WO2009105492A2 - Plantes transgéniques ayant des caractéristiques d'efficacité d'utilisation d'azote modifiées - Google Patents

Plantes transgéniques ayant des caractéristiques d'efficacité d'utilisation d'azote modifiées Download PDF

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WO2009105492A2
WO2009105492A2 PCT/US2009/034431 US2009034431W WO2009105492A2 WO 2009105492 A2 WO2009105492 A2 WO 2009105492A2 US 2009034431 W US2009034431 W US 2009034431W WO 2009105492 A2 WO2009105492 A2 WO 2009105492A2
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plant
nucleic acid
use efficiency
polypeptide
nitrogen use
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PCT/US2009/034431
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WO2009105492A3 (fr
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Richard Schneeberger
Abbey Pierson
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Ceres, Inc.
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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
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • 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

  • This document relates to methods and materials involved in modulating nitrogen use efficiency levels in plants. For example, this document provides plants having increased nitrogen use efficiency levels as well as materials and methods for making plants and plant products having increased nitrogen and nitrogen use efficiency levels.
  • Nitrogen is often the rate-limiting element in plant growth, and all field crops have a fundamental dependence on exogenous nitrogen sources.
  • Nitrogenous fertilizer which is usually supplied as ammonium nitrate, potassium nitrate, or urea, typically accounts for 40% of the costs associated with crops, such as corn and wheat in intensive agriculture. Increased efficiency of nitrogen use by plants should enable the production of higher yields with existing fertilizer inputs and/or enable existing yields of crops to be obtained with lower fertilizer input, or better yields on soils of poorer quality. Also, higher amounts of proteins in the crops could also be produced more cost-effectively.
  • Plants have a number of means to cope with nutrient deficiencies.
  • the nitrogen sensing mechanism relies on regulated gene expression and enables rapid physiological and metabolic responses to changes in the supply of inorganic nitrogen in the soil by adjusting nitrogen uptake, reduction, partitioning, remobilization and transport in response to changing environmental conditions.
  • Nitrate acts as a signal to initiate a number of responses that serve to reprogram plant metabolism, physiology and development (Redinbaugh et al. (1991) Physiol. Plant. 82, 640-650.; Forde (2002) Annual Review of Plant Biology 53, 203-224).
  • Nitrogen-inducible gene expression has been characterized for a number of genes in some detail.
  • nitrate reductase include nitrate reductase, nitrite reductase, 6-phosphoglucante dehydrogenase, and nitrate and ammonium transporters (Redinbaugh et al. (1991) Physiol. Plant. 82, 640-650; Huber et al. (1994) Plant Physiol 106, 1667-1674; Hwang et al. (1997) Plant Physiol. 113, 853-862; Redinbaugh et al. (1998) Plant Science 134, 129-140; Gazzarrini et al. (1999) Plant Cell 11, 937-948; Glass et al. (2002) J. Exp. Bot. 53, 855-864; Okamoto et al. (2003) Plant Ce// Physiol. 44, 304-317).
  • the present invention relates to a method for increasing growth potential, and/or increasing levels of nitrogen use efficiency in plants, characterized by expression of recombinant DNA molecules stably integrated into the plant genome.
  • This document provides methods and materials related to plants having modulated levels of nitrogen use efficiency.
  • this document provides transgenic plants and plant cells having increased levels of nitrogen use efficiency, nucleic acids ⁇ i.e. isolated polynucleotides), polypeptides encoded thereby used to generate transgenic plants and plant cells having increased levels of nitrogen use efficiency, and methods for making plants and plant cells having increased levels of nitrogen use efficiency.
  • Such plants and plant cells can be grown to produce, for example, plants having increased nitrogen use efficiency and/or increased levels of nitrogen content. Plants having increased levels of nitrogen use efficiency may be useful to produce biomass which may be converted to a liquid fuel or other chemicals and/or to produce food and feed having increased nitrogen content, which may benefit food nutrient value, feedstock and biofuels.
  • a method comprises growing a plant cell comprising an exogenous nucleic acid.
  • the exogenous nucleic acid comprises a regulatory region operably linked to a nucleotide sequence encoding a polypeptide.
  • the Hidden Markov Model (HMM) bit score of the amino acid sequence of the polypeptide is greater than about 1200 using an HMM generated from the amino acid sequences depicted in Figure 11 or 12.
  • the plant tissue has a difference in the level of nitrogen use efficiency as compared to the corresponding level in plant tissue from a control plant that does not comprise the exogenous nucleic acid.
  • a method comprises growing a plant cell comprising an exogenous nucleic acid.
  • the exogenous nucleic acid comprises a regulatory region operably linked to a nucleotide sequence encoding a polypeptide having 80, 85, 90, 95, 96, 97, 98 or 99 percent or greater sequence identity to an amino acid sequence set forth in SEQ ID NOs: 2, 4, 6, 8, 10, 12, 13, 14, 15, 16, 17, 20, 22, 24, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 40, 42, 44, 46, 47, 48, 49, 51, 52, 53, or 54.
  • a plant tissue of a plant produced from the plant cell has a difference in the level of nitrogen use efficiency as compared to the corresponding level in the corresponding tissue of a control plant that does not comprise the exogenous nucleic acid.
  • a method comprises growing a plant cell comprising an exogenous nucleic acid.
  • the exogenous nucleic acid comprises a regulatory region operably linked to a nucleotide sequence having 80, 85, 90, 95, 96, 97, 98 or 99 percent or greater sequence identity to at least a fragment of a nucleotide sequence set forth in SEQ ID NO: 1, 3, 5, 7, 9, 11, 18, 19, 21, 23, 25, 39, 41, 43, 45, or 50.
  • a tissue of a plant produced from the plant cell has a difference in the level of nitrogen use efficiency as compared to the corresponding level in tissue of a control plant that does not comprise the exogenous nucleic acid.
  • a method comprises introducing into a plant cell an exogenous nucleic acid, that comprises a regulatory region operably linked to a nucleotide sequence encoding a polypeptide.
  • the HMM bit score of the amino acid sequence of the polypeptide is greater than about 1200 using an HMM generated from the amino acid sequences depicted in one of Figures 11 and 12.
  • a tissue of a plant produced from the plant cell has a difference in the level of nitrogen use efficiency as compared to the corresponding level in tissue of a control plant that does not comprise the exogenous nucleic acid.
  • the amino acid sequence of the polypeptide has an exogenous nucleic acid, that comprises a regulatory region operably linked to a nucleotide sequence encoding a polypeptide.
  • the HMM bit score of the amino acid sequence of the polypeptide is greater than about 1200 using an HMM generated from the amino acid sequences depicted in one of Figures 11 and 12.
  • a tissue of a plant produced from the plant cell has a difference
  • the amino acid sequence of the polypeptide has an HMM score greater than about 1440, using an HMM generated from the amino acid sequences depicted in Figure 12, wherein the polypeptide comprises a PRT2 domain having 50, 60, 70, 80, 85, 90, 95, 96, 97, 98 or 99 percent or greater sequence identity to amino acid residues 96 to 494 of SEQ ID NO: 17, residues 96 to 494 of SEQ ID NO:20, residues 98 to 502 of SEQ ID NO:22, residues 110 to 517 of SEQ ID NO:24, residues 96 to 494 of SEQ ID NO:26, residues 105 to 503 of SEQ ID NO:27, residues 100 to 510 of SEQ ID NO:28, residues 100 to 513 of SEQ ID NO:29, residues 96 to 494 of SEQ ID NO:30, residues 100 to 510 of SEQ ID NO:31, residues 100 to 510 of SEQ ID NO:32, residues 100 to
  • a method comprises introducing into a plant cell an exogenous nucleic acid that comprises a regulatory region operably linked to a nucleotide sequence encoding a polypeptide having 80, 85, 90, 95, 96, 97, 98 or 99 percent or greater sequence identity to an amino acid sequence set forth in SEQ ID NO: 2, 4, 6, 8, 10, 12, 13, 14, 15, 16, 17, 20, 22, 24, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 40, 42, 44, 46, 47, 48, 49, 51 , 52, 53, or 54.
  • a tissue of a plant produced from the plant cell has a difference in the level of nitrogen use efficiency as compared to the corresponding level in tissue of a control plant that does not comprise the exogenous nucleic acid.
  • a method comprises introducing into a plant cell an exogenous nucleic acid that comprises a regulatory region operably linked to a nucleotide sequence having 80, 85, 90, 95, 96, 97, 98 or 99 percent or greater sequence identity to a nucleotide sequence set forth in SEQ ID NO: 1, 3, 5, 7, 9, 11, 18, 19, 21, 23, 25, 39, 41, 43, 45, or 50.
  • a tissue of a plant produced from the plant cell has a difference in the level of nitrogen use efficiency as compared to the corresponding level in tissue of a control plant that does not comprise the exogenous nucleic acid.
  • Plant cells comprising an exogenous nucleic acid are provided herein.
  • the exogenous nucleic acid comprises a regulatory region operably linked to a nucleotide sequence encoding a polypeptide.
  • the HMM bit score of the amino acid sequence of the polypeptide is greater than about 1200, using an HMM based on the amino acid sequences depicted in one of Figures 11 and 12.
  • the plant tissue has a difference in the level of nitrogen use efficiency as compared to the corresponding level in plant tissue of a control plant that does not comprise the exogenous nucleic acid.
  • the exogenous nucleic acid comprises a regulatory region operably linked to a nucleotide sequence encoding a polypeptide having 80, 85, 90, 95, 96, 97, 98 or 99 percent or greater sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 13, 14, 15, 16, 17, 20, 22, 24, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 40, 42, 44, 46, 47, 48, 49, 51, 52, 53, and 54.
  • a plant tissue of a plant produced from the plant cell has a difference in the level of nitrogen use efficiency as compared to the corresponding level in plant tissue of a control plant that does not comprise the exogenous nucleic acid.
  • the exogenous nucleic acid comprises a regulatory region operably linked to a nucleotide sequence having 80, 85, 90, 95, 96, 97, 98 or 99 percent or greater sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NO: 1, 3, 5, 7, 9, 11 , 18, 19, 21, 23, 25, 39, 41, 43, 45, and 50.
  • a tissue of a plant produced from the plant cell has a difference in the level of nitrogen use efficiency as compared to the corresponding level in tissue of a control plant that does not comprise the exogenous nucleic acid.
  • a transgenic plant comprising such a plant cell is also provided.
  • a food, feedstock or biofuel product is also provided.
  • the product comprises tissue from a transgenic plant with increased nitrogen content.
  • an isolated nucleic acid comprises a nucleotide sequence having 95% or greater sequence identity to the nucleotide sequence set forth in SEQ ID NO: 1, 3, 5, 7, 9, 11, 18, 19, 21, 23, 25, 39, 41, 43, 45, or 50.
  • an isolated nucleic acid comprises a nucleotide sequence encoding a polypeptide having 80% or greater sequence identity to the amino acid sequence set forth in SEQ ID NOs: 2, 4, 6, 8, 10, 12, 13, 14, 15, 16, 17, 20, 22, 24, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 40, 42, 44, 46, 47, 48, 49, 51, 52, 53, or 54.
  • methods of identifying a genetic polymorphism associated with variation in the level of nitrogen use efficiency include providing a population of plants, and determining whether one or more genetic polymorphisms in the population are genetically linked to the locus for a polypeptide selected from the group consisting of the polypeptides depicted in Figures 11 and 12, and functional homologs thereof.
  • the correlation between variation in the level of nitrogen use efficiency in a tissue in plants of the population and the presence of the one or more genetic polymorphisms in plants of the population is measured, thereby permitting identification of whether or not the one or more genetic polymorphisms are associated with such variation.
  • the invention provides a method of making a plant line by: a) determining whether one or more genetic polymorphisms in a population of plants is associated with the locus for a polypeptide selected from the group consisting of the polypeptides depicted in Figures 11 and 12 and functional homologs thereof; b) identifying one or more plants in the population in which the presence of at least one allele at said one or more genetic polymorphisms is associated with variation in a trait; c) crossing each of the one or more identified plants with itself or a different plant to produce seed; d) crossing at least one progeny plant grown from the seed with itself or a different plant; and e) repeating steps c) and d) for an additional 0-5 generations to make the plant line, wherein at least one allele is present in the plant line.
  • Figure 1 is a graph showing the results of an analysis of biomass of 35S-NRT1.3 plants grown hydroponically on 1OmM KNO 3 .
  • A Plant biomass.
  • B Root biomass. Standard deviation is indicated at statistical significance of P ⁇ 0.05.
  • WT refers to wild -type Arabidopsis thaliana control plants and (HH) to Arabidopsis thaliana plants transformed with and homozygous for the indicated gene.
  • Figure 2 is a graph showing the results of an analysis of nitrate levels in 35S-NRT1.3 media during experimental growth.
  • Figure 3 is a graph showing the results of an analysis of tissue nitrate levels for 35S- NRTl.3 plants grown hydroponically on 1OmM KNO 3 .
  • A Shoot nitrate.
  • B Root nitrate. Standard deviation is indicated
  • Figure 4 is a graph showing the results of an analysis of metabolite profiling, organic and amino acid levels, for shoot tissue of 35S-NRTL3 plants grown hydroponically.
  • Figure 5 is a graph showing the results of an analysis of carbon and nitrogen content in 35S-NRT1.3 shoots.
  • A Percent nitrogen.
  • B Percent carbon. Standard deviation is indicated.
  • Figure 6 is a graph showing the results of an analysis of biomass of 35S-NRT1.4 plants grown hydroponically on 1OmM KNO 3 .
  • A Shoot biomass.
  • B Root biomass. Standard deviation is indicated.
  • Figure 7 is a graph showing the results of an analysis of nitrate levels in 35S-NRT1.4 media during experimental growth.
  • Figure 8 is a graph showing the results of an analysis of tissue nitrate levels for 35S- NRTlA plants grown hydroponically on 1OmM KNO 3 .
  • A Shoot nitrate.
  • B Root nitrate. Standard deviation is indicated.
  • Figure 9 is a graph showing the results of an analysis of metabolite profiling, organic and amino acid levels, for shoot tissue of 35$-NRT1.4 plants grown hydroponically.
  • A Compounds showing increased levels in 35S-NRT1.4.
  • B Compounds showing reduced levels in 35S-NRT1.4. Standard deviation is indicated.
  • Figure 10 is a graph showing the results of an analysis of tissue carbon and nitrogen content for 35S-NRT1.4 plants grown hydroponically.
  • B Tissue carbon. Standard deviation is indicated.
  • Figure 11 is an alignment of functional homologues of NRTl .3 (At3g21670), SEQ ID NO:2.
  • a dash in an aligned sequence represents a gap, i.e., a lack of an amino acid at that position.
  • Identical amino acids or conserved amino acid substitutions among aligned sequences are identified by boxes.
  • Figure 11 and the other alignment figures provided herein were generated using the program MUSCLE version 3.52.
  • Figure 12 is an alignment of functional homologues of NRTl .4 (At2g26690), SEQ ID NO: 1 (At2g26690), SEQ ID NO: 1 (At2g26690), SEQ ID NO: 1 (At2g26690), SEQ ID NO: 1 (At2g26690), SEQ ID NO: 1 (At2g26690), SEQ ID NO: 1
  • a dash in an aligned sequence represents a gap, i.e., a lack of an amino acid at that position. Identical amino acids or conserved amino acid substitutions among aligned sequences are identified by boxes. Figure 12 and the other alignment figures provided herein were generated using the program MUSCLE version 3.52.
  • the invention features methods and materials related to modulating nitrogen use efficiency levels in plants.
  • the plants may also have modulated levels of nitrogen content.
  • the methods can include transforming a plant cell with a nucleic acid encoding a nitrogen use efficiency-modulating polypeptide, wherein expression of the polypeptide results in a modulated level of nitrogen use efficiency.
  • Plant cells produced using such methods can be grown to produce plants having an increased or decreased nitrogen content.
  • Such plants, and the seeds of such plants may be used to produce, for example, food products, feedstock and biofuels having an increased nitrogen content and/or nutritional value.
  • ABSORANS refers to the nitrogen concentration at which a given plant species will be adversely affected as evidenced by symptoms such as decreased chlorophyll (for example, measured by chlorophyll a/b absorbance) decreased photosynthesis (for example, measured by CO 2 fixation), membrane damage (for example, measured by electrolyte leakage), chlorosis (for example, via visual inspection), loss of biomass or seed yield, Since plant species vary in their capacity to tolerate abnormal nitrogen conditions, the precise environmental conditions that cause nitrogen stress can not be generalized. However, plants having increased nitrogen use efficiency and/or plants tolerant to nitrogen stress conditions are characterized by their ability to retain their normal appearance or recover quickly from abnormal nitrogen conditions. Such plants produce higher biomass and yield than plants that do not exhibit nitrogen use efficiency and/or tolerance to nitrogen stress conditions. Differences in physical appearance, recovery and yield can be quantified and statistically analyzed using well known measurement and analysis methods.
  • Plant seedlings vary considerably in their ability to grow under abnormal nitrogen conditions. Generally, seedlings of many plant species will not grow well at nitrogen concentration less than about 1 ppm or greater than about 750 ppm. High concentrations of ammoniac nitrogen are also inhibitory to seed germination and seedling growth and can occur when ammonium based fertilizer is used (Brenner and Krogmeier (1989) PNAS 86:8185- 8188).
  • seeds and seedlings having increased nitrogen use efficiency and/or Seeds and seedlings tolerant to nitrogen stress conditions during germination can survive for relatively long periods under which the nitrogen concentration is too high or too low for normal growth. Since plant species vary in their capacity to tolerate abnormal nitrogen conditions during germination, the precise environmental conditions that cause nitrogen stress during germination can not be generalized. However, seeds and seedlings that are nitrogen tolerant during germination are characterized by their ability to remain viable or recover quickly from low or high nitrogen conditions. Such plants germinate, become established, grow more quickly and ultimately produce more biomass and yield than plants that are not nitrogen tolerant. Differences in germination rate, appearance, recovery and yield can be quantified and statistically analyzed using well known measurement and analysis methods.
  • amino acid refers to one of the twenty biologically occurring amino acids and to synthetic amino acids, including D/L optical isomers.
  • Cell type-preferential promoter or “tissue-preferential promoter” refers to a promoter that drives expression preferentially in a target cell type or tissue, respectively, but may also lead to some transcription in other cell types or tissues as well.
  • Control plant refers to a plant that does not contain the exogenous nucleic acid present in a transgenic plant of interest, but otherwise has the same or similar genetic background as such a transgenic plant.
  • a suitable control plant can be a non-transgenic wild type plant, a non-transgenic segregant from a transformation experiment, or a transgenic plant that contains an exogenous nucleic acid other than the exogenous nucleic acid of interest.
  • Domains are groups of substantially contiguous amino acids in a polypeptide that can be used to characterize protein families and/or parts of proteins. Such domains have a "fingerprint” or “signature” that can comprise conserved primary sequence, secondary structure, and/or three-dimensional conformation. Generally, domains are correlated with specific in vitro and/or in vivo activities.
  • a domain can have a length of from 10 amino acids to 400 amino acids, e.g., 10 to 50 amino acids, or 25 to 100 amino acids, or 35 to 65 amino acids, or 35 to 55 amino acids, or 45 to 60 amino acids, or 200 to 300 amino acids, or 300 to 400 amino acids.
  • Down-regulation refers to regulation that decreases production of expression products (mRNA, polypeptide, or both) relative to basal or native states.
  • Exogenous with respect to a nucleic acid indicates that the nucleic acid is part of a recombinant nucleic acid construct, or is not in its natural environment.
  • an exogenous nucleic acid can be a sequence from one species introduced into another species, i.e., a heterologous nucleic acid. Typically, such an exogenous nucleic acid is introduced into the other species via a recombinant nucleic acid construct.
  • An exogenous nucleic acid can also be a sequence that is native to an organism and that has been reintroduced into cells of that organism.
  • exogenous nucleic acid that includes a native sequence can often be distinguished from the naturally occurring sequence by the presence of non-natural sequences linked to the exogenous nucleic acid, e.g., non-native regulatory sequences flanking a native sequence in a recombinant nucleic acid construct.
  • stably transformed exogenous nucleic acids typically are integrated at positions other than the position where the native sequence is found. It will be appreciated that an exogenous nucleic acid may have been introduced into a progenitor and not into the cell under consideration.
  • a transgenic plant containing an exogenous nucleic acid can be the progeny of a cross between a stably transformed plant and a non-transgenic plant. Such progeny are considered to contain the exogenous nucleic acid.
  • “Expression” refers to the process of converting genetic information of a polynucleotide into RNA through transcription, which is catalyzed by an enzyme, RNA polymerase, and into protein, through translation of mRNA on ribosomes.
  • Heterologous polypeptide refers to a polypeptide that is not a naturally occurring polypeptide in a plant cell, e.g. , a transgenic Panicum virgatum plant transformed with and expressing the coding sequence for a nitrogen transporter polypeptide from a Zea mays plant.
  • High Nitrogen Conditions refers to total nitrogen concentrations in soil and/or plant tissue that will result in growth retardation or tissue damage due to ionic or osmotioc stress. Growth medium concentrations of nitrogen that will lead to nitrogen stress can not be generalized. However, nitrogen concentrations that reduce germination rate by more than 20%, 25%, 30%, 35%, 40%, 45% or 50% are considered to be high and in excess.
  • isolated nucleic acid includes a naturally-occurring nucleic acid, provided one or both of the sequences immediately flanking that nucleic acid in its naturally-occurring genome is removed or absent.
  • an isolated nucleic acid includes, without limitation, a nucleic acid that exists as a purified molecule or a nucleic acid molecule that is incorporated into a vector or a virus.
  • Low Nitrogen Conditions refers to nitrogen concentrations in soil and/or plant tissue which lead to nitrogen deficiency symptoms such as pale green leaf color, chlorosis and reduced growth and vigor. These concentrations of nitrogen are generally less than 10 ppm nitrate in a soil nitrate test. Typically, low nitrogen conditions lead to a reduction of at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80% or 90% in growth and/or vigor.
  • Modulation of the level of nitrogen use efficiency refers to the change in the level of nitrogen stored in a plant that is observed as a result of expression of, or transcription from, an exogenous nucleic acid in a plant cell. The change in level is measured relative to the corresponding level in control plants. The modulation can be an increase or decrease in level as compared to control plants.
  • NUE Nonrogen Use Efficiency
  • NUE can also be measured as a change in nitrogen, nitrate, and/or metabolite content in a plant and/or plant tissue. NUE can also be represented as the product of two factors, uptake efficiency and utilization efficiency. Nitrogen uptake efficiency measures the efficiency with which a plant removes nitrogen from the soil while utilization efficiency measures the yield obtained per unit of nutrient absorbed by a plant. A number of different biological processes are involved in defining a particular plant's NUE and can independently affect processes involved in uptake efficiency and utilization efficiency. Many of these processes are genetically determined and can be improved by genetic or biotechnologic manipulation of the genes responsible for determining these traits.
  • Normal Nitrogen Conditions refers to the nitrogen concentration at which a given plant species will grow without damage. Since plant species vary in their capacity to tolerate nitrogen conditions, the precise environmental conditions that provide normal nitrogen conditions can not be generalized. However, the normal growth exhibited by nitrogen intolerant plants is characterized by the inability to retain a normal appearance or to recover quickly from abnormal nitrogen conditions. Such nitrogen intolerant plants produce lower biomass and yield less than plants that are nitrogen tolerant. Differences in physical appearance, recovery and yield can be quantified and statistically analyzed using well known measurement and analysis methods.
  • Plant seedlings vary considerably in their ability to grow under abnormal nitrogen conditions. Generally, seedlings of many plant species will not grow well at nitrogen concentration less than about 1 ppm or greater than about 750 ppm. High concentrations of ammoniac nitrogen are also inhibitory to seed germination and seedling growth and can occur when ammonium based fertilizer is used (Brenner and Krogmeier (1989) PNAS 86:8185- 8188).
  • Nucleic acid and polynucleotide are used interchangeably herein, and refer to both RNA and DNA, including cDNA, genomic DNA, synthetic DNA, and DNA or RNA containing nucleic acid analogs. Polynucleotides can have any three-dimensional structure. A nucleic acid can be double-stranded or single- stranded (i.e., a sense strand or an antisense strand).
  • Non-limiting examples of polynucleotides include genes, gene fragments, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, siRNA, micro- RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, nucleic acid probes and nucleic acid primers.
  • mRNA messenger RNA
  • transfer RNA transfer RNA
  • ribosomal RNA siRNA
  • micro- RNA micro- RNA
  • ribozymes cDNA
  • recombinant polynucleotides branched polynucleotides
  • nucleic acid probes and nucleic acid primers include genes, gene fragments, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, siRNA, micro- RNA, ribozymes, cDNA, recombinant polynucleotides,
  • operably linked refers to the positioning of a regulatory region and a sequence to be transcribed in a nucleic acid so that the regulatory region is effective for regulating transcription or translation of the sequence.
  • the translation initiation site of the translational reading frame of the coding sequence is typically positioned between one and about fifty nucleotides downstream of the regulatory region.
  • a regulatory region can, however, be positioned as much as about 5,000 nucleotides upstream of the translation initiation site, or about 2,000 nucleotides upstream of the transcription start site.
  • Polypeptide refers to a compound of two or more subunit amino acids, amino acid analogs, or other peptidomimetics, regardless of post-translational modification, e.g., phosphorylation or glycosylation.
  • the subunits may be linked by peptide bonds or other bonds such as, for example, ester or ether bonds.
  • Full-length polypeptides, truncated polypeptides, point mutants, insertion mutants, splice variants, chimeric proteins, and fragments thereof are encompassed by this definition.
  • Progeny includes descendants of a particular plant or plant line. Progeny of an instant plant include seeds formed on F 1 , F 2 , F 3 , F 4 , F 5 , F 6 and subsequent generation plants, or seeds formed on BCi, BC 2 , BC 3 , and subsequent generation plants, or seeds formed on F)BCi, FiBC 2 , F]BC 3 , and subsequent generation plants.
  • the designation Fj refers to the progeny of a cross between two parents that are genetically distinct.
  • the designations F 2 , F 3 , F 4 , F 5 and F 6 refer to subsequent generations of self- or sib-pollinated progeny of an Fi plant.
  • regulatory region refers to a nucleic acid having nucleotide sequences that influence transcription or translation initiation and rate, and stability and/or mobility of a transcription or translation product. Regulatory regions include, without limitation, promoter sequences, enhancer sequences, response elements, protein recognition sites, inducible elements, protein binding sequences, 5' and 3' untranslated regions (UTRs), transcriptional start sites, termination sequences, polyadenylation sequences, introns, and combinations thereof.
  • a regulatory region typically comprises at least a core (basal) promoter.
  • a regulatory region also may include at least one control element, such as an enhancer sequence, an upstream element or an upstream activation region (UAR).
  • a suitable enhancer is a cis-regulatory element (-212 to -154) from the upstream region of the octopine synthase (ocs) gene. Fromm et al. (1989) The Plant Cell, 1 :977-984. [0047] "Up-regulation” refers to regulation that increases the level of an expression product (mRNA, polypeptide, or both) relative to basal or native states.
  • Varying Nitrogen Conditions refers to growth conditions where the concentration of available nitrogen in soil and/or plant tissue fluctuates within and outside of the normal range. This phrase encompasses situations where the available nitrogen concentration is initially low, but increases to normal or high levels as well as situations where the initial available nitrogen concentration is high, but then falls to normal or low levels. Situations involving multiple changes in available nitrogen concentration, such as fluctuations from low to high to low levels, are also encompassed by this phrase. These available nitrogen concentration changes can occur in a gradual or punctuated manner.
  • Vector refers to a replicon, such as a plasmid, phage, or cosmid, into which another DNA segment may be inserted so as to bring about the replication of the inserted segment.
  • a vector is capable of replication when associated with the proper control elements.
  • the term “vector” includes cloning and expression vectors, as well as viral vectors and integrating vectors.
  • An "expression vector” is a vector that includes a regulatory region.
  • Polypeptides described herein include nitrogen use efficiency modulating polypeptides. Nitrogen use efficiency modulating polypeptides can be effective to modulate nitrogen uptake levels and/or nitrogen content when expressed in a plant or plant cell. Such polypeptides typically contain at least one domain indicative of nitrogen use efficiency- modulating polypeptides, as described in more detail herein. Nitrogen use efficiency- modulating polypeptides typically have an HMM bit score that is greater than 100, as described in more detail herein.
  • nitrogen use efficiency modulating polypeptides have greater than 80, 85, 90, 95, 96, 97, 97 or 99 % identity to SEQ ID NOs: 2, 4, 6, 8, 10, 12, 13, 14, 15, 16, 17, 20, 22, 24, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 40, 42, 44, 46, 47, 48, 49, 51, 52, 53, or 54 as described in more detail herein.
  • a nitrogen use efficiency modulating polypeptide can contain a PTR2 domain, which is predicted to be characteristic of a POT family member.
  • the POT (proton-dependent oligopeptide transport) proteins typically are proton dependent transporters (Paulsen and Skurray (1994) Trends Biochem Sci 19:404-404).
  • the PTR family of proteins is distinct from the ABC-type peptide transporters and was uncovered by sequence analyses of a number of peptide transport proteins. These proteins are involved in the intake of small peptides with the concomitant uptake of a proton and are integral membrane proteins predicted to typically comprise twelve transmembrane regions.
  • SEQ ID NOs: 2, 4, 6, 8, 10, 12, 13, 14, 15, 16, 17, 20, 22, 24, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 40, 42, 44, 46, 47, 48, 49, 51, 52, 53, or 54 set forth the amino acid sequence of polypeptides containing a putative PTR2 domain.
  • Examples are identified herein and in the Sequence Listing as Ceres NRT 1.3 (SEQ ID NO: 1), Ceres CLONE ID 111089 (SEQ ID NO:7), NRT 1.4 Genomic (SEQ ID NO: 18) and NRT 1.4 Predicted cDNA Sequence (SEQ ID NO: 19) that are predicted encode a polypeptide containing a putative PTR2 domain.
  • one or more functional homologs of a reference nitrogen use efficiency-modulating polypeptide defined by one or more of the pfam descriptions indicated above are suitable for use as nitrogen use efficiency-modulating polypeptides.
  • a functional homolog is a polypeptide that has sequence similarity to a reference polypeptide, and that carries out one or more of the biochemical or physiological function(s) of the reference polypeptide.
  • a functional homolog and the reference polypeptide may be natural occurring polypeptides, and the sequence similarity may be due to convergent or divergent evolutionary events. As such, functional homologs are sometimes designated in the literature as homologs, or orthologs, or paralogs.
  • Variants of a naturally occurring functional homolog such as polypeptides encoded by mutants of a wild type coding sequence, may themselves be functional homologs.
  • Functional homologs can also be created via site-directed mutagenesis of the coding sequence for a nitrogen use efficiency-modulating polypeptide, or by combining domains from the coding sequences for different naturally- occurring nitrogen use efficiency-modulating polypeptides ("domain swapping").
  • domain swapping domain swapping
  • Functional homologs can be identified by analysis of nucleotide and polypeptide sequence alignments. For example, performing a query on a database of nucleotide or polypeptide sequences can identify homologs of nitrogen use efficiency- modulating polypeptides. Sequence analysis can involve BLAST, Reciprocal BLAST, or PSI-BLAST analysis of nonredundant databases using a nitrogen use efficiency-modulating polypeptide amino acid sequence as the reference sequence. Amino acid sequence is, in some instances, deduced from the nucleotide sequence. Those polypeptides in the database that have greater than 40% sequence identity are candidates for further evaluation for suitability as a nitrogen use efficiency-modulating polypeptide.
  • Amino acid sequence similarity allows for conservative amino acid substitutions, such as substitution of one hydrophobic residue for another or substitution of one polar residue for another. If desired, manual inspection of such candidates can be carried out in order to narrow the number of candidates to be further evaluated. Manual inspection can be performed by selecting those candidates that appear to have domains present in nitrogen use efficiency-modulating polypeptides, e.g., conserved functional domains.
  • conserveed regions can be identified by locating a region within the primary amino acid sequence of a nitrogen use efficiency modulating polypeptide that is a repeated sequence, forms some secondary structure (e.g., helices and beta sheets), establishes positively or negatively charged domains, or represents a protein motif or domain. See, e.g., the Pfam web site describing consensus sequences for a variety of protein motifs and domains on the World Wide Web at sanger.ac.uk/Software/Pfam/ and pfam.janelia.org/. A description of the information included at the Pfam database is described in Sonnhammer et al. (1998) Nucl. Acids Res., 26:320-322; Sonnhammer et al.
  • conserveed regions also can be determined by aligning sequences of the same or related polypeptides from closely related species. Closely related species preferably are from the same family. In some embodiments, alignment of sequences from two different species is adequate.
  • polypeptides that exhibit at least about 40% amino acid sequence identity are useful to identify conserved regions.
  • conserved regions of related polypeptides exhibit at least 45% amino acid sequence identity ⁇ e.g., at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% amino acid sequence identity).
  • a conserved region exhibits at least 92%, 94%, 96%, 98%, or 99% amino acid sequence identity.
  • a functional homolog of SEQ ID NO:2 has an amino acid sequence with at least 40% sequence identity, e.g., 50%, 52%, 56%, 59%, 61%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity, to the amino acid sequence set forth in SEQ ID NO:2.
  • a functional homolog of SEQ ID NO: 17 has an amino acid sequence with at least 40% sequence identity, e.g., 50%, 52%, 56%, 59%, 61%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity, to the amino acid sequence set forth in SEQ ID NO: 17.
  • variants of nitrogen use efficiency-modulating polypeptide facilitates production of variants of nitrogen use efficiency- modulating polypeptides.
  • Variants of nitrogen use efficiency-modulating polypeptides typically have 10 or fewer conservative amino acid substitutions within the primary amino acid sequence, e.g., 7 or fewer conservative amino acid substitutions, 5 or fewer conservative amino acid substitutions, or between 1 and 5 conservative substitutions.
  • a useful variant polypeptide can be constructed based on one of the alignments set forth in Figure 11 or
  • Such a polypeptide includes the conserved regions, arranged in the order depicted in the Figure from amino-terminal end to carboxy-terminal end. Such a polypeptide may also include zero, one, or more than one amino acid in positions marked by dashes. When no amino acids are present at positions marked by dashes, the length of such a polypeptide is the sum of the amino acid residues in all conserved regions. When amino acids are present at all positions marked by dashes, such a polypeptide has a length that is the sum of the amino acid residues in all conserved regions and all dashes.
  • useful nitrogen use efficiency-modulating polypeptides include those that fit a Hidden Markov Model based on the polypeptides set forth in Figures 11 or 12.
  • a Hidden Markov Model is a statistical model of a consensus sequence for a group of functional homologs. See, Durbin et al. (1998) Biological Sequence Analysis: Probabilistic Models of Proteins and Nucleic Acids, Cambridge University Press, Cambridge, UK. An HMM is generated by the program HMMER 2.3.2 with default program parameters, using the sequences of the group of functional homologs as input. The multiple sequence alignment is generated by ProbCons (Do et al.
  • HMMER 2.3.2 was released October 3, 2003 under a GNU general public license, and is available from various sources on the World Wide Web such as hmmer.janelia.org; hmmer.wustl.edu; and fr.com/hmmer232/.
  • Hmmbuild outputs the model as a text file.
  • the HMM for a group of functional homologs can be used to determine the likelihood that a candidate nitrogen use efficiency-modulating polypeptide sequence is a better fit to that particular HMM than to a null HMM generated using a group of sequences that are not structurally or functionally related.
  • the likelihood that a candidate polypeptide sequence is a better fit to an HMM than to a null HMM is indicated by the HMM bit score, a number generated when the candidate sequence is fitted to the HMM profile using the HMMER hmmsearch program.
  • the default E-value cutoff (E) is 10.0
  • the default bit score cutoff (T) is negative infinity
  • the default number of sequences in a database (Z) is the real number of sequences in the database
  • the default E-value cutoff for the per-domain ranked hit list (domE) is infinity
  • the default bit score cutoff for the per-domain ranked hit list (domT) is negative infinity
  • a high HMM bit score indicates a greater likelihood that the candidate sequence carries out one or more of the biochemical or physiological function(s) of the polypeptides used to generate the HMM.
  • a high HMM bit score is at least 20, and often is higher. Slight variations in the HMM bit score of a particular sequence can occur due to factors such as the order in which sequences are processed for alignment by multiple sequence alignment algorithms such as the ProbCons program. Nevertheless, such HMM bit score variation is minor.
  • the nitrogen use efficiency-modulating polypeptides discussed below fit the indicated HMM with an HMM bit score greater than 20 (e.g., greater than 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, or 500 or even greater).
  • the HMM bit score of a nitrogen use efficiency-modulating polypeptide discussed below is about 50%, 60%, 70%, 80%, 90%, or 95% of the HMM bit score of a functional homolog provided in the Sequence Listing.
  • a nitrogen use efficiency-modulating polypeptide discussed below fits the indicated HMM with an HMM bit score greater than 20, and has a domain indicative of a nitrogen use efficiency-modulating polypeptide.
  • a nitrogen use efficiency-modulating polypeptide discussed below fits the indicated HMM with an HMM bit score greater than 20, and has 70% or greater sequence identity (e.g., at least 75%, 80%, 85%, 90%, 95%, or 100% sequence identity) to an amino acid sequence shown in Figures 11 or 12.
  • Polypeptides are shown in the Sequence Listing that have HMM bit scores greater than 1210 when fitted to an HMM generated from the amino acid sequences set forth in Figure 11.
  • Such polypeptides include NRT 1.3 (SEQ ID NO:2), Ceres ANNOT ID no. 1445187 (SEQ ID NO:4) , Ceres ANNOT ID no. 1498169 (SEQ ID NO:6), Ceres CLONE ID no. 111089 (SEQ ID NO:8), Ceres CLONE ID no. 1583036 (SEQ ID NO: 10), Ceres CLONE ID no. 1806436 (SEQ ID NO: 12), Public GI ID no. 15233123 (SEQ ID NO: 12), Public GI ID no.
  • Polypeptides are shown in the Sequence Listing that have HMM bit scores greater than 1440 when fitted to an HMM generated from the amino acid sequences set forth in Figure 12.
  • Such polypeptides include NRT 1.4 (SEQ ID NO: 17), Ceres ANNOT ID no. 1314724 (SEQ ID NO:20), Ceres ANNOT ID no. 1447247 (SEQ ID NO:22), Ceres CLONE ID no. 1777083 (SEQ ID NO:24), Ceres CLONE ID no. 1927461 (SEQ ID NO:26), Public GI ID no. 2760834 (SEQ ID NO:27), Public GI ID no. 9581817 (SEQ ID NO:28), Public GI ID no.
  • a nitrogen use efficiency-modulating polypeptide has an amino acid sequence with at least 40% sequence identity, e.g., at least 50%, 52%, 56%, 59%, 61%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity, to one of the amino acid sequences set forth in SEQ ID NOs: 2, 4, 6, 8, 10, 12, 13, 14, 15, 16, 17, 20, 22, 24, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 40, 42, 44, 46, 47, 48, 49, 51, 52, 53, or 54.
  • Polypeptides having such a percent sequence identity often have a domain indicative of a nitrogen use efficiency-modulating polypeptide and/or have an HMM bit score that is greater than 1200, as discussed above.
  • Amino acid sequences of nitrogen use efficiency-modulating polypeptides having at least 40% sequence identity to one of the amino acid sequences set forth in SEQ ID NOs: 2, 4, 6, 8, 10, 12, 13, 14, 15, 16, 17, 20, 22, 24, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 40, 42, 44, 46, 47, 48, 49, 51, 52, 53, or 54 are provided in Figures 11 and 12.
  • Percent sequence identity refers to the degree of sequence identity between any given reference sequence, e.g., SEQ ID NO:2 or SEQ ID NO: 17, and a candidate nitrogen use efficiency-modulating sequence.
  • a candidate sequence typically has a length that is from 80 percent to 200 percent of the length of the reference sequence, e.g., 82, 85, 87, 89, 90, 93, 95, 97, 99, 100, 105, 110, 115, 120, 130, 140, 150, 160, 170, 180, 190, or 200 percent of the length of the reference sequence.
  • a percent identity for any candidate nucleic acid or polypeptide relative to a reference nucleic acid or polypeptide can be determined as follows.
  • a reference sequence e.g., a nucleic acid sequence or an amino acid sequence
  • ClustalW version 1.83, default parameters
  • ClustalW calculates the best match between a reference and one or more candidate sequences, and aligns them so that identities, similarities and differences can be determined. Gaps of one or more residues can be inserted into a reference sequence, a candidate sequence, or both, to maximize sequence alignments.
  • word size 2; window size: 4; scoring method: percentage; number of top diagonals: 4; and gap penalty: 5.
  • word size 1 ; window size: 5; scoring method: percentage; number of top diagonals: 5; gap penalty: 3.
  • the ClustalW output is a sequence alignment that reflects the relationship between sequences.
  • ClustalW can be run, for example, at the Baylor College of Medicine Search Launcher site (searchlauncher.bcm.tmc.edu/multi-align/multi-align.html) and at the European Bioinformatics Institute site on the World Wide Web (ebi.ac.uk/clustalw).
  • the sequences are aligned using ClustalW, the number of identical matches in the alignment is divided by the length of the reference sequence, and the result is multiplied by 100. It is noted that the percent identity value can be rounded to the nearest tenth. For example, 78.11, 78.12, 78.13, and 78.14 are rounded down to 78.1, while 78.15, 78.16, 78.17, 78.18, and 78.19 are rounded up to 78.2.
  • a nitrogen use efficiency-modulating polypeptide has an amino acid sequence with at least 40% sequence identity, e.g., at least 50%, 52%, 56%, 59%, 61%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity, to the amino acid sequence set forth in SEQ ID NO:2.
  • Amino acid sequences of polypeptides having greater than 40% sequence identity to the polypeptide set forth in SEQ ID NO:2 are provided in Figure 1 1 and in the Sequence Listing.
  • Such polypeptides include Ceres ANNOT ID no. 1498169 (SEQ ID NO:6), Ceres CLONE ID no. 1583036 (SEQ ID NO: 10), Ceres CLONE ID no. 1806436 (SEQ ID NO:12), and Public GI ID no. 115446921 (SEQ ID NO:16).
  • a nitrogen use efficiency-modulating polypeptide has an amino acid sequence with at least 40% sequence identity, e.g., at least 50%, 52%, 56%, 59%, 61%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity, to the amino acid sequence set forth in SEQ ID NO: 17.
  • Amino acid sequences of polypeptides having greater than 40% sequence identity to the polypeptide set forth in SEQ ID NO: 17 are provided in Figure 12 and in the Sequence Listing.
  • Such polypeptides include Ceres ANNOT ID no. 1447247 (SEQ ID NO:22), Ceres CLONE ID no.
  • a nitrogen use efficiency-modulating polypeptide can include additional amino acids that are not involved in nitrogen use efficiency modulation, and thus such a polypeptide can be longer than would otherwise be the case.
  • a nitrogen use efficiency-modulating polypeptide can include a purification tag, a chloroplast transit peptide, a mitochondrial transit peptide, an amyloplast transit peptide, or a leader sequence added to the amino or carboxy terminus.
  • a nitrogen use efficiency-modulating polypeptide includes an amino acid sequence that functions as a reporter, e.g., a green fluorescent protein or yellow fluorescent protein.
  • Nucleic acids described herein include nucleic acids that are effective to modulate nitrogen use efficiency levels when transcribed in a plant or plant cell. Such nucleic acids include, without limitation, those that encode a nitrogen use efficiency- modulating polypeptide and those that can be used to inhibit expression of a nitrogen use efficiency-modulating polypeptide via a nucleic acid based method.
  • Nucleic acids encoding nitrogen use efficiency-modulating polypeptides are described herein. Such nucleic acids include SEQ ID NOs: 1, 3, 5, 7, 9, 11, 18, 19, 21, 23, 25, 39, 41, 43, 45, and 50, as described in more detail below. [0074] A nitrogen use efficiency-modulating nucleic acid can comprise the nucleotide sequence set forth in SEQ ID NO: 1.
  • a nitrogen use efficiency-modulating nucleic acid can be a variant of the nucleic acid having the nucleotide sequence set forth in SEQ ID NO: 1 .
  • a nitrogen use efficiency-modulating nucleic acid can have a nucleotide sequence with at least 80% sequence identity, e.g., 81%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity, to the nucleotide sequence set forth in SEQ ID NO: 1.
  • a nitrogen use efficiency-modulating nucleic acid can comprise the nucleotide sequence set forth in SEQ ID NO: 19 or SEQ ID NO: 18.
  • a nitrogen use efficiency-modulating nucleic acid can be a variant of the nucleic acid having the nucleotide sequence set forth in SEQ ID NO: 19 or SEQ ID NO: 18.
  • a nitrogen use efficiency-modulating nucleic acid can have a nucleotide sequence with at least 80% sequence identity, e.g., 81%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity, to the nucleotide sequence set forth in SEQ ID NO: 19 or SEQ ID NO: 18.
  • Isolated nucleic acid molecules can be produced by standard techniques. For example, polymerase chain reaction (PCR) techniques can be used to obtain an isolated nucleic acid containing a nucleotide sequence described herein. PCR can be used to amplify specific sequences from DNA as well as RNA, including sequences from total genomic DNA or total cellular RNA. Various PCR methods are described, for example, in PCR Primer: A Laboratory Manual Dieffenbach and Dveksler, eds., Cold Spring Harbor Laboratory Press, 1995. Generally, sequence information from the ends of the region of interest or beyond is employed to design oligonucleotide primers that are identical or similar in sequence to opposite strands of the template to be amplified.
  • PCR polymerase chain reaction
  • Isolated nucleic acids also can be chemically synthesized, either as a single nucleic acid molecule (e.g. , using automated DNA synthesis in the 3' to 5' direction using phosphoramidite technology) or as a series of oligonucleotides.
  • one or more pairs of long oligonucleotides can be synthesized that contain the desired sequence, with each pair containing a short segment of complementarity (e.g., about 15 nucleotides) such that a duplex is formed when the oligonucleotide pair is annealed.
  • DNA polymerase is used to extend the oligonucleotides, resulting in a single, double-stranded nucleic acid molecule per oligonucleotide pair, which then can be ligated into a vector.
  • Isolated nucleic acids of the invention also can be obtained by mutagenesis of, e.g., a naturally occurring DNA.
  • a nucleic acid encoding one of the nitrogen use efficiency-modulating polypeptides described herein can be used to express the polypeptide in a plant species of interest, typically by transforming a plant cell with a nucleic acid having the coding sequence for the polypeptide operably linked in sense orientation to one or more regulatory regions.
  • nucleic acids can encode a particular nitrogen use efficiency-modulating polypeptide; i.e., for many amino acids, there is more than one nucleotide triplet that serves as the codon for the amino acid.
  • codons in the coding sequence for a given nitrogen use efficiency-modulating polypeptide can be modified such that optimal expression in a particular plant species is obtained, using appropriate codon bias tables for that species.
  • expression of a nitrogen use efficiency-modulating polypeptide inhibits one or more functions of an endogenous polypeptide.
  • a nucleic acid that encodes a dominant negative polypeptide can be used to inhibit protein function.
  • a dominant negative polypeptide typically is mutated or truncated relative to an endogenous wild type polypeptide, and its presence in a cell inhibits one or more functions of the wild type polypeptide in that cell, i.e., the dominant negative polypeptide is genetically dominant and confers a loss of function.
  • the mechanism by which a dominant negative polypeptide confers such a phenotype can vary but often involves a protein-protein interaction or a protein-DNA interaction.
  • a dominant negative polypeptide can be an enzyme that is truncated relative to a native wild type enzyme, such that the truncated polypeptide retains domains involved in binding a first protein but lacks domains involved in binding a second protein. The truncated polypeptide is thus unable to properly modulate the activity of the second protein. See, e.g., US 2007/0056058.
  • a point mutation that results in a non-conservative amino acid substitution in a catalytic domain can result in a dominant negative polypeptide. See, e.g., US 2005/032221.
  • a dominant negative polypeptide can be a transcription factor that is truncated relative to a native wild type transcription factor, such that the truncated polypeptide retains the DNA binding domain(s) but lacks the activation domain(s).
  • a truncated polypeptide can inhibit the wild type transcription factor from binding DNA, thereby inhibiting transcription activation.
  • RNA interference RNA interference
  • TLS transcriptional gene silencing
  • Antisense technology is one well-known method. In this method, a nucleic acid segment from a gene to be repressed is cloned and operably linked to a regulatory region and a transcription termination sequence so that the antisense strand of RNA is transcribed. The recombinant construct is then transformed into plants, as described herein, and the antisense strand of RNA is produced.
  • the nucleic acid segment need not be the entire sequence of the gene to be repressed, but typically will be substantially complementary to at least a portion of the sense strand of the gene to be repressed. Generally, higher homology can be used to compensate for the use of a shorter sequence. Typically, a sequence of at least 30 nucleotides is used, e.g., at least 40, 50, 80, 100, 200, 500 nucleotides or more.
  • a nucleic acid in another method, can be transcribed into a ribozyme, or catalytic RNA, that affects expression of an mRNA.
  • Ribozymes can be designed to specifically pair with virtually any target RNA and cleave the phosphodiester backbone at a specific location, thereby functionally inactivating the target RNA.
  • Heterologous nucleic acids can encode ribozymes designed to cleave particular mRNA transcripts, thus preventing expression of a polypeptide.
  • Hammerhead ribozymes are useful for destroying particular mRNAs, although various ribozymes that cleave mRNA at site-specific recognition sequences can be used.
  • Hammerhead ribozymes cleave mRNAs at locations dictated by flanking regions that form complementary base pairs with the target mRNA. The sole requirement is that the target RNA contains a 5'-UG-3' nucleotide sequence.
  • the construction and production of hammerhead ribozymes is known in the art. See, for example, U.S. Patent No. 5,254,678 and WO 02/46449 and references cited therein.
  • Hammerhead ribozyme sequences can be embedded in a stable RNA such as a transfer RNA (tRNA) to increase cleavage efficiency in vivo.
  • tRNA transfer RNA
  • RNA endoribonucleases which have been described, such as the one that occurs naturally in Tetrahymena thermophila, can be useful. See, for example, U.S. Patent No. 4,987,071 and 6,423,885.
  • RNAi can also be used to inhibit the expression of a gene.
  • a construct can be prepared that includes a sequence that is transcribed into an RNA that can anneal to itself, e.g., a double stranded RNA having a stem-loop structure.
  • one strand of the stem portion of a double stranded RNA comprises a sequence that is similar or identical to the sense coding sequence of a nitrogen use efficiency- modulating polypeptide, and that is from about 10 nucleotides to about 2,500 nucleotides in length.
  • the length of the sequence that is similar or identical to the sense coding sequence can be from 10 nucleotides to 500 nucleotides, from 15 nucleotides to 300 nucleotides, from 20 nucleotides to 100 nucleotides, or from 25 nucleotides to 100 nucleotides.
  • the other strand of the stem portion of a double stranded RNA comprises a sequence that is similar or identical to the antisense strand of the coding sequence of the nitrogen use efficiency- modulating polypeptide, and can have a length that is shorter, the same as, or longer than the corresponding length of the sense sequence.
  • one strand of the stem portion of a double stranded RNA comprises a sequence that is similar or identical to the 3 ' or 5' untranslated region of an mRNA encoding a nitrogen use efficiency-modulating polypeptide
  • the other strand of the stem portion of the double stranded RNA comprises a sequence that is similar or identical to the sequence that is complementary to the 3 ' or 5' untranslated region, respectively, of the mRNA encoding the nitrogen use efficiency-modulating polypeptide.
  • one strand of the stem portion of a double stranded RNA comprises a sequence that is similar or identical to the sequence of an intron in the pre- mRNA encoding a nitrogen use efficiency-modulating polypeptide
  • the other strand of the stem portion comprises a sequence that is similar or identical to the sequence that is complementary to the sequence of the intron in the pre-mRNA.
  • the loop portion of a double stranded RNA can be from 3 nucleotides to 5,000 nucleotides, e.g., from 3 nucleotides to 25 nucleotides, from 15 nucleotides to 1 ,000 nucleotides, from 20 nucleotides to 500 nucleotides, or from 25 nucleotides to 200 nucleotides.
  • the loop portion of the RNA can include an intron.
  • a double stranded RNA can have zero, one, two, three, four, five, six, seven, eight, nine, ten, or more stem-loop structures.
  • Methods for using RNAi to inhibit the expression of a gene are known to those of skill in the art. See, e.g., U.S. Patents 5,034,323; 6,326,527; 6,452,067; 6,573,099; 6,753,139; and 6,777,588. See also WO 97/01952; WO 98/53083; WO 99/32619; WO 98/36083; and U.S. Patent Publications 20030175965, 20030175783, 20040214330, and 20030180945.
  • Constructs containing regulatory regions operably linked to nucleic acid molecules in sense orientation can also be used to inhibit the expression of a gene.
  • the transcription product can be similar or identical to the sense coding sequence of a nitrogen use efficiency-modulating polypeptide.
  • the transcription product can also be unpolyadenylated, lack a 5' cap structure, or contain an unsplicable intron.
  • a construct containing a nucleic acid having at least one strand that is a template for both sense and antisense sequences that are complementary to each other is used to inhibit the expression of a gene.
  • the sense and antisense sequences can be part of a larger nucleic acid molecule or can be part of separate nucleic acid molecules having sequences that are not complementary.
  • the sense or antisense sequence can be a sequence that is identical or complementary to the sequence of an mRNA, the 3 ' or 5' untranslated region of an mRNA, or an intron in a pre-mRNA encoding a nitrogen use efficiency-modulating polypeptide.
  • the sense or antisense sequence is identical or complementary to a sequence of the regulatory region that drives transcription of the gene encoding a nitrogen use efficiency-modulating polypeptide.
  • the sense sequence is the sequence that is complementary to the antisense sequence.
  • the sense and antisense sequences can be any length greater than about 12 nucleotides (e.g., 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more nucleotides).
  • an antisense sequence can be 21 or 22 nucleotides in length.
  • the sense and antisense sequences range in length from about 15 nucleotides to about 30 nucleotides, e.g., from about 18 nucleotides to about 28 nucleotides, or from about 21 nucleotides to about 25 nucleotides.
  • an antisense sequence is a sequence complementary to an mRNA sequence encoding a nitrogen use efficiency-modulating polypeptide described herein.
  • the sense sequence complementary to the antisense sequence can be a sequence present within the mRNA of the nitrogen use efficiency-modulating polypeptide.
  • sense and antisense sequences are designed to correspond to a 15-30 nucleotide sequence of a target mRNA such that the level of that target mRNA is reduced.
  • a construct containing a nucleic acid having at least one strand that is a template for more than one sense sequence can be used to inhibit the expression of a gene.
  • a construct containing a nucleic acid having at least one strand that is a template for more than one antisense sequence can be used to inhibit the expression of a gene.
  • a construct can contain a nucleic acid having at least one strand that is a template for two sense sequences and two antisense sequences.
  • the multiple sense sequences can be identical or different, and the multiple antisense sequences can be identical or different.
  • a construct can have a nucleic acid having one strand that is a template for two identical sense sequences and two identical antisense sequences that are complementary to the two identical sense sequences.
  • an isolated nucleic acid can have one strand that is a template for (1) two identical sense sequences 20 nucleotides in length, (2) one antisense sequence that is complementary to the two identical sense sequences 20 nucleotides in length, (3) a sense sequence 30 nucleotides in length, and (4) three identical antisense sequences that are complementary to the sense sequence 30 nucleotides in length.
  • the constructs provided herein can be designed to have any arrangement of sense and antisense sequences. For example, two identical sense sequences can be followed by two identical antisense sequences or can be positioned between two identical antisense sequences.
  • a nucleic acid having at least one strand that is a template for one or more sense and/or antisense sequences can be operably linked to a regulatory region to drive transcription of an RNA molecule containing the sense and/or antisense sequence(s).
  • a nucleic acid can be operably linked to a transcription terminator sequence, such as the terminator of the nopaline synthase (nos) gene.
  • two regulatory regions can direct transcription of two transcripts: one from the top strand, and one from the bottom strand. See, for example, Yan et al, Plant Physiol, 141 :1508-1518 (2006). The two regulatory regions can be the same or different.
  • the two transcripts can form double- stranded RNA molecules that induce degradation of the target RNA.
  • a nucleic acid can be positioned within a T-DNA or plant-derived transfer DNA (P-DNA) such that the left and right T-DNA border sequences, or the left and right border-like sequences of the P- DNA, flank or are on either side of the nucleic acid. See, US 2006/0265788.
  • the nucleic acid sequence between the two regulatory regions can be from about 15 to about 300 nucleotides in length.
  • the nucleic acid sequence between the two regulatory regions is from about 15 to about 200 nucleotides in length, from about 15 to about 100 nucleotides in length, from about 15 to about 50 nucleotides in length, from about 18 to about 50 nucleotides in length, from about 18 to about 40 nucleotides in length, from about 18 to about 30 nucleotides in length, or from about 18 to about 25 nucleotides in length.
  • nucleic-acid based methods for inhibition of gene expression in plants can be a nucleic acid analog.
  • Nucleic acid analogs can be modified at the base moiety, sugar moiety, or phosphate backbone to improve, for example, stability, hybridization, or solubility of the nucleic acid. Modifications at the base moiety include deoxyuridine for deoxythymidine, and 5-methyl-2'-deoxycytidine and 5-bromo-2'- deoxycytidine for deoxycytidine. Modifications of the sugar moiety include modification of the 2' hydroxyl of the ribose sugar to form 2'-O-methyl or 2'-O-allyl sugars.
  • the deoxyribose phosphate backbone can be modified to produce morpholino nucleic acids, in which each base moiety is linked to a six-membered morpholino ring, or peptide nucleic acids, in which the deoxyphosphate backbone is replaced by a pseudopeptide backbone and the four bases are retained. See, for example, Summerton and Weller (1997) Antisense Nucleic Acid Drug Dev., 7:187-195; Hyrup et al. (1996) Bioorgan. Med. Chem., 4:5-23.
  • the deoxyphosphate backbone can be replaced with, for example, a phosphorothioate or phosphorodithioate backbone, a phosphoroamidite, or an alkyl phosphotri ester backbone.
  • a recombinant nucleic acid construct can comprise a nucleic acid encoding a nitrogen use efficiency-modulating polypeptide as described herein, operably linked to a regulatory region suitable for expressing the nitrogen use efficiency-modulating polypeptide in the plant or cell.
  • a nucleic acid can comprise a coding sequence that encodes any of the nitrogen use efficiency-modulating polypeptides as set forth in SEQ ID NO: 2, 4, 6, 8, 10, 12, 13, 14, 15, 16, 17, 20, 22, 24, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 40, 42, 44, 46, 47, 48, 49, 51, 52, 53, or 54.
  • Examples of nucleic acids encoding nitrogen use efficiency-modulating polypeptides are set forth in 1, 3, 5, 7, 9, 11, 18, 19, 21, 23, 25, 39, 41, 43, 45, or 50.
  • the nitrogen use efficiency- modulating polypeptide encoded by a recombinant nucleic acid can be a native nitrogen use efficiency-modulating polypeptide, or can be heterologous to the cell.
  • the recombinant construct contains a nucleic acid that inhibits expression of a nitrogen use efficiency-modulating polypeptide, operably linked to a regulatory region. Examples of suitable regulatory regions are described in the section entitled "Regulatory Regions.”
  • Vectors containing recombinant nucleic acid constructs such as those described herein also are provided.
  • Suitable vector backbones include, for example, those routinely used in the art such as plasmids, viruses, artificial chromosomes, BACs, YACs, or PACs.
  • Suitable expression vectors include, without limitation, plasmids and viral vectors derived from, for example, bacteriophage, baculoviruses, and retroviruses.
  • the vectors provided herein also can include, for example, origins of replication, scaffold attachment regions (SARs), and/or markers.
  • a marker gene can confer a selectable phenotype on a plant cell.
  • a marker can confer biocide resistance, such as resistance to an antibiotic (e.g., kanamycin, G418, bleomycin, or hygromycin), or an herbicide (e.g., glyphosate, chlorsulfuron or phosphinothricin).
  • an expression vector can include a tag sequence designed to facilitate manipulation or detection (e.g. , purification or localization) of the expressed polypeptide.
  • Tag sequences such as luciferase, ⁇ -glucuronidase (GUS), green fluorescent protein (GFP), glutathione S-transferase (GST), polyhistidine, c-myc, hemagglutinin, or FlagTM tag (Kodak, New Haven, CT) sequences typically are expressed as a fusion with the encoded polypeptide.
  • GUS green fluorescent protein
  • GST glutathione S-transferase
  • polyhistidine c-myc
  • hemagglutinin hemagglutinin
  • FlagTM tag Kodak, New Haven, CT
  • regulatory regions to be included in a recombinant construct depends upon several factors, including, but not limited to, efficiency, selectability, inducibility, desired expression level, and cell- or tissue-preferential expression. It is a routine matter for one of skill in the art to modulate the expression of a coding sequence by appropriately selecting and positioning regulatory regions relative to the coding sequence. Transcription of a nucleic acid can be modulated in a similar manner.
  • Suitable regulatory regions initiate transcription only, or predominantly, in certain cell types.
  • Methods for identifying and characterizing regulatory regions in plant genomic DNA are known, including, for example, those described in the following references: Jordano et al. (1989) Plant Cell, 1 :855-866; Bustos et al. (1989) Plant Cell, 1 :839-854; Green et al. (1988) EMBO J., 7:4035-4044; Meier et al. (1991) Plant Cell, 3:309- 316; and Zhang et al. (1996) Plant Physiology, 110:1069-1079.
  • Examples of various classes of regulatory regions are described below. Some of the regulatory regions indicated below as well as additional regulatory regions are described in more detail in U.S.
  • PCT/US05/011105 PCT/US05/23639; PCT/US05/034308; PCT/US05/034343; and PCT/US06/038236; PCT/US06/040572; and PCT/US07/62762.
  • PCT/US05/034343 the sequences of regulatory regions PT0623, YP0388, YP0087, YP0093, YP0108, YP0022 and YP0080 are set forth in the sequence listing of U.S. Patent Application Ser. No. 11/172,703; the sequence of regulatory region PR0924 is set forth in the sequence listing of PCT/US07/62762; and the sequences of regulatory regions p530cl0, pOsFIE2-2, pOsMEA, pOsYpl 02, and pOsYp285 are set forth in the sequence listing of PCT/US06/038236.
  • a regulatory region may meet criteria for one classification based on its activity in one plant species, and yet meet criteria for a
  • a promoter can be said to be "broadly expressing" when it promotes transcription in many, but not necessarily all, plant tissues.
  • a broadly expressing promoter can promote transcription of an operably linked sequence in one or more of the shoot, shoot tip (apex), and leaves, but weakly or not at all in tissues such as roots or stems.
  • a broadly expressing promoter can promote transcription of an operably linked sequence in one or more of the stem, shoot, shoot tip (apex), and leaves, but can promote transcription weakly or not at all in tissues such as reproductive tissues of flowers and developing seeds.
  • Non-limiting examples of broadly expressing promoters that can be included in the nucleic acid constructs provided herein include the p326, YPO 144, YP0190, pl3879, YP0050, p32449, 21876, YP0158, YP0214, YPO38O, PT0848, and PT0633 promoters.
  • CaMV 35S promoter the cauliflower mosaic virus (CaMV) 35S promoter
  • MAS mannopine synthase
  • 1 ' or 2' promoters derived from T-DNA of Agrobacterium tumefaciens the figwort mosaic virus 34S promoter
  • actin promoters such as the rice actin promoter
  • ubiquitin promoters such as the maize ubiquitin-1 promoter.
  • the CaMV 35S promoter is excluded from the category of broadly expressing promoters. ii. Root Promoters
  • Root-active promoters confer transcription in root tissue, e.g., root endodermis, root epidermis, or root vascular tissues.
  • root-active promoters are root-preferential promoters, i.e., confer transcription only or predominantly in root tissue.
  • Root-preferential promoters include the YP0128, YP0275, PT0625, PT0660, PT0683, and PT0758 promoters.
  • Other root-preferential promoters include the PT0613, PT0672, PT0688, and PT0837 promoters, which drive transcription primarily in root tissue and to a lesser extent in ovules and/or seeds.
  • root-preferential promoters include the root-specific subdomains of the CaMV 35S promoter (Lam et al (1989) Proc. Natl. Acad. Sci. USA, 86:7890-7894), root cell specific promoters reported by Conkling et al. (1990) Plant Physiol, 93:1203-1211, and the tobacco RD2 promoter. iii. Maturing Endosperm Promoters
  • promoters that drive transcription in maturing endosperm can be useful. Transcription from a maturing endosperm promoter typically begins after fertilization and occurs primarily in endosperm tissue during seed development and is typically highest during the cellularization phase. Most suitable are promoters that are active predominantly in maturing endosperm, although promoters that are also active in other tissues can sometimes be used.
  • Non-limiting examples of maturing endosperm promoters that can be included in the nucleic acid constructs provided herein include the napin promoter, the Arcelin-5 promoter, the phaseolin promoter (Bustos et al.
  • zein promoters such as the 15 kD zein promoter, the 16 kD zein promoter, 19 kD zein promoter, 22 kD zein promoter and 27 kD zein promoter.
  • Osgt-1 promoter from the rice glutelin-1 gene (Zheng et al (1993) MoI. Cell Biol, 13:5829-5842), the beta-amylase promoter, and the barley hordein promoter.
  • Other maturing endosperm promoters include the YP0092, PT0676, and PT0708 promoters.
  • Promoters that are active in ovary tissues such as the ovule wall and mesocarp can also be useful, e.g. , a polygalacturonidase promoter, the banana TRX promoter, the melon actin promoter, YP0396, and PT0623.
  • promoters that are active primarily in ovules include YP0007, YPOl 11, YP0092, YP0103, YP0028, YP0121, YP0008, YP0039, YPOl 15, YPOl 19, YP0120, and YP0374.
  • Embryo Sac/Early Endosperm Promoters [00102] To achieve expression in embryo sac/early endosperm, regulatory regions can be used that are active in polar nuclei and/or the central cell, or in precursors to polar nuclei, but not in egg cells or precursors to egg cells. Most suitable are promoters that drive expression only or predominantly in polar nuclei or precursors thereto and/or the central cell. A pattern of transcription that extends from polar nuclei into early endosperm development can also be found with embryo sac/early endosperm-preferential promoters, although transcription typically decreases significantly in later endosperm development during and after the cellularization phase. Expression in the zygote or developing embryo typically is not present with embryo sac/early endosperm promoters.
  • Promoters that may be suitable include those derived from the following genes: Arabidopsis viviparous-1 (see, GenBank No. U93215); Arabidopsis atmycl (see, Urao (1996) Plant MoI. Biol, 32:571-57; Conceicao (1994) Plant, 5:493-505);
  • Arabidopsis FIE (GenBank No. AFl 29516); Arabidopsis MEA; Arabidopsis FIS2 (GenBank No. AF096096); and FIE 1.1 (U.S. Patent 6,906,244).
  • Other promoters that may be suitable include those derived from the following genes: maize MACl (see, Sheridan (1996) Genetics, 142:1009-1020); maize Cat3 (see, GenBank No. L05934; Abler (1993) Plant MoI Biol, 22:10131-1038).
  • promoters include the following Arabidopsis promoters: YP0039, YPOlOl, YP0102, YPOI lO, YPOl 17, YPOl 19, YP0137, DME, YP0285, and YP0212.
  • Other promoters that may be useful include the following rice promoters: p530cl 0, pOsFIE2-2, pOsMEA, pOsYpl02, and pOsYp285.
  • Embryo- preferential promoters include the barley lipid transfer protein (Ltpl) promoter (Plant Cell Rep (2001) 20:647-654), YP0097, YP0107, YP0088, YP0143, YP0156, PT0650, PT0695, PT0723, PT0838, PT0879, and PT0740.
  • Ltpl barley lipid transfer protein
  • Promoters active in photosynthetic tissue confer transcription in green tissues such as leaves and stems. Most suitable are promoters that drive expression only or predominantly in such tissues. Examples of such promoters include the ribulose-1,5- bisphosphate carboxylase (RbcS) promoters such as the RbcS promoter from eastern larch (Larix laricina), the pine cab6 promoter (Yamamoto et al.
  • RbcS ribulose-1,5- bisphosphate carboxylase
  • vascular tissue Promoters examples include YP0087, YP0093, YPOl 08, YP0022, and YP0080.
  • Other vascular tissue-preferential promoters include the glycine-rich cell wall protein GRP 1.8 promoter (Keller and Baumgartner (1991) Plant Cell, 30):1051-1061), the Commelina yellow mottle virus (CoYMV) promoter (Medberry et al (1992) Plant Cell, 4(2): 185-192), and the rice tungro bacilliform virus (RTBV) promoter (Dai et al (2004) Proc. Natl. Acad. Sci. USA, 101(2):687-692).
  • GRP 1.8 promoter Keller and Baumgartner (1991) Plant Cell, 30):1051-1061
  • CoYMV Commelina yellow mottle virus
  • RTBV rice tungro bacilliform virus
  • Inducible Promoters confer transcription in response to external stimuli such as chemical agents or environmental stimuli.
  • inducible promoters can confer transcription in response to hormones such as giberellic acid or ethylene, or in response to light or drought.
  • drought-inducible promoters include YPO38O, PT0848, YPO381, YP0337, PT0633, YP0374, PT0710, YP0356, YP0385, YP0396, YP0388, YP0384, PT0688, YP0286, YP0377, PDl 367, and PD0901.
  • nitrogen-inducible promoters examples include PT0863, PT0829, PT0665, and PT0886.
  • shade-inducible promoters examples include PR0924 and PT0678.
  • An example of a promoter induced by salt is rd29A (Kasuga et al. (1999) Nature Biotech 17: 287-291). x. Basal Promoters
  • a basal promoter is the minimal sequence necessary for assembly of a transcription complex required for transcription initiation.
  • Basal promoters frequently include a "TATA box” element that may be located between about 15 and about 35 nucleotides upstream from the site of transcription initiation.
  • Basal promoters also may include a "CCAAT box” element (typically the sequence CCAAT) and/or a GGGCG sequence, which can be located between about 40 and about 200 nucleotides, typically about 60 to about 120 nucleotides, upstream from the transcription start site.
  • a stem promoter may be specific to one or more stem tissues or specific to stem and other plant parts.
  • Stem promoters may have high or preferential activity in, for example, epidermis and cortex, vascular cambium, procambium, or xylem.
  • Examples of stem promoters include YPOOl 8 which is disclosed in US20060015970 and Cry ⁇ A(b) and Cry ⁇ A(c) (Braga et al. 2003, Journal of New Seeds 5:209-221). xii.
  • Promoters include, but are not limited to, shoot- preferential, callus-preferential, trichome cell-preferential, guard cell-preferential such as PT0678, tuber-preferential, parenchyma cell-preferential, and senescence-preferential promoters. Promoters designated YP0086, YPOl 88, YP0263, PT0758, PT0743, PT0829, YPOl 19, and YP0096, as described in the above-referenced patent applications, may also be useful. xiii. Other Regulatory Regions
  • a 5' untranslated region can be included in nucleic acid constructs described herein.
  • a 5' UTR is transcribed, but is not translated, and lies between the start site of the transcript and the translation initiation codon and may include the +1 nucleotide.
  • a 3' UTR can be positioned between the translation termination codon and the end of the transcript.
  • UTRs can have particular functions such as increasing mRNA stability or attenuating translation. Examples of 3' UTRs include, but are not limited to, polyadenylation signals and transcription termination sequences, e.g. , a nopaline synthase termination sequence.
  • more than one regulatory region may be present in a recombinant polynucleotide, e.g., introns, enhancers, upstream activation regions, transcription terminators, and inducible elements.
  • more than one regulatory region can be operably linked to the sequence of a polynucleotide encoding a nitrogen use efficiency-modulating polypeptide.
  • Regulatory regions such as promoters for endogenous genes, can be obtained by chemical synthesis or by subcloning from a genomic DNA that includes such a regulatory region.
  • a nucleic acid comprising such a regulatory region can also include flanking sequences that contain restriction enzyme sites that facilitate subsequent manipulation.
  • the invention also features transgenic plant cells and plants comprising at least one recombinant nucleic acid construct described herein.
  • a plant or plant cell can be transformed by having a construct integrated into its genome, i.e., can be stably transformed. Stably transformed cells typically retain the introduced nucleic acid with each cell division.
  • a plant or plant cell can also be transiently transformed such that the construct is not integrated into its genome. Transiently transformed cells typically lose all or some portion of the introduced nucleic acid construct with each cell division such that the introduced nucleic acid cannot be detected in daughter cells after a sufficient number of cell divisions. Both transiently transformed and stably transformed transgenic plants and plant cells can be useful in the methods described herein.
  • Transgenic plant cells used in methods described herein can constitute part or all of a whole plant. Such plants can be grown in a manner suitable for the species under consideration, either in a growth chamber, a greenhouse, or in a field. Transgenic plants can be bred as desired for a particular purpose, e.g., to introduce a recombinant nucleic acid into other lines, to transfer a recombinant nucleic acid to other species, or for further selection of other desirable traits. Alternatively, transgenic plants can be propagated vegetatively for those species amenable to such techniques. As used herein, a transgenic plant also refers to progeny of an initial transgenic plant, as long as the progeny inherits the transgene. Seeds produced by a transgenic plant can be grown and then selfed (or outcrossed and selfed) to obtain seeds homozygous for the nucleic acid construct.
  • Transgenic plants can be grown in suspension culture, or tissue or organ culture.
  • solid and/or liquid tissue culture techniques can be used.
  • transgenic plant cells can be placed directly onto the medium or can be placed onto a filter that is then placed in contact with the medium.
  • transgenic plant cells can be placed onto a flotation device, e.g., a porous membrane that contacts the liquid medium.
  • a solid medium can be, for example, Murashige and Skoog (MS) medium containing agar and a suitable concentration of an auxin, e.g. , 2,4-dichlorophenoxyacetic acid (2,4-D), and a suitable concentration of a cytokinin, e.g.
  • a reporter sequence encoding a reporter polypeptide having a reporter activity can be included in the transformation procedure and an assay for reporter activity or expression can be performed at a suitable time after transformation.
  • a suitable time for conducting the assay typically is about 1-21 days after transformation, e.g., about 1-14 days, about 1-7 days, or about 1-3 days.
  • the use of transient assays is particularly convenient for rapid analysis in different species, or to confirm expression of a heterologous nitrogen use efficiency-modulating polypeptide whose expression has not previously been confirmed in particular recipient cells.
  • nucleic acids into monocotyledonous and dicotyledonous plants are known in the art, and include, without limitation, Agrob ⁇ cterium- mediated transformation, viral vector-mediated transformation, electroporation and particle gun transformation, e.g., U.S. Patents 5,538,880; 5,204,253; 6,329,571 and 6,013,863. If a cell or cultured tissue is used as the recipient tissue for transformation, plants can be regenerated from transformed cultures if desired, by techniques known to those skilled in the art.
  • a population of transgenic plants can be screened and/or selected for those members of the population that have a trait or phenotype conferred by expression of the transgene. For example, a population of progeny of a single transformation event can be screened for those plants having a desired level of expression of a nitrogen use efficiency- modulating polypeptide or nucleic acid. Physical and biochemical methods can be used to identify expression levels.
  • RNA transcripts include Southern analysis or PCR amplification for detection of a polynucleotide; Northern blots, Sl RNase protection, primer-extension, or RT- PCR amplification for detecting RNA transcripts; enzymatic assays for detecting enzyme or ribozyme activity of polypeptides and polynucleotides; and protein gel electrophoresis, Western blots, immunoprecipitation, and enzyme-linked immunoassays to detect polypeptides.
  • Other techniques such as in situ hybridization, enzyme staining, and immunostaining also can be used to detect the presence or expression of polypeptides and/or polynucleotides. Methods for performing all of the referenced techniques are known.
  • a population of plants comprising independent transformation events can be screened for those plants having a desired trait, such as a modulated level of nitrogen use efficiency. Selection and/or screening can be carried out over one or more generations, and/or in more than one geographic location.
  • transgenic plants can be grown and selected under conditions which induce a desired phenotype or are otherwise necessary to produce a desired phenotype in a transgenic plant.
  • selection and/or screening can be applied during a particular developmental stage in which the phenotype is expected to be exhibited by the plant. Selection and/or screening can be carried out to choose those transgenic plants having a statistically significant difference in a nitrogen use efficiency level relative to a control plant that lacks the transgene. Selected or screened transgenic plants have an altered phenotype as compared to a corresponding control plant, as described in the "Transgenic Plant Phenotypes" section herein.
  • the polynucleotides and vectors described herein can be used to transform a number of monocotyledonous and dicotyledonous plants and plant cell systems, including species from one of the following families: Acanthaceae, Alliaceae, Alstroemeriaceae, Amaryllidaceae, Apocynaceae, Arecaceae, Asteraceae, Berberidaceae, Bixaceae, Brassicaceae, Bromeliaceae, Cannabaceae, Caryophyllaceae, Cephalotaxaceae, Chenopodiaceae, Colchicaceae, Cucurbitaceae, Dioscoreaceae, Ephedraceae,
  • Suitable species may include members of the genus Abelmoschus,
  • Suitable species include Panicum spp. or hybrid thereof, Sorghum spp. or hybrid thereof, sudangrass, Miscanthus spp. or hybrid thereof, Saccharum spp. or hybrid thereof, Erianthus spp., Populus spp., Andropogon gerardii (big bluestem), Pennisetum purpureum (elephant grass) or hybrid thereof (e.g., Pennisetum purpureum x Pennisetum typhoidum), Phalaris arundinacea (reed canarygrass), Cynodon dactylon (bermudagrass), Festuca arundinacea (tall fescue), Spartina pectinata (prairie cord-grass), Medicago sativa (alfalfa), Arundo donax (giant reed) or hybrid thereof, Secale cereale (rye), Salix spp.
  • Eucalyptus spp. eucalyptus
  • Triticosecale triticum - wheat X rye
  • Tripsicum dactyloides Eastern gammagrass
  • Leymus cinereus basic wildrye
  • Leymus condensatus giantt wildrye
  • a suitable species can be a wild, weedy, or cultivated sorghum species such as, but not limited to, Sorghum almum, Sorghum amplum, Sorghum angustum, Sorghum arundinaceum, Sorghum bicolor (such as bicolor, guinea, caudatum, kaf ⁇ r, and durra), Sorghum brachypodum, Sorghum bulbosum, Sorghum burmahicum, Sorghum controversum, Sorghum drummondii, Sorghum ecarinatum, Sorghum exstans, Sorghum grande, Sorghum halepense, Sorghum interjectum, Sorghum intrans, Sorghum laxiflorum, Sorghum leiocladum, Sorghum macrospermum, Sorghum matarankense, Sorghum miliaceum, Sorghum nigrum, Sorghum nitidum, Sorghum plumosum, Sorg
  • Carthamus tinctorius safflower
  • Jatropha curcas jatropha
  • Ricinus communis castor
  • Elaeis guineensis palm
  • Linum usitatissimum flax
  • Brassicajuncea Brassicajuncea
  • Suitable species also include Beta vulgaris (sugarbeet), and Manihot esculenta (cassava) [00126] Suitable species also include Lycopersicon esculentum (tomato),
  • Lactuca sativa (lettuce), Musa paradisiaca (banana), Solarium tuberosum (potato), Brassica oleracea (broccoli, cauliflower, brusselsprouts), Camellia sinensis (tea), Fragaria ananassa (strawberry), Theobroma cacao (cocoa), Coffea arabica (coffee), Vitis vinifera (grape), Ananas comosus (pineapple), Capsicum annum (hot & sweet pepper), Allium cepa (onion), Cucumis melo (melon), Cucumis sativus (cucumber), Cucurbita maxima (squash), Cucurbita moschata (squash), Spinacea oleracea (spinach), Citrullus lanatus (watermelon), Abelmoschus esculentus (okra), and Solanum melongena (eggplant).
  • Suitable species also include Papaver somniferum (opium poppy),
  • Papaver orientale, Taxus baccata, Taxus brevifolia, Artemisia annua, Cannabis sativa, Camptotheca acuminate, Catharanthus roseus, Vinca rosea, Cinchona officinalis, Colchicum autumnale, Veratrum californica, Digitalis lanata, Digitalis purpurea, Dioscorea spp., Andrographis paniculata, Atropa belladonna, Datura stomonium, Berberis spp,, Cephalotaxus spp., Ephedra sinica, Ephedra spp., Erythroxylum coca, Galanthus wornorii, Scopolia spp., Lycopodium serratum ( Huperzia serrata), Lycopodium spp., Rauwolfia serpentina, Rauwolfia spp., Sanguina ⁇ a canadensis, Hyoscyamus spp., Calendula officinalis,
  • Suitable species also include Parthenium argentatum (guayule), Hevea spp. (rubber), Mentha spicata (mint), Mentha piperita (mint), Bixa orellana, and Alstroemeria spp. [00129] Suitable species also include Rosa spp. (rose), Dianthus caryophyllus
  • Suitable species also include Nicotiana tabacum (tobacco), Lupinus albus (lupin), Uniola paniculata (oats), bentgrass (Agrostis spp.), Populus tremuloides (aspen), Pinus spp. (pine), Abies spp. (fir), Acer spp. (maple, Hordeum vulgare (barley), Poa pratensis (bluegrass), Lolium spp. (ryegrass) and Phleum pratense (timothy).
  • the methods and compositions can be used over a broad range of plant species, including species from the dicot genera Brassica, Carthamus, Glycine, Gossypium, Helianthus, Jatropha, Parthenium, Populus, and Ricinus; and the monocot genera Elaeis, Festuca, Hordeum, Lolium, Oryza, Panicum, Pennisetum, Phleum, Poa, Saccharum, Secale, Sorghum, Triticosecale, Triticum, and Zea.
  • a plant is a member of the species Panicum virgatum (switchgrass), Sorghum bicolor (sorghum, sudangrass), Miscanthus giganteus (miscanthus), Saccharum sp. (energycane), Populus balsamifera (poplar), Zea mays (corn), Glycine max (soybean), Brassica napus (canola), Triticum aestivum (wheat), Gossypium hirsutum (cotton), Oryza sativa (rice), Helianthus annuus (sunflower), Medicago sativa (alfalfa), Beta vulgaris (sugarbeet), or Pennisetum glaucum (pearl millet).
  • the polynucleotides and vectors described herein can be used to transform a number of monocotyledonous and dicotyledonous plants and plant cell systems, wherein such plants are hybrids of different species or varieties of a species ⁇ e.g., Saccharum sp. X Miscanthus sp., Panicum virgatum x Panicum amarum, Panicum virgatum x Panicum amarulum, and Pennisetum purpureum x Pennisetum typhoidum).
  • Saccharum sp. X Miscanthus sp. Panicum virgatum x Panicum amarum
  • Panicum virgatum x Panicum amarulum Panicum virgatum x Panicum amarulum
  • Pennisetum purpureum x Pennisetum typhoidum Pennisetum purpureum x Pennisetum typhoidum
  • a plant in which expression of a nitrogen use efficiency-modulating polypeptide is modulated can have increased levels of nitrogen.
  • a nitrogen use efficiency-modulating polypeptide described herein can be expressed in a transgenic plant and/or seedling, resulting in increased levels of nitrogen in tissue of plants and/or seedlings.
  • the nitrogen level can be increased by at least 2 percent, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, or more than 60 percent, as compared to the nitrogen level in a corresponding control plant that does not express the transgene.
  • a plant in which expression of a nitrogen use efficiency-modulating polypeptide is modulated can have decreased levels of seed nitrogen.
  • the nitrogen level can be decreased by at least 2 percent, e.g., 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, or more than 35 percent, as compared to the nitrogen level in a corresponding control plant that does not express the transgene.
  • a plant in which expression of a nitrogen use efficiency-modulating polypeptide is modulated can have increased or decreased levels of nitrogen in one or more tissues, e.g., leaf tissues, root tissues, stem tissues, reproductive tissues, petiole tissues, and/or seed tissues.
  • the nitrogen level can be increased by at least 2 percent, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, or more than 60 percent, as compared to the nitrogen level in a corresponding control plant that does not express the transgene.
  • a plant in which expression of a nitrogen use efficiency-modulating polypeptide is modulated can have decreased levels of nitrogen in one or more root tissues.
  • the nitrogen level can be decreased by at least 2 percent, e.g., 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, or more than 35 percent, as compared to the nitrogen level in a corresponding control plant that does not express the transgene.
  • a plant in which expression of a nitrogen use efficiency-modulating polypeptide is modulated can have increased levels of tissue nitrate levels.
  • a nitrogen use efficiency-modulating polypeptide described herein can be expressed in a transgenic plant and/or seedling, resulting in increased levels of nitrate in tissue of plants and/or seedlings.
  • the nitrate level can be increased by at least 2 percent, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, or more than 100 percent, as compared to the nitrate level in tissue of a corresponding control plant that does not express the transgene.
  • a plant in which expression of a nitrogen use efficiency-modulating polypeptide is modulated can have decreased levels of nitrate in shoot and/or root tissue.
  • the nitrate level can be decreased by at least 2 percent, e.g., 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, or more than 100 percent, as compared to the nitrate level in a corresponding control plant that does not express the transgene.
  • a plant in which expression of a nitrogen use efficiency-modulating polypeptide is modulated can have increased or decreased levels of nitrate in one or more tissues, e.g., leaf tissues, root tissues, stem tissues, reproductive tissues, petiole tissues, and/or seed tissues.
  • the nitrate level can be increased by at least 2 percent, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, or more than 100 percent, as compared to the nitrate level in a corresponding control plant that does not express the transgene.
  • a plant in which expression of a nitrogen use efficiency-modulating polypeptide is modulated can have decreased levels of nitrate in one or more root tissues.
  • the nitrate level can be decreased by at least 2 percent, e.g., 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, or more than 35 percent, as compared to the nitrate level in a corresponding control plant that does not express the transgene.
  • Increases in nitrogen and/or nitrate in such plants can provide improved nutritional content in geographic locales where the plant's intake of nitrogen and/or nitrate is often insufficient. Decreases in nitrogen and/or nitrate in such plants can be useful in situations where seedlings are not the primary plant part that is harvested for human or animal consumption.
  • a plant in which expression of a nitrogen use efficiency-modulating polypeptide is modulated can have increased levels of carbon.
  • a nitrogen use efficiency-modulating polypeptide described herein can be expressed in a transgenic plant and/or seedling, resulting in increased levels of carbon in tissue of plants and/or seedlings.
  • the carbon level can be increased by at least 2 percent, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, or more than 60 percent, as compared to the carbon level in a corresponding control plant that does not express the transgene.
  • a plant in which expression of a nitrogen use efficiency-modulating polypeptide is modulated can have increased or decreased levels of carbon in one or more tissues, e.g., leaf tissues, root tissues, stem tissues, reproductive tissues, petiole tissues, and/or seed tissues. Increases in carbon in such plants can provide improved nutritional content in edible roots, or improved grains.
  • a plant in which expression of a nitrogen use efficiency-modulating polypeptide is modulated can have increased levels of organic and amino acid metabolites.
  • a nitrogen use efficiency-modulating polypeptide described herein can be expressed in a transgenic plant and/or seedling, resulting in increased levels of metabolites, such as, L- Alanine, L-Glycinel, Phosphoric Acid, L-Threonine, L- Proline, Quinic Acid, a-ketoglutanic acid, L-Glutamic Acid, L-Glutamine, Ascorbic Acid, Sucrose, total Serine, Fructose, L- Valine, Glycerol, Phosphoric acid, L-Leucine, L-
  • a nitrogen use efficiency- modulating polypeptide described herein can be expressed in a transgenic plant and/or seedling, resulting in decreased levels of metabolites, such as, L- valine, Glycerol, L-Leucine, L-Isoleucine, pAminoButyric Acid, Malic Acid, L-Aspartic acid, L-Pjenylalanine, Arabinose, Galactose, Glucose, 2-hydroxybenoic acid, L-Glutamic acid, and/or sucrose in tissue of plants and/or seedlings.
  • metabolites such as, L- valine, Glycerol, L-Leucine, L-Isoleucine, pAminoButyric Acid, Malic Acid, L-Aspartic acid, L-Pjenylalanine, Arabinose, Galactose, Glucose, 2-hydroxybenoic acid, L-Glutamic acid, and/or sucrose in tissue of plants and/or seedlings.
  • the organic and amino acid levels can be modulated by at least 2 percent, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, or more than 60 percent, as compared to the organic and amino acid levels in a corresponding control plant that does not express the transgene.
  • a plant in which expression of a nitrogen use efficiency-modulating polypeptide is modulated can have increased or decreased levels of organic and amino acid metabolites in one or more tissues, e.g., leaf tissues, root tissues, stem tissues, reproductive tissues, petiole tissues, and/or seed tissues.
  • a difference in the amount of nitrogen in a transgenic plant or cell relative to a control plant or cell is considered statistically significant at p ⁇ 0.05 with an appropriate parametric or non-parametric statistic, e.g., Chi-square test, Student's t-test, Mann- Whitney test, or F-test.
  • a difference in the amount of nitrogen is statistically significant at p ⁇ 0.01 , p ⁇ 0.005, or p ⁇ 0.001.
  • a statistically significant difference in, for example, the amount of nitrogen in a transgenic plant compared to the amount in cells of a control plant indicates that the recombinant nucleic acid present in the transgenic plant results in altered nitrogen levels.
  • the phenotype of a transgenic plant is evaluated relative to a control plant.
  • a plant is said "not to express" a polypeptide when the plant exhibits less than 10%, e.g., less than 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.01%, or 0.001%, of the amount of polypeptide or mRNA encoding the polypeptide exhibited by the plant of interest.
  • Expression can be evaluated using methods including, for example, RT-PCR, Northern blots, S 1 RNase protection, primer extensions, Western blots, protein gel electrophoresis, immunoprecipitation, enzyme-linked immunoassays, chip assays, and mass spectrometry. It should be noted that if a polypeptide is expressed under the control of a tissue-preferential or broadly expressing promoter, expression can be evaluated in the entire plant or in a selected tissue. Similarly, if a polypeptide is expressed at a particular time, e.g., at a particular time in development or upon induction, expression can be evaluated selectively at a desired time period.
  • polymorphisms are discrete allelic sequence differences in a population. Typically, an allele that is present at 1 % or greater is considered to be a genetic polymorphism.
  • the discovery that polypeptides disclosed herein can modulate nitrogen content is useful in plant breeding, because genetic polymorphisms exhibiting a degree of linkage with loci for such polypeptides are more likely to be correlated with variation in a nitrogen use efficiency trait. For example, genetic polymorphisms linked to the loci for such polypeptides are more likely to be useful in marker-assisted breeding programs to create lines having a desired modulation in the nitrogen use efficiency trait.
  • one aspect of the invention includes methods of identifying whether one or more genetic polymorphisms are associated with variation in a nitrogen use efficiency trait. Such methods involve determining whether genetic polymorphisms in a given population exhibit linkage with the locus for one of the polypeptides depicted in Figures 11 and 12, and/or a functional homolog thereof. The correlation is measured between variation in the nitrogen use efficiency trait in plants of the population and the presence of the genetic polymorphism(s) in plants of the population, thereby identifying whether or not the genetic polymorphism(s) are associated with variation for the trait.
  • the allele is associated with variation for the trait and is useful as a marker for the trait. If, on the other hand, the presence of a particular allele is not significantly correlated with the desired modulation, the allele is not associated with variation for the trait and is not useful as a marker.
  • Such methods are applicable to populations containing the naturally occurring endogenous polypeptide rather than an exogenous nucleic acid encoding the polypeptide, i.e., populations that are not transgenic for the exogenous nucleic acid. It will be appreciated, however, that populations suitable for use in the methods may contain a transgene for another, different trait, e.g., herbicide resistance.
  • SSR polymorphisms that are useful in such methods include simple sequence repeats (SSRs, or microsatellites), rapid amplification of polymorphic DNA (RAPDs), single nucleotide polymorphisms (SNPs), amplified fragment length polymorphisms (AFLPs) and restriction fragment length polymorphisms (RFLPs).
  • SSR polymorphisms can be identified, for example, by making sequence specific probes and amplifying template DNA from individuals in the population of interest by PCR. If the probes flank an SSR in the population, PCR products of different sizes will be produced. See, e.g., U.S. Patent 5,766,847.
  • SSR polymorphisms can be identified by using PCR product(s) as a probe against Southern blots from different individuals in the population. See, U. H. Refseth et al. (1997) Electrophoresis 18: 1519. The identification of RFLPs is discussed, for example, in Alonso-Blanco et al. (Methods in Molecular Biology, vol.82, "Arabidopsis Protocols", pp. 137-146, J. M. Martinez-Zapater and J. Salinas, eds., c. 1998 by Humana Press, Totowa, NJ); Burr ("Mapping Genes with Recombinant Inbreds", pp. 249-254, in Freeling, M.
  • the methods are directed to breeding a plant line.
  • Such methods use genetic polymorphisms identified as described above in a marker assisted breeding program to facilitate the development of lines that have a desired alteration in the nitrogen use efficiency trait.
  • a suitable genetic polymorphism is identified as being associated with variation for the trait, one or more individual plants are identified that possess the polymorphic allele correlated with the desired variation. Those plants are then used in a breeding program to combine the polymorphic allele with a plurality of other alleles at other loci that are correlated with the desired variation.
  • Techniques suitable for use in a plant breeding program are known in the art and include, without limitation, backcrossing, mass selection, pedigree breeding, bulk selection, crossing to another population and recurrent selection.
  • each identified plants is selfed or crossed a different plant to produce seed which is then germinated to form progeny plants.
  • At least one such progeny plant is then selfed or crossed with a different plant to form a subsequent progeny generation.
  • the breeding program can repeat the steps of selling or outcrossing for an additional 0 to 5 generations as appropriate in order to achieve the desired uniformity and stability in the resulting plant line, which retains the polymorphic allele.
  • analysis for the particular polymorphic allele will be carried out in each generation, although analysis can be carried out in alternate generations if desired.
  • selection for other useful traits is also carried out, e.g., selection for fungal resistance or bacterial resistance. Selection for such other traits can be carried out before, during or after identification of individual plants that possess the desired polymorphic allele.
  • Transgenic plants provided herein have various uses in the agricultural and energy production industries.
  • transgenic plants described herein can be used to make animal feed and food products. Such plants, however, are often particularly useful as a feedstock for energy production.
  • Transgenic plants described herein often produce higher yields of grain and/or biomass per hectare, relative to control plants that lack the exogenous nucleic acid. In some embodiments, such transgenic plants provide equivalent or even increased yields of grain and/or biomass per hectare relative to control plants when grown under conditions of reduced inputs such as fertilizer and/or water. Thus, such transgenic plants can be used to provide yield stability at a lower input cost and/or under environmentally stressful conditions such as drought or abnormal soil nitrogen levels. In some embodiments, plants described herein have a composition that permits more efficient processing into free sugars, and subsequently ethanol, for energy production.
  • such plants provide higher yields of ethanol, other biofuel molecules, and/or sugar-derived co-products per kilogram of plant material, relative to control plants.
  • processing efficiencies are believed to be derived from the nitrate composition of the plant material.
  • Nitrogen stored in compounds in plant tissues may be critical if the plant encounters abnormal nitrogen conditions in the soil that would lead to nitrogen deficiency. For example leaf nitrate is used in many crop plants during grain filling as a nitrogen source.
  • Seeds from transgenic plants described herein can be conditioned and bagged in packaging material by means known in the art to form an article of manufacture.
  • Packaging material such as paper and cloth are well known in the art.
  • a package of seed can have a label, e.g., a tag or label secured to the packaging material, a label printed on the packaging material, or a label inserted within the package, that describes the nature of the seeds therein.
  • Arabidopsis transformation Ti: first generation transformant; T 2 : second generation, progeny of self-pollinated Ti plants; T 3 : third generation, progeny of self-pollinated T 2 plants; T 4 : fourth generation, progeny of self-pollinated T 3 plants.
  • Independent transformations are referred to as events.
  • Arabidopsis thaliana plants Clone NRT 1.3, Clone 1 11089 and NRTl .4.
  • the nucleic acids designated Ceres Annot ID No:1445187, Ceres Annot ID No:1498169 and Ceres Annot ID No: 1447247 were isolated from the species Populus balsamifera.
  • the nucleic acid designated Clone 1583036 was isolated from the species Zea mays and the nucleic acids designated Clone 1806436 and Clone 1777083 were isolated from the species Panicum virgatum.
  • the nucleic acid designated Clone 1927461 was isolated from the species Gossypium hirsutum.
  • Each isolated nucleic acid described above was cloned into a Ti plasmid vector containing a phosphinothricin acetyltransferase gene which confers FinaleTM resistance to transformed plants.
  • Constructs were made using SEQ ID NO: 1 , or SEQ ID NO: 18, each operably linked to a 35S promoter. Wild-type Arabidopsis thaliana ecotype Wassilewskija (Ws) or C24 plants were transformed separately with each construct. The transformations were performed essentially as described in Bechtold et al (1993), Ci?. Acad. Sci. Paris, 316:1194-1199.
  • NRT 1.3 or NRT 1.4, respectively.
  • the presence of each vector containing a nucleic acid described above in the respective transgenic Arabidopsis line transformed with the vector was confirmed by FinaleTM resistance, PCR amplification from green leaf tissue extract, and/or sequencing of PCR products.
  • wild-type Arabidopsis ecotype Ws plants were transformed with the empty vector.
  • Example 3 Screening for Nitrogen content in hydroponically grown transgenic Arabidopsis plants
  • Example 4 Screening for Seed Carbon and Nitrogen content in transgenic Arabidopsis seeds
  • 2 mg of transgenic Arabidopsis seeds are used to quantify the total nitrogen or carbon content using a CHN analyzer (Flash EA 11 12, Thermo Finnigan, San Jose, CA, USA). Wild-type C24 or WS seed is used as a control. 2 mg of shoots or roots can also be used to obtain values for these tissues.
  • plant tissue and seeds are combusted in ten 12 mm tin capsules (CE Elantech, New Jersey).
  • Data parameters (for NC Soil software) are as follows: LLOD: 0.25 mg standard, different for other materials; LLOQ: 3 mg for standard, 1 mg seed tissue, different for other materials.
  • Sample replicates are grouped together. Each sample is normalized to the average of the controls and average and standard deviation calculated for each set of samples based on the normalized values. Samples with a relative % standard deviation greater than 10 are excluded.
  • Example 5 Metabolite Profiling in transgenic Arabidopsis seeds
  • Freeze dried tissue is thoroughly homogenized ⁇ e.g. passes through a
  • the pellet is resuspended in 1 ml dichloromethane, vortexed, incubated for 30 minutes at 37 0 C under constant shaking at 250 rpm and centrifuged at 14,000 rpm for 4 minutes before removing the supernatant and adding it to that previously recovered.
  • the combined supernatants are then vortexed and incubated at room temperature for 15 minutes prior to centrifugation at 4000 rpm for 60 minutes for separation of phases.
  • the top layer containing polar metabolites (polar phase) is isolated from the bottom layer containing the nonpolar metabolites (nonpolar phase).
  • the speed-dried pellet is resuspended in 50/50 (v/v) Acetonitrile + 0.1 % formic acid/water + 0.1% formic acid prior to injecting a 50 ⁇ l sample.
  • Nonpolar metabolites are analyzed by adding 1 ml of 2.5% H 2 SO 4 (v/v in methanol) to 200 ⁇ l of the bottom layer (nonpolar phase) and heating at 9O 0 C for 4 hours. After cooling to room temperature, 900 ⁇ l of dichloromethane are added. Aqueous solvents are removed by adding 4 ml of water, vortexing, centrifuging at 4000 rpm for 15 minutes, discarding the top layer and repeating this process. Approximately 15 mg of anhydrous sodium sulfate is added to the dichloromethane layer to remove trace water, followed by centrifugation at 3000 rpm.
  • T 3 seed from five events of NRT 1.3 containing SEQ ID NO: 1 were analyzed for biomass as described in Example 2, Carbon and Nitrogen content as described in Examples 3 and 4, and Metabolite Profiling as described in Example 5.
  • Results for biomass are presented in Figure 1.
  • Results for nitrate and carbon content of plants grown hydroponically on 10 niM KNO 3 are presented in Figures 2, 3 and 5.
  • Results for increases in metabolite content are presented in Figure 4.
  • WT refers to wild -type Arabidopsis thaliana control plants
  • HH to Arabi ⁇ opsis thaliana plants transformed with and homozygous for the indicated gene.
  • T 3 seed from five events of NRT 1.4 containing SEQ ID NO: 18 was analyzed for biomass as described in Example 2, Carbon and Nitrogen content as described in Examples 3 and 4, and Metabolite Profiling as described in Example 5.
  • Results for biomass are presented in Figure 6.
  • Results for nitrate and carbon content of plants grown hydroponically on 10 mM KNO 3 are presented in Figures 7, 8 and 10.
  • Results for increases in metabolite content are presented in Figure 9.
  • a candidate sequence was considered a functional homolog of a reference sequence if the candidate and reference sequences encoded proteins having a similar function and/or activity.
  • a process known as Reciprocal BLAST (Rivera et al. (1998) Pr OC. Natl. Acad. Sci. USA, 95:6239-6244) was used to identify potential functional homolog sequences from databases consisting of all available public and proprietary peptide sequences, including NR from NCBI and peptide translations from Ceres clones.
  • the BLASTP version 2.0 program includes the following parameters: 1) an E-value cutoff of 1.0e-5; 2) a word size of 5; and 3) the -postsw option.
  • the BLAST sequence identity was calculated based on the alignment of the first BLAST HSP (High-scoring Segment Pairs) of the identified potential functional homolog sequence with a specific reference polypeptide. The number of identically matched residues in the BLAST HSP alignment was divided by the HSP length, and then multiplied by 100 to get the BLAST sequence identity. The HSP length typically included gaps in the alignment, but in some cases gaps were excluded.
  • the main Reciprocal BLAST process consists of two rounds of BLAST searches; forward search and reverse search.
  • a reference polypeptide sequence "polypeptide A”
  • top hits were determined using an E-value cutoff of 10 "5 and a sequence identity cutoff of 35%.
  • the sequence having the lowest E-value was designated as the best hit, and considered a potential functional homolog or ortholog. Any other top hit that had a sequence identity of 80% or greater to the best hit or to the original reference polypeptide was considered a potential functional homolog or ortholog as well. This process was repeated for all species of interest.
  • top hits identified in the forward search from all species were BLASTed against all protein sequences from the source species SA.
  • a top hit from the forward search that returned a polypeptide from the aforementioned cluster as its best hit was also considered as a potential functional homolog.
  • HMMs Hidden Markov Models
  • HMMER 2.3.2 To generate each HMM, the default HMMER 2.3.2 program parameters, configured for glocal alignments, were used. [00177] An HMM was generated using the sequences shown in Figure 11 as input. These sequences were fitted to the model and a representative HMM bit score for each sequence is shown in the Sequence Listing. Additional sequences were fitted to the model, and representative HMM bit scores for any such additional sequences are shown in the Sequence Listing. The results indicate that these additional sequences are functional homologs of SEQ ID NO:2.

Abstract

L'invention concerne des procédés et des matériels pour moduler les niveaux d'efficacité d'utilisation d'azote dans des plantes. Par exemple, des acides nucléiques codant pour des polypeptides de modulation de l'efficacité d'une utilisation d'azote sont décrits ainsi que des procédés pour utiliser de tels acides nucléiques pour transformer des cellules de plante. Des plantes ayant des niveaux d'efficacité d'utilisation d'azote accrus et des plantes produites à partir de plantes ayant des niveaux d'efficacité d'utilisation d'azote accrus sont également décrites.
PCT/US2009/034431 2008-02-19 2009-02-18 Plantes transgéniques ayant des caractéristiques d'efficacité d'utilisation d'azote modifiées WO2009105492A2 (fr)

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US8722072B2 (en) 2010-01-22 2014-05-13 Bayer Intellectual Property Gmbh Acaricidal and/or insecticidal active ingredient combinations
EP2563112A2 (fr) * 2010-04-28 2013-03-06 Evogene Ltd. Polynucléotides et polypeptides isolés et procédés d'utilisation de ceux-ci pour l'augmentation du rendement végétal et/ou des caractéristiques agricoles
US11542522B2 (en) 2010-04-28 2023-01-03 Evogene Ltd. Isolated polynucleotides and polypeptides for increasing plant yield and/or agricultural characteristics
EP2563112A4 (fr) * 2010-04-28 2014-03-05 Evogene Ltd Polynucléotides et polypeptides isolés et procédés d'utilisation de ceux-ci pour l'augmentation du rendement végétal et/ou des caractéristiques agricoles
US10689662B2 (en) 2010-04-28 2020-06-23 Evogene Ltd. Isolated polynucleotides and polypeptides for increasing plant yield and/or agricultural characteristics
US9265252B2 (en) 2011-08-10 2016-02-23 Bayer Intellectual Property Gmbh Active compound combinations comprising specific tetramic acid derivatives
CN104204210A (zh) * 2012-03-20 2014-12-10 英美烟草(投资)有限公司 具有叶中改变的硝酸盐水平的转基因植物
US9708622B2 (en) 2012-03-20 2017-07-18 British American Tobacco (Investments) Limited Transgenic plants with altered nitrate levels in leaves
US10017776B2 (en) 2012-03-20 2018-07-10 British American Tobacco (Investments) Limited Transgenic plants with altered nitrate levels in leaves
US10597666B2 (en) 2012-03-20 2020-03-24 British American Tobacco (Investments) Limited Transgenic plants with altered nitrate levels in leaves
WO2013140157A1 (fr) * 2012-03-20 2013-09-26 British American Tobacco (Investments) Limited Plantes transgéniques ayant des taux modifiés de nitrate dans les feuilles
WO2014164074A1 (fr) * 2013-03-13 2014-10-09 Pioneer Hi-Bred International, Inc. Amélioration de l'absorption et de la translocation des nitrates grâce à une surexpression des transporteurs de nitrates fonctionnels de faible affinité du maïs dans du maïs transgénique
CN115010796A (zh) * 2022-04-21 2022-09-06 济南大学 一种赖草生长素输出载体及其编码基因与应用
CN115010796B (zh) * 2022-04-21 2024-04-02 济南大学 一种赖草生长素输出载体及其编码基因与应用

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