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WO2015095186A2 - Nitrogen use efficiency in plants - Google Patents

Nitrogen use efficiency in plants Download PDF

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Publication number
WO2015095186A2
WO2015095186A2 PCT/US2014/070585 US2014070585W WO2015095186A2 WO 2015095186 A2 WO2015095186 A2 WO 2015095186A2 US 2014070585 W US2014070585 W US 2014070585W WO 2015095186 A2 WO2015095186 A2 WO 2015095186A2
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Prior art keywords
plant
seed
nitrogen
enhanced
expression
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WO2015095186A3 (en
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Sugin CAI
Hans Holtan
T. Lynne Reuber
Robert A. Creelman
Oliver J. Ratcliffe
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Koch Biological Solutions LLC
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Koch Biological Solutions LLC
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
    • C12N15/8222—Developmentally regulated expression systems, tissue, organ specific, temporal or spatial regulation
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8262—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield involving plant development
    • C12N15/8266—Abscission; Dehiscence; Senescence
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • 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

  • the present description relates to composit ons and methods for improving nitrogen use efficiency in plants, for example, by improving nitrogen uptake or assimilation, efficiency.
  • Nitrogen is a critical limiting nutrient for plants. Nitrogen fertilizer is a significant contributor to the yield increases obtained in the last several decades. However, these yield benefits have monetary and en vironmental costs, and nitrogen-based fertilizer represents a significant fraction of a farmer ' s input costs. Furthermore, crops only use a fraction of applied nitrogen. For exampie, it has been estimated that 50-70% of the nitrogen provided to the sod is lost (Masc!aux-Daubresse et a!., 2010, Ann. Boi. 105: 1.1.41-1 .157; Hodge et al, 2000, Trends Plan/ ScL 5: 304-308).
  • Maize production i the US is reported to have a nitrogen fertilizer recovery efficiency of 37% (Cassman et ah, 2002, Amhio 31 : 132- 140), and increased fertilizer application rates are subject, to diminishing returns.
  • a hectare of corn for example, retains 39% of the first 100 kilograms of nitrogen applied, as fertilizer, but only 1.3% of the second kilogram of nitrogen applied fSocolow, 1999, Proa Nail Acad Set. USA 96: 6001 -6008).
  • nitrogen fertilizer that is not taken up by plants is generally lost as runoff or converted to nitrogen gases by microbial action* contributing to water and air pollution.
  • NUE Nitrogen Use Efficiency
  • Plants obtain nitrogen through the processes of uptake and assimilation (Buchanan et al.,
  • Uptake refers to the transport of nitrogen into the plant, and assimilation is the conversion of nitrate and ammonia to amino acids. Plants generally take up nitrogen from the soil in. the form o nitrate or ammonium. Plants contain both low affi nity and high affinity transport systems for these ions. In the case of nitrate, there is both a constitutive and an inducible high affinity transport system (Glass ei al, 2002, , Exp. Boi.. 53; 855-864). Nitrate is transported throughout the plant and stored in the vacuole. Nitrate is reduced to ammonia through the action of nitrate reductase and nitrite reductase.
  • Assimilation of ammonia takes place through the glutamine synfhetase giutamine-2-oxoglutarate aminotransferase. (GS/GOGAT) pathway.
  • Glu amine synthetase (GS) adds an mino group to glutamate to make glutamine, and GOGAT transfers the amino grou to -ketogliitarate to make a second, molecule of glutamate.
  • Photosynthesis provides the fixed carbon and rediictarit necessary for assimilation.
  • Nitrogen utilization or assimilation efficiency is the fraction of plant-acquired nitrogen to he converted to total plant biomass or grain yield; (Xu et al, 2012, J mm. Rev.
  • a third mechanism could be i ncreased capacity to store nitrogen when it. is available. N itrogen is stored in the form of nitrate in cell vacuoles, but stored nitrate supplies are exhausted in a matter of days (Glass et al , 2002, supra). Nitrogen is also stored in the form of amino acids and protein, and this storage is dependent upon sufficient carbon availability. Control, of nitrogen losses is also possible. Nitrate and ammonia exit as well as enter root cells. Photorespiration is another source of ammonia loss. Ammonia released through photorespiration is recycled through the GS/GOGAT pathway, but this process may not be fully efficient.
  • Nitrogen remobi.lization within the plants is also an important component of UE, Mobilization of existing nitrogen, plays an important role in. seed filling (Rajcan and T/oHenaar, 1999, Field Crops Res. 60: 255-265: Schilte et al, 2004, Plam Physiol. 135: 2241-2260: Schite et alirri 2005, Plant Physiol. .137: 1463-1473) and germination in annual crop plants, and is critical for sustainabiiity in perennials. Nitrogen mobility between, source and sink, leaves m Ambidopsis has been shown to be associated with early senescence, whereas during seed set nitrogen
  • the present description relates to methods and compositions tor producing transgenic plants with modified traits, particularly traits that address agricultural and food needs by improving nitrogen use efficiency, in addition to reducing the demand for nitrogen ' application, it is expected that improving nitrogen use efficiency will improve yield and may provide significant value by allowing the plant to thrive in hostile environments, where, for example, low nutrient availability may limit yield or diminish or prevent growth of ' non-transgenic plants, in so doing, we have identified important polynucleotide and polypeptide sequences the expression levels of which may be manipulated to produce improved yield in commercially valuable plants and crops as well as the methods for making them and using them. Other aspects and
  • the present disclosure is directed to a method for producing a plant thai has improved or enhanced nitrogen remobilization in the plant or in. a part of the plant (for example, in. a leaf or a seed-bearing structure) relative to a control plant or its corresponding or analogous part.
  • a plant . is grown in a medium that contains either a limiting concentration of nitrogen tli t limits growth of the plant (for example, 2 rnM total nitrogen in the medium) or an ample concentration of nitrogen that does not limit growth of the plant, (for example, 1 raM total nitrogen in the medium).
  • Expression analysis of the plant may then, reveal the presence of one or more polypeptides (the "instant polypeptides") that have a higher level of expression in senescing leaves when the plant is grown in the limiting nitrogen medium as compared to plants grown in the medium with, ample nitrogen.
  • the expression of a polypeptide identified in this manner may be regulated by a developmeatal!y-reguiated promoter.
  • the promoter may be a seed-enhanced promoter or a seed-bearing structure enhanced promoter (the activity of which is enhanced in a seed or seed-bearing structure, respectively, relative to other plant tissues), or a green tissue promoter, in another embodiment, the deve!opmentaSly-reguiaied promoter is a senescence-enhanced or senescence-induced promoter, the act ivity of which is enhanced when the plant or a part of the plant is senescing.
  • transformed plants that comprise one or more nucleic acid constructs that contain the developmentally- regulated promoter and a polynucleotide that encodes one of the instant polynucleotide will have greater nitrogen re obitization relative to a control plant when the transformed plant exhibits senescence and as a result of the expression of the polynucleotide.
  • the one or more nucleic acid constructs may be introduced into the plant by, lor example, transformation or breeding.
  • a regulator of gene expression thai can enhance nitrogen immobilization during senescence of the plant or a part of the p ant may be identified.
  • a transformed plant may be selected thai has greater nitrogen remobili/atSon than the control plant.
  • RNAi molecule an siR A molecule, an antisense molecule, a ribozyme molecule, a deoxyrihosyme molecule (a "DNAzyme”) or a triple helix molecule that decreases the expression of the endogenous polypeptide.
  • Gene expression suppressors may be introduced into a plant by breeding plants with a parental line ' that contains an instant gene expression suppressor, or by direct application or, in a desirable embodiment, by way of a nucleic acid construct that encodes the suppressor. It is anticipated that plants that comprise nucleic acid constructs encoding one or more of the instant suppressors will suppress or inhibit the activity of an instant polypeptide in the plant during senescence and thereby enhance nitrogen remobilization in the plant. In this manner, regulator of gene expression that can suppress protei expression or protein activity during senescence of the plant or a part of the plant may be identified.
  • the .instant description also pertains to a method for producing a plant that has greater nitrogen remobilizaiion in the plant or a part of the plant relati ve to a control plant or its corresponding part.
  • a plant is grown in a medium that contains either a limiting concentration of nitrogen that limits growth of the plant (for example, 2 mM total nitrogen in the medium) or an ample concentration, of nitrogen that does not limit growth of the plant (for example, 1.0 rn . total nitrogen in. the medium).
  • Expression analysis of the plant may then reveal the presence of one or more polypeptides (the "instant polypeptides") that have a higher level of expression in seeds or seed-bearing structures when the plant is grown in the limiting nitrogen medium as compared to plants grown in the medium with ample ni trogen.
  • the expression of a polypeptide identified in this manner may be regulated by a developraen tally-regulated
  • the activity of the developmentaSly-regulated promoter is enhanced in seed or seed-bearing structures. It is anticipated that transformed plants that comprise one or more nucleic acid constructs that contain the developmentally-regulated promoter and a polynucleotide that encodes one of the instant polynucleotide will have greater nitrogen remobiiizaiion relative to a control plan when the transformed plant exhibits
  • the one or more nucleic acid constructs may be introduced into the plant by, for example, transformation or breeding.
  • a regulator of gene expression that can enhance nitrogen remohilization during senescence of the plant or a part of the plant may be identified, in this method, transformed plant may be selected thai has greater nitrogen remobilkaticai than the control plant.
  • the instant disclosure is also directed to a method for enhancing nitrogen re obilization in a crop plant relative to a control plant by providing a transformed crop plant that comprises at least one of the instant recombinant nucleic acid constructs, and the construct or constructs comprise a senescence-enhanced, or a promoter enhanced in green tissue or seeds or seed- hearing structures, and in the same construct or a separate construct, an operahly-iinked polynucleotide the expression of which is regulated by the promoter.
  • providing may refer to, for example, any one of the art-recognized means to introduce a nucleic acid construct into a plant or plant cell, such as by transformation or breeding where at least one parent line comprises at least one of the instant nucleic acid constructs (two parental lines may each contain an instant nucleic acid construct, as in the case when one plant line comprises a develepmentaUy-induced promoter that regulates expression of a polynucleotide comprised within a second promoter comprised within a different parental plant line).
  • the polynucleotide encodes a polypeptide that is at least 30%, at least 3 i%, at least 32%, at least 33%, at least 34%, at least 35%, at least 36%, at least 37%, at least 38%, at least 39%, at least 40%, at least 41%, at least 42%, at least 43%, at least 44%, at least 45%, at least 46%, at least 47%, at least 48%, at least 49%, at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%,
  • SEQ ID NO:2n where n » to 2300, or alternatively expressed as any of SEQ ID NOs: 2, 4, 6, 8, or any even integer up to and including 5600.
  • the senescence-enhanced promoter, seed-enhanced promoter, or seed-bearing-strucrnre- enhanced promoter preferentially enhances expression of the pol nucleotide during senescence and/or seed development in the transformed plant or hi a part of die transformed plant, and the preferential enhancement of expression results in increased nitrogen remobiiization in the transformed plant relative to the control plant during seed development and senescence of the transformed, plant or plant part.
  • Another aspect of the instant disclosure is a method of producing a crop plant with enhanced nitrogen, remobiiization by providing a crop plant that has a stably-integrated,
  • .recombinant DNA construct comprising a promoter that is functional in plant cells and operably linked to DNA that encodes or suppresses a polypeptide presented, n. the Sequence Listing, or any of SEQ ID NOs: 2n, where n ⁇ 1 to 2300, wherein the expression and activity of the polypeptide confers enhanced nitrogen remobiiization relative to a control plant.
  • the methods further comprise producing seed and a progeny plant from the crop plant with enhanced nitroge remobiiization, wherein the seed or progeny plant comprise the stably-integrated, recofflbiiiant DNA construct and the progeny plant or a plant grown from the seed exhibit enhanced nitrogen remobiiization relative to a control plant.
  • the instant disclosure also pertains to a recombinant nucleic acid construct comprising senescence-enhanced, a seed-enhanced, a seed-bearing-stnteture-enhanced promoter, or a green tissue promoter that regulates expression of a polynucleotide, wherein the polynucleotide encodes a polypeptide is at.
  • the instant disclosure also pertains to a transformed crop plant produced by any of t he above described, methods, wherein the crop plant has enhanced nitrogen remobiiization relative to a control plant when the expression, of an introduced, or endogenous polypeptide provided in the sequence listing is enhanced. or inhibited, respectively.
  • Figures JA-!D present a strategy to increase nitrogen, remobiiistation from leaves.
  • the expression pattern of two different genes in leaf ? of an A bidopsis plant: is analyzed by RNA sequencing from 22 to 42 days after sowing in. limiting (2 ittM, grey line) or ample (10 tnM, black line) nitrogen, conditions.
  • Figure I.C indicates the expression distribution of all the genes in the experiment.
  • Figure 1 D represents an expression construct to improve nitrogen remobilization in plants under ample nitrogen conditions, where the promoter of a gene such as the one in Fig, 1 A is used to drive a putative regulator of nitrogen remobilization such as the gene in Fig. .IB. The construct would increase the expression of the regulator to high levels under either low or high nitrogen conditions at the appropriate developmental stage.
  • Figures 2A-2B display nitrogen use data obtained with a remobilization defective mutant, aigS'l grown under limited N (top, Fig. 2A) or high N (bottom, Fig. 2B) conditions after a pulse labeling with L' N. Plotted in each is a ratio of the value obtained for the atgS-l ' plant normalized to the appropriaie control plant. A value of I indicates that the values for both atg5 ⁇ l and the control plants were identical. The amount of nitrogen, present in dry remains (%NDR) for the atg5-I plant was higher than ' controls, particularly when the plants were grown in low N (Fig. 2A).
  • seed yield weight of seeds (g)
  • NHI nitrogen harvest index (nitrogen in seeds/nitrogen in entire plant)
  • NUE nitrogen use efficiency measured, as NH1/HI
  • RSA Relative specific abundance ( dilution by 14 N).
  • the present description relates to polynucleotides and polypeptides for modifying phenot pes of plants, particularly those associated with increased p ' hotosynthetic resource use efficiency and increased yield with respect to a control plant (for example, a wild-type plant).
  • various information sources are referred to and/or are specifically incorporated.
  • the information sources include scientific journal articles, patent documents, textbooks, and " internet entries. While the reference to these information sources clearly indicates that they can he used by one of skill in the art, each and every one of the information sources cue-d herein are specifically incorporated in their entirety, whether or not a specific mention, of "incorporation by reference " " is noted.
  • the contents and teachings of each and every one of the information sources can be relied on and used to make and use embodiments of the instant description.
  • a host cell includes a plurality of such host cells
  • a reference to "a plant” is a reference to one or more plants, and so forth.
  • Upreguladon or “up-reguiation” refers to a process in which a cell or an organism (e.g., a plant) increases the quantity of a cellular component, such as RNA or protein, in response to an interna! or external signal. Upregnlation may result in a greater activity of interest occurring in the cell or organism, for example, an increase in nitrogen remobilizat on. Conversely,
  • downregulation refers to a process by which a cell decreases the quantity of a cellular component, such, as RNA or protein, in response to an internal or external signal.
  • internal or external signal may refer to, for example, an environmental variable such as a particular stress or a developmental marker such as moiecuie that signals the onset or occurrence of senescence.
  • Tissue-specific, tissue-enhanced (that is, tissue-preferred), cell type-specific, and inducible promoters constitute non-constitutive promoters. Promoters under developmental control include promoters that preferentially initiate transcription in certain tissues, such as xyieni, leaves, roots, or seeds. Suc promoters are examples of tissue-enhanced or tissue- preferred promoters (see US patent 7,365,186). Tissue-enhanced promoters can be found upstream and operatively linked to DNA sequences normal ly transcribed in higher levels in
  • Cell-enhanced s certain plant tissues or specifically in certain plant tissues, respectively.
  • tissue-enhanced tissue-enhanced regulation thus refer to the control of gene or protein expression, for example, by a promoter, which dri ves expression that is not necessarily totally restricted to a single type of cell, or tissue, hu t where expressi on i s elevated in particular cells or tissues to a greater extent than in other cells or tissues within the organism, and in the case of tissue-specific regulation, in a manner that is primarily elevated in a specific tissue.
  • Tissue- enhanced or preferred promoters have been described in, for example, US patent 7,365,186, .
  • seed-specific promoters are transcriptionally active entirely or almost entirely in seed tissue.
  • “Seed-preferred” or “seed-enhanced” promoters are transcriptionally active predominantly in seed tissue, but are not necessaril expressed only in seed, tissue,
  • a seed-specific, enhanced or preferred promoter may be preferentially active during seed development and/or during germination. Examples of seed-specific promoters are found in the present Sequence Listing, in Table 3, or have been taught in, for example, US patent publication US2013030541.4 or by Qing Qu and Takaiwa, 2004. Plant B iechnol. J. 2:1 13-125).
  • a seed-bearing-structure-enhanced promoter may be preferentially active during seed-bearing structure development.
  • An 'inducible promoter initiates transcription in response to an environmental stimulus such as a. an external physical stimulus, for example, abiotic stimuli including energy or a particular chemical or class of chemicals, or a biotie stimulus, for example, a pathogen, or an interna), stimulus such as one or more markers that signal a stage of developmen Examples include "pathogen-inducible" promoters that initiate transcription in response to the presence of various pathogenic organisms or their products, and developmentally-induced promoters thai are activated when a plant or plant part is at a particular growth stage, for example, "senescence- enhanced” (also referred to as "senescence- inducible”) promoters.
  • an environmental stimulus such as a. an external physical stimulus, for example, abiotic stimuli including energy or a particular chemical or class of chemicals, or a biotie stimulus, for example, a pathogen, or an interna
  • stimulus such as one or more markers that signal a stage of developmen Examples
  • Senescence-enhanced promoters are active late in the life cycle of a plant duri ng or near the time o f senescence (Noh and Amasino, 1 99. supra), and preferentially regulate expression of one or more genes (and any encoded polypeptides ⁇ during senescence of a plant cell from a leaf Slower, fruit, or other organ or plant part with respect to the level of expression of that gene in a non-senescing, i.e., a growing or mature (but pre-se»esce»t) cell.
  • endogenous refers to a molecule that naturally originates from, within a plant, plant tissue, or plant ceil.
  • endogenous polypeptide refers to a natural or native pol peptide that is encoded by a plant's native gene and thus it originates from within the plant, plant tissue, or plant ceil upon its translation.
  • A. "recombinant polynucleotide” is a. polynucleotide that is not in its native state, e.g., the polynucleotide comprises a nucleotide sequence not found in nature, or the polynucleotide is in a context other than that in which it is naturally found, e.g., separated from nucleotide sequences wit whic it typically is in proximity in nature, or adjacent (or contiguous with) nucleotide sequences with which it typically is not in proximity.
  • the sequence at issue can be cloned into a vector, or otherwise recombined with one or more additional nucleic acids.
  • An expression vector or cassette is ao example of a "recombinant nucleic acid construct".
  • A. plant refers to a whole plant as well as to a plant pari, such as seed, fruit, leaf, or root, plant tissue, plant ceils or any other plant material, e.g., a plant explain, as well as to progeny thereof, and to in vitro systems that mimic biochemical or cellular components or processes in a cell.
  • a “recombinant polypeptide” is a polypeptide produced by translation of a recombinant polynucleotide.
  • a “synthetic polypeptide' "5 is a polypeptide created by consecutive
  • isolated polypeptide whether a naturally occurring or a recombinant polypeptide, is more enriched in (or out of) a cell than the polypeptide in its natural state in a wild-type cell, e.g., more than about 5% enriched, more than about 10% enriched, or more than about 20%, or more than about 50%, or more, enriched, i.e., alternatively denoted; 105%, 110%, 120%, 150% or more, enriched relative to wild type standardized at 100%. Such an enrichment is not the result ; of a natural response of a wild-type plant.
  • the isolated polypeptide is separated from other DCiular components with which it is typically associated, e.g., b an of the various protein purification methods herein.
  • Constant domains are recurring units in molecular evolution, the extents of which can be determined by sequence and structure analysis.
  • a “conserved domain” or “conserved region” as used herein refers to a region in heterologous polynucleotide or polypeptide sequences where there is a relatively high degree of sequence identity between the distinct sequences.
  • conserveed domains contain conserved sequence patterns or motifs that allow for their detection in, and identification and characterization of, polypeptide sequences.
  • a DNA-binding domain is an example of a conserved domain:.
  • Identity or similarity refers to sequence similarity between two polynucleotide sequences or between two polypeptide sequences, with identity being a more strict comparison.
  • the phrases “percent identity” and “% identity” refer to the percentage of sequence similarity found in a comparison of two or more polynucleotide sequences or two or more polypeptide sequences.
  • Sequence similarity refers to the percen similarity in base pair sequence (as determined by any suitable method) between two or more polynucleotide sequences. Two or more sequences can be anywhere from 0-100% similar or identical, or any integer value between 0-100%. identity or similarity can be determined by comparing a position in each sequence that may be aligned for purposes of comparison.
  • a degree of similarity or identity between polynucleotide sequences is a function of the number of identical, matching or corresponding nucleotides at positions shared by the polynucleotide sequences.
  • a degree of identity of polypeptide sequences is a function of the number of identical, amino acids at corresponding positions shared b the polypeptide sequences.
  • a degree of homology or similarity of polypeptide sequences is a function of the number of amino acids at corresponding positions shared b the polypeptide sequences. The fraction or percentage of components in common is related to the homology or identity between the sequences.
  • An alignment may suitably be determined by means of computer programs known in the art, such as MACVECTO software, 1999 (Accelrys®, inc., San Diego, CA).
  • Nitrogen remob.ilization refers to the movement of nitrogenous compounds from one plant part to another, generally from senescing organs for the purpose of supplementing the nutrition of growing organs such, as new leaves and seeds.
  • Leaf proteins including
  • a “transgenic plant” or “transformed plant” refers to a plant that contains genetic material not found in a wi ld-type plant of the same species, variety or cultivar.
  • the genetic material may include an expression vector or cassette, a transgene, an insertions! mutagenesis event ⁇ such as by transposes! or T-DNA insertional mutagenesis), an activation tagging sequence, a mutated sequence, a homologous recombination event or a sequence modified by chimeraplasty.
  • the foreign genetic material has been introduced into the plant by human manipulation, but any method can be used as one of skill in the art recognizes.
  • a transgenic line or transgenic plant line refers to the progeny plant or plants deriving from the stable integration of heterologous genetic material into a speci fic location or locations wiihiii the genome of the original transformed cell.
  • An expression vector or cassette typically comprises a polypeptide-encodtng sequence operably linked (i.e., under regulatory control of) io appropriate inducible, tissue-enhanced, tissue-specific. deveiopmentaUy-enhanced, or constitutive regulatory sequences thai allow for the controlled expression of the polypeptide.
  • the expression cassette can be introduced into a plant by transformation or by breeding after transformation of a parent plant.
  • a plant refers to a whole plant as well as to a plant part; such as seed, fruit, leaf, or root, plant tissue, plant cells or any other plant material, e.g., a plant explant, as well as to progeny thereof, and to in vitro systems that mimic biochemical or cellular components or processes in a cell.
  • a transgenic or transformed plant of the instant disclosure may have enhanced or greater nitrogen remobfeation relative to a control plant when the transgenic plant is transformed with a recombinant polynucleotide encoding any of the listed sequences, or when the transgenic plant contains or expresses a lis ted polypeptide, and as a consequence of the expression of the listed polypeptide within the transgenic or transformed plant
  • a “seed-bearing structure”, as used herein, refers to a plant part that comprises a developing or mature seed, and may include, but is not limited to, an achene, berry, capsule, caryopsis or grain, ctreumcissiJe capsule, cypsela, drupe, ear, fruit or ripened pericarp, follicle, grain, kernel, legume, !ocylicidai capsule, lome tum, nut, pistil, pod, porfckla! capsule, samara, schkocarp, seed capsule, septicidal capsule, septifragal capsule, silicola, siliqua, siiique or strobilus.
  • overexpression refers to a greater expression level of a gene in a plant, plant cell or plant tissue, compared to expression in a wild-type plant, cell or tissue, at any developmental or temporal stage for the gene, Overexpression can occur when, for example, the genes encoding one or more transcription factors are under the control of a strong expression signal, such as one of the promoters described herein (for example, the cauliflower mosaic virus 35S transcription initiation region), Overexpression may occur throughout a plant or in specific tissues of the plant, depending on the promoter used, as described below.
  • Overexpression may take place in plant cells normally lacking expression of . polypeptides functionally equivalent or identical to the present transcription factors. Overexpression may also occur in plant cells where endogenous expression of the present transcriptio factors or
  • control plant refers to la.it such as a cell, seed, plant component, plant tissue, plant organ or whole plant used to compare against an altered or experimental plant such as a transgenic or genetically modified plant for the purpose of identifying an enhanced phenotype in the altered or experimental plant.
  • a control plant may i some cases be a transgenic plant line thai comprises an empty vector or marker gene, bat does not contain the recombinant polynucleotide of the present description that is expressed in the transgenic or genetically modified plant being evaluated, in general, a control plant is a plant of the same line or variety as the experimental or altered plant being tested.
  • a suitable control plant would include a genetically unaltered or ndn-traasgenic plant of the parental line used t generate a transgenic plant herein.
  • Wild type or "wi -type”, as used herein, refers to a plant cell, seed, plant component, plant tissue, plant organ or whole plant that has not been genetically modified or treated in an experimental sense. Wild-type cells, seed, components, tissue, organs or whole plants may be used as contols to compare levels of expression and the extent and. nature of trait modification with cells, tissue or plants of the same species in which a polypeptide's expression is altered, e.g., in that it has been knocked out, overexpressed, or eetopically expressed.
  • a seed-bearing structure or organ refers to a organ of a plan that comprises a seed such as, for example, achene, berry, capsule, caryopsts or grain, circumeissile capsule, cypsela, drupe, ear, fruit or ripened pericarp, follicle, grain, kernel, legume, locultcidal capsule, !omenium, nut, pistil, pod, poricidal capsule, samara, schkocarp, seed capsule, septieidai capsule, septifragal capsule, stlicuia, siliqua, silique, strobilus, etc.
  • a seed such as, for example, achene, berry, capsule, caryopsts or grain, circumeissile capsule, cypsela, drupe, ear, fruit or ripened pericarp, follicle, grain, kernel, legume, locultcidal capsule, !omenium,
  • overexpression refers to a greater expression level of a gene in a plant, plant cell or plant tissue, compared to expression of that gene in a wild-type plant, cell or tissue, at any developmental or temporal stage. Overexpression can occur when, for example, the genes encoding one or more polypeptides are under the control of a strong promoter (e.g. , the cauliflower mosaic virus 35$ transcription initiation region), Overexpression may also be achieved by placing a gene of interest under the control of an inducible or tissue specific promoter, or may be achieved through, integration, of transposons or engineered T-DNA.
  • a strong promoter e.g. , the cauliflower mosaic virus 35$ transcription initiation region
  • telomeres may be overexpressed by creating an artificial transcriptional activator targeted to hind specifically to its promoter sequences, comprising an engineered sequence-specific DNA binding domain such as a z nc finger protein or TAL -effector protein fused to a. transcriptional activation domain.
  • overexpression may occur throughout a plant, in specific tissues of the plant, or i n the presence or absence of particular environmental signals, depending on the promoter or overexpression approach, used.
  • Overexpression may take place in plant cells normally lacking expression of polypeptides functionally eqiiivaieni or identical to the instant polypeptides. Overexpression may also occur in plant cells where endogenous expression of the instant polypeptides or functionally equivalent molecules normally occurs, but such normal expression is at a lower level. Overexpression thus results in a greater than normal production, or "overproduction" of the polypeptide in the plant, cell or tissae.
  • Yield or “plant yield' 5 refers to increased plant growth, increased crop growth, increased biomass, and/or increased plant product production (including grain:), and is dependent to some extent on temperature, plant size, organ size, planting density, light, water and nutrient availability, and how the plant copes with various stresses, such as through temperature
  • Increased or improved yield may be measured as increased seed yield, increased plant product yield (plant products include, for example, plant tissue, including ground or otherwise broken-up plant tissue, and products derived from one or more types of plant tissue), or increased vegetative yield.
  • plant products include, for example, plant tissue, including ground or otherwise broken-up plant tissue, and products derived from one or more types of plant tissue, or increased vegetative yield.
  • Senescence is the last stage of development of an annual (monocarpic) plant, and is an active and tightly regulated process. Recycling of nutrients, particularly nitrogen, from the leaves to the seed, during senescence is a key contributor to grain yield.
  • Nitrogen remobilization is the process by which nitrogen is relocated to different plant organs at different times of development, for example, from senescing leaves to grain or seeds at the reproductive stage.
  • Nitrogen, remobilization efficiency may be defined as the proportion of nitrogen that is remobihzed from source or senescent leaves to siok leaves or developing grains (seeds). The efficiency of nitrogen remobilization. is controlled both by genetic and environmental factors. Genetic variability in NRE exists among crop plants. However, genetically identical plants grown on limiting nitrogen remobiifee nitrogen more efficiently from senescing leaves to seeds than when grown on ample nitrogen. Improving NRE either in limiting or ample nitrogen conditions is expected to increase the nitrogen available to the seeds and thereby increase yield.
  • the .nitrogen that is remobiiized to the seeds comes largely from the breakdown of chloroplasts. If leaves senesce too early in the seed fill period, the plants will lose photosynthetic capacity and the ability to fix carbon, and therefore yield will be decreased, indeed, a mild "stay-green" phenotype, where the plants show a delay in senescence, can be beneficial, io yield. However, if senescence is delayed indefinitely, the nitrogen available to the seed will be limited and yield will be reduced.
  • an increase in remobilization would be timed for when senescence would normally begin, and quantitatively enhance the process.
  • the efficiency of nitrogen remobilization may be regulated either in the senescing leaves that are the source of the nitrogen. or by signals coming from the sink (i.e. seeds) driving the developmental processes that induce senescence and nitrogen t emobilization.
  • the present description includes increased or ectopic expression of putative regulatory polypeptides (i.e., regulators or effectors of re obiikation) and isolated or recombinant polynucleotides encoding the polypeptides, or novel sequence variant polypeptides or
  • polynucleotides encoding novel variants of polypeptides derived from the specific sequences provided m the Sequence Listing.
  • the polynucleotides of the instant description ma be incorporated in expression vectors for the purpose of producing transformed, plants.
  • the claimed sequences will typically share at. least about 40% nucleotide sequence identity, or at least 41%, at least 42%, at least 43%, at least 44%, at least 45%, at least 46%, at least 47%, at least 48%, at least 49%, at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at.
  • the claimed nucleotide sequences will typically encode a polypeptide that is at least at least 30%, at least 31%, at least 32%, at least 33%, at least 34%, at least 35%, at least 36%, at least 37%, at least 38%, at least 39%, at least 40%., at least 41%, at least 42%, at least 43%., at least 44%, at least 45%, at least 46%., at least 47%, at least 48%, at least 49%, at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%o, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%,
  • These methods are based on the ability to alter the expression of critical regulatory molecules that may be conserved between diverse plant species.
  • Related conserved regulatory molecules may be originally discovered in a model system such as Ar bidopsis and homologous, functional molecules then discovered in other plant species. The latter may then be used to confer increased yield, or photosyntlietic resource use efficiency in diverse plant species.
  • Sequences in the Sequence Listing may be ectopically expressed in overexpressor plants.
  • the changes in the characteristic.(s) or txaii(s) of the plants may then be observed and found to confer increased yield and/or increased nitrogen use efficiency and/or nitrogen remobilization. Therefore, the polynucleotides and polypeptides can be used to improve desirable characteristics of plants.
  • polynucleotides of the instant description are also ectopically expressed in
  • polynucleotides and polypeptides can be used to change expression levels of genes, polynucleotides, and/or proteins of plants or plant cells.
  • polynucleotides and polypeptides that may be expressed in plants for the purpose of increasing yield that arises from improved nitrogen use efficiency and/or nitrogen remobilization.
  • the polynucleotides and polypeptides of the instant description, thai when expressed in plants or crop plant have the capacity to enhance nitrogen remobilization in a plant or a pari of a plant relative to a control plant or a corresponding part, of the control plant, include:
  • SEQ ID NO: 661 and 662 AT2G401 10 J (G5160 ⁇ and clade member sequences SEQ ID NOs: 639-702;
  • ATSG25J 10.1 (G5 76) and clade member sequences SEQ ID NOs:405 -4098;
  • SEQ ID NO: 4547 and 4548 AT3G1 1 1 10.1 (G2253) and clade member sequences SEQ ID NOs:4539-4566; SEQ ID NO: 4583 and 4584, AT2G44745.1 (G 180 ⁇ and. clade member sequences SEQ ID NOs: 4567-4600;
  • variants of the disclosed sequences are also within the scope of the instant description, that, is, one having a sequence that differs from the one of the polynucleotide sequences in the Sequence Listing, or a
  • variant nucleic acid may, for example, encode the same polypeptide but differ in sequence from the sequence in the Sequence Listing due to degeneracy in the genetic code. Included within this definition are polymorphisms thai may or may not be readily delectable using a particular oligonucleotide probe of the polynucleotide encoding polypeptide, and improper or unexpected hybridization to allelic variants, with a locus other than the norma! chromosomal locos for the polynucleotide sequence encoding polypeptide.
  • polypeptide sequence variants Differences between presently disclosed polypeptides and polypeptide variants are limited so that the sequences of the former and the latter are closely similar overall and, in many regions . , identical.
  • Presently disclosed polypeptide sequences and similar polypeptide variants may differ in amino acid sequence by one or more substitutions, additions, deletions, .fusions and truncations, which may be present in any combination. These differences may produce silent changes and result in functionally equivalent polypeptides.
  • a polypeptide sequence variant may have "conservative" changes, wherein a substituted amino acid has similar structural or chemical properties.
  • Conservative substitutions include substitutions in which at least one residue in the amino acid sequence has been removed and a different residue inserted in its place. Such substitutions generally are made in accordance with the Table 1 when it is desired to maintain the activity of the protein. Table 1 shows amino acids whic can be substituted for an amino acid in a protein and which are typically regarded as conservati ve substitutions.
  • polypeptides provided in the Sequence Listing have a novel activity, such as, for example, a regulatory activity.
  • ail conservative amino acid substitutions (for exampi one basic amino acid substituted for another basic amino acid) in a polypeptide will not necessarily result in the polypeptide retaining its activity, it is expected that many of these conservative imitations would result in the polypeptide retaining its activity.
  • Most mutations, conservative or non-conservative, made to a protein but outside of a conserved domain required for function and protein activity will not affect the activity of the protein to any great extent.
  • Deliberate amino aci d substitutions may thus be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathie nature of the resid ues, as long as s significant amount of the functional or biological activity of the
  • negatively charged amino acids may include aspartie acid and glutamic acid
  • positively charged amino acids may include lysine and arginine
  • amino acids with uncharged polar head groups having similar hydropbiiicity values may include leucine, isoleucine, and valine; glycine and alanine; asparagme and giutamine; serine and threonine; and phenylalanine and tyrosine.
  • a variant may have "non-conservative" changes, e.g., replacement of a glycine with a tryptophan.
  • Similar minor variations may also include amino acid deletions or insertions,, or both.
  • Related polypeptides may comprise, for example, additions and/or deletions of one or more N-linked or O-lmked giycosylation sites, or an addition and/or a deletion of one or more cysteine residues.
  • Guidance in determining which and how many amino acid residues may be substituted, .inserted or deleted without abolishing functional or biological activity ma be found using computer programs well known in the art, for example, DNASTA ' software (see US patent 5,840,544),
  • conserved domains are recurring functional and/or structural units of a protein sequence within a proiein family (for example, a family of regulatory proteins), and distinct conserved domains have been used as building blocks In molecular evolution and recombiiied in various arrangements to make proteins of different protein families with different functions. conserveed domains often correspond to the 3 -dimensional domains of proteins and contain conserved, sequence patterns or motifs, which allow for their detection in polypeptide sequences with, for example, the use of a conserveed Domain Database (.for example, at www.ncbi,nlni.nih.gov cdd).
  • conserved Domain Database The National Center for Biotechnology information conserveed Domain Database defines conserved domains as recurring units in molecular evolution, the extents of which can be determined by sequence and structure analysis. conserveed domains contain conserved sequence patterns or .motifs, which allow for their detection in polypeptide sequences (Conserved Domain Database; wvvw:ncbi.nlmj «3 ⁇ 4.gov/Stnict «re c-dd cdd.shtml).
  • a "conserved domain” or “conserved region” as used herein refers to a region in heterologous polynucleotide or polypeptide sequences where there is a relatively high degree of sequence identity between the distinct sequences.
  • a 'Myb DNA binding domain 1 ' is an example of a conserved domain. conserveed domains may also be identified as regions or domains of identity to a specific consensus sequeiice (see, for example, Riechmami et al. dislike 2000, Science 290, 2105-21 10;
  • the conserved domains of the plant polypeptides for example, for the first or second Myb DNA binding domain proteins may be determined.
  • the polypeptides in Table 2 have conserved domains specifically indicated by amino acid coordinate start and stop si es. A comparison of the regions of these polypeptides allows one of skill in the art (see, for example. Reeves and Nissen, 1 90. J, Biol Chem, 265, 8573-8582; Reeves and Nissen, 1995. Prog. Cell Cycle Res, I: 339-349) to .identify domains or conserved domains for any of the polypeptides listed or referred to in this disclosure.
  • conserved domain models are generally identified with multiple sequence alignments of rel ated proteins spanning a v ariety of organisms. These alignments reveal sequence regions containing the same, or similar, patterns of amino acids. Multiple sequence alignments, three- dimensional structure and three-dimensional struc ture superposition of conserved domains can be used to infer sequence, structure, and functional relationships (Conserved Domain Database, supra). Since the presence of a particular conserved domain within, a polypeptide is highly correlated with, an evO.hitionari.iy conserved function, a conserved domain database may be used to identify the amino acids in a protein sequence that are piuatively involved in functions such as binding or catalysis, as mapped from conserved domain annotations to the query sequence. For example, the presence in. a protein of a DNA binding domain that, is structurally and
  • phyiogeneticaily similar to one or more domains found in the sequence listing would be a strong indicator of a related function in plants (e.g., the function of regulating and/or improving nitrogen use efficiency, nitrogen remobi!ization, and/or yield, i.e., a polypeptide with such a domain is expected to confer enhanced nitrogen use efficiency, nitrogen remobi ligation, and/or yiel d when its expressi on leve l is increased under the regul atory control of a senescence- enhanced promoter).
  • a related function in plants e.g., the function of regulating and/or improving nitrogen use efficiency, nitrogen remobi!ization, and/or yield, i.e., a polypeptide with such a domain is expected to confer enhanced nitrogen use efficiency, nitrogen remobi ligation, and/or yiel d when its expressi on leve l is increased under the regul atory control of a senescence- enhanced promoter).
  • Said polypeptides may, when their expression level is altered by enhancing their expression during senescence and to a lesser extent prior to senescence, confer at least one regulatory activity selected from the group consisting of increased nitrogen use efficiency, greater nitrogen reraobiiixation, greater yield, greater size, greater biomass, and or greater vigor as compared to a control plant.
  • Methods using manual alignment of sequences similar or homologous to one or more polynucleotide sequences or one or more polypeptides encoded by the polynucleotide sequences may be used to identify regions of similarity and conserved domains (e.g., DMA binding domains, activation domains, localization domains, repression domains, oiigomerization domains, or other domains, that are recognizably related across plant, species.
  • conserved domains e.g., DMA binding domains, activation domains, localization domains, repression domains, oiigomerization domains, or other domains, that are recognizably related across plant, species.
  • Such manual methods are well-known of those of skill in the art and can include, for example, comparisons of tertiar structure between a polypeptide sequence encoded by a polynucleotide that comprises a known function, and a polypeptide sequence encoded by a .polynucleotide sequence that has a function not yet determined.
  • tertiary structure may comprise predicted - heS tees, ⁇ -sheets, amphipathic helices, leucine zipper motifs, zinc finger molife, prolme-rich regions, cysteine repeat motifs, and the like.
  • a conserved domain refers to a subsequence within a polypeptide family the presence of which is correlated with at least one function exhibited by members of the polypeptide family, and which exhibits a high degree of sequence homology, such, as at least 30%, at least 31 %, at least 32%, at least 33%, at least 34%, at least 35%, at least 36%, at least 37%, at least 38%, at least 39%, at least 40%, at least 41%, at least 42%, at least 43%, at least 44%, at least 45%, at least 46%, at least 47%, at least 48%, at least 49%, at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least
  • Table 2 lists Afabidopsis sequence identifiers and the descriptions .of various domains found within the respective proteins, including the domain names, the linear coordinates of the amino acids at the beginning and end of the respective domains, and the SEQ ID NOs: of the domains listed in this table. It is expected that domains of clade member polypeptides of each of these sequences, examples of said polypeptides being provided in the Sequence Listing, function similarly in plants and their presence is indicative of and correlated with t he same functions as the domains in the Arabidopsis sequences. Table 2, Various Arabidopsis domains found in polypeptide sequences that enhance nitrogen remobilization in plants
  • HSH Helix-loop-heiix domain
  • Homologous sequences as described above can comprise ort o!ogoiis or paralogous sequences.
  • Several different methods are know by those of skill in the art for identifying and defining these fractionally homologous sequences.
  • General methods for identifying orthologs and paralogs, including pliyiogeneiic .methods, sequence similarity and 'hybridization methods, are described herein; an ortlio!og orparaiog, including equivalogs, may be identified by one or more of the methods described below.
  • gene duplication may cause two copies of a particular gene, giving rise to two or more genes with similar sequence and often similar function ' known as paralogs.
  • a paralog is therefore a similar gene formed by duplication within the same species.
  • Paralogs typically cluster together or in the same clade (a group of similar genes) when a gene family phytogeny is analyzed- using programs such as CLUSTAL (Thompson, et al., 1994, Nucleic Acids Res. 22: 673-4680; Higgins et a!., ! 996, Methods, Emy oL 266; 383-402).
  • genes within a clade may contain paralogous sequences, or ortho!ogous sequences that share the same function (see also, for example, Mount, 2001 , in Bioinformatics: Sequence and Genome Analysis. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, p. 543)
  • Orthologous sequences can also be identified by a reciprocal BLAST strategy. Onc an orthologous sequence has been identified, the function of the orthoiog can be deduced from the identified function of the reference sequence.
  • polypeptides sequences belon to distinct clades of polypeptides that include members from diverse species.
  • most or ah of the c!ade member sequences derived from both eudicots and monocots have been shown and are predicted to enhance nitrogen remobilkation to confer increased yield when the sequences were overexpressed under the regulatory control of lor example, a senescence-enhanced promoter, a seed-enhanced promoter, a seed-bearing structure enhanced promoter, or a green tissue promoter.
  • Orthologs and paralogs of presentl disclosed polypeptides may be cloned using compositions provided by the present description according to methods well known in the art.
  • cDNAs can be cloned using rtiR A from a plant cell or tissue that expresses one of the presen sequences.
  • Appropriate mR ' NA sources may be identified by interrogating Northern blots with probes designed from the present sequences, after which a library is prepared from the niRNA obtained irom a positive ceil, or tissue.
  • Polypep kle-eucodmg cDNA is then isolated using, for example, PCR, using primers designed from a presently disclosed gene sequence, or by probing with a partial or complete cDNA or with one or more sets of degenerate probes based on the disclosed sequences.
  • the cDNA library may be used to transform plant cells. Expression of the cDNAs of interest is detected using, for example, mieroarrays, .Northern blots, quantitative PCR, or any other technique for monitoring changes in expression. Genomic clones may be isolated using similar techniques to those.
  • orthologs of the Ambidopsis polypeptide sequences and their functionally similar orthologs are listed, in the present Sequence Listing.
  • these orthologs are phyiogeneticalty and structurally similar to the sequences in the Sequence Listing and can also function in a plant by increasing nitrogen use efficiency, nitrogen reruobili ation, yield., vigor, and/or biornass when ectopically and preferentially expressed during senescence of a plant or in a plant organ.
  • polypeptide would also perform similar functions when ectopkaliy expressed under the regulatory control of the disclosed promoters or oth er senescence-enhanced promoters , seed-enhanced promoters, seed-bearing structure enhanced promoters, or green tissue promoters.
  • the instant senescence-enhanced promoters which may be found in Table 3 may be developmental ⁇ regulated in that transcription is initiated at the promoter site primarily during senescence, and the promoters are relatively insensitive to the concentration of nitrogen in the plant or its environment.
  • the instant senescence-enhanced promoters preferentially initiate gene expression at the onset of senescence and/or during plant senescence and can be used to regulate the expression of extrinsic genes in various ' plant or crop organs as the plants or organs enter and experience senescence, ' without affecting me normal (i. e.
  • seed-enhanced promoters and seed-bearing, structure enhanced promoters i Table 3 regulate gene expression in a manner that is enhanced in seeds or seed-bearing structures, respectively, relative to other tissues in a plant.
  • seed-enhanced promoters may also be found in the Sequence listing as SEQ ID NOs; 5742 to 5830.
  • the choice of promoter may also include a constitutive promoter or a promoter with enhanced acti vity in a tissue capable of photosynthesis (also referred to herein as a "green tissue promoter", a “photosynihetic promoter” or a “photosynthetic tissue-enhanced promoter”) such as a leaf tissue or other green tissue.
  • a tissue capable of photosynthesis also referred to herein as a "green tissue promoter", a “photosynihetic promoter” or a “photosynthetic tissue-enhanced promoter”
  • a leaf tissue or other green tissue such as a leaf tissue or other green tissue.
  • tissue promoters include for example, an RBCS3 promoter (SEQ ID NO: 5831), an RSCS4 promoter (SEQ ID NO: 5832) others such as the At4g01060 promoter (SEQ ID NO: 5833), the latter regulating expression in a guard cell, or rice sequences SEQ ID NOs: 5834 to 5857, shown in Table 3 or in the Sequence Listing.
  • Os03g30650 5846 enhanced Green tissue-
  • Example ⁇ Identification of regulators of remobillataiion through gene expression profiting Plants grown under limiting nitrogen conditions remobilize a greater fraction of the nitrogen in their vegetative organs to the seed than do plants grown under ample nitrogen. To elucidate the regulatory networks controlling these differences, a transcriptional profiling experiment, was performed on Ar hidopsis plants grown under conditions of limiting and ample nitrogen. Leaf and silique tissue were harvested beginning in the vegetative phase and continuing through seed development to create a developmental time series under these two different nutrient regimes.
  • Plant growth and tissue isolation Plants were grown in pots containing three volumes of fritted clay at bottom and one vol ume of .fine sand on top. Pots were pre-soaked in nutrient solution containing either 2 ra (low N) or 10 mM (high N) nitrate solution. Phosphate (0.25 mM), sulfate (0.25 raM), magnesium (0.25 .mM), and sodium (0.20 aiM) were present in both solutions at the same concentration. The.
  • Leaf 7 (the seventh leaf formed by each plant) was tagged with thread 21 days after sowing ("days after sowing" abbreviation; DAS). Collection of leaf 7 was started at 22 DAS and continued every other day until 42 DAS, for a total of 1 1 time points. Sampling of siiiques started when siliques reached stage 1 (floral organs withering) and 2, , 6, and 8 days post stage 1 , for a total of five time points. At each time point, leaf 7 and siliques were harvested from 10- 1.2 plants and eight plants, respectively, with plants being selected to minimize any potential effects of position wi thin, the hydroponic tub and growth room.
  • DAS day after sowing
  • RNA isolation Leaves were pulverized in liquid nitrogen with a mortar and pestle.
  • RNA yields were too low to satisfy the target of 2 pg for making adapter libraries. In these cases a second aliquot of 100 pL tissue was taken to perform an additional RNA extraction. RNA from ' the second extraction was used if the yield was sufficient Otherwise, RNA was . pooled from both extractions.
  • Silique RNA was extracted following a modified protocol I ' Meng and Feldinan., 2010, BiotechnoL J. 5: 1 3-186). This procedure employs a modified, high. pH (pH 9.5) extraction. buffer. An RNAeasy kit (Quiagen, Hilden, Germany ⁇ was subsequently used to purif the RNA.
  • RNA Sequence Library Creation The starting total. RNA. concentration was measured using a NanoDrop Spectrophotometer (Thermo Scientific, W ' al hani, MA) and 2 ⁇ % of total RNA was used as the entry point to the TraSeq RNA. Sample Prep method ⁇ 1 008136 A, Illumina#, San Diego, CA). AH steps were performed according to manufacturer specifications as indicated in the High-Throughput (HT) Protocol but implemented as a custom method on a Biomek FX 1 ' liquid handling workstation. In brief, poly-A containing mRNA molecules were purified using poly-T oligo-atlaehed.
  • HT High-Throughput
  • Double stranded cDNA was produced and indexed adapters were attached by ligation (illumina). Library size distribution was assessed by capillary gel electrophoresis, and then normalized based, on quantification based o absorbaace, fluorescence and quantitative polymerase chain reaction (qPCR) using primers targeting the adapter sequences.
  • transcript profiles of leaves at each of the 1 1 time points (22, 24, 26, 28, 30, 32, 34, 36, 38, 40 and 42 DAS) and silique (stage 16, 2, , 6 and S days post stage 16) were obtained, comparisons were made (i) between plants supplied with low and high nitrate at matched time point and (it) against the initial time poin (22 DAS and stage 1 6 for leaf and. siiique, respecti el ).
  • the expression profiles of the low N and high N grown plants were relatively similar until 30 DAS, corresponding to the time when seed filling began, when a large number of genes began to be differentially expressed in low N conditions and visual evidence of senescence initiation was first noted in leaf 7 in low .
  • the transcriptional profiling d ta described above in Example i was also mined to identify promoters that are expressed (enhanced) during leaf senescence.
  • the criteria, for selection of these promoters were as follows; ⁇ Induced during senescence in leaves, with low expression initially. Promoters were chosen ra three groups: induced before 30 DAS (pre-senescenc-e), induced at 30 DAS (at senescence onset) and induced after 30 DAS (late senescence).
  • SplineC!uster analysis was performed using the gene set from the low N condition to identify groups of genes that were similarly regulated. Clusters with the desired regulatory patterns were identified, then genes in these clusters were compared with public and proprietary data sets to eliminate those thai showed strong expression in tissues other than senescing leaves or induction during stress conditions. Exemplary putative regulatory sequences for these genes were identified and are listed as SEQ ID NO: 5724-574.1 , although it is anticipated that other senescence-enhanced or senescence- induced promoters may function in a similar manner. In.
  • a promoter region may include a fragment of the promoter sequences provided in the Sequence Listing or in this Example, or a complement thereof wherein the promoter sequence, or the fragment thereof or the complement thereof, regulates expression of a polypeptide in a plant cell for example, in a manner that is enhanced or preferred durin certain periods of development, e.g. , in senescing plant cells and tissues.
  • Example III Identification of seed-enhanced and seed-bearing-structure-enhanced promoters.
  • Genes with enhanced expression in seed and siiique were identified from our dataset, and compared to a number of public and proprietary datasets to prioritize -strongly seed-enhanced genes and eliminate those that showed stron expression in tissues other than seed or siiique, or induction during stress conditions.
  • Exemplary putative regulatory sequences for these genes were identified and are listed as SEQ I ' D NO; 5742-5830, although, it is anticipated that other senescence-enhanced or -induced promoters may function in a similar manner.
  • a promoter region may include a fragment of the promoter sequences provided in the Sequence Listing or in this Example, or a complement, thereof, wherein the promoter sequence, or the fragment thereof or the complement thereof, regulates expression of a polypeptide in a plant cell, for example, in a manner that is enhanced or preferred in certain plant tissues, e.g. , seed tissues or the tissues of seed-bearing structures.
  • Green tissue-enhanced promoters thai may be used io d ri ve expression of polynucleotides and polypeptides found in the Sequence Listing and stroetarally and functionally-related sequences have also been described in U.S. patent publication no. 201 J i ) .S 7952 A L incorporated ' herein by reference.
  • Such promoters include SEQ ID NOs: 5831-5857.
  • a promoter region may include a .fragment of the promoter sequences provided in the Sequence Listing or in this E ample, or a complement thereof, wherein the promoter sequence, or the fragment thereof, or the complement thereof regulates expression of a polypeptide in a plant cell, for example, in response to a biotic or abiotic stress, or in a manner that i enhanced or preferred in certain plant tissues, e.g., green or photosynihetic tissues.
  • the above-identified promoters and regulatory genes may be used to create constructs to transform plants.
  • Transformed plant may be prepared using the following methods, although these examples are not intended to limit the description or claims.
  • Promoter cloning For genes showing appropriate patterns of regulation, typically approximately 1.2 kb of upstream sequence are cloned by polymerase chain reaction (unless litis region contains another gene, in which case the upstream sequence up to the next gene is cloned). Each promoter is cloned into a nucleic acid construct (e.g., an expression vector or cassette) in front of either a polynucleotide encoding green fluorescent protein (G.FP) or another marker of gene expression, or i front of a polynucleotide encoding a polypeptide or a regulatory molecule of interest, for example, a polypeptide found in.
  • G.FP green fluorescent protein
  • the promoter may be used to regulate the expression of a polynucleotide that is expected to cause beneficial traits by reducing or eliminating the activity of a target gene or group of genes through, antisense or A.i based approaches.
  • the promoter may also be incorporated into antisense or RNAt constructs which target genes encoding homologs of the transcription factors.
  • Transformation. Transformation of Ambidop is typically performed by an
  • the Agrobticteriwn solution is poured into dipping containers. All flower birds and rosette leaves of the plants are immersed in this solution for 30 seconds. The plants are laid on iheir side and wrapped to keep the humidity high. The plants are kept this way overnight at 22 °C and then the pots are unwrapped, turned upright,, and moved to the growth racks.
  • the plants are maintained, on the growth rack under 24-hour light, until seeds are ready to be harvested. .Seeds are harvested when 80% of the siliques of the transformed plants are ripe (approximately five weeks after the initial transformation). This seed is deemed TO seed, since it is obtained from the TO generation, and is later plated on selection plates (kanam cin.,
  • Resistant plants that are identified on such selection plates comprise the Ti generation.
  • RT-PC may be performed to confirm the ability of cloned promoter fragments to drive expression of the polypeptide transgene in plants transformed with the vectors.
  • Ti plants transformed with promoter-TF combinations comprised within, a nucleic acid construct are subjected to morphological analysis.
  • Promoter's that produce a. substantial amelioration of the negative effects of TF overexpression are subjected to further analysis by propagation into the T2 generation, where the plants are analyzed for an altered trait relative to a control plant.
  • Example VI Methods for determining nitrogen immobilization
  • concentrations can be determined using iS N pulse-chase assays. Plants are grown under a defined fertilization regime (limiting N, e.g., 2 raM nitrate and ample N e.g. 10 m.M nitrate) conditions in an ebb and flow hydroponic system as described above. At a defined period in the plant life prior to reproductive growth, the plants are allowed to uptake l5 N twice (e.g. for Arahidopst 32 and 34 days after sowing when grown under 10: 14 fight.dark conditions). This is done by replacing the uniabeiied utrient solution with a solution that has the same composition except that l *N0.? is replaced by ' ' 'NO. ? at 10% enrichment.
  • l *N0.? is replaced by ' ' 'NO. ? at 10% enrichment.
  • Plants are then transferred to long day conditions (L D 24:0) to induce .flowering. Plants are harvested at the end of their life cycle when, all seeds are matured and the rosette is dr '. Samples are then separated into seeds and dry remains (rosette, stem, caulme leaves, and empty siliqaes) and seed weight and dry weight are determined. Four to six replicates are harvested for uptake and remobilization experiments.
  • sample After drying and weighing each sample, the dry remains are ground in a bead mil) to obtain a homogenous fine powder. A subsample of 2 mg to 3 mg of the fine powder from the dry remains or intact seeds are carefully weighed into tin capsules to determine the total N content and ! 5 N abundance. Samples are analyzed for S 5 N isotopes using a PDZ Europa ANCA-GSL elemental analyzer interfaced to a PDZ Europa 20-20 isotope ratio mass spectrometer (Sercon Ltd., Cheshire, UK). Samples are combusted at. K ) 0°C in a reactor packed with chromium oxide and silvered cobaltous cobaltic oxide. Following combustion, oxides are removed in a reduction reactor (reduced copper at 650°C), The helium carrier then flows through a water trap
  • the t5 N abundance is calculated as atom per cent ⁇ A ::: ⁇ ⁇ > ⁇ )/( ⁇ * ⁇ ⁇ !4 ) x 100) and for unlabelled plant controls f A% tX>B j ⁇ ;) was 0.3660.
  • a number of traits related to nitrogen remobilizatiori can be deri ved from seed weight (seedd3 ⁇ 4 ) > tissue dry weight (rosette dry matter minus seeds, or dry remains (DR*)).
  • indicators for yield are calculated from the data obtained from plants used in remobilteation experiments. From the dry weights for seeds and dry remains we calculate harvest index (Hi ⁇ see is eddw + D &), which is an indicator for individual plant yield. Tissue dry weight and nitrogen concentrations (N%) are also combined to determine the nitrogen harvest index (NHI + %DR X
  • DRiK winch estimates the extent, of seed filling with nitrogen.
  • the ratio Hi Hl is monitored in order to compare nitrogen use efficiency ( UE).
  • the dry weight of seed and D , %, and E% are combined to determine the partition of ° in seeds which represents the proportion of " absorbed at the vegetative stage and remobilized to the seeds at the ' reproductive stage ( i:' harvest index ( ,;> NHi) ( ⁇ % ⁇ x N3 ⁇ 4 Si;e a x seed f ⁇ w ⁇ /(E% ss:3 ⁇ 4i i x % ss£i i x seed t 3 ⁇ 4v -i- E DR x %D X DRd.
  • the ratio °NRl Rl is monitored in order io compare .nitrogen remo ' hiUsKUion efficiency (NRE),
  • the relative specific abundance (RSA) ratio is an indicator of the allocation of the nitrogen absorbed during seed fill This value is related to ts N enrichment and is an indicator of 3 ⁇ 4 dilution by the natural U N absorbed by roots after labeling and until harvest (RSA ratio - x N% Siii3 ⁇ 4s x $ee ⁇ i w ⁇ E% sied x N%DK x DR i !«) ( 3 ⁇ 4!
  • the RSA ratio can be equal to 1 when S 5 N dilution is similar in seeds and dry remains, lower than i when the N absorbed after labeling is routed to the seeds while " is retained in DR., or higher than ! when seeds are preferentially loaded with. ! N remobilteed while th i N absorbed after labeling is allocated to vegetative tissues.
  • Example VII identification of crop plants with enhanced nitrogen remobilization
  • seeds are the major sink for ni trogen remobilized from leaves after anth.es.is and daring seed fill
  • the nitrogen accumulated in the vegetative parts of the plant is remobilized and. translocated, to grain.
  • 60-95% of the grain nitrogen comes from the re-mobilization of nitrogen stored in roots and shoots before an the is while in maize 45-65% of the grain nitrogen is provided from pre-existing nitrogen in the stover before silking.
  • the remainder of the grain nitrogen originates from post- flowering nitrogen uptake.
  • a less biased and more accurate estimate of N flux than the "balance method" estimates nitrogen remobilization i plants grown under limiting and ample nitrogen concentrations can be using i 5 N pulse-chase assays.
  • l 5 N can be applied to Irydropomcai!y grown plants at the beginning of stem elongation, with a single sampling of grain and remaining plan! material occurring at maturity.
  • a second 3 ⁇ 4 pulse-chase can be applied at -auihesis to estimate of the proportion of post-flowering N uptake allocated to the grain.
  • the application of two ⁇ N pulse-chase techniques at different times is required to obtain a complete picture of N management and recycling during the entire developmental cycle of the crop.
  • traits related to nitrogen remobiIi3 ⁇ 4ation and harvest parameters can be defined as described above.

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Abstract

This disclosure provides transgenic plants, including crop plants and methods for their production. The transgenic plants comprise recombinant DNA constructs for the expression of polypeptides encoded by the DNA constructs. The polypeptides are capable of conferring improved traits to the transgenic plants when expressed under the control of a developmentally-regulated promoter. This disclosure also pertains to transgenic plants and their progeny plants, wherein the transgenic or progeny plants comprise the recombinant DNA constructs and are selected for having enhanced nitrogen use efficiency and/or nitrogen remobilization. Seed of the transgenic plants that can be grown into a plant that comprise the disclosed recombinant DNA constructs and exhibits having enhanced nitrogen use efficiency, and which may be selected for this trait, are also envisioned.

Description

NITROGEN USE EFFICIENCY IN PLANTS
FIELD OF TOE INVENTION
The present description relates to composit ons and methods for improving nitrogen use efficiency in plants, for example, by improving nitrogen uptake or assimilation, efficiency. BACKGROUND
Nitrogen is a critical limiting nutrient for plants. Nitrogen fertilizer is a significant contributor to the yield increases obtained in the last several decades. However, these yield benefits have monetary and en vironmental costs, and nitrogen-based fertilizer represents a significant fraction of a farmer's input costs. Furthermore, crops only use a fraction of applied nitrogen. For exampie, it has been estimated that 50-70% of the nitrogen provided to the sod is lost (Masc!aux-Daubresse et a!., 2010, Ann. Boi. 105: 1.1.41-1 .157; Hodge et al, 2000, Trends Plan/ ScL 5: 304-308). Maize production i the US is reported to have a nitrogen fertilizer recovery efficiency of 37% (Cassman et ah, 2002, Amhio 31 : 132- 140), and increased fertilizer application rates are subject, to diminishing returns. A hectare of corn, for example, retains 39% of the first 100 kilograms of nitrogen applied, as fertilizer, but only 1.3% of the second kilogram of nitrogen applied fSocolow, 1999, Proa Nail Acad Set. USA 96: 6001 -6008). As a consequence, nitrogen fertilizer that is not taken up by plants is generally lost as runoff or converted to nitrogen gases by microbial action* contributing to water and air pollution.
Thus, improving their efficiency of a crop plant's nitrogen use (i.e., its Nitrogen Use Efficiency or NUB) would have the benefit of improving yield and agricultural sastainability while reducing negative environmental impact NUE has been defined as increased grain yield per unit nitrogen available from the soil ( asclaux-Danbresse et al.. .201.0, supra), and thus it is judicious to identify means to increase the grain yield that may be obtained per unit nitrogen available from the soil.
Plants obtain nitrogen through the processes of uptake and assimilation (Buchanan et al.,
2000, Bioehettmiiy and Molecular iol gy of Plants, American Society of Plant Physiologists, Rockyiife, Maryland; Masetaux-Dauhresse et al, 20 W, supra). Uptake refers to the transport of nitrogen into the plant, and assimilation is the conversion of nitrate and ammonia to amino acids. Plants generally take up nitrogen from the soil in. the form o nitrate or ammonium. Plants contain both low affi nity and high affinity transport systems for these ions. In the case of nitrate, there is both a constitutive and an inducible high affinity transport system (Glass ei al, 2002, , Exp. Boi.. 53; 855-864). Nitrate is transported throughout the plant and stored in the vacuole. Nitrate is reduced to ammonia through the action of nitrate reductase and nitrite reductase.
Assimilation of ammonia takes place through the glutamine synfhetase giutamine-2-oxoglutarate aminotransferase. (GS/GOGAT) pathway. Glu amine synthetase (GS) adds an mino group to glutamate to make glutamine, and GOGAT transfers the amino grou to -ketogliitarate to make a second, molecule of glutamate. Photosynthesis provides the fixed carbon and rediictarit necessary for assimilation.
Plant nitrogen use efficiency cou!d conceivably be increased by several mechanisms
(Law lor 2002, J. Exp. Bat. 53; 773-787). One mechanism could, be increasing nitrogen uptake (which ca be defined, as the percentage of applied nitrogen taken op by plants (Maust and Williamson, 1 94, J. Amer. S . Hon. Set., 119: 195-201), through higher root surface area, deeper penetration into the soil, or more high affinity nitrate or anunottium transporters, A second, mechanism could be increased assimilation, possibly by increased activity of assimilatory enzymes or removal of negative regulation. Nitrogen utilization or assimilation efficiency, NUtE, is the fraction of plant-acquired nitrogen to he converted to total plant biomass or grain yield; (Xu et al, 2012, J mm. Rev. Plan ι ' Biol '. 63:153-182). A third mechanism could be i ncreased capacity to store nitrogen when it. is available. N itrogen is stored in the form of nitrate in cell vacuoles, but stored nitrate supplies are exhausted in a matter of days (Glass et al , 2002, supra). Nitrogen is also stored in the form of amino acids and protein, and this storage is dependent upon sufficient carbon availability. Control, of nitrogen losses is also possible. Nitrate and ammonia exit as well as enter root cells. Photorespiration is another source of ammonia loss. Ammonia released through photorespiration is recycled through the GS/GOGAT pathway, but this process may not be fully efficient. Overexpression of cytosolic g!oiamine synthetase in tobacco increased biomass produced, presumably through increased, efficiency of ammonia recycling (Ollveira et aL 2002, Plant Physiol 1.29: 1.170-1 180). The intrinsic nitrogen use efficiency (defined as biomass produced per unit ) could be changed by changing the plant's fundamental carbon/nitrogen ratio, improving the HUE of crop plants has the potential to reduce fertil izer application rates, providing both cost savings and environmental benefits.
Another means to increase plant N includes making remobi!ization more efficient- Nitrogen remobi.lization within the plants is also an important component of UE, Mobilization of existing nitrogen, plays an important role in. seed filling (Rajcan and T/oHenaar, 1999, Field Crops Res. 60: 255-265: Schilte et al, 2004, Plam Physiol. 135: 2241-2260: Schite et al„ 2005, Plant Physiol. .137: 1463-1473) and germination in annual crop plants, and is critical for sustainabiiity in perennials. Nitrogen mobility between, source and sink, leaves m Ambidopsis has been shown to be associated with early senescence, whereas during seed set nitrogen
remobilization is associated with relative biomass in source and sink organs (Diaz et al, 2008, Plant Physiol 147; 1437-1449). In spite of the apparent advantages of improved NUE, deoad.es of research have not produced significant improvements in NUB in crops, and unproved NUE is largely an unmet need in agriculture today.
The present description relates to methods and compositions tor producing transgenic plants with modified traits, particularly traits that address agricultural and food needs by improving nitrogen use efficiency, in addition to reducing the demand for nitrogen' application, it is expected that improving nitrogen use efficiency will improve yield and may provide significant value by allowing the plant to thrive in hostile environments, where, for example, low nutrient availability may limit yield or diminish or prevent growth of 'non-transgenic plants, in so doing, we have identified important polynucleotide and polypeptide sequences the expression levels of which may be manipulated to produce improved yield in commercially valuable plants and crops as well as the methods for making them and using them. Other aspects and
embodiments of the description are described below and can be derived from the teachings of this disclosure as a whole.
SUMMARY OF THE DISCLOSURE
The present disclosure is directed to a method for producing a plant thai has improved or enhanced nitrogen remobilization in the plant or in. a part of the plant (for example, in. a leaf or a seed-bearing structure) relative to a control plant or its corresponding or analogous part. In this •method, a plant . is grown in a medium that contains either a limiting concentration of nitrogen tli t limits growth of the plant (for example, 2 rnM total nitrogen in the medium) or an ample concentration of nitrogen that does not limit growth of the plant, (for example, 1 raM total nitrogen in the medium). Expression analysis of the plant may then, reveal the presence of one or more polypeptides (the "instant polypeptides") that have a higher level of expression in senescing leaves when the plant is grown in the limiting nitrogen medium as compared to plants grown in the medium with, ample nitrogen. The expression of a polypeptide identified in this manner may be regulated by a developmeatal!y-reguiated promoter. For example, the promoter may be a seed-enhanced promoter or a seed-bearing structure enhanced promoter (the activity of which is enhanced in a seed or seed-bearing structure, respectively, relative to other plant tissues), or a green tissue promoter, in another embodiment, the deve!opmentaSly-reguiaied promoter is a senescence-enhanced or senescence-induced promoter, the act ivity of which is enhanced when the plant or a part of the plant is senescing. It is anticipated that transformed plants that comprise one or more nucleic acid constructs that contain the developmentally- regulated promoter and a polynucleotide that encodes one of the instant polynucleotide will have greater nitrogen re obitization relative to a control plant when the transformed plant exhibits senescence and as a result of the expression of the polynucleotide. The one or more nucleic acid constructs may be introduced into the plant by, lor example, transformation or breeding. In this method, a regulator of gene expression thai can enhance nitrogen immobilization during senescence of the plant or a part of the p ant may be identified. In this .method, a transformed plant may be selected thai has greater nitrogen remobili/atSon than the control plant.
The converse observation, in which expression, analysis of the plant identifies one or more endogenous polypeptides that have a higher level of expression in senescing leaves when the plant is grown in the ample nitrogen medium as compared to plants grown in the medium with limiting nitrogen, may be used to identify one or more endogenous polypeptides that may enhance nitrogen remobilizaiion when expression of the endogenous polypepiide(s) is/are down- regulated. Down-regulation of expression may be accomplished with means that suppresses transcription or translation of the endogenous polypeptide. Of some interest ar suppressors o gene expression such as. for example, an. RNAi molecule, an siR A molecule, an antisense molecule, a ribozyme molecule, a deoxyrihosyme molecule (a "DNAzyme") or a triple helix molecule that decreases the expression of the endogenous polypeptide. Gene expression suppressors may be introduced into a plant by breeding plants with a parental line 'that contains an instant gene expression suppressor, or by direct application or, in a desirable embodiment, by way of a nucleic acid construct that encodes the suppressor. It is anticipated that plants that comprise nucleic acid constructs encoding one or more of the instant suppressors will suppress or inhibit the activity of an instant polypeptide in the plant during senescence and thereby enhance nitrogen remobilization in the plant. In this manner, regulator of gene expression that can suppress protei expression or protein activity during senescence of the plant or a part of the plant may be identified.
The .instant description, also pertains to a method for producing a plant that has greater nitrogen remobilizaiion in the plant or a part of the plant relati ve to a control plant or its corresponding part. In this method, a plant is grown in a medium that contains either a limiting concentration of nitrogen that limits growth of the plant (for example, 2 mM total nitrogen in the medium) or an ample concentration, of nitrogen that does not limit growth of the plant (for example, 1.0 rn . total nitrogen in. the medium). Expression analysis of the plant may then reveal the presence of one or more polypeptides (the "instant polypeptides") that have a higher level of expression in seeds or seed-bearing structures when the plant is grown in the limiting nitrogen medium as compared to plants grown in the medium with ample ni trogen. The expression of a polypeptide identified in this manner may be regulated by a developraen tally-regulated
promoter. In one embodiment, the activity of the developmentaSly-regulated promoter is enhanced in seed or seed-bearing structures. It is anticipated that transformed plants that comprise one or more nucleic acid constructs that contain the developmentally-regulated promoter and a polynucleotide that encodes one of the instant polynucleotide will have greater nitrogen remobiiizaiion relative to a control plan when the transformed plant exhibits
senescence and as a resul of the expression of the polynucleotide in a seed or seed-hearing structure. The one or more nucleic acid constructs may be introduced into the plant by, for example, transformation or breeding. In this method, a regulator of gene expression that can enhance nitrogen remohilization during senescence of the plant or a part of the plant may be identified, in this method, transformed plant may be selected thai has greater nitrogen remobilkaticai than the control plant.
The instant disclosure is also directed to a method for enhancing nitrogen re obilization in a crop plant relative to a control plant by providing a transformed crop plant that comprises at least one of the instant recombinant nucleic acid constructs, and the construct or constructs comprise a senescence-enhanced, or a promoter enhanced in green tissue or seeds or seed- hearing structures, and in the same construct or a separate construct, an operahly-iinked polynucleotide the expression of which is regulated by the promoter. In this context; "providing" may refer to, for example, any one of the art-recognized means to introduce a nucleic acid construct into a plant or plant cell, such as by transformation or breeding where at least one parent line comprises at least one of the instant nucleic acid constructs (two parental lines may each contain an instant nucleic acid construct, as in the case when one plant line comprises a develepmentaUy-induced promoter that regulates expression of a polynucleotide comprised within a second promoter comprised within a different parental plant line). The polynucleotide encodes a polypeptide that is at least 30%, at least 3 i%, at least 32%, at least 33%, at least 34%, at least 35%, at least 36%, at least 37%, at least 38%, at least 39%, at least 40%, at least 41%, at least 42%, at least 43%, at least 44%, at least 45%, at least 46%, at least 47%, at least 48%, at least 49%, at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 90%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95% or 96%, at least 97%, at least 98%, or at least 99%, or about 100% identical to. SEQ ID NO:2n, where n» to 2300, or alternatively expressed as any of SEQ ID NOs: 2, 4, 6, 8, or any even integer up to and including 5600. The senescence-enhanced promoter, seed-enhanced promoter, or seed-bearing-strucrnre- enhanced promoter preferentially enhances expression of the pol nucleotide during senescence and/or seed development in the transformed plant or hi a part of die transformed plant, and the preferential enhancement of expression results in increased nitrogen remobiiization in the transformed plant relative to the control plant during seed development and senescence of the transformed, plant or plant part.
Another aspect of the instant disclosure is a method of producing a crop plant with enhanced nitrogen, remobiiization by providing a crop plant that has a stably-integrated,
.recombinant DNA construct comprising a promoter that is functional in plant cells and operably linked to DNA that encodes or suppresses a polypeptide presented, n. the Sequence Listing, or any of SEQ ID NOs: 2n, where n ~1 to 2300, wherein the expression and activity of the polypeptide confers enhanced nitrogen remobiiization relative to a control plant. The methods further comprise producing seed and a progeny plant from the crop plant with enhanced nitroge remobiiization, wherein the seed or progeny plant comprise the stably-integrated, recofflbiiiant DNA construct and the progeny plant or a plant grown from the seed exhibit enhanced nitrogen remobiiization relative to a control plant.
The instant disclosure also pertains to a recombinant nucleic acid construct comprising senescence-enhanced, a seed-enhanced, a seed-bearing-stnteture-enhanced promoter, or a green tissue promoter that regulates expression of a polynucleotide, wherein the polynucleotide encodes a polypeptide is at. least 30%, at least 31%, at least 32%, at least 33%, at least 34%, at least 35%, at least 36%, at least 37%, at least 38%, at least 39%, at least 40%, at least 41%, at least 42%, at least 43%, at least 44%, at least 45%, at least 46%, at least 47%, at least 48%>, at least 49%, at least 50%, at least 51 %, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%>, at least 65%, at least 66%, at least 67%>, at least 68%, at least 69%, at least 70%>, at least 71%, at least 72%, at least 73%>, at least 74%, at least 75%, at least 76%>, at least 77%, at least 78%, at least 79%, at least 90%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85?/*, at least 86%, at least 87%, at least 88%, at least 89%, at. least 90%, at least 91 %, at least 92%, at least .93%, at least 94%, at least 95% or 96%, at least 97%, at leas 98%, or at least 99%, or about 100% .identical, to any of SEQ ID NO: n where n. is2, 4, 6, 8, or an even, integer between 2 and 5600, inclusive.
The instant disclosure also pertains to a transformed crop plant produced by any of t he above described, methods, wherein the crop plant has enhanced nitrogen remobiiization relative to a control plant when the expression, of an introduced, or endogenous polypeptide provided in the sequence listing is enhanced. or inhibited, respectively.
Brief Description of the Sequence Listing and Drawings
δ The Sequence Listing provides exemplary polynucleotide and polypeptide sequences of the instant description. The traits associated with the use of the sequences are included in the Examples,
Figure imgf000008_0001
Listing provides exemplar
polynucleotide and polypeptide sequences. The copy of the Sequence Listing, being submitted electronically with, this patent application, provided under 3.7 CFR §.1.821-1. 25, is a read-only memory computer-readable file in ASCII text format. The Sequence Listing is named " PS- 0230P. ST25.txt" the electronic file of the Sequence Listing was created on December 5, 2013, and is 17,3 14,435 bytes in size (16.3 megabytes in si/.e as measured in MS-WINDOWS). The Sequence Listing is herein incorporated by reference in its entirety.
Figures JA-!D present a strategy to increase nitrogen, remobiiistation from leaves. The expression pattern of two different genes in leaf ? of an A bidopsis plant: is analyzed by RNA sequencing from 22 to 42 days after sowing in. limiting (2 ittM, grey line) or ample (10 tnM, black line) nitrogen, conditions. Figure I.C indicates the expression distribution of all the genes in the experiment. Figure 1 D represents an expression construct to improve nitrogen remobilization in plants under ample nitrogen conditions, where the promoter of a gene such as the one in Fig, 1 A is used to drive a putative regulator of nitrogen remobilization such as the gene in Fig. .IB. The construct would increase the expression of the regulator to high levels under either low or high nitrogen conditions at the appropriate developmental stage.
Figures 2A-2B display nitrogen use data obtained with a remobilization defective mutant, aigS'l grown under limited N (top, Fig. 2A) or high N (bottom, Fig. 2B) conditions after a pulse labeling with L'N. Plotted in each is a ratio of the value obtained for the atgS-l ' plant normalized to the appropriaie control plant. A value of I indicates that the values for both atg5~l and the control plants were identical. The amount of nitrogen, present in dry remains (%NDR) for the atg5-I plant was higher than' controls, particularly when the plants were grown in low N (Fig. 2A).
Explanation of abbreviations and terms used in Figures 2A-2B:
DR : weight of above-ground dry remains less seeds (g)
seed yield: weight of seeds (g)
% D ; percentage of nitrogen present in the dry remains
%N seeds: percentage of nitrogen in the seeds
HI: harvest index (seed weight entire plant weight)
NHI: nitrogen harvest index (nitrogen in seeds/nitrogen in entire plant)
NUE: nitrogen use efficiency measured, as NH1/HI
15NHI: harvest index (seed 55N/ entire plant "N) QlEM; immobilization efficiency (,5NHI/HI)
RSA: Relative specific abundance ( dilution by 14N).
DETAILED. DESCRIPTION
The present description relates to polynucleotides and polypeptides for modifying phenot pes of plants, particularly those associated with increased p'hotosynthetic resource use efficiency and increased yield with respect to a control plant (for example, a wild-type plant). Throughout this disclosure, various information sources are referred to and/or are specifically incorporated. The information sources include scientific journal articles, patent documents, textbooks, and "internet entries. While the reference to these information sources clearly indicates that they can he used by one of skill in the art, each and every one of the information sources cue-d herein are specifically incorporated in their entirety, whether or not a specific mention, of "incorporation by reference"" is noted. The contents and teachings of each and every one of the information sources can be relied on and used to make and use embodiments of the instant description.
As used herein and in the appended claims, the singular forms "a", "an", and "the" include the plural reference unless the context clearly dictates otherwise. Thus, for example, a reference to "a host cell" includes a plurality of such host cells, and a reference to "a plant" is a reference to one or more plants, and so forth.
'DEFINITIONS
"Upreguladon" or "up-reguiation" refers to a process in which a cell or an organism (e.g., a plant) increases the quantity of a cellular component, such as RNA or protein, in response to an interna! or external signal. Upregnlation may result in a greater activity of interest occurring in the cell or organism, for example, an increase in nitrogen remobilizat on. Conversely,
"downregulation" or "down-regulation" refers to a process by which a cell decreases the quantity of a cellular component, such, as RNA or protein, in response to an internal or external signal. A.n internal or external signal may refer to, for example, an environmental variable such as a particular stress or a developmental marker such as moiecuie that signals the onset or occurrence of senescence.
Tissue-specific, tissue-enhanced (that is, tissue-preferred), cell type-specific, and inducible promoters constitute non-constitutive promoters. Promoters under developmental control include promoters that preferentially initiate transcription in certain tissues, such as xyieni, leaves, roots, or seeds. Suc promoters are examples of tissue-enhanced or tissue- preferred promoters (see US patent 7,365,186). Tissue-enhanced promoters can be found upstream and operatively linked to DNA sequences normal ly transcribed in higher levels in
s certain plant tissues or specifically in certain plant tissues, respectively. "Cell-enhanced",, "tissue-enhanced", or "tissue-specific" regulation thus refer to the control of gene or protein expression, for example, by a promoter, which dri ves expression that is not necessarily totally restricted to a single type of cell, or tissue, hu t where expressi on i s elevated in particular cells or tissues to a greater extent than in other cells or tissues within the organism, and in the case of tissue-specific regulation, in a manner that is primarily elevated in a specific tissue. Tissue- enhanced or preferred promoters have been described in, for example, US patent 7,365,186,. US patent 7,619,133, and by Noh and Ainasino, 1999, Plant Moke. Biol. 41 : 181-194. Generally, "seed-specific" promoters are transcriptionally active entirely or almost entirely in seed tissue. "Seed-preferred" or "seed-enhanced" promoters are transcriptionally active predominantly in seed tissue, but are not necessaril expressed only in seed, tissue, A seed-specific, enhanced or preferred promoter may be preferentially active during seed development and/or during germination. Examples of seed-specific promoters are found in the present Sequence Listing, in Table 3, or have been taught in, for example, US patent publication US2013030541.4 or by Qing Qu and Takaiwa, 2004. Plant B iechnol. J. 2:1 13-125). A seed-bearing-structure-enhanced promoter may be preferentially active during seed-bearing structure development.
An 'inducible promoter" initiates transcription in response to an environmental stimulus such as a. an external physical stimulus, for example, abiotic stimuli including energy or a particular chemical or class of chemicals, or a biotie stimulus, for example, a pathogen, or an interna), stimulus such as one or more markers that signal a stage of developmen Examples include "pathogen-inducible" promoters that initiate transcription in response to the presence of various pathogenic organisms or their products, and developmentally-induced promoters thai are activated when a plant or plant part is at a particular growth stage, for example, "senescence- enhanced" (also referred to as "senescence- inducible") promoters. Senescence-enhanced promoters are active late in the life cycle of a plant duri ng or near the time o f senescence (Noh and Amasino, 1 99. supra), and preferentially regulate expression of one or more genes (and any encoded polypeptides} during senescence of a plant cell from a leaf Slower, fruit, or other organ or plant part with respect to the level of expression of that gene in a non-senescing, i.e., a growing or mature (but pre-se»esce»t) cell.
In the instant description, "endogenous" refers to a molecule that naturally originates from, within a plant, plant tissue, or plant ceil. The term ""endogenous polypeptide" refers to a natural or native pol peptide that is encoded by a plant's native gene and thus it originates from within the plant, plant tissue, or plant ceil upon its translation.
A. "recombinant polynucleotide" is a. polynucleotide that is not in its native state, e.g., the polynucleotide comprises a nucleotide sequence not found in nature, or the polynucleotide is in a context other than that in which it is naturally found, e.g., separated from nucleotide sequences wit whic it typically is in proximity in nature, or adjacent (or contiguous with) nucleotide sequences with which it typically is not in proximity. For example, the sequence at issue can be cloned into a vector, or otherwise recombined with one or more additional nucleic acids. An expression vector or cassette is ao example of a "recombinant nucleic acid construct".
A. plant refers to a whole plant as well as to a plant pari, such as seed, fruit, leaf, or root, plant tissue, plant ceils or any other plant material, e.g., a plant explain, as well as to progeny thereof, and to in vitro systems that mimic biochemical or cellular components or processes in a cell.
A "recombinant polypeptide" is a polypeptide produced by translation of a recombinant polynucleotide. A "synthetic polypeptide'"5 is a polypeptide created by consecutive
polymerization of isolated amino acid residues using methods well known in the art. An
"isolated polypeptide," whether a naturally occurring or a recombinant polypeptide, is more enriched in (or out of) a cell than the polypeptide in its natural state in a wild-type cell, e.g., more than about 5% enriched, more than about 10% enriched, or more than about 20%, or more than about 50%, or more, enriched, i.e., alternatively denoted; 105%, 110%, 120%, 150% or more, enriched relative to wild type standardized at 100%. Such an enrichment is not the result; of a natural response of a wild-type plant. Alternatively, or additionally, the isolated polypeptide is separated from other ceiiular components with which it is typically associated, e.g., b an of the various protein purification methods herein.
"Conserved domains" are recurring units in molecular evolution, the extents of which can be determined by sequence and structure analysis. A "conserved domain" or "conserved region" as used herein refers to a region in heterologous polynucleotide or polypeptide sequences where there is a relatively high degree of sequence identity between the distinct sequences. Conserved domains contain conserved sequence patterns or motifs that allow for their detection in, and identification and characterization of, polypeptide sequences. A DNA-binding domain is an example of a conserved domain:.
"Identity" or "similarity" refers to sequence similarity between two polynucleotide sequences or between two polypeptide sequences, with identity being a more strict comparison. The phrases "percent identity" and "% identity" refer to the percentage of sequence similarity found in a comparison of two or more polynucleotide sequences or two or more polypeptide sequences. "Sequence similarity" refers to the percen similarity in base pair sequence (as determined by any suitable method) between two or more polynucleotide sequences. Two or more sequences can be anywhere from 0-100% similar or identical, or any integer value between 0-100%. identity or similarity can be determined by comparing a position in each sequence that may be aligned for purposes of comparison. When a position in the compared sequence is occupied by the same nucleotide base or amino acid, then the molecules are identical at that position. A degree of similarity or identity between polynucleotide sequences is a function of the number of identical, matching or corresponding nucleotides at positions shared by the polynucleotide sequences. A degree of identity of polypeptide sequences is a function of the number of identical, amino acids at corresponding positions shared b the polypeptide sequences. A degree of homology or similarity of polypeptide sequences is a function of the number of amino acids at corresponding positions shared b the polypeptide sequences. The fraction or percentage of components in common is related to the homology or identity between the sequences. An alignment may suitably be determined by means of computer programs known in the art, such as MACVECTO software, 1999 (Accelrys®, inc., San Diego, CA).
Nitrogen remob.ilization refers to the movement of nitrogenous compounds from one plant part to another, generally from senescing organs for the purpose of supplementing the nutrition of growing organs such, as new leaves and seeds. Leaf proteins, including
photosynthetic proteins of plastids, are extensively degraded during senescence. Thus, nitrogen can be remobilized from senescing leaves to expanding leaves at the vegetative stage as well as from, senescing leaves to seeds at the reproductive stage ( asclaux-Daubresse ef. al.., 2010, Ann. Bat, 1 5: 1 141 -Π 57)
A "transgenic plant" or "transformed plant" refers to a plant that contains genetic material not found in a wi ld-type plant of the same species, variety or cultivar. The genetic material, may include an expression vector or cassette, a transgene, an insertions! mutagenesis event {such as by transposes! or T-DNA insertional mutagenesis), an activation tagging sequence, a mutated sequence, a homologous recombination event or a sequence modified by chimeraplasty. Typically, the foreign genetic material has been introduced into the plant by human manipulation, but any method can be used as one of skill in the art recognizes.
A transgenic line or transgenic plant line refers to the progeny plant or plants deriving from the stable integration of heterologous genetic material into a speci fic location or locations wiihiii the genome of the original transformed cell.
An expression vector or cassette typically comprises a polypeptide-encodtng sequence operably linked (i.e., under regulatory control of) io appropriate inducible, tissue-enhanced, tissue-specific. deveiopmentaUy-enhanced, or constitutive regulatory sequences thai allow for the controlled expression of the polypeptide. The expression cassette can be introduced into a plant by transformation or by breeding after transformation of a parent plant. A plant refers to a whole plant as well as to a plant part; such as seed, fruit, leaf, or root, plant tissue, plant cells or any other plant material, e.g., a plant explant, as well as to progeny thereof, and to in vitro systems that mimic biochemical or cellular components or processes in a cell.
It i anticipated that a transgenic or transformed plant of the instant disclosure may have enhanced or greater nitrogen remobfeation relative to a control plant when the transgenic plant is transformed with a recombinant polynucleotide encoding any of the listed sequences, or when the transgenic plant contains or expresses a lis ted polypeptide, and as a consequence of the expression of the listed polypeptide within the transgenic or transformed plant
A "seed-bearing structure", as used herein, refers to a plant part that comprises a developing or mature seed, and may include, but is not limited to, an achene, berry, capsule, caryopsis or grain, ctreumcissiJe capsule, cypsela, drupe, ear, fruit or ripened pericarp, follicle, grain, kernel, legume, !ocylicidai capsule, lome tum, nut, pistil, pod, porfckla! capsule, samara, schkocarp, seed capsule, septicidal capsule, septifragal capsule, silicola, siliqua, siiique or strobilus.
The tetrn "overexpression" as used, herei n refers to a greater expression level of a gene in a plant, plant cell or plant tissue, compared to expression in a wild-type plant, cell or tissue, at any developmental or temporal stage for the gene, Overexpression can occur when, for example, the genes encoding one or more transcription factors are under the control of a strong expression signal, such as one of the promoters described herein (for example, the cauliflower mosaic virus 35S transcription initiation region), Overexpression may occur throughout a plant or in specific tissues of the plant, depending on the promoter used, as described below.
Overexpression may take place in plant cells normally lacking expression of .polypeptides functionally equivalent or identical to the present transcription factors. Overexpression may also occur in plant cells where endogenous expression of the present transcriptio factors or
functionally equivalent molecules normally occurs, but such normal expression is at a lower level. Overexpressio thus results in a greater than normal production, or "overproduction" of the transcription factor in the plant, cell or tissue.
A "control plant" as used in the present disclosure refers to la.it such as a cell, seed, plant component, plant tissue, plant organ or whole plant used to compare against an altered or experimental plant such as a transgenic or genetically modified plant for the purpose of identifying an enhanced phenotype in the altered or experimental plant. A control plant may i some cases be a transgenic plant line thai comprises an empty vector or marker gene, bat does not contain the recombinant polynucleotide of the present description that is expressed in the transgenic or genetically modified plant being evaluated, in general, a control plant is a plant of the same line or variety as the experimental or altered plant being tested. A suitable control plant would include a genetically unaltered or ndn-traasgenic plant of the parental line used t generate a transgenic plant herein.
"Wild type" or "wi -type", as used herein, refers to a plant cell, seed, plant component, plant tissue, plant organ or whole plant that has not been genetically modified or treated in an experimental sense. Wild-type cells, seed, components, tissue, organs or whole plants may be used as contols to compare levels of expression and the extent and. nature of trait modification with cells, tissue or plants of the same species in which a polypeptide's expression is altered, e.g., in that it has been knocked out, overexpressed, or eetopically expressed.
A seed-bearing structure or organ refers to a organ of a plan that comprises a seed such as, for example, achene, berry, capsule, caryopsts or grain, circumeissile capsule, cypsela, drupe, ear, fruit or ripened pericarp, follicle, grain, kernel, legume, locultcidal capsule, !omenium, nut, pistil, pod, poricidal capsule, samara, schkocarp, seed capsule, septieidai capsule, septifragal capsule, stlicuia, siliqua, silique, strobilus, etc.
The term "overexpression" as used, herein refers to a greater expression level of a gene in a plant, plant cell or plant tissue, compared to expression of that gene in a wild-type plant, cell or tissue, at any developmental or temporal stage. Overexpression can occur when, for example, the genes encoding one or more polypeptides are under the control of a strong promoter (e.g. , the cauliflower mosaic virus 35$ transcription initiation region), Overexpression may also be achieved by placing a gene of interest under the control of an inducible or tissue specific promoter, or may be achieved through, integration, of transposons or engineered T-DNA.
molecules into regulatory regions of a target gene. Other means for inducing overexpression may include making targeted changes in a gene's native promoter, e.g. through elimination of negative regulatory sequences or engineering positive regulatory sequences, though the use of targeted nuclease acti vity (such as zinc finger .nucleases or TAL effector nucleases) for genome editing. Elimination of micro-RNA binding sites in. a gene' transcript may also result in overexpression of that gene. Additionally, a gene may be overexpressed by creating an artificial transcriptional activator targeted to hind specifically to its promoter sequences, comprising an engineered sequence-specific DNA binding domain such as a z nc finger protein or TAL -effector protein fused to a. transcriptional activation domain. Thus, overexpression may occur throughout a plant, in specific tissues of the plant, or i n the presence or absence of particular environmental signals, depending on the promoter or overexpression approach, used.
Overexpression may take place in plant cells normally lacking expression of polypeptides functionally eqiiivaieni or identical to the instant polypeptides. Overexpression may also occur in plant cells where endogenous expression of the instant polypeptides or functionally equivalent molecules normally occurs, but such normal expression is at a lower level. Overexpression thus results in a greater than normal production, or "overproduction" of the polypeptide in the plant, cell or tissae.
"Yield" or "plant yield'5 refers to increased plant growth, increased crop growth, increased biomass, and/or increased plant product production (including grain:), and is dependent to some extent on temperature, plant size, organ size, planting density, light, water and nutrient availability, and how the plant copes with various stresses, such as through temperature
acclimation and water Or nutrient use efficiency. Increased or improved yield may be measured as increased seed yield, increased plant product yield (plant products include, for example, plant tissue, including ground or otherwise broken-up plant tissue, and products derived from one or more types of plant tissue), or increased vegetative yield.
DESCRIPTION OF THE SPECIFIC EMBODIMENTS
Senescence is the last stage of development of an annual (monocarpic) plant, and is an active and tightly regulated process. Recycling of nutrients, particularly nitrogen, from the leaves to the seed, during senescence is a key contributor to grain yield. (Fischer, 201 . Critical Rev. Plant Set., 31 :124-147; Bieker and Zentgraf, 2013. dx.doi.org/l0.5772/54392; DDI;
10.5772/54392).. Nitrogen remobilization is the process by which nitrogen is relocated to different plant organs at different times of development, for example, from senescing leaves to grain or seeds at the reproductive stage. Nitrogen, remobilization efficiency (NRE) may be defined as the proportion of nitrogen that is remobihzed from source or senescent leaves to siok leaves or developing grains (seeds). The efficiency of nitrogen remobilization. is controlled both by genetic and environmental factors. Genetic variability in NRE exists among crop plants. However, genetically identical plants grown on limiting nitrogen remobiifee nitrogen more efficiently from senescing leaves to seeds than when grown on ample nitrogen. Improving NRE either in limiting or ample nitrogen conditions is expected to increase the nitrogen available to the seeds and thereby increase yield.
Nitrogen remobilization occurs during leaf senescence, and any attempts to engineer improved NRE most take into account the appropriate developmental timing of this process. The .nitrogen that is remobiiized to the seeds comes largely from the breakdown of chloroplasts. If leaves senesce too early in the seed fill period, the plants will lose photosynthetic capacity and the ability to fix carbon, and therefore yield will be decreased, indeed, a mild "stay-green" phenotype, where the plants show a delay in senescence, can be beneficial, io yield. However, if senescence is delayed indefinitely, the nitrogen available to the seed will be limited and yield will be reduced. Ideally, an increase in remobilization would be timed for when senescence would normally begin, and quantitatively enhance the process. The efficiency of nitrogen remobilization may be regulated either in the senescing leaves that are the source of the nitrogen. or by signals coming from the sink (i.e. seeds) driving the developmental processes that induce senescence and nitrogen t emobilization.
Potential, strategies to improve N' 'E with the instantly listed sequences or other elade member sequences include increased expression of regulators (or effectors) of remobilization during a plant's raid to iate reproductive phase. There are three types of strategies;
1.) Increasing the expression of a positive regulator of nitrogen rernobilization in leaves only during leaf senescence. An example of this strategy is diagrammed in Figure 1, 2) Decreasing the expression of a negative regulator of nitrogen rernobilization in leaves only during leaf senescence.
3) Increasing the expression of a positive regulator of sink strength in seeds or seed- bearing-structures .
Polypeptides and Polynucleotides of the Present Description,
The present description includes increased or ectopic expression of putative regulatory polypeptides (i.e., regulators or effectors of re obiikation) and isolated or recombinant polynucleotides encoding the polypeptides, or novel sequence variant polypeptides or
polynucleotides encoding novel variants of polypeptides derived from the specific sequences provided m the Sequence Listing. The polynucleotides of the instant description ma be incorporated in expression vectors for the purpose of producing transformed, plants.
Because of their relatedness a t the nucleotide le vel, the claimed sequences will typically share at. least about 40% nucleotide sequence identity, or at least 41%, at least 42%, at least 43%, at least 44%, at least 45%, at least 46%, at least 47%, at least 48%, at least 49%, at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at. least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95% or at least 96%, at least 97%, at least 98%, at least 99%, or about 100% sequence identity to one or more of the listed sequences or to the full-length listed sequences (e.g., any of SEQ ID NO: 2n- 1 , where n:= 1 to 2300), or to a listed sequence within or outside of the region(s) encoding a known consensus sequence or consensus DNA-binding site, or to listed conserved domain sequence, or within or outside of the regionfs) encoding one or all -conserved domains. The degeneracy of the genetic code enables major variations in the nucleotide sequence of a polynucleotide while maintaining the amino acid sequence of the encoded -protein.
Because of their relatedness at the protein level, the claimed nucleotide sequences will typically encode a polypeptide that is at least at least 30%, at least 31%, at least 32%, at least 33%, at least 34%, at least 35%, at least 36%, at least 37%, at least 38%, at least 39%, at least 40%., at least 41%, at least 42%, at least 43%., at least 44%, at least 45%, at least 46%., at least 47%, at least 48%, at least 49%, at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%o, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 90%, at least 81%, at least 82%,, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%,, at least 90%, at least 91%, at least 92%,, at least 93%, at least 94%, at least 95%, or 96%, at least 97%, at least 98%, or at least 99%, or about 100% identical in its amino acid sequence to any of, or the entire length of any of, SEQ ID NOs: 2η, where n-1 to 2300, or 2, , 6, 8, or any even integer up to and including 5600.
Also provided are methods for modifying yield from a plant by enhancing the .ni trogen use efficiency or a plant's mitogen remobilixation of a plant by controlling a number of cellular processes by, for example, introducing into a target plant a gene thai encodes a polypeptide thai confers enhanced nitrogen remobilization. These methods are based on the ability to alter the expression of critical regulatory molecules that may be conserved between diverse plant species. Related conserved regulatory molecules may be originally discovered in a model system such as Ar bidopsis and homologous, functional molecules then discovered in other plant species. The latter may then be used to confer increased yield, or photosyntlietic resource use efficiency in diverse plant species.
Sequences in the Sequence Listing, derived from diverse plant species, may be ectopically expressed in overexpressor plants. The changes in the characteristic.(s) or txaii(s) of the plants may then be observed and found to confer increased yield and/or increased nitrogen use efficiency and/or nitrogen remobilization. Therefore, the polynucleotides and polypeptides can be used to improve desirable characteristics of plants.
The polynucleotides of the instant description are also ectopically expressed in
overexpressor plant cells and the changes in the expression levels of a number ofgen.es, polynucleotides, and/or proteins of the plant cells observed. Therefore, the polynucleotides and polypeptides can be used to change expression levels of genes, polynucleotides, and/or proteins of plants or plant cells.
The data presented herein represent the results obtained in experiments with
pol nucleotides and polypeptides that may be expressed in plants for the purpose of increasing yield that arises from improved nitrogen use efficiency and/or nitrogen remobilization. The polynucleotides and polypeptides of the instant description, thai, when expressed in plants or crop plant have the capacity to enhance nitrogen remobilization in a plant or a pari of a plant relative to a control plant or a corresponding part, of the control plant, include:
SEQ I'D NO:43 and 44, AT1G08320.1 (G ! 198) and clade member sequences SEQ ID NOs: 1-76;
SEQ ID NO:83 and 84, AT1G681S0.1 (G2 I.37) and clade member sequence's SEQ ID NOs: 77-98;
SEQ ID NO: 101 and 102, AT3G 1 5500.1 (G773) and ciade member sequences SEQ ID NOs: 99- 132;
SEQ I'D NO: 139 and 140, AT4G39780J (G36) and clade member sequences SEQ ID
NOs: 133-168;
SEQ ID NO: .1 9 and 380, AT 1 G7 ! 520.1 (G2133) and e!ade member sequences SEQ ID NOs: 169-192;
SEQ ID NO: 203 and 204, AT5G66870.I {04123) and clade member sequences SEQ ID NOs: 193-232;
SEQ ID NO: 265 and 264, AT4G28530.1 (G509) and clade member sequences SEQ ID NOs:233-282;
SEQ ID NO: 321 and 322, AT 1069490, 1 (GS03) and clade member sequences SEQ ID NOs: 283-358;
SEQ ID NO: 399 and 400, AT5G.151 0.1 (G2574) and. clade member sequences SEQ ID
NOs: 359-430;
SEQ ID NO: 533 and 534, AT3G 4070.1 (G2533) and clade member sequences SEQ ID NOs: 431 -554;
SEQ ID NO: 615 and 616, AT3GQ2150.2 (G2124) and clade member sequences SEQ ID NOs: 555-638;
SEQ ID NO: 661 and 662 AT2G401 10 J (G5160} and clade member sequences SEQ ID NOs: 639-702;
SEQ ID NO: 721 and 722, AT5G43170.I (G35I) nd clade member sequences SEQ ID
NOs: 703-756;
SEQ ID NO; 867 and 868, AT1G51800J and clade member sequences SEQ ID NOs;
757-976;
SEQ ID NO: 983 and 984, AT5G23750. I (G1398) and clade member sequences SEQ ID NOs: 977- 1008;
SEQ ID NO: 1 57 and 1 58, AT3G.1 910.1 (G.1 23) and clade member sequences SEQ ID NOs: 1009-1092; SEQ ID NO: 1097 and 1098 ATJG02230.1 (0517) and clade member sequences SEQ ID NOs: 1093-1100;
SEQ ID NO: 11 1.3 ami 1 1 14. AT2G28710. i (01889) and clade member sequences SEQ ID NOs: l 10.1 -1 140;
SEQ ID NO: 11 1 and 1 192, AT5G60470.1 (G30 1 ) and clade member sequences SEQ
ID NOs: 1 141 -1 1 8;
SEQ ID NO: 1 199 mid 1200, ATiG29i60.1 (02432) and clade member sequences SEQ ID NOs: 1 199-1220;
SEQ ID NO; 1331 and 1332, AT5G396I0J (G525) arid clade member sequences SEQ ID NOs: 1221-1376;
SEQ ID NO 1385 and 1386, AT2G20030J (G1 99) and clade member sequences SEQ ID NOs: 1 377-1424;
SEQ D NO: 1463 and 1464, AT3G01650. I (G3159) and clade member sequences SEQ ID NOs: 1425-1 78:
SEQ ID NO: 1631 and 1632, AT2G40180.1 and clade member sequences SEQ ID
NOs: 1579-1644;
SEQ ID NO; 1647 and 1648, AT2G261.50.1 {G266} and clade member sequences SEQ ID NOs: 1645- 1674;
SEQ ID NO: 1 83 and 1684, ATI Gl 6150.1 and clade member sequences SEQ ID NOs: 1675-1722;
SEQ ID NO: 1729 and 1730, AT5G62260.1 (GI 500) and clade member sequences SEQ ID NOs: 1723-1798;
SEQ ID NO: 1825 and 1826, AT4G38340J (G2188) and clade member sequences SEQ ID NOs: 1799-1890;
SEQ I'D NO: 1 33 and 1 34, ATIG0450D. i (G4609) and clade member sequences SEQ
ID NOs: 1891 -1958;
SEQ ID NO: 1979 and 1 80, AT2G38090.1 (G1638) and clade member sequences SEQ LD NOs: 1959-2060;
SEQ ID NO; 2075 and 2076, AT1G0549(U (G1869) aad clade member sequences SEQ ID NOs;2061 -2086;
SEQ ID NO; 2097 and 2098, ATIG02670. i (G2 18) and clade member sequences SEQ lD NOs:2087-2136;
SEQ ID NO: 2149 and 2150, AT3G02290.1 (G2275) and clade member sequences SEQ ID NOs:2137-2192; SEQ ID NO: 2199 and 2200, AT5G63750.1 (G 1920) aad ciade member sequences SEQ ID NOs: 2193-2200;
SEQ ID NO:2209 and 2210, AT3G27330.1 f G3189) and ciade member sequences SEQ ID Os: 2201-2224;
SEQ ID NO: 2225 and 2226 ATI G62370.1 (031 18) aad ciade member sequences SEQ
ID NOs: 2225-2266;
SEQ ID NO: 2299 and 2300, AT3G43230.1 and ciade member sequences SEQ ID Os:2267-2320;
SEQ ID NO; 2367 and 2368, ATI G48260, i aad ciade member sequences SEQ ID NOs: 2321-2404;
SEQ ID NO: 2495 and 2496, AT G05KXU (G5997) or SEQ ID NO: 2597 and 2498 AT2G325 10.1 ((55717) and ciade member sequences SEQ ID NOs; 2405-2534;
SEQ ED NO: 2535 and 2536, AT2G40670.2 and c ide member sequences SEQ ID NOs:2535-2556;
SEQ ID NO: 2591 and 2592, AT5G458 i 0.1 {05796} and ciade member sequences SEQ
ID NOs: 2557-2608;
SEQ ID NO; 2631 and 2632. AT3G1 360.2 and ciade member sequences SEQ ID NOs: 2609-2650:
SEQ ID NO: 2689 and 2690, AT3G63280J and ciade member sequences SEQ ID .NOs:265.1 -2744;
SEQ ID NO: 2757 and 2758, AT1G614 0.1 and SEQ ID NO: 2771 and 2772
ATI G61550.1 and ciade member sequences SEQ ID NOs;2745-2936;
SEQ ID NO: 2969 and 2970, A.T4G 1 1470.1 and SEQ ID NO: 2971 and 2972
AT4G1 1480.1 and ciade member sequences SEQ ID NOs: 2937-31 8;
SEQ ID NO: 3345 and 3346, AT5G14640. i and ciade member sequences SEQ ID
NOs:3199-3360;
SEQ ED NO: 3363 and 3364, ATiG66390J (G663) and ciade member sequences SEQ ID NOs: 3361 -3444;
SEQ ID NO; 3453 and 3454, AT5G 6840.5 (G665) and ciade member sequences SEQ ID NOs;3445-3490;
SEQ ID NO: 3511 and 3512, AT1 G28470. i (G2531 ) and c!ade member sequences SEQ ID Os;3491 -3566;
SEQ ID NO; 3583 and 3584, AT5G07680J (G523) and ciade member sequences SEQ ID NOs: 3567-3628; SEQ ID NO: 3629 and 3630, AT2G33480, 1 (G524) and clade member sequences SEQ ID NOs: 3629-3678;
SEQ I'D NO: 3695 and 3696, AT3G04060J (G765) and clade me ber sequences SEQ ID NOs: 3679-3714;
SEQ ID NO: 3725 and 3726, ATI G ! 9510.1 (02723) and clade member sequences SEQ
ID NOs:3715-3740;
SEQ ID NO: 3743 and 3744, AT2G38490.1 (06000) and clade member sequences SEQ ID NOs: 3741-3784;
SEQ ID NO; 3837 and 3838, AT5G355S0. i and clade member sequences SEQ ID NOs: 3785-3892;
SEQ 'D NO: 3927 and 3928, AT1G71830. i and clade member sequences SEQ ID Os;3893-3996;
SEQ iD NO: 4007 and 4008, AT4G32250. ! and clade member sequences SEQ ID . Os:39 7-4052;
SEQ ID NO: 4069 and 4068, AT5G 10930 (G6022) and SEQ ID NO: 4069 and 4070
ATSG25J 10.1 (G5 76) and clade member sequences SEQ ID NOs:405 -4098;
SEQ ID NO; 4111 and 41 12. AT5G59220.1 {G6094) and clade member sequences SEQ ID NOs: 4099-41 0;
SEQ ID NO: 4191 and 4192, AT5G01820.1 (G5763) and clade member sequences SEQ ID NOs: 416.1-4204:
SEQ ID NO: 4267 and 4268, AT5G02400. ! and clade member sequences SEQ ID NOs:4205-4276;
SEQ ID NO: 4277 and 4278, ATI.G22S00J (G 05) and clade member sequences SEQ ID NOs: 4277-431 6;
SEQ ID NO: 4351 and 4352, ATIG764I0. i (G22 J ) and clade member sequences SEQ
ID NQs:4317-4378;
SEQ ID NO: 4427 and 4428, AT3G49940J (G 1 12) and clade member sequences SEQ ID NOs: 4379-4454;
SEQ ID NO; 4485 and 4486, AT5G50915.1 (G5490) and clade member sequences SEQ ID NOs; 4455-4 1 0;
SEQ ID NO; 45 1.1 and 4512, AT5G42200. i (G321.7) and clade member sequences SEQ ID NOs: 4511 -4538;
SEQ ID NO: 4547 and 4548, AT3G1 1 1 10.1 (G2253) and clade member sequences SEQ ID NOs:4539-4566; SEQ ID NO: 4583 and 4584, AT2G44745.1 (G 180} and. clade member sequences SEQ ID NOs: 4567-4600;
SEQ ID NO: 4613 and 4614. AT5G0651.0J (GI 334) and clade member sequences SEQ ID NOs:460.J -465.2:
SEQ ID NO: 4685 and 4686, ATI G21240. \ and clade member sequences SEQ ID
NOs:4653-4758;
SEQ ID NO: 4769 and 4770, AT5G15500.2 (02878) and clade member sequences SEQ ID NOs:47S9-4834;
SEQ ID NO; 4887 and 4888, AT2G39 1.0, i and clade member sequences SEQ ID NOs:483S-4902;
SEQ I'D NO: 5013 and 5014, AT5G59670J and clade manber sequences SEQ ID NOs:4903-51?6;
SEQ ED NO: 5177 and 5178, AT3G55890 (G5163), and clade member sequences SEQ ID NOs: 177-5254;
SEQ ID NO: 5277 and 5278, AT5G25890 (0991) and clade member sequences SEQ ID
NOs:5255~5278;
SEQ ID NO; 5375 and 5376, A.T4G09100 (G321 1) an clade member sequences SEQ ID NOs: 5279-5460;
SEQ ED NO: 5529 and 5530, AT4G00305 (01738) and clade membe sequences SEQ ID NOs: 546.1 -5546;
SEQ ID NO: 5547 and 5548, AT5G43380 and clade member sequences SEQ ID NOs:554?-5598; and
SEQ ID NO: 5599 and 5600, AT5G14000 (G1670).
Variants of the disclosed sequences. Also within the scope of the instant description is a variant of a nucleic acid provided in the Sequence Listing, that, is, one having a sequence that differs from the one of the polynucleotide sequences in the Sequence Listing, or a
complementary sequence, that encodes a functionally equivalent polypeptide (i.e., a polypeptide .having some degree of equivalent or similar biological activity). The variant nucleic acid may, for example, encode the same polypeptide but differ in sequence from the sequence in the Sequence Listing due to degeneracy in the genetic code. Included within this definition are polymorphisms thai may or may not be readily delectable using a particular oligonucleotide probe of the polynucleotide encoding polypeptide, and improper or unexpected hybridization to allelic variants, with a locus other than the norma! chromosomal locos for the polynucleotide sequence encoding polypeptide. Differences between presently disclosed polypeptides and polypeptide variants are limited so that the sequences of the former and the latter are closely similar overall and, in many regions., identical. Presently disclosed polypeptide sequences and similar polypeptide variants may differ in amino acid sequence by one or more substitutions, additions, deletions, .fusions and truncations, which may be present in any combination. These differences may produce silent changes and result in functionally equivalent polypeptides. Thus, it will be readily appreciated by those of skill in the art, thai any of a variety of polynucleotide sequences is capable of encoding the polypeptides and homolog polypeptides of the instant description. A polypeptide sequence variant may have "conservative" changes, wherein a substituted amino acid has similar structural or chemical properties.
Conservative substitutions include substitutions in which at least one residue in the amino acid sequence has been removed and a different residue inserted in its place. Such substitutions generally are made in accordance with the Table 1 when it is desired to maintain the activity of the protein. Table 1 shows amino acids whic can be substituted for an amino acid in a protein and which are typically regarded as conservati ve substitutions.
Figure imgf000023_0001
The polypeptides provided in the Sequence Listing have a novel activity, such as, for example, a regulatory activity. Although ail conservative amino acid, substitutions (for exampi one basic amino acid substituted for another basic amino acid) in a polypeptide will not necessarily result in the polypeptide retaining its activity, it is expected that many of these conservative imitations would result in the polypeptide retaining its activity. Most mutations, conservative or non-conservative, made to a protein but outside of a conserved domain required for function and protein activity will not affect the activity of the protein to any great extent.
Deliberate amino aci d substitutions may thus be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathie nature of the resid ues, as long as s significant amount of the functional or biological activity of the
polypeptide is retained. For example, negatively charged amino acids may include aspartie acid and glutamic acid, positively charged amino acids may include lysine and arginine, and amino acids with uncharged polar head groups having similar hydropbiiicity values may include leucine, isoleucine, and valine; glycine and alanine; asparagme and giutamine; serine and threonine; and phenylalanine and tyrosine. More rarely, a variant may have "non-conservative" changes, e.g., replacement of a glycine with a tryptophan. Similar minor variations may also include amino acid deletions or insertions,, or both. Related polypeptides may comprise, for example, additions and/or deletions of one or more N-linked or O-lmked giycosylation sites, or an addition and/or a deletion of one or more cysteine residues. Guidance in determining which and how many amino acid residues may be substituted, .inserted or deleted without abolishing functional or biological activity ma be found using computer programs well known in the art, for example, DNASTA' software (see US patent 5,840,544),
Conserved domains. Conserved domains are recurring functional and/or structural units of a protein sequence within a proiein family (for example, a family of regulatory proteins), and distinct conserved domains have been used as building blocks In molecular evolution and recombiiied in various arrangements to make proteins of different protein families with different functions. Conserved domains often correspond to the 3 -dimensional domains of proteins and contain conserved, sequence patterns or motifs, which allow for their detection in polypeptide sequences with, for example, the use of a Conserved Domain Database (.for example, at www.ncbi,nlni.nih.gov cdd). The National Center for Biotechnology information Conserved Domain Database defines conserved domains as recurring units in molecular evolution, the extents of which can be determined by sequence and structure analysis. Conserved domains contain conserved sequence patterns or .motifs, which allow for their detection in polypeptide sequences (Conserved Domain Database; wvvw:ncbi.nlmj«¾.gov/Stnict«re c-dd cdd.shtml). A "conserved domain" or "conserved region" as used herein refers to a region in heterologous polynucleotide or polypeptide sequences where there is a relatively high degree of sequence identity between the distinct sequences. A 'Myb DNA binding domain 1 ' is an example of a conserved domain. Conserved domains may also be identified as regions or domains of identity to a specific consensus sequeiice (see, for example, Riechmami et al.„ 2000, Science 290, 2105-21 10;
Riechrnann et aL 2000, C rr Optn Plant Biol 3: 423-434). Thus, by using alignment methods well known, in. the art, the conserved domains of the plant polypeptides, for example, for the first or second Myb DNA binding domain proteins may be determined. The polypeptides in Table 2 have conserved domains specifically indicated by amino acid coordinate start and stop si es. A comparison of the regions of these polypeptides allows one of skill in the art (see, for example. Reeves and Nissen, 1 90. J, Biol Chem, 265, 8573-8582; Reeves and Nissen, 1995. Prog. Cell Cycle Res, I: 339-349) to .identify domains or conserved domains for any of the polypeptides listed or referred to in this disclosure.
Conserved domain models are generally identified with multiple sequence alignments of rel ated proteins spanning a v ariety of organisms. These alignments reveal sequence regions containing the same, or similar, patterns of amino acids. Multiple sequence alignments, three- dimensional structure and three-dimensional struc ture superposition of conserved domains can be used to infer sequence, structure, and functional relationships (Conserved Domain Database, supra). Since the presence of a particular conserved domain within, a polypeptide is highly correlated with, an evO.hitionari.iy conserved function, a conserved domain database may be used to identify the amino acids in a protein sequence that are piuatively involved in functions such as binding or catalysis, as mapped from conserved domain annotations to the query sequence. For example, the presence in. a protein of a DNA binding domain that, is structurally and
phyiogeneticaily similar to one or more domains found in the sequence listing would be a strong indicator of a related function in plants (e.g., the function of regulating and/or improving nitrogen use efficiency, nitrogen remobi!ization, and/or yield, i.e., a polypeptide with such a domain is expected to confer enhanced nitrogen use efficiency, nitrogen remobi ligation, and/or yiel d when its expressi on leve l is increased under the regul atory control of a senescence- enhanced promoter). Sequences herein referred to as functionally-related and/or closely-related to the sequences or domains provided in the Sequence Listing, including polypeptides thai are closely related to the polypeptides of the instant description., may have conserved domains that share at least at. least nine base pairs (bp) in length and at least 30%, at least 31%, at least 32%, at least 33%, at least 34%, at least 35%, at least 36%, at least 37%, at least 38%, at least 39%, at least 40%, at least 41%, at least 42%, at least 43%, at least 44%, at least 45%, at least 46%, at least 47%, at least 48%, at least 49%, at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 90%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95% or 96%, at least 97%, at least 98%, or at least 99%, or about 100% amino ackl sequence identity to the sequences provided in the Sequence Listing, and have similar functions in that the polypeptides of the instant description, where the presence of the listed or claimed domains in said polypeptides is positively correlated, or associated with the function(s) of said polypeptides in plants. Said polypeptides may, when their expression level is altered by enhancing their expression during senescence and to a lesser extent prior to senescence, confer at least one regulatory activity selected from the group consisting of increased nitrogen use efficiency, greater nitrogen reraobiiixation, greater yield, greater size, greater biomass, and or greater vigor as compared to a control plant.
Methods using manual alignment of sequences similar or homologous to one or more polynucleotide sequences or one or more polypeptides encoded by the polynucleotide sequences may be used to identify regions of similarity and conserved domains (e.g., DMA binding domains, activation domains, localization domains, repression domains, oiigomerization domains, or other domains, that are recognizably related across plant, species. Such manual methods are well-known of those of skill in the art and can include, for example, comparisons of tertiar structure between a polypeptide sequence encoded by a polynucleotide that comprises a known function, and a polypeptide sequence encoded by a .polynucleotide sequence that has a function not yet determined. Such examples of tertiary structure may comprise predicted - heS tees, β-sheets, amphipathic helices, leucine zipper motifs, zinc finger molife, prolme-rich regions, cysteine repeat motifs, and the like.
With respect to polynucleotides encoding presently disclosed polypeptides, a conserved domain refers to a subsequence within a polypeptide family the presence of which is correlated with at least one function exhibited by members of the polypeptide family, and which exhibits a high degree of sequence homology, such, as at least 30%, at least 31 %, at least 32%, at least 33%, at least 34%, at least 35%, at least 36%, at least 37%, at least 38%, at least 39%, at least 40%, at least 41%, at least 42%, at least 43%, at least 44%, at least 45%, at least 46%, at least 47%, at least 48%, at least 49%, at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 90%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95% or 96%, at least 97%, at least 98%, or at least 99%, or about 100% identity to a conserved domain (e.g., any of SEQ ID NOs: 5601-5723} of a polypeptide (e.g., any of SEQ ID NOs: 2u, where n^I to 2300) of the Sequence Listing. Sequences that possess or encode for conserved domains thai meet these criteria of percentage identity, and that have comparable biological and regulatory activity to the instant polypeptide sequences. Sequences having lesser degrees of identity hut comparable biological activity are considered to be equivalents.
Table 2 lists Afabidopsis sequence identifiers and the descriptions .of various domains found within the respective proteins, including the domain names, the linear coordinates of the amino acids at the beginning and end of the respective domains, and the SEQ ID NOs: of the domains listed in this table. It is expected that domains of clade member polypeptides of each of these sequences, examples of said polypeptides being provided in the Sequence Listing, function similarly in plants and their presence is indicative of and correlated with t he same functions as the domains in the Arabidopsis sequences. Table 2, Various Arabidopsis domains found in polypeptide sequences that enhance nitrogen remobilization in plants
Figure imgf000027_0001
AT2G33480
MA 15 137 5608 . ?
AT2G40670 Response regulator receiver
32 160 5609 domain
AT2G2 150
HSF D A-bi»ding domain 44 136 5610
J
Serine/threonine phosphatases,
AT2G40J 80
family 2C, catalytic domain 128 373 5611 .1
(PP2Cc)
Protein S erine/ Threonine
Kinase, Mitogen-Activated
AT2G325I0
Protein Kinase Kinase Ki nase 2 259 5612 .1
catalytic domain
(STKc_MAPK K)
AT2G3849G Serine/Threonine protein kinase
52 306 5613 J catalytic domain (S TKc)
AT2G38490 C-ienninal regulatory domain
333 445 5614 .1 of ClPKs (CIPK. C)
AT4GOG3Q5
R1 G/C3HC4 zinc finger 71 1 17 5615
J
AT4G39780
AP2 91 149 5616
. ]
AT4GO 10O
RING C3HC4 zinc finger 84 130 5617 .1
AT4G28530
NAM 10 154 5618 .1
AT4G11480
Salt stress reSpoose/antifiragal 23 126 5619
.1
AT4G11480
Salt stress response/antifungal 142 236 5620
J
AT4G11480 protein kinase catalytic
324 601 5621 .1 (STYKc) domain
AT4G38340
PB1 NLP 674 758 5622 .1
Figure imgf000029_0001
Figure imgf000030_0001
Figure imgf000031_0001
AT1G02670 Heiicase supei amiiy e-terminal
502 623 5671 Λ domain (HELICc)
AT3G04060
NAM 20 147 5672 .1
AT3G12910
NAM 22 151 5673 .1
AT3G02150
TCP 71 197 5674 .2
AT3G04070
NAM 10 141 5675 .1
AT3G27330
Giycosykransferase family 92 276 481 5676
Λ
AT3G27330
R1NG/C3HC4 zinc finger 724 766 5677 Λ
AT3G016S0 Yon W'illebrand factor type A
133 383 5678 .1 (vW A) superfamiiy domain
AT3G01650
RING/C3HC4 zinc finger 446 482 5679
J
AT3G i m <>
RING linger 109 152 5680 .1
AT3GO2290
RING/G3HC4 zinc finger 181 221 5681 Λ
AT3GH 6360 histi.dine-containi.ng
39 122 5682
.2 phosphotransfer (HPt) domain
AT3G49940 domain of unknown functio
2 107 5683 .1 (DUF260)
Catalytic domain of the
Serine/Threonine Kinases
AT3G63280
(ST s), Never in Mitosis gene 3 258 5684 .3
A f lMA}-re1aied kinase (Nek)
family (STKc Nek)
AT3G558 0
Yippee 1 108 5685 .1
AT3G15500 NAM 14 140 5686
Figure imgf000033_0001
PP1,PP L(PP1 and kcich-
AT5G43380 like) enzymes, and related
6 296 5703
J proteins, metallophosphaiase
domain (MPP PP 1 PPKL)
AT5G25890 AlJX/iAA family domain
47 170 5704
.1 (AUX IAA)
AGC family Protein
AT5G45810
Serine/Threonine Kinase 34 282 5705
.1
catal tic domain (ST c^AGC)
AT5G45810 Oterminal regulatory domain
346 458 5706 .1 ofCIPKs (CIPK C)
AT5G56840
SANT 92 137 5707 .1
AT5G15130
WRKY 227 284 5708 .1
AT5G42200
RING/C3HC4 zinc finger 103 149 5709
Λ
AT5G63750
RING finger (zf-RI G_2) 86 123 5710 J
AT5G63750 In Between Ring fingers (IBR)
158 230 5711 .1 domain
AT5G5D915
Helix-loop-heiix domain (HLH) 140 197 5712
J
AGC family Protein
AT5.G25H
Serine/Threonine Kinase 49 297 5713 .1
catalytic domain (STKc AGC)
AT5G25110 C-terminal regulatory domain
342 455 5714
.1 ofCiPKs(CIP .C)
Serine/threonine phosphatases,
AT5G59220
family 2C, catalytic domain. 111 349 5715 J
(PP2CC)
ATSG14000
NAM 15 141 5716 .}
AT5G59670 Malectmjike 31 354 5717
Figure imgf000035_0001
Qrtbplo us and Paralogs , Homologous sequences as described above can comprise ort o!ogoiis or paralogous sequences. Several different methods are know by those of skill in the art for identifying and defining these fractionally homologous sequences. General methods for identifying orthologs and paralogs, including pliyiogeneiic .methods, sequence similarity and 'hybridization methods, are described herein; an ortlio!og orparaiog, including equivalogs, may be identified by one or more of the methods described below.
As described by Eisen, 1998. Genome Res. 8: 163-167, evolutionary information may be used to predict gene function. It is common for groups of genes that are homologous in sequence to have diverse, although usually related, functions. However, in many cases, the identification o homologs is not sufficient to make specific predictions because not all homologs have the same function. Thus, an initial analysis of functional, elatedness based on sequence similarity alone may not provide one with a means to determine where similarity ends and functional relatedness begins. Fortunately, it is well known in the art that protein function can be classified using phySogeneiic analysis of gene trees combined with the corresponding species. Functional predictions can be greatly improved by focusing on how the genes became similar in sequence, (i.e., by evolutionary processes) rather than on the sequence similarity itself (Eisen, supra), in fact, many specific examples exist in which gene function has been shown to correlate well with gene phy!ogeny (Eisen, supra). Thus, "(tjhe first step in. making functional predictions is the generation of a phyioge etic tree representing the evolutionary history of the gene of interest and its homologs. Such trees are distinct from clusters and other means of characterizing sequence similarity because (hey are inferred by techniques that help convert patterns of similarity into evolutionary relationships After the gene tree is inferred, biologically determined functions of the various homologs are overlaid onto the tree. Finally, the structure of the tree and the relative p'hylogenetic positions of genes of different functions are used to trace the history of functional changes, which is then used to predict functions of [as yet) uncharaeterized genes" (Fisen, . supra) .
Within a single plant species, gene duplication may cause two copies of a particular gene, giving rise to two or more genes with similar sequence and often similar function 'known as paralogs. A paralog is therefore a similar gene formed by duplication within the same species. Paralogs typically cluster together or in the same clade (a group of similar genes) when a gene family phytogeny is analyzed- using programs such as CLUSTAL (Thompson, et al., 1994, Nucleic Acids Res. 22: 673-4680; Higgins et a!., ! 996, Methods, Emy oL 266; 383-402).
Groups of similar genes can also be identified with pair- ise BLAST analysis (Feng and
.Dooltttie, 1987, J. Mol Evol. 25: 351-360). For example, a clade of very similar MADS domain transcription factors from Ar hfdopsis all share a common function in flowering time ( atcliffe et al, 2001, Plant Physiol. 126: 122-132), and a group of very similar AP2 domain transcription factors from Arabidop are involved in tolerance of plants to freezing (Gilmour et al, 1998, supra). Analysis of groups of similar genes with similar function that fall within one clade can yield sub-sequences that .are particular to the clade. These sub-sequences, known as consensus sequences, can not. only be used to define the sequences within each clade, but define the functions of these genes; genes within a clade may contain paralogous sequences, or ortho!ogous sequences that share the same function (see also, for example, Mount, 2001 , in Bioinformatics: Sequence and Genome Analysis. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, p. 543)
Regulatory polypeptide gene sequences are conserved across diverse eukaryotic species lines (Goodnch elai, 1 93. Cell 75:519-530; Lin et al, 1991 , Nature 353:569-571 ; Sadowski et al, 1 88, Nature 335: 563-564), Plants are no exception to this observation; diverse plant species possess regulatory polypeptides that have simitar sequences and functions. Speciadon, the production of new species from a parental species, gives rise to two or more genes with similar sequence and similar function. These genes, termed orthologs, often have an identical function within their host plants and are. often interchangeable between species without losing function. Because plants have common ancestors, many genes in any plant species will have a
corresponding orth'ologous gene in another plant species. Once a phylogentc tree lor a gene family of one species has been constructed using a program such as CLUSTAL (Thompson et al., 1994, supra; Higgins et al., 1996, supra) potential ormologous sequences can be placed into 'the phylogenetic free and their relationship to genes from the species of interest can be
determined. Orthologous sequences can also be identified by a reciprocal BLAST strategy. Onc an orthologous sequence has been identified, the function of the orthoiog can be deduced from the identified function of the reference sequence.
The polypeptides sequences belon to distinct clades of polypeptides that include members from diverse species. In each case, most or ah of the c!ade member sequences derived from both eudicots and monocots have been shown and are predicted to enhance nitrogen remobilkation to confer increased yield when the sequences were overexpressed under the regulatory control of lor example, a senescence-enhanced promoter, a seed-enhanced promoter, a seed-bearing structure enhanced promoter, or a green tissue promoter. These studies each demonstrate that evohttionari!y conserved genes from diverse species are likely to function similarly (i.e., by regulating similar target sequences and controlling the same traits), and that polynucleotides irom one species may be transformed into closely-related or distantly-related plant species to confer or improve traits.
Orthologs and paralogs of presentl disclosed polypeptides may be cloned using compositions provided by the present description according to methods well known in the art. cDNAs can be cloned using rtiR A from a plant cell or tissue that expresses one of the presen sequences. Appropriate mR'NA sources may be identified by interrogating Northern blots with probes designed from the present sequences, after which a library is prepared from the niRNA obtained irom a positive ceil, or tissue. Polypep kle-eucodmg cDNA is then isolated using, for example, PCR, using primers designed from a presently disclosed gene sequence, or by probing with a partial or complete cDNA or with one or more sets of degenerate probes based on the disclosed sequences. The cDNA library may be used to transform plant cells. Expression of the cDNAs of interest is detected using, for example, mieroarrays, .Northern blots, quantitative PCR, or any other technique for monitoring changes in expression. Genomic clones may be isolated using similar techniques to those.
Examples of orthologs of the Ambidopsis polypeptide sequences and their functionally similar orthologs are listed, in the present Sequence Listing. In additio to the Amhidop polypeptide sequences in the Sequence Listing, these orthologs are phyiogeneticalty and structurally similar to the sequences in the Sequence Listing and can also function in a plant by increasing nitrogen use efficiency, nitrogen reruobili ation, yield., vigor, and/or biornass when ectopically and preferentially expressed during senescence of a plant or in a plant organ. Since a significant number of these sequences are phylogenetically and sequentially related to each other and may be shown to increase yield from a plant, and/or nitrogen remobilization, one skilled in the art would predict that other similar, phylogenetically related sequences, including those 'falling within the present clades of polypeptides or having the same consensus sequences or which are sequentially similar, having a disclosed .minimum percentage identity to one another or the listed A bkiop. polypeptide, would also perform similar functions when ectopkaliy expressed under the regulatory control of the disclosed promoters or oth er senescence-enhanced promoters , seed-enhanced promoters, seed-bearing structure enhanced promoters, or green tissue promoters.
Promoters, The instant senescence-enhanced promoters, which may be found in Table 3 may be developmental^ regulated in that transcription is initiated at the promoter site primarily during senescence, and the promoters are relatively insensitive to the concentration of nitrogen in the plant or its environment. Thus, the instant senescence-enhanced promoters preferentially initiate gene expression at the onset of senescence and/or during plant senescence and can be used to regulate the expression of extrinsic genes in various 'plant or crop organs as the plants or organs enter and experience senescence, 'without affecting me normal (i. e. , wild-type or control- like) development -of the plants at the seedling or at a presenescem mature- stage. Examples of senescence-enhanced promoters may be found in the' Sequence Listing as SEQ ID NOs: 5724 to 5741.
The seed-enhanced promoters and seed-bearing, structure enhanced promoters i Table 3 regulate gene expression in a manner that is enhanced in seeds or seed-bearing structures, respectively, relative to other tissues in a plant. Examples of seed-enhanced promoters may also be found in the Sequence listing as SEQ ID NOs; 5742 to 5830.
The choice of promoter may also include a constitutive promoter or a promoter with enhanced acti vity in a tissue capable of photosynthesis (also referred to herein as a "green tissue promoter", a "photosynihetic promoter" or a "photosynthetic tissue-enhanced promoter") such as a leaf tissue or other green tissue. Examples of photosynthefic or green, tissue promoters include for example, an RBCS3 promoter (SEQ ID NO: 5831), an RSCS4 promoter (SEQ ID NO: 5832) others such as the At4g01060 promoter (SEQ ID NO: 5833), the latter regulating expression in a guard cell, or rice sequences SEQ ID NOs: 5834 to 5857, shown in Table 3 or in the Sequence Listing.
Table 3.. Exeni iary promo ters
Figure imgf000038_0001
prAT3G25240 Senescence-enhanced 5727 prAT2G 11810 Senescence-enhanced 5728 prAT3G52820 Senescence-enhanced 5729 prAT2G3421(> Senescence-enhanced 5730 prAT3G04530 Senescence-enhanced 5731 prAT2G4 5 Senescence-enhanced 5732 prAT4G32810 Senescence-enhanced 5733 prATl G231 10 Senescence-enhanced 5734 prATl 0(58310 Senescence-enhanced 5735 prAT'2G29470 Senescence-enhanced 5736 prATSG 11210 Senescence-enhanced 5737 prAT5G67310 Senescence-enhanced 5738 prATIG 19200 Senescence-enhanced 5739 prAT] G62760 Senescence-enhanced 5740 prAT3G44510 Senescence-enhanced 5741 prAT5G45890 Seed-enhanced 5742 prAT3 G03880 Seed-enhaneed 5743 prATi G03890 Seed-enhanced 5744 prATlG04330 Seed-enhanced 5745 prATlG17810 Seed-enhanced 5746 prATl G27990 Seed-enhanced 5747 pr T.! G28640 Seed-enhanced 5748 prATlG47540 Seed-enhanced 5749 prATlG4S130 Seed-enhanced 5750 prATlG48470 Seed-enhanced 5751 prAT 1053690 Seed-enhanced 5752 pfAT] G54860 Seed-enhanced 5753 prATiG65090 Seed-enhanced 5754 prAT.? G658 0 Seed-enhanced 5755 prATlG67lOG Seed-enhanced 5756 prAT! 068510 Seed-enhanced 5757 prAT 1 G716 1 Seed-enhanced 5758 prATlG72100 Seed-enhanced 5759 prATl G731 0 Seed-enhanced 5760 prAT.lG77950 Seed-enhanced 5761 prATlG78500 Seed-enhanced 5762 prAT2G05580 Seed-enhanced 5763 prAT2G 14520 Seed-enhanced 5764 prAT2G 18540 Seed-enhanced 5765 prAT2G 19320 Seed-enhanced 5766 prAT2G23230 Seed-enhanced 5767 prAT2G23640 Seed-enhanced 5768 prAT2G25890 Seed-enhanced 5769 prAl'2G28490 Seed-enhanced 5770 prAT2G3 810 Seed-enhanced 5771 prAT2G34700 Seed-enhanced
prAT2G47120 Seed-enhanced
prAT3G0i S70 Seed-enhanced 5774 prAT3G 12060 Seed-enhanced 5775 prAT3G 12203 Seed-enhanced 5776 prAT3G 18570 Seed-enhanced 5777 prAT3Gl9920 Seed-enhanced 5778 prAT3G22640 Seed-enhanced 5779 prAT3G24250 Seed-enhanced 5780 prAT3G24340 Seed-enhanced 5781 prAT3G24650 Seed-enhanced 5782 prAT3G26 20 Seed-enhanced 5783 prAT3G27660 Seed-enhanced 5784 prAT3G29300 Seed-enhanced 5785 prAT3G44460 Seed-enhanced 5786 prA'T3G5i 200 Seed-enhanced 5787 prAT3G54 40 Seed-enhanced 5788 prAT3G60730 Seed-enhanced 5789 prAT3G63040 Seed-enhanced 5790 prAT4G03050 Seed-enhanced 5791 prAT4G09940 Seed-enhanced 5792 prAT4G 10020 Seed-enhanced 5793 prAT4G12130 Seed-enhanced 5794 prAT4G1(SI60 Seed-enhanced 5795 prAT4G2!020 Seed-enhanced 5796 prAT4G25140 Seed-enhanced 5797 prAT4G26740 Seed-enhanced 5798 prAT4G271 0 Seed-enhanced 5799 prAT4G27I50 Seed-enhanced 5800 prAT4G27l60 Seed-enhanced 5801 prAT4G27170 Seed-enhanced 5802 prAT4G2S36S Seed-enhanced 5803 prAl'4G3f.6S0 Seed-enhanced 5804 prAT4G32490 Seed-enhanced 5805 prAT4G3328C> Seed-enhanced 5806 prAT4G34520 Seed-enhanced 5807 pfAT4G3572S Seed-enhanced 5808 prAT4G36700 Seed-enhanced 5809 prAT5G071 O Seed-enhanced 581 prAT5G07200 Seed-enhanced 581 1 prAT5G08460 Seed-enhanced 5812 prAT5GI3790 Seed-enhanced 5813 prATSG 16460 Seed-enhanced 5814 prAT5G21 150 Seed-enhanced 5815 prAT5G228J0 Seed-enhanced 5816 prAT5G24l30 Seed-enhanced 5817 prAT5G27200 Seed-enhanced 5818 prAT5G38 l60 Seed-enhanced 5819 prAT5G38170 Seed-enhanced 5820 prAT5G40420 Seed-enhanced 5821 prAT5G44120 Seed-enhanced 5822 prAT5G4583() Seed-enhanced 5823 prAT5G4767Q Seed-enhanced 5824 prAT5G4 1 0 Seed-enhanced 5825 prAT5G50770 Seed-enhanced 5826 prAT5G5_210 Seed-enhanced 5827 prAT5G55240 Seed-enhanced 5828 ■prAT5G57810 Seed-enhanced 5829 prAT5G62850 Seed-enhanced 5830
Green tissue- pr BCS3 5831 enhanc d
Green tissue- prR.BCS 5832 enhaueed
Green tissue- prAt4g()]060 5833 enhanced
Green tissue-
Os02g0 720 5834 enhanced
Green tissue-
Os05g:34510 5835 enhanced
Green tissue-
Osl 1 g08230 5836 enhanced
Green tissue-
Os01g64390 5837 enhanced
Green tissue-
Os06gi 5760 5838 enhanced
Green tissue-
Gsi2g3756G 5839 enhanced
Green tissue-
Os03g 57420 5840 enhanced
Green tissue-
Os04g5l000 5841 enhanced
Green tissue-
OsOl §01960 5842 enhanced
Green tissue-
Os05gC)499D 5843 enhanced
Green tissue-
OsO2g44970 5844 enhanced
Gree tissue-
Os01g25S3'0 5845 enhanced
Green tissue-
Os03g30650 5846 enhanced Green tissue-
Os01g64 1 5847
enhanced
Green tissue-
Os0?g268i0 5848
enhanced
Green tissue-
Os07g2 82D 5849
enhanced
Green tissue-
Os09gU220 5850
enhanced
Green tissue-
€>s04g2 i80D 5851
enhanced
Green tissue-
Osl0g23840 5852
enhanced
Green tissue-
Os08gi385 5853
enhanced
Green tissue-
Osl2g42980 5854
enhaneed
Green tissue-
Os03g2928O 5855
enhanced
Green tissue-
Os03g206S0 5856
enhanced
Green tissue-
Os06g:43920 5857
enhanced
EXAMPLES
It is to be understood that this description 5s not limited to the particular devices, machines, materials and methods described. Although particular embodiments are described, equivalent: embodiments ma be used to practice the claims.
The specification, now being generally described, will be more readily understood by reference to the following examples, which are included merely for purposes of illustration of certain aspects and embodiments of the present description and are not. intended to limit the claims or description. It will be recognized by one of skill in the art that -a polypeptide that is associated with a particular first trait may also be associated with at least one other, unrelated and inherent second trait which was not predicted by the first trait.
Example Ϊ. Identification of regulators of remobillataiion through gene expression profiting Plants grown under limiting nitrogen conditions remobilize a greater fraction of the nitrogen in their vegetative organs to the seed than do plants grown under ample nitrogen. To elucidate the regulatory networks controlling these differences, a transcriptional profiling experiment, was performed on Ar hidopsis plants grown under conditions of limiting and ample nitrogen. Leaf and silique tissue were harvested beginning in the vegetative phase and continuing through seed development to create a developmental time series under these two different nutrient regimes.
Plant growth and tissue isolation. Plants were grown in pots containing three volumes of fritted clay at bottom and one vol ume of .fine sand on top. Pots were pre-soaked in nutrient solution containing either 2 ra (low N) or 10 mM (high N) nitrate solution. Phosphate (0.25 mM), sulfate (0.25 raM), magnesium (0.25 .mM), and sodium (0.20 aiM) were present in both solutions at the same concentration. The. difference between low and high N solutions affects only potassium (5.25 raM and 2.75 raM in high and low N solutions, respectively), calcium (2,50 mM and 0.50 mM, respectively), and chloride ions (0.25 mM and OJO raM, respectively). Pots were placed in Conviron growth chambers at a. day temperature of 22°C (19°C night) with a 1 bx photoperiod at an initial light intensity of-ΊΟΟ μιηοί m-2 s- 1 and a final Sight intensity of •--140 prnol m-2 s-1 at plant, height. After two weeks, flats were moved io a commercial ebb and flow hydroponic system (Bigfoot, American Hydroponics, Areata, CA) in an Enconair growth chamber with a i 6 hr photoperiod at a light intensity of 140 pmoi m-2 s-1 at plant height, and a 22eC day temperature (19X night). Plants were supplied with nutrient solution containing 2 mM or !OmM nitrate through a pumping system once every 8 hrs.
Leaf 7 (the seventh leaf formed by each plant) was tagged with thread 21 days after sowing ("days after sowing" abbreviation; DAS). Collection of leaf 7 was started at 22 DAS and continued every other day until 42 DAS, for a total of 1 1 time points. Sampling of siiiques started when siliques reached stage 1 (floral organs withering) and 2, , 6, and 8 days post stage 1 , for a total of five time points. At each time point,, leaf 7 and siliques were harvested from 10- 1.2 plants and eight plants, respectively, with plants being selected to minimize any potential effects of position wi thin, the hydroponic tub and growth room.
RNA isolation. Leaves were pulverized in liquid nitrogen with a mortar and pestle.
Approximately 100 pi of frozen tissue was combined with buffer RA1 with 1 % β- mercapioethanol (NucieoSpin® 96 RNA, Macherey- geL Bethlehem, PA), and total RNA. was isolated according to the manufacturer's specifications and implemented as a custom method on a Biomek FXP (Beckman Coulter, Brea, CA) liquid handling workstation. In some eases, RNA yields were too low to satisfy the target of 2 pg for making adapter libraries. In these cases a second aliquot of 100 pL tissue was taken to perform an additional RNA extraction. RNA from 'the second extraction was used if the yield was sufficient Otherwise, RNA was .pooled from both extractions.
Silique RNA was extracted following a modified protocol I'Meng and Feldinan., 2010, BiotechnoL J. 5: 1 3-186). This procedure employs a modified, high. pH (pH 9.5) extraction. buffer. An RNAeasy kit (Quiagen, Hilden, Germany} was subsequently used to purif the RNA.
Sequence Library Creation . The starting total. RNA. concentration was measured using a NanoDrop Spectrophotometer (Thermo Scientific, W'al hani, MA) and 2 μ% of total RNA was used as the entry point to the TraSeq RNA. Sample Prep method {1 008136 A, Illumina#, San Diego, CA). AH steps were performed according to manufacturer specifications as indicated in the High-Throughput (HT) Protocol but implemented as a custom method on a Biomek FX1' liquid handling workstation. In brief, poly-A containing mRNA molecules were purified using poly-T oligo-atlaehed. magnetic beads (2-ronnds), fragmented, with, heat to a size of 120-200 bp, and then reverse transcribed using random hexaraer primers. Double stranded cDNA was produced and indexed adapters were attached by ligation (illumina). Library size distribution was assessed by capillary gel electrophoresis, and then normalized based, on quantification based o absorbaace, fluorescence and quantitative polymerase chain reaction (qPCR) using primers targeting the adapter sequences.
Sequencing. Normalized libraries were pooled proportionally based on the results of the RT-PCR quantification and prepared for sequencing. Cluster formation on the floweelf was performed with the lllumina Cluster Station system and the TruSeq SR Cluster Kit v5 (Illumina). Flowcells were sequenced on the GA'l'lx system using TmSeq SBS Kit v5 (illumina) to produce single end 29bp reads, plus a 7~eycle index read. At least. 15 million reads per sample was acquired.
Data processing.. Output from the GAlix was pre-processed using Oiumina's CASAVA software v 1 .8.1 to produce one file of short-read basecail. profiles for each sample. Short 'reads in basecall profiles were aligned, to transcript elements (Ν=41 71) in the genomic reference Arohidopsis ttanscriptome (TAiR i.0_G.FF3_genes.gff) using the TopHat program v.2.0.4 with def ult parameters. Resulting TopHat alignment, profiles were converted, to SAM format using SAMTools vO, 1.18. For each sample, absolute expression profiles were quantified using HTSeq v0.5,3p to produce counts for each "gene", aggregating across all aligned transcripts of the gene as structurally defined in the OFF. HTSeq was run in "union" mode in non-strand specific- fashion. Gene expression profiles were then analyzed with EdgeR. v3.0 to account for batch, and treatment effects. Only genes that had more than two reads per million, total reads in three or more sample were modeled.. Contrasts were created to compare the combiaatioas of experimental factors against appropriate controls using a log-likelihood ratio test with p-values calculated using a χ2 distribution. Since these analyses produce results using many statistical tests, a Befijamini- Hochberg false discovery rate (FDR) multiple test correction was applied to p* values withi -each profile. To be considered differentially expressed between experimental conditions, a given gene was required to show at least a 1 .3 fold difference of expression with a statistical significance of p-FDR < .05.
Selection of genes. The transcript profiles of leaves at each of the 1 1 time points (22, 24, 26, 28, 30, 32, 34, 36, 38, 40 and 42 DAS) and silique (stage 16, 2, , 6 and S days post stage 16) were obtained, comparisons were made (i) between plants supplied with low and high nitrate at matched time point and (it) against the initial time poin (22 DAS and stage 1 6 for leaf and. siiique, respecti el ). The expression profiles of the low N and high N grown plants were relatively similar until 30 DAS, corresponding to the time when seed filling began, when a large number of genes began to be differentially expressed in low N conditions and visual evidence of senescence initiation was first noted in leaf 7 in low . The resulting data were analyzed through a number of computational approaches, e.g., Spline-Cluster (Heard et a!., 2006, </. Am. Statist, Assoc. 101 : 18-29: Heard 201 1 , J. Camput Graph. Stai. 20: 920-936, Comet
(cornet.psb.ugent.be X Gene ontology analysis, and various network inference approaches) to identify potential regulators of nitrogen immobiliza tion. Differentially expressed regulatory genes (transcription factors, kinases, phosphatases, RING uhiquitin hgases) were also individually examined. Three t pes of genes were selected for experimental analysts:
1 ) Regulatory genes consistently more highly expressed in leaves of plants grown in low N than in leaves of plants grow in high N beginning 30 DAS or later. These are candidate genes for up-regulation during senescence. An example of such an expression pattern is shown in Figure I B.
2) Regulatory genes consistently more highly expressed in leaves of plants grown in high N than in leaves of plants grown in low N beginning 30 DAS or later. These are candidate genes for down-regtilaiion during leaf senescence.
3) Regulatory genes more highly expressed in siliques grown in low N at any time point. These are candidates, for up-regulation. in seeds or seed-bearing-struciures.
Example 11. Identification of developmental!} regulated promoters
The transcriptional profiling d ta described above in Example i was also mined to identify promoters that are expressed (enhanced) during leaf senescence. The criteria, for selection of these promoters were as follows; ♦ Induced during senescence in leaves, with low expression initially. Promoters were chosen ra three groups: induced before 30 DAS (pre-senescenc-e), induced at 30 DAS (at senescence onset) and induced after 30 DAS (late senescence).
• Ideally a similar expression pattern in both low N and high N. Genes that showed a
quantitative difference between expression in low N and high N„ but similar expression kinetics, were also considered.
* Low expressio in other tissues (roots, seeds etc.).
An example expression pattern of such a promoter is shown in Figure 1A.
SplineC!uster analysis (Heard et al, 2006. supra: Heard, 201 1 , supra) was performed using the gene set from the low N condition to identify groups of genes that were similarly regulated. Clusters with the desired regulatory patterns were identified, then genes in these clusters were compared with public and proprietary data sets to eliminate those thai showed strong expression in tissues other than senescing leaves or induction during stress conditions. Exemplary putative regulatory sequences for these genes were identified and are listed as SEQ ID NO: 5724-574.1 , although it is anticipated that other senescence-enhanced or senescence- induced promoters may function in a similar manner. In. addition to these sequences, a promoter region may include a fragment of the promoter sequences provided in the Sequence Listing or in this Example, or a complement thereof wherein the promoter sequence, or the fragment thereof or the complement thereof, regulates expression of a polypeptide in a plant cell for example, in a manner that is enhanced or preferred durin certain periods of development, e.g. , in senescing plant cells and tissues.
Example III. Identification of seed-enhanced and seed-bearing-structure-enhanced promoters.
Genes with enhanced expression in seed and siiique were identified from our dataset, and compared to a number of public and proprietary datasets to prioritize -strongly seed-enhanced genes and eliminate those that showed stron expression in tissues other than seed or siiique, or induction during stress conditions. Exemplary putative regulatory sequences for these genes were identified and are listed as SEQ I'D NO; 5742-5830, although, it is anticipated that other senescence-enhanced or -induced promoters may function in a similar manner. In addition to these sequences, a promoter region may include a fragment of the promoter sequences provided in the Sequence Listing or in this Example, or a complement, thereof, wherein the promoter sequence, or the fragment thereof or the complement thereof, regulates expression of a polypeptide in a plant cell, for example, in a manner that is enhanced or preferred in certain plant tissues, e.g. , seed tissues or the tissues of seed-bearing structures. Example IV. Green-tissue enhanced promoters
Green tissue-enhanced promoters thai may be used io d ri ve expression of polynucleotides and polypeptides found in the Sequence Listing and stroetarally and functionally-related sequences have also been described in U.S. patent publication no. 201 J i).S 7952 A L incorporated 'herein by reference. Such promoters include SEQ ID NOs: 5831-5857. In. addition to these sequences, a promoter region, may include a .fragment of the promoter sequences provided in the Sequence Listing or in this E ample, or a complement thereof, wherein the promoter sequence, or the fragment thereof, or the complement thereof regulates expression of a polypeptide in a plant cell, for example, in response to a biotic or abiotic stress, or in a manner that i enhanced or preferred in certain plant tissues, e.g., green or photosynihetic tissues.
Example V. Production of transgenic plants
The above-identified promoters and regulatory genes may be used to create constructs to transform plants. Transformed plant may be prepared using the following methods, although these examples are not intended to limit the description or claims.
Promoter cloning. For genes showing appropriate patterns of regulation, typically approximately 1.2 kb of upstream sequence are cloned by polymerase chain reaction (unless litis region contains another gene, in which case the upstream sequence up to the next gene is cloned). Each promoter is cloned into a nucleic acid construct (e.g., an expression vector or cassette) in front of either a polynucleotide encoding green fluorescent protein (G.FP) or another marker of gene expression, or i front of a polynucleotide encoding a polypeptide or a regulatory molecule of interest, for example, a polypeptide found in. the Sequence listing, such as SEQ ID NOs: 2n, where n::: .l to 2 00, among others, in some instances the promoter may be used to regulate the expression of a polynucleotide that is expected to cause beneficial traits by reducing or eliminating the activity of a target gene or group of genes through, antisense or A.i based approaches. The promoter may also be incorporated into antisense or RNAt constructs which target genes encoding homologs of the transcription factors.
Transformation. Transformation of Ambidop is is typically performed by an
Agrobacterium-m.edhw . protocol based on the method of Bechiold and Pelletier, 1.998, Methods Mol Biol. 82:259-266.
P a t preparati n . Arahidopsis seeds are sown on. mesh covered pots. The seedlings axe thinned so that 6- 10 evenly spaced plants remain on each pot .10 days after planting. The primary bolts are cut off a week before trans omia tion. to break apical, dominance and encourage axillary shoots to form. Transformation is t pically performed, at 4-5 weeks after sowing. Bacterial culture preparation, AgrobacteriuM stocks are inoculated from single colony plates or from glycerol stocks and grown with the appropriate antibiotics and grown until saturation. On the morning of transformation,, the saturated cultures are cenirifuged and bacterial pellets are re-suspended in. infiltration Media ( .5X MS, I X B5 Vitamins, 5% sucrose, 1 mg ml be»2yiarai»opurine riboside, 200 ul/L Siiwet L77) until a A600 reading of 0.8 is reached.
Transf formation and seed harvest. The Agrobticteriwn solution is poured into dipping containers. All flower birds and rosette leaves of the plants are immersed in this solution for 30 seconds. The plants are laid on iheir side and wrapped to keep the humidity high. The plants are kept this way overnight at 22 °C and then the pots are unwrapped, turned upright,, and moved to the growth racks.
The plants are maintained, on the growth rack under 24-hour light, until seeds are ready to be harvested. .Seeds are harvested when 80% of the siliques of the transformed plants are ripe (approximately five weeks after the initial transformation). This seed is deemed TO seed, since it is obtained from the TO generation, and is later plated on selection plates (kanam cin.,
sulfonamide or glyphosate). Resistant plants that are identified on such selection plates comprise the Ti generation.
For polynucleotides encoding polypeptides used in these experiments, RT-PC may be performed to confirm the ability of cloned promoter fragments to drive expression of the polypeptide transgene in plants transformed with the vectors.
Ti plants transformed with promoter-TF combinations comprised within, a nucleic acid construct are subjected to morphological analysis. Promoter's that produce a. substantial amelioration of the negative effects of TF overexpression are subjected to further analysis by propagation into the T2 generation, where the plants are analyzed for an altered trait relative to a control plant.
Example VI. Methods for determining nitrogen immobilization
Nitrogen remobilization in plants rown under limiting and ample nitrogen
concentrations can be determined using iSN pulse-chase assays. Plants are grown under a defined fertilization regime (limiting N, e.g., 2 raM nitrate and ample N e.g. 10 m.M nitrate) conditions in an ebb and flow hydroponic system as described above. At a defined period in the plant life prior to reproductive growth, the plants are allowed to uptake l5N twice (e.g. for Arahidopst 32 and 34 days after sowing when grown under 10: 14 fight.dark conditions). This is done by replacing the uniabeiied utrient solution with a solution that has the same composition except that l*N0.? is replaced by '''NO.? at 10% enrichment. All pots are watered for 24 h by immersing the base of the pot with a volume of labeled solutions sufficient t cover the lower 35 mm of the pot. After labeling, plant .roots and sand are rinsed with deiom'zed. water. A second NCh pulse is given 24 his later followed by extensive rinsing with deionized water to remove residual i 5NO-f that may remain. Plants are grown for a further 14 days with unlabelled nutrient solution ti der short day conditions ( 10 h: 14 h lightidarfc) to promote the incorporation, of i5N into nitrogen containing compounds such as protein and chlorophyll. Plants are then transferred to long day conditions (L D 24:0) to induce .flowering. Plants are harvested at the end of their life cycle when, all seeds are matured and the rosette is dr '. Samples are then separated into seeds and dry remains (rosette, stem, caulme leaves, and empty siliqaes) and seed weight and dry weight are determined. Four to six replicates are harvested for uptake and remobilization experiments.
After drying and weighing each sample, the dry remains are ground in a bead mil) to obtain a homogenous fine powder. A subsample of 2 mg to 3 mg of the fine powder from the dry remains or intact seeds are carefully weighed into tin capsules to determine the total N content and ! 5N abundance. Samples are analyzed for S 5N isotopes using a PDZ Europa ANCA-GSL elemental analyzer interfaced to a PDZ Europa 20-20 isotope ratio mass spectrometer (Sercon Ltd., Cheshire, UK). Samples are combusted at. K )0°C in a reactor packed with chromium oxide and silvered cobaltous cobaltic oxide. Following combustion, oxides are removed in a reduction reactor (reduced copper at 650°C), The helium carrier then flows through a water trap
(magnesium perchlorate). r% and CO? are resolved on a Carbosieve gas chromatography column (65 *C, 65 mL min) before entering the isotope ratio mass spectrometer. During analysis, samples are interspersed with several replicates of at least two different laboratory standards. These laboratory standards (selected to be compositional!y similar to the samples being analyzed) are calibrated against NIS " Standard Reference Materials (lAEA-Nl, IAEA-N2, ΪΑΕΑ-Ν3, USGS- 40, and USGS-41).
The t5N abundance is calculated as atom per cent {A ::: ίι>Ν)/(ι*Ν ÷ !4 ) x 100) and for unlabelled plant controls f A%tX>Bj∞;) was 0.3660. The enrichment (E%) of the plant material was then defined as E% = Α%«η¾>¾ - A%c«et«>v
A number of traits related to nitrogen remobilizatiori can be deri ved from seed weight (seedd¾ )> tissue dry weight (rosette dry matter minus seeds, or dry remains (DR*)). seed and tissue % nitrogen ( ¾), and seed and tissue E%. indicators for yield are calculated from the data obtained from plants used in remobilteation experiments. From the dry weights for seeds and dry remains we calculate harvest index (Hi ~ see is eddw + D &), which is an indicator for individual plant yield. Tissue dry weight and nitrogen concentrations (N%) are also combined to determine the nitrogen harvest index (NHI
Figure imgf000050_0001
+ %DR X
DRiK )) winch estimates the extent, of seed filling with nitrogen. The ratio Hi Hl is monitored in order to compare nitrogen use efficiency ( UE). In a similar manner, the dry weight of seed and D , %, and E% are combined to determine the partition of ° in seeds which represents the proportion of " absorbed at the vegetative stage and remobilized to the seeds at the' reproductive stage (i:' harvest index (,;>NHi) (Ε%^ x N¾Si;ea x seedf}w}/(E%ss:¾ii x %ss£ii x seedt¾v -i- E DR x %D X DRd.w)). The ratio °NRl Rl is monitored in order io compare .nitrogen remo'hiUsKUion efficiency (NRE), The relative specific abundance (RSA) ratio is an indicator of the allocation of the nitrogen absorbed during seed fill This value is related to tsN enrichment and is an indicator of ¾ dilution by the natural UN absorbed by roots after labeling and until harvest (RSA ratio -
Figure imgf000051_0001
x N%Siii¾s x $ee <iw ÷ E%sied x N%DK x DRi!«) ( ¾!¾«i x see hv ÷ N%¾R X D¾k)|, The RSA ratio can be equal to 1 when S 5N dilution is similar in seeds and dry remains, lower than i when the N absorbed after labeling is routed to the seeds while " is retained in DR., or higher than ! when seeds are preferentially loaded with. ! N remobilteed while th i N absorbed after labeling is allocated to vegetative tissues.
The above method was applied to an Arahidopsis mutant deficient in aatopSiagy, aigS-I. This .mutant showed an increase m the percentage of n itrogen in the dry remains, and a reduction in dry remains, seed yield, harvest index, nitrogen, harvest index, ϋΕ, ,5N harvest index, remobili/.alion efficiency, and relative specific abundance under both limiting and ample nitrogen conditions,. -with stronger effects being observed under ample nitrogen (Figure 1 ). These results confirm that the assay can detect alterations in remobilizaiion.
Example VII. identification of crop plants with enhanced nitrogen remobilization
In crops, seeds are the major sink for ni trogen remobilized from leaves after anth.es.is and daring seed fill After -flowering, the nitrogen accumulated in the vegetative parts of the plant, is remobilized and. translocated, to grain. For example, in wheat, 60-95% of the grain nitrogen comes from the re-mobilization of nitrogen stored in roots and shoots before an the is while in maize 45-65% of the grain nitrogen is provided from pre-existing nitrogen in the stover before silking. The remainder of the grain nitrogen originates from post- flowering nitrogen uptake.
Because reraobthzation and post-flowering uptake are essential, for grain production, it is important io evaluate the contribution of both N sources. Two methods exist for measuring N flux within a crop plant. Classically, a "balance method" has been used to compare the content at anthesis and then in grain and the remaining plant material at maturity. This method can produce biased results as it neglects the contribution of roots to N remobiiizaiion, and it assumes thai all post-flowering N uptake is allocated to grain.
A less biased and more accurate estimate of N flux than the "balance method" estimates nitrogen remobilization i plants grown under limiting and ample nitrogen concentrations can be using i 5N pulse-chase assays. Depending on. the crop, l 5N can be applied to Irydropomcai!y grown plants at the beginning of stem elongation, with a single sampling of grain and remaining plan! material occurring at maturity. A second ¾ pulse-chase can be applied at -auihesis to estimate of the proportion of post-flowering N uptake allocated to the grain. The application of two { N pulse-chase techniques at different times is required to obtain a complete picture of N management and recycling during the entire developmental cycle of the crop. Then, traits related to nitrogen remobiIi¾ation and harvest parameters can be defined as described above.

Claims

( bums
What is claimed is:
1 , A method for producing a plant that has greater seed yield and/or greater nitrogen
remobilizaiion in the plan* or a part of the plant relati ve to a control plan t or a corresponding part of the control plant, the method comprising:
a. growing a plant in a medium containing:
a limiting concentration of nitrogen that limits growth of the plant; or
an ample concen tration of ni trogen that does not li mit growth of the plant:
b. identifying a polypeptide the expression of which is higher in a senescing plant part when the plant is grown in the limiting concentration of nitrogen as compared to the same part of a plant grown in the ample concentratio of nitrogen;
c. identifying senescence-enhanced promoter that is capable of up-regulating gene
expression during senescence of the plant or a part of the plant;
d. identifying a polynucleotide that encodes the polypeptide:
e. providing a transgenic plant comprising at least one recombinant nucleic acid construct- wherein the at least one recombinant nucleic acid construct comprises the senescence- enhanced promoter and the polynucleotide;
wherein the senescence-enhanced promoter regulates expression of the polypeptide, said regulation of expression is preferentially enhanced during senescence in the transgenic plant, and said regulation of expression increases nitrogen remobilization in the transgenic plant relative to the control plant; and
f. idemifying-a selected, transgenic plant that has greater nitrogen remobiiization and/or greater seed yield than, the conirol plant.
2, A method for producing a plant that has greater nitrogen remobiiization in the plant or a part of the plant relative to a control plant or a corresponding pari of the control plant, the method comprising;
a, growing a plant in. a medium containing:
a limiting concentration of nitrogen that limits growth of the plant; or
an ample concentration of nitrogen, that does not limi growth of the plant;
b, identifying an endogenous polypeptide the expression of which Is higher i a senescing plant part when the plant is grown in the ample concentration of nitrogen as compared to the same part of a plant grown in the limiting concentratio of nitrogen;
c. identifying a senescence-enhanced promoter that is capable of up-regulating gene
expression during senescence of the plant or a part of the plant; d. identifying a suppressor of gene ex pression capable of suppressing expression of an genous polynucleotide and its encoded endogenous polypeptide
e. introducing into a target plant at least one recombinant nucleic acid construct to produce a transgenic plant, wherein the at least one nucleic acid, construct comprises the senescence-enhanced promoter and the suppressor of gene expression;
wherein the senescence-enhanced promoter increases expression of the suppresso r of gene expression during senescence or onset of senescence of the transgenic plant or the part of the transgenic plant, which results in decreased expression of the endogenous polynucleotide and its encoded endogenous polypeptide; and said decreased expression of the endogenous polypeptide increases nitrogen immobilization in the transgenic plant relative to the control plant; and
f, selecting a transgenic plant that has greater nitrogen re.mobslkaiio.rs than the control pla t.
3. The method of cl im 2, wherein the suppressor of gene expression is an RNAi (RNA
interference) molecule, a small interfering RNA. (siRNA) molecule, a small hairpin RNA
(shRNA) molecule, a microRNA (miRNA) molecule, an antisense molecule, a cosuppression directing nucleic acid, a nucleic acid encoding a ribozyme, a nucleic acid encoding a
deoxyribozyme (DNAzyme), a nucleic acid encoding a transcription factor suppressor, or a triple helix oligonucleotide thai decreases the expression of the polynucleotide.
4. The method of any of claims 1 to 3, wherein the senescence-enhanced promoter is selected from the group consisting of SEQ ID NOs: 5724 to 5741 ,
5. A method for producing a plant that has greater nitrogen reraobili ation in the plant or a part of the plant relative to a con trol plant or a corresponding part of the con trol plant, the method comprising:
a. growing a plant in a medi um containing:
a limiting concentration of nitrogen thai limits growth of the plant: or
an ample concentration, of nitrogen that does not limit growth of the plant;
identifying a polypeptide the expression of which is higher in a seed or seed-bearing structure of a plant grown in the limiting concentration of nitrogen than in a
corresponding seed or seed-bearing structure of a plant grown in the ample concentration of nitrogen;
identifying a seed-enhanced promoter or a seed hearing sintciure-enhat ced promoter that is capable of up-regulating protein expression in the seed or seed-bearing structure;
identifying a polynucleotide that encodes the polypeptide; e. introducing into a target plant at least one recombinant nueleic acid construct to produce a transgenic plant, and the at least one recombinant nucleic acid construct comprises the polynucleotide and the seed-enhanced or the seed bearing structure-enhanced promoter; wherein the seed-enhanced or the seed bearing stracture-enhanced promoter regulates transcription of the polynucleotide, and said transcriptional regulation is preferentiall enhanced in. the seed-bearing structure of the transgenic plant;
wherein the preferentially enhanced, expression of the polypeptide in the seed or seed- bearing structure of the transgenic plant increases nitrogen remobiiizaiion in the transgenic plant .relative to the control plant; and
f. selecting a transgenic plant that has greater nitrogen remobllization than the control plant,
6. The method of claim 5, wherein the seed-bearing structure is a achene, berry, capsule, caryopsis or grain, circumcissile capsule, cypsela, drupe, ear, fruit or ripened pericarp, follicle, grain, kernel, legume, Iocuiicidal capsule, loraemura, nui, pistil, pod, porieidal capsule, samara, schizocarp, seed capsule, septtcidai. capsule, septifragal capsule, silicula, siliqua, silique or strohiius.
7. The method of any of claims 1 to 6, wherein the limiting concentration of nitrogen in. the medium is a total nitrogen content of 2 mM nitrogen and the ample concentration of nitrogen is a total nitrogen content of 10 nuYI nitrogen.
8. The method of any of claims 1 to 7, wherein the polypeptide is at least 30%, at least 31%, at least 32%, at least 33%, at least 34%, at least 35%, at least 36%, at least 37%, at least 38%, at least 39%, at least 40%, at least 41%, at least 42%, at least 43%, at least 44%, at least 45%, at least 46%, at least 47%, at least 48%», at least 49%, at least 50%, at least 51%», at least 52%, at least 53%, at least 54%>, at least 55%, at least 56%, at least 57%>, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%>, at least 63%, at least 64%>, at least 65%>, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 90%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95% or 96%, at least 97%, at least 98%, or at least 99%, ox about 100% identical io SEQ ID NO:2n, where n-l to 2300.
9. The method of any of claims 1 to 8, wherein the plant has a higher ratio of nitrogen remobilization from a source or senescent leaf to a sink leaf or a developing seed or grain than the control plant, or fee plant has greater seed protein content or grain protein content than the control plant.
1 0. A method for enhancing nitrogen remobilization in a crop plant relative to a control plant, the method comprising:
s providing a transgenic crop plant that comprises at least one recombinant nucleic acid construct, wherein the nucleic acid construct comprises a promoter and a -polynucleotide; and the promoter is a senescence-enhanced promoter, a seed-enhanced promoter, or a seed bearing struct ore-enhanced promoter;
wherein the polynucleotide encodes a polypeptide is at least 30%, at least 31%, at least0 32%, at least 33%, at least 34¾>, at least 35%, at least 36%, at least 37%., at least 38%., at least 39%, at least 40%, at least 41%, at least 42%, at least 43%, at least 44%, at least 45%, at least 46%, at least 47%, at least 48%, at least 49%, at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least5 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least
74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 90%., at least 81%, at least 82%, at least 83%., at least 84%, at least 85%, at least 86%., at least 87%,, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95% or 96%, at least 97%, at least 98%, or at least 99%, or about- 100% identical to SEQ ID0 NO:2«, where n=::'l to 2300; and
wherein the promoter preferentially enhances expression of the polynucleotide during senescence in the transgenic plant or a part of the transgenic plant, or in a seed or seed- bearing strucutre of the transgenic plant, and said preferential enhancement of expression increases nitrogen remobilization in the transgenic plant relative to the control plant during5 senescence or in said part, seed or seed-bearing structure of the transgenic plant.
1 1. The method of claim 1.0, wherein the seed-bearing structure is an achene, berry, capsule, caryopsis or grain, cireuroc-issile capsule, cypsela, drape, ear, fruit or ripened pericarp, follicle, grain, kernel, legume, !oculicidai capsule, lomentura, nut, pistil, pod, porieidal capsule, samara, schizocarp, seed capsule,, septieklai capsule, septifragal capsule, silicula, siliqua, silique or0 strobilus.
12. A method for enhancing nitrogen remobilization in a crop plant relative to a control plant, the method comprising: providin a transgenic crop plant thai comprises at least one recombinant nucleic acid construct, wherein the nucleic acid construct comprises a senescence-enhanced promoter and a suppressor of gene expression capable of suppressing expression of an endogenous polynucleotide;
wherein the suppressor of gene expression inhibits expression of the polynucleotide and its encoded endogenous polypeptide, and the -endogenous polypeptide is at least 30%, at least 31%, at least 32%, at least 33%, at least 34%>, at least 35%, at least 36%, at least 37%, at least 38%, at least 39%, at least 40%, at least 41%, at least 42%, at least 43%, at least 44%, at least 45%, at least 46%, at least 47%,, at least 48%, at least 49%, at least 50%,, at least 51 %, at least 52%, at least 53%», at least 54%, at least 55%, at least 56%», at least 57%, at least 58%, at least 59%, at least 60%», at least 61%, at least 62%, at least 63%», at least 64%, at least 65%, at least 66%, at least 67%,, at least 68%, at least 69%, at least 70%,, at least 71%, at least 72%, at least 73%, at least 74%,, at least 75%, at least 76%, at least 77%,, at least 78%, at least 79%, at least 90%», at least 81%, at least 82%, at least 83%», at least 84%, at least 85%, at least 86%», at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%», at least
94%, at least 95%, at least 96%,, at least 97%, at least 98%, at least 99%,, or about 100% identical to SEQ ID NO:2rt, where u-i to 2300; and
wherein the -senescence-enhanced promoter increases expression of the suppressor of gene expression during senescence or onset of senescence of the transgenic plant or the part of the transgenic plant, which results in decreased expression -of the endogenous
polynucleotide and its encoded endogenous polypeptide; and
said decreased expression of ihe endogenous polypeptide increases nitrogen
immobilization in the transgenic plant relative ιο the control, plant,
1 3. The method of claim 12, wherein the suppressor of gene expression is an RNAi (RNA interference) molecule, a small interfering- .R A isi.R A) molecule, a small hairpin RNA.
(shR A) molecule, a microRNA (miRNA) molecule, an antisense molecule, a cosuppression directing nucleic acid, a nucleic acid encoding a riboz me, a nucleic acid encoding a
dteoxynbozyme (DN Azyme), a nucleic acid encoding a transcription, factor suppressor, or a triple, helix oligonucleotide that decreases the expression of the polynucleotide. 14, The method of any of claims 10 to 13, wherein the plant has a higher ratio of nitrogen remobilkatton from a source or senescent leaf to a sink leaf or a developin seed or grain than the control plant, or the plant has greater seed protein content or grain protein content than the control plant.
1 5. The method, of any of claims i 0 to 14, wherein the senescence-enhanced promoter is selected from the grou consisting of SEQ ID NOs: 5724 to 5741.
1 . A recombinant nucleic acid construct comprising a se»escenc«-enhanced promoter that regulates expression of a polynucleotide, wherein the polynucleotide encodes a polypeptide is at least 30%, at least 31%, at least 32%, at least 33%, at least 34%, at least 35%, at least 36%, at least 37%, at least 38%, at least 39%, at least 40%, at least 41%, at least 42%, at least 43%, at least 44%, at least 45%, at least 46%, at least 47%, at least 48%, at least 49%, at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%>, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 90%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95% or 96%, at least 97%, at least 98%, or at least 99%, or about 100%, identical to SEQ I'D NO:2n, where n=i to 2300,
17. A. recombinant nucleic acid constmci comprising a senescence-enhanced promoter that regulates expression of a polynucleotide,, wherein the polynucleotide, wherein, the polynucleotide inhibits expression of an endogenous polypeptide that is at least 30%, at least 31%, at least 32%, at !east 33%, at least 34%, at least 35%, at least 36%, at least 37%, at leas 38%, at least 39%, at least 40%, at least 41%, at least 42%, at. least 43%, at least 44%, at least 45%, at least 46%, at least 47%, at least 48%, at least 49%, at least 50%, at least 51%, at least 52%», at least 53%. at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%», at least 66%>, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%», at least 76%, at least 77%, at least 78%», at least 79%, at least 90%, at least 81%», at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 1%, at least 92%, at least 93%, at least 94%, at least 95% or 96%, at least 97%», at least 98%, or at least 99%, or about 100% identical to SEQ ID NO:2n, where n===l to 2300. 18. A transgenic crop plant produced by the method of any of claims 1 to 15 or comprising a recombinant nucleic acid, construct of claims 1.6 or 17, wherein the transgenic plant has enhanced nttrogen remobilizalion relative to a control plant .
s? 19, The transgenic crop plant of claim 18, wherein the transgenic piam is selected from the group consisting of; a monocoi plant, a cereal plant, a maize (com) plant, a rice plant a wheat plant, a bariey plant, a sorghum plant, a millet plant, an oat plant, a iriticale plant, a rye plant, a buckwheat plant, a fonio plant, and. a quiooa plant.
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