WO2015193653A1

WO2015193653A1 - Oxidative resistance chimeric genes and proteins, and transgenic plants including the same

Info

Publication number: WO2015193653A1
Application number: PCT/GB2015/051759
Authority: WO
Inventors: Elina WELCHEN; Daniel H. Gonzalez; Gabriel CECCOLI; Francisco COLOMBATTI
Original assignee: Consejo Nacional De Investigaciones Cientificas Y Tecnicas; Universidad Nacional Del Litoral; Plant Bioscience Limited
Priority date: 2014-06-16
Filing date: 2015-06-16
Publication date: 2015-12-23
Also published as: AR100874A1

Abstract

A chimeric protein contains a first polypeptide portion of a first OXR (Oxidative Resistance) protein; and a second polypeptide portion of a second OXR protein, the second polypeptide portion being joined to the first polypeptide portion, such that 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. Isolated OXR polynucleotides and polynucleotides that encode the chimeric protein, as well as plants transformed by the polynucleotides, are also provided.

Description

OXIDATIVE RESISTANCE CHIMERIC GENES AND PROTEINS, AND TRANSGENIC

PLANTS INCLUDING THE SAME

BACKGROUND

[0001] The ever-increasing world population and the dwindling supply of arable land available for agriculture fuels research towards increasing the efficiency of agriculture and providing food security. Conventional means for crop and horticultural improvements utilise selective breeding techniques to identify plants having desirable characteristics. However, such selective breeding techniques have several drawbacks, namely that these techniques are typically labour intensive and result in plants that often contain heterogeneous genetic components that may not always result in the desirable trait being passed on from parent plants. Advances in molecular biology have allowed mankind to modify the germplasm of animals and plants. Genetic engineering of plants entails the isolation and manipulation of genetic material (typically in the form of DNA or RNA) and the subsequent introduction of that genetic material into a plant. Such technology has the capacity to deliver crops or plants having various improved economic, agronomic or horticultural traits, including increased yield.

[0002] Traits of particular economic interest are increased yield and stress tolerance.

[0003] 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.

[0004] Most plants have evolved protective mechanisms against dehydration caused by abiotic stress. However, if the severity and duration of the abiotic stress conditions are too great, the effects on development, growth, and yield of most crop plants are profound. Developing plants efficient in water use is therefore a strategy that has the potential to benefit human life. Many agricultural companies have attempted to identify genes that could confer tolerance to abiotic stress responses, in an effort to develop transgenic abiotic stress-tolerant crop plants. Although some genes that play a role in stress responses in plants have been characterized, the characterization and cloning of plant genes that confer the desired stress tolerance characteristics remain largely fragmented and incomplete.

[0005] Therefore, there is a need to identify genes expressed in stress tolerant plants that have the capacity to confer stress resistance and yield related traits to its host plant and to other plant species. The present invention is aimed at meeting this need.

SUMMARY OF THE INVENTION

[0006] 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. The various embodiments of the chimeric protein according to the aspects of the invention are described herein. Preferably, the chimeric protein is a plant protein.

[0007] In preferred embodiments, 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.

[0008] In some embodiments, 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.

[0009] 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.

[0010] In some embodiments, 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.

[0011] In some embodiments, 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. In some embodiments, 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.

[0012] The chimeric protein may comprise an amino acid sequence having at least 50%,

60%, 70%, 80% or 90% sequence identity with the full-length amino acid sequence of SEQ ID NO: 14. In some embodiments, the joined first and second polypeptide portions of the chimeric protein may constitute the full-length amino acid sequence of SEQ ID NO: 14.

[0013] In other embodiments of the invention the chimeric polypeptide includes other combinations of first and second polypeptide portions. For example, 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. [0014] Also within the scope of the invention are combinations other than OXR4 and

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.

[0015] 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.

[0016] Embodiments of the present invention also relate to an isolated polynucleotide that encodes the chimeric protein defined herein.

[0017] In one embodiment, the isolated polynucleotide comprises a first portion of a first

OXR polynucleotide and a second portion of a second OXR polynucleotide, the second portion being joined to the first polynucleotide portion. OXR polynucleotide portions from OXR polynucleotides of the same or different plant species can be joined together.

[0018] In one embodiment, 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.

[0019] 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.

[0020] In some embodiments of the invention, 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. For example, 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. In the polynucleotide of the present invention, the first and second regions are joined together through a covalent bond. In some embodiments, 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.

[0021] In other embodiments of the invention the chimeric polynucleotide includes other combinations of first and second polynucleotide portions. For example, 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.

[0022] Also within the scope of the invention are combinations other than OXR4 and

OXR2 polynucleotide sequences. 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. In the recombinant expression cassette, the promoter may be a tissue-preferred promoter, a constitutive promoter, and/or an inducible promoter. In some embodiments, the expression cassette may comprise the polynucleotide operably linked to a 35SCaMV constitutive promoter.

[0023] Also provided are host cells and transgenic plants (and/or seeds or products thereof or plant cells) comprising a nucleic acid construct or recombinant expression cassette that comprises the polynucleotide encoding the chimeric protein described herein. In some embodiments, the transgenic plant may be selected from the group consisting of maize, soybean, sorghum, canola, wheat, alfalfa, cotton, rice, brassica and barley. In embodiments, 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.

[0024] 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. In embodiments, 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.

[0025] 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.

BRIEF DESCRIPTION OF THE DRAWINGS

[0026] 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*.

[0027] Fig. 2A is a photographic showing rosette sizes of a AtQ42 transgenic plant and a

WT control plant; and 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.

[0028] 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).

[0029] 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; and 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).

[0030] 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.

[0031] Fig. 6 shows differences in yield (mg seeds/plant) between 2 homozygous lines of

AtQ42 transgenic plants and of WT control plants grown under salinity conditions.

[0032] 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).

[0033] 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; and 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). [0034] 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; and 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).

[0035] Fig. 10A shows differences in C0₂ assimilation between 4 homozygous lines of

Q42 transgenic plants and of WT control plants; 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).

[0036] 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 : Shoot 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. Plants: single blue circle: Wt plants (circle as marked with arrow); single red circle: oeOXR2 plants (as marked with arrow); remaining (green) circles: lines of different Q42 plants. PCI : principal component 1 ; PC2: principal component 2

[0037] 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). In rice and maize, two potential homologues of AtOxr4 were found. At: Arabidopsis thaliana, Vv: Vitis vinifera; Os: Oryza sativa; Zm: Zea mays, HV: Hordeum vulare, GM: Glycine max, Ta: Triticum aestivum, Br: Brassica rapa, Bo Brassica oleracea. 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:49), ZmOx2 (SEQ ID NO:50), OsOx2 (SEQ ID N0:51), AtOx2.1 (At2g05590.1, corresponds to OXR2 splice variant: SEQ ID NO:19), BoOx2 (SEQ ID NO:52), BrOx2 (SEQ ID NO:53), HvOx2 (SEQ ID NO:54), GmOx2 (SEQ ID NO:55), TaOx2 (SEQ ID NO:56), AtOx2.2 (At2g05590.2, corresponds to OXR2: SEQ ID NO:8) VvOx2 (SEQ ID NO:57), AtOxl (SEQ ID NO:20), VvOxl (SEQ ID NO:58), ZmOxl (SEQ ID NO:59), OsOxl l (SEQ ID NO:60) and OsOxl2 (SEQ ID NO:61).

[0038] 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.

[0039] 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.

[0040] 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.

[0041] 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).

[0042] Fig 17. Phylogenetic tree of Oxr proteins from selected plants. An alignment of

Oxr protein sequences with ClustalW (Thompson et al., 1994) 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.

[0043] 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.

[0044] Fig 19 OXR chimeric polypeptides and nucleic acid sequences. Q42 protein sequence (SEQ ID NO: 14), Q42 nucleotide sequence (SEQ K) 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).

DEFINITIONS

[0045] As used herein, 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).

[0046] Unless otherwise indicated, 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.

[0047] The term "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).

[0048] As used herein, the term "environmental stress" refers to sub-optimal conditions associated with salinity, drought, temperature, metal, chemical, pathogenic and oxidative stresses, or combinations thereof.

[0049] 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, while "down-regulation" or "repression" refers to regulation that decreases production relative to basal or native states.

[0050] 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). In embodiments, introducing a nucleotide sequence into a plant cell results in transformation of the plant cell to cause stable or transient expression of the sequence.

[0051] The term "isolated" as used herein 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. [0052] For example, 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. Likewise, a naturally occurring nucleic acid (e.g., a promoter) becomes isolated if it is introduced by non- naturally occurring means to a locus of the genome not native to that nucleic acid, as discussed further below. Preferably, 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.

[0053] As used herein, the term "nucleic acid" (or "polynucleotide") 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. Thus, 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. The term 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. The term "nucleic acid" (or "polynucleotide") may be used in place of, inter alia, gene, cDNA, mRNA.

[0054] As used herein, the term "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. Generally, 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.

[0055] The term "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. Thus, 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. As used herein, 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.

[0056] The terms "polypeptide" and "protein" as used herein 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.

[0057] As described herein, polypeptides may be "chimeric" in the sense that they are arranged in a configuration not normally found in nature. For example, the amino acid sequence of one or more of the segments can be a non-naturally occurring sequence. As another example, 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. Thus, a

"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. The term "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. As described herein, the chimeric protein comprises the TLDc domain and this domain can also be used for identification of other OXR protein members.

[0058] A "chimeric gene" (or recombinant 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. For example 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).

[0059] As used herein, the term "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.

[0060] As used herein, the term "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. For example, 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).

[0061] As used herein, the term "recombinant expression cassette" (or "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. Typically, the recombinant expression cassette portion of an expression vector includes a nucleic acid to be transcribed and a promoter.

[0062] As used herein, the term "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.

[0063] The terms "residue", "amino acid residue", and "amino acid" are used interchangeably herein to refer to an amino acid that is incorporated into a protein, polypeptide, or peptide. The amino acid may be a naturally occurring amino acid and, unless otherwise limited, may encompass non-natural analogs of natural amino acids that can function in a similar manner as naturally occurring amino acids.

[0064] As used herein, "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.

. . . number of identical positions

% sequence identity = _x ioo

total number of positions [0065] When percentage of sequence identity is used in reference to proteins, it is recognized that residue positions that are not identical often differ by conservative amino acid substitutions. A "conservative amino acid substitution" is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include 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), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Where 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.

[0066] As used herein, the term "transgenic plant" refers to a plant that includes within its genome a heterologous polynucleotide. Generally, 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.

[0067] As used herein, the term "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. One type of vector is a "plasmid," which refers to a circular double stranded DNA loop into which additional DNA segments can be ligated. 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. [0068] Certain vectors 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. The term "regulatory sequence" is intended to include promoters, enhancers, and other expression control elements.

[0069] As used herein, the term 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. In another embodiment, a control plant may be a plant overexpressing the wild type OXR protein.

[0070] As used herein, the term "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. The term "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. Individual plant parts directly contribute to yield based on their number, size and/or weight, or the actual yield is the yield per square meter for a crop and year, which is determined by dividing total production (includes both harvested and appraised production) by planted square metres. The term "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. Thus, according to the invention, 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. In one embodiment, the control plant is a plant that overexpresses the wild type gene.

[0071] 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. The term "gene" or "gene sequence" is used broadly to refer to a DNA nucleic acid associated with a biological function. Thus, 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.

DETAILED DESCRIPTION OF EMBODIMENTS

[0072] The invention may be more readily understood by reference to the following detailed description of preferred embodiments and the Examples included herein. However, it is to be understood that the invention is not limited to specific nucleic acids, specific polypeptides, specific cell types, specific host cells, specific conditions, or specific methods, etc., as such may vary, and the numerous modifications and variations will be apparent to those skilled in the art. The Examples are included for purposes of illustrating certain embodiments described herein and are not intended to limit the scope of the invention. Furthermore, the terminology used herein with respect to specific embodiments is not intended to limit the scope of the invention.

[0073] Thus, in the following passages, different aspects of the invention are defined in more detail. Each aspect so defined may be combined with any other aspect or aspects unless clearly indicated to the contrary. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature or features indicated as being preferred or advantageous.

[0074] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of botany, microbiology, tissue culture, molecular biology, chemistry, biochemistry and recombinant DNA technology, bioinformatics which are within the skill of the art. Such techniques are explained fully in the literature.

[0075] 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.

[0076] The present invention identifies 5 putative members of the OXR protein family in

Arabidopsis thaliana: Atlg32520 (OXRl) At2g05590 (OXR2: two splice variants exist encoding different proteins: 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).

[0077] Of these members, two were studied in order to elucidate the function of the OXR proteins in plants, namely: AtOXR2 (At2g05590.2) and AtOXR4 (At4g39870). Using RT- qPCR, it was observed that AtOXRl and AtOXRA are induced by stress conditions, such as heat, UV-B, and following the treatment of plants with salt. Like OXR human proteins, 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.

[0078] 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 οΐ 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).

[0079] The inventors have surprisingly shown that chimeric OXR polypeptides have improved characteristics compared to wild type polypeptides. This is shown in Figs. 11 and 17. Thus, 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.

[0080] By using standard genetic engineering methods, including PCR reactions followed by ligations and subsequent molecular cloning, a construct containing a fusion of two different DNA regions of.4iOXR4 and.4iOXR2 was generated. As shown in Fig. 1, 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 οΐΑίΟΧ 2 including the TLDc domain.

[0081] 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.

[0082] In other aspects, 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. In one embodiment, 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. [0083] 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. In some embodiments, 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.

[0084] As demonstrated in the examples, 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.

[0085] However, as a skilled person will understand, 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,.

[0086] Thus, as set out in the various aspects of the invention, functional variants and homologs of polypeptide and nucleic acid sequences disclosed herein, for example homologs of polypeptide and nucleic acid sequences from Arabidopsis, are also within the scope of the aspects of the invention.

[0087] 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. (2011) as the cysteine scavenging ROS compounds. In OXR2 from Arabidopsis that cysteine is not conserved; it is replaced by a serine residue. The TLDc from Arabidopsis OXR2 has however 6 cysteine residues (residues 182, 211, 229, 270, 286, 306).

[0088] 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. Also encompassed is a variant that is 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.

[0089] The term 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%,

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 reference sequence. In certain embodiments, the reference sequence is an Arabidopsis thaliana OXR polynucleotide or polypeptide sequence as set out herein.

[0090] For example, 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%, or at least 99% overall sequence identity to the amino acid represented by the reference sequence, for example SEQ ID NO: 1 , 15 or 16.

[0091] 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 identity to the amino acid represented by the reference sequence, for example SEQ ID NO: 2.

[0092] 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 99% overall sequence identity to the amino acid of the reference sequence, for example represented by SEQ ID NO: 7, 17 or 18.

[0093] 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 identity to the amino acid of the reference sequence, for example represented by SEQ ID NO: 8.

[0094] 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.

[0095] 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.

[0096] For example, 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 ID NO:48), OsOx42 (SEQ ID NO:49), ZmOx2 (SEQ ID NO:50), OsOx2 (SEQ ID NO:51), AtOx2.1 (At2g05590.1, corresponds to OXR2 splice variant: SEQ ID NO: 19), BoOx2 (SEQ ID NO:52), BrOx2 (SEQ ID NO:53), HvOx2 (SEQ ID NO: 54), GmOx2 (SEQ ID NO: 55), TaOx2 (SEQ ID NO: 56), AtOx2.2 (At2g05590.2, corresponds to OXR2: SEQ ID NO:8) VvOx2 (SEQ ID NO:57), AtOxl (SEQ ID NO:20), VvOxl (SEQ ID NO:58), ZmOxl (SEQ ID NO:59), OsOxl l (SEQ ID NO:60) and OsOxl2 (SEQ ID NO:61) and functional variants and polypeptide parts thereof.

[0097] 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. Appropriate 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.

[0099] In embodiments, 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. In embodiments, 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.

[00100] In some embodiments, 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,

50, 60, 100, 200, 300, or 500 nucleotides or up to the full length of for example SEQ ID NO: 1 or 7. Such 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.

[00101] 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.

[00102] Stringent hybridization conditions and stringent hybridization wash conditions in the context of nucleic acid hybridization experiments, such as Southern and Northern blot analysis, 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). Generally, 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. Typically, under stringent conditions a probe will hybridize specifically to its target sequence, but not to other sequences.

[00103] 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). Typically, 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. For short probes (e.g., about 10 to 50 nucleotides), 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.

[00104] 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. Accordingly, 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.

[00105] 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. See Batzer et al., Nucleic Acids Res, 19:5081 (1991), Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1991); and Rossolini et al., Mol. Cell. Probes 8:91 -98 (1994).

[00106] 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.

[00107] 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.

[00108] In one aspect, 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.

[00109] In one embodiment, 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. For example, 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. Similarly, a first portion of a plant OXR4, for example from a crop plant, may be joined to a second portion of a AtOXR2 polynucleotide.

[00110] In one embodiment, the first OXR polynucleotide comprises SEQ ID NO: 1, 15 or

16 or a functional variant or homolog thereof; and the second polynucleotide comprises SEQ ID NO: 7, 17 or 18, a functional variant or homolog thereof. In one embodiment, 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. In one embodiment, 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.

[00111] In another embodiment, 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.

[00112] In embodiments, 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 according to some embodiments 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 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 OXR2 nucleotide sequence of SEQ K) NO: 11.

[00113] In some embodiments, 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.

[00114] Also within the scope of the invention are combinations other than OXR4 and

OXR2. 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. [00115] 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. Thus, 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.

[00116] In one embodiment, 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.

[00117] In some embodiments, the chimeric protein is a Q42 polypeptide wherein 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. 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

80% sequence identity with the 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.

[00118] In preferred embodiments, 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.

[00119] In one embodiment, the chimeric protein comprises SEQ ID NO: 14 (encoded by

SEQ ID NO: 13), a functional variant or homolog thereof.

[00120] Also within the scope of the invention are combinations other than OXR4 and

OXR2. Any amino-terminal part from any plant OXR polypeptide may be combined with any carboxy-terminal part from a second plant OXR polypeptide.

[00121] Five OXR encoding nucleic acid sequences (including splice variants) have been identified in Arabidopsis thaliana: AtOXRl, AtOXR2, AtOXR4 and AtOXR5. For example, 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. Thus, possible combinations are: 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.

[00122] Nucleic acid sequences and polypeptide sequences for AtOXRl, AtOXR2,

AtOXR4 and AtOXR5, their N and C terminal parts and constructs are shown herein, see Fig 19.

[00123] In other embodiments of the invention the chimeric polypeptide includes other combinations of first and second polypeptide portions. For example, 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

50%, 60%, 70%, 80% or 90% sequence identity with the AtOXR4 carboxyl-terminal encoding sequence of SEQ ID NO: 5 resulting in a AtQ24 polypeptide construct. [00124] For example, 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.

[00125] For example, 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.

[00126] Other constructs according to the invention are selected from Q12, Q21 , Q41,

Q14, Q51, Q15, for example AtQ12, AtQ21, AtQ41, AtQ14, AtQ51, AtQ15 and homologs or variants thereof.

[00127] Thus, in some embodiments 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).

[00128] 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).

[00129] In embodiments, the coding region for the chimeric protein, for example a Q42 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).

[00131] 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.

[00132] 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. Generally, plants with hardy stems are less sensitive to lodging and breakage that causes losses of yield. Furthermore, 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).

[00133] It will be understood by persons skilled in the art that 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.

[00134] 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.

[00135] The invention also relates to sequences and constructs as described in the accompanying figures.

[00136] As will be explained below, the polynucleic acid sequences can be used in methods for producing plants and in methods for modulating a plant phenotype.

Expression Constructs

[00137] 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.

[00138] 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. In some embodiments, 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.

[00139] 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. For example, 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. In eukaryotes, this typically involves secretion into the Golgi apparatus, with some resulting glycosylation. 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

[00140] Suitable constructs may also contain 5' and 3' untranslated regions. A 3' untranslated region is a polynucleotide located downstream of a coding sequence. Polyadenylation signal sequences and other sequences encoding regulatory signals capable of affecting the addition of polyadenylic acid tracts to the 3' end of the mRNA precursor or the 3' untranslated regions. A 5' untranslated region is a polynucleotide located upstream of a coding sequence.

[00141] 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. 5:141-149 (1991); Mogen et al., Plant Cell 2:1261-1272 (1990); Munroe et al., Gene 91 : 151-158 (1990); Ballas et al., Nucleic Acids Res. 17:7891 -7903 (1989); and Joshi et al., Nucleic Acids Res. 15:9627-9639 (1987). These vectors are plant integrating vectors in that upon transformation, the vectors integrate a portion of vector DNA into the genome of the host plant.

[00142] In embodiments, 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.

[00143] In embodiments, 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).

[00144] 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).

[00145] Stress-regulated regulatory elements, which regulate expression of an operatively linked nucleotide sequence in a plant in response to a stress condition, are also provided. 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. By performing such assays, 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. Preferably, 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.

[00146] Regulatory elements, as described and defined herein, 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. In some embodiments, 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.

[00147] Plant expression vectors suitable for use in embodiments may comprise one or more DNA vector(s) for achieving plant transformation. For example, it is common practice for persons skilled in the art to utilize 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.

[00148] In some embodiments, 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.

[00149] 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. Often a second plasmid vectors the trans-acting factors that mediate T-DNA transfer from Agrobacterium to the target (host) plant cells. This plasmid 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). 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.

[00150] It will be appreciated by those skilled in the art that 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. In embodiments, 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.

[00151] In embodiments, regulatory elements (or regulatory sequences) 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. [00152] In some embodiments, 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).

[00153] To define a minimal promoter region, 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. To ensure that the expression level is due to the promoter, rather than the stability of the mRNA, the level of the reporter mRNA can be measured directly (e.g., by Northern blot analysis).

[00154] Once enzyme activity is detected, 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. Although promoters from dicotyledons have been shown to be operational in monocotyledons, and vice versa, dicotyledonous promoters are ideally selected for expression in dicotyledons, and monocotyledonous promoters are ideally selected for expression in monocotyledons. There is no restriction as to 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.).

[00156] 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.

[00157] In embodiments, 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. Moreover, 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).

[00158] 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. See e.g., Odell et al., Nature 313:810-812 (1985); McElroy et al., Plant Cell 2:163-171 (1990); Christensen et al., Plant Mol. Biol. 12:619-632 (1989); Christensen et al, Plant Mol. Biol. 18:675-689 (1992); Last et al., Theor. Appl. Genet. 81 :581-588 (1991); Velten et al., EMBO J. 3:2723-27310 (1984); Lepetit et al., Mol. Gen. Genet. Ι^'Μ .ΙΊβ-Ι^ (1992); and U.S. Patents Nos: 4,771,002; 5,102, 796; 5,182,200; 5,428,147; 5,850,019; 5,563,328; 5,378,619.

[00159] 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

In2 gene that responds to benzenesulfonamide herbicide safeners, and the promoter of the Tet repressor from TnlO. See Mett et al., Proc. Natl. Acad. Sci., 90:4567-4571, USA (1993);

Hershey et al., Mol. Gen. Genet. 227:229-237 (1991); and Gatz et al., Mol. Gen. Genet. 243:32-

38 (1994). 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

(1991); Zuo et al., Plant J. 24:265-273 (2000). Other inducible promoters suitable for use in embodiments may be selected from promoters described in EP 332104, PCT International

Publications Nos. W093/21334 and WO 97/06269. Promoters composed of portions of other promoters and partially or totally synthetic promoters may also be used. See e.g., Ni et al, Plant J. 7:661 -676 (1995); and PCT International Publication No. 95/14098 (describing use of such promoters in plants).

[00160] The promoter may include, or be modified to include, one or more enhancer elements to thereby provide for higher levels of transcription. Examples of suitable 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.

[00161] In embodiments, 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.

[00162] In embodiments, one or more plant expression cassette {i.e., a Q42 open reading frame operably linked to a promoter) may be inserted into 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.

Plants

[00163] 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. Thus, the construct comprises a chimeric nucleic acid, encoding a chimeric OXR polypeptide as described herein, for example a Q42 polypeptide.

[00164] Constructs comprising the polynucleotide, vectors and expression cassettes are described herein and include, for example, a Q42 chimeric construct, as described herein.

[00165] In one embodiment, the plant polynucleotide comprises a first portion of a first

OXR polynucleotide and a second portion of a second OXR polynucleotide.

[00166] In one embodiment, 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.

[00167] In one embodiment, 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.

[00168] In one embodiment, 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.

[00169] In one embodiment, the first region comprises a sequence with at least

50%, 60%, 70%, 80% or 90% sequence identity with the OXR4 amino-terminal encoding nucleotide sequence represented by SEQ ID NO: 3; and 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.

[00170] In one embodiment, 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.

[00171] Other combinations are also described herein. [00172] 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.

[00173] 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. For example, 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.

[00174] A monocot plant may, for example, be selected from the families Arecaceae,

Amaryllidaceae or Poaceae. For example, 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.

[00175] Also included are 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. Also included are crops for silage (maize), grazing or fodder (grasses, clover, sanfoin, alfalfa), fibres (e.g. cotton, flax), building materials (e.g. pine, oak), pulping (e.g. poplar), feeder stocks for the chemical industry (e.g. 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).

[00176] Preferably, the plant is a crop plant. By crop plant is meant any plant which is grown on a commercial scale for human or animal consumption or use. In a preferred embodiment, the plant is a cereal.

[00177] 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. [00178] In one embodiment, 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. In another embodiment, the transgene is a chimeric polynucleotide which includes OXR sequences of said plant. For example, a transgenic maize plant according to the invention may express a Zm chimeric OXR polynucleotide.

[00179] In one embodiment, the plant includes in its genome a stably integrated transgene which comprises the chimeric gene sequence.

[00180] In some embodiments, 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. 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.

[00181] Cells that have been transformed may be grown into plants in conventional ways.

See e.g., McCormick et al, Plant Cell Rep. 5:81-84 (1986). The plants may be grown, and either pollinated with the same transformed strain or different strains, the resulting hybrid having constitutive expression of the desired phenotypic characteristic identified. Two or more generations may be grown to ensure that expression of the desired phenotypic characteristic is stably maintained and inherited, and then seeds harvested to ensure expression of the desired phenotypic characteristic has been achieved. In this manner, 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.

[00182] The various aspects of the invention described herein clearly extend to any plant cell or any plant produced, obtained or obtainable by any of the methods described herein, and to all plant parts and propagules thereof unless otherwise specified. The present invention extends further to encompass the progeny of a primary transformed or transfected cell, tissue, organ or whole plant that has been produced by any of the aforementioned methods, the only requirement being that progeny exhibit the same genotypic and/or phenotypic characteristic(s) as those produced by the parent in the methods according to the invention.

[00183] 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

[00184] 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. Successful examples of modifications of plant characteristics by transformation with cloned sequences, which serve to illustrate current knowledge in the art and are herein incorporated by reference, include: U.S. Patents Nos: 5,571,706; 5,677,175; 5,510,471; 5,750,386; 5,597,945; 5,589,615; 5,750,871 ; 5,268,526; 5,780,708; 5,538,880; 5,773,269; 5,736,369; and 5,610,042.

[00185] The generation of transgenic plants according to embodiments 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.

[00186] Generally, there are three types of conventional Agrobacterium-mediated transformation methods. 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.

[00187] The efficiency of 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. See Shahla et al., Plant Molec. Biol. 8:291-298 (1987). Alternatively, 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).

[00188] In some embodiments, 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.

[00189] In some embodiments, 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. In cases where 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. However in cases where only the coding sequence or a portion thereof is inserted, heterologous translational control signals that include the ATG initiation codon may be provided.

[00190] Using polynucleotides disclosed herein, a protein may be expressed in 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.

[00191] In embodiments, 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. For example, to obtain a high level of expression of a cloned gene, it is generally desirable to construct 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. Persons skilled in the art would recognize that modifications could be made to a protein of the present invention without diminishing its biological activity.

[00192] In general, 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 general description of techniques and methods for generating transgenic plants may be found in Ayres et al., CRC Crit. Rev. Plant Sci. 13:219-239 (1994), and Bommineni et al., Maydica 42:107-120 (1997).

[00193] In general, since 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. Often, 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. For example, 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. Alternatively, particularly in considering the safety of the transgenic plants, the transformed plants can be selected under environmental stresses avoiding the incorporation of any selectable marker genes.

[00194] Using polynucleotides disclosed herein, a protein may be expressed in 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.

[00195] In embodiments, 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. For example, to obtain a high level of expression of a cloned gene, it is generally desirable to construct 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. Persons skilled in the art would recognize that modifications could be made to a protein of the present invention without diminishing its biological activity.

[00196] Where appropriate, 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.

[00197] For example, polynucleotides of interest can be targeted to the chloroplast for expression. In this manner, where the polynucleotide of interest is not directly inserted into the chloroplast, an expression cassette according to some embodiments may additionally contain a polynucleotide encoding a transcription factor polypeptide to direct the nucleotide of interest to the chlorop lasts. Such 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. 264: 17544- 17550 (1989); Della-Cioppa et al., Plant Phsyiol. 84:965-968 (1987); Romer et al., Biochem. Biophys. Res. Commun. 196: 1414-1421 (1993); and Shah et al., Science 233:478-481 (1986). 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.

[00198] For example, 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. 82:8524-5828 (1985); Hohn et al., Molecular Biology of Plant Tumors, Academic Press, New York, NY. pp. 549-560 (1982); U.S. Patent No. 4,407,956; Klein et al., Nature 327:70-73 (1987); PCT International Publication No. WO 85/01856. The 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). [00199] 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. As will be understood by persons skilled in the art, 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.

[00200] 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.

[00201] 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.

[00202] 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. As another example, 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).

Host Cells

[00203] 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. In some embodiments, 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.

[00204] In preferred embodiments, the host cell is a plant cell and may be a monocot or a dicot. Specifically, 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.

[00205] 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.

[00206] Following transformation, the plants are preferably selected using a dominant selectable marker incorporated into the transformation vector. After transformed plants are selected and grown to maturity, those plants showing a modified trait are identified. The modified train can be any of the traits described herein. Additionally, to confirm that the modified trait is due to changes in expression levels or activity of the polypeptide or polynucleotide disclosed herein, the mRNA expression may be analyzed using Northern blots, RT-PCR or microarrays, or protein expression using immunoblots or Western blots.

[00207] 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.

Methods

[00208] 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.

[00209] Constructs comprising the polynucleotide, vector and expression cassette are described herein and include, for example, a Q42 chimeric construct, as described herein.

[00210] For example, in one embodiment, the plant polynucleotide comprises a first portion of a first OXR polynucleotide and a second portion of a second OXR polynucleotide.

[00211] In one embodiment, 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.

[00212] In another embodiment, 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.

[00213] In one embodiment, 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. [00214] In one embodiment, the first region comprises a sequence with at least 50%, 60%,

70%, 80% or 90% sequence identity with the OXR4 amino -terminal encoding nucleotide sequence represented by SEQ ID NO: 3; and 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.

[00215] In one embodiment, 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.

[00216] Other combinations are also described herein.

[00217] Preferred plants are also listed herein.

[00218] In one embodiment, 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.

[00219] In one embodiment, said phenotype is increased yield.

[00220] The method may comprise the steps of assessing a yield related trait compared to a control plant.

[00221] 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.

[00222] In one embodiment, 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.

[00223] 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. [00224] 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.

[00225] 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.

[00226] The invention also relates to nucleic acid and polypeptide sequences and constructs described in the figures.

[00227] While the foregoing invention provides a general description of the subject matter encompassed within the scope of the present invention, including methods, as well as the best mode thereof, of making and using this invention, the following examples are provided to further enable those skilled in the art to practice this invention and to provide a complete written description thereof. However, those skilled in the art will appreciate that the specifics of these examples should not be read as limiting on the invention, the scope of which should be apprehended from the claims and equivalents thereof appended to this invention. Various further aspects and embodiments of the present invention will be apparent to those skilled in the art in view of the present invention.

[00228] All documents mentioned in this specification are incorporated herein by reference in their entirety, including references to gene and protein accession numbers.

[00229] "and/or" where used herein is to be taken as specific invention of each of the multiple specified features or components with or without the other at each combination unless otherwise dictated. For example "A, B and/or C" is to be taken as specific invention of each of (i) A, (ii) B, (iii) C, (iv) A and B, (v) B and C or (vi) A and B and C, just as if each is set out individually herein.

[00230] Unless context dictates otherwise, the descriptions and definitions of the features set out above are not limited to any particular aspect or embodiment of the invention and apply equally to all aspects and embodiments which are described.

[00231] The invention will be further described in the following non-limiting examples. EXAMPLES

SUMMARY:

[00232] Different chimeric proteins have been made as shown in Fig 1 and 19 using the techniques described herein. 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.

Background information about the OXR protein family.

[00233] 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. 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).

[00234] We identified yeast and humans OXR homologous proteins across the plant kingdom and especially in Arabidopsis thaliana.

[00235] We identified the protein sequence and inspected the characteristics of these sequences using several bioinformatics proteins tools available in the public domain: Plant proteome database (http://ppdb.tc.cornell.edu/dbsearch/searchacc.aspx) Sun Q, Zybailov B, Majeran W, Friso G, Olinares PD, van Wijk KJ. (2008) PPDB, the Plant Proteomics Database at Cornell. Nucleic Acids Res), UniProt database (http://www.uniprot.org/) (UniProt: a hub for protein information; Nucleic Acids Res. 43: D204-D212 (2015)). [00236] We identified 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).

[00237] By PCR strategy we built different chimeric cDNA fragments that were cloned into the binary vector pLBM, a modified version of the binary vector pBil21. pBil21 : Chen, P. Y., Wang, C. K., Soong, S. C, & To, K. Y. (2003). Complete sequence of the binary vector pBI121 and its application in cloning T-DNA insertion from transgenic plants. Molecular breeding, 11(4), 287-293.

[00238] In Arabidopsis thaliana, we identified five putative members of the OXR family. In order to elucidate the function of these proteins in plants, we started studying two of these members, which we called AtOXR2 (At2g05590) and AtOXR4 (At4g39870). By 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. Like OXR human proteins, AtOXR2 and AtOXR4 suppress the oxidative mutant phenotype when expressed in an E. coli strain that lacks enzymes for the DNA repair system. These previous results suggested us that AtOXR proteins would be excellent candidates to evaluate their role in the mechanisms of plant adaptation and growth under unfavorable conditions. For these purpose, we overexpressed AtOXR2 and AtOXR4 under the control of the 35SCaMV constitutive promoter in Arabidopsis Col-0 plants, thus obtaining oeOXR2 and oeOXR4 plants.

[00239] 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).

[00240] We then generated Arabidopsis plants expressing chimeric proteins with parts of AtOXR2 and AtOXR4. We observed that plants that express the chimeric protein Q42 (composed of the N- terminal half of AtOXR4 fused to the C-terminal half of AtOXR2) show interesting characteristics like increased shoot and root biomass, increased seed production and increased stem width. [00241] Main objectives: To generate chimeric (Q) proteins by fusing two different regions of Arabidopsis proteins containing TLDc domains. To characterize the effect of expressing these Q-proteins in Arabidopsis plants. To evaluate the possibility of using this technology for the improvement of economically important traits in crops.

I. Methodology used to generate plants expressing a chimeric protein:

1. Generation of the chimeric protein:

[00242] By using specific genetic engineering methods, including PCR reactions followed by ligations and subsequent molecular cloning, a construct containing a fusion of two different cDNA regions of AtOXR genes were generated (Figures 1 and 19). In one construct, a fusion of AtOXR4 and AtOXR2 was generated.

[00243] The complete coding region for the chimeric proteins, including AtQ42 were cloned in a modified version of the binary vector pBil21 (with prior changes made in our laboratory). This vector contains a strong constitutive promoter (35SCaMV), which generates high expression levels of the protein of interest in most plant tissues.

[00244] Plants were transformed with the constructs using standard transformation techniques.

2. Obtaining plants expressing AtQ42:

[00245] 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 μΕ 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. 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. For statistical treatments we used InfoStat software (2009). Data were analysed by ANOVA, using the statistical test LSD (Least Significant Differences) with a level of significance of 5%.

Results: I. Main results:

Phenotypic characterization of Q42 plants:

Q42 plants show increased shoot and root biomass

[00246] 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).

[00247] In order to quantify the differences in biomass, the dry weight of different tissues from control (Wt) and AtQ42 plants was determined. Roots and aerial tissues (stems and leaves separately) were harvested, weighted and placed at 70°C in an oven. Tissues were weighted regularly until constant values were obtained, indicative of total water loss. The results are shown in Figure 3.

[00248] According to Figure 3, it can be concluded that:

1. At the same developmental stage, the dry weight of AtQ42 leaves is 22% higher than in Wt leaves.

2. The dry weight of stems (SDW) in AtQ42 plants increases 160% respective to Wt plants.

3. The dry weight of the AtQ42 aerial portion (TSDW: total shoot dry weight) increases 50% respective to Wt plants.

4. The root dry weight of AtQ42 plants is significantly higher (about 4 times) than that of Wt plants.

5. The total dry weight (TDW) of AtQ42 plants is more than two-fold higher than that of Wt plants.

Leaf parameters:

Several parameters related to leaves (i.e.: number, mass) were analysed in plants grown under control conditions. Figure 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:

[00249] 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).\

b. Yield under water deficit or salt stress:

[00250] Seed production was also analysed in plants under salinity growth conditions.

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.

[00251] A relative increase in seed production per plant, ranging from 40% to 65%, was observed in lines expressing AtQ42 respective to Wt plants also under these conditions (Figure 6).

[00252] In addition, seed production was analysed in Q42 and Wt plants under water deficit conditions imposed between weeks 3 to 5 of growth according to Hummel et al. (2010). We determined that AtQ42 lines show about 2-fold increased seed yield compared to Wt (Figure V).

Parameters defining seed yield per plant:

[00253] In order to assess which parameters are responsible for generating the increased seed yield per plant in AtQ42 plants, we measured the number of branches (Fig. 8 A), the number of secondary stems (Fig. 8B), the number of siliques per plant (Fig. 8C), the number of seeds per silique (Fig. 8D), and the weight of seeds (Fig. 8E). The results indicate that AtQ42 plants exhibit an increased number of branches per plant and that this component is probably responsible for an increased number of siliques per plant.

I. Secondary outcomes:

Architecture and lignin content of the main stem:

[00254] Histological preparations were performed on cross-sections of main stems of

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).

[00255] In parallel, histological stem sections were stained with safranin-fast green dye. Safranin selectively stains lignified tissues, adopting a reddish-brownish colour (Figure 9B). Thus, using an image analysis program (Image J ®) we estimated the proportion of lignified (woody) tissue in the stem. We observed that AtQ42 plants have 54% more lignin content in their stem tissues than Wt plants (Figure 9E).

[00256] 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).

Photosynthetic parameters:

[00257] C0₂ fixation, gas exchange and stomatal closure were measured in Wt and AtQ42 plants. Values were obtained using an IRGA (Infra-Red Gas Analyser).

[00258] According to the analysis, it can be concluded that 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). Moreover, AtQ42 plants also showed lower stomatal conductance (Fig. 10B), thus resulting in increased water use efficiency (Fig. IOC).

Analysis of a minimum spanning tree:

[00259] Using several parameters analysed in different 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.

[00260] Q42 plants show higher content of ANTIOXIDANT ENZYMES :

[00261] We measured SOD (Superoxide Dismutase) activity in gel. By this technique, it is possible differentiate all isoforms of the enzyme localized in different cell compartments. Thus, MnSOD is a typical enzyme present in mitochondria, FeSOD is localized in chloroplasts and Cu/ZnSOD is involved in ROS detoxification both in cytosol and peroxisomes. We observed that 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.

[00262] Expression of chimeric proteins in soybean, rice, maize.

[00263] We made the following constructs.

1. 35S: :AtQ42::NOS-ter; BastaR(plasmid pTFlOl):

2. pUBI: :AtQ42::NOS-ter; BastaR (plasmid pTFlOl):

3. 35S: :AtQ42::NOS-ter; HygroR (plasmid pCAMBIA1802)

We introduced those constructs in different plants of agricultural interest: soybean, rice, maize using standard transformation techniques known in the art.

[00264] We are analysing transgenic plants thus obtained.

CONCLUSIONS

[00265] According to the results present here, plants expressing the chimeric construct

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:

Increased production of grain or seeds per plant and/or area.

[00267] Increased biomass, useful for fodder crops and plants used for biofuel production. Examples: alfalfa (Medicago sativa) and various clover species (white and red, Trifolium repens and Trifolium pratense, respectively).

[00268] Increased root biomass. Important in legumes (soybean, alfalfa) for the rapid development of a root system that allows faster nodulation for nitrogen fixation and soil nutrition. Furthermore, the development of the root system is a characteristic of interest for any crop, as this promotes irrigation and aeration of the soil and also prevents erosion. An example is the cultivation of sunflower (Helianthus annuus L ), which is cultivated in marginal areas (displaced by soybean). These areas are often characterized by having long periods of water shortage. The greater root biomass could circumvent problems of water stress, allowing the exploration of soil horizons with greater humidity. Increased root development is also important to prevent lodging of the plants.

[00269] 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. In general, plants with hardy stems are less sensitive to lodging and breakage that cause losses of yield. Furthermore, 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.

REFERENCES:

1. Berlyn G.P, Micksche P (1976). Botanical microtechnique and cytochemistry. Iowa State University Press, Ames, IA. Doerks T, Copley RR, Schultz J (2002) Systematic identification of novel protein domain families associated with nuclear functions. Genome Res 12: 47-56. 2. Durand M, Kolpak A, Farrell T, Elliott NA, Shao W, Brown M, Volkert MR (2007) The Oxr domain defines a conserved family of eukaryotic oxidation resistance proteins. BMC

3. Elliot NA, Volkert MR (2004) Stress induction and mitochondrial localization of Oxrl proteins in yeast and humans. Mol Cell Biol 24: 3180-3187.

4. Fischer H, Zhang XU, O'Brien KP, Kylsten P, Engvall E (2001) C7, a novel nucleolar protein, is the mouse homologue of the Drosophila Late Puff Product L82 and an isoform of human OXRl. Biochem Biophys Res Commun 281 :795-803.

5. Hoagland, D. R., Arnon, D. I. (1950): The water-culture method for growing plants without soil. California Agricultural Experiment Station Circular, pp. 1-32.

6. Hummel, I., Pantin, F., Sulpice, R, Piques, M., Rolland, G., Dauzat, M., Christophe, A., Pervent, M., Bouteille M., Stitt, M., Muller, B. 2010. Arabidopsis plants acclimate to water deficit at low cost through changes on carbon usage: an integrative perspective using growth, metabolite, enzyme, and gene expression analysis. Plant Physiology 154: 357-372.

7. Jaramillo-Gutierrez G, Molina-Cruz A, Kumar S, Barillas-Mury C (2010) The Anopheles gambiae Oxidation Resistance 1 (OXRl) gene regulates expression of enzymes that detoxify reactive oxygen species. PLoS ONE 5: el 1168. doi: 10.1371/journal.pone.0011168.

8. Natoli R, Pro vis J, Valter K, Stone J (2008) Expression and role of the early-response gene Oxrl in the hyperoxia- challenged mouse retina. Invest Ophth & Vis Sci 49: 4561- 4567.

9. Volkert MR, Elliott NA, Housman DE (2000) Functional genomics reveals a family of eukaryotic oxidation protection genes. Proc Natl Acad Sci USA 97: 14530-14535.

10. Oliver PL, Finelli MJ, Edwards B, Bitoun E, Butts DL, Becker EB, Cheeseman MT, Davies B, Davies KE. Oxrl Is Essential for Protection against Oxidative Stress-Induced Neurodegeneration. PLoS Genet. 2011

SEQUENCE INFORMATION

SEQ ID NO: 1 AtOXR4 - complete gene sequence CDS

SEQ ID NO: 2 AtOXR4 - complete protein sequence SEQ ID NO: 3 AtOXR4 - gene sequence of the amino-terminal fragment of SEQ ID NO: 1

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: 7 AtOXR2 - complete gene sequence CDS (splice variant At2g05590.2)

SEQ ID NO: 8 AtOXR2 - complete protein sequence

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: 13 AtQ42 - gene sequence (corresponding to SEQ ID NO: 3 + SEQ ID NO: 11)

SEQ ID NO: 14 AtQ42 - protein sequence (corresponding to SEQ ID NO: 4 + SEQ ID NO: 12)

SEQ ID NO: 15 AtOXR4 - complete gene sequence genomic

SEQ ID NO: 16 AtOXR4 - complete gene sequence cDNA

SEQ ID NO: 17 AtOXR2 - complete gene sequence genomic

SEQ ID NO: 18 AtOXR2 - complete gene sequence cDNA

SEQ ID NO: 19 AtOXR2 - complete protein sequence splice variant

SEQ ID NO: 20 AtOXRl complete protein sequence

SEQ ID NO: 21 AtOXR5 complete protein sequence

SEQ ID NO: 22 AtOXR5D complete protein sequence

SEQ ID NO: 23 AtOXRl complete gene sequence CDS

SEQ ID NO: 24 AtOXR5 complete gene sequence CDS

SEQ ID NO: 25 AtOXR5D complete gene sequence CDS

SEQ ID NO: 1 OXR4 CDS

ATGGGGAAACACAAATCTTTCAGAAGCAAAGCAGTGCACTTTGTCACCGATCTCACCGCTGG TCTTCTTAATCCCATCTCTGATAAACCCTCCTCCGCTCATCCTCCTCCTCCTCTTCCGGATGAG GAAGATGAGTCTAAGAGAAATCAGCTAGAGTCTACAACTGCAGAGCAGCCTAAGGATTTGG TTGACGAACCAGACACTTCTTCTTTCTCTGCATTCCTTGGTTCACTGTTATCTTCAGATCCCAA AGACAAAAGGAAGGATCAGGATCCAGAGGACGAAGAAGACGAGGAAGAAGACGAAGAAG

AAGATTCTGAGGCAGAAACAAGCGATACATCATCATCATCTGCCAATCCTACTAGAACTATG

AAGGAGACTACTAGTGGTGGTGCTGCAAAGAAGAGTTTTTTGTCCAAGTACAAACAGCATTT

TAGGAATTTTTACCAGGCTGTTAAGTTTCCCGGGGTCAAGGAGCGAAAGGGAAATTCTGATG

TGATACCAGATGATGAGGAGACTGAATATTATGATGGCTTAGAGATGAAGCCGATGCAGAA

TAATAATGTCAAAGAGGAAGTGACAGTAGTAGTGCAAGCAATTATACCTGAAATTTCAGAA

CCCTCTTTGCTTTTATCGGAGCAGTCTAGACGCTCCCTTTATACTTCACTTCCTGCGCTTGTTC

AAGGGAGGAAATGGATATTGCTTTATAGTACATGGAGGCATGGTATATCACTATCCACGCTA

TACAGGAAAAGCCTTCTCTGGCCAGGTCTTAGCCTACTGGTTGTTGGAGACAGAAAAGGTTC

GGTTTTTGGTGGTCTTGTTGAGGCACCTTTGATACCAACTGATAAAAAGTATCAGGGAACCA

ACAGTACCTTTGTTTTCACAAATAAATCAGGACAACCCACAATATACCGTCCCACAGGCGCA

AATCGGTTCTACACTTTATGCTCCAAGGAATTCCTAGCTCTGGGCGGTGGTGGACGGTTTGCT

CTCTATCTAGACAGTGAGCTGCTAAGCGGATCGAGCGCTTACTCAGAGACGTATGGGAACTC

TTGTCTCGCAGACTCTCAGGACTTTGATGTTAAGGAAGTGGAGCT

SEQ ID NO: 2 OXR4 - complete protein sequence TLDC domain in bold

MGKHKSFRSK AVHFVTDLTA GLLNPISDKP SSAHPPPPLP DEEDESKRNQ LESTTAEQPK DLVDEPDTSS FSAFLGSLLS SDPKDKRKDQ DPEDEEDEEE DEEEDSEAET SDTSSSSANP TRTMKETTSG GAAKKSFLSK YKQHFRNFYQ AVKFPGVKER KGNSDVIPDD EETEYYDGLE MKPMQNNNVK EEVTVVVQAI IPEISEPSLL LSEQSRRSLY TSLPALVQGR KWILLYSTWR HGISLSTLYR KSLLWPGLSL LWGDRKGSV FGGLVEAPLI PTDKKYQGTN STFVFTNKSG QPTIYRPTGA NRFYTLCSKE FLALGGGGRF ALYLDSELLS GSSAYSETYG NSCLADSQDF DVKEVELWGF VYGSKYDEIL AHSKTMEPGL CRWS

SEQ ID NO: 3 0XR4 - gene sequence of the amino-terminal fragment of SEQ ID NO: 1

ATGGGGAAACACAAATCTTTC AGAAGCAAAG CAGTGCACTT TGTCACCGAT CTCACCGCTGGTCTTCTTAA TCCCATCTCT GATAAACCCT CCTCCGCTCA TCCTCCTCCT CCTCTTCCGG ATGAGGAAGA TGAGTCTAAG AGAAATCAGC TAGAGTCTAC AACTGCAGAG CAGCCTAAGG ATTTGGTTGA CGAACCAGAC ACTTCTTCTT TCTCTGCATT CCTTGGTTCA CTGTTATCTT CAGATCCCAA AGACAAAAGG AAGGATCAGG ATCCAGAGGA CGAAGAAGAC GAGGAAGAAG ACGAAGAAGA AGATTCTGAG GCAGAAACAA GCGATACATC ATCATCATCT GCCAATCCTA CTAGAACTAT GAAGGAGACT ACTAGTGGTG GTGCTGCAAA GAAGAGTTTT TTGTCCAAGT ACAAACAGCA TTTTAGGAAT TTTTACCAGG CTGTTAAGTT TCCCGGGGTC AAGGAGCGAA AGGGAAATTC TGATGTGATA CCAGATGATG AGGAGACTGA ATATTATGAT GGCTTAGAGA TGAAGCCGAT GCAGAATAAT AATGTCAAAG AGGAAGTGAC AGTAGTAGTG CAAGCAATTA TACCT

SEQ ID NO: 4 OXR4 - protein sequence of the amino-terminal fragment of SEQ ID NO: 2

MGKHKSFRSK AVHFVTDLTA GLLNPISDKP SSAHPPPPLP DEEDESKRNQ LESTTAEQPK DLVDEPDTSS FSAFLGSLLS SDPKDKRKDQ DPEDEEDEEE DEEEDSEAET SDTSSSSANP TRTMKETTSG GAAKKSFLSK YKQHFRNFYQ AVKFPGVKER KGNSDVIPDD EETEYYDGLE MKPMQNNNVK EEVTVVVQAI IP

SEQ ID NO: 5 OXR4 - gene sequence of the domain region of SEQ ID NO: 1

GAAATTTCAGAACCCTCTTTGCTTTTATCGGAGCAGTCTAGACGCTCCCTTTATACTTCACTT CCTGCGCTTGTTCAAGGGAGGAAATGGATATTGCTTTATAGTACATGGAGGCATGGTATATC ACTATCCACGCTATACAGGAAAAGCCTTCTCTGGCCAGGTCTTAGCCTACTGGTTGTTGGAG ACAGAAAAGGTTCGGTTTTTGGTGGTCTTGTTGAGGCACCTTTGATACCAACTGATAAAAAG

TATCAGGGAACCAACAGTACCTTTGTTTTCACAAATAAATCAGGACAACCCACAATATACCG

TCCCACAGGCGCAAATCGGTTCTACACTTTATGCTCCAAGGAATTCCTAGCTCTGGGCGGTG

GTGGACGGTTTGCTCTCTATCTAGACAGTGAGCTGCTAAGCGGATCGAGCGCTTACTCAGAG

ACGTATGGGAACTCTTGTCTCGCAGACTCTCAGGACTTTGATGTTAAGGAAGTGGAGCT

SEQ ID NO: 6 OXR4 - protein sequence of the domain region of SEQ ID NO: 2

EISEPSLL LSEQSRRSLY TSLPALVQGR KWILLYSTWR HGISLSTLYR KSLLWPGLSL LVVGDRKGSV FGGLVEAPLI PTDKKYQGTN STFVFTNKSG QPTIYRPTGA NRFYTLCSKE FLALGGGGRF ALYLDSELLS GSSAYSETYG NSCLADSQDF DVKEVELWGF VYGSKYDEIL AHSKTMEPGL CRWS

SEQ ID NO: 7 OXR2 CDS

ATGCATGCTCTCAAGGACAAAGTCTCACAAAAACTCTCTAATCTCTTCGCCGATTCTCCAAG

CCAATCTGCTTCTCCTCGTTATTCCAACTCCGATTCTCCAAAGGCTAGGTTGAATTCAAGTGT

AGGCAAATCATTGTCTTCGTATTTCTCATTTGTTGTTCCTCAATCTGGGAACGAGGAGGATTC

TGAGTTATGTCCACCTCTACCCATTAGAACAGAGAGCTATGAGTGTATTGAGAATTGCAAAT

CTGCTAATGGGCAAGCTAAAGCTGGAACCTTTATCTCAATTGGTGAAGATAAGGATTGTGAG

TTACGTGTTTCTGCGAAAGTAGAAGAAAGTGGTAATGATTACTTTGATGGGGTGAAGAAGAT

GAGGGAATTAACAGAGAGCTCTGTGTTTATTACTGCTAACTTGTTTGAATTCTTGCATGCGA

GTCTTCCTAATATTGTAAGAGGATGCAAATGGATCTTGTTGTATAGTACGTTGAAACATGGT

ATATCACTTCGTACGCTTTTGCGAAGAAGTGGAGAGCTTCCTGGTCCTTGTTTACTGGTTGCT

GGAGACAAACAAGGTGCAGTGTTTGGAGCTCTGTTGGAATGTCCACTGCAACCTACTCCTAA

GAGGAAATATCAGGGTACAAGCCAGACATTCTTGTTCACAACTATTTATGGTGAACCACGCA

TATTTAGACCTACCGGTGCCAACCGATATTACCTGATGTGTATGAATGAATTTTTGGCGTTTG

GAGGTGGAGGAAACTTTGCACTATGTTTAGACGAAGATTTGTTGAAGGCAACAAGTGGACC

TTCTGAAACATTTGGGAACGAATGTTTGGCTAGCAGCACGGAGTTTGAGTTAAAGAATGTCG

AGCTCTGGGGATTTGCACATGCGTCTCAATACCTCTCGTAA

SEQ ID NO: 8 OXR2 (also termed OXR 2.2)- complete protein sequence TLDC domain in bold

MHALKDKVSQ KLSNLFADSP SQSASPRYSN SDSPKARLNS SVGKSLSSYF SFVVPQSGNE EDSELCPPLP IRTESYECIE NCKSANGQAK AGTFISIGED KDCELRVSAK VEESGNDYFD GVKKMRELTE SSVFITANLF EFLHASLPNI VRGCKWILLY STLKHGISLR TLLRRSGELP GPCLLVAGDK QGAVFGALLE CPLQPTPKRK YQGTSQTFLF TTIYGEPRIF RPTGANRYYL MCMNEFLAFG GGGNFALCLD EDLLKATSGP SETFGNECLA SSTEFELKNV ELWGFAHASQYLS

SEQ ID NO: 9 OXR2 - gene sequence of the amino-terminal fragment of SEQ ID NO: 7

ATGCATGCTCTCAAGGACAAAGTCTCACAAAAACTCTCTAATCTCTTCGCCGATTCTCCAAG

CCAATCTGCTTCTCCTCGTTATTCCAACTCCGATTCTCCAAAGGCTAGGTTGAATTCAAGTGT

AGGCAAATCATTGTCTTCGTATTTCTCATTTGTTGTTCCTCAATCTGGGAACGAGGAGGATTC

TGAGTTATGTCCACCTCTACCCATTAGAACAGAGAGCTATGAGTGTATTGAGAATTGCAAAT

CTGCTAATGGGCAAGCTAAAGCTGGAACCTTTATCTCAATTGGTGAAGATAAGGATTGTGAG

TTACGTGTTTCTGCGAAAGTAGAAGAAAGTGGTAATGATTACTTTGATGGGGTGAAGAAGAT

GAGGGAA SEQ ID NO: 10 OXR2 - protein sequence of the amino-terminal fragment of SEQ ID NO:8

MHALKDKVSQ KLSNLFADSP SQSASPRYSN SDSPKARLNS SVGKSLSSYF SFVVPQSGNE EDSELCPPLP IRTESYECIE NCKSANGQAK AGTFISIGED KDCELRVSAK VEESGNDYFD GVKKMRE

SEQ ID NO: 11 OXR2 - gene sequence of the domain region of SEQ ID NO: 7

TTAACAGAGA GCTCTGTGTT TATTACTGCT AACTTGTTTG AATTCTTGCA TGCGAGTCTT CCTAATATTG TAAGAGGATG CAAATGGATC TTGTTGTATA GTACGTTGAA ACATGGTATA TCACTTCGTA CGCTTTTGCG AAGAAGTGGA GAGCTTCCTG GTCCTTGTTT ACTGGTTGCT GGAGACAAAC AAGGTGCAGT GTTTGGAGCT CTGTTGGAAT GTCCACTGCA ACCTACTCCT AAGAGGAAAT ATCAGGGTAC AAGCCAGACA TTCTTGTTCA CAACTATTTA TGGTGAACCA CGCATATTTA GACCTACCGG TGCCAACCGA TATTACCTGA TGTGTATGAA TGAATTTTTG GCGTTTGGAG GTGGAGGAAA CTTTGCACTA TGTTTAGACG AAGATTTGTT GAAGGCAACA AGTGGACCTT CTGAAACATT TGGGAACGAA TGTTTGGCTA GCAGCACGGA GTTTGAGTTA AAGAATGTCG AGCTCTGGGG ATTTGCACAT GCGTCTCAAT ACCTCTCGTAA

SEQ ID NO: 12 OXR2 - protein sequence of the domain region of SEQ ID NO: 8

LTESSVFITANLF EFLHASLPNI VRGCKWILLY STLKHGISLR TLLRRSGELP GPCLLVAGDK QGAVFGALLE CPLQPTPKRK YQGTSQTFLF TTIYGEPRIF RPTGANRYYL MCMNEFLAFG GGGNFALCLD EDLLKATSGP SETFGNECLA SSTEFELKNV ELWGFAHASQ YLS

SEQ ID NO: 13 Q42 gene sequence (corresponding to SEQ ID NO: 3 + SEQ ID NO: 11 (in bold))

ATGGGGAAACACAAATCTTTC AGAAGCAAAG CAGTGCACTT TGTCACCGAT CTCACCGCTGGTCTTCTTAA TCCCATCTCT GATAAACCCT CCTCCGCTCA TCCTCCTCCT CCTCTTCCGG ATGAGGAAGA TGAGTCTAAG AGAAATCAGC TAGAGTCTAC AACTGCAGAG CAGCCTAAGG ATTTGGTTGA CGAACCAGAC ACTTCTTCTT TCTCTGCATT CCTTGGTTCA CTGTTATCTT CAGATCCCAA AGACAAAAGG AAGGATCAGG ATCCAGAGGA CGAAGAAGAC GAGGAAGAAG ACGAAGAAGA AGATTCTGAG GCAGAAACAA GCGATACATC ATCATCATCT GCCAATCCTA CTAGAACTAT GAAGGAGACT ACTAGTGGTG GTGCTGCAAA GAAGAGTTTT TTGTCCAAGT ACAAACAGCA TTTTAGGAAT TTTTACCAGG CTGTTAAGTT TCCCGGGGTC AAGGAGCGAA AGGGAAATTC TGATGTGATA CCAGATGATG AGGAGACTGA ATATTATGAT GGCTTAGAGA TGAAGCCGAT GCAGAATAAT AATGTCAAAG AGGAAGTGAC AGTAGTAGTG CAAGCAATTA TACCTTTAACAGAGA GCTCTGTGTT TATTACTGCT AACTTGTTTG AATTCTTGCA TGCGAGTCTT CCTAATATTG TAAGAGGATG CAAATGGATC TTGTTGTATA GTACGTTGAA ACATGGTATA TCACTTCGTA CGCTTTTGCG AAGAAGTGGA GAGCTTCCTG GTCCTTGTTT ACTGGTTGCT GGAGACAAAC AAGGTGCAGT GTTTGGAGCT CTGTTGGAAT GTCCACTGCA ACCTACTCCT AAGAGGAAAT ATCAGGGTAC AAGCCAGACA TTCTTGTTCA CAACTATTTA TGGTGAACCA CGCATATTTA GACCTACCGG TGCCAACCGA TATTACCTGA TGTGTATGAA TGAATTTTTG GCGTTTGGAG GTGGAGGAAA CTTTGCACTA TGTTTAGACG AAGATTTGTT GAAGGCAACA AGTGGACCTT CTGAAACATT TGGGAACGAA TGTTTGGCTA GCAGCACGGA GTTTGAGTTA AAGAATGTCG AGCTCTGGGG ATTTGCACAT GCGTCTCAAT ACCTCTCGTAA

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 ELWGFAHASQ YLS

SEQ ID NO: 15 OXR4 cDNA

CGTCTTTTTAGTTAAGCTGTCTCAAAAACACTCGGCATAAAATTCAGCTCTTTAAGAAAAAA

ATTCAGAGGAAAATTAAAAAAAGTGGATATAATAGAGAAAAAGGTAAAGGAAAGGAATAG

TGAGAGAGAGAGAGGGAGAGATGGTCTTCTTGTTTAGAACATGTTGTTTCCAATTATTCTAA

GAATGGCGTTTTAGGTAAATGATCACAGCTTATTACAGATGGAAGAGAAGCTGACTCAATTT

GTGGTTTAGATTTGTCAAAGAGGATCGTGTGGAATCTGAAAGTTTTCAATTTTCACTAAAAA

TAGGATTAGAGTTAAAAAGGAGGAAGAAGAAATGGGGAAACACAAATCTTTCAGAAGCAA

AGCAGTGCACTTTGTCACCGATCTCACCGCTGGTCTTCTTAATCCCATCTCTGATAAACCCTC

CTCCGCTCATCCTCCTCCTCCTCTTCCGGATGAGGAAGATGAGTCTAAGAGAAATCAGCTAG

AGTCTACAACTGCAGAGCAGCCTAAGGATTTGGTTGACGAACCAGACACTTCTTCTTTCTCT

GCATTCCTTGGTTCACTGTTATCTTCAGATCCCAAAGACAAAAGGAAGGATCAGGATCCAGA

GGACGAAGAAGACGAGGAAGAAGACGAAGAAGAAGATTCTGAGGCAGAAACAAGCGATAC

ATCATCATCATCTGCCAATCCTACTAGAACTATGAAGGAGACTACTAGTGGTGGTGCTGCAA

AGAAGAGTTTTTTGTCCAAGTACAAACAGCATTTTAGGAATTTTTACCAGGCTGTTAAGTTTC

CCGGGGTCAAGGAGCGAAAGGGAAATTCTGATGTGATACCAGATGATGAGGAGACTGAATA

TTATGATGGCTTAGAGATGAAGCCGATGCAGAATAATAATGTCAAAGAGGAAGTGACAGTA

GTAGTGCAAGCAATTATACCTGAAATTTCAGAACCCTCTTTGCTTTTATCGGAGCAGTCTAG

ACGCTCCCTTTATACTTCACTTCCTGCGCTTGTTCAAGGGAGGAAATGGATATTGCTTTATAG

TACATGGAGGCATGGTATATCACTATCCACGCTATACAGGAAAAGCCTTCTCTGGCCAGGTC

TTAGCCTACTGGTTGTTGGAGACAGAAAAGGTTCGGTTTTTGGTGGTCTTGTTGAGGCACCTT

TGATACCAACTGATAAAAAGTATCAGGGAACCAACAGTACCTTTGTTTTCACAAATAAATCA

GGACAACCCACAATATACCGTCCCACAGGCGCAAATCGGTTCTACACTTTATGCTCCAAGGA

ATTCCTAGCTCTGGGCGGTGGTGGACGGTTTGCTCTCTATCTAGACAGTGAGCTGCTAAGCG

GATCGAGCGCTTACTCAGAGACGTATGGGAACTCTTGTCTCGCAGACTCTCAGGACTTTGAT

GTTAAGGAAGTGGAGCTATGGGGATTTGTGTATGGCTCAAAGTATGATGAGATTCTAGCTCA

CAGCAAAACAATGGAGCCTGGTCTTTGCAGATGGTCATGATCATTATAAAGAAAACCCAAA

TGAATTCAACTGAGATTCGTCTTTTTGTTGTTGTTGTTGTTGTTGTTGTATAAATCTGTAGTAA

AAGAAATCTTGTTGTCTTGTAACTTGGCTAAAATCACATATATGTTACTCTCTCTCACTTGTA

ATGATGATTTTGAAATAACAAAATAAAACCACCTATTAGGCTC

SEQ ID NO: 16 OXR4 genomic DNA

CGTCTTTTTAGTTAAGCTGTCTCAAAAACACTCGGCATAAAATTCAGCTCTTTAAGAAAAAA

ATTCAGAGGAAAATTAAAAAAAGTGGATATAATAGAGAAAAAGGTAAAGGAAAGGAATAG

TGAGAGAGAGAGAGGGAGAGATGGTCTTCTTGTTTAGAACATGTTGTTTCCAATTATTCTAA

GAATGGCGTTTTAGGTAAATGATCACAGCTTATTACAGATGGAAGAGAAGCTGACTCAATTT

GTGGTTTAGATTTGTCAAAGAGGATCGTGTGGAATCTGAAAGTTTTCAATTTTCACTAAAAA

TAGGATTAGAGTTAAAAAGGAGGAAGAAGAAATGGGGAAACACAAATCTTTCAGAAGCAA

AGCAGTGCACTTTGTCACCGATCTCACCGCTGGTCTTCTTAATCCCATCTCTGATAAACCCTC

CTCCGCTCATCCTCCTCCTCCTCTTCCGGTACCCAAAGTTCTCATCTTTTTGTGTGTTTTGTGT

TGACCCAATCCCACAATTATTCTTAAAAAGGGTTCTTCTGGATTAAAGTCTTTATCTCTGTTC TTGACTCTGGGAATTTCTGGTTTTTCTTGGCTTTTGTGTTTATGCTTAGTTGTTTCAGGATTTT

GAGTGTTTCTAGGTGGGAGTTTCAATTTGTGTGATCATTTAATGAGAGCTCTTGTTAATTTTC

AATTGTTTTTTTCTTAATTAGGATGAGGAAGATGAGTCTAAGAGAAATCAGCTAGAGTCTAC

AACTGCAGAGCAGCCTAAGGATTTGGTTGACGAACCAGACACTTCTTCTTTCTCTGCATTCCT

TGGTTCACTGTTATCTTCAGATCCCAAAGACAAAAGGAAGGATCAGGATCCAGAGGACGAA

GAAGACGAGGAAGAAGACGAAGAAGAAGATTCTGAGGCAGAAACAAGCGATACATCATCA

TCATCTGCCAATCCTACTAGAACTATGAAGGAGACTACTAGTGGTGGTGCTGCAAAGAAGA

GTTTTTTGTCCAAGTACAAACAGCATTTTAGGAATTTTTACCAGGCTGTTAAGTTTCCCGGGG

TCAAGGAGCGAAAGGGAAATTCTGATGTGATACCAGATGATGAGGAGACTGAATATTATGA

TGGCTTAGAGATGAAGCCGATGCAGAATAATAATGTCAAAGAGGAAGTGACAGTAGTAGTG

CAAGCAATTATACCTGAAATTTCAGAACCCTCTTTGCTTTTATCGGAGCAGTCTAGACGCTCC

CTTTATACTTCACTTCCTGCGCTTGTTCAAGGGAGGAAATGGATATTGCTTTATAGGTGAGAC

AACACATTCAAAGCATTTCGAGCTGATTATAAATCTTAAGAATGGTTTATTTCATATATTCTA

TCTGTGTGTGTGTGTGATCATTTTTCTGAAATCTTGCAATTTTCTAGTACATGGAGGCATGGT

ATATCACTATCCACGCTATACAGGAAAAGCCTTCTCTGGCCAGGTCTTAGCCTACTGGTAAA

ATGAAATTCTTGTTCTGATTCAAATTGTAAAAGTGTGAACCTTTTGTTGAGCTTTATGGAAAC

TGCCGGTGTTTCAGGTTGTTGGAGACAGAAAAGGTTCGGTTTTTGGTGGTCTTGTTGAGGCA

CCTTTGATACCAACTGATAAAAAGTATCAGGTTTGGTTCTAAAGATCCAATCTTTGTATATAG

ATTCCATCTATCCAGTTTTCTTTCTTTTCACTGTAATTAACATTCTTTTGTGTTGGGTTGTTTTG

GCAGGGAACCAACAGTACCTTTGTTTTCACAAATAAATCAGGACAACCCACAATATACCGTC

CCACAGGTATTTATTATGCATTACTTGGATACATAGAACTTGTTGGGAGTTGTTTCATTATTC

ACCTGTAATTATGGATCAGGCGCAAATCGGTTCTACACTTTATGCTCCAAGGAATTCCTAGC

TCTGGGCGGTGGTGGACGGTTTGCTCTCTATCTAGACAGTGAGCTGTAAGATTTCCTTACCA

AATCTCTTAAAATCATATTTGCATTTTTATTATATTGCAAGATTGCGACAGACAACAAGGCAT

TTTTCCTCAAATCGATTGATCCATTGTGTTCTTATAGGCTAAGCGGATCGAGCGCTTACTCAG

AGACGTATGGGAACTCTTGTCTCGCAGACTCTCAGGACTTTGATGTTAAGGAAGTGGAGGTA

GTTAAATTTCATCCCTCTCCTTGAAAAATAAAAGCTTTATATTTCAATATTTTGCAACACCTT

TTAACTGAAACAAGCAAACAAACACACCATTGGTTGATTGATTGTTGCAGCTATGGGGATTT

GTGTATGGCTCAAAGTATGATGAGATTCTAGCTCACAGCAAAACAATGGAGCCTGGTCTTTG

CAGATGGTCATGATCATTATAAAGAAAACCCAAATGAATTCAACTGAGATTCGTCTTTTTGT

TGTTGTTGTTGTTGTTGTTGTATAAATCTGTAGTAAAAGAAATCTTGTTGTCTTGTAACTTGG

CTAAAATCACATATATGTTACTCTCTCTCACTTGTAATGATGATTTTGAAATAACAAAATAAA

ACCACCTATTAGGCTC

SEQ ID NO: 17 OXR2 cDNA

ATCTCCGAGCATCTGGCTTCTCTGTCGCATCTTCTCTTCCCTTTCCCCCACCGACAATTCTATT

CATCTCCGATCAAAAACTATTCCTTTCTGATCACAATGCATGCTCTCAAGGACAAAGTCTCA

CAAAAACTCTCTAATCTCTTCGCCGATTCTCCAAGCCAATCTGCTTCTCCTCGTTATTCCAAC

TCCGATTCTCCAAAGGCTAGGTTGAATTCAAGTGTAGGCAAATCATTGTCTTCGTATTTCTCA

TTTGTTGTTCCTCAATCTGGGAACGAGGAGGATTCTGAGTTATGTCCACCTCTACCCATTAGA

ACAGAGAGCTATGAGTGTATTGAGAATTGCAAATCTGCTAATGGGCAAGCTAAAGCTGGAA

CCTTTATCTCAATTGGTGAAGATAAGGATTGTGAGTTACGTGTTTCTGCGAAAGTAGAAGAA

AGTGGTAATGATTACTTTGATGGGGTGAAGAAGATGAGGGAATTAACAGAGAGCTCTGTGT

TTATTACTGCTAACTTGTTTGAATTCTTGCATGCGAGTCTTCCTAATATTGTAAGAGGATGCA

AATGGATCTTGTTGTATAGTACGTTGAAACATGGTATATCACTTCGTACGCTTTTGCGAAGA

AGTGGAGAGCTTCCTGGTCCTTGTTTACTGGTTGCTGGAGACAAACAAGGTGCAGTGTTTGG

AGCTCTGTTGGAATGTCCACTGCAACCTACTCCTAAGAGGAAATATCAGGGTACAAGCCAGA

CATTCTTGTTCACAACTATTTATGGTGAACCACGCATATTTAGACCTACCGGTGCCAACCGAT

ATTACCTGATGTGTATGAATGAATTTTTGGCGTTTGGAGGTGGAGGAAACTTTGCACTATGTT TAGACGAAGATTTGTTGAAGGCAACAAGTGGACCTTCTGAAACATTTGGGAACGAATGTTTG

GCTAGCAGCACGGAGTTTGAGTTAAAGAATGTCGAGCTCTGGGGATTTGCACATGCGTCTCA

ATACCTCTCGTAATCTTGGAGATCCCTTACTTTGTCGAGAATCAAGATAACTCAATAGAAAG

AAAAGCTTACTATCGTTTTTTAATCAGTTAATTGTTTCAAAGTTATGATTAGGCTTAAGACAC

TTTCTCAGGCTTTAATCTGTTGAAAGAGTGACAATATTATATGTTTGTATCATCTGTGAATTA

CTGAGAATACTAATTCAGCTAAGACCTTATTGAACATTTATTTGACTTGTAATTATATGATAG

TGCTTGTGTTGCTAAGA

SEQ ID NO: 18 OXR2 genomic

ATCTCCGAGCATCTGGCTTCTCTGTCGCATCTTCTCTTCCCTTTCCCCCACCGACAATTCTATT

CATCTCCGATCAAAAACTATTCCTTTCTGATCACAATGCATGCTCTCAAGGACAAAGTCTCA

CAAAAACTCTCTAATCTCTTCGCCGATTCTCCAAGCCAATCTGCTTCTCCTCGTTATTCCAAC

TCCGATTCTCCAAAGGTATGATATTTTCTTATCAGAAACCCTAATTGTTGTTTTCCTTAGATG

GGTCTGGATTTGAATCTCGTTTGGTTGATTTGTGTAGGCTAGGTTGAATTCAAGTGTAGGCAA

ATCATTGTCTTCGTATTTCTCATTTGTTGTTCCTCAATCTGGGAACGAGGAGGATTCTGAGTT

ATGTCCACCTCTACCCATTAGAACAGAGAGCTATGAGTGTATTGAGAATTGCAAATCTGCTA

ATGGGCAAGCTAAAGCTGGAACCTTTATCTCAATTGGTGAAGATAAGGATTGTGAGTTACGT

GTTTCTGCGAAAGTAGAAGAAAGTGGTAATGATTACTTTGATGGGGTGAAGAAGATGAGGG

AATTAACAGAGAGCTCTGTGTTTATTACTGCTAACTTGTTTGAATTCTTGCATGCGAGTCTTC

CTAATATTGTAAGAGGATGCAAATGGATCTTGTTGTATAGGTAAGGAAGTTTGTTTTCGTAT

CTTGTTTGGAGGTTTTGGAATTGTGGTTTTTGGTGTTAACTTTTTTATCGGATGAAGGGCAGT

ACGTTGAAACATGGTATATCACTTCGTACGCTTTTGCGAAGAAGTGGAGAGCTTCCTGGTCC

TTGTTTACTGGTTTGTTTGCTTACCTCTATGCTCTTTGAATGGGGTAGATCTTAGTTTGTGATG

ATTTGGTGTGGTGCAGGTTGCTGGAGACAAACAAGGTGCAGTGTTTGGAGCTCTGTTGGAAT

GTCCACTGCAACCTACTCCTAAGAGGAAATATCAGGCAGGCTAGCTAATTGTATTAGTGTT

TGTCTGTGGAGATTGATTAGTTTCCTTATTACTGATTCTCTTTAGGTGTTTTTGATGATAGGGT

ACAAGCCAGACATTCTTGTTCACAACTATTTATGGTGAACCACGCATATTTAGACCTACCGG

TAATCATTTGAACCCCTTCTTTTATTATTTACTTCTCAAGAAATCGCGTATAGTTAGCTAATG

GATTTATGGGAGGAAGAGTATAGAGAAAATTGAAGAGAATTCTTAAGTCTCTTCAAAATCA

GATCTTAAGAATAAGAACATTTGGTTGCAGCTGGTTTTTATCCTAATCCTTCTTGGTTCTTGT

TATCAAGACTTGATCCACACTTGTTTTAGTTAGTTGTATTGTTGATATAAACCATCTAGTGAA

AGAAGAGTAACTTGTTTAGGAGAAGTTGACATCCACTCAAGTATGTGTACTTAACTCAGGTA

CCTCTCTCATGAATTTGTGCGCAGGTGCCAACCGATATTACCTGATGTGTATGAATGAATTTT

TGGCGTTTGGAGGTGGAGGAAACTTTGCACTATGTTTAGACGAAGATTTGTAAGGCACAATT

TGGCTCAATCTCATTTAGAAAATTTCCCATTTTAATCGTTTTGATGATACTCAAACCAAACTT

TGCTACATGAACTAGGTTGAAGGCAACAAGTGGACCTTCTGAAACATTTGGGAACGAATGTT

TGGCTAGCAGCACGGAGTTTGAGTTAAAGAATGTCGAGGTATCGCTATATACATATATTTAG

TTTGTGCTACTATCTTACTTGGGTCTTGGAAGTAATCAGTGAAAGTTCATGGTGTTAAATCTG

CAGCTCTGGGGATTTGCACATGCGTCTCAATACCTCTCGTAATCTTGGAGATCCCTTACTTTG

TCGAGAATCAAGATAACTCAATAGAAAGAAAAGCTTACTATCGTTTTTTAATCAGTTAATTG

TTTCAAAGTTATGATTAGGCTTAAGACACTTTCTCAGGCTTTAATCTGTTGAAAGAGTGACA

ATATTATATGTTTGTATCATCTGTGAATTACTGAGAATACTAATTCAGCTAAGACCTTATTGA

ACATTTATTTGACTTGTAATTATATGATAGTGCTTGT

GTTGCTAAGA

SEQ ID NO: 19 OXR2 (also termed OXR 2.1), OXR2 protein splice variant At2g05590.1

MHALKDKVSQKLSNLFADSPSQSASPRYSNSDSPKARLNSSVGKSLSSYFSFVVPQSGNEEDSEL CPPLPIRTESYECIENCKSANGQAKAGTFISIGEDKDCELRVSAKVEESGNDYFDGVKKMRELTE SSVFITANLFEFLHASLPNIVRGCKWILLYSTLKHGISLRTLLRRSGELPGPCLLVAGDKQGA VFGALLECPLQPTPKRKYQGTSQTFLFTTIYGEPRIFRPTGANRYYLMCMNEFLAFGGGGN FALCLDEDL

SEQ ID NO: 20 AtOXRl complete protein sequence TLDc in bold

MASCAISNSFHTVTFKTLKRISPYNSLFGWNSGKKIDNIRPPQQPAYHDDVEIPFSLSMVNKTFLK GRELKCCYKASIDGFGATKFHERCDFKGPCVIIAYTKDKSFKFGGFSPEGYRSTDDYYDTFD AFLFYWLEDCDDPIVLPKVGGSGAALFDYARGGPQFGADGLLIGPPLAPVMGGFAGPDTNS

GIGDLRVAKSRLGLSYAKRKDGKESIFGDENKVSLDDVLVFCSPYIASLY

SEQ ID NO: 21 AtOXR5 complete protein sequence TLDc in bold

MGASSSTDDKESSEKREIESLAASTGALPLLKRSFSKLVDSQTNTVPFQSLKQSFGLSYDTITTEGE

QKVSDLFPKLLEHLGSSLVDLFFVPDKEGLSWVEFASGYVKCCGRMSNSMSFNTLLRVYYVTAK

NAGFSPKLEFESDEADCKINGSISVSELLVFLWMCWTMSWDGRSSKAAEMKGCLFLPDISHLILS

AVVSCTDSESGNSLDVWETDVSGLELELPIGKFLTWALMTVPCLTECLSHFCNSRLQNVTSAED

GSGPSKSTAVDDSASKTSENTLLTCGRAWAISLTSKSTISEEILSSCFPGNSGEPNEHLLYRSYYH

GKGMNRLWSNVEGYHAPILVIISASCKVEHEATSSERKWVIGAILQQGFENRDAFYGSSGN

LFSISPVFHAFSSSGKEKNFAYSHLHPAGGVYDAHPKPVGIGFGGTLGNERIFIDEDFAKITV

RHHAVDKTYQSGSLFPNQGYLPVEALVLDIEAWGLGGNKAREIQQKYQKREELFTNQRRKID

LKTFTNWEDSPEKMMMDMMGNPNAPRKEER

SEQ ID NO: 22 AtOXR5D complete protein sequence TLDc in bold

MGNSNSSSVDHRFISASRAFTQKKLDDLKSLFVSLASNSQSNDQYVSYPVFQEYFGLSGSLGERM

FDMVTQRRKDDKMTYEDLVIAKATYEKGTDDEIAEFIYQTLDVNGNGVLSRSDLESFLVVILKS

VFSTESSDAESSDYKKMVDALLNAATFSKSDDGSEKGMSFEDFRSWCSFVPTIRKFLGSLLMPPS

TVRPGYQVPHLLYEDSVSSDRLLLKKEYAWHIGGALPHHELVEWKLLYHSSVHGQSFNTFLGH

TSNTGMSASVLIIKDTEGYVYGGYASQPWERYSDFYGDMKSFLFQLNPKAAIYRPTGANTN

IQWCATNFTSENIPNGIGFGGKINHFGLFISASFDQGQTFECTTFGSPSLSKTSRIQPEVIECW

GIVQASNEQDTKHNAMKGTVLERFKEDRNMLKLVGMAGNSND

SEQ ID NO: 23 AtOXRl complete gene sequence CDS

ATGGCTTCGTGTGCCATCTCAAACTCATTTCATACTGTAACCTTCAAGACCTTGAAGAGAAT

AAGTCCTTACAACAGTCTATTCGGTTGGAACTCCGGTAAGAAAATAGACAATATCCGGCCGC

CGCAACAACCGGCTTACCACGACGATGTCGAAATTCCTTTCTCTCTTTCAATGGTAAACAAA

ACCTTCCTAAAAGGAAGAGAGTTAAAATGTTGCTACAAGGCAAGCATTGATGGGTTTGGTGC

AACAAAGTTCCACGAGCGTTGTGACTTCAAAGGTCCATGTGTTATCATAGCTTACACTAAAG

ACAAGTCTTTCAAGTTTGGTGGATTTAGTCCGGAAGGATATAGAAGTACTGATGATTATTAC

GATACATTTGATGCTTTCCTCTTCTATTGGCTCGAGGATTGTGATGACCCAATCGTCCTTCCT

AAGGTTGGAGGAAGTGGAGCAGCACTATTTGATTATGCTCGCGGAGGGCCTCAATTTGGAG

CTGACGGGCTTCTTATCGGACCGCCTTTAGCTCCCGTGATGGGCGGATTTGCGGGTCCGGAT

ACGAACTCGGGGATTGGTGATTTGAGGGTGGCTAAATCAAGATTGGGATTGTCATATGCTAA

GAGGAAAGATGGAAAAGAATCTATATTTGGAGATGAAAATAAGGTTAGTCTTGATGATGTA

TTGGTCTTTTGTAGTCCTTATATTGCTTCCTTGTATTAA SEQ ID NO: 24 AtOXR5 complete gene sequence CDS

ATGGGAAATTCGAATTCGTCTTCTGTTGATCATCGCTTCATTTCTGCTTCCAGAGCTTTTACTC

AGAAGAAGCTCGATGATCTCAAATCTCTCTTCGTCTCCCTCGCTTCCAATTCTCAGAGCAATG

ACCAATACGTCTCGTACCCTGTTTTCCAGGAGTATTTTGGTCTGAGTGGTTCTTTAGGAGAAA

GGATGTTTGATATGGTCACTCAACGCAGGAAAGATGATAAAATGACTTATGAAGATCTTGTT

ATTGCTAAGGCAACATACGAGAAAGGAACTGACGATGAGATTGCTGAGTTTATTTACCAGA

CTTTGGATGTTAATGGCAATGGGGTCTTGTCAAGGTCTGATTTAGAGTCGTTCCTGGTGGTGA

TTTTGAAGAGTGTGTTCTCCACTGAGAGTTCTGATGCGGAATCAAGTGATTACAAGAAGATG

GTGGATGCGCTACTCAATGCCGCTACTTTCTCAAAATCTGATGATGGTTCTGAAAAAGGAAT

GTCTTTTGAAGACTTCAGAAGCTGGTGCTCATTCGTTCCAACTATCAGAAAGTTCCTCGGAA

GCTTGCTTATGCCCCCGAGTACAGTGAGACCTGGATATCAAGTCCCGCATCTGCTATATGAA

GATAGTGTGAGTTCAGATAGGCTACTATTAAAGAAGGAATACGCTTGGCATATTGGAGGAG

CTCTTCCTCACCATGAGCTTGTAGAGTGGAAGCTGTTGTATCACAGTTCCGTACACGGTCAA

AGCTTCAACACATTCCTCGGACACACATCAAACACTGGTATGTCGGCATCTGTGCTAATCAT

CAAAGACACAGAAGGATATGTGTATGGAGGGTACGCCTCTCAACCTTGGGAGAGGTACAGC

GATTTCTATGGAGACATGAAGTCTTTTCTTTTCCAGCTTAACCCTAAAGCAGCCATTTACCGA

CCAACTGGAGCAAACACCAACATTCAATGGTGTGCTACTAATTTCACATCGGAGAACATTCC

GAATGGGATAGGATTTGGAGGTAAGATAAATCACTTTGGCCTGTTTATATCAGCAAGCTTCG

ATCAAGGCCAGACATTCGAATGCACAACGTTTGGTAGCCCGAGCCTTTCCAAGACGAGCAG

AATTCAGCCAGAAGTGATAGAATGCTGGGGAATCGTTCAGGCCTCAAATGAACAAGACACC

AAACATAACGCCATGAAAGGTACTGTTCTGGAGAGGTTTAAGGAAGATCGCAACATGCTCA

AACTAGTCGGCATGGCAGGCAATTCAAATGATTGA

SEQ ID NO: 25 AtOXR5D complete gene sequence CDS

ATGGGTGCTTCATCTTCTACAGACGACAAAGAATCATCGGAGAAACGAGAAATCGAAAGCC

TTGCTGCTTCAACCGGAGCTCTTCCTCTGCTTAAACGATCCTTCTCCAAACTCGTCGATTCTC

AAACCAATACTGTTCCTTTTCAATCCTTGAAGCAAAGTTTTGGTTTGAGCTACGATACAATTA

CTACTGAAGGAGAGCAAAAAGTTTCAGACTTGTTTCCCAAATTGTTGGAGCATTTAGGATCA

TCTTTAGTAGATTTGTTCTTCGTGCCTGATAAAGAAGGATTGAGTTGGGTTGAGTTTGCTAGT

GGTTATGTCAAATGTTGTGGAAGAATGTCGAATTCGATGTCTTTCAACACTTTGTTGAGAGTT

TACTATGTGACAGCTAAAAATGCAGGCTTTTCTCCAAAGCTTGAGTTTGAATCTGATGAAGC

TGATTGTAAGATTAACGGGTCGATTTCGGTTAGTGAATTGCTTGTGTTTCTTTGGATGTGTTG

GACAATGTCTTGGGATGGTCGAAGCTCTAAAGCTGCTGAAATGAAAGGGTGTTTGTTTCTTC

CGGATATTAGTCATTTGATTCTATCAGCAGTGGTGTCTTGCACTGATTCTGAATCTGGAAATA

GTTTGGATGTTTGGGAGACTGATGTTTCTGGTTTAGAACTTGAACTTCCTATCGGGAAATTCT

TAACGTGGGCTTTGATGACAGTTCCTTGCCTCACTGAGTGCCTTTCTCACTTTTGTAACTCGA

GGCTTCAAAATGTGACAAGTGCAGAGGATGGATCTGGACCTTCAAAGTCCACTGCTGTAGAT

GATTCTGCGTCCAAGACAAGCGAAAACACCCTTCTAACATGTGGAAGGGCATGGGCGATTTC

TTTGACGAGCAAAAGTACAATAAGCGAGGAGATTTTGAGCTCATGTTTTCCCGGCAATAGCG

GTGAACCAAATGAACATCTTCTATACCGCTCATATTATCACGGGAAAGGCATGAATCGACTG

TGGTCCAACGTTGAAGGGTATCATGCTCCTATACTAGTGATAATTTCTGCAAGTTGTAAAGT

CGAACATGAGGCTACTTCAAGCGAGAGGAAGTGGGTCATTGGTGCAATCTTGCAGCAAGGT

TTTGAGAACAGAGATGCATTTTATGGAAGCTCTGGGAACTTGTTCTCCATAAGTCCAGTCTTT

CACGCATTCTCATCTTCTGGTAAGGAGAAAAACTTTGCGTATAGCCATCTGCACCCTGCTGG

CGGAGTGTACGACGCACATCCCAAGCCTGTTGGAATTGGATTCGGGGGAACACTTGGAAAT

GAGAGAATTTTCATTGATGAAGACTTTGCTAAAATCACAGTCCGTCATCATGCAGTTGATAA AACTTACCAGTCAGGCTCTCTTTTCCCAAACCAGGGTTATTTACCAGTAGAGGCTTTGGTTCT

AGATATTGAAGCATGGGGGTTAGGTGGAAACAAGGCTAGGGAAATTCAACAAAAATACCAG

AAAAGAGAAGAGCTTTTCACTAACCAACGTCGAAAGATCGACTTGAAAACGTTTACGAACT

GGGAAGATTCACCTGAGAAAATGATGATGGATATGATGGGTAATCCTAATGCTCCTAGAAA

GGAAGAGAGGTAA

Claims

1. An isolated chimeric protein comprising

a first polypeptide portion of a first OXR (Oxidative Resistance) protein; and a second polypeptide portion of a second OXR protein.

2. The chimeric protein according to claim 1 , wherein

the amino acid sequence of the first polypeptide portion includes an amino- terminal region and the amino acid sequence of the second polypeptide portion includes a TLDc domain in a carboxyl-terminal region; and

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.

3. The chimeric protein according to claim 1 or 2 wherein the first OXR protein is selected from AtOXRl, AtOXR2, AtOXR4 or AtOXR5, a functional variant or homolog thereof and the second OXR protein is a different protein selected from AtOXRl, AtOXR2, AtOXR4 or AtOXR5, a functional variant or homolog thereof.

4. The chimeric protein according to 3, wherein

the first OXR protein comprises an AtOXR4 (AT4G39870) amino acid sequence of SEQ ID NO: 2, a functional variant or homolog thereof; and

the second OXR protein comprises an AtOXR2 (AT2G05590) amino acid sequence of SEQ ID NO: 8, a functional variant or homolog thereof.

5. The chimeric protein according to claim 4, wherein

the first OXR protein comprises an amino acid sequence having at least 50%, 60%, 70%, 80% or 90% sequence identity with the full-length AtOXR4 protein amino acid sequence of SEQ ID NO: 2; and

the second OXR protein comprises an amino acid sequence having at least 50%, 60%, 70%, 80% or 90% sequence identity with the full-length AtOXR2 protein amino acid sequence of SEQ ID NO: 8.

The chimeric protein according to a preceding claim, wherein

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 chimeric protein according to a preceding claim, wherein

the first polypeptide portion has at least 50%, 60%, 70%, 80% or 90% sequence identity with the AtOXR4 amino-terminal amino acid sequence of SEQ ID NO: 4; and the second polypeptide portion has at least 50%, 60%, 70%, 80% or 90% sequence identity with the AtOXR2 carboxy-terminal amino acid sequence of SEQ ID NO: 12.

The chimeric protein according to any of claims 1 to 3, comprising an amino acid sequence having at least 50%, 60%, 70%, 80% or 90% sequence identity with the full- length amino acid sequence of SEQ K) NO: 14, 26, 28, 30 or 31.

The chimeric protein according to a preceding claim, wherein

the first polypeptide portion comprises the amino-terminal amino acid sequence AtOXR4 represented by SEQ ID NO: 4; and

the second polypeptide portion comprises the TLDc domain of the carboxy- terminal amino acid sequence _^4iOXR2 represented by SEQ ID NO: 12.

An isolated polynucleotide that encodes the chimeric protein according to any of claims 1 -9.

An isolated chimeric polynucleotide comprising

a first portion of a first OXR polynucleotide; and

a second portion of a second OXR polynucleotide.

The chimeric polynucleotide according to claim 11 wherein the first OXR polynucleotide is selected from AtOXRl, AtOXR2, AtOXR4 or AtOXR5, a functional variant or homolog thereof and the second OXR polynucleotide is a different polynucleotide selected from AtOXRl, AtOXR2, AtOXR4 or AtOXR5, a functional variant or homolog thereof.

13. An isolated polynucleotide of claim 11 or 12, wherein

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.

14. An isolated polynucleotide of claim 11 to 13, wherein

the first polynucleotide comprises a nucleotide sequence having at least 50%, 60%, 70%, 80% or 90% sequence identity with the full-length AtOXR4 sequence of SEQ ID NO: l ; and

the second polynucleotide comprises a nucleotide sequence having at least 50%, 60%, 70%, 80% or 90% sequence identity with the full-length AtOXR2 sequence of SEQ ID NO: 7.

15. The polynucleotide of any of claims 11 to 14, wherein

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.

16. The polynucleotide of any of claims 11 to 15, wherein

the first region comprises at least 50%, 60%, 70%, 80% or 90% sequence identity with the OXR4 amino-terminal encoding nucleotide sequence represented by SEQ ID NO: 3; and

the second region comprises at least 50%, 60%, 70%, 80% or 90 sequence identity with the OXR2 carboxy-terminal encoding nucleotide sequence represented by SEQ ID NO: 11.

17. The polynucleotide of claim 11 or 12, wherein the polynucleotide has the full-length nucleotide sequence of SEQ ID NO: 13, 27 29, 31 or 33 or sequence with at least 50%, 60%, 70%, 80% or 90 sequence identity thereto.

18. A vector comprising the polynucleotide encoding the chimeric protein of any of claims 1- 10.

19. A vector comprising the polynucleotide according to any of claims 10 to 17.

20. A recombinant expression cassette comprising the polynucleotide encoding the chimeric protein of any of claims 1 to 9 or the polynucleotide according to any of claims 10 to 17, wherein the polynucleotide is operably linked to a promoter.

21. The recombinant expression cassette according to claim 20, wherein the promoter is selected from the group consisting of a tissue-preferred promoter, a constitutive promoter, and an inducible promoter.

22. The recombinant expression cassette according to claim 21, wherein the promoter is a 35SCaMV constitutive promoter.

23. A host cell comprising the vector of claim 18 or 19 or the recombinant expression cassette of claim 20 or 21.

24. A transgenic plant comprising a nucleic acid construct comprising the polynucleotide of any of claims 10 to 17, the vector of claim 18 or 19 or the recombinant expression cassette of claim 20 or 21.

25. The transgenic plant of claim 24 wherein said plant is selected from the group consisting of maize, soybean, sorghum, canola, wheat, alfalfa, cotton, rice, brassica, and barley.

26. The transgenic plant of claims 24 and 25 wherein said plant expresses the recombinant polynucleotide encoding the chimeric protein of any of claims 1 to 9.

27. The transgenic plant of any of claims 24 to 26, wherein the transgenic plant has increased biomass and seed production compared to a corresponding control plant that does not express the chimeric protein encoded by the recombinant polynucleotide.

28. A seed or other harvestable part or product of a transgenic plant of any of claims 24 to 27.

29. A method of producing a transgenic plant, comprising

introducing into a plant cell a nucleic acid construct comprising a polynucleotide of any of claims 10 to 17, the vector of claim 18 or 19 or the expression cassette of claim 20 or 21 ; and

generating from the plant cell a transgenic plant that expresses the polynucleotide.

30. A method of modulating a plant phenotype, comprising

introducing and expressing in a plant a polynucleotide of any of claims 10 to 17, the vector of claim 18 or 19 or the expression cassette of claim 20 or 21.

31. A method according to claim 30 wherein said phenotype is selected from one or more of increased root biomass, increased shoot biomass, increased seed production, early flowering time, increased lignification, improved efficiency in water use, improved stress tolerance and increased photosynthetic performance.

32. A method according to claim 30 wherein said phenotype is increased yield.

33. A method for increasing abiotic stress tolerance in a plant comprising introducing and expressing in a plant a nucleic acid construct comprising a polynucleotide of any of claims 10 to 17, the vector of claim 18 or 19 or the expression cassette of claim 20 or 21.

34. The method of claim 33 wherein said stress is selected from water deficit or salt stress.

35. The use of a polynucleotide of any of claims 10 to 17, the vector of claim 18 or 19 or the expression cassette of claim 20 or 21 in modulating a plant phenotype.