(12) United States Patent (10) Patent No.: US 8,252,977 B2 Tanksley Et Al

US008252977B2

(12) United States Patent (10) Patent No.: US 8,252,977 B2 Tanksley et al. (45) Date of Patent: Aug. 28, 2012

(54) POLYNUCLEOTIDES ENCODING Czerny, M. and Grosch, W., “Potent Odorants of Raw Arabica Coffee. CAROTENOID AND APOCARTENOID Their Changes During Roasting.” J. Agric. Food Chem... vol. 48, pp. BIOSYNTHETIC PATHWAY ENZYMES IN 868-872, 2000, XP-002431388. Iuchi, S. et al., “Regulation of Drought Tolerance by Gene Manipu COFFEE lation of 9-cis-Epoxycarotenoid Biosynthesis in Arabidopsis,” The (75) Inventors: Steven D. Tanksley, Dryden, NY (US); Plant Journal, vol. 27(4), pp. 325-333, 2001. XP-002973637. Chenwei Lin, Auburndale, MA (US); Ortiz, A. et al., “Volatile Composition of Coffee Berries at Different Andrew Simkin, Tours (FR); James Stages of Ripeness and Their Possible Attraction to the Coffee Berry Gérard McCarthy, Noizay (FR): Borer Hypothenemus Hampei (Coleoptera: Curculionidae).” J. Agric. Food Chem., vol. 52, pp. 5914-5918, 2004. XP-002431391. Vincent Petiard, Tours (FR) Variyar, P.S. et al., “Flavoring Components of Raw Monsooned (73) Assignees: Nestec S.A., Vevey (CH); Cornell Arabica Coffee and Their Changes During Radiation Processing.” University, Ithaca, NY (US) Agricultural and Food Chemistry, vol. 31, pp. 7945-7950, 2003. XP-OO2431390. (*) Notice: Subject to any disclaimer, the term of this Agrawal, G.K., et al., “Screening o The Rice Viviparous Mutants patent is extended or adjusted under 35 Generated by Endogenous Retrotransposon ToS 17 Insertion. Tagging U.S.C. 154(b) by 854 days. of a Zeaxanthin Epoxidase Gene and a Novel OsTATC Gene.” Plant (21) Appl. No.: 11/990,835 Physiol., vol. 125, pp., Vol., pp. 1248-1257, 2001. Akiyama, M. et al. "Analysis of Volatile Compounds Released Dur (22) PCT Filed: Sep. 1, 2006 ing the Grinding of Roasted Coffee Beans Using Solid-Phase (86). PCT No.: PCT/US2O06/0344O2 Microextraction.” J Agric Food Chem... vol. 51(7): pp. 1961-1969, 2003. S371 (c)(1), Albrecht, M. etal. “Molecular Cloning and Functional Expression in (2), (4) Date: Feb. 27, 2009 E. Coli of a Novel Plant Enzyme Mediating -Carotene Desatura (87) PCT Pub. No.: WO2007/028115 tion.” FEBS Letters, vol. 372:, pp. 199-202, 1995. Al-Babili, S. et al. “Identification of a Novel Gene Encoding for PCT Pub. Date: Mar. 8, 2007 Neoxanthin Synthase From Sola tuberosum," FEBS Letters, vol. 485, (65) Prior Publication Data pp. 168-172, 2000. Al-Babili, S. etal. “Biosynthesis of Beta-Carotene (Provitamin A) in US 2009/0178156A1 Jul. 9, 2009 Rice Endosperm Achieved by Genetic Engineering.” Novartis Found Related U.S. Application Data Symp., vol. 236, pp. 219-232, 2001. Arrach, N. et al., “A Single Gene for Lycopene Cyclase, Phytoene (60) Provisional application No. 60/714,106, filed on Sep. Synthase, and Regulation ofCarotene Biosynthesis in Phycomyces,". 2, 2005. PNAS 98(4): 1687-1692, 2001. Aust. O. et al., “Supplementation With Tomato-Based Products (51) Int. Cl. Increases Lycopene, Phytofluene, and Phytoene Levels in Human AOIH 5/00 (2006.01) Serum and Protects Against UV-Light Induced Erythema.” Int. J. AOIH 5/10 (2006.01) Vitam. Nutr. Res. 75:54-60, 2005. CI2N 15/29 (2006.01) Baldwin, E.A. et al., “Quantitative Analysis of Flavor Parameters in CI2N 15/52 (2006.01) Six Florida Tomato Cultivars (Lycopersicon esculentum).” J. Agri, CI2N 15/82 (2006.01) Food. Chem. 39: 1135-1140, 1991. (52) U.S. Cl...... 800/287: 800/285: 800/298; 536/23.2: Baldwin, E.A. et al., “Flavor Trivia and Tomato Aroma: Biochemistry and Possible Mechanisms for Control of Important Aroma Compo 536/23.6:435/320.1; 435/419 nents.” Hort. Sci. 35(6): 1013-1021, 2000. (58) Field of Classification Search ...... 800/285, Bartley, G.E. et al., “A Tomato Gene Expressed During Fruit Ripen 800/287, 298; 536/23.2, 23.6; 435/419, ing Encodes an Enzyme of the Carotenoid Biosynthesis Pathway.”J. 435/320.1 Biol. Chem. 267: 5036-5039, 1992. See application file for complete search history. Bartley, G.E. and Ishida, B.K., "Zeta-Carotene Desaturase From Tomato.” Plant Physiol. 121:1383, 1999. (56) References Cited (Continued) U.S. PATENT DOCUMENTS Primary Examiner — Russell Kallis 6,653,530 B1 * 1 1/2003 Shewmaker et al...... 800,282 2002/0128464 A1* 9, 2002 Busch et al...... 536,236 (74) Attorney, Agent, or Firm — Potter Anderson & Corroon LLP FOREIGN PATENT DOCUMENTS DE 19909 637 A1 3, 1999 (57) ABSTRACT EP 1 156 117 A2 4/2001 WO WO98,06862 2, 1998 Polynucleotides encoding polypeptides that comprise the WO WOOOf 11,199 3, 2000 biosynthetic pathway for carotenoids and apocarotenoids in WO WOO3,O2OO15 A2 3, 2003 the coffee plant are disclosed. Also disclosed are a promoter WO WO 2004/003208 A2 1, 2004 sequence from a coffee carotenoid gene, and methods for using these polynucleotides, polypeptides, and promoter OTHER PUBLICATIONS sequences for gene regulation and the manipulation of flavor, Akiyama, M. et al., “Analysis of Volatile Compounds Released Dur aroma, and other features of coffee beans, as well as the ing the Grinding of Roasted Coffee Beans Using Solid-Phase manipulation of photosynthesis in the coffee plant. Microextraction.” J. Agric. Food Chem... vol. 51, pp. 1961-1969, 2003. XP-OO243.1389. 20 Claims, 30 Drawing Sheets US 8,252,977 B2 Page 2

OTHER PUBLICATIONS Cunningham, F.X. et al. “Molecular Structure and Enzymatic Func tion of Lycopene Cyclase From the Cyanobacterium synechococcus Bäumlein, H. et al., “Cis-Analysis of a Seed Protein Gene Promoter: sp Strain PCC7942.” Plant Cell, 6: 1107-1121, 1994. The Conservative RY Repeat CATGCATGWithin the Legumin Box Czerny, M. and Grosch, W. "Potent Odorants of Raw Arabica Coffee. Is Essential for Tissue-Specific Expression of a Legumin Gene.” Their Changes During Roasting.” J Agric Food Chem. 48(3):868 Plant J. 2: 233-239, 1992. 872, 2000. Beyer, P. et al., “Golden Rice: Introducing the Beta-Carotene Czerny, M. et al., “Sensory Study on the Character Impact Odorants Biosynthesis Pathway Into Rice Endosperm by Genetic Engineering of Roasted Arabica Coffee,” J Agric Food Chem. 47(2): 695-699, to Defeat Vitamin A Deficiency.”J Nutr. 132(3):506S-510S, 2002. 1999. Boelsma, E., et al. 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Metabolism GGPP SYNTHASE o plate-ly VitaminDiterpenoids/Gibberellins El Vitamin K/Tocopherols

PDS és PTOxés, B) Ss. SS S. 'SS. SSMS SS. PDH *Sarx. S- - was Qox ZDS so PTOX C S. SYS-S-S s s s-s s's

F. LCY fe in-r 8-carotene D (?caroene)-Vitamin A CHY rised: (E) B-c ryptoxanthin B BCHY M is CHY OH B Zeinoxanthin ------a. OH ECHY G ("(h) OH ZEP WDE -- OH OH J Antheraxanthin B O --- OH ZEP VDE --OH K Violaxanthin NCED 2,3,5,6 OH O is > -s; Xanthoxin (N) OH L ris Noahind." OH ABA FIG. 1 OH U.S. Patent Aug. 28, 2012 Sheet 2 of 30 US 8,252,977 B2

OH I s-so s9. s 10 -----O as O tozeaxanthin : 19 3 -nydroxy-7-iononeF. 2

OH IV 9. 15 10 d -b so O 10 9 HO HO violaxanthin 3-hydroxy-5,6-epoxy-2-ionone

9. S. 19 s. 2 - Onone 10 9 h : a-carotene : VI orsos's os-s-s-s-s-s O O-OOE C dialdehydyde

: V : -s-s-s u-ul-3-I-S-Ug---10 9 ------Y geranyacetone phytoene ----- XI r) ------O C dialdehyde U.S. Patent Aug. 28, 2012 Sheet 3 of 30 US 8,252,977 B2

W A v is so 3-hydroxy-5,6-epoxy-2-ionone

94-9Q- OH V 9-cis neoxanthine grasshopper ketone w X s X e- OH eO 2-damascenone 3-hydroxy-2-damascenone

B XI

-r N-N-N-N-N---- OH pill---s S. -- crocetin dialdehyde HO ls zeaxanthin ;7 ra so X 3-hydroxy-2-cyclocitral

v Safron C ----- XV

1-9 6als---~~~~ S s -Y MHO lyCopene A XV

bixin dialdehyde

v FIG 3 Bixin (Annatto) U.S. Patent Aug. 28, 2012 Sheet 4 of 30 US 8,252,977 B2

: IWAYVRRRELVDGFNSHTAREARRDEREFMDASDISKFFDCCFSY 143AETELEMPERATIVERRIEELVIGENSHIP REEE APS M12123 132 ATEGIMITERAWAYVRIEDGENSTFAIRE REVERPEDIALSDTVSEEDLePSY1 X6041 144 AKTFYIGIMMEPERRKALWAYVRCRRTEINDGPNASHTTPALDRERLEDWESGRPFDMDALSDTVSKFPWD CaPSY1 X6801 23632EREMIEGARDLKKSRYKNEDELYLYCYNAGTYGLMSVPVIGIAPESKAVESVNALALGIANITNILRDVGECCPSY 223-PERATEGRIDERSRNEYCINGVINSYERGSKriSINAAGIANOINIRDEAEPSYNM.21129 21292FRIEGIRDLKSRYKNEDEYEYCYNAGTYGLMSVEGIAEESKATESYNALALGIANQITNILRVCE le:311880441 RMIKRYNEDEYEYCYAGIVESVEIAPESKATESYNAIALGIANITNIIROCEI Caby YGaol LEDELAGSIEDIERGRVIER MERRREEDEAERGIENSASRWEWSLLYRIDEIECCEST 303RRRYLEAGISEEDIFACSTERNIRAFEDEAEKGTEISASREWSLLERRIEIAtPSYNM121729 RRRYIPORIAGISIEDIFACTISIRINROAREEDEAEKGIEISASWSYLEI LePS(1880441 394 ARRGRWYLPODELAOAGSDEDIFACRVEEKRIEMKORARKFFDEAEKGVTELSASRPASLLLYRRILDEI CaPSY1 X68017 395 ADENRYSKERALAYASIVERS:CHS CCPSY 383 C. . At PSY NM121729 372 ADNEKRYSEDITIS, RAF LeRS X6441 334 ADNEKRAWSKPKAYERSIVESRI CPS1 X6801; FIG 4A U.S. Patent Aug. 28, 2012 Sheet 5 of 30 US 8,252,977 B2

1 WWECNYSEAN PRE---FEAS------GSFR SCALT i EGSES III RRL 1 SHRKTRNPSIRR

YLEARDVLGGKVAEDDDGCCPDS ? AKPLWTAGAGLGISTAKYLADA KLEARNIGAD AtPDS TC26.1857 79 (DYPRPELINTYNYLEAFISSERSPRPIKEIFICAGIGISTRIAAGKPILLEARDVLGKVAIDI LePDS X59948 is DYPRPEIDNEYNYLEAAFSSSFRSSPRPTKPLETACACI g|SFAKYLADACHKPILLEARDVAGKVAWKDDI CaPDS X6805 $1 SETGIEEEGAYPMNLEGEIGINDRLERSIFAMAEGEESREDELPAPINGAILKNMLWPERCCPDS 140 EETCHIEEGAYP NLEGEIGINDRIREESTEAMPSKEGEESREDFILPAPING ARNETE AtPDS TC26 1857 159 EEGLHIEEGAYER NLEGEIGINLRIEEESEARSKEGEFSREDEALPAPINGILATIENELTAPER (PDS X59948 58 NYETCHIEEGAYPNONLEGEIGINDRIOFFESTEAMKPCEFSREDEPEALPADINGIAIKNEXLRPEK CaPDS X6858

TVKDFMRKEDRTDEVEAMSKALNEINEDELSOCILIALNRFLEKHGSKMFCCPDS EAGIP ENEROnviroVEVFASKANEINEDESOCIAREIGCSKAtPDS TC261857 RFAIGLLPAMGGSYEADGISVKDRRGVEIRTDEVFIAMSKALNEINEDEISOCILIALNRELEKHGSKMA LePDS X59948 KEAIGLLPAMGGSYWEADGISVKMRKOCYPDRTDEVEIAMSKALNEINPDELSECILIALNRELEKHGSKMA CalDS X68038 1LIGNPEERIOMEIVEHIESGNSRIKIENIGSEPLSGDVEEDIK GPEGEPCCPDS IGNEERICET in SEGD'EAEDIKILLEP AtPDS TC261.85 319 IGERevisconstates SDGSEGAFVEARDI KILIPEDIREIPY LePDS 859948 313 LINPPERICMPIVEHESKCORLSRIKIENDSVEICSTIEGIAEVERYDILLIPEDIREIPYCaPDS; 38.98

FG. 4B (Page 1 of 2) U.S. Patent Aug. 28, 2012 Sheet 6 of 30 US 8,252,977 B2

321 ERKLEKLGVN IREDRINT HESRS CPDS 38O (RSGPINEERIKNYDELFSRSISVY SCKEYY AtPDS TC26 1857 399 EOK. RCSELNEAEAERISRSLSEIF LePDS X59948 IFSRSPISYArts TOKEYDP) CaPDS X6858

359 CCPDS HELEEDEISASKAKIKEWKPRSVYKTCEPCRPLRSPIEGELAGDTKOKYASEGAVISCAEPDS TO26.185 MEL FEDEISADSKAKILKYVRTPRSVKTVFCCEPCRPQRSPIEGELAGDSTKOKYLASAs V EGALSGKC LePDS 5998 478 RKEAK EPCRESP EGEYLAGDYKKYLASE ALSGKC CapDS X68058

CCPDS APDS TC361857 LePDS X59948 CPES X68058

FIG. 4B (Page 2 of 2) U.S. Patent Aug. 28, 2012 Sheet 7 of 30 US 8,252,977 B2

SOSNAERCLPPAGER VATSAYXCPATSASERER (SVLRADISYSIMSNRPGLEPPEPERCPK Ca2DS X8989 TSSAYs KVE KSVRADLDSVSDMSTNAPKGLFPPEERGPKL e2DS AF195.50

------ELLEGEDIESSIGGKVGSFVDRGNESCLEVECCINLERLENGENLVRCC's 58 KAIGGAGSIAVELLEVDISREIGKGSEVEREIE GERVERCYNLERVGAERNL el SVAIGAGAGSTAVELLEGEDIYESRTFIGCKWGSFWDERGNHEMGLEVERGCNNLERLMREVGAERNLVE 81 kVAIGAGAGSTAVELIDOGENDIYESRTFIGCKCSEVERGHTENGIEVECCYNLERKVGERNLIVE Le2)3 AE19507 THIEVNGEIGFIDEREPVGAPIHGINALSINIKIYDKARNAVAIAWRALVDPDCARIRDLRISESDE 1 tries credicing tressessive RISSESL At DS 3855 1. THEVNECEIGELDEREPVGAPIRGINAELSINOLKYDKARNAVALASPWRALVDPDCALOIRDLESSESL Ca2S X8989 1

CCES LRAtEUS U3853) CaLS X89897 LeSS AF195.50

CCEDS

296 GCRLY SIGE le.GLASKARKIVKAYVACNPGIKRI - NIYCWPWTOLRING ACDSU3835) 321 REVIETSSDGSEVS LASKEIOKTVKAVAWPCIKRIVPOREI FENIYKGVPVTVORYNGCa2DS X39897 32 GCREWEYETSSDGSMYWSGLAMSKATOKKIVKADAYWACDWPGIKRLWPOKERELEFEL IYKYPWTVORYNGLeZDSA 195507

FIG. 4C (Page 1 of 2) U.S. Patent Aug. 28, 2012 Sheet 8 of 30 US 8,252,977 B2

171 CCED S 378 FIELD GDNLLYTPDADFSCEADLALASPDYEG ICVLFPGDPERENDKIEM-T At ZDSDS J3 TEIDIFRSR.R GENETPIADESCEALALASPDYLEGSLOVEPCDPIMPLEEI KOW CaZDSS X89897 TELODIERSROKRA Lé2)SAS

171 CCS

FIG 4C (Page 2 of 2) U.S. Patent Aug. 28, 2012 Sheet 9 Of 30 US 8,252,977 B2

ge:DSCEsotersvikinistriarskyisraveya CCPTO 1. ESEKAETSESEKAETSGTEPLE---ERN SSSIELTSVIKILITEREEVIEIARYPEAF AtPTOK AJO)4881 71 SEAPKSEPGAWGGG III. THENT CaPOX AF1981 80 SEAFKSEENGSPDSSSGIERVIKEOSN. LeRTO: AFTT98)

38 SPRAYHESECWE CCPTC 148 SVLEEGRADEVHEAESNEMELLIEEIG AtPTOX AJC (4881 150 SVLELYESEGRADYLKVHEAESNEMHLLIMEELGGEWERELAOLAVEYMTVSEASPRAYESECVECaPTO. AF177981 159 SVLE O :YALSPRMAYESECVE. ePTO AE1798)

SHAYRTYFIA REPAIRVEYTGDIYLEDEISENTRRENLIVENIRDEAECRTC. ESIristic Dioclet." AtPTO: AJ064881 230 HAETYPEIREEPERIAITGDIYLEDEISREENTREDLIVENIRDEREFORTRC CaPTC, AF177991 239SHAYETYDKETEDGEELKNLPAPKIAWTGDLYLEDEFTSREPTRRPKIDNLYDEMRIRDDEAEECKTMEAC. LePTCX AE177986

31 Y. CCFTC. 3) GSIRSPES AtPTC AJC 881 31 THGSLRSPENE-SED PC CaPTOX AF7981 33 TEGSERSPETPC ADIGIVDCIKKSWR. LePTO AE17798)

FIG. 4D U.S. Patent Aug. 28, 2012 Sheet 10 of 30 US 8,252,977 B2

5363

79 suspekarstratiyassetskyi Rogy CCECH AtBCEY NM 124 636 68 LA R ASSEGISHAYRESRES LeRCE 14.809

EEWAREALEASLESHEREGENDEAIAEALALLIGEEGLIGICEGGIGIVEGAME CORE ERRIESINESHKPREENDERITNAPRICENGIGICRGGIGIGAME AtBCEY N 124636 15EFARWEEASEASIEEESHKPROPERDFAITAYPAGEEKGLIORAGIGIVENAME. LeRCHY (14.809

RFAAHOLESDKEGEEGFCPKELIGCSELEERRIKI, CCECH EGLEREEGRIAEIRAEKEYEG. GPREVEECEELEESRRIKINKGSSTS AtECHY 1246.36 ESENGEYC.E.D. EEEEENTRETR LECH 1489 U.S. Patent Aug. 28, 2012 Sheet 11 of 30 US 8,252,977 B2

9 , W. 156DEFIGIGIECIEDYLDPRIGRAGRERIEEELIRRCESSTSSRVERTRAPN CY AF321338 138DEFIGIGIECCIEHVRDFWYLDENDPILIGRAYGRVSRDLEELROSGVSYISSKVERITEAPNG TeLeC AE25.1016 177 SIPCRIAIVASGASGKLOYEIGERYOTAYEVEVENPYDE MEMORDERESIEFPIELY COLeCY 236 IPORLIYASASGREIGERVCEAGLEVEVENPY in A SLCY AF321.538 218 NIIPORLATVASGASGKLOYEIGGERVCVOTAYGIEVEVESIPYDEMEDYRD LeC AF51. 257 SPIFFECASKAMPFELIRIXSR.R.TYEEENSYIPGGSLPNEQRNAGASMVHPAIGYS Colecy 316SEFECIASAMPLE IMSRLGIRITEENSIPGSLPNEOKNAGASHERGYSislecY AF321538

TR-Isa tworkRaleiai. IEREEccleCY 96 VRSLSEANYAVIAKI ISKORETLSPIERRAFFIECIS SIRTEERTELsleC A321.538 378 VVRSLSEAPNYAVIAKILGRG ISKOREPERPIERKRORAEFLEGLALIVODEGREEERIEETeleCY AF251016

LSLeCY A321536 TLCY AF25)1 U.S. Patent Aug. 28, 2012 Sheet 12 of 30 US 8,252,977 B2

------CCEEP 1 O GANG) LeZEP SE3833 1 FASTLFNSMSA Es-- Pd2? AF15994's 1 --MASATAPAKRSSLESHEE -- (S2EP AEO5(384 PCSINESSELETRTHWESSKOELSSFSGGGSG LGVKATEKEKRVE - At 2EP NH 88.1285

| L:SEP 2,838.33 76 --TOKLEVAGGGIGGLVEALAKKKGFD E. PZEP AF159948 ! --GRERYLVAGGGIGLVELARGE ESARGECORGPIOISAAAAIDSAEEMEC 032EP AE050384 -k3 RVLVAGGGIGVFAIAKKKGFDYLVERDISRGEGRGPIOIOSNAIAEAIAEIG

154GDRINGLVDSGIEEEDIFPERCEPTRISSOLARAVINDSNWEEKENgRINGLVDGISCR.EDIFIP ERGLEVTRISRIOLARVCEEISNWDFEDIRTYLENCO Pd2EP AP159948 151 GDRINGEVICISS EDIFPAERGINRISTIOOILARVDEWDF EVER 033EP AED50934 TGDRINGLVDGISGTYVKFDTFPPARGIPVTRVISRMTIOOILARAVGEDE

239DLLVGADGSEVRNLEG-SEATSGYICTGIADEYEADIVGRVEIGREVSSDVGGKNOYAFEEP Le2EP 2838.35 234 LPsyTCIGIADFEDSCRVELSEYEYSSDVgg EGAFESP3EPAF159948 231EGDLVGADGISKYRESERTYSTCYCLADEVICIRVEIGEQEVSSDVGKMEAEEKEPOSTEP AB050384 RSEATYSGYTCYTGIADEPADESVGRWEIGHKOYFWSSDVGGGKNOWAFFEPA At ZEP NM851235 1 ------ELLATEDAIRDIYDRESSGRTLCDSHAMPNigGMAIEDSCCEP 319CDPNGKKERLICINVIDIATEDAILRRDIYDRESERTLEGDSVEAMQPNg3GMAIEDS LeZEP (83835 KCETLDSVERMP30AIEPGEP A 314 GGIPEGKNKR ...In VHAMOPNGOCGCMATEDCs2EP IARPARTE IAMOPNGOcGCMALEDSAt 2EPP FIG. 4G (Page 1 of 2) U.S. Patent Aug. 28, 2012 Sheet 13 of 30 US 8,252,977 B2

ESARRAIEGER ASSIGGGBS, KFRPHEGR CCEEP

RVAIHSARA ASTRAYIGIGIGPSF. KERIPHFGR. PdAEP RF159948 it r RPHPCR OSEEP REOS33 3. SHGIA GG RRA RASTEELYi . RFRPHPGR. At2EP NM 85123)

R. CCEEP

SRD LEEP E8383

CRISEORE AERIDGEECODNESOLCLNR PEEP F1599.8 CRREDDDALEAMCELETSS-SORLIRD OSEEP BO3O88

Sir LGNSEGREESCRIKARREEEDDDALERTGECDCCSETICTE. At EP NM 85.1285

OWS AFEVIDERSEEGEDEGRRRTSEEPERFEPSIVE LEEP (83835 35A EK SSIAIERPOSEMEARISEDCATDLRSEEGI. NFPARERPSAEI PdVEP AF159948 SSPLPSNARE EGRRYERSELECPPS----L OS2EP AEO5883 AtEEP NM 51285

2 Nesk- CCEEP KKLSERKE--EREAVE LSEP E83835

ARKESS SVE--KEGI PEP AF1599 OSEEP AE5884 6 f At EP NH 351285

FIG. 4G (Page 2 of 2) U.S. Patent Aug. 28, 2012 Sheet 14 of 30 US 8,252,977 B2

KC CCNDE SR At WDEAYO636

RL------ICRISC VTE 7. i a - a-- - - - LELLERL- PSADAWDATKTOCISCRIE. At DE A1063067 SHLWTTG------ACAPSAAA 12 FEI NeKNSKEER.A.E.ARTALEISpaakonouko NEVDE 034917

NSTEENECAVSRKCVPRKSDWGEEPRPDEAVERNEDIKCCVDE ARCIAEACANV CLCTCNRPDEECIKODLEENSWDEENECEWSRKCVPRES I are st RPETECOIKGDLENAW SAARENA (SYDE AF411133 NVDE U3481

16DESGRFYISSGNPTFEDCOEEEEETESKLVGNRLREGFFIRS:211 DRYS.NETEDOEGRIGSRSGEFIRSvg.V.I.P.E.NEDEYEYQAtVDEA106306? 196 BRISSGETEDOLERGKNIRIRIPSGFFIRVDLINEDEROOSYDEAE41113 3 -ENKLVGNSRIRTPLFFTRSAWOKYOPEPCTLYRIDNEYTONEYDE U3481 2445.) ISSENKPD) RAKSGRDFSKFIREDNICGPEPELVECcVDE 29 DYISSEENEDIFYRRNAIGYGWIRa DNTCGPEEEa EtWDE AYO 636 275 NYISSKWEREDDYIFWYYRCRNDERGYGGAVTRSKESIVE IRT NIOSPEPPLE OSVDE AF41113 1. f RSIP ECGPEpply NtyDE U34.17

FIG. 4H (Page 1 of 2) U.S. Patent Aug. 28, 2012 Sheet 15 Of 30 US 8,252,977 B2

KDIEELEERITEGEKEKDEEEE CCDE

EGER VREVELERVER G TFERC NELKODEENE

RTVE REVIEGEEEEEEETERIOMESKEE, MEYER (SV)3 AE411133

CCVDE AtWDEA)636 OSVDE AF411133 NtWDE U3481

FIG. 4H (Page 2 of 2) U.S. Patent Aug. 28, 2012 Sheet 16 of 30 US 8,252,977 B2

------LeNCED1 CAD30202 SNINSO StCED1 AAT551

PFTS.------WvNCED1 AAR11133

65 ------K------SFORASIAFE ARRAtNCED5 NM12743 65 ------Y SEESS,--SSISNALAYATE. LeNCED1 CAD30202 65 ------O c. SNARALAVALE StNCED1 AAT75.15) 72 ------SEGARTTPKEKLDESS------ARE, VyNCED1 AAR111.93 4. 5 EPLEKTADEISGNEAPVEEOPVRP PK A. D IGNREAPLEACHEDIRESFSCCNCED3 110 PIPEADO.NEAPEQVIEEPs VIRGANIEPHERDINHESS 100 SELPRDEISAPIPESISKIPO YRANDLEEDEVEVENS, AtNCED5 M102749 113IEEERISAFE EPLPKIADERVOISCREAVERSLFVGKIGERNANPLEGEDGDMVEVOKNSAS SINGEORGERGEARSStdE) StNCED1 AT75151

PDCIOGENANETPAGEDIGAAVOE Svy CED1 AR1119 R.ERSIGRPVFPRAIGEIHGHSGIARMFYAREGHSGIGANAGEYENNRIASEDDPY CONCED3 130CRFORGREEKAGEGIARMFIAEGGANGERIASDLP, AtNCED3N11230 180 RFEERVESPRAGEIHGSIARKEYARLEGIGANGEVERLASLEPI. AtNCED5 M102.74 1950RFEFEREEKARPvEPKAGEIHGSIRTFIERI FGTHSTANGVINRIASEDIPlaNCED1 CAD3820? 193RREEERiggie EPKAGEIHGSIARTEYARGEGHS GANGEVINRIAEDP StNCED1 AT75151 201 ACRETERRDERPVEPKAGEIHGHSIARLFYAFGLEGEWHSHGTGVANAG WYERRIASEDDLPC Wv CED1 AAR1193 FG. 4 (Page 1 of 3) U.S. Patent Aug. 28, 2012 Sheet 17 Of 30 US 8,252,977 B2

303/PSSDKISSENGOKSIMRPRLDP PYKYERESE 266RDD2) EP Ringlethis PELEASDAPEKEESPDEISTEDEAtNCED8 DIMEDEAtNCED3 NM102749NM 12303 25WTPGDKTEGREDEDGIRSTIAHPRLDPWSGELEALSYDKP EKYERESEN Int LeCED CAD3.02.2 23VVTESDKEGREDFIGOKSIMAPKLIFVSELEALSYDs IRSGrier E. StMCE) AAT5151 281RVTPSCDLVGDESQRSTMIAEDEVSGASYDWKPTKYERESESPDYELIMEDEVyNCED1 AAR11193

S 33 NEWIPDWEKSEMERGGSPWYDREKVSREGDKAESSATEVEVPDCECEHLFAWEEPETITWIGSCCNCED3 350 TENEVIONEERGSEWLKKREGIDKISSIOHNWEEPETEWIG, AtNCED3 M112304 340 TENEWIPOWEKISSPTSSRECIPRAKSMVSECHLEAEEEWIG, AtNCED3 M102749 353 (TENEWIPOWESEMIRGSEWERISREGIDRAKESDKNEVEDCOELANETFWIC LeNCED1 CAD3020: 353 connect WTGS StNCED1 AATIS 151 361 ESWOWEK Y is WWNCED1 AAR11193

465 RYALAIAEPKISGEAE CONCED3 430 MPPISIEEENESISERINRISR For al AtNCED3 NM112304 42 I. ? RAYLAIAEPEKSGEA AtNCED5 NM 102749

435 MPPISIENECEGLSVISEIRINRIGKSIESINEDEVNEAGNREIGREALAIEEEKVSGFA LeNCED1 CAD3020 33 AISPDENIEAGVNREIGRTOYAMAIAEEEWSSEAF St CEDl AT5151 d;1 CMEPSIEECEGLKSSEIRKGKSRRL WiNCED1 AAR1119

FIG. 4 (Page 2 of 3) U.S. Patent Aug. 28, 2012 Sheet 18 of 30 US 8,252,977 B2

543VD FIGV (IGEIGGEPLFIPRENFEDIGYLAVIEREWSELVNRTELES PSRWPGHG CCNCED3 - EGT EC AtNCED3 NM1123)4 499DSCENIEEKCCEFIPLEDICYSVIDE EYNTEERIVKPSRPGHG AtNCED5 NM 10249 LeNCED1 CAD322 SEIVERSEVPEG KEATKTPSRWPGHG StNCED1 AAT,5151 515 El INTEKTPSRPYGEHC WvNCED1 AAR111.93

CCNCED3 AtNCED3 NM112304 AtCED5 NM 102.49 LeNCED1 CADSO 202 StNCED1 AAT5151 WWNCED1 AAR1193

FIG. 4 (Page 3 of 3) U.S. Patent Aug. 28, 2012 Sheet 19 of 30 US 8,252,977 B2

WNEKENARINEK KPLAYESGNERENDEPEKDYKGHLECCCDl ori DETPPDLCHL PhCCD A576) J3

G-WNEKRRGITAKIDIEGVKLHDSSKPTHYLNEAAR AET DEEP, His LeCCD1ALiCCD1E AY576001EYS O2

ECLCEFRGENEKSEWAG RIRGKRTYSRITSRKQEEGKMGDIKGLEGIF CCCCD1 RePEKiev.GiEDGGERIKEGTSRRSREEEKEKGDEGRGIF CINEERGENREAPIRGEEDGEMEGIGIGINSERISREGARICOLEGLEGE. LeCODiE AY376002 CINGEFVRCPNPKEPAGEEDGEGRKDKATYSRRISREEEEEEKESDICEGETY LeCCD1A AY576001 3 ECLICEFRICPNPKDWFGTHREIGDGIHGRIKEGKATYVSRYSR. KEEEFCAKEMKIGDKGFGMVN ALCCD1 AJ) 3813

ISIRAKELM LEEDLIDIKRITISEIAERVDIGECCCD1 KIASEAKERLEDGEOIADYERSEAERDFGE PhCCD A1576003

nicipatigristorestarter CCD8 A500

A.V. ASEADKPKVIEEDIOIADYKRTHSFIAERVIVIGE LeCCDIA AY576001 B.N.GNGEANTALYEGKLIAEAKPVKWLEDGDIQICIDYDKRITHSFTAHPKWDFWTGEM AECCDl AJ)(381

DPVPISPIEDEAITEYASDLP. FIFC:TIRISKDEDEVEITEREDEATENTED2 236 ETEGS C. AITEDIPE REVENIITKRRE LeCCD1B AY576002

237TEGTP2YIRISLExEVPIEEPIMEFA KXELEEKRELECCDIA AY576001 TFCSHIP25IRISKolk MEETYSSRPKKARE AECCD1 AO05813

FIG 4J (Page 1 of 2) U.S. Patent Aug. 28, 2012 Sheet 20 of 30 US 8,252,977 B2

313 TPRSAIRELENCEIENNECEWIREDLARESNEYERENSGS, 316 IPRAkhil EIPNCFIFENAECWLISL PDL EERINEERENES

317313 TPRYSRELPNCFIFENANEWEEDEWLITCinto AIKEKLEECELYERENKSGAASs

399 SKISESADEPRNETGROWYGLISARVIGIXEDLEAEPEIGKREVGNOEDIGEGREGSEAVE CCCCD1 39 KKSES DEERINETGRRYIGNSIRTGIIRED EAEPEIGEGNOEDIGEGREGSEV PCCD1 A57603 LeCCD1E A56CO2 396 RISESADEFRIENTGRORYGISIRTREDEAEPEIGIII.ii.9A. EvGNoSIFDIGPGREGSEV E. (CCD1A A 5600 392 KISSAYDEERINEIORNGRISARVIGILKEDIAEEIGEREGGIEDIRESEAR At CCD1 i? 5813

CCCCI PCCD1 AY576CO3 (CCD1B AY56OO2 OAKL LeCCD1A 5600 EETI ACCD1 AO (5813

FG. 4U (Page 2 of 2) U.S. Patent Aug. 28, 2012 Sheet 21 of 30 US 8,252,977 B2

IE-EDGE AtFIE1a RM-1664() KEEGPE-LE C3FIE XT29

FIESGN-U213593 '''PEEELKSEIGIDRGISSERENELITOLENETPAPEALTLINGIWILF AEEATERFETERIE I tral LITOLESKNETPAPTEALINGSILA At FIB1b NY-113350 70 RWEISSSTIS TERKRSA S too

152TSEGIFPI. servisors 142 ISFSFP SRG I TVONS C AFIE NM-1183.50 150YISEYCIFPI. SE EPIVR isitingss AFTE: M-11664

EISTIEYNSWEAG ISISINEERSE SEEGIRLCaFIBX7729 VEISOTIDSESETYONSWEAGPLETTSISTNAKEEVRSPKRVQIKEEEGEOID LeFIBSGN-U21359: resy. GKIDNEKGILTSVIDIASSVASISSEEKESIS 22? Sir Evig IDENISOASYSPIRESSETI RISRCDGSEWAFIBM-11350 23C SIESVEVISIDENPIKGILTSVITASSVFISOPPIKES, D&LITILIRISREGST. At FIB1a M-11640 234 SEE SKIDGISYOTSVASISPESS G.V. CaFIRX7729) SPEEE LGOEIDEKGITSVODTASSVAKSISSIPEKEPS RCGSVELLE FIESGN-U21359

{ C E At FIE 1 RM-11835) AFIE NM-1166. CaFIB X79. FIESGN-1213598 FIG. 4K U.S. Patent Aug. 28, 2012 Sheet 22 of 30 US 8,252,977 B2

0.10 PTOX 0.08

0.06

FRTO5 T2308 FRTO5 T2308 FRTO5 T2308 FRTO5 T2508 F.G. 5A FIG 5B FIG. 5C FIG 5D

0.30 bCHY 0.25

0.20 0, 15 0.10 . Ill. In l.T. - v2 is fra of FRTO5 T2308 FRTO5 T2308 FRTO5 T2308 FRTO5 T2308 FIG. 5E FIG. 5F FIG 5G FIG 5H U.S. Patent Aug. 28, 2012 Sheet 23 of 30 US 8,252,977 B2

Ko. K. vide to FRTO5 T2308 FRTO5 T2308 FRTO5 T2308 FIG. 5J FIG. 5K FIG 5L U.S. Patent Aug. 28, 2012 Sheet 24 of 30 US 8,252,977 B2

0.2 CCD1 O Arabico 0, 10 Robusto 0.08 0.06 0.04 --

O.OO $29 $22 3 $22 $33 $33 Sp BP409 FRTO5 FRT64 T2308

F.G. 6A

Lycopene f-carotene Zeaxanthin

300 NI NI S 5 200 3 100 C) (C) 0 ge 100

it -T---, ------O 10 20 30 40 0 10 20 30 400 10 20 30 40 Min Min Min

F.G. 6C U.S. Patent Aug. 28, 2012 Sheet 25 Of 30 US 8,252,977 B2

U.S. Patent Aug. 28, 2012 Sheet 26 of 30 US 8,252,977 B2

U.S. Patent Aug. 28, 2012 Sheet 27 of 30 US 8,252,977 B2

700

100

30 20 30 20 Mir Min

FIG. 7B

U.S. Patent Aug. 28, 2012 Sheet 28 of 30 US 8,252,977 B2

FIG. 8A

FIG. 8B U.S. Patent Aug. 28, 2012 Sheet 29 Of 30 US 8,252,977 B2

E S3 r

70 Min FIG. 9A

3. 600 C. canephora E 500 3 b S3 w 3 3 5

O 10 20 JO 40 70 Min FIG 9B

8 5

O O 20 30 40 50 60 70 Min FIG. 9C U.S. Patent Aug. 28, 2012 Sheet 30 of 30 US 8,252,977 B2

Control Drought

2 as a 5 & 2 as 5 s 14 - - 1.2 PSY Control PSY Control o 10 ODroughtroug ODroughtroug

0.6 0.4 - 0.2 US 8,252,977 B2 1. 2 POLYNUCLEOTDESENCODING alternative, the expression of genes encoding naturally occur CAROTENOID AND APOCARTENOID ring coffee proteins that positively contribute to coffee flavor BIOSYNTHETIC PATHWAY ENZYMES IN may be enhanced. Conversely, the expression of genes encod COFFEE ing naturally occurring coffee proteins that negatively con tribute to coffee flavor may be suppressed. This is a U.S. National Phase of International Application Coffees from different varieties and origins exhibit signifi No. PCT/US2006/034402, filed Sep. 1, 2006, which claims cant flavor and aroma quality variations when the green grain benefit of U.S. Provisional Application No. 60/714,106, filed samples are roasted and processed in the same manner. The Sep. 2, 2005, the entire contents of each of which are incor quality differences are a manifestation of chemical and physi porated by reference herein. 10 cal variations within the grain samples that result mainly from differences in growing and processing conditions, and also FIELD OF THE INVENTION from differences in the genetic background of both the mater The present invention relates to the field of agricultural nal plant and the grain. At the level of chemical composition, biotechnology. In particular, the invention features poly 15 at least part of the flavor quality can be associated with varia nucleotides from coffee plants that encode enzymes respon tions in the levels of small metabolites, such as Sugars, acids, sible for carotenoid and apocarotenoid synthesis, promoter phenolics, and caffeine found associated with grain from sequences from coffee carotenoid genes, as well as methods different varieties. It is accepted that there are other less well for using these polynucleotides, polypeptides, and promoters characterized flavor and flavor-precursor molecules. In addi for gene regulation and manipulation of flavor, aroma and tion, it is likely that structural variations within the grain also other features of coffee beans. contribute to differences in coffee quality. One approach to finding new components in the coffee grain linked to coffee BACKGROUND OF THE INVENTION quality is to study the genes and proteins differentially expressed during the maturation of grain samples in different Various publications, including patents, published applica 25 varieties that possess different quality characteristics. Simi tions and scholarly articles, are cited throughout the specifi larly, genes and proteins that participate in the biosynthesis of cation. Each of these publications is incorporated by refer flavor and flavor-precursor molecules may be studied. ence herein, in its entirety. Citations not fully set forth within Carotenoids are one candidate class of flavor and flavor the specification may be found at the end of the specification. precursor molecules. Carotenoids have been identified in all Coffee aroma and flavor are key components in consumer 30 plants, as well as in a wide range of algae, certain fungi and preference for coffee varieties and brands. The characteristic bacteria (Fraser et al., 1999). Their 40-carbon structure con aroma and flavor of coffee stems from a complex series of fers particular properties, allowing them to absorb light chemical reactions involving flavor precursors (Maillard between about 400 and 500 nm. Carotenoids are liphophilic, reactions) that occur during the roasting of the bean. Flavor a characteristic that, along with their coloration, makes them precursors include chemical compounds and biomolecules 35 sensitive to oxidative degradation (Britton, 1988). Caro present in the green coffee bean. To date, over 800 chemicals tenoids represent the largest group of pigments in nature, with and biomolecules have been identified as contributing to cof some 600 different carotenoids identified to date (Cunning fee flavor and aroma. (Flament, I., 2002 “Coffee Flavor ham and Grant, 1998). In fact, carotenoid-derived apocaro Chemistry' J. Wiley et al. U.K.) Because coffee consumers tenoids conceivably constitute one of the largest classes of are becoming increasingly sophisticated, it is desirable to 40 molecules in nature. Some of the apocarotenoids are essential produce coffee with improved aroma and flavor in order to and valuable constituents of color, flavor, and aroma in edible meet consumer preferences. Both aroma and flavor may be plants. (Winterhalter and Rouseff, 2002). artificially imparted into coffee products through chemical In plants, the carotenoid pigments are synthesized in the means. See, for example, U.S. Pat. No. 4.072,761 (aroma) plastids. The biosynthetic pathway takes place on membranes and U.S. Pat. No. 3.962,321 (flavor). However, to date, there 45 in the plastid compartment of the cell, and the corresponding is little information concerning the influence of natural coffee genes are located in the nucleus. In chloroplasts, carotenoids grain components such as polysaccharides, proteins, pig accumulate primarily in the photosynthetic membranes in ments, and lipids, on coffee aroma and flavor. One approach association with the light-harvesting complexes. In the chro is to select varieties from the existing germplasm that have moplasts of ripening fruits and flower petals and in the chlo Superior flavor characteristics. A disadvantage to this 50 roplasts of senescing leaves, the carotenoids may be found in approach is that, frequently, the highest quality varieties also membranes or in oil bodies such as plastoglobules, or in other possess significant negative agronomics traits. Such as poor structures within the stroma. yield and low resistance to diseases and environmental The first true carotenoid is formed by the condensation of stresses. It is also possible to select new varieties from breed two molecules of geranylgeranyl diphosphate into phytoene ing trials in which varieties with different industrial and agro 55 (FIG. 1A). This reaction is catalyzed by the enzyme phytoene nomic traits are crossed and their progeny are screened for synthase (geranylgeranyl-diphosphate geranylgeranyl trans both high quality and good agronomic performance. How ferase PSY: EC 2.5.1.32). Phytoene, which is not a true pig ever, this latter approach is very time consuming, with one ment since it is unable to absorb light at visible wavelengths, crossing experiment and selection overthree growing seasons undergoes four consecutive desaturation steps. The first two taking a minimum of 7-8 years. Thus, an alternative approach 60 steps are carried out by the enzyme phytoene desaturase to enhancing coffee quality would be to use techniques of (PDS; EC 1.3.99), resulting in the formation of -carotene molecular biology to enhance those elements responsible for (FIG. 1B) via the intermediate phytofluene (Bartley et al., the flavor and aroma that are naturally found in the coffee 1992; Hugueney et al., 1992). The second two steps are cata bean, or to add aroma and flavor-enhancing elements that do lyzed by the enzyme -carotene desaturase (ZDS; EC not naturally occur in coffee beans. Genetic engineering is 65 1.14.99.30) to form lycopene (FIG. 1C) via the intermediate particularly suited to achieve these ends. For example, coffee neurosporene (Albrecht et al., 1995). These desaturation proteins from different coffee species may be swapped. In the steps require the presence of a plastid terminal oxidase US 8,252,977 B2 3 4 (PTOX) as a co-factor (Carol et al., 1999; Josse et al., 2000; tanatawee et al., 2005). B-damascenone also contributes to Josse et al., 2003; for review see Kuntz, 2004). the aroma of grapefruit juice (Lin et al., 2002). PDS and ZDS yield lycopene (FIG.1C), the main pigment Peak levels of B-ionone and geranylacetone emissions found in red tomatos. Lycopene serves as the Substrate for the from ripe tomato fruit were calculated to be 1.25 pg/g fow' formation of both C- and B-carotene via two cyclization reac hr' and 40 pg/gfw' hr', respectively. Although B-ionone tions. B-carotene (B.B-carotene) (FIG. 1D) is formed by the and geranylacetone are found in low concentrations when enzyme lycopene B-cyclase (LECY. Cunningham et al., compared to other more abundant Volatiles such as cis-3- 1996), which introduces two B-ring structures at the ends of hexenal and hexenal, which have been detected at levels the carbon chain. This reaction also results in the formation of 10,000-fold higher, B-ionone and geranylacetone have odor 10 thresholds of 0.007 mL/L and 60 mL/L respectively the intermediate Y-carotene (B.lp-carotene) containing one (Baldwin et al., 2000). These odor thresholds are significantly B-ring and one uncyclized end, referred to as psi (). Alpha lower than that observed for many of the other more abundant carotene (Be-carotene) (FIG. 1E) is formed by the enzymes volatiles. Thus, carotenoid-derived volatiles have the poten lycopene e-cyclase (LeCY; Ronen et al., 1999) and LRCY. tial to greatly impact aroma and flavor at low concentrations. which introduce one e-ring and one B-ring respectively. The 15 B-ionone is considered to be the second most important Vola activity of LeCY also results in the formation of the interme tile contributor to tomato fruit flavor (Baldwin et al., 2000). diate 6-carotene (e.up-carotene) having one e-ring and one The biosynthetic routes leading to the formation of apoc uncyclized psi end. In plants such as Lactuca sativa (lettuce), arotenoid have remained obscure. Based on their chemical LeCY introduces two e-ring structures at the ends of the structures and studies of Volatile production in tomato vari carbon chain, resulting in the formation of e-carotene (e.e- eties with unusual carotenoid accumulation, Buttery et al. carotene; Cunningham and Gantt 2001) (FIG.1F). (1988) predicted that these compounds are likely derived Oxygenated carotenoids are formed by two Successive from oxidative carotenoid cleavage. In recent years, a family hydroxylation steps. B-carotene (B.f3-carotene) is first con of carotenoid cleavage dioxygenases (CCDs) that cleave verted to cryptoxanthine and then zeaxanthine (3,3'-dihy carotenoid substrates at a variety of double bonds have been droxy-f, B-carotene) (FIG. 1G) by the action of the enzyme 25 identified (for review see Bouvier et al., 2005). The first B-carotene hydroxylase (BCHY: EC 1.14.13-; Sandmann., member of the family to be identified was VP14 from Arabi 1994). Alpha-carotene (Be-carotene) is also twice hydroxy dopsis thaliana, a 9-cis-epoxycarotenoid dioxygenase lated; first the B-ring is hydroxylated by BCHY to form the involved in synthesis of xanthoxin (FIG.1N), the precursor of intermediate Zienoxanthine (FIG.1M), and then the e-ring is the phytohormone abscisic acid (ABA: Tanet al., 1997). ABA hydroxylated by e-caroteine hydroxylase (eCHY) to form 30 controls embryo growth potential and endosperm cap weak lutein (dihydroxy-Be-carotene) (FIG. 1H). eCHY has only ening during coffee seed germination (da Silva et al., 2004). recently been cloned (Tian at al., 2004; Tian and DellaPenna, Other members of the dioxygenase family, including an 2004; for review see Inoue, 2004), and a lutein deficient Arabidopsis carotenoid cleavage dioxygenase, AtCCD1, that mutant (lut1) has been characterized (Pogson et al., 1996; symmetrically cleaves the 9,10(9'10") double bonds of mul Tian and DellaPenna, 2001). 35 tiple carotenoid substrates into a C dialdehyde and two C. The hydroxylated B-rings of zeaxanthine are epoxylated in cyclohexone derivatives in vitro have been identified two steps to give antheraxanthine (FIG. 1J) and violaxanthine (Schwartz et al., 2001). Orthologs of AtCCD1 have been (FIG. 1K). This reaction is catalyzed by the enzyme zeaxan found in a variety of species including Phaseolus vulgaris thine epoxidase (ZEP; Marin et al., 1996: Bouvier et al., (Schwartz et al., 2001), Capsicum annuum (Bouvier et al., 1996). During light stress, violaxanthine can be converted 40 2003a), Crocus sativus (Bouvier et al., 2003a) and Petunia back into antheraxanthine and Zeaxanthine due to the activity hybrida (Simkin et al., 2004a). More recently, Simkin et al. of violaxanthine de-epoxidase (VDE). ZEP and VDE partici (2004b) demonstrated in transgenic tomato plants that CCD1 pate in the Xanthophyll cycle, which is implicated in the enzymes are responsible for the formation of a variety of C. adaptation of plastids to changing environmental light condi cyclohexones in vivo. The potential relationships between tions (for review see Hieber et al., 2000). 45 these volatiles and their carotenoid precursors are shown in Another carotenoid in higher plants is neoxanthine (FIG. FIG. 2. Carotenoid cleaved at the 9,10(9'10") bond results in 1L). Neoxanthine is synthesized from violaxanthine by a the formation of the corresponding apocarotenoid. Because reaction catalyzed by neoxanthine synthase (NYS). NYS was CCD1 enzymes have 9,10(9'10") cleavage specificities, spe originally cloned from tomato and potato (Bouvier et al., cific products would be generated based on the carotenoid 2000: Al-Babili et al., 2000). 50 precursor that is present. AtCCD1 and its tomato orthologues All of the carotenoid substrates described above are avail are responsible for the formation of geranylacetone and able for both oxidative and enXymatic cleavage resulting in C-ionone (Schwartz et al., 2001; Simkin et al., 2004a and the formation of diverse volatile and non-volatile apocaro 2004b) and likely 3-damascenone (Suzuki et al., 2002). tenoids. Terpenoid flavor Volatile compounds are generally Schwartz et al. (2001) and Simkin et al. (2004b) showed that present in plants at relatively low levels, but possess strong 55 CCD1s were also capable of forming a number of other effects on the overall human appreciation of the flavor, for important carotenoid-derived volatiles example, intomatoes (Buttery et al., 1971 and 1987: Baldwin Schwartz et al. (2001) and Simkin et al. (2004b) purified et al., 1991 and 2000), carrots (Kjeldsen et al., 2003), quince recombinant AtCCD1 and LeCCD1A enzyme respectively (Lutz and Winterhalter, 1992), and Averrhoa carambola and assayed multiple carotenoid substrates in vitro. The assay (Winterhalter and Schreier, 1995). Among the more impor 60 products were characterized by thin-layer chromatography tant carotenoid derived volatile compounds are B-ionone, and HPLC. In assays containing either B-carotene, Zeaxan C-ionone, geranylacetone (6,10-dimethyl-5.9-undecadien-2- thine, lutein, violaxanthine and neoxanthine, the central Ca one), pseudoionone (6,10-dimethyl-3.5.9-undecatrien-2- dialdehyde cleavage product (4.9-dimethyldodeca-2,4,6,8. one) and B-damascenone. Alpha-ionone, B-ionone, B-cy 10-pentaene-1,12-dial: I) was the major compound resulting clocitral, and B-damascenone have been shown to contribute 65 from symmetrical cleavage at the 9,10 and 9', 10' positions approximately 8% of the total aroma intensity and 78% of the (see FIG. 2). In assays containing B-carotene, Zeaxanthine total floral aroma category of Valencia orange juice (Mahat and lycopene, 3-ionone (9-apo-B-caroten-9-one; II), 3-hy US 8,252,977 B2 5 6 droxy-f-ionone (3-hydroxy-9-apo-B-caroten-9-one: III) and apocarotenoid content in the coffee plant has implications for pseudoionone (V) were formed respectively, whereas C-caro optimizing photosynthesis in conditions of excess or insuffi teneled to the production of both B-ionone (II) and C.-ionone cient Sunlight. Accordingly, a need exists to identify, isolate (VI), while 6-carotene led to C-ionone (9-apo-O-caroten-9- and utilize genes and enzymes from coffee that are involved one; VI) and pseudoionone (6,10-dimethyl-3.5.9-undec in the biosynthesis of carotenoids and apocarotenoids. atrien-2-one; V). In assays containing violaxanthine or neox anthine, 5'6-epoxy-3-hydroxy-3-ionone (5.6-epoxy-3- SUMMARY OF THE INVENTION hydroxy-9-apo-f-caroten-9-one: IV) was formed. Asymmetric cleavage also led to the formation of a C, The invention described herein features genes encoding epoxy-apocarotenal with these Substrates. Several linear 10 enzymes responsible for carotenoid and apocarotenoid bio carotenoids including phytoene and -carotene are thought to synthesis in coffee plants, their encoded polypeptides, pro be the precursors of geranylacetone (6,10-dimethyl-5.9-un moter sequences from coffee carotenoid and apocarotenoid decatrien-2-one; VII), an important flavor volatile in tomato biosynthetic pathway enzyme genes, and methods for using fruit, and precursors for a second C dialdehyde (4.9-dim these polynucleotides, polypeptides and promoters for gene ethyldodeca-4,6,8-triendial; XI). 15 Inassays containing neoxanthine, the asymmetric cleavage regulation and manipulation of flavor, aroma and other fea also led to the formation of a C, allenic-apocarotenal and the tures of coffee beans. C grasshopper ketone (3,5-dihdroxy-6,7-didehydro-9-apo One aspect of the invention features a nucleic acid mol B-caroten-9-one; VIII) (see FIG.3a). The grasshopper ketone ecule isolated from coffee (Coffea spp.), having a coding is postulated to be the precursor for the formation of B-dama sequence that encodes a caroteonid or apocarotenoid biosyn scenone (IX) and 3-hydroxy-3-damascenone (X; Suzuki et thetic pathway enzyme. In one embodiment, the enzyme is a al., 2002). In assays containing lutein as Substrate, symmetri phytoene synthase that is at least 76% identical to SEQ ID cal cleavage at the 9,10 and 9'10" positions leads to the NO:13. In another embodiment, the enzyme is a phytoene formation of both 3-hydroxy-3-ionone (VI) and 3-hydroxy desaturase that is at least 76% identical to SEQID NO:14. In C-ionone. 25 another embodiment, the enzyme is a plastidterminal oxidase Additionally, Bouvier et al (2003b) identified a zeaxan that is at least 61% identical to SEQ ID NO:15. In another thine-specific 7.8(7,8)-cleavage dioxygenase (CszCD) from embodiment, the enzyme is a B-carotene hydroxylase that is Crocus sativus encoding an enzyme capable of forming of at least 73% identical to SEQID NO:16. In another embodi crocetin dialdehyde (XII) and 3-hydroxy-f-cyclocitral in ment, the enzyme is a lycopene S-cyclase that is at least 86% vitro (XIII; see FIG. 3b). Crocetin dialdehyde is known to 30 identical to SEQ ID NO:17. In another embodiment, the accumulate in the flowers of Jacquinia angustifolia (Eugster enzyme is a zeaxanthine epoxidase that is at least 26% iden et al., 1969) and the roots of Coleus forskohlii (Tandon et al., tical to SEQID NO:18. In another embodiment, the enzyme 1979).3-hydroxy-f-cyclocitral is believed to be the first com is a violaxanthine de-epoxidase that is at least 74% identical mitted step in the formation of Safranal, a constituent of the to SEQ ID NO:19. In another embodiment, the enzyme is a spice saffron in C. sativus (Bouvier et al., 2003a). The 7.8(7", 35 8)-cleavage of B-caroteine by a tomato ZCD orthologue is 9-cis-epoxycarotenoid dioxygenase that is at least 75% iden suspected of being responsible for the formation off-cycloci tical to SEQID NO:20. In another embodiment, the enzyme tral, contributing to tomato aroma. Bouvier et al. (2003b) is a carotenoid cleavage dioxygenase that is at least 83% have also identified a lycopene-specific 5,6(5'6")-cleavage identical to SEQ ID NO:21. In another embodiment, the dioxygenase (BoICD) from Bixa Orellana (see FIG. 3c), 40 enzyme is a fibrillin that is at least 72% identical to SEQID responsible for the formation of bixin dialdehyde (XIV) and NO:22. In another embodiment, the enzyme is a phytoene a C, cleavage product previously identified as 6-methyl-5- dehydrogenase-like enzyme that is at least 72% identical to hepten-2-one (MHO; XV; Fayet al., 2003). Bixin dialdehyde SEQ ID NO:23. In another embodiment, the enzyme is a is the precursor for the formation of the dye bixin/annatto. Zeta-carotene desaturase that is at least 82% identical to SEQ MHO has been identified as an important contributor to 45 ID NO:24. tomato flavor (Buttery et al., 1990; Baldwin et al., 2000). In certain embodiments, the nucleic acid molecule is a gene Despite this extensive knowledge, little work has been having an open reading frame that comprises the coding done to characterize Such volatile molecules in green and sequence. Alternatively, it may comprise an mRNA molecule roasted coffee. Roasted and un-roasted coffee has been shown produced by transcription of that gene, or a cDNA molecule to contain two carotenoid derived flavor components, C.-ion 50 produced by reverse transcription of the mRNA molecule. one and B-damascenone (CZemy et al., 2000; Akiyama et al., The invention also features an oligonucleotide between 8 and 2003; Variyar et al., 2003). The latter component has been 100 bases in length, which is complementary to a segment of identified as a major component of coffee both before and the aforementioned nucleic acid molecule. after roasting (Ortiz et al., 2004). These and other carotenoid Another aspect of the invention features a vector compris derived volatile compounds, due to their low odor threshold, 55 ing the above-described carotenoid or apocarotenoid biosyn require only small amounts to cause a change in aroma. thetic pathway enzyme-encoding nucleic acid molecules. In From the foregoing discussion, it will be appreciated that certain embodiments, the vector is an expression vector modulating carotenoid and apocarotenoid content in coffee selected from the group of vectors consisting of plasmid, grain by genetically modulating the production of the pro phagemid, cosmid, baculovirus, bacmid, bacterial, yeast and teins responsible for carotenoid and apocarotenoid biosyn 60 viral vectors. In certain embodiments, the vector contains the thesis would be of great utility to enhance the aroma and coding sequence of the nucleic acid molecule operably linked flavor of coffee beverages and coffee products produced from to a constitutive promoter. In other embodiments, the coding Such genetically engineered coffee beans. Enhanced caro sequence is operably linked to an inducible promoter. In other tenoid and apocarotenoid content in the coffee bean may also embodiments, the coding sequence of the nucleic acid mol positively contribute to the overall health and wellness of 65 ecule is operably linked to a tissue specific promoter, Such as consumers of coffee beverages and products produced from a seed specific promoter, preferably a coffee seed specific Such coffee beans. In addition, modulating carotenoid and promoter. In specific embodiments, the tissue specific pro US 8,252,977 B2 7 8 moter is a coffee carotenoid or apocarotenoid biosynthetic as cells transformed with the vector and fertile transgenic pathway enzyme-gene promoter, such as the promoter con plants produced by regenerating a plant cell transformed with tained in SEQID NO:25. the vector. According to another aspect of the invention, a host cell Other features and advantages of the present invention will transformed with the aforementioned vector is provided. The be understood from the drawings, detailed description and host cell may be a plant, bacterial, fungal, insect or mamma examples that follow. lian cell. In certain embodiments, the host cell is a plant cell selected from any one of coffee, tobacco, Arabidopsis, maize, BRIEF DESCRIPTION OF THE DRAWINGS wheat, rice, soybean barley, rye, oats, Sorghum, alfalfa, clo 10 FIG. 1. Overview of the biosynthesis of isoprenoids in ver, canola, safflower, Sunflower, peanut, cacao, tomatotoma plastids. Schematic of biosynthetic pathway for plant caro tillo, potato, pepper, eggplant, Sugar beet, carrot, cucumber, tenoids. Abbreviations used are as follows: PSY: Phytoene lettuce, pea, aster, begonia, chrysanthemum, delphinium, Zin synthase. PDS: phytoene desaturase ZDS: -carotene desatu nia, and turfgrasses. The invention also features a fertile trans rase. PTOX: plastid terminal oxidase. LBCY: lycopene f3-cy genic plant produced by regenerating the transformed plant 15 clase. LeCY: lycopene e-cyclase. BCHY: B-carotene cell. In a specific embodiment, the fertile transgenic plant is a hydroxylase. eCHY: e-carotene hydroxylase. ZEP: zeaxan Coffea species. thine epoxidase. VDE: violaxanthine de-epoxidase. NYS: Another aspect of the invention features a method to modu neoxanthine synthase. CCD1: carotenoid cleavage dioxyge late flavor or aroma of coffee beans. The method comprises nase 1. FIB: CCD4: carotenoid cleavage dioxygenase 4. modulating production of one or more caroteonid or apocaro Fibrillin. NCED3: 9-cis-epoxycarotenoid dioxygenase. tenoid biosynthetic pathway enzymes within coffee seeds. In PDHY: phytoene dehydrogenase-like. Some embodiments, the method comprises increasing pro FIG. 2. Scheme for the reactions catalyzed CCD1 proteins. duction of the one or more caroteonid or apocarotenoid bio The carotenoid substrates (left) when cleaved at the 9,10 and synthetic pathway enzymes, e.g., by increasing expression of 9'10" positions (indicated by dotted line) would yield two one or more endogenous caroteonidor apocarotenoid biosyn 25 monoaldehydes and a central dialdehyde product. I, 4.9-dim thetic pathway enzyme-encoding genes within the coffee ethyldodeca-2,4,6,8,10-pentaene-1,12-dial (Cadialdehyde). seeds, or by introducing a caroteonid or apocarotenoid bio II, 9-apo-?3-caroten-9-one (B-ionone). III, 3-hydroxy-9-apo synthetic pathway enzyme-encoding transgene into the plant. B-caroten-9-one (3-hydroxy-C-ionone). IV, 3-hydroxy-5,6- In other embodiments, the method comprises decreasing pro epoxy-9-apo-C-caroten-9-one (3-hydroxy-5'6-epoxy-3-ion duction of the one or more caroteonid or apocarotenoid bio 30 one). V. 6,10-dimethyl-3,5.9-undecatrien-2-one synthetic pathway enzymes, e.g., by introducing a nucleic (pseudoionone). VI, 9-apo-C-caroten-9-one (C-ionone). VII, acid molecule into the coffee that inhibits the expression of 6,10-dimethyl-5.9-undecatrien-2-one (geranylacetone). one or more of the caroteonid or apocarotenoid biosynthetic FIG. 3. Schematic for CCD-catalyzed formation of aroma pathway enzyme-encoding genes. compounds. A) Neoxanthine is cleaved to form VIII, (3S.5R, Another aspect of the invention features a method to modu 35 6R)-3,5-dihydroxy-6,7-didehydro-5,6-dihydro-9-apo-f- late photosynthesis in a plant, especially in conditions of caroten-9-one (grasshopper ketone; Schwartz et al., 2001) by excess or insufficient light, comprising modulating produc the 9,10(9'10) cleavage dioxygenase CCD1. The grasshopper tion of one or more polypeptides that comprise the carotenoid ketone is the precursor for the formation of IX, B-dama or apocarotenoid biosynthetic pathway within coffee seeds. scenone and X, 3-hydroxy-3-damascenone (Suzuki et al., This method comprises increasing production of one or more 40 2002). B) CszCD is a 7.8(78) cleavage dioxygenase resulting caroteonid or apocarotenoid biosynthetic pathway enzymes in the formation of 3-hydroxy-3-cyclocitral from zeaxan within the plant, e.g., by increasing expression of one or more thine. C) BoLCD is a lycopene specific 5,6(5'6) cleavage endogenous caroteonid or apocarotenoid biosynthetic path dioxygenase resulting in the formation of the C, cleavage way enzyme-encoding genes within the coffee seeds, or by product previously identified as 6-methyl-5-hepten-2-one introducing a caroteonid or apocarotenoid biosynthetic path 45 (MHO). way enzyme-encoding transgene into the plant. FIG. 4. Optimal alignment of Coffea canephora protein According to another aspect of the invention, a promoter sequences with the closest databank sequences. A) CoPSY isolated from a caroteonid or apocarotenoid biosynthetic (SEQ ID NO.: 13) aligned with AtPSY (SEQ ID NO.:94), pathway enzyme-encoding coffee plant gene is provided. In LePSY1 (SEQID NO.:95), and CaPSY1 (SEQID NO.:96): certain embodiments, the caroteonid or apocarotenoid bio 50 B) CePDS (SEQ ID NO.14) aligned with AtPDS (SEQ ID synthetic pathway enzyme-encoding coffee gene encodes a NO.:97), LePDS (SEQ ID NO.: 98), and CaPDS (SEQ ID caroteonid or apocarotenoid biosynthetic pathway enzyme NO.:99); C) CeZDS (SEQID NO.:24) aligned with AtZDS having the one or more of the features described above. In (SEQID NO.100), CaZDS (SEQID NO.101), and LeZDS certain embodiments, the promoter comprises one or more (SEQ ID NO.:102); D) CePTOX (SEQ ID NO.:15) aligned regulatory sequences selected from the group consisting of a 55 with AtPTOX (SEQ ID NO.:103), CaPTOX (SEQ ID NO.: TATA box, an abscisic acid responsive element, an RY repeat 104), and CaPTOX (SEQID NO.: 105); E) Ce(CHY (SEQID (CATGCA(T/a)(A/g) of a leguminin box for regulating NO.:16) aligned with At?CHY (SEQID NO.106), and Lef expression of leguminin-type proteins, at least one dehydra CHY (SEQ ID NO.107); F) CeLeCY (SEQ ID NO.:17) tion responsive element/C-repeat cis-acting sequence motif aligned with LSLeCY (SEQID NO.108), and TeleCY (SEQ (G/ACCGAC) and at least one E-box motif (CANNTG). In a 60 ID NO.108); G) CeZEP (SEQ ID NO.: 18) aligned with specific embodiment, the promoter comprises SEQ ID LeZEP (SEQ ID NO.109), PdzEP (SEQ ID NO.110), NO:25. OsZEP (SEQID NO.:111), and AtZEP (SEQID NO.112); The invention also features a chimeric gene comprising a H) CcVDE (SEQID NO.19) aligned with AtVDE (SEQ ID promoter of a coffee caroteonid or apocarotenoid biosyn NO.:113), OsvDE (SEQID NO.:114), and NtWDE (SEQID thetic pathway enzyme-encoding gene, operably linked to 65 NO.:115): I) CeNCED3 (SEQ ID NO.:20) aligned with one or more coding sequences. A vector for transforming a AtNCED3 (SEQ ID NO.116), AtNCED5 (SEQ ID NO.: cell, comprising the chimeric gene, is also provided, as well 117), LeNCED1 (SEQ ID NO.118), StNCED1 (SEQ ID US 8,252,977 B2 9 10 NO.: 119), and VvNCED1 (SEQ ID NO.120): J) CCCCD1 transcript levels in two well-watered controls. Transcript lev (SEQID NO.:21) aligned with PhCCD1 (SEQID NO.121), els in two independent water-stressed plants are shown in LeCCD1B (SEQ ID NO.122), LeCCD1A (SEQ ID NO.: grey bars. 123), and AtCCD1 (SEQID NO.124); and K) CeFIB (SEQ ID NO.:22) aligned with AtEIB1b (SEQ ID NO.125), DETAILED DESCRIPTION OF ILLUSTRATIVE AtFIB1a (SEQID NO.126), CaFIB (SEQID NO.127), and EMBODIMENTS LeFIB (SEQ ID NO.128). The alignments were generated with the clustalW program in the Lasergene Software pack Definitions age (DNASTAR) and then adjusted manually to optimize the Various terms relating to the biological molecules and alignment. 10 other aspects of the present invention are used throughout the specification and claims. FIG. 5. Expression of carotenoid and apocarotenoid bio The term “carotenoid and apocarotenoid biosynthetic path synthetic genes during seed maturation: Comparison way refers to polypeptides that participate in carotenoid or between Robusta FRT-05 and Arabica T2308. Transcript lev apocarotenoid biosynthesis in plants, and more specifically, els for A) PSY, B) PDS, C) ZDS, D) PTOX, E) LeCY, F) 15 in coffee plants. This term encompasses the specific mecha BCHY, G) ZEP, H) VDE, J) CCD1, K) NCED3 and L) FIB1, nism of action of each respective protein in the pathway, in the grain of C. canephora, (FRT05; black bars) and C. including the enzyme-mediated derivation of apocarotenoids arabica (T2308; grey bars). The expression levels are deter from carotenoids. The polypeptides include without limita mined relative to the expression of transcripts of the consti tion, phytoene synthase, phytoene desaturase, C-carotene tutively expressed RPL39 gene in the same samples. SG, desaturase, plastid terminal oxidase, lycopene B-cyclase, Small green grain, LG, large grain; YG, yellow grain; RG, lycopene e-cyclase, 3-carotene hydroxylase, e-carotene ripe grain. hydroxylase, Zeaxanthine epoxidase, violaxanthine de-ep FIG. 6. Expression of CCD1 transcripts during seed devel oxidase, neoxanthine synthase, carotenoid cleavage dioxyge opment and activity of CcCCD1 in E. Coli. nase 1, carotenoid cleavage dioxygenase 4, Fibrillin, 9-cis A) Transcript levels of CCD1 in the grain of three C. 25 epoxycarotenoid dioxygenase, phytoene dehydrogenase, and canephora genotypes, (BP409, FRT05, FRT64; black bars) the like, as exemplified herein. and C. arabica (T2308; grey bars) determined by quantitative “Isolated” means altered “by the hand of man” from the PCR. Reverse transcription was carried out with equal natural State. If a composition or Substance occurs in nature, amounts of total RNA. S.G. Small green grain; LG, large it has been "isolated if it has been changed or removed from grain; YG, yellow grain; RG, ripe grain. 30 its original environment, or both. For example, a polynucle B) Activity of CCD1 following over-expression in E. coli otide or a polypeptide naturally present in a living plant or previously engineered to accumulate lycopene, B-carotene or animal is not "isolated,” but the same polynucleotide or Zeaxanthine. NI, non-induced; I, induced. polypeptide separated from the coexisting materials of its C) HPLC characterisation of Lyc, B-C and Zealines before natural state is "isolated’, as the term is employed herein. and after induction. The arrow indicates the loss of the caro 35 “Polynucleotide.” also referred to as “nucleic acid mol tenoid peak following induction of CCD1. Astaxanthin (3) ecule', generally refers to any polyribonucleotide or was added to samples and used to normalise results. polydeoxyribonucleotide, which may be unmodified RNA or FIG. 7. Activity of BCHY in E. coli previously engineered DNA or modified RNA or DNA. “Polynucleotides” include, to accumulate B-carotene or zeaxanthine. A) E. coli lines without limitation single- and double-stranded DNA, DNA engineered to accumulate B-carotene or Zeaxanthin trans 40 that is a mixture of single- and double-stranded regions, formed with pPEST17-Cc(BCHY either before (NI); or after single- and double-stranded RNA, and RNA that is mixture of (I), induction. B) HPLC characterization of C-caroteine pro single- and double-stranded regions, hybrid molecules com ducing cell lines before and after induction. B-C, B-carotene; prising DNA and RNA that may be single-stranded or, more Zea, Zeaxanthin. typically, double-stranded or a mixture of single- and double FIG.8. HPLC analysis of Coffea canphora and C. arabica 45 stranded regions. In addition, “polynucleotide' refers to green and red grain. Absorbance profiles at 450 nm. Peaks triple-stranded regions comprising RNA or DNA or both are: (1) neoxanthin, (2) violaxanthin, (3) lutien, (4) C-caro RNA and DNA. The term polynucleotide also includes DNAS tene, (5) B-carotene, (a) chlorophyll A, (b) chlorophyll B. or RNAs containing one or more modified bases and DNAS or Astaxanthin (9) was added prior to extraction and used to RNAs with backbones modified for stability or for other normalise results. Green grain from large green pericarp 50 reasons. “Modified” bases include, for example, tritylated stage (LG). Redgrain from mature red pericarp stage (RG). bases and unusual bases such as inosine. A variety of modi FIG. 9. HPLC analysis of Coffea arabica, C. canephora fications can be made to DNA and RNA; thus, “polynucle and Arabidopsis thaliana mature leaves. Absorbance was otide' embraces chemically, enzymatically or metabolically monitored at 450 nm. 60 mg samples were extracted as modified forms of polynucleotides as typically found in described in materials and methods. Peaks are: (1) neoxan 55 nature, as well as the chemical forms of DNA and RNA thin, (2) violaxanthin, (3) lutein, (4) C-carotene, (5) B-caro characteristic of viruses and cells. “Polynucleotide' also tene, (a) chlorophyll A, (b) chlorophyll B. Astaxanthin (3) embraces relatively short polynucleotides, often referred to as was added to coffee samples prior to extraction and used to oligonucleotides. normalise results. “Polypeptide' refers to any peptide or protein comprising FIG. 10. Expression of carotenoid biosynthetic genes and 60 two or more amino acids joined to each other by peptide carotenoid cleavage dioxygenase genes in the leaves of Cof bonds or modified peptide bonds, i.e., peptide isosteres. fea arabica (catimor) under drought stress. Transcript levels “Polypeptide' refers to both short chains, commonly referred for PSY, PDS, ZDS, PTOX, LeCY, BCHY,ZEP. VDE,CCD1, to as peptides, oligopeptides or oligomers, and to longer NCED3 and FIB1 were determined by quantitative RT-PCR. chains, generally referred to as proteins. Polypeptides may Expression was determined relative to the expression of tran 65 contain amino acids other than the 20 gene-encoded amino scripts of the constitutively expressed RPL39 gene in the acids. "Polypeptides' includeamino acid sequences modified same samples. The black bars in each case represent the mean either by natural processes, such as post-translational pro US 8,252,977 B2 11 12 cessing, or by chemical modification techniques which are gene, and may be changes of individual residues, or insertions well known in the art. Such modifications are well described or deletions of regions of nucleic acids. These mutations may in basic texts and in more detailed monographs, as well as in also occur in the coding and/or regulatory regions of other a voluminous research literature. Modifications can occur genes, which may regulate or control a gene and/or encoded anywhere in a polypeptide, including the peptide backbone, protein, so as to cause the protein to be non-functional or the amino acid side-chains and the amino or carboxyl termini. largely absent. It will be appreciated that the same type of modification may The term “substantially the same' refers to nucleic acid or be present in the same or varying degrees at several sites in a amino acid sequences having sequence variations that do not given polypeptide. Also, a given polypeptide may contain materially affect the nature of the protein (i.e. the structure, many types of modifications. Polypeptides may be branched 10 stability characteristics, Substrate specificity and/or biologi as a result of ubiquitination, and they may be cyclic, with or cal activity of the protein). With particular reference to without branching. Cyclic, branched and branched cyclic nucleic acid sequences, the term "substantially the same is polypeptides may result from natural posttranslational pro intended to refer to the coding region and to conserved cesses or may be made by synthetic methods. Modifications sequences governing expression, and refers primarily to include acetylation, acylation, ADP-ribosylation, amidation, 15 degenerate codons encoding the same amino acid, or alternate covalent attachment of flavin, covalent attachment of a heme codons encoding conservative Substitute amino acids in the moiety, covalent attachment of a nucleotide or nucleotide encoded polypeptide. With reference to amino acid derivative, covalent attachment of a lipid or lipid derivative, sequences, the term "substantially the same' refers generally covalent attachment of phosphotidylinositol, cross-linking, to conservative Substitutions and/or variations in regions of cyclization, disulfide bond formation, demethylation, forma the polypeptide not involved in determination of structure or tion of covalent cross-links, formation of cystine, formation function. of pyroglutamate, formylation, gamma-carboxylation, glyco The terms “percent identical and “percent similar are Sylation, GPI anchor formation, hydroxylation, iodination, also used herein in comparisons among amino acid and methylation, myristoylation, oxidation, proteolytic process nucleic acid sequences. When referring to amino acid ing, phosphorylation, prenylation, racemization, selenoyla 25 sequences, “identity” or “percent identical refers to the per tion, sulfation, transfer-RNA mediated addition of amino cent of the amino acids of the Subject amino acid sequence acids to proteins such as arginylation, and ubiquitination. See, that have been matched to identical amino acids in the com for instance, Proteins—Structure and Molecular Properties, pared amino acid sequence by a sequence analysis program. 2nd Ed., T. E. Creighton, W. H. Freeman and Company, New “Percent similar refers to the percent of the amino acids of York, 1993 and Wold, F., Posttranslational Protein Modifica 30 the Subject amino acid sequence that have been matched to tions: Perspectives and Prospects, pgs. 1-12 in Posttransla identical or conserved amino acids. Conserved amino acids tional Covalent Modification of Proteins, B. C. Johnson, Ed., are those which differ in structure but are similar in physical Academic Press, New York, 1983: Seifter et al., Analysis for properties such that the exchange of one for another would Protein Modifications and Nonprotein Cofactors, Meth Enzy not appreciably change the tertiary structure of the resulting mol (1990) 182:626-646 and Rattan et al., Protein Synthesis: 35 protein. Conservative substitutions are defined in Taylor Posttranslational Modifications and Aging, Ann NY Acad Sci (1986, J. Theor. Biol. 119:205). When referring to nucleic (1992) 663:48-62. acid molecules, “percent identical refers to the percent of the “Variant as the term is used herein, is a polynucleotide or nucleotides of the Subject nucleic acid sequence that have polypeptide that differs from a reference polynucleotide or been matched to identical nucleotides by a sequence analysis polypeptide respectively, but retains essential properties. A 40 program. typical variant of a polynucleotide differs in nucleotide “Identity” and “similarity' can be readily calculated by sequence from another, reference polynucleotide. Changes in known methods. Nucleic acid sequences and amino acid the nucleotide sequence of the variant may or may not alter sequences can be compared using computer programs that the amino acid sequence of a polypeptide encoded by the align the similar sequences of the nucleic or amino acids and reference polynucleotide. Nucleotide changes may result in 45 thus define the differences. In preferred methodologies, the amino acid substitutions, additions, deletions, fusions and BLAST programs (NCBI) and parameters used therein are truncations in the polypeptide encoded by the reference employed, and the DNAStar system (Madison, Wis.) is used sequence, as discussed below. A typical variant of a polypep to align sequence fragments of genomic DNA sequences. tide differs in amino acid sequence from another, reference However, equivalent alignments and similarity/identity polypeptide. Generally, differences are limited so that the 50 assessments can be obtained through the use of any standard sequences of the reference polypeptide and the variant are alignment software. For instance, the GCG Wisconsin Pack closely similar overall and, in many regions, identical. A age version 9.1, available from the Genetics Computer Group variant and reference polypeptide may differ in amino acid in Madison, Wis., and the default parameters used (gap cre sequence by one or more substitutions, additions, deletions, ation penalty 12, gap extension penalty 4) by that program fusions or truncations in any combination. A substituted or 55 may also be used to compare sequence identity and similarity. inserted amino acid residue may or may not be one encoded Antibodies' as used herein includes polyclonal and by the genetic code. A variant of a polynucleotide or polypep monoclonal antibodies, chimeric, single chain, and human tide may be naturally occurring, such as an allelic variant, or ized antibodies, as well as antibody fragments (e.g., Fab., Fab'. it may be a variant that is not known to occur naturally. F(ab') and F), including the products of a Fab or other Non-naturally occurring variants of polynucleotides and 60 immunoglobulin expression library. With respect to antibod polypeptides may be made by mutagenesis techniques or by ies, the term, “immunologically specific' or “specific' refers direct synthesis. to antibodies that bind to one or more epitopes of a protein of In reference to mutant plants, the terms “null mutant” or interest, but which do not substantially recognize and bind “loss-of-function mutant are used to designate an organism other molecules in a sample containing a mixed population of or genomic DNA sequence with a mutation that causes a gene 65 antigenic biological molecules. Screening assays to deter product to be non-functional or largely absent. Such muta mine binding specificity of an antibody are well known and tions may occur in the coding and/or regulatory regions of the routinely practiced in the art. For a comprehensive discussion US 8,252,977 B2 13 14 of Such assays, see Harlow et al. (Eds.), ANTIBODIES A LABO A“vector” is a replicon, Such as plasmid, phage, cosmid, or RATORY MANUAL; Cold Spring Harbor Laboratory; Cold Spring virus to which another nucleic acid segment may be operably Harbor, N.Y. (1988), Chapter 6. inserted so as to bring about the replication or expression of The term “substantially pure' refers to a preparation com the segment. prising at least 50-60% by weight the compound of interest The term “nucleic acid construct” or “DNA construct” is (e.g., nucleic acid, oligonucleotide, protein, etc.). More pref Sometimes used to refer to a coding sequence or sequences erably, the preparation comprises at least 75% by weight, and operably linked to appropriate regulatory sequences and most preferably 90-99% by weight, the compound of interest. inserted into a vector for transforming a cell. This term may Purity is measured by methods appropriate for the compound be used interchangeably with the term “transforming DNA 10 or “transgene'. Such a nucleic acid construct may contain a of interest (e.g. chromatographic methods, agarose or poly coding sequence for a gene product of interest, along with a acrylamide gel electrophoresis, HPLC analysis, and the like). selectable marker gene and/or a reporter gene. With respect to single-stranded nucleic acid molecules, the A "marker gene' or “selectable marker gene' is a gene term “specifically hybridizing” refers to the association whose encoded gene product confers a feature that enables a between two single-stranded nucleic acid molecules of suffi 15 cell containing the gene to be selected from among cells not ciently complementary sequence to permit such hybridiza containing the gene. Vectors used for genetic engineering tion underpre-determined conditions generally used in the art typically contain one or more selectable marker genes. Types (sometimes termed “substantially complementary'). In par of selectable marker genes include (1) antibiotic resistance ticular, the term refers to hybridization of an oligonucleotide genes, (2) herbicide tolerance or resistance genes, and (3) with a Substantially complementary sequence contained metabolic or auxotrophic marker genes that enable trans within a single-stranded DNA or RNA molecule, to the sub formed cells to synthesize an essential component, usually an stantial exclusion of hybridization of the oligonucleotide with amino acid, which the cells cannot otherwise produce. single-stranded nucleic acids of non-complementary A “reporter gene' is also a type of marker gene. It typically Sequence. encodes a gene product that is assayable or detectable by A “coding sequence' or “coding region” refers to a nucleic 25 standard laboratory means (e.g., enzymatic activity, fluores acid molecule having sequence information necessary to pro cence). duce a gene product, when the sequence is expressed. The The term “express.” “expressed,” or “expression' of a gene coding sequence may comprise untranslated sequences (e.g., refers to the biosynthesis of a gene product. The process introns) within translated regions, or may lack Such interven involves transcription of the gene into mRNA and then trans 30 lation of the mRNA into one or more polypeptides, and ing untranslated sequences (e.g., as in cDNA). encompasses all naturally occurring post-translational modi “Intron” refers to polynucleotide sequences in a nucleic fications. acid that do not code information related to protein synthesis. "Endogenous” refers to any constituent, for example, a Such sequences are transcribed into mRNA, but are removed gene or nucleic acid, or polypeptide, that can be found natu before translation of the mRNA into a protein. 35 rally within the specified organism. The term “operably linked' or “operably inserted” means A "heterologous' region of a nucleic acid construct is an that the regulatory sequences necessary for expression of the identifiable segment (or segments) of the nucleic acid mol coding sequence are placed in a nucleic acid molecule in the ecule within a larger molecule that is not found in association appropriate positions relative to the coding sequence so as to with the larger molecule in nature. Thus, when the heterolo enable expression of the coding sequence. By way of 40 gous region comprises a gene, the gene will usually be flanked example, a promoter is operably linked with a coding by DNA that does not flank the genomic DNA in the genome sequence when the promoter is capable of controlling the of the Source organism. In another example, a heterologous transcription or expression of that coding sequence. Coding region is a construct where the coding sequence itself is not sequences can be operably linked to promoters or regulatory found in nature (e.g., a cDNA where the genomic coding sequences in a sense orantisense orientation. The term “oper 45 sequence contains introns, or synthetic sequences having ably linked' is sometimes applied to the arrangement of other codons different than the native gene). Allelic variations or transcription control elements (e.g. enhancers) in an expres naturally-occurring mutational events do not give rise to a sion vector. heterologous region of DNA as defined herein. The term Transcriptional and translational control sequences are “DNA construct’, as defined above, is also used to refer to a DNA regulatory sequences, such as promoters, enhancers, 50 heterologous region, particularly one constructed for use in polyadenylation signals, terminators, and the like, that pro transformation of a cell. vide for the expression of a coding sequence in a host cell. A cell has been “transformed’ or “transfected by exog The terms “promoter”, “promoter region' or “promoter enous or heterologous DNA when such DNA has been intro sequence” refer generally to transcriptional regulatory duced inside the cell. The transforming DNA may or may not regions of a gene, which may be found at the 5' or 3' side of the 55 be integrated (covalently linked) into the genome of the cell. coding region, or within the coding region, or within introns. In prokaryotes, yeast, and mammalian cells for example, the Typically, a promoter is a DNA regulatory region capable of transforming DNA may be maintained on an episomal ele binding RNA polymerase in a cell and initiating transcription ment Such as a plasmid. With respect to eukaryotic cells, a of a downstream (3' direction) coding sequence. The typical 5' stably transformed cell is one in which the transforming DNA promoter sequence is bounded at its 3' terminus by the tran 60 has become integrated into a chromosome so that it is inher Scription initiation site and extends upstream (5' direction) to ited by daughter cells through chromosome replication. This include the minimum number of bases or elements necessary stability is demonstrated by the ability of the eukaryotic cell to initiate transcription at levels detectable above back to establish cell lines or clones comprised of a population of ground. Within the promoter sequence is a transcription ini daughter cells containing the transforming DNA. A “clone” is tiation site (conveniently defined by mapping with nuclease 65 a population of cells derived from a single cell or common S1), as well as protein binding domains (consensus ancestor by mitosis. A “cell line' is a clone of a primary cell sequences) responsible for the binding of RNA polymerase. that is capable of stable growth in vitro for many generations. US 8,252,977 B2 15 16 “Grain.”99 “seed,”&g or “bean.” refers to a flowering plants unit herein, this invention is intended to encompass nucleic acids of reproduction, capable of developing into another Such and encoded proteins from other Coffea species that are Suf plant. As used herein, especially with respect to coffee plants, ficiently similar to be used interchangeably with the C. cane the terms are used synonymously and interchangeably. phora polynucleotides and proteins for the purposes As used herein, the term “plant includes reference to 5 described below. Accordingly, when the term polypeptides or whole plants, plant organs (e.g., leaves, stems, shoots, roots), proteins that “comprise the carotenoid and apocarotenoid seeds, pollen, plant cells, plant cell organelles, and progeny biosynthetic pathway' is used herein, it is intended to encom thereof. Parts of transgenic plants are to be understood within the scope of the invention to comprise, for example, plant pass all Coffea proteins that have the general physical, bio cells, protoplasts, tissues, callus, embryos as well as flowers, chemical, and functional features described herein, as well as stems, seeds, pollen, fruits, leaves, or roots originating in 10 the polynucleotides that encode them. transgenic plants or their progeny. Considered in terms of their sequences, the polynucle Description: otides of the invention that encode proteins that comprise the In one of its aspects the present invention features nucleic carotenoid and apocarotenoid biosynthetic pathway include acid molecules from coffee that encode a variety of proteins allelic variants and natural mutants of SEQ ID NOs: 1-12, involved in the carotenoid and apocarotenoid biosynthetic 15 which are likely to be found in different varieties of C. cane pathway. Representative examples of nucleic acid molecules phora, and homologs of SEQID NOs: 1-12 likely to be found encoding proteins that comprise the carotenoid and apocaro in different coffee species. Because such variants and tenoid biosynthetic pathway were identified from databases homologs are expected to possess certain differences in of over 47,000 expressed sequence tags (ESTs) from several nucleotide and amino acid sequence, this invention provides Coffea canephora (robusta) cDNA libraries made with RNA isolated polynucleotides encoding proteins that comprise the isolated from young leaves and from the grain and pericarp carotenoid and apocarotenoid biosynthetic pathway that have tissues of cherries harvested at different stages of develop at least about 60%, preferably at least about 61%. 62%. 63%, ment. Overlapping ESTs were identified and “clustered into 64%. 65%, 66%, 67%, 68%, 69% or 70%, more preferably at unigenes (contigs) comprising complete coding sequences. least about 71%, 72%, 73%, 74%, 75%, 76%, 77%. 78%, The unigene sequences were annotated by performing a 25 79%, or 80%, even more preferably 81%, 82%, 83%, 84%, BLAST search of each individual sequence against the NCBI 85%. 86%, 87%, 88%, 89%, and even more preferably 90%, (National Center for Biotechnology Information) non-redun dant protein database. 91%, 92%, 93%, 94%, 95%, and most preferably 96%, 97%, BLAST searches of the coffee EST databases using bio 98% and 99% or more identity with any one of SEQ ID chemically characterized protein sequences from public data NOS:13-24, and comprise a nucleotide sequence having bases revealed gene sequences representing several important 30 equivalent ranges of identity to any one of SEQID NOs: 1-12. enzymes of the carotenoid biosynthetic pathway in the coffee Because of the natural sequence variation likely to exist plant, and sequences coding for two enzymes (NCED3 and among proteins that comprise the carotenoid and apocaro CCD1) that participate in the generation of apocarotenoids. tenoid biosynthetic pathway, and the genes encoding them in The full open reading frames (ORFs) of several of these different coffee varieties and species, one skilled in the art sequences were obtained, and a partial sequence of two other 35 would expect to find this level of variation, while still main carotenoid biosynthetic enzyme (PDS and ZDS) was also taining the unique properties of the polypeptides and poly cloned using degenerate primers and non-degenerate primers. nucleotides of the present invention. Such an expectation is These cDNAs and their encoded proteins are referred to due in part to the degeneracy of the genetic code, as well as to herein as follows: the known evolutionary Success of conservative amino acid

Enzyme cDNA (SEQID NO:) encoded protein (SEQID NO:) Phytoene synthase CcPSY 1 CcPSY 13 Phytoene desaturase CCPDS 2 CCPDS 14 Plastid terminal oxidase CCPTOX 3 CCPTOX 15 3-caroteine hydroxylase CcpCHY 4 CcpCHY 16 Lycopene e-cyclase CcLeCY 5 CcLeCY 17 Zeaxanthine epioxidase CCZEP 6 CCZEP 18 Violaxanthine de-epoxidase CcVDE 7 CcVDE 19 9-cis-epoxycarotenoid dioxygenase CCNCED3 8 CCNCED3 2O Carotenoid cleavage dioxygenase CoCCD1 9 CCCCD1 21 Fibrillin CCFIB 10 CCFIB 22 Phytoene dehydrogenase-like CCPDH 11 CCPDH 23 Zeta-caroteine desaturase CCZDS 12 CCZDS 24 Promoter Region (SEQ ID NO:) Promoter CCNCED3 pNCED3 25

Another aspect of the invention features promoter sequence variations, which do not appreciably alter the nature sequences and related elements that control expression of of the encoded protein. Accordingly, such variants and carotenoid and apocarotenoid biosynthetic pathway genes in 60 homologs are considered substantially the same as one coffee. As described in greater detail in the examples, a pro another and are included within the scope of the present moter sequence (contained in SEQ ID NO:25), from invention. CcNCED3 was identified by PCR-assisted primer walking, as The gene regulatory sequences associated with genes described in the examples. encoding proteins that comprise the carotenoid and apocaro Although polynucleotides encoding proteins that catalyze 65 tenoid biosynthetic pathway are of practical utility and are key steps the carotenoid and apocarotenoid biosynthetic path considered within the scope of the present invention. The C. way from Coffea canephora are described and exemplified canephora NCED3 promoter is exemplified herein. The US 8,252,977 B2 17 18 upstream region of the C. canephora NCED3 genomic other gene regulatory sequences associated with genes sequence is set forth herein as SEQID NO:25, and contains encoding proteins that comprise the carotenoid and apocaro part or all of an exemplary promoter of the invention, though tenoid biosynthetic pathway, even though the regulatory other portions of the promoter may be foundat other locations sequences themselves may not share sufficient homology to in the gene, as explained in the definition of “promoter set enable suitable hybridization. Moreover, the annotation of at forth hereinabove. However, promoters and other gene regu least a partial coding sequence will enable the skilled artisan latory sequences of genes encoding proteins that comprise the to determine the remaining coding sequence, as well the carotenoid and apocarotenoid biosynthetic pathway from any promoter or other gene regulatory sequences associated with coffee species may be obtained by the methods described the carotenoid or apocarotenoid protein of interest by the below, and may be utilized in accordance with the present 10 technique of upstream or downstream genome walking. Such invention. Promoters and regulatory elements governing tis techniques are established in the art. (Mishra RN et al., 2002: Sue specificity and temporal specificity of the expression of Rishi A Set al., 2004). genes encoding proteins that comprise the carotenoid and As a typical illustration, hybridizations may be performed apocarotenoid biosynthetic pathway may be used to advan according to the method of Sambrook et al., using a hybrid tage, alter or modify the expression of proteins that comprise 15 ization solution comprising: 5xSSC, 5xDenhardt’s reagent, the carotenoid and apocarotenoid biosynthetic pathway 1.0% SDS, 100 ug/ml denatured, fragmented salmon sperm toward the goal of enhancing the flavor and aroma of coffee DNA, 0.05% sodium pyrophosphate and up to 50% forma products produced from coffee beans comprising Such modi mide. Hybridization is carried out at 37-42°C. for at least six fications, among other utilities. hours. Following hybridization, filters are washed as follows: The following sections set forth the general procedures (1) 5 minutes at room temperature in 2xSSC and 1% SDS.; (2) involved in practicing the present invention. To the extent that 15 minutes at room temperature in 2xSSC and 0.1% SDS; (3) specific materials are mentioned, it is merely for the purpose 30 minutes-1 hour at 37°C. in 2XSSC and 0.1% SDS; (4) 2 of illustration, and is not intended to limit the invention. hours at 45-55° C. in 2xSSC and 0.1% SDS, changing the Unless otherwise specified, general biochemical and molecu solution every 30 minutes. lar biological procedures. Such as those set forth in Sambrook 25 One common formula for calculating the Stringency con et al., Molecular Cloning, Cold Spring Harbor Laboratory ditions required to achieve hybridization between nucleic (1989) or Ausubeletal. (eds), Current Protocols in Molecular acid molecules of a specified sequence homology (Sambrook Biology, John Wiley & Sons (2005) are used. et al., 1989): Nucleic Acid Molecules, Proteins and Antibodies: Nucleic acid molecules of the invention may be prepared 30 Tm=81.5°C..+16.6 Log Na++0.41(% G+C)- by two general methods: (1) they may be synthesized from 0.63 (% formamide)-600, #bp in duplex appropriate nucleotide triphosphates, or (2) they may be iso As an illustration of the above formula, using Na+= lated from biological sources. Both methods utilize protocols 0.368 and 50% formamide, with GC content of 42% and an well known in the art. average probe size of 200 bases, the Tm is 57°C. The Tm of The availability of nucleotide sequence information, Such 35 a DNA duplex decreases by 1-1.5°C. with every 1% decrease as the cDNA having SEQ ID NOS:1-12, or the regulatory in homology. Thus, targets with greater than about 75% sequence of SEQ ID NO:25, enables preparation of an iso sequence identity would be observed using a hybridization lated nucleic acid molecule of the invention by oligonucle temperature of 42°C. In one embodiment, the hybridization otide synthesis. Synthetic oligonucleotides may be prepared is at 37° C. and the final wash is at 42°C.; in another embodi by the phosphoramidite method employed in the Applied 40 ment the hybridization is at 42°C. and the final wash is at 50° Biosystems 38A DNA Synthesizer or similar devices. The C.; and in yet another embodiment the hybridization is at 42 resultant construct may be purified according to methods C. and final wash is at 65° C., with the above hybridization known in the art, Such as high performance liquid chroma and wash Solutions. Conditions of high Stringency include tography (HPLC). Long, double-stranded polynucleotides, hybridization at 42°C. in the above hybridization solution such as a DNA molecule of the present invention, must be 45 and a final wash at 65° C. in 0.1XSSC and 0.1% SDS for 10 synthesized in stages, due to the size limitations inherent in minutes. current oligonucleotide synthetic methods. Thus, for Nucleic acids of the present invention may be maintained example, a long double-stranded molecule may be synthe as DNA in any convenient cloning vector. In a preferred sized as several Smaller segments of appropriate complemen embodiment, clones are maintained in plasmid cloning/ex tarity. Complementary segments thus produced may be 50 pression vector, such as pGEM-T (Promega Biotech, Madi annealed Such that each segment possesses appropriate cohe son, Wis.).pBluescript (Stratagene, La Jolla, Calif.), pCR4 sive termini for attachment of an adjacent segment. Adjacent TOPO (Invitrogen, Carlsbad, Calif.) or plT28a+(Novagen, segments may be ligated by annealing cohesive termini in the Madison, Wis.), all of which can be propagated in a suitable presence of DNA ligase to construct an entire long double E. coli host cell. stranded molecule. A synthetic DNA molecule so constructed 55 Nucleic acid molecules of the invention include cDNA, may then be cloned and amplified in an appropriate vector. genomic DNA, RNA, and fragments thereof which may be In accordance with the present invention, nucleic acids single-, double-, or even triple-stranded. Thus, this invention having the appropriate level sequence homology with part or provides oligonucleotides (sense orantisense Strands of DNA all of the coding and/or regulatory regions genes encoding or RNA) having sequences capable of hybridizing with at proteins that comprise the carotenoid and apocarotenoid bio 60 least one sequence of a nucleic acid molecule of the present synthetic pathway may be identified by using hybridization invention. Such oligonucleotides are useful as probes for and washing conditions of appropriate Stringency. It will be detecting genes encoding proteins that comprise the caro appreciated by those skilled in the art that the aforementioned tenoid and apocarotenoid biosynthetic pathway or mRNA in strategy, when applied to genomic sequences, will, in addi test samples of plant tissue, e.g., by PCR amplification, or for tion to enabling isolation coding sequences for genes encod 65 the positive or negative regulation of expression genes encod ing proteins that comprise the carotenoid and apocarotenoid ing proteins that comprise the carotenoid and apocarotenoid biosynthetic pathway, also enable isolation of promoters and biosynthetic pathway at or before translation of the mRNA US 8,252,977 B2 19 20 into proteins. Methods in which oligonucleotides or poly a multigene family, then member-specific antibodies made to nucleotides may be utilized as probes for Such assays include, synthetic peptides corresponding to nonconserved regions of but are not limited to: (1) in situ hybridization; (2) Southern the protein can be generated. hybridization (3) northern hybridization; and (4) assorted Kits comprising an antibody of the invention for any of the amplification reactions such as polymerase chain reactions purposes described herein are also included within the scope (PCR) (including RT-PCR) and ligase chain reaction (LCR). of the invention. In general. Such a kit includes a control Polypeptides encoded by nucleic acids of the invention antigen for which the antibody is immunospecific. may be prepared in a variety of ways, according to known Carotenoids and apocarotenoids play a role in many methods. If produced in situ the polypeptides may be purified aspects of human health and wellness. Carotenoids have been 10 demonstrated to be powerful antioxidants (DiMascio, Petal. from appropriate sources, e.g., seeds, pericarps, or other plant 1991), are efficient in protection from ultraviolet light (Sies, parts. H et al. 2004), have immunoprotective roles and may facili Alternatively, the availability of isolated nucleic acid mol tate immune cell proliferation (Watzl, B. et al. 1999; ecules enables production of the proteins using in vitro Boelsma, E et al. 2001: Aust, O et al. 2005), may be used to expression methods known in the art. For example, a cDNA or 15 treat photosensitivity diseases (Matthews-Roth, M. et al. gene may be cloned into an appropriate in vitro transcription 1993), are protective against age-related macular degenera vector, such a pSP64 or pSP65 for in vitro transcription, tion (Seddon J M. etal. 1994), are protective against cataracts followed by cell-free translation in a suitable cell-free trans (Taylor A 1993), may guard against cardiovascular disease lation system, such as wheat germ or rabbit reticulocytes. In (Gaziano JMetal. 1993), and demonstrate a preventative and vitro transcription and translation systems are commercially possible chemotherapeutic effect against many human can available, e.g., from Promega Biotech, Madison, Wis., BRL, cers such as lung, orolaryngeal, colorectal, breast, prostate, Rockville, Md. or Invitrogen, Carlsbad, Calif. and cervical cancers, among others (Mayne, S1996). In addi According to a preferred embodiment, larger quantities of tion, carotenoids Such as alpha- and beta-carotene play a key polypeptides that comprise the carotenoid and apocarotenoid role in vitamin A synthesis. (Britton G, 1995). This list of biosynthetic pathway may be produced by expression in a 25 health benefits attributable to carotenoids and apocarotenoids Suitable prokaryotic or eukaryotic system. For example, part is meant to be illustrative and not exhaustive, and it is pre or all of a DNA molecule, such as the cDNAs having SEQID sumed that there are many other beneficial health effects NOs: 1-12, may be inserted into a plasmid vector adapted for attributable to carotenoids presently unknown. Accordingly, expression in a bacterial cell (such as E. coli) or a yeast cell the coffee polypeptides that comprise the biosynthetic path 30 way of carotenoids and apocarotenoids described and exem (such as Saccharomyces cerevisiae), or into a baculovirus plified herein are expected to find utility in a variety of food, vector for expression in an insect cell. Such vectors comprise health, and wellness applications. For example, the coffee the regulatory elements necessary for expression of the DNA polypeptides that comprise the biosynthetic pathway of caro in the host cell, positioned in Such a manner as to permit tenoids and apocarotenoids, or their respective carotenoid or expression of the DNA in the host cell. Such regulatory ele 35 apocarotenoid products, may be utilized as dietary Supple ments required for expression include promoter sequences, ments. In addition, the antioxidant and photoprotective prop transcription initiation sequences and, optionally, enhancer erties of carotenoids and apopcarotenoids may prove advan Sequences. tageous in both food and cosmetic products. The polypeptides that comprise the carotenoid and apoc One or more of the aforementioned applications for the arotenoid biosynthetic pathway produced by gene expression 40 polypeptides that comprise the carotenoid and apocarotenoid in a recombinant prokaryotic or eukaryotic system may be biosynthetic pathway may be pursued by exploiting the avail purified according to methods known in the art. In a preferred ability of the polynucleotides encoding polypeptides that embodiment, a commercially available expression/secretion comprise the carotenoid and apocarotenoid biosynthetic system can be used, whereby the recombinant protein is pathaway described herein to generate significant quantities expressed and thereafter secreted from the host cell, to be 45 of pure protein using recombinant organisms (e.g., in the easily purified from the Surrounding medium. If expression/ yeast Picia pastoris or in food compatible Lactobacilli, or in secretion vectors are not used, an alternative approach plant cells), and then testing the proteins in new or established involves purifying the recombinant protein by affinity sepa assays for antioxidant potential, immunoproliferative poten ration, such as by immunological interaction with antibodies tial, chemoprotective or chemotherapeutic potential, and the that bind specifically to the recombinant protein. Such meth 50 like. Similar testing may be carried out using the carotenoids ods are commonly used by skilled practitioners. or apocarotenoids produced by these proteins according to The polypeptides that comprise the carotenoid and apoc suitable means established or developed in the art. If specific arotenoid biosynthetic pathway of the invention, prepared by purified proteins, or carotenoid or apocarotenoid products the aforementioned methods, may be analyzed according to produced by such proteins are found to be particularly useful, standard procedures. 55 natural versions of those proteins and their carotenoid or Polypeptides that comprise the carotenoid and apocaro apocarotenoid products also may be isolated from coffee tenoid biosynthetic pathway purified from coffee, or pro grains or other plant parts, or from tissues and organs of other duced recombinantly, may be used to generate polyclonal or plants enriched in those carotenoid and apocarotenoid bio monoclonal antibodies, antibody fragments or derivatives as synthetic pathway enzymes. defined herein, according to known methods. Antibodies that 60 Vectors, Cells, Tissues and Plants: recognize and bind fragments of the polypeptides that com Also featured in accordance with the present invention are prise the carotenoid and apocarotenoid biosynthetic pathway vectors and kits for producing transgenic host cells that con of the invention are also contemplated, provided that the tain a polynucleotide encoding polypeptides that comprise antibodies are specific for polypeptides that comprise the the carotenoid and apocarotenoid biosynthetic pathway, oran carotenoid and apocarotenoid biosynthetic pathway. For 65 oligonucleotide, or homolog, analog or variant thereof in a example, if analyses of the proteins or Southern and cloning sense or antisense orientation, or a reporter gene and other analyses (see below) indicate that the cloned genes belongs to constructs under control of cell or tissue-specific promoters, US 8,252,977 B2 21 22 particularly carotenoid- or apocarotenoid-encoding gene pro but are not limited to, BIN19 and derivatives thereof, the pBI moters as described herein, and other regulatory sequences. vector series, and binary vectors pGA482, pGA492, Suitable host cells include, but are not limited to, plant cells, pLH7000 (GenBank Accession AY234330) and any suitable bacterial cells, yeast and other fungal cells, insect cells and one of the pCAMBIA vectors (derived from the pPZP vectors mammalian cells. Vectors for transforming a wide variety of 5 constructed by Hajdukiewicz, Svab & Maliga, (1994) Plant these host cells are well known to those of skill in the art. They Mol Biol 25: 989-994, available from CAMBIA, GPO Box include, but are not limited to, plasmids, phagemids, cosmids, 3200, Canberra ACT 2601, Australia or via the worldwide baculoviruses, bacmids, bacterial artificial chromosomes web at CAMBIA.org). For transformation of monocot spe (BACs), yeast artificial chromosomes (YACs), as well as cies, biolistic bombardment with particles coated with trans other bacterial, yeast and viral vectors. Typically, kits for 10 forming DNA and silicon fibers coated with transforming producing transgenic host cells will contain one or more DNA are often useful for nuclear transformation. Alterna appropriate vectors and instructions for producing the trans tively, Agrobacterium "superbinary” vectors have been used genic cells using the vector. Kits may further include one or Successfully for the transformation of rice, maize and various more additional components, such as culture media for cul other monocot species. turing the cells, reagents for performing transformation of the 15 DNA constructs for transforming a selected plant comprise cells and reagents for testing the transgenic cells for gene a coding sequence of interest operably linked to appropriate 5' expression, to name a few. (e.g., promoters and translational regulatory sequences) and The present invention includes transgenic plants compris 3' regulatory sequences (e.g., terminators). In a preferred ing one or more copies of a gene encoding a polypeptide that embodiment, a coding sequence encoding a polypeptide that comprises the carotenoid and apocarotenoid biosynthetic comprises the carotenoid and apocarotenoid biosynthetic pathway, or nucleic acid sequences that inhibit the production pathway under control of its natural 5' and 3' regulatory ele or function of a plants endogenous polypeptides that com ments is utilized. In other embodiments, coding and regula prise the carotenoid and apocarotenoid biosynthetic pathway. tory sequences are swapped (e.g., CcPSY1 coding sequence This is accomplished by transforming plant cells with a trans operably linked to LRCY promoter) to alter the protein con gene that comprises part of all of a coding sequence for a 25 tent of the seed of the transformed plant for a phenotypic polypeptide that comprises the carotenoid and apocarotenoid improvement, e.g., in flavor, aroma or other feature. biosynthetic pathway, or mutant, antisense or variant thereof, In an alternative embodiment, the coding region of the gene including RNA, controlled by either native or recombinant is placed under a powerful constitutive promoter. Such as the regulatory sequences, as described below. Transgenic plants Cauliflower Mosaic Virus (CaMV) 35S promoter or the fig coffee species are preferred, including, without limitation, C. 30 wort mosaic virus 35S promoter. Other constitutive promot abeokutae, C. arabica, C. arnoldiana, C. aruweniensis, C. ers contemplated for use in the present invention include, but bengalensis, C. Canephora, C. congensis C. dewevrei, C. are not limited to: T-DNA mannopine synthetase, nopaline excelsa, C. eugenioides, and C. heterocalyx, C. kapakata, C. synthase and octopine synthase promoters. In other embodi khasiana, C. liberica, C. moloundou, C. rasenosa, C. Salva ments, a strong monocot promoter is used, for example, the trix, C. sessiflora, C. Stenophylla, C. travencorensis, C. 35 maize ubiquitin promoter, the rice actin promoter or the rice wightiana and C. Zanguebariae. Plants of any species are also tubulin promoter (Jeon et al., Plant Physiology. 123: 1005-14. included in the invention; these include, but are not limited to, 2000). tobacco, Arabidopsis and other “laboratory-friendly' spe Transgenic plants with coding sequences to express cies, cereal crops such as maize, wheat, rice, soybean barley, polypeptides that comprise the carotenoid and apocarotenoid rye, oats, Sorghum, alfalfa, clover and the like, oil-producing 40 biosynthetic pathway under an inducible promoter are also plants such as canola, safflower, Sunflower, peanut, cacao and contemplated to be within the scope of the present invention. the like, vegetable crops such as tomato tomatillo, potato, Inducible plant promoters include the tetracycline repressor/ pepper, eggplant, Sugar beet, carrot, cucumber, lettuce, pea operator controlled promoter, the heat shock gene promoters, and the like, horticultural plants such as aster, begonia, chry stress (e.g., wounding)-induced promoters, defense respon Santhemum, delphinium, petunia, Zinnia, lawn and turf 45 sive gene promoters (e.g. phenylalanine ammonia lyase grasses and the like. genes), wound induced gene promoters (e.g. hydroxyproline Transgenic plants can be generated using standard plant rich cell wall protein genes), chemically-inducible gene pro transformation methods known to those skilled in the art. moters (e.g., nitrate reductase genes, glucanase genes, chiti These include, but are not limited to, Agrobacterium vectors, nase genes, etc.) and dark-inducible gene promoters (e.g., polyethylene glycol treatment of protoplasts, biolistic DNA 50 asparagine synthetase gene) to name only a few. delivery, UV laser microbeam, gemini virus vectors or other Tissue specific and development-specific promoters are plant viral vectors, calcium phosphate treatment of proto also contemplated for use in the present invention, in addition plasts, electroporation of isolated protoplasts, agitation of cell to the carotenoid or apocarotenoid protein promoters of the Suspensions in Solution with microbeads coated with the invention. Non-limiting examples of seed-specific promoters transforming DNA, agitation of cell Suspension in Solution 55 include Cim 1 (cytokinin-induced message). cz19B1 (maize with silicon fibers coated with transforming DNA, direct 19 kDa Zein), milps (myo-inositol-1-phosphate synthase), DNA uptake, liposome-mediated DNA uptake, and the like. and celA (cellulose synthase) (U.S. application Ser. No. Such methods have been published in the art. See, e.g., Meth 09/377,648), bean beta-phaseolin, napin, beta-conglycinin, ods for Plant Molecular Biology (Weissbach & Weissbach, soybean lectin, cruciferin, maize 15 kDa Zein, 22 kDa Zein, 27 eds., 1988); Methods in Plant Molecular Biology (Schuler & 60 kDa Zein, g-Zein, waxy, shrunken 1, shrunken 2, and globulin Zielinski, eds., 1989); Plant Molecular Biology Manual 1, soybean 11S legumin (Bäumlein et al., 1992), and C. cane (Gelvin, Schilperoort, Verma, eds., 1993); and Methods in phora 11S seed storage protein (Marraccini et al., 1999, Plant Plant Molecular Biology—A Laboratory Manual (Maliga, Physiol. Biochem. 37: 273-282). See also WO 00/12733, Klessig, Cashmore, Gruissem & Varner, eds., 1994). where seed-preferred promoters from endl and end2 genes The method of transformation depends upon the plant to be 65 are disclosed. Other Coffea seed specific promoters may also transformed. Agrobacterium vectors are often used to trans be utilized, including but not limited to the oleosin gene form dicot species. Agrobacterium binary vectors include, promoter described in commonly-owned, co-pending Provi US 8,252,977 B2 23 24 sional Application No. 60/696,445 and the dehyrdin gene well known in the art, and include the cis-acting derivative promoter described in commonly-owned, co-pending Provi (omega') of the 5' leader sequence (omega) of the tobacco sional Application No. 60/696,890. Examples of other tissue mosaic virus, the 5' leader sequences from brome mosaic specific promoters include, but are not limited to: the ribulose virus, alfalfa mosaic virus, and turnip yellow mosaic virus. bisphosphate carboxylase (RuBisCo) small subunit gene pro Plants are transformed and thereafter screened for one or moters (e.g., the coffee Small subunit promoter as described more properties, including the presence of the transgene by Marracini et al., 2003) or chlorophyll a?b binding protein product, the transgene-encoding mRNA, or an altered pheno (CAB) gene promoters for expression in photosynthetic tis type associated with expression of the transgene. It should be Sue; and the root-specific glutamine synthetase gene promot recognized that the amount of expression, as well as the ers where expression in roots is desired. 10 The coding region is also operably linked to an appropriate tissue- and temporal-specific pattern of expression of the 3' regulatory sequence. In embodiments where the native 3' transgenes in transformed plants can vary depending on the regulatory sequence is not use, the nopaline synthetase poly position of their insertion into the nuclear genome. Such adenylation region may be used. Other useful 3' regulatory positional effects are well known in the art. For this reason, regions include, but are not limited to the octopine synthase 15 several nuclear transformants should be regenerated and polyadenylation region. tested for expression of the transgene. The selected coding region, under control of appropriate Methods: regulatory elements, is operably linked to a nuclear drug The nucleic acids and polypeptides of the present invention resistance marker, Such as kanamycin resistance. Other useful can be used in any one of a number of methods whereby the selectable marker systems include genes that confer antibi protein products can be expressed in coffee plants in order otic or herbicide resistances (e.g., resistance to hygromycin, that the proteins may play a role in photosynthesis, and in the Sulfonylurea, phosphinothricin, or glyphosate) or genes con enhancement of flavor and/or aroma of the coffee beverage or ferring selective growth (e.g., phosphomannose isomerase, coffee products ultimately produced from the bean of the enabling growth of plant cells on mannose). Selectable coffee plant expressing the protein. Similarly, the polypep marker genes include, without limitation, genes encoding 25 tides of the invention can be used in any one of a number of antibiotic resistance. Such as those encoding neomycin phos methods whereby the carotenoids, apocarotenoids, and other photransferase II (NEO), dihydrofolate reductase (DHPR) Such phytochemical products synthesized from the polypep and hygromycin phosphotransferase (HPT), as well as genes tides may play a role in photosynthesis, and in the enhance that confer resistance to herbicidal compounds. Such as gly ment of flavor and/or aroma of the coffee beverage or coffee phosate-resistant EPSPS and/or glyphosate oxidoreducatase 30 products ultimately produced from the bean of the coffee (GOX), Bromoxynil nitrilase (BXN) for resistance to bro plant containing the carotenoids and apocarotenoids. moxynil, AHAS genes for resistance to imidazolinones, sul With respect to photosynthesis, carotenoids play a role in fonylurea resistance genes, and 2,4-dichlorophenoxyacetate photoprotection under light stress, and in light collection in (2,4-D) resistance genes. shady environments. In fact, many plants alter their caro In certain embodiments, promoters and other expression 35 tenoid composition in response to shady conditions versus regulatory sequences encompassed by the present invention full sunlight. (Demmig-Adams et al., 1996). Therefore, the are operably linked to reporter genes. Reporter genes contem ability to manipulate production of polypeptides that com plated for use in the invention include, but are not limited to, prise the biosynthetic pathway for carotenoids and apocaro genes encoding green fluorescent protein (GFP), red fluores tenoids in a plant, or even to use the polynucleotides and cent protein (DSRed), Cyan Fluorescent Protein (CFP), Yel 40 proteins of the invention to monitor Such gene expression, low Fluorescent Protein (YFP), Cerianthus Orange Fluores will enable study and manipulation of the response of the cent Protein (cCFP), alkaline phosphatase (AP), B-lactamase, coffee plant to varying levels of Sunlight. This knowledge chloramphenicol acetyltransferase (CAT), adenosine deami enables the generation of modified coffee plants that are nase (ADA), aminoglycoside phosphotransferase (neo, better equipped for photosynthesis in Sub-optimal conditions G418) dihydrofolate reductase (DHFR), hygromycin-B- 45 Such as excess light, where carotenoids protect photosyn phosphotransferase (FPH), thymidine kinase (TK), lacZ (en thetic pigments, or insufficient light, where a decrease in coding O-galactosidase), and Xanthine guanine phosphoribo accessory pigments enables more efficient light harvesting. syltransferase (XGPRT), Beta-Glucuronidase (gus), With respect to flavor and aroma of roasted coffee grain, it Placental Alkaline Phosphatase (PLAP), Secreted Embryonic is expected that the polypeptides that comprise the carotenoid Alkaline Phosphatase (SEAP), or Firefly or Bacterial 50 and apocarotenoid biosynthetic pathway exert Some influ Luciferase (LUC). As with many of the standard procedures ence on the generation of coffee flavors via the Maillard associated with the practice of the invention, skilled artisans reaction that occurs during roasting, by means of the content will be aware of additional sequences that can serve the func of the proteins themselves, or the products Such as caro tion of a marker or reporter. tenoids or apocarotenoids they produce. Proteins, and par Additional sequence modifications are known in the art to 55 ticularly protein degradation products (peptides and amino enhance gene expression in a cellular host. These modifica acids), represent an important group of flavor precursors tions include elimination of sequences encoding Superfluous (Spanier et al., 2004). Therefore, relatively abundant proteins polyadenylation signals, exon-intron splice site signals, Such as those that comprise the carotenoid and apocarotenoid transposon-like repeats, and other such well-characterized biosynthetic pathway can be expected to make some contri sequences that may be deleterious to gene expression. Alter 60 bution to the flavor generating reactions that occur during natively, if necessary, the G/C content of the coding sequence coffee roasting. Such a contribution may stem from the con may be adjusted to levels average for a given coffee plant cell centration of the proteins themselves in the coffee bean, or the host, as calculated by reference to known genes expressed in concentration of the carotenoids or apocarotenoids ultimately a coffee plant cell. Also, when possible, the coding sequence produced from the proteins. The ability to monitor (e.g., is modified to avoid predicted hairpin secondary mRNA 65 through marker-assisted breeding) or manipulate protein structures. Another alternative to enhance gene expression is expression profiles for polypeptides that comprise the caro to use 5' leader sequences. Translation leader sequences are tenoid or apocarotenoid biosynthetic pathway is provided by US 8,252,977 B2 25 26 the polynucleotides of the present invention, in accordance the undesirable diterpenoids (cafestol and kahweol) by with the methods described herein. diverting metabolites for the formation of health beneficial Thus, one aspect of the present invention features methods carotenoids. to alter the profile of polypeptides that comprise the caro The capacity for light collection in shady environments in tenoid or apocarotenoid biosynthetic pathway in a plant, pref a plant Such as the coffee plant may be improved by decreas erably coffee, comprising increasing or decreasing an amount ing production of one or more of the polypeptides that com or activity of one or more polypeptides that comprise the prise the carotenoid or apocarotenoid biosynthetic pathway in carotenoid or apocarotenoid biosynthetic pathway in the the plant, by Screening naturally-occurring variants for plant. For instance, in one embodiment of the invention, a decreased expression of polypeptides that comprise the caro gene encoding a polypeptide that comprises the carotenoid or 10 tenoid or apocarotenoid biosynthetic pathway, or by Screen apocarotenoid biosynthetic pathway under control of its own ing naturally-occurring variants for decreased levels of the expression-controlling sequences is used to transform a plant various carotenoids or apopcarotenoids. For instance, loss for the purpose of increasing production of that polypeptide of-function (null) mutant plants may be created or selected in the plant. Alternatively, a coding region for a polypeptide from populations of plant mutants currently available. It will that comprises the carotenoid or apocarotenoid biosynthetic 15 also be appreciated by those of skill in the art that mutant plant pathway is operably linked to heterologous expression con populations may also be screened for mutants that over-ex trolling regions, such as constitutive or inducible promoters. press a particular polypeptide that comprises the carotenoid Increasing carotenoid and apocarotenoid production in or apocarotenoid biosynthetic pathway, utilizing one or more coffee seeds is expected to have a variety of beneficial effects. of the methods described herein. Mutant populations can be Coffee has been shown to contain two carotenoid derived made by chemical mutagenesis, radiation mutagenesis, and flavor components, C.-ionone and B-damascenone (Czerny et transposon or T-DNA insertions, or targeting induced local al., 2000; Akiyama et al., 2003; Variyaret al., 2003), the latter lesions in genomes (TILLING, see, e.g., Henikoffet al., 2004, being identified as major components of coffee both before Plant Physiol. 135(2): 630-636; Gilchrist & Haughn, 2005, and after roasting (Ortiz et al., 2004). Czemy et al. (2000) Curr. Opin. Plant Biol. 8(2): 211-215). The methods to make identified a 800-fold increase in B-damascenone after roast 25 mutant populations are well known in the art. ing. Due to their low odor threshold, only small amounts of The nucleic acids of the invention can be used to identify these and other carotenoid derived volatile compounds are mutant polypeptides that comprise the carotenoid and apoc needed to alter the aroma of roasted coffee. arotenoid biosynthetic pathway in various plant species. In Increasing carotenoid content in coffee grain is therefore species such as maize or Arabidopsis, where transposon expected to lead to an increase in carotenoid derived volatiles 30 insertion lines are available, oligonucleotide primers can be implicated in aroma during the roasting process. The 800-fold designed to Screen lines for insertions in the genes encoding increase in B-damascenone during roasting is likely due to the polypeptides that comprise the carotenoid and apocarotenoid thermal breakdown of the carotenoid precursor. Increased biosynthetic pathway. Through breeding, a plant line may production of carotenoids may lead to an increase in B-dama then be developed that is heterozygous or homozygous for the scenone as well as the formation of new apocarotenoids not 35 interrupted gene. previously detected in coffee, Such as B-ionone, pseudoion A plant also may be engineered to display a phenotype one, geranylacetone, B-cyclocitral, citral and 6-methyl-5- similar to that seen in null mutants created by mutagenic hepten-2-one. In other systems, e.g., tomato, the aroma of the techniques. A transgenic null mutant can be created by a “high beta’ mutant, which accumulates C-carotene, is domi expressing a mutant form of a selected polypeptide that com nated by the B-carotene derived B-ionone, whereas the Jubilee 40 prises the carotenoid and apocarotenoid biosynthetic path mutant, which produces mainly acyclic carotenes has been way to create a “dominant negative effect.” While not limiting shown to produce geranylacetone (Stevens, 1970). Acyclic the invention to any one mechanism, this mutant protein will carotenes Such as 1-carotene have been shown to be the pre compete with wild-type protein for interacting proteins or cursors of geranylacetone (Simkin et al., 2004b). Thus, modi other cellular factors. Examples of this type of “dominant fications in carotenoid content or of specific carotenoids can 45 negative effect are well known for both insect and vertebrate lead to changes in related carotenoid derived volatiles. systems (Radke et al., 1997, Genetics 145: 163-171; Kolchet The polynucleotides and methods provided in accordance al., 1991, Nature 349: 426-428). with the present invention also enable the overproduction of Another kind of transgenic null mutant can be created by novel ketocarotenoids, such as astaxanthin (3.3'dihydroxy-4, inhibiting the translation of mRNA encoding the polypep 4'diketo-Bf3-carotene) formed from B-carotene and Zeaxan 50 tides that comprise the carotenoid and apocarotenoid biosyn thin, by the overexpression of Adonis aestivalis ketocaro thetic pathway by “post-transcriptional gene silencing.” The tenoid biosynthetic genes (Cunningham and Gantt, 2005). gene from the species targeted for down-regulation, or a Astaxanthin is involved in cancer prevention (Tanaka et al. fragment thereof, may be utilized to control the production of 1994) and has been described as an immune system enhancer the encoded protein. Full-length antisense molecules can be (Jyonouchi et al., 1993) Astaxanthin is used as a food supple 55 used for this purpose. Alternatively, antisense oligonucle ment and in animal and fish feed. Its presence in coffee could otides targeted to specific regions of the mRNA that are impart a health benefit and could result in the formation of critical for translation may be utilized. The use of antisense novel apocarotenoids during roasting. molecules to decrease expression levels of a pre-determined The over-expression of phytoene synthase (PSY) in trans gene is known in the art. Antisense molecules may be pro genic tomatoes redirected metabolites from the diterpenoid 60 vided in situ by transforming plant cells with a DNA construct pathway for the formation of carotenoids (Fray et al., 1995). which, upon transcription, produces the antisense RNA All diterpenoids are derived from geranylgeranyl diphos sequences. Such constructs can be designed to produce full phate, the precursor of the first carotenoid-phytoene (Rich length or partial antisense sequences. This gene silencing man et al., 1998). Two diterpenoids of interest are cafestol and effect can be enhanced by transgenically over-producing both kahweol, which have been associated with negative health 65 sense and antisense RNA of the gene coding sequence so that impacts of espresso coffee. Accordingly, seed-specific over a high amount of dsRNA is produced (for example see Water expression of PSY in coffee seeds may lead to a decrease in house et al., 1998, PNAS 95: 13959-13964). In this regard, US 8,252,977 B2 27 28 dsRNA containing sequences that correspond to part or all of utility as research tools for the further elucidation of the at least one intron have been found particularly effective. In participation of polypeptides that comprise the carotenoid one embodiment, part or all of the coding sequence antisense and apocarotenoid biosynthetic pathway in flavor, aroma and Strand is expressed by a transgene. In another embodiment, other features of coffee seeds associated with pigments and hybridizing sense and antisense strands of part or all of the photosynthesis. Plants containing one transgene or a speci coding sequence for polypeptides that comprise the caro fied mutation may also be crossed with plants containing a tenoid and apocarotenoid biosynthetic pathway are transgeni complementary transgene or genotype in order to produce cally expressed. plants with enhanced or combined phenotypes. In another embodiment, carotenoid and apocarotenoid The present invention also features compositions and genes may be silenced through the use of a variety of other 10 methods for producing, in a seed-preferred or seed-specific post-transcriptional gene silencing (RNA silencing) tech manner, any selected heterologous gene product in a plant. A niques that are currently available for plant systems. RNA coding sequence of interest is placed under control of a seed silencing involves the processing of double-stranded RNA specific coffee promoter such as a carotenoid or apocaro (dsRNA) into small 21-28 nucleotide fragments by an RNase tenoid protein-encoding gene promoter, and other appropri H-based enzyme (“Dicer or “Dicer-like'). The cleavage 15 ate regulatory sequences, to produce a seed-specific chimeric products, which are siRNA (small interfering RNA) or gene. The chimeric gene is introduced into a plant cell by any miRNA (micro-RNA) are incorporated into protein effector of the transformation methods described herein or known in complexes that regulate gene expression in a sequence-spe the art. These chimeric genes and methods may be used to cific manner (for reviews of RNA silencing in plants, see produce a variety of gene products of interest in the plant, Horiguchi, 2004, Differentiation 72: 65-73; Baulcombe, including but not limited to: (1) detectable gene products Such 2004, Nature 431:356-363; Herr, 2004, Biochem. Soc. Trans. as GFP or GUS, as enumerated above; (2) gene products 32:946-951). conferring an agronomic or horticultural benefit, such as Small interfering RNAs may be chemically synthesized or those whose enzyme activities result in production of micro transcribed and amplified in vitro, and then delivered to the nutrients (e.g., pro-Vitamin A, also known as beta-carotene) cells. Delivery may be through microinjection (Tuschl Tet 25 or antioxidants (e.g., ascorbic acid, omega fatty acids, lyco al., 2002), chemical transfection (Agrawal Net al., 2003), pene, isoprenes, terpenes); or (3) gene products for control electroporation or cationic liposome-mediated transfection ling pathogens or pests, such as described by Mourgues et al., (Brummelkamp T Ret al., 2002: Elbashir S Metal., 2002), or (1998), TibTech 16:203-210 or others known to be protective any other means available in the art, which will be appreciated to plant seeds or detrimental to pathogens. by the skilled artisan. Alternatively, the siRNA may be 30 The following examples are provided to illustrate the expressed intracellularly by inserting DNA templates for invention in greater detail. The examples are intended illus siRNA into the cells of interest, for example, by means of a trate, not to limit, the invention. plasmid, (Tuschl T et al., 2002), and may be specifically targeted to select cells. Small interfering RNAs have been EXAMPLE 1. successfully introduced into plants. (Klahre Uet al., 2002). 35 A preferred method of RNA silencing in the present inven Materials and Methods for Subsequent Examples tion is the use of short hairpin RNAs (shRNA). A vector containing a DNA sequence encoding for a particular desired Plant material. Freshly harvested roots, young leaves, siRNA sequence is delivered into a target cell by any common stems, flowers and fruit at different stages of development means. Once in the cell, the DNA sequence is continuously 40 were harvested from Coffea arabica L. cv. Caturra T-2308 transcribed into RNA molecules that loop back on themselves and Coffea canephora var. BP409 grown under greenhouse and form hairpin structures through intramolecular base pair conditions (25°C., 70 RH) and also from Coffea canephora ing. These hairpin structures, once processed by the cell, are BP-409 grown in the field in East Java, Indonesia. The devel equivalent to siRNA molecules and are used by the cell to opment stages are defined as follows: Small green fruit (SG), mediate RNA silencing of the desired protein. Various con 45 large green fruit (LG), yellow fruit (Y) and red fruit (R). Fresh structs of particular utility for RNA silencing in plants are tissues were frozen immediately in liquid nitrogen, then described by Horiguchi, 2004, supra. Typically, such a con stored at -80° C. until used for RNA extraction. struct comprises a promoter, a sequence of the target gene to Cloning of full-length cDNA Sequences. The 5' upstream be silenced in the “sense' orientation, a spacer, the antisense region of phytoene synthase (PSI) from Coffea Canephora of the target gene sequence, and a terminator. 50 was recovered using the Genewalker kit (3DBiosciences) and Yet another type of synthetic null mutant can also be cre the primers GWPSY1 (5'-ACTTCACCGCAGCGATCAT ated by the technique of “co-suppression” (Vaucheret et al., AAGCTTCAC-3) (SEQID NO.:26) followed by nested PCR 1998, Plant J. 16(6): 651-659). Plant cells are transformed using primer GWPSY2 (5'-TTCACGTCCCAATCTTCTC with a copy of the endogenous gene targeted for repression. In GAGATCTC-3) (SEQID NO.:27). PCR reactions contained many cases, this results in the complete repression of the 55 1x buffer and 5 mM MgCl, 200 uM each of dATP, dCTP, native gene as well as the transgene. In one embodiment, a dGTP and dTTP, and 1 units of LA Taq polymerase (Takara, gene encoding a polypeptide that comprises the carotenoid Combrex Bio, Belgium) and 200 nM primer GWPSY1 and and apocarotenoid biosynthetic pathway from the plant spe 200 nM primer AP1 (5'-GTAATACGACTCACTATAGGGC cies of interest is isolated and used to transform cells of that 3'; Genewalker kit) (SEQ ID NO.:28). The reaction mixture same species. 60 was incubated for 10 min at 94°C., followed by 7 amplifica Mutant or transgenic plants produced by any of the fore tion cycles of 25 sec at 94° C./4 min at 72° C. and then 32 going methods are also featured in accordance with the amplification cycles of 25 sec at 94° C./4 min at 67°C. The present invention. Preferably, the plants are fertile, thereby PCR reaction was diluted 1/200 with Sterile distilled water being useful for breeding purposes. Thus, mutant or plants and the used for a second PCR reaction using 200 nM of that exhibit one or more of the aforementioned desirable 65 nested primer GWPSY2 and 200 nM of nested primer AP2 phenotypes can be used for plant breeding, or directly in (5'-ACTATAGGGCACGCGTGGT-3'; Genewalker kit) agricultural or horticultural applications. They will also be of (SEQ ID NO.:29). Nested PCR was incubated for 10 min at US 8,252,977 B2 29 30 94° C., followed by 5 amplification cycles of 25 sec at 94° into pENTR/D (Invitrogen) to generate pENTR-CcNCED3, C./4 min at 72° C. and then 22 amplification cycles of 25 sec and this clone will then be verified by sequencing. at 94° C./4 min at 67°C. A band of approximately 1.8 kb was The full-length B-carotene hydroxylase cDNA was recov recovered following PCR and cloned into pCR4-TOPO to ered from grain by RT-PCR using primers BCHYFWR (5'- make pCR4-GWPSY1, and the insert of this plasmid was CACCATGGCTGCCGGAATTGCCGTC-3) (SEQID NO.: sequenced resulting in the recovery of the full-length ORF 44) and BCHYREV (5'-CAAGTTGCGTAAGGGTTCA PSY after the in silico assembly of the sequences. TAA-3) (SEQID NO.:45) and cloned into pENTR/D (Invit Using the Genewalker kit, the 5' region of Violaxanthin rogen) to generate pENTR-Cc(BCHY. This clone was verified de-epoxidase was recovered from Coffea canephora using by sequencing. primers GWVDE1 (5'-ACATTTCTTTCGTGAGACTGCA 10 cDNA Isolation Using Degenerate Primers: CACTC-3) (SEQID NO.:30), followed by nested PCR using A search of the EST database for PDS revealed no corre primer GWVDE2 (5'-ATCACCACATTTGATCTGGCAT sponding sequences. However, a partial length cDNA of 897 TCAGTC-3) (SEQIDNO.:31). Aband of approximately 2.3 bp was recovered from yellow robusta grain by RT-PCR using kb was recovered and cloned into pCR4-TOPO to generate degenerate primers DegPDS2 FWR (5'-GGTG pCR4-GWVDE, and the insert of this clone was sequenced 15 GAAAGRTAGCTGCATGGA-3) (SEQ ID NO.:46) and resulting in the generation of the full-length ORF for VDE. DegPDS4 REV (5'-TGTTACRGACATGTCAGCATACAC The ORF contained two introns of 128 bp and 841 bp at 3') (SEQ ID NO.47), which correspond to the conserved positions 282 and 447, respectively, of the ORF. After the peptide sequences GGKVAAW (SEQ ID NO.48) and in-silico assembly of the sequences, the 1248 bp ORF was VYADMSVT (SEQ ID NO.:49) found in LePDS re-cloned by RT-PCR using the primers VDEFWR (5'-CACC (CAA55078), CaPDS (CAA48195) and AtPDS ATGGCTTCTGCTTTGCATTCAGC-3) (SEQ ID NO.:32) (AAA20109) orthologues. To generate the cDNA, PCR was and VDEREV (5'-ACTACCTTAGCTTCCTAATTGG-3') carried out as follows: 3 min 94°C., followed by 10 cycles of (SEQ ID NO.:33), and cloned into pENTR/D (Invitrogen) to 1 min 94° C.; 30 sec 60-50° C. (temperature decreasing by 1 make pENTR-CcVDE. This clone was then verified by C. per cycle); 2 min 72° C., followed by an additional 25 sequencing. 25 cycles of 1 min 94° C.; 30 sec 50° C.; 2 min 72° C. Using the Genewalker kit, the missing 5' region of CCD1 Using the Genewalker kit, the partial coding sequence of was recovered using nested primers GWCCD11 (5'-AA PDS was extended using nested primers GWPDS1 (5'-AT CAATCCGAACAGCCCCTTGAGATCCC-3') (SEQ ID CATTGAATGCTCCTTCCACTGCAAC-3') (SEQ ID NO.: NO.:34) and GWCCD12 (5'-GTTTAAGCCTTGATGTCT 50) and GWPDS2 (5-TCATTAATTCCTAGTTCTCCAAA TCACGTACCG-3) (SEQ ID NO.:35). A fragment 2475 bp 30 CAGG-3") (SEQID NO.:51). This generated of a fragment of was recovered and cloned into pCR4-TOPO to generate 2066 bp. This fragment was cloned into pCR4-TOPO to pCR4-GWCCD1 #1, and the insert of this clone was generate pCR4-GWPDSi5. The insert of this clone was sequenced, resulting in the generation of an extended-length sequenced, resulting in an additional 225 bp of the coding coding sequence for CCD1 containing three introns of 1604 sequence and a partial ORF of 1077 bp containing 3 introns of bp. 267 bp and 605bp at positions 237, 309 and 324, respec 35 616 bp, 373 bp and 609bp at positions 122 bp, 215bp and 272 tively, of the ORF. Because this step did not produce a full bp of the partial ORF, respectively. length coding sequence, a second round of genome walking No corresponding sequences were detected in the EST was carried out using the nested primers GWCCD13 (5'- database for ZDS, however, a partial cDNA clone of 472 bp TGCCAAGTTACTGTTCAATGACTAGGC-3) (SEQ ID was recovered using the non-degenerate primers DegZDS1 NO.:36) and GWCCD14 (5'-AAGCAATTTAATCCCGTC 40 FWR (5'-TTGCAGGCATGTCGACTGCTG-3') (SEQ ID CTTAATCTGG-3") (SEQID NO.:37). This genome walking NO.:52) and DegZDS3 REV (5'-GTGGGATCCTGTTG step resulted in a fragment 1045 bp, which was cloned into CATATGCTCT-3") (SEQID NO.:50), which encode the con pCR4-TOPO to generate pCR4-GWCCD1 #2, and the insert served amino acid sequences LAGMSTAV (SEQID NO.:54) of this clone was sequenced. The full-length coding sequence and MWDPVAYAL (SEQ ID NO.:55) in the Zeta-carotene was generated from the two over-lapping genewalker 45 desaturase orthologues LeZDS (AF195507), CaZDS sequences and the EST sequence (cccwc22w2a6). After the (X89897) and AtzDS (U38550). The coffee cDNA used to in-silico assembly of the sequences, the ORF of 1647 bp was recover this partial cDNA clone was generated from robusta re-cloned by RT-PCR using primers CCD1FWR (5'-CACC grain at the yellow stage. The PCR conditions are the same as ATGGGTAGGCAAGAAGGAGAAG-3') (SEQ ID NO.:38) used for cloning the PDS cDNA (described above). The par and CCD1REV (5'-ACTCTCCAGGACATGGTCCAGC-3) 50 tial clNA fragment obtained was cloned into pCR4-TOPO to (SEQ ID NO.:39), and cloned into pENTR/D (Invitrogen) to generate pCR4-CczDSi 1. generate peNTR-CcCCD1. This clone was then sequenced. Extraction of Total RNA and Generation of cDNA: Using the Genewalker kit, the full coding sequence of Plant tissue samples stored at -80°C. were ground into a NCED3 was obtained using nested primers GWNCED3F powder and total RNA was extracted from this powder using (5'-AAGCAGAAGCAGTCAGGGACTTCTACC-3') (SEQ 55 the method described by Lepelley et al. (this is the HQT/HCT ID NO.:40) and GWNCED3R (5'-TATCCAGTACAC reference). To remove DNA, samples were treated with CGAATCTTGACACC-3') (SEQID NO.:41). This generated DNase using the kit “Oiagen RNase-Free DNase' according a 2.5 kb fragment, which was cloned into pCR4-TOPO to to the manufacturers instructions. All RNA samples were generate pCR4-GWNCED#4. The insert of this clone was analysed by formaldehyde agarose gel electrophoresis. Ribo sequenced, resulting in the full-length open reading frame of 60 somal RNA bands were visualized with ethidium bromide 1908bp and 1104 bp upstream of the ATG. Consistent with staining. other reported NCEDs, CcNCED3 contains no introns. Using oligo (dTo) as a primer, cDNA was prepared from The in-silico assembly of the ORF will be confirmed by approximately 4 Lugtotal RNA according to the protocol in the RT-PCR using primers NCED3FWR (5'-CACCAT Superscript II Reverse Transcriptase kit (Invitrogen, Carls GATGGGCTTGGGTTTGGGTTGC-3) (SEQ ID NO.:42) 65 bad, Calif.). To test for the presence of contaminating and NCED3REV (5-TCACAAGTTTCTTTCaGTTC genomic DNA in the cDNA preparations, a primer pair was CAGGC-3') (SEQID NO.:43). The CeNCED3 will be cloned designed spanning a known intron of a specific ubiquitously US 8,252,977 B2 31 32 expressed coffee cDNA, chalcone isomerase (CHI). RT-PCR Table 1 - continued was carried out using 10-fold dilution of cDNA correspond Primers and probes used in real-time ing to 0.1 g of original RNA. Conventional-PCR reactions quantitative RT-PCR. The right-hand column contained 1x buffer and 5 mM MgCl2, 200 uM each of dATP, indicates size of the amplicon. dCTP, dGTP and dTTP, and 1 unit of polymerase and 800 nM 5 of each gene specific primers—FWD-CCCACCTGGAGC Gene Sequence SEO ID NO. : CTCTATTCTGTT (SEQ ID NO.:56) and REV CCD1 Forward CCTAGGACCAGGAAGGTTTGG 82 CCCCGTCGGCCTCAAGTTTC (SEQ ID NO.:57) for 35 Rewerse CCAGGCTGGCGTGGAA 83 cycles. AcDNA band of 272 bp was observed following PCR. Probe CGGAGGCTATCTTT 84 10 A second band which would correspond to the cDNA+intron NCED3, Forward GGAAATCGGAGCTTAGAATTGTCA 85 at 750 bp was not observed, indicating an absence of genomic Reverse CAGCTGCACTGATGCCTCTAAT 86 DNA in the samples (data not shown). These RNA samples Probe CGCCATGACATTGG 87 were later used to generate full-length ORF sequences FIB1 Forward CTGTCCAGGACACAGCATCCT 88 according to the methods described above. These ORFs were 15 Rewerse TCAGTGGTGGTCGGCTAGAAA 89 subsequently cloned into pENTR/D (Invitrogen). Probe GTCGCAAAGTCC 90 Quantitative TaqMan-PCR was carried out with cDNA RPL39 Forward GAACAGGCCCATCCCTTATTG 91 using the protocol recommended by the manufacturer (Ap Reverse CGGCGCTTGGCAATTGTA 92 plied Biosystems, Perkin-Elmer). All reactions contained 1 x Probe ATGCGCACTGACAACA 93 Taq Man buffer (Perkin-Elmer) and 5 mM MgCl, 200 uM All sequences are given 5' to 3'. each of dATP, dCTP, dGTP and dTTP, and 0.625 units of 'MGB Probes were labelled at the 5' end with fluorescent AmpliTaq Gold polymerase. PCR was carried out using 800 reporter dye 6-carboxyfluorescein (FAM) and at the 3' end with Sincher dye 6-carboxy-tetramethyl-rhodamine (TAMRA). nM of each gene specific primers, forward and reverse, and RPL39 probe were labelled at the 5' end with fluorescent 200 nM for the TaqMan probe and 1000-fold dilution of reporter dye WIC and at the 3' end with gluencher TAMRA. cDNA corresponding to 0.001 ug of original RNA. Primers 25 Functional Evaluation of Selected cDNAs in E. coli: and probes were designed using PRIMER EXPRESS Software (Ap The functionality of some encoded proteins was tested by plied Biosystems: see Table 1). The cross specificity of the co-expression of the corresponding cDNA containing com primers and probes is summarized in Table 4. The reaction plete ORF sequences in carotenoid accumulating strains of E. mixture was incubated for 2 minat 50° C., then 10 min at 95° coli. The full-length Coffea CCD1 was recovered from grain C., followed by 40 amplification cycles of 15 sec at 95°C./1 by RT-PCR using the primers CCD1FWR (5'-CACC min at 60° C. Samples were quantified in the GeneAmp 7500 ATGGGTAGGCAAGAAGGAGAAG-3') (SEQ ID NO.:38) Sequence Detection System (Applied Biosystems). Tran and CCD1REV (5'-ACTCTCCAGGACATGGTCCAGC-3') script levels were normalized to the levels of the control gene (SEQ ID NO.:39), and cloned into the gateway vector rp139. pENTR/D (Invitrogen) to make pENTR-CcCCD1. The 35 sequence of clone pENTR-CcCCD1 was identical to that of Table 1 the previous in-silico sequence. The CCD1 ORF was trans ferred into the gateway bacterial expression vectorp)EST17 Primers and probes used in real-time quantitative RT-PCR. The right-hand column (Invitrogen) by recombination as described by the manufac indicates size of the amplicon. turer (Invitrogen) to make plEST17-CcCCD1. 40 At the same time, the full-length Coffea BCHY was recov Gene Sequence SEO ID NO. : ered from grain by RT-PCR using primers BCHYFWR (5'- CACCATGGCTGCCGGAATTGCCGTC-3) (SEQID NO.: PSY Forward TGATGAGGCAGAGAAAGGAGTGA 58 44) and BCHYREV (5'-CAAGTTGCGTAAGGGTTCA Rever Se GATGCCCATACAGGCCATCT 59 TAA-3) (SEQID NO.:45) and cloned into pENTR/D (Invit Probe CGAGCTCAACTCTG 60 rogen) to make pl’NTR-CcBCHY, and then sequenced. The PDS Forward TGGTAACCCTCCAGAGAGACTTTG 61 45 BCHY ORF was transferred into the gateway bacterial Reverse TCTGCCTCCTCGTGACT CAA 62 expression vector ploEST17 (Invitrogen) by recombination Probe ATGCCGATTGTTGAGCA 63 as described by the manufacturer (Invitrogen) to make ZDS Forward GCTGATAAAAATTTGCTCGTGAAG 64 pDEST17-Cc(CHY. Reverse CACCAATTTCACCCCCTTTG 65 Plasmids pAC-LYC, p.AC-BETA and pAC-ZEAX contain Probe ATCATACT CACACATTTGTT 66 50 ing specific sets of functional carotenoid biosynthetic genes of Erwinia herbicola responsible for the formation of lyco PTOX Forward AAACGGAGAGCCACCTGATG 67 pene, B-carotene and Zeaxanthine, respectively (Cunningham Reverse TGCTCAATCTTTACAACCCATTTC 68 et al., 1994; Cunningham et al., 1996; Sun et al., 1996; Cun Probe TCATCCTCTAGTGGTTTGG 69 ningham and Gantt, 2001) were co-transformed into E. coli LeCY Forward GCCGCAAGAGAGGAAACG 70 55 strain BL21-AI with either the pDEST17-CcCCD1 or Rewerse GCAAAATAAGTGCCAATCCAAAA 71. pDEST17-Cc(BCHY constructs described above. An over Probe CAGAGAGCATTCTTC 72 night 3 ml culture of each strain, which is selected to contain BCHY Forward CGCCGTCCCTGCCATA 73 both sets of plasmids by the addition of chloramphenicol Rewerse AATGAGGCCCTTGTGGAAGA 74 (pAC plasmids) and amplicillin (pDEST17 plasmid), was Probe CCCTCCTTTCTTATGGC 7s 60 used to inoculate 50 ml of LB medium containing the appro priate antibiotics and 0.2% glucose. The cultures were grown ZEP Forward TTGGTTCTGACAAGGGTGCAT 76 at 28°C. until a cell density corresponding to an absorbance Rewerse CGAGAACGGTGGCTGGTT 77 at 600 nm of 0.5 was reached. Expression of the protein was Probe CCGGGTAAAGGTCA 78 induced by addition of 0.2% arabinose and the cultures were WDE Forward CCCCTTGTCGAGAGATTGGA 79 grown at 28°C. overnight. 2 ml of the E. coli culture was Reverse ACCTCCCTTACGATTGTCCTTTC 8O 65 centrifuged, and the cell pellet was photographed (FIGS. 6A Probe AAGACAGTGGAAGAAGG 81 and 7A). The remaining E. coli culture was centrifuged, and the cell pellet was resuspended in an equal volume of form US 8,252,977 B2 33 34 aldehyde. An equal Volume of methanol was then added, 2.5.1.32). A partial-length cDNA clone encoding PSY followed by two volumes of ethyl acetate. The phases were (cccs30w7e6) was identified in the Cornell Coffea canephora separated by the addition of water, and the ethyl acetate phase EST database. Because this clone was missing the 5' end of was retained for HPLCanalysis. The ethyl acetate was evapo the ORF, genome walking was employed to recover the miss rated under vacuum and analysed by HPLC as described ing 258 bp 5" sequence. Using nested primers GWPSY1 (5'- below. Carotenoid Analysis: ACTTCACCGCAGCGATCATAAGCTTCAC-3) (SEQ ID The method used to analyze and quantify carotenoids is NO.:26) and GWPSY2 (5'-TTCACGTCCCAATCTTCTC detailed in Fraser et al. (2000). Typically, grain tissues were GAGATCTC-3) (SEQID NO.:27)a fragment approximately ground into a powder and were freeze-dried and, 10-50 mg 1800 bp in length was recovered, cloned into pCR4-TOPO to aliquots were extracted using methanol: chloroform (1:3 by 10 make pCR4-GWPSY1, and sequenced. In “silico' assembly Vol) and partitioned against 50 mM Tris-HCl pH 7.0 (2 vols) of the sequence revealed the full-length ORF of PSY. As The aqueous phase was re-extracted twice. HPLC separa shown in Table 3, the deduced amino acid sequence of CcPSY tions were performed on a C30 reverse-phase column (250x was determined to have 76% identity to the Capsicum 4.6 mm) purchased from YMC, Wilmington, N.C. The mobile annuum PSY1 (X68017: Romeret al., 1993), 74% identity to phases used were methanol (A), water/methanol (20/80 by 15 the Lycopersion esculentum PSY1 (X60441; Ray et al., 1992) Vol) containing 0.2% ammonium acetate (B) and tert-methyl and 70% identity to Arabidopsis thaliana PSY (NM butyl ether (C). The gradient used was 95% A/5% B isocrati 121729). cally for 12 minutes, a step to 80% A/5% B/ and 15% Cat 12 minutes, followed by a linear gradient to 30% A/5% B/65% C TABLE 3 for 30 min (Fraser et al., 2002). Identity of the Coffea canephora phytoene synthase EXAMPLE 2 amino acid sequence with the most homologous GenBank sequences. NP = Not published. Isolation and Identification of Carotenoid Gene name (accession number) Publication % identity Biosynthetic Pathway Genes from Coffea canephora 25 Coffea canephora NP2 100 ESTs representing the longest available cDNA for genes of Capsicum annuum (X68017) Romer et al., 1993 76 the carotenoid biosynthetic pathway and those involved in the Lycopersicon esculentum (X60441) Ray et al., 1992 74 formation of the carotenoid-derived apocarotenoids were iso Arabidopsis thaliana (NM 121729) NP 70 lated from the appropriate library and sequenced. The fully Identities were individually calculated with clustalW using default parameters with the sequenced cDNAs were then analyzed for homologies with 30 full-length ORF known sequences, and named as follows: phytoene synthase *NP = not published (PSY) (cccs30w7e6), B-carotene hydroxylase (BCH) The condensation of two molecules of geranylgeranyl (cccs46w21b8), lycopene e-cyclase (LeCY) (cccp8f16), diphosphate into phytoene by PSY has been found to be a zeaxanthin epoxidase (ZEP) (cccp129g 15), violaxanthin de rate-limiting reaction in several different plant species and epoxidase (VDE) (cccp13a9), and carotenoid cleavage 35 tissues at different stages of development. The over-expres dioxygenase 1 (CCD1) (cccwc22w2a6). Additional ESTs encoding for the Fibrillin (FIB) structural protein implicated sion of PSY in a grain specific manner is thus expected to be in carotenoid stockage (cccs.16w 15e14), and an ortholog of useful for the overproduction of carotenoids and carotenoid the plastid terminal oxidase, a co-factor for carotenoid derived flavor molecules in the coffee grain. Seed-specific desaturation (PTOX; cccI24o 10) and a putative lycopene 40 over expression of PSY has been carried out in other species. e-cyclase (LeCY) (cccp8f16) were also identified. The num For example, the seed-specific over-expression of PSY in ber and distribution of the associated ESTs are shown in Table Arabidopsis led to increased carotenoids, increased chloro 2. phyll content, a delay in germination and an increase in ABA (Lindgren et al., 2003). Seed specific over-expression of PSY TABLE 2 and a bacterial phytoene desaturase (CRTI, from Erwinia 45 uredovora) in rice endosperm was shown to drive B-carotene Number and distribution of ESTs in the unigene synthesis as well as the formation of further downstream xanthophylls (Beyer et al., 2002: Paine et al., 2005). Overex Number of ESTs pression of PSY in canola seeds led to a 50-fold increase in Seed Seed Seed 50 carotenoid production (Shewmaker et al., 1999). Unigene 18w 3Ow 46w pericarp leaf Total FIG. 4A shows the Coffea canephora (CCPSY) amino acid CcPSY 123321 O 2 O 1 O 3 sequence aligned with the most homologous sequences in the CCPTOX 121182 O O O O 3 3 GenBank non-redundant protein database. CcpCHY 123117 O 1 1 3 3 8 B. Phytoene desaturase, C-carotene desaturase and lyco CcLeCY 131043 O O O 1 O 1 pene cyclases CCZEP 112969 O O O 2 1 3 55 CcVDE 13O4S4 O O O 1 O 1 Phytoene can undergo four consecutive desaturation steps CCNCED3 130641 O O O 1 O 1 catalyzed by the enzymes phytoene desaturase (PDS; EC CCCCD1 121850 O 3 O O O 3 1.3.99) and 4-carotene desaturase (ZDS: EC 1.14.99.30) CCFIB 119688 O O 1 1 O 2 CCPDH 125598 O O O O 1 1 (Bartley et al., 1992, Hugueney et at., 1992; Albrecht et al., 60 1995). These desaturation steps require the presence of a PSY: Phytoene synthase; PTOX: plastid terminal oxidase; BCHY: B-carotene hydroxylase; plastid terminal oxidase (PTOX) as a co-factor. LeCY: lycopene e-cyclase; ZEP: zeaxanthine epoxidase;VDE: violaxanthine de-epoxidase; CED3: 9-cis-epoxycarotenoid dioxygenase; CCD1: carotenoid cleavage dioxygenase 1; i) Phytoene Dehydrogenase FIB: Fibrillin; PDH: phytoene dehydrogenase-like. No corresponding sequences were detected in the EST A. Phytoene Synthase database for PDS or ZDS. However, two partial length clones The first true carotenoid is formed by the condensation of 65 (PDH, ccc.131g22; PDH2, cccs46w 13 n19) encoding phy two molecules of geranylgeranyl diphosphate into phytoene toene dehydrogenase-like (PDH) proteins were detected. The and is catalyzed by the enzyme phytoene synthase (PSY: EC coffee PDH1 was found to have 72% homology to an Arabi US 8,252,977 B2 35 36 dopsis PDH-like and 23% homology to the Phycomyces conserved peptide sequences LAGMSTAV (SEQID NO.:54) blakesleeanus PDH protein. The Phycomyces blakesleeanus and MWDPVAYAL (SEQ ID NO.:55) of LeZDS PDH protein is capable of introducing the four double bonds (AF195507), CaZDS (X89897) and AtZDS (U38550) ortho into phytoene to form lycopene, thereby replacing PDS and logues. ZDS in Phycomyces (Arrach et al., 2001; see FIG. 1). It is The PCR product of 472 bp was cloned in pCR4-TOPO to believed that this protein carries out a similar function in one generate pCR4-ZDSi 1. FIG. 4C shows the Coffea canephora or more coffee tissues. (CcŽDS) partial ZDS amino acid sequence deduced from the ii) Phytoene Desaturase plasmid pCR4-CcŽDSi 1 in an alignment with the most Because clone for PDS was found in the coffee EST librar 10 homologous sequences in the GenBank non-redundant pro ies, experiments were carried out to generate a PDS cDNA tein database. Table 5 shows the '% identity of the sequences clone de-novo. These experiments resulted in the generation in FIG. 4C in the regions of overlap. The high levels of of a partial PDS cDNA of 897 bp using RT-PCR with cDNA homology are consistent with the notion that pCR4-Cc generated from yellow robusta grain. RT-PCR was carried out 15 ZDSi1 encodes a partial cDNA encoding the Coffea cane using the degenerate primers DegPDS2 FWR (5'-GGTG phora ZDS gene CeZDS. The remaining 5' and 3' coding GAAAGRTAGCTGCATGGA-3) (SEQ ID NO.:46) and regions of this gene can be obtained using the well-known DegPDS4 REV (5'-TGTTACRGACATGTCAGCATACAC techniques of 5' and 3' RACE and primer assisted walking. 3') (SEQ ID NO.47), which were designed from the con served peptide sequences GGKVAAW (SEQID NO.:48) and TABLE 5 VYADMSVT (SEQ ID NO.:49) found in LePDS Identity of the Coffea canephora Zeta-caroteine desaturase (CAA55078), CaPDS (CAA48195) and AtPDS amino acid sequence with the most homologous GenBank (AAA20109). The PCR product of 897 bp was cloned in sequences. NP = Not published. pCR4-TOPO to generate pCR4-PDS. Using the Genewalker 25 Gene name (accession number) Publication % identity kit, the partial coding sequence of PDS was extended using Coffea canephora NP2 100 nested primers GWPDS1 (5'-ATCATTGAATGCTCCTTC Capsicum annuum (X89897) Albrecht et al., 1995 92 CACTGCAAC-3) (SEQ ID NO.:50) and GWPDS2 (5'- Lycopersicon escientiin Bartley and Ishida, 92 (AF195507) 1999 TCATTAATTCCTAGTTCTCCAAACAGG-3) (SEQ ID 30 Arabidopsis thaliana Scolnik and Bartley, 85 NO.:51), resulting in the generation of a fragment of 2066 bp. (U38550) 1995 This fragment was cloned into pCR4-TOPO to generate Identities were individually calculated with clustalW using default parameters with partial pCR4-GWPDSi5. The resulting clone was sequenced result ZDS sequence from Coffea canephora and the corresponding regions from orthologues ing in an additional 225 bp of the coding sequence and a *NP = not published partial open-reading frame of 1077 bp containing 3 introns of 35 iv) Plastid Terminal Oxidase 616 bp, 373 bp and 609bp at positions 122bp.215bp and 272 A full-length cDNA clone encoding PTOX (ccc 124o 10), a bp of the partial ORF, respectively. FIG. 4B shows the Coffea co-factor for phytoene desaturation (Carol et al., 1999; Josse canephora (CCPDS) partial PDS amino acid sequence et al., 2000; for review see Kuntz, 2004), was detected in the deduced from plasmids pCR4-CcPDS and pCR4-GW C. canephora EST database. Alignment of PTOX with the PDSi5. The partial ORF was aligned with the most homolo 40 most homologous GenBank sequences revealed 60% homol gous sequences in the GenBank non-redundant protein data ogy to the tomato (AF177980), and 61% homology to the base pepper (AF177981), and 46% homology to the Arabidopsis (AJO04881) PTOX proteins (see Table 6). The C-terminal TABLE 4 45 amino acid sequence was aligned with the three closest Identity of the Coffea canephora phytoene desaturase amino sequences in the NCBI database. This alignment is set forth in acid Sequence with the most homologous GenBank Sequences FIG 4D.

Gene name (accession number) Publication % identity TABLE 6 Coffea canephora (*****) NP2 100 50 Capsicum annuum (X68058) Hugueney et al., 1992 89 Identity of the Coffea canephora plastid terminal oxidase amino Lycopersicon esculentum (X59948) Pecker et al., 1992 89 acid sequence with the most honologous GenBank sequences. Arabidopsis thaliana (TC261857) TIGR 85 Gene name (accession number) Publication % identity 'Identities were individually calculated with clustalW using default parameters with partial PDS sequence from Coffea canephora and the corresponding regions from orthologues 55 Coffea canephora NP2 100 *NP = not published Capsicum annuum (AF177981) Josee et al., 2000 61 Lycopersicon esculentum (AF177980) Josse et al., 2000 60 iii) Zeta-Carotene Desaturase Arabidopsis thaliana (AJOO4881) Carol et al., 1999 46 Because no clone for ZDS was found in the coffee EST 'Identities were individually calculated with clustalW using default parameters with the libraries, experiments were carried out to generate a ZDS full-length ORF cDNA clone de-novo. These experiments resulted in the gen 60 *NP = not published eration of a partial ZDS cDNA of 472 bp using RT-PCR with Lycopene itself is the precursor of pseudoionone (FIG.2V) cDNA generated from yellow robusta grain. The RT-PCR was via a 9' 10 cleavage dioxygenase (CCD1; Simkin et al., carried out using the non-degenerate primers DegZDS1 FWR 2004b) and 6-methyl-5-hepten-2-one (MHO) via a 5'6 cleav (5'-TTGCAGGCATGTCGACTGCTG-3') (SEQID NO.:52) 65 age dioxygenase (CCD4; Bouvier et al., 2004a). Both MHO and DegZDS3 REV (5'-GTGGGATCCTGTTGCATAT and pseudoionone are potent contributors to tomato fruit fla GCTCT-3') (SEQID NO.:53), which were designed from the vor (Buttery et al., 1990; Baldwin et al., 2000). Without US 8,252,977 B2 37 38 intending to be bound to any particular theory, it is believed TABLE 8 that although neither has been detected in coffee grain to date, Identity of the Coffea canephora Lycopene e-cyclase these carotenoid-derived products may go undetected C-terminal amino acid sequence with the most homologous because they co-elute with other more abundant volatiles in GenBank sequences. NP = Not published. coffee. 5 B-carotene is formed by the enzyme lycopene B-cyclase Gene Gene name (accession number) Publication % identity (LBCY: Cunningham et al., 1996), which introduces two Coffea canephora NP2 100 Tagetes erecta (AF251016) Moehs et al. 2001 86 B-ring structures at the ends of the carbon chain and C-caro Lactuca sativa (AF321538) Cunningham et al., 77 tene is formed by the enzymes lycopene e-cyclase (LeCY: 10 2001 Ronenet al., 1999) and LRCY, which introduce one e-ring and Identities were individually calculated with clustalW using default parameters with with partial LeCY sequence from Coffea canephora and the corresponding regions from ortho one B-ring respectively. A corresponding cDNA for a putative logues coffee LeCY (cccp8f16) has been identified in the Nestlé *NP = not published Cornell C. canephora EST database. 15 FIG. 4F shows the C-terminal partial CeLeCYamino acid C. B-Carotene Hydroxylase sequence aligned with the most homologous sequences in the Oxygenated carotenoids are formed by two Successive GenBank non-redundant protein database. hydroxylation steps. B-carotene is converted Zeaxanthin by E. Zeaxanthin Epoxidase and Violaxanthin De-Epoxidase the action of the enzyme B-carotene hydroxylase (BCHY: EC The hydroxylated B-rings of zeaxanthine are epoxylated by 1.14.13-; Sandmann, 1994) and C-carotene is converted to the enzyme zeaxanthine epoxidase (ZEP; Marinet al., 1996; lutein by the actions of BCHY and e-carotene hydroxylase Bouvier et al., 1996) and de-epoxylated by the activity of (eCHY) together. eCHY has only recently been cloned (Tian violaxanthine de-epoxidase (VDE) in a reversible cycle at al., 2004; Tian and DellaPenna, 2004; for review see Inoue, implicated in the adaptation of plastids to changing environ 2004), and a lutein deficient mutant (lut1) has been charac mental light conditions. Partial clNAs clones for both VDE terised (Pogson et al., 1996; Tian and DellaPenna, 2001). 25 (cccp13a9) and ZEP (ccc.129g 15) were identified in the C. A full-length clone (cccs46w21b8)+intron encoding a pro canephora EST database. The deduced C-terminal amino acid sequence of Coffea tein with 73% identity to L. esculentum (Y14809; Hirshberg canephora ZEP (CCZEP) was aligned with the most homolo et al., 1998) ACHY, and 67% identity to the A. thaliana gous GenBank sequences, and found to encode a protein with (NM 124636) BCHY has been identified in the C. cane 30 72% homology to the C-terminal amino acid sequence of L. phora EST database (see Table 7). esculentum (Z83835; Burbridge et al., 1997) ZEP, 71% homology to the C-terminal amino acid sequence of Prunus TABLE 7 armeniaca (AF159948; Mbeguie-A-Mbeguie and Fils-Ly Identity of the Coffea canephora B-caroteine hydroxylase caon, 2000) ZEP, 60% homology to the C-terminal amino complete amino acid sequence with the most homologous 35 acid sequence of Oryza sativa (AB050884; Agrawal et al., GenBank sequences. NP = Not published. 2001) ZEP, and 52% homology to the C-terminal amino acid sequence of Arabidopsis thaliana (NM 126103) ZEP (Table Gene name (accession number) Publication % identity 9). Coffea canephora NP2 100 Lycopersicon esculentum (Y14809) Hirschberg et al., 73 40 TABLE 9 1998 Arabidopsis thaliana (NM 124636) NP2 67 Identity of the Coffea Canephora zeaxanthin epoxidase C-terminal amino acid sequence with the most homologous Identities were individually calculated with clustalW using default parameters with the GenBank sequences. NP = Not published. full-length ORF *NP = not published 45 Gene name (accession number) Publication % identity FIG. 4E shows the CCBCHYamino acid sequence aligned Coffea canephora NP2 100 with the most homologous sequences in the GenBank non Lycopersicon escientiin Burbidge, 1997 72 redundant protein database shown in Table 7. (Z83835) D. Lycopene e-Cyclase Prunus armeniaca (AF159948) Mbeguie-A-Mbeguie, 71 Lycopene is converted to C-carotene (B.e-carotene, FIG. 50 Oryza sativa (AB050884) Agrawal et al., 2001 60 1E) and B-carotene by the activity of two enzymes, lycopene Arabidopsis thaliana NP 52 8-cyclase (LeCY; Ronen et al., 1999) and lycopene B-cyclase (NM 851285) Identities were individually calculated with clustalW using default parameters with with (LBCY). LeCY introduces one e-ring and LRCY introduces partial ZEP sequence from Coffea canephora and the corresponding regions from ortho logues. one f-ring to form C-carotene. The activity of LeCY also 55 *NP = not published results in the formation of the intermediate 8-carotene (e.up carotene) having one e-ring and one uncyclized psi end. In FIG. 4G shows the N-terminal partial CeZEP amino acid plants such as Lactuca sativa (lettuce), LeCY introduces two sequence aligned with the most homologous sequences in the e-ring structures at the ends of the carbon chain, resulting in GenBank non-redundant protein database. 60 A partial length cDNAs clone of VDE (ccc129g 15, insert the formation of e-carotene (e.e-carotene; Cunningham and 1132 bp) was identified in the C. canephora Nestlé-Cornell Gantt 2001) (FIG.1F). EST database. The coding sequence of this gene was A partial cDNA (pcccp8f16) representing the C-terminal extended using the GenomeWalker kit. The remaining part of domain of a protein with 86% identity to T. erecta the ORF of CcVDE was obtained using GenomeWalker kit (AF251016: Moehs et al., 2001) BLeCY and 77% identity to 65 and nested primers GWVDE1 (5'-ACATTTCTTTCGT the L. sativa (AF321538; Cunningham et al., 2001) LeCY has GAGACTGCACACTC-3) (SEQID NO.:30) and GWVDE2 been identified in the C. canephora EST database (Table 8). (5'-ATCACCACATTTGATCTGGCATTCAGTC-3) (SEQ US 8,252,977 B2 39 40 ID NO.:31). This genome walk resulted in the generation of a TABLE 11 2.3 kb fragment. This fragment was cloned into pCR4-TOPO Identity of the Coffea canephora 9-cis-epoxycarotenoid to generate pCR4-GWVDE. The insert of this clone was dioxygenase 1 amino acid sequence with the most homologous sequenced resulting in the full length ORF of 1248 bp after GenBank sequences. NP = Not published. in-silico reconstruction. 5 The deduced full-length amino acid sequence of Coffea Gene name (accession number) Publication % identity* canephora VDE (CCVDE) was aligned with the most Coffea canephora NP2 100 homologous GenBank sequences, and found to encode a Solanum tuberosum (AAT75151) NP? 75 protein with 74% homology to the amino acid sequence of Lycopersicon escientiin Thompson et al 2004 75 10 (CAD30202) Nicotiana tabacum (U34817: Bugos et al., 1998) VDE, 67% Vitis vinifera (AAR11193) Soar et al., 2004 70 homology to the amino acid sequence of Oryza sativa Arabidopsis thaliana (NM112304) Sato et al., 2000 66 (AF411133) VDE, and 67% homology to the Arabidopsis Arabidopsis thaliana (NM102749) Sato et al., 2000 63 thaliana (AY063067) VDE (Table 10). 1. Identities were individually calculated with clustalW using default parameters with the full-length ORF 15 *NP = not published TABLE 10 Identity of the Coffea canephora violaxanthin de-epoxidase FIG. 4I shows the CCNCED3 full amino acid sequence amino acid sequence with the most homologous GenBank aligned with the four most homologous sequences in the sequences. NP = Not published. GenBank non-redundant protein database. G. Carotenoid Cleavage Dioxygenase 1 Gene name (accession number) Publication % identity Carotenoid Cleavage Dioxygenase 1 (CCD1) has been Coffea canephora NP2 1OO shown to be involved in the formation of B-ionone, C.-ionon Nicotiana tabacum (U34817) Bugos et al., 1998 74 egeranylacetone, and pseudoionone in Vivo (Simkin et al., Oryza sativa (AF411133) NP 67 2004b; see FIG.2). This gene is of particular interest due to its Arabidopsis thaliana (AYO63067) NP 67 25 role in the formation of the C grasshopper ketone from Identities were individually calculated with clustalW using default parameters with the Neoxanthin (FIG. 3). Without being bound to any particular full-length ORF theory or mechanism of action, the grasshopper ketone is *NP = not published postulated to be the precursor for the formation of B-dama FIG. 4H shows the full CoVDE amino acid sequence scenone and 3-hydroxy-3-damascenone (Suzuki et al., 2002) aligned with the three most homologous sequences in the 30 (FIG. 2), which are important flavor volatiles of green and roasted coffee. GenBank non-redundant protein database. A partial cDNA sequence (pcccwc22w2a6) for the Coffea F. 9-Cis-Epoxycarotenoid Dioxygenase 1 canephora CCD1 (CCCCD1) was identified in the C. cane A partial clone of a 9-cis-epoxycarotenoid dioxygenase phora EST database. The missing 5' sequence was recovered (NCED3; cccwc22w23o20), involved in synthesis of the phy using the GenomeWalker kit and the primers GWCCD11 tohormone abscisic acid from neoxanthine (Tan et al. 1997) 35 (5'-AACAATCCGAACAGCCCCTTGAGATCCC-3) (SEQ was detected in the C. canephora EST database. In Arabidop ID NO.:34) and GWCCD12 (5'-GTTTAAGCCTTGAT sis thaliana, 5 cDNAs are responsible for this 9-cis cleavage GTCTTCACGTACCG-3") (SEQID NO.:35). A fragment of reaction, NCED2 (NM117945), NCED3 (NM112304), 2475bp was recovered and cloned into pCR4-TOPO to gen NCED5 (NM102749), NCED6 (NM113327), and NCED9 erate pCR4-GWCCD1 #1, and the insert of this clone was (NM106486). The C. canephora cDNA identified here 40 sequenced. Because this step did not produce a full length showed highest orthology to the AtNCED3 (NM112304) and coding sequence, a second round of genome walking was was thus named CcNCED3. ABA is a carotenoid-derived carried out, using the nested primers GWCCD13 (5'-TGC apocarotenoid formed by the action of NCED3 (Tan et al., CAAGTTACTGTTCAATGACTAGGC-3) (SEQ ID 1997). NCEDs are 11,12-carotenoid cleavage dioxygenases, NO.:36) and GWCCD14 (5'-AAGCAATTTAATCCCGTC which cleave neoxanthin, in a similar reaction to the forma 45 CTTAATCTGG-3") (SEQ ID NO.:37), and a fragment of tion of a variety of carotenoid derived apocarotenoids impli 1045bp was recovered. This fragment was cloned into pCR4 TOPO to generate pCR4-GWCCD1 #2. The insert was cated in flavor and aroma of various plant foods, to form sequenced resulting in the full-length CCD1 ORF after in xanthoxin, the precursor of ABA. silico assembly of the appropriate sequences. CeCCD1 was CcNCED3 full coding sequence was obtained using the then aligned with the most homologous GenBank sequences, GenomeWalker kit and nested primers GWNCED3F (5'- 50 and found to encode a protein with 83% homology to L. AAGCAGAAGCAGTCAGGGACTTCTACC-3') (SEQ ID esculentum (AY576001) and 78% homology to L. esculentum NO.:40) and GWNCED3R (5'-TATCCAGTACAC (AY576002; Simkinet al., 2004b) CCD1s, 82% homology to CGAATCTTGACACC-3') (SEQ ID NO.41), generating a the Petuniaxhybrida (AY576003; Simkin et al., 2004a) fragment of 2.5 kb. This fragment was cloned into pCR4 CCD1, and 79% homology to Arabidopsis thaliania TOPO to generate pCR4-GWNCEDH4. The insert of this 55 (AJO05813: Neill et al., 1998) CCD1 (Table 12). clone was sequenced resulting in the full-length ORF of 1908 bp and 1104 bp, upstream of the ATG. Like all reported TABLE 12 NCEDs, CcNCED3 contains no introns. The deduced N-ter minal amino acid sequence of Coffea canephora NCED3 Identity of the Coffea camphora carotenoid cleavage (CcNCED3) was aligned with the most homologous Gen 60 dioxygenase 1 full amino acid sequence with the Bank sequences, and found to encode a protein with 75% most homologous GenBank sequences. homology to the amino acid sequence of L. esculentum Gene name (accession number) Publication % identity (CAD30202; Thompson et al., 2004) NCED1, 75% homol Coffea canephora NP2 100 ogy to the Solanum tuberosum (AAT75151) NCED1, 70% Lycopersicon escientiin Simkin et al., 2004b 83 homology to the Vitis vinifera (AF159948; Sato et al., 2004) 65 (AY576001) NCED1, and 66% homology to the Arabidopsis thaliana Petuniax hybrida (AY576.003) Simkin et al., 2004a 82 (BAB01336) NCED3 (Table 11). US 8,252,977 B2 41 42 TABLE 12-continued EXAMPLE 3 Identity of the Coffea Camphora carotenoid cleavage Transcript Expression in Coffee Grain dioxygenase 1 full amino acid sequence with the most homologous GenBank sequences. Using the TaqMan assays, relative amounts of transcripts for PSY, PDS, ZDS, PTOX, LeCY, BCHY,ZEP. VDE, CCD1, Gene name (accession number) Publication % identity NCED3 and FIB1 were quantified in coffee grain from Coffea Arabidopsis thaliana (AJO05813) Neill et al., 1998 79 canephora and C. arabica. Lycopersicon escientiin Simkin et al., 2004b 78 The results shown in FIG. 5 provide a comparison of the (AY576002) 10 QRT-PCR expression data obtained for C. Canephora (FRT05) with those from C. arabica (T2308). Apart from 'Identities were individually calculated with clustalW using default parameters with the full-length ORF Some clear expression differences for the Small green stage, *NP = not published the two robusta samples showed relatively similar expression patterns but showed significant expression pattern differences FIG. 4J shows the CCCCD1 full amino acid sequence 15 with the single arabica sample analysed. These data Suggest aligned with the four most homologous sequences in the that there are potentially significant differences in the expres GenBank non-redundant protein database. sion patterns of the carotenoid pathway genes in the coffee II. Fibrillin grain of arabica and robusta varieties. Moreover, as can be seen in FIG. 5 confirm that each Proteins referred to as plant fibrillins or plastid lipid asso transcript exhibits expression in grain during development. ciated proteins are widespread from cyanobacteria to higher Low expression of PSY, the first biosynthetic enzyme, in plants (Laizet et al., 2004), and in the latter case in diverse coffee grain may be important for carotenoid accumulation in tissues, in association with a variety of different lipidic struc coffee grain (FIG.5A). The condensation of two molecules of tures. Without being bound to any particular theory or mecha geranylgeranyl diphosphate into phytoene by PSY has been nism of action, fibrillin proteins are believed to be involved in 25 found to be a rate limiting reaction in several different plant the stabilization of lipid structures in an aqueous environment species and tissues at different stages of development. A (Vishnevetsky et al., 1999). In addition, lipoproteinstructures significant difference between arabica and robusta grain was in which fibrillins have been found may contain carotenoids observed at the level of PTOX transcript accumulation (FIG. (in greater or lesser quantities). The overexpression of fibril 5D). PTOX transcripts have been shown to be significantly lin in tobacco chloroplasts was reported to lead to an increase 30 higher in all grain stages in arabica when compared to robusta in plastoglobule number, lipoprotein structures implicated in grain. Given the importance of PTOX in phytoene desatura carotenoid sequestration (Rey et al., 2000). It may be, there tion, determined in the arbidopsis mutant immutans (Carolet al., 1999) and tomato ghost mutant (Josse et al., 2000), it is fore, that fibrillin can lead to modifications in the carotenoid believed that PTOX may play an important role in carotenoid “sink”. This is supported by the results of Lietal. (2001) who 35 biosynthesis during grain development. Another important, reported that carotenoid over-accumulation might be associ difference between arabica and robusta grain is observed with ated with the proliferation of deposition structures rather than CCD1 transcript levels, which appear to increase in arabica changes in the expression of the carotenogenic genes or the grain during development in contrast to a decrease observed abundance of the enzymes in the Brassica oleracea mutant in robusta (FIG.5J). Given the importance of CCD1 in flavor Or. Likewise, overexpression of the fibrillin protein in coffee 40 volatile formation, a more detailed analysis of CCD1 tran grain may lead to carotenoid stockage during grain develop Script levels in the grain of three C. canephora genotypes ment. (BP409, FRT05, FRT64) and one C. arabica (T2308) geno A full-length cDNA clone (cccs.16w 15e 14) showing 70%, type was carried out. In all three C. Canephora genotypes, 61% and 60% homology with the fibrillin proteins of Capsi CCD1 transcript either decreased or remained low during cuin annuum (S56633: Deruere et al., 1994a) and Arabidop 45 development (FIG. 6A). In contrast, CCD1 transcript in C. sis thaliana (NM-118350 and NM-116640; Laizet et al., arabica (T2308) increased 4-fold early on in development 2004) respectively has been identified in the C. Canephora and remained high throughout development (FIG. 6A). At the end of the maturation period, CCD1 transcript levels were EST database (Table 13). approximately 4-fold (BP409) to 20-fold (FRT05) higher in 50 TABLE 13 C. arabica when compared to C. Canephora. Identity of the Coffea canephora fibrillin amino acid EXAMPLE 4 sequence with the most homologous GenBank sequences. Activity of CCD1 and BCHY in E. coli Gene name (accession number) Publication % identity 55 Coffea canephora NP2 100 Plasmids expressing recombinant CoCCD1 protein (pD Lycopersicon escientiin TIGR 72 EST17-CcCCD1) were introduced into strains of E. coli pre (SGN-U213598) Capsicum annuum (S.56.633) Deruere et al., 1994a 70 viously engineered to accumulate different carotenoid com Arabidopsis thaliana (NM-118350) Laizet et al., 2004 61 pounds (Cunningham et al. 1994; Cunningham et al. 1996; Arabidopsis thaliana (NM-116640) Laizet et al., 2004 60 60 Sun et al. 1996). The carotenoids that accumulate in these strains impart color upon the cells and a change or loss of Identities were individually calculated with clustalW using default parameters with the full-length ORF color indicates that the carotenoids have been modified by the *NP = not published new gene product introduced. When each of the two recom binant proteins was expressed in cells producing lycopene, FIG. 4K shows the Coffea canephora fibrillin amino acid 65 B-caroteine or zeaxanthin, loss of color was observed (FIG. sequence aligned with the three most homologous sequences 6B). These observations are consistent with the results previ in the GenBank non-redundant protein database. ously reported by Simkin et al. (2004b). These results confirm US 8,252,977 B2 43 44 that the coffee CCD1 enzyme can catabolize a range of linear degradation products in the grain, molecules that are both and cyclic carotenoid substrates resulting in the formation of normal cell metobolites and, in some cases, potential coffee a range of apocarotenoids depending on the carotenoid Sub aroma/aroma precursors. strate provided. Plasmids expressing recombinant CeBCHY protein (pD EXAMPLE 5 EST17-Cc?BCHY) were introduced into strains of E. coli pre Analysis of the Carotenoids in Immature and Mature viously engineered to accumulate B-carotene or zeaxanthin. Green Grain of Coffee The loss of colour observed in FIG. 6B was confirmed by HPLC-PDA analysis (FIG. 6C). To assess the loss of colour It has recently been shown that mature arabica and robusta observed, carotenoids were analysed and quantified as fol 10 coffee grain contain low levels of the carotenoids lutein and lows: zeaxanthin (Degenhardt et al., 2004). As it is known that Analysis and detection of carotenoids from E. coli by carotenoid levels in some seeds diminish as maturation HPLC-PDA. The method of Fraser et al. (2002) was used to progresses (Bonham-Smith et al., 2006), we examined the perform HPLC-PDA analyses. The separations were per carotenoid levels found in immature arabica and robustagrain formed on a C30 reverse-phase column (250x4.6 mm) manu 15 and compared these to the levels found in mature grain from factured by YMC and purchased from Interchim (France). the same varieties. The HPLC profiles obtained are presented The mobile phases used were methanol (A), water/methanol in FIG. 8 and the quantified levels are given in Table 14. (20/80 by Vol) containing 0.2% ammonium acetate (B) and The presence of diverse carotenoids in the coffee grain was tert-methylbutyl ether (C). The gradient used was 95% A/5% shown by the HPLC analysis. As shown in FIGS. 8A and B, B isocratically for 12 minutes, a step to 80% A/5% B/15% C a chromatogram taken at 450 nm shows the presence of neoxanthin (1), violaxanthin (2), lutein (3), C-carotene (4), at 12 minutes, followed by a linear gradient to 30% A/5% and B-carotene (5) in the green coffee grain. Significant level B/65% C for 30 min. Fraser et al. (2000) of chlorophylls A and B (peaks a and b) were also detected, Analysis and detection of carotenoids from Coffea by which is likely responsible for the green color of non-roasted HPLC. The method used to analyse and quantify carotenoids coffee beans. Lycopene was not detected in coffee grain, from C. arabica (T2308) and C. canephoia (FRT05) grain is 25 possibly due to the rapid turnover of this intermediate in the detailed in Senger et al. (1993). Briefly, 8 to 9 grains were pathway. A chromatogram taken at 280 nm shows a peak that ground in liquid nitrogen and freeze dried. A known quantity may represent phytoene, the first true carotenoid and a poten of astaxanthin dissolved in methanol was added to an empty tial precursor for the formation of geranylacetone (VI) (data tube and lyophilised. Sixty milligrams (60 mg) of freeze dried not shown). material was added to the tube and was extracted using 30 As anticipated, significantly higher levels of lutein was methanol: chloroform (1:3 by Vol) and partitioned against 50 found in the immature grain versus the mature grain. Inter mM Tris-HCl pH 7.0 (2 vols). HPLC separations were per estingly, the experiments presented here also detected several formed on a C18 reverse-phase column (Macherey-Nagel). other carotenoids in the coffee grain. In the immature grain, The solvent consisted initially of 85% acetonitrilelmethanol low levels of four other carotenoids can be clearly seen, (75:25) and 15% water, followed by a gradient decreasing 35 neoxanthin, violaxanthin, C-carotene and B-carotene. As water content to 8% in 12 min, to 5% over the next 10 minand noted previously for coffee leaves (below), the presence of then to 0% over the next 3 min. 100% acetonitrile/methanol C-carotene was unexpected, but the identity of this peak was was then kept until the end of the run. confirmed when its spectrum and retention time was found to Quantification of carotinoids was achieved from dose-re be identical to an O-carotene standard extracted from carrot sponse curves and identification of carotenoids was achieved 40 root (data not shown). by co-chromatography and comparative spectral properties We observed no significant quantities of Zeaxanthin in either mature arabica or robusta grain samples used in con acquired on-line. A color change from a deep-orangeyyellow trast to the previous report (Degenhardt et al., 2004). To to a bright yellow was observed in the presence of C-carotene confirm the observation that Zeaxanthin is not present, due to the activity of BCHY, which converts -carotene to samples were reanalyzed on a C30 column as described by zeaxanthin via the intermediate B-cryptoxanthin (FIG. 1D to 45 Fraser et al. (2000), providing a more complete separation of 1G). These observations are consistent with the results pre lutein and Zeaxanthin. Even using this method, no zeaxanthin viously reported by Sun et al. (1996). No such color change was detected in these samples (data not shown). was observed in Zeaxanthin accumulating strains, which were ESTs for PSY and BCH were detected in the grain at 30 bright yellow before and after induction (FIG. 7A). To con weeks of development. Given the presence of the terminal firm the color change, carotenoids were extracted from the 50 cells and analyzed by HPLC. FIG. 7B confirms the presence carotenoids lutein and neoxanthin in coffee grain (see FIG. 1 off-carotene as the principal carotenoid prior to induction of and FIG. 8), it is believed that all the enzymes involved in pDEST17-CcBCHY. Following the induction, the peak rep carotenoid biosynthesis are expressed at sufficient levels to resenting B-caroteine has been reduced and two new peaks maintain flux through the pathway. The CCD1 transcript was representing zeaxanthin and the intermediate B-cryptoxan also detected in grain at 30 weeks development and is likely thin are observed. These data show that CofCHY has a 55 responsible for the formation of C-ionone (VI) and B-dama B-carotene hydroxylase activity resulting in the formation of scenone (IX) in vivo. B-cryptoxanthin and Zeaxanthin from B-carotene. Also, as can be seen in FIG. 6C, in the non-induced cultures TABLE 1.4 (NI), peaks representinglycopene, B-carotene and Zeaxanthin 60 Carotenoids content (ugg dw) in Coffea arabica (T2308) are observed. Following induction of CCD1, the accumula and C. Canephora (FRTOS) grain. tion of each of these carotenoids was significantly reduced (I), whilst the peak of astaxanthin added to the extract as a loading peak carotenoid T2308 FRTO5 control remains the same size. These data indicate that the coffee CCD1 protein can catabolize both linear and cyclic LARGE GREEN GRAIN carotenoid substrates. Significant expression of CCD1 in cof 65 4 C-Caroteine 7.7 (+1.7) 2.5 (+0.3) fee grain (particularly arabica grain) has been detected indi 5 B-Carotene 2.6 (+0.6) 4.1 (+0.7) cating that this enzyme is probably generating carotenoid US 8,252,977 B2 45 46 TABLE 14-continued EXAMPLE 6 Carotenoids content (Igg dw) in Coffea arabica (T2308) Promoter Isolation and Vector Construction and C. Canephora (FRTOS) grain. peak carotenoid T2308 FRTO5 5 The 5' upstream region of NCED3 from Coffea canephora 3 Lutein 24.2 (+4.8) 29.4 (+1.8) was recovered using the Genomewalker kit (BD Bio 2 Violaxanthin 1.9 (+0.3) 2.7 (+0.3) sciences). Briefly, 2.5 ug of Coffea canephora BP409 DNA 1 Neoxanthin 4.6 (+0.9) 5.3 (+0.3) was cut independently with Dra, EcoRV, PVul and Stul to Total Igg dw 41.O 44.0 10 form GenomeWalk banks DL1, DL2, DL3 and DL4, respec 8. Chlorophyll a 180.9 (+17.0) 156.7 (9.3) tively. Following purification, 0.5ug DNA was ligated to the b Chlorophyll b 61.6 (+11.8) 76.3 (+6.9) GenomeWalker adaptators (25 uM) following the manufac RED GRAIN turer's protocol. Subsequent PCR reactions contained 1 x 4 -Caroteine 0.3 (+0.1) 0.1 (+0.0) buffer and 5 mM MgCl, 200 uMeach of dATP, dCTP, dGTP 5 -Caroteine 0.1 (0.0) 0.2 (0.0) 15 and dTTP, and 1 units of LA Taq polymerase (Takara, Com 3 Lutein 3.4 ((0.9) 2.1 (0.3) brex Bio, Belgium), and 200 nM primer GWNCED3F (5'- 2 Violaxanthin 0.2 (0.1) ND 1 Neoxanthin 0.4 (( ND AAGCAGAAGCAGTCAGGGACTTCTACC-3') (SEQ ID NO.:40) and 200 nM of the GenomeWalker adaptator primer Total Igg dw 4.5 2.5 AP1(5'-GTAATACGACTCACTATAGGGC-3'; Genom 8. Chlorophyll a 12.7 (+3.9) 6.7 (+2.4) b Chlorophyll b 8.1 (+2.4) 5.2 (+1.1) 2O ewalker kit) (SEQ ID NO.:28). The reaction mixture was incubated for 10 min at 94° C., followed by 7 amplification Numbers represent the peaks in FIG.8. Values are means of 3 to 4 determinations, Standard deviations are shown in brackets, 60mg samples were extracted as described inmaterials and cycles of 25 sec at 94° C./4 min at 72° C., and then 32 methods. NTD = Not detected amplification cycles of 25 sec at 94° C./4 min at 67°C. The PCR reaction was diluted 1/200 and the used for a second The results presented here show that green coffee seeds PCR reaction using 200 nM of nested primer GWNCED3R contains at least five different carotenoids. The presence of (5'-TATCCAGTACACCGAATCTTGACACC-3') (SEQ ID the terminal carotenoids—lutein and neoxanthin in coffee NO.:41) and 200 nM of nested GenomeWalker adaptator grain (see FIG. 8 and Table 14) indicates that all the enzymes primer AP2 (5'-ACTATAGGGCACGCGTGGT-3'; Genom involved in carotenoid biosynthesis are expressed at sufficient ewalker kit) (SEQIDNO.:29). Nested PCR was incubated for levels to maintain flux through the pathway. Coffee grains 30 10 min at 94°C., followed by 5 amplification cycles of 25 sec develop within small cherries surrounded by a thin pericarp, at 94° C./4 min at 72°C. and then 22 amplification cycles of thus they are shielded from direct sunlight. However, coffee 25 sec at 94° C.f4 min at 67°C. A 1075bp genomic fragment grain were found to contain both chlorophylla and b, which was recovered from DL2 and cloned into the pCR4-TOPO is likely responsible for the green colour of un-roasted coffee vector (Invitrogen) to make pCR4-GWNCED#4. The insert beans. 35 of this clone was sequenced resulting in a 1104 bp sequence We also detected the presence of a quantity of C-carotene at upstream of the ATG start codon of NCED3 (SEQID NO:25). a concentration of 2-18% of total carotenoids depending on the variety. Generally, green tissues such as leaves accumu EXAMPLE 7 late significant quantities off-carotene and lutein (for review see Frank and Cogdell, 1996 and references therein) but not 40 Carotenoid Content in Coffee Leaves C-carotene. However, C-carotene can be found in the leaves of some plants such as carrot (Kock and Goldman, 2005). The Analysis of coffee leaf extracts by HPLC showed that C. presence of C-carotene in mature green coffee grain is con canephora contained a higher content of carotenoids than C. sistent with the previous report of C-carotene in coffee leaves arabica. Quantification of each carotenoid (Table 15) (Simkin et al., Submitted), with the mature green grain of C. 45 revealed that the two species show similar relative distribu arabica containing a higher relative level than C. canephora. tions of the carotenoids neoxanthin, violaxanthin and B-caro These higher C-carotene levels in the mature greengrain of C. tene (approximately. 12-13% B-carotene, 14-16% violaxan arabica may be related to higher LeCY transcript levels, thin and 13-14% neoxanthin). One of the most striking which we found to be significantly higher in C. arabica when observations from Table 15 is the presence of a significant compared to C. canephora (see FIG. 5). LeCY along with 50 amount of C-carotene (peak 4: FIG. 9), an intermediate in LRCY is responsible for the formation of C-carotene from lutein synthesis, which does not normally accumulate in lycopene (Ronen et al., 1999). It should be mentioned that leaves from most species including Arabidopsis (FIG.9C). To lower relative lutein levels in C. arabica versus C. Canephora confirm the identity of the peak as C-carotene, the spectrum accompany a higher relative C-caroteine content of C. arabica and retention time were compared and found to be identical to grain. This result is consistent with that previously observed 55 those of an O-carotene standard extracted from carrot root. A in coffee leaves (below). Thus, though other possibilities comparison of C-carotene levels in C. arabica and C. cane exist, one possibility may be that the accumulation of C-caro phora showed that C. arabica leaves contained a higher rela tene at the expense of lutein is due to lower transcript levels or tive C-carotene levels (12% and 3.5% of total carotenoids enzyme activity of e-carotene hydroxylase, which along with respectively). In contrast, C. Canephora had higher relative BCHY is responsible for the formation of lutein from OL-caro- 60 lutein levels (54%, as compared to 48% in C. arabica). The tene (Tian et al., 2004; Tian and DellaPenna, 2004). higher C-carotene levels may be related to higher LeCY tran It remains possible that the global difference of the caro Script levels, which were shown to be significantly higher in tenoid profile between the C. arabica versus C. Canephora C. arabica than in C. canephora. Additionally, since C. cane presented here could be the result of species-specific or vari phora leaves had higher relative lutein levels than those from ety-specific variations. It is also possible some of the differ- 65 C. arabica, and since C-carotene is the directlutein precursor, ences observed are at least partially the result of the different it may be that the higher lutein content in C. canephora leaves growth conditions under which the trees were grown. could be directly related to the lower levels of C-carotene and, US 8,252,977 B2 47 48 conversely, the lower lutein content in C. arabica leaves is drought. The amount of CCD1 transcript increased under related to the higher C-carotene content. conditions of water deficit and increased slowly throughout the 6 week sampling period. In contrast, transcript for TABLE 1.5 NCED3 increased significantly early on, before decreasing throughout the remaining sampling period. Carotenoids content (mg/g dw) in Coffea arabica (T2308) Small differences in transcript levels were observed in the and C. Canephora (FRTOS) leaves. control samples collected on different days (although peak carotenoid T238 FRTO5 samples were all collected at the same time each day). This observation is probably a manifestation that minor changes in 4 C-Caroteine 232.4 (+24.6) 104.5 (+6.1) 10 5 B-Carotene 238.8 (+26.6) 387.4 (+10.6) the environmental conditions between the two plants can 3 Lutein 987.3 (+135.6) 1629.5 (+48.4) slightly affect the expression of carotenoid genes. Similarly, 2 Violaxanthin 292.4 (+34.1) 473.4 (+5.6) the differences in transcript levels changes produced in each 1 Neoxanthin 287.8 (+39.4) 3749 (+3.51 of the stressed plants were possibly due to sample-specific Total mgg dw 2O38.7 2969.7 differences in the rate of dehydration following the removal 8. Chlorophyll A 9640 (+1028) 11572 (+140) 15 of irrigation. The plant set on the left of the histograms b Chlorophyll B 3.096 (+408) 4524 (+169) showed the most severe physical symptoms of water deficitat the end of the test period (data not shown). Values are means of 3 to 4 determinations, Standard deviations are shown in brackets, 60 mg samples were extracted as described inmaterials and methods. Numbers represent the peaks Water deficit can result in an increased production of reac in FIG, 7. tive oxygen species. Carotenoids, being involved in the pro Although the connection between carotenoid metabolism tection against oxidative stresses, may be Submitted to higher in the leaves and that in the grain is not precisely clear at this turnover rates under drought stress. This suggests that higher time, the data above again Support the idea that there are small synthesis rates may be needed to maintain Suitable pigment but significant differences in the carotenoid profiles of arabica levels in water-deprived plants (see Simkin et al., 2003b). and robusta coffees. It is believed that these profile differ Hence, we examined the expression of some carotenoid bio 25 synthetic genes in coffee leaves under water deficit condi ences contribute to some of the differences in the environ tions. Because the FIB1/CDSP34 gene is known to respond to mental stress tolerance and grain qualities that generally exist stressors, such as water deficit, which affects thylakoid func between these two coffee species. tion (Manach and Kuntz, 1999; Langenkämperet al., 2001), EXAMPLE 8 we examined the FIB1 expression levels in this experiment as 30 a clear control for water stress. As expected, the expression of FIB1 was induced strongly in coffee by drought stress (FIG. Quantitative Expression Analysis of Coffee Genes 10). This result confirmed that the experimental conditions Involved in Carotenoid Biosynthesis and Storage used here led to a strong stress response within the plastid. Under Drought Stress Conditions This stress response is also detected in other parts of the cell 35 because additional experiments using the same samples We evaluated the changes of carotenoid gene expression in clearly demonstrated that the water stress-inducible gene leaves of 3-year old C. arabica (catimor) plants caused by dehydrin CcDH1a (DQ323987) was very strongly induced in prolonged drought stress. For this experiment, leaf samples the water-stressed samples and not in the watered controls (G. were taken at the different times (T=O, 3, 4, 5, and 6 weeks) Pagny and J. McCarthy; unpublished results). from two well-watered control plants and from two parallel 40 We note that lycopene epsilon cyclase transcript level in un-watered plants. RNA was prepared from each of the coffee leaves decreased under conditions of water deficit to samples. For water deficit samples, total RNA from leaf tissue undetectable levels. This apparent down-regulation could re was isolated using the RNeasy Plant Mini Kit (Qiagen, Valen direct the metabolic flux from the lutein branch of the path cia, Calif.). RNA samples were treated with RNase-free way into the Xanthophyll (Zeaxanthin, antheraxanthin, and DNase (Qiagen) and purified using the Qiagen mini-column. 45 violaxanthin) branch of the pathway. The xanthophylls con Concentration and purity of total plant RNA was determined tribute to the dissipation of excess energy from photosynthe by spectrophotometric analysis. The quantification was veri sis through a mechanism known as the Xanthophyll cycle. fied for all RNA samples in each experiment by formaldehyde Two enzymes are involved in this cycle, ZEP and VDE. ZEP agarose gel electrophoresis and visual inspection of rRNA catalyses the conversion of zeaxanthin to antheraxanthin and bands upon ethidium bromide staining. cDNA was prepared 50 then to violaxanthin. VDE catalyses the reverse reaction. from approximately 2 ug total RNA using poly-dT primer Under normal conditions, ZEP activity results in a high vio according to the protocol in the Superscript II Reverse Tran laxanthin and low zeaxanthin content, whilst VDE activity scriptase Kit (Invitrogen, Carlsbad, Calif.). (and Zeaxanthin accumulation) is induced under excess light The RNA was then used for quantitative RT-PCR expres conditions (Ruban et al., 1994: Woitsch and Römer, 2003). sion analysis of the carotenoid and FIB1 genes (FIG. 10). 55 Our results show that, under normal growth conditions, the Expression analysis showed that the level of FIB1, PSY,ZDS, amount of ZEP transcript is approximately 10-fold greater PTOX, BCHY, and VDE transcripts each increased during the than that of VDE transcript in C. arabica. Interestingly, the drought period. In contrast, the amount of ZEP transcript data obtained using the water stressed plants indicate that increased under the initial stress before decreasing, whereas ZEP transcript level first increases during the early part of the transcript for LeCY decreased rapidly to undetectable levels 60 stress response, then decreases as the stress signal(s) become in some samples. No significant increase in the amount of stronger. The decrease in the amount of ZEP transcript is PDS transcript was observed during the first 5 weeks of accompanied by a concomitant steady increase in the amount drought stress, and only one of the two plants showing the of VDE transcript in water-stressed plants. It may be that this most severe stress symptoms showed an increase in PDS after gene expression regulation contributes to the optimal opera 6 weeks post drought. 65 tion of the xanthophyll cycle under water stress. Overall, We also evaluated the changes of carotenoid cleavage these results suggest that one or more of the following strat dioxygenase gene expression in leaves caused by prolonged egies could be used to improve the protection of green plant US 8,252,977 B2 49 50 tissues, including those of coffee, from water stress: 1) Beyer P. Al-Babili S. Ye X, Lucca P. Schaub P. Welsch R, increase synthesis of carotenoids in general, for example by Potrykus I. (2002). Golden Rice: introducing the beta increasing coffee PSY expression earlier in the stress carotene biosynthesis pathway into rice endosperm by response (more responsive to signal), 2) reduce the expres genetic engineering to defeat vitamin A deficiency. J Nutr. sion of LeCY earlier in the stress response to improve caro 5 132(3):506S-510S. tenoid entry into the Xanthophyll synthesis pathway, or 3) Boelsma E, Hendriks H F J. Roza L. (2001) Nutritional skin improve the functional capacity of the xanthophyll cycle (for care: health effects of micronutrients andfatty acids. Am. J. example by increasing VDE expression). These and other Clin. Nut: 73:853-864. potential improvements in the stress response in coffee are Bouvier F, d'Harlingue A, Hugueney P. Marin E. Marion-Poll now made possible by the isolation of the full length cDNA 10 sequences for the coffee carotenoid and apocarotenoid bio A, Camara B. (1996). Xanthophyll biosynthesis. 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M., Ajani U A, Sperduto R D, Hiller R, Blair N, Neill SJ, Burnett E. C. Desikan R, Hancock J. T. (1998) 35 Burton TC, Farber MD, Garoudos E. S. Haller J, Miller D Cloning of a wilt-responsive cDNA from Arabidopsis T, Yannuzzi L. A. Willet W. (1994). Dietary carotenoids, thaliana suspension culture cDNA library that encodes a Vitamins A, C, and E, and advanced age-related macular putative 9-cic-epoxy-carotenoid dioxygenase. J. Exp. Bot. degeneration. J. Am. Med. Assoc. 272: 1413-1420. 49: 1893-1894. Shewmaker C. K. Sheehy J. A. Daley M, Colburn S. Ke DY. Ortiz A. Ortiz A. Vega F E. Posada F. (2004). Volatile com 40 (1999). Seed-specific overexpression of phytoene syn position of coffee berries at different stages of ripeness and thase: increase in carotenoids and other metabolic effects. their possible attraction to the coffee berry borer Hypoth Plant J. 20(4): 401-412. enemus hanpei (Coleoptera: Curculionidae). JAgric Food Sies H. Stahl W. (2004). Carotenoids and UV protection. 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(1995) The generation of noriso Oxford University Press. prenoid volatiles in starfruit (Averrhoa carambola L.): A Tandon JS, Katti SB, Riedi P. Eugster C H. (1979) Crocetin review. Food Rev. International 11: pp. 237-254. dialdehyde from Coleus forskoli Briq., Labiatae. Helv. Winterhalter P. Rouseff R. (2002). Carotenoid-derived aroma Chin. Acta 274: 27062707. compounds. (Washington DC: American Chemical Soci Taylor A (1993). Cataract: relationship between nutrition and 30 ety). oxidation. J. Am. Coll. Nutr. 12: 138-146. Vishnevetsky M, Ovadis M. Vainstein A. (1999). Carotenoid Thompson, A.J., Thorne, E.T., Burbridge, A., Jackson, A.C., sequestration in plants: the role of carotenoid-associated Sharp, R. E. and Taylor, I. B. (2004). complementation of proteins. Trends Plant Sci. 4(6): 232-235. notabilis, an abscisic acid-deficient mutant of tomato: The present invention is not limited to the embodiments importance of sequence context and utility of partial described and exemplified above, but is capable of variation complementation. Plant Cell Environ. 27 (4): 459-471. and modification within the scope of the appended claims.

SEQUENCE LISTING

<16 Os NUMBER OF SEO ID NOS: 93

<21 Os SEQ ID NO 1 &211s LENGTH: 132O &212s. TYPE: DNA <213> ORGANISM: Coffea canephora

<4 OOs SEQUENCE: 1 atgtctgttg ctittgctatg ggttgttitta cctatot cag aggt cacgaa cago attgca 60 titcCtggaac cqgtacggga aggaa.gc.cgg Cttcttgatt cqtcCaggitt cqtggg.tc.gg 12O ggtaaaaact gcttgttgcaa tigcagacitt gagaaaggca agcaacaaag gtggaattict 18O

ggittatctta atggagattic gagaaactgt tottgggag gctic taggitt galagalaccga 24 O

ggcaaattitt Ctgttgatt.cc caatgtagtg gttagcc cag Ctggagaaat to catgtct 3 OO tdagagcaaa aggtt tatga tigtggittittg aagcaggcgg ccttggittaa taga caattg 360

agatctggag aagattggga citgaag.ccc gatattgttc. tccCaggaala tittaaacata 42O ttalagtgaag ct tatgatcg ctg.cggtgaa gtatgtgctgaatatgcc.ca gacttitttac 48O

ttgggaacaa tictaatgac acctgagaga agaa.gagct a tittgggcgat atatgtttgg 54 O

tgcaggagaa cagatgagct cqttgatggg cctaatgcat cacatataac to Caactgca 6 OO ttggataggit gggaa.gc.gcg attggaagat gt ctittagag gt catcCttt tatatgctt 660

gatgctgctic titt cagatac tdttt coaag titt.ccagttg acatccagoc attcagagac 72O US 8,252,977 B2 57 58 - Continued atgattgaag gaatgaga at ggacctaaag aagtcaagat acaaaaactt tdatgagcta tacctic tact gttactatgt gig.ccggtacc gttggattga tgagtgtc.cc agittatgggc 84 O attgcaccag aatcaaaag.c aac agtagaa agtgtctata atgcagc cct ggcattaggg 9 OO attgctaacc agctgactaa catactaagg gatgttggag aagatgctac aagaggalagg 96.O atctacctac cccaagatga attagcacag gCagggctitt Cagatgagga t at atttgct ggaaaggit ca Ctgaccalatg gagga atttic atgaagcagc aaatgaaaag agcaaggaag 108 O ttctttgatg aggcagagaa aggagtgacc gagct caact Ctgctagoag atggcctgta 114 O tgggcatcgc tattgctgta t cqtcaaata Ctcgacgaga ttgaa.gc.caa tact acaac 12 OO aattittgaca ggagagctta tottagcaaa ccgaagaagt tacttgct cit gccaatggca 126 O tatgcaaagt ct cittgtacct coaagaa.ca t catcto cqc tagcaaaagg catgagctga 132O

SEQ ID NO 2 LENGTH: 1077 TYPE: DNA ORGANISM: Coffea canephora FEATURE: NAMEAKEY: misc feature LOCATION: (42) ... (43) OTHER INFORMATION: Unknown nucleotide FEATURE: NAMEAKEY: misc feature LOCATION: (825) . . (827) OTHER INFORMATION: Unknown nucleotide

SEQUENCE: 2 attgacitat c caaggccaga gcttgaaaat gcc.gt caact atttggaagic tdott attta 60 t catcaac at tcc.gtact tc. tcc to atcca aataaac Cat tagaggtggit gat.cgc.cggit 12 O gCaggitttgg Ctggtttgtc. tactgcaaag tatttggcc.g atgcagg to a taalacctata 18O gtgttggaag Ctagggatgt t ctgggagga alaggttgctg Catggaaaga tigatgatgga 24 O gactgg tatg agactggcct gcacg tatto tittggggctt acccaaatat gcagaacct g 3OO tittggagaac taggaattaa tdatcggttg Cagtggalagg agcattcaat gat atttgca 360 atgccaaata agcctggaga gttcagticga. tttgattittc Ctgaggtgct accagcacca ttaaatggaa tatgggcc at Cttgaagaat aatgacatgc ttacttggcc agagaaagtic 48O aaatttgcaa ttggactic tit gcc agcaatt Ctgggtggac aatct tatgt tdaggcacala 54 O gatggtataa citgtcaaaga Ctggatgaga alagcaaggca taccagat.cg ggtgactgat gaagtatt ct ttgc.catgtc. aaaggcactg aactt Catala atccagatga actitt caatg 660 cagtgcattt taatagottt gaaccattt Ctt Caggaga agcatggat C caaaatggca 72 O tttittagatg gtaaccotcc agagagacitt tgcatgc.cga ttgttgagca cattgagtica cgaggaggca gagtacacct taact calaga att cagaaaa ttgagct caa tatgc.cgga 84 O agtgttgaaa acttcttgct gagtaatgga actgtgatta gaggagatgc titatgtattt 9 OO gccact coag ttgatat cott gaagcttctt ttgcctgagg attggaaaga gatgc catac 96.O ttcagaaagt tagaaatt agttggagtt cctgttataa atgtgcacat atggtttgac aggaagctica ggalacacata catcatctt Ctttittagca gaagt cc act tct tagt 1077

SEQ ID NO 3 LENGTH: 1056 TYPE: DNA ORGANISM: Coffea canephora

US 8,252,977 B2 71 72 - Continued t ctittggttg ctacaacaac Cttitt cogcc tgatgaaaaa gg taggtgct gataaaaatt 18O tgctctgaa ggat catact cacacatttg ttaacaaagg gggtgaaatt ggtgaacttg 24 O attt cogctt tccagttggg gCacctttac atggaattaa tgcattcttg totaccalat C 3OO agctaaagat titatgataag gcaagaaatg cc.gtggctict cgc.gcttggit c cagttgtac 360 gggctctggt tgatcctat ggagcgctga gggagatacg ggatttagac aggataagct tcticagattg gttctt at CC aaaggaggga Ctcgc.gcaag tatacagagg at 472

<210s, SEQ ID NO 13 &211s LENGTH: 439 212. TYPE : PRT <213> ORGANISM: Coffea canephora <4 OOs, SEQUENCE: 13

Met Ser Val Ala Lieu Lleu Trp Val Wall Lieu Pro Ile Ser Glu Wall. Thir 1. 5 1O 15

Asn Ser Ile Ala Phe Lieu. Glu Pro Val Arg Glu Gly Ser Arg Lieu. Luell 2O 25 3O

Asp Ser Ser Arg Phe Val Gly Arg Gly Lys ASn Lell Asin Gly 35 4 O 45

Arg Luell Glu Lys Gly Lys Glin Glin Arg Trp ASn Ser Gly Luell Asn SO 55 6 O

Gly Asp Ser Arg Asn. Cys Cys Lieu. Gly Gly Ser Arg Lell Asn Arg 65 70 8O

Gly Phe Ser Wall Ile Pro Asn Wall Wall Wall Ser Pro Ala Gly Glu 85 90 95

Ile Ala Met Ser Ser Glu Gln Lys Val Tyr Asp Wall Wall Luell Lys Glin 1OO 105 11 O

Ala Ala Luell Val Asn Arg Glin Lieu. Arg Ser Arg Glu Asp Trp Asp Wall 115 12 O 125

Pro Asp Ile Val Lieu Pro Gly Asn Luell ASn Ile Lell Ser Glu Ala 13 O 135 14 O

Tyr Asp Arg Cys Gly Glu Val Cys Ala Glu Tyr Ala Glin Thir Phe Tyr 145 150 155 160

Lell Gly Thir Met Leu Met Thr Pro Glu Arg Arg Arg Ala Ile Trp Ala 1.65 17O 17s

Ile Wall Trp. Cys Arg Arg Thr Asp Glu Luell Wall Asp Gly Pro Asn 18O 185 19 O

Ala Ser His Ile Thr Pro Thir Ala Lieu. Asp Arg Trp Glu Ala Arg Lieu. 195 2OO

Glu Asp Wall Phe Arg Gly His Pro Phe Asp Met Lell Asp Ala Ala Lieu 21 O 215

Ser Asp Thir Val Ser Lys Phe Pro Val Asp Ile Glin Pro Phe Arg Asp 225 23 O 235 24 O

Met Ile Glu Gly Met Arg Met Asp Lieu Lys Ser Arg Lys Asn 245 250 255

Phe Asp Glu Leu Tyr Lieu. Tyr Cys Tyr Tyr Wall Ala Gly Thir Val Gly 26 O 265 27 O

Lell Met Ser Val Pro Val Met Gly Ile Ala Pro Glu Ser Ala Thr 27s 28O 285

Wall Glu Ser Val Tyr Asn Ala Ala Lieu Ala Luell Gly Ile Ala ASn Glin 29 O 295 3 OO

Lell Thir Asn Ile Lieu. Arg Asp Val Gly Glu Asp Ala Thir Arg Gly Arg 3. OS 310 315 32O US 8,252,977 B2 73 - Continued Ile Tyr Lieu Pro Glin Asp Glu Lieu Ala Glin Ala Gly Lieu. Ser Asp Glu 3.25 330 335 Asp Ile Phe Ala Gly Llys Val Thr Asp Glin Trp Arg Asn. Phe Met Lys 34 O 345 35. O Glin Gln Met Lys Arg Ala Arg Llys Phe Phe Asp Glu Ala Glu Lys Gly 355 360 365 Val Thr Glu Lieu. Asn. Ser Ala Ser Arg Trp Pro Val Trp Ala Ser Lieu 37 O 375 38O Lieu. Lieu. Tyr Arg Glin Ile Lieu. Asp Glu Ile Glu Ala Asn Asp Tyr Asn 385 390 395 4 OO Asn Phe Asp Arg Arg Ala Tyr Val Ser Llys Pro Llys Llys Lieu. Lieu Ala 4 OS 41O 415 Lieu Pro Met Ala Tyr Ala Lys Ser Leu Val Pro Pro Arg Thr Ser Ser 42O 425 43 O Pro Lieu Ala Lys Gly Met Ser 435

<210s, SEQ ID NO 14 &211s LENGTH: 359 212. TYPE: PRT <213> ORGANISM: Coffea canephora <4 OOs, SEQUENCE: 14 Ile Asp Tyr Pro Arg Pro Glu Lieu. Glu Asn Ala Val Asn Tyr Lieu. Glu 1. 5 1O 15 Ala Ala Tyr Lieu Ser Ser Thr Phe Arg Thr Ser Pro His Pro Asn Lys 2O 25 3O Pro Lieu. Glu Val Val Ile Ala Gly Ala Gly Lieu Ala Gly Lieu. Ser Thr 35 4 O 45 Ala Lys Tyr Lieu Ala Asp Ala Gly. His Llys Pro Ile Val Lieu. Glu Ala SO 55 6 O Arg Asp Val Lieu. Gly Gly Llys Val Ala Ala Trp Lys Asp Asp Asp Gly 65 70 7s 8O Asp Trp Tyr Glu Thr Gly Lieu. His Val Phe Phe Gly Ala Tyr Pro Asn 85 90 95 Met Glin Asn Lieu. Phe Gly Glu Lieu. Gly Ile Asn Asp Arg Lieu Gln Trp 1OO 105 11 O Lys Glu. His Ser Met Ile Phe Ala Met Pro Asn Llys Pro Gly Glu Phe 115 12 O 125 Ser Arg Phe Asp Phe Pro Glu Val Lieu Pro Ala Pro Leu. Asn Gly Ile 13 O 135 14 O Trp Ala Ile Lieu Lys Asn. Asn Asp Met Lieu. Thir Trp Pro Glu Lys Val 145 150 155 160

Llys Phe Ala Ile Gly Lieu. Lieu Pro Ala Ile Lieu. Gly Gly Glin Ser Tyr 1.65 17O 17s

Val Glu Ala Glin Asp Gly Ile Thr Val Lys Asp Trp Met Arg Lys Glin 18O 185 19 O

Gly Ile Pro Asp Arg Val Thr Asp Glu Val Phe Phe Ala Met Ser Lys 195 2OO 2O5

Ala Lieu. Asn. Phe Ile Asn Pro Asp Glu Lieu. Ser Met Glin Cys Ile Lieu 21 O 215 22O

Ile Ala Lieu. Asn Arg Phe Lieu. Glin Glu Lys His Gly Ser Lys Met Ala 225 23 O 235 24 O

Phe Lieu. Asp Gly Asn Pro Pro Glu Arg Lieu. Cys Met Pro Ile Val Glu 245 250 255 US 8,252,977 B2 75 - Continued His Ile Glu Ser Arg Gly Gly Arg Val His Lieu. Asn. Ser Arg Ile Glin 26 O 265 27 O Lys Ile Glu Lieu. Asn Asp Ala Gly Ser Val Glu Asn. Phe Lieu. Lieu. Ser 27s 28O 285 Asn Gly Thr Val Ile Arg Gly Asp Ala Tyr Val Phe Ala Thr Pro Val 29 O 295 3 OO Asp Ile Lieu Lys Lieu Lleu Lleu Pro Glu Asp Trp Llys Glu Met Pro Tyr 3. OS 310 315 32O Phe Arg Llys Lieu. Glu Lys Lieu Val Gly Val Pro Val Ile Asin Val His 3.25 330 335 Ile Trp Phe Asp Arg Llys Lieu. Arg Asn. Thir Tyr Asp His Lieu. Lieu. Phe 34 O 345 35. O

Ser Arg Ser Pro Lieu Lleu Ser 355

<210s, SEQ ID NO 15 &211s LENGTH: 351 212. TYPE: PRT <213> ORGANISM: Coffea canephora <4 OOs, SEQUENCE: 15 Met Ala Thr Ser Thr Ser Ser Val Val Phe Gly Ile Ser Val Ser Ser 1. 5 1O 15 Ser Thr Ser Leu Lys Ile Arg Ser Phe Arg Asn Val Pro Thr Val Lieu. 2O 25 3O Asn Ser His Thr Pro Ser Gly Lieu. Asn Val Val Thr Ser Pro His His 35 4 O 45 Arg His Thir Ala Thr Glin Arg Pro Lieu. Ser Arg Asn. Ser Phe Arg Val SO 55 6 O Glin Ala Thr Val Lieu. Glin Glu Asp Glu Gln Llys Val Val Val Glu Glu 65 70 7s 8O Ser Phe Glin Ser Lys Ser Tyr Pro Glu Asin Gly Gly Gly Gly Asn Gly 85 90 95 Glu Pro Pro Asp Ala Ser Ser Ser Ser Gly Lieu. Glu Lys Trp Val Val 1OO 105 11 O Lys Ile Glu Glin Ser Ile Asn. Ile Phe Lieu. Thir Asp Ser Val Ile Llys 115 12 O 125 Ile Lieu. Asp Thir Lieu. Tyr His Asp Arg His Tyr Ala Arg Phe Phe Val 13 O 135 14 O Lieu. Glu Thir Ile Ala Arg Val Pro Tyr Phe Ala Phe Met Ser Val Lieu. 145 150 155 160 His Lieu. Tyr Glu Ser Phe Gly Trp Trp Arg Arg Ala Asp Lieu. Ser Glu 1.65 17O 17s

Val His Phe Ala Glu Ser Trp Asn Glu Met His His Leu Lieu. Ile Met 18O 185 19 O

Glu Glu Lieu. Gly Gly Asn. Ser Trp Trp Phe Asp Arg Phe Lieu Ala Glin 195 2OO 2O5

His Ile Ala Val Phe Tyr Tyr Phe Met Thr Val Phe Met Tyr Met Leu 21 O 215 22O

Ser Pro Arg Met Ala Tyr His Phe Ser Glu. Cys Val Glu Ser His Ala 225 23 O 235 24 O

Phe Glu Thir Tyr Asp Llys Phe Ile Lys Asp Glin Gly Glu Gln Lieu Lys 245 250 255

Llys Lieu Pro Ala Ser Asn. Wall Ala Val Lys Tyr Tyr Thr Glu Gly Asn 26 O 265 27 O US 8,252,977 B2 77 - Continued Lieu. Tyr Lieu Phe Asp Glu Phe Glin Thr Ala Arg Pro Pro Thr Ser Arg 27s 28O 285 Arg Pro Lys Ile Glu Asn Met Tyr Asp Val Phe Lieu. Asn. Ile Arg Asp 29 O 295 3 OO Asp Glu Ala Glu. His Cys Llys Thr Met Lys Ala Cys Glin Thr His Gly 3. OS 310 315 32O Gly Lieu. Arg Ser Pro His Ser Tyr Thr Asp Asp Ala Cys Glu Glu Asp 3.25 330 335

Ala Gly Tyr Gly Lieu Pro Glin Ala Asp Cys Glu Glu Lieu. Thr Glin 34 O 345 35. O

<210s, SEQ ID NO 16 &211s LENGTH: 310 212. TYPE: PRT <213> ORGANISM: Coffea canephora

<4 OOs, SEQUENCE: 16 Met Ala Ala Gly Ile Ala Wall Ala Ala Gly Ala Glin Thr Val Cys Phe 1. 5 1O 15

Arg Val Asn. Ser Phe Lieu. Thir Arg Llys Pro Thir Ser Lieu Val Ala Asp 2O 25 3O

Ser Lieu. Thir Lieu Ser Pro Leu Ala Glin Glin Phe Ser Thr Thr Arg Arg 35 4 O 45 His Arg Arg Llys Pro Arg Lieu. Thr Val Cys Phe Val Lieu. Glu Asp Glu SO 55 6 O

Glu Lieu Lys Ala Glin Lieu Val Thir Ser Glu Glu Glu Ala Arg Glu Arg 65 70 7s 8O

Glu Lys Ala Met Ala Lys Arg Ile Ser Asp Ala Arg Thr Ala Glu Lys 85 90 95

Lieu Ala Arg Lys Arg Ser Glu Arg Phe Thr Tyr Lieu Val Ala Ala Val 1OO 105 11 O

Met Ser Ser Phe Gly Ile Thir Ser Met Ala Val Lieu Ala Val Tyr Tyr 115 12 O 125

Arg Phe Val Trp Gln Met Glu Gly Gly Glu Val Pro Tyr Ser Glu Met 13 O 135 14 O

Phe Gly Thr Phe Ala Leu Ser Val Gly Ala Ala Val Gly Met Glu Phe 145 150 155 160 Trp Ala Arg Trp Ala His Lys Ala Lieu. Trp His Ala Ser Lieu. Trp His 1.65 17O 17s

Met His Glu Ser His His Arg Pro Arg Glu Gly Pro Phe Glu Lieu. Asn 18O 185 19 O

Asp Val Phe Ala Ile Ile Asn Ala Val Pro Ala Ile Ala Lieu. Lieu. Ser 195 2OO 2O5 Tyr Gly Phe Phe His Lys Gly Lieu. Ile Pro Gly Lieu. Cys Phe Gly Ala 21 O 215 22O Gly Lieu. Gly Ile Ile Val Phe Gly Met Ala Tyr Met Phe Val His Asp 225 23 O 235 24 O

Gly Lieu Val His Lys Arg Phe Pro Val Gly Pro Ile Ala Asn Val Pro 245 250 255 Tyr Phe Arg Arg Val Ala Ala Ala His Glin Lieu. His His Ser Asp Llys 26 O 265 27 O

Phe Asin Gly Val Pro Phe Gly Lieu Phe Leu Gly Pro Lys Glu Lieu. Glu 27s 28O 285 US 8,252,977 B2 79 - Continued Llys Val Gly Gly Lieu. Glu Glu Lieu. Glu Lys Glu Ile Asn Arg Arg Ile 29 O 295 3 OO Llys Lieu. Arg Lys Gly Ser 3. OS 310

<210s, SEQ ID NO 17 &211s LENGTH: 469 212. TYPE: PRT <213> ORGANISM: Coffea canephora

<4 OOs, SEQUENCE: 17 Cys Val Val Asp Llys Glu Glu Lys Phe Ala Asp Glin Glu Asp Tyr Ile 1. 5 1O 15 Lys Ala Gly Gly Ser Glu Lieu. Lieu. Tyr Val Glin Met Glin Glin Arg Llys 2O 25 3O Gln Met Asp Glin Glin Ser Lys Phe Ser Asp Llys Met Pro Glu Ile Ser 35 4 O 45 Ala Gly Asn. Ser Ile Lieu. Asp Lieu Val Val Ile Gly Cys Gly Pro Ala SO 55 6 O Gly Lieu Ala Lieu Ala Ala Glu Ser Ala Lys Lieu. Gly Lieu. Thr Val Gly 65 70 7s 8O Lieu. Ile Gly Pro Asp Val Pro Phe Thr Asn Asn Tyr Gly Val Trp Glu 85 90 95 Asp Glu Phe Lys Asp Lieu. Gly Lieu Ala Gly Cys Ile Glu. His Val Trip 1OO 105 11 O Arg Asp Thr Val Val Tyr Lieu. Asp Asp Asin Asp Pro Ile Lieu. Ile Gly 115 12 O 125 Arg Ala Tyr Gly Arg Phe Ser Arg His Lieu. Lieu. His Glu Glu Lieu. Lieu 13 O 135 14 O Arg Arg Cys Val Glu Ser Gly Val Ser Tyr Lieu. Ser Ser Val Glu Arg 145 150 155 160 Ile Val Glu Ala Ala Thr Gly. His Ser Lieu Val Glu. Cys Glu Gly Ser 1.65 17O 17s Ile Val Ile Pro Cys Arg Lieu Ala Thr Val Ala Ser Gly Ala Ala Ser 18O 185 19 O Gly Lys Lieu. Lieu. Glin Tyr Glu Lieu. Gly Gly Pro Arg Val Ser Val Glin 195 2OO 2O5 Thr Ala Tyr Gly Val Glu Val Glu Val Glu Asn Asn Pro Tyr Asp Pro 21 O 215 22O Asn Lieu Met Val Phe Met Asp Tyr Arg Asp Tyr Met Arg Gly Llys Val 225 23 O 235 24 O Glu Ser Lieu. Glu Ala Glu Phe Pro Thr Phe Leu Tyr Ala Met Pro Met 245 250 255

Ser Pro Thr Arg Val Phe Phe Glu Glu Thr Cys Lieu Ala Ser Lys Asp 26 O 265 27 O

Ala Met Pro Phe Glu Lieu Lleu Lys Llys Llys Lieu Met Ser Arg Lieu. Asp 27s 28O 285

Thr Lieu. Gly Val Arg Ile Ile Llys Thr Tyr Glu Glu Glu Trp Ser Tyr 29 O 295 3 OO

Ile Pro Val Gly Gly Ser Lieu Pro Asn Thr Glu Gln Lys Asn Lieu Ala 3. OS 310 315 32O

Phe Gly Ala Ala Ala Ser Met Val His Pro Ala Thr Gly Tyr Ser Val 3.25 330 335

Val Arg Ser Lieu. Ser Glu Ala Pro Llys Tyr Ala Ser Ala Ile Ala Asn 34 O 345 35. O US 8,252,977 B2 81 - Continued Ile Lieu Lys Glin Gly Glin Ala Lys Asp Met Met Thr Arg Asn. Ile Ser 355 360 365 Ala Glin Ala Trp Asn. Thir Lieu. Trp Pro Glin Glu Arg Lys Arg Glin Arg 37 O 375 38O Ala Phe Phe Lieu. Phe Gly Lieu Ala Lieu. Ile Lieu Gln Lieu. Asp Ile Glu 385 390 395 4 OO Gly Ile Arg Thr Phe Phe Glin Thr Phe Phe Arg Lieu Pro Asn Trp Met 4 OS 41O 415 Ser Glin Gly Phe Lieu. Gly Ser Ser Lieu. Ser Ser Thr Asp Lieu. Lieu. Lieu. 42O 425 43 O Phe Ala Phe Tyr Met Phe Val Ile Ala Pro Asn Asp Lieu. Arg Lys Cys 435 44 O 445 Lieu. Ile Gln His Lieu Lleu Ser Asp Pro Thr Gly Ala Thr Met Val Arg 450 45.5 460 Thr Tyr Lieu Ala Ile 465

<210s, SEQ ID NO 18 &211s LENGTH: 343 212. TYPE: PRT <213> ORGANISM: Coffea canephora <4 OOs, SEQUENCE: 18 Gly Lys Lys Glu Arg Lieu Lleu Lys Ile Phe Asp Gly Trp Cys Asp Llys 1. 5 1O 15 Val Met Glu Lieu Lleu Lieu Ala Thr Asp Glu Asp Ala Ile Lieu. Arg Arg 2O 25 3O Asp Ile Tyr Asp Arg Thr Pro Ser Phe Ser Trp Gly Arg Gly Arg Val 35 4 O 45 Thir Lieu. Lieu. Gly Asp Ser Ile His Ala Met Gln Pro Asn Lieu. Gly Glin SO 55 6 O Gly Gly Cys Met Ala Ile Glu Asp Ser Tyr Glin Lieu Ala Lieu. Glu Lieu. 65 70 7s 8O Asp Lys Ala Trp Glu Glin Ser Ile Llys Ser Gly Ser Pro Met Asp Wall 85 90 95 Val Ser Ala Lieu Lys Ser Tyr Glu Ser Ala Arg Llys Lieu. Arg Val Ala 1OO 105 11 O Ile Ile His Gly Lieu Ala Arg Lieu Ala Ala Ile Met Ala Ser Thr Tyr 115 12 O 125 Llys Pro Tyr Lieu. Gly Val Gly Lieu. Gly Pro Leu Ser Phe Lieu. Thir Lys 13 O 135 14 O Phe Arg Ile Pro His Pro Gly Arg Val Gly Gly Arg Ile Phe Ile Asp 145 150 155 160

Ile Gly Met Pro Leu Met Leu Ser Trp Val Lieu. Gly Gly Asn Gly Ser 1.65 17O 17s Llys Lieu. Glu Gly Arg Pro Lieu. His Cys Arg Lieu. Thir Asp Lys Ala Ser 18O 185 19 O

Asp Gln Lieu. Glin Llys Trp Phe Glin Asp Asp Asp Ser Lieu. Glu Arg Ala 195 2OO 2O5

Lieu. Asn Gly Glu Trp Phe Lieu. Phe Pro Ile Gly Glin Ala Asn Pro Asp 21 O 215 22O

Pro Val Ala Ile Phe Lieu. Gly Arg Asp Glu Lys Asn. Ile Cys Thir Ile 225 23 O 235 24 O

Gly Ser Ala Ser His Pro Asp Ile Lieu. Gly Ala Ser Ile Ile Ile Asn 245 250 255 US 8,252,977 B2 83 84 - Continued

Ser Pro Glin Wall Ser Lell His Ala Glin Ile Ser Tyr Lys Asp Gly 26 O 265 27 O

Lell Phe Phe Luell Thir Asp Lell Glin Ser Glu His Gly Thir Trp Ile Thir 27s 28O 285

Asp Asn Asp Gly Arg Arg Tyr Arg Luell Pro Pro Asn Ser Pro Ala Arg 29 O 295 3 OO

Phe His Pro Tyr Asp Ile Ile Glu Phe Gly Ser Asp Ala Ala Phe 3. OS 310 315

Arg Wall Wall Thir Asn Glin Pro Pro Phe Ser Gly Arg Glu 3.25 330 335

Thir Wall Luell Ser Ala Wall 34 O

<210s, SEQ ID NO 19 &211s LENGTH: 415 212. TYPE : PRT &213s ORGANISM: Coffea canephora

<4 OOs, SEQUENCE: 19

Met Ala Ser Ala Lell His Ser Ala Phe His Ser Asn Asp Glu Gly Ile 1. 5 15

Arg Phe Tyr Ile Arg Ser Glin His Arg Ile Gly Gly Arg Cys Ser Asn 25

Gly Gly Ala Arg Pro Glin Asn Ala Luell Phe Ser Wall Lys Met Trp Ser 35 4 O 45

Arg Trp Gly Ser Arg Tyr Ile Glin Luell Glin Arg Ala Pro Arg Ile SO 55 6 O

Luell Ser Luell Gly Ser Thir Arg Luell Phe Lell Asn Gly Ile 70

Arg Glu Lell Ala Ile Ala ASn Pro Ser Ala Ala 85 90 95

Asn Wall Cys Lell Glin Thir Asn Asn Arg Pro Asp Glu Thir Glu 1OO 105 11 O

Glin Cys Gly Asp Luell Phe Glin ASn Ser Wall Wall Asp Glu 12 O 125

Phe Asn Ala Wall Ser Arg Wall Pro Arg Ser 13 O 135 14 O

Asp Wall Glu Phe Pro Ala Pro Pro Ala Wall Lell Wall Asn 145 150 155 160

Phe Asp Asp Phe Ser Gly Trp Ile Ser Ser Gly Luell 1.65 17O 17s

Asn Pro Thir Phe Asp Thir Phe Asp Cys Glin Luell His Glu Phe His Thir 18O 185 19 O

Glu Ser Gly Lys Lell Wall Gly Asn Luell Thir Trp Arg Ile Arg Thir Pro 195 2OO

Asp Thir Gly Phe Phe Thir Arg Ser Ala Luell Glin Arg Phe Wall Glin Asp 21 O 215

Pro Pro Gly Ile Lell Tyr Asn His Asp Asn Glu Tyr Luell His 225 23 O 235 24 O

Glin Asp Trp Ile Luell Ser Ser Ile Glu Asn Lys Pro 245 250 255

Asp Asp Ala Phe Wall Arg Gly Arg Asn Asp Ala Trp Asp 26 O 265 27 O

Gly Gly Gly Ala Wall Wall Tyr Thir Arg Ser Ala Wall Luell Pro Glu 27s 28O 285 US 8,252,977 B2 85 - Continued Ser Ile Val Pro Glu Lieu. Glin Arg Ala Ala Lys Ser Ile Gly Arg Asp 29 O 295 3 OO Phe Ser Llys Phe Ile Arg Thr Asp Asn Thr Cys Gly Pro Glu Pro Pro 3. OS 310 315 32O Lieu Val Glu Arg Lieu. Glu Lys Thr Val Glu Glu Gly Glu Arg Thir Ile 3.25 330 335 Val Arg Glu Val Glu Glu Ile Glu Gly Glu Ile Glu Gly Glu Val Glu 34 O 345 35. O Llys Val Lys Asp Thr Glu Met Thr Lieu. Phe Glu Arg Lieu. Thr Glu Gly 355 360 365 Phe Lys Glu Lieu Lys Lys Asp Glu Glu Phe Phe Lieu. Arg Glu Lieu. Ser 37 O 375 38O Lys Glu Glu Lieu. Asp Val Lieu. Asp Ala Lieu Lys Met Glu Ala Ser Glu 385 390 395 4 OO Val Glu Lys Lieu. Phe Gly Arg Ser Lieu Pro Ile Arg Llys Lieu. Arg 4 OS 41O 415

<210s, SEQ ID NO 2 O &211s LENGTH: 635 212. TYPE: PRT <213> ORGANISM: Coffea canephora <4 OOs, SEQUENCE: 2O Met Pro Met Thr Met Ile Leu Pro Pro Ser Ser Arg Glu Met Met Gly 1. 5 1O 15 Lieu. Gly Lieu. Gly Cys Ser Pro Ser Ser Lys Thr Lieu Ala Phe Arg His 2O 25 3O Pro Asn Thr Lieu Pro Asn Tyr Ile Asn Cys Ser Leu Gln Thr Pro Ser 35 4 O 45 Ile Lieu. His Phe Pro Lys Glin Ser Ser Ala Thr Thr Ser Ser Pro Pro SO 55 6 O Ser Ser Ser Ser Ala Lys Thr Ala Tyr Pro Ala Leu Phe Leu Pro Gly 65 70 7s 8O Ser Ser Ala Ala Ile Ala Thr Pro Ser Lys Thr Pro Thr Gly Thr Ala 85 90 95

Thir Wall Pro Thr Pro Ser Pro Ser Ile Ser Ala Ser Pro Ser Pro Ser 1OO 105 11 O Arg Ser Pro Ser Thr Thr Pro Gln Trp Asn Val Lieu. Glin Arg Ala Ala 115 12 O 125 Ala Met Ala Lieu. Asp Ala Val Glu Thir Ala Lieu. Thir Ala Arg Glu Lieu 13 O 135 14 O Glu Gln Pro Lieu Pro Llys Thr Ala Asp Pro Arg Ile Glin Ile Ser Gly 145 150 155 160

Asn Phe Ala Pro Val Pro Glu Gln Pro Val Arg His Ala Leu Pro Val 1.65 17O 17s

Thr Gly Lys Ile Pro Asn Ser Ile Glin Gly Val Tyr Val Arg Asn Gly 18O 185 19 O

Ala Asn Pro Leu Phe Glu Pro Ala Ala Gly His His Phe Phe Asp Gly 195 2OO 2O5

Asp Gly Met Ile His Ala Lieu. Glin Phe Glin Asn Gly Ser Ala Ser Tyr 21 O 215 22O

Ala Cys Arg Phe Thr Glu Thr Glin Arg Lieu Ala Glin Glu Arg Ser Lieu 225 23 O 235 24 O Gly Arg Pro Val Phe Pro Lys Ala Ile Gly Glu Lieu. His Gly His Ser 245 250 255 US 8,252,977 B2 87 - Continued Gly Ile Ala Arg Lieu Met Lieu. Phe Tyr Ala Arg Gly Val Phe Gly Lieu. 26 O 265 27 O Lieu. Asp His Ser Glin Gly Thr Gly Val Ala Asn Ala Gly Lieu Val Tyr 27s 28O 285 Phe Asn. Asn Arg Lieu. Lieu Ala Met Ser Glu Asp Asp Lieu Pro Tyr His 29 O 295 3 OO Val Arg Ile Thr Pro Ser Gly Asp Leu Lys Thr Val Glu Arg Tyr Ser 3. OS 310 315 32O Phe Asin Gly Glin Lieu Lys Ser Thr Met Ile Ala His Pro Llys Lieu. Asp 3.25 330 335 Pro Val Thr Gly Glu Lieu Phe Ala Leu Ser Tyr Asp Val Ile Gln Lys 34 O 345 35. O Pro Tyr Lieu Lys Tyr Phe Arg Phe Ser Lys Ala Gly Glu Lys Ser Lys 355 360 365 Asp Ile Glu Ile Pro Val Pro Glu Pro Thr Met Met His Asp Phe Ala 37 O 375 38O Ile Thr Asp Asin Phe Val Val Ile Pro Asp Gln Glin Val Val Phe Lys 385 390 395 4 OO

Met Ser Glu Met Ile Arg Gly Gly Ser Pro Val Val Tyr Asp Llys Glu 4 OS 41O 415

Llys Val Ser Arg Phe Gly Val Lieu. Asp Llys Tyr Ala Glu Asp Ser Ser 42O 425 43 O Ala Ile Llys Trp Val Glu Val Pro Asp Cys Phe Cys Phe His Leu Trp 435 44 O 445

Asn Ala Trp Glu Glu Pro Glu Thir Asp Glu Ile Val Val Ile Gly Ser 450 45.5 460

Cys Met Thr Pro Pro Asp Ser Ile Phe Asin Glu. Cys Asp Glu Gly Lieu. 465 470 47s 48O

Llys Ser Val Lieu. Ser Glu Ile Arg Lieu. Asn Lieu Lys Thr Gly Lys Ser 485 490 495

Thir Arg Arg Ala Ile Ile Ser Asn Pro Glu Asp Glin Val Asn Lieu. Glu SOO 505 51O

Ala Gly Met Val Asn Arg Asn Llys Lieu. Gly Arg Llys Thr Arg Tyr Ala 515 52O 525

Tyr Lieu Ala Ile Ala Glu Pro Trp Pro Llys Val Ser Gly Phe Ala Lys 53 O 535 54 O

Val Asp Lieu. Phe Thr Gly Glu Val Arg Llys Phe Ile Tyr Gly Asp Glu 5.45 550 555 560

Llys Tyr Gly Gly Glu Pro Lieu. Phe Lieu Pro Arg Asp Pro Asn. Cys Glu 565 st O sts Ala Glu Asp Asp Gly Tyr Ile Lieu Ala Phe Val His Asp Glu Lys Glu 58O 585 59 O

Trp Llys Ser Glu Lieu. Arg Ile Val Asn Ala Met Thr Lieu. Glu Lieu. Glu 595 6OO 605

Ala Ser Val Glin Leu Pro Ser Arg Val Pro Tyr Gly Phe His Gly Thr 610 615 62O

Phe Ile Ser Ala Lys Asp Lieu Ala Ser Glin Ala 625 630 635

<210s, SEQ ID NO 21 &211s LENGTH: 548 212. TYPE: PRT <213> ORGANISM: Coffea canephora US 8,252,977 B2 89 90 - Continued

<4 OOs, SEQUENCE: 21

Met Gly Arg Glin Glu Gly Glu Glu Wall Wall Glu Ile Glu Gly 1. 15

Glin Glu Wall Wall Wall Wall Asn Pro Lys Pro ASn Asn Gly Phe Thir Ala 25

Luell Ile Asp Trp Wall Glu Lys Ala Wall Wall Lell Met Asp 35 4 O 45

Ser Lys Glin Pro Lell His Tyr Luell Ser Gly ASn Phe Ala Pro Wall Asp SO 55 6 O

Glu Thir Pro Pro Cys Lys Asp Luell Luell Wall Lys Gly His Luell Pro Glu 65 70

Luell Asn Gly Glu Phe Wall Arg Wall Gly Pro Asn Pro Phe Ser 85 90 95

Pro Wall Ala Gly Tyr His Trp Phe Asp Gly Asp Gly Met Ile His Gly 105 11 O

Ile Arg Ile Asp Gly Ala Thir Wall Ser Arg Wall 115 12 O 125

Thir Ser Arg Luell Lys Glin Glu Glu Phe Gly Gly Ser Phe Met 13 O 135 14 O

Lys Wall Gly Asp Lell Lys Gly Luell Phe Gly Luell Phe Met Wall Asn Met 145 150 155 160

Glin Ile Luell Arg Ala Lell Wall Luell Asp Met Thir Gly Ile 1.65 17O 17s

Gly Thir Ala Asn Thir Ala Lell Ile Tyr His His Gly Luell Luell Ala 18O 185 19 O

Lell Glin Glu Ala Asp Pro Tyr Wall Luell Arg Wall Lell Glu Asp Gly 195 2O5

Asp Luell Glin Thir Lell Gly Lell Luell Asp Asp Lys Arg Luell Thir His 21 O 215 22O

Ser Phe Thir Ala His Pro Wall Asp Pro Phe Thir Gly Glu Met Phe 225 23 O 235 24 O

Thir Phe Gly Ser His Thir Pro Pro Tyr Ile Thir Arg Wall Ile 245 250 255

Ser Glu Gly Wall Met Asp Asp Pro Wall Pro Ile Thir Ile Ser Asp 26 O 265 27 O

Pro Ile Met Met His Asp Phe Ala Ile Thir Glu Asn Tyr Ala Ile Phe 285

Met Asp Luell Pro Lell Phe Arg Pro Lys Glu Met Wall Asp 29 O 295 3 OO

Lys Luell Ile Phe Thir Phe Asp Pro Thir Lys Lys Ala Arg Phe Gly Wall 3. OS 310 315 32O

Lell Pro Arg Ser Asn Asp Ala Luell Ile Trp Phe Glu Luell 3.25 330 335

Pro Asn Phe Ile Phe His Asn Ala Asn Ala Trp Glu Glu Gly Asp 34 O 345 35. O

Glu Wall Ile Luell Ile Thir Arg Luell Glin ASn Pro Asp Luell Asp Met 355 360 365

Wall Ser Gly Ile Wall Lys Luell Glu ASn Phe Ser Asn Glu Luell 37 O 375

Tyr Glu Met Arg Phe Asn Lell Thir Gly Luell Ala Ser Glin Lys 385 390 395 4 OO

Lell Ser Glu Ser Ala Wall Asp Phe Pro Arg Wall Asn Glu Ser Tyr Thir 4 OS 41O 415 US 8,252,977 B2 91 - Continued Gly Arg Lys Glin Glin Tyr Val Tyr Gly. Thir Ile Lieu. Asp Ser Ile Ala 42O 425 43 O Llys Val Thr Gly Ile Ala Lys Phe Asp Lieu. His Ala Glu Pro Glu Thir 435 44 O 445 Gly Lys Thir Lys Ile Glu Val Gly Gly Asn Val Glin Gly Val Phe Asp 450 45.5 460 Lieu. Gly Pro Gly Arg Phe Gly Ser Glu Ala Ile Phe Val Pro Arg Glin 465 470 47s 48O Pro Gly Ile Thr Ser Glu Glu Asp Asp Gly Tyr Lieu. Ile Phe Phe Val 485 490 495 His Asp Glu Ser Thr Gly Llys Ser Ala Val Asn Val Ile Asp Ala Lys SOO 505 51O Thir Met Ser Ala Asp Pro Val Ala Val Val Glu Lieu Pro Asn Arg Val 515 52O 525 Pro Tyr Gly Phe His Ala Phe Phe Val Thr Glu Glu Gln Leu Glu Glu 53 O 535 54 O Glin Ala Lys Lieu 5.45

<210s, SEQ ID NO 22 &211s LENGTH: 32O 212. TYPE: PRT <213> ORGANISM: Coffea canephora <4 OOs, SEQUENCE: 22 Met Ala Ser Ile Thr Ser Phe Asin Glin Phe Ser Tyr Thr Val Lys Ser 1. 5 1O 15 Lys Thr Phe Gln His Pro Glin Phe Gly Thr Llys Val Ser Asn Ser Ala 2O 25 3O Val Asin Phe Thr Asp Phe Gly Lieu Lys Llys Pro Leu Gln Ser Ser Ile 35 4 O 45 Ser Ile Lys Glu Ser Ser Llys Lys Arg Pro Gly Phe Val Val Lieu Val SO 55 6 O Ala Ala Gly Asp Asp Tyr Gly Pro Glu Glu Glu Ala Ala Gly Val Ala 65 70 7s 8O Val Ala Glu Glu Pro Pro Pro Lys Glu Pro Arg Glu Ile Asp Ile Lieu 85 90 95 Llys Lys Arg Lieu Val Asp Ser Phe Tyr Gly Thr Asp Arg Gly Lieu. Asn 1OO 105 11 O Ala Ser Ser Glu Thir Arg Ala Glu Val Val Glu Lieu. Ile Thr Glin Lieu 115 12 O 125 Glu Ala Lys Asn Pro Thr Pro Ala Pro Thr Glu Ala Lieu. Thir Lieu. Lieu. 13 O 135 14 O

Asn Gly Lys Trp Ile Leu Ala Tyr Thr Ser Phe Ile Gly Lieu. Phe Pro 145 150 155 160

Lieu. Lieu. Ser Arg Gly Thr Lieu Pro Lieu Val Llys Val Glu Glu Ile Ser 1.65 17O 17s

Gln Thr Ile Asp Ser Glu Ala Phe Ser Val Glu Asn Val Val Glin Phe 18O 185 19 O

Ala Gly Pro Leu Ala Thr Thr Ser Ile Thr Thr Asn Ala Lys Phe Glu 195 2OO 2O5

Val Arg Ser Pro Lys Arg Val Glin Ile Llys Phe Glu Glu Gly Val Ile 21 O 215 22O

Gly Thr Pro Gln Lieu. Thr Asp Ser Ile Glu Lieu Pro Glu Ser Val Glu 225 23 O 235 24 O US 8,252,977 B2 93 - Continued Lieu. Lieu. Gly Glin Lys Ile Asp Lieu. Asn Pro Wall Lys Gly Lieu. Lieu. Thir 245 250 255 Ser Val Glin Asp Thr Ala Ser Ser Val Ala Lys Ser Ile Ser Ser Arg 26 O 265 27 O Pro Pro Lieu Lys Phe Ser Lieu. Ser Asn Arg Asn Ala Glu Ser Trp Lieu. 27s 28O 285 Lieu. Thir Thr Tyr Lieu. Asp Asp Glu Lieu. Arg Ile Ser Arg Gly Asp Gly 29 O 295 3 OO Gly Ser Ile Phe Val Lieu. Ile Lys Glu Gly Cys Pro Lieu. Lieu Lys Pro 3. OS 310 315 32O

<210s, SEQ ID NO 23 &211s LENGTH: 382 212. TYPE: PRT <213> ORGANISM: Coffea canephora

<4 OOs, SEQUENCE: 23 Llys Ser Ala Phe Trp Ala His Cys Met Arg Arg Ala Val Ser Met Gly 1. 5 1O 15 Glin Arg Glu Met Val Asp Phe Met Asp Lieu. Lieu Lleu Ser Pro Ala Ser 2O 25 3O Llys Val Lieu. Asn. Asn Trp Phe Glu Thr Glu Val Lieu Lys Ala Thr Lieu. 35 4 O 45 Ala Thr Asp Ala Val Ile Gly. Thir Thr Ala Ser Val His Thr Pro Gly SO 55 6 O Thr Gly Tyr Val Lieu. Lieu. His His Ile Met Gly Glu Thr Asp Gly Asp 65 70 7s 8O Arg Gly Ile Trp Ser Tyr Val Glu Gly Gly Met Gly Ser Val Ser Leu 85 90 95 Ala Val Gly Ser Ala Ala Glin Glu Ala Gly Ala Thir Ile Val Thir Lys 1OO 105 11 O Ala Glu Val Ser Llys Lieu. Lieu. Ile Gly Asp Ser Gly Arg Val Asp Gly 115 12 O 125 Val Lieu. Leu Pro Asp Gly Thr Glu Val Glin Ser Ser Val Val Lieu. Ser 13 O 135 14 O Asn Ala Thr Pro Tyr Lys Thr Phe Met Glu Lieu Val Pro Glu. His Val 145 150 155 160 Lieu Pro Asp Asp Phe Lieu. Glin Ala Ile Lys Cys Ser Asp Tyr Ser Ser 1.65 17O 17s Ala Thir Thir Lys Ile Asn Lieu Ala Val Glu Arg Val Pro Glin Phe Glin 18O 185 19 O Cys Cys Lys Ile Asn His Pro Asn Ala Gly Pro Gln His Met Gly Thr 195 2OO 2O5

Ile His Ile Gly Ser Glu Arg Met Glu Glu Val Asp Ser Ala Cys Glin 21 O 215 22O

Glu Ala Val Asn Gly Phe Pro Ser Lys Arg Pro Ile Ile Glu Met Thr 225 23 O 235 24 O

Ile Pro Ser Val Lieu. Asp Llys Thr Ile Ser Pro His Gly Lys His Ile 245 250 255

Ile Asn Lieu Phe Ile Glin Tyr Thr Pro Tyr Llys Pro Lieu. Asp Gly Ser 26 O 265 27 O

Trp Glu Asp Pro Ala Tyr Arg Glu Ser Phe Ala Glin Arg Cys Phe Ser 27s 28O 285

Lieu. Ile Asp Asp Tyr Ala Pro Gly Phe Ser Ser Ser Ile Lieu. Gly Tyr 29 O 295 3 OO US 8,252,977 B2 95 96 - Continued Asp Met Luell Thr Pro Pro Asp Leu Glu Arg Glu Ile Gly Lieu. Thr Gly 3. OS 310 315

Gly Asn Ile Phe His Gly Ala Met Gly Lieu. Asp Ser Lell Phe Luell Met 3.25 330 335

Arg Pro Wall Lys Gly Trp Ser Asn Tyr Arg Thr Pro Wall Glin Gly Lieu. 34 O 345 35. O

Luell Cys Gly Ser Gly Ala His Pro Gly Gly Gly Wall Met Gly Ala 355 360 365

Ala Gly Arg Asn Ala Ala Gly Thr Wall Ile Glin Asp Trp 37 O 375 38O

SEQ ID NO 24 LENGTH: 156 TYPE : PRT ORGANISM: Coffea canephora

< 4 OOs SEQUENCE: 24

Glu Lieu. Lieu. Asp Glin Gly. His Glu Val Asp Ile Glu Ser His Ser 1. 1O 15

Phe Ile Gly Gly Llys Val Gly Ser Phe Val Asp Arg Gly Asn His 25

Ile Gly Met Gly Lieu. His Val Phe Phe Gly Cys Asn Asn Lieu. Phe 35 4 O 45

Arg Luell Met Llys Llys Val Gly Ala Asp Lys Asn Lell Lell Wall SO 55 6 O

His Thir His Thr Phe Val Asn Lys Gly Gly Glu Ile Gly Glu Lieu. Asp 65 70 8O

Phe Arg Phe Pro Val Gly Ala Pro Lieu. His Gly Ile Asn Ala Phe Lieu. 85 90 95

Ser Thir Asn Glin Lieu Lys Ile Tyr Asp Lys Ala Arg Asn Ala Wall Ala 105 11 O

Lell Ala Luell Gly Pro Val Val Arg Ala Lieu Wall Asp Pro Asp Gly Ala 115 12 O 125

Lell Arg Glu Ile Arg Asp Lieu. Asp Arg Ile Ser Phe Ser Asp Trp Phe 13 O 135 14 O

Lell Ser Gly Gly Thr Arg Ala Ser Ile Glin Arg 145 150 155

<210s, SEQ ID NO 25 &211s LENGTH: 1143 &212s. TYPE: DNA <213> ORGANISM: Coffea canephora

<4 OOs, SEQUENCE: 25 cc.cgggctgg taaagtaata gatgagataa ttagaaagta cagaggaata act citt catc. 6 O tggtctacaa gtacaagttt ttggataact gtc.ttatcta ttatat cittg gcatgitatgc 12 O citatgct cqt CCCtaatact ttgtgg tatt agt attagtt a 999999999 ggttctgaata 18O ttaaatacac at cataatgt ggaccattga caaaaggctic acttgcgtgc ctaaagtaaa 24 O attaagaaaa ttaa.gc.caaa gggcgatcct agittaactta actacct tag tagcct cact 3OO ttitt catc. ca. talacatttitt tttitt tttitt ttata attcC tcc cttgcac gatact caac 360 toalaccCaac CCaact Caac attittgctga gttittaatta agtatttgaa agaacaaagg

Caaaaaattic atcggaaata attittggtca ggtgttgttgttg tgattgttga gtagaatgaa

aattggctga tggggtttgg gcattatatt attatt atta ttatt at Cta 54 O aagcgc.gcta tattataggc tggggaaaga gagagaggto gttggaggat gatt.cgtgtc. US 8,252,977 B2 97 - Continued aaaggttgaa gaalaccatgt cc.ccc.gc.ccc CtaCCCCCaC alaggt caatg ctaattggca 660 aatcct coct tcgagcttct citct tcct ct to CCC Calaat titt coat tita tcaaacacgt. 72 O gggctt Cacc tacacgittag aggtggcctic Cat CCCCaCa ct tcc ct cita tatatactict citct cit cact c cott citt to CCCCCCtcaa. ggcacacaca Cactcaaatc. c to tact act 84 O c ct citataac cct ct c tectic toaaatct Ct citotic tect ca. aaaactaaaa. cattt Caaaa. 9 OO aaaaaaaaaa aaaaaaaaac to CCtactac tgccactgga cgacgacgac ttctact aca 96.O c tag tagt cc at attggaaa at Caatcaat cgcaccalacg cgataaagat agcgaaaaac to CCCCCCCC aaaaaaaaaa. agc.cagtatg atc cctdctt CCaCt CCtac aaatt Catat 108 O t catgggitta atccaaaatc c cc catgc cc atgac catga ttttgcctcc titcgt caaga 114 O gag 1143

<210s, SEQ ID NO 26 &211s LENGTH: 28 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: GWPSY1 primer <4 OOs, SEQUENCE: 26 actt caccgc agcigat cata agctt cac 28

<210s, SEQ ID NO 27 &211s LENGTH: 27 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: GWPSY2 primer <4 OOs, SEQUENCE: 27 ttcacgt.ccc aatcttct cq agat citc 27

<210s, SEQ ID NO 28 &211s LENGTH: 22 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: AP1 primer <4 OOs, SEQUENCE: 28 gtaatacgac toactatagg gC 22

<210s, SEQ ID NO 29 &211s LENGTH: 19 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: AP2 primer

<4 OOs, SEQUENCE: 29 actatagggc acgcgtggit 19

<210s, SEQ ID NO 3 O &211s LENGTH: 27 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: GWVDE1 primer

<4 OOs, SEQUENCE: 30 acatttctitt cqtgag actg. cacactic 27 US 8,252,977 B2 99 100 - Continued <210s, SEQ ID NO 31 &211s LENGTH: 28 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: GWVDE2 primer <4 OOs, SEQUENCE: 31 atcaccacat ttgatctggc attcagtic 28

<210s, SEQ ID NO 32 &211s LENGTH: 27 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: VDEFWR primer <4 OOs, SEQUENCE: 32 caccatggct tctgctittgc attcago 27

<210s, SEQ ID NO 33 &211s LENGTH: 22 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: VDEREV primer <4 OOs, SEQUENCE: 33 actacct tag citt cotaatt gg 22

<210s, SEQ ID NO 34 &211s LENGTH: 28 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: GWCCD11 primer <4 OOs, SEQUENCE: 34 aacaatccga acagoc cctt gagat coc 28

<210s, SEQ ID NO 35 &211s LENGTH: 28 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: GWCCD12 primer

<4 OOs, SEQUENCE: 35 gtttaa.gc.ct tdatgtctitc acgtaccg 28

<210s, SEQ ID NO 36 &211s LENGTH: 27 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: GWCCD13 primer

<4 OOs, SEQUENCE: 36 tgccaagtta citgttcaatg act aggc 27

<210s, SEQ ID NO 37 &211s LENGTH: 28 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: GWCCD14 primer US 8,252,977 B2 101 102 - Continued

<4 OO > SEQUENCE: 37 aagcaattta atc.ccgt.cct taatctgg 28

<210s, SEQ ID NO 38 &211s LENGTH: 26 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: CCD1FWR primer

<4 OOs, SEQUENCE: 38

Caccatgggt aggcaagaag gagaag 26

<210s, SEQ ID NO 39 &211s LENGTH: 22 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: CCD1REV primer

<4 OOs, SEQUENCE: 39 actict c cagg acatggtc.ca gC 22

<210s, SEQ ID NO 4 O &211s LENGTH: 27 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: GWNCED3F primer

<4 OOs, SEQUENCE: 4 O alagcagaa.gc agt cagggac ttctacc 27

<210s, SEQ ID NO 41 &211s LENGTH: 26 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: GWNCED3R primer

<4 OOs, SEQUENCE: 41 tatic cagtac accogaatctt gacacc 26

<210s, SEQ ID NO 42 &211s LENGTH: 28 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: NCED3FWR primer

<4 OOs, SEQUENCE: 42

Caccatgatg ggcttgggitt tdgttgc 28

<210s, SEQ ID NO 43 &211s LENGTH: 25 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: NCED3REV primer

<4 OOs, SEQUENCE: 43 t cacaagttt ctitt cagttc caggc 25 US 8,252,977 B2 103 104 - Continued <210s, SEQ ID NO 44 &211s LENGTH: 25 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: BCHYFWR primer <4 OOs, SEQUENCE: 44

Caccatggct gcc.ggaattig C cqtc 25

<210s, SEQ ID NO 45 &211s LENGTH: 22 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: BCHYREV primer <4 OOs, SEQUENCE: 45

Caagttgcgt aagggttcat aa 22

<210s, SEQ ID NO 46 &211s LENGTH: 22 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: DegPDS2 FWR primer

<4 OOs, SEQUENCE: 46 ggtggaaagir tagctgcatgga 22

<210s, SEQ ID NO 47 &211s LENGTH: 24 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: DegPDS2 REV primer

<4 OOs, SEQUENCE: 47 tgttacrgac atgtcago at acac 24

<210s, SEQ ID NO 48 &211s LENGTH: 7 212. TYPE: PRT <213> ORGANISM: Lycopersicon esculentum <4 OOs, SEQUENCE: 48 Gly Gly Llys Val Ala Ala Trp 1. 5

<210s, SEQ ID NO 49 &211s LENGTH: 8 212. TYPE: PRT <213> ORGANISM: Lycopersicon esculentum <4 OOs, SEQUENCE: 49 Val Tyr Ala Asp Met Ser Val Thr 1. 5

<210s, SEQ ID NO 50 &211s LENGTH: 27 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: GWPDS1 primer <4 OOs, SEQUENCE: 50 at cattgaat gct cottcca citgcaac 27 US 8,252,977 B2 105 106 - Continued <210s, SEQ ID NO 51 &211s LENGTH: 27 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: GWPDS2 primer <4 OOs, SEQUENCE: 51 t cattaattic ctagttct co aaa.cagg 27

<210s, SEQ ID NO 52 &211s LENGTH: 21 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: DegZDS1 FWR primer <4 OOs, SEQUENCE: 52 ttgcaggcat gtcgactgct g 21

<210s, SEQ ID NO 53 &211s LENGTH: 24 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: DegZDS3 REV primer

<4 OOs, SEQUENCE: 53 gtgggat.cct gttgcatatg Ctct 24

<210s, SEQ ID NO 54 &211s LENGTH: 8 212. TYPE: PRT <213> ORGANISM: Lycopersicon esculentum <4 OOs, SEQUENCE: 54 Lieu Ala Gly Met Ser Thr Ala Val 1. 5

<210s, SEQ ID NO 55 &211s LENGTH: 9 212. TYPE: PRT <213> ORGANISM: Lycopersicon esculentum

<4 OO > SEQUENCE: 55 Met Trp Asp Pro Val Ala Tyr Ala Lieu. 1. 5

<210s, SEQ ID NO 56 &211s LENGTH: 24 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 56 cccacctgga gcct ct attctgtt 24

<210s, SEQ ID NO 57 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OO > SEQUENCE: 57 cc.ccgt.cggc ct caagtttc US 8,252,977 B2 107 108 - Continued <210s, SEQ ID NO 58 &211s LENGTH: 23 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 58 tgatgaggca gagaaaggag ta 23

<210s, SEQ ID NO 59 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OO > SEQUENCE: 59 gatgcc cata caggc.cat ct

<210s, SEQ ID NO 60 &211s LENGTH: 14 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: PSY probe <4 OOs, SEQUENCE: 60 cgagct caac totg 14

<210s, SEQ ID NO 61 &211s LENGTH: 24 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 61 tggit aaccct C cagagagac tittg 24

<210s, SEQ ID NO 62 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: primer

<4 OOs, SEQUENCE: 62 tctgcct c ct cqtgacticaa

<210s, SEQ ID NO 63 &211s LENGTH: 17 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: PDS probe

<4 OOs, SEQUENCE: 63 atgc.cgattg ttgagca 17

<210s, SEQ ID NO 64 &211s LENGTH: 24 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: primer US 8,252,977 B2 109 110 - Continued

<4 OOs, SEQUENCE: 64 gctgataaaa atttgctcgt gaag 24

<210s, SEQ ID NO 65 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: primer

<4 OOs, SEQUENCE: 65 caccaatttic accc.cc tittg

<210s, SEQ ID NO 66 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: ZDS probe

<4 OOs, SEQUENCE: 66 at catactica cacatttgtt

<210s, SEQ ID NO 67 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: primer

<4 OO > SEQUENCE: 67 aaacggagag ccacct gatg

<210s, SEQ ID NO 68 &211s LENGTH: 24 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: primer

<4 OOs, SEQUENCE: 68 tgct caatct ttacaa.ccca ttt c 24

<210s, SEQ ID NO 69 &211s LENGTH: 19 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: PTOX probe

<4 OOs, SEQUENCE: 69 t catcc tota gtggtttgg 19

<210s, SEQ ID NO 70 &211s LENGTH: 18 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: primer

<4 OO > SEQUENCE: 7 O gcc.gcaa.gag aggaaacg 18 US 8,252,977 B2 111 112 - Continued <210s, SEQ ID NO 71 &211s LENGTH: 23 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 71 gcaaaataag togccaatcca aaa 23

<210s, SEQ ID NO 72 &211s LENGTH: 15 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: LeCY probe <4 OOs, SEQUENCE: 72 cagaga.gcat t ctitc 15

<210s, SEQ ID NO 73 &211s LENGTH: 16 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OO > SEQUENCE: 73 cgc.cgt.ccct gccata 16

<210s, SEQ ID NO 74 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 74 aatgaggc cc ttgttggaaga

<210s, SEQ ID NO 75 &211s LENGTH: 17 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: BCHY probe

<4 OO > SEQUENCE: 75 c cct cottt c titatggc 17

<210s, SEQ ID NO 76 &211s LENGTH: 21 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: primer

<4 OO > SEQUENCE: 76 ttggttctga Caaggctgca t 21

<210s, SEQ ID NO 77 &211s LENGTH: 18 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: primer US 8,252,977 B2 113 114 - Continued

<4 OO > SEQUENCE: 77 cgagaacggt ggctggitt 18

<210s, SEQ ID NO 78 &211s LENGTH: 14 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: ZEP probe

<4 OO > SEQUENCE: 78 ccgggtaaag gtca 14

<210s, SEQ ID NO 79 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: primer

<4 OO > SEQUENCE: 79 CCCCttgtcg agagattgga

<210s, SEQ ID NO 8O &211s LENGTH: 23 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: primer

<4 OOs, SEQUENCE: 80 acct coctita cqattgtc.ct titc 23

<210s, SEQ ID NO 81 &211s LENGTH: 17 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: VDE probe

<4 OOs, SEQUENCE: 81 alagacagtgg aagaagg 17

<210s, SEQ ID NO 82 &211s LENGTH: 21 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: primer

<4 OOs, SEQUENCE: 82

Cctaggacca ggalaggtttgg 21

<210s, SEQ ID NO 83 &211s LENGTH: 16 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: primer

<4 OOs, SEQUENCE: 83 cCaggctggc gtggaa 16 US 8,252,977 B2 115 116 - Continued SEQ ID NO 84 LENGTH: 14 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: CCD1 probe SEQUENCE: 84 cggaggct at Cttt 14

SEO ID NO 85 LENGTH: 24 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: primer SEQUENCE: 85 ggaaatcgga gct tagaatt gtca 24

SEQ ID NO 86 LENGTH: 22 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: primer SEQUENCE: 86 cagotgcact gatgcc ticta at 22

SEO ID NO 87 LENGTH: 14 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: NCED3 probe SEQUENCE: 87 cgc.catgaca ttgg 14

SEO ID NO 88 LENGTH: 21 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: primer

SEQUENCE: 88 ctgtc.cagga cacago atcc t 21

SEO ID NO 89 LENGTH: 21 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: primer

SEQUENCE: 89 t cagtggtgg toggctagaa a 21

SEO ID NO 9 O LENGTH: 13 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: FIB1 probe US 8,252,977 B2 117 118 - Continued <4 OOs, SEQUENCE: 90 agtc.gcaaag. tcc 13

SEQ ID NO 91 LENGTH: 21 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: primer <4 OOs, SEQUENCE: 91 gaac aggc cc atc cct tatt g 21

SEQ ID NO 92 LENGTH: 18 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: primer

< 4 OOs SEQUENCE: 92 cggcgcttgg caattgta 18

SEO ID NO 93 LENGTH: 16 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: RPL39 probe <4 OOs, SEQUENCE: 93 atgcgcactg acaa.ca 16

35 What is claimed: ghum, alfalfa, clover, canola, Safflower, Sunflower, peanut, 1. A nucleic acid molecule isolated from coffee (Coffea cacao, tomatillo, potato, pepper, eggplant, Sugar beet, carrot, spp.), having a coding sequence that encodes a phytoene cucumber, lettuce, pea, aster, begonia, chrysanthemum, del synthase, wherein the phytoene synthase has an amino acid phinium, Zinnia, and turfgrasses. 40 12. A fertile plant produced from the plant cell of claim 11. sequence at least about 85% identical to SEQID NO:13. 13. A method of modulating flavor or aroma of coffee 2. The nucleic acid molecule of claim 1, wherein the phy beans, comprising modulating production or activity of one toene synthase has an amino acid sequence at least about 90% or more phytoene synthase enzymes within coffee seeds, identical to SEQID NO:13. wherein the phytoene synthase has an amino acid sequence at 3. The nucleic acid molecule of claim 1, wherein the coding 45 least about 85% identical to SEQID NO:13. sequence is an open reading frame of a gene, or a mRNA 14. The method of claim 13, comprising increasing pro molecule, or a cDNA molecule. duction or activity of the one or more phytoene synthase 4. A vector comprising the coding sequence of the nucleic enzymes. acid molecule of claim 1. 15. The method of claim 13, comprising decreasing pro 5. The vector of claim 4, which is an expression vector 50 duction or activity of the one or more phytoene synthase selected from the group of vectors consisting of plasmid, enzymes. phagemid, cosmid, baculovirus, bacmid, bacterial, yeast and 16. The method of claim 14, comprising increasing expres viral vectors. sion of one or more endogenous genes encoding phytoene 6. The vector of claim 4, wherein the coding sequence of synthase enzymes within the coffee seeds. the nucleic acid molecule is operably linked to a constitutive 55 17. The method of claim 14, comprising introducing a promoter, or an inducible promoter, or a tissue-specific pro phytoene synthase-encoding transgene into the plant. moter. 18. The method of claim 15, comprising introducing a 7. The vector of claim 4, wherein the tissue specific pro nucleic acid molecule into the coffee that inhibits the expres moter is a coffee seed specific promoter. sion of one or more genes encoding phytoene synthase. 8. The vector of claim 7, wherein the coffee seed specific 60 19. The nucleic acid molecule of claim 1, wherein the promoter is a carotenoid or apocarotenoid gene promoter. phytoene synthase has an amino acid sequence at least about 9. The vector of claim 8, wherein the carotenoid or apoc 95% identical to SEQID NO:13. arotenoid gene promoter comprises SEQID NO:25. 20. The nucleic acid molecule of claim 13, wherein the 10. A host cell transformed with the vector of claim 4. phytoene synthase has an amino acid sequence at least about 11. The host cell of claim 10, which is a plant cell selected 65 from the group of plants consisting of coffee, tobacco, Ara 95% identical to SEQID NO:13. bidopsis, maize, wheat, rice, soybean barley, rye, oats, Sor k k k k k