WO2005014794A2

WO2005014794A2 - Generation of low phytate plants by molecular disruption of inositol polyphosphate kinases

Info

Publication number: WO2005014794A2
Application number: PCT/US2004/025756
Authority: WO
Inventors: John D. York; Jill Stevenson-Paulik
Original assignee: Duke University
Priority date: 2003-08-07
Filing date: 2004-08-09
Publication date: 2005-02-17
Also published as: WO2005014794A3

Abstract

The present subject matter provides novel inositol phosphate kinase polypeptides (Ipk1 and Ipk2) and nucleic acid molecules encoding the same. The present subject matter also provides methods of using the novel polypeptides and5 nucleic acid molecules. In some embodiments, the methods provide steps for producing a low phytate plant.

Description

DESCRIPTION GENERATION OF LOW PHYTATE PLANTS BY MOLECULAR DISRUPTION OF INOSITOL POLYPHOSPHATE KINASES

RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application Serial No. 60/493,267, filed August 7, 2003; the disclosure of which is incorporated herein by reference in its entirety.

GOVERNMENT INTEREST This invention was made with Government support under Grant No.ROI #HL- 55672-07 and R01 #HL-55672-05 awarded by National Heart, Lung, and Blood Institute, National Institutes of Health. The Government has certain rights in the invention.

TECHNICAL FIELD The presently disclosed subject matter pertains to nucleic acid molecules encoding inositol polyphosphate kinase polypeptides. The presently disclosed subject matter also relates to methods of using the nucleic acid molecules and/or polypeptides and mutants thereof in transgenic plants to confer desirable agronomic traits, and more specifically to produce transgenic plants having reduced phytate levels, and/or increase the levels of non-phytate phosphorous in the transgenic plants. Low phytate content is a preferred trait in plants used for food or animal feed.

TABLE OF ABBREVIATIONS (1 ,3,4,6)P - inositoM ,3,4,6 tetrakisphosphate 5-ptase - inositol polyphosphate 5-phosphatase AMV - Alfalfa Mosaic Virus ATCC - America Type Culture Collection BiP - binding polypeptide bp - basepair(s) CAB chlorophyll a/b binding

CaMV cauliflower mosaic virus carboxylase/oxygenase cDNA complementary DNA

CDPK calcium-dependent protein kinase cM centimorgan(s)

CMV cytomegalovirus

ColE1 a synthetic E. coli origin of replication

DEAE di-ethyl-amino-ethyl

DHFR dihydrofolate reductase dsRNA double-stranded RNA

EDTA ethylene diamine tetraacetic acid

ELISA enzyme-linked immunosorbent assay

EMCV encephalomyocarditis virus

EPSP 5-enol-pyruvyl shikimate-3-phosphate

ER endoplasmic reticulum

EST expressed sequence tag

GS2 glutamine synthase 2

GST glutathione S-transferase

GUS β-glucuronidase

HPLC high performance liquid chromatography

HSV herpes simplex virus l(1 ,2,3,4,6)P₅ inositol 1 ,2,3,4,6 pentakisphosphate l(1 ,2,4,5,6)P₅ inositol 1 ,2,4,5,6 pentakisphosphate l(1 -3-4)P₃ inositol 1 ,3,4 trisphosphate l(1 ,3,4,5)P₄ inositol 1 ,3,4,5 tetrakisphosphate l(1 ,3,4,5.6)P₅ inositol 1 ,3,4,5,6 pentakisphosphate l(1 ,4,5)P₃ inositol 1 ,4,5 trisphosphate l(1 ,4,5,6)P₄ inositol 1 ,4,5,6 tetrakisphosphate

IP inositol polyphosphate

IPs inositol trisphosphate

IP4 inositol tetrakisphosphate IPs inositol pentakisphosphate IPs inositol hexakisphosphate kb kilobase(s) kcat catalytic constant Km Michaelis Constant MCMV Maize Chlorotic Mottle Virus MDMV Maize Dwarf Mosaic Virus MTL metallothionein-like ORF open reading frame PCR polymerase chain reaction PEG polyethylene glycol PEPC phosphoenol carboxylase pgk phosphoglycerate kinase Pi inorganic phosphate PMI phosphomannose isomerase Protox protoporphyrinogen oxidase QTL quantitative trait locus/loci RFLP restriction fragment length polymorphism RNAi RNA interference RUBISCO ribulose-1 ,5-bisphosphate SELEX Systematic Evolution of Ligands by Exponential Enrichment Sf9 Spodoptera frugiperda cell line TBE tris-borate-EDTA TEV Tobacco Etch Virus tk thymidine kinase TMTD tetramethylthiuram disulfide TMV Tobacco Mosaic Virus

Amino Acid Abbreviations, Codes, and Functionallv Equivalent Codons

Amino Acid 3-Letter 1 -Letter Codons

Alanine Ala A GCA GCC GCG GCU Arginine Arg R AGA AGG CGA CGC CGG CGU Asparagine Asn N AAC AAU Aspartic Acid Asp D GAC GAU Cysteine Cys C UGC UGU Glutamic acid Glu E GAA GAG Glutamine Gin Q CAA CAG Glycine Gly G GGA GGC GGG GGU Histidine His H CAC CAU Isoleucine lie 1 AUA AUC AUU Leucine Leu L UUA UUG CUA CUC CUG CUU Lysine Lys K AAA AAG Methionine Met M AUG Phenylalanine Phe F UUC UUU Proline Pro P CCA CCC CCG CCU Serine Ser S ACG AGU UCA UCC UCG UCU Threonine Thr T ACA ACC ACG ACU Tryptophan Trp w UGG Tyrosine Tyr Y UAC UAU Valine Val V GUA GUC GUG GUU

BACKGROUND As a relatively cheap source of energy-dense and nutrient-rich foodstuff, cereal grains contribute more than 50% of worldwide energy intake and thus offer the most important food supply to the world population. The high nutritional value of seeds comes from the deposition of starch, lipids, proteins, and essential minerals during seed development. In this process, there is a corresponding accumulation of phytate, or myo-inositol hexakisphosphate (IP₆), which typically comprises >1 % of the dry weight and is responsible for at least two-thirds of total seed phosphate. Although the role of phytate reserves in the plant seed is not understood, its abundance in grain feed is known to cause nutritional and environmental problems.

Phytate is largely undigestible by monogastric animals and it chelates essential minerals such as iron, zinc, and calcium, rendering them unavailable for absorption. In developing countries, high phytate, grain-based diets are feared to exacerbate iron and zinc malnutrition and substitution of low-phytate maize for normal maize in the diet shows promise in improving zinc and iron absorption in human subjects. In developed countries where livestock is fed primarily grain-based feed, the excreted phytate contributes to environmental phosphorus pollution by washing into surface waters where it accelerates eutrophication. The problem of unavailable seed phosphate in animal feed is usually overcome by supplementation with bioavailable forms of phosphate or with fungal phytase, an enzyme that degrades phytate. Various other strategies have been used to deal with the high phytate content of feed, including the production of transgenic animals (Golovan et al., 2001 ) and plants (Holm et al., 2002) that express increased levels of phytase. In another approach, to avoid phytate synthesis in the plant seed altogether, there have been low-phytate mutants generated by random mutagenesis in maize (Raboy et al. 2000), barley (Larson et al., 1998; and Hatzack et al., 2000), rice (Larson et al., 2000), and soybean (Wilcox et al., 2000; and Hitz et al., 2002). In most cases, the site ofthe mutation is not known, although based on inositol phosphate analyses, many of the mutations are predicted to occur in early steps of phytate synthesis. Since precursors of phytate are known to participate in important events such as cytosolic and nuclear signaling, it is predicted that plants with mutations early in the phytate pathway could have pleitropic defects that would lead to decreased productivity. Therefore, there is a need for the identification of genes encoding peptides involved late in the phytate production pathway that can be manipulated to decrease phytate production in food and animal feed plants. This and other needs in the art are addressed by the present disclosure.

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 ofthe 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 ofthe 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 nucleic acids and polypeptides involved with inositol phosphorylation and phytate production. In one embodiment, an isolated nucleic acid molecule encoding an inositol phosphate kinase polypeptide is provided. The nucleic acid molecule can be selected from the group consisting of: (a) a nucleic acid molecule encoding a polypeptide comprising an amino acid sequence of one of even numbered SEQ ID NOs: 2-10 or a polypeptide at least 40% identical to even numbered SEQ ID NOs: 2-10 and having inositol phosphate kinase activity; (b) a nucleic acid molecule comprising a nucleic acid sequence ofone of odd numbered SEQ ID NOs:1-9; (c) a nucleic acid molecule that has a nucleic acid sequence at least 90% identical to the nucleic acid sequence of the nucleic acid molecule of (a) or (b); (d) a nucleic acid molecule that hybridizes to (a) or (b) under stringent hybridization conditions; (e) a nucleic acid molecule comprising a nucleic acid sequence complementary to (a); and (f) a nucleic acid molecule comprising a nucleic acid sequence that is the full reverse complement of (a). In one embodiment, a vector is provided comprising the nucleic acids disclosed herein. In another embodiment, an expression cassette comprising at least one of the nucleic acids disclosed herein is provided. In some embodiments, the expression cassette is operably linked to a promoter. In some embodiments the promoter is a plant promoter. In some embodiments the promoter is a constitutinve promoter. In some embodiments the promoter is a tissue-specific or a cell type-specific promoter. In some embodiments, the tissue-specific or cell type-specific promoter directs expression ofthe expression cassette in a location selected from the group consisting of epidermis, root, vascular tissue, meristem, cambium, cortex, pith, leaf, flower, seed, and combinations thereof. In another embodiment, an isolated inositol phosphate kinase polypeptide having inositol phosphate kinase activity is provided. The polypeptide is selected from the group consisting of: (a) an amino acid sequence of one of even numbered SEQ ID NOs: 2-10; (b) an amino acid sequence that is at least 40% identical to (a); (c) an amino acid sequence encoded by a nucleotide sequence substantially identical to a nucleotide sequence of one of odd numbered SEQ ID NOs: 1-9; and (d) an amino acid sequence encoded by a nucleic acid molecule capable of hybridizing under stringent conditions to a nucleic acid molecule ofone of odd numbered SEQ ID NOs: 1-9 or to a sequence fully complementary thereto. A method for for producing the polypeptides disclosed herein is also provided. The method comprises growing cells comprising an expression cassette under suitable growth conditions, the expression cassette comprising a nucleic acid molecule disclosed herein; and isolating the polypeptide from the cells. In yet another embodiment ofthe presently disclosed subject matter, an isolated polypeptide having or comprising at least about 90% amino acid identity with a polypeptide as disclosed in Examples 6-11 of the subject application, over an entire sequence or functional fragment thereof, wherein the polypeptide functions as an inosoitol phosphate kinase is provided. In yet another embodiment of the presently disclosed subject matter, a transgenic plant cell comprising a homozygous disruption in at least one endogenous inositol phosphate kinase gene homologous to the nucleic acid molecules disclosed herein, wherein the disruption substantially inhibits the expression of a functional inositol phosphate polypeptide is provided. A transgenic plant comprising at least one of the transgenic plant cells is further provided herein. In some embodiments, the transgenic plant comprises a decreased level of phytic acid and/or an increased level of non-phytic acid when compared to a non-transformed parental plant. In some embodiments, the homozygous disruption is tissue-specific. In some embodiments, the tissue-specific disruption is in a tissue selected from the group consisting of epidermis, root, vascular tissue, meristem, cambium, cortex, pith, leaf, flower, seed, and combinations thereof. In some embodiments, the tissue is seed and the seed comprises a decreased level of phytic acid and/or an increased level of non-phytic acid when compared to a non-transformed parental plant. In some embodiments, the transgenic plant is a plant selected from the group consisting of Arabidopsis thaliana, corn (Zea mays), Brassica sp., alfalfa (Medicago sativa), rice (Oryza sativa ssp.), rye (Secale cereale), sorghum (Sorghum bicolor, Sorghum vulgare), pearl millet (Pennisetum glaucum), proso millet (Panicum miliaceum), foxtail millet (Setaria italica), finger millet (Eleusine coracana), sunflower (Helianthus annuus), safflower (Carthamus tinctorius), wheat (Triticum aestivum), soybean (Glycine max), tobacco (Nicotiana tabacum), potato (Solanum tuberosum), peanut (Arachis hypogaea), cotton, sweet potato (Ipomoea batatus), cassava (Manihot esculenta), coffee (Cofea spp.), coconut (Cocos nucifera), pineapple (Ananas comosus), citrus trees (Citrus spp.), cocoa (Theobroma cacao), tea (Camellia sinensis), banana (Musa spp.), avocado (Persea ultilane), fig (Ficus casica), guava (Psidium guajava), mango (Mangifera indica), olive (Olea europaea), papaya (Carica papaya), cashew (Anacardium occidentale), macadamia (Macadamia integrifolia), almond (Prunus amygdalus), sugar beets (Beta vulgaris), sugarcane (Saccharum spp.), oats, duckweed (Lemna), and barley. In some embodiments, the plant is selected from the group consisting of a vegetable, an ornamental, and a conifer. In some embodiments, the vegetable is selected from the group consisting of tomatoes, lettuce, guar, locust bean, fenugreek, soybean, garden beans, cowpea, mungbean, lima bean, fava bean, lentils, chickpea, green bean, lima bean, pea, and members of the genus Cucumis. In some embodiments, the ornamental is selected from the group consisting of impatiens, Begonia, Pelargonium, Viola, Cyclamen, Verbena, Vinca, Tagetes, Primula, Saint Paulia, Agertum, Amaranthus, Antihirrhinum, Aquilegia, Cineraria, Clover, Cosmo, Cowpea, Dahlia, Datura, Delphinium, Gerbera, Gladiolus, Gloxinia, Hippeastrum, Mesembryanthemum, Salpiglossos, and Zinnia, azalea, hydrangea, hibiscus, rose, tulip, daffodil, petunia, carnation, poinsettia, and chrysanthemum. In some embodiments, the conifer is selected from the group consisting of loblolly pine, slash pine, ponderosa pine, lodgepole pine, Monterey pine, Douglas-fir, Western hemlock, Sitka spruce, redwood, silver fir, balsam fir, Western red cedar, and Alaska yellow-cedar. In some embodiments, the homozygous disruption to the at least one endogenous inositol phosphate kinase gene is to at least a gene encoding an Ipk1 polypeptide and a gene encoding an Ipk2 polypeptide In still another embodiment of the presently disclosed subject matter, a method of modulating production of phytate in a plant is provided. The method comprises, modulating the enzymatic activity of at least one inositol phosphate kinase polypeptide selected from the group consisting of Ipk1 and Ipk2. In another embodiment of the presently disclosed subject matter, a method of producing a plant with low levels of phytate is provided. The method comprises modulating in the plant the enzymatic activity of at least one insotiol phosphate kinase polypeptide selected from the group consiting of Ipk1 and Ipk2. In another embodiment of the presently disclosed subject matter, a method of screening a plurality of compounds for a modulator of the enzymatic activity of an inositol phosphate kinase polypeptide is provided. The method comprises in some embodiments: (a) providing a library of test compounds; (b) contacting an inositol phosphate kinase polypeptide selected from the group consisting of Ipk1 and Ipk2 with each test compound; (c) detecting an interaction between a test compound and the inositol phosphate kinase polypeptide; (d) identifying a test compound that interacts with the inositol phosphate kinase polypeptide; and (e) isolating the test compound that interacts with the inositol phosphate kinase polypeptide, whereby a plurality of compounds is screened for a modulator of inositol phosphate kinase polypeptide enzymatic activity. Some of the objects of the presently disclosed subject matter having been stated hereinabove, and which are addressed in whole or in part by the present subject matter, other objects will become evident as the description proceeds when taken in connection with the accompanying drawings as best described hereinbelow. BRIEF DESCRIPTION OF THE DRAWINGS Figures 1A through 1C disclose the identification and sequence alignment of plant IP₅ 2-kinases. Figure 1A is a schematic depicting the combined IP kinase activities of Atlpkl (black arrows), Atlpk2 (grey arrows), and 1(1 ,3,4)P₃5/6 kinase (white arrows) described herein can synthesize IP₆ in vitro through the indicated routes from l(1 ,4,5)P₃ and l(1 -3,4)P₃. Figure 1 B is a multi-sequence alignment ofthe family of Ipk1 proteins. GenBank accession numbers for the plant sequences are: Oslpkl - AK102842; Zmlpkl - AY104429; Atlpkl - AY093362; At1 g22100 - NM_102060; At1 g59312 - NM J 04639; Hslpkl - AF520811. Figure 1C is digital image of gel electrophoresis showing that AtlPKI is expressed throughout Arabidopsis tissues. Semi-quantitative, non-competitive RT-PCR was performed from 250 ng total RNA from the indicated tissues. As a control for RNA quantification and comparison, actin (ACT2) was amplified. Figures 2A-2C depict that Atlpkl is anlP₄ and IP₅ 2-kinase. Figure 2A is a data readout of an 1(1 ,3,4,5,6)P₅ substrate assay. 10 μM of [³H]- 1(1 ,3,4,5,6)P₅ (7000 CPM) was incubated with 0.7 pmol purified recombinant GST-

Atlpkl for 30 min at 37°C. The reaction products were resolved by HPLC on a

Partisphere strong anion exchange (SAX) HPLC column. IPs were identified based on comparison to known standards. Figure 2B is a data readout of an l(1 ,3,4,6)P₄ substrate assay. 10 μWΛ was incubated with 0.7 pmol GST-Atlpk1 and then analyzed by HPLC. Figure 2C is a data readout of an 1(1 ,4,5,6)P substrate assay. [³H]-I(1 ,4,5,6)P₄ and ³H-I(1 ,3,4,5,6)P₅ was incubated with 0.7 pmol GST-Atlpk1R for 30 min at 37 °C and then separated by HPLC. Both substrates were phosphorylated to produce l(1 ,2,4,5,6)P₅ and IP₆₎ respectively. Figures 3A-3D depict dose dependence of specific IP species formation. GST-

Atlpkl and GST-Atlpk2R were incubated with 10 μM [³H]-I(1 ,4,5)P₃. Figure 3A is a data readout of an experiment wherein 1.6 pmol Atlpk2 ? and 1.3 pmol Atlpkl were incubated with 1(1 ,4,5)P₃ (top panel) for 2 hr at 37°C. The reaction (lower panel) was stopped by boiling for 1 min and then was separated by Partisphere SAX HPLC. Figure 3B is a data readout of an experiment wherein 3.2 pmol Atlpk2 ? and 1.3 pmol Atlpkl was incubated with 1(1 ,4,5)P₃ as above for 1 hr at 37°C, then stopped and analyzed by HPLC. Figure 3C is a data readout of an experiment wherein 8 pmol Atlpk2R- and 1.3 pmol Atlpkl were incubated with l(1 ,4,5)P₃ as above for 1 hr at 37°C, and then analyzed by HPLC. Figure 3D is a data readout showing in vitro synthesis of IP₆ from 1(1 ,3,4)P₃ by l(1 ,3,4)P₃ 5/6-kinase, Atlpk2, and Atlpkl . 10 μM [³H]-I(1 ,3,4)P₃ (3,500 CPM) (top panel) was incubated with 2-4 pmol each of purified recombinant mammalian 1(1 ,3,4)P₃ 5/6-kinase, Atlpk2R, and Atlpkl for 1 hr at 37° C (lower panel). Figures 4A-4C show the molecular characterization of an AtlPKI T-DNA insertional mutant. Figure 4A is a schematic of the T-DNA insertional element of SALK D65337 mapped 77 nt upstream of the stop codon of AtlPKI. Figure 4B is a digital image of a Northern blot analysis of seedling RNA from wild type and atipk1-1 Arabidopsis (ecotype Columbio-0) mutants probed with ³²P-labeled AtlPKI cDNA or actin (ACT2) cDNA. Figure 4C is a digital image of a Southern blot of T-DNA insertion. Ten μg of genomic DNA was digested with Spel. Left panel is the Southern blot probed with the 5' UTR of AHPK1, which yields a 6.4 kb band and indicates complete digestion of genomic DNA. The right panel is the same Southern probed with the sequence for the T-DNA. A single T-DNA element would yield a 6.8 kb band. The band identified here is greater than 15 kb, indicating at least three T-DNA's are inserted into the same site of the AtlPKI gene. Lane 1 is wild type genomic DNA. Lanes 2-5 are the genomic DNA from five independent T5 atipk1-1 plants. Figures 5A-5C show the effect of T-DNA insertion on IP synthesis. Figure 5A is a data readout of an experiment wherein wild type and atipk1-1 T seeds were germinated in liquid MS salts with 0.4 mCi/ml [³H]-myo-inositol for 6 days and inositol phosphates were harvested and analyzed by Partisphere SAX HPLC. IP₃x is an unknown species. Figure 5B is a data readout of an experiment wherein individual mature siliques (containing embryos in the late bent-cotyledon stage from wild type (top panel) and atipk1-1 plants (lower panel) were labeled with [³H]-myo-inositol as described in the methods and the IPs were harvested and analyzed by Partisphere SAX HPLC. Figure 5C is a data readout of an experiment wherein individual immature seeds containing embryos in the late bent cotyledon stage of development were dissected from siliques that had been labeled for 2 days with 2 mCi/ml [³H]-myo-inositol. The IPs were harvested from the seeds and analyzed by HPLC as described. Figures 6A and 6B are data readouts of a comparison of IP₆ synthesis in developing Arabidopsis seeds. Individual seeds were dissected from wild type (Figure 6A) and atipk1-1 (Figure 6B) siliques that had been labeled with [³H]-myo-inositol for two days. IPs were extracted and analyzed by Partisphere SAX HPLC and identified based on known standards. Top panels are the IP profiles from seeds with embryos in the early-bent cotyledon stage and the bottom panels are from seeds with embryos in the late-bent cotyledon stage. Figures 7A and 7B are data readouts of an identification of accumulated IP species in the atipk1-1 mutant. [³H]-IP extracts from atipk1-1 siliques were incubated with 1.3 pmol GST-Atlpk1 (Figure 7A), or the l(3,4,5,6)P₄ 1-kinase (Figure 7B) for 60 min at 37 °C. The reaction products were separated by Partisphere SAX HPLC. Since IP₅ was phosphorylated by Atlpkl , its identity is 1(1 , 3,4,5, 6)P₅. Since IP₄ was phosphorylated by Atlpkl to make IP₅x and the 1(3,4, 5,6)P 1-kinase to make l(1 ,3.4,5.6)P₅, it is mainly comprised of l(3,4,5,6)P₄. Neither IP₄ nor IP₅ were phosphorylated by active Atlpk2β, indicating that IP₄x is not 1(1 ,4,5,6)P₄, 1(1 ,3,4,5)P₄, or l(1 ,3,4,6)P₄, which are all substrates of Atlpk2R (Stevenson-Paulik et al., 2002). Figure 8 is a data readout from the non-radioactive mass IP analysis of mature desiccated seed extracts. Wild type (top panel) or T₄ atipk1-1 (bottom panel) seed extracts were separated by an lonPac AS7 anion-exchange column and detected by metal-dye chelation and measured at 550 nm. Data points are plotted as the negative absorbance values. Figures 9A and 9B show the affect of AtlPKI T-DNA insertion on vegetative growth. T seeds were germinated on a mixture of sand and vermiculite through sub irrigation until the cotyledons had emerged. Plants were then top-fed half-strength Hoagland's solution daily and were grown at 20°C under a 14-hr light (240 umol/m² s^"1) and 10-hr dark cycle in the Duke University National Phytotron. Figure 9A is a series of photographs of a top view of 4 week-old plant that is heterozygous for the AtlPKI T-DNA insertion (left panel, left plant), wild type (left panel, right plant), atipk1-1 (middle panel), atipk1-1 plant constitutively expressing AtlPKI (right panel). Figure 9B is a data readout and a series of photographs of a complementation of

IP synthesis and growth phenotype in atipk1-1 by constitutive expression of AtlPKI. AtlPKI expression is driven by the constitutive 35S promoter. Left panels are HPLC chromatograms of IP profiles from [³H]-inositol-labeled 5-day-old seedlings. Right panel are leaves from 5-week-old plants grown in half-strength Hoagland's solution as described. Top panel and leaf row are wild type plants transformed with the empty pBART expression vector. Middle panel and leaf row are atipk1-1 plants transformed with the pBART expression vector harboring the AtlPKI gene. Bottom panel and leaf row are atipk1-1 plants transformed with the empty pBART expression vector. Figures 10A and 10B show a conditional growth defect and altered phosphate levels of atipk1-1. Figure 10A is a photograph of leaves from 5-week-old wild type and atipk1-1 plants grown as described above in half-strength Hoagland's solution with variable phosphate concentrations as indicated. Figure 10B is a bar graph of inorganic phosphate concentrations of the leaves from plants grown in conditions described in Figure 10A. Figures 11A and 11 B show root hair growth in response to variable phosphate levels. Figure 11 A is a set of representative images of main root and root hairs from wild type (left panel) and atipk1-1 plants grown vertically on MS-agar plates containing 10 μM phosphate. Bar indicates 0.2 mm. Figure 11 B is a bar graph showing root hair lengths of wild type and atipk1-1 plants grown vertically on MS-agar plates containing various phosphate concentrations as indicated. n=12 for each genotype. * indicates p < 0.001. Figure 12A is a schematic of T-DNA insertion into atlpk2β locus. Figure 12B is a Northern Blot analysis of mRNA isolated from wild-type, atipk2β-

1 , or atipklβ -1 plants. Figure 13 is a schematic of IP determination methods used in Example 5. Figures 14A and 14B are spectral data and a schematic, respectivlely, of a non- radioactive method of measuring Phytate (IP₆) levels in mature seeds derived from wild- type or kinase mutant plants. Figure 15 is a series of photographs of Ipk1/ipk2 double mutant plants. Figure 16 is a bar graph of total seed phytate (IP₆) levels calculated from ipkl , ipk2 and ipk1/ipk2 double mutant seeds. Figures 17A and 17B are HPLC data and a bar graph, respectively, of total seed extracts as described in Example 5. Figures 18-23 depict various alignments between translated reading frames from various plants and the genes disclosed herein, as outlined in Examples 6-11.

DETAILED DESCRIPTION The presently disclosed subject matter will be now be described more fully hereinafter with reference to the accompanying Examples, in which representative embodiments of the presently disclosed subject matter are shown. The presently disclosed subject matter can, however, be embodied in different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the presently disclosed subject matter to those skilled in the art. All publications, patent applications, patents, and other references cited herein are incorporated by reference in their entireties. The presently disclosed subject matter relates, in part, to newly identified polynucleotides and polypeptides; variants and derivatives of these polynucleotides and polypeptides; processes for making these polynucleotides and these polypeptides, and their variants and derivatives; molecules that bind these polynucleotides and polypeptides including antagonists; methods of screening for these molecules; transgenic plants incorporating these polynucleotides and polypeptides as well as transgenic plants having endogenous homologues of these polynucleotides disrupted; and uses of these polynucleotides, polypeptides, variants, derivatives and antagonists. In particular, in these and in other regards, the presently disclosed subject matter relates to polynucleotides and polypeptides of the phytate metabolic pathway, most particularly with the enzymes inositol phosphate kinase 1 (Ipk 1 ) and inositol phosphate kinase 2 (Ipk 2) and genes encoding same. Described herein is the molecular and biochemical characterization of the machinery plants need to produce phytate, inositol hexakisphosphate (IP6). As phytate is a major antinutrient in grains used for feeding livestock, thus it has been a longstanding goal to generate a plant seed that lacks phytate. Disclsoed herein is disruption of a single gene, IPK1 , which is required for the last step in the biosynthetic pathway, resulting in seeds with greater that a 90% reduction in phytate levels. Disruption of Ipk2β, an inositol polyphosphate kinase that functions as the penultimate step in the synthesis of phytate, also results in low phytate seeds. Furthermore, simultaneous disruption of both Atlpkl and Atlpk2b, as described herein, results in the 95% ablation of phytate and other inositol polyphosphates in seeds with minimal yield effects. In one example of the presently disclosed subject matter, the gene and gene product of inositol polyphosphate 2-kinase, designated AtlPKI , from Arabidopsis thaliana has been identified, isolated, and characterized. As the enzyme that catalyzes the last step in inositol hexakisphosphate (IP₆ or phytate) biosynthesis, Ipk1 , is an attractive target for the genetic manipulation of phytate synthesis in plant seeds. To generate a low-phytate plant, a mutant Arabidopsis thaliana line with a foreign DNA element (T-DNA) stably inserted in the AtlPKI b, gene, has been identified and obtained, and shown herein to interrupt transcription of the gene by at least 90%. The mutation also causes a greater than 90% loss of phytate in seeds (developing and mature), and whole seedlings. This approach to generating a low-phytate plant by disrupting the last step in phytate synthesis is novel and can be applied to agriculturally relevant plants to alleviate the negative impact of high phytate grain diets on animal nutrition and environmental phosphorus pollution. Additionally, gene and gene products of two related inositol polyphosphate 6-/3- /5-kinases, designated AtlPK2α and AtlPK2β, from Arabidopsis thaliana have been identified, isolated, and characterized and are disclosed herein. As partially redundant enzymes that catalyze the penultimate step in phytate biosynthesis both AtlPK2α and Atlpk2β are attractive targets for the genetic manipulation of phytate synthesis in plant seeds. To generate a low-phytate plant, a mutant Arabidopsis thaliana line with a foreign DNA element (T-DNA) stably inserted in the AtlPKI b, gene, has been identified and obtained, which interrupts transcription of the gene by at least 90%. The mutation causes a significant loss of phytate in seeds (developing and mature), but not seedlings, thereby appearing seed specific. The mutant atipk2β plant has no detectable adverse growth phenotype and seed yields are normal. This approach to generating a low-phytate plant by disrupting the penultimate step in phytate synthesis is novel and may be applied to agriculturally relevant plants to alleviate the negative impact of high phytate grain diets on animal nutrition and environmental phosphorus pollution. Plants with T-DNA disruptions in both AtlPKI , and AtlPK2β are also disclosed herein and produced through breeding of single mutant lines described above. The double mutant plants are fertile, germinate normally and have seeds of nearly normal size. Examination of seed inositol phosphates demonstrates a 95% reduction of phytate with a minimal accumulation of IP , IP , IP₃ and IP₂. Collectively, these phenotypes represent an improvement over other described low phytate plants, especially in the area of IPe reduction and seed yield. Identification of the gene sequence and corresponding amino acid sequence of the enzyme involved in the last step of phytate synthesis in Arabidopsis is useful to identify orthologs in other plant species. Furthermore, now identification of the biosynthetic pathway of IP6 production, provides an advantage over random mutagenesis strategies in that a targeted approach in engineering altered gene expression (through antisense or post-transcriptional gene-silencing technologies) at multiple steps of the pathway and at specific developmental stages and tissues where phytate synthesis is greatest is possible. By spatiotemporally controlling the gene inactivation, a plant can be engineered to produce seed with reduced phytate, but avoid the loss of inositol phosphate production in other tissues where it may be vital for normal plant growth.

L 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 enzymatic 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. Sequence homology (sequence identity) of unknown sequences to known sequences provides suggestion of a possible function of the polypeptide encoded by the gene. In functional genomics, any or all ofthe above approaches are utilized, often in concert, to confirm suggested function provided by sequence homology to known genes, allowing functions to be assigned 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 one embodiment from a single methodology, in another embodiment from two methodologies, and in still another embodiment 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 ofthe strength ofthe 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 either through the upregulation or downregulation ofthe genes. 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, 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 et al., 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. Phytate (inositol hexakisphosphatejlPβ), is present in all eukaryotic cells and is highly abundant in plant seeds where it compromises animal nutrition and the environment. As a molecular approach for reducing seed phytate and studying IP₆ signaling function in plants, disclosed herein is the gene encoding the terminal kinase in Arabidopsis phytate synthesis, inositol polyphosphate 2-kinase (AtlPKI). Atlpkl specifically phosphorylates the D-2 position of the inositol ring and uses l(1 ,4,5,6)P , l(1 ,3,4,6)P₄, l(3,4,5,6)P₄, and 1(1 ,3,4,5,6^₅ as substrates. Together with Atlpk2, an IP₃/IP₄ 6-/3-/5-kinase, Atlpkl can generate multiple inositol polyphosphate products including IP₆ from an 1(1 ,4,5)P₃ precursor. A partial disruption of AtlPKI causes an 80- 90% loss of IPβ in seeds and all tissues tested. The low-phytate atipkl mutant plants are conditionally growth compromised and have defective root phosphate sensation; however no defects in germination are observed. Described herein are the identification, isolation, and characterization ofthe gene and gene products of two inositol polyphosphate 6-/3-/5-kinases, designated AtlPK2α and AtlPK2β, from Arabidopsis thaliana. As partially redundant enzymes that catalyze the penultimate step in inositol hexakisphosphate (IP₆ or phytate) biosynthesis both AtlPK2 and Atlpk2βare attractive targets for the genetic manipulation of phytate synthesis in plant seeds. To generate a low-phytate plant, a mutant Arabidopsis thaliana line with a foreign DNA element (T- DNA) stably inserted in the AtlPKI β, gene ahs been identified and obtained, which interrupts transcription of the gene by at least 90%. The mutation causes a significant loss of phytate in seeds (developing and mature), but not seedlings, thereby appearing seed specific. The mutant atipk2β plant has no detectable adverse growth phenotype and seed yields are normal. Also, plants have been generated with T-DNA disruptions in both AtlPKI, and AtlPKlβ through breeding of single mutant lines described above. The double mutant plants are fertile, germinate normally and have seeds of nearly normal size. Of significant interest, examination of seed inositol phosphates demonstrates a 95% reduction of phytate with a minimal accumulation of IP₅, IP₄, IP₃ and IP₂. Collectively, these phenotypes represent a significant improvement over other described low phytate plants, especially in the area of IP₆ reduction and seed yield. Targeting the last two enzymatic steps of phytate synthesis has advantages over targeting earlier steps, such as D-myo-inositol 3-phosphate synthase (sometimes referred to as L-myo-inositol 1 -phosphate synthase) or inositol 1 ,3,4-trisphosphate 5/6- kinase, both in reported efficacy of phytate reduction and markedly reduced adverse growth effects on plant growth and seed yield. The presently disclosed subject matter indicates a signaling role for phytate in the regulation of phosphate biology and demonstrate that inositol phosphate kinases, AtlPKI and Atlpk2, are commercially relevant targets for generating low-phytate cereals.

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 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 "associated with" a DNA sequence that encodes an RNA or a polypeptide if the two sequences are operatively linked, or situated such that the regulator DNA sequence will affect the expression level of the coding or structural DNA sequence. As used herein, the term "chimera" refers to a polypeptide that comprises domains or other features that are derived from different polypeptides or are in a position relative to each other that is not naturally occurring. As used herein, the term "chimeric construct" refers to a recombinant nucleic acid molecule in which a promoter or regulatory nucleic acid sequence is operatively linked to, or associated with, a nucleic acid sequence that codes for an mRNA or which is expressed as a polypeptide, such that the regulatory nucleic acid sequence is able to regulate transcription or expression of the associated nucleic acid sequence. The regulatory nucleic acid sequence of the chimeric construct is not normally operatively linked to the associated nucleic acid sequence as found in nature. 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 one embodiment, 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 occurwithin 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" and "feature", when used in reference to a polypeptide or amino acid sequence, refers to a subsequence of an amino acid sequence that has a particular biological function. Domains 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", or "feature" is that subsequence ofthe nucleic acid sequence that encodes a domain or feature of a polypeptide. As used herein, the term "enzymatic activity" or "enzyme activity", for example the activity of the novel functional inositol phosphate kinases disclosed herein, refers to the ability of an enzyme to catalyze the conversion of a substrate into a product. In the case of the inositol phosphate kinases disclosed herein, the enzyme activity is the addition of phosphate groups at specific locations on the backbone of a specific inositol substrate species. A substrate for the enzyme can comprise the natural substrate of the enzyme but also can comprise analogues of the natural substrate, which can also be converted by the enzyme into a product or into an analogue of a product. The activity ofthe enzyme is measured for example by determining the amount of product in the reaction after a certain period of time, or by determining the amount of substrate remaining in the reaction mixture after a certain period of time. The activity of the enzyme can also be measured by determining the amount of an unused co-factor of the reaction remaining in the reaction mixture after a certain period of time or by determining the amount of used co-factor in the reaction mixture after a certain period of time. The activity ofthe enzyme can also be measured by determining the amount of a donor of free energy or energy-rich molecule (e.g. ATP, phosphoenolpyruvate, acetyl phosphate, or phosphocreatine) remaining in the reaction mixture after a certain period of time or by determining the amount of a used donor of free energy or energy-rich molecule (e.g. ADP, pyruvate, acetate, or creatine) in the reaction mixture after a certain period of time. 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, organ, or stage of development, such as for example, specific for seed tissue. 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. Similarly, a functional fragment of a receptor tyrosine kinase includes, but is not limited to a ligand binding domain, a kinase domain, an ATP binding domain, and combinations thereof. As used herein, the term "gene" is used broadly to refer to any segment of DNA associated with a biological function. Thus, genes include, but are not limited to, nucleic acid molecules that are 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, for example, the use of DNA shuffling or other 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 cell into which it is introduced. As used herein, the term "disruption" refers to partial or complete reduction ofthe expression of at least a portion of a nucleic acid or a polypeptide encoded by one or more endogenous genes of a single cell, selected cells, or all of the cells of a plant. The plant can have a "heterozygous disruption," wherein one allele of one or more endogenous genes have been disrupted. Alternatively, the plant can have a "homozygous disruption," wherein both alleles of one or more endogenous genes have been disrupted. 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 ofthe hybridization media to achieve the desired detection of the target nucleic acid sequence. As used herein, the term "inhibitor" or "antagonist" refers to a chemical substance that inactivates or decreases the biological activity of a polypeptide such as a biosynthetic and catalytic activity, receptor, signal transduction polypeptide, structural gene product, or transport polypeptide. The term "herbicide" (or"herbicidal compound") is used herein to define an inhibitor applied to a plant at any stage of development, whereby the herbicide inhibits the growth of the plant or kills the plant. 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 "mature polypeptide" refers to a polypeptide from which the transit peptide, signal peptide, and/or propeptide portions have been removed. 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 "modified enzyme activity" refers to enzyme activity that is different from that which naturally occurs in a plant (i.e. enzyme activity that occurs naturally in the absence of direct or indirect manipulation of such activity by man). A "modulator of enzymatic activity" is a compound that modifies the activity of an enzyme.

As used herein, the term "native" refers to a gene that is endogenous, i.e. 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 ("nucleic acid molecules") 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", "nucleic acid molecule", "polynucleotide" 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 protein that perform the same biological function. For example, the inositol phosphate kinase 1 and 2 genes from, for example, Arabidopsis, maize, wheat, sorghum and rice, are orthologs. Typically, orthologous nucleic acid sequences are characterized by a degree of sequence similarity (in some examples, at least about 40% identity over a complete polypeptide sequence, in some examples, at least about 80% sequence identity over at least shared functional domains). A nucleic acid sequence of an ortholog in one species (for example, Arabidopsis) can be used to isolate the nucleic acid sequence of the ortholog in another species (for example, maize) using standard molecular biology techniques. This can be accomplished, for example, using techniques described in more detail below (see also 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" or "percent identity" in the context of two nucleic acid or polypeptide sequences, refers to two or more sequences or subsequences that have in one embodiment 40%, in one embodiment 45%, in one embodiment 50%, in one embodiment 55%, in one embodiment 60%, in another embodiment 75%, in another embodiment 70%, in another embodiment 80%, in another embodiment 90%, in another embodiment 95%, and in still another embodiment 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 one embodiment over a region of the sequences, for example a functional domain, that is at least about 50 residues in length, in another embodiment over a region of at least about 100 residues, and in another embodiment, the percent identity exists over at least about 150 residues. In still another embodiment, 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 over the internet through 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 ofthe 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 wordlength (W) of 11 , an expectation (E) of 10, a cutoff of 100, M = 5, N = -4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix. See Henikoff & Henikoff, 1992. In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis ofthe 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 ofthe 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 one embodiment less than about 0.1 , in another embodiment less than about 0.01 , and in still another embodiment less than about 0.001. As used herein, the term "phytic acid" refers to myo-inositol hexaphosphoric acid, also referred to herein as "IP₆". As a salt with cations, phytic acid is usually referred to as "phytate", however the two terms are used interchangeably herein. As used herein, the term "shuffled nucleic acid" refers to a recombinant nucleic acid molecule in which the nucleotide sequence comprises a plurality of nucleotide sequence fragments, wherein at least one of the fragments corresponds to a region of a nucleotide sequence listed in odd numbered sequences of SEQ ID NOs: 1-9, and wherein at least two of the plurality of sequence fragments are in an order, from 5' to 3', which is not an order in which the plurality of fragments naturally occur in a nucleic acid. The term "substantially identical", in the context of two nucleotide or amino acid sequences, can refer to two or more sequences or subsequences that have in one embodiment at least about 40% nucleotide or amino acid identity, at least about 45% nucleotide or amino acid identity, at least about 50% nucleotide or amino acid identity, at least about 55% nucleotide or amino acid identity, at least about 60% nucleotide or amino acid identity, in another embodiment at least about 65% nucleotide or amino acid identity, in another embodiment at least about 70% nucleotide or amino acid identity, in another embodiment at least about 75% nucleotide or amino acid identity, in another embodiment at least about 80% nucleotide or amino acid identity, in another embodiment at least about 85% nucleotide or amino acid identity, in another embodiment at least about 90% nucleotide or amino acid identity, and in yet another embodiment at least about 95% nucleotide or amino acid identity, when compared and aligned for maximum correspondence, as measured using one ofthe above-referenced sequence comparison algorithms or by visual inspection. In one example, the substantial identity exists in nucleotide or amino acid sequences of at least 50 residues, in another example in nucleotide or amino acid sequence of at least about 100 residues, in another example in nucleotide or amino acid sequences of at least about 150 residues, and in yet another example in nucleotide or amino acid sequences comprising complete coding sequences or complete amino acid sequences. In one example, the substantial identity exists in nucleotide or amino acid sequences of a conserved motif of the polypeptides compared. In one embodiment, for example, the conserved motif of Ipk2 is PxxxDxKxG, wherein 'x' is any amino acid, and polypeptides compared and each having this conserved motif would be considered substantially identical. Likewise, in another embodiment, the art recognized conserved motif of Ipk1 (as discussed in York et al. Science 1999; Odom et al. 2000 J. Biol. Chem.; and Stevenson-Paulik et al. J. Biol. Chem. 2002), if shared by compared polypeptides, then the polypeptides would be considered substantially identitical. 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 one embodiment at least an about 14 to 40 nucleotide sequence of a nucleic acid molecule ofthe presently disclosed subject matter. In one example, 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 the odd numbered SEQ ID NOs: 1-9. 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. "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 one embodiment 8°C, in another embodiment 10°C, in another embodiment 12°C, and in still another embodiment 15°C lower than the T_m for the specific sequence at a defined ionic strength and pH. The T_m is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. 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°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 40°C. For short probes (e.g., about 10 to 50 nucleotides), stringent conditions typically involve salt concentrations of less than about 1M Na⁺ ion, typically about 0.01 to 1M 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 one example to a target nucleotide sequence in 7% sodium dodecyl sulfate (NaDS), 0.5M NaP0 , 1 mm ethylene diamine tetraacetic acid (EDTA) at 50°C followed by washing in 2X SSC, 0.1% NaDS at 50°C; in another example, a probe and target sequence hybridize in 7% NaDS, 0.5 M NaP0 , 1 mm EDTA at 50°C followed by washing in 1X SSC, 0.1 % NaDS at 50°C; in another example, a probe and target sequence hybridize in 7% NaDS, 0.5 M NaP0 , 1 mm EDTA at 50°C followed by washing in 0.5X SSC, 0.1 % NaDS at 50°C; in another example, a probe and target sequence hybridize in 7% NaDS, 0.5 M NaP0 , 1 mm EDTA at 50°C followed by washing in 0.1 X SSC, 0.1 % NaDS at 50°C; in yet another example, a probe and target sequence hybridize in 7% NaDS, 0.5 M NaP0₄, 1 mm EDTA at 50°C followed by washing in 0.1X SSC, 0.1 % NaDS at 65°C. In one embodiment, 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 one embodiment at least about 50% pure, in another embodiment at least about 85% pure, and in still another embodiment 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 ofthe 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 1 sequence. As used herein, the term "significant increase" refers to an increase in activity (for example, enzymatic activity) that is larger than the margin of error inherent in the measurement technique, in one embodiment an increase by about 2-fold or greater over a baseline activity (for example, the activity of the wild-type enzyme in the presence of the inhibitor), in another embodiment an increase by about 5-fold or greater, and in still another embodiment an increase by about 10-fold or greater. As used herein, the terms "significantly less", "significantly inhibited" and "significantly reduced" refer to a result (for example, an amount of a product of an enzymatic reaction) that is reduced by more than the margin of error inherent in the measurement technique, in one embodiment a decrease by about 2-fold or greater with respect to a baseline activity (for example, the activity of the wild-type enzyme in the absence ofthe inhibitor), in another embodiment, a decrease by about 5-fold or greater, and in still another embodiment a decrease by about 10-fold or greater. As used herein, the terms "specific binding" and "immunological cross-reactivity" refer to an indicator that two molecules are substantially identical. An indication that two nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically cross reactive with, or specifically binds to, the polypeptide encoded by the second nucleic acid. Thus, a polypeptide is typically substantially identical to a second polypeptide, for example, where the two polypeptides differ only by conservative substitutions. The phrase "specifically (or selectively) binds to an antibody," or "specifically (or selectively) immunoreactive with," when referring to a polypeptide or peptide, refers to a binding reaction which is determinative of the presence of the polypeptide in the presence of a heterogeneous population of polypeptides and other biologies. Thus, under designated immunoassay conditions, the specified antibodies bind to a particular polypeptide and do not bind in a significant amount to other polypeptides present in the sample. Specific binding to an antibody under such conditions can require an antibody that is selected for its specificity for a particular polypeptide. For example, antibodies raised to the polypeptide with the amino acid sequence encoded by any of the nucleic acid sequences of the presently disclosed subject matter can be selected to obtain antibodies specifically immunoreactive with that polypeptide and not with other polypeptides except for polymorphic variants. A variety of immunoassay formats can be used to select antibodies specifically immunoreactive with a particular polypeptide. For example, solid-phase ELISA immunoassays, Western blots, or immunohistochemistry are routinely used to select monoclonal antibodies specifically immunoreactive with a polypeptide. See Harlow & Lane, 1988, for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity. Typically a specific or selective reaction will be at least twice background signal or noise and more typically more than 10 to 100 times background. 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 ofthe 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. As used herein, the term "viability" refers to a fitness parameter of a plant. Plants are assayed for their homozygous performance of plant development, indicating which polypeptides are essential for plant growth.

ML Nucleic Acid Molecules and Polypeptides The presently disclosed subject matter encompasses the identification and isolation of cDNAs encoding genes of interest in the expression of inositol kinase phosphates. Inositol kinase phosphates are a class of enzymes that facilitate production of inositol hexakisphosphate (IP₆), also known as phytate, along the phytate biosynthetic pathway through the serial addition of phosphates at specific carbons to myo-inositol. Altering the expression of genes related to these traits can be used to improve or modify plants, and in particular improve or modify plants producing seed utilized as food or animal feed. Examples describe the isolated genes of interest and methods of analyzing the alteration of expression and their effects on the plant characteristics. III.A. Nucleic Acid Molecules Embodiments of the presently disclosed subject matter encompass isolated nucleic acid molecules corresponding to genes that encode inositol phosphate kinases. In particular, the presently disclosed subject matter encompasses isolated nucleic acids encoding inositol phosphate kinase 2 (Ipk2) and inositol phosphate kinase 1

(Ipk1 ). As discussed herein, odd numbered SEQ ID NOs: 1-9 are nucleotide sequences encoding inositol phosphate kinases from Arabidopsis thaliana that have been isolated and identified using the methods and compositions disclosed herein. As discussed herein, even numbered SEQ ID NOs: 2-10 are inositol phosphate kinase polypeptide sequences encoded by the immediately preceding nucleotide sequence. For example, SEQ ID NO: 2 is the polypeptide encoded by the nucleotide sequence of SEQ ID NO: 1 , SEQ ID NO: 4 is the polypeptide encoded by the nucleotide sequence of SEQ ID NO: 3, etc. In one embodiment, an isolated nucleic acid molecule ofthe presently disclosed subject matter comprises a nucleic acid encoding a polypeptide comprising an amino acid sequence of one of even numbered SEQ ID NOs: 2-10. In another embodiment, an isolated nucleic acid molecule ofthe presently disclosed subject matter comprises a nucleic acid encoding a polypeptide comprising an amino acid sequence at least 40% identical, in some embodiments at least about 45% amino acid identity, in some embodiments at least about 50% amino acid identity, in some embodiments at least about 55% amino acid identity, in some embodiments at least about 60% amino acid identity, in some embodiments at least about 65% amino acid identity, in some embodiments at least about 70% amino acid identity, in some embodiments at least about 75% amino acid identity, in some embodiments at least about 80% amino acid identity, in some embodiments at least about 85% amino acid identity, in some embodiments at least about 90% amino acid identity, in some embodiments at least about 95% amino acid identiy, and in some embodiments at least about 95% 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, to a complete sequence, functional domain, or other subsequence of one of even numbered SEQ ID NOs: 2-10 and having inositol phosphate kinase activity. In one embodiment, an isolated nucleic acid molecule ofthe presently disclosed subject matter comprises a nucleotide sequence that hybridizes under stringent conditions of hybridization of 45°C in 1 M NaCI, followed by a final washing step at 50°C in 0.1 M NaCI to a nucleotide sequence as set forth in odd numbered sequences SEQ ID NOs: 1-9, or a fragment, domain, or feature thereof. In another embodiment, an isolated nucleic acid molecule of the presently disclosed subject matter comprises a nucleotide sequence having substantial identity to a nucleotide sequence that hybridizes under stringent conditions of hybridization of 45°C in 1 M NaCI, followed by a final washing step at 50°C in 0.1 M NaCI to a nucleotide sequence as set forth in odd numbered sequences SEQ ID NOs: 1-9, or a fragment, domain, or feature thereof. Another embodiment of the presently disclosed subject matter encompasses an isolated nucleic acid molecule comprising a nucleotide sequence that is complementary to, or the reverse complement of, a nucleotide sequence that hybridizes under stringent conditions of hybridization of 45°C in 1 M NaCI, followed by a final washing step at 50°C in 0.1 M NaCI to a nucleotide sequence listed in odd numbered sequences of SEQ ID NOs: 1-9, or a fragment, domain, or feature thereof. Still another embodiment ofthe presently disclosed subject matter encompasses an isolated nucleic acid molecule comprising a nucleotide sequence that is complementary to, or the reverse complement of, a nucleotide sequence that has substantial identity to, or is capable of hybridizing specifically to, a nucleotide sequence that hybridizes under stringent conditions of hybridization of 45°C in 1 M NaCI, followed by a final washing step at 50°C in 0.1 M NaCI to a nucleotide sequence listed in odd numbered sequences of SEQ ID NOs: 1-9, or a fragment, domain, or feature thereof. In a representative embodiment, the substantial identity is at least about 60% identity, in another embodiment at least about 65% identity, in another embodiment at least about 70% identity, in another embodiment at least about 75% identity, in another embodiment about 80% identity, in another embodiment at least about 85% identity, in another embodiment about 90% identity, and in still another embodiment at least about 95% identity to the nucleotide sequence listed in odd numbered sequences of SEQ ID NOs: 1-9, or a fragment, domain, or feature thereof. In another embodiment, the nucleotide sequence having substantial identity comprises an allelic variant of the nucleotide sequence that hybridizes under stringent conditions of hybridization of 45°C in 1 M NaCI, followed by a final washing step at 50°C in 0.1 M NaCI to a nucleotide sequence listed in odd numbered sequences of SEQ ID NOs: 1-9, or a fragment, domain, or feature thereof. In yet another embodiment, the nucleotide sequence having substantial identity comprises a naturally occurring variant. In another embodiment, the nucleotide sequence having substantial identity comprises a polymorphic variant of the nucleotide sequence that hybridizes under stringent conditions of hybridization of 45°C in 1 M NaCI, followed by a final washing step at 50°C in 0.1 M NaCI to a nucleotide sequence listed in odd numbered sequences of SEQ ID NOs: 1-9, or a fragment, domain, or feature thereof. In another embodiment, the nucleic acid having substantial identity comprises a deletion or insertion of at least one nucleotide. In one embodiment, the deletion or insertion comprises more than half the nucleotides. In one embodiment, the deletion or insertion comprises less than about thirty nucleotides. In another embodiment, the deletion or insertion comprises less than about five nucleotides. In another embodiment, the sequence of the isolated nucleic acid having substantial identity comprises a substitution in at least one codon. In one embodiment, the substitution is conservative. In another embodiment, the isolated nucleic acid comprises a plurality of regions having a nucleotide sequence that hybridizes under stringent conditions of hybridization of 45°C in 1 M NaCI, followed by a final washing step at 50°C in 0.1 M NaCI to a nucleotide sequence listed in odd numbered sequences of SEQ ID NOs: 1-9, or an exon, domain, or feature thereof. In one embodiment, the sequence having substantial identity to the nucleotide sequence that hybridizes under stringent conditions of hybridization of 45°C in 1 M NaCI, followed by a final washing step at 50°C in 0.1 M NaCI to a nucleotide sequence listed in odd numbered sequences of SEQ ID NOs: 1-9, or a fragment, domain, or feature thereof, is from a plant. In one embodiment, the plant is a dicot. In another embodiment, the plant is a gymnosperm. In another embodiment, the plant is a monocot. In one embodiment, the monocot is a cereal. In one embodiment, the cereal can be, for example, maize, wheat, barley, oats, rye, millet, sorghum, triticale, secale, einkorn, spelt, emmer, teff, milo, flax, gramma grass, Tripsacum sp., or teosinte. In another embodiment, the cereal is rice. In one embodiment, the nucleic acid is expressed in a specific location or tissue of a plant. In one embodiment, the location or tissue includes, but is not limited to, epidermis, root, vascular tissue, meristem, cambium, cortex, pith, leaf, flower, and combinations thereof. In another embodiment, the location or tissue is a seed. In one embodiment, the nucleic acid encodes a polypeptide involved in a function including, but not limited to, inositol phosphate kinase activity. Embodiments of the presently disclosed subject matter further relate to an isolated polynucleotide comprising a nucleotide sequence of at least 10 bases, which sequence is identical, complementary (for example, fully complementary), or substantially identical to a region of any sequence that hybridizes under stringent conditions of hybridization of 45°C in 1 M NaCI, followed by a final washing step at 50°C in 0.1 M NaCI to a nucleotide sequence of odd numbered sequences of SEQ ID NOs: 1-9, and wherein the polynucleotide is adapted for any of numerous uses. In one embodiment, the polynucleotide is used as a chromosomal marker. In another embodiment, the polynucleotide is used as a marker for restriction fragment length polymorphism (RFLP) analysis. In another embodiment, the polynucleotide is used as a marker for quantitative trait-linked breeding. In another embodiment, the polynucleotide is used as a marker for marker-assisted breeding. In another embodiment, 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 another embodiment, the polynucleotide is used as a diagnostic indicator for genotyping or identifying an individual or population of individuals. In yet another embodiment, the polynucleotide is used for genetic analysis to identify boundaries of genes or exons. Embodiments of the presently disclosed subject matter also relate to a shuffled nucleic acid molecule comprising a plurality of nucleotide sequence fragments, wherein at least one of the fragments corresponds to a region of a nucleotide sequence that hybridizes under stringent conditions of hybridization of 45°C in 1 M NaCI, followed by a final washing step at 50°C in 0.1 M NaCI to a nucleotide sequence listed in odd numbered sequences of SEQ ID NOs: 1-9, and wherein at least two of the plurality of sequence fragments are in an order, from 5' to 3', which is not an order in which the plurality of fragments naturally occur. In one embodiment, all of the fragments in a shuffled nucleic acid comprising a plurality of nucleotide sequence fragments are from a single gene. In another embodiment, the plurality of fragments is derived from at least two different genes. In one embodiment, the shuffled nucleic acid is operatively linked to a promoter sequence. In another embodiment, the shuffled nucleic acid comprises a chimeric polynucleotide comprising a promoter sequence operatively linked to the shuffled nucleic acid. In still another embodiment, the shuffled nucleic acid is contained within a host cell. ■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 et al., 1994; Reiter ef al., 1992; Schultz ef a/., 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 one embodiment, the monocot is a cereal. A more inclusive, but not intended to be limiting, list of plant genera relevant to the presently disclosed subject matter include Arabidopsis thaliana, corn (Zea mays), Brassica sp., alfalfa (Medicago sativa), rice (Oryza sativa ssp.), rye (Secale cereale), sorghum (Sorghum bicolor, Sorghum vulgare), pearl millet (Pennisetum glaucum), proso millet (Panicum miliaceum), foxtail millet (Setaria italica), finger millet (Eleusine coracana), sunflower (Helianthus annuus), safflower (Carthamus tincto us), wheat (Triticum aestivum), soybean (Glycine max), tobacco (Nicotiana tabacum), potato (Solanum tuberosum), peanut (Arachis hypogaea), cotton, sweet potato (Ipomoea batatus), cassava (Manihot esculenta), coffee (Cofea spp.), coconut (Cocos nucifera), pineapple (Ananas comosus), citrus trees (Citrus spp.), cocoa (Theobroma cacao), tea (Camellia sinensis), banana (Musa spp.), avocado (Persea ultilane), fig (Ficus casica), guava (Psidium guajava), mango (Mangifera indica), olive (Olea europaea), papaya (Carica papaya), cashew (Anacardium occidentale), macadamia (Macadamia integ folia), almond (Prunus amygdalus), sugar beets (Beta vulgaris), sugarcane (Saccharum spp.), oats, duckweed (Lemna), and barley. Still other plants relevant to the presently disclosed subject matter include vegetables, ornamentals, and conifers. Relevant vegetables include, but are not limited to, tomatoes, lettuce, guar, locust bean, fenugreek, soybean, garden beans, cowpea, mungbean, lima bean, fava bean, lentils, chickpea, green bean, lima bean, pea, and members of the genus Cucumis. Relevant ornamentals include, but are not limited to, impatiens, Begonia, Pelargonium, Viola, Cyclamen, Verbena, Vinca, Tagetes, Primula, Saint Paulia, Agertum, Amaranthus, Antihirrhinum, Aquilegia, Cineraria, Clover, Cosmo, Cowpea, Dahlia, Datura, Delphinium, Gerbera, Gladiolus, Gloxinia, Hippeastrum, Mesembryanthemum, Salpiglossos, and Zinnia, azalea, hydrangea, hibiscus, rose, tulip, daffodil, petunia, carnation, poinsettia, and chrysanthemum. Relevant conifers include, but are not limited to, loblolly pine, slash pine, ponderosa pine, lodgepole pine, Monterey pine, Douglas-fir, Western hemlock, Sitka spruce, redwood, silver fir, balsam fir, Western red cedar, and Alaska yellow-cedar. The presently disclosed subject matter also provides a method for genotyping a plant or plant part comprising a nucleic acid molecule ofthe presently disclosed subject matter. Optionally, the plant is a monocot such as, but not limited to, grains such as corn, 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 ofthe 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 one embodiment within 50 centimorgans (cM), in another embodiment within 40 cM, in another embodiment within 30 cM, in another embodiment within 20 cM, in another embodiment within 10 cM, and in still other embodiments within 5, 3, 2, or 1 cM of the nucleic acid of the presently disclosed subject matter. Embodiments ofthe 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 one embodiment, the nucleotide sequence encodes a polypeptide that is an ortholog of a polypeptide comprising a polypeptide sequence listed in even numbered sequences of SEQ ID NOs: 2-10, or a fragment, domain, repeat, feature, or chimera thereof. In another embodiment, the nucleotide sequence encodes a polypeptide that is an ortholog of a polypeptide comprising a polypeptide sequence having at least about 40% identity to a polypeptide sequence listed in even numbered sequences of SEQ ID NOs: 2-10, or a fragment, domain, repeat, feature, or chimera thereof. In another embodiment, 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 odd numbered sequences of SEQ ID NOs: 1-9, or a fragment, domain, or feature thereof, or a sequence complementary thereto. In another embodiment, the nucleotide sequence encodes a polypeptide comprising a polypeptide sequence encoded by a nucleotide sequence that hybridizes under stringent conditions of hybridization of 45°C in 1 M NaCI, followed by a final washing step at 50°C in 0.1 M NaCI to a nucleotide sequence listed in odd numbered sequences of SEQ ID NOs: 1-9, orto a sequence complementary thereto. In still another embodiment, the nucleotide sequence encodes a functional fragment of a polypeptide of the presently disclosed subject matter. In one embodiment, the isolated nucleic acid comprises a polypeptide-encoding sequence. In one embodiment, the polypeptide-encoding sequence encodes a polypeptide that is an ortholog of a polypeptide comprising a polypeptide sequence listed in even numbered sequences of SEQ ID NOs: 2-10, or a fragment thereof. Representative orthologs are disclosed in Examples 6-11 herein below. In some embodiments, an isolated nucleic acid of the presently disclosed subject encodes a polypeptide has or comprises at least about having substantial identity (in some embodiments 90% amino acid identity) with a polypeptide as disclosed in Examples 6- 11, over an entire sequence or functional fragment thereof. In one embodiment, the polypeptide functions as an inosoitol phosphate kinase. More specifically, in some embodiments, the polypeptide is an Ipk2 polypeptide and functions as a IP₃/IP-₄/IP₅6-/3- /5-kinase generating multiple 1P₄, IP₅ and IP₆ products. More specifically, in some other embodiments, the polypeptide is an Ipk1 polypeptide and functions as an 1(1 ,4,5,6)P₄, l(1 ,3,4,6)P₄ and l(1 ,3,4,5,6)P₅ 2-kinase generating l(1 ,2,4,5,6)P₅, l(1 ,2,3,4.6)P₅ and phytate (IP_β). In another embodiment, the polypeptide is a plant polypeptide. In one embodiment, the plant is a dicot. In another embodiment, the plant is a gymnosperm. In another embodiment, the plant is a monocot. In one embodiment, the monocot is a cereal. In one embodiment, the cereal includes, but is not limited to, maize, wheat, barley, oats, rye, millet, sorghum, triticale, secale, einkorn, spelt, emmer, teff, miloflax, gramma grass, Tripsacum, and teosinte. In another embodiment, the cereal is sorghum. In one embodiment, the polypeptide functions as an inositol phosphate kinase. Embodiments ofthe 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), encoding a polypeptide selected from a group comprising one or more of: (a) a polypeptide sequence encoded by a nucleotide sequence that hybridizes under conditions of hybridization of 45°C in 1 M NaCI, followed by a final washing step at 50°C in 0.1 M NaCI to a nucleotide sequence listed in odd numbered sequences of SEQ ID NOs: 1-9, or a fragment, domain, or feature thereof, or a sequence complementary thereto; and (b) a functional fragment of (a). In one embodiment, the polypeptide having substantial identity comprises an allelic variant of a polypeptide that is an ortholog of a polypeptide having an amino acid sequence listed in even numbered sequences of SEQ ID NOs: 2-10, or a fragment, domain, repeat, feature, or chimera thereof. In one embodiment, the isolated nucleic acid comprises a plurality of regions from the polypeptide sequence encoded by a nucleotide sequence that hybridizes under stringent conditions of hybridization of 45°C in 1 M NaCI, followed by a final washing step at 50°C in 0.1 M NaCI to a nucleotide sequence listed in odd numbered sequences of SEQ ID NOs: 1-9, or a fragment, domain, or feature thereof, or a sequence complementary thereto. In another embodiment, the sequence of the isolated nucleic acid encodes a polypeptide useful for generating an antibody having immunoreactivity against a polypeptide encoded by a nucleotide sequence that hybridizes under stringent conditions of hybridization of 45°C in 1 M NaCI, followed by a final washing step at 50°C in 0.1 M NaCI to a nucleotide sequence listed in odd numbered sequences of SEQ ID NOs: 1-9, or a fragment, domain, or feature thereof. ■C. Polypeptides The presently disclosed subject matter further relates to isolated polypeptides that are orthologs ofthe polypeptides comprising the amino acid sequences set forth in even numbered SEQ ID NOs: 2-10, including biologically active polypeptides. In one embodiment, the polypeptide comprises a polypeptide sequence of an ortholog of a polypeptide listed in even numbered sequences of SEQ ID NOs: 2-10. In another embodiment, the polypeptide comprises a functional fragment or domain of an ortholog of a polypeptide comprising a polypeptide sequence listed in even numbered sequences of SEQ ID NOs: 2-10. In yet another embodiment, the polypeptide comprises a chimera of an ortholog of the polypeptide sequence listed in even numbered sequences of SEQ ID NOs: 2-10, where the chimera can comprise functional polypeptide motifs, including domains, repeats, post-translational modification sites, or other features. Representative orthologs are disclosed in Examples 6-11 herein below. In some embodiments, a polypeptide ofthe presently disclosed subject matter has or comprises substantial identity (in some embodiments at least about 90% amino acid identity) with a polypeptide as disclosed in Examples 6-11 , over an entire sequence or functional fragment thereof. In one embodiment, the polypeptide functions as an inosoitol phosphate kinase. More specifically, in some embodiments, the polypeptide is an Ipk2 polypeptide and functions as a IP₃/IP₄/IP₅6-/3-/5-kinase generating multiple IP₄, IP₅ and IPe products. More specifically, in some other embodiments, the polypeptide is an Ipk1 polypeptide and functions as an l(1 ,4,5,6)P₄, l(1 ,3,4,6)P₄ and l(1 ,3,4,5,6)P₅ 2-kinase generating l(1 ,2,4,5,6)P₅, l(1 ,2,3,4,6)P₅ and phytate (IP₆). In another embodiment, the polypeptide is a plant polypeptide. In one embodiment, the plant is a dicot. In another embodiment, the plant is a gymnosperm. In another embodiment, the plant is a monocot. In one embodiment, the monocot is a cereal. A more inclusive, but not limited to, list of plant genera relevant to the presently disclosed subject matter include Arabidopsis thaliana, corn (Zea mays), Brassica sp., alfalfa (Medicago sativa), rice (Oryza sativa ssp.), rye (Secale cereale), sorghum (Sorghum bicolor, Sorghum vulgare), pearl millet (Pennisetum glaucum), proso millet (Panicum miliaceum), foxtail millet (Setaria italica), finger millet (Eleusine coracana), sunflower (Helianthus annuus), safflower (Carthamus tinctorius), wheat (Triticum aestivum), soybean (Glycine max), tobacco (Nicotiana tabacum), potato (Solanum tuberosum), peanut (Arachis hypogaea), cotton, sweet potato (Ipomoea batatus), cassava (Manihot esculenta), coffee (Cofea spp.), coconut (Cocos nucifera), pineapple (Ananas comosus), citrus trees (Citrus spp.), cocoa (Theobroma cacao), tea (Camellia sinensis), banana (Musa spp.), avocado (Persea ultilane), fig (Ficus casica), guava (Psidium guajava), mango (Mangifera indica), olive (Olea europaea), papaya (Carica papaya), cashew (Anacardium occidentale), macadamia (Macadamia iniegrifolia), almond (Prunus amygdalus), sugar beets (Beta vulgaris), sugarcane (Saccharum spp.), oats, duckweed (Lemna), and barley. Still other plants relevant to the presently disclosed subject matter include vegetables, ornamentals, and conifers. Relevant vegetables include, but are not limited to, tomatoes, lettuce, guar, locust bean, fenugreek, soybean, garden beans, cowpea, mungbean, lima bean, fava bean, lentils, chickpea, green bean, lima bean, pea, and members ofthe genus Cucumis. Relevant ornamentals include, but are not limited to, impatiens, Begonia, Pelargonium, Viola, Cyclamen, Verbena, Vinca, Tagetes, Primula, Saint Paulia, Agertum, Amaranthus, Antihirrhinum, Aquilegia, Cineraria, Clover, Cosmo, Cowpea, Dahlia, Datura, Delphinium, Gerbera, Gladiolus, Gloxinia, Hippeastrum, Mesembryanthemum, Salpiglossos, and Zinnia, azalea, hydrangea, hibiscus, rose, tulip, daffodil, petunia, carnation, poinsettia, and chrysanthemum. Relevant conifers include, but are not limited to, loblolly pine, slash pine, ponderosa pine, lodgepole pine, Monterey pine, Douglas-fir, Western hemlock, Sitka spruce, redwood, silver fir, balsam fir, Western red cedar, and Alaska yellow-cedar. In one embodiment, the polypeptide is expressed in a specific location or tissue of a plant. In one embodiment, the location or tissue includes, but is not limited to, epidermis, root, vascular tissue, meristem, cambium, cortex, pith, leaf, flower, and combinations thereof. In another embodiment, the location or tissue is a seed. In one embodiment, the polypeptide functions as an inosoitol phosphate kinase. More specifically, in some embodiments, the polypeptide is an Ipk2 polypeptide and functions as a I P₃/l P4 I 5 6-/3-/5-kinase generating multiple IP , IP₅ and IP₆ products. More specifically, in some other embodiments, the polypeptide is an Ipk1 polypeptide and functions as an l(1 ,4,5,6)P , l(1 ,3,4,6)P₄ and l(1 ,3,4,5,6)P₅ 2-kinase generating l(1.2,4.5,6)P₅, l(1 ,2,3,4.6)P₅ and phytate (IP₆). In one embodiment, isolated polypeptides comprise the amino acid sequences of orthologs of the polypeptides comprising the amino acid sequences set forth in even numbered SEQ ID NOs: 2-10, 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 ef al., 1991 ; Ohtsuka ef a/., 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. Substitutions of amino acids involve amino acids for which the hydropathic indices are in one embodiment within ±2 of the original value, in another embodiment within ±1 of the original value, and in still another embodiment within ±0.5 ofthe original value in making changes based upon the hydropathic index. 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. Pat. 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. Pat. 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). Substitutions of amino acids involve amino acids for which the hydrophilicity values are in one embodiment within ±2 of the original value, in another embodiment within ±1 ofthe original value, and in still another embodiment within ±0.5 ofthe original value in making changes based upon similar hydrophilicity values. 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 an alternative embodiment, the sequence having substantial identity contains a deletion or insertion of at least one amino acid. In another embodiment, the deletion or insertion is of less than about ten amino acids. In still another embodiment, the deletion or insertion is of less than about three amino acids. In one embodiment, the sequence having substantial identity encodes a substitution in at least one amino acid. Embodiments of the presently disclosed subject matter also provide an isolated polypeptide comprising a polypeptide sequence selected from the group consisting of: (e) an amino acid sequence of one of even numbered SEQ ID NOs: 2-10; (f) an amino acid sequence that is at least 40% identical to (a); (g) an amino acid sequence encoded by a nucleotide sequence substantially identical to a nucleotide sequence of one of odd numbered SEQ ID NOs: 1-9; (h) an amino acid sequence encoded by a nucleic acid molecule capable of hybridizing under stringent conditions to a nucleic acid molecule ofone of odd numbered SEQ ID NOs: 1-9 or to a sequence fully complementary thereto; and (i) a functional fragment of (a), (b), (c) or (d). In one embodiment, a polypeptide having substantial identity to a polypeptide sequence listed in even numbered SEQ ID NO: 2-10, or a domain or feature thereof, is an allelic variant of the polypeptide sequence listed in even numbered SEQ ID NO: 2-

10. In another embodiment, a polypeptide having substantial identity to a polypeptide sequence listed in even numbered SEQ ID NO: 2-10, or a domain or feature thereof, is a naturally occurring variant ofthe polypeptide sequence listed in even numbered SEQ ID NO: 2-10. In another embodiment, a polypeptide having substantial identity to a polypeptide sequence listed in even numbered SEQ ID NO: 2-10, or a domain or feature thereof, is a polymorphic variant of the polypeptide sequence listed in even numbered SEQ ID NO: 2-10. In one embodiment, the polypeptide is an ortholog of a polypeptide comprising one of the amino acid sequences listed in even numbered SEQ ID NO: 2-10. In another embodiment, the polypeptide is a functional fragment or domain of an ortholog of a polypeptide comprising one of the amino acid sequences listed in even numbered SEQ ID NOs: 2-10. In yet another embodiment, the polypeptide is a chimera, where the chimera comprises a functional polypeptide domain, including, but not limited to, a domain, a repeat, a post-translational modification site, and combinations thereof. Representative orthologs are disclosed in Examples 6-11 herein below. In some embodiments, a polypeptide of the presently disclosed subject matter has or comprises substantial identity (in some embodiments at least about 90% amino acid identity) with a polypeptide as disclosed in Examples 6-11 , over an entire sequence or functional fragment thereof. In one embodiment, the polypeptide functions as an inosoitol phosphate kinase. More specifically, in some embodiments, the polypeptide is an Ipk2 polypeptide and functions as a dual-specificity IP₃/IP 6-/3-kinase generating IP₅. More specifically, in some other embodiments, the polypeptide is an Ipk1 polypeptide and functions as an 1(1 ,3,4,5,6)P₅ 2-kinase generating phytate (IP₆). In one embodiment, the polypeptide is a plant polypeptide. In one embodiment, the plant is a dicot. In another embodiment, the plant is a gymnosperm. In another embodiment, the plant is a monocot. In one embodiment, the monocot is a cereal. A more inclusive, but not intended to be limiting, list of plant genera relevant to the presently disclosed subject matter include Arabidopsis thaliana, corn (Zea mays), Brassica sp., alfalfa (Medicago sativa), rice (Oryza sativa ssp.), rye (Secale cereale), sorghum (Sorghum bicolor, Sorghum vulgare), pearl millet (Pennisetum glaucum), proso millet (Panicum miliaceum), foxtail millet (Setaria italica), finger millet (Eleusine coracana), sunflower (Helianthus annuus), safflower (Carthamus tinctorius), wheat (Triticum aestivum), soybean (Glycine max), tobacco (Nicotiana tabacum), potato (Solanum tuberosum), peanut (Arachis hypogaea), cotton, sweet potato (Ipomoea batatus), cassava (Manihot esculenta), coffee (Cofea spp.), coconut (Cocos nucifera), pineapple (Ananas comosus), citrus trees (Citrus spp.), cocoa (Theobroma cacao), tea (Camellia sinensis), banana (Musa spp.), avocado (Persea ultilane), fig (Ficus casica), guava (Psidium guajava), mango (Mangifera indica), olive (Olea europaea), papaya (Carica papaya), cashew (Anacardium occidentale), macadamia (Macadamia integrifolia), almond (Prunus amygdalus), sugar beets (Beta vulgaris), sugarcane (Saccharum spp.), oats, duckweed (Lemna), and barley. Still other plants relevant to the presently disclosed subject matter include vegetables, ornamentals, and conifers. Relevant vegetables include, but are not limited to, tomatoes, lettuce, guar, locust bean, fenugreek, soybean, garden beans, cowpea, mungbean, lima bean, fava bean, lentils, chickpea, green bean, lima bean, pea, and members ofthe genus Cucumis. Relevant ornamentals include, but are not limited to, impatiens, Begonia, Pelargonium, Viola, Cyclamen, Verbena, Vinca, Tagetes, Primula, Saint Paulia, Agertum, Amaranthus, Antihirrhinum, Aquilegia, Cineraria, Clover, Cosmo, Cowpea, Dahlia, Datura, Delphinium, Gerbera, Gladiolus, Gloxinia, Hippeastrum, Mesembryanthemum, Salpiglossos, and Zinnia, azalea, hydrangea, hibiscus, rose, tulip, daffodil, petunia, carnation, poinsettia, and chrysanthemum. Relevant conifers include, but are not limited to, loblolly pine, slash pine, ponderosa pine, lodgepole pine, Monterey pine, Douglas-fir, Western hemlock, Sitka spruce, redwood, silver fir, balsam fir, Western red cedar, and Alaska yellow-cedar. In one embodiment, the polypeptide is expressed in a specific location or tissue of a plant. In one embodiment, the location or tissue includes, but is not limited to, epidermis, vascular tissue, meristem, cambium, cortex, or pith. In another embodiment, the location or tissue is leaf or sheath, root, flower, and developing ovule or seed. In another embodiment, the location or tissue can be, for example, epidermis, root, vascular tissue, meristem, cambium, cortex, pith, leaf, or flower. In yet another embodiment, the location or tissue is a seed. In one embodiment, the polypeptide sequence is encoded by a nucleotide sequence that hybridizes under stringent conditions of hybridization of 45°C in 1 M NaCI, followed by a final washing step at 50°C in 0.1 M NaCI to a nucleotide sequence listed in odd numbered SEQ ID NO: 1-9 or a fragment, domain, or feature thereof or a sequence complementary thereto, wherein the nucleotide sequence includes a deletion or insertion of at least one nucleotide. In one embodiment, the deletion or insertion is of less than about thirty nucleotides. In another embodiment, the deletion or insertion is of less than about five nucleotides. In another embodiment, the polypeptide sequence encoded by a nucleotide sequence that hybridizes under stringent conditions of hybridization of 45°C in 1 M NaCI, followed by a final washing step at 50°C in 0.1 M

NaCI to a nucleotide sequence listed in odd numbered SEQ ID NO: 1-9, or a fragment, domain, or feature thereof or a sequence complementary thereto, includes a substitution of at least one codon. In one embodiment, the substitution is conservative. In another embodiment, the polypeptide sequences having substantial identity to the polypeptide sequence listed in even numbered SEQ ID NO: 2-10, or a fragment, domain, repeat, feature, or chimera thereof, includes a deletion or insertion of at least one amino acid. The polypeptides ofthe presently disclosed subject matter, fragments thereof, or variants thereof, can comprise any number of contiguous amino acid residues from a polypeptide ofthe 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 one embodiment, 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 one embodiment 20%, in another embodiment 30%, in another embodiment 40%, in another embodiment 50%, in another embodiment 60%, in another embodiment 70%, in another embodiment 80%, in another embodiment 90%, and in still another embodiment 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 one embodiment 30%, in another embodiment 40%, in another embodiment 50% of the native, endogenous polypeptide; and in another embodiment at least 60%, in another embodiment 70%, in another embodiment 80%, and in yet another embodiment 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. The isolated polypeptides of the presently disclosed subject matter can elicit production of an antibody specifically reactive to a polypeptide of the presently disclosed subject matter when presented as an immunogen. Therefore, the polypeptides of the presently disclosed subject matter can be employed as immunogens for constructing antibodies immunoreactive to a polypeptide of the presently disclosed subject matter for such purposes including, but not limited to, immunoassays or polypeptide purification techniques. Immunoassays for determining binding are well known to those of skill in the art and include, but are not limited to, enzyme-linked immunosorbent assays (ELISAs) and competitive immunoassays. Embodiments of the presently disclosed subject matter also relate to chimeric polypeptides encoded by the isolated nucleic acid molecules ofthe present disclosure including a chimeric polypeptide containing a polypeptide sequence encoded by an isolated nucleic acid containing a nucleotide sequence selected from the group consisting of: (a) a nucleotide sequence that hybridizes under stringent conditions of hybridization of 45°C in 1 M NaCI, followed by a final washing step at 50°C in 0.1 M NaCI to a nucleotide sequence listed in odd numbered SEQ ID NO: 1-9, or an exon, domain, or feature thereof; (b) a nucleotide sequence complementary (for example, fully complementary) to (a); and (c) a nucleotide sequence which is the reverse complement (for example, full reverse complement) of (a); (d) or a functional fragment thereof.

IV. Controlling and Altering the Expression of Nucleic Acid Molecules IV.A. General Considerations One aspect ofthe presently disclosed subject matter provides compositions and methods for altering or modulating (i.e. increasing or decreasing) the level of nucleic acid molecules and/or polypeptides ofthe 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. The isolated nucleic acid molecules ofthe presently disclosed subject matter are useful for expressing a polypeptide of the presently disclosed subject matter in a recombinantly 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 as disclosed herein above, the isolated nucleic acid encoding a polypeptide as disclosed herein above. In some embodiments the nucleic acid comprise one of: (a) a nucleic acid molecule encoding a polypeptide comprising an amino acid sequence of one of even numbered SEQ ID NOs: 2-10 or a polypeptide at least 40% identical to even numbered SEQ ID NOs: 2-10 and having inositol phosphate kinase activity; (b) a nucleic acid molecule comprising a nucleic acid sequence ofone of odd numbered SEQ ID NOs:1-9; (c) a nucleic acid molecule that has a nucleic acid sequence that is substantially identical to the nucleic acid sequence of the nucleic acid molecule of (a) or (b); (d) a nucleic acid molecule that hybridizes to (a) or (b) under stringent hybridization conditions; (e) a nucleic acid molecule comprising a nucleic acid sequence complementary to (a); and (f) a nucleic acid molecule comprising a nucleic acid sequence that is the full reverse complement of (a). 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 transgenic plant cells comprising expression cassettes according to the present disclosure, and transgenic plants comprising these plant cells. In one embodiment, the plant is a dicot. In another embodiment, the plant is a gymnosperm. In another embodiment, the plant is a monocot. In one embodiment, the monocot is a cereal. A more inclusive, but not intended to be limiting, list of plant genera relevant to the presently disclosed subject matter include Arabidopsis thaliana, corn (Zea mays), Brassica sp., alfalfa (Medicago sativa), rice (Oryza sativa ssp.), rye (Secale cereale), sorghum (Sorghum bicolor, Sorghum vulgare), pearl millet (Pennisetum glaucum), proso millet (Panicum miliaceum), foxtail millet (Setaria italica), finger millet (Eleusine coracana), sunflower (Helianthus annuus), safflower (Carthamus tinctorius), wheat (Triticum aestivum), soybean (Glycine max), tobacco (Nicotiana tabacum), potato (Solanum tuberosum), peanut (Arachis hypogaea), cotton, sweet potato (Ipomoea batatus), cassava (Manihot esculenta), coffee (Cofea spp.), coconut (Cocos nucifera), pineapple (Ananas comosus), citrus trees (Citrus spp.), cocoa (Theobroma cacao), tea (Camellia sinensis), banana (Musa spp.), avocado (Persea ultilane), fig (Ficus casica), guava (Psidium guajava), mango (Mangifera indica), olive (Olea europaea), papaya (Carica papaya), cashew (Anacardium occidentale), macadamia (Macadamia integrifolia), almond (Prunus amygdalus), sugar beets (Beta vulgaris), sugarcane (Saccharum spp.), oats, duckweed (Lemna), and barley. Still other plants relevant to the presently disclosed subject matter include vegetables, ornamentals, and conifers. Relevant vegetables include, but are not limited to, tomatoes, lettuce, guar, locust bean, fenugreek, soybean, garden beans, cowpea, mungbean, lima bean, fava bean, lentils, chickpea, green bean, lima bean, pea, and members of the genus Cucumis. Relevant ornamentals include, but are not limited to, impatiens, Begonia, Pelargonium, Viola, Cyclamen, Verbena, Vinca, Tagetes, Primula, Saint Paulia, Agertum, Amaranthus, Antihirrhinum, Aquilegia, Cineraria, Clover, Cosmo, Cowpea, Dahlia, Datura, Delphinium, Gerbera, Gladiolus, Gloxinia, Hippeastrum, Mesembryanthemum, Salpiglossos, and Zinnia, azalea, hydrangea, hibiscus, rose, tulip, daffodil, petunia, carnation, poinsettia, and chrysanthemum. Relevant conifers include, but are not limited to, loblolly pine, slash pine, ponderosa pine, lodgepole pine, Monterey pine, Douglas-fir, Western hemlock, Sitka spruce, redwood, silver fir, balsam fir, Western red cedar, and Alaska yellow- cedar. In one embodiment, the expression cassette is expressed throughout the plant.

In another embodiment, the expression cassette is expressed in a specific location or tissue of a plant. In one embodiment, the location or tissue includes, but is not limited to, epidermis, root, vascular tissue, meristem, cambium, cortex, pith, leaf, flower, and combinations thereof. In another embodiment, the location or tissue is a seed. In one embodiment, the expression cassette is involved in a function including, but not limited to, inositol phosphate kinase activity resulting in phytate production. In one embodiment, the chimeric polypeptide functions as an inositol phosphate kinase, or replaces the endogenous inositol phosphate kinase polypeptide as a non-functional or reduced function homolog. Embodiments of the presently disclosed subject matter also relate to an expression vector comprising an isolated nucleic acid as disclosed herein above, the isolated nucleic acid encoding a polypeptide as disclosed herein above. In some embodiments the nucleic acid comprises one of: (a) a nucleic acid molecule encoding a polypeptide comprising an amino acid sequence of one of even numbered SEQ ID NOs: 2-10 or a polypeptide at least 40% identical to even numbered SEQ ID NOs: 2-10 and having inositol phosphate kinase activity; (b) a nucleic acid molecule comprising a nucleic acid sequence of one of odd numbered SEQ ID NOs: 1-9; (c) a nucleic acid molecule that has a nucleic acid sequence that is substantially identical to the nucleic acid sequence ofthe nucleic acid molecule of (a) or (b); (d) a nucleic acid molecule that hybridizes to (a) or (b) under stringent hybridization conditions; (e) a nucleic acid molecule comprising a nucleic acid sequence complementary to (a); and (f) a nucleic acid molecule comprising a nucleic acid sequence that is the full reverse complement of (a). In one embodiment, 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 one embodiment, the promoter-enhancer sequence comprises, for example, the cauliflower mosaic virus (CaMV) 35S promoter, the CaMV 19S promoter, the tobacco PR-1a promoter, the ubiquitin promoter, or the phaseolin promoter. In one embodiment, the promoter is operable in plants, and in another embodiment, the promoter is a constitutive or inducible promoter. In one embodiment, the selection marker sequence encodes an antibiotic resistance gene. In one embodiment the affinity purification tag sequence encodes a polyamino acid sequence or a polypeptide. In one embodiment, the polyamino acid sequence comprises polyhistidine. In one embodiment, the polypeptide is chitin-binding domain or glutathione-S-transferase. In another embodiment, the affinity purification tag sequence comprises an intein encoding sequence. In one embodiment, the expression vector comprises a eukaryotic expression vector, and in another embodiment, the expression vector comprises a prokaryotic expression vector. In one embodiment, the eukaryotic expression vector comprises a tissue-specific promoter. In another embodiment, 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 even numbered sequences of SEQ ID NOs: 2-10, or a nucleic acid sequence that hybridizes under stringent conditions of hybridization of 45°C in 1 M NaCI, followed by a final washing step at 50°C in 0.1 M NaCI to a nucleotide sequence listed in odd numbered sequences of SEQ ID NOs: 1-9, or a subsequence thereof, in combination with a heterologous sequence. Representative orthologs are disclosed in Examples 6- 11 herein below. In some embodiments, a polypeptide of the presently disclosed subject matter has or comprises substantial identity (in some embodiments at least about 90% amino acid identity) with a polypeptide as disclosed in Examples 6-11 , over an entire sequence orfunctional fragment thereof. In one embodiment, the polypeptide functions as an inosoitol phosphate kinase. More specifically, in some embodiments, the polypeptide is an Ipk2 polypeptide and functions as a dual-specificity IP₃/!P₄ 6-/3- kinase generating IP₅. More specifically, in some other embodiments, the polypeptide is an Ipk1 polypeptide and functions as an l(1 ,3,4,5,6)P₅ 2-kinase generating phytate

(IPs). In one embodiment, the cell is a bacterial cell, a fungal cell, a plant cell, or an animal cell. In one embodiment, the polypeptide is expressed in a specific location or tissue of a plant. In one embodiment, the location or tissue includes, but is not limited to, epidermis, root, vascular tissue, meristem, cambium, cortex, pith, leaf, flower, and combinations thereof. In an alternative embodiment, the location ortissue is a seed. In one embodiment, the polypeptide functions as an inositol phosphate kinase. 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 ofthe 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 ofthe 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 et al., 1990). Embodiments of the presently disclosed subject matter provide a method for producing a recombinant polypeptide in which 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 one embodiment, the nucleic acid construct comprises an epitope tag-encoding sequence and the isolating step employs an antibody specific for the epitope tag. In another embodiment, the nucleic acid construct comprises a polyamino acid-encoding sequence and the isolating step employs a resin comprising a polyamino acid binding substance, in one embodiment where the polyamino acid is polyhistidine and the polyamino acid binding resin is nickel- charged agarose resin. In yet another embodiment, the nucleic acid construct comprises a polypeptide-encoding sequence and the isolating step employs a resin comprising a polypeptide binding substance. In one embodiment, 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 modifying (i.e. increasing or decreasing) the concentration or composition or activity of a polypeptide of the presently disclosed subject matter in a plant or part thereof. Modification can be effected by increasing or decreasing the concentration, the composition, and/or the activity (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 or repressing 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. Inducing or repressing the nucleic acids disclosed herein encoding Ipk1 or 2 correlates with an observable modification in the amount of phytate produced in the plant or plant part relative to a non-transformed parental plant. A plant or plant part having modified expression of a nucleic acid molecule ofthe presently disclosed subject matter can be analyzed and selected using methods known to those skilled in the art including, but not limited to, Southern blotting, 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 one embodiment 5%, in another embodiment 10%, in another embodiment 20%, in another embodiment 30%, in another embodiment 40%, in another embodiment 50%, in another embodiment 60%, in another embodiment 70%, in another embodiment 80%, and in still another embodiment greater than 90% relative to a non-transformed native control plant, plant part, or cell lacking the expression cassette. IV.B. Alteration of Expression of Nucleic Acid Molecules The alteration in expression of the nucleic acid molecules of the presently disclosed subject matter can be achieved, for example, in one of the following ways: IV.B.1. "Sense" Suppression Alteration of the expression of a nucleotide sequence of the presently disclosed subject matter, in one embodiment reduction of its expression, is obtained by "sense" suppression (referenced in e.g. Jorgensen et al., 1996). In this case, the entirety or a portion of a nucleotide sequence ofthe presently disclosed subject matter is comprised in a DNA molecule. The DNA molecule can be operatively linked to a promoter functional in a cell comprising the target gene, in one embodiment 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 one embodiment, 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 another embodiment, the nucleotide sequence is partially translatable and a short peptide is translated. In one embodiment, this is achieved by inserting at least one premature stop codon in the nucleotide sequence, which brings translation to a halt. In another embodiment, 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 a further embodiment, the DNA molecule comprising the nucleotide sequence, or a portion thereof, is stably integrated in the genome ofthe plant cell. In another embodiment, the DNA molecule comprising the nucleotide sequence, or a portion thereof, is comprised in an extrachromosomally replicating molecule. In transgenic plants containing one ofthe DNA molecules disclosed immediately above, the expression of the nucleotide sequence corresponding to the nucleotide sequence comprised in the DNA molecule can be reduced. The nucleotide sequence in the DNA molecule in one embodiment is at least 70% identical to the nucleotide sequence the expression of which is reduced, in another embodiment is at least 80% identical, in another embodiment is at least 90% identical, in another embodiment is at least 95% identical, and in still another embodiment is at least 99% identical. IV.B.2. "Antisense" Suppression In another embodiment, the alteration of the expression of a nucleotide sequence of the presently disclosed subject matter, for example the reduction of its expression, is obtained by "antisense" suppression. The entirety or a portion of a nucleotide sequence of the presently disclosed subject matter is comprised in a DNA molecule. The DNA molecule can be operatively linked to a promoter functional in a plant cell, and introduced in a plant cell, in which the nucleotide sequence is expressible. The nucleotide sequence is inserted in the DNA molecule in the "antisense orientation", meaning that the reverse complement (also called sometimes non-coding strand) of the nucleotide sequence can be transcribed. In one embodiment, the DNA molecule comprising the nucleotide sequence, or a portion thereof, is stably integrated in the genome of the plant cell. In another embodiment the DNA molecule comprising the nucleotide sequence, or a portion thereof, is comprised in an extrachromosomally replicating molecule. Several publications describing this approach are cited for further illustration (Green et al., 1986; van der Krol et al., 1991 ; Powell et al., 1989; Ecker & Davis, 1986). In transgenic plants containing one ofthe DNA molecules disclosed immediately above, the expression of the nucleotide sequence corresponding to the nucleotide sequence comprised in the DNA molecule can be reduced. The nucleotide sequence in the DNA molecule is in one embodiment at least 70% identical to the nucleotide sequence the expression of which is reduced, in another embodiment at least 80% identical, in another embodiment at least 90% identical, in another embodiment at least 95% identical, and in still another embodiment at least 99% identical. IV.B.3. Homologous Recombination In another embodiment, 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 al., 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 one embodiment, 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 another embodiment, the nucleotide sequence is modified by deletion of a part of the nucleotide sequence or the entire 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) and are generally known in the art. In one embodiment, 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'-0-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 al., 1999. IV.B.4. Ribozymes In a further embodiment, an RNA coding for a polypeptide of the presently disclosed subject matter is cleaved by a catalytic RNA, or ribozyme, specific for such RNA. The ribozyme is expressed in transgenic plants and results in reduced amounts of RNA coding for the polypeptide of the presently disclosed subject matter in plant cells, thus leading to reduced amounts of polypeptide accumulated in the cells. This method is further illustrated in U.S. Patent No. 4,987,071. IV.B.5. Dominant-Negative Mutants In another embodiment, the activity of a polypeptide encoded by the nucleotide sequences of the presently disclosed subject matter is changed. This is achieved by expression of dominant negative mutants of the polypeptides in transgenic plants, leading to the loss of activity of the endogenous polypeptide. IV.B.6. Aptamers In a further embodiment, the activity of polypeptide of the presently disclosed subject matter is inhibited by expressing in transgenic plants nucleic acid ligands, so- called aptamers, which specifically bind to the polypeptide. Aptamers can be obtained by the SELEX (Systematic Evolution of Ligands by Exponential Enrichment) method. In the SELEX method, a candidate mixture of single stranded nucleic acids having regions of randomized sequence is contacted with the polypeptide and those nucleic acids having an increased affinity to the target are partitioned from the remainder of the candidate mixture. The partitioned nucleic acids are amplified to yield a ligand-enriched mixture. After several iterations a nucleic acid with optimal affinity to the polypeptide is obtained and is used for expression in transgenic plants. This method is further illustrated in U.S. Patent No. 5,270,163. IV.B.7. 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 ofthe nucleotide sequence. In alternative embodiments, transcription ofthe nucleotide sequence is reduced or increased. Zinc finger polypeptides are disclosed in, for example, Beerli et al., 1998, or in WO 95/19431 , WO 98/54311 , or WO 96/06166, all incorporated herein by reference in their entirety. IV.B.8. dsRNA Alteration of the expression of a nucleotide sequence of the presently disclosed subject matter can also be obtained by double stranded RNA (dsRNA) interference (RNAi) as disclosed, for example, in WO 99/32619, WO 99/53050, or WO 99/61631 , all incorporated herein by reference in their entireties. RNAi as used herein encompasses both plasmid-based and genomic-based strategies. In one embodiment, the alteration ofthe expression of a nucleotide sequence of the presently disclosed subject matter, in one embodiment the reduction of its expression, is obtained by dsRNA interference. The entirety, or in one embodiment a portion, of a nucleotide sequence of the presently disclosed subject matter, can be comprised in a DNA molecule. The size of the DNA molecule is in one embodiment from 100 to 1000 nucleotides or more; the optimal size to be determined empirically. Two copies of the identical DNA molecule are linked, separated by a spacer DNA molecule, such that the first and second copies are in opposite orientations. In one embodiment, the first copy ofthe DNA molecule is the reverse complement (also known as the non-coding strand) and the second copy is the coding strand; in another embodiment, the first copy is the coding strand, and the second copy is the reverse complement. The size of the spacer DNA molecule is in one embodiment 200 to 10,000 nucleotides, in another embodiment 400 to 5000 nucleotides, and in yet another embodiment 600 to 1500 nucleotides in length. The spacer is in one embodiment a random piece of DNA, in another embodiment a random piece of DNA without homology to the target organism for dsRNA interference, and in still another embodiment a functional intron that is effectively spliced by the target organism. The two copies of the DNA molecule separated by the spacer are operatively linked to a promoter functional in a plant cell, and introduced in a plant cell in which the nucleotide sequence is expressible. In one embodiment, the DNA molecule comprising the nucleotide sequence, or a portion thereof, is stably integrated in the genome of the plant cell. In another embodiment, the DNA molecule comprising the nucleotide sequence, or a portion thereof, is comprised in an extrachromosomally replicating molecule. Several publications describing this approach are cited for further illustration (Waterhouse et al., 1998; Chuang & Meyerowitz, 2000; Smith et al., 2000). In transgenic plants containing one ofthe DNA molecules disclosed immediately above, the expression of the nucleotide sequence corresponding to the nucleotide sequence comprised in the DNA molecule is in one embodiment reduced. In one embodiment, the nucleotide sequence in the DNA molecule is at least 70% identical to the nucleotide sequence the expression of which is reduced, in another embodiment it is at least 80% identical, in another embodiment it is at least 90% identical, in another embodiment it is at least 95% identical, and in still another embodiment it is at least 99% identical. IV.B.9. Insertion of a DNA Molecule (Insertional Mutagenesis) In one embodiment, a DNA molecule is inserted into a chromosomal copy of a nucleotide sequence of the presently disclosed subject matter, or into a regulatory region thereof. In one embodiment, such DNA molecule comprises a transposable element capable of transposition in a plant cell, such as, for example, Ac/Ds, Em/Spm, mutator. Alternatively, in another embodiment, the DNA molecule comprises a T-DNA border of an Agrobacterium T-DNA. The DNA molecule can also comprise a recombinase or integrase recognition site that can be used to remove part of the DNA molecule from the chromosome of the plant cell. Methods of insertional mutagenesis using T-DNA, transposons, oligonucleotides, or other methods known to those skilled in the art are also encompassed. Methods of using T-DNA and transposon for insertional mutagenesis are disclosed in Winkler & Feldmann, 1989, and Martienssen, 1998, incorporated herein by reference in their entireties. 1V.B.10. Deletion Mutagenesis In yet another embodiment, a mutation of a nucleic acid molecule of the presently disclosed subject matter is created in the genomic copy of the sequence in the cell or plant by deletion of a portion of the nucleotide sequence or regulator sequence. Methods of deletion mutagenesis are known to those skilled in the art. See e.g., Miao & Lam, 1995. In yet another embodiment, a deletion is created at random in a large population of plants by chemical mutagenesis or irradiation and a plant with a deletion in a gene of the presently disclosed subject matter is isolated by forward or reverse genetics.

Irradiation with fast neutrons or gamma rays is known to cause deletion mutations in plants (Silverstone et al., 1998; Bruggemann et al., 1996; Redei & Koncz, 1992).

Deletion mutations in a gene ofthe presently disclosed subject matter can be recovered in a reverse genetics strategy using PCR with pooled sets of genomic DNAs as has been shown in C. elegans (Liu et al., 1999). A forward genetics strategy involves mutagenesis of a line bearing a trait of interest followed by screening the M2 progeny for the absence of the trait. Among these mutants would be expected to be some that disrupt a gene of the presently disclosed subject matter. This could be assessed by

Southern blotting or PCR using primers designed for a gene of the presently disclosed subject matter with genomic DNA from these mutants. IV.B.11. Overexpression in a Plant Cell In yet another embodiment, 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 one embodiment, the expression ofthe nucleotide sequence ofthe presently disclosed subject matter is altered in every cell of a plant. This can be obtained, for example, through homologous recombination or by insertion into a chromosome. This can also be obtained, for example, by expressing a sense or antisense RNA, zinc finger polypeptide or ribozyme under the control of a promoter capable of expressing the sense or antisense RNA, zinc finger polypeptide, or ribozyme 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 ofthe expression of a nucleotide sequence of the presently disclosed subject matter in the plant cell. Constructs for expression of the sense or antisense RNA, zinc finger polypeptide, or ribozyme, 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. IV.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. IV.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 (seeds, roots, leaves, or flowers, for example) and the selection can reflect the desired location for accumulation ofthe gene product. Alternatively, the selected promoter can drive expression ofthe 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. IV.C.1.a. 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 et al., 1989; and Arabidopsis - Callis 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 LubrizoJ) 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 ofthe 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. IV.C.1.b. Constitutive Expression: the CaMV 35S Promoter Construction of the plasmid pCGN1761 is disclosed in the published patent application EP 0 392 225, 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 pCGN1761 is constructed which has a modified polylinker that 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-f/r?/ terminator cassette of such a construction can be excised by Hindlll, Sph\, Sail, and X-bal sites 5' to the promoter and X-bal, 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, Notl or Xhol) 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 ofthe translational initiation site as disclosed in Example 37 of U.S. Patent No. 5,639,949, incorporated herein by reference. IV.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 Acf/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 >4cf/-intron 1 , Adhl 5' flanking sequence (from the maize alcohol dehydrogenase gene) and Adhl-^'iniron 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 ef a/., 1993). IV.C.1.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-1 a promoter, can replace the double 35S promoter. Alternately, the Arabidopsis PR-1 promoter disclosed in Lebel etal., 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-1 a 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 pCGN1761ENX (Uknes et al., 1992). pCIB1004 is cleaved with A/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 /-//tic/Ill and the resultant PR-1 a 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 Xhol and blunting with T4 polymerase, followed by cleavage with Hindlll, and isolation ofthe larger vector-terminator containing fragment into which the pCIB1004 promoter fragment is cloned. This generates a pCGN1761 ENX derivative with the PR-1 a promoter and the tml terminator and an intervening polylinker with unique EcoRI and Notl 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. IV.C.1.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 AlcR 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. IV.C.1.f. Inducible Expression: a Glucocorticoid-lnducible 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 one embodiment from 0.1 mM to 1 mM, and in another embodiment from 10 mM to 100 mM. For the purposes of the presently disclosed subject matter, the luciferase gene sequences Aoyama & Chua are replaced by a nucleic acid sequence ofthe 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 etal., 1986) fused to the transactivating domain ofthe herpes viral polypeptide VP16 (Triezenberg etal., 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 GAL4/minimal promoter is also provided. Thus, tissue-specificity of the fusion polypeptide is achieved leading to inducible tissue-specificity of the nucleic acid sequence to be expressed. IV.C.1.g. 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. IV.C.1.h. Wound-lnducible Promoters Wound-inducible promoters can also be suitable for gene expression. Numerous such promoters have been disclosed (e.g. Xu et al., 1993; Logemann et al., 1989; Rohrmeier & Lehle, 1993; Firek et al. , 1993; Warner et al. , 1993) and all are suitable for use with the presently disclosed subject matter. Logemann et al. describe the 5' upstream sequences of the dicotyledonous potato wunl gene. Xu et al. show that a wound-inducible promoter from the dicotyledon potato (pin2) is active in the monocotyledon rice. Further, Rohrmeier & Lehle describe the cloning ofthe maize Wipl cDNA that is wound induced and which can be used to isolate the cognate promoter using standard techniques. Similarly, Firek et al. and Warner et al. have disclosed a wound-induced gene from the monocotyledon Asparagus officinalis, which is expressed at local wound and pathogen invasion sites. Using cloning techniques well known in the art, these promoters can be transferred to suitable vectors, fused to the genes pertaining to the presently disclosed subject matter, and used to express these genes at the sites of plant wounding. IV.C.1.i. Pith-Preferred Expression PCT International Publication WO 93/07278, which is herein incorporated by reference, describes the isolation of the maize trpA gene, which is preferentially expressed in pith cells. The gene sequence and promoter extending up to -1726 basepairs (bp) from the start of transcription are presented. Using standard molecular biological techniques, this promoter, or parts thereof, can be transferred to a vector such as pCGN1761 where it can replace the 35S promoter and be used to drive the expression of a foreign gene in a pith-preferred manner. In fact, fragments containing the pith-preferred promoter or parts thereof can be transferred to any vector and modified for utility in transgenic plants. IV.C.1.j. Leaf-Specific Expression A maize gene encoding phosphoenol carboxylase (PEPC) has been disclosed by

Hudspeth & Grula, 1989. Using standard molecular biological techniques, the promoter for this gene can be used to drive the expression of any gene in a leaf-specific manner in transgenic plants. IV.C.1.k. Pollen-Specific Expression WO 93/07278 describes the isolation of the maize calcium-dependent protein kinase (CDPK) gene that is expressed in pollen cells. The gene sequence and promoter extend up to 1400 bp from the start of transcription. Using standard molecular biological techniques, this promoter or parts thereof can be transferred to a vector such as pCGN1761 where it can replace the 35S promoter and be used to drive the expression of a nucleic acid sequence of the presently disclosed subject matter in a pollen-specific manner. IV.C.1.1. Seed-Specific Expression In some embodiments of the presently disclosed subject matter, a lack of expression or reduced expression in seeds can be desired. This can optionally accomplished by knocking expression in the plant and then re-establishing expression in one or more other tissues (including all tissues) except seeds. Example 5 below describes an ipk1/ipk2 beta double knockout plant. The double mutant grows about the same as the single mutant disclosed in the Examples. Thus, the seed specific knockdown of all IPs is not detrimental to seed yield and plant growth. The term "seed specific" is used because analysis of the plant tissue from the atlpk2 beta mutant shows no defect in IP metabolism whereas the seeds do. Further the ipkl ipk2 beta double mutant appears to synthesize IPs except for IP5 in the tissue. The growth issue and yield problems of ipkl mutant and ipk1/ipk2 beta double mutant plants ar expected to be corrected with the tissue (but not seed) expression of ipkl . Complementation of the ipkl mutant plant with pBART Ipkl complements the growth. The ipk1/ipk2 beta double mutant can be complemented with Ipkl under one or more tissue specific promoters as disclosed herein above, e.g. one or more promoters that can drive expression in one or more tissues except seeds. IV.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. IV.C.3. Seguences 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

-12- 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 et al., 1987). In the 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' noncoding region; see Elroy-Stein etal., 1989); potyvirus leaders, for example, from Tobacco Etch Virus (TEV; see Allison etal., 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 et al., 1987. In addition to incorporating one or more ofthe 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 bronzel gene of maize. The Bz1 core promoter is obtained from the "myc" mutant Bz1 -luciferase construct pBz1LucR98 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' 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. IV.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. IV.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 etal., 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). IV.D.1. Vectors Suitable for Aprobacterium 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.

IV.D.1.a. PC1B200 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 Accl fragment from pUC4K carrying an NPTH sequence (Messing & Vieira, 1982: Bevan et al., 1983: McBride & Summerfelt. 1990). Xhol linkers are ligated to the EcoR fragment of PCIB7 which contains the left and right T-DNA borders, a plant selectable nos/nptll chimeric gene and the pUC polylinker (Rothstein et al., 1987), and the Xhol- 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: EcoRI, Sstl, Kpnl, Bgll , Xba\, and Sail. pCIB2001 is a derivative of pClB200 created by the insertion into the polylinker of additional restriction sites. Unique restriction sites in the polylinker of pCIB2001 are EcoRI, Sstl, Kpnl, Bglll, Xbal, Sail, Mlul, Bell, Avήl, Apa\, Hpa , and Stul. pCIB2001 , in addition to containing these unique restriction sites, also has plant and bacterial kanamycin selection, left and right T-DNA borders for /Agro-bacter/t/tn-mediated transformation, the RK2-derived trfA function for mobilization between E. coli and other hosts, and the OriT and OriV functions also from RK2. The pCIB2001 polylinker is suitable for the cloning of plant expression cassettes containing their own regulatory signals. IV.D.1.b. pCIBIO and Hygromvcin 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 et al., 1987. Various derivatives of pCIB10 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). IV.D.2. Vectors Suitable for non-Aprobacterium Transformation Transformation without the use of Agrobacterium tumefaciens 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. IV.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 promoterof this vector contains two ATG sequences 5' ofthe start site. These sites are mutated using standard PCR techniques in such a way as to remove the ATGs and generate the restriction sites Sspl and Pvull. The new restriction

-71- 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 Sail and Sacl, the termini rendered blunt and religated to generate plasmid pCIB3060. The plasmid pJIT82 is obtained from the John Innes Centre, Norwich, England, and the 400 bp Smal 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 Sphl, Pstl, HindlU, and BamHI. This vector is suitable for the cloning of plant expression cassettes containing their own regulatory signals. IV.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 pSOG10. A 250- bp fragment encoding the E. coli dihydrofolate reductase type II gene is also amplified by PCR and these two PCR fragments are assembled with a Sacl-Psfl 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 HindlU, Sphl, Pstl, and EcoRI sites available for the cloning of foreign substances. IV.D.3. Vector Suitable for Chloroplast Transformation For expression of a nucleotide sequence of the presently disclosed subject matter in plant plastids, plastid transformation vector pPH143 (PCT International Publication WO 97/32011 , example 36) can be used. The nucleotide sequence is inserted into pPH143 thereby replacing the protoporphyrinogen oxidase (Protox) coding sequence. This vector can then be used for plastid transformation and selection of transformants for spectinomycin resistance. Alternatively, the nucleotide sequence can be inserted in pPH143 so that it replaces the aadH gene. In this case, transformants are selected for resistance to PROTOX inhibitors. IV.E. Transformation Once a nucleic acid sequence ofthe presently disclosed subject matter has been cloned into an expression system, it is transformed into a plant cell. The receptor and target expression cassettes ofthe 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 Agrobacterium 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. IV.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 Agrobacterium. 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 al., 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. Agrobacterium-medlated transformation is a useful technique for transformation of dicotyledons because of its high efficiency of transformation and its broad utility with many different species. Agrobacterium 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 Agrobacterium 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 ofthe target plant species by recombinant Agrobacterium 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 etal. 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. IV.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 et al., 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 ef al., 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 Japo-π/ca-types and Indica-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 et al., 1991). Furthermore, WO 93/21335 describes techniques for the transformation of rice via electroporation. Casas et al., 1993 discloses the production of transgenic sorghum plants by microprojectile bombardment. Patent Application EP 0 332 581 describes techniques for the generation, transformation, and regeneration of Pooideae protoplasts. These techniques allowthe transformation of Dactylis and wheat. Furthermore, wheat transformation has been disclosed in Vasil etal., 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 (i.e. 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 et al., 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 ODβoo 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 et al., 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/liter 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 (To generation) grown to maturity and the Ti seed is harvested.

IV.E.3. Transformation of Plastids As one example of plastid transformation, seeds of Nicotiana tabacum c.v.

'Xanthi nc' 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, Biorad, 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/m²/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). BamHMEcoRl -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 ³²P-labeled random primed DNA sequences corresponding to a 0.7 kb BamHllHindlll DNA fragment from pC8 containing a portion of the rps7/12 plastid targeting sequence. Homoplasmic shoots are rooted aseptically on spectinomycin-containing MS/IBA medium (McBήde etal., 1994) and transferred to the greenhouse.

V. Plants, Breeding, and Seed Production VA Plants The presently disclosed subject matter also provides plant cells and plants comprising the disclosed compositions. The presently disclosed subject matter further provides plant cells and plants comprising homozygous disruptions in endogenous nucleic acids homologous to the novel nucleic acid molecules disclosed herein. In one embodiment, the plant is characterized by a modification of a phenotype or measurable characteristic of the plant, the modification being attributable to the expression cassette comprising a novel nucleic acid disclosed herein. In one embodiment, the modification involves modulation of the expression of a functional inositol phosphate kinase polypeptide. In some embodiments, the functional inositol phosphate kinase polypeptide is Ipkl or 2. In some embodiments, the modification results in a change in the level of phytate and/or non-phytate phosphorous when compared to a non-transformed parental plant. In some embodiments, the modulation of functional inositol phosphate kinase polypeptide is an inhibition of the expression of the polypeptide resulting in a subsequent decreased level of phytate and/or a subsequent increase in non-phytate phosphorous. In some embodiments, the phytate levels are decreased by at least 90% when compared to a non-transformed parental plant. In another embodiment, the modification includes overexpression, underexpression, antisense modulation, sense suppression, inducible expression, inducible repression, or inducible modulation of a gene. V.B. 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 forthe presently disclosed subject matter are selected in one embodiment from the list of agronomically important target crops set forth hereinabove. The expression of a gene ofthe 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. 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 i 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 improved nutritional value, tolerance of pests, herbicides, or abiotic stress, 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). V.C. Seed Production Some embodiments ofthe 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 as disclosed herein above, the isolated nucleic acid encoding a polypeptide as disclosed herein above. In some embodiments the nucleic acid comprises one of: (a) a nucleic acid molecule encoding a polypeptide comprising an amino acid sequence of one of even numbered SEQ ID NOs: 2-10 or a polypeptide at least 40% identical to even numbered SEQ ID NOs: 2-10 and having inositol phosphate kinase activity; (b) a nucleic acid molecule comprising a nucleic acid sequence of one of odd numbered SEQ ID NOs: 1-9; (c) a nucleic acid molecule that has a nucleic acid sequence that is substantially identical to the nucleic acid sequence of the nucleic acid molecule of (a) or (b); (d) a nucleic acid molecule that hybridizes to (a) or (b) under stringent hybridization conditions; (e) a nucleic acid molecule comprising a nucleic acid sequence complementary to (a); and (f) a nucleic acid molecule comprising a nucleic acid sequence that is the full reverse complement of (a). Embodiments of the presently disclosed subject matter also relate to seed and isolated products produced by expression of an isolated nucleic acid comprising one of: (a) a nucleic acid molecule encoding a polypeptide comprising an amino acid sequence of one of even numbered SEQ ID NOs: 2-10 or a polypeptide at least 40% identical to even numbered SEQ ID NOs: 2-10 and having inositol phosphate kinase activity; (b) a nucleic acid molecule comprising a nucleic acid sequence ofone of odd numbered SEQ ID NOs: 1-9; (c) a nucleic acid molecule that has a nucleic acid sequence that is substantially identical to the nucleic acid sequence ofthe nucleic acid molecule of (a) or (b); (d) a nucleic acid molecule that hybridizes to (a) or (b) under stringent hybridization conditions; (e) a nucleic acid molecule comprising a nucleic acid sequence complementary to (a); and a nucleic acid molecule comprising a nucleic acid sequence that is the full reverse complement of (a). Embodiments further include seed and isolated products resulting from a homozygous disruption of an endogenous gene homologous to a nucleic acid molecule described herein. In one embodiment, the product is produced in a plant. In another embodiment, the product is produced in cell culture. In another embodiment, the product is produced in a cell-free system. In one embodiment, the product comprises an inositol phosphate kinase polypeptide. In another embodiment, the polypeptide is Ipkl or 2 and homologs and orthologs thereof. In another embodiment, the product is a polypeptide comprising an amino acid sequence listed in even numbered sequences of SEQ ID NOs: 2-10, or orthologs thereof, or a polypeptide encoded by a nucleic acid sequence that hybridizes under stringent conditions of hybridization of 45°C in 1 M NaCI, followed by a final washing step at 50°C in 0.1 M NaCI to a nucleotide sequence listed in odd numbered sequences of SEQ ID NOs: 1-9, or a subsequence thereof. Representative orthologs are disclosed in Examples 6-11 herein below. In some embodiments, a polypeptide of the presently disclosed subject matter has or comprises substantial identity (in some embodiments at least about 90% amino acid identity) with a polypeptide as disclosed in Examples 6-11 , over an entire sequence or functional fragment thereof. In one embodiment, the polypeptide functions as an inosoitol phosphate kinase. More specifically, in some embodiments, the polypeptide is an Ipk2 polypeptide and functions as a dual-specificity IP₃/IP₄6-/3-kinase generating IP₅. More specifically, in some other embodiments, the polypeptide is an Ipkl polypeptide and functions as an 1(1 ,3,4,5,6)P5 2-kinase generating phytate (IP₆). 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 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.

VI. Other Applications The presently disclosed subject matter also provides a method for producing a polypeptide as disclosed herein, the method comprising (a) growing cells comprising an expression cassette under suitable growth conditions, the expression cassette comprising a nucleic acid molecule encoding the polypeptide; and (b) isolating the polypeptide from the cells. The presently disclosed subject matter also provides a method for modulating production of phytate in a plant, the method comprising modulating the enzymatic activity of at least one inositol phosphate kinase polypeptide, wherein the polypeptide is Ipkl and/or lpk2. In one embodiment, the method reduces the enzymatic activity. In another embodiment, the enzymatic activity is reduced by inhibiting expression of a functional form of at least one inositol phosphate kinase polypeptide, using techniques as disclosed herein. In some embodiments, inhibiting expression of the functional polypeptide results in a decrease in the phytate content of the plant and/or an increase in the non-phytate phosphorous content, in some embodiments by at least 90%. In one embodiment, the polypeptide expression is modulated in a predetermined location or tissue of a plant. In one embodiment, the location or tissue is selected from the group consisting of epidermis, root, vascular tissue, meristem, cambium, cortex, pith, leaf, flower, seed, and combinations thereof. In one embodiment, the tissue is seed. The presently disclosed subject matter also provides a method of producing a plant with low phytate levels. The method comprises modulating in the plant the enzymatic activity of at least one inositol phosphate kinase polypeptide, wherein the polypeptide is Ipkl and/or 2. In one embodiment, the method reduces the enzymatic activity. In another embodiment, the enzymatic activity is reduced by inhibiting expression of a functional form of at least one inositol phosphate kinase polypeptide, using techniques as disclosed herein, in some embodiments, inhibiting expression of the functional polypeptide results in a significant decrease in the phytate content ofthe plant and/or a significant increase in the non-phytate phosphorous content, in some embodiments by at least 90%. In one embodiment, the polypeptide expression is modulated in a predetermined location or tissue of a plant. In one embodiment, the location or tissue is selected from the group consisting of epidermis, root, vascular tissue, meristem, cambium, cortex, pith, leaf, flower, seed, and combinations thereof. In one embodiment, the tissue is seed. The presently disclosed subject matter also provides a method for decreasing the expression of an isolated nucleic acid molecule as disclosed herein in a plant, the method selected from the group consisting of (a) expressing in said plant a molecule of the presently disclosed subject matter or a portion thereof in "sense" orientation; (b) expressing in said plant a molecule of the presently disclosed subject matter or a portion thereof in "antisense" orientation; (c) expressing in said plant a ribozyme capable of specifically cleaving a messenger RNA transcript encoded by an endogenous gene corresponding to an isolated nucleic acid molecule of the presently disclosed subject matter; (d) expressing in a plant an aptamer specifically directed to a polypeptide encoded by an isolated nucleic acid molecule of the presently disclosed subject matter; (e) expressing in a plant a mutated or a truncated form of an isolated nucleic acid molecule of the presently disclosed subject matter; (f) modifying by homologous recombination in a plant at least one chromosomal copy of the gene corresponding to an isolated nucleic acid molecule of the presently disclosed subject matter; (g) modifying by homologous recombination in a plant at least one chromosomal copy of the regulatory elements of a gene corresponding to an isolated nucleic acid molecule of the presently disclosed subject matter; and (h) expressing in said plant an isolated nucleic acid molecule of the presently disclosed subject matter or a portion thereof in the "sense" and "antisense" orientation. The presently disclosed subject matter also provides a method for screening a plurality of compounds for a modulator of the enzymatic activity of a polypeptide disclosed herein. The method comprises: (f) providing a library of test compounds; (g) contacting an inositol phosphate kinase polypeptide selected from the group consisting of Ipkl and Ipk2 with each test compound; (h) detecting an interaction between a test compound and the inositol phosphate kinase polypeptide; (i) identifying a test compound that interacts with the inositol phosphate kinase polypeptide; and (j) isolating the test compound that interacts with the inositol phosphate kinase polypeptide, whereby a plurality of compounds is screened for a modulator of inositol phosphate kinase polypeptide enzymatic activity. In some embodiments, the test compounds are bound to a substrate. In some embodiments, the test compounds are also synthesized directly on the substrate. In some embodiments, yeast knockouts for Ipk 1 and/or 2 are complemented with plant orthologs of interest. Yeast strains used rely on the plant orthologs to survive. As such, test compounds can be screened as Ipk inhibitors by contacting the compound with the yeast knockouts compelemtned with the Ipk of interest. Compounds that inhibit the Ipk of interest will adversely affect the yeast. In some embodiments, the modulator of the inositol phosphate kinase polypeptide enzymatic activity is an antagonist of the inositol phosphate kinase polypeptide enzymatic activity. An antagonist is a compound that decreases the natural biological functions of the enzymes. Potential antagonists include small organic molecules, peptides, polypeptides and antibodies that bind to a polypeptide of the invention and thereby inhibit or extinguish its activity. Potential antagonists also may be small organic molecules, a peptide, a polypeptide such as a closely related protein or antibody that binds the same sites on a binding molecule, such as a binding molecule, without inducing phytate biosynthetic enzyme-induced activities, thereby preventing the action ofthe enzyme by excluding the enzyme from binding. Potential antagonists include a small molecule that binds to and occupies the binding site ofthe polypeptide thereby preventing binding to cellular binding molecules, such as binding molecules, such that normal biological activity is prevented. Examples of small molecules include but are not limited to small organic molecules, peptides or peptide-like molecules.

EXAMPLES The following Examples have been included to illustrate representative modes of the presently disclosed subject matter. In light ofthe 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 in that numerous changes, modification, and alterations can be employed without departing from the spirit and scope of the invention.

Example 1 AtlPKI encodes an IP and IPs 2-kinase In order to identify the Arabidopsis ortholog of yeast Ipkl the Arabidopsis genome database was searched for candidate genes with sequence similarity to the known IP₅ 2-kinases from S. cerevisiae, S. pombe, and C. albicans. E. B. Ives, J. Nichols, S. R. Wente, J. D. York, J. Biol. Chem. 275, 36575 (2000). Three putative IP₅ 2-kinase genes at loci At1 g22100, At5g42810, and At1 g59312, were identified and had regions of similarity to the known human and fungal IP₅-2 kinases and to a rice and maize sequence (Figure 1 B). Of the three Arabidopsis genes, only At5g42810, designated AtlPKI, appeared to be expressed in major tissues throughout the plant (Figure 1C). Expression ofthe other two putative Arabidopsis IPK1 genes could not be detected and there are currently no ESTs available for these in the database, indicating that they are pseudogenes, or are selectively expressed in a spatio-temporal manner thereby limiting there detection by Northern and reverse-transcriptase PCR. To verify that the putative AtlPKI gene encoded a functional IP₅ 2-kinase, purified recombinant Atlpkl was tested in vitro for the ability to phosphorylate [³H]- l(1,3,4,5,6)P₅. As shown in Figure 2A, in the presence of ATP, recombinant Atlpkl completely phosphorylated IP₅ to IP₆. Itwas also found that Atlpkl could phosphorylate l(1 ,3,4,6)P₄ (Figure 2B) and l(1 ,4,5,6)P₄ (Figure 2C) on the D-2 position to produce 1(1 ,2,3,4,6)P₅ and 1(1 ,2,4,5,6)P₅, respectively. In an effort to recapitulate IP₆ synthesis in vitro from an l(1 ,4,5)P₃ precursor, nearly equal amounts of Atlpk2 ? (1.6 pmol) and Atlpkl (1.3 pmol) were initially used, and surprisingly it was found that l(1 ,2,4,5,6)P₅ was the only major product (Figure 3A). Interestingly, the end product of the reaction could be changed to IPβ with different relative doses of Atlpk2/?. For instance, when 2.5 times as much Atlpk2 ? as Atlpkl (3.2 pmol and 1.3 pmol, respectively) was incubated with 1(1 ,4,5)P₃, 1(1 ,2,4,5,6)P₅ formation was diminished and IPe was produced (Figure 3B). When the ratio of Atlpk2R- (8 pmol) to Atlpkl (1.3 pmol) was increased further to approximately 6:1 , the final product was only IPβ (Figure 3C). The differential product formation based on the relative dose of each enzyme may be due to different substrate affinities and rates of catalysis. The K_m of l(1 ,4,5,6)P for Atlpkl (7.6 μM) is approximately equal to its K_m of 1(1 , 3,4,5, 6)P₅ (7.1 μM) and one-half of the K_m value of l(1 ,4,5,6)P₄ for Atlpk2/? (15 μM) (J. Stevenson-Paulik, A. R. Odom, J. D. York, J. Biol. Chem. 277, 42711 (2002)). Since Atlpkl has a higher apparent affinity for 1(1 ,4,5,6)P than does Atlpk2R, this could explain why, when the enzymes are present at nearly equal concentrations, the majority of the 1(1 ,4,5,6)P₄ synthesized by Atlpk2R- is converted to 1(1 ,2,4,5,6)P₅ by Atlpkl . This result also indicates that although Atlpk2 ? performs two phosphorylation events to convert 1(1 ,4,5)P₃ to 1(1 ,3,4,5,6)P₅, it does so in a non-processive manner in which the l(1 ,4,5,6)P₄ intermediate is released in between phosphorylation steps. When the ratio of Atlpk2j-3 to Atlpkl is increased from 1 :1 to 6:1 , Atlpk2β appears to out-compete Atlpkl for 1(1 ,4,5,6)P₄ phosphorylation and produces 1(1 ,3,4,5,6)P₅ which is rapidly converted to IP₆ by Atlpkl . Another explanation for the dose-dependent changes in end-product formation is that 1(1 ,2,4,5,6)P₅ inhibits Atlpk2R- and this can be overcome with an increased dose of Atlpk2 ? to the reaction. Together these data indicate that IP₆ can be synthesized from an 1(1 ,4,5)P3 precursor by the activity of just two Arabidopsis gene-products and that the relative dose of each enzyme could influence the major IP formed. Additionally, it has been found that, along with the activity of a previously characterized 1(1 ,3,4)P₃5/6-kinase (M.P. Wilson, P.W. Majerus, Biochem. Biophys. Res. Commun., 232, 678-681 (1997); M. P. Wilson, P. W. Majerus. J. Biol. Chem. 271 , 11904 (1996)), these enzymes can convert 1(1 ,3,4)P₃ to IP₆ in vitro (Figure 3D) and that these three gene-products can synthesize up to 7 different IP species as outlined in Figure 1A. Example 2 Atlpkl is necessary for IP₆ synthesis in vivo To study the effect of a loss of AtlPKI function on IPβ production in the plant, publicly available populations of Arabidopsis T-DNA insertional mutants were searched for a disruption of AtlPKI, and identified a mutant (SALK_065337; designated here as atipk1-1) from the Salk Institute Genome Analysis Laboratory (SIGnAL) population of mapped insertions, with an insertion 77 nucleotides upstream of the stop codon in the last exon ofthe open reading frame (Figure 4A). The disruption causes a greater than 70% reduction in transcription of the AtlPKI gene as indicated by Northern blot analysis (Figure 4B). Southern blot analysis revealed that there were at least three T-DNA insertions in tandem in the same site of the AtlPKI gene (Figure 4C). To assess the effect of the disruption of AtlPKI expression on IPβ synthesis, IP synthesis in fourth generation (T₄) seed ofthe T-DNA transformed line was monitored. Seeds were germinated in liquid Murashige and Skoog (MS) media containing 400 μCi/ml [³H]-myo-inositol. The soluble inositol phosphates were harvested from 6 day- old seedlings and analyzed by Partisphere SAX HPLC. Underthese conditions, the IP₆ content of wild type seedlings was 9.6% ofthe total labeled IP species. Conversely, the IP₆ content of atipk1-1 seedlings was 0.71% of the total labeled IP species, representing a 93% reduction from wild type (Figure 5A). The decrease in IP₆ was accompanied by a corresponding accumulation of IP5 (3.4% of total), IP₄ (1.3% of total) and IP₃(1.3% of total) species, which are undetectable in wild type seedlings. Of interest, the 1(1 ,2,4,5,6)P₅ peak was no longer present in the atipk1-1 seedlings, which is consistent with the in vitro data that this IP is AtlPKI -dependent. To determine if there is an effect of the loss-of-fu notion of the AtlPKI gene on inositol phosphate production in seed development, the siliques of T atipk1-1 plants were labeled and compared to wild type. Similar to seedlings, there was an 86% reduction of IP₆, a loss of 1(1 ,2,4,5, 6)P₅, and a correlative increase in IP₅, IP₄ and IP₃ in atipk1-1 as compared to wild type siliques (Figure 5B). To take a closer look at individually developing seeds, the immature seeds from the labeled siliques were dissected and the IP profile was analyzed (Figure 5C). The reduction of IPβ was 90% in atipk1-1 as compared to wild type. In performing these labeling studies, it was found that although IPβ synthesis occurred in the silique tissue, the incorporation of [³H]- inositol into IP₆ was barely detectable in the developing seeds of wild type and atipk1-1 until the final stages of embryogenesis, in the late bent-cotyledon stage (Figure 6), just prior to desiccation where the majority of the endosperm has been consumed and the bulk of the developing seed is comprised of the embryo itself. This could indicate that the majority of IP₆ synthesis occurs in the last stages of seed development, or it could indicate that inositol uptake into the seed is not efficient until the siliques and seeds are developmentally mature. To identify the species of IP and IP₅ that accumulates in the ipk1-1 mutant, in vitro kinase experiments were performed with recombinant Atlpkl , Atlpk2, and 1(3,4,5, 6)P 1 -kinase to determine if the peaks were substrates for these kinases. As indicated in Figure 7A, the IP₅ peak is completely phosphorylated by Atlpkl , indicating that it is 1(1 ,3,4,5,6)P₅. In this reaction the IP peak was also completely phosphorylated by Atlpkl , indicating that it is an IP with a 2-position hydroxyl. Active Atlpk2/? was unable to phosphorylate the IP₃, IP₄, or IP₅ peaks in the extract indicating that the IP₃ is not 1(1 ,4,5)P₃, and that the IP is not 1(1 ,4,5,6)P₄, 1(1 ,3,4,6)P₄, or 1(1 ,3,4,5)P₅. However, the 1(3,4, 5,6)P₄ 1-kinase was able to phosphorylates the majority of the IP₄ peak, indicating that the peak is mostly 1(3,4, 5,6)P₄ (Figure 7B). The nature of the IP₃x species was not identified, other than to determine that it is not l(1 ,4,5)P₃. The accumulated 1(3,4, 5, 6)P could result from the build-up of IP₆ precursors due to a change in equilibrium, or the dephosphorylation ofthe accumulated 1(1 ,3,4,5,6)P₅ by an IPs 1-ptase which has been previously described to exist in other systems (M. W. Ho, X. Yang, M. A. Carew, T. Zhang, L. Hua, Y. U. Kwon, S. K. Chung, S. Adelt, G. Vogel, A. M. Rileey, B. V. Potter, S. B. Shears. Curr Biol. 12, 477 (2002)) and whose cognate gene has been identified in Arabidopsis (M.P. Wilson, P.W. Majerus, Biochem. Biophys. Res. Commun., 232, 678-681 (1997)). To measure mass levels of IPs in mature, desiccated seeds, a non-radioactive method to analyze bulk seed extracts was used. As indicated in Figure 8, IP₆ comprises 100% of the detected IPs in wild type mature seeds and equals approximately 22.3 +/- 0.4 nmol IPβ/mg seed. By comparison, the atipk1-1 seeds contain 3.9 +/- 0.9 nmol IPβ/mg, 29.7 +/- 0.6 nmol IP₅/mg and 3.3 +/- 0.3 nmol IP₄/mg seed. Additionally, there is a 2-fold increase in the concentration of Pi in the atipk1-1 (15.4 +/- 1.2 nmol/mg) seeds over wild type (7.5 +/- 1.7 nmol/mg). These data reveal that not only are the final seed concentrations of IP₆ reduced by over 80% in mature atipk1-1 seeds, but also that there is a net increase of total inositol phosphates in the mutant. These data were further corroborated by results indicating that recombinant Atlpkl can complement phytate production in Ipkl null yeast.

Example 3 atipk1-1 has conditional growth effects and intracellular Pi levels To assess the effect of a disruption of IP₆ synthesis on plant growth, seeds were germinated in a mixture of sand and vermiculite, watered daily with half-strength Hoagland's solution, and monitored the growth over the life cycle of the plant. No discernible differences could be seen in the timing of germination or in the appearance of the cotyledons of wild type, heterozygotes, or homozygotes for the T-DNA insertion. After three weeks under these conditions, the rosette leaves of the atipk1-1 plants became abaxially curled and this epinastic phenotype was more pronounced at four weeks where the leaves appeared to have necrotic margins and were approximately a third of the size of wild type and heterozygotes. This growth phenotype as well as the IP production was rescued by constitutive expression of AtlPKI (Figures 9A and 9B). Because the phenotype appeared to be the result of some toxicity from the Hoagland's solution, each nutrient was systematically removed from the solution and it was found that only the reduction of phosphate could alleviate the leaf epinasty and necrosis. At Pi concentrations below 1 mM, the leaves of the atipk1-1 mutants were not epinastic and the mass differential between the wild type and atipk1-1 leaves was reduced. At Pi concentrations of 1 mM or higher, the leaves of the atipk1-1 plants were epinastic and were one-third to one-quarter the size of wild type (Figure 10A). The leaf epinasty also corresponded with an increased intracellular concentration of Pi in the atipk1-1 mutants (Figure 10B). Thus, the atipk1-1 plants are unable to maintain normal intracellular phosphate concentrations and appear to suffer from phosphate toxicity when administered phosphate levels that are typically optimal for plant growth. This increase in leaf intracellular phosphate cannot be explained by a redistribution of the phosphate from the IP₆ pool to the inorganic phosphate pool. In wild type leaves, one finds that Pi concentrations are typically 5-10 nmol/mg fresh weight while the IP₆ concentration is less than 0.250 nmol/mg. This difference represents at least a 20 to 40-fold higher level of Pi than IP₆ in leaves, which would indicate that the contribution of the redistributed phosphate that resulted from the change in IPβ, IPs and IP concentrations in atipk1-1 would be insignificant and would not account for a 2 to 3-fold increase in intracellular Pi levels. This argument suggests that the altered concentrations of IP₆, IPs, and/or IP₄ in the atipk1-1 mutant could cause an aberration in signaling that disrupts normal phosphate metabolism, such as uptake, distribution, or retention.

Example 4 atipk1-1 roots have a decreased sensitivity to environmental phosphate Because the atipk1-1 mutant appears to have altered phosphate homeostasis, it was tested to see if it also had an altered sensitivity to extracellular Pi levels. Plants respond to a sensed change in phosphate by increasing or decreasing the surface area of the roots to alter uptake. One of the ways that a change in surface area is achieved is by altering the length ofthe root hairs. Thus, for instance, when phosphate is limiting, Arabidopsis seedlings produce longer root hairs to scavenge more Pi (Lopez-Bucio et al., 2003). Seeds from wild type, atipk1-1, and atipk1-1 plants transformed with pAtlPKI were plated onto MS agar containing variable concentrations of Pi and grown in a vertical position under constant light. After 12 days, the roots were imaged and the root hair length was measured. It was found that the atipk1-1 roots had longer root hairs than wild type at lower concentrations of Pi and a decreased ability to sense the increase in Pi as indicated by the inability to form shorter root hairs at higher Pi concentrations (Figure 11 ). The Pi insensitive root hair growth could be rescued in the atipk1-1 mutant by complementation of AtlPKI, indicating that the effect is due to the loss of AtlPKI. This result suggests an inability of the mutants to sense the extracellular phosphate levels as readily as wild type.

Discussion of Examples 1-4 Given the negative effect of phytate accumulation in seeds on animal nutrition and the environment and the general lack of understanding of its role in plant biology, it is of great interest to delineate phytate biosynthesis and function. The results of Examples 1-4 support the notion that there is a connection between phosphate homeostasis and inositol phosphate production. Multiple low-phytate mutants have been generated by random mutagenesis in various crop plants and these mutants have a reported molar equivalent increase of Pi that compensates for the loss of phosphate from the IP pool. S. Larson, K. Young, A. Cook, T. Blake, V. Raboy, Theor. Appl. Genet, 97, 141-146 (1998); F. Hatzack, K.S. Johansen, S.K. Rasmussen, J. Agric. Food Chem. 48, 6074-6080, (2000); S. Larson, J. Rutger, K. Young, V. Raboy, Crop Sci. 40, 1397-1405, (2000); J. R. Wilcox, G. S. Premachandra, K. S. Young, V. Raboy, Crop Sci. 40, 1601 (2000); W. D. Hitz, T. J. Carlson, P.S. Kerr, S. A. Sebastian, Plant Physiol. 128, 650 (2002); M. S. Otegui, R. Capp, L. A. Staehelin, The Plant Cell 14, 1311 (2002). The present data differ from these studies in that even though there is a net increase of IPs in the atipk1-1 mutant, there is still an increase in Pi, which may indicate that perturbation ofthe IP₄, IP₅, and/or IP₆ levels causes a defect in phosphate metabolism and signaling resulting in a detrimental effect on growth when the plants are provided with adequate levels of environmental phosphate. The Ipkl and 2 double knockout mutants described herein bleow further reduces phytate to 7% that ofthe wild- type organisms, and further, other inositol phosphates do not accumulate either. IPβ has been implicated in the ABA-induced regulation of stomatal pore closure, thus controlling water conservation in leaves (J. D. York, A. R. Odom, R. Murphy, E. B. Ives, S. R. Wente, Science 285, 96 (1999)). It was tested if the atlpkl -1 leaves had altered guard cell responses to ABA or increased water loss due to improper regulation of stomatal closure, but no differences between wild type and the mutant were found. This either indicates that I P₆ is not a mediator of this process or that the residual 10% of IP₆ in the mutant cells is adequate to maintain this signaling pathway. IP₆ was shown to be necessary for efficient mRNA export in S. cerevisiae (Y. Feng, S. R. Wente, P. W. Majerus, Proc. Natl. Acad. Sci. USA 98, 875 (2001 )) and human embryonic cells (X. Shen, H., Xiao, R. Ranallo, W-H., Wu, C, Science 299, 112 (2003)) and to be associated with the mammalian DNA-PK complex as a necessity for the non- homologous end-joining activity of the complex (L. A. Hanakahi, S. C. West, Embo J. 21 , 2038 (2002); Y. Ma, M. R. Lieber. J. Biol. Chem. 277, 10756 (2002); R. F. Irvine, M. J. Schell. Nature Reviews 2, 327 (2001)). Since some of these functions appear to be conserved across species, the atipk1-1 plants might suffer from a defect in mRNA translocation and/or an inability to repair DNA damage, although altered mRNA export or any significant differences in the sensitivity of atipk1-1 seed germination or seedling growth to methyl methane sulfonate, a strong mutagen that induces double strand breaks, were not detected. The effect of the AtlPKI loss-of-f unction on vegetative growth could be the result of a specific defect such as an increased cellular Pi concentration, or a pleiotropic one such as altered concentrations of multiple IP species, each of which might have specific regulatory functions. For instance, the phenotype might not be due to the loss of IP₆, but actually a loss of 1(1 ,2,4,5,6)P₅ or another IP either produced by Atlpkl or an IP for which IP₆ is a precursor, such as a pyrophosphorylated inositol species. Additionally, since IP₃x, 1(3,4, 5,6)P₄, and l(1 ,3,4,5,6)P₅, accumulate in these tissues the aberrant growth of the mutant plants could be due to the gain-of-function of these IP species. Hence, one hypothesis is that the perturbation of synthesis of higher IPs in the atipk1-1 mutant causes an uncontrolled response to environmental phosphate. Also, proper control of transcription and chromatin remodeling of the PH05 locus depends on IPK2, the kinase that generates IP₄ and IP₅ in yeast (L. A. Hanakahi, M. Barlet-Jones, C. Chappell, D. Pappin, S. C. West, Cell 102, 721 (2000)). PH05 is induced during phosphate starvation and encodes a secreted acid phosphatase that hydrolyzes phosphate from extracellular organic sources. Thus, a link is suggested between the maintenance of intracellular phosphate levels and IP production. Indeed, the present findings support the notion that there is communication between inositol phosphate production and phosphate homeostasis within the cell. The present findings that the IP₆ content in seeds can be reduced by more than 80% without compromising seed development, germination, and seedling development challenges the hypothesis that IPβ itself is an essential nutrient store of phosphate in the seed. Thus, specific inactivation of the IPK1 gene in developing seeds appears to spare the defects in vegetative growth and signaling and thus, this gene is a viable target for engineering low-phytate crops to enhance the nutritional value of feed grains and reduce phytate-induced phosphorus pollution.

Materials and Methods for Examples 1-4 Inositol phosphate kinase assays - Unlabeled IPs were purchased from Cell Signals, Inc. Tritiated [³H]-I(1 ,4,5)P₃ was purchased from Perkin Elmer Life Sciences. ³H-I(1 ,3,4,6)P₄was synthesized as previously described (B. Q. Phillippy, A. H. Ullah, K. C. Ehrlich. J. Biol. Chem. 269, 28393 (1994)). ³H-(1 ,4,5,6)P₄ was synthesized by incubating 10 μM [³H]-I(1 ,4,5)P₃ with 1.6 pmol GST-Atlpk2R for 10 min at 37°C. [³H]- 1(1 ,3,4,5,6)P₅ was synthesized by incubating 10 μM [³H]-I(1 ,4,5)P₃ with 1.6 pmol GST- Atlpk2/? for 30 min. The reaction was stopped by heat-inactivation at 100°C for 1 min. 1.3 pmol Atlpkl was then incubated with 10 M [³H]-I(1 ,3,4,6)P₄, [³H]-I(1 ,4,5,6)P₄₎ and [³H]-I(1 ,3,4,5,6)P₅ for 30 min at 37°C in a buffer containing 50 mM Hepes, pH 7.5, 50 mM KCI, 2 mM ATP, 10 mM MgCI₂. The reaction products were separated and analyzed by HPLC over a Partisphere strong anion exchange (SAX) column and a linear gradient from 10 mM to 1.7 M NH H₂P0₄ (pH 3.5) over 12 min, followed by elution for 25 min with 1.7 M NH H₂P0 . IPs were identified based on comparison to the elution of known IP species. Semi-quantitative RT-PCR. This was performed exactly as previously described (Stevenson-Paulik et al., 2002). The primers used were designed to amplify the 5' half of the open reading frame (kb) of AtlPKI and the entire open reading frame of ACT2 (kb) (GenBank accession U41998). AtlPKI primers used were constructed as follows. AtlPKI sense primer: 5' GGA GAT GAT TTT GGA GGA GAA AGA TGC AT 3' AtlPKI antisense primer: 5' ATG CAT TTT GAA ACG GCT TAC GCT TGT TTT G 3' ACT2 sense primer: 5'ATG GCT GAG GCT GAT GAT ATT CAA C 3' ACT2 antisense primer: 5' TGT GAA CGA TTC CTG GAC CTG CCT C 3' fHj myo-inositol labeling of Arabidopsis seedlings and siliques. Individual wild type and atipk1-1 seeds were stratified at 4°C for 2 days and then germinated in 25-50 μl liquid Murashige and Skoog (MS) salts media with 0.4 mCi/ml [³H]myo-inositol for 6 days at 20°C and constant light. The seedlings were washed twice with cold dH₂0 and then disrupted by agitation twice for 2 min with acid-washed glass beads in the presence of 100 μl 0.5 N HCI, 125 μl 2M KCI, 125 μl CHCI₃, 372 μ\ CHCI₃:MeOH (1 :2). Disrupted tissue was centrifuged for 5 min and the soluble layer was removed and analyzed by Partisphere SAX HPLC as described above. Siliques were labeled by standing vertically in tubes containing 50 μl MS salts and 100μCi [³H] myo-inositol for 2 days at 20°C, constant light. IPs were harvested either from whole siliques or from individually dissected developing seeds exactly as described for seedlings. Non-radioactive, mass seed IP analysis. For each sample, approximately 50 μl seeds were weighed and 10 volumes of 0.4 N HCI and 50 μl acid-washed glass beads (425-600 microns) were combined for pulverization by a "bead beater" for 4 minutes. The seeds were then boiled for 5 minutes and beaten again for 4 minutes. The seed extract was then centrifuged for 10 minutes at maximum speed and the supernatant was frozen on dry ice, thawed to room temperature and centrifuged again. The supernatant was loaded onto an lonPac AS7 anion exchange column (Dionex, Sunnyvale, California, United States of America) equilibrated with 10 mM methyl piperazine pH 4.0 (Buffer A). The IPs were eluted with a linear gradient from 0 to 100% buffer B (1 M NaN0₃ pH 4.0) with a flow rate of 0.3 ml/min using an HP190 pump. Eluate was mixed with color reagent (0.015% [w/v] FeCIs: 0.15% [w/v] sulfosalicylic acid) and detected with a photodiode array detector at an absorbance of 550 nm. IPIant growth conditions. Plants were grown in a mixture of vermiculite and sand in a controlled growth chamber with constant temperature of 21 °C. The plants were exposed to 14 hr light (135-150 μmol/m^"2 s^"1) and 10 hr dark cycles and were watered with half-strength Hoagland's solution every morning and with dH₂0 every evening. Pi assay. Leaf samples were homogenized in 0.4 N HCI, centrifuged, and the supernatant was incubated 1 :1 with color reagent (3 M H₂S0₄:2.5%NH₄Mo: 10% ascorbate: dH₂0; 1 :1 :1 :1). A standard curve was generated with K₂HP0 and found to be linear between 0-100 nmol phosphate. Example 5 Double knockout eliminates most IP production including IP4, IP5 and IP6 This Example describes an ipk1/ipk2 beta double knockout plant. The double mutant grows about the same as the single mutant discloshed herein above. Thus, the seed specific knockdown of all IPs is not detrimental to seed yield and plant growth. The term "seed specific" is used because analysis of the plant tissue from the atlpk2 beta mutant shows no defect in IP metabolism whereas the seeds do. Further the ipkl ipk2 beta double mutant appears to synthesize IPs except for IP5 in the tissue. The growth issue and yield problems of ipkl mutant and ipk1/ipk2 beta double mutant plants ar expected to be corrected with the tissue (but not seed) expression of ipkl . Complementation of the ipkl mutant plant with pBART Ipkl complements the growth. The ipk1/ipk2 beta double mutant can be complemented with Ipkl under one or more tissue specific promoters as disclosed herein above, e.g. one or more promoters that can drive expression in one or more tissues except seeds. Figure 12A is a schematic of T-DNA insertion into atlpk2β locus. AtlPK2 is a single exon and the T-DNA inserts at nucleotide 400 ofthe 901 nucleotide open reading frame (ORF). Figure 12B depicts a Northern blot analysis of mRNA isolated from wild- type, atipk2β -1 , or atipklβ -1 plants confirms that the T-DNA insertion disrupts over 90% of the expression of aflpk2R levels. Probing for aflpk2R- shows that its expression is unaffected by the loss of atlpMβ and atl K2β. Figure 13 is a schematic of IP determination methods used. Using non-radioactive method of measuring Phytate (IP₆) levels in mature seeds derived from wild-type or kinase mutant plants (single deletion) shows that both Ipk2 and Ipkl result in lowered phytate in seeds. See Figures 14A and 14B. Given there are two genes encoding lpk2 it is likely that the alpha gene partially compensates for the loss of beta. A double knockout of Ipk2 alpha and beta should ablate IP₃, IP₄, IP₅ and IPβ synthesis. IP analysis from the Ipkl and Ipk2 beta double mutant is shown in Figures 14A and 14B. The ipk1/ipk2 double mutant plants are viable, are partially growth compromised and develop normally as compared to wild type. See Figure 15, where this is shown as ipk1-1 complemented with plpkl . Restoration of seed excluded expression of plPKI with a specific promoter will likely restore full growth of the double mutant. Total seed phytate (IP₆) levels calculated from wild-type, ipkl , ipk2, and ipkl ipk2 double mutant seeds are 100%, 12%, 66% and 7%, respectively. See Figure 16. HPLC analysis of total seed extracts is listed and quantified in Figures 17A and 17B. It is noted that deletion of both ipkl and ipk2 results in lowering total IP production, specifically IP₅ and nearly ablating IPβ. Additionally, IP2, IP3 and IP4 levels do not accumulate to high levels of phytate seen in wild-type seeds. The reduction of phytate and total IP molelecules is greater than in the ipk1-1 mutant alone. Wild-type HPLC analysis is provided in accordance with techniques disclosed herein above. Example 6 Atlpk2 beta TBLASTN of rice (Oryza sativa) The amino acid sequence of the A. thaliana Atlpk2 beta gene product was compared to the Oryza sativa genomic sequence database available at the website of The Institute for Genomic Research using the program TBLASTN. The results are presented in Figure 18. An unspliced genomic clone identified as "9630.t02959" in the Oryza sativa genomic database had high homology (Identities = 153/297 (51 %), Positives = 195/297 (65%), Frame = +1). Example 7 Atlpk2 beta TBLASTN of Maize [Zea mays) The amino acid sequence of the A. thaliana Atlpk2 beta gene product was compared to a Zea mays genomic sequence database available at the website of The Institute for Genomic Research using the program TBLASTN. The results are presented in Figure 19. Several genomic clones were identified as showing high homology to the A. thaliana Atlpk2 beta gene product. These included several translational frames of clones AZM4_23277 (Figure 19A), AZM4_23275 (Figures 19B-D), and AZM4_23276 (Figures 19E-G). Example 8 Atlpk2 beta TBLASTN of All Plant Sequences at GENBANK^® The amino acid sequence of the A. thaliana Atlpk2 beta gene product was compared to the GENBANK^® sequence database available at the website of The National Center for Biotechnology Information using the program TBLASTN. The results are presented in Figure 20. Several homologous sequences were identified, including Accession No. AB010069.1 (Figure 20A), Accession No. AL163912.1 (Figure 20B), and Accession No. AP006476.1 (Figure 20C). Example 9 Atlpk2 alpha TBLASTN of Maize (Zea mays) at TIGR The amino acid sequence of the A. thaliana Atlpk2 alpha gene product was compared to a Zea mays genomic sequence database available at the website of The Institute for Genomic Research using the program TBLASTN. The results are presented in Figure 21. This BLAST search identified three genes encoding Ipk2 orthologs - note all three have PxxxDxKxG motif (shaded in Figure 21 ) found in all Ipk2 family members. Actual

DNA sequence and homology are shown separately for all three loci in Figure 21. These include subsequences of AZM4_23277 (Figures 21 A and B), AZM4_23275

(Figures 21 C and D), and AZM4_23276 (Figures 21 E-G). Example 10 Atlpk2 alpha TBLASTN of All Plant Sequences at GENBANK^® The amino acid sequence of the A. thaliana Atlpk2 alpha gene product was compared to the GENBANK^® sequence database available at the website of The National Center for Biotechnology Information using the program TBLASTN. The results are presented in Figure 22. Several homologous sequences were identified, including Accession No. AL163912 (Figure 22A), Accession No. AB010069.1 (Figure 22B), and Accession No. AP006476.1 (Figure 22C). Example 11 TBLASTN of all Plant genomes at GENBANK^® This Example pertains to a TBLASTN search of all plant genomes (excluding

Arabidopsis). The search obtained 1 hit from rice genome (Oryza sativa) - 1 gene product found with 42% overall identity to Atlpkl beta protein. The gene comprises at least 7 exons as outlined in Figure 23A. The sequences comprise signature motifs of Ipkl protein sequences. Hits from wheat are also shown in Figure 23. Figures 23B-F depict alignments ofthe Atlpkl beta protein sequence with various translational frames of GENBANK^® Accession No. AL606608.3. Figures 23G and H depict alignments ofthe Atlpkl beta protein sequence with various translational frames of GENBANK^® Accession No. BE498028.1 and BQ169513.1 , clones from a wheat cDNA library. REFERENCES The references listed below as well as all references cited in the specification are incorporated herein by reference to the extent that they supplement, explain, provide a background for or teach methodology, techniques, and/or compositions employed herein.

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

Claims

CLAIMS What is claimed is: 1. An isolated nucleic acid molecule encoding an inositol phosphate kinase polypeptide, wherein the nucleic acid molecule is selected from the group consisting of: (a) a nucleic acid molecule encoding a polypeptide comprising an amino acid sequence of one of even numbered SEQ ID NOs: 2-10 or a polypeptide at least 40% identical to even numbered SEQ ID NOs: 2-10 and having inositol phosphate kinase activity; (b) a nucleic acid molecule comprising a nucleic acid sequence ofone of odd numbered SEQ ID NOs:1-9; (c) a nucleic acid molecule that has a nucleic acid sequence at least 90% identical to the nucleic acid sequence of the nucleic acid molecule of (a) or (b); (d) a nucleic acid molecule that hybridizes to (a) or (b) under stringent hybridization conditions; (e) a nucleic acid molecule comprising a nucleic acid sequence complementary to (a); and (f) a nucleic acid molecule comprising a nucleic acid sequence that is the full reverse complement of (a). 2. A vector, comprising the nucleic acid of claim 1. 3. An expression cassette, comprising the nucleic acid of claim 1 operably linked to a promoter. 4. The expression cassette of claim 3, wherein the promoter is a plant promoter. 5. The expression cassette of claim 3, wherein the promoter is a constitutive promoter. 6. The expression cassette of claim 3, wherein the promoter is a tissue-specific or a cell type-specific promoter. 7. The expression cassette of claim 6, wherein the tissue-specific or cell type- specific promoter directs expression of the expression cassette in a location selected from the group consisting of epidermis, root, vascular tissue, meristem, cambium, cortex, pith, leaf, flower, seed, and combinations thereof. 8. The expression cassette of claim 7, wherein the tissue-specific or cell type- specific promoter directs expression of the expression cassette in seed. 9. An isolated inositol phosphate kinase polypeptide having inositol phosphate kinase activity selected from the group consisting of: (j) an amino acid sequence of one of even numbered SEQ ID NOs: 2-10; (k) an amino acid sequence that is at least 40% identical to (a); (I) an amino acid sequence encoded by a nucleotide sequence substantially identical to a nucleotide sequence of one of odd numbered SEQ ID NOs: 1-9; and (m) an amino acid sequence encoded by a nucleic acid molecule capable of hybridizing under stringent conditions to a nucleic acid molecule ofone of odd numbered SEQ ID NOs: 1-9 or to a sequence fully complementary thereto. 10. A method for producing a polypeptide of claim 9, comprising the steps of: (a) growing cells comprising an expression cassette under suitable growth conditions, the expression cassette comprising a nucleic acid molecule of claim 1 ; and (b) isolating the polypeptide from the cells. 11.A transgenic plant cell comprising a homozygous disruption in at least one endogenous inositol phosphate kinase gene homologous to the nucleic acid molecules of claim 1 , wherein the disruption substantially inhibits the expression of a functional inositol phosphate polypeptide. 12. A transgenic plant comprising at least one of the transgenic plant cells of claim 11. 13. The transgenic plant of claim 12, wherein the plant comprises a decreased level of phytic acid when compared to a non-transformed parental plant. 14. The transgenic plant of claim 12, wherein the plant comprises an increased level of non-phytic acid phosphorous when compared to a non-transformed parental plant. 15. The transgenic plant of claim 12, wherein the homozygous disruption is tissue-specific. 16. The transgenic plant of claim 15, wherein the tissue is selected from the group consisting of epidermis, root, vascular tissue, meristem, cambium, cortex, pith, leaf, flower, seed, and combinations thereof. 17. The transgenic plant of claim 16, wherein the tissue is seed. 18. The transgenic plant of claim 17, wherein the seed comprises a decreased level of phytic acid when compared to a seed from a non-transformed parental plant. 19. The transgenic plant of claim 17, wherein the seed comprises an increased level of non-phytic acid phosphorous when compared to a seed from a non-transformed parental plant. 20. The transgenic plant of claim 12, wherein the plant is selected from the group consisting of Arabidopsis thaliana, corn (Zea mays), Brassica sp., alfalfa (Medicago sativa), rice (Oryza sativa ssp.), rye (Secale cereale), sorghum (Sorghum bicolor, Sorghum vulgare), pearl millet (Pennisetum glaucum), proso millet (Panicum miliaceum), foxtail millet (Setaria italica), finger millet (Eleusine coracana), sunflower (Helianthus annuus), safflower (Carthamus tinctorius), wheat (Triticum aestivum), soybean (Glycine max), tobacco (Nicotiana tabacum), potato (Solanum tuberosum), peanut (Arachis hypogaea), cotton, sweet potato (Ipomoea batatus), cassava (Manihot esculenta), coffee (Cofea spp.), coconut (Cocos nucifera), pineapple (Ananas comosus), citrus trees (Citrus spp.), cocoa (Theobroma cacao), tea (Camellia sinensis), banana (Musa spp.), avocado (Persea ultilane), fig (Ficus casica), guava (Psidium guajava), mango (Mangifera indica), olive (Olea europaea), papaya (Carica papaya), cashew (Anacardium occidentale), macadamia (Macadamia integrifolia), almond (Prunus amygdalus), sugar beets (Beta vulgaris), sugarcane (Saccharum spp.), oats, duckweed (Lemna), and barley. 21.The transgenic plant of claim 20, wherein the plant is Arabidopsis thaliana. 22. The transgenic plant of claim 12, wherein the plant is selected from the group consisting of a vegetable, an ornamental, and a conifer. 23. The transgenic plant of claim 22, wherein the vegetable is selected from the group consisting of tomatoes, lettuce, guar, locust bean, fenugreek, soybean, garden beans, cowpea, mungbean, lima bean, fava bean, lentils, chickpea, green bean, lima bean, pea, and members of the genus Cucumis. 24. The transgenic plant of claim 22, wherein the ornamental is selected from the group consisting of impatiens, Begonia, Pelargonium, Viola, Cyclamen, Verbena, Vinca, Tagetes, Primula, Saint Paulia, Agertum, Amaranthus, Antihirrhinum, Aquilegia, Cineraria, Clover, Cosmo, Cowpea, Dahlia, Datura, Delphinium, Gerbera, Gladiolus, Gloxinia, Hippeastrum, Mesembryanthemum, Salpiglossos, and Zinnia, azalea, hydrangea, hibiscus, rose, tulip, daffodil, petunia, carnation, poinsettia, and chrysanthemum. 25. The transgenic plant of claim 22, wherein the conifer is selected from the group consisting of loblolly pine, slash pine, ponderosa pine, lodgepole pine, Monterey pine, Douglas-fir, Western hemlock, Sitka spruce, redwood, silver fir, balsam fir, Western red cedar, and Alaska yellow-cedar. 26. The transgenic plant of claim 11 , wherein the homozygous disruption to the at least one endogenous inositol phosphate kinase gene is to at least a gene encoding an

Ipkl polypeptide and a gene encoding an Ipk2 polypeptide 27. A method of modulating production of phytate in a plant, comprising modulating the enzymatic activity of at least one inositol phosphate kinase polypeptide selected from the group consisting of Ipkl and Ipk2. 28. The method of claim 27, wherein modulating the enzymatic activity comprises reducing the enzymatic activity of the at least one inositol phosphate kinase polypeptide. 29. The method of claim 28, wherein reducing the enzymatic activity of the at least one inositol phosphate kinase polypeptide comprises substantially inhibiting the expression of a functional form of the at least one inositol phosphate kinase polypeptide. 30. The method of claim 29, wherein substantially inhibiting the expression ofthe functional form of the at least one inositol phosphate kinase polypeptide results in at least about a 90% reduction in the phytate content of the plant. 31. The method of claim 29, wherein substantially inhibiting the expression ofthe functional inositol phosphate polypeptide comprises homozygous disruption of an endogenous gene encoding the at least one inositol phosphate kinase polypeptide. 32. The method of claim 31 , wherein disrupting the endogenous inositol phosphate kinase gene comprises stably inserting a foreign DNA element into the endogenous inositol phosphate kinase gene. 33. The method of claim 31 , wherein the foreign DNA element is T-DNA. 34. The method of claim 29, wherein substantially inhibiting the expression ofthe functional inositol phosphate polypeptide comprises post-transcriptionally silencing expression of the at least one inositol phosphate polypeptide. 35. The method of claim 34, wherein post-transcriptionally silencing expression of the functional inositol phosphate polypeptide comprises introducing a ribonucleic acid (RNA) into at least one cell of the plant in an amount sufficient to inhibit expression of the at least one inositol phosphate polypeptide, wherein the RNA comprises a ribonucleotide sequence which corresponds to a coding strand of a gene encoding the at least one inositol phosphate polypeptide. 36. The method of claim 34, wherein post-transcriptionally silencing expression of the functional inositol phosphate polypeptide comprises RNAi. 37. The method of claim 27, wherein the modulating the enzymatic activity of the at least one inositol phosphate polypeptide is a tissue-specific modulating. 38. The method of claim 37, wherein the tissue specifically modulated is selected from the group consisting of epidermis, root, vascular tissue, meristem, cambium, cortex, pith, leaf, flower, seed, and combinations thereof. 39. The method of claim 38, wherein the tissue specifically modulated is seed. 40. The method of claim 27, wherein the at least one inositol phosphate polypeptide is lpk-1 and 2. 41. A method of producing a plant with low levels of phytate comprising modulating in the plant the enzymatic activity of at least one inositol phosphate kinase polypeptide selected from the group consisting of Ipkl and Ipk2. 42. The method of claim 41 , wherein modulating the enzymatic activity comprises reducing the enzymatic activity ofthe at least one inositol phosphate kinase polypeptide. 43. The method of claim 42, wherein reducing the enzymatic activity of the at least one inositol phosphate kinase polypeptide comprises substantially inhibiting the expression of a functional form of the at least one inositol phosphate kinase polypeptide. 44. The method of claim 43, wherein substantially inhibiting the expression ofthe functional form of the at least one inositol phosphate kinase polypeptide results in at least about a 90% reduction in the phytate content of the plant. 45. The method of claim 44, wherein substantially inhibiting the expression ofthe functional inositol phosphate polypeptide comprises homozygous disruption of an endogenous gene encoding the at least one inositol phosphate kinase polypeptide. 46. The method of claim 45, wherein disrupting the endogenous inositol phosphate kinase gene comprises stably inserting a foreign DNA element into the endogenous inositol phosphate kinase gene. 47. The method of claim 45, wherein the foreign DNA element is T-DNA. 48. The method of claim 43, wherein substantially inhibiting the expression ofthe functional inositol phosphate polypeptide comprises post-transcriptionally silencing expression of the at least one inositol phosphate polypeptide. 49. The method of claim 48, wherein post-transcriptionally silencing expression of the functional inositol phosphate polypeptide comprises introducing a ribonucleic acid (RNA) into at least one cell of the plant in an amount sufficient to inhibit expression of the at least one inositol phosphate polypeptide, wherein the RNA comprises a ribonucleotide sequence which corresponds to a coding strand of a gene encoding the at least one inositol phosphate polypeptide. 50. The method of claim 48, wherein post-transcriptionally silencing expression of the functional inositol phosphate polypeptide comprises RNAi. 51. The method of claim 41 , wherein modulating the enzymatic activity of the at least one inositol phosphate polypeptide is a tissue-specific modulating. 52. The method of claim 51 , wherein the tissue specifically modulated is selected from the group consisting of epidermis, root, vascular tissue, meristem, cambium, cortex, pith, leaf, flower, seed, and combinations thereof. 53. The method of claim 52, wherein the tissue specifically modulated is seed. 54. The method of claim 41 , wherein the at least one inositol phosphate polypeptide is lpk-1 and 2. 55. A method of screening a plurality of compounds for a modulator of the enzymatic activity of an inositol phosphate kinase polypeptide, the method comprising: (k) providing a library of test compounds; (I) contacting an inositol phosphate kinase polypeptide selected from the group consisting of Ipkl and Ipk2 with each test compound; (m) detecting an interaction between a test compound and the inositol phosphate kinase polypeptide; (n) identifying a test compound that interacts with the inositol phosphate kinase polypeptide; and (o) isolating the test compound that interacts with the inositol phosphate kinase polypeptide, whereby a plurality of compounds is screened for a modulator of inositol phosphate kinase polypeptide enzymatic activity. 56. The method of claim 55, wherein the test compounds are bound to a substrate. 57. The method of claim 56, wherein the test compounds are synthesized directly on the substrate. 58. The method of claim 55, wherein the modulator of the inositol phosphate kinase polypeptide enzymatic activity is an antagonist ofthe inositol phosphate kinase polypeptide enzymatic activity. 59. An isolated polypeptide having or comprising at least about 90% amino acid identity with a polypeptide as disclosed in Examples 6-11 , over an entire sequence or functional fragment thereof, wherein the polypeptide functions as an inosoitol phosphate kinase. 60. An isolated polynucleotide encoding a polypeptide of claim 59.