# Patent

## Title
Transcription factor sequences for conferring advantageous properties to plants

## Abstract

The invention relates to plant homeobox (HB) protein family 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 or control plant, including increased yield.

## Background
RELATIONSHIP TO COPENDING APPLICATIONS
This application is a continuation-in-part application of prior U.S. application Ser. No. 11/375,241, filed Mar. 13, 2006 (pending); which claims the benefit of U.S. Provisional Application No. 60/713,952, filed Aug. 31, 2005; and U.S. application Ser. No. 11/375,241 is a continuation-in-part of prior U.S. application Ser. No. 10/225,067, filed Aug. 9, 2002 (issued as U.S. Pat. No. 7,135,616) which claims the benefit of U.S. Provisional Application No. 60/336,049, filed Nov. 19, 2001, U.S. Provisional Application No. 60/310,847, filed Aug. 9, 2001 and U.S. Provisional Application No. 60/338,692, filed Dec. 11, 2001; and, prior U.S. application Ser. No. 10/225,067 is a continuation-in-part of U.S. Non-provisional application Ser. No. 09/837,944, filed Apr. 18, 2001 (now abandoned), and U.S. Non-provisional application Ser. No. 10/171,468, filed Jun. 14, 2002 (now abandoned); and, this application is a continuation-in-part of prior U.S. application Ser. No. 10/714,887, filed Nov. 13, 2003 (pending); and, U.S. application Ser. No. 11/375,241 is a continuation-in-part of prior U.S. application Ser. No. 10/666,642, filed Sep. 18, 2003 (issued as U.S. Pat. No. 7,196,245) which claims the benefit of U.S. Provisional Application No. 60/465,809, filed Apr. 24, 2003, U.S. Provisional Application No. 60/434,166, filed Dec. 17, 2002 and U.S. Provisional Application No. 60/411,837, filed Sep. 18, 2002. This application is also a continuation-in-part of prior U.S. application Ser. No. 11/632,390, filed Dec. 17, 2008 (pending), which is a National Stage Entry of PCT/US2005/025010, filed Jul. 14, 2005, which claims the benefit of U.S. Provisional Application No. 60/588,405, filed Jul. 14, 2004. This application is also a continuation-in-part of prior U.S. application Ser. No. 12/338,024, filed Dec. 18, 2008 (pending), which is a divisional application of prior U.S. application Ser. No. 10/374,780, filed Feb. 25, 2003 (issued as U.S. Pat. No. 7,511,190), which is a continuation-in-part of prior U.S. application Ser. No. 09/713,994, filed Nov. 16, 2000 (abandoned); and, U.S. application Ser. No. 10/374,780 is also a continuation-in-part of prior U.S. application Ser. No. 09/934,455, filed Aug. 22, 2001 (abandoned), which is a continuation-in-part of prior U.S. application Ser. No. 09/713,994, filed Nov. 16, 2000 (abandoned); and, this application is continuation-in-part of prior U.S. application Ser. No. 11/479,226, filed Jun. 30, 2006 (pending), which is a continuation-in-part of prior U.S. application Ser. No. 09/713,994, filed Nov. 16, 2000 (abandoned). All of the above applications are hereby incorporated by reference in their entirety.


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, said agreement having been 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
Transgenic plants with improved traits, including enhanced yield, environmental stress tolerance, pest resistance, herbicide tolerance, improved seed compositions, and the like are desired by both farmers and consumers. Although considerable efforts in plant breeding have provided significant gains in desired traits, the ability to introduce specific DNA into plant genomes provides further opportunities for generation of plants with improved and/or unique traits. Fortunately, 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. Polynucleotides encoding transcription factors have been identified, transformed into transgenic plants, and the plants have been analyzed for a variety of important improved traits. In so doing, important polynucleotide and polypeptide sequences for producing commercially valuable plants and crops as well as the methods for making and using them were identified. In some cases, because of epigenetic effects, positional effects, or the like, introducing recombinant DNA into a plant genome does not result in a transgenic plant having the desired phenotype with the enhanced agronomic trait. Therefore, methods to select individual transgenic events from a population may be required to identify those transgenic events that are characterized by the enhanced agronomic trait.
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
The present invention pertains to transgenic plants, and methods for producing the transgenic plant, that have desirable characteristics relative to wild-type or control plants. The desirable characteristics in the transgenic plants, which have been transformed with a sequence that is closely or phylogenetically related to G47, polynucleotide SEQ ID NO: 65 and polypeptide SEQ ID NO: 66, include increased size and/or biomass, tolerance to osmotic stress or drought, and/or increased lignification. The transgenic plants may also be delayed in their flowering, relative to a control or wild-type plant of the same species. The transgenic plants are made by first producing an expression vector that comprises a nucleotide sequence encoding a polypeptide with a conserved domain, said domain having at least 69%, or at least 73%, or at least 80%, or at least 87% amino acid identity to the conserved domain of G47 (amino acid coordinates 11-80 of G47 or SEQ ID NO: 66). The expression vector is next introduced into a suitable target plant, and the polypeptide is overexpressed in this now transgenic plant. This results in the transgenic plant having increased size and/or biomass, tolerance to the osmotic stress or drought, delayed flowering, and/or increased lignification.
Methods for increasing plant size and/or biomass, increasing osmotic stress or drought tolerance of a plant, increasing lignin content, or causing a delay in development or flowering are also encompassed by the invention.



BRIEF DESCRIPTION OF THE SEQUENCE LISTING, TABLES, AND FIGURES
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.
Incorporation of the Sequence Listing.
The copy of the Sequence Listing, being submitted electronically with this patent application, provided under 37 CFR §1.821-1.825, is a read-only memory computer-readable file in ASCII text format. The Sequence Listing is named “MBI-0036-3CIP2-ST25.txt”, the electronic file of the Sequence Listing was created on Oct. 2, 2009, and is 527,112 bytes in size, or 515 kilobytes in size measured in MS-Windows. The Sequence Listing is herein incorporated by reference in its entirety.
FIG. 1 shows a phylogenic tree of related plant families adapted from Daly et al. (2001 Plant Physiology 127: 1328-1333).
FIG. 2 shows a phylogenic dendogram depicting phylogenetic relationships of higher plant taxa, including clades containing tomato and Arabidopsis; adapted from Ku et al. (2000) Proc. Natl. Acad. Sci. USA 97: 9121-9126; and Chase et al. (1993) Ann. Missouri Bot. Gard. 80: 528-580.
FIG. 3 shows a phylogenetic tree and multiple sequence alignments of G47 and related full length proteins were constructed using ClustalW (CLUSTAL W Multiple Sequence Alignment Program version 1.83, 2003) and MEGA2 (http://www.megasoftware.net) software. Sequences closely related to G47, SEQ ID NO: 66, fall within the G47 clade and descend from a common ancestral sequence represented by the arrow at an ancestral node of the tree. These phylogenetically-related sequences within the G47 clade that have thus far been shown to have a transcriptional regulatory activity of G47 by conferring similar morphological and physiological characteristics have conserved domains that are at least 69% identical to the conserved domain of G47 (amino acid coordinates 11-80). The percentage identity was determined by BLASTp analysis against a database containing G47 homologs, with default settings of 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: 10915-10919). ClustalW multiple alignment parameters for FIG. 3 were as follows:
Gap Opening Penalty: 10.00; Gap Extension Penalty: 0.20; Delay divergent sequences: 30%; DNA Transitions Weight: 0.50; Protein weight matrix: Gonnet series; DNA weight matrix: IUB; Use negative matrix: OFF.
A FastA formatted alignment was then used to generate a phylogenetic tree in MEGA2 using the neighbor joining algorithm and a p-distance model. A test of phylogeny was done via bootstrap with 1000 replications and Random Seed set to default. Cut off values of the bootstrap tree were set to 50%. Orthologs of G47 are considered as being those proteins within the node of the tree below with a bootstrap value of 93, bounded by G3644 and G47, as indicated by the sequences within the box.
FIG. 4 shows a Clustal W alignment of the AP2 domains of the G47 Glade and other representative AP2 proteins. The three residues indicated by the boxes define the G47 Glade; Glade members (indicated by the vertical line at left) have a two valines and a histidine residue at these positions, respectively. The SEQ ID NOs. of the sequences listed in FIG. 4 appear in parentheses.
FIG. 5 shows the conserved domain of G47 (SEQ ID NO: 66) aligned against the conserved domains of Arabidopsis paralog sequence G2133 (SEQ ID NO: 152; 62 of 71 or 87% identical residues) and three orthologs, soy G3643 (SEQ ID NO: 158; 45 of 65 or 69% of residues are identical), rice G3649 (SEQ ID NO: 154; 35 of 44 or 80% of residues identical) and rice G3644 (SEQ ID NO: 156; 35 of 48 or 73% of residues identical). Alignments and percentage identity were determined from BLASTp analysis in which the conserved domain of G47, amino acid coordinates 11-80, were queried against a database containing the G47 homologs, with default settings of a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff (1989) supra).
FIGS. 6A-6C show Arabidopsis G47, SEQ ID NO: 66 (FIG. 6A, plant at left), soy G3649, SEQ ID NO: 154 (FIG. 6B, plants at left and center), and rice G3643, SEQ ID NO: 158 (FIG. 6C, plants at left and center) overexpressors at 58, 44, and 33 days after planting, respectively. The overexpressors generally developed later, and some lines had larger rosettes and an increased amount of vegetative tissue compared to the control plants at the right of each photograph.
FIGS. 7A-7B compare seedlings ectopically expressing rice sequence G3644, SEQ ID NO: 156 (FIG. 7A) and wild-type seedling controls. The 35S::G3644 seedlings (FIG. 7A) were generally larger and greener after germination in a 150 mM NaCl than the wild-type control seedlings exposed to the same conditions (FIG. 7B). The small pale seedlings in FIG. 7A represent wild-type segregants, based on kanamycin resistance segregation data from the same population.
As shown in FIGS. 8A-8B, seedlings ectopically expressing rice sequence G3649, SEQ ID NO: 154 (FIG. 8A) were generally larger and greener after germination in a medium containing 0.3 μM abscisic acid than the wild-type control seedlings exposed to the same conditions (FIG. 8B).
FIG. 9 illustrates a dramatic example of osmotic-stress tolerance. Seedlings overexpressing Arabidopsis G2133, SEQ ID NO: 152, in the pot at the left were significantly greener and more vigorous than the wild-type control seedlings, seen at right, after both sets of plants had been exposed to the same severe drought conditions and rewatered. The overexpressors readily recovered from the severe treatment after resumption of watering, whereas the few control plants at right that survived had been severely and adversely affected by the drought treatment.



DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS
The present invention relates to polynucleotides and polypeptides for modifying phenotypes of plants, particularly those associated with increased biomass, increased disease resistance, and/or abiotic stress tolerance. 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. While the reference to these information sources clearly indicates that they can be used by one of skill in the art, each and every one of the information sources cited herein are specifically incorporated 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.
As used herein and in the appended claims, the singular forms “a”, “an”, and “the” include the plural reference unless the context clearly dictates otherwise. Thus, for example, a reference to “a host cell” includes a plurality of such host cells, 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.
DEFINITIONS
“Nucleic acid molecule” refers to an oligonucleotide, polynucleotide or any fragment thereof. It may be DNA or RNA of genomic or synthetic origin, double-stranded or single-stranded, and combined with carbohydrate, lipids, protein, or other materials to perform a particular activity such as transformation or form a useful composition such as a peptide nucleic acid (PNA).
“Polynucleotide” is a nucleic acid molecule comprising a plurality of polymerized nucleotides, e.g., at least about 15 consecutive polymerized nucleotides. A polynucleotide may be a nucleic acid, oligonucleotide, nucleotide, or any fragment thereof. 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 be combined with carbohydrate, lipids, protein, or other materials to perform a particular activity such as transformation or form a useful composition such as a peptide nucleic acid (PNA). The polynucleotide can comprise a sequence in either sense or antisense orientations. “Oligonucleotide” is substantially equivalent to the terms amplimer, primer, oligomer, element, target, and probe and is preferably single-stranded.
“Gene” or “gene sequence” refers to the partial or complete coding sequence of a gene, its complement, and its 5′ or 3′ untranslated regions. A gene is also a functional unit of inheritance, and in physical terms is a particular segment or sequence of nucleotides along a molecule of DNA (or RNA, in the case of RNA viruses) involved in producing a polypeptide chain. The latter may be subjected to subsequent processing such as chemical modification or folding to obtain a functional protein or polypeptide. A gene may be isolated, partially isolated, or found with an organism's genome. By way of example, a transcription factor gene encodes a transcription factor polypeptide, which may be functional or require processing to function as an initiator of transcription.
Operationally, genes may be defined by the cis-trans test, a genetic test that determines whether two mutations occur in the same gene and that may be used to determine the limits of the genetically active unit (Rieger et al. (1976)). A gene generally includes regions preceding (“leaders”; upstream) and following (“trailers”; downstream) the coding region. A gene may also include intervening, non-coding sequences, referred to as “introns”, located between individual coding segments, referred to as “exons”. Most genes have an associated promoter region, a regulatory sequence 5′ of the transcription initiation codon (there are some genes that do not have an identifiable promoter). The function of a gene may also be regulated by enhancers, operators, and other regulatory elements.
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. 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: (i) a localization domain; (ii) an activation domain; (iii) a repression domain; (iv) an oligomerization domain; (v) 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.
“Protein” refers to an amino acid sequence, oligopeptide, peptide, polypeptide or portions thereof whether naturally occurring or synthetic.
“Portion”, as used herein, refers to any part of a protein used for any purpose, but especially for the screening of a library of molecules which specifically bind to that portion or for the production of antibodies.
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.
“Homology” refers to sequence similarity between a reference sequence and at least a fragment of a newly sequenced clone insert or its encoded amino acid sequence.
“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. “Sequence similarity” refers to the percent similarity in base pair sequence (as determined by any suitable method) between two or more polynucleotide sequences. Two or more sequences can be anywhere from 0-100% similar, or any integer value therebetween. 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, matching or corresponding 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 corresponding positions shared by the polypeptide sequences. A degree of homology or similarity of polypeptide sequences is a function of the number of amino acids at corresponding positions shared by the polypeptide sequences.
“Alignment” refers to a number of nucleotide bases or amino acid residue sequences aligned by lengthwise comparison so that components in common (i.e., nucleotide bases or amino acid residues at corresponding positions) may be visually and readily identified. The fraction or percentage of components in common is related to the homology or identity between the sequences. Alignments such as those of FIG. 4 or FIG. 5 may be used to identify conserved domains and relatedness within these domains. An alignment may suitably be determined by means of computer programs known in the art, such as MACVECTOR software (1999) (Accelrys, Inc., San Diego, Calif.).
A “conserved domain” or “conserved region” as used herein refers to a region in heterologous polynucleotide or polypeptide sequences where there is a relatively high degree of sequence identity between the distinct sequences. For example, an “AT-hook” domain”, such as is found in a polypeptide member of AT-hook transcription factor family, is an example of a conserved domain. An “AP2” domain”, such as is found in a polypeptide member of AP2 transcription factor family, is another example of a conserved domain. With respect to polynucleotides encoding presently disclosed transcription factors, a conserved domain is preferably at least nine base pairs (bp) in length. A conserved domain (for example, a DNA binding domain) with respect to presently disclosed polypeptides refers to a domain that exhibits at least about 38% sequence identity, or at least about 55% sequence identity, or at least about 62% sequence identity, or at least about 69%, or at least about 70%, or at least about 73%, or at least about 76%, or at least about 78%, or at least about 80%, or at least about 82%, or at least about 85%, or at least about 87%, or at least about 89%, or at least about 95%, amino acid residue sequence identity, to a conserved domain of a polypeptide of the invention. Sequences that possess or encode for conserved domains that meet these criteria of percentage identity, and may have comparable biological activity to the present transcription factor sequences. This is particularly true for sequences that derive from a common ancestral sequence that had the same or similar function, and for which the function has been retained. These sequences, which are closely and phylogenetically related, being members of a particular clade of transcription factor polypeptides, are encompassed by the invention. A fragment or domain can be referred to as outside a conserved domain, 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. Sequences having lesser degrees of identity but comparable biological activity are considered to be equivalents.
As one of ordinary skill in the art recognizes, conserved domains may be identified as regions or domains of identity to a specific consensus sequence (see, for example, Riechmann et al. (2000) Science 290: 2105-2110, Riechmann et al. (2000b) Curr. Opin. Plant Biol. 3: 423-434). Thus, by using alignment methods well known in the art, the conserved domains of the plant transcription factors, for example, for the AT-hook proteins (Reeves and Beckerbauer (2001) Biochim. Biophys. Acta 1519: 13-29; and Reeves (2001) Gene 277: 63-81), may be determined.
The conserved domains for many of the transcription factor sequences of the invention are listed in Table 4. A comparison of the regions of these polypeptides allows one of skill in the art (see, for example, Reeves and Nissen (1995) Prog. Cell Cycle Res. 1: 339-349) to identify domains or conserved domains for any of the polypeptides listed or referred to in this disclosure.
“Complementary” refers to the natural hydrogen bonding by base pairing between purines and pyrimidines. For example, the sequence A-C-G-T (5′->3′) forms hydrogen bonds with its complements A-C-G-T (5′->3′) or A-C-G-U (5′->3′). Two single-stranded molecules may be considered partially complementary, if only some of the nucleotides bond, or “completely complementary” if all of the nucleotides bond. The degree of complementarity between nucleic acid strands affects the efficiency and strength of hybridization and amplification reactions. “Fully complementary” refers to the case where bonding occurs between every base pair and its complement in a pair of sequences, and the two sequences have the same number of nucleotides.
The terms “highly stringent” or “highly stringent condition” refer to conditions that permit hybridization of DNA strands whose sequences are highly complementary, wherein these same conditions exclude hybridization of significantly mismatched DNAs. Polynucleotide sequences capable of hybridizing under stringent conditions with the polynucleotides of the present invention may be, for example, variants of the disclosed polynucleotide sequences, including allelic or splice variants, or sequences that encode orthologs or paralogs of presently disclosed polypeptides. Nucleic acid hybridization methods are disclosed in detail by Kashima et al. (1985) Nature 313: 402-404, Sambrook et al. (1989) Molecular Cloning—A Laboratory Manual (2nd Ed.), Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989 (“Sambrook”), and by Haymes et al. (1985) Nucleic Acid Hybridization: A Practical Approach, IRL Press, Washington, D.C., which references are incorporated herein by reference.
In general, stringency is determined by the temperature, ionic strength, and concentration of denaturing agents (e.g., formamide) used in a hybridization and washing procedure (for a more detailed description of establishing and determining stringency, see the section “Identifying Polynucleotides or Nucleic Acids by Hybridization”, below). The degree to which two nucleic acids hybridize under various conditions of stringency is correlated with the extent of their similarity. Thus, similar nucleic acid sequences from a variety of sources, such as within a plant's genome (as in the case of paralogs) or from another plant (as in the case of orthologs) that may perform similar functions can be isolated on the basis of their ability to hybridize with known transcription factor sequences. Numerous variations are possible in the conditions and means by which nucleic acid hybridization can be performed to isolate transcription factor sequences having similarity to transcription factor sequences known in the art and are not limited to those explicitly disclosed herein. Such an approach may be used to isolate polynucleotide sequences having various degrees of similarity with disclosed transcription factor sequences, such as, for example, encoded transcription factors having 38% or greater identity with the conserved domain of disclosed transcription factors.
The terms “paralog” and “ortholog” are defined below in the section entitled “Orthologs and Paralogs”. In brief, orthologs and paralogs are evolutionarily related genes that have similar sequences and functions. Orthologs are structurally related genes in different species that are derived by a speciation event. Paralogs are structurally related genes within a single species that are derived by a duplication event.
The term “equivalog” describes members of a set of homologous proteins that are conserved with respect to function since their last common ancestor. Related proteins are grouped into equivalog families, and otherwise into protein families with other hierarchically defined homology types. This definition is provided at the Institute for Genomic Research (TIGR) World Wide Web (www) website, “tigr.org” under the heading “Terms associated with TIGRFAMs”.
In general, the term “variant” refers to molecules with some differences, generated synthetically or naturally, in their base or amino acid sequences as compared to a reference (native) polynucleotide or polypeptide, respectively. These differences include substitutions, insertions, deletions or any desired combinations of such changes in a native polynucleotide of amino acid sequence.
With regard to polynucleotide variants, differences between presently disclosed polynucleotides and polynucleotide variants are limited so that the nucleotide sequences of the former and the latter are closely similar overall and, in many regions, identical. Due to the degeneracy of the genetic code, differences between the former and latter nucleotide sequences may be silent (i.e., the amino acids encoded by the polynucleotide are the same, and the variant polynucleotide sequence encodes the same amino acid sequence as the presently disclosed polynucleotide. Variant nucleotide sequences may encode different amino acid sequences, in which case such nucleotide differences will result in amino acid substitutions, additions, deletions, insertions, truncations or fusions with respect to the similar disclosed polynucleotide sequences. These variations may result in polynucleotide variants encoding polypeptides that share at least one functional characteristic. The degeneracy of the genetic code also dictates that many different variant polynucleotides can encode identical and/or substantially similar polypeptides in addition to those sequences illustrated in the Sequence Listing.
Also within the scope of the invention is a variant of a transcription factor nucleic acid listed in the Sequence Listing, that is, one having a sequence that differs from the one of the polynucleotide sequences in the Sequence Listing, or a complementary sequence, that encodes a functionally equivalent polypeptide (i.e., a polypeptide having some degree of equivalent or similar biological activity) but differs in sequence from the sequence in the Sequence Listing, due to degeneracy in the genetic code. 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.
“Allelic variant” or “polynucleotide 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 may be “silent” or may encode polypeptides having altered amino acid sequence. “Allelic variant” and “polypeptide allelic variant” may also be used with respect to polypeptides, and in this case the terms refer to a polypeptide encoded by an allelic variant of a gene.
“Splice variant” or “polynucleotide splice variant” as used herein refers to alternative forms of RNA transcribed from a gene. Splice variation naturally occurs as a result of alternative sites being spliced within a single transcribed RNA molecule or between separately transcribed RNA molecules, and may result in several different forms of mRNA transcribed from the same gene. Thus, splice variants may encode polypeptides having different amino acid sequences, which may or may not have similar functions in the organism. “Splice variant” or “polypeptide splice variant” may also refer to a polypeptide encoded by a splice variant of a transcribed mRNA.
As used herein, “polynucleotide variants” may also refer to polynucleotide sequences that encode paralogs and orthologs of the presently disclosed polypeptide sequences. “Polypeptide variants” may refer to polypeptide sequences that are paralogs and orthologs of the presently disclosed polypeptide sequences.
Differences between presently disclosed polypeptides and polypeptide variants are limited so that the sequences of the former and the latter are closely similar overall and, in many regions, identical. Presently disclosed polypeptide sequences and similar polypeptide variants may differ in amino acid sequence by one or more substitutions, additions, deletions, fusions and truncations, which may be present in any combination. These differences may produce silent changes and result in a functionally equivalent transcription factor. Thus, it will be readily appreciated by those of skill in the art, that any of a variety of polynucleotide sequences is capable of encoding the transcription factors and transcription factor homolog polypeptides of the invention. A polypeptide sequence variant may have “conservative” changes, wherein a substituted amino acid has similar structural or chemical properties. Deliberate amino acid substitutions may thus be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues, as long as a significant amount of the functional or biological activity of the transcription factor 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. More rarely, a variant may have “non-conservative” changes, e.g., replacement of a glycine with a tryptophan. Similar minor variations may also include amino acid deletions or insertions, or both. Related polypeptides may comprise, for example, additions and/or deletions of one or more N-linked or O-linked glycosylation sites, or an addition and/or a deletion of one or more cysteine residues. Guidance in determining which and how many amino acid residues may be substituted, inserted or deleted without abolishing functional or biological activity may be found using computer programs well known in the art, for example, DNASTAR software (see U.S. Pat. No. 5,840,544).
“Fragment”, with respect to a polynucleotide, refers to a clone or any part of a polynucleotide molecule that retains a usable, functional characteristic. Useful fragments include oligonucleotides and polynucleotides that may be used in hybridization or amplification technologies or in the regulation of replication, transcription or translation. A “polynucleotide fragment” refers to any subsequence of a polynucleotide, typically, of at least about 9 consecutive nucleotides, preferably at least about 30 nucleotides, more preferably at least about 50 nucleotides, of any of the sequences provided herein. Exemplary polynucleotide fragments are the first sixty consecutive nucleotides of the transcription factor polynucleotides listed in the Sequence Listing. Exemplary fragments also include fragments that comprise a region that encodes an conserved domain of a transcription factor. Exemplary fragments also include fragments that comprise a conserved domain of a transcription factor. Exemplary fragments include fragments that comprise an conserved domain of a transcription factor, for example, amino acid residues 11-80 of G47 (SEQ ID NO: 66).
Fragments may also include subsequences of polypeptides and protein molecules, or a subsequence of the polypeptide. Fragments may have uses in that they may have antigenic potential. 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, and may initiate transcription. Fragments can vary in size from as few as 3 amino acid residues to the full length of the intact polypeptide, but are preferably at least about 30 amino acid residues in length and more preferably at least about 60 amino acid residues in length.
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.
“Derivative” refers to the chemical modification of a nucleic acid molecule or amino acid sequence. Chemical modifications can include replacement of hydrogen by an alkyl, acyl, or amino group or glycosylation, pegylation, or any similar process that retains or enhances biological activity or lifespan of the molecule or sequence.
The term “plant” includes whole plants, shoot vegetative organs/structures (for example, leaves, stems and tubers), roots, flowers and floral organs/structures (for example, bracts, sepals, petals, stamens, carpels, anthers and ovules), seed (including embryo, endosperm, and seed coat) and fruit (the mature ovary), plant tissue (for example, vascular tissue, ground tissue, and the like) and cells (for example, 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) supra, FIG. 2, adapted from Ku et al. (2000) supra; and see also Tudge (2000) in The Variety of Life, Oxford University Press, New York, N.Y. pp. 547-606.
A “control plant” as used in the present invention refers to a plant cell, seed, plant component, plant tissue, plant organ or whole plant used to compare against transgenic or genetically modified plant for the purpose of identifying an enhanced phenotype in the transgenic or genetically modified plant. A control plant may in some cases be a transgenic plant line that comprises an empty vector or marker gene, but does not contain the recombinant polynucleotide of the present invention that is expressed in the transgenic or genetically modified plant being evaluated. In general, a control plant is a plant of the same line or variety as the transgenic or genetically modified plant being tested. A suitable control plant would include a genetically unaltered or non-transgenic plant of the parental line used to generate a transgenic plant herein.
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 controlled 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.
“Wild type” or “wild-type”, as used herein, refers to a plant cell, seed, plant component, plant tissue, plant organ or whole plant that has not been genetically modified or treated in an experimental sense. Wild-type cells, seed, components, tissue, organs or whole plants may be used as controls to compare levels of expression and the extent and nature of trait modification with cells, tissue or plants of the same species in which a transcription factor expression is altered, e.g., in that it has been knocked out, overexpressed, or ectopically expressed.
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 tolerance to water deprivation or particular salt or sugar concentrations, 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 hyperosmotic stress tolerance or yield. 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.
As used herein an “enhanced trait” means a characteristic of a transgenic plant that includes, but is not limited to, an enhance agronomic trait characterized by enhanced plant morphology, physiology, growth and development, yield, nutritional enhancement, disease or pest resistance, or environmental or chemical tolerance. In more specific aspects of this invention enhanced trait is selected from group of enhanced traits consisting of enhanced water use efficiency, enhanced cold tolerance, increased yield, enhanced nitrogen use efficiency, enhanced seed protein and enhanced seed oil. In an important aspect of the invention the enhanced trait is enhanced yield including increased yield under non-stress conditions and increased yield under environmental stress conditions. Stress conditions may include, for example, drought, shade, fungal disease, viral disease, bacterial disease, insect infestation, nematode infestation, cold temperature exposure, heat exposure, osmotic stress, reduced nitrogen nutrient availability, reduced phosphorus nutrient availability and high plant density. “Yield” can be affected by many properties including without limitation, plant height, pod number, pod position on the plant, number of internodes, incidence of pod shatter, grain size, efficiency of nodulation and nitrogen fixation, efficiency of nutrient assimilation, resistance to biotic and abiotic stress, carbon assimilation, plant architecture, resistance to lodging, percent seed germination, seedling vigor, and juvenile traits. Yield can also affected by efficiency of germination (including germination in stressed conditions), growth rate (including growth rate in stressed conditions), ear number, seed number per ear, seed size, composition of seed (starch, oil, protein) and characteristics of seed fill.
Increased yield of a transgenic plant of the present invention can be measured in a number of ways, including plant volume, plant biomass, test weight, seed number per plant, seed weight, seed number per unit area (i.e. seeds, or weight of seeds, per acre), bushels per acre (bu/a), tonnes per acre, tons per acre, and/or kilo per hectare. For example, maize yield may be measured as production of shelled corn kernels per unit of production area, for example in bushels per acre or metric tons per hectare, often reported on a moisture adjusted basis, for example at 15.5 percent moisture. Increased yield may result from improved utilization of key biochemical compounds, such as nitrogen, phosphorous and carbohydrate, or from improved responses to environmental stresses, such as cold, heat, drought, salt, and attack by pests or pathogens. Recombinant DNA used in this invention can also be used to provide plants having improved growth and development, and ultimately increased yield, as the result of modified expression of plant growth regulators or modification of cell cycle or photosynthesis pathways. Also of interest is the generation of transgenic plants that demonstrate enhanced yield with respect to a seed component that may or may not correspond to an increase in overall plant yield. Such properties include enhancements in seed oil, seed molecules such as tocopherol, protein and starch, or oil particular oil components as may be manifest by an alteration in the ratios of seed components.
“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, or an even greater difference, in an observed trait as compared with a control or 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 and magnitude of the trait in the plants as compared to control or wild-type plants.
When two or more plants have “similar morphologies”, “substantially similar morphologies”, “a morphology that is substantially similar”, or are “morphologically similar”, the plants have comparable forms or appearances, including analogous features such as overall dimensions, height, width, mass, root mass, shape, glossiness, color, stem diameter, leaf size, leaf dimension, leaf density, internode distance, branching, root branching, number and form of inflorescences, and other macroscopic characteristics, and the individual plants are not readily distinguishable based on morphological characteristics alone.
“Modulates” refers to a change in activity (biological, chemical, or immunological) or lifespan resulting from specific binding between a molecule and either a nucleic acid molecule or a protein.
The term “transcript profile” refers to the expression levels of a set of genes in a cell in a particular state, particularly by comparison with the expression levels of that same set of genes in a cell of the same type in a reference state. For example, the transcript profile of a particular transcription factor in a suspension cell is the expression levels of a set of genes in a cell knocking out or overexpressing that transcription factor compared with the expression levels of that same set of genes in a suspension cell that has normal levels of that transcription factor. The transcript profile can be presented as a list of those genes whose expression level is significantly different between the two treatments, and the difference ratios. Differences and similarities between expression levels may also be evaluated and calculated using statistical and clustering methods.
With regard to transcription factor gene knockouts as used herein, the term “knockout” refers to a plant or plant cell having a disruption in at least one transcription factor gene in the plant or cell, where the disruption results in a reduced expression or activity of the transcription factor encoded by that gene compared to a control cell. The knockout can be the result of, for example, genomic disruptions, including transposons, tilling, and homologous recombination, antisense constructs, sense constructs, RNA silencing constructs, or RNA interference. A T-DNA insertion within a transcription factor gene is an example of a genotypic alteration that may abolish expression of that transcription factor gene.
“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.
The term “overexpression” as used herein refers to a greater expression level of a gene in a plant, plant cell or plant tissue, compared to expression in a wild-type plant, cell or tissue, at any developmental or temporal stage for the gene. Overexpression can occur when, for example, the genes encoding one or more transcription factors are under the control of a strong promoter (e.g., the cauliflower mosaic virus 35S transcription initiation region). Overexpression may also under the control of an inducible or tissue specific promoter. Thus, overexpression may occur throughout a plant, in specific tissues of the plant, or in the presence or absence of particular environmental signals, depending on the promoter used.
Overexpression may take place in plant cells normally lacking expression of polypeptides functionally equivalent or identical to the present transcription factors. Overexpression may also occur in plant cells where endogenous expression of the present transcription factors or functionally equivalent molecules normally occurs, but such normal expression is at a lower level. Overexpression thus results in a greater than normal production, or “overproduction” of the transcription factor in the plant, cell or tissue.
The term “transcription regulating region” refers to a DNA regulatory sequence that regulates expression of one or more genes in a plant when a transcription factor having one or more specific binding domains binds to the DNA regulatory sequence. Transcription factors of the present invention possess an conserved domain. The transcription factors of the invention also comprise an amino acid subsequence that forms a transcription activation domain that regulates expression of one or more abiotic stress tolerance genes in a plant when the transcription factor binds to the regulating region.
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 Develop. 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, which 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. (2001) supra.)
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.
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 4. 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 under-expression 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. (2000a) supra). 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 (ENBib; 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); Takatsuji (1998) Cell. Mol. Life. Sci. 54: 582-596); the homeobox (HB) protein family (Buerglin (1994) in Guidebook to the Homeobox Genes, Duboule (ed.) 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 (Abel et al. (1995) J. Mol. Biol. 251: 533-549); 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 (Goodrich et al. (1997) Nature 386: 44-51); 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 GARP 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 (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. (1989) Virology 168: 320-329), the PCF family (Cubas et al. Plant J. (1999) 18: 215-22), the SRS (SHI-related) family (Fridborg et al. (1999) Plant Cell 11: 1019-1032), the CPP (cysteine-rich polycomb-like) family (Cvitanich et al. (2000) Proc. Natl. Acad. Sci. 97: 8163-8168), the ARF (auxin response factor) family (Ulmasov et al. (1999) Proc. Natl. Acad. Sci. 96: 5844-5849), the SWI/SNF family (Collingwood et al. (1999) J. Mol. Endocrinol. 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: 733-742), 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, protein methyltransferases, 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.
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. supra, 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), Qβ-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.
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, cassava, 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 clade (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 clade 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. Transcription factor gene sequences are thus conserved across diverse eukaryotic species lines (Goodrich et al. (1993) Cell 75: 519-530; Lin et al. (1991) Nature 353: 569-571; Sadowski et al. (1988) Nature 335: 563-564). Plants are no exception to this observation; diverse plant species possess transcription factors that have similar sequences and functions. It is well known in the art that protein function can be classified using phylogenetic analysis of gene trees combined with the corresponding species. Functional predictions can be greatly improved by focusing on how the genes became similar in sequence (i.e., evolution) rather than on the sequence similarity itself (Eisen, (1998) Genome Res. 8: 163-167): “[t]he first step in making functional predictions is the generation of a phylogenetic tree representing the evolutionary history of the gene of interest and its homologs. Such trees are distinct from clusters and other means of characterizing sequence similarity because they are inferred by techniques that help convert patterns of similarity into evolutionary relationships . . . . After the gene tree is inferred, biologically determined functions of the various homologs are overlaid onto the tree. Finally, the structure of the tree and the relative phylogenetic positions of genes of different functions are used to trace the history of functional changes, which is then used to predict functions of [as yet] uncharacterized genes” (Eisen, supra). Thus, 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 be 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. It is then a matter of routine to align sequences that are most closely related by virtue of their presence in a related clade (e.g., a group of sequences descending from a strong node of a phylogenetic tree representing a common ancestral sequence) using BLAST or similar analysis, or compare similarity or identity of the amino acid residues of these sequences and/or their conserved domains or motifs that confer and correlate with conserved function.
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 (for example, a DNA binding domain) within a transcription factor family may exhibit a high degree of sequence homology, such as at least about at least about 65%, or at least about 69%, or at least about 70%, or at least about 73%, or at least about 76%, or at least about 78%, or at least about 80%, or at least about 82%, or at least about 85%, or at least about 87%, or at least about 89%, or at least about 95%, amino acid residue sequence identity, to a conserved domain of a transcription factor polypeptide of the invention listed in the Sequence Listing. 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 and 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 (Shpaer (1997) Methods Mol. Biol. 70: 173-187). 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 (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 (1993) J. Mol. Evol. 36: 290-300; Altschul et al. (1990) supra), BLOCKS (Henikoff and Henikoff (1991) Nucl. Acids Res. 19: 6565-6572), Hidden Markov Models (HMM; Eddy (1996) Curr. 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 α helices, β-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 polynucleotide sequences, listed in the Sequence Listing; and fragments, thereof under various conditions of stringency. (See, e.g., Wahl and 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 Table 4, 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 (Tm) for the specific sequence at a defined ionic strength and pH. The Tm 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.
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 G47, SEQ ID NO: 66, represents a single transcription factor; allelic variation and alternative splicing may be expected to occur. Allelic variants of SEQ ID NO: 65 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: 65, 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: 66. 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.
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).
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.
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. 126: 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) Plant Mol. Biol. 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.
The following represent specific examples of expression constructs used to overexpress sequences of the invention. The choice of promoters may include, for example, the constitutive CaMV 35S promoter, the STM shoot apical meristem-specific promoter, the CUT1 epidermal-specific promoter, the LTP1 epidermal-specific promoter, the SUC2 vascular-specific promoter, the RBCS3 leaf-specific promoter, the ARSK1 root-specific promoter, the RD29A stress inducible promoter, the AP1 floral meristem-specific promoter (SEQ ID NO: 209-217, respectively). Many of these examples have been used to produce transgenic plants. These or other inducible or tissue-specific promoters may be incorporated into an expression vector comprising a transcription factor polynucleotide of the invention, where the promoter is operably linked to the transcription factor polynucleotide, can be envisioned and produced.
P894 (SEQ ID NO: 218) contained a 35S::G47 direct fusion and carries KanR. The construct contains a G47 cDNA clone.
An alternative means of overexpressing G47 makes use of the two constructs P6506 (SEQ ID NO: 233; 35S::LexA-GAL4TA) and P3853 (SEQ ID NO: 224; opLexA::G47), which together constituted a two-component system for expression of G47 from the 35S promoter. A kanamycin resistant transgenic line containing P6506 was established, and this was then supertransformed with the P3853 construct containing a cDNA clone of G47 and a sulfonamide resistance marker.
P1572 (SEQ ID NO: 219) comprised a 35S::G2133 direct promoter fusion and carries KanR. The construct contains a cDNA clone of G2133
P23456 (SEQ ID NO: 220) contained a 35S::G3649 direct promoter fusion and carries KanR. The construct contains a cDNA clone of G3649.
P23455 (SEQ ID NO: 221) contained a 35S::G3644 direct promoter fusion and carries KanR. The construct contains a cDNA clone of G3644.
P23465 (SEQ ID NO: 222) contained a 35S::G3643 direct fusion and carries KanR. The construct harbors a cDNA clone of G3643.
P25402 (SEQ ID NO: 223) contained a 35S::G3650 direct fusion and carries KanR. The construct contains a cDNA clone.
The two constructs P5318 (SEQ ID NO: 225; STM::LexA-GAL4TA) and P3853 (SEQ ID NO: 224; opLexA::G47) together constitute a two-component system for expression of G47 from the STM promoter. Kanamycin resistant transgenic lines containing P5318 were established (lines #5 and #10), and these were then supertransformed with the P3853 construct containing a cDNA clone of G47 and a sulfonamide resistance marker.
The two constructs P5288 (SEQ ID NO: 226; CUT1::LexA-GAL4TA) and P3853 (SEQ ID NO: 224; opLexA::G47) together constitute a two-component system for expression of G47 from the CUT1 promoter. A kanamycin resistant transgenic line containing P5288 was established, and this was then supertransformed with the P3853 construct containing a cDNA clone of G47 and a sulfonamide resistance marker.
The two constructs P5284 (SEQ ID NO: 235; RBCS3::LexA-GAL4TA) and P3853 (SEQ ID NO: 224; opLexA::G47) together constituted a two-component system for expression of G47 from the RBCS3 promoter. A kanamycin resistant transgenic line containing P5284 was established, and this was then supertransformed with the P3853 construct containing a cDNA clone of G47 and a sulfonamide resistance marker.
The two constructs P5290 (SEQ ID NO: 234; SUC2::LexA-GAL4TA) and P3853 (SEQ ID NO: 224; opLexA::G47) together constitute a two-component system for expression of G47 from the SUC2 promoter. A kanamycin resistant transgenic line containing P5290 was established, and this was then supertransformed with the P3853 construct containing a cDNA clone of G47 and a sulfonamide resistance marker.
The two constructs P5311 (SEQ ID NO: 236; ARSK1::LexA-GAL4TA) and P3853 (SEQ ID NO: 224; opLexA::G47) together constitute a two-component system for expression of G47 from the ARSK1 promoter. A kanamycin resistant transgenic line containing P5311 was established, and this was then supertransformed with the P3853 construct containing a cDNA clone of G47 and a sulfonamide resistance marker.
The two constructs P9002 (SEQ ID NO: 237; RD29A::LexA-GAL4TA) and P3853 (SEQ ID NO: 224; opLexA::G47) together constitute a two-component system for expression of G47 from the RD29A promoter. A kanamycin resistant transgenic line (#5) containing P9002 was established, and this was then supertransformed with the P3853 construct containing a cDNA clone of G47 and a sulfonamide resistance marker.
The two constructs P5326 (SEQ ID NO: 238; AP1::LexA-GAL4TA) and P3853 (SEQ ID NO: 224; opLexA::G47) together constitute a two-component system for expression of G47 from the AP1 promoter. A kanamycin resistant transgenic line containing P5326 was established, and this was then supertransformed with the P3853 construct containing a cDNA clone of G47 and a sulfonamide resistance marker.
P25186 (SEQ ID NO: 239) contains a 35S::GAL4-G47 fusion and carries KanR (addition to the G47 protein of a strong transcription activation domain from the yeast GAL4 gene). SEQ ID NO: 240 is the predicted polypeptide that results expression of the vector comprising SEQ ID NO: 239.
P25279 (SEQ ID NO: 241) carries a 35S::G47-GFP fusion directly fused to the 35S promoter and a KanR marker. SEQ ID NO: 242 is the predicted polypeptide that results expression of the vector comprising SEQ ID NO: 239.
Similar to constructs made with G47, other vectors may be produced that incorporate a promoter and other transcription factor polynucleotide combination. For example, the two constructs P9002 (SEQ ID NO: 237; RD29A::LexA-GAL4TA) and P4361 (SEQ ID NO: 227; opLexA::G2133) together constitute a two-component system for expression of G2133 from the RD29A promoter. A kanamycin resistant transgenic line containing P9002 was established, and this was then supertransformed with the P4361 construct containing a cDNA clone of G2133 and a sulfonamide resistance marker.
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: 5824-5828, 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: 4803-4807).
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.
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.
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 phenotype 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 Biotechnol. 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.
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 Biotechnol., 14: 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 Chem. Eng. News 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.
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.
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, N.J., 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 Develop. 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. 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.
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 Tables 4 and 6.
The first column of Table 4 shows the polynucleotide SEQ ID NO; the second column shows the Mendel Gene ID No., GID; the third column shows the transcription factor family to which the polynucleotide belongs; the fourth column shows the category of the trait; the fifth column shows the trait(s) resulting from the knock out or overexpression of the polynucleotide in the transgenic plant; 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.
The first column (Col. 1) of Table 4 lists the SEQ ID NO: of presently disclosed polynucleotide sequences. The second column lists the corresponding GID number. The third column shows the transcription factor family in which each of the respective sequences is found. The fourth column lists the conserved domains in amino acid coordinates of the respective encoded polypeptide sequences. The fifth and sixth columns list the trait category and specific traits observed for plants overexpressing the respective sequences (except where noted as “KO” in Col. 2 for plants in which the respective sequence was knocked out).







TABLE 4







Sequences of the invention and the traits they confer in plants












Col. 1


Col. 4




SEQ ID
Col. 2
Col. 3
Conserved
Col. 5
Col. 6


NO:
GID No.
Family
domains
Trait Category
Observed trait(s)















1
G1272
PAZ
800-837 
Seed glucosinolates
Decrease in seed







glucosinolate M39497


3
G1506
GATA/Zn
7-33
Seed glucosinolates
Increase in glucosinolates







M39502 and M39498


5
G1897
Z-Dof
34-62 
Seed glucosinolates
Increase in seed







glucosinolates M39491







and M39493


7
G1946
HS
37-128
Seed glucosinolates
Increase in seed







glucosinolate M39501







Increased tolerance to







phosphate-free media


9
G2113
AP2
55-122
Seed glucosinolates
Decrease in seed







glucosinolate M39497,







increase of glucosinolates







M39501, M39494 and







M39478


11
G2117
bZIP
46-106
Seed glucosinolates
Decrease in M39496


13
G2155
AT-hook
18-38 
Seed glucosinolates
Increase in M39497






Plant size
Large plant size


15
G2290
WRKY
147-205 
Seed glucosinolates
Increase in M39496


17
G2340
MYB-
14-120
Seed glucosinolates
Altered glucosinolate




(R1)R2R3


profile


21
G353
Z-C2H2
41-61,
Seed glucosinolates
Increase in M39494





84-104


23
G484
CAAT
11-104
Seed glucosinolates
Altered glucosinolate



(KO)



profile


25
G674
MYB-
20-120
Seed glucosinolates
Increase in M39501




(R1)R2R3


27
G1052
bZIP
201-261 
Seed prenyl lipids
Decrease in lutein and







increase in xanthophyll 1


29
G1328
MYB-
14-119
Seed prenyl lipids
Decreased seed lutein




(R1)R2R3


31
G1930
AP2
59-124,
Seed prenyl lipids
Increased chlorophyll a





179-273

and b content






C/N sensing
Increased tolerance to







low nitrogen conditions







in C/N sensing assay


33
G214
MYB-related
25-71 
Seed prenyl lipids;
Increased seed lutein;






leaf fatty acids;
increased leaf fatty acids;






prenyl lipids
increased chlorophyll,







carotenoids






Plant size
Larger biomass







(increased leaf number







and size






Prenyl lipids
Darker green in







vegetative and







reproductive tissues due







to a higher chlorophyll







content in the later stages







of development


35
G2509
AP2
89-156
Seed prenyl lipids
Increase in α-tocopherol


37
G2520
HLH/MYC
139-197 
Seed prenyl lipids;
Increase in seed δ-






leaf glucosinolates
tocopherol and decrease







in seed γ-tocopherol.;







increase in M39478






C/N sensing
Increased tolerance to







low nitrogen conditions







in C/N sensing assay


39
G259
HS
40-131
Seed prenyl lipids
Increase in α-tocopherol


41
G490
CAAT
48-143
Seed prenyl lipids
Increase in seed δ-







tocopherol


43
G652
Z-CLDSH
28-49,
Seed prenyl lipids;
Increase in α-tocopherol;





137-151,
leaf glucosinolates
increase in M39480





182-196


45
G748
Z-Dof
112-140 
Seed prenyl lipids
Increased lutein content


47
G883
WRKY
245-302 
Seed prenyl lipids
Decreased seed lutein


49
G20
AP2
68-144
Seed sterols
Increase in campesterol


51
G974
AP2
80-147
Seed oil content
Altered seed oil content


53
G2343
MYB-
14-116
Seed oil content
Increased seed oil content




(R1)R2R3


55
G1777
RING/C3HC4
124-247 
Seed oil and protein
Increased seed oil content






content
and decreased seed







protein


57
G229
MYB-
14-120
Biochemistry: other
Up-regulation of genes




(R1)R2R3


involved in secondary







metabolism; Genes







coding for enzymes







involved in alkaloid







biosynthesis including







indole-3-glycerol







phosphatase and







strictosidine synthase







were induced; genes for







enzymes involved in







aromatic amino acid







biosynthesis were also







up-regulated including







tryptophan synthase and







tyrosine transaminase.







Phenylalanine ammonia







lyase, chalcone synthase







and trans-cinnamate







mono-oxygenase,







involved in







phenylpropenoid







biosynthesis, were also







induced


59
G663
MYB-
 9-111
Biochemistry: other
Increased anthocyanins in




(R1)R2R3


leaf, root, seed


61
G362
Z-C2H2
62-82 
Biochemistry: other
Increased trichome







density and trichome







products; increased







anthocyanins in various







tissues


63
G2105
TH
100-153 
Biochemistry: other
Increased trichome







density and trichome







products


65
G47
AP2
11-80 
Flowering Time
Increased lignin content






Biochemistry: other
Increased cold tolerance






Abiotic stress
Increased drought






tolerance
tolerance







Increased desiccation







tolerance







Increased salt tolerance







Late flowering







Dark green







Increased leaf size, larger







rosettes and/or increased







amount of vegetative







tissue


67
G2123
GF14
99-109
Biochemistry: other
Putative 14-3-3 protein


69
G1266
AP2
79-147
Leaf fatty acids,
Changes in leaf fatty






insoluble sugars;
acids, insoluble sugars,







decreased sensitivity to







ABA






C/N sensing
Increased tolerance to







low nitrogen conditions







in C/N sensing assay


71
G1337
Z-CO-like
9-75
Leaf fatty acids
Increase in the amount of






Sugar sensing
oleic acid







Decreased tolerance to







sucrose


73
G1399
AT-hook
86-93 
Leaf fatty acids
Increase of the percentage







of the 16:0 fatty acid


75
G1465
NAC
242-306 
Leaf fatty acids
Increases in the







percentages of 16:0, 16:1,







18:0 and 18:2 and







decreases in 16:3 and







18:3 fatty acids


77
G1512
RING/C3HC4
39-93 
Leaf fatty acids
Increase in 18:2 fatty







acids


79
G1537
HB
14-74 
Leaf fatty acids
Altered leaf fatty acid







composition


81
G2136
MADS
43-100
Leaf fatty acids
Decrease in 18:3 fatty







acid


83
G2147
HLH/MYC
163-220 
Leaf fatty acids
Increase in 16:0, increase







in 18:2 fatty acids


85
G377
RING/C3H2C3
85-128
Leaf fatty acids
Increased 18:2 and







decreased 18:3 leaf fatty







acids


87
G962
NAC
53-175
Leaf fatty acids
Increased 16:0 and







decreased 18:3 leaf fatty







acids


89
G975
AP2
4-71
Leaf fatty acids
Increased wax in leaves







Increased C29, C31, and







C33 alkanes increased up







to 10-fold compared to







control plants;







More drought tolerant







than controls






C/N sensing
Increased tolerance to







low nitrogen conditions







in C/N sensing assay


91
G987
SCR
395-462,
Leaf fatty acids; leaf
Reduction in 16:3 fatty





525-613,
prenyl lipids
acids; altered chlorophyll,





1027-1102,

tocopherol, carotenoid





1162-1255


93
G1069
AT-hook
67-74 
Leaf and seed
Altered leaf glucosinolate






glucosinolates
composition







Increased seed







glucosinolate M39497







Increased 16:0 fatty acid,







decreased 18:2 fatty acids,







decreased sensitivity to







ABA






C/N sensing
Increased tolerance to







low nitrogen conditions







in C/N sensing assay


95
G1198
bZIP
173-223 
Leaf glucosinolates
Increase in M39481


97
G1322
MYB-
26-130
Leaf glucosinolates
Increase in M39480




(R1)R2R3

C/N sensing
Increased tolerance to







low nitrogen conditions







in C/N sensing assay


99
G1421
AP2
74-151
Leaf glucosinolates
Increased leaf content of







glucosinolate M39482


101
G1794
AP2
182-249 
Leaf glucosinolates
Increased leaf content of







glucosinolate M39480


103
G2144
HLH/MYC
207-265 
Leaf glucosinolates
Increased leaf content of







glucosinolate M39480






C/N sensing
Increased tolerance to







low nitrogen conditions







in C/N sensing assay


105
G2512
AP2
79-147
Leaf glucosinolates
Increased leaf content of







glucosinolate M39481






C/N sensing
Increased tolerance to







low nitrogen conditions







in C/N sensing assay


107
G2552
HLH/MYC
124-181 
Leaf glucosinolates
Increased leaf content of







glucosinolate M39480


109
G264
HS
23-114
Leaf glucosinolates
Increased leaf content of







glucosinolate M39481


111
G681
MYB-
14-120
Leaf glucosinolates
Increased leaf content of




(R1)R2R3


glucosinolate M39480


113
G1012
WRKY
30-86 
Leaf insoluble sugars
Decreased rhamnose


115
G1309
MYB-
 9-114
Leaf insoluble sugars
Increased mannose




(R1)R2R3


117
G158
MADS
2-57
Leaf insoluble sugars
Increased rhamnose


119
G1641
MYB-related
32-82,
Leaf insoluble sugars
Increased rhamnose





141-189


121
G1865
GRF-like
45-162
Leaf insoluble sugars
Increased galactose,







decreased xylose


123
G2094
GATA/Zn
43-68 
Leaf insoluble sugars
Increase in arabinose


125
G211
MYB-
24-137
Leaf insoluble sugars
Increase in xylose




(R1)R2R3


127
G242
MYB-
 6-105
Leaf insoluble sugars
Increased arabinose




(R1)R2R3


129
G2589
MADS
1-57
Leaf insoluble sugars
Increase in arabinose


131
G274
AKR
94-600
Leaf insoluble sugars
Increased leaf arabinose


133
G598
DBP
205-263 
Leaf insoluble sugars
Altered insoluble sugars;







(increased galactose







levels)


135
G1543
HB
135-195 
Leaf prenyl lipids
Increase in chlorophyll a







and b







Increased biomass


137
G280
AT-hook
97-104, 130-
Leaf prenyl lipids
Increased δ- and γ-





137-155-162,

tocopherol





185-192


139
G2131
AP2
50-121,
Leaf sterols
Increase in campesterol





146-217 
C/N sensing
Increased tolerance to







low nitrogen conditions







in C/N sensing assay


141
G2424
MYB-
107-219 
Leaf sterols
Increase in stigmastanol




(R1)R2R3


143
G2583
AP2
4-71
Leaf wax
Glossy leaves, increased






Flowering time
epicuticular wax content







or altered composition







Late developing, late







flowering time


147
G977
AP2
5-72
Leaf wax
Altered epicuticular wax







content or composition


151
G2133
AP2
11-82 
Flowering Time
Increased cold tolerance






Biochemistry: other
Increased drought






Abiotic stress
tolerance






tolerance
Increased desiccation







tolerance







Increased salt tolerance







Late flowering







Dark green







Increased leaf size and/or







larger rosette







Increased seed size


157
G3643
AP2
14-79 
Flowering Time
Increased cold tolerance






Biochemistry: other
Increased drought






Abiotic stress
tolerance






tolerance
Increased desiccation







tolerance







Increased heat tolerance







Late flowering







Dark green







Larger plants


155
G3644
AP2
55-102
Flowering Time
Increased salt tolerance






Biochemistry: other
Late flowering






Abiotic stress
Dark green






tolerance
Large seedlings







Large rosettes with long,







broad leaves


153
G3649
AP2
18-61 
Flowering Time
Increased cold tolerance






Biochemistry: other
Increased drought






Abiotic stress
tolerance







Increased desiccation







tolerance







Decreased heat tolerance






tolerance
Late flowering







Dark green







Larger rosettes







Large cauline leaves


145
G1387
AP2
4-68

Few lines of







overexpressors have been







produced or examined


149
G4294
AP2
5-72

Overexpressors not yet







produced or examined





Abbreviations: KO—knockout






Table 5 lists a summary of orthologous and homologous sequences identified using BLAST (tblastx program). The first column shows the polynucleotide sequence identifier (SEQ ID NO), the second column shows the corresponding cDNA identifier (Gene ID), the third column shows the orthologous or homologous polynucleotide GenBank Accession Number (Test Sequence ID), the fourth column shows the calculated probability value that the sequence identity is due to chance (Smallest Sum Probability), the fifth column shows the plant species from which the test sequence was isolated (Test Sequence Species), and the sixth column shows the orthologous or homologous test sequence GenBank annotation (Test Sequence GenBank Annotation).
Of the identified sequences homologous to the Arabidopsis sequences provided in Table 5, 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 those listed in the Sequence Listing, using the Washington University TBLASTX algorithm (version 2.0a19MP) at the default settings using gapped alignments with the filter “off”. For each sequence listed in the Sequence Listing, 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. The identified homologous polynucleotide and polypeptide sequences and homologs of the Arabidopsis polynucleotides and polypeptides may be orthologs of the Arabidopsis polynucleotides and polypeptides and/or closely, phylogenetically-related sequences.







TABLE 5







Sequences phylogenetically related to Arabidopsis sequences shown to confer useful traits in plants













Col. 2
Col. 3





Col. 1
GID or Related
Related to
Col. 4
Col. 5
Col. 6


SEQ ID
Sequence Identifier
GID/SEQ ID
Smallest Sum
Species from which
Test Sequence


NO
(Accession No.)
NO
Probability
Sequence is Derived
GenBank Annotation















19
G671
G2340/17
1.0E−103
Arabidopsis thaliana




BG269414
G2340/17
1.60E−45
Mesembryanthemum
L0-3478T3 Ice plant






crystallinum
Lambda Un



BG448527
G2340/17
5.30E−41
Medicago truncatula
NF036F04RT1F1032







Developing root







Medica



AI730649
G2340/17
1.10E−40
Gossypium hirsutum
BNLGHi7595 Six-







day Cotton fiber







Gossypiu



AW706006
G2340/17
1.20E−39
Glycine max
sk64f05.yl Gm-







c1016 Glycine max







cDNA clone GENO



PHMYBPH31
G2340/17
1.60E−39
Petunia × hybrida
P.hybrida myb.Ph3







gene encoding protein



AI491024
G2340/17
4.10E−39
Lycopersicon
EST241733 tomato






esculentum
shoot, Cornell Lyc



AMMIXTA
G2340/17
1.40E−38
Antirrhinum majus
A.majus mixta







mRNA.



OSMYB1355
G2340/17
2.40E−38
Oryza sativa
O.sativa mRNA for







myb factor, 1355 bp.



BE495300
G2340/17
2.80E−37
Secale cereale
WHE1268_F02_K04ZS







Secale cereale







anther cDNA



BG300704
G2340/17
4.70E−36
Hordeum vulgare
HVSMEb0018B03f







Hordeum vulgare







seedling sho



gi2605617
G2340/17
1.50E−44
Oryza sativa
OSMYB1.



gi20563
G2340/17
7.30E−42
Petunia × hybrida
protein 1.



gi485867
G2340/17
4.00E−41
Antirrhinum majus
mixta.



gi437327
G2340/17
2.00E−39
Gossypium hirsutum
MYB A; putative.



gi19051
G2340/17
3.10E−39
Hordeum vulgare
MybHv1.



gi227030
G2340/17
3.10E−39
Hordeum vulgare
myb-related gene






var. distichum
Hv1.



gi1101770
G2340/17
6.40E−38
Picea mariana
MYB-like







transcriptional factor







MBF1.



gi1430846
G2340/17
6.30E−36
Lycopersicon
myb-related






esculentum
transcription factor.



gi5139814
G2340/17
2.50E−35
Glycine max
GmMYB29B2.



gi6651292
G2340/17
1.70E−34
Pimpinella
myb-related






brachycarpa
transcription factor.


257
G1752
G1266/69
1E−60
Arabidopsis thaliana


258
G2512
G1266/69
5E−41
Arabidopsis thaliana


259
G5185
G1266/69
7E−34
Zea mays


260
G5183
G1266/69
7E−39
Oryza sativa


261
G5184
G1266/69
1E−60
Glycine max


262
G5186
G1266/69
7E−38
Glycine max


263
G3972
G1266/69
2E−40
Nicotiana tabacum


264
G3973
G1266/69
2E−56
Nicotiana tabacum


265
G3979
G1266/69
1E−57
Solanum






lycopersicum


266
G5170
G1266/69
5E−39
Solanum






lycopersicum


145
G1387
G2583/143
6.00E−72
Arabidopsis thaliana


89
G975
G2583/143
3.00E−56
Arabidopsis thaliana


149
G4294
G2583/143
2.00E−49
Oryza sativa



AW928465
G2583/143
1.40E−43
Lycopersicon
EST337253 tomato






esculentum
flower buds 8 mm t



BE023297
G2583/143
2.40E−42
Glycine max
sm80e10.y1 Gm-







c1015 Glycine max







cDNA clone GENO



AP003615
G2583/143
1.60E−30
Oryza sativa
chromosome 6 clone







P0486H12, ***







SEQUENCING IN



AU088998
G2583/143
2.90E−21
Lotus japonicus
AU088998 Lotus







japonicus flower bud







cDNA Lo



AT001828
G2583/143
4.60E−20
Brassica rapa subsp.
AT001828 Flower






pekinensis
bud cDNA Br



BG415973
G2583/143
2.40E−18
Hordeum vulgare
HVSMEk0009E06f







Hordeum vulgare







testa/perica



BF647090
G2583/143
3.80E−17
Medicago truncatula
NF007A06EC1F1038







Elicited cell culture



BG560598
G2583/143
2.90E−16
Sorghum
RHIZ2_59_D07.b1_A003






propinquum
Rhizome2







(RHIZ2) So



AW011200
G2583/143
6.60E−16
Pinus taeda
ST17H08 Pine







TriplEx shoot tip







library Pinus ta



BF479478
G2583/143
1.60E−15
Mesembryanthemum
L48-3155T3 Ice plant






crystallinum
Lambda U



gi19507
G2583/143
1.40E−16
Lupinus polyphyllus
put. pPLZ2 product







(AA 1-164).



gi10798644
G2583/143
1.00E−12
Nicotiana tabacum
AP2 domain-







containing







transcription fac



gi8571476
G2583/143
4.70E−12
Atriplex hortensis
apetala2 domain-







containing protein.



gi2213783
G2583/143
8.40E−12
Lycopersicon
Pti5.






esculentum



gi8809573
G2583/143
5.30E−11
Nicotiana sylvestris
ethylene-responsive







element binding



gi4099914
G2583/143
8.40E−11
Stylosanthes hamata
ethylene-responsive







element binding p



gi6478845
G2583/143
8.90E−11
Matricaria
ethylene-responsive






chamomilla
element binding



gi15290041
G2583/143
9.40E−11
Oryza sativa
hypothetical protein.



gi12225884
G2583/143
1.70E−10
Zea mays
unnamed protein







product.



gi3264767
G2583/143
3.40E−10
Prunus armeniaca
AP2 domain







containing protein.


242
G361
G362/61
7.0Ee−17
Arabidopsis thaliana


244
G2826
G362/61
5.0E−14
Arabidopsis thaliana


246
G2838
G362/61
2.0E−12
Arabidopsis thaliana


248
G1995
G362/61
5.0E−10
Arabidopsis thaliana


250
G370
G362/61
5.0E−10
Arabidopsis thaliana



BG581135
G362/61
1.70E−19
Medicago truncatula
EST482865 GVN







Medicago truncatula







cDNA



BI206903
G362/61
7.70E−18
Lycopersicon
EST524943 cTOS






esculentum
Lycopersicon esculen



BG047435
G362/61
7.30E−17
Glycine max
saa71c12.y1 Gm-







c1060 Glycine max







cDNA clone GEN



AP003214
G362/61
3.00E−12
Oryza sativa
chromosome 1 clone







OSJNBa0083M16,







*** SEQUENCI



BE366047
G362/61
6.40E−12
Sorghum bicolor
PI1_30_G05.b2_A002







Pathogen induced 1







(PI1)



BF616974
G362/61
1.90E−05
Hordeum vulgare
HVSMEc0014C08f







Hordeum vulgare







seedling sho



BG444243
G362/61
3.70E−05
Gossypium arboreum
GA_Ea0023L22f







Gossypium arboreum







7-10 d



BE500265
G362/61
0.00015
Triticum aestivum
WHE0981_F11_L20ZS







Wheat pre-anthesis







spik



AB006604
G362/61
0.00023
Petunia × hybrida
mRNA for ZPT2-9,







complete cds.



AI163084
G362/61
0.0004
Populus tremula ×
A031p65u Hybrid






Populus tremuloides
aspen



gi15528588
G362/61
4.20E−15
Oryza sativa
hypothetical protein.



gi2346984
G362/61
3.80E−08
Petunia × hybrida
ZPT2-9.



gi7228329
G362/61
0.012
Medicago sativa
putative TFIIIA (or







kruppel)-like zinc fi



gi1763063
G362/61
0.016
Glycine max
SCOF-1.



gi485814
G362/61
0.026
Triticum aestivum
WZF1.



gi4666360
G362/61
0.03
Datisca glomerata
zinc-finger protein 1.



gi2058504
G362/61
0.079
Brassica rapa
zinc-finger protein-1.



gi861091
G362/61
0.08
Pisum sativum
putative zinc finger







protein.



gi2981169
G362/61
0.42
Nicotiana tabacum
osmotic stress-







induced zinc-finger







prot



BM110736
G2105/63
3.70E−45
Solanum tuberosum
EST558272 potato







roots Solanum







tuberosum



BF646615
G2105/63
6.60E−36
Medicago truncatula
NF066C08EC1F1065







Elicited cell culture



AB052729
G2105/63
9.50E−30
Pisum sativum
mRNA for DNA-







binding protein DF1,







complete cd



OSJN00022
G2105/63
1.10E−26
Oryza sativa
chromosome 4 clone







OSJNBa0011L07, ***







SEQUENC



AI777252
G2105/63
4.20E−25
Lycopersicon
EST258217 tomato






esculentum
resistant, Cornell



BM500043
G2105/63
6.70E−24
Zea mays
952036C09.y1 952 -







BMS tissue from







Walbot Lab (red



AP004839
G2105/63
1.90E−23
Oryza sativa
( ) chromosome 2 clo






(japonica cultivar-






group)



AW596787
G2105/63
2.30E−20
Glycine max
sj16f10.y1 Gm-c1032







Glycine max cDNA







clone GENO



AV410715
G2105/63
8.70E−20
Lotus japonicus
AV410715 Lotus







japonicus young







plants (two-



BM357046
G2105/63
3.10E−14
Triphysaria
16I-G5 Triphysaria






versicolor
versicolor root-t



gi13646986
G2105/63
7.50E−32
Pisum sativum
DNA-binding protein







DF1.



gi20249
G2105/63
1.30E−27
Oryza sativa
gt-2.



gi18182311
G2105/63
8.20E−22
Glycine max
GT-2 factor.



gi8096269
G2105/63
0.24
Nicotiana tabacum
KED.


167
G3645
G47/65
9.0E−64
Brassica rapa subsp.






Pekinensis


151
G2133
G47/65
1.0E−47
Arabidopsis thaliana


165
G3646
G47/65
2.0E−46
Brassica oleracea


163
G3647
G47/65
2.0E−33
Zinnia elegans


157
G3643
G47/65
1.0E−29
Glycine max


155
G3644
G47/65
9.0Ee−26
Oryza sativa






(japonica cultivar-






group)


159
G3650
G47/65
1.0E−23
Zea mays


153
G3649
G47/65
1.0E−23
Oryza sativa






(japonica cultivar-






group)


161
G3651
G47/65
9.0E−21
Oryza sativa






(japonica cultivar-






group)



BE320193
G47/65
5.90E−23
Medicago truncatula
NF024B04RT1F1029







Developing root







Medica



AP003379
G47/65
8.90E−20
Oryza sativa
chromosome 1 clone







P0408G07, ***







SEQUENCING IN



AW220454
G47/65
7.90E−16
Lycopersicon
EST302937 tomato






esculentum
root during/after



BI434553
G47/65
8.90E−16
Solanum tuberosum
EST537314 P.







infestans-challenged







leaf So



BF610198
G47/65
1.30E−15
Pinus taeda
NXSI_055_H04_F







NXSI (Nsf Xylem







Side wood Inclin



BE659994
G47/65
2.50E−15
Glycine max
4-G2 GmaxSC







Glycine max cDNA,







mRNA sequence.



BG446456
G47/65
5.00E−15
Gossypium arboreum
GA_Eb0034M18f







Gossypium arboreum







7-10 d



BG321374
G47/65
1.10E−14
Descurainia sophia
Ds01_06d08_R







Ds01_AAFC_ECORC—







cold_stress



AI728590
G47/65
2.40E−14
Gossypium hirsutum
BNLGHi11133 Six-







day Cotton fiber







Gossypi



gi14140155
G47/65
2.90E−16
Oryza sativa
putative AP2 domain







transcription factor.



gi5616086
G47/65
7.90E−14
Brassica napus
dehydration







responsive element







binding pro



gi12225916
G47/65
8.70E−14
Zea mays
unnamed protein







product.



gi8571476
G47/65
1.30E−13
Atriplex hortensis
apetala2 domain-







containing protein.



gi8980313
G47/65
9.00E−13
Catharanthus roseus
AP2-domain DNA-







binding protein.



gi6478845
G47/65
5.00E−12
Matricaria
ethylene-responsive






chamomilla
element binding



gi1208498
G47/65
6.40E−12
Nicotiana tabacum
EREBP-2.



gi8809573
G47/65
2.20E−11
Nicotiana sylvestris
ethylene-responsive







element binding



gi7528276
G47/65
3.40E−11
Mesembryanthemum
AP2-related






crystallinum
transcription f



gi3342211
G47/65
4.50E−11
Lycopersicon
Pti4.






esculentum


149
G4294
G975/89
2.0E−65
Oryza sativa


143
G2583
G975/89
3.0E−56
Arabidopsis thaliana


145
G1387
G975/89
5.0E−54
Arabidopsis thaliana



AP003615
G975/89
1.10E−51
Oryza sativa
chromosome 6 clone







P0486H12, ***







SEQUENCING IN



BG642554
G975/89
1.10E−50
Lycopersicon
EST356031 tomato






esculentum
flower buds, anthe



AW705973
G975/89
3.20E−45
Glycine max
sk64c02.y1 Gm-







c1016 Glycine max







cDNA clone GENO



AT001828
G975/89
4.80E−34
Brassica rapa subsp.
AT001828 Flower






pekinensis
bud cDNA Br



BG415973
G975/89
3.70E−29
Hordeum vulgare
HVSMEk0009E06f







Hordeum vulgare







testa/perica



AU088998
G975/89
2.10E−27
Lotus japonicus
AU088998 Lotus







japonicus flower bud







cDNA Lo



AL377839
G975/89
8.40E−21
Medicago truncalula
MtBB34C04F1







MtBB Medicago







truncatula cD



BF479478
G975/89
2.20E−18
Mesembryanthemum
L48-3155T3 Ice plant






crystallinum
Lambda U



BG560598
G975/89
3.40E−18
Sorghum
RHIZ2_59_D07.b1_A






propinquum
003 Rhizome2







(RHIZ2) So



L46408
G975/89
5.90E−18
Brassica rapa
BNAF1258 Mustard







flower buds Brassica







rapa cD



gi19507
G975/89
2.10E−19
Lupinus polyphyllus
put. pPLZ2 product







(AA 1-164).



gi2213783
G975/89
1.80E−15
Lycopersicon
Pti5.






esculentum



gi8571476
G975/89
2.80E−14
Atriplex hortensis
apetala2 domain-







containing protein.



gi4099914
G975/89
7.90E−14
Stylosanthes hamata
ethylene-responsive







element binding p



gi6478845
G975/89
3.40E−13
Matricaria
ethylene-responsive






chamomilla
element binding



gi12225884
G975/89
5.70E−13
Zea mays
unnamed protein







product.



gi8809573
G975/89
7.00E−13
Nicotiana sylvestris
ethylene-responsive







element binding



gi15290041
G975/89
1.20E−12
Oryza sativa
hypothetical protein.



gi8980313
G975/89
1.20E−12
Catharanthus roseus
AP2-domain DNA-







binding protein.



gi7528276
G975/89
1.30E−12
Mesembryanthemum
AP2-related






crystallinum
transcription f


252
G680
G214/33
1.0E−116
Arabidopsis thaliana



AW979367
G214/33
4.40E−35
Lycopersicon
EST310415 tomato






esculentum
root deficiency, C



BG156656
G214/33
1.80E−33
Glycine max
sab31d11.y1 Gm-







c1026 Glycine max







cDNA clone GEN



BE597638
G214/33
5.40E−28
Sorghum bicolor
PI1_72_C05.b1_A002







Pathogen induced 1







(PI1)



BI272895
G214/33
5.70E−26
Medicago truncatula
NF091A11FL1F1084







Developing flower







Medi



BE129981
G214/33
3.90E−23
Zea mays
945034C05.X1 945 -







Mixed adult tissues







from Walbot



BF889434
G214/33
7.50E−14
Oryza sativa
EST003 Magnaporthe







grisea infected 16-







day-old



gi15528628
G214/33
7.40E−14
Oryza sativa
hypothetical







protein~similar to







Oryza sativa



gi7677132
G214/33
0.41
Secale cereale
c-myb-like







transcription factor.



gi13676413
G214/33
0.43
Glycine max
hypothetical protein.



gi12406993
G214/33
0.57
Hordeum vulgare
MCB1 protein.



gi940288
G214/33
0.85
Pisum sativum
protein localized in







the nucleoli of pea nu



gi1279563
G214/33
0.92
Medicago sativa
nuM1.



gi12005328
G214/33
0.98
Hevea brasiliensis
unknown.



gi7688744
G214/33
0.99
Lycopersicon
asc1.






esculentum



gi1070004
G214/33
0.99
Brassica napus
Biotin carboxyl







carrier protein.



gi5326994
G214/33
1
Daucus carota
DNA topoisomerase







I.


254
G5
G974/51
1.0E−76
Arabidopsis thaliana



BI421315
G974/51
7.10E−54
Lycopersicon
EST531981 tomato






esculentum
callus, TAMU Lycop



AI966402
G974/51
9.40E−47
Glycine max
sc38e09.y1 Gm-







c1014 Glycine max







cDNA clone GENO



AF274033
G974/51
1.70E−43
Atriplex hortensis
apetala2 domain-







containing protein







mRNA,



BG592917
G974/51
8.40E−43
Solanum tuberosum
EST491595 cSTS







Solanum tuberosum







cDNA clo



AI166481
G974/51
6.20E−42
Populus balsamifera
xylem.est.309 Poplar






subsp. trichocarpa



AW776927
G974/51
2.10E−41
Medicago truncatula
EST335992 DSIL







Medicago truncatula







cDNA



AP004119
G974/51
2.70E−41
Oryza sativa
chromosome 2 clone







OJ1288_G09, ***







SEQUENCING



BE918036
G974/51
6.60E−38
Sorghum bicolor
OV1_1-B03.b1_A002







Ovary 1 (OV1)







Sorghum bic



gi8571476
G974/51
7.00E−45
Atriplex hortensis
apetala2 domain-







containing protein.



gi14140155
G974/51
4.40E−20
Oryza sativa
putative AP2 domain







transcription factor.



gi3342211
G974/51
9.10E−20
Lycopersicon
Pti4.






esculentum



gi1208498
G974/51
1.50E−19
Nicotiana tabacum
EREBP-2.



gi12225884
G974/51
1.50E−19
Zea mays
unnamed protein







product.



gi7528276
G974/51
3.90E−19
Mesembryanthemum
AP2-related






crystallinum
transcription f



gi8809571
G974/51
3.90E−19
Nicotiana sylvestris
ethylene-responsive







element binding



gi1688233
G974/51
3.50E−18
Solanum tuberosum
DNA binding protein







homolog.



gi3264767
G974/51
9.40E−18
Prunus armeniaca
AP2 domain







containing protein.



gi6478845
G974/51
2.00E−17
Matricaria
ethylene-responsive






chamomilla
element binding



BI311137
G2343/53
4.00E−45
Medicago truncatula
EST5312887 GESD







Medicago truncatula







cDN



BG130765
G2343/53
5.10E−45
Lycopersicon
EST463657 tomato






esculentum
crown gall Lycoper



AW672062
G2343/53
2.30E−44
Sorghum bicolor
LG1_354_G05.b1_A002







Light Grown 1







(LG1) Sor



AV421932
G2343/53
2.70E−42
Lotus japonicus
AV421932 Lotus







japonicus young







plants (two-



BE611938
G2343/53
9.10E−42
Glycine max
sr01h04.y1 Gm-







c1049 Glycine max







cDNA clone GENO



BF484214
G2343/53
1.90E−37
Triticum aestivum
WHE2309_F07_K13ZS







Wheat pre-anthesis







spik



BG301022
G2343/53
4.30E−35
Hordeum vulgare
HVSMEb0019E16f







Hordeum vulgare







seedling sho



AP003018
G2343/53
3.20E−34
Oryza sativa
genomic DNA,







chromosome 1, BAC







clone: OSJNBa000



BE495300
G2343/53
3.30E−34
Secale cereale
WHE1268_F02_K04ZS







Secale cereale







anther cDNA



AI657290
G2343/53
3.50E−34
Zea mays
486093A08.y1 486 -







leaf primordia cDNA







library fro



gi1167486
G2343/53
9.50E−53
Lycopersicon
transcription factor.






esculentum



gi13366181
G2343/53
1.30E−48
Oryza sativa
putative transcription







factor.



gi2130045
G2343/53
1.50E−37
Hordeum vulgare
MybHv33 protein -







barley.



gi82310
G2343/53
1.60E−34
Antirrhinum majus
myb protein 330 -







garden snapdragon.



gi1732247
G2343/53
4.20E−34
Nicotiana tabacum
transcription factor







Myb1.



gi1841475
G2343/53
7.80E−33
Pisum sativum
Myb26.



gi5139814
G2343/53
2.80E−31
Glycine max
GmMYB29B2.



gi13346178
G2343/53
4.90E−31
Gossypium hirsutum
BNLGHi233.



gi6651292
G2343/53
2.70E−30
Pimpinella
myb-related






brachycarpa
transcription factor.



gi8247759
G2343/53
1.10E−29
Triticum aestivum
GAMyb protein.



AF272573
G2123/67
1.30E−50
Populus alba ×
clone INRA717-1-B4






Populus tremula
14-3-3 pr



BG581482
G2123/67
3.70E−49
Medicago truncatula
EST483216 GVN







Medicago truncatula







cDNA



BG351501
G2123/67
9.60E−49
Solanum tuberosum
109A12 Mature tuber







lambda ZAP Solanum







tu



LETFT7
G2123/67
1.20E−48
Lycopersicon
mRNA for 14-3-3






esculentum
protein, TFT7.



AF228501
G2123/67
4.50E−44
Glycine max
14-3-3-like protein







mRNA, complete cds.



BE643058
G2123/67
5.30E−44
Ceratopteris
Cri2_7_M14_SP6






richardii
Ceratopteris Spore Li



AF222805
G2123/67
7.00E−43
Euphorbia esula
14-3-3-like protein







mRNA, complete cds.



PSA238682
G2123/67
1.30E−42
Pisum sativum
mRNA for 14-3-3-







like protein, sequence







2.



BG443252
G2123/67
1.80E−40
Gossypium arboreum
GA_Ea0020A13f







Gossypium arboreum







7-10 d



AI727536
G2123/67
9.70E−40
Gossypium hirsutum
BNLGHi8338 Six-







day Cotton fiber







Gossypiu



gi8515890
G2123/67
1.80E−52
Populus alba ×
14-3-3 protein.






Populus tremula



gi8099061
G2123/67
3.70E−52
Populus × canescens
14-3-3 protein.



gi7576887
G2123/67
1.00E−50
Glycine max
14-3-3-like protein.



gi3925703
G2123/67
8.90E−50
Lycopersicon
14-3-3 protein.






esculentum



gi6752903
G2123/67
8.90E−50
Euphorbia esula
14-3-3-like protein.



gi913214
G2123/67
2.10E−47
Nicotiana tabacum
T14-3-3.



gi11138322
G2123/67
3.40E−47
Vicia faba
vf14-3-3d protein.



gi2879818
G2123/67
8.50E−46
Solanum tuberosum
14-3-3 protein.



gi1015462
G2123/67
8.90E−46
Chlamydomonas
14-3-3 protein.






reinhardtii



gi2921512
G2123/67
1.10E−45
Fritillaria agrestis
GF14 protein.



AC091246
G1777/55
3.50E−96
Oryza sativa
chromosome 3 clone







OSJNBa0002I03, ***







SEQUENCI



BG136684
G1777/55
1.10E−67
Lycopersicon
EST477126 wild






pennellii
tomato pollen Lycoper



AW703793
G1777/55
2.50E−65
Glycine max
sk12f08.y1 Gm-







c1023 Glycine max







cDNA clone GENO



BE051040
G1777/55
6.60E−59
Zea mays
za71g01.b50 Maize







Glume cDNAs







Library Zea mays







cDN



AW933922
G1777/55
2.90E−53
Lycopersicon
EST359765 tomato






esculentum
fruit mature green



BG600834
G1777/55
3.40E−53
Solanum tuberosum
EST505729 cSTS







Solanum tuberosum







cDNA clo



BF440069
G1777/55
3.20E−39
Thellungiella
Sc0136 Thellungiella






salsuginea
salsuginea ZA



BF587440
G1777/55
4.20E−25
Sorghum
FM1_36_D07.b1_A003






propinquum
Floral-Induced







Merist



BI267961
G1777/55
2.10E−23
Medicago truncatula
NF118E09IN1F1071







Insect herbivory







Medic



BE415217
G1777/55
2.50E−22
Triticum aestivum
MWL025.F02F00020







8 ITEC MWL Wheat







Root Lib



gi1666171
G1777/55
7.50E−24
Nicotiana
unknown.






plumbaginifolia



gi643082
G1777/55
1
Fragaria × ananassa
unknown.



AW928317
G2520/37
4.60E−27
Lycopersicon
EST307050 tomato






esculentum
flower buds 8 mm t



BF271147
G2520/37
2.60E−26
Gossypium arboreum
GA_Eb0010K15f







Gossypium arboreum







7-10 d



BE329654
G2520/37
2.60E−26
Glycine max
so67c05.y1 Gm-







c1040 Glycine max







cDNA clone GENO



BG103016
G2520/37
4.40E−23
Sorghum
RHIZ2_36_A10.b1_A003






propinquum
Rhizome2







(RHIZ2) So



BE606980
G2520/37
1.00E−22
Triticum aestivum
WHE0914_F04_K08ZS







Wheat 5-15 DAP







spike cD



BG048756
G2520/37
1.60E−22
Sorghum bicolor
OV1_22_F05.b1_A002







Ovary 1 (OV1)







Sorghum bi



AI162779
G2520/37
2.10E−22
Populus tremula ×
A023P62U Hybrid






Populus tremuloides
aspen



BI270049
G2520/37
2.90E−22
Medicago truncatula
NF004D04FL1F1042







Developing flower







Medi



BE921054
G2520/37
3.90E−22
Solanum tuberosum
EST424823 potato







leaves and petioles







Sola



BF200249
G2520/37
9.10E−22
Triticum
WHE2254_F11_L22ZE






monococcum
Triticum







monococcum s



gi11862964
G2520/37
4.50E−16
Oryza sativa
hypothetical protein.



gi5923912
G2520/37
6.30E−16
Tulipa gesneriana
bHLH transcription







factor GBOF-1.



gi6166283
G2520/37
0.69
Pinus taeda
helix-loop-helix







protein 1A.



gi1086538
G2520/37
1
Oryza rufipogon
transcriptional







activator Rb homolog.









For many of the traits listed in Table 6 that may be conferred to plants by ectopically expressing transcription factors of the invention, 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.
The first and second columns of Table 6 provide the Trait category and specific trait were generally observed in plants overexpressing the listed transcription factor sequence of the invention, or, where noted, in plants in which a specific transcription factor has been knocked out (KO). The third column lists the sequences for which a specific trait was observed when the expression of the sequence was altered, and the last column provides the utility and specific observations, relative to controls, for each of the sequences.







TABLE 6







Genes, traits and utilities that affect plant characteristics












Transcription factor genes
Utility/


Trait Category
Traits
that impact traits
Observations





Environmental
Increased osmotic stress
G353, G1069, G1930
Enhanced germination rate,


stress resistance
tolerance

survivability, yield


and tolerance


G47 (in a root growth assay





on PEG-containing media,





G47 overexpressing





seedlings were larger and





had more root growth





compared to the wild-type)





G353 (on PEG containing





media, overexpressing





seedlings were larger and





greener than the wild-type)





G1069 (overexpressing





lines showed more





tolerance to osmotic stress





on high sucrose media)





G1930 (with more seedling





vigor on high sucrose than





wild-type control plants)



Altered C/N sensing and
G975, G1069, G1266,
Improved yield, less fertilizer



tolerance to low
G1322, G1930, G2131,
required, improved stress



nitrogen conditions
G2144, G2512, G2520
tolerance and quality





G975 (less anthocyanin





accumulation on low





nitrogen media, better





tolerance to low nitrogen





conditions than controls)





G1069 (less anthocyanin





accumulation on low





nitrogen media, better





tolerance to low nitrogen





conditions than controls)





G1266 (less anthocyanin





accumulation on low





nitrogen media, better





tolerance to low nitrogen





conditions than controls)





G1322 (accumulation on





low nitrogen media, better





tolerance to low nitrogen





conditions than controls)





G1930 (less anthocyanin





accumulation on low





nitrogen media, better





tolerance to low nitrogen





conditions than controls)





G2131 (less anthocyanin





accumulation on low





nitrogen media, better





tolerance to low nitrogen





conditions than controls)





G2144 (less anthocyanin





accumulation on low





nitrogen media, better





tolerance to low nitrogen





conditions than controls)





G2512 (less anthocyanin





accumulation on low





nitrogen media, better





tolerance to low nitrogen





conditions than controls)





G2520 (less anthocyanin





accumulation on low





nitrogen media, better





tolerance to low nitrogen





conditions than controls)



Increased tolerance to
G1946
Improved yield, less fertilizer



phosphate-limitation

required, improved stress





tolerance and quality





G1946 (more secondary





root growth on phosphate-





free media than wild-type





controls)



Increased salt tolerance
G47, G1930, G3644
G1930 (with more seedling





vigor on high salt media





than wild-type control





plants)





G47 and G3644 (homologs;





more seedling vigor on high





salt media than wild-type





control plants)



Increased cold stress
G47, G1322, G1930,
Enhanced germination,



resistance and/or
G2133, G3643, G3649
growth, earlier planting



improved germination in

G1322 (at 8° C.,



cold conditions

overexpressor seedlings





were slightly larger and had





longer roots than wild type)





G1930 (increased tolerance





to 8° C. in a germination





assay)





G47 (with leaf RBCS3 or





shoot apical meristem





promoters) and closely-





related homologs G2133,





G3643 and G3649 (35S





promoter) conferred





increased tolerance to 8° C.





in a germination assay





relative to controls)



Increased drought or
G47, G353, G975, G1069,
Improved survivability, yield,



desiccation tolerance
G2133, G3643, G3644,
extended range




G3649
G353 (overexpressors had





greater tolerance to drought





than wild type in a soil-





based assay)





G975 (overexpressors had





greater tolerance to





desiccation in plate-based





assays, and greater





tolerance to drought than





wild type in a soil-based





assay)





G1069 (overexpressors had





greater tolerance to drought





than wild type in a soil-





based assay)





G47 and homologs G2133,





G3643 and G3649





conferred increased water





deprivation when





overexpressed compared to





controls (another homolog,





G3644, was not tested in





drought assays)



Altered light response
G377, G1069, G1322,
Enhanced germination,



and shade tolerance
G1794, G2144, G2520
growth, development,





flowering time, greater





planting density and





improved yield





G377 (overexpressors had





altered leaf orientation)





G1322 (overexpressors





exhibited constitutive





photomorphogenesis)





G1069 (overexpressors





exhibited altered leaf





orientation)





G1794 (overexpressors





exhibited constitutive





photomorphogenesis)





G2144 overexpressors





exhibited long hypocotyls





G2520 (overexpressors had





long hypocotyls)


Sugar sensing
Altered plant response
G1337
Photosynthetic rate,



to sugars

carbohydrate accumulation,





biomass production, source-





sink relationships, senescence





G1337 (G1337





overexpressors germinated





poorly on high glucose





compared to controls, thus





G1337 may be involved in





sugar sensing, transport, or





metabolism)


Hormonal
Altered hormone
G47, G1069, G1266
Seed dormancy, drought



sensitivity

tolerance; plant form, fruit





ripening





G47 (overexpressors had





decreased sensitivity to





ABA)





G1069 (overexpressors had





decreased sensitivity to





ABA)





G1266 (overexpressors had





decreased sensitivity to





ABA)


Development,
Altered overall plant
G47, G353, G1543; G1794,
Altered vascular tissues,


morphology
architecture
G2509
increased lignin content;





altered cell wall content;





and/or appearance





G47 (increased lignin





content, stems were wider





with a much greater number





of xylem vessels than wild





type)





G353 (overexpressors had





short pedicels, downward





pointing siliques, leaves





had short petioles, were





rather flat, rounded, and





sometimes showed changes





in coloration)





G1543 (some G1543





overexpressors exhibited





contorted, stunted carpels;





35S::G1543 plants also





exhibited altered branching





pattern, and apical





dominance was reduced)





G1794 (overexpressors





exhibited decreased apical





dominance)





G2509 (overexpressors





exhibited decreased apical





dominance)



Increased size, stature
G47; G377, G1052, G1543,
Improved yield



and/or biomass
G2133, G2155, G3643,
G47 (stem sections were of




G3644, G3649
wider diameter and vascular





bundles were larger,





sometimes multiple cauline





leaves were present at each





node; overexpression of





G47 and its homologs





G2133, G3643, G3644 and





G3649, resulted in some





lines that produced larger





plants than controls with





larger rosettes, seedlings





and/or seeds)





G377 (some lines had





broader, fuller rosette





leaves than wild type)





G214 (larger biomass,





increased leaf number and





size compared to controls)





G1052 (larger leaves and





were generally more sturdy





than wild type)





G1543 (some





overexpressors exhibited





increased biomass,





including tomato plants





overexpressing this





sequence)





G2155 (late in





development, 35S::G2155





plants became very large





relative to controls)



Size: reduced stature or
G280; G353; G362; G652;
Ornamental utility (creation



dwarfism
G674; G962; G977;
of dwarf varieties); small




G1198; G1266; G1309;
stature also provides wind




G1322; G1421; G1537;
resistance




G1641; G1794; G2094;




G2144; G2147



Flower structure,
G47, G259, G353, G1543
Ornamental horticulture;



inflorescence

production of saffron or other





edible flowers





G47 (thick and fleshy





inflorescences)





G259 (rosette leaves were





longer, narrower, darker





green than controls, sepals





were longer, narrower, and





often fused at the tips)





G353 (35S::G353 plants





had a reduction in flower





pedicel length and





downward pointing





siliques)





G1543 (some lines showed





contorted, stunted carpels)



Number and
G362, G1930, G2105
Improved resistance to pests



development of

and desiccation; essential oil



trichomes

production





G362 (increased trichome





density)





G1930 (decreased trichome





density)





G2105 (adaxial leaf





surfaces had a somewhat





‘lumpy’ appearance caused





by trichomes being raised-





up on small mounds of





epidermal cells)



Seed size, color, and
G652; G2105
Improved yield



number

G652 (seeds produced by





knockouts of G652 plants





were somewhat wrinkled





and misshapen)





G2105 (pale, larger seeds





than controls)



Leaf shape, color,
G377; G674; G977;
Appealing shape or shiny



modifications
G1198; G2094; G2105;
leaves for ornamental




G2113; G2117; G2144;
agriculture, increased




G2155, G2583
biomass or photosynthesis





G377 (during later rosette





stage, leaves were rounder,





darker green, and shorter





than wild type. After





flowering, 35S::G377





leaves had a greater blade





area than wild-type)





G674 (rounded, dark green





leaves that sometimes





pointed upward)





G977 (dark green leaves





that were generally





wrinkled or curled)





G1198 (smaller, narrower





leaves)





G2094 (leaves pf





overexpressors were short,





wide, and slightly yellowed





compared to wild type.,





occasionally the leaves also





showed mild serrations on





their margins)





G2105 (uneven leaf





surface)





G2113 (long petioles,





vertical leaf orientation,





leaves appeared narrow and





were downward curling at





the margins compared to





controls)





G2155 (slightly small,





rounded, leaves that





became dark green, very





large and senesced later





than wild type late in





development)





G2144 (pale, narrow, flat





leaves that had long





petioles and sometimes





positioned in a vertical





orientation)





G2583 (narrow, curled





leaves)



Altered stem
G47, G748
Ornamental; digestibility



morphology

G47 (stems of wider





diameter with large





irregular vascular bundles





containing greater number





of xylem vessels than wild





type; some xylem vessels





within the bundles appeared





narrow and more lignified)





G748 (thicker and more





vascular bundles in stems





than controls)


Pigment
Production of
G214; G259; G362, G490;
Antioxidant activity, vitamin



anthocyanin and prenyl
G652, G748; G883; G977,
E



lipids
G1052; G1328; G1930;
G214 (darker green in




G2509, G2520
vegetative and reproductive





tissues due to a higher





chlorophyll content in the





later stages of development;





increased seed lutein)





G259 (increase in seed α-





tocopherol)





G362 (increased pigment





production compared to





controls, seeds developed





patches of dark purple





pigmentation, increased





anthocyanin in seedling





leaves; late flowering lines





also became darkly





pigmented.)





G490 (increased seed δ-





tocopherol)





G652 (increase in seed α-





tocopherol)





G748 overexpressors





consistently produced





greater root content than





controls)





G883 (decreased seed





lutein)





G1328 (decreased seed





lutein)





G977 (darker green leaves





than controls)





G1052 (overexpressors had





decreased lutein and





increased xanthophyll 1





relative to controls)





G1930 (increased





chlorophyll content)





G2509 (increase in α-





tocopherol)





G2520 (increase in seed δ-





tocopherol and a decrease





in seed γ-tocopherol)


Seed biochemistry
Production of seed
G20
Precursors for human steroid



sterols

hormones; cholesterol





modulators





G20 (increased





campesterol)



Production of seed
G353; G484; G674;
Defense against insects;



glucosinolates
G1069; G1272 (KO);
putative anticancer activity;




G1506; G1897; G1946;
undesirable in animal feeds




G2113; G2117; G2155;
G353 (increased M39494)




G2290, G2340
G484 (altered glucosinolate





profile)





G674 (increased M39501)





G1069 (increased M39497)





G1272 (decreased M39497)





G1506 (increased M39502





and M39498)





G1897 (increased M39491





and M39493)





G1946 (increased M39501)





G2113 (decreased M39497,





increased M39501 and





M39494)





G2117 (increased M39497,





decreased M39496)





G2155 (increased M39497)





G2290 (increased M39496)





G2340 (extreme alteration





in seed glucosinolate





profile)



Modified seed oil
G229, G652, G663, G974;
Vegetable oil production;



content
G1198; G1543; G1777;
increased caloric value for




G1946; G2117, G2123;
animal feeds; lutein content




G2343
G229 (increased seed oil)





G652 (decreased seed oil)





G663 (decreased seed oil)





G1198 (increased seed oil)





G1543 (decreased seed oil





observed in Arabidopsis





overexpressors, increased





seed oil observed in soy)





G1777 (increased seed oil)





G1946 (increased seed oil)





G2117 (decreased seed oil)





G2123 (increased seed oil)



Modified seed protein
G229, G663, G1641;
Reduced caloric value for



content
G1777; G1946; G2117;
humans




G2509
G229 (decreased seed





protein)





G663 (increased seed





protein)





G1641 (increased seed





protein)





G1777 (decreased seed





protein)





G1946 (decreased seed





protein)





G2117 (increased seed





protein)





G2509 (increased seed





protein)



Modified seed fatty acid
G1069, G1421
Altered nutritional value;



content

increase in waxes for disease





resistance





G1069 (increased 16:0 fatty





acids and decreased 18:2





fatty acids)





G1421 (increased 18:1 and





decreased 18:3 seed fatty





acids)


Leaf biochemistry
Production of leaf
G264; G353; G652; G681;
Defense against insects;



glucosinolates
G1069; G1198; G1322;
putative anticancer activity;




G1421; G1794; G2113, ;
undesirable in animal feeds




G2144; G2512; G2520;
G264 (increased M39481)




G2552
G353 (increased M39494)





G652 (increased M39480)





G681 (increased M39480)





G1069 ( )





G1198 (increased M3948)





G1322 (increased M39480)





G1421 (increased M39482)





G1794 (increased M39480)





G2113 (increased M39478)





G2144 (increased M39480)





G2512 (increased M39481)





G2520 (increased M39478)





G2552 (increased M39480)



Production of leaf
G2131; G2424
Precursors for human steroid



phytosterols, inc.

hormones; cholesterol



stigmastanol,

modulators



campesterol

G2131 (Increase in leaf





campesterol)





G2424 (increase in





stigmastanol)



Leaf fatty acid
G214; G377; G962; G975;
Altered nutritional value;



composition
G987 (KO); G1266;
increase in waxes for disease




G1337; G1399, G1465;
resistance




G1512; G2136; G2147,
G214 (increased leaf fatty




G2583
acids)





G377 (increase in leaf 18:2





fatty acids and decrease in





leaf 18:3 fatty acids)





G962 (increase in 16:0 leaf





fatty acids, decrease in 18:3





leaf fatty acids)





G987 KO (reduction in





16:3 fatty acids relative to





controls)





G975 (increased leaf fatty





acids, glossy leaves)





G1337 (increased leaf oleic





acids)





G1399 (increased leaf 16:0





fatty acid)





G1465 (increased in 16:0,





16:1, 18:0 and 18:2 and





decreased 16:3 and 18:3





leaf fatty acids)





G1512 (increased 18:2 leaf





fatty acids)





G2136 (decreased 18:3 leaf





fatty acids)





G2147 increased 16:0 and





18:2 3 leaf fatty acids)





G2583 (glossy leaves)



Production of prenyl
G214; G259; G280; G362,
Antioxidant activity, vitamin



lipids, including
G652; G987 (KO), G1543;
E



tocopherol
G1930, G2509; G2520
G214 (increased leaf





chlorophyll and





carotenoids)





G259 (increased seed α-





tocopherol)





G280 (increased leaf δ and





γ tocopherol)





G362 (increased





anthocyanin levels in





various tissues at different





stages of growth.; seedlings





showed high levels of





pigment in first true leaves,





late flowering lines became





darkly pigmented., seeds





from developed patches of





dark purple pigmentation)





G652 (increased seed α-





tocopherol)





G987 (overexpressors had





two xanthophylls not





present in wild-type leaves,





γ-tocopherol (which





normally accumulate in





seed tissue), and reduced





levels of chlorophyll a and





chlorophyll b in leaves)





G1543 (dark green color,





increased levels of





carotenoids and





chlorophylls a and b in





leaves)





G1930 (increased levels of





chlorophyll a and





chlorophyll b in seeds





compared to controls)





G2509 (increased seed α-





tocopherol)





G2520 (increase in seed δ-





tocopherol and a decrease





in seed γ-tocopherol)



Sugar, starch,
G158; G211; G242; G274;
Improved food digestibility,



hemicellulose
G1012; G1266; G1309;
increased hemicellulose &



composition,
G1641; G1865; G2094;
pectin content; increased




G2589
fiber content; increased plant





tensile strength, wood





quality, pathogen resistance,





pulp production and/or tuber





starch content





G158 (increased leaf





rhamnose)





G211 (increased leaf





xylose)





G242 (increased leaf





arabinose)





G274 (increased leaf





arabinose)





G1012 (decreased leaf





rhamnose)





G1266 (alterations in





rhamnose, arabinose,





xylose, and mannose, and





galactose)





G1309 (increased leaf





mannose)





G1641 (increased leaf





rhamnose)





G1865 (increased galactose,





decreased xylose)





G2094 (increased leaf





arabinose)





G2589 (increased leaf





insoluble sugars - increased





arabinose)


Growth,
Plant growth rate and
G1543
Faster growth, increased


Reproduction
development

biomass or yield, improved





appearance; delay in bolting





G1543 (faster growth of





seedlings)



Senescence; cell death
G652, G1897, G2155,
Altered yield, appearance;




G2340
response to pathogens





(potential protective response





without the potentially





detrimental consequences of





a constitutive systemic





acquired resistance)





G652 (premature





senescence of rosette





leaves)





G1897 (later senescence





than controls





G2155 (senesced much





later than controls)





G2340 (overexpressors





showed necrosis of blades





of rosette and cauline





leaves, necrotic lesions)



Modified fertility
G652; G962; G977;
Prevents or minimizes escape




G1266; G1421; G2094;
of the pollen of genetically




G2113; G2147
modified plants





G652 (poor fertility)





G962 (poor fertility)





G977 (poor fertility)





G1266 (poor fertility)





G1421 (poor fertility)





G2094 (poor fertility)





G2113 (poor fertility)





G2094 (poor fertility)





G2147 (poor fertility)



Early flowering
G490; G1946; G2144;
Faster generation time;




G2509
synchrony of flowering;





potential for introducing new





traits to single variety



Delayed flowering
G47; G214; G362; G748;
Delayed time to pollen




G1052; G1865; G1930,
production of GMO plants;




G2155, G2133, G3643,
synchrony of flowering;




G3644, G3649
increased yield



Flower and leaf
G259; G353; G377; G652;
Ornamental applications;



development
G1865; G1897; G2094
decreased fertility





G259 (rosette leaves were





longer and narrow, dark





green and curled compared





to control plants, sepals





were long, narrow, and





often fused at the tips)





G353 (reduction in flower





pedicel length and





downward pointing





siliques)





G377 (inflorescence stems





were shorter than wild-type,





during late rosette stage,





leaves were rounder, darker





green, and slightly shorter





than those of wild type)





G652 (reduced number of





stamens: 4-5 of these





organs rather than 6)





G1865 (short, thick,





inflorescence stems, greatly





increased number of leaves;





visible flower buds up to a





month after wild type,





continuous light conditions,





by which time rosette





leaves had become rather





large and contorted)





G1897 (narrow, dark-green





rosette and cauline leaves,





inflorescences had short





internodes with various





abnormalities, perianth





organs were typically rather





long and narrow., stamens





were short, silique





formation was poor)





G2094 (inflorescence stems





were often thin and carried





short flowers, mild





serrations on leaf margins)



Flower abscission
G1897
Ornamental: longer retention





of flowers





G1897 (delayed abscission





of floral organs)





* When co-expressed with G669 and G663






Significance of Modified Plant Traits

The sequences of the Sequence Listing, those in Tables 4-6, 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 Tables 4-6 provides exemplary polynucleotide and polypeptide sequences of the invention.
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.
Another potential utilities for sequences that increase trichome number is to increase the density of cotton fibers in cotton bolls. Cotton fibers are modified unicellular trichomes that are produced from the ovule epidermis. However, typically only 30% of the epidermal cells take on a trichome fate (Basra and Malik, 1984). Thus, cotton yields might be increased by inducing a greater proportion of the ovule epidermal cells to become fibers.
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 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 et al. (1995) Nature 377: 522-524; Weigel and Nilsson (1995) Nature 377: 495-500; and 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 Tables 4 and 6.
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.
Suppression of endogenous transcription factor gene expression can also be achieved using RNA interference, or RNAi. RNAi is a post-transcriptional, targeted gene-silencing technique that uses double-stranded RNA (dsRNA) to incite degradation of messenger RNA (mRNA) containing the same sequence as the dsRNA (Constans, (2002) The Scientist 16: 36). Small interfering RNAs, or siRNAs are produced in at least two steps: an endogenous ribonuclease cleaves longer dsRNA into shorter, 21-23 nucleotide-long RNAs. The siRNA segments then mediate the degradation of the target mRNA (Zamore, (2001) Nature Struct. Biol. 8: 746-50). RNAi has been used for gene function determination in a manner similar to antisense oligonucleotides (Constans, (2002) The Scientist 16: 36). Expression vectors that continually express siRNAs in transiently and stably transfected have been engineered to express small hairpin RNAs (shRNAs), which get processed in vivo into siRNAs-like molecules capable of carrying out gene-specific silencing (Brummelkamp et al. (2002) Science 296: 550-553, and Paddison et al. (2002) Genes & Dev. 16: 948-958). Post-transcriptional gene silencing by double-stranded RNA is discussed in further detail by Hammond et al. (2001) Nature Rev Gen 2: 110-119, Fire et al. (1998) Nature 391: 806-811 and Timmons and Fire (1998) Nature 395: 854.
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 Devel. 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 dicotyledonous plants. Suitable protocols are available for Leguminosae (alfalfa, soybean, clover, etc.), Umbelliferae (carrot, celery, parsnip), Cruciferae (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.
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: 482-489, 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 be 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.
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 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 32P 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 NaPO4 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 MarathonTM 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 MarathonTM 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 Res. 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 DH5α 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 (A600) 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 A 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 (A600) of >2.0 is reached. Cells were then harvested by centrifugation at 4,000×g for 10 min, and resuspended in infiltration medium (1⁄2× 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 A600 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/m2/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 H2O 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 H2O. 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/m2/sec) at 22-23° C. After 7-10 days of growth under these conditions, kanamycin resistant primary transformants (T1 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 (T2) 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
Morphological Analysis
Morphological analysis was performed to determine whether changes in transcription factor levels affect plant growth and development. This was primarily carried out on the T1 generation, when at least 10-20 independent lines were examined. However, in cases where a phenotype required confirmation or detailed characterization, plants from subsequent generations were also analyzed.
Primary transformants were selected on MS medium with 0.3% sucrose and 50 mg/l kanamycin. T2 and later generation plants were selected in the same manner, except that kanamycin was used at 35 mg/l. In cases where lines carry a sulfonamide marker (as in all lines generated by super-transformation), seeds were selected on MS medium with 0.3% sucrose and 1.5 mg/l sulfonamide. KO lines were usually germinated on plates without a selection. Seeds were cold-treated (stratified) on plates for 3 days in the dark (in order to increase germination efficiency) prior to transfer to growth cabinets. Initially, plates were incubated at 22° C. under a light intensity of approximately 100 microEinsteins for 7 days. At this stage, transformants were green, possessed the first two true leaves, and were easily distinguished from bleached kanamycin or sulfonamide-susceptible seedlings. Resistant seedlings were then transferred onto soil (Sunshine potting mix). Following transfer to soil, trays of seedlings were covered with plastic lids for 2-3 days to maintain humidity while they became established. Plants were grown on soil under fluorescent light at an intensity of 70-95 microEinsteins and a temperature of 18-23° C. Light conditions consisted of a 24-hour photoperiod unless otherwise stated. In instances where alterations in flowering time was apparent, flowering was re-examined under both 12-hour and 24-hour light to assess whether the phenotype was photoperiod dependent. Under 24-hour light growth conditions, the typical generation time (seed to seed) was approximately 14 weeks.
Because many aspects of Arabidopsis development are dependent on localized environmental conditions, in all cases plants were evaluated in comparison to controls in the same flat. Controls for transgenic lines were generally wild-type plants or, where specifically indicated, transgenic plants harboring an empty transformation vector selected on kanamycin or sulfonamide. Careful examination was made at the following stages: seedling (1 week), rosette (2-3 weeks), flowering (4-7 weeks), and late seed set (8-12 weeks). Seed was also inspected. Seedling morphology was assessed on selection plates. At all other stages, plants were macroscopically evaluated while growing on soil. All significant differences (including alterations in growth rate, size, leaf and flower morphology, coloration and flowering time) were recorded, but routine measurements were not be taken if no differences were apparent. In certain cases, stem sections were stained to reveal lignin distribution. In these instances, hand-sectioned stems were mounted in phloroglucinol saturated 2M HCl (which stains lignin pink) and viewed immediately under a dissection microscope.
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 (Koornneef 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.
Example VIII
Biochemical Analysis
Experiments were also 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, β-carotene, xanthophyll-1, xanthophyll-2, chlorophylls A or B, or α-, δ- or γ-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 H2SO4 and partitioned into hexane from methanolic brine. For seed fatty acids, seeds were pulverized and extracted in methanol:heptane:toluene:2,2-dimethoxypropane:H2SO4 (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 μl 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 μl 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 J. 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 NaBH4, 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).
Example IX
Plate-Based Physiology Experimental Methods
Plate Assays.
Twelve different plate-based physiological assays (shown below), representing a variety of drought-stress related conditions, are used as a pre-screen to identify top performing lines from each project (i.e. lines from transformation with a particular construct), that will be tested in subsequent soil based assays. Typically, ten lines are subjected to plate assays, from which the best three lines are selected for subsequent soil based assays. However, in projects where significant stress tolerance is not obtained in plate based assays, lines are not submitted for soil assays.
In addition, some projects are subjected to nutrient limitation studies. A nutrient limitation assay is intended to find genes that allow more plant growth upon deprivation of nitrogen. Nitrogen is a major nutrient affecting plant growth and development that ultimately impacts yield and stress tolerance. These assays monitor primarily root but also rosette growth on nitrogen deficient media. In all higher plants, inorganic nitrogen is first assimilated into glutamate, glutamine, aspartate and asparagine, the four amino acids used to transport assimilated nitrogen from sources (e.g. leaves) to sinks (e.g. developing seeds). This process is regulated by light, as well as by C/N metabolic status of the plant. We use a C/N sensing assay to look for alterations in the mechanisms plants use to sense internal levels of carbon and nitrogen metabolites which could activate signal transduction cascades that regulate the transcription of N-assimilatory genes. To determine whether these mechanisms are altered, we exploit the observation that wild-type plants grown on media containing high levels of sucrose (3%) without a nitrogen source accumulate high levels of anthocyanins. This sucrose induced anthocyanin accumulation can be relieved by the addition of either inorganic or organic nitrogen. We use glutamine as a nitrogen source since it also serves as a compound used to transport N in plants.
Germination Assays.
NaCl (150 mM), mannitol (300 mM), sucrose (9.4%), ABA (0.3 μM), Heat (32° C.), Cold (8° C.), −N is basal media minus nitrogen plus 3% sucrose and −N/+Gln is basal media minus nitrogen plus 3% sucrose and 1 mM glutamine.
Growth Assays.
Severe dehydration (drought), heat (32° C. for 5 days followed by recovery at 22° C.), chilling (8° C.), root development (visual assessment of lateral and primary roots, root hairs and overall growth). For the nitrogen limitation assay, all components of MS medium remain constant except N is reduced to 20 mg/L of NH4NO3. Note that 80% MS has 1.32 g/L NH4NO3 and 1.52 g/L KNO3.
Unless otherwise stated, all experiments are performed with the Arabidopsis thaliana ecotype Columbia (col-0). Assays are usually performed on non-selected segregating T2 populations (in order to avoid the extra stress of selection). Control plants for assays on lines containing direct promoter-fusion constructs are Col-0 plants transformed an empty transformation vector (pMEN65). Controls for 2-component lines (generated by supertransformation) are the background promoter-driver lines (i.e. promoter::LexA-GAL4TA lines), into which the supertransformations were initially performed.
All assays are performed in tissue culture. Growing the plants under controlled temperature and humidity on sterile medium produces uniform plant material that has not been exposed to additional stresses (such as water stress) which could cause variability in the results obtained. All assays were designed to detect plants that are more tolerant or less tolerant to the particular stress condition and were developed with reference to the following publications: Jang et al. (1997) Plant Cell 9: 5-19; Smeekens (1998) Curr. Opin. Plant Biol. 1: 230-234; Liu and Zhu (1997) Proc. Natl. Acad. Sci. U.S.A. 94: 14960-14964; Saleki et al. (1993) Plant Physiol. 101: 839-845; Wu et al. (1996) Plant Cell 8: 617-627; Zhu et al. (1998) Plant Cell 10: 1181-1191; Alia et al. (1998) Plant J. 16: 155-161; Xin and Browse, (1998) Proc. Natl. Acad. Sci. U.S.A. 95: 7799-7804; Leon-Kloosterziel et al. (1996) Plant Physiol. 110: 233-240. Where possible, assay conditions were originally tested in a blind experiment with controls that had phenotypes related to the condition tested.
Procedures
Prior to plating, seed for all experiments are surface sterilized in the following manner: (1) 5 minute incubation with mixing in 70% ethanol, (2) 20 minute incubation with mixing in 30% bleach, 0.01% triton-X 100, (3) 5× rinses with sterile water, (4) Seeds are re-suspended in 0.1% sterile agarose and stratified at 4° C. for 3-4 days.
All germination assays follow modifications of the same basic protocol. Sterile seeds are sown on the conditional media that has a basal composition of 80% MS+Vitamins. Plates are incubated at 22° C. under 24-hour light (120-130 μE m−2s−1) in a growth chamber. Evaluation of germination and seedling vigor is done 5 days after planting. For assessment of root development, seedlings germinated on 80% MS+Vitamins+1% sucrose are transferred to square plates at 7 days. Evaluation is done 5 days after transfer following growth in a vertical position. Qualitative differences are recorded including lateral and primary root length, root hair number and length, and overall growth.
For chilling (8° C.) and heat sensitivity (32° C.) growth assays, seeds are germinated and grown for 7 days on MS+Vitamins+1% sucrose at 22° C. and then are transferred to chilling or heat stress conditions. Heat stress is applied for 5 days, after which the plants are transferred back to 22° C. for recovery and evaluated after a further 5 days. Plants are subjected to chilling conditions (8° C.) and evaluated at 10 days and 17 days.
For severe dehydration (drought) assays, seedlings are grown for 14 days on MS+Vitamins+1% Sucrose at 22° C. Plates are opened in the sterile hood for 3 hr for hardening and then seedlings are removed from the media and let dry for 2 h in the hood. After this time they are transferred back to plates and incubated at 22° C. for recovery. Plants are evaluated after 5 days.
Experiments were also 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.
Example X
Soil Drought Experimental Methods
The soil drought assay (performed in clay pots) is based on that described by Haake et al. (2002). In the current procedure, seedlings were first germinated on selection plates containing either kanamycin or sulfonamide. Seeds were sterilized by a 2 minute ethanol treatment followed by 20 minutes in 30% bleach/0.01% Tween and five washes in distilled water. Seeds are sown to MS agar in 0.1% agarose and stratified for 3 days at 4° C., before transfer to growth cabinets with a temperature of 22° C. After 7 days of growth on selection plates, seedlings are transplanted to 3.5 inch diameter clay pots containing 80 g of a 50:50 mix of vermiculite:perlite topped with 80 g of ProMix. Typically, each pot contains 14 seedlings, and plants of the transgenic line being tested are in separate pots to the wild-type controls. Pots containing the transgenic line versus control pots were interspersed in the growth room, maintained under 24-hour light conditions (18-23° C., and 90-100 μE m−2s−1) and watered for a period of 14 days. Water was then withheld and pots were placed on absorbent diaper paper for a period of 8-10 days to apply a drought treatment. After this period, a visual qualitative “drought score” from 0-6 is assigned to record the extent of visible drought stress symptoms. A score of “6” corresponds to no visible symptoms whereas a score of “0” corresponds to extreme wilting and the leaves having a “crispy” texture. At the end of the drought period, pots are re-watered and scored after 5-6 days; the number of surviving plants in each pot is counted, and the proportion of the total plants in the pot that survived is calculated.
Split-Pot Method.
A variation of the above method was sometimes used, whereby plants for a given transgenic line were compared to wild-type controls in the same pot. For those studies, 7 wild-type seedlings were transplanted into one half of a 3.5 inch pot and 7 seedlings of the line being tested were transplanted into the other half of the pot.
Analysis of Results.
In a given experiment, we typically compare 6 or more pots of a transgenic line with 6 or more pots of the appropriate control. (In the split pot method, 12 or more pots are used.) The mean drought score and mean proportion of plants surviving (survival rate) are calculated for both the transgenic line and the wild-type pots. In each case a p-value* is calculated, which indicates the significance of the difference between the two mean values. The results for each transgenic line across each planting for a particular project are then presented in a results table. Results where the lines show a significantly better or worse performance versus the control are highlighted.
Calculation of P-Values.
For the assays where control and experimental plants are in separate pots, survival is analyzed with a logistic regression to account for the fact that the random variable is a proportion between 0 and 1. The reported p-value is the significance of the experimental proportion contrasted to the control, based upon regressing the logit-transformed data.
Drought score, being an ordered factor with no real numeric meaning, is analyzed with a non-parametric test between the experimental and control groups. The p-value is calculated with a Mann-Whitney rank-sum test.
For the split-pot assays, matched control and experimental measurements are available for both variables. In lieu of a direct transformed regression technique for this data, the logit-transformed proportions are analyzed by parametric methods. The p-value is derived from a paired-t-test on the transformed data. For the paired score data, the p-value from a Wilcoxon test is reported.
Measurement of Photosynthesis.
Photosynthesis was measured using a LICOR LI-6400. The LI-6400 uses infrared gas analyzers to measure carbon dioxide to generate a photosynthesis measurement. It is based upon the difference of the CO2 reference (the amount put into the chamber) and the CO2 sample (the amount that leaves the chamber). Since photosynthesis is the process of converting CO2 to carbohydrates, we expect to see a decrease in the amount of CO2 sample. From this difference, a photosynthesis rate can be generated. In some cases, respiration may occur and an increase in CO2 detected. To perform measurements, the LI-6400 is set-up and calibrated as per LI-6400 standard directions. Photosynthesis is measured in the youngest most fully expanded leaf at 300 and 1000 ppm CO2 using a metal halide light source. This light source provides about 700 μE m−2s−1.
Fluorescence was measured in dark and light adapted leaves using either a LI-6400 (LICOR) with a leaf chamber fluorometer attachment or an OS-1 (Opti-Sciences) as described in the manufacturer's literature. When the LI-6400 was used, all manipulations were performed under a dark shade cloth. Plants were dark adapted by placing in a box under this shade cloth until used. The OS-30 utilized small clips to create dark adapted leaves.
Chlorophyll/Carotenoid Determination.
For some experiments, chlorophyll was estimated in methanolic extracts using the method of Porra et al. (1989) Biochim. et Biophys. Acta 975: 384-394. Carotenoids were estimated in the same extract at 450 nm using an A(1%) of 2500. We currently are measuring chlorophyll using a SPAD-502 (Minolata). When the SPAD-502 is being used to measure chlorophyll, both carotenoid and chlorophyll content and amount can also be determined via HPLC. Pigments are extracted from leave tissue by homogenizing leaves in acetone:ethyl acetate (3:2). Water was added, the mixture centrifuged, and the upper phase removed for HPLC analysis. Samples are analyzed using a Zorbax C18 (non-endcapped) column (250×4.6) with a gradient of acetonitrile:water (85:15) to acetonitrile:methanol (85:15) in 12.5 minutes. After holding at these conditions for two minutes, solvent conditions were changed to methanol:ethyl acetate (68:32) in two minutes. Carotenoids and chlorophylls are quantified using peak areas and response factors calculated using lutein and β-carotene as standards.
Example XI
Experimental Results
G2340: (SEQ ID NOs. 17 and 18)
G2340 was analyzed using transgenic plants in which the gene was expressed under the control of the 35S promoter. Overexpression of G2340 produced a spectrum of deleterious effects on Arabidopsis growth and development. 35S::G2340 primary transformants were generally smaller than controls, and at early stages some displayed leaves that were held in a vertical orientation. The most severely affected lines died at early stages. Others survived, but displayed necrosis of the blades in later rosette leaves and cauline leaves. Inflorescence development was also highly abnormal; stems were typically shorter than wild type, often ‘kinked’ at nodes, and the tissue had a rather fleshy succulent appearance. Flower buds were frequently poorly formed, failed to open and withered away without siliques developing. Additionally, secondary shoot growth frequently failed the tips of such structures sometimes senesced. Due to these abnormalities, many of the primary transformants were completely infertile. Three T1 lines (#1,5,20) with a relatively weak phenotype, which did set some seed, were selected for further study. Plants from the T2-20 population displayed a strong phenotype, and died early in development. The other two T2 populations were slightly small, but the effects were much weaker than those seen in the parental plants, suggesting that activity of the transgene might have become reduced between the generations. It should be noted that G2340 and G671 (SEQ ID NO: 19) are part of the same clade and that they had very similar morphological phenotypes and a similar expression pattern. These two genes may have overlapping or redundant phenotypes in the plant. Small, pale seedlings with strap-like leaves that held a vertical orientation were found in the mixed line populations of 35S::G2340 transgenic seedlings when grown under sterile conditions, similar to those observed in soil grown plants in the T1 generation. The necrotic lesions observed on the T1 plants grown in soil were not observed on the plants grown in culture leaving uncertainty as to whether the necrotic lesion phenotype is a classic lesion mimic phenotype that would suggest that G2340 is involved in cell death responses or if the G2340 overexpressor plants are simply hyper-sensitive to stresses. One class of lesion mimic forms progressive lesions following an inductive stress. Lesion formation may be induced in G2340 overexpressing plants grown in culture. In addition to the morphological changes, overexpression of G2340 resulted in an extreme alteration in seed glucosinolate profile. This phenotype was observed in one line, line 1, in seed from two independent plantings. According to RT-PCR analysis, G2340 was expressed primarily in roots and was slightly induced in leaf tissue in response to auxin and heat treatments. G2340 can be used to engineer plants with an inducible cell death response. A gene that regulates cell death in plants can be used to induce a pathogen protective hyper-response (HR) in plants without the potentially detrimental consequences of a constitutive systemic acquired resistance (SAR). Other potential utilities include the creation of novel abscission zones or inducing death in reproductive organs to prevent the spread of pollen, transgenic or otherwise. In the case of necrotrophic pathogens that rely on dead plant tissue as a source of nutrients, prevention of cell death could confer tolerance to these diseases. Overexpression of G2340 in Arabidopsis also resulted in an extreme alteration in seed glucosinolate profile. Therefore, the gene 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 can 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.
G2583: (SEQ ID NOs. 143 and 144)
G2583 was studied using transgenic plants in which the gene was expressed under the control of the 35S promoter. Most notably, 35S::G2583 plants exhibited extremely glossy leaves. At early stages, 35S::G2583 seedlings appeared normal, but by about two weeks after sowing, the plants exhibited very striking shiny leaves, which were apparent until very late in development. In addition to this phenotype, it should be noted that many lines displayed a variety of other effects such as a reduction in overall size, narrow curled leaves, or various non-specific floral abnormalities, which reduced fertility. These effects on leaf appearance were observed in 18/20 primary transformants, and in all the plants from 4/6 of the T2 lines (#2, 4, 9 and 15) examined. The glossy nature of the leaves from 35S::G2583 plants can be a consequence of changes in epicuticular wax content or composition. G2583 belongs to a small clade within the large AP2/EREBP Arabidopsis family that also contains G975 (SEQ ID NO: 89), G1387 (SEQ ID NO: 145), and G977 (SEQ ID NO: 147). Overexpression of G975 (SEQ ID NO: 89) caused a substantial increase in leaf wax components, as well as morphological phenotypes resembling those observed in 35S::G2583 plants. G2583 was ubiquitously expressed (at higher levels in root, flower, embryo, and silique tissues). G2583 can be used to modify plant appearance (shiny leaves). In addition, it can be used to manipulate wax composition, amount, or distribution, which in turn can modify plant tolerance to drought and/or low humidity or resistance to insects.
G362: (SEQ ID NOs. 61 and 62)
G362 was analyzed using transgenic plants in which G362 was expressed under the control of the 35S promoter. 35S::G362 had a number of developmental effects with the most prominent result being an increase in trichome number as well as the ectopic formation of trichomes. Overexpression of G362 also increased anthocyanin levels in various tissues at different stages of growth. Seedlings sometimes showed high levels of pigment in the first true leaves. Late flowering lines also became darkly pigmented. Seeds from a number of lines were observed to develop patches of dark purple pigmentation. Inflorescences from 35S::G362 plants were thin, and flowers sometimes displayed poorly developed organs. The seed yield from many lines was somewhat poor. As determined by RT-PCR, G362 is expressed in roots, and is expressed at significantly lower levels in siliques, seedlings and shoots. No expression of G362 was detected in the other tissues tested. G362 expression was induced in rosette leaves by heat stress. G362 can be used to alter trichome number and distribution in plants. Trichome glands on the surface of many higher plants produce and secrete exudates which 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 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. Another use for G362 is to increase the density of cotton fibers in cotton bolls. Cotton fibers are modified unicellular trichomes that are produced from the ovule epidermis. However, typically only 30% of the epidermal cells take on a trichome fate (Basra and Malik (1984) Int. Rev. Cytol. 89: 65-113). Thus, cotton yields can be increased by inducing a greater proportion of the ovule epidermal cells to become fibers. 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. 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. Thus, the use of G362 and its homologs to increase trichome density, size or type can have profound utilities in molecular farming practices (for example, the use of trichomes as a manufacturing system for complex secondary metabolites), and in producing insect resistant and herbivore resistant plants. In addition, G362 can be used to alter a plant's time to flowering.
G2105: (SEQ ID NOs. 63 and 64)
The ORF boundary of G2105 was determined and G2105 was analyzed using transgenic plants in which G2105 was expressed under the control of the 35S promoter. Two of four T2 lines examined appeared dark green and were smaller than wild type at all stages of development. Additionally, the adaxial leaf surfaces from these plants had a somewhat ‘lumpy’ appearance caused by trichomes being raised-up on small mounds of epidermal cells. Two lines of G2105 overexpressing plants had larger seed. G2105 expression was root specific and induced in leaves by auxin, abscisic acid, high temperature, salt and osmotic stress treatments. On the basis of the analyses, G2105 can be used to manipulate some aspect of plant growth or development, particularly in trichome development. In addition, G2105 can be used to modify seed size and/or morphology, which can have an impact on yield. The promoter of G2105 can have some utility as a root specific promoter.
G47 (SEQ ID NOs. 65 and 66)
G47 was studied using transgenic plants in which the gene was expressed under the control of the 35S promoter. Overexpression of G47 resulted in a variety of morphological and physiological phenotypic alterations. 35S::G47 plants showed enhanced tolerance to osmotic stress. In a root growth assay on PEG-containing media, G47 overexpressing transgenic seedlings were larger and had more root growth compared with wild-type controls. G47 expression levels may be altered by environmental conditions, in particular reduced by salt and osmotic stresses. In addition to the phenotype observed in the osmotic stress assay, germination efficiency for the seeds from G47 overexpressor plants was low. Overexpression of G47 also produced a substantial delay in flowering time and caused a marked change in shoot architecture. 35S::G47 transformants were small at early stages and switched to flowering more than a week later than wild-type controls (continuous light conditions). The inflorescences from these plants appeared thick and fleshy, had reduced apical dominance, and exhibited reduced internode elongation leading to a short compact stature. The branching pattern of the stems also appeared abnormal, with the primary shoot becoming ‘kinked’ at each coflorescence node. Additionally, the plants showed slightly reduced fertility and formed rather small siliques that were borne on short pedicels and held vertically, close against the stem. Additional alterations were detected in the inflorescence stems of 35S::G47 plants. Stem sections from T2-21 and T2-24 plants were of wider diameter, and had large irregular vascular bundles containing a much greater number of xylem vessels than wild type. Furthermore, some of the xylem vessels within the bundles appeared narrow and were possibly more lignified than were those of controls. G47 was expressed at higher levels in rosette leaves, and transcripts were detected in other tissues (flower, embryo, silique, and germinating seedling). G47 can be used to manipulate flowering time, to modify plant architecture and stem structure (including development of vascular tissues and lignin content) and to improve plant performance under osmotic stress. The use of G47 or of G47 orthologs from tree species can be used to modulate lignin content of a plant. This allows the quality of wood used for furniture or construction to be improved. Lignin is energy rich; increasing lignin composition could therefore be valuable in raising the energy content of wood used for fuel. Conversely, the pulp and paper industries seek wood with a reduced lignin content. Currently, lignin must be removed in a costly process that involves the use of many polluting chemicals. Consequently, lignin is a serious barrier to efficient pulp and paper production. In addition to forest biotechnology applications, changing lignin content might increase the palatability of various fruits and vegetables. A wide variety of applications exist for systems that either lengthen or shorten the time to flowering.
Closely-related homologs of G47, determined by BLAST, alignment and phylogeneitc analysis, include G2133 (SEQ ID NO: 152), G3643 (SEQ ID NO: 158), G3644 (SEQ ID NO: 156), and G3649 (SEQ ID NO: 154). Each of these sequences has conferred a transcriptional regulatory activity of G47 in that when any of these sequences were overexpressed in plants, they have each produced some lines that were larger, later in their development and flowering, and more tolerant to water-deprivation, cold or salt, similar to plants overexpressing G47 (Table 4), as compared to controls.
G975: (SEQ ID NOs. 89 and 90)
G975 was identified as a new member of the AP2/EREBP family (EREBP subfamily) of transcription factors. G975 was expressed in flowers and, at lower levels, in shoots, leaves, and siliques. GC-FID and GC-MS analyses of leaves from G975 overexpressing plants showed that the levels of C29, C31, and C33 alkanes were substantially increased (up to 10-fold) compared with control plants. A number of additional compounds of similar molecular weight, presumably also wax components, also accumulated to significantly higher levels in G975 overexpressing plants. C29 alkanes constituted close to 50% of the wax content in wild-type plants (Millar et al. (1998) Plant Cell 11: 1889-1902), suggesting that a major increase in total wax content occurred in the G975 transgenic plants. However, the transgenic plants had an almost normal phenotype (although small morphological differences are detected in leaf appearance), indicating that overexpression of G975 was not deleterious to the plant. Overexpression of G975 did not cause the dramatic alterations in plant morphology that had been reported for Arabidopsis plants in which the FATTY ACID ELONGATION1 gene was overexpressed (Millar et al. (1998) supra). G975 may regulate the expression of some of the genes involved in wax metabolism. One Arabidopsis AP2 sequence (G1387; SEQ ID NO: 145) that is significantly more closely related to G975 than the rest of the members of the AP2/EREBP family is predicted to have a function and a use related to that of G975. G975 can be used to manipulate wax composition, amount, or distribution, which in turn can modify plant tolerance to drought and/or low humidity or resistance to insects, as well as plant appearance (shiny leaves). G975 can also be used to specifically alter wax composition, amount, or distribution in those plants and crops from which wax is a valuable product.
A non-Arabidopsis gene that is related to G975 is L46408 BNAF1258 Mustard flower buds Brassica rapa cDNA clone F1258. The similarity between G975 and the Brassica rapa gene represented by EST L46408 extends beyond the conserved AP2 domain that characterizes the AP2/EREBP family. This Brassica rapa gene appeared to be more closely related to G975 than Arabidopsis G1387, indicating that EST L46408 may represent a true G975 ortholog. The similarity between G975 and Arabidopsis G1387 (SEQ ID NO: 145) also extends beyond the conserved AP2 domain.
G2583 (SEQ ID NO: 143 and 144), a closely-related homolog of G975 determined by BLAST, alignment and phylogenetic analysis, has been shown to confer a transcriptional regulatory activity of G975 in that when the polypeptide sequences were overexpressed in plants and produced some lines that were later in their development and flowering, and produced shiny leaves, indicating more wax production, similar to plants overexpressing G975 (Table 4), as compared to controls. Other closely related sequences include G1387 (SEQ ID NO: 145 and 146), and G4294 (SEQ ID NO: 149 and 150). Although detailed analyses with plants overexpressing these sequence have not yet been performed, plants overexpressing these related sequences are likely to confer some similar transcriptional regulatory activity and traits as G975.
G214: (SEQ ID NOs. 33 and 34)
G214 overexpressing lines were late bolting, showed larger biomass (increased leaf number and size), and were darker green in vegetative and reproductive tissues due to a higher chlorophyll content in the later stages of development. In these later stages, the overexpressor plants also had higher insoluble sugar, leaf fatty acid, and carotenoid content per unit area. Line 11 also showed a significant, repeatable increase in lutein levels in seeds. Micro-array data was consistent with the morphological and biochemical data in that the genes that were highly induced included chloroplast localized enzymes, and light regulated genes such as Rubisco, carbonic anhydrase, and the photosystem 1 reaction center subunit precursor. A chlorophyll biosynthetic enzyme was also highly induced, consistent with the dark green color of the adult leaves and perhaps a higher photosynthetic rate. A measurement of leaf fatty acid in the older overexpressors suggested that the overall levels were higher than wild-type levels (except for the percent composition of 16:3 in line 11). Percent composition of 16:1 and 16:3 (fatty acids found primarily in plastids) is similar to wild-type arguing against an increase in chloroplast number as an explanation for increase chlorophyll content in the leaves. G214 overexpressing lines 3, 11, and 15 were sensitive to germination on high glucose showing less cotyledon expansion and hypocotyl elongation suggesting the late bolting and dark green phenotype could be tied into carbon sensing which has been shown to regulate phytochrome A signaling. Sugars are key regulatory molecules that affect diverse processes in higher plants including germination, growth, flowering, senescence, sugar metabolism and photosynthesis. Glucose-specific hexose-sensing has also been described in plants and implicated in cell division and the repression of famine genes (photosynthetic or glyoxylate cycles). Potential utilities of G214 include increasing chlorophyll content allowing more growth and productivity in conditions of low light. With a potentially higher photosynthetic rate, fruits can have higher sugar content. Increased carotenoid content can be used as a nutraceutical to produce foods with greater antioxidant capability. Also G214 can be used to manipulate seed composition which is very important for the nutritional value and production of various food products.
G214 is homologous to a tomato (Cornell Lycopersicon esculentum) EST (cLER12A11) generated from a Pseudomonas resistant line.
G974: (SEQ ID NOs. 51 and 52)
The complete sequence of G974 was obtained and G974 was studied using transgenic plants in which G974 was expressed under the control of the 35S promoter. Constitutive expression of G974 produced deleterious effects: the majority of 35S::G974 primary transformants showed a reduction in overall size and developed rather slowly compared to wild type controls. These phenotypic alterations were not observed in the T2 generation, perhaps indicating silencing of the transgene. The T2 plants were wild-type in the physiological and biochemical analyses performed. G974 was ubiquitously expressed. 35S::G974 had altered seed oil content
Several AP2 proteins from a variety of species (Atriplex hortensis, Lycopersicon esculentum, Glycine max, Populus balsamifera, Medicago truncatula) exhibited some sequence similarity with G974 outside of the signature AP2 domain sequence, and bear nearly identical AP2 domains. These proteins may be related.
G2343: (SEQ ID NOs. 53 and 54)
The complete sequence of G2343 was determined and G2343 was analyzed using transgenic plants in which G2343 was expressed under the control of the 35S promoter. The phenotype of these transgenic plants was wild-type in all assays performed. As determined by RT-PCR, G2343 is expressed in shoots, embryos and siliques. G2343 expression is induced in rosette leaves by auxin, heat stress, and infection by Fusarium oxysporum. 35S::G2343 had an altered seed oil content
G2343 is a related tomato gene LETHM1 (CAA64615). Similarity between G2343 and LETHM1 extends beyond the signature motif of the family to a level that would suggest the genes are orthologs.
G2123: (SEQ ID NOs. 67 and 68)
G2123 was analyzed using transgenic plants in which G2123 was expressed under the control of the 35S promoter. The phenotype of these transgenic plants was wild-type in all assays performed. G2123 was expressed primarily in developing seeds and silique tissue in wild-type plants. G2123 corresponds to a predicted putative 14-3-3 protein in annotated BAC clone T11I11 (AC012680), from chromosome 1 of Arabidopsis. 
G1777: (SEQ ID NOs. 55 and 56)
G1777 (SEQ ID NO: 55) was analyzed using transgenic plants in which G1777 was expressed under the control of the 35S promoter. Overexpression of G1777 in Arabidopsis resulted in an increase in seed oil content and a decrease in seed protein content in T2 lines 1 and 13. The change in seed oil in line 1 was just below the significance cutoff, but the seed protein change was significant. G1777 was expressed in all examined tissue of Arabidopsis. G1777 was induced by auxin and ABA treatment, and by heat stress. G1777 has utility in manipulating seed oil and protein content.
G2520: (SEQ ID NOs. 37 and 38)
G2520 was analyzed using transgenic plants in which G2520 was expressed under the control of the 35S promoter. At early stages, 35S::G2520 transformants displayed abnormal curled cotyledons, long hypocotyls, and rather short roots. During the vegetative phase, these plants formed somewhat small flat leaves. Following the switch to reproductive growth, 35S::G2520 inflorescences were typically very spindly, slightly pale colored, and stems often split open at late stages. Flowers were frequently small with narrow organs and showed poor pollen production. As a result, the seed yield from 35S::G2520 plants was low compared to wild-type controls. These effects were observed in the majority of primary transformants, and to varying extents, in all three of the T2 populations. Overexpression of G2520 also resulted in an increase in the leaf glucosinolate M39478 in lines 11 and 14. In addition, these lines showed an increase in seed δ-tocopherol and a decrease in seed γ-tocopherol. No altered phenotypes were detected in any of the physiological assays. G2520 was expressed throughout the plant and was induced by ABA, heat, salt, drought and osmotic stress. G2520 is useful for manipulating plant development and altering leaf glucosinolate composition. Increases or decreases in specific glucosinolates or total glucosinolate content are be 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 plant's natural defense against insects. Modification of glucosinolate composition or quantity can 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. G2520 can also be used to modify seed tocopherol composition. Tocopherols have anti-oxidant and vitamin E activity.
Example XII
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 and Henikoff (1992) Proc. Natl. Acad. Sci. USA 89: 10915-10919).
The polynucleotide and polypeptide sequences derived from monocots (e.g., the rice or maize sequences) may be used to transform both monocot and dicot plants, and those derived from dicots (e.g., the Arabidopsis and soy sequences) may be used to transform either group, although it is expected that some of these sequences will function best if the gene is transformed into a plant from the same class as that from which the sequence is derived.
Example XIII
Transformation of Dicots to Produce Improved Biochemical and Other Traits
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) supra; and Altschul et al. (1997) supra). The tblastx sequence analysis programs were employed using the BLOSUM-62 scoring matrix (Henikoff and Henikoff (1992) supra).
Crop species including tomato and soybean plants that overexpress any of a considerable number of the transcription factor polypeptides of the invention have been shown experimentally to produce plants with increased drought tolerance and/or biomass in field trials. For example, tomato plants overexpressing the G2153 polypeptide have been found to be larger than wild-type control tomato plants. For example, soy plants overexpressing a number of G481, G682, G867 and G1073 orthologs have been shown to be more drought tolerant than control plants. These observations indicate that these genes, when overexpressed, will result in larger yields than non-transformed plants in both stressed and non-stressed conditions.
Thus, transcription factor polynucleotide sequences listed in the Sequence Listing recombined into, for example, one of the expression vectors of the invention, or another suitable expression vector, may be transformed into a plant for the purpose of modifying plant traits for the purpose of improving yield and/or quality. The expression vector may contain a constitutive, tissue-specific or inducible promoter operably linked to the transcription factor polynucleotide. The cloning vector may be introduced into a variety of 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.
Numerous protocols for the transformation of tomato and soy plants have been previously described, and are well known in the art. Gruber et al. (1993) in Methods in Plant Molecular Biology and Biotechnology, p. 89-119, and Glick and Thompson (1993) Methods in Plant Molecular Biology and Biotechnology, eds., CRC Press, Inc., Boca Raton, describe several expression vectors and culture methods that may be used for cell or tissue transformation and subsequent regeneration. For soybean transformation, methods are described by Miki et al. (1993) in Methods in Plant Molecular Biology and Biotechnology, p. 67-88, Glick and Thompson, eds., CRC Press, Inc., Boca Raton; and U.S. Pat. No. 5,563,055, (Townsend and Thomas), issued Oct. 8, 1996.
There are a substantial number of alternatives to Agrobacterium-mediated transformation protocols, other methods for the purpose of transferring exogenous genes into soybeans or tomatoes. One such method is microprojectile-mediated transformation, in which DNA on the surface of microprojectile particles is driven into plant tissues with a biolistic device (see, for example, Sanford et al. (1987) Part. Sci. Technol. 5: 27-37; Christou et al. (1992) Plant. J. 2: 275-281; Sanford (1993) Methods Enzymol. 217: 483-509; Klein et al. (1987) Nature 327: 70-73; U.S. Pat. No. 5,015,580 (Christou et al), issued May 14, 1991; and U.S. Pat. No. 5,322,783 (Tomes et al.), issued Jun. 21, 1994).
Alternatively, sonication methods (see, for example, Zhang et al. (1991) Bio/Technology 9: 996-997); direct uptake of DNA into protoplasts using CaCl2 precipitation, polyvinyl alcohol or poly-L-ornithine (see, for example, Hain et al. (1985) Mol. Gen. Genet. 199: 161-168; Draper et al. (1982) Plant Cell Physiol. 23: 451-458); liposome or spheroplast fusion (see, for example, Deshayes et al. (1985) EMBO J., 4: 2731-2737; Christou et al. (1987) Proc. Natl. Acad. Sci. USA 84: 3962-3966); and electroporation of protoplasts and whole cells and tissues (see, for example, Donn et al. (1990) in Abstracts of VIIth International Congress on Plant Cell and Tissue Culture IAPTC, A2-38: 53; D'Halluin et al. (1992); and Spencer et al. (1994) Plant Mol. Biol. 24: 51-61) have been used to introduce foreign DNA and expression vectors into plants.
After a plant or plant cell is transformed (and the latter regenerated into a plant), the transformed plant may be crossed with itself or a plant from the same line, a non-transformed or wild-type plant, or another transformed plant from a different transgenic line of plants. Crossing provides the advantages of producing new and often stable transgenic varieties. Genes and the traits they confer that have been introduced into a tomato or soybean line may be moved into distinct line of plants using traditional backcrossing techniques well known in the art. Transformation of tomato plants may be conducted using the protocols of Koornneef et al (1986) In Tomato Biotechnology: Alan R. Liss, Inc., 169-178, and in U.S. Pat. No. 6,613,962, the latter method described in brief here. Eight day old cotyledon explants are precultured for 24 hours in Petri dishes containing a feeder layer of Petunia hybrida suspension cells plated on MS medium with 2% (w/v) sucrose and 0.8% agar supplemented with 10 μM α-naphthalene acetic acid and 4.4 μM 6-benzylaminopurine. The explants are then infected with a diluted overnight culture of Agrobacterium tumefaciens containing an expression vector comprising a polynucleotide of the invention for 5-10 minutes, blotted dry on sterile filter paper and cocultured for 48 hours on the original feeder layer plates. Culture conditions are as described above. Overnight cultures of Agrobacterium tumefaciens are diluted in liquid MS medium with 2% (w/v/) sucrose, pH 5.7) to an OD600 of 0.8.
Following cocultivation, the cotyledon explants are transferred to Petri dishes with selective medium comprising MS medium with 4.56 μM zeatin, 67.3 μM vancomycin, 418.9 μM cefotaxime and 171.6 μM kanamycin sulfate, and cultured under the culture conditions described above. The explants are subcultured every three weeks onto fresh medium. Emerging shoots are dissected from the underlying callus and transferred to glass jars with selective medium without zeatin to form roots. The formation of roots in a kanamycin sulfate-containing medium is a positive indication of a successful transformation.
Transformation of soybean plants may be conducted using the methods found in, for example, U.S. Pat. No. 5,563,055 (Townsend et al., issued Oct. 8, 1996), described in brief here. In this method soybean seed is surface sterilized by exposure to chlorine gas evolved in a glass bell jar. Seeds are germinated by plating on 1/10 strength agar solidified medium without plant growth regulators and culturing at 28° C. with a 16 hour day length. After three or four days, seed may be prepared for cocultivation. The seedcoat is removed and the elongating radicle removed 3-4 mm below the cotyledons.
Overnight cultures of Agrobacterium tumefaciens harboring the expression vector comprising a polynucleotide of the invention are grown to log phase, pooled, and concentrated by centrifugation. Inoculations are conducted in batches such that each plate of seed was treated with a newly resuspended pellet of Agrobacterium. The pellets are resuspended in 20 ml inoculation medium. The inoculum is poured into a Petri dish containing prepared seed and the cotyledonary nodes are macerated with a surgical blade. After 30 minutes the explants are transferred to plates of the same medium that has been solidified. Explants are embedded with the adaxial side up and level with the surface of the medium and cultured at 22° C. for three days under white fluorescent light. These plants may then be regenerated according to methods well established in the art, such as by moving the explants after three days to a liquid counter-selection medium (see U.S. Pat. No. 5,563,055).
The explants may then be picked, embedded and cultured in solidified selection medium. After one month on selective media transformed tissue becomes visible as green sectors of regenerating tissue against a background of bleached, less healthy tissue. Explants with green sectors are transferred to an elongation medium. Culture is continued on this medium with transfers to fresh plates every two weeks. When shoots are 0.5 cm in length they may be excised at the base and placed in a rooting medium.
Example XIV
Transformation of Cereal Plants with an Expression Vector
Cereal plants such as, but not limited to, corn, wheat, rice, sorghum, or barley, may be transformed with the present polynucleotide sequences, including monocot or dicot-derived sequences such as those presented in Tables 4-6, cloned into a vector such as pGA643 and containing a kanamycin-resistance marker, and expressed constitutively under, for example, the CaMV 35S or COR15 promoters, or with tissue-specific or inducible promoters. The expression vectors may be one found in the Sequence Listing, or any other suitable expression vector may be similarly used. 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 including direct DNA transfer or Agrobacterium tumefaciens-mediated transformation. The latter approach may be accomplished by a variety of means, including, for example, that of U.S. Pat. No. 5,591,616, in which monocotyledon callus is transformed by contacting dedifferentiating tissue with the Agrobacterium containing the cloning vector.
The sample tissues are immersed in a suspension of 3×10−9 cells of Agrobacterium containing the cloning vector for 3-10 minutes. The callus material is cultured on solid medium at 25° C. in the dark for several days. The calli grown on this medium are transferred to Regeneration medium. Transfers are continued every 2-3 weeks (2 or 3 times) until shoots develop. Shoots are then transferred to Shoot-Elongation medium every 2-3 weeks. Healthy looking shoots are transferred to rooting medium and after roots have developed, the plants are placed into moist potting soil.
The transformed plants are then analyzed for the presence of the NPTII gene/kanamycin resistance by ELISA, using the ELISA NPTII kit from 5Prime-3Prime Inc. (Boulder, Colo.).
It is also routine to use other methods to produce transgenic plants of most cereal crops (Vasil (1994) Plant Mol. Biol. 25: 925-937) such as corn, wheat, rice, sorghum (Cassas et al. (1993) Proc. Natl. Acad. Sci. 90: 11212-1121), and barley (Wan and Lemeaux (1994) Plant Physiol. 104: 37-48). DNA transfer methods such as the microprojectile method can be used for corn (Fromm et al. (1990) supra; Gordon-Kamm et al. (1990) supra; Ishida (1990) Nature Biotechnol. 14: 745-750), wheat (Vasil et al. (1992) Bio/Technol. 10: 667-674; Vasil et al. (1993a) Bio/Technology 10: 667-674; Vasil et al. (1993b) Bio/Technol. 11: 1553-1558; Weeks et al. (1993) supra), and rice (Christou (1991) Bio/Technology 9: 957-962; Hiei et al. (1994) Plant J. 6: 271-282; Aldemita and Hodges (1996) Planta 199: 612-617; and Hiei et al. (1997) Plant Mol. Biol. 35: 205-218). For most cereal plants, embryogenic cells derived from immature scutellum tissues are the preferred cellular targets for transformation (Hiei et al. (1997) supra; Vasil (1994) supra). For transforming corn embryogenic cells derived from immature scutellar tissue using microprojectile bombardment, the A188XB73 genotype is the preferred genotype (Fromm et al. (1990) supra; Gordon-Kamm et al. (1990) supra). After microprojectile bombardment the tissues are selected on phosphinothricin to identify the transgenic embryogenic cells (Gordon-Kamm (1990) Plant Cell 2: 603-618). Transgenic plants are regenerated by standard corn regeneration techniques (Fromm et al. (1990) supra; Gordon-Kamm et al. (1990) supra).
Example XV
Transcription Factor Expression and Analysis of Improved Traits
Biochemical assays such as those disclosed above may be used to identify improved characteristics in any of the transgenic or knock plants produced with sequences of the invention, such as polynucleotides SEQ ID NO: 2n−1, wherein n=1-84, or SEQ ID NO: 2n, wherein n=121-127.
Northern blot analysis, RT-PCR or microarray analysis of the regenerated, transformed plants may also be used to show expression of a transcription factor polypeptide or the invention and related genes that are capable of inducing improved biochemical characteristics, abiotic stress tolerance, and/or larger size.
To verify the ability to confer stress resistance, mature plants overexpressing a transcription factor of the invention, or alternatively, seedling progeny of these plants, may be challenged by a stress such as drought, heat, cold, high salt, or desiccation. Alternatively, these plants may challenged in a hyperosmotic stress condition that may also measure altered sugar sensing, such as a high sugar condition. By comparing control plants (for example, wild type) and transgenic plants similarly treated, the transgenic plants may be shown to have greater tolerance to the particular stress.
After a dicot plant, monocot plant or plant cell has been transformed (and the latter regenerated into a plant) and shown to have improved biochemical characteristics, greater size or tolerance to abiotic stress, or produce greater yield relative to a control plant under the stress conditions, the transformed monocot plant may be crossed with itself or a plant from the same line, a non-transformed or wild-type monocot plant, or another transformed monocot plant from a different transgenic line of plants.
These experiments would demonstrate that transcription factor polypeptides of the invention can be identified and shown to confer improved biochemical characteristics, larger size, greater yield, and/or abiotic stress tolerance in dicots or monocots, including multiple improved biochemical characteristics and/or tolerance to multiple stresses.
It is expected that the same methods may be applied to identify other useful and valuable sequences of the present transcription factor clades, and the sequences may be derived from a diverse range of species.
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.

## Claims

1. A transgenic plant comprising a recombinant construct encoding a polypeptide fragment of SEQ ID NO:136 that has a conserved domain comprising amino acids 135-195 of SEQ ID NO:136, wherein the polypeptide fragment confers increased yield in the transgenic plant as compared to a control plant that does not contain the recombinant construct.

2. A transgenic seed produced by the transgenic plant according to claim 1, wherein the transgenic seed comprises the recombinant construct.

3. A method for increasing plant yield, the method comprising:
introducing a recombinant construct into a target plant to produce a transgenic plant, wherein the recombinant construct encodes a polypeptide fragment of SEQ ID NO:136 that has a conserved domain comprising amino acids 135-195 of SEQ ID NO:136;
wherein expression of the polypeptide fragment in the transgenic plant results in the transgenic plant producing an increased yield as compared to a control plant that does not contain the recombinant construct.

4. The method of claim 3, wherein the transgenic plant produces a transgenic seed that comprises the recombinant construct.

5. A plant part of the transgenic plant of claim 1, wherein the plant part comprises an expression cassette that comprises the recombinant construct, and wherein the plant part is selected from seed, fruit, leaf, root, plant tissue, a plant explant, vascular tissue, ground tissue, or plant cells.

6. A plant part of the transgenic plant produced by the method of claim 3, wherein the plant part comprises an expression cassette that comprises the recombinant construct, and wherein the plant part is selected from seed, fruit, leaf, root, plant tissue, a plant explant, vascular tissue, ground tissue, or plant cells.

7. The method of claim 3, wherein the method further comprises selecting a transgenic plant comprising the polypeptide fragment and having increased yield as compared to a control plant that does not contain the recombinant construct.

8. The transgenic plant of claim 1, wherein the plant is a corn plant.