WO2015193653A1 - Gènes et protéines chimériques de résistance à l'oxydation et plantes transgéniques les comprenant - Google Patents

Gènes et protéines chimériques de résistance à l'oxydation et plantes transgéniques les comprenant Download PDF

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WO2015193653A1
WO2015193653A1 PCT/GB2015/051759 GB2015051759W WO2015193653A1 WO 2015193653 A1 WO2015193653 A1 WO 2015193653A1 GB 2015051759 W GB2015051759 W GB 2015051759W WO 2015193653 A1 WO2015193653 A1 WO 2015193653A1
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seq
polynucleotide
plant
sequence
protein
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PCT/GB2015/051759
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English (en)
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Elina WELCHEN
Daniel H. Gonzalez
Gabriel CECCOLI
Francisco COLOMBATTI
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Consejo Nacional De Investigaciones Cientificas Y Tecnicas
Universidad Nacional Del Litoral
Plant Bioscience Limited
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—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

  • Abiotic environmental stresses such as drought, salinity, wind, heat, and cold, are major limiting factors of plant growth and crop yield. Prolonged or continuous exposure to drought conditions causes major alterations in the plant metabolism that ultimately lead to cell death and, consequently, losses in crop yield. High salt content in some soils results in less water being available for cell intake; thus, high salt concentration has an effect on plants similar to the effect of drought on plants. Under freezing temperatures, plant cells lose water as a result of ice formation within the plant. Because crop damage from abiotic stresses is predominantly due to dehydration, water availability is an important aspect of the abiotic stresses and their effects on plant growth. Losses in crop yield of major crops caused by these stresses represent a major economic factor and contribute to food shortages in many underdeveloped countries.
  • the present invention relates to a chimeric protein comprising a first polypeptide portion of a first OXR (Oxidative Resistance) protein; and a second polypeptide portion of a second OXR protein and to related methods and uses.
  • the second polypeptide portion is joined to the first polypeptide portion, and the first and second polypeptide portions are combined in the chimeric protein in an order or in a spacing that does not occur in nature.
  • OXR Oxidative Resistance
  • the various embodiments of the chimeric protein according to the aspects of the invention are described herein.
  • the chimeric protein is a plant protein.
  • the amino acid sequence of the first polypeptide portion includes an amino(N)-terminal region of a first OXR protein and the amino acid sequence of the second polypeptide portion includes a TLDc domain of a carboxyl (C)-terminal region of a second OXR protein.
  • the first and second polypeptide portions are covalently attached by the amino-terminal region of the first polypeptide portion and the carboxyl-terminal region of the second polypeptide portion.
  • the first OXR protein comprises an AtOXR4 amino acid sequence of SEQ ID NO: 2, a functional variant or homolog thereof; and the second OXR protein comprises an AtOXR2 amino acid sequence of SEQ ID NO: 8, a functional variant or homolog thereof.
  • the first OXR protein or polypeptide portion of the chimeric protein may comprise an amino acid sequence having at least 50%, 60%, 70%, 80% or 90% sequence identity with the full-length OXR4 protein amino acid sequence of SEQ ID NO: 2, a functional variant or homolog thereof; and the second OXR protein or polypeptide portion may comprise an amino acid sequence having at least 50%, 60%, 70%, 80% or 90% sequence identity with the full- length OXR2 protein amino acid sequence of SEQ ID NO: 8, a functional variant or homolog thereof.
  • the first polypeptide portion comprises an AtOXR4 amino- terminal amino acid sequence of SEQ ID NO: 4, a functional variant or homolog thereof; and the second polypeptide portion comprises an AtOXR2 carboxy-terminal amino acid sequence of SEQ ID NO: 12, a functional variant or homolog thereof.
  • the first polypeptide portion of the chimeric protein may have at least 50%, 60%, 70%, 80% or 90% sequence identity with the AtOXR4 amino-terminal amino acid sequence of SEQ ID NO: 4, a functional variant or homolog thereof; and the second polypeptide portion may have at least 50%, 60%, 70%, 80% or 90% sequence identity with the OXR2 carboxy-terminal amino acid sequence of SEQ ID NO: 12, a functional variant or homolog thereof.
  • the first polypeptide portion of the chimeric protein may comprise the amino-terminal amino acid sequence AtOXRA (AT4G39870) represented by SEQ ID NO: 4; and the second polypeptide portion may comprise the TDLc domain of the carboxy-terminal amino acid sequence AtOXR2 (AT2G05590) represented by SEQ ID NO: 12.
  • the chimeric protein may comprise an amino acid sequence having at least 50%,
  • the joined first and second polypeptide portions of the chimeric protein may constitute the full-length amino acid sequence of SEQ ID NO: 14.
  • the chimeric polypeptide includes other combinations of first and second polypeptide portions.
  • said first OXR polypeptide may comprise a polypeptide sequence having at least 50%, 60%, 70%, 80% or 90% sequence identity with the AtOXR2 amino-terminal encoding nucleotide sequence of SEQ ID NO: 9 and said second OXR polypeptide may be encoded by a nucleotide sequence having at least 50%, 60%, 70%, 80% or 90% sequence identity with the full-length AtOXR4 carboxyl-terminal encoding nucleotide sequence of SEQ ID NO: 5 resulting in a Q24 polypeptide chimeric polypeptide.
  • OXR2 polypeptide portions Any amino-terminal part from any plant OXR polypeptide may be combined with any carboxy-terminal part from a different second plant OXR polypeptide.
  • Other examples include Q25 and Q52 as shown in Fig lb.
  • Embodiments of the present invention also relate to isolated C or N terminal polypeptide parts of a full protein as defined herein and polynucleotides encoding such parts. Examples are SEQ ID NO: 3, 4, 5, 6, 9, 10, 11 or 12.
  • Embodiments of the present invention also relate to an isolated polynucleotide that encodes the chimeric protein defined herein.
  • the isolated polynucleotide comprises a first portion of a first
  • OXR polynucleotide portions from OXR polynucleotides of the same or different plant species can be joined together.
  • the first polynucleotide comprises SEQ ID NO: 1 , a functional variant or homolog thereof and the second polynucleotide comprises SEQ ID NO: 7, a functional variant or homolog thereof.
  • An isolated, non -naturally occurring polynucleotide according to the invention may also comprise: a first OXR polynucleotide or first region comprising a nucleotide sequence having at least 50%, 60%, 70%, 80% or 90% sequence identity with the full-length AtOXR4 carboxyl-terminal encoding nucleotide sequence of SEQ ID NO: 1, a functional variant or homolog thereof; and a second OXR polynucleotide or second region comprising a nucleotide sequence having at least 80% sequence identity with the full-length AtOXR2 carboxyl-terminal encoding nucleotide sequence of SEQ ID NO: 7, a functional variant or homolog thereof.
  • the first region comprises the AtOXR4 amino-terminal encoding nucleotide sequence represented by SEQ ID NO: 3, a functional variant or homolog thereof and the second region comprises the AtOXR2 carboxy-terminal encoding nucleotide sequence represented by SEQ ID NO: 11, a functional variant or homolog thereof.
  • the first region may comprise at least 50%, 60%, 70%, 80% or 90% sequence identity with the OXR4 amino-terminal encoding nucleotide sequence represented by SEQ ID NO: 3, a functional variant or homolog thereof; and the second region may comprise at least 50%, 60%, 70%, 80% or 90% sequence identity with the OXR2 carboxyl-terminal encoding nucleotide sequence represented by SEQ ID NO: 11, a functional variant or homolog thereof.
  • the first and second regions are joined together through a covalent bond.
  • the polynucleotide may have the full-length nucleotide sequence of SEQ ID NO: 13 or a sequence with at least 50%, 60%, 70%, 80% or 90% sequence identity thereto.
  • the chimeric polynucleotide includes other combinations of first and second polynucleotide portions.
  • said first OXR polypeptide may comprise be encoded by a nucleotide sequence having at least 50%, 60%, 70%, 80% or 90% sequence identity with the full-length AtOXR2 amino-terminal encoding nucleotide sequence of SEQ ID NO: 7 and said second OXR polypeptide may be encoded by a nucleotide sequence having at least 50%, 60%, 70%, 80% or 90% sequence identity with the full-length AtOXR4 carboxyl-terminal encoding nucleotide sequence of SEQ ID NO: 1 resulting in a Q24 polypeptide construct.
  • Embodiments of the present invention also relate to a vector or expression cassette comprising the polynucleotide encoding the chimeric protein defined herein.
  • a recombinant expression cassette may comprise the polynucleotide encoding the chimeric protein defined herein, wherein the polynucleotide is operably linked to a promoter and is in sense or antisense orientation.
  • the promoter may be a tissue-preferred promoter, a constitutive promoter, and/or an inducible promoter.
  • the expression cassette may comprise the polynucleotide operably linked to a 35SCaMV constitutive promoter.
  • transgenic plants comprising a nucleic acid construct or recombinant expression cassette that comprises the polynucleotide encoding the chimeric protein described herein.
  • the transgenic plant may be selected from the group consisting of maize, soybean, sorghum, canola, wheat, alfalfa, cotton, rice, brassica and barley.
  • the transgenic plant may comprise the recombinant expression cassette, the transgenic plant being selected from the group consisting of maize, soybean, sorghum, canola, wheat, alfalfa, cotton, rice, brassica and barley.
  • the transgenic plant according to embodiments may have improved traits, for example yield related traits, including increased biomass and/or seed production and increased stress tolerance compared to a corresponding control plant, for example a wild-type control plant, that does not express the chimeric protein encoded by the recombinant polynucleotide.
  • a control plant may be a wild type or a plant that overexpresses the wild type protein, but not the chimeric protein.
  • aspects of the present invention also relate to a method for producing a transgenic plant, comprising introducing into a plant cell a nucleic acid construct or an expression vector or cassette comprising the polynucleotide encoding the chimeric protein as defined herein; and generating from the plant cell a transgenic plant that expresses the polynucleotide.
  • the method for producing a transgenic plant comprises introducing into a plant cell an expression vector comprising a polynucleotide as described herein and generating from the plant cell a transgenic plant that expresses the polynucleotide.
  • a recombinant expression cassette according to embodiments of the invention may comprise an isolated polynucleotide operably linked to a promoter, wherein the polynucleotide is a member selected from the group consisting of (a) a polynucleotide that encodes the polypeptide of SEQ K) NO: 14, and (b) the polynucleotide of SEQ ID NO: 13.
  • Fig. la is a schematic drawing of the chimeric protein Q42 obtained by fusion of the amino -terminal region of the OXR4 protein and the carboxyl-terminal region of the OXR2 protein (containing the TLDc domain).
  • Fig. lb is a schematic drawing of the chimeric proteins Q25, Q24, Q52 and Q52*.
  • Fig. 2A is a photographic showing rosette sizes of a AtQ42 transgenic plant and a
  • Fig. 2B is a photograph showing the root biomass of a AtQ42 transgenic plant and a WT control plant after removal from the growth medium.
  • Fig. 3A shows the difference in leaf dry weight between AtQ42 transgenic plants and WT control plants
  • Fig. 3B shows the difference in stem dry weight between AtQ42 transgenic plants and WT control plants
  • Fig. 3C shows the difference in total shoot dry weight between AtQ42 transgenic plants and WT control plants
  • Fig. 3D shows the difference in root dry weight between AtQ42 transgenic plants and WT control plants
  • Fig. 3E shows the difference in total dry weight (aerial and roots) between AtQ42 transgenic plants and WT control plants.
  • Different letters indicate samples that are significantly different (p value ⁇ 0.05).
  • Fig. 4A is a photograph showing differences in leaf numbers between AtQ42 transgenic plants and WT control plants
  • Fig. 4B shows the difference in leaf numbers between 4 homozygous lines of AtQ42 transgenic plants and of WT control plants
  • Fig. 4C shows the difference in the major axis of rosettes obtained from 4 homozygous lines of AtQ42 transgenic plants and of WT control plants
  • Fig. 4D shows the difference in the leaf area obtained from 4 homozygous lines of AtQ42 transgenic plants and of WT control plants
  • Fig. 4E shows the difference in leaf dry weight between 4 homozygous lines of AtQ42 transgenic plants and of WT control plants.
  • Different letters indicate samples that are significantly different (p value ⁇ 0.05).
  • Figs. 5A and 5B show differences in yield (mg seeds/plant) between 4 homozygous lines of AtQ42 transgenic plants and of WT control plants. Different letters indicate samples that are significantly different (p value ⁇ 0.05). Figs. 5A and 5B correspond to two different assays, with Fig. 5B showing the results of seed yield after adverse conditions being introduced into the growth chamber.
  • Fig. 6 shows differences in yield (mg seeds/plant) between 2 homozygous lines of
  • Fig. 7 shows differences in yield (mg seeds/plant) between 3 homozygous lines of
  • AtQ42 transgenic plants and of WT control plants grown under water deficit conditions Different letters indicate samples that are significantly different (p value ⁇ 0.05).
  • Fig. 8A shows differences in the number of branches between 4 homozygous lines of AtQ42 transgenic plants and of WT control plants
  • Fig. 8B shows differences in the number of secondary stem branches between 4 homozygous lines of Q42 transgenic plants and of WT control plants
  • Fig. 8C shows differences in the number of siliques per plant between 4 homozygous lines of AtQ42 transgenic plants and of WT control plants
  • 8D shows differences in the number of seeds per silique between 4 homozygous lines of AtQ42 transgenic plants and of WT plants
  • 8E shows differences in the weight of 1000 seeds from 4 homozygous lines of AtQ42 transgenic plants and from WT control plants.
  • Different letters indicate samples that are significantly different (p value ⁇ 0.05).
  • Fig. 9A shows the difference in the shoot (stem) area between AtQ42 transgenic plants and of WT control plants
  • Fig. 9B is a photograph showing the difference in the transversal shoot area between a AtQ42 transgenic plant and a WT control plants
  • Fig. 9C shows the difference in xylem vessel diameter between AtQ42 transgenic plants and WT control plants
  • Fig. 9D shows the difference in xylem vessel area between AtQ42 transgenic plants and WT control plants
  • Fig. 9E shows the percentage of lignified tissue in AtQ42 transgenic plants and in stems of WT control plants. Different letters indicate samples that are significantly different (p value ⁇ 0.05).
  • Fig. 10A shows differences in C0 2 assimilation between 4 homozygous lines of
  • Fig. 10B shows differences in stomatal conductance between 4 homozygous lines of AtQ42 transgenic plants and of WT control plants
  • Fig. IOC shows differences in the water use efficiency between 4 homozygous lines of AtQ42 transgenic plants and of WT plants. Different letters indicate samples that are significantly different (p value ⁇ 0.05).
  • Fig. 11 is a diagram showing parameters of various characteristics observed in
  • AtQ42 transgenic plants compared to those observed in WT control plants or plants that express AtOXR2.
  • Parameters grey boxes: 1 : Plant height (mm), 2: Number of stem branches, 3: Number of secondary stems branches, 4: Rosette major axis, 5: Leaf number, 6: Seed yield (mg seeds/plant), 7: Photosynthesis (A), 10: Water use efficiency, 11 : Leaf dry weight, 12. Leaf area, 13. Specific leaf area.
  • PCI principal component 1
  • PC2 principal component 2
  • Fig 12 a and b are alignments of OXR protein sequences identified from different plants.
  • the proteins are termed AtOx2 (At2g05590), AtOx4 (At4g39870), AtOx51 (At5g06260), AtOx52 (At5g39590), AtOxl (Atlg32520).
  • AtOx2 At2g05590
  • AtOx4 AtOx4g39870
  • AtOx51 At5g06260
  • AtOx52 AtOx52
  • AtOxl AtOxlg32520
  • AtOx5.2 (At5g06260, SEQ ID NO:22), VvOx52 (SEQ ID NO:34), ZmOx52 (SEQ ID NO:35), OsOx52 (SEQ ID NO:36), AtOx5.1 (At5g39590, SEQ ID NO:21), VvOx51 (SEQ ID NO:37), OsOx51 (SEQ ID NO:38), ZmOx51 (SEQ ID NO:39), AtOx4 (SEQ ID NO:2), TaOx4 (SEQ ID NO:40), BoOX4 (SEQ ID NO:41), HvOx4 (SEQ ID NO:42), BrOx4 (SEQ ID NO:43), Gmox4 (SEQ ID NO:44), VvOx4 (SEQ ID NO:45), OsOx41 (SEQ ID NO:46), ZmOx41 (SEQ ID NO:47), ZmOx42 (SEQ ID NO:48), OsOx42 (SEQ ID NO:
  • Fig. 13 Activity of Superoxide dismutase (SOD). Native gel stained for SOD activity present in total protein extracts prepared from 30-day-old Arabidopsis rosette leaves. (A) SDS-PAGE gel stained with Coomassie Blue. (B) SOD activity assay. Wells 1 to 4 correspond to WT plants and AtQ42 transgenic lines L25, L27 and L6, respectively.
  • SOD Superoxide dismutase
  • Fig 14 Percentage of plant tissue stained with Nitroblue Tetrazolium (NBT).
  • Plants of 30 days were placed in a 0,1 % NBT solution during 6 hours at room temperature. Then, they were destained by incubation in 80% EtOH at 70°C during 2 hours. Images were processed using ImageJ® software. Data are expressed as percentage of rosette leaf area stained with NBT. NBT detects superoxide anion.
  • Fig. 15 Ion leakage values in three AtQ42 lines (L25, L27 and L6) and WT plants at day 33 after sowing. Membrane damage was evaluated by measuring ion leakage (electrical conductivity of the solution after incubating leaves during 20 hours in water). Ion leakage is an indirect measure of cell death.
  • Fig. 16 Proline content in 25-day-old Arabidopsis plants under control (A) and water deficit conditions (B). Proline is an osmolyte and a ROS scavenger. Different letters indicate significant differences (p ⁇ 0.05, LSD Fisher's test, ANOVA. InfoStat® program).
  • Fig 17. Phylogenetic tree of Oxr proteins from selected plants. An alignment of
  • Oxr protein sequences with ClustalW was used to construct a phylogenetic tree with the PHYLIPgroup of programs (Felsenstein, 1989).
  • the tree is a neighbor-joining consensus generated by Consensus after bootstrap analysis of 100 trees performed with Protdist (with Dayhoff s PAM matrix) followed by Neighbor. Numbers indicate bootstrap values for each of the groups.
  • the arrow indicates the branch that separates Oxr2 proteins from the rest.
  • Fig. 18 Flowering time AtQ42 vs WT plants.
  • Q42 plants reach the reproductive stage earlier than WT plants under long (16 h light/8 h darkness) days. They elongate the flowering stem 4-5 days earlier than WT plants. The passage to reproductive phase was measured by the day of emergence of the flowering stem in individual AtQ42 (lines L25.1, L27.4 and L6.3), AtOX2 (line 10 1 and 2) and WT plants (1 and 2 correspond to different batches of WT seeds).
  • AtOX2 is a line that overexpr esses OXR2.
  • Fig 19 OXR chimeric polypeptides and nucleic acid sequences.
  • numeric ranges are inclusive of the numbers defining the range and include each integer within the defined range. It is also to be understood that “a” or “an” can mean one or more, depending upon the context in which it is used (e.g., reference to "a cell” can mean that at least one cell can be utilized).
  • nucleic acids are written left to right in 5' to 3' orientation, and amino acid sequences are written left to right in amino to carboxy orientation, respectively.
  • Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes.
  • amplified refers to the construction of multiple copies of a nucleic acid sequence or multiple copies complementary to the nucleic acid sequence using at least one of the nucleic acid sequences as a template.
  • Amplification systems include, but are not limited to, polymerase chain reaction (PCR), ligase chain reaction (LCR) system, nucleic acid sequence based amplification (NASBA), Q-Beta Replicase systems, transcription-based amplification system (TAS), and strand displacement amplification (SDA). See, e.g., D. H. Persing et al., "Diagnostic Molecular Microbiology: Principles and Applications," American Society for Microbiology, Washington D.C. (1993).
  • the term “environmental stress” refers to sub-optimal conditions associated with salinity, drought, temperature, metal, chemical, pathogenic and oxidative stresses, or combinations thereof.
  • the term "expression” refers to the process of converting genetic information encoded in a polynucleotide into RNA through transcription of the polynucleotide (i.e., via the enzymatic action of an RNA polymerase), and into protein, through translation of mRNA.
  • Up- regulation” or “activation” refers to regulation that increases the production of expression products relative to basal or native states
  • down-regulation or “repression” refers to regulation that decreases production relative to basal or native states.
  • the term "introduced” or "introducing” as used herein in the context of inserting into a cell refers to the incorporation of a nucleic acid into a target cell, such as a plant cell, such that the nucleic acid may be incorporated into the genome of the cell (e.g., chromosome, plasmid, plastid or mitochondrial DNA), converted into an autonomous replicon, or transiently expressed (e.g., transfected mRNA).
  • introducing a nucleotide sequence into a plant cell results in transformation of the plant cell to cause stable or transient expression of the sequence.
  • isolated refers to material, such as a nucleic acid or a protein, which is substantially or essentially free of components that normally accompany or interact with the material within its naturally occurring environment.
  • the isolated material may include a material not found with the material in its natural environment, or if the material is in its natural environment, the material has been synthetically (i.e., non-naturally) altered by deliberate human intervention to form a composition and/or be found in a location in the cell (e.g., genome or subcellular organelle) not native to the material found in that environment.
  • the alteration forming the synthetic material can be performed on the material within or removed from its natural state.
  • a naturally occurring nucleic acid becomes an isolated nucleic acid if it is altered, by means of human intervention on the cell from which it originates.
  • a naturally occurring nucleic acid e.g., a promoter
  • an isolated nucleic acid is free of some of the sequences, which naturally flank the nucleic acid (i.e., sequences located at the 5' and 3' ends of the nucleic acid) in its naturally occurring replicon.
  • a cloned nucleic acid is considered isolated.
  • nucleic acid refers to a deoxyribonucleotide or a ribonucleotide polymer, or analog thereof, that has the essential nature of natural nucleotides in that it hybridizes, under stringent hybridization conditions, to substantially the same nucleotide sequence as naturally occurring nucleotides and/or allows translation into the same amino acid(s) as the naturally occurring nucleotide(s).
  • a polynucleotide can be full-length or a sub-sequence of a native or heterologous structural or regulatory gene. Unless otherwise indicated herein, the term refers to a specified sequence, as well as the complementary sequence thereof.
  • DNAs or RNAs with backbones modified for stability or for other reasons, as well as DNAs and RNAs comprising unusual or modified bases, are polynucleotides as the term is defined herein.
  • polynucleotide also encompasses chemically, enzymatically, or metabolically modified forms of polynucleotides, as well as the chemical forms of DNA and RNA characteristic of simple and complex cells.
  • nucleic acid (or “polynucleotide”) may be used in place of, inter alia, gene, cDNA, mRNA.
  • operably linked refers to a functional linkage between sequences, such as a promoter and a second sequence, wherein the promoter sequence initiates and mediates transcription of the DNA sequence corresponding to the second sequence.
  • operably linked means that the nucleic acid sequences being linked are contiguous and, where necessary to join two protein coding regions, contiguous in the same reading frame.
  • plant is used broadly herein to describe a plant at any stage of development, to a part of a plant (e.g., plant cell, plant cell culture, plant organ, plant seed, etc.), and to progeny thereof.
  • a "plant cell” is the structural and physiological unit of the plant, comprising a protoplast and a cell wall.
  • a plant cell can be in the form of an isolated single cell or a cultured cell, or can be part of a higher organized unit, such as plant tissue, a plant organ, or a plant.
  • a plant cell can be a protoplast, a gamete-producing cell, or a cell or collection of cells that can regenerate into a whole plant.
  • a "seed” comprises multiple plant cells and is capable of regenerating into a whole plant, and may therefore be considered a plant cell.
  • a plant tissue or organ can be a seed, protoplast, callus, or any other group of plant cells that is organized into a structural or functional unit.
  • Parts of a plant that are particularly useful in embodiments include harvestable parts and parts used for propagation of progeny plants.
  • a harvestable part of a plant may include the flowers, pollen, seedlings, tubers, leaves, stems, fruit, seeds, roots, and the like.
  • Parts of the plant used for propagation include, e.g., seeds, fruits, cuttlings, seedlings, tubers, rootstocks, and the like.
  • the class of plants that may be used is generally as broad as the class of higher plants amenable to transformation techniques, including both monocotyledonous and dicotyledonous plants.
  • polypeptide and protein refer to a polymer of amino acid residues.
  • the terms encompass amino acid polymers, in which one or more amino acid residue is an artificial chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers.
  • the essential nature of such analogues of naturally occurring amino acids is that, when incorporated into a protein, the protein is specifically reactive to antibodies elicited to the same protein but consisting entirely of naturally occurring amino acids.
  • the polypeptide group includes, but is not limited to, DNA binding proteins, protein kinases, protein phosphatases, GTP-binding proteins, and receptors.
  • polypeptides may be "chimeric" in the sense that they are arranged in a configuration not normally found in nature.
  • the amino acid sequence of one or more of the segments can be a non-naturally occurring sequence.
  • the amino acid sequence of one segment may be a naturally occurring sequence found in one species, whereas the amino acid sequences of remaining segments may be naturally occurring sequences from different species or from-different alleles of the same species.
  • Chimeric polypeptides can include any naturally occurring amino acid or derivative thereof.
  • chimeric protein or “hybrid protein” is a protein composed of various protein "domains” (or motifs) which is not found as such in nature but which a joined to form a functional protein, which displays the functionality of the joined domains.
  • domain as used herein means any part(s) or domain(s) of the protein with a specific structure or function that can be transferred to another protein for providing a new hybrid protein with at least the functional characteristic of the domain. Specific domains can also be used to identify other OXR protein members, such as orthologs from other plant species.
  • the chimeric protein comprises the TLDc domain and this domain can also be used for identification of other OXR protein members.
  • a "chimeric gene” refers to any gene, which is not normally found in nature in a species, in particular a gene in which one or more parts of the nucleic acid sequence are present that are not associated with each other in nature.
  • the promoter is not associated in nature with part or all of the transcribed region or with another regulatory region.
  • the term “chimeric gene” is understood to include expression constructs in which a promoter or transcription regulatory sequence is operably linked to one or more coding sequences or to an antisense (reverse complement of the sense strand) or inverted repeat sequence (sense and antisense, whereby the RNA transcript forms double stranded RNA upon transcription).
  • promoter refers to a region of DNA that is upstream from the start of transcription and that is involved in recognition and binding of RNA polymerase and other proteins to initiate transcription.
  • the promoter may be any polynucleotide sequence that shows transcriptional activity in the host (target) plant cells, plant parts, or plants.
  • recombinant refers to a cell or vector that has been modified by the introduction of a heterologous nucleic acid or a cell that is derived from a cell so modified.
  • recombinant cells express genes that are not found in identical form within the native (non-recombinant) form of the cell or express native genes that are otherwise abnormally expressed, under-expressed, or not expressed at all as a result of deliberate human intervention.
  • the term does not encompass the alteration of the cell or vector by naturally occurring events (e.g., spontaneous mutation, or natural transformation, transduction, or transposition).
  • the term "recombinant expression cassette” refers to a nucleic acid construct that is recombinantly or synthetically generated with a series of specified nucleic acid elements, and that permits transcription of a particular nucleic acid in a target cell.
  • the recombinant expression cassette can be incorporated into a plasmid, chromosome, mitochondrial DNA, plastid DNA, virus, or nucleic acid fragment.
  • the recombinant expression cassette portion of an expression vector includes a nucleic acid to be transcribed and a promoter.
  • regulatory element means a nucleotide sequence that, when operatively linked to a coding region of a gene, effects transcription of the coding region such that a ribonucleic acid (RNA) molecule is transcribed from the coding region.
  • Regulatory elements include promoters, enhancers, silencers, 3'-untranslated or 5 '-untranslated sequences of transcribed sequences, e.g., a poly-A signal sequence or other protein or RNA stabilizing element, or other gene expression control elements known to regulate gene expression or the amount of expression of a gene product.
  • amino acid residue amino acid residue
  • amino acid amino acid
  • sequence identity in the context of two polynucleotide or polypeptide sequences refers to the residues in the two sequences that are the same when aligned for maximum correspondence over a comparison window of a contiguous and specified segment of a polynucleotide sequence.
  • the "percentage” of sequence identity means the value determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may include additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences.
  • the percentage of sequence identity between two sequences is therefore calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison, and multiplying the result by 100 to yield the percentage of sequence identity.
  • amino acids with basic side chains e.g., lysine, arginine, histidine
  • acidic side chains e.g., aspartic acid, glutamic acid
  • uncharged polar side chains e.g., glycine, asparagine, glutamine, serine, threonine, methionine, tryptophan
  • beta-branched side chains e.g., threonine, valine, isoleucine
  • aromatic side chains e.g., tyrosine, phenylalanine, tryptophan, histidine
  • sequences differ in conservative substitutions the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Sequences differing by such conservative mutations are said to have "sequence similarity.” Methods for making this adjustment are well known to persons skilled in the art.
  • transgenic plant refers to a plant that includes within its genome a heterologous polynucleotide.
  • the heterologous polynucleotide is stably integrated within the genome of the transgenic plant, such that the polynucleotide is passed on to successive generations.
  • the term “transgenic” is used herein to describe any cell, cell line, callus, tissue, plant part or plant (also referred to herein as a "target cell” or "host cell”) the genotype of which has been altered by the presence of a heterologous nucleic acid, and includes transgenic plants that have been initially altered, as well as those created by sexual crosses or asexual propagation from the initial transgenic plants.
  • the term “transgenic” as used herein does not encompass the alteration of the genome by naturally occurring events, such as random cross- fertilization or spontaneous mutation.
  • vector refers to a nucleic acid used to transport another nucleic acid (to which it has been linked) in the transfection of a target cell.
  • plasmid refers to a circular double stranded DNA loop into which additional DNA segments can be ligated.
  • viral vector Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome.
  • Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin). Other vectors are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome.
  • inventions permit transcription of genes to which they are operatively linked, and are referred to herein as "expression vectors.”
  • Recombinant expression vectors described herein may comprise a nucleic acid of the invention in a form suitable for expression of the nucleic acid in a host cell.
  • the recombinant expression vectors therefore include one or more regulatory sequences, selected on the basis of the host cell used for expression, which is operatively linked to the nucleic acid sequence to be expressed.
  • regulatory sequence is intended to include promoters, enhancers, and other expression control elements.
  • control plant refers to a plant that is not modified according to the invention.
  • a control plant may be a wild type plant.
  • Wild-type (or “WT") refers to a cell or plant that has not been genetically modified to over-express polypeptides according to embodiments of the present invention. Wild-type cells or plants may be used as controls to compare levels of expression and the extent and nature of trait modification in genetically modified (i.e., transgenic) cells or plants in which polypeptide expression is altered or ectopically expressed by, for example, knocking out or over-expressing a gene.
  • a control plant may be a plant overexpressing the wild type OXR protein.
  • yield related traits refers to traits or features which are related to plant yield. Yield-related traits may comprise one or more of the following non- limitative list of features: early flowering time, yield, biomass, seed yield, seed viability and germination efficiency, seed/grain size, starch content of grain, early vigour, greenness index, increased growth rate, delayed senescence of green tissue.
  • yield in general means a measurable produce of economic value, typically related to a specified crop, to an area, and to a period of time.
  • yield of a plant may relate to vegetative biomass (root and/or shoot biomass), to reproductive organs, and/or to propagules (such as seeds) of that plant.
  • yield comprises one or more of and can be measured by assessing one or more of: increased seed yield per plant, increased seed filling rate, increased number of filled seeds, increased harvest index, increased viability/germination efficiency, increased number or size of seeds/capsules/pods, increased growth or increased branching, for example inflorescences with more branches, increased biomass or grain fill.
  • Yield and yield related traits can be increased by at least 5%, at least 10%, 20% or more compared to a control plant.
  • the control plant is a plant that overexpresses the wild type gene.
  • nucleic acid As used herein, the words "nucleic acid”, “nucleic acid sequence”, “nucleotide”,
  • nucleic acid molecule or “polynucleotide” are intended to include DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., mRNA), naturally occurring, mutated, synthetic DNA or RNA molecules, and analogues of the DNA or RNA generated using nucleotide analogues. It can be single-stranded or double-stranded.
  • Such nucleic acids or polynucleotides include, but are not limited to, coding sequences of structural genes, anti-sense sequences, and non-coding regulatory sequences that do not encode mRNAs or protein products. These terms also encompass a gene.
  • genes or “gene sequence” is used broadly to refer to a DNA nucleic acid associated with a biological function.
  • genes may include introns and exons as in the genomic sequence, or may comprise only a coding sequence as in cDNAs, and/or may include cDNAs in combination with regulatory sequences.
  • the OXR (Oxidative Resistance) protein family members have a conserved region located near the C-terminal end called the TLDc domain. Proteins from this family have been studied in yeast (see Elliott and Volkert, Mol. Cell Biol. 24: 3180-3187 (2004)), Drosophila (see Fischer et al., Biochem. Biophys. Res. Immun. 281 : 795-803 (2001)), Anopheles (see Jaramillo-Gutierrez et al., 2010), mice (see Natoli et al., Invest. Ophth. & Vis. Sci. 49: 4561- 4567 (2008)), and humans (see Durand et al, BMC Cell Biol. 8: 13 (2007)).
  • the TLDc domain has been implicated in the prevention and/or repair of oxidative damage to DNA under stress conditions. See Elliott and Volkert, 2004; Durand et al., 2007; and Jaramillo-Gutierrez et al, 2010.
  • the present invention identifies 5 putative members of the OXR protein family in
  • Atlg32520 At2g05590
  • At2g05590 At2g05590.1 and At2g05590.2
  • A4g39870 (OXR4: two splice variants exist, At4g39870.1 and At4g39870.2, these encode the same protein)
  • At5g39590 OXR5
  • At5g06260 OXR5D
  • AtOXR2 At2g05590.2
  • AtOXR4 AtOXR4
  • stress conditions such as heat, UV-B
  • the AtOXR2 and AtOXR4 members were found to suppress the oxidative mutator phenotype when expressed in an E. coli strain that lacks enzymes for the DNA repair system.
  • AtOXRl Over-expression of AtOXRl in plants resulted in the plants having increased shoot biomass. A similar - although not statistically significant - trend was observed as a result of over-expression ⁇ AtOXRA in plants. Moreover, IRGA analysis indicated that the resulting oeOXR2 plants (and to a lesser extent oeOXR4 plants) showed higher values of net photosynthesis and electron transfer rate (ETR). The obtained oeOXR2 and oeOXR4 plants further showed decreased water loss by dehydration (measured as cut rosette water loss vs. time).
  • chimeric OXR polypeptides have improved characteristics compared to wild type polypeptides. This is shown in Figs. 11 and 17.
  • plants that overexpress the chimeric polypeptide have improved characteristics both compared to those that overexpress the wild type polypeptide and to wild type control plants that are not modified.
  • the chimeric protein AtQ42 confers new characteristics that are clearly different from those provided by the overexpression of AtOXR2. The most remarkable benefits are: increased seed yield, shoot height and number of stem branches. AtQ42 plants also show high photosynthetic performance and efficiency in water use and higher specific leaf area and early flowering (Fig 17).
  • the improved characteristics can be additive or synergistic.
  • the "AtQ42" protein was obtained by fusion of the amino-terminal part of the AtOXR4 protein and the carboxyl-end (containing the TLDc domain) of AtOXR2. That is, the AtQ42 protein is preferably composed of the N-terminal half of AtOXBA and the C-terminal half of AtOXR2, the C-terminal half ⁇ 2 including the TLDc domain.
  • AtQ42 chimeric protein Expression of the AtQ42 chimeric protein in Arabidopsis thaliana generated plants with increased shoot and root biomass, early flowering time as well as increased seed production per plant as shown in the examples. Additional advantageous characteristics of plants expressing the AtQ42 protein include thicker stems with increased lignin content, increased development of the root system, and improved photosynthetic performance, as discussed further below. As described in connection with the various embodiments and specific Examples provided herein, the chimeric AtQ42 protein unexpectedly results in plants (expressing the chimeric Q42 construct) having the highly advantageous of combination of (a) increased shoot and root biomass, and (b) increased seed production.
  • the invention relates to an isolated chimeric protein comprising a first polypeptide portion of a first OXR protein and a second polypeptide portion of a second OXR protein.
  • said first and second OXR protein is a plant OXR protein.
  • the invention also relates to an isolated polynucleotide that encodes such a chimeric protein. Such polypeptides and proteins are not naturally occurring. Also within the scope of the invention are uses of such polynucleotides in altering a plant phenotype and related methods.
  • Certain embodiments of the various aspects of the invention relate to Q42 plant chimeric polypeptides or Q42 plant polynucleotides.
  • Q42 chimeric polypeptides designate a polypeptide with a N-terminal plant OXR4 sequence and a C terminal plant OXR2 sequence that comprises the TLDc domain.
  • the polynucleotides described herein include nucleotide sequences that encode such chimeric polypeptides, including Q42 polypeptides, and variant and homolog polypeptides, as well as unique fragments of a coding sequence, or a sequence complementary thereto.
  • the polynucleotides may be, e.g., DNA or RNA, such as mRNA, cRNA, synthetic RNA, genomic DNA, cDNA, synthetic DNA, oligonucleotides, etc.
  • the polynucleotides may include the coding sequence of a homolog polypeptide, in isolation, in combination with additional coding sequences, in combination with non-coding sequences (e.g., introns, regulatory elements such as promoters, enhancers, terminators, and the like), and /or in a vector or host environment in which the polynucleotide encoding a homolog polypeptide is an endogenous or exogenous gene.
  • non-coding sequences e.g., introns, regulatory elements such as promoters, enhancers, terminators, and the like
  • the AtQ42 chimeric protein according to the present invention was generated using genetic engineering tools by combining DNA fragments from two genes encoding AtOXR proteins, AtOXR4 and AtOXR2.
  • the OXR2 sequence comprises the TLDc domain.
  • the invention is not limited to nucleic acid and polypeptide sequences from Arabidopsis and their related methods and uses, but extends to nucleic acid and polypeptide sequences from other plant species, for example those shown in Fig. 12, as well as related methods and uses,.
  • the OXR protein family members can be identified by the presence of a conserved region located near the C-terminal end called the TLDc domain. Accordingly, as used herein, a plant OXR protein is characterized by the presence of a TLDc domain.
  • the TLDc domain has a sequence as set forth in SEQ ID NO. 6 or 12 or a sequence with at least 70%, 80%, 90% or 95% sequence identity thereto.
  • the TLDc domain has been characterised in zebrafish.
  • the TLDc domains from Arabidopsis OXR2 and the zebrafish protein share 38% identity, therefore their tridimensional structures are likely to be very similar. The most interesting difference is the absence of the conserved Cys776 that was described in Oliver et al.
  • the term "functional variant” as used herein and with reference to the various aspects and embodiments of the invention refers to a variant gene or polypeptide sequence or part of the gene or polypeptide sequence which retains the biological function of the full non- variant OXR sequence.
  • a functional variant also comprises a variant of the OXR polynucleotide encoding a polypeptide which has sequence alterations that do not affect function of the resulting protein, for example in non-conserved residues.
  • a “functional variant” has at least 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% overall sequence identity to the non-variant sequence.
  • variants that are substantially identical, i.e. has only some sequence variations, for example in non-conserved residues. Variations may be one or more, for example 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 deletion, addition or substitution of a residue in the sequence.
  • Substantially identical sequences may be polymorphic sequences, i.e., alternative sequences or alleles in a population in which the allelic difference may be as small as one base pair.
  • Substantially identical polynucleotides may also comprise mutagenized sequences, including sequences comprising silent mutations.
  • homolog of a polynucleotide or polypeptide sequence as used herein and with reference to the various aspects and embodiment of the invention encompasses homologs, orthologs and paralogs. Such homologs have at least 25%, 26%, 27%, 28%, 29%, 30%, 31%,
  • the reference sequence is an Arabidopsis thaliana OXR polynucleotide or polypeptide sequence as set out herein.
  • a homolog of a reference sequence for example AtOXR4 polynucleotide has, in increasing order of preference, at least 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%
  • a homolog of a reference sequence for example AtOXR4 polypeptide has, in increasing order of preference, at least 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% overall sequence
  • a homolog of a reference sequence for example AtOXR2 polynucleotide has, in increasing order of preference, at least 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least
  • a homologue of a reference sequence for example AtOXR2 polypeptide has, in increasing order of preference, at least 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% overall sequence
  • the reference sequence is any plant OXR sequences described herein for example
  • OAtOXRl 2, 4 or 5 (including 5D) nucleotide or protein sequences or parts thereof.
  • Other reference sequences include those listed in Fig. 12.
  • Preferred homologs of an Arabidopsis OXR polynucleotide or polypeptide sequences include homologs from maize, rice, wheat, oilseed rape/canola, sorghum, soybean, sunflower, alfalfa, potato, tomato, tobacco, grape, barley, pea, bean, field bean, lettuce, cotton, sugar cane, sugar beet, broccoli or other vegetable brassicas or poplar.
  • the homologous sequence may be selected from one of the sequences shown in Fig. 12.
  • Examples include, but are not limited to, VvOx52 (SEQ ID NO:34), ZmOx52 (SEQ ID NO:35), OsOx52 (SEQ ID NO:36), AtOx5.1 (At5g39590, SEQ ID NO:21), VvOx51 (SEQ ID NO:37), OsOx51 (SEQ ID NO:38), ZmOx51 (SEQ ID NO:39), AtOx4 (SEQ ID NO:2), TaOx4 (SEQ ID NO:40), BoOX4 (SEQ ID NO:41), HvOx4 (SEQ ID NO:42), BrOx4 (SEQ ID NO:43), Gmox4 (SEQ ID NO:44), VvOx4 (SEQ ID NO:45), OsOx41 (SEQ ID NO:46), ZmOx41 (SEQ ID NO:47), ZmOx42 (SEQ
  • Suitable homologs can be identified by sequence comparisons and the presence of conserved domains, specifically the TLDc domain. Hybridization of such sequences may be carried out under stringent conditions. By “stringent conditions” or “stringent hybridization conditions” is intended conditions under which a probe will hybridize to its target sequence to a detectably greater degree than to other sequences (e.g., at least 2-fold over background). [0098] Homologs, orthologs and paralogs of polypeptides described herein may be cloned according to conventional methods. For example, cDNAs can be cloned using mRNA from a plant cell or tissue that expresses one of the polypeptides described herein.
  • mRNA sources may be identified by interrogating Northern blots with probes designed from amino acid sequences within the scope of the present invention, after which a library is prepared from the mRNA obtained from a positive cell or tissue.
  • cDNA is then isolated by, for example, PCR, using primers designed from a gene sequence disclosed herein, or by probing with a partial or complete cDNA or with one or more sets of degenerate probes based on sequences disclosed herein.
  • the cDNA library may be used to transform plant cells, as discussed further below, and expression of the cDNAs of interest is detected using, for example, methods known or described herein, such as microarrays, Northern blots, quantitative PCR, or any other technique for monitoring changes in expression. Genomic clones may also be isolated using similar techniques.
  • the polynucleotides may be cloned, synthesized, altered, mutagenized, or combinations thereof.
  • a nucleic acid can be isolated using standard molecular biology techniques and the sequence information provided herein. Standard recombinant DNA and molecular cloning techniques used to isolate nucleic acids are known to persons skilled in the art.
  • a nucleic acid molecule can be amplified using cDNA or, alternatively, genomic DNA, as a template and appropriate oligonucleotide primers according to standard PCR amplification techniques. The nucleic acid molecule so amplified can be cloned into an appropriate vector and characterized by DNA sequence analysis.
  • the polynucleotides include primers and primer pairs that allow specific amplification of the disclosed polynucleotides or of any specific parts thereof, and probes that selectively or specifically hybridize to nucleic acid molecules of the invention or to any part thereof.
  • Primers may also be used as probes and can be labeled with a detectable marker, such as, for example, a radioisotope, fluorescent compound, bioluminescent compound, a chemiluminescent compound, metal chelator or enzyme.
  • a particular nucleotide sequence employed for hybridization studies or assays may include probe sequences that are complementary to at least about 14-40 nucleotide sequence of a nucleic acid molecule described herein. Probes may comprise 14-20 nucleotides, or even longer where desired, such as 30, 40,
  • fragments may be readily prepared, for example by chemical synthesis of the fragment, by application of nucleic acid amplification technology, or by introducing selected sequences into recombinant vectors for recombinant production.
  • Specific hybridization refers to the binding, duplexing, or hybridizing of a molecule only to a particular nucleotide sequence under stringent conditions when that sequence is present in a complex nucleic acid mixture (e.g., total cellular DNA or RNA). Specific hybridization may accommodate mismatches between the probe and the target sequence depending on the stringency of the hybridization conditions.
  • a complex nucleic acid mixture e.g., total cellular DNA or RNA
  • Stringent hybridization conditions and stringent hybridization wash conditions in the context of nucleic acid hybridization experiments are both sequence- and environment-dependent. Longer sequences hybridize specifically at higher temperatures. An extensive guide to the hybridization of nucleic acids is found in Tijssen, Laboratory Techniques in Biochemistry and Molecular Biology - Hybridization with Nucleic Acid Probes, Part I, Chapter 2, Elsevier, New York, N.Y. (1993).
  • stringent hybridization and wash conditions are selected to be about 5°C below the thermal melting point for the specific sequence at a defined ionic strength and pH.
  • a probe will hybridize specifically to its target sequence, but not to other sequences.
  • the Tm is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe.
  • Stringent conditions are selected to be equal to the Tm for a particular probe.
  • An example of stringent hybridization conditions for Southern or Northern Blot analysis of complementary nucleic acids having more than about 100 complementary residues is overnight hybridization in 50% formamide with 1 mg of heparin at 42°C.
  • An example of highly stringent conditions is 15 minutes in O. lxSSC at 65°C, whereas an example of stringent wash conditions is 15 minutes in 0.2xSSC buffer at 65°C. See Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989).
  • a high stringency wash is preceded by a low stringency wash to remove background probe signal.
  • An example of medium stringency conditions for a duplex of more than about 100 nucleotides is 15 minutes in lxSSC at 45°C.
  • An example of low stringency for a duplex of more than about 100 nucleotides is 15 minutes in 4 to 6xSSC at 40°C.
  • stringent conditions typically involve salt concentrations of less than about 1M Na + ion, typically about 0.01 to 1M Na + ion concentration (or other salts) at pH 7.0-8.3, and the temperature is typically at least about 30°C.
  • Stringent conditions may also be achieved with the addition of destabilizing agents, such as formamide. Additional variations of these conditions will be readily apparent to those skilled in the art.
  • Stringency conditions may be selected such that an oligonucleotide that is perfectly complementary to the coding oligonucleotide hybridizes the coding oligonucleotide with at least about 5 to 10 times higher signal to noise ratio than the ratio for hybridization of the perfectly complementary oligonucleotide to a reference nucleic acid. It may be desirable to select conditions for a particular assay such that a higher signal to noise ratio (e.g., about 15x or more) is obtained.
  • a higher signal to noise ratio e.g., about 15x or more
  • a subject nucleic acid will hybridize to a unique coding oligonucleotide with at least 2 times (2x) or greater signal to noise ratio as compared to hybridization of the coding oligonucleotide to a nucleic acid encoding a known polypeptide.
  • the particular signal will depend on the label used in the relevant assay, e.g., a fluorescent label, a colorimetric label, a radioactive label, or the like.
  • Labeled hybridization or PCR probes for detecting related polynucleotide sequences may be produced by oligolabeling, nick translation, end-labeling, or PCR amplification using a labeled nucleotide.
  • a further indication that two nucleotide sequences are substantially identical is that proteins encoded by the polynucleotides are substantially identical, share an overall three- dimensional structure, or are biologically functional equivalents. Nucleic acid molecules that do not hybridize to each other under stringent conditions are still substantially identical if the corresponding proteins are substantially identical. This may occur, for example, when two nucleotide sequences comprise conservatively substituted variants as permitted by the genetic code. Conservatively substituted variants refer to nucleotide sequences having degenerate codon substitution wherein the third position of one or more (or all) codons is/are substituted with mixed-base and/or deoxyinosine residues.
  • 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.
  • Such manual methods are well known to persons skilled in the art and can include, for example, comparisons of the tertiary structure between a polypeptide sequence encoded by a polynucleotide that comprises a known function with a polypeptide sequence encoded by a nucleotide sequence that has a function not yet determined.
  • Examples of tertiary structure may include predicted alpha helices, beta-sheets, amphipathic helices, leucine zipper motifs, zinc finger motifs, proline-rich regions, cysteine repeat motifs, and the like.
  • Constructs are made by standard methods as shown in the examples by generating a fusion of the N terminal part of the first polynucleotide with the C terminal part of the second polynucleotide.
  • the latter comprises the TLDc domain.
  • the invention relates to an isolated chimeric plant polynucleotide comprising a first portion of a first OXR polynucleotide; and a second portion of a second OXR polynucleotide.
  • the second portion is joined to the first polynucleotide portion.
  • the second portion comprises the TLDc domain.
  • the isolated plant polynucleotide comprises a first portion of a first OXR4 polynucleotide and a second portion of a second OXR2 polynucleotide.
  • the second portion is joined to the first polynucleotide portion.
  • the two portions can be sequences of the same or different plant species.
  • a first portion of a AtOXR4 polynucleotide may be joined to a second portion of a OXR2 polynucleotide from another plant, for example a crop plant.
  • a first portion of a plant OXR4 for example from a crop plant, may be joined to a second portion of a AtOXR2 polynucleotide.
  • the first OXR polynucleotide comprises SEQ ID NO: 1, 15 or
  • said second polynucleotide comprises SEQ ID NO: 7, 17 or 18, a functional variant or homolog thereof.
  • said a functional variant or homolog of SEQ ID NO: 1 , 15 or 16 has at least 50%, 60%, 70%, 80% or 90% sequence identity to SEQ ID NO: 1, 15 or 16.
  • said a functional variant or homolog of SEQ ID NO: 7 has at least 50%, 60%, 70%, 80% or 90% sequence identity to SEQ ID NO: 7, 17 or 18.
  • the first polynucleotide comprises a first region comprising the full-length nucleotide sequence of SEQ ID NO: 1, a functional variant or homolog thereof; and the second polynucleotide comprising the full-length nucleotide sequence of SEQ ID NO: 7, a functional variant or homolog thereof.
  • an isolated, non-naturally occurring Q42 polynucleotide may comprise: a first region comprising the full-length OXR4 amino-terminal encoding nucleotide sequence represented by SEQ ID NO: 3, a functional variant or homolog thereof and a second region comprising the full-length OXR2 carboxyl-terminal encoding nucleotide sequence represented by SEQ ID NO: 11, a functional variant or homolog thereof.
  • Variant Q42 polynucleotides or homologs of AtQ42 may comprise a first region having at least 80% sequence identity with the full-length OXR4 amino-terminal carboxyl-encoding nucleotide sequence represented by SEQ ID NO: 3, the first region preferably having at least 85% sequence identity, or at least 90% sequence identity, or at least 91% sequence identity, or at least 92% sequence identity, or at least 93% sequence identity, or at least 94% sequence identity, or at least 95% sequence identity, or at least 96% sequence identity, or at least 97% sequence identity, or at least 98% sequence identity, or at least 99% sequence identity with the full-length OXR4 nucleotide sequence of SEQ ID NO: 3; and a second region having at least 80% sequence identity with the full-length OXR2 amino-terminal carboxyl-encoding nucleotide sequence represented by SEQ ID NO: 11, the second region preferably having at least 85% sequence identity, or at least 90% sequence identity, or at least 91% sequence
  • the isolated polynucleotides may also include polynucleotides encoding the OXR4 polypeptide of SEQ ID NO: 4 or 6, a functional variant or homolog thereof and the OXR2 polypeptide of SEQ ID NO: 12 or 14, a functional variant or homolog thereof.
  • Any amino-terminal encoding part from any plant OXR polynucleotide may be combined with any carboxy-terminal part from a second plant OXR polynucleotide. Examples are shown in Fig. 19.
  • Aspects of the invention and embodiments of certain aspects of the invention also relate to isolated chimeric polypeptides encoded by the polynucleotide sequences described herein. Representative polypeptides according to embodiments include the full-length amino acid sequences of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 12, and/or SEQ ID NO: 14, and functional variants and homologs of these sequences.
  • aspects of the invention and embodiments of certain aspects of the invention also relate to an isolated chimeric plant protein comprising a first polypeptide portion of a first OXR protein and a second polypeptide portion of a second OXR protein.
  • the second polypeptide portion is joined to the first polypeptide portion.
  • the isolated chimeric protein comprises a first polypeptide portion of a first OXR protein wherein said first OXR protein comprises AtOXR4 amino acid sequence of SEQ ID NO: 2, a functional variant or homolog thereof and a second polypeptide portion of a second OXR protein, wherein the second OXR protein comprises an AtOXR2 amino acid sequence of SEQ ID NO: 8, a functional variant or homolog thereof.
  • Percentage identity values of variant and homologs are specified elsewhere herein, but in one embodiment, the sequence identity is at least 80% or 90% sequence identity.
  • the chimeric protein is a Q42 polypeptide wherein the first polypeptide portion comprises an AtOXR4 amino-terminal amino acid sequence of SEQ ID NO:
  • AtOXR2 carboxy-terminal amino acid sequence of SEQ ID NO: 12, a functional variant or homolog thereof.
  • Examples include a first region having at least 80% sequence identity with the amino -terminated AtOXR4 polypeptide of SEQ ID NO: 4, the first region preferably having at least 85% sequence identity, or at least 90% sequence identity, or at least 91% sequence identity, or at least 92% sequence identity, or at least 93% sequence identity, or at least 94% sequence identity, or at least 95% sequence identity, or at least 96% sequence identity, or at least 97% sequence identity, or at least 98% sequence identity, or at least 99% sequence identity with the amino -terminated AtOXR4 polypeptide of SEQ ID NO: 4; and a second region having at least
  • AtOXR2 carboxyl-terminated polypeptide of SEQ ID NO: 12 the second region preferably having at least 85% sequence identity, or at least 90% sequence identity, or at least 91% sequence identity, or at least 92% sequence identity, or at least 93% sequence identity, or at least 94% sequence identity, or at least 95% sequence identity, or at least 96% sequence identity, or at least 97% sequence identity, or at least 98% sequence identity, or at least 99% sequence identity with the carboxyl-terminated AtOXR2 polypeptide of SEQ ID NO: 12.
  • the chimeric protein comprises: (1) a first polypeptide portion that comprises the amino-terminal amino acid sequence AtOXRA (AT4G39870) represented by SEQ ID NO: 4; and (2) a second polypeptide portion that comprises the TLDc domain of the carboxyl-terminal amino acid sequence AtOXR2 (AT2G05590) represented by SEQ ID NO: 12.
  • the chimeric protein comprises SEQ ID NO: 14 (encoded by
  • Any amino-terminal part from any plant OXR polypeptide may be combined with any carboxy-terminal part from a second plant OXR polypeptide.
  • the chimeric polypeptide may comprise any combination of a N-terminal part of a first polypeptide selected from AtOXRl, AtOXR2 AtOXR4 and AtOXR5, a variant or homolog thereof and a C- terminal part from a second polypeptide selected from AtOXRl, AtOXR2, AtOXR4 and AtOXR5 a variant or homolog thereof.
  • Q12, Q21, Q41, Q14, Q51, Q15, Q24, Q42, Q25 and Q52 for example AtQ12, AtQ21, AtQ41, AtQ14, At Q51, AtQ15, AtQ24, AtQ42, AtQ25 and AtQ52 and homologs or variants thereof.
  • AtOXR4 and AtOXR5 their N and C terminal parts and constructs are shown herein, see Fig 19.
  • the chimeric polypeptide includes other combinations of first and second polypeptide portions.
  • said first OXR polypeptide may comprise a polypeptide portion which has a sequence having at least 50%, 60%, 70%, 80% or 90% sequence identity with the AtOXR2 amino-terminal encoding nucleotide sequence of
  • SEQ ID NO: 9 and a second OXR polypeptide portion which has a sequence having at least
  • said first OXR polypeptide may comprise a polypeptide portion which has a sequence having at least 50%, 60%, 70%, 80% or 90% sequence identity with the AtOXR2 of SEQ ID NO: 8 and a second OXR polypeptide which has a sequence having at least 50%, 60%, 70%, 80% or 90% sequence identity with the AtOXR5 sequence of SEQ ID NO: 21 or 22 resulting in an AtQ25 polypeptide construct.
  • said first OXR polypeptide may comprise a polypeptide portion which has a sequence having at least 50%, 60%, 70%, 80% or 90% sequence identity with the AtOXR5 sequence of SEQ ID NO: 21 or 22 and a second OXR polypeptide portion which has a sequence having at least 50%, 60%, 70%, 80% or 90% sequence identity with the AtOXR2 sequence of SEQ ID NO: 8 resulting in a AtQ52 polypeptide construct.
  • Q14, Q51, Q15 for example AtQ12, AtQ21, AtQ41, AtQ14, AtQ51, AtQ15 and homologs or variants thereof.
  • the chimeric polypeptide or polynucleotide is selected from the following Q42 protein sequence (SEQ ID NO: 14), Q42 nucleotide sequence (SEQ ID NO:13) Q25 protein sequence (SEQ ID NO:26), Q25 nucleotide sequence (SEQ ID NO:27), Q24 protein sequence (SEQ ID NO:28), Q24 nucleotide sequence (SEQ ID NO:29), Q52 protein sequence (SEQ ID NO:30), Q52 nucleotide sequence (SEQ ID NO:31), Q52* (based on AT5G06260.1 OXR5D) protein sequence (SEQ ID NO:32), Q52 nucleotide sequence (SEQ ID NO: 33).
  • Isolated polypeptides according to embodiments may be purified and characterized using a variety of standard techniques that are known to persons skilled in the art. See Schroder et al, The Peptides, Academic Press, New York, NY (1965).
  • the coding region for the chimeric protein may be cloned in a vector containing a strong constitutive promoter in order to generate high expression levels of the protein of interest in most plant tissues.
  • An AtQ42 construct was further used to transform Arabidopsis plants to produce transgenic plants exhibiting different expression levels of the chimeric protein. Plants produced according to the methods described herein were analyzed both in standard growth conditions and in growth conditions in which they were subjected to abiotic stress factors. [00130] The inventors found that, when grown under optimal and stress (water deficit and salt stress) conditions, 35S:AtQ42 transgenic plants have increased shoot and root biomass, seed production, and photosynthetic efficiency. Notably, transgenic plants bearing the construct 35S:AtQ42 not only demonstrated improved tolerance to the various stress conditions, but also exhibited higher yields than the corresponding WT control plants (yield evaluated as a measure of seed production).
  • the chimeric protein confers improved and desirable agronomical traits on transgenic plants produced according to methods described herein.
  • Increased root biomass is a trait particularly important in legumes (e.g., soybean, alfalfa) for the rapid development of a root system in order to allow faster modulation for nitrogen fixation and soil nutrition.
  • the development of the root system is also a desirable characteristic for any crop, as this promotes irrigation and aeration of the soil and prevents erosion.
  • An example is the cultivation of sunflower (Helianthus annuus L.), which is cultivated in marginal areas (and displaced by soybean). Areas of sunflower cultivation are often characterized by having extended periods of water shortage. Not only does greater root biomass appear to circumvent problems of water stress, but increased root development is also important to prevent lodging of the plants.
  • Increased biomass in plants resulting from the expression of chimeric protein described herein is of particular importance for fodder crops and plants used for biofuel production.
  • Increased stem diameter (accompanied by an increase in the diameter of conductor vessels) and increased lignification are properties of particular interest for crops, such as sugarcane, or for growing trees that are used for wood production.
  • crops such as sugarcane, or for growing trees that are used for wood production.
  • plants with hardy stems are less sensitive to lodging and breakage that causes losses of yield.
  • plants with more lignified stems could be more resistant to pathogen attack (e.g., fungi of the genus Sclerotinia sp. for sunflower or larvae of Lepidoptera for leaf miners or stem borers in maize).
  • embodiments of the invention relate to, among other things, the isolation and functional characterization of: the chimeric polypeptides and sequences complementary thereto; nucleotide sequences that encode the polypeptides and sequences complementary thereto; and unique fragments of a coding sequence of a sequence complementary thereto.
  • Embodiments of the invention also relate to transforming a host cell using the nucleotides and polypeptides described herein, and modifying plant traits or conferring desirable traits upon host plants to produce transgenic plants having improved stress tolerance and yield in optimal and stress conditions compared to corresponding WT control plants. Transgenic plants transformed with constructs as described herein are also within the scope of the present invention.
  • the invention also relates to isolated nucleic acid and polypeptide sequences as identified in any of SEQ ID NO. 1 to 12 and vectors comprising such sequences.
  • polynucleic acid sequences can be used in methods for producing plants and in methods for modulating a plant phenotype.
  • the invention also relates to nucleic acid constructs, for example comprising the chimeric polynucleotides as described herein, for example a Q42 polynucleotide.
  • Nucleic acid constructs can comprise a plant OXR polynucleotide or part thereof. In one embodiment, the nucleic acid construct comprises any of SEQ ID NO. 1 to 12.
  • Embodiments include recombinant constructs comprising one or more of the polynucleotide sequences described herein.
  • the constructs typically comprise a vector, such as a plasmid, a cosmid, a phage, a virus, a bacterial artificial chromosome (BAC), a yeast artificial chromosome (YAC), or the like, into which a polynucleotide sequence as described herein has been inserted, in a forward or reverse orientation.
  • the constructs may further comprise regulatory sequences, including, e.g., a promoter that is operably linked to the sequence.
  • Vectors and promoters suitable for recombinant constructs of the present invention may include those generally known to persons having skill in the art and/or described herein.
  • Constructs suitable for use in embodiments may contain a "signal sequence" or
  • leader sequence to facilitate co-translational or post-translational transport of the polypeptide of interest to certain intracellular structures, such as the chloroplast (or other plastid), endoplasmic reticulum, or Golgi apparatus, or to be secreted.
  • Such sequences include leader sequences targeting transport and/or glycosylation by passage into the endoplasmic reticulum, vacuoles, plastids including chloroplasts, mitochondria, and the like.
  • the constructs may be engineered to contain a signal peptide to facilitate transfer of the peptide to the endoplasmic reticulum.
  • a signal sequence is known or suspected to result in co-translational or post-translational peptide transport across the cell membrane.
  • Leader sequence refers to any sequence that, when translated, results in an amino acid sequence sufficient to trigger co-translational transport of the peptide chain to a sub-cellular organelle.
  • Plant expression cassettes may also contain an intron, such that mRNA processing of the intron is required for expression
  • Suitable constructs may also contain 5' and 3' untranslated regions.
  • a 3' untranslated region is a polynucleotide located downstream of a coding sequence.
  • a 5' untranslated region is a polynucleotide located upstream of a coding sequence.
  • the termination region may be native to the transcriptional initiation region, the sequence described herein, or may be derived from another source. Suitable termination regions may be derived from the Ti-plasmid of A. tumefaciens, such as the octopine synthase and nopaline synthase termination regions, or the termination region of a plant gene, such as soybean storage protein. See Guerineau et al., Mol. Gen. Genet. 262: 141-144 (1991); Proudfoot, Cell 64:671-674 (1991); Sanfacon et al., Genes Dev.
  • vectors are plant integrating vectors in that upon transformation, the vectors integrate a portion of vector DNA into the genome of the host plant.
  • an isolated recombinant expression vector may comprise a polynucleotide as described herein, wherein expression of the vector in a host cell results in the plant's increased growth and yield under normal or water-limited conditions and/or increased tolerance to environmental stress as compared to a wild type variety of the host cell.
  • recombinant expression vectors comprise a polynucleotide described herein in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vectors may include one or more regulatory sequence (or sequences) selected on the basis of the host cell to be used for expression, which is operatively linked to the nucleic acid sequence to be expressed. That is, the nucleotide sequence of interest is linked to the regulatory sequence(s) in a manner that allows for expression of the nucleotide sequence (e.g., in a bacterial or plant host cell when the vector is introduced into the host cell).
  • a regulatory element generally can increase or decrease the amount of transcription of a nucleotide sequence operatively linked to the element with respect to the level at which the nucleotide sequence would be transcribed absent the regulatory element.
  • Such regulatory sequences are described, for example, in: Goeddel, "Gene Expression Technology: Methods in Enzymology,” Academic Press, San Diego, Calif. (1990); and Gruber and Crosby, Methods in Plant Molecular Biology and Biotechnology, eds. Glick and Thompson, Chapter 7:89-108, CRC Press, Boca Raton, Fla. (including the references cited therein).
  • Stress-regulated regulatory elements which regulate expression of an operatively linked nucleotide sequence in a plant in response to a stress condition.
  • the plant stress-regulated regulatory elements may be isolated from a polynucleotide sequence of a plant stress-regulated gene. Methods for identifying and isolating a stress-regulated regulatory element from the polynucleotides, or genomic DNA clones corresponding thereto, are known to persons skilled in the art. For example, methods of making deletion constructs or linker-scanner constructs can be used to identify nucleotide sequences that are responsive to a stress condition. Generally, such constructs include a reporter gene operatively linked to the sequence to be examined for regulatory activity.
  • a plant stress-regulated regulatory element can be defined within a sequence of about 500 nucleotides or fewer, generally at least about 200 nucleotides or fewer, or about 50 to 100 nucleotides.
  • the minimal (core) sequence required for regulating a stress response of a plant is identified.
  • the nucleotide sequences of the genes of a cluster can also be examined using a homology search engine to identify sequences of conserved identity, particularly in the nucleotide sequence upstream of the transcription start site.
  • Regulatory elements may be isolated from a naturally occurring genomic DNA sequence or can be synthetic (e.g., a synthetic promoter).
  • the regulatory elements can be constitutively expressed so as to maintain gene expression at a relative level of activity (basal level), or can be regulated.
  • Constitutively expressed regulatory elements can be expressed in any cell type, or can be tissue specific (expressed only in particular cell types), or phase specific (expressed only during particular developmental or growth stages of a plant cell).
  • Regulatory elements e.g., a tissue specific, phase specific, or inducible regulatory element
  • useful in constructing a recombinant polynucleotide or in practicing methods described herein include regulatory elements that are found in a plant genome.
  • the regulatory elements may be from an organism other than a plant, such as a plant or animal virus, or an animal or other multicellular organism.
  • Plant expression vectors suitable for use in embodiments may comprise one or more DNA vector(s) for achieving plant transformation.
  • plant transformation vectors that include one or more cloned plant coding sequences (genomic or cDNA) under the transcriptional control of 5' and 3' regulatory sequences as a dominant selectable marker.
  • Such plant transformation vectors typically also contain a promoter, a transcription initiation start site, an RNA processing signal, a transcription termination site, and/or a polyadenylation signal.
  • expression vectors include RNA processing signals that can be positioned within, upstream or downstream of the coding sequence.
  • the expression vectors may include additional regulatory sequences from the 3 '-untranslated region of plant genes.
  • Initiation signals may also be used to aid in efficient translation of coding sequences. These signals can include, e.g., an ATG initiation codon and adjacent sequences. In cases where a coding sequence, its initiation codon and upstream sequences are inserted into the appropriate expression vector, no additional translational control signals may be needed. However, in cases where only coding sequences, or a portion thereof, are inserted, exogenous transcriptional control signals including the ATG initiation codon can be separately provided. The initiation codon is provided in the correct reading frame to facilitate transcription. Exogenous transcription elements and initiation codons can be of various origins, both natural and synthetic. The efficiency of expression can be enhanced by including enhancers appropriate for the cell system in use.
  • Binary vectors are plant transformation vectors that utilize two non-contiguous
  • DNA vectors to encode all requisite cis- and trans-acting functions for transformation of plant cells See Hellens et al., Trends in Plant Science 5:446-451 (2000).
  • Binary vectors, as well as vectors with helper plasmids, are most often used for Agrobacterium-mediated transformations, in which the size and complexity of DNA segments needed to achieve efficient transformation is large, and in which it is therefore advantageous to separate functions among separate DNA molecules.
  • Binary vectors also typically contain a plasmid vector that contains the cis-acting sequence required for T-DNA transfer (such as left border and right border), a selectable marker that is engineered to be capable of expression in a plant cell, and a polynucleotide of interest (i.e., a polynucleotide engineered to be capable of expression in a plant cell for which generation of transgenic plants is desired). Sequences required for bacterial replication may also be present on this plasmid vector.
  • the cis-acting sequences are arranged in a fashion to allow for efficient transfer into plant cells and expression therein. For example, a selectable marker sequence and a sequence of interest are typically located between the left and right borders.
  • a second plasmid vector typically contains virulence functions (Vir genes) that allow infection of plant cells by Agrobacterium, and transfer of DNA by cleavage at border sequences and vir-mediated DNA transfer, as understood by persons skilled in the art. See e.g., Hellens et al, Trends in Plant Science 5:446-451 (2000).
  • virulence functions Vir genes
  • Several types of Agrobacterium strains ⁇ e.g., LBA4404, GV3101, EHA101, EHA105, etc.) may be used for plant transformation.
  • the second plasmid vector is typically not necessary for introduction of polynucleotides into plants by other methods, such as by microprojection, microinjection, electroporation, etc.
  • the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of polypeptide desired, etc.
  • the expression vectors can be introduced into host cells to thereby produce polypeptides or peptides, including fusion polypeptides or peptides, encoded by nucleic acids as described herein.
  • regulatory elements may include promoters, enhancers, and other expression control elements (e.g., polyadenylation signals). Regulatory sequences are well known in the art and include those that direct constitutive expression of nucleotide sequence in many types of host cells and those that direct expression of the nucleotide sequence only in certain host cells or under certain conditions.
  • the regulatory element may be a promoter. Several domains within a plant promoter region are necessary for the full function of the promoter. The first of these domains within the promoter region lies immediately upstream of the structural gene and forms the "core promoter region" containing consensus sequences.
  • the core promoter region represents a transcription initiation sequence that defines the transcription start point for the structural gene.
  • the presence of the core promoter region defines a sequence as being a promoter; that is, if the region is absent, the promoter is non-functional.
  • the core promoter region on its own is, however, insufficient to provide full promoter activity.
  • a series of regulatory sequences upstream of the core constitute the remainder of the promoter. These regulatory sequences determine expression levels, the spatial and temporal patterns of expression and, for the specific subset of promoters, the expression level under inductive conditions (e.g., light, temperature, chemicals, hormones).
  • a DNA segment representing the promoter region is removed from the 5 '-region of the gene of interest and operably linked to the coding sequence of a marker (reporter) gene by recombinant DNA techniques known to persons skilled in the art.
  • the reporter gene is operably linked downstream of the promoter, so that transcripts initiating at the promoter proceed through the reporter gene. Reporter genes generally encode proteins that are easily measured.
  • the construct containing the reporter gene under the control of the promoter is then introduced into an appropriate plant cell by transfection techniques known to persons skilled in the art.
  • the level of enzyme activity corresponds to the amount of enzyme produced, which, in turn, reveals the level of expression from the promoter of interest.
  • This level of expression can be compared to that achieved using other promoters to determine the relative strength of the promoter under study.
  • the level of the reporter mRNA can be measured directly (e.g., by Northern blot analysis).
  • mutational and/or deletional analyses may be performed to determine the minimal region and/or sequences required to initiate transcription. Sequences may be deleted at the 5'-end of the promoter region and/or at the 3 '-end of the promoter region, and nucleotide substitutions may be introduced. These constructs may then be introduced into cells and their activity determined. [00155]
  • the promoter selection depends on the temporal and spatial requirements for expression, as well as on the target species. In some embodiments, expression in multiple tissues may be desirable, while in others, tissue-specific (e.g., leaf-specific, seed-specific, petal-specific, anther-specific, or pith-specific) expression is desirable.
  • promoters from dicotyledons have been shown to be operational in monocotyledons, and vice versa
  • dicotyledonous promoters are ideally selected for expression in dicotyledons
  • monocotyledonous promoters are ideally selected for expression in monocotyledons.
  • the origin or source of the promoter selected it is sufficient that the selected promoter is operational in driving the expression of a nucleotide sequence described herein in the particular cell. That is, the promoter used in embodiments of the present invention may be any nucleotide sequence that shows transcriptional activity in the target (host) plant (cell, seed, etc.).
  • Useful promoters include, but are not limited to, constitutive, inducible, temporally regulated, developmentally regulated, spatially-regulated, chemically regulated, stress-responsive, tissue-specific, viral and synthetic promoters.
  • Promoter sequences are generally understood to be strong or weak.
  • a strong promoter provides for a high level of gene expression, whereas a weak promoter provides for a low level of gene expression.
  • An inducible promoter is a promoter that allows gene expression to be turned on and off in response to an exogenously added agent, or to an environmental or developmental stimulus.
  • An isolated promoter sequence that is a strong promoter for heterologous nucleic acid is typically advantageous because it provides for a sufficient level of gene expression to allow for easy detection and selection of transformed cells, while providing a high level of gene expression when desired.
  • the promoter may be native or analogous, or foreign or heterologous, to the plant host and/or to the DNA sequence disclosed herein. Where the promoter is native or endogenous to the plant host, it is intended that the promoter is found in the native plant into which the promoter is introduced. Where the promoter is foreign or heterologous to the DNA sequence disclosed herein, the promoter is not the native or naturally occurring promoter for the operably linked DNA sequence disclosed herein.
  • the promoter selected in embodiments may be "inducible" or "constitutive.” An inducible promoter is a promoter that is under environmental control, whereas a constitutive promoter is a promoter that is active under most environmental conditions.
  • the promoter may be naturally occurring, composed of portions of various naturally occurring promoters, or partially or totally synthetic.
  • Guidance for the design of promoters is provided by studies of promoter structure in Harley et al., Nucleic Acids Res. 15:2343-61 (1987). Additionally, the location of the promoter relative to the transcription start position may be optimized. See e.g., Roberts et al., Proc. Natl. Acad. Sci. 76:760-764, USA (1979).
  • Exemplary constitutive promoters for use in plants according embodiments may include promoters from plant viruses, such as the peanut chlorotic streak caulimovirus (PC1SV) promoter, the 35S promoter from cauliflower mosaic virus (CaMV), promoters of Chlorella virus methyltransferase genes, the full-length transcript promoter from figwort mosaic virus (FMV); the promoters from such genes as rice actin, ubiquitin, pEMU, MAS, maize H4 histone, Brassica napus ALS4; and promoters of various Agrobacterium genes.
  • PC1SV peanut chlorotic streak caulimovirus
  • CaMV cauliflower mosaic virus
  • FMV figwort mosaic virus
  • Exemplary inducible promoters for use in plants according to embodiments may include the promoter from the ACE1 system that responds to copper, the promoter of the maize
  • Another inducible promoter that may be used in plants described herein is one that responds to an inducting agent to which plants do not normally respond.
  • An inducible promoter of this type may be the inducible promoter from a steroid hormone gene, the transcriptional activity of which is induced by glucocorticosteroid hormone, or the recent application of a chimeric transcription activator, SVE, for use in an estrogen receptor-based inducible plant expression system activated by estradiol. See Schena et al., Proc. Natl. Acad. Sci. 88: 104-21
  • inducible promoters suitable for use in embodiments may be selected from promoters described in EP 332104, PCT International
  • the promoter may include, or be modified to include, one or more enhancer elements to thereby provide for higher levels of transcription.
  • enhancer elements for use in plants described herein include, for example, the PC1SV enhancer element, the CaMV 35S enhancer element and the FMV enhancer element. See Maiti et al., Transgenic Res. 6:143-156 (1997); PCT International Publication No. WO 96/23898; and U.S. Patents Nos: 5,850,019; 5,106,739; and 5,164,316.
  • the introduced polynucleotide may be maintained in the plant cell stably if it is incorporated into a non-chromosomal autonomous replicon or integrated into the plant chromosomes. Homozygous plants that are stably transformed are preferred. Any of the sequences described herein may be incorporated into a cassette or vector for expression in plants.
  • a plant expression cassette according to embodiments preferably contains regulatory sequences capable of riving gene expression in plant cells that are operatively linked so that each sequence can fulfill its function, e.g., termination of transcription by polyadenylation signals.
  • Embodiments include an expression cassette comprising at least: (1) a constitutive, inducible, or tissue-specific promoter; and (2) a recombinant polynucleotide having a polynucleotide sequence, or a complementary polynucleotide sequence thereof, selected from the group consisting of a polynucleotide sequence encoding: (a) a polypeptide sequence having a sequence as described herein; (b) a polynucleotide sequence selected from the polynucleotides described herein; or sequence variants ⁇ e.g., allelic or splice variants) of the polynucleotide sequences referenced in (a) or (b) above.
  • one or more plant expression cassette ⁇ i.e., a Q42 open reading frame operably linked to a promoter
  • a plant transformation vector which allows for the transformation of DNA into a cell.
  • Such expression cassettes may be organized into more than one vector DNA molecule.
  • the invention also relates to transgenic plants or parts thereof comprising a nucleic acid construct comprising a polynucleotide, a vector or expression cassette as described herein.
  • the construct comprises a chimeric nucleic acid, encoding a chimeric OXR polypeptide as described herein, for example a Q42 polypeptide.
  • Constructs comprising the polynucleotide, vectors and expression cassettes are described herein and include, for example, a Q42 chimeric construct, as described herein.
  • the plant polynucleotide comprises a first portion of a first
  • OXR polynucleotide and a second portion of a second OXR polynucleotide.
  • the first polynucleotide comprises SEQ ID NO: 1 , a functional variant or homolog thereof and the second polynucleotide comprises SEQ ID NO: 7, a functional variant or homolog thereof.
  • the first polynucleotide comprises a nucleotide sequence having at least 50%, 60%, 70%, 80% or 90% sequence identity with the full-length AtOXR4 amino-terminal encoding nucleotide sequence of SEQ ID NO: 1 ; and the second polynucleotide comprises a nucleotide sequence having at least 50%, 60%, 70%, 80% or 90% sequence identity with the full-length AtOXR2 carboxy -terminal encoding nucleotide sequence of SEQ ID NO: 7.
  • the first region comprises the AtOXR4 amino- terminal encoding nucleotide sequence represented by SEQ ID NO: 3, a functional variant or homolog thereof; and the second region comprises the AtOXR2 carboxy-terminal encoding nucleotide sequence represented by SEQ ID NO: 11, a functional variant or homolog thereof.
  • the first region comprises a sequence with at least
  • the second region comprises a sequence with at least 50%, 60%, 70%, 80% or 90% sequence identity with the OXR2 carboxy-terminal encoding nucleotide sequence represented by SEQ ID NO: 11.
  • the polynucleotide has the full-length nucleotide sequence of SEQ ID NO: 13 or sequence with at least 50%, 60%, 70%, 80% or 90% sequence identity thereto.
  • a plant according to the various aspects of the invention, including the transgenic plants, methods and uses described herein may be a monocot or a dicot plant.
  • a dicot plant may be selected from the families including, but not limited to Asteraceae, Brassicaceae (e.g. Brassica napus), Chenopodiaceae, Cucurbitaceae, Leguminosae (Caesalpiniaceae, Aesalpiniaceae Mimosaceae, Papilionaceae or Fabaceae), Malvaceae, Rosaceae or Solanaceae.
  • the plant may be selected from lettuce, sunflower, Arabidopsis, broccoli, spinach, water melon, squash, cabbage, tomato, potato, yam, capsicum, tobacco, cotton, okra, apple, rose, strawberry, alfalfa, bean, soybean, field (fava) bean, pea, lentil, peanut, chickpea, apricots, pears, peach, grape vine, bell pepper, chilli, sunflower or citrus species.
  • a monocot plant may, for example, be selected from the families Arecaceae,
  • the plant may be a cereal crop, such as maize, wheat, rice, barley, oat, sorghum, rye, millet, buckwheat, or a grass crop such as Lolium species or Festuca species, or a crop such as sugar cane, onion, leek, yam or banana.
  • a cereal crop such as maize, wheat, rice, barley, oat, sorghum, rye, millet, buckwheat, or a grass crop such as Lolium species or Festuca species, or a crop such as sugar cane, onion, leek, yam or banana.
  • biofuel and bioenergy crops such as rape/canola, sugar cane, sweet sorghum, Panicum virgatum (switchgrass), linseed, lupin and willow, poplar, poplar hybrids, Miscanthus or gymnosperms, such as loblolly pine.
  • high erucic acid oil seed rape, linseed and for amenity purposes (e.g. turf grasses for golf courses), ornamentals for public and private gardens (e.g. snapdragon, petunia, roses, geranium, Nicotiana sp.) and plants and cut flowers for the home (African violets, Begonias, chrysanthemums, geraniums, Coleus spider plants, Dracaena, rubber plant).
  • the plant is a crop plant.
  • crop plant is meant any plant which is grown on a commercial scale for human or animal consumption or use.
  • the plant is a cereal.
  • Most preferred plants are maize, rice, wheat, oilseed rape/canola, sorghum, soybean, sunflower, alfalfa, potato, tomato, tobacco, grape, barley, pea, bean, field bean, lettuce, cotton, sugar cane, sugar beet, broccoli or other vegetable brassicas or poplar.
  • the plant is selected from a plant as defined above, for example maize, rice, wheat, oilseed rape/canola, sorghum, soybean, sunflower, alfalfa, potato, tomato, tobacco, grape, barley, pea, bean, field bean, lettuce, cotton, sugar cane, sugar beet, broccoli or other vegetable brassicas or poplar and the transgene is a chimeric At polynucleotide as described herein, for example AtQ42.
  • the transgene is a chimeric polynucleotide which includes OXR sequences of said plant.
  • a transgenic maize plant according to the invention may express a Zm chimeric OXR polynucleotide.
  • the plant includes in its genome a stably integrated transgene which comprises the chimeric gene sequence.
  • the plants may be homozygous for the polynucleotide disclosed herein, i.e., a transgenic plant that contains two added sequences, one sequence at the same locus on each chromosome of a chromosome pair.
  • a homozygous transgenic plant according to these embodiments can be obtained by sexually mating (selfing) an independent segregating transgenic plant that contains the added sequences disclosed herein, germinating some of the seed produced and analyzing the resulting plants produced for enhanced enzyme activity and/or increased plant yield relative to a control (native, non-transgenic) or an independent segregant transgenic plant.
  • a control native, non-transgenic
  • an independent segregant transgenic plant Persons skilled in the art will understand that two different transgenic plants may be mated to produce offspring that contain two independently segregating added heterologous polynucleotides.
  • the present invention provides for transformed seeds (also referred to as transgenic seeds) having a polynucleotide as disclosed herein ⁇ e.g., an expression cassette as disclosed herein) stably incorporated into their genome.
  • the invention also extends to harvestable parts of a plant of the invention as described above such as, but not limited to seeds, leaves, fruits, flowers, stems, roots, rhizomes, tubers and bulbs.
  • the invention furthermore relates to products derived, preferably directly derived, from a harvestable part of such a plant, such as dry pellets or powders, oil, fat and fatty acids, starch or proteins.
  • the invention also relates to food products and food supplements comprising the plant of the invention or parts thereof.
  • Transformation and regeneration of both monocotyledonous and dicotyledonous plant cells has become routine, and the selection of the most appropriate transformation technique can be readily determined by the person skilled in the art.
  • the choice of method will typically vary based on the type of plant to be transformed, as persons skilled in the art will recognize the suitability of particular methods for given plant types.
  • Suitable methods for use in embodiments may include, but are not limited to: electroporation of plant protoplasts; liposome- mediated transformation; polyethylene glycol (PEG) mediated transformation; transformation using viruses; micro-injection of plant cells; micro-projectile bombardment of plant cells; vacuum infiltration; and Agrobacterium tumefaciens mediated transformation.
  • transgenic plants may be performed by methods known to persons skilled in the art, including: introduction of heterologous DNA by Agrobacterium into plant cells (Agrobacterium-mediated transformation); bombardment of plant cells with heterologous foreign DNA adhered to particles; and various other non-particle direct- mediated methods, such as microinjection, electroporation, application of Ti plasmid, Ri plasmid, or plant virus vector, and direct DNA transformation.
  • Agrobacterium-mediated transformation introduction of heterologous DNA by Agrobacterium into plant cells
  • bombardment of plant cells with heterologous foreign DNA adhered to particles and various other non-particle direct- mediated methods, such as microinjection, electroporation, application of Ti plasmid, Ri plasmid, or plant virus vector, and direct DNA transformation.
  • the first method involves co-cultivation of Agrobacterium with cultured isolated protoplasts. This method requires an established culture system that allows culturing protoplasts and plant regeneration from cultured protoplasts.
  • the second method involves transformation of cells or tissues with Agrobacterium. This method requires that the plant cells or tissues can be transformed by Agrobacterium, and that the transformed cells or tissues can be induced to regenerate into whole plants.
  • the third method involves the transformation of seeds, apices or meristems with Agrobacterium. This method requires micropropagation.
  • Agrobacterium-mediated transformation methods may be enhanced by, e.g., including in the Agrobacterium culture a natural wound response molecule, such as acetosyringone (AS), which has been shown to enhance transformation efficiency with Agrobacterium tumefaciens.
  • AS acetosyringone
  • transformation efficiency may be enhanced by wounding the target tissue to be transformed by, e.g., punching, maceration, bombardment with microprojectiles, etc. See e.g., Bidney et al, Plant Molec. Biol. 18:301 -313 (1992).
  • plant cells may be transfected with vectors via particle bombardment ⁇ e.g., with a gene gun).
  • Particle mediated gene transfer methods are known in the art, are commercially available, and include, e.g., the gas driven gene delivery instrument described in U.S. Patent No. 5,584,807, the contents of which are incorporated by reference herein. This method involves coating the polynucleotide sequence of interest onto heavy metal particles, and accelerating the coated particles under the pressure of compressed gas for delivery to the target tissue.
  • specific initiation signals may be used to achieve more efficient translation of sequences encoding a polypeptide described herein, such as, e.g., the ATG initiation codon and adjacent sequences.
  • sequences encoding the polypeptide of interest, its initiation codon, and upstream sequences are inserted into the appropriate expression vector, no additional transcriptional or translational control signals may be needed.
  • heterologous translational control signals that include the ATG initiation codon may be provided.
  • a protein may be expressed in a recombinantly engineered cell, such as a plant cell.
  • a recombinantly engineered cell such as a plant cell.
  • Persons skilled in the art are knowledgeable about various expression systems available for expression of a polynucleotide encoding a protein according to embodiments described herein.
  • the expression of isolated polynucleotides encoding a polypeptide (protein) described herein will typically be achieved by operably linking, for example, the DNA or cDNA to a promoter, followed by incorporation thereof into an expression vector.
  • Typical expression vectors include those described herein, and may contain transcription and translation terminators, initiation sequences, and promoters useful for regulation of the expression of the DNA encoding a polypeptide (protein) described herein.
  • an expression vector that contains, at minimum, a strong promoter to direct transcription, a ribosome binding site for translational initiation, and a transcription/translation termination site.
  • modifications could be made to a protein of the present invention without diminishing its biological activity.
  • plant transformation methods involve transferring heterologous DNA into target plant cells, followed by applying a maximum threshold level of appropriate selection to recover the transformed plant cells from an untransformed cell mass. Subsequently, the transformed cells are differentiated into shoots after being placed on a regeneration medium supplemented with a maximum threshold level of selecting agent (e.g., temperature, herbicide, etc.). The shoots are then transferred to a selective rooting medium for recovering the rooted shoot or plantlet. The transgenic plantlet is then grown into a mature plant that produces fertile seeds.
  • a maximum threshold level of selecting agent e.g., temperature, herbicide, etc.
  • the transformed material contains many cells, both transformed and non-transformed cells are present in any piece of subjected target callus, tissue, or group of cells.
  • the ability to kill non-transformed cells and allow transformed cells to proliferate results in transformed plant cultures.
  • the ability to remove non-transformed cells is a limitation to rapid recovery of transformed plant cells and successful generation of transgenic plants. Therefore, molecular and biochemical methods may be used for confirming the presence of the integrated nucleotide(s) of interest in the genome of the transgenic plant.
  • selectable markers such as enzymes resulting in a change of color or luminescent molecules (e.g., GUS and luciferase), antibiotic-resistant genes (e.g., gentamicin and kanamycin -resistance genes) and chemical-resistant genes (e.g., herbicide-resistance genes) may be used to confirm the integration of the nucleotide(s) of interest in the genome of the transgenic plant.
  • antibiotic-resistant genes e.g., gentamicin and kanamycin -resistance genes
  • chemical-resistant genes e.g., herbicide-resistance genes
  • a protein may be expressed in a recombinantly engineered cell, such as a plant cell.
  • a recombinantly engineered cell such as a plant cell.
  • Persons skilled in the art are knowledgeable about various expression systems available for expression of a polynucleotide encoding a protein according to embodiments described herein.
  • the expression of isolated polynucleotides encoding a polypeptide (protein) described herein will typically be achieved by operably linking, for example, the DNA or cDNA to a promoter, followed by incorporation thereof into an expression vector.
  • Typical expression vectors include those described herein, and may contain transcription and translation terminators, initiation sequences, and promoters useful for regulation of the expression of the DNA encoding a polypeptide (protein) described herein.
  • an expression vector that contains, at minimum, a strong promoter to direct transcription, a ribosome binding site for translational initiation, and a transcription/translation termination site.
  • modifications could be made to a protein of the present invention without diminishing its biological activity.
  • the vector and sequences disclosed herein may be optimized for increased expression in the transformed host cell. That is, the sequences may be synthesized using host cell-preferred codons for improved expression, or may be synthesized using codons at a host-preferred codon usage frequency. Generally, the GC content of the polynucleotide will be increased. See e.g., Campbell et al., Plant Physiol. 92:1-11 (1990). Methods for synthesizing host-preferred polynucleotides are known by persons skilled in the art. See e.g., Murray et al., Nucleic Acids Res. 17:477-498 (1989); U.S. Patent Application Publications Nos. 2004/0005600 and 2001/0003849; and U.S. Patents Nos: 6,320,100; 6,075,185; 5,380,831; and 5,436,391, the entire inventions of which are incorporated by reference herein.
  • polynucleotides of interest can be targeted to the chloroplast for expression.
  • an expression cassette may additionally contain a polynucleotide encoding a transcription factor polypeptide to direct the nucleotide of interest to the chlorop lasts.
  • transit peptides are known by persons skilled in the art. See e.g., Von Heyne et al., Plant Mol. Biol. Rep. 9:104-126 (1991); Clark et al., J. Biol. Chem.
  • the polynucleotides of interest to be targeted to the chloroplast may further be optimized for expression in the chloroplast to account for differences in codon usage between the plant nucleus and this organelle. In this manner, the polynucleotides of interest may be synthesized using chloroplast-preferred codons. See U.S. Patent No. 5,380,831, which is incorporated by reference herein.
  • the DNA fragments may be introduced into plant tissues, cultured plant cells or plant protoplasts by standard methods, including, e.g., electroporation, infection by viral vectors such as cauliflower mosaic virus (CaMV), high velocity ballistic penetration by small particles with the nucleic acid either within the matrix of small beads or particles, or on the surface, use of pollen as a vector, or using Agrobacterium tumefaciens or A. rhizogenes carrying a T-DNA plasmid in which DNA fragments are cloned. See Fromm et al, Proc. Natl. Acad. Sci.
  • viral vectors such as cauliflower mosaic virus (CaMV)
  • CaMV cauliflower mosaic virus
  • Agrobacterium tumefaciens or A.
  • rhizogenes carrying a T-DNA plasmid in which DNA fragments are cloned See Fromm et al, Proc. Natl. Acad. Sci.
  • T-DNA plasmid is transmitted to plant cells upon infection by Agrobacterium tumefaciens, and a portion is stably integrated into the plant genome. See Horsch et al., Science 233:496-498 (1984); and Fraley et al., Proc. Natl. Acad. Sci. 80:4803-4807 (1983).
  • telomeres For long-term, high-yield production of recombinant proteins, stable expression may be used.
  • Host cells transformed with a nucleotide sequence encoding a polypeptide as disclosed herein are optionally cultured under conditions suitable for the expression and recovery of the encoded protein from cell culture.
  • the protein or fragment thereof produced by a recombinant cell may be secreted, membrane-bound, or contained intracellularly, depending on the sequence and/or the vector used.
  • expression vectors according to embodiments containing polynucleotides that encode mature proteins can be designed with signal sequences that direct secretion of the mature polypeptides through a prokaryotic or eukaryotic cell membrane.
  • Some embodiments relate to recombinant expression of a Q42 polypeptide in a stable cell line.
  • Methods for generating a stable cell line following transformation of a heterologous construct into a host cell are known in the art.
  • the transformed cells, tissues, and plants are therefore understood to encompass not only the end product of a transformation process, but also transgenic progeny or propagated forms thereof.
  • Polynucleotides disclosed herein are favorably employed to produce transgenic plants with various traits or characteristics that have been modified in a desirable manner, e.g., to improve the seed characteristics of the plant. For example, altering the expression levels or patterns of one or more of the homologues disclosed herein, as compared with the levels of the same protein found in a control wild-type plant, can be used to modify a plant's traits. Illustrative examples of trait modification and improved characteristics resulting from altering expression levels of the disclosed Q42 amino acid sequences are explained in more detail in the various Examples below.
  • Polynucleotides and polypeptides disclosed herein can also be expressed in a plant in the absence of an expression cassette by manipulating the activity or expression level of the endogenous gene by other means, e.g., by ectopically expressing a gene by T-DNA activation tagging. See Ichikawa et al., Nature 390:698-701 (1997); and Kakimoto et al., Science 274:982- 985 (1996). This method entails transforming a plant with a gene tag containing multiple transcriptional enhancers and, once the tag has inserted into the genome, expression of a flanking gene coding sequence becomes deregulated.
  • the transcriptional machinery in a plant can be modified so as to increase transcription levels of a polynucleotide disclosed herein. See e.g., PCT International Publications Nos. WO 96/06166 and WO 98/53057 (describing modifications of DNA binding specificity of zinc finger proteins by changing particular amino acids in the DNA binding motif).
  • Embodiments also relate to host cells, for example isolated host cells, into which a nucleic acid construct or recombinant expression vector as described herein has been introduced.
  • the host cell may include a nucleic acid that encodes a polypeptide as described herein, such that the cell expresses the polypeptide of interest.
  • a host cell such as a prokaryotic or eukaryotic host cell in culture, can be used to express polypeptides described herein.
  • the host cell may be a plant or bacterial (for example Agrobacterium) cell.
  • a kit comprising such cell is also provided.
  • the host cell is a plant cell and may be a monocot or a dicot.
  • a polynucleotide sequence as described herein can be expressed in plants and plant cells, such as unicellular plant cells ⁇ see Falciatore et al., Marine Biotechnology 1(3):239- 251 and references cited therein) and cells from higher plants, such as crop plants. Preferred target plants within the scope of the methods described herein are listed elsewhere herein.
  • Embodiments include host ⁇ i.e., target) cells transduced with vectors described herein, and the production of polypeptides by recombinant techniques.
  • Host cells are genetically engineered ⁇ i.e., nucleic acids are introduced by transduction, transformation or transfection) with the vectors, which may be, e.g., a cloning vector or an expression vector comprising the relevant nucleic acids described herein.
  • the vector may optionally be, for example, a plasmid, a viral particle, a phage, a naked nucleic acid, etc.
  • the engineered host cells can be cultured in conventional nutrient media modified as appropriate for activating promoters, selecting transformants or amplifying the relevant gene.
  • the culture conditions such as temperature, pH and the like, may be those previously used with the host cell selected for expression, and will be apparent to those skilled in the art.
  • the plants are preferably selected using a dominant selectable marker incorporated into the transformation vector.
  • a dominant selectable marker incorporated into the transformation vector.
  • the modified train can be any of the traits described herein.
  • the mRNA expression may be analyzed using Northern blots, RT-PCR or microarrays, or protein expression using immunoblots or Western blots.
  • the invention also relates to a method of producing a transgenic plant, comprising introducing into a plant cell a nucleic acid construct, polynucleotide, a vector or an expression cassette described herein; and generating from the plant cell a transgenic plant that expresses the polynucleotide.
  • the invention also relates to a method for modulating a plant phenotype comprising introducing and expressing in a plant a nucleic acid construct comprising a polynucleotide described herein, a vector described herein or tan expression cassette described herein.
  • Constructs comprising the polynucleotide, vector and expression cassette are described herein and include, for example, a Q42 chimeric construct, as described herein.
  • the plant polynucleotide comprises a first portion of a first OXR polynucleotide and a second portion of a second OXR polynucleotide.
  • the first polynucleotide comprises SEQ ID NO: 1 , a functional variant or homolog thereof and the second polynucleotide comprises SEQ ID NO: 7, a functional variant or homolog thereof.
  • the first polynucleotide comprises a nucleotide sequence having at least 50%, 60%, 70%, 80% or 90% sequence identity with the full-length AtOXR4 amino -terminal encoding nucleotide sequence of SEQ ID NO: 1; and the second polynucleotide comprises a nucleotide sequence having at least 50%, 60%, 70%, 80% or 90% sequence identity with the full-length AtOXR2 carboxy-terminal encoding nucleotide sequence of SEQ ID NO: 7.
  • the first region comprises the AtOXR4 amino-terminal encoding nucleotide sequence represented by SEQ ID NO: 3, a functional variant or homolog thereof; and the second region comprises the AtOXR2 carboxy-terminal encoding nucleotide sequence represented by SEQ ID NO: 11, a functional variant or homolog thereof.
  • the first region comprises a sequence with at least 50%, 60%,
  • the second region comprises a sequence with at least 50%, 60%, 70%, 80% or 90% sequence identity with the OXR2 carboxy-terminal encoding nucleotide sequence represented by SEQ ID NO: 11.
  • the polynucleotide has the full-length nucleotide sequence of SEQ ID NO: 13 or sequence with at least 50%, 60%, 70%, 80% or 90% sequence identity thereto.
  • said phenotype is selected from one or more of increased root biomass, increased shoot biomass, increased seed production, early flowering time, increased lignification, increased stem diameter, improved efficiency in water use and/or increased photosynthetic performance compared to a control plant.
  • said phenotype is increased yield.
  • the method may comprise the steps of assessing a yield related trait compared to a control plant.
  • the invention also relates to a method for increasing abiotic or biotic stress tolerance in a plant comprising introducing and expressing in a plant a polynucleotide or a construct comprising a polynucleotide described herein, the vector described herein or the expression cassette described herein.
  • said stress is abiotic. This may be selected from one or more of chilling, freezing, water deficit, for example drought, or salt stress. In one embodiment, said stress is water deficit. In one embodiment, said stress is salt stress.
  • Drought stress can be measured through leaf water potentials. Generally speaking, moderate drought stress is defined by a water potential of between -1 and -2 Mpa.
  • the method may comprise the steps of exposing the plant to stress conditions and/or comparing the stress response of the transgenic plant to a control plant. Transgenic plants are more resistant to the stress than the control plant and show improved yield and survival.
  • the invention also relates to a use of a construct comprising a polynucleotide described herein, the vector described herein or the expression cassette described herein in modulating a plant phenotype.
  • the invention also relates to nucleic acid and polypeptide sequences and constructs described in the figures.
  • the AtQ42 protein is a chimeric protein constructed using genetic engineering techniques by fusing two cDNA fragments coding for two different proteins of the OXR family from Arabidopsis thaliana. Expression of AtQ42 using a strong constitutive promoter (35SCaMV) in Arabidopsis thaliana generated plants with increased shoot and root biomass, photosynthetic performance and seed production. These characteristics were maintained after subjecting plants to salt stress or water deficit conditions. This technology may be used to obtain plants with improved agronomical traits.
  • the AtQ42 protein is a chimeric protein generated using genetic engineering tools by combining cDNA fragments from two genes encoding OXR (Oxidative Resistance) proteins from Arabidopsis thaliana.
  • OXR Oxidative Resistance
  • the family members have a conserved region located near the C-terminal end called the TLDc domain. Proteins from this family were studied in yeast (Elliott and Volkert, 2004), Drosophila (Fischer et al., 2001), Anopheles (Jaramillo-Gutierrez et al, 2010), mice (Natoli et al., 2008) and humans (Durand et al., 2007).
  • the TLDc domain has been implicated in the prevention and/or repair of oxidative damage to DNA under stress conditions (Elliott and Volkert, 2004; Durand et al, 2007; Jaramillo-Gutierrez et al, 2010).
  • TLDc domains in each AtOXR protein candidate and design oligonucleotides for PCR amplification of different cDNA fragments, from different OXR proteins (e.g. AtOXR2, AtOXR4, AtOXR5).
  • AtOXR2 Arabidopsis thaliana
  • AtOXR4 Arabidopsis thaliana
  • RT- qPCR we observed that AtOXR2 and PA.OXR4 are induced by stress conditions like heat, UV-B and after the treatment of plants with salt.
  • AtOXR2 and AtOXR4 suppress the oxidative mutant phenotype when expressed in an E. coli strain that lacks enzymes for the DNA repair system.
  • AtOXR proteins would be excellent candidates to evaluate their role in the mechanisms of plant adaptation and growth under unfavorable conditions.
  • AtOXR2 and AtOXR4 under the control of the 35SCaMV constitutive promoter in Arabidopsis Col-0 plants, thus obtaining oeOXR2 and oeOXR4 plants.
  • AtOXR2 Overexpression of AtOXR2 originated plants with increased shoot biomass. A similar trend, although not statistically significant, was observed with AtOXR4. IRGA analysis indicated that oeOXR2 plants (and oeOXR4 in less extent) showed higher values of net photosynthesis (A) and electron transfer rate (ETR). Also, these plants showed decreased water loss by dehydration (measured as cut rosette water loss vs. time).
  • Plants were grown in a growth chamber (20-24°C, night-day temperature, humidity 50-70%) under a long-day (LD) photoperiod (16 h light / 8 h dark) and a light intensity of 100 ⁇ m "2 s "1 .
  • the substrate used was a mix of peat/perlite/vermiculite at a 2/2/1 ratio. Plants were watered regularly with a solution of 0.5X Hoagland (Hoagland and Arnon, 1950). Alternatively, they were grown in sand with sub-irrigation with Hoagland solution 0.5X to evaluate root biomass. All reported results were repeated at least three times, using 4-5 replicates per genotype, treatment, and parameter measured.
  • LD long-day
  • Transgenic lines used in different experiments correspond to plants with intermediate expression levels of AtQ42 and were identified using the following numbers: 4, 6, 25, 26, 27 and 28.
  • InfoStat software (2009). Data were analysed by ANOVA, using the statistical test LSD (Least Significant Differences) with a level of significance of 5%.
  • Figure 2A shows photographs of 35-day-old AtQ42 (line 25) and control (Wt) plants. It can be observed that AtQ42 plants have larger rosettes. After removal of plants from the sand, it was observed that Q42 plants also show increased development of root tissues when compared to Wt ( Figure 2B).
  • AtQ42 leaves is 22% higher than in Wt leaves.
  • the dry weight of stems (SDW) in AtQ42 plants increases 160% respective to Wt plants.
  • the dry weight of the AtQ42 aerial portion increases 50% respective to Wt plants.
  • AtQ42 plants The root dry weight of AtQ42 plants is significantly higher (about 4 times) than that of Wt plants.
  • the total dry weight (TDW) of AtQ42 plants is more than two-fold higher than that of Wt plants.
  • FIG. 4 shows that AtQ42 plants have a higher number of leaves (A,B) and a larger rosette diameter (C), resulting from an increased leaf area (D). AtQ42 leaves also show increased dry weight compared to Wt (E).AtQ42 plants show increased seed yield: a. Yield under control growth conditions:
  • AtQ42 plants grown under normal conditions show an increase in the production of seeds per plant. Increases in yield ranged from 35% to 62% compared to Wt plants under optimal conditions (Fig. 5 A), and up to 150% in an experiment in which seed yield was affected by changes in culture chamber conditions (Fig. 5B). ⁇
  • Salinization of the substrate was achieved by adding NaCl to the Hoagland solution.
  • the salt was applied in the irrigation solution after the third week of growth using irrigations with solutions of increasing concentration, from 50 to 130 mM NaCl.
  • AtQ42 and Wt plants following the methodology proposed by Berlin and Miksche (1976). Quantification of anatomical parameters indicated that the transversal area of the main stem was approximately doubled in Q42 with respect to Wt plants ( Figure 9 A and B). Moreover, the area and diameter of the conducting xylem vessels were found to be 36% and 12% higher in AtQ42 plants ( Figure 9C and D).
  • the larger size of the main stem may allow AtQ42 plants to support their high biomass content.
  • the larger diameter of xylem vessels may allow plants to have a greater hydraulic conductivity, thereby supporting higher growth rates. This is probably one of the reasons of the performance observed in AtQ42 plants and explains why they can support higher yields even under conditions of decreased soil water potential (water deficit and presence of NaCl in the nutrient solution).
  • C0 2 fixation, gas exchange and stomatal closure were measured in Wt and AtQ42 plants. Values were obtained using an IRGA (Infra-Red Gas Analyser).
  • AtQ42 plants have higher photosynthetic performance compare to Wt plants (Figure 10).
  • Photosynthetic C02 assimilation rates were 16% to 40% higher in AtQ42 plants (Fig. 10A).
  • AtQ42 plants also showed lower stomatal conductance (Fig. 10B), thus resulting in increased water use efficiency (Fig. IOC).
  • AtQ42 lines (6, 25, 26, 27 and 28), we compared these lines with wild-type plants and a representative line with high expression levels of AtOXR2. For this purpose, we made a principal component analysis followed by the construction of a minimum spanning. According to this, it can be concluded that the chimeric protein AtQ42 confers new characteristics that are clearly different from those provided by the overexpression of OXR2. The most remarkable benefits related to the AtQ42 technology are: increase seed yield (6), shoot height (1) and number of stem branches (2). AtQ42 plants also show high photosynthetic performance and efficiency in water use (7, 10) and higher specific leaf area (12, 13). Several important parameters like dry weight and lignin content (see Figures 3 and 9, respectively) were not included in the analysis because these parameters were not available for all lines.
  • SOD Superoxide Dismutase
  • MnSOD is a typical enzyme present in mitochondria
  • FeSOD is localized in chloroplasts
  • Cu/ZnSOD is involved in ROS detoxification both in cytosol and peroxisomes.
  • transgenic Arabidopsis plants expressing Q42 have higher SOD levels compared to WT plants at the same stage of development. An increase in Cu/Zn SOD is especially observed.
  • AtQ42 have increased shoot and root biomass, production of seeds per plant, and photosynthetic efficiency. They also show thicker stems with increased lignification. These characteristics indicate that the AtQ42 technology can be used to obtain plants with improved agronomical traits. [00266]
  • the exemplary applications of this technology are:
  • sunflower Helianthus annuus L
  • Increased stem diameter (accompanied by an increase in the diameter of conductor vessels) and increased lignification are properties of interest for crops such as sugarcane or for growing trees that are used for wood production.
  • crops such as sugarcane or for growing trees that are used for wood production.
  • plants with hardy stems are less sensitive to lodging and breakage that cause losses of yield.
  • plants with more lignified stems could be more resistant to pathogen attack, like fungi of the genus Sclerotinia sp (for sunflower) or larvae of Lepidoptera (leaf miners or stem borers in maize). More specifically, the highest percentage of lignification protects the stems of maize against the attack of Diatraea saccharalis or "sugarcane borer", which also attacks sugarcane, as its name suggests.
  • SEQ ID NO: 4 AtOXR4 - protein sequence of the amino-terminal fragment of SEQ ID NO: 2
  • SEQ ID NO: 5 AtOXR4 - gene sequence of the domain region of SEQ ID NO: 1
  • SEQ ID NO: 6 AtOXR4 - protein sequence of the domain region of SEQ ID NO: 2
  • SEQ ID NO: 9 AtOXR2 - gene sequence of the amino-terminal fragment of SEQ ID NO: 7
  • SEQ ID NO: 10 AtOXR2 - protein sequence of the amino-terminal fragment of SEQ ID NO: 8
  • SEQ ID NO: 11 AtOXR2 - gene sequence of the domain region of SEQ ID NO: 7
  • SEQ ID NO: 12 AtOXR2 - protein sequence of the domain region of SEQ ID NO: 8
  • SEQ ID NO: 14 AtQ42 - protein sequence (corresponding to SEQ ID NO: 4 + SEQ ID NO: 12)
  • SEQ ID NO: 3 0XR4 - gene sequence of the amino-terminal fragment of SEQ ID NO: 1
  • SEQ ID NO: 4 OXR4 - protein sequence of the amino-terminal fragment of SEQ ID NO: 2
  • SEQ ID NO: 5 OXR4 - gene sequence of the domain region of SEQ ID NO: 1
  • SEQ ID NO: 6 OXR4 - protein sequence of the domain region of SEQ ID NO: 2
  • SEQ ID NO: 9 OXR2 - gene sequence of the amino-terminal fragment of SEQ ID NO: 7
  • SEQ ID NO: 12 OXR2 - protein sequence of the domain region of SEQ ID NO: 8
  • SEQ ID NO: 14 Q42 protein sequence (corresponding to SEQ ID NO: 4 + SEQ ID NO: 12 (in bold)) MGKHKSFRSK AVHFVTDLTA GLLNPISDKP SSAHPPPPLP DEEDESKR Q LESTTAEQPK DLVDEPDTSS FSAFLGSLLS SDPKDKRKDQ DPEDEEDEEE DEEEDSEAET SDTSSSSANP TRTMKETTSG GAAKKSFLSK YKQHFR FYQ AVKFPGVKER KGNSDVIPDD EETEYYDGLE MKPMQN VK EEVTVVVQAI IPLTE SSVFITANLF EFLHASLPNI VRGCKWILLY STLKHGISLR TLLRRSGELP GPCLLVAGDK QGAVFGALLE CPLQPTPKRK YQGTSQTFLF TTIYGEPRIF RPTGANRYYL MCMNEFLAFG GGGNFALCLD EDLLKATSGPSETFGNECLA SSTEFELKNV
  • OXR2 (also termed OXR 2.1), OXR2 protein splice variant At2g05590.1

Abstract

L'invention concerne une protéine chimérique qui contient une première partie polypeptidique d'une première protéine OXR (de résistance à l'oxydation) ; et une seconde partie polypeptidique d'une seconde protéine OXR, la seconde partie polypeptidique étant liée à la première partie polypeptidique, de façon telle que les première et seconde parties polypeptidiques sont combinées dans la protéine chimérique dans un ordre ou dans un agencement spatial qui n'existe pas dans la nature. L'invention concerne également des polynucléotides d'OXR isolés et des polynucléotides qui codent pour la protéine chimérique, ainsi que des plantes transformées par les polynucléotides.
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