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WO2005084331A2 - Établissement de profils du gène de sorgho - Google Patents

Établissement de profils du gène de sorgho Download PDF

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Publication number
WO2005084331A2
WO2005084331A2 PCT/US2005/006729 US2005006729W WO2005084331A2 WO 2005084331 A2 WO2005084331 A2 WO 2005084331A2 US 2005006729 W US2005006729 W US 2005006729W WO 2005084331 A2 WO2005084331 A2 WO 2005084331A2
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Prior art keywords
plant
nucleic acid
polypeptide
nucleotide sequence
expression
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WO2005084331A3 (fr
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Henry T. Nguyen
Joel A. Kreps
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Syngenta Participations AG
Texas Tech University System
University of Missouri Columbia
University of Missouri St Louis
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Syngenta Participations AG
Texas Tech University System
University of Missouri Columbia
University of Missouri St Louis
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8273—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for drought, cold, salt resistance

Definitions

  • the presently disclosed subject matter pertains to nucleic acid molecules from sorghum [Sorghum bicolor (L) Moench] comprising nucleotide sequences that encode polypeptides for drought resistance.
  • the presently disclosed subject matter also relates to methods of using nucleic acid molecules and/or polypeptides from Sorghum spp. in transgenic plants to confer the desired agronomic traits, and to use such nucleic acids to assist germplasm enhancement by breeding.
  • NAD(P) nicotinamide adenine dinucleotide (phosphate)
  • Amino Acid Abbreviations, Codes, and Functionally E ⁇ uivalent 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 GGA; GGC; GGG; GGU Histidine His H CAC; CAU Isoleucine He 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
  • cultivars are needed with altered nutrient composition to enhance human and animal nutrition. To enable more efficient food and feed processing, cultivars can be designed for specific end-uses.
  • genes controlling phenotypic expression of traits of interest will play a role in accelerating development of superior crop germplasm by conventional or transgenic approaches.
  • a number of approaches are available to assist identification of genes playing key roles in expression of agronomically important traits. These include genetics, genomics, bioinformatics, and functional genomics. Genetics is the scientific study of the mechanisms of inheritance. By identifying mutations that alter a pathway or response of interest, classical (or forward) genetics can help to identify the genes involved in these pathways or responses. Genetics is also the central component in improvement of germplasm by breeding. Through molecular and phenotypic analysis of genetic crosses, loci controlling traits of interest can be mapped and followed in subsequent generations.
  • Genomics is the system-level study of an organism's genome, including genes and the corresponding gene products: RNA and polypeptides.
  • genomic approaches have provided large datasets of sequence information from diverse plant species, including full- length and partial cDNA sequences and the complete genomic sequence of a model plant species, Arabidopsis thaliana.
  • the first draft sequence of a crop plant's genome, that of rice (Oryza sativa) has also become available (see Goff et al., 2002).
  • Bioinformatics approaches interface directly with first-level genomic datasets in allowing for the uncovering of sequences of interest by annotative or other approaches. Using, for example, similarity searches, alignments, and phylogenetic analyses, bioinformatics can often identify homologs of a gene product of interest.
  • Functional genomics can be defined as the assignment of functions to genes and their products. Functional genomics draws from genetics, genomics, and bioinformatics to derive a path toward identifying genes important in a particular pathway or response of interest. Expression analysis, for example, uses high density DNA microarrays (often derived from genome-scale organismal sequencing) to monitor the mRNA expression of thousands of genes in a single experiment. Experimental treatments can include those eliciting a response of interest, such as the drought resistance response in plants subjected to low water conditions.
  • mRNA expression levels can be monitored in distinct tissues over a developmental time course, or in mutants affected in a response of interest.
  • Proteomics can also help to assign function by assaying the expression and post-translational modifications of hundreds of polypeptides in a single experiment. Proteomics approaches are in many cases analogous to the approaches taken for monitoring mRNA expression in microarray experiments.
  • Polypeptide-polypeptide interactions can also help to assign polypeptides to a given pathway or response by identifying polypeptides which interact with known components of the pathway or response. For functional genomics, polypeptide-polypeptide interactions are often studied using large-scale yeast two-hybrid assays.
  • Another approach to assigning gene function is to express the corresponding polypeptide in a heterologous host, for example the bacterium Escherichia coli, followed by purification and enzymatic assays of the purified polypeptide.
  • a heterologous host for example the bacterium Escherichia coli
  • the gene can alternatively be either overexpressed (via transgenesis) or underexpressed ("knocked out"), thereby increasing the chances of observing a phenotype linking the gene to a pathway or response of interest.
  • transgenic functional genomics Two aspects of transgenic functional genomics help lend a high level of confidence to functional assignments derived from this approach.
  • phenotypic observations are carried out in the context of the living plant.
  • the range of phenotypes observed can be checked and correlated with the observed expression levels of the introduced transgene.
  • Transgenic functional genomics is especially valuable in improved cultivar development. Only genes that function in a pathway or response of interest and that in addition are able to confer a desired trait-based phenotype are promoted as candidate genes for crop improvement efforts. In some cases, transgenic lines developed for functional genomics studies can be directly utilized in the initial stages of product development. Another approach towards plant functional genomics involves first identifying plant lines with mutations in specific genes of interest, followed by phenotypic evaluation of the consequences of such gene knockouts on the trait under study. Such an approach reveals genes essential for the expression of specific traits. Genes identified through functional genomics can be directly employed in efforts towards germplasm improvement by transgenic approaches as disclosed above, or used to develop markers for identification and tracking of alleles-of-interest in mapping and breeding populations.
  • the presently disclosed subject matter provides nucleic acids and polypeptides from genus Sorghum.
  • the presently disclosed subject matter provides a polypeptide selected from the group consisting of (a) a polypeptide comprising an amino acid sequence encoded by a nucleotide sequence that hybridizes under conditions of hybridization of 45°C in 1 M NaCl, followed by a final washing step at 50°C in 0.1 M NaCl, to a nucleic acid comprising a nucleotide sequence of one of odd numbered SEQ ID NOs: 1-105; and (b) a functional fragment of the polypeptide of (a).
  • the polypeptide is involved in abiotic stress tolerance.
  • the abiotic stress tolerance is drought resistance.
  • the presently disclosed subject matter also provides an isolated nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of (a) a nucleotide sequence encoding a polypeptide as disclosed above, or a fragment, domain, or feature thereof; (b) a sequence fully complementary to (a); and (c) a nucleotide sequence which is the full reverse complement of (a).
  • the presently disclosed subject matter also provides an isolated nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of (a) a nucleotide sequence that hybridizes under conditions of hybridization of 45°C in 1 M NaCl, followed by a final washing step at 50°C in 0.1 M NaCl, to a nucleic acid comprising a nucleotide sequence of one of odd numbered SEQ ID NOs: 1-105; (b) a nucleotide sequence fully complementary to (a); and (c) a nucleotide sequence which is the full reverse complement of (a).
  • the presently disclosed subject matter also provides an expression cassette comprising a promoter operatively linked to one or more of the disclosed nucleic acid molecules.
  • the presently disclosed subject matter provides a recombinant vector comprising the disclosed expression cassette. In some embodiments, the presently disclosed subject matter also provides a cell comprising an expression cassette. In some embodiments, the presently disclosed subject matter provides a transgenic plant comprising the disclosed expression cassette. In some embodiments, the expression cassette is expressed in a tissue selected from the group consisting of the epidermis, root, vascular tissue, meristem, cambium, cortex, pith, leaf, flower, and combinations thereof. In some embodiments, the transgenic plant is a monocot.
  • the monocot is selected from the group consisting of rice, maize, wheat, barley, oats, rye, millet, sorghum, trticale, secale, einkorn, spelt, emmer, teff, milo, flax, gramma grass, Tripsacum, and teosinte.
  • the transgenic plant is selected from the group consisting of rice, wheat, barley, rye, corn, potato, canola, soybean, sunflower, carrot, sweet potato, sugarbeet, bean, pea, chicory, lettuce, cabbage, cauliflower, broccoli, turnip, radish, spinach, asparagus, onion, garlic, eggplant, pepper, celery, squash, pumpkin, cucumber, apple, pear, quince, melon, plum, cherry, peach, nectarine, apricot, strawberry, grape, raspberry, blackberry, pineapple, avocado, papaya, mango, banana, soybean, tobacco, tomato, sorghum and sugarcane.
  • the transgenic plant is sorghum.
  • the plant has altered abiotic stress tolerance.
  • the altered abiotic stress tolerance is enhanced drought resistance.
  • the presently disclosed subject matter also provides a transgenic plant comprising a disclosed nucleic acid molecule. Additionally, the presently disclosed subject matter provides progeny and seed from the disclosed transgenic plants. The presently disclosed subject matter also provides a method for altering an abiotic stress tolerance of a plant, the method comprising expressing in the plant an expression cassette comprising a nucleic acid molecule encoding a disclosed polypeptide.
  • the presently disclosed subject matter also provides a shuffled nucleic acid 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 conditions of hybridization of
  • nucleic acid comprising a nucleotide sequence listed in odd numbered sequences of SEQ ID NOs: 1-105, 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 presently disclosed subject matter also provides a method for producing a polypeptide encoded by a disclosed nucleic acid molecule, comprising the steps of: (a) growing cells comprising an expression cassette under suitable growth conditions, the expression cassette comprising a nucleic acid molecule encoding a disclosed polypeptide; and (b) isolating the polypeptide from the cells.
  • the presently disclosed subject matter also provides a method for decreasing the expression of a disclosed nucleic acid molecule in a plant, the method selected from the group consisting of: (a) expressing in said plant a nucleic acid molecule encoding a disclosed polypeptide or a portion thereof in "sense" orientation; (b) expressing in said plant a nucleic acid encoding a disclosed polypeptide molecule 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 a disclosed nucleic acid molecule; (d) expressing in a plant an aptamer specifically directed to a polypeptide encoded by a disclosed isolated nucleic acid molecule; (e) expressing in a plant a mutated or a truncated form of a disclosed isolated nucleic acid molecule; (f) modifying by homologous recombination in a plant at
  • the presently disclosed subject matter also provides a method for increasing the expression of a disclosed isolated nucleic acid molecule in a plant, the method comprising: (a) inserting into the plant an expression cassette comprising a disclosed isolated nucleic acid molecule; and (b) growing the plant comprising the expression cassette under suitable growth conditions, wherein the expression of the disclosed isolated nucleic acid molecule is increased.
  • the presently disclosed subject matter also provides a method for enhancing the drought resistance of a plant, the method comprising: (a) inserting into the plant an expression cassette comprising at least one disclosed isolated nucleic acid molecule; and (b) growing the plant comprising the expression cassette under suitable growth conditions, wherein the drought resistance of the plant is enhanced.
  • the presently disclosed subject matter also provides an antibody that specifically binds to a disclosed polypeptide.
  • the presently disclosed subject matter also provides a method for altering the expression of a disclosed polypeptide in a plant, the method comprising expressing an expression cassette encoding a disclosed polypeptide in the plant.
  • the polypeptide is expressed in a predetermined location or tissue of a plant.
  • 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.
  • the presently disclosed subject matter also provides an isolated product from a.
  • nucleic acid comprising a nucleotide sequence selected from the group consisting of (a) a nucleotide sequence that hybridizes under conditions of hybridization of 45°C in 1 M NaCl, followed by a final washing step at 50°C in 0.1 M NaCl, to a nucleic acid comprising a nucleotide sequence listed in odd numbered sequences of SEQ ID NOs: 1-105, or a fragment, domain, or feature thereof;
  • the presently disclosed subject matter also provides a method for producing a recombinant polypeptide, the method comprising (a) growing recombinant cells comprising a nucleic acid construct under suitable growth conditions, the construct comprising an expression vector and a nucleic acid comprising a nucleotide sequence selected from the group consisting of: (i) a nucleotide sequence that hybridizes under conditions of hybridization of 45°C in 1 M NaCl, followed by a final washing step at 50°C in 0.1 M NaCl, to a nucleic acid comprising a nucleotide sequence listed in odd numbered nucleotide sequences of SEQ ID NOs: 1-105; and (ii) a subsequence of (i); and (b) isolating from the recombinant cells the recombinant polypeptide expressed there
  • the expression vector comprises one or more elements selected from the group consisting of a promoter- enhancer sequence, a selection marker sequence, an origin of replication, an epitope tag-encoding sequence, an affinity purification tag-encoding sequence, a polyamino acid binding substance, and chitin-binding domain, and combinations thereof.
  • Figures 1A and 1 B depict a comparison of the transcriptional response of the parental plant lines to dehydration stress over time.
  • Figure 1A depicts the number of transcripts induced in the parental lines at various time points
  • Figure 1 B depicts the number of transcripts repressed at these same time points.
  • Figures 2A-2E depict graphical representations of the expression patterns presented in Table 6.
  • Figure 2A-2E the first four points on the x-axis correspond to 7, 14, 21 , and 7RW treatments for Tx7000, and points 5 to 8 corresponds to 7, 14, 21 , and 7RW treatments for B35 respectively.
  • the Y-axis corresponds to the level of expression.
  • Figure 2A depicts a graphical representation of an expression pattern that is seen in a transient and early response in B35.
  • Figure 2B depicts a graphical representation of an expression pattern that is seen in a sustained response to dehydration.
  • Figure 2C depicts a graphical representation of an expression pattern that is seen in a transient and early response in Tx7000.
  • Figure 2D depicts a graphical representation of an expression pattern that is seen in a sustained response to dehydration.
  • Figure 2E depicts a graphical representation of a bi-modal expression pattern that is seen in certain genes that are induced in B35 at 7 and 21 days after drought.
  • Odd numbered SEQ ID NOs: 1-105 are nucleotide sequences from Oryza sativa that have been identified using the methods and compositions disclosed herein. Even numbered SEQ ID NOs: 2-106 are 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.
  • SEQ ID NO: 107 is the amino acid sequence of the V5 epitope from paramyxovirus.
  • SEQ ID NO: 108 is the amino acid sequence of an epitope tag.
  • 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.
  • 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.
  • functional genomics any or all of these approaches are utilized, often in concert, to assign functions to genes across any of a number of organismal phenotypes.
  • these different methodologies can each provide data as evidence for the function of a particular gene, and that such evidence is stronger with increasing amounts of data used for functional assignment: in some embodiments from a single methodology, in some embodiments from two methodologies, and in some embodiments from more than two methodologies.
  • 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.
  • a datum of biochemical, genetic, or molecular genetic evidence is considered stronger than a datum of bioinformatic or gene expression evidence.
  • a single datum from a single methodology can differ in terms of the strength of the evidence provided by each distinct datum for the assignment of the function of these different genes.
  • the objective of crop trait functional genomics is to identify crop trait genes of interest, for example, genes capable of conferring useful agronomic traits in crop plants.
  • Such agronomic traits include, but are not limited to, enhanced yield, whether in quantity or quality; enhanced nutrient acquisition and metabolic efficiency; enhanced or altered nutrient composition of plant tissues used for food, feed, fiber, or processing; enhanced utility for agricultural or industrial processing; enhanced resistance to plant diseases; enhanced tolerance of adverse environmental conditions (abiotic stresses) including, but not limited to, 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 nontransgenic 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.
  • the mechanisms of plant adaptations to drought include both avoidance and tolerance.
  • Drought avoidance mechanisms are generally constitutive phenotypic properties such as epicuticular wax structure and root thickness or root depth (Svenningson, 1988, Price et al., 2002), which are expressed in the presence or absence of stress conditions.
  • drought tolerance is an adaptive response that enables plants to maintain metabolism even at low water potential (Ingram & Bartels, 1996). These plants respond to water stress through wide spread changes in cellular metabolism which involves changes in thousands of genes (Hazen et al., 2003b). The functions of those gene products modulated in response to drought define the plant drought response phenotype.
  • Two well-known traits influencing drought tolerance are antioxidant capacity and osmotic adjustment.
  • Osmotic adjustment results from the accumulation of compatible solutes within cells, which lowers the osmotic potential and helps, maintain turgor as plants experience water stress (Sanchez et al., 2002).
  • Antioxidant capacity refers to ability of plants to detoxify reactive oxygen species that cause cellular injury such as lipid peroxidation or protein and nucleic acid modifications (McKersie & Leshem, 1994).
  • Sorghum has a strong pre-flowering and post-flowering drought response which makes it an excellent crop model for evaluating mechanisms of drought resistance (Rosenow et al., 1983; Rosenow, 1987). Post- flowering drought response is expressed when dehydration occurs during grain development where rapid and premature leaf senescence can occur.
  • sorghum accessions are able to resist premature leaf senescence by maintaining greater functional photosynthetic leaf area under moisture stress. This capacity is known as the "stay-green" (stg) trait and is an important component of post-flowering drought tolerance in sorghum (Rosenow & Clark, 1981 ).
  • stg stay-green
  • a recent study has highlighted the contribution of both onset and rate of leaf senescence relative to the nitrogen dynamics of the plant or leaf in sorghum to explain the mechanism leading to the stg phenotype in sorghum (Borrel et al., 2000a, 2000b; Borrel & Hammer, 2000).
  • the sorghum accessions B35 and Tx7000 differ phenotypically with respect to post-flowering drought tolerance and were used to develop a recombinant inbred line (RIL) population (Sanchez et al., 2002).
  • RIL recombinant inbred line
  • Tx7000 is an elite, non-stg line and susceptible to post-flowering drought.
  • a rice GENECHIP® array was used to monitor the gene expression profile of B35, Tx7000, and four derived RILs. This is the first report of 1 ) hybridization of sorghum to a GENECHIP®; 2) heterologous microarray hybridization in plants; and 3) parallel expression profiling of two contrasting genotypes and their recombinant progeny under gradual dehydration stress.
  • the presently disclosed subject matter relates to the screening of a rice GENECHIP® microarray (Affymetrix, Santa Clara, California, United States of America; see Zhu et al., 2003) with cDNAs isolated from sorghum (Sorghum bicolor (L.) Moench) to identify changes in gene expression in sorghum when exposed to drought conditions.
  • rice genes that exhibit different levels of hybridization to sorghum sequences isolated from plants under various conditions of water availability likely correspond to orthologs of sorghum genes that play a role in drought resistance.
  • the identified rice orthologs can be used to isolate the corresponding sorghum genes.
  • the sorghum genes can be used for the construction of vectors designed for altering expression of these genes in transgenic plants using plant molecular genetic methodologies, which are disclosed in detail below.
  • Alteration of plant phenotype through overexpression or underexpression of key trait genes in transgenic plants is a robust and established method for assigning functions to plant genes.
  • Assays to identify transgenic plants with alterations in traits of interest are to be used to unambiguously assign the usefulness of these genes for the improvement of rice, and by extension, other cereals, either by transgenic or classical breeding methods.
  • altered abiotic stress tolerance comprises enhanced drought resistance, wherein “enhanced drought resistance” is defined as an increased ability of a plant (for example, a recombinant or transgenic plant) to withstand periods of low water abundance as compared to a native plant of the same species.
  • altering an abiotic stress tolerance refers in some embodiments to a manipulation of a plant's genome to produce a recombinant or transgenic plant in which the manipulation results in a change in the plant's abiotic stress tolerance.
  • altering an abiotic stress tolerance comprises enhancing a plant's resistance to drought.
  • 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
  • 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.
  • 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.
  • co-factor refers to a natural reactant, such as an organic molecule or a metal ion, required in an enzyme-catalyzed reaction.
  • a co-factor can be, for example, NAD(P), riboflavin (including FAD and FMN), folate, molybdopterin, thiamin, biotin, lipoic acid, pantothenic acid and coenzyme A, S-adenosylmethionine, pyridoxal phosphate, ubiquinone, and menaquinone.
  • a co-factor can be regenerated and reused.
  • 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.
  • the RNA is then translated in vivo or in vitro to produce a polypeptide.
  • 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.
  • 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.
  • two sequences that hybridize to each other under a given set of conditions do not necessarily have to be 100% fully complementary.
  • the terms “fully complementary” and “100% complementary” refer to sequences for which the complementary regions are 100% in Watson-Crick base-pairing, i.e., that no mismatches occur within the complementary regions.
  • certain of these molecules can have non-complementary overhangs on either the 5' or 3' ends that result from the cloning event.
  • 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.
  • domains and features 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.
  • a "domain", or “feature” is that subsequence of the nucleic acid sequence that encodes a domain or feature of a polypeptide.
  • enzyme activity refers to the ability of an enzyme to catalyze the conversion of a substrate into a product.
  • 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 of the 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 of the 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.
  • a donor of free energy or energy-rich molecule e.g. ATP, phosphoenolpyruvate, acetyl phosphate, or phosphocreatine
  • 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.
  • 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
  • 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.
  • 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.
  • a functional fragment of a transcription factor can include, but is not limited to, a DNA binding domain, a transactivating domain, or both.
  • a functional fragment of a receptor tyrosine kinase includes, but is not limited to a ligand binding domain, a kinase domain, an ATP binding domain, and combinations thereof.
  • the term "gene” is used broadly to refer to any segment of DNA associated with a biological function.
  • genes include, but are not limited to, coding sequences and/or the regulatory sequences required for their expression.
  • Genes can also include non-expressed DNA segments that, for example, form recognition sequences for a polypeptide.
  • Genes can be obtained from a variety of sources, including cloning from a source of interest or synthesizing from known or predicted sequence information, and can include sequences designed to have desired parameters.
  • heterologous 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • bind(s) substantially refers to complementary hybridization between a probe nucleic acid and a target nucleic acid and embraces minor mismatches that can be accommodated by reducing the stringency of the hybridization media to achieve the desired detection of the target nucleic acid sequence.
  • the term “inhibitor” 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.
  • 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.
  • 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.
  • the term "mature polypeptide” refers to a polypeptide from which the transit peptide, signal peptide, and/or propeptide portions have been removed.
  • 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.
  • 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 modified enzyme activity is displayed by a non-naturally occurring enzyme that is tolerant to inhibitors that inhibit the cognate naturally occurring enzyme activity.
  • native refers to a gene that is naturally present in the genome of an untransformed plant cell.
  • a “native polypeptide” is a polypeptide that is encoded by a native gene of an untransformed plant cell's genome.
  • naturally occurring refers to an object that is found in nature as distinct from being artificially produced by man.
  • 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.
  • 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.
  • nucleic acid refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g. degenerate codon substitutions) and complementary sequences and as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al,
  • nucleic acid or nucleic acid sequence
  • gene cDNA
  • orthologs refers to genes in different species that encode protein that perform the same biological function.
  • glucose-6-phosphate dehydrogenase genes from, for example, sorghum and rice, are orthologs.
  • orthologous nucleic acid sequences are characterized by a high degree of sequence similarity (for example, at least about 90% sequence identity).
  • a nucleic acid sequence of an ortholog in one species can be used to isolate the nucleic acid sequence of the ortholog in another species (for example, sorghum) using standard molecular biology techniques. This can be accomplished, for example, using techniques 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).
  • the phrase "percent identical" in the context of two nucleic acid or polypeptide sequences, refers to two or more sequences or subsequences that have in some embodiments 60%, in some embodiments 70%, in some embodiments 80%, in some embodiments 90%, in some embodiments 95%, and in some embodiments at least 99% nucleotide or amino acid residue identity, respectively, when compared and aligned for maximum correspondence, as measured using one of the following sequence comparison algorithms or by visual inspection.
  • the percent identity exists in some embodiments over a region of the sequences that is at least about 50 residues in length, in some embodiments over a region of at least about 100 residues, and In some embodiments, the percent identity exists over at least about 150 residues.
  • the percent identity exists over the entire length of the sequences.
  • sequence comparison typically one sequence acts as a reference sequence to which test sequences are compared.
  • test and reference sequences are input into a computer, subsequence coordinates are designated if necessary, and sequence algorithm program parameters are designated.
  • 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 &
  • HSPs high scoring sequence pairs
  • 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).
  • M forward score for a pair of matching residues
  • N penalty score for mismatching residues; always ⁇ 0.
  • 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.
  • W wordlength
  • E expectation
  • W wordlength
  • E probability density function
  • test nucleic acid sequence is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid sequence to the reference nucleic acid sequence is in some embodiments less than about 0.1 , in some embodiments less than about 0.01 , and in some embodiments less than about 0.001.
  • the term “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: 1-105, 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
  • substantially identical in the context of two nucleotide or amino acid sequences, refers to two or more sequences or subsequences that have in some embodiments at least about 60% nucleotide or amino acid identity, in some embodiments at least about 65% nucleotide or amino acid identity, in some embodiments at least about 70% nucleotide or amino acid identity, in some embodiments at least about 75% nucleotide
  • 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.
  • 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
  • a "'target sequence” is a test nucleic acid molecule, often found within a heterogeneous population of nucleic acid molecules.
  • a “target sequence” is synonymous with a "test sequence”.
  • An exemplary nucleotide sequence employed for hybridization studies or assays includes probe sequences that are complementary to or mimic in some embodiments at least an about 14 to 40 nucleotide sequence of a nucleic acid molecule of the presently disclosed subject matter.
  • 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: 1-105.
  • 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.
  • hybridizing substantially to refers to complementary hybridization between a probe nucleic acid molecule and a target nucleic acid molecule and embraces minor mismatches (for example, polymorphisms) that can be accommodated by reducing the stringency of the hybridization and/or wash media to achieve the desired hybridization.
  • Stringent hybridization conditions and “stringent hybridization wash conditions” in the context of nucleic acid hybridization experiments such as Southern and Northern blot analysis are both sequence- and environment- dependent. Longer sequences hybridize specifically at higher temperatures. An extensive guide to the hybridization of nucleic acids is found in Tijssen, 1993.
  • 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.
  • T m thermal melting point
  • medium stringency hybridization and wash conditions are selected to be more than about 5°C lower than the T m for the specific sequence at a defined ionic strength and pH.
  • Exemplary medium stringency conditions include hybridizations and washes as for high stringency conditions, except that the temperatures for the hybridization and washes are in some embodiments 8°C, in some embodiments 10°C, in some embodiments 12°C, and in some embodiments 15°C lower than the T m for the specific sequence at a define'd 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.
  • medium stringency wash conditions for a duplex of more than about 100 nucleotides is 15 minutes in 1X 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.
  • stringent conditions typically involve salt concentrations of less than about 1 M Na + ion, typically about 0.01 to 1 M Na + ion concentration (or other salts) at pH 7.0-8.3, and the temperature is typically at least about 30°C.
  • Stringent conditions can also be achieved with the addition of destabilizing agents such as formamide.
  • a signal to noise ratio of 2-fold (or higher) than that observed for an unrelated probe in the particular hybridization assay indicates detection of a specific hybridization.
  • the following are examples of hybridization and wash conditions that can be used to clone homologous nucleotide sequences that are substantially identical to reference nucleotide sequences of the presently disclosed subject matter: a probe nucleotide sequence hybridizes in some embodiments to a target nucleotide sequence in 7% sodium dodecyl sulfate (NaDS), 0.5M NaP0 4 , 1 mm ethylene diamine tetraacetic acid (EDTA) at 50°C followed by washing in 2X SSC, 0.1 % NaDS at 50°C; in some embodiments, a probe and target sequence hybridize in 7% NaDS, 0.5 M NaP0 4 , 1 mm EDTA at 50°C followed by washing in 1X SSC, 0.1 % NaDS at 50°C; in some embodiments, a probe and target sequence
  • a probe and target sequence hybridize in 7% NaDS, 0.5 M NaP0 , 1 mm EDTA at 50°C followed by washing in 0.1X SSC, 0.1 % NaDS at 50°C; in some embodiments, a probe and target sequence hybridize in 7% NaDS, 0.5 M NaP0 4 , 1 mm EDTA at 0.1X SSC, 0.1 % NaDS at 50°C; in some embodiments, a probe and target sequence hybridize in 7% NaDS, 0.5 M NaP0 4 , 1 mm EDTA at
  • hybridization conditions comprise hybridization in a roller tube for at least 12 hours at 42°C.
  • pre-polypeptide refers to a polypeptide that is normally targeted to a cellular organelle, such as a chloroplast, and still comprises a transit peptide.
  • 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.
  • a polypeptide that is the predominant species present in a preparation is substantially purified.
  • the term "purified” denotes that a nucleic acid or polypeptide gives rise to essentially one band in an electrophoretic gel. Particularly, it means that the nucleic acid or polypeptide is in some embodiments at least about 50% pure, in some embodiments at least about 85% pure, and in some embodiments at least about 99% pure.
  • Two nucleic acids are "recombined" when sequences from each of the two nucleic acids are combined in a progeny nucleic acid.
  • Two sequences are “directly” recombined when both of the nucleic acids are substrates for recombination.
  • Two sequences are “indirectly recombined” when the sequences are recombined using an intermediate such as a cross-over oligonucleotide.
  • an intermediate such as a cross-over oligonucleotide.
  • no more than one of the sequences is an actual substrate for recombination, and in some cases, neither sequence is a substrate for recombination.
  • regulatory elements refers to nucleotide sequences involved in controlling the expression of a nucleotide sequence. Regulatory elements can comprise a promoter operatively linked to the nucleotide sequence of interest and termination signals.
  • Regulatory sequences also include enhancers and silencers. They also typically encompass sequences required for proper translation of the nucleotide sequence.
  • 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 some embodiments 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 some embodiments an increase by about 5-fold or greater, and in some embodiments an increase by about 10-fold or greater.
  • the terms “significantly less” 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 some embodiments 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 of the inhibitor), In some embodiments, a decrease by about 5-fold or greater, and in some embodiments a decrease by about 10-fold or greater.
  • the terms “specific binding” and “immunological cross-reactivity” refer to an indicator that two molecules are substantially identical.
  • 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.
  • a polypeptide is typically substantially identical to a second polypeptide, for example, where the two polypeptides differ only by conservative substitutions.
  • 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.
  • 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
  • sequence 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.
  • the term “substrate” refers to a molecule that an enzyme naturally recognizes and converts to a product in the biochemical pathway in which the enzyme naturally carries out its function; or is a modified version of the molecule, which is also recognized by the enzyme and is converted by the enzyme to a product in an enzymatic reaction similar to the naturally-occurring reaction.
  • 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.
  • 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.
  • nucleic acid molecule refers 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.
  • 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.
  • JJLL- Nucleic Acid Molecules and Polypeptides
  • Abiotic stresses include, but are not limited to, cold, heat, drought, and salt stress, and can significantly affect the growth and/or yield of plants.
  • an abiotic stress is drought. Altering the expression of genes related to these traits can be used to improve or modify the rice plants, grain, or both plants and grain, as desired. Examples describe the isolated genes of interest and methods of analyzing the alteration of expression and their effects on the plant characteristics. III.A.
  • an isolated nucleic acid molecule of the presently disclosed subject matter comprises a nucleotide sequence that hybridizes under conditions of hybridization of 45°C in 1 M NaCl, followed by a final washing step at 50°C in 0.1 M NaCl to a nucleotide sequence as set forth in odd numbered sequences SEQ ID NOs: 1-105, or a fragment, domain, or feature thereof.
  • 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 conditions of hybridization of 45°C in 1 M NaCl, followed by a final washing step at 50°C in 0.1 M NaCl to a nucleotide sequence as set forth in odd numbered sequences SEQ ID NOs: 1-105, 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 conditions of hybridization of 45°C in 1 M NaCl, followed by a final washing step at 50°C in 0.1 M NaCl to a nucleotide sequence listed in odd numbered sequences of SEQ ID NOs: 1-105, or a fragment, domain, or feature thereof.
  • Some embodiments of the presently disclosed subject matter encompass 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 to, a nucleotide sequence that hybridizes under conditions of hybridization of 45°C in 1 M NaCl, followed by a final washing step at 50°C in 0.1 M NaCl to a nucleotide sequence listed in odd numbered sequences of SEQ ID NOs: 1-105, or a fragment, domain, or feature thereof.
  • the substantial identity is at least about 60% identity, in some embodiments at least about 65% identity, in some embodiments at least about 70% identity, in some embodiments at least about 75% identity, in some embodiments about 80% identity, in some embodiments at least about 85% identity, in some embodiments about 90% identity, and in some embodiments at least about 95% identity to the nucleotide sequence listed in odd numbered sequences of SEQ ID NOs: 1- 105, or a fragment, domain, or feature thereof.
  • the nucleotide sequence having substantial identity comprises an allelic variant of the nucleotide sequence that hybridizes under conditions of hybridization of 45°C in 1 M NaCl, followed by a final washing step at 50°C in 0.1 M NaCl to a nucleotide sequence listed in odd numbered sequences of SEQ ID NOs: 1-105, or a fragment, domain, or feature thereof.
  • the nucleotide sequence having substantial identity comprises a naturally occurring variant.
  • the nucleotide sequence having substantial identity comprises a polymorphic variant of the nucleotide sequence that hybridizes under conditions of hybridization of 45°C in 1 M NaCl, followed by a final washing step at 50°C in 0.1 M NaCl to a nucleotide sequence listed in odd numbered sequences of SEQ ID NOs: 1-105, or a fragment, domain, or feature thereof.
  • the nucleic acid having substantial identity comprises a deletion or insertion of at least one nucleotide. In some embodiments, the deletion or insertion comprises less than about thirty nucleotides. In some embodiments, the deletion or insertion comprises less than about five nucleotides.
  • the sequence of the isolated nucleic acid having substantial identity comprises a substitution in at least one codon. In some embodiments, the substitution is conservative.
  • the isolated nucleic acid comprises a plurality of regions having a nucleotide sequence that hybridizes under conditions of hybridization of 45°C in 1 M NaCl, followed by a final washing step at 50°C in 0.1 M NaCl to a nucleotide sequence listed in odd numbered sequences of SEQ ID NOs: 1-105, or an exon, domain, or feature thereof.
  • the sequence having substantial identity to the nucleotide sequence that hybridizes under conditions of hybridization of 45°C in 1 M NaCl, followed by a final washing step at 50°C in 0.1 M NaCl to a nucleotide sequence listed in odd numbered sequences of SEQ ID NOs: 1-105, or a fragment, domain, or feature thereof, is from a plant.
  • the plant is a dicot.
  • the plant is a gymnosperm.
  • the plant is a monocot.
  • the monocot is a cereal.
  • the cereal can be, for example, maize, wheat, barley, oats, rye, millet, sorghum, triticale, secale, einkorn, spelt, emmer, teff, milo, flax, gramma grass,
  • the cereal is rice.
  • the nucleic acid is expressed in a specific location or tissue of a plant.
  • the location or tissue includes, but is not limited to, epidermis, root, vascular tissue, meristem, cambium, cortex, pith, leaf, flower, and combinations thereof.
  • the location or tissue is a seed.
  • the nucleic acid encodes a polypeptide involved in a function including, but not limited to, carbon metabolism, photosynthesis, signal transduction, cell growth, reproduction, disease processes, gene regulation, and differentiation.
  • the nucleic acid encodes a polypeptide involved in abiotic stress tolerance, enhanced yield, disease resistance, or nutritional content.
  • 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 conditions of hybridization of 45°C in 1 M NaCl, followed by a final washing step at 50°C in 0.1 M NaCl to a nucleotide sequence of odd numbered sequences of SEQ ID NOs: 1-105, and wherein the polynucleotide is adapted for any of numerous uses.
  • the polynucleotide is used as a chromosomal marker. In some embodiments, the polynucleotide is used as a marker for restriction fragment length polymorphism (RFLP) analysis. In some embodiments, the polynucleotide is used as a marker for quantitative trait- linked breeding. In some embodiments, the polynucleotide is used as a marker for marker-assisted breeding. In some embodiments, the polynucleotide is used as a bait sequence in a two-hybrid system to identify sequence-encoding polypeptides interacting with the polypeptide encoded by the bait sequence.
  • RFLP restriction fragment length polymorphism
  • the polynucleotide is used as a diagnostic indicator for genotyping or identifying an individual or population of individuals. In some embodiments, the polynucleotide is used for genetic analysis to identify boundaries of genes or 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 conditions of hybridization of 45°C in 1 M NaCl, followed by a final washing step at 50°C in 0.1 M NaCl to a nucleotide sequence listed in odd numbered sequences of SEQ ID NOs: 1-105, 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.
  • all of the fragments in a shuffled nucleic acid comprising a plurality of nucleotide sequence fragments are from a single gene. In some embodiments, the plurality of fragments is derived from at least two different genes. In some embodiments, the shuffled nucleic acid is operatively linked to a promoter sequence. In some embodiments, the shuffled nucleic acid comprises a chimeric polynucleotide comprising a promoter sequence operatively linked to the shuffled nucleic acid. In some embodiments, the shuffled nucleic acid is contained within a host cell. MLB.
  • the isolated nucleic acids and polypeptides of the presently disclosed subject matter are usable over a range of plants - monocots and dicots - in particular monocots such as sorghum, rice, wheat, barley, and maize. In some embodiments, the monocot is a cereal.
  • the cereal can be, for example, maize, wheat, barley, oats, rye, millet, sorghum, triticale, secale, einkorn, spelt, emmer, teff, milo, flax, gramma grass, Thpsacum sp., or teosinte.
  • the cereal is sorghum.
  • Plant genera relevant to the presently disclosed subject matter include, but are not limited to, Cucurbita, Rosa, Vitis, Juglans, Gragaria, Lotus, Medicago, Onobrychis, Thgonella, Vigna, Citrus, Linum, Geranium, Manihot, Daucus, Arabidopsis, Brassica, Raphanus, Sinapis, Atropa, Capsicum, Datura, Hyoscyamus, Lycopersicon, Nicotiana, Solanum,
  • Petunia Digitalis, Majorana, Ciahorium, Helianthus, Lactuca, Bromus, Asparagus, Antirrhinum, Heterocallis, Nemesis, Pelargonium, Panieum, Pennisetum, Ranunculus, Senecio, Salpiglossis, Cucumis, Browaalia, Glycine, Pisum, Phaseolus, Lolium, Oryza, Avena, Hordeum, Secale, Allium, and Triticum.
  • the presently disclosed subject matter also provides a method for genotyping a plant or plant part comprising a nucleic acid molecule of the presently disclosed subject matter.
  • the plant is a monocot such as, but not limited to, sorghum, rice or wheat.
  • Genotyping provides a methodology for distinguishing homologs of a chromosome pair and can be used to differentiate segregants in a plant population.
  • Molecular marker methods can be used in phylogenetic studies, characterizing genetic relationships among crop varieties, identifying crosses or somatic hybrids, localizing chromosomal segments affecting monogenic traits, mapping based cloning, and the study of quantitative inheritance (see Clark, 1997;
  • the method for genotyping can employ any number of molecular marker analytical techniques including, but not limited to, restriction length polymorphisms (RFLPs).
  • RFLPs are produced by differences in the DNA restriction fragment lengths resulting from nucleotide differences between alleles of the same gene.
  • the presently disclosed subject matter provides a method for following segregation of a gene or nucleic acid of the presently disclosed subject matter or chromosomal sequences genetically linked by using RFLP analysis.
  • Linked chromosomal sequences are in some embodiments within 50 centimorgans (cM), in some embodiments within 40 cM, in some embodiments within 30 cM, in some embodiments within 20 cM, in some embodiments within 10 cM, and in some embodiments within 5, 3, 2, or 1 cM of the nucleic acid of the presently disclosed subject matter.
  • Embodiments of the presently disclosed subject matter also relate to an isolated nucleic acid molecule comprising a nucleotide sequence, its complement (for example, its full complement), or its reverse complement (for example, its full reverse complement), the nucleotide sequence encoding a polypeptide (for example, a biologically active polypeptide or biologically active fragment).
  • 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: 2-106, or a fragment, domain, repeat, feature, or chimera thereof. In some embodiments, the nucleotide sequence encodes a polypeptide that is an ortholog of a polypeptide comprising a polypeptide sequence having substantial identity to a polypeptide sequence listed in even numbered sequences of SEQ ID NOs: 2-106, or a fragment, domain, repeat, feature, or chimera thereof.
  • 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: 1-105, or a fragment, domain, or feature thereof, or a sequence complementary thereto.
  • the nucleotide sequence encodes a polypeptide comprising a polypeptide sequence encoded by a nucleotide sequence that hybridizes under conditions of hybridization of 45°C in 1 M NaCl, followed by a final washing step at 50°C in 0.1 M NaCl to a nucleotide sequence listed in odd numbered sequences of SEQ ID NOs: 1-105, or to a sequence complementary thereto.
  • the nucleotide sequence encodes a functional fragment of a polypeptide of the presently disclosed subject matter.
  • the isolated nucleic acid comprises a polypeptide-encoding sequence.
  • 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: 2-106, or a fragment thereof.
  • the polypeptide is an ortholog of a polypeptide disclosed in Table 4 or 5.
  • the polypeptide is a plant polypeptide.
  • the plant is a dicot.
  • the plant is a gymnosperm.
  • the plant is a monocot.
  • the monocot is a cereal.
  • 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.
  • the cereal is sorghum.
  • the polypeptide is involved in a function such as abiotic stress tolerance, enhanced yield, disease resistance, or nutritional content.
  • the polypeptide is involved in drought resistance.
  • Embodiments of the presently disclosed subject matter also relate to an isolated nucleic acid molecule comprising a nucleotide sequence, its complement (for example, its full complement), or its reverse complement
  • 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 NaCl, followed by a final washing step at 50°C in 0.1 M NaCl to a nucleotide sequence listed in odd numbered sequences of SEQ ID NOs: 1-105, or a fragment, domain, or feature thereof, or a sequence complementary thereto; and (b) a functional fragment of (a).
  • 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: 2-106, or a fragment, domain, repeat, feature, or chimera thereof.
  • the isolated nucleic acid comprises a plurality of regions from the polypeptide sequence encoded by a nucleotide sequence that hybridizes under conditions of hybridization of 45°C in 1 M NaCl, followed by a final washing step at 50°C in 0.1 M NaCl to a nucleotide sequence listed in odd numbered sequences of SEQ ID NOs: 1-105, or a fragment, domain, or feature thereof, or a sequence complementary thereto.
  • 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 conditions of hybridization of 45°C in 1 M NaCl, followed by a final washing step at 50°C in 0.1 M NaCl to a nucleotide sequence listed in odd numbered sequences of SEQ ID NOs: 1-105, or a fragment, domain, or feature thereof.
  • III.C. Polypeptides The presently disclosed subject matter further relates to isolated polypeptides that are orthologs of the polypeptides comprising the amino acid sequences set forth in even numbered SEQ ID NOs: 2-106, including biologically active polypeptides.
  • the polypeptide comprises a polypeptide sequence of an ortholog of a polypeptide listed in even numbered sequences of SEQ ID NOs: 2-106. In some embodiments, 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: 2-106. In some embodiments, the polypeptide comprises a chimera of an ortholog of the polypeptide sequence listed in even numbered sequences of SEQ ID NOs: 2-106, where the chimera can comprise functional polypeptide motifs, including domains, repeats, post- translational modification sites, or other features. In some embodiments, the polypeptide is a plant polypeptide.
  • the plant is a dicot. In some embodiments, the plant is a gymnosperm. In some embodiments, the plant is a monocot. In some embodiments, the monocot is a cereal. In some embodiments, the cereal is, for example, maize, wheat, barley, oats, rye, millet, sorghum, triticale, secale, einkorn, spelt, emmer, teff, milo, flax, gramma grass, Thpsacum, or teosinte. In some embodiments, the cereal is sorghum. In some embodiments, the polypeptide is expressed in a specific location or tissue of a plant.
  • the location or tissue includes, but is not limited to, epidermis, root, vascular tissue, meristem, cambium, cortex, pith, leaf, flower, and combinations thereof.
  • the location or tissue is a seed.
  • the polypeptide is involved in a function such as abiotic stress tolerance, disease resistance, enhanced yield or nutritional quality or composition.
  • the polypeptide is involved in drought resistance.
  • 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: 2-106, and variants having conservative amino acid modifications.
  • conservative modified variants refers to polypeptides that can be encoded by nucleic acid sequences having degenerate codon substitutions wherein at least one position of one or more selected (or all) codons is substituted with mixed- base and/or deoxyinosine residues (Batzer et al., 1991 ; Ohtsuka et al., 1985;
  • 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.
  • 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 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.
  • 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.
  • 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).
  • hydropathic amino acid index in conferring interactive biological function on a protein is generally understood in the art (Kyte & Doolittle, 1982, incorporated herein by reference). It is known that certain amino acids can be substituted for other amino acids having a similar hydropathic index or score and still retain a similar biological activity. In making changes based upon the hydropathic index, substitutions of amino acids can involve amino acids for which the hydropathic indices are in some embodiments within ⁇ 2 of the original value, in some embodiments within ⁇ 1 of the original value, and in some embodiments within ⁇ 0.5 of the original value. It is also understood in the art that the substitution of like amino acids can be made effectively on the basis of hydrophilicity.
  • 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 can involve amino acids for which the hydrophilicity values are in some embodiments within ⁇ 2 of the original value, in some embodiments within ⁇ 1 of the original value, and in some embodiments within ⁇ 0.5 of the original value. While discussion has focused on functionally equivalent polypeptides arising from amino acid changes, it will be appreciated that these changes can be effected by alteration of the encoding DNA, taking into consideration also that the genetic code is degenerate and that two or more codons can code for the same amino acid.
  • the sequence having substantial identity contains a deletion or insertion of at least one amino acid. In some embodiments, the deletion or insertion is of less than about ten amino acids.
  • the deletion or insertion is of less than about three amino acids.
  • 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: (a) a polypeptide sequence having substantial identity to a polypeptide sequence listed in even numbered SEQ ID NO: 2- 106, or a domain or feature thereof; (b) a polypeptide sequence encoded by a nucleotide sequence identical to or having substantial identity to a nucleotide sequence that hybridizes under conditions of hybridization of 45°C in 1 M NaCl, followed by a final washing step at 50°C in 0.1 M NaCl listed in odd numbered SEQ ID NO: 1-105, or an exon, domain, or feature thereof, or a sequence complementary thereto; (c) a polypeptide sequence encoded by a nucleotide sequence capable of hybridizing under medium stringency conditions to a nucle
  • a polypeptide having substantial identity to a polypeptide sequence listed in even numbered SEQ ID NO: 2-106, or a domain or feature thereof is an allelic variant of the polypeptide sequence listed in even numbered SEQ ID NO: 2-106.
  • a polypeptide having substantial identity to a polypeptide sequence listed in even numbered SEQ ID NO: 2-106, or a domain or feature thereof is a naturally occurring variant of the polypeptide sequence listed in even numbered SEQ ID NO: 2-106.
  • a polypeptide having substantial identity to a polypeptide sequence listed in even numbered SEQ ID NO: 2-106, or a domain or feature thereof is a polymorphic variant of the polypeptide sequence listed in even numbered SEQ ID NO: 2-106.
  • the polypeptide is an ortholog of a polypeptide comprising one of the amino acid sequences listed in even numbered SEQ ID NO: 2-106.
  • 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: 2-106.
  • 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.
  • the polypeptide is a plant polypeptide.
  • the plant is a dicot.
  • the plant is a gymnosperm.
  • the plant is a monocot.
  • the monocot is a cereal.
  • the cereal can be, for example, maize, wheat, barley, oats, rye, millet, sorghum, triticale, secale, einkorn, spelt, emmer, teff, milo, flax, gramma grass, Tripsacum, or teosinte.
  • the cereal is sorghum.
  • the polypeptide is expressed in a specific location or tissue of a plant.
  • the location or tissue includes, but is not limited to, epidermis, vascular tissue, meristem, cambium, cortex, or pith.
  • the location or tissue is leaf or sheath, root, flower, and developing ovule or seed.
  • the location or tissue can be, for example, epidermis, root, vascular tissue, meristem, cambium, cortex, pith, leaf, or flower.
  • the location or tissue is a seed.
  • the polypeptide sequence is encoded by a nucleotide sequence that hybridizes under conditions of hybridization of 45°C in 1 M NaCl, followed by a final washing step at 50°C in 0.1 M NaCl to a nucleotide sequence listed in odd numbered SEQ ID NO: 1-105 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.
  • the deletion or insertion is of less than about thirty nucleotides. In some embodiments, the deletion or insertion is of less than about five nucleotides.
  • the polypeptide sequence encoded by a nucleotide sequence that hybridizes under conditions of hybridization of 45°C in 1 M NaCl, followed by a final washing step at 50°C in 0.1 M NaCl to a nucleotide sequence listed in odd numbered SEQ ID NO: 1-105, or a fragment, domain, or feature thereof or a sequence complementary thereto, includes a substitution of at least one codon. In some embodiments, the substitution is conservative.
  • the polypeptide sequences having substantial identity to the polypeptide sequence listed in even numbered SEQ ID NO: 2-106, or a fragment, domain, repeat, feature, or chimera thereof includes a deletion or insertion of at least one amino acid.
  • polypeptides of the presently disclosed subject matter, fragments thereof, or variants thereof can comprise any number of contiguous amino acid residues from a polypeptide of the presently disclosed subject matter, wherein the number of residues is selected from the group of integers consisting of from 10 to the number of residues in a full-length polypeptide of the presently disclosed subject matter.
  • the portion or fragment of the polypeptide is a functional polypeptide.
  • the presently disclosed subject matter includes active polypeptides having specific activity of at least in some embodiments 20%, in some embodiments 30%, in some embodiments 40%, in some embodiments 50%, in some embodiments 60%, in some embodiments 70%, in some embodiments 80%, in some embodiments 90%, and in some embodiments 95% that of the native (non- synthetic) endogenous polypeptide.
  • the substrate specificity k ca t/Km
  • the substrate specificity can be substantially identical to the native (non-synthetic), endogenous polypeptide.
  • the K m will be at least in some embodiments 30%, in some embodiments 40%, in some embodiments 50% of the native, endogenous polypeptide; and in some embodiments at least 60%, in some embodiments 70%, in some embodiments 80%, and in some embodiments 90% of the native, endogenous polypeptide.
  • Methods of assaying and quantifying measures of activity and substrate specificity are well known to those of skill in the art.
  • 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.
  • 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 (ELISA) and competitive immunoassays.
  • Embodiments of the presently disclosed subject matter also relate to chimeric polypeptides encoded by the isolated nucleic acid molecules of the 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 conditions of hybridization of 45°C in 1 M NaCl, followed by a final washing step at 50°C in 0.1 M NaCl to a nucleotide sequence listed in odd numbered SEQ ID NO: 1-105, 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.
  • 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 of the 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.
  • a non-natural condition e.g. in quantity, composition, location and/or time
  • Embodiments of the presently disclosed subject matter provide an expression cassette comprising a promoter sequence operatively linked to an isolated nucleic acid, the isolated nucleic acid comprising: (a) a nucleotide sequence that hybridizes under conditions of hybridization of 45°C in 1 M NaCl, followed by a final washing step at 50°C in 0.1 M NaCl to a nucleotide sequence listed in odd numbered sequences of SEQ ID NOs: 1-105, or a fragment, domain, or feature thereof; (b) a nucleotide sequence complementary (for example, fully complementary) to (a); and (c) a nucleotide sequence that is the reverse complement (for example, the full reverse complement) of (a).
  • a recombinant vector comprising an expression cassette according to the embodiments of the presently disclosed subject matter.
  • plant cells comprising expression cassettes according to the present disclosure, and plants comprising these plant cells.
  • the plant is a dicot.
  • the plant is a gymnosperm.
  • the plant is a monocot.
  • the monocot is a cereal.
  • the cereal is, for example, maize, wheat, barley, oats, rye, millet, sorghum, triticale, secale, einkorn, spelt, emmer, teff, milo, flax, gramma grass, Tripsacum or teosinte.
  • the cereal is sorghum.
  • the expression cassette is expressed throughout the plant.
  • the expression cassette is expressed in a specific location or tissue of a plant.
  • the location or tissue includes, but is not limited to, epidermis, root, vascular tissue, meristem, cambium, cortex, pith, leaf, flower, and combinations thereof.
  • the location or tissue is a seed.
  • the expression cassette is involved in a function including, but not limited to, disease resistance, yield, abiotic stress resistance, nutritional quality, carbon metabolism, photosynthesis, signal transduction, cell growth, reproduction, disease processes (for example, pathogen resistance), gene regulation, and differentiation.
  • the chimeric polypeptide is involved in a function such as abiotic stress tolerance, enhanced yield, disease resistance, or nutritional composition.
  • the abiotic stress tolerance is drought resistance.
  • Embodiments of the presently disclosed subject matter also relate to an expression vector comprising a nucleic acid molecule selected from the group consisting of: (a) a nucleic acid encoding a polypeptide as listed in even numbered sequences of SEQ ID NOs: 2-106, or ortholog thereof; (b) a fragment, domain, or featured region of a nucleic acid sequence that hybridizes under conditions of hybridization of 45°C in 1 M NaCl, followed by a final washing step at 50°C in 0.1 M NaCl to a nucleotide sequence listed in odd numbered sequences of SEQ ID NOs: 1-105; and (c) a complete nucleic acid sequence that hybridizes under conditions of hybridization of 45°C in 1 M NaCl, followed by a final washing step at 50°C in 0.1 M NaCl to a nucleotide sequence listed in odd numbered sequences of SEQ ID NOs: 1- 105, or a fragment thereof, in combination with a heterologous sequence.
  • 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.
  • 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.
  • the promoter is operable in plants, and in some embodiments, the promoter is a constitutive or inducible promoter.
  • the selection marker sequence encodes an antibiotic resistance gene.
  • the epitope tag sequence encodes V5 (GKPIPNPLLGLDST; SEQ ID NO: 107; Southern et al., 1991 ), the peptide Phe-His-His-Thr-Thr (SEQ ID NO: 108), hemaglutinin, or glutathione-S-transferase.
  • the affinity purification tag sequence encodes a polyamino acid sequence or a polypeptide.
  • the polyamino acid sequence comprises polyhistidine.
  • the polypeptide is chitin-binding domain or glutathione-
  • the affinity purification tag sequence comprises an intein encoding sequence.
  • the expression vector comprises a eukaryotic expression vector, and in some embodiments, the expression vector comprises a prokaryotic expression vector. In some embodiments, the eukaryotic expression vector comprises a tissue-specific promoter. In some embodiments, the expression vector is operable in plants.
  • Embodiments of the presently disclosed subject matter also relate to a cell comprising a nucleic acid construct comprising an expression vector and a nucleic acid comprising a nucleic acid encoding a polypeptide that is an ortholog of a polypeptide as listed in even numbered sequences of SEQ ID NOs: 2-106, or a nucleic acid sequence that hybridizes under conditions of hybridization of 45°C in 1 M NaCl, followed by a final washing step at 50°C in 0.1 M NaCl to a nucleotide sequence listed in odd numbered sequences of SEQ ID NOs: 1-105, or a subsequence thereof, in combination with a heterologous sequence.
  • the cell is a bacterial cell, a fungal cell, a plant cell, or an animal cell.
  • the polypeptide is expressed in a specific location or tissue of a plant.
  • the location or tissue includes, but is not limited to, epidermis, root, vascular tissue, meristem, cambium, cortex, pith, leaf, flower, and combinations thereof.
  • the location or tissue is a seed.
  • the polypeptide is involved in a function such as, for example, carbon metabolism, photosynthesis, signal transduction, cell growth, reproduction, disease processes, gene regulation, and differentiation.
  • the polypeptide is involved in a function such as abiotic stress tolerance, enhanced yield, disease resistance, or nutritional composition.
  • 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.
  • 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,
  • 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.
  • a promoter e.g., the CMV promoter, a Herpes Simplex Virus thymidine kinase (HSV-tk) promoter or phosphoglycerate kinase (pgk) promoter
  • polypeptides 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.
  • suitable insect cell lines are available including, but not limited to, mosquito larvae, silkworm, armyworm (for example, Spodoptera frugiperda), moth, and Drosophila cell lines. Methods of transforming animal and lower eukaryotic cells are known.
  • Numerous methods can be used to introduce exogenous DNA into eukaryotic cells including, but not limited to, calcium phosphate precipitation, fusion of the recipient cell with bacterial protoplasts containing the DNA, treatment of the recipient cells with liposomes containing the DNA, DEAE dextran, electroporation, biolistics, and microinjection of the DNA directly into the cells.
  • Transformed cells are cultured using means well known in the art (see Kuchler, 1997).
  • Once a polypeptide of the presently disclosed subject matter is expressed it can be isolated and purified from the expressing cells using methods known to those skilled in the art. The purification process can be monitored using Western blot techniques, radioimmunoassay, or other standard immunoassay techniques.
  • Embodiments of the presently disclosed subject matter provide a method for producing a recombinant polypeptide in which the expression vector comprise one or more elements including, but not limited to, a promoter-enhancer sequence, a selection marker sequence, an origin of replication, an epitope tag-encoding sequence, and an affinity purification tag-encoding sequence.
  • the nucleic acid construct comprises an epitope tag-encoding sequence and the isolating step employs an antibody specific for the epitope tag.
  • the nucleic acid construct comprises a polyamino acid-encoding sequence and the isolating step employs a resin comprising a polyamino acid binding substance, in some embodiments where the polyamino acid is polyhistidine and the polyamino acid binding resin is nickel-charged agarose resin.
  • the nucleic acid construct comprises a polypeptide- encoding sequence and the isolating step employs a resin comprising a polypeptide binding substance.
  • the polypeptide is a chitin-binding domain and the resin contains chitin-sepharose.
  • the presently disclosed subject matter further provides a method for modifying (i.e. increasing or decreasing) the concentration or composition 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 and/or the composition (i.e. the ration 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.
  • a plant or plant part having modified expression of a nucleic acid molecule of the presently disclosed subject matter can be analyzed and selected using methods known to those skilled in the art including, but not limited to, Southern blotting, DNA sequencing, or PCR analysis using primers specific to the nucleic acid molecule and detecting amplicons produced therefrom.
  • a concentration or composition is increased or decreased by at least in some embodiments 5%, in some embodiments 10%, in some embodiments 20%, in some embodiments 30%, in some embodiments 40%, in some embodiments 50%, in some embodiments 60%, in some embodiments 70%, in some embodiments 80%, and in some embodiments 90% relative to a native control plant, plant part, or cell lacking the expression cassette.
  • 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.
  • 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 cell comprising the target gene, in some embodiments a plant cell, and introduced into the cell, in which the nucleotide sequence is expressible.
  • the nucleotide sequence is inserted in the DNA molecule in the "sense orientation", meaning that the coding strand of the nucleotide sequence can be transcribed.
  • the nucleotide sequence is fully translatable and all the genetic information comprised in the nucleotide sequence, or portion thereof, is translated into a polypeptide.
  • the nucleotide sequence is partially translatable and a short peptide is translated. In some embodiments, this is achieved by inserting at least one premature stop codon in the nucleotide sequence, which brings translation to a halt.
  • the nucleotide sequence is transcribed but no translation product is made. This is usually achieved by removing the start codon, i.e.
  • the DNA molecule comprising the nucleotide sequence, or a portion thereof is stably integrated in the genome of the plant cell. In some embodiments, the DNA molecule comprising the nucleotide sequence, or a portion thereof, is comprised in an extrachromosomally replicating molecule. In transgenic plants containing one of the 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 some embodiments is at least 70% identical to the nucleotide sequence the expression of which is reduced, in some embodiments is at least 80% identical, in some embodiments is at least 90% identical, in some embodiments is at least 95% identical, and in some embodiments is at least
  • the alteration of the expression of a nucleotide sequence of the presently disclosed subject matter 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.
  • the DNA molecule comprising the nucleotide sequence, or a portion thereof is stably integrated in the genome of the plant cell. In some embodiments the DNA molecule comprising the nucleotide sequence, or a portion thereof, is comprised in an extrachromosomally replicating molecule.
  • 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 of the 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 some embodiments at least 70% identical to the nucleotide sequence the expression of which is reduced, in some embodiments at least 80% identical, in some embodiments at least 90% identical, in some embodiments at least 95% identical, and in some embodiments at least 99% identical.
  • IV.B.3. Homologous Recombination
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • this technique is further illustrated in U.S. Patent No. 5,501 ,967 and Zhu et al., 1999. IV.B.4.
  • Ribozvmes In some embodiments, 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 some embodiments, 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.
  • 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
  • Zinc Finger Polypeptides A zinc finger polypeptide that binds a nucleotide sequence of the presently disclosed subject matter or to its regulatory region can also be used to alter expression of the nucleotide sequence. In alternative embodiments, transcription of the nucleotide sequence is reduced or increased.
  • Zinc finger polypeptides are disclosed in, for example, Beerli et 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,
  • the alteration of the expression of a nucleotide sequence of the presently disclosed subject matter, in some embodiments the reduction of its expression, is obtained by dsRNA interference.
  • the entirety, or in some embodiments 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 some embodiments 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.
  • the first copy of the DNA molecule is the reverse complement (also known as the non-coding strand) and the second copy is the coding strand; In some embodiments, the first copy is the coding strand, and the second copy is the reverse complement.
  • the size of the spacer DNA molecule is in some embodiments 200 to 10,000 nucleotides, in some embodiments 400 to 5000 nucleotides, and in some embodiments 600 to 1500 nucleotides in length.
  • the spacer is in some embodiments a random piece of DNA, in some embodiments a random piece of DNA without homology to the target organism for dsRNA interference, and in some embodiments 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.
  • the DNA molecule comprising the nucleotide sequence, or a portion thereof is stably integrated in the genome of the plant cell.
  • the DNA molecule comprising the nucleotide sequence, or a portion thereof is comprised in an extrachromosomally replicating molecule.
  • the expression of the nucleotide sequence corresponding to the nucleotide sequence comprised in the DNA molecule is in some embodiments reduced.
  • the nucleotide sequence in the DNA molecule is at least 70% identical to the nucleotide sequence the expression of which is reduced, in some embodiments it is at least 80% identical, in some embodiments it is at least 90% identical, in some embodiments it is at least 95% identical, and in some embodiments it is at least 99% identical. IV.B.9.
  • 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.
  • such DNA molecule comprises a transposable element capable of transposition in a plant cell, such as, for example, Ac/Ds, Em/Spm, mutator.
  • 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.
  • 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.
  • 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 of the 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 some embodiments, a nucleotide sequence of the presently disclosed subject matter encoding a polypeptide is over-expressed. Examples of nucleic acid molecules and expression cassettes for over- expression of a nucleic acid molecule of the presently disclosed subject matter are disclosed above.
  • nucleotide sequence of the presently disclosed subject matter is altered in every cell of a plant. This can be obtained, for example, though homologous recombination or by insertion into a chromosome. This can also be obtained, for example, by expressing a sense 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 of the 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.
  • 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.
  • 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 (roots, leaves, or flowers, for example) and the selection can reflect the desired location for accumulation of the gene product.
  • the selected promoter can drive expression of the gene under various inducing conditions. Promoters vary in their strength; i.e., their abilities to promote transcription Depending upon the host cell system utilized, any one of a number of suitable promoters can be used, including the gene's native promoter. The following are non-limiting examples of promoters that can be used in expression cassettes. IV.C.1.a.
  • Ubiquitin Promoter is a gene product known to accumulate in many cell types and its promoter has been cloned from several species for use in transgenic plants (e.g. sunflower - Binet et al, 1991 ; maize - Christensen & Quail, 1989; and Arabidopsis - 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 Lubrizol) which is herein incorporated by reference.
  • Taylor et al, 1993 describes a vector (pAHC25) that comprises the maize ubiquitin promoter and first intron and its high activity in cell suspensions of numerous monocotyledons when introduced via microprojectile bombardment.
  • the Arabidopsis ubiquitin promoter is suitable for use with the nucleotide sequences of the presently disclosed subject matter.
  • the ubiquitin promoter is suitable for gene expression in transgenic plants, both monocotyledons and dicotyledons.
  • Suitable vectors are derivatives of pAHC25 or any of the transformation vectors disclosed herein, modified by the introduction of the appropriate ubiquitin promoter and/or intron sequences. IV.C.1.b.
  • 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 seq ence-tml terminator cassette of such a construction can be excised by Hind ⁇ , Sph ⁇ , Sal ⁇ , and Xba ⁇ sites 5' to the promoter and Xba ⁇ , BamH ⁇ and ⁇ g/l sites 3' to the terminator for transfer to transformation vectors such as those disclosed below.
  • the double 35S promoter fragment can be removed by 5' excision with Hind ⁇ , Sph ⁇ , Sal ⁇ , Xba ⁇ , or Pst ⁇ , and 3' excision with any of the polylinker restriction sites (EcoRI, Not ⁇ or Xho ⁇ ) for replacement with another promoter.
  • 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.
  • pCGN1761 ENX can be modified by optimization of the translational initiation site as disclosed in Example 37 of U.S. Patent No. 5,639,949, incorporated herein by reference. IV.C.1.c.
  • the actin promoter can be used as a constitutive promoter.
  • the promoter from the rice Actl gene has been cloned and characterized (McElroy et al, 1990).
  • a 1.3 kilobase (kb) fragment of the promoter was found to contain all the regulatory elements required for expression in rice protoplasts.
  • numerous expression vectors based on the Actl promoter have been constructed specifically for use in monocotyledons (McElroy et al, 1991 ).
  • GUS GUS reporter gene
  • 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.
  • the rice Actl promoter with its first intron has also been found to direct high expression in cultured barley cells (Chibbar e al., 1993). IV.C.1.d.
  • 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.
  • 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.
  • the Arabidopsis PR-1 promoter disclosed in Lebel et al., 1998 can be used.
  • the promoter of choice can be excised from its source by restriction enzymes, but can alternatively be PCR-amplified using primers that carry appropriate terminal restriction sites.
  • 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 pCGN1761 ENX (Uknes et al, 1992).
  • pCIB1004 is cleaved with Nco ⁇ and the resulting 3' overhang of the linearized fragment is rendered blunt by treatment with T4 DNA polymerase.
  • the fragment is then cleaved with Hind ⁇ and the resultant PR-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 Xho ⁇ and blunting with T4 polymerase, followed by cleavage with Hind ⁇ , and isolation of the larger vector-terminator containing fragment into which the pCIB1004 promoter fragment is cloned. This generates a pCGN1761 ENX derivative with the PR-1 a promoter and the tml terminator and an intervening polylinker with unique EcoR ⁇ and Not ⁇ sites.
  • the selected coding sequence can be inserted into this vector, and the fusion products (i.e. promoter-gene- terminator) can subsequently be transferred to any selected transformation vector, including those disclosed herein.
  • fusion products i.e. promoter-gene- terminator
  • 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).
  • the alcA gene encodes alcohol dehydrogenase I, the expression of which is regulated by the AlcR transcription factors in presence of the chemical inducer.
  • the CAT coding sequences in plasmid palcA:CAT comprising a alcA gene promoter sequence fused to a minimal 35S promoter (Caddick et al,
  • a glucocorticoid-mediated induction system is used (Aoyama & Chua, 1997) and gene expression is induced by application of a glucocorticoid, for example a synthetic glucocorticoid, for example dexamethasone, at a concentration ranging in some embodiments from 0.1 mM to 1 mM, and in some embodiments from 10 mM to 100 mM.
  • a glucocorticoid for example a synthetic glucocorticoid, for example dexamethasone
  • the luciferase gene sequences Aoyama & Chua, 1997 are replaced by a nucleic acid sequence of the presently disclosed subject matter to form an expression cassette having a nucleic acid sequence of the presently disclosed subject matter under the control of six copies of the GAL4 upstream activating sequences fused to the 35S minimal promoter. This is carried out using methods known in the art.
  • the trans-acting factor comprises the GAL4 DNA-binding domain (Keegan et al., 1986) fused to the transactivating domain of the herpes viral polypeptide VP16 (Triezenberg et al, 1988) fused to the hormone-binding domain of the rat glucocorticoid receptor (Picard et al, 1988).
  • the expression of the fusion polypeptide is controlled either by a promoter known
  • 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.
  • tissue- or organ-specificity of the fusion polypeptide is achieved leading to inducible tissue- or organ-specificity of the nucleic acid sequence to be expressed.
  • 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.
  • a suitable vector such as pCGN1761 ENX
  • Wound-lnducible 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.
  • 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.
  • 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.
  • fragments containing the pith-preferred promoter or parts thereof can be transferred to any vector and modified for utility in transgenic plants.
  • Leaf-Specific Expression A maize gene encoding phosphoenol carboxylase (PEPC) has been disclosed by Hudspeth & Grula, 1989.
  • 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 the promoter for this gene can be used to drive the expression of any gene in a leaf-specific manner in transgenic plants.
  • WO 93/07278 describes the isolation of the maize calcium-dependent protein kinase (CDPK) gene that is expressed in pollen cells.
  • CDPK calcium-dependent protein kinase
  • the gene sequence and promoter extend up to 1400 bp from the start of transcription.
  • 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.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. Sequences for the Enhancement or Regulation of Expression Numerous sequences have been found to enhance gene expression from within the transcriptional unit and these sequences can be used in conjunction with the genes of the presently disclosed subject matter to increase their expression in transgenic plants. Various intron sequences have been shown to enhance expression, particularly in monocotyledonous cells.
  • 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).
  • 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.
  • TMV Tobacco Mosaic Virus
  • MCMV Maize Chlorotic Mottle Virus
  • AMV Alfalfa Mosaic Virus
  • leader sequences known in the art include, but are not limited to, picomavirus leaders, for example, EMCV (encephalomyocarditis virus) leader (5' noncoding region; see Elroy-Stein et al, 1989); potyvirus leaders, for example, from Tobacco Etch Virus (TEV; see Allison et al, 1986); Maize Dwarf Mosaic Virus (MDMV; see Kong & Steinbiss 1998); human immunoglobulin heavy-chain binding polypeptide (BiP) leader (Macejak & Sarnow, 1991 ); untranslated leader from the coat polypeptide mRNA of alfalfa mosaic virus (AMV; RNA 4; see Jobling & Gehrke, 1987); tobacco mosaic virus (TMV) leader (Gallie et al, 1989); and Maize Chlorotic Mottle Virus (MCMV) leader (Lommel et al, 1991 ).
  • picomavirus leaders for example, EMCV (encephalomyocarditis virus) leader (5'
  • Such elements include, but are not limited to, a minimal promoter.
  • 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.
  • Bz1 minimal promoter which is obtained from the bronzel gene of maize.
  • the Bz1 core promoter is obtained from the "myc" mutant
  • the derived Bz1 core promoter fragment thus extends from positions -53 to +227 and includes the Bz1 intron-1 in the 5' untranslated region.
  • 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.
  • 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
  • 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.
  • 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
  • the transgene product 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.
  • 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.
  • 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.
  • Selection markers used routinely in transformation include the nptll gene, which confers resistance to kanamycin and related antibiotics (Messing & Vieira, 1982; Bevan et al, 1983); the bar gene, which confers resistance to the herbicide phosphinothricin (White et al, 1990; Spencer et al, 1990); the hph gene, which confers resistance to the antibiotic hygromycin (Blochinger & Diggelmann, 1984); the dhfr gene, which confers resistance to methotrexate (Bourouis & Jarry, 1983); the EPSP synthase gene, which confers resistance to glyphosate (U.S. Patent Nos. 4,940,935 and 5,188,642); and the mannose-6-phosphate isomerase gene, which provides the ability to metabolize mannose (U.S. Patent Nos.
  • IV.D.1. Vectors Suitable for Aprobactehum 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. PCIB200 and pCIB2001 The binary vectors pCIB200 and pCIB2001 are used for the construction of recombinant vectors for use with Agrobacterium and are constructed in the following manner.
  • pTJS75kan is created by Na ⁇ digestion of pTJS75 (Schmidhauser & Helinski, 1985) allowing excision of the tetracycline-resistance gene, followed by insertion of an Acc ⁇ fragment from pUC4K carrying an NPTII sequence (Messing & Vieira, 1982: Bevan et al., 1983: McBride & Summerfelt. 1990).
  • Xho ⁇ linkers are ligated to the EcoRV fragment of PCIB7 which contains the left and right T-DNA borders, a plant selectable nos/nptll chimeric gene and the pUC polylinker (Rothstein et al, 1987), and the X ⁇ ol-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: EcoR ⁇ , Sst ⁇ , Kpn ⁇ , ⁇ g/ll, Xba ⁇ , and Sail.
  • pCIB2001 is a derivative of pCIB200 created by the insertion into the polylinker of additional restriction sites.
  • Unique restriction sites in the polylinker of pCIB2001 are EcoR ⁇ , Sst ⁇ , Kpn ⁇ , ⁇ g/ll, Xba ⁇ , Sal ⁇ , Mlu ⁇ , Bcl ⁇ , Av ⁇ , Apa ⁇ , Hpa ⁇ , and Stu ⁇ .
  • pCIB2001 in addition to containing these unique restriction sites, also has plant and bacterial kanamycin selection, left and right T-DNA borders for
  • the pCIB2001 polylinker is suitable for the cloning of plant expression cassettes containing their own regulatory signals.
  • IV.D.Lb. pCIBI O and Hygromycin Selection Derivatives Thereof The binary vector pCIBI O 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.
  • pCIBIO Various derivatives of pCIBIO can be constructed which incorporate the gene for hygromycin B phosphotransferase disclosed by Gritz & Davies, 1983. These derivatives enable selection of transgenic plant cells on hygromycin only (pCIB743), or hygromycin and kanamycin (pCIB715, pCIB717). 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.
  • PEG polyethylene glycol
  • the choice of vector depends largely on the species being transformed. Below, the construction of typical vectors suitable for non- Agrobacterium transformation is disclosed.
  • 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.
  • the 35S promoter of this vector contains two ATG sequences 5' of the start site. These sites are mutated using standard PCR techniques in such a way as to remove the ATGs and generate the restriction sites Ssp ⁇ and PvuU. The new restriction sites are 96 and 37 bp away from the unique Sail site and 101 and 42 bp away from the actual start site.
  • the resultant derivative of pCIB246 is designated pCIB3025.
  • the GUS gene is then excised from pCIB3025 by digestion with Sail and Sac ⁇ , 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 Sma ⁇ fragment containing the bar gene from Streptomyces viridochromogenes is excised and inserted into the Hpal 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.
  • 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.
  • DHFR E. coli dihydrofolate reductase
  • 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-Ps-1 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.
  • MCMV Maize Chlorotic Mottle Virus
  • pSOG19 and pSOG35 carry the pUC gene for ampicillin resistance and have Hind ⁇ , Sph ⁇ , Pst ⁇ , and EcoR ⁇ sites available for the cloning of foreign substances.
  • IV.D.3. Vector Suitable for Chloroplast Transformation
  • plastid transformation vector pPH143 PCT International Publication WO 97/32011 , example 36
  • the nucleotide sequence is inserted into pPH143 thereby replacing the protoporphyrinogen oxidase (Protox) coding sequence.
  • This vector is then used for plastid transformation and selection of transformants for spectinomycin resistance.
  • nucleotide sequence is inserted in pPH143 so that it replaces the aadH gene.
  • transformants are selected for resistance to Protox inhibitors.
  • IV.E. Transformation Once a nucleic acid sequence of the presently disclosed subject matter has been cloned into an expression system, it is transformed into a plant cell.
  • the receptor and target expression cassettes of the presently disclosed subject matter can be introduced into the plant cell in a number of art-recognized ways. Methods for regeneration of plants are also well known in the art. For example, Ti plasmid vectors have been utilized for the delivery of foreign DNA, as well as direct DNA uptake, liposomes, electroporation, microinjection, and microprojectiles.
  • bacteria from the genus Agrobacterium can be utilized to transform plant cells.
  • Transformation of Dicotyledons Transformation techniques for dicotyledons are well known in the art and include yAgrofoacfer/ ' -vm-based techniques and techniques that do not require Agrobacterium.
  • on-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.
  • y4grobacter/i/.7.-mediated 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.
  • the recombinant binary vector can be transferred to Agrobacterium by DNA transformation (H ⁇ fgen & Willmitzer, 1988). Transformation of the 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 et al. Generally, this procedure involves propelling inert or biologically active particles at the cells under conditions effective to penetrate the outer surface of the cell and afford incorporation within the interior thereof.
  • the vector can be introduced into the cell by coating the particles with the vector containing the desired gene.
  • 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
  • Biologically active particles can also be propelled into plant cell tissue.
  • 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.
  • a disadvantage of the use of co-transformation is the less than 100% frequency with which separate DNA species are integrated into the genome
  • 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.
  • WO 93/07278 and Koziel et al, 1993 describe techniques for the transformation of elite inbred lines of maize by particle bombardment. This technique utilizes immature maize embryos of
  • Transformation of rice can also be undertaken by direct gene transfer techniques utilizing protoplasts or particle bombardment.
  • Protoplast- mediated transformation has been disclosed for Japonica-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 ).
  • WO 93/21335 describes techniques for the transformation of rice via electroporation.
  • embryos 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.
  • 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.
  • 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.
  • psi pounds per square inch
  • 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.
  • Rice Oryza sativa
  • Various rice cultivars can be used (Hiei et al, 1994; Dong et al, 1996; Hiei et al, 1997).
  • 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.
  • 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
  • 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 resuspended in liquid MS-CIM medium. The Agrobacterium culture is diluted to an OD 6 oo of 0.2-0.3 and acetosyringone is added to a final concentration of 200 ⁇ M. 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.
  • MS-CIM medium with ticarcillin 400 mg/liter
  • 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
  • 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 ⁇ mol photons/m 2 /s) on plates of RMOP medium (Svab et al, 1990) containing 500 ⁇ g/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.
  • the presently disclosed subject matter also provides plants comprising the disclosed compositions.
  • the plant is characterized by a modification of a phenotype or measurable characteristic of the plant, the modification being attributable to the expression cassette.
  • the modification involves, for example, nutritional enhancement, increased nutrient uptake efficiency, enhanced production of endogenous compounds, or production of heterologous compounds.
  • the modification includes having increased or decreased resistance to an herbicide, an abiotic stress, or a pathogen.
  • the modification includes having enhanced or diminished requirement for light, water, nitrogen, or trace elements.
  • the modification includes being enriched for an essential amino acid as a proportion of a polypeptide fraction of the plant.
  • the polypeptide fraction can be, for example, total seed polypeptide, soluble polypeptide, insoluble polypeptide, water-extractable polypeptide, and lipid-associated polypeptide.
  • the modification includes overexpression, underexpression, antisense modulation, sense suppression, inducible expression, inducible repression, or inducible modulation of a gene. V.B.
  • the plants obtained via transformation with a nucleic acid sequence of the presently disclosed subject matter can be any of a wide variety of plant species, including monocots and dicots; however, the plants used in the method for the presently disclosed subject matter are selected in some embodiments from the list of agronomically important target crops set forth hereinabove.
  • the expression of a gene of the presently disclosed subject matter in combination with other characteristics important for production and quality can be incorporated into plant lines through breeding. Breeding approaches and techniques are known in the art. See e.g., Welsh, 1981 ; Wood, 1983; Mayo, 1987; Singh, 1986; Wricke & Weber, 1986.
  • 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.
  • 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.
  • measures are undertaken to control weeds, plant diseases, insects, nematodes, and other adverse conditions to improve yield.
  • transgenic plants and seeds 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.
  • agrochemicals such as herbicides, fungicides, gametocides, nematicides, growth regulants, ripening agents, and insecticides.
  • Use of the advantageous genetic properties of the transgenic plants and seeds according to the presently disclosed subject matter can further be made in plant breeding, which aims at the development of plants with improved properties such as tolerance of pests, herbicides, or abiotic stress, improved nutritional value, increased yield, or improved structure causing less loss from lodging or shattering.
  • the various breeding steps are characterized by well-defined human intervention such as selecting the lines to be crossed, directing pollination of the parental lines, or selecting appropriate progeny plants.
  • 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.
  • Some embodiments of the presently disclosed subject matter also provide seed and isolated product from plants that comprise an expression cassette comprising a promoter sequence operatively linked to an isolated nucleic acid, the nucleic acid sequence being selected from the group consisting of: (a) a nucleotide sequence that hybridizes under conditions of hybridization of 45°C in 1 M NaCl, followed by a final washing step at 50°C in 0.1 M NaCl to a nucleotide sequence listed in odd numbered sequences of SEQ ID NOs: 1-105, or fragment, domain, or feature thereof; .
  • nucleotide sequence encoding a polypeptide that is an ortholog of a polypeptide of even numbered sequences of SEQ ID NOs: 2-106, or a fragment, domain or feature thereof;
  • a nucleotide sequence complementary for example, fully complementary
  • a nucleotide sequence that is the reverse complement for example, its full reverse complement
  • the isolated product comprises an enzyme, a nutritional polypeptide, a structural polypeptide, an amino acid, a lipid, a fatty acid, a polysaccharide, a sugar, an alcohol, an alkaloid, a carotenoid, a propanoid, a steroid, a pigment, a vitamin, or a plant hormone.
  • Embodiments of the presently disclosed subject matter also relate to isolated products produced by expression of an isolated nucleic acid containing a nucleotide sequence selected from the group consisting of: (a) a nucleotide sequence that hybridizes under conditions of hybridization of 45°C in 1 M NaCl, followed by a final washing step at 50°C in 0.1 M NaCl to a nucleotide sequence listed in odd numbered sequences of SEQ ID NOs: 1-105, or a fragment, domain, or feature thereof; (b) a nucleotide sequence encoding a polypeptide that is an ortholog of a polypeptide listed in even numbered sequences of SEQ ID NOs: 2-106, or a fragment, domain, or feature thereof; (c) a nucleotide sequence complementary (for example, fully complementary) to (a) or (b); and (d) a nucleotide sequence that is the reverse complement (for example, its full reverse complement) of (a) or (b) according to the present disclosure
  • the product is produced in a plant. In some embodiments, the product is produced in cell culture. In some embodiments, the product is produced in a cell-free system. In some embodiments, the product comprises an enzyme, a nutritional polypeptide, a structural polypeptide, an amino acid, a lipid, a fatty acid, a polysaccharide, a sugar, an alcohol, an alkaloid, a carotenoid, a propanoid, a steroid, a pigment, a vitamin, or a plant hormone. In some embodiments, the product is polypeptide comprising an amino acid sequence listed in even numbered sequences of SEQ ID NOs: 2-106, or ortholog thereof. In some embodiments, the polypeptide comprises an enzyme.
  • Propagation material to be used as seeds is customarily treated with a protectant coating comprising herbicides, insecticides, fungicides, bactericides, nematicides, molluscicides, or mixtures thereof.
  • 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
  • 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.
  • the presently disclosed subject matter also provides a method for altering an abiotic stress tolerance of a plant, the method comprising expressing in the plant an expression cassette comprising a nucleic acid molecule encoding a polypeptide as disclosed herein. In some embodiments, the method alters the drought resistance of the plant. Also provided is a method for enhancing the drought resistance of a plant, the method comprising (a) inserting into the plant an expression cassette comprising at least one isolated nucleic acid molecule of the presently disclosed subject matter; and (b) growing the plant comprising the expression cassette under suitable growth conditions, wherein the drought resistance of the plant is enhanced.
  • a method for producing a plant comprising a modification thereto including the steps of: (1 ) providing a nucleic acid comprising a nucleotide sequence selected from the group consisting of: (a) a nucleotide sequence that hybridizes under conditions of hybridization of 45°C in 1 M NaCl, followed by a final washing step at 50°C in 0.1 M NaCl to a nucleotide sequence listed in odd numbered sequences of SEQ ID NOs: 1-105, or 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, its full reverse complement) of (a); and
  • the modification comprises an altered trait in the plant, wherein the trait corresponds to the nucleic acid introduced into the plant.
  • the altered trait is related to a feature listed in any of Tables 1-6.
  • the trait can correspond to disease resistance, yield, abiotic stress tolerance, nutritional composition, carbon metabolism, photosynthesis, signal transduction, cell growth, reproduction, a disease process, or differentiation.
  • the modification comprises an increased or decreased expression or accumulation of a product of the plant.
  • the product comprises a natural product of the plant.
  • the product comprises a new or altered product of the plant.
  • the product comprises an enzyme, a nutritional polypeptide, a structural polypeptide, an amino acid, a lipid, a fatty acid, a polysaccharide, a sugar, an alcohol, an alkaloid, a carotenoid, a propanoid, a steroid, a pigment, a vitamin, or a plant hormone.
  • the presently disclosed subject matter provides a method for increasing drought resistance by delivering an effective amount of a product resulting from modification of the plant.
  • Also encompassed within the presently disclosed subject matter is a method for producing a recombinant polypeptide, the method comprising: (a) growing recombinant cells comprising a nucleic acid construct under suitable growth conditions, the 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 nucleotide sequences of SEQ ID NOs: 2-106, or a nucleic acid sequence that hybridizes under conditions of hybridization of 45°C in 1 M NaCl, followed by a final washing step at 50°C in 0.1 M NaCl to a nucleotide sequence listed in odd numbered nucleotide sequences of SEQ ID NOs: 1-105, or a subsequence thereof; and (b) isolating from the recombinant cells the recombinant polypeptide expressed thereby.
  • 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.
  • the nucleic acid construct comprises an epitope tag-encoding sequence and the isolating step employs an antibody specific for the epitope tag.
  • the nucleic acid construct comprises a polyamino acid-encoding sequence and the isolating step employs a resin comprising a polyamino acid binding substance.
  • the polyamino acid comprises polyhistidine and the polyamino acid binding resin comprises nickel-charged agarose resin.
  • the nucleic acid construct comprises a polypeptide-encoding sequence and the isolating step employs a resin containing a polypeptide binding substance.
  • the polypeptide comprises a chitin binding domain and the resin comprises chitin-sepharose.
  • the presently disclosed subject matter also provides a method for altering the expression of a polypeptide of the presently disclosed subject matter in a plant, the method comprising expressing an expression cassette encoding a polypeptide of the presently disclosed subject matter in the plant.
  • the polypeptide is expressed in a predetermined location or tissue of a plant.
  • 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.
  • 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 recombin
  • Embodiments of the presently disclosed subject matter also relate to a method for increasing the expression of an isolated nucleic acid molecule of the presently disclosed subject matter in a plant, the method comprising (a) inserting into the plant an expression cassette comprising an isolated nucleic acid molecule of the presently disclosed subject matter; and (b) growing the plant comprising the expression cassette under suitable growth conditions, wherein the expression of the isolated nucleic acid molecule of the presently disclosed subject matter is increased.
  • Embodiments of the presently disclosed subject matter also relate to a plant modified by a method that comprises introducing into the plant a nucleic acid where the nucleic acid is expressible in the plant in an amount effective to effect the modification.
  • the modification can be, for example, nutritional enhancement, increased nutrient uptake efficiency, enhanced production of endogenous compounds, and production of heterologous compounds.
  • the modified plant has increased or decreased resistance to a herbicide, an abiotic stress, or a pathogen.
  • the modified plant has enhanced or diminished requirement for light, water, nitrogen, or trace elements.
  • the modified plant is enriched for an essential amino acid as a proportion of a polypeptide fraction of the plant.
  • the polypeptide fraction can be, for example, total seed polypeptide, soluble polypeptide, insoluble polypeptide, water-extractable polypeptide, and lipid-associated polypeptide.
  • the modification can include, but is not limited to, overexpression, underexpression, antisense modulation, sense suppression, inducible expression, inducible repression, or inducible modulation of a gene.
  • the presently disclosed subject matter further relates to a seed from a modified plant or an isolated product of a modified plant, wherein the product includes, but is not limited to, an enzyme, a nutritional polypeptide, a structural polypeptide, an amino acid, a lipid, a fatty acid, a polysaccharide, a sugar, an alcohol, an alkaloid, a carotenoid, a propanoid, a steroid, a pigment, a vitamin, or a plant hormone.
  • the presently disclosed subject matter will be further disclosed by reference to the following detailed examples. These examples are provided for purposes of illustration only, and are not intended to be limiting unless otherwise specified.
  • Example 1 Plant Materials and Stress Treatment A recombinant inbred population was developed from the cross of B35, an inbred sorghum line with a stay-green, drought tolerant phenotype, and Tx7000, an elite, high yielding sorghum accession with a non-stay- green, post-flowering drought susceptible phenotype (Xu et al, 2000).
  • the two parental lines, two stay-green (stg) recombinant inbred lines (RILs; STG40 and STG62), and two non-stg RILs (NSG41 and NSG95) were used. Seeds of these lines were kindly provided by Dr. Darrell T. Rosenow of the Texas Agricultural Experiment Station in Lubbock, Texas. Plants were grown in the greenhouse in a randomized complete block design with three replications. Two sorghum plants were grown in five-gallon plastic pots filled with Ball Growing Mix (Ball Seed Co., Chicago, Illinois, United States of America), with three pots per treatment, a single pot serving as a replicate. Plants were watered every alternate day and fertilized (MIRACLE-GRO®) every two weeks.
  • Final irrigation was administered 3 days after anthesis to impose gradual water deficit treatment and to simulate natural post-flowering drought conditions.
  • Leaf samples were collected at 7, 14 and 21 days after withholding irrigation.
  • Relative water content (RWC) was monitored for each sampling date.
  • RWC of the plants was allowed to drop to ⁇ 65% and then watering was resumed.
  • Leaf sampling for the fourth treatment (7RW) was done 7 days after resuming irrigation.
  • Corresponding fully irrigated plants were sampled at 7, 14, 21 and 7RW and the leaf tissues pooled to serve as fully irrigated control.
  • Average greenhouse day conditions were 32°C, 43% humidity, 13-hour day length, 825 mE m-2 sec-1 light intensity.
  • Average greenhouse night temperature was 27°C.
  • the GENECHIP® microarray contained probe sets for over 20,000 rice genes designed from the O. sativa spp. japonica genome sequence (Goff et al, 2002). Expression data were normalized globally prior to data analysis. Genes with accurately detectable transcript levels were defined by probe sets showing averaged expression levels equal to or greater than 50 and those with lesser signals were adjusted to 50. Change in expression was calculated as fold- change difference from well-watered plants to corresponding dehydration stress treatments. While a two-fold change in gene expression cut-off was used by Zhu et al, 2002 a 1.5-fold or greater change cut-off was employed to compensate for expected lower signal intensities due to cross-species hybridization (Chismar e- ' a/., 2002).
  • Each transcript was assigned to functional categories based on the MIPS classification scheme (Schoof et al, 2002).
  • the scheme includes hierarchical structures of protein functions, enzymatic activities, and pathways.
  • the sequence was compared to currently classified protein sequences using FASTX and FASTX-i. Top hits from FASTA searches were parsed based on a 200 cut-off z-score. The sequence was then assigned with the same hierarchical classification as the top hits.
  • Adaptive clustering of expression profiles of B35 and Tx7000 was performed using an adaptive, quality-based algorithm to cluster gene expression data and find groups of genes that have a similar expression profile (see Smet et al, 2002).
  • the software uses an adaptive, quality- based clustering algorithm to cluster gene expression data to find groups of genes that have a similar expression profile.
  • MIN_NR_GENES define the minimum number of genes that can belong to a gene cluster and S defines the probability of a gene belonging to a specific cluster.
  • Table 1 General Trends in Expression Profile: Number of Transcripts that Exhibited 50% Change in Expression Level Relative to Well-irrigated Control at Corresponding Days After Initiation of Water Deficit Stress Treatments Induced Repressed
  • NSG41 569 176 222 139 585 170 153 285 NSG95 632 615 620 131 311 360 386 86 STG lines (in total) 1204 911 1036 808 1262 727 1382 1 130 NSG lines 0 (in total) 1156 932 843 372 883 700 563 434
  • transcripts in the parents under each sampling time point were classified according to MAtDB functional categories (Table 2; Schoof et al, 2002). Many of the identified transcripts were assigned to multiple categories and considered synonymous with
  • Example 5 Transcriptional Response of the RILs
  • Two stay-green lines with defined tolerant response to post-flowering drought stress and two non-stay-green lines susceptible to post-flowering drought were selected from the RIL population derived from a cross between B35 and Tx7000.
  • a greater number of drought-modulated transcripts were found in the two derived RILs than in the stay-green parent. More differentially regulated transcripts were also observed in the two non-stay- green RIL than in Tx7000 (Table 1 ).
  • fewer modulated transcripts were observed in the non-stg lines compared to the two stg lines.
  • Forty-three up-regulated genes were found common in all the stg lines and 25 in all the non-stg lines.
  • Twenty transcripts were commonly down regulated in all the stg lines and 9 in the non-stg lines (Tables 4 and
  • HNSb 38 a homeobox gene and HNSb 17, a phospholipid transfer protein
  • HNSb 39 a homeobox gene and HNSb 17, a phospholipid transfer protein
  • Annotation of the 43 induced transcripts suggests various potential roles related to stress response.
  • the 43 transcripts with expected stress-related functions are heat shock protein (HNSb 8) and a cation transport protein (OS018250.1_S_AT).
  • Transcripts with known stress-related functions include HNSb . 40 and HNSb 43 (detoxification), HNSb 31 (cellular reorganization), HNSb 45 (transcription regulation).
  • HNSb 30, HNSb 36, HNSb 48 A number of stress-related transcripts with unknown functions were also observed (HNSb 30, HNSb 36, HNSb 48).
  • transcripts commonly induced across all the stg lines are genes specific to photosystem II reaction center. Repressed functions common to all stg lines include protein degradation (HNSb 34) and signal transduction (HNSb 4, HNSb 29).
  • HNSb 14 2.15 2.20 1.85 24-methylene lophenol C24(1 )methyltransferase (O. sativa) HNSb 16 4.84 3.00 1.69 Thiozole biosynthetic enzyme 1-1 (Z. mays) HNSb 18 2.01 2.40 4.63 Ribosomal protein (A. thaliana) HNSb 21 1.89 1.54 3.39 Ribosomal protein (A. thaliana)
  • HNSb 23 2.32 1.74 1.63 Unknown H. vulgare cDNA
  • HNSb 24 1.67 2.36 3.56 ATP synthase (0. sativa)
  • HNSb 31 2.07 1.56 2.64 Histone H1 protein (Z. mays)
  • HNSb 37 1.58 1.62 2.11 ATHSAR1 EST (A. thaliana)
  • HNSb 40 2.61 4.08 4.26 NADH plastoquinone oxidoreductase (O. sativa)
  • HNSb 44 3.08 2.93 2.32 Cysteine protease component (A thaliana)
  • HNSb 51 1.68 1.54 1.53 Pistil extensin like protein (N. alata)
  • HNSb 59 1.70 1.55 3.57 Purple acid phosphatase precursor (A. thaliana) induced at 14 days
  • HNSb 8 2.93 8.94 2.59 Heat shock protein (O. sativa)
  • HNSb 15 1.62 1.55 2.64 Phenylalanine ammonia-lyase (H. vulgare)
  • HNSb 40 2.13 2.04 2.08 NADH plastoquinone oxidoreductase (O. sativa)
  • HNSb 9 2.13 2.09 1.94 Lipid transfer protein (O. sativa)
  • HNSb 14 1.64 1.71 1.74 24-methylene lophenol C24(1 )methyltransferase (O. sativa)
  • HNSb 17 4.90 2.48 1.71 Phospholipid transfer protein (O. sativa) induced at 7 days after rewatering
  • HNSb 39 1.54 1.52 1.96 Glucose:flavonoid 3-o- glucosyltransferase -like protein (Z. mays)
  • HNSb 46 2.01 2.90 3.39 Enolase (2-phospho-D-glycerate hydroylase) (Z. mays)
  • HNSb 53 1.54 1.76 1.60 Unknown H. vulgare cDNA repressed at 7 days
  • HNSb 60 -1.99 -1.65 -1.53 Unknown L. luteus cDNA repressed at 21 i days HNSb 2 -1.54 -1.54 -1.81 Unknown H. vulgare cDNA HNSb 7 -6.19 -2.20 -1.67 ATP synthase and tRNA-Met (A. crassa) HNSb 26 -1.85 -1.64 -1.64 Unknown G. max cDNA HNSb 29 -2.25 -1.70 -2.21 Calmodulin (A. thaliana) HNSb 34 -2.63 -2.55 -2.25 Ubiquitin conjugating enzyme (A.
  • HNSb 17 1.62 3.63 4.83 phospholipid transfer protein (O. sativa
  • HNSb 38 1.75 1.79 5.35 homeobox gene 13 protein (A. thaliana
  • Example 6 Gene Clusters with Common Expression Patterns Microarray analysis can generate a vast amount of data which help elucidate global gene expression which can infer cellular behavior. Clustering of genes with similar expression profile is one way of analyzing massive amount of data.
  • the parental expression profile was analyzed with an adaptive, quality-based software (Adapt_Cluster; Smet et al, 2002) to identify genes that share similar expression patterns. After global normalization of the expression data and removing probes showing unaltered levels of hybridization, a final set of 9056 probes was analyzed. A total of 2487 probes were assigned into 16 gene clusters at the set parameters.
  • Adapt_Cluster adaptive, quality-based software
  • Cluster 1 has the greatest number of genes (583 probes) followed by cluster 3 that contains 267 genes.
  • Cluster 1 contains genes which are continuously induced in the non-stay-green parent but continuously down-regulated in the stay-green parent across the duration of the imposed dehydration stress.
  • Cluster 3 shows the reverse pattern: continuously induced in B35 and continuously repressed in Tx7000 during water deficit.
  • Clusters 6 and 16 represent drought-responsive genes expressed only in B35 (Table 6).
  • Cluster 6 genes were induced only in the early stage of dehydration while cluster 9 shows two expression peaks at 7 and 21 days respectively.
  • Cluster 8 represents genes showing faster response to dehydration in B35 (7 days) but induced slower in Tx7000 (14 days).
  • Examples 1-6 By pair-wise comparison of samples, gene expression profiling can indicate possible association between a phenotype and changes in gene expression.
  • the association between genes and the resulting traits as revealed by transcription profiling could be used to develop plants with increased yields or with more resistance to diseases and tolerance to abiotic stresses (Zhu et al, 2003).
  • the variety of organisms for which transcription profiling can be carried out is limited by the lack of a genomic sequence data.
  • One solution to this limitation is to use microarrays designed for one species to profile RNA samples from a closely related species.
  • the Affymetrix U95A human gene array is effective at measuring the transcript profiles of chimpanzee, orangutan, and rhesus macaque as is the Mus musculus gene array able to measure the transcript profile of M. caroli and M. spretus (Chismar et al, 2002; Enard et al, 2002).
  • the presently disclosed subject matter measured the transcript profile of dehydration stressed sorghum using a rice GENECHIP® microarray.
  • the major cereal crops are highly related (Kellogg, 1998) and their transcriptomes likely detectable by the rice GENECHIP®.
  • a large number of the rice probes detect genes expressed in maize and barley (Zhu et al, 2001 a).
  • the present disclosure also shows that conservation of gene sequences between sorghum and rice was sufficient to generate good quality data. Even though the overall gene sequences of sorghum and rice and very similar, it was expected that not all of the 25-mer oligonucleotides that make up a probe set for a single gene will be alike. Therefore, a 1.5- fold change threshold was used rather than 2.0. The number of modulated transcripts with at least a 1.5 fold change in expression level in response to gradual water deficit ranges from a low of
  • the present disclosure indicates that in the drought resistant parent B35, transcriptional induction in response to drought was faster. However, general transcript down-regulation was found slower than in the susceptible parent. Taken together, these indicate a longer window of active and functional cellular processes in the resistant lines under drought conditions. The wide array of functional changes that occur in response to drought had been widely reviewed (Bray, 2002, Ramanjulu & Bartels 2002). Down-regulation of genes involved in photosynthesis or chloroplast organization has been reported (Parry et al, 2002; Lawlor & Comic 2002).
  • Down-regulated transcripts such as protease inhibitors and protein ubiquitination found in the stay-green lines indicate less protein degradation and turnover and probably less damage to proteins.
  • the markedly contrasting response of rice and sorghum to dehydration especially with respect to photosynthetic functions could indicate that each utilizes a different drought response mechanism.
  • RNA is isolated as described in Example 2, and double-stranded cDNA is prepared.
  • the cDNA is purified and cloned into a cDNA vector (for example, the SUPERSCRIPTTM Double-Stranded cDNA Synthesis Kit from Invitrogen) to produce a cDNA library.
  • the cDNA library is then screened using probes corresponding to odd numbered SEQ ID NOs: 1-105 under the conditions used to screening the GENECHIP® array.
  • the library is transferred to a solid support (for example, nitrocellulose or nylon) and hybridized to detectably-labeled probes corresponding to odd numbered SEQ ID NOs: 1-105 in a hybridization buffer consisting of 0.1 mg/mL sonicated and denatured herring sperm DNA, 100 mM 2-N-morpholino- ethane-sulphonic acid (MES), 1 M NaCl, 20 mM EDTA, 0.01 % Tween 20.
  • the hybridization is performed 45 °C for 16 hours on a rotisserie at 60 r.p.m.
  • the solid support is washed with a first wash buffer consisting of 6X SSPE (0.9 M NaCl, 0.06 M NaH 2 P0 4 , 0.006 M EDTA),
  • Binary destination vectors for plant transformation comprise or optionally consist of a binary backbone and a T-DNA portion.
  • the binary backbone contains the sequences necessary for selection and growth in
  • Escherichia coli DH-5 ⁇ (Invitrogen Corp., Carlsbad, California, United States of America) and Agrobacterium tumefaciens LBA4404, including the bacterial spectinomycin antibiotic resistance aadA gene from E. coli transposon Tn7, origins of replication for E. coli (ColEI ) and A. tumefaciens (VS1 ), and the A. tumefaciens virG gene.
  • the T-DNA portion is flanked by the right and left border sequences and includes the POSITECHTM (Syngenta Corp., Wilmington, Delaware, United States of America) plant selectable marker and a gene expression cassette that varies depending on the application.
  • the POSITECHTM plant selectable marker in this instance consists of a rice actin (ACT1) promoter driving expression of the phosphomannose isomerase (PMI) gene, followed by the cauliflower mosaic virus transcriptional terminator, and confers resistance to mannose.
  • the gene expression cassette portion of the binary destination vectors varies depending on the application.
  • the cassette comprises and optionally consists of a promoter designed to express the gene in certain tissues of the plant, followed by cloning sites (in some cases interrupted by a segment of spacer DNA), and finally by the A. tumefaciens nos 3' end transcriptional terminator.
  • the promoters used are designed to express the gene of interest in specific target tissues (e.g.
  • endosperm rice RS-4, wheat glutelin, maize ADPgpp or ⁇ -zein, or barley ⁇ -thionin; e.g. embryo: maize globulin or oleosin; e.g. aleurone: barley ⁇ -amylase; e.g. root: maize MSR1 and MRS3; e.g. green tissue: maize PEPC) or constitutively (e.g. maize UBI plus intron), depending on the gene of interest.
  • endosperm rice RS-4, wheat glutelin, maize ADPgpp or ⁇ -zein, or barley ⁇ -thionin
  • embryo maize globulin or oleosin
  • aleurone barley ⁇ -amylase
  • root maize MSR1 and MRS3
  • green tissue maize PEPC
  • constitutively e.g. maize UBI plus intron
  • the cloning site contains either unique restriction enzyme sites (for conventional cloning) and/or a GATEWAYTM recombination-based cloning cassette (Invitrogen Corp., Carlsbad, California, United States of America), in either the forward or reverse orientation.
  • dsRNA double-stranded interfering RNA
  • the cloning site is divided by a spacer region (e.g. first intron of the rice SH1 gene). The spacer permits the cloning of two gene fragments: one in the forward and one in the reverse orientation.
  • Antisense reverse orientation expression
  • Transformation of the nucleic acid molecules of the presently disclosed subject matter into plants is performed using methods disclosed herein above.
  • Crop Sci 40 1037-1048. Borrel AK & Hammer GL (2000) Nitrogen dynamics and physiological basis of stay-green in sorghum. Crop Sci 40: 1295-1307. Bourouis M & Jarry B (1983) Vectors containing a prokaryotic dihydrofolate reductase gene transform Drosophila cells to methotrexate- resistance. EMBO J 2:1099-1104. Bray EA (2002). Classification of genes differentially expressed during water- deficit stress in Arabidopsis thaliana: an analysis using Microarray and differential expression data. Ann Bot 89:803-811.
  • Gallie DR Kado Cl
  • Hershey JWB Wilson MA & Walbot V (1989) in Molecular Biology of RNA (Cech TR, ed.), Alan R. Liss, Inc., New York, New York, United States of America, pp. 237-256.
  • Goff SA Ricke D, Lan TH, Presting G, Wang RL, Dunn M,
  • Hiei Y Komari T & Kubo T (1997) Transformation of rice mediated by Agrobacterium tumefaciens. Plant Mol Biol 35:205-18. Hiei Y, Ohta S, Komari T & KumashiroY (1994) Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA. Plant J 6(2):271-282. H ⁇ fgen R & Willmitzer L (1988) Storage of competent cells for Agrobacterium transformation. Nucl Acids Res 16:9877.
  • Arabidopsis Genome Methods for Mapping with Recombinant Inbreds and Random Amplified Polymorphic DNAs (RAPDs) in Methods in Arabidopsis
  • Tuinstra MR, Ejeta G & Goldsbough PB (1998) Evaluation of near- isogenic sorghum lines contrasting for QTL markers associated with drought tolerance. Crop Sci. 38:835-842.
  • Vasil V Srivastava V, Castillo AM, Fromm MR & Vasil IK (1993) Rapid production of transgenic plants by direct bombardment of cultured immature embryos. Bio/Technology 11 :1553-1558. Wang RL, Stec A, Hey J, Lukens L & Doebley J (1999). The limits of selection during maize domestication. Nature 398:236-239. Warner SA, Scott R, Draper J (1993) Isolation of an asparagus intracellular PR gene (AoPRI) wound-responsive promoter by the inverse polymerase chain reaction and its characterization in transgenic tobacco. Plant J 3:191-201. Wasmann CC et al. (1986) Mol Gen Genet 205:446-453.
  • Hd1 a major photoperiod sensitivity quantitative trait locus in rice, is closely related to the Arabidopsis flowering time gene CONSTANS. Plant Cell 12:2473- 2483. Zhang HM, Yang H, Rech EL, Golds TJ, Davis AS , Mulligan BJ, Cocking EC & Davey MR (1988) Transgenic rice plants produced by electroporation-mediated plasmid uptake into protoplasts. Plant Cell Reports 7: 379-384. Zhang J, Zheng HG, Aarti A, Pantuwan G, Nguyen TT, Tripathy JN,

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Abstract

La présente invention a trait à un procédé pour l'amélioration de la résistance à la sécheresse d'une plante. Dans certains modes de réalisation, l'invention a également trait à des polynucléotides dérivés de Sorghum bicolor d'hybridation aux polynucléotides isolés de riz (Oryzasativa) et codant pour des polypeptides pour la tolérance au stress abiotique.
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CN102775481A (zh) * 2011-05-10 2012-11-14 中国农业大学 耐旱相关蛋白dt1及其编码基因和应用
EP2440033A4 (fr) * 2009-06-10 2013-03-20 Evogene Ltd Polynucléotides et polypeptides isolés, et procédés d'utilisation de ceux-ci pour augmenter l'efficacité d'utilisation de l'azote, le rendement, le taux de croissance, la vigueur, la biomasse, la teneur en huile, et/ou la tolérance au stress abiotique
WO2015119681A1 (fr) * 2014-02-04 2015-08-13 University Of Florida Research Foundation, Inc. Sequences de nucléotides et d'acides aminés de phytase de pteris vittata et leurs procédés d'utilisation
US9631000B2 (en) 2006-12-20 2017-04-25 Evogene Ltd. Polynucleotides and polypeptides involved in plant fiber development and methods of using same
US9670501B2 (en) 2007-12-27 2017-06-06 Evogene Ltd. Isolated polypeptides, polynucleotides useful for modifying water user efficiency, fertilizer use efficiency, biotic/abiotic stress tolerance, yield and biomass in plants
US10457954B2 (en) 2010-08-30 2019-10-29 Evogene Ltd. Isolated polynucleotides and polypeptides, and methods of using same for increasing nitrogen use efficiency, yield, growth rate, vigor, biomass, oil content, and/or abiotic stress tolerance
US10760088B2 (en) 2011-05-03 2020-09-01 Evogene Ltd. Isolated polynucleotides and polypeptides and methods of using same for increasing plant yield, biomass, growth rate, vigor, oil content, abiotic stress tolerance of plants and nitrogen use efficiency

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AU2005234725B2 (en) 2003-05-22 2012-02-23 Evogene Ltd. Methods of Increasing Abiotic Stress Tolerance and/or Biomass in Plants and Plants Generated Thereby
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AU2008236316B2 (en) 2007-04-09 2013-05-02 Evogene Ltd. Polynucleotides, polypeptides and methods for increasing oil content, growth rate and biomass of plants
CN102037127A (zh) 2007-07-24 2011-04-27 伊沃基因有限公司 多核苷酸及其编码的多肽和用于在表达所述多核苷酸、多肽的植物中用所述多核苷酸、多肽提高非生物胁迫耐受性和/或生物量和/或产量的方法
MX367882B (es) 2008-05-22 2019-09-10 Evogene Ltd Polinucleótidos y polipéptidos aislados y métodos para usarlos para incrementar el rendimiento de plantas, biomasa, velocidad de crecimiento, vigor, contenido de aceite, tolerancia al estrés abiótico de las plantas y eficiencia de uso de nitrógeno.
BR122021014193B1 (pt) 2008-08-18 2022-08-30 Evogene Ltd Método para aumentar a eficiência de uso do nitrogênio, eficiência de uso de fertilizantes, produção, taxa de crescimento, vigor, biomassa e/ou tolerância ao estresse por deficiência de nutriente de uma planta
MX389590B (es) 2008-10-30 2025-03-20 Evogene Ltd Polinucleotidos y polipeptidos aislados y metodos para utilizarlos para aumentar el rendimiento de la planta, biomasa, tasa de crecimiento, vigor, contenido de aceite, tolerancia al estres abiotico de las plantas y eficacia en el uso de nitrogeno.
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US9631000B2 (en) 2006-12-20 2017-04-25 Evogene Ltd. Polynucleotides and polypeptides involved in plant fiber development and methods of using same
US9670501B2 (en) 2007-12-27 2017-06-06 Evogene Ltd. Isolated polypeptides, polynucleotides useful for modifying water user efficiency, fertilizer use efficiency, biotic/abiotic stress tolerance, yield and biomass in plants
EP2440033A4 (fr) * 2009-06-10 2013-03-20 Evogene Ltd Polynucléotides et polypeptides isolés, et procédés d'utilisation de ceux-ci pour augmenter l'efficacité d'utilisation de l'azote, le rendement, le taux de croissance, la vigueur, la biomasse, la teneur en huile, et/ou la tolérance au stress abiotique
US10006040B2 (en) 2009-06-10 2018-06-26 Evogene Ltd. Isolated polynucleotides and polypeptides, and methods of using same for increasing nitrogen use efficiency, yield, growth rate, vigor, biomass, oil content, and/or abiotic stress tolerance
US10791690B2 (en) 2009-06-10 2020-10-06 Evogene Ltd. Isolated polynucleotides and polypeptides, and methods of using same for increasing nitrogen use efficiency, yield, growth rate, vigor, biomass, oil content, and/or abiotic stress tolerance
US11286495B2 (en) 2009-06-10 2022-03-29 Evogene Ltd. Isolated polynucleotides and polypeptides, and methods of using same for increasing nitrogen use efficiency, yield, growth rate, vigor, biomass, oil content, and/or abiotic stress tolerance
US10457954B2 (en) 2010-08-30 2019-10-29 Evogene Ltd. Isolated polynucleotides and polypeptides, and methods of using same for increasing nitrogen use efficiency, yield, growth rate, vigor, biomass, oil content, and/or abiotic stress tolerance
US10760088B2 (en) 2011-05-03 2020-09-01 Evogene Ltd. Isolated polynucleotides and polypeptides and methods of using same for increasing plant yield, biomass, growth rate, vigor, oil content, abiotic stress tolerance of plants and nitrogen use efficiency
CN102775481A (zh) * 2011-05-10 2012-11-14 中国农业大学 耐旱相关蛋白dt1及其编码基因和应用
CN102775481B (zh) * 2011-05-10 2014-04-02 中国农业大学 耐旱相关蛋白dt1及其编码基因和应用
WO2015119681A1 (fr) * 2014-02-04 2015-08-13 University Of Florida Research Foundation, Inc. Sequences de nucléotides et d'acides aminés de phytase de pteris vittata et leurs procédés d'utilisation

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