US007868155B2

(12) United States Patent (10) Patent No.: US 7,868,155 B2 Lu et al. (45) Date of Patent: Jan. 11, 2011

(54) PROMOTER, PROMOTER CONTROL OTHER PUBLICATIONS ELEMENTS, AND COMBINATIONS, AND Kim et al., “A 20 Nucleotide Upstream Element is Essential for the USES THEREOF Nopaline Synthase (nos) Promoter Activity.” Plant Molecular Biol (75) Inventors: Yu-Ping Lu, Camarillo, CA (US); Roger ogy, vol. 24, pp. 105-117, 1994. Donald et al., “Mutation of Either G Box or I Box Sequences Pro Pennell, Malibu, CA (US); Jack foundly Affects Expression From the Arabidopsis rbcS-1A Pro Okamuro, Oak Park, CA (US); Richard moter.” EMBO Journal, vol. 9, No. 6, pp. 1717-1726, 1990. Schneeberger, Carlsbad, CA (US); Dolferus et al., “Differential Interactions of Promoter Elemtns in Yiwen Fang, Los Angeles, CA (US); Stress Responses of the Arabidopsis Adh Gene.” Plant Physiology, Shing Kwok, Woodland Hills, CA (US) vol. 105, pp. 1075-1087, 1994. Database Genbank Online Feb. 14, 2004, "Arabidopsis thaliana (73) Assignee: Ceres, Inc., Thousand Oaks, CA (US) genomic DNA, chromosome 5, P1 clone: MNC 17” retrieved from NCBI accession No. AB016890 BA000015. (*) Notice: Subject to any disclaimer, the term of this Database EMBL Nov. 24, 2004 WiscDsLox 262D09 Arabidopsis patent is extended or adjusted under 35 thaliana T-DNA insertion flanking sequences XP002372669. U.S.C. 154(b) by 574 days. Database EMBL Mar. 21, 2002 Arabadopsis thaliana cDNA clone: RAFL09-89-K19,5-end. XPO02372670. (21) Appl. No.: 11/603,542 Database EMBL Apr. 23, 2002, Arabidopsis thaliana putative photoassimilate-responsive protein PAR (At5g52390) mRNA, com (22) Filed: Nov. 22, 2006 plete cds. XP002372671. Database Geneseq Oct. 18, 2000 Arabadopsis thaliana protein frag (65) Prior Publication Data ment SEQ ID No. 62052, XP002372672. Shahmuradov I A et al., PlantProm: a database of plant promoter US 2008/OO44898A1 Feb. 21, 2008 sequences, Nucleic Acids Research, Oxford University Press, Surrey GB vol. 31, No. 1, 2003, pp. 114-117 XP002993034. (51) Int. Cl. Database EMBLNov. 16, 1998 Arabidopsis thaliana genomic DNA, CI2N 15/82 (2006.01) chromosome 5, TAC clone: K24M7 XP002372668 retrieved from CI2N 15/63 (2006.01) EBI accession No. EM PRO: AB019226 Database accession No. ABO 19226. CI2N IS/II (2006.01) Benfey, et al. Science, (1990), vol. 250, pp. 959-966. AOIH 5/00 (2006.01) Sato, etal, NCBI GenBank Sequence Accession No. AB022216, pp. (52) U.S. Cl...... 536/24.1:536/23.1: 800/278: 1-31 (Dec. 27, 2000). 800/287; 435/320.1 Tyagi, et al. Current Science, (2001), vol. 80. pp. 161-169.

(58) Field of Classification Search ...... None Maniatis, et al. Molecular Cloning: A Laboratory Manual, Cold See application file for complete search history. Spring Harbor Laboratory (1982). GenBank Accession No. NM 112618 (Apr. 20, 2007). (56) References Cited Goto, et al., Genes Genet. Syst., (2002), vol. 77, pp. 89-95. Shen, et al., Plant J. (2002), vol. 29(3), pp. 371-380. U.S. PATENT DOCUMENTS Doerks, et al., TIG, (1998), vol. 14, pp. 248-250. Smith, et al., Nature Biotechnology, (1997), vol. 15, pp. 1222-1223. 5,362,865 A 11/1994 Austin Bork, et al., TIG, (1996), vol. 12, pp. 425-427. 5,424,412 A 6, 1995 Brown Guo, et al., PNAS, (2004), vol. 101, pp. 9205-9210. 5,659,122 A 8, 1997 Austin Keskin, et al., Protein Science, (2004), vol. 13, pp. 1043-1055. 5,754,888 A 5/1998 Yang et al. Thornton, et al., Nature structural Biology, structural genomics 5,764,903. A 6, 1998 Yu supplement, (Nov. 2000). 5,857,208 A 1, 1999 Ofek Wells, Biochemistry, (1990), vol. 29, pp. 8509-8517. 5,974,563 A 10/1999 Beeler, Jr. Miyoshi, et al., Plant J, (2003), vol. 36, pp. 532-540. 6,092,066 A 7, 2000 Ofek Ngo, et al., The Protein Folding Problem and Tertiary Structure 6,448.476 B1 9/2002 Barry Prediction, K. Merz and S. LeGrand (eds.), (1994), pp. 492-495. 2003,0226166 A1 12, 2003 Falco Office Action dated Jul. 28, 2008 in U.S. Appl. No. 11/058,689. 2004/0163144 A1* 8, 2004 Kriz et al...... 800,279 Office Action dated May 7, 2009 in U.S. Appl. No. 1 1/097.589. 2005/004.8556 A1 3, 2005 Heck Office Action dated May 13, 2009 in U.S. Appl. No. 11/058,689. 2005, OO86718 A1 4/2005 Heard 2006, OOO881.6 A1 1/2006 Lu * cited by examiner 2006,004 1952 A1 2/2006 Cook Primary Examiner Michele KJoike 2006, O137034 A1 6/2006 Schneeberger (74) Attorney, Agent, or Firm Birch, Stewart, Kolasch and 2006, O150283 A1 7/2006 Alexandrov Birch, LLP 2006, O150285 A1 7/2006 Nadzan 2006, O168696 A1 7/2006 Feldmann (57) ABSTRACT 2006/0195934 A1 8/2006 Apuya 2006/0195943 A1 8, 2006 Feldmann The present invention is directed to promoter sequences and 2006/0236421 A1 10, 2006 Pennell promoter control elements, polynucleotide constructs com 2007,0006335 A1 1, 2007 Cook prising the promoters and control elements, and methods of 2007,0006345 A1 1/2007 Alexandrov identifying the promoters, control elements, or fragments 2007/O199090 A1 8/2007 Apuya thereof. The invention further relates to the use of the present promoters or promoter control elements to modulate tran FOREIGN PATENT DOCUMENTS script levels. EP 1033405 9, 2000 WO WOO1f9848O 12/2001 10 Claims, 2 Drawing Sheets

U.S. Patent Jan. 11, 2011 Sheet 2 of 2 US 7,868,155 B2

Figure 2

Upstream TATA RNA promoter elements box initation

XYXYXY US 7,868,155 B2 1. 2 PROMOTER, PROMOTER CONTROL (3) a polynucleotide having a nucleotide sequence which ELEMENTS, AND COMBINATIONS, AND hybridizes to those shown in Table 1 under a condition USES THEREOF establishing a Tm-20°C. It is another object of the present invention to provide This non-provisional application claims priority under 35 5 isolated polynucleotides that are promoter control element U.S.C. S 119(e) on U.S. to Application No: 10-981,334, filed sequences. These promoter control element sequences com Nov. 4, 2004, and Application No. 60/518,075 filed on Nov. 6, prise, for example, 2003 and Provisional Application No. 60/527,611 filed on (1) a polynucleotide having a nucleotide sequence accord Dec. 4, 2003, the entire contents of which are hereby incor ing to Table 1 or fragment thereof; porated by reference. 10 (2) a polynucleotide having a nucleotide sequence having at least 80% sequence identity to those shown in Table 1 FIELD OF THE INVENTION or fragment thereof, and (3) a polynucleotide having a nucleotide sequence which The present invention relates to promoters and promoter hybridizes to those shown in Table 1 under a condition control elements that are useful for modulating transcription 15 establishing a Tm-20°C. of a desired polynucleotide. Such promoters and promoter Promoter or promoter control element sequences of the control elements can be included in a polynucleotide con present invention are capable of modulating preferential tran struct, expression cassettes, vectors, or inserted into the chro Scription. mosome or as an exogenous element, to modulate in Vivo and In another embodiment, the present promoter control ele in vitro transcription of a polynucleotide. Host cells, includ- 20 ments are capable of Serving as or fulfilling the function, for ing plant cells, and organisms, such as regenerated plants example, as a core promoter, a TATA box, a polymerase therefrom, with desired traits or characteristics using poly binding site, an initiator site, a transcription binding site, an nucleotides comprising the promoters and promoter control enhancer, an inverted repeat, a locus control region, or a elements of the present invention. scaffold/matrix attachment region. 25 It is yet another object of the present invention to provide a BACKGROUND OF THE INVENTION polynucleotide that includes at least a first and a second promoter control element. The first promoter control element This invention relates to the field of biotechnology and, in is a promoter control element sequence as discussed above, particular, to specific promoter sequences and promoter con and the second promoter control element is heterologous to trol element sequences which are useful for the transcription 30 the first control element. Moreover, the first and second con of polynucleotides in a host cell or transformed host organ trol elements are operably linked. Such promoters may modu 1S late transcript levels preferentially in a tissue or under par One of the primary goals of biotechnology is to obtain ticular conditions. organisms, such as plants, mammals, yeast, and prokaryotes In another embodiment, the present isolated polynucle having particular desired characteristics or traits. Examples 35 otide comprises a promoter or a promoter control element as of these characteristic or traits abound and may include, for described above, wherein the promoter or promoter control example, in plants, virus resistance, insect resistance, herbi element is operably linked to a polynucleotide to be tran cide resistance, enhanced Stability or additional nutritional scribed. value. Recent advances in genetic engineering have enabled In another embodiment of the present vector, the promoter researchers in the field to incorporate polynucleotide 40 and promoter control elements of the instant invention are sequences into host cells to obtain the desired qualities in the operably linked to a heterologous polynucleotide that is a organism of choice. This technology permits one or more regulatory sequence. polynucleotides from a source different than the organism of It is another object of the present invention to provide a host choice to be transcribed by the organism of choice. If desired, cell comprising an isolated polynucleotide or vector as the transcription and/or translation of these new polynucle 45 described above or fragment thereof. Host cells include, for otides can be modulated in the organism to exhibit a desired instance, bacterial, yeast, insect, mammalian, and plant. The characteristic or trait. Alternatively, new patterns of transcrip host cell can comprise a promoter or promoter control ele tion and/or translation of polynucleotides endogenous to the ment exogenous to the genome. Such a promoter can modu organism can be produced. Both approaches can be used at late transcription in cis- and in trans-. 50 In yet another embodiment, the present host cell is a plant the same time. cell capable of regenerating into a plant. It is yet another embodiment of the present invention to SUMMARY OF THE INVENTION provide a plant comprising an isolated polynucleotide or vec tor described above. The present invention is directed to isolated polynucleotide 55 It is another object of the present invention to provide a sequences that comprise promoters and promoter control ele method of modulating transcription in a sample that contains ments from plants, especially Arabidopsis thaliana, Glycine either a cell-free system of transcription or host cell. This max, Oryza sativa, and Zea mays, and other promoters and method comprises providing a polynucleotide or vector promoter control elements functional in plants. according to the present invention as described above, and It is an object of the present invention to provide isolated to contacting the sample of the polynucleotide or vector with polynucleotides that are promoter sequences. These promoter conditions that permit transcription. sequences comprise, for example, In another embodiment of the present method, the poly (1) a polynucleotide having a nucleotide sequence accord nucleotide or vector preferentially modulates ing to Table 1 or fragment thereof; (a) constitutive transcription, (2) a polynucleotide having a nucleotide sequence having 65 (b) stress induced transcription, at least 80% sequence identity to sequences shown in (c) light induced transcription, Table 1 or fragment thereof; and (d) dark induced transcription, US 7,868,155 B2 4 (e) leaf transcription, tions. The column headed"cDNA ID' provides the identifier (f) root transcription, number for the cDNA tracked in the experiment. Using Table (g) stem or shoot transcription, 2, these numbers can be used to correlate the differential (h) silique transcription, expression pattern observed and produced by the endogenous (i) callus transcription, promoter with the isolated promoters of the invention. () flower transcription, The column headed “EXPT REP ID provides an identi (k) immature bud and inflorescence specific transcription, fier number for the particular experiment conducted. The O column “SHORT NAME gives a brief description of the (1) senescing induced transcription experimental conditions or the developmental stage used. The (m) germination transcription. 10 values in the column headed “Differential indicate whether Other and further objects of the present invention will be expression of the cDNA was increased (+) or decreased (-) made clear or become apparent from the following descrip compared to the control. tion. Table 5 BRIEF DESCRIPTION OF THE TABLES 15 Table 5 links the “short name' from Table 4 with a short Table 1 description of the experiment, the parameters and the utility. Table 1 identifies nucleic acid promoter sequences using FIG. 1 the headings “SEQ ID NO” and “construct.” The “SEQ ID FIG. 1 is a schematic representation of the vector pNew NO” is a number that identifies the sequence of the candidate Bina-HAP1-GFP. The definitions of the abbreviations used in promoter used in the experiments, while the “construct” text the vector map are as follows: identifies the construct used to produce a specific plant line. Ori the origin of replication used by an E. coli host 25 RB sequence for the right border of the T-DNA from Table 2 pMOG800 Table 2 consists of the Expression Reports and provides BstXI—restriction enzyme cleavage site used for cloning details for expression driven by each of the nucleic acid HAP1VP16—coding sequence for a fusion protein of the promoter sequences as observed in transgenic plants. The HAP1 and VP16 activation domains results are presented as Summaries of the spatial expression, 30 NOS terminator region from the nopaline synthase gene which provides information as to gross and/or specific HAP1UAS the upstream activating sequence for HAP1 expression in various plant organs and tissues. The observed 5ERGFP the green fluorescent protein gene that has been expression pattern is also presented, which gives details of optimized for localization to the endoplasmic reticulum expression during different generations or different develop OCS2 the terminator sequence from the octopine synthase mental stages within a generation. Additional information is 35 2 gene provided regarding the associated gene, the GenBank refer OCS the terminator sequence from the octopine synthase ence, the Source organism of the promoter, and the vector and gene marker genes used for the construct. The following symbols p28716 (a.k.a 28716 short) promoter used to drive expres are used consistently throughout the Table: sion of the PAT (BAR) gene T1: First generation transformant 40 PAT (BAR)—a marker gene conferring herbicide resistance T2: Second generation transformant LB sequence for the left border of the T-DNA from T3: Third generation transformant pMOG800 (L): low expression level Spec—a marker gene conferring spectinomycin resistance (M): medium expression level TrfA—transcription repression factor gene (H): high expression level 45 RK2-OriV—origin of replication for Agrobacterium FIG. 2 is a schematic representation of a common configu Table 3 ration of the promoter control elements in RNA polymerase II promoters. Table 3 lists the co-ordinates of nucleotides of the promoter that represent optional promoter fragments. The optional pro 50 DETAILED DESCRIPTION OF THE INVENTION moter fragments comprise the 5' UTR and any exon(s) of the endogenous coding region. The optional promoter fragments 1. Definitions may also comprise any exon(s) and the 3' or 5' UTR of the gene residing upstream of the promoter (that is, 5' to the Chimeric: The term "chimeric' is used to describe poly promoter). The optional promoter fragments also include any 55 nucleotides or genes, as defined Supra, or constructs wherein intervening sequences that are introns or sequence occurring at least two of the elements of the polynucleotide or gene or between exons or an exon and the UTR. construct, Such as the promoter and the polynucleotide to be The information in Table 3 can be used to generate either transcribed and/or other regulatory sequences and/or filler reduced promoter sequences or “core” promoters. A reduced sequences and/or complements thereof, are heterologous to promoter sequence is generated when at least one optional 60 each other. promoter fragment is deleted. Deletion of all optional pro Constitutive Promoter: Promoters referred to herein as moter fragments generates a “core” promoter. “constitutive promoters' actively promote transcription under most, but not necessarily all, environmental conditions Table 4 and states of development or cell differentiation. Examples of 65 constitutive promoters include the cauliflower mosaic virus Table 4 presents the results of microarray experiments that (CaMV) 35S transcript initiation region and the 1" or 2 pro track expression of particular cDNAS under specific condi moter derived from T-DNA of Agrobacterium tumefaciens, US 7,868,155 B2 5 6 and other transcription initiation regions from various plant Technology 13:486 (1995)), biolistic methods (Armaleo et genes, such as the maize ubiquitin-1 promoter, known to al., Current Genetics 17:97 1990)), electroporation, in planta those of skill. techniques, and the like. Such a plant containing the exog Core Promoter: This is the minimal stretch of contiguous enous nucleic acid is referred to here as a To for the primary DNA sequence that is sufficient to direct accurate initiation of 5 transgenic plant and T for the first generation. The term transcription by the RNA polymerase II machinery (for “exogenous” as used herein is also intended to encompass review see: Struhl, 1987, Cell 49: 295-297; Smale, 1994. In inserting a naturally found element into a non-naturally found Transcription: Mechanisms and Regulation (eds R. C. Con location. away and J. W. Conaway), pp 63-81/Raven Press, Ltd., New All of the references cited in this section are hereby incor York; Smale, 1997, Biochim. Biophys. Acta 1351: 73-88: 10 porated by reference. Smale et al., 1998, Cold Spring Harb. Symp. Ouant. Biol. 58: Gene: The term “gene, as used in the context of the current 21-31; Smale, 2001, Genes & Dev. 15: 2503-2508; Weis and invention, encompasses all regulatory and coding sequence Reinberg, 1992, FASEB.J. 6: 3300-3309: Burke et al., 1998, contiguously associated with a single hereditary unit with a Cold Spring Harb. Symp. Ouant. Biol 63: 75-82). There are genetic function (see SCHEMATIC 1). Genes can include several sequence motifs, including the TATA box, initiator 15 non-coding sequences that modulate the genetic function that (Inr), TFIIB recognition element (BRE) and downstream core include, but are not limited to, those that specify polyadeny promoter element (DPE), that are commonly found in core lation, transcriptional regulation, DNA conformation, chro promoters, however not all of these elements occur in all matin conformation, extent and position of base methylation promoters and there are no universal core promoter elements and binding sites of proteins that control all of these. Genes (Butler and Kadonaga, 2002, Genes & Dev. 16:2583-2592). encoding proteins are comprised of “exons' (coding All of the references cited in this section are hereby incor sequences), which may be interrupted by “introns' (non porated by reference. coding sequences). In some instances complexes of a plural Domain: Domains are fingerprints or signatures that can be ity of protein or nucleic acids or other molecules, or of any used to characterize protein families and/or parts of proteins. two of the above, may be required for a gene's function. On Such fingerprints or signatures can comprise conserved (1) 25 the other hand a gene's genetic function may require only primary sequence, (2) secondary structure, and/or (3) three RNA expression or protein production, or may only require dimensional conformation. A similar analysis can be applied binding of proteins and/or nucleic acids without associated to polynucleotides. Generally, each domain has been associ expression. In certain cases, genes adjacent to one another ated with either a conserved primary sequence or a sequence may share sequence in Such a way that one gene will overlap motif. Generally these conserved primary sequence motifs 30 the other. A gene can be found within the genome of an have been correlated with specific in vitro and/or in vivo organism, in an artificial chromosome, in a plasmid, in any activities. A domain can be any length, including the entirety other sort of vector, or as a separate isolated entity. of the polynucleotide to be transcribed. Examples of domains Heterologous sequences: “Heterologous sequences' are include, without limitation, AP2, helicase, homeobox, Zinc those that are not operatively linked or are not contiguous to finger, etc. 35 each other in nature. For example, a promoter from corn is Endogenous: The term “endogenous, within the context considered heterologous to an Arabidopsis coding region of the current invention refers to any polynucleotide, sequence. Also, a promoter from a gene encoding a growth polypeptide or protein sequence which is a natural part of a factor from corn is considered heterologous to a sequence cellor organisms regenerated from said cell. In the context of encoding the corn receptor for the growth factor. Regulatory promoter, the term “endogenous coding region' or "endog 40 element sequences, such as UTRS or 3' end termination enous cDNA refers to the coding region that is naturally sequences that do not originate in nature from the same gene operably linked to the promoter. as the coding sequence originates from, are considered het Enhancer/Suppressor: An "enhancer is a DNA regulatory erologous to said coding sequence. Elements operatively element that can increase the steady state level of a transcript, linked in nature and contiguous to each other are not heter usually by increasing the rate of transcription initiation. 45 ologous to each other. Enhancers usually exert their effect regardless of the distance, Homologous: In the current invention, a “homologous' upstream or downstream location, or orientation of the gene or polynucleotide or polypeptide refers to a gene or enhancer relative to the start site of transcription. In contrast, polynucleotide or polypeptide that shares sequence similarity a “Suppressor is a corresponding DNA regulatory element with the gene or polynucleotide or polypeptide of interest. that decreases the steady state level of a transcript, again 50 This similarity may be in only a fragment of the sequence and usually by affecting the rate of transcription initiation. The often represents a functional domain Such as, examples essential activity of enhancer and Suppressor elements is to including without limitation a DNA binding domain or a bind a protein factor(s). Such binding can be assayed, for domain with tyrosine kinase activity. The functional activities example, by methods described below. The binding is typi of homologous polynucleotide are not necessarily the same. cally in a manner that influences the steady state level of a 55 Inducible Promoter: An “inducible promoter in the con transcript in a cell or in an in vitro transcription extract. text of the current invention refers to a promoter, the activity Exogenous: As referred to within, "exogenous” is any of which is influenced by certain conditions, such as light, polynucleotide, polypeptide or protein sequence, whether temperature, chemical concentration, protein concentration, chimeric or not, that is introduced into the genome of a host conditions in an organism, cell, or organelle, etc. A typical cellor organism regenerated from said host cell by any means 60 example of an inducible promoter, which can be utilized with other than by a sexual cross. Examples of means by which this the polynucleotides of the present invention, is PARSK1, the can be accomplished are described below, and include Agro promoter from an Arabidopsis gene encoding a serine-threo bacterium-mediated transformation (of dicots—e.g. nine kinase enzyme, and which promoter is induced by dehy Salomon et al. EMBO.J. 3:141 (1984); Herrera-Estrella et al. dration, abscissic acid and Sodium chloride (Wang and Good EMBO J. 2:987 (1983); of monocots, representative papers 65 man, Plant J. 8:37 (1995), which is hereby incorporated by are those by Escudero et al., Plant J. 10:355 (1996), Ishida et reference). Examples of environmental conditions that may al., Nature Biotechnology 14:745 (1996), May et al., Bio/ affect transcription by inducible promoters include anaerobic US 7,868,155 B2 7 8 conditions, elevated temperature, the presence or absence of parison may be conducted by the local homology algorithm a nutrient or other chemical compound or the presence of of Smith and Waterman Add. APL. Math. 2:482 (1981), by the light. homology alignment algorithm of Needleman and Wunsch J. Modulate Transcription Level: As used herein, the phrase Mol. Biol. 48:443 (1970), by the search for similarity method “modulate transcription” describes the biological activity of a of Pearson and Lipman Proc. Natl. Acad. Sci. (USA)85: 2444 promoter sequence or promoter control element. Such modu (1988), by computerized implementations of these algo lation includes, without limitation, includes up- and down rithms (GAP, BESTFIT, BLAST, PASTA, and TFASTA in the regulation of initiation of transcription, rate of transcription, Wisconsin Genetics Software Package, Genetics Computer and/or transcription levels. Group (GCG), 575 Science Dr. Madison, Wis.), or by inspec Mutant: In the current invention, "mutant” refers to a heri 10 tion. Given that two sequences have been identified for com table change in nucleotide sequence at a specific location. parison, GAP and BESTFIT are preferably employed to Mutant genes of the current invention may or may not have an determine their optimal alignment. Typically, the default val associated identifiable phenotype. ues of 5.00 for gap weight and 0.30 for gap weight length are Operable Linkage: An "operable linkage' is a linkage in used. All of the references discussed in this paragraph are which a promoter sequence or promoter control element is 15 hereby incorporated by reference. connected to a polynucleotide sequence (or sequences) in All of the references cited in this section are hereby incor Such a way as to place transcription of the polynucleotide porated by reference. sequence under the influence or control of the promoter or Plant Promoter: A “plant promoter is a promoter capable promoter control element. Two DNA sequences (such as a of initiating transcription in plant cells and can modulate polynucleotide to be transcribed and a promoter sequence transcription of a polynucleotide. Such promoters need not be linked to the 5' end of the polynucleotide to be transcribed) are of plant origin. For example, promoters derived from plant said to be operably linked if induction of promoter function viruses, such as the CaMV35S promoter or from Agrobacte results in the transcription of mRNA encoding the polynucle rium tumefaciens such as the T-DNA promoters, can be plant otide and if the nature of the linkage between the two DNA promoters. A typical example of a plant promoter of plant sequences does not (1) result in the introduction of a frame 25 origin is the maize ubiquitin-1 (ubi-1) promoter known to shift mutation, (2) interfere with the ability of the promoter those of skill sequence to direct the expression of the protein, antisense Plant Tissue: The term “plant tissue' includes differenti RNA or ribozyme, or (3) interfere with the ability of the DNA ated and undifferentiated tissues or plants, including but not template to be transcribed. Thus, a promoter sequence would limited to roots, stems, shoots, cotyledons, epicotyl, hypo be operably linked to a polynucleotide sequence if the pro 30 cotyl, leaves, pollen, seeds, tumor tissue and various forms of moter was capable of effecting transcription of that poly cells in culture Such as single cells, protoplast, embryos, and nucleotide sequence. callus tissue. The plant tissue may be in plants or in organ, Optional Promoter Fragments: The phrase “optional pro tissue or cell culture. moter fragments' is used to refer to any Sub-sequence of the Preferential Transcription: “Preferential transcription' is promoter that is not required for driving transcription of an 35 defined as transcription that occurs in a particular pattern of operationally linked coding region. These fragments com cell types or developmental times or in response to specific prise the 5' UTR and any exon(s) of the endogenous coding stimuli or combination thereof. Non-limitive examples of region. The optional promoter fragments may also comprise preferential transcription include: high transcript levels of a any exon(s) and the 3' or 5' UTR of the gene residing upstream desired sequence in root tissues; detectable transcript levels of the promoter (that is, 5' to the promoter). Optional pro 40 of a desired sequence in certain cell types during embryogen moter fragments also include any intervening sequences that esis; and low transcript levels of a desired sequence under are introns or sequence that occurs between exons or an exon drought conditions. Such preferential transcription can be and the UTR. determined by measuring initiation, rate, and/or levels of Orthologous: “Orthologous is a term used herein to transcription. describe a relationship between two or more polynucleotides 45 Promoter: A "promoter' is a DNA sequence that directs the or proteins. Two polynucleotides or proteins are “ortholo transcription of a polynucleotide. Typically a promoter is gous' to one another if they serve a similar function in dif located in the 5' region of a polynucleotide to be transcribed, ferent organisms. In general, orthologous polynucleotides or proximal to the transcriptional start site of Such polynucle proteins will have similar catalytic functions (when they otide. More typically, promoters are defined as the region encode enzymes) or will serve similar structural functions 50 upstream of the first exon; more typically, as a region (when they encode proteins or RNA that form part of the upstream of the first of multiple transcription start sites; more ultrastructure of a cell). typically, as the region downstream of the preceding gene and Percentage of sequence identity: “Percentage of sequence upstream of the first of multiple transcription start sites; more identity, as used herein, is determined by comparing two typically, the region downstream of the polyA signal and optimally aligned sequences over a comparison window, 55 upstream of the first of multiple transcription start sites; even where the fragment of the polynucleotide or amino acid more typically, about 3,000 nucleotides upstream of the ATG sequence in the comparison window may comprise additions of the first exon; even more typically, 2,000 nucleotides or deletions (e.g., gaps or overhangs) as compared to the upstream of the first of multiple transcription start sites. The reference sequence (which does not comprise additions or promoters of the invention comprise at least a core promoter deletions) for optimal alignment of the two sequences. The 60 as defined above. Frequently promoters are capable of direct percentage is calculated by determining the number of posi ing transcription of genes located on each of the complemen tions at which the identical nucleic acid base or amino acid tary DNA strands that are 3' to the promoter. Stated differ residue occurs in both sequences to yield the number of ently, many promoters exhibit bidirectionality and can direct matched positions, dividing the number of matched positions transcription of a downstream gene when present in either by the total number of positions in the window of comparison 65 orientation (i.e. 5' to 3' or 3' to 5' relative to the coding region and multiplying the result by 100 to yield the percentage of of the gene). Additionally, the promoter may also include at sequence identity. Optimal alignment of sequences for com least one control element Such as an upstream element. Such US 7,868,155 B2 10 elements include UARs and optionally, other DNA sequences ture of hybridization or wash conditions. Stringency is typi that affect transcription of a polynucleotide Such as a syn cally compared by the parameter T, which is the temperature thetic upstream element. at which 50% of the complementary molecules in the hybrid Promoter Control Element: The term “promoter control ization are hybridized, in terms of a temperature differential element as used herein describes elements that influence the from T. High Stringency conditions are those providing a activity of the promoter. Promoter control elements include condition of T-5° C. to T-10° C. Medium or moderate transcriptional regulatory sequence determinants such as, but stringency conditions are those providing T-20° C. to not limited to, enhancers, scaffold/matrix attachment regions, T-29° C. Low Stringency conditions are those providing a TATA boxes, transcription start locus control regions, UARS, condition of T-40° C. to T-48° C. The relationship of URRs, other transcription factor binding sites and inverted 10 hybridization conditions to T. (in C.) is expressed in the repeats. mathematical equation Public sequence: The term “public sequence, as used in the context of the instant application, refers to any sequence T=81.5-16.6(logo Na'"0.41(% G+C)–(600/N) (1) that has been deposited in a publicly accessible database prior where N is the length of the probe. This equation works well to the filing date of the present application. This term encom 15 for probes 14 to 70 nucleotides in length that are identical to passes both amino acid and nucleotide sequences. Such the target sequence. The equation below for T of DNA-DNA sequences are publicly accessible, for example, on the hybrids is useful for probes in the range of 50 to greater than BLAST databases on the NCBI FTP web site (accessible at 500 nucleotides, and for conditions that include an organic incbi.nlm.nih.gov/ftp). The database at the NCBI FTP site solvent (formamide). utilizes “gi' numbers assigned by NCBI as a unique identifier for each sequence in the databases, thereby providing a non redundant database for sequence from various databases, (2) including GenBank, EMBL, DBBJ, (DNA Database of where L is the length of the probe in the hybrid. (P. Tijessen, Japan) and PDB (Brookhaven Protein Data Bank). “Hybridization with Nucleic Acid Probes’ in Laboratory Regulatory Sequence: The term “regulatory sequence, as 25 Techniques in Biochemistry and Molecular Biology, P. C. used in the current invention, refers to any nucleotide vand der Vliet, ed., c. 1993 by Elsevier, Amsterdam.) The T. sequence that influences transcription or translation initiation of equation (2) is affected by the nature of the hybrid; for and rate, or stability and/or mobility of a transcript or DNA-RNA hybrids T is 10-15° C. higher than calculated, polypeptide product. Regulatory sequences include, but are for RNA-RNA hybrids T is 20-25° C. higher. Because the not limited to, promoters, promoter control elements, protein 30 T, decreases about 1° C. for each 1% decrease in homology binding sequences, 5' and 3' UTRS, transcriptional start sites, when a long probe is used (Bonner et al., J. Mol. Biol. 81: 123 termination sequences, polyadenylation sequences, introns, (1973)), stringency conditions can be adjusted to favor detec certain sequences within amino acid coding sequences Such tion of identical genes or related family members. as secretory signals, protease cleavage sites, etc. Equation (2) is derived assuming equilibrium and there Related Sequences: “Related sequences’ refer to either a 35 fore, hybridizations according to the present invention are polypeptide or a nucleotide sequence that exhibits some most preferably performed under conditions of probe excess degree of sequence similarity with a reference sequence. and for sufficient time to achieve equilibrium. The time Specific Promoters: In the context of the current invention, required to reach equilibrium can be shortened by inclusion “specific promoters' refers to a subset of promoters that have of a hybridization accelerator such as dextran sulfate or a high preference for modulating transcript levels in a specific 40 another high volume polymer in the hybridization buffer. tissue or organ or cell and/or at a specific time during devel Stringency can be controlled during the hybridization reac opment of an organism. By “high preference' is meant at least tion or after hybridization has occurred by altering the salt and 3-fold, preferably 5-fold, more preferably at least 10-fold still temperature conditions of the wash solutions used. The for more preferably at least 20-fold, 50-fold or 100-fold increase mulas shown above are equally valid when used to compute in transcript levels under the specific condition over the tran 45 the stringency of a wash solution. Preferred wash solution Scription under any other reference condition considered. stringencies lie within the ranges stated above; high Strin Typical examples of temporal and/or tissue or organ specific gency is 5-8°C. below T. medium or moderate stringency is promoters of plant origin that can be used with the polynucle 26-29° C. below T. and low stringency is 45-48° C. below otides of the present invention, are: PTA29, a promoter which T is capable of driving gene transcription specifically in tape 50 r tum and only during another development (Koltonow et al., All of the references cited in this section are hereby incorpo Plant Cell 2:1201 (1990); RCc2 and RCc3, promoters that rated by reference. direct root-specific gene transcription in rice (Xu et al., Plant Substantially free of: A composition containing A is “sub Mol. Biol. 27:237 (1995); TobRB27, a root-specific promoter stantially free of B when at least 85% by weight of the total from tobacco (Yamamoto et al., Plant Cell 3:371 (1991)). 55 A+B in the composition is A. Preferably, A comprises at least Examples of tissue-specific promoters under developmental about 90% by weight of the total of A+B in the composition, control include promoters that initiate transcription only in more preferably at least about 95% or even 99% by weight. certain tissues or organs, such as root, ovule, fruit, seeds, or For example, a plant gene can be substantially free of other flowers. Other specific promoters include those from genes plant genes. Other examples include, but are not limited to, encoding seed storage proteins or the lipid body membrane 60 ligands Substantially free of receptors (and vice versa), a protein, oleosin. A few root-specific promoters are noted growth factor substantially free of other growth factors and a above. See also “Preferential transcription'. transcription binding factor Substantially free of nucleic All of the references cited in this section are hereby incor acids. porated by reference. Suppressor: See “Enhancer/Suppressor Stringency: “Stringency” as used herein is a function of 65 TATA to start: “TATA to start” shall mean the distance, in probe length, probe composition (G+C content), and salt number of nucleotides, between the primary TATA motif and concentration, organic solvent concentration, and tempera the start of transcription. US 7,868,155 B2 11 12 Transgenic plant: A “transgenic plant is a planthaving one Specifically, promoters and control elements of the inven or more plant cells that contain at least one exogenous poly tion can be used to modulate transcription of a desired poly nucleotide introduced by recombinant nucleic acid methods. nucleotide, which includes without limitation: Translational start site: In the context of the present inven (a) antisense; tion, a “translational start site' is usually an ATG or AUG in a (b) ribozymes; transcript, often the first ATG or AUG. A single protein encod (c) coding sequences; or ing transcript, however, may have multiple translational start (d) fragments thereof. sites. The promoter also can modulate transcription in a host Transcription start site: “Transcription start site' is used in 10 genome in cis- or in trans-. the current invention to describe the point at which transcrip In an organism, Such as a plant, the promoters and promoter tion is initiated. This point is typically located about 25 nucle control elements of the instant invention are useful to produce otides downstream from a MID binding site, such as a TATA preferential transcription which results in a desired pattern of box. Transcription can initiate at one or more sites within the transcript levels in a particular cells, tissues, or organs, or gene, and a single polynucleotide to be transcribed may have 15 under particular conditions. multiple transcriptional start sites, some of which may be specific for transcription in a particular cell-type or tissue or 3. Table of Contents organ. “+1 is stated relative to the transcription start site and The following description of the present invention is out indicates the first nucleotide in a transcript. lined in the following table of contents. Upstream Activating Region (UAR): An "Upstream Acti A. Identifying and Isolating Promoter Sequences of the vating Region' or “UAR’ is a position or orientation depen Invention dent nucleic acid element that primarily directs tissue, organ, (1) Cloning Methods cell type, or environmental regulation of transcript level, usu (2) Chemical Synthesis ally by affecting the rate of transcription initiation. Corre B. Generating a "core promoter sequence sponding DNA elements that have a transcription inhibitory 25 C. Isolating Related Promoter Sequences effect are called herein “Upstream Repressor Regions” or (1) Relatives Based on Nucleotide Sequence Identity “URR's. The essential activity of these elements is to bind a (2) Relatives Based on Coding Sequence Identity protein factor. Such binding can be assayed by methods (3) Relatives based on Common Function described below. The binding is typically in a manner that D. Identifying Control Elements influences the steady state level of a transcript in a cell or in 30 (1) Types of Transcription Control Elements vitro transcription extract. (2) Those Described by the Examples Untranslated region (UTR): A “UTR’ is any contiguous (3) Those Identifiable by Bioinformatics series of nucleotide bases that is transcribed, but is not trans (4) Those Identifiable by In Vitro and InVivo Assays lated. A 5' UTR lies between the start site of the transcript and (5) Non-Natural Control Elements the translation initiation codon and includes the +1 nucle 35 E. Constructing Promoters and Control Elements otide. A 3' UTR lies between the translation termination (1) Combining Promoters and Promoter Control Elements codon and the end of the transcript. UTRs can have particular (2) Number of Promoter Control Elements functions such as increasing mRNA message stability or (3) Spacing Between Control Elements translation attenuation. Examples of 3' UTRs include, but are F. Vectors not limited to polyadenylation signals and transcription ter 40 (1) Modification of Transcription by Promoters and Pro mination sequences. moter Control Elements Variant: The term “variant' is used herein to denote a (2) Polynucleotide to be Transcribed polypeptide or protein or polynucleotide molecule that differs (3) Other Regulatory Elements from others of its kind in Some way. For example, polypeptide (4) Other Components of Vectors and protein variants can consist of changes in amino acid 45 G. Insertion of Polynucleotides and Vectors Into a Host sequence and/or charge and/or post-translational modifica Cell tions (such as glycosylation, etc) Likewise, polynucleotide (1) Autonomous of the Host Genome variants can consist of changes that add or delete a specific (2) Integrated into the Host Genome H. Utility UTR or exon sequence. It will be understood that there may 50 be sequence variations within sequence or fragments used or A. Identifying and Isolating Promoter Sequences of the disclosed in this application. Preferably, variants will be such Invention that the sequences have at least 80%, preferably at least 90%, The promoters and promoter control elements of the at least 95%, at least 97%, at least 98%, or at least 99% sequence identity. Variants preferably measure the primary present invention presented in Table 1 were identified from 55 Arabidopsis thaliana or Oryza sativa. Additional promoter biological function of the native polypeptide or protein or sequences encompassed by the invention can be identified as polynucleotide. described below. (1) Cloning Methods 2. Introduction Isolation from genomic libraries of polynucleotides com 60 prising the sequences of the promoters and promoter control The polynucleotides of the invention comprise promoters elements of the present invention is possible using known and promoter control elements that are capable of modulating techniques. transcription. For example, polymerase chain reaction (PCR) can Such promoters and promoter control elements can be used amplify the desired polynucleotides utilizing primers in combination with native or heterologous promoter frag 65 designed from sequences in Table 2. Polynucleotide libraries ments, control elements or other regulatory sequences to comprising genomic sequences can be constructed according modulate transcription and/or translation. to Sambrook et al., (Molecular Cloning: A Laboratory US 7,868,155 B2 13 14 Manual, 2" Ed. (1989) Cold Spring Harbor Press, Cold tutions, insertions and deletions can be tolerated in these Spring Harbor, N.Y., for example. spacer regions to a certain degree without loss of function. Other procedures for isolating polynucleotides comprising In contrast, less variation is permissible in the functionally the promoter sequences of the invention include, without important regions, since changes in the sequence can interfere limitation, tail-PCR, and 5' rapid amplification of cDNA ends 5 with protein binding. Nonetheless, some variation in the func (RACE). See, for tail-PCR, for example, Liu et al., Plant J tionally important regions is permissible so longas function is 8(3): 457-463 (September, 1995); Liu et al., Genomics 25: conserved. 674-681 (1995); Liu et al., Nucl. Acids Res. 21 (14): 3333 The effects of substitutions, insertions and deletions to the 3334 (1993); and Zoe et al., BioTechniques 27(2): 240-248 promoter sequences or promoter control elements may be to (1999); for RACE, see, for example, PCR Protocols: A Guide 10 increase or decrease the binding of relevant DNA binding to Methods and Applications, (1990) Academic Press, Inc. proteins to modulate transcript levels of a polynucleotide to All of the references cited in this section are hereby incor be transcribed. Effects may include tissue-specific or condi porated by reference. tion-specific modulation of transcript levels of the polypep (2) Chemical Synthesis tide to be transcribed. Polynucleotides representing changes In addition, the promoters and promoter control elements 15 to the nucleotide sequence of the DNA-protein contact region described in Table 1 can be chemically synthesized according by insertion of additional nucleotides, changes to identity of to techniques in common use. See, for example, Beaucage et relevant nucleotides, including use of chemically-modified al., Tet. Lett. (1981) 22: 1859 and U.S. Pat. No. 4,668,777, bases, or deletion of one or more nucleotides are considered both of which are hereby incorporated by reference. encompassed by the present invention. Such chemical oligonucleotide synthesis can be carried out (1) Relatives Based on Nucleotide Sequence Identity using commercially available devices, such as, BioSearch Included in the present invention are promoters exhibiting 4600 or 8600 DNA synthesizer, by Applied Biosystems, a nucleotide sequence identity to those described in Table 1. division of Perkin-Elmer Corp., Foster City, Calif., USA; and Expedite by Perceptive Biosystems, Framingham, Mass. DEFINITION USA. 25 Synthetic RNA, including natural and/or analog building Typically, such related promoters exhibit at least 80% blocks, can be synthesized on the Biosearch 8600 machines, sequence identity, preferably at least 85%, more preferably at see above. least 90%, and most preferably at least 95%, even more pref Oligonucleotides can be synthesized and then ligated erably, at least 96%, at least 97%, at least 98% or at least 99% together to construct the desired polynucleotide. 30 sequence identity compared to those shown in Table 1. Such sequence identity can be calculated by the algorithms and B. Generating Reduced and “Core” Promoter Sequences computers programs described above. Included in the present invention are reduced and “core” Usually, such sequence identity is exhibited in an align promoter sequences. The reduced promoters can be isolated ment region that is at least 75% of the length of a sequence from the promoters of the invention by deleting at least one 5' 35 shown in Table 1 or corresponding full-length sequence; UTR, exon or 3' UTR sequence present in the promoter more usually at least 80%; more usually, at least 85%, more sequence that is associated with a gene or coding region usually at least 90%, and most usually at least 95%, even more located 5' to the promoter sequence or in the promoters usually, at least 96%, at least 97%, at least 98% or at least 99% endogenous coding region. of the length of a sequence shown in Table 1. Similarly, the “core promoter sequences can be generated 40 The percentage of the alignment length is calculated by by deleting all 5' UTRs, exons and 3' UTRs present in the counting the number of residues of the sequence in region of promoter sequence and the associated intervening sequences strongest alignment, e.g., a continuous region of the sequence that are related to the gene or coding region 5' to the promoter that contains the greatest number of residues that are identical region and the promoter's endogenous coding region. to the residues between two sequences that are being aligned. This data is presented in Table 3. 45 The number of residues in the region of strongest alignment is C. Isolating Related Promoter Sequences divided by the total residue length of a sequence in Table 1. Included in the present invention are promoter and pro These related promoters may exhibit similar preferential moter control elements that are related to those described in transcription as those promoters described in Table 1. Table 1. Such related sequence can be isolated utilizing 50 Construction of Polynucleotides (a) nucleotide sequence identity; Naturally occurring promoters that exhibit nucleotide (b) coding sequence identity; or sequence identity to those shown in Table 1 can be isolated using the techniques as described above. More specifically, (c) common function or gene products. Such related promoters can be identified by varying stringen Relatives can include both naturally occurring promoters and 55 cies, as defined above, in typical hybridization procedures non-natural promoter sequences. Non-natural related pro such as Southern blots or probing of polynucleotide libraries, moters include nucleotide Substitutions, insertions or dele for example. Non-natural promoter variants of those shown in tions of naturally-occurring promoter sequences that do not Table 1 can be constructed using cloning methods that incor substantially affect transcription modulation activity. For porate the desired nucleotide variation. See, for example, Ho, example, the binding of relevant DNA binding proteins can 60 S. N., et al. Gene 77:51-59 1989, describing a procedure site still occur with the non-natural promoter sequences and pro directed mutagenesis using PCR, which is hereby incorpo moter control elements of the present invention. rated by reference. According to current knowledge, promoter sequences and Any related promoter showing sequence identity to those promoter control elements exist as functionally important shown in Table 1 can be chemically synthesized as described regions, such as protein binding sites, and spacer regions. 65 above. These spacer regions are apparently required for proper posi Also, the present invention includes non-natural promoters tioning of the protein binding sites. Thus, nucleotide Substi that exhibit the above-sequence identity to those in Table 1 US 7,868,155 B2 15 16 The promoters and promoter control elements of the larger than 500 bases; even more usually, no more than any present invention may also be synthesized with 5' or 3' exten one of the following: 250, 200, 150 or 100 bases. sions, to facilitate additional manipulation, for instance. Relatives Based on Nucleotide Sequence Identity The present invention also includes reduced promoter Included in the present invention are promoter control ele sequences. These sequences have at least one of the optional 5 ments exhibiting nucleotide sequence identity to those promoter fragments deleted. described in Table 1 of fragments thereof. Core promoter sequences are another embodiment of the Typically, such related promoters exhibit at least 80% present invention. The core promoter sequences have all of sequence identity, preferably at least 85%, more preferably at the optional promoter fragments deleted. least 90%, and most preferably at least 95%, even more pref Testing of Polynucleotides 10 erably, at least 96%, at least 97%, at least 98% or at least 99% Polynucleotides of the invention were tested for activity by sequence identity compared to those shown in Table 1. Such cloning the sequence into an appropriate vector, transforming sequence identity can be calculated by the algorithms and plants with the construct and assaying for marker gene computers programs described above. expression. Recombinant DNA constructs were prepared Promoter Control Element Configuration which comprise the polynucleotide sequences of the inven 15 A common configuration of the promoter control elements tion inserted into a vector suitable for transformation of plant in RNA polymerase II promoters is shown below: cells. The construct can be made using standard recombinant DNA techniques (Sambrook et al. 1989) and can be intro For more description, see, for example, “Models for predic duced to the species of interest by Agrobacterium-mediated tion and recognition of eukaryotic promoters’. T. Werner, transformation or by other means of transformation as refer Mammalian Genome, 10, 168-175 (1999). enced below. Promoters are generally modular in nature. Promoters can The vector backbone can be any of those typical in the art consist of a basal promoter which functions as a site for Such as plasmids, viruses, artificial chromosomes, BACs, assembly of a transcription complex comprising an RNA YACs and PACs and vectors of the sort described by polymerase, for example RNA polymerase II. A typical tran (a) BAC: Shizuya et al., Proc. Natl. Acad. Sci. USA 89: 25 Scription complex will include additional factors such as 8794-8797 (1992); Hamilton et al., Proc. Natl. Acad. Sci. TFB, TFD, and TFE. Of these, TFD appears to be the USA 93:9975-9979 (1996); only one to bind DNA directly. The promoter might also (b)YAC: Burke et al., Science 236:806-812 (1987); contain one or more promoter control elements such as the (c) PAC: Sternberg N. et al., Proc Natl AcadSci USA. Janu elements discussed above. These additional control elements ary: 87(1):103-7 (1990); 30 may function as binding sites for additional transcription (d) Bacteria-Yeast Shuttle Vectors: Bradshaw et al., Nucl factors that have the function of modulating the level of Acids Res 23: 4850-4856 (1995): transcription with respect to tissue specificity and of tran (e) Lambda Phage Vectors: Replacement Vector, e.g., Scriptional responses to particular environmental or nutri Frischaufetal., J. Mol. Biol 170: 827-842 (1983); or Inser tional factors, and the like. tion vector, e.g., Huynh et al., In: Glover NM (ed) DNA 35 One type of promoter control element is a polynucleotide Cloning: A practical Approach, Vol. 1 Oxford: IRL Press sequence representing a binding site for proteins. Typically, (1985); T-DNA gene fusion vectors: Walden et al., Mol within a particular functional module, protein binding sites Cell Biol 1: 175-194 (1990); and constitute regions of 5 to 60, preferably 10 to 30, more pref (g) Plasmid vectors: Sambrook et al., infra. erably 10 to 20 nucleotides. Within such binding sites, there Typically, the construct comprises a vector containing a 40 are typically 2 to 6 nucleotides which specifically contact sequence of the present invention operationally linked to any amino acids of the nucleic acid binding protein. marker gene. The polynucleotide was identified as a promoter The protein binding sites are usually separated from each by the expression of the marker gene. Although many marker other by 10 to several hundred nucleotides, typically by 15 to genes can be used, Green Fluorescent Protein (GFP) is pre 150 nucleotides, often by 20 to 50 nucleotides. ferred. The vector may also comprise a marker gene that 45 Further, protein binding sites in promoter control elements confers a selectable phenotype on plant cells. The marker often display dyad symmetry in their sequence. Such ele may encode biocide resistance, particularly antibiotic resis ments can bind several different proteins, and/or a plurality of tance, Such as resistance to kanamycin, G418, bleomycin, sites can bind the same protein. Both types of elements may hygromycin, or herbicide resistance, such as resistance to be combined in a region of 50 to 1,000 base pairs. chlorosulfuron or phosphinotricin. Vectors can also include 50 Binding sites for any specific factor have been known to origins of replication, Scaffold attachment regions (SARs), occur almost anywhere in a promoter. For example, func markers, homologous sequences, introns, etc. tional AP-1 binding sites can be located far upstream, as in the All of the references cited in this section are hereby incor rat bonesialoprotein gene, where an AP-1 site located about porated by reference. 900 nucleotides upstream of the transcription start site Sup Promoter Control Elements of the Invention 55 presses expression. Yamauchi et al., Matrix Biol. 15, 119-130 The promoter control elements of the present invention (1996). Alternatively, an AP-1 site located close to the tran include those that comprise a sequence shown in Table 1 and Scription start site plays an important role in the expression of fragments thereof. The size of the fragments of Table 1 can Moloney murine leukemia virus. Sap et al., Nature, 340, range from 5 bases to 10 kilobases (kb). Typically, the frag 242-244. (1989). All of the references cited in this section are ment size is no Smaller than 8 bases; more typically, no 60 hereby incorporated by reference. Smaller than 12; more typically, no Smaller than 15 bases; (2) Those Identifiable by Bioinformatics more typically, no smaller than 20 bases; more typically, no Promoter control elements from the promoters of the Smaller than 25 bases; even more typically, no more than any instant invention can be identified utilizing bioinformatic or one of the following: 30, 35, 40 or 50 bases. computer driven techniques. Usually, the fragment size in no larger than 5 kb bases; 65 One method uses a computer program AlignACE to iden more usually, no larger than 2 kb, more usually, no larger than tify regulatory motifs in genes that exhibit common prefer 1 kb; more usually, no larger than 800 bases; more usually, no ential transcription across a number of time points. The pro US 7,868, 155 B2 17 18 gram identifies common sequence motifs in Such genes. See, Immunology 99:101 (2000); V. Kolla et al., Biochem. Bio Roth et al., Nature Biotechnol. 16:949-945 (1998); Tavazoie phys. Res. Comm. 266:5 (1999)) or “mobility-shift' assays et al., Nat Genet. 1999 July; 22(3):281-5: (E. D. Fabiani et al., J. Biochem. 347:147 (2000): N. Sugiura Genomatix, also makes available a GEMS Launcher pro et al., J. Biochem 347: 155 (2000)) or fluorescence polariza gram and other programs to identify promoter control ele- 5 tion (e.g. Royer et al., U.S. Pat. No. 5,445,935). Both mobility ments and configuration of Such elements. Genomatix is shift and DNA footprinting assays can also be used to identify located in Munich, Germany. portions of large DNA fragments that are boundby proteins in Other references also describe detection of promoter mod unpurified transcription extracts prepared from tissues or ules by models independent of overall nucleotide sequence organs of interest. similarity. See, for instance, Klingenhoffet al., Bioinformat- 10 Cell-free transcription extracts can be prepared and used to ics 15, 180-186 (1999). directly assay in a reconstitutable system (Narayan et al., Protein binding sites of promoters can be identified as Biochemistry 39:818 (2000)). reported in “Computer-assisted prediction, classification, and All of the references cited in this section are hereby incor delimination of protein binding sites in nucleic acids'. Frech, porated by reference. et al., Nucleic Acids Research, Vol. 21, No. 7, 1655-1664, 15 In-Vivo Assays 1993. Promoter control elements can be identified with reporter Other programs used to identify protein binding sites genes in in-vivo assays with the use of fragments of the instant include, for example, Signal Scan, Prestridge et al., Comput. promoters or variants of the instant promoter polynucle Appl. Biosci. 12: 157-160 (1996); Matrix Search, Chen et al., otides. Comput. Appl. Biosci. 11:563-566 (1995), available as part 20 For example, various fragments can be inserted into a of Signal Scan 4.0; Matinspector, Ghosh et al., Nucl. Acid vector, comprising a basal or “core” promoter, for example, Res. 21:3117-3118 (1993) available on the internet, ConsIn operably linked to a reporter sequence, which, when tran spector, Frechet al., Nucl. Acids Res. 21: 1655-1664 (1993), scribed, can produce a detectable label. Examples of reporter available at on the internet; TFSearch; and TESS. genes include those encoding luciferase, green fluorescent Frech et al., “Software for the analysis of DNA sequence 25 protein, GUS. neo, cat and bar. Alternatively, reporter elements of transcription', Bioinformatics & Sequence sequence can be detected utilizing AFLP and microarray Analysis, Vol. 13, no. 1, 89-97 (1997) is a review of different techniques. software for analysis of promoter control elements. This paper also reports the usefulness of matrix-based approaches In promoter probe vector systems, genomic DNA frag to yield more specific results. 30 ments are inserted upstream of the coding sequence of a For other procedures, see, Fickett et al., Curr. Op. Biotech reporter gene that is expressed only when the cloned fragment nol. 11:19-24 (2000); and Quandt et al., Nucleic Acids Res., contains DNA having transcription modulation activity 23,4878-4884 (1995). (Neve, R. L. et al., Nature 277:324-325 (1979)). Control All of the references cited in this section are hereby incor elements are disrupted when fragments or variants lacking porated by reference. 35 any transcription modulation activity. Probe vectors have (3) Those Identifiable by In-Vitro and In-Vivo Assays been designed for assaying transcription modulation in E. coli Promoter control elements also can be identified with in (An, G. et al., J. Bact. 140:400-407 (1979)) and other bacterial vitro assays, Such as transcription detection methods; and hosts (Band, L. et al., Gene 26:313-315 (1983); Achen, M.G., with in-vivo assays, such as enhancer trapping protocols. Gene 45:45-49 (1986)), yeast (Goodey, A. R. et al., Mol. Gen. In-Vitro Assays 40 Genet. 204:505-511 (1986)) and mammalian cells (Pater, M. Examples of in-vitro assays include detection of binding of M. et al., J. Mol. App. Gen. 2:363-371 (1984)). protein factors that bind promoter control elements. Frag A different design of a promoter/control element trap ments of the instant promoters can be used to identify the includes packaging into retroviruses for more efficient deliv location of promoter control elements. Another option for ery into cells. One type of retroviral enhancer trap was obtaining a promoter control element with desired properties 45 described by von Melchner et al. (Genes Dev. 1992; U.S. Pat. is to modify known promoter sequences. This is based on the No. 5,364,783). The basic design of this vector includes a fact that the function of a promoter is dependent on the inter reporter protein coding sequence engineered into the U3 por play of regulatory proteins that bind to specific, discrete tion of the 3' LTR. No splice acceptor consensus sequences nucleotide sequences in the promoter, termed motifs. Such are included, limiting its utility to work as an enhancer trap interplay Subsequently affects the general transcription 50 only. A different approach to a gene trap using retroviral machinery and regulates transcription efficiency. These pro vectors was pursued by Friedrich and Soriano (Genes Dev. teins are positive regulators or negative regulators (repres 1991), who engineered a lacz-neofusion protein linked to a sors), and one protein can have a dual role depending on the splicing acceptor. Lacz-neo fusion protein expression from context (Johnson, P. F. and McKnight, S. L. Annu. Rev. Bio trapped loci allows not only for drug selection, but also for chem. 58:799-839 (1989)). 55 visualization off-galatactosidase expression using the chro One type of in-vitro assay utilizes a known DNA binding mogenic Substrate, X-gal. factor to isolate DNA fragments that bind. If a fragment or A general review of tools for identifying transcriptional promoter variant does not bind, then a promoter control ele regulatory regions of genomic DNA is provided by J. W. ment has been removed or disrupted. For specific assays, see, Fickett et al. (Curr. Opn. Biotechnol. 11:19 (2000). for instance, B. Luo et al., J. Mol. Biol. 266:470 (1997), S. 60 All of the references cited in this section are hereby incor Chusacultanachai et al., J. Biol. Chem. 274:23.591 (1999), D. porated by reference. Fabbro et al., Biochem. Biophys. Res. Comm. 213:781 (4) Non-Natural Control Elements (1995)). Non-natural control elements can be constructed by insert Alternatively, a fragment of DNA suspected of conferring ing, deleting or Substituting nucleotides into the promoter a particular pattern of specificity can be examined for activity 65 control elements described above. Such control elements are in binding transcription factors involved in that specificity by capable of transcription modulation that can be determined methods such as DNA footprinting (e.g. D. J. Cousins et al., using any of the assays described above. US 7,868,155 B2 19 20 D. Constructing Promoters with Control Elements maize; see Marrs and Walbot, 1997, Plant Physiol 113:93 (1) Combining Promoters and Promoter Control Elements 102), HSP32 (hyperthermia; rat; see Raju and Maines, 1994, The promoter polynucleotides and promoter control ele Biochim Biophys Acta 1217:273-80); MAPKAPK-2 (heat ments of the present invention, both naturally occurring and shock; Drosophila; see Larochelle and Suter, 1995, Gene synthetic, can be combined with each other to produce the 163:209-14). desired preferential transcription. Also, the polynucleotides In addition, the following examples of promoters are of the invention can be combined with other known sequences induced by the presence or absence of light can be used in to obtain other useful promoters to modulate, for example, combination with those of the present invention: Topoi tissue transcription specific or transcription specific to certain somerase II (pea; see Reddy et al., 1999, Plant Mol Biol conditions. Such preferential transcription can be determined 10 41:125-37), chalcone synthase (soybean; see Wingender et using the techniques or assays described above. al., 1989, Mol Gen Genet. 218:315-22), mdm2 gene (human Fragments, variants, as well as full-length sequences those tumor; see Saucedo et al., 1998, Cell Growth Differ 9:119 shown in Table 1 and relatives are useful alone or in combi 30), Clock and BMAL1 (rat; see Namihira et al., 1999, Neu nation. rosci Lett 271:1-4), PHYA (Arabidopsis; see Canton and The location and relation of promoter control elements 15 Quail, 1999, Plant Physiol 121:1207-16), PRB-1b (tobacco: within a promoter can affect the ability of the promoter to see Sessa et al., 1995, Plant Mol Biol 28:537-47) and Ypr10 modulate transcription. The order and spacing of control ele (common bean; see Walter et al., 1996, Eur J. Biochem 239: ments is a factor when constructing promoters. 281-93). (2) Number of Promoter Control Elements The promoters and control elements of the following genes Promoters can contain any number of control elements. For can be used in combination with the present invention to example, a promoter can contain multiple transcription bind confertissue specificity: MipE (iceplant; Yamada et al., 1995, ing sites or other control elements. One element may confer Plant Cell 7:1129-42) and SUCS (root nodules; broadbean: tissue or organ specificity; another element may limit tran Kuster et al., 1993, Mol Plant Microbe Interact 6:507-14) for Scription to specific time periods, etc. Typically, promoters roots, OsSUT1 (rice: Hirose et al., 1997. Plant Cell Physiol will contain at least a basal or core promoter as described 25 38:1389-96) for leaves, Msg (soybean: Stomvik et al., 1999, above. Any additional element can be included as desired. For Plant Mol Biol 41:217-31) for siliques, cell (Arabidopsis: example, a fragment comprising a basal or “core promoter Shani et al., 1997. Plant Mol Biol34(6):837-42) and ACT11 can be fused with another fragment with any number of (Arabidopsis: Huang et al., 1997, Plant Mol Biol 33:125-39) additional control elements. for inflorescence. (3) Spacing Between Control Elements Spacing between 30 Still other promoters are affected by hormones or partici control elements or the configuration or control elements can pate in specific physiological processes, which can be used in be determined or optimized to permit the desired protein combination with those of present invention. Some examples polynucleotide or polynucleotide interactions to occur. are the ACC synthase gene that is induced differently by For example, if two transcription factors bind to a promoter ethylene and brassinosteroids (mung bean: Yi et al., 1999, simultaneously or relatively close in time, the binding sites 35 Plant Mol Bio 141:443-54), the TAPG1 gene that is active are spaced to allow each factor to bind without steric hinder during abscission (tomato; Kalaitzis et al., 1995, Plant Mol ance. The spacing between two Such hybridizing control ele Biol 28:647-56), and the 1-aminocyclopropane-1-carboxy ments can be as Small as a profile of a protein bound to a late synthase gene (carnation; Jones et al., 1995, Plant Mol control element. In some cases, two protein binding sites can Biol 28:505-12) and the CP-2/cathepsin L gene (rat; Kim and be adjacent to each other when the proteins bind at different 40 Wright, 1997, Biol Reprod 57:1467-77), both active during times during the transcription process. SeSCCC. Further, when two control elements hybridize the spacing All of the references cited in this section are hereby incor between such elements will be sufficient to allow the pro porated by reference. moter polynucleotide to hairpin or loop to permit the two elements to bind. The spacing between two such hybridizing 45 E. Vectors control elements can be as Small as a t-RNA loop, to as large Vectors area useful component of the present invention. In as 10 kb. particular, the present promoters and/or promoter control ele Typically, the spacing is no smaller than 5 bases; more ments may be delivered to a system such as a cell by way of typically, no smaller than 8; more typically, no Smaller than a vector. For the purposes of this invention, such delivery may 15 bases; more typically, no smaller than 20 bases; more 50 range from simply introducing the promoter or promoter typically, no smaller than 25 bases; even more typically, no control element by itself randomly into a cell to integration of more than one of the following: 30, 35, 40 or 50 bases. a cloning vector containing the present promoter or promoter Usually, the fragment size in no larger than 5 kb bases; control element. Thus, a vector need not be limited to a DNA more usually, no larger than 2 kb, more usually, no larger than molecule Such as a plasmid, cosmid or bacterial phage that 1 kb; more usually, no larger than 800 bases; more usually, no 55 has the capability of replicating autonomously in a host cell. larger than 500 bases; even more usually, no more than one of All other manner of delivery of the promoters and promoter the following: 250, 200, 150 or 100 bases. control elements of the invention are envisioned. The various Such spacing between promoter control elements can be T-DNA vector types are a preferred vector for use with the determined using the techniques and assays described above. present invention. Many useful vectors are commercially (4) Other Promoters 60 available. The following are promoters that are induced under stress It may also be useful to attach a marker sequence to the conditions and can be combined with those of the present present promoter and promoter control element in order to invention: ldhl (oxygen stress; tomato; see Germain and determine activity of such sequences. Marker sequences typi Ricard, 1997, Plant Mol Biol 35:949-54), GPx and CAT cally include genes that provide antibiotic resistance, Such as (oxygen stress; mouse; see Franco et al., 1999, Free Radic 65 tetracycline resistance, hygromycin resistance or amplicillin Biol Med 27:1122-32), ciT (cold stress; potato; see Kirch et resistance, or provide herbicide resistance. Specific select al., 1997, Plant Mol. Biol. 33:897-909), BZ2 (heavy metals: able marker genes may be used to confer resistance to herbi US 7,868,155 B2 21 22 cides Such as glyphosate, glufosinate or broxynil (Comai et (4) Other Components of Vectors al., Nature 317: 741-744 (1985); Gordon-Kamm et al., Plant Translation of eukaryotic mRNA is often initiated at the Cell 2: 603-618 (1990); and Stalker et al., Science 242: 419 codon that encodes the first methionine. Thus, when con 423 (1988)). Other marker genes exist which provide hor structing a recombinant polynucleotide according to the mone responsiveness. present invention for expressing a protein product, it is pref All of the references cited in this section are hereby incor porated by reference. erable to ensure that the linkage between the 3' portion, pref (1) Modification of Transcription by Promoters and Pro erably including the TATA box, of the promoter and the moter Control Elements polynucleotide to be transcribed, or a functional derivative The promoter or promoter control element of the present 10 thereof, does not contain any intervening codons which are invention may be operably linked to a polynucleotide to be capable of encoding a methionine. transcribed. In this manner, the promoter or promoter control The vector of the present invention may contain additional element may modify transcription by modulate transcript components. For example, an origin of replication allows for levels of that polynucleotide when inserted into a genome. replication of the vector in a host cell. Additionally, homolo However, prior to insertion into a genome, the promoter or 15 gous sequences flanking a specific sequence allows for spe promoter control element need not be linked, operably or cific recombination of the specific sequence at a desired loca otherwise, to a polynucleotide to be transcribed. For example, tion in the target genome. T-DNA sequences also allow for the promoter or promoter control element may be inserted insertion of a specific sequence randomly into a target alone into the genome in front of a polynucleotide already genome. present in the genome. In this manner, the promoter or pro moter control element may modulate the transcription of a The vector may also be provided with a plurality of restric polynucleotide that was already present in the genome. This tion sites for insertion of a polynucleotide to be transcribed as polynucleotide may be native to the genome or inserted at an well as the promoter and/or promoter control elements of the earlier time. present invention. The vector may additionally contain select Alternatively, the promoter or promoter control element 25 able marker genes. The vector may also contain a transcrip may be inserted into a genome alone to modulate transcrip tional and translational initiation region, and a transcriptional tion. See, for example, Vaucheret, Hetal. (1998) Plant J 16: and translational termination region functional in the host 651-659, which is hereby incorporated by reference. Rather, cell. The termination region may be native with the transcrip the promoter or promoter control element may be simply tional initiation region, may be native with the polynucleotide inserted into a genome or maintained extrachromosomally as 30 to be transcribed, or may be derived from another source. a way to divert transcription resources of the system to itself. Convenient termination regions are available from the Ti This approach may be used to down-regulate the transcript plasmid of A. tumefaciens, such as the octopine synthase and levels of a group of polynucleotide(s). nopaline synthase termination regions. See also, Guerineau et (2) Polynucleotide to be Transcribed al., (1991) Mol. Gen. Genet. 262:141-144; Proudfoot (1991) The nature of the polynucleotide to be transcribed is not 35 Cell 64:671-674; Sanfacon et al. (1991) Genes Dev. 5:141 limited. Specifically, the polynucleotide may include 149; Mogen et al. (1990) Plant Cell 2:1261-1272; Munroe et sequences that will have activity as RNA as well as sequences al. (1990) Gene 91:151-158; Ballas et al. 1989) Nucleic Acids that result in a polypeptide product. These sequences may Res. 17:7891-7903; Joshi et al. (1987) Nucleic Acid Res. include, but are not limited to antisense sequences, ribozyme 15:9627-9639. sequences, spliceosomes, amino acid coding sequences, and 40 Where appropriate, the polynucleotide to be transcribed fragments thereof. may be optimized for increased expression in a certain host Specific coding sequences may include, but are not limited cell. For example, the polynucleotide can be synthesized to endogenous proteins or fragments thereof, or heterologous using preferred codons for improved transcription and trans proteins including marker genes or fragments thereof. lation. See U.S. Pat. Nos. 5,380,831, 5,436,391; see also and Promoters and control elements of the present invention 45 are useful for modulating metabolic or catabolic processes. Murray et al., (1989) Nucleic Acids Res. 17:477-498. Such processes include, but are not limited to, secondary Additional sequence modifications include elimination of product metabolism, amino acid synthesis, seed protein Stor sequences encoding spurious polyadenylation signals, exon age, oil development, pest defense and nitrogen usage. Some intron splice site signals, transposon-like repeats, and other examples of genes, transcripts and peptides or polypeptides 50 Such sequences well characterized as deleterious to expres participating in these processes, which can be modulated by sion. The G-C content of the polynucleotide may be adjusted the present invention: are tryptophan decarboxylase (tdc) and to levels average for a given cellular host, as calculated by strictosidine synthase (strl), dihydrodipicolinate synthase reference to known genes expressed in the host cell. The (DHDPS) and aspartate kinase (AK), 2S albumin and alpha-, polynucleotide sequence may be modified to avoid hairpin beta-, and gamma-Zeins, ricinoleate and 3-ketoacyl-ACP syn 55 secondary mRNA structures. thase (KAS), Bacillus thuringiensis (Bt) insecticidal protein, A general description of expression vectors and reporter cowpea trypsin inhibitor (CpTI), asparagine synthetase and genes can be found in Gruber, et al., “Vectors for Plant Trans nitrite reductase. Alternatively, expression constructs can be formation, in Methods in Plant Molecular Biology & Bio used to inhibit expression of these peptides and polypeptides technology” in Glich et al., (Eds. pp. 89-119, CRC Press, by incorporating the promoters in constructs for antisense 60 1993). Moreover GUS expression vectors and GUS gene use, co-suppression use or for the production of dominant cassettes are available from Clonetech Laboratories, Inc., negative mutations. Palo Alto, Calif, while luciferase expression vectors and (3) Other Regulatory Elements luciferase gene cassettes are available from Promega Corp. As explained above, several types of regulatory elements (Madison, Wis.). GFP vectors are available from Aurora Bio exist concerning transcription regulation. Each of these regu 65 Sciences. latory elements may be combined with the present vector if All of the references cited in this section are hereby incor desired. porated by reference. US 7,868,155 B2 23 24 F. Polynucleotide Insertion Into A Host Cell the exemplary promoters of Table 1 will also be capable of The polynucleotides according to the present invention can Sustaining expression in Some tissues or organs after the ini be inserted into a host cell. A host cell includes but is not tiation or completion of regeneration. Examples of these tis limited to a plant, mammalian, insect, yeast, and prokaryotic Sues or organs are somatic embryos, cotyledon, hypocotyl, cell, preferably a plant cell. epicotyl, leaf stems, roots, flowers and seed. The method of insertion into the host cell genome is chosen Integration into the host cell genome also can be accom based on convenience. For example, the insertion into the host plished by methods known in the art, for example, by the cell genome may either be accomplished by vectors that homologous sequences or T-DNA discussed above or using integrate into the host cell genome or by vectors which exist the cre-lox system (A. C. Vergunst et al., Plant Mol. Biol. independent of the host cell genome. 10 38:393 (1998)). (1) Polynucleotides Autonomous of the Host Genome All of the references cited in this section are hereby incor The polynucleotides of the present invention can exist porated by reference. autonomously or independent of the host cell genome. Vec tors of these types are known in the art and include, for G. Utility Common Uses example, certain type of non-integrating viral vectors, 15 autonomously replicating plasmids, artificial chromosomes, In yet another embodiment, the promoters of the present and the like. invention can be used to further understand developmental Additionally, in some cases transient expression of a poly mechanisms. For example, promoters that are specifically nucleotide may be desired. induced during callus formation, Somatic embryo formation, (2) Polynucleotides Integrated into the Host Genome shoot formation or root formation can be used to explore the The promoter sequences, promoter control elements or effects of overexpression, repression or ectopic expression of vectors of the present invention may be transformed into host target genes, or for isolation of trans-acting factors. cells. These transformations may be into protoplasts or intact The vectors of the invention can be used not only for tissues or isolated cells. Preferably expression vectors are expression of coding regions but may also be used in exon introduced into intact tissue. General methods of culturing 25 trap cloning, or promoter trap procedures to detect differen plant tissues are provided for example by Maki et al. “Proce tial gene expression in various tissues, K. Lindsey et al., 1993 dures for Introducing Foreign DNA into Plants' in Methods "Tagging Genomic Sequences That Direct Transgene in Plant Molecular Biology & Biotechnology, Glich et al. Expression by Activation of a Promoter Trap in Plants', (Eds. pp. 67-88 CRC Press, 1993); and by Phillips et al. Transgenic Research 2:3347. D. Auch & Reth, et al., “Exon “Cell-Tissue Culture and In-Vitro Manipulation' in Corn & 30 Trap Cloning: Using PCR to Rapidly Detect and Clone Exons Corn Improvement, 3rd Edition 10Sprague et al. (Eds. pp. from Genomic DNA Fragments. Nucleic Acids Research, 345-387) American Society of Agronomy Inc. et al. 1988. Vol. 18, No. 22, p. 674. Methods of introducing polynucleotides into plant tissue Entrapment vectors, first described for use in bacteria include the direct infection or co-cultivation of plant cell with (Casadabanand Cohen, 1979, Proc. Nat. Aca. Sci. U.S.A., 76: Agrobacterium tumefaciens, Horsch et al., Science, 227: 1229 35 4530; Casadaban et al., 1980, J. Bacteriol., 143: 971) permit (1985). Descriptions of Agrobacterium vector systems and selection of insertional events that lie within coding methods for Agrobacterium-mediated gene transfer provided sequences. Entrapment vectors can be introduced into pluri by Gruber et al. supra. potent ES cells in culture and then passed into the germline Alternatively, polynucleotides are introduced into plant via chimeras (Gossler et al., 1989, Science, 244: 463; Skar cells or other plant tissues using a direct gene transfer method 40 nes, 1990, Biotechnology, 8: 827). Promoter or gene trap such as microprojectile-mediated delivery, DNA injection, vectors often contain a reporter gene, e.g., lac7, lacking its electroporation and the like. More preferably polynucleotides own promoter and/or splice acceptor sequence upstream. are introduced into plant tissues using the microprojectile That is, promoter gene traps contain a reporter gene with a media delivery with the biolistic device. See, for example, splice site but no promoter. If the vector lands in a gene and is Tomes et al., “Direct DNA transfer into intact plant cells via 45 spliced into the gene product, then the reporter gene is microprojectile bombardment In: Gamborg and Phillips expressed. (Eds.) Plant Cell, Tissue and Organ Culture: Fundamental Recently, the isolation of preferentially-induced genes has Methods, Springer Verlag, Berlin (1995). been made possible with the use of sophisticated promoter In another embodiment of the current invention, expression traps (e.g. IVET) that are based on conditional auxotrophy constructs can be used for gene expression in callus culture 50 complementation or drug resistance. In one IVET approach, for the purpose of expressing marker genes encoding peptides various bacterial genome fragments are placed in front of a or polypeptides that allow identification of transformed necessary metabolic gene coupled to a reporter gene. The plants. Here, a promoter that is operatively linked to a poly DNA constructs are inserted into a bacterial strain otherwise nucleotide to be transcribed is transformed into plant cells lacking the metabolic gene, and the resulting bacteria are used and the transformed tissue is then placed on callus-inducing 55 to infect the host organism. Only bacteria expressing the media. If the transformation is conducted with leaf discs, for metabolic gene Survive in the host organism; consequently, example, callus will initiate along the cut edges. Once callus inactive constructs can be eliminated by harvesting only bac growth has initiated, callus cells can be transferred to callus teria that survive for some minimum period in the host. At the shoot-inducing or callus root-inducing media. Gene expres same time, constitutively active constructs can be eliminated sion will occur in the callus cells developing on the appropri 60 by screening only bacteria that do not express the reporter ate media: callus root-inducing promoters will be activated on gene under laboratory conditions. The bacteria selected by callus root-inducing media, etc. Examples of such peptides or Such a method contain constructs that are selectively induced polypeptides useful as transformation markers include, but only during infection of the host. The WET approach can be are not limited to barstar, glyphosate, chloramphenicol modified for use in plants to identify genes induced in either acetyltransferase (CAT), kanamycin, spectinomycin, strepto 65 the bacteria or the plant cells upon pathogen infection or root mycin or other antibiotic resistance enzymes, green fluores colonization. For information on WET see the articles by cent protein (GFP), and B-glucuronidase (GUS), etc. Some of Mahan et al. in Science 259:686-688 (1993), Mahan et al. in US 7,868,155 B2 25 26 PNAS USA92:669-673 (1995), Heithoffetal. in PNAS USA stress can protect cells against damage caused by oxidative 94:934-939 (1997), and Wang et al. in PNAS USA. 93:10434 agents, such as hydrogen peroxide and other free radicals. (1996). All of the references cited in this section are hereby Drought induction of genes, transcripts, and/or polypep incorporated by reference. tides are useful to increase the viability of a plant, for Constitutive Transcription example, when water is a limiting factor. In contrast, genes, Use of promoters and control elements providing consti transcripts, and/or polypeptides induced during oxygen stress tutive transcription is desired for modulation of transcription can help the flood tolerance of a plant. in most cells of an organism under most environmental con The promoters and control elements of the present inven ditions. In a plant, for example, constitutive transcription is tion can modulate stresses similar to those described in, for useful for modulating genes involved in defense, pest resis 10 example, stress conditions are VuPLD1 (drought stress; Cow tance, herbicide resistance, etc. pea; see Pham-Thiet al., 1999, Plant Mol Biol 1257-65), Constitutive up-regulation and transcription down-regula pyruvate decarboxylase (oxygen stress; rice; see Rivosal et tion is useful for these applications. For instance, genes, tran al., 1997, Plant Physiol 114(3): 1021–29), chromoplast spe Scripts, and/or polypeptides that increase defense, pest and cific carotenoid gene (oxidative stress; capsicum; see Bouvier herbicide resistance may require constitutive up-regulation of 15 et al., 1998, J Biol Chem 273: 30651-59). transcription. In contrast, constitutive transcriptional down Promoters and control elements providing preferential regulation may be desired to inhibit those genes, transcripts, transcription during wounding or induced by methyl jas and/or polypeptides that lower defense, pest and herbicide monate can produce a defense response in host cells or organ resistance. isms. In a plant, for example, preferential modulation of Typically, promoter or control elements that provide con genes, transcripts, and/or polypeptides under Such conditions stitutive transcription produce transcription levels that are is useful to induce a defense response to mechanical wound statistically similar in many tissues and environmental con ing, pest or pathogen attack or treatment with certain chemi ditions observed. cals. Calculation of P-value from the different observed tran Promoters and control elements of the present invention Script levels is one means of determining whether a promoter 25 also can trigger a response similar to those described for cf) or control element is providing constitutive up-regulation. (viral pathogen; tomato; see O’Donnell et al., 1998, Plant J P-value is the probability that the difference of transcript 14(1): 137-42), hepatocyte growth factor activator inhibitor levels is not statistically significant. The higher the P-value, type 1 (HAI-1), which enhances tissue regeneration (tissue the more likely the difference of transcript levels is not sig injury; human; Koono et al., 1999, J. Histochem Cytochem 30 47: 673-82), copper amine oxidase (CuAO), induced during nificant. One formula used to calculate P-value is as follows: ontogenesis and woundhealing (wounding; chick-pea; Rea et ?p(x).dx, integrated from a to Oo, al., 1998, FEBS Ltr 437: 177-82), proteinase inhibitor II where p(x) is a normal distribution; (wounding: potato; see Pena-Cortes et al., 1988, Planta 174: 84-89), protease inhibitor II (methyl jasmonate; tomato; see 35 Farmer and Ryan, 1990, Proc Natl AcadSci USA 87: 7713 where a = —t - 7716), two vegetative storage protein genes Visp A and VspB TO (all Samples except Sv) (wounding, jasmonic acid, and water deficit; soybean; see Mason and Mullet, 1990, Plant Cell 2: 569-579). where SX=the intensity of the sample of interest Up-regulation and transcription down-regulation are use 40 ful for these applications. For instance, genes, transcripts, and/or polypeptides that increase oxidative, flood, or drought where u = is the average of the intensities of all samples tolerance may require up-regulation of transcription. In con except Sx, trast, transcriptional down-regulation may be desired to (XS1...Sn) - Sx inhibit those genes, transcripts, and/or polypeptides that 45 lower Such tolerance. in - 1 Typically, promoter or control elements, which provide preferential transcription in wounding or under methyl jas where O(S1 ... S11, not including SX)=the standard devia monate induction, produce transcript levels that are statisti tion of all sample intensities except SX. cally significant as compared to cell types, organs or tissues 50 under other conditions. The P-value from the formula ranges from 1.0 to 0.0. For preferential up-regulation of transcription, promoter Usually, each P-value of the transcript levels observed in a and control elements produce transcript levels that are above majority of cells, tissues, or organs under various environ background of the assay. mental conditions produced by the promoter or control ele All of the references cited in this section are hereby incor ment is greater than 10; more usually, greater than 107; 55 porated by reference. even more usually, greater than 10"; even more usually, Light Induced Preferential Transcription greater than 10 or 10. Promoters and control elements providing preferential For up-regulation of transcription, promoter and control transcription when induced by light exposure can be utilized elements produce transcript levels that are above background to modulate growth, metabolism, and development; to of the assay. 60 increase drought tolerance; and decrease damage from light Stress Induced Preferential Transcription stress for host cells or organisms. In a plant, for example, Promoters and control elements providing modulation of modulation of genes, transcripts, and/or polypeptides in transcription under oxidative, drought, oxygen, wound, and response to light is useful methyl jasmonate stress are particularly useful for producing (1) to increase the photosynthetic rate; host cells or organisms that are more resistant to biotic and 65 (2) to increase storage of certain molecules in leaves or abiotic stresses. In a plant, for example, modulation of genes, green parts only, e.g., Silage with high protein or starch transcripts, and/or polypeptides in response to oxidative content; US 7,868,155 B2 27 28 (3) to modulate production of exogenous compositions in host cells or organisms. In a plant, for example, preferential green tissue, e.g., certain feed enzymes; modulation of genes, transcripts, and/or polypeptide in a leaf. (4) to induce growth or development, Such as fruit devel is useful, for example, opment and maturity, during extended exposure to light; (1) to modulate leaf size, shape, and development; (5) to modulate guard cells to control the size of stomata in (2) to modulate the number of leaves; or leaves to prevent water loss, or (3) to modulate energy or nutrient usage in relation to other (6) to induce accumulation of beta-carotene to help plants organs and tissues cope with light induced stress. Up-regulation and transcription down-regulation is useful The promoters and control elements of the present invention for these applications. For instance, genes, transcripts, and/or also can trigger responses similar to those described in: absci 10 polypeptides that increase growth, for example, may require sic acid insensitive3 (ABI3) (dark-grown Arabidopsis seed up-regulation of transcription. In contrast, transcriptional lings, see Rohde et al., 2000, Plant Cell 12:35-52), asparagine down-regulation may be desired to inhibit energy usage in a synthetase (pea root nodules, see Tsai and Coruzzi, 1990, leaf to be directed to the fruit instead, for instance. EMBO J.9: 323-32), mdm2 gene (human tumor; see Saucedo Typically, promoter or control elements, which provide et al., 1998, Cell Growth Differ 9: 119-30). 15 preferential transcription in the cells, tissues, or organs of a Up-regulation and transcription down-regulation are use leaf, produce transcript levels that are statistically significant ful for these applications. For instance, genes, transcripts, as compared to other cells, organs or tissues. and/or polypeptides that increase drought or light tolerance For preferential up-regulation of transcription, promoter may require up-regulation of transcription. In contrast, tran and control elements produce transcript levels that are above Scriptional down-regulation may be desired to inhibit those background of the assay. genes, transcripts, and/or polypeptides that lower Such toler Root Preferential Transcription aCC. Promoters and control elements providing preferential Typically, promoter or control elements, which provide transcription in a root can modulate growth, metabolism, preferential transcription in cells, tissues or organs exposed to development, nutrient uptake, nitrogen fixation, or modulate light, produce transcript levels that are statistically significant 25 energy and nutrient utilization in host cells or organisms. In a as compared to cells, tissues, or organs under decreased light plant, for example, preferential modulation of genes, tran exposure (intensity or length of time). Scripts, and/or in a leaf, is useful For preferential up-regulation of transcription, promoter (1) to modulate root size, shape, and development; and control elements produce transcript levels that are above (2) to modulate the number of roots, or root hairs; background of the assay. 30 (3) to modulate mineral, fertilizer, or water uptake: All of the references cited in this section are hereby incor (4) to modulate transport of nutrients; or porated by reference. (4) to modulate energy or nutrient usage in relation to other Dark Induced Preferential Transcription organs and tissues. Promoters and control elements providing preferential Up-regulation and transcription down-regulation is useful transcription when induced by dark or decreased light inten 35 for these applications. For instance, genes, transcripts, and/or sity or decreased light exposure time can be utilized to time polypeptides that increase growth, for example, may require growth, metabolism, and development, to modulate photo up-regulation of transcription. In contrast, transcriptional synthesis capabilities for host cells or organisms. In a plant, down-regulation may be desired to inhibit nutrient usage in a for example, modulation of genes, transcripts, and/or root to be directed to the leaf instead, for instance. polypeptides in response to dark is useful, for example, 40 Typically, promoter or control elements, which provide (1) to induce growth or development, such as fruit devel preferential transcription in cells, tissues, or organs of a root, opment and maturity, despite lack of light; produce transcript levels that are statistically significant as (2) to modulate genes, transcripts, and/or polypeptide compared to other cells, organs or tissues. active at night or on cloudy days; or For preferential up-regulation of transcription, promoter 45 and control elements produce transcript levels that are above (3) to preserve the plastid ultrastructure present at the onset background of the assay. of darkness. Stem/Shoot Preferential Transcription The present promoters and control elements can also trigger Promoters and control elements providing preferential response similar to those described in the section above. transcription in a stem or shoot can modulate growth, metabo Up-regulation and transcription down-regulation is useful 50 lism, and development or modulate energy and nutrient uti for these applications. For instance, genes, transcripts, and/or lization in host cells or organisms. In a plant, for example, polypeptides that increase growth and development may preferential modulation of genes, transcripts, and/or polypep require up-regulation of transcription. In contrast, transcrip tide in a stem or shoot, is useful, for example, tional down-regulation may be desired to inhibit those genes, (1) to modulate stem/shoot size, shape, and development; transcripts, and/or polypeptides that modulate photosynthesis 55 O capabilities. (2) to modulate energy or nutrient usage in relation to other Typically, promoter or control elements, which provide organs and tissues preferential transcription under exposure to dark or decrease Up-regulation and transcription down-regulation is useful light intensity or decrease exposure time, produce transcript for these applications. For instance, genes, transcripts, and/or levels that are statistically significant. 60 polypeptides that increase growth, for example, may require For preferential up-regulation of transcription, promoter up-regulation of transcription. In contrast, transcriptional and control elements produce transcript levels that are above down-regulation may be desired to inhibit energy usage in a background of the assay. stem/shoot to be directed to the fruit instead, for instance. LeafPreferential Transcription Typically, promoter or control elements, which provide Promoters and control elements providing preferential 65 preferential transcription in the cells, tissues, or organs of a transcription in a leaf can modulate growth, metabolism, and stem or shoot, produce transcript levels that are statistically development or modulate energy and nutrient utilization in significant as compared to other cells, organs or tissues. US 7,868,155 B2 29 30 For preferential up-regulation of transcription, promoter Flower Specific Transcription and control elements produce transcript levels that are Promoters and control elements providing preferential above background of the assay. transcription inflowers can modulate pigmentation; or modu Fruit and Seed Preferential Transcription late fertility in host cells or organisms. In a plant, for example, Promoters and control elements providing preferential 5 preferential modulation of genes, transcripts, and/or polypep transcription in a silique or fruit can time growth, develop tides in a flower, is useful, ment, or maturity; or modulate fertility; or modulate energy (1) to modulate petal color; or and nutrient utilization in host cells or organisms. In a plant, (2) to modulate the fertility of pistil and/or stamen. for example, preferential modulation of genes, transcripts, Up-regulation and transcription down-regulation is useful and/or polypeptides in a fruit, is useful 10 for these applications. For instance, genes, transcripts, and/or (1) to modulate fruit size, shape, development, and matu polypeptides that increase pigmentation, for example, may rity: require up-regulation of transcription. In contrast, transcrip (2) to modulate the number of fruit or seeds; tional down-regulation may be desired to inhibit fertility, for (3) to modulate seed shattering; instance. (4) to modulate components of seeds. Such as, storage 15 Typically, promoter or control elements, which provide molecules, starch, protein, oil, vitamins, anti-nutritional preferential transcription in flowers, produce transcript levels components, such as phytic acid; that are statistically significant as compared to other cells, (5) to modulate seed and/or seedling vigor or viability; organs or tissues. (6) to incorporate exogenous compositions into a seed, For preferential up-regulation of transcription, promoter Such as lysine rich proteins; 2O and control elements produce transcript levels that are above (7) to permit similar fruit maturity timing for early and late background of the assay. blooming flowers; or Immature Bud and Inflorescence Preferential Transcrip (8) to modulate energy or nutrient usage in relation to other tion organs and tissues. Promoters and control elements providing preferential Up-regulation and transcription down-regulation is useful 25 transcription in a immature bud or inflorescence can time for these applications. For instance, genes, transcripts, and/or growth, development, or maturity; or modulate fertility or polypeptides that increase growth, for example, may require viability in host cells or organisms. In a plant, for example, up-regulation of transcription. In contrast, transcriptional preferential modulation of genes, transcripts, and/or polypep down-regulation may be desired to inhibit late fruit maturity, tide in a fruit, is useful, for instance. 30 (1) to modulate embryo development, size, and maturity; Typically, promoter or control elements, which provide (2) to modulate endosperm development, size, and compo preferential transcription in the cells, tissues, or organs of sition; siliques or fruits, produce transcript levels that are statisti (3) to modulate the number of seeds and fruits; or cally significant as compared to other cells, organs or tissues. (4) to modulate seed development and viability. For preferential up-regulation of transcription, promoter 35 Up-regulation and transcription down-regulation is useful and control elements produce transcript levels that are above for these applications. For instance, genes, transcripts, and/or background of the assay. polypeptides that increase growth, for example, may require Callus Preferential Transcription up-regulation of transcription. In contrast, transcriptional Promoters and control elements providing preferential down-regulation may be desired to decrease endosperm size, transcriptionina callus can be useful to modulating transcrip- 40 for instance. tion in dedifferentiated host cells. In a plant transformation, Typically, promoter or control elements, which provide for example, preferential modulation of genes, transcripts, in preferential transcription in immature buds and inflores callus is useful to modulate transcription of a marker gene, cences, produce transcript levels that are statistically signifi which can facilitate selection of cells that are transformed cant as compared to other cell types, organs or tissues. with exogenous polynucleotides. 45 For preferential up-regulation of transcription, promoter Up-regulation and transcription down-regulation is useful and control elements produce transcript levels that are above for these applications. For instance, genes, transcripts, and/or background of the assay. polypeptides that increase marker gene detectability, for Senescence Preferential Transcription example, may require up-regulation of transcription. In con Promoters and control elements providing preferential trast, transcriptional down-regulation may be desired to 50 transcription during senescencing can be used to modulate increase the ability of the calluses to later differentiate, for cell degeneration, nutrient mobilization, and scavenging of instance. free radicals in host cells or organisms. Other types of Typically, promoter or control elements, which provide responses that can be modulated include, for example, Senes preferential transcription in callus, produce transcript levels cence associated genes (SAG) that encode enzymes thought that are statistically significant as compared to other cell 55 to be involved in cell degeneration and nutrient mobilization types, tissues, or organs. Calculation of P-value from the (arabidopsis: see Hensel et al. 1993. Plant Cell 5: 553-64), different observed transcript levels is one means of determin and the CP-2/cathepsin L gene (rat; Kim and Wright. 1997. ing whether a promoter or control element is providing Such Biol Reprod 57: 1467-77), both induced during senescence. preferential transcription. In a plant, for example, preferential modulation of genes, Usually, each P-value of the transcript levels observed in 60 transcripts, and/or polypeptides during senescence is useful callus as compared to, at least one other cell type, tissue or to modulate fruit ripening. organ, is less than 10"; more usually, less than 10: even Up-regulation and transcription down-regulation is useful more usually, less than 10: even more usually, less than 107 for these applications. For instance, genes, transcripts, and/or or 10. polypeptides that increase Scavenging of free radicals, for For preferential up-regulation of transcription, promoter 65 example, may require up-regulation of transcription. In con and control elements produce transcript levels that are above trast, transcriptional down-regulation may be desired to background of the assay. inhibit cell degeneration, for instance. US 7,868,155 B2 31 32 Typically, promoter or control elements, which provide sequences. In general, transformants with the constructs were preferential transcription in cells, tissues, or organs during grown to an appropriate stage, and tissue samples were pre senescence, produce transcript levels that are statistically sig pared for the microarray differential expression analysis. In nificant as compared to other conditions. this manner it is possible to determine the differential expres For preferential up-regulation of transcription, promoter sion for the cDNAs under the control of the endogenous and control elements produce transcript levels that are above promoter under various conditions. background of the assay. Germination Preferential Transcription Microarray Experimental Procedures and Results Procedures Promoters and control elements providing preferential 1. Sample Tissue Preparation transcription in a germinating seed can time growth, devel 10 Tissue samples for each of the expression analysis experi opment, or maturity; or modulate viability in host cells or ments were prepared as follows: organisms. In a plant, for example, preferential modulation of (a) Roots genes, transcripts, and/or polypeptide in a germinating seed, Seeds of Arabidopsis thaliana (Ws) were sterilized in full is useful, strength bleach for less than 5 min., washed more than 3 times (1) to modulate the emergence of they hypocotyls, cotyle 15 in sterile distilled deionized water and plated on MS agar dons and radical; or plates. The plates were placed at 4°C. for 3 nights and then (2) to modulate shoot and primary root growth and devel placed vertically into a growth chamber having 16 hr light/8 opment; hr dark cycles, 23° C., 70% relative humidity and -11,000 Up-regulation and transcription down-regulation is useful LUX. After 2 weeks, the roots were cut from the agar, flash for these applications. For instance, genes, transcripts, and/or frozen in liquid nitrogen and stored at -80°C. polypeptides that increase growth, for example, may require (b) Rosette Leaves, Stems, and Siliques up-regulation of transcription. In contrast, transcriptional Arabidopsis thaliana (Ws) seed was vernalized at 4°C. for down-regulation may be desired to decrease endosperm size, 3 days before sowing in Metro-mix soil type 350. Flats were for instance. placed in a growth chamber having 16 hr light/8 hr dark, 80% Typically, promoter or control elements, which provide 25 relative humidity, 23° C. and 13,000 LUX for germination preferential transcription in a germinating seed, produce tran and growth. After 3 weeks, rosette leaves, stems, and siliques Script levels that are statistically significant as compared to were harvested, flash frozen in liquid nitrogen and stored at other cell types, organs or tissues. -80°C. until use. After 4 weeks, siliques (<5 mm, 5-10 mm For preferential up-regulation of transcription, promoter and >10 mm) were harvested, flash frozen in liquid nitrogen and control elements produce transcript levels that are above 30 and stored at -80°C. until use. 5 week old whole plants (used background of the assay. as controls) were harvested, flash frozen in liquid nitrogen Microarray Analysis and kept at -80° C. until RNA was isolated. A major way that a cell controls its response to internal or (c) Germination external stimuli is by regulating the rate of transcription of 35 Arabidopsis thaliana seeds (ecotype Ws) were sterilized in specific genes. For example, the differentiation of cells during bleach and rinsed with sterile water. The seeds were placed in organogenesis into forms characteristic of the organ is asso 100 mm petri plates containing soaked autoclaved filter ciated with the selective activation and repression of large paper. Plates were foil-wrapped and left at 4°C. for 3 nights numbers of genes. Thus, specific organs, tissues and cells are to vernalize. After cold treatment, the foil was removed and functionally distinct due to the different populations of 40 plates were placed into a growth chamber having 16 hr light/8 mRNAS and protein products they possess. Internal signals hr dark cycles, 23° C., 70% relative humidity and -11,000 program the selective activation and repression programs. For lux. Seeds were collected 1 d, 2 d, 3 d and 4 d later, flash example, internally synthesized hormones produce Such sig frozen in liquid nitrogen and stored at -80°C. until RNA was nals. The level of hormone can be raised by increasing the isolated. level of transcription of genes encoding proteins concerned 45 (d) Abscissic Acid (ABA) with hormone synthesis. Seeds of Arabidopsis thaliana (ecotype Wassilewskija) To measure how a cell reacts to internal and/or external were sown in trays and left at 4°C. for 4 days to vernalize. stimuli, individual mRNA levels can be measured and used as They were then transferred to a growth chamber having an indicator for the extent of transcription of the gene. Cells grown 16 hr light/8 hr dark, 13,000 LUX, 70% humidity, and can be exposed to a stimulus, and mRNA can be isolated and 50 20° C. and watered twice a week with 1 L of 1x Hoagland's assayed at different time points after stimulation. The mRNA solution. Approximately 1,000 14 day old plants were spayed from the stimulated cells can be compared to control cells that with 200-250 mls of 100 uMABA in a 0.02% solution of the were not stimulated. The mRNA levels that are higher in the detergent Silwet L-77. Whole seedlings, including roots, stimulated cell versus the control indicate a stimulus-specific were harvested within a 15 to 20 minute time period at 1 hr response of the cell. The same is true of mRNA levels that are 55 and 6 hr after treatment, flash-frozen in liquid nitrogen and lower in stimulated cells versus the control condition. Stored at -80° C. Similar studies can be performed with cells taken from an Seeds of maize hybrid 35A (Pioneer) were sown in water organism with a defined mutation in their genome as com moistened sand in flats (10 rows, 5-6 seed/row) and covered pared with cells without the mutation. Altered mRNA levels with clear, plastic lids before being placed in a growth cham in the mutated cells indicate how the mutation causes tran 60 ber having 16hr light (25°C.)/8 hr dark (20°C.), 75% relative Scriptional changes. These transcriptional changes are asso humidity and 13,000-14,000 LUX. Covered flats were ciated with the phenotype that the mutated cells exhibit that is watered every three days for 7 days. Seedlings were carefully different from the phenotype exhibited by the control cells. removed from the sand and placed in 1-liter beakers with 100 Applicants have utilized microarray techniques to measure uM ABA for treatment. Control plants were treated with the levels of mRNAs in cells from plants transformed with a 65 water. After 6 hr and 24 hr, aerial and root tissues were construct containing the promoter or control elements of the separated and flash frozen in liquid nitrogen prior to storage at present invention together with their endogenous clNA -80° C. US 7,868,155 B2 33 34 (e) Brassinosteroid Responsive (high nitrate modified 1XMS media). Plates were then grown Two separate experiments were performed, one with epi for 7 days in a Percival growth chamber at 22°C. with 16 hr. brassinolide and one with the brassinosteroid biosynthetic light/8 hr dark. inhibitor brassinazole. In the epi-brassinolide experiments, Germinated seedlings were then transferred to a sterile flask containing 50 mL of high nitrate modified 1xMS liquid seeds of wild-type Arabidopsis thaliana (ecotype Was silews media. Seedlings were grown with mild shaking for 3 addi kija) and the bras sinosteroid biosynthetic mutant dwf4-1 tional days at 22°C. in 16 hr. light/8 hr dark (in a Percival were sown in trays and left at 4°C. for 4 days to vernalize. growth chamber) on the high nitrate modified 1xMS liquid They were then transferred to a growth chamber having 16 hr media. light/8 hr dark, 11,000 LUX, 70% humidity and 22°C. tem 10 After three days of growth on high nitrate modified 1xMS perature. Four week old plants were spayed with a 1 uM liquid media, seedlings were transferred either to a new sterile Solution of epi-brassinolide and shoot parts (unopened floral flask containing 50 mL of high nitrate modified 1xMS liquid primordia and shoot apical meristems) harvested three hours media or to low nitrate modified 1xMS liquid media (con later. Tissue was flash-frozen in liquid nitrogen and stored at taining 20 uM KNO). Seedlings were grown in these media -80°C. In the brassinazole experiments, seeds of wild-type 15 conditions with mild shaking at 22°C. in 16 hr light/8 hr dark Arabidopsis thaliana (ecotype Wassilewskija) were grown as for the appropriate time points and whole seedlings harvested described above. Four week old plants were spayed with a 11 for total RNA isolation via the Trizol method (LifeTech.). The uM solution of brassinazole and shoot parts (unopened floral time points used for the microarray experiments were 10 min. primordia and shoot apical meristems) harvested three hours and 1 hour time points for both the high and low nitrate later. Tissue was flash-frozen in liquid nitrogen and stored at modified 1XMS media. -80° C. Alternatively, seeds that were surface sterilized in 30% In addition to the spray experiments, tissue was prepared bleach containing 0.1% Triton X-100 and further rinsed in from two different mutants: (1) a dwf4-1 knock out mutant sterile water, were planted on MSagar, (0.5% sucrose) plates and (2) a mutant overexpressing the dwf4-1 gene. containing 50 mM KNO (potassium nitrate). The seedlings Seeds of wild-type Arabidopsis thaliana (ecotype 25 were grown under constant light (3500 LUX) at 22°C. After Wassilewskija) and of the dwf4-1 knockout and over-expres 12 days, seedlings were transferred to MS agar plates con sor mutants were sown in trays and left at 4°C. for 4 days to taining either 1 mMKNO or 50 mMKNO. Seedlings trans vernalize. They were then transferred to a growth chamber ferred to agarplates containing 50 mMKNO, were treated as having 16 hr light/8 hr dark, 11,000 LUX, 70% humidity and controls in the experiment. Seedlings transferred to plates 22°C. temperature. Tissue from shoot parts (unopened floral 30 with 1 mM KNO, were rinsed thoroughly with sterile MS primordia and shoot apical meristems) was flash-frozen in solution containing 1 mM KNO. There were ten plates per liquid nitrogen and stored at -80° C. transfer. Roottissue was collected and frozen in 15 mL Falcon Another experiment was completed with seeds of Arabi tubes at various time points which included 1 hour, 2 hours, 3 dopsis thaliana (ecotype Wassilewskija) were Sown in trays hours, 4 hours, 6 hours, 9 hours, 12 hours, 16 hours, and 24 and left at 4° C. for 4 days to vernalize. They were then 35 hours. transferred to a growth chamber. Plants were grown under Maize 35A19 Pioneer hybrid seeds were sown on flats long-day (16 hr light: 8 hr. dark) conditions, 13,000 LUX containing sand and grown in a Conviron growth chamber at light intensity, 70% humidity, 20°C. temperature and watered 25°C., 16 hr light/8 hr dark, -13,000 LUX and 80% relative twice a week with 1 L1x Hoagland's solution (recipe recited humidity. Plants were watered every three days with double in Feldmann et al., (1987) Mol. Gen. Genet. 208: 1-9, hereby 40 distilled deionized water. Germinated seedlings are allowed incorporated by reference) and described as complete nutri to grow for 10 days and were watered with high nitrate modi ent solution). Approximately 1,000 14 day old plants were fied 1xMS liquid media (see above). On day 11, young corn spayed with 200-250 mls of 0.11 uM Epi-Bras sinolite in seedlings were removed from the sand (with their roots intact) 0.02% solution of the detergent Silwet L-77. At 1 hr. and 6hrs. and rinsed briefly in high nitrate modified 1xMS liquid after treatment aerial tissues were harvested within a 15 to 20 45 media. The equivalent of half a flat of seedlings were then minute time period and flash-frozen in liquid nitrogen. Submerged (up to their roots) in a beaker containing either 500 mL of high or low nitrate modified 1xMS liquid media Seeds of maize hybrid 35A (Pioneer) were sown in water (see above for details). moistened sand in flats (10 rows, 5-6 seed/row) and covered At appropriate time points, seedlings were removed from with clear, plastic lids before being placed in a growth cham 50 their respective liquid media, the roots separated from the ber having 16hr light (25°C.)/8 hr dark (20°C.), 75% relative shoots and each tissue type flash frozen in liquid nitrogen and humidity and 13,000-14,000 LUX. Covered flats were stored at -80°C. This was repeated for each time point. Total watered every three days for 7 days. Seedlings were carefully RNA was isolated using the Trizol method (see above) with removed from the sand and placed in 1-liter beakers with 0.1 root tissues only. uM epi-brassinolide for treatment. Control plants were 55 Corn root tissues isolated at the 4 hr and 16 hr time points treated with distilled deionized water. After 24 hr, aerial and were used for the microarray experiments. Both the high and root tissues were separated and flash frozen in liquid nitrogen low nitrate modified 1xMS media were used. prior to storage at -80° C. (g) Nitrogen: Low to High (f) Nitrogen: High to Low Arabidopsis thaliana ecotype Wis seeds were sown on flats Wild type Arabidopsis thaliana seeds (ecotype Ws) were 60 containing 4 L of a 1:2 mixture of Grace Zonolite vermiculite surface sterilized with 30% Clorox, 0.1% Triton X-100 for 5 and soil. Flats were watered with 3 L of water and vernalized minutes. Seeds were then rinsed with 4-5 exchanges of sterile at 4°C. for five days. Flats were placed in a Conviron growth double distilled deionized water. Seeds were vernalized at 4° chamber having 16 hr light/8 hr dark at 20°C., 80% humidity C. for 2-4 days in darkness. After cold treatment, seeds were and 17.450 LUX. Flats were watered with approximately 1.5 plated on modified 1XMS media (without NHNO or 65 L of water every four days. Mature, bolting plants (24 days KNO), 0.5% sucrose, 0.5 g/L MES pH5.7, 1% phytagar and after germination) were bottom treated with 2 L of either a supplemented with KNO to a final concentration of 60 mM control (100 mM mannitol pH 5.5) or an experimental (50 US 7,868,155 B2 35 36 mM ammonium nitrate, pH 5.5) solution. Roots, leaves and days post germination plants were sprayed with 2 mMSA, siliques were harvested separately 30, 120 and 240 minutes 0.02% SilwettL-77 or control solution (0.02% SilwettL-77. after treatment, flash frozen in liquid nitrogen and stored at Aerial parts or flowers were harvested 1 hr, 4 hr, 6 hr, 24hr and -80° C. 3 weeks post-treatment flash frozen and stored at -80° C. Hybrid maize seed (Pioneer hybrid 35A19) were aerated 5 Seeds of maize hybrid 35A (Pioneer) were sown in water overnight in deionized water. Thirty seeds were plated in each moistened sand in flats (10 rows, 5-6 seed/row) and covered flat, which contained 4 liters of Grace Zonolite vermiculite. with clear, plastic lids before being placed in a growth cham Two liters of water were bottom fed and flats were kept in a ber having 16hr light (25°C.)/8 hr dark (20°C.), 75% relative Conviron growth chamber with 16 hr light/8 hr dark at 20°C. humidity and 13,000-14,000 LUX. Covered flats were and 80% humidity. Flats were watered with 1 L of tap water 10 watered every three days for 7 days. Seedlings were carefully every three days. Five day old seedlings were treated as removed from the sand and placed in 1-liter beakers with 2 described above with 2 L of either a control (100 mM man mMSA for treatment. Control plants were treated with water. nitol pH 6.5) solution or 1 L of an experimental (50 mM After 12 hr and 24 hr, aerial and root tissues were separated ammonium nitrate, pH 6.8) solution. Fifteen shoots per time and flash frozen in liquid nitrogen prior to storage at -80° C. point per treatment were harvested 10, 90 and 180 minutes 15 (j) Drought Stress after treatment, flash frozen in liquid nitrogen and stored at Seeds of Arabidopsis thaliana (Wassilewskija) were sown -80° C. in pots and left at 4°C. for three days to vernalize before being Alternatively, seeds of Arabidopsis thaliana (ecotype transferred to a growth chamber having 16 hr light/8 hr dark, Wassilewskija) were left at 4°C. for 3 days to vernalize. They 150,000-160,000 LUX, 20° C. and 70% humidity. After 14 were then Sown on Vermiculite in a growth chamber having 16 days, aerial tissues were cut and left to dry on 3mMWhattman hours light/8 hours dark, 12,000-14,000 LUX, 70% humidity, paper in a Petri-plate for 1 hour and 6 hours. Aerial tissues and 20°C. They were bottom-watered with tap water, twice exposed for 1 hour and 6 hours to 3 mM Whattman paper weekly. Twenty-four days old plants were sprayed with either wetted with 1x Hoagland's solution served as controls. Tis water (control) or 0.6% ammonium nitrate at 4L/cm of tray Sues were harvested, flash-frozen in liquid nitrogen and Surface. Total shoots and some primary roots were cleaned of 25 Stored at -80° C. vermiculite, flash-frozen in liquid nitrogen and stored at -80° Alternatively, Arabidopsis thaliana (Ws) seed was vernal C. ized at 4°C. for 3 days before sowing in Metromix soil type (h) Methyl Jasmonate 350. Flats were placed in a growth chamber with 23°C., 16 hr Seeds of Arabidopsis thaliana (ecotype Wassilewskija) light/8 hr. dark, 80% relative humidity, ~13,000 LUX for were sown in trays and left at 4°C. for 4 days to vernalize 30 germination and growth. Plants were watered with 1-1.5 L of before being transferred to a growth chamber having 16 hr water every four days. Watering was stopped 16 days after light/8 hr. dark, 13,000 LUX, 70% humidity, 20° C. tempera germination for the treated samples, but continued for the ture and watered twice a week with 1 L of a 1x Hoaglands control samples. Rosette leaves and stems, flowers and siliq solution. Approximately 1,000 14 day old plants were spayed ues were harvested 2d, 3d, 4d, 5 d, 6d and 7d after watering with 200-250 mls of 0.001% methyl jasmonate in a 0.02% 35 was stopped. Tissue was flash frozen in liquid nitrogen and solution of the detergent Silwet L-77. At 1 hr and 6 hrs after kept at -80° C. until RNA was isolated. Flowers and siliques treatment, whole seedlings, including roots, were harvested were also harvested on day 8 from plants that had undergone within a 15 to 20 minute time period, flash-frozen in liquid a 7 d drought treatment followed by 1 day of watering. Con nitrogen and stored at -80° C. trol plants (whole plants) were harvested after 5 weeks, flash Seeds of maize hybrid 35A (Pioneer) were sown in water 40 frozen in liquid nitrogen and stored as above. moistened sand in flats (10 rows, 5-6 seed/row) and covered Seeds of maize hybrid 35A (Pioneer) were sown in water with clear, plastic lids before being placed in a growth cham moistened sand in flats (10 rows, 5-6 seed/row) and covered ber having 16hr light (25°C.)/8 hr dark (20°C.), 75% relative with clear, plastic lids before being placed in a growth cham humidity and 13,000-14,000 LUX. Covered flats were ber having 16hr light (25°C.)/8 hr dark (20°C.), 75% relative watered every three days for 7 days. Seedlings were carefully 45 humidity and 13,000-14,000 LUX. Covered flats were removed from the sand and placed in 1-liter beakers with watered every three days for 7 days. Seedlings were carefully 0.001% methyljasmonate for treatment. Control plants were removed from the sand and placed in empty 1-liter beakers at treated with water. After 24 hr, aerial and root tissues were room temperature for treatment. Control plants were placed separated and flash frozen in liquid nitrogen prior to storage at in water. After 1 hr., 6 hr, 12hr and 24 hr aerial and root tissues -80° C. 50 were separated and flash frozen in liquid nitrogen prior to (i) Salicylic Acid storage at -80°C. Seeds of Arabidopsis thaliana (ecotype Wassilewskija) (k) Osmotic Stress were sown in trays and left at 4°C. for 4 days to vernalize Seeds of Arabidopsis thaliana (Wassilewskija) were sown before being transferred to a growth chamber having 16 hr in trays and left at 4°C. for three days to vernalize before light/8 hr. dark, 13,000 LUX, 70% humidity, 20° C. tempera 55 being transferred to a growth chamber having 16 hr light/8.hr ture and watered twice a week with 1 L of a 1x Hoaglands dark, 12,000-14,000 LUX, 20° C., and 70% humidity. After solution. Approximately 1,000 14 day old plants were spayed 14 days, the aerial tissues were cut and placed on 3 mM with 200-250 mls of 5 mM salicylic acid (solubilized in 70% Whattman paper in a Petri-plate wetted with 20% PEG (poly ethanol) in a 0.02% solution of the detergent Silwet L-77. At ethylene glycol-M, 8,000) in 1x Hoagland's solution. Aerial 1 hr and 6 hrs after treatment, whole seedlings, including 60 tissues on 3 mM Whattman paper containing 1x Hoagland's roots, were harvested within a 15 to 20 minute time period Solution alone served as the control. Aerial tissues were har flash-frozen in liquid nitrogen and stored at -80°C. vested at 1 hour and 6 hours after treatment, flash-frozen in Alternatively, seeds of wild-type Arabidopsis thaliana liquid nitrogen and stored at -80° C. (ecotype Columbia) and mutant CS3726 were sown in soil Seeds of maize hybrid 35A (Pioneer) were sown in water type 200 mixed with osmocote fertilizer and Marathon insec 65 moistened sand in flats (10 rows, 5-6 seed/row) and covered ticide and left at 4° C. for 3 days to vernalize. Flats were with clear, plastic lids before being placed in a growth cham incubated at room temperature with continuous light. Sixteen ber having 16hr light (25°C.)/8 hr dark (20°C.), 75% relative US 7,868,155 B2 37 38 humidity and 13,000-14,000 LUX. Covered flats were and 22°C. temperature. 3-4 siliques (fruits) bearing develop watered every three days for 7 days. Seedlings were carefully ing seeds were selected from at least 3 plants and were hand removed from the sand and placed in 1-liter beakers with 10% dissected to determine what developmental stage(s) were rep PEG (polyethylene glycol-M, 8,000) for treatment. Control resented by the enclosed embryos. Description of the stages plants were treated with water. After 1 hr and 6hr aerial and 5 of Arabidopsis embryogenesis used in this determination root tissues were separated and flash frozen in liquid nitrogen were summarized by Bowman (1994). Silique lengths were prior to storage at -80° C. then determined and used as an approximate determinant for Seeds of maize hybrid 35A (Pioneer) were sown in water embryonic stage. Siliques 0-5 mm in length containing post moistened sand in flats (10 rows, 5-6 seed/row) and covered fertilization through pre-heart stage 0-72 hours after fertili with clear, plastic lids before being placed in a growth cham 10 zation (HAF) embryos were harvested and flash frozen in ber having 16hr light (25°C.)/8 hr dark (20°C.), 75% relative liquid nitrogen. humidity and 13,000-14,000 LUX. Covered flats were Fruits (pod+seed) 5-10 mm watered every three days for 7 days. Seedlings were carefully Seeds of Arabidopsis thaliana (ecotype Wassilewskija) removed from the sand and placed in 1-liter beakers with 150 were sown in pots and left at 4°C. for two to three days to mM NaCl for treatment. Control plants were treated with 15 vernalize. They were then transferred to a growth chamber. water. After 1 hr., 6 hr, and 24 hr aerial and root tissues were Plants were grown under long-day (16 hr light: 8 hr dark) separated and flash frozen in liquid nitrogen prior to storage at conditions, 7000-8000 LUX light intensity, 70% humidity, -80° C. and 22°C. temperature. 3-4 siliques (fruits) bearing develop (1) Heat Shock Treatment ing seeds were selected from at least 3 plants and were hand Seeds of Arabidopsis Thaliana (Wassilewskija) were sown dissected to determine what developmental stage(s) were rep in trays and left at 4°C. for three days to vernalize before resented by the enclosed embryos. Description of the stages being transferred to a growth chamber with 16 hr light/8 hr of Arabidopsis embryogenesis used in this determination dark, 12,000-14,000 Lux, 70% humidity and 20° C., fourteen were summarized by Bowman (1994). Silique lengths were day old plants were transferred to a 42° C. growth chamber then determined and used as an approximate determinant for and aerial tissues were harvested 1 hr and 6 hr after transfer. 25 embryonic stage. Siliques 5-10 mm in length containing Control plants were left at 20° C. and aerial tissues were heart—through early upturned-U-stage 72-120 hours after harvested. Tissues were flash-frozen in liquid nitrogen and fertilization (HAF) embryos were harvested and flash frozen Stored at -80° C. in liquid nitrogen. Seeds of maize hybrid 35A (Pioneer) were sown in water Fruits (pod+seed)>10 mm moistened sand in flats (10 rows, 5-6 seed/row) and covered 30 Seeds of Arabidopsis thaliana (ecotype Wassilewskija) with clear, plastic lids before being placed in a growth cham were sown in pots and left at 4°C. for two to three days to ber having 16hr light (25°C.)/8 hr dark (20° C.), 75% relative vernalize. They were then transferred to a growth chamber. humidity and 13,000-14,000 LUX. Covered flats were Plants were grown under long-day (16 hr light: 8 hr dark) watered every three days for 7 days. Seedlings were carefully conditions, 7000-8000 LUX light intensity, 70% humidity, removed from the sand and placed in 1-liter beakers contain 35 and 22°C. temperature. 3-4 siliques (fruits) bearing develop ing 42°C. water for treatment. Control plants were treated ing seeds were selected from at least 3 plants and were hand with water at 25°C. After 1 hr and 6hr aerial and root tissues dissected to determine what developmental stage(s) were rep were separated and flash frozen in liquid nitrogen prior to resented by the enclosed embryos. Description of the stages storage at -80°C. of Arabidopsis embryogenesis used in this determination (m) Cold Shock Treatment 40 were summarized by Bowman (1994). Silique lengths were Seeds of Arabidopsis thaliana (Wassilewskija) were sown then determined and used as an approximate determinant for in trays and left at 4°C. for three days to vernalize before embryonic stage. Siliques >10 mm in length containing being transferred to a growth chamber having 16 hr light/8.hr green, late upturned-U-stage D120 hours after fertilization dark, 12,000-14,000 LUX, 20° C. and 70% humidity. Four (HAF)-9 days after flowering (DAF) embryos were har teen day old plants were transferred to a 4° C. dark growth 45 Vested and flash frozen in liquid nitrogen. chamber and aerial tissues were harvested 1 hour and 6 hours Green Pods 5-10 mm (Control Tissue for Samples 72-74) later. Control plants were maintained at 20° C. and covered Seeds of Arabidopsis thaliana (ecotype Wassilewskija) with foil to avoid exposure to light. Tissues were flash-frozen were sown in pots and left at 4°C. for two to three days to in liquid nitrogen and stored at -80° C. vernalize. They were then transferred to a growth chamber. Seeds of maize hybrid 35A (Pioneer) were sown in water 50 Plants were grown under long-day (16 hr light: 8 hr dark) moistened sand in flats (10 rows, 5-6 seed/row) and covered conditions, 7000-8000 LUX light intensity, 70% humidity, with clear, plastic lids before being placed in a growth cham and 22°C. temperature. 3-4 siliques (fruits) bearing develop ber having 16hr light (25°C.)/8 hr dark (20°C.), 75% relative ing seeds were selected from at least 3 plants and were hand humidity and 13,000-14,000 LUX. Covered flats were dissected to determine what developmental stage(s) were rep watered every three days for 7 days. Seedlings were carefully 55 resented by the enclosed embryos. Description of the stages removed from the sand and placed in 1-liter beakers contain of Arabidopsis embryogenesis used in this determination ing 4°C. water for treatment. Control plants were treated with were summarized by Bowman (1994). Silique lengths were water at 25°C. After 1 hr and 6hr aerial and root tissues were then determined and used as an approximate determinant for separated and flash frozen in liquid nitrogen prior to storage at embryonic stage. Green siliques 5-10 mm in length contain -80° C. 60 ing developing seeds 72-120 hours after fertilization (HAF) (n) Arabidopsis Seeds were opened and the seeds removed. The remaining tissues Fruits (pod--seed) 0-5 mm (green pods minus seed) were harvested and flash frozen in Seeds of Arabidopsis thaliana (ecotype Wassilewskija) liquid nitrogen. were sown in pots and left at 4°C. for two to three days to Green Seeds from Fruits >10 mm vernalize. They were then transferred to a growth chamber. 65 Seeds of Arabidopsis thaliana (ecotype Wassilewskija) Plants were grown under long-day (16 hr light: 8 hr dark) were sown in pots and left at 4°C. for two to three days to conditions, 7000-8000 LUX light intensity, 70% humidity, vernalize. They were then transferred to a growth chamber. US 7,868,155 B2 39 40 Plants were grown under long-day (16 hr light: 8 hr dark) Seeds were vernalized at 4°C. for 3 days before being plated conditions, 7000-8000 LUX light intensity, 70% humidity, onto GM agar plates at a density of about 144 seeds per plate. and 22°C. temperature. 3-4 siliques (fruits) bearing develop Plates were incubated in a Percival growth chamber having 16 ing seeds were selected from at least 3 plants and were hand hr light/8 hr dark, 80% relative humidity, 22°C. and 11,000 dissected to determine what developmental stage(s) were rep LUX for 14 days. resented by the enclosed embryos. Description of the stages of Arabidopsis embryogenesis used in this determination Plates were sprayed (-0.5 mls/plate) with water, Finale were summarized by Bowman (1994). Silique lengths were (1.128 g/L), Glean (1.88 g/L), RoundUp (0.01 g/L) or Trimec then determined and used as an approximate determinant for (0.08 g/L). Tissue was collected and flash frozen in liquid embryonic stage. Green siliques >10 mm in length containing 10 nitrogen at the following time points: 0, 1, 2, 4, 8, 12 and 24 developing seeds up to 9 days after flowering (DAF) were hours. Frozen tissue was stored at -80° C. prior to RNA opened and the seeds removed and harvested and flash frozen isolation. in liquid nitrogen. (p) Root Tips Brown Seeds from Fruits >10 mm Seeds of Arabidopsis thaliana (ecotype Ws) were placed Seeds of Arabidopsis thaliana (ecotype Wassilewskija) 15 on MS plates and vernalized at 4°C. for 3 days before being were sown in pots and left at 4°C. for two to three days to placed in a 25° C. growth chamber having 16 hr light/8 hr vernalize. They were then transferred to a growth chamber. dark, 70% relative humidity and about 3 W/m. After 6 days, Plants were grown under long-day (16 hr light: 8 hr dark) young seedlings were transferred to flasks containing B5 conditions, 7000-8000 LUX light intensity, 70% humidity, liquid medium, 1% sucrose and 0.05 mg/l indole-3-butyric and 22°C. temperature. 3-4 siliques (fruits) bearing develop acid. Flasks were incubated at room temperature with 100 ing seeds were selected from at least 3 plants and were hand rpm agitation. Media was replaced weekly. After three weeks, dissected to determine what developmental stage(s) were rep roots were harvested and incubated for 1 hr with 2% pecti resented by the enclosed embryos. Description of the stages nase, 0.2% cellulase, pH 7 before straining through a #80 of Arabidopsis embryogenesis used in this determination (Sigma) sieve. The root body material remaining on the sieve were summarized by Bowman (1994). Silique lengths were 25 (used as the control) was flash frozen and stored at -80°C. then determined and used as an approximate determinant for until use. The material that passed through the #80 sieve was embryonic stage. Yellowing siliques >10 mm in length con strained through a #200 (Sigma) sieve and the material taining brown, dessicating seeds >11 days after flowering remaining on the sieve (root tips) was flash frozen and stored (DAF) were opened and the seeds removed and harvested at -80° C. until use. Approximately 10 mg of root tips were and flash frozen in liquid nitrogen. 30 collected from one flask of root culture. Green/Brown Seeds from Fruits >10 mm Seeds of maize hybrid 35A (Pioneer) were sown in water Seeds of Arabidopsis thaliana (ecotype Wassilewskija) moistened sand in flats (10 rows, 5-6 seed/row) and covered were sown in pots and left at 4°C. for two to three days to with clear, plastic lids before being placed in a growth cham vernalize. They were then transferred to a growth chamber. ber having 16hr light (25°C.)/8 hr dark (20°C.), 75% relative Plants were grown under long-day (16 hr light: 8 hr dark) 35 humidity and 13,000-14,000 LUX. Covered flats were conditions, 7000-8000 LUX light intensity, 70% humidity, watered every three days for 8 days. Seedlings were carefully and 22°C. temperature. 3-4 siliques (fruits) bearing develop removed from the sand and the root tips ( 2 mm long) were ing seeds were selected from at least 3 plants and were hand removed and flash frozen in liquid nitrogen prior to storage at dissected to determine what developmental stage(s) were rep -80° C. The tissues above the root tips (—1 cm long) were resented by the enclosed embryos. Description of the stages 40 cut, treated as above and used as control tissue. of Arabidopsis embryogenesis used in this determination were summarized by Bowman (1994). Silique lengths were (q) Imbibed Seed then determined and used as an approximate determinant for Seeds of maize hybrid 35A (Pioneer) were sown in water embryonic stage. Green siliques >10 mm in length containing moistened sand in covered flats (10 rows, 5-6 seed/row) and both green and brown seeds >9 days after flowering (DAF) 45 covered with clear, plastic lids before being placed in a were opened and the seeds removed and harvested and flash growth chamber having 16 hr light (25°C.)/8 hr dark (20°C.), frozen in liquid nitrogen. 75% relative humidity and 13,000-14,000 LUX. One day Mature Seeds (24 Hours after Imbibition) after Sowing, whole seeds were flash frozen in liquid nitrogen Mature dry seeds of Arabidopsis thaliana (ecotype prior to storage at -80°C. Two days after sowing, embryos Wassilewskija) were sown onto moistened filter paper and 50 and endosperm were isolated and flash frozen in liquid nitro left at 4°C. for two to three days to vernalize. Imbibed seeds gen prior to storage at -80° C. On days 3-6, aerial tissues, were then transferred to a growth chamber 16 hr light: 8 hr roots and endosperm were isolated and flash frozen in liquid dark conditions, 7000-8000 LUX light intensity, 70% humid nitrogen prior to storage at -80° C. ity, and 22° C. temperature, the emerging seedlings har (r) Flowers (Green, White or Buds) vested after 48 hours and flash frozen in liquid nitrogen. 55 Approximately 10 ul of Arabidopsis thaliana seeds Mature Seeds (Dry) (ecotype Ws) were sown on 350 soil (containing 0.03% mara Seeds of Arabidopsis thaliana (ecotype Wassilewskija) thon) and vernalized at 4C for 3 days. Plants were then grown were sown in pots and left at 4°C. for two to three days to at room temperature under fluorescent lighting until flower vernalize. They were then transferred to a growth chamber. ing. Flowers were harvested after 28 days in three different Plants were grown under long-day (16 hr light: 8 hr dark) 60 categories. Buds that had not opened at all and were com conditions, 7000-8000 LUX light intensity, 70% humidity, pletely green were categorized as “flower buds' (also referred and 22° C. temperature and taken to maturity. Mature dry to as green buds by the investigator). Buds that had started to seeds are collected, dried for one week at 28°C., and vernal open, with white petals emerging slightly were categorized as ized for one week at 4°C. before used as a source of RNA. “green flowers” (also referred to as white buds by the inves (o) Herbicide Treatment 65 tigator). Flowers that had opened mostly (with no silique Arabidopsis thaliana (Ws) seeds were sterilized for 5 min. elongation) with white petals completely visible were catego with 30% bleach, 50 ul Triton in a total volume of 50 ml. rized as “white flowers” (also referred to as open flowers by US 7,868,155 B2 41 42 the investigator). Buds and flowers were harvested with for Seeds of maize hybrid 35A (Pioneer) were sown in water ceps, flash frozen in liquid nitrogen and stored at -80C until moistened sand in flats (10 rows, 5-6 seed/row) and covered RNA was isolated. with clear, plastic lids before being placed in a growth cham s) Ovules ber having 16hr light (25°C.)/8 hr dark (20°C.), 75% relative Seeds of Arabidopsis thaliana heterozygous for pistillata humidity and 13,000-14,000 LUX. Covered flats were (pi) ecotype Landsberg erecta (Ler) were sown in pots and watered every three days for 7 days. Seedlings were carefully left at 4°C. for two to three days to vernalize. They were then removed from the sand and placed in 1-liter beakers with 5 transferred to a growth chamber. Plants were grown under mM nitroprusside for treatment. Control plants were treated long-day (16 hr light: 8 hr dark) conditions, 7000-8000 LUX with water. After 1 hr., 6 hr and 12 hr., aerial and root tissues light intensity, 76% humidity, and 24°C. temperature. Inflo 10 were separated and flash frozen in liquid nitrogen prior to rescences were harvested from seedlings about 40 days old. storage at -80°C. The inflorescences were cut into Small pieces and incubated v) Root Hairless Mutants in the following enzyme solution (pH 5) at room temperature Plants mutant at the rhl gene locus lack root hairs. This for 0.5-1 hr.: 0.2% pectolyase Y-23, 0.04% pectinase, 5 mM mutation is maintained as a heterozygote. MES, 3% Sucrose and MS salts (1900 mg/1 KNO, 1650 mg/1 15 Seeds of Arabidopsis thaliana (Landsberg erecta) mutated NHNO, 370 mg/l MgSO.7H2O, 170 mg/l KHPO, 440 at the rhl gene locus were sterilized using 30% bleach with 1 mg/l CaCl2.H2O, 6.2 mg/l HBO, 15.6 mg/l MnSO4HO, ul/ml 20% Triton-X 100 and then vernalized at 4° C. for 3 8.6 mg/l ZnSO.7H2O, 0.25 mg/l NaMoO2HO, 0.025 mg/1 days before being plated onto GM agar plates. Plates were CuCO.5H2O, 0.025 mg/l CoC1.6H2O, 0.83 mg/l KI, 27.8 placed in growth chamber with 16 hr light/8 hr. dark, 23°C., mg/l FeSO.7H2O, 37.3 mg/l Disodium EDTA, pH 5.8). At 14,500-15,900 LUX, and 70% relative humidity for germi the end of the incubation the mixture of inflorescence mate nation and growth. rial and enzyme solution was passed through a size 60 sieve After 7 days, seedlings were inspected for root hairs using and then through a sieve with a pore size of 125um. Ovules a dissecting microscope. Mutants were harvested and the greater than 125 um in diameter were collected, rinsed twice cotyledons removed so that only root tissue remained. Tissue in B5 liquid medium (2500 mg/l KNO. 250 mg/1 25 was then flash frozen in liquid nitrogen and stored at -80C. MgSO.7H2O, 150 mg/l NaH2PO4.H.O. 150 mg/1 Arabidopsis thaliana (Landsberg erecta) seedlings grown CaCl2.H2O, 134 mg/l (NH4). CaCl.S.O.3 mg/l HBO, 10 and prepared as above were used as controls. mg/l MnSO.4H2O, 2 ZnSO.7H2O, 0.25 mg/1 Alternatively, seeds of Arabidopsis thaliana (Landsberg NaMoO2HO, 0.025 mg/l CuCO.5HO, 0.025 mg/1 erecta), heterozygous for the rhill (root hairless) mutation, CoCl2.6H2O, 0.75 mg/1 KI, 40 mg/l EDTA sodium ferric salt, 30 were surface-sterilized in 30% bleach containing 0.1% Triton 20 g/l sucrose, 10 mg/l Thiamine hydrochloride, 1 mg/l Pyri X-100 and further rinsed in sterile water. They were then doxine hydrochloride, 1 mg/l Nicotinic acid, 100 mg/l myo vernalized at 4°C. for 4 days before being plated onto MS inositol, pH 5.5)), rinsed once in deionized water and flash agar plates. The plates were maintained in a growth chamber frozen in liquid nitrogen. The Supernatant from the 125 um at 24°C. with 16 hr light/8 hr dark for germination and sieving was passed through Subsequent sieves of 50 um and 35 growth. After 10 days, seedling roots that expressed the phe 32 Lum. The tissue retained in the 32 um sieve was collected notype (i.e. lacking root hairs) were cut below the hypocotyl and mRNA prepared for use as a control. junction, frozen in liquid nitrogen and stored at -80°C. Those t) Wounding seedlings with the normal root phenotype (heterozygous or Seeds of Arabidopsis thaliana (Wassilewskija) were sown wt) were collected as described for the mutant and used as in trays and left at 4°C. for three days to vernalize before 40 controls. being transferred to a growth chamber having 16 hr light/8.hr w) Ap2 dark, 12,000-14,000 LUX, 70% humidity and 20°C. After 14 Seeds of Arabidopsis thaliana (ecotype Landesberg days, the leaves were wounded with forceps. Aerial tissues erecta) and floral mutant apetala2 (Jofuku et al., 1994, Plant were harvested 1 hour and 6 hours after wounding. Aerial Cell 6:1211-1225) were sown in pots and left at 4°C. for two tissues from unwounded plants served as controls. Tissues 45 to three days to vernalize. They were then transferred to a were flash-frozen in liquid nitrogen and stored at -80° C. growth chamber. Plants were grown under long-day (16 hr Seeds of maize hybrid 35A (Pioneer) were sown in water light, 8 hr dark) conditions 7000-8000 LUX light intensity, moistened sand in flats (10 rows, 5-6 seed/row) and covered 70% humidity and 22°C. temperature. Inflorescences con with clear, plastic lids before being placed in a growth cham 50 taining immature floral buds (stages 1-7: Bowman, 1994) as ber having 16hr light (25°C.)/8 hr dark (20°C.), 75% relative well as the inflorescence meristem were harvested and flash humidity and 13,000-14,000 LUX. Covered flats were frozen. Polysomal polyA+ RNA was isolated from tissue watered every three days for 7 days. Seedlings were wounded according to Cox and Goldberg, 1988). (one leaf nicked by scissors) and placed in 1-liter beakers of x) Salt water for treatment. Control plants were treated not wounded. 55 Arabidopsis thaliana ecotype Wis seeds were vernalized at After 1 hr and 6hr aerial and root tissues were separated and 4°C. for 3 days before sowing in flats containing vermiculite flash frozen in liquid nitrogen prior to storage at -80°C. soil. Flats were placed at 20°C. in a Conviron growth cham u) Nitric Oxide Treatment ber having 16 hr light/8 hr dark. Whole plants (used as con Seeds of Arabidopsis thaliana (Wassilewskija) were sown trols) received water. Other plants were treated with 100 mM in trays and left at 4°C. for three days to vernalize before 60 NaCl. After 6 hr and 72 hr., aerial and root tissues were being transferred to a growth chamber having 16 hr light/8.hr harvested and flash frozen in liquid nitrogen prior to storage dark, 12,000-14,000 LUX, 20° C. and 70% humidity. Four at 80° C. teen day old plants were sprayed with 5 mM sodium nitro y) Petals prusside in a 0.02% Silwett L-77 solution. Control plants Arabidopsis thaliana ecotype Wis seeds were vernalized at were sprayed with a 0.02% Silwett L-77 solution. Aerial 65 4°C. for 3 days before sowing in flats containing vermiculite tissues were harvested 1 hour and 6 hours after spraying, soil. Flats were watered placed at 20°C. in a Conviron growth flash-frozen in liquid nitrogen and stored at -80°C. chamber having 16 hr light/8 hr dark. Whole plants (used as US 7,868,155 B2 43 44 the control) and petals from inflorescences 23-25 days after pre-flower stage. Stems and rosette leaves were harvested germination were harvested, flash frozen in liquid nitrogen from the mutants and the wild-type segregants, flash frozen and stored at -80° C. and stored at -80° C. Z) Pollen hh) Caf Arabidopsis thaliana ecotype Wis seeds were vernalized at Carple factory (Caf) is a double-stranded RNAse protein 4°C. for 3 days before sowing in flats containing vermiculite that is hypothesized to process small RNAs in Arabidopsis. soil. Flats were watered and placed at 20° C. in a Conviron The protein is closely related to a Drosophila protein named growth chamber having 16 hr light/8 hr dark. Whole plants DICER that functions in the RNA degradation steps of RNA (used as controls) and pollen from plants 38 dap was har interference. Arabidopsis thaliana Caf mutant seeds were vested, flash frozen in liquid nitrogen and stored at -80°C. 10 vernalized at 4°C. for 3 days before sowing inflats of Metro aa) Interploidy Crosses Mix 200. Flats were placed in the greenhouse, watered and Interploidy crosses involving a 6x parent are lethal. grown to the 8 leaf, pre-flower stage. Stems and rosette leaves Crosses involving a 4xparent are complete and analyzed. The were harvested from the mutants and the wild-type seg imbalance in the maternal/paternal ratio produced from the regants, flash frozen and stored at -80° C. cross can lead to big seeds. Arabidopsis thaliana ecotype WS 15 ii) Drought Reproduction seeds were vernalized at 4°C. for 3 days before sowing. Small Arabidopsis thaliana (ecotype Wassilewskija) seeds are siliques were harvested at 5 days after pollination, flash fro kept at 4°C. in dark for three days and then sown in soil mix Zen in liquid nitrogen and stored at -80° C. (Metromix 200) with a regular watering schedule (1.5-2 L per bb) Line Comparisons flat per week). Drought treatment by withholding water starts Alkaloid 35S over-expressing lines were used to monitor when plants are 30-days-old. The control samples are watered the expression levels of terpenoid/alkaloid biosynthetic and as before. Rosettes, flowers (with siliques less than 5mm) and P450 genes to identify the transcriptional regulatory points I siliques (>5 mm) are harvested separately on day 5, 7 and 10 the biosynthesis pathway and the related P450 genes. Arabi post-drought-treatment (PDT). By day 10 PDT, the majority dopsis thaliana ecotype Wis seeds were vernalized at 4°C. for of drought plants are wilted and unable to recover after re 3 days before sowing in vermiculite soil (Zonolite) supple 25 watering and the experiment is terminated. The samples are mented by Hoagland solution. Flats were placed in Conviron harvested between 2-5 PM. Plants are grown in a walk-in growth chambers under long day conditions (16 hr light, 23° growth chamber under these conditions: 16 hlight/8 hr dark, C./8 hr dark, 20° C.). Basta spray and selection of the over 70% relative humidity, 20° C. light/18°C. dark for the first 10 expressing lines was conducted about 2 weeks after germi days, and under 22° C. light/20° C. dark for the following nation. Approximately 2-3 weeks after bolting (approxi 30 days. mately 5-6 weeks after germination), Stem and siliques from (ii) Drought Stress the over-expressing lines and from wild-type plants were Seeds of Arabidopsis thaliana (ecotype Wassilewskija) are harvested, flash frozen in liquid nitrogen and stored at -80°C. sown in pots and left at 4°C. for three days to vernalize before cc) DMT-II being transferred to a growth chamber having 16 hr light/8.hr Demeter (dmt) is a mutant of a methyltransferase gene and 35 dark, 150,000-160,000 LUX, 20° C. and 70% humidity. After is similar to fie. Arabidopsis thaliana ecotype Wis seeds were 14 days, aerial tissues are cut and left to dry on 3 mM What vernalized at 4°C. for 3 days before sowing. Cauline leaves man paper in a petri-plate for 1 hour and 6 hours. Aerial and closed flowers were isolated from 35S::DMT and dimt-f- tissues exposed for 1 hour and 6 hours to 3 mM Whatman plant lines, flash frozen in liquid nitrogen and stored at -80° paper wetted with 1x Hoagland's solution serve as controls. C. 40 Tissues are harvested, flash-frozen in liquid nitrogen and dd) CS6630 Roots and Shoots Stored at -80° C. Arabidopsis thaliana ecotype Wis seeds were vernalized at Alternatively, Arabidopsis thaliana (ecotype Wassilews 4°C. for 3 days before sowing on MS media (1%) sucrose on kija) seed is vernalized at 4°C. for 3 days before sowing in bacto-agar. Roots and shoots were separated 14 days after Metromix soil type 350. Flats are placed in a growth chamber germination, flash frozen in liquid nitrogen and stored at -80° 45 with 23° C., 16 hr light/8 hr. dark, 80% relative humidity, C. ~13,000 LUX for germination and growth. Plants are watered ee) CS237 with 1-1.5 L of water every four days. Watering is stopped 16 CS237 is an ethylene triple response mutant that is insen days after germination for the treated Samples, but continues sitive to ethylene and which has an etrl-1 phenotype. Arabi for the control samples. Rosette leaves and stems, flowers and dopsis thaliana CS237 seeds were vernalized at 4°C. for 3 50 siliques are harvested 2 d. 3 d, 4d, 5 d, 6d and 7 d after days before sowing. Aerial tissue was collected from mutants watering is stopped. Tissue is flash frozen in liquid nitrogen and wild-type Columbia ecotype plants, flash frozen in liquid and kept at -80° C. until RNA is isolated. Flowers and siliq nitrogen and stored at -80° C. ues are also harvested on day 8 from plants that had under f) Guard Cells gone a 7 d drought treatment followed by 1 day of watering. Arabidopsis thaliana ecotype Wis seeds were vernalized at 55 Control plants (whole plants) are harvested after 5 weeks, 4° C. for 3 days before sowing. Leaves were harvested, flash frozen in liquid nitrogen and stored as above. homogenized and centrifuged to isolate the guard cell con Seeds of maize hybrid 35A (Pioneer) are sown in water taining fraction. Homogenate from leaves served as the con moistened sand in flats (10 rows, 5-6 seed/row) and covered trol. Samples were flash frozen in liquid nitrogen and stored at with clear, plastic lids before being placed in a growth cham -80°C. Identical experiments using leaf tissue from canola 60 ber having 16hr light (25°C.)/8 hr dark (20°C.), 75% relative were performed. humidity and 13,000-14,000 LUX. Covered flats are watered gg) 3642-1 every three days for 7 days. Seedlings are carefully removed 3642-1 is a T-DNA mutant that affects leaf development. from the sand and placed in empty 1-liter beakers at room This mutant segregates 3:1, wild-type:mutant. Arabidopsis temperature for treatment. Control plants are placed in water. thaliana 3642-1 mutant seeds were vernalized at 4°C. for 3 65 After 1 hr., 6 hr, 12 hr and 24 hr aerial and root tissues are days before sowing in flats of MetroMix 200. Flats were separated and flash frozen in liquid nitrogen prior to storage at placed in the greenhouse, watered and grown to the 8 leaf, 800 C. US 7,868,155 B2 45 46 (kk) Far-Red-Enriched ber having 16hr light (25°C.)/8 hr dark (20°C.), 75% relative Seeds from wildtype Arabidopsis thaliana (ecotype humidity and 13,000-14,000 LUX. Covered flats were Columbia) are vernalized in sterile water for 4 days at 4°C. watered every three days for 7 days. Seedlings are carefully prior to planting. Seeds are then sterilized and evenly planted removed from the sand and placed in 1-liter beakers with 100 on 0.5% sucrose MS media plates. Plates are sealed with uMBA for treatment. Control plants are treated with water. Scotch micropore tape to allow for gas exchange and prevent After 6 hr, aerial and root tissues are separated and flash contamination. Plates are grown in a growth room (16 h frozen in liquid nitrogen prior to storage at 80°C. light/8 h dark, 22°C.; 6 bulbs total Gro-Lux); light measure 2. Microarray Hybridization Procedures ments are as follows: Red=646.4 uW/cm, Blue=387 Microarray technology provides the ability to monitor uW/cm, Far Red=158.7 uW/cm. At 7 days after germina 10 mRNA transcript levels of thousands of genes in a single tion, the plates containing the seedlings are transferred to Far experiment. These experiments simultaneously hybridize Red light only (Far Red=525 uW/cm) for various durations two differentially labeled fluorescent cDNA pools to glass of exposure time (1, 4, 8, and 24 hrs). After timed exposure, slides that have been previously spotted with cDNA clones of tissue is flash frozen with liquid nitrogen and stored at -80°C. the same species. Each arrayed cDNA spot will have a corre Control seedlings are not transferred, but are collected at 15 sponding ratio of fluorescence that represents the level of same time as the corresponding far-red exposed experimental disparity between the respective mRNA species in the two samples. sample pools. Thousands of polynucleotides can be spotted (l) Far-Red-Induction-Adult Wildtype Arabidopsis thaliana (ecotype Columbia) seeds on one slide, and each experiment generates a global expres are planted on soil and vernalized for 4 days at 4°C. Soil sown sion pattern. plants are grown in a growth room (16hlight/8h dark, 22°C.; Coating Slides 4 bulbs total alternating Gro-Lux and cool whites); light The microarray consists of a chemically coated micro measurements areas follows: Red=330.9W/cm, Blue=267 scope slide, referred herein as a “chip” with numerous poly uW/cm, Far Red=56.1 W/cm. At 4 weeks after germina nucleotide samples arrayed at a high density. The poly-L- tion, the soil pots are transferred to shade environment (16 h 25 lysine coating allows for this spotting at high density by light/8 h dark; Red=376 uW/cm, Blue=266 uW/cm, Far providing a hydrophobic Surface, reducing the spreading of Red=552 uW/cm) for various durations of exposure time (1, spots of DNA solution arrayed on the slides. Glass micro 4, 8, 16, 24, 48, and 72 hrs). After timed exposure, above scope slides (Gold Seal #3010 manufactured by Gold Seal ground tissue is flash frozen with liquid nitrogen and stored at Products, Portsmouth, N.H., USA) were coated with a 0.1% -80° C. Control seedlings are not transferred, but are col 30 W/V solution of Poly-L-lysine (Sigma, St. Louis, Mo.) using lected at same time as the corresponding shade-exposed the following protocol: experimental samples. 1. Slides were placed in slide racks (Shandon Lipshaw #121). (mm) Shoots The racks were then put in chambers (Shandon Lipshaw Sterilized wild-type Arabidopsis thaliana seeds (ecotype #121). Wassilewskija) are sown on MS plates (0.5% sucrose, 1.5% 35 2. Cleaning Solution was prepared: agar) after 3 day-cold treatment. The plates are placed verti 70 g NaOH was dissolved in 280 mL ddH.O. cally in a Percival growth chamber (16:8 light cycles, 22°C.) 420 mL 95% ethanol was added. The total volume was 700 so that roots grow vertically on the agar Surface. The shoots or mL (2x350 mL); it was stirred until completely mixed. aerials, harvested after 7 d- and 14 d-growth in the chamber, If the solution remained cloudy, ddHO was added until are used as the experimental samples. The control sample is 40 clear. derived from tissues harvested from 3 week-old plants that 3. The solution was poured into chambers with slides; the are grown in Soil in a Conviron chamber (16:8 light cycles, chambers were covered with glass lids. The solution was 22°C.), including rosettes, roots, stems, flowers, and siliques. mixed on an orbital shaker for 2 hr. (nn.) Siliques 4. The racks were quickly transferred to fresh chambers filled Wild type Arabidopsis thaliana (ecotype Wassilewskija) 45 with ddH2O. They were rinsed vigorously by plunging seeds are sown in moistened soil mix, metromix 200 with racks up and down. Rinses were repeated 4x with fresh osmocote, and stratified at 4°C. for 3 days in dark. Flats are ddHO each time, to remove all traces of NaOH-ethanol. placed in a Conviron growth chamber maintained at 16 hlight 5. Polylysine solution was prepared: (22°C.), 8 h dark (20°C.) and 70% humidity. After 3 weeks, 0 mL poly-L-lysine+70 mL tissue culture PBS in 560 mL siliques (<5 mm long) are collected in liquid nitrogen. The 50 water, using plastic graduated cylinder and beaker. control samples are 3-week old whole plants (including all 6. Slides were transferred to polylysine solution and shaken tissue types) grown in the same Conviron growth chamber. for 1 hr. (oo) Cytokinin (BA) 7. The rack was transferred to a fresh chambers filled with Seeds of Arabidopsis thaliana (ecotype Wassilewskija) are ddHO. It was plunged up and down 5x to rinse. sown in trays and left at 4°C. for 4 days to vernalize. They are 55 8. The slides were centrifuged on microtiter plate carriers then transferred to a growth chamber having 16 hr light/8 hr (paper towels were placed below the rack to absorb liquid) dark, 13,000 LUX, 70% humidity, 20° C. temperature and for 5 min. (a) 500 rpm. The slide racks were transferred to watered twice a week with 1 L of 1x Hoagland's solution. empty chambers with covers. Approximately 1,000 14 day old plants are spayed with 200 9. Slide racks were dried in a 45C oven for 10 min. 250 mls of 100 uM BA in a 0.02% solution of the detergent 60 10. The slides were stored in a closed plastic slide box. Silwet L-77. Aerial tissues (everything above the soil line) are 11. Normally, the surface of lysine coated slides was not very harvested within a 15 to 20 minute time period 1 hr and 6 hrs hydrophobic immediately after this process, but became after treatment, flash-frozen in liquid nitrogen and stored at increasingly hydrophobic with storage. A hydrophobic 800 C. surface helped ensure that spots didn’t run together while Seeds of maize hybrid 35A (Pioneer) are sown in water 65 printing at high densities. After they aged for 10 days to a moistened sand in flats (10 rows, 5-6 seed/row) and covered month the slides were ready to use. However, coated slides with clear, plastic lids before being placed in a growth cham that have been sitting around for long periods of time were US 7,868,155 B2 47 48 usually too old to be used. This was because they developed Following this, the slide rack was gently plunge in the 95C opaque patches, visible when held to the light, and these water (just stopped boiling) for 2 min. Then the slide rack was resulted in high background hybridization from the fluo plunged 5x in 95% ethanol. The slides and rack were centri rescent probe. Alternatively, pre-coated glass slides were fuged for 5 min. (a) 500 rpm. The slides were loaded quickly purchased from TeleChem International, Inc. (Sunnyvale, and evenly onto the carriers to avoid streaking. The arrays Calif., 94089; catalog number SMM-25, Superamine sub were used immediately or store in slide box. strates). The Hybridization process began with the isolation of PCR Amplification Of cDNA Clone Inserts mRNA from the two tissues (see “Isolation of total RNA' and Polynucleotides were amplified from Arabidopsis cloNA "Isolation of mRNA, below) in question followed by their 10 conversion to single stranded cDNA (see “Generation of clones using insert specific probes. The resulting 100 u, PCR probes for hybridization', below). The cDNA from each tis reactions were purified with Qiaquick 96 PCR purification sue was independently labeled with a different fluorescent columns (Qiagen, Valencia, Calif., USA) and eluted in 30 ul dye and then both samples were pooled together. This final of 5 mM Tris. 8.5 u of the elution were mixed with 1.5ul of differentially labeled cDNA pool was then placed on a pro 20xSSC to give a final spotting solution of DNA in 3xSSC. 15 cessed microarray and allowed to hybridize (see "Hybridiza The concentrations of DNA generated from each clone varied between 10-100 ng/ul, but were usually about 50 ng/ul. tion and wash conditions', below). Isolation of Total RNA Arraying of PCR Products on Glass Slides Approximately 1 g of plant tissue was ground in liquid PCR products from cDNA clones were spotted onto the nitrogen to a fine powder and transferred into a 50-ml centri poly-L-Lysine coated glass slides using an arrangement of fuge tube containing 10 ml of Trizol reagent. The tube was quill-tip pins (ChipMaker 3 spotting pins; Telechem, Interna vigorously Vortexed for 1 min and then incubated at room tional, Inc., Sunnyvale, Calif., USA) and a robotic arrayer temperature for 10-20 min. on an orbital shaker at 220 rpm. (PixSys 3500, Cartesian Technologies, Irvine, Calif., USA). Two ml of chloroform was added to the tube and the solution Around 0.5 nil of a prepared PCR product was spotted at each vortexed vigorously for at least 30-sec before again incubat location to produce spots with approximately 100 um diam 25 ing at room temperature with shaking. The sample was then eters. Spot center-to-center spacing was from 180 um to 210 centrifuged at 12,000xg (10,000 rpm) for 15-20 min at 4°C. um depending on the array. Printing was conducted in a The aqueous layer was removed and mixed by inversion with chamber with relative humidity set at 50%. 2.5 ml of 1.2 M NaCl/0.8 M Sodium Citrate and 2.5 ml of Slides containing maize sequences were purchased from isopropyl alcohol added. After a 10 min. incubation at room Agilent Technology (Palo Alto, Calif. 94304). 30 temperature, the sample was centrifuged at 12,000xg (10,000 Post-Processing of Slides rpm) for 15 min at 4°C. The pellet was washed with 70% After arraying, slides were processed through a series of ethanol, re-centrifuged at 8,000 rpm for 5 min and then air steps—rehydration, UV cross-linking, blocking and denatur dried at room temperature for 10 min. The resulting total ation—required prior to hybridization. Slides were rehy 35 RNA was dissolved in either TE (10 mM Tris-HCl, 1 mM drated by placing them over a beaker of warm water (DNA EDTA, pH 8.0) or DEPC (diethylpyrocarbonate) treated face down), for 2-3 sec, to distribute the DNA more evenly deionized water (RNAse-free water). For subsequent isola within the spots, and then snap dried on a hot plate (DNA side, tion of mRNA using the Qiagen kit, the total RNA pellet was face up). The DNA was then cross-linked to the slides by UV dissolved in RNAse-free water. irradiation (60-65 m.J; 2400 Stratalinker, Stratagene, LaJolla, 40 Isolation of mRNA Calif., USA). mRNA was isolated using the Qiagen Oligotex mRNA Following this a blocking step was performed to modify Spin-Column protocol (Qiagen, Valencia, Calif.). Briefly, remaining free lysine groups, and hence minimize their abil 500 ul OBB buffer (20 mM Tris-C1, pH 7.5, 1M NaCl, 2 mM ity to bind labeled probe DNA. To achieve this the arrays were EDTA, 0.2% SDS) was added to 500 ul of total RNA (0.5- placed in a slide rack. An empty slide chamber was left ready 45 0.75 mg) and mixed thoroughly. The sample was first incu on an orbital shaker. The rack was bent slightly inwards in the bated at 70° C. for 3 min, then at room temperature for 10 middle, to ensure the slides would not run into each other minutes and finally centrifuged for 2 min at 14,000-18,000xg. while shaking. The blocking Solution was prepared as fol The pellet was resuspended in 400 ul OW2 buffer (10 mM lows: Tris-C1, pH 7.5, 150 mM. NaCl, 1 mM EDTA) by vortexing, 50 the resulting solution placed on a small spin column in a 1.5 3x350-ml glass chambers (with metal tops) were set to one ml RNase-free microcentrifuge tube and centrifuged for 1 side, and a large round Pyrex dish with dHO was placed min at 14,000-18,000xg. The spin column was transferred to ready in the microwave. At this time, 15 ml sodium borate was a new 1.5 ml RNase-free microcentrifuge tube and washed prepared in a 50 ml conical tube. with 400 ul of OW2 buffer. To release the isolated mRNA 6-g succinic anhydride was dissolved in approx. 325-350 55 from the resin, the spin column was again transferred to a new mL 1-methyl-2-pyrrolidinone. Rapid addition of reagent was RNase-free 1.5 ml microcentrifuge tube, 20-100 ul 70° C. crucial. OEB buffer (5 mM Tris-Cl, pH 7.5) added and the resin a. Immediately after the last flake of the succinic anhydride resuspended in the resulting solution via pipeting. The dissolved, the 15-mL sodium borate was added. mRNA solution was collected after centrifuging for 1 min at b. Immediately after the sodium borate solution mixed in, 60 14,000-18,000xg. the solution was poured into an empty slide chamber. Alternatively, mRNA was isolated using the Stratagene c. The slide rack was plunged rapidly and evenly in the Poly(A) Quik mRNA. Isolation Kit (Startagene, La Jolla, Solution. It was vigorously shaken up and down for a few Calif.). Here, up to 0.5 mg of total RNA (maximum volume of seconds, making Sure slides never left the solution. 1 ml) was incubated at 65° C. for 5 minutes, snap cooled on d. It was mixed on an orbital shaker for 15-20 min. Mean 65 ice and 0.1x volumes of 10x sample buffer (10 mM Tris-HCl while, the water in the Pyrex dish (enough to cover slide rack) (pH 7.5), 1 mM EDTA (pH 8.0) 5 MNaCl) added. The RNA was heated to boiling. sample was applied to a prepared push column and passed US 7,868,155 B2 49 50 through the column at a rate of ~1 drop every 2 sec. The fluorescent dye to the cDNA by reaction with the amino Solution collected was reapplied to the column and collected functional groups. Briefly, 5 Jug of oligo(dT)s primer as above. 200ul of high saltbuffer (10 mM Tris-HCl (pH 7.5), d(TTTTTTTTTTTTTTTTTTV) (SEQ ID NO: 67) was 1 mM EDTA, 0.5 NaCl) was applied to the column and passed mixed with Poly A+mRNA (1.5-2 ug mRNA isolated using through the column at a rate of 1 drop every 2 sec. This step the Qiagen Oligotex mRNA Spin-Column protocol or —the was repeated and followed by three low salt buffer (10 mM Stratagene Poly(A) Quik mRNA. Isolation protocol (Strat Tris-HCl (pH 7.5), 1 mM EDTA, 0.1 M NaCl) washes pre agene, La Jolla, Calif., USA)) in a total volume of 25ul. The formed in a similar manner. mRNA was eluted by applying to sample was incubated in a thermocycler at 70° C. for 5 min, the column four separate 200 aliquots of elution buffer (10 cooled to 48° C. and 10 ul of 5xcDNA Synthesis Buffer (kit mM Tris-HCl (pH 7.5), 1 mM EDTA) preheated to 65° C. 10 supplied), 5ul 10xdNTP mix (dATP, dCTP, dGTP, dTTP and Here, the elution buffer was passed through the column at a aminoallyl-dUTP; kit supplied), 7.5ul deionized water and rate of 1 drop/sec. The resulting mRNA solution was precipi 2.5 ul MMLV Reverse Transcriptase (500 U) added. The tated by adding 0.1x volumes of 10x sample buffer, 2.5 vol reaction was then incubated at 48° C. for 30 minutes, fol umes of ice-cold 100% ethanol, incubating overnight at -20° lowed by 1 hr incubation at 42°C. At the end of the incubation C. and centrifuging at 14,000-18,000xg for 20-30 min at 4°C. 15 the reaction was heated to 70° C. for 10 min, cooled to 37° C. The pellet was washed with 70% ethanol and air dried for 10 and 0.5ul (5U) RNase Hadded, before incubating for 15 min min. at room temperature before resuspension in RNase-free at 37° C. The solution was vortexed for 1 min after the deionized water. addition of 0.5ul 0.5 M EDTA and 5ul of QuickClean Resin Preparation of Yeast Controls (kit supplied) then centrifuged at 14,000-18,000xg for 1 min. Plasmid DNA was isolated from the following yeast clones After removing the supernatant to a 0.45 um spin filter (kit using Qiagen filtered maxiprep kits (Qiagen, Valencia, Supplied), the sample was again centrifuged at 14,000-18, Calif.): YAL022c(Fun26), YAL031c(Fun21), YBR032w, 000xg for 1 min, and 5.5ul 3M sodium acetate and 137.5 ul YDL131 w, YDL182w,YDL194w, YDL196w, YDR050c and of 100% ethanol added to the sample before incubating at YDR116c. Plasmid DNA was linearized with either BSrBI -20°C. for at least 1 hr. The sample was then centrifuged at 25 14,000-18,000xg at 4° C. for 20 min, the resulting pellet (YAL022c(Fun26), YAL031c(Fun21), YDL131w, washed with 500 ul 70% ethanol, air-dried at room tempera YDL182w, YDL194w, YDL196w, YDR050c) or AflIII ture for 10 min and resuspended in 10 ul of 2x fluorescent (YBRO32w, YDR116c) and isolated. labeling buffer (kit provided). 10 ul each of the fluorescent In Vitro Transcription of Yeast Clones dyes Cy3 and Cy5 (Amersham Pharmacia (Piscataway, N.J., The following solution was incubated at 37°C. for 2 hours: 30 USA); prepared according to AtlasTM kit directions of Clon 17 ul of isolated yeast insert DNA (1 lug), 20 ul 5x buffer, 10 tech) were added and the sample incubated in the darkat room ul 100 mM DTT, 2.5 ul (100 U) RNasin, 20 ul 2.5 mM (ea.) temperature for 30 min. rNTPs, 2.7 (40 U) SP6 polymerase and 27.8 pul RNase-free The fluorescently labeled first strand cDNA was precipi deionized water. 2 (2 U) Ampli DNase I was added and the tated by adding 2 ul 3M sodium acetate and 50 ul 100% incubation continued for another 15 min. 10ul 5 MNHOAC 35 ethanol, incubated at -20°C. for at least 2 hrs, centrifuged at and 100 ul phenol: chloroform:isoamyl alcohol (25:24:1) 14,000-18,000xg for 20 min, washed with 70% ethanol, air were added, the solution vortexed and then centrifuged to dried for 10 min and dissolved in 100 ul of water. separate the phases. To precipitate the RNA, 250 ul ethanol Alternatively, 3-4 ug mRNA, 2.5 (-8.9 ng of in vitro trans was added and the solution incubated at -20°C. for at least lated mRNA) ul yeast control and 3 ug oligo dTV one hour. The sample was then centrifuged for 20 min at 4°C. 40 (TTTTTTTTTTTTTTTTTT(A/C/G) (SEQID NO: 68) were at 14,000-18,000xg, the pellet washed with 500 ul of 70% mixed in a total volume of 24.7 ul. The sample was incubated ethanol, air dried at room temperature for 10 min and resus in a thermocycler at 70° C. for 10 min. before chilling on ice. pended in 100 ul of RNase-free deionized water. The precipi To this, 8 ul of 5x first strand buffer (SuperScript II RNase tation procedure was then repeated. H Reverse Transcriptase kit from Invitrogen (Carlsbad, Alternatively, after the two-hour incubation, the solution 45 Calif. 92008); cat no. 18064022), 0.8° C. ofaa-duTP/dNTP was extracted with phenol/chloroform once before adding 0.1 mix (50x; 25 mM dATP 25 mM dGTP, 25 mM dCTP 1.5 mM Volume 3M sodium acetate and 2.5 volumes of 100% ethanol. dTTP, 10 mMaminoallyl-dUTP), 4 ul of 0.1 M DTT and 2.5 The solution was centrifuged at 15,000 rpm, 4° C. for 20 ul (500 units) of Superscript R.T. II enzyme (Stratagene) were minutes and the pellet resuspended in RNase-free deionized added. The sample was incubated at 42°C. for 2 hours before water. The DNase I treatment was carried out at 37° C. for 30 50 a mixture of 10° C. of 1M NaOH and 10° C. of 0.5 M EDTA minutes using 2U of Ampli DNase I in the following reaction were added. After a 15 minute incubation at 65°C., 25ul of 1 condition: 50 mM Tris-HCl (pH 7.5), 10 mM MgCl2. The M Tris pH 7.4 was added. This was mixed with 450 ul of water DNase I reaction was then stopped with the addition of in a Microcon 30 column before centrifugation at 11,000xg NHOAC and phenol: chloroform:isoamyl alcohol (25:24:1), for 12 min. The column was washed twice with 450 ul (cen and RNA isolated as described above. 55 trifugation at 11,000 g, 12 min.) before eluting the sample by 0.15-2.5 ng of the in vitro transcript RNA from each yeast inverting the Microcon column and centrifuging at 11,000xg clone were added to each plant mRNA sample prior to label for 20 seconds. Sample was dehydrated by centrifugation ing to serve as positive (internal) probe controls. under vacuum and stored at -20°C. Generation of Probes for Hybridization Each reaction pellet was dissolved in 9 ul of 0.1 M carbon 60 ate buffer (0.1M sodium carbonate and sodium bicarbonate, Generation of Labeled Probes for Hybridization from First pH-8.5-9) and 4.5 ul of this placed in two microfuge tubes. Strand cDNA 4.5 ul of each dye (in DMSO) were added and the mixture Hybridization probes were generated from isolated mRNA incubated in the dark for 1 hour. 4.5 of 4 M hydroxylamine using an AtlasTM Glass Fluorescent Labeling Kit (Clontech was added and again incubated in the dark for 15 minutes. Laboratories, Inc., Palo Alto, Calif., USA). This entails a two 65 Regardless of the method used for probe generation, the step labeling procedure that first incorporates primary ali probe was purified using a Qiagen PCR cleanup kit (Qiagen, phatic amino groups during cDNA synthesis and then couples Valencia, Calif., USA), and eluted with 100 ul EB (kit pro US 7,868,155 B2 51 52 vided). The sample was loaded on a Microcon YM-30 (Mil Results lipore, Bedford, Mass., USA) spin column and concentrated Table 4 presents the results of the differential expression to 4-5 ul in volume. Probes for the maize microarrays were experiments for the mRNAs, as reported by their correspond generated using the Fluorescent Linear Amplification Kit ing cDNA ID number, that were differentially transcribed (cat. No. G2556A) from Agilent Technologies (Palo Alto, under a particular set of conditions as compared to a control Calif.). sample. The cDNA ID numbers correspond to those utilized. Increases in mRNA abundance levels in experimental plants Hybridization and Wash Conditions Versus the controls are denoted with the plus sign (+). Like The following Hybridization and Washing Condition were wise, reductions in mRNA abundance levels in the experi developed: 10 mental plants are denoted with the minus (-) sign. Hybridization Conditions: The Table is organized according to the clone number with Labeled probe was heated at 95°C. for 3 min and chilled on each set of experimental conditions being denoted by the term ice. Then 25 DL of the hybridization buffer which was “Expt Rep ID:” followed by a “short name. Table 5 links warmed at 42C was added to the probe, mixing by pipeting, to each “short name' with a short description of the experiment 15 and the parameters. give a final concentration of The sequences showing differential expression in a par 50% formamide ticular experiment (denoted by either a "+” or '-' in the 4XSSC Table) thereby shows utility for a function in a plant, and these O.03% SDS functions/utilities are described in detail below, where the title of each section (i.e. a “utility section') is correlated with 5xDenhardt's solution the particular differential expression experiment in TABLE 5. 0.1 ug/ml single-stranded salmon sperm DNA Organ-Affecting Genes, Gene Components, Products (In The probe was kept at 42C. Prior to the hybridization, the cluding Differentiation and Function) probe was heated for 1 more min., added to the array, and then covered with a glass cover slip. Slides were placed in hybrid 25 Root Genes ization chambers (Telechem, Sunnyvale, Calif.) and incu The economic values of roots arise not only from harvested bated at 42°C. overnight. adventitious roots or tubers, but also from the ability of roots to funnel nutrients to support growth of all plants and increase Washing Conditions: their vegetative material, seeds, fruits, etc. Roots have four A. Slides were washed in 1XSSC+0.03% SDS Solution at 30 main functions. First, they anchor the plant in the soil. Sec room temperature for 5 minutes, ond, they facilitate and regulate the molecular signals and B. Slides were washed in 0.2xSSC at room temperature for 5 molecular traffic between the plant, soil, and soil fauna. minutes, Third, the root provides a plant with nutrients gained from the C. Slides were washed in 0.05xSSC at room temperature for soil or growth medium. Fourth, they condition local soil 5 minutes. 35 chemical and physical properties. After A, B, and C, slides were spun at 800xg for 2 min. to Root genes are active or potentially active to a greater dry. They were then scanned. extent in roots than in most other organs of the plant. These Maize microarrays were hybridized according to the genes and gene products can regulate many plant traits from instructions included Fluorescent Linear Amplification Kit yield to stress tolerance. Root genes can be used to modulate (cat. No. G2556A) from Agilent Technologies (Palo Alto, 40 root growth and development. Calif.). Differential Expression of the Sequences in Roots The relative levels of mRNA product in the root versus the SCANNING OF SLIDES aerial portion of the plant was measured. Specifically, mRNA The chips were scanned using a Scan Array 3000 or 5000 was isolated from roots and root tips of Arabidopsis plants (General Scanning, Watertown, Mass., USA). The chips were 45 and compared to mRNA isolated from the aerial portion of the scanned at 543 and 633 nm, at 10 um resolution to measure plants utilizing microarray procedures. the intensity of the two fluorescent dyes incorporated into the samples hybridized to the chips. Root Hair Genes, Gene Components and Products Root hairs are specialized outgrowths of single epidermal Data Extraction and Analysis 50 cells termed trichoblasts. In many and perhaps all species of The images generated by scanning slides consisted of two plants, the trichoblasts are regularly arranged around the 16-bit TIFF images representing the fluorescent emissions of perimeter of the root. In Arabidopsis, for example, tricho the two samples at each arrayed spot. These images were then blasts tend to alternate with non-hair cells or atrichoblasts. quantified and processed for expression analysis using the This spatial patterning of the root epidermis is under genetic data extraction software ImageneTM (Biodiscovery, Los 55 control, and a variety of mutants have been isolated in which Angeles, Calif., USA). Imagene output was Subsequently this spacing is altered or in which root hairs are completely analyzed using the analysis program GenespringTM (Silicon absent. Genetics, San Carlos, Calif., USA). In Genespring, the data The root hair development genes of the instant invention was imported using median pixel intensity measurements are useful to modulate one or more processes of root hair derived from Imagene output. Background subtraction, ratio 60 structure and/or function including (1) development; (2) calculation and normalization were all conducted in Gene interaction with the soil and soil contents; (3) uptake and spring. Normalization was achieved by breaking the data into transport in the plant; and (4) interaction with microorgan 32 groups, each of which represented one of the 32 pin print isms. ing regions on the microarray. Groups consist of 360 to 550 1.) Development spots. Each group was independently normalized by setting 65 The Surface cells of roots can develop into single epidermal the median of ratios to one and multiplying ratios by the cells termed trichoblasts or root hairs. Some of the root hairs appropriate factor. will persist for the life of the plant; others will gradually die US 7,868,155 B2 53 54 back; some may cease to function due to external influences. fertilization and finally seed and fruit development. These These genes and gene products can be used to modulate root genes are of great economic interest as well as biological hair density or root hair growth; including rate, timing, direc importance. The fruit and vegetable industry grosses over S1 tion, and size, for example. These genes and gene products billion USD a year. The seed market, valued at approximately can also be used to modulate cell properties Such as cell size, S15 billion USD annually, is even more lucrative. cell division, rate and direction and number, cell elongation, Inflorescence and Floral Development Genes, Gene Compo cell differentiation, lignified cell walls, epidermal cells (in nents and Products cluding trichoblasts) and root apical meristem cells (growth During reproductive growth the plant enters a program of and initiation); and root hair architecture Such as leaf cells floral development that culminates in fertilization, followed under the trichome, cells forming the base of the trichome, 10 by the production of seeds. Senescence may or may not trichome cells, and root hair responses. follow. The flowerformation is a precondition for the sexual In addition these genes and gene products can be used to propagation of plants and is therefore essential for the propa modulate one or more of the growth and development pro gation of plants that cannot be propagated vegetatively as well cesses in response to internal plant programs or environmen as for the formation of seeds and fruits. The point of time at tal stimuli in, for example, the seminal system, nodal system, 15 which the merely vegetative growth of plants changes into hormone responses, Auxin, root cap abscission, root senes flower formation is of vital importance for example in agri cence, gravitropism, coordination of root growth and devel culture, horticulture and plant breeding. Also the number of opment with that of other organs (including leaves, flowers, flowers is often of economic importance, for example in the seeds, fruits, and stems), and changes in soil environment case of various useful plants (tomato, cucumber, Zucchini, (including water, minerals, Ph, and microfauna and flora). cotton etc.) with which an increased number of flowers may 2.) Interaction with Soil and Soil Contents lead to an increased yield, or in the case of growing ornamen Root hairs are sites of intense chemical and biological tal plants and cut flowers. activity and as a result can strongly modify the Soil they Flowering plants exhibit one of two types of inflorescence contact. Roots hairs can be coated with Surfactants and muci architecture: indeterminate, in which the inflorescence grows lage to facilitate these activities. Specifically, roots hairs are 25 indefinitely, or determinate, in which a terminal flower is responsible for nutrient uptake by mobilizing and assimilat produced. Adult organs of flowering plants develop from ing water, reluctant ions, organic and inorganic compounds groups of stem cells called meristems. The identity of a mer and chemicals. In addition, they attract and interact with istem is inferred from structures it produces: Vegetative mer beneficial microfauna and flora. Root hairs also help to miti 30 istems give rise to roots and leaves, inflorescence meristems gate the effects of toxic ions, pathogens and stress. Thus, root give rise to flower meristems, and flower meristems give rise hair genes and gene products can be used to modulate traits to floral organs such as sepals and petals. Not only are mer Such as root hair Surfactant and mucilage (including compo istems capable of generating new meristems of different iden sition and secretion rate and time); nutrient uptake (including tity, but their own identity can change during development. water, nitrate and other sources of nitrogen, phosphate, potas 35 For example, a vegetative shoot meristem can be transformed sium, and micronutrients (e.g. iron, copper, etc.); microbe and into an inflorescence meristem upon floral induction, and in nematode associations (such as bacteria including nitrogen Some species, the inflorescence meristem itself will eventu fixing bacteria, mycorrhizae, nodule-forming and other ally become a flower meristem. Despite the importance of nematodes, and nitrogen fixation); oxygen transpiration; meristem transitions in plant development, little is known detoxification effects of iron, aluminum, cadmium, mercury, about the underlying mechanisms. salt, and other soil constituents; pathogens (including chemi 40 Following germination, the shoot meristem produces a cal repellents) glucosinolates (GSL1), which release patho series of leaf meristems on its flanks. However, once floral gen-controlling isothiocyanates; and changes in Soil (such as induction has occurred, the shoot meristem switches to the Ph, mineral excess and depletion), and rhizosheath. production of flower meristems. Flower meristems produce floral organ primordia, which develop individually into 3.) Transport of Materials in Plants 45 sepals, petals, stamens or carpels. Thus, flowerformation can Uptake of the nutrients by the root and root hairs contrib be thought of as a series of distinct developmental steps, i.e. utes a source-sink effect in a plant. The greater source of floral induction, the formation of flower primordia and the nutrients, the more sinks, such as stems, leaves, flowers, production of flower organs. Mutations disrupting each of the seeds, fruits, etc. can draw Sustenance to grow. Thus, root hair steps have been isolated in a variety of species, suggesting development genes and gene products can be used to modu 50 that a genetic hierarchy directs the flowering process (see for late the vigor and yield of the overall plant as well as distinct review, Weigel and Meyerowitz. In Molecular Basis of Mor cells, organs, or tissues of a plant. The genes and gene prod phogenesis (ed. M. Bernfield). 51st Annual Symposium of the ucts, therefore, can modulate plant nutrition, growth rate Society for Developmental Biology, pp. 93-107, New York, (such as whole plant, including height, flowering time, etc., 1993). seedling, coleoptile elongation, young leaves, stems, flowers, 55 Expression of many reproduction genes and gene products seeds and fruit) and yield, including biomass (fresh and dry is orchestrated by internal programs or the Surrounding envi weight during any time in plant life, including maturation and ronment of a plant. These genes can be used to modulate traits senescence), number of flowers, number of seeds, seed yield, such as fruit and seed yield number, size, weight and harvest index (content and compo sition, e.g. amino acid, jasmonate, oil, protein and starch) and 60 Seed and Fruit Development Genes, Gene Components and fruit yield (number, size, weight, harvest index, and post Products harvest quality). The ovule is the primary female sexual reproductive organ of flowering plants. At maturity it contains the egg cell and Reproduction Genes, Gene Components and Products one large central cell containing two polar nuclei encased by Reproduction genes are defined as genes or components of 65 two integuments that, after fertilization, develops into the genes capable of modulating any aspect of sexual reproduc embryo, endosperm, and seed coat of the mature seed, respec tion from flowering time and inflorescence development to tively. As the ovule develops into the seed, the ovary matures US 7,868,155 B2 55 56 into the fruit or silique. As such, seed and fruit development germination takes place within twenty-four (24) hours after requires the orchestrated transcription of numerous poly imbibition. As such, germination requires the rapid and nucleotides, some of which are ubiquitous, others that are orchestrated transcription of numerous polynucleotides. Ger embryo-specific and still others that are expressed only in the mination is followed by expansion of the hypocotyland open endosperm, seed coat, or fruit. Such genes are termed fruit ing of the cotyledons. Meristem development continues to development responsive genes and can be used to modulate promote root growth and shoot growth, which is followed by seed and fruitgrowth and development such as seed size, seed yield, seed composition and seed dormancy. early leaf formation. Differential Expression of the Sequences in Siliques, Inflo Imbibition and Germination Genes rescences and Flowers 10 Imbibition and germination includes those events that The relative levels of mRNA product in the siliques relative commence with the uptake of water by the quiescent dry seed to the plant as a whole was measured. Differential Expression of the Sequences in Hybrid Seed and terminate with the expansion and elongation of the shoots Development and roots. The germination period exists from imbibition to when part of the embryo, usually the radical, extends to The levels of mRNA product in the seeds relative to those 15 penetrate the seed coat that Surrounds it. Imbibition and ger in a leaf and floral stems was measured. mination genes are defined as genes, gene components and Development Genes, Gene Components and Products products capable of modulating one or more processes of Imbibition and Germination Responsive Genes, Gene Com imbibition and germination described above. They are useful ponents and Products to modulate many plant traits from early vigor to yield to Seeds are a vital component of the world's diet. Cereal stress tolerance. grains alone, which comprise -90% of all cultivated seeds, Differential Expression of the Sequences in Germinating contribute up to half of the global per capita energy intake. Seeds and Imbibed Embryos The primary organ system for seed production in flowering The levels of mRNA product in the seeds versus the plant plants is the ovule. At maturity, the ovule consists of a haploid 25 as a whole was measured. female gametophyte or embryo sac Surrounded by several layers of maternal tissue including the nucleus and the integu Hormone Responsive Genes, Gene Components and Prod ments. The embryo sac typically contains seven cells includ lucts ing the egg cell, two synergids, a large central cell containing Abscissic Acid Responsive Genes, Gene Components and two polar nuclei, and three antipodal cells. That pollination 30 Products results in the fertilization of both egg and central cell. The Plant hormones are naturally occurring Substances, effec fertilized egg develops into the embryo. The fertilized central tive in very Small amounts, which act as signals to stimulate or cell develops into the endosperm. And the integuments inhibit growth or regulate developmental processes in plants. mature into the seed coat. As the ovule develops into the seed, Abscisic acid (ABA) is a ubiquitous hormone in vascular the ovary matures into the fruit or silique. Late in develop 35 plants that has been detected in every major organ or living ment, the developing seed ends a period of extensive biosyn tissue from the root to the apical bud. The major physiological thetic and cellular activity and begins to desiccate to complete responses affected by ABA are dormancy, stress stomatal its development and entera dormant, metabolically quiescent closure, water uptake, abscission and senescence. In contrast state. Seed dormancy is generally an undesirable character to Auxins, cytokinins and gibberellins, which are principally istic in agricultural crops, where rapid germination and 40 growth are required. However, some degree of dormancy is growth promoters, ABA primarily acts as an inhibitor of advantageous, at least during seed development. This is par growth and metabolic processes. ticularly true for cereal crops because it prevents germination Changes in ABA concentration internally or in the Sur of grains while still on the ear of the parent plant (preharvest rounding environment in contact with a plant results in modu sprouting), a phenomenon that results in major losses to the 45 lation of many genes and gene products. These genes and/or agricultural industry. Extensive domestication and breeding products are responsible for effects on traits such as plant of crop species have ostensibly reduced the level of dormancy vigor and seed yield. While ABA responsive polynucleotides mechanisms present in the seeds of their wild ancestors, and gene products can act alone, combinations of these poly although under some adverse environmental conditions, dor nucleotides also affect growth and development. Useful com mancy may reappear. By contrast, weed seeds frequently 50 binations include different ABA responsive polynucleotides mature with inherent dormancy mechanisms that allow some and/or gene products that have similar transcription profiles seeds to persist in the soil for many years before completing or similar biological activities, and members of the same or germination. similar biochemical pathways. Whole pathways or segments Germination commences with imbibition, the uptake of of pathways are controlled by transcription factor proteins water by the dry seed, and the activation of the quiescent 55 and proteins controlling the activity of signal transduction embryo and endosperm. The result is a burst of intense meta pathways. Therefore, manipulation of Such protein levels is bolic activity. At the cellular level, the genome is transformed especially useful for altering phenotypes and biochemical from an inactive state to one of intense transcriptional activ activities of plants. In addition, the combination of an ABA ity. Stored lipids, carbohydrates and proteins are catabolized responsive polynucleotide and/or gene product with another fueling seedling growth and development. DNA and 60 environmentally responsive polynucleotide is also useful organelles are repaired, replicated and begin functioning. Cell because of the interactions that exist between hormone-regu expansion and cell division are triggered. The shoot and root lated pathways, stress and defense induced pathways, nutri apical meristem are activated and begin growth and organo tional pathways and development. genesis. Schematic 4 Summarizes some of the metabolic and Differential Expression of the Sequences in ABA Treated cellular processes that occur during imbibition. Germination 65 Plants is complete when a part of the embryo, the radical, extends to The relative levels of mRNA product in plants treated with penetrate the structures that Surround it. In Arabidopsis, seed ABA versus controls treated with water were measured. US 7,868,155 B2 57 58 Brassinosteroid Responsive Genes, Gene Components and Differential Expression of the Sequences in Whole Seed Products lings, Shoots and Roots Plant hormones are naturally occurring Substances, effec The relative levels of mRNA product in whole seedlings, tive in very Small amounts, which act as signals to stimulate or shoots and roots treated with either high or low nitrogen inhibit growth or regulate developmental processes in plants. 5 media were compared to controls. Brassinosteroids (BRs) are the most recently discovered, and Viability Genes, Gene Components and Products least studied, class of plant hormones. The major physiologi Plants contain many proteins and pathways that when cal response affected by BRs is the longitudinal growth of blocked or induced lead to cell, organ or whole plant death. young tissue via cell elongation and possibly cell division. Gene variants that influence these pathways can have pro Consequently, disruptions in BR metabolism, perception and 10 found effects on plant Survival, vigor and performance. The critical pathways include those concerned with metabolism activity frequently result in a dwarf phenotype. In addition, and development or protection against stresses, diseases and because BRs are derived from the sterol metabolic pathway, pests. They also include those involved in apoptosis and any perturbations to the sterol pathway can affect the BR necrosis. Viability genes can be modulated to affect cell or pathway. In the same way, perturbations in the BR pathway 15 plant death. can have effects on the laterpart of the sterol pathway and thus Herbicides are, by definition, chemicals that cause death of the sterol composition of membranes. Changes in BR con tissues, organs and whole plants. The genes and pathways that centration in the Surrounding environment or in contact with are activated or inactivated by herbicides include those that a plant result in modulation of many genes and gene products. cause cell death as well as those that function to provide These genes and/or products are responsible for effects on protection. traits such as plant biomass and seed yield. These genes were Differential Expression of the Sequences in Herbicide discovered and characterized from a much larger set of genes Treated Plants and Herbicide Resistant Mutants by experiments designed to find genes whose mRNA abun The relative levels of mRNA product in plants treated with dance changed in response to application of BRS to plants. heribicide and mutants resistant to heribicides were com While BR responsive polynucleotides and gene products 25 pared to control plants. can act alone, combinations of these polynucleotides also Stress Responsive Genes, Gene Components and Products affect growth and development. Useful combinations include different BR responsive polynucleotides and/or gene prod Wounding Responsive Genes, Gene Components and Prod ucts that have similar transcription profiles or similar biologi lucts cal activities, and members of the same or functionally related 30 Plants are continuously subjected to various forms of biochemical pathways. Whole pathways or segments of path wounding from physical attacks including the damage cre ways are controlled by transcription factors and proteins con ated by pathogens and pests, wind, and contact with other trolling the activity of signal transduction pathways. There objects. Therefore, survival and agricultural yields depend on fore, manipulation of Such protein levels is especially useful constraining the damage created by the wounding process for altering phenotypes and biochemical activities of plants. 35 and inducing defense mechanisms against future damage. In addition, the combination of a BR responsive polynucle Plants have evolved complex systems to minimize and/or otide and/or gene product with another environmentally repair local damage and to minimize Subsequent attacks by responsive polynucleotide is useful because of the interac pathogens or pests or their effects. These involve stimulation tions that exist between hormone-regulated pathways, stress of cell division and cell elongation to repair tissues, induction pathways, nutritional pathways and development. Here, in 40 of programmed cell death to isolate the damage caused addition to polynucleotides having similar transcription pro mechanically and by invading pests and pathogens, and files and/or biological activities, useful combinations include induction of long-range signaling systems to induce protect polynucleotides that may have different transcription profiles ing molecules, in case of future attack. The genetic and bio but which participate in common or overlapping pathways. chemical systems associated with responses to wounding are 45 connected with those associated with other stresses such as Differential Expression of the Sequences in Epi-brassino pathogen attack and drought. lide or Brassinozole Plants Wounding responsive genes and gene products can be used The relative levels of mRNA product in plants treated with to alter or modulate traits such as growth rate; whole plant either epi-brassinolide or brassinozole were measured. height, width, or flowering time; organ development (Such as Metabolism Affecting Genes, Gene Components and Prod 50 coleoptile elongation, young leaves, roots, lateral roots, tuber lucts formation, flowers, fruit, and seeds); biomass; fresh and dry weight during any time in plant life, such as at maturation; Nitrogen Responsive Genes, Gene Components and Products number of flowers; number of seeds; seed yield, number, size, Nitrogen is often the rate-limiting element in plant growth, weight, harvest index (such as content and composition, e.g., and all field crops have a fundamental dependence on exog 55 amino acid, nitrogen, oil, protein, and carbohydrate); fruit enous nitrogen Sources. Nitrogenous fertilizer, which is usu yield, number, size, weight, harvest index, post harvest qual ally supplied as ammonium nitrate, potassium nitrate, or urea, ity, content and composition (e.g., amino acid, carotenoid, typically accounts for 40% of the costs associated with crops, jasmonate, protein, and starch); seed and fruit development; Such as corn and wheat in intensive agriculture. Increased germination of dormant and non-dormant seeds; seed viabil efficiency of nitrogen use by plants should enable the produc 60 ity, seed reserve mobilization, fruit ripening, initiation of the tion of higher yields with existing fertilizer inputs and/or reproductive cycle from a vegetative state, flower develop enable existing yields of crops to be obtained with lower ment time, insect attraction for fertilization, time to fruit fertilizer input, or better yields on soils of poorer quality. maturity, senescence; fruits, fruit drop; leaves; stress and Also, higher amounts of proteins in the crops could also be disease responses; drought; heat and cold; wounding by any produced more cost-effectively. “Nitrogen responsive' genes 65 Source, including wind, objects, pests and pathogens: uv and and gene products can be used to alter or modulate plant high light damage (insect, fungus, virus, worm, nematode growth and development. damage). US 7,868,155 B2 59 60 Cold Responsive Genes, Gene Components and Products These Substances are synthesized from linolenic acid in a The ability to endure low temperatures and freezing is a lipoxygenase-dependent biosynthetic pathway. Jasmonates major determinant of the geographical distribution and pro are signaling molecules which have been shown to be growth ductivity of agricultural crops. Even in areas considered Suit regulators as well as regulators of defense and stress able for the cultivation of a given species or cultivar, can give 5 responses. As such, jasmonates represent a separate class of rise to yield decreases and crop failures as a result of aberrant, plant hormones. Jasmonate responsive genes can be used to freezing temperatures. Even modest increases (1-2°C.) in the modulate plant growth and development. freezing tolerance of certain crop species would have a dra Differential Expression of the Sequences in Methyl Jas matic impact on agricultural productivity in some areas. The monate Treated Plants development of genotypes with increased freezing tolerance 10 would provide a more reliable means to minimize crop losses The relative levels of mRNA product in methyl jasmonate and diminish the use of energy-costly practices to modify the treated plants were compared to control plants. microclimate. Salicylic Acid Responsive Genes, Gene Components and Sudden cold temperatures result in modulation of many Products genes and gene products, including promoters. These genes 15 Plant defense responses can be divided into two groups: and/or products are responsible for effects on traits such as constitutive and induced. Salicylic acid (SA) is a signaling plant vigor and seed yield. molecule necessary for activation of the plant induced Manipulation of one or more cold responsive gene activi defense system known as Systemic acquired resistance or ties is useful to modulate growth and development. SAR. This response, which is triggered by prior exposure to Differential Expression of the Sequences in Cold Treated avirulent pathogens, is long lasting and provides protection Plants against a broad spectrum of pathogens. Another induced The relative levels of mRNA product in cold treated plants defense system is the hypersensitive response (HR). HR is far were compared to control plants. more rapid, occurs at the sites of pathogen (avirulent patho Heat Responsive Genes, Gene Components and Products gens) entry and precedes SAR. SA is also the key signaling The ability to endure high temperatures is a major deter molecule for this defense pathway. minant of the geographical distribution and productivity of Differential Expression of the Sequences in Salicylic Acid agricultural crops. Decreases in yield and crop failure fre Treated Plants quently occur as a result of aberrant, hot conditions even in The relative levels of mRNA product in salicylic acid areas considered Suitable for the cultivation of a given species treated plants were compared to control plants. or cultivar. Only modest increases in the heat tolerance of Osmotic Stress Responsive Genes, Gene Components and crop species would have a dramatic impact on agricultural Products productivity. The development of genotypes with increased The ability to endure and recover from osmotic and salt heat tolerance would provide a more reliable means to mini related stress is a major determinant of the geographical dis mize crop losses and diminish the use of energy-costly prac tribution and productivity of agricultural crops. Osmotic tices to modify the microclimate. 35 stress is a major component of stress imposed by Saline soil Changes in temperature in the Surrounding environment or and water deficit. Decreases in yield and crop failure fre in a plant microclimate results in modulation of many genes quently occur as a result of aberrant or transient environmen and gene products. tal stress conditions even in areas considered suitable for the Differential Expression of the Sequences in Heat Treated 40 cultivation of a given species or cultivar. Only modest Plants increases in the osmotic and salt tolerance of a crop species The relative levels of mRNA product in heat treated plants would have a dramatic impact on agricultural productivity. were compared to control plants. The development of genotypes with increased osmotic toler ance would provide a more reliable means to minimize crop Drought Responsive Genes, Gene Components and Products losses and diminish the use of energy-costly practices to The ability to endure drought conditions is a major deter 45 modify the Soil environment. Thus, osmotic stress responsive minant of the geographical distribution and productivity of genes can be used to modulate plant growth and development. agricultural crops. Decreases in yield and crop failure fre Differential Expression of the Sequences in PEG Treated quently occur as a result of aberrant, drought conditions even Plants in areas considered Suitable for the cultivation of a given The relative levels of mRNA product in PEG treated plants species or cultivar. Only modest increases in the drought 50 were compared to control plants. tolerance of crop species would have a dramatic impact on agricultural productivity. The development of genotypes with Shade Responsive Genes, Gene Components and Products increased drought tolerance would provide a more reliable Plants sense the ratio of Red (R):Far Red (FR) light in their means to minimize crop losses and diminish the use of environment and respond differently to particular ratios. A energy-costly practices to modify the microclimate. 55 low R:FR ratio, for example, enhances cell elongation and Drought conditions in the Surrounding environment or favors flowering over leaf production. The changes in R:FR within a plant, results in modulation of many genes and gene ratios mimic and cause the shading response effects in plants. products. The response of a plant to shade in the canopy structures of Differential Expression of the Sequences in Drought agricultural crop fields influences crop yields significantly. Treated Plants and Drought Mutants 60 Therefore manipulation of genes regulating the shade avoid The relative levels of mRNA product in drought treated ance responses can improve crop yields. While phytochromes mediate the shade avoidance response, the down-stream fac plants and drought mutants were compared to control plants. tors participating in this pathway are largely unknown. One Methyl Jasmonate (Jasmonate) Responsive Genes, Gene potential downstream participant, ATHEB-2, is a member of Components and Products 65 the HD-Zip class of transcription factors and shows a strong Jasmonic acid and its derivatives, collectively referred to as and rapid response to changes in the R:FR ratio. ATHEB-2 jasmonates, are naturally occurring derivatives of plant lipids. over-expressors have a thinner root mass, Smaller and fewer US 7,868,155 B2 61 62 leaves and longer hypocotyls and petioles. This elongation Rapid, efficient establishment of a seedling is very impor arises from longer epidermal and cortical cells, and a decrease tant in commercial agriculture and horticulture. It is also vital in secondary vascular tissues, paralleling the changes that resources are approximately partitioned between shoot observed in wild-type seedlings grown under conditions and root to facilitate adaptive growth. Phototropism and geot simulating canopy shade. On the other hand, plants with ropism need to be established. All these require post-germi reduced ATHEB-2 expression have a thick root mass and many nation process to be Sustained to ensure that vigorous seed larger leaves and shorter hypocotyls and petioles. Here, the lings are produced. Early seedling phase genes, gene changes in the hypocotyl result from shorter epidermal and components and products are useful to manipulate these and cortical cells and increased proliferation of vascular tissue. other processes. Interestingly, application of Auxin is able to reverse the root 10 Guard Cell Genes, Gene Components and Products phenotypic consequences of high ATHEB-2 levels, restoring Scattered throughout the epidermis of the shoot are minute the wild-type phenotype. Consequently, given that ATHE-2 is pores called stomata. Each stomal pore is surrounded by two tightly regulated by phytochrome, these data Suggest that guard cells. The guard cells control the size of the stomal ATHEB-2 may link the Auxin and phytochrome pathways in pore, which is critical since the stomata control the exchange the shade avoidance response pathway. 15 of carbon dioxide, oxygen, and water vapor between the Shade responsive genes can be used to modulate plant interior of the plant and the outside atmosphere. Stomata open growth and development. and close through turgor changes driven by ion fluxes, which Differential Expression of the Sequences in Far-red Light occur mainly through the guard cell plasma membrane and Treated Plants tonoplast. Guard cells are known to respond to a number of The relative levels of mRNA product in far-red light treated external stimuli Such as changes in light intensity, carbon plants were compared to control plants. dioxide and water vapor, for example. Guard cells can also Viability Genes, Gene Components and Products sense and rapidly respond to internal stimuli including changes in ABA, auxin and calcium ion flux. Plants contain many proteins and pathways that when Thus, genes, gene products, and fragments thereof differ blocked or induced lead to cell, organ or whole plant death. 25 entially transcribed and/or translated in guard cells can be Gene variants that influence these pathways can have pro useful to modulate ABA responses, drought tolerance, respi found effects on plant Survival, vigor and performance. The ration, water potential, and water management as examples. critical pathways include those concerned with metabolism All of which can in turn affect plant yield including seed and development or protection against stresses, diseases and yield, harvest index, fruit yield, etc. pests. They also include those involved in apoptosis and 30 To identify such guard cell genes, gene products, and frag necrosis. The applicants have elucidated many Such genes ments thereof. Applicants have performed a microarray and pathways by discovering genes that when inactivated lead experiment comparing the transcript levels of genes in guard to cell or plant death. cells versus leaves. Experimental data is shown below. Herbicides are, by definition, chemicals that cause death of tissues, organs and whole plants. The genes and pathways that 35 Nitric Oxide Responsive Genes, Gene Components and Prod are activated or inactivated by herbicides include those that lucts cause cell death as well as those that function to provide The rate-limiting element in plant growth and yield is often protection. The applicants have elucidated these genes. its ability to tolerate suboptimal or stress conditions, includ The genes defined in this section have many uses including ing pathogen attack conditions, wounding and the presence of manipulating which cells, tissues and organs are selectively 40 various other factors. To combat such conditions, plant cells killed, which are protected, making plants resistant to herbi deploy a battery of inducible defense responses, including cides, discovering new herbicides and making plants resistant synergistic interactions between nitric oxide (NO), reactive to various stresses. oxygen intermediates (ROS), and salicylic acid (SA). NO has Viability genes were also identified from a much larger set been shown to play a critical role in the activation of innate of genes by experiments designed to find genes whose mRNA 45 immune and inflammatory responses in animals. At least part products changed in concentration in response to applications of this mammalian signaling pathway is present in plants, of different herbicides to plants. Viability genes are charac where NO is known to potentiate the hypersensitive response teristically differentially transcribed in response to fluctuat (HR). In addition, NO is a stimulator molecule in plant pho ing herbicide levels or concentrations, whether internal or tomorphogenesis. external to an organism or cell. The MA diffTable reports the 50 Changes in nitric oxide concentration in the internal or changes in transcript levels of various viability genes. Surrounding environment, or in contact with a plant, results in modulation of many genes and gene products. Early Seedling-Phase Specific Responsive Genes, Gene In addition, the combination of a nitric oxide responsive Components and Products polynucleotide and/or gene product with other environmen One of the more active stages of the plant life cycle is a few 55 tally responsive polynucleotides is also useful because of the days after germination is complete, also referred to as the interactions that exist between hormone regulated pathways, early seedling phase. During this period the plant begins stress pathways, pathogen stimulated pathways, nutritional development and growth of the first leaves, roots, and other pathways and development. organs not found in the embryo. Generally this stage begins Nitric oxide responsive genes and gene products can func when germination ends. The first sign that germination has 60 tion either to increase or dampen the above phenotypes or been completed is usually that there is an increase in length activities either in response to changes in nitric oxide concen and fresh weight of the radicle. Such genes and gene products tration or in the absence of nitric oxide fluctuations. More can regulate a number of plant traits to modulate yield. For specifically, these genes and gene products can modulate example, these genes are active or potentially active to a stress responses in an organism. In plants, these genes and greater extent in developing and rapidly growing cells, tissues 65 gene products are useful for modulating yield under stress and organs, as exemplified by development and growth of a conditions. Measurements of yield include seed yield, seed seedling 3 or 4 days after planting a seed. size, fruit yield, fruit size, etc. US 7,868,155 B2 63 64 Shoot-Apical Meristem Genes, Gene Components and Prod Add 1 Tbsp. Peters fertilizer to 3 gallons of water and add to lucts Soil mixture and mix thoroughly. New organs, stems, leaves, branches and inflorescences Fill 4-inch pots with soil mixture and round the surface to develop from the stem apical meristem (SAM). The growth create a slight dome. structure and architecture of the plant therefore depends on 5 Cover pots with 8-inch squares of nylon netting and fasten the behavior of SAMs. Shoot apical meristems (SAMs) are using rubber bands. comprised of a number of morphologically undifferentiated, Place 14 4-inch pots into each no-hole utility flat. dividing cells located at the tips of shoots. SAM genes eluci dated here are capable of modifying the activity of SAMs and 4. Planting. thereby many traits of economic interest from ornamental 10 Using a 60 ml syringe, aspirate 35 ml of the seed mixture. leaf shape to organ number to responses to plant density. Exude 25 drops of the seed mixture onto each pot. In addition, a key attribute of the SAM is its capacity for Repeat until all pots have been seeded. self-renewal. Thus, SAM genes of the instant invention are Place flats on greenhouse bench, cover flat with clear useful for modulating one or more processes of SAM struc propagation domes, place 55% shade cloth on top of flats ture and/or function including (I) cell size and division; (II) 15 and subirrigate by adding 1 inch of water to bottom of cell differentiation and organ primordia. The genes and gene each flat. components of this invention are useful for modulating any . Plant Maintenance. one or all of these cell division processes generally, as in timing and rate, for example. In addition, the polynucleotides 3 to 4 days after planting, remove clear lids and shade cloth. and polypeptides of the invention can control the response of Subirrigate flats with water as needed. these processes to the internal plant programs associated with After 7-10 days, thin pots to 20 plants perpot using forceps. embryogenesis, and hormone responses, for example. After 2 weeks, Subirrigate all plants with Peters fertilizer at Because SAMs determine the architecture of the plant, a rate of 1 Tsp per gallon water. modified plants will be useful in many agricultural, horticul When bolts are about 5-10 cm long, clip them between the tural, forestry and other industrial sectors. Plants with a dif 25 first node and the base of stem to induce secondary bolts. ferent shape, numbers of flowers and seed and fruits will have 6 to 7 days after clipping, perform dipping infiltration. altered yields of plant parts. For example, plants with more . Preparation of Agrobacterium. branches can produce more flowers, seed or fruits. Trees Add 150 ml freshYEB to 250 ml centrifuge bottles and cap without lateral branches will produce long lengths of clean each with a foam plug (Identi-Plug). timber. Plants with greater yields of specific plant parts will 30 Autoclave for 40 min at 121°C. be useful sources of constituent chemicals. After cooling to room temperature, uncap and add 0.1 ml GFP Experimental Procedures and Results each of carbenicillin, spectinomycin and rifampicin stock solutions to each culture vessel. Procedures Obtain Agrobacterium starter block (96-well block with The polynucleotide sequences of the present invention 35 Agrobacterium cultures grown to an ODoo of approxi were tested for promoter activity using Green Fluorescent mately 1.0) and inoculate one culture vessel per con Protein (GFP) assays in the following manner. struct by transferring 1 ml from appropriate well in the Approximately 1-2 kb of genomic sequence occurring starter block. immediately upstream of the ATG translational start site of Cap culture vessels and place on Lab-Line incubator the gene of interest was isolated using appropriate primers 40 shaker set at 27° C. and 250 RPM. tailed with BstXI restriction sites. Standard PCR reactions Remove after Agrobacterium cultures reach an ODoo of using these primers and genomic DNA were conducted. The approximately 1.0 (about 24 hours), cap culture vessels resulting product was isolated, cleaved with BstXI and cloned with plastic caps, place in Sorvall SLA 1500 rotor and into the BstXI site of an appropriate vector, such as pNew centrifuge at 8000 RPM for 8 min at 4°C. Bina-HAP1-GFP (see FIG. 1). 45 Pour out supernatant and put bottles on ice until ready to Transformation SC. The following procedure was used for transformation of Add 200 ml Infiltration Media (1M) to each bottle, resus plants pend Agrobacterium pellets and store on ice. 1. Stratification of WS-2 Seed. 50 . Dipping Infiltration. Add 0.5ml WS-2 (CS2360) seed to 50 ml of 0.2%Phytagar Pour resuspended Agrobacterium into 16 oz. polypropy in a 50 ml Corning tube and vortex until seeds and lene containers. Phytagar form a homogenous mixture. Invert 4-inch pots and Submerge the aerial portion of the Cover tube with foil and stratify at 4°C. for 3 days. plants into the Agrobacterium suspension and let stand 55 for 5 min. 2. Preparation of Seed Mixture. Pour out Agrobacterium Suspension into waste bucket Obtain stratified seed from cooler. while keeping polypropylene container in place and Add seed mixture to a 1000 ml beaker. return the plants to the upright position. Add an additional 950 ml of 0.2% Phytagar and mix to 60 Place 10 covered pots per flat. homogenize. Fill each flat with 1-inch of water and cover with shade 3. Preparation of Soil Mixture. cloth. Mix 24 L SunshineMix #5 soil with 16 L Therm-O-Rock Keep covered for 24 hr and then remove shade cloth and vermiculite in cement mixer to make a 60:40 soil mix polypropylene containers. ture 65 Resume normal plant maintenance. Amend soil mixture by adding 2 Tbsp Marathon and 3 When plants have finished flowering cover each pot with a Tbsp Osmocote and mix contents thoroughly. ciber plant sleeve. US 7,868,155 B2 65 66 After plants are completely dry, collect seed and place into Cover the entire set of flats with 55% shade cloth. The cloth 2.0 ml micro tubes and store in 100-place cryogenic is necessary to cut down the light intensity during the boxes. delicate germination period. Recipes: The cloth and domes should remain on the flats until the cotyledons have fully expanded. This usually takes 0.2% Phytagar about 4-5 days under standard greenhouse conditions. 2g Phytagar 1 L nanopure water 7. Remove 55% Shade Cloth and Propagation Domes. Shake until Phytagar suspended After the cotyledons have fully expanded, remove both the Autoclave 20 min 10 55% shade cloth and propagation domes. YEB (for 1 L) 8. Spray Plants with Finale Mixture. Wear Gloves and Pro 5 g extract of meat tective Clothing at all Times. 5 g Bacto peptone Prepare working Finale mixture by mixing 3 ml concen 1 g yeast extract 15 trated Finale in 48 oz of water in the Poly-TEK sprayer. 5 g Sucrose Completely and evenly spray plants with a fine mist of the 0.24 g magnesium sulfate Finale mixture. While stiffing, add ingredients, in order, to 900 ml nan Repeat Finale spraying every 3-4 days until only transfor opure water mants remain. (Approximately 3 applications are nec When dissolved, adjust pH to 7.2 essary.) Fill to 1 L with nanopure water When satisfied that only transformants remain, discontinue Autoclave 35 min Finale spraying. Infiltration Medium (IM) (for 1 L) 9. Weed Out Excess Transformants. 2.2 g MS salts 25 Weed out excess transformants such that a maximum number 50 g. Sucrose of five plants per pot exist evenly spaced throughout the pot. 5ul BAP solution (stock is 2 mg/ml) While stiffing, add ingredients in order listed to 900 ml GFP Assay nanopure water Tissues are dissected by eye or under magnification using When dissolved, adjust pH to 5.8. 30 INOX 5 grade forceps and placed on a slide with water and Volume up to 1 L with nanopure water. coversliped. An attempt is made to record images of observed Add 0.02% Silwet L-77 just prior to resuspending Agro expression patterns at earliest and latest stages of develop bacterium ment of tissues listed below. Specific tissues will be preceded High Throughput Screening T1 Generation with High (H), Medium (M), Low (L) designations. 35 1. Soil Preparation. Wear Gloves at all Times. In a large container, mix 60% autoclaved SunshineMix #5 with 40% vermiculite. Flower pedicel receptacle hectary sepal petal Add 2.5 Tbsp of Osmocote, and 2.5 Tbsp of 1% granular filament anther pollen carpel 'style papillae vascular epidermis stomata trichome Marathon per 25 L of soil. 40 Silique stigma style carpel 'septum placentae Mix thoroughly. transmitting tissue vascular epidermis stomata 2. Fill Com-Packs With Soil. abscission zone ovule Loosely fill D601 Com-Packs level to the rim with the Ovule Pre-fertilization: inner integument outer prepared soil. integument embryo sac funiculus chalaza Place filled pot into utility flat with holes, within a no-hole 45 micropyle gametophyte utility flat. 'Embryo Post-fertilization: zygote inner integument Repeat as necessary for planting. One flat set should con outer integument seed coat primordia tain 6 pots. chalaza micropyle early endosperm mature endosperm embryo suspensor preglobular 3. Saturate Soil. 50 globular heart torpedo late mature Evenly water all pots until the soil is saturated and water is provascular hypophysis radicle cotyledons collecting in the bottom of the flats. hypocotyl After the Soil is completely saturated, dump out the excess s tel epidermis cortex vascular xylem phloem Water. pith stomata trichome 55 4. Plant the Seed. 'Leaf petiole mesophyll vascular lepidermis trichome primordia stomata stipule margin 5. Stratify the Seeds. After sowing the seed for all the flats, place them into a dark 4° C. cooler. T1 Mature: These are the T1 plants resulting from inde Keep the flats in the cooler for 2 nights for WS seed. Other 60 pendent transformation events. These are screened between ecotypes may take longer. This cold treatment will help stage 6.50–6.90 (means the plant is flowering and that 50-90% of the flowers that the plant will make have developed) which promote uniform germination of the seed. is 4-6 weeks of age. At this stage the mature plant possesses 6. Remove Flats from Cooler and Cover with Shade Cloth. flowers, siliques at all stages of development, and fully (Shade Cloth is Only Needed in the Greenhouse) 65 expanded leaves. We do not generally differentiate between After the appropriate time, remove the flats from the cooler 6.50 and 6.90 in the report but rather just indicate 6.50. The and place onto growth racks or benches. plants are initially imaged under UV with a Leica Confocal US 7,868,155 B2 67 68 microscope. This allows examination of the plants on a global Objectives level. If expression is present, they are imaged using scanning laser confocal micSirocopy. HCPL FLUOTAR 5x/O.5 T2 Seedling: Progeny are collected from the T1 plants HCPL APO 10x/0.4 IMM water/glycerol/oil giving the same expression pattern and the progeny (T2) are sterilized and plated on agar-solidified medium containing HCPL APO 20x/0.7 IMM water/glycerol/oil M&S salts. In the event that there was no expression in the T1 HCXL APO 63x/1.2 IMM water/glycerol/oil plants, T2 seeds are planted from all lines. The seedlings are grown in Percival incubators under continuous light at 22°C. Leica TCS SP2 Confocal Scanner for 10-12 days. Cotyledons, roots, hypocotyls, petioles, 10 Spectral range of detector optics 400-850 nm. leaves, and the shoot meristem region of individual seedlings were screened until two SHE were observed to have E. Variable computer controlled pinhole diameter. same pattern. Generally found the same expression pattern Optical Zoom 1-32x. was found in the first two seedlings. However, up to 6 seed- Four Simultaneous Detectors: lings were screened before “no expression pattern' was 15 recorded. All constructs are screened as T2 seedlings even if Three channels for collection of fluorescence or reflected they did not have an expression pattern in the T1 generation. light. T2 Mature: The T2 mature plants were screened in a simi lar manner to the T1 plants. E. T2 seeds were planted in the One channel for transmitted light detector. greenhouse, exposed to selection and at least one plant 20 Laser Sources: screened to confirm the T1 expression pattern. In instances where there were any subtle changes in expression, multiple N.Y.,W. 8/5 mW, 476 mm/5 mW, 488 mm/20 mW, 514 plants were examined and the changes noted in the tables. T3 Seedling: This was done similar to the T2 seedlings Green HeNe 543 nm/1.2 mW except that only the plants for which we are trying to confirm 25 Red HeNe 633 nm/10 mW the pattern are planted. Image Data: Results Images are collected by Scanning laser confocal micros- Table 2 presents the results of the GFP assays as reported copy. Scanned images are taken as 2-D optical sections or 3-D by their COesponding cDNA ID number, construct number images generated by stacking the 2-D optical sections col- 30 and line number. Unlike the microarray results, which mea lected in series. All scanned images are saved as TIFF files by sure the difference in expression of the endogenous cDNA imaging software, edited in Adobe Photoshop, and labeled in under various conditions, the GFP data gives the location of Powerpoint specifying organ and specific expressing tissues. expression that 1S visible under the Imaging parameters. The invention being thus described, it will be apparent to Instrumentation: 35 one of ordinary skill in the art that various modifications of Microscope the materials and methods for practicing the invention can be made. Inverted Leica DMIRB Such modifications are to be considered within the scope of Fluorescence Filter Blocks: the invention as defined by the following claims. 40 Each of the references from the patent and periodical lit Blue excitation BP 450-490; long pass emission LP 515. erature cited herein is hereby expressly incorporated in its Green excitation BP 515-560; long pass emission LP 590 entirety by Such citation.

TABL E 1. >SEQ ID NO 1 construct YPOOO1 ctgcattcacacatattittgggct ct cacgtgtttgttgaatttaatatatttgact acacgatctittcaacg tatgaa aaagttittatact act attitt cqtttgagtgggaaataaacaaatgatagot acagttatctatatggtataattitta cacttittatalactaataatgatgagtgatgacaatcgagtgtcggatataac aggccaacaagtggaatggactitatg taactttittaatcacgggattaaatcacgtaacccaatgtc.ctaattgg tatttaattittgattatctogatgctaca tattgt cataggact catat ctittgat cacgtgcc.gctaccaatccaga cattt tagtatacaaaaaaaaagaagata caaacttaagatatggaatatatat cagaactat cagttittagactittaataattic gaattgaataactacgatcaat at at aaattggcaaatagattggtcaattgtagtgcaagaaatttgttgaactitt attacagtacgaagagagtaagag aa.gcaagat.ccggitttittaggcaacaagta acatttittgagttcagagagtttgcttct tactittaagttacgtcact

acaaaa.gc.caagttcct acttct taggit ctaaagt caattitt.cgaat attcagaaaaattgtact citactagat.cgaa

tagttitt caccggtgaaacgatatataaatgaag act acaatatttitttaattitttittaa.gcg tatgagttctagacc

tttggcacgtaaatttctic cqgtacctgggaccaatcgttgataatat cacgtttalagatttaat catc catcc.caag

tagagttgaactagtaaccttgagcactttitt ct cq agacaactaaaccatcatcc acttagtgcaataaag.cgt cat US 7,868,155 B2 69 70

TABLE 1 - continued t ctitttittittcttittcaaaaatt cqtatttaattittaatttattaaaaatatttcttttgttittaaattgggacagaa titat catttalacatatttaaaattitat atttittaattaaaaataggg taaaatatatttittcaaacaaaaattcaaaa atagggcaattittcaaaat catc catt cittaaatctaaagt cqgctacagt cittitt cqttgttttgttgctaattitca atttatatacatgcaaattacaaaatataatagitttittgggggataattat cittcttgcgcc tttitt attaaattaat atgct catatagcagttcttacaattaatataac tagggittittaaatttcaat atcgagttgacaaaatgaattgttt acaagtttittittcttitt caatatgcattgttcat cacg tattogtag tdatgcaaaaacaaactataaattataattg cact agtgagattagcaagaagtgttataaattagaataaacggalactatoaaactgttgttatgtacac catttattt ttgttaaagaatatgtgtag tagttagaaaactgat caaattaaactgaaaatticacattacggagat caagttacat tgtc. tattgatgaaaaaaacaaaataaatccaaatggcactaaaagttgtagaaattgaaagaagaaaatagatttitt gtctaggaataaaagttcaaaatgggaaaga caaaaaaaagagaggcaaataag cagtgatggagct aaa.gcaacgctt tact cittittaattatgaattatttgatttgacct coacticgcc tiggctttittittggttgttctittatagaaaagtaaa ataacacaattagcacatalacatgagttatcgagaalacca attct citttgtggtgttittagttaatttctata actta tgaaac cattttct cagttitat catgataattgatcct ct atttaaaac cctaaagtttatattttgtttgttcaaac ac

>SEQ ID NO 2 construct YPOOO7 agcagaacaactatatttattgttgtca cataaatctgagat catttata accaccaaagaac ctatacacagtaaatg acaaatgitat ct coct citat citctattgcc catatgtagatgctaaagtaagatttct ctitttittittaatgtacttitt ttttgtataaagtatatt coataagaaaaaggaaaagcttgtt tatggat caattgaccc caaaaaaagtttittagat caaagcc.caatataaaaaaaaaacacagtagtgacacaaaggaacttaaataaaccatgaattgatctataaacagta gagat.cgataaggcgaacatttitccatgtgaagtgtc.ttctitt catctataat atttittgacat coaataattitcctic tataatat catt cacataattgatagaaac attatgttagaattgtc. cacat catttgagctgtaatatattotgttt taacaaattatatgg tagttgcttaatct tatgtc.cat cittcttctatogcatcgttitt cqcgcc tagttgtc.cagt cc atttcaactacctacct ctaatt cittatcttaaaacaa.cattttittaatttaagtattatgctcaaagactaact aga tagaaaac cqtt attaaac attaaacgaattaaaagtc.ttacatggaaaatgtaggitttataaaccacgagittatgat tgacaataaaaaaaatgcaaatcatcaatcaaaagagacittgagtgcgactictatat caaccattgcaattaaaatta t citat cacaaaaattittagacagattaagttaatttagt ctaaatt cactaatttattittctataattagtaattaac tatatttatttatttacacatttitctgata atttagaaatttgcatgaataacaaatataagattittggaaattagta gcaaatttaattaataattatttittgcctaaatgaaccaaacTATAAAacctic cacatacaccagt catcaaatttac agagacaacaaactaaagt

>SEQ ID NO 3 construct GOO13 atcttgttgatacacaattt attact atttggtacattttgaagtatttgtttittgcatgatatatgacgittaatttga actgat attagt caattitatggg tacaaaagttgaaagtttagagcactatgttggattt attaaaaatgatat cata caatggttcaatatatatatatttitttitccacgtttittaataa catttittgtaaacaagt ctitc tact attgtc.ttta ttgttaatgagtttctagtacctaattaggaattittgaggatatacgatacattaatgagttacattatcc.cgaaaac aaaatc.ttgaaaacgaacaaagataatttgga cattact.cgittatgtatacgitatggaattggatagagcc.gttgaac catcaagtgggit cittcaagt caacgaactgaatttgattittacact catgtacatcggccacaattittatt cacacac tactaacaccitctggtgtc. cacttittittctitt ct ctagattgatgtgttaagattitttgttgcaatt catttatt cag gtatttittatatatatatatatataaattagaataaactaatttaaagaaagatatagdaattatgttt cacatttta acattct caat catttataaaactaatgtggtgatgaatggtatatatatatatatatatatatatatatatatatat tttgttgttgaactaatggtaaat atttaaaataaga catacgtacataaatccacggact cittaaagt catgatgcgg ttaataaatgttcacataacggtaaccaagtggctcaaaatcatgaaacaacgtcacataattitat cittataatgtgg US 7,868,155 B2 71 72

TABLE 1 - continued ataattagtaccgcattatttgtaagaaaattaaattaattatagatt cacagctaagaaaatacgaaaagacagctic aacacttitt.ccactitct attccc cactgtctataaact citgataaataatctotgat citcticc

>SEQ ID NO. 4 construct YPOO97 ttcatctittatatttaa.gagtttaaaaactgcaactitttgtttitt ctitt cactaagtic titatggccacagttaattaa alagcagatgaaaggtggtc.caatggaalaaggagaatgtgattgggct agttgggagagttctgatgtct agtgttggg tacacgtgtc.cgtcagttacacatago attaaat cagacggcatgtcattatt caaatctagttca catagitacgact aatagotgataaattaatgattatacagoatatgaattatgaattcaaaaaaaaaaaaaaattgaaaatgttaaggag atgctatattitt acaaaatt catcgcaatgctttctactaatttgctaagtggtottct c cagittagt cittgtcgatt c caag.cgatatt attaaatcttgaagcatcgctcaaag cattatagottaagataaccaaattgtt attaaaaac acc tagtgaaatttittaaattaaaacaattittgatat citttgtaatat ctaatact act ctittctgttgtctaaaaggatta attittcaaaaattt cacacat attaaaaaaaaaaaaaaattac tagctaaacaattittcaataatcataaaacaatag taacttaataattittitttittattittcaaaatagt ccttcaagtttacaatt cattt tagt attataatcaacaaaatt tg tattaaaaagttggaaaattaatctttgtggaacaaaaaaatctagaaatcatttitttagaattagagagaggittt gataaaaaaaaataaaaaaaaatagagagagg tag tacatact aaacgatgtgatact act attgacaaaatc.ttaat t ct cagtttagtagaataaactagaaggaatgaatgaagtaaatgcgaatccaact actaacaaac cct acttagtica t catattitt.cccatatgaaatcc cTATATA Aacc catcatCatct cocacttittitt catat coa >SEQ ID NO 5 construct YPO111 tatatagtttittatgcattct cotcttgtgtaatacataaaccaaatatgagataggittaatctgt attt cagataat attaaatticcaaacaat atttitt acttgttataagaaggcaattaatat ct ct ctottaatggcaagtggtaccaagt agtattaaact attaatgcaatggaagagtactgttggaaattataatcct citat cacacattcaaacagatctoctd aaatcttctic titccaaacttgtact tctctgatccaaatgtaggctic caaaatataga catttaccatttactaagtic cacaactic ctitt cittgtct cottcaaaaatgact cittgttgtaaccat catatgact cogacagttcggcattgc.catg atgaga.gcttaaaaatt cacct tcc tdagcatttcaagtc.ttcactic ccttagcttgacctgaaccaagataaaatgc Ctttgtcgt.ccc.gtaatat coat cotgctittggacggcatcat agttacatt.cgatccatcc tatttacaatgttatt ttagt attaaaaacatgacaataaatttgttgttaalacat attcaaatacaatatgattggatttataagtaattgta atatgaaatgtc.ct tagtaatatgttaaaaaatacatagatacacacacgtactaaaagaggcaacg.cgggagatgtc attagaggaagaactaggaag cagagcgttcatgcaaaatgctaccaaaaacgittaatgcaat atctgaactaat cag cacagt ccattt catactgagaatgtaaaaac caat cago atcgt.ccatttitt totatic taattatttgttaact citta attggccacaact tccaac cacatgacgct ctittctattocctittatatatt cocatctoaaatgttcttggaga cac aaaatat cataaacatataaa cataaacgc.caatcgcagottttgtacttittgg.cggitttaca

>SEQ ID NO 6 construct YPO104 tittattittatttitttgaatgaaaatgtc.ttctittatt cqtaattittaaact cactggtggtggatatattgttatgtc cc caatticgtctggcaact ct cqtat attagtgagaaaaatttgtcc attatt tactgcact attaccctgtgttaat ttitttgtattgaaattgttttittagtaatt cacgt.catatagogaatgatt ctittaattittaaaaatticagt cittaag tttacaaattaaataacgctact.gtaaccaactctgtacgaccaa.catgttctgagttitttgtatatacggc catatat gtacat attt tactataaag.cgaaaaaatc catalaattatttaattaatatataaaggtgcc attctattt coaatgt gcttaggaaaatgcagaac ct cqtgctatat citctgtc.gc.cacgtgcaaatataacaatatgaaatagalactagoaaa t cittgaaatctaact cittaagactaattcaag cacatacgtagagaaagttgaccaacggittat cago attittaacat ggaccittatcaa.cattittaacaaagtic cacaaacaaccagt citta caatcgcattggtacaagataatcga attcatc titccatataacaaaacctaaaccttggtgtgaaaaggagaagatatgtatgttaaaggcc.gc.ctatgcctic toggtttg gggtatatgattictaagattagggitttgaatattitt cqttagcctgc catgagatatatt tatgtgata attagagcc US 7,868,155 B2 73 74

TABLE 1 - continued t cittatgcattaatgcatalaccgactagat catgtgg tatt cagotaat cagtacacacalagacaaagtag taaatga gtttgatgaagactgtggtctgataatticcitat caacgttaaatctgtcggggcCagg cago Cagcaac attittgcct aacaacgctctgaattcaattgaac ctaggctatataatagoaggctaact talactaagagtt

>SEQ ID NO 7 construct YPOO75 tggattacaaat cattaagctaatat citt.cgatgaattaagaagataagtggataacaagtaccta accogcaatagt c catalaattaaaacattaatgt atttgtcgttgaaaatttgg.ccgacttittatttgttatt ctagtttccacatcaaaa atgtttgtactitcqtagcaatccatccaccitaaaccc.caaatc.ttaatttatatttgttgcgtttalaatttgggtgag atttgattictaagtagttgagataaattgatatt ct attcattagtaaaatgatagagaaattggtttataataattit taccctagaacatgacatgat attggtaac cattaatcaaagaaagagcaaag catttaatttaccctact ct coaac cact coagcc titt attagttgcagttgggaat catttctittatgatt cittatgtcattgtct cotaaat caatgaagt gccttgaccttgttactaatt cqaa catagcaaag.ccaactacatagat cotttacaaagttctaaaaa.caggttgtt taggcgt.ctaga caaacaaaaccattttgtacgattcaacaaattgg to catagaatgttattgat ctittcttgttta ggcatt cqataaatcggctaatacattatttittttgttittgctttitt cott attaaaaatatgcaaagtattatgatg titta acctgaactgaattitta catttalactggatataggaaaatattgggttgaatttaataattaa.gcaattgtcac gtaaatcaaattgggcttaatatatattgttgattt cagdaaaga caaagttgggcc.gtttcaatagt citt cacgcga tgtaagcgttcactaac caactagagaaga caatcaaatgaatacgttccacgtgacgcttacgaacttgtcagt cac tittggtaatatgacagacagtaaccagtaaac tactaatctottt cqctaacgaacacacaaaa

>SEQ ID NO 8 construct YPOO16 aaacatgttt tatgtaact actttgct tatgtgattgcct gaggatact attattotctgtc.tttattotctt cacac cacatttaaatagtttalagag catagaaattaattattittcaaaaaggtgattatatgcatgcaaaatagdacac cat titatgtttatattittcaaattatttaatacatttcaat attt cataagtgtgattitttitttitttitttgtcaattt cat aagtgtgatttgtcatttgtatt aaacaattgttatcgc.gcagtacaaataaac agtgggagaggtgaaaatgcagtta taaaactgtc. caataatttactalacacatttaaatat ctaaaaagagtgtttcaaaaaaaattcttittgaaataagaa aagtgatagatatttittacgctitt.cgtctgaaaataaaacaataatagittt attagaaaaatgttat caccgaaaatt attic tagtgccact cqct cqgatcqaaatt cqaaagttatatt ctitt ct citttacctaatataaaaatcacaagaaaa atcaatcc gaatatat citat caa.catagtatatgcc citta catattgtttctgacttittct citatic cqaatttct cqc tt catggtttittttittaa.cat attct catttaattitt cattact attatataactaaaagatggaaataaaataaagt gtc.tttgagaatcgaacgt.ccatat cagtaagatagitttgttgttgaaggtaaaatctaaaagatttaagttccaaaaac agaaaataatat attacgctaaaaaagaagaaaataattaaatacaaaacagaaaaaaataatatacgacagacacgt. gtcacgaagataccctacgctatagacacagotctgttitt citcttittctatgcct caaggct ct cittaact tcactgt citcc tott cqgataatccitat cottct citt cotataaatacct ct coactic titcct ctitcct co

>SEQ ID NO 9 construct YPOO94 taaagat Cagaagaggalaggttt cqc.cgcggcggttgcatctt Caccgt.cgattt catcgttacagcgacgc.cggtaa titcc taggttgcttagttcc cattct citctictaaaattagggct cqaaatgaattgttgaacaagatagagat cittitt tctgat coccgt.cgaacatttatt caaggccaaaaaaag.cacacgggaatttagagtaccaatacatat caaaac cta atgggctittgaatggttgcatgtgtgtgtttatttctgatatgcaaag.cgatcgatagt cittitt coatacaagtgtaa actgtaaacaacgtaattaag cataacaatacaact ctitt cittct ctitttittitttgtaaacacaaaataaaattacat caatt catgcttitt cotagttcatctgacatttitccaaaatt catgttc cattgagtic cctaat acttgttcatatt c at attaggg tacatgaataaaagttat cattcttgaaact act aaattitt catagtttatttitt cittcttitt.cgttt c actitt.cgaacaaaacactacgcgtggcatttgcaatgaatticcacattatatggaataacac catgatgaacattcta catatataattattatgtttalag cact tag acagcatalaattic tittctaattatataaatctaaccttgttacattgt US 7,868,155 B2 75 76

TABLE 1 - continued a catctataaattacttgaagaaataacgagttctatttctttittaaaaattaaaaatactataccatat citcagtga ttaagttgaaccaaaaggtacggaggagaaacaa.gcatttgattctitcc ttattittattittatt catct ct cactaat gatggtggagaaaaaaagaaaatacctaacaaacaaatatatattgt catacaaaaat atttctatatttittagttaa ttagtttatatt cotcacttitt cagggcttatataagaaagtgagcaaacacaaatcaaaatgc

>SEQ ID NO 10 construct YPOO33 aaact tccaaatttctaaacggatgcaataagaact tacatatt citctitt cattagt catttattggccagatttatt aaaaaaagttt tact caatgaccaaggattagagittaaagataatatagattattacatatatt attcgaaaaaatat acgcatgtc.cgactittittaaacct caaaaat atcaaaaccagaaaagatgataccacacaaaaaaacaataaaataat aagtggaagagatat catcggacaa.cagtacaagtacago accagctctgccaaaa.gc.caaaac catttgttcaattac agaaagatact attgtttgcaattactaaattaccc ct cqgactittacaaaag catct ctaacttatccacgtgtcag t catctattgattgtttcaataccaccittg tattaacgc.cccacgatt.cgtggttggg tacacctgatagt cc gagga tatttaaatctoacgc.gct cqtgtctataatt cqactgtact.cgcttitt cittgtcgtgattittagcaatttacgaagt caaatgtttgact caat cagacittgcgcataagaga.gcgagtataaatgtt tactatact cacgcaagtggggctitta ttgaaact actic titttgtaataaaaccagoagtggttttgttctgaatcc.gct citcttgc catatataccacaaacag aaac cacagaagatat cittittgagaaggaaaaaaaaaaagaagcttct c ct ct tcc totgcc ttcttctitt coattta ttgcaaac cctdatcaagtaagt caaatctt cacgaacacatatgtatataaattcaatccaagaalactaggagaaat citatgaaagaggacaaatctaagttcaagtttgaatcaggaagattatctagatttgat cattttgacatttacgatgt gctt actt attcttgataaactittgatgcagttggttittggtgttagticttittggggagaga.g

>SEQ ID NO 11 construct YPOO49 acat caatttgcctgcttgtagggtgatt.cgtcaaatc tattatcaggittittaaatatact.cgaattgact tccaaat t cittagt ct c tagtgtaatgattittgagaat cacttaact coaaaaatataatccacgatcc.cgtgttaattattgaa gaat caatcgtttittaatttct caccalatagatgttgct cittatt acttaaaacaaattgtttaga caaatgtagcaa. gtgtgatact tagtgggat cittaaagacgatttct c ctataacagagga caaacaggit cqgt caattacaatgtcatc cct citttaccctgtc.tttittttitt citt cittaaaacctaac catttgattgtttctaaagg tatttcaagaatatatga t caatctagatgaatactataccgacgatgactacacacacaaggaaatatatatat cagctttcttitt caccitaaaa gtggtc.ccggitttagaatctaattic ctittatctot catttitctitctgct tcacatt cocqctagt caaatgttaataa gtgcacacaacgttittct cqaag cattagaatgtcc to ct cittaattaatcto cittctgattagattct caatagagt ttaaatttgttaatggagagatatattggg accct caaggcttctaattataccacgtttgg cataattct citat cqt ttggggccacat ctitt cacactt cattacctitat caccaaaacataaaatcaatcaacttittttittgccittattgatt gtgttggat.ccctic caaaattaaaacttgttgttc.cccacaaaagcttacccaattt cactitcaatc.ttaacaaatagg accaccactaccacgtacggtttgcatcatacaaac cacaaactic cttctt cattac

>SEQ ID NO 12 construct YPOO6 O tggagctt tattgaaatgcaagaaagtaaacaaaggaagat Ctttagattgtcaccalaga.gtggtctgaaact ct cat aacact caatcc toctic ct cotcat caccaccactacaaaatatt at attctictat ct ct caatctatogaggagatgt attctatoaa.gcatttgaaatgataagaaactggcgat catcc to tacgtcac cat cact coaaaattatcct ctitt c taggitttaagttttgtaatgatcgc.ctittatttgttgagatct ctaactitct cqcatttic caaaatgttaagt ccaat aactgcattggittaagttggggggttactagt cqgcttaaatcCaaatatggatttgatt CCatatgtatgtgacagt ttcttaacgttcat attacaatgaatgatggat.ccttgactagacaaagagaaaatggattgtcactitcgtaggaaaa atagaaattct c cacgaaggctggit ct cottt atttaacgacaaatt cact catagt ct cattcacaatttgaacttg totalacacaatgtgttatatact cqcgaaaagaa.gcataataggct cittaagggitaatccacga aaccaaaacacata taaaac attaat atttittct ctaaatttatt catat caataataaagtttacaaaaaatataaaacaataatc catac

US 7,868,155 B2 79 80

TABLE 1 - continued

Ctaaattitta agggittt Cttgtacgaalacaggga caat cagagattaagaaactictatgtggaaaaggc catgcgcat tttgttatgtgattcaacaaataagatgaggaggcaaagt catggttctgttctaattaacaaatctactatgggggc cgttgctic cc tattgtccacgct cottttctt catttct citcc tigcaggatat cittgtc.ttittgattctt cattt tag gtc.ttataaatat cacgtggttcaggcctic caatgtcaaattat cattacgtggaact ct cittagatgcttgagaaaa gttagcticttacctgtcCatagaagct coaaggaag.cgagaat agtagatactittggttggCC

>SEQ ID NO 16 construct YPO102 atttggttgataacgttitt cact cqactaattatatactt cagaaggatagtaatagaataccaaaataattaaatga ttggittagtgcct tagtggagacitttittaa.ccgattictaatagactaatgatgtagctaa.gcatttatttgggat cat cactgtttgaaaacgtgaaatgtgataaaagttatgaaacgattaaaatataaaataaccotacaaaac attatgtac cgtttittittct ctdttcttittggcgatttggtttagttcgttacactictaaatgttattgcagatatatatataatga tgcatttgcatctgaggaacatataattic.cggittaa.cact tccaaatcttatat cogit ctagg tagggattittataaa t catttgttgtcatcatgcgittatgcttgtcggctittgaccatalacgcagagatatagaactagottt tact taactitt tagatt tatt atttgat Ctagagittaagtggagatatatagtgtttttgttagattattggtggatgtgagagtttgt Ctttagtttcaagttgagaatataaggcaa.gaggagacitctgaggcaat cagaggttittgattggcaaaat atccaaa agg.cccaaac caagttctgaag.cccatct cqtacaaaaaaagaaagagatctgtaagaaaaaat attctittgatatt citt acaaaaataagtgtaaaacttitt attagt caaaatc.ttcaatctittaaaaact ct cat cact cotacgaaag.cgcgtg agagittatgaga cattcct taatagcattact cacaagt cacaagttcaaaacgtctgactgaaacagaaacaagcct ttgttgaagttcttgaagaa.gaga cattagtact.cgt.cgtatagccataaaagg taatatacgaaatttctitcqctaat citct tcacct tcct citacgcgttt cactitt cactittataaatccaaatc tocc titcgaaaac at

>SEQ ID NO 17 construct YPO 103 gttittgaagaacaatctggat.cgaaatcta acataagg to atcg tatt caagttacgcagt caagg acttgacat cat cc tact ctdgtotgaggittaccact tccaaagatgggatttitt coacticgg tatgct tcc taagaaatt cqttittatt gaacct agcaaatat cttgtaatgtaagattic ctgagatgatgaagaaaaaacaaact tttgttacagcaggagaacg gagagaaagaaaacagagaac Caaatgct Cttgaagcaaacagaagaagaaga cacaaatccaaacttgagacitt Ctt Ctacaccagaaaaccgcagcattctgggacaacgcaaaac acgaaagtgaaacgggcaatgatatatatgtcttgggt gcqttaca aggcatcgtttgcatgttgagttggataagttcaactgtc.ttcttittcttittggttgtagtagctgcc titt tttitt.cctttgttgctittaagaaatagc.ccgaaaaaaagaatgttctacattt cqgagcagaaaactaaccogaatgag tttittggit cqgatcatcggat.cgat cagatatattittgagttacgaactgttataaaaaaagccataattittgttgttg agtttgcaaaataccttataacttgttatttgagattgcacct coatatat attaatt cqtaagag tattt attaagt aagctittagtataaatc ctitttitt.cctittaaagtaagttaatgttctactaaataatagtaaagttgaagaac cqctic cgttitt acac catgcacgtgttatctaacaaagaaaatatgg tacaccitaatggctaatgcaaaggacaacacaatga aactaacttgactctgtgttatacaaaccolataga catctgcatacatcc tag tatttgTATAAAttggactcaaatt cctgaggacaat catagdaaacaat cacat catcgcaatatacataaacaaaagaggaagaaaaa

>SEQ ID NO 18 construct YPO107 taacaatccttgggaac attgcatc catagatat coggittaagat.cgat ctittgaact cataaaaactagtagattgg ttggttggitttccatgtaccagaaggcttaccct attagttgaaagttgaaactttgttc cc tact caatticcitagtt gtgtaaatgtatgtatatgtaatgtgtataaaacgtag tacttaaatgact aggagtggttcttgaga.ccgatgaga.g atgggagcagaactaaagatgatgacataattaagaacga atttgaaaggct Cttaggitttgaatcctatt.cgagaat gttitttgtcaaagatagtgg.cgattittgaaccaaagaaaacatttaaaaaatcagtat coggittacgttcatgcaaat agaaagtggtctaggatctgattgtaattittagacittaaagagt ct cittaagattcaatcctggctgtgtacaaaact acaaataatatattittagact atttggccittaactaaact tcc act cattatt tactgaggittagagaatagacittgc US 7,868,155 B2 81 82

TABLE 1 - continued gaataaacacattccc.gagaaatact catgat cocataattagt cagaggg tatgccaat cagatctaagaacacaca titcc ct caaattittaatgcacatgtaatcatagitttagcacaattcaaaaataatgtag tattaaagacagaaatttg tagacttittttittgg.cgittaaaagaagactaagtttatacgtacattittattittaagtggaaaaccaaattitt.ccat cgaaatatatgaatttagtatatatatttctgcaatgtact attittgct attittggcaactitt cagtggac tact act ttattacaatgtgtatggatgcatgagtttgagtatacacatgtctaaatgcatgctttgtaaaacgtaacggaccac aaaagaggat.ccatacaaata catctoatagott cotc cattattitt cogacacaaacagagca

>SEQ ID NO 19 construct YPO110 t catctgctaggcgattaggttt catacacacatgagtaaactgcactatotagttcatata cact coat cittattga tgat atttcaattittaaatagtaact catatacttitt cagtatttaatttatt atttic cittaaaccaaatttcaatct tacaattt cqaatttgcaatacaatttaaatat c tattittatgataataaaaataaaatttaatttgattgtataaaa ttcaaatacaatticgattittgcaatagaaaacaatttaattictatacactic catctact attaattitt.ccattatagt tataaattagtatatgtaaatttgtttatttitttittaggittttitt ct cittctaagagaaaaaaaaaaagttaaaatct titt.ccgatacatgtcaaaatataagat.cgatagatttgc.catgtgttacgatcg tatgagtt atta actittgaaaatc at actittatataatacaaaacatgtaaata catgtttata catatatttacaactaaaaacatgtgtaaaatctaatg gatttittaaatacatgcttittagct cqaaaaaaatttgatacggagaaaaaaatttgacgggaaataacatacgtaaa tatctgat caaattatctatagtacgattittgacgggaaaaaaaattattittaaaggaagagctta actittgaat ct c actaaaccagat catacataatcaatic ctittcttittat cittitttittittcttitt cattacgtgtaatcgtgttgttgtct aatatat cagtttgatttgtaataatttgaataaaaaagggagtgttgttatctittaagtttgcc.caaaatctatagt catgttcgatgtaaacgitat cittaaacaaaattattaaatgttaaagatagta acatacaattattaatgaataaatg tttalactaattaaatat catttagtgattgtccTATAAAatct cittgttitt cittgttt catatc

>SEQ ID NO 20 construct YPO112 titatgtgc cctgatgtc.ctatgcagatggtgcaact actgcttittggtgagaa.gct tcgcgaacaagttgaggaaagg Ctagaattitt atgacaaaggtgttgc.cccacgcaagaacgtggatgtaatgaaggaggtgatagagaatctaaagcaa. gg tatttcttgtagctgtttittttittggttgtaatcagagt cct citt tatgatggcaaacticagtgtttittittatctg titcCtcCtttagaa.gagga agggaaggagc.cagttgatgccticggtgaagaaaagcaagaagaagaaggcaaagggtg aagaagaagaagaggtggtggcaatggaggagga caagttcagagaaaaagaagaagaaagagaagaggaagatggaga Ctgcagaggagaacgagaaat cagagaagaagaaga caaagaagagtaaagctggaggagaagaggagactgatgatg gtcacagcaccalagaagaagaagaagaagttctalaga.gc.gctgaatagaaagggatgcaac attaacaaaccctgt att gtatttitttittittgagctaaattaatgtcgtctgtttitt.cgtag togalacat cqgagaattitttgttittggtctggaaa cgattcaaggtttggcaatat cittaagtttgtttaggittitt cact attittgacgtttgcaa.ccgtgaaggaggct cot c cattittataaaatacaattaccaatticcagtgctittgcaaatgtttcaataatagotaalactalactaccalaattgga aaactagottaacaagtttgttgaaaatgaatttggagc catatgatttatt attitt acccaaatggagtaatagaaga agagcagctcgc.gtttgaatggt cagttalacattaacaaaaggtaaaattgaatagatgttaaaacttgttgtaagtaa acaatagagctacct cottttgagaaggatagataaacticgtgaccalaccacatt.cccagtic cc

>SEOID NO 21 construct YPO116 aaacgcct ctitcggtccacgctgtcgttittattgaaggaattatattittattittaattgggcctgcaggctaalactat aagt cc.gtctgatatgggit cqggttgggct tatgagtt atgggtctgg taggggtcaattagcttaattitcgatatgt gccc tact ct cq acctaacgttittgaacacgtaagagagagtttctaat attgagttgtctaattaact cqataggct tatacaaagtgttt cogcattttacct tcttaataact catcatt cactaactaagaaaagttt tact cagaccatat citt.ccgcttcttgattattgtcaatttgttgtcact caattitat citcttgcaaaatttagttgaaatcatttggttt c atctittggct cittgaatagttgcatgtgtg tatttagtaagttcttittcaattaagaaggaagaataaaacaaattgt US 7,868,155 B2 83 84

TABLE 1 - continued ggcc agaaacaattatgttgagttittatct catacgttggct cattcatc.cccatctotctgcttittgaat cattcta citcc toccatttitttgatcgtcc titttitcc tigcttctgaacatggat cattgttgcatgttcggatgttcct coat cqt gctgaaactcaaagtctgaatcgattaccatagact ct caacccatctittgatatataaaaaagagccittaac ccatc tottct actic tocct ct ctagaaacaaacacat cacgtgatgatctgtttic cc cccatacttacgggatgat cagaat gtgg catgaggaaaaagccaagaaataagttgataaatttaaggtttaatttaacaaaaatgagagattaatcttitt c attittagggit cqcacgcggtgttttgttgcaa.ccgcagaaact tcc TATAAAtaccgatacaatgtgcatgctittcta

>SEQ ID NO 22 construct YPO117 aatcacagtic ctittatgataaaacgaact cataattatto caccgacaa catgcqttittaaattatttittt cittaaat tatatt at attatattgatat caac ctagotaaaataatt cqgatggcgaaatcggacaatttittaatagaaaaaatg gg tatgaagatagt ctatgatt.ccgttcttagcgactagagggacctgctcaaatctg.ccgggtgatacgcgatgtca agct caatagaaccccaca accqacga gaccgagaaatcc ttgatttgggctagaagattittgaaatgaatttaatat attctaagta acttgcttaaatttitttittcaaactictaaagacataactaacataaagtaaaaaaaaaaagttaatac atgggaagaaaaaaattaalactaatgattagctic totaacgtgtttaatct cqtat caagtttittttittaaaaattat attgct attaaaac attgtact attgtttctattttgtttagctatt attcttgttgaaatgaaaagttgttgtttatt c aattactaaatggcaat atttat cittggaaaactatacct ctaattggattaggcc ctagacatcct ctittagctitat tgacgittaaaattatt cocaaaact attaaagtttagtag tittgaaagatgcatcaag acct acticagatagg taaaa gtagaaaact acagttagtgttgattatattittaaaatatataaaacaat cittattaaactaaat attcaagatatata citcaaatggaagataaaaacatttagt ctdttaccactaccagcctago tagt cactaatagt cactittggaact gag tagatatttgcatcttgagttaccatggactcaaaagt ccaaaaagaga.ccc.cgagtgaaaatgctaccaacttaata acaaagaa.gcatttacagogg to aaaaagtat cTATAAAtgtttacaca acagtag to ataagc

>SEQ ID NO 23 construct YPO118 aattgagaaaggtgcct caattt cagtagaac citgacgcaaaattitcgcgat catgcatgactcaaattggitt tatt c acttaaataaaaaagttgttt coctatictagttgaagttct caattcaaacgcaacttct tacttitttctttittattt at actggaatgaatttitt cqt caatgctag acct caat atttggtgattaagt coaaaaaattatagdaatatt catt agittaaat cataataat atttgttatttctgctaaatatattagttittaaattggtaaatatat cagt cat catactt tatatatgtgcacaagaaaaagaggaaaaaaaacta acttittaataaattgaacgctatoctictatat citcgt.cctgg tccaaatgtaaact tcaatat cottttgattittattgctgattgctittaaaaaattt cacaaac acttittatcattct tittatt coaccaaaatctacaga cataatactttgtaattittatgtaaaaatc.ttcaaaatttgggaaaagaaaaatc atttaaaatcaatttgcattaactggatttattt coaaaggtgtgg tattgttgtttatatatgtggagttgttggcta gtaatataataaggaaaagagtgaaacatatgtagtataacgt atttctagtttittitt citctgt attaatgaat cact aattaagtag tatgcattaattgaattatcagaa.gctggit cacaaaagttctaccaaaaaaaacaaaaaaattggit cag aagaaaatgaaaataatgagaataaaaaagggaaaaaaaataagaagctagdaaacaaagcaattalacatttcaaggc agittaatt catcatgcaaggtgctitatgtgtgacaacgt.catgcgttactittittgcgt.ctacact catctotctaacg caatccactaattctgg taatggattctgctatttagaccago cagtttctitcgt.ct citcaatc

>SEOID NO 24 construct YPO126 cattgt atctgagatgtgactgttgaagaacaaagatt catgacatgg tattgttaa.gc.cgcc cattggatgat catala c caaactic titcc toagatt tact caacagttgttgaaacaaaggctggitttaagtatgaaac cqgcaccacatat ct c ttctitc.ttctgat cattct ct cota catagaccoccatgaatcct cittggtgtcgacgatgatt coctitcgaataatt tgcttagcacccaagaaactic ct caaaaaag.c catattitt coctitatgtttitcctgaagcttaaatgtttcttagt ct tggagaaagctittgagattittaaaattggat.cttctittagtttgttgaatctaaaggggtttagttacttgttatataa acgaacg tatgaaagaaatgattaagtatttittgaggtttittctttittaattacagag cacatggctittgggttgtag US 7,868,155 B2 85 86

TABLE 1 - continued atactaaaccaagaacaaatcaataaatggtgtctgagaagttagtgtctaatgatgtcc tacatgata actt cattg gggcttatttgtct caaagacat cacatgccaaatctotctatagattatgtagggacatgaagttgtgtacctaatg aaccacaagttctictato actgattaagt catacctt cittct caatgatatt caaaaga caggacca catgatttgatt atatactgacaaagt cacaaaagcc ttcaaaaaaattctgtggcaagaaaggaaaatttgac tagttatagtgtctat ctaacaaacaagtggtcat attgatttctgtc.tt cacatcagaaat catgaagattgat cactataggg.ccct tactt atcatgcc.gtggtc.cggcaaagc catgtgcttgcttgttggtgtaaaaatt tatgagctgaaacttittgaalaccalata aagggittatctacaagtaatgttcttatcTATATAtact catcactgactic ctittctgctctgc

>SEQ ID NO 25 construct YPO127 acgtttaaagttgagacataaaa.ca.gtgatttcaaatttgtattagggtggtc.ttattgttgttgtctagotact agcta gagaatact agaagaagaatacgtagcaagatacgcacaa.catttggtcct ct cittitttitt tactittcttitta acaca ttgtcc tott atgatttgct tattgattitcagtat cittitttgtat caataatticcict c caaatgattaaac cctaaaa aaatgtgatt catt caccaccc.gaagattagcatcatcaagta acacacaataactaccalataacctagttitt cattt ttctatactaaaatcctaalacat cocataaaaatacaaacaactctgaaccaataattitcct ctaatccacgtgcacc c catcgt.ctic ctdacgtaagatttgtctataacttatcaaatcc.caaatticagotttgttitt cattatatagitacgta citcttataaaaaagagaagagtaca catctittaatactittaacttaaaagaagaaagtaatactaatataagaggagt ctgagt cagogacaagtgttcgcggagaaacggaaacgct citc.tttct citctic titcc.cccaacgc.caatacctittgga atcc ct coctaact ctdtcctgtcc titt.cgt.cct cactitt citct cittitt tacatttitctacacaccaataaaattgaa accagdaacttataaat caact caagtttgaattaatgat cqaaaaact agtttatttgttgttcaatatgacccattct ttatt cacataagtattittaacttittcaaaatgttatctoaatct cotttgagtttctgtct tccc cataataaattit caaataattaatacacatggttttittaattagaaataatggaaaagaaaggacaaaggaataaaaaagaaacacaagt tggcacactic tott tattatt cact cocct cTATAAAt ct catactatottct ct catcttcttaaatattggatata tittctttittcaaattitcggaaaagaaa

>SEOID NO 26 construct YPO128 gataaactgataatggaaaagaacaaagaaac cagtttittaactatttgcatatgtaatttatttgttgcaaattata tittagttaaaatgttitcct ct atttatatatatat cagt caag cactatgtataagaaatgtcaatttataaatttitt acatgtcc titta acagaaagaaaatgaatttitta catgtcatt catagagagt cactic gtttattt cittatatagaga ataacacact cacatgcatatgcatgcaatatgata cattt tatgacaaagataat caacggaaacggit caagacata atttgataaacaacttgcacgatgcacagatctgat caaatatataact ctittaa.catat coaaaatatt caaaaaga aaaact catccaaactagoa acat cacgct cacgc.gtaggctaaaaattt attaatctocaaaagt ctitt cittatga acactgcaaacacaacaacttgaaaagt catataggitttagatgatgacgc.gt attggct atcgct taccggagtggc t cataaatacaataaacaatacgtaaaagt caaagt caaatatatttagt caactataac cattaatcgggcaaaacc tittagotgtcaaaacaacgtgaaaacgatatttgtatatat catcaagaat cagtagataagagaatgatttaatcc c ctgact attacaattittggtgtaataalacagt ct ct attggtttittatt citttgttittaatttct catgacctataga gaga attagg tagtttcgaaaattggctaatcaacttittgaaaac tactgtct actittgcttaa attct ct acactta gttt cqgataagataattgtcggactaatagittaatcc cttgacaat ctittgat attataaaaggtttagttaatct c ttctictatataaatatt catacaccagctttcaaaaaTA

>SEOID NO 27 construct YPOO2O cagagcagtgcatatttitttitttitttitttittittggtgttagtgcatat citatatatatag tact attataatatattt caatatatatattittaagaaaat atctgatt cittaagtttggact tatttgtcaacaatago cagtaaaaaacaaaag cgaagttt cactaacttaaaaaataac cacatttgtatatttcgaatacatactataaattaataaatttatcaaaac aactatagaaactgttattitccaat caatttctittatcaagattatat citgaaatatatttattaaaattaatagitta US 7,868,155 B2 87 88

TABLE 1 - continued tttacaagaact attitt tatgaaagtgtaagaact citctgaaaacttgataagt caat atttitt totaa catcgtaaa cataaact agattcaaatt cqaatc tagttattoaaaaacttataaaaacataaaaatgaaatactgttacttcaa.ca aaaaaacattatt attattttgtttaaatat ctaaatttattoat caac agcaaaatatttaaaagagtgggaaacaa ataaaaattaaact ctdttittgg tatgataaaattatt tactaaactaaact caattitttitt tagtat cacggittata actataacaataatcgaactttgttattitt cittgg tactggttt tag tagtatagatagatattittagt catalactica taagatacatgtacaaatatttgctatatatgat cagtgataactgaattt cqtgctgaaaattgc catagitttgctt attt tact cittgaaacaataacgatatggit cqttacttaaaacaa.cattittaaaaacgaagaaaattaalacagagttt gttaaaataaattaaataccatalaatttct ctittgactict tcc tatatagtaaaatct ct catc.cccttct ct ct ct c t ct catagcatgttggit ctittaggttcctatataaacaacgc.cacacacacccatttagt ccc

>SEQ ID NO 28 construct YPOO22 tagttc cattacaattt coaaatgatttgttacaaagctacaagattatt cqaaataggattt catccatalagagaga atggtgtggtcgacgct acaatgttgattt attggttgttggtttgcatcttggggatgtcaaatcc taagtttcaagt t cittgtaaaaacgttitt caggitttctittaatatattittaat attaatgtaaaaagaaaagatatagcttttgtacaaa aaaatttgtttaat cactato taggaggatgcgatcaaatt catggaatgatgtatt attagcttittctat cotcact ctaaaaacaatactatagtgagttaaataatttgat catttcaatgtagattaaaattitt attaaaagaagaaaaatt taaaagcctataacaaaataaaaaaggaggct Cagg tatgatgggtgtag cagaa.gagctggcaa.ca.gct at Cact gagtgattacgaacticagtacticagtgttct cagct cacacactic titttitttgttctic tittcttittgga cagotttca ttitt ct cittittcttttittctattttgtttcaaaatticcatccatattaaaataggcctgat catgagaataaaggaaa tactaatgatgagtttct caataatgcaataagatgcaattattatgagctatttact attgaaaatgagcaaataaa tgtcaaaacacaatctggittaagttagagcaact coattgtataggatt catgtag tittctaagaaaacaaaatgitat taat attt tactitt tacat coaaaaaacca acttatatgagtaatagaaacgatcc taat attaggaattittagagat tittct ct catctgtttcttaacttittcaat atttittatttitttaaaattgtatgagtttctact aagaaac tactgct ggagttggtc.ttagct tcc caatgcttct c caccitatatatatgcatat ct cottcttaaaac

>SEQ ID NO 29 construct YPOO24 tgttaagggalaggtttgcaccitaagaattittgaaggaattittgcgg.cgatatat cagtaagtaactittcttct tagt c tcaaaatttaagttgccataaaagtatat cagtttggagttgttalacct cittgttt tatt atttct cagctgactacg t catttgccttggttgcaa.gagcc.ca.gacaccattct citccaaggagaaccotct cittctittctgagatgtgaaaagg tataagttaatctaattagt cct gatc.ttgatatgcattcctttgtttctgttttacagttt tactittctg.cgcaa.ca aagtaataaagt attttgttgttgtttgaatttgctaatgtgattaacgagtgggctacatggitttittgcagtgtggat C t caacgatctgtggctic.cgatcaaaac agggitttgttgct cqtgttagt cqcaggalagacittgagaaattagalaggtg aagtgaccttgg tatggagtttggagctatt ctactgcttctgtatgagtt tatgagttgaagaaatacttgtcttgt tttittt tattttgttittggaatatgattatgacittgacttittaaaatgggataggatcaaaacctitt tact citgtcag gttcatgtggtcaccittgaaggttgatttagtaaatccatggactitctttitttgttgttaagattatt cittagttcaaa attaatagactaatgat attaacgt.ccacaggcattgcgttcaa.cat citcaaattaaag.cgtggaaggct cagaaagt c caatatacactatgtttatctacagttacaat catactacaaaaaacaaataatgtatacggitttggit ctaatatag cc.gcatacgatt tag tatttaccaacaaaaaattggtc.tcaaaccaaac cqaacaattgg taattaacaattgttctt ttggtottgaac cqaac caaaccogalactgaactatattaa.ccgaccg actt.cgtcc titt.cctic

>SEQ ID NO 3 O construct YPOO28 tag tacttgaaacacttggttggittt catgitatttggcctatatataaacaaacat cqtaattatatacggattittitt tcqgaattittacgc.cat atctgtaagtatatata acatgcatgtcgttittcaaatt catatgatgaacgatccacgta agtgctac tact cotacaatattgcatgagagagatatgt atttataaattittattittgaagaagaaataagagggaa US 7,868,155 B2 89 90

TABLE 1 - continued ggitt acttgggtggat.cgatgtgaaaacaaaagaagaaaaag.cgaalacc Cactaagcc attacatgat atcgacctt C titat citttitt cotctittattittatttittct cagg acttittittctact taatgaaac ct coaaactatotaactaatac actic ccatgtagaataaagaaaattatataagatattgttgat attttgtaactagaaaatatatttgctctgtaatt titt.cgtaagttaaatcaacatttitt cagtagaaacaaatattact.gcaaaaagtaggat cattattitttgtc.caaaat cticagttagctatagggttgtag taaaaacaaaacacattcttgatttgc.cccaaaaaataaagagagagaagaatat tgttcaaaagtggit ct cittct ct ct ctaattatgttitt cactaaacccaattagattcaaac agtic tacaaagtic caa aagataaacatgggacaacaatt cqatgcaaaaaatcc to ttitt catgct cittitttittatt citc tagt cittittaaatt actaataaaaact cacaaatccaccaaacccattct ctacaact cacct tcatctagatttacc cactic ccaccgaga aacacaagaaaaaaaatatacatatataaatatacaagacaacacatgatgctgatgcaatatacacaacaaagtatt aaatcttagatattgttggg to tcc ctittcttctatt cattttct tatto attaaaaaaaaaaa >SEOID NO 31 construct YPOO3 O tacttgcct catgtgtttggatacgagattactgaacgttgtggtgt attittatagt catgggitttgttaattgttat catgcttgcc tact taactagogtaattatgtttittttgtactacct cqgaagtagct attttgtc.gct tattgacaa cgagatactittaagatgttccacat coacgt.cgtaatcggttgat cqaatggtgcc taatagat caaagttatcct ca acaaatat cqatgtgtagtatatacgtgaatatatagtag tot cittgcatgcatat catatacaacttaaatact citt tttgtttcaaaataaataatgttittaggaaaaagattattgttgtcaaattaagtgttggit ct attcatccaaacaaga aagaaaaaaaatacgaatttgttittatatat cattgacgaacaatgtttagctaataataaataattatttatttata aaaattaaaagttagatagitttcttaatttaggtgcatataagttctittaacaaaaaaaacatttaggtgcataagtic ttaaatat caaatattittggalacagtaattittatgtataacttittitt cqtacctat citt cacaccgcataaattgc.ca aagttcaac ctitttgatattt cattcct cacaaaaccatattaatttatacacct caat attgtttaatagt attatca tgttggctitt cqctgaatttatcaaagtgcaa.catgttittatc.ttacaaaaaaataaaaagaaatt cacgttgttgttga t ctitgagagttgacttittaaatatat caca acttatataaatacgcagdaacattccaat ct ct caagaaaatctaca gttcct coaaataataataccct coctictaaggtttaaaactatacct cattalacacattaagaagctagt cattact t catttctatattittaaataatgtttattgataacaattgcaggcaactaattitt cagcaatc

>SEOID NO 32 construct YPOO54 agct tattttgttctatt citat cqtatttgattcttctitt cqtttittttitttgtttgacittaagaalaccgattgttta tagtag taaacatttgtttittaatgttgct cqattic cagtgcacatgtc.caggctaga cacttgtcgittataaaggtt gctittggttcaatattgat coactagagatgttacaactattgttga catctgagattgttgttgataagaaaatatgaa actggatttagtgaaagttacaatatataatcatacat catagataggaaataaggaaatgtcagatatacttgaaga atacatcaaatagacaaggtoctittitt cittattgtcgact attatagagcc.gtacagaacct titt cacgtc.tttagta attagtacattct c cattt cqgct citct cittatttitttitt coat citcttitt acttctic caaataataacaataaaagc titcgattttgttgttgttgtttgt atttacatc.ttga catcgatatt cittitt catcaatttitttaccaaaaatgtaataaa aacaaaaaaaaaccaacgctgaacacagacatggitttctic catcc.gtttatattolatcgtttgtatgtt tacttaa.ca actitatttcaaaatagtacatat catggttgttgtttittaaaaaaagtatacagaacagaaaag.cacatggtag acaaa ataatgaa.gc.caaaattaatacaaagaagaagttcaacttgtatttattaa.ca catttitctitt.ccttgtcaaaga cat gcaaattggttttgttitt cittatt cocatttittitttittataataaaaagaagaagagtaaaacaaaaaaactato att tott cittatcgcaaaactic titat ctaa.gcaagaaac cqacaaaac ctatat ctacatatatt ct catcaiacat ct citt gaga catatt cattttggittaaa.gcaaaagattittaa.gagagaaagggggagaagtgagaga.g

>SEOID NO 34 construct YPOO50 tacttgagggaaacat catatttittaalaccittgtct cagtaagctaacacacaccc cttgttgattactitat coatgtt tatic cacaagaatgcagttggattgagatattitt cittctttgttgaaat caggcct caaggtgttcatgtggtctgca US 7,868,155 B2 91 92

TABLE 1 - continued aaaaaatticc caaaaataaagatagtgacatctgaaatcgataatggattagacgaagagtttcgtgttattoctitgg tatgggcgggtttggggacagat attittggcacagacgaggactaggcc actgtggtc.ctgcagcattaggtgtc.cct tccatgtc.ctgcattacattt tattgatggattcat cacccitat c tact acaacggctacacaaactatgaagagttt tgtt tactaataaatgcc.caagtgagggg.tc.gatcgaacccgggacacgtttitt cagtttaccatatagaattatcct tggaac ccttgatactic cataaaac at caccaccitctgttgtcatct catgaatcCaggttcaaacctagt ct ct ct c t ccctagtgggaggtatatggcc actgggccalatgatgacaaaatgcaaaaaaaataaaatacatttgggttcattat Ctaaaatat ct cttgttgtttgtaagttittggttgcacact cqtgtggttgaagttgttgttgtgagagg tactatacaata cactctgcttttgttttgtacct at ct ctittctic ttct coacatat coaag actittggggataaagctgagat cattg gttgccatttggttgtgtagaa.gcaat cacccatttgctittatcc gaggttgataaattitcct cqggttct cottctg acacg tatgacaaattictaatagtatatt cotcgtagatattacctatatatt citcaatagttgcagg tacttaaggc tttgtc.ttggcatcct cqtcct citt cagdaaaactic git ct citc.ttgcactic caaaaagcaacc

>SEQ ID NO 35 construct YPOO4 O cc catcacatgtaa.cat cattgggctatocaaaagtictaaccaataatgtcaatctataalaccacattaagtagttca tttitttttgtag togtgtttagcttgttaalacct cataaaatatgttitt cacttacgittaacaaaacaaatat citt.ca cgaaaaaaaataaaataaaatat citttittgataccaaaaaataaaataaaataattitt.ccctitt.cgat cataaaatg cgtagataagagaaactgtgtttgaggctic cattt catgttcaccitaccagtic taccacgt.cattt citcaaagacgca aattittctaattagggatgtgct citttitta catatagat caatat cotaaaaaaatttaagatatt catattitt.cgta catatatat coagttitcc.cgaaaaatc cataaaatgggtataatgatagt cctttitt cacctittaataataatttctg aacaaaattatat cataataaacttgttgattittatacaaaatttatttgtatatataattitt actalaccaacgtgaac gataaaaataat attct cataaaatgttgattaaaaattacttaaaataaataattatttaggattatgtatt agtag tact cqaaccatttittt tagttatctgcatgaag accctaatttitt cacatatat cqaaactaaaactittggatatac actgtaatttgaaaacgcttggaacggataatgtagttacct cacaagattttgtacatc cctogacatttitat attca ttaaagtgttgtttittitt ctitcagaaaagaaaacacttitttitttitttgtgcttittagtttalaattaacaaaaaaatgga caccatgagatt coactaact catgtgtatataa cattagggaagicagt caatt cattt cagoatccacacacactitt gaatgct caatcaaagctt citt catagittaaact tccacacaacgtcaaaact coagaagaag

>SEQ ID NO 37 construct YPOO56 ataggaatctgctt.cgg tagaagattic gagagaggagaggaagcatcggtggttittggagttcCttatt Cttctictt C titt.ccaaagttttgtcatt cqccaagattic cittaaaaacttgttcacacat cataattatgcaccalataggittataaa t cataatccaacaagttagt cattggctittaattittaaaaaatcc catalagagtaaaatcttittagaaagttaat caa cc cacacatgggctagaaaac caaaaacco cacgaacattgagattacaagaaacatttittaagtic ctaaatgagcc c aagagcattgcttaatgaagaagaactgat attaattalactaat attaggacacataaaaaaatacgaaaacaccaat citt catgccacaaaatcaaacaaaaacgaaaaaatcaattitt catgaaatggataaagagagagcgtaattat cagga atttgattgagtacggttgttatgatgat cattcacaattatc.tttgat cittgagatttagcaatagittaattitt cqg atgtttittttgttacttgctgct cactitcttg tatgcagattaatttataagagagaccagttacaact ctitt ctitat ttgaataagattittataagatgtagttggcc atgtgggtttattgcatgcagct Ctctg.cgttggtcc caagtccac gacaatagagagtttctgcactt cacggitat cqtcqtcgt cacaagttctttacctitat cattggcacaagttagc.ca ccgt.ctittgcgcaagttagcatgttgttgctacatacgtgt catgaactgattggtcaaatttggatatattittatt co cgt.cggittatgtttggataaaaatataaaacggaaatttctgttt cago: ct tccttggtc.ccaaagaaaaatacgcac acct actic cctt cattctictatoctict coact cataatatata catctaaatgcaatc to tcc

>SEQ ID NO 38 construct YPOO68 aaattggggagtggggagatgtttggittat attc ccttct catcgatggtctagatgtgcgaggtgact ct catggag

US 7,868,155 B2 95 96

TABLE 1 - continued >SEQ ID NO 42 construct YPO 087 tgaattgagtaaaatgtgttittcaaac agittaggtggtagaaggtaaaggtaataa catcatgatct tact aaaagaa ttgttgcatactaactato aatatt ct caacaacataatataatgtttittt taggtaattitt coattittaattittittg tgattaaacaattaaacaact cqaatgatgatgataaaaaaaaaaaattaacaact cqaataagttaaagtag caata cacatgtcgttcaattcaaccaataaagtaagacittatatttittaagaagttgactaatagottaataagttggaaaa cittgttgtag titt cittaatticc cacgtgcagtaagaaataaaaatgaaaaaaattattatat cottcc.cactctg.cgac ttittcttittattittatcaaat attaaaaagatt catat cacagtttacacattgaaat cataaacgataattatgtat tttgtaataaaaagttagttctgaagct catactittggatagt cqctagt cqctaatatgct cottgtaataattaaa gtcactacgacgcacgtcaaag.ccgatatttagggcttaattgatgcgtgtttittcttitt catataatagtaatataa attagtactaataaagtatgatggatggttgagacagaaaagaaaaaagatgactgtatggit catc attacaaagaag aatgtatt citt catgttcttaagaataataaaatgtcacttgtaaatcaagttggtaa.gcattttgagaactttgttc gatgcaacg tatgatgatt tatgtaga caaaagataaaac cqtat cittcaact attgccaagaaaagataaaacctaa tctagt cagt ct ct caa.cataaataca acccaatagccaaactgtgtc.caatt cqgagagaaactaaactaaaacaaa acacaaaag.cccaa.cataag.cccaataaaaccoattittataaacagaac attactaac actica

>SEQ ID NO 43 construct YPO18O ttattgttgaaacggatggitatic cagatt catagagittatagttgttgacct cqtaaggatgaatt cattatc.ttctt Cttcttittgcagcatggaggtgatcgatgg tatgactittgatgatagcc atgtccaccalaat cagccaagaaaagat C alagacct cqgctgcttacgttctgttctataaacgc.cttgtag actaaagaaactgaagcggaaaagacaagaaagtg gtatttgcatttittgc.cgggitttggct tatttaaaaacat cattggcttgattictaatt cactacaagat caagatga alagcagctctg.cgttgaggctaatttacagaa.gaga.gaga.gagagttgggaagaagagcaaaagaccgagaggacatg ttgcggggaatttattittatt cittacaaaaattggt atctgattattitt attalaccatatt caattagagaatagaag aatagagaaaag.cccttttgttgggatatggttctaaattgttgtttagttcttgttgttgtcagttittggct citcgt.cga c caaagaagattaaagaaacctic tacct tattittaact caattcttttgtttittgcaatgtc.ctittgctitt coaaaat tgttagt citt acttitt cact actittgatagacattgcc tittgcgttt coct gattaataa.gc.ca.gagtact taaatca aaattgactgttttgttgcatcctgcatcacgttt coaatcagaac catagtgttgtcgttgttgtcattatc.cgaattit aagtggagacattggtaagttatttataaactaattacaatct atttittctaattatttcaaataa.cat atttaa.gct ctgtagct tccact agacggtgaagatttgaagtgaga.gct ct ctittgcattgct cacccaccalatggat.ctacctac ccttcttcttcttcticctic cttittaaaccotaaaagtttct ctitt cottcaa.ca

>SEQ ID NO 44 construct YPO186 tgga caattact cittgttgttgtat cottggagttgctgttt catatgtaagtggacaattact cittgtgtgtag ccttg gagtttittittatttacgittattittggit cagcc tittaattattittgcaaaaaatgitatctgtttittgccacatgcc cac ataatacattt cqcaaatttgatacattatgctittggc ccttgtatatt cqgtaaaaaaaaaagct caggctact ct c aaaaccoggctctgagtatt cqtaggccacaatcqaagaaaaaaagtgc.cgatttacat atttitt catacaaaaaatta aaactgttatgt attatt caaaagctatttacatatgttt tactaac acgttittcaat attittcttaatcc titttcaa aatttalactaagtataatacttittitttgttgttgttattt cqttgttittggittaaagaaaaacgaaaaaaagagagagtt attcatcc ttgcagataaggctagggttggttgaataaagatgtgcatat cittataccactagaccaaagaaacagtic acaagtaaaaggc.cgaatcCtttittataaaatataalacagacgaaagctaatgctt catgggcttggcc caagtgcag gct citcgctagt cqctacgctacaact atcc.cat atttaattagtgaagag tattittatt attittggtoaacgggcta totttgttgacaaaactat co cattgg taaagaaatagoaaaataggcgttt cattctictatatttaaacttgattitt atgaagagttgaatagotgaaccaggaagatatttalagaa.gc.ccgtact tcacgctittaactgtcaatcgatagat.ca taataaatgactatictatoggataggaactata actgaatt cagaaagaatc tact act actataaatactaaaagagt US 7,868,155 B2 97 98

TABLE 1 - continued attaatacaacggaaaaaacaaaacaaaaaaaagggggaacaagggagttt catgttaaaaag

>SEOID NO 45 construct YPO121 ttggatttitttittttgttgagt cagcagac catctaatct citc.tttitt.ccaccacagoctogctittctatgaag cattt gggcttacggttgttggaat caatgacttgttgcact.cccaacgtttgctacctgtcatggtggaccc.gcagagattatic gaaaacggagtttctgggttcCacattgacccatat catccagacCaggttgcagctaccttggtcagcttctittgag acct gtaacaccaatcCaaat cattgggittaaaatct ctgaaggagggct Caagcgaatctatgaaaggttggcc cat tctic cttgacaggcttaacaatacaacttgtatcgcttcaacaagatgatggcttaataaggatttittgcatgtatag gtacacatggaagaagtacticagagagactgctt accctggctggagtictatgcattctggaalacatgtgtctaa.gct cgaaaggagagaalacacgacgttacct agagatgttitt act cattgaaattitcgtgatttggittagtgtaacccactg ttatt cittittgatgtctacat ct actt tact tacattatt cittitt ctitcggitttgcaggcca attcaatcc.cgctggc aacagatgagaactgat catgacaggg taggattittattt cotgcactittctittagat ctitttgtttgttgttatcttg aataaaaattgttgggttttgtttic ctitcagtggitttgattittggacittatttgttgttaatgttgttittggctgttct cittaatat caataacaaataaatttactggttggitatictaagatctaacaatagittac tatttittagaggtaaagaca c caaccttgttatattggit cagaga.gctaaaaccttgacittgttgggaaaacaaaactictaatgacagaaaatctgac atgatgccittataattagcct catgttctacataaatcctaacaatago actttgtttct

>SEOID NO 46 construct YPOO96 tgcaaaattgaaaaattgaagggtgagacaaatttaaagataatat c tattaaatcct ctaattittaaaaatttagca aaaattgt attittctitatggat cagttagttcacacgitat cittagttagtatcaaatcatat citaatgattagtgata aaac tagttagatat ctatatgtgtc.tttaccatttaacttgaatcc ttcttctitttitttacgitaaacaacttgaatc citt.cgittaatatataaatttaaagcatttitttctittaattictattgatcggtatatatt tactataagttittagctica tatgcaatttcaaatgatatgcttittaaattttgtc.taggtgttgatagttgtatictittaa cataaatcttatagdaaa actatacttgat attctaaattitatictatttgct cittgttgaac ct catattagt ctagagaaactittgaaatc ctitt c aattagttgtatgtccaatacatttitt actaacatttattagt citttittaattaagattattgttagaaaaaaaaaga ttttittaaaaataaataatatgttittagatacaatgtgagttaggctitc.ttatattittaaaaaataaatttattt cat acttaaaaatagitttggaatttcaatttatttggctgaataccataaaatatgtcaatttgaac ctitat acco attga ctatttggtgttagaaaccctittaacaaaaaaaaactatttggtgttagatat caaaataaaaaaaaattaac cattg gttt cittatattgaattggatattgttacatgitattaaagttitttittggitttaattittgaaacgttgatagaaactat taagtttaagtttgg tagtatatttatttgtggaaaatttaattgcc attaaatataacgtcaacttittitttgtttitt ttittgagaagttacgttgttgattittgattt cotatataaaagttagattacgt cattttittaa

>SEOID NO 47 construct YPO 098 tatttittataaattat cittagtaaaagtatgt attittctaatagatctgttagttcatacatat cittaattagtgtta aattagat ctaatgattagtgataaagtttittagatat cqatataggtgtc.tttaccattta acttgaatcctttgtt aatgtaaaattittaaaatattittgctittgatt citact tattggtatataattittaa catat caatccaatgcc actict taaattat catgtacttitt cqatatatgttatgact cacttgttatgaaacgatggattitt caccalattittggittatt tattaact agaagttittagctictagtgcaattittaaataatatgcttittaaaattggit ctagittataatagttgtatic tata acataaaacttataacaaaactatacttgatatt caaaaattattgattittctic ttgttgaactt cat attagcc tagagaaactittgaaaacctittcaataaattig tatgtcgaataaagttttacaaacattt attagc cattt cq attaa gact attgtgagcaaaagtttitttittattataaaataaataatttgtttaagataaattgttgaattaggcttcttata ttittaaaaattatataaattitat actgaaaaattgttagaattittcaaattittaaatttatttggcttaagaacataa atatgtcaatttgaacctitat acccactaaat attc catgttagatat ctaaataaaagaaaattalactattgatttic ttatattgaattggatattgttacttg tatt tatgtttitttgttt catttittaaacgttgataaaatcattaalactaa US 7,868,155 B2 99 100

TABLE 1 - continued agttttgtagtatatttatttgtcgaaaatttatt cocattaaatataacgittaaatttatttgtc.ttt attaaaaaa gttactttgttgattittgattt cotatataaaatttagata acttcaattittcaaataaaaaat

>SEQ ID NO 48 construct YPO108 ttagctgaac caggaaattgat citcttataccagtttccgggitttagattggtttgatggcgatttgattaaaccc.cc gaaattittatgtcg tagttgttgcatag tatt attatt ctittgcggacaatagacgitat cqggaccaagttctgtagca aaattgtataagcttaagtttgatgaaatttaaagg taat cactaaaac ccaaatgggacaataaaccogtgaagatt tagagtttittaattittgact catgaatctggagaaagagc cct cittaaaaggagtgaat caatccataggggaaaaa gttttgtc.tttittaaaaactaaagaac caaac cittaatagaagicagotcaatgtgtgacaactitt.ccactggcactaa gataaagtgactagogatgagtgcaattattgaaatagtagatgg taaat attacatacaagagtaaaaatat ctitta tgtcaatgcttaatticagtgtttctggittaacaa.gagaaacttctictaactitt cqtaattgggit cittataaaattitta tgcaattatgattittaccc.ttt tact acttitt cattagctitt cacgaatctattittgacaagagaaatcattagaggit aaacatgctttittggtcaagggc citta acagttccaccaatcaagct caaaagttgtact taac coacatc.ttctgtg aaaacatataattacatgtacaaatcaaaactacct tatgaaataaatagaaatattgcagttcatttctaatttaac citct tcaacttittaaaact atttacatttctt tatgtcatttctagt cattttgatgcaaattgtaccatttatggat tat citt cacaaatttittaagttggtgaaaacttitttggtggg tagttaaaacttgaaatagaaatt tactittaccaaa ataaactaatgaaaagtaat cactic cactic cctataataagattt coaacgttcc.cactaagc

>SEQ ID NO 49 construct YPO134 cc tactittaggcttaaacaagaagaaaatatgactgctaagt catatttittcaact ct catgagcaa.ccgtaaagttg caccgcaat atccaacaaatgacattctgttatctacaatctaatgttgaaaatttggct catctaataaaggagac aaaagttatat ct ctitt cacacacacgittaatggaagtgtaaaggcggtgaga.gtgtgggagagacittggggaacaag aagaaggacgcggtcaaaaagtgacggtgggctacggcttitt Cttgg tagcagttggalaatticcattaatgacttaaa aagtgtaaatcttatcttctttittattttgttgatttgatatgcacatt cattt catgaaaat atttgtatagitttgat gat catacgacaaacttatagggitt cacaaagtagatgcaatagttgcatacctctgtttaaatgttcttgttaatat tatacttgatgatgaaact cqtgaatgttatt caaaatgtc.catgitaatacaagat catgcactataataagtaatct atcaattt cago acaacaattittgacaaaaagtaaaaataaaataaaataaactgatat catattt cogaattatatg taaacgttittctgtttct caatggit ct ctitt cacticttgttgttittctaatattt catttaaacct atttctaalactaa gcacat citttgttgattgattgcatttcaaccaaaatcgataaccalat cattgtttittt tatgttittattt cagctt accacacacgtttagaattittaaaaataaaacaaaaaaaagttaact cqttacaaatgaaaatgat atttittaattgg actic gatggaaaggaccaatttatt caacact attgtttagtic cqaacacttgcc.gcgtaagttitt coaactic cc ccc attgacctitt cqcactitt cacaaactic cqtatatatataatggatacactict citctittgatct

>SEQ ID NO 50 construct YPO138 tgttgttgtc.ctaaatagtttctttittaaaatttgtaaataccaagacgcg tatttaa.gagtattittgaaaagatatttg attataaaaagaaagaaaaagagaaggctgaggattaactgcaacgt.ct accgttggaaaagaaaaacgat Cagaaaa Cacagaaattaataaaaagagagaaaaaaaaatagagtatgagagatgcacatgggtgcctgcaaaaaaaagg tagaa gaaatttgtctgaaagtgtcacaggcacactict citcqaac cacatttaacaac act coaaac actic ttcttct actitt gtaccctt cagtacattactic titt.ccaaagtc.cgtgatttacgct ctitcgatgacacct citcaiacagagagagacitac atgtgtacatttitcttctaccattaaattittgaagattitt catgattcaatttagtatatatatggaagataaaatt tt cattgtc.tttctacatgatagtaacggttittagaagggtggittat cacttatag tatttgagttaagaaatataaa aatatacgtgactgtttitt cottgtaalactatttittagg.ccct tatttittatt caagtag to acatacgtgtttgaag tgtatttalactaagaaaaagaaagtaggaaatgaaaaggatatag tatt tatggtgtaatcttggtaaggaccaggag atcagaagggg.ccacaatgtcacaaagaggaccaacaatgaaattaaatcct cagotggc ctittaa cattttggcticc US 7,868,155 B2 101 102

TABLE 1 - continued caccatct cott coacacatatgcacatgtc.ttcatgtct citct ct citctatacgttacctacacaaatatgtacaga caaatagoccattacaaaatctittatttataaatatatacticcitcaact coct caat atccacc catct cottct coa taact citct citct citct coctaalacacaac caaagacttittat ct ct caggaacco caaaaac >SEOID NO 52 construct YPO192 t cct cotactgtctgctacgt caacaagtggattgcaatcagacggtgattgttgtc.tc.ttitt cattct ct citc.ttitta ctaatttctic tdataattaaact gagaatgtatattaagaaaaaaaaacaaaaacaagagaggaattitt catacacac taacttaagactictttgtaagttitt cocaaatatggattittctagtataaatatgagttcattagttt caccaagcct acaa.gcatct ct coat citcaaat catatt caccitaaaaat caggit coccitctic tittatat ct citaa cattcttatatic agat catatttitttggatttcttgttalagtaacaccaatc.ttittaaaagtgttitt caggittaatataaaagaataatg atgtttitcggtgacggttgcgatcc ttgtttgtc.ttattggctacatttaccgat catttaa.gc.ct coaccaccg.cga atctg.cggc.catcc taacggit cotc.cggittacttct cogagaatcaa.gcticagtgatggaagatat cittgcttataga gaatctggggttgatagagacaatgctaactacaagat cattgtcgttcatggcttcaac agct coaaaga cactgaa titt.cccatcc ctaaggttcact cittatt ct caat attaact ct cqtacatgtcacatgcc catttt caccattttaga tatacagttittgat actitt actittgcatttattittgctatatgtaattgaggatattgttittaatttctittgggttitt tttitttggctaaatgagaatticagtgtc.tttggttcttaaaaaaaaagtatttgttaatggtaaacgctaaacgctat ttgagtttatgtttitttcaagaactgaaaacgttittattgaaaatatacacttitttittgctatttatagaaaggcata t cacatctagacgcaaacgcaaaattgacttittgaa.gcaaccacaat cittaaatgcaatgaaa

>SEOID NO 53 construct YPO2O4 aactaattaggit cqttaattgtc.ca agggitttitt catagttgatatagttctgttcaaatatagc.catcct taatcga tt catgggat.cgtaaattact actitcqagtgttgtaaaaaaaaatgaaactitctacattacaaact cqaatttaatgc atctggagtgat actataaaagtagggatgct ct caggtogcatttgagagacacagaaatgattittaatggaattaa tatattitt cagtttitt cacaaaaaaaaattgttgtttataacaactgcagattcaatgctgattittatgagt ct cacct atagaatttatatttctatatt catagaggcagtataggtgttgacccaa.catcqaaagaac actt cqtaaaaaattic tittggaacaaggctgaaaatt tact cocaaatttagctat cogatgaagataaatcatttaccgttt attaaagaatt atcgagattittagt ccaaaccaaaagagattatgagcc taagattittgaatttgtattgg taaaagaaattgaacgaa aattitcagaaaaaaat attaataaattgaacgatagagttcact tactacatagt caact agtgcc tagctataatag tittcaaaaga caaaaaaaaacaaaatcggittaac tact tcc.gtgacataattctgattittgatttittgaatccagtict aatttgaaaagtatatt caaaatctittaaatc cattaatgata acttittataatacgttgacacacgcaattgtatat acaatatt cittgaattittaaatgtaaattictagaatatattgcgat caccacactaatcaaaatctittgggacaactt gaac ccacatttgacttitt cittggtcaaat attittggcatcatgcatgat citt citctataaaaaccaaaaggcct caa cgacatt cataaacticagt cattatatttattitttgttgt atttcaacgttcaatctotgaaa >SEQ ID NO 54 construct BIN2A2/28716-HY2 gtct cittaaaaaggatgaacaaacacgaaactggtggattatacaaatgtc.gc.cttatacatatat cqgittattggcc aaaagagctattttacct tatggataatggtgct actatggttggagttggaggtgtagttcaggctt Cacct tctgg tittaagcc ct coaatgggtaatggtaaattitc.cggcaaaaggit cotttgagat cago catgttitt.ccaatgttgatgt Cttatatt CCaagtatgagaaaggtaaaataaatgcgttt cot at agtggagttgctagatagtag tagatgttatgg gctacgaattggtaagagagttcgattittggact agtic cacticggat actttitt caattatggtggtcCt99aggaat citcttgttggagtttgat atttgcgagtataatctittgaacttgttgtagattgtacc caaaac cqaaaacatat cotat ataaattt cattatgagagtaaaattgtttgttt tatgtat catttct caactgttgattgagttgactattgaaaa.ca tat cittagataagttitcgittatgagagittaatgatgattgatgacatacacacticcitt tatgatggtgattcaacgtt ttggagaaaatttatttataatctot catalaattct cogittattagttgaataaaatc.ttaaatgtctic ctittaacca

US 7,868,155 B2 105 106

TABLE 1 - continued atggcgaagact agtgggcttgaac catalaggtttggc.cccaatacggatt coaaacaacaa.gc.ctagogcagtictitt tgggatgcatalagactaaactgtc.gcagtgatagacgtaagatatat cacttgattggaatcgt.ctaa.gctaataag tttaccttgaccgtttatagttgcgtcaacgtcc titatggagattgatgcc catcaaataaacctgaaaatc.cat cac catgaccaccataaact cocttgctg.ccgctgctittggcttgagcaaggtgtttcc ttgtaaagct cogatctittgga taaagttgttccacttitttgcaagtagctctgacc cct citcagagatgtcaccggaatc.ttagacagaacct cotctgc caaat cacttggaagat cqgacaatgtcat catttittgcaggtaatttct c ctitcgttgctgctittggcttgagcacg gtgctt Ctttgtaaagctic.cgat Ctttggatalagagcggat.cggaatcct c taggaggtgc.cagtic ccttgacct att aatttatagaaggttittagtg tattttgttccaatttcttct ctaacttaacaaataacaactgcct catagt catgg gcttcaaattittatcgcttggtg tattitcgittatttgcaaggccttggcc cattttgagcc.caataactaaatctago cittitt cagaccgga catgaactt cqcatattgg.cgtaactgtgcagttttacctttitt cqgatcagacaagat cagat ttagaccacccaacaatagt cagt catatttgacaacctaagctago coacac tactaaaaa.gcaaacaaaagaagaa ttctatgttgtcattttaccggtggcaagtgg accottctataaaagagtaaagagacagcc tdtgtgtgtataatct ctaattatgttcaccgacacaat cacacaaac ccttct ctaat cacaca acttctt catgatttacgacattaattat cattaact ctittaaatt cactitt acatgct caaaaat atctaatttgcago attaatttgagtaccgataact attat tataatcgt.cgtgatt.cgcaatc.ttctt cattagatgctgtcaagttgtact.cgcacgcggtggtc.cagtgaa.gcaaa tccaacggitttaaaaccttcttacatttctagat ctaatctgaac cqtcagat atctagat citcattgtctgaacaca gttagatgaaactgggaatgaatctggacgaaattacgat cittacacca accc cct coacgagct cqtatatataaag cittatacgct cotcctt caccitt.cgtactact actaccaccacatttctittagctcaacctt cattactaatctoctt ttaagg tatgttcacttitt ctitcgatt catactittct caagattic ct gcatttctgtagaatttgaaccaagtgtcga ttitttgtttgagagaagttgttgatttatagatctggittattgaatctagattic caatttittaattgatt coagtttgt tatgtgttgtttatact acttct cattgat cittgtttgatttctictogctctg tattaggitttctitt.cgtgaatcagat c ggaa

>SEQ ID NO: 57 construct YPOO18 tagaaacct caacttgaatataatagtttgtttgtttgcttgaagittaatctotct cittttittatcagotaaagctgc atttataaaaattic tagttta acttittaccatttgctataatttagagattittaacaagaaatctggcc tagt cc.gca aaacttatagaataaatcaaacattcttcaat attttaca catccacaatacc caatccaagaaatggattgcagttg caccagagattacatgtct cqttittagtttgctagt cact caaact cacaaag cataaattgtaatagaaaatagaac tittatttaaatctgaagaaagatatatataaaaaaaaaaaaaaaagaagaa.gcaggct coagttittgatgggaga aga aaagagagctggcaa.ca.gctatt cactgatagaccgat cactic tottctgtc.ccgcacticttitt cittcttttgtttct ttcttitt.cga-cagottt catttitt.cct coatttittaaatttgaattattitt acagt cataaaagtacttcaaacgitat atgtaaataacgagcaacaaaacaattaactacaacaaaactagttctagota agaga attagttagaaatttittatt ataatagittagtatatgtttatt catalacacaattaattalacacacaaaatacatgtaattt cotcitat accotcttic acatataattagagtag togctittaatttaagattaattatcgatttacat cattaatgat catc tagt cittacacaga gagttt cagtatctgcatgagattatataaaggaatgtatt catgttitt tact tctttittatt catggittaaggatga tacattataattataaatc cataatctatogaact caactattott TATAaaaaaggaattaa attctgaaaataaaca actgtagttggct tcc.caaggctgctgctt caccitataaatacccitatcct ctittgaaaactic

>SEQ ID NO: 58 construct YPOO71 ataggc cc tact tctaattaaagcc catttacttct ct cottgtc.ttct tatt cotcttittctic cc cat cacgtgacg acgatgctataaacgc.cgt.cggattatata actggtgc.cgttgacaagacggcgacagaagaaagaaagaagaalacca caggctictagggaacgtaacgittatgtc.ctgtctatago atttataacggit cagat caacgc.cgtttagataaagat c US 7,868,155 B2 107 108

TABLE 1 - continued tgtcaatgttaaagaagagatgcatct ctacaccgittaaatttaaaacgc.cgtgaacct cittatctattgattitttgt ttgatgaa.gc.caaaacaaatcgtgtcagaagacittatcagagaagaagaaaacgacgacgttc.ccgtttct coatgtc. taataagtgtag tagtgg.cggctactaaaaactictaaagtttgactic cagtaaaactgcc tittctagtgtaattic cag tgattittagagtttgaatagtgtgtgaccalaatttgaaagtacaatctoragcaat attattgat cacticgittataaaa gaatcqaatgtaaaaatagocaatgagagact gagacg tatgtgtttgaccataagttcgtatagitttgtatictaticta cctgcaagat cagoagatggttctic tdatcaattgtacct taattat cittittatttitcgtaaaatttctictatt caca aatgataaatct acttalagacagtaac cataacaagatttacaagataatttgaaaaatgaacacataaaagtattitt gg.cgcattatttittaataataacaatatt tatgtaaagt cacataaaagta TATAttcgct cacaaagt cittacggta tittagaacagtag taccacat cq attct citt catcttcttctt cataatatgc cattgttcatgtc.tctgttgtccitat cgcatalacact cacgctat cittatt attitt ct ct cqct ctittct cactgagaggacactaaaa

>SEQ ID NO: 59 construct YPOO8O aagcggcaatttagtaagaagtact caaagtat catttaccaaaagtatatggittittgggaagagttgttagggatgt attctittctaaacagatgatatgacgatgttcttgaaaactaatgttaaagacggaat citctggcatctt cacticggg agatat attaaaccogttgattgtagttagc catgtacttagottagtgcacaaataatctgctgcaagaaatc.tttitt ctattataatat ct ct catttaaac attagaacatattgttta acttgttcttctagaaataaaactgctaattt citt atgg taaact atttitcc tittagattgcacaatcqaact cqaaaatctagtggagacitatgtgactattittatatata tgaaac ctaaatcaaattatcc.caataattgggaga cacaaaagaaaaattacgaaagaaaacaggaaatcaaat caa aagataaagaga aggtaaaaaaaggcaagaag cactaatgtttaatatttatagittitt ct coattaaagaaaaag.cga tgatgtgtgttctgatcttttgttgaaagtatatatattgcttittgctttitctoaaaagcaaaag acticatccaacaag aacaaaaaaaaaaactaaagct caatccaaaagacgaagaatgcattggatactacaactitctttitt cacttittctitt caaatttacaattatgattitt cacaatacagtttatt caaaaataaataaaaaaacgaggcatgaaaataatgattat cct citt cact tattaagcc acticacTATAagcagagcaacticcagaacatagtgagcc cc caaaac attaaag catga tgatgtctaatgatgatgat cittctitcgttccatttct ctaaatttittgggatttctg.cgaaga cc cittctitct ctitt citctitctctgaact tcaagatt.cgtgtcggacaaattitttgtttittatttittctgatgttaca

>SEQ ID NO: 60 construct YPO12O tagtttittgatttaatctacgtttitt cittaat cataaatggg taatt attagtttittgcaaaatcaaaatccaaaaat tgttctaaac actgcaaccatttaaggcctatat cact cagaaaatttctggtgggagaactaatcgtttgtc.ctitt c taaatc to acat attagaatttagaattagtgtgctacataaaaat attagttcagotcggaacaactatttitttggit aaaacagagaacttaaacaaatgcattattittat caacatgcattttgaattgaatataaaattt cataattgtaaag acataaattacataaaattittacatgaaaaaatagatatagaaagaaaatgaaactaact gatgatatgct ct ctaaa ttttittaatctoataacaagaattcaaattaattagttcatatttittggittaatataa catttacctgtctaagttgg aactitt catttitttittctgttttgtttagt cagtatt cittaatgtgaaacggaaagttgaatttatt caaacttaaat t caatago attaattaaaggcgaaagct attatctotacatgtggttcaaactaga catccaatttaattagct tatt gacgttgaaatgttitt.ccaaaac tactatagtttggcaatttgaaagatgcatcagaact acticagacagg taaaagt agaacctictagotgttgttgaattig tatgttagt ccataaagaac at cittgtaaactt catact taagatatatattaca atatatactitgaatggtagataaaaacgattagt ctdattgctagdatact cacaact atttggaaatgagtaagata ttggcatctagagttact act atggagacaaaagtic gaataaaagagacct cacgtgaaaatgttacgagctagtaac taaagcatttacactaacggtaaaaaaagtat cTATAaatgtttacaca aggtag tagt catt

>SEQ ID NO : 61 construct YPO122 agtttaattatttgttatctato caat caatttitttittctaaactgtttggaccaatgtacgitacgtaccatccttitt tgattitttitttgtaaactaaattitt cqaattagcaggttcttaataattgaacgaagaaaataaagaatagaggtaga US 7,868,155 B2 109 110

TABLE 1 - continued cacctgtag tattittcttggit cagaccaataatttata attcaacgtcaaagaagaagaaaaatataaaccattattt cattatgacttacgtataccaaaatacacaaattaaatgtataattgtgaggcattittatatgcgggaaaaaataaaa taaaaagaat attaatatttcttittgaaaattgtaaag cattttgacccacttgttgatatatatatatatatagatat atatagagagagagattaaaacattgatggctagot at agagtictatggcagggit catgat cacctgtc.ttctgatct ctgaagagataccaatctgattittittct ct tcc taggtttaattittattittaccattittata attctittatttittgcc tgtag tacaatttacagacccatactaaaagaaaaattaaattttgttcaaagtacaaaacaaagagagaggtgaagcc acacaatctottttcttct ct ct ct citctgttatat citcttctgtttaattcttittattottctitcgtctatottctic cTATAatctottct citctic cct citt cacctaaagaataagaagaaaaataatt cacat ctittatgcaaact actittct tgtagggittittaggagctatotic tattgtc.ttggttctgatacaaagttttgtaattitt catgg tatgagaagatttg c ctittctattttgtttattggttctttitta actttittcttggagatgggttcttgtagat cittaatgaaacttctgtt tttgtc.ccaaaaagagttittcttttitt cittct cittctttittgggttittcaatt cittgagagac

>SEQ ID NO: 62 construct YPO 216 Ttttgtttctaatagtttgatgtttatat caa.catt attatttactitt catttgttac.cgatagaaagaggagaaaat tgttgacaaaaacaaagaaaaaagtaaaattaat attattaaattaataaaaataacaaactgtaaaagct atttitta aaaatttittcttgtaaaac atctaaaaattattottgtagaaacagaggaatat cattgaagataatagtgtgaaatt atatatatatatagaaatatataaagtaggattitttittctgtatacaaatatacgttt coaattittatcaaaaactgt aaagattitttittctttgtcagtacctgctaaacttgttaattitttitt attaaaaaaaaatcaaattacaattcttcta taat cattittaaatticcatttctittataccacaaaagattatattgc ctittatcgt.ctittgg tatgtatgcgtgaata tatttatttattittcttittctitt cattttcttitttaaagaactittataaatgaaataaggaacaaacaatatacacat gtactaacgtatataaataatat catcaatat citat coaaaacttggattt catggttgacgtgg.cccaac caaaatc t caagttctic togcggatgacgaac catctoac catctotttttitt ct ct ct ctitttitttitttittaatat catcagcac ggittacataaaatt cqtgatc catgaagttggctitt cittgtcgttitt actt catcaccc.catttittittaaagt ct coa t ctittatact tc.ttcaact ct coaccaccaccattgtcaccaccacatttaaacacacactitt cacttgtag togggat tcqaaagtgcgttittatt catttgttt tactgtttittgataac ct caaaatttgcc taaattittatt ct cTATAaatc cittatatgttitt acttacatt cotaaagttittcaactittcttgagct tcaaaaagtacct coa

>SEQ ID NO: 63 construct YPO23 O ataaaattatctataaatcattaaatctittgatgagaaat atccaatctactaatgtatat cqatgatttaaatgaaa ttact tatttgaacacaaaaataaatgaatttactaataaataaatagogtagttggagcaa.gtggctaaaaaaatta caaatc tagttt coatt ct cagogt cqgctgcttggaacgtcaccgttittctggaaaacgcaat cittct coct tcc.gt gacgt.ct caccoggaattitt ct cqcttttgttctact citcct coat citcc.gaggttct coaa.gcticagotcct ct tcc.ca t catt catc.cgaccgcc titat cogg to agatc ctittacg tatttctattitt cotgatcgt.cgatttittgagaaatgta aaaacagat.cgTATAaggcct cqaagtttittaatttgaaagtggitat cqaaattittittggtc.tttgattaggittaggg Caccgtagctctggg tattgaatttgtagggitttitcct ctggittattggtc.tttggagcttggtaatttctgctgaat tgattgat coctitt to catcttittgaagtaaagt ct cqagctitt.cgtgtct cqatgtagatgaattictattittgaata tgagatttgatalagacgtcaattgctgata atttggagtictttgttgtctgaatttgttcatatgaagttittctgaggg atgtgaattittattgttctgctaattittgaaacgttccttittggaatttggtttgttgaggagt cc tagat citttittctg tgaagtttcttgcttgtaagttittctggat cacttgattgagtictagaatctagatagattacatgitacggitttgatt c ctittggctgattitt coaaagttttgttcaaattitcaggagaact acaaagaggaaac caagattgttttgttttgtt agactic tacc ccttitt.ccgatt cacatggtaaggacattgagg tagagaataatactaaaaag

>SEQ ID NO: 64 construct YPO261 gtcgattggttgtaaattagttittatcgtagaagtaccaaatcaagtgattcaatggittaaattaagg tattaagtta US 7,868,155 B2 111 112

TABLE 1 - continued catttgat atttaaaagtatic cagacct tcattatagotcataagggittaaaattttgtcgttcttttgtatatt cat ggcaa.gctictaatt catgactaagt cacatttittcaaatatgtttittagttitt tactitatgttggtaattagtggatt tatagittaagttaaaaagttgg.cgagttctagotttgaaact catttagaaatatatatatatatatatatatt caat tittagtaaattgttaatct attctaatggtgtaactgtaacaaatgagaatgaaaaaaatatact attgttgaataaaa c cccacacaa.ca cattactataataagttaaactitctttittittatagg.cgc.ctggaaaaaaaagaaaagcaacaagag ggstgtgaggacgcatcaccinggitttcgtag cacacatgtgcatttgtc.tc.tttgctttitt.cggitttttitt cittgc.ca atcaatttattttgttcct cagaaaaaagaaaatctaaaaccaaaatatat attataacct catttaataaacaacaa aaatgtttgttgaaaaaaaaaaagtttittatttatc.ttgacct tatttctittgaagaaaataaagcttggittattaaa gaagtic caagttagttgccaccatcagtgg catalacggtaaattaaa.gc.ca acttic ct ctaactaaagtttitcTATAa attcaaccactic acctic cc actictaaaacccaacaa cataattt cacatat ct citctittctittct cittgaaggaaaga cgaagat citccaagttcc caagtacgtaact actittct c catctacattcaattgtttct c cittaatttctic tagtaca tatt tacttgtgctataagtaattgattittatat cacc catgtgcaggttgttaacacaaga

>SEQ ID NO: 65 construct YPO263 tattatatatacgattaaataaataaaaaaattgtaatgtgaaaatat catagt cdagaggggaactgacaagtgtac atatgt at Ctagctgtggatt CCaC caaaattctggcagggc.catgatctaaaaactgagactg.cgcgtgttgttittg cagtgatttgtattt catatttgcaccatcct acacagtic cacttggitat cqtaac caaacataaggagaacctaatt acattattgttittaattitcgtcaaactggtttittacct tittagttacat agttgattctt catttgttt tag tagtta tggagcacaataatgtgcaacaaagaaagatcat agtggattaatatgttgagaggltdagaaattcttggittaacaaa aaaaaaaaagttacaaggactgagattittgggtgggagaaagc catagottttaaaac atgattgaact taaaagtga tgtt atggtttgaggggaaaaaggttgatgtcaact aagatagttgaagtaatgtc.ttaalactaaagtaalaccaccgg tccaaacgtggit coggaag catctotgg tatgattitat cotaaaaatcaaaatagtagaalacatactittaaatatata cattgatcggacgaaaattgtaaac tagtatagitttcaaaaac tagttgaacaggittatgtaccttaaacatttattt caaacttaaacactaaagaacatatatgaatagaagtttatataaattactatatat citaccataaatctottataat tatgatgtcacgatgaggaagtgttgaaacgittaaaatgccaaaatataag catgcgacggaattittggcagaagatt gtagagttgtaatctgtc.gcaat cattact catgctago atttitt catttitcc citt catttgttggataacgcacgata taac attctacacaccaacaagatt cTATAaaaacgcaaaggttgtc.t.c catagaatat cqtc

>SEQ ID NO: 66 construct YPO 271 atct ctdatttitttittatcaggaacaagtaaataaatagotttgagttitttgtttitttittctacattctitcgc.ccaaa agatgtaagaaaataaaggatttgaaaccttgttctgttgttact cotttaaattcttaaaaactataaat cattata t ctittgatctgttt cacaaactaat catatt cqttgcaaagtgagaatt cqtcc.cactitt actic tttacac cq atact agtattatagatgtacagdatag tatt coat atc tagttatttagt caaaactictatatattaa.gagg taggittaatt aattaaggagtaattgaagattatagaaagaataaaaaataccatttaatggacagaaccaaagataactalactato a tactataatgttgaatttct tccacgatccaatgcatggataacaa.cat caatcaaat catacatt catgctatataa catagttitt cagttacaaact ct ctitttittatttattt cagttgttccttitt catgaccatattaa catcaaataatg catttittittcaacgt.ct cittgacttacacc cactaatattgacaaattgaacatctatacgactatacacacataagt taaaaatgcatgcaagtgctaagggaatttataa catctaaggittaataagactaagaaagtataaaataagaatacg tatt atgaattitatgat at actt tactaatctttittgaaaaatactittaatttaatctactataggggg taaaaagta aaaaagaaataaagatacgtttatcc.gcatatag tacctggaaataacagaaaataaaaacacagg taagtactittgc ctgagctagIATAtgaacactaaagagatacacacacacaaaaagaga.gcagaaacaaaacacacacacttaaagctt tcqtctttacct ct tcc cttct citct ct citat citaaaaagagttcc.gagaagaagatcat cat US 7,868,155 B2 113 114

TABLE 1 - continued

>SEQ ID NO: 67 construct YPO279 cgctitt attataggitttaacaattgatttitt cattattttgttitt caat ct coaaatcatttct caataactic toaaa cattgtttaaagcttitttitt cittaattaac attataacaaaaaaataaatagagaaatttactittgattcaaacacca gtcattgtagattagccaagagttitt cagtaacaaaatttaccttataa acct tittgaatggct atttctgaaatgga atagaaatctittagt cqtggaagitat citctat coataagaaaact cqttttacaaagtaattittaaatcaatacaaaa agtgaaaaaatccactggtgg accc catt cattccagaattgc.cgattacgagctatottgtcc cttctt cac catt c gctcactictotctotct ct ct citcqtcttcttct tcccaccactictotctgtttct coacaactitctottctoaaagt taaaattacc cctaaac caaaaaaaaaaaaaacgct citt cact atttatt tactaaact citcctttgtttgttactaa gctcitcactaaaaccotaatctttctoctottaTATAtct cqtgacticttctttct cotccaatctotctotcccitct t cacaaac caattagcttctittctgtaaaacct cactic gttggccaattcttittggittitt catacacataaatct cag attic caaatgggttitt cittagct ctitt ctittcaaatgatgaacatttgttagcagaatct tcct ctitt.ccctaaagtt ttgatc.tttitttitcc.ccct tcaattttgtattitt ct caccaaataaaaaaaggttt citt cagtgggittittaagggittt attatt at cittaaaattaalacacaattctittaatcaaaaggcaaaaatc.ttaattt catcactic tottct cact caca aaagttcttacaat cittcaaagttittggtc.ttgtttcttitt.ccgattitcaccggacaaaaaaa

>SEQ ID NO: 68 construct YPO285 gggattatatatgatagacgattgt atttgcgggacattgagatgtttc.cgaaaatagt catcaaatat caaaccaga atttgatgtgaaaacactaattaaaacatataattgacaactagactatat catttgttaagttgagcgttgaaagaa aatgaaagagtgtag actgtag tatgt atgagtttcc.caaaggatggtgcttgaat attattgggaagagactittggit tggttcggttgaatgaagattitt tacctgc catgttgatagagaaaggcaaataaatgtagggg.tc.gatgtctaacgt. aaag actggat caaccalagagt cct cotcct cqt ctitcaccaaaaaaaaagagt cct c ct cqtggaaacttattt citt citccagccaagat citcatctoat ct citt cactictatgaaatataaaggaat cittatggitttittctaaaaactatagta cgt.ctatataccaaaggaaacaatataaaatcagttaatctgataaattittgagtaaataataaagttaactttgtac ttacctatat caaactaatticacaaaataaagtaataataacaaagaatttittagtagat coacaatatacacacaca citatgagaaat cataatagagaattittaatgattttgtctaact catagcaacaagtc.gctittgg.ccgagtggittaag gcgtgtgcctgctaagtacatgggctctgc.ccgc.gagagttctgaatctotcaggcgacgttt Cttttgtttitcggc.ca taaaggaaaaagcc caattaa.cacgtc.tc.gcttataagcc cataaagcaaacaatgggctgtctctgtc.t.c acticaca cacgcgttitt cotactittittgactattitt'TATAaccggcgggtctgacittaattagggittitt ctittaataatcagaca citct ct cact cqtttcqtcaa.cattgaacacagacaaaac cqcgt cacaaaacaaaactic got

>SEQ ID NO: 69 construct YPOOO3 tggatctgctagatatatgagaacggaaagaaccagaa.gct attagaggcgggaggagatatgtggggatgattt cag tgcaattic cacgacgcaccattt coactitt cqtaac acctaaacgaccqctitcgg.ccgtataaaatcgcaaatgtttg gtct cagtgt atttitt.ccaattt coaaata catcaattcaaattatataatat ctagtggcaattataagtatat cat at attittcaaaattaattaaaaagattactaaattatgtttgact acaact attataatagittaaaaacataaacaaa aacaaagaaact attittaataaaaaaatcaagtaaacattaaaacataagcaaaaaataatgttaaagaaatt attaa ttattaatttactaataattaatacct ctataaattaattgttagaggitttaacgtaatttataaggaaaactaaaga agactittaac coataaagaaaaaaacaaag actgaattgaaggcc catatttagaagaagagaaagaag accolaaata tgatataaaatc.ca.gcc catttatatatttittattttgtttctggaaggaaaataagaaaatggcaaaaacgaaataa tctgaaaaagtaaggtottttaccaaaaaggatatttittitt'TATAaacagagcataaagttitt cacttittcttctgct c ctittct cqt citctgtc.ttctitcgt.cct catt cqttittaaagcatcaaaattt catcaac ccaaaatagattaaaaaa atctgtagctitt cqcatgtaaatct ct ctittgaaggttcc taact cqttaatcgtaact cacagtgact cqtt coagt caaagttct ctdt ctittagctcaaa US 7,868,155 B2 115 116

TABLE 1 - continued

>SEQ ID NO: 70 construct YPOO15 Ttgagcct tattgttgttattgacttittagcc-aatagaaagagatggaaattcaataattatccacaaaattic caaat cattggtgtacaaaaagat ctaaggctgttatattittcaaaaaagaaagaaaagaaatgcaacaaatatggattaaac tgtggtttgtaaattgagctittgcatgaaaactittat cactatgattt cact actic catatttattgactaaagtggc actaatgaattt cittaat catgaaatc.ttgtatcaaaaagtactaaaataaacatgacattggcaattaggaaaattic taaattagaaattagtaaaaatgaaaggtgaaagggaaagatgatgatatgaattggttggtgacCaggagaaatgta tcc.cgatttittgcagacactitt cagtgtc.cccatt catataattatgg.cccaccitcqttalagatttitt cattcaccac cataacaagatctaagcttagattt catgtaattaalacatataatat acttgccaatact at citaataaagtatactt aagcaaaaattatt actictagtgtaaggcgatgaaatataagtttagttgaaaattitatgtcgatataacaaagtata atgaattaag accttggittitt cqattaacaaactaattaalacact agttittgcctaataaaaccoggaatcg tatt ca aaac cqaacgacaaaacaagggacaagttgagagacaaaaccalaatcagcatctittct tccagaaatgtcatgaccac atgacgtcatcttgacc ctitctt cattgttgat atctgtggataaag.cgcacgtgtttaattcacga acctitcqtagta acgaaaaatccacaactitt catatttitttaattacc cactaaactaaaacaaatttggaaaaacatgaaaaactttitt cittitttitt coaggttcgtgaacct cqtaccct citat ATAaacct cittaaccacct tccacata

TABLE 2 Promoter Expression Report # 1 Report Date: Jan. 31, 2003; Revised Aug. 15, 2003 Promoter Tested In: Arabidopsis thaliana, WS ecotype Spatial expression Summary: Flower (M)upper part of receptacle, (M)base of ovary Flower (M)pedicel, (M) receptacle, silique, (M)carpel Stem (H)cortex, (H)pith Hypocotyl (M)cortex Primary Root (H)vascular, (M)cap Observed expression pattern: T1 mature: Expression was specific to the top of the receptacle and base of gynoecium of immature flowers. Not detected in any other organs. T2 seedlings: No expression observed. T2 mature: In addition to the original expression observed in T1 mature plants, expression is observed in pith cells near the apex of the inflorescence meristem and stem-pediceljunctions. T3 Seedling: Expressed at cotyledon-hypocotyl junction, root vascular, and root tip epidermis. This expression is similar to the original 2-component line CS9107. Expected expression pattern: The candidate was selected from a 2-component line with multiple inserts. The target expression pattern was lateral root cap and older vascular cells, especially in hypocotyls. Selection Criteria: Arabidopsis 2-component line CS9107 (J1911) was selected to test promoter reconstitution and validation. T-DNA flanking sequences were isolated by TAIL-PCR and the fragment cloned into pNewBin4-HAP1-GFP vector to validate expression. Gene: 2 kb seq. is in 7 kb repeat region on Chr.2 where no genes are annotated. GenBank: NM 127894 Arabidopsis thaliana leucine-rich repeat transmembrane protein kinase, putative (At2g23300) mRNA, complete colsgil 18400232|refNM 127894.1||18400232) Source Promoter Organism: Arabidopsis thaliana WS Vector: pNewBin4-HAP1-GFP Marker Type: X GFP-ER Generation Screened: XT1 Mature X T2 Seedling X T2 Mature XT3 Seedling Bidirectionality: NO Exons: NO Repeats: none noted Promoter utility Trait-Subtrait Area: Among other uses this promoter sequence could be useful to improve: PG&D- abscission, plant size Nutrients- nitrogen utilization Utility: Promoter may be useful in fruit abscission but as it appears the expression overlaps the base of the gynoecium, it may be useful to overexpress genes thought to be important in Supplying nutrients to the gynoecium or genes important in development of carpel primordia. Construct: YPOOO1 Promoter Candidate I.D: 13148168 (Old ID: CS9107-1) cDNA ID: 1273.6079 T1 lines expressing (T2 seed): SR00375-01, -02, -03, -04, -05 US 7,868,155 B2 117 118

TABLE 2-continued Promoter Expression Report # 2 Report Date: Jan. 31, 2003 Promoter Tested In: Arabidopsis thaliana, WS ecotype Spatial expression Summary: Ovule Pre-fertilization: (H)inner integument Post-fertilization: (M)seed coat, (M)endothelium Root (H)epidermis, (H)atrichoblast Cotyledons (L)epidermis Observed expression pattern: T1 mature: GFP expression exists in the inner integument of ovules. T2 Seedling: Expression exists in root epidermal atrichoblast cells. T2 mature: Same expression exists as T1 mature. T3 seedlings: Same expression, plus additional weak epidermal expression was observed in cotyledons. Expected expression pattern: flower buds, ovules, mature flower, and silique Selection Criteria: Arabidopsis 2-component line CS9180(J2592). Gene: water channel-like protein' major intrinsic protein (MIP) family GenBank: NM 118469 Arabidopsis thaliana major intrinsic protein (MIP) family (Atag23400) mRNA, complete cols gi30686182|refNM 118469.2|30686.182) Source Promoter Organism: Arabidopsis thaliana WS Vector: pNewBin4-HAP1-GFP Marker Type: X GFP-ER Generation Screened: XT1 Mature X T2 Seedling X T2 Mature XT3 Seedling Bidirectionality: NO Exons: NO Repeats: None Noted Promoter utility Utility: Promoter could be used to misexpress any genes playing a role in seed size. It will also have utility in misexpressing genes important in root hair initiation to try to get the plant to generate more or ewer root hairs to enhance nutrient utilization and drought tolerance. Construct: YPOOO7 Promoter Candidate I.D: 13148318 (Old ID: CS9180-3) cDNA ID: 12703,041 (Old ID: 12332468) T1 lines expressing (T2 seed): SR00408-01, -02, -05 Promoter Expression Report #3 Report Date: Jan. 31, 2003 Promoter Tested In: Arabidopsis thaliana, WS ecotype Spatial expression Summary: Leaf (L)vascular Hypocotyl (L)epidermis Primary Root (H)epidermis, (H)cap Lateral root (H)epidermis, (H)cap Observed expression pattern: T1 mature: Low GFP expression was detected throughout the vasculature of leaves of mature plants. T2 seedling: No expression was detected in the vasculature of Seedlings. T2 mature: Transformation events which expressed as T1 plants were screened as T2 plants and no expression was detected. This line was re-screened as T1 plants and leaf expression was not detected in 3 independent events. T3 seedling: New expression was observed in T3 seedlings which was not observed in T2 seedlings. Strong primary and lateral root tip expression and weak hypocotyl epidermal expression exists. Expected expression pattern: High in leaves. Low in tissues like roots or flowers Selection Criteria: Arabidopsis Public; Sauer N. EMBO J 1990 9:3045-3050 Gene: Glucose transporter (Sugar carrier) STP1 GenBank: NM 100998 Arabidopsis thaliana glucose transporter (Atlg11260) mRNA, complete cols, gi30682126 refNM 100998.230682126 Source Promoter Organism: Arabidopsis thaliana WS Vector: pNewBin4-GFP Direct fusion construct Marker Type: X GFP-ER Generation Screened: XT1 Mature X T2 Seedling X T2 Mature XT3 Seedling Bidirectionality: NO Exons: NO Repeats: None Noted Promoter utility Trait-subtrait Area: Among other uses this promoter sequence could be useful to improve: Source-CN partitioning, transport of amino acids, source enhancement Yield- Total yield Quality- Amino acids, carbohydrates, Optimize C3-C4 transition Utility: Sequence most useful to overexpress genes important in vascular maintenance and transport in and out of the phloem and xylem. Construct: GOO13 Promoter Candidate I.D.: 1768610 (Old ID: 35139302) cDNA ID: 12679922 (Old IDs: 12328210,4937586.) T1 lines expressing (T2 seed): SR00423-01, -02, -03, -04, -05 Promoter Expression Report # 4 Report Date: Mar. 6, 2003 Promoter Tested In: Arabidopsis thaliana, WS ecotype Spatial expression Summary: Flower (H)sepal, (L)epidermis Embryo (H)Suspensor, (HOpreglobular, (H)globular, (M)heart, (M)torpedo, (L)late, (L)mature, (L)hypophysis Ovule Pre fertilization: (M)outer integument, (H) funiculus Post fertilization: (M)outer integument, (H)Zygote US 7,868,155 B2 119 120

TABLE 2-continued Embryo (H)hypocotyl, (H)epidermis, (H)cortex, (H)stipules, (L)lateral root, (H)initials, (H)lateral root cap Stem (L)epidermis Observed expression patterns: T1 Mature: Strong expression was seen in 4-cell through heart stage embryo with decreasing expression in the torpedo stage; preferential expression in the root and shoot meristems of the mature embryo. Strong expression was seen in the outer integument and funiculus of developing seed. T2 Seedling: Strong expression was seen in epidermal and cortical cells at the base of the hypocotyl. Strong expression was seen in stipules flanking rosette leaves. Low expression was seen in lateral root initials with increasing expression in the emerging lateral root cap. T2 Mature Same expression patterns were seen as T1 mature plants with weaker outer integument expression in Second event. Both lines show additional epidermal expression at the inflorescence meristem, pedicels and tips of sepals in developing flowers. T3 seedling expression - same expression Expected expression pattern: Expression in ovules Selection Criteria: Greater than 50x up in pi ovule microarray Gene: Lipid transfer protein-like GenBank: NM 125323 Arabidopsis thaliana lipid transfer protein 3 (LTP3) (At 5g59320) mRNA, complete cols, gi30697205 refNM 125323.230697205) Source Promoter Organism: Arabidopsis thaliana WS Vector: pNewbina-HAP1-GFP Marker Type: X GFP-ER Generation Screened: XT1 Mature X T2 Seedling X T2 Mature XT3 Seedling Bidirectionality: NO Exons: NO Repeats: None noted Promoter utility Trait-subtrait Area: Among other uses this promoter sequence could be useful to improve: Water use efficiency - Moisture stress, water use efficiency, ovuleseed abortion Seed - test weight, seed size Yield - harvest index, total yield Quality - amino acids, carbohydrate, protein total oil, total seed composition Construct: YPO097 Promoter Candidate I.D: 11768657 (Old ID: 35.139702) cDNA ID 12692181 (Old IDs: 12334169, 1021642) T1 lines expressing (T2 seed): SR00706-01, -02 Promoter Expression Report #5 Report Date: Mar. 6, 2003 Promoter Tested In: Arabidopsis thaliana, WS ecotype Spatial expression Summary: Ovule Pre-fertilization: (L)inner integument Post-fertilization: (H)inner integument, (M)endothelium Primary Root (H)endodermis Observed expression pattern: GFP is expressed in the endosperm of developing seeds and pericycle cells of seedling roots. GFP level rapidly increases following fertilization, through mature endosperm cellularization. GFP is also expressed in individual pericycle cells.T1 and T2 mature: Same expression pattern was observed in T1 and T2 mature plants. Closer examination of the images reveals that GFP is expressed in the endothelium of ovules which is derived from the inner most layer of the inner integuments. Lower levels of expression can be seen in the maturing seeds which is consistent with disintegration of the endothelium layer as the embryo enters maturity. T2 seedling: Expression appears o be localized to the endodermis which is the third cell layer of seedling root not pericycle as previously noted. T3 seedlings: Low germination. No expression was observed in the few Surviving seedlings. Expected expression pattern: Expression in ovules Selection Criteria: Greater than 50x up in pi ovule microarray Gene: palmitoyl-protein thioesterase GenBank: NM 124106 Arabidopsis thaliana palmitoyl protein thioesterase precursor, putative (AtSg47350) mRNA, complete cols gi30695 161|refNM 124106.2|30695 161 Source Promoter Organism: Arabidopsis thaliana WS Vector: pNewbina-HAP1-GFP. Marker Type: (X) GFP-ER Generation Screened: (X) T1 Mature (X) T2 Seedling (X) T3 Mature (X) T3 Seedling Marker Intensity: (X) High Med Low Bidirectionality: NO Exons: NO Repeats: None Noted Promoter utility Trait - Sub-trait Area: Among other uses this promoter sequence could be useful to improve: Seed - ovule?seed abortion, seed size, test weight, total seed Composition -amino acids, carbohydrate, protein to oil composition Utility: Promoter useful for increasing endosperm production or affecting compositional changes in the developing seed. Should also have utility in helping to control seed size. Construct: YPO111 Promoter Candidate I.D: 11768845 (Old ID: 4772159) cDNA ID 13619323 (Old IDs: 12396.169, 4772159) T1 lines expressing (T2 seed): SR00690-01, -02 Promoter Expression Report # 6 Report Date: Mar. 6, 2003 Promoter Tested In: Arabidopsis thaliana, WS ecotype Spatial expression Summary: Stem (H)epidermis, (H)cortex Hypocotyl (H)epidermis, (H)cortex US 7,868,155 B2 121 122

TABLE 2-continued Silique (H)style, (H)carpel, (H)septum, (H)epidermis Lea (M)mesophyll, (M)epidermis Observed expression patterns: Strong GFP expression exists throughout stem epidermal and cortical cells in T1 mature plants. GFP expression exhibits polarity in T2 seedling epidermal cells. First, it appears in the upper part of the hypocotyl near cotyledonary petioles, increasing toward the root, and in the abaxial epidermal cells of the petiole. An optical section of the seedling reveals GFP expression in the cortical cells of the hypocotyl. T2 mature: Same expression pattern was seen as in T1 mature with extension of cortex and epidermal expression through to siliques. No expression was seen in placental tissues and ovules. Additional expression was observed in epidermis and mesophyll of cauline leaves. T3 seedling: Same as T2. Expected expression pattern: Expression in ovules Selection Criteria: Greater than 50x up in pi ovule microarray Gene: cytochrome P450 homolog GenBank: NM 104570 Arabidopsis thaliana cytochrome P450, putative (Atlg57750) mRNA, complete cols, gi30696174|refNM 104570.230696174) Source Promoter Organism: Arabidopsis thaliana WS Vector: pNewbina-HAP1-GFP Marker Type: X GFP-ER Generation Screened: XT1 Mature X T2 Seedling XT3 Mature XT3 Seedling Bidirectionality: NO Exons: NO Repeats: None Noted Promoter utility Trait - Sub-trait Area: Among other uses this promoter sequence could be useful to improve: Water use efficiency - moisture stress, water use efficiency, ovule?seed abortion Seed - test weight, seed size Yield - harvest index, total yield Composition -amino acids, carbohydrate, protein total oil, total seed Utility: Useful when expression is predominantly desired in stems, in particular, the epidermis. Construct: YPO104 Promoter Candidate ID: 11768842 cDNA ID: 13612879 (Old IDs: 12371683, 1393104) T1 lines expressing (T2 seed): SR00644-01, -02, -03 Promoter Expression Report # 7 Report Date: Mar. 6, 2003 Promoter Tested In: Arabidopsis thaliana, WS ecotype Spatial expression Summary: Flower (L)sepal, (L)petal, (L)Silique, (L)vascular, (H)stomata, (L)pedicel Silique (L)vascular, (L)epidermis Cotyledon (H)stomata, (L) root hair Observed expression patterns: GFP expressed in the vasculature and guard cells of sepals and pedicels in mature plants. GFP expressed in the guard cells of seedling cotyledons. T2 mature: Stronger expression extended into epidermal tissue of siliques in proximal-distal fashion. T3 seedling: Weak root hair expression was observed which was not observed in T2 seedlings; no guard cell expression observed. All epidermal tissue type expression was seen with the exception of weak vasculature in siliques. Expected expression pattern: Drought induced Selection Criteria: Expression data (cDNAChip), >10 fold induction under drought condition. Screened under non-induced condition. Gene: Unknown protein; AtSg43750 GenBank: NM 123742 Arabidopsis thaliana expressed protein (AtSg43750) mRNA, complete cols, gi30694366 refNM 123742.2|30694366) Source Promoter Organism: Arabidopsis thaliana WS Vector: pNewbina-HAP1-GFP Marker Type: X GFP-ER Generation Screened: XT1 Mature X T2 Seedling XT3 Mature XT3 Seedling Bidirectionality: NO Exons: NO Repeats: None noted Promoter utility Trait - Subtrait Area: Among other uses this promoter sequence could be useful to improve: Water use efficiency - Heat Construct: YPOO75 Promoter Candidate I.D: 11768.626 (Old ID: 3513.9358) cDNA ID: 13612919 (Old IDs: 12694633,5672796) T1 lines expressing (T2 seed): SR00554-01, -02 Promoter Expression Report # 8 Report Date: Mar. 6, 2003 Promoter Tested In: Arabidopsis thaliana, WS ecotype Spatial expression Summary: Flower (L) receptacle, (L)vascular Leaf (H)vascular, (H)epidermis Root (M)phloem Cotyledon (M)vascular, (M)hydathode Primary Root (L)epidermis, (M) vascular Observed expression patterns: Expression was seen at the receptacle and vasculature of immature flower and leaf, and phloem of seedling root. T2 mature: Similar to T1 expression. Strong expression was seen in vascular tissues on mature leaves. Vascular expression in flowers was not observed as in T1. T3 seedling: Similar to T2 seedling expression. Expected expression pattern: Vascular tissues: The SUC2 promoter directed expression of GUS activity with US 7,868,155 B2 123 124

TABLE 2-continued high specificity to the phloem of all green tissues of Arabidopsis such as rosette leaves, stems, and sepals. Selection Criteria: Arabidopsis public; Planta 1995; 196:564-70 Gene: “Sugar Transport SUC2 GenBank: NM 102118 Arabidopsis thaliana Sucrose transporter SUC2 (sucrose-proton transporter) (Atlg22710) mRNA, complete cols, gi30688004|refNM 102118.230688004 Source Promoter Organism: Arabidopsis thaliana WS Vector: Newbina-HAP1-GFP Marker Type: X GFP-ER Generation Screened: XT1 Mature X T2 Seedling XT3 Mature XT3 Seedling Bidirectionality: NO Exons: NO Repeats: None Noted Promoter utility Trait - Sub-trait Area: Among other uses this promoter sequence could be useful to improve: Source - Source enhancement, CN partitioning Utility: Useful for loading and unloading phloem. Construct: YPOO16 Promoter Candidate I.D: 11768612 (Old ID: 35139304) cDNA ID 13491988 (Old IDs: 6434453, 12340314) T1 lines expressing (T2 seed): SR00416-01, -02, -03, -04, -05 Promoter Expression Report # 9 Report Date: Mar. 6, 2003 Promoter Tested In: Arabidopsis thaliana, WS ecotype Spatial expression Summary: Flower (L)inflorescence, (H)pedicel, (H)vascular Stem (L)phloem Leaf (L)vascular Owule Pre fertilization: (H)chalaza end of embryo sac Hypocotyl (M)vascular, (M)phloem Cotyledon (M)vascular, (M)phloem Roo (H)vascular, (H)pericycle, (H)phloem Observed expression patterns: GFP expressed in the stem, pedicels and leaf vasculature of mature plants and in Seedling hypocotyl, cotyledon, petiole, primary leaf and root. Expected expression pattern: Phloem of the stem, xylem-to-phloem transfer tissues, veins of Supplying seeds, vascular strands of siliques and in funiculi. Also expressed in the vascular system of the cotyledons in developing seedlings. T2 mature: Same as T1 mature. T3 seedling: Same as T2 seedling. Selection Criteria: Arabidopsis public PNAS 92, 12036-12040 (1995) Gene: AAP2 (X95623) GenBank: NM 120958 Arabidopsis thaliana amino acid permease 2 (AAP2) (At 5g09220) mRNA, complete cols, gi30682579 refNM 120958.230682579) Source Promoter Organism: Arabidopsis thaliana WS Vector: pNewbina-HAP1-GFP Marker Type: X GFP-ER Generation Screened: XT1 Mature X T2 Seedling XT3 Mature XT3 Seedling Bidirectionality: FAILS Exons: FAILS Repeats: None Noted Promoter Utility Trait - Sub-trait Area: Among other uses this promoter sequence could be useful to improve: Trait Area: Seed - Seed enhancement Source - transport amino acids Yield - harvest index, test weight, seed size, Quality - amino acids, carbohydrate, protein, total seed composition Utility: Construct: YPOO94 Promoter Candidate I.D: 11768.636 (Old ID: 35139638) cDNA ID: 136098.17 (Old IDs: 7076261, 12680497) T1 lines expressing (T2 seed): SR00641-01, -02 Promoter Expression Report # 10 Report Date: Mar. 6, 2003 Promoter Tested In: Arabidopsis thaliana, WS ecotype Spatial expression Summary: Flower (L)sepal, (L)pedicel, (L)vascular Silique (H)stomata Hypocotyl (M)epidermis Primary Leaf (H)stomata Root (H)epidermis, (H) root hairs Observed expression pattern: T1 mature: GFP expression was seen in the guard cells of pedicles and mature siliques. Weak expression was seen in floral vasculature.T2 seedling: Strong expression observed in epidermis and root hairs of seedling roots (not in lateral roots) and guard cells of primary leaves. T2 mature: Similar to T1 plants. T3 seedling: Similar to T2 seedling. Screened under non-induced conditions. Expected expression pattern: As described by literature. Expressed preferentially in the root, not in mature stems or leaves of adult plants (much like AGL 17); induced by KNO3 at 0.5 hr with max at 3.5 hr Selection Criteria: Arabidopsis Public: Science 279, 407-409 (1998) Gene: ANR1, putative nitrate inducible MADS-box protein; GenBank: NM 126990 Arabidopsis thaliana MADS-box protein ANR1 (At2g 14210) mRNA, complete cols gi22325672|refNM 126990.2122325672) Source Promoter Organism: Arabidopsis thaliana WS US 7,868,155 B2 125 126

TABLE 2-continued Vector: pNewbina-HAP1-GFP Marker Type: X GFP-ER Generation Screened: XT1 Mature X T2 Seedling X T2 Mature XT3 Seedling Bidirectionality: NO Exons: NO Repeats: None Noted Promoter Utility Trait - Sub-trait Area: Among other uses this promoter sequence could be useful to improve: Yield - Heterosis, general combining ability, specific combining ability Construct: YPOO33 Promoter Candidate I.D: 13148.205 (Old ID: 35139684) cDNA ID: 12370148 (Old IDs: 7088230, 12729537) T1 lines expressing (T2 seed): SRXXXXX-01, Promoter Expression Report # 11 Report Date: Mar. 6, 2003 Promoter Tested In: Arabidopsis thaliana, WS ecotype Spatial expression Summary: Flower (H)epidermis, (H)sepal, (H)petal, (H)vascular Stem (L)vascular Hypocotyl (L)epidermis, (H)phloem Cotyledon (L)epidermis, (M)stomata, (L)vascular Root (H)phloem Observed expression pattern: Strong GFP expression was seen in the epidermal layer and vasculature of the sepals and petals of developing flowers in mature plants and seedlings. T2 mature: Expression was similar to T1 mature plants. Vascular expression in the stem was not observed in T1 mature. T3 Seedling: Same expression seen as T2 seedling expression Expected expression pattern: Predominantly expressed in the phloem. Selection Criteria: Arabidopsis public: Deeken, R. The Plant J. (2000) 23(2), 285-290 Geiger, D. Plant Cell (2002) 14, 1859-1868 Gene: potassium channel protein AKT3 GenBank: NM 118342 Arabidopsis thaliana potassium channel (K+ transporter 2)(AKT2) (Atag22200) mRNA, complete cols, gi30685723 refNM 118342.2|30685723 Source Promoter Organism: Arabidopsis thaliana WS Vector: pNewbina-HAP1-GFP Marker Type: X GFP-ER Generation Screened: XT1 Mature XT2 Seedling XT3 Mature XT3 Seedling Bidirectionality: NO Exons: NO Repeats: None Noted Trait - Sub-trait Area: Among other uses this promoter sequence could be useful to improve: Nutrient - Low nitrogen tolerance: Nitrogen use efficiency; Nitrogen utilization Utility: Construct: YPOO49 Promoter Candidate I.D: 11768643 (Old ID: 6452796) cDNA ID 1266.0077 (Old IDs: 7095446,6452796) T1 lines expressing (T2 seed): SR00548-01, -02, -03 Promoter Expression Report # 12 Report Date: Mar. 6, 2003 Promoter Tested In: Arabidopsis thaliana, WS ecotype Spatial expression Summary: Flower (L)pedicel, (L)sepal, (L)vascular Leaf (M)petiole, (M)vascular Cotyledon (H)stomata, (M)petiole, (H)vascular Primary Leaf (L)vascular, (L)petiole Root (H) root hair Observed expression pattern: GFP expression was detected in the vasculature of sepals, pedicel, and leaf petiole of immature flowers. Also weak guard cell expression existed in sepals. Strong GFP expression was seen in guard cells and phloem of cotyledons, and upper root hairs at hypocotyl root transition Zone. T2 mature: Same as T1 mature. T3 seedling: Same as T2 seedling. Expected expression pattern: Shoot apical meristems Selection Criteria: Greater than 5X down in stim microarray Gene: AP2 domain transcription factor GenBank: NM 129594 Arabidopsis thaliana AP2 domain transcription factor, putative(DRE2B) (At2g40340) mRNA, complete cols, gi30688235 refNM 129594.2|30688235) Source Promoter Organism: Arabidopsis thaliana WS Vector: pNewbina-HAP1-GFP Marker Type: X GFP-ER Generation Screened: XT1 Mature X T2 Seedling XT3 Mature XT3 Seedling Bidirectionality: NO Exons: FAILS Repeats: None Noted Promoter Utility Trait Area: Among other uses this promoter sequence could be useful to improve: Cold, PG&D, Sub-trait Area: Cold germination & vigor, plant size, growth rate, plant development Utility: Construct: YPOO60 Promoter Candidate I.D: 11768797 (Old ID: 35139885) cDNA ID: 13613553 (Old IDs: 4282588, 1242.1894) T1 lines expressing (T2 seed): SR00552-02, -03 US 7,868,155 B2 127 128

TABLE 2-continued Promoter Expression Report # 13 Report Date: Mar. 6, 2003 Promoter Tested In: Arabidopsis thaliana, WS ecotype Spatial expression Summary: Ovule Post-fertilization: (H)endothelium, (H)micropyle, (H)chalaza Observed expression pattern: T1 and T2 mature: Strong expression was seen in the mature inner integument cell layer, endothelium, micropyle and chalaza ends of maturing ovules. Expression was not detected in earlier stage ovules. T2 and T3 seedling expression: None Expected expression pattern: Primarily in developing seeds Selection Criteria: Arabidopsis public; Mol. Gen. Genet. 244, 572-587 (1994) Gene: plasma membrane H(+)-ATPase isoform AHA10; GenBank: NM 101587 Arabidopsis thaliana ATPase 10, plasma membrane type (proton pump 10) (proton-exporting ATPase), putative (Atlg17260) mRNA, complete cols, gi|18394.459 Source Promoter Organism: Arabidopsis thaliana WS Vector: pNewbina-HAP1-GFP. Marker Type: X GFP-ER Generation Screened: XT1 Mature X T2 Seedling XT3 Mature XT3 Seedling Bidirectionality: FAILS Exons: FAILS Repeats: None Note Trait Area: Among other uses this promoter sequence could be useful to improve: Seed - Endosperm cell number and size, endosperm granule numbersize, seed enhancement Yield - harvest index, test weight, seed size Quality - protein, total oil, total seed composition, composition Utility: Construct: YPO092 Promoter Candidate I.D: 13148.193 (Old ID: 35139598) cDNA ID 1266.1844 (Old ID: 4993117) T1 lines expressing (T2 seed): SR00639-01, -02, -03 Promoter Expression Report # 14 Report Date: Mar. 6, 2003 Promoter Tested In: Arabidopsis thaliana, WS ecotype Spatial expression Summary: Flower (L)silique Silique (L)medial vasculature, (L)lateral vasculature Observed expression pattern: GFP expressed in the medial and lateral vasculature of pre-fertilized siliques. Expression was not detected in the older siliques or in T2 seedlings. T2 mature: Weak silique vasculature expression was seen in one of two events. T3 seedling: Same as T2 seedling, no expression was seen. Expected expression pattern: Expression in ovules Selection Criteria: Greater than 50x up in pi ovule microarray Gene: expressed protein; protein id: Atag1575.0.1, hypothetical protein GenBank: NM 117666 Arabidopsis thaliana expressed protein (Atag15750) mRNA, complete cols gi|18414516 refNM 117666.1||18414516) Source Promoter Organism: Arabidopsis thaliana WS Vector: pNewbina-HAP1-GFP Marker Type: X GFP-ER Lines Screened: n = 3 Lines Expressing: n = 3 Generation Screened: XT1 Mature X T2 Seedling XT3 Mature XT3 Seedling Bidirectionality: NO Exons: NO Repeats: None Noted Promoter utility Trait - Sub-trait Area: Among other uses this promoter sequence could be useful to improve: Water use efficiency - Moisture stress at seed set, Moisture stress at seed fill, water use efficiency, Ovuleseed abortion Seed - test weight, seed size Yield - harvest index, total yield Quality - amino acids, carbohydrate, protein, total oil, total seed composition Construct: YPO113 Promoter Candidate I.D: 13148162 (Old ID: 35139698) cDNA ID: 12332135 (Old ID: 56638.09) T1 lines expressing (T2 seed): SR00691-01, -03 Promoter Expression Report # 15 Report Date: Mar. 6, 2003 Promoter Tested In: Arabidopsis thaliana, WS ecotype Spatial expression Summary: Flower (L)silique Silique (L)medial vasculature, (L)lateral vasculature, (H)guard cells Rosette leaf (H)guard cell Observed expression pattern: GFP expressed in the medial and lateral vasculature of pre-fertilized siliques. Expression was not detected in older siliques. Guard cell expression was seen throughout pre-fertilized and ertilized siliques. T2 seedling: No expression was seen. T2 mature expression: Similar to T1 mature expression. T3 seedling: Guard cell expression not seen in T2 seedlings, however it is in the same issue type observed in mature plants of previous generation. Expected expression pattern: Strong activity in the inner endosperm tissue of developing seeds and weak activity in root tips. Selection Criteria: Arabidopsis public; Plant Mol. Biol. 39, 149-159 (1999) US 7,868,155 B2 129 130

TABLE 2-continued Gene: Alanine aminotransferase, AlaAT GenBank: NM 103859 Arabidopsis thaliana abscisic acid responsive elements-binding factor (Atlg49720) mRNA, complete cols gi30694628 |refNM 103859.2|30694628) INCORRECT (L. M. 10/14/03) AAK92629 - CORRECT (L. M. 10/1403) Putative alanine aminotransferase Oryza satival gil 15217285 Igb|AAK92629.1|ACO79633 9.15217285) Source Promoter Organism: Rice Vector: pNewbina-HAP1-GFP. Marker Type: X GFP-ER Generation Screened: XT1 Mature X T2 Seedling XT3 Mature XT3 Seedling Bidirectionality: NO Exons: NO Repeats: None Noted Promoter utility Trait Area: Among other uses this promoter sequence could be useful to improve: Seed, source, yield, quality Sub-trait Area: Seed enhancement, transport amino acids, harvest index, test weight, seed size, amino acids, carbohydrate, protein, total seed composition Construct: YPO095 Promoter Candidate ID: 13148198 (Old ID: 35139658) cDNA ID: 6795099 in rice T1 lines expressing (T2 seed): SR00642-02, -03 Promoter Expression Report # 16 Report Date: Mar. 6, 2003 Promoter Tested In: Arabidopsis thaliana, WS ecotype Spatial expression Summary: Ovule Pre-fertilization: (M)gametophyte, (M)embryo sac Root (H)epidermis, (M)pericycle, (H) root hairs Lateral root (H)flanking cells Observed expression patterns: GFP expressed in the egg cell and synergid cell of female gametophyte in early ovule development. It expressed in polarizing embryo sac in later stages of pre-fertilized ovule development. No expression was seen in fertilized ovules. GFP expressed throughout the epidermal cells of seedling roots. It also expressed in flanking cells of lateral root primordia. T2 mature: Same as T1 mature. T3 seedling: Same as T2 seedling Expected expression pattern: Expression in ovules Selection Criteria: Greater than 50x up in pi ovule microarray Gene: Senescence-associated protein homolog GenBank: NM 119189 Arabidopsis thaliana senescence-associated protein family (Atag30430) mRNA, complete cals, gi|18417592|refNM 119189.1||18417592) Source Promoter Organism: Arabidopsis thaliana WS Vector: pNewbina-HAP1-GFP Marker Type: X GFP-ER Generation Screened: XT1 Mature X T2 Seedling XT3 Mature XT3 Seedling Bidirectionality: NO Exons: NO Repeats: None Noted Promoter utility Trait Area: Among other uses this promoter sequence could be useful to improve: Water use efficiency, seed, yield Sub-trait Area: Moisture stress, water use efficiency, ovule?seed abortion, harvest index, test weight, seed size, total yield, amino acids, carbohydrate, proteintotail oil, total seed composition Construct: YPO102 Promoter Candidate I.D: 11768651 (Old ID: 35139696) cDNA ID: 13613954 (Old IDs: 12329268, 1382001) T1 lines expressing (T2 seed): SR00643-01, -02 Promoter Expression Report # 17 Report Date: Mar. 6, 2003 Promoter Tested In: Arabidopsis thaliana, WS ecotype Spatial expression Summary: Ovule Pre-fertilization: (H)inner integument Post-fertilization: (H)inner integument, (M)outer integument, (M)seed coat Primary Root (L) root hair Observed expression pattern: GFP expressed in the inner integuments of pre-fertilized and fertilized ovules. Female gametophyte vacuole seen as dark Oval. T2 mature: Same expression was seen as T1 with additional expression observed in similar tissue. GFP expressed in the outer integument and seed coat of developing ovules and seed. T3 seedling expression: GFP expression was seen in a few root hairs. Expected expression pattern: Expression in ovules Selection Criteria: Greater than 50x up in pi ovule microarray Gene: putative protease inhibitor GenBank: NM 129447 Arabidopsis thaliana protease inhibitor - related (At2g38900) mRNA, complete cols, gi30687699 refNM 129447.2|30687699) Source Promoter Organism: Arabidopsis thaliana WS Vector: pNewbina-HAP1-GFP Marker Type: X GFP-ER Generation Screened: XT1 Mature X T2 Seedling XT3 Mature XT3 Seedling Bidirectionality: NO Exons: FAILS Repeats: None Noted Promoter utility US 7,868,155 B2 131 132

TABLE 2-continued Trait Area: Among other uses this promoter sequence could be useful to improve: Water use efficency, seed, yield Sub-trait Area: Moisture stress, water use efficiency, ovule?seed abortion, harvest index, test weight, seed size, otal yield, amino acids, carbohydrate, proteinto tail oil, total seed composition. Construct: YPO103 Promoter Candidate I.D: 13148.199 (Old ID: 35.139718) cDNA ID: 4905097 (Old ID: 12322121, 1387372) T1 lines expressing (T2 seed): SR00709-01, -02, -03 Promoter Expression Report # 18 Report Date: Mar. 6, 2003 Promoter Tested In: Arabidopsis thaliana, WS ecotype Spatial expression Summary: Embryo (H)mature, (H)late Ovule (H)endothelium Primary root (L) root hair Observed expression pattern: Low levels of GFP expression were detected in late torpedo stage with highest evels in the mature and late embryo. High GFP expression was detected in late endosperm stage in endothelium ayer of developing seed. T2 mature: Same as T1 mature. T3 seedling: GFP was detected in a few root hairs not observed in T2 seedlings. Expected expression pattern: Embryo and seed Selection Criteria: Arabidopsis public; Rossak, M. Plant Mol. Bio. 2001.46:717 Gene: fatty acid elongase 1: FAE1 GenBank: NM 119617 Arabidopsis thaliana fatty acid elongase 1 (FAE 1) (Atag34520) mRNA, complete cols, gi30690063|refNM 119617.230690063) Source Promoter Organism: Arabidopsis thaliana WS Vector: pNewbina-HAP1-GFP Marker Type: X GFP-ER Generation Screened: XT1 Mature X T2 Seedling X T2 Mature XT3 Seedling Bidirectionality: NO Exons: NO Repeats: Not Done Promoter utility Trait - Sub-trait Area: Among other uses this promoter sequence could be useful to improve: Seed - Ovule?seed abortion, seed enhancement, seed size Yield Construct: YPO107 Promoter Candidate I.D: 13148252 (Old ID: 35139824) cDNA ID: 12656458 (Old ID: 1815714) T1 lines expressing (T2 seed): SR00646-01, -02 Promoter Expression Report # 19 Report Date: Mar. 6, 2003 Promoter Tested In: Arabidopsis thaliana, WS ecotype Spatial expression Summary: Ovule Pre-fertilization: (M)gametophyte, (M)embryo sac Post-fertilization: (H)Zygote Observed expression pattern: GFP expressed in the developing female gametophyte of unfertilized ovules and the degenerated synergid cell of the fertilized ovule hours after fertilization. No expression was observed in T2 Seedlings. T2 mature: Similar expression as T1 mature. T3 seedling: Root expression in one of two events was not observed in T2 seedlings. No expression was observed in the second line which is consistent with T2 seedling expression. Expected expression pattern: Expression in ovules Selection Criteria: Greater than 50x up in pi ovule microarray Gene: Hypothetical protein GenBank: NM 112033 Arabidopsis thaliana expressed protein (At3g 11990) mRNA, complete cols gi|18399438 |refNM 112033.1||18399438) Source Promoter Organism: Arabidopsis thaliana WS Vector: pNewbina-HAP1-GFP Marker Type: X GFP-ER Generation Screened: XT1 Mature X T2 Seedling XT3 Mature XT3 Seedling Bidirectionality: NO Exons: FAILS Repeats: None Noted Promoter utility Trait Area: Among other uses this promoter sequence could be useful to improve: Water use efficiency, seed, yield Sub-trait Area: Moisture stress, water use efficiency, ovule?seed abortion, harvest index, test weight, Seed size, total yield, amino acids, carbohydrate, proteintotal oil, total seed composition. Construct: YPO110 Promoter Candidate I.D: 13148.212 (Old ID: 35139697) cDNA ID: 13604221 (Old IDs: 12395.818, 4772042) T1 lines expressing (T2 seed): SR00689-02, -03 Promoter Expression Report # 20 Report Date: Mar. 6, 2003 Promoter Tested In: Arabidopsis thaliana, WS ecotype Spatial expression Summary: Flower (L)silique Silique (M) medial vasculature, (M)lateral vasculature, (M)guard cells Observed expression pattern: GFP expressed in the medial and lateral vasculature of pre-fertilized siliques. US 7,868,155 B2 133 134

TABLE 2-continued Expression was not detected in older siliques. Guard cell expression was seen throughout pre-fertilized and fertilized siliques. T2 Mature: Same as T1 Mature. T2 seedling: Same as T2 seedling. Expected expression pattern: Expression in ovules Selection Criteria: Greater than 50x up in pi ovule microarray Gene: hypothetical protein GenBank: NM 104488 Arabidopsis thaliana hypothetical protein (Atlg56100) mRNA, complete cols gi|18405686 refNM 104488.1||18405686) Source Promoter Organism: Arabidopsis thaliana WS Vector: pNewbina-HAP1-GFP Marker Type: X GFP-ER Generation Screened: XT1 Mature X T2 Seedling X T2 Mature XT3 Seedling Bidirectionality: NO Exons: FAILS Repeats: None Noted Promoter Utility Trait Area: Among other uses this promoter sequence could be useful to improve: Water use efficiency, seed, yield Sub-trait Area: Moisture stress at seed set, moisture stress at seed fill, water use efficiency, ovuleseed abortion, harvest index, test weight, seed size, total yield, amino acids, carbohydrate, protein, total oil, total seed composition, composition Utility: Construct: YPO112 Promoter Candidate I.D: 13148226 (Old ID: 35.139719) cDNA ID: 12321680 (Old ID: 5662775) T1 lines expressing (T2 seed): SR00710-01, -02, -03 Promoter Expression Report # 21 Report Date: Mar. 6, 2003 Promoter Tested In: Arabidopsis thaliana, WS ecotype Spatial expression Summary: Silique (H)stigma, (H)transmitting tissue Observed expression pattern: GFP expression was seen in the stigma and pollen transmitting tract spanning the entire silique. No expression was detected in the T2 seedlings. T2 Mature: Same as T1. T3 seedlings: No data Expected expression pattern: Expression in ovules Selection Criteria: Greater than 50x up in pi ovule microarray Gene: putative drought induced protein GenBank: NM 105888 Arabidopsis thaliana drought induced protein - related (Atlg72290) mRNA, complete cols gil 18410044|refNM 105888.1||18410044) Source Promoter Organism: Arabidopsis thaliana WS Vector: pNewbina-HAP1-GFP Marker Type: X GFP-ER Generation Screened: XT1 Mature X T2 Seedling XT3 Mature XT3 Seedling Bidirectionality: NO Exons: NO Repeats: None Noted Promoter utility Trait - Sub-trait Area: Among other uses, this promoter sequence could be useful to improve: Water use efficiency - Moisture stress at seed set, Moisture stress at seed fill, water use efficiency, Ovule?seed abortion Utility: Interesting to think about using this promoter to drive a gene that would select against a specific pollen type in a hybrid situation. Construct: YPO116 Promoter Candidate I.D: 13148262 (Old ID: 35139699) cDNA ID: 12325134 (Old ID: 6403538) T1 lines expressing (T2 seed): SR00693-02, -03 Promoter Expression Report # 22 Report Date: Mar. 8, 2003 Promoter Tested In: Arabidopsis thaliana, WS ecotype Spatial expression Summary: Flower (H)pedicle Silique (M)vascular Stem (H)cortex Ovule Pre-fertilization: (H)Outer integument, (M)chalaza Hypocotyl (H)cortex Root (H)epidermis, (H)atrichoblast, (H)cortex Observed expression pattern: Strong GFP expression was seen in the adaxial Surface of the pedicel and secondary inflorescence meristem internodes. High magnification reveals expression in 2-3 cell layers of the cortex. GFP expressed in the vasculature of silique, inner integuments, and chalazal region of ovule. Expression was highest in the Outer integuments of pre fertilized ovules decreasing to a few cells at the micropylar pole at maturity. Specific expression was in the chalazal bulb region where mineral deposits are thought to be accumulated for seed storage. GFP expressed in 2 cortical cell layers of the hypocotyl from root transition Zone to apex. At the apex, GFP is expressed at the base of the leaf primordial and cotyledon. Root expression is specific to the epidermis and cortex. T2 Mature: Same as T1 mature. T3 seedling: Same expression as in T2 seedlings. Expression is different in one seedling which has with weak root epidermal, weak hypocotyl and stronger lateral root expression. This expression is variable within siblings in this family. Expected expression pattern: Expressed in ovules and different parts of seeds Selection Criteria: Greater than 50x up in pi ovule microarray Gene: hypothetical protein T20K18.24 GenBank: NM 117358 Arabidopsis thaliana expressed protein (Atag12890) US 7,868,155 B2 135 136

TABLE 2-continued mRNA, complete cols gi30682271 refNM 117358.230682271 Source Promoter Organism: Arabidopsis thaliana WS Vector: pNewbina-HAP1-GFP Marker Type: X GFP-ER Generation Screened: XT1 Mature X T2 Seedling X T2 Mature XT3 Seedling Bidirectionality: NO Exons: NO Repeats: NO Promoter utility Trait - Sub-trait Area: Among other uses this promoter sequence could be useful to improve: Water use efficiency - Moisture stress at seed set, Moisture stress at seed fill, water use efficiency, ovule?seed abortion Seed - harvest index, test weight, seed size Yield - total yield Quality - amino acids, carbohydrate, protein, total oil, total seed composition Construct: YPO117 Promoter Candidate I.D: 11768655 (Old ID: 3513.9700) cDNA ID: 13617054 (Old IDs: 12322571, 7074452) T1 lines expressing (T2 seed): SR00694-01, -02 Promoter Expression Report # 23 Report Date: Mar. 8, 2003 Promoter Tested In: Arabidopsis thaliana, WS ecotype Spatial expression Summary: Flower (L)silique Silique (L)carpel, (L)vascular Observed expression pattern: Low levels of GFP expressed in the medial and lateral vasculature of developing pre-fertilized siliques. T2 mature: No Expression. T3 seedling: No Expression. Expected expression pattern: Expressed in ovules and different parts of seeds. Selection Criteria: Greater than 50x up in pi ovule microarray Gene: Putative vacuolar processing enzyme GenBank: NM 112912 Arabidopsis thaliana vacuolar processing enzyme asparaginyl endopeptidase - related (At3g20210) mRNA, complete cols gi30685671|refNM 112912.2|30685671) Source Promoter Organism: Arabidopsis thaliana WS Vector: pNewbina-HAP1-GFP Marker Type: X GFP-ER Generation Screened: XT1 Mature XT2 Seedling XT2 Mature XT3 Seedling Bidirectionality: NO Exons: NO Repeats: None Noted Promoter utility Trait Area: Among other uses this promoter sequence could be useful to improve: Water use efficiency - Moisture stress at seed set, Moisture stress at seed fill, water use efficiency, ovule?seed abortion Seed - harvest index, test weight, seed size Yield - total yield Quality - amino acids, carbohydrate, protein, total oil, total seed composition Construct: YPO118 Promoter Candidate I.D: 11768691 (Old ID: 35.139754) cDNA ID: 12329827 (Old ID: 4908.806) T1 lines expressing (T2 seed): SR00711-01, -02, -03 Promoter Expression Report # 24 Report Date: Mar. 9, 2003 Promoter Tested In: Arabidopsis thaliana, WS ecotype Spatial expression Summary: Flower sepal, petal, silique Silique epidermis Leaf mesophyll, vascular, epidermis, margin Hypocotyl epidermis Cotyledon mesophyll, vascular epidermis Observed expression pattern: Screened under non-induced conditions. Strong GFP expression was seen in epidermal and vasculature tissue of mature floral organs and leaves including photosynthetic cells. GFP is expressed in two cell layers of the margin and throughout mesophyll cells of mature leaf. GFP expressed in the epidermal cells of hypocotyl and cotyledons and mesophyll cells. GFP expression in the leaf is non guard cell, epidermal specific. Expected expression pattern: N induced, source tissue. Selection Criteria: arabidopsis microarray-nitrogen Gene: hypothetical protein, auxin-induced protein-like GenBank: NM 120044 Arabidopsis thaliana auxin-induced (indole-3-acetic acid induced) protein, putative (Atag38840) mRNA, complete cols gi|18420319|refNM 120044.1|18420319 Source Promoter Organism: Arabidopsis thaliana WS Vector: pNewbina-Hap1-GFP Marker Type: X GFP-ER Generation Screened: XT1 Mature X T2 Seedling XT3 Mature XT3 Seedling Bidirectionality: FAILS Exons: FAILS Repeats: None Noted Promoter utility Trait - Sub-trait Area: Among other uses this promoter sequence could be useful to improve: Source - Photosynthetic efficiency Yield - seed size US 7,868,155 B2 137 138

TABLE 2-continued Construct: YPO126 Promoter Candidate I.D: 11768662 (Old ID: 35.139721) cDNA ID: 12713856 (Old IDs: 12580379,4767659) T1 lines expressing (T2 seed): SR00715-01, -02 Promoter Expression Report # 25 Report date: Mar. 23, 2003 Promoter Tested In: Arabidopsis thaliana, WS ecotype Spatial expression Summary: Flower (H)sepal, (H)anther Silique (M)vascular Ovule Post-fertilization: (M)inner integument, (M)chalaza, (M)micropyle Stem (H)Pith Hypocotyl (H)phloem Cotyledon (M)epidermis Rosette Leaf (H)hydathode Primary Root (H)phloem, (H)pericycle Lateral root (H)phloem Observed expression pattern: Expressed in the vasculature of sepal and connective tissue of anthers in pre ertilized flowers, inner integuments restricted to micropyle region, and chalazal bulb of post-fertilized ovules. GFP expressed throughout the phloem of hypocotyl and root and in pericycle cells in root differentiation Zone. Screened under non-induced conditions. T2 mature: Same expression as observed in T1 mature. In addition, silique vascular expression was not observed in T1 mature.T3 seedling: Same expression as observed in T2 seedlings. In addition, expression was observed in cotyledon epidermal and rosette leafhydathode secretory gland cells. Expected expression pattern: nitrogen induced Selection Criteria: Arabidopsis microarray Gene: probable auxin-induced protein GenBank: NM 119918 Arabidopsis thaliana lateral organ boundaries (LOB) domain family (Atag37540) mRNA, complete cols gil 18420067 refNM 119918.1|18420067 Source Promoter Organism: Arabidopsis thaliana WS Vector: pNewBin4-HAP1-GFP Marker Type: X GFP-ER Generation Screened: XT1 Mature X T2 Seedling X T2 Mature XT3 Seedling Bidirectionality: NO Exons: NO Repeats: None Noted Promoter Utility Trait - Sub-trait Area: Among other uses this promoter sequence could be useful to improve: Source - Photosynthetic efficiency Yield - seed size Utility: Construct: YPO127 Promoter Candidate I.D: 13148.197 (Old ID: 11768663) cDNA ID: 13617784 (Old IDs: 12712729,4771741) T1 lines expressing (T2 seed): SR00716-01, -02 Promoter Expression Report # 26 Report Date: Mar. 17, 2003 Promoter Tested In: Arabidopsis thaliana, WS ecotype Spatial expression Summary: Silique (L)vascular Rosette Leaf (H)stipule Primary Root (H)trichoblast, (H)atrichoblast Cotyledon (L)hydathode Observed expression pattern: Weak expression in vasculature of pre ertilized siliques. Expressed throughout epidermal cells of seedling root. T2 mature: Expression not confirmed. T3 seedlings: Same expression as observed in T2 seedlings. In addition, expression was observed in cotyledon epidermal and hydathode secretory gland cells. Expected expression pattern: Inducible promoter - induced by different forms of stress (e.g., drought, heat, cold). Selection Criteria: Arabidopsis microarray-Nitrogen Gene: similar to SPIP30986 reticuline oxidase precursor (Berberine-bridge-forming enzyme: Tetrahydroprotoberberine synthase) contains PFO1565 FAD binding domain product = FAD-linked oxidoreductase family GenBank: NM 102808 Arabidopsis thaliana FAD-linked oxidoreductase family (Atlg30720) mRNA, complete cols gi30692034|refNM 1028.08.2| 30692034 Source Promoter Organism: Arabidopsis thaliana WS Vector: pNewBin4-HAP1-GFP Marker Type: X GFP-ER Generation Screened: XT1 Mature X T2 Seedling XT(2 Mature XT3 Seedling Bidirectionality: NO Exons: NO Repeats: NO Promoter utility Trait - Sub-trait Area: Among other uses this promoter sequence cou d be useful to improve: Water use efficiency - Heat Utility: This promoter is useful for root nutrient uptake. Construct: YPO128 Promoter Candidate I.D: 13148257 (Old ID: 11769664) cDNA ID: 13610584 (Old IDs: 12327909,4807730) T1 lines expressing (T2 seed): SR00717-01, -02 US 7,868,155 B2 139 140

TABLE 2-continued Promoter Expression Report # 27 Report Date: Mar. 23, 2003 Promoter Tested In: Arabidopsis thaliana, WS ecotype Spatial expression Summary: Flower (L)stomata Silique (M)stomata Stem (L)stomata Cotyledon (L)mesophyll, (L)vascular, (M)hydathode Rosette Leaf (H)stomata, (H)hydathode Primary Root (L) root hairs Observed expression pattern: Expression specific to upper root hairs at hypocotyl root transition Zone and hydathode secretory cells of the distal cotyledon. T1 mature: No T1 mature expression by old screening protocol T2 mature: Guard cell and Hydathode expression same as T1 mature expression (new protocol), T2 and T3 seedling expression. Expected expression pattern: Shoot and root meristem Selection Criteria: Literature. Plant Cell 1998 10 231-243 Gene: CYP90B1, Arabidopsis steroid 22-alpha-hydroxylase (DWF4) GenBank: NM 113917 Arabidopsis thaliana cytochrome p450, putative (At3.g30180) mRNA, complete cols gi30689806 refNM 113917.2|30689806) Source Promoter Organism: Arabidopsis thaliana WS Vector: pNewBin4-HAP1-GFP Marker Type: X GFP-ER Generation Screened: XT1 Mature XT2 Seedling XT2 Mature XT3 Seedling Bidirectionality: NO Exons: NO Repeats: None Noted Promoter utility Trait - Sub-trait Area: Among other uses, this promoter sequence could be useful to improve: PG&D - Plant size, growth rate Utility: Useful to increase biomass, root mass, growth rate, seed set Construct: YPOO20 Promoter Candidate I.D: 11768639 (Old ID: 11768639) cDNA ID: 12576899 (Old ID: 7104529) T1 lines expressing (T2 seed): SR00490-01, -02, -03, -04 Promoter Expression Report # 28 Report Date: Mar. 23, 2003 Promoter Tested In: Arabidopsis thaliana, WS ecotype Spatial expression Summary: Flower (L)pedicel, (M)vascular Stem (H)vascular, (H)pith Silique (H)Septum, (H)vascular Cotyledon (H)vascular, (H)epidermis Rosette Leaf (H)vascular, (H)phloem Primary Root (H)vascular: (H)phloem Lateral root (H)vascular Observed expression pattern: T1 mature (old protocol - screened target tissue): No expression observed. T2 Seedling: Strong expression throughout phloem of hypocotyl, cotyledons, primary rosette leaves and roots. Also ound in epidermal cells of upper root hairs at root transition Zone. GFP expressed in a few epidermal cells of distal cotyledon. T1 mature: (new protocol - Screened all tissues): High expression found in silique vasculature. T2 mature: Strong expression detected in inflorescence meristem and silique medial vasculature. T3 seedling: Same expression as T2 Seedlings, however no cotyledon vascular expression was detected. Expected expression pattern: Shoot and root meristem Selection Criteria: Plant Physiol. 2002 129:1241-51 Gene: brassinosteroid-regulated protein (Xyloglucan endotransglycosylase related protein GenBank: NM 117490 Arabidopsis thaliana Xyloglucan endotransglycosylase (XTR7) (Atag14130) mRNA, complete cols gi30682721 refNM 117490.2|30682721 Source Promoter Organism: Arabidopsis thaliana WS Vector: pNewBin4-HAP1-GFP Marker Type: X GFP-ER Generation Screened: XT1 Mature XT2 Seedling XT2 Mature XT3 Seedling Bidirectionality: NO Exons: NO Repeats: None Noted Promoter utility Trait Area: Among other uses this promoter sequence could be useful to improve: PG&D - Plant size, growth rate Utility: Useful to increase biomass, root mass, growth rate Construct: YPOO22 Promoter Candidate I.D: 11768614 cDNA ID: 12711515 (Old ID: 5674312) T1 lines expressing (T2 seed): SR00492-02, -03 Promoter Expression Report #29 Report Date: Mar. 23, 2003 Promoter Tested In: Arabidopsis thaliana, WS ecotype Spatial expression Summary: Flower (M)sepal, (L)stomata Silique (M)stomata US 7,868,155 B2 141 142

TABLE 2-continued Rosette Leaf (H)stomata Primary Root (H)epidermis, (H)trichoblast, (H)root hair Observed expression pattern: Strong GFP expression in stomata of primary rosette leaves and epidermal root hair trichoblast cells of seedlings. T1 mature: No expression observed. T2 seedling: Same as T2 seedling expression. T2 mature: Guard cell and weak vascular expression in flowers. Expected expression pattern: embryo Selection Criteria: Plant J 2000 21:143-55 Gene: ABI3 - interacting protein 2. AIP2 Arabidopsis thaliana GenBank: NM 122099 Arabidopsis thaliana zinc finger (C3HC4-type RING finger) protein family (At 5g20910) mRNA, complete cols gi30688046 refNM 122099.2|30688046 Source Promoter Organism: Arabidopsis thaliana, WS Vector: pNewBin4-HAP1-GFP Marker Type: X GFP-ER Generation Screened: XT1 Mature XT2 Seedling XT2 Mature XT3 Seedling Bidirectionality: NO Exons: FAILS Repeats: None Noted Promoter utility Trait - Sub-trait Area: Among other uses this promoter sequence could be useful to improve: Water use efficiency - Drought, heat Utility: This promoter might be useful for enhancing recovery after growth under water deprivation Also could be useful for nutrition uptake Construct: YPOO24 Promoter Candidate I.D: 11768616 cDNA ID: 13614559 (Old IDs: 12324998,5675795) T1 lines expressing (T2 seed): SR00494-01, -03 Promoter Expression Report # 30 Report Date: Mar. 17, 2003 Promoter Tested In: Arabidopsis thaliana, WS ecotype Spatial expression Summary: Silique (H)Ovule Ovule Pre-fertilization: (H)Outer integument, (H) funiculus Post-fertilization: (H)Outer integument, (H) funiculus Rosette Leaf (H)vascular Primary Root (H)epidermis, (H)trichoblast, (H)root hair Lateral root (H)pericycle Observed expression pattern: Strong GFP expression in upper root hairs at root transition Zone and in distal vascular bundle of cotyledon. Low expression in pericycle cells of seedling root. T1 mature: No expression observed. T3 seedling: Same as T2 seedling expression. T2 mature: GFP expression in funiculus of ovules as in connective tissue between locules of anther. Expected expression pattern: Root vasculature Selection Criteria: Helariutta, et al. 2000 Cell 101:555-567 Gene: SHR (Short-root gene) GenBank: NM 119928 Arabidopsis thaliana short-root transcription factor (SHR) (Atag37650) mRNA, complete cols gi30691 190|refNM 119928.2|30691190) Source Promoter Organism: Arabidopsis thaliana WS Vector: pNewBin4-HAP1-GFP Marker Type: X GFP-ER Generation Screened: XT1 Mature XT2 Seedling XT2 Mature XT3 Seedling Bidirectionality: NO Exons: NO Repeats: None Noted Promoter utility Trait - Sub-trait Area: Among other uses this promoter sequence could be useful to improve: Water use efficiency - Increase leaf water potential PG&D - increase root biomass, plantsize Nutrient - nitrogen use efficiency, nitrogen utilization, low nitrogen tolerance Construct: YPOO28 Promoter Candidate I.D: 11768648 cDNA ID: 1256,1142 (Old ID: 7093615) T1 lines expressing (T2 seed): SR00586-03, -04 Utility: This promoter might be a good promoter for root nutrition uptake, root biomass. Promoter Expression Report # 31 Report Date: Mar. 23, 2003 Promoter Tested In: Arabidopsis thaliana, WS ecotype Spatial expression Summary: Flower (L)stomata Primary Root (H)epidermis, (H)trichoblast, (H)atrichoblast, (H) root hairs Observed expression pattern: Strong GFP expression specific to epidermal root hair trichoblast and atrichoblast cells throughout seedling root. Not expressed in lateral root. T1 mature: No expression observed. T2 mature: Low guard cell expression in flower not observed in T1 mature. T3 seedling expression: Same as T2 seedlings. Expected expression pattern: localized to the lateral root cap, root hairs, epidermis and cortex of roots. Selection Criteria: Arabidopsis public: The roles of three functional sulfate transporters involved in uptake and translocation of sulfate in Arabidopsis thaliana. Plant J. 2000 23:171-82 Gene: Sulfate transporter GenBank: NM 116931 Arabidopsis thaliana Sulfate transporter-related (Atago8620) mRNA, complete cols gi30680813|refNM 116931.2|30680813 Source Promoter Organism: Arabidopsis thaliana WS US 7,868,155 B2 143 144

TABLE 2-continued Vector: pNewBin4-HAP1-GFP Marker Type: X GFP-ER Generation Screened: XT1 Mature XT2 Seedling XT2 Mature XT3 Seedling Bidirectionality: NO Exons: NO Repeats: None Noted Promoter utility Sub-trait Area: Among other uses this promoter sequence could be useful to improve: Water use efficiency - Water potential, drought, moisture stress at seed set and seed fill, water use efficiency Nutrient - nitrogen use efficiency Utility: This is good promoter rootnutrient uptake, increase root mass and water use efficiency Construct: YPOO3O Promoter Candidate I.D: 117686.42 cDNA ID: 12664333 (Old ID: 7079065) T1 lines expressing (T2 seed): SR00545-01, -02 Promoter Expression Report # 32 Report Date: Mar. 24, 2003 Promoter Tested In: Arabidopsis thaliana, WS ecotype Spatial expression Summary: Cotyledon (L)epidermis Primary Root (H)epidermis, (H)trichoblast, (H)atrichoblast Observed expression pattern: High GFP expression in epidermal cells of seedling root from hypocotyl root transition to differentiation Zone. Not observed in root tip. Low GFP expression in epidermal cells of distal cotyledon. T1 mature: No expression detected. T2 mature: Guard cell expression in stem, pedicles. Low silique vascular expression. T3 seedling: Same as T2 seedlings. Expected expression pattern: predominantly expressed in the phloem Selection Criteria: Ceres microarray data Gene: putative glucosyltransferase Arabidopsis thaliana GenBank: BTO10327 Arabidopsis thaliana At2g43820 mRNA, complete cols gi3394205Ogb|BTO10327.1133942050 Source Promoter Organism: Arabidopsis thaliana WS Vector: pNewBin4-HAP1-GFP Marker Type: X GFP-ER Generation Screened: XT1 Mature XT2 Seedling XT2 Mature XT3 Seedling Bidirectionality: NO Exons: NO Repeats: None Noted Promoter utility Trait - Sub-trait Area: Among other uses this promoter sequence could be useful to improve: Nutrient - nitrogen and phosphate uptake and transport Growth and Development - plant size, growth rate Utility: Promoter should be useful where expression in the root epidermis is important. Expression appears to be in expanded or differentiated epidermal cells. Construct: YPOO54 Promoter ID: 13148233 (Old ID: 11768.644) cDNA ID: 12348737 (Old ID: 1609253) T1 lines expressing (T2 seed): SR00549-01, -02 Promoter Expression Report # 34 Report Date: Jan. 31, 2003 Promoter Tested In: Arabidopsis thaliana, WS ecotype Spatial expression Summary: Flower (M)sepal, (M)style, (M)epidermis Stem (M)epidermis, (H)endodermis, (H)cortex Leaf (H)mesophyll, (H)epidermis Hypocotyl (H)epidermis, (H)vascular Cotyledon (H)epidermis, (H)mesophyll Primary Root (H)epidermis, (H)trichoblast, (H)atrichoblast, (H)vascular phloem, (H)Root cap, (H) root hairs Lateral root (H)vascular, (H)cap Observed expression pattern: GFP expressed in sepals, style of silique in immature flowers, mesophyll, and epidermis of mature leaves. GFP expressed throughout epidermal layers of seedling including root issue. Also expressed in mesophyll and epidermal tissue in distal primary leaf, and vasculature of root. Specific expression in meristematic Zone of primary and lateral root. T2 Mature: Same expression as T1 mature: Additional images taken of stem expression. T3 Seedling expression: Same as T2 seedling expression. Expected expression pattern: Shoot apical meristem Selection Criteria: Greater than 5X down in stim microarray Gene: Fructose-bisphosphate aldolase GenBank: NM 118786 Arabidopsis thaliana fructose-bisphosphate aldolase, putative (Atag26530) mRNA, complete cols gi30687252|refNM 118786.2|30687252) Source Promoter Organism: Arabidopsis thaliana WS Vector: pNewBin4-HAP1-GFP Marker Type: X GFP-ER Generation Screened: XT1 Mature X T2 Seedling X T2 Mature XT3 Seedling Bidirectionality: NO?? Exons: NO?? Repeats: None Noted Promoter Utility Trait - Sub-trait Area: Among other uses this promoter sequence could be useful to improve: PG&D - Plantsize, growth rate, plant development Water use efficiency - US 7,868,155 B2 145 146

TABLE 2-continued Utility: Construct: YPOOSO Promoter Candidate I.D: 13148170 (Old ID: 11768794) cDNA ID: 4909806 (Old IDs: 12340148, 1017738) T1 lines expressing (T2 seed): SR00543-01, -02 Promoter Expression Report # 35 Report Date: Mar. 24, 2003 Promoter Tested In: Arabidopsis thaliana, WS ecotype Spatial expression Summary: Flower (H)pedicel, (H)anther, (H)pollen, (H)vascular, (H)epidermis Stem (H)cortex, (L)vascular Hypocotyl (H)epidermis, (H)vascular, (H)phloem Cotyledon (H)vascular Primary Root (H)vascular, (H)phloem, (H)pericycle Observed expression pattern: High GFP expression throughout seedling vasculature including root. Low Expression at the base of hypocotyls. Not detected in rosette leaves. T1 mature: No expression observed. T3 Seedling: Same as T2 seedling expression. T2 mature: Strong vascular and epidermal expression in floral pedicels and in developing pollen sacs of anthers. Expected expression pattern: xylem parenchyma cells of roots and leaves and in the root pericycles and leaf phloem. Selection Criteria: Arabidopsis public: The roles of three functional sulfate transporters involved in uptake and translocation of sulfate in Arabidopsis thaliana. Plant J. 2000 23:171-82 Gene: Sulfate transport GenBank: NM 121056 Arabidopsis thaliana Sulfate transporter (AtSg10180) mRNA, complete cols gi30683.048 refNM 121056.2|30683.048) Source Promoter Organism: Arabidopsis thaliana WS Vector: pNewBin4-HAP1-GFP Marker Type: X GFP-ER Generation Screened: XT1 Mature X T2 Seedling XT2 Mature XT3 Seedling Bidirectionality: NO Exons: NO Repeats: None Noted Promoter utility Trait Area: Among other uses this promoter sequence could be useful to improve: Water use efficiency - Nutrient - nitrogen use, Nutrient efficiency Plant Growth and Development - growth rate Utility: Useful for root nutrient uptake and metabolism manipulation Construct: YPOO40 Promoter Candidate I.D: 11768694 cDNA ID: 12670159 (Old ID: 11020088) T1 lines expressing (T2 seed): SR00588-01, -02, -03 Promoter Expression Report # 37 Report Date: Jan. 31, 2003 Promoter Tested In: Arabidopsis thaliana, WS ecotype Spatial expression Summary: Flower (L)pedicel, (L)stomata Stem (L)stomata Leaf (L)vascular, (L)stomata Cotyledon (H)mesophyll, (H)vascular, (H)epidermis Primary Root (H) root hairs Observed expression pattern: Low GFP expression in stomatal cells of stem, pedicels, and vasculature of leaves in mature plants. High GFP expression in root hairs, epidermis and mesophyll cells of seedling cotyledon. Not seen in rosette leaves. T2 mature: Same as T1 mature expression. T3 seedling: Same as T2 seedling expression. Expected expression pattern: Constitutively expressed in all green tissues Selection Criteria: Arabidopsis microarray Gene: Expressed protein Arabidopsis thaliana GenBank: NM 119524 Arabidopsis thaliana expressed protein (Atag33666) mRNA, complete cols gi30689773|refNM 119524.2|30689773) Source Promoter Organism: Arabidopsis thaliana WS Vector: pNewBin4-HAP1-GFP Marker Type: X GFP-ER Generation Screened: XT1 Mature X T2 Seedling XT2 Mature XT3 Seedling Bidirectionality: Exons: Repeats: Promoter utility Trait Area: Among other uses this promoter sequence could be useful to improve: Sub-trait Area: Plant size, growth rate, stay green, Utility: Useful for C/N partitioning, photosynthetic efficiency, source enhancement and seedling establishment Construct: YPOO56 Promoter Candidate I.D: 1176864.5 cDNA ID: 12396394 (Old ID: 7083850) T1 lines expressing (T2 seed): SR00550-01 US 7,868,155 B2 147 148

TABLE 2-continued Promoter Expression Report # 38 Report Date: Mar. 24, 2003 Promoter Tested In: Arabidopsis thaliana, WS ecotype Spatial expression Summary: Primary root (H) root hairs Observed expression pattern: GFP expression specific to epidermal root hairs at hypocotyl root transition Zone. This line was not screened in T2 mature and T3 seedlings. Expected expression pattern: Shoot apical meristem Selection Criteria: Greater than 5X down in stim microarray Gene: hypothetical protein GenBank: NM 118575 Arabidopsis thaliana RNA recognition motif (RRM)-containing protein (Atag24420) mRNA, complete cols gi|18416342|refNM 118575..1||18416342 Source Promoter Organism: Arabidopsis thaliana WS Vector: pNewBin4-HAP1-GFP Marker Type: X GFP-ER Generation Screened: XT1 Mature X T2 Seedling T2 Mature T3 Seedling Bidirectionality: Exons: Fail Repeats: Promoter utility Trait Area: Among other uses this promoter sequence could be useful to improve: Water use efficiency: Nutrient Sub-trait Area: Plant size, growth rate, drought, water use efficiency, nitrogen utilization Utility: early establishment of Rhizobium infection by increasing expression of elicitors Construct: YPOO68 Promoter Candidate I.D: 11768798 cDNA ID: 12678173 (Old ID: 1022896) T1 lines expressing (T2 seed): SR00598-01, -02 Promoter Expression Report # 39 Report Date: Mar. 24, 2003 Promoter Tested In: Arabidopsis thaliana, WS ecotype Spatial expression Summary: Primary root (H) root hairs Observed expression pattern: High GFP expression specific to epidermal root hair at hypocotyls root transition Zone. Screened under non-induced condition. T1 mature: No expression detected. T2 mature: No expression detected. T3 seedling: Same expression as T2 seedlings. GFP specific to root hairs. Expected expression pattern: Heat inducible. Selection Criteria: Expression data (full chip) >30 fold induction at 42 C. at 1 h and 6 Gene: LMW heat shock protein - mitochondrial GenBank: NM 118652 Arabidopsis thaliana mitochondrion-localized Small heat shock protein (Atag25200) mRNA, complete cols gi30686795 refNM 118652.2|30686795) Source Promoter Organism: Arabidopsis thaliana WS Vector: pNewBin4-HAP1-GFP Marker Type: X GFP-ER Generation Screened: XT1 Mature X T2 Seedling X T2 Mature XT3 Seedling Bidirectionality: NO Exons: NO Repeats: NO Promoter utility Trait Area: Among other uses this promoter sequence could be useful to improve: Water use efficiency: Nutrient Sub-trait Area: Increase plant growth or seed yield under heat stress conditions, nitrogen utilization, low N tolerance Utility: Useful for root nutrient uptake Construct: YPOO82 Promoter Candidate I.D: 13148250 (Old ID: 11768604) cDNA ID: 13609100 (Old IDs: 12678209,6462494) T1 lines expressing (T2 seed): SR00606-01, -02, -03 Promoter Expression Report # 40 Report Date: Mar. 24, 2003 Promoter Tested In: Arabidopsis thaliana, WS ecotype Spatial expression Summary: Hypocotyl (H)epidermis Primary Root (H)epidermis, (H)trichoblast, (H)root hairs Observed expression pattern: High GFP expression throughout epidermal layer of hypocotyl and upper root including root hairs. Not detected in lower root. No expression observed in T1 mature plants. T2 mature: No expression observed. T3 seedling: Same expression as T2 seedlings. Expected expression pattern: Root Selection Criteria: Genome annotation Gene: ABI3-interacting protein 2 homolog (but recent annotation changed as hypothetical protein and promoter position is opposite orientation in the hypothetical protein, see map below); unknown protein GenBank: NM 101286 Arabidopsis thaliana zinc finger (C3HC4-type RING finger) protein family (Atlg14200) mRNA, complete cols gi30683647 refNM 101286.2|30683647 Source Promoter Organism: Arabidopsis thaliana WS Vector: pNewBin4-HAP1-GFP Marker Type: X GFP-ER US 7,868,155 B2 149 150

TABLE 2-continued Generation Screened: XT1 Mature X T2 Seedling X T2 Mature XT3 Seedling Bidirectionality: Fail Exons: Fail Repeats: NO Promoter utility Trait Area: Among other uses this promoter sequence could be useful to improve: Sub-trait Area: Nitrogen utilization; plant size, growth rate Utility: Useful for nutrient uptake e.g., root hairs root epidermis Construct: YPOO19 Promoter Candidate I.D: 117686.13 cDNA ID: 4909291 T1 lines expressing (T2 seed): SR00489-01, -02 Promoter Expression Report # 42 Report Date: Mar. 22, 2003 Promoter Tested In: Arabidopsis thaliana, WS ecotype Spatial expression Summary: Flower (L) receptacle, (L)vascular Silique (L)vascular Stem (L)vascular, (L)phloem Primary root: (H)phloem Observed expression pattern: High GFP expression specific to the seedling root phloem tissue. T1 mature: No expression was observed. T2 mature: Low expression in flower and stem vascular tissues was not observed in T1 mature. T3 seedlings: Same vascular expression exists as T2 seedlings. Expected expression pattern: Constitutive in all green tissues Selection Criteria: cDNA cluster Gene: 40S ribosomal protein S5 GenBank: NM 129283 Arabidopsis thaliana 40S ribosomal protein S5 (RPS5A) (At2g37270) mRNA, complete cols gi30687090|refNM 129283.2|30687090) Source Promoter Organism: Arabidopsis thaliana WS Vector: pNewBin4-HAP1-GFP Marker Type: X GFP-ER Generation Screened: XT1 Mature X T2 Seedling X T2 Mature XT3 Seedling Bidirectionality: NO Exons: NO Repeats: NO Promoter utility Trait Area: Among other uses this promoter sequence could be useful to improve: PG&D, Nutrient economy Sub-trait Area: Plant size, growth rate, low nitrogen tolerance, NUE Utility: Useful for root nutrient uptake, sourcelsink relationships, root growth Construct: YPOO87 Promoter Candidate I.D: 12748731 cDNA ID: 13580795 (Old IDs: 11006078, 1258.1302) T1 lines expressing (T2 seed): SR00583-01, -02 Promoter Expression Report # 43 Report Date: Mar. 25, 2003 Promoter Tested In: Arabidopsis thaliana, WS ecotype Spatial expression Summary: Screened under non-induced conditions Flower (H)petal, (H)epidermis, (H)anther Stem (H)epidermis Cotyledon (H)epidermis Hypocotyl (L)epidermis, (L)stomata Rosette Leaf (L)petiole, (L)stomata Primary Root (H)phloem, (H)vascular Observed expression pattern: T1 mature: High GFP expression in petals of developing to mature flowers and in and pollen nutritive lipid rich ameboid tapetum cells in developing anthers. T2 seedling: High GFP expression in rootphloem with weak expression in epidermal tissues of seedlings. T2 mature: Same as T1 mature with additional stem epidermal expression was not observed in T1 mature plants. T3 seedling: Same as T2 seedling, however, no expression was seen in epidermal cells of hypocotyls as in T2 seedlings. Expected expression pattern:: Inducible promoter - was induced by different forms of stress (e.g., drought, heat, cold) Selection Criteria Arabidopsis microarray Gene: Putative strictosidine synthase GenBank: NM 147884 Arabidopsis thaliana strictosidine synthase family (At 5g22020) mRNA, complete cols gi30688266 refNM 147884.2|30688266 Source Promoter Organism: Arabidopsis thaliana WS Vector: pNewBin4-HAP1-GFP Marker Type: X GFP-ER Generation Screened: XT1 Mature X T2 Seedling XT2 Mature XT3 Seedling Bidirectionality: NO Exons: FAILS Repeats: NO Promoter utility Trait Area: PD&G, Nutrient, seed, water use efficiency Sub-trait Area: Nutrient uptake, CN partitioning, Source enhancement, Source? sink Utility: Useful for nutrient uptake and transport in root, transport or mobilization of steroid reserves Construct: YPO18O Promoter Candidate I.D: 11768712 cDNA ID: 5787483 (Old IDs: 2918666, 12367001) T1 lines expressing (T2 seed): SR00902-01, -02, -03 US 7,868,155 B2 151 152

TABLE 2-continued Promoter Expression Report # 44 Report Date: Mar. 22, 2003 Promoter Tested In: Arabidopsis thaliana, WS ecotype Spatial expression Summary: Hypocotyl (L)epidermis Observed expression pattern: Low GFP expression in the epidermal cells of hypocotyl. Screened under non induced conditions. No T1 mature expression was observed. T2 mature: No expression was observed. T3 Seedling: Same expression as the T2 seedling seen in one of two events. Guard cell expression was observed in second event. Expected expression pattern: Induced by different forms of stress (e.g., drought, heat, cold). Selection Criteria: Arabidopsis microarray. Induced by different forms of stress (e.g., drought, heat, cold) Gene: Berberine bridge enzyme GenBank: NM 100078 Arabidopsis thaliana FAD-linked oxidoreductase family (Atlgo 1980) mRNA, complete cols gi|18378.905 refNM 100078.1||18378.905 Source Promoter Organism: Arabidopsis thaliana WS Vector: pNewBin4-HAP1-GFP Marker Type: X GFP-ER Generation Screened: XT1 Mature X T2 Seedling X T2 Mature XT3 Seedling Bidirectionality: NO Exons: NO Repeats: NO Promoter utility Trait Area: Among other uses this promoter sequence could be useful to improve: Water use efficiency; PG&D Sub-trait Area: Heat Utility: Seedling establishment, Construct: YPO186 Promoter Candidate I.D: 11768854 cDNA ID: 13647840 (Old IDs: 12689527, 11437778) T1 lines expressing (T2 seed): SR00906-02, -03 Promoter Expression Report # 45 Report Date: Mar. 25, 2003 Promoter Tested In: Arabidopsis thaliana, WS ecotype Spatial expression Summary: Ovule Pre-fertilization: (H)inner integument Post-fertilization: (H)inner integument, (H)Outer integument Observed expression pattern: High GFP expression specific to the inner integuments of developing pre ertilized ovules and Outer integuments at the mycropylar end of post fertilized ovules. GFP detected throughout inner integument of developing seed at mature embryo stage. T2 seedling: No expression observed. T2 Mature: Same expression as observed in T1 mature. T3 seedling: Not screened. Expected expression pattern: Expressed in ovules and different parts of seeds Selection Criteria: Greater than 50x up in pi ovule microarray Gene: pectin methylesterase Arabidopsis thaliana. GenBank: NM 124295 Arabidopsis thaliana pectinesterase family (AtSg49180) mRNA, complete cols gi30695612|refNM 1242.95.2|30695612) Source Promoter Organism: Arabidopsis thaliana WS Vector: pNewBin4-HAP1-GFP Marker Type: X GFP-ER Generation Screened: XT1 Mature X T2 Seedling XT2 Mature T3 Seedling Bidirectionality: NO Exons: FAILS Repeats: NO Promoter utility Trait Area: Seed, Yield, Nutrient, cold, water use efficiency Sub-trait Area: Ovuleseed abortion, seed enhamcement, seed number, seed size, total yield, seed nitrogen, cold germination and vigor Utility: Useful for improvement for seed yield, composition, moisture stress at seed set, moisture stress during seed fill Construct: YPO121 Promoter Candidate I.D: 11768686 cDNA ID: 12646933 (Old IDs: 12370661, 7080188) T1 lines expressing (T2 seed): SR00805-01, -02, -03 Promoter Expression Report # 46 Report Date: Mar. 25, 2003 Promoter Tested In: Arabidopsis thaliana, WS ecotype Spatial expression Summary: Silique (H)Ovule Ovule Pre-fertilization: (H)embryo sac, (H)gametophyte Post-fertilization: (H)Zygote Observed expression pattern: GFP expression is specific to female gametophyte and Surrounding sporophytic tissue of pre-fertilized ovules and Zygote of fertilized ovule 0-5 hours after fertilization (HAF). Not detected in developing embryos. T2 mature: Did not germinate. T3 seedlings: No seeds available. Expected expression pattern: Expressed in ovules and different parts of seeds Selection Criteria: Greater than 50x up in pi ovule microarray Gene: hypothetical protein GenBank: NM 123661 Arabidopsis thaliana expressed protein (AtSg42955) mRNA, complete cols gi|18422274|refNM 123661.1||18422274) Source Promoter Organism: Arabidopsis thaliana WS Vector: pNewBin4-HAP1-GFP Marker Type: X GFP-ER US 7,868,155 B2 153 154

TABLE 2-continued Generation Screened: XT1 Mature X T2 Seedling T2 Mature T3 Seedling Bidirectionality: NO Exons: NO Repeats: NO Promoter utility Trait Area: Among other uses this promoter sequence could be useful to improve: Seed, yield, quality Sub-trait Area: Ovule?seed abortion, harvest index, test weight, seed size, total yield, amino acid, protein, total oil, total seed composition Utility: This is promoter is useful for enhance of seed composition, seed size, seed number and yield, etc. Construct: YPO096 Promoter Candidate I.D: 13148242 (Old ID: 11768682) cDNA ID: 4949423 (Old IDs: 12325608, 1007532) T1 lines expressing (T2 seed): SR00775-01, -02 Promoter Expression Report # 47 Report Date: Mar. 25, 2003 Promoter Tested In: Arabidopsis thaliana, WS ecotype Spatial expression Summary: Flower (H)pedicel, (H)stomata Silique (M)stomata Stem (M)stomata Rosette Leaf (L)stomata Primary Root (H) root hairs Observed expression pattern: Guard cell expression throughout stem, pedicels, and siliques. High GFP preferential expression to root hairs of seedlings and medium to low expression in primary rosette leaves and petioles and stems. T2 mature: Same expression as T1 mature. T3 seedlings: Same expression as T2 seedlings. Expected expression pattern: Expressed in ovules and different parts of seeds Selection Criteria: Greater than 50x up in pi ovule microarray Gene: hypothetical protein GenBank: NM 122878 Arabidopsis thaliana expressed protein (AtSg34885) mRNA, complete cols gi30692647 refNM 122878.2|30692647) Source Promoter Organism: Arabidopsis thaliana WS Vector: pNewBin4-HAP1-GFP Marker Type: X GFP-ER Generation Screened: XT1 Mature X T2 Seedling XT2 Mature XT3 Seedling Bidirectionality: NO Exons: NO Repeats: NO Promoter utility Trait Area: Among other uses this promoter sequence could be useful to improve: Water use efficiency, PG&D, nutrient Sub-trait Area: Drought, heat, water use efficiency, plantsize, low nitrogen utilization Utility: Useful for root nutrient uptake, plant growth under drought, heat Construct: YPOO98 Promoter Candidate I.D: 127584.79 cDNA ID: 4906343 (Old IDs: 12662283, 1024001) T1 lines expressing (T2 seed): SR00896-01, -02 Promoter Expression Report # 48 Report Date: Mar. 25, 2003 Promoter Tested In: Arabidopsis thaliana, WS ecotype Spatial expression Summary: Flower (H)pedicel, (H)sepal, (H)vascular Silique (H)Septum, (H)vascular Stem (H)vascular Leaf (H)petiole, (H)vascular, (H)phloem Hypocotyl (H)vascular Primary Root (H)vascular, (H)phloem Observed expression pattern: High GFP expression throughout mature and seedling vascular tissue. T2 mature and T3 seedling: Not screened. Expected expression pattern: Expressed in ovules and different parts of seeds Selection Criteria: Greater than 50x up in pi ovule microarray Gene: unknown protein; expressed protein GenBank: NM 129068 Arabidopsis thaliana expressed protein (At2g35150) mRNA, complete cols gi30686319|refNM 129068.230686319) Source Promoter Organism: Arabidopsis thaliana WS Vector: pNewBin4-HAP1-GFP Marker Type: X GFP-ER Generation Screened: XT1 Mature X T2 Seedling T2 Mature T3 Seedling Bidirectionality: NO Exons: FAILS Repeats: NO Promoter utility Trait Area: Among other uses this promoter sequence could be useful to improve: PG&D, nutrient, seed Sub-trait Area: Growth rate, plant size, low nitrogen use efficiency, nitrogen utilization, seed size and yield Utility: Useful for root nutrient uptake and transport, enhance plant growth rate under low nitrogen condition. Enhance plant to use water efficiently. Might be also useful for seed program. Sourcelsink Construct: YPO108 Promoter Candidate I.D: 11768683 US 7,868,155 B2 155 156

TABLE 2-continued cDNA ID: 13601936 (Old IDs: 12339941,4768.517) T1 lines expressing (T2 seed): SR00778-01, -02 Promoter Expression Report # 49 Report Date: Mar. 25, 2003 Promoter Tested In: Arabidopsis thaliana, WS ecotype Spatial expression Summary: Screened under non-induced conditions. Flower (H)septum, (H)epidermis Silique (L)carpel, (H)Septum, (H)epidermis, (M)vascular Stem (M)epidermis Hypocotyl (L)epidermis, (L)stomata Cotyledon (L)epidermis, (L)guard cell Primary Root (H)epidermis, (H)trichoblast, (H)atrichoblast, (H) root hairs Observed expression pattern: High preferential GFP expression in septum epidermal cells in siliques and root hair cells of seedlings. Low expression in cotyledon and hypocotyl epidermal cells. T2 mature: Stem epidermal and silique vascular expression observed in addition to expression observed in T1 mature. Expression in stem epidermal cells appears variable. T3 seedling: Same expression as T2 seedlings with additional guard cell expression in siliques. Expected expression pattern: Root Selection Criteria: Greater than 10x induced by Roundup. Induced in Arabidopsis microarray at 4 hours Gene: Hypothetical protein GenBank: NM 111930 Arabidopsis thaliana expressed protein (At3g10930) mRNA, complete cols gi3068.1550 refNM 111930.2|3068.1550) Source Promoter Organism: Arabidopsis thaliana WS Vector: pNewBin4-HAP1-GFP Marker Type: X GFP-ER Generation Screened: XT1 Mature X T2 Seedling XT2 Mature XT3 Seedling Bidirectionality: NO Exons: NO Repeats: NO Promoter utility Trait Area: Among other uses this promoter sequence could be useful to improve: Water use efficiency, PG&D, nutrient, yield Sub-trait Area: Drought, growth rate, plant size, low nitrogen use efficiency, nitrogen utilization; seed yield Utility: Useful for root nutrient uptake, enhance plant growth rate under low nitrogen condition. Enhance plant to use water efficiency, useful for pod shatter Construct: YPO134 Promoter Candidate I.D: 11768684 cDNA ID: 13489977 (Old IDs: 12332605, 6403797) T1 lines expressing (T2 seed): SR00780-02, -03 Promoter Expression Report # 50 Report Date: Mar. 25, 2003 Promoter Tested In: Arabidopsis thaliana, WS ecotype Spatial expression Summary: Screened under non-induced conditions Flower (H)pedicel, (L)petal, (H)silique Silique (H)carpel, (H)cortex, (H)epidermis Ovule Post-fertilization: (L)outer integument Embryo (L)mature Stem (M)epidermis, (H)cortex, (H)endodermis Leaf (H)petiole, (H)mesophyll, (H)epidermis Cotyledon (H)mesophyll, (H)epidermis Rosette Leaf (H) mesophyll, (L)vascular, (H)epidermis Primary Root (H)cortex Lateral root (H)cortex, (H) flanking cells Observed expression pattern: High preferential GFP expression in photosynthetic, cortical and epidermal tissues in mature plants and seedlings. T2 mature: Weak outer integument expression in mature ovules and mature embryo in addition to expression observed in T1 mature plants. T3 seedling: Same expression observed as T2 Seedlings (seen in one event). Weak epidermal and high lateral root flanking cell expression observed in second event. Expected expression pattern: Root hairs Selection Criteria: Ceres Microarray 2.5-5X down in rhl (root hair less) mutant Gene: probable auxin-induced protein GenBank: NM 119642 Arabidopsis thaliana auxin-induced (indole-3-acetic acid induced) protein family (Atag34760) mRNA, complete cols gi30690121 refNM 119642.2|30690121 Source Promoter Organism: Arabidopsis thaliana WS Vector: pNewBin4-HAP1-GFP Marker Type: X GFP-ER Generation Screened: XT1 Mature X T2 Seedling XT2 Mature XT3 Seedling Bidirectionality: NO Exons: NO Repeats: NO Promoter utility Trait Area: Among other uses this promoter sequence could be useful to improve: PG&D, Nutrient; C3-C4 optimization Sub-trait Area: Low nitrogen use efficiency, nitrogen utilization, low nitrogen tolerance, plant size, growth rate, water use efficiency; manipulate expression of C3-C4 enzymes in leaves Utility: Useful for root nutrient uptake and transport, enhance plant growth rate, also for enhance of plant water use efficiency Construct: YPO138 Promoter Candidate I.D: 13148247 (Old ID: 11768685) US 7,868,155 B2 157 158

TABLE 2-continued cDNA ID: 12333534 (Old ID: 7077536) T1 lines expressing (T2 seed): SR00781-01, -02, -03 Promoter Expression Report # 52 Report Date: Mar. 25, 2003 Promoter Tested In: Arabidopsis thaliana, WS ecotype Spatial expression Summary: Flower (L)sepal, (L)vascular Rosette Leaf (L)vascular, (L)stomata Observed expression pattern: Weak GFP expression in sepal vasculature of developing flower buds. Weak expression in vasculature and guard cells of rosette leaves. Not detected in mature flowers. T2 mature: Same expression as T1 mature detected in one of two events. Vascular expression in pedicels of developing flowers. T3 Seedlings: No expression detected. Expected expression pattern: Shoot apex including leaf primordia and parts of leaves Selection Criteria: Greater than 5x up in stim microarray Gene: unknown protein GenBank: NM 122151 Arabidopsis thaliana esterase/lipase? thioesterase family (At 5g22460) mRNA, complete cols gi30688485 refNM 122151.2|30688485 Source Promoter Organism: Arabidopsis thaliana WS Vector: pNewBin4-HAP1-GFP Marker Type: X GFP-ER Generation Screened: XT1 Mature X T2 Seedling XT2 Mature XT3 Seedling Bidirectionality: NO Exons: FAILS Repeats: NO Promoter utility Trait Area: Among other uses this promoter sequence could be useful to improve: Water use efficiency Sub-trait Area: Water use efficiency Construct: YPO192 Promoter Candidate ID: 11768715 cDNA ID: 12688453 (Old IDs: 12384618, 3434328) T1 lines expressing (T2 seed): SR00908-01, -02 Utility: This is weak promoter expressed in guard cell and flower. Might be useful for water use efficiency Promoter Expression Report #53 Report Date: Mar. 25, 2003 Promoter Tested In: Arabidopsis thaliana, WS ecotype Spatial expression Summary: Flower (H)pedicel, (H)vascular Primary Root (H)epidermis, (H)trichoblast, (H)atrichoblast, (L) root hair Observed expression pattern: High GFP expression specific in floral pedicel vascular tissue of developing flowers. Not detected in pedicels and stems of mature plants. High GFP expression throughout epidermal layers of primary seedling root. T2 mature: No expression in 3 plants observed. T3 seedling: Same as T2 seedling expression. Expected expression pattern: Inducible promoter - induced by different forms of stress (e.g., drought, heat, cold). Selection Criteria: Arabidopsis microarray Gene: Reticuline oxidase; berberine bridge enzyme GenBank: NM 102806 Arabidopsis thaliana FAD-linked oxidoreductase family (Atlg30700) mRNA, complete cols gi30692021|refNM 1028.06.2|30692021) Source Promoter Organism: Arabidopsis thaliana WS Vector: pNewBin4-HAP1-GFP Marker Type: X GFP-ER Generation Screened: XT1 Mature X T2 Seedling XT2 Mature XT3 Seedling Bidirectionality: NO Exons: NO Repeats: NO Promoter utility Trait Area: PG&D, Nutrient. Seed development, yield Sub-trait Area: Plant size, growth rate, nitrogen use efficiency and utilization Utility: Very useful for rootnutrient uptake, enhancement for plant growth under low nitrogen condition Construct: YPO204 Promoter Candidate I.D: 11768721 cDNA ID: 1266.9615 (Old ID: 7089815) T1 lines expressing (T2 seed): SR00914-02, -03, -04 Promoter Expression Report # 54 Report Date: Mar. 31, 2003 Promoter Tested In: I. Arabidopsis thaliana, WS ecotype II. Oryza sativa III. Lycopersicon escientiin. Spatial expression Summary: . Arabidopsis thaliana Flower (H)pedicel, (H)receptacle, (H)nectary, (H)sepal, (H)petal, (H)filament, (H)anther, (H)carpel, (H)style, (H)stigma, (H)epidermis Silique (H)stigma, (H)style, (H)carpel, (H)Septum, (H)placentae, (H)epidermis, (H)Ovule Ovule Pre-fertilization: (H)inner integument, (H)Outer integument, (H)embryo sac, (H) funiculus, (H)chalaza, (H)micropyle Post-fertilization: (H)inner integument, (H)Outer integument, (H)Seed coat, (H)chalaza, (H)micropyle, (H)embryo Embryo (H)late, (H)mature US 7,868,155 B2 159 160

TABLE 2-continued Stem (H)epidermis, (H)cortex, (H)vascular Leaf (H)petiole, (H)mesophyll, (H)epidermis Hypocotyl (M)epidermis Cotyledon (H)mesophyll, (H)epidermis Primary Root (H)epidermis, (H)atrichoblas, (H)vascular, (H)cap Lateral root (H)epidermis, (H)initials, (H)cap I. Oryza sativa Leafsheath epidermis, vascular, cortex Leaf mesophyll, vascular Lateral root initials, cap Primary root cap Embryo 5 day II. Lycopersicon escientiin Leaf mesophyll Flower ovules, stamen, pollen Root epidermis Fruit peel tissue Observed expression patterns: T2 mature and T2 seedling: Expressed throughout mature and seedling issues. High expression in L1, L2, and L3 layers of shoot apical meristem. Expected expression pattern: Constitutive Selection Criteria: cDNA cluster Gene: Arabidopsis Elongation Factor 1-C. GenBank: NM 125432 Arabidopsis thaliana elongation factor 1-alpha (EF-1-alpha) (AtSg60390) mRNA, complete cols gi30697365 refNM 125432.2|30697365) Source Promoter Organism: Arabidopsis thaliana WS Vector: CRS-BIN2A2 Marker Type: Histone-YFP Generation Screened: I. Arabidopsis thaliana T1 Mature XT2 Seedling XT2 Mature T3 Seedling II. Oryza sativa XT1 Mature T2 Seedling T2 Mature T3 Seedling III. Lycopersicon esculentum XT1 Mature T2 Seedling T2 Mature T3 Seedling Criteria: Bidirectionality: NO Exons: NO Repeats: NO Trait Area: Among other uses, this promoter sequence could be useful to improve: Water use efficiency, PG&D, Seed, Nutrient, Yield Construct: BIN2A2,28716-HY2 Promoter Candidate I.D: 12786308 cDNA ID: 12739224 (Old ID: 12731344) Promoter Expression Report # 55 Report Date: Mar. 23, 2003 Promoter Tested In: I. Arabidopsis thaliana, WS ecotype II. Oryza sativa Spatial expression Summary: I. Arabidopsis thaliana, WS ecotype Flower (H)pedicel, (H)receptacle, (H)nectary, (H)sepal, (H)petal, (H)filament, (H)anther, (H)pollen, (H)carpel, (H)style, (H)papillae, (H)epidermis, (H)SAM Silique (H)stigma, (H)style, (H)carpel, (H)Septum, (H)placentae, (H)transmitting (H)tissue, (H)epidermis, (H)Ovule Ovule Pre-fertilization: (H)inner integument, (H)Outer integument, (H)embryo sac, (H) funiculus, (H)chalaza, (H) micropyle Post-fertilization: (H)Zygote, (H)inner integument, (H)Outer integument, (H)seed coat, (H)chalaza, (H)micropyle, (H)early endosperm, (H)mature endosperm, (H)embryo Embryo (H)Suspensor, (HOpreglobular, (H)globular, (H)heart, (H) torpedo, (H)late, (H)mature, (H)hypophysis, (H)radicle, (H)cotyledons, (H)hypocotyl Stem (H)epidermis, (H)cortex, (H)vascular, (H)pith Leaf (H)petiole, (H)mesophyll, (H)epidermis Hypocotyl (L)epidermis, (L)cortex, (L)vascular Rosette Leaf (H)mesophyll, (H)epidermis, (H)petiole Primary Root (H)epidermis, (H)trichoblast, (H)atrichoblast, (H)cortex, (H)cap, (H) root hairs Lateral Root (H)epidermis, (H)initials, (H)cap II. Oryza sativa Flower Pollen Leafsheath Observed expression patterns: Constitutive. Expression observed throughout mature and seedling plants. Expected expression pattern: Constitutive Selection Criteria: cDNA cluster Gene: Arabidopsis ADP-Ribosylation Factor 1 GenBank: NM 130285 Arabidopsis thaliana ADP-ribosylation factor 1 (ARF1) (At2g47170) mRNA, complete cols gi|18407284|refNM 130285.1||18407284) Source Promoter Organism: Arabidopsis thaliana WS Vector: CRS-Bin1A1 Marker Type: XHistone-YFP Generation Screened: I. Arabidopsis thaliana T1 Mature XT2 Seedling XT2 Mature T3 Seedling II. Oryza sativa XT1 Mature T2 Seedling T2 Mature T3 Seedling US 7,868,155 B2 161 162

TABLE 2-continued Bidirectionality: NO Exons: NO Repeats: NO Promoter utility Trait Area: Among other uses, this promoter sequence could be useful to improve: Water use efficiency, PG&D, Seed, Nutrient, Yield Construct: BINA1-34414-HY2 Promoter Candidate I.D: 12786307 cDNA ID: 13609583 (Old ID: 12394813) Promoter Expression Report #56 Report Date: Mar. 23, 2003 Promoter Tested In: I. Arabidopsis thaliana, WS ecotype II. Oryza sativa Spatial expression Summary: . Arabidopsis thaliana Flower (H)pedicel, (H)receptacle, (H)nectary, (H)sepal, (H)anther, (H)phloem, (H)cap, (H) root hairs, (H)pollen, (H)carpel, (H)style, (H)epidermis Silique (H)style, (H)carpel, (H)septum, (H)placentae, (H)vascular, (H)epidermis, (H)Ovule Ovule Pre-fertilization: (H)Outer integument, (H) funiculus Post-fertilization: (H)Outer integument, (H)seed coat Stem (H)epidermis, (H)cortex, (H)vascular, (H)xylem, (H)phloem, (H)pith Leaf (M)mesophyll, (H)vascular Hypocotyl (H)epidermis, (H)vascular Cotyledon (H)mesophyll, (H)epidermis Primary Root (H)epidermis, (H)trichoblast, (H)atrichoblast, (H)vascular, (H)xylem, (H)phloem, (H)cap, (H)root hairs I. Oryza sativa Flower (L)vascular Sheath (H)all cells Leaf tip (H)all cells Leaflower blade (H)vascular Root (M) vascular, (L)epidermis Lateral root (H)epidermis Ovule (H)all structures mmature seed (M)connective tissue Observed expression patterns: . Arabidopsis thaliana: Expressed throughout most mature tissues screened. Not detected in shoot apical meristem and stage 1 and 2 flower buds. Not detected in stamen and siliques of stage 4 flowers. Not detected in the Stigma, which has abnormal development. Aborted embryos. Not detected in developing embryos. High Expression in epidermal, vascular and photosynthetic tissue of seedling. Lines characterized have gone through several generations. Not Screened in Successive generation. II. Oryza Saiva: High expression throughout leaf sheath, leaf, root, lateral root tip, anther filament, ovule, stem and connection point between seed and pedicel. Not detectable in developing seeds. Not expressed in organs of developing flowers. Expected expression pattern: Constitutive expression Selection Criteria: From Ceres, Inc. and Stanford microarray data. Selected for constitutive expression. Gene: S-Adenosylmethionine Synthetase 2 GenBank: NM 112618 Arabidopsis thaliana S-adenosylmethionine synthetase - related (At3g17390) mRNA, complete cols gi30684.501 refNM 112618.2|30684.501) Source Promoter Organism: Arabidopsis thaliana WS Vector: . Arabidopsis- CRS-HT1 (Construct: CR13-GFP-ER) I. Oryza sativa- CRS-HT1 (Construct: CR 13-GFP-ER), CRS-BIN1A (Construct: CR14-hYFP) Marker Type: . Arabidopsis- GFP-ER I. Oryza sativa- GFP-ER, hYFP Generation Screened: . Arabidopsis- T1 Mature X T2 Seedling XT2 Mature T3 Seedling I. Oryza sativa- XT1 Mature X T2 Seedling T2 Mature T3 Seedling Bidirectionality: FAILS'?? Exons: FAILS'?? Repeats: NO Promoter utility Trait Area: Among other uses this, promoter sequence could be useful to improve: Water use efficiency, PG&D, seeds; nutrients Sub-trait Area: Drought, water use efficiency, growth rate, plant size, low nitrogen olerance, nitrogen use efficiency, seed enhancement Utility: Useful for root nutrient uptake and transport, water use efficiency, and improvement of seed size, yield, etc. Construct: CR13 (GFP-ER) CR14 (H-YFP) Promoter ID: 12786.306 cDNA ID: 13614841 (Old ID: 12331556) Promoter Expression Report # 98 Report Date: Dec. 3, 2003 Promoter Tested In: Arabidopsis thaliana, WS ecotype Spatial expression Summary: Flower H pedicel, H receptacle, H sepal, Hepidermis, Hendodermis Silique H placenta Stem Hendodermis Leaf Hendodermis US 7,868,155 B2 163 164

TABLE 2-continued Hypocotyl M epidermis, L vascular Cotyledon L vascular Rosette Leaf H vascular, H epidermis, Hmidrib Primary Root H pericycle, Hendodermis, L root hairs Lateral root H initials Observed expression pattern: T1 mature: Strong GFP expression in rib vein support tissue in flowers, leaves and endodermis of stems. Appears not to be expressed within vascular tissue. T2 seedling: Expressed throughout epidermal and vascular tissues of seedling. Expressed in both mid-vein ground tissue and vasculature of developing leaves. Expression in ground tissues of roots. Not observed in root vascular. Expected expression pattern: Shoot meristem Selection Criteria: Arabidopsis public Gene: Xyloglucan endotransglycosylase. GenBank: NM 113277 Arabidopsis thaliana Xyloglucan endotransglycosylase, putative (At3g23730) mRNA, complete colsgil 18403866 refNM 113277.1||18403866) Source Promoter Organism: Arabidopsis thaliana WS Vector: pNewbina-HAP1-GFP Marker Type: GFP-ER Generation Screened: XT1 Mature X T2 Seedling T2 Mature T3 Seedling Criteria: Bidirectional: PASS Exons: PASS Repeats: PASS Table 3. Promoter utility Utility: Translocation, seed fill. Improved loading of phloem, increased source capacity. Increased seed yield. Notes: The polysaccharide xyloglucan is thought to play an important structural role in the primary cell wall of dicotyledons. Endodermis: Recent studies have implicated these cell types in gravity perception by sedimentation of starch within these cells. Gravity perception by dicot organs involves primarily the sedimentation of amyloplasts within specialized cells (statocytes) located in the columella region of the root cap and in the starch sheath, which constitutes the endodermis of hypocotyls and inflorescence stems (Kiss et al., 1996; Kuznetsov and Hasenstein, 1996: Blancafior et al., 1998; Weise et al., 2000). In shoots, sedimentable amyloplasts and the curvature response to gravistimulation occur along the elongation Zone (for review, see Masson et al., 2002). After amyloplast sedimentation, signals are likely transduced within the endodermal cells, and physiological signals are transported laterally to affect elongation of cortical and epidermal cells. In roots, sites of gravity perception and curvature response may be physically separated (Poff and Martin, 1989). Construct: YPOO18 Promoter candidate I.D: 11768673 cDNA ID: 1264.7555 Lines expressing: YPOO18-01;YPOO18-02 plant date 7/28/03 Promoter Expression Report # 99 Report Date: Dec. 3, 2003 Promoter Tested In: Arabidopsis thaliana, WS ecotype Spatial expression Summary: Flower L. pedicel, L receptacle, L sepal, L. petal, L filament, Lepidermis Stem L vascular Leaf M vascular, L. rib Hypocotyl Lepidermis, L cortex, Hvascular Cotyledon L mesophyll, Lepidermis Rosette Leaf L mesophyll, L vascular, Lepidermis, H petiole Primary Root Hvascular Observed expression pattern: T1 mature: Weak vascular expression throughout inflorescence meristem and flowers. Variable levels of expression in cells at receptacle of flowers. Expressed in both vascular and Supporting ground tissue in leaves. T2 Seedling: Strong expression observed throughout vasculature of root and hypocotyl. Expression in a few epidermal and cortex cells of hypocotyl at cotyledonjunction. Weak epidermis and mesophyll expression in developing leaves. Expected expression pattern: Stem cell population in center of shoot apical, inflorescence and floral meristem. Selection Criteria: Arabidopsis public. Clark SE, Williams RW, Meyerowitz E. M., The CLAVATA1 gene encodes a putative receptor kinase that controls shoot and floral meristem size in Arabidopsis. Cell. 1997 May 16; 89(4):575-85. Gene: CLAVATA1 receptor kinase (CLV1) GenBank: NM 106232 Arabidopsis thaliana CLAVATA1 receptor kinase (CLV1) (Atlg75820) mRNA, complete cols gi30699119 refNM 106232.2|306991.19) Source Promoter Organism: Arabidopsis thaliana WS Vector: pNewbina-HAP1-GFP Marker Type: GFP-ER Generation Screened: XT1 Mature X T2 Seedling T2 Mature T3 Seedling Criteria: Bidirectionality: PASS Exons: PASS Repeats: PASS Table 3. Promoter utility Utility: Translocation, seed fill. Improved loading of phloem, increased source capacity. Increased seed yield. Cotyledon angle, improved seedling Survival. Notes: Extensive studies on plant signaling molecules over the past decade indicate that plant cell-to-cell communication, as is the case with animal systems, makes use of Small peptide signals and specific receptors. To date, four peptide-ligand-receptor pairs have been identified and shown to be involved in a variety of processes. Matsubayashi. Ligand-receptor pairs in plant peptide signaling. J Cell Sci. 2003 Oct 1; 116(Pt. 19):3863-70. Construct: YPOO71 Promoter candidate I.D: 11768674 US 7,868,155 B2 165 166

TABLE 2-continued cDNA ID: 12721583 (OCKHAM3-C) Lines expressing: YPO071-01, YPO071-02 plant date 7/28/03 Promoter Expression Report # 101 Report Date: Dec. 3, 2003 Promoter Tested In: Arabidopsis thaliana, WS ecotype Spatial expression Summary: Flower H pedicel, H receptacle Silique H placentae Stem H epidermis H cortex H vascular, L. pith Hypocotyl H epidermis, H vascular Cotyledon H mesophyll, H vascular, H epidermis, H hydathode Rosette Leaf H mesophyll, H vascular, H epidermis, H primordia Primary Root H epidermis, H cortex, H vascular Lateral root H epidermis, H cortex Observed expression pattern: T1 mature: High expression in epidermis and cortical cells of stem and pedicles near inflorescence shoot apex. Weakens near floral organs except in the placenta where GFP is also highly expressed. Not expressed in ovules or embryos. High GFP expression in vasculature of stem. T2 seedling: High expression throughout leaves and epidermis of hypocotyl. No expression observed in ground tissues of hypocotyl. High epidermal, cortex and vascular expression in root. Expected expression pattern: Enzyme located in chloroplasts, >4 old high in seedlings Selection Criteria: Ceres Arabidopsis microarray Gene: product = “DEF (CLA1) protein' CLA1 (for “cloroplastos alterados, or “altered chloroplasts) CLA1 encodes 1-deoxy-d-xylulose 5-phosphate synthase, which catalyses the first step of the non-mevalonate isoprenoid biosynthetic pathway. GenBank: NM 117647 Arabidopsis thaliana DEF (CLA1) protein (Atag15560) mRNA, complete cols gi306833.16|refNM 117647.2|30683316) Source Promoter Organism: Arabidopsis thaliana WS Vector: pNewbina-HAP1-GFP Marker Type: GFP-ER Generation Screened: XT1 Mature X T2 Seedling T2 Mature T3 Seedling Criteria: Bidirectionality: PASS Exons: PASS Repeats: PASS Table 3. Promoter utility Utility: Increased photosynthetic capacity and source capacity. Larger plants. Altered plant morphology. Altered plant metabolism. Increased seed loading and seed yield. Notes: CLA1 encodes 1-deoxy-d-xylulose 5-phosphate synthase, which catalyses the first step of the non-mevalonate isoprenoid biosynthetic pathway. Crowell DN, Packard CE, Pierson CA, Giner J. L., Downes BP, Chary SN. Identification of an allele of CLA1 associated with variegation in Arabidopsis thaliana. Physiol Plant. 2003 May: 118(1):29-37. Construct: YPO216 Promoter candidate I.D: 13148171 cDNA ID: 1257582O Lines expressing: YPO216-01, -02, -03, -04 plant date 05/05/03; Promoter Expression Report # 102 Report Date: Oct. 30, 2003 Promoter Tested In: Arabidopsis thaliana, WS ecotype Spatial expression Summary: Ovule Pre-fertilization: L primordia Linner integument L outer integument Post-fertilization: H suspensor Observed expression pattern: T1 mature: Weak expression observed throughout ovule primordia including mother megaspore cell. Post fertilization expression specific to Suspensor cells of embryo. Degeneration of expression in Suspensor attorpedo stage. T2 Seedling: No expression. Expected expression pattern: Nucellus and megaspore mother cell Selection Criteria: Literature. Yang W C, Ye D, Xu J, Sundaresan V. The SPOROCYTELESS gene of Arabidopsis is required for initiation of sporogenesis and encodes a novel nuclear protein. Genes Dev. 1999 Aug 15; 13(16):2108-17. Gene: Nozzle Sporocyteles GenBank: NM 118867 Arabidopsis thaliana NOZZLE SPOROCYTELESS (Atag27330) RNA, complete colsgil 18416968 refNM 118867.1||18416968) Source Promoter Organism: Arabidopsis thaliana WS Vector: pNewbina-HAP1-GFP Marker Type: GFP-ER Generation Screened: XT1 Mature XT2 Seedling T2 Mature T3 Seedling Criteria: Bidirectionality: Exons: Repeats: Table 5. Promoter utility Utility: Better embryo fill, larger embryo and seed. Altered seed composition. Increased seed weight and yield. Better performing seedlings. Seedlings tolerant to stress. Altered source-sink balance. Notes: Balasubramanian S, Schneitz K. NOZZLE links proximal-distal and adaxial-abaxial pattern formation during ovule development in Arabidopsis thaliana. Development. 2002 Sep; 129(18):4291 Construct: YPO271 Promoter candidate I.D: 11768757 cDNA ID: 12658070 Lines expressing: YPO271-01, -02 plant date 4/14/03 US 7,868,155 B2 167 168

TABLE 2-continued Promoter Expression Report # 103 Report Date: Oct. 30, 2003 Promoter Tested In: Arabidopsis thaliana, WS ecotype Spatial expression Summary: Silique Lovule Ovule Post-fertilization: M Zygote L embryo sac L embryo Embryo M Suspensor L torpedo L. radicle Rosette Leaf M mesophyll Hepidermis H stomata Primary Root H pericycle Lateral root H initials H flanking cells H primordia Observed expression pattern: T1 mature: High expression throughout mature female gametophyte at fertilization and in embryo from Zygote to orpedo stage embryo. Expression in embryo restricted to radicle. Not observed in leaf, however this may coincide with severe yellowing of leaves in plants screened during this time. T2 seedling: High GFP expression in mesophyll and epidermal cells of rosette leaves. Expression in root is specific to pericycle cells and lateral rootprimordia. Expected expression pattern: Leaf Selection Criteria: Literature. Leaf-Specific Upregulation of Chloroplast Translocon Genes by a CCT Motif Containing Protein, CLA 2. Sun CW, Chen LJ, Lin L. C. Li H M. Plant Cell. 2001 Sep: 13(9): 2053-2062, PMCID: 39451 (Abstract Full Text) PDF Gene: CIA2 GenBank: NM 125100 Arabidopsis thaliana CIA2 (CLA2) (AtSg57180) mRNA, complete cols gi30696839|refNM 125100.2|30696839 Source Promoter Organism: Arabidopsis thaliana WS Vector: pNewbina-HAP1-GFP Marker Type: GFP-ER Generation Screened: XT1 Mature XT2 Seedling T2 Mature T3 Seedling Criteria: Bidirectionality: Exons: Repeats: Table 5. Promoter utility Utility: Nutrition. Imprint modulation through female, heavier seed, lighter seed, seedless fruits. Increased lateral root growth. More lateral roots, larger lateral roots. Improved drought tolerance. Improved performance in low nitrogen soil, improved source capacity. Notes: Construct: YPO279 Promoter candidate I.D: 11768839 cDNA ID: 12600234 (OCKHAM3-C) Lines expressing: YPOO279-01, -02, -03 plant date 4/14/03 Promoter Expression Report # 105 Report Date: Dec. 3, 2003 Promoter Tested In: Arabidopsis thaliana, WS ecotype Spatial expression Summary: Silique Lovule Ovule Membryo sac Leaf L vascular Hypocotyl L vascular Cotyledon L vascular, M hydathode Primary Root Lepidermis, M vascular, M pericycle Observed expression pattern: T1 mature: GFP expression decreasing in female gametophyte. Low expression in leaf vasculature. T2 seedling: Low expression in cotyledon and hypocotylvasculature. Low expression in root vasculature and pericycle cells. Expected expression pattern: PEG-inducible Selection Criteria: Ceres, Inc. Arabidopsis Expression data Gene: Arabidopsis thaliana mitochondrial carrier protein family GenBank: NM 118590 Arabidopsis thaliana mitochondrial carrier protein family (Atag24570) mRNA, complete cals gi30686585 refNM 118590.2|30686585 Source Promoter Organism: Arabidopsis thaliana WS Vector: pNewbina-HAP1-GFP Marker Type: GFP-ER Generation Screened: XT1 Mature X T2 Seedling T2 Mature T3 Seedling Criteria: Bidirectionality: PASS Exons: PASS Repeats: PASS Table 3. Promoter utility Utility: Imprint modulation through female, larger (heavier) seeds, Smaller (lighter) seeds, seedless fruits. Altered endosperm and seed composition, improved drought tolerance. Improved performance in low-nitrogen soil. Notes: Construct: YPO285 Promoter candidate I.D: 11768588 cDNA ID: 13609092 Lines expressing: YPO285-01, -02, -04 plant date 6/04/03 Promoter Expression Report # 106 Report Date: Oct. 31, 2003 Promoter Tested In: Arabidopsis thaliana, WS ecotype Spatial expression Summary: Flower Hvascular Silique Hvascular Stem Hvascular US 7,868,155 B2 169 170

TABLE 2-continued Leaf Hvascular Hypocotyl Hvascular Cotyledon Hvascular Rosette Leaf Hvascular Primary Root H vascular, H pericycle Lateral root H pericycle H vascular Observed expression pattern: T1 mature: Very high expression in vasculature of flowers, stems, and leaves. Not detected in reproductive tissues in silique. T2 seedling: Very high expression throughout Seedling vasculature. Expression in root extending into pericycle cells. Expected expression pattern: Shoot apical meristem Selection Criteria: Greater than 5X down in stim microarray Gene: Leucine-rich repeat transmembrane protein kinase GenBank: NM 118146 Arabidopsis thaliana leucine-rich repeat transmembrane protein kinase, putative (Atag20270) mRNA, complete cols gi30685044|refNM 118146.2|30685044 Source Promoter Organism: Arabidopsis thaliana WS Vector: pNewbina-HAP1-GFP Marker Type: GFP-ER Generation Screened: XT1 Mature XT2 Seedling 2 T2 Mature ? T3 Seedling Criteria: Bidirectionality: Exons: Repeats: Table 5. Promoter utility Utility: Improved translocation, improved source capacity and seed fill. Heavier seeds. More seeds. Larger siliques. Improved seed yield. Moderate nitrate and/or amino acid transport. Increased transport to floorsink. Notes: Construct: YPOO8O Promoter candidate I.D: 11768676 cDNA ID: 12603755 (OCKHAM3-C) Lines expressing: YPO080-01, -02, -03 plant date 7/28/03 Promoter Expression Report # 107 Report Date: Oct. 31, 2003 Promoter Tested In: Arabidopsis thaliana, WS ecotype Spatial expression Summary: Rosette Leaf L vascular M epidermis Primary Root H epidermis M. root hairs Observed expression pattern: T1 mature: No expression observed. Predicted expression in ovule primordium. T2 seedling: High expression throughout root epidermal cells. Low epidermal and vasculature expression at leaf margins. Expected expression pattern: Integument. Selection Criteria: Arabidopsis public: The BELL1 gene encodes a homeodomain protein involved in pattern formation in the Arabidopsis ovule primordium. Gene: = “homeodomain protein, BELL 1 (BEL1) GenBank: NM 123506 Arabidopsis thaliana homeodomain protein, BELL1 (BEL1) (At 5g41410) mRNA, complete cols gi30693794|refNM 123506.230693794) Source Promoter Organism: Arabidopsis thaliana WS Vector: pNewbina-HAP1-GFP Marker Type: GFP-ER Generation Screened: XT1 Mature XT2 Seedling 2 T2 Mature ? T3 Seedling Criteria: Bidirectionality: Exons: Repeats: Table 5. Promoter utility Utility: Improve ion uptake in roots. Notes: Construct: YPO122 Promoter candidate I.D: 11768849 cDNA ID: 13593439 (OCKHAM3-C) Lines expressing: YPO122-01, -02 plant date 1/17/03 Promoter Expression Report # 116 Report Date: Nov. 4, 2003 Promoter Tested In: Arabidopsis thaliana, WS ecotype Spatial expression Summary: Flower L. pedicel, L. sepal, H carpel, H epidermis, L stomata, Hsilique Silique H carpel, H epidermis, L stomata, Labscission Zone Leaf H mesophyll, L. vascular, H epidermis Primary Root L root hairs Observed expression pattern: T1 mature: GFP expression at the base of sepals at abscission Zone of developing and mature flowers. High expression specific to carpels of developing and mature siliques. T2 seedling: Weak root hair expression at hypocotyl transition Zone observed in 1 in 6 Seedlings and in only 1 of 2 events screened. Expected expression pattern: Flowers, seed, roots. Selection Criteria: Arabidopsis public; containing AP2 DNA binding domain. Gene:EREBP-2 GenBank: NM 124093 Arabidopsis thaliana ethylene responsive element binding factor 2 (EREBP-2) (At 5g47220) mRNA, complete cols gi30695135|refNM 124093.2|30695135 Source Promoter Organism: Arabidopsis thaliana WS US 7,868,155 B2 171 172

TABLE 2-continued Vector: pNewbina-HAP1-GFP Marker Type: GFP-ER Generation Screened: XT1 Mature XT2 Seedling 2 T2 Mature ? T3 Seedling Criteria: Bidirectionality: PASS Exons: PASS Repeats: Table 5. Promoter utility Utility: Increased leaf size and volume, increased source capacity. Tolerance to drought. Improved performance in low nitrogen conditions. Larger siliques, increased seed number. Increased seed yield. Altered dehiscence and seed scatter. Notes: Construct: YPOO15 Promoter candidate I.D: 1768611 cDNA ID: 361.238O Lines expressing: YPOO15-03, -04 plant date 9/8/03 Promoter Expression Report # 118 Report Date: Nov. 4, 2003 Promoter Tested In: Arabidopsis thaliana, WS ecotype Spatial expression Summary: Flower Hanther H. pollen L vascular H stomata Silique Movule Ovule Pre-fertilization: Houter integument H embryo sac H gametophyte Post-fertilization: M Outer integument H seed coat H embryo Embryo H Suspensor H heart H late H mature L radicle L cotyledons Stem H epidermis H stomata H trichome Leaf Histomata Hypocotyl H epidermis L. cortex H stomata Cotyledon H mesophyll H vascular H epidermis H stomata Rosette Leaf Histomata Primary Root H cortex Observed expression pattern: T1 mature: Expressed in pollen cells throughout development. Expression visible during pollination when dehisced pollen attaches to stigma resulting in extension of the cell wall to establish an attachment site or “foot'. Once attached, pollen is hydrated and germination of pollen tubes follows through the Stigma at the attachment site. These processes are likely targets in regulation of self-incompatibility and species-specific pollen recognition. No expression is observed after hydration. Expressed in egg sac of pre-fertilized ovules, inner integument, endosperm, heart stage embryo and Suspensor cells of developing ovules. High specific expression in and epidermal cell files flanking trichomes of stem and guard cells throughout mature plant. T2 seedling: High expression in epidermal and guard cells throughout seedling. High expression epidermal, vascular, and mesophyll cells of cotyledons. Not observed in leaf primordia. High expression specific to cortical cells of root. Expected expression pattern: Induced prior to cell division and usually associated with dividing cells. Selection Criteria: Ceres BLAST search homology; CDC2-like protein Gene: putative protein kinase/note = similar to cyclin-dependent kinase codc2MsE Medicago Saiiva GenBank: NM 125756 Arabidopsis thaliana protein kinase, putative (AtSg63610) mRNA, complete cols gi30697871 refNM 125756.230697871 Source Promoter Organism: Arabidopsis thaliana WS Vector: bNewbina-HAP1-GFP Marker Type: GFP-ER Generation Screened: XT1 Mature XT2 Seedling T2 Mature T3 Seedling Criteria: Bidirectionality: Exons: Repeats: Table 5. Promoter utility Utility: Imprint modulation through male, larger (heavier) seeds, Smaller (lighter) seeds. Malesterility and altered breeding barriers. Altered pollen composition. Altered fertility. Resistance to drought. Notes: Abstracts; Elucidating the complete molecular determination of a self-incompatibility (SI) system in plants has grown significantly with recent investigations of the sporophytic incompatibility system in the genus Brassica. Male (pollen) and female (stigma) components of the recognition incompatibility reaction appear to be controlled by separate genes that reside in a Small genomic region (the S-locus; see YU et al. 1996 ; SCHOPFER et al. 1999). The interaction between male and female components is not completely understood, but it is thought that a pollen Surface protein acts as a ligand that is recognized by a transmembrane protein in the papillary cells on the Surface of the stigma. When the pollen and pistill specificities are from the same S-allele, pollen tube growth is inhibited. The Stigma component of this recognition system is now thought to be the S-locus receptor kinase, encoded by the SRK gene. This protein has an extracellular glycoprotein domain and an intracellular serine-threonine protein kinase (STEIN et al. 1991 ) and has been shown to be necessary, and perhaps sufficient, for determining specificity (CUI et al. 2000 ; TAKASAKI et al. 2000). A Second protein, S-locus glycoprotein, encoded by the closely linked SLG gene, is not in itself sufficient for determining specificity, although it may be necessary for proper rejection of incompatible pollen (SHIBA et al. 2000 : TAKASAKI et al. 2000). SLG sequences show homology to those of the first exon of SRK (the S-domain). A pollen coat protein, encoded by the linke SCR gene, has recently been shown to be necessary and sufficient for de ermination of the pollen speci ficity (SCHOPFER et al. 1999 : TAKAYAMA et al. 2000). Genetics, Vol. 158, 387-399, May 200 , Copyright (C) 2001 SCHIERUP, M. H., B. K. MABLE, P AWADALLA, and D. CHARLESWORTH, 2001 Identification and characterization of a polymorphic receptor kinase gene linked to the self-incompatibility locus of Arabidopsis lyrata. Genetics 158:387-399. Abstract/Free Full Text SCHOPFER, C. R., M. E. NASRALLAH, and J. B. NASRALLAH, 1999 The male determinant of self incompatibility in Brassica. Science 286:1697-1700Abstract Full Text. STEIN, J., B. HOWLETT, D. C. BOYES, M. E. NASRA LLAH, and J. B. NASRALLAH, 1991 Molecular cloning of a putative receptor protein kinase gene encoded at the self-incompa ibility locus of Brassica oleracea. Proc. Natl. Acad. Sci. USA 88:8816-8820Abstract). US 7,868,155 B2 173 174

TABLE 2-continued Construct: YPO230 Promoter candidate I.D: 131482O1 cDNA ID: 12676237 Lines expressing: YPO230-02, -03 (9/08/03) Promoter Expression Report # 119 Report Date: Dec. 3, 2003 Promoter Tested In: Arabidopsis thaliana, WS ecotype Spatial expression Summary: Silique Hovule Ovule Pre-fertilization: Houter integument Post-fertilization: Houter integument, Hseed coat Observed expression pattern: T1 Mature: GFP expressed in outer integument early in ovule development through seed coat of mature seeds. T2 Seedling: No expression observed. Expected expression pattern: Expressed in ovules and different parts of seeds Selection Criteria: Greater than 50x up in pi ovule microarray Gene: “hypothetical protein product = “expressed protein' GenBank: NM 117365 Arabidopsis thaliana expressed protein (Atag12960) mRNA, complete cols gi30682287|refNM 117365.230682287) Source Promoter Organism: Arabidopsis thaliana WS Vector: bNewbina-HAP1-GFP Marker Type: GFP-ER Generation Screened: XT1 Mature XT2 Seedling T2 Mature T3 Seedling Criteria: Bidirectionality: PASS Exons: PASS Repeats: PASS Table 3. Promoter utility Utility: Seed size determination. Increased seed size. Altered seed composition. Tolerance of seeds to desiccation. Resistance of seeds to abortion. Increase sink strength by expression of A.A. transporters Construct: YPO120 Promoter candidate I.D: 1768656 cDNA ID: 2370095 Lines expressing: YPO120-01, -02 Plant date 9/8/03 Promoter Expression Report # 120 Report Date: Nov. 4, 2003 Promoter Tested In: Arabidopsis thaliana, WS ecotype Spatial expression Summary: Flower H sepal Rosette Leaf H epidermis H stomata Observed expression pattern: T1 Mature: High epidermal expression in petals of developing and mature flowers. Not detected in other organs. T2 Seedlings: High expression in epidermal cells of initial leaf primordia. Expected expression pattern: Emerging true leaves. Selection Criteria: Literature; Cho HT, Cosgrove DJ Altered expression of expansin modulates leaf growth and pedicel abscission in Arabidopsis thaliana. Proc Natl Acad Sci USA. 2000 Aug 15; 97 (17):9783-8. Gene: product = “expansin, putative (EXP10) GenBank: NM 102440 Arabidopsis thaliana expansin, putative (EXP10) (Atlg26770) mRNA, complete cols gi30689629 refNM 102440.2|30689629) Source Promoter Organism: Arabidopsis thaliana WS Vector: pNewbina-HAP1-GFP Marker Type: GFP-ER Generation Screened: XT1 Mature XT2 Seedling 2 T2 Mature ? T3 Seedling Criteria: Bidirectionality: Exons: Repeats: Table 5. Promoter utility Utility: Leafsize and photosynthetic capacity. Increased source strength. Increased Sucrose loading. increased leaf expansion, resulting in improved seedling stress tolerance. Modulate size of organs, young leaf specific exp. Notes: Construct: YPO261 Promoter candidate I.D: 11768750 cDNA ID: 12385291 Lines expressing: YPO261-01, -03 plant date 09/08/03 Promoter Expression Report # 12 Report Date: Nov. 5, 2003 Promoter Tested In: Arabidopsis thaliana, WS ecotype Spatial expression Summary: Flower H pedicel H receptacle H filament Hanther H carpel H vascular H silique Silique Hvascular Stem Hvascular Leaf Hvascular Hypocotyl Lepidermis Hvascular Cotyledon Hvascular Lepidermis Rosette Leaf H epidermis Primary Root H cortex H root cap US 7,868,155 B2 175 176

TABLE 2-continued Observed expression pattern: T1 Mature: High GFP expression in vasculature of stem and leaves, also pedicles, siliques and stamen of flowers. Not detected in sepals and petals. Expression in silique specific to medial vasculature. T2 Seedling: High GFP expression in hypocotyl and cotyledon vasculature and cortex of root. Not observed in root vasculature. Expected expression pattern: Stem and root elongation Zones. Selection Criteria: Hanzawa Y. Takahashi T, Michael AJ, Burtin D, Long D, Pineiro M, Coupland G, Komeda Y. ACAULIS5, an Arabidopsis gene required for stem elongation, encodes aspermine synthase. EMBO J. 2000 Aug 15; 19(16):4248-56. Gene: Arabidopsis ACAULIS5 (ACL5) GenBank: NM 121958 Arabidopsis thaliana spermine synthase (ACL5)(AtSg19530) mRNA, complete cols gi30687363 refNM 121958.230687363) Source Promoter Organism: Arabidopsis thaliana WS Vector: pNewbina-HAP1-GFP Marker Type: GFP-ER Generation Screened: XT1 Mature XT2 Seedling T2 Mature T3 Seedling Criteria: Bidirectionality: Exons: Repeats: Table 5. Promoter utility Utility: Translocation to the flower, seed fill. Sucrose loading and transport. Improved source capacity, lending o larger plant organs, larger plants, Increased biomass, increased yield. Improved root growth and soil penetration. Resistance to drought, improved uptake of nitrogen and phosphate. Modify nitrate uptake and translocation to Xylem. Construct: YPO263 Promoter candidate I.D: 11768752 cDNA ID: 1264.0578 Lines expressing: YPO263-10, -11, -12 plant date 9/8/03 Promoter Expression Report # 123 Report Date: Nov. 5, 2003 Promoter Tested In: Arabidopsis thaliana, WS ecotype Spatial expression Summary: Flower Mstomata Silique Mstomata Ovule Post-fertilization: L embryo Embryo L provascular, L. cotyledons Primary Root Lepidermis, L xylem Observed expression pattern: T1 mature: Strong expression in embryonic vascular tissue of cotyledons. Weak guard cell expression in flower pedicle and silique. T2 seedling: Weak degrading root epidermis expression near transition Zone. Weak root vascular expression in elongation Zone. Expression in verythin cell layer appears to be xylem. Expected expression pattern: root, flowers, ovules, young silique Selection Criteria: Arabidopsis Two component line CS9135 (see notes). Gene: Hypothetical protein containing helix-loop-helix DNA binding domain. GenBank: NM 116493 Arabidopsis thaliana bHLH protein (Atago2590) mRNA, complete cols gi30679204|refNM 116493.2|30679204) Source Promoter Organism: Arabidopsis thaliana WS Vector: pNewbina-HAP1-GFP Marker Type: GFP-ER Generation Screened: XT1 Mature XT2 Seedling 2 T2 Mature ? T3 Seedling Criteria: Bidirectionality: Exons: Repeats: Table 5. Promoter utility Utility: Translocation to cotyledons, seed fill. Increased embryo and seed weight. Altered embryo and seed composition. Improved seedling vigor, seedling resistance to drought, cold, cold? wet conditions. Construct: YPOOO3 Promoter candidate I.D: 131482.13 cDNA ID: 12649228 Lines expressing: YPO003-04, -06 plant date 9/8/03

TABLE 3 TABLE 3-continued Optional Promoter Fragments 55 Optional Promoter Fragments SEQ INTER- SEQ INTER ID CONSTRUCT VENING ID CONSTRUCT VENING NO. NO EXON UTR SEQUENCE NO. NO EXON UTR SEQUENCE

1 11 YPOO49 395-915 2 YPOOO7 1013-1033 60 12 YPOO60 26S-343 1-264 3 13 YPO092 1-764 5 YPO111 935-999 14 6 15 YPO095 1-214 7 16 8 YPOO16 746-1OOO 935-1000 17 YPO103 224-362; 1-128 950-1001 129-223 9 YPO094 1-28: 1-101 65 18 YPO107 1-55 10 19 US 7,868,155 B2 177 178

TABLE 3-continued TABLE 4-continued Optional Promoter Fragments Dif fer SEQ INTER cDNA ID Expt Rep ID Short Name ential ID CONSTRUCT VENING NO. NO EXON UTR SEQUENCE 4909291 2OOOOO92 42deg Heat 361.2879 2OOOO18S Roots 2O YPO112 245-640 1-245 3489977 2OOOO234 Siliques 21 3610584 2OOOO794 22 10 361.2879 2OOOO438 23 26696.15 2OOOO234 24 YPO126 1-355 3489977 2OOOO264 25 YPOO24 1-56; 148-233; 57-147; 26696.15 2OOOO286 381-670 234-280 23298.27 2OOOO439 26 3610584 2OOOO286 27 15 3610584 2OOOO234 28 361.2879 2OOOO439 29 2688453 2OOOO439 30 26696.15 2OOOO794 31 2688453 2OOOO18S 32 POO54 948-999 23298.27 2OOOO18S Roots 33 269218.1 2OOOO314 34 POOSO 1-44; 131-339 940-999 45-130 3489977 2OOOO286 35 POO40 933-999 4909291 2OOOO457 36 237O148 2OOOO234 37 3489977 2OOOO26S 38 POO68 1-119; 271-725 932-999 120-270 36098.17 108434 39 POO82 2348.737 2OOOO794 40 25 2713856 2OOOO439 41 2333534 2OOOO794 42 361.2879 2OOOO184 43 91-234; 302-656 1-90; 235-301 3489977 2OOOO438 44 269218.1 108457 Diversity Expt 45 PO121 55-294; 389-530; 1-54; 295-388: 3489977 2OOOO235 Siliques 604-640 531-603 30 26696.15 2OOOO235 46 3489977 2OOOO326 47 3489977 2OOOO236 48 PO108 1-216 2713856 2OOOO18S 49 361.2879 20000527 50 361.2879 2OOOO794 51 35 49.09806 10843S 52 Y 257-640 641 3610584 2OOOO235 53 Y 947-999 237O148 2OOOO235 S4 B 14-4O6 1260-1322 1-14 26696.15 2OOOO438 2 2322657 2OOOO438 55 900-1056 269218.1 2OOOO315 4day Shoots-Roots 56 237O148 2OOOO26S 178-735; 761-813 1660-1723: 1-177; 736-760; 40 Open Flower 1928-1954 1724-1927 2679922 2OOOO439 Roots 57 2678,173 2OOO1654 interploidy Crosses 58 3612919 108595 Ler-pi. Ovule 59 237O148 2OOOO286 Open Flower 60 26701.59 2OOOO18S Roots 61 YPO122 709-999 2333534 2OOO1654 interploidy Crosses 62 YPO216 925-999 45 2713856 2OOOOO71 OOuM ABA Mutants 63 271.1515 2OOOO214 64 YPO261 806-880 2322657 2OOOO326 65 2679922 2OOOO234 66 YPO271 983-999 2679922 2OOOO18S 237O148 2OOOO236 67 Siliques 50 26.60077 2OOO1248 Far-red-induction 68 2713856 108595 Ler-pi. Ovule cDNA P 69 YPOOO3 700-882 3612919 2OOOO326 Pollen 70 2679922 2OOOO26S Open Flower 2348.737 2OOOO264 Open Flower 26696.15 2OOOO236 Siliques 55 2679922 2OOO1556 Drought Soil Dry TABLE 4 271.1515 2OOO1554 Drought Soil Dry 271.1515 2OOOO264 Open Flower Dif 2678,173 2OOOO223 CS6632 Shoots-Roots cDNA P fer 2713856 2OOOO117 10OuM ABA Mutants cDNA P cDNA ID Expt Rep ID Short Name ential 361.2879 2OOOO458 42deg Heat 23298.27 2OOOO245 Caf Knockout 13610584 2OOOO264 At Open Flower 60 3612919 2OOOO439 Roots 12656458 2OOOO708 At Fis1 Siliques 2322657 2OOOO184 Shoots 49.09806 2OOOO439 At Roots 2646.933 2OOOO18S Roots 49.09806 2OOOO18S At Roots YF 2646.933 2OOOO438 Shoots 1266.9615 2OOOO264 At Open Flower 2679922 2OOOO286 Open Flower 49.09806 2OOOO794 At Petals 2576899 2OOOO458 42deg Heat 1271.1515 2OOOO794 At Petals 65 2348.737 2OOOO235 Siliques 1266.9615 2OOOO26S At Open Flower 2348.737 108595 Ler-pi. Ovule cDNA P US 7,868,155 B2 179 180

TABLE 4-continued TABLE 4-continued

Dif Dif fer fer cDNA ID Expt Rep ID Short Name ential cDNA ID Expt Rep ID Short Name ential 36098.17 108457 Diversity Expt cDNA P 2713856 2OOOO326 Pollen 26.60077 2OOOO18S Roots 2679922 2OOO1559 Drought Soil Dry 2664333 20000527 10% PEG 2679922 2OOOO214 4deg Cold 3613553 2OOOO18O Germinating Seeds 3647840 2OOOO245 Caf Knockout 3647840 2OOOO438 Shoots 10 27O3O41 2OOO1556 Drought Soil Dry 2679922 2OOO1555 Drought Soil Dry 27O3O41 108595 Ler-pi. Ovule cDNA P 237O148 2OOOO439 Roots 237O148 20000573 10OuM ABA Mutants 2348.737 2OOOO708 Fis1 Siliques 361.2879 2OOOO326 Pollen 271.1515 2OOO1556 Drought Soil Dry 2348.737 2OOOO234 Siliques 2333534 108577 42deg Heat cDNA P 49.09806 108585 SmM NaNP cDNA P 23298.27 2OOOO438 Shoots 15 2348.737 2OOOO26S Open Flower 2348.737 10843S stim Mutants cDNA P 3614559 2OOOO18O Germinating Seeds 2679922 2OOOO236 Siliques 361.2879 108668 2m MSA cDNA P 2713856 2OOO1248 Far-red-induction 3489977 20000573 OOuM ABA Mutants 49.09806 2OOO1557 Drought Soil Dry 3613553 2OOOO184 Shoots 271.1515 2OOO1555 Drought Soil Dry 2713856 2OOOOO70 OOuM ABA Mutants cDNAP 3619323 2OOOO438 Shoots 26701.59 2OOOO264 Open Flower 2679922 2OOOO264 Flower 3491988 2OOOO227 Root-Tips-vs-Tops 3613553 2OOO1247 Far-red-induction 36098.17 2OOOO234 Siliques 24554.36 108462 Germinating Seeds cDNA P 23298.27 108462 Germinating Seeds cDNA P 3612919 2OOO1450 Far-red-induction 27O3O41 108434 Root Tips cDNA P 2332135 2OOOO439 36.17784 2OOOO286 Open Flower 2332135 2OOOO438 27O3O41 2OOOO437 Drought 3647840 2OOOO18S 25 3653114 2OOOO3O7 Germinating Seeds 2576899 2OOO1555 26701.59 2OOOO439 Roots 24554.36 108464 26S6458 2OOO1653 interploidy Crosses 3612919 2OOO1248 Far-red-induction 271.1515 2OOOO437 Drought 3647840 2OOOO708 Fis1 Siliques 3489977 2OOOO179 Germinating Seeds 237O148 2OOOO264 Flower 2396394 108595 Ler-pi. Ovule cDNAP 36098.17 2OOO1558 Drought Soil Dry 30 3491988 2OOOO18S Roots 23298.27 2OOOO794 Petals 271.1515 108668 2m MSA cDNA P 3489977 2OOOO184 Shoots 3610584 2OOOO495 Guard Cells 3489977 2OOOO18O Germinating Seeds 36.17784 2OOOO236 Siliques 3613553 108464 Germinating Seeds cDNA P 2348.737 2OOOO286 Open Flower 49.09806 2OOO1560 Drought Soil Dry 2688453 2OOOO326 Pollen 2678,173 108457 Diversity Expt cDNA P 35 36.17784 2OOOO264 Open Flower 3613553 2OOOO438 Shoots 237O148 2OOOO173 42deg Heat 237O148 2OOOO18S Roots 36.17784 2OOOO26S Open Flower 3613553 2OOOO18S Roots 237O148 2OOOOS74 10OuM ABA Mutants 49.09806 2OOO1558 Drought Soil Dry 2333534 2OOOO111 42deg Heat cDNA P 2576899 2OOOO173 42deg Heat 3610584 2OOO1557 Drought Soil Dry 237O148 2OOOO438 Shoots 4909291 108488 40 50mM NH4NO3 L-to 2332135 2OOOO26S Open Flower Rosette cDNA P 2333534 2OOOO18S Roots 2333534 2OOOO227 Root-Tips-vs-Tops 3489977 2OOOO439 Roots 2396394 2OOO1248 Far-red-induction 36.17784 2OOOO234 Siliques 2713856 2OOOOO87 OOuM ABA Mutants cDNAP 49.09806 108577 42deg Heat cDNA P 2332135 2OOOO18S Roots 49.09806 108595 Ler-pi. Ovule cDNA P 3612919 2OOOO18S 24554.36 108463 Germinating Seeds cDNA P 45 2576899 2OOOO214 27O3O41 2OOO1559 Drought Soil Dry 36.17784 2OOOO235 Siliques 2713856 2OOOOO86 OOuM ABA Mutants cDNA P 3489977 2OOOO708 Fis1 Siliques 271.1515 2OOOO213 26.60077 20000573 OOuM ABA Mutants 27O3O41 2OOOO234 2322657 2OOOO18S Roots 3489977 2OOOO18S 237O148 2OOOO171 42deg Heat 26696.15 20000527 O% PEG 50 26696.15 2OOOO458 42deg Heat 2679922 1085O1 ap2 floral buds cDNA P 3619323 108474 Drought Flowers cDNA P 3612919 10843S stim Mutants cDNA P 2333534 2OOOO173 42deg Heat 2333534 108579 4deg Cold cDNA P 49.09806 2OOO1459 50mM NH4NO3 L-to-H 26701.59 2OOOO26S Open Flower 3647840 2OOOO439 Roots 2396394 2OOO1557 Drought Soil Dry 23298.27 108463 Germinating Seeds cDNA P 2646.933 2OOOO439 Roots 55 2679922 108577 42deg Heat cDNA P 2735519 2OOO1248 Far-red-induction 2679922 2OOOO268 OOmM NaCl 36098.17 2OOO1557 Drought Soil Dry 237O148 2OOOO18O Germinating Seeds 2348.737 2OOOO236 Siliques 237O148 108434 Root Tips cDNA P 2713856 2OOOOO69 OOuM ABA Mutants cDNA P 2322657 2OOO1247 Far-red-induction 2713856 108573 Drought cDNA P 3610584 2OOOO438 237O148 2OOOO184 Shoots 3647840 2OOOO184 3619323 2OOOO184 Shoots 60 3613553 2OOO1451 Far-red-induction 3610584 2OOOO236 Siliques 4905097 2OOO1654 interploidy Crosses 3610584 2OOOO437 Drought 27O3O41 2OOOO26S Open Flower 2713856 2OOOOO88 OOuM ABA Mutants cDNA P 2396394 108579 4deg Cold cDNA P 36098.17 2OOOO26S Open Flower 2713856 2OOOO794 Petals 271.1515 2OOOO236 Siliques 2646.933 2OOOO245 Caf Knockout 2333534 108595 Ler-pi. Ovule cDNA P 65 237O148 2OOOO268 100mM NaCl 36098.17 2OOOO227 Root-Tips-vs-Tops 2333534 2OOOO437 Drought US 7,868,155 B2 181 182

TABLE 4-continued TABLE 4-continued

Dif Dif fer fer cDNA ID Expt Rep ID Short Name ential cDNA ID Expt Rep ID Short Name ential 2713856 108499 DMT-II cDNA P 3613553 108462 Germinating Seeds cDNA P 2678,173 2OOOOO3O CS6630 Roots cDNA P 2576899 2OOOO213 4deg Cold 26696.15 2OOOO18O Germinating Seeds 36O1936 2OOO1556 Drought Soil Dry 49.09806 108668 2m MSA cDNA P 2396394 2OOOOO86 OOuM ABA Mutants cDNAP 27O3O41 2OOO1555 Drought Soil Dry 10 4909291 2OOOO26S Flower 3491988 2OOOO439 Roots 271.1515 2OOOO234 3610584 2OOOO4S1 CS6879 Shoots-Roots 27O3O41 2OOOOO87 OOuM ABA Mutants cDNAP 271.1515 108499 DMT-II cDNA P 27O3O41 2OOOO267 Drought 361.2879 2OOO1556 Drought Soil Dry 26701.59 2OOO1451 Far-red-induction 2332135 2OOOO184 Shoots 237O148 2OOOOO46 CS237-vs-Columbia cDNA P 26701.59 2OOOO794 Petals 15 2679922 2OOO1558 Drought Soil Dry 2646.933 2OOO1654 Interploidy Crosses 271.1515 108463 Germinating Seeds cDNA P 2348.737 108579 4deg Cold cDNA P 2332135 108474 Drought Flowers cDNA P 237O148 2OOOO111 42deg Heat cDNA P 237O148 2OOOO437 Drought 2576899 2OOO1556 Drought Soil Dry 3621692 2OOOO173 42deg Heat 23298.27 2OOOO184 Shoots 2396394 10843S stm Mutants cDNA P 2333534 2OOOO439 Roots 26.60077 2OOOO439 237O148 2OOOO708 Fis1 Siliques 4909291 2OOOO234 3613553 2OOOO439 Roots 2396394 2OOOOO90 2mM SA CS3726 2322657 2OOOO439 Roots olumb ia cDNA P 3610584 108434 Root Tips cDNA P 26.60077 2OOOOS74 OOuM ABA Mutants 2713856 108584 SmM NaNP cDNA P 26696.15 2OOOO179 Germinating Seeds 2333534 108499 DMT-II cDNA P 2396394 108589 SmM NH4NO3 L-to-H. cDNA P 36098.17 2OOO1560 Drought Soil Dry 25 237O148 1085O1 ap2 floral buds cDNA P 26701.59 2OOOO286 Open Flower 36.17784 108457 Diversity Expt cDNA P 4909291 2OOOO286 Open Flower 4909291 2OOOO127 2396394 2OOOOO70 OOuM ABA Mutants cDNA P Siliques cDNA P 2679922 2OOOO18O Germinating Seeds 2455436 2OOOO326 Pollen 271.1515 10843S stim Mutants cDNA P 237O148 108608 OOuM ABA cDNA P 49.09806 108576 42deg Heat cDNA P 30 361.2879 1086.67 2mM SA cDNA P 2332135 20000527 237O148 2OOO1317 interploidy Crosses 49.09806 2OOOO264 Open Flower 3489977 2OOOO171 42deg Heat 271.1515 2OOOO235 Siliques 237O148 2OOOO166 OOuM ABA 271.1515 108461 Germinating Seeds cDNA P 36098.17 2OOO1556 Drought Soil Dry 3489977 2OOOOST4 OOuM ABA Mutants 2679922 2OOO1554 Drought Soil Dry 3614841 2OOOO437 Drought 35 36.17784 2OOOO456 OOuM BA 271.1515 2OOOO438 Shoots 2333534 2OOOOO88 OOuM ABA Mutants cDNAP 27O3O41 2OOOO214 4deg Cold 49.09806 2OOOOS76 OOuM ABA Mutants 2646.933 2OOOO184 Shoots 49.09806 20000573 OOuM ABA Mutants 3610584 2OOO1300 Line Comparisons 49.09806 2OOOOS74 OOuM ABA Mutants 2333534 2OOOO117 OOuM ABA Mutants cDNA P 2576899 2OOOO171 42deg Heat 26S6458 2OOO1316 2679922 108462 interploidy Crosses 40 Germinating Seeds cDNA P 2679922 2OOOO3O8 OOmM NaCl 3621692 2OOOO184 Shoots 2678,173 O843S stm Mutants cDNA P 2396394 2OOO1556 Drought Soil Dry 26696.15 Caf Knockout 4909291 2OOOO264 Open Flower 2333534 OOuM ABA Mutants cDNA P 2322657 108577 42deg Heat cDNA P 23298.27 Open Flower 49.09806 20000575 OOuM ABA Mutants 36 3553 Germinating Seeds cDNA P 2333534 2OOOOO70 OOuM ABA Mutants cDNAP Ler-rhil Root cDNAP 45 36098.17 108595 Ler-pi. Ovule cDNA P 36 77.84 Pollen 2348.737 1085O1 ap2 floral buds cDNA P 2333534 Drought Soil Dry 2396394 2OOOOO71 OOuM ABA Mutants cDNAP 2396394 Drought Soil Dry 3613553 108461 Germinating Seeds cDNA P 23298.27 Germinating Seeds cDNA P 49.09806 1086O7 OOuM ABA cDNA P 2332135 interploidy Crosses 3610584 2OOOO267 Drought 27 1515 42deg Heat 50 2713856 108579 4deg Cold cDNA P 2679922 Drought Soil Dry 36098.17 2OOOO708 Fis1 Siliques 2679922 2688453 2OOOO438 Shoots 36 3553 3489977 2OOOOSO6 Wounding Ro sette cDNA P 2348.737 2OOOO179 Germinating Seeds 232S134 Drought Flowers cDNA P 271.1515 O8579 4deg Cold cDNA P 36 2919 OOuM ABA Mutants cDNA P 55 2713856 O86O7 10OuM ABA cDNA P 2333534 SmM NaNP cDNA P 2679922 O8461 Germinating Seeds cDNA P 36 2919 Petals 3614841 2OOO1451 Far-red-induction 2679922 Line Comparisons 49.09806 2OOOO437 Drought 36 Shoot Apices cDNA P 232S134 0.001% Me.JA cDNA P 237O148 OOuM ABA cDNA P 4909291 Siliques 36 2919 OOuM ABA Mutants cDNA P 2713856 10OuM ABA cDNA P 36 3553 stm Mutants cDNA P 60 2321680 Roots 2396394 OOuM ABA Mutants cDNA P 36O1936 10OuM ABA Mutants cDNA P 27O3O41 DMT-II cDNA P 3610584 Pollen 27 3856 Drought cDNA P 2679922 DMT-II cDNA P 2333534 Drought 2322657 42deg Heat cDNA P 27 1515 OOuM BA 3619323 Interploidy Crosses 36 3553 Root Tips cDNA P 65 2713856 stm Mutants cDNA P 2OOOO794 Petals 2348.737 50mM NH4NO3 L-to US 7,868,155 B2 183 184

TABLE 4-continued TABLE 4-continued

Dif Dif fer fer cDNA ID Expt Rep ID Short Name ential cDNA ID Expt Rep ID Short Name ential H. Rosette cDNA P 36091OO 2OOO1248 Far-red-induction 2576899 2OOO1554 Drought Soil Dry 237O148 108579 4deg Cold cDNA P 36O1936 2OOOO438 Shoots 36.17784 2OOOO269 mM KNO3 L-vs-H. Roots 3614841 2OOOO794 Petals 2735519 20000573 OOuM ABA Mutants 24554.36 2OOOO794 Petals 10 2646.933 2OOOO794 Petals 2333534 2OOO1555 Drought Soil Dry 3489977 2OOOO211 0.001% Me.JA 237O148 2OOOO3O8 100mM NaCl 2713856 108608 10OuM ABA cDNA P 2713856 108577 42deg Heat cDNA P 2348.737 2OOOO173 42deg Heat 271.1515 2OOOO171 42deg Heat 271.1515 2OOOO267 Drought 2713856 2OOOO436 Drought 27O3O41 2OOO1554 Drought Soil Dry 271.1515 2OOOOO46 CS237-vs-Columbia cDNA P 15 4996264 2OOOO264 Open Flower 2333534 108478 Shoot Apices cDNA P 3612919 2OOOO437 Drought 27O3O41 2OOOOO90 2m MSA CS3726 3610584 2OOOO268 olumbia cDNAP 3653114 2OOOO306 3509244 2OOOO184 Shoots 2322657 108434 Tips cDNA P 3610584 2OOOO709 SmM NH4NO3 L-to-H 27O3O41 108457 Diversity Expt cDNA P 271.1515 20000527 10% PEG 49.09806 2OOO1556 Drought Soil Dry 237O148 108575 Wounding cDNA P 237O148 2OOOOSO6 Wounding 3612919 2OOO1451 Far-red-induction 3489977 108434 Root Tips cDNA P 2333534 2OOO1559 Drought Soil Dry 3612919 108585 SmM NaNP cDNA P 4905097 108461 Germinating Seeds cDNA P 26.60077 2OOOO184 Shoots 26696.15 2OOOO173 42deg Heat 2679922 2OOOO179 Germinating Seeds 3491988 2OOOO4S1 CS6879 Shoots-Roots 3614559 108464 Germinating Seeds cDNA P 26696.15 2OOO1555 Drought Soil Dry 25 3610584 108574 Wounding cDNA P 271.1515 2OOOO111 2646.933 2OOO1449 Line Comparisons 4909291 2OOOO213 36.17784 2OOOOO86 OOuM ABA Mutants cDNAP 2333534 2OOOOO72 49.09806 2OOO1316 interploidy Crosses 2713856 2OOOO437 Drought 27O3O41 2OOOO268 OOmM NaCl 3621692 2OOOO26S Open Flower 2576899 2OOO1557 Drought Soil Dry 2679922 108463 Germinating Seeds cDNA P 30 2333534 2OOOOO93 42deg Heat cDNA P 3491988 2OOO1558 Drought Soil Dry 3491988 2OOO1560 Drought Soil Dry 49.09806 108512 3642-1 cDNA P 4996264 2OOOO26S Open Flower 237O148 108461 Germinating Seeds cDNA P 2679922 2OOOO794 Petals 36.17784 108595 Ler-pi. Ovule cDNA P 49.09806 2OOOO169 OOuM ABA 2332135 2OOO1397 Line Comparisons 2688453 20000573 271.1515 2OOOO144 42deg Heat cDNA P 35 49.09806 2OOOO234 4909291 108579 4deg Cold cDNA P 271.1515 2OOOO436 3610584 2OOOO184 Shoots 3619323 108SOO DMT-II cDNA P 2679922 2OOOO113 42deg Heat cDNA P 2713856 2OOO1450 Far-red-induction 2321680 2OOO1654 interploidy Crosses 2333534 2OOOOO71 OOuM ABA Mutants cDNAP 2713856 2OOOO286 Open Flower 2348.737 108594 Ler-rhil Root cDNAP 3489977 2OOOO227 Root-Tips-vs-Tops 4996264 2OOOO184 40 Shoots 271.1515 O8464 Germinating Seeds cDNA P 36098.17 2OOOOO90 2m MSA CS3726 26921.81 O848O Shoot Apices cDNA P olumbia cDNAP 26701.59 Drought Soil Dry 2321680 2OOOO438 Shoots 237O148 Drought 237O148 2OOOO227 Root-Tips-vs-Tops 26701.59 Fis1 Siliques 4909291 2OOO1558 Drought Soil Dry 4905097 Drought Flowers cDNA P 49.09806 2OOOO286 Open Flower 2396394 OOuM ABA Mutants cDNA P 45 2S61142 2OOO1556 Drought Soil Dry 3614559 Germinating Seeds cDNA P 36.17784 108499 DMT-II cDNA P 26701.59 Drought Soil Dry 2321680 2OOO1397 Line Comparisons 3509244 Pollen 2679922 2OOO1449 Line Comparisons 4996264 Open Flower 3491988 108577 42deg Heat cDNA P 3614559 Germinating Seeds cDNA P 2333534 1086O7 10OuM ABA cDNA P 26921.81 stm Mutants cDNA P 50 4909291 2OOOO214 4deg Cold 2333534 OOuM ABA Mutants cDNA P 2396394 2OOO1555 Drought Soil Dry 24554.36 Germinating Seeds cDNA P 27O3O41 108489 50mM NH4NO3 L-to 26701.59 Root-Tips-vs-Tops Rosette cDNA P 26696.15 42deg Heat 5787483 108577 42deg Heat cDNA P 27O3O41 CS6879 Shoots-Roots 4996264 108434 Root Tips cDNA P 2333534 OOuM ABA cDNA P 55 3612919 2OOOO4S1 CS6879 Shoots-Roots 2332135 0.001% Me.JA cDNA P 26.60077 2OOOO460 10% PEG 3621692 Shoots 3612919 2OOOO213 4deg Cold 23298.27 interploidy Crosses 4905097 2OOOO113 42deg Heat cDNA P 2646.933 Line Comparisons 2713856 2OOOOO66 CS3071 vs Columbia cDNA P 26696.15 Shoots 2713856 108590 15 mM NH4NO3 L-to-H. cDNA P 5787483 42deg Heat cDNA P 3489977 2OOOO223 CS6632 Shoots-Roots cDNA P 271.1515 2OOOO286 Open Flower 60 2576899 2OOOO286 Open Flower 2678,173 2OOOOO68 CS3824 vs Landsberg cDNA P 2333534 2OOO1560 Drought Soil Dry 3612919 2OOO1557 Drought Soil Dry 4909291 2OOOO496 Guard Cells 3612919 2OOOOO90 2m MSA CS3726 2713856 2OOO1397 Line Comparisons olumbia cDNAP 2739224 2OOO1558 Drought Soil Dry 3614559 108462 Germinating Seeds cDNA P 494.9423 2OOOO264 Open Flower 2333534 2OOOOO86 10OuM ABA Mutants cDNA P 65 26696.15 2OOOO439 Roots 271.1515 2OOOO26S Open Flower 3613553 20000573 10OuM ABA Mutants

US 7,868,155 B2 189 190

TABLE 4-continued TABLE 4-continued

Dif Dif fer fer cDNA ID Expt Rep ID Short Name ential cDNA ID Expt Rep ID Short Name ential 26701.59 2OOOO495 Guard Cells 2678,173 2OOOO286 Open Flower 3489977 2OOOO441 uM BR-BRZ 4905097 2OOOO234 Siliques 237O148 2OOOO441 uM BR-BRZ 2321680 2OOOO234 Siliques 2646.933 108591 SmM NH4NO3 L-to-H. cDNA P 3489977 2OOO1308 Line Comparisons 2348.737 2OOOOO70 OOuM ABA Mutants cDNA P 10 269218.1 2OOOOO86 OOuM ABA MutantscDNA P 3489977 2OOOOO69 OOuM ABA Mutants cDNA P 2455436 108434 Root TipscDNA P 2348.737 108470 2m MSA CS3726 36.17784 108588 SmM NH4NO3 L-to-H. cDNA P olumbia cDNA P 2322657 2OOOOO70 OOuM ABA Mutants cDNAP 3610584 108464 Germinating Seeds cDNA P 3614559 108585 SmM NaNP cDNA P 3613553 2OOO1557 Drought Soil Dry 3610584 2OOO1248 Far-red-induction 3614841 2OOO1557 Drought Soil Dry 15 271.1515 2OOOO441 uM BR-BRZ 237O148 2OOOO443 uM BR-BRZ 23298.27 2OOOO234 Siliques 2322657 2OOOOO72 269218.1 2OOOOO69 OOuM ABA Mutants cDNAP 266.1844 2OOOO236 3619323 2OOOO708 Fis1 Siliques 4905097 2OOOO236 2322657 2OOO1248 Far-red-induction 271.1515 2OOOO117 OOuM ABA Mutants cDNA P 2332135 2OOOO235 Siliques 27O3O41 108462 Germinating Seeds cDNA P 269218.1 2OOOO349 OOuM ABA 2322657 2OOOOO86 OOuM ABA Mutants cDNA P 3621692 2OOOO326 Pollen 237O148 108481 Shoot Apices cDNA P 3489977 2OOOO460 O% PEG 27O3O41 108573 Drought cDNA P 2576899 2OOOO794 Petals 26921.81 108608 OOuM ABA cDNA P 271.1515 2OOOO453 OOuM ABA 2321680 108591 SmM NH4NO3 L-to-H. cDNA P 23298.27 2OOOO179 Germinating Seeds 26921.81 108464 Germinating Seeds cDNA P 2322657 2OOOOO71 OOuM ABA Mutants cDNAP 4909291 2OOOO111 42deg Heat cDNA P 25 2692 81 OOuM ABA cDNA P 49.09806 108464 Germinating Seeds cDNA P 23298.27 Fis1 Siliques 3613553 2OOOO709 SmM NH4NO3 L-to-H 4909291 42deg Heat cDNA P 26921.81 108572 Drought cDNA P 2678 73 Pollen 49.09806 2OOOO184 Shoots 2348.737 2mM SA cDNA P 361.2879 2OOOO234 2670 59 CS6879 Shoots-Roots 494.9423 2OOOO437 30 2692 81 OOuM ABA Mutants cDNAP 4905097 2OOOO235 Siliques 271.1515 Shoot Apices cDNA P 26921.81 2OOOOO89 2m MSA CS3726 2332 35 Siliques olumbia cDNA P 36098.17 Germinating Seeds cDNA P 271.1515 108609 OOuM ABA cDNA P 2348.737 Drought cDNA P 2688453 2OOOO436 Drought 2692 81 OOuM ABA cDNA P 3647840 2OOOO794 Petals 35 2322657 OOuM ABA Mutants cDNAP 2322657 2OOOOO90 2m MSA CS3726 2321680 Fis1 Siliques olumbia cDNA P 2646.933 Siliques 3489977 108595 Ler-pi. Ovule cDNA P 2325 34 ap2 floral buds cDNA P 36.17784 108591 SmM NH4NO3 L-to-H. cDNA P 271.1515 Far-red-induction 49.09806 108462 Germinating Seeds cDNA P 36098.17 Germinating Seeds cDNA P 2664333 2OOO1451 Far-red-induction 266.1844 Siliques 40 2333534 2OOOO495 Guard Cells 3613553 SmM NaNP cDNA P 3612919 2OOOO18O Germinating Seeds 269218.1 OOuM ABA cDNA P 36098.17 108463 Germinating Seeds cDNA P 36091OO 15 mM NH4NO3 L-to-H 2396394 108573 Drought cDNA P 4909291 42deg Heat cDNA P 2332135 2OOOO264 Open Flower 271.1515 Shoot Apices cDNA P 2321680 2OOOO236 Siliques YF 269218.1 OOuM ABA cDNA P 3647840 2OOOO264 Open Flower 45 2679922 2OOO1247 induction 49.09806 2OOOO708 Fis1 Siliques 2646.933 2OOOO236 361.2879 2OOOO708 Fis1 Siliques 2646.933 2OOOO234 2332135 108589 SmM NH4NO3 L-to-H. cDNA P 2322657 2OOOOO87 OOuM ABA Mutants cDNAP 26921.81 1085O1 ap2 floral buds cDNA P 2679922 2OOO1248 induction 2678,173 2OOOO264 Open Flower 2692 81 2OOOOO88 OOuM ABA Mutants cDNAP 36.17784 2OOOO458 42deg Heat 50 2646.933 1085O1 ap2 floral buds cDNA P 23298.27 2OOOO18O Germinating Seeds 49.09806 108488 50mM NH4NO3 L-to 3619323 2OOOO234 Siliques Rosette cDNA P 2348.737 2OOO1654 Interploidy Crosses 2692 81 2OOOOO70 10OuM ABA Mutants cDNA P 23298.27 15mM NH4NO3 L-to-H. cDNA P 4909291 2OOOO171 42deg Heat 2688453 10OuM ABA 2396394 108572 Drought cDNA P 2713856 Germinating Seeds cDNA P 55 2692 81 108512 3642-1 cDNA P 3491988 Drought 2322657 2OOOOO69 OOuM ABA Mutants cDNAP 2396394 Shoots 23298.27 2OOOO236 2348.737 Drought cDNA P 3647840 2OOOO326 2348.737 Diversity Expt cDNA P 2332 35 2OOOO234 3610584 Far-red-induction 2679922 2OOO1450 Far-red-induction 26.60077 Petals 271.1515 2OOO1248 Far-red-induction 3619323 15mM NH4NO3 L-to-H. cDNA P 60 4909291 2OOOOO93 42deg Heat cDNA P 26921.81 10OuM ABA Mutants cDNA P 2692 81 1086OS OOuM ABA cDNA P 4905097 Fis1 Siliques 2692 81 1086O7 10OuM ABA cDNA P 2321680 Siliques 4909291 2OOOO144 42deg Heat cDNA P 27O3O41 Germinating Seeds cDNA P 3619323 108595 Ler-pi. Ovule cDNA P 266.1844 SiliquesP 2692 81 2OOOO117 OOuM ABA Mutants cDNAP 2348.737 SmM NaNP cDNA P 65 2321680 1085O1 ap2 floral buds cDNA P 3612919 Germinating Seeds cDNA P 2679922 2OOO1451 Far-red-induction

US 7,868,155 B2 193 194

TABLE 4-continued TABLE 4-continued

Dif Dif fer fer cDNA ID Expt Rep ID Short Name ential cDNA ID Expt Rep ID Short Name ential 2395532 108462 Germinating Seeds cDNA P 264.7555 20000436 At Drought P 2395532 108561 10OuM ABA cDNA P 264.7555 20000437 At Drought P 2395532 108575 Wounding cDNA P 264.7555 20000439 At Roots P 2395532 108577 42deg Heat cDNA P 264.7555 20000451. At CS6879 Shoots-Roots P 2395532 108606 10OuM ABA cDNA P - 10 264.7555 20001554. At Drought Soil Dry P 2395532 2OOOOO46 CS237-vs-Columbia cDNA P 264.7555 20001555 At Drought Soil Dry P 2395532 2OOOOO70 10OuM ABA Mutants cDNA P 264.7555 20001556 At Drought Soil Dry P 2395532 2OOOOO89 2m MSA CS3726- 2649228 108434. At Root Tips cDNA P olumbia cDNAP 2649228 20000326 At Pollen P 2395532 2OOOO144 42deg Heat cDNA P 26S8070 20000439 At Roots P 2395532 2OOOO171 42deg Heat P 15 2721583 20000173 At 42deg Heat P 2395532 2OOOO184 Shoots P 2721583 20000265 At Open Flower P 2395532 2OOOO18S Roots P 2721583 20000458 At 42deg Heat P 2395532 2OOOO234 Siliques P 2721583 20000794. At Petals P 2395532 2OOOO236 Siliques P 2721583 20001555 At Drought Soil Dry P 2395532 2OOOO264 Open Flower P 2721583 20001556 At Drought Soil Dry P 2395532 2OOOO26S Openp Flower P 2O 2721583 20001557 At Droughtg Soil Dryry P 2395532 2OOOO268 100mM NaCl P 2721583 20001558 At Drought Soil Dry P 2395532 2OOOO286 Open Flower P 2721583 20001559 At Drought Soil Dry P 2395532 2OOOO326 Pollen P 2721583 20001560 At Drought Soil Dry P 2395532 2OOOO437 Drought P 3593439 20000173 At 42deg Heat P 2395532 2OOOO438 Shoots P 3593439 20000184 At Shoots P 2395532 2OOOO439 Roots P 3593439 20000185. At Roots P 2395532 2OOOO495 Guard Cells P 25 3593439 2000 1247 At Far-red-induction P 2395532 2OOOOSO6 Wounding P 3593439 2000.1248. At Far-red-induction P 2395532 20000573 10OuM ABA Mutants P 3593439 20001560 At Drought Soil Dry P 2395532 2OOOOST4 10OuM ABA Mutants2 P 361238O 108434. At Root Tips cDNA P 2395532 2OOOO794 Petals P 361238O 108577 At 42deg Heat cDNA P 2395532 2OOO1554 Drought Soil Dry P 361238O 108606. At 10OuM ABA cDNA P 2395532 2OOO1760 50mM NH4NO3 L-to-H. P - 30 361238O 20000046. At CS237-vs-Columbia cDNA P 257582O 108579 4deg Cold cDNA P 361238O 20000111 At 42deg Heat cDNA P 257582O 108590 15mM NH4NO3 L-to-H. cDNA P 361238O 20000113 At 42deg Heat cDNA P 257582O 2OOOO326 Pollen P 361238O 20000144. At 42deg Heat cDNA P 257582O 2OOOO439 Roots P 361238O 20000166 At 10OuM ABA P 257582O 2OOOO495 Guard Cells P 361238O 20000169 At 10OuM ABA P 257582O 2OOO1248 Far-red-induction P 35 361238O 20000173 At 42deg Heat P 257582O 2OOO1557 Drought Soil Dry P 361238O 20000179 At Germinating Seeds P 260O234 2OOO1248 Far-red-induction P 361238O 20000180 At Germinating Seeds P 2603755 2OOOO326 Pollen P 361238O 20000184 At Shoots P 264.0578 2OOOO227 Root-Tips-vs-Tops P 361238O 20000185. At Roots P 264.0578 2OOOO264 Open Flower P 361238O 20000234. At Siliques P 264.0578 2OOOO26S Open Flower P - 40 1361238.0 20000236 At Siliques P 264.0578 2OOOO286 9pen Flower P 361238.0 20000264. At Open Flower P 264.0578 2OOOO326 Pollen 361238.0 20000265 At Open Flower P 264.0578 2OOOO4S1 So shoots-Roots 361238.0 20000286 At Open Flower P 264.0578 2OOOO794 (8IS 361238O 20000436 At Drought P 264.7555 108473 264.7555 108572 E. EDNA - 361238.0 2000.0438. At Shoots P 264.7555 108573 5. cDNA P 45 1361238.0 20000439 At Roots P 264.7555 108577 42deg Heat P 361238O 20000458 At 42deg Heat P 264.7555 108668 2m MSA cDNA P 361238.0 20000495 At Guard Cells P 264.7555 2OOOO111 42deg Heat cDNA P 361238.0 20000573 At 10OuM ABA Mutants P 264.7555 2OOOO173 42deg Heat P 361238.0 20000574 At 10OuM ABA Mutants P 264.7555 2OOOO18S Roots P 361238O 20000794. At Petals P 264.7555 2OOOO236 Siliques P - SO 361238O 20001555 At Drought Soil Dry P 264.7555 2OOOO268 100mM NaCl P

TABLE 5 Utility Section Expt Rep ID Short Name Parameter Value Viability 107881 At Herbicide v2 cDNAP Timepoint (hr) 4 107881 At Herbicide v2 cDNAP Treatment Glean vs. No Treatment 107891 At Herbicide v2 cDNAP Timepoint (hr) 12 107891 At Herbicide v2 cDNAP Treatment Trimec vs. No Treatment Root 108429 At Tissue Specific Expression cDNA P Probe Amount 50 108429 At Tissue Specific Expression cDNA P Probe Method operon 108429 At Tissue Specific Expression cDNA P Tissue Green Flower vs. Whole Plant