US 201 10252501A1 (19) United StateS (12) IPatent Application IPublication (10) Pub. No?: US 2011/0252501A1 Abad etal. (43) IPub. Date: Oct. 13, 2011

(54) TRANSGENICPLANTSWITHENHANCED Savidge, Davis, CA (US): IDale L. AC ()N()MIC "I"|A|("I"S Val, Zamora, CA(US):Wei Zheng, Davis, CA (US): Thomas J. Savage, Sacramento, CA (US): (75) Inventors: Mark Abad, Webster Groves, MO) IRajani Monnanda Somaiah, St. (US):Molian Deng, Grover, MO Louis, MO) (US): Sireekanta Suma, (US):Sephen Duff St. Louis, MO Bangalore(IN): Jindong Sum, (US): Mary Hermandes, St. Louis, Urbana, IIL (US): Tymagondlu MO)(US):Karen K. Gabbert, St. Venkatesh, St. Louis, MC)(US): Louis, MC) (US): Julie A. Alvarez, Nanfei Xu, Cary, NC(US): Woodland, CA (US): Kristen A. Mahindra Amuradha, Bangalore IBennett, Davis, CA (US): IPaolo (IN): Alice Clara Augustine, Castiglioni, Davis, CA (US): Jill Karnataka (IN): IFarah Deeba, Deikman, Davis, CA (US): Jason Bangalore(IN): IRamachandra IFenner, Sacramento, CA (US): IDhanalakshmi, Karnataka (IN): IDanyang Ke, Sacramento, CA Savitha Madappa, Bangalore(IN): IBadami S.IPramesh, Karnataka (US): IBarry S. Goldman, St. (IN): M.S.IRajani, Karnataka (IN): Louis, MC) (US): Qungang Qi, G. IRamamoham, Karnataka (IN): Balwin, MO)(US): Thomas G. S. Sangeetha, Karnataka (IN): Ruff Wildwood, MO) (US): Char Shobha, Karnataka (IN): IRebecca L.Thompason-Mize, St. IPadmini Sudarshana, Bangalore Charles, MO)(US):JingruiWu, (IN): K. R. Vidva, Karnataka (IN): Chesterfield, MO) (US): Thomas R. Srikanth Venkatachalayya, Adams, Stonigton, CT (US): Karnataka (IN): Tymagondlu Robert Bensen, Niantic, CT(US): Venkatesh, St. Louis, MC)(US) Jacqueline IE. Heard, Stonington, ASSignee: MI()NSAN"I'() "I" (CHN()?()(Y CT(US): Donald E.Nelson, (73) Stonington, CT(US): Erin Bell, St. ILLC, ST LOUIS, MO (US) Louis,MO)(US): R.Eric Cerny, (21) Appl. No.: 11/893.915 Chesterfield, MO) (US): Jaishiree M. Filed: Aug. 17,2007 Chittoor-Vijayanath, Wildwood, (22) MO)(US): Bradon J. Fabbri, IRelated U.S.Application Data Chesterfield, MO) (US): (60) Provisional application No. 60/838.415. filed on Aug. IBalasulojini Karumamandaa, 17, 2006. Creve Coeur, MO) (US): John R. LeDeaux, Creve Coeur, MO)(US): IPublication Classification Xianfeng Chen, Christianburg, VA (51) (US): Meghan (2006.01) Galligan-Dommarummo. Westerly, (2006.01) RI(US):Deborah J.Hawkins, (2006.01) Davis, CA (US): Garrett J.Lee. (2006.01) Wayne, NJ(US): Obed Patty, (2006.01) Marysvile, CA(US): Rick A. (52) U.S. Cl...... 800/275:435/419:800/298:800/3201: Sanders, Davis, CA(US): Beth 800/314:800/3203:800/3202: 435/6.1

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plantSare Selected forhaving an enhanced traitSelected from IhiSinvention provideStransgenic plant Cells With recombi the group of traits ConSiSting of enhanced Water use efi Imant DNA for expression of proteins that are useful for ciency, enhanced Cold tolerance, increased yield, enhanced imparting enhanced agronomic trait(S) to transgenic Crop Imitrogenuse eficiency, enhanced Seed protein and enhanced plantS. Ihis invention alSo provides transgenic plants and Seed Oil. AlSo diSClOSed aremethods formanufacturing tranS progeny Seed CompriSing the transgenic plant Cells Where the genic Seed and plants With enhanced traitS. Patent Application Publication Oct. 13,2011 Sheet 1 of 6 US 2()11/()2525()I A1

Figure#1 Page1 of2 SEO ID INO : homo1.og SEQ ID NOs 15651 ------MTsssPTORTHVSIPPEPGGKSLTOEANEPPVPISVSSPCGKRTRDPEDE-V 21.36 FIFVILCKYCSRYCHESIVSCILELINSSGGKSITQEANEPPVPISVSSPCGKRTRDPEDE-V 3530 ------MTPASPIOGTHVSDPPEPRGKTITRESNEAALPISDSSACGNRTIDAEDD-V 1.3661 ------MGPE:SRSTPISASSPASSSSPSGKIRTRIDPEDDEV 25924 ------MGSDSINILGGSPSASSPSAKRTRIDPEEE-V 1.5894 ------MGSDSNILGCSPSASSPSAKRTRIDPEEE-V 16409 ------MGFIDSNILGGSPSSSSPS SKRI'RDPEEE-V 7383 ------MGFDSNILGGSPSSSSPTAKRTRIDPEEE-V 1.6880) ------MVGGGGGGEAAMSPPS------GCGKRGRIDPIEED-V 1824 ------MAGGSGEGAAMSPPSSSGVGGGGGGGKIRGRIDPEED-V 561 ------MAGGSGIEAAMSPIPS SS------S(KRCRIDPEED-V COn Sern S11S ------xxxxxxxxxxxxxxxxxxxxSXSSXXXkRt.1:DOEXX-V 30 328

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"I"|ANS(NIC IPIANTIS \WITI"H INIHANCII) from a population are required toidentity thOSetransgenic AC ()N()MIC "I AIS eventSthatarecharacterized bythe enhancedagronomictrait. SUMMARY (DF "I"HH INVIENTII(DN CRC)SS RFIFFIRINCF "I"() RFIAIHID) APPI,ICAII(DNS |0007|| This invention provides plant cell nuclei with recombinant IDNA thatimpartSenhanced agronomic traitSin [0001] This application claimsbenefit under 35USCS119 transgenic plants having the Inuclei in their CellS, e.g. (e) of U.S.provisional application Ser No. 60/838.415. filed enhanced Water use eficiency, enhanced Cold tolerance, Aug. 17,2006. herein incorporated by reference. increased yield, enhanced Initrogen use efficiency, enhanced seed proteinorenhanced Seed oil. Such recombinant DNA in Incorporation Of Sequence Listing a plant Cell InucleuS Of this invention iSprovided in as a ConStruct CompriSing a promoter thatis functional in plant |0002| Two copies of the Sequence listing (Copy 1 and cells and thatis operably linked to DNA that encodes a Copy2) anda computerreadableform(CRF) ofthe sequence protein. Such DNA in the constructis Sometimes defined by listing, all On CD-RS, each Containing the text file Inamed protein domains of an encoded protein targeted for produc 38-21(54146)B_seqListing.txt, which is 103.067,648 bytes tion OrSuppreSSion, e.g.a"Piam domain module”(as defined (measured in MS-WINDOWS), were created on Aug. 16. hereinbelow) from the group of Pfamdomain modulesiden 2007and are herein incorporated by reference. tified in Table 9. Alternatively, e?g. Wherea Pfam domain Imoduleismotavailable, Such IDNA in the constructis defined Incorporation Of Computer Program Listing a ConSenSuSamino acid Sequence Ofan encoded protein that is targeted for production e.g. a protein having amino acid |0003|| Two copies ofthe Computer Program Listing(Copy Sequence Withatleast 90%identity to a ConSenSuSaminoacid 1 and Copy2) and a computerreadable form (CRF) contain sequence in the group of SEQ IDNO: 30328, and SEQ ID ing foldershmmer-2.3.2 and 248pfamiDir, all on CD-RSare NO: 30377 through SEQ IDNO: 30418. Alternatively, in incorporated herein by reference in their entirety. Folder Other CaSeSWhere Ineither a Piam domain module InOra Con hmmer-2.3.2 Contains the Source Code and Other aSSOciated SenSuSamino acid Sequence is available, Such IDNA in the fileforimplementingthe HMMersoftwarefor Pfamanalysis. ConStruct is defined by the Sequence of a Specific encoded Folder248pfamiDircontains248Pfam Hidden Markov Mod and/Orits homologouSproteimS. els, Both folders Were created on CD-R on Aug, 16, 2007. |[0008|| Other aspects of the invention are specifically having a total size of 20.594.688 bytes (measured in MS directed to transgenic plant Cells CompriSing therecombinant WINDOWS) IDNA Ofthe invention, transgenic plants CompriSing a plural ity Of Such plant Cells, progeny transgenic Seed, embryo and Incorporation ofTable transgenic pollen from Such plantS. Such plant Cells are Selected from a population oftransgenic plants regenerated |0004|| Two copies of Table 9 (Copy 1 and Copy 2) and a fromplant cellstransformed with recombinant DNA and that computerreadable form (CRF), al on CD-RS, each contain expreSS the protein by Screening transgenic plants in the ing the file named 38-21(54146)B_table9.txt, Which is 319. populationforamenhanceditraitaScompared to Controliplants 488 bytes (measured in MS-WINDOWS), were created on that do Inothave Said recombinant IDNA, Where the enhanced Aug. 16. 2007, and comprise 74 pages when Viewed in MS trait iS Selected from group ofenhanced traits ConSiSting of Word, are herein incorporated by reference. enhanced Water use eficiency, enhanced Cold tolerance, increased yield, enhanced Initrogen use efficiency, enhanced FIFII) (DF "I"HH INVIENTII(DN Seed protein and enhanced Seed Oil. |0009|| Inyetanotheraspect oftheinventiontheplant cells. |0005| Disclosed herein areinventionsin the field ofplant plants, Seeds, embryo and pollen further comprise DNA genetics and developmental biology More Specifically, the expreSSing a protein thatprovideStolerance from expOSure to presentinventionsprovideplant cellswithrecombinant DNA an herbicide applied at levels thatarelethal to a Wild type of for providing an enhanced trait in a transgenic plant, plants Said plant Cell. Such toleranceis eSpeciallyuSeful Inot Onlyas CompriSing Such Cells, Seed and pollen derived from Such an advantageous trait in Such plants but is alSo uSeful in a plants, methods ofmakingand uSing Such CellS, plants, SeedS Selection Step in the methods of the invention. In aspects of and pollem. the invention the agent of Such herbicide is a glyphOSate, dicamba, OrglufOSinate Compound. IBACKCRC)UNI) (DF "I"HH INVIENTII(DN |0010|| Yetotheraspects oftheinventionprovidetransgenic plants whichare homozygous forthe recombinant DNA and |0006|| Transgenic plants with improved agronomic traits transgenic Seed of the invention from Corn, Soybean, Cotton, Such aSyield, enVirommental StreSS tolerance, peStreSiStance, Canola, alialfa, Wheat OrriceplantS. herbicidetolerance.improved Seed CompOSitionS, andthelike |0011] This inventionalsoprovides methods formanufac are deSired byboth farmerSand ConSumerS. Although ConSid turing InOn-natural, transgenic Seed that Can be uSed to pro erable efforts in plant breeding have provided Significant duce a Crop oftransgenic plants With an enhanced traitreSult gains in desired traits, the ability to introduce Specific DNA ing from expreSSion of Stably-integrated, recombinant IDNA intoplantgenomeSprovides furtheropportunities forgenera in thenucleus oftheplant cells.More specificallythemethod tion of plants with improved and/or unique traits. Merely CompriSeS (a) Screening a population Of plants for an introducing recombinant IDNA into a plant genome doesn't enhanced trait and recombinant IDNA, Where individual alwaySproduce a transgenic plant With an enhanced agro plantSin thepopulation CanexhibitthetraitatalevelleSSthan, Inomic trait. Methods to Select individual transgenic events essentiallythe Sameas orgreaterthan thelevelthatthetraitis US 201 1/0252501 A1 Oct. 13, 2011

exhibited in control plants Which do not express the recom IDNA imparting an enhanced trait in plant Cells, i.e. each binant DNA: (b) Selecting from the population one or more repreSents a Coding Sequence for a protein: plants thatexhibit thetraitatalevelgreaterthan thelevel that 10017 SEQIDNO: 359-716areaminoacid sequences of Said traitis exhibited in ControlplantSand (c) Collecting Seed the Cognate protein of the ‘genes” With nucleotide Coding froma Selected plant. Such method further comprises steps sequences 1-358: (di)verifyingthattherecombinant DNAlisstablyintegratedin 10018|| SEQ IDNO: 717-30327areamino acid sequences Said Selected plants: and (e) analyZing tiSSue of a Selected OflhomologouSproteins: plant to determine the production of a protein having the 10019|| SEQIDNO: 30328isaconsensussequence ofSEQ function ofa protein encoded by a recombinant DNA with a IDNO: 561 and its homologs: sequence ofone of SEQ IDNO: 1-358: In oneaspect ofthe 10020|| SEQIDNO: 30329is thenucleotidesequence ofa invention theplants in thepopulation further comprise DNA plasmid basevectorpMON93039 useful forcorntransforma expreSSing a protein that provideStolerance to expOSure to an tion: herbicide applied at levels that are lethal to Wild typeplant 10021] SEQIDNO: 30330is thenucleotidesequence ofa CellSand Where the Selectingis effected by treating thepopu plasmid basevectorpMON92705useful forcorntransforma lation With the herbicide, e.g.a glyphOSate, dicamba, Orglu tion: foSinate Compound. In another aspect of the invention the 10022| SEQIDNO: 30331 is thenucleotidesequence ofa transgenic plants areSelected by identifying plants With the plasmid base vector pMON82053 useful for Soybean and enhanced trait. The methodSare eSpecially uSeful formanu Canola transformation: facturing Corn, Soybean, Cotton, alialia, Wheat Or rice Seed [0023|| SEQ ID NO: 30332-30375 are nucleotide selectedashavingone oftheenhanced traits describedabove. Sequences of the regulatory elementSin base Vectors: |0012| Another aspect ofthe invention provides a method 10024|| SEQIDNO: 30376is thenucleotidesequence ofa Of producing hybrid Corn Seed CompriSing acquiring hybrid plasmid basevectorpMON99053 useful for cotton transfor corn Seed fromaherbicide tolerant cormplant whichalsohas Stably-integrated, recombinant IDNA CompriSing a promoter Imation: and SEQ ID NO: 30377-30418 are consensus thatis(a) functionalinplant cells and(b)isoperablylinked to SequlenCeS. DNA thatencodes a protein. Such proteinis defined by pro 10025 Table 11ists the protein SEQ IDNosand theircor teindomainS Ofan encoded proteintargeted forproduction Or responding consensus SEQ IID Nos. SuppreSSion, e.g.a"Piam domain module”(as defined herein below) fromthegroup of Pfamdomain modulesidentifiedin "IABI, 1 Table 9. Alternatively, e.g. Wherea Pfam domain moduleis PEP COnSenSUS Imot available, Such protein is defined by a ConSenSuSamino SEQID SEQID acid Sequence Ofan encoded protein thatiStargeted forpro N() Ceme IID) N() duction e.g. a protein having amino acid Sequence With at 371 PHE0002860_7494 30377 least 90%, identity to a ConSenSuSamino acid Sequencein the 372 PHE0002860_8694 30378 groupofSEQIDNO:30328, and SEQIDNO: 30377through 378 PHE0004013_9281 30379 401 PHE0004780_5752 30380 SEQIDNO: 30418. Alternatively, inother cases wheremei 402 PHE0004782_5754 30381 ther a Pfam domain module Inor a ConSenSuSamino acid 420 PHE0004859_5896 30382 sequenceisavailable, Such DNA intheconstructis defined by 421 PHE0004859_5917 30383 the Sequence Of a Specific encoded and/or its homologous 427 PHE0004889_7961 30384 436 PHE0004903_5960 30385 proteimS. Ihe methods further Comprise producing Corn 446 PHE0004948_6003 30386 plants from Said hybrid corn Seed, Wherein a fraction ofthe 470 PHE0006047_7234 30387 plants produced from Said hybrid Corn Seed is homOZygous 471 PHE0006047_8766 30388 for Said recombinant DNA, a fraction oftheplantsproduced 474 PHE0006049_7107 30389 480 PHE0006062_7058 30390 from Said hybrid Corn Seed is hemiZygous for Said recombi 485 PHE0006072_7071 30391 Imant DNA, and a fraction of the plants produced from Said 486 PHE0006074_7060 30392 hybrid corn Seed has mone of Said recombinant DNA: Select 487 PHE0006076_7052 30393 ing Corn plants Which are homOZygouSand hemiZygous for 488 PHE0006076_7331 30394 514 PHE0006176_7147 30395 SaidrecombinantDNAbytreating withanherbicide:collect 544 PHE0006286_7314 30396 ing Seed from herbicide-treated-SurViving Corn plants and 545 PHE0006286_8011 30397 planting Said Seed to produce further progeny Corn plants: 546 PHE0006288_7310 30398 repeating the Selecting and Collecting Steps at least Once to 547 PHE0006288_8023 30399 558 PHE0006346_8132 30400 produce aninbred Cornline: and CrOSSing theinbred Corn line 561 PHE0006351_8200 30328 with a Second corn linetoproduce hybrid Seed. 562 PHE0006353_8098 30401 563 PHE0006355_8084 30402 IBRIFF IDFSCIRIPTII(DN (DF "I"HH IDRAWINCS 567 PHE0006378_7667 30403 568 PHE0006378_8715 30404 615 PHE0006593_8245 30405 |0013|| FIG. 1 is a consensus aminoacid Sequence of SEQ 616 PHE0006593_8256 30406 IDNO: 561 and its homologs. 654 PHE0006740_8446 30407 |0014) FIGS. 2-5areplasmid maps. 655 PHE0006740_8596 30408 679 PHE0006816_8560 30409 680 PHE0006844_8839 304.10 IDFTIAIIHI) IDFSCIRIPTII(DN (DF "I"HH INVIENTII(DN 683 PHE0006908_9016 304.11 699 PHE0006941_9117 30412 |0015| In theattached Sequence listing: 707 PHE0006954_9154 304.13 [0016|| SEQ IDNO:1-358 arenucleotide sequences ofthe 708 PHE0006954_9161 30414 coding Strand of IDNA for ‘genes” used in the recombinant US 201 1/0252501 A1 Oct. 13, 2011

find Similar Sequences, and the Summary Expectation Value IABLE 1-continued (E-Value) uSed to meaSure the Sequence base Similarity ASa proteinhitWithithebestE-ValuleforaparticularorganiSmmay PEP COnSenSUIS SEQID SEQID Imot IneceSSarily bean Ortholog Or the Only Ortholog, a recip N() Ceme IID) N() rocal query iS uSed in the preSent invention to filter hit Sequences With Significant E-Values for Orthologidentifica 712 PHE0006970_9141 304.15 714 PHE0006986_9183 30416 tion."Ilhereciprocal queryentails Search ofthe Significanthits 715 PHE0006992_9140 304.17 against a database Of amino acid Sequences from the base 716 PHE0006992_9184 304.18 Organism thatare Similarto the Sequence ofthe queryprotein. Ahitisalikely ortholog.whenthereciprocal query’s besthit |0026|| As used hereina ‘plant cell’meansaplant cell that iSthe queryproteinitSelforaprotein encoded bya duplicated is transformed With Stably-integrated, InOn-natural, recombi gene afier Specification. A further aspect of the invention Imant IDNA, e.g. by 4grohoicierin-mediated transformation Comprises functional homolog proteins that differ in One Or Orby bombardiment uSing microparticles Coated With recom ImoreaminoacidsfromithOSeofdisclosed proteinaSthereSult binant DNAorothermeans. Aplant cell ofthisinvention can OfconSerVative aminoacid SubstitutionS, forexample Substi be an Originally-transformed plant Cell thatexistSaSamicro tutions areamong: acidic (negatively Charged) amino acids Organism Oras a progeny plant Cell thatis regenerated into Such as aspartic acid and glutamic acid: baSic (pOSitively differentiated tiSSue, e.g. into a transgenic plant With Stably Charged) aminoacidSSuch aSarginine, histidine, and lySine: integrated, InOn-natural recombinant IDNA, Or Seed Orpollen Ineutral polaramino acidSSuch as glycine, Serine, threonine, derived from a progeny transgenic plant. CySteine, tyroSine, asparagine, and glutamine:Ineutral InOnpo |0027 As used herein a ‘transgenic plant” means a plant WhOSegenome haSbeen altered by the Stable integration of lar (hydrophobic) amino acids Such as alanine, leucine, iSO recombinant IDNA. Atransgenic plantincludeSaplantregen leucine, Valine, proline, phenylalanine, tryptophan, and erated froman Originally-transformed plant Celland progeny Imethionine:amino acidShavingaliphatic Side ChainSSuch as transgenic plants from latergenerationS Or CroSSeS Ofa tranS glycine, alanine, Valine, leucine, and iSoleucine:aminoacids formed plant. having aliphatic-hydroxyl Side Chains Such as Serine and |0028|| As used herein “recombinant DNA” means DNA threonine:amino acidShaving amide-containing Side Chains Which has been a genetically engineered and ConStructed Such aSasparagine and glutamine; amino acidShaving aro outside ofacell including DNA containing maturally occur Imatic Side ChainSSuch aSphenylalanine, tyrOSine, and tryp ring DNA orcDNAor synthetic DNA. tophan:amino acidShaving baSic Side ChainSSuch as lySine, |0029|| Asusedherein‘consensussequence’meansanarti arginine, and histidine:amino acidShaving Sulfur-Containing ficial Sequence Ofamino acids in a ConSerVed region Of an Side ChainSSuch aS CySteine and methionine; Imaturally Con alignment Ofamino acid Sequences of homologouSproteimS. Servative amino acids Such aS Valine-leucine, Valine-isoleu e?g.as determined bya CLUSTALWalignmentofaminoacid Cine, phenylalanine-tyroSine, lySine-arginine, alanine-Valine, Sequence of homolog proteimS. aspartic acid-glutamic acid, and asparagine-glutamine.A fur |0030]] As used herein “homolog” means a protein in a ther aspect of the homologs encoded by DNA useful in the group of proteins that perform the Same biological function, transgenic plantSoftheinventionarethOSeproteinSthat differ e.g. proteinSthatbelong to the Same Pfamproteinfamily and froma disclosed protein as the result of deletion orinsertion that provide a Common enhanced traitin transgenic plants of Of One Ormoreamino acidSin a Imative Sequence. this invention. Homologs are expreSSed by homologous geneS. HomologouSgeneSinclude Imaturally Occurring alleles |0032| As used herein, ‘percentidentity”means the extent and artificially-Created VariantS. IDegeneracy of the genetic to Which tWo Optimallyaligned IDNA Orprotein SegmentSare codeprovides thepossibility to Substituteatleastone base of invariant throughout a Window Ofalignment of ComponentS. theprotein encoding Sequence Ofagene With a differentbase forexamplenucleotide Sequence Oraminoacid Sequence. An Without CauSing the amino acid Sequence ofthe polypeptide “identity fraction"foralignedSegments OfatestSequenceand produced from the gene to be Changed. Hence, a polynucle a reference Sequenceis the Inumber Ofidentical Components Otide uSeful in the present invention Imay have any base that are Shared by Sequences of the tWo aligned Segments sequence thathas beenchanged from SEQ IIDNO:1 through divided by the total Inumber Of Sequence Components in the SEQIDNO:358substitutioninaccordance with degeneracy reference Segment OVer a Window Ofalignment Whichis the Of the genetic Code. HomologSareproteins that, When Opti Smaller of the ful test Sequence or the ful reference Imally aligned, have at least 60%, identity, more preferably sequence. ‘Percent identity”(“%, identity”)is the identity about 70% or higher, morepreferably at least 80%, and even fraction times 100 Imorepreferablyatleast 90%identity overthe full length ofa |0033|| The ‘Pfam’database is a large collection of mul protein identified as being aSSociated With imparting an tiple Sequence alignmentSand hidden MarkOVImodels Cover enhanced trait When expreSSed in plant CellS. Homologs ing Imany Common protein families, e.g. Pfam Version 19.0 includeproteins With anamino acid Sequence thathaSatleast (December 2005) containsalignments and models for 8183 90%, identity to a ConSenSuSamino acid Sequence Ofproteins protein families and is based on the Swissprot 47.0 and SP and homologs disclosed hereim. TrEMIBL 300 protein Sequence databases. See S. R. Eddy, |0031] Homologsarebeidentified by comparison ofamino ‘Profile Hidden Markov Models", Bioin/inicy 14:755 acid Sequence, e.g. Imanually Orby use of a Computer-based 763. 1998. The Pfam database is currently maintained and tooluSing knownhomology-based Search algorithmSSuch as updated by the Pfam Consortium. Thealignments represent those commonly known and referred to as BLAST IFASTA, SOme eVolutionary ConSerVed Structure thathaSimplications and Smith-Waterman. Alocal Sequence alignment program, for the protein's function. Profile hidden Markov models e.g. BLASI Cambeused to Search a database OfSequenceSto (profile HMMs)built from the protein family alignments are US 201 1/0252501 A1 Oct. 13, 2011

uSeful for automatically recognizing that a Inew protein LRR_1:LRR_1:LRR_1, Bromodomain, IDUF1365. belongSto an existing proteinfamily eVenifthehomology by PTS_2-RNA, Pkinase:UBA:KA1, MATH:BTB, DUF6: alignmentappearSto be loW. TPT Cyclin_N:Cyclin_C, Zf-AN1, Methyltransf_6. |0034|| A “Pfam domain module’is a representation of Thioredoxin, DNA_photolyase:IFAID_binding_7, VATP Pfam domains in a protein, in order from N terminus to C Synt_E. Bac_globin, B_lectin:S_locuS_glycop:PAN_2: terminuS.Ima Piamdomainmoduleindividual Piamdomains Pkinase_Tyr, Sigma70_r2:Sigma70_r3:Sigma70_r4. Ribo areseparated by double colons ‘:”. The order and copy somal_L10, Zf-C3HC4:WD40:WD40:WD40, IPGM_ number of the Piam domains from N to C terminus are PMM_I:PGM_PMM_II:PGM_PMM_II:PGM_PMM_IV attributes ofa Pfamdomain module. Althoughthe copynum Hydrolase, Peptidase_C1, IDS, Carotene_hydrox, Aa_trans. ber Ofrepetitive domainSiSimportant, Varying Copy Inumber Mov34, Zf-MYND:UCH, Heme_oxygenase, S6PP SSB, ofiemenables a Similarfunction."Ilhus, a Piamdomainmodule Peptidase_M16:Peptidase_M16_C, Bet_V_I, Auxin_induc With multiple Copies of a domain Should define an equivalent ible, Response_reg, Di19, DUF125, GDC-P Pyr_redox_2: Pfamdomain module with varianceinthenumberofmultiple Fer2_BFD:NIR_SIR_ferr:NIR_SIR. KOW:eIF-5a. CopieS. A Pfam domain module iS Inot Specific for distance MtN3_slv:MtN3_slv, Ribul_P_3_epim, NPH3, DnaJ: betWeen adjacent domains, but Contemplates matural dis DnaJ_C, UQ_con, RRM_1:RRM_1:RRM_1, F-box, tanceSand VariationSindistance thatprovide equivalentfunc COA_binding:Ligase_COA, adh_Short, RibOSomal_L22. tion.The Pfamdatabasecontainsbothinarrowly-andbroadly AA_permease, Acyltransferase, AMPKBI, RRM_1, Chal defined domainS, leading toidentification of OVerlapping cone, GAIase_2:ASn_synthase, Peptidase_M24. DUF498. domains On SOme proteimS. A Pfam domain module iS Char DAGAT PFK, DUF1677, Glyco_transf_43, Zf-DNIL, acterized by mon-overlapping domains. Where thereis over DHBP_synthase:GTP_cyclohydro-2, PseudoU_synth_2, lap, the domain having a function thatis more Closely aSSO Glyoxalase, DUF21:CBS, Ribosomal_S30AE, Glycolytic, ciatedwith thefunction oftheprotein(basedonthe Evalue of Chloroa_b-bind, 2F-HD_dimer, Usp. Ferrochelatase, Pyri the Pfam Imatch)is Selected. doxal_deC, Glyco_transf_8. Pyr_redox_2:Glutaredoxin, |0035| Once one DNA isidentified as encoding a protein Epimerase, UPF0113, RNase_PH.AIG1, Phi_1, CorA, HD: WhichimpartSamenhanced traitWhen expreSSedintransgenic RelA_SpoT P-II, GSHIPx, PGAM, PGI, DUF868, Lung_7- plants, other DNA encoding proteins with the Same Pfam TM_R,F-box:FBA_1, TPP_enzyme_N:TPP_enzyme_M: domain module areidentified by Cluerying the amino acid TPP_enzyme_C, DnaJ:Zf-CSL, DEAD:Helicase_C, 2OG Sequence of protein encoded by Candidate IDNA against the FeII_Oxy HMGIL-like:LeuA_dimer VQ, DUF298.DREPP. Hidden MarkOV Models Which characterizes the Pfam ketoacyl-synt:Keloacyl-synt_C, THF_IDHG_CYH:THIF_ domainsusing HMMER Software.acurrent version of which DHG_CYH_C, DNA_pol_E_B, UPF0051, Pkinase:ethan iSprovided in the appended Computerlisting. Candidatepro d::efthand::ethand::efthand, Imalic:Malic_M, ThiF."Iransket_ teins meeting the Same Pfam domain modulearein the pro pyr:Transketolase_C, Ribosomal_L37ae, PEPcase, Glyco_ teinfamilyandhavecognate DNA thatis useful in construct hydro_32N:Glyco_hydro_32C. GASA, IDImaJ. ing recombinant DNA for the use in the plant cels of this AA_kinase:ACT:ACT Plkinase_Tyr, Cupin_1, Zf-LSD1: invention. Hidden MarkoV Model databases for use With Zf-LSD1:Zf-LSD1, Cupin_3, GAF:HisKA:HATPase_c: HMMER Software in identifying DNA expressing protein ReSpOnSe_reg, Methyltransf_12:Mg-por_mtran_C. With a common Piam domain moduleforrecombinant IDNA DUF516, IPTR2, Ammonium_transp, eIF-5a, ECH, Aldedih, in theplant cels of this invention arealso included in the Zf-C3HC4. SAM_decarbox, X8. Mg_chelatase, PurA, Ribo appended Computer listing. Somal_S6e. Molybdop_Fe4S4:Molybdopterin:Molydop_ |0036|| Version 19.0 of the HMMER Software and Pfam binding, CP12, Biotin_lipoyl:E3_binding:2-oxoacid_dh, databases Were uSed toidentity known domains in the pro NOI, Tubulin:Tubulin_C, V-SNARE, AP2, ELFV_dehy teins corresponding toamino acid Sequence of SEQ IDINC): drog_N:ELFV_dehydrog, Ribosomal_L32e, and FAID_ 359 through SEQ IDNO: 716. All DNA encoding proteins binding_3. thathave SCOreShigherthan the gathering CutOffdisclosed in |0037|| As used herein‘promoter”means regulatory DNA Table 16 by Pfam analysis disclosed herein can be used in forinitializingtranScription.A"plantpromoter”iSapromoter recombinant DNA ofthe plant cells ofthisinvention, e?g. for Capable ofinitiatingtranScriptioninplanticellSWhetherormot Selectingtransgenic plantShavingenhancedagronomictraitS. its Originis a plant Cell, e.g. iSit Well known that4grohicie Iherelevant Piams modules foruseinthisinvention, asmore 7in promotersarefunctionalin plant CellS.ThuS, plant pro specifically disclosed below, are Gp_dh_N:Grp_dh_C. Imoters includepromoter DNA obitained fromplants, plant Mg_chelatase:VWA, Zf-CCCH:Zif-CCCH:Zf-CCCH:Zif ViruSeSand bacteria Such aS 4grohicierin and Liroir/lizo CCCH:zf-CCCH, WD40, tRNA-synt_2b:HGTP_antic ?)in bacteria. Examples ofpromoters under developmental odon, RNase_PH:RNase_PH_C, F-box:Kelch_1:Kelch_ control include promoters that preferentially initiate tran 1. Peptidase_C54. Iso_dh, Metallophos, OTU, Rotamase. Scription in Certain tiSSules, Such as leaveS, roots, Or SeedS. Sugar_tr, Glyoxalase:Glyoxalase, Ras, Brix, S6PP:S6PP_ Suchpromotersarereferred toas ‘tissuepreferred”. Promot C, PsbR, Pkinase.p450, PP2C, CH:EB1, DUF537, Histone, ers that initiate tranScription Only in Certain tiSSules are PPR:PPR:PPR:PPR:PPR, TFIS_M:TFIS_. DUF751. referred to as ‘tissue Specific’. A ‘cell type’specific pro RRM_1:RRM_1, ETC_C1_NDUIFA4. SRF-TF. CCT. Imoterprimarilydrives expreSSionin Certain Cell typeSin One Globin:FAD_binding_6:NAD_binding_1, FAE1_CUT1 Ormore OrganS, forexample, VaScularcellSin roots OrleaveS. RippA:ACP_syn_I_C, Frataxim_Cyay, F-box:LRR_2, An“inducible’or“repressible’promoterisapromoterwhich Tryp_alpha_amyl, PFK:PFK, Dehydrin, RLI:Fer4:ABC_ iS under enVirommental Control. EXamples ofenVirommental tran:ABC_tran, CTP_transf_2, GTP_EFTU:GTP_EFTU_ ConditionSthatmayeffect tranScription byinduciblepromot D2:GTP_EFTU_ID3, PfkB, IPT TPR_1:TPR_2:TPR_ erSinclude anaerobic ConditionS, Orcertain ChemicalS, Orthe 1:TPR_2:TPR_1:TPR_1:TPR_1:TPR_1:TPR_1, presence Oflight. IISSue Specific, tiSSue preferred, Cell type Globin, Porphobil_deam:Porphobil_deamC, NB-ARC: Specific, andinduciblepromoterSCOnStitute the ClaSS Of"non US 201 1/0252501 A1 Oct. 13, 2011 constitutive’promoters. A ‘constitutive’promoteris a pro invention CanalSobeused toprovideplantShavingimproved Imoter Which iSactive undermiOSt ConditionS. growth and development, and ultimately increased yield, as |0038|| As used herein “operably linked” means theasso thereSult ofmodifiedexpreSSion ofplantgrowthregulatorS Or ciation oftwoormore DNAfragments ina DNA constructso Imodification of Cell Cycle OrphotoSyntheSiSpathWayS. AlSO that the function of One, e.g. protein-encoding IDNA, iS Con OfintereStis the generation oftransgenic plants that demon trolled by the Other, e.g.a promoter. Strate enhanced yield With reSpect to a Seed Component that |0039|| As used herein “expressed”means produced, e?g.a Imay Or Imay Inot Correspond to an increase in OVeral plant protein is expreSSed in a plant Cell Whenits Cognate IDNA iS yield. Such propertieSinclude enhancementSin Seed Oil, Seed transcribed to mRNA thatistranslated to the protein. Imolecules Such as tocopherol, protein and Starch, Or Oil par |0040|| As used hereina ‘control plant”means a plant that ticularoil ComponentSaSImaybe ImanifeStbyanalterationSin does not contain the recombinant DNA thatexpressed a pro the ratiOS OfSeed ComponentS. teinthatimpartamenhanced trait.A ControliplantiStoidentify |0043|| A Subset ofthe nucleic molecules ofthis invention and Select a transgenic plant that has an enhance trait. A includes fragments ofthe disclosed recombinant DNA con Suitable Control plant Can be a InOn-transgenic plant of the sisting ofoligonucleotides ofatleast 15.preferablyatleast 16 parentallineused togenerateatransgeniciplant, i.e.deVoid of or 17, more preferably at least 18 or 19. and even more recombinant DNA. A Suitable control plant may in Some preferablyat least 20ormore.consecutive nucleotides.Such CaSeS be a progeny OfahemizygouStransgenic plant line that OligonucleotideSarefragmentSofthelargermoleculeShaving is does Inot Contain the recombinant IDNA, known as a mega a Sequence Selected from the group ConSiSting of SEQ IID tive Segregant. NO:1 through SEQIDNO: 358, andifinduse, forexample as |0041|| AS used herein an ‘enhanced trait”means a charac probes andprimers fordetection ofthepolynucleotides ofthe teristic Ofatransgeniciplantithatincludes.butiSnotlimited to. presentinvention. an enhance agronomic trait Characterized by enhanced plant |0044|| DNA constructsareassembled using methods well Imorphology, physiology, growth and development, yield, known toperSons of Ordinary Skill in the art and typically Imultritional enhancement, disease Orpest reSiStance, Or envi comprisea promoteroperably linked to DNA, the expression ronmental Orchemical tolerance. In more Specific aspectSof Of Which provides the enhanced agronomic trait. Other Con this invention enhanced trait is Selected from group of Struct Components Imay include additional regulatory ele enhanced traits ConSiSting ofenhanced Wateruse eficiency, ImentS.SuchaS 5'leaderSandintronsforenhancing tranScrip enhanced Cold tolerance, increased yield, enhanced Initrogen tion, 3'untranslated regionS(Such aSpolyadenylation SignalS use efficiency, enhanced Seed protein and enhanced Seed Oil. and Sites), DNA fortransitor Signal peptides. Iman important aspect Of theinvention the enhanced traitiS |0045| Numerous promoters that areactive in plant cells enhanced yield including increased yield under InOn-StreSS havebeendescribedintheliterature.Theseincludepromoters Conditions and increased yield under enVironmental StreSS present in plant genomes as Well as promoters from Other ConditionS. StreSS ConditionS Imay include, for example, Sources, including Inopaline Synthase (NOS) promoter and drought, Shade, fungal diSease, Viral diSease, bacterial dis Octopine Synthase (OCS) promoterS Carried On tumor-induc ease, inSectinfeStation, Inematode infeStation, Cold tempera ing plaSmids Of 4grohoicierin he/licieny, Caulimovirus ture expoSure, heat expOSure, OSmotic StreSS, reduced Initro promoterSSuch as the CauliflowermiOSaic ViruS. HorinStance, gen Inutrient availability, reduced phOSphoruS Inutrient See U.S. Pat. Nos. 5.858.742 and 5.322.938.which disclose availabilityandhighiplantdensity. “Yield”canbeaffected by versions of the constitutive promoter derived from cauli Imany properties including Without limitation, plant height, flower mosaic virus (CaMV35S), U.S. Pat. No. 5.641.876. pod number, pod pOSition On theplant, Inumber ofinternodeS. whichdisclosesariceactinpromoter.U.S. PatentApplication incidence of pod Shatter, grain Size, efficiency Of modulation Publication 2002/0192813A1, which discloses 5', 3' and and Initrogen fixation, efficiency Of Inutrient aSSimilation, intron elementSuSefulin the deSign ofeffectiveplantexpreS reSiStance to biotic and abiotic StreSS, Carbon aSSimillation, sion vectors, U.S. patent application Ser No. 09/757.089. plantarchitecture, reSiStance to lodging, percent Seed germi Which discloses a Imaize Chloroplastaldolasepromoter, U.S. Imation, Seedling Vigor, and juVenile traitS. Yield Can alSO patentapplication Ser No. 08/706.946.which disclosesarice affected byeficiency ofgermination (includinggermination glutelin promoter, U.S. patent application Ser. No. 09/757. in StreSSed Conditions), growth rate(including growth ratein 089.which discloses a maizealdolase (FDA) promoter, and StreSSed Conditions), ear Inumber, Seed Inumberper ear, Seed U.S.patentapplication Ser. No. 60/310.370, Which discloses Size, CompiOSition OfSeed (Starch, Oil, protein)and Character a Imaize Inicotianamine Synthase promoter, all of Which are istics of Seed incorporated hereinby reference. Theseand numerous other |0042| Increased yield ofa transgenic plant ofthe present promoters that function in plant Cells are known to thOSe invention CambemeaSuredina numberof WayS, includingtest skilled in theart and availablefor usein recombinant poly Weight, Seed numberperplant, Seed Weight, Seed numberper nucleotides ofthepresentinvention toprovide forexpression unitarea(i.e. SeedS. Or Weight OfSeedS, peracre), buShelSper Of deSired genes in transgenic plant CellS. acre, tons per acre, tons per acre, kilo per hectare. Hor |0046|| In other aspects of the invention, preferential example, Imaize yield Imay be meaSured as production of expreSSion in plant green tiSSules is deSired. Promoters of Shelled Corn kernelSperunit Ofproduction area, for example interest for Such uSeSinclude thOSe from genes Such aS 4/ol in buShelsperacre Ormetric tonSper hectare, Ofien reported ?)ioDyis, 1/i/ini ribulOSe-1.5-bisphOSphate Carboxylase On a moistureadjuSted baSiS, for example at 15.5 percent (Rubisco)smal subunit (Fischhoffetal. (1992) Plant Mol. Imoisture. Increased yield Imay reSult from improved utiliza Biol. 20:81-93), aldolaseand pyruvate orthophosphate diki tion ofkey biochemical Compounds, SuchaSInitrogen, phOS Imase (PPDK) (Taniguchi et al. (2000) Pin Ce/ Pil0sio/ phorous and Carbohydrate, Or from improved responSes to 41(1):42-48). enVirommental StreSSeS, Such aS Cold, heat, drought, Salt, and |0047 Furthermore, the promoters may bealtered to con attackbypests orpathogens. Recombinant DNAused in this tain multiple ‘enhancerSequences”toaSSiStinelevatinggene US 201 1/0252501 A1 Oct. 13, 2011

expreSSion. Such enhancerSare known in the art. By includ 3-phosphate Synthase (EPSPS) disclosed in U.S. Pat. Nos. ing an enhancer Sequence With Such ConStructS, the expreS 5.094.945:5.627061:5.633.435and6.040.497forimparting Sion ofthe Selected protein Imaybeenhanced."Ilhese enhanc glyphOSate tolerance: pOlynucleotide molecules encoding a ers ofienarefound 5'to the Start oftranscriptioninapromoter glyphosate oxidoreductase (GOX) disclosed in U.S. Pat. No. that functions in eukaryotic cells, but can ofien be inserted 5.463.175 and a glyphosate-N-acetyl transferase(GAT) dis upStream (5) OrdownStream (3) to the Coding Sequence. In closedin U.S. Patent Applicationpublication 2003/0083480 SOmeinStances, these 5'enhancingelementSareintronS. Par A1 alSo for imparting glyphOSate tolerance: dicamba ticularlyusefulasenhancersarethe 5'introns oftherice actin ImonOOxygenase disclosed in U.S. Patent Application publi 1 (See U.S. Pat. No. 5.641.876) and rice actin 2 genes, the cation 2003/0135879A1 forimparting dicambatolerance: a Imaize alcohol dehydrogenaSegene intron, the Imaize heat polynucleotide molecule encoding bromOxymil Initrilase shock protein 70 gene intron (U.S. Pat. No. 5.593874) and (Bxn) disclosed in U.S. Pat. No. 4.810.648 for imparting the Imaize:Shrunken 1 gene. bromoxymil tolerance: a polymucleotide molecule encoding |0048|| In other aspects ofthe invention, Sufficient expres phytoene desaturase (crtl) described in Misawa et al, (1993) Sionimplant Seed tiSSueSiSdeSired to effectimprovementSin Pin/ 4:833-840 and Misawa etal, (1994) Pin/ 6:481 Seed CompiOSition. EXemplary promoters for use for Seed 489 for norfurazon tolerance: a polynucleotide molecule Composition modificationincludepromotersfromSeedgenes encoding acetohydroxyacid Synthase (AHAS, aka ALS) Such as napin (U.S. Pat. No. 5.420034), maize L3 oleosin described in Sathasivan et al. (1990) AVic/ 4cis Rey. (U.S. Pat. No. 6433.252), Zein Z27 (Russel et al. (1997) 18:2188-2193 forimparting tolerance to Sulfonylurea herbi 17onsgenic Res. 6(2):157-166), globulin 1 (Belanger et al cides: polynucleotide molecules known as bar genes dis (1991) Genetics 129:863-872), glutelin 1 (Russell (1997) closed in DeBlock, etal. (1987) ZM9() / 6:2513-2519 for Supra), and peroxiredoxin antioxidant (Perl) (Stacy et al. imparting glufOSinate and bialaphOS tolerance: polynucle (1996) Pin Mo/ Bio/ 31(6):1205-1216). otide molecules disclosed in U.S. Patent Application Publi |0049|| Recombinant DNA constructs prepared in accor cation 2003/010609A1 forimparting N-aminomethylphos dance with the invention Will also generally include a 3 phonic acid tolerance:polynucleotidemolecules disclosed in element that typically Contains a polyadenylation Signal and U.S. Pat. No. 6.107.549 for imparting pyridine herbicide site. Well-known 3' elements include those from 4groinc1e reSiStance:ImoleculeSand methods forimpartingtolerance to 7in he/licieny geneSSuchas InOS 3', tml 3', tmr 3', timS 3'. Imultiple herbicideSSuch as glyphOSate, atrazine, ALSinhibi ocs 3', tr73', for example disclosed in U.S. Pat. No. 6.090, tors, iSOXafilutole and glufOSinate herbicideSare disclosed in 627, incorporated hereinby reference: 3'elements fromplant U.S. Pat. No. 6.376.754 and U.S. Patent Application Publi geneSSuchas Wheat (17ich Mileyeyin) heat Shockprotein cation 2002/0112260, al of Said U.S. patents and Patent 17(Hsp173"), a wheat ubiquitingene.a wheat fructose-1.6- Application Publications are incorporated herein by refer biphOSphataSegene, a rice glutelingene a rice lactate dehy ence. MoleculeSand methods forimpartinginSect/nematode/ drogenaSegene and a rice beta-tubulingene, all of Whichare Virus resistance is disclosed in U.S. Pat. NOS. 5.250.515: disclosedin U.S. published patentapplication 2002/0192813 5.880.275:6.506.599:5.986.175and U.S.PatentApplication A1. incorporated herein by reference: and the pea (Pisin Publication 2003/0150017A1, al of whichareincorporated Sin)ribulOSebiphOSphate CarboxylaSegene (rbS 3'), and herein by reference. 3' elements from the genes within the host plant. |0050) Constructs and vectors may also include a transit Plant Cell Iransformation Methods peptide for targeting of a gene target to a plant Organelle, |0052| Numerous methods for transforming plant cells particularly to a chloroplast, leucoplast Or Other plastid with recombinantDNA areknown in the artand maybeused organelle. For descriptions of the use of chloroplast transit in the present invention. IWo Commonly uSed methods for peptidessee U.S. Pat. No. 5.188642and U.S. Pat.No. 5.728. plant transformation are 4grohoicierin-mediated transfor 925. incorporated hereinby reference.Fordescription ofthe Imation and microprojectile bombardiment. Microprojectile transitipeptideregion ofan4/o/iopsis EPSPSgeneusefulin bombardimentimethods areillustratedinU.S.IPat.NOS. 5.015. the present invention, See Klee, H. J. et al (MGG (1987) 580 (soybean): 5.550.318 (corn): 5.538.880 (corn): 5.914, 210:437-442). 451 (soybean): 6.160.208 (corn): 6.399.861 (corn): 6.153. |0051] Transgenic plants comprising orderived fromplant 812 (Wheat) and 6.365.807 (rice) and 4groborc1erin cells of this invention transformed With recombinant IDNA mediated transformationis describedinU.S.IPat.NOS. 5.159. Can be further enhanced With Stacked traits, e.g.a Cropplant 135(cotton): 5.824.877 (soybean): 5.463.174 (canola): having an enhanced trait reSulting from expreSSion of IDNA 5.591,616 (corn): 6.384.301 (soybean), 7026.528 (wheat) disclosed herein in combination with herbicide and/or pest and 6329571 (rice), all ofwhichare incorporated herein by reSiStance traitS. Hor example.genes of the Currentinvention reference.For?4grohicierin he/licieny based plant tranS Cambe Stacked With Othertraits Ofagronomic interest, Such as formation SyStems, additional elementSpreSent On transfor a trait providing herbicide reSiStance, Or inSect reSiStance, Imation constructs Will include T-DNA left and right border Such aS uSing a gene from Lilicily hingenyis to provide SequenceSto facilitateincorporation Oftherecombinantipoly reSiStance against lepidopteran, Coliopteran, homopteran, Inucleotide into the plant genome. hemiopteran, and other insects. Herbicides for Which trans |0053|| IngeneralitisusefultointroducerecombinantDNA genic plant tolerance has been demonStrated and the method randomily i.e.ata InOn-Specific location, in the genome of a Of the present invention Can be applied include, but are Inot target plant line. In Special CaSes it Imay be uSeful to target limited to, glyphOSate, dicamba, glufOSinate, Sulfonylurea, recombinant DNA insertion in order to achieve Site-specific bromoxynil and norfurazonherbicides.Polynucleotidemol integration, for example, to replace an existing gene in the ecules encoding proteinSinVolved in herbicide toleranceare genome, to uSean existing promoterin theplant genome, Or Well-knoWin in theart and include, but are Inot limited to, a toinSerta recombinantpOlynucleotideatapredetermined Site polynucleotide molecule encoding 5-enolpyruVylShikimate known to beactive forgene expreSSion. Several Site Specific US 201 1/0252501 A1 Oct. 13, 2011

recombination SyStems exist Which are known to function in ImarkergeneSprovide Selective Imarkers Which Confer reSiS plants including cre-loxas disclosed in U.S. Pat. No. 4.959. tance to a Selective agent, SuchaSan antibiotic Oraherbicide. 317 and FLP-FRTas disclosed in U.S. Pat. No. 5.527.695. Any oftheherbicidesto whichiplants ofthisinvention maybe both incorporated herein by reference. resistant are usefulagents forselective markers. Potentially |0054) Transformation methods ofthis invention arepref transformed Cells are exposed to the Selective agent. In the erablypracticed intiSSuleculture On media andin a Controlled population Of SurViving Cells Will be thOSe Cells Where, gen enViromment. “Media”refers to the numerous mutrient mix erally, the reSiStance-conferring gene is integrated and tures thatareused togrow cells invitro.thatis.outside ofthe expressed at Sufficient levels topermit cell Survival. Cells intact living Organism. Recipient Cell targetSinclude, butare Imay be tested further to confirm Stable integration of the Imot limited to, meristem Cells, hypOCotylS, Calli, immature exogenouS IDNA. Commonly uSed Selective Imarker genes embryOS and gametic Cells Such aS ImicrosporeS, pollen, include thOSe Conferring reSiStance to antibiotics Such as Sperm and egg CellS. It iS Contemplated that any Cell from kanamyCin and paromomyCin (Inptill), hygromyCin B (aph Which a fertileplant Imayberegeneratedis uSeflas a recipi IV), SpectinomyCin(aadA) andgentamyCin(aac3 andaacC4) ent cell. Callus Imay beinitiated from tissue Sources includ Or reSiStance to herbicides Such as glufOSinate (bar Or pat), ing, but Inot limited to, immature embryOS, hypocotylS, Seed dicamba (DMO) and glyphosate(aroAor EPSPS). Examples lingapical meristemS. microsporeSand thelike. CellScapable of Such Selectable Imarkers areillustrated in U.S. Pat. NOS. Of proliferating aS CalluSare alSo recipient Cells forgenetic 5.550.318: 5.633.435: 5.780.708 and 6.118.047, al of which transformation. Practical transformation methods and Imate are incorporated herein by reference. Selectable markers rials for Imaking transgenic plantS Of this invention, for which provide an ability to Visually identity transformants example VariouSmedia and recipient target CellS, transforma Can alSo be employed, for example.agene expreSSing a Col tion of immature embryo CellSand Subsequent regeneration Ored OrfluoreScent protein Such as a luciferase Orgreen fluo offertile transgenic plants are disclosed in U.S. Pat. Nos. reScent protein (CHIP) Or a gene expreSSing a beta-glucul 6.194.636 and 6.232.526. Whichareincorporated herein by ronidase oruidAgene(GUS) forwhich various chromogenic reference. SubstrateSare known. |0055| The Seeds of transgenic plants can be harvested 10057 Plant cells that survive exposure to the selective from fertile transgenic plants and be uSed to groW progeny agent, Orplant CellSthathavebeen Scored pOSitiveina Screen generations oftransformed plants ofthiSinvention including ingaSSay, Imaybe Culturedin regeneration media and allowed hybrid plants line for Selection of plantShaving an enhanced to ImatureintoplantS. IDeveloping plantlets regenerated from trait. In addition to direct transformation of a plant With a transformed plant cells can be transferred to plant growth recombinant IDNA, transgenic plantS Can be prepared by Imix, and hardened Oft; for example, in an enVironmentally CroSSing a first plant having a recombinant IDNA With a Sec controlled chamberatabout85%, relative humidity, 600ppm ond plant lacking the DNA. For example, recombinant DNA CO, and 25-250 microeinsteins mT? ST of light, prior to can be introduced into a first plant line thatis amenable to transfer to a greenhouse Orgrowth Chamber for Imaturation. transformation to produce a transgenic plant Which Can be Plantsare regenerated fromabout 6weeks to 10 monthsafter CroSSed With a SecondiplantlinetointrogreSStherecombinant a transformant iSidentified, depending On the initial tiSSue, DNA into the Second plant line. A transgenic plant with and plant SpecieS. Plants Imay be pollinated uSing Conven recombinant IDNAprovidingamenhanced trait, e.g.enhanced tional plant breeding methods known to those of Skill in the yield, Can be CroSSed With transgenic plant line having other art and Seed produced, for example Self-pollination is Com recombinant DNA that confers another trait, for example Imonly uSed With transgenic Corm. Ihe regenerated tranS herbicide reSiStance Orpest reSiStance, to produce progeny formed plant Orits progeny Seed Orplants Can be tested for plants having recombinant DNA that confers both traits. expression of the recombinant DNA and Selected for the Ilypically in Such breeding for Combining traits the tranS presence ofenhanced agronomic trait. genic plant donating theadditional traitiSamale line and the transgenic plantCarryingthebaSetraitSiSthefemale line."Ilhe Transgenic Plants and Seeds progeny of this CrOSS Will Segregate Such that SOme of the plants Will carry the DNA for both parental traits and Some |0058|| Transgenic plants derivedfromtheplantcells ofthis Will carry DNA for one parental trait: Such plants can be invention aregroWin to generate transgenic plantShaving an identified by Imarkers associated with parental recombinant enhanced trait as Compared to a Control plant and produce DNA, e?g. Imarkeridentification by analysis forrecombinant transgenic Seed and haploid pollen Of this invention. Such IDNAor, inthe case Where a Selectablemarkeris linked to the plantswithenhanced traitsareidentifiedbyselection oftrans recombinant, by application of the Selecting agent Such as a formed plants Or progeny Seed for the enhanced trait. Hor herbicide for use with a herbicide tolerance marker, or by eficiency a Selection methodis deSigned to evaluatemultiple selectionfortheenhanceditrait.Progenyplantscarrying DNA transgenic plants (events) CompriSing therecombinant IDNA, for both parental traits can be crossed back into the female for example multipleplants from 2 to 20Ormore transgenic parent linemultipletimes, forexampleuSually 6 to8genera eventS. Iransgenic plants groWin from transgenic Seed pro tionS, toproduce a progeny plant With SubStantially the Same Vided herein demonStrate improved agronomic traits that genotypeas One Original transgenic parental line but for the contribute to increased yield or other trait that provides recombinant DNA ofthe othertransgenic parental line increased plant Value, including, forexample, improved Seed |0056|| In the practice oftransformation DNA istypically quality Of particular intereStare plants having enhanced introduced into Only a Smalpercentage oftargetiplant CellSin Water use eficiency, enhanced Cold tolerance, increased any Onetransformation experiment. MarkergeneSareuSed to yield, enhanced Initrogen uSeeficiency, enhanced Seed pro provide an efficient System for identification of those cells tein and enhanced Seed Oil. that are Stably transformed by receiving and integrating a 10059|| Table 2 provides a list of protein encoding DNA recombinant DNA molecule into their genomes. Preferred ("genes”) thatareusefulas recombinant DNA forproduction US 201 1/0252501 A1 Oct. 13, 2011

Oftransgenic plants With enhanced agronomic trait, the ele ‘Gene Name’whichisa common name forprotein encoded ments ofTable 2 are described by reference to: by the recombinant DNA. “Annotation” refers to a description of the top hit protein ‘PEPSEQ IDNO”identifies anaminoacid sequence from obtained from an amino acid Sequence query of each PEP SEQIDNO: 359 to 716 SEQIDNOto GenBankdatabase ofthe National Centerfor “NUCSEQIDNO”identifiesaDNAsequencefromSEQID Biotechnology Information (Incbi). Moreparticularly, ‘gi’is the GenBank ID number forthetop BLASThit: N(): 1 tC) 358. “description’refersto the description ofthe top BLASThit: “IBVid’isa reference to theidentifying numberinTable 4 of ‘%id’refersto thepercentage ofidenticallymatched amino base Vectors used forconstruction ofthe transformation Vec acid reSiduleSalong the length Oftheportion ofthe Sequences tors oftherecombinant DNA. Construction ofplant transfor whichisaligned by BLAST(-FT)between the Sequence of Imation ConStructSiSilluStrated in EXample 1. interest provided herein and the hit Sequence in GenBank:

"IABI, 2

Annotation NC) IID) NC) Ceme IID) BVid Geme Name %id GenBankid desciption 1 359 PHE0001295_7469 4 rice Cryptochrome 1 95 gil50909767|| refXP_466372.1||cryptochrome AB073546 1a IC727 Siz (?1927icri Cultivar-group)] 2 360 PHE0002129_8308 16 Vosioc sp. PCC7120 93 gil17133998|| refNP_488901.1|| phOSphoemolpyruvate phosphoenolpyruvate Carboxylase Carboxylase IVOSiOC Sp. PCC 7120| 3 361 PHE0002132_4965 4 Imalize 80 gi59803710 b|AAX07936.1phosphoenolpyruvate phOSphoemolpyruvate arboxylase Carboxylase kinase 2 inase 2 ||2lei ini)"S] 4 362 PHE0002132_8653 19 Imalize 80 gi59803710 b|AAX07936.1phosphoenolpyruvate phOSphoemolpyruvate arboxylase Carboxylase kinase 2 inase 2 ||2lei ini)"S] 5 363 PHE0002133_7497 4 Imalize 82 gi59803708 b|AAX07935.1phosphoenolpyruvate phOSphoemopyruvate arboxylase Carboxylase kinase 3 inase 1 ||2lei ini)"S] 6 364 PHE0002693_8516 17 Wheat geranylgeranyl 93 gil23397035 b|AAN31803.1 ||putative Ireductase like 1 eranylgeranyl reductase SequleilCe 4/7)iopSis ini) 7 365 PHE0002777_7490 4 Imalize ferrochelatase-II 85 g|50725080|| dbjBAID33213.1putative ike 2 Sequence errochelatase IOryzz Siz (i)(2nicri Cultivar group)] 8 366 PHE0002777_8472 6 Imalizeferrochelatase-II 85 g|50725080|| dbjBAID33213.1putative ike 2 Sequence errochelatase IOryzz Siz (i)(2nicri Cultivar group)] 9 367 PHE0002777_8726 19 Imalize ferrochelatase-II 85 g|50725080|| dbjBAID33213.1putative ike 2 Sequence errochelatase IOryzz Siz (i)(2nicri Cultivar group)] 10 368 PHE0002779_7478 4 Soybean 89 gi6272281|| emb|CAB60127.1||cytosolic phOSphoglulcomutase phosphoglulcomultase ike 1 Sequence Pisin Sin) 11 369 PHE0002810_5803 9 Imalize cytochrome 92 g|1870201|| emb|CAA72208.1||cytochrome P450 monooxygenase p450 [Zein ini)'s) (CYP71B3)like 4 emb|CAA57423.1 || SequleilCe cytochrome P450[Zei1 inis) 12 370 PHE0002857_7502 4 2lein Mil)'s putative low 61 g|50934635| refXP_476845.1||putative Imolecularearly light OVImolecular ImaSS early inducible protein ight-induced protein, Chloroplast precursor (ELIP)[Onza Siz (i)(2nicri Cultivar group)] 13 371 PHE0002860_7494 4 2lein Mil)'s Unknown 25 gil15237638|| refNP_201222.1|unknown protein protein I4/7)iopSiS ini) 14 372 PHE0002860_8694 19 Zein Mil)'s Unknown 25 gil15237638|| refNP_201222.1|unknown protein protein I4/7)iopSiS ini) 15 373 PHE0003814_7802 17 rice PsibS like 85 gill34908652| refNP_915673.1||putative photosystem ISubunit (22 KDa) precursor [Onza

US 201 1/0252501 A1 Oct. 13, 2011 20

IABLE 2-continued

Annotation

NC) IID) NC) Ceme IID) BVid Geme Name %, id GenBank id eSciption 208 566 PHE0006377_8683 19 Sincoinon)"ces Cene)"isine Rirp44p EXOSome Complex exonuclease RRP43 (Ribosomal RNA proceSSing protein 43) 209 567 PHE0006378_7667 4 Sirccinon)"ces Cene)"isine Rirp40p Sirccinon)"ces Cereisize) 210 568 PHE0006378_8715 19 Sincoinon)"ces Cene)"isine Rirp40p Sirccinon)"ces Cereisize) 21 1 569 PHE0006380_7658 4 Sirccinon)"ces 100 gil1323143|| sp|P53256||RRP46_YEAST Cene)"isine Rirp46p EXOSome Complex exonuclease RRP46 (Ribosomal RNA proceSSing protein 46) 212 570 PHE0006380_8719 19 Sincoinon)"ces 100 gil1323143|| sp|P53256IRRP46_YEAST Cene)"isine Rirp46p EXOSome Complex exonuclease RRP46 (Ribosomal RNA proceSSing protein 46) 213 571 PHE0006381_7655 4 Sirccinon)"ces 88 gil1045263 refiNP_011674.1|3"5" Cene)"isine mtr3p exoribonuclease, exOSome Subunit: Inucleolarprotein involved in export of ImRNA and Iribosomal Subunits: homologousto the E. Coli exonuclease RNase PH:Mtr3p [Sinccinon)"ces Cereisize) 214 572 PHE0006381_8695 19 Sincoinon)"ces 88 gil1045263 refiNP_011674.1|3"5" Cene)"isine mtr3p exoribonuclease, exOSome Subunit: Inucleolarprotein involved in export of ImRNA and Iribosomal Subunits: homologous to the E. Coli exonuclease RNase PH:Mtr3p [Sinccinon)"ces Cereisize) 215 573 PHE0006382_7652 4 PHE0006382_Saccharomyces 100 gi6321225| Iref|NP_011302.1||Proteim Cene)"Sie inVolved in exOSome SKI8 Imediated 3' to 5'ImRNA degradation and tramslation imhibition of InOn-poly(A) ImRNASas Well as double-Strand break formation during Imelotic recombination: 1equired for repreSSing propagation of dSRNA viruses: Ski8p [Sinccinon)"ces Cereisize) 216 574 PHE0006382_8678 19 Sincoinon)"ces 100 gi6321225| Iref|NP_011302.1||Proteim Ce/eisile SKI8 inVolved in exOSome Imediated 3' to 5'ImRNA degradation and tramslation imhibition of InOn-poly(A) ImRNASas Well as double-Strand break formation during Imelotic recombination: 1equired for repreSSing propagation of dSRNA viruses: Ski8p [Sinccinon)"ces Cereisize)

US 201 1/0252501 A1 Oct. 13, 2011 29)

IABLE 2-continued

Seq pep IID) Seq Annotation

NC) IID) NC) Ceme IID) BVid Geme Name %, id GenBank id deSciption 347 705 PHE0006952_9233 22 Gipm1p Withctp 95 gi407495| refiNP_012770.1|| Tetrameric phOSphoglycerate Inutase, [Sinccinon)"ces Cereisize) 348 706 PHE0006953_9121 22 universal Stress 93 gi30678807 Iref|NP_850506.1||lumknown protein (USP) family protein I4/i/2)SiS protein in) 349 707 PHE0006954_9154 22 unnamed protein 80 gil22327694 reflNP_680415.1|unknown product protein I4/i/2)SiS in) 350 708 PHE0006954_9161 9 unnamed protein 80 gil22327694 Iref|NP_680415.1||lumknown product protein I4/i/2)SiS in) 351 709 PHE0006962_9114 5 Imitrate reductase 94 gi85675313 dbil|BAA15989.2|mitrate 1eductase, periplaSmic, large subunit|[Escierici1 coli W3110) 352 710 PHE0006963_9131 5 Imitrite reductase 97 gi85676675 dbil|BAE77925.1||mitrite 1eductase, large Subunit, NAD(P)H-binding EScierici Coli W3110) 353 711 PHE0006965_9119 7 glutaminyl-tRNA 86 gi77554943 gb|ABA97739.1|| prolyl Synthetase RNA synthetase [Onza Siz (?1927icil Cultivar group) 354 712 PHE0006970_9141 9 DNAbinding/ 100 gil15242227|| refiNP_197020.1DNA tranScription factor binding/tranScription actor|4/i/2)SiS in) 355 713 PHE0006977_9163 9 ribulose-phosphate 3 96 gil15221735| refiNP_176518.1||ribulose epinerase phOSphate 3-epinerase 4/i/2)Sis ini) 356 714 PHE0006986_9183 9 Unknown protein 82 gil50932819|| refXP_475937.1|unknown protein[Onza Sinz (?1907icri Cultivar-group)] 357 715 PHE0006992_9140 22 unknoVIn protein 93 gil15234800|| refiNP_194792.1|unknown protein I4/i/2)SiS in) 358 716 PHE0006992_9184 19 Unknown protein 93 gil15234800|| refiNP_194792.1|unknown protein I4/i/2)SiS in)

Selection Methods for Transgenic Plants with Enhanced pOSition Of grain Can be detected by analySiS Of the Seed Agronomic Irait Composition and Contentofprotein, free aminoacids, Oil, free |0060|| Within a population of transgenic plants regener fatty acidS, Starch Or tocopherolS. Changes in biomaSS Char ated fromplant cellstransformedwith therecombinant DNA acteristics Can be Imade On greenhouse Orfield groWin plants Imany plants that SurVive to fertiletransgenic plants that pro and Can includeplant height, Stem diameter, root and Shoot duce Seeds and progeny plants Will Inot exhibit an enhanced dry Weights: and, for Corn plants, ear length and diameter. agronomictrait. Selectionfrom thepopulationis IneceSSaryto Changes in physiological properties Can be identified by identify One Ormore transgenic plant Cells that Can provide evaluating reSponSeSto StreSS ConditionS, forexample aSSayS plants With the enhanced trait. Iransgenic plants having uSing imposed StreSS ConditionSSuch as Water deficit, Initro enhanced traitSare Selected frompopulationS Ofplants regen gen deficiency, Cold groWing ConditionS, pathogen OrinSect erated or derived fromplant cells transformed as described attack Orlight deficiency, Orincreased plant denSity. Changes hereinby eValuating theplantSina Variety OfaSSaySto detect in morphology Can be meaSured by Visual Observation of an enhanced trait, e.g. enhanced Water use eficiency, tendency Ofa transformed plant With an enhanced agronomic enhanced Cold tolerance, increased yield, enhanced Initrogen trait to alSO appear to be a Inormal plant as Compared to use efficiency, enhanced Seed protein and enhanced Seed Oil. ChangeStoWard buShy, taller thicker, ImarrowerleaveS, Striped Ihese aSSaySalSO Imay take Imany forms including, but Inot leaveS, knotted trait, ChloroSiS, albino, anthocyanin produc limited to, directScreening forthetraitinagreenhouSeorfield tion, Oraltered taSSelS, earS OrrootS. Other Selection proper trial Orby Screening fora Surrogatetrait.SuchanalySeSCambe tieSinclude dayStopollen Shed, daySto Silking, leafeXtension directed to detecting Changes in the Chemical Composition, rate, Chlorophyll Content, leaftemperature, Stand, Seedling biomaSS, phySiological properties, morphology of the plant. Vigor, internode length, plant height, leafnumber, leafarea, Changes in chemical CompiOSitionSSuch aS Inutritional Com tillering, brace roots, Stay green, Stalk lodging, root lodging, US 201 1/0252501 A1 Oct. 13, 2011

planthealth, barreneSS/prolificacy.green Smap, andipeStreSiS closed and stillobtainalike orsimilarresults without depart tance. In addition, phenotypic Characteristics of harVeSted ing from the Spiritand Scope ofthe invention. grain Imaybe eValuated, including Inumber ofkernelSperrow on the ear, Inumber of rows of kernels on the ear, kernel Example 1 abortion, kernel Weight,kernel Size, kerneldenSityandiphySi calgrain quality Although theplant CellSand methodSOfthiS Plant Expression Constructs invention Can be applied to any plant Cell, plant, Seed Or |0062| This exampleillustrates the construction of plas pollen, e.g. any fruit, Vegetable, graSS, tree Or Ornamental Imidsfortransferringrecombinant DNAintoplantcells which plant, the Various aspectS Of the invention are preferably Can beregenerated into transgenic plants ofthiSinvention applied to Corn, Soybean, Cotton, Canola, alialia, Wheat and rice plantS. In Imany CaSes the invention is applied to Corn plants thatareinherently resistant to disease from the Mal de A. Plant Expression Constructs for Corn Transformation Rio Cuarto Virus orthe Piccin Sorghifungus orboth. |0063|| A base corn plant transformation vector |0061] The following examples are included to demon pMON93039, assetforthin SEQIDNO:30329.illustratedin strateaspects oftheinvention, those ofskillin theart Should, Table3 and FIG. 2. Was fabricated foruseinpreparingrecom in light of the preSent diSClOSure, appreciate that Imany binantDNAfor?4groincierin-mediateditransformationinto Changes Can be Imade in the Specific aspects Which are dis Corn tiSSue.

"IABI) 3

Coordimates of SEQIDNO: function annotation 30329 Agro B-AGRturight border Agro right border 1 1364-1 1720 transforantion Sequence, eSSential fortransfer of T-DNA. Geme of upStream promoter 19-775 interest Iregion of the Irice actin expression 1 gele C3SSette duplicated35SA1-B3 788-1 120 domain Without TAIA box promoter region of the 1125-1204 Irice actin 1 gene 5' untranslated leader 1210-1270 of Wheat Imajor chlorophyl a/bbinding protein firstintron and fianking 1287-1766 UTR eXOn Sequences from the Irice actim 1 gele T-St.Pis4 3' InOn-tranSlated Iregion 1838-2780 ofthe potato proteinase inhibitor Igene which functionsto direct polyadenylation ofthe ImRNA Plant Promoter from the Irice 2830-3670 Selectable actin 1 gene Imarker firstexon ofthe Irice 3671-3750 expression actin 1 gene C3SSette firstintron and fianking 3751-4228 UTR eXOn Sequences from the Irice actim 1 gele TS-At.Shk C-CTP2 Transit peptide region 4238-4465 of4/ailopsis EPSPS CR-ACRitu.arOA Synthetic CP4 coding 4466-5833 CP4.Imat Iregion With dicot preferred Codon uSage. I-AGRitl.nOS A 3'Imon-tramslated 5849-6101 Iregion of the Illopaline Synthase gene of 4g/0)icierin tumefacienS TiplaSmid Which functions to direct polyadenylation of the mRNA. Agro B-AGRitl.left border Agroleft border 6168-6609 transformation Sequence, eSSential for transfer of T-DNA. US 201 1/0252501 A1 Oct. 13, 2011 31

"IABLH 3-continued

Coordimates of SEQIDNO: function annotation 30329 Maintemance The Vegetative Origin of 6696-7092 in E. Co/ Ireplication from plasmid RK2. CR-Ec.top Coding Iregion for 8601-8792 1epressor ofprimer from the ColE1 plaSmid. IExpression of this gene product interferes with primer binding at the Origin of Ireplication, keeping plaSmid Copy Inumber loW. CDR-Ec.ort-ColE1 The Imininal Origin of 9220-9808 Ireplication from the E. Coliplasmid ColE1. P-Ec.aadA-SPC/STR Iromoter for Tim7 10339-10380 adenylyltransferaSe (AAD(3")) CR-Ec.aadA-SPC/STR Coding region for Tin7 10381-1 1 169 adenylyltransferaSe (AAD(3")) conferring SpectinomyCin and Streptomycin IreSiStance. T-Ec.aadA-SPC/STR 3' UTRI from the Tim7 1 1170-1 1227 adenylyltransferaSe (AAD(3")) gene of E. Coli.

|0064) Another embodiment of corn plant transformation base Vector is pMON92705, as set forth in SEQ ID NO: 30330, illustratedinTable4 and FIG. 3.whichwas fabricated for uSein preparing recombinant IDNA for 4grohoicierin Imediated transformation into Corn tiSSue.

"IAHILH 4

Coordimates of SEQID function annotation N(): 30330 Agro B-AGRturight border Agro right border 5206-5526 transforantion Sequence, eSSential for transfer of T-DNA. Geme of P-(DS.ACitl Promoter from the Irice 5580-6423 interest actin 1 gene expression 5UTR ofriceActil gene 6424-6503 C3SSette Introm from the Irice 6504-6980 actiml gene T-St.Pis4 3' InOn-tranSlated Iregion of 7055-7997 he potatoproteinase inhibitor Igene which InctionSto direct polyadenylation ofthe ImRNA Plant Promoter from the Irice 8047-8887 Selectable actin 1 gene Imarker firstexon ofthe Irice actim 8888-8967 expression gele C3SSette firstintron and fianking 8968-9445 UTR eXOn Sequences ron the Irice actin 1 gene TS-At.Shk C-CTP2 Transit peptide region of 9455-9682 4/ailopsis EPSPS CR-ACRitu.arOA Synthetic CP4 coding 9683-1 1050 CP4.Imat Iregion With dicot preferred Codon uSage. US 201 1/0252501 A1 Oct. 13, 2011 32

IABLE 4-continued

Coordimates of SEQID function Il8IIll annotation N(): 30330 I-AGRitl.nOS A 3'InOn-translated region 1 1066-11318 ofthe Illopaline Synthase gene of4g/2)(1Cierin Imefaciens TiplaSmid Which functions to direct polyadenylation ofthe ImRNA. Agro B-AGRitl.left border Agroleft border 10-451 transformation Sequence, eSSential for transfer of T-DNA. Maintemance CDR-Ec.orfV-RK2 The Vegetative Origin Ofreplication 538-934 in E. Co/ romplaSmid RK2. CR-Ec.top Coding Iregion for 2443-2634 1epressor ofprimer from he ColE1 plasmid. Expression ofthis gene productinterferes With primerbinding at the Origin Ofreplication, keeping plaSmid Copy Imumber loW. CDR-Ec.ort-ColE1 The Imininal Origin of 3062-3650 1eplication from the E. Coliplasmid ColE1. P-Ec.aadA-SPC/STR Iromoter for Tim7 4181-4222 adenylyltransferaSe (AAD(3")) CR-Ec.aadA-SPC/STR Coding region for Tin7 4223-5011 adenylyltransferaSe (AAD(3")) conferring SpectinomyCin and Streptomycin IreSiStance. T-Ec.aadA-SPC/STR 3' UTRI from the Tim7 5012-5562 adenylyltransferaSe (AAD(3")) gene of E. Coli.

|0065| Otherbase Vectors Similarto those described above Werealso constructed as listed in Iable 5. See Iable 5 for a "IABLH 5-contimued Summary ofbasevectorplasmids and basevector IIDSwhich BaSe are referenced in Table2. Alsosee Table 5 fora Summary of Vector regulatory elementSuSed in the gene expreSSion CaSSette for IID) these basevectorsand SEQ DNOS for elements. 7 pMON92713 |0066|| Primers for PCR amplification of protein coding 8 pMON92714 nucleotides ofrecombinant DNA are designed atornearthe 9 pMON92715 Start and Stop CodonS Of the Coding Sequence, in Order to 10 pMON92716 eliminate most of the 5' and 3' untranslated regions. Each 11 pMON92717 12 pMON92718 recombinantDNAcoding foraproteinidentifiedinTable 2is 13 pMON92719 amplified by PCR prior to insertion into the insertion site 14 pMON92721 Within the gene Ofinterest eXpreSSion CaSSette of One of the 15 pMON92722 base Vectors as referenced in Iable2. 16 pMON92723 17 pMON92724 19 pMON93039 "IABI) 5 20 pMON93043 21 pMON94781 BaSe BaSe Vector Vector for IID) Soybean BaSe Vector for 1 pMON82053 Corm 2 pMON92671 3 pMON92672 4 pMON92705 18 pMON93007 5 pMON92708 22 pMON99006 6 pMON92709 US 201 1/0252501 A1 Oct. 13, 2011 33

"IABI) 6 SEQID SEQ SEQID Ve:CitOfr bitOIllOter NO) leader IDINC) introm N() 30332 NC)NE / N()NE / 30333 IL-At.SAMS3 30352 I-At.SAMS3 30371 30334 JL-At.Stml 30353 N()NE / 30335 JL-C)8.ACitl 30354 I-(DS.ACitl 30372 30336 IL-2/m1.CA4H 30355 N()NE / 30337 II)-()8. GT1 30356 I-2/IT1.IDI13K 30373 3033& JL-2/m1.39486 30357 I-2/IT1.IDI13K 30373 30339) I-RTBV 3035& I-2/IT1.IDI13K 30373 30340 JL-HV.JPer1 30359 I-2/IT1.IDI13K 30373 30341 JL-2/ml.FIDA 30360 I-2/IT1.IDI13K 30373 30334 JL-At.SAMS3 30352 I-At.SAMS3 30371 30342 IL-2m. Cik1 30361 I-2m. Cik1 30374 30343 IL-2/ml.JRAB17 30362 I-2/IT1.IDI13K 30373 30344 IL-2m.SZeimC1 30363 I-2/IT1.IDI13K 30373 30345 II)-CalMTV35S 30364 I-2m1.IDImaK 30373 30346 IL-2/ml.JNAS2 30365 I-2/IT1.IDI13K 30373

30347 II)-2m.JPPIDK 30366 I-2/IT1.IDI13K 30373

30348 IL-Atrd293 30367 N()NE / 30349 IL-Ta.ILhicb1 3036& I-(DS.ACitl 30375

30350 JL-2/ml.JEM 30369 I-2/IT1.IDI13K 30373 30351 IL-2m.Brittle-2 30370 I-2/IT1.IDI13K 30373 30332 NC)NE / N()NE /

B. PlaSmids for use in transformation of Soybean WerealSO prepared.ElementSOfan exemplary CommonexpreSSion Vec tor plasmid pMON82053 areshown in Table 7 below and HIC. 4.

"IABI) 7

Coordinates of SEQIDNO: function Il8IIll annotation 30331 Agro B-AGRitl.left Agro left border Sequence, eSSential 6144-6585 transforamtion border fortransfer of T-DNA. Plant P-At.ACit 7 Promoter from the arabidopsis actin Selectable 7 gene Imarker IL-At.Act 7 5UTR of4hilopsis Act7 gene expression I-At.Act 7 Intron from the 4hilopsis actin7 6624-7861 C3SSette gele TS-At.ShkG- Transit peptide region of A/lio)SiS 7864-8091 CTP2 EPSPS CR-AGRitu.aroA- Synthetic CP4 coding region with 8092-9459 CP4.IminO_At dicot preferred Codon uSage. I-AGRitl.nOS A3'Inon-translated region ofthe 9466-9718 Illopaline Synthase gene of 4g70)(1cierintumefaciens Ti plaSmid Which functionSto direct polyadenylation ofthe mRNA. Geme of P-CaMV35S-enh Promoter for 35S RNA from CaMV 1-613 interest Containing a duplication ofthe -90 to expression -350 region. C3SSette T-(b.JE6-3b 3'untranslated region from the fiber 688-1002 protein IE6 gene of Sea-island Cotton: Agro B-AGRturight Agro right border Sequence, eSSential transformation border fortransfer of T-DNA. 1033-1389 OR-Ec.oriV-RK2 The vegetative origin ofreplication 5661-6057 fromplasmid RK2. CR-Ec.top Coding Iregion for repreSSOr Ofprimer 3961-4152 from the ColE1 plasmid. Expression ofthis gene productinterferes with primerbinding at the Origin of 1eplication, keeping plaSmid Copy Imumber loW.

US 201 1/0252501 A1 Oct. 13, 2011

"IABLE 8-continued

Coordinates of SEQ IDNO: function Il8IIll annotation 30376

P-Ec.aadA-SPC/STR romoter for Tim7 6382-6423 adenylyltransferaSe (AAD(3")) CR-Ec.aadA-SPC/STR Coding region for Th7 6424-7212 adenylyltransferaSe (AAD(3")) conferring SpectinomyCin and Streptomycin IreSiStance. T-Ec.aadA-SPC/STR 3' UTRI from the Tim7 7213-7270 adenylyltransferaSe (AAD(3")) gene of E. coli.

Example2 bardiment, a SuSpension of gold particles iSprepared Onto which the desiredrecombinant DNA expressioncassettesare Corn Iransformation precipitated. DNA isintroducedintomaizecellsas described |0069|| This exampleillustrates plant cell transformation in U.S. Pat. Nos. 5.550.318and 6.399861 using the electric Imethods uSeful in producing transgenic Corn plant CellS. discharge particle accelerationgene delivery device. Hollow plants, SeedSand pollen ofthiSinvention and the production ing microprojectile bombardiment, tiSSue iS Cultured in the and identification oftransgenic Corn plants and Seed With an darkat 27degrees C.Additional transformation methods and enhanced trait, i.e. enhanced Water use efficiency, enhanced Imaterials for Imaking transgenic plants Of this invention, for Cold tolerance, increased yield, enhanced Initrogen use efi example, Various media and recipient target CellS, transfor ciency, enhanced Seed proteinandenhanced Seed Oil. PlaSmid Imation of immatureembryOS and Subsequence regeneration vectorswereprepared bycloning DNAidentifiedinTable 2in offertile transgenic plants are disclosed in U.S. Pat. Nos. the identified base Vectors for uSein Corn tranSformation of 6.194.636and 6.232.526and U.S.patentapplication Ser No. CormplanticellStoproducetransgenic CormplantSandprogeny 09/757089, whichareincorporated hereinby reference. plants, Seed and pollem. |0073|| To regenerate transgenic corn plants a callus of |0070) For 4gronc1erin-mediated transformation of transgenic plant CellSireSulting from transformationiSplaced corn embryo cells cormplants ofa readilytransformable line On media toinitiate Shoot developmentimplantletSWhichare (deSignated LH59)is grown in the greenhouSeand earShar transferred topotting SOil forinitialgrowthina growth Cham vested whentheembryosare1.5to2.0mminlength. Earsare berat 26 degrees C. folowed by a mist bench beforetrans Surface Sterilized by Spraying Or SOaking the ears in 80% planting to 5 inch pots Whereplants aregroWin to Imaturity ethanol, folowed by air drying. Immature embryOSare iSO Theregeneratediplantsareseliffertilizedand Seedisharvested lated fromindividual kernelson Surface Sterilized earS. Prior for uSein One Or more methods to Select Seed, SeedlingS Or toinoculation ofmaize Cells, 4gro)icierin CellSaregroWin progeny Second generation transgenic plants (R2 plants) Or OVernightatroomtemperature. Immaturemaizeembryo Cells hybrids, e.g. by Selecting transgenic plants exhibiting an areinoculated With4grohicierin Shortlyafierexcision, and enhanced traitaS Compared to a Control plant. incubated at room temperature with 4groncierin for 5-20 |0074) Transgenic corn plant cells are transformed with IminuteS. Immature embryo plant Cells are then Co-cultured recombinant DNA fromeach ofthe genesidentified in Table with 4groncierin for 1 to 3 days at 23° C. in the dark. 2. Progeny transgenic plants and Seed of the transformed Co-cultured embryosare transferred to Selection media and plant Cells are Screened for enhanced Water use eficiency, Cultured forapproximately tWOWeekSto allow embryogenic enhanced Cold tolerance, increased yield, enhanced Initrogen callusto develop. Embryogenic calusistransferreditoculture use efficiency, enhanced Seed protein and enhanced Seed Oill Imedium Containing 100mg/L paromomyCin and Subcultured as reported in Example 7. at about two week intervals. Transformed plant cells are recovered 6 to8 Weeks afierinitiation of Selection. Example 3 |0071] For 4gronc1erin-mediated transformation of Soybean Transformation Imaize Callus immature embryOS are Cultured for approxi Imately8-21 days afier excision to allow callus to develop. |0075| This exampleillustratesplant transformationuseful Calus is then incubated for about 30 minutes at room tem in producing the transgenic Soybeamplants of this invention perature With the 4grohicierin SuSpenSion, folowed by and the production and identification Oftransgenic Seed for removal Ofthe liquid by aspiration."Ilhe CalluSand 4grohoic transgenic Soybean having enhanced Water use eficiency, 1erin are co-cultured without Selection for 3-6 days fol enhanced Cold tolerance, increased yield, enhanced Initrogen lowed by Selection On paromomyCin for approximately 6 use efficiency, enhanced Seed protein and enhanced Seed Oil. weeks, with biweekly transfers to fresh media, and paromo |0076|| For 4groncierin mediated transformation, Soy ImyCin reSiStant Callus identified as Containing the recombi bean Seeds are germinated OVernight and the meristem Imant IDNA in an expreSSion CaSSette. explantSexcised."Ilhe meristems and the explantSareplaced |0072| For transformation by microprojectile bombard in a Wounding VeSSel. Soybean explants and induced 4gro Imentimmature Imaize embryOSareisolated and Cultured 3-4 Arc1erin cells from a Strain containing plasmid DNA with days prior to bombardiment. Prior to microprojectile bom the gene Of interest CaSSette and a plant Selectable Imarker US 201 1/0252501 A1 Oct. 13, 2011 36

CaSSette are mixed mo later than 14 hours from the time of Imodeand fruitinumberincluding mode above whiteflowerand initiation OfSeedgermination and WoundeduSing Sonication. Imode above Crackboll meaSurements: and Visual Wilt SCOring. HoloWing Wounding, explantSareplacedin Co-culturefor2-5 Cotton boll Samples aretaken and analyzed for lint fraction daysatWhichipoint theyaretransferred toselection media for and fiberquality.Thecottonisharvestedatthenormalharvest 6-8 WeekSto alloWSelection and growth oftransgenic ShootS. timeframe forthetrial area. Enhanced wateruse efficiencyis Trait positive Shoots are harvested approximately 6-8 Weeks indicated byincreasedyield, improvedrelative Watercontent, and placedintoselectiverootingmedia for2-3 Weeks. Shoots enhanced leafWaterpotential, increased biomaSS, enhanced producing roots are transferred to the greenhouSeand potted leafeXtension rates, and improved fiberparameterS. in Soil. Shoots that remain healthy on Selection, but do not |0079|| The transgenic cotton plants of this invention are produce roots are transferred to InOn-Selectiverooting media identified from among the transgenic Cotton plants by agro for an additional two weeks. Roots from any Shoots that Inomic trait ScreeningaShavingincreasedyield andenhanced produce roots offselection are tested for expression of the Wateruse efficiency. plant Selectable marker before they are transferred to the greenhouSeand potted in SOil. Additionally a IDNA ConStruct Example 5 Can be transferred into the genome of a Soybean Cell by particle bombardimentand the cell regenerated into a fertile Canola Iransformation Soybeanplantas describedin U.S. Pat.No. 5.015.580, herein |0080) This exampleillustratesplant transformationuseful incorporated by reference. inproducingthetransgenic CanolaplantSofthiSinventionand |0077 Transgenic Soybeamplant cellsaretransformedwith theproduction and identification oftransgenic Seed fortranS recombinant DNA fromeach ofthe genesidentified inTable genic Canola havingenhanced WateruSeefficiency, enhanced 2. Progeny transgenic plants and Seed of the transformed Cold tolerance, increased yield, enhanced Initrogen use efi plant Cells are Screened for enhanced Water use efficiency, ciency, enhanced Seed protein and enhanced Seed Oil. enhanced Cold tolerance, increased yield, enhanced Initrogen |0081] Tissues from in vitro grown canola Seedlings are use eficiency, enhanced Seed protein and enhanced Seed Oill prepared and inoculated With OVernight-groWin 4grohicie as reported in Example 7. 7incells containing plasmid DNA with the gene ofinterest CaSSette and a plant Selectable Imarker CaSSette. HoloWing Example 4 Co-cultivation With 4grohoicierin, the infected tiSSues are allowed togroWon Selectiontopromotegrowth oftransgenic Cotton Iransgenic Plants With Enhanced Agronomic Shoots, followed by growth of Iroots from the transgenic Iraits Shoots. The Selected plantlets are then transferred to the |0078|| Cotton transformation is performed as generally greenhouSeandpotted in Soil. Molecularcharacterization are described in WO0036911 and in U.S. Pat. No. 5.846.797. performed to Confirm thepreSence ofthe gene ofinterest, and Iransgenic Cotton plants Containing each Ofthe recombinant its expreSSion in transgenic plants and progenieS. Progeny DNAhaving a sequence of SEQ IDNO: 1 through SEQID transgenic plantSare Selected fromapopulation oftransgenic NO: 358 are obtained by transforming with recombinant Canola events under Specified groWing Conditions and are DNA from each of the genesidentified in Table2. Progeny Compared With Control Canola plantS. Control Canola plants transgenic plantSare Selected fromapopulation oftransgenic are SubStantially the Same Canola genotype but Without the Cotton eVents under Specified groWing Conditions and are recombinantDNA, forexample, eitheraparental Canolaplant Compared With Control Cotton plantS. Control Cotton plants Ofthe SamegenotypethatiSnottransformed Withitheidentical are Substantially the Same Cotton genotype but Without the recombinant DNA or a megative isoline of the transformed recombinantDNA, forexample, eitheraparentalcottomplant plant Iransgenic Canola plant Cells are transformed With Ofthe Samegenotype that WaSInot transformed With theiden recombinant DNA fromeach ofthe genesidentified in Table tical recombinant DNA or a megative isoline of the trans 2. Iransgenic progeny plants and Seed Of the transformed formed plant. Additionally, a Commercial Cotton Cultivar plant Cells are Screened for enhanced Water use eficiency, adapteditothe geographical regionand Cultivation ConditionS. enhanced Cold tolerance, increased yield, enhanced Initrogen i.e.cotton variety ST474. cotton variety FM 958. and cotton use efficiency, enhanced Seed protein and enhanced Seed Oill variety Siokra L-23. areused to compare the relative perfor as reported in Example 7. Imance Ofthe transgenic cottomplants Containing the recom binant DNA. The Specified culture conditionsaregrowing a Example 6 first Set oftransgenic and control plants under ‘wet’ condi Homolog Identification tions.i.e.irrigatedin therange of85to 100percent ofevapo transpiration to provide leafwater potential of -14 to -18 |0082| This example illustrates the identification of barS, and groWing a Second Set of transgenic and Control homologs ofproteinsencoded bythe DNAidentifiedinTable plantSunder"dry”ConditionS, i.e.irrigated in therange Of40 2 Which iSuSed toprovide transgenic Seed and plantShaving to 60 percent of evapotranspiration to provide a leafwater enhanced agronomic traitS. From the Sequence of the potential of-21 to -25 bars. Pest control, Suchas weed and homologS. homologouSIDNA Sequence Can beidentified for inSect Control is applied equally to both Wet and dry treat preparingadditionaltransgenic SeedSandiplantSofthiSinven Iments aS Ineeded. IData gathered during the trial includes tion Withenhanced agronomic traitS. Weather records throughout the groWing SeaSon including |0083|| An ‘All Protein Database’ was constructed of detailedrecords ofrainial: Soilicharacterizationinformation: known protein Sequences uSing a proprietary Sequence data any herbicide Or inSecticide applications: any groSS agro base and the National Centerfor Biotechnology Information Inomic differences ObserVed SuchaSleafmorphology branch (NCBI)mon-redundantaminoacid database (nraa). Foreach ing habit, leaf Color time to flowering, and fruiting pattern: Organism from Which a polymucleotide Sequence provided plantheightat VariouSpoints during the trial: Stand density: herein Was obtained, an ‘Organism Protein Database” Was

US 201 1/0252501 A1 Oct. 13, 2011 38

Level I.Transgenic plantsprovided bythepresentinvention "IABLE 10-continued areplanted infieldwith threelevels ofnitrogen(N) fertilizer being applied, i.e. low level (0 N), medium level (801b/ac) 2 mMNHNO 20mMNHNO3 (high (Low Nitrogen Growth Nitrogen Growth and highlevel (1801b/ac). Liquid 28% or 32%, UAN(Urea, Condition, Low IN) Condition, High N) Ammonium Nitrogen) areused as the N Source and apply by Nutrient Stock ImL/L ImL/L broadcast boom and incorporate with a field cultivator with rear rolling basket in the Same direction as intended Crop 1MCaCl2 2.5 2.5 rows. Although thereis no Napplied to the 0 Ntreatiment the 1MK2SO? 1 1 Soil Should Stilbedisturbedin the Samefashionasthetreated Note: area. Iransgenic plants and Control plants are grouped by Adjust pH to 5.6 with HClor KOH genotype and ConStruct With Controls arranged randomily |0092| (c) Harvest Measurements and Data Collection Within genotype blockS. Each type of transgenic plants iS After 28 days ofplant growth forlow Niruns and 23 days of testedby3 replicationSandacroSS4 locationS.Nitrogenlevels plantgrowth forhighNirunS, the folloWingmeaSurementSare in the fields areanalyzed in early April pre-planting by Col lecting 30 Sample Soil cores from 0-24" and 24 to 48"Soil taken(phenocodeSimparentheses): total Shoot fresh maSS (g) layer. Soil Samples are analyzed for Initrate-mitrogen, phOS (SFM) measured by Sartorius electronic balance, V6 leaf chlorophylmeasuredby Minolta SPADmeter(relativeunits) phoruS(P), PotaSSium(K), Organic Imatter and pH toprovide (LC), V6 leafarea(cm)(LA)measuredbya Li-Corleafarea baseline values. IP, K and micronutrients areapplied based meter, V6 leaffresh Imass (g)(LFM) measured by Sartorius upon SOil test recommendationS. electronic balance.andV6 leafdrymass(g)(LDMI)measured by Sartorius electronic balance. Raw data vereanalyzed by B. Selection for Increased YTield SAS Software. Results Shown are the comparison oftrans |0097 Many transgenic plants of this invention exhibit genic plants relative to the Wilditype ControlS. improved yield as Compared to a Control plant. Improved |0093|| To take a leafreading, Samples were excised from yield Can reSult from enhanced Seed Sink potential, i.e. the the V6 leaf Since chlorophyll meterreadings ofcorn leaves Inumber and Size Of endosperm Cells Or kernels and/or areaffected bythepart oftheleafand theposition ofthe leaf enhanced Sink Strength, i.e. the rate of Starch biOSyntheSiS. on theplant thatis Sampled, SPAD meterreadingswere done Sink potential can be established very early during kernel on leaf Six of the plants. Three measurements per leafwere development, as endosperm Cell Inumber and Sizeare deter taken, of Which the first reading Was taken from a point Imined Within the first few daySafierpollination. one-halifthe distance between the leaftipand the collar and |0098|| Much oftheincreaseincornyield ofthepastSeveral halfway from the leafmargin to the midrib Whiletwo were decades has reSulted from an increase in planting density takentowardtheleaftip.Themeasurementswererestrictedin IDuring that period, Cormyield haSbeen increaSingata rate of the area from /2 to 4 ofthe total length ofthe leaf(from the 2.1 buShelS/acre/year, but theplanting denSity haSincreased base) With approximately equal Spacing betWeen them. Ihe ata rate of 250plants/acre/year. A characteristic of modern average ofthe threemeasurements Wastaken from the SPAD hybrid corn is the ability of these varieties to be planted at Imachine. high density Many Studies have Shown that a higher than |0094|| Leaffresh mass is recorded for an excised V6 leaf Current planting denSity Should reSult in more biomaSSpro the leafiSplaced into a paperbag. IhepaperbagS Containing duction, but Current germplaSm does Inot perform. Well at the leavesarethemplaced into a forced airovenat 80° C. for these higher densitieS. One approach toincreaSingyield iSto 3 dayS. Afier 3 days, the paper bags are removed from the increase harvestindex(HI), theproportion ofbiomass thatis OVen and the leafdry ImaSS ImeaSurementSaretaken. allocated to the kernel Compared to total biomaSS, in high |0095| From the collected data, two derived measurements density plantingS. aremade: (1)ILeafichlorophyllarea (LCA), whichis aproduct |0099|| Effectiveyield Selection ofenhancedyieldingtrans of V6 relative chlorophyll contentand its leafarea (relative genic CorneventSuSeShybridprogeny ofthetransgenic event units). Leaf chlorophyll area=leaf chlorophyll X leafarea. OVermultiple locations With plantSgroWin underoptimal pro IhiSparametergives an indication of the Spread of chloro duction Imanagementpractices, andmaximumpeStControl. A phyll overthe entire leafarea: (2) specific leafarea (LSA)is useful target forimproved yield is a 5%, to 10%, increase in calculated as theratio ofV6 leafareatoits dry mass(cm/g yield as Compared to yield produced by plants groWin from dry ImaSS), aparameteralSorecognized aSameaSure of NUE. seed fora controlplant. Selection methods maybeappliedin Imultiple and diverSegeographic locationS, for example up to Nitrogen Use Field Efficacy Assay 16 Ormore locationS, OVer One Ormoreplating SeaSOnS, for exampleat least tWoplanting SeaSOnSto Statistically distin |0096|| Level I.Transgenic plants provided by the present guishiyield improvementfrommatural enVirommental effectS. invention are planted in field Without any Initrogen Source It iStoplant multipletransgenic plants, pOSitive and megative being applied. Iransgenic plants and Control plants are Control plants, and pollinator plants in Standard plots, for grouped by genotype and ConStruct With Controls arranged example 2 row plots, 20 feet long by 5 feet wide with 30 randomily Within genotype blockS. Each type of transgenic inches distance between rows and a 3 foot alley between plants are tested by 3 replicationSand acrOSS 5 locationS. rangeS. Iransgenic events Can be grouped by recombinant Nitrogen levels in the fields areanalyzed in early April pre DNA constructs with groups randomilyplaced in the field. A plantingby collecting 30Samplesoil cores from 0-24"and 24 pollinatorplot of a high Cuality Corn line iSplanted forevery to 48"Soil layer Soil Samplesareanalyzed fornitrate-nitro tWoplots to allow Open pollination When uSing Imale Sterile gen, phOSphoruS(P), PotaSSium(K), Organic Imatterand pH to transgenic events. A useful planting density is about 30,000 provide baseline values.P. Kand micronutrientsareapplied plants/acre. High planting density is greater than 30,000 based upon SOil test recommendationS. plants/acre, preferably about 40,000plants/acre, morepref US 201 1/0252501 A1 Oct. 13, 2011 39) erably about 42,000 plants/acre, most preferably about On live transgenic plants and the Wild type Counterparts a. 45.000 plants/acre. Surrogate indicators for yield improve Threetrifoliates from each plantareanalyzed, and each tri Iment include Source Capacity (biomaSS), Source Output (Su foliate were analyzed three times. Then 9 data points are Crose and photOSynthesis), Sink ComponentS (kernel Size, ear averaged to obitain the chlorophyll level.The number ofana Size, Starchin the Seed), development (light responSe, height, lyzed plants ofeach genotyperanges from 5 to 8. density tolerance), Imaturity, early flowering traitand phySi |0102| When Selecting foryield improvementa useful Sta Ological responSes to high density planting, for example at tistical meaSurement approach Comprises three ComponentS. 45.000plantsperacre, for example asillustrated in Table 11 i.e. modeling Spatial autocorrelation of the test field Sepa and 12. rately foreachlocation, adjusting traits ofrecombinant DNA

"IABIH 11 Timing Evaluation Description COIlIlle:IltiS V2-3 Earlystand Cambetaken anytime afer germination and priorto 1emoval of any plantS. Pollenshed GDUto 50%shed GDUto 50% plants shedding 50% tassel. Silking GDUto 50% silk GDUto 50%plantsShowing Silks. Maturity Plant height Height from Sol surface to 10plantsperplot-Yield fiagleafattachment (inches). team asSiStance Maturity Ear height Height from Sol surface to 10plantsperplot-Yield primary earattachment Imode. team assistance Maturity Leaves above ear Visual Scores:erect, Size, Irolling Maturity Tassel Size Visual Scores +/- VS. WT Pre-HarVest Final Stand Final Stand Count priorto harvest, exclude tillers Pre-Harvest Stalklodging No. of Stalks broken below the primary earattachment. Exclude leaningtlers Pre-Harvest Root lodging No. ofstalks leaning >45 angle fromperpendicular. Pre-Harvest Stay green Afterphysiological Imaturity and When diferences among genotypes are eVident: Scale 1 (90-100%tissue green)-9 (0-19%tissue green). HarVest GraimYTeld Grain yield/plot (Shel weight)

eventSforspatial dependence foreach location, and Conduct "IABI) 12 ing an acrOSS location analySiS. Ilhe first Step in modeling - - - - , - Spatial autocorrelation is eStimating the COVariance param Timing EValuation Description eterS Of the Semivariogram. A Spherical COVariance model iS V8-V12 Chlorophyll aSSumed to model the Spatial autocorrelation. Because ofthe V12-VT Earleafarea size and mature ofthe trial, itis likely that the spatialautocor V15-15IDAP Chl fillorescence relation Imay Change. Iherefore, anisotropy is alSo aSSumed V15-15IDAP CER along With-- Spherical-- COVariance-- Structure."Ilhe folloWing-- Set Pre-HarVest15-25 IDAP Carbohydrates1st intermode diameter SUlcroSe, Starch ofequations-- describes-- the Statistical- , - form of the anisotropic-- -- Pre-HarVest Base 3 intermode diameter Spherical COVariance model. Pre-HarVest Earintermode diameter Maturity Eartraits diameter, length, kernel Inumber, kernel Weight 3 1 C(Ai; 6) = yi(h = 0) + r - 51 -- "M < 1), |0100|| Electron transport rates (ETR) and CO2 exchange rates (CER): ETR and CER are measured with Li6400LCF (Licor, Lincolin, Nebr?) around V9-R1 Stages. Leaf chloro |0103|| where I()is the indicatorfunction, h-Vy and phyll fluoreScenceis a quick Wayto monitorthe Source activ ity and is reported to be highly correlated with CO assimi lation under varies conditions (Photosyn Research, 37: 89-102). The youngest fully expanded leafor 21eaves above the ear leafis measured with actinic light 1500 (with 10% Where S=(x,y1)are the Spatial Coordinates of One location bluelight)micromolimT?ST', CO2levels450ppm.Templants and S3=(x2, y2) are the Spatial COOrdinates Of the Second are meaSured in each event. Ihereare 2 readings for each location Thereare 5 covariance parameters, 6=(vp-0. plant. (0,0), wherevis the nuggetefect, p?is thepartial sill, pis [0101] A hand-held chlorophyll meter SPAD-502 (Mi a rotation in degrees ClockWise from Inorth, (0, is a Scaling Imolta—Japan)is uSed to meaSure the total Chlorophyll level parameterfortheminoraxis ando), is a scalingparameterfor

US 201 1/0252501 A1 Oct. 13, 2011 by Sub-irrigation and flats were rotated every third day in a length, and Whole Seedling length Of every individual Seed growth Chamber forgroWing Corn SeedlingS. ling and then Calculates theaverage ofeach rol. 10113|| Onthe 10"dayafierplantingthetransgenicpositive |0118|| Afterstatistical analysis, the events that Show a Sta and Wild-typenegative(WT)plantsarepositioned infiats in tistical Significanceat the p level ofless than 0.1 relative to an alternating patterm. Chlorophyll fluoreScence Of plants iS Wild-type ControlSWilladvanceto a Secondary Cold Selection. Ihe Secondary Cold Selectionis Conducted in the Same Iman measuredonthe 10"dayduringthedarkperiodofgrowthby Imer of the primary Selection Only increaSing the Inumber of using a PAM-2000portablefluorometerasper the manufac repetitions to five. Statistical analysis of the data from the turer’s instructions(Walz, Germany). Afterchlorophyll mea secondary Selection is conducted toidentity the events that Surements, leaf Samples from each event are Collected for Show a Statistical Significance at the plevel ofless than 0.05 Confirming the expreSSion ofgenes Ofthe preSent invention. relative to Wild-type controls. For expression analysis Six V1 leaftips fromeach Selection are randomily harvested. The flats are moved to a growth 4. Cold Field Efficacy Trial chamber Set at 5° C. All other conditions Such as humidity, day/inight Cycle and light intenSity are held ConStant in the |0119|| This example Sets forth a cold field efficacy trial to growth chamber The flats are Sub-irrigated every day after identify gene ConStructs that Confer enhanced Cold Vigor at transfer to the cold temperature. On the 4"day chlorophyl germination and early Seedling growth under early Spring fluorescence is measured. Plants are transferred to mormal planting field Conditions in Conventional-till and Simulated growth ConditionSafter Six daySOf Cold Shock treatiment and Imo-till enVirommentS. Seeds are planted into the ground allowed torecoverforthe next three dayS.During this recov around tWoWeeksbefore local farmerSarebeginningtoplant eryperiodthelength oftheV3leafismeasuredonthe 1"and Corn So that a Significant Cold StreSSiSexerted Onto the Crop. 3"days Aflertwo daysofrecovery V2leafdamageis deter Imamed aS Cold treatiment. SeedSalSo areplanted under local Imined Visually by eStimating percent OfgreenV2 leaf Optimal planting ConditionSSuch that the Crophas little Orno expOSure to Cold Condition, Inamed aSInormal treatiment. Ihe |0114|| Statistical differences in V3 leafgrowth, V2 leaf cold field efficacy trials are carried out in five locations. InecroSiSand fiuoreScence during pre-Shock and Cold Shock including Glyndon Minm. Mason Mich. Monmouth Ill. Can be uSed for eStimation of Cold Shock damage On Corn IDayton loWa, MyStic Comm. At each location, Seeds are plantS. (3) Early Seedling growth aSSay—Ihree SetS OfSeedS planted underboth cold and mormal conditionswith 3 repeti areused forthe experiment. The first set consists ofpositive tionSper treatiment, 20 kernelSper roW and Single roW per transgenic events (F1 hybrid)where the genes ofthepresent plot. Seeds areplanted 1.5 to 2 inch deep into Soil to avoid invention are expreSSed in the Seed. Ihe Second Seed Set iS Imuddy ConditionS. IWo temperature monitors are Set up at Imontransgenic, Wild-type Inegative Control Imade from the each location to monitor both airand SOil temperature daily Same genotype as the transgenic eventS.The third Seed Set |0120) Seed emergence is defined as the point when the ConSiSts oftwo Cold tolerant and tWo Cold Sensitive Commer growing Shoot breaks the Soil Surface. The number of cial checklines ofcorm. All Seedsaretreatedwith a fungicide emerged Seedling in each plot is Counted everyday from the ‘Captan", (3a.4.7.a-tetrahydro-2-(trichloromethly)thio day the earliest plot begins to emerge until Ino Significant 1H-isoindole-1.3(2H)-dione, Drex Chemical Co. Memphis. Changes in emergence Occur In addition, for each planting Ienn.). Captan (0.43 mL) WaSapplied per 45g of Corn SeedS date, the latest date When emergence iS 0 in all plots is alSO by mixingit Welland drying the fungicidepriorto the experi recorded. Seedling Vigoris also rated at V3-V4 Stage before I11ent. the average of Corn plant height reacheS 10 inches, With |0115| Seedsaregrown ingermination paper forthe early 1=excellent early growth, 5=Average growth and 9=poor seedling growth assay. Three12"×18"pieces ofgermination growth. IDaySto 50%, emergence, Imaximum percent emer paper(Anchor Paper#SD7606) areused foreachentryin the gence and Seedling Vigor are Calculated uSing SAS SOffiWare test (three repetitions per transgenic eVent). Ilhe papers are forthe data Within each location OracroSS al locationS. Wetted in a Solution of0.5%, KNC), and 0.1%, Thyram. E.ScreensforTransgenic Plant Seeds with Increased Protein |0116|| For each paper fifeen Seeds areplaced on the line and/or Oil Levels evenlyspaced down thelength ofthepaper The fifeen Seeds |0121] This examplesets forth a high-throughput selection arepOSitioned On thepaper Such that the radical Would grow foridentifying plant Seeds Withimprovementin Seed Compo doWinWard, for example longer distance to thepaper’s edge. sitionusing the Infratec1200Series Grain Analyzer, whichis Thewetpaperis roled up starting fromone ofthe Shortends. a Inear-infrared tranSmittance Spectrometerused to determine Thepaperis rolled evenly and tight enough to hold the Seeds the CompiOSition Ofabulk Seed Sample. Nearinfrared analySiS in place. Ihe rollis Secured intoplace With tWo large paper is a InOn-destructive, high-throughput method that Can ana clips, Oneat the top and oneat the bottom. The rolls are lyZemultiple traitSin a Single Sample Scan. An NIR Calibra incubated in a growth chamber at 23° C. for three days ina tion forthe analytes ofinterestisused topredict thevalues of randomized Complete block design Within an appropriate an unknown Sample. The NIR Spectrum is obtained for the container The chamberisset for 65%, humidity with nolight Sample and Compared to the Calibration uSing a CompleX cycle.Forthecoldstresstreatimentitherollsarethenincubated chemometric SoftWarepackage that provides a predicted Val in a growth chamberat 12°C. fortwelve dayS.The chamber ues as Wellasinformation on how Well the Sample fits in the isset for 65%, humidity with no light cycle. calibration. |0117|| Afterthe cold treatiment the germination papersare 10122| Infratec Model 1221, 1225, or 1227with transport unrolled and the Seeds that did Inot germinateare discarded. Imodule by Foss North America is used with cuvette, item The lengths of the radical and coleoptile for each Seed are #1000-4033. Foss North America or for Small Samples with ImeaSured through an automated imaging program thatauto Small cell cuvette, Foss Standard cuvette modified by Leon Imatically Collects and proceSSes the imageS. The imaging Girard Co. Corn and Soycheck Samples of Varying CompOSi program automatically meaSures the Shoot length, Iroot tion Imaintained in check Cell CulVetteSare Supplied by Leon US 201 1/0252501 A1 Oct. 13, 2011 42

Girard Co.NITcollection Softwareisprovided by Maximum Symbols for ConSenSuS Sequence are (1) upperCaSeletters for ConSulting Inc. SoftWare. CalculationSareperformed auto 100%identityinalpositions ofmultiplesequencealignment Imatically bythe Software. Seed Samplesarereceivedinpack output:(2) lowercaseletters for >=70%identity:Symbol: (3) ets or containers Withbarcode labels from the customer."Ilhe ‘X’ indicated <70%, identity: (4) dashes “-” meaning that Seed iSpoured into the CulVettes and analyzed aS received. gaps were in >=70%Sequences. |0125| The ConSenSuSamino acid Sequence Can be uSed to "IABI) 13 identiflyIDNAcorrespondingtothe fullscope ofthisinvention thatiSuSefilinprovidingtransgenic plants, forexample Corn Typical Sample(S): Whole grain Corn and Soybean Seeds Analytical time to rum Inethod: Less than 0.75 min persample and Soybean plants With enhanced agronomic traits, for Total elapsed time perrum: 1.5 minute per Sample example improved Initrogen use efficiency, improved yield, Typical and Iminimum Corn typical: 50cc: minimum 30cc improved WateruSeeficiency and/Orimproved growth under Sample Size: Soybeantypical: 50cc: minimum 5cc coldstress, due to the expressionin theplants of DNAencod Typical analytical Irange: Determined in part by the specific Calibration. ing a protein With amino acid Sequenceidentical to the Con Corm-moisture 5-15%, oil 5-20%, SenSuSamino acid Sequence. protein 5-30%, starch 50-75%, and density 1.0-1.3%. Example 9 Soybean-moisture 5-15%, oil 15-25%, and protein 35-50%. Identification of Amino Acid Domain by Pfam AnalySiS |0126|| This exampleillustratestheidentification ofdomain Example8 and domain module by Pfam analySiS. |0127 The amino acid Sequence ofthe expressed proteins ConSenSuS Sequence that Were Shown to beassociated With an enhanced trait Were |0123|| This exampleillustrates the identification of con analyzed for Pfam protein family against the current Pfam SenSuSamino acid Sequence for the proteins and homologs Collection Of multiple Sequence alignments and hidden encoded by DNA thatis used topreparethe transgenic Seed Markovmodels using the HMMER Softwarein theappended and plants of this invention having enhanced agronomic computer listing. The Pfam domain modules and individual traitS. proteindomain forthe proteins of SEQ IIDNO: 359 through |0124) ClustalW program Was Selected for multiple 716 are Shown in Table 14 and Table 15 respectively. The Sequencealignments oftheamino acid Sequence of SEQ ID Hidden Markov model databases for the identified protein NO: 561 and its 10 homologS.Three majorfactorsaffecting families arealSO in the appended Computer listing alloWing the Sequence alignments dramatically are (1) protein Weight identification ofotherhomologouSproteinSand theircognate Imatrices: (2) gap Open penalty: (3) gap eXtension penalty encoding DNA to enable the ful breadth oftheinvention for Protein Weight matrices available for ClustalW program a perSon of Ordinary Skill in the art. Certain proteins are include Blosum, Pamand Gionnet Series. Thoseparameters identified by a Single Pfam domain and others by multiple Withgap Openpenalty and gap eXtension penalty Were eXten Pfam domains. Forinstance, t For instance, the protein with sively tested. On the basis ofthe testresults, Blosum Weight aminoacids of SEQIDNO: 417ischaracterizedbytwo Pfam ImatriX, gap Openpenalty of 10and gap eXtenSion penalty of 1 domains.i.e. HDand RelA_Spot. Were ChOSen formultiple Sequence alignment. HIG. 1 ShoWS |0128|| In Table 15 “score’is the gathering Score for the the Sequences of SEQ IID NO: 561. its homologs and the Hidden MarkoV Model of the domain Which exceeds the consensus Sequence (SEQ ID NO: 30328) at the end. The gathering cutoffreported in Table 16.

"IAHILH 14

PEP SEQID NC) Ceme IID) Pfam domain Imodule domain Coordinates 591 PHE0006505_7871.pep Thioredoxim 69-174 541 PHE0006264_7285.pep IDAGAT 48-349 359 PHE0001295_7469.pep DNA_photolyase:FAD_binding_7 18-190:223-501 665 PHE0006760_8529.pep vATP-synt_E 16-225 639 PHE0006684_8413.pep Ribosomal_IL10 19-123 645 PHE0006715_8477.pep AMPKBI 197-287 626 PHE0006600_8249.pep Iso_dth 6-355 484 IPHE0006071_7068.pep PPR:PPR:PPR:PPR:PPR 30-63:64-98:99 132:138-172:173-207 417 PHE0004830_5828?pep HD:RelA_SpoT 233-337:427-537 576 PHE0006426_8056pep AA_pemease 2-454 571 PHE0006381_7655.pep RINase_IPHI 42-186 570 IPHE0006380_8719.pep RNase_PH:RNase_PH_C 1-126:129-201 713 PHE0006977_9163.pep Ribul_P_3_epim 7-207 466 PHE0006021_7077.pep Bet_V_I 1-155 596 PHE0006516_7887.pep CorA 90-474 632 PHE0006620_8462pep Epimerase 13-259 631 PHE0006617_8463.pep Cupin_1 65-215 585 PHE0006468_7903.pep F-box:FIBA_1 2-49:209-387 424 IPHE0004887_5939.pep IDUF516 49-310

US 201 1/0252501 A1 Oct. 13, 2011 54

"IABLE 15-continued

PEP SEQID Pfam domaim GENE IID) Il8IIll begin Stop Score E-Value

690 E0006931_9148. t) CID)C-P 3 443 700.9 8.30 -208 691 E0006931_9168. t) CID)C-P 3 443 700.9 8.30 -208 692 E0006932_9147. t) DUF498 56 164 153.1 6.90 -43 693 E0006932_9174. t) DUF498 56 164 153.1 6.90 -43 694 E0006933_9139. t) adhi_Short 30 212 5.7 1.50 -06 695 E0006934_9145. t) DNA_pol_E_B 178 389) 249.9 5.00 -72 696 E0006937_9126. t) DUJF298 127 242 222.4 9.20 -64 697 E0006938_9149. t) F-box 41 88 34.3 3.80 -07 698 E0006940_9122. t) Aldedh 18 477 674.3 8.70 -200 700 E0006943_9124. t) Aa_tranS 29) 428 51 6.5 2.80 - 152 701 E0006948_9125. t) RRM_1 38 109 98.5 1.80 -26 702 E0006948_9160. t) RRM_1 38 109 98.5 1.80 -26 703 E0006949_9133. t) Aldedh 19 478 778.3 4.10 -231 704 E0006949_9179. t) Aldedh 19 478 778.3 4.10 -231 705 E0006952_9233. t) PCAM 91 277 153.2 6.30 706 E0006953_9121. t) USp 3 157 85.3 1.80 709 ID) E0006962_9114. t) Molybdop_Fe4S4 39 93 88.2 2.40 709 E0006962_9114. t) Molybdopterin 96 568 478.2 9.20 -141 709 E0006962_9114. t) Molydop_binding 714 822 121.4 2.40 710 E0006963_9131. t) Pyr_redox_2 5 287 191.2 2.30 710 E0006963_9131. t) Pyr_redox 147 242 100.4 4.80 710 E0006963_9131. t) Fer2_IBFD 422 474 92.1 1.50 710 E0006963_9131. t) NIR_SIR_ferr 556 623 82 1.70 710 IPHE0006963_9131.pe NIR_SIR 631 777 166.4 6.70 711 PHE0006965_9119.jpe RNA-synt_2b 67 243 57.5 3.90 711 PHE0006965_9119.jpe HGTP_anticodom 312 409) 100 6.40 713 PHE0006977_9163.pe Ribul_P_3_epim 7 207 332.8 5.30

"IABIH 16

accession gathering Pfam domain Illame Inumber Cutof domain description 2-Hacid_dh PF00389.18 13.2 D-isomer specific 2-hydroxyacid dehydrogenase, Catalytic domain 2-Hacid_dh_C PF02826.6 -75.7 D-isomer specific 2-hydroxyacid dehydrogenase, NAD bindingdomain 3Beta_HSD PF01073.8 -135.9 3-beta hydroxysteroid dehydrogenase/iSOmerase family 3_5_eXOIllC PF01612.10 -32 3-5 exonuclease AAA PF00004.17 10 ATPase family associated with various CellularactiVities (AAA) AA_kinase PF00696.16 -40 Amino acid kinase family AA_permease PF00324.10 -120.8 Amino acid permease ABC 1 PF03109.6 -27.6 ABC1 family ABC_tram PF00005.14 8.6 ABCtransporter ADH_N PF08240. -14.5 Alcohol dehydrogenase GroES-like domain ADH_Zinc_N PF00107.15 23.8 2inc-binding dehydrogenase AMP-binding PF00501.15 0 AMP-bindingenzyme AMPKFBI PF04739.4 25 5-AMP-activated protein kinase, beta Subunit, Complex-interacting region AP2 PF00847.9 0 AP2 domaim APS_kimase PF01583.9 25 AdenylylSulphate kinase ARID) PF01388.10 -8 ARID/BRIGHTDNAbindingdomain AI_hook PF02178.7 14.2 AThook motif AUX_IAA PF02309.6 -83 AUX/IAA family Aa_tranS PF01490.7 -128.4 TranSmembrane amino acid transporter protein Abhydrolase_1 PF00561.9 5.5 alpha/beta hydrolase fold Acetyltransf_1 PF00583.12 18.6 Acetyltransferase (GNAT) family Acyltransferase PF01553.10 6 Acyltransferase Aldedh PF00171.11 -295 Aldehyde dehydrogenase family Aldo_ket_red PF00248.10 -97 Aldo/keto reductase family Alpha-amylase PF00128.1 1 -93 Alpha anylase, Catalytic domain Alpha_adaptinC2 PF02883.9 -12 Adaptin C-terminal domain Aminotran 1 2 PF00155.9 -57.5 Aminotransferase class I and II Aminotran 3 PF00202.10 -207.6 Aminotransferase class-II Aminotram 5 PF00266.8 -92.9 Aminotransferase class-V US 201 1/0252501 A1 Oct. 13, 2011 55

"IABLE 16-continued accession gathering Pfam domain Illame Inumber Cutof domain description Ammonium_transp PF00909.10 -144 Ammonium Transporter Family Ank PF00023.17 21.6 Ankyrin Irepeat Annexim PF00191.8 8 Annexim ArfGap PF01412.8 - 17 Putative GTPase activating protein for Arf ASIll_Synthase PF00733.10 -52.8 ASparagine Synthase ASp PF00026.13 -186.1 Eukaryotic aspartyl protease Auxim_inducible PF02519.4 - 15 Auxin reSponsive protein Auxim_resp PF06507.3 25 Auxin reSponse factor IB12ID) PF06522.1 25 B12D protein IB3 PF02362.1 1 26.5 B3 DNA bindingdomain IB56 PF01603.8 -210 Proteimphosphatase2Aregulatory B subunit (B56 family) IBAH PF01426.6 7 BAHI domaim IBRC)1 PF03097.6 25 BRO1-like domaim IBURIP PF03181.5 -52 IBURIP domaim Bromodomain PF00439.13 8.9 Bromodomaim CAF1 PF04857.8 -100.5 CAF1 family ribonuclease CBFD_NFYB_HIMF PF00808.12 184 Histone-liketranscription factor (CBF/NF Y) and archaeal histone CBS PF00571.16 15.8 CBS domain pair CCT PF06203.3 25 CCTImotif CHI PF00307.18 22.5 Calponin homology(CH) domain CMAS PF02353.9 -177.9 Cyclopropane-fatty-acyl-phospholipid SynLinase CN_hydrolase PF00795.11 -13.9 Carbon-nitrogen hydrolase CTP_synth_IN PF06418.2 25 CTPsynthase N-terminus CTP_transf_2 PF01467.15 -11.8 Cytidylyltransferase Carb_kinase PF01256.7 -66.3 Carbohydrate kinase Catalase PF00199.8 -229 Catalase Cation_effilux PF01545.10 -95.7 Cation eflux family Chal_Sti_Synt_C PF02797.5 -6.1 Chalcone and stilbene synthases, C terminal domain Chromo PF00385.11 27.5 ‘chromo’(CHIRIromatin Organisation MOdifier) domain Citrate_synt PF00285.10 -101.5 Citrate synthase CobW_C PF07683.3 18 Cobalamin synthesisprotein cobWC terminal domain ComA PF02679.5 25 (2R)-phospho-3-sulfolactate synthase (ComA) CorA PF01544.8 -61.3 CorA-like Mg2+ transporterprotein Cpn10 PF00166.1 1 -7.8 Chaperomin 10 Kd subunit Cpn60_TCP1 PF001 18.13 -223.4 TCP-1/cpn60chaperonin family Cul-oxidase PF00394.1 1 -18.9 Multicopper oxidase Cul-oxidase_2 PF07731.3 -5.8 Multicopper oxidase Cul-oxidase_3 PF07732.4 0 Multicopper oxidase Cyclin_C PF02984.7 -13 Cyclin, C-terminal domain Cyclin_N PF00134.12 -14.7 Cyclin, N-terminal domain Cyclotide PF03784.3 25 Cyclotide family Cys_Met_Meta_PP PF01053.9 -278.4 Cys/Met metabolism PLP-dependent enzyme Cystatim PF00031.10 7.5 Cystatin domain DAC) PF01266.1 1 -36.5 FAID dependent Oxidoreductase DNA_photolyase PF00875.7 -10 DNAphotolyase DSPC PF00782.9 -21.8 Dual Specificityphosphatase, Catalytic domain DUF125 PF01988.8 -10.1 Integral membrane protein DUF125 DUJIF1423 PF07227.1 25 Protein ofunknown function (DUF1423) DUF1530 PF07060.1 25 ProFARisomeraSeassociated DUJIF1685 PF07939.1 25 Protein ofunknown function (DUF1685) DUJF246 PF03138.4 - 15 Plant protein family DUF250 PF03151.6 125 Domain ofunknown function, IDUF250 DUJF296 PF03479.4 -11 Domain ofunknown function (DUF296) DUF393 PF04134.2 25 Protein ofunknown function, IDUF393 DUF581 PF04570.4 -3.1 Protein ofunknown function (DUF581) DUF6 PF00892.9 30 Integral membrane protein DUF6 DUF641 PF04859.2 25 Plant protein ofunknown function (DUF641) DUF760 PF05542.1 25 Protein ofunknown function (DUF760) DUF788 PF05620.1 25 Protein ofunknown function (DUF788) Dehydrin PF00257.8 -4.4 Dehydrin Di19 PF05605.2 25 Drought induced 19protein (Di19) Dirigent PF03018.4 25 Dirigent-like protein DimaJ PF00226.18 -8 Dina.J domain US 201 1/0252501 A1 Oct. 13, 2011 56

"IABLE 16-continued

accession gathering Pfam domain Illame Imumber cutof domain description ID) F00676.9 -90 IDehydrogenase IE1 Compoment ID) F02319.9 17 E2F/DPfamily winged-helix DNA binding domain F03271.6 25 EB1-like C-terminal Imotif EF1_GNE F00736.8 20 IEF-1guanine Illicleotide exchange domain ELFV_dehydrog F00208.1 0 -27 Glutamate/Leucine/Phenylalanine/Valine dehydrogenase ELFV_dehydrog_N F02812.7 31.8 Glu/Leu/iPhe/Val dehydrogenase, dineriSation domain ERC)1 F04137.5 - 179.5 EndoplaSmic Reticulum Oxidoreductim 1 (ERO1) ERip29 F07749.2 10.5 Endoplasmic reticulumprotein ERp29, C erminal domain Epinerase F01370). 0 -46.3 NAD dependentepimerase/dehydratase anily F-box F00646.2 0 12.4 F-box domain FAD_binding_3 F01494.8 -136.6 FAD bindingdomain FAID_binding_4 -8.1 FAD bindingdomain FAD_binding_7 25 FAD bindingdomain of DNAphotolyase FAE_3-kCoA_syn1 25 Fatty acid elongase 3-ketoacyl-COA Synthase 1 FA_deSaturase F00487. -46 Fatty acid deSaturase FBA_1 F07734.2 -39.4 F-boxassociated FBIPase F00316.9 - 170.3 Fructose-1-6-bisphosphatase FGGY_N F00370). 0 - 104.7 FGGYfamily ofcarbohydrate kinases, N erminal domain FHA F00498. 3 25 FHA domaim Fer4 F00037. 4 4Fe-4S bindingdomain CAF F01590. 4 23 GAF domain CAT F03127.4 GAI domaim CAIA F00320. 28.5 GATAzinc finger CAIaSe F001 17. -38.1 Ultamine amidotransferase ClaSS-II CAIaSe_2 F00310. -106.2 Ultamine amidotransferases ClaSS-II GFO_IDH_MocA F01408. -7.2 xidoreductase family, NAD-binding OSSmann fol GFO_IDH_MocA_C F02894.7 Xidoreductase family, C-terminal lpha/beta domaim CH3 F03321.3 -336 H3 aluxin-reSponsive promoter CIIDA F01134. 1 -226.7 ucose inhibited division protein A CIRAS F03514.4 -78 RAS family transcription factor CIRIM-19 F06212. 25 RIM-19protein GSHIPX F00255.9 -16 Ultathione peroxidase GST_C F00043. 3 22.3 utathione S-transferase, C-terminal C)Imain GST_N ID) F02798.8 14.6 utathione S-transferase, N-terminal C)Imain GTP_EFTU F00009. 4 ongation factor Tu GTP bindingdomain GTP_EFTU_ID2 F03144. 3 25 OIngation factor Tu domain 2 GTP_EFTU_ID3 F03143.6 14.3 OIngation factor Tu C-terminal domain Gamma-thionin F00304. 0 9.6 amma-thionin family In-Synt_C F00120. 3 -124 Ultamine Synthetase, Catalytic domain Im-synt_N F03951.8 Ultamine Synthetase, beta-Grasp domain obin F00042. 1 -8.8 obin yCO_hydro_1 F00232.8 -301.8 ycoSyl hydrolase family 1 yCO_hydro_14 F01373.7 -231.4 ycoSyl hydrolase family 14 yCO_hydro_16 F00722.9 -65 ycosyl hydrolases family 16 yco_hydro_38 F01074. 1 - 125.3 ycosyl hydrolases family 38N-terminal C)Imain F07748.2 -93.1 ycosyl hydrolases family 38 C-teminal Imain yCO_transf_20 F00982.9 -243.6 ycosyltransferase family 20 yCOgell_SyIll F05693.2 -492.3 ycogen Synthase ycolytic F00274.8 - 158 IructOSe-bisphosphate aldolase ClaSS-II yCOS_transf_1 F00534.9 -7.3 ycoSyl transferases group 1 yCOS_transf_2 F00535.1 4 17.6 ycoSyl transferase family 2 yoxalase F00903.1 4 12.1 yoxalase/BleomyCin Iresistance otein/Dioxygenase Superfamily C) t 1 ID) F04178.2 25 ot1 -like family F02800.8 -64.1 yceraldehyde 3-phosphate dehydrogenase, C-terminal domain F00044.1 1 -74.2 Glyceraldehyde 3-phosphate dehydrogenase, NAD bindingdomain HAILZ F02183.7 17 Homeobox aSSociated leucine Zipper US 201 1/0252501 A1 Oct. 13, 2011 57

"IABLE 16-continued

accession gathering Pfam domain Illame Imumber cutof domain description

IAMP ID) F00672.1 3 7 HAMP domain HAIPaSe_C ID) F02518.1 3 22.4 istidine kinase-, DNA gyrase B-, and SP90-like ATPase IFAT F02985.9 7.6 HEATrepea IFM4 F02602.5 -12 Uroporphyrinogen-Isynthase HemD HGTP_anticodom F03129.9 -2 Anticodon binding domain I0933like F03486.4 -255.8 I0933-like protein IIII F00010. 5 8.2 elix-loop-helix DNA-bindingdomain HMA F00403. 4 7.4 eavy-metal-aSSociated domain HMG_box F00505.8 4.1 MG (high mobility group)box HSF_IDNA-bind F00447.7 -70 SF-type DNA-binding ISP20 F00011.9 3 Sp20/alpha CryStallin family H_PPase -377 Imorganic H+ pyrophOSphatase 1eme_oxygenase -58 1eme Oxygenase 1exapep 20 Bacterial transferase hexapeptide (three Irepeats) Hexokinase_l -110.3 Hexokinase Hexokinase_2 -131.3 Hexokinase HiSKA is Kinase A (phosphoacceptor) domain ist_deacetyl F00850.9 Histone deacetylase domain Histome F00125. Core histome H2A/H2B/H3/H4 Homeobox F00046. Homeobox domain Hipt F01627. Hipt domain Hydrolase F00702. haloacid dehalogenase-like hydrolase CIL F00463.9 Socitrate lyase family F01652.8 Eukaryotic initiation factor 4E F03770.6 InOSitol polyphosphate kinase VC F01450.8 -33.8 Acetohydroxy acid iSOneroreductase, Catalytic domain VN F07991. -75.8 Acetohydroxy acid isomeroreductase, Catalytic domain Imhibitor_I29 F08246. 4.9 CathepSin propeptide inhibitordomain (I29) OIl_tralS F00520. 8 -4.5 On transport protein SOamylase_N F02922.7 -6.5 SOamylase N-terminal domain acalin F014.19.6 20 acalin-like lectin domain F02373. 1 InjC domain miN F02375.6 25 imjN domain K-box F01486.7 K-box 1egion KA1 F02149.9 25 Kinase aSSociated domain 1 F00013. 7 8.1 KHI domain Kelch_1 F01344. 3 20 Kelch Imotif Kelch_2 F07646.4 20 Kelch Imotif Ketoacyl-Synt_C F02801. 0 -54.9 Beta-ketoacyl Synthase, C-terminal domain KunitZ_legune F00197.8 -32 Trypsin and protease inhibitor LEA_5 F004777 25 Smal hydrophilic plant seed protein LIM F004.12. 0 0 LIMI domaim LRR_2 F07723.2 8.7 Leucine Rich Repeat LactamaSe_B F00753. 5 22.3 Metallo-beta-lactamase Superfamily Ldh_1_C F02866.6 -13 actate/malate dehydrogenase, alpha/beta C-terminal domain ID) F00056. -31.3 actate/malate dehydrogenase, NAD bindingdomain Lectim_legA F00138.7 19 Legume lectins alpha domain Lectim_legB F00139.9 -77 Legume lectins beta domain Lig_Chal F00060. 6 8.2 Ligand-gated iOn Channel Lipase_GDSL F00657. 1 10.9 GDSL-like Lipase/Acylhydrolase M20_dimer F07687.3 12 Peptidase dimeriSation domain MAP1_ILC3 F02991.5 -18.8 Microtubule associated protein 1A/1B, ight Chain 3 MIFMR F07777. -46.7 G-box binding protein MIFMR MFS_1 F07690.4 23.5 Major Facilitator Superfamily MIP F00230.8 -62 Majorintrinsic protein Malic_M F03949.4 -143.9 Malic enzyme, NAD bindingdomain Math F01554.8 59.6 Math Metallophos F00149.1 6 22 Calcineurin-like phOSphoesterase Metallothio_2 F01439.7 -3 Metallothiomeim Meth_synt_1 F08267.1 - 167.8 Cobalamin-independent Synthase, N terminal domain Meth_synt_2 F017177 - 155 Cobalamin-independent Synthase, Catalytic domain Methyltransf_11 F08241.1 17.1 Methyltransferase domain US 201 1/0252501 A1 Oct. 13, 2011 58

"IABLE 16-continued

accession gathering Pfam domain Illame Imumber cutof domain description Methyltransf_12 ID) F08242.1 21.4 Methyltransferase domain Methyltransf_2 ID) F00891.7 - 103.8 O-methyltransferase Methyltransf_3 ID) F015967 -120.6 O-methyltransferase Mpv17_PMP22 ID) F041 17.2 -5.4 Mpv17/IPMP22 family MtN3_slv ID) F03083.5 -0.8 MtN3/saliva family Myb_IDNA-binding ID) F00249.18 19.1 Myb-like DNA-bindingdomain NAC ID) F01849.6 0 NAC domaim NAD_binding_4 ID) F07993.1 -87.7 Male sterilityprotein NAF ID) F03822.4 25 NAF domain NAM ID) F02365.5 -19 No apical meristem (NAM) protein NIDK ID) F00334.8 -59.9 NucleOSide diphosphate kinase NIF ID) F03031.7 -81 NLI interacting factor-like phOSphatase NPH3 ID) F03000.4 25 NPH3 family NTF2 ID) F02136.10 6 Nucleartransport factor 2 (NTF2) domain NTP_transferase ID) F00483.12 -90.5 Nucleotidtyl transferase NUIDIX ID) F00293.16 0 NUIDIX domaim Na_Ca_ex ID) F01699.12 25 Sodiuml/calcium exchangerprotein NifU_N ID) F01592.6 -13 NifU-like Nterminal domaim NimirA ID) F05368.2 -90.6 NImrA-like family Orn_Arg_deC_N ID) F02784.6 -76 Pyridoxal-dependent decarboxylase, pyridoxal bindingdomain Orm_IDAP_Arg_deC F00278.1 1 -34.9 Pyridoxal-dependent decarboxylase, C erminal Sheet domain Oxidored_FMIN F00724.8 - 147.7 NADH:fiaVim Oxidoreductase /NADH Oxidase family PA F02225.10 13 PA domain PAD_porph F04371.4 - 180.8 Porphyromonas-type peptidyl-arginine deiminase PARIP F00644.9 -55.5 Poly(ADP-ribose) polymerase catalytic domain PAS F00989. 20 PAS fold PB1 F00564. 12.1 PB1 domaim BID) F00786. 12.1 P21-Rho-binding domain CI F01399. 25 PCI domain DZ, F00595. 12.1 PDZdomain (Also known as DHR or GLGF) EP-utilizers F00391.12 10 PEP-utilisingenzyme, mobile domain EP-utilizers_C F02896.7 - 173 PEP-utilisingenzyme, TIMbarrel domain EPCaSe F0031 1.7 25 Phosphoenolpyruvate Carboxylase CAM F00300.1 1 -3 Phosphoglycerate Inutase family IID) F00628.16 25.9 PHD-finger K F00224.10 -244 Pyruvate kinase, barrel domain KIC F02887.5 -44 Pyruvate kinase, alpha/beta domain MSR F01625.9 -62 Peptide methionine sulfoxide reductase P2C F00481.10 -44 Proteimphosphatase 2C PDK_N F01326.8 -87 Pyruvatephosphate dikinase, PEP/pyruvate bindingdomain RA1 F03208.8 25 PRA1 familyprotein SI_PsalF F02507.5 25 Photosystem Ireaction centre subunit II TR-2 F00854.1 1 -50 POTfamily UA F01472.8 2.2 PUA domaim Peptidase_A22B F04258.3 - 137.3 Signal peptide peptidaSe Peptidase_C1 F001 12.1 1 - 115.8 Papain family cySteine protease Peptidase_C15 F01470.7 -100 Pyroglutanyl peptidase Peptidase_M20 F01546.16 -14.4 Peptidase family M20/M25/M40 Peptidase_S10 F00450.1 1 - 198 Serine Carboxypeptidase Peptidase_S41 F03572.7 -25.8 Peptidase family S41 PflkB F00294.12 -67.8 pfikB family carbohydrate kinase Phytochrome F00360.9 11 Phytochrome region Pikimase F00069.14 -70.8 Proteinkinase domain Pikimase_C F00433.1 1 14 Proteim kimase Cterminal domaim Pikinase_Tyr F07714.4 65 Proteintyrosine kinase PolySacC_Synt_2 F02719.5 - 176 Polysaccharide biosynthesisprotein Pro_CA F00484.8 -45 Carbonic anhydrase Pro_dh F01619.7 - 120.5 Proline dehydrogenase Pyr_redox F00070.16 5 Pyridine nucleotide-disulphide Oxidoreductase Pyr_redox_2 F07992.2 -20 Pyridine nucleotide-disulphide Oxidoreductase Pyr_redox_dim F02852.1 1 -13 Pyridine nucleotide-disulphide Oxidoreductase, dineriSation domain Pyridoxal_deC F00282.8 - 158.6 Pyridoxal-dependent decarboxylase ConSerVed domain US 201 1/0252501 A1 Oct. 13, 2011 59)

"IABLE 16-continued

accession gathering Pfam domain Illame Imumber cutof domain description RHID3 ID) F05879.2 25 Roothair defective 3 GTP-binding protein (RHID3) RIC)1 F01163.1 1 -89.1 RIO1 family RRM_1 F00076.10 15.2 RNA recognition motif (a.k.a. RRM, RBD, or RNP domain) RTC F01137.1 1 -36.9 RNA 3-terminalphosphate cyclase RTC_imSert ID) F05189.3 25 RNA 3-terminalphosphate cyclase (RTC), insert domain RWIP-RIK F02042.5 25 RWIP-RK domaim Ran_BP1 F00638.8 -38 RamBPI domain RaS F00071.1 1 18 Ras family Remorim_C F03763.3 25 Remorin, C-terminal region ResponSe_reg F00072.1 1 -14.4 Response Iregulator receiver domain Reticulom F02453.7 -40 Reticulom Ribomuclease_T2 F00445.8 -53 Ribonuclease T2 family Ribosomal_IL1 F00687.10 -101 Ribosomal protein Lip/L10e family Ribosomal_IL10e F00826.7 25 Ribosomal IL10 Ribosomal_IL12 F00542.8 25 Ribosomal protein L7/L12 C-terminal domain Ribosomal_IL19e ID) F01280.9 -28 Ribosomal protein IL19e Ribosomal_L39 F00832.9 25 Ribosomal L39?protein Ribosomal_IL7Ae F01248.13 6 Ribosomal protein L7Ae/L30e/S12e/Gadd45 family Ribosomal_S11 F004.1 1.7 -4 Ribosomal protein S11 Ribosomal_S17 F00366.9 1.7 Ribosomal protein S17 Ribosomal_S2 F00318.9 -22 Ribosomal protein S2 Ribosomal_S27 F01599.8 50 Ribosomal protein S27a Rieske F00355.15 -7 Rieske [2Fe-2SI domain RImlID_Sub_bind F04321.6 - 171.8 RimillDSubstrate bindingdomain RuBisCO_Small F()()1()1.9 -20.1 Ribulose bisphosphate carboxylase, Smal Chain Rubrerythrin F02915.7 -4.8 Rubrerythrin SAM_1 F00536.17 1 1.3 SAMdomain (Sterile alpha motif) SAM_2 F07647.5 20 SAMdomain (Sterile alpha motif) SPC25 F06703.1 25 MicroSOImal Signal peptidase 25 kDa subunit (SPC25) SPX F03105.9 -20 SPX domaim SRF-TF F00319.8 11 SRF-type transcription factor (DNA binding and dimeriSation domain) START F01852.8 25 START domain SapB_1 F05184.4 20 SapoSin-like type B, Iregion 1 SapB_2 F03489.5 20 SapoSin-like type B, Iregion 2 SecY F00344.9 -210 eubacterial secYprotein SellR F01641.8 -66.5 Se1R domain Sigma70_r1_2 F00140.9 25 Sigma-70 factor, 1egion 1.2 Sigma70_r2 F04542.3 11 Sigma-701egion 2 Sigma7013 F04539.4 10 Sigma-701egion 3 Sigma70_r4 F04545.5 20.7 Sigma-70, Iregion 4 Sima F03145.6 -48.4 Sevenin absentia protein family Steroid_dh F02544.6 -44.7 3-OXO-5-alpha-Steroid 4-dehydrogenase Suc_Fer-like F06999.2 -42.4 Sucrase/ferredoxim-like Succ_IDH_fiaV_C F02910.9 Fumarate IreductaSe/Succinate ehydrogenase flavoprotein C-terminal OImain Sulcrose_Synth F00862.9 -134 Sulcrose Synthase Sugar_tr F00083.12 -85 Sugar (and other) transporter Synaptobrevin F00957.9 25 Synaptobrevin TPP_enzyme_C F02775.9 19.7 Thiamine pyrophosphate enzyme, C terminal TPPbinding domain TPP_enzyme_M F00205.1 1 -23.9 Thiamine pyrophosphate enzyme, Central OImain TPP_enzyme_N F02776.7 -70 Thiamine pyrophosphate enzyme, N erminal TPPbinding domain Thiolase_C F02803.6 -30.7 Thiolase, C-terminal domain Thiolase_N F00108.1 1 - 129.5 Thiolase, N-terminal domain Thioredoxim F00085.8 -25.7 Thioredoxim Tic22 F04278.2 25 Tic22-like family TranSaldolase F00923.8 TranSaldolase Transferase F02458.5 -161.2 Transferase family TranSket_pyr F02779.12 -50 TranSketolase, pyridine binding domain US 201 1/0252501 A1 Oct. 13, 2011 60

"IABLE 16-continued accession gathering Pfam domain Illame Inumber Cutof domain description Transketolase_C PF02780.9 -15.5 Transketolase, C-terminal domain Transketolase_N PF00456.10 -98 TranSketolase, thiamine diphOSphate binding domain Trehalase PF01204.8 25 Trehalase Trehalase_Ca-bi PF07492.1 20 Neutral trehalase Ca2+ binding domain Trehalose_PPase PF02358.6 -49.4 Trehalose-phosphatase Trp_Tyr_perm PF03222.3 -232.6 Tryptophan/tyrosine pemease family Trp_SyntA PF00290.10 -149.8 Tryptophan synthase alpha chain Trypsin PF00089.13 -33.2 Trypsin Tub PF01167.7 -98 Tub family Tubulim PF00091.14 -55.7 Tubulin/FtsZ family, GTPase domain Tubulim_C PF03953.6 -10 Tubulin/FtsZ family, C-teminal domain UIBA PF00627.18 20.5 UBA/TS-N domaim UIDPGP PF01704.7 -265.2 UTP--glucose-l-phosphate uridtylyltransferase UIDPGT PF00201.8 - 151 UDP-glucoronosyl and UDP-glucosyl transferase UPF0057 PF01679.7 25 Uncharacterized protein family UPF0057 UbiA PF01040.8 -45 UbiAprenyltransferase family Ubie_methyltran PF01209.8 - 117 ubiE/COQ5 methyltransferase family Usp PF00582.15 25.7 Universal stressprotein family VHS PF00790.8 -13.2 VHS domain VQ PF05678.3 25 VQ motif W72 PF02020.7 25 eIF4-gamma/eIF5/eIF2-epsilon WID40 PF00400.19 21.4 WD domain, G-beta repeat WHIEP-TRS PF00458.9 10 WHIEP-TRS domaim WRKY PF03106.5 25 WRKY DNA -bindingdomain Wzy_C PF04932.4 25 O-Antigen Polymerase XIET_C PF06955.2 11.4 Xyloglucan endo-transglycoSylase (XIET) C-terminus Xan_ur_permease PF00860.10 -151.2 Permease family YI, 1 PF05764.3 25 YIL1 muclearprotein YLI_C PF08265 18.6 YTL1 muclearprotein C-teminal domain YTH PF04146.5 25 YT521-B-like family Yippee PF03226.4 25 Yippeeputative Zinc-binding protein YeF_N PF03853.3 25 YjeF-related protein N-terminus 2F-HD_dimer PF04770.2 25 ZF-HD protein dimerisation region Zip PF02535.10 -28 ZIPZinctransporter adh Short PF00106.13 -46.6 Short Chain dehydrogenase bZIP_1 PF00170.10 6.5 bZIPtranscription factor bZIP_2 PF07716.4 5 Basic region leucine Zipper cNMIP_binding PF00027.17 20.6 Cyclic Inucleotide-binding domain cobW PF02492.8 -10 CobWHypB/UreG, Inucleotide-binding domain efthand PF00036.19 7.5 EF hand ketoacyl-Synt PF00109.14 -73.6 Beta-ketoacyl synthase, N-terminal domain Imalic PF00390.8 25 Malic enzyme, N-terminal domain p450 PF00067.11 - 105 Cytochrome P450 peroxidase PF00141.12 -10 Peroxidase RNA synthetase class Icore domain (G, tRNA-synt_2b PF00587.14 -40.5 H, P. Sand T) ubiquitin PF00240.12 9.4 Ubiquitin family Zf-B_box PF00643.13 1.1 B-box Zinc finger Zif-C2H2 PF00096.14 9 Zinc finger, C2H2 type Zif-C3H(C4 PF00097.12 6.9 Zinc finger, C3HC4 type (RING finger) Zf-Dof PF02701.5 25 Dof domain, Zinc finger Zf-LSD1 PF06943.2 25 LSD1 zinc finger

Example 10 in EXample 6."Iransgenic plant CellSOfcorn, Soybean, Cotton, Canola, Wheat and rice are transformed With recombinant Selection of Transgenic Plants with Enhanced Agro DNA for expressing each of the homologs identified in Inomic Irait(S) Example 6. Plants areregenerated from thetransformediplant |0129|| This exampleillustrates the preparation and identi Cells and uSed to produce progeny plants and Seed that are fication by Selection of transgenic Seeds and plants derived Screened for enhanced Water use efficiency, enhanced Cold from transgenic plant Cells ofthiSinvention Where theplants tolerance, increased yield, enhanced Initrogen use eficiency, and Seed are identified by Screening a having an enhanced enhanced Seed protein andenhanced Seed Oil. PlantSareiden agronomic traitimparted by expreSSion Ofa protein Selected tifiedexhibitingenhanced traitsimparted byexpression ofthe from the group including the homologouSproteinSidentified homologouSproteimS.

US 201 1/0252501 A1 Oct. 13, 2011 62

2. Theplant cell nucleus of claim 1 Wherein Said protein (b)Selecting from Said population One Ormoreplants that COding IDNA encodes a protein having an amino acid exhibit Said traitatalevel greaterthan thelevel that Said sequence Selected from the group consisting of SEQ IDINC): traitis exhibited in control plants: and 350through SEQIDNO: 30327. (c) Collecting Seed from Selected plantSSelected from Step 3. The plant cell nucleus of claim 1 further comprising |b IDNA expreSSingaprotein that provideStolerance from expo 11. Themethod ofclaim 10 further comprising Sure to an herbicide applied at levels thatare lethal to a wild type of Said plant Cell. (d) verifying that Said recombinant DNA is Stably inte 4. Theplant cell nucleus of claim 3 Wherein the agent of grated in Said Selected plants: and Said herbicide is a glyphOSate, dicamba, OrglufOSinate Com (e) analyZing tiSSule OfSaid Selected plant to determine the pound. expreSSion Or SuppreSSion Of a gene that encodes an 5. Atransgenic plant Cell Orplant CompriSingaplurality of proteinhaving the function Ofaproteinhavingan amino plant cells with theplant cell nucleus of claim 1. acid Sequence Selected from the group ConSiSting ofone 6. The transgenic plant cell or plant of claim 5 Which is ofSEQIDNO358-716 homozygous for Said recombinant DNA. 12. Amethod Ofproducing hybrid Corn Seed CompriSing: 7. A transgenic Seed CompriSing a plurality of plant Cells (a) acquiring hybrid Corn Seed from a herbicide tolerant with theplant cell nucleus of claim 1. Corn plant Which alSo has Stably-integrated, recombi 8. Ihe transgenic Seed of Claim 7 from a Corn, Soybean, Imant IDNA in a nucleus of claim 1: Cotton, Canola, alialia, Wheat Orriceplant. (b) producing Corn plants from Said hybrid Corn Seed, 9. A transgenic pollen grain CompriSing a haploid deriva wherein a fraction of the plants produced from Said tive ofthe plant cell nucleus ofclaim 1. hybrid Corn Seed is homOZygous for Said recombinant 10. A method for Imanufacturing InOn-matural, transgenic DNA, a fraction oftheplantsproduced from Said hybrid seed of claim 7that can be used toproduce a crop oftrans Corn SeediShemiZygouSforSaidrecombinant IDNA, and genic plants With an enhanced traitreSultingfrom expreSSion a fraction ofthe plants produced from Said hybrid corn of Stably-integrated recombinant DNA wherein Said method formanufacturing Said transgenic Seed CompriSing: Seed has mone of Said recombinant IDNA: (a) Screeningapopulation of plants for Said enhanced trait (c) Selecting Cormplants Which are homOZygouSand hem and Said recombinant DNA Whereinindividual plants in izygous for Said recombinant DNA by treating with an Said population Can exhibit Said traitat a level leSS than, herbicide: eSSentially the Same aSOrgreaterthan the level that Said (d) collecting Seed from herbicide-treated-surviving corn traitis exhibited in control plants which do not express plantSandiplanting Said Seed toproduce furtherprogeny the recombinant IDNA, Wherein Said enhanced trait is Corn plants: Selected from the group ofenhanced traits ConSiSting of (e) repeating StepS (c) and (d) at least Once toproduce an enhanced WateruSeeficiency, enhanced Cold tolerance, inbred Corn line: and enhanced heat tolerance, enhanced reSiStance to Salt (f) CrOSSing Said inbred Corn line With a Second Corn line to expOSure, enhanced Shade tolerance, increased yield, produce hybrid Seed. enhancedmitrogenuSeeficiency, enhanced Seed protein and enhanced Seed Oil: