agenes, clone, sequence them and functionally characterize them. To date, five potentialCYP98A genes have been identified in potato based on in silico analysis from an ESTdatabase. Gene specific primers have been designed, and tissue expression and suberininductionexplored.POSTER5PHYTOCHEMICALSCREENINGANDIMMUNOADJUVANTACTIVITYOFCALLIANDRAHAEMATOCEPHALAEXTRACTS AntonydePaulaBarbosa,BernadetePereiradaSilva,andJoséPazParenteLaboratoryofMedicinalPlantChemistry,NaturalProductsResearchNucleus,HealthSciencesCentre,FederalUniversityofRiodeJaneiro,RiodeJaneiro,BrazilCalliandra haematocephala (Leguminosae) is a native species found inTropical America.Thisplant iswidespreadandcultivatedwithornamentalpurposes ingardensandparks.Studies on the constituents of other species of this genus have been conducted in anattempt to isolate substanceswith immunomodulatory properties. However, a survey ofthe literature showed thatnophytochemical investigationshavebeencarriedouton theconstituentsofthisnativespecies.Inthiswork,wereportthephytochemicalscreeningand

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theimmunoadjuvantactivityofthebutanolicextractfromtheaerialpartsofthisplant.Thephytochemical screeningshowed thepresenceof tannins, flavonoidsandsaponins in thebutanolicextract.Theimmunoadjuvantpropertywasevaluatedagainstovalbuminantigen,since the delayed type hypersensitivity reaction was measured as an in vivo assay ofcellular immune response. Mice immunized with ovalbumin conjugated with extractsshowed remarkable responses greater than thosewhen the antigenwas combinedwithcommercialadjuvants.Thisresponsedevelopedrapidlyafterimmunizationandpersistedat lower levels forat least threedays.Theresultsobtainedsuggest therelevantadjuvantpotentialofthebutanolicextractfromCalliandrahaematocephalaincomparisonwiththecommercial extract of Quillaja saponaria, a commonly used adjuvant for experimentalvaccineformulations.POSTER6NONDESTRUCTIVEANALYSISOFPHYTOCHEMICALCOMPONENTSBYNEARINFARED(NIR)SPECTROSCOPY:MEASUREMENTOFROSMERINICACIDINPRUNELLAVULGARIS MarkA.Berhow,BrentTisserat,SandraDuval,MarkP.Widrlechner,andCandiceGardnerUSDA,ARS,NCAURUSDA,ARS,PlantIntroductionResearchUnit,Ames,IA NIRspectroscopyhasdevelopedintoarapidnondestructivemethodtoanalyzeinasingleevent an increasingly complex number of general and specific components in solid andliquidsamples,includingdissolvedsolids,acids,density,pH,microbialcontamination,andpercentoil,carbohydrate,protein,andmoisture,aswellastodetermineconcentrationsofspecificchemicalcomponents.Theadventofrapidcomputerprogramsthatutilizecomplexmathematical calculations, including Fourier transformation, has allowed for thecomputationofrelationshipsbetweenvariationofmultipleparametersandcorrespondingNIR spectra. In addition, the long pathlengths in NIR spectroscopy allow for samplingthrough glass and plastic, making NIR an evenmore efficientmeasuring process.Whencoordinated with proven chemical and physical analytical methods, the results of thesestandardchemicalanalysescanbetranslatedintoreliableNIRspectrometriccalibrations.We have developed methods to measure the levels of the phenolic phytochemicalrosmarinicacid,indriedleafpowdersofMenthaandPrunella.Theposterwilldiscusstheextensionofthismethodologytorapidlymeasureavarietyofphytochemicalcomponentsinplant tissuesandseeds.The long‐termgoalof this research is toprovide rapid, large‐scale analytical assessment of sample composition and nondestructively assess seed‐compositionalcomponentsforselectioninbreedingprograms.POSTER7GERMPLASMENHANCEMENT FORBLACKPODDISEASERESISTANCE INCACAO(TheobromacacaoL.)

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A. D. Iwaro, S.M. Bharath, V. Singh, C. Perez, L. Ali; The Cocoa Research Unit, TheUniversity of the West Indies, St. Augustine, Trinidad and Tobago, West Indies,cru@sta.uwi.eduBlack pod disease is caused by the fungus‐like organism Phytophthora. It is the mostwidespreaddiseaseofTheobromacacao.Of thesevenknownspeciesofPhytophthoraoncacao(P.palmivora,P.capsici,P.citrophthora,P.megakarya,P.megasperma,P.nicotianae,P.arecae)P.palmivora isthemostpredominant,beingfoundalmosteverywherecacaoisplanted.Lossesfromthisdiseasehavebeenestimatedtobearound30%ofaverageannualproduction. However, susceptible varieties and favorable environmental conditions canlead to losses ashigh as90%.Chemical control of thisdisease is expensive, increasinglyless effective and has significant environmental impact. Thus, developing plants withinherent resistance will affect progress in all the aforementioned areas. The CocoaResearchUnit,TrinidadandTobago,hasbeenpartofagermplasmenhancementprograminitiated in1998.TheprimarygoalhasbeentheaccumulationofresistancegenestothisdiseaseinseveraldiversegeneticgroupsderivedfrommaterialhousedattheInternationalCocoa Genebank, Trinidad. The program comprised two cycles involving evaluation ofdisease resistance in offspring derived from controlled crosses using a leaf discmethod.Cycle 1 of the laboratory evaluations showed that 63.7% (of 3,486 seedlings) weremoderate to highly resistant. This represented a considerable improvement from theparental genotypes. Field evaluations of the most promising types for black pod andwitches’broomresistance,earlyflowering,vigorandpodindexarecurrentlyinprogress.Cycle2 laboratoryevaluations (involving crossesmade frompromising typesof the firstcycle) have just been completed.An increase in thenumber of resistant andmoderatelyresistanttypesisexpected.Themostpromisingtypesinthiscyclewillbeselectedforfieldestablishmentandassessment.POSTER8PUTATIVESECONDARYPRODUCTGLUCOSYLTRANSFERASEEXPRESSIONDURINGCITRUSPARADISIGROWTHANDDEVELOPMENTJalaJ.Daniel1,DanielK.Owens2,andCeciliaA.McIntosh1,2,3.1Dept.Physiology,2Dept.Biol.Sci.,3SchoolofGrad.Studies,EastTennesseeStateUniversity,JohnsonCity,TN37614.mcintosc@etsu.eduFlavonoidsareoneofthemosttheabundantclassesofsecondarymetabolitesfoundinallplants.Severaldifferentkindsofflavonoidglucosyltransferases(GT)existinthetissuesofgrapefruit (Citrus paradisi) making it a model plant for studying their structure andfunction.EffortstounderstandhowflavonoidGTsinfluencesecondarymetabolismandtostudy secondary product GT structure and function has led to the isolation of severalputative secondary product GT clones (PGTs). This research was designed to test thehypothesis that PGT 2‐8 are expressed constitutively during grapefruit seedling growth

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and development. Alternatively, one or more could be expressed in a tissue‐specificmannerordevelopmentallyregulated. WedeterminedmRNAexpressionpatternsofPGT2‐8 during the grapefruit seedling growth and development by quantifying mRNAexpression levels in theroots, stems, leaves,and flowersusingsemi‐quantitativeRT‐PCRandImageJwasusedtoquantifyintensityofbandsstandardizedto18srRNAexpressionasacontrol.Sixgrowthstagesweredefined.FindingsshowthattherewerevariabledegreesofPGTexpressionbetweenthedifferenttissuesandstagesofdevelopment.Forexample,aflavonol‐specific 3‐O‐GTwas expressed in nearly all tissues butwith significantly higherlevelsinleaftissue.PGT8,aclonewith98%homologytoalimonoidGT,wasnotdetectableinvegetativetissuesorflowers,butwasexpressedinsomefruittissues.Additionalresultsare presented. Therefore, results weremore consistent with the alternative hypothesisthatputative secondaryproductGTexpression is tissue specific/andordevelopmentallyregulated.POSTER9BIOASSAY‐DIRECTEDISOLATIONOFHYPOTENSIVEALKALOIDFROMHOLARRHENAPUBESCENSKAftab,SBUsmani,SBegumandBSSiddiquiDepartmentofPharmacology&Therapeutics,PeshawarMedicalCollege,Peshawar.H.E.J.ResearchInstituteofChemistry,UniversityofKarachi,Karachi,Pakistan.

E­mail:aftabk@cyber.net.pk Fax#9291­5200980

Holarrhenapubescens belongs to the familyApocynacea, commonlyknownas “kurchi” ishighly reputed in traditional medicine as a remedy for amoebic dysentery and otherintestinalailment.Bioassay‐directedfractionation[1]oftheethanolicextractofHolarrhenapubescensresultedintheisolationofsteroidalalkaloidsi.e.HolamideandPubscinine.Holamideshowedathreeprotondoubletat1.45(J=6.56Hz)andtwoABdoublesat3.17and3.00eachforonproton(J=12.06Hz)inthe1HNMRspectrumsuggestedthatitbelongsto conanine series of alkaloid (A class of compoundwith the steroid nucleus and a fivemembers heterocyclic ringwith nitrogen). In contrast Pubscinine showed onemethyl at1.28 while the doublet is missing a three proton singlet was observed at 2.28 due to avinylicmethylindicatedadoublebondinthe18,20–epiminoringoftheconanineseriesofalkaloids.In anaesthetized rats, the Holamide and Pubscinine caused a fall in blood pressure in adose‐dependent manner. Pretreatment of animals Atropine completely abolished thehypotensive response of Acetycholine; whereas hypotensive effect of Holamide andPubscininewerenotmodifiedbyAtropine[1].SimilarlyAcetylcholineproducedcontractileeffect in guinea‐pig ileum,whichwas antagonized by atropine, however both (HolamideandPubscinine)failedtoproducedanystimulantresponseonguinea‐pigileum.Thesedataindicate that the steroidal alkaloids i.e. Holamide and Pubscinine from Holarrhena

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pubescens mediated hypotensive response through a mechanism different to that ofAcetycholine.1. Aftab, K., Shaheen, F., Mohammad, F. V., Noorwala, M., Ahmed, V. U. Phyto‐pharmacologyofsaponins fromSymphytumofficinale,AdvExpMedBiol1996;404:429‐442.POSTER10ISSABP2ARECEPTORFORACIBENZOLAR‐S‐METHYL(ASM):ACHEMICALINDUCEROFSYSTEMICACQUIREDRESISTANCE(SAR)INPLANTS?DiwakerTripathi1,Yu­LiJiang2,DhirendraKumar1DepartmentofBiologicalSciences1,DepartmentofChemistry2,EastTennesseeStateUniversity,JohnsonCity,TN­37614 Plants defend themselves by developing an enhanced resistance to a broad spectrum ofpathogensintheareaofprimaryinfectionaswellasinthedistal,uninoculatedparts.ThisinducedstateofresistanceiscalledSystemicAcquiredResistance(SAR).Salicylicacid(SA),aplanthormone,isessentialforthefulldevelopmentofSAR.TreatmentofplantswithSAmakes them resistant to pathogens. SA‐binding protein 2 (SABP2) is amethyl salicylateesterasethatcatalyzestheconversionoflipidmobilemethylsalicylate(MeSA),synthesizedin plants resisting pathogens, into SA. Plantswhich do not express SABP2 are unable toconvert MeSA into SA and fail to develop an effective SAR response. Various functionalanalogs of SA have been developed and used to activate plants own natural defensesagainst microbial pathogens. ASM (Acibenzolar‐S‐Methyl) is one such functional analogwhich inducesSAR invarious familiesofmonocotanddicotplants.ASM is commerciallyavailablebythenameofBIONandActigard.HowASMfunctionsinplantsismostlyunclear?WehypothesizethatASMactivitydependson its conversion into anacid form, a reaction catalyzedbySABP2.WeperformedHPLCstudiestoshowthatSABP2catalyzestheconversionofASMesterintoitsacidform.Totestourhypotheses inbiologicalsystem,weareusingtransgenic tobacco(NicotianatabacumcvXanthinc)SABP2‐silencedplants inwhichSABP2geneexpression is silencedbyRNAinterference and control plants, containing empty silenced vector. Using this system,wewilldetermineifSABP2catalyzedconversionofASMesterintoacidformisalsorequiredfor (1) SAR induction and (2) expression of SAR genes. Results showing the actionmechanismofASMwillbepresented.POSTER11CAROTENOGENESISOFORANGECOLORINCAPSICUMANNUUMFRUITIvetteGuzman,PaulBoslandandMaryO’Connell

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PlantandEnvironmentalSciencesDepartmentNewMexicoStateUniversityMSC3QP.O.Box30001LasCruces,NM88003­8001ivetteg@nmsu.eduPepper,Capsicum spp., is aworldwide crop valued for heat, nutrition, and rich pigmentcontent.Carotenoids, the largestgroupofplantpigments, functionasantioxidantsandasvitamin A precursors. The most abundant carotenoids in ripe peppers are β‐carotene,capsanthin, and capsorubin. The genetic and biochemical basis for the orange color inpeppershasnotbeen fullyexplained.Carotenoidprofilesandgenestudiesare lacking.Anovel UPLC method was developed to efficiently separate 6 carotenoid standards withuniqueelution times.Wehavedetermined the carotenoid chemicalprofilepresent in13orangepeppervarietiesusingUPLC;insomecasestheorangeappearanceofthefruitwasduetotheaccumulationofβ‐carotene,whileinothercases,noβ‐carotenewasdetected,rather red and yellow carotenoids were the basis for the color. Four carotenoidbiosyntheticgenes,Psy,Lcyb,Ca1,andCcswereclonedandsequencedfromthesecultivars.This data allowed us to test the hypothesis that different alleles for specific carotenoidbiosynthetic enzymes are associatedwith specific carotenoid profiles in orangepeppers.The results indicated polymorphic variation among the orange peppers analyzed.SupportedinpartbyNMAESandNIHR25‐GM61222.POSTER12IDENTIFICATIONANDFUNCTIONALCHARACTERIZATIONOFSUBERIN‐ASSOCIATEDOMEGA‐HYDROXYLASESINPOTATOM.L.HaggittandM.A.BernardsUniversityofWesternOntarioDepartmentofBiologySuberin isa complexplantbiopolymer that functionsasabarrieragainstwater lossandpathogen attack. Suberin biosynthesis involves the complex regulation of twometabolicpathways, lipid and phenolic metabolism, to form a macromolecule with two domains(poly(aliphatic) and poly(phenolic)) covalently linked together. In Solanum tuberosum L.(potato), 55% of the suberin poly(aliphatic) monomers are derived from the omega‐hydroxylationof fatty acids, indicating that theomega‐hydroxylase enzymehas a criticalroleinsuberinformation.Omega‐hydroxylasescatalyzethehydroxylationoftheterminalmethyl group of fatty acids, and belong exclusively to the large protein family ofcytochromeP450enzymes.Thusfar,twoplantsubfamilieshavebeenidentifiedasomega‐hydroxylases, CYP86A and CYP94A. Members from each of these subfamilies have beenshown toparticipate in cutinbiosynthesis, but their role in suberinbiosynthesishasnotbeeninvestigated.Inpotato,amodelsystemforstudyingsuberin,threecandidateomega‐hydroxylase sequences have been identified through database searching. This studywill

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focus on the functional characterization of omega‐hydroxylase sequences expressed insuberizing tissue, to determine if they are active in suberin biosynthesis. Functionalcharacterization of the potato omega‐hydroxylases will include testing for substratespecificity, enzyme kinetics and determining themolecularweight. Immunocytochemicallocalizationofthepotatoomega‐hydroxylaseswillbethefirstevidenceforthelocationofsuberin assembly, indicating whether the fatty acids are modified prior to‐ or post‐incorporation into the macromolecule. Finally, exploration of the promoters of theseomega‐hydroxylases will lead to further understanding of the regulation involved insuberin biosynthesis. Understanding the process of suberin biosynthesis involves thecharacterizationofakeyreaction,omega‐hydroxylation,whichwill leadtoanincreaseinourbasicunderstandingofthecooperativeregulationofmultiplemetabolicpathways.POSTER13GREENHOUSESTUDIESOFTHEALLELOPATHICPROPERTIESOFSORGHUM(SORGHUMBICOLOR)KellyHarrisonandJeffreyD.WeidenhamerDepartmentofChemistry,AshlandUniversity,Ashland,OH44805USAjweiden@ashland.edu

Sorghum (Sorghum bicolor L. Moench) is an allelopathic species that represses weedgrowth through the exudation of the phytotoxic benzoquinone, sorgoleone. It haspreviously been shown that small‐seeded weeds are most affected by sorgoleone, andredrootpigweed(AmaranthusretroplexusL.)hasshownparticularsensitivitytosorghuminpreviousstudies.Twoplantgrowthstudieshavebeencarriedouttoassesstheimpactofsorghumallelochemicalsonneighboringplantgrowth.Atarget‐neighbordesignwasusedin which differing densities of pigweed were planted around a single sorghum plant.Pigweedwasplantedatdensitiesof0,3,6,9,12plantsperpotandtheshootdrymasswascollectedafter25days.Theexpectedresultofresourcecompetitioninsuchastudywouldbethatpigweeddrymasswoulddecreaseproportionatelyasthenumberofplantsperpotincreased. However,giventhepresenceofphytotoxinsproducedbysorghum,deviationsin this expected pattern may occur. Allelopathic effects are density‐dependent, withgreater effects observed at lower plant densities and reduced when plants share theavailablepoolof toxinswithoneanother. Thusallelopathic inhibitionwillbegreatestatlowpigweeddensities,andresourcecompetitionmost intenseathighpigweeddensities.This could then lead to maximum size of the pigweed neighbors occurring at anintermediatedensity.Asecondstudywascarriedouttodeterminethepotentialimpactofsorghum on the competitive outcome of two neighboring species which differ in theirsensitivity to sorghum phytotoxins. In contrast to redroot pigweed, velvetleaf (AbutilontheophrastiMedic.) is less sensitive to the allelopathic effects of sorghum. In this study,four sorghum plants were established around a central zone containing either fourpigweedplants,fourvelvetleafplants,ortwoofeachtype.Inhalfofthepots,therootsofthe velvetleaf andpigweed target plantswere kept separated from sorghumbyplanting

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intoStyrofoamcupsburiedinthepots,preventingroot‐rootinteractionbetweensorghumand theweed species. Given the greater sensitivity of redroot pigweed to sorghum, it isexpectedthatthecompetitivebalancebetweenvelvetleafandpigweedwillbeshiftedwhentheplantsaregrownincontactwithsorghum.Thisisbeingassessedbymeasurementofshootdrymass.POSTER14UNRAVELING THE BIOSYNTHESIS OF VOLATILES IN PLANT DEFENSE: AMULTI‐CATALYTIC ARABIDOPSIS CYTP450 ENZYME IS RESPONSIBLE FOR THECONVERSIONOFGERANYLLINALOOL TO THE INSECT‐INDUCEDHOMOTERPENETMTTSungbeomLee1,MarcoHerde2,ChristianeGatz3,andDorotheaTholl11Department of Biological Sciences, Latham Hall, Virginia Tech, Blacksburg, VA24060;sungbeom@vt.edu;tholl@vt.edu2Department of Biochemistry and Molecular Biology, Michigan State University;mherde@msu.edu3Albrecht­von­Haller­InstitutforPlantSciences,UntereKarspuele1A,Georg­August­Universität,Göttingen,GermanyVolatile secondary metabolites such as homoterpenes released from vegetative planttissues have significant functions in plant defense by directly warding‐off herbivorousinsectsorattractingnaturalenemiesofherbivores.Thisintriguingself‐defensesystemhasdrawnincreasingattentiontothemetabolicengineeringofvolatilesasatoolindevelopingalternative pest controls. The C16‐homoterpene 4,8,12‐trimethyltrideca‐1,3,7,11‐tetraene(TMTT) is emitted from aerial parts ofmany plants including crops such asmaize, limabean,tomatoandalfalfaaswellasthemodelplantArabidopsisthaliana.TMTTemissionisinducedupon insectandmiteattackand functions inattractingparasitesorpredatorsofthese pests. Besides insect feeding, emission of TMTT can also be induced by fungalelicitorsandbacterialpathogeninfection.WeareinterestedinidentifyingthekeyenzymaticstepsintheTMTTmetabolicpathway.Here we demonstrate for the first time that CYP82G1 (At3g25180), a multi‐catalyticCytP450 enzyme of the Arabidopsis CYP82 family, is responsible for the single‐stepconversionof theC20‐precursor geranyllinalool toTMTT.Wedescribe that expressionoftheCYP82G1geneiscoordinatedwiththatofgeranyllinaloolsynthaseinArabidopsisleavesupon treatment with the fungal elicitor alamethicin, particularly in a COI‐1 dependentmanner. TMTT emission is absent in CYP82G1 loss‐of‐function mutants under elicitor‐induced conditions but can be restored by complementation with the CYP82G1 geneexpressedundercontrolof theconstitutiveCaMV35Spromoter.Notably, invitroenzymeassayswithrecombinantCYP82G1andinvivosubstrate­feedingassaysinyeastcellsover‐expressingCYP82G1revealabroadersubstratespecificityoftheenzymebyconvertingnotonly geranyllinalool but also its C15‐analog nerolidol to the respective C11‐homoterpeneDMNT.HistochemicalCYP82G1promoter‐GUSassaysconfirminducibleexpressionofthis

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geneinleavesuponinsectfeedingandshowthatconstitutiveexpressionislimitedtostemsandinflorescences.POSTER15SEEDCOATPHENOLICSANDTHEDEVELOPINGSILIQUETRANSCRIPTOMEOFYELLOW‐SEEDEDANDBROWN‐SEEDEDBRASSICACARINATAXiangLi,NeilWestcott,andMargaretY.GruberAUTHOREMAILADDRESS:XiangLi,lixi@agr.gc.ca;MargaretY.Gruber,gruberm@agr.gc.caAgricultureandAgri­FoodCanada,SaskatoonResearchCentre,107SciencePlace,Saskatoon,Saskatchewan,S7N0X2,CanadaLittle isknownabout themolecularbasisof theBrassica carinatadominantyellowseed,whichisuniqueamongthemyriadofrecessiveyellowseedtraitsofotherBrassicaspecies.In order to address this deficiency, seed coats from near‐isogenic B. carinata lines withbrownseedoryellowseedwereexaminedformoredetaileddifferencesintheirphenoliccompositionsthanpreviouslydetermined.Thestructureofninecompounds,includingfourflavonoids, three lignans and two phenylpropanoids was elucidated on the basis ofextensive 1D‐ and 2D‐NMR spectroscopy, as well as ESI‐MS‐MS. The yellow seed coatsaccumulated flavonoids, phenylpropanoids and lignans, but soluble and insolubleproanthocyanidins were strongly reduced, while brown seed coats accumulated largeamountsofbothtypesofproanthocyanidinsandphenylpropanoidsandlignansequivalenttotheamountsintheyellowseed,butlackedflavonoids.Bothlinesaccumulatedamixedcomponent containing quercetin and lignan derivatives and larger‐sized unidentifiedmaterialastheirmajorHPLC‐UVpeak,butthepeakwas0.4‐foldlowerintheyellowseedcoats. DNAmicroarray analysis using a Brassica napus 15, 000 expressed sequence tag(EST) array indicated 296 developing silique genes which were differentially expressedmore than 2‐fold, including proteins involved in defense, metabolism, transcription,transportation, signal transduction, cytochromeP450, aswell as proteinswith unknownfunctions.Fivegenes(F3’H,FLS,F3H,FOMTandDFR) involved in the flavonoidpathwaywere differentially expressed, but genes in the phenylpropanoids pathway were notchanged significantly. Regulatory factors and genes with unknown function wererepresented among genes with changed expression 2.4‐foldmore in developing yellow‐seededsiliquesthaninbrown‐seededsiliques.KEYWORDS:flavonoids,lignans,phenylpropanoids,B.carinata,microarray.POSTER16CLONINGANDEXPRESSIONOFAPUTATIVESECONDARYPRODUCTGLUCOSYLTRANSFERASECLONE,PGT10,FROMCITRUSPARADISIUSINGE.COLI

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EXPRESSIONANDAGROBACTERIUM‐MEDIATEDTRANSIENTEXPRESSIONINNICOTIANATOBACUM ZhangfanLin1,DanielK.Owens1,CeciliaA.McIntosh1,2.1linzhangfan@gmail.com

DepartmentofBiologicalSciences,2SchoolofGraduateStudies,EastTennesseeStateUniversity,JohnsonCity,TN,37614. FlavonoidsareaclassofplantsecondarymetabolitesthatfulfillmanyfunctionsincludingUV protection, producing pigmentation, having antipathogenic properties and acting asfeedingdeterrents.Glucosylationofflavonoidshelpflavonoidstoperformtheirimportantroles in planta and these reactions are catalyzed by glucosyltransferases (GTs). TheMcIntosh lab research focus is on flavonoid metabolism and structure/function ofsecondaryproductGTs.Citrusparadisi(grapefruit)isusedasthemodelplantsystemsinceitisveryactiveinflavonoidmetabolismandpossessesavarietyofsecondaryproductGTs.This research is designed to test the hypothesis that PGT10 is a flavonoidglucosyltransferase. PGT10wasclonedintopCD1vectorwhichcontainsthioredoxinand6His tags as well as a thrombin proteolytic cleavage site to remove these tags afterrecombinantexpression.ItwasheterologouslyexpressedandpurifiedfromE.coliextractsbyimmobilizedmetalionaffinitychromatography;howeverthemajorityoftheexpressedprotein was found in insoluble inclusion bodies. In another approach, we are using anAgrobacterium‐mediated transient expression system in Nicotiana tobacum which willlikely overcome solubility issues. PGT10 has been subcloned into binary vector pER8,transformed into Agrobacterium tumefaciens GV3101, and transfected into Nicotianatobacum leaves. Expression of the transgene in tobacco leaveswas induced by estradioltreatmentandPGT10proteinproductionisbeingmonitoredbywesternblot.Ifincreasedsoluble PGT10 expression levels are observed, we will perform enzyme assays andrigorous biochemical analyses of the protein. The plant expression system will beevaluatedforapplicabilityinexaminationofothergrapefruitPGTs.POSTER17VARIABILITYOFCYP3A4INHIBITIONBYGINSENGROOTSANDCOMMERCIALPRODUCTSAliceLuu1,KristinaMcIntyre1,TeresaW.Tam2,JohnT.Arnason1,BrianC.Foster2luua2@mcmaster.ca1DepartmentofBiology,UniversityofOttawa,30MarieCurie,Gendron283,OttawaOntario,K1N6N52TherapeuticProductsDirectorate,HPFB,HealthCanada,Ottawa,ONGinsenghasbeenusedinTraditionalChineseMedicine(TCM)totreatavarietyofailmentsincluding fatigue, insomnia, impotence, irritability, and even illnesses such as diabetes.

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There is growing concern for the potential of herb‐drug interactions with the use ofginseng, especially when taken with medications such as blood thinners andantidepressants.CytochromeP450(CYP)istheprimaryfamilyofenzymesinvolvedinthebiotransformationandmetabolismofdrugs.Inparticular,CYP3A4isknowntometabolizemore than50%of clinically‐prescribeddrugs.This studywasperformed todetermine ifmethanolic extracts of ground Panax quinquefolius and Panax ginseng roots, andcommercialproductshavethepotentialtocauseadverseeffectsthroughinteractionswithdrugsbyCYP3A4inhibitionusingahighthroughputfluorescence‐basedplatereaderassay.Two different response profiles were obtained in the dose response study. The testedcommercial products (Ontario Ginseng from Community Choice, Korean Ginseng, RedChineseGinsengfromSwissNaturalSources)hadaweakinhibitoryprofilewithIC50valuesgreaterthan200μg/mL.ThegroundrootsamplesofOntario‐grownPanaxquinquefolius,however,demonstratedahighpotential tocompetitively inhibitCYP3A4with IC50valuesrangingbetween34 to44μg/mL. Studies areunderway to correlate inhibitionpotentialwith ginsenoside content through HPLC‐DAD analysis. In particular, all samples will becharacterizedfortheirRg1,Re,Rb1,Rc,Rb2,Rd,andRfcontent.Overall,theresultssuggestthatsomeginsengproductsfromthePanaxgenusmayalternormaldrugmetabolismandconsequentlyresult inpotentialherb‐drug interactions.Furtherstudiesarewarranted todetermineiftheseeffectsareclinicallysignificant.POSTER18HETEROLOGOUSEXPRESSIONANDELUCIDATIONOFBIOCHEMICALFUNCTIONOFPUTATIVEFLAVONOIDGLUCOSYLTRANSFERASECLONESFROMCITRUSPARADISIVenkataS.Mallampalli1,DanielK.Owens1andCeciliaA.McIntosh1,21Dept.BiologicalSciencesand2SchoolofGraduateStudies,EastTennesseeStateUniversity,JohnsonCity,TN,37614email:mallampalli_siddhartha@yahoo.comFlavonoidsareamajorgroupofplantsecondarymetaboliccompounds.Flavonoidsplayavariety of roles for plants including protection againstUV light, pigmentation and flavorwhichcanbeimportantforpollinationandseeddispersal,sexualreproductionandpollendevelopment, antimicrobial and antifungal properties, and cues for microbial symbiontcolonization.Flavonoidsarealsoknowntohavebenefittohumanhealthduetoantioxidantandanticancerproperties.Glucosylationisaprominentmodificationreactioninflavonoidbiosynthesis and these reactions are catalyzed by glucosyltransferases (GTs). Citrusparadisi(grapefruit)isanexcellentmodelsystemforstudyingflavonoidGTsasitisknownto contain GTs capable of producing flavanone, flavone, and flavonol 7‐O‐glucosides,flavonol 3‐O‐glucosides, and chalcone glucosides. Current efforts are focused on cloning,expressionandcharacterizationofputativeGTsfromgrapefruit.Thisresearchisdesignedto test the hypothesis that grapefruit PGT2 and PGT9 clones are flavonoidglucosyltransferases. Results from experiments designed to optimize heterologousexpression,enrichsolublerecombinantprotein,andscreenforGTactivityusingasuiteof

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substratesarepresented. ResultsshowthatPGT2proteinexhibitsa low levelofactivitytowardquercetinandthereactionproductisbeingidentified.PGT2proteinhadnoactivitytoward any of the other substrates tested. Results of work on PGT9, a clone obtainedthroughabioinformaticapproachusingtheharvESTdatabase,willalsobepresented.POSTER19ALKALOIDSASFEEDINGDETERRENTSFORGYPSYMOTHLARVAE,LYMANTRIADISPAR(L.):ANEUROPHYSIOLOGICALANALYSISOFGUSTATORYNEURONSENSITIVITY

TimothyL.Martin,KatelynF.Beattie,andVonnieD.C.Shields

DepartmentofBiologicalSciences,TowsonUniversity,8000YorkRd,Towson,MD21252

Alkaloids are secondary nitrogen‐containing plant metabolites that generally havepharmacologicaleffects.Morethan3000alkaloidshavebeenidentifiedandarecommoninmore than 4000 plant species. Gypsy moth larvae, Lymantria dispar (L.), are highlypolyphagousanddisplayawidehostpreference, feedingonthe foliageofa fewhundredspeciesofplants.TheselarvaearemajorpestdefoliatorsintheUnitedStatesanddestroymillionsofacresoftreesannually. Theypossessgustatorysensoryorganslocatedonthemaxillae, namely themedial and lateral galeal styloconic sensilla. These sensillaplay animportant role in host‐plant selection through the detection of phytochemicals, such asalkaloids. The styloconic sensilla each house four taste receptor cells within them,includingasugar,salt,deterrent,andinositolcell. Usingasinglecellelectrophysiologicaltip‐recording method, our aim was to characterize the temporal firing patterns andsensitivitiesofthereceptorcellshousedwithineachsensillumwhenexposedtoapanelofselected phytochemicals. Our results revealed that these receptor cells responded toalkaloids, including strychnine, atropine, aristolochic acid, nicotine, and caffeine; sugarsandsugaralcohols,includingsucrose,fructose,andinositol;andsalt, includingpotassiumchloride. In general, the deterrent cell exhibited a robust temporal firing pattern andexhibitedvarying sensitivity to alkaloid stimulation. Thus, this studyoffers insights intotheroleofphytochemicals,especiallyalkaloids,inthetastephysiologyofthislarvalinsect.Italsoprovidesagatewayforthepossibleuseofalkaloidsasantifeedantswhendesigningbiocontrolagents.

ThisresearchwassupportedbyNIHgrant1R15DC007609‐01toV.D.S.

POSTER20SPATIALDISTRIBUTIONANDDIFFUSIONOFROOT‐EXUDEDTHIOPHENESFROMMARIGOLD(TAGETESSPP.)

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TriciaMatz,JessicaLaMoreaux,BrianMohneyandJeffreyWeidenhamerDepartmentofChemistry,AshlandUniversity,Ashland,OH44805USAjweiden@ashland.eduRoots of marigold (Tagetes erecta and Tagetes patula) are known to release the highlyphytotoxic thiophenes5‐(3‐buten‐1‐ynyl)‐2,2’‐bithienyl (BBT)andα‐terthienyl (α‐T) intosurroundingsoil.Recently,solidphaserootzoneextraction(SPRE)probesconstructedbyinsertingstainlesssteelwireintopolydimethylsiloxane(PDMS)tubinghavebeenshowntorecovernano‐tomicrogramquantitiesofthiophenesfromsoilbeneathgrowingmarigolds,using a24hour sampling time. Theobjectiveof these experimentswas to extend thesenewmethods tomap the spatial distribution of thiophenes in the root zone. Marigoldsweregrownina1‐cmthicklayerof1:1sand/growthmediummixmaintainedbetweentwofoil‐lined glass plates which were then clamped together. After plants were well‐established,athin,PDMSmembranewaspress‐appliedtotherootzoneandleftinplacefor24hours.Membraneswerethencutinto2.5x2.5cmsegmentsandanalyzedforthiophenecontent. Marigolds were also grown in large PVC pipes with access ports drilled forinsertion of SPRE probes at eight different depths. Following establishment of themarigolds, pipes were sampled weekly for three weeks to monitor thiophene content.Resultsthusfarshowthattheamountsofthiophenesdetectedarehighlyvariable,anddonot directly correlate to the amount of root mass in adjacent soil. Amounts of BBTrecovered with PDMS are often but not always greater than amounts of α‐T measured.Furthermore,analysesofmarigoldrootsshowvariableconcentrationsofthiophenesfromone rootportion toanother, althoughamountsofBBT foundare ingeneralmuchhigherthanα‐T. Inordertogaininsight intothebehaviorofthiophenesinsoil,diffusionofα‐TthroughagarandsandisbeingstudiedusingPDMSprobesloadedwithknownamountsofα‐T.Diffusionofα‐Thasbeenobservedoverdistancesupto5cminPhytagelagar,andupto2cminsand.Furtherstudiesarecontinuingtoexplorereasonsfortheheterogeneityofthiophenedistributionsbeneathmarigolds.POSTER21THEUTILITYOFCOUPLED‐HSQCSPECTRAFORDETERMININGTHEIDENTITIESOFSUGARUNITSINPROTONNMR‐SPECTRALLYCROWDEDSAPONINS

EugeneP.Mazzola,1AinsleyParkinson,2BruceCoxon,3DaronI.Freedberg,4andEdwardJ.Kennelly2

1.UniversityofMaryland­FDAJointInstitute,CollegePark,MD,20742,2.LehmanCollege,CUNY,Bronx,NY,10468,3.NIH,NICHD­LDMI,Bethesda,MD20892,4.FDA,CBER,Bethesda,MD20850

Saponins are glycosides of steroids, steroid alkaloids or triterpenes found in plants, especially in the plant skins where they form a waxy protective coating. The structures of

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three complex saponins, blighosides A-C from the tree Blighia sapida, were elucidated by one- and two-dimensional NMR and the H-1 and C-13 NMR spectra of these saponins assigned using only 1D and 2D NMR techniques. Blighoside A (MW 1086) has a tetrasaccharide linked to a triterpene aglycone, hederagenin. The four sugars in order of attachment at C-3 of hederagenin are arabinose, rhamnose, glucose, and another arabinose. Blighoside B (MW 1070) has the same tetrasaccharide linked to a different triterpene aglycone, oleanolic acid, which differs from hederagenin in that its C-23 is a methyl group rather than CH2OH. Blighoside C (MW 1504) has a hexasaccharide linked to oleanolic acid. The six sugars are attached at C-3 of oleanolic acid in the following linear array: two xyloses, rhamnose, glucose, a second rhamnose, and another glucose. Additionally, the first glucose is acetylated at positions 3 and 6 and the terminal glucose at positions 4 and 6. High-resolution coupled-HSQC spectra were especially useful for determining the identities of the monosaccharides in the three saponins. HMBC experiments enabled the linkages to be established between both the individual sugar units and C-3 of the two triterpenes. In all cases, complimentary 3-bond connectivities were observed between the anomeric protons and carbinol carbons and the respective anomeric carbons and carbinol protons. Both the H-1 and C-13 chemical shifts occur in relatively narrow spectral ranges, and the proton NMR spectra of all three saponins exhibit severe signal overlap. The coupled-HSQC spectra were of inestimable value in situations where protons on adjacent carbons had nearly coincident chemical shifts and the resulting strong H–H coupling made the measurement of vicinal couplings essentially impossible. Coupled-HSQC spectra elegantly obviated this problem because vicinal proton couplings are now determined from 1H – 13 C –12C—1H units, and the large one-bond C-H coupling effectively results in weak vicinal H–H coupling.

POSTER22PUTATIVESECONDARYPRODUCTGLUCOSYLTRANSFERASEEXPRESSIONDURINGCITRUSPARADISIGROWTHANDDEVELOPMENTJalaJ.Daniel1,DanielK.Owens2,andCeciliaA.McIntosh1,2,3.1Dept.Physiology,2Dept.Biol.Sci.,3SchoolofGrad.Studies,EastTennesseeStateUniversity,JohnsonCity,TN37614.mcintosc@etsu.eduFlavonoidsareoneofthemosttheabundantclassesofsecondarymetabolitesfoundinallplants.Severaldifferentkindsofflavonoidglucosyltransferases(GT)existinthetissuesofgrapefruit (Citrus paradisi) making it a model plant for studying their structure andfunction.EffortstounderstandhowflavonoidGTsinfluencesecondarymetabolismandtostudy secondary product GT structure and function has led to the isolation of severalputative secondary product GT clones (PGTs). This research was designed to test thehypothesis that PGT 2‐8 are expressed constitutively during grapefruit seedling growthand development. Alternatively, one or more could be expressed in a tissue‐specificmannerordevelopmentallyregulated. WedeterminedmRNAexpressionpatternsofPGT2‐8 during the grapefruit seedling growth and development by quantifying mRNAexpression levels in theroots, stems, leaves,and flowersusingsemi‐quantitativeRT‐PCR

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andImageJwasusedtoquantifyintensityofbandsstandardizedto18srRNAexpressionasacontrol.Sixgrowthstagesweredefined.FindingsshowthattherewerevariabledegreesofPGTexpressionbetweenthedifferenttissuesandstagesofdevelopment.Forexample,aflavonol‐specific 3‐O‐GTwas expressed in nearly all tissues butwith significantly higherlevelsinleaftissue.PGT8,aclonewith98%homologytoalimonoidGT,wasnotdetectableinvegetativetissuesorflowers,butwasexpressedinsomefruittissues.Additionalresultsare presented. Therefore, results weremore consistent with the alternative hypothesisthatputative secondaryproductGTexpression is tissue specific/andordevelopmentallyregulated.POSTER23GINSENOSIDEVARIATIONWITHINANDBETWEENONTARIO‐GROWNNORTHAMERICANGINSENG(PANAXQUINQUEFOLIUS)POPULATIONSKristinaL.McIntyre,AliceLuu,JohnT.ArnasonCentreforResearchinBiotechnologyandBiopharmaceuticals,DepartmentofBiologyUniversityofOttawa,Ottawa,CanadaNorthAmericanginseng(Panaxquinquefolius)isanincreasinglyimportantcropinOntario.CultivatedOntarioginsengpopulationshavebeengrowingseparatelyforseveraldecadeswith each population potentially possessing unique characteristics and phytochemicalproperties. Ginsenoside content and variation were assessed within and between fiveOntario ginseng farmpopulationsusingHPLC‐DAD. GinsenosidesRg1,Re,Rb1,Rc,Rb2,andRdwerequantifiedin20‐25rootsperpopulation.Ginsenosideprofileswereconsistentacross populations (Rb1> Rd> Re> Rc> Rg1> Rb2),which is unique in comparisonwithseveral reportsofP.quinquefolius grown inotherareaswhereRd isoftenpresent in thelowestquantity.Significantvariationwasshownwithineachpopulation,varyingby5‐12%(w/w). There was no significant difference inmean total ginsenoside content betweenpopulationsthoughtherewasadifferenceinRccontentbetweenpopulations.Thisintra‐population variation suggests potential for the development of cultivars with uniquephytochemicalprofiles. POSTER24SUBERINLAMELLAEBIOSYNTHESISINIRISGERMANICA'SMULTILAYEREDEXODERMIS.ChrisJ.Meyer1,CarolA.Peterson1,MarkA.Bernards2

1DepartmentofBiology,UniversityofWaterloo,Waterloo,ON,CanadaN2L3G1,and2DepartmentofBiology,UniversityofWesternOntario,London,ON,CanadaN6A5B7More than 90% of angiosperm species have roots with an exodermis. Of these species,approximately 18% develop a multilayered exodermis (MEX), including roots of Iris

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germanica. All of I. germanica's mature MEX walls contain suberin lamellae with apoly(aliphatic) domain (SPAD) that is important for regulating radial water and iontransport. In general, the SPAD has a known monomeric composition as revealed fromanalysisof fullymaturesuberinlamellae.However, itssynthesis inamaturingexodermishad never been examined. Our objectivewas to analyze SPAD biosynthesis by detectingand quantifying suberin‐specific compounds at particular stages ofMEXmaturation andunderdifferentgrowthconditions.Rootsweregrowninhydroponicculturewherethetankwaseithercompletelyfilledorpartiallyfilledtocreatea60mmhumidairgapbetweenthebase of the rhizome and solution surface.MEXmaturationwas restricted in submergedrootsbutacceleratedinrootareasexposedtotheairgap.Atspecificstagesofmaturation,the exodermis was isolated manually from the central cortex and flash frozen.Unpolymerized fattyacids,alkanes,andprimaryalcohols (soluble fraction)were isolatedfrom the exodermal tissue using exhaustive micro‐soxhlet extraction withchloroform:methanol (2:1). The remaining polymerized suberin monomers (insolublefraction), including18:1α,ω‐dioicacidsand18:1ω‐hydroxy fattyacids,were isolatedbytransesterificationusing2MMeOH/HClat80°Cfor2h.Theisolatedmonomersfromboththe soluble and insoluble fractions were TMS derivatized, and then quantified andidentifiedbyGC‐MS.Thedata tobepresentedwilldetail solubleand insolublemonomercompositions and amounts as the MEX matures in submerged or air gap‐exposed rootareas. Data collection over time revealed patterns of carbon flux into monomers, andalloweddetectionofmonomersastheywereactivelyincorporatedintotheSPAD.POSTER25THEANTICATARACTOGENESISPROPERTIESOFTRADITIONALVIETNAMESEANDCREEMEDICINESBYALDOSEREDUCTASEINHIBITION.SanNguyen1,DariusFarsi1,CoryHarris1,PierreHaddad2,3,JohnT.Arnason1.1CentreforResearchinBiotechnologyandBiopharmaceuticals,DepartmentofBiologyUniversityofOttawa,Ottawa,Canada.2DepartmentofPharmacology,UniversitédeMontréal,Montréal,Québec,CanadaH3C3J73Institutdesnutraceutiquesetdesalimentsfonctionnels,UniversitéLaval,Québec,CanadaG1K7P4Thisresearchexaminestheroleofnaturalproductsfromtraditionalmedicinesaspotentialcandidates inpreventing thedevelopmentofcataracts in type2diabetesby inhibitionofaldose reductase. The focus of the study is traditional VietnameseMedicinal (TVM) andCreeFirstNationmedicinesusedintreatingsymptomsofTypeIIDiabetes(T2D).Extractsof each plant were prepared by extraction with 80% ethanol and tested in an aldosereductase preparation obtained from pig eyes by monitoring NADPH consumptionspectrophotometrically.GlycyrrhizaeuralensisandCuscutachinensis,inparticularexhibitedover 90% inhibition of NADPH consumption at a concentration of 25 mg/ml of plantextract.Furtheranalysisofthesetwoplantsrevealedalineardose‐dependentrelationship.The IC50 was determined to be 9.5±5x10‐5μg extracts/mg protein and 5.5±5x10‐5μg of

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extracts/mgofproteinforC.chinensisandG.uralensisrespectively.BasedonthepositivefindingswithTVMplants,thecurrentstudyiscontinuingtheinvestigationofthepotentialofCreemedicinalplantsinpreventingcataractogenesisasaresultoftype2diabetes.Anewmicrotiterplatemethodhasbeendevelopedtoincreaseefficiencyofoutput.

POSTER26COMPARATIVEPHYTOCHEMICALPROFILINGOFTRADITIONALWATEREXTRACTSANDETHANOLEXTRACTSFROMCREEOFEEYOUISTCHEEANTI‐DIABETICBOTANICALTHERAPIESCarolinaOgrodowczyk1,BrendanWalshe­Roussel1,AmmarSaleem1,JoseAntonioGuerrero1,AsimMuhammad1,PierreHaddad2,JohnThorArnason11Ottawa­CarletonInstituteofBiology,UniversityofOttawa,Ottawa,Ontario,CanadaK1N6N52Départementdepharmacologie,Facultédemédecine,UniversitédeMontréal,C.P.

6128­Succursale"Centre­ville",Montréal,Quebec,CanadaH3C3J7Six boreal forest plant species used by the Cree of Eeyou Istchee in the treatment ofdiabetes were analyzed phytochemically. The phytochemical profiles of standardlaboratory ethanol extracts are comparedwithwater extractspreparedusing traditionalmethods. HPLC‐DAD‐ELSD and HPLC‐DAD‐MS were used to identify and quantifystandards and isolated compounds. Themajority of plant species investigated exhibitedverysimilarphytochemicalprofiles,withonlyminordifferencesoccurringinstronglypolarand non‐polar extremities of the chromatograms. However, important phytochemicaldifferences are observed in one species where substantial qualitative and quantitativediscrepanciesbetweenbothtypesofextractarereported.POSTER27ANTI‐DIABETICEFFECTOFELEPHANT‐FOOTYAM(AMORPHOPHALLUS

PAEONIIFOLIOUS(DENNST.)NICOLSON)INSTREPTOZOTOCIN‐INDUCED

DIABETICRATS

HarshavardhanReddyA*1,JamunaJ.Bhaskar2,

ParamahansV.Salimath2,AradhyaSM1

1DeptofFruitandVegetableTechnology;2DeptofBiochemistryandNutritionCentralFoodTechnologicalResearchInstitute(CSIR),Mysore­570020,India,ahvreddy11@rediffmail.com

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Elephant foot yam (Amorphophallus paeoniifolious (Dennst.)Nicolson) is a tropical tubercropusedinmanyayurvedicpreparationsandisrecommendedincaseofpiles,dysentery,asthma,swellingoflungsandasbloodpurifier.Thepresentinvestigationaimstostudytheanti‐diabetic effect of elephant‐foot yam. Dried powder of tuber was subjected tosequential extraction by using different solvents viz., hexane, chloroform, ethyl acetate,acetoneandmethanol.Amongthese,acetoneextractexhibitedhighestantioxidantcapacitybyDPPH(IC50value8µg/ml)/Superoxideradicalscavengingactivity(IC50value62µg/ml),total phenol (866 mg GAE/gm of extract) and flavonoid (586 mg GAE/gm of extract)content.Theeffectoffeedingacetoneextractat0.1and0.25%levelindietwasstudiedinstreptozotocininduceddiabeticrats.Thestudyinvolvedacomparisonbetweenstarchfeddiabetic(SFD),acetoneextractat0.1%feddiabetic(CFD0.1),acetoneextractat0.25%feddiabetic (CFD0.25) and aminoguanidine fed diabetic (AFD) groups. Fasting blood sugar ofCFD0.1 and CFD0.25 groups showed 23% and 37% reduction, respectively, whereas, AFDgroup showed 45% reduction in comparison to SFD group. Glomerular filtration rate ofexperimental rats in CFD0.1 and CFD0.25 groups showed 28% and 41% reduction,respectively, whereas, AFD group showed 54% reduction compared to SFD group.Ameliorationofintestinalmaltaseactivitiesby18%26%and48%wasobservedinCFD0.1,CFD0.25 and AFD groups respectively when compared to SFD group. Intestinal sucraseactivitywashigh inSFDgroupandwasameliorated inCFD0.1,CFD0.25andAFDgroupstoabout 28, 45 and 56% respectively. Similarly, intestinal lactase activitywas high in SFDgroup andwas ameliorated in CFD0.1, CFD0.25 andAFD groups to about 36, 52 and64%,respectively.Incontrastdecreaseinrenalmaltase,sucraseandlactasewereobservedinSFDgroupwhileamelioratedinCFD0.1,CFD0.25andAFDgroups.Thereductioninglycemicconditionsmaybeattributedtoantioxidant‐richphenolsparticularlyflavonoidspresentinacetoneextract.Theresultsclearlyindicatedthatacetoneextractofelephantfootyamisaneffectiveanti‐diabeticagenttostreptozotocininduceddiabeticrats. POSTER28ACTIVITYBASEDFRACTIONATIONOFGRAPESEEDPROANTHOCYANIDINSPERTAININGTOTHEIRPROTECTIVEEFFECTSAGAINSTALZHEIMER’SDISEASEVaishaliSharma,andRichardA.DixonPlantBiologyDivision,SamuelRobertsNobleFoundation,2510SamNobleParkway,Ardmore,Oklahoma73401,radixon@noble.orgGrapeseedextract(GSE)isrichinflavonoids,particularlyproanthocyanidins(PAs)whichareoligomericandpolymericflavan‐3‐olscontainingsubunitsofcatechin,epicatechinandepicatechingallate linkedbyC4‐C8orC4‐C6 interflavanbonds.PAsand theirmonomersarestrongantioxidantsandhavebeenascribedanumberofpotentialactivitiesbeneficialto health, including protection against cancers, cardiovascular disease and Alzheimer’s

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disease. However, little is known about the chemical nature and the concentration ofbioactivepolyphenolstowhichthebodyisexposedafteringestionofGSE.We are investigating the activity based fractionation of grape seed extract into itsrespectivemonomers,oligomersandpolymersinrelationtotheirprotectiveeffectsagainstAlzheimer’s disease. Several techniques ranging from fractionation using liquid‐liquidextraction, chromatography on Sephadex LH‐20 and Toyopearl resin, (Normal/Reversephase) HPLC and preparative HPLC have been optimized to separate and purifymonomeric,oligomericandpolymericPAsfromgrapeseedextract.Eachpurifiedfractionwillbeusedforbioavailabilitystudiesandinvestigationofeffectsoncognitivefunctionintransgenicmice, throughcollaborationwithresearchersatMtSinaiMedicalCenter(NewYork) and Purdue University. The extraction efficiency, together with the separationefficiency towards the resolution of monomers, oligomers and polymers with varioustechniqueswillbefurtherdiscussed.POSTER29BIOCHEMICAL AND MOLECULAR ANALYSIS OF THE FORMATION OF THE STRESS-INDUCED VOLATILE C11-HOMOTERPENE (E)-4,8-DIMETHYLNONA-1,3,7-TRIENE IN THE ARABIDOPSIS ROOT

Reza Sohrabi, Dorothea Tholl; Department of Biological Sciences, 427 Latham Hall, Virginia Tech, Blacksburg, VA 24061 , rsohrabi@vt.edu; tholl@vt.edu

Plants emit a mixture of volatile compounds in response to insect attack. These volatiles defend plants against herbivores through direct repellent activities and also function as indirect defense compounds by attracting natural enemies of herbivores. We investigate the formation of the acyclic C11-homoterpene (E)-4,8-dimethylnona-1,3,7-triene (DMNT) which is a primary constituent of insect-induced volatile blends with important indirect defense activity (Kappers et al., 2005). Interestingly, DMNT is also emitted from roots of Arabidopsis thaliana after treatment with the defense hormone jasmonic acid (JA) suggesting defense functions of DMNT in plant roots. Our goal is to elucidate key enzymatic steps in DMNT biosynthesis and use this knowledge to genetically engineer DMNT biosynthesis in developing alternative plant-pest controls. Previous analysis of other plant species has demonstrated that DMNT is derived from the C15-alcohol (E)-nerolidol. However, the enzymatic steps involved in the conversion of (E)-nerolidol to DMNT are not understood. We are using a combined biochemical and functional genomics approach to elucidate the biosynthetic pathway leading to DMNT formation. Our studies suggest a participation of cytochrome P450 enzymes in nerolidol degradation. Currently, few candidate CytP450 genes have been selected for screening of homozygous gene-knockout lines based on the analysis of publically available microarray datasets. Furthermore, we are investigating the subcellular organization of root-specific DMNT formation. Treatment with inhibitors such as lovastatin and fosmidomycin of the early cytosolic and plastidial terpenoid biosynthetic pathways, respectively, showed that DMNT formation is highly reduced only after lovastatin treatment, suggesting a major contribution of the cytosolic mevalonate pathway to DMNT formation.

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POSTER30BIOCHEMICALANDMOLECULARANALYSISOFTHEFORMATIONOFTHESTRESS‐INDUCEDVOLATILEC11‐HOMOTERPENE(E)‐4,8‐DIMETHYLNONA‐1,3,7‐TRIENEINTHEARABIDOPSISROOT

RezaSohrabi,DorotheaTholl;

Department ofBiological Sciences, 427 LathamHall, VirginiaTech,Blacksburg, VA24061,rsohrabi@vt.edu;tholl@vt.edu

Plants emit a mixture of volatile compounds in response to insect attack. These volatiles defend plants against herbivores through direct repellent activities and also function as indirect defense compounds by attracting natural enemies of herbivores. We investigate the formation of the acyclic C11-homoterpene (E)-4,8-dimethylnona-1,3,7-triene (DMNT) which is a primary constituent of insect-induced volatile blends with important indirect defense activity (Kappers et al., 2005). Interestingly, DMNT is also emitted from roots of Arabidopsis thaliana after treatment with the defense hormone jasmonic acid (JA) suggesting defense functions of DMNT in plant roots. Our goal is to elucidate key enzymatic steps in DMNT biosynthesis and use this knowledge to genetically engineer DMNT biosynthesis in developing alternative plant-pest controls. Previous analysis of other plant species has demonstrated that DMNT is derived from the C15-alcohol (E)-nerolidol. However, the enzymatic steps involved in the conversion of (E)-nerolidol to DMNT are not understood. We are using a combined biochemical and functional genomics approach to elucidate the biosynthetic pathway leading to DMNT formation. Our studies suggest a participation of cytochrome P450 enzymes in nerolidol degradation. Currently, few candidate CytP450 genes have been selected for screening of homozygous gene-knockout lines based on the analysis of publically available microarray datasets. Furthermore, we are investigating the subcellular organization of root-specific DMNT formation. Treatment with inhibitors such as lovastatin and fosmidomycin of the early cytosolic and plastidial terpenoid biosynthetic pathways, respectively, showed that DMNT formation is highly reduced only after lovastatin treatment, suggesting a major contribution of the cytosolic mevalonate pathway to DMNT formation.

POSTER31GENE‐METABOLITERELATIONSHIPSINBLACKCOHOSH(ACTAEARACEMOSAL) MartinJSpiering,1DonaldLNuss,2LoriUrban,3AmyCheng,1ArlinStoltzfus,1andEdwardEisenstein1

1CenterforAdvancedResearchinBiotechnology,2CenterforBiosystemsResearch,and3PlantTransformationFacility,UniversityofMarylandBiotechnologyInstitute,9600GudelskyDr,Rockville,MD20850.Email:spiering@umbi.umd.edu

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Black cohosh (Actaea racemosa L.) is a Ranunculaceae species native to the eastern parts of North America. Preparations from the rhizome and roots of black cohosh have been used medicinally by Native Americans to treat a variety of illnesses, including rheumatism and gynecological complaints. Today black cohosh is used chiefly as dietary supplement and non-estrogenic alternative to hormone-replacement therapy to alleviate menopausal vasomotor symptoms. However, very little is known about the active principles and about their biosynthesis in black cohosh. Initial results are presented from a study aimed at identifying gene content and diversity in A. racemosa and in an effort to pinpoint natural product genes for production of black cohosh signature compounds (triterpene glycosides and phenolic acids) and serotonergic agents (serotonin derivatives). cDNA libraries were constructed from young leaves and from rhizomes and roots of greenhouse-grown black cohosh plants and sequenced to obtain a collection of expressed sequence tags (ESTs). BLASTX analysis of 1500 ESTs from young leaves identified a large number of general metabolic genes (e.g., for RuBisCo, ribosomal proteins, translation elongation factor 1-α), several genes encoding 2,3 oxidosqualene cyclase (cycloartenol synthase) an entry-point enzyme in triterpene (TT) biosynthesis, and BAHD-type acetyltransferases in phenylpropanoid (PP) biosynthesis. Among 400 rhizome/root ESTs, several gave matches to additional PP pathway enzymes (e.g., hydroxycinnamoyl transferase). A black cohosh gene with similarity to genes for tryptophan decarboxylase—a key plant enzyme for the biosynthesis of serotonin—was cloned by degenerate PCR, enabling more detailed studies by in vitro protein expression of the encoded enzyme along with enzymes encoded by the candidate genes in TT and PP biosynthesis identified in the EST screens. POSTER32ANTIOXIDANTACTIVITYOFTHEFRUITPULPOFADANSONIADIGITATA(BAOBABTREE)ChungKi Sung,1Ah­ReumHan,1HeebyungChai,1William J.Keller,2 andA.DouglasKinghorn1, 1College of Pharmacy, The Ohio State University, 500 West 12th Ave.,Columbus,Ohio43210and 2Nature’s SunshineProducts, Inc., 1655NorthMain St.,SpanishFork,UT84660,E­mailsung.116@osu.eduBaobab,AdansoniadigitataL.(Malvaceae),isanunusuallyshapedtreenativetoAfrica.Inrecent years, there has been increasing interest in developing baobab as a nutrient‐richbotanicaldietarysupplementintheUnitedStates.Thefruitorfruitpulp,themajorpartsofboababused,containvariouschemicalcomponentsincludingaminoacids,carbohydrates,flavonoidsandotherphenols,inorganicsubstances,lipids,organicacids,andvitamins.Fromabioactivitypointofview,thereareseveralreportsontheeffectsofcrudeextractsand partially purified fractions of A. digitata, such as those having antioxidant,antibacterial, antifungal, anti‐inflammatory, anti‐sickling, and hepatitis C virus proteaseinhibition activities. The antioxidant activity has been tested by ABTS, DPPH, FRAP,photochemiluminescence, and trolox‐based procedures, using various plant parts ofbaobab. As a result, undefined polyphenol and water‐soluble flavonoid glycosideconstituents have been implicated with antioxidant activity, but individual antioxidantshavenotbeenreportedtodatefromA.digitata.

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For the purpose of investigating the antioxidant components of baobab, our group hasextractedthefruitpulpofbaobabinitiallywithmethanol,andpartitionedthisdriedextractwith a series of solvents (hexane, chloroform, ethyl acetate, butanol and water) ofincreasing polarity. The antioxidant activities of these extractswere determined using ahydroxyl‐radicalscavengingactivitytestmethod,with2′,7′‐dichlorofluoresceinasaprobe.Theethylacetate‐solublepartitionshowedthemostpotentactivity(ED500.2μg/ml)andthis fractionwas further fractionated by silica gel chromatography. From fraction BBE3(ED50 0.2 μg/mL) derived from this extract, several compounds have been isolated, andtestedforantioxidantactivity.Preliminaryresultsofthisinvestigationwillbereported.POSTER33INHIBTIONOFGABA‐TRANSAMINASEBYPIPERAMIDES:QUANTITATIVESTRUCTURE‐ACTIVITYRELATIONSHIP(QSAR)ChieuAnhTa,RosalieAwad,TonyDurst,JohnArnasonCentreforResearchinBiotechnologyandBiopharmaceuticals,DepartmentofBiologyUniversityofOttawa,Ottawa,CanadaThe objective of this study was to investigate the structure‐activity relationship of 25natural piperamides and their synthetic analogs. An in vitro bioassaymeasuring GABA‐transaminaseactivityinratbraintissuewasused. Thepiperamidesweregroupedbasedon thepresence or absence of 3,4‐methylenedioxyphenyl (MDP) andmethoxy functionalgroups.QSARmodelsweregeneratedusingregressionoflog(1/Activity)asthedependentvariable and either log partition coefficient (log P) or molar refractivity (MR) as theindependent variable. Piperine, antiepilepsirine, and peepuloidin were most active withfiftypercentinhibition(IC50)valuesof81μM,59μM,and79μM,respectively. Structure‐activityrelationshipanalysisshowedasignificantpositivecorrelationbetweentheGABA‐Tinhibitory activity and the number ofmethoxy (‐OCH3) groups on the piperamides (p <0.001;r2=0.404).Thebestfitmodelwasaquadraticfunctiondefinedbytheequations:

Log(1/Activity)=aLogP+bLogP2+c (1)Log(1/Activity)=aMR+bMR2+c (2)

To our knowledge, this is the first QSAR report of piperamides and their effects on theGABAergicsystem,bothofwhichcouldleadtointerestingpharmaceuticalleads.POSTER34ISSABP2ARECEPTORFORACIBENZOLAR‐S‐METHYL(ASM):ACHEMICALINDUCEROFSYSTEMICACQUIREDRESISTANCE(SAR)INPLANTS?DiwakerTripathi1,Yu­LiJiang2,DhirendraKumar1

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DepartmentofBiologicalSciences1,DepartmentofChemistry2,EastTennesseeStateUniversity,JohnsonCity,TN­37614 Plants defend themselves by developing an enhanced resistance to a broad spectrum ofpathogensintheareaofprimaryinfectionaswellasinthedistal,uninoculatedparts.ThisinducedstateofresistanceiscalledSystemicAcquiredResistance(SAR).Salicylicacid(SA),aplanthormone,isessentialforthefulldevelopmentofSAR.TreatmentofplantswithSAmakes them resistant to pathogens. SA‐binding protein 2 (SABP2) is amethyl salicylateesterasethatcatalyzestheconversionoflipidmobilemethylsalicylate(MeSA),synthesizedin plants resisting pathogens, into SA. Plantswhich do not express SABP2 are unable toconvert MeSA into SA and fail to develop an effective SAR response. Various functionalanalogs of SA have been developed and used to activate plants own natural defensesagainst microbial pathogens. ASM (Acibenzolar‐S‐Methyl) is one such functional analogwhich inducesSAR invarious familiesofmonocotanddicotplants.ASM is commerciallyavailablebythenameofBIONandActigard.HowASMfunctionsinplantsismostlyunclear?WehypothesizethatASMactivitydependson its conversion into anacid form, a reaction catalyzedbySABP2.WeperformedHPLCstudiestoshowthatSABP2catalyzestheconversionofASMesterintoitsacidform.Totestourhypotheses inbiologicalsystem,weareusingtransgenic tobacco(NicotianatabacumcvXanthinc)SABP2‐silencedplants inwhichSABP2geneexpression is silencedbyRNAinterference and control plants, containing empty silenced vector. Using this system,wewilldetermineifSABP2catalyzedconversionofASMesterintoacidformisalsorequiredfor (1) SAR induction and (2) expression of SAR genes. Results showing the actionmechanismofASMwillbepresented.POSTER35EFFECTSOFAUXINSONGENEEXPRESSIONINVOVLEDINTHEBIOSYNTHESISOFANTHOCYANINSINPAP1‐TRANSGENICCALLUSPatrickT.Walker1LydiaMeador1,2,Ming­ZhuShi1,andDe­YuXie1*1:DepartmentofPlantBiology,NorthCarolinaStateUniversity,Raleigh,NC27695;ptwalker@ncsu.edu,dxie@ncsu.edu2:REDstudentfromDepartmentofBotany,OklahomaStateUniversity,Stillwater,OK74077PAP1 is a positive regulator of the biosynthetic pathway of anthocyanins. We haveestablished red and white callus cultures of the PAP1‐transgene. Our previous workreported thatbyvarying theconcentrationsof2,4‐DandNAA in themedium, the levelsandprofilesofanthocyaninsincalliareaffected.Inthisstudy,wereporttheeffectsofIAA,NAA,and2,4‐Dongeneexpressioncontrollingthebiosynthesisofanthocyanin inPAP1‐transgenic red calli. Transgenicwhite callus andwild‐type calluswereusedas controls.Five concentrations of 0uM, 0.1uM, 1.0uM, 5.0uM, and 20.0uM for the tree auxins were

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compared.Preliminarydatashowedsignificantdifferentimpactsoftheseconcentrationsongeneexpression,biomass,andanthocyaninproduction.Thesedataprovideinsightintothe effects of auxins on the PAP1 transcription factor in themetabolic pathway. In thispresentation,wewilldiscussthemechanism(s)oftheeffectsofauxinsongeneexpressionand the biosynthesis of anthocyanins. This research is funded by USDA‐NRI (proposalnumber2006‐1334).POSTER36ANALAYISOFBIOACTIVEPOLYPHENOLICSINMUSCADINEGRAPESGROWINGINNORTHCAROLINADe­YuXie1*,PatrickWalker1,ConnieFisk2,SaraSpayd2,andKeithHarris31:TheDepartmentofPlantBiology;2:TheDepartmentofHorticulturalSciences;3:TheDepartmentofFood,Bioprocessing,andNutritionSciences;NorthCarolinaStateUniversity,NC27695;*:correspondent(dxie@ncsu.edu)Muscadinegrapes(Vitis rotundifolia)areanativewinegrapespecies to thesoutheasternUnitedStates.MuscadinegrapesaremainresourcesforwineproductioninNorthCarolina.Muscadine grapes are approximately 1 to 1.5 inches in diameter, have thick skin, canproduce up to five seeds per berry, and are known for their unique flavor and aroma.Muscadinegrapesproducemanypotentantioxidativecompounds(mostly found inseedsandskin)including:vitamins;phenolicacids;carotenoids;andflavonoids.Identifiedactivecompounds include anthocyanins, resveratrol, quercetin, gallic acid, ellagic acids etc.dependentuponcultivars.Inthisstudy,weinterestedintheproductionandstructuresofactive polyphenolics e.g. condensed tannins and hydrolyzable tannins in two cultivars,CarlosandNesbitt.Sampleswerecollectedfromdifferentdevelopmentalstagesofflowersand berries. We are using LC‐MS analysis to characterize the profiles of bioactivepolyphenolics.Inaddition,wecompareprofilesofpolyphenolicsofthetwocultivarswithtwoVitisviniferacultivars,CabernetFrancandChardonnay.Thedatageneratedfromthisstudywillformaplatformforgeneticbreedingormetabolicengineeringtoincreaselevelsof bioactive polyphenolics, e.g. resveratrol and oligomeric condensed tannins,which canimprovethequalityofmuscadinegrapeandwineproductsinNorthCarolina.ThisresearchisfundedbyNorthCarolinaWineandGrapeCouncil..POSTER37DEVELOPMENT‐DEPENDENTFORMATIONANDMETABOLISMOFANTHOCYANINSINACERPALMATUMSPECIESHai­NianZeng,Li­LiZhou,Ming­ZhuShi,De­YuXie*DepartmentofPlantBiology,NorthCarolinaStateUniversity,Raleigh,NC,27695Anthocyaninsareoneoftherichestpigmentsinplantkingdom.Theirbiologicalfunctionscanbesummarizedasfollows:antioxidativeactivityagainstoxidation‐induceddamageby

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differentenvironmental factors; attractiveeffectsonpollinators,pathogen resistanceetc.Overthepastmanyyears,numerouseffortshavebeenmadetodeterminethebiosyntheticpathwayofanthocyaninsandalsotoidentifyseveralregulatoryproteinsmainlyinflowersand fruits of model plants and crop plants. However, many questions concerning themetabolism of anthocyanins in foliage of flora remains unsolved. One example is “Howcandoes a developmental process impact on accumulation patterns of anthocyanins”. Inthis study,we choose several cultivars from one of themost popular ornamental plantsAcerpalmatumtounderstandthemechanismofdevelopmentalchangesofpigmentationinleaf. We propose that the metabolism of anthocyanins play an essential role in suchchanges.We used an integrated approach of phytochemistry, andmetabolic profiling todetermine thebiosynthesisandmetabolismofanthocyaninsand their impactson foliagecolor. Anthocyanins were extracted from different developmental stages of leaves for 6Acer palmatum cultivars, which have obviously different accumulation patterns ofanthocyanins in spring. Spectrometric analysis showed three major development‐dependenttrendsofanthocyaninlevelsinleavesgrowinginspring:1)gradualdecrease;2)constitutive accumulation; 3) lacking anthocyanins. HPLC‐MS analysis showed that thealterationofanthocyaninlevelsresultedfromeitherqualitativeorquantitativechangeorfrom both changes, while qualitative changes probably stem from structure changes ormodification or degradation. Proanthocyanidin analysis has beencarried out as well todetermine their relationshipwithboth anthocyaninproductionand foliar coloration.Wehave found that even for green leaveswithno/trace amountofdetectable anthocyanins,the biosynthetic pathway of anthocyanidin/proanthocyanidin is still activated. And ourresults indicate that metabolic channeling directing the anthocyanin pathway to theproanthocyanidinbiosynthesisplaysaveryimportantroleinpigmentationpatternchangealongdevelopmentalprocesses.(ThisprojectissupportedbyPolkCounty,NorthCarolina.)POSTER38PREDICTABLETRANSITIVERNAINTERFERENCEINDUCEDBYMRNAHAIRPINSINC.ELEGANS AlyssaPawlowicz,EvelynBrown,andTimothyM.DwyerStevensonUniversity,DepartmentofChemistry,1525GreenspringValleyRd.Stevenson,MD21153 ThespecificsilencingofgenesthroughRNAinterference(RNAi)bydouble‐strandedRNAisnowbeingstudiednotonlyasameanstounderstandcellularprocesses,butalsoasameans of treating patients in the pharmaceutical industry and controlling pests in theagriculturalsector.Whiletheproteinmachinerynecessarytocausethisphenomenonhasbeenwell characterized, it is not always possible to predict the silencing of one specificgene. At times,whenatargetgene issilenced, itcan leadtothesilencingofotherRNAs.Wearetryingtounderstandthisprocess,termedtransitiveRNAi,andtryingtodetermineif there are scientific rules governing it. Using a computer algorithmwe developed,wehavebeenabletocreatemapsofgenenetworksinwhichsuchinteractionsmightoccurdueto sequence homology. One of these networks is now being examined in the nematode

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Caenorhabditiselegans.Twoplasmidconstructshavecreated.Thefirsttargetsfourgeneswithinthenetworkforsilencingthroughacommonhairpinsequencefoundineachofthegenes.Thesecondconstructtargetsjustonethesegenesthroughsequencehomologyinanareaoutsideofthehairpin. Thetwoconstructs,aswellasacontrolconstruct,havebeenintroducedintobacteria, inducedtoexpressthedsRNAandfedtotheC.elgansto induceRNAi.Nematodesfedthefirstconstructarephenotypicallyverysimilartonematodesfedthe second construct. Both exhibit impaired movement as compared C. elegans fed thecontrol construct or no construct. This suggests that the pattern of silenced genes insimilar between the two groups and that the silencing of the one genewith the secondconstructmighthaveledtothesilencingoftheothergenesinmannerthatwecouldthenbegin to predict. We are currently examining RNA levels for all of the genes with theC.elegans network to determine if there is a discernible pattern with rules that we candetermine.POSTER39ONTHEORIGINOFMETABOLITEMODULARITYANDARRAYSINPLANTSPECIALIZEDMETABOLISMDavidR.GangDepartmentofPlantSciencesandBIO5Institute,TheUniversityofArizona,Tucson,AZ,85721gang@ag.arizona.eduManyplants,suchasginger,turmeric,sweetbasilandtomatoandrelatedspecies,producearraysof related secondaryor specializedmetabolites. Sweetbasil produces a varietyofmethylated flavones, tomato and related Solanum species produce a multitude of acylsugars. Turmeric produces dozens of diarylheptanoids related to the medicinally activecurcumin. Ginger produces not only a large array of diarylheptanoids, but biogeneticallyrelatedgingerols andother related compoundsaswell.All of these species alsoproducediverse collections of terpenoids, aswell as compounds from othermetabolic pathways.Howtheselargearraysofcompoundsareproducedandhowtheirproductioniscontrolledremainlargelyunansweredquestions,althoughrecentprogressisbeginningtoshedlightonthesequestions.Onefeatureofspecializedmetabolismthathasemergedrecentlyisthatitappearstobeorganizedattimesandincertainspecies(ifnotall)inamodularfashion.That is, groups of metabolites that are biosynthetically linked can accumulate in aconcerted manner, in biosynthetic modules. The existence of these modules suggestsconcertedcontrolofproductionofsuchcompounds.Inthecaseofthespecieslistedabove,the large collectionof relatedmetabolites that accumulatedonot all belong to the samemodule. Indeed, subgroups of compounds from these species accumulate in separatemodules. This suggests that multiple enzymes must be involved in production of thesecompounds. Identification of such enzymes and characterization of their individualfunctions are important steps in understandinghow such complex arrays ofmetabolitescanevolveinspecificplantlineages.

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POSTER40PROMOTERANALYSISOFPOTATO(Solanumtuberosum)FATTYACIDw‐HYDROXYLASE(FAωH1)DanielP.N.Lee,AbdullahB.MakhzoumandMarkA.BernardsDepartmentofBiologyandtheBiotronTheUniversityofWesternOntarioLondon,Ontario,Canada,N6A5B7dlee64@uwo.caSuberinisaplantbiopolymerthatprovidesaprotectivebarrierbetweenitsundergroundorgans and the external environment, preventingwater and nutrient loss and providingprotection against pathogen attack. Suberin is composed of two different polymericdomains; the poly‐aliphatic domain (SPAD) and the poly‐phenolic domain (SPPD).Suberization is induced by wounding, and requires the coordinate biosynthesis of newaliphaticandphenoliccomponents.Withrespecttothealiphatics,newlysynthesizedfattyacidscanundergooneoftwomainmetabolicfates:(1)theelongationofstearicacid(18:0)toverylongchain,saturatedfattyacids(VLCFA–C20toC28),withandwithoutthefurthermodification to n‐alkanes and 1‐alkanols and (2) the oxidation of oleic acid (18:1) toproduceω‐hydroxy‐fattyacids(18‐OH‐18:1)andα,ω‐dioicacids.Theconversionofoleicacidtoanω‐hydroxy‐fattyacidisametabolicstepuniquetosuberin(andcutin)formation,and thereforecanbeusedasamarker to studySPAD formation in tissues that suberize.ThisreactioniscatalyzedbymembersoftwodifferentcytochromeP450families;CYP86AandCYP94A,andarereferredtoasfattyacidω­hydroxylases(FAωH).InS.tuberosumonememberoftheCYP86Afamily(FAωH1)hasbeenshowntohavehighlevelsofexpressioninsuberizingroottissue.Inthepresentstudy,wehavesequencedaportionoftheFAwH1promoterregion.Usinginsilicoanalyses,putativeregulatorymotifshavebeenidentifiedintheFAωH1promoterthatmaycontrolitsexpression.

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POSTER41IDENTIFICATIONANDCHARACTERIZATIONOFANANTHOCYANIDIN/FLAVONOL3‐O‐GLUCOSYLTRANFERASEGENEISOLATEDFROMTHESEEDCOATOFBLACKSOYBEAN*NikKovinich1,2,AmmarSaleem3,JohnT.Arnason3andBrianMiki11AgricultureandAgri­FoodCanada,Ottawa,Canada.2CarletonUniversity,Ottawa,Canada.3UniversityofOttawa,Ottawa,Canada.*kovinichn@agr.gc.caTheseedcoatsofblacksoybean(Glycinemax(L.)Merr.)areknowntoaccumulateallanthocyaninsrequiredforthered(cyanidin3‐O‐glucoside),blue(delphinidin3‐O‐glucoside),purple(petunidin3‐O‐glucoside),andorange(pelargonidin3‐O‐glucoside)colorationofplanttissues1.Blacksoybeananthocyaninshaverecentlydemonstratedarangeofmedicinalactivitiessuchasanti‐inflammatory2,anti‐obesity3,anti‐apoptotic4andanti‐proliferative2activities,andtoenhanceimmuneresponse5.Almostallstructuralgenesinvolvedinanthocyaninbiosynthesisinsoybeanhavebeencharacterized6‐12,butthegenerequiredtocatalyzethefinalstep,namelyanthocyanidin3‐O‐glucosyltransferase,remainedtobeidentified.Inthisstudywehaveisolatedananthocyanidin/flavonol3‐O‐glucosyltransferasefromthesoybeanseedcoat.ComplementationofanthocyaninbiosynthesisinanArabidopsisthalianamutantwasusedtoverifytheinvivofunctionofthesoybeangene.Therecombinantenzymeusedonlyanthocyanidinsandflavonolsassubstratesinvitro.Cyanidin,butnotkaempferol,inhibitedrecombinantenzymeactivityatlevelsabove12μM.Thispropertymayfunctiontolimitcytoplasmicanthocyaninlevelsorfluxintotheanthocyaninpathwayinvivo.UDP‐galactosecouldalsobeusedasasugardonoratlowvelocityrelativetoUDP‐glucose.Thus,highlevelsofglucosylatedandlowlevelsofgalactosylatedanthocyaninsintheseedcoatofblacksoybeanmaybeattributedtotheactivitiesofasingleenzyme.References:1Leeetal2009.FoodChem112:226–231.2Kimetal2008.JAgricFoodChem56:8427–8433.3Kwonetal2007.JMedFood10:552–556.4Tsoyietal2008.JAgricFoodChem56:10600–10605.5Chanetal2009.IntJNanomed4:27–35.6ToddandVodkin1996.PlantCell8:687‐699.7ZabalaandVodkin2005.PlantCell17:2619–2632.8ZabalaandVodkin2003.Genetics163:295–309.9ZabalaandVodkin2007.PlantGenome47:S113–S124.10Iwashinaetal2007.JHeredity98:250–257.11Takahashietal2007.PlantMolBiol63:125–135.12Fasoulaetal1995.CropSci35:1028‐1031.

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13Tohgeetal2005.PlantJ42,218–235.

ContactInformation

Ahmed,Fida­fahmed074@uottawa.ca,UniversityofOttawa,30MarieCurie,Gendron283,Ottawa,ONT.K1N6N5613‐562‐5800Alejos­Gonzales,Fatima­fmalejos@ncsu.edu,NorthCarolinaStateUniversity,716CarlDrive,ChapelHill,NC27516919‐370‐7957Alkharouf,Nadim­nalkharouf@towson.edu,TowsonUniversity,8000YorkRoad,Towson,MD21252410‐704‐3149Anderson,Stephanie­sander74@uwo.ca,UniversityofWesternOntario,404NcampusBuilding,London,ONN6A5B7519‐661‐2111AnhTa,Chieu­kta074@uottawa.ca,UniversityofOttawa,30MarieCurie,Gendron283,Ottawa,ONT.K1N6N5613‐562‐5800Arnason,John­John.Arnason@uottawa.ca,UniversityofOttawa,30MarieCurie,Gendron283,Ottawa,ONT.K1N6N5613‐562‐5800Baker,Bill­bjbajer@cas.usf.edu,UniversityofSouthFlorida,4202EFowlerAveCHE205A,Tampa,FL33620813‐974‐1967Baker,Cedric­baker691@umn.edu,MercerUniversity,POBox13593,Atlanta,GA97331404‐536‐5661Berhow,Mark­mark.berhow@ars.usda.gov,USDA‐ARS‐NCAUR,1815NuniversitySt.,Peoria,IL61604,309‐681‐6347Bernards,Mark­bernards@uwo.ca,EnvironmentalStressBiologyGroup,UniversityofWesternOntario,London,ONN6A5B7519‐661‐2111Bharath,Sarah­Sarahbharath@yahoo.com,UniversityofWestIndies‐CocoaResearchUnit,410‐218‐0684Bozzo,Gale­gbozzo@upguelph.ca,UniversityOfGuelph,50StoneRoadE,Guelph,ONT,N1G2WIBrown,Evelyn­ebrown1502@stevenson.edu,StevensonUniversity,1525GreenspringValleyRoad,Stevenson,MD21153443‐334‐2423Dayan,Franck­fdayan@olemiss.edu,USDA‐ARS‐NPURU,POBox8048,University,MS38677662‐915‐1039dePaulaBarbosa,Antony­antonypb@hotmail.com,UniversidadFederaldeRiodeJaneiro,RuaRonalddeCarvalho250Apt802,Copacabana,RiodeJaneiro,MD2202102021‐343‐59879

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Dhaubhadel,Sangeeta­dhaubhadels@agr.gc.ca,SouthernCropProtection&FoodResearchCentre‐1391SanfordStreet,London,ONN5V4T3519‐457‐3997Dou,Jinhui­Jinhui.Dou@fda.hhs.gov,BotanicalReviewTeam‐FDA,10903NewHampshireAve,SilverSpring,MD20993301‐796‐1062Duke,James­JimDuke@comcast.net,theHerbalVillage‐8210MurphyRoad,Fulton,MD20759301‐498‐1175Dwyer,Timothy­tdwyer@stevenson.edu,StevensonUniversity,1525GreenspringValleyRoad,Stevenson,MD21153443‐334‐2423Esrey,Elizabeth­Elizabeth.Esrey@cgr.dupont.com,DuPontAgricultureandNutrition,RT141andHenryClayRoad,Wilmington,DE19880302‐695‐6622Fahey,Jed­jfahey@jhmi.edu,BloombergSchoolofHealth‐centerforHumanNutrition,725NWolfeStreet,Chicago,IL,60612410‐614‐2607Gang,David­gang@ag.arizona.edu,UniversityofArizona,1657EHelenStreet,Tucson,AZ85721520‐621‐7154Guzman,Ivette­herbalivette@gmail.com,NewMexicoStateUniversity,MSC3QPOBox30003,LasCruces,NM88003575‐646‐5169Haggitt,Meg­meg.haggitt@ow.ca,UniversityofWesternOntario,404NcampusBuilding,London,ONN6A5B7519‐661‐2111Harnly,James­James.harnly@ars.usda.gov,USDA,10300BaltimoreAveRM102A,Beltsville,MD20705301‐504‐8596Harrison,Kelly­kharris6@ashland.edu,AshlandUniversity,3791WilliamsCt,Avon,OH44011440‐937‐5850Hession,Aideen­aideen.hession@cgr.dupont.com,DuPontAgricultureandNutrition,RT141andHenryClayRoad,Wilmington,DE19880302‐695‐4387Ismail,Ahmed­nalkharouf@towson.edu,TowsonUniversity,8000YorkRoad,Towson,MD21252410‐704‐3149Khan,Ikhlas­ikhan@olemiss.edu,TheCochranResearchCenter,POBox1848,University,MS38677662‐915‐7821Kinghorn,Douglas­Kinghorn4@osu.edu,theOhioStateUniversity,Columbus,OH43210614‐247‐8094LaMoreaux,Jessica­jlamore1@ashland.edu,AshlandUniversity,1025WabashAve,Grafton,OH44044440‐315‐9182

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Lee,Daniel­dlee64@uwo.ca,UniversityofWesternOntario,404N.CampusBuildingLondon,ONN6A5B7519‐661‐2111Lewis,Norman­lewisn@wsu.edu,WashingtonStateUniversity,POBox646340,Pullman,WA99164509‐335‐2682Li,Xiang­lixi@agr.gc.ca,AgricultureandAgri‐foodCanada,107SciencePlace,Saskatoon,SKS7H3E6306‐956‐7632Lin,Zhanfan­linzhanfan@gmail.com,EastTennesseeStateUniversity,Biology,Box70703,JohnsonCity,TN37614423‐439‐5838Luu,Alice­luua2@mcmaster.ca,UniversityofOttawa,30MarieCurie,Gendron283,Ottawa,ONT.K1N6N5613‐562‐5800Mallampalli,Venkata­mallampali_siddhartha@yahoo.com,EastTennesseeStateUniversity,Biology,Box70703,JohnsonCity,TN37614423‐439‐5838Martin,Timothy­Tmarti7@students.towson.edu,TowsonUniversity,8000YorkRoad,Towson,MD21252240‐676‐8635Matz,Tricia­tmatx@ashland.edu,AshlandUniversity,401CollegeAveBox1915,Ashland,OH44805260‐438‐6708Maxwell,Carl­carl.a.maxwell@cgr.dupont.com,DuPontAgricultureandNutrition,RT141andHenryClayRoad,Wilmington,DE19880302‐695‐3195Mazzola,Eugene­emazzola@umd.edu,UniversityofMaryland/FDA,UniversityofMarylandCollegePark,MD20742301‐405‐1826McCormick,Susan­susan.mccormick@ars.usda.gov,USDA‐ARS‐NCAUR,1815UniversityRoad,Peonia,IL616043090‐681‐6381McIntosh,Cecilia­mcintosc@etsu.edu,EastTennesseeStateUniversity,Biology,Box70703,JohnsonCity,TN37614423‐439‐5838McInyre,Kristina­kmcin009@uottawa.ca,UniversityofOttawa,30MarieCurie,Gendron283,Ottawa,ONT.K1N6N5613‐562‐5800Meador,Lydia­lydiarm@okstate.edu,OklahomaStateUniversity,3108W.LansingCircle,BrokenArrow,OK74012918‐671‐8420Ming­ZhuShi­mshi@ncsu.edu,NorthCarolinaStateUniversity,Raleigh,NC27606919‐699‐7811Mohney,Brian­bmohney@ashland.edu,AshlandUniversity,401CollegeAve,Ashland,OH44805419‐289‐5962Moinuddin,SyedG.A­syed@wsu.edu,WashingtonStateUniversity,POBox646340,Pullman,WA,99164509‐335‐3428

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Nguyen,San­san.nguyen2005@gmail.com,UniversityofOttawa,30MarieCurie,Gendron283,Ottawa,ONT.K1N6N5613‐562‐5800Norman,Farnsworth­norman@uic.edu,UniversityofIllinoisatChicago,833SWoodStreet,Chicago,IL60612312‐996‐7253Nozzolilo,Constance­Cnozzoli@rogers.com,UniversityofOttawa,306CrestviewRd,Ottawa,ONT.K1H5G6613‐733‐0755Ogrodowczyk,Carolina­carolina07@gmail.com,UniversityofOttawa,30MarieCurie,Gendron283,Ottawa,ONT.K1N6N5613‐562‐5800Owens,Daniel­owensdk@gmail.com,EastTennesseeStateUniversity,Biology,Box70703,JohnsonCity,TN37614423‐439‐5838Owosela,Folashade­Folashade.owosela@cgr.dupont.com,DuPontAgricultureandNutrition,RT141andHenryClayRoad,Wilmington,DE19880302‐645‐6957Pawlowicz,Alyssa­apawlowic@stevenson.edu,StevensonUniversity,1525GreenspringValleyRoad,Stevenson,MD21153443‐334‐2423Pedras,Soledade­s.pedras@usask.ca,UniversityofSaskatchewan,110SciencePlace,Saskatoon,SKS7H3E6306‐966‐4772PhanikanthTurlapati­phani60@mail.wsu.edu,WashingtonStateUniversity,POBox646340,Pullman,WA,99164509‐335‐4643ReinhardJetter­jetter@interchange.ubc.ca,UniversityofBritishColumbia,6270UniversityBlvd,Vancouver,BCV6T1Z4301‐822‐2477Reynolds,William­wreynold@chem@utoronto.ca,UniversityofToronto,80StGeorgeSt,Toronto,ONM5S3H6416‐979‐3563Roberts,Roland­rroberts@towson.edu,TowsonUniversity,8000YorkRoad,Towson,MD21252410‐704‐3034Rodriguez,Eloy­er30@cornell.edu,CornellUniversity,412MannLibrary,Ithaca,NY14853607‐254‐2956Romeo,John­romeo@cas.usf.edu,UniversityofSouthFlorida,4202EFowlerAveCHE205A,Tampa,FL33620813‐974‐2336Ross,Samir­Sross@olemiss.edu,UniversityofMississippi‐SchoolOfPharmacy,University,MS38677662‐915‐1031Saunders,James­Jsaunders@towson.edu,TowsonUniversity‐8000YorkRoad,360SmithHallTowson,MD21252410‐704‐3491

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Sharma,Vaishali­vasharma@noble.org,TheSamuelRobertsNobleFoundation,2510SamNobleParkway,Ardmore,OK73401580‐224‐6112Shields,Vonnie­Vshields@towson.edu,TowsonUniversity‐8000YorkRoadTowson,MD21252410‐704‐3130Sohrabi,Reza­rsohrabi@vt.edu,VirginiaTech,427Lathamhall,Blacksburg,VA24060540‐231‐0914Spiering,Martin­spiering@umbi.umd.edu,CARBUniversityofMarylandBiotechnologyInstitute,9600GudelksyDrive,Rockville,MD20850240‐312‐6262Stevens,Fred­fred.stevens@oregonstate.edu,OregonStateUniversity,1601SWJefferson,Covallis,OR97331541‐737‐9534Sung,ChungKi­sung.116@osu.edu,OhioStateUniversity,Columbus,OH43210614‐882‐5615Tholl,Dorthea­tholl@vt.edu,VirginiaTech,408LathamHall,Blacksburg,VA24060540‐231‐54567Tims,Michael­michael.tims@nist.gov,NationalInstituteofStandardsandTechnology,100BereauDrive,Gaithersburg,MD20899,301‐975‐4026Tokuhisa,Jim­Tokuhisa@vt.net,VirginiaTech,408LathamHall,Blacksburg,VA24060540‐231‐5653Tripathi,Diwaker­tripathi@goldmail.etsu.edu,EastTennesseeStateUniversity,Biology,Box70703,JohnsonCity,TN37614423‐439‐5838Umezawa,Toshiaki­tumezawa@rish.kyoto­u.ac,KyotoUniversity,Gokasho,Uii,CH611‐001181774383625Walker,Patrick­ptwalker@ncsu.edu,NorthCarolinaStateUniversity,Raleigh,NC27607910‐297‐2670Weidenhamer,Jeffrey­jweiden@ashland.edu,AshlandUniversity,Ashland,OH,44805419‐289‐5281Xie,Deyu­dxie@ncsu.edu,NorthCarolinaStateUniversity,4219GardnerHall,Raleigh,NC27695919‐515‐2129Zeng,Hainian­hzeng2@ncsu.edu,NorthCarolinaStateUniversity,Raleigh,NC27606540‐231‐0914Zhou,Lili­lzhou3@ncsu.edu,NorthCarolinaStateUniversity,Raleigh,NC27607919‐297‐2670

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PSNA 2009