(en)The invention relates to plant transcription factor polypeptides, polynucleotides that encode them, homologs from a variety of plant species, and methods of using the polynucleotides and polypeptides to produce transgenic plants having advantageous properties compared to a reference plant. Sequence information related to these polynucleotides and polypeptides can also be used in bioinformatic search methods and is also disclosed.

1.ApplicationNumber: US-72856707-A
1.PublishNumber: US-7635800-B2
2.Date Publish: 20091222
3.Inventor: RATCLIFFE OLIVER
ADAM LUC
HEARD JACQUELINE E.
JIANG CAI-ZHONG
REUBER T. LYNNE
CREELMAN ROBERT A.

4.Inventor Harmonized: RATCLIFFE OLIVER(US)
ADAM LUC(US)
HEARD JACQUELINE E(US)
JIANG CAI-ZHONG(US)
REUBER T LYNNE(US)
CREELMAN ROBERT A(US)

5.Country: US
6.Claims:
(en)The invention relates to plant transcription factor polypeptides, polynucleotides that encode them, homologs from a variety of plant species, and methods of using the polynucleotides and polypeptides to produce transgenic plants having advantageous properties compared to a reference plant. Sequence information related to these polynucleotides and polypeptides can also be used in bioinformatic search methods and is also disclosed.

7.Description:
(en)This application claims the benefit of U.S. Non-provisional application Ser. No. 09/837,444, filed Apr. 18, 2001, U.S. Provisional Application No. 60/310,847, filed Aug. 9, 2001, U.S. Provisional Application No. 60/336,049, filed Dec. 5, 2001, U.S. Provisional Application No. 60/338,692, filed Dec. 11, 2001, and U.S. Non-provisional application Ser. No. 10/171,468, filed Jun. 14, 2002, the entire contents of which are hereby incorporated by reference.


JOINT RESEARCH AGREEMENT
The claimed invention, in the field of functional genomics and the characterization of plant genes for the improvement of plants, was made by or on behalf of Mendel Biotechnology, Inc. and Monsanto Company as a result of activities undertaken within the scope of a joint research agreement in effect on or before the date the claimed invention was made.
FIELD OF THE INVENTION
This invention relates to the field of plant biology. More particularly, the present invention pertains to compositions and methods for phenotypically modifying a plant.
INTRODUCTION
A plant's traits, such as its biochemical, developmental, or phenotypic characteristics, may be controlled through a number of cellular processes. One important way to manipulate that control is through transcription factors—proteins that influence the expression of a particular gene or sets of genes. Transformed and transgenic plants that comprise cells having altered levels of at least one selected transcription factor, for example, possess advantageous or desirable traits. Strategies for manipulating traits by altering a plant cell's transcription factor content can therefore result in plants and crops with commercially valuable properties. Applicants have identified polynucleotides encoding transcription factors, developed numerous transgenic plants using these polynucleotides, and have analyzed the plants for a variety of important traits. In so doing, applicants have identified important polynucleotide and polypeptide sequences for producing commercially valuable plants and crops as well as the methods for making them and using them. Other aspects and embodiments of the invention are described below and can be derived from the teachings of this disclosure as a whole.
BACKGROUND OF THE INVENTION
Transcription factors can modulate gene expression, either increasing or decreasing (inducing or repressing) the rate of transcription. This modulation results in differential levels of gene expression at various developmental stages, in different tissues and cell types, and in response to different exogenous (e.g., environmental) and endogenous stimuli throughout the life cycle of the organism.
Because transcription factors are key controlling elements of biological pathways, altering the expression levels of one or more transcription factors can change entire biological pathways in an organism. For example, manipulation of the levels of selected transcription factors may result in increased expression of economically useful proteins or metabolic chemicals in plants or to improve other agriculturally relevant characteristics. Conversely, blocked or reduced expression of a transcription factor may reduce biosynthesis of unwanted compounds or remove an undesirable trait. Therefore, manipulating transcription factor levels in a plant offers tremendous potential in agricultural biotechnology for modifying a plant's traits.
The present invention provides novel transcription factors useful for modifying a plant's phenotype in desirable ways.
SUMMARY OF THE INVENTION
In a first aspect, the invention relates to a recombinant polynucleotide comprising a nucleotide sequence selected from the group consisting of: (a) a nucleotide sequence encoding a polypeptide comprising a polypeptide sequence selected from those of the Sequence Listing, SEQ ID NOs:2 to 2N, where N=2-561, or those listed in Table 5, or a complementary nucleotide sequence thereof; (b) a nucleotide sequence encoding a polypeptide comprising a variant of a polypeptide of (a) having one or more, or between 1 and about 5, or between 1 and about 10, or between 1 and about 30, conservative amino acid substitutions; (c) a nucleotide sequence comprising a sequence selected from those of SEQ ID NOs:1 to (2N−1), where N=2-561, or those included in Table 5, or a complementary nucleotide sequence thereof; (d) a nucleotide sequence comprising silent substitutions in a nucleotide sequence of (c); (e) a nucleotide sequence which hybridizes under stringent conditions over substantially the entire length of a nucleotide sequence of one or more of: (a), (b), (c), or (d); (f) a nucleotide sequence comprising at least 10 or 15, or at least about 20, or at least about 30 consecutive nucleotides of a sequence of any of (a)-(e), or at least 10 or 15, or at least about 20, or at least about 30 consecutive nucleotides outside of a region encoding a conserved domain of any of (a)-(e); (g) a nucleotide sequence comprising a subsequence or fragment of any of (a)-(f), which subsequence or fragment encodes a polypeptide having a biological activity that modifies a plant's characteristic, functions as a transcription factor, or alters the level of transcription of a gene or transgene in a cell; (h) a nucleotide sequence having at least 31% sequence identity to a nucleotide sequence of any of (a)-(g); (i) a nucleotide sequence having at least 60%, or at least 70%, or at least 80%, or at least 90%, or at least 95% sequence identity to a nucleotide sequence of any of (a)-(g) or a 10 or 15 nucleotide, or at least about 20, or at least about 30 nucleotide region of a sequence of (a)-(g) that is outside of a region encoding a conserved domain; (j) a nucleotide sequence that encodes a polypeptide having at least 31% sequence identity to a polypeptide listed in Table 5, or the Sequence Listing; (k) a nucleotide sequence which encodes a polypeptide having at least 60%, or at least 70%, or at least 80%, or at least 90%, or at least 95% sequence identity to a polypeptide listed in Table 5, or the Sequence Listing; and (l) a nucleotide sequence that encodes a conserved domain of a polypeptide having at least 85%, or at least 90%, or at least 95%, or at least 98% sequence identity to a conserved domain of a polypeptide listed in Table 4 5, or the Sequence Listing. The recombinant polynucleotide may further comprise a constitutive, inducible, or tissue-specific promoter operably linked to the nucleotide sequence. The invention also relates to compositions comprising at least two of the above-described polynucleotides.
In a second aspect, the invention comprises an isolated or recombinant polypeptide comprising a subsequence of at least about 10, or at least about 15, or at least about 20, or at least about 30 contiguous amino acids encoded by the recombinant or isolated polynucleotide described above, or comprising a subsequence of at least about 8, or at least about 12, or at least about 15, or at least about 20, or at least about 30 contiguous amino acids outside a conserved domain.
In a third aspect, the invention comprises an isolated or recombinant polynucleotide that encodes a polypeptide that is a paralog of the isolated polypeptide described above. In one aspect, the invention is an paralog which, when expressed in Arabidopsis , modifies a trait of the Arabidopsis plant.
In a fourth aspect, the invention comprises an isolated or recombinant polynucleotide that encodes a polypeptide that is an ortholog of the isolated polypeptide described above. In one aspect, the invention is an ortholog which, when expressed in Arabidopsis , modifies a trait of the Arabidopsis plant.
In a fifth aspect, the invention comprises an isolated polypeptide that is a paralog of the isolated polypeptide described above. In one aspect, the invention is an paralog which, when expressed in Arabidopsis , modifies a trait of the Arabidopsis plant.
In a sixth aspect, the invention comprises an isolated polypeptide that is an ortholog of the isolated polypeptide described above. In one aspect, the invention is an ortholog which, when expressed in Arabidopsis , modifies a trait of the Arabidopsis plant.
The present invention also encompasses transcription factor variants. A preferred transcription factor variant is one having at least 40% amino acid sequence identity, a more preferred transcription factor variant is one having at least 50% amino acid sequence identity and a most preferred transcription factor variant is one having at least 65% amino acid sequence identity to the transcription factor amino acid sequence SEQ ID NOs:2 to 2N, where N=2-561, and which contains at least one functional or structural characteristic of the transcription factor amino acid sequence. Sequences having lesser degrees of identity but comparable biological activity are considered to be equivalents.
In another aspect, the invention is a transgenic plant comprising one or more of the above-described isolated or recombinant polynucleotides. In yet another aspect, the invention is a plant with altered expression levels of a polynucleotide described above or a plant with altered expression or activity levels of an above-described polypeptide. Further, the invention is a plant lacking a nucleotide sequence encoding a polypeptide described above or substantially lacking a polypeptide described above. The plant may be any plant, including, but not limited to, Arabidopsis , mustard, soybean, wheat, corn, potato, cotton, rice, oilseed rape, sunflower, alfalfa, sugarcane, turf, banana, blackberry, blueberry, strawberry, raspberry, cantaloupe, carrot, cauliflower, coffee, cucumber, eggplant, grapes, honeydew, lettuce, mango, melon, onion, papaya, peas, peppers, pineapple, pumpkin, spinach, squash, sweet corn, tobacco, tomato, watermelon, rosaceous fruits, vegetable brassicas, and mint or other labiates. In yet another aspect, the inventions is an isolated plant material of a plant, including, but not limited to, plant tissue, fruit, seed, plant cell, embryo, protoplast, pollen, and the like. In yet another aspect, the invention is a transgenic plant tissue culture of regenerable cells, including, but not limited to, embryos, meristematic cells, microspores, protoplast, pollen, and the like.
In yet another aspect the invention is a transgenic plant comprising one or more of the above described polynucleotides wherein the encoded polypeptide is expressed and regulates transcription of a gene.
In a further aspect the invention provides a method of using the polynucleotide composition to breed a progeny plant from a transgenic plant including crossing plants, producing seeds from transgenic plants, and methods of breeding using transgenic plants, the method comprising transforming a plant with the polynucleotide composition to create a transgenic plant, crossing the transgenic plant with another plant, selecting seed, and growing the progeny plant from the seed.
In a further aspect, the invention provides a progeny plant derived from a parental plant wherein said progeny plant exhibits at least three fold greater messenger RNA levels than said parental plant, wherein the messenger RNA encodes a DNA-binding protein which is capable of binding to a DNA regulatory sequence and inducing expression of a plant trait gene, wherein the progeny plant is characterized by a change in the plant trait compared to said parental plant. In yet a further aspect, the progeny plant exhibits at least ten fold greater messenger RNA levels compared to said parental plant. In yet a further aspect, the progeny plant exhibits at least fifty fold greater messenger RNA levels compared to said parental plant.
In a further aspect, the invention relates to a cloning or expression vector comprising the isolated or recombinant polynucleotide described above or cells comprising the cloning or expression vector.
In yet a further aspect, the invention relates to a composition produced by incubating a polynucleotide of the invention with a nuclease, a restriction enzyme, a polymerase; a polymerase and a primer; a cloning vector, or with a cell.
Furthermore, the invention relates to a method for producing a plant having a modified trait. The method comprises altering the expression of an isolated or recombinant polynucleotide of the invention or altering the expression or activity of a polypeptide of the invention in a plant to produce a modified plant, and selecting the modified plant for a modified trait. In one aspect, the plant is a monocot plant. In another aspect, the plant is a dicot plant. In another aspect the recombinant polynucleotide is from a dicot plant and the plant is a monocot plant. In yet another aspect the recombinant polynucleotide is from a monocot plant and the plant is a dicot plant. In yet another aspect the recombinant polynucleotide is from a monocot plant and the plant is a monocot plant. In yet another aspect the recombinant polynucleotide is from a dicot plant and the plant is a dicot plant.
In another aspect, the invention is a transgenic plant comprising an isolated or recombinant polynucleotide encoding a polypeptide wherein the polypeptide is selected from the group consisting of SEQ ID NOs: 2-2N, where N=2-561. In yet another aspect, the invention is a plant with altered expression levels of a polypeptide described above or a plant with altered expression or activity levels of an above-described polypeptide. Further, the invention is a plant lacking a polynucleotide sequence encoding a polypeptide described above or substantially lacking a polypeptide described above. The plant may be any plant, including, but not limited to, Arabidopsis , mustard, soybean, wheat, corn, potato, cotton, rice, oilseed rape, sunflower, alfalfa, sugarcane, turf, banana, blackberry, blueberry, strawberry, raspberry, cantaloupe, carrot, cauliflower, coffee, cucumber, eggplant, grapes, honeydew, lettuce, mango, melon, onion, papaya, peas, peppers, pineapple, pumpkin, spinach, squash, sweet corn, tobacco, tomato, watermelon, rosaceous fruits, vegetable brassicas, and mint or other labiates. In yet another aspect, the inventions is an isolated plant material of a plant, including, but not limited to, plant tissue, fruit, seed, plant cell, embryo, protoplast, pollen, and the like. In yet another aspect, the invention is a transgenic plant tissue culture of regenerable cells, including, but not limited to, embryos, meristematic cells, microspores, protoplast, pollen, and the like.
In another aspect, the invention relates to a method of identifying a factor that is modulated by or interacts with a polypeptide encoded by a polynucleotide of the invention. The method comprises expressing a polypeptide encoded by the polynucleotide in a plant; and identifying at least one factor that is modulated by or interacts with the polypeptide. In one embodiment the method for identifying modulating or interacting factors is by detecting binding by the polypeptide to a promoter sequence, or by detecting interactions between an additional protein and the polypeptide in a yeast two hybrid system, or by detecting expression of a factor by hybridization to a microarray, subtractive hybridization, or differential display.
In yet another aspect, the invention is a method of identifying a molecule that modulates activity or expression of a polynucleotide or polypeptide of interest. The method comprises placing the molecule in contact with a plant comprising the polynucleotide or polypeptide encoded by the polynucleotide of the invention and monitoring one or more of the expression level of the polynucleotide in the plant, the expression level of the polypeptide in the plant, and modulation of an activity of the polypeptide in the plant.
In yet another aspect, the invention relates to an integrated system, computer or computer readable medium comprising one or more character strings corresponding to a polynucleotide of the invention, or to a polypeptide encoded by the polynucleotide. The integrated system, computer or computer readable medium may comprise a link between one or more sequence strings to a modified plant trait.
In yet another aspect, the invention is a method for identifying a sequence similar or homologous to one or more polynucleotides of the invention, or one or more polypeptides encoded by the polynucleotides. The method comprises providing a sequence database, and querying the sequence database with one or more target sequences corresponding to the one or more polynucleotides or to the one or more polypeptides to identify one or more sequence members of the database that display sequence similarity or homology to one or more of the one or more target sequences.
The method may further comprise of linking the one or more of the polynucleotides of the invention, or encoded polypeptides, to a modified plant phenotype.



BRIEF DESCRIPTION OF THE SEQUENCE LISTING, AND FIGURE
The Sequence Listing provides exemplary polynucleotide and polypeptide sequences of the invention. The traits associated with the use of the sequences are included in the Examples.
CD-ROM1 (Copy 1) is a read-only memory computer-readable compact disc and contains a copy of the Sequence Listing in ASCII text format. The Sequence Listing is named “Seq Listing.txt” and is 923 kilobytes in size. The copies of the Sequence Listing on the CD-ROM disc are hereby incorporated by reference in their entirety.
CD-ROM2 (Copy 2) is an exact copy of CD-R1 (Copy 1).
CD-ROM3 contains a CRF copy of the Sequence Listing as a text (.txt) file and is 1846 kilobytes in size.
FIG. 1 shows a phylogenic tree of related plant families adapted from Daly et al. (2001 Plant Physiology 127:1328-1333).



DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS
In an important aspect, the present invention relates to polynucleotides and polypeptides, e.g. for modifying phenotypes of plants. Throughout this disclosure, various information sources are referred to and/or are specifically incorporated. The information sources include scientific journal articles, patent documents, textbooks, and World Wide Web browser-inactive page addresses, for example. While the reference to these information sources clearly indicates that they can be used by one of skill in the art, applicants specifically incorporate each and every one of the information sources cited herein, in their entirety, whether or not a specific mention of “incorporation by reference” is noted. The contents and teachings of each and every one of the information sources can be relied on and used to make and use embodiments of the invention.
It must be noted that as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural reference unless the context clearly dictates otherwise. Thus, for example, a reference to “a plant” includes a plurality of such plants, and a reference to “a stress” is a reference to one or more stresses and equivalents thereof known to those skilled in the art, and so forth.
The polynucleotide sequences of the invention encode polypeptides that are members of well-known transcription factor families, including plant transcription factor families, as disclosed in Table 5. Generally, the transcription factors encoded by the present sequences are involved in cell differentiation and proliferation and the regulation of growth. Accordingly, one skilled in the art would recognize that by expressing the present sequences in a plant, one may change the expression of autologous genes or induce the expression of introduced genes. By affecting the expression of similar autologous sequences in a plant that have the biological activity of the present sequences, or by introducing the present sequences into a plant, one may alter a plant's phenotype to one with improved traits. The sequences of the invention may also be used to transform a plant and introduce desirable traits not found in the wild-type cultivar or strain. Plants may then be selected for those that produce the most desirable degree of over- or underexpression of target genes of interest and coincident trait improvement.
The sequences of the present invention may be from any species, particularly plant species, in a naturally occurring form or from any source whether natural, synthetic, semi-synthetic or recombinant. The sequences of the invention may also include fragments of the present amino acid sequences. In this context, a “fragment” refers to a fragment of a polypeptide sequence which is at least 5 to about 15 amino acids in length, most preferably at least 14 amino acids, and which retain some biological activity of a transcription factor. Where “amino acid sequence” is recited to refer to an amino acid sequence of a naturally occurring protein molecule, “amino acid sequence” and like terms are not meant to limit the amino acid sequence to the complete native amino acid sequence associated with the recited protein molecule.
As one of ordinary skill in the art recognizes, transcription factors can be identified by the presence of a region or domain of structural similarity or identity to a specific consensus sequence or the presence of a specific consensus DNA-binding site or DNA-binding site motif (see, for example, Riechmann et al., (2000) Science 290: 2105-2110). The plant transcription factors may belong to one of the following transcription factor families: the AP2 (APETALA2) domain transcription factor family (Riechmann and Meyerowitz (1998) Biol. Chem. 379:633-646); the MYB transcription factor family (Martin and Paz-Ares, (1997) Trends Genet. 13:67-73); the MADS domain transcription factor family (Riechmann and Meyerowitz (1997) Biol. Chem. 378:1079-1101); the WRKY protein family (Ishiguro and Nakamura (1994) Mol. Gen. Genet. 244:563-571); the ankyrin-repeat protein family (Zhang et al. (1992) Plant Cell 4:1575-1588); the zinc finger protein (Z) family (Klug and Schwabe (1995) FASEB J. 9: 597-604); the homeobox (HB) protein family (Buerglin in Guidebook to the Homeobox Genes , Duboule (ed.) (1994) Oxford University Press); the CAAT-element binding proteins (Forsburg and Guarente (1989) Genes Dev. 3:1166-1178); the squamosa promoter binding proteins (SPB) (Klein et al. (1996) Mol. Gen. Genet. 1996 250:7-16); the NAM protein family (Souer et al. (1996) Cell 85:159-170); the IAA/AUX proteins (Rouse et al. (1998) Science 279:1371-1373); the HLH/MYC protein family (Littlewood et al. (1994) Prot. Profile 1:639-709); the DNA-binding protein (DBP) family (Tucker et al. (1994) EMBO J. 13:2994-3002); the bZIP family of transcription factors (Foster et al. (1994) FASEB J. 8:192-200); the Box P-binding protein (the BPF-1) family (da Costa e Silva et al. (1993) Plant J 4:125-135); the high mobility group (HMG) family (Bustin and Reeves (1996) Prog. Nucl. Acids Res. Mol. Biol. 54:35-100); the scarecrow (SCR) family (Di Laurenzio et al. (1996) Cell 86:423-433); the GF14 family (Wu et al. (1997) Plant Physiol. 114:1421-1431); the polycomb (PCOMB) family (Kennison (1995) Annu. Rev. Genet. 29:289-303); the teosinte branched (TEO) family (Luo et al. (1996) Nature 383:794-799; the ABI3 family (Giraudat et al. (1992) Plant Cell 4:1251-1261); the triple helix (TH) family (Dehesh et al. (1990) Science 250:1397-1399); the EIL family (Chao et al. (1997) Cell 89:1133-44); the AT-HOOK family (Reeves and Nissen (1990) J. Biol. Chem. 265:8573-8582); the S1FA family (Zhou et al. (1995) Nucleic Acids Res. 23:1165-1169); the bZIPT2 family (Lu and Ferl (1995) Plant Physiol. 109:723); the YABBY family (Bowman et al. (1999) Development 126:2387-96); the PAZ family (Bohmert et al. (1998) EMBO J. 17:170-80); a family of miscellaneous (MISC) transcription factors including the DPBF family (Kim et al. (1997) Plant J 11:1237-1251) and the SPF1 family (Ishiguro and Nakamura (1994) Mol. Gen. Genet. 244:563-571); the golden (GLD) family (Hall et al. (1998) Plant Cell 10:925-936), the TUBBY family (Boggin et al, (1999) Science 286:2119-2125), the heat shock family (Wu C (1995) Annu Rev Cell Dev Biol 11:441-469), the ENBP family (Christiansen et al (1996) Plant Mol Biol 32:809-821), the RING-zinc family (Jensen et al. (1998) FEBS letters 436:283-287), the PDBP family (Janik et al Virology . (1989) 168:320-329), the PCF family (Cubas P, et al. Plant J . (1999) 18:215-22), the SRS(SHI-related) family (Fridborg et al Plant Cell (1999) 11:1019-1032), the CPP (cysteine-rich polycomb-like) family (Cvitanich et al Proc. Natl. Acad. Sci . USA. (2000) 97:8163-8168), the ARF (auxin response factor) family (Ulmasov, et al. (1999) Proc. Natl. Acad. Sci. USA 96: 5844-5849), the SWI/SNF family (Collingwood et al J. Mol. End. 23:255-275), the ACBF family (Seguin et al (1997) Plant Mol. Biol. 35:281-291), PCGL (CG-1 like) family (da Costa e Silva et al. (1994) Plant Mol Biol. 25:921-924) the ARID family (Vazquez et al. (1999) Development. 126: 733-42), the Jumonji family, Balciunas et al (2000, Trends Biochem Sci. 25: 274-276), the bZIP-NIN family (Schauser et al (1999) Nature 402: 191-195), the E2F family Kaelin et al (1992) Cell 70: 351-364) and the GRF-like family (Knaap et al (2000) Plant Physiol. 122: 695-704). As indicated by any part of the list above and as known in the art, transcription factors have been sometimes categorized by class, family, and sub-family according to their structural content and consensus DNA-binding site motif, for example. Many of the classes and many of the families and sub-families are listed here. However, the inclusion of one sub-family and not another, or the inclusion of one family and not another, does not mean that the invention does not encompass polynucleotides or polypeptides of a certain family or sub-family. The list provided here is merely an example of the types of transcription factors and the knowledge available concerning the consensus sequences and consensus DNA-binding site motifs that help define them as known to those of skill in the art (each of the references noted above are specifically incorporated herein by reference). A transcription factor may include, but is not limited to, any polypeptide that can activate or repress transcription of a single gene or a number of genes. This polypeptide group includes, but is not limited to, DNA-binding proteins, DNA-binding protein binding proteins, protein kinases, protein phosphatases, GTP-binding proteins, and receptors, and the like.
In addition to methods for modifying a plant phenotype by employing one or more polynucleotides and polypeptides of the invention described herein, the polynucleotides and polypeptides of the invention have a variety of additional uses. These uses include their use in the recombinant production (i.e., expression) of proteins; as regulators of plant gene expression, as diagnostic probes for the presence of complementary or partially complementary nucleic acids (including for detection of natural coding nucleic acids); as substrates for further reactions, e.g., mutation reactions, PCR reactions, or the like; as substrates for cloning e.g., including digestion or ligation reactions; and for identifying exogenous or endogenous modulators of the transcription factors. A “polynucleotide” is a nucleic acid sequence comprising a plurality of polymerized nucleotides, e.g., at least about 15 consecutive polymerized nucleotides, optionally at least about 30 consecutive nucleotides, at least about 50 consecutive nucleotides. In many instances, a polynucleotide comprises a nucleotide sequence encoding a polypeptide (or protein) or a domain or fragment thereof. Additionally, the polynucleotide may comprise a promoter, an intron, an enhancer region, a polyadenylation site, a translation initiation site, 5′ or 3′ untranslated regions, a reporter gene, a selectable marker, or the like. The polynucleotide can be single stranded or double stranded DNA or RNA. The polynucleotide optionally comprises modified bases or a modified backbone. The polynucleotide can be, e.g., genomic DNA or RNA, a transcript (such as an mRNA), a cDNA, a PCR product, a cloned DNA, a synthetic DNA or RNA, or the like. The polynucleotide can comprise a sequence in either sense or antisense orientations.
A “recombinant polynucleotide” is a polynucleotide that is not in its native state, e.g., the polynucleotide comprises a nucleotide sequence not found in nature, or the polynucleotide is in a context other than that in which it is naturally found, e.g., separated from nucleotide sequences with which it typically is in proximity in nature, or adjacent (or contiguous with) nucleotide sequences with which it typically is not in proximity. For example, the sequence at issue can be cloned into a vector, or otherwise recombined with one or more additional nucleic acid.
An “isolated polynucleotide” is a polynucleotide whether naturally occurring or recombinant, that is present outside the cell in which it is typically found in nature, whether purified or not. Optionally, an isolated polynucleotide is subject to one or more enrichment or purification procedures, e.g., cell lysis, extraction, centrifugation, precipitation, or the like.
A “polypeptide” is an amino acid sequence comprising a plurality of consecutive polymerized amino acid residues e.g., at least about 15 consecutive polymerized amino acid residues, optionally at least about 30 consecutive polymerized amino acid residues, at least about 50 consecutive polymerized amino acid residues. In many instances, a polypeptide comprises a polymerized amino acid residue sequence that is a transcription factor or a domain or portion or fragment thereof. Additionally, the polypeptide may comprise a localization domain, 2) an activation domain, 3) a repression domain, 4) an oligomerization domain or 5) a DNA-binding domain, or the like. The polypeptide optionally comprises modified amino acid residues, naturally occurring amino acid residues not encoded by a codon, non-naturally occurring amino acid residues.
A “recombinant polypeptide” is a polypeptide produced by translation of a recombinant polynucleotide. A “synthetic polypeptide” is a polypeptide created by consecutive polymerization of isolated amino acid residues using methods well known in the art. An “isolated polypeptide,” whether a naturally occurring or a recombinant polypeptide, is more enriched in (or out of) a cell than the polypeptide in its natural state in a wild type cell, e.g., more than about 5% enriched, more than about 10% enriched, or more than about 20%, or more than about 50%, or more, enriched, i.e., alternatively denoted: 105%, 110%, 120%, 150% or more, enriched relative to wild type standardized at 100%. Such an enrichment is not the result of a natural response of a wild type plant. Alternatively, or additionally, the isolated polypeptide is separated from other cellular components with which it is typically associated, e.g., by any of the various protein purification methods herein.
“Identity” or “similarity” refers to sequence similarity between two polynucleotide sequences or between two polypeptide sequences, with identity being a more strict comparison. The phrases “percent identity” and “% identity” refer to the percentage of sequence similarity found in a comparison of two or more polynucleotide sequences or two or more polypeptide sequences. Identity or similarity can be determined by comparing a position in each sequence that may be aligned for purposes of comparison. When a position in the compared sequence is occupied by the same nucleotide base or amino acid, then the molecules are identical at that position. A degree of similarity or identity between polynucleotide sequences is a function of the number of identical or matching nucleotides at positions shared by the polynucleotide sequences. A degree of identity of polypeptide sequences is a function of the number of identical amino acids at positions shared by the polypeptide sequences. A degree of homology or similarity of polypeptide sequences is a function of the number of amino acids, i.e., structurally related, at positions shared by the polypeptide sequences.
“Altered” nucleic acid sequences encoding polypeptide include those sequences with deletions, insertions, or substitutions of different nucleotides, resulting in a polynucleotide encoding a polypeptide with at least one functional characteristic of the polypeptide. Included within this definition are polymorphisms that may or may not be readily detectable using a particular oligonucleotide probe of the polynucleotide encoding polypeptide, and improper or unexpected hybridization to allelic variants, with a locus other than the normal chromosomal locus for the polynucleotide sequence encoding polypeptide. The encoded polypeptide protein may also be “altered”, and may contain deletions, insertions, or substitutions of amino acid residues that produce a silent change and result in a functionally equivalent polypeptide. Deliberate amino acid substitutions may be made on the basis of similarity in residue side chain chemistry, including, but not limited to, polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues, as long as the biological activity of polypeptide is retained. For example, negatively charged amino acids may include aspartic acid and glutamic acid, positively charged amino acids may include lysine and arginine, and amino acids with uncharged polar head groups having similar hydrophilicity values may include leucine, isoleucine, and valine; glycine and alanine; asparagine and glutamine; serine and threonine; and phenylalanine and tyrosine. Alignments between different polypeptide sequences may be used to calculate “percentage sequence similarity”.
The term “plant” includes whole plants, shoot vegetative organs/structures (e.g., leaves, stems and tubers), roots, flowers and floral organs/structures (e.g., bracts, sepals, petals, stamens, carpels, anthers and ovules), seed (including embryo, endosperm, and seed coat) and fruit (the mature ovary), plant tissue (e.g., vascular tissue, ground tissue, and the like) and cells (e.g., guard cells, egg cells, and the like), and progeny of same. The class of plants that can be used in the method of the invention is generally as broad as the class of higher and lower plants amenable to transformation techniques, including angiosperms (monocotyledonous and dicotyledonous plants), gymnosperms, ferns, horsetails, psilophytes, lycophytes, bryophytes, and multicellular algae. (See for example, FIG. 1 , adapted from Daly et al. 2001 Plant Physiology 127:1328-1333; and see also Tudge, C., The Variety of Life , Oxford University Press, New York, 2000, pp. 547-606.)
A “transgenic plant” refers to a plant that contains genetic material not found in a wild type plant of the same species, variety or cultivar. The genetic material may include a transgene, an insertional mutagenesis event (such as by transposon or T-DNA insertional mutagenesis), an activation tagging sequence, a mutated sequence, a homologous recombination event or a sequence modified by chimeraplasty. Typically, the foreign genetic material has been introduced into the plant by human manipulation, but any method can be used as one of skill in the art recognizes.
A transgenic plant may contain an expression vector or cassette. The expression cassette typically comprises a polypeptide-encoding sequence operably linked (i.e., under regulatory control of) to appropriate inducible or constitutive regulatory sequences that allow for the expression of polypeptide. The expression cassette can be introduced into a plant by transformation or by breeding after transformation of a parent plant. A plant refers to a whole plant as well as to a plant part, such as seed, fruit, leaf, or root, plant tissue, plant cells or any other plant material, e.g., a plant explant, as well as to progeny thereof, and to in vitro systems that mimic biochemical or cellular components or processes in a cell.
“Ectopic expression or altered expression” in reference to a polynucleotide indicates that the pattern of expression in, e.g., a transgenic plant or plant tissue, is different from the expression pattern in a wild type plant or a reference plant of the same species. The pattern of expression may also be compared with a reference expression pattern in a wild type plant of the same species. For example, the polynucleotide or polypeptide is expressed in a cell or tissue type other than a cell or tissue type in which the sequence is expressed in the wild type plant, or by expression at a time other than at the time the sequence is expressed in the wild type plant, or by a response to different inducible agents, such as hormones or environmental signals, or at different expression levels (either higher or lower) compared with those found in a wild type plant. The term also refers to altered expression patterns that are produced by lowering the levels of expression to below the detection level or completely abolishing expression. The resulting expression pattern can be transient or stable, constitutive or inducible. In reference to a polypeptide, the term “ectopic expression or altered expression” further may relate to altered activity levels resulting from the interactions of the polypeptides with exogenous or endogenous modulators or from interactions with factors or as a result of the chemical modification of the polypeptides.
A “fragment” or “domain,” with respect to a polypeptide, refers to a subsequence of the polypeptide. In some cases, the fragment or domain, is a subsequence of the polypeptide which performs at least one biological function of the intact polypeptide in substantially the same manner, or to a similar extent, as does the intact polypeptide. For example, a polypeptide fragment can comprise a recognizable structural motif or functional domain such as a DNA-binding site or domain that binds to a DNA promoter region, an activation domain, or a domain for protein-protein interactions. Fragments can vary in size from as few as 6 amino acids to the full length of the intact polypeptide, but are preferably at least about 30 amino acids in length and more preferably at least about 60 amino acids in length. In reference to a polynucleotide sequence, “a fragment” refers to any subsequence of a polynucleotide, typically, of at least about 15 consecutive nucleotides, preferably at least about 30 nucleotides, more preferably at least about 50 nucleotides, of any of the sequences provided herein.
The invention also encompasses production of DNA sequences that encode transcription factors and transcription factor derivatives, or fragments thereof, entirely by synthetic chemistry. After production, the synthetic sequence may be inserted into any of the many available expression vectors and cell systems using reagents well known in the art. Moreover, synthetic chemistry may be used to introduce mutations into a sequence encoding transcription factors or any fragment thereof.
A “conserved domain”, with respect to a polypeptide, refers to a domain within a transcription factor family which exhibits a higher degree of sequence homology, such as at least 65% sequence identity including conservative substitutions, and preferably at least 80% sequence identity, and more preferably at least 85%, or at least about 86%, or at least about 87%, or at least about 88%, or at least about 90%, or at least about 95%, or at least about 98% amino acid residue sequence identity of a polypeptide of consecutive amino acid residues. A fragment or domain can be referred to as outside a consensus sequence or outside a consensus DNA-binding site that is known to exist or that exists for a particular transcription factor class, family, or sub-family. In this case, the fragment or domain will not include the exact amino acids of a consensus sequence or consensus DNA-binding site of a transcription factor class, family or sub-family, or the exact amino acids of a particular transcription factor consensus sequence or consensus DNA-binding site. Furthermore, a particular fragment, region, or domain of a polypeptide, or a polynucleotide encoding a polypeptide, can be “outside a conserved domain” if all the amino acids of the fragment, region, or domain fall outside of a defined conserved domain(s) for a polypeptide or protein. The conserved domains for each of polypeptides of SEQ ID NOs:2-2N, where N=2-561, are listed in Table 5 as described in Example VII. Also, many of the polypeptides of Table 5 have conserved domains specifically indicated by start and stop sites. A comparison of the regions of the polypeptides in SEQ ID NOs:2-2N, where N=2-561, or of those in Table 5, allows one of skill in the art to identify conserved domain(s) for any of the polypeptides listed or referred to in this disclosure, including those in Table 5.
A “trait” refers to a physiological, morphological, biochemical, or physical characteristic of a plant or particular plant material or cell. In some instances, this characteristic is visible to the human eye, such as seed or plant size, or can be measured by biochemical techniques, such as detecting the protein, starch, or oil content of seed or leaves, or by observation of a metabolic or physiological process, e.g. by measuring uptake of carbon dioxide, or by the observation of the expression level of a gene or genes, e.g., by employing Northern analysis, RT-PCR, microarray gene expression assays, or reporter gene expression systems, or by agricultural observations such as stress tolerance, yield, or pathogen tolerance. Any technique can be used to measure the amount of, comparative level of, or difference in any selected chemical compound or macromolecule in the transgenic plants, however.
“Trait modification” refers to a detectable difference in a characteristic in a plant ectopically expressing a polynucleotide or polypeptide of the present invention relative to a plant not doing so, such as a wild type plant. In some cases, the trait modification can be evaluated quantitatively. For example, the trait modification can entail at least about a 2% increase or decrease in an observed trait (difference), at least a 5% difference, at least about a 10% difference, at least about a 20% difference, at least about a 30%, at least about a 50%, at least about a 70%, or at least about a 100%, or an even greater difference compared with a wild type plant. It is known that there can be a natural variation in the modified trait. Therefore, the trait modification observed entails a change of the normal distribution of the trait in the plants compared with the distribution observed in wild type plant.
I. TRAITS WHICH MAY BE MODIFIED
Trait modifications of particular interest include those to seed (such as embryo or endosperm), fruit, root, flower, leaf, stem, shoot, seedling or the like, including: enhanced tolerance to environmental conditions including freezing, chilling, heat, drought, water saturation, radiation and ozone; improved tolerance to microbial, fungal or viral diseases; improved tolerance to pest infestations, including nematodes, mollicutes, parasitic higher plants or the like; decreased herbicide sensitivity; improved tolerance of heavy metals or enhanced ability to take up heavy metals; improved growth under poor photoconditions (e.g., low light and/or short day length), or changes in expression levels of genes of interest. Other phenotype that can be modified relate to the production of plant metabolites, such as variations in the production of taxol, tocopherol, tocotrienol, sterols, phytosterols, vitamins, wax monomers, anti-oxidants, amino acids, lignins, cellulose, tannins, prenyllipids (such as chlorophylls and carotenoids), glucosinolates, and terpenoids, enhanced or compositionally altered protein or oil production (especially in seeds), or modified sugar (insoluble or soluble) and/or starch composition. Physical plant characteristics that can be modified include cell development (such as the number of trichomes), fruit and seed size and number, yields of plant parts such as stems, leaves, inflorescences, and roots, the stability of the seeds during storage, characteristics of the seed pod (e.g., susceptibility to shattering), root hair length and quantity, internode distances, or the quality of seed coat. Plant growth characteristics that can be modified include growth rate, germination rate of seeds, vigor of plants and seedlings, leaf and flower senescence, male sterility, apomixis, flowering time, flower abscission, rate of nitrogen uptake, osmotic sensitivity to soluble sugar concentrations, biomass or transpiration characteristics, as well as plant architecture characteristics such as apical dominance, branching patterns, number of organs, organ identity, organ shape or size.
Transcription Factors Modify Expression of Endogenous Genes
Expression of genes which encode transcription factors that modify expression of endogenous genes, polynucleotides, and proteins are well known in the art. In addition, transgenic plants comprising isolated polynucleotides encoding transcription factors may also modify expression of endogenous genes, polynucleotides, and proteins. Examples include Peng et al. (1997, Genes and Development 11:3194-3205) and Peng et al. (1999, Nature, 400:256-261). In addition, many others have demonstrated that an Arabidopsis transcription factor expressed in an exogenous plant species elicits the same or very similar phenotypic response. See, for example, Fu et al. (2001, Plant Cell 13:1791-1802); Nandi et al. (2000, Curr. Biol. 10:215-218); Coupland (1995, Nature 377:482-483); and Weigel and Nilsson (1995, Nature 377:482-500).
In another example, Mandel et al. (1992, Cell 71-133-143) and Suzuki et al. (2001, Plant J. 28:409-418) teach that a transcription factor expressed in another plant species elicits the same or very similar phenotypic response of the endogenous sequence, as often predicted in earlier studies of Arabidopsis transcription factors in Arabidopsis (see Mandel et al., 1992, supra; Suzuki et al., 2001, supra).
Other examples include Müller et al. (2001, Plant J. 28:169-179); Kim et al. (2001, Plant J. 25:247-259); Kyozuka and Shimamoto (2002, Plant Cell Physiol. 43:130-135); Boss and Thomas (2002, Nature, 416:847-850); He et al. (2000, Transgenic Res., 9:223-227); and Robson et al. (2001, Plant J. 28:619-631).
In yet another example, Gilmour et al. (1998, Plant J. 16:433-442) teach an Arabidopsis AP2 transcription factor, CBF1, which, when overexpressed in transgenic plants, increases plant freezing tolerance. Jaglo et al (2001, Plant Physiol. 127:910-017) further identified sequences in Brassica napus which encode CBF-like genes and that transcripts for these genes accumulated rapidly in response to low temperature. Transcripts encoding CBF-like proteins were also found to accumulate rapidly in response to low temperature in wheat, as well as in tomato. An alignment of the CBF proteins from Arabidopsis, B. napus , wheat, rye, and tomato revealed the presence of conserved amino acid sequences, PKK/RPAGRxKFxETRHP and DSAWR, that bracket the AP2/EREBP DNA binding domains of the proteins and distinguish them from other members of the AP2/EREBP protein family. (See Jaglo et al., supra.)
III. POLYPEPTIDES AND POLYNUCLEOTIDES OF THE INVENTION
The present invention provides, among other things, transcription factors (TFs), and transcription factor homologue polypeptides, and isolated or recombinant polynucleotides encoding the polypeptides, or novel variant polypeptides or polynucleotides encoding novel variants of transcription factors derived from the specific sequences provided here. These polypeptides and polynucleotides may be employed to modify a plant's characteristic. Exemplary polynucleotides encoding the polypeptides of the invention were identified in the Arabidopsis thaliana GenBank database using publicly available sequence analysis programs and parameters. Sequences initially identified were then further characterized to identify sequences comprising specified sequence strings corresponding to sequence motifs present in families of known transcription factors. In addition, further exemplary polynucleotides encoding the polypeptides of the invention were identified in the plant GenBank database using publicly available sequence analysis programs and parameters. Sequences initially identified were then further characterized to identify sequences comprising specified sequence strings corresponding to sequence motifs present in families of known transcription factors. Polynucleotide sequences meeting such criteria were confirmed as transcription factors.
Additional polynucleotides of the invention were identified by screening Arabidopsis thaliana and/or other plant cDNA libraries with probes corresponding to known transcription factors under low stringency hybridization conditions. Additional sequences, including full length coding sequences were subsequently recovered by the rapid amplification of cDNA ends (RACE) procedure, using a commercially available kit according to the manufacturer's instructions. Where necessary, multiple rounds of RACE are performed to isolate 5′ and 3′ ends. The full length cDNA was then recovered by a routine end-to-end polymerase chain reaction (PCR) using primers specific to the isolated 5′ and 3′ ends. Exemplary sequences are provided in the Sequence Listing.
The polynucleotides of the invention can be or were ectopically expressed in overexpressor or knockout plants and the changes in the characteristic(s) or trait(s) of the plants observed. Therefore, the polynucleotides and polypeptides can be employed to improve the characteristics of plants.
The polynucleotides of the invention can be or were ectopically expressed in overexpressor plant cells and the changes in the expression levels of a number of genes, polynucleotides, and/or proteins of the plant cells observed. Therefore, the polynucleotides and polypeptides can be employed to change expression levels of a genes, polynucleotides, and/or proteins of plants.
IV. PRODUCING POLYPEPTIDES
The polynucleotides of the invention include sequences that encode transcription factors and transcription factor homologue polypeptides and sequences complementary thereto, as well as unique fragments of coding sequence, or sequence complementary thereto. Such polynucleotides can be, e.g., DNA or RNA, e.g., mRNA, cRNA, synthetic RNA, genomic DNA, cDNA synthetic DNA, oligonucleotides, etc. The polynucleotides are either double-stranded or single-stranded, and include either, or both sense (i.e., coding) sequences and antisense (i.e., non-coding, complementary) sequences. The polynucleotides include the coding sequence of a transcription factor, or transcription factor homologue polypeptide, in isolation, in combination with additional coding sequences (e.g., a purification tag, a localization signal, as a fusion-protein, as a pre-protein, or the like), in combination with non-coding sequences (e.g., introns or inteins, regulatory elements such as promoters, enhancers, terminators, and the like), and/or in a vector or host environment in which the polynucleotide encoding a transcription factor or transcription factor homologue polypeptide is an endogenous or exogenous gene.
A variety of methods exist for producing the polynucleotides of the invention. Procedures for identifying and isolating DNA clones are well known to those of skill in the art, and are described in, e.g., Berger and Kimmel, Guide to Molecular Cloning Techniques Methods in Enzymology volume 152 Academic Press, Inc., San Diego, Calif. (“Berger”); Sambrook et al., Molecular Cloning—A Laboratory Manual (2nd Ed.), Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989 (“Sambrook”) and Current Protocols in Molecular Biology , F. M. Ausubel et al., eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., (supplemented through 2000) (“Ausubel”).
Alternatively, polynucleotides of the invention, can be produced by a variety of in vitro amplification methods adapted to the present invention by appropriate selection of specific or degenerate primers. Examples of protocols sufficient to direct persons of skill through in vitro amplification methods, including the polymerase chain reaction (PCR) the ligase chain reaction (LCR), Qbeta-replicase amplification and other RNA polymerase mediated techniques (e.g., NASBA), e.g., for the production of the homologous nucleic acids of the invention are found in Berger (supra), Sambrook (supra), and Ausubel (supra), as well as Mullis et al., (1987) PCR Protocols A Guide to Methods and Applications (Innis et al. eds) Academic Press Inc. San Diego, Calif. (1990) (Innis). Improved methods for cloning in vitro amplified nucleic acids are described in Wallace et al., U.S. Pat. No. 5,426,039. Improved methods for amplifying large nucleic acids by PCR are summarized in Cheng et al. (1994) Nature 369: 684-685 and the references cited therein, in which PCR amplicons of up to 40 kb are generated. One of skill will appreciate that essentially any RNA can be converted into a double stranded DNA suitable for restriction digestion, PCR expansion and sequencing using reverse transcriptase and a polymerase. See, e.g., Ausubel, Sambrook and Berger, all supra.
Alternatively, polynucleotides and oligonucleotides of the invention can be assembled from fragments produced by solid-phase synthesis methods. Typically, fragments of up to approximately 100 bases are individually synthesized and then enzymatically or chemically ligated to produce a desired sequence, e.g., a polynucleotide encoding all or part of a transcription factor. For example, chemical synthesis using the phosphoramidite method is described, e.g., by Beaucage et al. (1981) Tetrahedron Letters 22:1859-1869; and Matthes et al. (1984) EMBO J. 3:801-805. According to such methods, oligonucleotides are synthesized, purified, annealed to their complementary strand, ligated and then optionally cloned into suitable vectors. And if so desired, the polynucleotides and polypeptides of the invention can be custom ordered from any of a number of commercial suppliers.
V. HOMOLOGOUS SEQUENCES
Sequences homologous, i.e., that share significant sequence identity or similarity, to those provided in the Sequence Listing, derived from Arabidopsis thaliana or from other plants of choice are also an aspect of the invention. Homologous sequences can be derived from any plant including monocots and dicots and in particular agriculturally important plant species, including but not limited to, crops such as soybean, wheat, corn, potato, cotton, rice, rape, oilseed rape (including canola), sunflower, alfalfa, sugarcane and turf; or fruits and vegetables, such as banana, blackberry, blueberry, strawberry, and raspberry, cantaloupe, carrot, cauliflower, coffee, cucumber, eggplant, grapes, honeydew, lettuce, mango, melon, onion, papaya, peas, peppers, pineapple, pumpkin, spinach, squash, sweet corn, tobacco, tomato, watermelon, rosaceous fruits (such as apple, peach, pear, cherry and plum) and vegetable brassicas (such as broccoli, cabbage, cauliflower, Brussels sprouts, and kohlrabi). Other crops, fruits and vegetables whose phenotype can be changed include barley, rye, millet, sorghum, currant, avocado, citrus fruits such as oranges, lemons, grapefruit and tangerines, artichoke, cherries, nuts such as the walnut and peanut, endive, leek, roots, such as arrowroot, beet, cassaya, turnip, radish, yam, and sweet potato, and beans. The homologous sequences may also be derived from woody species, such pine, poplar and eucalyptus, or mint or other labiates.
Orthologs And Paralogs
Several different methods are known by those of skill in the art for identifying and defining these functionally homologous sequences. Three general methods for defining paralogs and orthologs are described; a paralog or ortholog or homolog may be identified by one or more of the methods described below.
Orthologs and paralogs are evolutionarily related genes that have similar sequence and similar functions. Orthologs are structurally related genes in different species that are derived from a speciation event. Paralogs are structurally related genes within a single species that are derived by a duplication event.
Within a single plant species, gene duplication may cause two copies of a particular gene, giving rise to two or more genes with similar sequence and similar function known as paralogs. A paralog is therefore a similar gene with a similar function within the same species. Paralogs typically cluster together or in the same lade (a group of similar genes) when a gene family phylogeny is analyzed using programs such as CLUSTAL (Thompson et al. (1994) Nucleic Acids Res. 22:4673-4680; Higgins et al. (1996) Methods Enzymol. 266 383-402). Groups of similar genes can also be identified with pair-wise BLAST analysis (Feng and Doolittle (1987) J. Mol. Evol. 25:351-360). For example, a lade of very similar MADS domain transcription factors from Arabidopsis all share a common function in flowering time (Ratcliffe et al. (2001) Plant Physiol. 126:122-132), and a group of very similar AP2 domain transcription factors from Arabidopsis are involved in tolerance of plants to freezing (Gilmour et al. (1998) Plant J. 16:433-442). Analysis of groups of similar genes with similar function that fall within one clade can yield sub-sequences that are particular to the clade. These sub-sequences, known as consensus sequences, can not only be used to define the sequences within each clade, but define the functions of these genes; genes within a clade may contain paralogous or orthologous sequences that share the same function. (See also, for example, Mount, D. W. (2001) Bioinformatics: Sequence and Genome Analysis Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. page 543.)
Speciation, the production of new species from a parental species, can also give rise to two or more genes with similar sequence and similar function. These genes, termed orthologs, often have an identical function within their host plants and are often interchangeable between species without losing function. Because plants have common ancestors, many genes in any plant species will have a corresponding orthologous gene in another plant species. Once a phylogenic tree for a gene family of one species has been constructed using a program such as CLUSTAL (Thompson et al. (1994) Nucleic Acids Res. 22:4673-4680; Higgins et al. (1996) Methods Enzymol. 266:383-402), potential orthologous sequences can placed into the phylogenetic tree and its relationship to genes from the species of interest can be determined. Once the ortholog pair has been identified, the function of the test ortholog can be determined by determining the function of the reference ortholog.
Transcription factors that are homologous to the listed sequences will typically share at least about 30% amino acid sequence identity, or at least about 30% amino acid sequence identity outside of a known consensus sequence or consensus DNA-binding site. More closely related transcription factors can share at least about 50%, about 60%, about 65%, about 70%, about 75% or about 80% or about 90% or about 95% or about 98% or more sequence identity with the listed sequences, or with the listed sequences but excluding or outside a known consensus sequence or consensus DNA-binding site, or with the listed sequences excluding one or all conserved domain. Factors that are most closely related to the listed sequences share, e.g., at least about 85%, about 90% or about 95% or more % sequence identity to the listed sequences, or to the listed sequences but excluding or outside a known consensus sequence or consensus DNA-binding site or outside one or all conserved domain. At the nucleotide level, the sequences will typically share at least about 40% nucleotide sequence identity, preferably at least about 50%, about 60%, about 70% or about 80% sequence identity, and more preferably about 85%, about 90%, about 95% or about 97% or more sequence identity to one or more of the listed sequences, or to a listed sequence but excluding or outside a known consensus sequence or consensus DNA-binding site, or outside one or all conserved domain. The degeneracy of the genetic code enables major variations in the nucleotide sequence of a polynucleotide while maintaining the amino acid sequence of the encoded protein. Conserved domains within a transcription factor family may exhibit a higher degree of sequence homology, such as at least 65% sequence identity including conservative substitutions, and preferably at least 80% sequence identity, and more preferably at least 85%, or at least about 86%, or at least about 87%, or at least about 88%, or at least about 90%, or at least about 95%, or at least about 98% sequence identity. Transcription factors that are homologous to the listed sequences should share at least 30%, or at least about 60%, or at least about 75%, or at least about 80%, or at least about 90%, or at least about 95% amino acid sequence identity over the entire length of the polypeptide or the homolog. In addition, transcription factors that are homologous to the listed sequences should share at least 30%, or at least about 60%, or at least about 75%, or at least about 80%, or at least about 90%, or at least about 95% amino acid sequence similarity over the entire length of the polypeptide or the homolog.
Percent identity can be determined electronically, e.g., by using the MEGALIGN program (DNASTAR, Inc. Madison, Wis.). The MEGALIGN program can create alignments between two or more sequences according to different methods, e.g., the clustal method. (See, e.g., Higgins, D. G. and P. M. Sharp (1988) Gene 73:237-244.) The clustal algorithm groups sequences into clusters by examining the distances between all pairs. The clusters are aligned pairwise and then in groups. Other alignment algorithms or programs may be used, including FASTA, BLAST, or ENTREZ, FASTA and BLAST. These are available as a part of the GCG sequence analysis package (University of Wisconsin, Madison, Wis.), and can be used with or without default settings. ENTREZ is available through the National Center for Biotechnology Information. In one embodiment, the percent identity of two sequences can be determined by the GCG program with a gap weight of 1, e.g., each amino acid gap is weighted as if it were a single amino acid or nucleotide mismatch between the two sequences (see U.S. Pat. No. 6,262,333).
Other techniques for alignment are described in Methods in Enzymology, vol. 266: Computer Methods for Macromolecular Sequence Analysis (1996), ed. Doolittle, Academic Press, Inc., San Diego, Calif., USA. Preferably, an alignment program that permits gaps in the sequence is utilized to align the sequences. The Smith-Waterman is one type of algorithm that permits gaps in sequence alignments. See Methods Mol. Biol. 70: 173-187 (1997). Also, the GAP program using the Needleman and Wunsch alignment method can be utilized to align sequences. An alternative search strategy uses MPSRCH software, which runs on a MASPAR computer. MPSRCH uses a Smith-Waterman algorithm to score sequences on a massively parallel computer. This approach improves ability to pick up distantly related matches, and is especially tolerant of small gaps and nucleotide sequence errors. Nucleic acid-encoded amino acid sequences can be used to search both protein and DNA databases.
The percentage similarity between two polypeptide sequences, e.g., sequence A and sequence B, is calculated by dividing the length of sequence A, minus the number of gap residues in sequence A, minus the number of gap residues in sequence B, into the sum of the residue matches between sequence A and sequence B, times one hundred. Gaps of low or of no similarity between the two amino acid sequences are not included in determining percentage similarity. Percent identity between polynucleotide sequences can also be counted or calculated by other methods known in the art, e.g., the Jotun Hein method. (See, e.g., Hein, J. (1990) Methods Enzymol. 183:626-645.) Identity between sequences can also be determined by other methods known in the art, e.g., by varying hybridization conditions (see US Patent Application No. 20010010913).
Thus, the invention provides methods for identifying a sequence similar or paralogous or orthologous or homologous to one or more polynucleotides as noted herein, or one or more target polypeptides encoded by the polynucleotides, or otherwise noted herein and may include linking or associating a given plant phenotype or gene function with a sequence. In the methods, a sequence database is provided (locally or across an inter or intra net) and a query is made against the sequence database using the relevant sequences herein and associated plant phenotypes or gene functions.
In addition, one or more polynucleotide sequences or one or more polypeptides encoded by the polynucleotide sequences may be used to search against a BLOCKS (Bairoch et al. (1997) Nucleic Acids Res. 25:217-221), PFAM, and other databases which contain previously identified and annotated motifs, sequences and gene functions. Methods that search for primary sequence patterns with secondary structure gap penalties (Smith et al. (1992) Protein Engineering 5:35-51) as well as algorithms such as Basic Local Alignment Search Tool (BLAST; Altschul, S. F. (1993) J. Mol. Evol. 36:290-300; Altschul et al. (1990) supra), BLOCKS (Henikoff, S, and Henikoff, G. J. (1991) Nucleic Acids Research 19:6565-6572), Hidden Markov Models (HMM; Eddy, S. R. (1996) Cur. Opin. Str. Biol. 6:361-365; Sonnhammer et al. (1997) Proteins 28:405-420), and the like, can be used to manipulate and analyze polynucleotide and polypeptide sequences encoded by polynucleotides. These databases, algorithms and other methods are well known in the art and are described in Ausubel et al. (1997; Short Protocols in Molecular Biology, John Wiley & Sons, New York N.Y., unit 7.7) and in Meyers, R. A. (1995; Molecular Biology and Biotechnology, Wiley VCH, New York N.Y., p 856-853).
Furthermore, methods using manual alignment of sequences similar or homologous to one or more polynucleotide sequences or one or more polypeptides encoded by the polynucleotide sequences may be used to identify regions of similarity and conserved domains. Such manual methods are well-known of those of skill in the art and can include, for example, comparisons of tertiary structure between a polypeptide sequence encoded by a polynucleotide which comprises a known function with a polypeptide sequence encoded by a polynucleotide sequence which has a function not yet determined. Such examples of tertiary structure may comprise predicted alpha helices, beta-sheets, amphipathic helices, leucine zipper motifs, zinc finger motifs, proline-rich regions, cysteine repeat motifs, and the like.
VI. IDENTIFYING POLYNUCLEOTIDES OR NUCLEIC ACIDS BY HYBRIDIZATION
Polynucleotides homologous to the sequences illustrated in the Sequence Listing and tables can be identified, e.g., by hybridization to each other under stringent or under highly stringent conditions. Single stranded polynucleotides hybridize when they associate based on a variety of well characterized physical-chemical forces, such as hydrogen bonding, solvent exclusion, base stacking and the like. The stringency of a hybridization reflects the degree of sequence identity of the nucleic acids involved, such that the higher the stringency, the more similar are the two polynucleotide strands. Stringency is influenced by a variety of factors, including temperature, salt concentration and composition, organic and non-organic additives, solvents, etc. present in both the hybridization and wash solutions and incubations (and number thereof), as described in more detail in the references cited above. Encompassed by the invention are polynucleotide sequences that are capable of hybridizing to the claimed polynucleotide sequences, and, in particular, to those shown in SEQ ID NOs: 860; 802; 240; 274; 558; 24; 1120; 44; 460; 286; 120; 130; 134; 698; 832; 580; 612; 48, and fragments thereof under various conditions of stringency. (See, e.g., Wahl, G. M. and S. L. Berger (1987) Methods Enzymol. 152:399-407; Kimmel, A. R. (1987) Methods Enzymol. 152:507-511.) Estimates of homology are provided by either DNA-DNA or DNA-RNA hybridization under conditions of stringency as is well understood by those skilled in the art (Hames and Higgins, Eds. (1985) Nucleic Acid Hybridisation, IRL Press, Oxford, U.K.). Stringency conditions can be adjusted to screen for moderately similar fragments, such as homologous sequences from distantly related organisms, to highly similar fragments, such as genes that duplicate functional enzymes from closely related organisms. Post-hybridization washes determine stringency conditions.
In addition to the nucleotide sequences listed in Tables 4 and 5, full length cDNA, orthologs, paralogs and homologs of the present nucleotide sequences may be identified and isolated using well known methods. The cDNA libraries orthologs, paralogs and homologs of the present nucleotide sequences may be screened using hybridization methods to determine their utility as hybridization target or amplification probes.
An example of stringent hybridization conditions for hybridization of complementary nucleic acids which have more than 100 complementary residues on a filter in a Southern or northern blot is about 5° C. to 20° C. lower than the thermal melting point (T m ) for the specific sequence at a defined ionic strength and pH. The T m is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. Nucleic acid molecules that hybridize under stringent conditions will typically hybridize to a probe based on either the entire cDNA or selected portions, e.g., to a unique subsequence, of the cDNA under wash conditions of 0.2×SSC to 2.0×SSC, 0.1% SDS at 50-65° C. For example, high stringency is about 0.2×SSC, 0.1% SDS at 65° C. Ultra-high stringency will be the same conditions except the wash temperature is raised about 3 to about 5° C., and ultra-ultra-high stringency will be the same conditions except the wash temperature is raised about 6 to about 9° C. For identification of less closely related homologues washes can be performed at a lower temperature, e.g., 50° C. In general, stringency is increased by raising the wash temperature and/or decreasing the concentration of SSC, as known in the art.
In another example, stringent salt concentration will ordinarily be less than about 750 mM NaCl and 75 mM trisodium citrate, preferably less than about 500 mM NaCl and 50 mM trisodium citrate, and most preferably less than about 250 mM NaCl and 25 mM trisodium citrate. Low stringency hybridization can be obtained in the absence of organic solvent, e.g., formamide, while high stringency hybridization can be obtained in the presence of at least about 35% formamide, and most preferably at least about 50% formamide. Stringent temperature conditions will ordinarily include temperatures of at least about 30° C., more preferably of at least about 37° C., and most preferably of at least about 42° C. Varying additional parameters, such as hybridization time, the concentration of detergent, e.g., sodium dodecyl sulfate (SDS), and the inclusion or exclusion of carrier DNA, are well known to those skilled in the art. Various levels of stringency are accomplished by combining these various conditions as needed. In a preferred embodiment, hybridization will occur at 30° C. in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS. In a more preferred embodiment, hybridization will occur at 37° C. in 500 mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 100 μg/ml denatured salmon sperm DNA (ssDNA). In a most preferred embodiment, hybridization will occur at 42° C. in 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide, and 200 μg/ml ssDNA. Useful variations on these conditions will be readily apparent to those skilled in the art.
The washing steps that follow hybridization can also vary in stringency. Wash stringency conditions can be defined by salt concentration and by temperature. As above, wash stringency can be increased by decreasing salt concentration or by increasing temperature. For example, stringent salt concentration for the wash steps will preferably be less than about 30 mM NaCl and 3 mM trisodium citrate, and most preferably less than about 15 mM NaCl and 1.5 mM trisodium citrate. Stringent temperature conditions for the wash steps will ordinarily include temperature of at least about 25° C., more preferably of at least about 42° C. Another preferred set of highly stringent conditions uses two final washes in 0.1×SSC, 0.1% SDS at 65° C. The most preferred high stringency washes are of at least about 68° C. For example, in a preferred embodiment, wash steps will occur at 25° C. in 30 mM NaCl, 3 mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, wash steps will occur at 42° C. in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. In a most preferred embodiment, the wash steps will occur at 68° C. in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. Additional variations on these conditions will be readily apparent to those skilled in the art (see U.S. Patent Application No. 20010010913).
As another example, stringent conditions can be selected such that an oligonucleotide that is perfectly complementary to the coding oligonucleotide hybridizes to the coding oligonucleotide with at least about a 5-10× higher signal to noise ratio than the ratio for hybridization of the perfectly complementary oligonucleotide to a nucleic acid encoding a transcription factor known as of the filing date of the application. Conditions can be selected such that a higher signal to noise ratio is observed in the particular assay which is used, e.g., about 15×, 25×, 35×, 50× or more. Accordingly, the subject nucleic acid hybridizes to the unique coding oligonucleotide with at least a 2× higher signal to noise ratio as compared to hybridization of the coding oligonucleotide to a nucleic acid encoding known polypeptide. Again, higher signal to noise ratios can be selected, e.g., about 5×, 10×, 25×, 35×, 50× or more. The particular signal will depend on the label used in the relevant assay, e.g., a fluorescent label, a colorimetric label, a radioactive label, or the like.
Alternatively, transcription factor homolog polypeptides can be obtained by screening an expression library using antibodies specific for one or more transcription factors. With the provision herein of the disclosed transcription factor, and transcription factor homologue nucleic acid sequences, the encoded polypeptide(s) can be expressed and purified in a heterologous expression system (e.g., E. coli ) and used to raise antibodies (monoclonal or polyclonal) specific for the polypeptide(s) in question. Antibodies can also be raised against synthetic peptides derived from transcription factor, or transcription factor homologue, amino acid sequences. Methods of raising antibodies are well known in the art and are described in Harlow and Lane (1988) Antibodies: A Laboratory Manual , Cold Spring Harbor Laboratory, New York. Such antibodies can then be used to screen an expression library produced from the plant from which it is desired to clone additional transcription factor homologues, using the methods described above. The selected cDNAs can be confirmed by sequencing and enzymatic activity.
VII. SEQUENCE VARIATIONS
It will readily be appreciated by those of skill in the art, that any of a variety of polynucleotide sequences are capable of encoding the transcription factors and transcription factor homologue polypeptides of the invention. Due to the degeneracy of the genetic code, many different polynucleotides can encode identical and/or substantially similar polypeptides in addition to those sequences illustrated in the Sequence Listing. Nucleic acids having a sequence that differs from the sequences shown in the Sequence Listing, or complementary sequences, that encode functionally equivalent peptides (i.e., peptides having some degree of equivalent or similar biological activity) but differ in sequence from the sequence shown in the sequence listing due to degeneracy in the genetic code, are also within the scope of the invention.
Altered polynucleotide sequences encoding polypeptides include those sequences with deletions, insertions, or substitutions of different nucleotides, resulting in a polynucleotide encoding a polypeptide with at least one functional characteristic of the instant polypeptides. Included within this definition are polymorphisms which may or may not be readily detectable using a particular oligonucleotide probe of the polynucleotide encoding the instant polypeptides, and improper or unexpected hybridization to allelic variants, with a locus other than the normal chromosomal locus for the polynucleotide sequence encoding the instant polypeptides.
Allelic variant refers to any of two or more alternative forms of a gene occupying the same chromosomal locus. Allelic variation arises naturally through mutation, and may result in phenotypic polymorphism within populations. Gene mutations can be silent (i.e., no change in the encoded polypeptide) or may encode polypeptides having altered amino acid sequence. The term allelic variant is also used herein to denote a protein encoded by an allelic variant of a gene. Splice variant refers to alternative forms of RNA transcribed from a gene. Splice variation arises naturally through use of alternative splicing sites within a transcribed RNA molecule, or less commonly between separately transcribed RNA molecules, and may result in several mRNAs transcribed from the same gene. Splice variants may encode polypeptides having altered amino acid sequence. The term splice variant is also used herein to denote a protein encoded by a splice variant of an mRNA transcribed from a gene.
Those skilled in the art would recognize that G681, SEQ ID NO: 580, represents a single transcription factor; allelic variation and alternative splicing may be expected to occur. Allelic variants of SEQ ID NO: 579 can be cloned by probing cDNA or genomic libraries from different individual organisms according to standard procedures. Allelic variants of the DNA sequence shown in SEQ ID NO: 579, including those containing silent mutations and those in which mutations result in amino acid sequence changes, are within the scope of the present invention, as are proteins which are allelic variants of SEQ ID NO: 580. cDNAs generated from alternatively spliced mRNAs, which retain the properties of the transcription factor are included within the scope of the present invention, as are polypeptides encoded by such cDNAs and mRNAs. Allelic variants and splice variants of these sequences can be cloned by probing cDNA or genomic libraries from different individual organisms or tissues according to standard procedures known in the art (see U.S. Pat. No. 6,388,064).
For example, Table 1 illustrates, e.g., that the codons AGC, AGT, TCA, TCC, TCG, and TCT all encode the same amino acid: serine. Accordingly, at each position in the sequence where there is a codon encoding serine, any of the above trinucleotide sequences can be used without altering the encoded polypeptide.






TABLE 1



Amino acid
Possible Codons















Alanine
Ala
A
GCA
GCC
GCG
GCU



Cysteine
Cys
C
TGC
TGT

Aspartic acid
Asp
D
GAC
GAT

Glutamic acid
Glu
E
GAA
GAG

Phenylalanine
Phe
F
TTC
TTT

Glycine
Gly
G
GGA
GGC
GGG
GGT

Histidine
His
H
CAC
CAT

Isoleucine
Ile
I
ATA
ATC
ATT

Lysine
Lys
K
AAA
AAG

Leucine
Leu
L
TTA
TTG
CTA
CTC
CTG
CTT

Methionine
Met
M
ATG

Asparagine
Asn
N
AAC
AAT

Proline
Pro
P
CCA
CCC
CCG
CCT

Glutamine
Gln
Q
CAA
CAG

Arginine
Arg
R
AGA
AGG
CGA
CGC
CGG
CGT

Serine
Ser
S
AGC
AGT
TCA
TCC
TCG
TCT

Threonine
Thr
T
ACA
ACC
ACG
ACT

Valine
Val
V
GTA
GTC
GTG
GTT

Tryptophan
Trp
W
TGG

Tyrosine
Tyr
Y
TAC
TAT








































Sequence alterations that do not change the amino acid sequence encoded by the polynucleotide are termed “silent” variations. With the exception of the codons ATG and TGG, encoding methionine and tryptophan, respectively, any of the possible codons for the same amino acid can be substituted by a variety of techniques, e.g., site-directed mutagenesis, available in the art. Accordingly, any and all such variations of a sequence selected from the above table are a feature of the invention.
In addition to silent variations, other conservative variations that alter one, or a few amino acids in the encoded polypeptide, can be made without altering the function of the polypeptide, these conservative variants are, likewise, a feature of the invention.
For example, substitutions, deletions and insertions introduced into the sequences provided in the Sequence Listing are also envisioned by the invention. Such sequence modifications can be engineered into a sequence by site-directed mutagenesis (Wu (ed.) Meth. Enzymol . (1993) vol. 217, Academic Press) or the other methods noted below. Amino acid substitutions are typically of single residues; insertions usually will be on the order of about from 1 to 10 amino acid residues; and deletions will range about from 1 to 30 residues. In preferred embodiments, deletions or insertions are made in adjacent pairs, e.g., a deletion of two residues or insertion of two residues. Substitutions, deletions, insertions or any combination thereof can be combined to arrive at a sequence. The mutations that are made in the polynucleotide encoding the transcription factor should not place the sequence out of reading frame and should not create complementary regions that could produce secondary mRNA structure. Preferably, the polypeptide encoded by the DNA performs the desired function.
Conservative substitutions are those in which at least one residue in the amino acid sequence has been removed and a different residue inserted in its place. Such substitutions generally are made in accordance with the Table 2 when it is desired to maintain the activity of the protein. Table 2 shows amino acids which can be substituted for an amino acid in a protein and which are typically regarded as conservative substitutions.






TABLE 2




Conservative

Residue
Substitutions



Ala
Ser

Arg
Lys

Asn
Gln; His

Asp
Glu

Gln
Asn

Cys
Ser

Glu
Asp

Gly
Pro

His
Asn; Gln

Ile
Leu, Val

Leu
Ile; Val

Lys
Arg; Gln

Met
Leu; Ile

Phe
Met; Leu; Tyr

Ser
Thr; Gly

Thr
Ser; Val

Trp
Tyr

Tyr
Trp; Phe

Val
Ile; Leu




































Similar substitutions are those in which at least one residue in the amino acid sequence has been removed and a different residue inserted in its place. Such substitutions generally are made in accordance with the Table 3 when it is desired to maintain the activity of the protein. Table 3 shows amino acids which can be substituted for an amino acid in a protein and which are typically regarded as structural and functional substitutions. For example, a residue in column 1 of Table 3 may be substituted with residue in column 2; in addition, a residue in column 2 of Table 3 may be substituted with the residue of column 1.






TABLE 3



Residue
Similar Substitutions









Ala
Ser; Thr; Gly; Val; Leu; Ile


Arg
Lys; His; Gly

Asn
Gln; His; Gly; Ser; Thr

Asp
Glu, Ser; Thr

Gln
Asn; Ala

Cys
Ser; Gly

Glu
Asp

Gly
Pro; Arg

His
Asn; Gln; Tyr; Phe; Lys; Arg

Ile
Ala; Leu; Val; Gly; Met

Leu
Ala; Ile; Val; Gly; Met

Lys
Arg; His; Gln; Gly; Pro

Met
Leu; Ile; Phe

Phe
Met; Leu; Tyr; Trp; His; Val; Ala

Ser
Thr; Gly; Asp; Ala; Val; Ile; His

Thr
Ser; Val; Ala; Gly

Trp
Tyr; Phe; His

Tyr
Trp; Phe; His

Val
Ala; Ile; Leu; Gly; Thr; Ser; Glu







































Substitutions that are less conservative than those in Table 2 can be selected by picking residues that differ more significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain. The substitutions which in general are expected to produce the greatest changes in protein properties will be those in which (a) a hydrophilic residue, e.g., seryl or threonyl, is substituted for (or by) a hydrophobic residue, e.g., leucyl, isoleucyl, phenylalanyl, valyl or alanyl; (b) a cysteine or proline is substituted for (or by) any other residue; (c) a residue having an electropositive side chain, e.g., lysyl, arginyl, or histidyl, is substituted for (or by) an electronegative residue, e.g., glutamyl or aspartyl; or (d) a residue having a bulky side chain, e.g., phenylalanine, is substituted for (or by) one not having a side chain, e.g., glycine.
VIII. FURTHER MODIFYING SEQUENCES OF THE INVENTION
Mutation/Forced Evolution
In addition to generating silent or conservative substitutions as noted, above, the present invention optionally includes methods of modifying the sequences of the Sequence Listing. In the methods, nucleic acid or protein modification methods are used to alter the given sequences to produce new sequences and/or to chemically or enzymatically modify given sequences to change the properties of the nucleic acids or proteins.
Thus, in one embodiment, given nucleic acid sequences are modified, e.g., according to standard mutagenesis or artificial evolution methods to produce modified sequences. The modified sequences may be created using purified natural polynucleotides isolated from any organism or may be synthesized from purified compositions and chemicals using chemical means well know to those of skill in the art. For example, Ausubel, supra, provides additional details on mutagenesis methods. Artificial forced evolution methods are described, for example, by Stemmer (1994) Nature 370:389-391, Stemmer (1994) Proc. Natl. Acad. Sci. USA 91:10747-10751, and U.S. Pat. Nos. 5,811,238, 5,837,500, and 6,242,568. Methods for engineering synthetic transcription factors and other polypeptides are described, for example, by Zhang et al. (2000) J. Biol. Chem. 275:33850-33860, Liu et al. (2001) J. Biol. Chem. 276:11323-11334, and Isalan et al. (2001) Nature Biotechnol. 19:656-660. Many other mutation and evolution methods are also available and expected to be within the skill of the practitioner.
Similarly, chemical or enzymatic alteration of expressed nucleic acids and polypeptides can be performed by standard methods. For example, sequence can be modified by addition of lipids, sugars, peptides, organic or inorganic compounds, by the inclusion of modified nucleotides or amino acids, or the like. For example, protein modification techniques are illustrated in Ausubel, supra. Further details on chemical and enzymatic modifications can be found herein. These modification methods can be used to modify any given sequence, or to modify any sequence produced by the various mutation and artificial evolution modification methods noted herein.
Accordingly, the invention provides for modification of any given nucleic acid by mutation, evolution, chemical or enzymatic modification, or other available methods, as well as for the products produced by practicing such methods, e.g., using the sequences herein as a starting substrate for the various modification approaches.
For example, optimized coding sequence containing codons preferred by a particular prokaryotic or eukaryotic host can be used e.g., to increase the rate of translation or to produce recombinant RNA transcripts having desirable properties, such as a longer half-life, as compared with transcripts produced using a non-optimized sequence. Translation stop codons can also be modified to reflect host preference. For example, preferred stop codons for Saccharomyces cerevisiae and mammals are TAA and TGA, respectively. The preferred stop codon for monocotyledonous plants is TGA, whereas insects and E. coli prefer to use TAA as the stop codon.
The polynucleotide sequences of the present invention can also be engineered in order to alter a coding sequence for a variety of reasons, including but not limited to, alterations which modify the sequence to facilitate cloning, processing and/or expression of the gene product. For example, alterations are optionally introduced using techniques which are well known in the art, e.g., site-directed mutagenesis, to insert new restriction sites, to alter glycosylation patterns, to change codon preference, to introduce splice sites, etc.
Furthermore, a fragment or domain derived from any of the polypeptides of the invention can be combined with domains derived from other transcription factors or synthetic domains to modify the biological activity of a transcription factor. For instance, a DNA-binding domain derived from a transcription factor of the invention can be combined with the activation domain of another transcription factor or with a synthetic activation domain. A transcription activation domain assists in initiating transcription from a DNA-binding site. Examples include the transcription activation region of VP16 or GAL4 (Moore et al. (1998) Proc. Natl. Acad. Sci. USA 95: 376-381; and Aoyama et al. (1995) Plant Cell 7:1773-1785), peptides derived from bacterial sequences (Ma and Ptashne (1987) Cell 51; 113-119) and synthetic peptides (Giniger and Ptashne, (1987) Nature 330:670-672).
IX. EXPRESSION AND MODIFICATION OF POLYPEPTIDES
Typically, polynucleotide sequences of the invention are incorporated into recombinant DNA (or RNA) molecules that direct expression of polypeptides of the invention in appropriate host cells, transgenic plants, in vitro translation systems, or the like. Due to the inherent degeneracy of the genetic code, nucleic acid sequences which encode substantially the same or a functionally equivalent amino acid sequence can be substituted for any listed sequence to provide for cloning and expressing the relevant homologue.
X. VECTORS, PROMOTERS, AND EXPRESSION SYSTEMS
The present invention includes recombinant constructs comprising one or more of the nucleic acid sequences herein. The constructs typically comprise a vector, such as a plasmid, a cosmid, a phage, a virus (e.g., a plant virus), a bacterial artificial chromosome (BAC), a yeast artificial chromosome (YAC), or the like, into which a nucleic acid sequence of the invention has been inserted, in a forward or reverse orientation. In a preferred aspect of this embodiment, the construct further comprises regulatory sequences, including, for example, a promoter, operably linked to the sequence. Large numbers of suitable vectors and promoters are known to those of skill in the art, and are commercially available.
General texts that describe molecular biological techniques useful herein, including the use and production of vectors, promoters and many other relevant topics, include Berger, Sambrook and Ausubel, supra. Any of the identified sequences can be incorporated into a cassette or vector, e.g., for expression in plants. A number of expression vectors suitable for stable transformation of plant cells or for the establishment of transgenic plants have been described including those described in Weissbach and Weissbach, (1989) Methods for Plant Molecular Biology , Academic Press, and Gelvin et al., (1990) Plant Molecular Biology Manual , Kluwer Academic Publishers. Specific examples include those derived from a Ti plasmid of Agrobacterium tumefaciens , as well as those disclosed by Herrera-Estrella et al. (1983) Nature 303: 209, Bevan (1984) Nucl Acid Res. 12: 8711-8721, Klee (1985) Bio/Technology 3: 637-642, for dicotyledonous plants.
Alternatively, non-Ti vectors can be used to transfer the DNA into monocotyledonous plants and cells by using free DNA delivery techniques. Such methods can involve, for example, the use of liposomes, electroporation, microprojectile bombardment, silicon carbide whiskers, and viruses. By using these methods transgenic plants such as wheat, rice (Christou (1991) Bio/Technology 9: 957-962) and corn (Gordon-Kamm (1990) Plant Cell 2: 603-618) can be produced. An immature embryo can also be a good target tissue for monocots for direct DNA delivery techniques by using the particle gun (Weeks et al. (1993) Plant Physiol 102: 1077-1084; Vasil (1993) Bio/Technology 10: 667-674; Wan and Lemeaux (1994) Plant Physiol 104: 37-48, and for Agrobacterium -mediated DNA transfer (Ishida et al. (1996) Nature Biotech 14: 745-750).
Typically, plant transformation vectors include one or more cloned plant coding sequence (genomic or cDNA) under the transcriptional control of 5′ and 3′ regulatory sequences and a dominant selectable marker. Such plant transformation vectors typically also contain a promoter (e.g., a regulatory region controlling inducible or constitutive, environmentally- or developmentally-regulated, or cell- or tissue-specific expression), a transcription initiation start site, an RNA processing signal (such as intron splice sites), a transcription termination site, and/or a polyadenylation signal.
Examples of constitutive plant promoters which can be useful for expressing the TF sequence include: the cauliflower mosaic virus (CaMV) 35S promoter, which confers constitutive, high-level expression in most plant tissues (see, e.g., Odell et al. (1985) Nature 313:810-812); the nopaline synthase promoter (An et al. (1988) Plant Physiol 88:547-552); and the octopine synthase promoter (Fromm et al. (1989) Plant Cell 1: 977-984).
A variety of plant gene promoters that regulate gene expression in response to environmental, hormonal, chemical, developmental signals, and in a tissue-active manner can be used for expression of a TF sequence in plants. Choice of a promoter is based largely on the phenotype of interest and is determined by such factors as tissue (e.g., seed, fruit, root, pollen, vascular tissue, flower, carpel, etc.), inducibility (e.g., in response to wounding, heat, cold, drought, light, pathogens, etc.), timing, developmental stage, and the like. Numerous known promoters have been characterized and can favorably be employed to promote expression of a polynucleotide of the invention in a transgenic plant or cell of interest. For example, tissue specific promoters include: seed-specific promoters (such as the napin, phaseolin or DC3 promoter described in U.S. Pat. No. 5,773,697), fruit-specific promoters that are active during fruit ripening (such as the dru 1 promoter (U.S. Pat. No. 5,783,393), or the 2A11 promoter (U.S. Pat. No. 4,943,674) and the tomato polygalacturonase promoter (Bird et al. (1988) Plant Mol Biol 11:651), root-specific promoters, such as those disclosed in U.S. Pat. Nos. 5,618,988, 5,837,848 and 5,905,186, pollen-active promoters such as PTA29, PTA26 and PTA13 (U.S. Pat. No. 5,792,929), promoters active in vascular tissue (Ringli and Keller (1998) Plant Mol Biol 37:977-988), flower-specific (Kaiser et al, (1995) Plant Mol Biol 28:231-243), pollen (Baerson et al. (1994) Plant Mol Biol 26:1947-1959), carpels (Ohl et al. (1990) Plant Cell 2:837-848), pollen and ovules (Baerson et al. (1993) Plant Mol Biol 22:255-267), auxin-inducible promoters (such as that described in van der Kop et al. (1999) Plant Mol Biol 39:979-990 or Baumann et al. (1999) Plant Cell 11:323-334), cytokinin-inducible promoter (Guevara-Garcia (1998) Plant Mol Biol 38:743-753), promoters responsive to gibberellin (Shi et al. (1998) Plant Mol Biol 38:1053-1060, Willmott et al. (1998) 38:817-825) and the like. Additional promoters are those that elicit expression in response to heat (Ainley et al. (1993) Plant Mol Biol 22: 13-23), light (e.g., the pea rbcS-3A promoter, Kuhlemeier et al. (1989) Plant Cell 1:471, and the maize rbcS promoter, Schaffner and Sheen (1991) Plant Cell 3: 997); wounding (e.g., wunI, Siebertz et al. (1989) Plant Cell 1: 961); pathogens (such as the PR-1 promoter described in Buchel et al. (1999) Plant Mol. Biol. 40:387-396, and the PDF1.2 promoter described in Manners et al. (1998) Plant Mol. Biol. 38:1071-80), and chemicals such as methyl jasmonate or salicylic acid (Gatz et al. (1997) Plant Mol Biol 48: 89-108). In addition, the timing of the expression can be controlled by using promoters such as those acting at senescence (An and Amazon (1995) Science 270: 1986-1988); or late seed development (Odell et al. (1994) Plant Physiol 106:447-458).
Plant expression vectors can also include RNA processing signals that can be positioned within, upstream or downstream of the coding sequence. In addition, the expression vectors can include additional regulatory sequences from the 3′-untranslated region of plant genes, e.g., a 3′ terminator region to increase mRNA stability of the mRNA, such as the PI-II terminator region of potato or the octopine or nopaline synthase 3′ terminator regions.
Additional Expression Elements
Specific initiation signals can aid in efficient translation of coding sequences. These signals can include, e.g., the ATG initiation codon and adjacent sequences. In cases where a coding sequence, its initiation codon and upstream sequences are inserted into the appropriate expression vector, no additional translational control signals may be needed. However, in cases where only coding sequence (e.g., a mature protein coding sequence), or a portion thereof, is inserted, exogenous transcriptional control signals including the ATG initiation codon can be separately provided. The initiation codon is provided in the correct reading frame to facilitate transcription. Exogenous transcriptional elements and initiation codons can be of various origins, both natural and synthetic. The efficiency of expression can be enhanced by the inclusion of enhancers appropriate to the cell system in use.
Expression Hosts
The present invention also relates to host cells which are transduced with vectors of the invention, and the production of polypeptides of the invention (including fragments thereof) by recombinant techniques. Host cells are genetically engineered (i.e., nucleic acids are introduced, e.g., transduced, transformed or transfected) with the vectors of this invention, which may be, for example, a cloning vector or an expression vector comprising the relevant nucleic acids herein. The vector is optionally a plasmid, a viral particle, a phage, a naked nucleic acid, etc. The engineered host cells can be cultured in conventional nutrient media modified as appropriate for activating promoters, selecting transformants, or amplifying the relevant gene. The culture conditions, such as temperature, pH and the like, are those previously used with the host cell selected for expression, and will be apparent to those skilled in the art and in the references cited herein, including, Sambrook and Ausubel.
The host cell can be a eukaryotic cell, such as a yeast cell, or a plant cell, or the host cell can be a prokaryotic cell, such as a bacterial cell. Plant protoplasts are also suitable for some applications. For example, the DNA fragments are introduced into plant tissues, cultured plant cells or plant protoplasts by standard methods including electroporation (Fromm et al., (1985) Proc. Natl. Acad. Sci. USA 82, 5824, infection by viral vectors such as cauliflower mosaic virus (CaMV) (Hohn et al., (1982) Molecular Biology of Plant Tumors , (Academic Press, New York) pp. 549-560; U.S. Pat. No. 4,407,956), high velocity ballistic penetration by small particles with the nucleic acid either within the matrix of small beads or particles, or on the surface (Klein et al., (1987) Nature 327, 70-73), use of pollen as vector (WO 85/01856), or use of Agrobacterium tumefaciens or A. rhizogenes carrying a T-DNA plasmid in which DNA fragments are cloned. The T-DNA plasmid is transmitted to plant cells upon infection by Agrobacterium tumefaciens , and a portion is stably integrated into the plant genome (Horsch et al. (1984) Science 233:496-498; Fraley et al. (1983) Proc. Natl. Acad. Sci. USA 80, 4803).
The cell can include a nucleic acid of the invention which encodes a polypeptide, wherein the cells expresses a polypeptide of the invention. The cell can also include vector sequences, or the like. Furthermore, cells and transgenic plants that include any polypeptide or nucleic acid above or throughout this specification, e.g., produced by transduction of a vector of the invention, are an additional feature of the invention.
For long-term, high-yield production of recombinant proteins, stable expression can be used. Host cells transformed with a nucleotide sequence encoding a polypeptide of the invention are optionally cultured under conditions suitable for the expression and recovery of the encoded protein from cell culture. The protein or fragment thereof produced by a recombinant cell may be secreted, membrane-bound, or contained intracellularly, depending on the sequence and/or the vector used. As will be understood by those of skill in the art, expression vectors containing polynucleotides encoding mature proteins of the invention can be designed with signal sequences which direct secretion of the mature polypeptides through a prokaryotic or eukaryotic cell membrane.
XI. MODIFIED AMINO ACID RESIDUES
Polypeptides of the invention may contain one or more modified amino acid residues. The presence of modified amino acids may be advantageous in, for example, increasing polypeptide half-life, reducing polypeptide antigenicity or toxicity, increasing polypeptide storage stability, or the like. Amino acid residue(s) are modified, for example, co-translationally or post-translationally during recombinant production or modified by synthetic or chemical means.
Non-limiting examples of a modified amino acid residue include incorporation or other use of acetylated amino acids, glycosylated amino acids, sulfated amino acids, prenylated (e.g., farnesylated, geranylgeranylated) amino acids, PEG modified (e.g., “PEGylated”) amino acids, biotinylated amino acids, carboxylated amino acids, phosphorylated amino acids, etc. References adequate to guide one of skill in the modification of amino acid residues are replete throughout the literature.
The modified amino acid residues may prevent or increase affinity of the polypeptide for another molecule, including, but not limited to, polynucleotide, proteins, carbohydrates, lipids and lipid derivatives, and other organic or synthetic compounds.
XII. IDENTIFICATION OF ADDITIONAL FACTORS
A transcription factor provided by the present invention can also be used to identify additional endogenous or exogenous molecules that can affect a phentoype or trait of interest. On the one hand, such molecules include organic (small or large molecules) and/or inorganic compounds that affect expression of (i.e., regulate) a particular transcription factor. Alternatively, such molecules include endogenous molecules that are acted upon either at a transcriptional level by a transcription factor of the invention to modify a phenotype as desired. For example, the transcription factors can be employed to identify one or more downstream gene with which is subject to a regulatory effect of the transcription factor. In one approach, a transcription factor or transcription factor homologue of the invention is expressed in a host cell, e.g., a transgenic plant cell, tissue or explant, and expression products, either RNA or protein, of likely or random targets are monitored, e.g., by hybridization to a microarray of nucleic acid probes corresponding to genes expressed in a tissue or cell type of interest, by two-dimensional gel electrophoresis of protein products, or by any other method known in the art for assessing expression of gene products at the level of RNA or protein. Alternatively, a transcription factor of the invention can be used to identify promoter sequences (i.e., binding sites) involved in the regulation of a downstream target. After identifying a promoter sequence, interactions between the transcription factor and the promoter sequence can be modified by changing specific nucleotides in the promoter sequence or specific amino acids in the transcription factor that interact with the promoter sequence to alter a plant trait. Typically, transcription factor DNA-binding sites are identified by gel shift assays. After identifying the promoter regions, the promoter region sequences can be employed in double-stranded DNA arrays to identify molecules that affect the interactions of the transcription factors with their promoters (Bulyk et al. (1999) Nature Biotechnology 17:573-577).
The identified transcription factors are also useful to identify proteins that modify the activity of the transcription factor. Such modification can occur by covalent modification, such as by phosphorylation, or by protein-protein (homo or -heteropolymer) interactions. Any method suitable for detecting protein-protein interactions can be employed. Among the methods that can be employed are co-immunoprecipitation, cross-linking and co-purification through gradients or chromatographic columns, and the two-hybrid yeast system.
The two-hybrid system detects protein interactions in vivo and is described in Chien et al. ((1991), Proc. Natl. Acad. Sci. USA 88:9578-9582) and is commercially available from Clontech (Palo Alto, Calif.). In such a system, plasmids are constructed that encode two hybrid proteins: one consists of the DNA-binding domain of a transcription activator protein fused to the TF polypeptide and the other consists of the transcription activator protein's activation domain fused to an unknown protein that is encoded by a cDNA that has been recombined into the plasmid as part of a cDNA library. The DNA-binding domain fusion plasmid and the cDNA library are transformed into a strain of the yeast Saccharomyces cerevisiae that contains a reporter gene (e.g., lacZ) whose regulatory region contains the transcription activator's binding site. Either hybrid protein alone cannot activate transcription of the reporter gene. Interaction of the two hybrid proteins reconstitutes the functional activator protein and results in expression of the reporter gene, which is detected by an assay for the reporter gene product. Then, the library plasmids responsible for reporter gene expression are isolated and sequenced to identify the proteins encoded by the library plasmids. After identifying proteins that interact with the transcription factors, assays for compounds that interfere with the TF protein-protein interactions can be preformed.
XIII. IDENTIFICATION OF MODULATORS
In addition to the intracellular molecules described above, extracellular molecules that alter activity or expression of a transcription factor, either directly or indirectly, can be identified. For example, the methods can entail first placing a candidate molecule in contact with a plant or plant cell. The molecule can be introduced by topical administration, such as spraying or soaking of a plant, and then the molecule's effect on the expression or activity of the TF polypeptide or the expression of the polynucleotide monitored. Changes in the expression of the TF polypeptide can be monitored by use of polyclonal or monoclonal antibodies, gel electrophoresis or the like. Changes in the expression of the corresponding polynucleotide sequence can be detected by use of microarrays, Northerns, quantitative PCR, or any other technique for monitoring changes in mRNA expression. These techniques are exemplified in Ausubel et al. (eds) Current Protocols in Molecular Biology , John Wiley & Sons (1998, and supplements through 2001). Such changes in the expression levels can be correlated with modified plant traits and thus identified molecules can be useful for soaking or spraying on fruit, vegetable and grain crops to modify traits in plants.
Essentially any available composition can be tested for modulatory activity of expression or activity of any nucleic acid or polypeptide herein. Thus, available libraries of compounds such as chemicals, polypeptides, nucleic acids and the like can be tested for modulatory activity. Often, potential modulator compounds can be dissolved in aqueous or organic (e.g., DMSO-based) solutions for easy delivery to the cell or plant of interest in which the activity of the modulator is to be tested. Optionally, the assays are designed to screen large modulator composition libraries by automating the assay steps and providing compounds from any convenient source to assays, which are typically run in parallel (e.g., in microtiter formats on microtiter plates in robotic assays).
In one embodiment, high throughput screening methods involve providing a combinatorial library containing a large number of potential compounds (potential modulator compounds). Such “combinatorial chemical libraries” are then screened in one or more assays, as described herein, to identify those library members (particular chemical species or subclasses) that display a desired characteristic activity. The compounds thus identified can serve as target compounds.
A combinatorial chemical library can be, e.g., a collection of diverse chemical compounds generated by chemical synthesis or biological synthesis. For example, a combinatorial chemical library such as a polypeptide library is formed by combining a set of chemical building blocks (e.g., in one example, amino acids) in every possible way for a given compound length (i.e., the number of amino acids in a polypeptide compound of a set length). Exemplary libraries include peptide libraries, nucleic acid libraries, antibody libraries (see, e.g., Vaughn et al. (1996) Nature Biotechnology, 14(3):309-314 and PCT/US96/10287), carbohydrate libraries (see, e.g., Liang et al. Science (1996) 274:1520-1522 and U.S. Pat. No. 5,593,853), peptide nucleic acid libraries (see, e.g., U.S. Pat. No. 5,539,083), and small organic molecule libraries (see, e.g., benzodiazepines, Baum C & EN January 18, page 33 (1993); isoprenoids, U.S. Pat. No. 5,569,588; thiazolidinones and metathiazanones, U.S. Pat. No. 5,549,974; pyrrolidines, U.S. Pat. Nos. 5,525,735 and 5,519,134; morpholino compounds, U.S. Pat. No. 5,506,337) and the like.
Preparation and screening of combinatorial or other libraries is well known to those of skill in the art. Such combinatorial chemical libraries include, but are not limited to, peptide libraries (see, e.g., U.S. Pat. No. 5,010,175; Furka, (1991) Int. J. Pept. Prot. Res. 37:487-493; and Houghton et al. (1991) Nature 354:84-88). Other chemistries for generating chemical diversity libraries can also be used.
In addition, as noted, compound screening equipment for high-throughput screening is generally available, e.g., using any of a number of well known robotic systems that have also been developed for solution phase chemistries useful in assay systems. These systems include automated workstations including an automated synthesis apparatus and robotic systems utilizing robotic arms. Any of the above devices are suitable for use with the present invention, e.g., for high-throughput screening of potential modulators. The nature and implementation of modifications to these devices (if any) so that they can operate as discussed herein will be apparent to persons skilled in the relevant art.
Indeed, entire high throughput screening systems are commercially available. These systems typically automate entire procedures including all sample and reagent pipetting, liquid dispensing, timed incubations, and final readings of the microplate in detector(s) appropriate for the assay. These configurable systems provide high throughput and rapid start up as well as a high degree of flexibility and customization. Similarly, microfluidic implementations of screening are also commercially available.
The manufacturers of such systems provide detailed protocols the various high throughput. Thus, for example, Zymark Corp. provides technical bulletins describing screening systems for detecting the modulation of gene transcription, ligand binding, and the like. The integrated systems herein, in addition to providing for sequence alignment and, optionally, synthesis of relevant nucleic acids, can include such screening apparatus to identify modulators that have an effect on one or more polynucleotides or polypeptides according to the present invention.
In some assays it is desirable to have positive controls to ensure that the components of the assays are working properly. At least two types of positive controls are appropriate. That is, known transcriptional activators or inhibitors can be incubated with cells/plants/etc. in one sample of the assay, and the resulting increase/decrease in transcription can be detected by measuring the resulting increase in RNA/protein expression, etc., according to the methods herein. It will be appreciated that modulators can also be combined with transcriptional activators or inhibitors to find modulators that inhibit transcriptional activation or transcriptional repression. Either expression of the nucleic acids and proteins herein or any additional nucleic acids or proteins activated by the nucleic acids or proteins herein, or both, can be monitored.
In an embodiment, the invention provides a method for identifying compositions that modulate the activity or expression of a polynucleotide or polypeptide of the invention. For example, a test compound, whether a small or large molecule, is placed in contact with a cell, plant (or plant tissue or explant), or composition comprising the polynucleotide or polypeptide of interest and a resulting effect on the cell, plant, (or tissue or explant) or composition is evaluated by monitoring, either directly or indirectly, one or more of: expression level of the polynucleotide or polypeptide, activity (or modulation of the activity) of the polynucleotide or polypeptide. In some cases, an alteration in a plant phenotype can be detected following contact of a plant (or plant cell, or tissue or explant) with the putative modulator, e.g., by modulation of expression or activity of a polynucleotide or polypeptide of the invention. Modulation of expression or activity of a polynucleotide or polypeptide of the invention may also be caused by molecular elements in a signal transduction second messenger pathway and such modulation can affect similar elements in the same or another signal transduction second messenger pathway.
XIV. SUBSEQUENCES
Also contemplated are uses of polynucleotides, also referred to herein as oligonucleotides, typically having at least 12 bases, preferably at least 15, more preferably at least 20, 30, or 50 bases, which hybridize under at least highly stringent (or ultra-high stringent or ultra-ultra-high stringent conditions) conditions to a polynucleotide sequence described above. The polynucleotides may be used as probes, primers, sense and antisense agents, and the like, according to methods as noted supra.
Subsequences of the polynucleotides of the invention, including polynucleotide fragments and oligonucleotides are useful as nucleic acid probes and primers. An oligonucleotide suitable for use as a probe or primer is at least about 15 nucleotides in length, more often at least about 18 nucleotides, often at least about 21 nucleotides, frequently at least about 30 nucleotides, or about 40 nucleotides, or more in length. A nucleic acid probe is useful in hybridization protocols, e.g., to identify additional polypeptide homologues of the invention, including protocols for microarray experiments. Primers can be annealed to a complementary target DNA strand by nucleic acid hybridization to form a hybrid between the primer and the target DNA strand, and then extended along the target DNA strand by a DNA polymerase enzyme. Primer pairs can be used for amplification of a nucleic acid sequence, e.g., by the polymerase chain reaction (PCR) or other nucleic-acid amplification methods. See Sambrook and Ausubel, supra.
In addition, the invention includes an isolated or recombinant polypeptide including a subsequence of at least about 15 contiguous amino acids encoded by the recombinant or isolated polynucleotides of the invention. For example, such polypeptides, or domains or fragments thereof, can be used as immunogens, e.g., to produce antibodies specific for the polypeptide sequence, or as probes for detecting a sequence of interest. A subsequence can range in size from about 15 amino acids in length up to and including the full length of the polypeptide.
To be encompassed by the present invention, an expressed polypeptide which comprises such a polypeptide subsequence performs at least one biological function of the intact polypeptide in substantially the same manner, or to a similar extent, as does the intact polypeptide. For example, a polypeptide fragment can comprise a recognizable structural motif or functional domain such as a DNA binding domain that binds to a specific DNA promoter region, an activation domain or a domain for protein-protein interactions.
XV. PRODUCTION OF TRANSGENIC PLANTS
Modification of Traits
The polynucleotides of the invention are favorably employed to produce transgenic plants with various traits, or characteristics, that have been modified in a desirable manner, e.g., to improve the seed characteristics of a plant. For example, alteration of expression levels or patterns (e.g., spatial or temporal expression patterns) of one or more of the transcription factors (or transcription factor homologues) of the invention, as compared with the levels of the same protein found in a wild type plant, can be used to modify a plant's traits. An illustrative example of trait modification, improved characteristics, by altering expression levels of a particular transcription factor is described further in the Examples and the Sequence Listing.
Arabidopsis as a Model System
Arabidopsis thaliana is the object of rapidly growing attention as a model for genetics and metabolism in plants. Arabidopsis has a small genome, and well documented studies are available. It is easy to grow in large numbers and mutants defining important genetically controlled mechanisms are either available, or can readily be obtained. Various methods to introduce and express isolated homologous genes are available (see Koncz, et al., eds. Methods in Arabidopsis Research . et al. (1992), World Scientific, New Jersey, New Jersey, in “Preface”). Because of its small size, short life cycle, obligate autogamy and high fertility, Arabidopsis is also a choice organism for the isolation of mutants and studies in morphogenetic and development pathways, and control of these pathways by transcription factors (Koncz, supra, p. 72). A number of studies introducing transcription factors into A. thaliana have demonstrated the utility of this plant for understanding the mechanisms of gene regulation and trait alteration in plants. See, for example, Koncz, supra, and U.S. Pat. No. 6,417,428).
Arabidopsis Genes in Transgenic Plants.
Expression of genes which encode transcription factors modify expression of endogenous genes, polynucleotides, and proteins are well known in the art. In addition, transgenic plants comprising isolated polynucleotides encoding transcription factors may also modify expression of endogenous genes, polynucleotides, and proteins. Examples include Peng et al. (1997, Genes and Development 11:3194-3205) and Peng et al. (1999, Nature, 400:256-261). In addition, many others have demonstrated that an Arabidopsis transcription factor expressed in an exogenous plant species elicits the same or very similar phenotypic response. See, for example, Fu et al. (2001, Plant Cell 13:1791-1802); Nandi et al. (2000, Curr. Biol. 10:215-218); Coupland (1995, Nature 377:482-483); and Weigel and Nilsson (1995, Nature 377:482-500).
Homologous Genes Introduced into Transgenic Plants.
Homologous genes that may be derived from any plant, or from any source whether natural, synthetic, semi-synthetic or recombinant, and that share significant sequence identity or similarity to those provided by the present invention, may be introduced into plants, for example, crop plants, to confer desirable or improved traits. Examples of non- arabidopsis sequences homologous to arabidopsis are listed in Table 4. Consequently, transgenic plants may be produced that comprise a recombinant expression vector or cassette with a promoter operably linked to one or more sequences homologous to presently disclosed sequences. The promoter may be, for example, a plant or viral promoter.





TABLE 4



Orthologs of arabidopsis sequences











Smallest

Test Sequence

SEQ ID

Test
Sum
Test Sequence
GenBank

NO
GID
Sequence ID
Probability
Species
Annotation



859
G192
AW596933
7.70E-40
[Glycine max]
sj84f07.yl Gm-c1034 Glycine max cDNA clone GENO

859
G192
AV423663
2.40E-39
[Lotus japonicus]
AV423663 Lotus japonicus young plants (two-

859
G192
B1422074
4.50E-34
[Lycopersicon esculentum]
EST532740 tomato callus, TAMU Lycop

859
G192
AW447931
1.40E-27
[Triticum aestivum]
BRY_1082 BRY Triticum aestivum cDNA clone

859
G192
BE998060
2.60E-24
[Medicago truncatula]
EST429783 GVSN Medicago truncatula cDNA

859
G192
AC018727
1.70E-23
[Oryza sativa]
chromosome 10 clone OSJNBa0056G17,*** SEQUENC

859
G192
BG600477
1.00E-20
[Solanum tuberosum]
EST505372 cSTS Solanum tuberosum cDNA clo

859
G192
BG356878
2.80E-16
[Sorghum bicolor]
OV2_11_B04.g1_A002 Ovary 2 (OV2) Sorghum bi

859
G192
gi12039364
1.10E-31
[Oryza sativa]
putative DNA-binding protein.

859
G192
gi4894963
3.30E-14
[Avena sativa]
DNA-binding protein WRKY3.

859
G192
gi1432056
5.80E-14
[Petroselinum crispum]
WRKY3.

859
G192
gi4760596
2.60E-13
[Nicotiana tabacum]
DNA-binding protein NtWRKY3.

859
G192
gi11993901
1.40E-12
[Dactylis glomerata]
somatic embryogenesis related protein.

859
G192
gi927025
7.60E-09
[Cucumis sativus]
SPF 1-like DNA-binding protein.

859
G192
gi13620227
8.40E-09
[Lycopersicon esculentum]
hypothetical protein.

859
G192
gi3420906
2.80E-08
[Pimpinella brachycarpa]
zinc finger protein; WRKY1.

859
G192
gi1159877
4.70E-08
[Avena fatua]
DNA-binding protein.

859
G192
gi484261
1.60E-07
[Ipomoea batatas]
SPF1 protein.

801
G1946
LPHSF8
1.10E-119
[Lycopersicon peruvianum]
L.peruvianum Lp-hsf8 mRNA for heat

801
G1946
AC087771
4.10E-112
[Medicago truncatula]
clone 8D15,*** SEQUENCING IN PROGRESS

801
G1946
LEHSF8
5.90E-103
[Lycopersicon esculentum]
L.esculentum Le-hsf8 gene for heat

801
G1946
AW569138
3.10E-75
[Glycine max]
si63g09.yl Gm-r1030 Glycine max cDNA clone GENO

801
G1946
BG890899
1.30E-70
[Solanum tuberosum]
EST516750 cSTD Solanum tuberosum cDNA clo

801
G1946
AC027658
4.60E-53
[Oryza sativa]
subsp. japonica BAC nbxb0006I13, chromosome 10

801
G1946
AV833112
4.90E-52
[Hordeum vulgare subsp.vulgare]
AV833112 K. Sato unpublished

801
G1946
gi19492
2.80E-121
[Lycopersicon peruvianum]
heat shock transcription factor 8

801
G1946
gi19260
5.10E-106
[Lycopersicon esculentum]
heat stress transcription factor

801
G1946
gi662924
2.00E-47
[Glycine max]
heat shock transcription factor 21.

801
G1946
gi5821138
9.70E-46
[Nicotiana tabacum]
heat shock factor.

801
G1946
gi11761077
2.90E-40
[Oryza sativa]
putative heat shock factor protein 1 (HSF 1)

801
G1946
gi886742
3.20E-40
[Zea mays]
heat shock factor.

801
G1946
gi7158882
2.70E-38
[Medicago sativa]
heat shock transcription factor.

801
G1946
gi3550588
1.90E-30
[Pisum sativum]
heat shock transcription factor (HSFA).

801
G1946
gi100546
0.46
[Avena sativa]
avenin precursor - oat.

801
G1946
gi14190783
1
[Apium graveolens]
putative phloem transcription factor M1.

239
G375
AW696439
3.40E-33
[Medicago truncatula]
NF106B07ST1F1060 Developing stem Medica

239
G375
BG595870
1.90E-31
[Solanum tuberosum]
EST494548 cSTS Solanum tuberosum cDNA clo

239
G375
A1899263
3.70E-31
[Lycopersicon esculentum]
EST268706 tomato ovary, TAMU Lycope

239
G375
NTBBF3
4.00E-31
[Nicotiana tabacum]
N.tabacum mRNA for zinc finger protein, B

239
G375
BG405482
2.70E-30
[Glycine max]
sac44a11.y1 Gm-c1062 Glycine max cDNA clone GEN

239
G375
AB028130
3.30E-30
[Oryza sativa]
mRNA for Dof zinc finger protein, complete cds

239
G375
AB026297
7.30E-28
[Pisum sativum]
mRNA for elicitor-responsive Dof protein ERDP

239
G375
HVBPBF
1.10E-27
[Hordeum vulgare]
mRNA for DNA binding protein BPBF.

239
G375
BG263089
1.70E-27
[Triticum aestivum]
WHE2337_A02 A03ZS Wheat pre-anthesis spik

239
G375
ZMU82230
4.20E-27
[Zea mays]
endosperm-specific prolamin box binding factor (PB

239
G375
gi4996640
1.90E-37
[Oryza sativa]
Dof zinc finger protein.

239
G375
gi3777436
8.10E-35
[Hordeum vulgare]
DNA binding protein.

239
G375
gi2393775
1.10E-33
[Zea mays]
prolamin box binding factor.

239
G375
gi1360088
2.00E-33
[Nicotiana tabacum]
Zn finger protein.

239
G375
gi3790264
4.30E-32
[Triticum aestivum]
PBF protein.

239
G375
gi6092016
1.30E-29
[Pisum sativum]
elicitor-responsive Dof protein ERDP.

239
G375
gi7688355
5.60E-29
[Solanum tuberosum]
Dof zinc finger protein.

239
G375
gi1669341
4.60E-20
[Cucurbita maxima]
AOBP (ascorbate oxidase promoter-binding

239
G375
gi3929325
5.50E-18
[Dendrobium grex Madame Thong-In]
putative DNA-binding prot

239
G375
gi19547
5.50E-06
[Medicago sativa subsp. falcata]
environmental stress and a

273
G1255
AC087181
1.60E-46
[Oryza sativa]
chromosome 3 clone OSJNBa0018H01, *** SEQUENCI

273
G1255
BG239774
4.50E-33
[Glycine max]
clone GEN

273
G1255
BG321336
1.70E-32
[Descurainia sophia]
Ds01_06h10_A DsO1_AAFC ECORC_cold_stress

273
G1255
A1772841
2.90E-30
[Lycopersicon esculentum]
E5T253941 tomato resistant, Cornell

273
G1255
BF480245
4.60E-29
[Mesembryanthem um crystallinum]
L0-2152T3 Ice plant Lambda Un

273
G1255
AW688119
2.10E-28
[Medicago truncatula]
NF002E075T1F1000 Developing stem Medica

273
G1255
BF266327
1.80E-26
[Hordeum vulgare]
HV_CEa00l4N02fHordeum vulgare seedling gre

273
G1255
AW671538
5.80E-25
[Sorghum bicolor]
LG1_348_B08.bl_A002_Light Grown 1 (LG1)Sor

273
G1255
B1072021
5.30E-20
[Populus tremula x
C067P76U Populus stra





Populus tremuloides]

273
G1255
BG273908
4.90E-19
[Vitis vinifera]
EST 110 Green Grape berries Lambda Zap II Li

273
G1255
gi13702811
3.70E-52
[Oryza sativa]
putative zinc finger protein.

273
G1255
gi11037311
4.00E-21
[Brassica nigra]
constans-like protein.

273
G1255
gi2303683
1.10E-19
[Brassica napus]
unnamed protein product.

273
G1255
gi4091804
2.30E-18
[Malus x domestica]
CONSTANS-like protein 1.

273
G1255
gi3341723
4.30E-18
[Raphanus sativus]
CONSTANS-like 1 protein.

273
G1255
gi10946337
5.20E-17
[Ipomoea nil]
CONSTANS-like protein.

273
G1255
gi4557093
3.30E-15
[Pinus radiata]
zinc finger protein.

273
G1255
gi8132543
0.97
[Chloroplast Zamia furfuracea]
cytochrome b559 alpha subuni

273
G1255
gi11795
0.99
[Nicotiana tabacum]
put. psbE protein (aa 1-83).

273
G1255
gi65646
0.99
[Chloroplast Nicotiana tabacum]
cytochrome b559 component p

557
G865
BE419451
3.70E-32
[Triticum aestivum]
WWS012.C2R000101 ITEC WWS Wheat Scutellum

557
G865
AW560968
1.10E-28
[Medicago truncatula]
EST316016 DSIR Medicago truncatula cDNA

557
G865
AW782252
1.20E-26
[Glycine max]
sm03d11.y1 Gm-c1027 Glycine max cDNA clone GENO

557
G865
B1421895
3.60E-25
[Lycopersicon esculentum]
E5T532561 tomato callus, TAMU Lycop

557
G865
BE642320
1.60E-24
[Ceratopteris richardii]
Cri2_5_L17_SP6 Ceratopteris Spore Li

557
G865
BE494041
1.60E-24
[Secale cereale]
WHE1277_B09_D17ZS Secale cereale anther cDNA

557
G865
D39914
2.60E-24
[Oryza sativa]
RJCS1576A Rice shoot Oryza sativa cDNA, mRNA s

557
G865
AV428124
9.00E-23
[Lotus japonicus]
AV428124 Lotus japonicus young plants (two-

557
G865
TOBBY4D
1.80E-21
[Nicotiana tabacum]
Tobacco mRNA for EREBP-2, complete cds.

557
G865
gi1208495
2.40E-23
[Nicotiana tabacum]
ERF1.

557
G865
gi8809571
5.10E-23
[Nicotiana sylvestris]
ethylene-responsive element binding

557
G865
gi3342211
1.40E-22
[Lycopersicon esculentum]
Pti4.

557
G865
gi7528276
1.70E-22
[Mesembryanthem um crystallinum]
AP2-related transcription f

557
G865
gi15217291
7.80E-22
[Oryza sativa]
Putative AP2 domain containing protein.

557
G865
gi3264767
2.70E-21
[Prunus armeniaca]
AP2 domain containing protein.

557
G865
gi8980313
2.10E-20
[Catharanthus roseus]
AP2-domain DNA-binding protein.

557
G865
gi8571476
9.30E-20
[Atriplex hortensis]
apetala2 domain-containing protein.

557
G865
gi1688233
1.40E-19
[Solanum tuberosum]
DNA binding protein homolog.

557
G865
gi6478845
1.80E-19
[Matricaria chamomilla]
ethylene-responsive element binding

23
G2509
BH577856
2.50E-29
[Brassica oleracea]
BOHOJ67TR BOHO Brassica oleracea genomic

23
G2509
BM269574
5.90E-28
[Glycine max]
sak01eO8.yl Gm-c1074 Glycine max cDNA clone SOY

23
G2509
BE419451
2.20E-27
[Triticum aestivum]
WWS012.C2R000101 ITEC WWS Wheat Scutellum

23
G2509
A1483636
7.80E-27
[Lycopersicon esculentum]
E5T249507 tomato ovary, TAMU Lycope

23
G2509
AW560968
8.90E-27
[Medicago truncatula]
EST316016 DSIR Medicago truncatula cDNA

23
G2509
BE642320
4.30E-26
[Ceratopteris richardii]
Cri2_S_L17_SP6 Ceratopteris Spore Li

23
G2509
AP003286
1.00E-25
[Oryza sativa]
chromosome 1 clone P0677H08, *** SEQUENCING IN

23
G2509
BE494041
3.20E-25
[Secale cereale]
WHE1277_B09_D17ZS Secale cereale anther cDNA

23
G2509
BE602106
1.10E-24
[Hordeum vulgare]
HVSMEh0102I06f Hordeum vulgare 5-45 DAP spi

23
G2509
AV428124
1.00E-23
[Lotus japonicus]
AV428124 Lotus japonicus young plants (two-

23
G2509
gi3264767
4.00E-27
[Prunus armeniaca]
AP2 domain containing protein.

23
G2509
gi12003376
1.40E-23
[Nicotiana tabacum]
Avr9/Cf-9 rapidly elicited protein 1.

23
G2509
gi14140141
2.30E-23
[Oryza sativa]
putative AP2-related transcription factor.

23
G2509
gi1688233
5.40E-23
[Solanum tuberosum]
DNA binding protein homolog.

23
G2509
gi4099921
2.60E-22
[Stylosanthes hamata]
EREBP-3 homolog.

23
G2509
gi8809571
7.80E-22
[Nicotiana sylvestris]
ethylene-responsive element binding

23
G2509
gi3342211
1.00E-21
[Lycopersicon esculentum]
Pti4.

23
G2509
gi7528276
2.70E-21
[Mesembryanthemum crystallinum]
AP2-related transcription f

23
G2509
gi17385636
1.90E-20
[Matricaria chamomilla]
ethylene-responsive element binding

23
G2509
gi1849606
3.30E-20
[Fagus sylvatica]
ethylene responsive element binding prote

1119
G2347
B1931517
5.30E-31
[Lycopersicon esculentum]
E5T551406 tomato flower, 8 mm to pr

1119
G2347
BE058432
4.20E-29
[Glycine max]
sn16a06.yl Gm-c1016 Glycine max eDNA clone GENO

1119
G2347
AMSPB1
1.80E-28
[Antirrhinum majus]
A.majus mRNA for squamosa-promoter bindin

1119
G2347
BG525285
5.70E-28
[Stevia rebaudiana]
48-3 Stevia field grown leaf cDNA Stevia

1119
G2347
L38193
4.60E-27
[Brassica rapa]
BNAF1025E Mustard flower buds Brassica rapa c

1119
G2347
BG455868
6.40E-27
[Medicago truncatula]
NF068F05PL1F1045 Phosphate starved leaf

1119
G2347
BG097153
1.70E-24
[Solanum tuberosum]
E5T461672 potato leaves and petioles Sola

1119
G2347
BF482644
1.60E-23
[Triticum aestivum]
WHE2301-2304_A21_A21ZS Wheat pre-anthesis

1119
G2347
AW747167
2.30E-23
[Sorghum bicolor]
WS1_66_F11.b1_A002 Water-stressed 1 (WS1) S

1119
G2347
BG442540
2.50E-23
[Gossypium arboreum]
GA_Ea0017G06f Gossypium arboreum 7-10 d

1119
G2347
gi1183864
1.50E-31
[Antirrhinum majus]
squamosa-promoter binding protein 2.

1119
G2347
gi5931786
3.40E-25
[Zea mays]
SBP-domain protein 5.

1119
G2347
gi8468036
1.40E-21
[Oryza sativa]
Similar to Arabidopsis thaliana chromosome 2

1119
G2347
gi9087308
6.60E-09
[Mitochondrion Beta vulgaris
orf102a.





var. altissima]

1119
G2347
gi7209500
0.83
[Brassica rapa]
S-locus pollen protein.

43
G988
CRU303349
3.10E-208
[Capsella rubella]
ORF1, ORF2, ORF3, ORF4, ORF5 and ORF6(pa

43
G988
A84072
4.50E-86
[Lycopersicon esculentum]
Sequence 1 from Patent WO9846759.

43
G988
A84080
3.30E-85
[Solanum tuberosum]
Sequence 9 from Patent WO9846759.

43
G988
AP003944
1.30E-57
[Oryza sativa]
chromosome 6 clone OJ1126_F05, *** SEQUENCING

43
G988
AX081276
2.80E-43
[Brassica napus]
Sequence 1 from Patent WO0109356.

43
G988
ZMA242530
1.50E-40
[Zea mays]
partial d8 gene for gibberellin response modulato

43
G988
AX005804
2.50E-37
[Triticum aestivum]
Sequence 13 from Patent WO9909174.

43
G988
AB048713
9.10E-33
[Pisum sativum]
PsSCR mRNA for SCARECROW, complete cds.

43
G988
AW774515
2.00E-29
[Medicago truncatula]
E5T333666 KV3 Medicago truncatula cDNA

43
G988
BE822458
1.20E-27
[Glycine max]
GM700017A20H12 Gm-r1070 Glycine max cDNA clone

43
G988
gi13620166
8.00E-211
[Capsella rubella]
hypothetical protein.

43
G988
gi4160441
1.40E-87
[Lycopersicon esculentum]
lateral suppressor protein.

43
G988
gi10178637
2.20E-48
[Zea mays]
SCARECROW.

43
G988
gi6970472
1.20E-47
[Oryza sativa]
OsGAI.

43
G988
gi5640157
2.80E-45
[Triticum aestivum]
gibberellin response modulator.

43
G988
gi13170126
7.10E-45
[Brassica napus]
unnamed protein product.

43
G988
gi13365610
1.10E-40
[Pisum sativum]
SCARECROW.

43
G988
gi14318115
1.10E-14
[Zea mays subsp. mays]
gibberellin response modulator.

43
G988
gi14318165
7.30E-14
[Tripsacum dactyloides]
gibberellin response modulator.

43
G988
gi347457
2.40E-05
[Glycine max]
hydroxyproline-rich glycoprotein.

459
G2346
AMA011622
3.10E-35
[Antirrhinum majus]
mRNA for squamosa promoter binding

459
G2346
AW691786
1.80E-26
[Medicago truncatula]
NF044B06ST1F1000 Developing stem Medica

459
G2346
AQ273505
7.00E-25
[Oryza sativa]
nbxb0030O03f CUGI Rice BAC Library Oryza sativ

459
G2346
AW932595
7.90E-24
[Lycopersicon esculentum]
EST358438 tomato fruit mature green

459
G2346
BG593787
9.50E-24
[Solanum tuberosum]
EST492465 cSTS Solanum tuberosum cDNA clo

459
G2346
BG442540
1.00E-23
[Gossypium arboreum]
GA__Ea00l7G06fGossypium arboreum 7-10 d

459
G2346
A7919034
1.90E-23
[Zea mays]
1006013G02.x3 1006 - RescueMu GridGZea mays geno

459
G2346
B596165
2.70E-23
[Sorghum bicolor]
PI1_50_D04.b1 —






A002 Pathogen induced 1 (PI1)

459
G2346
A1443033
2.30E-22
[Glycine max]
sa31a08.yl Gm-c1004 Glycine max cDNA clone GENO

459
G2346
BF482644
4.30E-22
[Triticum aestivum]
WHE2301-2304_A21_A21ZS Wheat pre-anthesis

459
G2346
gi5931643
6.20E-45
[Antirrhinum majus]
squamosa promoter binding protein-homol

459
G2346
gi5931786
4.20E-26
[Zea mays]
SBP-domain protein 5.

459
G2346
gi8468036
3.30E-14
[Oryza sativa]
Similar to Arabidopsis thaliana chromosome 2

459
G2346
gi9087308
8.30E-08
[Mitochondrion Beta vilgaris var. altissima]
orf102a.

285
G1354
BG128374
2.90E-58
[Lycopersicon esculentum]
E5T474020 tomato shoot/meristem Lyc

285
G1354
BE202831
1.90E-56
[Medicago truncatula]
E5T402853 KV1 Medicago truncatula cDNA

285
G1354
A1161918
6.60E-55
[Populus tremula x Populus tremuloides]
A009P50U Hybrid aspen

285
G1354
AB028186
1.20E-53
[Oryza sativa]
mRNA for OsNAC7 protein, complete cds.

285
G1354
BE060921
8.00E-50
[Hordeum vulgare]
HVSMEg0013N15f Hordeum vulgare pre-anthesis

285
G1354
AF402603
1.50E-42
[Phaseolus vulgaris]
NAC domain protein NAC2 mRNA, complete c

285
G1354
BE357920
1.60E-42
[Sorghum bicolor]
DG1_23_F03.b1_A002 Dark Grown 1 (DG1) Sorgh

285
G1354
PHRNANAM
3.60E-42
[Petunia x hybrida]
P.hybrida mRNA encoding NAM protein.

285
G1354
AW185617
5.30E-40
[Glycine max]
se80b05.y1 Gm-c 1023Glycine max cDNA clone GENO

285
G1354
gi6006373
4.50E-63
[Oryza sativa]
Similar to NAM like protein (AC005310).

285
G1354
gi5148914
2.30E-44
[Phaseolus vulgaris]
NAC domain protein NAC2.

285
G1354
gi14485513
3.50E-44
[Solanum tuberosum]
putative NAC domain protein.

285
G1354
gi1279640
5.90E-44
[Petunia x hybrida]
NAM.

285
G1354
gi6175246
5.20E-41
[Lycopersicon esculentum]
jasmonic acid 2.

285
G1354
gi4218535
5.10E-39
[Triticum sp.]
GRAB1 protein.

285
G1354
gi6732158
5.10E-39
[Triticum monococcum]
unnamed protein product.

285
G1354
gi7716952
3.30E-35
[Medicago truncatula]
NACi.

285
G1354
gi4996349
2.50E-26
[Nicotiana tabacum]
NAC-domain protein.

285
G1354
gi2982275
3.10E-14
[Picea mariana]
ATAF1-like protein.

119
G1063
BH700922
4.50E-90
[Brassica oleracea]
BOMMZ07TR BO_2_3_KB Brassica oleraceagen

119
G1063
BE451174
2.40E-41
[Lycopersicon esculentum]
E5T402062 tomato root, plants pre-a

119
G1063
AW832545
2.00E-40
[Glycine max]
sm12e10.y1 Gm-c1027 Glycine max cDNA clone GENO

119
G1063
AP004693
5.90E-37
[Oryza sativa]
chromosome 8 clone P0461F06 *** SEQUENCING IN

119
G1063
AP004462
4.40E-32
[Oryza sativa (japonica
( ) chromosome 8 clo





cultivar-group)]

119
G1063
AT002234
8.90E-32
[Brassica rapa subsp.pekinensis]
AT002234 Flower bud cDNA Br

119
G1063
BF263465
5.40E-25
[Hordeum vulgare]
HV_CEa0006N02f Hordeum vulgare seedling gre

119
G1063
BG557011
4.20E-22
[Sorghum bicolor]
EM1_41_E02.gl_A002 Embryo 1 (EM1) Sorghum b

119
G1063
BG842856
3.10E-21
[Zea mays]
MEST40-H05.T3 ISUM4-TN Zea mays cDNA clone MEST40-

119
G1063
BG559930
1.40E-18
[Sorghum propinquum]
RHIZ2_75_D09.gl_A003 Rhizome2 (RHIZ2) So

119
G1063
gi15528743
4.20E-26
[Oryza sativa]
contains EST C74560(E31855)~unknown protein.

119
G1063
gi6166283
8.10E-10
[Pinus taeda]
helix-loop-helix protein lA.

119
G1063
gi11045087
8.80E-09
[Brassica napus]
putative protein.

119
G1063
gi10998404
7.10E-08
[Petunia x hybrida]
anthocyanin 1.

119
G1063
gi99441
2.60E-07
[Volvox carteri]
sulfated surface glycoprotein 185 - Volvox

119
G1063
gi1142621
5.00E-07
[Phaseolus vulgaris]
phaseolin G-box binding protein PG2.

119
G1063
gi166428
8.10E-07
[Antirrhinum majus]
DEL.

119
G1063
gi1247386
9.50E-07
[Nicotiana alata]
PRP2.

119
G1063
gi82091
1.00E-06
[Lycopersicon esculentum]
hydroxyproline-rich glycoprotein

119
G1063
gi1486263
1.40E-06
[Catharanthus roseus]
extensin.

129
G2143
BH650724
3.00E-88
[Brassica oleracea]
BOMIW43TR BO_2_3_KB Brassica oleracea gen

129
G2143
AW832545
1.50E-40
[Glycine max]
sm12e10.y1 Gm-c1027 Glycine max cDNA clone GENO

129
G2143
BE451174
3.50E-40
[Lycopersicon esculentum]
E5T402062 tomato root, plants pre-a

129
G2143
AP004693
4.00E-38
[Oryza sativa]
chromosome 8 clone P0461F06, *** SEQUENCING IN

129
G2143
AP004584
6.30E-33
[Oryza sativa(japonica cultivar-group)]
( )chromosome 8 clo

129
G2143
AT002234
3.00E-31
[Brassica rapa subsp. pekinensis]
AT002234 Flower bud cDNA Br

129
G2143
BF263465
2.90E-26
[Hordeum vulgare]
HV_CEa0006N02fHordeum vulgare seedling gre

129
G2143
BG557011
2.60E-22
[Sorghum bicolor]
EM1_41_E02.g1_A002 Embryo 1 (EM1) Sorghum b

129
G2143
BG842856
3.50E-20
[Zea mays]
MEST4O-H05.T3 ISUM4-TN Zea mays cDNA clone MEST4O-

129
G2143
BG559930
6.10E-18
[Sorghum propinquum]
RHIZ2_75_D09.g1_A003 Rhizome2 (RHIZ2) So

129
G2143
gi15528743
5.50E-26
[Oryza sativa]
contains EST C74560(E31855)~unknown protein.

129
G2143
gi1086538
7.60E-09
[Oryza rufipogon]
transcriptional activator Rb homolog.

129
G2143
gi6166283
1.10E-08
[Pinus taeda]
helix-loop-helix protein 1A.

129
G2143
gi1142621
4.60E-07
[Phaseolus vulgaris]
phaseolin G-box binding protein PG2.

129
G2143
gi3399777
5.20E-07
[Glycine max]
symbiotic ammonium transporter; nodulin.

129
G2143
gi5923912
6.10E-07
[Tulipa gesneriana]
bHLH transcription factor GBOF-1.

129
G2143
gi10998404
9.20E-07
[Petunia x hybrida]
anthocyanin 1.

129
G2143
gi4321762
5.20E-06
[Zea mays]
transcription factor MYC7E.

129
G2143
gi166428
6.00E-06
[Antirrhinum majus]
DEL.

129
G2143
gi527665
7.40E-06
[Sorghum bicolor]
myc-like regulatory R gene product.

133
G2557
BH511840
6.70E-62
[Brassica oleracea]
BOGRJ19TR BOGR Brassica oleracea genomic

133
G2557
BE347811
3.70E-46
[Glycine max]
sp05h10.y1 Gm-c1041 Glycine max cDNA clone GENO

133
G2557
AP003141
2.40E-33
[Oryza sativa]
genomic DNA, chromosome 1, PAC clone:P0002B05,

133
G2557
BF263465
3.00E-31
[Hordeum vulgare]
HV_CEa0006N02fHordeum vulgare seedling gre

133
G2557
AT002234
6.60E-27
[Brassica rapa subsp. pekinensis]
AT002234 Flowerbud cDNA Br

133
G2557
BG557011
6.40E-26
[Sorghum bicolor]
EM1_41_E02.g1_A002 Embryo 1 (EM1) Sorghum b

133
G2557
AP004462
7.90E-26
[Oryza sativa
( )chromosome 8 clo





(japonica cultivar-group)]

133
G2557
BE451174
3.90E-25
[Lycopersicon esculentum]
E5T402062 tomato root, plants pre-a

133
G2557
BG842856
5.60E-22
[Zea mays]
MEST40-H05.T3 ISUM4-TN Zea mays cDNA clone MEST4O-

133
G2557
BG559930
7.00E-14
[Sorghum propinquum]
RH1Z2_75_D09.g1 A003 Rhizome2 (RHIZ2) So

133
G2557
gi15289790
2.40E-36
[Oryza sativa]
contains EST C74560(E31855)~unknown protein.

133
G2557
gi3399777
2.60E-06
[Glycine max]
symbiotic ammonium transporter; nodulin.

133
G2557
gi4206118
1.10E-05
[Mesembryanthemum crystallinum]
transporter homolog.

133
G2557
gi6166283
1.30E-05
[Pinus taeda]
helix-loop-helix protein 1A.

133
G2557
gi527655
3.70E-05
[Pennisetum glaucum]
myc-like regulatory R gene product.

133
G2557
gi5923912
3.70E-05
[Tulipa gesneriana]
bHLH transcription factor GBOF-1.

133
G2557
gi527661
7.80E-05
[Phyllostachys acuta]
myc-like regulatory R gene product.

133
G2557
gi527665
9.50E-05
[Sorghum bicolor]
myc-like regulatory R gene product.

133
G2557
gi1086538
0.0001
[Oryza rufipogon]
transcriptional activator Rb homolog.

133
G2557
gi5669656
0.00013
[Lycopersicon esculentum]
ER33 protein.

697
G2430
BF632520
1.90E-14
[Medicago truncatula]
NF039A08DT1F10541054 Drought Medicago trunc

697
G2430
AW396912
1.20E-13
[Glycine max]
sg64g09.y1 Gm-c1007 Glycine max cDNA clone GENO

697
G2430
D41804
4.50E-13
[Oryza sativa]
RICS4626A Rice shoot Oryza sativa cDNA, mRNA s

697
G2430
BE214029
2.60E-10
[Hordeum vulgare]
HV_CEb0001P06fHordeum vulgare seedling gre

697
G2430
AW564570
2.70E-10
[Sorghum bicolor]
LG1_296_E01.b1_A002 Light Grown 1 (LG1) Sor

697
G2430
BG129795
5.40E-10
[Lycopersicon esculentum]
EST475441 tomato shoot/meristem Lyc

697
G2430
AB060130
5.40E-09
[Zea mays]
ZmRR8 mRNA for response regulator 8, complete cds.

697
G2430
BF587105
2.50E-05
[Sorghum propinquum]
FM1_32_C0Sbi_A003 Floral-Induced Merist

697
G2430
AI163121
0.3
[Populus tremula x Populus tremuloides]
A033P70U Hybrid aspen

697
G2430
BG595628
0.46
[Solanum tuberosum]
EST494306 cSTS Solanum tuberosum cDNA clo

697
G2430
gi13661174
5.40E-18
[Zea mays]
response regulator 8.

697
G2430
gi15289981
0.028
[Oryza sativa]
hypothetical protein.

697
G2430
gi6942190
0.12
[Mesembryanthemum crystallinum]
CDPK substrate protein 1; C

697
G2430
gi4519671
0.2
[Nicotiana tabacum]
transfactor.

831
G1478
BF275913
1.50E-20
[Gossypium arboreum]
GA__Eb0025C07fGossypium arboreum 7-10 d

831
G1478
BG157399
6.50E-19
[Glycine max]
sab36g12.y1 Gm-c1026 Glycine max cDNA clone GEN

831
G1478
C95300
2.20E-10
[Citrus unshiu]
C95300 Citrus unshiu Miyagawa-wase maturation

831
G1478
AW034552
2.70E-10
[Lycopersicon esculentum]
EST278168 tomato callus, TAMU Lycop

831
G1478
B1070429
3.40E-10
[Populus tremula x Populus tremuloides]
C037P68U Populus stra

831
G1478
AF016011
5.10E-09
[Brassica napus]
CONSTANS homolog (Bn9C0N10) gene, complete c

831
G1478
BE598912
6.20E-09
[Sorghum bicolor]
P11_84_H11.bi_A002 Pathogen induced 1 (PI1)

831
G1478
BG605313
6.80E-09
[Triticum aestivum]
WHE2331_C04_F07ZS Wheat pre-anthesis spik

831
G1478
BE558327
8.90E-09
[Hordeum vulgare]
HV_CEb0017Dl9fHordeum vulgare seedling gre

831
G1478
BG647091
1.20E-08
[Medicago truncatula]
EST508710 HOGA Medicago truncatula cDNA

831
G1478
gi2895188
4.70E-11
[Brassica napus]
CONSTANS homolog.

831
G1478
gi3618308
1.50E-09
[Oryza sativa]
zinc finger protein.

831
G1478
gi11037308
4.70E-09
[Brassica nigra]
constans-like protein

831
G1478
gi3341723
1.30E-08
[Raphanus sativus]
CONSTANS-like 1 protein.

831
G1478
gi4091806
1.50E-07
[Malus x domestica]
CONSTANS-like protein 2.

831
G1478
gi10946337
3.10E-07
[Ipomoea nil]
CONSTANS-like protein.

831
G1478
gi4557093
1.40E-05
[Pinus radiata]
zinc finger protein.

831
G1478
gi619312
0.9
[Capparis masaikai]
mabinlin III B-chain = sweet protein mabi

831
G1478
gi4732091
1
[Zea mays]
bundle sheath defective protein 2.

831
G1478
gi4699629
1
[Nicotiana alata]
Chain A, Putative Ancestral Protein Encod

579
G681
BG128147
6.80E-41
[Lycopersicon esculentum]
E5T473793 tomato shoot/meristem Lyc

579
G681
BF054497
1.50E-39
[Solanum tuberosum]
E5T439727 potato leaves and petioles Sola

579
G681
BE054276
8.40E-39
[Gossypium arboreum]
GA__Ea0002018f






Gossypium arboreum 7-10 d

579
G681
BG269414
4.00E-38
[Mesembryanthemum
L0-3478T3 Ice plant Lambda Un





crystallinum]

579
G681
BF620286
7.40E-38
[Hordeum vulgare]
HVSMEc0019F08f Hordeum vulgare seedling sho

579
G681
BE490032
1.00E-37
[Triticum aestivum]
WHE0364_C04_E08ZS Wheat cold-stressed see

579
G681
B1542536
1.40E-36
[Zea mays]
949021A03.y1 949 - Juvenile leaf and shoot cDNA fr

579
G681
BF425254
7.20E-36
[Glycine max]
su42c10.y1 Gm-c1068 Glycine max cDNA clone GENO

579
G681
AW672062
3.20E-34
[Sorghum bicolor]
LG1_354_G05b1_A002 Light Grown 1 (LG1) Sor

579
G681
BG448527
1.00E-33
[Medicago truncatula]
NF036F04RT1F1032 Developing root Medica

579
G681
gi13346188
9.10E-37
[Gossypium hirsutum]
GHMYB25.

579
G681
gi20563
6.30E-36
[Petunia x hybrida]
protein 1.

579
G681
gi485867
1.20E-34
[Antirrhinum majus]
mixta.

579
G681
gi2605617
1.70E-32
[Oryza sativa]
OSMYB1.

579
G681
gi1430846
2.00E-31
[Lycopersicon esculentum]
myb-related transcription factor.

579
G681
gi6651292
2.20E-30
[Pimpinella brachycarpa]
myb-related transcription factor.

579
G681
gi15042116
4.90E-30
[Zea mays subsp. parviglumis]
CI protein.

579
G681
gi82730
6.10E-30
[Zea mays]
transforming protein (myb) homolog (clone Zm38)

579
G681
gi5139806
8.30E-30
[Glycine max]
GmMYB29A2.

579
G681
gi19055
1.10E-29
[Hordeum vulgare]
MybHv5.

611
G878
AF096299
6.20E-90
[Nicotiana tabacum]
DNA-binding protein 2 (WRKY2) mRNA, compl

611
G878
CUSSLDB
1.80E-83
[Cucumis sativus]
SPF 1-like DNA-binding protein mRNA, complet

611
G878
AF193802
3.50E-63
[Oryza sativa]
zinc finger transcription factor WRKY1 mRNA, c

611
G878
AX192162
2.20E-62
[Glycine max]
Sequence 9 from Patent WO0149840.

611
G878
IPBSPF1P
3.80E-58
[Ipomoea batatas]
Sweet potato mRNA for SPF1 protein, complet

611
G878
AFABF1
2.00E-56
[Avena fatua]
A.fatua mRNA for DNA-binding protein(clone ABF

611
G878
LE5303343
7.20E-55
[Lycopersicon esculentum]
mRNA for hypothetical protein (ORF

611
G878
AX192164
4.00E-54
[Triticum aestivum]
Sequence 11 from Patent WO0149840.

611
G878
AF080595
2.10E-53
[Pimpinella brachycarpa]
zinc finger protein (ZFP1) mRNA, com

611
G878
PCU48831
2.30E-53
[Petroselinum crispum]
DNA-binding protein WRKY1 mRNA, comple

611
G878
gi4322940
3.30E-128
[Nicotiana tabacum]
DNA-binding protein 2.

611
G878
gi927025
1.10E-109
[Cucumis sativus]
SPF1-like DNA-binding protein.

611
G878
gi6689916
1.50E-74
[Oryza sativa]
zinc finger transcription factor WRKY1

611
G878
gi484261
1.10E-66
[Ipomoea batatas]
SPF1 protein.

611
G878
gi1159877
2.30E-63
[Avena fatua]
DNA-binding protein.

611
G878
gi13620227
4.60E-63
[Lycopersicon esculentum]
hypothetical protein.

611
G878
gi5917653
1.70E-56
[Petroselinum crispum]
zinc-finger type transcription facto

611
G878
gi4894965
5.00E-56
[Avena sativa]
DNA-binding protein WRKY1.

611
G878
gi3420906
8.70E-56
[Pimpinella brachycarpa]
zinc finger protein; WRKY1.

611
G878
gi13620168
4.20E-22
[Capsella rubella]
hypothetical protein.

47
G374
AP004457
1.20E-73
[Oryza sativa (japonica cultivar group)]
( )chromosome 8 clo

47
G374
AP004693
1.90E-73
[Oryza sativa]
chromosome 8 clone P0461F06 *** SEQUENCING IN

47
G374
BH552835
1.30E-62
[Brassica oleracea]
BOHGT56TR BOHG Brassica oleracea genomic

47
G374
BG128229
6.50E-55
[Lycopersicon esculentum]
EST473875 tomato shoot/meristem Lyc

47
G374
BG646959
3.20E-46
[Medicago truncatula]
E5T508578 HOGA Medicago truncatula cDNA

47
G374
BG890162
8.70E-41
[Solanum tuberosum]
EST516013 cSTD Solanum tuberosum cDNA clo

47
G374
AW179366
6.00E-38
[Zea mays]
618046G06.y1 618 - Inbred Tassel cDNA Library Zea

47
G374
BF473206
1.50E-32
[Triticum aestivum]
WHE0922_G12_M24ZS Wheat 5-15 DAPspike cD

47
G374
AW761011
2.90E-29
[Glycine max]
s161g11.y1 Gm-c1027 Glycine max cDNA clone GENO

47
G374
AJ436050
1.50E-27
[Hordeum vulgare]
AJ436050 S00007 Hordeum vulgare cDNA clone

47
G374
gi422012
0.8
[Sorghum bicolor]
lipid transfer protein - sorghum (fragmen

47
G374
gi1827893
1
[Zea mays]
Maize Nonspecific Lipid Transfer Protein Complex





































































































































































































































































































































































The invention thus provides for methods for preparing transgenic plants, and for modifying plant traits. These methods include introducing into a plant a recombinant expression vector or cassette comprising a functional promoter operably linked to one or more sequences homologous to presently disclosed sequences. Plants and kits for producing these plants that result from the application of these methods are also encompassed by the present invention.
Traits of Interest
Examples of some of the traits that may be desirable in plants, and that may be provided by transforming the plants with the presently disclosed sequences, are listed in Table 5 and 6.





TABLE 5



Genes, traits, transcription factor families and conserved domains










Poly-





Poly-


nucleotide





peptide

SEQ
GID




SEQ ID
Conserved

ID NO:
No.
Trait
Category
Family
Comment
NO:
domains












1
G1275
Architecture;
Dev and
WRKY
Reduced apical
2
(113-169)



size
morph

dominance; small






plant

3
G1411
Architecture
Dev and morph
AP2
Loss of apical dominance
4
(87-154)

5
G1488
Architecture;
Dev and
GATA/Zn
Reduced apical
6
(221-246)



light
morph; seed

dominance, shorter



response;
biochemistry

stems; constitutive



size; seed


photomorphogenesis;



protein


reduced size; altered



content


seed protein content

7
G1499
Architecture;
Dev and
HLH/MYC
Altered plant
8
(118-181)



flower;
morph

architecture; altered



morphology:


floral organ identity



other


and development; dark






green color

9
G1543
Architecture;
Dev and
HB
Altered plant
10
(135-195)



flower;
morph; seed

architecture; altered



morphology:
biochemistry

carpel shape; dark



other; seed oil


green color; decreased






seed oil

11
G1635
Architecture;
Dev and
MYB-
Reduced apical
12
(44-104)



morphology:
morph
related
dominance; pale



other; fertility


green, smaller plants;






reduced fertility

13
G1794
Architecture;
Dev and
AP2
Altered plant
14
(182-248)



light
morph; seed

architecture;



response;
biochemistry

constitutive



seed oil and


photomorphogenesis;



protein content


altered seed oil and






protein content

15
G1839
Architecture;
Dev and
AP2
Altered plant
16
(118-184)



size
morph

architecture; reduced






size

17
G2108
Architecture
Dev and morph
AP2
Altered inflorescence
18
(18-85)






structure

19
G2291
Architecture;
Dev and
AP2
Altered plant
20
(TBD)



flowering
morph;

architecture; late



time
flowering

flowering




time

21
G2452
Architecture;
Dev and
MYB-
Reduced apical
22
(27-213)



leaf
morph
related
dominance; pale green






color

23
G2509
Architecture;
Dev and morph;
AP2
Reduced apical
24
(89-156)



seed oil and
seed biochemistry

dominance; altered



protein content


seed oil and protein






content

25
G390
Architecture
Dev and
HB
Altered shoot
26
(18-81)




morph

development

27
G391
Architecture
Dev and
HB
Altered shoot
28
(25-85)




morph

development

29
G438
Architecture;
Dev and
HB
Reduced branching,
30
(22-85)



stem
morph

reduced lignin

31
G47
Architecture;
Dev and morph;
AP2
Altered architecture
32
(11-80)



stem;
flowering

and inflorescence



flowering
time; seed

development, structure



time; altered
biochemistry

of vascular tissues;



seed oil


late flowering; altered



content


seed oil content

33
G559
Architecture;
Dev and
bZIP
Loss of apical
34
(203-264)



fertility
morph

dominance; reduced






fertility

35
G568
Architecture;
Dev and
bZIP
Altered branching; late
36
(215-265)



flowering
morph;

flowering



time
flowering




time

37
G580
Architecture;
Dev and
bZIP
Altered inflorescences;
38
(162-218)



flower
morph

altered flower






development

39
G615
Architecture;
Dev and
TEO
Altered plant
40
(88-147)



fertility
morph

architecture; little or






no pollen production,






poor filament






elongation

41
G732
Architecture;
Dev and
bZIP
Reduced apical
42
(31-91)



flower; seed
morph; seed

dominance; abnormal



oil and
biochemistry

flowers; altered seed



protein


oil and protein content



content

43
G988
Architecture;
Dev and
SCR
Reduced lateral
44
(178-195)



fertility;
morph; seed

branching; reduced



flower; stem;
biochemistry

fertility; enlarged



seed oil and


floral organs, short



protein


pedicels; thicker stem,



content


altered distribution of






vacular bundles;






altered seed oil and






protein content

45
G1519
Embryo lethal
Dev and morph
RING/C3HC4
Embryo lethal
46
(327-364)

47
G374
Embryo lethal
Dev and morph
Z-ZPF
Embryo lethal
48
(35-67,








245-277)

49
G877
Embryo lethal
Dev and morph
WRKY
Embryo lethal
50
(272-328,








487-603)

51
G1000
Fertility; size;
Dev and
MYB-
Reduced fertility;
52
(14-117)



flower; stem
morph
(R1)R2R3
small plant; reduced or






absent petals and






sepals; reduced






inflorescence, stem






elongation

53
G1067
Fertility; leaf
Dev and
AT-hook
Reduced fertility,
54
(86-93)



size
morph

altered leaf shape;






small plant

55
G1075
Fertility; flower;
Dev and
AT-hook
Reduced fertility,
56
(78-85)



leaf; size
morph

reduced or absent






petals, sepals and






stamens; altered leaf






shape; small plant

57
G1266
Fertility; size
Dev and
AP2
Reduced fertility;
58
(79-147)




morph

small plant

59
G1311
Fertility; size
Dev and
MYB-
Reduced fertility;
60
(11-112)




morph
(R1)R2R3
small plant

61
G1321
Fertility; flower
Dev and
MYB-
Poor fertility; altered
62
(4-106)




morph
(R1)R2R3
flower morphology

63
G1326
Fertility; flower;
Dev and
MYB-
Reduced fertility;
64
(18-121)



size
morph
(R1)R2R3
petals and sepals are






smaller; small plant

65
G1367
Fertility; size
Dev and
AT-hook
Reduced fertility;
66
(179-201,




morph

reduced size

262-285,








298-319,








335-357)

67
G1386
Fertility; size;
Dev and
AP2
Reduced fertility;
68
(TBD)



seed oil and
morph; seed

and reduced size; altered



protein
biochemistry

seed oil and protein



content


content

69
G1421
Fertility; size;
Dev and
AP2
Reduced fertility;
70
(74-151)



seed oil
morph; seed

small plant; altered



content
biochemistry

seed oil content

71
G1453
Fertility;
Dev and
NAC
Reduced fertility;
72
(13-160)



morphology:
morph

altered inflorescence



other


development

73
G1560
Fertility;
Dev and
HS
Reduced fertility;
74
(62-151)



flower; size
morph

altered flower






development; reduced






size

75
G1594
Fertility; leaf;
Dev and
HB
Reduced fertility;
76
(343-308)



seed
morph

altered leaf shape and






and development; large






pale seed

77
G1750
Fertility; size
Dev and
AP2
Reduced fertility;
78
(107-173)



seed oil
morph; seed

reduced size;



content
biochemistry

increased seed oil






content

79
G1947
Fertility; flower;
Dev and
HS
Reduced fertility;
80
(37-120)



seed protein
morph; seed

and extended period of



content
biochemistry

flowering; altered seed






protein content

81
G2011
Fertility; size
Dev and
HS
Reduced fertility;
82
(56-147)



seed oil and
morph; seed

and reduced size; altered



protein content
biochemistry

seed oil and protein






content

83
G2094
Fertility; leaf;
Dev and
GATA/Zn
Reduced fertility;
84
(43-68)



size
morph

altered leaf






and development; reduced






size

85
G2113
Fertility; leaf;
Dev and
AP2
Reduced fertility; long
86
(TBD)



seed protein
morph; seed

petioles, altered



content
biochemistry

orientation; altered






seed protein content

87
G2115
Fertility; size
Dev and morph
AP2
Reduced fertility;
88
(46-115)






reduced size

89
G2130
Fertility; size;
Dev and morph
AP2
Reduced fertility;
90
(93-160)



senescence


reduced size; early






senescence

91
G2147
Fertility; size
Dev and morph
HLM/MYC
Reduced fertility;
92
(160-234)






reduced size

93
G2156
Fertility; size;
Dev and morph;
AT-hook
Reduced fertility;
94
(66-86)



seed protein
seed biochemistry

reduced size; altered



content


seed protein content

95
G2294
Fertility; size
Dev and morph
AP2
Reduced fertility;
96
(32-102)






reduced size

97
G2510
Fertility; size
Dev and morph
AP2
Reduced fertility;
98
(41-108)






reduced size

99
G2893
Fertility; flower;
Dev and morph
MYB-(R1)
Reduced fertility;
100
(19-120)



size

R2R3
altered flower






development; reduced






size

101
G340
Fertility; size
Dev and morph
Z-C3H
Reduced fertility, size
102
(37-154)

103
G39
Fertility; size
Dev and morph
AP2
Reduced fertility,
104
(24-90)






small plant

105
G439
Fertility; size
Dev and morph
AP2
Reduced fertility;
106
(110-177)






small plant

107
G470
Fertility
Dev and morph
ARF
Short stamen filaments
108
(61-393)

109
G652
Fertility; seed;
Dev and morph;
Z-CLDSH
Reduced fertility;
110
(28-49,



flower; size;
seed biochemistry

irregular shaped seed

137-151,



seed oil content


altered flower

182-196)






development; reduced






size, slow growth;






altered seed oil content

111
G671
Fertility; flower;
Dev and morph
MYB-(R1)
Reduced fertility;
112
(15-115)



leaf size; stem

R2R3
abscission; altered leaf






shape; small plant;






altered inflorescence






stem structure

113
G779
Fertility; flower
Dev and morph
HLH/MYC
Reduced fertility,
114
(126-182)






homoerotic






transformations

115
G962
Fertility; size
Dev and morph
NAC
Reduced fertility;
116
(53-175)






small plant

117
G977
Fertility; leaf;
Dev and morph
AP2
Reduced fertility;
118
(5-72)



morphology; other


altered leaf shape;



size


dark green; small plant

119
G1063
Flower; leaf;
Dev and morph;
HLM/MYC
Altered flower
120
(131-182)



inflorescence;
seed biochemistry

development, ectopic



seed oil and


carpel tissue; altered



protein content


leaf shape, dark altered






color; altered






inflorescence






development; altered






seed oil and protein






content

121
G1140
Flower
Dev and morph
MADS
Altered flower development
122
(2-57)

123
G1425
Flower
Dev and morph
NAC
Altered flower and
124
(20-173)






inflorescence

125
G1449
Flower
Dev and morph
IAA
Altered flower structure
126
(48-53,








74-107,








122-152)

127
G1897
Flower; leaf;
Dev and morph
Z-Dof
Altered flower
128
(34-62)



seed protein
seed biochemistry

development;



content


altered leaf






development altered






seed protein content

129
G2143
Flower; leaf;
Dev and morph
HLM/MYC
Altered flower
130
(128-179)



inflorescence


development, ectopic






carpel tissue; altered






leaf shape, dark green






color; altered






inflorescence






development

131
G2535
Flower; seed
Dev and morph;
NAC
Altered flower
132
(11-114)



protein content
seed biochemistry

development; altered






seed protein






content

133
G2557
Flower; leaf
Dev and morph
HLM/MYC
Altered flower
134
(278-328)






development






ectopic carpel






tissue; altered leaf






shape, dark green






color

135
G259
Flower; leaf
Dev and morph
HS
Altered flower
136
(27-131)






development;






altered leaf






development

137
G353
Flower; leaf; size;
Dev and morph;
Z-C2H2
Short pedicels,
138
(41-61,



seed protein
seed biochemistry

downward pointing

84-104)



content


siliques; altered leaf






development;






reduced size;






altered seed






protein content

139
G354
Flower; light
dev and morph
Z-C2H2
Short pedicels,
140
(42-62



respond; size


downward pointing

88-109)






siliques; morphogenesis;






reduced size

141
G638
Flower; morphology:
Dev and morph
TH
Altered flower
142
(119-206)



other


development; multiple






developmental defects

143
G869
Flower; morphology:
Dev and morph;
AP2
Abnormal anther
144
(109-177)



other; seed oil
seed biochemistry

development; small






and spindly plant;






altered seed fatty acids

145
G1645
Inflorescence; leaf
Dev and morph
MYB-(R1)
Altered inflorescence
146
(90-210)





R2R3
structure; altered leaf






development

147
G1038
Leaf
Dev and morph
GARP
Altered leaf shape
148
(198-247)

149
G1073
Leaf; size;
Dev and morph;
AT-hook
Serrated leaves;
150
(33-42,



flowering time
flowering time

increased plant size;

18-175)






flowering appears






to be slightly delayed

151
G1146
Leaf
Dev and morph
PAZ
Altered leaf
152
(886-896)






development

153
G1267
Leaf; size
Dev and morph
WRKY
Dark green shiny
154
(70-127)






leaves; small plant

155
G1269
Leaf
Dev and morph
MYB-related
Long petioles,
156
(27-83)






upturned leaves

157
G1452
Leaf; trichome;
Dev and morph;
NAC
Altered leaf shaped,
158
(30-177)



flowering time
flowering time

dark green color;






reduced trichome






density; late flowering

159
G14949
Leaf; size; light
Dev and morph
HLH/MYC
Pale green leaves,
160
(261-311)



response; seed


altered leaf shape;






reduced size; long






hypocotyls; large,






pale seeds

161
G1548
Leaf
Dev and morph
HB
Altered leaf development
162
(17-77)

163
G1574
Leaf
Dev and morph
SWI/SNF
Altered leaf development
164
(28-350)

165
G1586
Leaf; size
Dev and morph
HB
Narrow leaves; small
166
(21-81)






plants

167
G1786
Leaf; light
Dev and morph
MYB-(R1)
Dark green, small
168
(TBD)



response; size

R2R3
leaves with short






petioles; photo-






photomorphogensis in






the dark; small plant

169
G1792
Leaf; seed oil
Dev and morph;
AP2
Dark green, shiny
170
(17-85)



and protein content
seed biochemistry

leaves; altered seed






oil and protein content

171
G1865
Leaf; seed oil
Dev and morph;
GRF-like
Altered leaf
172
(124-149)



and protein content
seed biochemistry

development; altered






seed oil and protein content

173
G1886
Leaf; size
Dev and morph
Z-Dof
Chlorotic patches in
174
(17-59)






leaves; reduced size

175
G1933
Leaf; size; seed
Dev and morph;
WRKY
Altered leaf
176
(205-263,



protein content
seed biochemistry

development; reduced size

344-404)






altered seed protein






content

177
G2059
Leaf; seed oil
Dev and morph;
AP2
Smaller, curled leaves;
178
(184-254)



and protein content
seed

altered seed oil,




biochemistry

protein content

179
G2105
Leaf; seed
Dev and morph
TH
Alteration in leaf
180
(100-153)






surface; large,






pale seeds

181
G2117
Leaf; seed oil
Dev and morph;
bZIP
Small, dark green
182
(46-106)



and protein content
seed biochemistry

leaves; altered seed oil






and protein content

183
G2124
Leaf; seed protein
Dev and morph;
TEO
Altered leaf
184
(75-132)



content
seed

development; altered




biochemistry

seed protein content

185
G2140
Leaf; root
Dev and morph
HLH/MYC
Altered leaf
186
(167-242)






development; short roots

187
G2144
Leaf; light response;
Dev and morph
HLH/MYC
Pale green leaves,
188
(203-283)



size; seed oil content
seed

altered leaf shape;




biochemistry

long hypocotyls; reduced






size; altered seed oil






content

189
G2431
Leaf
Dev and morph
GARP
Dark green leaves;
190
(38-88)






reduced size

191
G2465
Morphology; other;
Dev and morph
GARP
Slowed development;
192
(219-269)



leaf


altered leaf color






and shape

193
G2583
Leaf; seed oil
Dev and morph
AP2
Glossy, shiny leaves;
194
(4-71)



and protein content
seed biochemistry

altered seed oil






and protein content

195
G2724
Leaf
Dev and morph
MYB-(R1)R2R3
Dark green leaves
196
(7-113)

197
G377
Leaf; morphology:
Dev and morph
RING/C3H2C3
Altered leaf
198
(85-128)



other


development; slow






growth

199
G428
Leaf
Dev and morph
HB
Altered leaf shape
200
(229-292)

201
G447
Leaf; morphology:
Dev and morph
ARF
Dark green leaves;
202
(22-356)



other; size


altered cotyledon






shape; reduced size

203
G464
Leaf
Dev and morph
IAA
Altered leaf shape
204
(20-28,








71-82,








126-142,








187-224)

205
G557
Leaf; size
Dev and morph
bZIP
Dark green color;
206
(90-150)






small plant

207
G577
Leaf
Dev and morph
BZIPT2
Reduced size,
208
(TBD)






increased






anthocyanins

209
G674
Leaf; size
Dev and morph
MYB-(R1)
Dark green leaves,
210
(20-120)





R2R3
upwardly oriented;






reduced size

211
G736
Leaf; flowering time
Dev and morph;
Z-Dof
Altered leaf shape;
212
(54-111)




flowering time

later flowering

213
G903
Leaf
Dev and morph
Z-C2H2
Altered leaf
214
(68-92)






morphology

215
G917
Leaf; seed oil
Dev and morph
MADS
Altered leaf development;
216
(2-57)



and protein content
seed biochemistry

altered seed oil and protein






content

217
G921
Leaf
Dev and morph
WRKY
Serrated leaves
218
(146-203)

219
G922
Leaf; size
Dev and morph
SCR
Altered development,
220
(225-242)






dark green color;






reduced size

221
G932
Leaf; size
Dev and morph
MYB-(R1)R2R3
Altered development,
220
(225-242)






dark green color;






reduced size

223
G599
Leaf; size
Dev and morph
DBP
Altered leaf shape;
224
(187-219,






small plant

264-300)

225
G804
Leaf; size
Dev and morph
PCF
Altered leaf shape,
226
(54-117)






small plant

227
G1062
Light response;
Dev and morph
HLH/MYC
Constitutive
228
(308-359)



morphology;


photomorphogenesis;



other; seed


slow growth; altered






seed shape

229
G1322
Light response; size
Dev and morph
MYB-(R1)R2R3
Photomorphogenesis
230
(26-130)






in the dark; reduced






size

231
G1331
Light response;
Dev and morph;
MYB-(R1)
Constitutive
232
(8-109)



morphology; other;
seed biochemistry
R2R3
photomorphogenesis;



seed oil and


multiple developmental



protein content


alterations; altered






seed oil and protein






content

233
G1521
Light response
Dev and morph
RING/C3HC4
Constitutive
234
(39-80)






photomorphogenesis

235
G183
Light response;
Dev and morph;
WRKY
Constitutive
236
(307-363)



seed protein content
seed biochemistry

photomorphogenesis;






altered seed






protein content

237
G2555
Light response
Dev and morph
HLH/MYC
Constitutive
238
(175-245)






photomorphogenesis

239
G375
Light response
Dev and morph
Z-Dof
Upward pointing leaves
240
(75-103)

241
G1007
Morphology; other
Dev and morph
AP2
Multiple
242
(TBD)






developmental alterations

243
G1010
Morphology; other
Dev and morph
ABI3/VP-1
Multiple
244
(33-122)






developmental alterations

245
G1014
Morphology; other
Dev and morph
ABI3/VP-1
Multiple
246
(90-172)



trichome


developmental defects;






reduced trichomes

247
G1035
Morphology; other
Dev and morph
bZIP
Multiple
248
(39-91)






developmental






alterations

249
G1046
Morphology; other
Dev and morph
bZIP
Multiple
250
(79-138)






developmental






alterations

251
G1049
Morphology;
Dev and morph;
bZIP
Multiple
252
(77-132)



other; seed
seed biochemistry

developmental



protein content


alterations; altered






seed protein content

253
G1069
Morphology: other
Dev and morph;
AT-hook
Multiple
254
(67-74)



seed oil content
seed biochemistry

developmental






alterations; altered






seed oil content

255
G1070
Morphology: other
Dev and morph
AT-hook
Several developmental
256
(98-120)






defects

257
G1076
Morphology; other
Dev and morph
AT-hook
Lethal when
258
(82-89)






overexpressed

259
G1089
Morphology; other
Dev and morph
BZIPT2
Developmental defects
260
(425-500)






at seeding stage

261
G1093
Morphology: other
Dev and morph
RING/C3H2C3
Multiple morphological
262
(105-148)






alterations

263
G1127
Morphology: other
Dev and morph
AT-hook
Multiple developmental
264
(103-110,






alterations

155-162)

265
G1131
Morphology: other;
Dev and morph;
HLH/MYC
Multiple developmental
266
(173-220)



seed protein content


alterations; altered






seed protein content

267
G1145
Morphology; other;
Dev and morph;
bZIP
Multiple developmental
268
(227-270)



seed oil and
seed biochemistry

alterations; reduced



protein content


seed size, altered seed






shape; altered seed oil






and protein content

269
G1229
Morphology: other;
Dev and morph;
HLH/MYC
Several developmental
270
(102-160)



seed oil and
seed biochemistry

defects; altered seed



protein content
content

oil and protein

271
G1246
Morphology; other
Dev and morph;
MYB-(R1)
Multiple developmental
272
(27-139)



seed protein
seed biochemistry
R2R3
alterations; altered



content


seed protein content

273
G1255
Morphology; other
Dev and morph
Z-CO-like
Reduced apical
274
(18-56)



seed


dominance; increased






seed size

275
G1304
Morphology: other
Dev and morph
MYB-(R1)
Lethal when
276
(13-118)





R2R3
overexpressed

277
G1318
Morphology: other
Dev and morph
MYB-(R1)
Multiple developmental
378
(20-123)





R2R3
alterations

279
G1320
Morphology: other
Dev and morph
MYB-(R1)
Multiple developmental
280
(5-108)





R2R3
alterations

281
G1330
Morphology: other
Dev and morph
MYB-(R1)
Multiple developmental
282
(28-134)





R2R3
alterations

283
G1352
Morphology: other
Dev and morph
Z-C2H2
Multiple developmental
284
(108-129,






alterations

167-188)

285
G1354
Morphology: other
Dev and morph
NAC
Multiple developmental
286
(TBD)






alterations

287
G1360
Morphology: other
Dev and morph
NAC
Lethal when overexpressed
288
(18-174)

289
G1364
Morphology: other
Dev and morph
CAAT
Lethal when overexpressed
290
(29-120)

291
G1379
Morphology: other
Dev and morph
AP2
Multiple developmental
292
(18-85)






alterations

293
G1384
Morphology: other
Dev and morph
AP2
Abnormal inflorescence
294
(TBD)






and flower development

295
G1399
Morphology: other
Dev and morph
AT-hook
Multiple developmental
296
(86-93)






alterations

297
G1415
Morphology: other
Dev and morph
AP2
Multiple developmental
298
(TBD)






alterations

299
G1417
Morphology: other;
Dev and morph;
WRKY
Reduced seeding
300
(239-296)



seed oil
seed biochemistry

germination and vigor;






increases in 18:2,






decrease in 18:3

301
G1442
Morphology: other
Dev and morph
GRF-like
Multiple developmental
302
(172-223)






alterations

303
G1454
Morphology: other;
Dev and morph;
NAC
Multiple developmental
304
(9-178)



seed oil and
seed biochemistry

alteration; altered



protein content


seed oil and protein






content

305
G1459
Morphology; other
Dev and morph
NAC
Multiple developmental
306
(10-152)






alterations

307
G1460
Morphology: other;
Dev and morph
NAC
Multiple developmental
308
(TBD)



seed protein content
seed biochemistry

alterations; altered






seed protein content

309
G147
Morphology: other
Dev and morph
MADS
Multiple developmental
310
(2-57)






defects

311
G1471
Morphology: other;
Dev and morph;
Z-C2H2
Multiple developmental
312
(49-70)



seed oil
seed biochemistry
alterations;
defects





increased
seed oil content

313
G1475
Morphology: other
Dev and morph
Z-C2H2
Multiple developmental
314
(51-73)






alterations

315
G1477
Morphology: other
Dev and morph
Z-C2H2
Multiple developmental
316
(29-48)






alterations

317
G1487
Morphology: other;
Dev and morph:
GATA/Zn
Multiple developmental
318
(251-276)



seed oil and
seed biochemistry

alterations; altered



protein content


seed oil and






protein content

319
G1492
Morphology: other
Dev and morph
GARP
Multiple developmental
320
(34-83)






alterations

321
G1531
Morphology: other;
Dev and morph;
RING/C3HC4
Multiple developmental
322
(41-77)



seed; seed protein
seed biochemistry

alterations; pale seed;



content


altered seed protein






content

323
G1540
Morphology: other
Dev and morph
HB
Reduced cell
324
(35-98)






differentiation






in meristem

325
G1544
Morphology: other
Dev and morph
HB
Multiple developmental
326
(64-124)






alterations

327
G156
Morphology: other;
Dev and morph
MADS
Multiple developmental
328
(2-57)



seed


defects; seed color






alteration

329
G1584
Morphology: other
Dev and morph
HB
Multiple developmental
330
(TBD)






alteration

331
G1587
Morphology: other
Dev and morph
HB
Multiple developmental
332
(61-121)






alteration

333
G1588
Morphology: other
Dev and morph
HB
Multiple developmental
334
(66-124)






alteration

335
G1589
Morphology: other
Dev and morph
HB
Multiple developmental
336
(384-448)



seed protein content
seed biochemistry

alterations; altered






seed protein content

337
G160
Morphology: other
Dev and morph
MADS
Multiple developmental
338
(7-62)






defects

339
G1636
Morphology: other
Dev and morph
MYB-
Pale green, smaller
340
(100-165)





related
plants

341
G1642
Morphology: other
Dev and morph
MYB-
Multiple developmental
342
(TBD)





(R1)R2R3
alterations

343
G1747
Morphology: other;
Dev and morph;
MYB-(R1)
Multiple developmental
344
(11-114)



seed protein contcent
seed biochemistry
R2R3
alterations; altered






seed protein content

345
G1749
Morphology: other
Dev and morph
AP2
Multiple developmental
346
(84-155)






alterations; formation






of necrotic lesions

347
G1751
Morphology: other
Dev and morph
AP2
Multiple developmental
348
(TBD)






alterations

349
G1752
Morphology: other
Dev and morph
AP2
Multiple developmental
350
(83-151)






alterations

351
G1763
Morphology: other
Dev and morph
AP2
Lethal when overexpressed
352
(140-209)

353
G1766
Morphology: other
Dev and morph
NAC
Multiple developmental
354
(10-153)






alterations

355
G1767
Morphology: other
Dev and morph;
SCR
Multiple developmental
356
(255-272)



seed oil content
seed biochemistry

alterations; altered






seed oil content

357
G1778
Morphology: other
Dev and morph
GATA/Zn
Lethal when overexpressed
358
(94-119)

359
G1789
Morphology: other;
Dev and morph;
MYB-related
Delayed development;
360
(1-50)



seed protein content
seed biochemistry

altered seed protein






content

361
G1790
Morphology: other
Dev and morph
MYB(R1)R2R3
Lethal when overexpressed
362
(217-316)

363
G1791
Morphology: other
Dev and morph
AP2
Multiple developmental
364
(TBD)






alterations

365
G1793
Morphology: other;
Dev and morph;
AP2
Multiple developmental
366
(179-255,



seed oil
seed biochemistry

alterations; increased

281-349)






seed oil content

367
G1795
Morphology: other;
Dev and morph
AP2
Multiple developmental
368
(12-80)



triichome


alterations; reduced






trichomes

369
G1800
Morphology: other
Dev and morph
AP2
Multiple developmental
370
(TBD)






alterations

371
G1806
Morphology: other
Dev and morph
bZIP
Multiple developmental
372
(165-225)






alterations

373
G1811
Morphology: other
Dev and morph
ABI3/VP-1
Multiple developmental
374
(TBD)






alterations

375
G182
Morphology: other
Dev and morph
WRKY
Multiple developmental
376
(217-276)






alterations

377
G1835
Morphology: other
Dev and morph
GATA/Zn
Small, spindly plant
378
(224-296)

379
G1836
Morphology: other
Dev and morph
CAAT
Pale green
380
(30-164)

381
G1838
Morphology: other;
Dev and morph;
AP2
Multiple developmental
382
(229-305,



seed oil content
seed biochemistry

alterations; increaed

330-400)






seed oil content

383
G1843
Morphology: other
Dev and morph
MADS
Multiple developmental
384
(2-57)






alterations

385
G1853
Morphology: other
Dev and morph
AKR
Lethal when overexpressed
386
(entire








protein)

387
G1855
Morphology: other
Dev and morph
AKR
Slow growth
388
(entire








protein)

389
G187
Morphology: other
Dev and morph
WRKY
Variety of
390
(172-228)






morphological






alterations

391
G1881
Morphology: other
Dev and morph
Z-CO-like
Multiple developmental
392
(5-28,






alterations

56-79)

393
G1882
Morphology: other
Dev and morph
Z-Dof
Lethal when overexpressed
394
(97-125)

395
G1883
Morphology: other
Dev and morph
Z-Dof
Multiple developmental
396
(82-124)






alterations

397
G1884
Morphology: other
Dev and morph
Z-Dof
Multiple developmental
398
(43-71)






alterations

399
G1891
Morphology: other
Dev and morph
Z-Dof
Multiple developmental
400
(27-69)






alterations

401
G1896
Morphology: other
Dev and morph
Z-Dof
Multiple developmental
402
(43-85)






alterations

403
G1898
Morphology: other
Dev and morph
Z-Dof
Lethal when overexpressed
404
(31-59)

405
G1902
Morphology: other;
Dev and morph;
Z-Dof
Multiple developmental
406
(31-59)



seed oil content
seed biochemistry

alterations; increased






seed oil content

407
G1904
Morphology: other
Dev and morph
Z-Dof
Multiple developmental
408
(53-95)






alterations

409
G1906
Morphology: other
Dev and morph
Z-Dof
Multiple developmental
410
(19-47)






alterations

411
G1913
Morphology: other
Dev and morph
Z-Dof
Lethal when overexpressed
412
(27-55)

413
G1914
Morphology: other
Dev and morph
Z-C2H2
Multiple developmental
414
(195-216,






alterations

245-266)

415
G1925
Morphology: other
Dev and morph
NAC
Multiple developmental
416
(6-150)






alterations

417
G1929
Morphology: other
Dev and morph
Z-CO-like
Slow growth, delayed
418
(31-53)






development

419
G1930
Morphology: other
Dev and morph
AP2
Multiple developmental
420
(59-124)






alterations

421
G195
Morphology: other
Dev and morph
WRKY
Multiple developmental
422
(183-239)






defects

423
G1954
Morphology: other
Dev and morph
HLH/MYC
Lethal when overexpressed
424
(187-259)

425
G1958
Morphology: other;
Dev and morph;
GARP
Reduced size and root
426
(230-278)



seed protein content
seed biochemistry

mass in plates; altered






seed protein content

427
G196
Morphology: other;
Dev and morph;
WRKY
Multiple developmental
428
(223-283)



seed protein content
seed biochemistry

alterations; altered






seed protein content

429
G1965
Morphology: other
Dev and morph
Z-Dof
Lethal when overexpressed
430
(27-55)

431
G1976
Morphology: other
Dev and morph
Z-C2H2
Multiple developmental
432
(219-323)






alterations

433
G2057
Morphology: other
Dev and morph
TEO
Multiple developmental
434
(TBD)






alterations

435
G2107
Morphology: other
Dev and morph
AP2
Multiple developmental
436
(TBD)






alterations

437
G211
Morphology: other
Dev and morph
MYB-(R1)
Multiple developmental
438
(24-137)





R2R3
alterations

439
G2133
Morphology: other;
Dev and morph;
AP2
Multiple developmental
440
(11-83)



flowering time;
flowering time

alterations; late



seed protein


flowering; altered seed



content


protein content

441
G2134
Morphology: other
Dev and morph
AP2
Multiple developmental
442
(TBD)






alterations

443
G2151
Morphology: other;
Dev and morph;
AT-hook
Multiple developmental
444
(93-113,



seed oil and
seed biochemistry

alterations; altered

124-144)



protein content


seed oil and protein






content

445
G2154
Morphology: other
Dev and morph
AT-hook
Multiple developmental
446
(97-119)






alterations

447
G2157
Morphology: other
Dev and morph
AT-hook
Multiple developmental
448
(82-120,






alteration

164-107)

449
G2181
Morphology: other
Dev and morph
NAC
Multiple developmental
450
(22-169)






alterations

451
G221
Morphology: other
Dev and morph
MYB-(R1)
Multiple developmental
452
(21-125)





R2R3
alteration

453
G2290
Morphology: other
Dev and morph
WRKY
Multiple developmental
454
(147-205)






alterations

455
G2299
Morphology: other
Dev and morph
AP2
Multiple developmental
456
(48-115)






alterations

457
G2340
Morphology: other;
Dev and morph;
MYB-(R1)
Tissue necrosis; multiple
458
(14-120)



seed oil and
seed biochemistry
R2R3
developmental alterations;



protein content


altered seed oil






and protein content

459
G2346
Morphology: other
Dev and morph
SBP
Enlarged seedlings
460
(59-135)

461
G237
Morphology; other
Dev and morph
MYB-(R1)
Multiple developmental
462
(11-113)





R2R3
alterations

463
G2373
Morphology: other;
Dev and morph;
TH
Multiple developmental
464
(290-350)



seed protein content
seed biochemistry

alterations; altered






seed protein content

465
G2276
Morphology: other;
Dev and morph;
TH
Seeding lethality;
466
(79-178,



seed oil
seed biochemistry

altered seed protein

336-408)



protein


content

467
G24
Morphology: other
Dev and morph
AP2
Reduced size and
468
(25-93)






necrotic patches

469
G2424
Morphology: other
Dev and morph
MYB-(R1)
Multiple developmental
470
(107-219)





R2R3
alterations

471
G2505
Morphology: other
Dev and morph
NAC
Lethal when overexpressed
472
(10-159)

473
G2512
Morphology: other
Dev and morph
AP2
Multiple developmental
474
(79-139)






alterations

475
G2513
Morphology: other
Dev and morph
AP2
Multiple developmental
476
(TBD)






alterations

477
G2519
Morphology: other
Dev and morph
HLH/MYC
Multiple developmental
478
(1-65)






alterations

479
G2520
Morphology: other;
Dev and morph;
HLH/MYC
Multiple developmental
480
(135-206)



seed oil and
seed biochemistry

alterations; altered



protein content


seed oil and






protein content

481
G2533
Morphology: other;
Dev and morph;
NAC
Multiple developmental
482
(11-186)



seed protein content
seed biochemistry

alterations; altered






seed protein content

483
G2534
Morphology: other
Dev and morph
NAC
Lethal when overexpressed
484
(10-157)

485
G2573
Morphology: other;
Dev and morph;
AP2
Multiple developmental
486
(31-98)



seed oil and
seed biochemistry

alterations; altered



protein content


seed oil and






protein content

487
G2589
Morphology: other
Dev and morph
MADS
Multiple developmental
488
(2-57)






alterations

489
G2687
Morphology: other
Dev and morph
AP2
Multiple developmental
490
(51-120)






alterations

491
G27
Morphology: other
Dev and morph
AP2
Abnormal development,
492
(37-104)






small

493
G2720
Morphology: other;
Dev and morph;
MYB-(R1)
Multiple development
494
(10-114)



seed oil and
seed biochemistry
R2R3
alteration; altered



protein content


seed oil and






protein content

495
G2787
Morphology: other;
Dev and morph;
AT-hook
Multiple developmental
496
(172-192,



seed oil content
seed biochemistry

alterations; altered

226-247,






seed oil content

256-276,








290-311,








245-366)

497
G2789
Morphology: other
Dev and morph
AT-hook
Multiple developmental
498
(53-73,






alterations

121-165)

449
G31
Morphology: other
Dev and morph
AP2
Multiple developmental
500
(TBD)






alterations

501
G33
Morphology: other
Dev and morph
AP2
Multiple developmental
502
(50-117)






defects

503
G342
Morphology: other;
Dev and morph;
GATA/Zn
Multiple developmental
504
(155-190)



seed oil and
seed biochemistry

alterations; altered



protein content


seed oil and






protein content

505
G352
Morphology: other
Dev and morph
Z-C2H2
Multiple developmental
506
(99-119,






alterations

166-186)

507
G357
Morphology: other
Dev and morph
Z-C2H2
Developmental defect
508
(7-29)

509
G358
Morphology: other
Dev and morph
Z-C2H2
Lethal when overexpressed
510
(124-135,








188-210)

511
G360
Morphology: other
Dev and morph
Z-C2H2
Multiple development
512
(42-62)






alterations

513
G362
Size; Morphology:
Dev and morph;
Z-C2H2
Reduced size; increased
514
(62-82)



other; trichome;
flowering time;

pigmentation in seed



flowering time;
seed biochemistry

embryos and other



seed protein


organs; ectopic



content


trichome formation;






increased trichome






number; late






flowering; altered






protein content

515
G364
Morphology: other
Dev and morph
Z-C2H2
Developmental defect
516
(54-76)

517
G365
Morphology: other
Dev and morph
Z-C2H2
Multiple developmental
518
(70-90)






alterations

519
G367
Morphology: other
Dev and morph
Z-C2H2
Lethal when overexpressed
520
(63-84)

521
G373
Morphology: other
Dev and morph
RING/
Multiple developmental
522
(129-168)





C3HC4
alterations

523
G396
Morphology: other;
Dev and morph
HB
Altered leaf coloration
524
(159-220)



size


and shape reduced






fertility; small plant

525
G431
Morphology: other
Dev and morph
HB
Developmental defect,
526
(286-335)






sterile

527
G479
Morphology: other
Dev and morph
SBP
Multiple developmental
528
(70-149)






alterations

529
G546
Morphology: other
Dev and morph
RING/C3H2C3
Slow growth and
530
(114-155)






development; increased






anthocyanin






pigmentation

531
G551
Morphology: other
Dev and morph
HB
Multiple developmental
532
(73-133)






alterations

533
G578
Morphology: other
Dev and morph
bZIP
Lethal when overexpressed
534
(36-96)

535
G596
Morphology: other
Dev and morph
AT-hook
Multiple developmental
536
(89-96)






alterations

537
G617
Morphology: other
Dev and morph
TEO
Multiple developmental
538
(64-118)






alterations

539
G620
Morphology: other;
Dev and morph;
CAAT
Multiple developmental
540
(20-118)



seed protein content
seed biochemistry

alterations; altered






seed protein content

541
G625
Morphology: other
Dev and morph
AP2
Lethal when
542
(52-119)






overexpressed

543
G658
Morphology: other
Dev and morph
MYB-(R1)
Developmental defect
544
(2-105)





R2R3

545
G716
Morphology: other
Dev and morph
ARF
Multiple developmental defects
546
(24-355)

547
G725
Morphology: other
Dev and morph
GARP
Developmental defect
548
(39-87)

549
G727
Morphology: other
Dev and morph
GARP
Multiple morphological
550
(226-269)






alterations

551
G740
Morphology: other
Dev and morph
Z-CLDSH
Slow growth
552
(24-42,








232-268)

553
G770
Morphology: other
Dev and morph
NAC
Multiple developmental
554
(19-162)






alterations

555
G858
Morphology: other
Dev and morph
MADS
Multiple developmental
556
(2-57)






alterations

557
G865
Morphology: other;
Dev and morph;
AP2
Altered morphology
558
(36-103)



seed protein content
seed biochemistry

increased see protein

559
G872
Morphology: other
Dev and morph
AP2
Multiple developmental
560
(18-85)






alterations

561
G904
Morphology: other
Dev and morph
RING/C3H2C3
Multiple developmental
562
(117-158)






alterations

563
G910
Morphology: other;
Dev and morph;
Z-CO-like
Multiple developmental
564
(14-37,



flowering time
flowering time

alterations; late

77-103)






flowering

565
G912
Morphology: other;
Dev and morph;
AP2
Dark green color;
566
(51-118)



size; sugar
sugar sensing;

small plant; reduced



sensing;
flowering

cotyledon expansion



flowering
time

in glucose; late



time


flowering

567
G290
Morphology: other
Dev and morph
WRKY
Multiple developmental
568
(152-211)






alterations

569
G939
Morphology: other;
Dev and morph
EIL
Pale seedlings on agar;
570
(97-106)



size


reduced size

571
G963
Morphology: other;
Dev and morph;
NAC
Slowed growth rate;
572
(TBD)



seed protein
seed biochemistry

altered seed protein



content


content

573
G979
Morphology: other;
Dev and morph
AP2
Several development
574
(63-139,



seed


defects; altered seed

165-233)






development, ripening






and germination

575
G987
Morphology: other
Dev and morph
SCR
Developmental defects
576
(428-432,








704-708)

577
G993
Morphology: other
Dev and morph;
AP2
Multiple developmental
578
(69-134)



seed protein content
seed biochemistry

alterations; altered






seed protein content

579
G681
Morphology: other;
Dev and morph;
MYB-(R1)
Multiple developmental
580
(14-120)



leaf glucosinolates
leaf biochemistry
R2R3
alterations;






overexpression results






in an increased in M39480

581
G1482
Root
Dev and morph
Z-CO-like
Increased root growth
582
(5-63)

583
G225
Root; trichome
Dev and morph
MYB-related
Increased root hairs;
584
(39-76)






glabrous, lack






of trichomes

585
G226
Root; trichome;
Dev and morph;
MYB-related
Increased root hair;
586
(28-78)



seed protein content
seed biochemistry

glabrous, lack of






trichomes; increased






seed protein

587
G9
Root
Dev and morph
AP2
Increased root mass
588
(62-127)

589
G1040
Seed
Dev and morph
GARP
Smaller and more
590
(109-158)






rounded seeds

591
G2114
Seed
Dev and morph
AP2
Increased seed size
592
(221-297,








323-393)

593
G450
Seed; size;
Dev and morph;
IAA
Increased seed size;
594
(TBD)



seed protein
seed biochemistry

reduced plant size;



content


altered seed protein






content

595
G584
Seed
Dev and morph
HLH/MYC
Large seeds
596
(401-494)

597
G668
Seed
Dev and morph
MYB-(R1)
Reduced seed color
598
(13-113)





R2R3

599
G1050
Senescence
Dev and morph
bZIP
Delayed senescence
600
(372-425)

601
G1463
Senescence
Dev and morph
NAC
Premature senescence
602
(9-156)

603
G1944
Senescence; size;
Dev and morph;
AT-hook
Early senescence;
604
(87-100)



seed protein content
seed biochemistry

reduced size; altered






seed protein content

605
G2383
Senescence; seed
Dev and morph;
TEO
Early senescence;
606
(89-149)



protein content
seed biochemistry

altered seed protein






content

607
G571
Senescence;
Dev and morph;
bZIP
Delayed senescence;
608
(160-220)



flowering time
flowering time

late flowering

609
G636
Senescence; size
Dev and morph
TH
Premature senescence;
610
(55-145,






reduced size

405-498)

611
G878
Senescence;
Dev and morph;
WRKY
Delayed senescence;
612
(250-305,



flowering time
flowering time

late flowering

415-475)

613
G1134
Silique
Dev and morph
HLH/MYC
Siliques with altered
614
(198-247)






shape

615
G1008
Size
Dev and morph
AP2
Small plant
616
(96-163)

617
G1020
Size
Dev and morph
AP2
Very small T1 plants
618
(28-95)

619
G1023
Size
Dev and morph
AP2
Reduced size
620
(128-195)

621
G1053
Size
Dev and morph
bZIP
Small plant
622
(74-120)

623
G1137
Size
Dev and morph
HLH/MYC
Small T1 plants
624
(264-314)

625
G1181
Size
Dev and morph
HS
Small T1 plants
626
(24-114)

627
G1228
Size
Dev and morph
HLH/MYC
Reduced size
628
(179-233)

629
G1277
Size
Dev and morph
AP2
Small plant
630
(18-85)

631
G1309
Size
Dev and morph
MYB-(R1)
Small plant
632
(9-114)





R2R3

633
G1314
Size; sugar
Dev and morph;
MYB-(R1)
Reduced size; reduced
634
(14-116)



sensing; seed
sugar sensing;
R2R3
seedling vigor on high



protein content
seed biochemistry

glucose; altered seed






protein content

635
G1317
Size
Dev and morph
MYB-(R1)
Reduced size
636
(13-118)





R2R3

637
G1323
Size; seed oil
Dev and morph;
MYB-(R1)
Small T1 plants, dark
638
(15-116)



and protein content
seed biochemistry
R2R3
green; decreased seed






oil, increased seed






protein

639
G1332
Size; trichome; seed
Dev and morph;
MYB-(R1)
Reduced size; reduced
640
(13-116)



oil and protein
seed biochemistry
R2R3
trichome densitry;



content


altered seed oil






and protein content

641
G1334
Size
Dev and morph
CAAT
Small, dark green
642
(18-190)

643
G1381
Size
Dev and morph
AP2
Reduced size
644
(68-135)

645
G1382
Size
Dev and morph
WRKY
Small plant
646
(210-266.








385-437)

647
G1435
Size; flowering
Dev and morph;
GARP
Increased plant size;
648
(146-194)



time
flowering time

late flowering

649
G1537
Size
Dev and morph
HB
Small T1 plants with
650
(14-74)






altered development

651
G1545
Size
Dev and morph
HB
Reduced size
652
(54-117)

653
G1641
Size; seed oil
Dev and morph;
MYB- related
Small plant; altered
654
(139-200)



and protein content
seed biochemistry

seed oil and protein






content

655
G165
Size; seed
Dev and morph;
MADS
Reduced size; altered
656
(7-62)



content
seed biochemistry

seed protein content

657
G1652
Size; seed oil
Dev and morph;
HLH/MYC
Reduced size; altered
658
(143-215)



and protein content
seed biochemistry

seed oil and protein content

659
G1655
Size
Dev and morph
HLH/MYC
Small plant
660
(134-192)

661
G1671
Size
Dev and morph
NAC
Reduced size
662
(TBD)

663
G1756
Size; seed
Dev and morph
WRKY
Reduced size; altered
664
(TBD)



protein content
seed biochemistry

seed protein content

665
G1757
Size; seed
Dev and morph;
WRKY
Small plant; altered
666
(158-218)



protein content
seed biochemistry

seed protein content

667
G1782
Size
Dev and morph
CAAT
Small, spindly plant
668
(166-238)

669
G184
Size
Dev and morph
WRKY
Small plant
670
(295-352)

671
G1845
Size
Dev and morph
AP2
Small plant
672
(140-207)

673
G1879
Size; seed oil
Dev and morph;
HLH/MYC
Reduced size; altered
674
(107-176)



and protein content
seed biochemistry

seed oil and protein






content

675
G1888
Size
Dev and morph
Z-CO-like
Reduced size, dark
676
(5-50)






green leaves

677
G189
Size; seed protein
Dev and morph;
WRKY
Increased leaf size;
678
(240-297)



content
seed biochemistry

altered seed protein






content

679
G1939
Size
Dev and morph
PCF
Reduced size
680
(40-102)

681
G194
Size
Dev and morph
WRKY
Small plant
682
(174-230)

683
G1973
Size
Dev and morph
HLH/MYC
Reduced size
684
(335-406)

685
G21
Size; seed oil
Dev and morph;
AP2
Reduced size; altered
686
(97-164)



and protein content
seed biochemistry

seed oil and protein






content

687
G2132
Size; seed oil
Dev and morph;
AP2
Reduced size; altered
688
(TBD)



and protein content
seed biochemistry

seed oil and protein






content

689
G2145
Size
Dev and morph
HLJH/MYC
Reduced size
690
(166-243)

691
G23
Size
Dev and morph
AP2
Small T1 plants
692
(61-117)

693
G2313
Size
Dev and morph
MYB-related
Reduced size
694
(TBD)

695
G2344
Size
Dev and morph
CAAT
Reduced size, slow growth
696
(TBD)

697
G2430
Size
Dev and morph
GARP
Increased leaf size,
698
(425-478)






faster development

699
G2517
Size
Dev and morph
WRKY
Reduced size
700
(118-234)

701
G2521
Size
Dev and morph
HLH/MYC
Reduced size
702
(145-213)

703
G258
Size; seed oil
Dev and morph;
MYB-(R1)
Reduced size; altered
704
(24-124)



and protein content
seed biochemistry
R2R3
seed oil and protein






content

705
G280
Size; seed
Dev and morph;
AT-hook
Reduced size; altered
706
(97-104,



protein content
seed biochemistry

seed protein content

130-137-








155-162,








185-192)

707
G3
Size
Dev and morph
AP2
Small plant
708
(28-95)

709
G343
Size
Dev and morph
GATA/Zn
Small plant
710
(178-214)

711
G363
Size
Dev and morph
Z-C2H2
Small plant
712
(87-108)

713
G370
Size
Dev and morph
Z-C2H2
Reduced size, shiny
714
(97-117)






leaves

715
G385
Size
Dev and morph
HB
Small plant, short
716
(60-123)






inflorescence stems,






dark green

717
G439
Size
Dev and morph
AP2
Small plant
718
(110-177)

719
G440
Size
Dev and morph
AP2
Small plant
720
(122-189)

721
G5
Size
Dev and morph
AP2
Small plant
722
(149-216)

723
G550
Size
Dev and morph
Z-Dof
Small plant
724
(134-180)

725
G670
Size
Dev and morph
MYB-(R1)
Small plant
726
(14-122)





R2R3

727
G760
Size
Dev and morph
NAC
Reduced size
728
(12-156)

729
G831
Size
Dev and morph
AKR
Reduced size
730
(470-591)

731
G864
Size
Dev and morph
AP2
Small plant
732
(119-186)

733
G884
Size
Dev and morph
WRKY
Reduced size
734
(227-285,








407-465)

735
G898
Size; seed oil
Dev and morph;
RING/C3HC4
Reduced size; altered
736
(148-185)



and protein content
seed biochemistry

seed oil and protein



content

737
G900
Size
Dev and morph
Z-CO-like
Reduced size
738
(6-28,








48-74)

739
G913
Size; flowering
Dev and morph;
AP2
Small plant; late
740
(62-128)



time
flowering time

flowering

741
G937
Size
Dev and morph
GARP
Slightly reduced size
742
(197-246)

743
G960
Size
Dev and morph
NAC
Small plant
744
(13-156)

745
G991
Size; seed oil
Dev and morph;
IAA
Slightly reduced size;
746
(7-14,48-



and protein content
seed biochemistry

altered seed oil and

59,82-115,






protein content

128-164)

747
G748
Stem; flowering
Dev and morph;
Z-Dof
More vascular bundles
748
(112-140)



time
flowering time

in stem; late flowering

749
G247
Trichome; seed
Dev and morph;
MYB-(R1)
Altered trichome
750
(15-116)



seed protein
seed biochemistry
R2R3
distribution; altered



content


seed protein content

751
G585
Trichome
Dev and morph
HLH/MYC
Reduced trichome denisty
752
(436-501)

753
G634
Trichome; seed
Dev and morph;
TH
Increased trichome
754
(62-147,



protein content
seed biochemistry

density and size;

189-245)






altered seed protein






content

755
G676
Trichome
Dev and morph
MYB-(R1)
reduced trichomes
756
(17-119)





R2R3

757
G682
Trichome
Dev and morph
MYB-related
Glabrous, lack of
758
(27-63)






trichomes

759
G635
Variegation
Dev and morph
TH

760
(239-323)

761
G1068
Sugar sensing
Sugar sensing
AT-hook
Reduced cotyledon
762
(143-150)






expansion in glucose

763
G1225
Sugar sensing; seed
Sugar sensing;
HLH/MYC
Better germination on
764
(78-147)



oil and protein
biochemistry

sucrose and glucose



content


media; altered seed oil






and protein content

765
G1337
Sugar sensing
Sugar sensing
Z-CO-like
Decreased germination
766
(9-75)






on sucrose medium

767
G1759
Sugar sensing
Sugar sensing
MADS
Reduced germination
768
(2-57)






on high glucose

769
G1804
Sugar sensing;
Sugar sensing;
bZIP
Altered sugar sensing;
770
(357-407)



flowering time
flowering time

late flowering

771
G207
Sugar sensing
Sugar sensing
MYB-(R1)
Decreased germination
772
(6-106)





R2R3
on glucose medium

773
G218
Sugar sensing;
Sugar sensing;
MYB-(R1)
Reduced cotyledon
774
(TBD)



seed oil content
seed biochemistry
R2R3
expansion in glucose;






altered seed oil content

775
G241
Sugar sensing; seed
Sugar sensing;
MYB-(R1)
Decreased germination
776
(14-114)



oil and protein
seed biochemistry
R2R3
and growth on glucose



content


medium; decreased






seed oil, altered






protein content

777
G254
Sugar sensing
Sugar sensing
MYB-related
Decreased germination
778
(62-106)






and growth on glucose medium

779
G26
Sugar sensing
Sugar sensing
AP2
Decreased germination
780
(67-134)






and growth on glucose






medium

781
G263
Sugar sensing
Sugar sensing
HS
Decreased root growth
728
(TBD)






on sucrose medium,






root specific






expression

783
G308
Sugar sensing
Sugar sensing
SCR
No germination on
784
(270-247)






glucose medium

785
G38
Sugar sensing
Sugar sensing
AP2
Reduced germination on
786
(76-143)






glucose medium

787
G43
Sugar sensing
Sugar sensing
AP2
Decreased germination
788
(104-172)






and growth on glucose






medium

789
G536
Sugar sensing
Sugar sensing
GF14
Decreased germination
790
(226-233)






and growth on glucose medium

791
G567
Sugar sensing; seed
Sugar sensing; seed
b-ZIP
Decreased seedling
792
(210-270)



oil and
biochemistry

vigor on high glucose;



protein content


altered seed oil and






protein content

793
G680
Sugar sensing;
Sugar sensing;
MYB-related
Reduced germination
794
(24-70)



flowering time
flowering time

on glucose medium;






late flowering

795
G867
Sugar sensing
Sugar sensing
AP2
Better seedling vigor
796
(59-124)






on sucrose medium

797
G956
Sugar sensing
Sugar sensing
NAC
Reduced germination
798
(TBD)






on glucose medium

799
G996
Sugar sensing
Sugar sensing
MYB-(R1)
Reduced germination
800
(14-114)





R2R3
on glucose medium

801
G1946
Seed glucosinolates
Seed biochemistry
HS
Increased in M3950;
802
(32-130)



oil protein


increased oil content;



content


decreased protein content

803
G217
Seed oil composition
Seed biochemistry
MYB-related
Increased in 20:2
804
(8-67)

805
G2192
Seed oil composition
Seed biochemistry
bZIP-NIN
Altered composition
806
(600-700)

807
G504
Seed oil
Seed biochemistry
NAC
Altered seed oil
808
(TBD)



composition; seed


composition and content;



protein content


altered seed protein






content

809
G622
Seed oil composition
Seed biochemistry
ABI3/VP-1
Decreased 18:2 fatty acid
810
(TBD)

811
G778
Seed oil composition
Seed biochemistry
HLH/MYC
Increased seed 18:1 fatty acid
812
(220-267)

813
G791
Seed oil composition
Seed biochemistry
HLH/MYC
Altered seed fatty acid
814
(75-143)






composition

815
G861
Seed oil composition;
Seed biochemistry
MADS
Increase in 16:1;
816
(2-57)



seed oil content


altered seed oil content

817
G938
Seed oil composition
Seed biochemistry
EIL
Altered seed fatty acid
818
(96-104)






composition

819
G965
Seed oil composition
Seed biochemistry
HB
Increased in 18:1
820
(423-486)

821
G1143
Seed oil and
Seed biochemistry
HLH/MYC
Altered seed oil and
822
(33-82)



protein content


protein content

823
G1190
Seed oil content
Seed biochemistry
AKR
Increased content
824
(entire protein)

825
G1198
Seed oil and
Seed biochemistry
bZIP
Altered seed oil and
826
(173-223)



protein content


protein content

827
G1226
Seed oil and protein
Seed biochemistry
HLH/MYC
Altered seed oil and
828
(115-174)



content


protein content

829
G1451
Seed oil content
Seed biochemistry
ARF
Altered seed oil content
830
(22-357)

831
G1478
Seed oil and
Seed biochemistry;
Z-CO-like
Altered seed oil,
832
(32-76)



protein content;
flowering time

protein content; late



flowering time


flowering

833
G1496
Seed oil content
Seed biochemistry
HLH/MYC
Altered seed oil
834
(184-248)






content

835
G1526
Seed oil content
Seed biochemistry
SWI/SNF
Increased seed oil
836
(493-620,






content

864-1006)

837
G1543
Seed oil content
Seed biochemistry
HB
Decreased seed oil
838
(135-195)

839
G162
Seed oil and
Seed biochemistry
MADS
Altered seed oil
840
(2-57)



protein content


content; altered seed






oil and protein content

841
G1640
Seed oil content
Seed biochemistry
MYB-(R1)
Increased seed oil
842
(14-115)





R2R3

843
G1644
Seed oil and
Seed biochemistry
MYB-(R1)
Altered seed oil,
844
(39-102)



protein content

R2R3
protein content

845
G1646
Seed oil content
Seed biochemistry
CAAT
Altered seed oil content
846
(72-162)

847
G1672
Seed oil content
Seed biochemistry
NAC
Altered seed oil content
848
(41-194)

849
G1677
Seed oil and
Seed biochemistry
NAC
Altered seed oil,
850
(17-181)



protein content


protein content

851
G1765
Seed oil and
Seed biochemistry
NAC
Altered seed oil and
852
(20-140)



protein content


protein content

853
G1777
Seed oil and
Seed biochemistry
RING/C3HC4
Increased oil,
854
(124-247)



protein content


decreased protein






content

855
G1793
Seed oil content
Seed biochemistry
AP2
Increased seed oil
856
(179-255,






content

281-349)

857
G180
Seed oil content
Seed biochemistry
WRKY
Decreased seed oil
858
(118-174)






content

859
G192
Seed oil and
Seed biochemistry;
WRKY
Altered seed oil and
860
(128-185)



protein content;
flowering time

protein content; late



flowering time


flowering

861
G1948
Seed oil and
Seed biochemistry
AKR
Altered seed oil and
862
(entire protein)



protein content


protein content

863
G2123
Seed oil and
Seed biochemistry
GF14
Altered seed oil and
864
(99-109)



protein content


protein content

865
G2138
Seed oil content
Seed biochemistry
AP2
Increased seed oil
866
(TBD)






content

867
G2139
Seed oil content
Seed biochemistry
MADS
Increased seed content
868
(14-69)

869
G2343
Seed oil content
Seed biochemistry
MYB-(R1)
Altered seed oil content
870
(14-116)





R2R3

871
G265
Seed oil and
Seed biochemistry
HS
Altered seed oil
872
(11-105)



protein content


and protein content

873
G2792
Seed oil content
Seed biochemistry
HLH/MYC
Increased seed oil content
874
(190-258)

875
G2830
Seed oil and
Seed biochemistry
Z-C2H2
Altered seed oil and
876
(245-266)



protein content


protein content

877
G286
Seed oil and
Seed biochemistry
ENBP
Altered seed oil and
878
(TBD)



protein content


protein content

879
G291
Seed oil content
Seed biochemistry
MISC
Increased seed oil content
880
(132-160)

881
G427
Seed oil and
Seed biochemistry
HB
Increased oil content;
882
(307-370)



protein content


decreased protein content

883
G509
Seed oil and protein
Seed biochemistry
NAC
Altered seed oil and
884
(13-169)



content


protein content

885
G519
Seed oil and protein
Seed biochemistry
NAC
Altered seed oil and
886
(11-104)



content


protein content

887
G561
Seed oil content
Seed biochemistry
bZIP
Altered seed oil content
888
(248-308)

889
G590
Seed oil and
Seed biochemistry
HLH/MYC
Altered seed oil and
890
(202-254)



protein content


protein content

891
G818
Seed oil content
Seed biochemistry
HS
Increased content
892
(70-162)

893
G849
Seed oil and
Seed biochemistry
BPF-1
Increased seed oil,
894
(324-413.



protein content


altered protein content

504-583)

895
G892
Seed oil and
Seed biochemistry
RING/C3H2C3
Altered seed oil,
896
(177-270)



protein content


protein content

897
G961
Seed oil content
Seed biochemistry
NAC
Altered seed oil,
896
(15-140)






content

899
G1465
Seed oil and protein
Seed biochemistry
NAC
Altered seed oil and
900
(242-306)



content


protein content

901
G425
Seed oil content
Seed biochemistry
HB
Altered seed oil
902
(TBD)






content

903
G347
Seed oil and
Seed biochemistry
Z-LSDlike
Altered seed oil
904
(9-39,50-



protein content


protein content

70,80-127)

905
G1512
Seed oil and
Seed biochemistry
RING/C3HC4
Altered seed oil and
906
(39-93)



protein content


protein content

907
G2069
Seed oil and
Seed biochemistry
bZIP
Altered seed oil and
908
(TBD)



protein content


protein content

909
G1852
Seed oil content
Seed biochemistry
AKR
Altered seed oil content
910
(1-601)

911
G1793
Seed oil content
Seed biochemistry
AP2
Altered seed oil content
912
(179-255,








281-349)

913
G761
Seed oil and
Seed biochemistry
NAC
Altered seed oil and
914
(10-156)



protein content


protein content

915
G1056
Seed oil content
Seed biochemistry
bZIP
Altered seed oil content
916
(183-246)

917
G1447
Seed oil content
Seed biochemistry
MISC
Altered seed oil content
918
(3-54,








124-156)

919
G323
Seed oil and
Seed biochemistry
RING/C3HC4
Altered seed oil and
920
(48-96)



protein content


protein content

921
G176
Seed oil content
Seed biochemistry
WRKY
Altered seed oil content
922
(117-173,








234-290)

923
G174
Seed oil and protein
Seed biochemistry
WRKY
Altered seed oil and
924
(111-166,



content


protein content

283-339)

925
G715
Seed oil content
Seed biochemistry
CAAT
Altered seed oil content
926
(60-132)

927
G588
Seed oil and protein
Seed biochemistry
HLH/MYC
Altered seed oil and
928
(309-376)



content


protein content

929
G1758
Seed oil and
Seed biochemistry
WRKY
Altered seed oil and
930
(109-165)



protein content


protein content

931
G2148
Seed oil content
Seed biochemistry
HLH/MYC
Altered seed oil
932
(130-268)






content

933
G2379
Seed oil content
Seed biochemistry
TH
Altered seed oil
934
(19-110,






content

173-232)

935
G1462
Seed oil content
Seed biochemistry
NAC
Altered seed oil content
936
(TBD)

937
G1211
Seed oil and
Seed biochemistry
MISC
Altered seed oil and
938
(123-179)



protein content


protein content

939
G1048
Seed oil content
Seed biochemistry
bZIP
Altered seed oil content
940
(138-190)

941
G986
Seed oil content
Seed biochemistry
WRKY
Altered seed oil content
942
(146-203)

943
G789
Seed oil content
Seed biochemistry
HLH/MYC
Altered seed oil content
944
(253-313)

945
G2085
Seed oil and
Seed biochemistry
RING/C3HC4
Altered seed oil and
946
(TBD)



protein content


protein content

947
G1783
Seed oil and protein
Seed biochemistry
MYB-related
Altered seed oil and
948
(81 . . . 129)



content


protein content

949
G2072
Seed oil and
Seed biochemistry
bZIP
Altered seed oil and
950
(90-149)



protein content


protein content

951
G931
Seed oil and protein
Seed biochemistry
CAAT
Altered seed oil and
952
(TBD)



content


protein content

953
G278
Seed oil and
Seed biochemistry
AKR
Altered seed oil and
954
(2-593)




protein content

protein content

955
G2421
Seed oil content
Seed biochemistry
MYB-(R1)
Altered seed oil content
956
(9-110)





R2R3

957
G2032
Seed oil content
Seed biochemistry
AKR
Altered seed oil content
958
(entire protein)

959
G1396
Seed oil and protein
Seed biochemistry
S1FA
Altered seed oil and
960
(TBD)



content


protein content

961
G619
Seed oil and
Seed biochemistry
ARF
Altered seed oil and
962
(64-406)



protein content


protein content

963
G2295
Seed oil content
Seed biochemistry
MADS
Altered seed oil content
964
(2-57)

965
G312
Seed oil content
Seed biochemistry
SCR
Altered seed oil
966
(320-336)






content

967
G1444
Seed oil and
Seed biochemistry
GRF-like
Altered seed oil and
968
(168-193)



protein content


protein content

969
G801
Seed oil content
Seed biochemistry
PCF
Altered seed oil content
970
(32-93)

971
G1950
Seed oil content
Seed biochemistry
AKR
Altered seed oil content
972
(65-228)

973
G958
Seed oil and protein
Seed biochemistry
NAC
Altered seed oil and
974
(7-156)



content


protein content

975
G1037
Seed oil and protein
Seed biochemistry
GARP
Altered seed oil and
976
(11-134,



content


protein content

200-248)

977
G2065
Seed oil content
Seed biochemistry
MADS
Altered seed oil content
978
(TBD)

979
G2137
Seed oil and protein
Seed biochemistry
WRKY
Altered seed oil and
980
(109-168)



content


protein content

981
G746
Seed oil content
Seed biochemistry
RING/C3HC4
Altered seed oil content
982
(139-178)

983
G2701
Seed oil and
Seed biochemistry
MYB-related
Altered seed oil and
984
(33-81,



protein content


protein content

129-183)

985
G1819
Seed oil content
Seed biochemistry
CAAT
Altered seed oil content
986
(46-188)

987
G1227
Seed oil and protein
Seed biochemistry
HLH/MYC
Altered seed oil and
988
(183-244)



content


protein content

989
G2417
Seed oil content
Seed biochemistry
GARP
Altered seed oil content
990
(235-285)

991
G2116
Seed oil content
Seed biochemistry
bZIP
Altered seed oil content
992
(150-210)

993
G647
Seed oil content
Seed biochemistry
Z-C3H
Altered seed oil content
994
(77-192)

995
G974
Seed oil and protein
Seed biochemistry
AP2
Altered seed oil and
996
(81-140)



content


protein content

997
G1419
Seed protein content
Seed biochemistry
AP2
Increased seed protein
998
(69-137)

999
G1634
Seed protein content
Seed biochemistry
MYC-related
Altered seed protein
1000
(129-180)






content

1001
G1637
Seed protein content
Seed biochemistry
MYB-related
Altered seed protein
1002
(109-173)






content

1003
G1818
Seed protein content;
Seed biochemistry;
CAAT
Increased protein
1004
(36-113)



flowering time
flowering time

content; late flowering

1005
G1820
Seed oil and protein
Seed biochemistry
CAAT
Altered seed oil,
1006
(70-133)



content


protein content

1007
G1903
Seed oil and protein
Seed biochemistry
Z-Dof
Altered seed oil and
1008
(134-180)



content


protein content

1009
G371
Seed oil and protein
Seed biochemistry
RING/C3HC4
Altered seed oil and
1010
(21-74)



content


protein content

1011
G597
Seed protein content
Seed biochemistry
AT-hook
Altered seed protein content
1012
(97-104,








137-144)

1013
G1009
Seed protein content
Seed biochemistry
AP2
Altered seed protein content
1014
(201-277,








303-371)

1015
G170
Seed protein content
Seed biochemistry
MADS
Altered seed protein content
1016
(2-57)

1017
G1768
Seed protein content
Seed biochemistry
SCR
Altered seed protein content
1018
(54-413)

1019
G185
Seed protein content
Seed biochemistry
WRKY
Altered seed protein content
1020
(113-172)

1021
G1931
Seed protein content
Seed biochemistry
WRKY
Altered seed protein content
1022
(114-170)

1023
G2543
Seed protein content
Seed biochemistry
HB
Altered seed protein content
1024
(31-991)

1025
G264
Seed protein content
Seed biochemistry
HS
Altered seed protein content
1026
(24-114)

1027
G32
Seed protein content
Seed biochemistry
AP2
Altered seed protein content
1028
(17-84)

1029
G436
Seed protein content
Seed biochemistry
HB
Altered seed protein content
1030
(22-85)

1031
G556
Seed protein content
Seed biochemistry
bZIP
Altered seed protein content
1032
(83-143)

1033
G1420
Seed protein content
Seed biochemistry
WRKY
Altered seed protein content
1034
(221-280)

1035
G1412
Seed protein content
Seed biochemistry
NAC
Altered seed protein content
1036
(17-159)

1037
G738
Seed protein content
Seed biochemistry
Z-Dof
Altered seed protein content
1038
(351-393)

1039
G2426
Seed protein content
Seed biochemistry
MYB-(R1)
Altered seed protein content
1040
(14-114)





R2R3

1041
G1524
Seed protein content
Seed biochemistry
RING/C3HC3
Altered seed protein content
1042
(49-110)

1043
G1243
Seed protein content
Seed biochemistry
SWI/SNF
Altered seed protein content
1044
(216-609)

1045
G631
Seed protein content
Seed biochemistry
bZIP
Altered seed protein content
1046
(TBD)

1047
G1909
Seed protein content
Seed biochemistry
Z-Dof
Altered seed protein content
1048
(23-51)

1049
G1663
Seed protein content
Seed biochemistry
PCF
Altered seed protein content
1050
(TBD)

1051
G1231
Seed protein content
Seed biochemistry
Z-C4HC3
Altered seed protein content
1052
(TBD)

1053
G227
Seed protein content
Seed biochemistry
MYB-(R1)
Altered seed protein content
1054
(13-112)





R2R3

1055
G1842
Seed protein content
Seed biochemistry
MADS
Altered seed protein content
1056
(2-57)

1057
G1505
Seed protein content
Seed biochemistry
GATA/Zn
Altered seed protein content
1058
(TBD)

1059
G657
Seed protein content
Seed biochemistry
MYB-(R1)
Altered seed protein content
1060
(TBD)





R2R3

1061
G1959
Seed protein content
Seed biochemistry
GARP
Altered seed protein content
1062
(46-97)

1063
G2180
Seed protein content
Seed biochemistry
NAC
Altered seed protein content
1064
(7-156)

1065
G1817
Seed protein content
Seed biochemistry
PMR
Altered seed protein content
1066
(47-331)

1067
G1649
Seed protein content
Seed biochemistry
HLH/MYC
Altered seed protein content
1068
(225-295)

1069
G2131
Seed protein content
Seed biochemistry
AP2
Altered seed protein content
1070
(50-186,








112-183)

1071
G215
Seed protein content
Seed biochemistry
MYB-related
Altered seed protein content
1072
(TBD)

1073
G1508
Seed protein content
Seed biochemistry
GATA/Zn
Altered seed protein content
1074
(38-63)

1075
G2110
Seed protein content
Seed biochemistry
WRKY
Altered seed protein content
1076
(239-298)

1077
G2442
Seed protein content
Seed biochemistry
RING/C3HC4
Altered seed protein content
1078
(220-246)

1079
G1051
Flowering time
Flowering time
bZIP
Late flowering
1080
(189-250)

1081
G1052
Flowering time
Flowering time
bZIP
Late flowering
1082
(201-261)

1083
G1079
Flowering
Flowering
BZIPT2
Late flowering; altered
1084
(1-50)



time; seed
time; seed

seed protein content



protein
biochemistry



content

1085
G1335
Flowering
Flowering
Z-CLDSH
Late flowering, slow
1086
(24-43,



time
time

growth

131-144,








185-203)

1087
G157
Flowering
Flowering
MAPS
Altered flowering;
1088
(2-57)



time
time

significant






overexpression delays






flowering time

1089
G1895
Flowering
Flowering
Z-Dof
Late flowering
1090
(55-110)



time
time

1091
G1900
Flowering
Flowering
Z-Dof
Late flowering
1092
(54-106)



time
time

1093
G2007
Flowering
Flowering
MYB-(R1)
Late flowering; altered
1094
(TBD)



time; seed
time; seed
R2R3
seed protein content



protein
biochemistry



content

1095
G214
Flowering time
Flowering time
MYB-related
Late flowering
1096
(22-71)

1097
G2155
Flowering time
Flowering time
AT-hook
Late flowering
1098
(18-38)

1099
G234
Flowering
Flowering
MYB-
Late flowering, small
1100
(14-115)



time
time
(R1)R2R3
plant

1101
G361
Flowering
Flowering
Z-C2H2
Late flowering
1102
(43-63)



time
time

1103
G562
Flowering
Flowering
bZIP
Late flowering
1104
(253-315)



time
time

1105
G591
Flowering
Flowering
HLH/MYC
Late flowering
1106
(143-240)



time
time

1107
G8
Flowering
Flowering
AP2
Late flowering
1108
(151-217,



time
time



243-296)

1109
G859
Flowering
Flowering
MADS
Late flowering; altered
1110
(TBD)



time; seed
time; seed

seed protein content



protein
biochemistry



content

1111
G878
Flowering time
Flowering time
WRKY
Late flowering
1112
(250-305,








415-475)

1113
G971
Flowering
Flowering
AP2
Late flowering
1114
(120-186)



time
time

1115
G975
Flowering
Flowering
AP2
Late flowering;
1116
(4-71)



time;
time; dev and

glossy leaves



morphology:
morph



other

1117
G994
Flowering
Flowering
MYB-
Late flowering, small
1118
(14-123)



time
time
(R1)R2R3

1119
G2347
Flowering
Flowering
SBP
Late flowering, small
1120
(60-136)



time
time

1121
G2010
Flowering
Flowering
SBP
Late flowering
1122
(53-127)



time
time

























































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































TABLE 6 Genes, traits and utilities that affect plant characteristics Transcription factor genes that Utility Trait Category Traits impact traits Gene effect on: Resistance and Salt stress resistance G22; G196; G226; G303; G312; Germination rate, tolerance G325; G353; G482; G545; G801; survivability, yield; G867; G884; G922; G926; G1452; extended growth G1794; G1820; G1836; G1843; range G1863; G2053; G2110; G2140; G2153; G2379; G2701; G2713; G2719; G2789 Osmotic stress G47; G175; G188; G303; G325; Germination rate, resistance G353; G489; G502; G526; G921; survivability, yield G922; G926; G1069; G1089; G1452; G1794; G1930; G2140; G2153; G2379; G2701; G2719; G2789; Cold stress resistance; G256; G394; G664; G864; G1322; Germination, cold germination G2130 growth, earlier planting Tolerance to freezing G303; G325; G353; G720; G912; Survivability, yield, G913; G1794; G2053; G2140; appearance, G2153; G2379; G2701; G2719; extended range G2789 Heat stress resistance G3; G464; G682; G864; G964; Germination, G1305; G1645; G2130 G2430 growth, later planting Drought, low humidity G303; G325; G353; G720; G912; Survivability, yield, resistance G926; G1452; G1794; G1820; extended range G1843; G2053; G2140; G2153; G2379; G2583; G2701; G2719; G2789 Radiation resistance G1052 Survivability, vigor, appearance Decreased herbicide G343; G2133; G2517 Resistant to sensitivity increased herbicide use Increased herbicide G374; G877; G1519 Use as a herbicide sensitivity target Oxidative stress G477; G789; G1807; G2133; Improved yield, G2517 appearance, reduced senescence Light response G183; G354; G375; G1062; Germination, G1322; G1331; G1488; G1494; growth, G1521; G1786; G1794; G2144; development, G2555; flowering time Development, Overall plant G24; G27; G31; G33; G47; G147; Vascular tissues, morphology architecture G156; G160; G182; G187; G195; lignin content; cell G196; G211; G221; G237; G280; wall content; G342; G352; G357; G358; G360; appearance G362; G364; G365; G367; G373; G377; G396; G431; G447; G479; G546; G546; G551; G578; G580; G596; G615; G617; G620; G625; G638; G658; G716; G725; G727; G730; G740; G770; G858; G865; G869; G872; G904; G910; G912; G920; G939; G963; G977; G979; G987; G988; G993; G1007; G1010; G1014; G1035; G1046; G1049; G1062; G1069; G1070; G1076; G1089; G1093; G1127; G1131; G1145; G1229; G1246; G1304; G1318; G1320; G1330; G1331; G1352; G1354; G1360; G1364; G1379; G1384; G1399; G1415; G1417; G1442; G1453; G1454; G1459; G1460; G1471; G1475; G1477; G1487; G1487; G1492; G1499; G1499; G1531; G1540; G1543; G1543; G1544; G1548; G1584; G1587; G1588; G1589; G1636; G1642; G1747; G1749; G1749; G1751; G1752; G1763; G1766; G1767; G1778; G1789; G1790; G1791; G1793; G1794; G1795; G1800; G1806; G1811; G1835; G1836; G1838; G1839; G1843; G1853; G1855; G1865; G1881; G1882; G1883; G1884; G1891; G1896; G1898; G1902; G1904; G1906; G1913; G1914; G1925; G1929; G1930; G1954; G1958; G1965; G1976; G2057; G2107; G2133; G2134; G2151; G2154; G2157; G2181; G2290; G2299; G2340; G2340; G2346; G2373; G2376; G2424; G2465; G2505; G2509; G2512; G2513; G2519; G2520; G2533; G2534; G2573; G2589; G2687; G2720; G2787; G2789; G2893 Size: increased stature G189; G1073; G1435; G2430 Size: reduced stature or G3; G5; G21; G23; G39; G165; Ornamental; small dwarfism G184; G194; G258; G280; G340; stature provides G343; G353; G354; G362; G363; wind resistance; G370; G385; G396; G439; G440; creation of dwarf G447; G450; G550; G557; G599; varieties G636; G652; G670; G671; G674; G729; G760; G804; G831; G864; G884; G898; G900; G912; G913; G922; G932; G937; G939; G960; G962; G977; G991; G1000; G1008; G1020; G1023; G1053; G1067; G1075; G1137; G1181; G1198; G1228; G1266; G1267; G1275; G1277; G1309; G1311; G1314; G1317; G1322; G1323; G1326; G1332; G1334; G1367; G1381; G1382; G1386; G1421; G1488; G1494; G1537; G1545; G1560; G1586; G1641; G1652; G1655; G1671; G1750; G1756; G1757; G1782; G1786; G1794; G1839; G1845; G1879; G1886; G1888; G1933; G1939; G1943; G1944; G2011; G2094; G2115; G2130; G2132; G2144; G2145; G2147; G2156; G2294; G2313; G2344; G2431; G2510; G2517; G2521; G2893; G2893 Fruit size and number G362 Biomass, yield, cotton boll fiber density Flower structure, G47; G259; G353; G354; G671; Ornamental inflorescence G732; G988; G1000; G1063; horticulture; G1140; G1326; G1449; G1543; production of G1560; G1587; G1645; G1947; saffron or other G2108; G2143; G2893 edible flowers Number and G225; G226; G247; G362; G585; Resistance to pests development of G634; G676; G682; G1014; and desiccation; trichomes G1332; G1452; G1795; G2105 essential oil production Seed size, color, and G156; G450; G584; G652; G668; Yield number G858; G979; G1040; G1062; G1145; G1255; G1494; G1531; G1534; G1594; G2105; G2114; Root development, G9; G1482; G1534; G1794; modifications G1852; G2053; G2136; G2140 Modifications to root G225; G226 Nutrient, water hairs uptake, pathogen resistance Apical dominance G559; G732; G1255; G1275; Ornamental G1411; G1488; G1635; G2452; horticulture G2509 Branching patterns G568; G988; G1548 Ornamental horticulture, knot reduction, improved windscreen Leaf shape, color, G375; G377; G428; G438; G447; Appealing shape or modifications G464; G557; G577; G599; G635; shiny leaves for G671; G674; G736; G804; G903; ornamental G977; G921; G922; G1038; agriculture, G1063; G1067; G1073; G1075; increased biomass G1146; G1152; G1198; G1267; or photosynthesis G1269; G1452; G1484; G1586; G1594; G1767; G1786; G1792; G1886; G2059; G2094; G2105; G2113; G2117; G2143; G2144; G2431; G2452; G2465; G2587; G2583; G2724; Silique G1134 Ornamental Stem morphology G47; G438; G671; G748; G988; Ornamental; G1000 digestibility Shoot modifications G390; G391 Ornamental stem bifurcations Disease, Pathogen Bacterial G211; G347; G367; G418; G525; Yield, appearance, Resistance G545; G578; G1049 survivability, extended range Fungal G19; G28; G28; G28; G147; Yield, appearance, G188; G207; G211; G237; G248; survivability, G278; G347; G367; G371; G378; extended range G409; G477; G545; G545; G558; G569; G578; G591; G594; G616; G789; G805; G812; G865; G869; G872; G881; G896; G940; G1047; G1049; G1064; G1084; G1196; G1255; G1266; G1363; G1514; G1756; G1792; G1792; G1792; G1792; G1880; G1919; G1919; G1927; G1927; G1936; G1936; G1950; G2069; G2130; G2380; G2380; G2555 Nutrients Increased tolerance to G255; G226; G1792 nitrogen-limited soils Increased tolerance to G419; G545; G561; G1946 phosphate-limited soils Increased tolerance to G561; G911 potassium-limited soils Hormonal Hormone sensitivity G12; G546; G926; G760; G913; Seed dormancy, G926; G1062; G1069; G1095; drought tolerance; G1134; G1330; G1452; G1666; plant form, fruit G1820; G2140; G2789 ripening Seed biochemistry Production of seed G214; G259; G490; G652; G748; Antioxidant activity, prenyl lipids, including G883; G1052; G1328; G1930; vitamin E tocopherol G2509; G2520 Production of seed G20 Precursors for sterols human steroid hormones; cholesterol modulators Production of seed G353; G484; G674; G1272; Defense against glucosinolates G1506; G1897; G1946; G2113; insects; putative G2117; G2155; G2290; G2340 anticancer activity; undesirable in animal feeds Modified seed oil G162; G162; G180; G192; G241; Vegetable oil content G265; G286; G291; G427; G509; production; G519; G561; G567; G590; G818; increased caloric G849; G892; G961; G974; G1063; value for animal G1143; G1190; G1198; G1226; feeds; lutein content G1229; G1323; G1451; G1471; G1478; G1496; G1526; G1543; G1640; G1644; G1646; G1672; G1677; G1750; G1765; G1777; G1793; G1838; G1902; G1946; G1948; G2059; G2123; G2138; G2139; G2343; G2792; G2830 Modified seed oil G217; G504; G622; G778; G791; Heat stability, composition G861; G869; G938; G965; G1417; digestibility of seed G2192 oils Modified seed protein G162; G226; G241; G371; G427; Reduced caloric content G509; G567; G597; G732; G849; value for humans G865; G892; G963; G988; G1323; G1323; G1419; G1478; G1488; G1634; G1637; G1641; G1644; G1652; G1677; G1777; G1777; G1818; G1820; G1903; G1909; G1946; G1946; G1958; G2059; G2117; G2417; G2509 Leaf biochemistry Production of flavonoids G1666* Ornamental pigment production; pathogen resistance; health benefits Production of leaf G264; G353; G484; G652; G674; Defense against glucosinolates G681; G1069; G1198; G1322; insects; putative G1421; G1657; G1794; G1897; anticancer activity; G1946; G2115; G2117; G2144; undesirable in G2155; G2155; G2340; G2512; animal feeds G2520; G2552 Production of diterpenes G229 Induction of enzymes involved in alkaloid biosynthesis Production of G546 Ornamental pigment anthocyanin Production of leaf G561; G2131; G2424 Precursors for phytosterols, inc. human steroid stigmastanol, hormones; campesterol cholesterol modulators Leaf fatty acid G214; G377; G861; G962; G975; Nutritional value; composition G987; G1266; G1337; G1399; increase in waxes G1465; G1512; G2136; G2147; for disease G2192 resistance Production of leaf G214; G259; G280; G652; G987; Antioxidant activity, prenyl lipids, including G1543; G2509; G2520 vitamin E tocopherol Biochemistry, Production of G229; G663 general miscellaneous secondary metabolites Sugar, starch, G158; G211; G211; G237; G242; Food digestibility, hemicellulose G274; G598; G1012; G1266; hemicellulose & composition, G1309; G1309; G1641; G1765; pectin content; fiber G1865; G2094; G2094; G2589; content; plant G2589 tensile strength, wood quality, pathogen resistance, pulp production; tuber starch content Sugar sensing Plant response to sugars G26; G38; G43; G207; G218; Photosynthetic rate, G241; G254; G263; G308; G536; carbohydrate G567; G567; G680; G867; G912; accumulation, G956; G996; G1068; G1225; biomass production, G1314; G1314; G1337; G1759; source-sink G1804; G2153; G2379; relationships, senescence Growth, Plant growth rate and G447; G617; G674; G730; G917; Faster growth, Reproduction development G937; G1035; G1046; G1131; increased biomass G1425; G1452; G1459; G1492; or yield, improved G1589; G1652; G1879; G1943; appearance; delay in G2430; G2431; G2465; G2521 bolting Embryo development G167 Seed germination rate G979; G1792; G2130 Yield Plant, seedling vigor G561; G2346 Survivability, yield Senescence; cell death G571; G636; G878; G1050; Yield, appearance; G1463; G1749; G1944; G2130; response to G2155; G2340; G2383 pathogens; Modified fertility G39; G340; G439; G470; G559; Prevents or G615; G652; G671; G779; G962; minimizes escape of G977; G988; G1000; G1063; the pollen of GMOs G1067; G1075; G1266; G1311; G1321; G1326; G1367; G1386; G1421; G1453; G1471; G1453; G1560; G1594; G1635; G1750; G1947; G2011; G2094; G2113; G2115; G2130; G2143; G2147; G2294; G2510; G2893 Early flowering G147; G157; G180; G183; G183; Faster generation G184; G185; G208; G227; G294; time; synchrony of G390; G390; G390; G391; G391; flowering; potential G427; G427; G490; G565; G590; for introducing new G592; G720; G789; G865; G898; traits to single G898; G989; G989; G1037; variety G1037; G1142; G1225; G1225; G1226; G1242; G1305; G1305; G1380; G1380; G1480; G1480; G1488; G1494; G1545; G1545; G1649; G1706; G1760; G1767; G1767; G1820; G1841; G1841; G1842; G1843; G1843; G1946; G1946; G2010; G2030; G2030; G2144; G2144; G2295; G2295; G2347; G2348; G2348; G2373; G2373; G2509; G2509; G2555; G2555 Delayed flowering G8; G47; G192; G214; G234; Delayed time to G361; G362; G562; G568; G571; pollen production of G591; G680; G736; G748; G859; GMO plants; G878; G910; G912; G913; G971; synchrony of G994; G1051; G1052; G1073; flowering; increased G1079; G1335; G1435; G1452; yield G1478; G1789; G1804; G1865; G1865; G1895; G1900; G2007; G2133; G2155; G2291; G2465 Extended flowering G1947 phase Flower and leaf G259; G353; G377; G580; G638 Ornamental development G652; G858; G869; G917; G922; applications; G932; G1063; G1075; G1140; decreased fertility G1425; G1452; G1499; G1548; G1645; G1865; G1897; G1933; G2094; G2124; G2140; G2143; G2535; G2557 Flower abscission G1897 Ornamental: longer retention of flowers *When co-expressed with G669 and G663
Significance of Modified Plant Traits

Currently, the existence of a series of maturity groups for different latitudes represents a major barrier to the introduction of new valuable traits. Any trait (e.g. disease resistance) has to be bred into each of the different maturity groups separately, a laborious and costly exercise. The availability of single strain, which could be grown at any latitude, would therefore greatly increase the potential for introducing new traits to crop species such as soybean and cotton.
For many of the traits, listed in Table 6 and below, that may be conferred to plants, a single transcription factor gene may be used to increase or decrease, advance or delay, or improve or prove deleterious to a given trait. For example, overexpression of a transcription factor gene that naturally occurs in a plant may cause early flowering relative to non-transformed or wild-type plants. By knocking out the gene, or suppressing the gene (with, for example, antisense suppression) the plant may experience delayed flowering. Similarly, overexpressing or suppressing one or more genes can impart significant differences in production of plant products, such as different fatty acid ratios. Thus, suppressing a gene that causes a plant to be more sensitive to cold may improve a plant's tolerance of cold.
Salt stress resistance. Soil salinity is one of the more important variables that determines where a plant may thrive. Salinity is especially important for the successful cultivation of crop plants, particular in many parts of the world that have naturally high soil salt concentrations, or where the soil has been over-utilized. Thus, presently disclosed transcription factor genes that provide increased salt tolerance during germination, the seedling stage, and throughout a plant's life cycle would find particular value for imparting survivability and yield in areas where a particular crop would not normally prosper.
Osmotic stress resistance. Presently disclosed transcription factor genes that confer resistance to osmotic stress may increase germination rate under adverse conditions, which could impact survivability and yield of seeds and plants.
Cold stress resistance. The potential utility of presently disclosed transcription factor genes that increase tolerance to cold is to confer better germination and growth in cold conditions. The germination of many crops is very sensitive to cold temperatures. Genes that would allow germination and seedling vigor in the cold would have highly significant utility in allowing seeds to be planted earlier in the season with a high rate of survivability. Transcription factor genes that confer better survivability in cooler climates allow a grower to move up planting time in the spring and extend the growing season further into autumn for higher crop yields.
Tolerance to freezing. The presently disclosed transcription factor genes that impart tolerance to freezing conditions are useful for enhancing the survivability and appearance of plants conditions or conditions that would otherwise cause extensive cellular damage. Thus, germination of seeds and survival may take place at temperatures significantly below that of the mean temperature required for germination of seeds and survival of non-transformed plants. As with salt tolerance, this has the added benefit of increasing the potential range of a crop plant into regions in which it would otherwise succumb. Cold tolerant transformed plants may also be planted earlier in the spring or later in autumn, with greater success than with non-transformed plants.
Heat stress tolerance. The germination of many crops is also sensitive to high temperatures. Presently disclosed transcription factor genes that provide increased heat tolerance are generally useful in producing plants that germinate and grow in hot conditions, may find particular use for crops that are planted late in the season, or extend the range of a plant by allowing growth in relatively hot climates.
Drought, low humidity tolerance. Strategies that allow plants to survive in low water conditions may include, for example, reduced surface area or surface oil or wax production. A number of presently disclosed transcription factor genes increase a plant's tolerance to low water conditions and provide the benefits of improved survivability, increased yield and an extended geographic and temporal planting range.
Radiation resistance. Presently disclosed transcription factor genes have been shown to increase lutein production. Lutein, like other xanthophylls such as zeaxanthin and violaxanthin, are important in the protection of plants against the damaging effects of excessive light. Lutein contributes, directly or indirectly, to the rapid rise of non-photochemical quenching in plants exposed to high light. Increased tolerance of field plants to visible and ultraviolet light impacts survivability and vigor, particularly for recent transplants. Also affected are the yield and appearance of harvested plants or plant parts. Crop plants engineered with presently disclosed transcription factor genes that cause the plant to produce higher levels of lutein therefore would have improved photoprotection, leading to less oxidative damage and increase vigor, survivability and higher yields under high light and ultraviolet light conditions.
Decreased herbicide sensitivity. Presently disclosed transcription factor genes that confer resistance or tolerance to herbicides (e.g., glyphosate) may find use in providing means to increase herbicide applications without detriment to desirable plants. This would allow for the increased use of a particular herbicide in a local environment, with the effect of increased detriment to undesirable species and less harm to transgenic, desirable cultivars.
Increased herbicide sensitivity. Knockouts of a number of the presently disclosed transcription factor genes have been shown to be lethal to developing embryos. Thus, these genes are potentially useful as herbicide targets.
Oxidative stress. In plants, as in all living things, abiotic and biotic stresses induce the formation of oxygen radicals, including superoxide and peroxide radicals. This has the effect of accelerating senescence, particularly in leaves, with the resulting loss of yield and adverse effect on appearance. Generally, plants that have the highest level of defense mechanisms, such as, for example, polyunsaturated moieties of membrane lipids, are most likely to thrive under conditions that introduce oxidative stress (e.g., high light, ozone, water deficit, particularly in combination). Introduction of the presently disclosed transcription factor genes that increase the level of oxidative stress defense mechanisms would provide beneficial effects on the yield and appearance of plants. One specific oxidizing agent, ozone, has been shown to cause significant foliar injury, which impacts yield and appearance of crop and ornamental plants. In addition to reduced foliar injury that would be found in ozone resistant plant created by transforming plants with some of the presently disclosed transcription factor genes, the latter have also been shown to have increased chlorophyll fluorescence (Yu-Sen Chang et al. Bot. Bull. Acad. Sin. (2001) 42: 265-272).
Heavy metal tolerance. Heavy metals such as lead, mercury, arsenic, chromium and others may have a significant adverse impact on plant respiration. Plants that have been transformed with presently disclosed transcription factor genes that confer improved resistance to heavy metals, through, for example, sequestering or reduced uptake of the metals will show improved vigor and yield in soils with relatively high concentrations of these elements. Conversely, transgenic transcription factors may also be introduced into plants to confer an increase in heavy metal uptake, which may benefit efforts to clean up contaminated soils.
Light response. Presently disclosed transcription factor genes that modify a plant's response to light may be useful for modifying a plant's growth or development, for example, photomorphogenesis in poor light, or accelerating flowering time in response to various light intensities, quality or duration to which a non-transformed plant would not similarly respond. Examples of such responses that have been demonstrated include leaf number and arrangement, and early flower bud appearances.
Overall plant architecture. Several presently disclosed transcription factor genes have been introduced into plants to alter numerous aspects of the plant's morphology. For example, it has been demonstrated that a number of transcription factors may be used to manipulate branching, such as the means to modify lateral branching, a possible application in the forestry industry. Transgenic plants have also been produced that have altered cell wall content, lignin production, flower organ number, or overall shape of the plants. Presently disclosed transcription factor genes transformed into plants may be used to affect plant morphology by increasing or decreasing internode distance, both of which may be advantageous under different circumstances. For example, for fast growth of woody plants to provide more biomass, or fewer knots, increased internode distances are generally desirable. For improved wind screening of shrubs or trees, or harvesting characteristics of, for example, members of the Gramineae family, decreased internode distance may be advantageous. These modifications would also prove useful in the ornamental horticulture industry for the creation of unique phenotypic characteristics of ornamental plants.
Increased stature. For some ornamental plants, the ability to provide larger varieties may be highly desirable. For many plants, including t fruit-bearing trees or trees and shrubs that serve as view or wind screens, increased stature provides obvious benefits. Crop species may also produce higher yields on larger cultivars.
Reduced stature or dwarfism. Presently disclosed transcription factor genes that decrease plant stature can be used to produce plants that are more resistant to damage by wind and rain, or more resistant to heat or low humidity or water deficit. Dwarf plants are also of significant interest to the ornamental horticulture industry, and particularly for home garden applications for which space availability may be limited.
Fruit size and number. Introduction of presently disclosed transcription factor genes that affect fruit size will have desirable impacts on fruit size and number, which may comprise increases in yield for fruit crops, or reduced fruit yield, such as when vegetative growth is preferred (e.g., with bushy ornamentals, or where fruit is undesirable, as with ornamental olive trees).
Flower structure inflorescence and development. Presently disclosed transgenic transcription factors have been used to create plants with larger flowers or arrangements of flowers that are distinct from wild-type or non-transformed cultivars. This would likely have the most value for the ornamental horticulture industry, where larger flowers or interesting presentations generally are preferred and command the highest prices. Flower structure may have advantageous effects on fertility, and could be used, for example, to decrease fertility by the absence, reduction or screening of reproductive components. One interesting application for manipulation of flower structure, for example, by introduced transcription factors could be in the increased production of edible flowers or flower parts, including saffron, which is derived from the stigmas of Crocus sativus.
Number and development of trichomes. Several presently disclosed transcription factor genes have been used to modify trichome number and amount of trichome products in plants. Trichome glands on the surface of many higher plants produce and secrete exudates that give protection from the elements and pests such as insects, microbes and herbivores. These exudates may physically immobilize insects and spores, may be insecticidal or ant-microbial or they may act as allergens or irritants to protect against herbivores. Trichomes have also been suggested to decrease transpiration by decreasing leaf surface air flow, and by exuding chemicals that protect the leaf from the sun.
Seed size, color and number. The introduction of presently disclosed transcription factor genes into plants that alter the size or number of seeds may have a significant impact on yield, both when the product is the seed itself, or when biomass of the vegetative portion of the plant is increased by reducing seed production. In the case of fruit products, it is often advantageous to modify a plant to have reduced size or number of seeds relative to non-transformed plants to provide seedless or varieties with reduced numbers or smaller seeds. Presently disclosed transcription factor genes have also been shown to affect seed size, including the development of larger seeds. Seed size, in addition to seed coat integrity, thickness and permeability, seed water content and by a number of other components including antioxidants and oligosaccharides, may affect seed longevity in storage. This would be an important utility when the seed of a plant is the harvested crops, as with, for example, peas, beans, nuts, etc. Presently disclosed transcription factor genes have also been used to modify seed color, which could provide added appeal to a seed product.
Root development, modifications. By modifying the structure or development of roots by transforming into a plant one or more of the presently disclosed transcription factor genes, plants may be produced that have the capacity to thrive in otherwise unproductive soils. For example, grape roots that extend further into rocky soils, or that remain viable in waterlogged soils, would increase the effective planting range of the crop. It may be advantageous to manipulate a plant to produce short roots, as when a soil in which the plant will be growing is occasionally flooded, or when pathogenic fungi or disease-causing nematodes are prevalent.
Modifications to root hairs. Presently disclosed transcription factor genes that increase root hair length or number potentially could be used to increase root growth or vigor, which might in turn allow better plant growth under adverse conditions such as limited nutrient or water availability.
Apical dominance. The modified expression of presently disclosed transcription factors that control apical dominance could be used in ornamental horticulture, for example, to modify plant architecture.
Branching patterns. Several presently disclosed transcription factor genes have been used to manipulate branching, which could provide benefits in the forestry industry. For example, reduction in the formation of lateral branches could reduce knot formation. Conversely, increasing the number of lateral branches could provide utility when a plant is used as a windscreen, or may also provide ornamental advantages.
Leaf shape, color and modifications. It has been demonstrated in laboratory experiments that overexpression of some of the presently disclosed transcription factors produced marked effects on leaf development. At early stages of growth, these transgenic seedlings developed narrow, upward pointing leaves with long petioles, possibly indicating a disruption in circadian-clock controlled processes or nyctinastic movements. Other transcription factor genes can be used to increase plant biomass; large size would be useful in crops where the vegetative portion of the plant is the marketable portion.
Siliques. Genes that later silique conformation in brassicates may be used to modify fruit ripening processes in brassicates and other plants, which may positively affect seed or fruit quality.
Stem morphology and shoot modifications. Laboratory studies have demonstrated that introducing several of the presently disclosed transcription factor genes into plants can cause stem bifurcations in shoots, in which the shoot meristems split to form two or three separate shoots. This unique appearance would be desirable in ornamental applications.
Diseases, pathogens and pests. A number of the presently disclosed transcription factor genes have been shown to or are likely to confer resistance to various plant diseases, pathogens and pests. The offending organisms include fungal pathogens Fusarium oxysporum, Botrytis cinerea, Sclerotinia sclerotiorum , and Erysiphe orontii . Bacterial pathogens to which resistance may be conferred include Pseudomonas syringae . Other problem organisms may potentially include nematodes, mollicutes, parasites, or herbivorous arthropods. In each case, one or more transformed transcription factor genes may provide some benefit to the plant to help prevent or overcome infestation. The mechanisms by which the transcription factors work could include increasing surface waxes or oils, surface thickness, local senescence, or the activation of signal transduction pathways that regulate plant defense in response to attacks by herbivorous pests (including, for example, protease inhibitors).
Increased tolerance of plants to nutrient-limited soils. Presently disclosed transcription factor genes introduced into plants may provide the means to improve uptake of essential nutrients, including nitrogenous compounds, phosphates, potassium, and trace minerals. The effect of these modifications is to increase the seedling germination and range of ornamental and crop plants. The utilities of presently disclosed transcription factor genes conferring tolerance to conditions of low nutrients also include cost savings to the grower by reducing the amounts of fertilizer needed, environmental benefits of reduced fertilizer runoff; and improved yield and stress tolerance. In addition, this gene could be used to alter seed protein amounts and/or composition that could impact yield as well as the nutritional value and production of various food products.
Hormone sensitivity. One or more of the presently disclosed transcription factor genes have been shown to affect plant abscisic acid (ABA) sensitivity. This plant hormone is likely the most important hormone in mediating the adaptation of a plant to stress. For example, ABA mediates conversion of apical meristems into dormant buds. In response to increasingly cold conditions, the newly developing leaves growing above the meristem become converted into stiff bud scales that closely wrap the meristem and protect it from mechanical damage during winter. ABA in the bud also enforces dormancy; during premature warm spells, the buds are inhibited from sprouting. Bud dormancy is eliminated after either a prolonged cold period of cold or a significant number of lengthening days. Thus, by affecting ABA sensitivity, introduced transcription factor genes may affect cold sensitivity and survivability. ABA is also important in protecting plants from drought tolerance.
Several other of the present transcription factor genes have been used to manipulate ethylene signal transduction and response pathways. These genes can thus be used to manipulate the processes influenced by ethylene, such as seed germination or fruit ripening, and to improve seed or fruit quality.
Production of seed and leaf prenyl lipids, including tocopherol. Prenyl lipids play a role in anchoring proteins in membranes or membranous organelles. Thus modifying the prenyl lipid content of seeds and leaves could affect membrane integrity and function. A number of presently disclosed transcription factor genes have been shown to modify the tocopherol composition of plants. Tocopherols have both anti-oxidant and vitamin E activity.
Production of seed and leaf phytosterols: Presently disclosed transcription factor genes that modify levels of phytosterols in plants may have at least two utilities. First, phytosterols are an important source of precursors for the manufacture of human steroid hormones. Thus, regulation of transcription factor expression or activity could lead to elevated levels of important human steroid precursors for steroid semi-synthesis. For example, transcription factors that cause elevated levels of campesterol in leaves, or sitosterols and stigmasterols in seed crops, would be useful for this purpose. Phytosterols and their hydrogenated derivatives phytostanols also have proven cholesterol-lowering properties, and transcription factor genes that modify the expression of these compounds in plants would thus provide health benefits.
Production of seed and leaf glucosinolates. Some glucosinolates have anti-cancer activity; thus, increasing the levels or composition of these compounds by introducing several of the presently disclosed transcription factors might be of interest from a nutraceutical standpoint. (3) Glucosinolates form part of a plants natural defense against insects. Modification of glucosinolate composition or quantity could therefore afford increased protection from predators. Furthermore, in edible crops, tissue specific promoters might be used to ensure that these compounds accumulate specifically in tissues, such as the epidermis, which are not taken for consumption.
Modified seed oil content. The composition of seeds, particularly with respect to seed oil amounts and/or composition, is very important for the nutritional value and production of various food and feed products. Several of the presently disclosed transcription factor genes in seed lipid saturation that alter seed oil content could be used to improve the heat stability of oils or to improve the nutritional quality of seed oil, by, for example, reducing the number of calories in seed, increasing the number of calories in animal feeds, or altering the ratio of saturated to unsaturated lipids comprising the oils.
Seed and leaf fatty acid composition. A number of the presently disclosed transcription factor genes have been shown to alter the fatty acid composition in plants, and seeds in particular. This modification may find particular value for improving the nutritional value of, for example, seeds or whole plants. Dietary fatty acids ratios have been shown to have an effect on, for example, bone integrity and remodeling (see, for example, Weiler, H. A., Pediatr Res (2000) 47:5 692-697). The ratio of dietary fatty acids may alter the precursor pools of long-chain polyunsaturated fatty acids that serve as precursors for prostaglandin synthesis. In mammalian connective tissue, prostaglandins serve as important signals regulating the balance between resorption and formation in bone and cartilage. Thus dietary fatty acid ratios altered in seeds may affect the etiology and outcome of bone loss.
Modified seed protein content. As with seed oils, the composition of seeds, particularly with respect to protein amounts and/or composition, is very important for the nutritional value and production of various food and feed products. A number of the presently disclosed transcription factor genes modify the protein concentrations in seeds would provide nutritional benefits, and may be used to prolong storage, increase seed pest or disease resistance, or modify germination rates.
Production of flavonoids in leaves and other plant parts. Expression of presently disclosed transcription factor genes that increase flavonoid production in plants, including anthocyanins and condensed tannins, may be used to alter in pigment production for horticultural purposes, and possibly increasing stress resistance. Flavonoids have antimicrobial activity and could be used to engineer pathogen resistance. Several flavonoid compounds have health promoting effects such as the inhibition of tumor growth and cancer, prevention of bone loss and the prevention of the oxidation of lipids. Increasing levels of condensed tannins, whose biosynthetic pathway is shared with anthocyanin biosynthesis, in forage legumes is an important agronomic trait because they prevent pasture bloat by collapsing protein foams within the rumen. For a review on the utilities of flavonoids and their derivatives, refer to Dixon et al. (1999) Trends Plant Sci. 4:394-400.
Production of diterpenes in leaves and other plant parts. Depending on the plant species, varying amounts of diverse secondary biochemicals (often lipophilic terpenes) are produced and exuded or volatilized by trichomes. These exotic secondary biochemicals, which are relatively easy to extract because they are on the surface of the leaf, have been widely used in such products as flavors and aromas, drugs, pesticides and cosmetics. Thus, the overexpression of genes that are used to produce diterpenes in plants may be accomplished by introducing transcription factor genes that induce said overexpression. One class of secondary metabolites, the diterpenes, can effect several biological systems such as tumor progression, prostaglandin synthesis and tissue inflammation. In addition, diterpenes can act as insect pheromones, termite allomones, and can exhibit neurotoxic, cytotoxic and antimitotic activities. As a result of this functional diversity, diterpenes have been the target of research several pharmaceutical ventures. In most cases where the metabolic pathways are impossible to engineer, increasing trichome density or size on leaves may be the only way to increase plant productivity.
Production of anthocyanin in leaves and other plant parts. Several presently disclosed transcription factor genes can be used to alter anthocyanin production in numerous plant species. The potential utilities of these genes include alterations in pigment production for horticultural purposes, and possibly increasing stress resistance in combination with another transcription factor.
Production of miscellaneous secondary metabolites. Microarray data suggests that flux through the aromatic amino acid biosynthetic pathways and primary and secondary metabolite biosynthetic pathways are up-regulated. Presently disclosed transcription factors have been shown to be involved in regulating alkaloid biosynthesis, in part by up-regulating the enzymes indole-3-glycerol phosphatase and strictosidine synthase. Phenylalanine ammonia lyase, chalcone synthase and trans-cinnamate mono-oxygenase are also induced, and are involved in phenylpropenoid biosynthesis.
Sugar, starch, hemicellulose composition. Overexpression of the presently disclosed transcription factors that affect sugar content resulted in plants with altered leaf insoluble sugar content. Transcription factors that alter plant cell wall composition have several potential applications including altering food digestibility, plant tensile strength, wood quality, pathogen resistance and in pulp production. The potential utilities of a gene involved in glucose-specific sugar sensing are to alter energy balance, photosynthetic rate, carbohydrate accumulation, biomass production, source-sink relationships, and senescence.
Hemicellulose is not desirable in paper pulps because of its lack of strength compared with cellulose. Thus modulating the amounts of cellulose vs. hemicellulose in the plant cell wall is desirable for the paper/lumber industry. Increasing the insoluble carbohydrate content in various fruits, vegetables, and other edible consumer products will result in enhanced fiber content. Increased fiber content would not only provide health benefits in food products, but might also increase digestibility of forage crops. In addition, the hemicellulose and pectin content of fruits and berries affects the quality of jam and catsup made from them. Changes in hemicellulose and pectin content could result in a superior consumer product.
Plant response to sugars and sugar composition. In addition to their important role as an energy source and structural component of the plant cell, sugars are central regulatory molecules that control several aspects of plant physiology, metabolism and development. It is thought that this control is achieved by regulating gene expression and, in higher plants, sugars have been shown to repress or activate plant genes involved in many essential processes such as photosynthesis, glyoxylate metabolism, respiration, starch and sucrose synthesis and degradation, pathogen response, wounding response, cell cycle regulation, pigmentation, flowering and senescence. The mechanisms by which sugars control gene expression are not understood.
Because sugars are important signaling molecules, the ability to control either the concentration of a signaling sugar or how the plant perceives or responds to a signaling sugar could be used to control plant development, physiology or metabolism. For example, the flux of sucrose (a disaccharide sugar used for systemically transporting carbon and energy in most plants) has been shown to affect gene expression and alter storage compound accumulation in seeds. Manipulation of the sucrose signaling pathway in seeds may therefore cause seeds to have more protein, oil or carbohydrate, depending on the type of manipulation. Similarly, in tubers, sucrose is converted to starch which is used as an energy store. It is thought that sugar signaling pathways may partially determine the levels of starch synthesized in the tubers. The manipulation of sugar signaling in tubers could lead to tubers with a higher starch content.
Thus, the presently disclosed transcription factor genes that manipulate the sugar signal transduction pathway may lead to altered gene expression to produce plants with desirable traits. In particular, manipulation of sugar signal transduction pathways could be used to alter source-sink relationships in seeds, tubers, roots and other storage organs leading to increase in yield.
Plant growth rate and development. A number of the presently disclosed transcription factor genes have been shown to have significant effects on plant growth rate and development. These observations have included, for example, more rapid or delayed growth and development of reproductive organs. This would provide utility for regions with short or long growing seasons, respectively. Accelerating plant growth would also improve early yield or increase biomass at an earlier stage, when such is desirable (for example, in producing forestry products).
Embryo development. Presently disclosed transcription factor genes that alter embryo development has been used to alter seed protein and oil amounts and/or composition which is very important for the nutritional value and production of various food products. Seed shape and seed coat may also be altered by these genes, which may provide for improved storage stability.
Seed germination rate. A number of the presently disclosed transcription factor genes have been shown to modify seed germination rate, including when the seeds are in conditions normally unfavorable for germination (e.g., cold, heat or salt stress, or in the presence of ABA), and may thus be used to modify and improve germination rates under adverse conditions.
Plant, seedling vigor. Seedlings transformed with presently disclosed transcription factors have been shown to possess larger cotyledons and appeared somewhat more advanced than control plants. This indicates that the seedlings developed more rapidly that the control plants. Rapid seedling development is likely to reduce loss due to diseases particularly prevalent at the seedling stage (e.g., damping off) and is thus important for survivability of plants germinating in the field or in controlled environments.
Senescence, cell death. Presently disclosed transcription factor genes may be used to alter senescence responses in plants. Although leaf senescence is thought to be an evolutionary adaptation to recycle nutrients, the ability to control senescence in an agricultural setting has significant value. For example, a delay in leaf senescence in some maize hybrids is associated with a significant increase in yields and a delay of a few days in the senescence of soybean plants can have a large impact on yield. Delayed flower senescence may also generate plants that retain their blossoms longer and this may be of potential interest to the ornamental horticulture industry.
Modified fertility. Plants that overexpress a number of the presently disclosed transcription factor genes have been shown to possess reduced fertility. This could be a desirable trait, as it could be exploited to prevent or minimize the escape of the pollen of genetically modified organisms (GMOs) into the environment.
Early and delayed flowering. Presently disclosed transcription factor genes that accelerate flowering could have valuable applications in such programs since they allow much faster generation times. In a number of species, for example, broccoli, cauliflower, where the reproductive parts of the plants constitute the crop and the vegetative tissues are discarded, it would be advantageous to accelerate time to flowering. Accelerating flowering could shorten crop and tree breeding programs. Additionally, in some instances, a faster generation time might allow additional harvests of a crop to be made within a given growing season. A number of Arabidopsis genes have already been shown to accelerate flowering when constitutively expressed. These include LEAFY, APETALA1 and CONSTANS (Mandel, M. et al., 1995, Nature 377, 522-524; Weigel, D. and Nilsson, O., 1995, Nature 377, 495-500; Simon et al., 1996, Nature 384, 59-62).
By regulating the expression of potential flowering using inducible promoters, flowering could be triggered by application of an inducer chemical. This would allow flowering to be synchronized across a crop and facilitate more efficient harvesting. Such inducible systems could also be used to tune the flowering of crop varieties to different latitudes. At present, species such as soybean and cotton are available as a series of maturity groups that are suitable for different latitudes on the basis of their flowering time (which is governed by day-length). A system in which flowering could be chemically controlled would allow a single high-yielding northern maturity group to be grown at any latitude. In southern regions such plants could be grown for longer, thereby increasing yields, before flowering was induced. In more northern areas, the induction would be used to ensure that the crop flowers prior to the first winter frosts.
In a sizeable number of species, for example, root crops, where the vegetative parts of the plants constitute the crop and the reproductive tissues are discarded, it would be advantageous to delay or prevent flowering. Extending vegetative development with presently disclosed transcription factor genes could thus bring about large increases in yields. Prevention of flowering might help maximize vegetative yields and prevent escape of genetically modified organism (GMO) pollen.
Extended flowering phase. Presently disclosed transcription factors that extend flowering time have utility in engineering plants with longer-lasting flowers for the horticulture industry, and for extending the time in which the plant is fertile.
Flower and leaf development. Presently disclosed transcription factor genes have been used to modify the development of flowers and leaves. This could be advantageous in the development of new ornamental cultivars that present unique configurations. In addition, some of these genes have been shown to reduce a plant's fertility, which is also useful for helping to prevent development of pollen of GMOs.
Flower abscission. Presently disclosed transcription factor genes introduced into plants have been used to retain flowers for longer periods. This would provide a significant benefit to the ornamental industry, for both cut flowers and woody plant varieties (of, for example, maize), as well as have the potential to lengthen the fertile period of a plant, which could positively impact yield and breeding programs.
A listing of specific effects and utilities that the presently disclosed transcription factor genes have on plants, as determined by direct observation and assay analysis, is provided in Table 5.
XVI. ANTISENSE AND CO-SUPPRESSION
In addition to expression of the nucleic acids of the invention as gene replacement or plant phenotype modification nucleic acids, the nucleic acids are also useful for sense and anti-sense suppression of expression, e.g., to down-regulate expression of a nucleic acid of the invention, e.g., as a further mechanism for modulating plant phenotype. That is, the nucleic acids of the invention, or subsequences or anti-sense sequences thereof, can be used to block expression of naturally occurring homologous nucleic acids. A variety of sense and anti-sense technologies are known in the art, e.g., as set forth in Lichtenstein and Nellen (1997) Antisense Technology: A Practical Approach IRL Press at Oxford University Press, Oxford, U.K. In general, sense or anti-sense sequences are introduced into a cell, where they are optionally amplified, e.g., by transcription. Such sequences include both simple oligonucleotide sequences and catalytic sequences such as ribozymes.
For example, a reduction or elimination of expression (i.e., a “knock-out”) of a transcription factor or transcription factor homologue polypeptide in a transgenic plant, e.g., to modify a plant trait, can be obtained by introducing an antisense construct corresponding to the polypeptide of interest as a cDNA. For antisense suppression, the transcription factor or homologue cDNA is arranged in reverse orientation (with respect to the coding sequence) relative to the promoter sequence in the expression vector. The introduced sequence need not be the full length cDNA or gene, and need not be identical to the cDNA or gene found in the plant type to be transformed. Typically, the antisense sequence need only be capable of hybridizing to the target gene or RNA of interest. Thus, where the introduced sequence is of shorter length, a higher degree of homology to the endogenous transcription factor sequence will be needed for effective antisense suppression. While antisense sequences of various lengths can be utilized, preferably, the introduced antisense sequence in the vector will be at least 30 nucleotides in length, and improved antisense suppression will typically be observed as the length of the antisense sequence increases. Preferably, the length of the antisense sequence in the vector will be greater than 100 nucleotides. Transcription of an antisense construct as described results in the production of RNA molecules that are the reverse complement of mRNA molecules transcribed from the endogenous transcription factor gene in the plant cell.
Suppression of endogenous transcription factor gene expression can also be achieved using a ribozyme. Ribozymes are RNA molecules that possess highly specific endoribonuclease activity. The production and use of ribozymes are disclosed in U.S. Pat. No. 4,987,071 and U.S. Pat. No. 5,543,508. Synthetic ribozyme sequences including antisense RNAs can be used to confer RNA cleaving activity on the antisense RNA, such that endogenous mRNA molecules that hybridize to the antisense RNA are cleaved, which in turn leads to an enhanced antisense inhibition of endogenous gene expression.
Vectors in which RNA encoded by a transcription factor or transcription factor homologue cDNA is over-expressed can also be used to obtain co-suppression of a corresponding endogenous gene, e.g., in the manner described in U.S. Pat. No. 5,231,020 to Jorgensen. Such co-suppression (also termed sense suppression) does not require that the entire transcription factor cDNA be introduced into the plant cells, nor does it require that the introduced sequence be exactly identical to the endogenous transcription factor gene of interest. However, as with antisense suppression, the suppressive efficiency will be enhanced as specificity of hybridization is increased, e.g., as the introduced sequence is lengthened, and/or as the sequence similarity between the introduced sequence and the endogenous transcription factor gene is increased.
Vectors expressing an untranslatable form of the transcription factor mRNA, e.g., sequences comprising one or more stop codon, or nonsense mutation) can also be used to suppress expression of an endogenous transcription factor, thereby reducing or eliminating it's activity and modifying one or more traits. Methods for producing such constructs are described in U.S. Pat. No. 5,583,021. Preferably, such constructs are made by introducing a premature stop codon into the transcription factor gene. Alternatively, a plant trait can be modified by gene silencing using double-strand RNA (Sharp (1999) Genes and Development 13: 139-141).Another method for abolishing the expression of a gene is by insertion mutagenesis using the T-DNA of Agrobacterium tumefaciens . After generating the insertion mutants, the mutants can be screened to identify those containing the insertion in a transcription factor or transcription factor homologue gene. Plants containing a single transgene insertion event at the desired gene can be crossed to generate homozygous plants for the mutation. Such methods are well known to those of skill in the art. (See for example Koncz et al. (1992) Methods in Arabidopsis Research World Scientific.)
Alternatively, a plant phenotype can be altered by eliminating an endogenous gene, such as a transcription factor or transcription factor homologue, e.g., by homologous recombination (Kempin et al. (1997) Nature 389:802-803).
A plant trait can also be modified by using the Cre-lox system (for example, as described in U.S. Pat. No. 5,658,772). A plant genome can be modified to include first and second lox sites that are then contacted with a Cre recombinase. If the lox sites are in the same orientation, the intervening DNA sequence between the two sites is excised. If the lox sites are in the opposite orientation, the intervening sequence is inverted.
The polynucleotides and polypeptides of this invention can also be expressed in a plant in the absence of an expression cassette by manipulating the activity or expression level of the endogenous gene by other means. For example, by ectopically expressing a gene by T-DNA activation tagging (Ichikawa et al. (1997) Nature 390 698-701; Kakimoto et al. (1996) Science 274: 982-985). This method entails transforming a plant with a gene tag containing multiple transcriptional enhancers and once the tag has inserted into the genome, expression of a flanking gene coding sequence becomes deregulated. In another example, the transcriptional machinery in a plant can be modified so as to increase transcription levels of a polynucleotide of the invention (See, e.g., PCT Publications WO 96/06166 and WO 98/53057 which describe the modification of the DNA-binding specificity of zinc finger proteins by changing particular amino acids in the DNA-binding motif).
The transgenic plant can also include the machinery necessary for expressing or altering the activity of a polypeptide encoded by an endogenous gene, for example by altering the phosphorylation state of the polypeptide to maintain it in an activated state.
Transgenic plants (or plant cells, or plant explants, or plant tissues) incorporating the polynucleotides of the invention and/or expressing the polypeptides of the invention can be produced by a variety of well established techniques as described above. Following construction of a vector, most typically an expression cassette, including a polynucleotide, e.g., encoding a transcription factor or transcription factor homologue, of the invention, standard techniques can be used to introduce the polynucleotide into a plant, a plant cell, a plant explant or a plant tissue of interest. Optionally, the plant cell, explant or tissue can be regenerated to produce a transgenic plant.
The plant can be any higher plant, including gymnosperms, monocotyledonous and dicotyledenous plants. Suitable protocols are available for Leg minosae (alfalfa, soybean, clover, etc.), Umbelliferae (carrot, celery, parsnip), Cr ciferae (cabbage, radish, rapeseed, broccoli, etc.), Curcurbitaceae (melons and cucumber), Gramineae (wheat, corn, rice, barley, millet, etc.), Solanaceae (potato, tomato, tobacco, peppers, etc.), and various other crops. See protocols described in Ammirato et al. (1984) Handbook of Plant Cell Culture - Crop Species , Macmillan Publ. Co. Shimamoto et al. (1989) Nature 338:274-276; Fromm et al. (1990) Bio/Technology 8:833-839; and Vasil et al. (1990) Bio/Technology 8:429-434.
Transformation and regeneration of both monocotyledonous and dicotyledonous plant cells is now routine, and the selection of the most appropriate transformation technique will be determined by the practitioner. The choice of method will vary with the type of plant to be transformed; those skilled in the art will recognize the suitability of particular methods for given plant types. Suitable methods can include, but are not limited to: electroporation of plant protoplasts; liposome-mediated transformation; polyethylene glycol (PEG) mediated transformation; transformation using viruses; micro-injection of plant cells; micro-projectile bombardment of plant cells; vacuum infiltration; and Agrobacterium tumefaciens mediated transformation. Transformation means introducing a nucleotide sequence into a plant in a manner to cause stable or transient expression of the sequence.
Successful examples of the modification of plant characteristics by transformation with cloned sequences which serve to illustrate the current knowledge in this field of technology, and which are herein incorporated by reference, include: U.S. Pat. Nos. 5,571,706; 5,677,175; 5,510,471; 5,750,386; 5,597,945; 5,589,615; 5,750,871; 5,268,526; 5,780,708; 5,538,880; 5,773,269; 5,736,369 and 5,610,042.
Following transformation, plants are preferably selected using a dominant selectable marker incorporated into the transformation vector. Typically, such a marker will confer antibiotic or herbicide resistance on the transformed plants, and selection of transformants can be accomplished by exposing the plants to appropriate concentrations of the antibiotic or herbicide.
After transformed plants are selected and grown to maturity, those plants showing a modified trait are identified. The modified trait can be any of those traits described above. Additionally, to confirm that the modified trait is due to changes in expression levels or activity of the polypeptide or polynucleotide of the invention can be determined by analyzing mRNA expression using Northern blots, RT-PCR or microarrays, or protein expression using immunoblots or Western blots or gel shift assays.
XVII. INTEGRATED SYSTEMS
Sequence Identity
Additionally, the present invention may be an integrated system, computer or computer readable medium that comprises an instruction set for determining the identity of one or more sequences in a database. In addition, the instruction set can be used to generate or identify sequences that meet any specified criteria. Furthermore, the instruction set may be used to associate or link certain functional benefits, such improved characteristics, with one or more identified sequence.
For example, the instruction set can include, e.g., a sequence comparison or other alignment program, e.g., an available program such as, for example, the Wisconsin Package Version 10.0, such as BLAST, FASTA, PILEUP, FINDPATTERNS or the like (GCG, Madison, Wis.). Public sequence databases such as GenBank, EMBL, Swiss-Prot and PIR or private sequence databases such as PHYTOSEQ sequence database (Incyte Genomics, Palo Alto, Calif.) can be searched.
Alignment of sequences for comparison can be conducted by the local homology algorithm of Smith and Waterman (1981) Adv. Appl. Math. 2:482, by the homology alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443-453, by the search for similarity method of Pearson and Lipman (1988) Proc. Natl. Acad. Sci. U.S.A. 85:2444-2448, by computerized implementations of these algorithms. After alignment, sequence comparisons between two (or more) polynucleotides or polypeptides are typically performed by comparing sequences of the two sequences over a comparison window to identify and compare local regions of sequence similarity. The comparison window can be a segment of at least about 20 contiguous positions, usually about 50 to about 200, more usually about 100 to about 150 contiguous positions. A description of the method is provided in Ausubel et al., supra.
A variety of methods for determining sequence relationships can be used, including manual alignment and computer assisted sequence alignment and analysis. This later approach is a preferred approach in the present invention, due to the increased throughput afforded by computer assisted methods. As noted above, a variety of computer programs for performing sequence alignment are available, or can be produced by one of skill.
One example algorithm that is suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm, which is described in Altschul et al. J. Mol. Biol. 215:403-410 (1990). Software for performing BLAST analyses is publicly available, e.g., through the National Center for Biotechnology Information (see internet website at ncbi.nlm.nih.gov). This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) of 10, a cutoff of 100, M=5, N=−4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915). Unless otherwise indicated, “sequence identity” here refers to the % sequence identity generated from a tblastx using the NCBI version of the algorithm at the default settings using gapped alignments with the filter “off” (see, for example, internet website at ncbi.nlm.nih.gov).
In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5787). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence (and, therefore, in this context, homologous) if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.1, or less than about 0.01, and or even less than about 0.001. An additional example of a useful sequence alignment algorithm is PILEUP. PILEUP creates a multiple sequence alignment from a group of related sequences using progressive, pairwise alignments. The program can align, e.g., up to 300 sequences of a maximum length of 5,000 letters.
The integrated system, or computer typically includes a user input interface allowing a user to selectively view one or more sequence records corresponding to the one or more character strings, as well as an instruction set which aligns the one or more character strings with each other or with an additional character string to identify one or more region of sequence similarity. The system may include a link of one or more character strings with a particular phenotype or gene function. Typically, the system includes a user readable output element that displays an alignment produced by the alignment instruction set.
The methods of this invention can be implemented in a localized or distributed computing environment. In a distributed environment, the methods may implemented on a single computer comprising multiple processors or on a multiplicity of computers. The computers can be linked, e.g. through a common bus, but more preferably the computer(s) are nodes on a network. The network can be a generalized or a dedicated local or wide-area network and, in certain preferred embodiments, the computers may be components of an intra-net or an internet.
Thus, the invention provides methods for identifying a sequence similar or homologous to one or more polynucleotides as noted herein, or one or more target polypeptides encoded by the polynucleotides, or otherwise noted herein and may include linking or associating a given plant phenotype or gene function with a sequence. In the methods, a sequence database is provided (locally or across an inter or intra net) and a query is made against the sequence database using the relevant sequences herein and associated plant phenotypes or gene functions.
Any sequence herein can be entered into the database, before or after querying the database. This provides for both expansion of the database and, if done before the querying step, for insertion of control sequences into the database. The control sequences can be detected by the query to ensure the general integrity of both the database and the query. As noted, the query can be performed using a web browser based interface. For example, the database can be a centralized public database such as those noted herein, and the querying can be done from a remote terminal or computer across an internet or intranet.
XVIII. EXAMPLES
The following examples are intended to illustrate but not limit the present invention. The complete descriptions of the traits associated with each polynucleotide of the invention is fully disclosed in Table 4 5 and Table 6.
Example I
Full Length Gene Identification and Cloning
Putative transcription factor sequences (genomic or ESTs) related to known transcription factors were identified in the Arabidopsis thaliana GenBank database using the tblastn sequence analysis program using default parameters and a P-value cutoff threshold of −4 or −5 or lower, depending on the length of the query sequence. Putative transcription factor sequence hits were then screened to identify those containing particular sequence strings. If the sequence hits contained such sequence strings, the sequences were confirmed as transcription factors.
Alternatively, Arabidopsis thaliana cDNA libraries derived from different tissues or treatments, or genomic libraries were screened to identify novel members of a transcription family using a low stringency hybridization approach. Probes were synthesized using gene specific primers in a standard PCR reaction (annealing temperature 60° C.) and labeled with 32 P dCTP using the High Prime DNA Labeling Kit (Boehringer Mannheim). Purified radiolabelled probes were added to filters immersed in Church hybridization medium (0.5 M NaPO 4 pH 7.0, 7% SDS, 1% w/v bovine serum albumin) and hybridized overnight at 60° C. with shaking. Filters were washed two times for 45 to 60 minutes with 1×SCC, 1% SDS at 60° C.
To identify additional sequence 5′ or 3′ of a partial cDNA sequence in a cDNA library, 5′ and 3′ rapid amplification of cDNA ends (RACE) was performed using the Marathon™ cDNA amplification kit (Clontech, Palo Alto, Calif.). Generally, the method entailed first isolating poly(A) mRNA, performing first and second strand cDNA synthesis to generate double stranded cDNA, blunting cDNA ends, followed by ligation of the Marathon™ Adaptor to the cDNA to form a library of adaptor-ligated ds cDNA.
Gene-specific primers were designed to be used along with adaptor specific primers for both 5′ and 3′ RACE reactions. Nested primers, rather than single primers, were used to increase PCR specificity. Using 5′ and 3′ RACE reactions, 5′ and 3′ RACE fragments were obtained, sequenced and cloned. The process can be repeated until 5′ and 3′ ends of the full-length gene were identified. Then the full-length cDNA was generated by PCR using primers specific to 5′ and 3′ ends of the gene by end-to-end PCR.
Example II
Construction of Expression Vectors
The sequence was amplified from a genomic or cDNA library using primers specific to sequences upstream and downstream of the coding region. the expression vector was pMEN20 or pMEN65, which are both derived from pMON316 (Sanders et al, (1987) Nucleic Acids Research 15:1543-1558) and contain the caMV 35S promoter to express transgenes. To clone the sequence into the vector, both pmen20 and the amplified DNA fragment were digested separately with Sali and Noti restriction enzymes at 37° C. for 2 hours. The digestion products were subject to electrophoresis in a 0.8% agarose gel and visualized by ethidium bromide staining. The DNA fragments containing the sequence and the linearized plasmid were excised and purified by using a Qiaquick gel extraction kit (Qiagen, Valencia Calif.). The fragments of interest were ligated at a ratio of 3:1 (vector to insert). Ligation reactions using t4 DNA ligase (New England Biolabs, Beverly, Mass.) were carried out at 16° C. for 16 hours. The ligated DNAs were transformed into competent cells of the E. coli strain DH5alpha by using the heat shock method. The transformations were plated on LB plates containing 50 mg/l kanamycin (Sigma, St. Louis, Mo.). Individual colonies were grown overnight in five milliliters of LB broth containing 50 mg/l kanamycin at 37° c. Plasmid DNA was purified by using Qiaquick Mini Prep kits (Qiagen).
Example III
Transformation of Agrobacterium with the Expression Vector
After the plasmid vector containing the gene was constructed, the vector was used to transform Agrobacterium tumefaciens cells expressing the gene products. The stock of Agrobacterium tumefaciens cells for transformation were made as described by Nagel et al. (1990) FEMS Microbiol Letts. 67: 325-328. Agrobacterium strain ABI was grown in 250 ml LB medium (Sigma) overnight at 28° C. with shaking until an absorbance (A 600 ) of 0.5-1.0 was reached. Cells were harvested by centrifugation at 4,000×g for 15 min at 4° C. Cells were then resuspended in 250 μl chilled buffer (1 mM HEPES, pH adjusted to 7.0 with KOH). Cells were centrifuged again as described above and resuspended in 125 μl chilled buffer. Cells were then centrifuged and resuspended two more times in the same HEPES buffer as described above at a volume of 100 μl and 750 μl, respectively. Resuspended cells were then distributed into 40 μl aliquots, quickly frozen in liquid nitrogen, and stored at −80° C.
Agrobacterium cells were transformed with plasmids prepared as described above following the protocol described by Nagel et al. For each DNA construct to be transformed, 50-100 ng DNA (generally resuspended in 10 mM Tris-HCl, 1 mM EDTA, pH 8.0) was mixed with 40 μl of Agrobacterium cells. The DNA/cell mixture was then transferred to a chilled cuvette with a 2 mm electrode gap and subject to a 2.5 kV charge dissipated at 25 μF and 200 μF using a Gene Pulser II apparatus (Bio-Rad, Hercules, Calif.). After electroporation, cells were immediately resuspended in 1.0 ml LB and allowed to recover without antibiotic selection for 2-4 hours at 28° C. in a shaking incubator. After recovery, cells were plated onto selective medium of LB broth containing 100 μg/ml spectinomycin (Sigma) and incubated for 24-48 hours at 28° C. Single colonies were then picked and inoculated in fresh medium. The presence of the plasmid construct was verified by PCR amplification and sequence analysis.
Example IV
Transformation of Arabidopsis Plants with Agrobacterium tumefaciens with Expression Vector
After transformation of Agrobacterium tumefaciens with plasmid vectors containing the gene, single Agrobacterium colonies were identified, propagated, and used to transform Arabidopsis plants. Briefly, 500 ml cultures of LB medium containing 50 mg/l kanamycin were inoculated with the colonies and grown at 28° C. with shaking for 2 days until an optical absorbance at 600 nm wavelength over 1 cm (A 600 ) of >2.0 is reached. Cells were then harvested by centrifugation at 4,000×g for 10 min, and resuspended in infiltration medium (½×Murashige and Skoog salts (Sigma), 1× Gamborg's B-5 vitamins (Sigma), 5.0% (w/v) sucrose (Sigma), 0.044 μM benzylamino purine (Sigma), 200 μl/l Silwet L-77 (Lehle Seeds) until an A 600 of 0.8 was reached.
Prior to transformation, Arabidopsis thaliana seeds (ecotype Columbia) were sown at a density of ˜10 plants per 4″ pot onto Pro-Mix BX potting medium (Hummert International) covered with fiberglass mesh (18 mm×16 mm). Plants were grown under continuous illumination (50-75 μE/m 2 /sec) at 22-23° C. with 65-70% relative humidity. After about 4 weeks, primary inflorescence stems (bolts) are cut off to encourage growth of multiple secondary bolts. After flowering of the mature secondary bolts, plants were prepared for transformation by removal of all siliques and opened flowers.
The pots were then immersed upside down in the mixture of Agrobacterium infiltration medium as described above for 30 sec, and placed on their sides to allow draining into a 1′×2′ flat surface covered with plastic wrap. After 24 h, the plastic wrap was removed and pots are turned upright. The immersion procedure was repeated one week later, for a total of two immersions per pot. Seeds were then collected from each transformation pot and analyzed following the protocol described below.
Example V
Identification of Arabidopsis Primary Transformants
Seeds collected from the transformation pots were sterilized essentially as follows. Seeds were dispersed into in a solution containing 0.1% (v/v) Triton X-100 (Sigma) and sterile H 2 O and washed by shaking the suspension for 20 min. The wash solution was then drained and replaced with fresh wash solution to wash the seeds for 20 min with shaking. After removal of the second wash solution, a solution containing 0.1% (v/v) Triton X-100 and 70% ethanol (Equistar) was added to the seeds and the suspension was shaken for 5 min. After removal of the ethanol/detergent solution, a solution containing 0.1% (v/v) Triton X-100 and 30% (v/v) bleach (Clorox) was added to the seeds, and the suspension was shaken for 10 min. After removal of the bleach/detergent solution, seeds were then washed five times in sterile distilled H 2 O. The seeds were stored in the last wash water at 4° C. for 2 days in the dark before being plated onto antibiotic selection medium (1× Murashige and Skoog salts (pH adjusted to 5.7 with 1M KOH), 1× Gamborg's B-5 vitamins, 0.9% phytagar (Life Technologies), and 50 mg/l kanamycin). Seeds were germinated under continuous illumination (50-75 μE/m 2 /sec) at 22-23° C. After 7-10 days of growth under these conditions, kanamycin resistant primary transformants (T 1 generation) were visible and obtained. These seedlings were transferred first to fresh selection plates where the seedlings continued to grow for 3-5 more days, and then to soil (Pro-Mix BX potting medium).
Primary transformants were crossed and progeny seeds (T 2 ) collected; kanamycin resistant seedlings were selected and analyzed. The expression levels of the recombinant polynucleotides in the transformants varies from about a 5% expression level increase to a least a 100% expression level increase. Similar observations are made with respect to polypeptide level expression.
Example VI
Identification of Arabidopsis Plants with Transcription Factor Gene Knockouts
The screening of insertion mutagenized Arabidopsis collections for null mutants in a known target gene was essentially as described in Krysan et al (1999) Plant Cell 11:2283-2290. Briefly, gene-specific primers, nested by 5-250 base pairs to each other, were designed from the 5′ and 3′ regions of a known target gene. Similarly, nested sets of primers were also created specific to each of the T-DNA or transposon ends (the “right” and “left” borders). All possible combinations of gene specific and T-DNA/transposon primers were used to detect by PCR an insertion event within or close to the target gene. The amplified DNA fragments were then sequenced which allows the precise determination of the T-DNA/transposon insertion point relative to the target gene. Insertion events within the coding or intervening sequence of the genes were deconvoluted from a pool comprising a plurality of insertion events to a single unique mutant plant for functional characterization. The method is described in more detail in Yu and Adam, U.S. application Ser. No. 09/177,733 filed Oct. 23, 1998.
Example VII
Identification of Modified Phenotypes in Overexpression or Gene Knockout Plants
Experiments were performed to identify those transformants or knockouts that exhibited modified biochemical characteristics. Among the biochemicals that were assayed were insoluble sugars, such as arabinose, fucose, galactose, mannose, rhamnose or xylose or the like; prenyl lipids, such as lutein, beta-carotene, xanthophyll-1, xanthophyll-2, chlorophylls A or B, or alpha-, delta- or gamma-tocopherol or the like; fatty acids, such as 16:0 (palmitic acid), 16:1 (palmitoleic acid), 18:0 (stearic acid), 18:1 (oleic acid), 18:2 (linoleic acid), 20:0, 18:3 (linolenic acid), 20:1 (eicosenoic acid), 20:2, 22:1 (erucic acid) or the like; waxes, such as by altering the levels of C29, C31, or C33 alkanes; sterols, such as brassicasterol, campesterol, stigmasterol, sitosterol or stigmastanol or the like, glucosinolates, protein or oil levels.
Fatty acids were measured using two methods depending on whether the tissue was from leaves or seeds. For leaves, lipids were extracted and esterified with hot methanolic H 2 SO 4 and partitioned into hexane from methanolic brine. For seed fatty acids, seeds were pulverized and extracted in methanol:heptane:toluene:2,2-dimethoxypropane:H 2 SO 4 (39:34:20:5:2) for 90 minutes at 80° C. After cooling to room temperature the upper phase, containing the seed fatty acid esters, was subjected to GC analysis. Fatty acid esters from both seed and leaf tissues were analyzed with a Supelco SP-2330 column.
Glucosinolates were purified from seeds or leaves by first heating the tissue at 95° C. for 10 minutes. Preheated ethanol:water (50:50) is and after heating at 95° C. for a further 10 minutes, the extraction solvent is applied to a DEAE Sephadex column which had been previously equilibrated with 0.5 M pyridine acetate. Desulfoglucosinolates were eluted with 300 ul water and analyzed by reverse phase HPLC monitoring at 226 nm.
For wax alkanes, samples were extracted using an identical method as fatty acids and extracts were analyzed on a HP 5890 GC coupled with a 5973 MSD. Samples were chromatographically isolated on a J&W DB35 mass spectrometer (J&W Scientific).
To measure prenyl lipids levels, seeds or leaves were pulverized with 1 to 2% pyrogallol as an antioxidant. For seeds, extracted samples were filtered and a portion removed for tocopherol and carotenoid/chlorophyll analysis by HPLC. The remaining material was saponified for sterol determination. For leaves, an aliquot was removed and diluted with methanol and chlorophyll A, chlorophyll B, and total carotenoids measured by spectrophotometry by determining optical absorbance at 665.2 nm, 652.5 nm, and 470 nm. An aliquot was removed for tocopherol and carotenoid/chlorophyll composition by HPLC using a Waters uBondapak C18 column (4.6 mm×150 mm). The remaining methanolic solution was saponified with 10% KOH at 80° C. for one hour. The samples were cooled and diluted with a mixture of methanol and water. A solution of 2% methylene chloride in hexane was mixed in and the samples were centrifuged. The aqueous methanol phase was again re-extracted 2% methylene chloride in hexane and, after centrifugation, the two upper phases were combined and evaporated. 2% methylene chloride in hexane was added to the tubes and the samples were then extracted with one ml of water. The upper phase was removed, dried, and resuspended in 400 ul of 2% methylene chloride in hexane and analyzed by gas chromatography using a 50 m DB-5 ms (0.25 mm ID, 0.25 um phase, J&W Scientific).
Insoluble sugar levels were measured by the method essentially described by Reiter et al., (1999) Plant Journal 12:335-345. This method analyzes the neutral sugar composition of cell wall polymers found in Arabidopsis leaves. Soluble sugars were separated from sugar polymers by extracting leaves with hot 70% ethanol. The remaining residue containing the insoluble polysaccharides was then acid hydrolyzed with allose added as an internal standard. Sugar monomers generated by the hydrolysis were then reduced to the corresponding alditols by treatment with NaBH 4 , then were acetylated to generate the volatile alditol acetates which were then analyzed by GC-FID. Identity of the peaks was determined by comparing the retention times of known sugars converted to the corresponding alditol acetates with the retention times of peaks from wild-type plant extracts. Alditol acetates were analyzed on a Supelco SP-2330 capillary column (30 m×250 um×0.2 um) using a temperature program beginning at 180° C. for 2 minutes followed by an increase to 220° C. in 4 minutes. After holding at 220° C. for 10 minutes, the oven temperature is increased to 240° C. in 2 minutes and held at this temperature for 10 minutes and brought back to room temperature.
To identify plants with alterations in total seed oil or protein content, 150 mg of seeds from T2 progeny plants were subjected to analysis by Near Infrared Reflectance Spectroscopy (NIRS) using a Foss NirSystems Model 6500 with a spinning cup transport system. NIRS is a non-destructive analytical method used to determine seed oil and protein composition. Infrared is the region of the electromagnetic spectrum located after the visible region in the direction of longer wavelengths. ‘Near infrared’ owns its name for being the infrared region near to the visible region of the electromagnetic spectrum. For practical purposes, near infrared comprises wavelengths between 800 and 2500 nm. NIRS is applied to organic compounds rich in O—H bonds (such as moisture, carbohydrates, and fats), C—H bonds (such as organic compounds and petroleum derivatives), and N—H bonds (such as proteins and amino acids). The NIRS analytical instruments operate by statistically correlating NIRS signals at several wavelengths with the characteristic or property intended to be measured. All biological substances contain thousands of C—H, O—H, and N—H bonds. Therefore, the exposure to near infrared radiation of a biological sample, such as a seed, results in a complex spectrum which contains qualitative and quantitative information about the physical and chemical composition of that sample.
The numerical value of a specific analyte in the sample, such as protein content or oil content, is mediated by a calibration approach known as chemometrics. Chemometrics applies statistical methods such as multiple linear regression (MLR), partial least squares (PLS), and principle component analysis (PCA) to the spectral data and correlates them with a physical property or other factor, that property or factor is directly determined rather than the analyte concentration itself. The method first provides “wet chemistry” data of the samples required to develop the calibration.
Calibration for Arabidopsis seed oil composition was performed using accelerated solvent extraction using 1 g seed sample size and was validated against certified canola seed. A similar wet chemistry approach was performed for seed protein composition calibration.
Data obtained from NIRS analysis was analyzed statistically using a nearest-neighbor (N—N) analysis. The N—N analysis allows removal of within-block spatial variability in a fairly flexible fashion which does not require prior knowledge of the pattern of variability in the chamber. Ideally, all hybrids are grown under identical experimental conditions within a block (rep). In reality, even in many block designs, significant within-block variability exists. Nearest-neighbor procedures are based on assumption that environmental effect of a plot is closely related to that of its neighbors. Nearest-neighbor methods use information from adjacent plots to adjust for within-block heterogeneity and so provide more precise estimates of treatment means and differences. If there is within-plot heterogeneity on a spatial scale that is larger than a single plot and smaller than the entire block, then yields from adjacent plots will be positively correlated. Information from neighboring plots can be used to reduce or remove the unwanted effect of the spatial heterogeneity, and hence improve the estimate of the treatment effect. Data from neighboring plots can also be used to reduce the influence of competition between adjacent plots. The Papadakis N—N analysis can be used with designs to remove within-block variability that would not be removed with the standard split plot analysis (Papadakis, 1973, Inst. d'Amelior. Plantes Thessaloniki (Greece) Bull. Scientif., No. 23; Papadakis, 1984, Proc. Acad. Athens, 59, 326-342).
Experiments were performed to identify those transformants or knockouts that exhibited an improved pathogen tolerance. For such studies, the transformants were exposed to biotropic fungal pathogens, such as Erysiphe orontii , and necrotropic fungal pathogens, such as Fusarium oxysporum. Fusarium oxysporum isolates cause vascular wilts and damping off of various annual vegetables, perennials and weeds (Mauch-Mani and Slusarenko (1994) Molecular Plant - Microbe Interactions 7: 378-383). For Fusarium oxysporum experiments, plants grown on Petri dishes were sprayed with a fresh spore suspension of F. oxysporum . The spore suspension was prepared as follows: A plug of fungal hyphae from a plate culture was placed on a fresh potato dextrose agar plate and allowed to spread for one week. 5 ml sterile water was then added to the plate, swirled, and pipetted into 50 ml Armstrong Fusarium medium. Spores were grown overnight in Fusarium medium and then sprayed onto plants using a Preval paint sprayer. Plant tissue was harvested and frozen in liquid nitrogen 48 hours post infection.
Erysiphe orontii is a causal agent of powdery mildew. For Erysiphe orontii experiments, plants were grown approximately 4 weeks in a greenhouse under 12 hour light (20° C., ˜30% relative humidity (rh)). Individual leaves were infected with E. orontii spores from infected plants using a camel's hair brush, and the plants were transferred to a Percival growth chamber (20° C., 80% rh.). Plant tissue was harvested and frozen in liquid nitrogen 7 days post infection.
Botrytis cinerea is a necrotrophic pathogen. Botrytis cinerea was grown on potato dextrose agar in the light. A spore culture was made by spreading 10 ml of sterile water on the fungus plate, swirling and transferring spores to 10 ml of sterile water. The spore inoculum (approx. 105 spores/ml) was used to spray 10 day-old seedlings grown under sterile conditions on MS (minus sucrose) media. Symptoms were evaluated every day up to approximately 1 week.
Infection with bacterial pathogens Pseudomonas syringae pv maculicola (Psm) strain 4326 and pv maculicola strain 4326 was performed by hand inoculation at two doses. Two inoculation doses allows the differentiation between plants with enhanced susceptibility and plants with enhanced resistance to the pathogen. Plants were grown for 3 weeks in the greenhouse, then transferred to the growth chamber for the remainder of their growth. Psm ES4326 was hand inoculated with 1 ml syringe on 3 fully-expanded leaves per plant (4½ wk old), using at least 9 plants per overexpressing line at two inoculation doses, OD=0.005 and OD=0.0005. Disease scoring occurred at day 3 post-inoculation with pictures of the plants and leaves taken in parallel.
In some instances, expression patterns of the pathogen-induced genes (such as defense genes) was monitored by microarray experiments. cDNAs were generated by PCR and resuspended at a final concentration of ˜100 ng/ul in 3×SSC or 150 mM Na-phosphate (Eisen and Brown (1999) Methods Enzymol. 303:179-205). The cDNAs were spotted on microscope glass slides coated with polylysine. The prepared cDNAs were aliquoted into 384 well plates and spotted on the slides using an x-y-z gantry (OmniGrid) purchased from GeneMachines (Menlo Park, Calif.) outfitted with quill type pins purchased from Telechem International (Sunnyvale, Calif.). After spotting, the arrays were cured for a minimum of one week at room temperature, rehydrated and blocked following the protocol recommended by Eisen and Brown (1999; supra).
Sample total RNA (10 ug) samples were labeled using fluorescent Cy3 and Cy5 dyes. Labeled samples were resuspended in 4×SSC/0.03% SDS/4 ug salmon sperm DNA/2 ug tRNA/50 mM Na-pyrophosphate, heated for 95° C. for 2.5 minutes, spun down and placed on the array. The array was then covered with a glass coverslip and placed in a sealed chamber. The chamber was then kept in a water bath at 62° C. overnight. The arrays were washed as described in Eisen and Brown (1999) and scanned on a General Scanning 3000 laser scanner. The resulting files are subsequently quantified using Imagene, a software purchased from BioDiscovery (Los Angeles, Calif.).
Experiments were performed to identify those transformants or knockouts that exhibited an improved environmental stress tolerance. For such studies, the transformants were exposed to a variety of environmental stresses. Plants were exposed to chilling stress (6 hour exposure to 4-8° C.), heat stress (6 hour exposure to 32-37° C.), high salt stress (6 hour exposure to 200 mM NaCl), drought stress (168 hours after removing water from trays), osmotic stress (6 hour exposure to 3 M mannitol), or nutrient limitation (nitrogen, phosphate, and potassium) (Nitrogen: all components of MS medium remained constant except N was reduced to 20 mg/l of NH 4 NO 3 , or Phosphate: All components of MS medium except KH 2 PO 4 , which was replaced by K 2 SO 4 , Potassium: All components of MS medium except removal of KNO 3 and KH 2 PO 4 , which were replaced by NaH 4 PO 4 ).
Experiments were performed to identify those transformants or knockouts that exhibited a modified structure and development characteristics. For such studies, the transformants were observed by eye to identify novel structural or developmental characteristics associated with the ectopic expression of the polynucleotides or polypeptides of the invention.
Experiments were performed to identify those transformants or knockouts that exhibited modified sugar-sensing. For such studies, seeds from transformants were germinated on media containing 5% glucose or 9.4% sucrose which normally partially restrict hypocotyl elongation. Plants with altered sugar sensing may have either longer or shorter hypocotyls than normal plants when grown on this media. Additionally, other plant traits may be varied such as root mass.
Flowering time was measured by the number of rosette leaves present when a visible inflorescence of approximately 3 cm is apparent Rosette and total leaf number on the progeny stem are tightly correlated with the timing of flowering (Koomneef et al (1991) Mol. Gen. Genet. 229:57-66. The vernalization response was measured. For vernalization treatments, seeds were sown to MS agar plates, sealed with micropore tape, and placed in a 4° C. cold room with low light levels for 6-8 weeks. The plates were then transferred to the growth rooms alongside plates containing freshly sown non-vernalized controls. Rosette leaves were counted when a visible inflorescence of approximately 3 cm was apparent.
Modified phenotypes observed for particular overexpressor or knockout plants are provided in Table 5. For a particular overexpressor that shows a less beneficial characteristic, it may be more useful to select a plant with a decreased expression of the particular transcription factor. For a particular knockout that shows a less beneficial characteristic, it may be more useful to select a plant with an increased expression of the particular transcription factor.
The sequences of the Sequence Listing or those in Tables 4, Table 5 or those disclosed here can be used to prepare transgenic plants and plants with altered traits. The specific transgenic plants listed below are produced from the sequences of the Sequence Listing, as noted. Table 5 provides exemplary polynucleotide and polypeptide sequences of the invention. Table 5 includes, from left to right for each sequence: the first column shows the polynucleotide SEQ ID NO; the second column shows the Mendel Gene ID No., GID; the third column shows the trait(s) resulting from the knock out or overexpression of the polynucleotide in the transgenic plant; the fourth column shows the category of the trait; the fifth column shows the transcription factor family to which the polynucleotide belongs; the sixth column (“Comment”), includes specific effects and utilities conferred by the polynucleotide of the first column; the seventh column shows the SEQ ID NO of the polypeptide encoded by the polynucleotide; and the eighth column shows the amino acid residue positions of the conserved domain in amino acid (AA) co-ordinates.
Seed of plants overexpressing sequences G265 (SEQ ID NOs:871 and 872), G715 (SEQ ID NOs:925 and 926), G1471 (SEQ ID NOs:311 and 312), G1793 (SEQ ID NOs:365 and 366), G1838 (SEQ ID NOs:381 and 382), G1902 (SEQ ID NOs:405 and 406), G286 (SEQ ID NOs:877 and 878), G2138 (SEQ ID NOs:865 and 866) and G2830 (SEQ ID NOs:875 and 876) was subjected to NIR analysis and a significant increase in seed oil content compared with seed from control plants was identified.
G192: G192 (SEQ ID NO: 859) was expressed in all plant tissues and under all conditions examined. Its expression was slightly induced upon infection by Fusarium . G192 was analyzed using transgenic plants in which this gene was expressed under the control of the 35S promoter. G192 overexpressors were late flowering under 12 hour light and had more leaves than control plants. This phenotype was manifested in the three T2 lines analyzed. Results of one experiment suggest that G192 overexpressor was more susceptible to infection with a moderate dose of the fungal pathogen Erysiphe orontii . The decrease in seed oil observed for one line was replicated in an independent experiment. G192 overexpression delayed flowering. A wide variety of applications exist for systems that either lengthen or shorten the time to flowering, or for systems of inducible flowering time control. In particular, in species where the vegetative parts of the plants constitute the crop and the reproductive tissues are discarded, it will be advantageous to delay or prevent flowering. Extending vegetative development can bring about large increases in yields. G192 can be used to manipulate the defense response in order to generate pathogen-resistant plants. G192 can be used to manipulate seed oil content, which can be of nutritional value.
Closely Related Genes from Other Species
G192 had some similarity within the conserved WRKY domain to non-Arabidopsis plant proteins.
G1946: G1946 (SEQ ID NO: 801) was studied using transgenic plants in which the gene was expressed under the control of the 35S promoter. Overexpression of G1946 resulted in accelerated flowering, with 35S::G1946 transformants producing flower buds up to a week earlier than wild-type controls (24-hour light conditions). These effects were seen in 12/20 primary transformants and in two independent plantings of each of the three T2 lines. Unlike many early flowering Arabidopsis transgenic lines, which are dwarfed, 35S::G1946 transformants often reached full-size at maturity, and produced large quantities of seeds, although the plants were slightly pale in coloration and had slightly flat leaves compared to wild-type. In addition, 35S::G1946 plants showed an altered response to phosphate deprivation. Seedlings of G1946 overexpressor plants showed more secondary root growth on phosphate-free media, when compared to wild-type control. In a repeat experiment, all three lines showed the phenotype. Overexpression of G1946 in Arabidopsis also resulted in an increase in seed glucosinolate M39501 in T2 lines land 3. An increase in seed oil and a decrease in seed protein was also observed in these two lines. G1946 was ubiquitously expressed, and does not appear to be significantly induced or repressed by any of the biotic and abiotic stress conditions tested at this time, with the exception of cold, which repressed G1946 expression. G1946 can be used to modify flowering time, as well as to improve the plant's performance in conditions of limited phosphate, and to alter seed oil, protein, and glucosinolate composition.
Closely Related Genes from Other Species
A comparison of the amino acid sequence of G1946 with sequences available from GenBank showed strong similarity with plant HSFs of several species ( Lycopersicon peruvianum, Medicago truncatula, Lycopersicon esculentum, Glycine max, Solanum tuberosum, Oryza sativa and Hordeum vulgare subsp. vulgare ).
G375: The sequence of G375 (SEQ ID NO:239) was experimentally determined and G375 was analyzed using transgenic plants in which G375 was expressed under the control of the 35S promoter. Overexpression of G375 produced marked effects on leaf development. At early stages of growth, 35S::G375 seedlings developed narrow, upward pointing leaves with long petioles (possibly indicating a disruption in circadian-clock controlled processes or nyctinastic movements). Additionally, some seedlings were noted to have elongated hypocotyls, and some were rather small compared to wild-type controls. Comparable phenotypes were obtained by overexpression of an AP2 family gene, G2113 (SEQ ID NO: 85). Following the switch to flowering, 35S::G375 plants showed reduced fertility, which possibly arose from a failure of stamens to fully elongate. One of the three T2 lines, (#41) was later flowering than wild-type controls, and also developed large numbers of small secondary rosette leaves in the axils of the primary rosette. Although these effects were not noted in the other two lines, the phenotypes obtained in line 41 were somewhat similar to those produced by overexpression of another Z-dof gene, G736 (SEQ ID NO: 211). G375 was expressed in all tissues, although at different levels. It was expressed at low levels in the root and germinating seed, and expressed at high levels in the embryo. The effects of G375 on leaf architecture are of potential interest to the ornamental horticulture industry.
Closely Related Genes from Other Species
G375 showed some homology to non-Arabidopsis plant proteins within the conserved D of domain.
G1255: The sequence of G1255 (SEQ ID NO: 273) was experimentally determined and G1255 was analyzed using transgenic plants in which G1255 was expressed under the control of the 35S promoter. Plants overexpressing G1255 had alterations in leaf architecture, a reduction in apical dominance, an increase in seed size, and showed more disease symptoms following inoculation with a low dose of the fungal pathogen Botrytis cinerea . G1255 was constitutively expressed and not significantly induced by any conditions tested. On the basis of the phenotypes produced by overexpression of G1255, G1255 can be used to manipulate the plant's defense response to produce pathogen resistance, alter plant architecture, or alter seed size.
Closely Related Genes from Other Species
G1255 showed strong homology to a putative rice zing finger protein represented by sequence AC087181 — 3. Sequence identity between these two protein extended beyond the conserved domain, and therefore, these genes can be orthologs.
G865: The complete cDNA sequence of G865 (SEQ ID NO: 557) was determined. G865 was ubiquitously expressed in Arabidopsis tissues. G865 was analyzed using transgenic plants in which G865 was expressed under the control of the 35S promoter. Plants overexpressing G865 were early flowering, with numerous secondary inflorescence meristems giving them a bushy appearance. G865 overexpressors were more susceptible to infection with a moderate dose of the fungal pathogens Erysiphe orontii and Botrytis cinerea . In addition, seeds from G865 overexpressing plants showed a trend of increased protein and reduced oil content, although the observed changes were not beyond the criteria used for judging significance except in one line. G865 can be used to control flowering time. G865 can be used to manipulate the defense response in order to generate pathogen-resistant plants. G865 can be used to alter seed oil and protein content of a plant.
Closely Related Genes from Other Species
G865 and other non-Arabidopsis AP2/EREBP proteins were similar within the conserved AP2 domain.
G2509: G2509 (SEQ ID NO: 23) was studied using transgenic plants in which the gene was expressed under the control of the 35S promoter. Overexpression of G2509 caused multiple alterations in plant growth and development, most notably, altered branching patterns, and a reduction in apical dominance, giving the plants a shorter, more bushy stature than wild type. Twenty 35S::G2509 primary transformants were examined; at early stages of rosette development, these plants displayed a wild-type phenotype. However, at the switch to flowering, almost all T1 lines showed a marked loss of apical dominance and large numbers of secondary shoots developed from axils of primary rosette leaves. In the most extreme cases, the shoots had very short internodes, giving the inflorescence a very bushy appearance. Such shoots were often very thin and flowers were relatively small and poorly fertile. At later stages, many plants appeared very small and had a low seed yield compared to wild type. In addition to the effects on branching, a substantial number of 35S::G2509 primary transformants also flowered early and had buds visible several days prior to wild type. Similar effects on inflorescence development were noted in each of three T2 populations examined. The branching and plant architecture phenotypes observed in 35S::G2509 lines resemble phenotypes observed for three other AP2/EREBP genes: G865 (SEQ ID NO: 557), G1411 (SEQ ID NO: 3), and G1794 (SEQ ID NO: 13). G2509, G865, and G1411 form a small clade within the large AP2/EREBP family, and G1794, although not belonging to the clade, is one of the AP2/EREBP genes closest to it in the phylogenetic tree. It is thus likely that all these genes share a related function, such as affecting hormone balance. Overexpression of G2509 in Arabidopsis resulted in an increase in alpha-tocopherol in seeds in T2 lines 5 and 11. G2509 was ubiquitously expressed in Arabidopsis plant tissue. G2509 expression levels were altered by a variety of environmental or physiological conditions. G2509 can be used to manipulate plant architecture and development. G2509 can be used to alter tocopherol composition. Tocopherols have anti-oxidant and vitamin E activity. G2509 can be useful in altering flowering time. A wide variety of applications exist for systems that either lengthen or shorten the time to flowering.
Closely Related Genes from Other Species
G2509 showed some sequence similarity with known genes from other plant species within the conserved AP2/EREBP domain.
G2347: G2347 (SEQ ID NO: 1119) was analyzed using transgenic plants in which G2347 was expressed under the control of the 35S promoter. Overexpression of G2347 markedly reduced the time to flowering in Arabidopsis . This phenotype was apparent in the majority of primary transformants and in all plants from two out of the three T2 lines examined. Under continuous light conditions, 35S::G2347 plants formed flower buds up a week earlier than wild type. Many of the plants were rather small and spindly compared to controls. To demonstrate that overexpression of G2347 could induce flowering under less inductive photoperiods, two T2 lines were re-grown in 12 hour conditions; again, all plants from both lines bolted early, with some initiating flower buds up to two weeks sooner than wild-type. As determined by RT-PCR, G2347 was highly expressed in rosette leaves and flowers, and to much lower levels in embryos and siliques. No expression of G2347 was detected in the other tissues tested. G2347 expression was repressed by cold, and by auxin treatments and by infection by Erysiphe . G2347 is also highly similar to the Arabidopsis protein G2010 (SEQ ID NO: 1121). The level of homology between these two proteins suggested they could have similar, overlapping, or redundant functions in Arabidopsis . In support of this hypothesis, overexpression of both G2010 and G2347 resulted in early flowering phenotypes in transgenic plants.
Closely Related Genes from Other Species
The closest relative to G2347 is the Antirrhinum protein, SBP2 (CAA63061). The similarity between these two proteins is extensive enough to suggest they might have similar functions in a plant.
G988: G988 (SEQ ID NO: 43) was analyzed using transgenic plants in which G988 was expressed under the control of the 35S promoter. Plants overexpressing G988 had multiple morphological phenotypes. The transgenic plants were generally smaller than wild-type plants, had altered leaf, inflorescence and flower development, altered plant architecture, and altered vasculature. In one transgenic line overexpressing G988 (line 23), an increase in the seed glucosinolate M39489 was observed. The phenotype of plants overexpressing G988 was wild-type in all other assays performed. In wild-type plants, G988 was expressed primarily in flower and silique tissue, but was also present at detectable levels in all other tissues tested. Expression of G988 was induced in response to heat treatment, and repressed in response to infection with Erysiphe . Based on the observed morphological phenotypes of the transgenic plants, G988 can be used to create plants with larger flowers. This can have value in the ornamental horticulture industry. The reduction in the formation of lateral branches suggests that G988 can have utility on the forestry industry. The Arabidopsis plants overexpressing G988 also had reduced fertility. This could actually be a desirable trait in some instances, as it can be exploited to prevent or minimize the escape of GMO (genetically modified organism) pollen into the environment.
Closely Related Genes from Other Species
The amino acid sequence for the Capsella rubella hypothetical protein represented by GenBank accession number CRU303349 was significantly identical to G988 outside of the SCR conserved domains. The Capsella rubella hypothetical protein is 90% identical to G988 over a stretch of roughly 450 amino acids. Therefore, it is likely that the Capsella rubella gene is an ortholog of G988.
G2346: G2346 (SEQ ID NO: 459) was analyzed using transgenic plants in which the gene was expressed under the control of the 35S promoter. 35S::G2346 seedlings from all three T2 populations had slightly larger cotyledons and appeared somewhat more advanced than controls. This indicated that the seedlings developed more rapidly that the control plants. At later stages, however, G2346 overexpressing plants showed no consistent differences from control plants. The phenotype of these transgenic plants was wild-type in all other assays performed. According to RT-PCR analysis, G2346 is expressed ubiquitously.
Closely Related Genes from Other Species
G2346 shows some sequence similarity with known genes from other plant species within the conserved SBP domain.
G1354: The complete sequence of G1354 (SEQ ID NO: 285) was determined. G1354 was analyzed using transgenic plants in which G1354 was expressed under the control of the 35S promoter. Overexpression of G1354 produced highly deleterious effects on growth and development. Only three 35S::G1354 T1 plants were obtained; all were extremely tiny and slow developing. After three weeks of growth, each of the plants comprised a completely disorganized mass of leaves and root that had no clear axis of growth. Since these individuals would not have survived transplantation to soil, they were harvested for RT-PCR analysis; all three plants showed moderate levels of G1354 overexpression compared to whole wild-type seedlings of an equivalent size. Only a very small number of transformants were obtained from two selection attempts on separate batches of T0 seed. Usually between 15 and 120 transformants are obtained from each aliquot of 300 mg T0 seed from wild-type plants. The low transformation frequency obtained in this experiment suggests that high levels of G1354 overexpression might have completely lethal effects and prevent transformed seeds from germinating. As determined by RT-PCR, G1354 was uniformly expressed in all tissues and under all conditions tested in RT-PCR. However, the gene was repressed in leaf tissue in response to Erysiphe infection.
Closely Related Genes from Other Species
G1354 is closely related to a NAM protein encoded by polynucleotide from rice (AC005310). Similarity between G1354 and this rice protein extends beyond the signature motif of the family to a level that would suggest the genes are orthologs.
G1063: G1063 (SEQ ID NO: 119) is a member of a clade of highly related HLH/MYC proteins that also includes G779 (SEQ ID NO: 113), G1499 (SEQ ID NO: 7), G2143 (SEQ ID NO: 129), and G2557 (SEQ ID NO: 133). All of these genes caused similar pleiotropic phenotypic effects when overexpressed, the most striking of which was the production of ectopic carpelloid tissue. These genes can be considered key regulators of carpel development. A spectrum of developmental alterations was observed amongst 35S::G1063 primary transformants and the majority were markedly small, dark green, and had narrow curled leaves. The most severely affected individuals were completely sterile and formed highly abnormal inflorescences; shoots often terminated in pin-like structures, and flowers were replaced by filamentous carpelloid structures. In other cases, flowers showed internode elongation between floral whorls, with a central carpel protruding on a pedicel-like organ. Additionally, lateral branches sometimes failed to develop and tiny patches of carpelloid tissue formed at axillary nodes of the inflorescence. In lines with an intermediate phenotype, flowers contained defined whorls of organs, but sepals were converted to carpelloid structures or displayed patches of carpelloid tissue. In contrast, lines with a weak phenotype developed relatively normal flowers and produced a reasonable quantity of seed. Such plants were still distinctly smaller than wild-type controls. Since the strongest 35S::G1063 lines were sterile, three lines with a relatively weak phenotype, that had produced sufficient seed for biochemical and physiological analysis, were selected for further study. Two of the T2 populations (T2-28,37) were clearly small, darker green and possessed narrow leaves compared to wild type. Plants from one of these populations (T2-28) also produced occasional branches with abnormal flowers like those seen in the T1. The final T2 population (T2-30) displayed a very mild phenotype. Overexpression of G1063 in Arabidopsis resulted in a decrease in seed oil content in T2 lines 28 and 37. No altered phenotypes were detected in any of the physiological assays, except that the plants were noted to be somewhat small and produce anthocyanin when grown in Petri plates. G1063 was expressed at low to moderate levels in roots, flowers, rosette leaves, embryos, and germinating seeds, but was not detected in shoots or siliques. It was induced by auxin. G1063 can be used to manipulate flower form and structure or plant fertility. One application for manipulation of flower structure can be in the production of saffron, which is derived from the stigmas of Crocus sativus. G1063 has utility in manipulating seed oil and protein content.
Closely Related Genes from Other Species
G1063 protein shared extensive homology in the basic helix loop helix region with a protein sequence encoded by Glycine max cDNA clone (AW832545) as well as a tomato root, plants pre-anthesis Lycopersicon sculentum cDNA (BE451174).
G2143: G2143 (SEQ ID NO: 129) is a member of a clade of highly related HLH/MYC proteins that also includes G779 (SEQ ID NO: 113), G1063 (SEQ ID NO: 119), G1499 (SEQ ID NO: 7), and G2557 (SEQ ID NO: 133). All of these genes caused similar pleiotropic phenotypic effects when overexpressed, the most striking of which was the production of ectopic carpelloid tissue. These genes can be considered key regulators of carpel development. Twelve out of twenty 35S::G2143 T1 lines showed a very severe phenotype; these plants were markedly small and had narrow, curled, dark-green leaves. Such individuals were completely sterile and formed highly abnormal inflorescences; shoots often terminated in pin-like structures, and flowers were replaced by filamentous carpelloid structures, or a fused mass of carpelloid tissue. Furthermore, lateral branches usually failed to develop, and tiny patches of stigmatic tissue often formed at axillary nodes of the inflorescence. Strongly affected plants displayed the highest levels of transgene expression (determined by RT-PCR). The remaining T1 lines showed lower levels of G2143 overexpression; these plants were still distinctly smaller than wild type, but had relatively normal inflorescences and produced seed. Since the strongest 35S::G2143 lines were sterile, three lines with a relatively weak phenotype, that had produced sufficient seed for biochemical analysis, were selected for further study. T2-11 plants displayed a very mild phenotype and had somewhat small, narrow, dark green leaves. The other two T2 populations, however, appeared wild-type, suggesting that transgene activity might have been reduced between the generations. Reduced seedling vigor was noted in the physiological assays. G2143 expression was detected at low levels in flowers and siliques, and at higher levels in germinating seed. G2143 can be used to manipulate flower form and structure or plant fertility. One application for manipulation of flower structure can be in the production of saffron, which is derived from the stigmas of Crocus sativus.
Closely Related Genes from Other Species
G2143 protein shared extensive homology in the basic helix loop helix region with a protein encoded by Glycine max cDNA clones (AW832545, BG726819 and BG154493) and a Lycopersicon esculentum cDNA clone (BE451174). There was lower homology outside of the region.
G2557: G2557 (SEQ ID NO: 133) is a member of a clade of highly related HLH/MYC proteins that also includes G779 (SEQ ID NO: 113), G1063 (SEQ ID NO: 119), G1499 (SEQ ID NO: 7), and G2143 (SEQ ID NO: 129). All of these genes caused similar pleiotropic phenotypic effects when overexpressed, the most striking of which was the production of ectopic carpelloid tissue. These genes can be considered key regulators of carpel development. The flowers of 35S::G2557 primary transformants displayed patches of stigmatic papillae on the sepals, and often had rather narrow petals and poorly developed stamens. Additionally, carpels were also occasionally held outside of the flower at the end of an elongated pedicel like structure. As a result of such defects, 35S::G2557 plants often showed very poor fertility and formed small wrinkled siliques. In addition to such floral abnormalities, the majority of primary transformants were also small and darker green in coloration than wild type. Approximately one third of the T1 plants were extremely tiny and completely sterile. Three T1 lines (#7,9,12), that had produced some seeds, and showed a relatively weak phenotype, were chosen for further study. All three of the T2 populations from these lines contained plants that were distinctly small, had abnormal flowers, and were poorly fertile compared to controls. Stigmatic tissue was not noted on the sepals of plants from these three T2 lines. Another line (#4) that had shown a moderately strong phenotype in the T1 was sown for only morphological analysis in the T2 generation. T2-4 plants were small, dark green, and produced abnormal flowers with ectopic stigmatic tissue on the sepals, as had been seen in the parental plant. G2557 expression was detected at low to moderate levels in all tissues tested except shoots. It was induced by cold, heat, and salt, and repressed by pathogen infection. G1063 can be used to manipulate flower form and structure or plant fertility. One application for manipulation of flower structure can be in the production of saffron, which is derived from the stigmas of Crocus sativus.
Closely Related Genes from Other Species
G2557 protein shows extensive sequence similarity in the region of basic helix loop helix with a protein encoded by Glycine max cDNA clone (BE347811).
G2430: The complete sequence of G2430 (SEQ ID NO: 697) was determined. G2430 is a member of the response regulator class of GARP proteins (ARR genes), although one of the two conserved aspartate residues characteristic of response regulators is not present. The second aspartate, the putative phosphorylated site, is retained so G2430 can have response regulator function. G2430 is specifically expressed in embryo and silique tissue. In morphological analyses, plants overexpressing G2430 showed more rapid growth than control plants at early stages, and in two of three lines examined produced large, flat leaves. Early flowering was observed for some lines, but this effect was inconsistent between plantings. G2430 can regulate plant growth. Overexpression of G2430 in Arabidopsis also resulted in seedlings that are slightly more tolerant to heat in a germination assay. Seedlings from G2430 overexpressing transgenic plants were slightly greener than the control seedlings under high temperature conditions. In a repeat experiment on individual lines, G2430 line 15 showed the strongest heat tolerant phenotype. G2430 can be useful to promote faster development and reproduction in plants.
Closely Related Genes from Other Species
G2430 had some similarity within of the conserved GARP and response-regulator domains to non-Arabidopsis proteins.
G1478: The sequence of G1478 (SEQ ID NO: 831) was determined and G1478 was analyzed using transgenic plants in which G1478 was expressed under the control of the 35S promoter. Plants overexpressing G1478 had a general delay in progression through the life cycle, in particular a delay in flowering time. G1478 is expressed at higher levels in flowers, rosettes and embryos but otherwise expression is constitutive. Based on the phenotypes produced through G1478 overexpression, G1478 can be used to manipulate the rate at which plants grow, and flowering time.
Closely Related Genes from Other Species
G1478 shows some homology to non-Arabidopsis proteins within the conserved domain.
G681: G681 (SEQ ID NO: 579) was analyzed using transgenic plants in which the gene was expressed under the control of the 35S promoter. Approximately half of the 35S::G681 primary transformants were markedly small and formed narrow leaves compared to controls. These plants often produced thin inflorescence stems, had rather poorly formed flowers with low pollen production, and set few seeds. Three T1 lines with relatively weak phenotypes, which had produced reasonable quantities of seed, were selected for further study. Plants from one of the T2 populations were noted to be slightly small, but otherwise the T2 lines displayed no consistent differences in morphology from controls. In leaves of two of the T2 lines, overexpression of G681 resulted in an increase in the percentage of the glucosinolate M39480. According to RT-PCR analysis, G681 expression was detected at very low levels in flower and rosette leaf tissues. G681 was induced by drought stress. G681 can be used to alter glucosinolate composition in plants. Increases or decreases in specific glucosinolates or total glucosinolate content are desirable depending upon the particular application. For example: (1) Glucosinolates are undesirable components of the oilseeds used in animal feed, since they produce toxic effects. Low-glucosinolate varieties of canola have been developed to combat this problem. (2) Some glucosinolates have anti-cancer activity; thus, increasing the levels or composition of these compounds might be of interest from a nutraceutical standpoint. (3) Glucosinolates form part of a plants natural defense against insects. Modification of glucosinolate composition or quantity could therefore afford increased protection from predators. Furthermore, in edible crops, tissue specific promoters can be used to ensure that these compounds accumulate specifically in tissues, such as the epidermis, which are not taken for consumption.
Closely Related Genes from Other Species
G681 shows some sequence similarity with known genes from other plant species within the conserved Myb domain.
G878: G878 (SEQ ID NO: 611) was studied using transgenic plants in which the gene was expressed under the control of the 35S promoter. Analysis of primary transformants revealed that overexpression of G878 delays the onset of flowering in Arabidopsis. 11/20 of the 35S::G878 T1 plants flowered approximately one week later than wild type under continuous light conditions. These plants were also darker green, had shorter stems, and senesced later than controls. G878 was ubiquitously expressed. G878 can be used to modify flowering time and senescence, and a wide variety of applications exist for systems that either lengthen or shorten the time to flowering.
Closely Related Genes from Other Species
G878 was highly related to other WRKY proteins from a variety of plant species, such as the Nicotiana tabacum DNA-binding protein 2 (WRKY2) (AF096299), and a Cucumis sativus SPF1-like DNA-binding protein (L44134).
G374: G374 (SEQ ID NO: 47) was expressed at low levels throughout the plant and was induced by salicylic acid. G374 was investigated using lines carrying a T-DNA insertion in this gene. The T-DNA insertion was approximately three quarters of the way into the protein coding sequence and should result in a null mutation. Homozygosity for a T-DNA insertion within G374 caused lethality at early stages of embryo development. In an initial screen for G374 knockouts, heterozygous plants were identified. Seed from those individuals was sown to soil and eleven plants were PCR-screened to identify homozygotes. No homozygotes were obtained; 6 of the progeny were heterozygous whilst the other 5 were wild type. This raised the prospect that homozygosity for the G374 insertion was lethal. To examine this possibility further, heterozygous KO.G374 plants were re-grown. These individuals looked wild type, but their siliques were examined for seed abnormalities. When green siliques were dissected, around 25% of developing seeds were white or aborted. Embryos from these siliques were cleared using Hoyers solution, and examined under the microscope. It was apparent that embryos from the white seeds had arrested at early (globular or heart) stages of development, whilst embryos from the normal seeds were fully developed. Such arrested or aborted seeds most likely represented homozygotes for the G374 insertion. To support this conclusion, seed was collected from heterozygous plants and sown to kanamycin plates (the T-DNA insertion carried the NPT marker gene). Of the seedlings that germinated, 160 were kanamycin resistant and 107 were kanamycin sensitive. These data more closely fitted a 2:1 (chi-sq., 1df, =5.5, 0.05>P>0.01) than a 3:1 (chi-sq., 1df, =32, P<0.001) ratio. Such a segregation ratio suggested that a homozygous class of kanamycin resistant seedlings was absent from the progeny of KO.G374 plant. G374 can be a herbicide target.
Closely Related Genes from Other Species
Similar sequences to G374 are present in tomato and Medicago truncatula , and these sequences can be orthologs.
Example VIII
Identification of Homologous Sequences
Homologous sequences from Arabidopsis and plant species other than Arabidopsis were identified using database sequence search tools, such as the Basic Local Alignment Search Tool (BLAST) (Altschul et al. (1990) J. Mol. Biol. 215:403-410; and Altschul et al. (1997) Nucl. Acid Res. 25: 3389-3402). The tblastx sequence analysis programs were employed using the BLOSUM-62 scoring matrix (Henikoff, S, and Henikoff, J. G. (1992) Proc. Natl. Acad. Sci. USA 89: 10915-10919).
Identified non-Arabidopsis sequences homologous to the Arabidopsis sequences are provided in Table 4. The percent sequence identity among these sequences can be as low as 47%, or even lower sequence identity. The entire NCBI GenBank database was filtered for sequences from all plants except Arabidopsis thaliana by selecting all entries in the NCBI GenBank database associated with NCBI taxonomic ID 33090 (Viridiplantae; all plants) and excluding entries associated with taxonomic ID 3701 ( Arabidopsis thaliana ). These sequences are compared to sequences representing genes of SEQ IDs NOs:2-2N, where N=2-561, using the Washington University TBLASTX algorithm (version 2.0a19 MP) at the default settings using gapped alignments with the filter “off”. For each gene of SEQ IDs NOs:2-2N, where N=2-561, individual comparisons were ordered by probability score (P-value), where the score reflects the probability that a particular alignment occurred by chance. For example, a score of 3.6e-40 is 3.6×10 −40 . In addition to P-values, comparisons were also scored by percentage identity. Percentage identity reflects the degree to which two segments of DNA or protein are identical over a particular length. Examples of sequences so identified are presented in Table 4. Homologous or orthologous sequences are readily identified and available in GenBank by Accession number (Table 4; Test sequence ID) The identified homologous polynucleotide and polypeptide sequences and homologues of the Arabidopsis polynucleotides and polypeptides may be orthologs of the Arabidopsis polynucleotides and polypeptides. (TBD: to be determined.)
Example IX
Introduction of Polynucleotides into Dicotyledonous Plants
SEQ ID NOs:1-(2N−1), wherein N=2-561, paralogous, orthologous, and homologous sequences recombined into pMEN20 or pMEN65 expression vectors are transformed into a plant for the purpose of modifying plant traits. The cloning vector may be introduced into a variety of cereal plants by means well-known in the art such as, for example, direct DNA transfer or Agrobacterium tumefaciens -mediated transformation. It is now routine to produce transgenic plants using most dicot plants (see Weissbach and Weissbach, (1989) supra; Gelvin et al., (1990) supra; Herrera-Estrella et al. (1983) supra; Bevan (1984) supra; and Klee (1985) supra). Methods for analysis of traits are routine in the art and examples are disclosed above.
Example X
Transformation of Cereal Plants with an Expression Vector
Cereal plants such as corn, wheat, rice, sorghum or barley, may also be transformed with the present polynucleotide sequences in pMEN20 or pMEN65 expression vectors for the purpose of modifying plant traits. For example, pMEN020 may be modified to replace the NptII coding region with the BAR gene of Streptomyces hygroscopicus that confers resistance to phosphinothricin. The KpnI and BglII sites of the Bar gene are removed by site-directed mutagenesis with silent codon changes.
The cloning vector may be introduced into a variety of cereal plants by means well-known in the art such as, for example, direct DNA transfer or Agrobacterium tumefaciens -mediated transformation. It is now routine to produce transgenic plants of most cereal crops (Vasil, I., Plant Molec. Biol. 25: 925-937 (1994)) such as corn, wheat, rice, sorghum (Cassas, A. et al., Proc. Natl. Acad Sci USA 90: 11212-11216 (1993) and barley (Wan, Y. and Lemeaux, P. Plant Physiol. 104:37-48 (1994). DNA transfer methods such as the microprojectile can be used for corn (Fromm. et al. Bio/Technology 8: 833-839 (1990); Gordon-Kamm et al. Plant Cell 2: 603-618 (1990); Ishida, Y., Nature Biotechnology 14:745-750 (1990)), wheat (Vasil, et al. Bio/Technology 10:667-674 (1992); Vasil et al., Bio/Technology 11: 1553-1558 (1993); Weeks et al., Plant Physiol. 102:1077-1084 (1993)), rice (Christou Bio/Technology 9:957-962 (1991); Hiei et al. Plant J. 6:271-282 (1994); Aldemita and Hodges, Planta 199:612-617; Hiei et al., Plant Mol. Biol. 35:205-18 (1997)). For most cereal plants, embryogenic cells derived from immature scutellum tissues are the preferred cellular targets for transformation (Hiei et al., Plant Mol. Biol. 35:205-18 (1997); Vasil, Plant Molec. Biol. 25: 925-937 (1994)).
Vectors according to the present invention may be transformed into corn embryogenic cells derived from immature scutellar tissue by using microprojectile bombardment, with the A188XB73 genotype as the preferred genotype (Fromm, et al., Bio/Technology 8: 833-839 (1990); Gordon-Kamm et al., Plant Cell 2: 603-618 (1990)). After microprojectile bombardment the tissues are selected on phosphinothricin to identify the transgenic embryogenic cells (Gordon-Kamm et al., Plant Cell 2: 603-618 (1990)). Transgenic plants are regenerated by standard corn regeneration techniques (Fromm, et al., Bio/Technology 8: 833-839 (1990); Gordon-Kamm et al., Plant Cell 2: 603-618 (1990)).
The plasmids prepared as described above can also be used to produce transgenic wheat and rice plants (Christou, Bio/Technology 9:957-962 (1991); Hiei et al., Plant J. 6:271-282 (1994); Aldemita and Hodges, Planta 199:612-617 (1996); Hiei et al., Plant Mol. Biol. 35:205-18 (1997)) that coordinately express genes of interest by following standard transformation protocols known to those skilled in the art for rice and wheat Vasil, et al. Bio/Technology 10:667-674 (1992); Vasil et al., Bio/Technology 11:1553-1558 (1993); Weeks et al., Plant Physiol. 102:1077-1084 (1993)), where the bar gene is used as the selectable marker.
All references, publications, patent documents, web pages, and other documents cited or mentioned herein are hereby incorporated by reference in their entirety for all purposes. Although the invention has been described with reference to specific embodiments and examples, it should be understood that one of ordinary skill can make various modifications without departing from the spirit of the invention. The scope of the invention is not limited to the specific embodiments and examples provided.