US 2015O1841 89A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2015/0184189 A1 Abad et al. (43) Pub. Date: Jul. 2, 2015

(54) GENES ENCODING GLUTAMINE (60) Provisional application No. 60/679,917, filed on May SYNTHETASE AND USES FOR PLANT 10, 2005, provisional application No. 60/723,596, IMPROVEMENT filed on Oct. 4, 2005. (71) Applicant: Monsanto Technology LLC, St. Louis, Publication Classification MO (US) (51) Int. Cl. (72) Inventors: Mark Scott Abad, Webster Groves, MO CI2N 5/82 (2006.01) (US); Marie Coffin, Cary, NC (US); (52) U.S. Cl. Barry S. Goldman, St. Louis, MO (US) CPC ...... CI2N 15/8273 (2013.01); C12N 15/8261 (2013.01); C12N 15/8251 (2013.01); CI2N (21) Appl. No.: 14/544,259 15/8247 (2013.01) (22) Filed: Dec. 12, 2014 (57) ABSTRACT O O Transgenic seed for crops with improved traits are provided Related U.S. Application Data by trait-improving recombinant DNA in the nucleus of cells (63) Continuation of application No. 13/694,398, filed on of the seed where plants grown from Such transgenic seed Nov. 28, 2012, now abandoned, which is a continu exhibit one or more improved traits as compared to a control ation of application No. 12/459,621, filed on Jul. 2, plant. Of particular interest are transgenic plants that have 2009, now Pat. No. 8,343,764, which is a continuation increased yield. The present invention also provides recom of application No. 1 1/431,855, filed on May 10, 2006, binant DNA molecules for expression of a protein, and now abandoned. recombinant DNA molecules for suppression of a protein. Patent Application Publication Jul. 2, 2015 Sheet 1 of 3 US 2015/O184189 A1

Figure 1. Consensus sequence of SEQ ID NO:601 and its homologs SEQ ID NO 601 ------MDITVRISGLKSSNLIRITPRFS--SSSRFRCSNNEPPRKGNESNAGGGGDK 32524 ------MDITVRISGLKSSNLIRIT PRFS.--SSSRFRCSNNEPPRKGNESNGGGG-DK 34O4 MQMDVYTHISLPSFISHRFSSSRFQCSSLSS-YSPFKLVCSNRDSKESQRNDDNNNKGDK 2616 ---MQMDIVYTPSFISHRFSSSRHQCPSLSSSYSPFKLFCSNRDFKESQRNDDNNNKGDK 8168 ------MDVLLGSGRFLARRPP-LALVPRCSRGSPDKSG-SDKGET 8488 ------MDVLLGSGRFLARRPP-LALVPRCSRGSPDKSG-SDKGET 8557 ------MDVLLGSGRFLARRPP-LALVPRCSRGSPDKSG-SDKGET 7495 ------MDVLLGSGRFLARRPP-LALVPRCSRGSPDKSG-SDKGET 8625 ------MDILLGSGRFLARRTP-LMLVPRCSRGSRDRSG-SDKGET 8626 ------MDILLGSGRFLARRTP-LMLVPRCSRGSRDRSG-SDKGET 4535 ------MDILLGSGRFLARRLPPLALVPRCSRGSPDKSG-SDKGET 62.55 ------MGILLVSGRFL.ARRPP-LALAPRCSRGSPDKRG-RDEGDT 44 58 ------MDTLLGSGRFLARRPP-LALAPRCSRGSPEKGGGSDKGDT 5207 ------MISSDT 6640 ------Consensus S ------XXXXXXXXrfxXXXXXX lxxxxrxxxx SXxxxxxxxxgxx ASTDWDKAWKNFKKQSKKSLFSQFNVDKYVTWNPPRSEFDLSEEVDPIKRT-ERSNLMLW ASTDWDKAWKNFKKOSKKSLFSQFNVDKYVTWNPPRSEFDLSEEVDPIKRT-ERSNLMLW SSTDWDKAWSKFKKOGGKKPFSKF-SDKYVSWNPKRSEFPLSEEVDPIKRT-ERSNLSFW SSTDWDKAWSKFKKOGGKKPFSKF-SDKYVSWNPRRSEFPLSEEVDPIKRT-ERSNLSFW S-ADWDKAWSAFKKKGKRTLFSDFSPDKYVTWNPRRSEYPLSEEVDPIKRS-ERSNLMLW S-ADWDKAWSAFKKKGKRTLFSDFSPDKYVIWNPRRSEYPLSEEVDPIKRS-ERSNLMLW S-ADWDKAWSAFKKKGKRTLFSDFSPDKYVTWNPRRSEYPLSEEVDPIKRT-ERSNLMLW S-ADWDKAWSAFKKKGKRTLFSDFSPDKYVTWNPRRSEYPLSEEVDPIKRT-ERSNLMLW S-ADWDKAWAKFKKKGKRTLFSDFSPDKYVTWNPRRSEYPLSEEVDPIKRTERSN-LMLW S-ADWDKAWAKFKKKGKRTLFSDFSPDKYVT WNPRRSEY PLSEEVDPIKSOLKDLNLMRC S-TDWDKAWASFKNKGKRTIFSDFSPNKYVTWNPRRSEY PLSEEWDPIKRT-ERSNLMW SSTDWDKAWSTFKKKGKKTLFSEFSPNKYVTWNPRRSEY PLSEEVDPIKRA-ERSNLMLW SSTDWDKAWSTFKKKGKKTLFSEFSPNKYWSWNPRRSEYPLSEEWDPIRRT-ERSNLMLW SSTEWDKAWSSLRKKGKKTLFSEFSPNKYVTCNPRREY PLSGRSRSXRAXRSNLML--

------FHTTKLTSSLFHDFSRIKLVTCNPWPTONPLPOOVDPIKRS-OTSQLXLW sxxdwdkawxxfkkkgkxxl Fs xF sixxKyVtwNPrrs expliseevdplkrx-ersnlmlw

TSPRFTLVGAIVIVSFLLLYTILAPVK------k TSPRFTLVGAIVIVSFLLLYTLAPVK------k NSPTFTLGGAI.IVLFILLLYTILAPIK------NSPTFTLGGAIIIWTFLILLYTILAPIK------k TSPQFTLVGAIIIVLTLLIYTLVVPPPK------se TSPQFTLVGAIIIVLTLLIYTLVVPPPK------TSPQFTLVGAIII VLTLLIYTLVVPPPK------r TSPQFTLVGAIII------TSPQFTLVGAIII VLTLLIYTLVVPPK------TSPQFRWVRRSSSSCHN------k TSPOFTLVGAI.I.IVLTLLIYTLVVPPPK------r TSPRFTLVGAIIIVSALLYTLVWPPK------sk TSPKFTLVMAIVIVSTLLYTVWPPK------k TSPQFPLDGAIII VSPPLINTOVWPPPKKLHRSQPPPPLTCNLAEKKKSSLHCTFST* tspxftlvgaixivxxllxytxxxpxxx------Patent Application Publication Jul. 2, 2015 Sheet 2 of 3 US 201S/O1841.89 A1

Figure 2.

G-St.Pisa

-AGRtu.nos B-AGRtu.left border

OR-EcoriV-RK2 CR-Ec.npt-Tn5 s S -CaMV.35S P-CaMW.35S

Corn transformation base vector CR-Ec.rop

OR-Ecori-CoIE1

P-Ec.aadA-SPCfSTR R-ECaadA-SPCISTR -OS.Act 1 -ECaadA-SPCfSTR

L-OS.Act 1 P-Os. Act 1

B-AGRtu.right bdrder Patent Application Publication Jul. 2, 2015 Sheet 3 of 3 US 201S/O1841.89 A1

Figure 3.

T-AGRtu.nos P-CaMV.35S-enh

B-AGRtu, right border

T-EcaadA-SPC/STR TS-Alt.ShkCG-CTP2 CR-Ec.aadA-SPC/STR I-At. Act 7 P-Ec.aadA-SPCASTR L-At. Act 7

OR-322 B-AGRtu.left border OR-Ecori-Col. 1

OR-Ec, oriV-RK2 US 2015/O 1841 89 A1 Jul. 2, 2015

GENESENCOOING GLUTAMINE BACKGROUND OF THE INVENTION SYNTHETASE AND USES FOR PLANT IMPROVEMENT 0006 Transgenic plants with improved traits such as improved yield, environmental stress tolerance, pest resis CROSS REFERENCE TO RELATED tance, herbicide tolerance, modified seed compositions, and APPLICATIONS the like are desired by both farmers and consumers. Although considerable efforts in plant breeding have provided signifi 0001. This application is a continuation of and claims the cant gains in desired traits, the ability to introduce specific benefit under 35 U.S.C. S 120 to U.S. application Ser. No. DNA into plant genomes provides further opportunities for 13/694,398, filed Nov. 28, 2012, which is a continuation of generation of plants with improved and/or unique traits. The and claims the benefit of priority under 35 U.S.C. S 120 to ability to develop transgenic plants with improved traits U.S. application Ser. No. 12/459,621, filed on Jul. 2, 2009, depends in part on the identification of useful recombinant which is a continuation of U.S. application Ser. No. 1 1/431, DNA for production of transformed plants with improved 855 filed on May 10, 2006, which claims benefit under 35 properties, e.g. by actually selecting a transgenic plant from a USC S119(e) of U.S. provisional application Ser. No. 60/679, screen for Such improved property. 917, filed May 10, 2005, and U.S. provisional application Ser. No. 60/723,596, filed Oct. 4, 2005, the benefit of priority of which are claimed hereby, and all of which are incorporated SUMMARY OF THE INVENTION herein by reference in their entirety. 0007. This invention provides plant cell nuclei with recombinant that imparts enhanced agronomic traits in trans INCORPORATION OF SEQUENCE LISTING genic plants having the nuclei in their cells. Recombinant 0002. Two copies of the sequence listing (Copy 1 and DNA in this invention is provided in a construct comprising a Copy 2) and a computer readable form (CRF) of the sequence promoter that is functional in plant cells and that is operably listing, all on CD-R's, each containing the file named 38-21 linked to DNA that encodes a protein having at least one (53708)D seqListing..txt, which is 97.937,408 bytes (mea amino acid domain in a sequence that exceeds the Pfam sured in MS-WINDOWS) and recorded on Dec. 10, 2014, are gathering cutoff for amino acid sequence alignment with a incorporated herein by reference in their entirety. protein domain family identified by a Pfam name in the group of Pfam domain names identified in Table 17. In more specific INCORPORATION OF TABLES embodiments of the invention plant cells are provided which

LENGTHY TABLES The patent application contains a lengthy table section. A copy of the table is available in electronic form from the USPTO web site (http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20150184189A1). An electronic copy of the table will also be available from the USPTO upon request and payment of the fee set forth in 37 CFR 1.19(b)(3).

0003. Two copies of Table 2 (Copy 1 and Copy 2) and a express a protein having amino acid sequence with at least computer readable form (CRF), all on CD-R's, each contain 90% identity to a consensus amino acid sequence in the group ing the file named 38-21 (53708)C table2.txt.txt, which is of consensus amino acid sequences consisting of the consen 331,776 bytes when measured in MS-WINDOWS(R) operat sus amino acid sequence constructed for SEQIDNO: 426 and ing system, was recorded on Dec. 10, 2014, are incorporated homologs thereof listed in Table 2 through the consensus herein by reference in their entirety. amino acid sequence constructed for SEQID NO: 850 and homologs thereof listed in Table 2. Amino acid sequences of INCORPORATION OF COMPUTER PROGRAM homologs are SEQID NO: 851 through 33634. In even more LISTING specific embodiments of the invention the protein expressed in plant cells is a protein selected from the group of proteins 0004 A Computer Program Listing with folders “hmmer identified in Table 1 by annotation to a related protein in 2.3.4” and “288pfamIDir' are contained on a CD-R and are Genbank and alternatively identified in Table 16 by identifi incorporated herein by reference in their entirety. Folder cation of protein domain family. himmer-2.3.2 contains the source code and other associated file for implementing the HMMer software for Pfam analysis. 0008. Other aspects of the invention are specifically Folder 288pfamDir contains 288 Pfam Hidden Markov Mod directed to transgenic plant cells, and transgenic plants com els. Both folders were recorded on the disk on Dec. 10, 2014, prising a plurality of plant cells with Such nuclei, progeny having a total size of 23.205,888 bytes when measured in transgenic seed, embryo and transgenic pollen from Such MS-WINDOWS(R) operating system. plants. Such plant cell nuclei are selected from a population of transgenic plants regenerated from plant cells with a nucleus FIELD OF THE INVENTION transformed with recombinant DNA by screening the trans genic plants in the population for an enhanced trait as com 0005 Disclosed herein are transgenic plant cells, plants pared to control plants that do not have the recombinant DNA and seeds comprising recombinant DNA and methods of in their nucleus, where the enhanced trait is enhanced water making and using Such plant cells, plants and seeds. use efficiency, enhanced cold tolerance, enhanced heat toler US 2015/O 1841 89 A1 Jul. 2, 2015

ance, enhanced shade tolerance, enhanced tolerance to salt nant DNA. The method further comprises producing corn exposure, increased yield, enhanced nitrogen use efficiency, plants from said hybrid corn seed, where a fraction of the enhanced seed protein and enhanced seed oil. In some aspects plants produced from said hybrid corn seed is homozygous of the invention the recombinant DNA expresses a protein for said recombinant DNA, a fraction of the plants produced that imparts the enhanced trait; in other aspects of the inven from said hybrid corn seed is hemizygous for said recombi tion the recombinant DNA expresses RNA for suppressing nant DNA, and a fraction of the plants produced from said the level of an endogenous protein. In yet another aspect of hybrid corn seed has none of said recombinant DNA; select the invention the nucleus of plant cells in plants, seeds, ing corn plants which are homozygous and hemizygous for embryo and pollen further comprise DNA expressing a pro tein that provides tolerance from exposure to an herbicide said recombinant DNA by treating with an herbicide; collect applied at levels that are lethal to a wildtype of said plant cell. ing seed from herbicide-treated-surviving corn plants and Such tolerance is especially useful not only as an advanta planting said seed to produce further progeny corn plants; geous trait in Such plants but is also useful in a selection step repeating the selecting and collecting steps at least once to in the methods of the invention. In aspects of the invention the produce an inbred corn line; and crossing the inbred corn line agent of such herbicide is a glyphosate, dicamba, or glufosi with a second corn line to produce hybrid seed. nate compound. 0012 Another aspect of the invention provides a method 0009. Yet other aspects of the invention provide nuclei is of selecting a plant comprising a nucleus of this invention in cells of transgenic plants which are homozygous for the its plant cells by using an immunoreactive antibody to detect recombinant DNA and transgenic seed of the invention from the presence of protein expressed by recombinant DNA in corn, Soybean, cotton, canola, alfalfa, wheat or rice plants. In seed or plant tissue. Another aspect of the invention provides other important embodiments for practice of various aspects anti-counterfeit milled seed having, as an indication of origin, of the invention in Argentina the recombinant DNA in the a nucleus of this invention with unique recombinant DNA. nucleus is provided in plant cells derived from corn lines that that are and maintain resistance to a virus Such as the Mal de 0013 Aspects of the invention relating to nucleus in plant Rio Cuarto virus or a fungus Such as the Puccina sorghi cells having recombinant DNA for Suppressing the expres fungus or to both. sion of a protein are identified in Table 1 and Table 16. More 0010. This invention also provides methods for manufac specific aspects of the invention provide plant cells having turing non-natural, transgenic seed that can be used to pro recombinant DNA for Suppressing the expression of a protein duce a crop of transgenic plants with an enhanced trait result having the function in a plant of the protein with amino acid ing from expression of stably-integrated, recombinant DNA sequence of SEQID NO: 426,428,429, 430, 524,525, 541, in the nucleus of the plant cells. In some aspects of the 601, 602, 650, 651, 654, 655, 657, 660, 694,698,772,801 or invention the recombinant DNA can express a protein having the corresponding Pfam identified in Table 16, i.e. Histone, at least one domain of amino acids in a sequence that exceeds WD40, NPH3, FHA, PB1, ADH zinc N, NAPRTase, ADK the Pfamgathering cutoff for amino acid sequence alignment lid, p450, B56, DUF231, C2, DUF568, WD40, F-box, Pki with a protein domain family identified by a Pfam name in the nase, Terpene synth, respectively. Such suppression can be group of Pfam names identified in Table 17; in other aspects effected by any of a number of ways known in the art, e.g. the recombinant DNA suppresses the level of a such a protein anti-sense Suppression, RNAi or mutation knockout and the More specifically the method comprises (a) screening a popu like. lation of plants for an enhanced trait and recombinant DNA, 0014) Another aspect of this invention relates to growing where individual plants in the population can exhibit the trait transgenic plants with enhanced water use efficiency or at a level less than, essentially the same as or greater than the enhanced nitrogen use efficiency. For instance, this invention level that the trait is exhibited in control plants which do not provides methods of growing a corn, cotton or soybean crop express the recombinant DNA; (b) selecting from the popu without irrigation water comprising planting seed having lation one or more plants that exhibit the trait at a level greater plant cells of the invention which are selected for enhanced than the level that said trait is exhibited in control plants; (c) water use efficiency. Alternatively methods comprise apply verifying that the recombinant DNA is stably integrated in ing reduced irrigation water, e.g. providing up to 300 milli said selected plants; (d) analyzing tissue of a selected plant to meters of ground water during the production of a corn crop. determine the production of a protein having the function of a This invention also provides methods of growing a corn, protein encoded by nucleotides in a sequence of one of SEQ cotton or soybean crop without added nitrogen fertilizer com ID NO:1-425; and (e) collecting seed from a selected plant. In prising planting seed having plant cells of the invention which one aspect of the invention the plants in the population further are selected for enhanced nitrogen use efficiency. Alterna comprise DNA expressing a protein that provides tolerance to tively methods comprise applying reduced amount of nitro exposure to an herbicide applied at levels that are lethal to gen input as compared to the conventional input during the wild type plant cells and where the selecting is effected by production of a corn crop. treating the population with the herbicide, e.g. a glyphosate, dicamba, or glufosinate compound. In another aspect of the 0015 The various aspects of this invention are especially invention the plants are selected by identifying plants with the useful for transgenic plant cells in seeds and transgenic plants enhanced trait. The methods are especially useful for manu having any of the above-described enhanced traits in crop facturing corn, soybean, cotton, alfalfa, wheat or rice seed plants such as corn (maize), soybean, cotton, canola (rape), selected as having one of the enhanced traits described above. wheat, Sunflower, Sorghum, alfalfa, barley, millet, rice, 0011. Another aspect of the invention provides a method tobacco, fruit and vegetable crops, and turfgrass. of producing hybrid corn seed comprising acquiring hybrid 0016. The invention also provides recombinant DNA con corn seed from a herbicide tolerant corn plant which also has structs comprising the DNA useful in the nuclei in plant cells a nucleus of this invention with stably-integrated, recombi for imparting enhanced traits in plants having those cells. US 2015/O 1841 89 A1 Jul. 2, 2015

BRIEF DESCRIPTION OF THE DRAWINGS may exist outside of the cell, for example as a PCR fragment, or integrated into a genome. Such as a plant genome. 0017 FIG. 1 is a consensus amino acid sequence of SEQ 0030. “Trait” means a physiological, morphological, bio ID NO: 601 and homologs. chemical, or physical characteristic of a plant or particular 0018 FIGS. 2 and 3 are plasmid maps. plant material or cell. In some instances, this characteristic is visible to the human eye. Such as seed or plant size, or can be DETAILED DESCRIPTION OF THE INVENTION measured by biochemical techniques, such as detecting the 0019. In the attached sequence listing: protein, starch, or oil content of seed or leaves, or by obser 0020 SEQID NO:1-425 are nucleotide sequences of the Vation of a metabolic or physiological process, e.g., by mea coding strand of DNA for “genes' used in the recombinant suring uptake of carbon dioxide, or by the observation of the DNA imparting an enhanced trait in plant cells, i.e. each expression level of a gene or genes, e.g., by employing North represents a coding sequence for a protein; ern analysis, RT-PCR, microarray gene expression assays, or 0021 SEQ ID NO:426-850 are amino acid sequences of reporter gene expression systems, or by agricultural observa the cognate protein of the 'genes' with nucleotide coding tions such as stress tolerance, yield, or pathogen tolerance. sequence 1-425; 0031. A “control plant' is a plant without trait-improving 0022 SEQID NO:851-33634 areamino acid sequences of recombinant DNA in its nucleus. A control plant is used to homologous proteins; measure and compare trait improvement in a transgenic plant with such trait-improving recombinant DNA. A suitable con 0023 SEQ ID NO:33635 is a consensus amino acid trol plant may be a non-transgenic plant of the parental line Sequence. used to generate a transgenic plant herein. Alternatively, a 0024 SEQ ID NO:33636 is a nucleotide sequence of a control plant may be a transgenic plant that comprises an plasmid base vector useful for corn transformation; and empty vector or marker gene, but does not contain the recom 0025 SEQID NO:33637 is a DNA sequence of a plasmid binant DNA that produces the trait improvement. A control base vector useful for soybean transformation. plant may also be a negative segregant progeny of hemizy 0026. The nuclei of this invention are identified by screen gous transgenic plant. In certain demonstrations of trait ing transgenic plants for one or more traits including improvement, the use of a limited number of control plants improved drought stress tolerance, improved heat stress tol can cause a wide variation in the control dataset. To minimize erance, improved cold stress tolerance, improved high salin the effect of the variation within the control dataset, a “refer ity stress tolerance, improved low nitrogen availability stress ence' is used. As use herein a “reference' is a trimmed mean tolerance, improved shade stress tolerance, improved plant of all data from both transgenic and control plants grown growth and development at the stages of seed imbibition under the same conditions and at the same developmental through early vegetative phase, and improved plant growth stage. The trimmed mean is calculated by eliminating a spe and development at the stages of leaf development, flower cific percentage, i.e., 20%, of the Smallest and largest obser production and seed maturity. Vation from the data set and then calculating the average of the 0027 “Gene” refers to chromosomal DNA, plasmid DNA, remaining observation. cDNA, synthetic DNA, or other DNA that encodes a peptide, 0032 “Trait improvement’ means a detectable and desir polypeptide, protein, or RNA molecule, and regions flanking able difference in a characteristic in a transgenic plant relative the coding sequences involved in the regulation of expres to a control plant or a reference. In some cases, the trait Sion. In aspects of the invention where an improved trait is improvement can be measured quantitatively. For example, provided by expression of a protein, “gene’ refers at least to the trait improvement can entail at least a 2% desirable dif coding nucleotide sequence for a protein or a function ference in an observed trait, at least a 5% desirable difference, polypeptide fragment of a protein that imparts the trait. In at least about a 10% desirable difference, at least about a 20% aspects of the invention where an improved trait is provided desirable difference, at least about a 30% desirable differ by Suppression of expression of an endogenous protein, ence, at least about a 50% desirable difference, at least about 'gene' refers to any part of the gene that can be a target for a 70% desirable difference, or at least about a 100% differ Suppression. ence, or an even greater desirable difference. In other cases, 0028 “Transgenic seed' means a plant seed whose the trait improvement is only measured qualitatively. It is nucleus has been altered by the incorporation of recombinant known that there can be a natural variation in a trait. There DNA, e.g., by transformation as described herein. The term fore, the trait improvement observed entails a change of the “transgenic plant' is used to refer to the plant produced from normal distribution of the trait in the transgenic plant com an original transformation event, or progeny from later gen pared with the trait distribution observed in a control plant or erations or crosses of a plant to a transformed plant, so long as a reference, which is evaluated by statistical methods pro the progeny contains a nucleus with the recombinant DNA in vided herein. Trait improvement includes, but is not limited its genome. to, yield increase, including increased yield under non-stress 0029) “Recombinant DNA” a polynucleotide having a conditions and increased yield under environmental stress genetically engineered modification introduced through conditions. Stress conditions may include, for example, combination of endogenous and/or exogenous elements in a drought, shade, fungal disease, viral disease, bacterial dis transcription unit, manipulation via mutagenesis, restriction ease, insect infestation, nematode infestation, cold tempera enzymes, and the like or simply by inserting multiple copies ture exposure, heat exposure, osmotic stress, reduced nitro of a native transcription unit. Recombinant DNA may com gen nutrient availability, reduced phosphorus nutrient prise DNA segments obtained from different sources, or DNA availability and high plant density. segments obtained from the same source, but which have 0033. Many agronomic traits can affect "yield, including been manipulated to join DNA segments which do not natu without limitation, plant height, pod number, pod position on rally exist in the joined form. A recombinant polynucleotide the plant, number of internodes, incidence of pod shatter, US 2015/O 1841 89 A1 Jul. 2, 2015 grain size, efficiency of nodulation and nitrogen fixation, ing densities per acre of a few examples of crop plants in the efficiency of nutrient assimilation, resistance to biotic and USA in the year 2000 were: wheat 1,000,000-1,500,000; rice abiotic stress, carbon assimilation, plant architecture, resis 650,000-900,000; soybean 150,000-200,000, canola 260, tance to lodging, percent seed germination, seedling vigor, 000-350,000, Sunflower 17,000-23,000 and cotton 28,000 and juvenile traits. Other traits that can affect yield include, 55,000 plants per acre (Cheikh, e.g., (2003) U.S. Patent efficiency of germination (including germination in stressed Application No. 20030101479). conditions), growth rate (including growth rate in stressed 0040 “Increased yield of a transgenic plant of the present conditions), ear number, seed number per ear, seed size, com invention is evidenced and measured in a number of ways, position of seed (starch, oil, protein) and characteristics of including test weight, seed number per plant, seed weight, seed fill. Also of interest is the generation of transgenic plants seed number per unit area (i.e., seeds, or weight of seeds, per that demonstrate desirable phenotypic properties that may or acre), bushels per acre, tons per acre, tons per acre, kilo per may not confer an increase in overall plant yield. Such prop hectare. For example, maize yield can be measured as pro erties include enhanced plant morphology, plant physiology duction of shelled corn kernels per unit of production area, or improved components of the mature seed harvested from e.g., in bushels per acre or metric tons per hectare, often the transgenic plant. reported on a moisture adjusted basis, e.g., at 15.5% moisture. 0034) “Yield-limiting environment’ means the condition Increased yield can result from improved utilization of key under which a plant would have the limitation on yield includ biochemical compounds, such as nitrogen, phosphorous and ing environmental stress conditions. carbohydrate, or from improved tolerance to environmental 0035 “Stress condition” means a condition unfavorable stresses, such as cold, heat, drought, salt, and attack by pests for a plant, which adversely affect plant metabolism, growth or pathogens. Trait-improving recombinant DNA can also be and/or development. A plant under the stress condition typi used to provide transgenic plants having improved growth cally shows reduced germination rate, retarded growth and and development, and ultimately increased yield, as the result development, reduced photosynthesis rate, and eventually of modified expression of plant growth regulators or modifi leading to reduction in yield. Specifically, “water deficit cation of cell cycle or photosynthesis pathways. stress' used herein preferably refers to the sub-optimal con ditions for water and humidity needed for normal growth of 0041) “Expression” means transcription of DNA to pro natural plants. Relative water content (RWC) can be used as a duce RNA. The resulting RNA may be without limitation physiological measure of plant water deficit. It measures the mRNA encoding a protein, antisense RNA, or a double effect of osmotic adjustment in plant water status, when a stranded RNA for use in RNAi technology. Expression also plant is under stressed conditions. Conditions which may refers to production of encoded protein from mRNA. result in water deficit stress include heat, drought, high salin 0042. A “plant promoter' is a promoter capable of initiat ity and PEG induced osmotic stress. ing transcription in plant cells whether or not its origin is a 0036 “Cold stress” means the exposure of a plant to a plant cell. Exemplary plant promoters include, but are not temperatures below (two or more degrees Celsius below) limited to, those that are obtained from plants, plant viruses, those normal for a particular species or particular strain of and bacteria which comprise genes expressed in plant cells plant. Such Agrobacterium or Rhizobium. Examples of promoters 0037 “Nitrogen nutrient’ means any one or any mix of the under developmental control include promoters that prefer nitrate salts commonly used as plant nitrogen fertilizer, entially initiate transcription in certain tissues, such as leaves, including, but not limited to, potassium nitrate, calcium roots, or seeds. Such promoters are referred to as “tissue nitrate, sodium nitrate, ammonium nitrate. The term ammo preferred. Promoters which initiate transcription only in cer nium as used herein means any one or any mix of the ammo tain tissues are referred to as “tissue specific''. A "cell type' nium salts commonly used as plant nitrogen fertilizer, e.g., specific promoter primarily drives expression in certain cell ammonium nitrate, ammonium chloride, ammonium Sulfate, types in one or more organs, for example, Vascular cells in etc roots or leaves. An “inducible' or “repressible' promoter is a 0038 “Low nitrogen availability stress' means a plant promoter which is under environmental control. Examples of growth condition that does not contain sufficient nitrogen environmental conditions that may effect transcription by nutrient to maintain a healthy plant growth and/or for a plant inducible promoters include anaerobic conditions, or certain to reach its typical yield under a Sufficient nitrogen growth chemicals, or the presence of light. Tissue specific, tissue condition. For example, a limiting nitrogen condition can preferred, cell type specific, and inducible promoters consti refers to a growth condition with 50% or less of the conven tute the class of “non-constitutive' promoters. A "constitu tional nitrogen inputs. “Sufficient nitrogen growth condition' tive' promoter is a promoter which is active under most means a growth condition where the soil or growth medium conditions. As used herein, “antisense orientation' includes contains or receives optimal amounts of nitrogen nutrient to reference to a polynucleotide sequence that is operably linked Sustain a healthy plant growth and/or for a plant to reach its to a promoter in an orientation where the antisense strand is typical yield for a particular plant species or a particular transcribed. The antisense Strand is sufficiently complemen strain. One skilled in the art would recognize what constitute tary to an endogenous transcription product such that trans Such soil, media and fertilizer inputs for most plant species. lation of the endogenous transcription product is often inhib 0039. “Shade stress' means a growth condition that has ited. limited light availability that triggers the shade avoidance 0043. As used herein, “operably linked refers to the asso response in plant. Plants are subject to shade stress when ciation of two or more nucleic acid fragments on a single localized at lower part of the canopy, or in close proximity of nucleic acid fragment so that the function of one is affected by neighboring vegetation. Shade stress may become exacer the other. For example, a promoter is operably linked with a bated when the planting density exceeds the average prevail coding sequence when it is capable of affecting the expres ing density for a particular plant species. The average prevail sion of that coding sequence (i.e., that the coding sequence is US 2015/O 1841 89 A1 Jul. 2, 2015 under the transcriptional control of the promoter). Coding constructing recombinant DNA for the use in the plant cells of sequences can be operably linked to regulatory sequences in this invention. Hidden Markov Model databases for use with sense or antisense orientation. HMMER software in identifying DNA expressing protein in 0044. A “consensus sequence” refers to an artificial, a common Pfam for recombinant DNA in the plant cells of amino acid sequence of conserved parts of the proteins this invention are also included in the appended computer encoded by homologous genes, e.g., as determined by a listing. The HMMER software and Pfam databases are ver CLUSTALW alignment of amino acid sequence of homolog sion 18.0 and were used to identify known domains in the proteins. proteins corresponding to amino acid sequence of SEQ ID 0.045 Homologous genes are genes which encode pro NO: 426 through SEQID NO: 850. All DNA encoding pro teins with the same or similar biological function to the pro teins that have scores higher than the gathering cutoff dis tein encoded by the second gene. Homologous genes may be closed in Table 17 by Pfam analysis disclosed herein can be generated by the event of speciation (see ortholog) or by the used in recombinant DNA of the plant cells of this invention, event of genetic duplication (see paralog). "Orthologs' refer e.g. for selecting transgenic plants having enhanced agro to a set of homologous genes in different species that evolved nomic traits. The relevant Pfams for use in this invention, as from a common ancestral gene by specification. Normally, more specifically disclosed below, are Mito carr, 6PGD, orthologs retain the same function in the course of evolution; UBX, iPGM N, WD40, Fer4, Enolase C, DUF1639, PBP and “paralogs' refer to a set of homologous genes in the same PLAC8, Acyl-CoA dh 1, Isoamylase N. Acyl-CoA dh 2, species that have diverged from each other as a consequence PC4, Sugar tr, UCH, Enolase N, HATPase c, PRA-PH, Pki of genetic duplication. Thus, homologous genes can be from nase, SBP56, PEP-utilizers, SIS, PCI, DUF1644, Terpene the same or a different organism. As used herein, "homolog” synth, Acyl-CoA dh M. Acyl-CoA dh N. Glutaminase, means a protein that performs the same biological function as Lectin legB, Dehydrin, Mat, Ank, 2-Hacid dh C, Chal a second protein including those identified by sequence iden sti synt C, DUF1070, ATP-grasp 2, Arginase, HABP4 tity search. PAI-RBP1, ABC2 membrane, DUF1723, Glyco hydro 1, 0046 Percent identity refers to the extent to which two MFS 1, ARD, PDT, HMA, Pro isomerase, Ferric reduct, optimally aligned DNA or protein segments are invariant PRA-CH, Aa trans, ACT, LisH, PGM PMM II, Spermine throughout a window of alignment of components, e.g., synth, Zf-MYND, LRRNT 2, Ribul P 3 epim, PGM PM nucleotide sequence or amino acid sequence. An "identity M IV, NPH3, Dapb C, TPR 1, TPR 2, FAE1 CUT1 fraction' for aligned segments of a test sequence and a refer RppA, Ktrans, F-box, Cyclin C, ADK, NUDIX, NIR SIR, ence sequence is the number of identical components which PEPCK ATP, La, DapB N, MtN3 silv, FMO-like, TIM, are shared by sequences of the two aligned segments divided FKBP C, PMEI, Peptidase C12, Cyclin N, DUF568, Meth by the total number of sequence components in the reference yltransf 11, Methyltransf 12, DUF1677, DnaJ C, BRAP2, segment over a window of alignment which is the Smaller of IF2 N, Carboxyl trans, mTERF, Glyoxalase, TMEM14, the full test sequence or the full reference sequence. “Percent Mlo, Beta elim lyase, Pyr redox dim, Glyco transf 8, identity” (“% identity”) is the identity fraction times 100. “% Nicastrin, Flavodoxin 1, Epimerase, PTPA, Lipase 3, Pyr identity to a consensus amino acid sequence' is 100 times the redox 2, GSHPx, ELM2, PGI, Aminotran 1, 2, ABC tran, identity fraction in a window of alignment of an amino acid GRP PGK, Oleosin, Sulfotransfer 1, EXS, DUF1325, sequence of a test protein optimally aligned to consensus AMP-binding, Arm, NTP transferase, LSM, Metalloen amino acid sequence of this invention. Zyme, Molybdop Fe4S4, MFAP1 C, Aminotran 3, PHD, 0047 Arabidopsis” means plants of Arabidopsis B56, DUF588, PSI PsaF, Zf-CCCH, HEAT, PALP, FH2, thaliana. SapB 1, Ammonium transp, MannoseP isomer, NOP5NT, 0048 “Pfam” refers to a large collection of multiple SapB 2, Pyr redox, Pollen allerg 1, Asp, DUF662, FHA, sequence alignments and hidden Markov models covering YieF N, COX5C, GTP EFTU D2, Ion trans 2, PK, many common protein families, e.g. Pfam version 18.0 (Au DUF231, FAD binding 1, Hrf1, FAD binding 4, FAD gust 2005) contains alignments and models for 7973 protein binding 6, FAD binding 8, CBS, Smr, aPHC, DUF241, families and is based on the Swissprot 47.0 and SP-TREMBL Brix, Ras, Acetyltransf1, NAF, SPX. Na Ca ex, C2, p450, 30.0 protein sequence databases. See S. R. Eddy, “Profile PP2C, Histone, 2-Hacid dh, SBF, CCT, BCNT, PKC, Miro, Hidden Markov Models”, Bioinformatics 14:755-763, 1998. CH, PfkB, ACP syn III C, Sterol desat, ADH zinc N, CS, Pfam is currently maintained and updated by a Pfam Consor Cys Met Meta PP. Lactamase B. Bromodomain, CDI. tium. The alignments represent some evolutionary conserved Linker histone, DAO, Dicty CAR, Aldo ket red, Zf-AN1, structure that has implications for the protein's function. Pro Methyltransf 6, DUF1005, LEA 2, NIR SIR fen, file hidden Markov models (profile HMMs) built from the DUF260, Oxidored FMN, DUF26, Lectin C, Pec lyase C, Pfam alignments are useful for automatically recognizing Nop, TB2 DP1 HVA22, ADH N, YGGT, NAPRTase, that a new protein belongs to an existing protein family even NAD binding 1, DUF914, PGM PMM I, NAD binding if the homology by alignment appears to be low. Once one 2, AICARFT IMPCHas, Auxin inducible, NAD binding DNA is identified as encoding a protein which imparts an 6, Anti-silence, RuBisCO large, Response reg, FeThRed enhanced trait when expressed in transgenic plants, other A, Di19, SNARE, PGM PMM III, Molydop binding, DNA encoding proteins in the same protein family are iden efhand, Zf-CCHC, GTP EFTU, ARID, adh short, Fibril tified by querying the amino acid sequence of protein larin, RuBisCO large N, WWE, AA permease, PABP encoded by candidate DNA against the Hidden Markov OMPdecase, RRM 1, U-box, OPT, TBC, MGS, DUF786, Model which characterizes the Pfam domain using HMMER 3Beta HSD, Zf-UBP, ZfA20, DPBB 1. GDPD, PI-PLC-X, software, a current version of which is provided in the SEP, PI-PLC-Y, NOSIC, Glycolytic, SET, ADK lid, Alpha appended computer listing. Candidate proteins meeting the amylase, EB1, PGAM, Abhydrolase 1, Glyco hydro 14, gathering cutoff for the alignment of a particular Pfam are in Lung 7-TM R, Abhydrolase 3, TCTP, GATase 2, Gln the protein family and have cognate DNA that is useful in synt C, 20G-FeH Oxy. Pribosyltran, MIF, CoAtrans, RCC1, US 2015/O 1841 89 A1 Jul. 2, 2015

Pkinase Tyr, MIP. DnaJ, HSCB C, Trehalose PPase, LRR TABLE 1 1, Cupin 2. LRR 2, Glyco hydro 28, Yip 1, Trp syntA, Sedlin N, SGS, Aldedh, CK II beta, Zf-C3HC4, GIDA, NUC PEP PB1, IMPDH, Carb kinase, PurA, Molybdopterin, Nodulin- s s Construct like, Tim17, Xanur permease, Hist, deacetyl. RNA poll NO NO Gene ID ID orientation Rpb8, Agenet, Myb DNA-binding, Glyoxal oxid N. Ribo phorin I, and FAE 3-kCoA syn1. 1 426 CGPG699 10919 ANTI-SENSE 2 427 COPG567 11142 SENSE 3 428 CGPG267 11310 ANTI-SENSE Recombinant DNA Constructs 4 429 CGPG1179 11866 ANTI-SENSE 5 430 CGPG959 11937 ANTI-SENSE 0049. The present invention provides recombinant DNA 6 431 CGPG2158 16218 SENSE constructs comprising one or more polynucleotides disclosed 7 432 CGPG24.46 1741 O SENSE herein for imparting one or more improved traits to transgenic 8 433 CG C 1862 18401 S RENS R plantlant when iincorporated ted into the nucleus of the pplant cells 109 434435 CGPG3014CGPG1674 1866S19141 SENSE Such constructs also typically comprise a promoter opera- 11 436 CGPG268O 191S6 SENSE tively linked to said polynucleotide to provide for expression 12 437 CGPG3577 19621 SENSE in the plant cells. Other construct components may include 13 438 CG C S46 19760 SENSER R additionaldditional regulatorylat elements,1 t suchhas as 5'3 or 3'3' untranslateuntranslated 1514 440439 CGPG2488CGPG3929 7051019857 SENSE regions (such as polyadenylation sites), intron regions, and 16 4.41 CGPG3012 70605 SENSE transit or signal peptides. Such recombinant DNA constructs 17 442 CGPG3162 7O607 SENSE can be assembled using methods known to those of ordinary 18 443 CG C 6. 70812 SENSER R kill in the art 19 444 CGPG4084 70933 SENSE SK1 18. 2O 445 CGPG3917 70939 SENSE 0050. In a preferred embodiment, a polynucleotide of the 21 446 CGPG4414 71319 SENSE present invention is operatively linked in a recombinant DNA 22 447 CGPG18S 71.446 SENSE 23 448 CGPG1679 71609 SENSE construct to a promoter functional- in a plant to provide for 24 449 CGPG271 71649 SENSE- - - - - expression of the polynucleotide 1n the sense orientation such 25 450 CGPG4434 71814 SENSE that a desired protein or polypeptide fragment of a protein is 26 451 CGPG21.98 71945 SENSE produced. Also provided are embodiments wherein a poly- 27 42 CESS; 2005 SENSE nucleotide is operatively linked to a promoter functional in a 3. 2. : 5. 2. s NS plant to provide for expression of gene suppression RNA to 30 455 CGPG4859 72637 SENSE Suppress the level of an endogenous protein. 31 456 CGPG1589 72782 SENSE

0051 Recombinant constructs prepared in accordance 3233 457458 CGCGPG3899 g 7291912794 SENSE with the present invention also generally include a 3' untrans- 34 459 CGPGS610 72983 SENSE lated DNA region (UTR) that typically contains a polyade- 35 460 CGPGS66S 73025 SENSE nylation sequence following the polynucleotide coding 36 461 CGPGS697 73050 SENSE C R R region. Examples of useful 3' UTRs include those from the 3738 462463 CGPG5695CGPG4862 73.33813092 SENSE nopaline synthase gene of Agrobacterium tumefaciens (nos), 39 464 CGPG4910 73344 SENSE a gene encoding the Small subunit of a ribulose-1,5-bisphos- 40 46S CGPG6541 73536 SENSE phate carboxylase-oxygenase (rbcS), and the T7 transcript of 41 466 CGPG1756 73644 SENSE C RNTQR Agrobacterium tumefaciens. Constructs and vectors may also 4342 468467 CGPGS106CGPG4927 7367273662 SENSE include a transit peptide for targeting of agene target to a plant 44 469 CGPGS167 73733 SENSE organelle, particularly to a chloroplast, leucoplast or other 45 470 CGPG1924 73846 SENSE plastid organelle. For descriptions of the use of chloroplast 46 471 CGPGS2O1 73863 SENSE C R R transit peptides, see U.S. Pat. No. 5,188,642 and U.S. Pat. No. 4847 473472 CGPGSO89CGPG1884 7420514059 SENSE 5,728,925, incorporated herein by reference. 49 474 CGPG5870 74321 SENSE 0052 Table 1 provides a list of genes that provide recom- 50 47S CGPGS888 74337 SENSE binant DNA that was used in a model plant to discover asso- 5251 477476 CGPG6743CGPG1461 7441214388 SENSE ciated improved traits and that can be used with homologs to 53 478 CGPG6722 74445 SENSE define a consensus amino acid sequence for characterizing S4 479 CGPG82 74522 SENSE recombinant DNA for use in the nuclei of this invention. An 55 480 CGPG6761 74526 SENSE understanding of Table 1 is facilitated- by the following 56 481 CG C 6. 14576 SENSER R description of the headings: 57 482 CGPG4914 74707 SENSE escripuOn OI une head1ngs: 58 483 CGPGS840 74743 SENSE 0053 “NUC SEQ ID NO” refers to a SEQ ID NO. for 59 484 CGPGS980 74755 SENSE particular DNA sequence in the Sequence Listing. s 2. : A. 2. SENSE 0054 “PEP SEQ ID NO refers to a SEQ ID NO. in the 62 487 CGPGS824 75226 SENSE Sequence Listing for the amino acid sequence of a protein 63 488 CGPGS879 75231 SENSE cognate to a particular DNA 64 489 CGPGS949 75239 SENSE & G 99 65 490 CGPG6096 75258 SENSE (0.055 construct id refers to an arbitrary number used tO 66 491 CGPG6218 7527 O SENSE identify a particular recombinant DNA construct comprising 67 492 CGPG6226 75271 SENSE the particular DNA. 68 493 CGPG-7518 75355 SENSE 69 494 CGPG7654 7546O SENSE (0056 "Gene ID' refers to al arbitrary aC used tO iden- 70 495 CGPG6875 75847 SENSE tify the particular DNA. “orientation” refers to the orientation 71 496 CGPG8259 75907 SENSE of the particular DNA in a recombinant DNA construct rela- 72 497 CGPG8229 75927 SENSE tive to the promoter.

US 2015/O 1841 89 A1 Jul. 2, 2015

TABLE 1-continued through SEQ ID NO:425, as well as the homologs of such DNA molecules. A subset of the DNA for gene suppression NUC PEP aspects of the invention includes fragments of the disclosed SEQ SEQ ID ID Construct full polynucleotides consisting of oligonucleotides of 21 or NO NO Gene ID ID orientation more consecutive nucleotides. Oligonucleotides the larger molecules having a sequence selected from the group con 361 786 CGPG6SO2 73578 SENSE 362 787 CGPG 6630 74112 SENSE sisting of SEQID NO:1 through SEQID NO:425 are useful 363 788 CGPGS484 74249 SENSE as probes and primers for detection of the polynucleotides 364 789 CGPGS394 74278 SENSE used in the invention. Also useful in this invention are variants 365 790 CGPG-6058 74359 SENSE of the DNA. Such variants may be naturally occurring, 366 791 CGPG2090 74393 SENSE 367 792 CGPG6664 74414 SENSE including DNA from homologous genes from the same or a 368 793 CGPG77O6 75530 SENSE different species, or may be non-natural variants, for example 369 794 CGPG7884 75752 SENSE DNA synthesized using chemical synthesis methods, or gen 370 795 CGPG6965 75867 SENSE erated using recombinant DNA techniques. Degeneracy of 371 796 CGPGI2S6 7591O SENSE 372 797 CGPG2297 76.101 SENSE the genetic code provides the possibility to substitute at least 373 798 CGPG6246 76431 SENSE one base of the protein encoding sequence of a gene with a 374 799 CGPG9037 76832 SENSE different base without causing the amino acid sequence of the 375 800 CGPG1941 16O23 SENSE polypeptide produced from the gene to be changed. Hence, a 376 801 CGPG2OSS 16424 ANTI-SENSE 377 802 CGPG2427 17807 SENSE DNA useful in the present invention may have any base 378 8O3 CGPG386 70802 SENSE sequence that has been changed from the sequences provided 379 804 CGPG393 70817 SENSE herein by Substitution in accordance with degeneracy of the 380 80S CGPG609 70819 SENSE genetic code. 381 806 CGPG4022 70908 SENSE 382 807 CGPG942 72351 SENSE 0.058 Homologs of the genes providing DNA demon 383 808 CGPG1800 73O87 SENSE strated as useful in improving traits in model plants disclosed 384 809 CGPG4783 73223 SENSE herein will generally have significant identity with the DNA 385 810 CGPG3257 73709 SENSE 386 811 CGPG1696 73804 SENSE disclosed herein. DNA is substantially identical to a reference 387 812 CGPG1982 73819 SENSE DNA if, when the sequences of the polynucleotides are opti 388 813 CGPG-378O 73851 SENSE mally aligned there is about 60% nucleotide equivalence; 389 814 CGPG66O2 741S6 SENSE more preferably 70%: more preferably 80% equivalence; 390 815 CGPG6621 74194 SENSE 391 816 CGPGS493 74252 SENSE more preferably 85% equivalence; more preferably 90%; 392 817 CGPGS811 743.17 SENSE more preferably 95%; and/or more preferably 98% or 99% 393 818 CGPGS902 74328 SENSE equivalence over a comparison window. A comparison win 394 819 CGPG-6791 74506 SENSE dow is preferably at least 50-100 nucleotides, and more pref 395 820 CGPG6778 74S4O SENSE 396 821 CGPG6166 746SO SENSE erably is the entire length of the polynucleotide provided 397 822 CGPG3735 74718 SENSE herein. Optimal alignment of sequences for aligning a com 398 823 CGPGS854 74745 SENSE parison window may be conducted by algorithms; preferably 399 824 CGPG7451 74956 SENSE by computerized implementations of these algorithms (for 400 825 CGPGSO24 75076 SENSE 401 826 CGPG6054 75.255 SENSE example, the Wisconsin Genetics Software Package Release 402 827 CGPG62O7 75269 SENSE 7.0-10.0, Genetics Computer Group, 575 Science Dr. Madi 403 828 CGPG-762O 75432 SENSE son, Wis.). The reference polynucleotide may be a full-length 404 829 CGPG-7623 75468 SENSE molecule or a portion of a longer molecule. Preferentially, the 40S 830 CGPG7775 75686 SENSE 406 831 CGPG73 75911 SENSE window of comparison for determining polynucleotide iden 407 832 CGPG2100 76064 SENSE tity of protein encoding sequences is the entire coding region. 408 833 CGPG 6026 76121 SENSE 0059 Proteins useful for imparting improved traits are 409 834 CGPG-7269 76.193 SENSE 410 835 CGPG-6361 76237 SENSE entire proteins or at least a sufficient portion of the entire 411 836 CGPG6993 76281 SENSE protein to impart the relevant biological activity of the pro 412 837 CGPG8899 76388 SENSE tein. Proteins useful for generation of transgenic plants hav 413 838 CGPG6926 76557 SENSE ing improved traits include the proteins with an amino acid 414 839 CGPG7172 76567 SENSE 415 840 CGPG-7129 76624 SENSE sequence provided herein as SEQID NO: 426 through SEQ 416 841 CGPG7276 76764 SENSE ID NO: 850, as well as homologs of such proteins. 417 842 CGPG9031 76855 SENSE 0060 Homologs of the proteins useful in the invention are 418 843 CGPG91OS 76976 SENSE 419 844 CGPG9082 76985 SENSE identified by comparison of the amino acid sequence of the 420 845 CGPG6212 77O14 SENSE protein to amino acid sequences of proteins from the same or 421 846 CGPG92O6 77112 SENSE different plant sources, e.g., manually or by using known 422 847 CGPG9151 77117 SENSE homology-based search algorithms such as those commonly 423 848 CGPG9129 77138 SENSE 424 849 CGPG9224 77226 SENSE known and referred to as BLAST, FASTA, and Smith-Water 425 850 CGPG93S8 77418 SENSE man. As used herein, a homologis a protein from the same or a different organism that performs the same biological func tion as the polypeptide to which it is compared. An ortholo gous relation between two organisms is not necessarily mani Recombinant DNA fest as a one-to-one correspondence between two genes, because a gene can be duplicated or deleted after organism 0057 DNA for use in the present invention to improve phylogenetic separation, such as speciation. For a given pro traits in plants have a nucleotide sequence of SEQ ID NO:1 tein, there may be no ortholog or more than one ortholog. US 2015/O 1841 89 A1 Jul. 2, 2015

Other complicating factors include alternatively spliced tran 70% sequence identity or higher, e.g., at least about 80% Scripts from the same gene, limited gene identification, sequence identity with an amino acid sequence of SEQ ID redundant copies of the same gene with different sequence NO: 426 through SEQID NO: 850. Of course useful proteins lengths or corrected sequence. A local sequence alignment also include those with higher identity, e.g., 90% to 99% program, e.g., BLAST, can be used to search a database of identity. Identity of protein homologs is determined by opti sequences to find similar sequences, and the Summary Expec mally aligning the amino acid sequence of a putative protein tation value (E-value) used to measure the sequence base homolog with a defined amino acid sequence and by calcu similarity. As a protein hit with the best E-value for a particu lating the percentage of identical and conservatively substi lar organism may not necessarily be an ortholog or the only tuted amino acids over the window of comparison. The win ortholog, a reciprocal BLAST search is used in the present dow of comparison for determining identity can be the entire invention to filter hit sequences with significant E-values for amino acid sequence disclosed herein, e.g., the full sequence ortholog identification. The reciprocal BLAST entails search of any of SEQ ID NO: 426 through SEQID NO: 850. of the significant hits against a database of amino acid 0063 Genes that are homologous to each other can be sequences from the base organism that are similar to the grouped into families and included in multiple sequence sequence of the query protein. A hit is a likely ortholog, when alignments. Then a consensus sequence for each group can be the reciprocal BLAST's best hit is the query protein itself or derived. This analysis enables the derivation of conserved and a protein encoded by a duplicated gene after speciation. Thus, class- (family) specific residues or motifs that are function homolog is used herein to describe proteins that are assumed ally important. These conserved residues and motifs can be to have functional similarity by inference from sequence base further validated with 3D protein structure if available. The similarity. The relationship of homologs with amino acid consensus sequence can be used to define the full scope of the sequences of SEQID NO: 851 to SEQID NO:33634 to the invention, e.g., to identify proteins with a homolog relation proteins with amino acid sequences of SEQID NO: to 426 to ship. Thus, the present invention contemplates that protein SEQ ID NO: 850 is found in the listing of Table 2. homologs include proteins with an amino acid sequence that 0061. Other functional homolog proteins differ in one or has at least 90% identity to Such a consensus amino acid more amino acids from those of a trait-improving protein Sequence Sequences. disclosed herein as the result of one or more of the well known conservative amino acid Substitutions, e.g., valine is a Promoters conservative Substitute for alanine and threonine is a conser 0064. Numerous promoters that are active in plant cells Vative Substitute for serine. Conservative substitutions for an have been described in the literature. These include promoters amino acid within the native sequence can be selected from present in plant genomes as well as promoters from other other members of a class to which the naturally occurring Sources, including nopaline synthase (NOS) promoter and amino acid belongs. Representative amino acids within these octopine synthase (OCS) promoters carried on tumor-induc various classes include, but are not limited to: (1) acidic ing plasmids of Agrobacterium tumefaciens, caulimovirus (negatively charged) amino acids such as aspartic acid and promoters such as the cauliflower mosaic virus or Figwort glutamic acid; (2) basic (positively charged) amino acids Such mosaic virus promoters. For instance, see U.S. Pat. Nos. as arginine, histidine, and lysine; (3) neutral polar amino 5,858,742 and 5,322,938 which disclose versions of the con acids such as glycine, serine, threonine, cysteine, tyrosine, stitutive promoter derived from cauliflower mosaic virus asparagine, and glutamine; and (4) neutral nonpolar (hydro (CaMV35S), U.S. Pat. No. 5,378,619 which discloses a Fig phobic) amino acids such as alanine, leucine, isoleucine, wort Mosaic Virus (FMV)35S promoter, U.S. Pat. No. 6,437, Valine, proline, phenylalanine, tryptophan, and methionine. 217 which discloses a maize RS81 promoter, U.S. Pat. No. Conserved substitutes for an amino acid within a native 5,641,876 which discloses a rice actin promoter, U.S. Pat. No. amino acid sequence can be selected from other members of 6.426,446 which discloses a maize RS324 promoter, U.S. Pat. the group to which the naturally occurring amino acid No. 6,429,362 which discloses a maize PR-1 promoter, U.S. belongs. For example, a group of amino acids having ali Pat. No. 6,232,526 which discloses a maize A3 promoter, phatic side chains is glycine, alanine, Valine, leucine, and U.S. Pat. No. 6,177,611 which discloses constitutive maize isoleucine; a group of amino acids having aliphatic-hydroxyl promoters, U.S. Pat. No. 6,433,252 which discloses a maize side chains is serine and threonine; a group of amino acids L3 oleosin promoter, U.S. Pat. No. 6,429.357 which discloses having amide-containing side chains is asparagine and a rice actin 2 promoter and intron, U.S. Pat. No. 5,837,848 glutamine; a group of amino acids having aromatic side which discloses a root specific promoter, U.S. Pat. No. 6,084. chains is phenylalanine, tyrosine, and tryptophan; a group of 089 which discloses cold inducible promoters, U.S. Pat. No. amino acids having basic side chains is lysine, arginine, and 6.294.714 which discloses light inducible promoters, U.S. histidine; and a group of amino acids having Sulfur-contain Pat. No. 6,140,078 which discloses salt inducible promoters, ing side chains is cysteine and methionine. Naturally conser U.S. Pat. No. 6,252,138 which discloses pathogen inducible Vative amino acids Substitution groups are: Valine-leucine, promoters, U.S. Pat. No. 6,175,060 which discloses phospho Valine-isoleucine, phenylalanine-tyrosine, lysine-arginine, rus deficiency inducible promoters, U.S. Patent Application alanine-Valine, aspartic acid-glutamic acid, and asparagine Publication 2002/01928.13A1 which discloses 5', 3' and glutamine. A further aspect of the invention comprises pro intron elements useful in the design of effective plant expres teins that differ in one or more amino acids from those of a sion vectors, U.S. patent application Ser. No. 09/078.972 described protein sequence as the result of deletion or inser which discloses a coixin promoter, U.S. patent application tion of one or more amino acids in a native sequence. Ser. No. 09/757,089 which discloses a maize chloroplast 0062 Homologs of the trait-improving proteins disclosed aldolase promoter, and U.S. patent application Ser. No. provided herein will generally demonstrate significant 10/739,565 which discloses water-deficit inducible promot sequence identity. Of particular interest are proteins having at ers, all of which are incorporated herein by reference. These least 50% sequence identity, more preferably at least about and numerous other promoters that function in plant cells are US 2015/O 1841 89 A1 Jul. 2, 2015

known to those skilled in the art and available for use in Mutant plants produced by Agrobacterium or transposon recombinant polynucleotides of the present invention to pro mutagenesis and having altered expression of a polypeptide vide for expression of desired genes in transgenic plant cells. of interest can be identified using the polynucleotides of the 0065. Furthermore, the promoters can include multiple present invention. For example, a large population of mutated "enhancer sequences' to assist in elevating gene expression. plants may be screened with polynucleotides encoding the Such enhancers are known in the art. By including an polypeptide of interest to detect mutated plants having an enhancer sequence with Such constructs, the expression of the insertion in the gene encoding the polypeptide of interest. selected protein may be enhanced. These enhancers often are found 5' to the start of transcription in a promoter that func Gene Stacking tions in eukaryotic cells, but can often be inserted in the forward or reverse orientation 5' or 3' to the coding sequence. 0070 The present invention also contemplates that the In some instances, these 5' enhancing elements are introns. trait-improving recombinant DNA provided herein can be Deemed to be particularly useful as enhancers are the 5' used in combination with other recombinant DNA to create introns of the rice actin1 and rice actin 2 genes. Examples of plants with a multiple desired traits. The combinations gen other enhancers that can be used in accordance with the erated can include multiple copies of any one or more of the invention include elements from the CaMV 35S promoter, recombinant DNA constructs. These stacked combinations octopine synthase genes, the maize alcohol dehydrogenase can be created by any method, including but not limited to gene, the maize shrunken 1 gene and promoters from non cross breeding of transgenic plants, or multiple genetic trans plant eukaryotes. formation. 0066. In some aspects of the invention it is preferred that the promoter element in the DNA construct be capable of Transformation Methods causing sufficient expression to result in the production of an 0071 Numerous methods for producing plant cell nuclei effective amount of a polypeptide in water deficit conditions. with recombinant DNA are known in the art and may be used Such promoters can be identified and isolated from the regu in the present invention. Two commonly used methods for latory region of plant genes that are over expressed in water plant transformation are Agrobacterium-mediated transfor deficit conditions. Specific water-deficit-inducible promoters mation and microprojectile bombardment. Microprojectile for use in this invention are derived from the 5' regulatory bombardment methods are illustrated in U.S. Pat. No. 5,015, region of genes identified as a heat shock protein 17.5 gene 580 (soybean): U.S. Pat. No. 5,550,318 (corn); U.S. Pat. No. (HSP17.5), an HVA22 gene (HVA22), a Rab 17 gene and a 5,538,880 (corn); U.S. Pat. No. 5,914,451 (soybean); U.S. cinnamic acid 4-hydroxylase (CA4H) gene (CA4H) of Zea Pat. No. 6,160,208 (corn): U.S. Pat. No. 6,399,861 (corn)and maize. Such water-deficit-inducible promoters are disclosed U.S. Pat. No. 6,153,812 (wheat) and Agrobacterium-medi in U.S. application Ser. No. 10/739,565, incorporated herein ated transformation is described in U.S. Pat. No. 5,159,135 by reference. (cotton); U.S. Pat. No. 5,824,877 (soybean): U.S. Pat. No. 0067. In some aspects of the invention, sufficient expres 5,591,616 (corn); and U.S. Pat. No. 6,384.301 (soybean), all sion in plant seed tissues is desired to effect improvements in of which are incorporated herein by reference. For Agrobac seed composition. Exemplary promoters for use for seed terium tumefaciens based plant transformation system, addi composition modification include promoters from seed genes tional elements present on transformation constructs will such as napin (U.S. Pat. No. 5,420,034), maize L3 oleosin include T-DNA left and right border sequences to facilitate (U.S. Pat. No. 6,433,252), Zein Z27 (Russell et al., (1997) incorporation of the recombinant polynucleotide into the Transgenic Res. 6(2): 157-166), globulin 1 (Belanger et al., plant genome. (1991) Genetics 129:863-872), glutelin 1 (Russell (1997) 0072. In general it is preferred to introduce heterologous Supra), and peroxiredoxin antioxidant (Perl) (Stacy et al., DNA randomly, i.e., at a non-specific location, in the genome (1996) Plant Mol Biol. 31(6):1205-1216). of a target plant line. In special cases it may be useful to target 0068. In some aspects of the invention, preferential heterologous DNA insertion in order to achieve site-specific expression in plant green tissues is desired. Promoters of integration, e.g., to replace an existing gene in the genome, to interest for such uses include those from genes such as SSU use an existing promoter in the plant genome, or to insert a (Fischhoff, et al., (1992) Plant Mol Biol. 20:81-93), aldolase recombinant polynucleotide at a predetermined site known to and pyruvate orthophosphate dikinase (PPDK) (Taniguchi, et be active for gene expression. Several site specific recombi al., (2000) Plant Cell Physiol. 41(1):42-48). nation systems exist which are known to function implants 0069 Gene suppression includes any of the well-known include cre-lox as disclosed in U.S. Pat. No. 4,959,317 and methods for Suppressing transcription of a gene or the accu FLP-FRT as disclosed in U.S. Pat. No. 5,527,695, both incor mulation of the mRNA corresponding to that gene thereby porated herein by reference. preventing translation of the transcript into protein. Posttran 0073 Transformation methods of this invention are pref Scriptional gene Suppression is mediated by transcription of erably practiced in tissue culture on media and in a controlled RNA that forms double-stranded RNA (dsRNA) having environment. “Media' refers to the numerous nutrient mix homology to a gene targeted for Suppression. Suppression can tures that are used to grow cells in vitro, that is, outside of the also be achieved by insertion mutations created by transpos intact living organism. Recipient cell targets include, but are able elements may also prevent gene function. For example, not limited to, meristem cells, callus, immature embryos and in many dicot plants, transformation with the T-DNA of Agro gametic cells such as microspores, pollen, sperm and egg bacterium may be readily achieved and large numbers of cells. It is contemplated that any cell from which a fertile plant transformants can be rapidly obtained. Also, Some species may be regenerated is useful as a recipient cell. Callus may be have lines with active transposable elements that can effi initiated from tissue sources including, but not limited to, ciently be used for the generation of large numbers of inser immature embryos, seedling apical meristems, microspores tion mutations, while some other species lack Such options. and the like. Cells capable of proliferating as callus are also US 2015/O 1841 89 A1 Jul. 2, 2015 recipient cells for genetic transformation. Practical transfor uct, e.g., by immunological means (ELISAS and Western mation methods and materials for making transgenic plants of blots) or by enzymatic function; plant part assays, such as leaf this invention, e.g., various media and recipient target cells, or root assays; and also, by analyzing the phenotype of the transformation of immature embryos and Subsequent regen whole regenerated plant. eration of fertile transgenic plants are disclosed in U.S. Pat. Nos. 6,194.636 and 6.232,526 and U.S. patent application Discovery of Trait-Improving Recombinant DNA Ser. No. 09/757,089, which are incorporated herein by refer CCC. (0077. To identify nuclei with recombinant DNA that con 0074. In practice DNA is introduced into only a small fer improved traits to plants, Arabidopsis thaliana was trans percentage of target cell nuclei in any one experiment. Marker formed with a candidate recombinant DNA construct and genes are used to provide an efficient system for identification screened for an improved trait. of those cells with nuclei that are stably transformed by 0078 Arabidopsis thaliana is used a model for genetics receiving and integrating a transgenic DNA construct into and metabolism in plants. Arabidopsis has a small genome, their genomes. Preferred marker genes provide selective and well-documented Studies are available. It is easy to grow markers that confer resistance to a selective agent, Such as an in large numbers and mutants defining important genetically antibiotic or herbicide. Potentially transformed cells with a controlled mechanisms are either available, or can readily be nucleus of the invention are exposed to the selective agent. In obtained. Various methods to introduce and express isolated the population of surviving cells will be those cells where, homologous genes are available (see Koncz, e.g., Methods in generally, the resistance-conferring gene has been integrated Arabidopsis Research e.g., (1992), World Scientific, New and expressed at sufficient levels to permit cell survival. Cells Jersey, New Jersey, in “Preface”). may be tested further to confirm stable integration of the 0079 A two-step screening process was employed which exogenous DNA in the nucleus. Useful selective marker comprised two passes of trait characterization to ensure that genes include those conferring resistance to antibiotics Such the trait modification was dependent on expression of the as kanamycin (nptII), hygromycin B (aph IV) and gentamy recombinant DNA, but not due to the chromosomal location cin (aac3 and aacC4) or resistance to herbicides such as of the integration of the transgene. Twelve independent trans glufosinate (bar orpat) and glyphosate (EPSPS). Examples of genic lines for each recombinant DNA construct were estab such selectable are illustrated in U.S. Pat. Nos. 5,550,318; lished and assayed for the transgene expression levels. Five 5,633,435; 5,780,708 and 6,118,047, all of which are incor transgenic lines with high transgene expression levels were porated herein by reference. Screenable markers which pro used in the first pass screen to evaluate the transgene’s func vide an ability to visually identify transformants can also be tion in T2 transgenic plants. Subsequently, three transgenic employed, e.g., a gene expressing a colored or fluorescent events, which had been shown to have one or more improved protein such as a luciferase or green fluorescent protein (GFP) traits, were further evaluated in the second pass screen to or a gene expressing a beta-glucuronidase or uidA gene confirm the transgene’s ability to impart an improved trait. (GUS) for which various chromogenic substrates are known. The following Table 3 summarizes the improved traits that It is also contemplated that combinations of screenable and have been confirmed as provided by a recombinant DNA selectable markers will be useful for identification of trans COnStruct. formed cells. See PCT publication WO99/61129 which dis 0080. In particular, Table 3 reports: closes use of a gene fusion between a selectable marker gene and a screenable marker gene, e.g., an NPTII gene and a GFP I0081) “PEP SEQID” which is the amino acid sequence of gene. the protein cognate to the DNA in the recombinant DNA 0075 Cells that survive exposure to the selective agent, or construct corresponding to a protein sequence of a SEQ ID cells that have been scored positive in a screening assay, may NO. in the Sequence Listing. be cultured in regeneration media and allowed to mature into I0082 “construct id' is an arbitrary name for the recom plants. Developing plantlets can be transferred to soil less binant DNA describe more particularly in Table 1. plant growth mix, and hardened off, e.g., in an environmen I0083) “annotation” refers to a description of the top hit tally controlled chamber at about 85% relative humidity, 600 protein obtained from an amino acid sequence query of each ppm CO, and 25-250 microeinsteins mis' of light, prior to PEP SEQID NO to GenBank database of the National Center transfer to a greenhouse or growth chamber for maturation. for Biotechnology Information (ncbi). More particularly, “gi” Plants are preferably matured either in a growth chamber or is the GenBank ID number for the top BLAST hit. greenhouse. Plants are regenerated from about 6 wk to 10 I0084 “description” refers to the description of the top months after a transformant is identified, depending on the BLAST hit. initial tissue. During regeneration, cells are grown to plants on solid media at about 19 to 28°C. After regenerating plants I0085 “e-value” provides the expectation value for the have reached the stage of shoot and root development, they BLAST hit. may be transferred to a greenhouse for further growth and I0086) “identity” refers to the percentage of identically testing. Plants may be pollinated using conventional plant matched amino acid residues along the length of the portion breeding methods known to those of skill in the art and seed of the sequences which is aligned by BLAST between the produced. sequence of interest provided herein and the hit sequence in 0076 Progeny may be recovered from transformed plants GenBank. and tested for expression of the exogenous recombinant poly I0087 “traits” identify by two letter codes the confirmed nucleotide. Useful assays include, for example, "molecular improvement in a transgenic plant provided by the recombi biological assays, such as Southern and Northern blotting nant DNA. The codes for improved traits are: and PCR; “biochemical assays, such as detecting the pres I0088. “CK' which indicates cold tolerance improvement ence of RNA, e.g., double stranded RNA, or a protein prod identified under a cold shock tolerance screen; US 2015/O 1841 89 A1 Jul. 2, 2015 13

I0089. “CS” which indicates cold tolerance improvement 0094 “LN” which indicates nitrogen use efficiency identified by a cold germination tolerance screen; improvement identified by a limited nitrogen tolerance 0090 “DS” which indicates drought tolerance improve Screen; ment identified by a soil drought stress tolerance screen; 0.095 “LL' which indicates attenuated shade avoidance response identified by a shade tolerance screen under a low 0091 “PEG' which indicates osmotic stress tolerance light condition; improvement identified by a PEG induced osmotic stress (0096 “PP” which indicates improved growth and devel tolerance screen; opment at early stages identified by an early plant growth and 0092 “HS which indicates heat stress tolerance improve development screen; ment identified by a heat stress tolerance screen; (0097 “SP” which indicates improved growth and devel 0093 “SS” which indicates high salinity stress tolerance opment at late stages identified by a late plant growth and improvement identified by a salt stress tolerance Screen; development screen provided herein. TABLE 3

PEP SEQ ID annotation

NO e value % identity GenBank id description trait 426 gi21553646| histone H2A CK 427 gi7327824 refNP 195785.1| CK macrophage migration inhibitory factor family protein/MIF family protein Arabidopsis thaliana 428 O 99 gi284.16663| refNP 180648.1 CK transducin family protein WD-40 repeat family protein 429 2.OOE-78 1OO gi23198326| gb|AAN15690.1 unknown CK protein Arabidopsis thaliana 430 O 1OO gi23297810 refNP 194910.2 CK LN phototropic-responsive NPH3 family protein 431 8.OOE-48 1OO gil 12248017| refNP 173542.1|Small CK nuclear ribonucleoprotein, putative, snRNP. putative? Sm protein, putative 432 1.OOE-126 1OO gil 16323514| refNP 187673.1| CK CS PP HS diadenosine 5'5"-P1-P4 etraphosphate hydrolase, putative Arabidopsis thaliana 433 O 1OO gi30679613| refNP 1721 13.1| DNA CK SS PEG binding bromodomain containing protein Contains similarity to a Ring3 protein 434 2.OOE-48 1OO gb|AAC17827.1| similar to CK PP ate embryogenesis abundant proteins gi2O466169. refNP 564623.2 CK Sodium calcium exchanger amily protein calcium binding EF hand family protein 436 2.OOE-87 1OO gl 1931O829 refNP 1793.96. histone H1-3 (HIS1-3) 437 1.OOE-99 1OO gl 21593872 refNP 563.973. amily protein actoylglutathione lyase amily protein glyoxalase I 438 1.OOE-159 66 gl 1522O367 refNP 176890.1|F-box CS HS amily protein Arabidopsis thaliana 439 O 78 gl 19757954 refNP 99024.11 expressed protein Arabidopsis thaliana 440 S.OOE-51 1OO gl 21618.078 refNP 176371. HS SS postsynaptic pro ein-related Arabidopsis thaliana 441 O 1OO gli24030234 refNP O27420.1 Zinc CS SS LL PEG finger (CCCH-type) family protein Arabidopsis thaliana US 2015/O 1841 89 A1 Jul. 2, 2015 14

TABLE 3-continued

PEP SEQ ID annotation

NO e value % identity GenBank id description trait 442 O gi21618116| refNP 567671.1|bile acid: sodium symporter family protein 443 1.OOE-168 gil 16930405| refNP 850182.1|PUR PP HS SS alpha-1 protein Arabidopsis thaliana 444 83 gi23397029 | refNP 567709.1| 26S CS PP proteasome regulatory subunit, putative (RPN7) 445 83 refNP 850094.1| CBL SS PEG interacting protein kinase 3 (CIPK3) 446 gi192942.91. db|BAB02193.1|cytochrome PP 4SO 447 gi7270365 emb|CAB80133.1|cyclin PP HS SS PEG delta-3 448 gi31580811| gb|AAP51420.1 ferric chelate reductase Arabidopsis thaliana 449 99 gi18439909. refNP 563791.1 | protein phosphatase 2C family protein/PP2C family protein 450 OO gil 15238701 refNP 197894.1|cytochrome DS P450 family protein Arabidopsis thaliana 451 1.OOE-178 OO gi21553567 gb|AAM62660.1 unknown Arabidopsis thaliana 452 O OO gi231983.54 refNP 193140.1 selenium HS binding protein, putative Arabidopsis thaliana 453 OO gi22655117 refNP 565411.2 calcium CS HS SS LL dependent protein kinase isoform 6 (CPK6) 454 4.OOE-83 OO gi21537110 refNP 197849.11 expressed protein Arabidopsis thaliana 455 1.OOE-122 OO gi23397082 refNP 174418. PEG photosystem I reaction center subunit III family protein 456 OO gi3068O112 refNP 179369.2 expressed protein Arabidopsis thaliana 457 OO refNP 190390.1| expressed protein Arabidopsis thaliana 458 OO gi3169180| refNP 179889.1 casein kinase II alpha chain, putative Arabidopsis thaliana 459 OO gi30725494. refNP 851 182.1 | protein SS kinase family protein Arabidopsis thaliana 460 OO gi26986957| refNP 742382.1|4- DS aminobutyrate aminotransferase Pseudomonas puttida KT2440 461 91 gi37526249. refNP 92.9593.14 CK aminobutyrate aminotransferase (gamma amino-N-butyrate transaminase) (GABA transaminase) 462 83 gi37527865 refNP 93.1210.11glutamate CK synthase NADPH Small chain (glutamate synthase beta subunit) (NADPH GOGAT) (GLTS beta chain) US 2015/O 1841 89 A1 Jul. 2, 2015 15

TABLE 3-continued

PEP SEQ ID annotation

NO e value % identity GenBank id description trait 463 gl 233O8229 refNP 175649.11glycosyl PP hydrolase family 1 protein beta-glucosidase, putative a 464 gl 30685484 refNP 849681.1 serine? LL LN hreonine protein phosphatase 2A (PP2A).55 kDa regulatory Subunit B Arabidopsis thaliana 465 99 gli16080446 refNP 391273.1 phospho PP glycerate kinase Bacilius subtilis subsp. subtilis str. 68) 466 OO gl16729O16 gb|AAF27012.1 putative SAR DNA-binding protein-1 Arabidopsis thaliana 467 OO gl |42569691 refNP 181253.2 transducin HS amily protein? WD-40 repeat family protein 468 OO gl 386O3990 refNP 177237.1| C2 domain-containing protein 469 O OO gliI22136622 refNP 193273.1 PP cytochrome P450 family protein Arabidopsis thaliana 470 OO gl 2233.1742 refNP 190771.2F-box amily protein? WD-40 repeat family protein Arabidopsis thaliana 471 1.OOE-162 OO gl 7021738 refNP 566517.1|expressed CS SS protein Arabidopsis thaliana 472 98 gl 10176804 db|BABO9992.1 serine? SS LL hreonine protein kinase-like Arabidopsis thaliana 473 1OO gl 28827.534 refNP 568971.1 leucine rich repeat transmembrane protein kinase, putative 474 gli12321664 gb|AAG50866.1 protein kinase, putative Arabidopsis thaliana 475 1.OOE-177 gl 2651299| refNP 181590.1 | protein kinase family protein 476 O gl S638.1947 refNP 564161. SS phosphoglycerate bisphosphoglycerate mutase amily protein 477 gl 15075789 refNP 38.6871. PROBABLE PHOSPHOGLYCERATE KINASE PROTEIN 478 96 gl 28872422 refNP 795.041.11glutamine CS synthetase 479 OO gl 21595073 refNP 192046.11 mitogen SS PEG activated protein kinase, putative/MAPK, putative (MPK4) 480 OO gl 10174347 refNP 242595.11 glutamate synthase (Small Subunit) 481 OO gli16081086 refNP 391914.1ornithine PP aminotransferase 482 OO gl 184OOOO3 refNP 564469.1 WD-40 HS SS LL PEG repeat family protein Arabidopsis thaliana 483 OO gl 4559336 refNP 181783.1 | protein kinase family protein Arabidopsis thaliana 484 OO gli11994,319 refNP 188976.1 casein CS PP SS PEG kinase, putative Arabidopsis thaliana 485 OO gl 235.07789 refNP 176390. LN mitochondrial transcription US 2015/O 1841 89 A1 Jul. 2, 2015 16

TABLE 3-continued

PEP SEQ ID annotation

NO e value % identity GenBank id description trait termination factor-related mTERF-related Arabidopsis thaliana 486 1.OOE-120 OO gl 10177463 db|BAB10854.1 unnamed LL protein product Arabidopsis thaliana 487 OO gl 15221219 refNP 177573.1 protein LL kinase, putative Arabidopsis thaliana 488 OO gl 15218854 refNP 174217.1|CBL CS LL PEG interacting protein kinase 18 (CIPK18) 489 OO gl 2213678O refNP 189420. PP LL monodehydroascorbate reductase, putative Arabidopsis thaliana 490 1.OOE-132 OO gl 17104619 gb|AAL34.198.1 putative glutathione peroxidase Arabidopsis thaliana 491 OO gl 726.8589 refNP 1921.4.11 Sugar transporter, putative Arabidopsis thaliana 492 OO gl S6381949 refNP 200733.2 sugar transporter family protein Arabidopsis thaliana 493 7.OOE-60 83 gl 3143336S refNP 922597.1 unknown SS LL protein 494 1.OOE-171 79 gl 349.10712 refNP 916703.1 putative nuclear RNA binding protein A Oryza sativa 495 1.OOE-139 1OO gl S1536564 refNP 189139.1| zinc finger PP (C3HC4-type RING finger) family protein 496 1.OOE-136 98 gl 22995109 refZP OOO39591.1|COGO14 PEG 9: Triosephosphate isomerase 497 74 gli32488446 refixP 473139.1| OSJNBa0004NO5.3 (Oryza Saiiva (japonica cultivar group) 498 gliI23126040 refZP 00107950.1|COGO15 5: Sulfite reductase, beta Subunit (hemoprotein) Nostoc punctiforme PCC 73102) 499 gi7287983| refNP 191594.1| CK PEG armadillo, beta-catenin repeat family protein/F-box family protein Arabidopsis thaliana 500 19758604 db|BAB09237.1beta amylase Arabidopsis thaliana gl 23463OS1 refNP 178065.1 inosine-5'- monophosphate dehydrogenase Arabidopsis thaliana 502 1.OOE-100 77 gl SS1682SO gb|AAV44116.1 unknown protein Oryza Saiiva (japonica cultivar-group) 503 83 gl SO942175 refixP 480615.1putative aminoimidazolecarboximide ribonucleotide transformylase SO4 1.OOE-159 gl 10177927 refNP 1996O2.1| respiratory burst oxidase protein D (RbohD), NADPH oxidase 505 1.OOE-172 86 gl S7899142 db|BAD87004.1 unknown protein Oryza Saiiva (japonica cultivar-group) US 2015/O 1841 89 A1 Jul. 2, 2015 17

TABLE 3-continued

PEP SEQ ID annotation

NO e value % identity GenBank id description trait SO6 gil 15223469 | refNP 171679.1 protein CK kinase family protein Arabidopsis thaliana 507 gi3482918 refNP 17241.4.11 ATP CK SS citrate synthase (ATP-citrate (pro-S-)-lyase/citrate cleavage enzyme), putative SO8 gi23297 150| refNP 567724.1 fibrillarin CK 2 (FIB2) Arabidopsis thaliana 509 refNP 193573.1|F-box CK LL PEG family protein Arabidopsis thaliana 510 6.OOE-87 79 gi21553981| refNP 564267.11 peptidyl CK prolyl cis-trans isomerase cyclophilin-type family protein Arabidopsis thaliana 1.OOE-151 68 34913784 refNP 918239.1 pectate lyase-like protein 4.OOE-91 75 gl 53793523 db|BAD54684.1 unknown protein Oryza Saiiva (japonica cultivar-group) 1.OOE-41 63 gl ISO948.309 refixP 483.682.1 putative LL auxin induced protein 1.OOE-53 58 gl 562O2166 db|3AD73644.1|60S CS PEG ribosomal protein L18A-like 93 gli34910948 db|BAB84492.11 nuclear movement protein-like O gl 6513938 refNP 566157.1 modulin amily protein Arabidopsis thaliana 1.OOE-171 gl 6957722 refNP 186908.1 delta 7 LL sterol-C5-desaturase, putative Arabidopsis thaliana gl 17132051 refNP 486997. PP hypothetical protein alr2957 Nostoc sp. PCC 7120 O 71 gli34911116 refNP916905.1putative glyoxal oxidase 520 6.OOE-24 42 gl refNP 565.499.11 expressed SS protein Arabidopsis thaliana 521 O 81 gl 50934555 refixP 4768.05.1 unknown SS PEG protein 522 8.OOE-94 gl 26453028 refNP 197956.1| expressed PP SS PEG protein 523 1.OOE-167 gl 19795142 emb|CAB67657.2 splicing PP actor-like protein Arabidopsis thaliana refNP 190918.3 Zinc knuckle (CCHC-type) amily protein Arabidopsis thaliana 524 1.OOE-146 gi|6642639. refNP 187382.1 forkhead CS PEG associated domain containing protein FHA domain-containing protein 525 gi927.9657 refNP 1884.51.1| CS PP HS octicosapeptide/PhoX/Bem1 p (PB1) domain-containing protein Arabidopsis thaliana 526 gi19759010 db|BABO9537.1 unnamed CS protein product Arabidopsis thaliana 527 gil 18390671| refNP 563769.1|F-box CS PP HS SS family protein 528 gil 12248033) refNP 172880.1| CS phytochrome kinase, US 2015/O 1841 89 A1 Jul. 2, 2015 18

TABLE 3-continued

PEP SEQ ID annotation

NO e value % identity GenBank id description trait putative Arabidopsis thaliana 529 gi21554.404| refNP 179646.1| DNAJ CS PP SS heat shock family protein Arabidopsis thaliana 530 1.OOE-111 refNP 201441.1| dehydrin CS PEG (RAB18) rab18 protein Arabidopsis thaliana sp|P30185|DHR18 ARATH Dehydrin Rab18 531 db|BAB70612.1| CS SS anthocyanin-related membrane protein 1 532 O gi21553568 refNP 849480.11 expressed CS PP PEG protein 533 O gil21553616| refNP 568089.1| expressed CS protein 534 1.OOE-158 gi21554.409| refNP 174469.1 histidine CS biosynthesis bifunctional protein (HISIE) Arabidopsis thaliana pirT51812 phosphoribosyl AMP cyclohydrolase (EC 3.5.4.19) 535 1.OOE-156 68 refixP 493889.1 putative CS protein kinase Oryza Saiiva 536 76 gi31455393 | emb|CAD92450.1amino CS SS acid permease 6 Brassica naptis 537 gil 6324421 | sp|Q12068|GRE2 YEAST CS NADPH-dependent methylglyoxal reductase GRE2 (Genes de respuesta a estres protein 2) 538 99 18401915 refNP 5666.12.1 histone CS deacetylase family protein Arabidopsis thaliana 539 1OO gl 30685903 refNP 851041.1|zinc finger CS PEG (CCCH-type) family protein Arabidopsis thaliana 1.OOE-134 68 gli31432120 refNP 92.1503.1 | putative CS PP polyprotein Oryza sativa (japonica cultivar-group) S41 1OO gl 22136.556 emb|CAB80464.1 CS cinnamyl-alcohol dehydrogenase ELI3-2 Arabidopsis thaliana S42 gl 74.13528 refNP 196086. CS cytochrome P450, putative Arabidopsis thaliana 543 99 gli4256.8440 refNP 199845.2DNA CS PEG repair protein-related Arabidopsis thaliana 544 1OO gl 3.0102478 refNP 195917.1| hydrolase, CS PEG alpha/beta fold family protein Arabidopsis thaliana 545 gl 2128O881 refNP 176968. 11 glycerate CS HS SS PEG dehydrogenase/NADH dependent hydroxypyruvate (CC8Se. S46 gl 10177967 refNP 198904.1|WD-40 CS PEG repeat Family protein zfwd3 protein (ZFWD3) Arabidopsis thaliana 547 gl 7269306 refNP 567705.1| ubiquitin CS specific protease 16, putative (UBP16) Jul. 2, 2015 19

TABLE 3-continued

PEP SEQ ID annotation NO e value % identity GenBank id description trait S48 O 99 gi|6324.066 | sp|P53845|YNO3 YEAST CS Hypothetical 35.5 kDa protein in PIK1-POL2 intergenic region 549 O 99 gi|6323.074 refNP 013146.1Microtubule CS associated protein (MAP) of the XMAP215/Dis1 family; regulates microtubule dynamics during spindle orientation and metaphase chromosome alignment; interacts with spindle pole body component Spc.72p Saccharomyces cerevisiae 550 O 100 giló324990| refNP O15058.1|Dicarboxylic CS PP amino acid permease, mediates high-affinity and high-capacity transport of L glutamate and L-aspartate; also a transporter for Gln, ASn, Ser, Ala, and Gly Saccharomyces cerevisiae 551 O 100 gil51013603. sp|P50947|YNJ7 YEAST CS PEG Hypothetical 37.0 kDa Protein in RAS2-RPS7B intergenic region 552 O 100 giG319413| refNP 009495.1 Shplp CS Saccharomyces cerevisiae emb|CAA80789.1 YBLO515 Saccharomyces cerevisiae emb|CAA84878.1 SHP1 553 O 100 gi22136824| refNP 1974.55.11 expressed CS PP protein Arabidopsis thaliana 554. O 100 gi22655036| refNP 566453.1|WD-40 CS repeat family protein Arabidopsis thaliana 555 O 100 gi48771699 | refZP 00276041.1|COG106 CS 2: Zn-dependentalcohol dehydrogenases, class III Ralstonia metallidurans CH34) 556 O 1OO gi49176169| refNP 416429.3|D-cysteine CS desulfhydrase, PLP dependent enzyme cysteine desulfhydrase, PLP dependent enzyme 557 O 100 gi9294512. gb|AAK21273.1|aberrant CS PP SS lateral root formation 5 Arabidopsis thaliana refNP 566730.1|MATE efflux family protein Arabidopsis thaliana 558 O 1OO gi31711972| refNP 175641.1 | protein CS SS kinase family protein C type lectin domain containing protein SS9 8.OOE-61 1OO gi21592962| refNP 564579.11 expressed CS SS protein Arabidopsis thaliana S60 O 100 gi301.02452 refNP 195770. CS LL mitochondrial substrate carrier family protein Arabidopsis thaliana 561 O 100 giló056406 gb|AAF02870.1|Hypothetical CS protein Arabidopsis thaliana US 2015/O 1841 89 A1 Jul. 2, 2015 20

TABLE 3-continued

PEP SEQ ID annotation

NO e value % identity GenBank id description trait S62 1.OOE-143 gi21689635 | emb|CAB78986.11 lectin like CS SS protein Arabidopsis thaliana 563 gi22135769. refNP 188127.1| CS oxidoreductase, zinc-binding dehydrogenase family protein S64 refNP 199669.1| FK506 CS PEG binding protein 2-2 (FKBP15-2)/immunophilin? peptidyl-prolyl cis-trans isomerase/rotamase 565 1.OOE-151 gi21592742| refNP 195534.1|expansin, CS PP putative (EXP20) 566 8.OOE-64 gi215371251 refNP 196975.11 expressed CS protein Arabidopsis thaliana 567 1.OOE-49 gil 16331623| refNP442351.1|hypothetical CS protein SS10353 Synechocystis sp. PCC 6803 568 gi216180541 refNP 564383.1| ABC CS transporter family protein Arabidopsis thaliana 569 gi7321037 refNP192879.1 CS PP SS ARIDBRIGHT DNA binding domain-containing protein ELM2 domain containing protein Myb ike DNA-binding domain containing protein 570 1.00E-44 emb|CAA48415.1 unnamed CS protein product synthetic construct 571 99 gi| 10172815| db|BABO3922.11glycine CS PEG betaine aldehyde dehydrogenase Bacilius halodurans C-125 572 1.OOE-165 1OO gi3928.103. refNP 181434.1|acquaporin, CS PP putative Arabidopsis thaliana 573 91 gi37524-1261 refNP 927470.1 phosphoenol CS pyruvate carboxykinase ATP Photorhabdus luminescens subsp. iatinondi TTO1 574 OO gl 192938.60 refNP 564217.1| lysine and CS histidine specific transporter, putative Arabidopsis thaliana 575 OO gl 101753.58 refNP 243603.11 CS pyrroline-5-carboxylate dehydrogenase Bacilius halodurans C-125 576 OO gl 10175785 refNP 244029.1| pyruvate CS PP kinase Bacilius halodhirans C-125 577 8.OOE-45 OO gl 19758890 db|BABO9466.1 auxin CS induced protein-like Arabidopsis thaliana 578 1.OOE-108 OO gl 17104761 refNP 193699.1 Ras CS related GTP-binding protein, putative Arabidopsis thaliana 579 OO gl 5915859 sp|O22203|C98A3 ARATH CS Cytochrome P450 98A3 OO gl16729O15 refNP 187156.1 | protein CS SS PEG kinase family protein Arabidopsis thaliana US 2015/O 1841 89 A1 Jul. 2, 2015 21

TABLE 3-continued

PEP SEQ ID annotation

NO e value % identity GenBank id description trait 581 gi21689671| refNP 195397.1| CS PP SS transporter-related Arabidopsis thaliana 582 gi20148451| refNP 564797.1 flavin CS SS PEG containing monooxygenase family protein/FMO family protein 583 gi21280977| gb|AAM45011.1|putative CS protein kinase Arabidopsis thaliana 1.OOE-116 82 gi27452901 | gb|AAO15285.1|Putative CS dihydrodipicolinate reductase-like protein Oryza Saiva (japonica cultivar-group) 585 1.OOE-123 99 23113896 refZP OOO99233.1|COGOO3 CS 6: Pentose-5-phosphate-3- epimerase Desulfitobacterium hafniense DCB-2 S86 99 gl 16330731 YCF3 SYNY3 CS Photosystem I assembly protein yef3 587 1OO gl 217OO831 refNP 191332.2 protein CS DS SS PEG kinase, putative Arabidopsis thaliana 588 gli4415911 gb|AAD20142.1 putative CS poly(A) binding protein Arabidopsis thaliana 589 gl 573.3869 gb|AAD49757.1|Contains F CS box domain PF100646. Arabidopsis thaliana 590 1.OOE-149 gliI29824243 refNP 192170.11 tryptophan CS synthase, alpha Subunit, putative Arabidopsis thaliana 591 gliI26449849 refNP 198924.1| CS HS glycerophosphoryl diester phosphodiesterase family protein 592 78 gl 56783874 db|BAD81286.1 putative CS dual specificity kinase 1 593 1OO gl16321857 refNPO11933.1|Ssflip CS DS Saccharomyces cerevisiae sp|P38789 ISSF1 YEAST Ribosome biogenesis protein SSF1 594 1.OOE-18O 89 gl ISO906987 refixP 464982.1 lipase class CS 3-like Oryza sativa (japonica cultivar-group) db|BAD21500.1| lipase class 3-like Oryza sativa (japonica cultivar-group) 595 1OO gl 2558659 gb|AAB81672.1putative CS HS PEG protein kinase Arabidopsis thaliana 596 gl 19758547 refNP 201503.11 expressed CS protein Arabidopsis thaliana 597 gl 4678.335 refNP 190376.1| L CS SS galactono-1,4-lactone dehydrogenase, putative 598 1.OOE-173 gl 21553608 refNP 564800.11 expressed CS protein Arabidopsis thaliana 599 79 gl ISO912531 refixP 467673.1 putative CS DS SS PEG nicastrin Oryza Saiiva (japonica cultivar-group) 600 gl 50898642 refixP 45.0109.1 modulation CS PP SS receptor kinase-like protein Jul. 2, 2015 22

TABLE 3-continued

PEP SEQ ID annotation NO e value % identity GenBank id description trait 601 S.OOE-74 00 gi30693828 refNP 850693.1|expressed DS protein Arabidopsis thaliana 602 O 00 gi25054896. refNP 179923.2 nicotinate DS phosphoribosyltransferase family protein. NAPRTase family protein Arabidopsis thaliana 603 1.OOE-121 00 giló006854| gb|AAF00630.1 hypothetical DS LN protein Arabidopsis thaliana 604 O 00 giló862917| refNP 566273.1| heavy DS metal-associated domain containing protein 60S O 00 gil56382003. refNP 177794.1| 12 DS Oxophytodienoate reductase (OPR1) Arabidopsis thaliana 606 O 00 gi21280825| gb|AAM45040.1 putative DS AtMlo-h1 protein Arabidopsis thaliana 607 O 61 gi22136432. refNP 191438.2 glycosyl DS SS transferase family 8 protein Arabidopsis thaliana 608 O 69 gi22136270| refNP 190235.1 DS armadillo, beta-catenin repeat family protein U-box domain-containing family protein 609 O 100 gi4678377 refNP 193O87.1 DS ammonium transporter 1, member 1 (AMT1.1) 610 1.OOE-143 99 gi27754217 refNP 1874.13.1| expressed DS LN PEG protein Arabidopsis thaliana 611 1.OOE-123 1OO gi21554344 refNP 198627.1|ASF1 DS PP HS ike anti-Silencing family protein Arabidopsis thaliana 612 O 77 gi25402857 pirA86318 protein DS PP F15H18.1|imported Arabidopsis thaliana gb|AAF25996.1|F15H18.1| 613 1.OOE-139 99 gil 1632995.0 refNP440678.1 hypothetical DS protein slr1900 Synechocystis sp. PCC 6803) 614 O 1OO gi30725526 gb|AAP37785.1|AtAg24520 DS SS Arabidopsis thaliana emb|CAB79362.1 NADPH errihemoprotein reductase ATR1 615 O 98 gi33353.45| gb|AAC27147.1|Contains DS LL PEG similarity to ABC transportergb|1651790 from Synechocystis sp. 616 1.OOE-120 1OO gi38603932| refNP 193578.1| Ras DS related GTP-binding protein, putative Arabidopsis thaliana 617 O 1OO gil 15810337| refNP 565310.11 expressed DS PEG protein Arabidopsis thaliana 618 O 1OO gi|8978.074 refNP 199518.11 protein DS kinase, putative Arabidopsis thaliana 619 O 75 gi19294678 db|BAB03027.11glutamine DS ructose-6-phosphate transaminase 2 Arabidopsis thaliana US 2015/O 1841 89 A1 Jul. 2, 2015 23

TABLE 3-continued

PEP SEQ ID annotation

NO e value % identity GenBank id description trait O gi42567142| refNP 194265.2|EXS DS amily protein ERD1/XPR1/SYG1 family protein Arabidopsis thaliana gb|AAR99486.1| PHO1-like protein Arabidopsis thaliana 621 1.OOE-151 89 gi50938765| refixP 478910.1 serine/ DS SS PEG hreonine protein kinase PKPA ike protein Oryza Saiiva 622 1OO gil 16323410| gb|AAD49980.1| Similar to HS gb|AF10333 PrMC3 protein from Pinus radiata and is a member of PFIOO135 Carboxylesterases amily. 623 gi233971281 refNP 178620.11 glycine HS SS rich protein (GRP) Arabidopsis thaliana 624 1.OOE-104 gi24417354| refNP 564833.1| expressed HS protein Arabidopsis thaliana 625 1.OOE-48 57 gi21553397 refNP 180326.1 Zinc HS finger (AN1-like) family protein Arabidopsis thaliana 626 1OO gil 16130575 refNP 417147.1 succinate HS semialdehyde dehydrogenase I, NADP dependent Escherichia coli K12) 627 gl 15218645 refNP 176713.1|cytochrome HS PEG P450, putative Arabidopsis thaliana 628 gl 16329.269 refNP 439997.1|hypothetical HS protein slrO731 Synechocystis sp. PCC 6803 629 99 gl 12322845 gb|AAG51407.1 putative HS cysteine synthase: 39489 37437 Arabidopsis thaliana 630 1.OOE-126 OO gl 288276.86 gb|AA050687.1 unknown HS PEG protein Arabidopsis thaliana 631 1.OOE-143 OO gl 23197714 refNP 196259.2 DNAJ HS SS LL heat shock N-terminal domain-containing protein 632 OO gl gb|AAM63172.1 putative integral membrane protein Arabidopsis thaliana 633 OO gl 16329756 refNP440484.1|formaldehyde dehydrogenase (glutathione) 634 OO gliI26450489 refNP 19014.8.1 transducin family protein WD-40 repeat family protein 635 OO gl 398,0410 refNP 180485.1 lectin LN protein kinase, putative Arabidopsis thaliana 636 1.OOE-153 OO gl 6572061 refNP 190708.11 expressed LN protein Arabidopsis thaliana 637 1.OOE-141 OO gliI22328141 refNP 201402.2 hetero LN geneous nuclear ribonucleoprotein, putative hnRNP. putative 638 OO gl 15219657 refNP176819.1 protein kinase family protein Arabidopsis thaliana US 2015/O 1841 89 A1 Jul. 2, 2015 24

TABLE 3-continued

PEP SEQ ID annota ion NO e value % identity GenBank id description trait 639 S.OOE-53 55 gi34906632 refNP 914663.1|PO431GO6. 11 Oryza Saiiva (japonica cultivar-group) 640 1.OOE-169 1OO gi21674686 refNP 662751.1|6- phosphogluconate dehydrogenase, decarboxylating, putative Chlorobium tepidum TLS) 641 1.OOE-69 96 gi32490260 refixP 473078.1 PEG OSJNBa0014K14.9 Oryza Saiiva (japonica cultivar group) 642 O 1OO gi52354393 gb|AAU44517.1 hypothetical PEG protein AT4G22980 Arabidopsis thaliana 643 2.OOE-90 1OO gi9758347 refNP 200586.1| expressed protein Arabidopsis thaliana 644 O 82 gi50946759. refixP 482907.1 putative LN glycoprotein 3-alpha-L- fucosyltransferase 645 1.OOE-131 83 gi7269121| emb|CAB79230.1 predicted protein Arabidopsis thaliana 646 1.OOE-130 88 gi50929089 refixP 474O72.1|OSJNBbOO LN 79B02.14 Oryza sativa (japonica cultivar-group) 647 O 00 gil 10176523 refNP 244766.1|acetyl CoA:acetoacetyl-CoA transferase Bacillus halodurans C-125 648 O OO gi34365735 refNP 566384. Xanthinefuracil permease amily protein Arabidopsis thaiana 649 1.OOE-84 OO gil 10177539 refNP 201459.1| expressed protein Arabidopsis thaiana 6SO 1.OOE-137 OO gi21537398 refNP 201145.1|adenylate LN kinase Arabidopsis thaiana 651 O OO gil 10177005 refNP 851 136. LN cytochrome P450 family protein Arabidopsis thaiana 652 O 99 gi9758597 refNP 851196.1 outward LN rectifying potassium channel (KCO1) Arabidopsis thaiana 653 1.OOE-126 1OO gi20453405| refNP 180901.11 plastid LN developmental protein DAG, putative Arabidopsis thaiana 654 O 1OO gi7378.631. refNP 195967. LN serine/threonine protein phosphatase 2A (PP2A) regulatory subunit B' (B'alpha) 655 O 1OO gi500,58955| gb|AAT69222.1|hypothetical LN protein At2g30900 Arabidopsis thaliana 656 3.OOE-36 1OO gi26453058| refNP 191804.1| LN expressed protein Arabidopsis thaliana 657 O 1OO gi28973131| refNP 1903.13.1| LN phosphoinositide-specific phospholipase C family protein US 2015/O 1841 89 A1 Jul. 2, 2015 25

TABLE 3-continued

PEP SEQ ID annotation NO e value % identity GenBank id description trait 658 O OO gi23297411| gb|AAN12963.1 lenolase (2- phospho-D-glycerate hydroylase) Arabidopsis thaliana 659 1.OOE-157 OO gi22136938| refNP 566528.1| expressed LN protein Arabidopsis thaliana 660 O OO gi19294186 refNP 566,763.1 auxin LN responsive family protein Arabidopsis thaliana 661 O OO gi7269372. refNP 19425.2.1] LN transporter, putative Arabidopsis thaliana 662 3.OOE-93 OO gi28393887| refNP182046.1 auxin LN responsive protein-related Arabidopsis thaliana 663 O OO gi4725942. refNP 192980.1 trehalose LN 6-phosphate phosphatase, putative 664 O OO gil 15074657 refNP 385829.1| LN PROBABLE AMINOTRANSFERASE

66S 1.OOE-179 81 gi35215000| refNP 927371.1| LN glutathione dependent ormaldehyde dehydrogenase 666 1.OOE-142 1OO gi23507799 refNP 565973.1| LOB LN domain protein 16/lateral organ boundaries domain protein 16 667 O 99 gi48731145| refZP 00264891.1|COG101 LN 2: NAD-dependent aldehyde dehydrogenases Pseudomonas fittorescens Pfo-1 668 O 99 gil 15223.033 refNP 177763.1 protein LN kinase, putative Arabidopsis thaliana 669 O 1OO gi22137136 refNP 181639.1|RNA LN recognition motif (RRM)- containing protein 670 O 1OO gi50904773 refixP 463875.1putative LN iron-phytosiderophore transporter protein yellow stripe 1 671 S.OOE-40 48 gil 15232064 refNP 189339.1|expressed LN protein Arabidopsis thaliana 672 O 88 gi51535369 db|BAD37240.1putative LN phosphotyrosyl phosphatase activator Oryza Saiiva (japonica cultivar-group) 673 8.OOE-93 1OO gi21555349 refNP 190798.1|ATP LN synthase D chain-related Arabidopsis thaliana 674 O 1OO gi9795597 refNP 173294. LN Sulfotransferase family protein Arabidopsis thaliana 675 O 1OO gi42567054| refNP 194058.2 protein LN kinase family protein Arabidopsis thaliana 676 O 1OO gi31711846 | refNP 177412.1|cinnamyl LN alcohol dehydrogenase, putative 677 O 1OO gi23308.375 refNP 851141.1|RNA LN recognition motif (RRM)- containing protein US 2015/O 1841 89 A1 Jul. 2, 2015 26

TABLE 3-continued

PEP SEQ ID annotation

NO e value % identity GenBank id description trait 678 O OO gl 73296.58 emb|CAB82755.1 protein LN kinase ATN1-like protein Arabidopsis thaliana 679 OO gl 22655386 refNP 197288.1 cation LN exchanger, putative (CAX7) Arabidopsis thaliana 1.OOE-162 OO gl gb|AAM14984.1 high LN affinity K+ transporter (AtKUP1AtKT1p) Arabidopsis thaliana 681 OO gl 15219169 refNP 175713.1 protein LN kinase family protein Arabidopsis thaliana 682 OO gl 19758.951 refNP 568809.2 protein LN kinase family protein Arabidopsis thaliana 683 OO gl 7573368 refNP 196762.11 expressed LN protein Arabidopsis thaiana 684 OO gli2O259.017 refNP 200323.1| expressed LN protein Arabidopsis thaiana 685 OO gl 3O793811 refNP 191519.1| DNA LN PEG directed RNA polymerase I, I, and III, putative 686 OO gl 7268609 refNP193550.1 outward LN rectifying potassium channel, putative (KCO6) 687 1.OOE-111 71 gl 7270315| refNP 195093.1| L LN galactose dehydrogenase (L- GalDH) Arabidopsis thaiana 688 S.OOE-98 gl129824277 refNP 568.016.1| expressed LN protein Arabidopsis thaiana 689 1.OOE-170 69 gl 7269325 emb|CAB79384.1 protein LN kinase (AFC2) Arabidopsis thaiana 690 3.OOE-96 79 gl 57899263 db|BAD87508.1 putative LN calcyclin-binding protein Oryza Saiva (japonica cultivar-group) 691 1.OOE-164 OO gl 1994.637 emb|CAD44270.1 LN monomeric G-protein Arabidopsis thaliana 692 1.OOE-109 OO gl 7269377 refNP 194256. LN invertase?pectin methylesterase inhibitor amily protein 693 1.OOE-57 OO gl refNP 195832.1|thylakoid LN membrane one helix protein (OHP) Arabidopsis thaliana 694 OO gl 21689839 gb|AAM67563.1 putative protein transport protein SEC12p Arabidopsis thaliana 695 1.OOE-36 OO gl 101 77015 refNP 199045.1| ubiquitin PEG amily protein Arabidopsis thaliana 696 1.OOE-106 OO gliI20466087 refNP 179457.1| zinc finger (C3HC4-type RING finger) amily protein 697 OO gl 72672O3 refNP 192355.11 aspartyl protease family protein Arabidopsis thaliana 698 OO gli46931342 refNP 85.0347.1|F-box amily protein Arabidopsis thaliana 699 60 gl |46931354 gb|AATO6481.1|At3g23540 Arabidopsis thaliana US 2015/O 1841 89 A1 Jul. 2, 2015 27

TABLE 3-continued

PEP SEQ ID annotation

NO e value % identity GenBank id description trait 700 1.OOE-161 gl 21594.556 gb|AAM66021.1 plasma membrane intrinsic protein SIMIP Arabidopsis thaliana 701 gl 1902923 db|BAA07547.1 phosphoin ositide specific phospholipase C Arabidopsis thaliana 702 gl 26449713 refNP 195154.2 transducin amily protein? WD-40 repeat family protein 703 94 gl 15232616 refNP 188176.1 phototropic responsive NPH3 family protein Arabidopsis thaliana 704 gl 2329,6692 refNP 189177.1 formin homology 2 domain containing protein FH2 domain-containing protein Arabidopsis thaliana 705 gl 51O13899 refNP 015238.1|Ydc1p PEG Saccharomyces cerevisiae gb|AAB68212.1| Lpg21p gb|AAG22594.1|alkaline ceramidase Yolclp 706 gl 23297.093 refNP 565508.1 fructose bisphosphate aldolase, putative Arabidopsis thaliana 707 99 gl16322722 refNP 012795.1|Pgm1p Saccharomyces cerevisiae emb|CAASO895.1 phosphoglucomutase 708 99 gl 10175338 refNP 243583.1| glutaminase Bacilius halodurans C-125 709 94 gl 7268277 refNP 193265.1 cytochrome P450 family protein Arabidopsis thaliana 710 gl16322296 sp|P3897OHAL5 YEAST Serine/threonine-protein kinase HALS 711 gl 17104779 refNP 564866.1 U-box domain-containing protein 712 99 gli1608.0934 refNP 391762.1|aldehyde dehydrogenase Bacilius subtilis subsp. subtilis str. 168) 713 87 gl 37524894 refNP 928.238.11glutamate 1-semialdehyde 2,1- aminomutase Photorhabdus luminescens subsp. laumondi TTO1 71.4 1OO gil 16077848| refNP 388662.1trehalose PEG 6-phosphate hydrolase Bacillus subtilis Subsp. subtilis str. 168) 715 1.OOE-81 gil 1922244| refNP 171654.1 late PEG embryogenesis abundant protein, putative/LEA protein, putative 716 1.OOE-112 gi7268505 refNP 193486.1 Ras PEG related GTP-binding protein, putative Arabidopsis thaliana 717 3.OOE-44 gi21592597 refNP 563987.1 |expressed PEG protein Arabidopsis thaliana gb|AAF18498.1 | Identical to gb|Y10291 GAG1 protein Jul. 2, 2015 28

TABLE 3-continued

PEP SEQ ID annota ion NO e value % identity GenBank id description trait 718, 3.OOE-97 44 gi52353666 gb|AAU44232.1 hypothetical PEG protein Oryza Saiiva (japonica cultivar-group) 719 1.OOE-175 63 gi53850529 gb|AAC31235.1| PEG hypothetical protein Arabidopsis thaliana gb|AAT71954.1| 72O 1.OOE-131 99 gi|16330417 refNP 441|45.1 hypothetical PEG protein sl1162 Synechocystis sp. PCC 6803) 721 O 100 gil 171298.62 refNP 484561.1 PEG fructokinase Nostoc sp. PCC 7120 722 O 100 gil 15293245 refNP 567109.11 COP9 PEG signaloSome complex subunit 1/CSN complex subunit 1 (CSN.1)/COP11 protein (COP11)/FUSCA protein (FUS6) Arabidopsis 723 O 100 gi2O259583 | refNP 180431.1 beta PEG ketoacyl-CoA synthase family protein Arabidopsis thaiana 724 O 100 gi20465963 | refNP 189034.1beta PEG amylase, putative? 14 alpha-D-glucan maltohydrolase, putative Arabidopsis thaliana 725 4.OOE-72 100 gi32815911 | refNP 201|00.11 expressed protein Arabidopsis thaiana 726 1.OOE-179 100 gi|8953402. refNP 196701.1 | protein kinase-related Arabidopsis thaiana 727 O 100 giló522600| emb|CAB61965.11 aminocyclopropane-1- carboxylic acid oxidase-like protein 728 1.OOE-145 85 gi57899179 db|BAD87231.1|membrane protein-like Oryza sativa (japonica cultivar-group) 729 O 87 gi50909427 refixP 466202.11putative DNAJ domain protein Oryza Saiva (japonica cultivar-group) 730 O 100 gil53828613 refNP 179479.11 protein kinase family protein Arabidopsis thaliana 731 1.OOE-93 100 gi23306374 refNP 200428.1| expressed protein Arabidopsis thaliana 732 1.OOE-172 100 gil56121884 refNP178191.1| major intrinsic family protein MIP family protein 733 O 98 gi24762199 gb|AAN64166.1 unknown protein Arabidopsis thaliana 734 O 100 gi21230100 refNP 636017.1 phosphoma innose isomerasef GDP mannose pyrophosphorylase 735 O 100 gi 10177277 db|BAB10630.11glucose-6- PP phosphate isomerase, cytosolic Arabidopsis thaliana 736 3.OOE-61 100 gi21537079 refNP 564835.1| expressed PP HS protein Arabidopsis thaliana 737 3.OOE-91 100 gi26452507 refNP 197149.1| PP PEG dimethylmenaquinone US 2015/O 1841 89 A1 Jul. 2, 2015 29

TABLE 3-continued

PEP SEQ ID annotation

NO e value % identity GenBank id description trait methyltransferase family protein 738 OO gl 5734730 refNP 172592.1| glucose transporter (STP1) Arabidopsis thaliana 739 1.OOE-113 OO gl 573.4748 refNP 564017.1| integral SS PEG membrane family protein Arabidopsis thaliana 740 1.OOE-153 OO gl 30693623 refNP 198871.2|expressed protein Arabidopsis thaliana 741 OO gl 5638.1993 refNP 177188.1 SS PEG spermidine synthase 2 (SPDSYN2), putrescine aminopropyltransferase 2 742 OO gl 4678359 emb|CAB41169.1|cytochrome P450-like protein Arabidopsis thaliana 743 OO gl 15220055 refNP 173165.1|translation initiation factor IF-2, chloroplast, putative Arabidopsis thaliana 744 1.OOE-168 OO gl 21593.654 refNP 181996.1 casein LL LIN PEG kinase II beta chain, putative Arabidopsis thaliana 745 OO gl 19758987| db|BABO9497.1|chloroplast nucleoid DNA-binding protein-like Arabidopsis thaliana refNP199325.1| aspartyl protease family protein Arabidopsis thaliana 746 gli12642918 refNP 180133.11 protein SS phosphatase 2C, putative PP2C, putative 747 gl 727.0935 refNP 195661.1 cytochrome P450 family protein Arabidopsis thaliana 748 1.OOE-138 gl 21554052 gb|AAM63133.1|delta onoplast integral protein delta-TIP Arabidopsis thaliana 749 85 gl 28872439 refNP 795058.1 phospho glycerate mutase, 2,3- bisphosphoglycerate independent 750 gliI25284116 pirA95262probable formate PEG dehydrogenase (EC 1.2.1.2) alpha chain FoloG imported SinoRhizobium meioti (strain 1021) magaplasmid pSymA 751 1.OOE-SS gi7629997 refNP 190945.1 late PP SS PEG embryogenesis abundant protein-related LEA protein-related 752 gi23126243) refZP 00108145.1|COG152 PP SS 3: Type II secretory pathway, pullulanase PulA and related glycosidases Nostoc punctiforme PCC 73102) 753 gi42563.087 refNP 564975.21CBS PP domain-containing protein Arabidopsis thaliana 754 gil 17104597| refNP 566716.1 | protein PP SS LL kinase, putative Arabidopsis thaliana US 2015/O 1841 89 A1 Jul. 2, 2015 30

TABLE 3-continued

PEP SEQ ID annotation

NO e value % identity GenBank id description trait 755 O 1OO gil 158105411 reflNP 566876.3| protein PP SS kinase family protein Arabidopsis thaliana 756 3.OOE-84 99 gil 16331120I refNP 441848.1|hypothetical PP protein SIO359 Synechocystis sp. PCC 803) 757 O 99 gi483.5235| emb|CAB42913.1putative PP protein Arabidopsis thaliana pirTO8405 hypothetical protein F18B3.120-Arabidopsis thaiana 758 O 1OO gil 18394.553| refNP 564042.1|expressed PP protein Arabidopsis thaliana 759 O 99 gi49176911| refNP 858035.1 mercuric PP reductase uncultured bacterium 760 O 1OO gi53794956 refZP 00356067.1|COG499 PP SS 2: Ornithinefacetylornithine aminotransferase Chloroflexits attrantiacus 761 O 1OO refNP 186761.1| PP LL cyStathionine gamma synthase, chloroplast O Succinylhomoserine (Thiol)- yase (CGS) 762 O 1OO 19755789 refNP 197266.1| expressed SS LL LIN PEG protein Arabidopsis thaliana 763 O 1OO gl 15810649 pirT48630 high affinity nitrate transporter-like protein-Arabidopsis thaiana 764 1.OOE-129 1OO gl 264518OO refNP 850901.1| expressed protein Arabidopsis thaliana 765 4.OOE-58 91 gl ISO94.8565 refixP 483810.1putative PEG Bet1 Sft1-related SNARE (AtBS14a) Oryza sativa 766 O 1OO gl 763 OO64 emb|CAB88286.1 serinef hreonine-specific protein kinase-like protein 767 O 92 gl ISO932239 refixP 475647.1 putative HS PEG glutaryl-CoA dehydrogenase 768 1.OOE-104 50 gl 30684716 refNP 196906.2|expressed SS protein Arabidopsis thaliana 769 2.OOE-87 89 gl ISO912633 refixP 467724.1 unknown PEG protein Oryza sativa (japonica cultivar-group) 770 9.OOE-13 94 gli6682226 gb|AAF23278.1 unknown HS SS PEG protein Arabidopsis thaliana 771 2.OOE-90 49 gl 31430516 refNP 920131.1| putative Magnaporihe grisea pathogenicity protein 772 O 1OO gl 23297753 refNP 192381.1 calcium HS PEG dependent protein kinase, putative/CDPK, putative 773 3.OOE-54 1OO gli4417292 refNP 179791.1| expressed CK PP PEG protein Arabidopsis thaliana 774 O 1OO gli2O148423 refNP 177550.1| Sulfotransferase family protein Arabidopsis thaliana US 2015/O 1841 89 A1 Jul. 2, 2015 31

TABLE 3-continued

PEP SEQ ID annotation

NO e value % identity GenBank id description trait 775 4.OOE-88 OO gl 21554O27 refNP 192830. SS transcriptional coactivator p15 (PC4) family protein (KELP) 776 1.OOE-100 OO gl 22136806 refNP 176726.1| flowering SS PEG locus T protein (FT) Arabidopsis thaliana 777 OO gliI21592464 refNP 565844. Zinc finger LN (AN1-like) fami y protein Arabidopsis thaliana 778 1.OOE-101 OO gl1877864.5 LN Arabidopsis thaliana 779 OO gl 28973.123 refNP 196373. glycine CK rich protein (GRP20) Arabidopsis thaliana OO gl 1994.377 refNP 188020. PP PEG exopolygalacturonase galacturan 1,4-alpha galacturonidase?pectinase 781 OO gl 20465689 refNP 200440. CK peroxisomal targeting signal type 1 receptor (PEX5) 782 OO gl 21536923 refNP 194774.1| glycine CK rich protein Arabidopsis thaliana 783 1.OOE-124 OO gl 30O23686 refNP 197608.1 leucine DS PEG rich repeat protein, putative Arabidopsis thaliana 784 3.OOE-76 OO gl 26453292 refNP 563653.2 SS LN NPR1/NIM1-interacting protein 1 (NIMIN-1) Arabidopsis thaliana 785 1.OOE-90 OO gl 1522OO22 refNP 173727.1|C2 CS PEG domain-containing protein Arabidopsis thaliana 786 2.OOE-97 OO gl S6479608 refNP 706078.2 hypoxanthine SS PEG phosphoribosyltransferase Shigella flexneri 2a str. 301 EDL933) 787 1.OOE-130 99 gl 16331548 refNP 442276.1 hypothetical SS protein slrO630 Synechocystis sp. PCC 6803 788 1OO gli2O259167 refNP 568786.1 | protein phosphatase 2C, putative PP2C, putative 789 gli6466946 refNP 974249. PEG prephenate dehydratase amily protein Arabidopsis thaliana 790 1.OOE-130 gliI20465721 refNP 1745.36. plastid CS PEG developmental protein DAG, putative 791 1.OOE-63 84 gl 1017762O db|BAB10767. unnamed SS protein product Arabidopsis thaliana 792 1.00E-44 gl 58299 emb|CAA48415.1 unnamed CK PEG protein product synthetic construct 793 1.OOE-108 S4 gl 3150411 refNP 180570. 1 GCNS CS related N-acetyltransferase (GNAT) family protein 794 gl 16331766 refNP 442494. 1|aldehyde SS PEG dehydrogenase Synechocystiss p. PCC 6803) 795 1.OOE-30 gl emb|CAB82954.1|cytochrome CK CS PEG c oxidase subunit 5c-like protein Arabidopsis thaliana US 2015/O 1841 89 A1 Jul. 2, 2015 32

TABLE 3-continued

PEP SEQ ID annotation

NO e value % identity GenBank id description trait 796 1.OOE-137 gl refNP 175374.2 expressed SP CS HS PEG protein Arabidopsis thaliana 797 1.OOE-93 gl 19310667 refNP 188286.1 SP PP SS translationally controlled tumor family protein Arabidopsis thaliana 798 gl 15293.235 refNP 194825.1| CBL SP interacting protein kinase 6 (CIPK6) Arabidopsis thaliana 799 1.OOE-165 62 gli4220476 gb|AAD12699.1 putative SP ribophorin I Arabidopsis thaliana 800 gl 20908080 refNP 200169.1 SS RabGAPTBC domain containing protein Arabidopsis thaliana 1.OOE-68 99 gl 51449987 refNP 189210.2 SS PEG myrcene/ocimene synthase, putative Arabidopsis thaliana 802 2.OOE-94 1OO gl 121592517 refNP 564266.1| expressed SS protein Arabidopsis thaliana 803 gl |42572237 reflNP 974213.1|ankyrin SS PEG repeat family protein regulator of chromosome condensation (RCC1) family protein Arabidopsis thaliana 804 1.OOE-115 gi21593862. refNP 190632.11 kip SS related protein 2 (KRP2), cyclin-dependent kinase inhibitor 2 (ICK2)/cdc2a interacting protein Arabidopsis thaliana 805 18843.792 refNP 200680.1| PRLI SS interacting factor, putative Arabidopsis thaliana 806 90 gl 21586474 gb|AAM55306.1|auxin SS influx carrier protein Medicago truncatula 807 99 gli2O7.99715 gb|ArefNP 190468.1 SS AMP-dependent synthetase and ligase family protein 808 1OO gl 15293255 refNP 564424.1| PHD SS finger family protein Arabidopsis thaliana 809 gli24030409 refNP 1942O7.1| SS LN expressed protein Arabidopsis thaliana 810 gl 6728973 refNP 186938.1 leucine SS PEG rich repeat transmembrane protein kinase, putative 811 1.OOE-133 gl 127754697 gb|AA022792.1 putative SS PEG cytochromec oxidoreductase Arabidopsis thaliana 812 91 gl 45394.45 refNP 1928.05.1| SS transcription elongation factor-related Arabidopsis thaliana 813 gl 232966OO refNP 568107.1 | pseudo SS response regulator 7 (APRR7) Arabidopsis thaliana sp|Q93WK5 IAPRR7 ARAT HTwo-component response regulator-like APRR7 (Pseudo-response regulator 7) US 2015/O 1841 89 A1 Jul. 2, 2015 33

TABLE 3-continued

PEP SEQ ID annotation

NO e value % identity GenBank id description trait 814 99 gil 16331392. refNP 442120.1|ribulose SS bisphosphate carboxylase large subunit 815 99 gil 16329673| refNP 440401.1|mercuric SS reductase Synechocystis sp. PCC 6803) 816 1OO gi23506207| gb|AAN31115.1|At2g26250/ SS T1D16.11 Arabidopsis thaliana gb|AAG60062.1| putative beta-ketoacyl-CoA synthase FIDDLEHEAD 817 99 refNP 186798.1 | protein SS kinase, putative Arabidopsis thaliana 818 1OO gi21593811| gb|AAM65778.1 putative SS NADPH quinone oxidoreductase Arabidopsis thaliana 819 gi23125809| refZP 00107727.1|COGO16 SS 6: Glucose-6-phosphate isomerase Nostoc punctiforme PCC 73102) gil 16077353| refNP 388166.1 hypothetical SS PEG protein BSUO2840 Bacillus subtilis Subsp. subtilis str. 168) 821 1.OOE-117 refNP194624.1 Rac-like SS GTP-binding protein (ARAC7) Arabidopsis thaliana 822 1.OOE-128 96 gi27808548 gb|AAO24554.1|Atlg61150 SS PEG Arabidopsis thaliana 823 O 97 gi30684071 refNP 85.0128.1 protein SS kinase family protein Arabidopsis thaliana 824 2.OOE-77 99 gil 1633O866 refNP 441594.1|hypothetical SS LL protein slr1273 Synechocystis sp. PCC 6803 825 1.OOE-177 gi23297050 refNP 194O73.2 short SS chain dehydrogenase/reductase (SDR) family protein 826 1.OOE-109 gil 12083284 refNP 173437.1 Rac-like SS GTP-binding protein (ARAC4), Rho-like GTP binding protein (ROP2) 827 gi20465939 refNP 850393.11 sugar SS transporter family protein Arabidopsis thaliana 828 98 gi2661128 gb|AAC04613.1 larginase SS Glycine max pirTO6222 probable arginase (EC 3.5.3.1)-soybean sp|O49046|ARGI SOYBN Arginase 829 64 gi40539114| gb|AAR87370.1|expressed SS protein Oryza Saiiva (japonica cultivar-group) 830 91 gi521376151 emb|CAH4.0838.1 protein SS O-fucosyltransferase 1 Saccharum officinarum 831 gi5733881| refNP 175261.1| G protein SS coupled receptor-related Arabidopsis thaliana 832 gil 15217930| refNP 176128.1|hypothetical SS protein Arabidopsis thaliana 833 1.OOE-29 75 gi20465895 refNP 974937.1| RNA SS LN recognition motif (RRM)- containing protein US 2015/O 1841 89 A1 Jul. 2, 2015 34

TABLE 3-continued

PEP SEQ ID annotation

NO e value % identity GenBank id description trait 834 S.OOE-79 100 gi3927837 refNP 180456.1| SS mitochondrial import inner membrane translocase Subunit Tim17/Tim22fTim23 family protein Arabidopsis thaliana 83S 1.OOE-115 1OO gl 21554O15 refN13 565766.1| glycolipid SS transfer protein-related Arabidopsis thaliana 836 1.OOE-131 1OO iI26452816 refNP 193484.1| ubiquitin SS carboxyl-terminal hydrolase 837 1.OOE-102 99 gl 10177705 refNP 1994.36.1 inward SS LN rectifying potassium channel (KAT1) 838 1.OOE-144 1OO gl 766995O refNP 190844.1| integral SS PEG membrane Yip1 family protein Arabidopsis thaliana 839 1OO gl 101775.95 refNP 201509.11 protein SS LN kinase family protein Arabidopsis thaliana 840 4.OOE-27 1OO gl 38566508 refNP 200137. SS arabinogalactan protein, putative (AGP22 Arabidopsis thaliana 841 1OO gl 18391117 refNP 563862.1|expressed SS PEG protein Arabidopsis thaliana 842 92 gl SO251322 db|BAD28194.1 putative SS MFAP1 protein Oryza Saiiva (japonica cultivar group) 843 1.OOE-112 95 gl 32O1969 gb|AAC19375.1 submergence SS induced protein 2A Oryza Saiiva 844 1.OOE-74 88 gl 29371519 gb|AA072703.1 unknown SS Oryza Saiva (japonica cultivar-group) 845 1OO gl 284.16475 refNP 187911.1| SS PEG transporter-related Arabidopsis thaliana 846 1OO gl 21323473 refNP 599939.1| detergent SS LN sensitivity rescuerdtsR2 Corynebacterium glutamicum ATCC 13032 847 82 gl 50928.705 gb|AAQ14479.11 putative SS aminotransferase Oryza Saiiva 848 99 gl 3695,005 gb|AAC63962.1 pyruvate SS dehydrogenase kinase isoform 2; PDK2 Zea mays 849 1OO gl 10175838 refNP 244081.1 SS PEG hypothetical protein BH3215 Bacilius halodurans C-125 8SO 1OO gl 33589668 refNP 564285.1| SS PEG calmodulin-binding protein Arabidopsis thaliana

Trait Improvement Screens The present invention provides recombinant DNA that can improve the plant Survival rate under Such Sustained drought 0098. DS-Improvement of Drought Tolerance Identified condition. Exemplary recombinant DNA for conferring such by a Soil Drought Stress Tolerance Screen: drought tolerance are identified as such in Table 3. Such 0099 Drought or water deficit conditions impose mainly recombinant DNA may find particular use in generating osmotic stress on plants. Plants are particularly Vulnerable to transgenic plants that are tolerant to the drought condition drought during the flowering stage. The drought condition in imposed during flowering time and in other stages of the plant the screening process disclosed in Example 1B started from life cycle. As demonstrated from the model plant screen, in the flowering time and was Sustained to the end of harvesting. Some embodiments of transgenic plants with trait-improving US 2015/O 1841 89 A1 Jul. 2, 2015

recombinant DNA grown under Such Sustained drought con were set up to look for transgenic plants that display visual dition can also have increased total seed weight per plant in growth advantage at lower temperature. In cold germination addition to the increased Survival rate within a transgenic tolerance screen, the transgenic Arabidopsis plants were population, providing a higher yield potential as compared to exposed to a constant temperature of 8°C. from planting until control plants. day 28 post plating. The trait-improving recombinant DNA 0100 PEG-Improvement of Drought Tolerance Identified identified by such screen are particular useful for the produc by PEG Induced Osmotic Stress Tolerance Screen: tion of transgenic plant that can germinate more robustly in a 0101 Various drought levels can be artificially induced by cold temperature as compared to the wild type plants. In cold using various concentrations of polyethylene glycol (PEG) to shock tolerance screen, the transgenic plants were first grown produce different osmotic potentials (Pilon-Smits e.g., under the normal growth temperature of 22°C. until day 8 (1995) Plant Physiol. 107:125-130). Several physiological post plating, and Subsequently were placed under 8°C. until characteristics have been reported as being reliable indica day 28 post plating. As demonstrated from the model plant tions for selection of plants possessing drought tolerance. screen, embodiments of transgenic plants with trait-improv These characteristics include the rate of seed germination and ing recombinant DNA identified in a cold shock stress toler seedling growth. The traits can be assayed relatively easily by ance screen and/or a cold germination stress tolerance Screen measuring the growth rate of seedling in PEG solution. Thus, can Survive better cold conditions providing a higher yield a PEG-induced osmotic stress tolerance screen is a useful potential as compared to control plants. Surrogate for drought tolerance screen. As demonstrated from 0.108 Improvement of Tolerance to Multiple Stresses: the model plant screen, embodiments of transgenic plants 0.109 Different kinds of stresses often lead to identical or with trait-improving recombinant DNA identified in the similar reaction in the plants. Genes that are activated or PEG-induced osmotic stress tolerance screen can survive bet inactivated as a reaction to stress can either act directly in a ter drought conditions providing a higher yield potential as way the genetic product reduces a specific stress, or they can compared to control plants. act indirectly by activating other specific stress genes. By 0102) SS-Improvement of Drought Tolerance Identified manipulating the activity of Such regulatory genes, i.e., mul by High Salinity Stress Tolerance Screen: tiple stress tolerance genes, the plant can be enabled to react 0103) Three different factors are responsible for salt dam to different kinds of stresses. For examples, PEP SEQID NO: ages: (1) osmotic effects, (2) disturbances in the mineraliza 459 can be used to improve both salt stress tolerance and cold tion process, (3) toxic effects caused by the salt ions, e.g., stress tolerance in plants. Of particular interest, plants trans inactivation of enzymes. While the first factor of salt stress formed with PEP SEQID NO: 440 can resist heat stress, salt results in the wilting of the plants that is similar to drought stress and cold stress. In addition to these multiple stress effect, the ionic aspect of salt stress is clearly distinct from tolerance genes, the stress tolerance conferring genes pro drought. The present invention provides genes that help vided by the present invention may be used in combinations to plants to maintain biomass, root growth, and/or plant devel generate transgenic plants that can resist multiple stress con opment in high salinity conditions, which are identified as ditions. such in Table 3. Since osmotic effect is one of the major 0110. PP-Improvement of Early Plant Growth and Devel components of salt stress, which is common to the drought opment: stress, trait-improving recombinant DNA identified in a high 0111. It has been known in the art that to minimize the salinity stress tolerance screen can also provide transgenic impact of disease on crop profitability, it is important to start crops with improved drought tolerance. As demonstrated the season with healthy vigorous plants. This means avoiding from the model plant screen, embodiments of transgenic seed and seedling diseases, leading to increased nutrient plants with trait-improving recombinant DNA identified in a uptake and increased yield potential. Traditionally early high salinity stress tolerance screen can Survive better planting and applying fertilizer are the methods used for drought conditions and/or high Salinity conditions providing promoting early seedling vigor. In early development stage, a higher yield potential as compared to control plants. plant embryos establish only the basic root-shoot axis, a 0104 HS-Improvement of Drought Tolerance Identified cotyledon Storage organ(s), and stem cell populations, called by Heat Stress Tolerance Screen: the root and shoot apical meristems, that continuously gen 0105 Heat and drought stress often occur simultaneously, erate new organs throughout post-embryonic development. limiting plant growth. Heat stress can cause the reduction in "Early growth and development used herein encompasses photosynthesis rate, inhibition of leaf growth and osmotic the stages of seed imbibition through the early vegetative potential in plants. Thus, genes identified by the present phase. The present invention provides genes that are useful to invention as heat stress tolerance conferring genes may also produce transgenic plants that have advantages in one or more impart improved drought tolerance to plants. As demon processes including, but not limited to, germination, seedling strated from the model plant screen, embodiments of trans vigor, root growth and root morphology under non-stressed genic plants with trait-improving recombinant DNA identi conditions. The transgenic plants starting from a more robust fied in a heat stress tolerance screen can survive better heat seedling are less Susceptible to the fungal and bacterial patho stress conditions and/or drought conditions providing a gens that attach germinating seeds and seedling. Further higher yield potential as compared to control plants. more, seedlings with advantage in root growth are more resis 0106 CK and CS-Improvement of Tolerance to Cold tant to drought stress due to extensive and deeper root Stress: architecture. Therefore, it can be recognized by those skilled 0107 Low temperature may immediately result in in the art that genes conferring the growth advantage in early mechanical constraints, changes in activities of macromol stages to plants may also be used to generate transgenic plants ecules, and reduced osmotic potential. In the present inven that are more resistant to various stress conditions due to tion, two screening conditions, i.e., cold shock tolerance improved early plant development. The present invention screen (CK) and cold germination tolerance screen (CS), provides such exemplary recombinant DNA that confer both US 2015/O 1841 89 A1 Jul. 2, 2015 36 the stress tolerance and growth advantages to plants, identi 0116 LN-Improvement of Tolerance to Low Nitrogen fied as such in Table 3, e.g., PEP SEQID NO: 529 which can Availability Stress improve the plant early growth and development, and impart 0117 Nitrogen is a key factor in plant growth and crop yield. The metabolism, growth and development of plants are salt and cold tolerance to plants. As demonstrated from the profoundly affected by their nitrogen supply. Restricted nitro model plant screen, embodiments of transgenic plants with gen Supply alters shoot to root ratio, root development, activ trait-improving recombinant DNA identified in the early ity of enzymes of primary metabolism and the rate of Senes plant development screen can grow better under non-stress cence (death) of older leaves. All field crops have a conditions and/or stress conditions providing a higher yield fundamental dependence on inorganic nitrogenous fertilizer. potential as compared to control plants. Since fertilizer is rapidly depleted from most soil types, it must be Supplied to growing crops two or three times during 0112 SP-Improvement of Late Plant Growth and Devel the growing season. Enhanced nitrogen use efficiency by opment: plants should enable crops cultivated under low nitrogen 0113 “Late growth and development used herein encom availability stress condition resulted from low fertilizer input passes the stages of leaf development, flower production, and or poor soil quality. 0118 According to the present invention, transgenic seed maturity. In certain embodiments, transgenic plants pro plants generated using the recombinant nucleotides, which duced using genes that confer growth advantages to plants confer enhanced nitrogen use efficiency, identified as such in provided by the present invention, identified as such in Table Table 3, exhibit one or more desirable traits including, but not 3, exhibit at least one phenotypic characteristics including, limited to, increased seedling weight, greener leaves, but not limited to, increased rosette radius, increased rosette increased number of rosette leaves, increased or decreased dry weight, seed dry weight, silique dry weight, and silique root length. One skilled in the art may recognize that the length. On one hand, the rosette radius and rosette dry weight transgenic plants provided by the present invention with are used as the indexes of photosynthesis capacity, and enhanced nitrogen use efficiency may also have altered amino thereby plant source strength and yield potential of a plant. acid or protein compositions, increased yield and/or better seed quality. The transgenic plants of the present invention On the other hand, the seed dry weight, silique dry weight and may be productively cultivated under low nitrogen growth silique length are used as the indexes for plant sink strength, conditions, i.e., nitrogen-poor soils and low nitrogen fertilizer which are considered as the direct determinants of yield. As inputs, which would cause the growth of wild type plants to demonstrated from the model plant screen, embodiments of cease or to be so diminished as to make the wild type plants transgenic plants with trait-improving recombinant DNA practically useless. The transgenic plants also may be advan identified in the late development screen can grow better tageously used to achieve earlier maturing, faster growing, and/or have improved development during leaf development and/or higher yielding crops and/or produce more nutritious and seed maturation providing a higher yield potential as foods and animal feedstocks when cultivated using nitrogen compared to control plants. non-limiting growth conditions. 0119 Stacked Traits: 0114 LL-Improvement of Tolerance to Shade Stress Iden 0.120. The present invention also encompasses transgenic tified in a Low Light Screen: plants with stacked engineered traits, e.g., a crop having an 0115 The effects of light on plant development are espe improved phenotype resulting from expression of a trait cially prominent at the seedling stage. Under normal light improving recombinant DNA, in combination with herbicide conditions with unobstructed direct light, a plant seeding and/or pest resistance traits. For example, genes of the current develops according to a characteristic photomorphogenic invention can be stacked with other traits of agronomic inter pattern, in which plants have open and expanded cotyledons est. Such as a trait providing herbicide resistance, for example and short hypocotyls. Then the plant's energy is devoted to a RoundUp Ready(R) trait, or insect resistance. Such as using a cotyledon and leaf development while longitudinal extension gene from Bacillus thuringensis to provide resistance against growth is minimized. Under low light condition where light lepidopteran, coliopteran, homopteran, hemiopteran, and quality and intensity are reduced by shading, obstruction or other insects. Herbicides for which resistance is useful in a high population density, a seedling displays a shade-avoid plant include glyphosate herbicides, phosphinothricin herbi ance pattern, in which the seedling displays a reduced coty cides, oxynil herbicides, imidazolinone herbicides, dinitroa ledon expansion, and hypocotyls extension is greatly niline herbicides, pyridine herbicides, sulfonylurea herbi increased. As the result, a plant under low light condition cides, bialaphos herbicides, sulfonamide herbicides and increases significantly its stem length at the expanse of leaf, gluphosinate herbicides. To illustrate that the production of seed or fruit and storage organ development, thereby transgenic plants with herbicide resistance is a capability of adversely affecting of yield. The present invention provides those of ordinary skill in the art, reference is made to U.S. recombinant DNA that enable plants to have an attenuated patent application publications 2003/0106096A1 and 2002/ shade avoidance response so that the source of plant can be O112260A1 and U.S. Pat. Nos. 5,034,322; 5,776,760, 6,107, contributed to reproductive growth efficiently, resulting 549 and 6,376,754, all of which are incorporated herein by higher yield as compared to the wild type plants. As demon reference. To illustrate that the production of transgenic strated from the model plant screen, embodiments of trans plants with pest resistance is a capability of those of ordinary genic plants with trait-improving recombinant DNA identi skill in the art reference is made to U.S. Pat. Nos. 5,250,515 fied in a shade stress tolerance Screen can have attenuated and 5,880,275 which disclose plants expressing an endotoxin shade response under shade conditions providing a higher of Bacillus thuringiensis bacteria, to U.S. Pat. No. 6,506.599 yield potential as compared to control plants. The transgenic which discloses control of invertebrates which feed on trans plants generated by the present invention may be suitable for genic plants which express dsRNA for Suppressing a target a higher density planting, thereby resulting increased yield gene in the invertebrate, to U.S. Pat. No. 5,986,175 which per unit area. discloses the control of viral pests by transgenic plants which US 2015/O 1841 89 A1 Jul. 2, 2015 37 express viral replicase, and to U.S. Patent Application Publi silking, leaf extension rate, chlorophyll content, leaftempera cation 2003/015.0017 A1 which discloses control of pests by ture, stand, seedling vigor, internode length, plant height, leaf a transgenic plant which express a dsRNA targeted to Sup number, leaf area, tillering, brace roots, stay green, stalk pressing a gene in the pest, all of which are incorporated lodging, root lodging, plant health, barreness/prolificacy, herein by reference. green Snap, and pest resistance. In addition, trait characteris 0121. Once one recombinant DNA has been identified as tics of harvested grain may be confirmed, including number conferring an improved trait of interest in transgenic Arabi of kernels per row on the ear, number of rows of kernels on the dopsis plants, several methods are available for using the ear, kernelabortion, kernel weight, kernel size, kernel density sequence of that recombinant DNA and knowledge about the and physical grain quality. protein it encodes to identify homologs of that sequence from 0.125 To confirm hybrid yield in transgenic corn plants the same plant or different plant species or other organisms, expressing genes of the present invention, it may be desirable e.g., bacteria and yeast. Thus, in one aspect, the invention to test hybrids over multiple years at multiple locations in a provides methods for identifying a homologous gene with a geographical location where maize is conventionally grown, DNA sequence homologous to any of SEQID NO: 1 through e.g., in Iowa, Illinois or other locations in the midwestern SEQ ID NO: 425, or a homologous protein with an amino United States, under “normal field conditions as well as acid sequence homologous to any of SEQ ID NO: 426 under stress conditions, e.g., under drought or population through SEQ ID NO: 850. In another aspect, the present density stress. invention provides the protein sequences of identified 0.126 Transgenic plants can be used to provide plant parts homologs for a sequence listed as SEQID NO: 851 through according to the invention for regeneration or tissue culture of SEQID NO:33634. In yet another aspect, the present inven cells or tissues containing the constructs described herein. tion also includes linking or associating one or more desired Plant parts for these purposes can include leaves, stems, roots, traits, or gene function with a homolog sequence provided flowers, tissues, epicotyl, meristems, hypocotyls, cotyledons, herein. pollen, ovaries, cells and protoplasts, or any other portion of 0122) The trait-improving recombinant DNA and meth the plant which can be used to regenerate additional trans ods of using Such trait-improving recombinant DNA forgen genic plants, cells, protoplasts or tissue culture. Seeds of erating transgenic plants with improved traits provided by the transgenic plants are provided by this invention can be used to present invention are not limited to any particular plant spe propagate more plants containing the trait-improving recom cies. Indeed, the plants according to the present invention binant DNA constructs of this invention. These descendants may be of any plant species, i.e., may be monocotyledonous are intended to be included in the scope of this invention if or dicotyledonous. Preferably, they will be agricultural useful they contain a trait-improving recombinant DNA construct of plants, i.e., plants cultivated by man for purposes of food this invention, whether or not these plants are selfed or production or technical, particularly industrial applications. crossed with different varieties of plants. Of particular interest in the present invention are corn and I0127. The various aspects of the invention are illustrated soybean plants. The recombinant DNA constructs optimized by means of the following examples which are in no way for soybean transformation and recombinant DNA constructs intended to limit the full breath and scope of claims. optimized for corn transformation are provided by the present invention. Other plants of interest in the present invention for EXAMPLES production of transgenic plants having improved traits include, without limitation, cotton, canola, wheat, Sunflower, Example 1 Sorghum, alfalfa, barley, millet, rice, tobacco, fruit and Veg etable crops, and turfgrass. Identification of Recombinant DNA that Confers 0123. In certain embodiments, the present invention con Improved Trait(s) to Plants templates to use an orthologous gene in generating the trans genic plants with similarly improved traits as the transgenic A. Expression Constructs for Arabidopsis Plant Arabidopsis counterpart. Improved physiological properties Transformation in transgenic plants of the present invention may be con I0128. Each gene of interest was amplified from a genomic firmed in responses to stress conditions, for example in assays or cDNA library using primers specific to sequences using imposed stress conditions to detect improved responses upstream and downstream of the coding region. Transforma to drought stress, nitrogen deficiency, cold growing condi tion vectors were prepared to constitutively transcribe DNA tions, or alternatively, under naturally present stress condi in either sense orientation (for enhanced protein expression) tions, for example under field conditions. Biomass measures or anti-sense orientation (for endogenous gene Suppression) may be made on greenhouse or field grown plants and may under the control of an enhanced Cauliflower Mosaic Virus include Such measurements as plant height, stem diameter, 35S promoter (U.S. Pat. No. 5,359,142) directly or indirectly root and shoot dry weights, and, for corn plants, ear length (Moore, e.g., PNAS95:376-381, 1998; Guyer, e.g., Genetics and diameter. 149: 633-639, 1998: International patent application NO. 0124 Trait data on morphological changes may be col PCT/EP98/07577). The transformation vectors also contain a lected by visual observation during the process of plant regen bar gene as a selectable marker for resistance to glufosinate eration as well as in regenerated plants transferred to soil. herbicide. The transformation of Arabidopsis plants was car Such trait data includes characteristics such as normal plants, ried out using the vacuum infiltration method known in the art bushy plants, taller plants, thicker Stalks, narrow leaves, (Bethtold, e.g., Methods Mol. Biol. 82:259-66, 1998). Seeds striped leaves, knotted phenotype, chlorosis, albino, antho harvested from the plants, named as T1 seeds, were Subse cyanin production, or altered tassels, ears or roots. Other quently grown in a glufosinate-containing selective medium enhanced traits may be identified by measurements taken to select for plants which were actually transformed and under field conditions, such as days to pollen shed, days to which produced T2 transgenic seed. US 2015/O 1841 89 A1 Jul. 2, 2015 38

B. Soil Drought Tolerance Screen ized for the transgenic plants and their controls and analyzed as a quantitative response according to example 1 M. 0129. This example describes a soil drought tolerance I0133. Two approaches were used for statistical analysis on screen to identify Arabidopsis plants transformed with the wilting response. First, the risk score was analyzed for recombinant DNA that wilt less rapidly and/or produce wilting phenotype and treated as a qualitative response higher seed yield when grown in Soil under drought condi according to the example 1L. Alternatively, the Survival tions analysis was carried out in which the proportions of wilted 0130 T2 seeds were sown inflats filled with Metro/Mix(R) and non-wilted transgenic and control plants were compared 200 (The Scotts(R) Company, USA). Humidity domes were over each of the six days under scoring and an overall log rank added to each flat and flats were assigned locations and placed test was performed to compare the two Survival curves using in climate-controlled growth chambers. Plants were grown S-PLUS statistical software (S-PLUS 6, Guide to statistics, under a temperature regime of 22°C. at day and 20° C. at Insightful, Seattle, Wash., USA). Table 4 provides a list of night, with a photoperiod of 16 hours and average light inten recombinant DNA constructs that improve drought tolerance sity of 170 umol/m/s. After the first true leaves appeared, in transgenic plants. TABLE 4

Time to PEP Drought Seed wilting SEQ SCO yield Risk ID Construct Nomination Delta P- Delta P- SCO P NO ID ID Orientation mean value le:8 value le:8 value

6O1 12116 CGPG1112 ANTI- O. 119 O496 O.736 O.O14 -O.O26 OOO SENSE 602 13053 CGPG1284 ANTI- O.122 O.S32 0.785 O.O28 -0.122 OOO SENSE 603 14733 CGPG1640 SENSE O469 O.O16 -O.225 O.363 O.263 OOO 604 16132 CGPG2136 SENSE O. 149 O.S29 O.472 O.046 f f 60S 18276 CGPG3542 SENSE O.32O O.O88 O.433 O.264 O.362 OOO 606 70851 CGPG1691 SENSE O.267 O.06S O.427 O.O17 f f 607 70941 CGPG4O67 SENSE O.254 O.OS6 O.S33 O.OO6 f f 450 71814 CGPG4434 SENSE O.241 O.186 O.719 O.O2O O.348 OOO 609 72326 CGPG27 SENSE O.300 O.149 O.674 O.036 O.152 OOO 608 72134 CGPG5335 SENSE O.239 O.169 O. 613 0.077 O.O10 OOO 783 72824 CGPG4998 SENSE O.254 O.18O O.608 O.046 f f 610 72978 CGPG3441 SENSE O.447 O.O94 -O.973 O.O71 f f 611 73619 CGPG4375 SENSE O.990 O.O22 -0.204 O.283 O.900 OOO 612 73737 CGPG5176 SENSE O.162 O.293 1.126 O.OOO O.333 OOO 460 73025 CGPG5665 SENSE O.06S O.731 O.489 O.09S f f 613 74196 CGPG 6637 SENSE O.172 O.230 1.533 O.OO1 f f 614 74S46 CGPG661 SENSE -0.052 O.727 0.777 O.O3O -0.226 OOO 615 74558 CGPG869 SENSE O.344 O.O72 -0.138 O.117 O.O33 0.955 616 74.643 CGPG-6159 SENSE O.369 O.042 -1.232 O.12S 0.556 OOO 617 75234 CGPG5931 SENSE 0.157 O.O38 -0.095 O.479 O.O26 O.881 618 75278 CGPG6282 SENSE O. 110 O.200 1.027 O.OO3 0.057 O.789 587 75289 CGPG6295 SENSE O. 627 O.OO)4 -0.074 O.267 O.196 O.883 619 75585 CGPG7671 SENSE O.474 O.OO8 -0.452 O.259 f f 62O 76063 CGPG1574 SENSE O. 105 O.637 O.98S O.062 O.O23 1.OOO 593 76318 CGPG8909 SENSE O.S.09 O.OO9 1.093 O.OOS O.471 O.952 599 76891 CGPG9034 SENSE O.186 O.115 O.796 O.OO3 O.128 1.OOO 621 774O1 CGPG9294 SENSE -O.O72 O643 O.SS4 O.061 -O.O90 1.OOO humidity domes were removed. The plants were sprayed with If p <0.05 and delta or risk score mean >0, the transgenic glufosinate herbicide and put back in the growth chamber for plants showed statistically significant trait improvement as 3 additional days. Flats were watered for 1 hour the week compared to the reference (p value, of the delta of a quanti following the herbicide treatment. Watering was continued tative response or of the risk score of a qualitative response, is every seven days until the flower bud primordia became the probability that the observed difference between the trans apparent, at which time plants were watered for the last time. genic plants and the reference occur by chance) If p <0.2 and 0131 To identify drought tolerant plants, plants were delta or risk score mean >0, the transgenic plants showed a evaluated for wilting response and seed yield. Beginning ten trend of trait improvement as compared to the reference. days after the last watering, plants were examined daily until 4 plants/line had wilted. In the next six days, plants were C. Heat Stress Tolerance Screen monitored for wilting response. Five drought scores were 0.134 Under high temperatures, Arabidopsis seedlings assigned according to the visual inspection of the phenotypes: become chlorotic and root growth is inhibited. This example 1 for healthy, 2 for dark green, 3 for wilting, 4 severe wilting, sets forth the heat stress tolerance screen to identify Arabi and 5 for dead. A score of 3 or higher was considered as dopsis plants transformed with the gene of interest that are wilted. more resistant to heat stress based on primarily their seedling 0.132. At the end of this assay, seed yield measured as seed weight and root growth under high temperature. T2 seeds weight per plant under the drought condition was character were plated on /2xMS salts, 17% phytagel, with 10 g/ml US 2015/O 1841 89 A1 Jul. 2, 2015 39

BASTA (7 per plate with 2 control seeds; 9 seeds total per gene of interest that are tolerant to high levels of salt based on plate). Plates were placed at 4°C. for 3 days to stratify seeds. their rate of development, root growth and chlorophyll accu Plates were then incubated at room temperature for 3 hours mulation under high salt conditions. and then held vertically for 11 additional days attemperature of 34° C. at day and 20° C. at night. Photoperiod was 16 h. 0.137 T2 seeds were plated on glufosinate selection plates Average light intensity was ~140 umol/m/s. After 14 days of containing 90 mM NaCl and grown understandard light and growth, plants were scored for glufosinate resistance, root temperature conditions. All seedlings used in the experiment length, final growth stage, visual color, and seedling fresh were grown at a temperature of 22°C. at day and 20° C. at weight. A photograph of the whole plate was taken on day 14. night, a 16-hour photoperiod, an average light intensity of 0135 The seedling weight and root length were analyzed approximately 120 umol/m. On day 11, plants were mea as quantitative responses according to example 1M. The final sured for primary root length. After 3 more days of growth grow stage at day 14 was scored as success if 50% of the (day 14), plants were scored for transgenic status, primary plants had reached 3 rosette leaves and size of leaves are root length, growth stage, visual color, and the seedlings were greater than 1 mm (Boyes, e.g., (2001) The Plant Cell 13, 1499-1510). The growth stage data was analyzed as a quali pooled for fresh weight measurement. A photograph of the tative response according to example 1 L. Table 5 provides a whole plate was also taken on day 14. list of recombinant DNA constructs that improve heat toler 0.138. The seedling weight and root length were analyzed ance in transgenic plants. as quantitative responses according to example 1M. The final TABLE 5

Growth stage Seedling Root length at day 14 weight at day PEP at day 14 Risk 14

Seq Construct Nomination Delta P- SCOe P- Delta P id ID ID Orientation (8. value (8. value (8. value

772 10923 CGPG414 ANTI-SENSE O.443 O.O32 f f f f 525 15419 CGPG1788 ANTI-SENSE O.385 0.040 -O.O70 f O416 0.076 527 17903 CGPG1908 SENSE O.125 0.091 f f f f 623 1833O CGPG3336 SENSE O.274 0.174 1.014 O.310 1.279 0.012 624 18409 CGPG3613 SENSE O.181 O.O98 f f f f 736 1841 O CGPG3614 SENSE O.398 0.094 f f f f 432 17410 CGPG2446 SENSE O.199 OO67 O.241 O.304 O.828 0.271 622 17808 CGPG2435 SENSE O.266 O.O40 f f f f 438 19760 CGPG4O6S SENSE O.O15 0.771 -0.111 f 1611 O.OOS 625 19813 CGPG4168 SENSE O.183 0.088 O.643 O.344 O.807 (0.038 440 70510 CGPG2488 SENSE O.607 O.OS6 O.171 O.184 O.863 O.O21 443 70812 CGPG6O7 SENSE O.319 O.O8O O.882 0.345 0.934 O.OSS 447 71.446 CGPG18S SENSE O.427 O.110 O.184 O.16O O.470 OO68 452 72005 COGPG5253 SENSE O.209 0.098 O.331 O.S86 1.178 O.122 545 726O3 CGPG646 SENSE O.476 O.O29 -0.17O 0.149 1.272 0.185 611 73619 CGPG4375 SENSE O.368 O.OS8 O.853 0.241 O.746 (0.102 467 73662 CGPG4927 SENSE O.205 O.OS3 f f f f 453 72026 CGPGS231 SENSE O.S89 O.O12 f f f f 626 73580 COGPG6517 SENSE -0.077 O.602 -0.195 f 1.106 0.052 628 74124 CGPG6631 SENSE O.2S6 O.OSS O46O O.O13 O-512 0.110 627 73935 COGPG993 SENSE O.284 O.O89 1102 O.262 1.098 0.115 482 74707 CGPG4914 SENSE O.077 O.68O O.O99 0.745 0.773 O.OS8 629 74725 CGPG5393 SENSE O.351 O.O38 f f f f 591 76220 CGPG6189 SENSE O.381 O.O39 f f f f 796 75910 CGPG1256 SENSE O.514 O.O81 f f f f 630 76403 CGPG1313 SENSE O.158 0.075 -0.033 0.864 O2S2 O.S81 595 76513 COGPG5892 SENSE O.281 O.OS1 f f f f 631 77366 CGPG815O SENSE O.259 O.O44 -0.116 0.058 O.354 (0.369 767 77115 COGPG9135 SENSE O-269 O.O94 f f f f 770 77159 CGPG9202 SENSE O.17O OSO2 -0.014 O.887 O.389 0.048

If p <0.05 and delta or risk score mean >0, the transgenic growth stage at day 14 was scored as Success if 50% of the plants showed statistically significant trait improvement as plants reached 3 rosette leaves and size of leaves are greater compared to the reference. If p <0.2 and delta or risk score mean >0, the transgenic plants showed a trend of trait than 1 mm (Boyes, D.C., et al., (2001), The Plant Cell 13, improvement as compared to the reference. 1499/1510). The growth stage data was analyzed as a quali D. Salt Stress Tolerance Screen tative response according to example 1L. Table 6 provides a 0136. This example sets forth the high salinity stress list of recombinant DNA constructs that improve high salinity screen to identify Arabidopsis plants transformed with the tolerance in transgenic plants

US 2015/O 1841 89 A1 Jul. 2, 2015 41

TABLE 6-continued Seedling PEP Root length at Root length at Growth stage weight at day SEQ day 11 day 14 at day 14

ID Construct Delta P- Delta P- RS P- Delta P NO ID Orientation mean value (8. value (8. value (8. value

755 74754 SENSE O.230 O.O88 O.228 O.O77 O966 O.353 O.407 O.118 484 74755 SENSE O.07O O.S74 -0.02O O.470 O.26S O.208 O4S5 O.O73 493 75355 SENSE O.290 O.OS7 O.305 O.OOO O440 O.425 O.360 O.301 828 75432 SENSE O.249 O.O2O O.178 O.100 O.475 O.196 O.216 O.454 829 75468 SENSE O.146 0.344 O.185 O.OS4 1.373 O.268 O311 O.141 794 75752 SENSE O.402 0.058 O.394 O.049 O.S21 O.335 O.672 O.O27 760 75931 SENSE O. 104 O239 O.O74 0.433 1.121 O.209 O.321 O.069 832 76.064 SENSE O.349 OO11 O.27O O.O46 O.152 O.360 O.2SO O.295 797 76101 SENSE O.S27 O.064 O.S42 0.107 O.724 O.367 1.480 O.O24 833 76121 SENSE -0.026 O.849 O.O19 O.829 2.841 O.059 O.069 O.822 834 76.193 SENSE O.262 O.O2O O.254 O.O21 O.834 O.097 0.441 O.OS4 835 76237 SENSE O.334 0.098 O.365 0.047 0.727 O.042 O.786 0.137 836 76281 SENSE O.167 0.154 O.181 0184 0.572 O.096 O.S98 O.O92 762 76293 SENSE O.O42 (0.763 -0.044 O.776 O.769 O.270 O610 O.O17 831 75911 SENSE O.179 0.041 O.143 O.O83 1.508 O.266 O464 O.OO1 830 75686 SENSE O.378 O.O36 O.358 0.039 1.039 0.057 O666 O.O14 837 76388 SENSE O.209 OOS4 O.163 O.136 O.853 O.367 O485 O.O13 838 76557 SENSE O.165 0.042 O.104 O.095 1943 O.O27 O.395 O.O31 839 76567 SENSE -0.046 (O.SS1 O.O67. O.196 1685 O.108 0.273 O.OS3 507 76575 SENSE O. 101 O3O2 O.183 OO67 O.121 O496 O.298 O.306 840 76624 SENSE O.399 O.O47 O.367 0.041 O.652 O.122 O622 O.069 597 76719 SENSE O.229 O.OS1 O.14O O.317 O.726 O.330 O487 O.083 841 76764 SENSE O.482 O.O36 O.376 O.O36 2.259 O.163 O.670 O.127 825 75076 SENSE O.186 O139 O.233 O.O16 2.197 O.062 O.S38 O.O31 843 76976 SENSE O.4SS O.O13 O.375 0.044 3.499 O.O2O 0.737 O.007 844 76985 SENSE O.313 O.OSO O.299 O.O37 3.310 O.O41 0.587 O.048 842 76855 SENSE O463 O.O26 O.636 OO1O O.188 O.O2O 1334 O.OO1 599 76891 SENSE O.325 O.OOO O.368 O.OO3 O.445 O.087 0.795 O.OO3 845 77014 SENSE O.OOS 0.889 -0.04O O.S97 O.O24 O.932 O.26S O.098 522 77351 SENSE O392 O.048 O.454 OO14 2.912 O.OS2 1340 O.OO6 631 77366 SENSE O.214 O.OO7 O.159 0.017 O.2OS O.O76 O.382 O.O29 846 77112 SENSE -0.001 O.986 O.O2O O.801 1.640 O.O2O O.S83 O.O2S 847 77117 SENSE O.219 O.OS8 O.234 0.116 1406 O.172 O.S16 O.O72 768 77128 SENSE O.239 O.O20 O.312 O.O19 O.324 O.S23 O.470 O.O28 52O 77135 SENSE O.153 O.O70 O.133 0.059 O.386 O.SO3 O.344 O.064 848 77138 SENSE O.171 O.O3O O.271 O.O12 1.211 O.256 O482 O.O3S 770 771.59 SENSE O.188 O. 115 O.22O O. 110 1.176 O.381 O.S89 O.O39 600 772.08 SENSE O.321 O.O75 O.254 0.154 O.183 0.578 O.S4O O.059 521 772.19 SENSE O.297 0.090 O413 OO18 2.299 O.O24 1.010 O.O14 849 77226 SENSE O.164 O.193 O.098 0.413 1.279 O. 142 O.264 O.043 621 774O1 SENSE O.317 O.O68 O.274 0.018 1134 O.258 OSO2 O.213 850 77418 SENSE O.204 0.096 O.201 O. 110 O.344 O.184 O626 O.OOS

If p <0.05 and delta or risk score mean >0, the transgenic 0140 T2 seeds were plated on BASTA selection plates plants showed statistically significant trait improvement as containing 3% PEG and grown understandard light and tem compared to the reference. If p <0.2 and delta or risk score perature conditions. Seeds were plated on each plate contain mean >0, the transgenic plants showed a trend of trait ing 3% PEG, /2xMS salts, 1% phytagel, and 10 ug/ml glufo improvement as compared to the reference. sinate. Plates were placed at 4°C. for 3 days to stratify seeds. On day 11, plants were measured for primary root length. After 3 more days of growth, i.e., at day 14, plants were E. Polyethylene Glycol (PEG) Induced Osmotic Stress scored for transgenic status, primary root length, growth Tolerance Screen stage, visual color, and the seedlings were pooled for fresh weight measurement. A photograph of the whole plate was 0139. There are numerous factors, which can influence taken on day 14. seed germination and Subsequent seedling growth, one being 0.141. Seedling weight and root length were analyzed as the availability of water. Genes, which can directly affect the quantitative responses according to example 1M. The final Success rate of germination and early seedling growth, are growth stage at day 14 was scored as success or failure based potentially useful agronomic traits for improving the germi on whether the plants reached 3 rosette leaves and size of nation and growth of crop plants under drought stress. In this leaves are greater than 1 mm. The growth stage data was assay, PEG was used to induce osmotic stress on germinating analyzed as a qualitative response according to example 1L. transgenic lines of Arabidopsis thaliana seeds in order to Table 7 provides a list of recombinant DNA constructs that screen for osmotically resistant seed lines. improve osmotic stress tolerance in transgenic plants.

US 2015/O 1841 89 A1 Jul. 2, 2015 45

TABLE 8-continued

Rosette area at PEP Rosette area day 28 Rosette area SEQ at day 8 Risk difference

ID Construct Nomination Delta P- SCOe P- Delta NO ID ID Orientation mean value (8. value mean P-value

489 75239 CGPG5949 SENSE 1.048 O.O11 O.S70 O.242 O.S47 0.290 490 752S8 CGPG6096 SENSE O.808 O.047 O.435 O.13S O.S85 O.O2S 491 7527O CGPG6218 SENSE O.S26 O.O11 O.746 O.141 O.866 O.193 492 75271 CCGPG6226 SENSE O.092 O.698 0.963 O.083 O.982 0.128 470 73846 CGPG1924 SENSE O.807 O.O32 0.175 O.623 O.O14 O.973 471 73863 CGPGS2O1 SENSE O.285 O404 O.740 O.O21 O.797 O.O16 472 74059 CGPG1884 SENSE O.364 O.O14 O.162 O.137 -0.131 O.424 477 74412 CGPG6743 SENSE O.163 O.S22 O.670 O.O26 O.626 O.131 478 74445 CGPG6722 SENSE O.805 O.065 O.361 O. 164 O.S84 O.145 482 74707 CGPG4914 SENSE O.S84 O.OOS 0453 O.248 O.239 O.415 483 74743 CGPGS84O SENSE O.187 O.245 0.843 O.O40 O.976 O.O45 484 74755 CGPG598O SENSE O.959 O.083 0.68O O.205 O.768 0.2O6 493 75355 CGPG7518 SENSE O.268 O.OO6 0.715 O.OS3 O.799 O.O38 494 7546O CGPG7654 SENSE O.214 O.12O O.649 O.OS3 O.713 O.O94 795 75867 CGPG6965 SENSE O.817 O.O4O O.707 O.OS8 O.S88 O.128 497 75927 COGPG8229 SENSE O.360 O.163 O.808 O.O17 0.955 0.007 498 75962 CGPG8224 SENSE O.337 -0.167 0.398 O.O61 O.7O6 O.O3O 499 75984 CGPG1927 SENSE 1.141 O.OSO 0.220 O.O73 O.O76 O.2O2 500 76119 CGPGS962 SENSE O.2O6 O.445 1.056 O.O11 1.364 OO15 5O1 76209 CGPG5375 SENSE O.S98 0.078 O.737 O. 112 O.834 0.075 496 75907 COGPG8259 SENSE O.730 O.OO2 0.376 O.O23 O494 OO65 495 75847 CGPG6875 SENSE O.S68 O.129 O.927 O.O2S 1.062 O.O33 502 76323 CGPG8949 SENSE O.S92 O.042 0.736 O.049 O844 0.048 503 76346 CGPG8943 SENSE O.1SO O.647 O.S88 O.O61 O.656 0.044 SO4 76352 CGPG8896 SENSE O464 O.S.S1 1.124 O. 109 1.375 0.049 505 76360 CGPG8960 SENSE O442 O.274 O.646 O.O72 O.83O O.O38 507 76575 CGPG726O SENSE 0.444 O.O3O 0.813 O.O23 1.123 O.OSO SO6 76512 CGPG5891 SENSE -O.O60 O.669 O.486 O.OOS O.799 O.O14 SO8 76733 CGPG2647 SENSE O3O4 O.312 1028 O.O19 O.976 O.O34 509 76741 CGPG6995 SENSE 1.007 O.040 O.344 O.OO8 O3O2 O.O33 512 76902 CGPG9083 SENSE O.343 O.O97 OSS1 O.O34 O496 O.O82 513 76913 CGPG9076 SENSE O.S24 O.O21 O.257 O.S30 O.149 0.719 S14 76917 CGPG9108 SENSE O.728 O.O63 O.804 O.O15 O.779 0.036 515 76929 CGPG9109 SENSE O.335 O.22O O.628 O.O31 O844 O.O27 510 76845 CGPG9046 SENSE O.123 0.751 0.877 O.O81 1.232 O.O27 511 76857 CGPG9047 SENSE 1.055 O.OS3 1.170 O.O23 1.286 O.O2O S16 77009 CGPG5933 SENSE O604 O.OS1 O.853 O. 104 O.864 O.12O 517 77022 CGPG6335 SENSE O.301 O.356 O.444 O.O24 O.SS4 O.OO1 522 77351 CGPG8O87 SENSE O.749 O.O23 1.023 O.O12 1.031 O.O32 S18 77108 CGPG9174 SENSE O.651 O.O2S O.788 O.O2O O.723 O.133 519 77125 CGPG912O SENSE O.945 O.049 1641 O.O13 1794 O.O11 52O 77135 COGPG9200 SENSE O648 O.253 1.299 O.O19 1486 O.O34 521 77219 CGPG9263 SENSE O636 O.OOS O.300 0.355 O.763 O.OO3

If p <0.05 and delta or risk score mean >0, the transgenic disinfested using chlorine gas and then seeded on assay plates plants showed statistically significant trait improvement as containing an aqueous solution of /2x Gamborg's B/5 Basal compared to the reference (p value, of the delta of a quanti Salt Mixture (Sigma/Aldrich Corp., St. Louis, Mo., USA tative response or of the risk score of a qualitative response, is G/5788), 1% PhytagelTM (Sigma-Aldrich, P-8169), and 10 the probability that the observed difference between the trans ug/ml glufosinate with the final pH adjusted to 5.8 using genic plants and the reference occur by chance) If p <0.2 and KOH. Test plates were held vertically for 28 days at a constant delta or risk score mean >0, the transgenic plants showed a temperature of 8°C., a photoperiod of 16 hr, and average light trend of trait improvement as compared to the reference. intensity of approximately 100 umol/m/s. At 28 days post plating, root length was measured, growth stage was G. Cold Germination Tolerance Screen observed, the visual color was assessed, and a whole plate 0144. This example sets forth a screen to identify Arabi photograph was taken. dopsis plants transformed with the genes of interests are 0146 The root length at day 28 was analyzed as a quanti resistant to cold stress based on their rate of development, root tative response according to example 1M. The growth stage at growth and chlorophyll accumulation under low temperature day 7 was analyzed as a qualitative response according to conditions. example 1L. Table 9 provides a list of recombinant DNA 0145 T2 seeds were plated and all seedlings used in the constructs that improve cold stress tolerance in transgenic experiment were grown at 8° C. Seeds were first surface plants.

US 2015/O 1841 89 A1 Jul. 2, 2015 47

TABLE 9-continued Root length at day Growth stage at day PEP 28 28 SEQ Construct Delta Delta ID ID Orientation le:8 P-value (3. P-value

S8O 74733 SENSE O.487 O.O2O 2.504 O.088 581 74749 SENSE O.SO1 O.O26 3.4O6 O.O29 582 74752 SENSE O.276 O.097 3.389 O.O31 484 74755 SENSE O.136 O.392 2.8.21 O.045 583 74773 SENSE O.376 O.OO)4 4.OOO O.OOO 795 75867 SENSE O401 O.061 4.OOO O.OOO 793 75530 SENSE -O.O32 O.760 2.385 O.098 S88 76003 SENSE O.239 O.O21 1611 O.315 589 76O74 SENSE O-269 O.O33 2.696 O.174 590 76137 SENSE O.316 0.075 2624 O.O81 591 7622O SENSE O.074 O.S16 2.585 O.O89 796 75910 SENSE O.225 O.O70 4.OOO O.OOO 592 763O1 SENSE O. 110 O.308 2.683 0.055 593 76318 SENSE O.OS2 O600 2.692 O.078 594 76347 SENSE O.088 O.362 3.2S4 O.049 596 76566 SENSE 0.144 O.OS2 2.408 O. 117 595 76513 SENSE O.316 O.019 3.347 O.036 597 76719 SENSE O.O99 O.O63 4.OOO 598 76757 SENSE O.251 O.O37 3.042 O.O86 S14 76917 SENSE O.16S 0.055 1834 O.254 599 76891 SENSE O.382 O.O70 O.OOO 1.OOO 600 772.08 SENSE O.408 O.O34 2.835 O.OS1

If p <0.05 and delta or risk score mean >0, the transgenic particular interest, we were looking for plants that didn't plants showed statistically significant trait improvement as extend their petiole length, had an increase in seedling weight compared to the reference. If p <0.2 and delta or risk score relative to the reference and had leaves that were more close mean >0, the transgenic plants showed a trend of trait to parallel with the plate surface. improvement as compared to the reference. 0148 T2 seeds were plated on glufosinate selection plates with /2 MS medium. Seeds were sown on /2xMS salts, 1% H. Shade Tolerance Screen Phytagel, 10 ug/ml BASTA. Plants were grown on vertical plates at a temperature of 22°C. at day, 20° C. at night and 0147 Plants undergo a characteristic morphological underlow light (approximately 30 uE/m/s, far/redratio (655/ response in shade that includes the elongation of the petiole, 665/725/735) -0.35 using PLAQ lights with GAM color filter a change in the leaf angle, and a reduction in chlorophyll #680). Twenty-three days after seedlings were sown, mea content. While these changes may confer a competitive Surements were recorded including seedling status, number advantage to individuals, in a monoculture the shade avoid of rosette leaves, status of flower bud, petiole leaf angle, ance response is thought to reduce the overall biomass of the petiole length, and pooled fresh weights. A digital image of population. Thus, genetic alterations that prevent the shade the whole plate was taken on the measurement day. Seedling avoidance response may be associated with higher yields. weight and petiole length were analyzed as quantitative Genes that favor growth under low light conditions may also responses according to example 1M. The number of rosette promote yield, as inadequate light levels frequently limit leaves, flowering bud formation and leafangel were analyzed yield. This protocol describes a screen to look for Arabidopsis as qualitative responses according to example 1L. plants that show an attenuated shade avoidance response and/ 0149 Table 10 provides a list of recombinant DNA con or grow better than control plants under low light intensity. Of structs that improve shade tolerance in plants TABLE 10 Leaf Seedling Petiole angle weight length PEP at day at day at day SEQ 23 23 23 ID Construct RS Delta Delta P NO ID Orientation mean P-value le:8 P-value (8. value

441 7O605 SENSE f f O424 O.061 O.352 O.O38 632 72414 SENSE O.OOO 1.OOO O.396 OO67 O.222 O.O47 464 73344 SENSE 2.667 0.057 O.179 0.444 O.289 O.OS8 744 73666 SENSE f f O.838 O.OO8 O.142 O.445 453 72026 SENSE O.OOO 1.OOO O469 O.042 O.313 O.O73 634 73723 SENSE 1.333 O423 -0.103 0.677 -0.406 O.O89 635 73747 SENSE 2.OOO O.225 -0.605 O.OO2 -O.S6S O.OSO 633 734.09 SENSE 2.667 0.057 O.365 O.OS2 O.283 O.O1O 636 74634 SENSE 0.667 O423 -0.636 O.109 -O.869 O.O17 615 74,558 SENSE 1.333 O.184 O.486 O.018 O.231 O.108 US 2015/O 1841 89 A1 Jul. 2, 2015 48

TABLE 10-continued Leaf Seedling Petiole angle weight length PEP at day at day at day SEQ 23 23 23 ID Construct RS Delta Delta P NO ID Orientation mean P-value (8. P-value le:8 value

824 74956 SENSE 1.333 0.423 O.154 O.O76 -0.052 O.289 486 75220 SENSE O.OOO 1.OOO -0.644 O.230 -O.716 O.099 487 75226 SENSE O.OOO 1.OOO -0.601 O.O67 -1.142 O.O86 488 75231 SENSE O.667 O.423 O.45S O.O39 O.258 O.O72 638 75232 SENSE 1.333 0.423 -1931 0.077 -1992 O.O38 489 75239 SENSE O.667 O.423 O.149 0.075 O.O60 0.444 S60 73855 SENSE 3.333 O.O38 -0.261 O.121 -0.361 O.215 472 74059 SENSE O.OOO 1.OOO O.409 O.O66 O.331 O.062 482 74707 SENSE 3.333 O.O38 O-269 O.O71 O.136 O.O16 754 74753 SENSE O.OOO 1.OOO O.365 O.O79 O.267 0.057 637 74769 SENSE 1.333 0.184 -0.141 O.346 -0.221 O.O29 493 75355 SENSE 1.333 0.423 -O.735 O.O69 -O-581 O.OS8 639 75429 SENSE 2.667 O.057 -0.453 O.151 -1319 O.O32 640 75965 SENSE O.667 O.423 O.O92 O.O86 O.109 O.122 642 76061 SENSE f f 0.373 0.075 O-110 O.330 643 76155 SENSE O.667 O.423 -O.473 O.O21 -0.608 O.OO)4 761 762O7 SENSE 2.667 O.057 -0.374 O.O60 -0.369 O.O88 641 75975 SENSE 2.OOO O.225 O.405 O.OS6 O.223 O.044 762 76293 SENSE 2.667 O.184 -0.412 O.OS2 -0.717 O.018 644 76322 SENSE O.667 O.423 -O.S33 O.O44 -0.559 O.O45 645 76440 SENSE f f -0.405 O.141 -O.S25 O.O45 509 76741 SENSE O.667 O.423 -O.793 O.O27 -O.803 O.018 513 76913 SENSE f f O.313 O.O73 O.336 O.129 646 76828 SENSE O.667 O.423 -0185 O.141 -1.1.89 O.OO1 517 77022 SENSE O.OOO 1.OOO -0.495 O.282 -O.475 O.O79 648 77303 SENSE 2.OOO O.225 -O.296 O.117 -0.176 O.O38 649 77352 SENSE 1.333 0.423 -O.298 O.127 -0.377 O.036 631 77366 SENSE O.667 O.423 O.611 O.O1S 0.327 O.044 647 77136 SENSE 1.333 0.423 -0.824 O.O76 -0.566 O.O89

For “seeding weight' and “leaf angle', if p <0.05 and delta or screen, we were looking for genes that confer advantages in risk score mean >0, the transgenic plants showed statistically the processes of germination, seedling vigor, root growth and significant trait improvement as compared to the reference. If root morphology under non-stressed growth conditions to p<0.2 and delta or risk score mean >0, the transgenic plants plants. The transgenic plants with advantages in seedling showed a trend of trait improvement as compared to the growth and development were determined by the seedling reference with p-0.2. weight and root length at day 14 after seed planting. For “petiole length', if p <0.05 and delta <0, the transgenic 0151 T2 seeds were plated on glufosinate selection plates plants showed statistically significant trait improvement as and grown under standard conditions (~100 uE/m/s, 16 h compared to the reference. If p <0.2 and delta <0, the trans photoperiod, 22° C. at day, 20° C. at night). Seeds were genic plants showed a trend of trait improvement as compared stratified for 3 days at 4°C. Seedlings were grown vertically to the reference. (at a temperature of 22°C. at day 20° C. at night). Observa tions were taken on day 10 and day 14. Both seedling weight I. Early Plant Growth and Development Screen and root length at day 14 were analyzed as quantitative 0150. This example sets forth a plate based phenotypic responses according to example 1 M. analysis platform for the rapid detection of phenotypes that 0152 Table 11 provides a list recombinant DNA con are evident during the first two weeks of growth. In this structs that improve early plant growth and development. TABLE 11 PEP Root length at day Root length at day Seedling weight at SEQ 10 14 day 14

ID Construct Delta Delta Delta NO ID Orientation le:8 P-value le:8 P-value (8. P-value

523 14810 SENSE 0.175 O.O27 O.049 O.231 -O.O89 0.337 773 14836 SENSE O. 105 O.OO7 O.O89 O.O29 O.162 O.217 735 14944 SENSE O.2OS 0.2O7 0.175 O.046 O.259 O.221 525 15419 ANTI-SENSE O.182 O.019 O.12S O.048 O.212 0.155 527 17903 SENSE O401 O.OO8 O.174 O.O11 O.330 O.OOS 529 18284 SENSE O.O31 0.795 O.O16 O.722 O.223 O.043 737 18536 SENSE O.158 O.222 O.104 O.118 O.149 O.122 736 1841O SENSE O.222 O.228 O.078 O.640 O.141 O.O8O

US 2015/O 1841 89 A1 Jul. 2, 2015 50

If p <0.05 and delta or risk score mean >0, the transgenic tally controlled rooms under a 16 halay length with an average plants showed statistically significant trait improvement as light intensity of ~200 umoles/m/s. Day and night tempera compared to the reference. If p <0.2 and delta or risk score ture set points were 22°C. and 20°C., respectively. Humidity mean >0, the transgenic plants showed a trend of trait was maintained at 65%. Plants were watered by sub-irrigation improvement as compared to the reference. every two days on average until mid-flowering, at which point the plants were watered daily until flowering was complete. J. Late Plant Growth and Development Screen 0.155. Application of the herbicide glufosinate was per 0153. This example sets forth a soil based phenotypic formed to select T2 individuals containing the target trans platform to identify genes that confer advantages in the pro gene. A single application of glufosinate was applied when cesses of leaf development, flowering production and seed the first true leaves were visible. Each pot was thinned to leave maturity to plants. a single glufosinate-resistant seedling ~3 days after the selec 0154 Arabidopsis plants were grown on a commercial tion was applied. potting mixture (Metro Mix 360, Scotts Co., Marysville, 0156 The rosette radius was measured at day 25. The Ohio) consisting of 30-40% medium grade horticultural ver silique length was measured at day 40. The plant parts were miculite, 35-55% sphagnum peat moss, 10-20% processed harvested at day 49 for dry weight measurements if flowering bark ash, 1-15% pine bark and a starter nutrient charge. Soil production was stopped. Otherwise, the dry weights of rosette was supplemented with Osmocote time-release fertilizer at a and silique were carried out at day 53. The seeds were har rate of 30 mg/ft. T2 seeds were imbibed in 1% agarose Vested at day 58. All measurements were analyzed as quan solution for 3 days at 4°C. and then sown at a density of -5 per titative responses according to example 1M. 2/4" pot. Thirty-two pots were ordered in a 4 by 8 grid in (O157 Table 12 provides a list of recombinant DNA con standard greenhouse flat. Plants were grown in environmen structs that improve late plant growth and development. TABLE 12

Rosette dry Seed net dry Silique dry PEP weight at day Rosette radius weight at day weight at day Silique length SEQ 53 at day 25 62 53 at day 40

ID Delta P Delta P- Delta P- Delta P- Delta P NO Orientation mean value le:8 value le:8 value le:8 value (8. value

772 ANTI- O.O72 0.189 f f O.846 OOO2 O.143 O.381 -0.012 O.803 SENSE 773 SENSE O.O94 O.S92 -O.238 0.098 O.665 0.059 O.247 O.474 -O.O29 O.184 775 SENSE O.134 0.019 f f O.675 0.061 O.S4O O.O10 O.093 0.008 774 SENSE -0.262 0.112 -0.407 O.O32 -0.393 O.120 O.357 0.081 O.OO3 0.922 776 SENSE -1.027 O.O44 f f 2.216 OOO4 0.351 0355 O.13S O.OO1 780 SENSE O.310 0.048 f f -0.245 0.629 O.149 0.157 O.149 0.043 781 SENSE O.219 OO69 f f O.779 O.OO1 -0.174 0.108 -0.015 0.595 777 SENSE O.S26 O.045 f f -0.388 O.478 O.131 O.281 -0.351 O.O36 778 SENSE O4O2 (0.004 -0.158 0.126 -2.159 O.O36 -0.410 OO16 -0.195 O.043 779 SENSE O454 O.OO6 -O.124 O.234 -0.112 O.328 O.228 O.O70 O.065 O.O24 782 SENSE O.195 0.2OS -0.153 0.019 O.352 O.OOS O.488 O.O31 -0.063 O.473 783 SENSE -0.388 O.381 f f 1.339 O.OO2 -1.147. O.126 O.601 OOOO 786 SENSE -0.107 O.28O O.091 O-148 O.S63 0.058 O.166 0.165 O.O42 O.O84 787 SENSE -0.290 O.138 -O.752 O.O2O OSO1 O.O44 -0.114 O.237 O.048 0.188 788 SENSE -0.064 O.709 -O.O60 O3O2 O.346 0.027 O.198. O.O82 O.O62 0.115 789 SENSE -0.06O O.S85 -0.204 0.076 O.930 OOO4 -0.243 O.109 O.O94 OO16 790 SENSE -0.299 O.O76 O.2O8 (0.181 O.683 0.008 O.140 O.652 -0.047 O.O70 792 SENSE O.47O O.O39 f f O.113 O.691 -0.118 OS 11 -O.OO3 O921 791 SENSE -O.O37 0.754 f f O484 O.O84 -0.163 O.366 O.O24 O.328 784 SENSE -0.182 0.070 -O.O79 0.243 O.649 O.O75 O.O70 0.659 -0.1SO 0.112 785 SENSE -0.057 0.216 -0.086 O.O16 1645 OOO8 O.278 O.O36 O.O15 0.364 795 SENSE O.62O O.O13 f f O.698 OO1O -0.102 O.794 O.O40 O247 793 SENSE O492 O.045 -O.232 O.O29 -0.505 O.O3O O.O2S O.881 794 SENSE -O.OS1 O.809 f f O.809 OOO3 -0.054 O.649 -O.O2O O.786 797 SENSE O.121 O.107 f f O.915 0.017 -O.287 O.OS4 O.OS4 O.120 796 SENSE O.324 O.O17 f f -O.289 O.468 O.331 O.O12 O.O4O O.476 798 SENSE O411 O.O38 f f O.809 OO18 O.231 O.286 -0.048 0.279 799 SENSE -0.014 O.720 f f O.337 OO64 -0.112 O.165 O.O18 O.S81 US 2015/O 1841 89 A1 Jul. 2, 2015 51

If p <0.05 and delta or risk score mean >0, the transgenic root length. After 21 days of growth, plants were scored for plants showed statistically significant trait improvement as BASTA resistance, visual color, seedling weight, number of compared to the reference. If p <0.2 and delta or risk score green leaves, number of rosette leaves, root length and for mean >0, the transgenic plants showed a trend of trait mation of flowering buds. A photograph of each plant was improvement as compared to the reference. also taken at this time point. 0160 The seedling weight and root length were analyzed K. Limited Nitrogen Tolerance Screen as quantitative responses according to example 1M. The num 0158 Under low nitrogen conditions, Arabidopsis seed ber green leaves, the number of rosette leaves and the flow lings become chlorotic and have less biomass. This example erbud formation were analyzed as qualitative responses sets forth the limited nitrogen tolerance screen to identify according to example 1L. The leaf color raw data were col Arabidopsis plants transformed with the gene of interest that lected on each plant as the percentages of five color elements are altered in their ability to accumulate biomass and/or retain (Green, DarkGreen, LightGreen, RedPurple, YellowChlo chlorophyll under low nitrogen condition. rotic) using a computer imaging system. A statistical logistic 0159 T2 seeds were plated on glufosinate selection plates regression model was developed to predict an overall value containing 0.5xN-Free Hoagland's T 0.1 mM NHNOT based on five colors for each plant. 0.1% Sucrose T 1% phytagel media and grown understandard 0.161 Table 13 provides a list of recombinant DNA con light and temperature conditions. At 12 days of growth, plants structs that improve low nitrogen availability tolerance in were scored for seedling status (i.e., viable or non-viable) and plants. TABLE 13

PEP Leaf color SEQ Root length Risk Rosette weight

ID Construct Delta P- SCO P- Delta P NO ID Orientation le:8 value le:8 value (8. value

6SO 10913 ANTI-SENSE -0.322 O.O72 O.S.S9 O.234 -0.052 0.209 651 10925 ANTI-SENSE -0.407 O.O47 5.828 O.342 -0.064 O416 652 11357 SENSE -0.436 OO16 O.996 O.O47 -0.093 0.233 653 11358 SENSE -O-521 OOOO 297 O.O12 -O.OO3 O.945 654 11432 ANTI-SENSE -0.215 O.049 O.669 O.O46 O.OO9 O.899 655 11927 ANTI-SENSE -0.416 0.028 O.S81 O.O21 O.O28 O.293 430 11937 ANTI-SENSE -0.3O2 O.O76 O.564 O.O39 -0.102 OO67 656 12OSO SENSE -0188 OOO3 323 O.O32 -0.031 O.245 657 12358 ANTI-SENSE -0.179 O.OSO O.278 O.06S O.O24 O.818 658 13809 SENSE -O.126 O.O24 O.796 O.303 -0.099 O.O73 603 14733 SENSE -0.161 O.131 2.267 0.085 -O.OSS O.676 525 15419 ANTI-SENSE -0.068 0.119 .607 O.OS1 O.O44 O451 659 16877 SENSE -0.207 O.158 2.516 0.057 O.O12 O.843 660 18117 ANTI-SENSE -0.221 O.OO4 O.906 OOO4 -0.146 OO67 805 70819 SENSE -O.258 0.057 2.275 O.O88 -0.103 0.231 777 7O639 SENSE -O.276 O.O71 -1.184 O.O99 O.132 O.OS2 778 7O668 SENSE -O.126 OO10 -0.521 O156 O.O67 O.S2O 661 71651 SENSE -O.O37 O.S99 O.485 0.042 -O.O2O O.682 662 72449 SENSE -O.O19 O.645 -0.738. O.043 O.324 O.O24 454 72533 SENSE -O.O11 O.866 131 O.O17 O.O68 0.237 663 72649 SENSE -0.154 O.309 O.181 O.O93 -0.046 (O.350 610 72978 SENSE -O3O4. O.184 O.367 0.12S O.144 OOO8 464 73344 SENSE -O.O68 OOO2 -0.547 O400 O.O4O O.612 744 73666 SENSE O.239 O.137 -2.192 O.O89 O.125 OO64 635 73747 SENSE -O.O31 O.568 0.671 O.O10 O.OO2 0.974 664 73107 SENSE -O.236 O.O39 O.196 O.OS7 O.OSO O.SS4 567 74135 SENSE O.173 O.O81 -2988 OO61 O.117 O.O90 636 74634 SENSE -1.049 O.019 O.805 O.O83 -O.O84 O.O34 578 74644 SENSE -O.224 0.027 O.247 O.353 -0.054 0.256 665 74875 SENSE -O.297 O2O1 1084 O.O89 O.201 O. 106 666 75079 SENSE -O.303 0.277 O.S83 0.071 -0.122 O.329 485 752O2 SENSE -O.277 O.093 O463 O.O49 -O.O2O O.570 668 75228 SENSE -O.838 (O.036 1.239 O.O33 -0.091 OSO3 669 7S246 SENSE -1192 OOO9 1314 O.O39 -0.136 O.205 809 73223 SENSE O.06O O.764 -0.249 O.442 O.O66 O.O84 784 73.283 SENSE O.2OO O.242 -2.819 O.O46 O.198. O.OS1 637 74769 SENSE -0.135 0.090 O.642 O.O24 O. 102 OO64 667 7518O SENSE -O.266 O.O31 O.602 O.O21 O.OO6 O.281 670 75.375 SENSE -0.647 OO12 1.618 OOO1 -0.156 O.043 671 75476 SENSE -0.563 OO11 1.101 OOO3 -0.091 O.162 673 75857 SENSE -O.2S6 OO67 2.472 OOO6 -0.125 O.O41 672 755O2 SENSE O.101 O.209 -0.147 O.482 O.156 0.066 675 75970 SENSE -0.125 O.191 O.963 O.O36 O.O46 0.391 676 76115 SENSE -0.499 O.113 O.619 O.O92 O.O18 O.752 833 76121 SENSE -O.707 O.O12 1.695 (OOO7 -0.199 O.049 643 76155 SENSE -0.059 O.454 O.779 0.022 -0.033 0.742 677 76217 SENSE -O.312 O.12O O.750 OOO1 -0.010 O.864 US 2015/O 1841 89 A1 Jul. 2, 2015 52

TABLE 13-continued

PEP Leaf color SEQ Root length Risk Rosette weight

ID Construct Delta P- SCO P Delta P NO ID Orientation le:8 value le:8 value 8 value

678 76287 SENSE -O.SO4 O.O3S 1.326 O.OO3 -0.147 0.018 679 76294 SENSE -O492 O.OO2 1.096 OOO6 -0.134 OO13 762 76293 SENSE O.102 0.330 -2.184 OOO8 O.358 0.049 674 75873 SENSE -O.191 (0.071 3.07O 0.093 O-109 O.197 68O 76305 SENSE -0.643 O.OO1 2.319 O.OOO -O-110 OO18 644 76322 SENSE -0.568 0.019 1.259 0.091 -0.205 O.O19 837 76388 SENSE O.O78 O.155 -0.032 0.862 O.130 O.O17 681 76391 SENSE -O.259 (0.114 1.043 O.O46 O.O36 O.806 798 76431 SENSE O.143 0.134 -0.383 0554 O.196 O.O34 682 76438 SENSE -0183 0.029 0.790 O.057 -O.O62 0.291 683 76452 SENSE -O.284 0.112 O.928 O.O17 -OOO2 0.969 684 76.563 SENSE -O.O32 0.100 -0.047 O2O3 -O.095 O153 839 76567 SENSE O.228 O.O57 -0.403 O.418 O.18O, O.O24 646 76828 SENSE -0.163 0.051 O.666 0.018 O. 1S6 O.078 685 77055 SENSE -O.261 (0.18O O.926 0.079 O.OS4 O.S16 686 77059 SENSE -0.380 OOOS 1188 O.O21 -0.138 0.012 687 77062 SENSE -0.494 O.O2O 1.284 O.O33 O.O1 O O.759 688 77063 SENSE -O.290 (0.018 O.814 O.O19 -O.O82 0.116 691 77314 SENSE -OSOO 0.046 O.369 O.OO8 -0.185 O.O29 692 77344 SENSE -0.163 O.O29 O.199 OO16 OO67 O.3SO 693 77348 SENSE -O.218 O.OO3 O.313 O.OOO OOO2 O.960 846 77112 SENSE -O.O2S O.839 -0.057 0.919 O.142 0.059 689 77218 SENSE -O.O72 0.687 0.679 0.008 O.OS4 O.336 521 772.19 SENSE O.183 0.071 O.134 O290 O.319 O.O38 690 772.56 SENSE -O.339 O.O15 O.216 0.2OS -0.058 0.302

For leaf color and rosette weight, if p <0.05 and delta or risk ment as compared to the reference. If p<0.2, the transgenic score mean >0, the transgenic plants showed statistically plants showed a trend of trait improvement as compared to the significant trait improvement as compared to the reference. If reference. p<0.2 and delta or risk score mean >0, the transgenic plants showed a trend of trait improvement as compared to the L. Statistic Analysis for Qualitative Responses reference with p-0.2. For root length, if p <0.05, the trans 0162 Table 14 provides a list of responses that were ana genic plants showed statistically significant trait improve lyzed as qualitative responses TABLE 1.4

response SCCEl categories (Success vs. failure)

wilting response Risk Soil drought tolerance screen non-wilted vs. wilted Score growth stage at day 14 heat stress tolerance screen 50% of plants reach stage1.03 vs. not growth stage at day 14 Salt stress tolerance screen 50% of plants reach stage1.03 vs. not growth stage at day 14 PEG induced osmotic stress 50% of plants reach stage1.03 vs. not tolerance screen growth stage at day 7 cold germination tolerance screen 50% of plants reach stage 0.5 vs. not number of rosette leaves Shade tolerance screen 5 leaves appeared vs. not at day 23 flower bud formation at Shade tolerance. Screen flower buds appear vs. not day 23 leaf angle at day 23 Shade tolerance screen >60 degree vs. <60 degree number of green leaves limited nitrogen tolerance screen 6 or 7 leaves appeared vs. not at day 21 number of rosette leaves limited nitrogen tolerance screen 6 or 7 leaves appeared vs. not at day 21 Flower bud formation at limited nitrogen tolerance screen flower buds appear vs. not day 21 US 2015/O 1841 89 A1 Jul. 2, 2015

0163 Plants were grouped into transgenic and reference sequence database and the National Center for Biotechnology groups and were scored as Success or failure according to Information (NCBI) non-redundant amino acid database (nr. Table 14. First, the risk (R) was calculated, which is the aa). For each organism from which a DNA sequence provided proportion of plants that were scored as of failure plants herein was obtained, an “Organism Protein Database' is con within the group. Then the relative risk (RR) was calculated structed of known protein sequences of the organism; the as the ratio of R (transgenic) to R (reference). Risk score (RS) Organism Protein Database is a subset of the All Protein was calculated as -log, “... Subsequently the risk scores from Database based on the NCBI taxonomy ID for the organism. multiple events for each transgene of interest were evaluated 0167. The All Protein Database is queried using amino for statistical significance by t-test using SAS statistical soft acid sequence of cognate protein for gene DNA used in trait ware (SAS 9, SAS/STAT User’s Guide, SAS Institute Inc, improving recombinant DNA, i.e., sequences of SEQID NO: Cary, N.C., USA). RS with a value greater than 0 indicates 426 through SEQ ID NO: 850 using “blastp” with E-value that the transgenic plants perform better than the reference. cutoff of 1e-8. Up to 1000 top hits were kept, and separated by RS with a value less than 0 indicates that the transgenic plants organism names. For each organism other than that of the perform worse than the reference. The RS with a value equal query sequence, a list is kept for hits from the query organism to 0 indicates that the performance of the transgenic plants itself with a more significant E-value than the best hit of the and the reference don’t show any difference. organism. The list contains likely duplicated genes, and is referred to as the Core List. Another list was kept for all the M. Statistic Analysis for Quantitative Responses hits from each organism, sorted by E-value, and referred to as 0164. Table 15 provides a list of responses that were ana the Hit List. lyzed as quantitative responses. 0.168. The Organism Protein Database is queried using amino acid sequences of SEQID NO: 426 through SEQ ID TABLE 1.5 NO: 850 using “blastp” with E-value cutoff of 1e-4. Up to 1000 top hits are kept. A BLAST searchable database is response SCEl constructed based on these hits, and is referred to as seed yield Soil drought stress tolerance screen “SubDB’. SubDB was queried with each sequence in the Hit Seedling weight at day 14 heat stress tolerance screen List using “blastp' with E-value cutoff of 1e-8. The hit with root length at day 14 heat stress tolerance screen Seedling weight at day 14 salt stress tolerance screen the best E-value is compared with the Core List from the root length at day 14 salt stress tolerance screen corresponding organism. The hit is deemed a likely ortholog root length at day 11 saltstress tolerance screen if it belongs to the Core List, otherwise it is deemed not a Seedling weight at day 14 PEG induced osmotic stress tolerance screen likely ortholog and there is no further search of sequences in root length at day 11 PEG induced osmotic stress tolerance screen root length at day 14 PEG induced osmotic stress tolerance screen the Hit List for the same organism. Likely orthologs from a rosette area at day 8 cold shock tolerance screen large number of distinct organisms were identified and are rosette area at day28 cold shock tolerance screen reported by amino acid sequences of SEQID NO: 851 to SEQ difference in rosette area cold shock tolerance screen ID NO: 33634. These orthologs are reported in Tables 2 as from day 8 to day 28 root length at day 28 cold germination tolerance screen homologs to the proteins cognate to genes used in trait-im Seedling weight at day 23 Shade tolerance screen proving recombinant DNA. petiole length at day 23 Shade tolerance screen root length at day 14 Early plant growth and development screen Example 3 Seedling weight at day 14 Early plant growth and development screen Rosette dry weight at day 53 Late plant growth and development screen rosette radius at day 25 Late plant growth and development screen Consensus Sequence Build seed dry weight at day 58 Late plant growth and development screen silique dry weight at day 53 Late plant growth and development screen 0169 ClustalW program is selected for multiple sequence silique length at day 40 Late plant growth and development screen alignments of an amino acid sequence of SEQ ID NO: 426 Seedling weight at day 21 Limited nitrogen tolerance screen and its homologs, through SEQID NO: 850 and its homologs. Root length at day 21 Limited nitrogen tolerance screen Three major factors affecting the sequence alignments dra matically are (1) protein weight matrices; (2) gap open pen 0.165. The measurements (M) of each plant were trans alty; (3) gap extension penalty. Protein weight matrices avail formed by log calculation. The Delta was calculated as log able for ClustalW program include Blosum, Pam and Gonnet M(transgenic)-log M(reference). Subsequently the mean series. Those parameters with gap open penalty and gap delta from multiple events of the transgene of interest was extension penalty were extensively tested. On the basis of the evaluated for statistical significance by t-test using SAS sta test results, Blosum weight matrix, gap open penalty of 10 tistical software (SAS9, SAS/STATUser's Guide, SAS Insti and gap extension penalty of 1 were chosen for multiple tute Inc, Cary, N.C., USA). The Delta with a value greater sequence alignment. The consensus sequence of SEQID NO: than 0 indicates that the transgenic plants perform better than 601 and its 13 homologs was derived according to the proce the reference. The Delta with a value less than 0 indicates that dure described above and is displayed in FIG. 1. the transgenic plants perform worse than the reference. The Delta with a value equal to 0 indicates that the performance of Example 4 the transgenic plants and the reference don’t show any differ 0170 This example illustrates the identification of amino CCC. acid domain by Pfam analysis. Example 2 0171 The amino acid sequence of the expressed proteins that were shown to be associated with an enhanced trait were Identification of Homologs analyzed for Pfam protein family against the current Pfam (0166 A BLAST searchable “All Protein Database' is con collection of multiple sequence alignments and hidden structed of known protein sequences using a proprietary Markov models using the HMMER software in the appended US 2015/O 1841 89 A1 Jul. 2, 2015 54 computer listing. The Pfam protein families for the proteins of Pfam domains. For instance, the protein with amino acids of SEQ ID NO: 425 through 850 are shown in Table 16. The SEQID NO: 488 is characterized by two Pfam domains, i.e. Hidden Markov model databases for the identified patent “NAF and “Pyr redox 2. See also the protein with amino families are also in the appended computer listing allowing acids of SEQ ID NO. 441 which is characterized by three identification of other homologous proteins and their cognate copies of the Pfam domain“zf-CCCH'. In Table 16"score” is encoding DNA to enable the full breadth of the invention for the gathering score for the Hidden Markov Model of the a person of ordinary skill in the art. Certain proteins are domain which exceeds the gathering cutoff reported in Table identified by a single Pfam domain and others by multiple 17. TABLE 16

PEP SEQ ID NO GENEID Pfam domain name begin stop score E-value 426 CGPG699 Histone 27 100 99.6 8.8OE-27 427 COPG567 MIF 2 115 694 1.OOE-17 428 CGPG267 WD40 46 83 28.3 24OE-OS 428 CGPG267 WD40 136 173 36.3 9.6OE-08 430 CGPG959 NPH3 209 444 429.9 3.2OE-126 431 CGPG2158 LSM 14 92 75.7 1.3OE-19 432 CGPG24.46 NUDIX 63 210 94.9 2.2OE-25 433 CGPG1862 Bromodomain 421 510 97.4 4.OOE-26 435 CGPG1674 efhand 303 331 23.1 O.OOO89 435 CGPG1674 Na Cal ex 441 575 58.5 1.90E-14 436 CGPG268O Linker histone 23 93 101 3.3OE-27 437 COPG3577 Glyoxalase 13 132 45.2 2.00E-10 438 CGPG406S LRR 2 150 174 17.3 O.OS1 439 CGPG3929 Lung 7-TM R 134 419 130.2 S.2OE-36 441 CGPG3012 Zf CCCH 30 57 7.7 O.12 441 CGPG3012 Zf CCCH 61 85 7.3 O.13 441 CGPG3012 Zf CCCH 115 140 19.3 O.0034 442 CGPG3162 SBF 128 313 239.7 5.6OE-69 443 CGPG6O7 PrA 28 275 44.4 S.8OE-12 444 CGPG4084 PCI 251 355 102 1.6OE-27 445 CGPG3917 Pkinase 13 268 3416 1.2OE-99 445 CGPG3917 NAF 307 367 124.7 2.3OE-34 446 CGPG4414 p450 32 502 364.5 150E-106 447 CGPG18S Cyclin N 62 187 117.7 3.OOE-32 447 CGPG18S Cyclin C 189 312 83.3 7.OOE-22 448 CGPG1679 Ferric reduct 183 304 126.7 6.OOE-3S 448 CGPG1679 FAD binding 8 334 435 1636 4.7OE-46 448 CGPG1679 FAD binding 6 336 435 -7.5 O.OO26 448 CGPG1679 NAD binding 6 441 709 348.5 9.90E-102 449 CGPG271 PP2C 269 629 56 1.10E-13 450 CGPG4434 p450 30 490 347.1 2.7OE-101 452 CGPG5253 SBP56 2O 487 1316.4 O 453 CGPGS231 Pkinase 85 343 344.4 1.7OE-100 453 CGPGS231 efhand 390 418 35.6 1. SOE-07 453 CGPGS231 efhand 426 454 31.4 3.OOE-06 453 CGPGS231 efhand 462 490 28.3 2. SOE-OS 453 CGPGS231 efhand 496 524 39.3 1.2OE-08 455 CGPG4859 PSI PsaF 47 221 443.9 2.OOE-130 456 CGPG1589 PLAC8 292 390 87.4 4.OOE-23 458 CGPG3899 Pkinase 132 417 294.1 24OE-85 460 CGPGS665 Aminotran 3 27 362 550.2 2.OOE-162 460 CGPGS665 Aminotran 12 42 417 -49.4 S4OE-OS 461 CGPGS697 Aminotran 3 25 360 SO3.2 2.7OE-148 461 CGPGS697 Aminotran 12 40 415 -51.6 7.OOE-OS 462 CGPGS695 Pyr redox 148 240 10.4 O.OO19 462 CGPGS695 Pyr redox 2 148 450 71.6 2.2OE-18 463 CGPG4862 Glyco hydro 1 40 52O 611 9.40E-181 46S CGPG6541 PGK 88 479 732.8 2.1OE-217 466 CGPG1756 NOPSNT 2 67 119.5 8.7OE-33 466 CGPG1756 NOSIC 160 212 127.9 2.7OE-3S 466 CGPG1756 Nop 252 400 332.4 7.OOE-97 467 CGPG4927 WD40 294 331 32.9 1.OOE-06 467 CGPG4927 WD40 336 373 24.7 O.OOO3 467 CGPG4927 WD40 378 428 22.1 O.OO18 468 CGPGS106 C2 17 96 91.7 2.1OE-24 469 CGPGS167 p450 43 5O1 265.6 9.3OE-77 470 CGPG1924 F-box 21 68 39.6 9.8OE-09 471 CGPGS2O1 Methyltransf 11 116 231 86.8 6.2OE-23 471 CGPGS2O1 Methyltransf 12 116 229 31.3 3.1OE-06 472 CGPG1884 Pkinase Tyr 88 365 137.7 2.90E-38 472 CGPG1884 Pkinase 88 368 1425 1.OOE-39

US 2015/O 1841 89 A1 Jul. 2, 2015 62

TABLE 16-continued

PEP SEQ ID NO GENEID Pfam domain name begin stop score E-value

847 CGPG9151 Aminotran 3 82 421. 269 8.9 OE-78 848 CGPG9129 HATPase c 228 356 83.7 5.2OE-22 850 CGPG9358 TPR 1 551 S84 1O.S O.28 850 CGPG9358 TPR 1 585 618 15.5 O.O68 850 CGPG9358 TPR 1 655 688 16.1 O.O59

TABLE 17 Pfam domain accession gathering l8le number cutoff domain description Zf-MYND PFO1753.8 11 MYND finger UCH PFOO443.18 -8.6 Ubiquitin carboxyl-terminal hydrolase MIF PFO1187.7 -17.6 Macrophage migration inhibitory factor (MIF) PurA PFO4845.3 25 Pura ssDNA and RNA-binding protein Gln-synt C PFOO120.14 -124 Glutamine synthetase, catalytic domain WD40 PFOO400.2O 21.5 WD domain, G-beta repeat Pkinase PFOOO69.14 -70.8 Protein kinase domain Pkinase PFOOO69.14 -70.8 Protein kinase domain NAF PFO3822.4 4.5 NAF domain Sterol desat PFO1598.7 -13 Sterol desaturase Pkinase PFOOO69.14 -70.8 Protein kinase domain Asp PFOOO26.13 -186.1 Eukaryotic aspartyl protease Glyco hydro 1 PFOO232.9 -301.8 Glycosyl hydrolase family 1 Oleosin PFO1277.7 -27 Oleosin Sugar tr PFOOO83.13 -85 Sugar (and other) transporter MFS 1 PFO7690.5 23.5 Major Facilitator Superfamily ATP-grasp 2 PFO8442. -118.8 ATP-grasp domain PLAC8 PFO4749.6 -1.1 PLAC8 family Ferric reduct PFO1794.8 -7 Ferric reductase like transmembrane component FAD binding 8 PFO8022. -10.4 FAD-binding domain FAD binding 6 PFOO970.13 -11.4 Oxidoreductase FAD-binding domain NAD binding 6 PFO8O3O. -23.6 Ferric reductase NAD binding domain LSM PFO1423.12 13.7 LSM domain Fibrillarin PFO1269.7 -86.6 Fibrillarin WD40 PFOO400.2O 21.5 WD domain, G-beta repeat WD40 PFOO400.2O 21.5 WD domain, G-beta repeat Linker histone PFOO538.8 -8 linker histone H1 and H5 family PP2C PFOO481.11 -44 Protein phosphatase 2C Sugar tr PFOOO83.13 -85 Sugar (and other) transporter MFS 1 PFO7690.5 23.5 Major Facilitator Superfamily SBF PFO1758.6 -27.8 Sodium Bile acid symporter family Glyoxalase PFOO903.14 12.1 Glyoxalase/Bleomycin resistance protein Dioxygenase Superfamily Pkinase PFOOO69.14 -70.8 Protein kinase domain NAF PFO3822.4 4.5 NAF domain LRR 2 PFO7723.2 6 Leucine Rich Repeat 4SO PFOOO67.11 -105 Cytochrome P450 WD40 PFOO400.2O 21.5 WD domain, G-beta repeat WD40 PFOO400.2O 21.5 WD domain, G-beta repeat WD40 PFOO400.2O 21.5 WD domain, G-beta repeat LRR 1 PFOOS60.21 7.7 Leucine Rich Repeat LRR 1 PFOOS60.21 7.7 Leucine Rich Repeat LRR 1 PFOOS60.21 7.7 Leucine Rich Repeat LRR 1 PFOOS60.21 7.7 Leucine Rich Repeat LRR 1 PFOOS60.21 7.7 Leucine Rich Repeat Pkinase PFOOO69.14 -70.8 Protein kinase domain C2 PFOO168.18 3.7 C2 domain SBP56 PFOS 694.1 25 56 kDa selenium binding protein (SBP56) MPDH PFOO478.13 -190.6 IMP dehydrogenase/GMP reductase domain MtN3 silv PFO3O83.5 -0.8 MtN3/saliva family MtN3 silv PFO3O83.5 -0.8 MtN3/saliva family Aminotran 3 PFOO2O2.10 -207.6 Aminotransferase class-III Aminotran 12 PFOO155.10 -57.5 Aminotransferase class I and II Aminotran 3 PFOO2O2.10 -207.6 Aminotransferase class-III Aminotran 12 PFOO155.10 -57.5 Aminotransferase class I and II Pkinase PFOOO69.14 -70.8 Protein kinase domain Pkinase PFOOO69.14 -70.8 Protein kinase domain US 2015/O 1841 89 A1 Jul. 2, 2015 63

TABLE 17-continued

Pfam domain accession gathering l8le number cutoff domain description Pkinase -70.8 Protein kinase domain Pkinase Tyr 65 Protein tyrosine kinase Pkinase FOOO69. -70.8 Protein kinase domain NAF FO3822.4 4.5 NAF domain Pkinase FOOO69. 4 -70.8 Protein kinase domain Pkinase FOOO69. 4 -70.8 Protein kinase domain Nodulin-like FO6813.3 -57.8 Nodulin-like Glyco hydro 14 FO1373.7 -231.4 Glycosyl hydrolase family 14 GSHPx FOO255. O -16 Glutathione peroxidase MFS 1 FO7690.5 23.5 Major Facilitator Superfamily Sugar tr FOOO83. 3 -85 Sugar (and other) transporter FOO162.9 -39.9 Phosphoglycerate kinase PGK FOO162.9 -39.9 Phosphoglycerate kinase Pyr redox FOOO70. 7 5 Pyridine nucleotide-disulphide oxidoreductase Pyr redox 2 FO7992.3 -20 Pyridine nucleotide-disulphide oxidoreductase Zf C3HC4 FOOO97. 3 16.9 Zinc finger, C3HC4 type (RING finger) MIP FOO230.9 -62 Major intrinsic protein Tim17 FO2466.8 2.7 Tim17/Tim22/Tim23 family HABP4. PAI-RBP1 FO4774.4 17.1 Hyaluronan mRNA binding family DUF1677 FO79114 25 Protein of unknown function (DUF1677) NIR SIR ferr FO3460.6 2.4 Nitrite Sulfite reductase ferredoxin-like half domain NIR SIR FO1077. 1 -25 Nitrite and Sulphite reductase 4Fe-4S domain NIR SIR ferr FO3460.6 2.4 Nitrite Sulfite reductase ferredoxin-like half domain NIR SIR FO1077. 1 -25 Nitrite and Sulphite reductase 4Fe-4S domain TIM FOO121.8 -97 Triosephosphate isomerase DnaJ FOO226. 9 -8 DnaJ domain Fer4 FOOO37. 5 9.3 4Fe-4S binding domain Pro isomerase FOO16O. O -37 Cyclophilin type peptidyl-prolyl cis-trans isomerasef CLD Pec lyase C FOOS44.8 -45 Pectate lyase CS FO4969.5 8.6 CS domain Glyoxal oxid N FO7250. 25 Glyoxal oxidase N-terminus YeF N FO3853.4 25 YeF-related protein N-terminus Carb kinase FO1256.7 -66.3 Carbohydrate kinase TPR1 FOO515. 6 7.7 Tetratricopeptide repeat TPR1 FOO515. 6 7.7 Tetratricopeptide repeat TPR1 FOO515. 6 7.7 Tetratricopeptide repeat DUF1644 FO780O.2 25 Protein of unknown function (DUF1644) TMEM14 FO3647.3 -1 Transmembrane proteins 14 C Aldo ket red -97 Aldo/keto reductase family Pkinase FOOO69. -70.8 Protein kinase domain BCNT FO7572.2 25 Bucentaur or craniofacial development Mito carr FOO153. O Mitochondrial carrier protein Mito carr FOO153. O Mitochondrial carrier protein Mito carr FOO153. O Mitochondrial carrier protein NUDIX FOO293. O NUDIX domain PRA-CH FO1502.9 25 Phosphoribosyl-AMP cyclohydrolase PRA-PH FO1 SO3.8 6 Phosphoribosyl-ATP pyrophosphohydrolase Pkinase FOOO69. -70.8 Protein kinase domain Pkinase Tyr 65 Protein tyrosine kinase Zf-CCCEH O Zinc finger C-x8-C-x5-C-X3-H type (and similar) WD40 FOO400.2O 21.5 WD domain, G-beta repeat WD40 FOO400.2O 21.5 WD domain, G-beta repeat WD40 FOO400.2O 21.5 WD domain, G-beta repeat CKII beta FO1214.9 -106 Casein kinase II regulatory subunit MFS 1 FO7690.5 23.5 Major Facilitator Superfamily FO4893.6 -6.4 Yip1 domain ARID FO1388.1 1 ARID/BRIGHT DNA binding domain ELM2 FO1448.1 2 12 ELM2 domain Myb DNA-binding FOO249.1 9 2.8 Myb-like DNA-binding domain PP2C FOO481.1 1 Protein phosphatase 2C Mat FO1554.8 59.6 Mat Mat FO1554.8 59.6 Mat Methyltransf 11 FO8241.1 17.1 Methyltransferase domain Methyltransf 12 FO8242.1 21.4 Methyltransferase domain FMO-like FOOf 43.9 -381.6 Flavin-binding monooxygenase-like DAO FO1266.1 2 -35.9 FAD dependent oxidoreductase Pyr redox 2 FO7992.3 -20 Pyridine nucleotide-disulphide oxidoreductase Auxin inducible -15 Auxin responsive protein Response reg FOOO72.1 2 Response regulator receiver domain CCT 25 CCT motif US 2015/O 1841 89 A1 Jul. 2, 2015 64

TABLE 17-continued

Pfam domain accession gathering l8le number cutoff domain description Pkinase FOOO69.14 -70.8 Protein kinase domain Pkinase FOOO69.14 -70.8 Protein kinase domain Pkinase Tyr FO7714.5 65 Protein tyrosine kinase RRM 1 FOOO76.11 20.7 RNA recognition motif. (a.k.a. RRM, RBD, or RNP domain) RRM 1 FOOO76.11 20.7 RNA recognition motif. (a.k.a. RRM, RBD, or RNP domain) RRM 1 FOOO76.11 20.7 RNA recognition motif. (a.k.a. RRM, RBD, or RNP domain) RRM 1 FOOO76.11 20.7 RNA recognition motif. (a.k.a. RRM, RBD, or RNP domain) PABP FOO658.8 25 Poly-adenylate binding protein, unique domain FOOO67.11 -105 Cytochrome P450 FO1490.7 -128.4 Transmembrane amino acid transporter protein FOO646.21 13.6 F-box domain FO7723.2 6 Leucine Rich Repeat LRR 2 FO7723.2 6 Leucine Rich Repeat FHA FOO498.14 25 FHA domain Bromodomain FOO439.14 8.9 Bromodomain PHD FOO628.17 25.9 PHD-finger SET FOO856.17 23.5 SET domain Abhydrolase 1 FOOS61.10 10.3 alpha/beta hydrolase fold Epimerase FO1370.11 -46.3 NAD dependent epimerase? dehydratase amily 3Beta HSD FO1073.8 -135.9 3-beta hydroxysteroid dehydrogenase isomerase family DUF231 CC FO3005.5 -58 Arabidopsis proteins of unknown function ABC tran FOOOO5.15 9.5 ABC transporter ABC2 membrane FO1061.12 -17.9 ABC-2 type transporter Dehydrin FOO257.9 -4.4 Dehydrin DnaJ FOO226.19 -8 DnaJ domain DnaJ C FO1556.9 -24 DnaJ C terminal region FOOO23.18 O Ankyrin repeat FOOO23.18 O Ankyrin repeat FOO415.8 19 Regulator of chromosome condensation (RCC1) RCC1 FOO415.8 19 Regulator of chromosome condensation (RCC1) Pkinase FOOO69.14 -70.8 Protein kinase domain Lung 7-TM R FO6814.3 25 Lung seven transmembrane receptor Pkinase Tyr FO7714.5 65 Protein tyrosine kinase Pkinase FOOO69.14 -70.8 Protein kinase domain Aa trans FO1490.7 -128.4 Transmembrane amino acid transporter protein PCI FO1399.15 25 PCI domain ADH N FO8240.2 -14.5 Alcohol dehydrogenase GroES-like domain ADH zinc N FOO107.16 23.8 Zinc-binding dehydrogenase 4SO FOOO67.11 -105 Cytochrome P450 FKBP C FOO254.17 -7.6 FKBP-type peptidyl-prolyl cis-trans isomerase WD40 FOO400.2O 21.5 WD domain, G-beta repeat Lectin C FOOOS9.10 -10.4 Lectin C-type domain Pkinase FOOO69.14 -70.8 Protein kinase domain Pkinase Tyr FO7714.5 65 Protein tyrosine kinase HEAT FO2985.10 9.9 HEAT repeat HEAT FO2985.10 9.9 HEAT repeat Zf-CCCEH FOO642.14 Zinc finger C-x8-C-x5-C-X3-H type (and similar) FOO642.14 Zinc finger C-x8-C-x5-C-X3-H type (and similar) FOO642.14 Zinc finger C-x8-C-x5-C-X3-H type (and similar) FOO642.14 Zinc finger C-x8-C-x5-C-X3-H type (and similar) FOO642.14 Zinc finger C-x8-C-x5-C-X3-H type (and similar) PHD FOO628.17 25.9 PHD-finger SEP FO8059.2 25 SEP domain UBX FOOf89.10 10 UBX domain AA permease FOO324.10 -120.8 Amino acid permease Pkinase Tyr FO7714.5 65 Protein tyrosine kinase Pkinase FOOO69.14 -70.8 Protein kinase domain FAD binding 4 FO1565.12 -8.1 FAD binding domain US 2015/O 1841 89 A1 Jul. 2, 2015 65

TABLE 17-continued

Pfam domain accession gathering l8le number cutoff domain description PALP FOO291.14 -70 Pyridoxal-phosphate dependent enzyme GIDA FO1134.11 -226.7 Glucose inhibited division protein A Pyr redox 2 -20 Pyridine nucleotide-disulphide oxidoreductase Pyr redox 5 Pyridine nucleotide-disulphide oxidoreductase Pyr redox dim -13 Pyridine nucleotide-disulphide oxidoreductase, dimerisation domain FO2325.7 YGGT family FOO171. Aldehyde dehydrogenase family PEPCK ATP FO1293. Phosphoenolpyruvate carboxykinase Trp syntA FOO29O. Tryptophan synthase alpha chain DapB N FO1113. Dihydrodipicolinate reductase, N-terminus DapB C FO5173.3 Dihydrodipicolinate reductase, C-terminus Ribul P 3 epim FOO834.8 Ribulose-phosphate 3 epimerase family OMPdecase FOO215.13 Orotidine 5'-phosphate decarboxylase HUMPS family Arginase FOO491.11 Arginase family Pkinase FOOO69.14 Protein kinase domain Ferric reduct FO1794.8 Ferric reductase like transmembrane component Lipase 3 FO1764.14 Lipase (class 3) Auxin inducible FO2S19.4 Auxin responsive protein TPR 2 FO7719.5 Tetratricopeptide repeat TPR1 Tetratricopeptide repeat TPR 2 Tetratricopeptide repeat TPR1 Tetratricopeptide repeat TPR1 Tetratricopeptide repeat TPR 2 Tetratricopeptide repeat TPR1 Tetratricopeptide repeat Anti-silence FO4729.4 Anti-silencing protein, ASF1-like p450 FOOO67.11 -105 Cytochrome P450 Pkinase FOOO69.14 -70.8 Protein kinase domain Pkinase Tyr FO7714.5 65 Protein tyrosine kinase TCTP FOO838.7 -70.7 Translationally controlled tumour protein NAPRTase FO4O95.5 -88.5 Nicotinate phosphoribosyltransferase (NAPRTase) family SPX FO3105.9 -20 SPX domain EXS FO3124.4 2O EXS family DUF1639 FO7797.3 25 Protein of unknown function (DUF1639) Milo FO3094.5 -263 Mb family Sulfotransfer 1 FOO685.16 -53.1 Sulfotransferase domain PI-PLC-X FOO388.8 18.8 Phosphatidylinositol-specific phospholipase C, X domain FOO387.8 -11 Phosphatidylinositol-specific phospholipase C.Y domain C2 FOO168.18 3.7 C2 domain HMA FOO403.14 17.4 Heavy-metal-associated domain Ammonium transp FOO909.10 -144 Ammonium Transporter Family Asp FOOO26.13 -186.1 Eukaryotic aspartyl protease SapB 2 FO3489.6 10.7 Saposin-like type B, region 2 SapB 1 FOS1844 Saposin-like type B, region 1 Oxidored FMN FOOf24.9 -147.7 NADH:flavin oxidoreductase,NADH oxidase amily Pyr redox Pyridine nucleotide-disulphide oxidoreductase Pyr redox 2 -20 Pyridine nucleotide-disulphide oxidoreductase Pkinase -70.8 Protein kinase domain Miro 28 Miro-like protein Ras -69.9 Ras family Miro 28 Miro-like protein Ras -69.9 Ras family Pkinase Tyr 65 Protein tyrosine kinase Pkinase FOOO69.14 -70.8 Protein kinase domain Sterol desat FO1598.7 -13 Sterol desaturase ADH N FO8240.2 -14.5 Alcohol dehydrogenase GroES-like domain ADH zinc N FOO107.16 23.8 Zinc-binding dehydrogenase Sulfotransfer 1 FOO685.16 -53.1 Sulfotransferase domain TPR1 7.7 Tetratricopeptide repeat TPR 2 20.1 Tetratricopeptide repeat TPR1 7.7 Tetratricopeptide repeat TPR 2 20.1 Tetratricopeptide repeat TPR1 7.7 Tetratricopeptide repeat GATase 2 -106.2 Glutamine amidotransferases class-II SIS SIS domain SIS C SIS domain Acetyltransf 1 18.6 Acetyltransferase (GNAT) family US 2015/O 1841 89 A1 Jul. 2, 2015 66

TABLE 17-continued

Pfam domain accession gathering l8le number cutoff domain description UF1005 FO6219.2 25 Protein of unknown function (DUF1005) UF231 FO3005.5 -58 Arabidopsis proteins of unknown function B2 DP1 HVA22 FO3134.9 -25.1 TB2/DP1, HVA22 family i19 FO5605.2 25 Drought induced 19 protein (Di19) MtN3 silv FO3O83.5 -0.8 MtN3/saliva family MtN3 silv FO3O83.5 -0.8 MtN3/saliva family Pkinase FOOO69.14 -70.8 Protein kinase domain FOOO67.11 -105 Cytochrome P450 PGI FOO342.8 -168.9 Phosphoglucose isomerase lyco hydro 28 FOO295.7 -97 Glycosyl hydrolases family 28 Pkinase FOOO69.14 -70.8 Protein kinase domain Soamylase N FO2922.7 -6.5 Isoamylase N-terminal domain Alpha-amylase FOO128.12 -93 Alpha amylase, catalytic domain PBP FO1161.9 -20.6 Phosphatidylethanolamine-binding protein FOO642.14 O Zinc finger C-x8-C-x5-C-X3-H type (and similar) FOO642.14 O Zinc finger C-x8-C-x5-C-X3-H type (and similar) Zf-CCCEH FOO642.14 O Zinc finger C-x8-C-x5-C-X3-H type (and similar) Aa trans FO1490.7 -128.4 Transmembrane amino acid transporter protein Smir FO1713. 1 10 Smr domain 2-Haciddh FOO389.19 11.2 D-isomer specific 2-hydroxyacid dehydrogenase, catalytic domain 2-Haciddh C FO2826.7 -82.2 D-isomer specific 2-hydroxyacid dehydrogenase, NAD binding domain DUF1070 FO6376.2 25 Protein of unknown function (DUF1070) AMP-binding FOOSO1.16 AMP-binding enzyme Enolase N FO3952.6 -3.3 Enolase, N-terminal domain Enolase C FOO113.12 -34 Enolase, C-terminal TIM barrel domain FAE 3-kCoA syn1 FO7168.2 25 Fatty acid elongase 3-ketoacyl-CoA synthase 1 efhand FOOO36.20 17.5 EF hand Na Cal ex FO1699.12 25 Sodium calcium exchanger protein NOPSNT FO8156.2 25 NOP5NT (NUC127) domain NOSIC FO806O.2 25 NOSIC (NUC001) domain Nop FO1798.6 25 Putative SnoRNA binding domain Terpene synth FO1397.10 -86 Terpene synthase, N-terminal domain PCI FO1399.15 25 PCI domain Abhydrolase 3 FO7859.2 25.8 alpha/beta hydrolase fold GRP FO7172. 16.8 Glycine rich protein family DUF914 FO6O27.2 -193 Eukaryotic protein of unknown function (DUF914) DUF1325 FO7039. 25 Protein of unknown function (DUF1325) ZfA20 FO1754.6 25 A20-like zinc finger ZfAN1 FO1428.6 AN1-like Zinc finger PALP FOO291.14 -70 Pyridoxal-phosphate dependent enzyme MFS 1 FO7690.5 23.5 Major Facilitator Superfamily DUF1723 FO8330. 17 Protein of unknown function (DUF1723) GDPD FO3009.7 -18 Glycerophosphoryl diester phosphodiesterase amily Pkinase FOOO69.14 -70.8 Protein kinase domain Pkinase Tyr FO7714.5 65 Protein tyrosine kinase Pkinase FOOO69.14 -70.8 Protein kinase domain ADH N FO8240.2 -14.5 Alcohol dehydrogenase GroES-like domain ADH zinc N FOO107.16 23.8 Zinc-binding dehydrogenase Pribosyltran FOO156.15 Phosphoribosyltransferase domain FOO171.11 -209.3 Aldehyde dehydrogenase family FO7576. 25 BRCA1-associated protein 2 FOOO97.13 16.9 Zinc finger, C3HC4 type (RING finger) FO2148.8 25 Zn-finger in ubiquitin-hydrolases and other protein FOO226.19 DnaJ domain FO7743.4 HSCB C-terminal oligomerisation domain FOOOO5.15 ABC transporter Acyl-CoA dh N FO2.7717 Acyl-CoA dehydrogenase, N-terminal domain Acyl-CoA dh M FO2770.9 Acyl-CoA dehydrogenase, middle domain Acyl-CoA dh 1 FOO441.13 Acyl-CoA dehydrogenase, C-terminal domain Acyl-CoA dh 2 FO8O28. Acyl-CoA dehydrogenase, C-terminal domain NTP transferase FOO483.12 Nucleotidyl transferase MannoseP isomer FO 1050.8 Mannose-6-phosphate isomerase Cupin 2 FO7883. Cupin domain Lectin legB FOO139.10 Legume lectin domain Pkinase FOOO69.14 Protein kinase domain US 2015/O 1841 89 A1 Jul. 2, 2015 67

TABLE 17-continued Pfam domain accession gathering l8le number cutoff domain description Pkinase Tyr PFO7714.5 65 Protein tyrosine kinase Pkinase Tyr PFO7714.5 65 Protein tyrosine kinase Pkinase PFOOO69.14 -70.8 Protein kinase domain PGAM PFOO3OO.12 -3 Phosphoglycerate mutase family WD40 PFOO400.2O 21.5 WD domain, G-beta repeat Alpha-amylase PFOO128.12 -93 Alpha amylase, catalytic domain PTPA PFO3095.4 -106 Phosphotyrosyl phosphate activator (PTPA) protein Sedlin N PFO4628.2 25 Sedlin, N-terminal conserved region NAD binding 2 PFO3446.4 -63.5 NAD binding domain of 6-phosphogluconate dehydrogenase 6PGD PFOO393.8 -232.3 6-phosphogluconate dehydrogenase, C erminal domain ADK PFOO4O6.11 24.2 Adenylate kinase ADKlid PFO51913 25 Adenylate kinase, active site lid DUF26 PFO1657.7 O Domain of unknown function DUF26 DUF26 PFO1657.7 O Domain of unknown function DUF26 Pkinase Tyr PFO7714.5 65 Protein tyrosine kinase Pkinase PFOOO69.14 -70.8 Protein kinase domain TERF PFO2S36.4 -60 TERF BS6 PFO1603.9 -210 Protein phosphatase 2A regulatory B subunit (B56 family) ZfAN1 PFO1428.6 O AN1-like Zinc finger Xan ur permease PFOO860.10 -151.2 Permease family MFS 1 PFO7690.5 23.5 Major Facilitator Superfamily Auxin inducible PFO2S19.4 -15 Auxin responsive protein Trehalose PPase PFO2358.6 -49.4 Trehalose-phosphatase DUF260 PFO3195.4 0.8 Protein of unknown function DUF260 Aminotran 12 PFOO155.10 -57.5 Aminotransferase class I and II Pkinase PFOOO69.14 -70.8 Protein kinase domain Pkinase Tyr PFO7714.5 65 Protein tyrosine kinase Pkinase PFOOO69.14 -70.8 Protein kinase domain Pkinase Tyr PFO7714.5 65 Protein tyrosine kinase ADH N PFO8240.2 -14.5 Alcohol dehydrogenase GroES-like domain ADH zinc N PFOO107.16 23.8 Zinc-binding dehydrogenase RRM 1 PFOOO76. 20.7 RNA recognition motif. (a.k.a. RRM, RBD, or RNP domain) RRM 1 PFOOO76. 20.7 RNA recognition motif. (a.k.a. RRM, RBD, or RNP domain) La PFO5383.5 25 La domain RRM 1 PFOOO76. 20.7 RNA recognition motif. (a.k.a. RRM, RBD, or RNP domain) RRM 1 PFOOO76. 20.7 RNA recognition motif. (a.k.a. RRM, RBD, or RNP domain) RRM 1 PFOOO76. 20.7 RNA recognition motif. (a.k.a. RRM, RBD, or RNP domain) RRM 1 PFOOO76. 20.7 RNA recognition motif. (a.k.a. RRM, RBD, or RNP domain) LEA 2 PFO31 68.3 25 Late embryogenesis abundant protein Miro PFO8477. 28 Miro-like protein Ras PFOOO71. -69.9 Ras family DUF241 PFO3O87.4 -53.6 Arabidopsis protein of unknown function Sugar tr PFOOO83.13 -85 Sugar (and other) transporter MFS 1 PFO7690.5 23.5 Major Facilitator Superfamily on trans 2 PFO7885.4 24.9 Ion channel on trans 2 PFO7885.4 24.9 Ion channel PI-PLC-X PFOO388.8 18.8 Phosphatidylinositol-specific phospholipase C, X domain C2 PFOO168.18 3.7 C2 domain Aldedh PFOO171.11 -209.3 Aldehyde dehydrogenase family RNA poll Rpb8 PFO3870.5 -31.2 RNA polymerase Rpb8 COX5C PFO5799.1 25 Cytochrome c oxidase subunit Vc (COX5C) on trans 2 PFO7885.4 24.9 Ion channel on trans 2 PFO7885.4 24.9 Ion channel Aldo ket red PFOO248.10 -97 Aldo/keto reductase family Pkinase PFOOO69.14 -70.8 Protein kinase domain Pkinase Tyr PFO7714.5 65 Protein tyrosine kinase Na Cal ex PFO1699.12 25 Sodium calcium exchanger protein Na Cal ex PFO1699.12 25 Sodium calcium exchanger protein Pkinase PFOOO69.14 -70.8 Protein kinase domain ADH N PFO8240.2 -14.5 Alcohol dehydrogenase GroES-like domain ADH zinc N PFOO107.16 23.8 Zinc-binding dehydrogenase US 2015/O 1841 89 A1 Jul. 2, 2015 68

TABLE 17-continued

Pfam domain accession gathering l8le number cutoff domain description OPT FO3169.6 -238.6 OPT oligopeptide transporter protein PMEI FO4043.5 25 Plant invertase?pectin methylesterase inhibitor K trans FO2705.6 -482 K+ potassium transporter Pkinase FOOO69.1 4 -70.8 Protein kinase domain CH FOO3O7.1 9 22.5 Calponin homology (CH) domain EB1 FO3271.6 25 EB1-like C-terminal motif Carboxyl trans FO1039.1 1 -262.3 Carboxyl transferase domain CoA trans FO1144.1 2 25 Coenzyme A transferase Pkinase FOOO69.1 4 -70.8 Protein kinase domain CS FO4969.5 8.6 CS domain SGS FO5002.5 5.2 SGS domain NPH3 FO3OOO.4 25 NPH3 family WD40 FOO400.2O 21.5 WD domain, G-beta repeat Spermine synth FO1564.6 -93.8 Spermine?spermidine synthase FeThRed A FO2941.5 25 Ferredoxin thioredoxin reductase variable alpha chain Alpha-amylase FOO128.1 2 -93 Alpha amylase, catalytic domain Cyclin N FOO134.1 3 -14.7 Cyclin, N-terminal domain Cyclin C FO2984.8 -13 Cyclin, C-terminal domain F-box FOO646.2 1 13.6 F-box domain F-box FOO646.2 1 13.6 F-box domain Arm FOOS14.1 1 17 Armadillofbeta-catenin-like repeat Arm FOOS14.1 1 17 Armadillofbeta-catenin-like repeat Arm FOOS14.1 1 17 Armadillofbeta-catenin-like repeat Arm FOOS14.1 1 17 Armadillofbeta-catenin-like repeat Arm FOOS14.1 1 17 Armadillofbeta-catenin-like repeat FOOS14.1 1 17 Armadillofbeta-catenin-like repeat FOOS14.1 1 17 Armadillofbeta-catenin-like repeat FOOS14.1 1 17 Armadillofbeta-catenin-like repeat FOO230.9 -62 Major intrinsic protein FO8263.2 18.6 Leucine rich repeat N-terminal domain FOOS60.21 7.7 Leucine Rich Repeat FOOS60.21 7.7 Leucine Rich Repeat FOOS60.21 7.7 Leucine Rich Repeat Pkinase Tyr FO7714.5 65 Protein tyrosine kinase Pkinase FOOO69.1 4 -70.8 Protein kinase domain WD40 FOO400.2O 21.5 WD domain, G-beta repeat Glycolytic FOO274.9 -174.5 Fructose-bisphosphate aldolase class-I PSI PsaE FO2507.5 25 Photosystem I reaction centre subunit III NT 2 FO8263.2 18.6 Leucine rich repeat N-terminal domain 1 FOOS60.21 7.7 Leucine Rich Repeat 1 FOOS60.21 7.7 Leucine Rich Repeat 1 FOOS60.21 7.7 Leucine Rich Repeat 1 FOOS60.21 7.7 Leucine Rich Repeat 2 FOO168. 3.7 C2 domain FOOO67. -105 Cytochrome P450 FO2181. -98.3 Formin Homology 2 Domain O-box 1O.S U-box domain FAE1 CUT1 RppA -1927 FAE1/Type III polyketide synthase-like protein Chal sti Synt C -6.1 Chalcone and stilbene synthases, C-terminal domain ACP syn III C C FO8541. -24.4 3-Oxoacyl-[acyl-carrier-protein (ACP) synthase III C terminal aPHC FO5875.2 25 Alkaline phytoceramidase (aPHC) Pkinase FOOO69. 4 -70.8 Protein kinase domain Pkinase FOOO69. 4 -70.8 Protein kinase domain Pkinase Tyr FO7714.5 65 Protein tyrosine kinase Glyco hydro 14 FO1373.7 -231.4 Glycosyl hydrolase family 14 Miro FO8477. 28 Miro-like protein Ras FOOO71. 1 -69.9 Ras family PGM PMM I FO2878.5 -37.5 Phosphoglucomutase/phosphomannomutase, alphabeta alpha domain I PGM PMM II FO2879.5 -20 Phosphoglucomutase/phosphomannomutase, alphabeta alpha domain II PGM PMM III -7.8 Phosphoglucomutase/phosphomannomutase, alphabeta alpha domain III PGM PMM IV FOO408.9 Phosphoglucomutase/phosphomannomutase, C-terminal domain Glutaminase -143.6 Glutaminase Aldedh FOO171.1 1 -209.3 Aldehyde dehydrogenase family Aminotran 3 FOO2O2.1 O -2O7.6 Aminotransferase class-III 2OG-Fell Oxy FO3171.9 11.5 20G-Fe(II) oxygenase Superfamily Histone FOO125.1 3 17.4 Core histone H2AH2B, H3/H4 US 2015/O 1841 89 A1 Jul. 2, 2015 69

TABLE 17-continued

Pfam domain accession gathering l8le number cutoff domain description

F-box C FOO646.2 1 3.6 F-box domain Agenet C FOS641.2 6.6 Agenet domain Agenet C FOS641.2 6.6 Agenet domain DnaJ C FOO226.1 DnaJ domain SNARE C FO5739.8 SNARE domain adh short C FOO106.1 4 short chain dehydrogenase Pyr redox 2 C FO7992.3 Pyridine nucleotide-disulphide oxidoreductase Pyr redox C FOOO70.1 7 Pyridine nucleotide-disulphide oxidoreductase NPH3 C FO3OOO.4 NPH3 family C FOOS64.1 3 PB1 domain MIP C FOO230.9 Major intrinsic protein F-box C FOO646.2 1 F-box domain LRR 2 C FO7723.2 Leucine Rich Repeat F-box C FOO646.2 1 F-box domain MIP C FOO230.9 Major intrinsic protein Methyltransf 6 C FO3737.5 Demethylmenaquinone methyltransferase Zf C3HC4 C FOOO97. 3 Zinc finger, C3HC4 type (RING finger) RRM 1 C FOOO76. 1 RNA recognition motif. (a.k.a. RRM, RBD, or RNP domain) Zf CCHC FOOO98. 2 7.9 Zinc knuckle Zf CCHC FOOO98. 2 7.9 Zinc knuckle NPH3 FO3OOO.4 25 NPH3 family p450 FOOO67. 1 -105 Cytochrome P450 p450 FOOO67. 1 -105 Cytochrome P450 DPBB 1. FO3330.7 5.3 Rare lipoprotein A (RlpA)-like double-psi beta-barrel Pollen allerg 1 FO1357. Pollen allergen p450 FOOO67. Cytochrome P450 p450 FOOO67. Cytochrome P450 CBS FOO571. CBS domain pair CBS FOO571. CBS domain pair Cys Met Meta PP FO1 OS3.9 Cys/Met metabolism PLP-dependent enzyme Beta elim lyase FO1212. Beta-eliminating lyase Aldedh FOO171. Aldehyde dehydrogenase family PK Pyruvate kinase, barrel domain PKC Pyruvate kinase, alphabeta domain PEP-utilizers PEP-utilising enzyme, mobile domain iPGM N FO6415.3 BPG-independent PGAMN-terminus (iPGM N) Metalloenzyme FO1676.7 Metalloenzyme Superfamily Molybdop Fe4S4 FO4879.5 Molybdopterin oxidoreductase Fe4S4 domain Molybdopterin FOO384.1 1 Molybdopterin oxidoreductase Molydop binding FO1568.1 O Molydopterin dinucleotide binding domain Aminotran 3 FOO2O2.1 O Aminotransferase class-III HMA FOO403.1 4 Heavy-metal-associated domain DAO FO1266.1 2 FAD dependent oxidoreductase Pyr redox 2 FO7992.3 Pyridine nucleotide-disulphide oxidoreductase Pyr redox FOOO70.1 7 Pyridine nucleotide-disulphide oxidoreductase Pyr redox dim FO2852.1 2 Pyridine nucleotide-disulphide oxidoreductase, dimerisation domain Aminotran 3 FOO2O2.1 O Aminotransferase class-III ADH N FO8240.2 Alcohol dehydrogenase GroES-like domain ADH zinc N FOO107.1 6 Zinc-binding dehydrogenase Hist deacetyl FOO850.9 Histone deacetylase domain ZfA20 FO1754.6 A20-like zinc finger ZfAN1 FO1428.6 AN1-like Zinc finger U-box FO4564.5 U-box domain Arm FOOS14.1 1 Armadillofbeta-catenin-like repeat Arm FOOS14.1 1 Armadillofbeta-catenin-like repeat Arm FOOS14.1 1 Armadillofbeta-catenin-like repeat Arm FOOS14.1 1 Armadillofbeta-catenin-like repeat Arm FOOS14.1 1 Armadillofbeta-catenin-like repeat PDT FOO800.8 Prephenate dehydratase ACT FO1842.1 3 ACT domain PP2C FOO481.1 1 Protein phosphatase 2C Aminotran 3 FOO2O2.1 O Aminotransferase class-III DUF568 FO4526.3 Protein of unknown function (DUF568) PHD FOO628.1 7 PHD-finger TBC FOOS66.8 TBC domain DUF786 FOS646.3 -31.3 Protein of unknown function (DUF786) PLAC8 FO4749.6 PLAC8 family IF2 N FO4760.6 Translation initiation factor IF-2, N-terminal region US 2015/O 1841 89 A1 Jul. 2, 2015 70

TABLE 17-continued Pfam domain accession gathering l8le number cutoff domain description GTP EFTU PFOOOO9.15 8 Elongation factor Tu GTP binding domain Ras PFOOO71.11 -69.9 Ras family GTP EFTU D2 PFO314414 25 Elongation factor Tu domain 2 PC4 PFO2229.5 4 Transcriptional Coactivator p15 (PC4) DUF588 PFO4535.2 25 Domain of unknown function (DUF588) LisH PFO8513.1 20.7 LisH CDI PFO2234.8 17 Cyclin-dependent kinase inhibitor Glyco transf 8 PFO1501.9 -43.2 Glycosyltransferase family 8 Pkinase PFOOO69.14 -70.8 Protein kinase domain efhand PFOOO36.20 17.5 EF hand efhand PFOOO36.20 17.5 EF hand efhand PFOOO36.20 17.5 EF hand efhand PFOOO36.20 17.5 EF hand WWE PFO2825.9 25 WWE domain FAE1 CUT1 RppA PF08392 -192.7 FAE1/Type III polyketide synthase-like protein Chal sti Synt C PFO2797.5 -6.1 Chalcone and stilbene synthases, C-terminal domain ACP syn III C PFO8541 -24.4 3-Oxoacyl-[acyl-carrier-protein (ACP) synthase III C terminal Hrf1 PFO3878.5 -81.2 Hrf1 family Yip1 PFO4893.6 -6.4 Yip1 domain Pkinase PFOOO69.14 -70.8 Protein kinase domain Pkinase Tyr PFO7714.5 65 Protein tyrosine kinase Pkinase PFOOO69.14 -70.8 Protein kinase domain ADH N PFO8240.2 -14.5 Alcohol dehydrogenase GroES-like domain ADH zinc N PFOO107.16 23.8 Zinc-binding dehydrogenase Miro PFO8477. 28 Miro-like protein Ras PFOOO71.11 -69.9 Ras family Miro PFO8477. 28 Miro-like protein Ras PFOOO71.11 -69.9 Ras family Sugar tr PFOOO83.13 -85 Sugar (and other) transporter MFS 1 PFO7690.5 23.5 Major Facilitator Superfamily RuBisCO large N. PFO2788.5 25 Ribulose bisphosphate carboxylase large chain, N-terminal domain RuBisCO large PFOOO16.9 -76 Ribulose bisphosphate carboxylase large chain, catalytic domain Flavodoxin 1 PFOO258.14 6.3 Flavodoxin FAD binding 1 PFOO667.9 -79 FAD binding domain NAD binding 1 PFOO175.10 -3.9 Oxidoreductase NAD-binding domain Lactamase B PFOO753.16 24.6 Metallo-beta-lactamase Superfamily PGI PFOO342.8 -168.9 Phosphoglucose isomerase Peptidase C12 PFO1088.10 -91.4 Ubiquitin carboxyl-terminal hydrolase, amily 1 Dicty CAR PFOS462.2 -39.7 Slime mold cyclic AMP receptor Aldedh PFOO171.11 -209.3 Aldehyde dehydrogenase family PfkB PFOO294.13 -67.8 pfkB family carbohydrate kinase Brix PFO4427.7 11.4 Brix domain MGS PFO2142.11 3 MGS-like domain AICARFT IMPCHas PFO1808.8 -98 AICARFT/IMPCHase bienzyme MFAP1 C PFO6991.1 25 Micro-fibrillar-associated protein 1 C-terminus Nicastrin PFOS450.4 -85.8 Nicastrin Ribophorin I PFO4597.4 -217 Ribophorin I DUF662 PFO4949.2 25 Family of unknown function (DUF662) ARD PFO3O79.4 25 ARD/ARD' family Cupin 2 PFO7883.1 16.6 Cupin domain HATPase c PFO2S18.14 22.4 Histidine kinase-, DNA gyrase B-, and HSP90-like ATPase Pkinase PFOOO69.14 -70.8 Protein kinase domain Pkinase Tyr PFO7714.5 65 Protein tyrosine kinase Pkinase PFOOO69.14 -70.8 Protein kinase domain

Example 5A DNA for the identified homologous genes, are cloned and amplified by PCR prior to insertion into the insertion site the 0172. This example illustrates the construction of plas- base vector. mids for transferring recombinant DNA into the nucleus of a 0173 Elements of an exemplary common expression vec plant cell which can be regenerated into a transgenic crop tor, pMON82060 are illustrated in Table 18. The exemplary plant of this invention. Primers for PCR amplification of base vector which is especially useful for corn transformation protein coding nucleotides of recombinant DNA are designed is illustrated in FIG. 2 and assembled using technology at or near the start and stop codons of the coding sequence, in known in the art. The DNA of interest are inserted in a expres order to eliminate most of the 5' and 3' untranslated regions. sion vector at the insertion site between the intron 1 of rice act DNA of interest, i.e. each DNA identified in Table 1 and the 1 gene and the termination sequence of Pin gene. US 2015/O 1841 89 A1 Jul. 2, 2015 71

TABLE 18 pMON82060

Coordinates of SEQID function l8le annotation NO:33,636 Agro B-AGRtu.right border Agro right border sequence, essential for 5235-55.91 transformation transfer of T-DNA. Gene of P-OS.Act1 Promoter from the rice actin gene act1. S609-7009 interest plant L-Os.Act1 Leader (first exon) from the rice actin 1 expression gene. caSSette I-Os.Act1 First intron and flanking UTR exon sequences from the rice actin 1 gene insertion site T-St.Pisa. The 3' non-translated region of the 7084-8026 potato proteinase inhibitor II gene which functions to direct polyadenylation of the mRNA Plant P-CaMV.35S CaMV 35S promoter 8075-8398 selectable L-CaMV.35S SUTR from the 3SS RNA of CaMV marker CR-Ec.nptII-Tn5 nptII selectable marker that confers 8432-9226 expression resistance to neomycin and kanamycin caSSette T-AGRtu.nos A 3' non-translated region of the 9255-9507 nopaline synthase gene of Agrobacterium tumefaciens Tiplasmid which functions to direct polyadenylation of the mRNA. Agro B-AGRtu.left border Agro left border sequence, essential for 39-48O transformation transfer of T-DNA. Maintenance OR-Ec.oriV-RK2 The vegetative origin of replication from S67-963 in E. coi plasmid RK2. CR-Ec.rop Coding region for repressor of primer 2472-2663 from the ColE1 plasmid. Expression of this gene product interferes with primer binding at the origin of replication, keeping plasmid copy number low. OR-Ec.ori-CoIE1 The minimal origin of replication from 3091-3679 the E. coli plasmid ColE1. P-Ec.aadA-SPC/STR promoter for TnT adenylyltransferase 421O-4251 (AAD(3")) CR-Ec.aadA- Coding region for TnT. 42S2-SO40 SPCfSTR adenylyltransferase (AAD(3")) conferring spectinomycin and streptomycin resistance. T-Ec.aadA-SPC/STR 3' UTR from the TnT adenylyltransferase 5041-5098 (AAD(3")) gene of E. coli.

0.174 Plasmids for use in transformation of soybean are and the DNA for the identified homologous genes, are cloned also prepared. Elements of an exemplary common expression and amplified by PCR prior to insertion into the insertion site vector plasmid pMON82053 are shown in Table 19 below. This exemplary soybean transformation base vector illus the base vector at the insertion site between the enhanced 35S trated in FIG.3 was assembled using the technology known in CaMV promoter and the termination sequence of cotton E6 the art. DNA of interest, i.e. each DNA identified in Table 1 gene. TABLE 19 pMON82053 Coordinates of SEQ function l8le annotation ID NO:33637 Agro B-AGRtu.left border Agro left border 6144-6585 transforamtion sequence, essential for transfer of T-DNA. Plant P-At-Act, Promoter from the 6624-7861 selectable arabidopsis actin 7 gene marker L-At.-Act 7 5' UTR of Arabidopsis expression Act7 gene caSSette I-At.-Act 7 Intron from the Arabidopsis actin7 gene TS-Alt.ShkC-CTP2 Transit peptide region of 7864-8091 Arabidopsis EPSPS US 2015/O 1841 89 A1 Jul. 2, 2015 72

TABLE 19-continued pMON82053 Coordinates of SEQ function l8le annotation ID NO:33637 CR-AGRtu.aroA Synthetic CP4 coding 8092-9459 CP4.nno At region with dicot preferred codon usage. T-AGRtu.nos A 3' non-translated region 946.6-9718 of the nopaline synthase gene of Agrobacterium tumefaciens Tiplasmid which functions to direct polyadenylation of the mRNA. Gene of Promoter for 35S RNA 1-613 interest from CaMV containing a expression duplication of the -90 to caSSette -350 region. insertion site TGb.E6-3b 3' untranslated region 688-1OO2 rom the fiber protein E6 gene of sea-island cotton; Agro B-AGRtu.right border Agro right border 1033-1389 transformation sequence, essential for transfer of T-DNA. Maintenance OR-Ec.oriV-RK2 The vegetative origin of S661-6057 in E. coi replication from plasmid RK2. CR-Ec.rop Coding region for 3961-4152 repressor of primer from he ColE1 plasmid. Expression of this gene product interferes with primer binding at the origin of replication, keeping plasmid copy number low. OR-Ec.ori-CoIE1 The minimal origin of 2945-3533 replication from the E. coli plasmid ColE1. P-Ec.aadA-SPCASTR romoter for Tnt 2373-2414 adenylyltransferase (AAD(3")) CR-Ec.aadA Coding region for TnT. 1584-2372 SPCfSTR adenylyltransferase (AAD(3")) conferring spectinomycin and streptomycin resistance. T-Ec.aadA-SPCSTR 3'UTR from the TnT. 1526-1583 adenylyltransferase (AAD(3")) gene of E. coi.

Example 5 B genic callus to develop. Embryogenic callus is transferred to culture medium containing 100 mg/L paromomycin and Sub 0.175. This example illustrates monocot plant transforma cultured at about two week intervals. Transformants are tion to produce nuclei of this invention in cells of a transgenic recovered 6 to 8 weeks after initiation of selection. plant by transformation of corn callus. Corn plants of a 0176 Plasmid vectors are prepared essentially as readily transformable line are grown in the greenhouse and described in Example 5 for transforming into corn callus each ears harvested when the embryos are 1.5 to 2.0 mm in length. of DNA identified in Table 1 and the corresponding DNA for Ears are surface sterilized by spraying or soaking the ears in the identified homologous genes identified in Table 2, by 80% ethanol, followed by air drying. Immature embryos are Agrobacterium-mediated transformation. isolated from individual kernels on surface sterilized ears. 0177. For Agrobacterium-mediated transformation of Prior to inoculation of maize cells, Agrobacterium cells are corn callus, immature embryos are cultured for approxi grown overnight at room temperature. Immature maize mately 8-21 days after excision to allow callus to develop. embryos are inoculated with Agrobacterium shortly after Callus is then incubated for about 30 minutes at room tem excision, and incubated at room temperature with Agrobac perature with the Agrobacterium suspension, followed by terium for 5-20 minutes. Immature embryos are then co removal of the liquid by aspiration. The callus and Agrobac cultured with Agrobacterium for 1 to 3 days at 23°C. in the terium are co-cultured without selection for 3-6 days fol dark. Co-cultured embryos are transferred to selection media lowed by selection on paromomycin for approximately 6 and cultured for approximately two weeks to allow embryo weeks, with biweekly transfers to fresh media, and paromo US 2015/O 1841 89 A1 Jul. 2, 2015 mycin resistant callus identified as containing the recombi Selection for Enhanced Nitrogen Use Efficiency nant DNA in an expression cassette. 0182 Transgenic corn plants with nuclei of the invention 0.178 Transgenic corn plants are regenerated from trans are planted in fields with three levels of nitrogen (N) fertilizer genic callus resulting from transformation on media to ini being applied, i.e. low level (0 pounds per acre N), medium tiate shoot development in plantlets which are transferred to level (80 pounds per acre N) and high level (180 pounds per potting soil for initial growth in a growth chamber at 26 acre N). Liquid 28% or 32% UAN (Urea, Ammonium Nitro degrees C. followed by a mist bench before transplanting to 5 gen) are used as the NSource and apply by broadcast boom inch pots where plants are grown to maturity. The plants are and incorporate with a field cultivator with rear rolling basket self fertilized and seed is harvested for screening as seed, in the same direction as intended crop rows. Although there is seedlings or progeny R2 plants or hybrids, e.g. for yield trials no Napplied in the low level treatment, the soil should still be in the screens indicated above. Populations of transgenic disturbed in the same fashion as the treated area. Transgenic plants and seeds produced form transgenic plant cells from plants and control plants can be grouped by genotype and each transgenic event are screened as described in Example 7 construct with controls arranged randomly within genotype below to identify the members of the population having the blocks. For improved statistical analysis each type of trans enhanced trait. genic plant can be tested by 3 replications and across 4 loca tions. Nitrogen levels in the fields are analyzed before plant Example 6 ing by collecting sample soil cores from 0-24" and 24 to 48" 0179 This example illustrates dicot plant transformation soil layer. Soil samples are analyzed for nitrate-nitrogen, to produce nuclei of this transgenic in cells of transgenic phosphorus (P), potassium (K), organic matter and pH to plants by transformation of soybean tissue. For Agrobacte provide baseline values. P. K and micronutrients are applied rium-mediated transformation, soybean seeds are germinated based upon Soil test recommendations. overnight and the meristem explants excised. The meristems 0183 Transgenic corn plants prepared in Example 5 and and the explants are placed in a wounding vessel. Soybean which exhibit a 2 to 5% yield increase as compared to control explants and induced Agrobacterium cells from a strain con plants when grown in the high nitrogen field are selected as taining plasmid DNA with the gene of interest cassette and a having nuclei of the invention. Transgenic corn plants which plant selectable marker cassette are mixed no later than 14 have at least the same or higher yield as compared to control hours from the time of initiation of seed germination and plants when grown in the medium nitrogen field are selected wounded using Sonication. Following wounding, explants are as having nuclei of the invention. Transgenic corn plants placed in co-culture for 2-5 days at which point they are having a nucleus with DNA identified in Table 3 as imparting transferred to selection media for 6-8 weeks to allow selection nitrogen use efficiency (LN) and homologous DNA are and growth of transgenic shoots. Trait positive shoots are selected from a nitrogen use efficiency Screen as having a harvested approximately 6-8 weeks post bombardment and nucleus of this invention. placed into selective rooting media for 2-3 weeks. Shoots producing roots are transferred to the greenhouse and potted Selection for Increased Yield in soil. Shoots that remain healthy on selection, but do not produce roots are transferred to non-selective rooting media 0184. Many transgenic plants of this invention exhibit for an additional two weeks. Roots from any shoots that improved yield as compared to a control plant. Improved produce roots off selection are tested for expression of the yield can result from enhanced seed sink potential, i.e. the plant selectable marker before they are transferred to the number and size of endosperm cells or kernels and/or greenhouse and potted in soil. Populations of transgenic enhanced sink strength, i.e. the rate of Starch biosynthesis. plants and seeds produced form transgenic plant cells from Sink potential can be established very early during kernel each transgenic event are screened as described in Example 7 development, as endosperm cell number and size are deter below to identify the members of the population having the mined within the first few days after pollination. enhanced trait. 0185. Much of the increase in cornyield of the past several decades has resulted from an increase in planting density. Example 7 During that period, cornyield has been increasing at a rate of 2.1 bushels/acre?year, but the planting density has increased 0180. This example illustrates identification of nuclei of at a rate of 250 plants/acre?year. A characteristic of modern the invention by Screening derived plants and seeds for an hybrid corn is the ability of these varieties to be planted at enhanced trait identified below. high density. Many studies have shown that a higher than 0181 Many transgenic events which survive to fertile current planting density should result in more biomass pro transgenic plants that produce seeds and progeny plants will duction, but current germplasm does not perform well at these not exhibit an enhanced agronomic trait. Populations of trans higher densities. One approach to increasing yield is to genic seed and plants prepared in Examples 5 and 6 are increase harvest index (HI), the proportion of biomass that is screened to identify those transgenic events providing trans allocated to the kernel compared to total biomass, in high genic plant cells with a nucleus having recombinant DNA density plantings. imparting an enhanced trait. Each population is screened for 0186 Effective yield selection of enhanced yielding trans enhanced nitrogen use efficiency, increased yield, enhanced genic corn events uses hybrid progeny of the transgenic event water use efficiency, enhanced tolerance to cold and heat, over multiple locations with plants grown under optimal pro increased level of oil and protein in seed using assays duction management practices, and maximum pest control. A described below. Plant cell nuclei having recombinant DNA useful target for improved yield is a 5% to 10% increase in with each of the genes identified in Table 1 and the identified yield as compared to yield produced by plants grown from homologs are identified in plants and seeds with at least one of seed for a control plant. Selection methods may be applied in the enhanced traits. multiple and diverse geographic locations, for example up to US 2015/O 1841 89 A1 Jul. 2, 2015 74

16 or more locations, over one or more planting seasons, for 0190. Transgenic corn plants and soybean plants prepared example at least two planting seasons to statistically distin in Examples 5 and 6 are screened for water use efficiency. guish yield improvement from natural environmental effects. Transgenic plants having at least a 1% increase in GRG and It is to plant multiple transgenic plants, positive and negative RWC as compared to control plants are identified as having control plants, and pollinator plants in Standard plots, for enhanced water used efficiency and are selected as having a example 2 row plots, 20 feet long by 5 feet wide with 30 nucleus of this invention. Transgenic corn and Soybean plants inches distance between rows and a 3 foot alley between having in their nucleus DNA identified in Table 3 as imparting ranges. Transgenic events can be grouped by recombinant drought tolerance improvement (DS) and homologous DNA DNA constructs with groups randomly placed in the field. A are identified as showing increased water use efficiency as pollinator plot of a high quality corn line is planted for every compared to control plants and are selected as having a two plots to allow open pollination when using male sterile nucleus of this invention. transgenic events. A useful planting density is about 30,000 plants/acre. High planting density is greater than 30,000 Selection for Growth Under Cold Stress plants/acre, preferably about 40,000 plants/acre, more pref 0191 Cold germination assay Three sets of seeds are erably about 42,000 plants/acre, most preferably about used for the assay. The first set consists of positive transgenic 45,000 plants/acre. events (F1 hybrid) where the genes of the present invention 0187. Each of the transgenic corn plants and soybean are expressed in the seed. The second seed set is nontrans plants with a nucleus of the invention prepared in Examples 5 genic, wild-type negative control made from the same geno and 6 are screened for yield enhancement. At least one event type as the transgenic events. The third set consisted of two from each of the corn and soybean plants is selected as having cold tolerant and one cold sensitive commercial checklines of at least between 3 and 5% increase in yield as compared to a corn. All seeds are treated with a fungicide “Captan’ (MAE control plant as having a nucleus of this invention. STROR 80DF Fungicide, Arvesta Corporation, San Fran cisco, Calif., USA). 0.43 mL Captain is applied per 45 g of Selection for Enhanced Water Use Efficiency (WUE) corn seeds by mixing it well and drying the fungicide prior to the experiment. 0188 The following is a high-throughput method for 0.192 Corn kernels are placed embryo side down on blot screening for water use efficiency in a greenhouse to identify ter paper within an individual cell (8.9x8.9 cm) of a germi the transgenic corn plants with a nucleus of this invention. nation tray (54x36 cm). Ten seeds from an event are placed This selection process imposes 3 drought/re-water cycles on into one cell of the germination tray. Each tray can hold 21 plants over a total period of 15 days after an initial stress free transgenic events and 3 replicates of wildtype (LH244SDms+ growth period of 11 days. Each cycle consists of 5 days, with LH59), which is randomized in a complete block design. For no water being applied for the first four days and a water every event there are five replications (five trays). The trays quenching on the 5th day of the cycle. The primary pheno are placed at 9.7 C for 24 days (no light) in a Convrion(R) types analyzed by the selection method are the changes in growth chamber (Conviron Model PGV36, Controlled Envi plant growth rate as determined by height and biomass during ronments, Winnipeg, Canada). Two hundred and fifty millil a vegetative drought treatment. The hydration status of the ters of deionized water are added to each germination tray. shoot tissues following the drought is also measured. The Germination counts are taken 10th, 11th, 12th, 13th, 14th, plant height are measured at three time points. The first is 17th, 19th, 21st, and 24th day after start date of the experi takenjust prior to the onset drought when the plant is 11 days ment. Seeds are considered germinated if the emerged radicle old, which is the shoot initial height (SIH). The plant height is size is 1 cm. From the germination counts germination index also measured halfway throughout the drought/re-water regi is calculated. men, on day 18 after planting, to give rise to the shoot mid 0193 The germination index is calculated as per: drought height (SMH). Upon the completion of the final drought cycle on day 26 afterplanting, the shoot portion of the Germination index=(XCIT-1-n7*IP-P)))/T plantis harvested and measured for a final height, which is the where T is the total number of days for which the germination shoot wilt height (SWH) and also measured for shoot wilted assay is performed. The number of days after planting is biomass (SWM). The shoot is placed in water at 40 degree defined by n. “i' indicated the number of times the germina Celsius in the dark. Three days later, the shoot is weighted to tion had been counted, including the current day. P is the give rise to the shoot turgid weight (STM). After drying in an percentage of seeds germinated during any given rating. Sta oven for four days, the shoots are weighted for shoot dry tistical differences are calculated between transgenic events biomass (SDM). The shoot average height (SAH) is the mean and wildtype control. After statistical analysis, the events that plant height across the 3 height measurements. The procedure show a statistical significance at the p level of less than 0.1 described above may be adjusted for +/-one day for each relative to wild-type controls will advance to a secondary cold step given the situation. selection. The secondary cold Screen is conducted in the same 0189 To correct for slight differences between plants, a manner of the primary selection only increasing the number size corrected growth value is derived from SIH and SWH. of repetitions to ten. Statistical analysis of the data from the This is the Relative Growth Rate (RGR). Relative Growth secondary selection is conducted to identify the events that Rate (RGR) is calculated for each shoot using the formula show a statistical significance at the p level of less than 0.05 RGR 96=(SWH-SIH)/((SWH+SIH)/2)x100. Relative relative to wild-type controls. water content (RWC) is a measurement of how much (%) of 0194 Transgenic corn plants and soybean plants prepared the plant was water at harvest. Water Content (RWC) is cal in Examples 5 and 6 are screened for water use efficiency. culated for each shoot using the formula RWC 96 (SWM Transgenic plants having at least a 5% increase in germina SDM)/(STM-SDM)x100. Fully watered corn plants of this tion index as compared to control plants are identified as age run around 98% RWC. having enhanced cold stress tolerance and are selected as US 2015/O 1841 89 A1 Jul. 2, 2015

having a nucleus of this invention. Transgenic corn and Soy TABLE 21-continued bean plants having in their nucleus DNA identified in Table 3 Corn-moisture 5-15%, oil 5-20%, as imparting cold tolerance improvement (CK or CS) and protein 5-30%, starch 50-75%, and homologous DNA are identified as showing increased cold density 1.0-1.3%. stress tolerance as compared to control plants and are selected Soybean—moisture 5-15%, oil as having a nucleus of this invention. 15-25%, and protein 35-50%. Screens for Transgenic Plant Seeds with Increased Protein and/or Oil Levels Transgenic corn plants and soybean plants prepared in 0.195 The following is a high-throughput selection Examples 5 and 6 are screened for increased protein and oil in method for identifying plant seeds with improvement in seed seed. Transgenic inbred corn and Soybean plants having an composition using the Infratec R1200 series Grain Analyzer, increase of at least 1 percentage point in the total percent seed which is a near-infrared transmittance spectrometer used to protein or at least 0.3 percentage point in total seed oil and determine the composition of a bulk seed sample. Near infra transgenic hybrid cornplants having an increase of at least 0.4 red analysis is a non-destructive, high-throughput method percentage point in the total percent seed protein as compared that can analyze multiple traits in a single sample scan. An to control plants are identified as having enhanced seed pro NIR calibration for the analytes of interest is used to predict tein or enhanced seed oil and are selected as having a nucleus the values of an unknown sample. The NIR spectrum is of this invention. obtained for the sample and compared to the calibration using Example 8 a complex chemometric Software package that provides a predicted values as well as information on how well the 0197) This example illustrates monocot and dicot plant sample fits in the calibration. transformation to produce nuclei of this invention in cells of a transgenic plant by transformation where the recombinant (0196) Infratec(R) Model 1221, 1225, or 1227 analyzer with DNA Suppresses the expression of an endogenous protein transport module by Foss North America is used with cuvette, identified by Pfam, Histone, WD40, NPH3, FHA, PB1, item #1000-4033, Foss North America or for small samples ADH zinc N, NAPRTase, ADK lid, p450, B56, DUF231, with small cell cuvette, Foss standard cuvette modified by C2, DUF568, WD40, F-box, Pkinase, or Terpene synth. Leon Girard Co. Corn and soy check samples of varying Corn callus and soybean tissue are transformed as describe in composition maintained in check cell cuvettes are Supplied Examples 5 and 6 using recombinant DNA in the nucleus by Leon Girard Co. NIT collection software is provided by with DNA that transcribes to RNA that forms double Maximum Consulting Inc. Calculations are performed auto stranded RNA targeted to an endogenous gene with DNA matically by the software. Seed samples are received in pack encoding the protein. The genes for which the double ets or containers with barcode labels from the customer. The Stranded RNAS are targeted are the native gene in corn and seed is poured into the cuvettes and analyzed as received. Soybean that are homolog of the genes encoding the protein with an amino acid sequence of SEQID NO: 426, 428, 429, TABLE 21 430, 524,525,541, 601, 602, 650, 651, 654, 655, 657, 660, Typical sample(s): Whole grain corn and soybean seeds 694,698, 772,801. Analytical time to run method: Less than 0.75 min per sample 0198 Populations of transgenic corn plants and soybean Total elapsed time per run: 1.5 minute per sample plants prepared in Examples 5 and 6 with DNA for suppress Typical and minimum sample Corn typical: 50 cc, minimum 30 cc size: Soybean typical: 50 cc, minimum 5 cc ing a gene identified in Table 3 as providing an enhanced trait Typical analytical range: Determined in part by the specific by gene Suppress10n are screened to identify an event from calibration. those plants with a nucleus of the invention by selecting the trait identified in this specification.

SEQUENCE LISTING The patent application contains a lengthy “Sequence Listing section. A copy of the “Sequence Listing is available in electronic form from the USPTO web site (http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20150184189A1). An electronic copy of the “Sequence Listing will also be available from the USPTO upon request and payment of the fee set forth in 37 CFR1.19(b)(3).

1. (canceled) group of Pfam names consisting of Mito can, 6PGD, UBX. iPGM N, WD40, Fer4, Enolase C, DUF1639, PBP, PLAC8, 2. A plant cell nucleus with stably integrated, recombinant Acyl-CoA dh 1, Isoamylase N. Acyl-CoA dh 2, PC4. DNA a) comprising a promoter that is functional in plant cells Sugar tr, UCH, Enolase N, HATPase c, PRA-PH, Pkinase, and that is operably linked to DNA from a plant, bacteria or SBP56, PEP-utilizers, SIS, PCI, DUF1644, Terpene synth, yeast that encodes a protein having at least one domain of Acyl-CoA dh M. Acyl-CoA dh N. Glutaminase, Lectin amino acids in a sequence that exceeds the Pfam gathering legB, Dehydrin, Mat, Ank, 2-Hacid dh C, Chal sti synt cutoff for amino acid sequence alignment with a protein C, DUF1070, ATP-grasp 2, Arginase, HABP4. PAI-RBP1, domain family identified by a Pfam name selected from the ABC2 membrane, DUF1723, Glyco hydro 1, MFS 1, US 2015/O 1841 89 A1 Jul. 2, 2015 76

ARD, PDT, HMA, Pro isomerase, Ferric reduct, PRA-CH, tified by Pfam name in the group of Pfam names con Aa trans, ACT, LisH, PGM PMM II, Spermine synth, Zf sisting of Histone, WD40, NPH3, FHA, PB1, ADH MYND, LRRNT 2, Ribul P 3epim, PGM PMM IV, zinc N, NAPRTase, ADK lid, p450, B56, DUF231, C2, NPH3, DapB C, TPR 1, TPR 2, FAE1 CUT1 Rpp.A, DUF568, WD40, F-box, Pkinase, and Terpene synth K trans, F-box, Cyclin C, ADK, NUDIX, NIR SIR, PEP wherein the Pfam gathering cutoff for said protein CK ATP, La, Dapb N, MtN3 silv, FMO-like, TIM, FKBP domain families is stated in Table 16; wherein said plant C, PMEI, Peptidase C12, Cyclin N, DUF568, Methyl cell nucleus is selected by Screening a population of transf 11, Methyltransf 12, DUF1677, DnaJ C, BRAP2, transgenic plants with said recombinant DNA and have IF2 N, Carboxyl trans, mTERF, Glyoxalase, TMEM14, Mlo, the level of said endogenous protein Suppressed for an Beta elim lyase, Pyr redox dim, Glyco transf 8, Nicas enhanced trait as compared to control plants that do not trin, Flavodoxin 1, Epimerase, PTPA, Lipase 3, Pyr re have said recombinant DNA; and wherein said enhanced dox 2, GSHPx, ELM2, PGI, Aminotran 1 2, ABC tran, trait is selected from the group of enhanced traits con GRP PGK, Oleosin, Sulfotransfer 1, EXS, DUF1325, sisting of enhanced water use efficiency, enhanced cold AMP-binding, Arm, NTP transferase, LSM, Metalloen tolerance, enhanced heat tolerance, enhanced resistance Zyme, Molybdop Fe4S4, MFAP1 C, Aminotran 3, PHD, to Salt exposure, enhanced shade tolerance, increased B56, DUF588, PSI PsaF, Zf-CCCH, HEAT, PALP, FH2, yield, enhanced nitrogen use efficiency, enhanced seed SapR 1, Ammonium transp, MannoseP isomer, NOP5NT, protein and enhanced seed oil; or SapR 2. Pyr redox, Pollen allerg 1, Asp, DUF662, FHA, c) comprising a promoter that is functional in plant cells YeF N, COX5C, GTP EFTU D2, Ion trans 2, PK, and that is operably linked to DNA from a plant, bacteria DUF231, FAD binding 1, Hrf1, FAD binding 4, FAD or yeast that encodes a protein having at least one binding 6, FAD binding 8, CBS, Smr, aPHC, DUF241, domain of amino acids in a sequence that exceeds the Brix, Ras, Acetyltransf 1, NAF, SPX. Na Ca ex, C2, p450, Pfam gathering cutoff of 16.9 for amino acid sequence PP2C, Histone, 2-Hacid dh, SBF, CCT, BCNT, PKC. Miro, alignment with a protein domain family identified by CH, PfkB, ACP syn III C, Sterol desat, ADH zinc N, CS, Pfam name Cyclin C; wherein said plant cell nucleus is Cys Met Meta PP. Lactamase B. Bromodomain, CDI. Selected from by Screening a population of transgenic Linker histone, DAO, Dicty CAR, Aldo ket red, Zf-AN1, plants that have said recombinant DNA in its nuclei and Methyltransf 6, DUF1005, LEA 2, NIR SIR ferr, express said protein for an enhanced trait as compared to DUF260, Oxidored FMN, DUF26, Lectin C, Pec lyase C, control plants that do not have said recombinant DNA in Nop, TB2_DP1 HVA22, ADH, N, YGGT, NAPRTase, their nuclei; and wherein said enhanced trait is selected NAD binding 1, DUF914, PGM PMM I, NAD binding from group of enhanced traits consisting of enhanced 2, AICARFT IMPCHas, Auxin inducible, NAD binding water use efficiency, enhanced cold tolerance, increased 6, Anti-silence, RuBisCO large, Response reg, FeThRed yield, enhanced nitrogen use efficiency, enhanced seed A, Di19, SNARE, PGM PMM III, Molydop binding, protein and enhanced seed oil. efhand, Zf-CCHC, GTP EFTU, ARID, adh short, Fibril 3. The plant cell nucleus of claim 2 wherein said protein has larin, RuBisCO large N, WWE, AA permease, PABP an amino acid sequence with at least 90% identity to a con OMPdecase, RRM 1, U-box, OPT, TBC, MGS, DUF786, sensus amino acid sequence in the group of consensus amino 3Beta HSD, Zf UBP. ZfA20, DPBB 1. GDPD, PI-PLC-X, acid sequences consisting of the consensus amino acid SEP, PI-PLC-Y, NOSIC, Glycolytic, SET, ADK lid, Alpha sequence constructed for SEQID NO: amylase, EB1, PGAM, Abhydrolase 1, Glyco hydro 14, 205 and homologs thereof listed in Table 2 through the Lung 7-TM R, Abhydrolase 3, TCTP, GATase 2, Gln consensus amino acid sequence constructed for SEQID synt C, 20G-FeII Oxy. Pribosyltran, MIF, CoA trans, NO:408 and homologs thereof listed in Table 2. RCC1, Pkinase Tyr, MIP. DnaJ, HSCB C, Trehalose PPase, 4. The plant cell nucleus of claim 2 wherein said protein is LRR 1, Cupin 2, LRR 2, Glyco hydro 28, Yip 1, Trp selected from the group of proteins identified in Table 1. syntA, Sedlin N, SGS, Aldedh, CK II beta, Zf-C3HC4, 5. (canceled) GIDA, PB1, IMPDH, Carb kinase, PurA, Molybdopterin, 6. The plant cell nucleus of claim 2 further comprising Nodulin-like, Tim17, Xan ur permease. Hist deacetyl, DNA expressing a protein that provides tolerance from expo RNA pol Rpb8, Agenet, Myb DNA-binding, Glyoxal ox sure to an herbicide applied at levels that are lethal to a wild id N. Ribophorin I and FAE 3-kCoA syn1 wherein said type of said plant cell. Pfam gathering cutoff for said protein domain families are 7. The plant cell nucleus of claim 6 wherein the agent of stated in Table 16; wherein said plant cell nucleus is selected said herbicide is a glyphosate, dicamba, or glufosinate com by Screening a population of transgenic plants that have said pound. recombinant DNA and express said protein for an enhanced 8. A transgenic plant cell or plant comprising a plurality of trait as compared to control plants that do not have said plant cells with a plant cell nucleus of claim 2. recombinant DNA in their nuclei; and wherein said enhanced 9. The transgenic plant cell or plant of claim 8 which is trait is selected from group of enhanced traits consisting of homozygous for said recombinant DNA. enhanced water use efficiency, enhanced cold tolerance, 10. A transgenic seed comprising a plurality of plant cells enhanced heat tolerance, enhanced resistance to salt expo with a plant cell nucleus of claim 2. Sure, enhanced shade tolerance, increased yield, enhanced 11. The transgenic seed of claim 10 from a corn, soybean, nitrogen use efficiency, enhanced seed protein and enhanced cotton, canola, alfalfa, wheat or rice plant. seed oil; 12. The transgenic corn seed of claim 11 wherein said seed b) to Suppress the level of an endogenous protein having at can produce corn plants that are resistant to disease from the least one domain of amino acids in a sequence that Mal de Rio Cuarto virus or the Puccina sorghi fungus or both. exceeds the Pfam gathering cutoff for amino acid 13. A transgenic pollen grain comprising a haploid deriva sequence alignment with a protein domain family iden tive of a plant cell nucleus of claim 2. US 2015/O 1841 89 A1 Jul. 2, 2015 77

14. A method for manufacturing non-natural, transgenic (b) producing corn plants from said hybrid corn seed, seed that can be used to produce a crop of transgenic plants wherein a fraction of the plants produced from said with an enhanced trait resulting from expression of stably hybrid corn seed is homozygous for said recombinant integrated, recombinant DNA in a nucleus of claim 2, said DNA, a fraction of the plants produced from said hybrid method for manufacturing said transgenic seed comprising: corn seed is hemizygous for said recombinant DNA, and (a) screening a population of plants for said enhanced trait a fraction of the plants produced from said hybrid corn and said recombinant DNA, wherein individual plants in seed has none of said recombinant DNA; said population can exhibit said trait at a level less than, (c) selecting corn plants which are homozygous and hem essentially the same as or greater than the level that said izygous for said recombinant DNA by treating with an trait is exhibited in control plants which do not express herbicide; the recombinant DNA, (d) collecting seed from herbicide-treated-Surviving corn (b) selecting from said population one or more plants that plants and planting said seed to produce further progeny exhibit said trait at a level greater than the level that said corn plants; trait is exhibited in control plants, (c) verifying that said recombinant DNA is stably inte (e) repeating steps (c) and (d) at least once to produce an grated in said selected plants, inbred corn line; (d) analyzing tissue of said selected plant to determine the (f) crossing said inbred corn line with a second corn line to production or Suppression of a protein having the func produce hybrid seed. tion of a protein encoded by nucleotides having a 20. A method of selecting a plant comprising cells with a sequence selected from the group consisting of one of plant cell nucleus of claim 2 wherein an immunoreactive SEQID NO:205-408; and antibody is used to detect the presence of said protein in seed (e) collecting seed from said selected plant. or plant tissue. 15. The method of claim 14 wherein plants in said popu 21. Anti-counterfeit milled seed having, as an indication of lation further comprise DNA expressing a protein that pro origin, a plant cell nucleus of claim 2. vides tolerance to exposure to an herbicide applied at levels 22. A method of growing a corn, cotton, Soybean, canola, that are lethal to wild type plant cells, and wherein said alfalfa or wheat, crop without irrigation water comprising selecting is effected by treating said population with said planting seed having the plant cells with a plant cell nucleus herbicide. of claim 2 which are selected for enhanced water use effi 16. The method of claim 15 wherein said herbicide com ciency. prises a glyphosate, dicamba, or glufosinate compound. 23. The method of claim 22 comprising providing up to 300 17. The method of claim 14 wherein said selecting is millimeters of ground water during the production of said effected by identifying plants with said enhanced trait. crop. 18. The method of claim 15 wherein said seed is corn, Soybean, cotton, alfalfa, wheat or rice seed. 24. A method of growing a corn, cotton, Soybean, canola, 19. A method of producing hybrid corn seed comprising: alfalfa or wheat crop without added nitrogen fertilizer com (a) acquiring hybrid corn seed from a herbicide tolerant prising planting seed having plant cells of claim 2 which are corn plant which also has stably-integrated, recombi selected for enhanced nitrogen use efficiency. nant DNA in a nucleus of claim 2: k k k k k