DESCRIPTION

METHODS AND COMPOSITIONS FOR MODULATING ROOT GROWTH IN PLANTS

CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application Serial No. 60/705,620, filed August 4, 2005; the disclosure of which is incorporated herein by reference in its entirety.

TECHNICAL FIELD

The presently disclosed subject matter relates to methods and compositions for modulating root growth in plants. In particular, the presently disclosed subject matter relates to modulating root growth in plants by modulating expression of Armadillo repeat-containing (ARC) proteins in plants.

BACKGROUND

Animal β-catenin and its Drosophila homologue Armadillo (Arm) are multifunctional proteins required for embryonic development and with roles throughout adult life, β-catenin and Armadillo specify cell fates by directly regulating gene expression. They are also essential for cell-cell adhesion, being components of actin-based intercellular junctions.

Cytosolic β-catenin/Armadillo protein is targeted for destruction by the proteasome unless stabilized by extracellular Wnt/Wingless signals. Wnt signals allow β-catenin to translocate to the nucleus and interact with LEF/TCF family transcription factors. This complex of proteins activates the expression of target genes. Direct β-catenin targets in animals include cell growth regulators such as cyclin D1, c-myc and c-jun; developmental regulators such as siamois, ultrabithorax, and LEF-1 itself; and cell adhesion regulators such as laminin, matrix metalloproteases, and Nr-CAM. β-catenin/Armadillo is also required for the formation of adherens junctions, sites of cell-cell adhesion where transmembrane cadherin molecules

are linked to the actin cytoskeleton via interactions with α- and-β-catenin. This adhesive function of Armadillo/β-catenin is required for normal development. The functions of β-catenin both in Wnt signaling and in cell adhesion are conserved throughout the animal kingdom, although its modes of regulation have changed during evolution. In the multicellular protist Dictyostelium discoideum, a β-catenin-related protein, Aardvark, promotes prespore gene expression and is also a component of actin-containing cell-cell junctions (Grimson et al. 2000). This demonstrates that β-catenin function is also conserved outside the animal kingdom. β-catenin/Armadillo is part of a large multifunctional family of metazoan

Armadillo-repeat-containing proteins that regulate cell signaling, the cytoskeleton, and protein-protein interactions. A family of over 100 Armadillo- repeat-containing (ARC) proteins is also present in the plant kingdom (Coates 2003, Mudgil et al. 2004). Very little is known about the functions of the vast majority of these proteins. The few plant ARC proteins studied so far have diverse functions, for example in light/gibberellin signaling in potato (Amador et al. 2001 ), the self-incompatibility response in Brassica (Stone eif al. 2003), trichome development and endoreduplication in Arabidopsis (Downes et al. 2003, El Refy et al. 2003), abscisic acid signaling (Kim et al. 2004) and putatively developmental signaling in Nicotiana (Kim et al. 2003). However, apart from ARC1 (Stone et al. 2003), the mechanisms by which these proteins function is largely unknown.

Therefore, there is presently an unmet need for further study and determination of the functions of plant ARC proteins, particularly of those ARC proteins having a role in plant development and/or phenotype.

SUMMARY

This Summary lists several embodiments of the presently disclosed subject matter, and in many cases lists variations and permutations of these embodiments. This Summary is merely exemplary of the numerous and varied embodiments. Mention of one or more representative features of a given embodiment is likewise exemplary. Such an embodiment can typically exist with or without the feature(s) mentioned; likewise, those features can be applied

to other embodiments of the presently disclosed subject matter, whether listed in this Summary or not. To avoid excessive repetition, this Summary does not list or suggest all possible combinations of such features.

The presently disclosed subject matter provides in some embodiments methods of modulating root growth of a plant. In some embodiments, the methods comprise modulating expression of an Armadillo repeat-containing

(ARC) polypeptide in the plant. In some embodiments, the methods comprise administering an effective amount of an ARC polypeptide to the plant.

The presently disclosed subject matter provides in some embodiments methods of improving abiotic stress tolerance in a plant. In some embodiments, the methods comprise providing a plant susceptible to abiotic stress and modulating expression of an ARC polypeptide in the plant. In some embodiments, the methods comprise providing a plant susceptible to abiotic stress and administering to the plant an effective amount of an ARC polypeptide.

The presently disclosed subject matter provides in some embodiments methods of producing a heterologous ARC polypeptide in a plant cell. In some embodiments, the methods comprise providing a plant cell comprising a nucleic acid sequence encoding a heterologous ARC polypeptide operatively linked to a promoter; and expressing in the plant cell the nucleic acid sequence encoding the heterologous ARC polypeptide, whereby the heterologous ARC polypeptide is produced in the plant cell.

In some embodiments of the methods, modulating expression comprises expressing in the plant an expression cassette comprising a nucleic acid molecule encoding the ARC polypeptide. In some embodiments, modulating expression comprises overexpressing in the plant the nucleic acid molecule encoding the ARC polypeptide. Further, in some embodiments, modulating expression comprises expressing the ARC polypeptide in the plant root.

In some embodiments of the methods, administering comprises contacting one or more roots of the plant with the ARC polypeptide.

In some embodiments of the methods, the ARC polypeptide comprises an F-box motif and at least one Armadillo repeat region. Further, in some embodiments, the polypeptide is selected from the group consisting of: (a) a

polypeptide encoded by a nucleic acid sequence as set forth in any of SEQ ID NOs: 3, 4, and 6; (b) a polypeptide encoded by a nucleic acid having at least about 75% identity to a DNA sequence as set forth in any of SEQ ID NOs: 3, 4, and 6 and having root growth modulating activity; (c) a polypeptide encoded by a nucleic acid capable of hybridizing under stringent conditions to a nucleic acid comprising a sequence or the complement of a sequence as set forth in any of SEQ ID NOs: 3, 4, and 6; (d) a polypeptide which is a biologically functional equivalent of a peptide as set forth in any of SEQ ID NOs: 1 , 2, and 5 and has at least about 75% identity to a peptide as set forth in any of SEQ ID NOs: 1 , 2, and 5; and (e) a polypeptide comprising a functional fragment of a polypeptide of (a), (b), (c), or (d). Further, in some embodiments, the ARC polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 , 2, and 5, or a functional fragment thereof.

In some embodiments of the methods, the plant is a monocot. Further, in some embodiments, the plant is selected from the group consisting of rice, maize, wheat, barley, rye, oats, millet, triticale, secale, einkorn, spelt, emmer, teff, milo, flax, gramma grass, Tήpsacum, teosinte, Arabidopsis, potato, canola,

' soybean, sunflower, carrot, sweet potato, sugarbeet, bean, pea, chicory, lettuce, cabbage, cauliflower, broccoli, turnip, radish, spinach, asparagus, onion, garlic, eggplant, pepper, celery, squash, pumpkin, cucumber, apple, pear, quince, melon, plum, cherry, peach, nectarine, apricot, strawberry, grape, raspberry, blackberry, pineapple, avocado, papaya, mango, banana, soybean, tobacco, tomato, sorghum and sugarcane.

The presently disclosed subject matter provides in some embodiments an expression cassette for modulating root growth of a plant, comprising an isolated nucleic acid molecule encoding an ARC polypeptide. The presently disclosed subject matter further provides a recombinant vector comprising the expression cassette. The presently disclosed subject matter still further provides a transgenic plant (and progeny therefrom), seed, plant cell, or tissue comprising the expression cassette. Further, in some embodiments, the transgenic plant has improved abiotic stress tolerance, which can improve in the plant tolerance to inappropriate amounts of nutrients, drought, excessive cold, excessive heat, excessive soil salinity, extreme acidity or alkalinity,

alterations in plant architecture, alteration in plant development, or combinations thereof.

Accordingly, it is an object of the presently disclosed subject matter to provide methods and compositions for modulating root growth in plants. This object is achieved in whole or in part by the presently disclosed subject matter. An object of the presently disclosed subject matter having been stated above, other objects and advantages will become apparent to those of ordinary skill in the art after a study of the following description of the presently disclosed subject matter and non-limiting examples.

BRIEF DESCRIPTION OF THE DRAWING

Figure 1 shows Arabidillo-1 (SEQ ID NO:1 ) and Arabidillo -2 (SEQ ID

NO:2) proteins. Arabidillo-1 and -2 are Arabidopsis ARC proteins. F-boxes

(residues 45-93 in Arabidillo-1 , 38-85 in Arabidillo-2) are shown in black; Arm repeats are shown in light gray. Nuclear-localization sequences (residues 3-8) are marked with an arrow. Numbers indicate amino acids.

BRIEF DESCRIPTION OF THE SEQUENCE LISTING SEQ ID NO:1 is the amino acid sequence of Arabidillo-1 from Arabidopsis thaliana.

SEQ ID NO:2 is the amino acid sequence of Arabidillo-2 from Arabidopsis thaliana.

SEQ ID NO:3 is the polynucleotide sequence encoding Arabidillo-1 from Arabidopsis thaliana. SEQ ID NO:4 is the polynucleotide sequence encoding Arabidillo-2 from

Arabidopsis thaliana.

SEQ ID NO:5 is the amino acid sequence of an ARC protein from Oryza sativa.

SEQ ID NO:6 is the polynucleotide sequence encoding the ARC protein of SEQ ID NO:5.

DETAILED DESCRIPTION

The details of one or more embodiments of the presently disclosed subject matter are set forth in the accompanying description below. Other features, objects, and advantages of the presently disclosed subject matter will be apparent from the detailed description and claims. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. Some of the polynucleotide and polypeptide sequences disclosed herein are cross-referenced to GENBANK ^® accession numbers. The sequences cross-referenced in the GENBANK ^® database are expressly incorporated by reference as are equivalent and related sequences present in GENBANK ^® or other public databases. Also expressly incorporated herein by reference are all annotations present in the GENBANK ^® database associated with the sequences disclosed herein. In case of conflict, the present specification, including definitions, will control. Armadillo/β-catenin and related proteins have important developmental functions in both animals and Dictyostelium, regulating cell differentiation, proliferation and adhesion. Armadillo repeat-containing (ARC) proteins also exist in plants, but the majority of them have unknown roles.

The present subject matter discloses the discovery through functional genomics studies of root development functions of a class of ARC plant proteins homologous to metazoan and Dictyostelium β-catenins. The ARC plant proteins disclosed herein having homology to /?-catenins are unique over the general class of plant Arm repeat proteins and can have an F-box motif as well as Arm repeat regions, and therefore fall into a phylogenetically distinct subgroup.

The presently disclosed subject matter discloses for the first time that ARC proteins can regulate root growth and branching in plants. A plant's root system is composed of a primary root that is formed during embryonic development, and lateral roots that emerge from the primary root of the adult plant throughout its life. The shape of a plant's root architecture determines its ability to acquire water and nutrients from the soil, and also its stability in the ground. Root architecture is regulated dynamically by many signals, including plant hormones and the nutritional status of the plant, allowing the plant to

adapt to its changing environment.

In a particular embodiment of the presently disclosed subject matter, it is demonstrated that exemplary ARC proteins from Arabidopsis, Arabidillo-1 and Arabidillo-2, promote lateral root development in Arabidopsis. As demonstrated by the experimental data disclosed in the Examples, arabidillo-1 /-2 mutant plants form fewer lateral roots than wild type, due to a lack of initiation of lateral root primordia. Conversely, Arabidillo-1 overexpressing plants produce an elevated number of lateral roots. Further, as disclosed in detail in the Examples, arabidillo-1 and -2 mutants and Arabidillo-overexpressing seedlings can respond normally to exogenous application of known lateral root regulating signals such as auxin, suggesting that they regulate root branching by a novel mechanism.

The Examples further demonstrate that Arabidillo-1 and -2 can localize to the nucleus. Protein deletion analysis studies disclosed in the Examples show that different parts of Arabidillo-1 protein can localize to different subcellular compartments, suggesting that its localization can depend on the availability of its interaction partners, similarly to the homologous β-catenin proteins.

Thus, the presently disclosed subject matter provides for the first time a discovered function for plant ARC proteins having homology to /?-catenins, such as for example Arabidillo-1 and Arabidillo-2 and related homologues and orthologs, showing for the first time that plant β-catenin-related proteins regulate root development. Like β-catenin, these ARC proteins can contain both nuclear and cytosolic targeting information. As Arabidillo-1 and -2, for example, do not appear to function directly within previously characterized lateral root regulatory pathways, the ARC proteins likely define a novel mechanism to promote root development and branching, which can provide significant agronomic benefits.

I General Considerations

A goal of functional genomics is to identify genes controlling expression of organismal phenotypes, and functional genomics employs a variety of methodologies including, but not limited to, bioinformatics, gene expression

studies, gene and gene product interactions, genetics, biochemistry, and molecular genetics. For example, bioinformatics can assign function to a given gene by identifying genes in heterologous organisms with a high degree of similarity (homology) at the amino acid or nucleotide level. Studies of the expression of a gene at the mRNA or polypeptide levels can assign function by linking expression of the gene to an environmental response, a developmental process, or a genetic (mutational) or molecular genetic (gene overexpression or underexpression) perturbation. Expression of a gene at the mRNA level can be ascertained either alone (for example, by Northern analysis) or in concert with other genes (for example, by microarray analysis), whereas expression of a gene at the polypeptide level can be ascertained either alone (for example, by native or denatured polypeptide gel or immunoblot analysis) or in concert with other genes (for example, by proteomic analysis). Knowledge of polypeptide/polypeptide and polypeptide/DNA interactions can assign function by identifying polypeptides and nucleic acid sequences acting together in the same biological process. Genetics can assign function to a gene by demonstrating that DNA lesions (mutations) in the gene have a quantifiable effect on the organism, including, but not limited to, its development; hormone biosynthesis and response; growth and growth habit (plant architecture); mRNA expression profiles; polypeptide expression profiles; ability to resist diseases; tolerance of abiotic stresses (for example, drought conditions); ability to acquire nutrients; photosynthetic efficiency; altered primary and secondary metabolism; and the composition of various plant organs. Biochemistry can assign function by demonstrating that the polypeptide(s) encoded by the gene, typically when expressed in a heterologous organism, possesses a certain activity, either alone or in combination with other polypeptides. Molecular genetics can assign function by overexpressing or underexpressing the gene in the native plant or in heterologous organisms, and observing quantifiable effects as disclosed in functional assignment by genetics above. In functional genomics, any or all of these approaches are utilized, often in concert, to assign functions to genes across any of a number of organismal phenotypes.

It is recognized by those skilled in the art that these different methodologies can each provide data as evidence for the function of a

particular gene, and that such evidence is stronger with increasing amounts of data used for functional assignment: in some embodiments from a single methodology, in some embodiments from two methodologies, and in some embodiments from more than two methodologies. In addition, those skilled in the art are aware that different methodologies can differ in the strength of the evidence provided for the assignment of gene function. Typically, but not always, a datum of biochemical, genetic, or molecular genetic evidence is considered stronger than a datum of bioinformatic or gene expression evidence. Finally, those skilled in the art recognize that, for different genes, a single datum from a single methodology can differ in terms of the strength of the evidence provided by each distinct datum for the assignment of the function of these different genes.

The objective of crop trait functional genomics is to identify crop trait genes of interest, for example, genes capable of conferring useful agronomic traits in crop plants. Such agronomic traits include, but are not limited to, enhanced yield, whether in quantity or quality; enhanced nutrient acquisition and metabolic efficiency; enhanced or altered nutrient composition of plant tissues used for food, feed, fiber, or processing; enhanced utility for agricultural or industrial processing; enhanced resistance to plant diseases; enhanced tolerance of adverse environmental conditions (abiotic stresses) including, but not limited to, inappropriate amounts of nutrients, drought, excessive cold, excessive heat, or excessive soil salinity or extreme acidity or alkalinity; and alterations in plant architecture or development, including changes in developmental timing. The deployment of such identified trait genes by either transgenic or non-transgenic approaches can materially improve crop plants for the benefit of agriculture.

Cereals are the most important crop plants on the planet in terms of both human and animal consumption. Genomic synteny (conservation of gene order within large chromosomal segments) is observed in rice, maize, wheat, barley, rye, oats, and other agriculturally important monocots including sorghum (see e.g., Kellogg, 1998; Song etal., 2001 , and references therein), which facilitates the mapping and isolation of orthologous genes from diverse cereal species based on the sequence of a single cereal gene. Rice has the smallest (about

420 Mb) genome among the cereal grains, and has recently been a major focus of public and private genomic and EST sequencing efforts. See Goff et al., 2002.

The mechanisms of plant adaptations to abiotic stresses include both avoidance and tolerance. Drought avoidance mechanisms, for example, are generally constitutive phenotypic properties such as epicuticular wax structure and root thickness or root depth (Svenningson, 1988, Price et al. , 2002), which are expressed in the presence or absence of stress conditions. On the other hand, drought tolerance is an adaptive response that enables plants to maintain metabolism even at low water potential (Ingram & Bartels, 1996). These plants respond to water stress through wide spread changes in cellular metabolism which involves changes in thousands of genes.

IL Definitions Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the presently disclosed subject matter pertains. For clarity of the present specification, certain definitions are presented hereinbelow.

Following long-standing patent law convention, the terms "a" and "an" mean "one or more" when used in this application, including in the claims.

As used herein, the term "Armadillo (Arm) repeat" protein, or region, refers to a protein region that contains one or more tandem copies of an approximately 40 amino acid long degenerate protein sequence motif that forms a conserved three-dimensional structure. Animal Arm repeat proteins function in various processes, including intracellular signaling and cytoskeletal regulation. A subset of these proteins are conserved across eukaryotic kingdoms, and non-metazoa such as Dictyostelium and Chlamydomonas possess homologues of members of the animal Arm repeat family. Higher plants also possess Arm repeat proteins, which, like their animal counterparts, can function in intracellular signaling. See also Coates 2003 for a further description of Arm repeats.

As used herein, the phrase "improved abiotic stress tolerance" refers to a state wherein a plant exhibits a different and preferred response to one or more

abiotic stresses (for example, but not limited to, inappropriate amounts of nutrients, drought, excessive cold, excessive heat, excessive soil salinity or extreme acidity or alkalinity; and alterations in plant architecture or development) than does another plant of the same species. As used herein, the phrase "improving an abiotic stress tolerance" refers in some embodiments to a manipulation of a plant's genome to produce a recombinant or transgenic plant in which the manipulation results in a change in the plant's abiotic stress tolerance.

As used herein, the terms "associated with" and "operatively linked" refer to two nucleic acid sequences that are related physically or functionally. For example, a promoter or regulatory DNA sequence is said to be "operatively linked" to a DNA sequence that encodes an RNA or a polypeptide if the two sequences are situated such that the regulator DNA sequence will affect the expression level of the coding or structural DNA sequence. As used herein, the terms "coding sequence" and "open reading frame"

(ORF) are used interchangeably and refer to a nucleic acid sequence that is transcribed into RNA such as mRNA, rRNA, tRNA, snRNA, sense RNA, or antisense RNA. In some embodiments, the RNA is then translated in vivo or in vitro to produce a polypeptide. As used herein, the term "complementary" refers to two nucleotide sequences that comprise antiparallel nucleotide sequences capable of pairing with one another upon formation of hydrogen bonds between the complementary base residues in the antiparallel nucleotide sequences. As is known in the art, the nucleic acid sequences of two complementary strands are the reverse complement of each other when each is viewed in the 5' to 3' direction.

As is also known in the art, two sequences that hybridize to each other under a given set of conditions do not necessarily have to be 100% fully complementary. As used herein, the terms "fully complementary" and "100% complementary" refer to sequences for which the complementary regions are 100% in Watson-Crick base-pairing, i.e., that no mismatches occur within the complementary regions. However, as is often the case with recombinant molecules (for example, cDNAs) that are cloned into cloning vectors, certain of

these molecules can have non-complementary overhangs on either the 5' or 3' ends that result from the cloning event. In such a situation, it is understood that the region of 100% or full complementarity excludes any sequences that are added to the recombinant molecule (typically at the ends) solely as a result of, or to facilitate, the cloning event. Such sequences are, for example, polylinker sequences, linkers with restriction enzyme recognition sites, etc.

As used herein, the terms "domain", "motif, and "feature", when used in reference to a polypeptide or amino acid sequence, refers to a subsequence or region of an amino acid sequence that has a particular biological function and/or primary sequence or secondary structure. Domains, motifs, and features that have a particular biological function include, but are not limited to, ligand binding, nucleic acid binding, catalytic activity, substrate binding, and polypeptide-polypeptide interacting domains. Similarly, when used herein in reference to a nucleic acid sequence, a "domain", "motif or "feature" is that subsequence of the nucleic acid sequence that encodes a domain or feature of a polypeptide. Exemplary domains, motifs and features particularly relevant to the presently disclosed subject matter include F-box motifs and Armadillo repeat regions.

As used herein, the term "expression cassette" refers to a nucleic acid molecule capable of directing expression of a particular nucleotide sequence in an appropriate host cell, comprising a promoter operatively linked to the nucleotide sequence of interest which is operatively linked to termination signals. It also typically comprises sequences required for proper translation of the nucleotide sequence. The coding region usually encodes a polypeptide of interest but can also encode a functional RNA of interest, for example antisense RNA or a non-translated RNA, in the sense or antisense direction. The expression cassette comprising the nucleotide sequence of interest can be chimeric, meaning that at least one of its components is heterologous with respect to at least one of its other components. The expression cassette can also be one that is naturally occurring but has been obtained in a recombinant form useful for heterologous expression. Typically, however, the expression cassette is heterologous with respect to the host; i.e., the particular DNA sequence of the expression cassette does not occur naturally in the host cell

and was introduced into the host cell or an ancestor of the host cell by a transformation event. The expression of the nucleotide sequence in the expression cassette can be under the control of a constitutive promoter or of an inducible promoter that initiates transcription only when the host cell is exposed to some particular external stimulus. In the case of a multicellular organism such as a plant, the promoter can also be specific to a particular tissue or stage of development.

As used herein, the term "F-box motif refers to a protein motif of approximately 50 amino acids that functions as a site of protein-protein interaction. There are few invariant amino acid positions that characterize the F-box motif. See e.g., Kipreos & Pagano Genome, 2000 for a review of F-box motifs. This lack of a strict consensus makes identification by eye difficult. However, computer search algorithms are readily available that can detect F- boxes. For example, two search algorithms that can be used for F-box detection are found in the Prosite and Pfam databases, which are well known to those of skill in the art and can be accessed, for example, at the web page of the ISREC ProfileScan Server. One or both databases can be used to search for F-box motifs. However, a more accurate determination of F-box motifs can sometimes be acquired using both databases as a check of accuracy upon one another.

As used herein, the term "fragment" refers to a sequence that comprises a subset of another sequence. When used in the context of a nucleic acid or amino acid sequence, the terms "fragment" and "subsequence" are used interchangeably. A fragment of a nucleic acid sequence can be any number of nucleotides that is less than that found in another nucleic acid sequence, and thus includes, but is not limited to, the sequences of an exon or intron, a promoter, an enhancer, an origin of replication, a 5' or 3' untranslated region, a coding region, and a polypeptide binding domain. It is understood that a fragment or subsequence can also comprise less than the entirety of a nucleic acid sequence, for example, a portion of an exon or intron, promoter, enhancer, etc. Similarly, a fragment or subsequence of an amino acid sequence can be any number of residues that is less than that found in a naturally occurring polypeptide, and thus includes, but is not limited to, domains, features, repeats,

etc. Also similarly, it is understood that a fragment or subsequence of an amino acid sequence need not comprise the entirety of the amino acid sequence of the domain, feature, repeat, etc. A fragment can also be a "functional fragment", in which the fragment retains a specific biological function of the nucleic acid sequence or amino acid sequence of interest. For example, a functional fragment of a transcription factor can include, but is not limited to, a DNA binding domain, a transactivating domain, or both. A functional fragment of an ARC protein, for example, retains at least some root growth modulating activity. As used herein, the term "gene" is used broadly to refer to any segment of DNA associated with a biological function, such as for example a root growth modulating activity. Thus, genes include, but are not limited to, coding sequences and/or the regulatory sequences required for their expression. Genes can also include non-expressed DNA segments that, for example, form recognition sequences for a polypeptide. Genes can be obtained from a variety of sources, including cloning from a source of interest or synthesizing from known or predicted sequence information, and can include sequences designed to have desired parameters.

The terms "heterologous", "recombinant", and "exogenous", when used herein to refer to a nucleic acid sequence (e.g. a DNA sequence) or a gene, refer to a sequence that originates from a source foreign to the particular host cell or, if from the same source, is modified from its original form. Thus, a heterologous gene in a host cell includes a gene that is endogenous to the particular host cell but has been modified through recombinant techniques. The terms also include non-naturally occurring multiple copies of a naturally occurring DNA sequence. Thus, the terms refer to a DNA segment that is foreign or heterologous to the cell, or homologous to the cell but in a position or form within the host cell in which the element is not ordinarily found. Similarly, when used in the context of a polypeptide or amino acid sequence, an exogenous polypeptide or amino acid sequence is a polypeptide or amino acid sequence that originates from a source foreign to the particular host cell or, if from the same source, is modified from its original form. Thus, exogenous DNA segments can be expressed to yield exogenous polypeptides.

A "homologous" nucleic acid (or amino acid) sequence is a nucleic acid (or amino acid) sequence naturally associated with a host ceil into which it is introduced.

The phrase "hybridizing specifically to" refers to the binding, duplexing, or hybridizing of a molecule only to a particular nucleotide sequence under stringent conditions when that sequence is present in a complex mixture (e.g., total cellular) DNA or RNA. The phrase "bind(s) substantially" refers to complementary hybridization between a probe nucleic acid and a target nucleic acid and embraces minor mismatches that can be accommodated by reducing the stringency of the hybridization media to achieve the desired detection of the target nucleic acid sequence.

As used herein, the term "isolated", when used in the context of an isolated DNA molecule or an isolated polypeptide, is a DNA molecule or polypeptide that, by the hand of man, exists apart from its native environment and is therefore not a product of nature. An isolated DNA molecule or polypeptide can exist in a purified form or can exist in a non-native environment such as, for example, in a transgenic host cell.

As used herein, the term "minimal promoter" refers to the smallest piece of a promoter, such as a TATA element, that can support any transcription. A minimal promoter typically has greatly reduced promoter activity in the absence of upstream or downstream activation. In the presence of a suitable transcription factor, a minimal promoter can function to permit transcription. As used herein, the term "native" refers to a gene that is naturally present in the genome of an untransformed plant cell. Similarly, when used in the context of a polypeptide, a "native polypeptide" is a polypeptide that is encoded by a native gene of an untransformed plant cell's genome.

As used herein, the term "naturally occurring" refers to an object that is found in nature as distinct from being artificially produced by man. For example, a polypeptide or nucleotide sequence that is present in an organism (including a virus) in its natural state, which has not been intentionally modified or isolated by man in the laboratory, is naturally occurring. As such, a polypeptide or nucleotide sequence is considered "non-naturally occurring" if it is encoded by or present within a recombinant molecule, even if the amino acid

or nucleic acid sequence is identical to an amino acid or nucleic acid sequence found in nature.

As used herein, the term "nucleic acid" refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g. degenerate codon substitutions) and complementary sequences and as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al., 1991 ; Ohtsuka et al., 1985; Rossolini et al. , 1994). The terms "nucleic acid" or "nucleic acid sequence" can also be used interchangeably with gene, cDNA, and mRNA encoded by a gene.

As used herein, the term "orthologs" refers to genes in different species that encode proteins that perform the same biological function. For example, the glucose-6-phosphate dehydrogenase genes from, for example, sorghum and rice, are orthologs. Typically, orthologous nucleic acid sequences are characterized by a high degree of sequence similarity (for example, at least about 75% sequence identity). Orthologous nucleic acid sequences may also be characterized by a high degree of similarity in protein functional domains. A nucleic acid sequence of an ortholog in one species (for example, rice) can be used to isolate the nucleic acid sequence of the ortholog in another species (for example, sorghum) using standard molecular biology techniques. This can be accomplished, for example, using techniques generally known to those of skill in the art (see e.g. Sambrook & Russell, 2001 for a discussion of hybridization conditions that can be used to isolate closely related sequences). As used herein, the phrase "percent identical"," in the context of two nucleic acid or polypeptide sequences, refers to two or more sequences or subsequences that have in some embodiments 60%, in some embodiments 70%, in some embodiments 80%, in some embodiments 90%, in some

embodiments 95%, and in some embodiments at least 99% nucleotide or amino acid residue identity, respectively, when compared and aligned for maximum correspondence, as measured using one of the following sequence comparison algorithms or by visual inspection. The percent identity exists in some embodiments over a region of the sequences that is at least about 50 residues in length, in some embodiments over a region of at least about 100 residues, and In some embodiments, the percent identity exists over at least about 150 residues. In some embodiments, the percent identity exists over the entire length of the sequences. For sequence comparison, typically one sequence acts as a reference sequence to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters.

Optimal alignment of sequences for comparison can be conducted, for example, by the local homology algorithm disclosed in Smith & Waterman, 1981 , by the homology alignment algorithm disclosed in Needleman & Wunsch, 1970, by the search for similarity method disclosed in Pearson & Lipman, 1988, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the GCG® Wisconsin Package®, available from Accelrys, Inc., San Diego, California, United States of America), or by visual inspection. See generally, Ausubel et al., 1994. One example of an algorithm that is suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm, which is described in Altschul et al., 1990. Software for performing BLAST analysis is publicly available through the website of the National Center for Biotechnology Information. 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. See generally, Altschul et al. , 1990.

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 word length (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 word length (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix. See Henikoff & Henikoff, 1992.

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). 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 test nucleic acid sequence is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid sequence to the reference nucleic acid sequence is in some embodiments less than about 0.1 , in some embodiments less than about 0.01 , and in some embodiments less than about 0.001. The term "substantially identical", in the context of two nucleotide or amino acid sequences, refers to two or more sequences or subsequences that have in some embodiments at least about 60% nucleotide or amino acid identity, in some embodiments at least about 65% nucleotide or amino acid

identity, in some embodiments at least about 70% nucleotide or amino acid identity, in some embodiments at least about 75% nucleotide or amino acid identity, in some embodiments at least about 80% nucleotide or amino acid identity, in some embodiments at least about 85% nucleotide or amino acid identity, in some embodiments at least about 90% nucleotide or amino acid identity, in some embodiments at least about 95% nucleotide or amino acid identity, in some embodiments at least about 97% nucleotide or amino acid identity, and in some embodiments at least about 99% nucleotide or amino acid identity, when compared and aligned for maximum correspondence, as measured using one of the above-referenced sequence comparison algorithms or by visual inspection. In some embodiments, the substantial identity exists in nucleotide or amino acid sequences of at least 50 residues, in some embodiments in nucleotide or amino acid sequence of at least about 100 residues, in some embodiments in nucleotide or amino acid sequences of at least about 150 residues, and in some embodiments in nucleotide or amino acid sequences comprising complete coding sequences or complete amino acid sequences.

In one aspect, polymorphic sequences can be substantially identical sequences. The term "polymorphic" refers to the two or more genetically determined alternative sequences or alleles in a population. An allelic difference can be as small as one base pair. Nonetheless, one of ordinary skill in the art would recognize that the polymorphic sequences correspond to the same gene.

Another indication that two nucleotide sequences are substantially identical is that the two molecules specifically or substantially hybridize to each other under conditions of medium or high stringency. In the context of nucleic acid hybridization, two nucleic acid sequences being compared can be designated a "probe sequence" and a "target sequence". A "probe sequence" is a reference nucleic acid molecule, and a "'target sequence" is a test nucleic acid molecule, often found within a heterogeneous population of nucleic acid molecules. A "target sequence" is synonymous with a "test sequence".

An exemplary nucleotide sequence employed for hybridization studies or assays includes probe sequences that are complementary to or mimic in some

embodiments at least an about 14 to 40 nucleotide sequence of a nucleic acid molecule of the presently disclosed subject matter. In some embodiments, probes comprise 14 to 20 nucleotides, or even longer where desired, such as 30, 40, 50, 60, 100, 200, 300, or 500 nucleotides or up to the full length (for example, the full complement) of any of the nucleic acid sequence set forth in SEQ ID NOs: 3, 4, and 6. Such fragments can be readily prepared by, for example, directly synthesizing the fragment by chemical synthesis, by application of nucleic acid amplification technology, or by introducing selected sequences into recombinant vectors for recombinant production. The phrase "hybridizing substantially to" refers to complementary hybridization between a probe nucleic acid molecule and a target nucleic acid molecule and embraces minor mismatches (for example, polymorphisms) that can be accommodated by reducing the stringency of the hybridization and/or wash media to achieve the desired hybridization. "Stringent hybridization conditions" and "stringent hybridization wash conditions" in the context of nucleic acid hybridization experiments such as Southern and Northern blot analysis are both sequence- and environment- dependent. Longer sequences hybridize specifically at higher temperatures. An extensive guide to the hybridization of nucleic acids is found in Tijssen, 1993. Generally, high stringency hybridization and wash conditions are selected to be about 5°C lower than the thermal melting point (T _m ) for the specific sequence at a defined ionic strength and pH. Typically, under "highly stringent conditions" a probe will hybridize specifically to its target subsequence, but to no other sequences. Similarly, medium stringency hybridization and wash conditions are selected to be more than about 5°C lower than the T _m for the specific sequence at a defined ionic strength and pH. Exemplary medium stringency conditions include hybridizations and washes as for high stringency conditions, except that the temperatures for the hybridization and washes are in some embodiments 8 ⁰ C, in some embodiments 1O ⁰ C, in some embodiments 12°C, and in some embodiments 15°C lower than the T _m 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. Very

stringent conditions are selected to be equal to the T _m for a particular probe. An example of highly stringent hybridization conditions for Southern or Northern Blot analysis of complementary nucleic acids having more than about 100 complementary residues is overnight hybridization in 50% formamide with 1 mg of heparin at 42°C. An example of highly stringent wash conditions is 15 minutes in 0.1x standard saline citrate (SSC), 0.1 % (w/v) SDS at 65°C. Another example of highly stringent wash conditions is 15 minutes in 0.2x SSC buffer at 65 ⁰ C (see Sambrook and Russell, 2001 for a description of SSC buffer and other stringency conditions). Often, a high stringency wash is preceded by a lower stringency wash to remove background probe signal.

An example of medium stringency wash conditions for a duplex of more than about 100 nucleotides is 15 minutes in 1 X SSC at 45°C. Another example of medium stringency wash for a duplex of more than about 100 nucleotides is 15 minutes in 4-6X SSC at 4O ⁰ C. For short probes (e.g., about 10 to 50 nucleotides), stringent conditions typically involve salt concentrations of less than about 1 M Na ⁺ ion, typically about 0.01 to 1 M Na ⁺ ion concentration (or other salts) at pH 7.0-8.3, and the temperature is typically at least about 30°C. Stringent conditions can also be achieved with the addition of destabilizing agents such as formamide. In general, a signal to noise ratio of 2-fold (or higher) than that observed for an unrelated probe in the particular hybridization assay indicates detection of a specific hybridization.

The following are examples of hybridization and wash conditions that can be used to clone homologous nucleotide sequences that are substantially identical to reference nucleotide sequences of the presently disclosed subject matter: a probe nucleotide sequence hybridizes in some embodiments to a target nucleotide sequence in 7% sodium dodecyl sulfate (NaDS), 0.5M NaPO ₄ , 1 mm ethylene diamine tetraacetic acid (EDTA) at 50 ⁰ C followed by washing in 2X SSC, 0.1 % NaDS at 50°C; in some embodiments, a probe and target sequence hybridize in 7% NaDS, 0.5 M NaPO ₄ , 1 mm EDTA at 5O ⁰ C followed by washing in 1X SSC, 0.1 % NaDS at 50 ⁰ C; in some embodiments, a probe and target sequence hybridize in 7% NaDS, 0.5 M NaPO ₄ , 1 mm EDTA at 5O ⁰ C followed by washing in 0.5X SSC, 0.1 % NaDS at 5O ⁰ C; in some embodiments, a probe and target sequence hybridize in 7% NaDS, 0.5 M NaPO ₄ , 1 mm EDTA

at 50 ⁰ C followed by washing in 0.1X SSC, 0.1 % NaDS at 5O ⁰ C; in some embodiments, a probe and target sequence hybridize in 7% NaDS, 0.5 M NaPO ₄ , 1 mm EDTA at 50°C followed by washing in 0.1 X SSC, 0.1 % NaDS at 65°C. In some embodiments, hybridization conditions comprise hybridization in a roller tube for at least 12 hours at 42°C.

As used herein, the term "purified", when applied to a nucleic acid or polypeptide, denotes that the nucleic acid or polypeptide is essentially free of other cellular components with which it is associated in the natural state. It can be in a homogeneous state although it can be in either a dry or aqueous solution. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography. A polypeptide that is the predominant species present in a preparation is substantially purified. The term "purified" denotes that a nucleic acid or polypeptide gives rise to essentially one band in an electrophoretic gel. Particularly, it means that the nucleic acid or polypeptide is in some embodiments at least about 50% pure, in some embodiments at least about 85% pure, and in some embodiments at least about 99% pure.

Two nucleic acids are "recombined" when sequences from each of the two nucleic acids are combined in a progeny nucleic acid. Two sequences are "directly" recombined when both of the nucleic acids are substrates for recombination. Two sequences are "indirectly recombined" when the sequences are recombined using an intermediate such as a cross over oligonucleotide. For indirect recombination, no more than one of the sequences is an actual substrate for recombination, and in some cases, neither sequence is a substrate for recombination.

As used herein, the term "regulatory elements" refers to nucleotide sequences involved in controlling the expression of a nucleotide sequence. Regulatory elements can comprise a promoter operatively linked to the nucleotide sequence of interest and termination signals. Regulatory sequences also include enhancers and silencers. They also typically encompass sequences required for proper translation of the nucleotide sequence.

As used herein, the term "root growth" refers to all manner of root development in a plant, including formation of root tissue and/or elongation of root tissue. "Modulating root growth" refers to either an increase or decrease in root development in a plant as compared to a comparable control plant. "Root", as the term is used herein refers to all types of root tissue found in a plant, including primary root tissue and lateral root tissue.

As used herein, the term "subsequence" refers to a sequence of nucleic acids or amino acids that comprises a part of a longer sequence of nucleic acids or amino acids (e.g., polypeptide), respectively. As used herein, the term "substrate" refers to a molecule that an enzyme naturally recognizes and converts to a product in the biochemical pathway in which the enzyme naturally carries out its function; or is a modified version of the molecule, which is also recognized by the enzyme and is converted by the enzyme to a product in an enzymatic reaction similar to the naturally-occurring reaction.

As used herein, the term "suitable growth conditions" refers to growth conditions that are suitable for a certain desired outcome, for example, the production of a recombinant polypeptide or the expression of a nucleic acid molecule. As used herein, the term "transformation" refers to a process for introducing heterologous DNA into a plant cell, plant tissue, or plant. Transformed plant cells, plant tissue, or plants are understood to encompass not only the end product of a transformation process, but also transgenic progeny thereof. As used herein, the terms "transformed", "transgenic", and "recombinant" refer to a host organism such as a bacterium or a plant into which a heterologous nucleic acid molecule has been introduced. The nucleic acid molecule can be stably integrated into the genome of the host or the nucleic acid molecule can also be present as an extrachromosomal molecule. Such an extrachromosomal molecule can be auto-replicating. Transformed cells, tissues, or plants are understood to encompass not only the end product of a transformation process, but also transgenic progeny thereof. A "non- transformed," "non-transgenic", or "non-recombinant" host refers to a wild-type

organism, e.g., a bacterium or plant, which does not contain the heterologous nucleic acid molecule.

HL Nucleic Acid Molecules and Polypeptides The presently disclosed subject matter encompasses the determination of functions of plant ARC proteins and methods of using the proteins. In particular, the presently disclosed subject matter discloses the identification and isolation of nucleic acid sequences (e.g., cDNAs) encoding genes of interest having root growth modulating activity, which in turn can affect, for example, abiotic stress tolerance.

Abiotic stresses include, but are not limited to, cold, heat, drought, and salt stress, and can significantly affect the growth and/or yield of plants. In some embodiments, an abiotic stress is drought. In some embodiments, an abiotic stress is a lack of appropriate nutrients in the soil. In some embodiments, an abiotic stress is high salinity. Altering the expression of genes related to these traits can be used to improve or modify plants as desired, including for example rice plants and grains.

The Examples describe exemplary genes isolated from Arabidopsis that encode ARC proteins having root growth modulating activities and methods of analyzing the alteration of expression and their effects on plant characteristics. However, the presently disclosed subject matter is intended to also encompasses other homologous genes and encoded polypeptides from Arabidopsis and orthologs that encode functionally equivalent ARC proteins, such as for example the homologous rice ARC gene set forth in SEQ ID NO:6 encoding the ARC protein set forth in SEQ ID NO:5. III.A. Nucleic Acid Molecules

Embodiments of the presently disclosed subject matter encompass methods for utilizing isolated nucleic acid molecules corresponding to genes that are related to root growth modulating activities, which can modulate tolerance for abiotic stress in plants, and in particular genes encoding ARC proteins related to root growth. In some embodiments, the polynucleotides encode ARC proteins that are homologous to mammalian /?-catenin and Drosophila Armadillo, such as for example Arabidopsis Arabidillo-1 and

Arabidillo-2.

In some embodiments, an ARC nucleic acid molecule of the presently disclosed subject matter comprises a nucleotide sequence encoding an ARC polypeptide comprising an F-box motif and at least one Armadillo repeat region. In some embodiments, the polynucleotide encodes a polypeptide comprising an amino acid sequence of any of SEQ ID NOs: 1 , 2, and 5. In some embodiments, the polynucleotide comprises a polynucleotide sequence of any of SEQ ID NOs: 3, 4, and 6.

In other embodiments, the polynucleotide comprises a polynucleotide sequence having substantial sequence identity to a DNA sequence as set forth in any of SEQ ID NOs:3, 4, and 6 and having root growth modulating activity. In some embodiments, the polynucleotide comprises a polynucleotide sequence capable of hybridizing under stringent conditions to a nucleic acid comprising a sequence or the complement of a sequence as set forth in any of SEQ ID NOs:3, 4, and 6.

In some embodiments, the substantial sequence identity is at least about 60% identity, in some embodiments at least about 65% identity, in some embodiments at least about 70% identity, in some embodiments at least about 75% identity, in some embodiments about 80% identity, in some embodiments at least about 85% identity, in some embodiments about 90% identity, and in some embodiments at least about 95%, 96%, 97%, 98% or 99% identity to the nucleotide sequence listed in SEQ ID NOs: 3, 4, and 6, or a fragment, domain, or feature thereof.

In some embodiments, the nucleotide sequence having substantial identity comprises an allelic variant of the nucleotide sequence that hybridizes under stringent hybridization conditions to a nucleotide sequence listed in SEQ ID NOs: 3, 4, and 6, or a fragment, domain, or feature thereof. In some embodiments, the nucleotide sequence having substantial identity comprises a naturally occurring variant. In some embodiments, the nucleotide sequence having substantial identity comprises a polymorphic variant of the nucleotide sequence that hybridizes under stringent conditions of hybridization to a nucleotide sequence listed in SEQ ID NOs: 3, 4, and 6, or a fragment, domain, or feature thereof.

In some embodiments, the nucleic acid having substantial identity comprises a deletion or insertion of at least one nucleotide. In some embodiments, the deletion or insertion comprises less than about thirty nucleotides. In some embodiments, the deletion or insertion comprises less than about five nucleotides. In some embodiments, the sequence of the isolated nucleic acid having substantial identity comprises a substitution in at least one codon. In some embodiments, the substitution is conservative.

In some embodiments, the isolated nucleic acid comprises a plurality of regions having a nucleotide sequence that hybridizes under stringent hybridization conditions to a nucleotide sequence listed in SEQ ID NOs: 3, 4, and 6, or an exon, domain, or feature thereof.

In some embodiments, the nucleic acid is expressed in a specific location or tissue of a plant. In some embodiments, the location or tissue includes, but is not limited to, epidermis, root, vascular tissue, meristem, cambium, cortex, pith, leaf, flower, and combinations thereof. In some embodiments, the location or tissue is a seed. In some embodiments, the location or tissue is a root, such as for example a lateral root.

In some embodiments, the polynucleotide is used as a chromosomal marker. In some embodiments, the polynucleotide is used as a marker for restriction fragment length polymorphism (RFLP) analysis. In some embodiments, the polynucleotide is used as a marker for quantitative trait- linked breeding. In some embodiments, the polynucleotide is used as a marker for marker-assisted breeding. In some embodiments, the polynucleotide is used as a bait sequence in a two-hybrid system to identify sequence-encoding polypeptides interacting with the polypeptide encoded by the bait sequence. In some embodiments, the polynucleotide is used as a diagnostic indicator for genotyping or identifying an individual or population of individuals. In some embodiments, the polynucleotide is used for genetic analysis to identify boundaries of genes or exon. III.B. Identifying, Cloning, and Sequencing cDNAs

The cloning and sequencing of the cDNAs of the presently disclosed subject matter is accomplished using techniques known in the art. See generally, Sambrook & Russell, 2001 ; Silhavy et al., 1984; Ausubel etal., 1994;

Reiter et al, 1992; Schultz et al., 1998.

The isolated nucleic acids and polypeptides of the presently disclosed subject matter are usable over a range of plants - monocots and dicots - in particular monocots such as sorghum, rice, wheat, barley, and maize. In some embodiments, the monocot is a cereal. In some embodiments, the cereal can be, for example, rice, maize, wheat, barley, oats, rye, millet, sorghum, triticale, secale, einkorn, spelt, emmer, teff, milo, flax, gramma grass, Tripsacum sp., or teosinte. In some embodiments, the cereal is rice. Other plant genera relevant to the presently disclosed subject matter include, but are not limited to, Cucurbita, Rosa, Vitis, Juglans, Gragaria, Lotus, Medicago, Onobrychis, Trigonella, Vigna, Citrus, Linum, Geranium, Manihot, Daucus, Arabidopsis, Brassica, Raphanus, Sinapis, Atropa, Capsicum, Datura, Hyoscyamus, Lycopersicon, Nicotiana, Solarium, Petunia, Digitalis, Majorana, Ciahorium, Helianthus, Lactuca, Bromus, Asparagus, Antirrhinum, Heterocallis, Nemesis, Pelargonium, Panieum, Pennisetum, Ranunculus, Senecio, Salpiglossis, Cucumis, Browaalia, Glycine, Pisum, Phaseolus, Lolium, Oryza, Avena, Hordeum, Secale, Allium, and Triticum.

The presently disclosed subject matter also provides a method for genotyping a plant or plant part comprising a nucleic acid molecule of the presently disclosed subject matter. Optionally, the plant is a monocot such as, but not limited to, sorghum, rice or wheat. Genotyping provides a methodology for distinguishing homologs of a chromosome pair and can be used to differentiate segregants in a plant population. Molecular marker methods can be used in phylogenetic studies, characterizing genetic relationships among crop varieties, identifying crosses or somatic hybrids, localizing chromosomal segments affecting monogenic traits, mapping based cloning, and the study of quantitative inheritance {see Clark, 1997; Paterson, 1996).

The method for genotyping can employ any number of molecular marker analytical techniques including, but not limited to, restriction length polymorphisms (RFLPs). As is well known in the art, RFLPs are produced by differences in the DNA restriction fragment lengths resulting from nucleotide differences between alleles of the same gene. Thus, the presently disclosed subject matter provides a method for following segregation of a gene or nucleic

acid of the presently disclosed subject matter or chromosomal sequences genetically linked by using RFLP analysis. Linked chromosomal sequences are in some embodiments within 50 centimorgans (cM), in some embodiments within 40 cM, in some embodiments within 30 cM, in some embodiments within 20 cM, in some embodiments within 10 cM, and in some embodiments within 5, 3, 2, or 1 cM of the nucleic acid of the presently disclosed subject matter. Other art-recognized methods of genotyping utilize simple sequence repeats (SSRs), amplification fragment length polymorphisms (AFLPs), or single nucleotide polymorphisms (SNPs). Embodiments of the presently disclosed subject matter also relate to an isolated nucleic acid molecule comprising a nucleotide sequence, its complement (for example, its full complement), or its reverse complement (for example, its full reverse complement), the nucleotide sequence encoding a polypeptide (for example, a biologically active polypeptide or biologically active fragment). In some embodiments, the nucleotide sequence encodes a polypeptide that is an ortholog of a polypeptide comprising a polypeptide sequence listed in SEQ ID NOs: 1 , 2, and 5, or a fragment, domain, repeat, feature, or chimera thereof. In some embodiments, the nucleotide sequence encodes a polypeptide that is an ortholog of a polypeptide comprising a polypeptide sequence having substantial identity to a polypeptide sequence listed in SEQ ID NOs: 1 , 2, and 5, or a fragment, domain, repeat, feature, or chimera thereof. In some embodiments, the nucleotide sequence encodes a polypeptide that is an ortholog of a polypeptide comprising a polypeptide sequence encoded by a nucleotide sequence identical to or having substantial identity to a nucleotide sequence listed in SEQ ID NOs: 3, 4, and 6, or a fragment, domain, or feature thereof, or a sequence complementary thereto. In some embodiments, the nucleotide sequence encodes a polypeptide comprising a polypeptide sequence encoded by a nucleotide sequence that hybridizes under stringent hybridization conditions to a nucleotide sequence listed in SEQ ID NOs: 3, 4, and 6, or to a sequence complementary thereto. In some embodiments, the nucleotide sequence encodes a functional fragment of a polypeptide of the presently disclosed subject matter.

In some embodiments, the isolated nucleic acid comprises a

polypeptidθ-encoding sequence. In some embodiments, the polypeptide- encoding sequence encodes a polypeptide that is an ortholog of a polypeptide comprising a polypeptide sequence listed in SEQ ID NOs: 1 , 2, and 5, or a fragment thereof. In some embodiments, the polypeptide is a plant polypeptide. In some embodiments, the plant is a dicot. In some embodiments, the plant is a gymnosperm. In some embodiments, the plant is a monocot. In some embodiments, the monocot is a cereal. In some embodiments, the cereal includes, but is not limited to, rice, maize, wheat, barley, oats, rye, millet, sorghum, triticale, secale, einkorn, spelt, emmer, teff, miloflax, gramma grass, Tripsacum, and teosinte. In some embodiments, the cereal is rice. III.C. Polypeptides

Embodiments of the presently disclosed subject matter encompass methods for utilizing ARC polypeptides that have root growth modulating activities for modulating root growth in plants and modulating tolerance for abiotic stress in plants. In some embodiments, the polypeptides comprise ARC proteins that are homologous to mammalian β-catenin and Drosophila Armadillo, such as for example Arabidopsis Arabidillo-1 and Arabidillo-2.

In some embodiments, an ARC protein of the presently disclosed subject matter comprises an ARC polypeptide comprising an F-box motif and at least one Armadillo repeat region. In some embodiments, the polypeptide comprises an amino acid sequence of any of SEQ ID NOs: 1 , 2, and 5 as well as homologues and orthologs.

In some embodiments, the polypeptide comprises a polypeptide encoded by a nucleic acid sequence as set forth in any of SEQ ID NOs:3, 4, and 6. In some embodiments, the polypeptide comprises a polypeptide encoded by a nucleic acid having at least about 75% or greater identity to a DNA sequence as set forth in any of SEQ ID NOs:3, 4, and 6 and having root growth modulating activity. In some embodiments, the polypeptide comprises a polypeptide encoded by a nucleic acid capable of hybridizing under stringent conditions to a nucleic acid comprising a sequence or the complement of a sequence as set forth in any of SEQ ID NOs:3, 4, and 6. In some embodiments, the polypeptide comprises a polypeptide which is a biologically functional equivalent of a peptide as set forth in any of SEQ ID NOs: 1 , 2, and 5.

Further, in some embodiments, the polypeptide comprises a functional fragment of the presently disclosed subject matter.

In some embodiments, the substantial sequence identity is at least about 60% identity, in some embodiments at least about 65% identity, in some embodiments at least about 70% identity, in some embodiments at least about 75% identity, in some embodiments about 80% identity, in some embodiments at least about 85% identity, in some embodiments about 90% identity, and in some embodiments at least about 95%, 96%, 97%, 98% or 99% identity to the nucleotide sequence listed in SEQ ID NOs: 3, 4, and 6, or a fragment, domain, or feature thereof.

The presently disclosed subject matter further relates to isolated polypeptides that are orthologs of the polypeptides comprising the amino acid sequences set forth in SEQ ID NOs: 1 , 2, and 5, including biologically active polypeptides. In some embodiments, the polypeptide comprises a functional fragment or domain of an ortholog of a polypeptide comprising a polypeptide sequence listed in SEQ ID NOs: 1 , 2, and 5. In some embodiments, the polypeptide is a plant polypeptide. In some embodiments, the plant is a dicot. In some embodiments, the plant is a gymnosperm. In some embodiments, the plant is a monocot. In some embodiments, the monocot is a cereal. In some embodiments, the cereal is, for example, rice, maize, wheat, barley, oats, rye, millet, sorghum, triticale, secale, einkorn, spelt, emmer, teff, milo, flax, gramma grass, Tripsacum, or teosinte. In some embodiments, the cereal is rice.

In some embodiments, the polypeptide is expressed in a specific location or tissue of a plant. In some embodiments, the location or tissue includes, but is not limited to, epidermis, root, vascular tissue, meristem, cambium, cortex, pith, leaf, flower, and combinations thereof. In some particular embodiments, the location or tissue is root.

In some embodiments, the polypeptide is involved in a function such as abiotic stress tolerance, disease resistance, enhanced yield or nutritional quality or composition.

In some embodiments, isolated polypeptides comprise the amino acid sequences of orthologs of the polypeptides comprising the amino acid sequences set forth in SEQ ID NOs: 1 , 2, and 5, and variants having

conservative amino acid modifications. The term "conservative modified variants" refers to polypeptides that can be encoded by nucleic acid sequences having degenerate codon substitutions wherein at least one position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al., 1991 ; Ohtsuka et al., 1985; Rossolini et al., 1994). Additionally, one skilled in the art will recognize that individual substitutions, deletions, or additions to a nucleic acid, peptide, polypeptide, or polypeptide sequence that alters, adds, or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a "conservative modification" where the modification results in the substitution of an amino acid with a chemically similar amino acid. Conservative modified variants provide similar biological activity as the unmodified polypeptide. Conservative substitution tables listing functionally similar amino acids are known in the art. See Creighton, 1984. The term "conservatively modified variant" also refers to a peptide having an amino acid residue sequence substantially identical to a sequence of a polypeptide of the presently disclosed subject matter in which one or more residues have been conservatively substituted with a functionally similar residue. Examples of conservative substitutions include the substitution of one non-polar (hydrophobic) residue such as isoleucine, valine, leucine or methionine for another; the substitution of one polar (hydrophilic) residue for another such as between arginine and lysine, between glutamine and asparagine, between glycine and serine; the substitution of one basic residue such as lysine, arginine or histidine for another; or the substitution of one acidic residue, such as aspartic acid or glutamic acid for another.

Amino acid substitutions, such as those which might be employed in modifying the polypeptides described herein, are generally based on the relative similarity of the amino acid side-chain, substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like. An analysis of the size, shape and type of the amino acid side-chain substituents reveals that arginine, lysine and histidine are all positively charged residues; that alanine, glycine and serine are all of similar size; and that phenylalanine, tryptophan and tyrosine all have a generally similar shape. Therefore, based upon these

considerations, arginine, lysine and histidine; alanine, glycine and serine; and phenylalanine, tryptophan and tyrosine; are defined herein as biologically functional equivalents. Other biologically functionally equivalent changes will be appreciated by those of skill in the art. In making biologically functional equivalent amino acid substitutions, the hydropathic index of amino acids can be considered. Each amino acid has been assigned a hydropathic index on the basis of their hydrophobicity and charge characteristics, these are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (- 3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).

The importance of the hydropathic amino acid index in conferring interactive biological function on a protein is generally understood in the art (Kyte & Doolittle, 1982, incorporated herein by reference). It is known that certain amino acids can be substituted for other amino acids having a similar hydropathic index or score and still retain a similar biological activity. In making changes based upon the hydropathic index, substitutions of amino acids can involve amino acids for which the hydropathic indices are in some embodiments within ±2 of the original value, in some embodiments within ±1 of the original value, and in some embodiments within ±0.5 of the original value.

It is also understood in the art that the substitution of like amino acids can be made .effectively on the basis of hydrophilicity. U.S. Patent No. 4,554,101 , incorporated herein by reference, states that the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with its immunogenicity and antigenicity, i.e. with a biological property of the protein. It is understood that an amino acid can be substituted for another having a similar hydrophilicity value and still obtain a biologically equivalent protein. As detailed in U.S. Patent No. 4,554,101 , the following hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0 ± 1); glutamate (+3.0 ± 1 ); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5 ± 1 ); alanine

(-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (- 1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4).

In making changes based upon similar hydrophilicity values, substitutions of amino acids can involve amino acids for which the hydrophilicity values are in some embodiments within ±2 of the original value, in some embodiments within ±1 of the original value, and in some embodiments within

±0.5 of the original value.

While discussion has focused on functionally equivalent polypeptides arising from amino acid changes, it will be appreciated that these changes can be effected by alteration of the encoding DNA, taking into consideration also that the genetic code is degenerate and that two or more codons can code for the same amino acid.

In some embodiments, the sequence having substantial identity contains a deletion or insertion of at least one amino acid. In some embodiments, the deletion or insertion is of less than about ten amino acids. In some embodiments, the deletion or insertion is of less than about three amino acids.

In some embodiments, the sequence having substantial identity encodes a substitution in at least one amino acid.

In some embodiments, a polypeptide having substantial identity to a polypeptide sequence listed in SEQ ID NO: 1 , 2, and 5, or a domain or feature thereof, is an allelic variant of the polypeptide sequence listed in SEQ ID NO: 1 ,

2, and 5. In some embodiments, a polypeptide having substantial identity to a polypeptide sequence listed in SEQ ID NO: 1 , 2, and 5, or a domain or feature thereof, is a naturally occurring variant of the polypeptide sequence listed in SEQ ID NO: 1 , 2, and 5. In some embodiments, a polypeptide having substantial identity to a polypeptide sequence listed in SEQ ID NO: 1 , 2, and 5, or a domain or feature thereof, is a polymorphic variant of the polypeptide sequence listed in SEQ ID NO: 1 , 2, and 5.

In some embodiments, the polypeptide is an ortholog of a polypeptide comprising one of the amino acid sequences listed in SEQ ID NO: 1 , 2, and 5.

In some embodiments, the polypeptide is a functional fragment or domain of an ortholog of a polypeptide comprising one of the amino acid sequences listed in

SEQ ID NOs: 1 , 2, and 5.

The polypeptides of the presently disclosed subject matter, fragments thereof, or variants thereof, can comprise any number of contiguous amino acid residues from a polypeptide of the presently disclosed subject matter, wherein the number of residues is selected from the group of integers consisting of from 10 to the number of residues in a full-length polypeptide of the presently disclosed subject matter. In some embodiments, the portion or fragment of the polypeptide is a functional polypeptide. The presently disclosed subject matter includes active polypeptides having specific activity of at least in some embodiments 20%, in some embodiments 30%, in some embodiments 40%, in some embodiments 50%, in some embodiments 60%, in some embodiments 70%, in some embodiments 80%, in some embodiments 90%, and in some embodiments 95% that of the native (non-synthetic) endogenous polypeptide. Further, the substrate specificity (k _cat /K _m ) can be substantially identical to the native (non-synthetic), endogenous polypeptide. Typically the K _m will be at least in some embodiments 30%, in some embodiments 40%, in some embodiments 50% of the native, endogenous polypeptide; and in some embodiments at least 60%, in some embodiments 70%, in some embodiments 80%, and in some embodiments 90% of the native, endogenous polypeptide. Methods of assaying and quantifying measures of activity and substrate specificity are well known to those of skill in the art.

IV. Methods for Modulating Root Growth and Improving Abiotic Stress Tolerance in Plants IVA Methods of Modulating Root Growth in Plants The presently disclosed subject matter provides methods of modulating root growth in a plant. In some embodiments, the methods comprise modulating expression of an ARC polypeptide disclosed herein in the plant. In some embodiments, the methods comprise administering an effective amount of an ARC polypeptide disclosed herein to the plant. In some embodiments, modulating expression of an ARC polypeptide in a plant comprises expressing in the plant an expression cassette comprising a nucleic acid molecule encoding the ARC polypeptide. Modulating expression of an ARC polypeptide in a plant refers to underexpressing or overexpressing in

tne plant a polynucleotide encoding the ARC polypeptide and/or the ARC polypeptide. By "underexpressing" or "overexpressing" is meant that a measurably lesser or greater amount, respectively, of the polynucleotide encoding the ARC polypeptide and/or the ARC polypeptide is expressed in the plant than would normally be present without the ARC polypeptide expression modulation. In some embodiments, the ARC polypeptide can be native to the plant but there may be more or less expression due to modulation of expression by the methods of the presently disclosed subject matter. In some embodiments, the ARC polypeptide can be a peptide not found in the native plant, in which case any measurable expression in the plant is considered overexpression. In some embodiments, the underexpression or overexpression of the ARC polypeptide in the plant will occur in a root of the plant.

In some embodiments, administering an effective amount of an ARC polypeptide to the plant comprises contacting one or more roots of the plant with the ARC polypeptide. In some embodiments, the administering can comprise applying an ARC polypeptide-containing solution to a root by applying the solution to the root or dipping or soaking the root in the solution. In some embodiments, the administering can comprise applying an ARC polypeptide containing solution or composition to soil near the base of the plant so that it can contact the root by diffusing through the soil and contacting the root or by the root growing toward the ARC polypeptide and contacting it.

The ARC polypeptide of the methods of modulating root growth in a plant comprises, in some embodiments, an ARC polypeptide comprising an F-box domain and at least one Armadillo repeat region. In some embodiments the ARC polypeptide is selected from the group consisting of (a) a polypeptide encoded by a nucleic acid sequence as set forth in any of SEQ ID NOs:3, 4, and 6; (b) a polypeptide encoded by a nucleic acid having at least about 75% identity to a DNA sequence as set forth in any of SEQ ID NOs:3, 4, and 6 and having root growth modulating activity; (c) a polypeptide encoded by a nucleic acid capable of hybridizing under stringent conditions to a nucleic acid comprising a sequence or the complement of a sequence as set forth in any of SEQ ID NOs:3, 4, and 6; (d) a polypeptide which is a biologically functional equivalent of a peptide as set forth in any of SEQ ID NOs:1 , 2, and 5 and has at

least about 75% identity to a peptide as set forth in any of SEQ ID N0s:1 , 2, and 5; and (e) a polypeptide comprising a functional fragment of a polypeptide of (a), (b), (c), or (d). In some embodiments the ARC polypeptide is selected from the group consisting of SEQ ID NOs:1 , 2, and 5. The plant in which the root growth is modulated can in some embodiments be a monocot. In some embodiments, the plant is rice, maize, wheat, barley, rye, oats, millet, triticale, secale, einkorn, spelt, emmer, teff, milo, flax, gramma grass, Tripsacum, teosinte, Arabidopsis, potato, canola, soybean, sunflower, carrot, sweet potato, sugarbeet, bean, pea, chicory, lettuce, cabbage, cauliflower, broccoli, turnip, radish, spinach, asparagus, onion, garlic, eggplant, pepper, celery, squash, pumpkin, cucumber, apple, pear, quince, melon, plum, cherry, peach, nectarine, apricot, strawberry, grape, raspberry, blackberry, pineapple, avocado, papaya, mango, banana, soybean, tobacco, tomato, sorghum or sugarcane. IV.B. Methods of Improving Abiotic Stress Tolerance

The presently disclosed subject matter further provides methods of improving abiotic stress tolerance in a plant. In some embodiments, the methods comprise providing a plant susceptible to abiotic stress and modulating expression of an ARC polypeptide in the plant. In some embodiments, the methods comprise providing a plant susceptible to abiotic stress and administering to the plant an effective amount of an ARC polypeptide. The abiotic stress to which the plant is provided improved tolerance can be, for example, inappropriate amounts of nutrients, drought, excessive cold, excessive heat, excessive soil salinity, extreme acidity or alkalinity, alterations in plant architecture, alteration in plant development, or combinations thereof.

In some embodiments, modulating expression of an ARC polypeptide in a plant comprises expressing in the plant an expression cassette comprising a nucleic acid molecule encoding the ARC polypeptide. Modulating expression of an ARC polypeptide in a plant refers to underexpressing or overexpressing in the plant a polynucleotide encoding the ARC polypeptide and/or the ARC polypeptide. By "underexpressing" or "overexpressing" is meant that a measurably lesser or greater amount, respectively, of the polynucleotide

encoding the ARC polypeptide and/or the ARC polypeptide is expressed in the plant than would normally be present without the ARC polypeptide expression modulation. In some embodiments, the ARC polypeptide can be native to the plant but there may be more or less expression due to modulation of expression by the methods of the present subject matter. In some embodiments, the ARC polypeptide can be a peptide not found in the native plant, in which case any measurable expression in the plant is considered overexpression. In some embodiments, the underexpression or overexpression of the ARC polypeptide in the plant will occur in a root of the plant. In some embodiments, administering an effective amount of an ARC polypeptide to the plant comprises contacting one or more roots of the plant with the ARC polypeptide. In some embodiments, the administering can comprise applying an ARC polypeptide-containing solution to a root by applying the solution to the root or dipping or soaking the root in the solution. In some embodiments, the administering may comprise applying an ARC polypeptide containing solution or composition to soil near the base of the plant so that it can contact the root by diffusing through the soil and contacting the root or by the root growing toward the ARC polypeptide and contacting it.

The ARC polypeptide of the methods of improving abiotic stress tolerance in a plant comprises, in some embodiments, an ARC polypeptide comprising an F-box domain and at least one Armadillo repeat region. In some embodiments the ARC polypeptide is selected from the group consisting of (a) a polypeptide encoded by a nucleic acid sequence as set forth in any of SEQ ID NOs:3, 4, and 6; (b) a polypeptide encoded by a nucleic acid having at least about 75% identity to a DNA sequence as set forth in any of SEQ ID NOs:3, 4, and 6 and having root growth modulating activity; (c) a polypeptide encoded by a nucleic acid capable of hybridizing under stringent conditions to a nucleic acid comprising a sequence or the complement of a sequence as set forth in any of SEQ ID NOs:3, 4, and 6; (d) a polypeptide which is a biologically functional equivalent of a peptide as set forth in any of SEQ ID NOs: 1 , 2, and 5 and has at least about 75% identity to a peptide as set forth in any of SEQ ID NOs:1 , 2, and 5; and (e) a polypeptide comprising a functional fragment of a polypeptide of (a), (b), (c), or (d). In some embodiments the ARC polypeptide is selected

from the group consisting of SEQ ID NOs:1 , 2, and 5.

The plant in which the abiotic stress tolerance is improved can in some embodiments be a monocot. In some embodiments, the plant is rice, maize, wheat, barley, rye, oats, millet, triticale, secale, einkorn, spelt, emmer, teff, milo, flax, gramma grass, Tripsacum, teosinte, Arabidopsis, potato, canola, soybean, sunflower, carrot, sweet potato, sugarbeet, bean, pea, chicory, lettuce, cabbage, cauliflower, broccoli, turnip, radish, spinach, asparagus, onion, garlic, eggplant, pepper, celery, squash, pumpkin, cucumber, apple, pear, quince, melon, plum, cherry, peach, nectarine, apricot, strawberry, grape, raspberry, blackberry, pineapple, avocado, papaya, mango, banana, soybean, tobacco, tomato, sorghum or sugarcane.

V. Controlling and Modulating the Expression of Nucleic Acid Molecules VA General Considerations One aspect of the presently disclosed subject matter provides compositions and methods for modulating (i.e. increasing or decreasing) the level of nucleic acid molecules and/or polypeptides of the presently disclosed subject matter in plants. In particular, the nucleic acid molecules and polypeptides of the presently disclosed subject matter are expressed constitutively, temporally, or spatially (e.g. at developmental stages), in certain tissues, and/or quantities, which are uncharacteristic of non-recombinantly engineered plants. Therefore, the presently disclosed subject matter provides utility in such exemplary applications as altering the specified characteristics identified above. In some embodiments, the presently disclosed subject matter provides methods of producing a heterologous ARC polypeptide in a plant cell. In some embodiments, the methods comprise generating a plant cell comprising a nucleic acid sequence encoding a heterologous ARC polypeptide as disclosed herein operatively linked to a promoter and expressing in the plant cell the nucleic acid sequence encoding the heterologous ARC polypeptide, whereby the heterologous ARC polypeptide is produced in the plant cell.

By expression of an ARC polypeptide in a plant cell is meant expressing or overexpressing in the plant cell the nucleic acid molecule encoding the ARC

polypeptide. In some embodiments, the expression or overexpression of the ARC in the plant will occur in a root cell.

The isolated nucleic acid molecules of the presently disclosed subject matter are useful for expressing a polypeptide of the presently disclosed subject matter in a recombinant^/ engineered cell such as a bacterial, yeast, insect, mammalian, or plant cell. Expressing cells can produce the polypeptide in a non-natural condition (e.g. in quantity, composition, location and/or time) because they have been genetically altered to do so. Those skilled in the art are knowledgeable in the numerous expression systems available for expression of nucleic acids encoding a polypeptide of the presently disclosed subject matter.

Embodiments of the presently disclosed subject matter provide an expression cassette comprising a promoter sequence operatively linked to an isolated nucleic acid, the isolated nucleic acid molecule encoding an ARC polypeptide. In some embodiments, the ARC polypeptide is selected from the group consisting of :

(a) a polypeptide encoded by a nucleic acid sequence as set forth in any of SEQ ID NOs:3, 4, and 6;

(b) a polypeptide encoded by a nucleic acid having at least about 75% identity to a DNA sequence as set forth in any of SEQ ID

NOs:3, 4, and 6 and having root growth modulating activity;

(c) a polypeptide encoded by a nucleic acid capable of hybridizing under stringent conditions to a nucleic acid comprising a sequence or the complement of a sequence as set forth in any of SEQ ID NOs:3, 4, and 6;

(d) a polypeptide which is a biologically functional equivalent of a peptide as set forth in any of SEQ ID NOs: 1 , 2, and 5; and

(e) a polypeptide comprising a functional fragment of a polypeptide of (a), (b), (C) ₁ or (d). Further encompassed within the presently disclosed subject matter is a recombinant vector comprising an expression cassette according to the embodiments of the presently disclosed subject matter. Also encompassed are plant cells comprising expression cassettes according to the present

disclosure, ana plants comprising these plant cells. In some embodiments, the plant is a dicot. In some embodiments, the plant is a gymnosperm. In some embodiments, the plant is a monocot. In some embodiments, the plant is selected from the group consisting of rice, maize, wheat, barley, rye, oats, millet, triticale, secale, einkorn, spelt, emmer, teff, milo, flax, gramma grass, Tripsacum, teosinte, Arabidopsis, potato, canola, soybean, sunflower, carrot, sweet potato, sugarbeet, bean, pea, chicory, lettuce, cabbage, cauliflower, broccoli, turnip, radish, spinach, asparagus, onion, garlic, eggplant, pepper, celery, squash, pumpkin, cucumber, apple, pear, quince, melon, plum, cherry, peach, nectarine, apricot, strawberry, grape, raspberry, blackberry, pineapple, avocado, papaya, mango, banana, soybean, tobacco, tomato, sorghum or sugarcane.

In some embodiments, the expression cassette is expressed throughout the plant. In some embodiments, the expression cassette is expressed in a specific location or tissue of a plant. In some embodiments, the location or tissue includes, but is not limited to, epidermis, root, vascular tissue, meristem, cambium, cortex, pith, leaf, flower, and combinations thereof. In some embodiments, the location or tissue is a root, such as for example a lateral root.

In some embodiments, the expression cassette is involved providing the plant with improved abiotic stress tolerance. Exemplary abiotic stresses include but are not limited to inappropriate amounts of nutrients, drought, excessive cold, excessive heat, excessive soil salinity or extreme acidity or alkalinity, and alterations in plant architecture or development.

Embodiments of the presently disclosed subject matter also relate to an expression vector comprising a nucleic acid molecule selected from the group consisting of:

(a) a polynucleotide encoding a polypeptide comprising an amino acid sequence of any of SEQ ID NOs: 1 , 2, and 5;

(b) a polynucleotide comprising a polynucleotide sequence of any of SEQ ID NOs: 3, 4, and 6;.

(c) a polynucleotide having substantial sequence identity to a DNA sequence as set forth in any of SEQ ID NOs:3, 4, and 6 and having root growth modulating activity;

(d) a polynucleotide comprising a polynucleotide sequence capable of hybridizing under stringent conditions to a nucleic acid comprising a sequence or the complement of a sequence as set forth in any of SEQ ID NOs:3, 4, and 6; and (e) a polynucleotide having at least about 75% sequence identity to a polynucleotide encoding an amino acid sequence of any of SEQ ID

NOs: 1 , 2, and 5 and hybridizing under stringent conditions to a polynucleotide of any of SEQ ID NOs: 3, 4, and 6.

In some embodiments, the expression vector comprises one or more elements including, but not limited to, a promoter-enhancer sequence, a selection marker sequence, an origin of replication, an epitope tag-encoding sequence, and an affinity purification tag-encoding sequence. In some embodiments, the promoter-enhancer sequence comprises, for example, the cauliflower mosaic virus (CaMV) 35S promoter, the CaMV 19S promoter, the tobacco PR-1 a promoter, the ubiquitin promoter, or the phaseolin promoter. In some embodiments, the promoter is operable in plants, and in some embodiments, the promoter is a constitutive or inducible promoter. In some embodiments, the selection marker sequence encodes an antibiotic resistance gene. In some embodiments, the epitope tag sequence encodes V5 (GKPIPNPLLGLDST; SEQ ID NO: 107; Southern etal., 1991 ), the peptide Phe-

His-His-Thr-Thr (SEQ ID NO: 108), hemagglutinin, orglutathione-S-transferase.

In some embodiments the affinity purification tag sequence encodes a polyamino acid sequence or a polypeptide. In some embodiments, the polyamino acid sequence comprises polyhistidine. In some embodiments, the polypeptide is chitin-binding domain or glutathione-S-transferase. In some embodiments, the affinity purification tag sequence comprises an intein encoding sequence.

In some embodiments, the expression vector comprises a eukaryotic expression vector, and in some embodiments, the expression vector comprises a prokaryotic expression vector. In some embodiments, the eukaryotic expression vector comprises a tissue-specific promoter. In some embodiments, the expression vector is operable in plants.

Embodiments of the presently disclosed subject matter also relate to a cell comprising a nucleic acid construct comprising an expression vector and a nucleic acid comprising a nucleic acid encoding a polypeptide that is an ortholog of a polypeptide as listed in SEQ ID NOs: 1 , 2, and 5, or a nucleic acid sequence that hybridizes under stringent conditions of hybridization to a nucleotide sequence listed in SEQ ID NOs: 3, 4, and 6, or a subsequence thereof, in combination with a heterologous sequence.

In some embodiments, the cell is a bacterial cell, a fungal cell, a plant cell, or an animal cell. In some embodiments, the polypeptide is expressed in a specific location or tissue of a plant. In some embodiments, the location or tissue includes, but is not limited to, epidermis, root, vascular tissue, meristem, cambium, cortex, pith, leaf, flower, and combinations thereof. In some embodiments, the location or tissue is a root. In some embodiments, the polypeptide is involved in a function such as abiotic stress tolerance, enhanced yield, disease resistance, or nutritional composition through modulation of root growth.

Prokaryotic cells including, but not limited to, Escherichia coli and other microbial strains known to those in the art, can be used a host cells. Methods for expressing polypeptides in prokaryotic cells are well known to those in the art and can be found in many laboratory manuals such as Sambrook & Russell, 2001. A variety of promoters, ribosome binding sites, and operators to control expression are available to those skilled in the art, as are selectable markers such as antibiotic resistance genes. The type of vector is chosen to allow for optimal growth and expression in the selected cell type. A variety of eukaryotic expression systems are available such as, for example, yeast, insect cell lines, plant cells, and mammalian cells. Expression and synthesis of heterologous polypeptides in yeast is well known (see Sherman et al., 1982). Yeast strains widely used for production of eukaryotic polypeptides are Saccharomyces cerevisiae and Pichia pastoris, and vectors, strains, and protocols for expression are available from commercial suppliers [e.g., Invitrogen Corp., Carlsbad, California, United States of America).

Mammalian cell systems can be transformed with expression vectors for production of polypeptides. Suitable host cell lines available to those in the art

include, but are not limited to, the HEK293, BHK21 and CHO cells lines. Expression vectors for these cells can include expression control sequences such as an origin of replication, a promoter, (e.g., the CMV promoter, a Herpes Simplex Virus thymidine kinase (HSV-tk) promoter or phosphoglycerate kinase (pgk) promoter), an enhancer, and polypeptide processing sites such as ribosome binding sites, RNA splice sites, polyadenylation sites, and transcription terminator sequences. Other animal cell lines useful for the production of polypeptides are available commercially or from depositories such as the American Type Culture Collection (Manassas, Virginia, United States of America).

Expression vectors for expressing polypeptides in insect cells are usually derived from baculovirus or other viruses known in the art. A number of suitable insect cell lines are available including, but not limited to, mosquito larvae, silkworm, armyworm (for example, Spodoptera frugiperda), moth, and Drosophila cell lines.

Methods of transforming animal and lower eukaryotic cells are known. Numerous methods can be used to introduce exogenous DNA into eukaryotic cells including, but not limited to, calcium phosphate precipitation, fusion of the recipient cell with bacterial protoplasts containing the DNA, treatment of the recipient cells with liposomes containing the DNA, DEAE dextran, electroporation, biolistics, and microinjection of the DNA directly into the cells. Transformed cells are cultured using means well known in the art (see Kuchler, 1997).

Once a polypeptide of the presently disclosed subject matter is expressed it can be isolated and purified from the expressing cells using methods known to those skilled in the art. The purification process can be monitored using Western blot techniques, radioimmunoassay, or other standard immunoassay techniques. Polypeptide purification techniques are commonly known and used by those skilled in the art (see Scopes, 1982; Deutscher, 1990).

Embodiments of the presently disclosed subject matter provide a method for producing a recombinant polypeptide in which the expression vector comprise one or more elements including, but not limited to, a promoter-

enhancer sequence, a selection marker sequence, an origin of replication, an epitope tag-encoding sequence, and an affinity purification tag-encoding sequence. In some embodiments, the nucleic acid construct comprises an epitope tag-encoding sequence and the isolating step employs an antibody specific for the epitope tag. In some embodiments, the nucleic acid construct comprises a polyamino acid-encoding sequence and the isolating step employs a resin comprising a polyamino acid binding substance, in some embodiments where the polyamino acid is polyhistidine and the polyamino acid binding resin is nickel-charged agarose resin. In some embodiments, the nucleic acid construct comprises a polypeptide-encoding sequence and the isolating step employs a resin comprising a polypeptide binding substance. In some embodiments, the polypeptide is a chitin-binding domain and the resin contains chitin-sepharose.

The polypeptides of the presently disclosed subject matter can be synthesized using non-cellular synthetic methods known to those in the art. Techniques for solid phase synthesis are disclosed in Barany & Merrifield, 1980; Merrifield et al., 1963; Stewart & Young, 1984.

The presently disclosed subject matter further provides a method for modulating (i.e. increasing or decreasing) the concentration or composition of a polypeptide of the presently disclosed subject matter in a plant or part thereof. Modulation can be effected by increasing or decreasing the concentration and/or the composition (i.e. the ratio of the polypeptides of the presently disclosed subject matter) in a plant. The method comprises introducing into a plant cell an expression cassette comprising a nucleic acid molecule of the presently disclosed subject matter as disclosed above to obtain a transformed plant cell or tissue, and culturing the transformed plant cell or tissue. The nucleic acid molecule can be under the regulation of a constitutive or inducible promoter. The method can further comprise inducing expression of a nucleic acid molecule of a sequence in the plant for a time sufficient to modify the concentration and/or composition in the plant or plant part.

A plant or plant part having modified expression of a nucleic acid molecule of the presently disclosed subject matter can be analyzed and selected using methods known to those skilled in the art including, but not

iimneα ιo, ^outnern Plotting, DNA sequencing, or PCR analysis using primers specific to the nucleic acid molecule and detecting amplicons produced therefrom.

In general, a concentration or composition is increased or decreased by at least in some embodiments 5%, in some embodiments 10%, in some embodiments 20%, in some embodiments 30%, in some embodiments 40%, in some embodiments 50%, in some embodiments 60%, in some embodiments 70%, in some embodiments 80%, and in some embodiments 90% relative to a native control plant, plant part, or cell lacking the expression cassette. V.B. Modulating Expression of Nucleic Acid Molecules

The modulation in expression of the nucleic acid molecules of the presently disclosed subject matter can be achieved, for example, in one of the following ways:

V.B.1. "Sense" Expression Alteration of the expression of a nucleotide sequence of the presently disclosed subject matter, in some embodiments increasing its expression, can be obtained by "sense" expression. In this case, the entirety or a portion of a nucleotide sequence of the presently disclosed subject matter is provided in a DNA molecule. The DNA molecule can be operatively linked to a promoter functional in a cell comprising the target gene, in some embodiments a plant cell, and introduced into the cell, in which the nucleotide sequence is expressible. The nucleotide sequence is inserted in the DNA molecule in the "sense orientation", meaning that the coding strand of the nucleotide sequence can be transcribed. In some embodiments, the nucleotide sequence is fully translatable and all the genetic information comprised in the nucleotide sequence, or portion thereof, is translated into a polypeptide. In some embodiments, the nucleotide sequence is partially translatable and a short peptide is translated. In some embodiments, this is achieved by inserting at least one premature stop codon in the nucleotide sequence, which brings translation to a halt. In some embodiments, the nucleotide sequence is transcribed but no translation product is made. This is usually achieved by removing the start codon, i.e. the "ATG", of the polypeptide encoded by the nucleotide sequence.

In some embodiments, the DNA molecule comprising the nucleotide sequence, or a portion thereof, is stably integrated in the genome of the plant cell. In some embodiments, the DNA molecule comprising the nucleotide sequence, or a portion thereof, is provided in an extrachromosomally replicating molecule.

In transgenic plants containing one of the DNA molecules disclosed herein above, the expression of the nucleotide sequence corresponding to the nucleotide sequence provided in the DNA molecule can be reduced. The nucleotide sequence in the DNA molecule in some embodiments is at least 70% identical to the nucleotide sequence the expression of which is reduced, in some embodiments is at least 80% identical, in some embodiments is at least 90% identical, in some embodiments is at least 95% identical, and in some embodiments is at least 99% identical.

V.B.2. Homologous Recombination In some embodiments, at least one genomic copy corresponding to a nucleotide sequence of the presently disclosed subject matter is modified in the genome of the plant by homologous recombination as further illustrated in Paszkowski et ai, 1988. This technique uses the ability of homologous sequences to recognize each other and to exchange nucleotide sequences between respective nucleic acid molecules by a process known in the art as homologous recombination. Homologous recombination can occur between the chromosomal copy of a nucleotide sequence in a cell and an incoming copy of the nucleotide sequence introduced in the cell by transformation. Specific modifications are thus accurately introduced in the chromosomal copy of the nucleotide sequence. In some embodiments, the regulatory elements of the nucleotide sequence of the presently disclosed subject matter are modified. Such regulatory elements are easily obtainable by screening a genomic library using the nucleotide sequence of the presently disclosed subject matter, or a portion thereof, as a probe. The existing regulatory elements are replaced by different regulatory elements, thus altering expression of the nucleotide sequence, or they are mutated or deleted, thus abolishing the expression of the nucleotide sequence. In some embodiments, the nucleotide sequence is modified by deletion of a part of the nucleotide sequence or the entire

I nucleotide sequence, or by mutation. Expression of a mutated polypeptide in a plant cell is also provided in the presently disclosed subject matter. Recent refinements of this technique to disrupt endogenous plant genes have been disclosed (Kempin et al., 1997 and Miao & Lam, 1995). In some embodiments, a mutation in the chromosomal copy of a nucleotide sequence is introduced by transforming a cell with a chimeric oligonucleotide composed of a contiguous stretch of RNA and DNA residues in a duplex conformation with double hairpin caps on the ends. An additional feature of the oligonucleotide is for example the presence of 2'-O-methylation at the RNA residues. The RNA/DNA sequence is designed to align with the sequence of a chromosomal copy of a nucleotide sequence of the presently disclosed subject matter and to contain the desired nucleotide change. For example, this technique is further illustrated in U.S. Patent No. 5,501 ,967 and Zhu et a/., 1999. V.B.3. Zinc Finger Polypeptides

A zinc finger polypeptide that binds a nucleotide sequence of the presently disclosed subject matter or to its regulatory region can also be used to alter expression of the nucleotide sequence. In alternative embodiments, transcription of the nucleotide sequence is reduced or increased. Zinc finger polypeptides are disclosed in, for example, Beerli et ai, 1998, or in WO 95/19431 , WO 98/54311 , or WO 96/06166, all incorporated herein by reference in their entirety.

V.B.4. Overexpression in a Plant Cell

In some embodiments, a nucleotide sequence of the presently disclosed subject matter encoding a polypeptide is over-expressed. Examples of nucleic acid molecules and expression cassettes for over-expression of a nucleic acid molecule of the presently disclosed subject matter are disclosed above. Methods known to those skilled in the art of over-expression of nucleic acid molecules are also encompassed by the presently disclosed subject matter. In some embodiments, the expression of the nucleotide sequence of the presently disclosed subject matter is altered in every cell of a plant. This can be obtained, for example, though homologous recombination or by insertion into a chromosome. This can also be obtained, for example, by expressing a sense

RNA or zinc finger polypeptide under the control of a promoter capable of expressing the sense RNA or zinc finger polypeptide in every cell of a plant.

Constitutive, inducible, tissue-specific, or developmentally-regulated expression are also within the scope of the presently disclosed subject matter and result in a constitutive, inducible, tissue-specific, or developmentally- regulated alteration of the expression of a nucleotide sequence of the presently disclosed subject matter in the plant cell. Constructs for expression of the sense RNA or zinc finger polypeptide, or for over-expression of a nucleotide sequence of the presently disclosed subject matter, can be prepared and transformed into a plant cell according to the teachings of the presently disclosed subject matter, for example, as disclosed herein. V.C. Construction of Plant Expression Vectors

Coding sequences intended for expression in transgenic plants can be first assembled in expression cassettes operatively linked to a suitable promoter expressible in plants. The expression cassettes can also comprise any further sequences required or selected for the expression of the transgene. Such sequences include, but are not limited to, transcription terminators, extraneous sequences to enhance expression such as introns, vital sequences, and sequences intended for the targeting of the gene product to specific organelles and cell compartments. These expression cassettes can then be easily transferred to the plant transformation vectors disclosed below. The following is a description of various components of typical expression cassettes. V.C.1. Promoters

The selection of the promoter used in expression cassettes can determine the spatial and temporal expression pattern of the transgene in the transgenic plant. Selected promoters can express transgenes in specific cell types (such as leaf epidermal cells, mesophyll cells, root cortex cells) or in specific tissues or organs (roots, leaves, or flowers, for example) and the selection can reflect the desired location for accumulation of the gene product. Alternatively, the selected promoter can drive expression of the gene under various inducing conditions. Promoters vary in their strength; i.e., their abilities to promote transcription. Depending upon the host cell system utilized, any one of a number of suitable promoters can be used, including the gene's native

promoter. The following are non-limiting examples of promoters that can be used in expression cassettes.

V.C.la. Constitutive Expression: the Ubiquitin Promoter

Ubiquitin is a gene product known to accumulate in many cell types and its promoter has been cloned from several species for use in transgenic plants

(e.g. sunflower - Binet et al., 1991 ; maize - Christensen & Quail, 1989; and

Arabidopsis - CaIMs et al., 1990; Norris et al., 1993). The maize ubiquitin promoter has been developed in transgenic monocot systems and its sequence and vectors constructed for monocot transformation are disclosed in the patent publication EP 0 342 926 (to Lubrizol) which is herein incorporated by reference. Taylor et al. , 1993, describes a vector (pAHC25) that comprises the maize ubiquitin promoter and first intron and its high activity in cell suspensions of numerous monocotyledons when introduced via microprojectile bombardment. The Arabidopsis ubiquitin promoter is suitable for use with the nucleotide sequences of the presently disclosed subject matter. The ubiquitin promoter is suitable for gene expression in transgenic plants, both monocotyledons and dicotyledons. Suitable vectors are derivatives of pAHC25 or any of the transformation vectors disclosed herein, modified by the introduction of the appropriate ubiquitin promoter and/or intron sequences. V.d .b. Constitutive Expression: the CaMV 35S Promoter

Construction of the plasmid pCGN1761 is disclosed in the published patent application EP 0 392 225 (Example 23), which is hereby incorporated by reference. pCGN1761 contains the "double" CaMV 35S promoter and the tml transcriptional terminator with a unique EcoRI site between the promoter and the terminator and has a pUC-type backbone. A derivative of pCGN 1761 is constructed which has a modified polylinkerthat includes Notl and Xhol sites in addition to the existing EcoRI site. This derivative is designated pCGN1761 ENX. pCGN1761 ENX is useful for the cloning of cDNA sequences or coding sequences (including microbial ORF sequences) within its polylinker for the purpose of their expression under the control of the 35S promoter in transgenic plants. The entire 35S promoter-coding sequence-frn/ terminator cassette of such a construction can be excised by Hind\\\, Sph\, Sal\, and Xba\ sites 5' to the promoter and Xbal, BamH\ and Bgl\ sites 3' to the terminator for

transfer to transformation vectors such as those disclosed below. Furthermore, the double 35S promoter fragment can be removed by 5' excision with Hind\\\, Sph\, Sal\, Xba\, or Pst\, and 3' excision with any of the polylinker restriction sites (EcoRI, Not\ or Xho\) for replacement with another promoter. If desired, modifications around the cloning sites can be made by the introduction of sequences that can enhance translation. This is particularly useful when overexpression is desired. For example, pCGN1761 ENX can be modified by optimization of the translational initiation site as disclosed in Example 37 of U.S. Patent No. 5,639,949, incorporated herein by reference. V.C.1.C. Constitutive Expression: the Actin Promoter

Several isoforms of actin are known to be expressed in most cell types and consequently the actin promoter can be used as a constitutive promoter. In particular, the promoter from the rice Actl gene has been cloned and characterized (McElroy et al., 1990). A 1.3 kilobase (kb) fragment of the promoter was found to contain all the regulatory elements required for expression in rice protoplasts. Furthermore, numerous expression vectors based on the Actl promoter have been constructed specifically for use in monocotyledons (McElroy et al., 1991 ). These incorporate the Actl-Mron 1 , Adhl 5' flanking sequence (from the maize alcohol dehydrogenase gene) and Adhl-Mron 1 and sequence from the CaMV 35S promoter. Vectors showing highest expression were fusions of 35S and Actl intron or the Actl 5' flanking sequence and the Actl intron. Optimization of sequences around the initiating ATG (of the β-glucuronidase (GUS) reporter gene) also enhanced expression. The promoter expression cassettes disclosed in McElroy et al., 1991 , can be easily modified for gene expression and are particularly suitable for use in monocotyledonous hosts. For example, promoter-containing fragments are removed from the McElroy constructions and used to replace the double 35S promoter in pCGN1761 ENX, which is then available for the insertion of specific gene sequences. The fusion genes thus constructed can then be transferred to appropriate transformation vectors. In a separate report, the rice Actl promoter with its first intron has also been found to direct high expression in cultured barley cells (Chibbar et al., 1993).

V.C.I .d. Inducible Expression: PR-1 Promoters

The double 35S promoter in pCGN1761 ENX can be replaced with any other promoter of choice that will result in suitably high expression levels. By way of example, one of the chemically regulatable promoters disclosed in U.S. Patent No. 5,614,395, such as the tobacco PR-Ia promoter, can replace the double 35S promoter. Alternately, the Arabidopsis PR-1 promoter disclosed in

Lebel et al. , 1998, can be used. The promoter of choice can be excised from its source by restriction enzymes, but can alternatively be PCR-amplified using primers that carry appropriate terminal restriction sites. Should PCR- amplification be undertaken, the promoter can be re-sequenced to check for amplification errors after the cloning of the amplified promoter in the target vector. The chemically/pathogen regulatable tobacco PR-Ia promoter is cleaved from plasmid pCIB1004 (for construction, see example 21 of EP 0 332 104, which is hereby incorporated by reference) and transferred to plasmid pCGN1761 ENX (Uknes et al., 1992). pCIB1004 is cleaved with λ/col and the resulting 3' overhang of the linearized fragment is rendered blunt by treatment with T4 DNA polymerase. The fragment is then cleaved with Hind\\\ and the resultant PR-Ia promoter-containing fragment is gel purified and cloned into pCGN1761 ENX from which the double 35S promoter has been removed. This is accomplished by cleavage with Xho\ and blunting with T4 polymerase, followed by cleavage with Hind\\\, and isolation of the larger vector-terminator containing fragment into which the pCIB1004 promoter fragment is cloned. This generates a pCGN1761 ENX derivative with the PR-Ia promoter and the tml terminator and an intervening polylinker with unique EcoR\ and Not\ sites. The selected coding sequence can be inserted into this vector, and the fusion products (i.e. promoter-gene-terminator) can subsequently be transferred to any selected transformation vector, including those disclosed herein. Various chemical regulators can be employed to induce expression of the selected coding sequence in the plants transformed according to the presently disclosed subject matter, including the benzothiadiazole, isonicotinic acid, and salicylic acid compounds disclosed in U.S. Patent Nos. 5,523,311 and 5,614,395.

V.C.I .e. Inducible Expression: an Ethanol-lnducible Promoter

A promoter inducible by certain alcohols or ketones, such as ethanol, can also be used to confer inducible expression of a coding sequence of the presently disclosed subject matter. Such a promoter is for example the alcA gene promoter from Aspergillus nidulans (Caddick et al. , 1998). In A. nidulans, the alcA gene encodes alcohol dehydrogenase I, the expression of which is regulated by the AIcR transcription factors in presence of the chemical inducer.

For the purposes of the presently disclosed subject matter, the CAT coding sequences in plasmid palcA:CAT comprising a alcA gene promoter sequence fused to a minimal 35S promoter (Caddick et al., 1998) are replaced by a coding sequence of the presently disclosed subject matter to form an expression cassette having the coding sequence under the control of the alcA gene promoter. This is carried out using methods known in the art.

V.C.I .f. Inducible Expression: a Glucocorticoid-Inducible Promoter Induction of expression of a nucleic acid sequence of the presently disclosed subject matter using systems based on steroid hormones is also provided. For example, a glucocorticoid-mediated induction system is used (Aoyama & Chua, 1997) and gene expression is induced by application of a glucocorticoid, for example a synthetic glucocorticoid, for example dexamethasone, at a concentration ranging in some embodiments from 0.1 mM to 1 mM, and in some embodiments from 10 mM to 100 mM.

For the purposes of the presently disclosed subject matter, the luciferase gene sequences are replaced by a nucleic acid sequence of the presently disclosed subject matter to form an expression cassette having a nucleic acid sequence of the presently disclosed subject matter under the control of six copies of the GAL4 upstream activating sequences fused to the 35S minimal promoter. This is carried out using methods known in the art. The trans-acting factor comprises the GAL4 DNA-binding domain (Keegan etai, 1986) fused to the transactivating domain of the herpes viral polypeptide VP16 (Triezenberg et al., 1988) fused to the hormone-binding domain of the rat glucocorticoid receptor (Picard et al., 1988). The expression of the fusion polypeptide is controlled either by a promoter known in the art or disclosed herein.

A plant comprising an expression cassette comprising a nucleic acid sequence of the presently disclosed subject matter fused to the 6x GαL4/minimal promoter is also provided. Thus, tissue- or organ-specificity of the fusion polypeptide is achieved leading to inducible tissue- or organ- specificity of the nucleic acid sequence to be expressed. V.C.I .q. Root Specific Expression

Another pattern of gene expression is root expression. A suitable root promoter is the promoter of the maize metallothionein-like (MTL) gene disclosed in de Framond, 1991 , and also in U.S. Patent No. 5,466,785, each of which is incorporated herein by reference. This "MTL" promoter is transferred to a suitable vector such as pCGN1761 ENX for the insertion of a selected gene and subsequent transfer of the entire promoter-gene-terminator cassette to a transformation vector of interest.

V.C.2. Transcriptional Terminators A variety of transcriptional terminators are available for use in expression cassettes. These are responsible for termination of transcription and correct mRNA polyadenylation. Appropriate transcriptional terminators are those that are known to function in plants and include the CaMV 35S terminator, the tml terminator, the nopaline synthase terminator, and the pea rbcS E9 terminator. These can be used in both monocotyledons and dicotyledons. In addition, a gene's native transcription terminator can be used.

V.C.3. Sequences for the Enhancement or Regulation of

Expression

Numerous sequences have been found to enhance gene expression from within the transcriptional unit and these sequences can be used in conjunction with the genes of the presently disclosed subject matter to increase their expression in transgenic plants.

Various intron sequences have been shown to enhance expression, particularly in monocotyledonous cells. For example, the introns of the maize Adhl gene have been found to significantly enhance the expression of the wild- type gene under its cognate promoter when introduced into maize cells. Intron 1 was found to be particularly effective and enhanced expression in fusion constructs with the chloramphenicol acetyltransferase gene (Callis etal., 1987).

in me same experimental system, the intron from the maize bronzel gene had a similar effect in enhancing expression. Intron sequences have been routinely incorporated into plant transformation vectors, typically within the non- translated leader. A number of non-translated leader sequences derived from viruses are also known to enhance expression, and these are particularly effective in dicotyledonous cells. Specifically, leader sequences from Tobacco Mosaic Virus (TMV; the "W-sequence"), Maize Chlorotic Mottle Virus (MCMV), and Alfalfa Mosaic Virus (AMV) have been shown to be effective in enhancing expression (see e.g. Gallie et al., 1987; Skuzeski et al., 1990). Other leader sequences known in the art include, but are not limited to, picornavirus leaders, for example, EMCV (encephalomyocarditis virus) leader (5 ¹ noncoding region; see Elroy-Stein et al., 1989); potyvirus leaders, for example, from Tobacco Etch Virus (TEV; see Allison et al., 1986); Maize Dwarf Mosaic Virus (MDMV; see Kong & Steinbiss 1998); human immunoglobulin heavy-chain binding polypeptide (BiP) leader (Macejak & Sarnow, 1991 ); untranslated leader from the coat polypeptide mRNA of alfalfa mosaic virus (AMV; RNA 4; see Jobling & Gehrke, 1987); tobacco mosaic virus (TMV) leader (Gallie et al., 1989); and Maize Chlorotic Mottle Virus (MCMV) leader (Lommel et al., 1991 ). See also, Della-Cioppa ef a/., 1987.

In addition to incorporating one or more of the aforementioned elements into the 5' regulatory region of a target expression cassette of the presently disclosed subject matter, other elements can also be incorporated. Such elements include, but are not limited to, a minimal promoter. By minimal promoter it is intended that the basal promoter elements are inactive or nearly so in the absence of upstream or downstream activation. Such a promoter has low background activity in plants when there is no transactivator present or when enhancer or response element binding sites are absent.

One minimal promoter that is particularly useful for target genes in plants is the Bz1 minimal promoter, which is obtained from the bronze 1 gene of maize. The Bz1 core promoter is obtained from the "myc" mutant Bz1-luciferase construct pBz1 LucR98 via cleavage at the Nhe\ site located at positions -53 to - 58 (Roth et al., 1991 ). The derived Bz1 core promoter fragment thus extends

from positions -53 to +227 and includes the Bz1 intron-1 in the 5 ¹ untranslated region.

Also useful for the presently disclosed subject matter is a minimal promoter created by use of a synthetic TATA element. The TATA element allows recognition of the promoter by RNA polymerase factors and confers a basal level of gene expression in the absence of activation (see generally,

Mukumoto et al., 1993; Green, 2000.

It can also be useful to optimize some or all of the codons of a gene disclosed herein for expression in a particular plant species. Expression of heterologous protein genes in plants can in some instances prove difficult. One postulated explanation for the cause of low expression is that fortuitous transcription processing sites produce aberrant forms of the mRNA transcript. These aberrantly processed transcripts are non-functional in a plant, in terms of producing the encoded protein. Thus, codon usage of a native heterologous gene can be significantly different from that which is typical of a plant gene naturally encoded by the host plant. This issue, if present, can be addressed through optimization of the codons of the heterologous gene for expression in the host plant. An optimized gene or DNA sequence refers to a gene in which the nucleotide sequence of a heterologous gene has been modified in order to utilize preferred codons for expression in the recipient plant. U.S. Patent No. 6,121 ,014, incorporated herein by reference in its entirety, teaches methods of codon optimization for expression of heterologous proteins in plants. V.C.4. Targeting of the Gene Product Within the Cell

Various mechanisms for targeting gene products are known to exist in plants and the sequences controlling the functioning of these mechanisms have been characterized in some detail. For example, the targeting of gene products to the chloroplast is controlled by a signal sequence found at the amino terminal end of various polypeptides that is cleaved during chloroplast import to yield the mature polypeptides (see e.g., Comai et al., 1988). These signal sequences can be fused to heterologous gene products to affect the import of heterologous products into the chloroplast (Van den Broeck et al., 1985). DNA encoding for appropriate signal sequences can be isolated from the 5' end of the cDNAs encoding the ribulose-1 ,5-bisphosphate carboxylase/oxygenase (RUBISCO)

polypeptide, the chlorophyll a/b binding (CAB) polypeptide, the 5-enol-pyruvyl shikimate-3-phosphate (EPSP) synthase enzyme, the GS2 polypeptide and many other polypeptides which are known to be chloroplast localized. See also, the section entitled "Expression With Chloroplast Targeting" in Example 37 of U.S. Patent No. 5,639,949, herein incorporated by reference.

Other gene products can be localized to other organelles such as the mitochondrion and the peroxisome (e.g. Unger et al., 1989). The cDNAs encoding these products can also be manipulated to effect the targeting of heterologous gene products to these organelles. Examples of such sequences are the nuclear-encoded ATPases and specific aspartate amino transferase isoforms for mitochondria. Targeting cellular polypeptide bodies has been disclosed by Rogers et al., 1985.

In addition, sequences have been characterized that control the targeting of gene products to other cell compartments. Amino terminal sequences are responsible for targeting to the endoplasmic reticulum (ER), the apoplast, and extracellular secretion from aleurone cells (Koehler & Ho, 1990). Additionally, amino terminal sequences in conjunction with carboxy terminal sequences are responsible for vacuolar targeting of gene products (Shinshi et al., 1990).

By the fusion of the appropriate targeting sequences disclosed above to transgene sequences of interest it is possible to direct the transgene product to any organelle or cell compartment. For chloroplast targeting, for example, the chloroplast signal sequence from the RUBISCO gene, the CAB gene, the EPSP synthase gene, or the GS2 gene is fused in frame to the amino terminal ATG of the transgene. The signal sequence selected can include the known cleavage site, and the fusion constructed can take into account any amino acids after the cleavage site that are required for cleavage. In some cases this requirement can be fulfilled by the addition of a small number of amino acids between the cleavage site and the transgene ATG or, alternatively, replacement of some amino acids within the transgene sequence. Fusions constructed for chloroplast import can be tested for efficacy of chloroplast uptake by in vitro translation of in vitro transcribed constructions followed by in vitro chloroplast uptake using techniques disclosed by Bartlett et al. , 1982 and Wasmann et al. ,

1986. These construction techniques are well known in the art and are equally applicable to mitochondria and peroxisomes.

The above-disclosed mechanisms for cellular targeting can be utilized not only in conjunction with their cognate promoters, but also in conjunction with heterologous promoters so as to effect a specific cell-targeting goal under the transcriptional regulation of a promoter that has an expression pattern different from that of the promoter from which the targeting signal derives. V.D. Construction of Plant Transformation Vectors Numerous transformation vectors available for plant transformation are known to those of ordinary skill in the plant transformation art, and the genes pertinent to the presently disclosed subject matter can be used in conjunction with any such vectors. The selection of vector will depend upon the selected transformation technique and the target species for transformation. For certain target species, different antibiotic or herbicide selection markers might be employed. Selection markers used routinely in transformation include the nptll gene, which confers resistance to kanamycin and related antibiotics (Messing & Vieira, 1982; Bevan et al., 1983); the bar gene, which confers resistance to the herbicide phosphinothricin (White et al., 1990; Spencer et al., 1990); the hph gene, which confers resistance to the antibiotic hygromycin (Blochinger & Diggelmann, 1984); the dhfr gene, which confers resistance to methotrexate (Bourouis & Jarry, 1983); the EPSP synthase gene, which confers resistance to glyphosate (U.S. Patent Nos. 4,940,935 and 5,188,642); and the mannose-6- phosphate isomerase gene, which provides the ability to metabolize mannose (U.S. Patent Nos. 5,767,378 and 5,994,629). V.D.1. Vectors Suitable for Agrobacterium Transformation

Many vectors are available for transformation using Agrobacterium tumefaciens. These typically carry at least one T-DNA border sequence and include vectors such as pBIN19 (Bevan, 1984). Below, the construction of two typical vectors suitable for Agrobacterium transformation is disclosed. V.D.I .a. PCIB200 and pCIB2001

The binary vectors pCIB200 and pCIB2001 are used for the construction of recombinant vectors for use with Agrobacterium and are constructed in the following manner. pTJS75kan is created by Naή digestion of pTJS75

(Schmidhauser& Helinski, 1985) allowing excision of the tetracycline-resistance gene, followed by insertion of an Acc\ fragment from pUC4K carrying an NPTII sequence (Messing & Vieira, 1982: Bevan et a/. , 1983: McBride & Summerfelt. 1990). Xho\ linkers are ligated to the EcoRV fragment of PCIB7 which contains the left and right T-DNA borders, a plant selectable nos/nptll chimeric gene and the pUC polylinker (Rothstein etal., 1987), and the Xλo I -digested fragment are cloned into Sa/l-digested pTJS75kan to create pCIB200 (see also EP 0 332 104, example 19). pCIB200 contains the following unique polylinker restriction sites: EcoRl, Sst\, Kpn\, BgIW, Xba\, and Sa/I. pCIB2001 is a derivative of pCIB200 created by the insertion into the polylinker of additional restriction sites. Unique restriction sites in the polylinker of pCIB2001 are EcoR\, Sst\, Kpn\, BgIW, Xba\, Sal\, Mlu\, BcIl, AvrW, Apa\, Hpa\, and Stu\. pCIB2001 , in addition to containing these unique restriction sites, also has plant and bacterial kanamycin selection, left and right T-DNA borders for v4grabacte/7i/m-mediated transformation, the RK2-derived trfA function for mobilization between E. coll and other hosts, and the OriT and OnV functions also from RK2. The pCIB2001 polylinker is suitable for the cloning of plant expression cassettes containing their own regulatory signals.

V.D.I .b. pCIBIO and Hvgromvcin Selection Derivatives Thereof The binary vector pCIBIO contains a gene encoding kanamycin resistance for selection in plants, T-DNA right and left border sequences, and incorporates sequences from the wide host-range plasmid pRK252 allowing it to replicate in both E. coli and Agrobacterium. Its construction is disclosed by Rothstein etal., 1987. Various derivatives of pCIBIO can be constructed which incorporate the gene for hygromycin B phosphotransferase disclosed by Gritz & Davies, 1983. These derivatives enable selection of transgenic plant cells on hygromycin only (pCIB743), or hygromycin and kanamycin (pCIB715, pCIB717).

V.D.2. Vectors Suitable for non-Aαrobacterium Transformation Transformation without the use of Agrobacterium tυmefaciens circumvents the requirement for T-DNA sequences in the chosen transformation vector, and consequently vectors lacking these sequences can be utilized in addition to vectors such as the ones disclosed above that contain

T-DNA sequences. Transformation techniques that do not rely on

Agrobacterium include transformation via particle bombardment, protoplast uptake (e.g. polyethylene glycol (PEG) and electroporation), and microinjection.

The choice of vector depends largely on the species being transformed. Below, the construction of typical vectors suitable for non-Agrobacterium transformation is disclosed.

V.D.2.a. PCIB3064 pCIB3064 is a pUC-derived vector suitable for direct gene transfer techniques in combination with selection by the herbicide BASTA ^® (glufosinate ammonium or phosphinothricin). The plasmid pCIB246 comprises the CaMV 35S promoter in operational fusion to the E. coli β-glucuronidase (GUS) gene and the CaMV 35S transcriptional terminator and is disclosed in the PCT International Publication WO 93/07278. The 35S promoter of this vector contains two ATG sequences 5' of the start site. These sites are mutated using standard PCR techniques in such a way as to remove the ATGs and generate the restriction sites Ssp\ and PvuU. The new restriction sites are 96 and 37 bp away from the unique Sail site and 101 and 42 bp away from the actual start site. The resultant derivative of pCIB246 is designated pCIB3025. The GUS gene is then excised from pCIB3025 by digestion with Sa// and Sac\, the termini rendered blunt and religated to generate plasmid pCIB3060. The plasmid pJIT82 is obtained from the John lnnes Centre, Norwich, England, and the 400 bp Sma\ fragment containing the bar gene from Streptomyces viridochromogenes is excised and inserted into the Hpa\ site of pCIB3060 (Thompson et al., 1987). This generated pCIB3064, which comprises the bar gene under the control of the CaMV 35S promoter and terminator for herbicide selection, a gene for ampicillin resistance (for selection in E. coli) and a polylinker with the unique sites Sph\, Pst\, Hind\\\, and BamH\. This vector is suitable for the cloning of plant expression cassettes containing their own regulatory signals. V.D.2.b. pSOG19 and pSOG35 pSOG35 is a transformation vector that utilizes the E. coli dihydrofolate reductase (DHFR) gene as a selectable marker conferring resistance to methotrexate. PCR is used to amplify the 35S promoter (-800 bp), intron 6 from

the maize Adh1 gene (-550 bp), and 18 bp of the GUS untranslated leader sequence from pSOGIO. A 250-bp fragment encoding the E. coli dihydrofolate reductase type Il gene is also amplified by PCR and these two PCR fragments are assembled with a Sac\-Pst\ fragment from pB1221 (BD Biosciences Clontech, Palo Alto, California, United States of America) that comprises the pUC19 vector backbone and the nopaline synthase terminator. Assembly of these fragments generates pSOG19 that contains the 35S promoter in fusion with the intron 6 sequence, the GUS leader, the DHFR gene, and the nopaline synthase terminator. Replacement of the GUS leader in pSOG19 with the leader sequence from Maize Chlorotic Mottle Virus (MCMV) generates the vector pSOG35. pSOG19 and pSOG35 carry the pUC gene for ampicillin resistance and have Hind\\\, Sph\, Pst\, and EcoR\ sites available for the cloning of foreign substances. V.E. Transformation Once a nucleic acid sequence of the presently disclosed subject matter has been cloned into an expression system, it is transformed into a plant cell.

The receptor and target expression cassettes of the presently disclosed subject matter can be introduced into the plant cell in a number of art-recognized ways.

Methods for regeneration of plants are also well known in the art. For example, Ti plasmid vectors have been utilized for the delivery of foreign DNA, as well as direct DNA uptake, liposomes, electroporation, microinjection, and microprojectiles. In addition, bacteria from the genus Agrobacteήum can be utilized to transform plant cells. Below are descriptions of representative techniques for transforming both dicotyledonous and monocotyledonous plants, as well as a representative plastid transformation technique. V.E.1. Transformation of Dicotyledons

Transformation techniques for dicotyledons are well known in the art and include Agrobacterium-based techniques and techniques that do not require Agrobacteήum. Non-Agrobacterium techniques involve the uptake of exogenous genetic material directly by protoplasts or cells. This can be accomplished by PEG or electroporation-mediated uptake, particle bombardment-mediated delivery, or microinjection. Examples of these techniques are disclosed in Paszkowski et a/., 1984; Potrykus et al., 1985;

Reich et al., 1986; and Klein et al., 1987. In each case the transformed cells are regenerated to whole plants using standard techniques known in the art. transformation is a useful technique for transformation of dicotyledons because of its high efficiency of transformation and its broad utility with many different species. Agrobacteήum transformation typically involves the transfer of the binary vector carrying the foreign DNA of interest (e.g. pCIB200 or pCIB2001 ) to an appropriate Agrobacterium strain which can depend on the complement of vir genes carried by the host Agrobacterium strain either on a co-resident Ti plasmid or chromosomally (e.g. strain CIB542 for pCIB200 and pCIB2001 (Uknes et al., 1993). The transfer of the recombinant binary vector to Agrobacteήum is accomplished by a triparental mating procedure using E. coli carrying the recombinant binary vector, a helper E. coli strain that carries a plasmid such as pRK2013 and which is able to mobilize the recombinant binary vector to the target Agrobacterium strain. Alternatively, the recombinant binary vector can be transferred to Agrobacterium by DNA transformation (Hόfgen & Willmitzer, 1988).

Transformation of the target plant species by recombinant Agrobacteήum usually involves co-cultivation of the Agrobacterium with explants from the plant and follows protocols well known in the art. Transformed tissue is regenerated on selectable medium carrying the antibiotic or herbicide resistance marker present between the binary plasmid T-DNA borders.

Another approach to transforming plant cells with a gene involves propelling inert or biologically active particles at plant tissues and cells. This technique is disclosed in U.S. Patent Nos. 4,945,050; 5,036,006; and 5,100,792; all to Sanford et al. Generally, this procedure involves propelling inert or biologically active particles at the cells under conditions effective to penetrate the outer surface of the cell and afford incorporation within the interior thereof. When inert particles are utilized, the vector can be introduced into the cell by coating the particles with the vector containing the desired gene. Alternatively, the target cell can be surrounded by the vector so that the vector is carried into the cell by the wake of the particle. Biologically active particles

(e.g., dried yeast cells, dried bacterium, or a bacteriophage, each containing DNA sought to be introduced) can also be propelled into plant cell tissue. V.E.2. Transformation of Monocotyledons

Transformation of most monocotyledon species has now also become routine. Exemplary techniques include direct gene transfer into protoplasts using PEG or electroporation, and particle bombardment into callus tissue. Transformations can be undertaken with a single DNA species or multiple DNA species (i.e. co-transformation), and both these techniques are suitable for use with the presently disclosed subject matter. Co-transformation can have the advantage of avoiding complete vector construction and of generating transgenic plants with unlinked loci for the gene of interest and the selectable marker, enabling the removal of the selectable marker in subsequent generations, should this be regarded as desirable. However, a disadvantage of the use of co-transformation is the less than 100% frequency with which separate DNA species are integrated into the genome (Schocher eif a/. , 1986).

Patent Applications EP 0 292 435, EP 0 392 225, and WO 93/07278 describe techniques for the preparation of callus and protoplasts from an elite inbred line of maize, transformation of protoplasts using PEG or electroporation, and the regeneration of maize plants from transformed protoplasts. Gordon- Kamm et al., 1990 and Fromm et al., 1990 have published techniques for transformation of A188-derived maize line using particle bombardment. Furthermore, WO 93/07278 and Koziel etal., 1993 describe techniques for the transformation of elite inbred lines of maize by particle bombardment. This technique utilizes immature maize embryos of 1.5-2.5 mm length excised from a maize ear 14-15 days after pollination and a PDS-1000He Biolistic particle delivery device (DuPont Biotechnology, Wilmington, Delaware, United States of America) for bombardment.

Transformation of rice can also be undertaken by direct gene transfer techniques utilizing protoplasts or particle bombardment. Protoplast-mediated transformation has been disclosed for Japon/ca-types and /nd/ca-types (Zhang et al., 1988; Shimamoto et al., 1989; Datta et al., 1990) of rice. Both types are also routinely transformable using particle bombardment (Christou etal., 1991 ).

Furthermore, WO 93/21335 describes techniques for the transformation of rice

via θlθctroporation. Casas et al., 1993 discloses the production of transgenic sorghum plants by microprojectile bombardment.

European Patent Application EP 0 332 581 describes techniques for the generation, transformation, and regeneration of Pooideae protoplasts. These techniques allow the transformation of Dactylis and wheat. Furthermore, wheat transformation has been disclosed in Vasil et al., 1992 using particle bombardment into cells of type C long-term regenerable callus, and also by Vasil et al., 1993 and Weeks et al., 1993 using particle bombardment of immature embryos and immature embryo-derived callus. A representative technique for wheat transformation, however, involves the transformation of wheat by particle bombardment of immature embryos and includes either a high sucrose or a high maltose step prior to gene delivery. Prior to bombardment, embryos (0.75-1 mm in length) are plated onto MS medium with 3% sucrose (Murashige & Skoog, 1962) and 3 mg/l 2,4- dichlorophenoxyacetic acid (2,4-D) for induction of somatic embryos, which is allowed to proceed in the dark. On the chosen day of bombardment, embryos are removed from the induction medium and placed onto the osmoticum (Ae. induction medium with sucrose or maltose added at the desired concentration, typically 15%). The embryos are allowed to plasmolyze for 2-3 hours and are then bombarded. Twenty embryos per target plate are typical, although not critical. An appropriate gene-carrying plasmid (such as pCIB3064 or pSG35) is precipitated onto micrometer size gold particles using standard procedures. Each plate of embryos is shot with the DuPont BIOLISTICS® helium device using a burst pressure of about 1000 pounds per square inch (psi) using a standard 80 mesh screen. After bombardment, the embryos are placed back into the dark to recover for about 24 hours (still on osmoticum). After 24 hours, the embryos are removed from the osmoticum and placed back onto induction medium where they stay for about a month before regeneration. Approximately one month later the embryo explants with developing embryogenic callus are transferred to regeneration medium (MS + 1 mg/liter NAA, 5 mg/liter GA), further containing the appropriate selection agent (10 mg/l BASTA® in the case of pCIB3064 and 2 mg/l methotrexate in the case of pSOG35). After approximately one month, developed shoots are transferred to larger sterile

containers known as "GA7s" which contain half-strength MS, 2% sucrose, and the same concentration of selection agent.

Transformation of monocotyledons using Agrobacterium has also been disclosed. See WO 94/00977 and U.S. Patent No. 5,591 ,616, both of which are incorporated herein by reference. See also Negrotto etal., 2000, incorporated herein by reference. Zhao et al., 2000 specifically discloses transformation of sorghum with Agrobacterium. See also U.S. Patent No. 6,369,298.

Rice (Oryza sativa) can be used for generating transgenic plants. Various rice cultivars can be used (Hiei et al., 1994; Dong et al., 1996; Hiei et al., 1997). Also, the various media constituents disclosed below can be either varied in quantity or substituted. Embryogenic responses are initiated and/or cultures are established from mature embryos by culturing on MS-CIM medium (MS basal salts, 4.3 g/liter; B5 vitamins (200 x), 5 ml/liter; Sucrose, 30 g/liter; proline, 500 mg/liter; glutamine, 500 mg/liter; casein hydrolysate, 300 mg/liter; 2,4-D (1 mg/ml), 2 ml/liter; pH adjusted to 5.8 with 1 N KOH; Phytagel, 3 g/liter).

Either mature embryos at the initial stages of culture response or established culture lines are inoculated and co-cultivated with the Agrobacterium tumefaciens strain LBA4404 {Agrobacterium) containing the desired vector construction. Agrobacterium is cultured from glycerol stocks on solid YPC medium (plus 100 mg/L spectinomycin and any other appropriate antibiotic) for about 2 days at 28°C. Agrobacterium is re-suspended in liquid MS-CIM medium. The Agrobacterium culture is diluted to an OD ₆ oo of 0.2-0.3 and acetosyringone is added to a final concentration of 200 μM. Acetosyringone is added before mixing the solution with the rice cultures to induce Agrobacterium for DNA transfer to the plant cells. For inoculation, the plant cultures are immersed in the bacterial suspension. The liquid bacterial suspension is removed and the inoculated cultures are placed on co-cultivation medium and incubated at 22°C for two days. The cultures are then transferred to MS-CIM medium with ticarcillin (400 mg/liter) to inhibit the growth of Agrobacterium. For constructs utilizing the PMI selectable marker gene (Reed etal., 2001 ), cultures are transferred to selection medium containing mannose as a carbohydrate source (MS with 2% mannose, 300 mg/liter ticarcillin) after 7 days, and cultured for 3-4 weeks in the dark. Resistant colonies are then transferred to

regeneration induction medium (MS with no 2,4-D, 0.5 mg/liter IAA, 1 mg/!iter zeatin, 200 mg/liter TIMENTIN®, 2% mannose, and 3% sorbitol) and grown in the dark for 14 days. Proliferating colonies are then transferred to another round of regeneration induction media and moved to the light growth room. Regenerated shoots are transferred to GA7 containers with GA7-1 medium (MS with no hormones and 2% sorbitol) for 2 weeks and then moved to the greenhouse when they are large enough and have adequate roots. Plants are transplanted to soil in the greenhouse (T ₀ generation) grown to maturity and the Ti seed is harvested. V.E.3. Transformation of Plastids

Seeds of Nicotiana tabacum c.v. 'Xanthi nc ^J are germinated seven per plate in a 1 " circular array on T agar medium and bombarded 12-14 days after sowing with 1 μm tungsten particles (M10, Bio-Rad Laboratories, Hercules, California, United States of America) coated with DNA from plasmids pPH143 and pPH145 essentially as disclosed (Svab & Maliga, 1993). Bombarded seedlings are incubated on T medium for two days after which leaves are excised and placed abaxial side up in bright light (350-500 μmol photons/m ² /s) on plates of RMOP medium (Svab et al., 1990) containing 500 μg/ml spectinomycin dihydrochloride (Sigma, St. Louis, Missouri, United States of America). Resistant shoots appearing underneath the bleached leaves three to eight weeks after bombardment are subcloned onto the same selective medium, allowed to form callus, and secondary shoots isolated and subcloned. Complete segregation of transformed plastid genome copies (homoplasmicity) in independent subclones is assessed by standard techniques of Southern blotting (Sambrook & Russell, 2001 ). βamHI/EcoRI-digested total cellular DNA (Mettler, 1987) is separated on 1 % Tris-borate-EDTA (TBE) agarose gels, transferred to nylon membranes (Amersham Biosciences, Piscataway, New Jersey, United States of America) and probed with ³² P-labeled random primed DNA sequences corresponding to a 0.7 kb BamH\/Hind\\\ DNA fragment from pC8 containing a portion of the φs7/12 plastid targeting sequence. Homoplasmic shoots are rooted aseptically on spectinomycin-containing MS/IBA medium (McBride et al., 1994) and transferred to the greenhouse.

VL Plants, Breeding, and Seed Production VIA Plants

The presently disclosed subject matter also provides plants comprising the disclosed compositions. In some embodiments, the plant is characterized by a modification of a phenotype or measurable characteristic of the plant, the modification being attributable to the expression cassette. In some embodiments, the modification involves root growth modification, which can affect nutritional enhancement, increased nutrient uptake efficiency, enhanced production of endogenous compounds, production and/or uptake of heterologous compounds, and/or increased, resistance to an herbicide, an abiotic stress, or a pathogen. In some embodiments, the modification involves root growth modification, which can result in the plant having enhanced or diminished requirement for light, water, nitrogen, or trace elements. In some embodiments, the modification involves root growth modification, which can result in the plant being enriched for an essential amino acid as a proportion of a polypeptide fraction of the plant. In some embodiments, the polypeptide fraction can be, for example, total seed polypeptide, soluble polypeptide, insoluble polypeptide, water-extractable polypeptide, and lipid-associated polypeptide. In some embodiments, the modification includes overexpression, underexpression, antisense modulation, sense suppression, inducible expression, inducible repression, or inducible modulation of a gene. VLB. Breeding

The plants obtained via transformation with a nucleic acid sequence of the presently disclosed subject matter can be any of a wide variety of plant species, including monocots and dicots; however, the plants used in the method for the presently disclosed subject matter are selected in some embodiments from the list of agronomically important target crops set forth hereinabove. The expression of a gene of the presently disclosed subject matter in combination with other characteristics important for production and quality can be incorporated into plant lines through breeding. Breeding approaches and techniques are known in the art. See e.g., Welsh, 1981 ; Wood, 1983; Mayo, 1987; Singh, 1986; Wricke & Weber, 1986; Varshney et al., 2004.

The genetic properties engineered into the transgenic seeds and plants disclosed above are passed on by sexual reproduction or vegetative growth and can thus be maintained and propagated in progeny plants. Generally, the maintenance and propagation make use of known agricultural methods developed to fit specific purposes such as tilling, sowing, or harvesting. Specialized processes such as hydroponics or greenhouse technologies can also be applied. As the growing crop is vulnerable to attack and damage caused by insects or infections as well as to competition by weed plants, measures are undertaken to control weeds, plant diseases, insects, nematodes, and other adverse conditions to improve yield. These include mechanical measures such as tillage of the soil or removal of weeds and infected plants, as well as the application of agrochemicals such as herbicides, fungicides, gametocides, nematicides, growth regulants, ripening agents, and insecticides. Use of the advantageous genetic properties of the transgenic plants and seeds according to the presently disclosed subject matter can further be made in plant breeding, which aims at the development of plants with improved properties such as tolerance of pests, herbicides, or abiotic stress, improved nutritional value, increased yield, or improved structure causing less loss from lodging or shattering. The various breeding steps are characterized by well- defined human intervention such as selecting the lines to be crossed, directing pollination of the parental lines, or selecting appropriate progeny plants.

Depending on the desired properties, different breeding measures are taken. The relevant techniques are well known in the art and include, but are not limited to, hybridization, inbreeding, backcross breeding, multiline breeding, variety blend, interspecific hybridization, aneuploid techniques, etc. Hybridization techniques can also include the sterilization of plants to yield male or female sterile plants by mechanical, chemical, or biochemical means. Cross- pollination of a male sterile plant with pollen of a different line assures that the genome of the male sterile but female fertile plant will uniformly obtain properties of both parental lines. Thus, the transgenic seeds and plants according to the presently disclosed subject matter can be used for the breeding of improved plant lines that, for example, increase the effectiveness of

conventional methods such as herbicide or pesticide treatment or allow one to dispense with said methods due to their modified genetic properties. Alternatively new crops with improved stress tolerance can be obtained, which, due to their optimized genetic "equipment", yield harvested product of better quality than products that were not able to tolerate comparable adverse developmental conditions (for example, drought). Vl. C. Seed Production

Some embodiments of the presently disclosed subject matter also provide seed and isolated product from plants that comprise an expression cassette comprising a promoter sequence operatively linked to an isolated nucleic acid, the nucleic acid sequence encoding an ARC polypeptide having root growth modulating activities as disclosed herein.

In some embodiments the isolated product comprises an enzyme, a nutritional polypeptide, a structural polypeptide, an amino acid, a lipid, a fatty acid, a polysaccharide, a sugar, an alcohol, an alkaloid, a carotenoid, a propanoid, a steroid, a pigment, a vitamin, or a plant hormone.

Embodiments of the presently disclosed subject matter also relate to isolated products produced by expression of an isolated nucleic acid comprising a nucleotide sequence encoding an ARC polypeptide having root growth modulating activities as disclosed herein.

In some embodiments, the product is produced in a plant. In some embodiments, the product is produced in cell culture. In some embodiments, the product is produced in a cell-free system.

In some embodiments, the product comprises an enzyme, a nutritional polypeptide, a structural polypeptide, an amino acid, a lipid, a fatty acid, a polysaccharide, a sugar, an alcohol, an alkaloid, a carotenoid, a propanoid, a steroid, a pigment, a vitamin, or a plant hormone. In some embodiments, the product is a polypeptide comprising an amino acid sequence of SEQ ID NOs: 1 , 2, and 5, or orthologs thereof. In seed production, germination quality and uniformity of seeds are essential product characteristics. As it is difficult to keep a crop free from other crop and weed seeds, to control seedbome diseases, and to produce seed with good germination, fairly extensive and well-defined seed production practices

f have been developed by seed producers who are experienced in the art of growing, conditioning, and marketing of pure seed. Thus, it is common practice for the farmer to buy certified seed meeting specific quality standards instead of using seed harvested from his own crop. Propagation material to be used as seeds is customarily treated with a protectant coating comprising herbicides, insecticides, fungicides, bactericides, nematicides, molluscicides, or mixtures thereof. Customarily used protectant coatings comprise compounds such as captan, carboxin, thiram (tetramethylthiuram disulfide; TMTD®; available from R. T. Vanderbilt Company, Inc., Norwalk, Connecticut, United States of America), methalaxyl (APRON XL®; available from Syngenta Corp., Wilmington, Delaware, United States of America), and pirimiphos-methyl (ACTELLIC ^® ; available from Agriliance, LLC, St. Paul, Minnesota, United States of America). If desired, these compounds are formulated together with further carriers, surfactants, and/or application-promoting adjuvants customarily employed in the art of formulation to provide protection against damage caused by bacterial, fungal, or animal pests. The protectant coatings can be applied by impregnating propagation material with a liquid formulation or by coating with a combined wet or dry formulation. Other methods of application are also possible such as treatment directed at the buds or the fruit.

EXAMPLES

The following Examples have been included to illustrate modes of the presently disclosed subject matter. In light of the present disclosure and the general level of skill in the art, those of skill will appreciate that the following Examples are intended to be exemplary only and that numerous changes, modifications, and alterations can be employed without departing from the scope of the presently disclosed subject matter.

MATERIALS AND METHODS FOR EXAMPLES 1-5 Phylogenetic analysis

Protein sequences were aligned using ClustalW 1.82 (available through the website of the European Bioinformatics Institute, Cambridge, United Kingdom) with standard parameters. A cladogram was generated from a

ClustalW alignment using the Phylip method. Plant growth conditions

Arabidopsis thaliana (Columbia ecotype) was germinated and seedlings grown in sterile conditions on plates containing 0.5 x Murashige and Skoog (MS) salts (Sigma Chemical Co., St. Louis, Missouri, U.S.A., M0404) pH 5.7 and 1 % agar. Seeds were surface sterilized with 20% PAROZONE™ and stratified at 4 ^° C in the dark for 2-3 days before germinating. Seedlings were grown under a 16 hours light/8 hours dark regime (Osram 4800 white, Munich, Germany and Sylvania GRO-LUX ^® Fluo 58 Watt T12 bulbs, Danvers, Massachusetts, U.S.A.). For mature plant growth and seed harvesting, seedlings were transferred to a 1 :1 mix of LEVINGTON M3 ^® compost (Scotts, Marysville, Ohio, U.S.A.) and vermiculite and grown under long day greenhouse conditions.

Root assays For lateral root assays, seedlings were grown vertically, as a single row of seeds planted ~1.5 cm from the top of the plate. Lateral roots visible to the naked eye were counted 7, 10, 11 , 12 and 13 days after germination. Digital photographs of all plates were taken. Root length was measured from these pictures using IMAGEJ™ software available from the National Institutes of Health, Research Services Branch, Bethesda, Maryland, U.S.A.

Root development was also assayed on 0.5x MS plates supplemented with the following plant growth regulators: 1 μM IAA, 2OnM 2,4D, 1-5μM NPA and 0.5μM ABA. Control plates were supplemented with the relevant concentration of solvent without hormone. Mutant analysis arabidillo-1 and -2 insertion lines (SAIL190_D02 and SAIL162__B11 , respectively) were obtained from the Syngenta SAIL collection (Sessions etal. 2002). Single insert lines were obtained by segregation analysis followed by PCR screening of genomic DNA with the recommended primers (Sessions et a/. 2002), and Southern blotting of individual genomic DNAs. Genomic DNA was prepared using a Qiagen DNEASY ^® plant miniprep kit (Qiagen, Valencia, California, U.S.A.) and Southern analysis was performed using the DIG™ system (Roche, Indianapolis, Indiana, U.S.A.).

W

uioning and construct generation

Putative arabidillo promoter regions were amplified from Arabidopsis genomic DNA, using PFUTURBO ^® DNA polymerase (Stratagene, La JoIIa California, U.S.A.). Long (1.7kb of upstream region for arabidillo-1 , 1.6kb of 5 upstream region for arabidillo-2) and short (1.5kb for arabidillo-1, 1.3kb for arabidillo-2) versions of the promoters were amplified, the same results were seen with both long and short versions of each promoter. The promoter fragments were cloned into pBI101 (Clontech, Palo Alto, California, U.S.A.) to make pArabidillor.GUS reporter constructs.

10 arabidillo coding regions were cloned into pGREEN 0029™ (Hellens et al. 2000; John lnnes Centre, Norwich, United Kingdom), to create Arabidillo- yellow fluorescent protein (YFP) protein fusions under the control of 35S or Arabidillo promoters, and the NOS terminator. The predicted full-length arabidillo-1 and -2 protein coding regions were amplified from CoI-O genomic 15 DNA, as were truncated versions of arabidillo-1. Site directed mutagenesis of the putative nuclear localization signal (NLS) of Arabidillo-1 was performed using the QUIKCHANGE ^® XL kit (Stratagene). Transgenic homozygous, single-insert T3 and T4 generation lines were examined for YFP expression.

Constructs were mobilized in Agrobacterium tumefaciens strain GV3101 20 by electroporation and transformed into Arabidopsis by floral dipping. Stable, single-insert homozygous lines were generated, as assayed by selection of seedlings on 50mg/l kanamycin.

RT-PCR

Total RNA was prepared from mature aerial tissues, whole seedlings and 5 seedling root tissue using a RNEASY ^® plant miniprep kit (Qiagen). cDNA was generated using SUPERSCRIPTII ^® reverse transcriptase (Invitrogen, Carlsbad,

California, U.S.A.) with both gene-specific and oligo-dT primers, arabidillo-1 or-

2 primers were used for gene-specific cDNA fragment amplification. Gene specificity of the amplified PCR products was confirmed by restriction digests. 0 β-glucuronidase (GUS) staining

Homozygous, single-insert T3 and T4 transformant seedlings were assayed for β-glucuronidase activity according to standard protocols (Wiegel and Glazebrook 2002). Briefly, seedlings were incubated in 0.5 mM potassium

ferrocyanide/0.5mM potassium ferricyanide/0.5mg/ml X-gluc pH 7 at 37 ⁰ C from 45 minutes to overnight. Seedling tissue was cleared through an ethanol series followed by clearing in 50% glycerol, 25% chloral hydrate for 15 min before mounting in Hoyer's medium for observation. Microscopy

Lateral root primordia of seedlings mounted in water on slides were counted at high magnification under the 10X objective of a Nikon E400 ECLIPSE™ microscope (Melville, New York, U.S.A.). GUS staining images were captured using a Leica FLUO III™ dissecting microscope (whole seedling images; Leica Microsystems GmbH, Wetzlar, Germany), or with the Differential interference contrast (DIC) optics of a Leica TCS SP confocal microscope and a modified COOLPIX ^® 950 digital camera (Nikon, Tokyo, Japan).

Optical sections of live tissue mounted in water were obtained using a Leica TCS SP confocal microscope and either 2OX or 63X (water immersion) objectives. The excitation wavelength was 512nm for both YFP and propidium iodide. For propidium iodide staining, seedlings were incubated in 5μg/L propidium iodide for 1 -1.5 min at room temperature and rinsed in distilled water.

EXAMPLE 1

ARABIDOPSIS GENES RELATED TO β-CATENIN/ARMADILLO ARE

EXPRESSED THROUGHOUT THE PLANT

As disclosed previously herein, Arabidopsis possesses two ARC protein- encoding genes, named arabidillo-1 (At2g44900; GENBANK ^® Accession Number NMJ 30054; SEQ ID NO:3) and arabidillo-2 (At3g60350; GENBANK ^® Accession Number NM_115899; SEQ ID NO:4), which show the greatest similarity of any Arabidopsis genes to Dictyostelium and metazoan β- caterim/ Armadillo (Coates 2003). There is also an ARC protein Arabidillo homologue in the model monocot, rice (Oryza spp.) (GENBANK ^® Accession Number NP_922746; SEQ ID NO. 5 and GENBANK ^® Accession Number NM_197764; SEQ ID NO. 6 for protein and polynucleotide sequences, respectively).

Arabidillo-1 and -2 contain at least nine Armadillo repeats (see Figure 1 )

and the two proteins share 80% amino acid identity (92% within the Arm repeat region). Like Dictyostelium β-catenin, both Arabidillo-1 and -2 contain a putative F-box motif (Figure 1 ). arabidillo-1 (At2g44900; SEQ ID NO:3) is also annotated as AtFBXδ (Xiao and Jang 2000). Arabidillo-1 and -2, the Oryza ARC protein and Arabidillo homologue, and Dictyostelium Aardvark each have an F-box/Armadillo repeat domain structure as demonstrated by database survey using CDART (available from the National Center for Biotechnology Information, National Institutes of Health).

Arabidillo-1 and -2 protein sequences contain a basic nuclear localization signal (Conti et al. 1998) at their amino-terminal ends (Figure 1 ). Both proteins also contain several putative leucine-rich nuclear export signals (Wen et al. 1995, Fornerod et al. 1997, Ossareh-Nazari et al. 1997, Fukuda et al. 1997, Neville et al. 1997) (Figure 1 ). cDNAs corresponding to the entire predicted open reading frames of both genes can be amplified from total RNA, indicating that the predicted splice sites and protein coding sequences in the databases are correct.

Expression of both arabidillo-1 and -2 mRNA were detected in all parts of the plant by RT-RCR. The same results were obtained using two independent pairs of gene-specific primers for both arabidillo-1 and -2. In addition, arabidillo-1 and -2 are both detected at a similar level in all plant parts in publicly available microarray datasets (GENEVESTIGATOR ^® Gene Atlas, Swiss Federal Institute of Technology, Zurich, Switzerland).

The spatial and temporal expression of arabidilllo-1 and -2 was analyzed further by fusing each of their putative promoters to the β-glucuronidase (GUS) reporter gene, to make pArabidillo-1::GUS and pArabidillo-2::GUS reporter constructs. 1.7kb and 1.5kb upstream regions of arabidillo-1 gave identical results, as did the corresponding 1.6 and 1.3kb upstream regions of arabidillo- 2. pArabidillo-1::GUS activity was detected throughout Arabidopsis seedlings, in both the root and shoot. GUS activity was strongest in the root tip, pericycle and vasculature. pArabidillo-2::GUS was also detected throughout the seedling, although strong expression was absent from both primary and lateral root tips; GUS activity was only detected in the columella region. Both

promoters were active in developing lateral root primordia.

EXAMPLE 2

ARABIDILLO-1 AND -2 PROTEINS LOCALIZE TO THE NUCLEUS To determine the subcellular localization of Arabidillo-1 and -2 proteins, the genomic coding regions of arabidillo-1 and -2 were both fused to YFP and driven from the cauliflower mosaic virus (CaMV) 35S promoter.

Both Arabidillo-1 -YFP and Arabidillo-2-YFP were detected in nuclei. Arabidillo-1 -YFP expressed more strongly than Arabidillo-2-YFP. Both fusion proteins were detected only in root cells by confocal microscopy, despite being expressed from the 35S promoter, and the transcripts being detected plant- wide. This could be due either to generally low levels of protein expression, or to specific degradation of Arabidillo proteins in certain cells.

EXAMPLE 3

ARABIDILLO-1 PROTEIN TARGETING

In order to understand how the different motifs present in the Arabidillo protein sequence contribute to protein function within the cell, a series of truncated Arabidillo-1 -YFP fusion proteins was generated and expressed stably in Arabidopsis. For each construct, the subcellular localization of YFP was identified.

A construct containing the first 93 amino acids of Arabidillo-1 fused to YFP (1-93) localized to the nucleus. Mutation of single residues in the NLS had no effect on the localization of construct 1-93, but mutation of pairs of residues (R4AR6A, R6AR7A, R6AK8A or R7AK8A) reduced the nuclear localization and increased the cytosolic localization of the protein. In addition, deletion of the first 31 or 45 amino acids of this sequence (constructs 31-93 and 45-93 respectively) lead to a relocalization of the fusion protein to the cytosol. Addition of a β-glucuronidase protein module to the F-box region (45-93) upstream of the YFP (45-93:GUS), relocated the F-box exclusively to the cytosol, suggesting that the nuclear localization seen with constructs 1-93 (mutant), 31- 93 and 45-93 was because their small size allows some diffusion into the nucleus.

Expression of the entire amino-terminus of Arabidillo-1 up to the start of Arm repeat domain (construct 1-376) or the amino terminus without the NLS (45-376) did not lead to reliably detectable YFP expression. It is possible that expression of these constructs at detectable levels is detrimental to seedlings. Expression of N-terminal regions of some yeast F-box proteins is toxic to cells (Dixon et al. 2003).

Expression of a construct containing the Arm repeat domain and C- terminal region but lacking the F-box (construct 221-930) localized to the cytosol, as did similar constructs with truncated C-termini (221-769 and 221- 829). However, a construct containing only the Arm domain and C-terminus (construct 375-930) localized both to the nucleus and cytosol. The N-terminal region of Arabidillo-1 lying between the F-box and the Arm domain contains at least three putative nuclear export signals (NES), which could account for the observed localization of construct 375-930 compared to 221-930. However, the cytosolic localization of constructs 221-930, 221-769 and 221-829 was not sensitive to leptomycin B, which inhibits CRM-1 mediated nuclear export (Fornerod et al. 1997; Ossareh-Nazari et al. 1997). The partial nuclear localization of construct 375-930 also demonstrates that Arabidillo-1 protein can localize to the nucleus in the absence of a classical NLS ₁ presumably via its Arm repeat domain, as is also seen with β-catenin (Fagotto et al. 1998).

EXAMPLE 4 ARABIDILLO-1 AND -2 ACT REDUNDANTLY TO PROMOTE LATERAL

ROOT DEVELOPMENT In order to examine the in plants functions of arabidillo-1 and -2,

Arabidopsis lines with T-DNA insertions within the coding region of each gene were obtained from the Syngenta Arabidopsis Insertion Line (SAIL) collection (Sessions et al. 2002). Homozygous, single insert lines were generated, and confirmed by Southern blotting, segregation analysis and PCR on genomic DNA. Neither the arabidillo-1 or -2 single mutant showed any obvious morphological phenotype. However, and without wishing to be limited by theory, as arabidillo-1 and -2 are very similar to each other and show overlapping expression within the plant, it is possible that the two genes

function redundantly. The single mutants were crossed and homozygous double mutant lines generated. arabidillo-1/-2 double mutants did not show gross morphological or fertility defects when grown on soil. However, when grown on vertical plates, arabidillo-1/-2 mutants develop fewer lateral roots than wild type plants, or either single mutant. The same results were obtained when 1% sucrose was included in the medium, and also when seedlings were grown on basal medium. This suggests that Arabidillo-1 and -2 have a positive function during lateral root development. To test whether overexpression of Arabidillo-1 or -2 could have the opposite effect to the double mutant, and increase the frequency of lateral root formation, Arabidopsis seedlings expressing Arabidillo-YFP fusion proteins under the control of the CaMV 35S promoter were assayed on vertical plates. Arabidillo-1 overexpression increased lateral root formation. When observed at high magnification, arabidillo double mutants did not contain an excess of unelongated or "arrested" visible lateral root primordia compared to wild type controls. In addition, arabidillo-1 /2 mutant lines stably expressing either Arabidillo-1 -YFP or -GFP or Arabidillo-2-YFP from their own promoters showed increased lateral root formation compared to the arabidillo-1 /2 mutant, despite an inability do detect fluorescent protein expression in these. Thus, it appears that arabidillo-1 and -2 have a redundant function promoting lateral root development in Arabidopsis and this appears to be due to promoting lateral root initiation rather than elongation.

In contrast to the full-length Arabidillo-1 -YFP overexpressing lines, none of the truncated Arabidillo-1 overexpressing lines tested had significantly different lateral root numbers to wild type. This suggests that the function of the full-length Arabidillo-1 protein is required to promote lateral root formation.

EXAMPLE 5 ARABIDILLO-1 AND -2 DO NOT ACT DIRECTLY IN KNOWN LATERAL

ROOT REGULATORY PATHWAYS

In order to understand how Arabidillo-1 and -2 function to promote lateral root development, their possible interaction with known lateral root

regulatory processes was tested. The plant hormone auxin has pleiotropic effects on development, including the promotion of lateral root formation. Mutants in components of the auxin signaling pathway, such as tir1 (Ruegger θt al. 1998), nad (Xie et al. 2000), slr1 (Fukaki et al. 2002) and sew (Pfluger and Zambryski 2004) have a reduced lateral root number. However, mutation or overexpression of these genes also generates additional phenotypes, such as reduced temperature-induced hypocotyl elongation in tir1 (Ruegger et al. 1998), larger shoot size in NAC1 overexpressing lines (Xie etal. 2000), shorter primary roots in plants overexpressing SINAT5 (Xie etal. 2002), reduced shoot size and agravitropic roots in slr1 (Fukaki et al. 2002) and aberrant floral development in seu (Pfluger and Zambryski 2004). These mutants are also less sensitive to the effects of exogenous auxins and auxin transport inhibitors (Ruegger et al. 1998, Xie et al. 2000, Fukaki et al. 2002, Pfluger and Zambryski 2004). arabidillo-1/2 mutants and Arabidillo-overexpressing lines do not exhibit obvious auxin-related shoot phenotypes, and have primary root lengths similar to wild type. The arabidillo-1/-2 mutant and the 35S::Arabidillo-1-YFP overexpressing lines were germinated and grown on medium containing the auxin analogues indole-3 acetic acid (IAA) and 2,4D. Both the arabidillo double mutant and the Arabidillo-overexpressing lines displayed identical responses to wild type in terms of primary root growth inhibition. In addition, arabidillo-1/-2 mutants and Arabidillo-1 overexpressors showed an increased lateral root density when treated with IAA and 2,4D, similarly to wild type. Notably, arabidillo-1 /-2 mutants grown on IAA and 2,4D had fewer lateral roots than their wild type counterparts, while Arabidillo-1 overexpressing lines had more lateral roots, as is seen with plants grown on medium without added growth regulators. arabidillo-1 /-2 mutants and Arabidillo-1 overexpressors were also sensitive to the auxin transport inhibitor N-1-Naphthylphtalamic acid (NPA).

The pArabidillo-1 :;GL/Stransgene was not responsive to IAA, and Arabidillo-1 -

YFP fusions did not change their localization or level in response to IAA, NPA or 2,4D. Together, these data suggest that Arabidillo-1 and -2 are not directly participating in auxin signaling during lateral root development.

Both arabidillo-1 /-2 mutant and Arabidillo-1 overexpressing seedlings showed reduced lateral root formation on 0.5μM abscisic acid (ABA, de Smet et

a/. 2003), similarly to wild type seedlings. However, ABA is postulated to affect lateral root elongation (de Smet et al. 2003, Deak and Ryan 2005), rather than initiation, so is likely to be working at a later developmental stage than arabidillo-1 and -2. This result also suggests that nitrate- or osmotica-sensing pathways are not dependent on arabidillo-1 and -2 (Signora et a/. 2001 , Deak and Malamy 2005).

The availability of macronutrients can profoundly affect the architecture of a plant's root system (Lopez-Bucio et a/. 2003 and references therein). arabidillo-1 /2 mutants and Arabidillo- overexpressing seedlings responded similarly to wild type when depleted of nutrients (nitrate, phosphate, or sulphate anions). As with auxin treatment, the actual lateral root density of the arabidillo- 1/-2 mutant is lower than wild type, while the Arabidillo-1 overexpressing lines attain a higher lateral root density, under all conditions tested.

Inspection of publicly available microarray data sets revealed no significant change in arabidillo gene expression in response to auxin, ABA or nutrient challenge. Thus, Arabidillo function defines a novel mechanism for promoting lateral root development.

DISCUSSION OF EXAMPLES 1-5 β-catenin related proteins promote lateral root development in Arabidopsis.

Arabidopsis possesses over 100 Armadillo repeat containing proteins

(Mudgil et al. 2004), of which two, Arabidillo-1 and -2, show the greatest similarity to metazoan and Dictyostelium β-catenins (Coates, 2003). As demonstrated in the Examples, Arabidillo-1 and -2 have a redundant function promoting lateral root development, arabidillo-1 /-2 double mutant seedlings form fewer lateral roots than wild type and arabidillo single mutants, while plants overexpressing Arabidillo-1 under the control of the CaMV 35S promoter form more lateral roots than wild type. The widespread gene expression pattern of both Arabidillo-1 and -2 suggests that they could have additional functions in other tissues.

The phenotype of the arabidillo-1 /-2 mutant is surprisingly subtle compared with the lethal effects of Armadillo/β-catenin loss of function in animals. In Drosophila, loss of embryonic Arm function leads to developmental

arrest and a segment polarity phenotype, due to the inability to specify cell fates correctly (Nusslein-Volhard and Wieschaus 1980). Zygotic Arm function is also required for cell adhesion during early morphogenesis (Cox et al. 1996). Mouse, Xenopus, zebrafish, ascidian and sea urchin embryos with loss or reduction of β-catenin function have severe axis-specification and patterning defects (Haegel et al. 1995, Kelly et al. 1995, Huelsken et al. 2000, Heasman et al. 1994, Logan et al. 1999, Wikramayake et al. 1998, lmai et al. 2000). However, in mammals, plakoglobin, a protein closely related to β-catenin, is able to substitute for β-catenin's adhesive functions (Huelsken et al. 2000). In C. elegans, three β-catenin homologues, wrm-1, hmp-2 and bar-1, are present.

Inhibition of wrm-1 function by RNAi is embryo-lethal, leading to a failure to specify endoderm at the four-cell stage (Rocheleau et al. 1997). Loss of hmp-2 arrests embryo development at dorsal closure due to a failure of cell-cell adhesion during gastrulation (Costa et al. 1998). However, loss of bar-1 leads to postembryonic cell specification defects in the vulva (Eisenmann et al. 1998) and certain neurons (Maloof et al. 1999).

By contrast, the Dictyostelium β-catenin (aardvark) loss of function mutant does not have a lethal phenotype. Although Aardvark functions analogously to β-catenin, regulating gene expression, cell differentiation, and actin-containing cell-cell contact formation (Grimson et al. 2000, Coates et al. 2002), the aar mutant manages to produce a viable, reproducing adult. The Dictyostelium multicellular developmental program is governed by responses to environmental stimuli including nutrient availability, light and temperature change. In this respect, it is can be compared to postembryonic development in plants.

Like Dictyostelium Aardvark, Arabidopsis Arabidillo-1 and -2 are not essential for embryonic development or development into an adult organism. Plants, being sessile, must reprogram their development to a great extent in response to changing environmental signals and stresses, leading to changes in overall morphology. Lateral root formation is a prime example of this process, as root architecture changes dramatically in response to a changing environment (eg Lopez-Bucio review, Malamy 2005). It is possible that a loss or gain of arabidillo function would be more easily noticeable under an as yet

untested set of environmental conditions.

Arabidillo-1 and -2 do not function within known lateral root signaling networks. arabidillo-1 /-2 mutants formed fewer lateral roots than wild type, while Arabidillo-1 overexpressing plants formed more, under each of the conditions tested. The fact that neither mutant nor overexpressing plants were more sensitive or resistant than wild type to a particular signal (hormonal or nutritional) suggests that Arabidillo-1 and -2 could regulate a competence or likelihood to form lateral roots, which could stem from the competence of pericycle cells within the main root to divide.

Arabidillo-1 and -2 fluorescent protein fusions localize to the nucleus, suggesting that like their non-plant counterparts they could function to directly regulate gene expression. It is known that β-catenin regulates cell proliferation in animals, and transcription of G1 phase cyclins (D1 and E1 ) is stimulated by β-catenin (Shtutman et al. 1999; Botrugno et al. 2004)

In Arabidopsis, overexpression of KRP2, a cyclin dependent kinase inhibitor, reduces, but does not abolish, lateral root formation (Himanen et al. 2002), similarly to the arabidillo double mutant. Overexpression of Arabidopsis cyclin B1 (cydAt) increases lateral root length rather than lateral root number (Doerner et al. 1996), suggesting other cyclins would be more likely candidate target genes for Arabidillo-1 and -2 in Arabidopsis.

The arabidillo mutant and overexpressing root phenotypes are also reminiscent of those of Arabidopsis heterotrimeric G-protein subunit mutants

(Ullah et al. 2003). Mutants in the β-subunit, agb-1, have pleiotropic developmental effects, including about twice as many lateral roots than wild type. Conversely, gpa-1 α-subunit mutants have about half the wild type number of lateral roots, like arabidillo-1 /-2 mutants. Like arabidillo-1 and -2, gpa-1 and agb-1 do not appear to directly couple to auxin signaling, gpa-1 and agb-1 have been implicated in integrating multiple signals that control cell proliferation during lateral root formation and other developmental processes

(Ullah 2001 , 2003).

Another protein that has been implicated in non-auxin-mediated lateral root formation is ALF4 (Di Donato et al. 2004). ALF4 is a novel nuclear plant-

specific protein that has widespread expression in non-root tissues. The alf4 mutant does not make lateral roots, and lateral root formation cannot be restored by auxin (DiDonato et al. 2004). It is possible that ALF4 is required to maintain the pericycle in a mitotically competent state for lateral root formation. Thus, it is possible there is a connection is between ALF4 and Arabidillo function.

Arabidillo-1 protein targeting.

As shown in the Examples, using truncated protein-YFP fusions to different regions of Arabidillo-1 protein can mediate targeting to different parts of the cell. The amino terminus of Arabidillo-1 contains a functional nuclear localization signal (NLS) that can direct the (otherwise cytoplasmic) F-box region to the nucleus, β-catenin does not contain a classical nuclear localization signal, but the Arm repeat domain can mediate nuclear targeting in an NLS-independent manner (Fagotto et al. 1998). The same is true of the Arm domain of Arabidillo-1. Whether the Arabidillo-1 NLS functions or is regulated in vivo is not yet known.

Arabidillo-1 has putative NESs between amino acids 221 and 378. Deletion of this region of the protein leads to increased nuclear localization of the remaining protein fragment. However, the cytoplasmic localization of Arabidillo-1 (221-930) is not altered by leptomycin B. Animal β-catenin undergoes LMB-independent nuclear export, which does not depend on CRM1 or classical NESs (Eleftheriou ef al. 2001 , Wiechens and Fagotto 2001 ). Although not wising to be bound by theory, It is possible that Arabidillo-1 is also regulated in this manner. Alternatively, and again without wishing to be bound by theory, the cytosolic localization of Arabidillo-1 (221-930) could be due to its ability to interact with cytosol-retaining binding partners that cannot bind to the shorter Arabidillo-1 (375-930) protein. Residues 221-378 of the Arabidillo-1 sequence do not show significant similarity to β-catenins. Concluding remarks. As disclosed herein in the Examples, two Arabidopsis β-catenin-related

ARC proteins, Arabidillo-1 and -2, function redundantly to promote the formation of lateral roots. At least Arabidillo-1 overexpressing plants, which have an increased lateral root number, but are not perturbed in other aspects of

development, can be of agronomic benefit. Different plant species expressing Arabidillo-1 or a homologous ARC protein can also be of agronomic benefit.

Arabidillo-1 and -2 are nuclear proteins, and by analogy with β-catenin it seems likely that they can regulate the expression of downstream target genes. However, both proteins also contain an F-box motif. Other F-box containing proteins act as specificity factors for multisubunit E3 ubiquitin ligases (reviewed in Moon et al. 2004). Thus, Arabidillo-1 and -2 could also function to target other proteins for degradation by the 26S proteasome in the nucleus. These two potential functions of Arabidillo-1 and -2 are not necessarily mutually exclusive: lateral root formation involves cell cycle regulation, which requires coordinated transcriptional regulation and degradation of specific protein targets. Arabidillo-1 and -2 could also have additional novel functions: for example, a yeast F-box protein, Rcyi p, has a non-proteasomal function in membrane recycling (Galan et al. 2001 ).

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It will be understood that various details of the presently disclosed subject matter may be changed without departing from the scope of the present subject matter. Furthermore, the foregoing description is for the purpose of illustration only, and not for the purpose of limitation.