Classifications

• • 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
  • 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
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
  • C07K14/70571—Receptors; Cell surface antigens; Cell surface determinants for neuromediators, e.g. serotonin receptor, dopamine receptor

Definitions

• the field of the invention relates to plant breeding and genetics and, in particular, relates to recombinant DNA constructs useful in plants for conferring tolerance to abiotic stress, such as drought, and for improving nitrogen use efficiency.
• Abiotic stress is the primary cause of crop loss worldwide, causing average yield losses more than 50% for major crops (Boyer, J.S. (1982) Science 218:443-448; Bray, E.A. et al. (2000) In Biochemistry and Molecular Biology of Plants, edited by Buchannan, B.B. et al., Amer. Soc. Plant Biol., pp. 1 158-1249).
• drought is the major factor that limits crop productivity worldwide, and exposure of plants to a water-limiting environment during various developmental stages appears to activate various physiological and developmental changes.
• Activation tagging can be utilized to identify genes with the ability to affect a trait, and this approach has been used in Arabidopsis thaliana (the model plant species) (Weigel, D., et al. (2000) Plant Physiol. 122:1003-1013). Insertions of transcriptional enhancer elements can dominantly activate and/or elevate the expression of nearby endogenous genes, so this method can be used to select genes involved in agronomically important phenotypes, including abiotic stress tolerance such as improved drought tolerance and NUE.
• Glutamate receptors can bind glutamate and function through ligand gated ion channels or G-protein coupled receptors in the major excitatory
• GLR-like genes have been identified in plants
• GLRs may function in nutrient uptake, transport, and signaling (Davenport, supra), so manipulation of GLR genes may improve plant tolerance to stresses, particularly abiotic stress such as drought, cold, or low nitrogen.
• An isolated polynucleotide comprising: (a) a polynucleotide with nucleotide sequence of at least 85% sequence identity, based on either Clustal V or GAP method of alignment, to SEQ ID NO: 3; (b) a polynucleotide with nucleotide sequence of at least 85% sequence identity, based on either Clustal V or Gap method of alignment, to SEQ ID NO: 5; (c) a polynucleotide encoding a polypeptide with amino acid sequence of at least of 90% sequence identity, based on either Clustal V or GAP method of alignment, to SEQ ID NO:4; (d) a polynucleotide encoding a polypeptide with amino acid sequence of at least 90% sequence identity, based on either Clustal V or GAP method of alignment, to SEQ ID NO:6; or (e) the full complement of the nucleotide sequence of (a) a polynucleotide with nucleot
• a recombinant DNA construct comprising the isolated polynucleotide of embodiment 1 operably linked to at least one regulatory sequence.
• a plant or seed comprising a recombinant DNA construct, wherein the recombinant DNA construct comprises the polynucleotide operably linked to at least one regulatory sequence, wherein said polynucleotide encodes GLR protein having amino acid sequence of at least 90% sequence identity, based on either Clustal V or GAP method of alignment, to SEQ ID NO:4 or SEQ ID NO:6.
• a plant comprising in its genome a recombinant DNA construct comprising a polynucleotide operably linked to at least one regulatory element, wherein said polynucleotide encodes GLR protein having amino acid sequence of at least 90% sequence identity, based on either Clustal V or GAP method of alignment, to SEQ ID NO:4 or SEQ ID NO:6, wherein said plant exhibits increased drought tolerance and/or improved nitrogen use efficiency (NUE) when compared to a control plant, and wherein said plant exhibits an increase in grain yield, biomass, or both, when compared to a control plant.
• NUE nitrogen use efficiency
• a rice plant comprising a recombinant transcriptional activator element enhancing the expression of an endogenous polynucleotide, wherein the
• polynucleotide encodes an amino acid sequence that is 90% identical to SEQ ID NO: 4 or SEQ ID NO: 6.
• a method of improving drought tolerance or NUE comprising: (a) crossing the plant of embodiment 8 with a second rice plant to produce progeny seed; (b) harvesting and planting the progeny seed to produce at least one progeny plant of a subsequent generation which comprises the insertion between positions 15760218 and 15760298 in Chromosome 9 of the rice genome; (c) crossing the progeny plant with the second rice plant to produce at least one backcross progeny seed; and optionally (d) repeating steps (b) and (c) for additional generations to produce a plant with improved drought tolerance or NUE when compared to the second rice plant.
• a method of increasing drought tolerance in a plant comprising: (a) introducing into a regenerable plant cell a recombinant DNA construct comprising a polynucleotide operably linked to at least one regulatory sequence, wherein the polynucleotide encodes a polypeptide having an amino acid sequence of at least 90% sequence identity, based on either Clustal V or GAP method of alignment, to SEQ ID NO: 4 or SEQ ID NO: 6; (b) regenerating a transgenic plant from the regenerable plant cell after step (a), wherein the transgenic plant comprises in its genome the recombinant DNA construct; and (c) obtaining a progeny plant derived from the transgenic plant of step (b), wherein said progeny plant comprises in its genome the recombinant DNA construct and exhibits increased drought tolerance when compared to a control plant.
• a method of evaluating drought tolerance in a plant comprising: (a) obtaining a transgenic plant, wherein the transgenic plant comprises in its genome a recombinant DNA construct comprising a polynucleotide operably linked to at least one regulatory element, wherein the polynucleotide encodes a polypeptide having an amino acid sequence of at least 90% sequence identity, based on either Clustal V or GAP method of alignment, to SEQ ID NO: 4 or SEQ ID NO: 6; (b) obtaining a progeny plant derived from the transgenic plant, wherein the progeny plant comprises in its genome the recombinant DNA construct; and (c) evaluating the progeny plant for drought tolerance compared to a control plant not comprising the recombinant DNA construct.
• a method of determining an alteration of grain yield, biomass, or both in a plant comprising: (a) obtaining a transgenic plant, wherein the transgenic plant comprises in its genome a recombinant DNA construct comprising a polynucleotide operably linked to at least one regulatory element, wherein the polynucleotide encodes a polypeptide having an amino acid sequence of at least 90% sequence identity, based on either Clustal V or GAP method of alignment, to SEQ ID NO: 4 or SEQ ID NO: 6; (b) obtaining a progeny plant derived from the transgenic plant, wherein the progeny plant comprises in its genome the recombinant DNA construct; and (c) measuring the grain yield and/or biomass of the progeny plant and comparing said measurement to the grain yield and/or biomass of a control plant.
• the present disclosure concerns a recombinant DNA construct comprising any of the isolated polynucleotides of the present disclosure operably linked to at least one regulatory sequence, and a cell, a plant, or a seed comprising the recombinant DNA construct.
• the cell may be eukaryotic, e.g., a yeast, insect or plant cell; or prokaryotic, e.g., a bacterial cell.
• Figure 1 provides a schematic of the A-vector used to generate the rice activation-tagged population.
• CaMV 35S enhancer 4X CaMV 35S enhancer
• LTP2::RFP red fluorescent protein
• LB left border
• UBLhygromycin resistance gene Hyg r
• Spacer 15-kb "spacer” is positioned between the 4X CaMV35S enhancer and the Hyg r .
• Amp r ampicillin resistance gene
• Hyg r hygromycin resistance gene
• UBI maize ubiquitin promoter
• LTP2 barley lipid transfer protein 2 promoter.
• Figures 2A and 2B show the activated expression levels of GLR1 and GLR2 genes in different tissues of line AH01486 plants as revealed by real-time PCR analyses.
• Zhonghual 1 -WT wild type of Zhonghual 1
• Zhonghual 1 -TC Zhonghual 1 from tissue culture without a construct
• R roots
• S stems
• L leaves.
• the numbers on top of the columns are the fold-changes compared to the control leaves.
• 2A. for GLR1 gene AH01486 fold-changes in expression levels are 17.74 for roots, 3.95 for stems, and 53.83 for leaves
• 2B. for GLR2 gene AH01486 fold-changes in expression levels are 1 .00 for roots, 1 .45 for stems, and 38.45 for leaves.
• Figure 3 provides GLR2 transgene expression levels in leaves of eight separate transgenic rice events compared to control (CK) by real-time PCR analyses.
• the base level of expression in CK event was set at 1 .00, and the expression levels in other GLR2 events are shown as fold-increases compared to control.
• Figure 4 provides GLR1 transgene expression levels in leaves of transgenic rice events compared to control (CK) by real-time PCR analyses.
• the base level of expression in CK event was set at 1 .00, and the expression levels in other GLR1 events are shown as fold-increases compared to control.
• Figure 5 provides photograph indicating more tillers of rice plants over- expressing GLR2 under CaMV 35S promoter (DP0015, T1 generation) than T1 rice plants introduced with vector control (CK, T1 generation), under low nitrogen conditions.
• Figure 6A-C provides photographs and figures indicating that over-expression of rice GLR2 gene under CaMV 35S promoter in Arabidopsis (AtDP0015.05)
• AtDP0015.06 can significantly increase drought tolerance.
• Figure 7A-C provides photographs and figures indicating that over-expression of rice GLR1 gene under CaMV 35S promoter in Arabidopsis (AtDP0025.13,
• AtDP0025.16 can significantly increase drought tolerance.
• C photographs taken on the 3 rd day after re-watering.
• WT wild type Columbia;
• VC vector control DP0009 transformant.
• the Sequence Listing contains the one-letter code for nucleotide sequences and the three-letter code for amino acid sequences as defined in conformity with the lUPAC-IUBMB standards described in Nucleic Acids Res. 73:3021 -3030 (1985) and in the Biochemical J. 219 (No.2 ⁇ :345-373 (1984) which are herein incorporated by reference.
• the symbols and format used for nucleotide and amino acid sequence data comply with the rules set forth in 37 C.F.R. ⁇ 1 .822.
• GLR polypeptide refers to a glutamate receptor polypeptide.
• GLR1 and GLR2 are examples of GLR polypeptides.
• GLR1 refers to the protein (SEQ ID NO: 4) encoded by SEQ ID NO: 3
• GLR2 refers to the protein (SEQ ID NO: 6) encoded by SEQ ID NO: 5.
• a monocot of the current invention includes plants of the Gramineae family.
• a dicot of the current invention includes the following families: Brassicaceae, Leguminosae, and Solanaceae.
• nucleotide sequence refers to a complement of a given nucleotide sequence, wherein the complement and the nucleotide sequence consist of the same number of nucleotides and are 100% complementary.
• EST is a DNA sequence derived from a cDNA library and therefore represents a sequence which has been transcribed.
• An EST is typically obtained by a single sequencing pass of a cDNA insert.
• the sequence of an entire cDNA insert is termed the "Full-Insert Sequence” (“FIS").
• FIS Frull-Insert Sequence
• a "Contig” sequence is a sequence assembled from two or more sequences that can be selected from, but not limited to, the group consisting of an EST, FIS and PCR sequence.
• a sequence encoding an entire or functional protein is termed a
• CCS Complete Gene Sequence
• trait refers to a physiological, morphological, biochemical, or physical characteristics of a plant or particular plant material or cell. In some instances, this characteristics is visible to the human eye, such as seed or plant size, or can be measured by biochemical techniques, such as detecting the protein, starch, or oil content of seed or leaves, or by observation of a metabolic or physiological process, e.g. by measuring tolerance to water deprivation or particular salt or sugar or nitrogen concentrations, or by the observation of the expression level of a gene or genes, or by agricultural observations such as osmotic stress tolerance or yield.
• Agronomic characteristic is a measurable parameter including but not limited to: greenness, grain yield, growth rate, total biomass or rate of accumulation, fresh weight at maturation, dry weight at maturation, fruit yield, seed yield, total plant nitrogen content, fruit nitrogen content, seed nitrogen content, nitrogen content in a vegetative tissue, total plant free amino acid content, fruit free amino acid content, seed free amino acid content, free amino acid content in a vegetative tissue, total plant protein content, fruit protein content, seed protein content, protein content in a vegetative tissue, drought tolerance, nitrogen uptake, root lodging, harvest index, stalk lodging, plant height, ear height, ear length, salt tolerance, tiller number, panicle size, early seedling vigor and seedling emergence under low temperature stress.
• Increased biomass can be measured, for example, as an increase in plant height, plant total leaf area, plant fresh weight, plant dry weight or plant seed yield, as compared with control plants.
• Crop cultivars may be developed to produce higher yield of the vegetative portion of the plant, to be used in food, feed, fiber, and/or biofuel.
• Increased leaf size may be of particular interest.
• Increased leaf biomass can be used to increase production of plant-derived pharmaceutical or industrial products.
• Increased tiller number may be of particular interest and can be used to increase yield.
• An increase in total plant photosynthesis is typically achieved by increasing leaf area of the plant.
• Additional photosynthetic capacity may be used to increase the yield derived from particular plant tissue, including the leaves, roots, fruits or seed, or permit the growth of a plant under decreased light intensity or under high light intensity.
• Modification of the biomass of another tissue, such as root tissue may be useful to improve a plant's ability to grow under harsh environmental conditions, including drought or nutrient deprivation, because larger roots may better reach or take up water or nutrients.
• Transgenic refers to any cell, cell line, callus, tissue, plant part or plant, the genome of which has been altered by the presence of a heterologous nucleic acid, such as a recombinant DNA construct, including those initial transgenic events as well as those created by sexual crosses or asexual propagation from the initial transgenic event.
• a heterologous nucleic acid such as a recombinant DNA construct
• the term “transgenic” used herein does not encompass the alteration of the genome (chromosomal or extra-chromosomal) by conventional plant breeding methods or by naturally occurring events such as random cross- fertilization, non-recombinant viral infection, non-recombinant bacterial
• a "control” or “control plant” or “control plant cell” provides a reference point for measuring changes in phenotype of a subject plant or plant cell in which genetic alteration, such as transformation, has been effected as to a gene of interest.
• a subject plant or plant cell may be descended from a plant or cell so altered and will comprise the alteration.
• a control plant or plant cell may comprise, for example: (a) a wild-type plant or cell, i.e., of the same genotype as the starting material for the genetic alteration which resulted in the subject plant or cell; (b) a plant or plant cell of the same genotype as the starting material but which has been transformed with a null construct (i.e., with a construct which has no known effect on the trait of interest, such as a construct comprising a marker gene); (c) a plant or plant cell which is a non-transformed segregant among progeny of a subject plant or plant cell; (d) a plant or plant cell genetically identical to the subject plant or plant cell but which is not exposed to a condition or stimulus that would induce expression of the gene of interest; or (e) the subject plant or plant cell itself, under conditions in which the gene of interest is not expressed.
• a wild-type plant or cell i.e., of the same genotype as the starting material for the genetic alteration which resulted in the subject plant or cell
• Gene as it applies to plant cells encompasses not only chromosomal DNA found within the nucleus, but also organelle DNA found within subcellular
• components e.g., mitochondria, plastid
• Plant includes reference to whole plants, plant organs, plant tissues, seeds and plant cells and progeny of the same.
• Plant cells include, without limitation, cells from seeds, suspension cultures, embryos, meristematic regions, callus tissues, leaves, roots, shoots, gametophytes, sporophytes, pollen, and microspores.
• Progeny comprises any subsequent generation of a plant.
• Transgenic plant includes reference to a plant which comprises within its genome a heterologous polynucleotide.
• heterologous polynucleotide For example, the heterologous
• polynucleotide is stably integrated within the genome such that the polynucleotide is passed on to successive generations.
• the heterologous polynucleotide may be integrated into the genome alone or as part of a recombinant DNA construct.
• a TO plant is directly recovered from the transformation and regeneration process.
• T1 first progeny generation
• T2 second progeny generation
• Heterologous with respect to sequence means a sequence that originates from a foreign species, or, if from the same species, is substantially modified from its native form in composition and/or genomic locus by deliberate human intervention.
• nucleic acid sequence refers to a polymer of RNA or DNA that is single- or double-stranded, optionally containing synthetic, non-natural or altered nucleotide bases. Nucleotides (usually found in their 5'-monophosphate form) are referred to by their single-letter designation as follows: “A” for adenylate or deoxyadenylate, “C” for cytidylate or deoxycytidylate, and “G” for guanylate or deoxyguanylate for RNA or DNA, respectively; "U” for uridylate; "T” for
• Polypeptide”, “peptide”, “amino acid sequence” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers.
• the terms “polypeptide”, “peptide”, “amino acid sequence”, and “protein” are also inclusive of modifications including, but not limited to, glycosylation, lipid attachment, and sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation and ADP-ribosylation.
• RNA essential RNA (mRNA) refers to the RNA which has no intron and can be translated into protein by the cell.
• cDNA refers to a DNA that is complementary to and synthesized from an mRNA template using reverse transcriptase.
• the cDNA can be single-stranded or converted into the double-stranded form using the Klenow fragment of DNA polymerase I.
• “Mature” protein refers to a post-translationally processed polypeptide; i.e., any pre- or pro-peptides present in the primary translation product has been removed.
• Precursor protein refers to the primary product of translation of mRNA; i.e., with pre- and pro-peptides still present. Pre- and pro-peptides may be and are not limited to intracellular localization signals.
• isolated refers to materials, such as nucleic acid molecules and/or proteins, which are substantially free or otherwise removed from components that normally accompany or interact with the materials in a naturally occurring environment.
• Isolated polynucleotides may be purified from a host cell in which they naturally occur. Conventional nucleic acid purification methods known to skilled artisans may be used to obtain isolated polynucleotides. The term also embraces recombinant polynucleotides and chemically synthesized polynucleotides.
• Recombinant refers to an artificial combination of two otherwise separated segments of sequence, e.g., by chemical synthesis or by the manipulation of isolated segments of nucleic acids by genetic engineering techniques. "Recombinant” also includes reference to a cell or vector, that has been modified by the introduction of a heterogonous nucleic acid or a cell derived from a cell so modified, but does not encompass the alteration of the cell or vector by naturally occurring events (e.g., spontaneous mutation, natural transformation/transduction/transposition) such as those occurring without deliberate human intervention.
• naturally occurring events e.g., spontaneous mutation, natural transformation/transduction/transposition
• Recombinant DNA construct refers to a combination of nucleic acid fragments that are not normally found together in nature. Accordingly, a recombinant DNA construct may comprise regulatory sequences and coding sequences that are derived from different sources, or regulatory sequences and coding sequences derived from the same source, but arranged in a manner different than that normally found in nature.
• regulatory sequences refer to nucleotide sequences located upstream (5' non- coding sequences), within, or downstream (3' non-coding sequences) of a coding sequence, and influencing the transcription, RNA processing or stability, or translation of the associated coding sequence. Regulatory sequences may include, but are not limited to, promoters, translation leader sequences, introns, and poly- adenylation recognition sequences. The terms “regulatory sequence” and “regulatory element” are used interchangeably herein.
• Promoter refers to a nucleic acid fragment capable of controlling transcription of another nucleic acid fragment.
• Promoter functional in a plant is a promoter capable of controlling transcription of genes in plant cells whether or not its origin is from a plant cell.
• tissue-specific promoter and “tissue-preferred promoter” may refer to a promoter that is expressed predominantly but not necessarily exclusively in one tissue or organ, but that may also be expressed in one specific cell or cell type.
• “Developmentally regulated promoter” refers to a promoter whose activity is determined by developmental events.
• “Operably linked” refers to the association of nucleic acid fragments in a single fragment so that the function of one is regulated by the other.
• a promoter is operably linked with a nucleic acid fragment when it is capable of regulating the transcription of that nucleic acid fragment.
• “Expression” refers to the production of a functional product.
• expression of a nucleic acid fragment may refer to transcription of the nucleic acid fragment (e.g., transcription resulting in mRNA or functional RNA) and/or translation of mRNA into a precursor or mature protein.
• Phenotype means the detectable characteristics of a cell or organism.
• recombinant DNA construct into a cell, means “transfection” or “transformation” or “transduction” and includes reference to the incorporation of a nucleic acid fragment into a eukaryotic or prokaryotic cell where the nucleic acid fragment may be incorporated into the genome of the cell (e.g., chromosome, plasmid, plastid or mitochondrial DNA), converted into an autonomous replicon, or transiently expressed (e.g., transfected mRNA).
• a nucleic acid fragment may be incorporated into the genome of the cell (e.g., chromosome, plasmid, plastid or mitochondrial DNA), converted into an autonomous replicon, or transiently expressed (e.g., transfected mRNA).
• a “transformed cell” is any cell into which a nucleic acid fragment (e.g., a recombinant DNA construct) has been introduced.
• Transformation refers to both stable transformation and transient transformation.
• “Stable transformation” refers to the introduction of a nucleic acid fragment into a genome of a host organism resulting in genetically stable inheritance. Once stably transformed, the nucleic acid fragment is stably integrated in the genome of the host organism and any subsequent generation.
• Transient transformation refers to the introduction of a nucleic acid fragment into the nucleus, or DNA-containing organelle, of a host organism resulting in gene expression without genetically stable inheritance.
• an "allele” is one of two or more alternative forms of a gene occupying a given locus on a chromosome.
• that plant is homozygous at that locus.
• that plant is heterozygous at that locus.
• a transgene is present on one of a pair of homologous chromosomes in a diploid plant, that plant is hemizygous at that locus.
• chloroplast transit peptide is an amino acid sequence which is translated in conjunction with a protein and directs the protein to the chloroplast or other plastid types present in the cell in which the protein is made.
• Chloroplast transit sequence refers to a nucleotide sequence that encodes a chloroplast transit peptide.
• a “signal peptide” is an amino acid sequence which is translated in conjunction with a protein and directs the protein to the secretory system (Chrispeels. (1991 ) >4nn. Rev. Plant Phys. Plant Mol. Biol. 42:21 -53).
• a vacuolar targeting signal can further be added, or if to the endoplasmic reticulum, an endoplasmic reticulum retention signal (supra) may be added.
• any signal peptide present should be removed and instead a nuclear localization signal included (Raikhel. (1992) Plant Phys. 100 ⁇ 627-1632).
• a "mitochondrial signal peptide” is an amino acid sequence which directs a precursor protein into the mitochondria (Zhang and Glaser. (2002) Trends Plant Sci 7:14-21 ). Methods to determine the relationship of various polynucleotide and polypeptide sequences are known.
• reference sequence is a defined sequence used as a basis for sequence comparison.
• a reference sequence may be a subset or the entirety of a specified sequence, such as a segment of a full-length cDNA or gene sequence, or may be the complete cDNA or gene sequence.
• comparison window makes reference to a contiguous and specified segment of a polynucleotide or polypeptide sequence, wherein the sequence in the comparison window may comprise additions or deletions (i.e., gaps) compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences.
• the comparison window is at least 20 contiguous nucleotides or amino acids in length, and optionally can be 30, 40, 50, 100 or longer.
• a gap penalty is typically introduced and is subtracted from the number of matches.
• Computer implementations of these mathematical algorithms can be utilized for comparison of sequences to determine sequence identity. Such implementations include, but are not limited to: CLUSTAL in the PC/Gene program (available from Intelligenetics, Mountain View, California); the ALIGN program (Version 2.0) and GAP, BESTFIT, BLAST, FASTA and TFASTA in the GCG Wisconsin Genetics Software Package, Version 10 (available from Accelrys Inc., 9685 Scranton Road, San Diego, California, USA); and the Megalign® program of the LASERGENE® bioinformatics computing suite (DNASTAR® Inc., Madison, Wl).
• Alignments using these programs can be performed using the default parameters.
• the CLUSTAL program is well described by Higgins, et al. (1988) Gene 73:237-244; Higgins, et al. (1989) CABIOS 5:151 -153; Corpet, et al. (1988) Nucleic Acids Res. 16:10881 -10890; Huang, et al. (1992) CABIOS 8: 155-165 and Pearson, et al. (1994) Meth. Mol. Biol. 24:307-331 .
• the ALIGN program is based on the algorithm of Myers and Miller, (1988) supra.
• a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4 can be used with the ALIGN program when comparing amino acid sequences.
• the BLAST programs of Altschul, et al. (1990) J. Mol. Biol. 215:403 are based on the algorithm of Karlin and Altschul. (1990) supra.
• Gapped BLAST in BLAST 2.0
• PSI-BLAST in BLAST 2.0
• the default parameters of the respective programs e.g., BLASTN for nucleotide sequences, BLASTX for proteins
• Alignment may also be performed by manual inspection.
• Paired sequence identity/similarity values can be obtained using GAP Version 10 with the following parameters: % identity and % similarity for a nucleotide sequence using GAP Weight of 50 and Length Weight of 3 and the nwsgapdna.cmp scoring matrix; % identity and % similarity for an amino acid sequence using GAP Weight of 8 and Length Weight of 2, and the BLOSUM62 scoring matrix; or any equivalent program thereof.
• equivalent program is intended any sequence comparison program that, for any two sequences in question, generates an alignment having identical nucleotide or amino acid residue matches and an identical percent sequence identity when compared to the corresponding alignment generated by GAP Version 10.
• GAP uses the algorithm of Needleman and Wunsch. (1970) J. Mol. Biol. 48:443- 453, to find the alignment of two complete sequences that maximizes the number of matches and minimizes the number of gaps. GAP considers all possible alignments and gap positions and creates the alignment with the largest number of matched bases and the fewest gaps. It allows for the provision of a gap creation penalty and a gap extension penalty in units of matched bases. GAP must make a profit of gap creation penalty number of matches for each gap it inserts. If a gap extension penalty greater than zero is chosen, GAP must, in addition, make a profit for each gap inserted of the length of the gap times the gap extension penalty.
• gap creation penalty values and gap extension penalty values in Version 10 of the GCG Wisconsin Genetics Software Package for protein sequences are 8 and 2, respectively.
• the default gap creation penalty is 50 while the default gap extension penalty is 3.
• the gap creation and gap extension penalties can be expressed as an integer selected from the group of integers consisting of from 0 to 200.
• the gap creation and gap extension penalties can be 0, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65 or greater.
• GAP presents one member of the family of best alignments. There may be many members of this family, but no other member has a better quality. GAP displays four figures of merit for alignments: Quality, Ratio, Identity and Similarity.
• the Quality is the metric maximized in order to align the sequences. Ratio is the Quality divided by the number of bases in the shorter segment.
• Percent Identity is the percent of the symbols that actually match.
• Percent Similarity is the percent of the symbols that are similar. Symbols that are across from gaps are ignored.
• a similarity is scored when the scoring matrix value for a pair of symbols is greater than or equal to 0.50, the similarity threshold.
• the scoring matrix used in Version 10 of the GCG Wisconsin Genetics Software Package is BLOSUM62 (Henikoff and Henikoff. (1989) Proc. Natl. Acad. Sci. USA 89:10915).
• sequence identity or “identity” in the context of two
• polynucleotides or polypeptide sequences makes reference to the residues in the two sequences that are the same when aligned for maximum correspondence over a specified comparison window.
• percentage of sequence identity is used in reference to proteins it is recognized that residue positions which are not identical often differ by conservative amino acid substitutions, where amino acid residues are substituted for other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity) and therefore do not change the functional properties of the molecule.
• sequences differ in conservative substitutions the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Sequences that differ by such conservative substitutions are said to have "sequence similarity" or “similarity”. Means for making this adjustment are well known to those of skill in the art. Typically this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing the
• percentage of sequence identity is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison, and multiplying the result by 100.
• Standard recombinant DNA and molecular cloning techniques used herein are well known in the art and are described more fully in Sambrook, J., Fritsch, E.F. and Maniatis, T. Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, 1989 (hereinafter "Sambrook”).
• Embodiments include isolated polynucleotides and polypeptides, and
• the present disclosure includes the following isolated polynucleotides and polypeptides:
• An isolated polynucleotide comprising: (i) a nucleic acid sequence encoding a polypeptide having at least 50%, 51 %, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61 %, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, based on either Clustal V or GAP method of alignment, to SEQ ID NO: 4 or 6; or (ii) a full complement of the nucleic acid sequence of (i), wherein the full complement and the nucleic acid sequence of (i)
• polynucleotides may be utilized in any recombinant DNA constructs (including suppression DNA constructs) of the present disclosure.
• Over-expression of the encoded polypeptide preferably increases plant drought tolerance activity and/or NUE.
• the polypeptide is preferably a GLR polypeptide. Over-expression of the polypeptide preferably increases plant drought tolerance activity and/or NUE.
• An isolated polynucleotide comprising (i) a nucleic acid sequence of at least
• the isolated polynucleotide may be utilized in any recombinant DNA constructs (including suppression DNA constructs) of the present disclosure.
• the isolated polynucleotide preferably encodes a GLR polypeptide. Over-expression of the GLR polypeptide preferably improves plant drought tolerance activity and/or NUE.
• the present disclosure includes recombinant DNA constructs (including suppression DNA constructs).
• a recombinant DNA construct comprises a polynucleotide operably linked to at least one regulatory sequence (e.g., a promoter functional in a plant), wherein the polynucleotide comprises (i) a nucleic acid sequence encoding an amino acid sequence of at least 50%, 51 %, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61 %, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, based on either Clustal V or GAP method of alignment, to
• a recombinant DNA construct comprises a
• polynucleotide operably linked to at least one regulatory sequence (e.g., a promoter functional in a plant), wherein said polynucleotide comprises (i) a nucleic acid sequence of at least 50%, 51 %, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61 %, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, based on either Clustal V or GAP method of alignment, to SEQ ID NO: 3 or 5; or (ii) a full complement of the nucle
• a recombinant DNA construct comprises a
• the GLR polypeptide preferably has drought tolerance activity and/or improved NUE.
• the GLR polypeptide may be from, for example, Oryza sativa, Arabidopsis thaliana, Zea mays, Glycine max, Glycine tabacina, Glycine soja or Glycine tomentella.
• the present disclosure includes suppression DNA constructs.
• a suppression DNA construct may comprise at least one regulatory sequence (e.g., a promoter functional in a plant) operably linked to (a) all or part of: (i) a nucleic acid sequence encoding a polypeptide having an amino acid sequence of at least 50%, 51 %, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61 %, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, based on either Clustal V or GAP method of alignment, to SEQ ID NO: 4 or 6, or (ii)
• the suppression DNA construct may comprise a cosuppression construct, antisense construct, viral-suppression construct, hairpin suppression construct, stem-loop suppression construct, double- stranded RNA-producing construct, RNAi construct, or small RNA construct (e.g., an siRNA construct or an miRNA construct).
• a codon for the amino acid alanine, a hydrophobic amino acid may be substituted by a codon encoding another less hydrophobic residue, such as glycine, or a more hydrophobic residue, such as valine, leucine, or isoleucine.
• “Suppression DNA construct” is a recombinant DNA construct which when transformed or stably integrated into the genome of the plant, results in “silencing” of a target gene in the plant.
• the target gene may be endogenous or transgenic to the plant.
• “Silencing,” as used herein with respect to the target gene, refers generally to the suppression of levels of mRNA or protein/enzyme expressed by the target gene, and/or the level of the enzyme activity or protein functionality.
• suppression include lowering, reducing, declining, decreasing, inhibiting, eliminating or preventing.
• RNAi-based approaches RNAi-based approaches
• small RNA-based approaches RNAi-based approaches
• a suppression DNA construct may comprise a region derived from a target gene of interest and may comprise all or part of the nucleic acid sequence of the sense strand (or antisense strand) of the target gene of interest.
• the region may be 100% identical or less than 100% identical (e.g., at least 50%, 51 %, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61 %, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical) to all or part of the sense strand (or antisense strand) of the
• Suppression DNA constructs are well-known in the art, are readily constructed once the target gene of interest is selected, and include, without limitation,
• RNAi RNA interference
• small RNA constructs such as siRNA (short interfering RNA) constructs and miRNA (microRNA) constructs.
• Antisense inhibition refers to the production of antisense RNA transcripts capable of suppressing the expression of the target gene or gene product.
• Antisense RNA refers to an RNA transcript that is complementary to all or part of a target primary transcript or mRNA and that blocks the expression of a target isolated nucleic acid fragment (for example, U.S. Patent No. 5,107,065).
• complementarity of an antisense RNA may be with respect to any part of the specific gene transcript, i.e., at the 5' non-coding sequence, 3' non-coding sequence, introns, or the coding sequence.
• Codon refers to the production of sense RNA transcripts capable of suppressing the expression of the target gene or gene product.
• Sense RNA refers to RNA transcript that includes the mRNA and can be translated into protein within a cell or in vitro. Cosuppression constructs in plants have been previously designed by focusing on over-expression of a nucleic acid sequence having homology to a native mRNA, in the sense orientation, which results in the reduction of all RNA having homology to the overexpressed sequence (Vaucheret et al. (1998) Plant J. 16:651 - 659; and Gura. (2000) Nature 404:804-808).
• RNA interference refers to the process of sequence-specific post- transcriptional gene silencing (PTGS) in animals mediated by short interfering RNAs (siRNAs) (Fire et al. (1998) Nature 391 :806).
• PTGS sequence-specific post- transcriptional gene silencing
• siRNAs short interfering RNAs
• quelling in fungi.
• the process of PTGS is thought to be an evolutionarily-conserved cellular defense mechanism used to prevent the expression of foreign genes and is commonly shared by diverse flora and phyla (Fire et al. (1999) Trends Genet.
• Small RNAs play an important role in controlling gene expression, for example, small RNAs regulate many developmental processes which include flowering. It is now possible to engineer changes in gene expression of plant genes by using transgenic constructs which produce small RNAs in the plant.
• Small RNAs appear to function by base-pairing to complementary RNA or DNA target sequences. When bound to RNA, small RNAs trigger either RNA cleavage or translational inhibition of the target sequence. When bound to DNA target
• MicroRNAs are noncoding RNAs of about 19 to 24 nucleotides (nt) in length that have been identified in both animals and plants (Lagos-Quintana et al. (2001 ) Science 294:853-858, Lagos-Quintana et al. (2002)Curr. Biol. 12:735-739; Lau et al. (2001 ) Science 294:858-862; Lee and Ambros. (2001 ) Science 294:862- 864; Llave et al. (2002) Plant Cell 14:1605-1619; Mourelatos et al. (2002) Genes Dev. 16:720-728; Park et al. (2002)Curr. Biol.
• miRNAs appear to regulate target genes by binding to complementary sequences located in the transcripts produced by these genes. It seems likely that miRNAs can enter at least two pathways of target gene regulation: (1 ) translational inhibition; and (2) RNA cleavage. miRNAs entering the RNA cleavage pathway are analogous to the 21 -25 nt siRNAs generated during RNAi in animals and PTGS in plants, and likely are incorporated into an RNA-induced silencing complex (RISC) that is similar or identical to that seen for RNAi.
• RISC RNA-induced silencing complex
• a recombinant DNA construct (including a suppression DNA construct) of the present disclosure may comprise at least one regulatory sequence.
• a regulatory sequence may be a promoter.
• promoters can be used in recombinant DNA constructs of the present disclosure.
• the promoters can be selected based on the desired outcome, and may include constitutive, tissue-specific, inducible, or other promoters for expression in the host organism.
• Promoters that cause a gene to be expressed in most cell types at most times are commonly referred to as “constitutive promoters”.
• Suitable constitutive promoters for use in a plant host cell include, for example, the core promoter of the Rsyn7 promoter and other constitutive promoters disclosed in WO 99/43838 and U.S. Patent No.
• tissue-specific or developmentally regulated promoter it may be desirable to use a tissue-specific or developmentally regulated promoter.
• a tissue-specific or developmentally-regulated promoter is a DNA sequence which regulates the expression of a DNA sequence selectively in the cells/tissues of a plant, such as in those cells/tissues critical to tassel development, seed set, or both, and which usually limits the expression of such a DNA sequence to the developmental period of interest (e.g. tassel development or seed maturation) in the plant.
• Any identifiable promoter which causes the desired temporal and spatial expression may be used in the methods of the present disclosure.
• Promoters which are seed or embryo-specific and may be useful in the invention include soybean Kunitz trypsin inhibitor (Kti3, Jofuku and Goldberg. (1989) Plant Cell 1 :1079-1093), convicilin, vicilin, and legumin (pea cotyledons) (Rerie, W.G., et al. (1991 ) Mol. Gen. Genet. 259:149-157; Newbigin, E.J., et al. (1990) Planta 180:461 - 470; Higgins, T.J.V., et al. (1988) Plant. Mol. Biol. 1 1 :683-695), zein (maize endosperm) (Schemthaner, J. P., et al. (1988) EMBO J. 7:1249-1255), phaseolin
• Promoters of seed- specific genes operably linked to heterologous coding regions in chimeric gene constructions maintain their temporal and spatial expression pattern in transgenic plants.
• Such examples include Arabidopsis 2S seed storage protein gene promoter to express enkephalin peptides in Arabidopsis and Brassica napus seeds
• Inducible promoters selectively express an operably linked DNA sequence in response to the presence of an endogenous or exogenous stimulus, for example by chemical compounds (chemical inducers) or in response to environmental, hormonal, chemical, and/or developmental signals.
• Inducible or regulated promoters include, for example, promoters regulated by light, heat, stress, flooding or drought, phytohormones, wounding, or chemicals such as ethanol, jasmonate, salicylic acid, or safeners.
• Promoters for use in certain embodiments include the following: 1 ) the stress- inducible promoter RD29A (Kasuga et al. (1999) Nature Biotechnol. 17:287-291 ); 2) the stress-inducible promoter Rab17 (Vilardell et al. (1991 ) Plant Mol. Bio. 17:985- 993; Kamp Busk et al. (1997) Plant J 1 1 (6):1285-1295); 3) the barley promoter B22E whose expression is specific to the pedicel in developing maize kernels ("Primary Structure of a Novel Barley Gene Differentially Expressed in Immature Aleurone Layers". Klemsdal, S.S. et al. (1991 ) Mol.
• Zag2 transcripts can be detected 5 days prior to pollination to 7 to 8 days after pollination ("DAP"), and directs expression in the carpel of developing female inflorescences and Ciml which is specific to the nucleus of developing maize kernels. Ciml transcript is detected 4 to 5 days before pollination to 6 to 8 DAP.
• Other useful promoters include any promoter which can be derived from a gene whose expression is maternally associated with developing female florets.
• promoters of particular interest include seed-preferred promoters, particularly early kernel/embryo promoters and late kernel/embryo promoters.
• Kernel development post-pollination is divided into approximately three primary phases.
• the lag phase of kernel growth occurs from about 0 to 10-12 DAP.
• the linear grain fill stage begins at about 10-12 DAP and continues to about 40 DAP.
• the kernel attains almost all of its final mass, and various storage products (i.e., starch, protein, oil) are produced.
• the maturation phase occurs from about 40 DAP to harvest. During this phase of kernel
• kernel/embryo promoters are promoters that drive expression principally in developing seed during the lag phase of development (i.e., from about 0 to about 12 DAP).
• Early kernel/embryo promoters include, for example, Cim1 that is active 5 DAP in particular tissues (WO 00/1 1 177), which is herein incorporated by reference.
• Other early kernel/embryo promoters include the seed-preferred promoters endl which is active 7-10 DAP, and enc/2, which is active 9-14 DAP in the whole kernel and active 10 DAP in the endosperm and pericarp. (WO 00/12733), herein incorporated by reference.
• Additional early kernel/embryo promoters that find use in certain methods of the present disclosure include the seed-preferred promoter Itp2 (U.S. Pat. No. 5,525,716); maize Zm40 promoter (U.S. Pat. No.
• Additional promoters for regulating the expression of the nucleotide sequences of the present disclosure in plants are stalk-specific promoters, including the alfalfa S2A promoter (GenBank Accession No. EF030816; Abrahams et al. (1995) Plant Mol. Biol. 27:513-528) and S2B promoter (GenBank Accession No. EF030817) and the like, herein incorporated by reference.
• Promoters may be derived in their entirety from a native gene, or be composed of different elements derived from different promoters found in nature, or even comprise synthetic DNA segments.
• Promoters for use in certain embodiments of the current invention may include: RIP2, ml_IP15, ZmCORI , Rab17, CaMV 35S, RD29A, B22E, Zag2, SAM
• Recombinant DNA constructs of the present disclosure may also include other regulatory sequences, including but not limited to, translation leader sequences, introns, and polyadenylation recognition sequences.
• a recombinant DNA construct further comprises an enhancer or silencer.
• An intron sequence can be added to the 5' untranslated region, the protein- coding region or the 3' untranslated region to increase the amount of the mature message that accumulates in the cytosol. Inclusion of a spliceable intron in the transcription unit in both plant and animal expression constructs has been shown to increase gene expression at both the mRNA and protein levels up to 1000-fold
• Any plant can be selected for the identification of regulatory sequences and GLR polypeptide genes to be used in recombinant DNA constructs of the present disclosure.
• suitable plant targets for the isolation of genes and regulatory sequences would include but are not limited to alfalfa, apple, apricot, Arabidopsis, artichoke, arugula, asparagus, avocado, banana, barley, beans, beet, blackberry, blueberry, broccoli, brussels sprouts, cabbage, canola, cantaloupe, carrot, cassava, castorbean, cauliflower, celery, cherry, chicory, cilantro, citrus, Clementines, clover, coconut, coffee, corn, cotton, cranberry, cucumber, Douglas fir, eggplant, endive, escarole, eucalyptus, fennel, figs, garlic, gourd, grape, grapefruit, honey dew, jicama, kiwifruit, lettuce, leeks, lemon, lime, Loblolly pine, linseed
• compositions are Compositions:
• composition of the present disclosure is a plant comprising in its genome any of the recombinant DNA constructs (including any of the suppression DNA
• compositions also include any progeny of the plant, and any seed obtained from the plant or its progeny, wherein the progeny or seed comprises within its genome the recombinant DNA construct (or suppression DNA construct).
• Progeny includes subsequent generations obtained by self-pollination or out-crossing of a plant.
• Progeny also includes hybrids and inbreds.
• mature transgenic plants can be self-pollinated to produce a homozygous inbred plant.
• the inbred plant produces seed containing the newly introduced recombinant DNA construct (or suppression DNA construct). These seeds can be grown to produce plants that would exhibit an altered
• agronomic characteristics e.g., an increased agronomic characteristics optionally under water limiting conditions
• the seeds may be maize seeds or rice seeds.
• the plant may be a monocotyledonous or dicotyledonous plant, for example, a rice or maize or soybean plant, such as a maize hybrid plant or a maize inbred plant.
• the plant may also be sunflower, sorghum, canola, wheat, alfalfa, cotton, rice, barley, millet, sugar cane or switchgrass.
• the recombinant DNA construct may be stably integrated into the genome of the plant.
• a plant for example, a rice or maize or soybean plant
• a recombinant DNA construct comprising a polynucleotide operably linked to at least one regulatory sequence, wherein said polynucleotide encodes a polypeptide having an amino acid sequence of at least 50%, 51 %, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61 %, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, based on either Clustal V or GAP method of alignment, to
• a plant for example, a rice or maize or soybean plant
• a recombinant DNA construct comprising a polynucleotide operably linked to at least one regulatory sequence, wherein said polynucleotide encodes a GLR polypeptide, and wherein said plant exhibits increased drought tolerance when compared to a control plant.
• the plant may further exhibit an alteration of at least one agronomic characteristic when compared to the control plant.
• a plant for example, a rice or maize or soybean plant
• a recombinant DNA construct comprising a polynucleotide operably linked to at least one regulatory sequence, wherein said polynucleotide encodes a GLR polypeptide, and wherein said plant exhibits an alteration of at least one agronomic characteristic when compared to a control plant.
• a plant for example, a rice or maize or soybean plant
• a recombinant DNA construct comprising a polynucleotide operably linked to at least one regulatory element, wherein said polynucleotide encodes a
• polypeptide having an amino acid sequence of at least 50%, 51 %, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61 %, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, based on either Clustal V or GAP method of alignment, to SEQ ID NO: 4 or 6, and wherein said plant exhibits an alteration of at least one agronomic characteristic when compared to a control plant.
• a plant for example, a rice or maize or soybean plant
• a suppression DNA construct comprising at least one regulatory element operably linked to a region derived from all or part of a sense strand or antisense strand of a target gene of interest, said region having a nucleic acid sequence of at least 50%, 51 %, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61 %, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%,85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, based on either Clustal V or GAP method of alignment, to
• a plant for example, a rice or maize or soybean plant
• a suppression DNA construct comprising at least one regulatory element operably linked to all or part of (a) a nucleic acid sequence encoding a polypeptide having an amino acid sequence of at least 50%, 51 %, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61 %, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, based on either Clustal V or GAP method of alignment, to SEQ ID NO: 4 or 6, or (b
• the GLR polypeptide may be from Oryza sativa, Arabidopsis thaliana, Zea mays, Glycine max, Glycine tabacina, Glycine soja or Glycine tomentella.
• the recombinant DNA construct (or suppression DNA construct) may comprise at least a promoter functional in a plant as a regulatory sequence.
• the alteration of at least one agronomic characteristic is either an increase or decrease.
• the at least one agronomic characteristic may be selected from the group consisting of greenness, grain yield, growth rate, biomass, fresh weight at maturation, dry weight at
• the alteration of at least one agronomic characteristic may be an increase in grain yield, greenness or biomass.
• the plant may exhibit the alteration of at least one agronomic characteristic when compared, under water limiting conditions, to a control plant.
• “Drought” refers to a decrease in water availability to a plant that, especially when prolonged or when occurring during critical growth periods, can cause damage to the plant or prevent its successful growth (e.g., limiting plant growth or seed yield).
• “Drought tolerance” reflects a plant's ability to survive under drought without exhibiting substantial physiological or physical deterioration, and/ or its ability to recover when water is restored following a period of drought.
• “Drought tolerance activity" of a polypeptide indicates that over-expression of the polypeptide in a transgenic plant confers increased drought tolerance of the transgenic plant relative to a reference or control plant.
• “Increased drought tolerance” of a plant is measured relative to a reference or control plant, and reflects ability of the plant to survive under drought conditions with less physiological or physical deterioration than a reference or control plant grown under similar drought conditions, or ability of the plant to recover more substantially and/or more quickly than would a control plant when water is restored following a period of drought.
• NUE is nitrogen utilization efficiency and refers to a plant's ability to utilize nitrogen in low or high levels of fertilizer. It reflects plant ability to uptake, assimilate, and/or otherwise utilize nitrogen.
• Soil plant analyses development (SPAD) value is SPAD reading which is measured by SPAD-502 plus (a chlorophyll meter, made by KONICA MINOLTA).
• the SPAD value is relative content of leaf chlorophyll and an important indicator of plant health.
• Many studies indicated that a significant and positive correlation was observed between leaf nitrogen content and SPAD value (Swain D.K. and Sandip S.J. (2010) Journal of Agronomy 9 (2):38-44), and leaf SPAD value is used as index of nitrogen status diagnosis in crops (Cai H.-G.et al. (2010) Acta metallurgica sinica 16 (4): 866-873).
• the SPAD value may be measured during low nitrogen treatment.
• control cell or control plant provides a reference point for measuring changes in phenotype of a subject plant or plant cell in which genetic alteration, such as transformation, has been effected as to a gene of interest.
• a subject plant or plant cell may be descended from a plant or cell so altered and will comprise the alteration.
• One of ordinary skill in the art would readily recognize a suitable control or reference plant to be utilized when assessing or measuring an agronomic characteristic or phenotype of a transgenic plant using compositions or methods as described herein. For example, by way of non-limiting illustrations:
• the progeny not comprising the recombinant DNA construct (or the suppression DNA construct) is the control or reference plant.
• the second hybrid line would typically be measured relative to the first hybrid line (i.e., the first hybrid line is the control or reference plant).
• a plant comprising a recombinant DNA construct (or suppression DNA construct) the plant may be assessed or measured relative to a control plant not comprising the recombinant DNA construct (or suppression DNA construct) but otherwise having a comparable genetic background to the plant (e.g., sharing at least 50%, 51 %, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61 %, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%,85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity of nuclear genetic material compared to the plant comprising the recombinant DNA construct (or suppression DNA construct)).
• RFLPs Randomly Amplified Polymorphic DNAs
• AP-PCR Arbitrarily Primed Polymerase Chain Reaction
• DAF DNA Amplification Fingerprinting
• SCARs Sequence Characterized Amplified Regions
• AFLP®s Amplified Fragment Length Polymorphisms
• SSRs Sequence Repeats
• a control plant or plant cell may comprise, for example: (a) a wild-type (WT) plant or cell, i.e., of the same genotype as the starting material for the genetic alteration which resulted in the subject plant or cell; (b) a plant or plant cell of the same genotype as the starting material but which has been transformed with a null construct (i.e., with a construct which has no known effect on the trait of interest, such as a construct comprising a marker gene); (c) a plant or plant cell which is a non-transformed segregant among progeny of a subject plant or plant cell; (d) a plant or plant cell genetically identical to the subject plant or plant cell but which is not exposed to conditions or stimulus that would induce expression of the gene of interest or (e) the subject plant or plant cell itself, under conditions in which the gene of interest is not expressed.
• a control may comprise numerous individuals representing one or more of the categories above; for example, a collection of the non-transformed segregants of category "c" is often
• Methods include but are not limited to methods for increasing drought tolerance in a plant, methods for evaluating drought tolerance in a plant, methods for altering an agronomic characteristic in a plant, methods for determining an alteration of an agronomic characteristic in a plant, methods for increasing NUE in a plant, and methods for producing seed.
• the plant may be a monocotyledonous or
• dicotyledonous plant for example, a rice, maize or soybean plant.
• the plant may also be sunflower, canola, wheat, alfalfa, cotton, rice, barley, millet, sugar cane or sorghum.
• the seed may be a maize or soybean or rice seed, for example, a maize hybrid seed or maize inbred seed.
• Methods include but are not limited to the following:
• a method for transforming a cell comprising transforming a cell with any one or more of the isolated polynucleotides of the present disclosure, wherein, in particular embodiments, the cell is a eukaryotic cell, e.g., a yeast, insect or plant cell; or a prokaryotic cell, e.g., a bacterial cell.
• the cell is a eukaryotic cell, e.g., a yeast, insect or plant cell; or a prokaryotic cell, e.g., a bacterial cell.
• a method for producing a transgenic plant comprising transforming a plant cell with any of the isolated polynucleotides or recombinant DNA constructs (including suppression DNA constructs) of the present disclosure and regenerating a
• transgenic plant from the transformed plant cell wherein, the transgenic plant and the transgenic seed obtained by this method may be used in other methods of the present disclosure.
• a method for isolating a polypeptide of the invention from a cell or culture medium of the cell wherein the cell comprises a recombinant DNA construct comprising a polynucleotide of the invention operably linked to at least one regulatory sequence, and wherein the transformed host cell is grown under conditions that are suitable for expression of the recombinant DNA construct.
• a method for altering the level of expression of a polypeptide of the invention in a host cell comprising: (a) transforming a host cell with a recombinant DNA construct of the present disclosure; and (b) growing the transformed host cell under conditions that are suitable for the expression of the recombinant DNA construct, wherein the expression of the recombinant DNA construct results in production of altered levels of the polypeptide of the invention in the transformed host cell.
• a method of increasing drought tolerance in a plant comprising: (a) introducing into a regenerable plant cell a recombinant DNA construct comprising a
• polynucleotide operably linked to at least one regulatory sequence (for example, a promoter functional in a plant), wherein the polynucleotide encodes a polypeptide having an amino acid sequence of at least 50%, 51 %, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61 %, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, based on either Clustal V or GAP method of alignment, to SEQ ID NO: 4 or 6;(b) regenerating a transgenic plant from the regener
• a method of evaluating drought tolerance in a plant comprising (a) obtaining a transgenic plant, which comprises in its genome a recombinant DNA construct comprising a polynucleotide operably linked to at least one regulatory sequence (for example, a promoter functional in a plant), wherein said polynucleotide encodes a polypeptide having an amino acid sequence of at least 50%, 51 %, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61 %, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or
• a method of evaluating drought tolerance in a plant comprising (a) obtaining a transgenic plant, wherein the transgenic plant comprises in its genome a
• suppression DNA construct comprising at least one regulatory sequence (for example, a promoter functional in a plant) operably linked to all or part of (i) a nucleic acid sequence encoding a polypeptide having an amino acid sequence of at least 50%, 51 %, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61 %, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, based on either Clustal V or GAP method of alignment, when compared to SEQ ID NO:4 or 6, or (ii) a full complement
• a method of evaluating drought tolerance in a plant comprising (a) obtaining a transgenic plant, wherein the transgenic plant comprises in its genome a
• suppression DNA construct comprising at least one regulatory sequence (for example, a promoter functional in a plant) operably linked to a region derived from all or part of a sense strand or antisense strand of a target gene of interest, said region having a nucleic acid sequence of at least 50%, 51 %, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61 %, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, based on either Clustal V or GAP method of alignment, when compared to said all or part of
• a method of determining an alteration of an agronomic characteristic in a plant comprising (a) obtaining a transgenic plant which comprises in its genome a recombinant DNA construct comprising a polynucleotide operably linked to at least one regulatory sequence (for example, a promoter functional in a plant), wherein said polynucleotide encodes a polypeptide having an amino acid sequence of at least 50%, 51 %, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61 %, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%,
• a method of determining an alteration of an agronomic characteristic in a plant comprising (a) obtaining a transgenic plant, wherein the transgenic plant comprises in its genome a suppression DNA construct comprising at least one regulatory sequence (for example, a promoter functional in a plant) operably linked to all or part of (i) a nucleic acid sequence encoding a polypeptide having an amino acid sequence of at least 50%, 51 %, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61 %, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%
• a method of determining an alteration of an agronomic characteristic in a plant comprising (a) obtaining a transgenic plant, wherein the transgenic plant comprises in its genome a suppression DNA construct comprising at least one regulatory sequence (for example, a promoter functional in a plant) operably linked to a region derived from all or part of a sense strand or antisense strand of a target gene of interest, said region having a nucleic acid sequence of at least 50%, 51 %, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61 %, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%
• a method of producing seed comprising any of the preceding methods, and further comprising obtaining seeds from said progeny plant, wherein said seeds comprise in their genome said recombinant DNA construct (or suppression DNA construct).
• the said regenerable plant cell may comprise a callus cell, an embryogenic callus cell, a gametic cell, a meristematic cell, or a cell of an immature embryo.
• the regenerable plant cells may derive from an inbred maize plant.
• said regenerating step may comprise the following: (i) culturing said transformed plant cell or cells in a medium comprising an embryogenic promoting hormone until callus organization is observed; (ii) transferring said transformed plant cell or cells of step (i) to a first medium which includes a tissue organization promoting hormone; and (iii) subculturing said transformed plant cell or cells after step (ii) onto a second medium, to allow for shoot elongation, root development or both.
• the at least one agronomic characteristic may be selected from the group consisting of greenness, grain yield, growth rate, biomass, fresh weight at maturation, dry weight at maturation, fruit yield, seed yield, total plant nitrogen content, fruit nitrogen content, seed nitrogen content, nitrogen content in a vegetative tissue, total plant free amino acid content, fruit free amino acid content, seed free amino acid content, amino acid content in a vegetative tissue, total plant protein content, fruit protein content, seed protein content, protein content in a vegetative tissue, drought tolerance, nitrogen uptake, root lodging, harvest index, stalk lodging, plant height, ear height, ear length, tiller number, panicle size, salt tolerance, early seedling vigor and seedling emergence under low temperature stress.
• the alteration of at least one agronomic characteristic may be an increase in grain yield, greenness or biomass.
• the plant may exhibit the alteration of at least one agronomic characteristic when compared, under water limiting conditions, to a control plant.
• a regulatory sequence such as one or more enhancers, optionally as part of a transposable element
• recombinant DNA constructs of the present disclosure into plants may be carried out by any suitable technique, including but not limited to direct DNA uptake, chemical treatment, electroporation, microinjection, cell fusion, infection, vector-mediated DNA transfer, bombardment, or Agrobacterium-mediated transformation.
• Techniques for plant transformation and regeneration have been described in International Patent Publication WO 2009/006276, the contents of which are herein incorporated by reference.
• methods to modify or alter the host endogenous genomic DNA are available. This includes altering the host native DNA sequence or a pre-existing transgenic sequence including regulatory elements, coding and non-coding sequences. These methods are also useful in targeting nucleic acids to pre- engineered target recognition sequences in the genome.
• the genetically modified cell or plant described herein is generated using "custom" meganucleases produced to modify plant genomes (e.g., WO 2009/1 14321 ; Gao et al. (2010) Plant Journal 1 :176-187).
• Another site-directed engineering is through the use of zinc finger domain recognition coupled with the restriction properties of restriction enzyme (e.g., Urnov, et al. (2010) Nat Rev Genet. 1 1 (9):636-46; Shukla, et al. (2009) Nature 459 (7245):437-41 ).
• the development or regeneration of plants containing the foreign, exogenous isolated nucleic acid fragment that encodes a protein of interest is well known in the art.
• the regenerated plants may be self-pollinated to provide homozygous transgenic plants. Otherwise, pollen obtained from the regenerated plants is crossed to seed- grown plants of agronomically important lines. Conversely, pollen from plants of these important lines is used to pollinate regenerated plants.
• a transgenic plant containing a desired polypeptide is cultivated using methods well known to one skilled in the art.
• Zhonghual 1 was cultivated by institute of crop sciences, Chinese Academy of Agricultural Sciences. The first batch of seeds used in this research was provided by Beijing Weiming Kaituo Agriculture Biotech Co., Ltd. Calli induced from embryos was transformed with Agrobacteria with the vector. The events generated with the T-vector were developed and the transgenic seeds were harvested to form the rice activation tagging population.
• NUE drought tolerance and nitrogen use efficiency
• T2 (or T1 ) seeds which showed red color under green fluorescent light were used for greenhouse screens including drought tolerance and NUE except as otherwise specifically noted.
• the primary positives were randomly inserted into the unscreened pools and screened again and the data were analyzed using SAS-ANOVA.
• each tagged line had 60 plants and was analyzed only with respect to the wild type plants, using SAS-ANOVA (P ⁇ 0.05).
• Survival rate was also used as a parameter for the screens, which is the percentage of survived plants over the total plant number.
• NUE Nitrogen Use Efficiency
• Field drought screens of mature rice were carried out in Hainan province. For each tagged line to be tested, about 100 positive seeds were soaked in water for 16 h at room temperature, germinated for 36 h at 35-37 °C in an incubator, and planted in a bedded field. At 3-leaf stage, the seedlings were transplanted into the testing field, with 4 replicates and 10 plants per replicate for each tagged line, and the 4 replicates were planted in the same block. The wild type Zhonghual 1 plants which were from the tissue culture procedure, were nearby the tagged lines in the same block, and were used as control in the statistical analysis.
• the rice plants were managed by normal practice using pesticides and fertilizers. Watering was stopped at the booting stage, so as to give drought stress at flowering stage depending on the weather conditions (temperature and humidity). In case the drought was too severe for the plants to survive, they were re-watered one or two times in order to keep a reasonable yield.
• the soil water content was measured every 3 days at about 10 sites per block using TDR30
• Plant phenotypes were observed and recorded during the experiments.
• the phenotypes include heading time, leaf rolling, drought sensitive and drought tolerant. Special attention was paid to leaf rolling at noontime. At the end of the season, 5 representative plants were harvested from the middle of the row per line, and grain weight per plant was measured.
• the grain weight data were statistically analyzed using SAS-ANOVA-mixed model by ASReml program. Positive lines were selected based on the analysis (P ⁇ 0.05). Similar to the greenhouse screens, the primary positives were rescreened twice and only those lines which passed all three screens were considered as positive lines.
• line AH01486 consistently showed enhanced drought tolerance compared to Zhonghual 1 control generated from tissue culture procedure.
• AH01486 gave an average grain yield of 23.93 g /plant (20.72, 18.38, and 32.70 g/plant for three replicates) compared to the Zhonghual 1 control grain yield of 17.70 g/plant (17.80, 18.29, and 17.00 g/plant for three replicates). On the average AH01486 exhibited 35% grain yield advantage over the control (p ⁇ 0.05).
• a successful result is one where a single DNA fragment contains a T-DNA border sequence and flanking genomic sequence.
• candidate genes are identified by alignment to publicly available rice genome sequence. Specifically, the annotated genes nearest the CaMV 35S enhancer elements/T-DNA RB and LB are candidates for genes that are activated.
• a diagnostic PCR on genomic DNA is done with one oligo in the T-DNA and one oligo specific for the local genomic DNA. Genomic DNA samples that give a PCR product are interpreted as representing a T-DNA insertion. This analysis also verifies a situation in which more than one insertion event occurs in the same line, e.g., if multiple differing genomic fragments are identified in Plasmid Rescue and/or Inverse-PCR analyses.
• Genomic DNA was isolated from the leaf tissues of the AH01486 line using CTAB method (Murray, M.G. and W.F. Thompson. (1980) Nucleic Acids Res.8:
• P2up-5389 5'-ACCCCAGGCTTTACACTTTATGCTTCC-3' (SEQ ID NO: 7)
• P2down-3534 5'-AACCCACTCGTGCACCCAACTGATC-3' (SEQ ID NO: 8)
• the LB flanking sequence of the T-DNA in AH01486 is listed in SEQ ID NO: 2.
• the right border (RB) of T-DNA is inserted in the 15760218 bp in rice
• the T-DNA insertion resulted in 79 bp rice genomic DNA deletion (based on rice genome database in June-July, 2009).
• the T-DNA in AH01486 has the LB::LB serial construction.
• the T-DNA has 22 bp deletion at RB end.
• On the left border end there is the UbiI ZM promoter with 65 bp deletion at the 3' end.
• the T-DNA inserted in the second intron of a glutamate receptor gene (GLR1 , LOC_Os09g26144.1 ).
• Upstream of the T-DNA RB there are two retrotransposon protein genes.
• Downstream of the T-DNA LB there are another glutamate receptor gene (GLR2, LOC_Os09g26160.1 ), a MYB family transcription factor gene, and a transcription initiation factor TFIID gene.
• a functional activation-tagged allele should result in up-regulation of the candidate gene in tissues where it is normally expressed, ectopic expression in tissues that do not normally express that gene, or both.
• Expression levels of the candidate gene in selected tissues of the cognate mutant line vs. wild-type are compared.
• a standard RT-PCR or a real-time PCR procedure such as the QuantiTect® Reverse Transcription Kit from Qiagen® and Real Time-PCR(SYBR R Premix Ex TaqTM, TaKaRa), is used.
• EF-1 gene is used as a control to show that the amplification and loading of samples from the mutant line and wild-type are similar.
• Assay conditions are optimized for each gene. If the activation-tagged allele results in ectopic expression in other tissues, it is not detected by this assay. As such, a positive result is useful but a negative result does not eliminate a gene from further analysis.
• RNAiso Plus kit TaKaRa
• the cDNA were prepared by RevertAidTM First Strand cDNA Synthesis Kit (Fermentas) and from 500 ng total RNA.
• the real-time-PCR (SYBR R Premix Ex TaqTM, TaKaRa) was conducted using 7,500 Fast real-time PCR equipment and according to the manual (ABI).
• the primers for real time PCR for the GLR1 gene and GLR2 gene are listed below:
• GLR1 -5'RT-F 5'-GTCAAGGTCCGGCAAGAAG-3' (SEQ ID NO: 13)
• GLR1 -5'RT-R 5'-CTTCCCTCTGTCCATGATGTTC-3' (SEQ ID NO: 14)
• GLR2-RT-F 5'-CTCCTAAAGATATTCCACG-3' (SEQ ID NO: 15)
• GLR2-RT-R 5'-ACGCTCTCCACATCACTGC-3' (SEQ ID NO: 16)
• GLR1 gene and GLR2 gene were significantly activated in AH01486 plants (root, stem, and leaf tissues) compared to both wild-type control and tissue-cultured Zhonghua1 1 control. In leaf tissues, the GLR1 gene and GLR2 gene expression levels were increased 53- and 38-fold compared to the control, respectively. Based on these results, GLR1 gene (SEQ ID NO: 3) and GLR2 gene (SEQ ID NO: 5) were cloned and validated as to their functions in drought tolerance and other agronomic trait improvement.
• PCR reaction was carried out using these two primers and pooled cDNA from leaf, stem, root tissues of Zhonghual 1 plants.
• a -2.8 kb PCR fragment was obtained, purified, and then digested by Sbf ⁇ .
• This DNA fragment was ligated with Psfl-digested vector DP0005 to generate an over-expression vector DP0025 (CaMV35S Pro::GLR1 ::Nos).
• the GLR1 gene sequence and orientation in DP0025 were confirmed by sequencing.
• the sequence of this DNA fragment is provided as SEQ ID NO: 3
• the encoded amino acid sequence is provided as SEQ ID NO: 4
• this gene is named GLR1 (glutamate receptor 1 ).
• the GLR2 (glutamate receptor 2) (LOC_Os09g26160.1 ) gene was amplified from Zhonghual 1 cDNA pool using the following primers which were designed based on the sequence of LOC_Os09g26160.1 in the rice genbank:
• DP0005 was constructed from pCAMBIA1300 (Cambia, Brisbane, Australia).
• the CaMV 35S promoter was amplified by PCR using template of pCAMBIA1300 and primers of 35sF and 35sR.
• the restriction enzyme sites H/ndlM and Pst ⁇ were added in 35sF primer (forward primer of CaMV 35S promoter) and 35sR primer (reverse primer of CaMV 35S promoter), respectively.
• This amplified CaMV 35S promoter fragment was digested by H/ndlM and Pst ⁇ and ligated into H/ndlll-Psil-digested pCAMBIA1300 to produce pCAMBIA1300-35s.
• Terminator NOS polyA was amplified by PCR using template of
• the restriction enzymes sites Pst ⁇ +Bgl ⁇ +Kpn ⁇ +Nco ⁇ +sal ⁇ and BamH ⁇ were designed in Terminator NOS polyA 5' and 3' ends of the primers, respectively.
• TnosF 5'-CTGCAGAGATCTGGTACCGGTCGCCACCATGGAAGTCG
• TnosR 5'- GGATCCTCTAGTCCCGATCTAGTAACATAG-3' (SEQ ID NO: 24) Terminator NOS polyA fragment was digested with Pst ⁇ and SamHI and ligated into Pst ⁇ -SamHI-digested pCAMBIA1300-35s to pCAMBIA1300-35s-Tnos.
• the AsRED gene was isolated from pAsRED2 (Clontech Laboratories Inc.) by Not ⁇ digestion, blunt-ended by T4 polymerase, and then Nco ⁇ digestion.
• the 703 bp- DNA fragment containing the AsRED gene was ligated with pCAMBIAI 300-35s - Tnos which was digested by Sa/I, blunt-ended by T4 polymerase, and then Nco ⁇ digested.
• the resulting construct was designated pCAMBIAI 300-35s -AsRED-Tnos.
• the new Terminator NOS polyA fragment has SamHI+/Vral+Sa/l sites at the 5' end and an EcoRI at the 3' end.
• the primers are:
• TnosF2 5'-CCGGGATCCTCGCGAGTCGACCTCCAAGCTGGGCCACAACTGAAG- 3' (SEQ ID NO: 25)
• TnosR2 5'-CGAGAATTCTCTAGTCCCGATCTAGTAACATAG-3' (SEQ ID NO: 26).
• the new Terminator NOS polyA fragment was amplified by PCR using pCAMBIAI 300-35s-AsRED-Tnos as a template. This DNA fragment was digested by SamHI and EcoRI, and ligated to pCAMBIAI 300-35s-AsRED-Tnos which was digested by SamHI and EcoRI to produce DP0005, an over-expression vector backbone for the candidate genes of GLR1 and GLR2. DP0005 was digested by BglW and SamHI, and then self-ligated for eliminating the AsRED gene from DP0005 to produce DP0009.
• DP0025 were transformed into Zhonghual 1 using Agrobacteria rice transformation methods as known to those of skill in the art.
• the TO plants were cultured in greenhouse to 3-leaf stage. Uniform healthy plants were selected for drought screens as described above. For the T1 seeds, the first selection was for hygromycin-resistance, by culturing the seedling (from 1 -2 cm in height) in 50 mg/L hygromycin solution. Only the surviving plants (hygromycin- resistant) were used in the drought and NUE assays as described above (examples 2, 3, and 4). Leaf samples were collected for real-time PCR analysis to verify that the events from DP0015 and DP0025 were transgenic and over-expressed the GLR2 or GLR1 transgene.
• Primer DP0018-F1 and DP0018-R1 were used, primers for detecting the expression levels of GLR1 transgene are as follow:
• DP0018-F1 5'-GACGCCGTACCTGAGGATCTTC-3' (SEQ ID NO: 27)
• T1 and T2 DP0015 transgenic seedlings were planted in greenhouse and treated as described in example 2.
• GLR1 in rice in drought tolerance is studied.
• Table 6 the GLR1 -transgenic rice plants show higher survival rate and recovery degree compared to the control under moderate drought stress in the greenhouse assays. These results show that over-expression of rice GLR1 enhanced the drought tolerance of the GLR1 -transgenic rice.
• An RNAi construct (DP0018) of GLR1 gene was constructed, and the transgenic rice (DP0018) was obtained. The gene validation revealed that the survival rate and the recovery degree of the DP0018 rice were lower than that of the control, which is correlated with the expression levels of GLR1 gene in the DP0018 transgenic events by real-time analysis (data not shown). Decrease of the GLR1 gene expression reduced the drought tolerance of the transgenic rice.
• NUE Nitrogen Use Efficiency
• Transformation of Arabidopsis with GLR1 or GLR2 Soybean, sorghum and Arabidopsis homologs to rice GLR1 and/or GLR2 can be identified (for example based on homology searches), and transformed into Arabidopsis and/or soybean under control of the CaMV 35S promoter for assessment of their ability to enhance drought tolerance.
• Vector construction, plant transformation and phenotypic analysis will be similar to that in previously described examples.
• rice GLR1 or GLR2 gene (vector DP0025 or DP0015) was transformed into Arabidopsis using standard known protocols.
• the Arabidopsis seeds were planted onto MS selection plates containing appropriate antibiotics. The seeds were vernalized by placing at 4 °C for 3 days; and then were moved to a growth chamber under long- day conditions. After growing on MS selection plates for 7-10 days, seedlings with healthy green cotyledons and true leaves and roots extend into the selection medium were considered as transformants. The transformants were then transplanted in planting soil to produce Arabidopsis seeds, and the transgenic plants were further confirmed by PCR analysis. The expression levels of GLR1 and GLR2 transgene in transgenic Arabidopsis were analyzed. The total RNA of the transformed Arabidopsis seedlings were extracted as described in example 6. Primers of GLR1 -5'RT-F and GLR1 -5'RT-R; and GLR2-RT-F and GLR2-RT-R are used for real-time PCR analysis.
• GLR2 transgene over-expressed in the transgenic AtDP0015 plants
• GLR1 transgene over-expressed in the transgenic AtDP0025 plants
• transgene are not detectable in the wild-type Arabidopsis.
• CaMV 35S Pro:: GLR2 (AtDP0015) and CaMV 35S Pro::GLR1 (AtDP0025) transgenic Arabidopsis plants under drought stress wild-type (Columbia), vector control plants (AtDP0009), two AtDP0015 events, and two AtDP0025 events were planted in planting soil to grow for 4 weeks under normal watering condition.
• the planting soils were watered to saturation before drought treatment, and the Arabidopsis plants grew without watering for about 12 days.
• the relative water content of the soil decreased to about 3%, the Arabidopsis plants were rewatered for 3 days. Three measurements were performed and 12 plants were used.

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