US 2002O174452A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2002/0174452 A1 Lewis et al. (43) Pub. Date: Nov. 21, 2002

(54) MONOCOT SEEDS WITH INCREASED Publication Classification LGNAN CONTENT (51) Int. Cl...... A01H 5/00 (76) Inventors: Norman G. Lewis, Pullman, WA (US); (52) U.S. Cl...... 800/284 Laurence B. Davin, Pullman, WA (US); Ning Huang, Davis, CA (US) Correspondence Address: (57) ABSTRACT CHRISTENSEN, O'CONNOR, JOHNSON, KINDNESS, PLLC 1420 FIFTHAVENUE The present invention provides methods for modifying lig SUTE 2800 nan content in plants by transforming plants with vectors SEATTLE, WA 98101-2347 (US) containing a DNA sequence encoding one or more proteins (21) Appl. No.: 09/944,160 integral to the pathway leading to G- formation. Such coding Sequences are expressed under the (22) Filed: Aug. 30, 2001 control of a Seed tissue specific or Seed developmental Stage Related U.S. Application Data Specific promoter. Expression of the DNA sequence results in a modification of the absolute and/or relative level of an (60) Provisional application No. 60/230,632, filed on Sep. intermediate metabolite leading to the production of G-lig 7, 2000. nans (e.g., diglucoside or matairesinol).

Patent Application Publication Nov. 21, 2002. Sheet 2 of 23 US 2002/0174452 A1

Fig. 2 Patent Application Publication Nov. 21, 2002. Sheet 3 of 23 US 2002/0174452 A1

PIO

Fig. Patent Application Publication Nov. 21, 2002. Sheet 4 of 23 US 2002/0174452 A1

Fig. 4B Patent Application Publication Nov. 21, 2002. Sheet 5 of 23 US 2002/0174452 Al Basic Cassette For G In l Developing Riceene ExpressiPression

Gt POIOter

pAPi188 (p.G11) Not 3754 bp G1 Signal Xhol - Xibal

Kanamycin 1 R NCS terminator

Fig. 5 Patent Application Publication Nov. 21, 2002. Sheet 6 of 23 US 2002/0174452 A1

Plasmid.for Lignan Used BiosynthesisTo Express Genes in Developing Rice Seeds

GE ProTotef

Not pAP1244 (pter 1-DIRG) Kanamyer PAP1245 (gGTi-LACcy Karanycin 422 bp Dirigent protein

Xol. Xa NOS Terminator NOS terminator x

Fig. 6A Fig. . 6B

n G. Peterot AsN Promoter No pAP124s (pGT-REDs) Nit pAP249 (pgti-DEHY Kanarycin 4605

eductase Kanamycin ba Dehydrogenase

s Xho al OS terriratar or Xbal NCS entiator Fig. 6C Fig. 6d Patent Application Publication Nov. 21, 2002. Sheet 7 of 23 US 2002/0174452 A1

Plasmid pa245 DNA sequence to express laccase Range: 1 to 5355

Sc s r t

o s t st st AArt ArtsAGTransgarcariccercase The Careercesters Terrs grearrerascissaar Gr "jois d s es AcAcAGAAAACTTGAACCARGCTArcGAAGGCAGAT targeteatatasarrettaGract a 4. s d 3. as

al is 4.is dis 4. ds g

W s sa es s sis d s s o

s sza s s 55 s s es

" s 77 g s

AA A f at s s s s s terrarier AGA'arragroggAAAAAAircraft ITAACR.AGRaccARRGCffragrakaAAIGArg ;h'ot, Sof seating resid: s gs sa 9so s s se sess g arristcarrorTrcrgical ArgeisaccGerrero carrcrg FAAzTATGARBTT retrorracArtAAAGGTTTTTTCrgcraga AGGA is ...... s. case S. t s SE s so s s GreenTrec CircrgerCATACCSCCATAGITGGTGARATACTATTeRAAGGCTGcGAAAAAGKTATTG v. E. v . P L a A. L. T R. F. v ...... R. L. S is K a F w As t s list o s TarikrisacakrraccaccAACAAzattaccAertgaggagAAAAAArcrgArgrict AAAACAAccGAAAAAGAActrica v v is G F r ; it y & T S G T p N P S E E W T Hs Aclass s izzt 23 D Re- est y w E' R Y P W S a Y ...... g . . A s is AegAS: ru-ra-rearra-eparterror arreror-wrat s s ess s less la s l arrarietirectarrestrict.cccaricciaregretreaccretaceoTCAA rest Arror CrazAras it is t it t w is w is . A i a W I R P K As . 4. 14 s S. es Earcriteraeace arcscsaacscarriaggarrigawaasraraticaggaaraargarrierra, r a a . . . g . . if x is a R A Y E. P. L. a AS s s s s s s s s s ises gagsarcargotterraraargarcarrcroATASCAAGAAcial sigra is a s . . v c F C S R a y Es

ppx As s 6a s ls se 7 s 59 17 re-regaragrarrrrrri. ATerrazresertarcraucaccaraterritcCATEGAAACAATGGAPCGTTorregATCAGAir a Y ...... A y T I F F is I A N H is v F W W G is De ------i.e. 4. s so s areer TAcaccacccer Aasrar TRT Accorrorceatagaki Racca railrt first AAGAAPrice Areacts a y T K P L x B D Y T s p G or I. D F i. e. g. A N G T P as Ji Y Yr

Fig. 7A Patent Application Publication Nov. 21, 2002 Sheet 8 of 23 US 2002/0174452 A1

ACRs Y CTGGTGCCCGIGCTTATc."orgetAGAAAcrTCGAARCACGACACAAGAGCTATArccestracArisatirArasercarcererscolic 2 t s:0 84 iss 86 87 iss s M. A. A R A Y v A a N F O N T if T r A I R Y x G N Y r A F is is P. ACCs s TrcCTCCAAATCTTCCAGGCSrCAATGATACAAAEGTCAGITAAfrcACATACAGAAAAAGTrrigatauaaATTxTccArctice9. 192. sa said sess less lso sa so 2 S F E. P G F N n 'E' as a s W N F Y R is is N. K. S. Y w v.

r ACASE 2. 23' 203 24 25 28 2O7 08C s s CTxAAAATGriargRIAAATTRTTTACRTTTCCATCAAirraacCecrgroTAATAATckTaTclTegcc TTTCAATGAGCGCreeRC P. K. N. W T K . I. F. T. F. S I N 1. T R C P N N is a G. F. F. N. E. R. F. R A ACCASE d Sl 220 2. ' 4. s s t s els AAggrega ATAACATIATIrrigg CTCCTCCCATIcicago TATEACCAAccAAAAATTCTAcGIAATAACTTTCCTAGAT s w N N I T R v P T A. I a Y Y o R. i. R x v Y S N N F P is Rs. r Acces s -Notsite o 23 224 C. 22 5 227 230 29 23 ccaccATTACITATATTACAAATCCTGAACTAGGAccTCAAGAAGAAGAAAAGTCTAATAATTACAG P P F. T. F. N. Y. T s d I e R. D. L. W. R. P. g. N. G T E W K V k y N. s r. LAASE 2. 230 30 23i 3S 235 3. 38 2 W E I w F : G L. A. g ...... R G s F Y V V G W G L G ku. Actase s

2 242 23. 4. 2s 248 247 2d 8 49 2. Criticiarcgcaa.carceN N A r F ' rGATTArcrgicoccercaret, Y is w F P AscanciscsarctiisaststreatcracoasticM N T w w is is T J W R Fa ERASE s SO 220 2so 254 s ass 257 2ss 59 SO AACRRGTAATCGGATTGAATTTAGAccer AAGGGGEAATGSACAGGATTArtRCCAAATGTAGGGAA x A is N P w til . . H C H R H a G M D M W F I g N G G. re- AASE s xiii-sie step-sing Nos. Tarnator 25 262o 263 G s SS O 27 258 SS 27C Argaara Cattacca fricalsTaTCATTTGATAGAGCCTCCAAccCGATCGTTCAARCRTTTCCCAATAAAQ R. I. L. R. F. F D M P : *r ass s 7. 272 ( ; 2736 A 27s 276 t 27so 27s 28 tractergaacetaccestriccartsATATATATAATTrcrgicaatacerra AccArcravitaar'TAACAGAATGAGACGITA 28. :8: 3. 24 2 as 285 87 o 289 - 250 TrTArcigarcargAfricccs. AAirspactatara TacceptaGaalaazrat AGCCaCAAATAragra TIACGCCG 2s 2s2 93 St 2ssig 2s. 27 9t 29 so Carcataract acrggcCGTCGTAATCGGGAAAAccrggogiccCAACTTARTCGGGTTGCRG 302D 3.030 349 s 37 303 39 careccc. reacAGanitarAge AAA acccaccaccredicata?tricksaccalarcCAttacGCCTRICCG a : 30 s 2. 3. 80 9. GTATTccer Acecareggarcacciarracracker reactors TortATGcccAAAAGCCAGCCccGAct 32s 3.27. 3. 3. 25 259 327 ad s2.so 3. coccoacaccCorgatacccCCTsAccGGCTTGrcareccCAircog tracksacagatorrigAccGTTCCGGGAGCTGCATGTGTAACGr g 3. 33 34t 35 35 s37 380 st Tracercargariacecrat AccariccircGigasco ATTTTTRI AargaretakaArgirarascGTAG TsetaertrocGeaarcrge34. 3420 GAAceCerrargrrrrrrrrroraAAACAATCAAAATGTArcGCFATAGACARTARCCTGATAAATGed 3d 4ts s a 45 347t 40 3ds 35 s e20 ass 355 355 ass 35 3590 300 erraaraararticacciac arrget Aceregsoc.cccTergilwcGTTGGGAAGCCottacAAAGAACTGATGGTTTC TrocC3CCA s C. 3. s s 37 est s s Atass CTGATGGCJCAGGGGArc Arcrgaroanacaccatasarcotticecarcial& Q righterstressG L H A G S As

Fig. 7B

Patent Application Publication Nov. 21, 2002. Sheet 10 of 23 US 2002/0174452 A1 Plasmid pAFT244 DNA sequence to express dirigent protein Range: 1 to 227

s 4. g 5 3. 10 GCGCCCAATCGCARACGCfCCCGCGCGTTGGCCGATTCATTAATGAGCTGGCACAt-Agstretcareg AadiccGreggcoche l, : 4. s l s CCAAFTArgrgAGTAttacTTAGGCACCCCAGGCTTACACTERGCTCC tactCTArcretiregarrierraGGGarta Tre SctiFroRkater l 2 23 240 s 2. 20 s s 30 ACACAAAACAGTArtsACAGATTACCCAATGRTRAftergrosaccArtArecac AccAAAAAAAARtrattractarrcrg d so 34 3s 30 37 st 4. ATCCrcersAgCCTAAASSITTCreACCCCGSATAAGAAACCCTAAGAAEGracAAAGTTTSCATTCrcCAercAdiATA Arach TAAGTAf alo 4t 46 440 4. 4, 6 470 ? 49 50 CATCGATsACATAGAAG8 rCrcRTArcasterroricacciarTCATTCACTATCTAcATAAgrArterrogorasatcTTGAACATA 5. s 40 s S5 7. s so o AccAAAGTATTGAAAGGGRAAAAAAAcA&AcaxaggravagalurgArtTGTRRRAAcTCAccer S2. St. S4 SS S. 70 SS s 7 AATTCATAretaAAAAGAGASATAAAAAGAAAAAAAIATTCCTTTCrgchaaaassaggcaa.car 7. 2 73 48 c g r 79. BO ArtCAATGAAAAGAAes AAAAAAACAATTGastria AATTACAATAAccASTActa so TAA 80 size so 8. s 87. s s sc Act TCAATAATAAccrCAGICTGAAaacARACAATAATGAAActareCACAAATGAGArg 5.8 otist- scoding regic so 2 S5 4. ssis sts g7& B S3 to M W S K I W. A. L. P. L. C. F. l. Recr's states sh l 1020 its 4. s t 98. O YrcaccrecciaccaccGCRAccratcarcicacegoric reakacorrectaretaccacciarraargaaaaaar s T S S A Y ; R. x. P R P R R P C K E L v. F. Y F is t w L. F. K. G. N. Ns Ricar FROrsay s id l la & s s 7 s 9. 2 AccAurcCAtticce, GrccCGTCCCCCARGGGGaARAgATGCATGGCCGGATTCAA TATGGRC grgstricSACA Y A : v is s r or w G a T A M R v F Y G E . V V F I) to pas ckg Pages 2 lo 3. Zs 4. ess i5 27 es 90 300 r N is L. ii. s r P v is a R C G M Y F. Y. K a G

w Rickar PRCEN - Lic 4. s o st 32 TreTorrrrrt TrewarTextrastriarurcGAAcerronacarracrccGrchTCCATIGErgAACAAGARAGACACAFArcAGreat rissa F is F. L. F is is Y W G T F as G. A. E. F...... 8 W T G

r Rt. Rox s al 42. 43's s s 40 17C as lic St. cacrocrgaretarggestigatarge acrosarcacca Tiger Traggggarcategic regrosaritairca G T G F F W A I, M T A E. g. C. W. Y F. R. L. R. Y d 2 x . Ya. orger Frient 0 xxhe 5M sessite posteriod catar Gl ,52t so 54 s sa S7 se sa g

60 s s s Gs es y s lis 7 certaircrxtretcharacSrraccaroraArahraxcArgio RT3CAAcGTATArtIAGANCGTTTTTATGATTAGAG 7. 7. o 70 17Go d 77 f s st Tccescaragarazrat AhazucharaxiscoggcAucracGATRAAATCOCGCGCarrorcroft ract AGATCGGA Patent Application Publication Nov. 21, 2002. Sheet 11 of 23 US 2002/0174452 A1

Bl ael 14 less AG 37 lso s s RTCASG ACACGTCUTGAcroG3AARACCCTGGCTACCAACTTArcGeorgcasescarccceercoccaccreoccra ls. lic 3. LS4 iss s isfy iss issu R TAGCEAAaAGSecCGCutcKittcecCroccaacActiTaPGAccord ArggesAxrcqcticcarostarr retres reorcrossessor st 2. 04 es g 2B ATTTGACCCGCTATG''CAcrCrassacKCTGCTTGATGCCSCATAGTAAAggiectaeacetic cogcraigogccorsal l 2. 2.9 24 as? Rlic o 29 s 22 GStre'rcrger cccGescecCTTACAGAAAGCrgTGAccGrercocesawgerceraggrerasercaccsircarcaccaccitcea al 2 224 so 226 7 228 s 3d AcGAARggecircGrisat, GCTATITAggiratorTAAAAAggTrraracticaggregdaircrgsgatanarctics 23;[. s A42 2ss 23A so 38 29 240 Accett Artigrate."'AATAAAAATRT's ArcCGCfcAteacACASAAccreat AATGorcharasarracaricchetics 2 24: ) 243 244 s 24 4. 24 st 9. ass TracCAescrgsggCo TGGIAAg3 ITGGGAAGccCGAAGAAACTGGA3G Trotscgc.caragrarcCGCAcciarcacar 25 25 25 254) asso se so s Ss: o CTGATAAGASAtcAGGRT at ATCGTTCGCAGRITAtalasarcatecitate TTC-recreaticCraggregacascarciccarate M t A G is r A A W R L. F G Y de RNRCs s as 251) 2. 264 sist s 257 2S3 es 27 Craccaracascarcircarriagecccsearcsecrgreaticackaggacierrerritoric Accacecsrdci A Q r. I ( C is A. R. W. F. R L. S. A G R F W . F W x T. it is . A an Kass > 27. 72 2 to 2& 2ss 275 277 2780 es 28 CTAGRAGCA3ACGGGCAGGGCGATSGGGGCAGACGGGTTCitraCiscASCTGGrcGAegrCAergy Accorages: N E E R f A. A. R L S W A T if G W c A R v W W T S A. G. R. - KNAMCs a 2028 28 2.84 BS 29 e3, 38 so 2 TrecciagarcaearcsecretricrocorroresticcessarisTATetscartraigekaTactice W L i I. G E P G G P. L. L. 5 a R. I. A P J S K V S E M A D A M R. R. L. o-rah-raa-muha-ol-va KANNYN 23 92 293 394d s seo 27 298 ess d catacicertarerock argcatcGAccaccAASCAxacArguagga As-RSAsgxissarcCargrecarcasAT H. T. L. a " C P F is a Q A x i R. J. R. R. A. R. T. R. M. E. R. G. L. v in as Kar Max sa a st e 3. stadt sts s abo ast 39 g . A A. S. L. F. A. R. T. K. R. & M P is E. w w

w salar r 3. 32 3a 38 s tled g sle 39s so to a . P is V E N G R F G N G. R. L. 5 A R Y f I A L KAAMYCIN as 32 to ; 323G 3.24.' s ses 27t R s god TecreatercretiricorrecogcrickstoreAccriticorrecTTAGAT caccacciccirca accroctric A. E. E. . . . is W. A R F is A is R. I. A FY Katyct s vula-a-ul-Hawwu-rhyme M as 232 eig did s 3360 sigg 39 as Tarcicer retrexcavarrorerqvarrawaihagavagaraguaraacrgaareer transAhrarchTGACCARXArceirah Y R. L. T. D. E. F. . . . KAN2-2 st 343 3.43 4 s & 3. 3.48 3&G so AirTeacAgra Accessagavataggar TTRACGcGATT3TACA 35 SP 39 S&c. s s 357 58 asso so accc.gc.ccgcy ITTetrictsATArcrack?tretreakagraaf GGrckgcASAgecharachaara ae 365.6 3S4) es 360 st 368 3es 31 go GTrcrxgrgiscess, AGGCAccords AcAAcrocracciscattacACercogracetarCarea.AccultzGo serg 3. 37.20 so sia sts 376 77 as G 7so s crascaragrarcticsgracicaragatarraccessarasceticAleesecessor AACGGCGgators capacitcA grging3. acticciacciacifiagarxtracAgrgaserArtAGAAAGCGCACGTCCCGAAGGAGAAGGGracharacter82 s 4. BS As By 33B sal s 3s 32). iss 9. 325e 396 ge", Asaf iss 4. Accessages reckacategizccoxaaacggagicTreece.cggRAACGCCTGGSATerrATAircraft correschrecreas

Fig. 8B Patent Application Publication Nov. 21, 2002. Sheet 12 of 23 US 2002/0174452 A1

circarrrrrarcatscraggggg.cgAGCCTAGGAAAAAGeMGAAcaciceiraserceresaccrrigercocerTargeral : d a s a st o t 4t d &l A. 4. as al s t RCATGTrcreergCGracecrgxTICTGiraaTAAccra TAccocerciaGrangcroRAccCrcCCCGCAGCCGRACGACCAGCCAG i. 4. crascocascaitiaasa Patent Application Publication Nov. 21, 2002 Sheet 13 of 23 US 2002/0174452 A1

Plasmid paPI 246 DNA sequence to express reductase Range: 1 to 4605

LC 40 so s 8 SO Geccee, ATACGCARAccost creeccGCGCGfrtsCCGATTC TrAATGCAGGTGGCxcexcacGrrrcecargo&xxGcGGGGAgrexcoccRac GCAATTAATGTGAGTTAGC (2 TCATThisGCACCCAGGCTTrACAcrrTArgorricccGerratrefrogrggruarTigreaGCGSaracarto 40 so s 70 so 2 Yat. Fromoter AcACAGAAACAGCTArcACCRIATAccAGorrariesTAATGorczAGCRTTATCGGAttacAAAraaaaaaaaaTTTTArchgrearer22 o zo 2. s 70 so 2s) 30 33 G 3. 35 g 39 RTCrgrgagic. TCAARSITT ACCCCG3RTAARucciagraargrgo AAGarrattorcaccaragaragra 4. 42 43 4. dis 45s 47 dia 49 s cATCATGAcAragAAcreatiscArchiArcArgercrore AccraTrcArcCracroreTAcaragrarc reager AhrerraGAxcar AccAAGTACGT3ATGWRTAGGGTGACAATAAATCACAGATARGoatiaTAAAGAAAARTTAAAAATAACs 52 53 550 St. s 59 59 c ATATSTATATGAAAAA3J.C.AGAGCTATAAGAAAGGCTATCAAAAAircacargottacGrgravaaaassacGGCLT3AATArcS.) 5. 3. s 3. es st S8 es 70 s t 75 7. 7. s 790 B CATerrATATTGAAAAGAAucla GAAAAGAACARAAATGarcCorea.TAtxtATIGirgiccarta AT carcicacitrogrTAcc -qt Ra alo se 83 d 5 8t 57 3. 8st s ACTTATATATATAAAir. ATACGTCCCACAT.VCAAATIATTCATAksaractic TAFCrcAAAAEcACT ps d:15DM *Coding Egion. ATATGTTCACARACGGCGACAAAAAAAAGAAGAAAAAAGAAAGTTGArcATsogacagarkadcso s s 34 agg so 7s s 990 M G R is K. v. L. G G T G L. G. RERSE w lo, 0.29 l 1960 lso le 07 8 so also AGAGAcetraacAAccratic ATGRAAAAATTCrgic Accreat TicciriakaArtsAAAAA R. L. W. K. A s ). A C G E E T Y R r G W D W E M its ResoterAs sa il i c 17 ls 9. , Targcrackercrgaretarrcassa TAAAGTGTGAccGAAgricTAGACGAGAATAGGAT S M A : I W 8 S F x to F is s r. v. E. A. W. K. L. W. E. v. W i s A Retus eit 220 23 24 25 ls l 28 29 ad B G V . . . R. S. F. I I, it g . K. L. W. E. A x x. E. W at R P S to Ridges o

e 32 13 4. s 7. as: as 4. F. G M D F A K F T. A. N. E. P G y it. K. M. W. W. E. K. A E. K. As Rotunatas a. 40 2 43 rida les As 48 90 s cGAT recTrackTrcircrgaazrocirrtcracGTTAETCrgg3AcGrcTergicTriggcAAAAirTrcCTCTAGASATFrTTAT F Y W S F A G F g c F K P S R F W retuals ar TATAATAAGAAAAgataka.T.R.EAAAAAAAAAAAAAAAAATAAAACAAAAG5. 52 5 St. G St. g S. iss s G G K R Y N E D A Y. A K N P R T Ke Rous f

s2 ls S- s ed f s less 7 AcAATerAAAGreer causearcTreat-AAcacial cycACATGGGAGAACTArtugaacRACTCCAGAAAACACT r I. Y i s p r x x . . s R. E. v v or T W E x L. I. G K E L Q K . T LP

Fig. 9A Patent Application Publication Nov. 21, 2002. Sheet 14 of 23 US 2002/0174452 A1

o 177 s lso s S K ERAA P. F. w E. Kl':Fe?tricts v K E L E Y A Q assistancerv is , s h Y Tretorcarcastecstasi t w y g g c l, r. s. {k eas

sea site a storicodon al l 8. last s e s s o frograGGAATGAargaggaroraACTTATCASAGF RalsTATxceAGGrissalsTaccreasco Tacgregarderer F E G D E E A S K Y S E V K Y T S v E E Y . K R Y Roys Y stroS feratirater lso s ls les g7 s s AGRCGGregAATTTecca rearrah ACATTGGAATAAAgarter FAAgrirrorcestroecorerraccareerArcaravarre

a ed as ea acts 2. tTGRATEACGRACESARFTARGAGATCRACGTIATAAGTATATArcoccalata a

al & s e s es GCSARCAA Acalaric, gogaraacGATAAATCGCGGCGGGTrrectrifacetsarraceTirra

223 .222 c al s e 7. 2. 2s AcGrecrgareika ActreggcrackstareCorsaceaexeccorrecciagartegratics accelegacar

23. R 23 235) s 2. 23s s accific craacAGEriocarctical GscCAccGractargescrgrocessfircacategoric A

- se s 24 4. 24 2. St. TTAGACAATSGrcrg SCCAGTTAGGAGCCSAACCCGAACAceCorgascaccrgAcGGTreccerciscar

Sl s 5. St. ass s 270 s Sg s

2. ests d s s s assa ckartageRTCFAAAAAGerarcataccartriccariccessarrascaceccaragrari Tr.

g r: s s g o 7 2. 29 2.

l se as saf t s s 2s. 2g AAGGTTGarageorgia AAAcresciaTocorre TccGCAAGGArcrgArggotscAaggaRTCARArcGArcAAGRGAccGArtsAGG

s eas ass s SS 25 7 Rae asso Agreer AAGASCAccorregg careccGAAGAAAAAAsc E f : A G S P A A T is es ARR s

3020 sq.3. as t s g s 3. Tucciarraceccrg essagestestseas actrexaceaeacacas C is D A A v. f. t. i. 5 A G R P v R, f w x T. D. E. s. G. A. L. f. E . q P R .- AA's s

e s R 7 s 2 R. A. R. , s: , . A W A A Y W G R . . . . . we Kaas Xr

er t 37. s 3. GcceeccAtercrecrgreaterriccarciercercoexaAAAgarter Arcareecratcoxaracticester Atarise racciggerAcc a S R . A a W S E M A a R. R. . . . . F A is are sists

also si s 2. 3. Bas ess "carricaceaeacaceaetaceaetic Federategic Arcaircrgarag K. R E R A R. M. E. A. g . W L. P K is ti > ------

si a 4. s as 47 Patent Application Publication Nov. 21, 2002 Sheet 15 of 23 US 2002/0174452 A1

GorcGCGCCRGCCAAgri Costaggotter KoccCGCATGCCCGACGGCGAGGArercercrerecroscsarcoarserraccharacar G L X P A E IF A. R. L. K. A R P R D G E D L. v v I. H. G D A c L r n I. Ms - KXNAMYc s

s s S. As 3ss 35 357 3S so as tgrgGRAAATGGeogCTFTTC FearthaccacierraGCGGCTGG3TGIGGCGGAccacArcAGGRATAGCT rockTcccGrattracrgh v 2 N G R F is C F N F C G R. i. G. v. R. E. R. Y Q to I A a R p it. A XANAMN r

lf 35. 30 364 3 is St. 3. ass Go ag GRCTGcGCGRATGSGC.Accorcorcoceri Tacts TATCGCccCrcoccaFrcercise Arcscertrarcescorterracguirer E. G. G. E. W. A. B. R W - Y G A A e D is q R. I. A. F. Y. R. L. L. D. E. Fs n KANAMYS s OrcaravaggAAgastracATTAGGGAAGArcCTTTCATATCTckTcaccAAATCCTTAAcircastTCGTicciotou37. 372o. 73 370 375 s 7 37sc 380 F .

38. so as 34 so B 37 B9 s coat accCTAGAAAgaaaaaTortAATCCTTTTTrcrgetscarcreertgcareeracia AAAAccaccesetaccaccc. GGTTTTTGcGGAraguirACAATTTTTCCGAAGAckgrgicagciscARRAccAaracticciagaragas 392n so 94. 35 3570 3S 399 & CG 4. 40 4 D30 4.04 dos 486d 407 40 4.09 4, TTAssoccaceae. TcAaaaac TsagaccGCCTAAAtracGrcrgrarcerraceAgeCicclescoag TGGCATAircretch

4. (20 & Al4 4ts 4. 7 4s 43 coercircacar accecataAcacTCGGGTAAgxacerTrcirca Acago ATTsAGAAccACCTACA

4. 220 t 424 2 2. 8. 429. o coacTag TAccoacCraccaravacAAAGCCCAC3CTCCAAGGRAAAAGCCACAGGRTCCGGAAGCGKCGTCGGAAccra. 42 M32 3. & s 4. 47s 48 439 44 categicaceaegracercategAAAccorregaTerraracecrecessarcoccaccort TTsAGCSTcaatarargascist

44 42 4& (sia) 4. wide 47 4 3 tids gSO AggaegerArg- Accocaccescoccer-rraccarceragcertacarcaccirrigercacarorterraccrgerrarc al 452 &Sigg 4.54 ass 4So dist s 459 Go riggscot accTTGATA3 grace EccescascaRAGAGAGCRTAGAccArtico ARA

Fig. 9C Patent Application Publication Nov. 21, 2002. Sheet 16 of 23 US 2002/0174452 A1

Plasmid pA(249 DNA sequence to express dehydrogenase Range: 1 to 1505 w

3. 4. 5 s As 9. GCGCCAAAcGCAAGiocreccCGCCGTTGGCCSATTCATAAGAGorgGcaccacycroccaccGAAGC sessegrgagcupad i. laity 5d. o i8 s 2 CAAAATGTGAGTAstroAccArragcheckstaggeracArcCarrcaststriargroTertisaargreat secracArre sce-Prote & alo so s s 2. es ACACAGAAACACTRT (ATCATTACGCCRCTCAtri.AGTAATGracascaTTArceaACCACCARIAAAAAeAT triargarcarct 3. 3. 3C 3& 35 3S t 3A s 4 do ArcGTGAGCCTCRA,TTCTTCACCCCGGRAGA ACCCTAPRATGirgraag TiccaTTCTCCACTGART3AARTAAGATA 4t 4. 40 4s t St. s s coats AAAATGarcircycottaccarcatracroArctical AAGATctrict rare ITAGAAAAA St. s S. s sis s7 g s so

s st s 64 es G es 8) ss 7 ATATCATAGAAAAACL seagoialaa AtascCRTCAccAAAAAATracrgccreatercaraagaaagtercarc T 7ter g 7 75 so 7 s so CATSir TACCAAAAAGAAAAAGAAAAAAATGCTGcircraaTATAATrciarrackTracATCActiTecretacca

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sal S2 53 SA sis 566 57 ass G5 St. sarcascalancertersecraccretarctracCoAgartaccagaTravaayakrcarguagACTTGGCG et q r a r R v - c L is P. F. G. L. F. F. E. A. K. K. F. T. G. I. E N p B D . A - ResENSE X AATGaharacAActreseacGarreravagaraCAAvnTrcRGGXTrchagATTTGCCAATGAGGGTTTrcTGGGEAGTGArgigoccuraTG5 53 63 64. 5. lso lso ass 7 to is g R. A. g . . x G at K L R if E t w A is A A F. L. A S A Ys -mm-rama-ra-ra-rp PPRE------Patent Application Publication Nov. 21, 2002. Sheet 17 of 23 US 2002/0174452 A1

Pacbsite sKhoist 7. 7. its. 75 7. so regro ACAAkircrg Tr ("ccATGGAGGGriekccGTGT3rari TortAATCAAA rercera recacaoreruarcircaeroser v S G O N L. F : E G G F S v c N S A r x , F y D is a - crisis s p-NOS rector

als es les a s ASAGGCGrcticsAATTCATCGTrear A.C.ArrogCAAAAAQTTrearrasaTrcarcererriccercTriggarstracAra Trrrr.

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282 4. s also e s g CCGAAAAAAAAAAttice:Caa AfraGSTAAA ArcucciscarcierciaTracAgriccircarceetsTrrata

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so e s s 29s ass 25 r iss s TGctogrescoccGTwTrris EgergroAGGGCAGGGGGCeror TTTrrrroAXgaegic TGtcGargergaria acco ScacGAGG is a y 3 r w , t w x T is as g ......

re '''"-a- s s g es us scs cacaoccerarcgregerggregaCCCGcGirrectricise AgrargerencerrarcRorexacccGGAAGGGAcresteracral riggerer A. A A W A w W T : A R ...... ses s zo 33 d s s S. G ...... A A E ...... A sms s

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l al s s g s s s

Fig. 10B Patent Application Publication Nov. 21, 2002. Sheet 18 of 23 US 2002/0174452 A1

TGGARATGGCGCfa"FittsgracGACTG2GGGGGGC resorter GcGGACCGeirreggattaractergracecrgrxtraorea

v N G R F an: o f it D C G R isKAAN y A D F : Q D a L. D. T. r. D : A E s

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Fig. 10C Patent Application Publication Nov. 21, 2002. Sheet 19 of 23 US 2002/0174452 A1

Basic Cassette For Gene Expression In Developing Rice Seeds

pAPI217(pCH126-GUS) Arpicillin 5691 bp

GUS coding region

For NOS terminator

Fig. 11 Patent Application Publication Nov. 21, 2002. Sheet 20 of 23 US 2002/0174452 A1 Procedure Of Rice Transformation

4N Expression Vector aliafewise

Callus Tissue

Transgenic Seedling

Transgenic R, 1 Plant Producing Transgenic R. Seed in Greenhouse Patent Application Publication Nov. 21, 2002. Sheet 21 of 23 US 2002/0174452 A1 Detection. Of Lignan Production In Half-seeds Of Transgenic Rice A 6 - 6 D & 6) B - 6) & 3 c 6: P 6...) 6' 6.) C s s s s 3) homogenate

A, B Positive C Negative

Fig. 13 Patent Application Publication Nov. 21, 2002 Sheet 22 of 23 US 2002/0174452 A1

Matairesinol (ng/100mg rice grain) C as S s is 5. 52 I al. M20 258 O TF30S 63 62E 37 28 82 3. SE

258 7 149S 37 26

6)

s

i-

Fig. 14 Patent Application Publication Nov. 21, 2002. Sheet 23 of 23 US 2002/0174452 A1 A Typical Picture for Multiplex PCR Screening of Transformed Rice Plants Bombarded with Four Lignan Genes

+ Controls E r Y as a stra e - d - 9 as s r ...... 3; S. .S.

s r i 5st &sit 55s sit is rif r s is is 5 A e.

Fig. 15 US 2002/0174452 A1 Nov. 21, 2002

MONOCOT SEEDS WITH INCREASED LGNAN 0022 Brandt, et al., 1985 (hordein B1 gene promoter, CONTENT GenBank Accession No. X87232). CROSS-REFERENCE TO RELATED 0023 Burr and Burr, J. Mol. Biol. 154, 33-49, 1982. APPLICATION 0024 Bustos, M., et al., EMBO J. 10:1469, 1991. 0001. This application claims benefit of priority from 0025 Davin L. B., and Lewis N. G., Plant Physiol United States Provisional Patent Application No. 60/230, 123(2):453-62, 2000. 632, filed Sep. 7, 2000, under 35 U.S.C. S 119, which is incorporated herein by reference. 0026 Davin, L., et al., Science 275(17):362-367, 1997. 0027 Depicker, et al., Mol. Appl. Genet. 1:561-573, FIELD OF THE INVENTION 1982. 0002 The present invention relates to a method of 0028) Eich, E., et al., J Med Chem 39:86-95, 1996. increasing guaiacyl or “G” lignan content in monocot Seeds, by way of the “G” lignan branch of the phenylpropanoid 0029 Price, K. R., and Fenwick, G. R., Food Addict pathway, and to plants and Seed compositions produced Contam 2:73-106, 1985. thereby. 0030 Evans, B. A. J., et al., J Endocrinol 147:295-302, 1995. REFERENCES 0031) Fotsis, T., et al., Proc Natl AcadSci USA 90:2690 0003) Abe, et al., 1989 (rice prepro-glutelin gene, Gen 2694, 1993. Bank Accession No. D00584) 0032) Fotsis, T., et al., J Nutr 125:S790-797, 1995. 0004 Adlercreutz, H., et al., Med Biol 59:259-261, 1981. 0033) Gielen, et al., EMBO J. 3:835-846, 1984. 0005 Adlercreutz, H., et al., Lancet 2:1295-9, 1982. 0034) Hanson, K. R. and Havir, E. A. in “The Biochem 0006 Adlercreutz, H., Gastroenterology 86:761-764, istry of Plants: A Comprehensive Treatise', Vol. 7, Second 1984. ary Plant Products, E. E. Conn, ed., Academic Press, New 0007 Adlercreutz, H., et al., in HORMONES AND CAN York, p. 577-625, 1981. CER 3, Raven Press, New York, 1988. 0035 Hrazdina, G. and Wagner, G. J., Arch. Biochem. 0008 Adlercreutz, H., Scand J Clin Lab Invest 50(Suppl Biophys. 237, 88-100 (1985). 201):3-23, 1990. 0036) Joannou et al., J Steroid Biochem Mol Biol 54: 0009 Adlercreutz, H., et al., Lancet 339:1233, 1992. 167-184, 1995. 0037 Jones, D. H. Phytochemistry 23, 1349-1359, 1984. 0010 Adlercreutz, H., et al., Scand J Clin Lab Invest 53:5-18, 1993. 0.038) Kim and Wu, 1990 (rice glutelin gene, GenBank 0011) Adlercreutz, H., et al., Lancet 342:1209-1210, Accession No. X52153). 1993. 0039 Kim, S.Y., and Wu, R., Nucl. Acids Res., 8:6845 0012 Adlercreutz, H., et al., J Steroid Biochem Mol Biol 6852, 1990. 44: 147-153, 1993. 0040 Knutzon, D. S., et al., Proc. Natl. Acad. Sci. USA 0013 Adlercreutz, H., et al., Cancer Detect Prev 18 89:2624, 1992. :259-271, 1994. 0041 Kostova, A. T., et al., J. Biol. Chen. 0014 Adlercreutz, C. H. T., et al., J Nutr 125:757S-770S, 272(46):29473-29482, 1996. 1995. 0042 Krol et al., Nature, 333:866-869, 1988. 0015 Adlercreutz et al., The Finnish Medical Society 0043 Kurzer, MS et al., Cancer Epidemiol Biomarkers DUODECIM, Ann. Med. 29:95-120 1997. Prey 4:353-358, 1995. 0016 Alber and Kawasaki, Mol. and Appl. Genet. 1:419 0044 Lam, E., and Chua, N. H., J. Biol. Chem. 434, 1982. 266:17131, 1991. 0017 Altschulet al., Nucl. Acids Res. 25(17) 3389-3402, 0045 Lampe, J W et al., Am J Clin Nutr 60:122-128, 1997. 1994. 0018) Atanassova et al., Plant Journal 8:465-477, 1995). 0046 Lampe J W et al., Cancer Epidemiol Biomarkers 0019 Ayres, D.C.; Loike, J. D. Chemistry and Pharma Prev 8(8):699-707, 1999. cology of Natural Products. . Chemical, Biological 0047 Lavery et al., Genetics 94:6831-6836, 1997. and Clinical Properties; Cambridge University Press: Cam bridge, England, 1990. 0048 Lee et al., Proc. Nat. Acad. Sci. 88:2825, 1991. 0020 Bannwart, C., et al., Clin Chim Acta 136:165-172, 0049) Lewis, N. and Yamamoto, E. Annu. Rev. Plant 1984. Physiol. Plant Mol. Biol., 1990, 41,455-496. 0021 Brandt et al., Carlsberg Res Commun 50:333-345, 0050 Li, D, et al., Cancer Lett 19:142(1): 91-6, 1999. 1985. 0051) Mazur et al., Anal Biochem 233: 169-180, 1996. US 2002/0174452 A1 Nov. 21, 2002

0.052 Miller and Knudsen, 1993, barley hor1-17 gene, 0077. Thompson LU, Baillieres Clin Endocrinol Metab GenBank Accession No. X60037) 12(4):691-705, 1998. 0053 Nilsson, M.; Aman, P. J. Sci. Food Agric. 0022 0078 Trowell, H. C., and Burkitt, D. P., in WESTERN 5142:143-148, 1997. DISEASES: THEIR EMERGENCE AND PREVENTION, 0054) Nishibe, S., et al., Chem. Pharmacol. Bull. Edward Arnold Ltd., London, 1981. 38:1763-1765, 1990. 0079 Whitten, P. L., and Naftolin, F., in NEW BIOLOGY 0055 Mäkelä, S., et al., Environ Health Perspect OFSTEROID HORMONES, Raven Press, New York, 1991. 102:572-578, 1994. 0080 Wolf, N., Mol. Gen. Genet. 234:33-42, 1992. 0056 Mills, P. K., et al., Cancer 64:598-604, 1989. 0081) Zhang, J-X., et al., Cancer Lett 114:1-2, 1997. 0057 Montesano, R., and Orci, L., Cell 42:469-477, 1985. BACKGROUND OF THE INVENTION 0.058 Nakase, M., et al., Plant Mol. Biol. 32(4):621-630, 0082 Lignans are a large structurally diverse class of 1996. plant metabolites. Lignans exist as dimeric compounds that 0059 Okita, T. W., et al., J. Biol. Chem. 264(21): 12573 are found throughout the plant kingdom. A variety of bio 12581, 1989. logical functions and pharmacological properties have been attributed to lignans (Ayres, 1990). Lignans, in addition to 0060 Okita, et al., 1989 (rice Gt2 gene, GenBank Acces neolignans and lignins, are formed in plants as part of a sion No. L36819 M28157; rice Gt3 gene, GenBank Acces biochemical process known as the phenylpropanoid path sion No. M28158; rice Gt1 gene, GenBank Accession No. way. One of the primary products of the phenylpropanoid M28156; rice glutelin gene, GenBank Accession Nos. pathway is lignin, a three dimensional polymer which is D26363, D26366, D26367, D26368 and D26369). intercalated with cellulose and hemicelluloses in the cell 0061 Pietinen, P., et al., Circulation 94:2720-2727, 1996. walls of vascular plants, to provide rigidity and compressive 0062 Pool-Zobel B L et al., Carcinogenesis 21(6):1247 Strength. LigninS are produced from monolignol precursors, 1252, 2000. as are lignans and neolignans. 0063 Reddy, B. S., and Cohen, L. A.(eds), in MACRO 0083. In contrast to the polymeric structure of lignins, NUTRIENT'S AND CANCER, CRC Press, Boca Raton Fla., lignans and neolignans are dimeric compounds, typically 1986. C6C 3 phenylpropanoid dimers, although a diversity of forms are known. While lignins are generally racemic 0064) Reddy, B. S., and Cohen, L. A.(eds), in MACRO (Lewis et al., 1990), lignans are typically optically active, NUTRIENTS AND CANCER, CRC Press, Boca Raton Fla., existing as Single enantiomeric forms, although racemic 1986. mixtures are occasionally encountered (e.g., +Syringares inols). Lignans are usually constructed via 8.8' carbon 0065 Rose, D. P., et al, Cancer 58:2363-2371, 1986. carbon linkages between the phenylpropanoid Subunits 0.066 Russell, et al., Transgenic Res., 6:157-168, 1997. (Ayres et al., 1990), while neolignans are defined as being 0067 Schmidt et al., Science 238,960-963, 1987. connected via linkages other than 8.8'. 0068 Schroder, H. C., et al., Z. Naturforsch 45d:1215 0084. A number of plant lignans of diverse structure and function are known (Ayres and Loike, 1990; Lewis et al., 1221, 1990. 1994). Examples include , a successfully 0069 Schubert et al., Nucl. Acids Res. 18: 377, 1990. employed plant anticancer agent (Ayres et al., 1990), arcti genenin and trachelogenin, which have been demonstrated 0070 Setchell, K. D. R., and Adlercreutz, H., in ROLE to be effective against HIV (Schroder et al., 1990), and OF THE GUT FLORA IN TOXICITY AND CANCER, Aca B-glucoside, which is reported to enhance demic Press, London, 1988. cardiovascular activity (Nishibe et al., 1990). Plant sources 0071 Shaw and Hannah, 1992, GenBank Accession No. of lignans include the gymnosperms (e.g., Araucariaceae, S48563. Podocarpaceae, Taxaceae, Taxodiaceae families) and the angiosperms (e.g., Magnoliiflorae, Nymphaeiflorae, Rosi 0072 Shutt, D. A., and Cox, R.I., J. Endocrinol 52:299 310, 1972. florae, Ariflorae, Commeliniflorae). 0073 Stayton, M., et al., Aust. J. Plant Physiol. 18:507, 0085 Lignans are found not only in plants, but have been 1991. identified in the biological fluids of humans and several animals as well. The two major mammalian lignans are 0074 Takaiwa, et al., 1991a (rice glutelin gene, GenBank and (FIG. 3). These lignans are Accession No. YO0687). excreted in the urine of Subjects on diets rich in whole grain products, berries or fruits, and have been detected in Sig 0075) Takaiwa, et al., 1991b (rice Glu-B gene, GenBank nificant levels in the plasma of Subjects in geographical Accession No. X54193; rice GluB-2 gene, GenBank Acces areas of low cancer occurrence (Adlercreutz, 1993a, 1993b; sion No. X54192; rice GluB-1 gene, Genbank Accession Adlercreutz, 1994). The mammalian lignans, enterolactone No. X54314). and enterodiol, are presumed to be formed by the metabolic 0076 Teicher, B. A., et al., Cancer Chemother Pharma action of intestinal bacteria in the gut on the plant lignans, col 38:169-177, 1996. Secoisolariciresinol diglucoside and matairesinol (FIG. 3), US 2002/0174452 A1 Nov. 21, 2002

respectively, which are released from plant food Sources onstrated to be the immediate precursor of enterolactone. during digestion by the action of intestinal bacteria. Enterodiol, enterolactone, matairesinol and Secoisolaricires 0.086 A number of positive health benefits have been inol diglucoside have been measured in human urine and associated with elevated intake of plant lignans (Adler feces by isotope dilution gas chromatography-mass Spec creutz, 1982, Adlercreutz, 1984, Adlercreutz 1990; Setchell trometry. (Adlercreutz et al., 1997). et al., 1988 (16)), and there is evidence to support the protective role of lignans in reducing the risk of breast SUMMARY OF THE INVENTION cancer (Adlercreutz et al., 1982; Adlercreutz et al., 1984; 0091. The present invention is based, in part, on the Bannwart et al., 1984; Setchell et al., 1988), and other discovery that modified expression of certain enzymes inte hormone-related cancers and colonic cancers (Adlercreutz et gral to the phenylpropanoid pathway in plants may be al., 1995; Adlercreutz et al., 1993; Adlercreutz et al., 1988; effective to generate plants exhibiting modified levels and/or Adlercreutz et al., 1992). Epidemiologic and in vitro studies modified relative proportions of G-lignans (or monolignol Support the role of lignans in decreasing the risk of prostate G-lignan precursors) in plants, and in particular, in Seeds of cancer (Mills et al., 1989; Zhang et al., 1997), and lignans Such plants. have also been implicated in the prevention of Osteoporosis. 0092. The present invention provides methods for modi Further, phyto-Oestrogens, and lignans in particular, have fying lignan content in plants by transforming plants with also been reported to reduce the risk of cardiovascular vectors containing a DNA sequence encoding one or more disease (Pietinen et al., 1996). proteins integral to the phenylpropanoid pathway leading to 0087 Unfortunately, the Western diet lacks foods which G-lignan formation. Such coding Sequences are expressed are lignan-rich, and the high incidence of certain major under the control of a Seed tissue Specific or Seed develop hormone-dependent cancers, colon cancer and coronary mental Stage specific promoter. heart disease in the United States, Western Europe and Canada, when compared to countries in Asia has, in part, 0093 Expression of the DNA sequence results in a modi been attributed to the Western diet, and in particular, to a fication of the absolute and/or relative level of an interme Western diet lacking certain protective compounds, i.e., diate metabolite leading to the production of G-lignans (e.g., lignans. (Trowell, 1981; Reddy, 1986a,b; Rose, 1986). Fac Secoisolariciresinol diglucoside or matairesinol). tors which have been attributed to low lignan levels in 0094. By placing, under the control of a tissue-specific Western populations are (i) low intake of cereal grains, and promoter, a gene Sequence encoding an enzyme in the in particular, whole grain bread, and (ii) increased produc phenylpropanoid pathway at a point following coniferyl tion and consumption of highly processed grains, which as alcohol, whose expression results in production of G-lign a result of extensive processing procedures, have greatly ans, or an intermediate metabolite leading to the production reduced levels of naturally occurring lignans and/or their of G-lignans (particularly, Secoisolariciresinol or mataires monolignol precursors. inol), it is possible to increase the G-lignan content specifi 0088. It has been demonstrated that increased dietary cally in Seeds, or within certain Seed tissues, while allowing consumption of plant lignans Such as those found in numer for normal plant growth and development. ous grains results in increased levels of mammalian lignans 0095 Accordingly, it is an object of the invention to (enterodiol and enterolactone) in the urine and feces (Joan provide a method of increasing the guaiacyl- (“G-)lignan nou et al., 1995). Taken together with the positive health content in Seeds of a monocot plant, including Selecting at benefits that have been associated with elevated intake of least one protein or enzyme integral to the pathway leading plant lignans, consumption of plants with increased levels of to G-lignan formation, and Stably transforming a monocot lignans can result in corresponding increases in circulating plant with one or more chimeric gene constructs having a levels of mammalian lignans thereby providing positive Seed-specific transcriptional regulatory region operably health benefits through normal dietary consumption of linked to a nucleic acid Sequence encoding at least one plants. protein or enzyme integral to the pathway leading to G-lig 0089. The prevalence of the above described diseases, nan formation. particularly in Western cultures, coupled with escalating 0096. In a related aspect, at least one protein or enzyme health care costs, Suggests a need to develop new and integral to the pathway leading to G-lignan formation is aggressive approaches aimed at preventing rather than Selected from the group consisting of (i) a dirigent protein, merely treating these diseases, particularly by increasing (ii) / reductase, (iii) Secosisolari-cir lignan intake. Ideally, this goal should be met in a manner esinol dehydrogenase, and (iv) laccase. which is readily adaptable to Western lifestyles and eating habits, does not require a drastic change or restriction in 0097. In another related aspect, (i) a dirigent protein, (ii) dietary intake or require consumption of dietary Supple pinoresinol/lariciresinol reductase, (iii) Secosisolari-cires ments, or adversely impact existing grain processing facili inol dehydrogenase, and (iv) laccase, are expressed at the ties or the quality or flavor of the resulting grains. Same time in Seeds of a monocot plant. 0090 Flaxseed (linseed) is the most abundant source of 0098. In yet another related aspect the amount of (-)- lignans in food. Other food Sources in which lignans have Secoisolariciresinol diglucoside or (-)-matairesinol accumu been detected include various grains, Seeds, fruits, berries lated in Said Seeds is greater than in Seeds of an untrans and vegetables. (Adlercreutz et al., 1997). The dietary lig formed monocot plant. nans Secoisolariciresinol diglucoside, isolariciresinol and 0099. In one embodiment,the seed-specific transcrip lariciresinol have been demonstrated to be the immediate tional regulatory region is derived from aleurone, pericarp, precursors of enterodiol, while matairesinol has been dem embryo or endosperm tissue. In another embodiment, the US 2002/0174452 A1 Nov. 21, 2002

Seed-specific transcriptional regulatory region is induced 0111. It is yet another aspect of the invention to provide during Seed development and corresponds to an endosperm a library comprising Seeds derived from one or more mono Specific Gt-1 promoter. cot plants produced by the method described above. 0100. In yet another embodiment, the seed-specific tran 0.112. It is yet another aspect to provide a method of Scriptional regulatory region is induced during Seed devel producing a progeny monocot plant by crossing one or more opment and corresponds to an aleurone-specific Chi26 pro parent monocot plants where the amount of G-lignans in the moter. Seeds of the progeny monocot plant resulting from the crossing is greater than the amount of G-lignans in the Seeds 0101. It is another object of the invention to provide a of the parent monocot plant. method of Stably transforming a monocotyledonous plant with one or more chimeric gene constructs, resulting in 0113. In another aspect, the G-lignan enriched seed com increased expression of the genes encoded by the chimeric position for use as a food additive includes a Seed prepara gene constructs. tion derived from Seeds of a transformed monocot plant wherein the amount of (-)-secoisolariciresinol diglucoside 0102) In one aspect, the gene in one or more chimeric or (-)-matairesinol accumulated in Said Seeds is two or more gene constructs is Selected from the group consisting of (i) times the amount detectable in Seeds of an untransformed a dirigent protein, (ii) pinoresinol/lariciresinol reductase, monocot plant. (iii) Secosisolari-ciresinol dehydrogenase, and (iv) laccase. 0114. In a related aspect, the G-lignan enriched seed 0103). In another aspect, the nucleic acid Sequence encod composition is used as a food additive. ing at least one protein or enzyme integral to the pathway leading to G-lignan formation has at least 90% sequence 0115 These and other objects and features of the inven identity to a Sequence Selected from the group consisting of tion will become more fully apparent when the following (i) a dirigent protein, (ii) pinoresinol/lariciresinol reductase, detailed description of the invention is read in conjunction (iii) Secosisolari-ciresinol dehydrogenase, and (iv) laccase. with the accompanying drawings. 0104. In yet another aspect, the nucleic acid sequence BRIEF DESCRIPTION OF THE DRAWINGS encoding Said protein or enzyme is capable of hybridizing under high Stringency conditions and Said protein or enzyme 0116. The foregoing aspects and many of the attendant has Substantially equivalent biological activity to the native advantages of this invention will become more readily protein or enzyme Selected from the group consisting (i) a appreciated as the Same become better understood by ref dirigent protein, (ii) pinoresinol/lariciresinol reductase, (iii) erence to the following detailed description, when taken in Secosisolari-ciresinol dehydrogenase, and (iv) laccase. conjunction with the accompanying drawings, wherein: 0117 FIG. 1 represents an overview of the plant Mono 0105. It is another aspect of the invention to provide a lignol Biosynthetic Pathway (front end of the phenylpro transformed monocot plant produced by the method panoid pathway) leading to para-coumaryl alcohol, coniferyl described above. alcohol, and Sinapyl alcohol. 0106. In a related aspect, the transformed monocot plant 0118 FIG. 2 presents an overview of the “back end” of is capable of producing Seeds where the amount of (-)- the phenylpropanoid pathway showing the Stereospecific Secoisolariciresinol diglucoside or (-)-matairesinol accumu lated in the Seeds of Said transformed monocot plant is two conversion of coniferyl alcohol to Secoisolariciresinol and or more times the amount detectable in Seeds of an untrans matairesinol (i.e., the formation of G lignans). formed monocot plant. 0119 FIG. 3 presents an overview of the metabolic conversion of the G lignans, Secoisolariciresinol and 0107. In another related aspect, the transformed monocot matairesinol to enterodiol and enterolactone which takes plant is capable of producing Seeds where the amount of place in the mammalian intestine and is mediated by intes (-)-secoisolariciresinol diglucoside or (-)-matairesinol tinal bacteria, with the Structures of the lignans presented in accumulated in the Seeds of the transformed monocot plant the figure as matairesinol, Secoisolariciresinol diglucoside, is five or more times the amount detectable in Seeds of an enterolactone, and enterodiol. untransformed monocot plant. 0120 FIGS. 4A and 4B presents an overview of the 0108. It is another aspect of the invention to provide a Synthesis of deuterated d6-matairesinol (3) and d8-secoiso Seed composition derived from a plant produced by the lariciresinol (4) from matairesinol (1) (FIG. 4A); and d6-an method described above. hydro-secoisolariciresinol (5) from anhydrosecoisolaricires 0109. In one embodiment, the seed composition includes inol (6) (FIG. 4B). an amount of (-)-secoisolariciresinol diglucoside or (-)- 0121 FIGS. 5 and 11 depict images of basic cassettes for matairesinol accumulated in the Seeds of Said transformed gene expression in developing rice Seeds. monocot plant which is two or more times the amount 0122 FIGS. 6A, 6B, 6C, and 6D present schematic detectable in Seeds of an untransformed monocot plant. depictions of the map of four plasmids used to express genes 0110. In another embodiment, the seed composition involved in lignan biosynthesis in developing rice Seeds, includes an amount of (-)-secoisolariciresinol diglucoside which contain the coding Sequences for various enzymes or (-)-matairesinol accumulated in the Seeds of the trans involved in lignan biosynthesis under the control of the rice formed monocot plant which is five or more times the endosperm-specific glutelin (Gt-1) promoter. pGt-1-DIRG amount detectable in Seeds of an untransformed monocot (FIG. 6A) contains the coding sequence for dirigent protein; plant. pGt-1-LACC (FIG. 6B) contains the coding sequence for US 2002/0174452 A1 Nov. 21, 2002 laccase; pGt-1-REDS (FIG. 6C) contains the coding region (promoter) operably linked to a heterologous protein Sequence for pinoresinol/lariciresinol reductase, and pCit-1- coding Sequence, or, in a Selectable-marker chimeric gene, DEHY (FIG. 6D) contains the coding sequence for seco to a Selectable marker gene encoding a protein conferring Sisolariciresinol dehydrogenase. antibiotic resistance to transformed plant cells. A typical chimeric gene of the present invention, for transformation 0123 FIGS. 7A, 7B, and 7C dipict the nucleotide into a plant, may include a transcriptional regulatory region Sequence of plasmid paPI245 used to express laccase. inducible during Seed development, a protein coding 0124 FIGS. 8A, 8B, and 8C depict the nucleotide Sequence, and a terminator Sequence. A chimeric gene Sequence of plasmid paPI244 used to express dirigent. construct may also include a Second DNA sequence encod 0125 FIGS. 9A, 9B, and 9C depict the nucleotide ing a if Secretion of the target protein is Sequence of plasmid paPI246 used to express pinoresinol/ desired. lariciresinol reductase. 0.134 Monocot” or “monocotyledonous plant”, refers to an angiosperm (i.e., a flowering plant) that has only a single 0126 Figs 10A, 10B, and 10C depict the nucleotide cotyledon (seed leaf) that is formed during embryogenesis. Sequence of plasmid pAPI249 used to express SecoSisolar Exemplary monocots include wheat, maize, barley, rice, iciresinol dehydrogenase. millet, oats, rye, triticale, Sorghum, and corn. 0127 FIG. 12 presents a flow diagram of the rice trans 0.135 “Seed' is meant to encompass all seed compo formation procedure where an expression vector is intro nents, including, for example, the coleoptile and leaves, duced into callus tissue, which is Selected, then regenerated radicle and coleorhiza, Scutulum, Starchy endosperm, aleu to produce transgenic Seedlings, which are grown to trans rone layer, pericarp and/or testa, either during Seed matura genic Ro plants that produce transgenic R. Seed. tion and Seed germin.ation. 0128 FIG. 14 is a graphic depiction of the results of an 0.136. A “phenylpropanoid” compound is a compound analysis of Matairesinol in numerous transgenic rice lines having a 3 carbon Side chain on an aromatic ring-a transformed with four vectors containing four lignan genes, common structural element shared by all of the metabolites relative to untransformed control M202 and TP309 plants. in the monolignol pathway. 0129 FIG. 13 is a schematic depiction of an exemplary 0.137. A nucleic acid is “operably linked” when it is procedure used to detect lignan production in half Seeds of placed into a functional relationship with another nucleic transgenic rice, as a part of the process of Selecting trans acid Sequence. For example, DNA for a presequence or genic plants for further propagation in the greenhouse of secretory leader is operably linked to DNA for a polypeptide field. if it is expressed as a preprotein that participates in the 0130 FIG. 15 depicts an image of an ethidium bromide Secretion of the polypeptide; a promoter or enhancer is stained gel showing the results of electrophoresis of PCR operably linked to a coding Sequence if it affects the tran products derived by amplification of DNA derived from rice Scription of the Sequence; or a ribosome binding Site is plants transformed with the four heterologous nucleic acid operably linked to a coding Sequence if it is positioned So as constructs presented in FIGS. 6A, 6B, 6C, and 6D, DNA to facilitate translation. Generally, "operably linked” means from non-transformed plants, or control DNA from reduc that the DNA sequences being linked are contiguous, and, in tase, laccase, dehydrogenase and dirigent protein encoding the case of a Secretory leader, contiguous and in reading DNA constructs, by performing PCR using primerS Specific phase. However, enhancers do not have to be contiguous. to the reductase, laccase, dehydrogenase and dirigent protein Linking is accomplished by ligation at convenient restriction coding Sequences at the same time. Sites. If Such sites do not exist, the Synthetic oligonucleotide adaptorS or linkers are used in accordance with conventional DETAILED DESCRIPTION OF THE practice. PREFERRED EMBODIMENT 0.138. By “promoter” or “transcriptional or translational 0131) I. DEFINITIONS regulatory region' is meant a Sequence of DNA that directs or regulates transcription of a downstream gene heterolo 0132 Unless otherwise indicated, all terms used herein gous to the promoter, and includes promoters derived by have the same meaning as they would to one skilled in the means of ligation with operator regions, random or con art of the present invention. Practitioners are particularly trolled mutagenesis, addition or duplication of enhancer directed to Sambrook et al. (1989) Molecular Cloning: A Sequences, addition or modification with Synthetic linkers, Laboratory Manual (Second Edition), Cold Spring Harbor and the like. Press, Plainview, N.Y. and Ausubel F Metal. (1993) Current Protocols in Molecular Biology, John Wiley & Sons, New 0.139. The transcriptional and translational regulatory York, N.Y., for definitions and terms of the art. It is to be nucleic acid will generally be appropriate to the host cell understood that this invention is not limited to the particular used to express the target protein; for example, transcrip methodology, protocols, and reagents described, as these tional and translational regulatory nucleic acid Sequences may vary. from Specific plant tissues are preferably used to express the protein (e.g. enzyme) intermediates of the phenylpropanoid 0.133 “Chimeric gene” or "heterologous nucleic acid pathway protein in various Seed tissues. Numerous types of construct”, as defined herein refers to a non-native gene (i.e., appropriate expression vectors, and Suitable regulatory one which has been introduced into a host) which may be composed of parts of different genes, including regulatory Sequences are known in the art for a variety of host cells. elements. A chimeric gene construct for plant/seed transfor 0140. In general, the transcriptional and translational mation is typically composed of a transcriptional regulatory regulatory Sequences may include, but are not limited to, US 2002/0174452 A1 Nov. 21, 2002 promoter Sequences, ribosomal binding Sites, transcriptional protein or enzyme'. Such a “protein or enzyme variant' Start and Stop Sequences, translational Start and Stop includes, for example, the amin.o acid Sequence for laccase Sequences, and enhancer or activator Sequences. In a pre wherein one or more amin.o acid residues are added, or ferred embodiment, the regulatory Sequences include a pro deleted, at the N or C ternin.us of the Sequence, or Substi moter and transcriptional Start and Stop Sequences. Specific tuted for one or more amin.o acid residues within the types of promoters are defined below. sequence of SEQ ID NO: 12. Ordinarily, a “protein or enzyme variant” will have at least about 80% amin.o acid 0141 Suitable sources for gene sequences useful in the Sequence identity, preferably at least about 85% amin.o acid present invention are dicot or monocot plants, or bacteria or sequence identity, more preferably at least about 90% fungi. amin.o acid Sequence identity and even more preferably at 0142 “Seed development” refers to any seed condition, least about 95 or 98% amin.o acid sequence identity with the from fertilization to late-stage germin.ation, characterized amin.o acid Sequence of the native protein or enzyme. by induction of one or more enzymes in the Seed. Seed development refers to both Seed maturation and Seed ger 0.148 AS used herein, the term “sequence identity” means nucleic acid or amino acid Sequence identity in two or more min.ation. aligned Sequences, aligned using a sequence alignment 0143 A “seed developmental stage specific promoter” or program. Sequence Searches are preferably carried out using “seed inducible promoter' is one which is inducible in seeds, the BLASTN program when evaluating the of a given generally at particular Stages of Seed development, either nucleic acid Sequence relative to nucleic acid Sequences in under the control of endogenous factors present in the Seed, the GenBank DNA Sequences and other public databases. plant hormones, Such as abscissic or gibberellic acid, or The BLASTX program is preferred for searching nucleic physical Stimuli, e.g., heat and moisture. A Seed-induced acid Sequences which have been translated in all reading promoter may be "seed-specific', meaning it is induced frames against amin.o acid Sequences in the GenBank Pro preferentially in Seeds relative to other tissue (Knutzon et al., tein Sequences and other public databases. Both BLASTN 1992; Bustos et al., 1991; Lam et al., 1991; and Stayton et and BLASTX are run using default parameters of an open al., 1991), “seed tissue specific', meaning it is induced gap penalty of 11.0, and an extended gap penalty of 1.0, and preferentially in certain Seed cells, e.g., testa-layer cells, or utilize the BLOSUM-62 matrix. See, Altschulet al., 1997. “Seed-stage-Specific', meaning it is induced during certain Stages of Seed maturation or germin.ation. 0149. A preferred alignment of selected sequences in order to determine “% identity” between two or more 0144) “Stably transformed” as used herein refers to a Sequences, is performed using the CLUSTAL-W program in plant cell or plant or Seed that has foreign nucleic acid MacVector, operated with default parameters, including an integrated into its genome which is maintained through at open gap penalty of 10.0, an extended gap penalty of 0.1, least two or more generations. and a BLOSUM 30 similarity matrix. 0145 A“gene encoding a protein” refers to DNA encod 0150. A nucleic acid sequence is considered to be “selec ing a protein having a specified function in plant cells. tively hybridizable' to a reference nucleic acid Sequence if Examples include the representative enzyme-coding the two Sequences Specifically hybridize to one another Sequences disclosed herein (e.g., a gene or gene family under high Stringency hybridization and wash conditions. A encoding an enzyme or protein which functions in the plant nucleic acid Sequence is considered to be “selectively phenylpropanoid pathway, including laccase (SEQ ID NO: hybridizable' to a reference nucleic acid Sequence if the two 11; FIGS. 7A, 7B, and 7C), a dirigent protein (SEQ ID NO: Sequences specifically hybridize to one another under mod 15; FIGS. 8A, 8B, and 8C), for pinoresinol/lariciresinol erate to high Stringency hybridization and wash conditions. reductase (SEQ ID NO: 19, FIGS. 9A, 9B, and 9C), and Hybridization conditions are based on the melting tempera secosisolariciresinol dehydrogenase (SEQID NO:23; FIGS. ture (Tm) of the nucleic acid binding complex or probe. For 10A, 10B, and 10C), and sequences have appropriate levels example, "maximum Stringency' typically occurs at about of Sequence identity to or capable of hybridizing under high Tm-5°C. (5° below the Tm of the probe); “high stringency” Stringency conditions to those described herein. at about 5-10 below the Tm; “intermediate stringency” at 0146 A“native protein or enzyme” means a protein and about 10-20 below the Tm of the probe; and “low strin enzyme represented by the amin.o acid Sequence and in the gency” at about 20-25 below the Tm. Functionally, maxi form endogenous to the plant tissue in which it is found in mum Stringency conditions may be used to identify nature. Such a protein or enzyme is encoded by the nucleic Sequences having Strict identity or near-strict identity with acid Sequences which encodes the native protein and has a the hybridization probe, while high Stringency conditions biological activity as it is found in nature. An example of are used to identify Sequences having about 80% or more Such a native enzyme is laccase found in docot or monocot Sequence identity with the probe. plants, having the amin.o acid Sequence Set forth in SEQ ID 0151 High stringency hybridization conditions are well NO: 12, encoded by the nucleic acid sequence set forth in known in the art (see, for example, Sambrook, et al. (1989) SEQ ID NO: 11, and which functions together with dirigent Chapters 9 and 11, and in Ausubel, F. M., et al., 1993, protein to facilitate the Stereospecific conversion of E-co expressly incorporated by reference herein). An example of niferyl alcohol to the 8.8-linked dimeric lignan, (+)-pinores high Stringency conditions includes hybridization at about inol. 42 C. in 50% formamide, 5xSSC, 5xDenhardt's solution, 0147 A “protein or enzyme variant” means a protein or 0.5% SDS and 100 lug/ml denatured carrier DNA followed enzyme which comprises the biological activity of the native by washing two times in 2xSSC and 0.5% SDS at room protein or enzyme and is further defined as having at least temperature and two additional times in 0.1xSSC and 0.5% about 80% amin.o acid sequence identity with the “native SDS at 43° C. US 2002/0174452 A1 Nov. 21, 2002

0152. As used herein, the term “expression” refers to the correspond to the terminology used to indicate lignan type. proceSS by which a polypeptide is produced based on the Thus, lignans derived from coumaryl alcohol are referred to nucleic acid Sequence of a gene. The proceSS includes both herein as H-lignans, lignans derived from coniferyl alcohol transcription and translation. are referred to as G-lignans, and lignans derived from 0153. As used herein, the terms “transformed”, “stably Sinapyl alcohol are referred to as S-lignans. Pathways com transformed” or “transgenic” with reference to a plant cell peting with the G-lignan pathway are discussed herein means the plant cell has a non-native (heterologous) nucleic primarily in the context of their impact on the formation of acid Sequence integrated into its genome which is main G-lignans. tained through two or more generations. 0.161. A summary of each of the pertinent enzymes in the 0154) “G-lignans', as referred to herein, are lignans Monolignol Pathway and the enzymes necessary for con derived from coniferyl alcohol or its precursors, as illus Verting phenylalanine to each of the monolignols, is pro trated in FIG. 1. The G-lignans are formed from coniferyl vided in below. alcohol in a Series of bimolecular radical coupling reactions, via a branch point in the biosynthetic pathway which leads 0162 A1. PHENYLALANINE AMMONIA to G-lignans rather than to H or Slignans, or the polymeric LYASE metabolite of any one of G, H or Slignans, namely, lignins. Preferred G-lignans include (+)-pinoresinol, (+)-laricires 0163 Phenylalanine ammonia-lyase, (PAL), is the inol, (-) Secoisolariciresinol-isolariciresinol, (-) Secoisolar enzyme which catalyzes the first reaction in the general iciresinol diglucoside, and (-)-matairesinol, that is, those phenylpropanoid pathway, and is often referred to as the lignans derived from coniferyl alcohol up to and including entry point into plant phenylpropanoid metabolism. PAL matairesinol. catalyzes the deamin.ation of phenylalanine to form cin namate and ammonia (which is most likely recaptured O155 II. LIGNAN PATHWAYS within the plant), as indicated in FIG. 1. 0156 The plant biochemical pathways leading to E-co niferyl alcohol has been described as the “front end” of the 0164. PAL exists as a tetramer in vascular plants (Hanson plant phenylpropanoid pathway (FIG. 1) and the pathway and Havir, 1981; Jones, 1984), and PAL subunits are typi from E-coniferyl alcohol to the lignans has been designated cally encoded by multigene families in angiosperms, the “back end” of the plant phenylpropanoid pathway (FIG. depending on the Species. Coding Sequences for PAL and 2). The present invention is directed to modifying the other plant enzymes described herein are discussed in phenylpropanoid pathway in monocots, particularly G-lig greater, below. nan expression in Seeds, by altering the expression of various genes which regulate levels of lignan precursors and 0165 A2. CINNAMATE 4-HYDROXYLASE lignans, Suitable for expression in developing, maturing or 0166 The enzyme, cinnamate 4-hydroxylase (C4H), is a germin.ating Seeds as further described below. cytochrome P-450-linked monooxygenase which functions O157 A. THE MONOLIGNAN BIOSYNTHETIC to hydroxylate cinnamate at the para ring position, to form PATHWAY FRONT END the mono-hydroxylated compound, para-coumarate. The enzyme acts by cleaving molecular oxygen, followed by 0158. The plant biochemical pathway leading to addition of one oxygen atom to the aromatic ring and coniferyl alcohol (and other monolignols as well) is referred reduction of the other oxygen atom to water. It has been to as the Monolignol Pathway, based on conversion involv Suggested that the Substrate cinnamate is transferred to C4H ing non-dimeric phenylpropanoid monomers. The Monoli from PAL by a process known as metabolic channeling gnol Pathway is part of a Series of enzyme-catalyzed reac (Hrazdina and Wagner, 1985), a mechanism in which inter tions in plants referred to as phenylpropanoid metabolism, mediates in a metabolic pathway are transferred between which is utilized by plants to synthesize a number of enzymes via a multi enzyme complex. different Secondary products or metabolites. 0167 A3. CAFFEATE O-METHYLTRANS 0159. In this multistep process, phenylalanine is con FERASE verted to coniferyl alcohol and two other monolignols, para-coumaryl alcohol and Sinapyl alcohol, as shown in 0168 Caffeate O-Methyltransferase (C-OMT) catalyzes FIG. 1. The Monolignol Pathway is the starting point for the methylation of the 3-hydroxy group of caffeic acid to forming not only monolignols, but also for forming lignins, form the corresponding 3-methoxy compound, ferulic acid. lignans, flavenoids, isoflavenoids, coumarins, Soluble esters The C-OMT-promoted methylation reaction limits the reac and Stilbenes, whose formation also proceeds through at tivity of the 3-hydroxy group, thereby reducing the number least a portion of this shared pathway. of Sites on the aromatic ring that can form bonds to other monolignols. 0160 AS indicated in FIG. 1, upon formation of each of the three monolignols, these compounds may either proceed 0169 C-OMT utilizes S-adenosylmethionine to provide to form dimeric lignans or may go on to polymerize to form the methyl donor group, and this Same enzyme is also lignin. The three monolignols each give rise to different believed to catalyze the methylation of 5-hydroxyferulate to types of lignin. Polymerization of para-coumaryl alcohol Sinapate, as indicated in FIG. 1. Thus, C-OMT catalyzes Subunits leads to formation of H-lignin, polymerization of reactions leading to the formation of both coniferyl and coniferyl alcohol leads to formation of G-lignin, and poly Sinapyl alcohols, with the preference for Substrate differing merization of Sinapyl alcohol leads to formation of S-lignin. among different plant species (Shumada et al., 1973; Kuroda The names of each of the lignan pathways as used herein et al., 1981). US 2002/0174452 A1 Nov. 21, 2002

0170 A4. FERULATE 5-HYDROXYLASE 0180 A8. CAFFEOYL-COENZYME A O-ME THYLTRANSFERASE 0171 Ferulate 5-hydroxylase, (F5H), is another cyto chrome P450-linked monooxygenase, functioning to 0181 Caffeoyl-Coenzyme A O-Methyltransferase, hydroxylate ferulate to the corresponding dihydroxy, meth (CCoA-OMT), plays a role in methylating both caffeoyl oxy-Substituted aromatic compound, 5-hydroxy ferrulate. CoA and 5-hydroxyferuloyl-CoA, thereby contributing to The reaction catalyzed by this enzyme represents a branch formation of both coniferyl and Sinapyl alcohol and their point in the monolignol pathway, leading to formation of corresponding downstream lignan products. non-G lignan products, and more Specifically, to Sinapyl 0182) A9. CINNAMOYL-COENZYME A alcohol derivatives (i.e. S-lignans). REDUCTASE 0172 A5. 4-COUMARIC ACID COENZYME 0183 The enzyme, cinnamoyl-Coenzyme A reductase, LIGASE (CCR), is a non-specific enzyme which catalyzes the reduc tion of hydroxycinnamoyl-CoA thioesters (e.g., para-cou 0173 The enzyme 4-coumaric acid coenzyme ligase maroyl-CoA, feruloyl-CoA, 5-hydroxyferuloyl-Coa, and (4Cl) catalyzes the formation of CoA thioesters of cinnamic Sinapoyl-CoA) to the corresponding aldehydes. In general, acids in the biosynthesis of a wide variety of phenolic this enzyme does not exhibit much Specificity for one derivatives, including benzoic acid, condensed tannins, fla thioester Substrate over another, although feruloyl-CoA is venoids, and the cinnamyl alcohols. The activity of 4C1 is reported to be the best substrate for CCR in Soybean dependent upon ATP, and thioester formation proceeds (Wengenmayer et al., 1976), with the product of this reaction through an intermediate acyl adenylate which reacts with (i.e., coniferaldehyde) being the immediate precursor of CoA to form the thioester (Whetten et al., 1995). As indi coniferyl alcohol. cated in FIG. 1, 4C1 catalyzes formation of CoA thioesters from the Substrates para-coumarate, caffeate, ferulate, and 0184 A10. CINNAMOYL ALCOHOL DEHY 5-hydroxyferulate, and Sinapate and thus contributes to DROGENASE forming each of the monolignols, para-coumaryl alcohol, 0185. The final step in the Monolignol Pathway, i.e., the coniferyl alcohol, and Sinapyl alcohol. reduction of hydroxycinnamaldehydes to hydroxycinnamyl alcohols (the monolignols), is catalyzed by cinnamoyl alco 0.174 A6. CHALCONE SYNTHASE hol dehydrogenase, (CAD). In addition to regulation by plant developmental pathways, CAD is expressed in 0175 Chalcone synthase, (CHS), represents the initial response to stress (Galliano et al., 1983, Campbell et al., enzyme for entry into the flavenoid pathway, at a branch 1992a) and wounding. In contrast to CCR, CAD has been point of the phenylpropanoid pathway which leads, not to reported to display different Substrate affinities depending lignans or lignins, but to flavenoid compounds composed of upon the plant Species, although in general, angiosperm a basic 15-carbon Skeleton. CAD preparations tend to exhibit equal activities with 0176 Specifically, chalcone synthase (CHS) catalyzes coniferaldehyde and sinapylaldehyde (Gross, 1985). the production of a chalcone via condensation of three 0186 B. THE PLANT “G-LIGNAN” PATHWAY molecules of malonyl-Coenzyme A and one molecule of para-coumaroyl-Coenzyme A. The ultimate products of this 0187. The back end of the G-lignan pathway involves a biosynthetic pathway are anthocyanins, glycosylated fla Series of reactions Subsequent to monolignol formation, Venoid pigments which play many diverse roles in plants. originating with the monolignol, coniferyl alcohol. An over View of the G-lignan pathway, and in particular, reactions 0177. The reaction of para-coumaroyl-CoA with CHS pertinent to the formation of matairesinol and Secoisolar represents a branch point in the phenylpropanoid pathway, iciresinol, are shown in FIG. 2. leading to formation of non-G lignan products. In fact, the 0188 Once formed, coniferyl alcohol can react via two Substrate para-coumaroyl-CoA can react with three different different metabolic pathways. One pathway, the lignin path enzymes, with the hydroxylation reaction catalyzed by way, leads to formation of lignins via Oxidation of coniferyl CCoA-3H leading to the eventual formation of the monoli alcohol, followed by a Series of coupling reactions which gnols, coniferyl and Sinapyl alcohol, and the reduction ultimately give rise to the macromolecular racemic catalyzed by cinnamoyl-Coenzyme A reductase (CCR, G-lignins. Lignins may contain mixtures of one or more of described in section A.9 below) leading to formation of the H, G and S monolignol subunits. The various types of third monolignol, para-coumaryl alcohol. ligninS are characterized by the different relative amounts of the monolignol Subunits, e.g. G-S lignins or S-lignins. 0.178 A7. COUMARYL-COA 3-HYDROXY LASE 0189 An alternative pathway, the G-lignan pathway, involves stereoselective dimerization of coniferyl alcohol to 0179 Coumaryl-CoA 3-hydroxylase, (CCoA-3H) is an form the lignan, (+)pinoresinol, followed by Subsequent enzyme which also acts upon the Substrate, para-coumaroyl Stereospecific conversions of (+)pinoresinol to form various CoA, facilitating incorporation of a hydroxyl group at the 3 optically active G-lignans (+)lariciresinol, (-)Secoisolar position of the aromatic ring to form the corresponding iciresinol, (-) Secoisolariciresinol-isolariciresinol digluco dihydroxy compound, caffeoyl-CoA. The intermediate Side and (-)matairesinol). Although not shown, further formed by action of this enzyme, caffeoyl-CoA, is a pre metabolism of matairesinol presumably affords lignans Such cursor to a metabolite which provides direct entry into the as (-)trachelogenin and (-)podophyllotoxin, and also G-lignan pathway. affords entry into various lignan Subclasses. US 2002/0174452 A1 Nov. 21, 2002

0.190 Proteins and enzymes specific to the biosynthesis 0202) III. MODIFIED G-LIGNAN LEVELS IN MONO of G-lignans, (i.e., those which act specifically on coniferyl COT SEEDS/STRATEGIES TO INCREASE LIGNAN alcohol or its lignan metabolites), and those at branch points PRODUCTION in the phenylpropanoid pathway leading to competing path 0203 As indicated in FIG. 3 human lignans are derived ways, find utility in the methods described herein. from plant lignans through the action of colonic bacteria. 0191 The sequential conversion of E-coniferyl alcohol to The plant lignan, Secoisolariciresinol, is converted to (+)pinoresinol, (+)lariciresinol, (-)Secoisolariciresinol, enterodiol and then to enterolactone while matairesinol is (-)secoisolariciresinol diglucoside, and finally (-)mataires converted to enterolactone directly. Recently, the pathway inol is facilitated by dirigent protein, pinoresinol/laricires for matairesinol production in Forsythia intermedia has inol reductase, Secoisolariciresinol diglucosyl transferase, been elucidated (FIG. 2) and a number of genes which and Secoisolariciresinol dehydrogenase, respectively. encode enzymes responsible for lignan biosynthesis in the phenylpropanoid pathway have been cloned, laying the 0.192 B1. “DIRIGENT PROTEIN" (DIRG) foundation for the metabolic-engineering of lignans in crop 0193 The first step in the G-lignan pathway is the plants. (See e.g., Davin et al., 2000.) stereospecific conversion of E-coniferyl alcohol to the 8.8- 0204. The present invention is applicable to the genera linked dimeric lignan, (+)-pinoresinol. The reaction is facili tion of monocots exhibiting modified levels of G-lignans in tated by a Stereospecific coupling agent referred to as a Seed tissues in which they are typically found. The Specific “dirigent protein', (Lewis et al., WO 98/20113), meaning a Seed tissue and/or maturation State in which G-lignans are protein which aligns or guides (Davin et al., 1997). The typically found may vary between plant species. Using the enzyme has been characterized as a 78-kD protein lacking a methods of the present invention, a G-lignan, e.g., mataires catalytically active oxidative center. The dirigent protein inol, is produced in Seeds of transformed plants in an amount serves to bind and orient two coniferyl alcohol-derived free greater than the amount found in Seeds of untransformed radicals arising from a one electron oxidation (typically plants. In other words, the concentration of any one of the resulting from action of an oxidase), which then undergo above-described G-lignans in transformed Seeds exceeds the Stereoselective coupling to form (+)-pinoresinol. In model in amount found in untransformed Seeds, when assayed by a Vitro Studies, the dirigent protein was shown to possess conventional assay System for detecting lignan(s), for marked substrate specificity for E-coniferyl alcohol over example, gas chromatography-mass spectroscopy (GC-MS, Sinapyl and para-coumaryl alcohols (Davin et al., 1997), GC-MS-SIM) or high performance liquid chromatography making this protein a preferred target for up-regulation in (HPLC). monocot Seeds according to the methods described herein. 0205. In practicing the method, chimeric genes or heter 0194 B2. LACCASE (LACC) ologous nucleic acid constructs containing the coding Sequence for one or more proteins or enzymes integral to the 0.195 The first step in the G-lignan pathway is the phenylpropanoid biosynthetic pathway for production of stereospecific conversion of E-coniferyl alcohol to the 8.8- G-lignans are introduced into a target plant to alter produc linked dimeric lignan, (+)-pinoresinol. The reaction is facili tion of G-lignans. In making Such alterations, it is generally tated by dirigent protein, together with laccase. preferred that the gene target, promoter and tissue be endog enous to the plant of interest, and that expression occur in a 0196) B3. PINORESINOL/LARICIRESINOL tissue and developmental Stage consistent with that normally REDUCTASE (REDS) found in nature. Therefore, preferred Strategies for increas 0197) The sequential conversion of (+)pinoresinol into ing lignan content in monocot Seeds take advantage of (+)lariciresinol and (-)-secoisolariciresinol is facilitated by existing biosynthetic pathways utilized by the native (wild the enzyme, pinoresinol/lariciresinol reductase. Two iso type) plant, that are Seed tissue and/or developmental stage forms of the reductase have been isolated from F intermedia Specific. Preferred protein and enzyme targets for inclusion (Kostova, et al., 1996; Lewis, et al., WO 98/20113), and both in chimeric gene constructs used to produce the transgenic act to catalyze the Sequential reduction of (+)pinoresinol to monocot plants and Seeds of the present invention are those (+)lariciresinol, and (+)lariciresinol to (-)-secoisolaricires which facilitate rate limiting Steps in the phenylpropanoid inol, i.e., each isoform is capable of catalyzing the reaction pathway leading to G-lignan formation, examples of which of both Substrates. The products of these reactions, laricir include dirigent protein; laccase; pinoresinol/lariciresinol esinol and Secoisolariciresinol, can be either (+) or (-). Both reductase (reductase) and Secosisolariciresinol dehydroge isoforms have comparable molecular masses of -34.9 kD. nase (dehydrogenase). 0206 Exemplary monocots for introduction of the chi 0198 B4. SECOISOLARICIRESINOL DEHY meric gene constructs of the present invention include, but DROGENASE (DEHY) are not limited to, wheat, maize, barley, rice, millet, oats, 0199 The conversion of (-)-secoisolariciresinol into (-)- rye, triticale, Sorghum, and corn. matairesinol is facilitated by the enzyme, Secoisolaricires 0207. The steps involved in modification of lignan levels inol dehydrogenase. in monocot Seeds include, but are not limited to, the fol 0200 B5. SECOISOLARICIRESINOL DIGLU lowing; (1) determining in which seed tissue G-lignans are COSYL TRANSFERASE found by analysis of G-lignans, e.g. lariciresinol and Sec oisolariciresinol diglucoside, in various plant tissues and at 0201 The conversion of (-)-secoisolariciresinol into (-)- various Stages of development for the target plant Species, Secoisolariciresinol diglucoside is facilitated by the enzyme, followed by; (2) Selecting an appropriate promoter based on Secoisolariciresinol diglucosyl transferase. the target Seed tissue and Seed developmental stage; and (3) US 2002/0174452 A1 Nov. 21, 2002 assembling a chimeric gene construct including the pro and endosperm, without Significantly interfering with plant moter, the target gene and appropriate regulatory elements growth and development, or phenotypic characteristics of necessary for expression in Seeds of a monocot plant. the plant or Seed. 0208 Candidate target genes include those which encode 0215. The present invention provides transformed mono proteins integral to the biosynthetic pathway leading to cot plants and methods of making them wherein the plants G-lignan formation, as described above, and will be found in produce higher levels of G-lignan precursors or G-lignans, the Seed tissue in which G-lignans are normally found. at levels and in Specific tissues that allow the plant to Exemplary target enzymes for modification include, but are develop normally and without interfering with Seed produc not limited to dirigent protein; laccase; pinoresinol/laricir tion levels. Chimeric genes including DNA sequences eSinol reductase, and SecoSisolariciresinol dehydrogenase. encoding one or more proteins integral to the phenylpro panoid pathway leading to G-lignan formation, plant expres 0209. A typical chimeric gene of the present invention, Sion vectors, monocot plant cells bearing Such chimeric for transformation into a plant, may include a transcriptional genes or transformed by Such expression vectors, trans regulatory region inducible during Seed development (e.g., a formed monocot plants and Seeds having elevated levels of Seed tissue Specific promoter Such as a Seed-induced pro G-lignans (e.g., Secoiso-lariciresinol, matairesinol), are thus moters include the barley B-amylase (Kreis, et al., 1987) and provided. Seeds from Such transformed monocot plants may f-glucanase gene promoters (Wolf, 1992), or a Seed devel upon mammalian consumption, provide associated positive opmental Stage specific promoter, Such as a promoter for the health benefits, as further detailed below. rice glutelin multigene family, Gt1, Gt2, Gt3, Glu A-3, and GluB-1, described, for example, in Takaiwa, et al., 1991a, 0216) More specifically, one aspect of the present inven 1991b; Okita, et al., 1989; Abe et al., 1989; Kim et al., 1990. tion is directed to a method of increasing G-lignan content Such a typical chimeric gene will also include a protein in monocot Seeds by up-regulating in developing, maturing, coding Sequence, and a termin.ator Sequence. or germin.ating Seeds, one or more of the following proteins or enzymes: dirigent protein; laccase; pinoresinol/laricires 0210 Preferred promoters for use in the present invention inol reductase (reductase); and SecoSisolariciresinol dehy include, for example, known tissue Specific promoters for drogenase (dehydrogenase), all of which are integral to the the Seed tissue and/or developmental Stage in which G-lig branch of the phenylpropanoid pathway leading to formation nans are normally found, preferably promoters derived from of monolignols or dimeric lignan precursors and G-lignans. the same plant species as the plant in which the chimeric Specifically, the production of one or more of the above gene construct will be introduced, and native (wild type) proteins is up-regulated by introducing into a monocot plant promoters for the gene encoding the target protein. or plant cell, a chimeric gene having (i) a Seed tissue or Seed 0211 Expression of native (wild type) genes for the developmental Stage specific transcriptional regulatory various enzymes and proteins of the phenylpropanoid bio region induced during Seed development, maturation or Synthetic pathway may yield varying levels of transcript germin.ation (e.g., a Seed Specific promoter), and (ii) a gene depending upon the tissue type and developmental Stage of encoding a protein integral to the lignan biosynthetic path the plant in which they are expressed. way operably linked to the transcriptional regulatory region. 0212. The methods of the invention include stably trans 0217. In an alternative approach, the production of one or forming a monocot plant with a chimeric gene having (1) an more of the above proteins may be up-regulated by intro appropriate promoter, as described above, (2) a first DNA ducing into a monocot plant or plant cell, a chimeric gene Sequence encoding a protein integral to the lignan biosyn having (i) the endogenous promoter for the target protein, thetic pathway, (3) control elements necessary for expres operably linked to (ii) multiple copies of a gene encoding Sion of the protein coding Sequence, and may further include one of the above-described proteins. The protein is prefer (4) a Second DNA sequence encoding a signal polypeptide. ably expressed in plant tissueS which normally produce In Such cases, the Second DNA sequence is operably linked lignans by using a transcription regulatory region specific to the transcriptional regulatory region and the first DNA for various Seed tissues, Such as the Seed testa layer, peri Sequence, and the Signal polypeptide are in translation-frame carp, aleurone and endosperm. with the protein coding Sequence. Expression of form of the 0218. In a preferred approach, the coding sequence for protein having a Signal Sequence is effective to facilitate multiple proteins integral to the lignan biosynthetic pathway Secretion of the protein across the aleurone or Scutellar are expressed in the same plant cell at the same time, e.g., epithelium layer of Seeds into the endosperm. the coding Sequence for dirigent protein; laccase; pinores 0213) IV. METHODS AND COMPOSITIONS OF THE inol/lariciresinol reductase (reductase); and SecoSisolaricir INVENTION esinol dehydrogenase (dehydrogenase). 0219. In a related aspect, the invention includes a mono 0214. The phenylpropanoid pathway that leads to lignan cotyledonous plant capable of producing Seeds with elevated formation has three branches at the point of formation of para-coumaryl alcohol, coniferyl alcohol and Sinapyl alco G-lignan content, and monocot Seeds produced by the plant. hol, leading to “H”, “G” and “S” lignans, respectively. Using 0220 Also disclosed is a seed composition for use in the methods of the present invention, it is possible to Stereospecifically converting E-coniferyl alcohol to G-lign introduce a chimeric gene construct containing a DNA ans, e.g. (+)-pinoresinol, (+)-lariciresinol, (-)-secoisolaricir Sequence encoding a protein integral to the phenylpropanoid esinol, (-)-secoisolariciresinol diglucoside, and in particu pathway leading to G-lignan formation into monocot plants, lar, to (-)-matairesinol (see FIG. 2). The seed composition and increase the G-lignan content in Seeds, or within certain contains Seeds from a monocot plant which has been Stably Seed tissues, Such as the Seed testa layer, pericarp, aleurone transformed and contain a gene encoding one or more of the US 2002/0174452 A1 Nov. 21, 2002 following proteins: a dirigent protein together with laccase 0229 Expression vectors for use in this aspect of the capable of converting E-coniferyl alcohol Stereospecifically invention comprise a chimeric gene construct (or expression to a Selected enantiomer of pinoresinol; pinoresinollaricir cassette), designed for operation in plants, particularly eSinol reductase, and SecoSisolariciresinol dehydrogenase, monocot plants, with companion Sequences upstream and operably linked to a Seed-specific promoter or their respec downstream from the expression cassette. The companion tive endogenous promoters. Due to Selective expression of Sequences will be of plasmid or viral origin and provide the transgenes in Seed tissues, the resulting Seed composition necessary characteristics to the vector to permit the vectors is effective to produce significant amounts of one or more of to move DNA from bacteria to the desired plant host. the above-described optically active G-lignans, which are 0230. A monocot plant may be stably transformed with a derived from coniferyl-alcohol. chimeric gene having (i) a promoter, for example, a native 0221) In a related aspect, a method of forming a lignan promoter for the target gene, a Seed tissue Specific promoter; enriched Seed composition is disclosed. or a seed-developmental Stage Specific promoter induced 0222. In one alternative approach, Over expression of during Seed development, maturation or germin.ation, and Selected gene products integral the G-lignan biosynthetic (ii) a gene sequence whose expression ultimately results in pathway is carried out together with inhibition of the expres production of G-lignans, or whose expression is effective to sion of proteins which facilitate formation of metabolites block formation of a metabolite of a competing biosynthetic other than G-lignans in order to produce elevated levels of pathway or branch point which does not lead to formation of G-lignans in the Seeds of transgenic monocot plants. G-lignans. 0223) In another alternative approach, the expression of 0231. The expression construct also utilizes additional Selected gene products integral to the front end of the regulatory DNA sequences e.g., preferred codons, termin.a- phenylpropanoid biosynthetic pathway is up-regulated by tion Sequences, to promote efficient translation of the gene introduction and Stable expression of chimeric gene con encoding the protein, as will be described. Structs as a means to increase G-lignan levels in monocot 0232. In addition to encoding the protein of interest, the Seeds including, but not limited to, one or more of (1) chimeric gene expression cassette may encode a signal phenylalanine ammonia lyase (PAL); (2) cinnamate-4-hy peptide that allows processing and translocation of the droxylase (C4H); and (3) caffeate-3-hydroxylase (C3H). protein, as appropriate. Preferred Signal Sequences are those 0224. In a further alternative approach, the expression of corresponding to the RAmy3D rice promoter and Ramy1A Selected gene products integral to the front end of the promoter, respectfully. A plant Signal Sequence is placed in phenylpropanoid biosynthetic pathway is downregulated, frame with a heterologous nucleic acid encoding a peptide or including, but not limited to: (1) ferulate-5-hydroxylase protein Such that Signal peptidase cleavage occurs precisely (F5H); and (2) chalcone synthase (CS). at the Start of the mature peptide. 0225. Following introduction of a chimeric gene con 0233. The expression cassette or chimeric gene(s) in the Struct containing the coding Sequence for a gene product transformation vector typically have a transcriptional termi integral to the G-lignan biosynthetic pathway, plant tissue nation region at the opposite end from the transcription may be analyzed for proteins and nucleic acids indicative of initiation regulatory region (promoter), essentially as modified G-lignan expression, as further described below. defined above. The transcriptional termin.ation region may 0226 C. CHIMERIC GENE CONSTRUCTS normally be associated with the transcriptional initiation 0227. In order to increase the absolute or relative con region or from a different gene. The transcriptional termi centrations of G-lignans in plant Seeds, the expression of nation region may be selected, particularly for Stability of various intermediates integral to the G-lignan biosynthetic the mRNA to enhance expression. Illustrative transcriptional pathway are modified by growing a plant transformed with termin.ation regions include the NOS termin.ator from an expression vector containing a chimeric gene construct Agrobacterium Tiplasmid and the rice B-amylase termin.a- under the appropriate conditions to induce or cause expres tor. Sion of the protein (e.g. dirigent protein or an enzyme). The 0234 Polyadenylation tails, (Alber et al., 1982) are also conditions appropriate, for example, for enzyme expression commonly added to the expression cassette to optimize high will vary with the choice of the expression vector, the target levels of transcription and proper transcription termin.ation, plant tissue and developmental Stage, and will be easily respectively. Polyadenylation Sequences include, but are not ascertained by one skilled in the art through routine experi limited to, the Agrobacterium octopine Synthetase signal, mentation. For example, the use of constitutive promoters in Gielen et al., 1984, or the nopaline Synthase of the same the expression vector will require optimizing the growth and Species, Depicker et al., 1982. proliferation of the plant cell, while the use of an inducible promoter requires the appropriate growth conditions for 0235 Since the ultimate expression of the chimeric gene induction. For example, if the promoter is developmental construct will be in a eukaryotic cell (in this case, a member Stage specific (e.g. inducible during Seed development), the of the grass family), it is desirable to determine whether any appropriate environmental Stimulus will be required for the portion of the cloned gene contains Sequences which will be promoter to be induced and the chimeric gene construct to processed out as introns by the host's splicing machinery. If be expressed, e.g., Sugar depletion in culture or water uptake So, Site-directed mutagenesis of the “intron” region may be followed by gibberellic acid production in germin.ating conducted to prevent losing a portion of the genetic message Seeds. as a false intron code (Reed et al., 1985). 0228 Numerous strategies may be pursued to increase 0236 Chimeric gene constructs containing a gene encod lignan production in Seeds, including, but not limited to, the ing a protein integral to the phenylpropanoid biosynthetic following. pathway may also include a Selectable marker for use in US 2002/0174452 A1 Nov. 21, 2002

plant cells. Such Selectable markers may be either on the heat or moisture, or the presence or absence of a Small Same plasmid or in the form of Separate plasmids, including, molecule, Such as the reduction or depletion of Sugar, Such but not limited to, the npt kanamycin resistance gene, for as Sucrose, in culture medium, or in plant tissues, Such as Selection in kanamycin-containing media, or the phosphi germin.ating Seeds. In this way, chimeric gene constructs nothricin acetyltransferase gene, for Selection in medium affecting plant regulatory and developmental pathways are containing phosphinothricin (PPT). expressed preferentially and preferably Solely in Seeds of the 0237) The vectors may also include sequences that allow plant, thereby allowing the plant to develop without signifi their Selection and propagation in a Secondary host, Such as, cantly interfering with the phenylpropanoid pathway in Sequences containing an origin of replication and a Select other plant tissues and organs. Seed-specific expression is able marker Such as antibiotic or herbicide resistance genes. also desirable since the naturally-occurring plant lignans, Typical Secondary hosts include bacteria and yeast. In a Secoisolariciresinol diglucoside and matairesinol, are found representative approach for forming an expression vector predominantly in the Outer Seed layers of representative for use in the present invention, the Secondary host is cereals such as rye (Nilsson et al., 1997). Thus, the patterns Escherichia coli, the origin of replication is a col. 1-type, of lignan production in transformed plants will be designed and the Selectable marker is a gene encoding amplicillin to closely follow those of wild-type plants, to produce as resistance. Such Sequences are well known in the art and are little phenotypic variation as possible (outside of enhanced commercially available as well (e.g., Clontech, Palo Alto, G-lignan biosynthesis) in transformed monocot seeds rela Calif.; Stratagene, La. Jolla, Calif.). tive to native Seeds. 0238 A vector for use in the present invention may also 0245 Promoters for use in the present invention are be modified to form an intermediate plant transformation preferably derived from cereals Such as rice, barley, wheat, plasmid that contains a region of homology to an Agrobac oat, rye, millet, triticale, Sorghum, and corn. terium tumefaciens vector, a T-DNA border region from 0246 Alternatively, a seed-specific promoter from a non Agrobacterium tumefaciens, and chimeric genes or expres cereal monocot may be used, and modified according to Sion cassettes (described above). Further, the vector may techniques well known in the art for Seed-Specific expression comprise a disarmed plant tumor inducing plasmid of Agro of the enzyme-coding Sequences described herein. Exem bacterium tumefaciens. plary promoters are described below. 0239). The appropriate nucleic acid sequence may be 0247. In determining how the chimeric gene constructs inserted into the vector by a variety of procedures known to encoding proteins integral to the phenylpropanoid pathway those of skill in the art. In general, DNA is inserted into an will be expressed, relevant factors include the type of appropriate restriction endonuclease site(s) using techniques promoter, the temporal pattern of the promoter (e.g. with known in the art. Construction of Suitable vectors containing respect to the developmental Stage of the plant or the plant the components Set forth above employs Standard ligation tissue), and the operation of the promoter based on its techniques which are known to the skilled artisan. position within the genome. A promoter which is expressed 0240 The construction of such expression vectors is concurrently with or prior to the normal activation of the generally known in the art. Specific examples provided here native gene Sequence is preferred. Alternatively, the protein include expression vectors comprising the coding Sequence may be made at a significantly higher concentration than is for laccase, dirigent protein, lariciresinol reductase and normally Seen, through the use of a inducible promoter or Secoisolariciresinol dehydrogenase, as further described in high expression promoter, Such that the protein is made at Example 2. increased concentration levels. 0241 B. PROMOTERS 0248 B2. SEED TISSUE SPECIFIC 0242 Transcriptional regulatory or promoter Sequences 0249. The present invention takes advantage of promot for use in the methods described herein may be either erS Specific to the various Seed tissues, including, but not constitutive or inducible promoters. The promoters may be limited to the pericarp, testa layer, aleurone and the either naturally occurring promoters or hybrid promoters. endosperm. Such Seed tissue specific promoters may also be Hybrid promoters, which combine elements of more than expressed at varying levels depending upon the develop one promoter, are known in the art, and may be used in mental stage of the seed (Okita et al., 1989). practicing the present invention. 0250 Promoters from seed tissue specific genes such as 0243 B 1. KNOWN PROMOTERS those described in Muller and Knudsen, 1993, the contents 0244 Promoters for regulating expression of any one of of which is expressly incorporated herein by reference, as the herein described genes are preferably Seed-specific pro well as the references contained therein, are Suitable for use moters, that is, promoters which induce transcription of a in the chimeric gene constructs of the present invention. downstream coding Sequence preferentially in Seeds, rela 0251 Representative seed tissue specific promoters tive to other types of plant tissues. Moreover, the Seed include promoters that direct endosperm-specific expression Specific promoter may be seed-tissue Specific, i.e., induced preferentially in a particular Seed tissue Such as the Seed testa (Takaiwa et al., 1991a, 1991b; Okita et al., 1989; Abe et al., layer, pericarp, aleurone or endosperm, and/or Seed devel 1989; Kim et al., 1990). opmental Stage Specific, i.e., induced in Seeds during a 0252). During germin.ation of the barley grain, two ul-glu certain Stage of Seed development, in response to either canase isozymes (E.C.3.2.1.73) are synthesized in the aleu endogenous factors present in the Seed, plant hormones, rone (isozyme II) and in the aleurone and Scutellum Such as abscissic or gibberellic acid, or physical Stimuli, e.g., (isozyme II), then secreted into the endosperm (Wolf, 1992). US 2002/0174452 A1 Nov. 21, 2002

0253) Additional seed-induced promoters include the which are active during Seed development and direct barley u-amylase promoter (Kreis et al., 1987) and promot endosperm-specific expression (Okita, et al., 1989, Kim et erS from the barley hordein gene family, e.g., B-, C-, and al., 1990). Promoters from the rice glutelin multigene family D-hordein genes (Sorensen et al., 1996 and references were first detected 6 days after flowering and reached a therein; Sorensen, 1992; Brandt et al., 1985; Entwistle et al., maximum at 14 days after flowering, then Subsequently 1991; Muller et al., 1993). B- and C-, hordein polypeptides declined (Takaiwa et al., 1991b). Various rice seed storage are major Storage proteins Synthesized in the endosperm proteins (albumin., prolamin., and type II glutelin) were (Brandt et al., 1985), and hordein gene promoters appear to shown to be expressed over a short time window after direct the Specific expression of the corresponding genes in flowering (i.e. maximum detection at 15 days after flower the endosperm. A 12S Seed Storage promoter, Such as that ing) Suggesting that they share transcriptional machinery isolated from oat (Avena sativa) may be used in the chimeric (Nakase et al., 1996). gene constructs of the present invention to direct endosperm 0261 Exemplary promoters for directing expression in specific expression (Schubert et al., 1990). germin.ating Seeds include the rice f3-amylase RAmy1A 0254 Additional seed-induced promoters which direct promoter, which is up-regulated by gibberellic acid; and the production of Seed Storage products for use in the invention rice B-amylase RAmy3D and RAmy3E promoters which are are the maize Zein gene promoter (Bianchi et al., 1988), and Strongly up-regulated by Sugar depletion in the culture. promoters from wheat glutenin genes, Such as those These promoters are also active during Seed germin.ation. described in Anderson et al., 1989 and Halford et al., 1989. Representative promoters further include the promoters The endosperm-specific expression regulated by both the from the rice B-amylase RAmy1A, RAmy 1B, RAmy2A, barley B-hordein promoter and the maize Zein promoter has RAmy3A, RAmy3B, RAmy3C, RAmy3D, and RAmy3E been correlated with hypomethylation of the corresponding genes, and from the pM/C, gkAmy141, gkAmy 155, structural genes (Sorensen et al., 1996; Bianchi et al., 1988). Amy32b, and HV18 barley B-amylase genes. These promot 0255 Also useful in the present invention are seed ers are described, for example, in ADVANCES IN PLANT induced corn promoters. Promoters from cloned Seed genes BIOTECHNOLOGY Ryu, D. D. Y., et al, Eds., Elsevier, expressed in Specific tissues in corn include: the corn Amsterdam, 1994, p.37, and references cited therein. O2-opaque 2 gene promoter, which regulates expression of 0262. Additional seed-induced promoters include the a Zein polypeptide expressed in the endosperm (Schmidt, et barley B22E gene promoter, which is differentially al., 1987); the corn Sh2-shrunken 2 gene promoter (Shaw et expressed in immature aleurone layers, but has been shown al., 1992) and the Bt2-brittle 2 gene promoter (Bae et al., to direct expression in both the aleurone and pericarp cells 1990), which together regulate starch synthesis in the of immature barley grains (Klemsdal, et al., 1991). endosperm; the Zp1 Zein gene promoter (Burr et al., 1982); the ZmZ27 Zein gene promoter; a rice Small subunit ADP 0263 Exemplar preferred promoters include the rice glu glucose phosphorylase gene promoter, OSAGP; and the rice telin Gt1 promoter and the aleurone-specific chitinase gene glutelin 1 gene promoter, all of which direct endosperm promoter (Chi26). Specific expression, as well as the promoter for maize granule-bound Starch Synthase (Waxy) gene, ZmCBS, which 0264 B4. PROMOTER ISOLATION directs expression in the pollen and the endosperm (Russell 0265. In addition, promoters for use in the methods of the et al., 1997). invention may be isolated from various tissues and/or at 0256 Also for use in the present invention are the Agp1 various Stages of Seed development by a variety of tech and Agp2 gene promoters (Giroux, M. J., Hannah, L. C., niques routinely used by those of Skill in the art. Target 1994), the corresponding genes for which are expressed in tissues for isolation of Such promoters include, but are not both the embryo and endosperm. limited to, the Seed testa layer, pericarp, aleurone and the endosperm. 0257 Additional seed-induced promoters include the barley B22E gene promoter, which is differentially 0266 The testa, or seed coat, is an outer layer of cells expressed in immature aleurone layers, but has been shown adjacent to the aleurone layer Surrounding the endosperm. to direct expression in both the aleurone and pericarp cells The testa is composed of an inner and outer cuticle, often of immature barley grains (Klemsdal et al., 1991). impregnated with waxes and fats, and one or more layers of 0258 Other suitable promoters include the sucrose syn thick-walled protective cells. The testa may contain layers of thase and Sucrose-6-phosphate-synthetase (SPS) promoters crystal containing cells composed of calcium oxalate or from rice and barley. A promoter directing expression of carbonate; the Seed coat may also contain mucilaginous cells Selectable markers used for plant transformation (e.g., npt) that burst upon contact with water, to provide a water should operate effectively in plant hosts. One Such promoter retaining barrier. The testa layer in Seeds functions to is the nos promoter from native Ti Plasmids (Herrera provide a protective barrier between the embryo and the Estrella et al., 1983). Others include the 35S and 19S external environment. promoters of cauliflower mosaic virus (Odell et al., 1985), 0267 The pericarp, is the outer covering of a grain or and the 2' promoter (Velten et al., 1984). Seed. The aleurone layer Surrounds the endosperm and is a living tissue which does not Store many reserves, but may be 0259) B3. SEED DEVELOPMENTAL STAGE responsible for the release and mobilization of enzymes, SPECIFIC such as proteinases. (Bewley, J D and Black, M., Eds., Seeds 0260 Representative seed developmental stage specific Physiology of Development and Germination, page 324, promoters include the promoters from the rice glutelin Plenum Press, New York, 1994). The endosperm is the major multigene family, Gt1, Gt2, Gt3, Glu A-3, and GluB-1, Storage reservoir for carbohydrate reserves in cereals. US 2002/0174452 A1 Nov. 21, 2002

0268 Various strategies for promoter isolation from RNA play of mRNAS expressed at different Stages of liver regen extracts of each target tissue at the appropriate developmen eration. The modified techniques has the added features of a tal Stage are employed to isolate a tissue specific promoter Specific PCR primer design, running Sequencing gels under which may then be incorporated into a chimeric gene non-denaturing conditions and automatic recording of the construct along with a gene encoding a protein which is cDNA pattern, allowing for more efficient analysis of integral to the biosynthetic pathway leading to G-lignan Sequence differences between 2 mRNA samples. formation. 0277. In an additional approach called selective amplifi 0269 Promoters may also be obtained from an alternative cation via biotin- and restriction-mediated enrichment monocot Species. For example, a Suitable promoter Such as (SABRE; Lavery et al., Genetics 94:6831-6836, 1997), the Gt1 gene promoter from rice may be isolated from Species more abundant in one double-Stranded DNA popu nucleic acid containing extracts derived from other cereals, lation (the tester) than in another (the driver) can be enriched e.g., wheat, oat, or the like, using conventional hybridization relative to species equally expressed in both by denaturing techniques routinely used by those of skill in the art. a mixture of both populations and allowing it to reasSociate, and then isolating only double-Stranded molecules of which 0270. Such techniques include, but are not limited to; (1) both Strands are derived from the tester population (tester use of conventional hybridization techniques and known homobybrids). promoters from a different Species, tissue and/or develop mental Stage to obtain related Sequences from the target 0278. Once a tissue-specific or developmental stage spe monocot Species, Seed tissue or Seed developmental Stage, cific gene is isolated, for example by the difference tech (2) subtractive hybridization (Lee et al., 1991), (3) differ niques discussed above, the full gene may then be sequenced ential display (Liang et al., 1992; Bauer et al., 1993), and (4) and the promoter region is identified. Identifying the pro Selective amplification via biotin-and restriction-mediated moter region is performed by Standard analysis, for example, enrichment (SABRE, Lavery et al., 1997). by identifying a start codon at the beginning of an open reading frame, and promoter-specific Sequences, e.g., a 0271 The present invention takes advantage of a seed tissue Specific or developmental Stage specific promoter, i.e., TATA box and transcription initiation site upstream of the a promoter that is Selectively induced in a particular Seed Start codon. The promoter can then be isolated and cloned by tissue or temporally expressed at various times during Seed Standard recombinant methods, for use in the chimeric gene development. of the invention. 0279. Following isolation of the promoter, expression 0272 Conventional hybridization techniques are applied Specificity can be further confirmed by fusing the promoter to identifying the Sequence of known promoters Sequences in-frame to a reporter Such as the Synthetic gene of the jelly from monocots other than the one in which they have been fish green fluorescence protein, engineered for high level found. By way of example, to identify a wheat endosperm expression in higher plants (Chiu, et al., 1996). The pro Specific promoter, a cDNA library from wheat Seeds, or moter-reporter gene construct is then delivered on gold wheat endosperm, is amplified using probes containing particles by the biolistic method into the target tissue(s) and consensus Sequences from, e.g. the known corn, promoter control tissues (e.g., leaf, Stem), as described below. Expres Sequence. The Sequence and location of the promoter sion specificity can also be confirmed by Northern blot Sequence for the corn Zein gene, Sh2 (Shaw et al., 1992) may analysis using mRNA preparations from a variety of barley be used to identify related promoter Sequences in wheat or tissues. other monocots. The identified cDNA is then sequenced, and may be incorporated into a chimeric gene construct with the 0280 B5. ANTISENSE STRATEGY appropriate target gene, according to the methods described 0281. As a general approach, an inhibitory transcript herein. RNA produces its biological effect by inhibiting expression 0273 Sequences present in one nucleic acid Sample and of an enzyme or protein that is a component integral to a not another may also be derived by Subtractive hybridization branch point of the phenylpropanoid pathway leading to where nucleic acid extracts are prepared from two Samples, formation of metabolites other than G-lignans. hybridized and Sequences that do not hybridize are amplified 0282. The sense strand of the inhibitory gene sequence (Lee et al., 1991). may be complementary to the transcribed region of the Sense 0274 Alternatively, cDNA difference techniques (e.g., a Strand of the target gene. That is, the orientation of the form of representational difference analysis for cDNA as inhibitory gene Sequence in the chimeric gene is in the described in Hubank et al., 1994) can be employed to reverse direction (3'-5'), so that RNA transcribed from the identify a gene that is expressed in one type of Seed tissue, plasmid is complementary in Sequence to the mRNA tran e.g. in the endosperm, but not in other Seed cells, Such as Scribed from the corresponding endogenous gene (i.e. anti aleurone cells. Sense). 0275. In another approach for identifying cDNAs for 0283 Antisense inhibition with an antisense nucleic acid testa-specific transcripts, a differential display technique is Sequence can take place in two independent ways: the utilized to systematically identify, by their cDNAS, the antisense Sequence can control the transcript level in the nucleus and/or the translation efficiency of the target mRNA messenger RNAS expressed in one type of tissue Sample and in the cytoplasm (Cornelissen et al., 1989; Cornelissen, not in another. (Liang et al., 1992; Bauer et al., 1993). 1989). It has been demonstrate that introduction of either 0276 The Bauer et al. variation of this technique, called Sense or anti-Sense constructs for an O-methyl transferase differential display reverse transcription polymerase chain (OMT) can result in altered lignin composition in transgenic reaction (DDRT-PCR) has been applied to differential dis tobacco plants (Atanassova et al., 1995). US 2002/0174452 A1 Nov. 21, 2002

0284. A monocot plant may be genetically altered by 0290 Construction of a chimeric gene construct contain transforming the plant with a chimeric gene having (i) a ing an inhibitory Sequence, e.g., an antisense nucleic acid transcriptional regulatory region (e.g., a promoter) Sequence, may be accomplished as described in Van der Krol expressed in a specific Seed tissue, or induced during Seed et al., 1988. development, (ii) an inhibitory gene Sequence operably linked to the transcriptional regulatory region for induction 0291 An exemplary procedure for preparing an antisense of an inhibitory transcript RNA, and (iii) additional regula construct of the present invention includes obtaining a tory DNA sequences and/or a sequence encoding a signal cDNA fragment from a known enzyme in a branch point in peptide, as appropriate to promote efficient expression of the the phenylpropanoid biosynthetic pathway, e.g., a chalcone inhibitory Sequence. Synthase (CHS) gene sequence. That sequence may be used to determine the complementary Sequence by preparing a 0285 Such an inhibitory nucleic acid sequence is capable lambda cDNA library as described in Kristiansen and Rohde of inhibiting expression of a gene encoding a protein or (1991) and screening with DNA derived from the target enzyme that normally is at a branch point in the phenylpro monocot Species. Positive clones are identified, and a cDNA panoid pathway that leads to formation of metabolites other insert about 20 nucleotides to 1.3 kb in length, correspond than G-lignans. By way of example, enzymes that occur at ing to a portion of the gene Sequence, may then be Subcloned Such branch points include, but are not limited to, ferulate into a Subcloning vector Such as puC9. 5-hydroxylase (F5H) which catalyzes the conversion of ferulate to 5-hydroxyferulate and chalcone synthase (CHS) 0292 A clone containing the complementary Sequence which catalyzes formation of flavenoid compounds com may then be partially digested with a restriction enzyme, posed of a basic 15-carbon flavenoid skeleton including e.g., e.g., Nicol, and EcoRI, and the Sticky ends made blunt using anthocyanins, glycosylated flavenoid pigments, Starting with the Klenow fragment of DNA polymerase I and dNTPs. para-coumaryl CoA. Buildup of ferulate resulting from a Various resulting blunt ended fragments are then Subcloned, lack of enzymatically active F5H or defective F5H will e.g., into the HindII site of phage M13 mp7, to allow favor the pathways leading to H and G-lignan formation by isolation of a BamHI fragment. This fragment can then be blocking the pathway leading to S-lignan formation. cloned into, e.g., the BamHI Site Separating the promoter and a nopaline Synthase 3' flanking region containing a polyA 0286 Inclusion of Such an inhibitory nucleic acid Sequence in a chimeric gene construct of the invention is tail, and inserted into the polylinker Site of a Suitable plant effective to prevent (i) transcription of the gene encoding transformation vector, to form an antisense construct. e.g. F5H or CHS; or (ii) translation of the F5H or CHS 0293 An antisense chimeric gene construct as described mRNA. Preferably, the inhibitory sequence is an antisense above, may be introduced into monocot plant cells by the F5H or CHS sequence effective to allow an accumulation of methods described below resulting in production of trans ferulate or para-coumaryl CoA within transformed cells. genic plants and Seeds wherein the level of a target protein Preferably, the inhibitory sequence is placed under the or enzyme is reduced or elimin.ated. control of a Seed tissue specific or Seed developmental Stage Specific promoter. In one aspect, the promoter is a stage 0294 V. PRODUCING TRANSGENIC PLANTS Specific promoter that is activated in Seed at the appropriate developmental Stage of the Seed. 0295) A. METHODS OF DNA INTRODUCTION 0287. It will be appreciated that the inhibitory transcript 0296. Following introduction of a chimeric gene con RNA may also be designed to block transcription by binding Struct encoding a protein integral to the biosynthetic path to one Strand of the nucleic acid during transcription. The way leading to G-lignan formation or an antisense chimeric transcript in this case may have either Sense or antisense gene construct encoding a protein which facilitates forma orientation with respect to the target gene Sequence. Further, tion of metabolites other than G-lignans, the coding the transcript can target any Sequence of the gene, including Sequence contained within the chimeric gene construct is transcription binding site(s) and intron regions, effective to expressed, either in cell culture, or in germin.ating Seeds. block transcription of a full-length mRNA. Such non-c)NA 0297. The plants to be transformed are monocot plants, Sequences can be identified by isolation and Sequencing of particularly the members of the taxonomic family known as the chalcone Synthase (CHS) gene from a genomic library, the Gramin.eae. This family includes all members of the according to Standard procedures. grass family of which the edible varieties are known as 0288 For example, constitutive expression of an anti cereals. The cereals include a wide variety of Species Such as Sense chalcone Synthase gene in transgenic petunia and wheat (Triticum sps.), rice (Oryza sps.) barley (Hordeum tobacco plants has been shown to reduce levels of both SpS.) oats, (Avena sps.) rye (Secale sps.), corn (Zea sps.) and mRNA for the enzyme and the enzyme itself (Van der Krol millet (Pennisettum sps.). In the present invention, preferred et al., Nature 333:866-869, 1988) family members are rice, corn, wheat and barley. 0289. The structure of the chalcone synthase (GenBank 0298 The transformation of plants may be carried out in Accession No. X92548) gene which encodes the CHS any of the various ways known to those skilled in the art of enzyme or protein in rye is presented herein by way of plant molecular biology. See, Wu and Grossman (1987) example. It will be understood that nucleotide differences in incorporated herein by reference. Such techniques include, the CHS gene may exist among different genotypes and but are not limited to, e.g., electroporation, protoplast fusion among various monocots, and that the invention is intended and microparticle bombardment. Various methods for direct to encompass Such variants, as defined above. It will be or vectored transformation of plant cells, Such as plant further understood that Sequences as Short as 10-20 bases, protoplast cells, have been described, e.g., in PCT applica can be used for antisense inhibition. tion WO95/14099. US 2002/0174452 A1 Nov. 21, 2002

0299. By transformation is meant alteration of the geno termed virulent region, is essential for the introduction of the type of a host plant by the introduction of a nucleic acid T DNA into plants. The transfer DNA region, which trans Sequence, e.g., a chimeric gene construct or heterologous fers to the plant genome, can be increased in size by the nucleic acid construct of the type described herein. The insertion of the foreign nucleic acid Sequence without its nucleic acid Sequence need not necessarily originate from a transferring ability being affected. By removing the tumor different Source, but it will, at Some point, have been causing genes So that they no longer interfere, the modified external to the cell into which it is to be introduced. Tiplasmid can then be used as a vector for the transfer of the 0300. In one approach, the nucleic acid is mechanically gene constructs of the invention into an appropriate plant transferred by microinjection directly into plant cells by use cell, such being a “disabled Ti vector'. All plant cells which of micropipettes. Alternatively, the foreign nucleic acid may can be transformed by Agrobacterium and whole plants be transferred into the plant cell by using polyethylene regenerated from the transformed cells can also be trans glycol, which forms a precipitation complex with the genetic formed according to the invention So as to produce trans material that is taken up by the cell (Paszkowski, et al., formed whole plants which contain the transferred target 1984). nucleic acid Sequence. 0307 There are presently at least three different ways to 0301 Achimeric gene construct may also be introduced transform plant cells with Agrobacterium: (i) co-cultivation into the plant cells by electroporation (Fromrnm, et al., of Agrobacterium with cultured isolated protoplasts, (ii) 1985). Electrical impulses of high field strength reversibly transformation of cells or tissues with Agrobacterium, or permeabilize biomembranes, allowing the introduction of (iii) transformation of Seeds, apices or meristems with Plasmids or nucleic acids containing the chimeric gene Agrobacterium. The first method requires an established constructs into plant protoplasts. Electroporated plant pro culture System that allows culturing protoplasts and plant toplasts reform the cell wall, divide, and form a plant callus. regeneration from cultured protoplasts. The Second method Selection of the transformed plant cells with the transformed requires that the plant cells or tissues can be transformed by gene can be accomplished using phenotypic markers. Agrobacterium, and that the transformed cells or tissues can 0302) Transformation of monocot plants is preferably be induced to regenerate into whole plants, while the third carried out as generally described in Jensen, et al., 1996, and method requires micropropagation. in Wan, et al., 1994. In this procedure, the chimeric gene 0308 Transgenic cells, e.g., callus cells, are broken into construct-plasmid is adsorbed to gold particles (e.g., 1.0 lim Small clumps of typically 1-20 or more cells, and Suspended particles, DuPont, Wilmington, Del.) and delivered into in a suitable cell culture medium. The cells are preferably immature embryos by particle bombardment. When two or cultured under conditions that favor plant cell growth, until more plasmid DNA constructs are used to transform plant the cells reach a desired cell density, then under conditions tissue, a mixture of the two or more plasmids may be that favor expression of the target gene under the control of adsorbed to the particles for co-injection. the appropriate promoter. 0303 Preferred methods of transforming wheat and sor 0309. A typical transformation protocol for rice generally ghum are generally described in Nehra et al., 1994. follows the methods detailed generally in Sivamani et al., 0304 Particle bombardment may be used alone or in 1996; Zhang et al., 1996; and Li, et al., 1993. Briefly, seeds combination with other methods Such as Agrobacterium are Sterilized by Standard methods, and callus induction mediated transformation. In a typical transformation meth from the seeds is carried out on MB media with 2,4-D. odology, the embryo and endosperm of mature Seeds are During a first incubation period, callus tissue forms around removed to expose Scutulum tissue cells. The cells may be the embryo of the seed. By the end of the incubation period, transformed by DNA bombardment or injection, or by (e.g., 14 days at 28°C.) the calli are about 0.25 to 0.5 cm in vectored transformation, e.g., by Agrobacterium infection diameter. Callus mass is then detached from the Seed, and after bombarding the Scuteller cells with microparticles to placed on fresh NB media, and incubated again for about 14 make them Susceptible to Agrobacterium infection (Bidney days at 28 C. After the second incubation period, satellite et al., 1992). calli develop around the original “mother callus mass. 0305 Another method of introducing the nucleic acid These Satellite calli are slightly Smaller, more compact and constructs of the invention into plant cells is to infect a plant defined than the original tissue, and are transferred to fresh cell, an explant, a meristem or a Seed with Agrobacterium media. tumefaciens transformed with the Segment. Under appropri 0310 Calli to be bombarded are selected from 14 day old ate conditions known in the art, the transformed plant cells Subcultures. The size, Shape, color and density are all are grown to form shoots, roots, and develop further into important in Selecting calli for transformation. The calli plants. The nucleic acid Segments can be introduced into typically should be between about 0.8 and 1.1 mm in appropriate plant cells, for example, by means of the Ti diameter and appear as Spherical masses with a rough plasmid of Agrobacterium tumefaciens. The Ti plasmid is exterior. transmitted to plant cells upon infection by Agrobacterium tumefaciens, and is stably integrated into the plant genome 0311. After transformation preferably by particle bom and preferred methods for Agrobacterium-mediated trans bardment, the cells are typically grown under conditions that favor Selection of transformants in the presence of the formation of corn and/or rice are generally disclosed in Substrate for the Selectable marker, e.g., hygromycin, the Horsch et al., 1984 and Fraley et al., 1983. bialophos resistance gene or phosphinotricin. Alternatively, 0306 Ti Plasmids contain two regions essential for the plasmid pEmuGN (Last, 1991), which directs high level production of transformed cells. One of these, named trans expression of B-glucoronidase in monocots, can be used as fer DNA (T DNA), induces tumor formation. The other, an internal control to monitor the efficiency of gene transfer; US 2002/0174452 A1 Nov. 21, 2002 substrate 5-bromo-4-chloro-3-indolyl is then used for his 0318 VI. EVALUATION OF TRANSGENIC PLANTS tochemical Staining of transformed tissue. 0319 A. PCR ANALYSIS 0312 Preferably, the transformed cells are cultured under 0320 DNA may be extracted from various plant tissues, multiple rounds of Selection to produce a uniform, stably particularly Seed tissueS or whole Seeds and analyzed for the transformed cell line. presence of a target gene by use of appropriate primers. (See, 0313 Reproducible transformation frequencies are typi e.g., Jensen, et al., 1996; FIG. 15). cally obtained by growing the plants over a period of 60-80 0321 B. RT PCR ANALYSIS days at temperature cycles of 18 C. for 16 hand 12 C. for 0322 RNA is extracted from various plant tissues, par 8 h using high light intensities above about 120 uE/m2. ticularly Seed tissues and from whole Seeds, reverse tran scribed and is analyzed for the presence of the mRNA 0314. A preferred transformation protocol for monocots corresponding to the target gene. mRNA from the same is as follows. Immature embryos of about 1.5-2.5 mm in tissues is also analyzed for Sequences encoding downstream length are isolated from plants and grown under controlled proteins or enzymes integral to the phenylpropanoid path conditions (day 0). The embryos are then bombarded with way leading to G-lignans for which altered expression is the target chimeric gene containing a Selectable marker, e.g., expected based on modified expression of the target gene, by a herbicide resistance gene (day 1) and placed on Selection use of appropriate primers (Jensen et al., 1996). In addition, media (day 2). During week 2, a first Subcultivation is if expression of dirigent protein is up-regulated, analyses carried out and most immature embryos have exhibited may include e.g., an evaluation of the expression of Sec growth. A Second Subcultivation is carried out at week 4, oisolariciresinol dehydrogenase (SD) in the same tissues. followed by a third subcultivation during week 6. During week 6, first callus is placed on shoot inducing medium. At 0323 C. SOUTHERN ANALYSIS about week 8, a fourth Subcultivation is carried out and the 0324 Transformation of each plant can be confirmed first clones are placed on regeneration media. At week 10, a using Southern blot analysis of genomic DNA. Typically, fifth Subcultivation is carried out, and clones are placed on total DNA is isolated from each transformant (e.g., Schwarz shoot inducing medium. By about week 12, a sixth Subcul Sommer, et al., 1984). The DNA is then digested with tivation is conducted on shoot inducing and regeneration restriction enzyme, fractionated in 1% agarose gels and media; typically, the first green plantlets appear during this transferred to nylon filters (e.g., HYBOND-N, Amersham) Stage. At about week 14, a Seventh Subcultivation is carried according to Standard techniques. The blot may then be out; this is followed during week 16 by an eighth subculti probed, e.g., with 'P-labeled target cDNA as described. Vation. During this period the first clones are then transferred to soil and allowed to grow for up until about month 7. 0325 D. NORTHERN ANALYSIS Nucleic acids are then isolated from plant tissue and ana 0326 RNA is isolated from specific seed tissues (e.g., lyzed by PCR for the presence of the chimeric gene and the Seed testa layer, pericarp, aleurone and endosperm) or at metabolic products impacted by altered expression of that Specific developmental Stages, Separated, e.g., in a 1.2% the target gene. By about month 8, the TO plants begin agarose gel containing 2.2M formaldehyde, and blotted to a Setting grains, and during month 9, the immature grains are nylon filter, e.g., Hybond-N, according to the Supplier's harvested and the T1 embryos germin.ated to establish the protocol. Strand specific RNA probes are synthesized by next generation. The T1 plants are grown to maturity, and the phage T7 and T3 RNA polymerases from the Ant18 cl)NA Success rate of the transformation determin.ed. Typical rates clone and hybridized to the RNA on the filter. This allows an of transformation are about 1% transformants per isolated estimation of the amount of endogenous Sense and trans immature Zygotic embryo. genic antisense mRNA for the target enzyme or protein. 0315 Plant regeneration from cultured protoplasts or 0327 E. HIGH PERFORMANCE LIQUID CHRO callus tissue is carried by Standard methods, e.g., as MATOGRAPHY (HPLC) described above and in Evans et al., HANDBOOK OF 0328 Extracts from seeds of transformed and native PLANT CELL CULTURES, Vol. 1: (MacMillan Publishing plants may be analyzed by high performance liquid chro Co. New York, 1983); and Vasil I. R. (Ed.), CELL CUL matography (HPLC), in order to detect the presence and/or TURE AND SOMATIC CELL GENETICS OF PLANTS, levels of particular intermediates in the phenylpropanoid Acad. Press, Orlando, Vol. I, 1984, and Vol. III, 1986. pathway, including, but not limited to L-phenylalanine, 0316. In a preferred method for producing plants with cinnamate, para-coumarate, caffeate, ferulate, Sinapate, elevated levels of G-lignans in Seeds, monocot plant cells are para-coumaryl CoA, caffeoyl CoA, feruloyl CoA, 5-hydroxy transformed and used to regenerate plants, Seeds from the feruloyl CoA, Sinapoyl CoA, para-coumarylaldehyde, plants are harvested, germin.ated, and the Seeds derived coniferylaldehyde, 5-hydroxy coniferylaldehyde, Sinapylal from Such transformed plants are processed as appropriate dehyde, para-coumaryl alcohol, coniferyl alcohol, and for use as food additives. Sinapyl alcohol. 0317 Following introduction of the chimeric gene and 0329. F. ENZYME ASSAYS growth of transformants, transformation of plant tissue can 0330 Enzyme extracts of seeds from monocot plants can be confirmed by a variety of methods. Exemplary methods also be assayed for activity of the various enzymes integral for confirming transformation and for analyzing proteins, to the phenylpropanoid pathway. For example, phenylala enzymes and metabolites in Seeds derived from transgenic nine amin.o lyase (PAL), cinnamate-4 hydroxylase (C4H), monocot plants, are described below. 4-coumaric acid coenzyme A ligase (4Cl), caffeate acid US 2002/0174452 A1 Nov. 21, 2002

O-methyl transferase (C-OMT), ferulate-5-hydroxylase tea, etc. (approximately 130, 610, 14, 221, 172, 468,240, (F5H), chalcone synthase (CHS), coumaroyl-coenzyme A 192, 414, 379, 1510, 333, 1590, and 2460 ug/ml, respec 3-hydroxylase (CCoA-3H), caffeoyl-CoA 3-O-methyltrans tively; Adlercreutz et al., 1997). ferase (CCoA-OMT), cinnamoyl-coenzyme A reductase 0338. The corresponding level of matairesinol in flaxseed (CCR), pinoresinol/lariciresinol reductase (PR), secosisola has been determined to be (1090-1300 ug/ml; Adlercreutz et riciresinol dehydrogenase (SD) and SecoSisolariciresinol al., 1997). Levels of matairesinol in various grains exem diglucosyl transferase (SDT) may be analyzed. plified by whole grain wheat, oat bran, whole grain rye, rye 0331] G. LIGNAN ANALYSIS bran, whole grain triticale, and triticale meal were deter 0332. In one exemplary approach, G-lignans are analyzed min.ed to be approximately 3, 155, 65, 167, 9, and 11 lug/ml, by carrying out the steps of (1) separating the appropriate respectively (Adlercreutz et al., 1997). The amount of plant tissues into components for comparative analysis, (2) matairesinol in poppy Seeds, caraway Seeds, mung bean releasing the lignans from the plant material by the action of Sprouts, urid dahl beans, carrots, broccoli, garlic, Earl Grey hydrolytic enzymes and acid, (3) extraction of lignans with Black tea, Japanese green tea, has been determined to be diethyl ether, (4) purification on DEAE cellulose and QAE approximately 12, 6, 1, 79, 3, 23, 4, 197 and 186 ug/ml, Sephadex columns, silylation and (4) quantitation by gas respectively (Adlercreutz et al., 1997). chromatography/mass SpectroScopy using isotope dilution 0339. In contrast to secoisolariciresinol, matairesinol has and selected ion monitoring (SIM; Nilsson et al., 1997; not been detected in pumpkin Seeds, wheat bran, whole grain Mazur et al.,1996). barley, barley bran, Soybean flour, Sunflower Seeds, or cran berries, and has been found in only trace amounts in 0333 An analysis of rye following roller-milling into 6 oatmeal, mung beans, and peanuts. (Adlercreutz et al., flour fractions, a short and a bran, indicated the highest 1997). No correlation between the concentrations of sec concentrations of the G-lignans, Secoisolariciresinol and oisolariciresinol and matairesinol was found in whole meal matairesinol, in the short and the bran, indicating that the majority of both lignans are present in the Outer layers of the rye flour (Nilsson et al., 1996). rye kernal, however lower levels of Secoisolariciresinol also 0340. The methods and compositions of the present appeared to be present in the Starchy endosperm (Nilsson et invention reflect an alteration in G-lignan concentration in al., 1997). the Seeds of monocot plants, particularly the G-lignans Secoisolariciresinol diglucoside and matairesinol. The intro 0334. In practicing the present invention, extracts taken duction of the chimeric gene constructs of the present from Seeds of transformed and native plants may be ana invention and the corresponding expression of proteins lyzed by gas chromatography, together with mass spectros integral to the phenylpropanoid pathway in monocot Seeds, copy using isotope dilution and Selected ion monitoring as yield a change in the concentration of G-lignans over the further described below, in order to detect the presence concentration of G-lignans found in the Seeds of monocot and/or levels of particular lignans Such as matairesinol or plants that have not been transformed with Such chimeric Secoisolariciresinol diglucoside. Detection of, or an increase gene constructs (i.e. plants in their native state). in, the level of matairesinol or Secoisolariciresinol digluco Side in transformed plants relative to native (non-trans 0341 Ordinarily, a “change in lignan concentration or formed) plants is an indication that introduction and stable lignan level” will correspond to at least about a 2x increase expression of the chimeric gene construct has resulted in in the G-lignan concentration or level detectable in the Seeds modifying the level of G-lignans in the transformed plants. of transformed plants relative to the Seeds of plants that have not been transformed with the chimeric gene constructs of 0335 H. ELEVATED LIGNAN CONTENT IN the present invention. Such increases in G-lignan concen SEEDS AND SEED PRODUCTS tration are preferably at least about 5x, and more preferably 0336 Secoisolariciresinol and matairesinol mainly occur at least about 10x or more relative to the G-lignan concen in the form of glycosides in plants. After hydrolysis of the tration present in the Seeds of transformed plants relative to glycosides, the concentrations of Secoisolariciresinol and the seeds of plants that have not been transformed with the lariciresinol have been evaluated in various Soybean prod chimeric gene constructs of the present. ucts, grains and other foods. (Adlercreutz et al., 1997). 0342. It will be understood that the invention provides 0337 The highest levels of secoisolariciresinol reported plants Seeds having elevated levels of G-lignans and that to date have been those detected in flaxseed and pumpkin Such Seeds may be used in their native State, and may serve seed (approximately 370-545 mg/100g and 21 mg/100 g, as the basis for Seed extracts and food additives. Such Seed respectively). See, e.g., Thompson, 1998; Adlercreutz et al., extracts and food additives may be prepared using methods 1997. In addition, moderate levels of secoisolariciresinol generally employed by those of skill in the art. have been detected in numerous grains examples of which 0343 VII. UTILITY/BIOLOGICAL EFFECTS OF LIG include, whole grain wheat, wheat bran, oat meal, oat bran, NANS whole grain barley, barley bran, whole grain rye, rye bran, whole grain triticale, and triticale meal (approximately 33, 0344) Dietary consumption of processed transgenic Seeds 110, 13, 24, 58, 63, 47, 132, 39, 21 ug/ml, respectively; containing increased levels of G-lignans can result in Adlercreutz et al., 1997). Further, secoisolariciresinol has increased levels of mammalian lignans (enterodiol and also been found to be present in a diverse list of food enterolactone) which are formed by bacterial metabolism of products including, but not limited to, Soybean flour, Sun plant lignans contained in the diet. (Adlercreutz et al., 1997; flower Seeds, poppy Seeds, caraway Seeds, mung beans, Lampe et al., 1999.) mung bean Sprouts, urid dahl beans, carrots, broccoli, garlic, 0345 Plant lignans and have been suggested as capable cranberries, peanuts, Earl Grey Black tea, Japanese green of impacting a wide variety of biological processes (Adler US 2002/0174452 A1 Nov. 21, 2002 creutz et al., 1997). For example, many plant lignans have 0355 C. LARGE SCALE CULTURES (MAX been shown to have anti-Viral, anti-bacterial and fungistatic IPREPS) activitie; alignan with close Structural Similarity to mataires inol has been found to have immunomodulatory activity; 0356. Large scale cultures of expression vectors (volume lignans have been shown to effect intracellular and Steroid of 500 ml) were prepared for rice transformation starting metabolic enzymes, protein Synthesis, numerous anti-cancer with 300 ul of min.iprep-culture cells containing the correct effects, inhibition of chemotaxis, inhibition of multiple steps plasmid. For future use, 700 ul of grown cells were added to of the cell cycle of vascular Smooth muscle cells and 300 ul of 50% glycerol and stored in -70° C. inhibition of endothelial cell proliferation. (See, e.g., Pool 0357 D. LIGNAN ANALYSIS Zobel B L et al., 2000; Li, et al., 1999; Adlercreutz, 1997; Ayres et al., 1990). 0358) D1. CHEMICALS AND REAGENTS. 0346 Epidemiological Studies Suggest that a lignan-rich 0359 Methanol and n-hexane (HPLC grade, glacial ace diet Such as that of American macrobiotics and vegetarians tic acid, NaOH, Et2O, ascorbic acid, hydrochloric acid were may be correlated with reduced incidence of breast cancer obtained from Aldrich. Tri-Sil reagent and Sillyl-8 were from and protective effects during the promotional phase of Pierce Chemical Co. Helix pomatia juice was purchased proState cancer. from BioSepra, Inc. 0347 The mammalian lignans along with the isofla 0360 D2. CHROMATOGRAPHIC MATERIAL. venoids are classified as phyto-Oestrogens (Adlercreutz, 1997). The phyto-Oestrogens are the hormone-like metabo 0361 DEAE- and QAE-Sephadex A-25 are washed with liteS produced by metabolic processing of lignans and isofla 20%, 50% and absolute ethanol and stored at 6 C. The venoids, which have the ability to bind with low affinity to hydroxyl form of DEAE-Sephadex A-25 is obtained by Oestrogen receptors as well as having weak oestrogen activ washing the gel successively with 0.1M NaOH in 70% CHOH, 70% CH-OH and then CH-OH. The DEAE-Sepha ity (Schutt, 1972; Setchell, 1988). Enterolactone, the most deX A-25 is used immediately after conversion. The acetate abundant mammalian lignan competes with the natural form of QAE-Sephadex A-25 is obtained by washing the gel Substrate for placental aromatase. Placental aromatase con successively with 0.1M NaOH in 70% CH-OH, 70% verts its natural Substrate androstenedione, into Oestrone CHOH, 0.5M Acetic Acid in 70% CHOH, 70% CH-OH which has been implicated in various types of cancers. and then CH-OH. The acetate form of QAE-Sephadex A is 0348 The lignans and isoflavenoids appear to stimulate stored at 6 C. sex hormone-binding globulin (SHBG) synthesis in the liver and may in this way influence the biological effects of SeX 0362) D3. STANDARDS hormones. Phyto-Oestrogens have been implicated in inhi 0363 Natural abundance matairesinol (1), secoisolaricir bition of numerous biosynthetic and metabolic enzymes, and esinol (2), and anhydrosecoisolariciresinol (6) were previ Such activities are a likely Source of their disease-preventive ously Synthesized according to known methods. Deuterated effects. (See, e.g., Adlercreutz et al., 1997.) d6-matairesinol (3), d8- Secoisolariciresinol (4) and d6-an 0349 From the foregoing, it will be appreciated that the hydrosecoisolariciresinol (5) were synthesized as described invention provides methods for increasing the concentra below. (See FIGS. 4A-4B) tions of G-lignans in the Seeds of monocots. The invention further provides plants and Seed compositions produced 0364) D4. SYNTHESIS OF D6-MATAIRES using Such methods. The following examples are intended to INOL (3) illustrate, but in no way limit the invention. 0365) Matairesinol (1) (0.356 g., 0.99 mmol) was dis Solved in deutero-labeled acetic acid. After Several hours at 0350 MATERIALS AND METHODS room temperature, the Solution was evaporated in vacuo to 0351) A TRANSFORMATION AND ISOLATION Small volume to give (FIG. 4A). Deutero-labeled acetic acid OF POSITIVE COLONIES was made from freshly distilled acetic anhydride (32.5g, 0352) 40 ul of competent cells (DH10B) are placed on ice 0.32 mol) to which heavy water (8.8 g., 0.44 mol) was slowly and 1-2 ul of ligated plasmid DNA added followed by gentle added with stirring under argon for 4 h. Predeuterated mixing and allowing the mixture to Stand on ice for 2 min. matairesinol was added under argon to labeled phosphoric The cells are then electroporated and 1 ml of SOC medium acid, which was prepared by adding DO (6 g) to POs (6 g). immediately added, followed by incubation at 37 C., 225 The reaction mixture was stirred at 80° C. for 3 days. Water rpm for 45 min. 25 ul, 50 ul and 100 ul of culture cells are (10 ml) was next added to the cooled reaction mixture, Spread on LB medium plates containing 0.1 mg/ml Ampi which was then extracted with ether (15 ml), followed by cillin and incubated at 37 C., 225 rpm, overnight. washing the ether layer with NaHCO (15 mlx2) and water 0353 B. SMALL-SCALE CULTURES (MINI (15 mlx2). The organic phase was dried with MgSO4 and PREPS) evaporated to dryneSS. The above deuteration and work-up 0354 Small-scale cultures are prepared by picking sev were repeated once again. The crude product was recrystal eral Single-colonies and inoculating them into 3 ml of LB lized from n-hexane-EtOAc to give (3; FIG. 4A). medium containing 0.1 mg/ml amplicillin and incubating at 0366 Spectral data (3; FIG. 4A) includes the following: 37 C., 225 rpm, overnight. 500 ul of culture cells are stored H-NMR (CDCl3): 6.80 -6.40 (6H, m, Ar-H), 5.42 (2H, s, in glycerol for large-scale culture and 60 ul of plasmids are OHx2), 4.16 and 3.90 (1H.m., H-9, H-9"), 3.82 (3H, s, isolated following conventional methods, then digested with OCH3), 3.81 (3H, s, OCH3), 2.92 (2H, m, H7), 2.55 (4H, restriction enzymes to confirm the presence of the lignan m, H-7, H-8, H-8); EI-MS: m/z. 358 (M+), 136. (4) 1 gene. H-NMR (CDC13): 5.50 (2H, s, OHx2), 4.17 and 3.90 (1H, US 2002/0174452 A1 Nov. 21, 2002 20 m, H-9, H9), 3.81 (3H, s, OCH3), 3.82 (3H, s, OCH3), 2.93 H-9"), 2.55 (4H, m, H-7, H-7), 2.19 (2H, m, H-8, H-8) and (2H, m, h-7), 2.57 (4H, m, H-7, H-8, H-8); EI-MS: m/z. 364 showed the absence of aromatic protons, EI-MS: m/Z 350 (M+), 140. (M+), 137. 0367) D5. SYNTHESIS OF D8-SECOISOLAR EXAMPLE 1. ICIRESINOL (4) 0368 d6-Matairesinol (3) (120 mg) was dissolved in An Efficient Rice Transformation System With a tetrahydrofuran (THF, 9 ml) freshly distilled over LiAIH Selectable Marker Driven by a Non-Constitutive under N. The resulting Solution was added dropwise, at Promoter room temperature, over a period of 15 min. to a Stirred 0374] A. CONSTRUCTION OF A SELECTABLE suspension of LiAID (106 mg) in dry THF (2.4 ml). MARKER PLASMID, PAPI76. Following Stirring for an additional hour at the same tem 0375. A selectable marker plasmid paPI76 was con perature, the reaction mixture was cooled to 0° C. Next, Structed in three StepS. First, a DNA fragment was amplified EtOAc (3 ml) was added dropwise, and the whole mixture from a rice alpha-amylase gene, RAmy1A, (Huang et al., poured onto dry ice. Distilled water (5 ml) was added to the 1990) and cloned into pBluescript KS+ at the SmaI/EcoRI resulting Suspension, with the organic Solvent removed in restriction sites. The primers used to amplify the fragment vacuo. The sample was reconstituted in distilled water (10 were 1AR1 (SEQ ID NO: 1) and 1Asma (SEQ ID NO:2). ml) with the whole extracted EtOAc (20 mlx6). The com The amplified fragment contained 297 bp of RAmy1A bined EtOAc Solubles were washed with a Saturated NaCl termin.ator. This resulting plasmid was called p1AT. Second, solution, dried with anhydrous MgSO4 and evaporated in a BamHI DNA fragment from pGL2 (Shimamoto et al., vacuo to yield crude d8-Secoisolariciresinol, which was 1989) was cloned into BamHI site of p1AT. The BamHI purified by preparative TLC (silica gel) to give d8-secoiso fragment contained most of the hygromycin phosphotrans lariciresinol (4; FIG. 4A). ferase (hph) gene with a deletion of four amin.o acids at the 0369 Spectral data (4; FIG. 4A) includes the following: C-termin.us. This plasmid was named paPI74. Third, a H-NMR (CD3 OD): 6.71 -6.44 (6H, m, Ar-H), 4.05 (2H, dd, SacI/Xbal fragment amplified from a glucanase gene, Gns9, H-9, H-9), 3.70 (3Hx2, s, O-CH3), 3.58 (2H, dd, H-9, H-9), was inserted into paPI74 cut with SacI/Xbal to form 2.65 (2H, dd, H-7, H-7), 1.86 (2H, m, H-8, H-8); EI-MS: pAPI76. The primer sequences used to amplify the Gns9 m/z. 362 (M+), 189, 137. (4). 1 H-NMR (CD3 CN): 3.74 promoter were GnsF (SEQ ID NO: 3) and GnsR (SEQ ID (6H, s, OCH3), 3.43-3.32 (2H, m, H-9, H-9"), 2.88 and 2.65 NO: 4). PCR fragments were confirmed by DNA sequenc (2H, d, J=8Hz, H-7, H-H-7), 1.87 (2H, m, H-8, H-8); ing. To visualize the tissue specific expression of the Gns9 EI-MS: m/z. 370 (M+), 140.7. promoter, a Gns9 promoter-GUS construct may be pre pared for rice transformation. The GUS gene and noS 0370 D6. SYNTHESIS OF D6-ANHYDROSEC terminator was obtained from pBI221 (ClonTech, CA) by OISOLARICIRESINOL (5) digestion with restriction enzyme EcoRI and BamHI. The 0371 Anhydrosecoisolariciresinol (40 mg) (6) was dis fragment was then inserted into paPI76, which was cut with Solved in deutero-labeled acetic acid. After Several hours at the same enzymes. Thus, the GUS gene replaced the hph room temperature, the Solution was evaporated in vacuo to gene, resulting in a plasmid paPI83. Small volume to give (8). Deutero-labeled acetic acid was 0376 B. RICE TRANSFORMATION made from freshly distilled acetic anhydride (3.65 g, 35 mmol) to which heavy water (0.98 g., 49 mmol) was slowly 0377 The procedure for microprojectile-mediated rice added with Stirring under argon 8 h. Predeuterated anhy transformation (Sivamani et al., 1996; Chen et al., 1998) was drosecoisolariciresinol (8) was added, under argon, to deu followed with modifications. Approximately 200 TP309 tero-labeled phosphoric acid, which was prepared by adding seeds were dehusked, sterilized in 50% commercial bleach D2O (1 g) to P2O5 (1 g). The reaction mixture was stirred for 25 min. and washed with sterile water three times for 5 at 80° C. for 3 days. Water (5 ml) was added to the cooled min. each. The Sterilized Seeds were placed plates containing reaction mixture, which was then extracted with ether (5 N6 semisolid medium supplemented with 2,4-D 2 mg/L, mlx6) and washed with NaHCO Solution (5 mlx2) and sealed with parafilm and incubated in the dark at 28 C. for water (5 mlx2). The organic phase was dried with MgSO 14 days to induce calli. and evaporated to dryneSS. The above deuteration and work 0378 Developing calli (less than 1 mm in diameter) were up was repeated once again to give a residue (35.2 mg), then transferred developing onto fresh N6 medium (35 calli which was purified by preparative TLC (silica gel) to give per plate) for 30 days and serve as bombardment material. (5, FIG. 4). The crude product was recrystallized from Calli were subcultured every 15 days. n-hexane-EtOAc. 0379 Prior to bombardment, calli from 30-day old sub 0372 Spectral data (6, FIG. 4) includes the following: cultures are Selected with size, shape, color, and density very 1H-NMR (CDC13): 6.82 (2H, d, J=8 Hz, H-5, H-5), 6.60 important factors for Successful transformation. It is pre and 6.57 (4H, m, H-6, H-6"), 6.5 (2H, br. S., H-2, H-2), 5.51 ferred that calli for transformation have the following char (2H, br.s, OHx2), 3.83 (3H, s, OCH3), 3.82 (3H, s, OCH3), acteristics: a Size of 1.0-3 mm in diameter, a spherical mass 3.92 and 3.54 (2H, dd, H-9, H-9), 2.55 (4H, m, H-7, H-7), with a rough exterior, a white and opaque color and a density 2.19 (2H, m, H-8, H-8); EI-MS: m/z 344 (M+),137. which is compact and hard. 0373) Spectral data (5; FIG. 4) includes the following: 0380 Prior to transformation, approximately 200 calli are 1H-NMR (CDC13); 5.50 (2H, br. s, OHx2), 3.82 (3H, s, placed together in the center of a plate (about 4 cm in OCH3), 3.81 (3H, s, OCH3), 3.92 and 3.54 (2H, dd, H9, diameter) on N6 semisolid medium and incubated at 28°C. US 2002/0174452 A1 Nov. 21, 2002 21 in the dark for 24 hours. The efficiency of rice co-transfor plasmid, paPI 141, contained the Gt1 promoter, the Gt1 mation over eight plasmids is presented in Table 1, with the Signal Sequence, a multiple cloning Site and a noS termin.a- procedure further detailed in Example 3. tor. In order to make a Not site at the 5' UTR of Gt1, two primers were generated. The first primer is Gt1SDMF (SEQ TABLE 1. ID NO:34), and the second primer is Gt1SDMR (SEQ ID NO: 35). The protocol from Stratagene's Quickchange kit, EFFICIENCY OF RICE CO-TRANSFORMATION using pAPI141, Gt1SDMF and Gt1SDMR, was followed to OVEREIGHT PLASMIDS create the Not Site. The resulting plasmid was named Selectable Selectable Marker Gene Target Gene pAPI188(Kan). The mutation was confirmed by sequencing. Target marker PCR PCR PCR PCR 0387. The coding sequences for selected genes which plasmid plasmid positive negative positive negative encode proteins integral to the lignan biosynthetic pathway pAPI65 pAPI76 33 O 33 O are presented in FIGS. 7A-C, 8A-C, 9A-C, and 10A-C. The pAPI72 pAPI76 27 O 27 O coding Sequences were full length for dirigent protein; pAPI96 pAPI76 3O O 3O O laccase; pinoresinol/lariciresinol reductase (reductase); pAPI35 pAPI76 26 O 22 4 while the coding Sequence for SecoSiSolariciresinol dehy pAPI98 pAPI76 13 O 13 O PAP90 pAPI76 13 O 13 O drogenase (dehydrogenase), lacks at least the Met codon. pAPI64 pAPI76 13 O 13 O 0388 1. CLONING THE LACCASE GENE pAPI78 pAPI76 28 O 26 2 (LACC) INTO AN EXPRESSION CASSETTE TOTALS 183 O 177 6 0389) Two oligonucleotide primers, LACCF (SEQ ID NO: 9) and LACCR (SEQ ID NO: 10) were synthesized based on the laccase gene Sequence. LACCF contains a Not EXAMPLE 2 site while LACCR contains an Xbal site. The 1745 bp PCR product generated from the two primers was digested with Promoter Cassette for Tissue-Specific Expression of Not and Xbal. The processed PCR products were then Lignan Genes in Rice. ligated to the paPI 188 vector, prepared as described above. After ligation, transformation and mini-Screening of colo 0381 A. EXPRESSION PLASMIDS CONTAIN nies, a positive clone was identified and the junction ING LIGNAN BIOSYNTHESIS GENES Sequence between the vector and the gene were confirmed as EXPRESSED UNDER THE CONTROL OF THE correct. A restriction map of the plasmid (FIG. 6B), its DNA ENDOSPERM-SPECIFIC GT1 PROMOTER Sequence and the coding Sequence and corresponding 0382 Four plasmids were generated and used to express amin.o acid Sequence for laccase are presented in FIGS. genes involved in lignan biosynthesis in developing rice 7A-C (SEQ ID NO: 11 and SEQ ID NO: 12, respectively). Seed The plasmids contain the coding Sequence for dirigent protein; laccase; pinoresinol/lariciresinol reductase; and 0390) 2. CLONING THE DIRIGENT PROTEIN SecoSisolariciresinol dehydrogenase under the control of the GENE (DIRG) INTO AN EXPRESSION CAS rice endosperm-specific glutelin (Gt-1) promoter: pGt-1- SETTE DIRG, dirigent protein; pGt-1-LACC, laccase; pGt-1-REDS 0391) Two oligonucleotide primers, DIRGF (SEQ ID pinoresinol/lariciresinol reductase; and pGt-1-DEHY seco NO: 13) and DIRGR (SEQ ID NO: 14) were synthesized Sisolariciresinol dehydrogenase. based on the dirigent protein gene Sequence. DIRGF con tains Not site while DIRGR contains XhoI site. The 612 bp 0383. In order to enhance the level of the plant lignans PCR product generated from these two primers was digested Secoisolariciresinol and matairesinol in rice endosperm, four with Not and XhoI. The processed PCR products were then key proteins in the G-lignan biosyntheitc pathway were ligated to the paPI 188 vector, prepared as described above. placed under the control of the rice glutelin 1 promoter, After ligation, transformation and mini-Screening of colo (Gt1) in preparing vector constructs for Subsequent trans nies, a positive clone was identified and the junction formation. Sequence between the vector and gene were confirmed as 0384 Promoter cassettes were developed for expressing correct. A restriction map of the plasmid (FIG. 6A), its DNA lignan genes in rice endosperm tissues using the endosperm Sequence and the coding Sequence and corresponding Specific glutelin (Gt-1) promoter. amin.o acid Sequence for dirigent protein are presented in FIG. 8A-C (SEQ ID NO: 15 and SEQ ID NO: 16, respec 0385) The glutelin 1 gene promoter was cloned using two tively). primers based on the glutelin 1 (Gt1) gene Sequence (Okita et al., 1989). The forward primer with a HindIII site was 0392) 3. CLONING THE PINORESINOL/LAR designated MV-Gt1-F1 (SEQ ID NO. 5), and the reverse ICIRESINOL REDUCTASE GENE (REDS) primer was designated Xba-gt1-R1 (SEQ ID NO: 6). Crude INTO AN EXPRESSION CASSETTE DNA was isolated from leaves of the rice variety M202 and 0393 Two oligonucleotide primers, REDSF (SEQ ID the PCR product amplified from the crude DNA cloned into NO: 17) and REDSR (SEQ ID NO: 18) were synthesized pCR2.1 (Invitrogen, CA) in both orientations. The resulting based on the reductase gene sequence. REDSF contains Not plasmid was named pCRGT1 or p API 134. site while REDSR contains a Sall site. The 1013 bp PCR 0386 To generate the Gt1 expression cassette plasmid, product generated from these two primerS was digested with pAPI 134 was digested with Hind III and Xba I and the Not and SalI. The processed PCR products were then fragment containing Gt1 promoter was cloned into ligated to the paPI 188 vector digested with Not and XhoI pAPI135, which contains the nos termin.ator. The resulting (The ends generated by SalI and XhoI are compatible). After US 2002/0174452 A1 Nov. 21, 2002 22 ligation, transformation and mini-Screening of colonies, a Seed. The plasmids contained the coding Sequence for diri positive clone was identified and the junction Sequence gent protein; laccase; pinoresinol/lariciresinol reductase; between the Vector and the gene were confirmed as correct. and SecoSisolariciresinol dehydrogenase under the control of A restriction map of the plasmid (FIG. 6C), its DNA the rice alleurone-specific Chitinase 26 (Chi26) promoter: Sequence and the coding Sequence and corresponding pChi26-DIRG, dirigent protein; pChi26-LACC, laccase; amin.o acid Sequence for pinoresinol/lariciresinol reductase pChi26-REDS pinoresinol/lariciresinol reductase; and are presented in FIGS. 9A-C (SEQ ID NO: 19 and SEQ ID pChi26-DEHY secosisolariciresinol dehydrogenase. NO: 20, respectively). 0400 An expression cassette (pAPI217) was constructed 0394 4. CLONING THE SECOISOLARICIR containing the Chi26 promoter, the NoS termin.ator and ESINOL DEHYDROGENASE (DEHY) GENE multiple cloning sites between the two for easy cloning INTO AN EXPRESSION CASSETTE (FIG. 11). The coding sequences for the laccase, dirigent 0395. Two oligonucleotide primers, REHYF (SEQ ID protein, reductase and dehydrogenase genes were removed NO: 21) and DEHYR (SEQ ID NO: 22) were synthesized from the Git-1 plasmids API244 (pGt1-DIRG), p.API245 based on the dehydrogenase gene Sequence. DEHYF con (p.G11-LACC), p.API246 (pCit1-REDS) and pAPI249 (pGt1 tains Not site whileDEHYR contains XhoI site. The for DEHY), and cloned into a Chi26 expression vector, ward primer contains ATG start codon which is followed by pAPI217. Large Scale preparations of each vector were the GCC ACT codons. According to Lewis Lab, the trun made. cated dehydrogenase produced in E coli showed correct 04.01. Once positive clones were identified and confirmed function. The 886 bp PCR product generated from these two by restriction digestion and DNA sequencing, large Scale primers was digested with Not and XhoI. The processed preparation of plasmids was carried out using a Qiagen PCR products were then ligated to the pPAI188 vector, Plasmid Preparation Kit. The plasmids were confirmed to prepared as described above. After ligation, transformation contain the correct fragments by restriction digestion as and mini-Screening of colonies, a positive clone was iden Summarized below in Table 3. tified and the junction Sequence between the vector and the gene were confirmed as correct. The restriction map of the TABLE 3 plasmid (FIG. 6D), its DNA sequence and the coding Sequence and corresponding amin.o acid Sequence for Sec PLASMIDS FOR EXPRESSION OF oisolariciresinol dehydrogenase are presented in FIG. LIGNAN BIOSYNTHESIS GENES 10A-C (SEQ ID NO: 23 and SEQID NO: 24, respectively). Pro- Termi- Diagnostic Diagnostic 0396 Table 2 presents characteristics of the plasmids Plasmid moter Gene nator enzyme fragments (bp) used to express lignan biosynthesis genes under the control pAPI260 Chi26 Laccase Nos EcoRI/ 2011/3524 of the endosperm-specific Gt-1 promoter. Xbal pAPI261 Chi26 Dirigent NOS Xba 6O4/38O3 pAPI262 Chi26 Reductase NOS Xba 414/568/38O3 TABLE 2 pAPI263 Chi26 Dehydro- NOS Xba 882/3803 genase PLASMIDS FOR EXPRESSION OF GENES INVOLVED IN LIGNAN BIOSYNTHESIS. Pro- Diagnostic Diagnostic 0402 Insert DNA was prepared by taking 5 lug of each Plasmid moter Gene Termin.ator Enzyme Fragment (bp) DNA plasmid (pAPI244: pGt1-DIRIG, pAPI245: pGt1 pAPI245 Gitl Laccase Nos EcoRI/ 689/4666 LACC, p.API246: pGT1-REDS and pAPI249: pgt1-DEHY) XhoI and digesting with 10 units of Not at 37 C. for 1 h under pPAI244 Gitl Dirigent Nos Not/XbaI 594/3633 Standard reaction conditions. pPAI246 Gitl Reductase Nos Not/Xbal 414/558/3633 pPAI249 Gitl Dehydro- Nos NotIXhoI 866/3639 0403 Vector DNA was prepared by taking 5 lug of genase Chi26-Gus plasmid (pAPI217) and digesting with 10 units of BarnHI at 37 C. for 1 h under standard reaction condi tions. Digested sites were filled in with T4 polymerase to 0397) 5. EXPRESSION PLASMIDS CONTAIN ING LIGNAN BIOSYNTHESIS GENES generate a blunt end under Standard reaction conditions. UNDER THE CONTROL OF THE ALEURONE 04.04 The DNA was then extracted with chloroform by SPECIFIC CHITINASE GENE PROMOTER mixing and inverting Several times, then centrifuging at (CH126) 14,000 rpm for 5 min. (RT), followed by transferring the Supernatant to a new tube, adding 50 ul of 7.5M NH4OAC, 0398 Promoter cassettes were also developed for 300 ul of 100% chilled ethanol and inverting the tube several expressing lignan genes in rice aleurone tissues under the times, then immediately centrifuging at 14,000 rpm for 20 control of the barley chitinase gene promoter (Chi26). The min. (4° C). The Supernatant was removed and 5001 ul of Barley chitinase 26 promoter was PCR-amplified from the 70% cold ethanol added, followed by centrifuging at 14,000 genomic DNA of barley (Hymalaya), using primers rpm for 5 min. The Supernatant was removed and the DNA Chi26FW (SEQ ID NO:36) and Chi26RV (SEQ ID NO: pellet air-dried. The pellet was dissolved in 35 ul of ddH2O 37). The resulting PCR product was digested with PstI and and digested with EcoRI for 37° C. for 1 h. A 1% CTC Xbal, and then used to replace the CaMV 35S promoter of agarose gel was run at 100V for 1 h using the undigested pBI221. plasmid as a control. The DNA fragments were cut from the 0399 Four plasmids were generated and used to express gels, put in a 1.5 ml centrifuge tube together with the same genes involved in lignan biosynthesis in developing rice amount (V/w) of Buffer QG (QIA quick Gel Extraction Kit), US 2002/0174452 A1 Nov. 21, 2002 then incubated at 50° C. for 10 min... to melt the gel ferred that calli for transformation have the following char completely. The same volume of 100% cold isopropanol was acteristics: a Size of 1.0-3 mm in diameter, a spherical mass ten added and the mixture spun down at 14,000 rpm for 1 with a rough exterior, a white and opaque color and a density min.., another 500 ul of QG buffer added, followed by which is compact and hard. pelleting at 14,000 rpm for 1 min.., addition of 750 ul of PE buffer and again pelleting at 14,000 rpm for 1 min. 50 ul of 0411 Prior to transformation, approximately 200 calli are EB buffer was then added to the center of filter tube, allowed placed together in the center of a plate (about 4 cm in to stand for 1 min., then pelleted at 14,000 rpm for 1 min..., diameter) on N6 semisolid medium and incubated at 28°C. followed by addition of 25ul of 7.5M NH4OAC, 175ul of in the dark for 24 hours. 100% cold ethanol and immediately centrifuging at 15,000 0412 60 mg 1.0 uM sized gold particles are weighed into rpm for 20 min. (4° C). The Supernatant was removed and a sterile 1.5 ml microcentrifuge tube, 1 ml of 100% EtOH 500 ul of 70% cold ethanol added, followed by centrifuging added, followed by vortexing 2 min.utes at 1600 rpm. The at 14,000 rpm for 20 min. The Supernatant was removed and particles are centrifuged for 10 Seconds at full Speed the the DNA pellet air-dried, then dissolved in 10 ul of dd H2O. Supernatant discarded, then washed with 1 ml of Sterile did 04.05 50 ng of each insert DNA was ligated into 5 ng of H2O 3 times, with vortexing for 2 min. at 1600 rpm each vector DNA under Standard reaction conditions overnight at time, followed by centrifuging for 10 Seconds and discard room temperature. The following day, 5 ul of ing the Supernatant. The gold particles are resuspended in 1 7.5MNH4OAC and 35ul of 100% cold ethanol was added, ml sterile dd HO, and stored at -20° C. until used. and the mixture was and immediately centrifuged at 15,000 0413 DNA from plasmids containing the Gtl promoter rpm for 20 min. (4° C). The Supernatant was removed and driven lignan genes: Reductase (PAPI 246), Laccase (pAPI 500 ul of 70% cold ethanol added, followed by centrifuging 245), Dehydrogenase (pAPI 249), and Dirigent (pAPI 244); at 14,000 rpm for 5 min. The Supernatant was removed and and the Chi26 promoter driven lignan genes: Reductase the DNA pellet air-dried, then dissolved in 3 ul of ddH.O. (pAPI 262), Laccase (pAPI 260), Dehydrogenase (pAPI 263) and Dirigent (pAPI 261) was prepared as described EXAMPLE 4 above and the concentration determined for transformation of calli. Rice Transformation With Lignan Gene Expression Constructs Containing the GT1 Promoter or the 0414 50 ul of washed gold particles were pipeted into a CHI26 Promoter sterile 1.5 ml centrifuge tube, 5-20 lug of DNA added, the mixture is vortexed 3 seconds at 1300 rpm with 20 ul of 0406 To elevate the lignan concentration in transgenic 0.1M spermidine added while vortexing (1200 rpm) and 50 rice Seeds, eight high expression plasmids containing lignan All of 2.5M CaCl2 added in a dropwise fashion while biosynthesis genes were constructed including paPI244 Vortexing, followed by Vortexing an additional 10 min.utes, (pGt1-DIRG), p.API245 (pGt1-LACC), p.API246 (pGt1 incubating at room temperature for 10 min.utes, centrifuging REDS), pAPI249 (pGt1-DEHY), each with the lignan bio for 10 seconds, and discarding the Supernatant. The DNA Synthesis gene expressed under the control of the rice gold particles were then resuspended in 20 ul of cold 100% endosperm-specific glutelin (Gt-1) promoter which becomes ethanol and incubated on ice before loading onto microcar active in endosperm during the rice Seed maturation; and CS. pAPI260 (pChi26-LACC), p.API261 (pChi26-DIRG), pAPI262 (pChi26-REDS) and pAPI263 (pChi26-DEHY), 0415) DNA-gold particles were loaded onto a microcar each with the lignan biosynthesis gene expressed under the rier by pipeting 10 ul of DNA-gold particles onto the center control of the Chi26 promoter or the barley Chitinase 26 (about 1 cm) of microcarriers, spreading as evenly as (Chi26) gene promoter which becomes active in the alleu possible, then allowing them to air dry. rone layer during Seed maturation. 0416 Biolistic bombardment was carried out with the Biolistic PDC-1000/He system (BIO-RAD). The procedure 0407 A. RICE TRANSFORMATION AND required 1.5 mg of gold particles (60 ug/ul) coated with 2.5 PLANT REGENERATION lug paPI76 DNA and cotransfer plasmid DNA at a ratio of 0408. The procedure for microprojectile-mediated rice 1 to 6. DNA-coated gold particles were bombarded into the transformation (Sivamani et al., 1996; Chen et al., 1998) was rice callus with a helium pressure of 1100 psi. followed with modifications. (See FIG. 12.) Approximately 0417 Exemplary bombardment conditions include: 200 TP309 seeds were dehusked, sterilized in 50% com vacuum-27 inches Hg, rupture disk-1100 psi; Cell (tar mercial bleach for 25 min. and washed with sterile water get) position-8 cm from Stopping Screen; and two bom three times for 5 min. each. The sterilized seeds were placed plates containing N6 Semisolid medium Supplemented with bardments per plate. 2,4-D 2 mg/L, Sealed with parafilm and incubated in the dark 0418. After bombardment, the calli were allowed to at 28 C. for 14 days to induce calli. recover on the same plate for 48 hrs and then transferred to N6 media containing 20 mg/l Hygromycin B. The bom 04.09 Developing calli (less than 1 mm in diameter) were barded calli were incubated on the Selection media in the then transferred developing onto fresh N6 medium (35 calli dark at 26 C. for 45 days. At this time, transformants were per plate) for 30 days and serve as bombardment material. Selected based on their white, opaque, compact appearance Calli were subcultured every 15 days. and transferred to pre-regeneration media consisting of N6 0410 Prior to bombardment, calli from 30-day old Sub with 5 mg/l ABA, 2 mg/l BAP, 1 mg/l NAA and 20 mg/1 cultures are Selected with size, shape, color, and density very Hygromycin B for 9 to 12 days. Transformants are easily important factors for Successful transformation. It is pre distinguished from non-transformants which appear yellow US 2002/0174452 A1 Nov. 21, 2002 24 ish or brown, soft, and watery. The transformants were then 0422 A. PCR ANALYSIS OF TRANSGENIC transferred to regeneration media (RN) consisting of N6 PLANTS (without 2,4-D) 3 mg/l BAP, and 0.5 mg/l NAA without Hygromycin B and cultured under continued lighting con 0423 Crude DNA suitable for PCR analysis was obtained ditions for about two weeks. using a simplified procedure was. In carrying out the 0419 When the regenerated plants were 1 to 3 cm high, method, about 2-3 cm of young leaf tissue was collected in plantlets were transferred to rooting media containing 0.05 a 1.5 ml eppendorf tube and the DNA extracted in the same mg/l NAA which was half the strength of MS media. After tube and used as template DNA. The PCR reaction mixture approximately two weeks in rooting media, plantlets devel contained about 50 ng of template DNA, 50 ng of each of oped roots shoots of 10 cm or more. The plants are then primers, 0.05 mM dNTPs, 1x PCR buffer (10 mM Tris pH transferred to a 2.5 inch pot containing 50% commercial 8.4, 50 mM KC1, 2.0 mM MgCl2, and 0.01 mg/ml gelatin) soil, Sunshine #1 (Sun Gro Horticulture Inc, WA) and 50% and 1 unit of Taq DNA polymerase in a volume of 50 ul. The natural Soil from rice fields. The pots are placed within a template DNA was initially denatured at 94 C. for 2 plastic container which is covered by another transparent min.utes followed by 28 cycles of PCR amplification with plastic container to maintain 100% humidity and cultured the following parameters: 30 seconds denaturation at 94 C., under lighting conditions for 1 week, after which the trans parent plastic cover was opened little by little during one day 90 seconds primer annealing at 58 C., and 90 seconds to gradually reduce the humidity. Afterwards, the plastic primer extension at 70° C. A final 5 minutes incubation at cover was removed completely, and water and fertilizers 72 C. allowed for completion of primer extension. The added as necessary. When the plants grew to approximately amplified products were electrophoretically resolved on a 12 cm tall, they were transferred to a greenhouse and 1.5% agarose gel in 1xTAE buffer. allowed to grow to maturity. During potting, typically 2-3 0424 Four pairs of PCR primers were designed to detect cm of leaf tissue is taken for PCR analysis to determine the presence of all four lignan genes in a single plant (Table whether the target genes were present in the plantlets. 4). The resulting PCR reactions indicated that all four lignan 0420. In order to obtain large number of transformants, biosynthesis genes could be Simultaneously detected in a 173 plates of explants were bombarded with Gt1 constructs. single PCR reaction (FIG. 15).

TABLE 4

PCR PRIMERS

Product Size Promoter Plasmid Primers (5' to 3') (bp) Gtl Reductase TCATTGGGGGTACAGGGTACTTAG (SEQ ID NO:38) 5 OO (pAPI 246) TGCCAAATTGACAGAGACCTCC (SEQ ID NO:39

Gtl Laccase TGCTAGTGCTTCCTCTTCATGCTGC (SEQ ID No.: 40) 452 (pAPI 245) CCCCTAATATGATTGTCGCTTCCGC (SEQ ID NO: 41) Gt Dehydrogenase GAGCCAGTGGAGTTGGAGAAGTC (SEQ ID NO: 42) 423 (pAPI 249) GCATGTGAAGAACCACCACCC (SEQ ID NO: 43) Gt Dirigent CAATGCCACTTCCGCCATAG (SEQ ID NO:44) 321 (pAPI 244) CGCCATGAAAAAGTCACCAGTTCC (SEQ ID NO: 45)

After selection more than 3500 transgenic calli were 0425 The transformation for eight different lignan gene Selected and placed on regeneration medium, resulting in the containing plasmids was determined and the results indi generation of over 700 transgenic plants, and over 140 plant cated that almost all the plants expressed the target gene and Seed Sets. the Selectable marker gene. 0426. After hygromycin-based selection, 100 randomly EXAMPLE 5 chosen plantlets were analyzed by PCR to confirm the presence of those genes in rice cells. A comparison of Analysis of Transgenic Rice Genomic DNA by positive (+control) and negative controls (untransformed/- PCR and Southern Blot control) revealed that out of 100 plants derived from trans 0421 Hundreds of transgenic plants were produced by genic calli 59 plants (59%) were positive for all four co-transformation of rice with the four lignan gene expres transgenes, 22 plants carried three genes, 7 plants were Sion constructs. PCR analysis of Seedling tissues from these positive for two transgenes, 7 plants were positive for one plants indicated that many of the plants carried the lignan transgene and 5 plants were negative for all four transgenes. biosynthesis genes. The transgenic Seedlings were trans Given that the PCR primers were targeted to a portion of planted into larger pots and moved to the greenhouse where each of the four transgenes, Southern blot analysis was they developed into healthy mature plants. An analysis of carried out on the PCR positive plants to confirm that full transgenic rice genomic DNA indicated that the majority of length transgenes were present in the genome of those the plants carried multiple lignan biosynthesis genes. plants. US 2002/0174452 A1 Nov. 21, 2002 25

0431 FIG. 15 shows the PCR banding pattern of lignan TABLE 5 genes in transgenic rice plants. Each plant carries a different number of genes. PCR RESULTS 0432 C. LIGNAN PRODUCTION IN TRANS Plants Plants Plants Plants Plants GENIC RICE Plant # with with with with with 0433 Lignan production was evaluated in transgenic rice analyzed four genes three genes two genes one gene no genes plants which expressed lignan biosynthesis genes. Mataires 1OO 59 (59%) 22 (22%) 7 (7%) 7 (7%) 5 (5%) inol concentrations in 142 transgenic Seeds were measured, and analysis revealed that 21 transgenic plants produced Seeds with elevated lignan levels. 0427 B. SOUTHERN BLOT ANALYSIS OF TRANSGENIC PLANTS TABLE 6. MATAIRESINOL (NG/100MG) ING 0428. About 3 gm of young leaves were collected from TRANSGENIC RICE SEEDS. RO transgenic plants and ground with liquid nitrogen into 0434 Total number of samples (R1 seeds) is 142. Cat very fine powder. The powder was then placed in a 50 ml egory 5 is selected for advancing to R2/R3 and other tube and 25 ml of extraction buffer (100 mM Tris-HCL, pH Subsequent generations (see Table 7). 8.0, 50 mM EDTA, pH 8.0, 500 mM NaCl, 1.25% SDS, 0.38% sodium bisulfite) was added. The mixture was incu TABLE 6 bated 15 min.utes at 65° C. with repeated shaking. Ten ml of 5M potassium acetate was added and the Samples were MATAIRESINOL (NG/100 MG) IN TRANSGENIC RICE SEEDS. incubated on ice for 20 min.utes with shaking. The tubes Total number of samples (R1 seeds) is 142. Category 5 is selected for were centrifuged at 3000 rpm for 20 min.utes and the advancing to R2/R3 and other subsequent generations (see Table 7). Supernatant was filtered into another 50 ml tube through a Category Number Range Mean SD miracloth. Two Volumes of isopropanol were added and the 1. 28 O.21-0.94 O.53 O.22 tubes were kept at room temperature for 30 min.utes, then 2 26 1.06-1.99 1.47 O.28 centrifuged at 3000 rpm for 10 minutes. The DNA pellets 3 42 2.04-4.99 3.37 O.98 were washed in 70% alcohol and resuspended in 5 ml of TE 4 25 5.05-8.73 6.42 1.09 5 21 10.54-48.9 19.8 8.33 buffer (with RNase) and incubated at 37° C. for 30 min. Non- 7 O.28-222 1.13 0.78 Then 0.5 ml of 3M Sodium acetate and 2 volumes of transgenic pre-chilled absolute alcohol were added to the tubes and incubated at 4 C. for 30 min. The DNA was hooked out, washed with 70% ethanol in a 1.5 ml tube and further 0435) resuspended in TE buffer. About 5 ug of HindIII/EcoRI digested DNA samples from each transgenic line were used TABLE 7 to make a blot for Southern analysis. 0429. The ECLTM direct nucleic acid labeling and detec SELECTED LIGNAN PRODUCING LINES IN CATEGORYS tion System (Amersham) was used. Briefly, it involves Line # Matairesinol (ng/100 mg) directly labeling DNA with horseradish peroxidase by com 4PE-2O-1 9.26 pletely denaturing the probe So that it is in a Single-Stranded 4PE-25-1 5.92 form. Then, peroxidase conjugated with a positively charged 4PE-57-1 32.6 polymer is added and forms a loose attachment to the nucleic 4PE-59-1 6.88 acid by charge attraction. Addition of gluteraldehyde causes 4PE-8O-1 O.54 4PE-94-1 3.31 the formation of chemical croSS-linkS So that the probe is 4PE-102-1 20.88 covalently labeled with enzyme. After hybridization the 4PE-103-1 9.71 detection Solution is comprised of two detection reagents 4PE-104-1 2012 4PE-112-1 9.62 (DRI and II). DR-I decays to hydrogen peroxide, the Sub 4PE-115-1 24.51 Strate for peroxidase. Reduction of hydrogen peroxide by the 4PE-131-1 2.26 enzyme is coupled to the light production reaction promoted 4PE-147-1 5.84 by DR-II. This contains lumin.ol, which upon oxidation 4PE-212-1 7.5 produces blue light. Hybridization is the re-annealing of a 4PE-213-1 26.22 4PE-230-1 5.12 labeled single stranded DNA probe with a complementary 4PE-245-1 8.64 Sequence of genomic DNA on the filter. 4PE-256-1 9.29 4PE-264-1 7.06 0430. About 300 ng of probe (plasmid digested with NotI 4PE-265-1 48.90 and Xhol) was used for each hybridization reaction. The 4PE-348-1 2.63 inserts were directly eluted from 0.7% LMP agarose and labeled with the labeling reagent. Pre-hybridization and hybridization (ECLTM GOLD buffer with 500 mM NaCl and 0436. In carrying out the analysis, rice R1 seeds har 5% nonfat milk) were performed at 42° C. Blots were Vested from transgenic plants were dissected into two washed with primary wash buffer (0.5xSSC, 0.1% SDS, 6N halves. Embryo halves were kept and used to recover R1 Urea) for 2x30 min. at 43° C. and 2x10 min. with 2xSSC at transgenic plants. Endosperm halves were used to deter room temperature with Shaking. Then the blots were min.e. the concentration of lignan. About 7 half Seeds from Shocked with the detection reagent for two min.utes and then each transgenic plant were prepared to obtain over 100 mg exposed to X-ray film. of material for lignan assay (FIG. 13). US 2002/0174452 A1 Nov. 21, 2002 26

0437 Embryo halves of transgenic rice seeds were ster in 1 ml CH-OH, was loaded onto the DEAE-Sephadex ilized in 50% w/v commercial bleach for 25 min. and washed (hydroxyl form) column. The column was eluted with 2.5 ml with sterile water three times for 5 min. each. Sterilized half CH-OH to first remove the neutral steroids. Lignans were Seeds were placed into test tube (one embryo/tube) contain obtained by eluting with 0.1 MHAc in CH-OH (10 ml). This ing MS media. The test tubes were then placed under Strong fraction was evaporated to dryneSS under N. light inorder to develop healthy plants from the embryos to obtain R2 seeds. 0444 4. CHROMATOGRAPHY ON QAE SEPHADEXAC. 0438) 1. ENZYMATIC HYDROLYSIS 0445 QAE-Sephadex Ac was used to remove used to 0439 1 ml of distilled HO was added to a freeze-dried remove organic acids and many chromogens. The residue B, Sample (100 mg) with the resulting slurry Stirred at room dissolved in 1 ml CH-OH, was loaded onto the QAE temperature overnight. Hydrolysis is carried out in a glass Sephadex (acetate form) column (0.5 i.d.x6 cm) equilibrated tube in 2 ml of 0.3 M acetate buffer (pH 4.1) with 5000 in CH-OH. The column was eluted with 8 ml CH-OH to Fishman units of Helix pomatia juice. Before hydrolysis, 2.5 give a fraction (9 ml) containing the lignans of interest. After mg ascorbic acid was added for the protection of labile evaporation to dryneSS under N2, the residue was ready for metabolites. The sample was gently mixed with a Vortex TMS-derivatization. mixer and incubated at 60° C. in a Dri-Block, for 2 h. After hydrolysis, the Sample was cooled to room temperature, 0446 5. PREPARATION OF TMS-DERIVA extracted with cold Et2O (3 mlx5). The EtO extracts were TIVES combined and evaporated to dryneSS under nitrogen with gentle heating (45 C.) in a Dri-Block to give residue A, 0447 The dry samples were silylated by adding 100 ul of which was stored at -20° C. The water phase was then the silanization reagent (Tri-Sil, Pierce Co.). After incuba Subjected to acid hydrolysis. tion at 60° C. for 15 min., the solvent was evaporated to dryneSS under N. The resulting residue was then dissolved 0440 2. ACID HYDROLYSIS in 50 ul n-hexane containing 3% Sillyl-8 (column condi 0441 To the water phase (2 ml) was added 6 M HCl (1 tioned). The n-hexane extracts were transferred to Small ml) to obtain a final concentration 2 M. The sample was vials with 1 ul of which submitted to GC-MS analysis. mixed with Vortex mixer and incubated at 100 C. in the 0448 6. GC-MS-SIM ANALYSIS Dri-Bock. After 2.5 h, the sample was cooled to room temperature and the pH adjusted to 6 with 0.6 ml 10 M 0449 The GC-MS instrument consisted of a gas chro NaOH. Deuterium-labeled secoisolariciresinol (4) (67 ng), matograph (HP 6890 Series) and a HP 5973 quadrupole anhydrosecoisolariciresinol (5) (72 ng) and matairesinol (3) mass spectrometer equipped with a HP 7683 autoinjector. A (55.2 ng) were next added (in CH-OH, 20 ul) as internal 30 mx0.25 mm column (0.25 um film thickness) was used standards for GC-MS analysis. The sample was then (HP-5MS, crosslinked 5% PHME Siloxane). The carrier gas extracted with EtO (4 mlx5). This fraction was combined was helium (flow rate: 1.4 ml/min.). The initial temperature with residue A obtained after enzymatic hydrolysis. Finally, of the oven was 160° C., it was then increased by 30° C. after evaporation of the Solvent, the dry Sample was re every min.ute, to a final temperature of 280 C. and held at dissolved in 1 ml CH-OH. this temperature for 10 min. The calculation of the results is carried out by comparing the peak area ratioS of the ions 0442. 3. CHROMATOGRAPHY ON DEAE for the plant-derived compounds and the deuterated internal SEPHADEX OH Standards d8-secoisolariciresinol (4), d6-anhydrosecoisola 0443) The free base form of DEAE-Sephadex was used to riciresinol (5) and d6-matairesinol (3) with the same peak remove neutral steroids, etc. The DEAE-Sephadex was area ratioS of the calibrators used for preparing the Standard packed in CH-OH in a Pasteur pipette (0.5 i.d.x3 cm) with curve. Standard curves were obtained with the lignans (1) - a Small piece of cotton in the bottom. The Sample, dissolved (3) using between 5 and 200 ng of each.

TABLE 8

SEQUENCE LISTING TABLE

Description EQ ID NO

1AR1 : 5' AAC AAT ACT GGA ATT CGA GAA GTA AAA AG 3'

1Asma : 5' CTA CGC AAC CCG GGA GAA AAT C 3'

GnsF 5' GAC TTA ACT, TTA GTC ATA TTT AG 3'

GnsR 5 TTC GCT. CTT, GCT, GCT, GCT CACT 3'

MV-Gt1-F1 : 5' ATCGAAGCTTC ATGAGTAATGTGTGAGC ATTATGGGACCACG-3' 5 US 2002/0174452 A1 Nov. 21, 2002 27

TABLE 8-continued SEQUENCE LISTING TABLE Description EQ ID NO

Xba-gt1-R1 : 5' - CTAGTCTAGA CTCGAGCCACGGCC ATGGGGCCGGCTAGGGA 6 GCCATCGCACAAGAGGAA-3' Gt- 1 promoter sequence 7 Chi26 promoter sequence 8

LACCF CATATGAACAGCGGCCGCTTGTGAACATTGAGTTAAATATG 9

LACCR: GGAGGTCTATACGGAGGTACAACTAGATCTGATC O nucleic acid sequence encoding Lac case (FIGS. 7A-C) 1 Laccase amin. o acid sequence (FIGS. 7A-C) 2

DIRGF: GGCTCAAGAAGCGGCCGCGGCACGAGATTAAACCAAACATGG 3

DIRGR: GCTATAATTAAACATACTTACAACCATTGAGCTCGCCGT 4 nucleic acid sequence encoding dirigent protein (WO 98/20113) seq (FIGS. 8A-C) 5 dirigent protein amin. o acid sequence (FIGS. 8A-C) 6

REDSF GGCTCAAGAAGCGGCCGCCACGAGAAAAACAGAGAGAGATGGG 7

REDSR: ATGGAGTTCGCAATGCACATCAGCTGCGCA 8 nucleic acid sequence encoding pinoresinol/lariciresinol reductase (reductase) (FIGS. 9A-C) 9 pinoresinol/lariciresinol reductase amin. o acid sequence (FIGS. 9A-C) 2O

REHYF ACAAAAAGCGGCCGCTTCATTAGTCCTACAACAACATGG CCACTT 21 CACAGCTTCGAAC

DEHYR GTCTGAGAACTAGTAACGTGAGCTCAGATCT 22 nucleic acid sequence encoding secoisolariciresinol dehydrogenase (dehydrogenase) (FIGS. 10A-C) 23 secoisolariciresinol dehydrogenase amin. o acid sequence (FIGS 10A-C) 24 chalcone synthase coding sequence from rye (Genbank Accession No. X92548) 25 PCR primer designed to detect laccase (pAIPI 245) : TGCTAGTGCTTCCTCTTCATGCTGC 26 PCR primer designed to detect laccase (pAPI 245) : CCCCTAATATGAGATTGTCGCTTCCGC 27 PCR primer designed to detect dirigent protein (pAPI 244) : CAATGCCACTTCCGCCATAG 28 PCR primer designed to detect dirigent protein (pAPI 244) : CGCCATGAAAAAGTCACCAGTTCC 29 PCR primer designed to detect reductase (pAPI 246) : TCATTGGGGGTACAGGGTACTTAG 30 PCR primer designed to detect reductase (pAPI 246) : TGCCAAATTGACAGAGACCTCC 31 PCR primer designed to detect dehydrogenase (pAPI 249) : GAGCCAGTGGAGTTGGAGAAGTC 32 PCR primer designed to detect dehydrogenase (pAPI 249) : GCATGTGAAGAACCACCACCC 33

Gt1SDMF CTCATTGTTTCTCACAAAAAGCGGCCGCTTCATTAGTCCTACAACAACATGGC 34

Gt1SDMR GCCATGTTGTTGTAGGACTAATGAAGCGGCCGCTTTTTGTGAGAAACAATGAG 35

Chi26FW: AACCCTCTCTGCAGTCACCTCCTGTGAAGT 36

Chi26RW: CGGAGCGATCTAGATGTGCGAGCCAACAAA 37 US 2002/0174452 A1 Nov. 21, 2002 28

The embodiments of the invention in which an exclusive hybridizing under high Stringency conditions and Said pro property or privilege is claimed are defined as follows: tein or enzyme has Substantially equivalent biological activ 1. A method of increasing the guaiacyl- (“G-)lignan ity to the native protein or enzyme Selected from the group content in Seeds of a monocot plant, comprising consisting (i) a dirigent protein (SEQ ID NO:16), (ii) pinoresinol/lariciresinol reductase (SEQ ID NO:20), (iii) Selecting at least one protein or enzyme integral to the secosisolari-ciresinol dehydrogenase (SEQ ID NO: 24), and pathway leading to G-lignan formation (iv) laccase (SEQ ID NO:12). Stably transforming a monocot plant with one or more 12. A transformed monocot plant produced by the method chimeric gene constructs having a Seed-specific tran of claim 1. Scriptional regulatory region operably linked to a 13. The transformed monocot plant according to claim 15, nucleic acid Sequence encoding Said at least one protein capable of producing Seeds where the amount of (-)-sec or enzyme integral to the pathway leading to G-lignan oisolariciresinol diglucoside or (-)-matairesinol accumu formation. lated in the Seeds of Said transformed monocot plant is two 2. The method according to claim 1 wherein Said at least or more times the amount detectable in Seeds of an untrans one protein or enzyme integral to the pathway leading to formed monocot plant. G-lignan formation is Selected from the group consisting of 14. The transformed monocot plant according to claim 15, (i) a dirigent protein (SEQ ID NO: 16), (ii) pinoresinol/ capable of producing Seeds where the amount of (-)-sec lariciresinol reductase (SEQ ID NO: 20), (iii) secosisolari oisolariciresinol diglucoside or (-)-matairesinol accumu ciresinol dehydrogenase (SEQ ID NO: 24), and (iv) laccase lated in the Seeds of Said transformed monocot plant five or (SEQ ID NO:12). more times the amount the amount detectable in Seeds of an 3. The method according to claim 2 wherein (i) a dirigent untransformed monocot plant. protein (SEQ ID NO:16), (ii) pinoresinol/lariciresinol reduc 15. A seed composition derived from a plant produced by tase (SEQ ID NO:20), (iii) secosisolari-ciresinol dehydro the method of claim 1. genase (SEQ ID NO:24), and (iv) laccase (SEQ ID NO: 12), are expressed at the same time in Seeds of a monocot plant. 16. The Seed composition according to claim 15, where 4. The method according to claim 2 or 3 wherein the the amount of (-)-secoisolariciresinol diglucoside or (-)- amount of (-)-secoisolariciresinol diglucoside or (-)- matairesinol accumulated in the Seeds of Said transformed matairesinol accumulated in Said Seeds is greater than in monocot plant is two or more times the amount detectable in Seeds of an untransformed monocot plant. Seeds of an untransformed monocot plant. 5. The method according to claim 1 where said seed 17. The Seed composition according to claim 15, where Specific transcript-ional regulatory region is derived from the amount of (-)-secoisolariciresinol diglucoside or (-)- aleurone, pericarp, embryo or endosperm tissue. matairesinol accumulated in the Seeds of Said transformed 6. The method according to claim 1 where said Seed monocot plant is five or more times the amount detectable in Specific transcript-ional regulatory region is induced during Seeds of an untransformed monocot plant. Seed development and corresponds to an endosperm-specific 18. A library comprising seeds derived from one or more Gt-1 promoter. monocot plants produced by the method of claim 1. 7. The method according to claim 1 where said seed 19. A method of producing a progeny monocot plant by Specific transcriptional regulatory region is induced during crossing one or more parent monocot plants produced by the Seed development and corresponds to an aleurone-specific method of claim 1 where the amount of G-lignans in the Chi26 promoter. Seeds of Said progeny monocot plant resulting from Said 8. The method of stably transforming a monocotyledon crossing is greater than the amount of G-lignans in the Seeds ous plant with Said one or more chimeric gene constructs of Said parent monocot plant. according to claim 1 resulting in increased expression of 20. A G-lignan enriched Seed composition for use as a Said genes encoded by Said one or more chimeric gene food additive comprising a Seed preparation derived from COnStructS. Seeds of a transformed monocot plant wherein the amount of 9. The method according to claim 8 where the gene in said (-)-secoisolariciresinol diglucoside or (-)-matairesinol one or more chimeric gene constructs is Selected from the accumulated in Said Seeds is two or more times the amount group consisting of (i) a dirigent protein (SEQ ID NO: 15), detectable in Seeds of an untransformed monocot plant. (ii) pinoresinol/lariciresinol reductase (SEQ ID NO: 19), 21. The use of a G-lignan enriched Seed composition (iii) secosisolari-ciresinol dehydrogenase (SEQ ID NO. 23), according to claim 15 for use as a food additive. and (iv) laccase (SEQ ID NO:11). 10. The method according to claim 2 where the nucleic 22. The method of claim 1 wherein the monocot plant is acid Sequence encoding Said at least one protein or enzyme a rice plant. integral to the pathway leading to G-lignan formation has at 23. A transformed rice plant produced by the method of least 90% sequence identity to a Sequence Selected from the claim 1. group consisting of (i) a dirigent protein (SEQ ID NO:15), 24. A Seed composition derived from a rice plant produced (ii) pinoresinol/lariciresinol reductase (SEQ ID NO: 19), by the method of claim 1. (iii) secosisolari-ciresinol dehydrogenase (SEQ ID NO. 23), 25. A library comprising Seeds derived from rice plants and (iv) laccase (SEQ ID NO:11). produced by the method of claim 1. 11. The method according to claim 2 wherein the nucleic acid Sequence encoding Said protein or enzyme is capable of k k k k k