WO2017096527A2 - Procédés et compositions de régulation de l'amidon de maïs - Google Patents

Procédés et compositions de régulation de l'amidon de maïs Download PDF

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WO2017096527A2
WO2017096527A2 PCT/CN2015/096681 CN2015096681W WO2017096527A2 WO 2017096527 A2 WO2017096527 A2 WO 2017096527A2 CN 2015096681 W CN2015096681 W CN 2015096681W WO 2017096527 A2 WO2017096527 A2 WO 2017096527A2
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plant
zmhxk3a
gene
increased
polypeptide
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PCT/CN2015/096681
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WO2017096527A3 (fr
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Yingni XIAO
Xiaohong Yang
Jiansheng LI
Bailin Li
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China Agricultural University
E.I. Du Pont De Nemours And Company
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    • 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
    • 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
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
    • C12N15/8245—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine involving modified carbohydrate or sugar alcohol metabolism, e.g. starch biosynthesis
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1205—Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
    • Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants

Definitions

  • sequence listing is submitted electronically via EFS-Web as an ASCII formatted sequence listing with a file named RTS16368A_ST25_SequenceListing created on November 29, 2015 and having a size of 74 kilobytes and is filed concurrently with the specification.
  • the sequence listing contained in this ASCII formatted document is part of the specification and is herein incorporated by reference in its entirety.
  • the field relates to plant breeding and genetics andto recombinant DNA constructs useful in plants for increasing yield and/orconferring tolerance to abiotic stress tolerance.
  • Yield is a trait of particular economic interest, especially because of increasing world population and the dwindling supply of arable land available for agriculture.
  • Crops such as corn, wheat, rice, canola and soybean account for over half the total human caloric intake, whether through direct consumptionof the seeds themselves or through consumption of meat products raised on processed seeds.
  • starch In maize kernels, starch is the most abundant component, comprising about 70%of dry kernel weight. In addition to the direct consumption of food or feed, maize starch also plays an important role in industrial applications such as bio-ethanol production and the miller industry (Ellis et al. (1998) , J Sci Food Agr 77, 289-311) . Thus, enhancing starch content may not only lead to higher maize yields but could also increase the portion used in industrial applications. Starch biosynthesis initiates with the degradation of sucrose by sucrose synthase, then at least four classes of enzymes contribute to amylopectin or amylose biosynthesis. Although the general biochemical process of starch metabolism in maize is reasonably understood, its regulation remains unclear. In summary, maize plants that have increased kernel starch content are desirable.
  • the present disclosure includes:
  • a maize plant that produces maize kernels with increased starch content, wherein the maize plant comprises a gene encoding a variant ZmHXK3a hexokinase polypeptide, wherein the presence of the variant ZmHXK3a polypeptide results in increased starch content compared to a control maize plant.
  • the ZmHXK3a hexokinase comprises an amino acid sequence with at least 80%sequence identity to SEQ ID NO: 1.
  • the ZmHXK3a hexokinase comprises amino acid lysine at position 471 instead of glutamic acid.
  • the plant exhibits the phenotype of increased yield. In an embodiment, the plant exhibits the phenotype under drought stress conditions.
  • the variant ZmHXK3a hexokinase is caused by guided cas9 endonuclease.
  • the increase in endogenous ZmHXK3a hexokinase activity is caused by a mutation in the endogenous ZmHXK3a hexokinase gene.
  • the mutation in the endogenous ZmHXK3a hexokinase gene is caused by guided cas9 endonuclease.
  • the mutation in the endogenous ZmHXK3a hexokinase gene is caused by zinc finger nuclease, Transcription Activator-Like Effector Nuclease (TALEN) or meganuclease.
  • TALEN Transcription Activator-Like Effector Nuclease
  • a recombinant DNA construct comprising a polynucleotide, wherein the polynucleotide is operably linked in sense or antisense orientation, or both, to a heterologous promoter, wherein the construct is effective for increasing or reducing expression of an endogenous ZmHXK3a hexokinase gene gene in a plant, and wherein the polynucleotide comprises:
  • the DNA construct includes a polynucleotide sequence that has at least 90%sequence identity to SEQ ID NO: 2.
  • a method of increasing the starch content of maize kernels includes the steps of increasing the activity of ZmHXK3a hexokinase polypeptide, wherein the ZmHXK3a hexokinase polypeptide comprises amino acid lysine at position 471 of SEQ ID NO: 1 instead of a glutamic acid residue.
  • the activity of the ZmHXK3a hexokinase polypeptide is increased by increasing the activity of an endogenous ZmHXK3a hexokinase polypeptide.
  • the activity of the endogenous ZmHXK3a hexokinase polypeptide is increased by a mutation in the genome of the maize plant. In an embodiment, the activity of the endogenous ZmHXK3a hexokinase polypeptide is increased by a mutation in the genome of the maize plant caused by a guided cas9 endonuclease.
  • a method of increasing the starch content of maize kernels comprising, the method comprising modulating the expression level of one or more nucleotide sequences or modulating the activity of one or more of the polypeptide encoded by the one or more nucleotide sequences, wherein the one or more polypeptide comprises an amino acid sequence selected from the group consisting of:
  • a method of increasing yield of a maize plant, when compared to a control plant comprising modulating the expression level of one or more nucleotide sequences or modulating the activity of one or more of the polypeptide encoded by the one or more nucleotide sequences, wherein the one or more polypeptide comprises an amino acid sequence selected from the group consisting of:
  • the mutation is introduced using zinc finger nuclease, Transcription Activator-Like Effector Nuclease (TALEN) , guided cas9 endonuclease or meganuclease.
  • TALEN Transcription Activator-Like Effector Nuclease
  • a method of identifying one or more alleles associated with increased starch content in a population of maize plants comprising the steps of:
  • polypeptide comprises the amino acid sequence selected from the group consisting of SEQ ID NOS: 1, 3, 9, 11, 13, 15, and 17, wherein the one or more polymorphisms in the genomic region encoding the polypeptide or in the regulatory region controlling expression of the polypeptide is associated with increased starch content;
  • the one or more alleles associated with increased yield is used for marker assisted selection of a maize plant with increased yield.
  • the one or more polymorphisms is in the coding region of the polynucleotide.
  • the regulatory region is a promoter
  • FIG. 1 shows characteristics of NIL_Aand NIL_B.
  • A Genomic composition of NIL_A (left) and NIL_B (right) . Blue represents regions homozygous with B73; red represents homozygous regions from DHLoPro1; green represents heterozygous regions; grey represents missing data; yellow lines indicate the centromeres.
  • FIG. 2 shows the transcriptomic profiles of NILs.
  • A Euler diagrams of shared presence or absence of genes in NILs at two developmental stages.
  • B Clusters of NILs at different developmental stages identified by principal component analysis of gene expression level (RPKM) . Each spot represents an individual sample, with three biological replicates, indicated by the name of each line followed by the developmental stage.
  • RPKM principal component analysis of gene expression level
  • FIG. 3 shows comparative genomics and transcriptomics of NILs.
  • A Distribution of SNPs between NILs, and DEGs between NILs at different developmental stages. The three circles from inner to outer show the distribution of SNPs identified by RNA-Seq, DEGs at 21 DAP, and DEGs at 14 DAP, respectively. The dark red dots above of the line represent upregulated genes, while the blue dots under the line represent downregulated genes.
  • B Euler diagrams of DEGs between NILs at two developing stages.
  • C Association between gene function and DEGs at two different stages. Only genes encoding transcription factors and enzymes in metabolic pathways are shown.
  • FIG. 5 shows an overview of the coexpression network caused by qHS3 in maize kernel. Seven DEGs are highlighted in the starch biosynthesis pathway. Red represents upregulated genes, and blue represents downregulated genes. The cytosol and amyloplast compartments are indicated.
  • UDPG UDP-glucose
  • ADPG ADP-glucose
  • UGPase UDP glucosepyrophosphorylase
  • ⁇ -D-G1 P ⁇ -D glucose-1-phosphate
  • ⁇ -D-G6P ⁇ -D glucose-6-phosphate
  • ⁇ -D-G6P ⁇ -D glucose-6-phosphate
  • BT1 ADPglucose transporter
  • GPT glucose 1/6-phosphate transporter
  • GlcT glucose transporter.
  • FIG. 6 shows expression patterns of DEGs between NIL_Aand NIL_B at two stages.
  • FIG. 7 shows plots of the correlation between expression level estimated by RNA-Seq and RT-PCR.
  • the x axis represents relative expression values from RT-PCR.
  • the y axis represents the RPKM values from RNA-Seq. Both values are normalized with the logarithm to base 10.
  • the r value is a Pearson correlation coefficient.
  • Table 1 presents SEQ ID NOs for the CDSsequences of other ZMHXK3A family members fromZea mays.
  • the SEQ ID NOs for the corresponding amino acid sequences encoded by the cDNAs are also presented.
  • the Sequence Listing contains the one letter code for nucleotide sequence characters and the three letter codes for amino acids as defined in conformity with the IUPAC-IUBMB standards described in Nucleic Acids Res. 13: 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.
  • a monocot of the current disclosure includes the Gramineae.
  • a dicot of the current disclosure includes the following families: Brassicaceae, Leguminosae, and Solanaceae.
  • full complement and “full-length complement” are used interchangeably herein, and refer 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.
  • a “trait” generally refers to a physiological, morphological, biochemical, or physical characteristic of a plant or a particular plant material or cell. In some instances, this characteristic is visible to the human eye, such as seed or plant size, or can be measured by biochemical techniques, such as detecting the protein, starch, or oil content of seed or leaves, or by observation of a metabolic or physiological process, e.g. by measuring tolerance to water deprivation or particular salt or sugar concentrations, or by the observation of the expression level of a gene or genes, or by agricultural observations such as osmotic stress tolerance or yield.
  • “Agronomic characteristic” is a measurable parameter including but not limited to, abiotic stress tolerance, greenness, 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, 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, early seedling vigor and seedling emergence under low temperature stress.
  • Abiotic stress may be at least one condition selected from the group consisting of: drought, water deprivation, flood, high light intensity, high temperature, low temperature, salinity, etiolation, defoliation, heavy metal toxicity, anaerobiosis, nutrient deficiency, nutrient excess, UV irradiation, atmospheric pollution (e.g., ozone) and exposure to chemicals (e.g., paraquat) that induce production of reactive oxygen species (ROS) .
  • Nutrients include, but are not limited to, the following: nitrogen (N) , phosphorus (P) , potassium (K) , calcium (Ca) , magnesium (Mg) and sulfur (S) .
  • the abiotic stress may be drought stress, low nitrogen stress, or both.
  • Nitrogen limiting conditions or “low nitrogen stress” refers to conditions where the amount of total available nitrogen (e.g., from nitrates, ammonia, or other known sources of nitrogen) is not sufficient to sustain optimal plant growth and development. One skilled in the art would recognize conditions where total available nitrogen is sufficient to sustain optimal plant growth and development. One skilled in the art would recognize what constitutes sufficient amounts of total available nitrogen, and what constitutes soils, media and fertilizer inputs for providing nitrogen to plants. Nitrogen limiting conditions will vary depending upon a number of factors, including but not limited to, the particular plant and environmental conditions.
  • “Increased stress tolerance” of a plant is measured relative to a reference or control plant, and is a trait of the plant to survive under stress conditions over prolonged periods of time, without exhibiting the same degree of physiological or physical deterioration relative to the reference or control plant grown under similar stress conditions.
  • a plant with “increased stress tolerance” can exhibit increased tolerance to one or more different stress conditions.
  • Stress tolerance activity indicates that over-expression of the polypeptide in a transgenic plant confers increased stress tolerance to the transgenic plant relative to a reference or control plant.
  • 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 species may be generated that produce larger cultivars, generating higher yield in, for example, plants in which the vegetative portion of the plant is useful as food, biofuel or both.
  • Increased leaf size may be of particular interest.
  • Increasing leaf biomass can be used to increase production of plant-derived pharmaceutical or industrial products.
  • 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 water or nutrients or take up water or nutrients.
  • Neitrogen stress tolerance is a trait of a plant and refers to the ability of the plant to survive under nitrogen limiting conditions over prolonged periods of time, without exhibiting the same degree of physiological or physical deterioration relative to the reference or control plant grown under similar stress conditions.
  • “Increased nitrogen stress tolerance” of a plant is measured relative to a reference or control plant, and means that the nitrogen stress tolerance of the plant is increased by any amount or measure when compared to the nitrogen stress tolerance of the reference or control plant.
  • a “nitrogen stress tolerant plant” is a plant that exhibits nitrogen stress tolerance.
  • a nitrogen stress tolerant plant may be a plant that exhibits an increase in at least one agronomic characteristic relative to a control plant under nitrogen limiting conditions.
  • “Environmental conditions” refer to conditions under which the plant is grown, such as the availability of water, availability of nutrients (for example nitrogen) , or the presence of insects or disease.
  • “Stay-green” or “staygreen” is a term used to describe a plant phenotype, e.g., whereby leaf senescence (most easily distinguished by yellowing of leaf associated with chlorophyll degradation) is delayed compared to a standard reference or a control.
  • the staygreen phenotype has been used as selective criterion for the development of improved varieties of crop plants such as corn, rice and sorghum, particularly with regard to the development of stress tolerance, and yield enhancement (Borrell et al. (2000b) Crop Sci. 40:1037-1048; Spano et al, (2003) J. Exp. Bot. 54: 1415-1420; Christopher et al, (2008) Aust. J. Agric. Res. 59: 354-364, 2008, Kashiwagi et al (2006) Plant Physiology and Biochemistry 44: 152-157, 2006 and Zheng et al, (2009) Plant Breed 725: 54-62.
  • “Increase in staygreen phenotype” as referred to in here, indicates retention of green leaves, delayed foliar senescence and significantly healthier canopy ina plant, compared to control plant.
  • thermal time terms used herein to describe thermal time include “growing degree days” (GDD) , “growing degree units” (GDU) and “heat units” (HU) .
  • Gene as it applies to plant cells encompasses not only chromosomal DNA found within the nucleus, but organelle DNA found within subcellular components (e.g., mitochondrial, plastid) of the cell.
  • Plant includes reference to whole plants, plant organs, plant tissues, plant propagules, seeds and plant cells and progeny of same.
  • Plant cells include, without limitation, cells from seeds, suspension cultures, embryos, meristematic regions, callus tissue, leaves, roots, shoots, gametophytes, sporophytes, pollen, and microspores.
  • Propagule includes all products of meiosis and mitosis able to propagate a new plant, including but not limited to, seeds, spores and parts of a plant that serve as a means of vegetative reproduction, such as corms, tubers, offsets, or runners. Propagule also includes grafts where one portion of a plant is grafted to another portion of a different plant (even one of a different species) to create a living organism. Propagule also includes all plants and seeds produced bycloning or by bringing together meiotic products, or allowing meiotic products to come together to form an embryo or fertilized egg (naturally or with human intervention) .
  • Progeny comprises any subsequent generation of a plant.
  • gene stacking approach may encompass modulating of more than one ZMHXK3A gene, or may also refer to stacking of a suppression DNA construct with a recombinant DNA construct that leads to overexpression of a particular gene or polypeptide.
  • Gene stacking can be accomplished by many means including but not limited to co-transformation, retransformation, and crossing lines with different transgenes.
  • the suppression DNA constructs and nucleic acid sequences of the current disclosure may be used in combination ( “stacked” ) with other polynucleotide sequences of interest in order to create plants with a desired phenotype.
  • the desired combination may affect one or more traits; that is, certain combinations may be created for modulation of gene expression affecting ZMHXK3A gene activity or expression.
  • Other combinations may be designed to produce plants with a variety of desired traits including but not limited to increased yield and altered agronomic characteristics.
  • “Transgenic plant” also includes reference to plants which comprise more than one heterologous polynucleotide within their genome. Each heterologous polynucleotide may confer a different trait to the transgenic plant.
  • endogenous relates to any gene or nucleic acid sequence that is already present in a cell.
  • 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 RNA sequence
  • nucleotide sequence RNA sequence
  • nucleic acid fragment a polymer of RNA or DNA that is single-or double-stranded, optionally containing synthetic, non-natural or altered nucleotide bases.
  • Nucleotides are referred to by their single letter designation as follows: “A” for adenylate or deoxyadenylate (for RNA or DNA, respectively) , “C” for cytidylate or deoxycytidylate, “G” for guanylate or deoxyguanylate, “U” for uridylate, “T” for deoxythymidylate, “R” for purines (A or G) , “Y” for pyrimidines (C or T) , “K” for G or T, “H” for A or C or T, “I” for inosine, and “N” for any nucleotide.
  • 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, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation and ADP-ribosylation.
  • RNA generally refers to the RNA that is without introns and that can be translated into protein by the cell.
  • cDNA generally refers to a DNA that is complementary to and synthesized from a mRNA template using the enzyme reverse transcriptase.
  • the cDNA can be single-stranded or converted into the double-stranded form using the Klenow fragment of DNA polymerase I.
  • Coding region generally refers to the portion of a messenger RNA (or the corresponding portion of another nucleic acid molecule such as a DNA molecule) which encodes a protein or polypeptide.
  • Non-coding region generally refers to all portions of a messenger RNA or other nucleic acid molecule that are not a coding region, including but not limited to, for example, the promoter region, 5′untranslated region ( “UTR” ) , 3′UTR, intron and terminator.
  • the terms “coding region” and “coding sequence” are used interchangeably herein.
  • the terms “non-coding region” and “non-coding sequence” are used interchangeably herein.
  • “Mature” protein generally refers to a post-translationally processed polypeptide; i.e., one from which any pre-or pro-peptides present in the primary translation product have been removed.
  • Precursor protein generally 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 generally 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.
  • non-genomic nucleic acid sequence or non-genomic nucleic acid molecule generally refer to a nucleic acid molecule that has one or more change in the nucleic acid sequence compared to a native or genomic nucleic acid sequence.
  • the change to a native or genomic nucleic acid molecule includes but is not limited to: changes in the nucleic acid sequence due to the degeneracy of the genetic code; codon optimization of the nucleic acid sequence for expression in plants; changes in the nucleic acid sequence to introduce at least one amino acid substitution, insertion, deletion and/or addition compared to the native or genomic sequence; removal of one or more intron associated with a genomic nucleic acid sequence; insertion of one or more heterologous introns; deletion of one or more upstream or downstream regulatory regions associated with a genomic nucleic acid sequence; insertion of one or more heterologous upstream or downstream regulatory regions; deletion of the 5’and/or 3’untranslated region associated with a genomic nucleic acid sequence; and insertion of a heterologous 5’and/or 3’untranslated region.
  • “Recombinant” generally 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 heterologous 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 generally 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.
  • the terms “recombinant DNA construct” and “recombinant construct” are used interchangeably herein.
  • “Suppression DNA construct” is a recombinant DNA construct which when transformed or stably integrated into the genome of the plant, results in “modulating” of a target gene in the plant.
  • suppression DNA constructs include, but are not limited to, coconstructs, antisense constructs, viral constructs, hairpin constructs, stem-loop constructs, double-stranded RNA-producing constructs, RNA modulating constructs, RNA interference constructs , ribozyme constructs, constructs causing genomic disruptions/mutations (examples of which include but are not limited to transposons, CRISPR, Zinc Finger nucleases, meganucleases, homologous recombination, etc. ) .
  • the current disclosure provides for plants that have a disruption/mutation in at least one endogenous ZMHXK3A gene, that leads to modulating or reduction in expression or activity of the at least one ZMHXK3A polypeptide, in at least one tissuein at least one developmental stage, compared to a control plant that does not have any modulating or reduction in the ZMHXK3A gene expression or ZMHXK3A polypeptide activity, and lacks the disruption/mutation in the ZMHXK3A gene.
  • the at least one ZMHXK3A polypeptide comprises two or more ZMHXK3A polypeptides. In one aspect, the at least one ZMHXK3A polypeptide comprises three or more ZMHXK3A polypeptides.
  • control plant refers to aparent, null, or non-transgenic plant of the same species that lacks the disruption/mutation or modulating of theZMHXK3A gene.
  • a control plant as defined herein is a plant that is not made according to any of the methods disclosed herein.
  • a control plant can also be a parent plant that contains a wild-type allele of a ZMHXK3A gene.
  • a wild-type plant would be: (1) a plant that carries the unaltered or not modulated form of a gene or allele, or (2) the starting material/plant from which the plants produced by the methods described herein are derived.
  • Modulating, refers generally to the reduction or inhibition of levels of mRNA or protein/enzyme expressed by the target gene, and/or the level of the enzyme activity or protein functionality.
  • reduction includes lowering, reducing, declining, decreasing, inhibiting, eliminating or preventing.
  • Modulating does not specify mechanism and is inclusive, and not limited to, anti-sense, cosuppression, viral-suppression, hairpin suppression, stem-loop suppression, RNAi-based approaches, small RNA-based approaches, or genome disruption approaches.
  • Many techniques can be used for producing a plant having a disruption in at least one ZMHXK3A gene, where the disruption results in aincreased/reduced expression or activity of the ZMHXK3A polypeptide encoded by the ZMHXK3Agene compared to a control plant.
  • the disruption can be a result of introducing a suppression DNA construct that is effective for inhibiting the expression of the ZMHXK3A gene, or for mutagenizing the ZMHXK3A gene.
  • Down regulation of expression or activity of the ZMHXK3A gene or polypeptide is by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%or even complete (100%) loss of activity or expression.
  • assays for measuring gene expression are well known in the art and can be done at the protein level (examples include, but are not limited to, Western blot, ELISA) or at the mRNA level such as by RT-PCR.
  • the suppression DNA construct is sense or antisense suppression DNA construct.
  • Coconstructs in plants have been previously designed by focusing on overexpression of a nucleic acid sequence corresponding to all or part of a native mRNA, in the sense orientation, which results in the reduction of all RNA having homology to the overexpressed sequence (see Vaucheret et al., Plant J. 16: 651-659 (1998) ; and Gura, Nature 404: 804-808 (2000) ) .
  • the polynucleotide used for cosuppression may correspond to all or part of the sequence encoding the target gene, and coconstructs may contain sequences from coding regions or non-coding regions, e.g., introns, 5’-UTRs and 3’-UTRs, or both.
  • 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 nucleic acid and that blocks the expression of a target isolated nucleic acid fragment (U.S. Patent No. 5,107,065) .
  • the complementarity of an antisense RNA may be with any part of the specific gene transcript, i.e., at the 5'non-coding sequence, 3'non-coding sequence, introns, or the coding sequence.
  • a duplex can form between the antisense sequence and its complementary sense sequence, resulting in reducing or inhibiting expression from the gene (US Patent No. 7,763,773) .
  • antisense nucleic acids Use of antisense nucleic acids is well known in the art (U.S. Pat. Nos. US5,759,829, US6,242,258, US6,500,615 and US5,942,657) .
  • An antisense nucleic acid can be produced by a number of well-established techniques, examples include, but are not limited to, chemical synthesis of an antisense RNA or oligonucleotide of at least about 15 bases and complementary to unique regions of the mRNA transcript sequence encoding a ZMHXK3A polypeptide (ahomolog or a derivative thereof can be synthesized, e.g., by conventional phosphodiester techniques) , or in vitro transcription.
  • RNA interference RNA interference
  • RNA modulating RNA modulating
  • RNA interference refers to the process of sequence-specific post-transcriptional gene modulating in animals mediated by short interfering RNAs (siRNAs) (Fire et al., Nature 391: 806 (1998) ) .
  • siRNAs short interfering RNAs
  • PTGS post-transcriptional gene modulating
  • quelling in fungi.
  • RNAi refers to a mechanism through which presence of a double-stranded RNA in a cell results in reduction in expression of the corresponding target gene, for example, expression of a hairpin (stem-loop) RNA or of the two strands of an interfering RNA will lead to modulating of a target gene by RNA interference.
  • RNA interference is well described in the literature, as are methods for determining appropriate interfering RNA (s) to target a desired gene, e.g., a ZMHXK3A gene, and for generating such interfering RNAs.
  • RNA interference is described in (US patent publications US20020173478, US20020162126, and US20020182223) "RNA interference” Nature., July 11; 418 (6894) : 244-51; Ueda R. (2001) "RNAi: a new technology in the postgenomic sequencing era” J Neurogenet. ; 15 (3-4) : 193-204; Ullu et al (2002) "RNA interference: advances and questions" Philos Trans R SocLond B Biol Sci. January 29; 357 (1417) : 65-70; Fire et al., Trends Genet. 15: 358 (1999) ; US patent No. 7763773)
  • a polynucleotide sequence is said to “encode” a sense or antisense RNA molecule, or RNA modulating or interference molecule or a polypeptide, if the polynucleotide sequence can be transcribed (in spliced or unspliced form) and /or translated into the RNA or polypeptide, or a subsequence thereof.
  • “Expression of a gene” or “expression of a nucleic acid” means transcription of DNA into RNA (optionally including modification of the RNA, e.g., splicing) , translation of RNA into a polypeptide (possibly including subsequent modification of the polypeptide, e.g., posttranslational modification) , or both transcription and translation, as might be indicated by the context.
  • Small RNAs play an important role in controlling gene expression. Regulation of many developmental processes, including flowering, is controlled by small RNAs. 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 sequences, it is thought that small RNAs can mediate DNA methylation of the target sequence. The consequence of these events, regardless of the specific mechanism, is that gene expression is inhibited.
  • MicroRNAs are noncoding RNAs of about 19 to about 24 nucleotides (nt) in length that have been identified in both animals and plants (Lagos-Quintana et al., Science 294: 853-858 (2001) , Lagos-Quintana et al., Curr. Biol. 12: 735-739 (2002) ; Lau et al., Science 294: 858-862 (2001) ; Lee and Ambros, Science 294: 862-864 (2001) ; Llave et al., Plant Cell 14: 1605-1619 (2002) ; Mourelatos et al., Genes. Dev.
  • MicroRNAs 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. MicroRNAs entering the RNA cleavage pathway are analogous to the 21-25 nt short interfering RNAs (siRNAs) generated during RNA interference (RNAi) in animals and posttranscriptional gene modulating (PTGS) in plants, and likely are incorporated into an RNA-induced modulating complex (RISC) that is similar or identical to that seen for RNAi.
  • siRNAs short interfering RNAs
  • PTGS posttranscriptional gene modulating
  • the expression or activity of the ZMHXK3A gene and/or polypeptide can be increased/reduced by mutating the gene encoding the ZMHXK3A polypeptide.
  • the ZMHXK3A gene can be disrupted by any means known in the art. One way of disrupting a gene is by insertional mutagenesis. The gene can be disrupted by mutagenizing the plant or plant cell using random or targeted mutagenesis.
  • mutagenic methods can also be employed to introduce mutations in the ZMHXK3A gene.
  • Methods for introducing genetic mutations into plant genes and selecting plants with desired traits are well known.
  • seeds or other plant material can be treated with a mutagenic chemical substance, according to standard techniques.
  • chemical substances include, but are not limited to, the following: diethyl sulfate, ethylene imine, and N-nitroso-N-ethylurea.
  • ionizing radiation from sources such as X-rays or gamma rays can be used.
  • TILLING or “Targeting Induced Local Lesions IN Genomics” refers to a mutagenesis technology useful to generate and/or identify, and to eventually isolate mutagenised variants of a particular nucleic acid with modulated expression and/or activity (McCallum et al., (2000) , Plant Physiology 123: 439-442; McCallum et al., (2000) Nature Biotechnology 18: 455-457; and, Colbert et al., (2001) Plant Physiology 126: 480-484) .
  • TILLING combines high density point mutations with rapid sensitive detection of the mutations.
  • EMS ethylmethanesulfonate
  • M1 ethylmethanesulfonate
  • M2 next generation
  • TILLING also allows selection of plants carrying mutant variants. These mutant variants may exhibit modified expression, either in strength or in location or in timing (if the mutations affect the promoter for example) . These mutant variants may even exhibit lower ZMHXK3Aactivity than that exhibited by the gene in its natural form.
  • TILLING combines high-density mutagenesis with high-throughput screening methods. The steps typically followed in TILLING are: (a) EMS mutagenesis (Redei G P and Koncz C (1992) In Methods in Arabidopsis Research, Koncz C, Chua N H, Schell J, eds. Singapore, World Scientific Publishing Co, pp.
  • the plant containing the mutated ZMHXK3A gene can be crossed with other plants to introduce the mutation into another plant. This can be done using standard breeding techniques.
  • Homologous recombination allows introduction in a genome of a selected nucleic acid at a defined selected position. Homologous recombination has been demonstrated in plants. See, e.g., Puchta et al. (1994) , Experientia 50: 277-284; Swoboda et al. (1994) , EMBO J. 13: 484-489; Offringa et al. (1993) , Proc. Natl. Acad. Sci. USA 90: 7346-7350; Kempin et al. (1997) Nature 389: 802-803; and, Terada et al., (2002) Nature Biotechnology, 20 (10) : 1030-1034) .
  • the nucleic acid to be introduced (which may be ZMHXK3A nucleic acid or a variant thereof) need not be targeted to the locus of the ZMHXK3A gene, but may be introduced into, for example, regions of high expression.
  • the nucleic acid to be introduced may be a dominant negative allele used to replace the endogenous gene or may be introduced in addition to the endogenous gene.
  • DNA nuclease domains are another type of enzymes that can be used to introduce DNA damage or mutation.
  • a DNA nuclease domain is an enzymatically active protein or fragment thereof that causes DNA cleavage resulting in a DSB.
  • DNA binding domains include, for example, an array specific DNA binding domain or a site-specific DNA binding domain.
  • Site specific DNA binding domain include but are not limited to a TAL (Transcription Activator-Like Effector) or a zinc finger binding domain.
  • DNA-binding domains fused to DNA nucleases include but are not limited to TALEN and multiple TALENs.
  • Transcription Activator-Like Effector Nucleases are artificial restriction enzymes generated by fusing the TAL effector DNA binding domain to a DNA enzyme domain.
  • TAL proteins are produced by bacteria and include a highly conserved 33-34 amino acid DNA binding domain sequence (PCT publication No. WO2014127287; US Patent Publication No. US20140087426) .
  • TALEN The original TALEN chimera were prepared using the wild-type FokI endonuclease domain.
  • TALEN may also include chimera made from Fok1 endonuclease domain variants with mutations designed to improve cleavage specificity and cleavage activity.
  • multipleTALENs can be expressed to target multiple genomic regions.
  • a zinc finger is another type of DNA binding domain that can be used for introducing mutations into the target DNA.
  • Various protein engineering techniques can be used to alter the DNA-binding specificity of zinc fingers and tandem repeats of such engineered zinc fingers can be used to target desired genomic DNA sequences. Fusing a second protein domain such as a transcriptional repressor to a zinc finger that can bind near the promoter of the YEP gene can reduce the expression levels of ZMHXK3A gene.
  • the proteins of the CRISPR (clustered regularly interspaced short palindromic repeat) system are examples of other DNA-binding and DNA-nuclease domains.
  • the expression levels of ZMHXK3Agene or the activity of the ZMHXK3A polypeptide can be increased/reduced by introducing mutations through CRISPR (clustered regularly interspaced short palindromic repeat) /Cas9 system.
  • the bacterial CRISPR/Cas system involves the targeting of DNA with a short, complementary single stranded RNA (CRISPR RNA or crRNA) that localizes the Cas9 nuclease to the target DNA sequence (Burgess DJ (2013) Nat Rev Genet 14: 80; PCT publication No. WO2014/127287) .
  • the crRNA can bind on either strand of DNA and the Cas9 will cleave the DNA making a DSB.
  • the present disclosure encompasses variants and subsequences of the polynucleotides and polypeptides described herein.
  • variant with respect to a polynucleotide or DNA refers to a polynucleotide that contains changes in which one or more nucleotides of the original sequence is deleted, added, and/or substituted while substantially maintaining the function of the polynucleotide.
  • a variant of a promoter that is disclosed herein can have minor changes in its sequence without substantial alteration to its regulatory function.
  • variants refers to an amino acid sequence that is altered by one or more amino acids with respect to a reference sequence.
  • the variant can have “conservative changes, wherein a substituted amino acid has similar structural or chemical properties, for example, and replacement of leucine with isoleucine.
  • a variant can have “non-conservative” changes, for example, replacement of a glycine with a tryptophan.
  • Analogous minor variation can also include amino acid deletion or insertion, or both.
  • fragment and “subsequence” are used interchangeably herein, and refer to any portion of an entire sequence.
  • regulatory sequences refer to nucleotide sequences located upstream (5'non-coding sequences) , within, or downstream (3'non-coding sequences) of a coding sequence, and which influence 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 polyadenylation recognition sequences. The terms “regulatory sequence” and “regulatory element” are used interchangeably herein.
  • Promoter generally 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 in plant cells whether or not its origin is from a plant cell.
  • tissue-specific promoter and “tissue-preferred promoter” are used interchangeably, and 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.
  • “Developmentally regulated promoter” generally refers to a promoter whose activity is determined by developmental events.
  • “Operably linked” generally 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.
  • Phenotype means the detectable characteristics of a cell or organism.
  • “Introduced” in the context of inserting a nucleic acid fragment (e.g., a 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 e.g., a recombinant DNA construct
  • a “transformed cell” is any cell into which a nucleic acid fragment (e.g., a recombinant DNA construct) has been introduced.
  • Transformation generally refers to both stable transformation and transient transformation.
  • “Stable transformation” generally 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 generally 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.
  • Allele is one of several alternative forms of a gene occupying a given locus on a chromosome. When the alleles present at a given locus on a pair of homologous chromosomes in a diploid plant are the same that plant is homozygous at that locus. If the alleles present at a given locus on a pair of homologous chromosomes in a diploid plant differ that plant is heterozygous at that locus. If a transgene is present on one of a pair of homologous chromosomes in a diploid plant that plant is hemizygous at that locus.
  • Allelic variants encompass Single nucleotide polymorphisms (SNPs) , as well as Small Insertion/Deletion Polymorphisms (INDELs) .
  • SNPs Single nucleotide polymorphisms
  • INDELs Small Insertion/Deletion Polymorphisms
  • the size of INDELs is usually less than 100bp. SNPs and INDELs form the largest set of sequence variants in naturally occurring polymorphic strains of most organisms.
  • Plant breeding techniques known in the art and used in the maize plant breeding program include, but are not limited to, recurrent selection, bulk selection, mass selection, backcrossing, pedigree breeding, open pollination breeding, restriction fragment length polymorphism enhanced selection, genetic marker enhanced selection, double haploids and transformation. Often combinations of these techniques are used.
  • the Clustal W method of alignment may be used.
  • the Clustal W method of alignment (described by Higgins and Sharp, CABIOS. 5: 151-153 (1989) ; Higgins, D. G. et al., Comput. Appl. Biosci. 8: 189-191 (1992) ) can be found in the MegAlign TM v6.1 program of the bioinformatics computing suite ( Inc., Madison, Wis. ) .
  • 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, recombinant DNA constructs useful for conferring drought tolerance, compositions (such as plants or seeds) comprising these recombinant DNA constructs, and methods utilizing these recombinant DNA constructs.
  • the plant exhibits increased abiotic stress tolerance, and the abiotic stress is drought stress, low nitrogen stress, or both.
  • the plant exhibits the phenotype of increased yield and the phenotype is exhibited under non-stress conditions.
  • the plant exhibits the phenotype of increased yield and the phenotype is exhibited under stress conditions.
  • the plant exhibits the phenotype under drought stress conditions.
  • the endogenous ZMHXK3A polypeptide comprises an amino acid sequence with at least 80%sequence identity to SEQ ID NOS: 1, 3, 9, 11, 13, 15, and 17.
  • the plant is a monocot plant. In another embodiment, the plant is a maize plant.
  • the reduction in expression of the endogenous ZMHXK3A gene is caused by sense suppression, antisense suppression, miRNA suppression, ribozymes, or RNA interference. In one embodiment, the reduction in expression of the endogenous ZMHXK3A gene is caused by a mutation in the endogenous ZMHXK3A gene. In one embodiment, the mutation in the endogenous ZMHXK3A gene is caused by insertional mutagenesis. In one embodiment, the insertional mutagenesis is caused by transposon mutagenesis.
  • the mutation in the endogenous ZMHXK3A gene is caused by zinc finger nuclease, Transcription Activator-Like Effector Nuclease (TALEN) , CRISPR or meganuclease.
  • TALEN Transcription Activator-Like Effector Nuclease
  • the activity of the endogenous ZMHXK3A polypeptide is increased/reduced as a result of mutation of the endogenous ZMHXK3A gene.
  • the mutation of the endogenous ZMHXK3A gene is caused by zinc finger nuclease, Transcription Activator-Like Effector Nuclease (TALEN) , CRISPR or meganuclease.
  • the mutation in the endogenous ZMHXK3A gene is detected using the TILLING method.
  • One embodiment is a method of making a plant in which expression of an endogenous ZMHXK3A gene is increased/reduced, when compared to a control plant, and wherein the plant exhibits at least one phenotype selected from the group consisting of: increased yield, increased abiotic stress tolerance, increased staygreen and increased biomass, compared to the control plant, the method comprising the steps of: (a) introducing a mutation into an endogenous ZMHXK3A gene; and (b) detecting said mutation using the Targeted Induced Local Lesions In Genomics (TILLING) method, wherein said mutation results in reducing expression of the endogenous ZMHXK3A gene.
  • TILLING Targeted Induced Local Lesions In Genomics
  • the current disclosure includes a method of enhancing seed yield in a plant, when compared to a control plant, wherein the plant exhibits enhanced yield under either stress conditions, or non-stress conditions, or both, the method comprising the step of reducing expression of the endogenous ZMHXK3A gene in a plant.
  • One embodiment of the current disclosure is a method of making a plant in which expression of an endogenous ZMHXK3A gene is increased/reduced, when compared to a control plant, and wherein the plant exhibits at least one phenotype selected from the group consisting of: increased yield, increased abiotic stress tolerance, increased staygreen and increased biomass, compared to the control plant, the method comprising the step of utilizing a transposon to introduce an insertion into an endogenous ZMHXK3A gene in a plant, wherein the insertion is effective for reducing expression of an endogenous ZMHXK3A gene.
  • One embodiment of the current disclosure is a method of making a plant in which activity of an endogenous ZMHXK3A polypeptide is increased/reduced, when compared to the activity of wild-type ZMHXK3A polypeptide from a control plant, and wherein the plant exhibits at least one phenotype selected from the group consisting of: increased yield, increased staygreen, increased abiotic stress tolerance and increased biomass, compared to the control plant, wherein the method comprises the steps of introducing into a plant a suppression DNA construct comprising a polynucleotide operably linked to a heterologous promoter, wherein the polynucleotide encodes a fragment or a variant of a polypeptide having an amino acid sequence of at least 80%sequence identity, when compared to SEQ ID NOS: 1, 3, 9, 11, 13, 15, and 17, wherein the fragment or the variant confers a dominant-negative phenotype in the plant.
  • the method further comprises the step of detecting the mutation and the detection is done using the Targeted Induced Local Lesions IN Genomics (TILLING) method.
  • the mutation is introduced using zinc finger nuclease, Transcription Activator-Like Effector Nuclease (TALEN) , CRISPR or meganuclease.
  • TALEN Transcription Activator-Like Effector Nuclease
  • the current disclosure also includes the plant obtained by any of the methods disclosed herein, wherein the plant exhibits at least one phenotype selected from the group consisting of: increased yield, increased staygreen, increased abiotic stress tolerance and increased biomass, compared to the control plant.
  • One embodiment of the current disclosure includes the plant comprising any of the suppression DNA constructs disclosed herein, wherein expression or activity of the endogenous ZMHXK3A gene is increased/reduced in the plant, when compared to a control plant, and wherein the plant exhibits at least one phenotype selected from the group consisting of: increased yield, increased staygreen, increased abiotic stress tolerance and increased biomass, compared to the control plant.
  • the plant exhibits an increase in abiotic stress tolerance, and the abiotic stress is drought stress, low nitrogen stress, or both.
  • the plant exhibits the phenotype of increased yield and the phenotype is exhibited under non-stress conditions.
  • the phenotype is exhibited under stress conditions.
  • the plant is a monocot plant. In another embodiment, the monocot plant is a maize plant.
  • One embodiment of the current disclosure is a method of identifying one or more alleles associated with increased yield in a population of maize plants, the method comprising the steps of: (a) detecting in a population of maize plants one or more polymorphisms in (i) a genomic region encoding a polypeptide or (ii) a regulatory region controlling expression of the polypeptide, wherein the polypeptide comprises the amino acid sequence selected from the group consisting of SEQ ID NOS: 1, 3, 9, 11, 13, 15, and 17, or a sequence that is 90%identical to SEQ ID NOS: 1, 3, 9, 11, 13, 15, and 17, wherein the one or more polymorphisms in the genomic region encoding the polypeptide or in the regulatory region controlling expression of the polypeptide is associated with yield; and (b) identifying one or more alleles at the one or more polymorphisms that are associated with increased yield.
  • the one or more polymorphisms is in the coding region of the polynucleotide.
  • the regulatory region is a promoter element.
  • One embodiment encompasses the plants obtained by any of the methods disclosed herein, or comprising any of the suppression DNA constructs disclosed herein.
  • the current disclosure also encompasses any progeny, or seeds obtained from the plants disclosed herein.
  • the present disclosure includes the following isolated polynucleotides and polypeptides:
  • An isolated polynucleotide comprising: (i) a nucleic acid sequence encoding a ZMHXK3Apolypeptide having an amino acid sequence of at least 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 the Clustal V or Clustal W method of alignment, when compared to SEQ ID NOS: 1, 3, 9, 11, 13, 15, and 17, and combinations thereof; or (ii) a full complement of the nucleic acid sequence of (i) , wherein the full complement and the nucleic acid sequence of (i) consist of the same number of nucleotides and are 100%complementary. Any of the foregoing isolated polynucleotides or afragment or subsequence of the isolated polynucleotidesmay be utilized in any suppression DNA constructs of the present
  • polypeptide having an amino acid sequence of at least 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 the Clustal V or Clustal W method of alignment, when compared to SEQ ID NOS: 1, 3, 9, 11, 13, 15, and 17, and combinations thereof.
  • the polypeptide is preferably a ZMHXK3Apolypeptide.
  • An isolated polynucleotide comprising (i) a nucleic acid sequence of at least 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 the Clustal V or Clustal W method of alignment, when compared to SEQ ID NOS: 2, 4, 6-8, 10, 12, 14, 16 and 18 and combinations thereof; (ii) a full complement of the nucleic acid sequence of (i) ; or (iii) a fragment or subsequence of the nucleic acid sequence of (i) .
  • isolated polynucleotides preferably encodes a ZMHXK3Apolypeptide.
  • An isolated polynucleotide comprising a nucleotide sequence, wherein the nucleotide sequence is hybridizable under stringent conditions with a DNA molecule comprising the full complement of SEQ ID NOS: 2, 4, 6-8, 10, 12, 14, 16 and 18, or a subsequence thereof.
  • the isolated polynucleotide preferably encodes a ZMHXK3Apolypeptide.
  • An isolated polynucleotide comprising a nucleotide sequence, wherein the nucleotide sequence is derived from SEQ ID NOS: 2, 4, 6-8, 10, 12, 14, 16 and 18by alteration of one or more nucleotides by at least one method selected from the group consisting of: deletion, substitution, addition and insertion.
  • the isolated polynucleotide preferably encodes a ZMHXK3Apolypeptide.
  • An isolated polynucleotide comprising a nucleotide sequence, wherein the nucleotide sequence corresponds to an allele of SEQ ID NOS: 2, 4, 6-8, 10, 12, 14, 16 and 18.
  • 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.
  • the protein of the current disclosure may also be a protein which comprises an amino acid sequence comprising deletion, substitution, insertion and/or addition of one or more amino acids in an amino acid sequence presented in SEQ ID NOS: 1, 3, 9, 11, 13, 15, and 17.
  • the substitution may be conservative, which means the replacement of a certain amino acid residue by another residue having similar physical and chemical characteristics.
  • conservative substitution include replacement between aliphatic group-containing amino acid residues such as Ile, Val, Leu or Ala, and replacement between polar residues such as Lys-Arg, Glu-Asp or Gln-Asn replacement.
  • Proteins derived by amino acid deletion, substitution, insertion and/or addition can be prepared when DNAs encoding their wild-type proteins are subjected to, for example, well-known site-directed mutagenesis (see, e.g., Nucleic Acid Research, Vol. 10, No. 20, p. 6487-6500, 1982, which is hereby incorporated by reference in its entirety) .
  • site-directed mutagenesis see, e.g., Nucleic Acid Research, Vol. 10, No. 20, p. 6487-6500, 1982, which is hereby incorporated by reference in its entirety.
  • the term “one or more amino acids” is intended to mean a possible number of amino acids which may be deleted, substituted, inserted and/or added by site-directed mutagenesis.
  • Site-directed mutagenesis may be accomplished, for example, as follows using a synthetic oligonucleotide primer that is complementary to single-stranded phage DNA to be mutated, except for having a specific mismatch (i.e., a desired mutation) .
  • the above synthetic oligonucleotide is used as a primer to cause synthesis of a complementary strand by phages, and the resulting duplex DNA is then used to transform host cells.
  • the transformed bacterial culture is plated on agar, whereby plaques are allowed to form from phage-containing single cells.
  • 50%of new colonies contain phages with the mutation as a single strand, while the remaining 50%have the original sequence.
  • plaques hybridized with the probe are picked up and cultured for collection of their DNA.
  • Techniques for allowing deletion, substitution, insertion and/or addition of one or more amino acids in the amino acid sequences of biologically active peptides such as enzymes while retaining their activity include site-directed mutagenesis mentioned above, as well as other techniques such as those for treating a gene with a mutagen, and those in which a gene is selectively cleaved to remove, substitute, insert or add a selected nucleotide or nucleotides, and then ligated.
  • the protein of the present disclosure may also be a protein which is encoded by a nucleic acid comprising a nucleotide sequence comprising deletion, substitution, insertion and/or addition of one or more nucleotides in the nucleotide sequence of SEQ ID NOS: 2, 4, 6-8, 10, 12, 14, 16 and 18. Nucleotide deletion, substitution, insertion and/or addition may be accomplished by site-directed mutagenesis or other techniques as mentioned above.
  • the protein of the present disclosure may also be a protein which is encoded by a nucleic acid comprising a nucleotide sequence hybridizable under stringent conditions with the complementary strand of the nucleotide sequence of SEQ ID NOS: 2, 4, 6-8, 10, 12, 14, 16 and 18.
  • under stringent conditions means that two sequences hybridize under moderately or highly stringent conditions. More specifically, moderately stringent conditions can be readily determined by those having ordinary skill in the art, e.g., depending on the length of DNA. The basic conditions are set forth by Sambrook et al., Molecular Cloning: A Laboratory Manual, third edition, chapters 6 and 7, Cold Spring Harbor Laboratory Press, 2001 and include the use of a prewashing solution for nitrocellulose filters 5xSSC, 0.5%SDS, 1.0 mM EDTA (pH 8.0) , hybridization conditions of about 50%formamide, 2xSSC to 6xSSC at about 40-50 °C (or other similar hybridization solutions, such as Stark’s solution, in about 50%formamide at about 42 °C) and washing conditions of, for example, about 40-60 °C, 0.5-6xSSC, 0.1%SDS.
  • moderately stringent conditions include hybridization (and washing) at about 50 °C and 6xSSC. Highly stringent conditions can also be readily
  • such conditions include hybridization and/or washing at higher temperature and/or lower salt concentration (such as hybridization at about 65 °C, 6xSSC to 0.2xSSC, preferably 6xSSC, more preferably 2xSSC, most preferably 0.2xSSC) , compared to the moderately stringent conditions.
  • highly stringent conditions may include hybridization as defined above, and washing at approximately 65-68 °C, 0.2xSSC, 0.1%SDS.
  • SSPE (1xSSPE is 0.15 M NaCl, 10 mM NaH2PO4, and 1.25 mM EDTA, pH 7.4) can be substituted for SSC (1xSSC is 0.15 M NaCl and 15 mM sodium citrate) in the hybridization and washing buffers; washing is performed for 15 minutes after hybridization is completed.
  • hybridization kit which uses no radioactive substance as a probe.
  • Specific examples include hybridization with an ECL direct labeling &detection system (Amersham) .
  • Stringent conditions include, for example, hybridization at 42°C for 4 hours using the hybridization buffer included in the kit, which is supplemented with 5% (w/v) Blocking reagent and 0.5 M NaCl, and washing twice in 0.4%SDS, 0.5xSSC at 55 °C for 20 minutes and once in 2xSSC at room temperature for 5 minutes.
  • the present disclosure includes suppression DNA constructs.
  • One embodiment is a suppression DNA construct comprising a polynucleotide, wherein the polynucleotide is operably linked to a heterologous promoter in sense or antisense orientation, or both, wherein the construct is effective for reducing expression of an endogenous ZMHXK3A gene in a plant, and wherein the polynucleotide comprises: (a) the nucleotide sequence of SEQ ID NOS: 2, 4, 6-8, 10, 12, 14, 16 and 18; (b) a nucleotide sequence that has at least 80%sequence identity, when compared to SEQ ID NOS: 2, 4, 6-8, 10, 12, 14, 16 and 18; (c) a nucleotide sequence of at least 100 contiguous nucleotides of SEQ ID NOS: 2, 4, 6-8, 10, 12, 14, 16 and 18; (d) a nucleotide sequence that can hybridize under stringent conditions with the nucleotide sequence of (a) ; or (e) a modified plant miRNA precursor,
  • One embodiment of the current disclosure encompasses the suppression DNA construct, wherein the polynucleotide comprises at least 100 contiguous nucleotides of SEQ ID NOS: 2, 4, 6-8, 10, 12, 14, 16 and 18, and the suppression DNA construct is designed for RNA interference, and is effective for reducing expression of ZMHXK3A gene in a plant.
  • the ZMHXK3Apolypeptide may be from a monocot plant.
  • the ZMHXK3A polypeptide may be from Zea mays, Glycine max, Oryza sativa, Sorghum bicolor, Saccharumofficinarum, orTriticumaestivum.
  • the promoter may be a constitutive promoter, an inducible promoter, a tissue-specific promoter.
  • 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 the Clustal V or Clustal W method of alignment, when compared to SEQ ID NOS: 1, 3, 9, 11, 13, 15, and 17,
  • the suppression DNA construct may comprise a coconstruct, antisense construct, viral-construct, hairpin construct, stem-loop 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.
  • 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 gene of interest.
  • a suppression DNA construct may comprise 100, 200, 300, 400, 500, 600, 700, 800, 900 or 1000 contiguous nucleotides of the sense strand (or antisense strand) of the gene of interest, and combinations thereof.
  • Suppression DNA constructs are well-known in the art, are readily constructed once the target gene of interest is selected, and include, without limitation, coconstructs, antisense constructs, viral-constructs, hairpin constructs, stem-loop constructs, double-stranded RNA-producing constructs, and more generally, RNAi (RNA interference) constructs and small RNA constructs such as siRNA (short interfering RNA) constructs and miRNA (microRNA) constructs.
  • Suppression of gene expression may also be achieved by use of artificial miRNA precursors, ribozyme constructs and gene disruption.
  • a modified plant miRNA precursor may be used, wherein the precursor has been modified to replace the miRNA encoding region with a sequence designed to produce a miRNA directed to the nucleotide sequence of interest.
  • Gene disruption may be achieved by use of transposable elements or by use of chemical agents that cause site-specific mutations.
  • Antisense inhibition generally refers to the production of antisense RNA transcripts capable of suppressing the expression of the target gene or gene product.
  • Antisense RNA generally 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 (U.S. Patent No. 5,107,065) .
  • the complementarity of an antisense RNA may be with any part of the specific gene transcript, i.e., at the 5'non-coding sequence, 3'non-coding sequence, introns, or the coding sequence.
  • Sense suppression generally refers to the production of sense RNA transcripts capable of suppressing the expression of the target gene or gene product.
  • Sense RNA generally refers to RNA transcript that includes the mRNA and can be translated into protein within a cell or in vitro.
  • Sense constructs in plants have been previously designed by focusing on overexpression 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 (see Vaucheret et al., Plant J. 16: 651-659 (1998) ; and Gura, Nature 404: 804-808 (2000) ) .
  • RNA interference generally refers to the process of sequence-specific post-transcriptional gene modulating in animals mediated by short interfering RNAs (siRNAs) (Fire et al., Nature 391: 806 (1998) ) .
  • the corresponding process in plants is commonly referred to as post-transcriptional gene modulating (PTGS) or RNA modulating and is also referred to as quelling in fungi.
  • PTGS post-transcriptional gene modulating
  • the process of post-transcriptional gene modulating 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., Trends Genet. 15: 358 (1999) ) .
  • the RNA interference is achieved by hairpin RNA interference or intron containing hairpin RNA (hpRNA) (Waterhouse and Helliwell, (2003) Nat. Rev. Genet. 5: 29-38) .
  • hpRNA interference the expression cassette is designed to express an RNA molecule that hybridizes with itself to form a hairpin structure that comprises a single-stranded loop region and a base-paired stem.
  • the base-paired stem region comprises a sense sequence corresponding to all or part of the endogenous mRNA whose expression is to be inhibited, and an antisense sequence that is fully or partially complementary to the sense sequence.
  • Such kind of hairpin RNA interference is highly efficient at inhibiting the expression of endogenous genes (for example US Patent publication No.
  • the hpRNA molecule comprises an intron that is capable of being spliced in the cell in which the hpRNA is expressed.
  • the use of an intron minimizes the size of the loop in the hairpin RNA molecule following splicing, and this increases the efficiency of interference.
  • intron containing hpRNA interference constructs such as petunia chalcone synthase intron, rice waxy intron, Flavariatrinerviapyruvate orthophosphate dikinase intron, intron from potato LS1gene (Smith et al. (2000) Nature 407: 319-320; Preuss and Pikaard Targeted gene modulating in plants using RNA interference Pg. 23-36; from “RNA Interference ⁇ Nuts and bolts of siRNA technology” ; edited by David Engelke, Eckes et al (1986) Mol. Gen Genet. 205: 14-22) .
  • the intron could be the 2 nd intron from potato LS1 gene.
  • Small RNAs play an important role in controlling gene expression. Regulation of many developmental processes, including flowering, is controlled by small RNAs. 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 sequences, it is thought that small RNAs can mediate DNA methylation of the target sequence. The consequence of these events, regardless of the specific mechanism, is that gene expression is inhibited.
  • MicroRNAs 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. MicroRNAs entering the RNA cleavage pathway are analogous to the 21-25 nt short interfering RNAs (siRNAs) generated during RNA interference (RNAi) in animals and posttranscriptional gene modulating (PTGS) in plants, and likely are incorporated into an RNA-induced modulating complex (RISC) that is similar or identical to that seen for RNAi.
  • siRNAs short interfering RNAs
  • PTGS posttranscriptional gene modulating
  • miRNA-star sequence and “miRNA*sequence” are used interchangeably herein and they refer to a sequence in the miRNA precursor that is highly complementary to the miRNA sequence.
  • miRNA and miRNA*sequences form part of the stem region of the miRNA precursor hairpin structure.
  • a method for the suppression of a target sequence comprising introducing into a cell a nucleic acid construct encoding a miRNA substantially complementary to the target.
  • the miRNA comprises about 19, 20, 21, 22, 23, 24 or 25 nucleotides.
  • the miRNA comprises 21 nucleotides.
  • the nucleic acid construct encodes the miRNA.
  • the nucleic acid construct encodes a polynucleotide precursor which may form a double-stranded RNA, or hairpin structure comprising the miRNA.
  • the nucleic acid construct comprises a modified endogenous plant miRNA precursor, wherein the precursor has been modified to replace the endogenous miRNA encoding region with a sequence designed to produce a miRNA directed to the target sequence.
  • the plant miRNA precursor may be full-length of may comprise a fragment of the full-length precursor.
  • the endogenous plant miRNA precursor is from a dicot or a monocot.
  • the endogenous miRNA precursor is from Arabidopsis, tomato, maize, soybean, sunflower, sorghum, canola, wheat, alfalfa, cotton, rice, barley, millet, sugar cane or switchgrass.
  • the miRNA template (i.e. the polynucleotide encoding the miRNA) , and thereby the miRNA, may comprise some mismatches relative to the target sequence.
  • the miRNA template has > 1 nucleotide mismatch as compared to the target sequence, for example, the miRNA template can have 1, 2, 3, 4, 5, or more mismatches as compared to the target sequence. This degree of mismatch may also be described by determining the percent identity of the miRNA template to the complement of the target sequence.
  • the miRNA template may have a percent identity including about at least 70%, 75%, 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%as compared to the complement of the target sequence.
  • the miRNA template (i.e. the polynucleotide encoding the miRNA) and thereby the miRNA, may comprise some mismatches relative to the miRNA-star sequence.
  • the miRNA template has > 1 nucleotide mismatch as compared to the miRNA-star sequence, for example, the miRNA template can have 1, 2, 3, 4, 5, or more mismatches as compared to the miRNA-star sequence. This degree of mismatch may also be described by determining the percent identity of the miRNA template to the complement of the miRNA-star sequence.
  • the miRNA template may have a percent identity including about at least 70%, 75%, 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%as compared to the complement of the miRNA-star sequence.
  • 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. 6,072,050; the core CaMV 35S promoter (Odell et al., Nature 313: 810-812 (1985) ) ; rice actin (McElroy et al., Plant Cell 2: 163-171 (1990) ) ; ubiquitin (Christensen et al., Plant Mol. Biol. 12: 619-632 (1989) and Christensen et al., Plant Mol. Biol.
  • 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 critical to tassel development, seed set, or both, and limits the expression of such a DNA sequence to the period of tassel development or seed maturation in the plant. Any identifiable promoter may be used in the methods of the present disclosure which causes the desired temporal and spatial expression.
  • Promoters which are seed or embryo-specific and may be useful include soybean Kunitz trypsin inhibitor (Kti3, Jofuku and Goldberg, Plant Cell 1: 1079-1093 (1989) ) , patatin (potato tubers) (Rocha-Sosa, M., et al. (1989) EMBO J. 8: 23-29) , 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.
  • 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 Arabidopsisthaliana 2S seed storage protein gene promoter to express enkephalin peptides in Arabidopsis and Brassica napus seeds (Vanderkerckhove et al., Bio/Technology 7: L929-932 (1989) ) , bean lectin and bean beta-phaseolin promoters to express luciferase (Riggs et al., Plant Sci.
  • 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 include the following: 1) the stress-inducible RD29A promoter (Kasuga et al. (1999) Nature Biotechnol. 17: 287-91) ; 2) the barley promoter, B22E; expression of B22E 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., Mol. Gen. Genet.
  • 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 for use also include the following: Zm-GOS2 (maize promoter for “Gene from Oryza sativa” , US publication number US2012/0110700 Sb-RCC (Sorghum promoter for Root Cortical Cell delineating protein, root specific expression) , Zm-ADF4 (US7902428 ; Maize promoter for Actin Depolymerizing Factor) , Zm-FTM1 (US7842851; maize promoter for Floral transition MADSs) promoters.
  • Zm-GOS2 miize promoter for “Gene from Oryza sativa”
  • Sb-RCC Sorghum promoter for Root Cortical Cell delineating protein, root specific expression
  • Zm-ADF4 US7902428 ; Maize promoter for Actin Depolymerizing Factor
  • Zm-FTM1 US7842851; maize promoter for Floral transition MADSs
  • stalk-specific promoters include the alfalfa S2A promoter (GenBank Accession No. EF030816; Abrahams et al., Plant Mol. Biol. 27: 513-528 (1995) ) 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.
  • the at least one regulatory element may be an endogenous promoter operably linked to at least one enhancer element; e.g., a 35S, nos or ocs enhancer element.
  • Promoters for use may include: RIP2, mLIP15, ZmCOR1, Rab17, CaMV 35S, RD29A, B22E, Zag2, SAM synthetase, ubiquitin, CaMV 19S, nos, Adh, sucrose synthase, R-allele, the vascular tissue preferred promoters S2A (Genbank accession number EF030816) and S2B (Genbank accession number EF030817) , and the constitutive promoter GOS2 fromZea mays.
  • promoters include root preferred promoters, such as the maize NAS2 promoter, the maize Cyclo promoter (US 2006/0156439, published July 13, 2006) , the maize ROOTMET2 promoter (WO05063998, published July 14, 2005) , the CR1 BIO promoter (WO06055487, published May 26, 2006) , the CRWAQ81 (WO05035770, published April 21, 2005) and the maize ZRP2.47 promoter (NCBI accession number: U38790; GI No. 1063664) ,
  • Suppression 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 of the present disclosure further comprises an enhancer or silencer.
  • the promoters disclosed herein may be used with their own introns, or with any heterologous introns to drive expression of the transgene.
  • 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. Buchman and Berg, Mol. Cell Biol. 8: 4395-4405 (1988) ; Callis et al., Genes Dev. 1: 1183-1200 (1987) .
  • Transcription terminator refers to DNA sequences located downstream of a protein-coding sequence, including polyadenylation recognition sequences and other sequences encoding regulatory signals capable of affecting mRNA processing or gene expression.
  • the polyadenylation signal is usually characterized by affecting the addition of polyadenylic acid tracts to the 3′end of the mRNA precursor.
  • the use of different 3′non-coding sequences is exemplified by Ingelbrecht, I. L., et al., Plant Cell 1: 671-680 (1989) .
  • a polynucleotide sequence with “terminator activity” generally refers to a polynucleotide sequence that, when operably linked to the 3’end of a second polynucleotide sequence that is to be expressed, is capable of terminating transcription from the second polynucleotide sequence and facilitating efficient 3’end processing of the messenger RNA resulting in addition of poly A tail.
  • Transcription termination is the process by which RNA synthesis by RNA polymerase is stopped and both the processed messenger RNA and the enzyme are released from the DNA template.
  • RNA transcript Improper termination of an RNA transcript can affect the stability of the RNA, and hence can affect protein expression. Variability of transgene expression is sometimes attributed to variability of termination efficiency (Bieri et al (2002) Molecular Breeding 10: 107–117) .
  • terminators for use include, but are not limited to, PinII terminator, SB-GKAF terminator (US Appln. No. 61/514055) , Actin terminator, Os-Actin terminator, Ubi terminator, Sb-Ubi terminator, Os-Ubi terminator.
  • Any plant can be selected for the identification of regulatory sequences and ZMHXK3Apolypeptide genes to be used in suppression DNA constructs and other compositions (e.g. transgenic plants, seeds and cells) and methods of the present disclosure.
  • suitable plants for the isolation of genes and regulatory sequences and for compositions and methods of the present disclosure 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,
  • compositions are Compositions:
  • a composition of the present disclosure includes a transgenic microorganism, cell, plant, and seed comprising the suppression DNA construct.
  • the cell may be eukaryotic, e.g., a yeast, insect or plant cell, or prokaryotic, e.g., a bacterial cell.
  • composition of the present disclosure is a plant comprising in its genome any of the suppression DNA constructs of the present disclosure (such as any of the constructs discussed above) .
  • 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 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 suppression DNA construct.
  • These seeds can be grown to produce plants that would exhibit an altered agronomic characteristic (e.g., an increased agronomic characteristicoptionally under stress conditions) , or used in a breeding program to produce hybrid seed, which can be grown to produce plants that would exhibit such an altered agronomic characteristic.
  • the seeds may be maize seeds.
  • the stress condition may be selected from the group of drought stress, and nitrogen stress.
  • the plant may be a monocotyledonous or dicotyledonous plant, for example, a maize or soybean plant.
  • the plant may also be sunflower, sorghum, canola, wheat, alfalfa, cotton, rice, barley, millet, sugar cane or switchgrass.
  • the plant may be a hybrid plant or an inbred plant.
  • Particular embodiments include but are not limited to the following:
  • a plant for example, a maize, rice or sorghumplant
  • a plant comprising in its genome any of the suppression DNA constructs described herein.
  • a plant comprising a disruption or modulating of at least one of the ZMHXK3A genes.
  • a plant for example, a maize, rice or sorghum plant
  • said plant exhibits at least one phenotype selected from the group consisting of increased staygreen phenotype, increased yield, increased biomass and increased tolerance to abiotic stress, when compared to a control plant not comprising said recombinant DNA construct.
  • the abiotic stress may be drought stress, low nitrogen stress, or both.
  • the plant may further exhibit an alteration of at least one agronomic characteristic when compared to the control plant.
  • the plant of the current disclosure can have the reduction in expression of the endogenous ZMHXK3A gene caused by a mutation in the endogenous ZMHXK3A gene.
  • the mutation in the endogenous ZMHXK3A gene in the plant is caused by insertional mutagenesis.
  • the insertional mutagenesis is caused by transposon mutagenesis.
  • the plant encompassed by the current disclosure comprises a mutation in the endogenous ZMHXK3A gene, and the mutation is caused by zinc finger nuclease, Transcription Activator-Like Effector Nuclease (TALEN) , CRISPR or meganuclease.
  • TALEN Transcription Activator-Like Effector Nuclease
  • a plant for example, amaize, rice 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 the Clustal V or Clustal W method of alignment, when compared to SEQ ID NOS: 1, 3, 9, 11, 13, 15, and 17,
  • the ZMHXK3Apolypeptide may be from Zea mays, Glycine max, Glycine tabacina, Glycine soja, Glycine tomentella, Oryza sativa, Brassica napus, Sorghum bicolor, Saccharumofficinarum, orTriticumaestivum.
  • the 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: abiotic stress tolerance, greenness, 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, 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, early seedling vigor and seedling emergence under low temperature stress.
  • the alteration of at least one agronomic characteristic may be an increase in yield, greenness or biomass.
  • the plant encompassed by the current disclosure, and comprising disruption or modulating of at least one endogenous ZMHXK3A gene may exhibit the alteration of at least one agronomic characteristic when compared, under at least one stress condition, to a control plant.
  • the at least one stress condition may be either drought stress, low nitrogen stress, or both.
  • the plant is a hybrid plant exhibiting staygreen phenotype
  • the plant may exhibit less yield loss relative to the control plants, for example, at least 25%, at least 20%, at least 15%, at least 10%or at least 5%less yield loss, under water limiting conditions, or would have increased yield, for example, at least 5%, at least 10%, at least 15%, at least 20%or at least 25%increased yield, relative to the control plants under water non-limiting conditions.
  • the plant may exhibit less yield loss relative to the control plants, for example, at least 25%, at least 20%, at least 15%, at least 10%or at least 5%less yield loss, under stress conditions.
  • the stress may be either drought stress, low nitrogen stress, or both.
  • the plant may exhibit increased yield, for example, at least 5%, at least 10%, at least 15%, at least 20%or at least 25%increased yield, relative to the control plants under non-stress conditions.
  • Yield analysis can be done to determine whether plants that have downregulated expression levels of at least one of the ZMHXK3A genes have an improvement in yield performance under non-stress or stress conditions, when compared to the control plants that have wild-type expression levels and activity levels of the YEP gene and polypeptide, respectively.
  • Stress conditions can be water-limiting conditions, or low nitrogen conditions. Specifically, drought conditions or nitrogen limiting conditions can be imposed during the flowering and/or grain fill period for plants that contain the suppression DNA construct and the control plants.
  • the plant may exhibit increased staygreen phenotype, or an increase in biomass, relative to the control plants under non-stress conditions.
  • the plant may exhibit increased staygreen phenotype, or an increase in biomass, relative to the control plants under stress conditions.
  • yield can be measured in many ways, including, for example, test weight, seed weight, seed number per plant, seed number per unit area (i.e. seeds, or weight of seeds, per acre) , bushels per acre, tonnes per acre, tons per acre, kilo per hectare.
  • stress tolerance or “stress resistance” as used herein generally refers to a measure of a plants ability to grow under stress conditions that would detrimentally affect the growth, vigor, yield, and size, of a “non-tolerant” plant of the same species. Stress tolerant plants grow better under conditions of stress than non-stress tolerant plants of the same species. For example, a plant with increased growth rate, compared to a plant of the same species and/or variety, when subjected to stress conditions that detrimentally affect the growth of another plant of the same species would be said to be stress tolerant. A plant with “increased stress tolerance” can exhibit increased tolerance to one or more different stress conditions.
  • “Increased stress tolerance” of a plant is measured relative to a reference or control plant, and is a trait of the plant to survive under stress conditions over prolonged periods of time, without exhibiting the same degree of physiological or physical deterioration relative to the reference or control plant grown under similar stress conditions.
  • the reference or control plant does not comprise in its genome the recombinant DNA construct or suppression DNA construct.
  • Water limiting conditions generally refers to a plant growth environment where the amount of water is not sufficient to sustain optimal plant growth and development. The terms “drought” and “water limiting conditions” are used interchangeably herein.
  • “Drought tolerance” is a trait of a plant to survive under drought conditions over prolonged periods of time without exhibiting substantial physiological or physical deterioration.
  • “Drought tolerance activity” of a polypeptide indicates that over-expression of the polypeptide in a transgenic plant confers increased drought tolerance to 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 is a trait of the plant to survive under drought conditions over prolonged periods of time, without exhibiting the same degree of physiological or physical deterioration relative to the reference or control plant grown under similar drought conditions.
  • the reference or control plant does not comprise in its genome the recombinant DNA construct or suppression DNA construct.
  • a transgenic plant comprising a suppression DNA construct in its genome exhibits increased stress tolerance relative to a reference or control plant
  • the reference or control plant does not comprise in its genome the suppression DNA construct.
  • the range of stress and stress response depends on the different plants which are used, i.e., it varies for example between a plant such as wheat and a plant such as Arabidopsis.
  • One of ordinary skill in the art is familiar with protocols for simulating drought conditions and for evaluating drought tolerance of plants that have been subjected to simulated or naturally-occurring drought conditions. For example, one can simulate drought conditions by giving plants less water than normally required or no water over a period of time, and one can evaluate drought tolerance by looking for differences in physiological and/or physical condition, including (but not limited to) vigor, growth, size, or root length, or in particular, leaf color or leaf area size. Other techniques for evaluating drought tolerance include measuring chlorophyll fluorescence, photosynthetic rates and gas exchange rates.
  • a drought stress experiment may involve a chronic stress (i.e., slow dry down) and/or may involve two acute stresses (i.e., abrupt removal of water) separated by a day or two of recovery.
  • Chronic stress may last 8–10 days.
  • Acute stress may last 3–5 days. The following variables may be measured during drought stress and well watered treatments of transgenic plants and relevant control plants.
  • a suitable control or reference plant to be utilized when assessing or measuring an agronomic characteristic or phenotype of a transgenic plant would not include a plant that had been previously selected, via mutagenesis or transformation, for the desired agronomic characteristic or phenotype.
  • Methods include but are not limited to methods for increasing yield ina plant, method of increasing staygreen phenotype in a plant, method of increasing drought tolerance in a plant, methods for altering an agronomic characteristic in a plant, and methods for producing seed.
  • the plant may be a monocotyledonous or dicotyledonous plant, for example, a maize or soybean plant.
  • the plant may also be sunflower, sorghum, canola, wheat, alfalfa, cotton, rice, barley, millet, sugar cane or sorghum.
  • the seed may be a maize or soybean seed, for example, a maize hybrid seed or maize inbred seed.
  • Methods include but are not limited to the following:
  • the suppression DNA construct is selected from the group consisting of: sense construct, antisense construct, ribozyme construct, RNA interference construct and an miRNA construct.
  • the suppression DNA construct is an RNA interference construct and the RNA interference construct comprises at least 100 contiguous nucleotides of SEQ ID NOS: 2, 4, 6-8, 10, 12, 14, 16 and 18, and wherein the RNA interference construct is effective for reducing the expression of the endogenous ZMHXK3A gene.
  • the RNA interference construct comprises a polynucleotide sequence that has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100%sequence identity to SEQ ID NO: 2.
  • TILLING Targeted Induced Local Lesions In Genomics
  • the method further comprises the step of detecting the mutation and the detection is done using the Targeted Induced Local Lesions IN Genomics (TILLING) method.
  • the mutation is introduced using zinc finger nuclease, Transcription Activator-Like Effector Nuclease (TALEN) , CRISPR or meganuclease.
  • TALEN Transcription Activator-Like Effector Nuclease
  • the current disclosure also includes the plant obtained by any of the methods disclosed herein, wherein the plant exhibits at least one phenotype selected from the group consisting of: increased yield, increased staygreen, increased abiotic stress tolerance and increased biomass, compared to the control plant.
  • the current disclosure also includes a method for transforming a cell (or microorganism) comprising transforming a cell (or microorganism) with any of the isolated polynucleotides or suppression DNA constructs of the present disclosure.
  • the cell (or microorganism) transformed by this method is also included.
  • the cell is eukaryotic cell, e.g., a yeast, insect or plant cell, or prokaryotic, e.g., a bacterial cell.
  • the microorganism may be Agrobacterium, e.g. Agrobacterium tumefaciens or Agrobacterium rhizogenes.
  • a method for producing a transgenic plant comprising transforming a plant cell with any of the isolated polynucleotides or suppression DNA constructs of the present disclosure and regenerating a transgenic plant from the transformed plant cell.
  • the disclosure is also directed to the transgenic plant produced by this method, and transgenic seed obtained from this transgenic plant.
  • the transgenic plant obtained by this method may be used in other methods of the present disclosure.
  • a method for isolating a polypeptide of the disclosure from a cell or culture medium of the cell wherein the cell comprises a suppression DNA construct comprising a polynucleotide of the disclosure 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 suppression DNA construct.
  • a method of altering the level of expression of a polypeptide of the disclosure in a host cell comprising: (a) transforming a host cell with a DNA construct of the present disclosure; and (b) growing the transformed host cell under conditions that are suitable for expression of the DNA construct wherein expression of the DNA construct results in production of altered levels of expression or activity of the polypeptide of the disclosure in the transformed host cell.
  • the method may further comprise (c) obtaining a progeny plant derived from the transgenic plant, wherein said progeny plant comprises in its genome the suppression DNA construct and exhibits at least one phenotype selected from the group consisting of : increased yield, increased staygreen and increased stress tolerance, wherein the stress is selected from the group consisting of drought stress, and low nitrogen stress, when compared to a control plant not comprising the suppression DNA construct.
  • the progeny plant further exhibits aincreased/lower level of expression and/or activity of at least one ZMHXK3A gene and/or polypeptide.
  • a method of increasing stress tolerance wherein the stress is selected from the group consisting of drought stress, and low nitrogen stress, the method comprising: (a) introducing into a regenerable plant cell a suppression DNA construct comprising a polynucleotide operably linked to at least one regulatory element, wherein said polynucleotide comprises a nucleotide sequence, wherein the nucleotide sequence is: (a) hybridizable under stringent conditions with a DNA molecule comprising the full complement of SEQ ID NOS: 2, 4, 6-8, 10, 12, 14, 16 and 18; or (b) derived from SEQ ID NOS: 2, 4, 6-8, 10, 12, 14, 16 and 18, by alteration of one or more nucleotides by at least one method selected from the group consisting of: deletion, substitution, addition and insertion; and (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 andexhibits increased stress tolerance, wherein the
  • the method may further comprise (c) obtaining a progeny plant derived from the transgenic plant, wherein said progeny plant comprises in its genome the suppression DNA construct and exhibits increased stress tolerance, wherein the stress is selected from the group consisting of drought stress and low nitrogen stress, when compared to a control plant not comprising the recombinant DNA construct.
  • a method of selecting for (or identifying) increasedstress tolerance in a plant, wherein the stress is selected from the group consisting of drought stress and low nitrogen stress comprising (a) obtaining a transgenic plant, wherein the transgenic plant comprises in its genome a suppression 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%, 9
  • a method of selecting for (or identifying) an alteration of an agronomic characteristic in a plant comprising (a) obtaining a transgenic plant, wherein the transgenic plant comprises in its genome a DNA construct comprising a polynucleotide operably linked to at least one regulatory element, wherein said polynucleotide comprises a nucleotide sequence, wherein the nucleotide sequence is: (i) hybridizable under stringent conditions with a DNA molecule comprising the full complement of SEQ ID NOS: 2, 4, 6-8, 10, 12, 14, 16 and 18by alteration of one or more nucleotides by at least one method selected from the group consisting of: deletion, substitution, addition and insertion; (b) obtaining a progeny plant derived from said transgenic plant, wherein the progeny plant comprises in its genome the DNA construct; and (c) selecting (or identifying) the progeny plant that exhibits an alteration in at least one agronomic characteristic when compared, optionally under stress conditions, where
  • 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 DNA construct.
  • 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 cells in a media comprising an embryogenic promoting hormone until callus organization is observed; (ii) transferring said transformed plant cells of step (i) to a first media which includes a tissue organization promoting hormone; and (iii) subculturing said transformed plant cells after step (ii) onto a second media, to allow for shoot elongation, root development or both.
  • the at least one agronomic characteristic may be selected from the group consisting of: abiotic stress tolerance, greenness, 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, 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 yield, greenness or biomass.
  • the plant may exhibit the alteration of at least one agronomic characteristic when compared, under stress conditions, wherein the stress is selected from the group consisting of drought stress, and low nitrogen stress, to a control plant not comprising said suppression DNA construct.
  • a regulatory sequence such as one or more enhancers, optionally as part of a transposable element
  • suppression DNA constructs of the present disclosure 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.
  • 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.
  • 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 of the present disclosure containing a desired polypeptide is cultivated using methods well known to one skilled in the art.
  • NIL_A carries a ⁇ 26 Mb fragment from DHLoPro1 while NIL_B carries a 175 Mb fragment from DHLoPro1 on chromosome 3, with a 13 MB overlap (Fig. 1A) .
  • the two NILs differ from 15 Mb to 193 Mb on chromosome 3, with 4.1% (54/1323) of SNPs polymorphic between NIL_Aand NIL_B, containing the qHS3.
  • Kernel starch content is significantly higher in NIL_B (average 72.1%) than NIL_A (average 69.3%) (Fig. 1 B) .
  • No significant difference in kernel starch content between these NILs was observed at 14 DAP, whereas the starch content of NIL_B is significantly higher than that of NIL_Aat 21 DAP (3.2%) and at maturity (2.7%) (Fig. 1 B) .
  • RNA-Seq on 14 and 21 DAP maize kernels of both NILs was performed.
  • a total of 190 million 50-bp single-end reads were obtained for 12 samples, with reads for each sample ranging from 10 million to 23 million.
  • the read length for all samples combined is >9 Gb, representing a ⁇ 3.7-fold coverage of the maize genome. Approximately 81%of these short reads could be uniquely mapped to the reference B73 genome (Version 5b. 60) .
  • PCA principal component analysis
  • RNA-Seq data which identified 659 SNPs between the two NILs, with 658 SNPs distributed in the introgressed fragment on chromosome 3 and 1 SNP located on chromosome 6 (Fig. 3A) , consistent with results inferred using the MaizeSNP3K array (Fig. 1A) .
  • the two NILs were found to mainly differ in the 173-Mb genomic fragment ranging from 16,566,519 to 189,901,888 bp on chromosome 3 with 709 SNPs.
  • ZmHXK3a encodes a hexokinase, which converts glucose to glucose-6-phosphate (G6P; Fig. 5) , a step in starch synthesis.
  • cluster I the expression levels of DEGs between the two NILs at 14 DAP (e.g., GRMZM2G325920) decrease sharply in NIL_B whereas there is little change in NIL_Aduring development.
  • clusters II and III e.g., GRMZM2G127798 and GRMZM2G479110
  • the expression levels of the DEGs between the two NILs differ significantly at both stages, but the direction and degree of difference vary among clusters.
  • the genes in NIL_B are enriched in cluster IV but increased/reduced in cluster VI at both stages, whereas they are upregulated at 14 DAP but downregulated at 21 DAP in cluster V.
  • Type I with most of the genes related to starch biosynthesis, has higher expression levels at 14 DAP than at 21 DAP in both NILs.
  • type III which contains most of the genes related to starch degradation, has lower expression levels at 14 DAP than at 21 DAP.
  • ZmHXK3a (GRMZM2G068913) in the introgressed fragment interval
  • AGPLLZM (GRMZM2G391936) on chromosome 1
  • SSV (GRMZM2G130043) on chromosome 4
  • ZmGPE5 (GRMZM2G011662) on chromosome 6
  • GBSSII (GRMZM2G008263) on chromosome 7
  • ZmHXK3b (GRMZM2G467069) on chromosome 8
  • Du1 (GRMZM2G141399) on chromosome 10 (Fig.
  • GBSSII encoding a granule-bound starch synthase
  • Du1 and SSV catalyze 1-4- ⁇ -glucose to produce amylopectin
  • GBSSII catalyzes 1-4- ⁇ -glucose to produce amylose.
  • Maize plants can be transformed with the suppression DNA construct containing DNA construct in order to examine the resulting phenotype.
  • Agrobacterium-mediated transformation of maize is performed essentially as described by Zhao et al. in Meth. Mol. Biol. 318: 315-323 (2006) (see also Zhao et al., Mol. Breed. 8: 323-333 (2001) and U.S. Patent No. 5,981,840 issued November 9, 1999, incorporated herein by reference) .
  • the transformation process involves bacterium inoculation, co-cultivation, resting, selection and plant regeneration.
  • Immature maize embryos are dissected from caryopses and placed in a 2 mL microtube containing 2 mL PHI-Amedium.
  • PHI-Amedium of (1) is removed with 1 mL micropipettor, and 1 mL of Agrobacterium suspension is added. The tube is gently inverted to mix. The mixture is incubated for 5 min at room temperature.
  • the Agrobacterium suspension is removed from the infection step with a 1 mL micropipettor. Using a sterile spatula the embryos are scraped from the tube and transferred to a plate of PHI-B medium in a 100x15 mm Petri dish. The embryos are oriented with the embryonic axis down on the surface of the medium. Plates with the embryos are cultured at 20°C, in darkness, for three days. L-Cysteine can be used in the co-cultivation phase. With the standard binary vector, the co-cultivation medium supplied with 100-400 mg/L L-cysteine is critical for recovering stable transgenic events.
  • Embryonic tissue propagated on PHI-D medium is subcultured to PHI-E medium (somatic embryo maturation medium) , in 100x25 mm Petri dishes and incubated at 28°C, in darkness, until somatic embryos mature, for about ten to eighteen days.
  • PHI-E medium synthetic embryo maturation medium
  • Individual, matured somatic embryos with well-defined scutellum and coleoptile are transferred to PHI-F embryo germination medium and incubated at 28°C in the light (about 80 ⁇ E from cool white or equivalent fluorescent lamps) .
  • regenerated plants about 10 cm tall, are potted in horticultural mix and hardened-off using standard horticultural methods.
  • PHI-A 4g/L CHU basal salts, 1.0 mL/L 1000X Eriksson’s vitamin mix, 0.5 mg/L thiamin HCl, 1.5 mg/L 2, 4-D, 0.69 g/L L-proline, 68.5 g/L sucrose, 36 g/L glucose, pH 5.2. Add 100 ⁇ M acetosyringone (filter-sterilized) .
  • PHI-B PHI-Awithout glucose, increase 2, 4-D to 2 mg/L, reduce sucrose to 30 g/L and supplemented with 0.85 mg/L silver nitrate (filter-sterilized) , 3.0 g/L 100 ⁇ M acetosyringone (filter-sterilized) , pH 5.8.
  • PHI-C PHI-B without and acetosyringonee, reduce 2, 4-D to 1.5 mg/L and supplemented with 8.0 g/L agar, 0.5 g/L 2- [N-morpholino] ethane-sulfonic acid (MES) buffer, 100 mg/L carbenicillin (filter-sterilized) .
  • MES 2- [N-morpholino] ethane-sulfonic acid
  • PHI-D PHI-C supplemented with 3 mg/L bialaphos (filter-sterilized) .
  • PHI-E 4.3 g/L of Murashige and Skoog (MS) salts, (Gibco, BRL 11117-074) , 0.5 mg/L nicotinic acid, 0.1 mg/L thiamine HCl, 0.5 mg/L pyridoxine HCl, 2.0 mg/L glycine, 0.1 g/L myo-inositol, 0.5 mg/L zeatin (Sigma, Cat.
  • MS Murashige and Skoog
  • No.Z-0164 1 mg/L indole acetic acid (IAA) , 26.4 ⁇ g/L abscisic acid (ABA) , 60 g/L sucrose, 3 mg/L bialaphos (filter-sterilized) , 100 mg/L carbenicillin (filter-sterilized) , 8 g/L agar, pH 5.6.
  • PHI-F PHI-E without zeatin, IAA, ABA; reduce sucrose to 40 g/L; replacing agar with 1.5 g/L pH 5.6.
  • Plants can be regenerated from the transgenic callus by first transferring clusters of tissue to N6 medium supplemented with 0.2 mg per liter of 2, 4-D. After two weeks the tissue can be transferred to regeneration medium (Fromm et al., Bio/Technology 8: 833-839 (1990) ) .
  • Transgenic T0 plants can be regenerated and their phenotype determined.
  • T1 seed can be collected.
  • a suppression DNA construct can be introduced into an elite maize inbred line either by direct transformation or introgression from a separately transformed line.
  • Transgenic plants can undergo more vigorous field-based experiments to study yield enhancement and/or stability under water limiting and water non-limiting conditions.
  • Subsequent yield analysis can be done to determine whether plants that contain the increased/reduced/increased expression levels or increased/increased/reduced activity of the genes identified herein (e.g., SEQ ID NOS: 1-18) have an improvement in yield performance (under stress or non-stress conditions) , when compared to the control (or reference) plants that do not contain the desired allele or SNP or a recombinant DNA of interest.
  • the genes identified herein e.g., SEQ ID NOS: 1-18
  • a DNA construct comprising a gene encoding SEQ ID NO: 1 or any of the Zea mays genesidentified herein (e.g., SEQ ID NOS: 2-18) can be introduced into an elite maize inbred line either by direct transformation or introgression from a separately transformed line.
  • Transgenic plants either inbred or hybrid, can undergo more vigorous field-based experiments to study yield enhancement and/or stability under stress and non-stress conditions.
  • Subsequent yield analysis can be done to determine whether plants that have downregulated/upregulated expression levels of one or more of the genes have an improvement in yield performance under non-stress or stress conditions, when compared to the control plants that have wild-type expression levels and activity levels.
  • Stress conditions can be water-limiting conditions, or low nitrogen conditions. Specifically, drought conditions or nitrogen limiting conditions can be imposed during the flowering and/or grain fill period for plants that contain the suppression DNA construct and the control plants. Reduction in yield can be measured for both.
  • the above method may be used to select transgenic plants with increased yield, under non-stress conditions, when compared to a control plant.
  • RNA-Seq Whole-transcriptome sequencing approaches, such as RNA-Seq, as well as the whole-genome sequencing approaches have been a popular method for identifying the causal genetic factors controlling phenotypic variation (e.g., Ng et al. (2010) Nature Genetics 42 (1) : 30–35) . Because RNA-Seq provides information on both the genomic and transcriptomic variation of a mutant or QTL, it is an efficient strategy for mapping genes and understanding their transcriptional regulation. In this study, 76 genes with nonsynonymous SNPs and 384 DEGs in the introgressed fragment, with 22 genes having both nonsynonymous mutations and differential expression, are likely responsible for qHS3.
  • GRMZM2G068913 encodes a hexokinase involved in starch metabolism, making it a potential causative gene for elevated starch content in NIL_B.
  • a G-to-Atransition occurrs in the exon 9 of ZmHXK3a, which converts glutamic acid to lysine at amino acid residue 471.
  • the position of the SNP is at 152181419 (based on version 5b. 60 of the maize reference sequence) coding for GRMZM2G068913.
  • ZmHXK3a is upregulated in NIL_B at both 14 and 21 DAP (Fig. 7) , which also supports that ZmHXK3a might be one of the important candidate genes for qHS3.
  • ZmHXK3a encodes a key enzyme, converting glucose to G6P, and therefore may play important roles in adjusting the rate of starch synthesis, due to G6P’s role as an important intermediate product in starch metabolism.
  • Intensive investigations of hexokinases in numerous plant species have revealed their distinct roles in plant metabolism, including controlling starch content.
  • nine ZmHXK gene family members have been identified. Two of these genes, ZmHXK3a and ZmHXK3b, are homologous to OsHXK3 in rice. They are both upregulated in NIL_B kernels at 14 DAP, suggesting that both of these hexokinase genes could exhibit the same function in controlling starch content.
  • ZmHXKb has the same expression pattern as ZmHXKa but does not have a QTL associated with it indicates that the regulation of ZmHXKb is likely a secondary effect of a gene within the qHS3 interval.
  • ZmHXK3a controls qHS3
  • one direct way is to overexpress and knock down ZmHXK3a to see whether the starch content in maize kernel changes.
  • the mechanism of ZmHXK3a regulating starch variation could occur at the post-transcriptional or protein level.
  • analyzing the enzyme activity of different ZmHXK3a alleles may also validate whether ZmHXK3a controls qHS3.
  • the causal genes for qHS3 could include DEGs involved in starch metabolism in the qHS3 interval or genes in that interval regulating DEGs in starch metabolism outside the QTL interval at 14 DAP. Therefore, 25 transcription factors with differential expression at 14 DAP are the second set of genes that may be responsible for qHS3. To determine whether these genes are the real causal genes for qHS3, fine mapping of qHS3 is necessary to narrow down the QTL interval.
  • Transcriptional regulation is one of the molecular mechanisms by which genes affect phenotypic variation. Altering the regulation of some candidate genes in the qHS3 region could therefore lead to transcriptional responses in related genes.
  • the large fragment introgressed in NIL_B caused a large number of genes differentially expressed, which implies a complex regulatory framework involving the candidate genes in qHS3 region.
  • Five more DEGs, AGPLLZM, SSV, GBSSII and Du1, ZmGPE5 were identified besides ZmHXK3a and ZmHXK3b.
  • the expression pattern of all DEGs caused by the candidate genes for qHS3 is consistent with their activities in starch metabolism besides AGPLLZM, which is one large subunit of AGPase.
  • AGPase is a heterotetramer comprised of two large subunits and two small subunits, each of which is encoded by distinct genes (Hannah and James 2008) .
  • Shrunken2 (Sh2) , AGPLLZM, and AGPLEMZM encode the large subunits
  • Brittle2a (Bt2a) , Brittle2b (Bt2b) , AGPSEMZM, AGPSLZM, and AGPL3 encode the small subunits.
  • mutant characterization of sh2, bt2, agpsemzm and agpllzm in maize kernels provides evidence that downregulation of these three genes reduces AGPase activity and thus decreases starch content.
  • all seven available genes encoding subunits of AGPase are downregulated in NIL_B at 14 DAP except for AGPSLZM, although AGPLLZM was the only statistically significant DEG among these genes. This makes sense, given that AGPLLZM is the plastidial form of AGPase, and its downregulation has no effect on the total endosperm AGPase activity since around 95%of the total AGPase in maize endosperm is extraplastidial.
  • RNA-Seq also provides an opportunity to mine new genes controlling a target trait and explain their functions.
  • the total number of genes encoding starch synthase is unknown in maize, although eight genes, namely Du1, SS1, Su2, SS2b-2, SS2c, SS3b-1, SS3b-2, and SS4, were cloned using mutants in maize or via comparison with the rice genome.
  • a new starch synthase gene, SSV, identified herein is upregulated in NIL_B as well as the known starch synthase gene, Du1. Their similar expression patterns indicate that they may have similar functions in starch biosynthesis.
  • the BC5F2 NIL pair was grown with three biological replicates in the winter nursery of China Agricultural University in 2011 in SanYa, Hainan province, China.
  • the bulked leaves of each NIL were collected from at least six individuals for DNA extraction. All plants were self-pollinated, and four immature ears were harvested at 14 and 21 DAP for each NIL in each replicate.
  • Four kernels from each ear of one NIL within a replicate were randomly chosen and stored at–80°C for RNA extraction. The remaining kernels of each ear were dried for measurement of starch content. Mature ears of NILs were also collected for analysis of starch.
  • Starch content in maize kernels was determined with a fermentable carbohydrate assay. Fifteen immature or mature kernels from the middle section of each ear were ground to fine powder by Genogrinder 2010 (Spex, USA) . 120 mg powder was placed into a 2 ml snap cap tube. ⁇ -amylase solution was added to each tube, then the tubes were placed in a boiling water bath for 15 min. Tubes were then incubated at 500C for 1.5 hours, followed by addition of Glucoamylase. Finally, 3 mg yeast was added to ferment glucose into ethanol and carbon dioxide. Starch content was calculated as the weight loss due to fermentation (CO 2 ) and heat (ethanol) . All samples were analyzed in duplicates, and the average was used for subsequent analysis.
  • DNA was extracted from leaf tissue of both NILs using the well-known modified CTAB method and genotyped with the MaizeSNP3K array containing 3, 072 SNPs, a subset of the Illumina MaizeSNP50 Beadchip. SNP genotyping was performed on the Illumina GoldenGate SNP genotyping platform at the National Maize Improvement Center of China, China Agricultural University. The quality of each SNP was assessed manually, and SNPs with poor quality were excluded from further analysis.
  • RNA-Seq libraries with 150-to 200-bp insert size were prepared using the Illumina TruSeq mRNA-Seq kit and sequenced on the Illumina HiSeq 2000 system with TruSeq SBS v3 reagents based on the protocol from Illumina (San Diego, CA, USA) .
  • TopHat v2.0.9 was used to map the single-end clean reads against the B73 reference genome sequence (B73 AGPv2; http: //www. maizesequence. org/) . Only reads that mapped uniquely to the genome were retained for further analysis.
  • the uniquely mapped reads were sorted depending on their alignment position on each chromosome and then converted to SAM format using Picard tools and Samtools. Unpaired SNP calling was performed using the Genome Analysis Toolkit pipeline. SNPs detected as the same in all three biological replicates and kept consistent in the same NIL at two developmental stages were used for subsequent analysis. SNPs were categorized as intergenic, exonic, or intronic based on their position. The genetic codes in which exonic SNPs were located were extracted to classify them as synonymous or nonsynonymous.
  • DEGs differentially expressed genes
  • MapMan http: //mapman. gabipd. org
  • MapMan http: //mapman. gabipd. org
  • MapMan Gene ontology (GO) enrichment for DEGs was also analyzed with MapMan.
  • the number of genes in each Bin was counted. Fisher’s exact test was used to investigate the GO enrichment category. P-values were adjusted for multiple testing based on the false-discovery rate with the BH method. Bins for which the false-discovery rate was ⁇ 0.05 were considered to be enriched.
  • First-strand cDNA was synthesized using the ProtoScript cDNA Synthesis kit (New England Biolabs, UK) . Quantitative RT-PCR was carried out in triplicate for each sample using the SYBRGreen I kit (Takara Biotechnology, China) on a 7500 Real-Time PCR System (Applied Biosystems, USA) . Maize ACTIN was used as a control for normalization between samples. Relative quantification of genes was calculated using the comparative threshold cycle method.

Abstract

Cette invention concerne un procédé d'accroissement de la teneur en amidon du maïs par modulation de l'activité ou du niveau d'expression d'un ou plusieurs gènes du maïs dont les hexokinases. La variation allélique des gènes de maïs endogènes résultent de l'accroissement de la teneur en amidon. Un procédé et des compositions pour accroître et/ou moduler la teneur en amidon sont en outre décrits.
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