AU2009287445C1 - Plant glutamine phenylpyruvate transaminase gene and transgenic plants carrying same - Google Patents

Abstract

The invention relates to transgenic plants exhibiting enhanced growth rates, seed and fruit yields, and overall biomass yields, as well as methods for generating growth-enhanced transgenic plants. In one embodiment, transgenic plants engineered to over-express glutamine phenypyruvate transaminase (GPT) are provided.

Description

PLANT GLUTAMINE PHENYLPYRUVATE TRANSAMINASE GENE AND TRANSGENIC PLANTS CARRYING SAME

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under Contract No. W-7405-ENG-36 awarded by the United States Department of Energy to The Regents of The University of California, and Contract No. DE-AC52-06NA25396, awarded by the United States Department of Energy to Los Alamos National Security, LLC. The government has certain rights in this invention.

RELATED APPLICATIONS

This application claims priority to United States Provisional Application No. 61/190,581 filed August 29, 2008.

BACKGROUND OF THE INVENTION

As the human population increases worldwide, and available farmland continues to be destroyed or otherwise compromised, the need for more effective and sustainable agriculture systems is of paramount interest to the human race. Improving crop yields, protein content, and plant growth rates represent major objectives in the development of agriculture systems that can more effectively respond to the challenges presented.

In recent years, the importance of improved crop production technologies has only increased as yields for many well-developed crops have tended to plateau. Many agricultural activities are time sensitive, with costs and returns being dependent upon rapid turnover of crops or upon time to market. Therefore, rapid plant growth is an economically important goal for many agricultural businesses that involve high-value crops such as grains, vegetables, berries and other fruits.

Genetic engineering has and continues to play an increasingly important yet controversial role in the development of sustainable agriculture technologies. A large number of genetically modified plants and related technologies have been developed in recent years, many of which are in widespread use today (Factsheet: Genetically Modified Crops in the United States, Pew Initiative on Food and Biotechnology, August 2004, (pewagbiotech.org/resources/factsheets). The adoption of transgenic plant varieties is now very substantial and is on the rise, with approximately 250 million acres planted with transgenic plants in 2006.

While acceptance of transgenic plant technologies may be gradually increasing, particularly in the United States, Canada and Australia, many regions of the World remain slow to adopt genetically modified plants in agriculture, notably Europe. Therefore, consonant with pursuing the objectives of responsible and sustainable agriculture, there is a strong interest in the development of genetically engineered plants that do not introduce toxins or other potentially problematic substances into plants and/or the environment. There is also a strong interest in minimizing the cost of achieving objectives such as improving herbicide tolerance, pest and disease resistance, and overall crop yields. Accordingly, there remains a need for transgenic plants that can meet these objectives.

The goal of rapid plant growth has been pursued through numerous studies of various plant regulatory systems, many of which remain incompletely understood. In particular, the plant regulatory mechanisms that coordinate carbon and nitrogen metabolism are not fully elucidated. These regulatory mechanisms are presumed to have a fundamental impact on plant growth and development.

The metabolism of carbon and nitrogen in photosynthetic organisms must be regulated in a coordinated manner to assure efficient use of plant resources and energy. Current understanding of carbon and nitrogen metabolism includes details of certain steps and metabolic pathways which are subsystems of larger systems. In photosynthetic organisms, carbon metabolism begins with COa fixation, which proceeds via two major processes, termed C-3 and C-4 metabolism. In plants with C-3 metabolism, the enzyme ribulose bisphosphate carboxylase (RuBisCo) catalyzes the combination of CO2 with ribulose bisphosphate to produce 3-phosphoglycerate, a three carbon compound (C-3) that the plant uses to synthesize carbon-containing compounds. In plants with C-4 metabolism, CO2 is combined with phosphoenol pyruvate to form acids containing four carbons (C-4), in a reaction catalyzed by the enzyme phosphoenol pyruvate carboxylase. The acids are transferred to bundle sheath cells, where they are decarboxylated to release CO2, which is then combined with ribulose bisphosphate in the same reaction employed by C-3 plants.

Numerous studies have found that various metabolites are important in plant regulation of nitrogen metabolism. These compounds include the organic acid malate and the amino acids glutamate and glutamine. Nitrogen is assimilated by photosynthetic organisms via the action of the enzyme glutamine synthetase (GS) which catalyzes the combination of ammonia with glutamate to form glutamine. GS plays a key role in the assimilation of nitrogen in plants by catalyzing the addition of ammonium to glutamate to form glutamine in an ATP-dependent reaction (Miflin and Habash, 2002, Journal of Experimental Botany, Vol. 53, No. 370, pp. 979-987). GS also reassimilates ammonia released as a result of photorespiration and the breakdown of proteins and nitrogen transport compounds. GS enzymes may be divided into two general classes, one representing the cytoplasmic form (GS1) and the other representing the plastidic (i.e., chloroplastic) form (GS2).'

Previous work has demonstrated that increased expression levels of GS1 result in increased levels of GS activity and plant growth, although reports are inconsistent. For example, Fuentes et al. reported that CaMV S35 promoter driven overexpression of Alfalfa GS1 (cytoplasmic form) in tobacco resulted in increased levels of GS expression and GS activity in leaf tissue, increased growth under nitrogen starvation, but no effect on growth under optimal nitrogen fertilization conditions (Fuentes et al., 2001, J. Exp. Botany 52: 1071-81). Temple et al. reported that transgenic tobacco plants overexpressing the full length Alfalfa GS1 coding sequence contained greatly elevated levels of GS transcript, and GS polypeptide which assembled into active enzyme, but did not report phenotypic effects on growth (Temple et al., 1993, Molecular and General Genetics 236: 315-325). Corruzi et al. have reported that transgenic tobacco overexpressing a pea cytosolic GS1 transgene under the control of the CaMV S35 promoter show increased GS activity, increased cytosolic GS protein, and improved growth characteristics (U.S. Patent No. 6,107,547). Unkefer et al. have more recently reported that transgenic tobacco plants overexpressing the Alfalfa GS1 in foliar tissues, which had been screened for increased leaf-to-root GS activity following genetic segregation by selfing to achieve increased GS1 transgene copy number, were found to produce increased 2-hydroxy-5-oxoproline levels in their foliar portions, which was found to lead to markedly increased growth rates over wildtype tobacco plants (see, U.S. Patent Nos. 6,555,500; 6,593,275; and 6,831,040).

Unkefer et al. have further described the use of 2-hydroxy-5-oxoproline (also known as 2-oxoglutaramate) to improve plant growth (U.S. Patent Nos. 6,555,500; 6,593,275; 6,831,040). In particular, Unkefer et al. disclose that increased concentrations of 2-hydroxy-5-oxoproline in foliar tissues (relative to root tissues) triggers a cascade of events that result in increased plant growth characteristics. Unkefer et al. describe methods by which the foliar concentration of 2-hydroxy-5-oxoproline may be increased in order to trigger increased plant growth characteristics, specifically, by applying a solution of 2-hydroxy-5-oxoproline directly to the foliar portions of the plant and over-expressing glutamine synthetase preferentially in leaf tissues. A number of transaminase and hydrolyase enzymes known to be involved in the synthesis of 2-hydroxy-5-oxoproline in animals have been identified in animal liver and kidney tissues (Cooper and Meister, 1977, CRC Critical Reviews in Biochemistry, pages 281-303; Meister, 1952, J. Biochem. 197: 304). In plants, the biochemical synthesis of 2-hydroxy-5-oxoproline has been known but has been poorly characterized. Moreover, the function of 2-hydroxy-5-oxoproline in plants and the significance of its pool size (tissue concentration) are unknown. Finally, the art provides no specific guidance as to precisely what transaminase(s) or hydrolase(s) may exist and/or be active in catalyzing the synthesis of 2-hydroxy-5-oxoproline in plants, and no such plant transaminases have been reported, isolated or characterized.

SUMMARY OF THE INVENTION

The invention relates to transgenic plants exhibiting enhanced growth rates, seed and fruit yields, and overall biomass yields, as well as methods for generating growth-enhanced transgenic plants. In one embodiment, transgenic plants engineered to over-express glutamine phenylpyruvate transaminase (GPT) are provided. In general, these plants outgrow their wild-type counterparts by about 50%.

Applicants have identified the enzyme glutamine phenylpyruvate transaminase (GPT) as a catalyst of 2-hydroxy-5-oxoproline (2-oxoglutaramate) synthesis in plants. 2-oxoglutaramate is a powerful signal metabolite which regulates the function of a large number of genes involved in the photosynthesis apparatus, carbon fixation and nitrogen metabolism.

By preferentially increasing the concentration of the signal metabolite 2- oxoglutaramate (i.e., in foliar tissues), the transgenic plants of the invention are capable of producing higher overall yields over shorter periods of time, and therefore may provide agricultural industries with enhanced productivity across a wide range of crops. Importantly, unlike many transgenic plants described to date, the invention utilizes natural plant genes encoding a natural plant enzyme. The enhanced growth characteristics of the transgenic plants of the invention is achieved essentially by introducing additional GPT capacity into the plant. Thus, the transgenic plants of the invention do not express any toxic substances, growth hormones, viral or bacterial gene products, and are therefore free of many of the concerns that have heretofore impeded the adoption of transgenic plants in certain parts of the World. A first aspect of the invention provides for a method for generating and selecting transgenic plants having increased production of 2-oxo-glutaramate by transforming plants with transgenes that encode enzymes in the synthesis pathway for 2-oxo-glutaramate to thereby improve growth characteristics of the transgenic plant relative to an analogous wild type or untransformed plant, comprising: (a) . generating a plurality of transgenic plants by transforming a plurality of plants with a glutamine phenylpyruvate transaminase (GPT) transgene that encodes a GPT polypeptide having GPT catalytic activity; (b) . expressing the GPT transgene in the plurality of transgenic of plants or the progeny thereof, wherein GPT polypeptides are overexpressed in at least one of the plurality of transgenic of plants or said progeny relative to an analogous wild type or untransformed plant of the same species; and (c) . selecting a transgenic plant from said plurality of transgenic plants or said progeny that has increased production of 2-oxo-glutaramate relative to a wild type or untransformed plant of the same species. A second aspect of the invention provides for a method for generating and selecting transgenic plants having increased production of 2-oxo-glutaramate by transforming plant cells with transgenes that encode enzymes in the synthesis pathway for 2-oxo-glutaramate to thereby improve growth characteristics of the transgenic plants relative to an analogous wild type or untransformed plant, comprising: (a) . transforming a plurality of plant cells with a glutamine phenylpyruvate transaminase (GPT) transgene that encodes a polypeptide having GPT catalytic activity; (b) . generating a plurality of transgenic plants from the transformed plant cells; (c) . expressing the GPT transgene in the plurality of transgenic plants or the progeny thereof, wherein GPT polypeptides are overexpressed in the plurality of transgenic of plants or the progeny thereof relative to an analogous wild type or untransformed plant of the same species; (d) . selecting a transgenic plant from said plurality of transgenic plants or said progeny having increased production of 2-oxo-glutaramate relative to an analogous wild type or untransformed plant of the same species. A third aspect of the invention provides for a method for generating transgenic plants having increased production of 2-oxo-glutaramate by transforming plants with transgenes that encode enzymes in the synthesis pathway for 2-oxo-glutaramate to thereby improve growth characteristics of the transgenic plants relative to an analogous wild type or untransformed plant, comprising: (a) . generating a transgenic plant having a glutamine phenylpyruvate transaminase (GPT) transgene, wherein the GPT transgene is linked to a plant promoter and the GPT transgene is an exogenous GPT gene from a plant; and (b) . expressing the GPT transgene in the transgenic plant or the progeny thereof, wherein the polypeptide formed by the expression of the GPT transgene has GPT catalytic activity, and wherein said transgenic plant and the progeny thereof produce more 2-oxo-glutaramate relative to an analogous wild type or untransformed plant of the same species. A fourth aspect of the invention provides for a method of for generating and selecting transgenic plants having increased production of 2-oxo-glutaramate by transforming plants with transgenes that encode enzymes in the synthesis pathway for 2-oxo-glutaramate to thereby improve growth characteristics of the transgenic plants relative to an analogous wild type or untransformed plant, comprising: (a) generating a plurality of transgenic plants by transforming a plurality of plants with a GPT transgene; (b) expressing the GPT transgene in the plurality of transgenic plants or the progeny thereof, wherein GPT polypeptides are overexpressed in the plurality of transgenic of plants or the progeny thereof relative to a wild type or untransformed plant of the same species; and, (c) selecting at least one transgenic plant having an increased biomass yield relative to a wild type or untransformed plant of the same species.

An fifth aspect of the invention provides for a progeny of any generation of a transgenic plant generated and selected by the method of the first to fourth aspects of the invention, wherein said progeny comprises said GPT transgene. A sixth aspect of the invention provides for a seed of any generation of a transgenic plant generated and selected by the method according to the first to fourth aspects of the invention, wherein said seed comprises said GPT transgene.

In one embodiment, the invention provides a transgenic plant comprising a GPT transgene, wherein said GPT transgene is operably linked to a plant promoter. In a specific embodiment, the GPT transgene encodes a polypeptide having an amino acid sequence selected from the group consisting of (a) SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 10, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30 and SEQ ID NO: 31, and (b) an amino acid sequence that is at least 75% identical to any one of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 10, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO: 25. SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30 and SEQ ID NO: 31 and has GPT activity.

In some embodiments, the GPT transgene is incorporated into the genome of the plant. The transgenic plant of the invention may be a monocotyledonous or a dicotyledonous plant.

The invention also provides progeny of any generation of the transgenic plants of the invention, wherein said progeny comprises a GPT transgene, as well as a seed of any generation of the transgenic plants of the invention, wherein said seed comprises said GPT transgene. The transgenic plants of the invention may display one or more enhanced growth characteristics rate when compared to an analogous wild-type or untransformed plant, including without limitation increased growth rate, biomass yield, seed yield, flower or flower bud yield, fruit or pod yield, larger leaves, and may also display increased levels of GPT activity and/or increased levels of 2-oxoglutaramate. In some embodiments, the transgenic plants of the invention display increased nitrogen use efficiency.

Methods for producing the transgenic plants of the invention and seeds thereof are also provided, including methods for producing a plant having enhanced growth properties, increased nitrogen use efficiency and increased tolerance to germination or growth in salt or saline conditions, relative to an analogous wild type or untransformed plant.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1. Nitrogen assimilation and 2-oxoglutaramate biosynthesis: schematic of metabolic pathway. FIG. 2. Photograph showing comparison of transgenic tobacco plants overexpressing GPT, compared to wild type tobacco plant. From left to right: wild type plant, Alfalfa GS1 transgene, Arabidopsis GPT transgene. See Example 3, infra. FIG. 3. Photograph showing comparison of transgenic Micro-Tom tomato plants over-expressing GPT, compared to wild type tomato plant. (A) wild type plant; (B) Arabidopsis GPT transgene. See Example 4, infra. FIG. 4. Photograph showing comparisons of leaf sizes between wild type (top leaf) and GPT transgenic (bottom leaf) tobacco plants.

DETAILED DESCRIPTION OF THE INVENTION

DEFINITIONS

Unless otherwise defined, all terms of art, notations and other scientific terminology used herein are intended to have the meanings commonly understood by those of skill in the art to which this invention pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and/or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art. The techniques and procedures described or referenced herein are generally well understood and commonly employed using conventional methodology by those skilled in the art, such as, for example, the widely utilized molecular cloning methodologies described in Sambrook et al., Molecular Cloning: A Laboratory Manual 3rd. edition (2001) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Current Protocols in Molecular Biology (Ausbel et al., eds., John Wiley & Sons, Inc. 2001; Transgenic Plants: Methods and Protocols (Leandro Pena, ed., Humana Press, 1sl edition, 2004); and, Agrobacterium Protocols (Wan, ed., Humana Press, 2nd edition, 2006). As appropriate, procedures involving the use of commercially available kits and reagents are generally carried out in accordance with manufacturer defined protocols and/or parameters unless otherwise noted.

The term "nucleic acid" refers to deoxyribonucleotides or ribonucleotides and polymers thereof ("polynucleotides") in either single- or double-stranded form. Unless specifically limited, the term “polynucleotide" encompasses nucleic acids containing known analogues of natural nucleotides which have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g. degenerate codon substitutions) and complementary sequences and as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al., 1991, Nucleic Acid Res. 19: 5081; Ohtsuka et al., 1985 J. Biol. Chem. 260: 2605-2608; and Cassol et al., 1992; Rossolini et al., 1994, Mol. Cell. Probes 8: 91-98). The term nucleic acid is used interchangeably with gene, cDNA, and mRNA encoded by a gene.

The term "promoter” refers to an array of nucleic acid control sequences that direct transcription of an operably linked nucleic acid. As used herein, a "plant promoter" is a promoter that functions in plants. Promoters include necessary nucleic acid sequences near the start site of transcription, such as, in the case of a polymerase II type promoter, a TATA element. A promoter also optionally includes distal enhancer or repressor elements, which can be located as much as several thousand base pairs from the start site of transcription. A "constitutive" promoter is a promoter that is active under most environmental and developmental conditions. An "inducible" promoter is a promoter that is active under environmental or developmental regulation. The term "operably linked" refers to a functional linkage between a nucleic acid expression control sequence (such as a promoter, or array of transcription factor binding sites) and a second nucleic acid sequence, wherein the expression control sequence directs transcription of the nucleic acid corresponding to the second sequence.

The terms “polypeptide,” “peptide" and “protein” are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers.

The term “amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acid analogs refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an a carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid.

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 coles.

The term "plant" includes whole plants, plant organs (e.g., leaves, stems, flowers, roots, etc.), seeds and plant cells and progeny thereof. The class of plants which can be used in the method of the invention is generally as broad as the class of higher plants amenable to transformation techniques, including angiosperms (monocotyledonous and dicotyledonous plants), as well as gymnosperms. It includes plants of a variety of ploidy levels, including polyploid, diploid, haploid and hemizygous.

The terms "GPT polynucleotide" and “GPT nucleic acid” are used interchangeably herein, and refer to a full length or partial length polynucleotide sequence of a gene which encodes a polypeptide involved in catalyzing the synthesis of 2-oxoglutaramate, and includes polynucleotides containing both translated (coding) and un-translated sequences, as well as the complements thereof. The term “GPT coding sequence" refers to the part of the gene which is transcribed and encodes a GPT protein. The term "targeting sequence" refers to the amino terminal part of a protein which directs the protein into a subcellular compartment of a cell, such as a chloroplast in a plant cell. GPT polynucleotides are further defined by their ability to hybridize under defined conditions to the GPT polynucleotides specifically disclosed herein, or to PCR products derived therefrom. A “GPT transgene" is a nucleic acid molecule comprising a GPT polynucleotide which is exogenous to transgenic plant, or plant embryo, organ or seed, harboring the nucleic acid molecule, or which is exogenous to an ancestor plant, or plant embryo, organ or seed thereof, of a transgenic plant harboring the GPT polynucleotide.

Exemplary GPT polynucleotides of the invention are presented herein, and include GPT coding sequences for Arabidopsis, Rice, Barley, Bamboo, Soybean, Grape, and Zebra Fish GPTs.

Partial length GPT polynucleotides include polynucleotide sequences encoding N-or C-terminal truncations of GPT, mature GPT (without targeting sequence) as well as sequences encoding domains of GPT. Exemplary GPT polynucleotides encoding N-terminal truncations of GPT include Arabidopsis -30, -45 and -56 constructs, in which coding sequences for the first 30, 45, and 56 respectively, amino acids of the full length GPT structure of SEQ ID NO: 2 are eliminated.

In employing the GPT polynucleotides of the invention in the generation of transformed cells and transgenic plants, one of skill will recognize that the inserted polynucleotide sequence need not be identical, but may be only "substantially identical" to a sequence of the gene from which it was derived, as further defined below. The term “GPT polynucleotide" specifically encompasses such substantially identical variants. Similarly, one of skill will recognize that because of codon degeneracy, a number of polynucleotide sequences will encode the same polypeptide, and all such polynucleotide sequences are meant to be included in the term GPT polynucleotide. In addition, the term specifically includes those sequences substantially identical (determined as described below) with an GPT polynucleotide sequence disclosed herein and that encode polypeptides that are either mutants of wild type GPT polypeptides or retain the function of the GPT polypeptide (e.g., resulting from conservative substitutions of amino acids in a GPT polypeptide). The term “GPT polynucleotide" therefore also includes such substantially identical variants.

The term “conservatively modified variants" applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, conservatively modified variants refers to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are “silent variations,” which are one species of conservatively modified variations. Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of the nucleic acid. One of skill will recognize that each codon in a nucleic acid (except AUG, which is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan) can be modified to yield a functionally identical molecule. Accordingly, each silent variation of a nucleic acid which encodes a polypeptide is implicit in each described sequence.

As to amino acid sequences, one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a “conservatively modified variant” where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles of the invention.

The following eight groups each contain amino acids that are conservative substitutions for one another 1) Alanine (A), Glycine (G); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine <M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C), Methionine (M) (see, e.g., Creighton, Proteins (1984)).

Macromolecular structures such as polypeptide structures can be described in terms of various levels of organization. For a general discussion of this organization, see, e.g., Alberts et a!., Molecular Biology of the Cell (3rd ed., 1994) and Cantor and Schimmel, Biophysical Chemistry Part I: The Conformation of Biological Macromolecules (1980). “Primary structure” refers to the amino acid sequence of a particular peptide. “Secondary structure” refers to locally ordered, three dimensional structures within a polypeptide. These structures are commonly known as domains. Domains are portions of a polypeptide that form a compact unit of the polypeptide and are typically 25 to approximately 500 amino acids long. Typical domains are made up of sections of lesser organization such as stretches of β-sheet and α-helices. “Tertiary structure" refers to the complete three dimensional structure of a polypeptide monomer. “Quaternary structure” refers to the three dimensional structure formed by the noncovalent association of independent tertiary units. Anisotropic terms are also known as energy terms.

The term "isolated" refers to material which is substantially or essentially free from components which normally accompany the material as it is found in its native or natural state. However, the term "isolated" is not intended refer to the components present in an electrophoretic gel or other separation medium. An isolated component is free from such separation media and in a form ready for use in another application or already in use in the new application/milieu. An "isolated" antibody is one that has been identified and separated and/or recovered from a component of its natural environment. Contaminant components of its natural environment are materials that would interfere with diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In preferred embodiments, the antibody will be purified (1) to greater than 95% by weight of antibody as determined by the Lowry method, and most preferably more than 99% by weight, (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE under reducing or nonreducing conditions using Coomassie blue or, preferably, silver stain. Isolated antibody includes the antibody in situ within recombinant cells since at least one component of the antibody's natural environment will not be present. Ordinarily, however, isolated antibody will be prepared by at least one purification step.

The term “heterologous" when used with reference to portions of a nucleic acid indicates that the nucleic acid comprises two or more subsequences that are not found in the same relationship to each other in nature. For instance, a nucleic acid is typically recombinantly produced, having two or more sequences from unrelated genes arranged to make a new functional nucleic acid, e.g., a nucleic acid encoding a protein from one source and a nucleic acid encoding a peptide sequence from another source. Similarly, a heterologous protein indicates that the protein comprises two or more subsequences that are not found in the same relationship to each other in nature (e.g., a fusion protein).

The terms “identical" or percent “identity,” in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (i.e., about 70% identity, preferably 75%, 80%, 85%, 90%, or 95% identity over a specified region, when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using a sequence comparison algorithms, or by manual alignment and visual inspection. This definition also refers to the complement of a test sequence, which has substantial sequence or subsequence complementarity when the test sequence has substantial identity to a reference sequence. This definition also refers to the complement of a test sequence, which has substantial sequence or subsequence complementarity when the test sequence has substantial identity to a reference sequence.

When percentage of sequence identity is used in reference to polypeptides, it is recognized that residue positions that are not identical often differ by conservative amino acid substitutions, where amino acids residues are substituted for other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity) and therefore do not change the functional properties of the polypeptide. Where sequences differ in conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution.

For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters. A "comparison window”, as used herein, includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of from 20 to 600, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Methods of alignment of sequences for comparison are well-known in the art.

Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith &amp; Waterman, 1981, Adv. Appl. Math. 2:482, by the homology alignment algorithm of Needleman &amp; Wunsch, 1970, J. Mol. Biol. 48:443, by the search for similarity method of Pearson &amp; Lipman, 1988, Proc. Nat'l. Acad. Sci. USA 85:2444, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wl), or by manual alignment and visual inspection (see, e.g., Current Protocols in Molecular Biology (Ausubel et al., eds. 1995 supplement)). A preferred example of algorithm that is suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., 1977, Nuc. Acids Res. 25:3389-3402 and Altschul et al., 1990, J. Mol. Biol. 215:403-410, respectively. BLAST and BLAST 2.0 are used, typically with the default parameters described herein, to determine percent sequence identity for the nucleic acids and proteins of the invention. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequent», which either match or satisfy some positivevalued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul ef al., supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always > 0) and N (penalty score for mismatching residues; always < 0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a word length (W) of 11, an expectation (E) of 10, M=5, N=-4 and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a word length of 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff &amp; Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)) alignments (B) of 50, expectation (E) of 10, M=5, N=-4, and a comparison of both strands.

The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin &amp; Altschul, 1993, Proc. Nat’l. Acad. Sci. USA 90:5873-5787). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic add is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nudeic acid to the reference nudeic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001.

The phrase "stringent hybridization conditions" refers to conditions under which a probe will hybridize to its target subsequence, typically in a complex mixture of nucleic add, but to no other sequences. Stringent conditions are sequence-dependent and will be different in different drcumstances. Longer sequences hybridize specifically at higher temperatures. An extensive guide to the hybridization of nucleic adds is found in Tijssen, Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic Probes, "Overview of principles of hybridization and the strategy of nucleic acid assays" (1993). Generally, highly stringent conditions are selected to be about 5-10eC. lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength pH. Low stringency conditions are generally selected to be about 15-30°C. below the Tm. Tm is the temperature (under defined ionic strength, pH, and nucleic concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (as the target sequences are present in excess, at Tm, 50% of the probes are occupied at equilibrium). Stringent conditions will be those in which the salt concentration is less than about 1 .OM sodium ion, typically about 0.01 to 1.0M sodium ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30°C for short probes (e.g., 10 to 50 nucleotides) and at least about 60°C for long probes (e.g., greater than 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. For selective or specific hybridization, a positive signal is at least two times background, preferably 10 times background hybridization.

Nucleic acids that do not hybridize to each other under stringent conditions are still substantially identical if the polypeptides which they encode are substantially identical. This occurs, for example, when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code. In such cased, the nucleic acids typically hybridize under moderately stringent hybridization conditions.

Genomic DNA or cDNA comprising GPT polynucleotides may be identified in standard Southern blots under stringent conditions using the GPT polynucleotide sequences disclosed here. For this purpose, suitable stringent conditions for such hybridizations are those which include a hybridization in a buffer of 40% formamide, 1M NaCI, 1% SDS at 37CC, and at least one wash in 0.2 X SSC at a temperature of at least about 50°C, usually about 55°C to about 60°C, for 20 minutes, or equivalent conditions. A positive hybridization is at least twice background. Those of ordinary skill will readily recognize that alternative hybridization and wash conditions may be utilized to provide conditions of similar stringency. A further indication that two polynucleotides are substantially identical is if the reference sequence, amplified by a pair of oligonucleotide primers, can then be used as a probe under stringent hybridization conditions to isolate the test sequence from a cDNA or genomic library, or to identify the test sequence in, e.g., a northern or Southern blot. TRANSGENIC PLANTS:

The invention provides novel transgenic plants exhibiting substantially enhanced agronomic characteristics, including faster growth, greater mature plant fresh weight and total biomass, earlier and more abundant flowering, and greater fruit and seed yields. The transgenic plants of the invention are generated by introducing into a plant one or more expressible genetic constructs capable of driving the expression of one or more polynucleotides encoding glutamine phenylpyruvate transaminase (GPT). The invention is exemplified by the generation of transgenic tobacco plants carrying and expressing the heterologous Arabidopsis GPT gene (Example 2, infra). It is expected that all plant species also contain a GPT homolog which functions in the same metabolic pathway, namely the biosynthesis of the signal metabolite 2-hydroxy-5-oxoproline. Thus, in the practice of the invention, any plant gene encoding a GPT homolog or functional variants thereof may be useful in the generation of transgenic plants of this invention.

In stable transformation embodiments of the invention, one or more copies of the expressible genetic construct become integrated into the host plant genome, thereby providing increased GPT enzyme capacity into the plant, which serves to mediate increased synthesis of 2-oxoglutaramate, which in turn signals metabolic gene expression, resulting in increased plant growth and the enhancement other agronomic characteristics. 2-oxoglutaramate is a metabolite which is an extremely potent effector of gene expression, metabolism and plant growth (U.S. Patent No. 6,555,500), and which may play a pivotal role in the coordination of the carbon and nitrogen metabolism systems (Lancien et al., 2000, Enzyme Redundancy and the importance of 2-Oxoglutarate in Higher Piants Ammonium Assimilation, Plant Physiol. 123: 817-824). See, also, the schematic of the 2-oxoglutaramate pathway shown in FIG. 1.

In one aspect of the invention, applicants have isolated a nucleic acid molecule encoding the Arabidopsis glutamine phenylpyruvate transaminase (GPT) enzyme (see Example 1, infra), and have demonstrated for the first time that the expressed recombinant enzyme is active and capable of catalyzing the synthesis of the signal metabolite, 2-oxoglutaramate (Example 2, infra). Further, applicants have demonstrated for the first time that over-expression of the Arabidopsis glutamine transaminase gene in a transformed heterologous plant results in enhanced CO2 fixation rates and increased growth characteristics (Example 3, infra).

As disclosed herein (see Example 3, infra), over-expression of a transgene comprising the full-length Arabidopsis GPT coding sequence in transgenic tobacco plants also results in faster C02 fixation, and increased levels of total protein, glutamine and 2-oxoglutaramate. These transgenic plants also grow faster than wild-type plants (FIG. 2). Similarly, in preliminary studies conducted with tomato plants (see Example 4, infra), tomato plants transformed with the Arabidopsis GPT transgene showed significant enhancement of growth rate, flowering, and seed yield in relation to wild type control plants (FIG. 3 and Example 4, mfra).

In addition to the transgenic tobacco plants referenced above, various other species of transgenic plants comprising GPT and showing enhanced growth 1 characteristics have been generated in two species of Tomato, Pepper, Beans, Cowpea, Alfalfa, Cantaloupe, Pumpkin, Arabidopsis and Camilena (see coowned, co-pending application docket number S-112,983, filed August 31, 2009, the contents of which are incorporated herein by reference in its entirety). The foregoing transgenic plants were generated using a variety of transformation methodologies, including Agrobacterium-mediated callus, floral dip, seed inoculation, pod inoculation, and direct flower inoculation, as well as combinations thereof, and via sexual crosses of single transgene plants, using various GPT transgenes.

The invention also provides methods of generating a transgenic plant having enhanced growth and other agronomic characteristics. In one embodiment, a method of generating a transgenic plant having enhanced growth and other agronomic characteristics comprises introducing into a plant cell an expression cassette comprising a nucleic acid molecule encoding a GPT transgene, under the control of a suitable promoter capable of driving the expression of the transgene, so as to yield a transformed plant cell, and obtaining a transgenic plant which expresses the encoded GPT. In another embodiment, a method of generating a transgenic plant having enhanced growth and other agronomic characteristics comprises introducing into a plant cell one or more nucleic acid constructs or expression cassettes comprising nucleic acid molecules encoding a GPT transgene, under the control of one or more suitable promoters (and, optionally, other regulatory elements) capable of driving the expression of the transgenes, so as to yield a plant cell transformed thereby, and obtaining a transgenic plant which expresses the GPR transgene.

Any number of GPT polynucleotides may be used to generate the transgenic plants of the invention. GPT proteins are highly conserved among various plant species, and it is evident from the experimental data disclosed herein that closely related non-plant GPTs may be used as well (e.g., Danio rerio GPT). With respect to GPT, numerous GPT polynucleotides derived from different species have been shown to be active and useful as GPT transgenes.

In a specific embodiment, the GPT transgene is a GPT polynucleotide encoding an Arabidopsis derived GPT, such as the GPT of SEQ ID NO: 2, SEQ ID NO: 16 and SEQ ID NO: 25. The GPT transgene may be encoded by the nucleotide sequence of SEQ ID NO: 1; a nucleotide sequence having at least 75% and more preferably at least 80% identity to SEQ ID NO: 1, and encoding a polypeptide having GPT activity; a nucleotide sequence encoding the polypeptide of SEQ ID NO: 2, or a polypeptide having at least 75% and more preferably at least 80% sequence identity thereto which has GPT activity; and a nucleotide sequence encoding the polypeptide of SEQ ID NO: 2 truncated at its amino terminus by between 30 to 56 amino acid residues, or a polypeptide having at least 75% and more preferably at least 80% sequence identity thereto which has GPT activity.

In another specific embodiment, the GPT transgene is a GPT polynucleotide encoding a Grape derived GPT, such as the Grape GPTs of SEQ ID NO: 4 and SEQ ID NO: 26. The GPT transgene may be encoded by the nucleotide sequence of SEQ ID NO: 3; a nucleotide sequence having at least 75% and more preferably at least 80% identity to SEQ ID NO: 3, and encoding a polypeptide having GPT activity: a nucleotide sequence encoding the polypeptide of SEQ ID NO: 4 or SEQ ID NO: 26, or a polypeptide having at least 75% and more preferably at least 80% sequence identity thereto which has GPT activity.

In yet another specific embodiment, the GPT transgene is a GPT polynucleotide encoding a Rice derived GPT, such as the Rice GPTs of SEQ ID NO: 6 and SEQ ID NO: 27. The GPT transgene may be encoded by the nucleotide sequence of SEQ ID NO: 5; a nucleotide sequence having at least 75% and more preferably at least 80% identity to SEQ ID NO: 5, and encoding a polypeptide having GPT activity; a nucleotide sequence encoding the polypeptide of SEQ ID NO: 6 or SEQ ID NO: 27, or a polypeptide having at least 75% and more preferably at least 80% sequence identity thereto which has GPT activity.

In yet another specific embodiment, the GPT transgene is a GPT polynucleotide encoding a Soybean derived GPT, such as the Soybean GPTs of SEQ ID NO: 8, SEQ ID NO: 28 or SEQ ID NO: 28 with a further Isoleucine at the N-terminus of the sequence. The GPT transgene may be encoded by the nucleotide sequence of SEQ ID NO: 7; a nucleotide sequence having at least 75% and more preferably at least 80% identity to SEQ ID NO: 7, and encoding a polypeptide having GPT activity; a nucleotide sequence encoding the polypeptide of SEQ ID NO: 8 or SEQ ID NO: 28 or SEQ ID NO: 28 with a further Isoleucine at the N-terminus of the sequence, or a polypeptide having at least 75% and more preferably at least 80% sequence identity thereto which has GPT activity.

In yet another specific embodiment, the GPT transgene is a GPT polynucleotide encoding a Barley derived GPT, such as the Barley GPTs of SEQ ID NO: 10 and SEQ ID NO: 29. The GPT transgene may be encoded by the nucleotide sequence of SEQ ID NO: 9; a nucleotide sequence having at least 75% and more preferably at least 80% identity to SEQ ID NO: 9, and encoding a polypeptide having GPT activity; a nucleotide sequence encoding the polypeptide of SEQ ID NO: 10 or SEQ ID NO: 29, or a polypeptide having at least 75% and more preferably at least 80% sequence identity thereto which has GPT activity.

In yet another specific embodiment, the GPT transgene is a GPT polynucleotide encoding a Zebra fish derived GPT, such as the Zebra fish GPTs of SEQ ID NO: 12 and SEQ ID NO: 30. The GPT transgene may be encoded by the nucleotide sequence of SEQ ID NO: 11; a nucleotide sequence having at least 75% and more preferably at least 80% identity to SEQ ID NO: 11, and encoding a polypeptide having GPT activity; a nucleotide sequence encoding the polypeptide of SEQ ID NO: 12 or SEQ ID NO: 30, or a polypeptide having at least 75% and more preferably at least 80% sequence identity thereto which has GPT activity.

In yet another specific embodiment, the GPT transgene is a GPT polynucleotide encoding a Bamboo derived GPT, such as the Bamboo GPT of SEQ ID NO: 31. The GPT transgene may be encoded by a nucleotide sequence encoding the polypeptide of SEQ ID NO: 31, or a polypeptide having at least 75% and more preferably at least 80% sequence identity thereto which has GPT activity.

Other GPT polynucleotides suitable for use as GPT transgenes in the practice of the invention may be obtained by various means, as will be appreciated by one skilled in the art, tested for the ability to direct the expression of a GPT with GPT activity in a recombinant expression system (i.e., £. coli (see Examples 20-23), in a transient in planta expression system (see Example 19), or in a transgenic plant (see Examples 1-18).

TRANSGENE CONSTRUCTS/EXPRESSION VECTORS

In order to generate the transgenic plants of the invention, the gene coding sequence for the desired transgene(s) must be incorporated into a nucleic acid construct (also interchangeably referred to herein as a (transgene) expression vector, expression cassette, expression construct or expressible genetic construct) which can direct the expression of the transgene sequence in transformed plant cells. Such nucleic acid constructs carrying the transgene(s) of interest may be introduced into a plant cell or cells using a number of methods known in the art, including but not limited to electroporation, DNA bombardment or biolistic approaches, microinjection, and via the use of various DNA-based vectors such as Agrobacterium tumefaciens and Agrobacterium rhizogenes vectors. Once introduced into the transformed plant cell, the nucleic acid construct may direct the expression of the incorporated transgene(s) (i.e., GPT), either in a transient or stable fashion. Stable expression is preferred, and is achieved by utilizing plant transformation vectors which are able to direct the chromosomal integration of the transgene construct. Once a plant cell has been successfully transformed, it may be cultivated to regenerate a transgenic plant. A large number of expression vectors suitable for driving the constitutive or induced expression of inserted genes in transformed plants are known. In addition, various transient expression vectors and systems are known. To a large extent, appropriate expression vectors are selected for use in a particular method of gene transformation (see, infra). Broadly speaking, a typical plant expression vector for generating transgenic plants will comprise the transgene of interest under the expression regulatory control of a promoter, a selectable marker for assisting in the selection of transformants, and a transcriptional terminator sequence.

More specifically, the basic elements of a nucleic acid construct for use in generating the transgenic plants of the invention are: a suitable promoter capable of directing the functional expression of the transgene(s) in a transformed plant cell, the transgene(s) (i.e., GPT coding sequence) operably linked to the promoter, preferably a suitable transcription termination sequence (i.e., nopaline synthetic enzyme gene terminator) operably linked to the transgene, and typically other elements useful for controlling the expression of the transgene, as well as one or more selectable marker genes suitable for selecting the desired transgenic product (i.e., antibiotic resistance genes).

As Agrobacterium tumefaciens is the primary transformation system used to generate transgenic plants, there are numerous vectors designed for

Agrobacterium transformation. For stable transformation, Agrobacterium systems utilize “binary” vectors that permit plasmid manipulation in both E. coli and Agrobacterium, and typically contain one or more selectable markers to recover transformed plants (Hellene et al., 2000, Technical focus: A guide to Agrobacterium binary Ti vectors. Trends Plant Sci 5:446-451). Binary vectors for use in Agrobacterium transformation systems typically comprise the borders of T-DNA, multiple cloning sites, replication functions for Escherichia coli and A. tumefaciens, and selectable marker and reporter genes.

So-called “super-binary" vectors provide higher transformation efficiencies, and generally comprise additional virulence genes from a Ti (Komari et al., 2006, Methods Mol. Biol. 343:15-41). Super binary vectors are typically used in plants which exhibit lower transformation efficiencies, such as cereals. Such additional virulence genes include without limitation virB, virE, and virG (Vain et al., 2004, The effect of additional virulence genes on transformation efficiency, transgene integration and expression in rice plants using the pGreen/pSoup dual binary vector system. Transgenic Res. 13: 593-603; Srivatanakul et al., 2000, Additional virulence genes influence transgene expression: transgene copy number, integration pattern and expresshn. J. Plant Physiol. 157, 685-690; Park et al., 2000, Shorter T-DNA or additional virulence genes improve Agrobacterium-mediated transformation. Theor. Appl. Genet. 101, 1015-1020; Jin et al., 1987, Genes responsible for the supervirulence phenotype of Agrobacterium tumefaciens A281. J. Bacteriol. 169: 4417-4425).

In the embodiments exemplified herein (see Examples, infra), expression vectors which place the inserted transgene(s) under the control of the constitutive CaMV 35S promoter are employed. A number of expression vectors which utilize the CaMV 35S promoter are known and/or commercially available.

PLANT PROMOTERS

The term ‘promoter’ is used to designate a region in the genome sequence upstream of a gene transcription start site (TSS), although sequences downstream of TSS may also affect transcription initiation as well. Promoter elements select the transcription initiation point, transcription specificity and rate. Depending on the distance from the TSS, the terms of ‘proximal promoter’ (several hundreds nucleotides around the TSS) and 'distal promoter’ (thousands and more nucleotides upstream of the TSS) are also used. Both proximal and distal promoters include sets of various elements participating in the complex process of cell-, issue-, organ-, developmental stage and environmental factors-specific regulation of transcription. Most promoter elements regulating TSS selection are localized in the proximal promoter. A large number of promoters which are functional in plants are known in the art. In constructing GPT transgene constructs, the selected promoters) may be constitutive, non-specific promoters such as the Cauliflower Mosaic Virus 35S ribosomal promoter (CaMV 35S promoter), which is widely employed for the expression of transgenes in plants. Examples of other strong constitutive promoters include without limitation the rice actin 1 promoter, the CaMV 19S promoter, the Ti plasmid nopaline synthase promoter, the alcohol dehydrogenase promoter and the sucrose synthase promoter.

Alternatively, in some embodiments, it may be desirable to select a promoter based upon the desired plant cells to be transformed by the transgene construct, the desired expression level of the transgene, the desired tissue or subcellular compartment for transgene expression, the developmental stage targeted, and the like.

For example, when expression in photosynthetic tissues and compartments is desired, a promoter of the ribulose bisphosphate carboxylase (RuBisCo) gene may be employed. When the expression in seeds is desired, promoters of various seed .storage protein genes may be employed. For expression in fruits, a fruit-specific promoter such as tomato 2A11 may be used. Examples of other tissue specific promoters include the promoters encoding lectin (Vodkin et al., 1983, Cell 34:1023-31; Lindstrom et al., 1990, Developmental Genetics 11:160-167), corn alcohol dehydrogenase 1 (Vogel et al, 1989, J. Cell. Biochem. (Suppl. 0) 13:Part D; Dennis et al., 1984, Nucl. Acids Res., 12(9): 3983-4000), corn light harvesting complex (Simpson, 1986, Science, 233: 34-38; Bansal et al., 1992, Proc. Natl. Acad. Sci. USA, 89: 3654-3658), corn heat shock protein (Odell et al., 1985, Nature, 313: 810-812; Rochester et al., 1986, EMBO J., 5: 451-458), pea small subunit RuBP carboxylase (Poulsen et al., 1986, Mol. Gen. Genet., 205(2): 193-200; Cashmore et al., 1983, Gen. Eng. Plants, Plenum Press, New York, pp 29-38); Ti plasmid mannopine synthase and Ti plasmid nopaline synthase (Langridge et al., 1989, Proc. Natl. Acad. Sci. USA, 86: 3219-3223), petunia chalcone isomerase (Van Tunen et al., 1988, EMBO J. 7(5): 1257-1263), bean glycine rich protein 1 (Keller et al., 1989, EMBO J. 8(5): 1309-1314), truncated CaMV 35s (Odell et al., 1985, supra), potato patatin (Wenzler et al., 1989, Plant Mol. Biol. 12: 41-50), root cell (Conkling et al., 1990, Plant Physiol. 93: 1203-1211), maize zein (Reina et al., 1990, Nucl. Acids Res. 18(21): 6426; Kriz et al., 1987, Mol. Gen. Genet. 207(1): 90-98; Wandelt and Feix, 1989, Nuc. Acids Res. 17(6): 2354; Langridge and Feix, 1983, Cell 34: 1015-1022; Reina et al., 1990, Nucl. Acids Res. 18(21): 6426), globulin-1 (Belanger and Kriz, 1991, Genetics 129: 863-872), α-tubulin (Carpenter et al., 1992, Plant Cell 4(5): 557-571; Uribe et al., 1998, Plant Mol. Biol. 37(6): 1069-1078), cab (Sullivan, eta!., 1989, Mol. Gen. Genet. 215(3): 431-440), PEPCase (Hudspeth and Grula, 1989, Plant Mol. Biol. 12: 579-589), R gene complex (Chandler et al., 1989, The Plant Cell 1: 1175-1183), chalcone synthase (Franken et al., 1991, EMBO J. 10(9): 2605-2612) and glutamine synthetase promoters (U.S. Pat. No. 5,391,725; Edwards et al., 1990, Proc. Natl. Acad. Sci. USA 87: 3459-3463; Brears etal., 1991, Plant J. 1(2): 235-244).

In addition to constitutive promoters, various inducible promoter sequences may be employed in cases where it is desirable to regulate transgene expression as the transgenic plant regenerates, matures, flowers, etc. Examples of such inducible promoters include promoters of heat shock genes, protection responding genes (i.e., phenylalanine ammonia lyase; see, for example Bevan et al., 1989, EMBO J. 8(7): 899-906), wound responding genes (i.e., cell wall protein genes), chemically inducible genes (i.e., nitrate reductase, chitinase) and dark inducible genes (i.e., asparagine synthetase; see, for example U.S. Patent No. 5,256,558). Also, a number of plant nuclear genes are activated by light, including gene families encoding the major chlorophyll a/b binding proteins (cab) as well as the small subunit of ribulose-1,5-bisphosphate carboxylase (rbcS) (see, for example, Tobin and Silverthorne, 1985, Annu. Rev. Plant Physiol. 36: 569-593; Dean et al., 1989, Annu. Rev. Plant Physiol. 40:415-439.).

Other inducible promoters include ABA- and turgor-inducible promoters, the auxin-binding protein gene promoter (Schwob et al.. 1993, Plant J. 4(3): 423-432), the UDP glucose flavonoid glycosyl-transferase gene promoter (Ralston et al., 1988, Genetics 119(1): 185-197); the MPI proteinase inhibitor promoter (Cordero et al.. 1994, Plant J. 6(2): 141-150), the glyceraldehyde-3-phosphate dehydrogenase gene promoter (Kohler et al., 1995, Plant Mol. Biol. 29(6): 1293-1298; Quigley et al., 1989, J. Mol. Evol. 29(5): 412-421; Martinez et al.,1989, J. Mol. Biol. 208(4): 551-565) and light inducible plastid glutamine synthetase gene from pea (U.S. Pat. No. 5,391,725; Edwards et al., 1990, supra).

For a review of plant promoters used in plant transgenic plant technology, see Potenza et al., 2004, In Vitro Cell. Devel. Biol - Plant, 40(1): 1-22. For a review of synthetic plant promoter engineering, see, for example, Venter, M., 2007, Trends Plant Sci., 12(3): 118-124.

GLUTAMINE PHENYLPYRUVATE TRANSAMINASE (GPT) TRANSGENE

The present invention discloses for the first time that plants contain a glutamine phenylpyruvate transaminase (GPT) enzyme which is directly functional in the synthesis of the signal metabolite 2-hydroxy-5-oxoproline. Until now, no plant transaminase with a defined function has been described. Applicants have isolated and tested GPT polynucleotide coding sequences derived from several plant and animal species, and have successfully incorporated the gene into heterologous transgenic host plants which exhibit markedly improved growth characteristics, including faster growth, higher foliar protein content, and faster CO2 fixation rates.

It is expected that all plant species contain a GPT which functions in the same metabolic pathway, involving the biosynthesis of the signal metabolite 2-hydroxy-5-oxoproline. Thus, in the practice of the invention, any plant gene encoding a GPT homolog or functional variants thereof may be useful in the generation of transgenic plants of this invention. Moreover, given the structural similarity between various plant GPT protein structures and the putative ( and biologically active) GPT homolog from Danio rerio (Zebra fish) (see Example 22), other nonplant GPT homologs may be used in preparing GPT transgenes for use in generating the transgenic plants of the invention. When individually compared (by BLAST alignment) to the Arabidopsis mature protein sequence provided in SEQ ID NO: 25, the following sequence identities and homologies (BLAST "positives", including similar amino acids) were obtained for the following mature GPT protein sequences:

[SEQ ID] ORIGIN % IDENTITY %POSITIVE

[31] Grape 84 93 [32] Rice 83 91 [33] Soybean 83 93 [34] Barley 82 91 [35] Zebra fish 83 92 [36] Bamboo 81 90

Com 79 90

Castor 84 93

Poplar 85 93

Underscoring the conserved nature of the structure of the GPT protein across most plant species, the conservation seen within the above plant species extends to the non-human putative GPTs from Zebra fish and Chlamydomonas. In the case of Zebra fish, the extent of identity is very high (83% amino acid sequence identity with the mature Arabidopsis GPT of SEQ ID NO: 25, and 92% homologous taking similar amino acid residues into account). The Zebra fish mature GPT was confirmed by expressing it in E. coli and demonstrating biological activity (synthesis of 2-oxoglutaramate).

In order to determine whether putative GPT homologs would be suitable for generating the growth-enhanced transgenic plants of the invention, one need initially express the coding sequence thereof in E. coli or another suitable host and determine whether the 2-oxoglutaramate signal metabolite is synthesized at increased levels (see Examples 19-23). Where such an increase is demonstrated, the coding sequence may then be introduced into both homologous plant hosts and heterologous plant hosts, and growth characteristics evaluated. Any assay that is capable of detecting 2-oxoglutaramate with specificity may be used for this purpose, including without limitation the NMR and HPLC assays described in Example 2, infra. In addition, assays which measure GPT activity directly may be employed.

Any plant GPT with 2-oxoglutaramate synthesis activity may be used to transform plant cells in order to generate transgenic plants of the invention. There appears to be a high level of structural homology among plant species, which appears to extend beyond plants, as evidenced by the close homology between various plant GPT proteins and the putative Zebra fish GPT homolog. Therefore, various plant GPT genes may be used to generate growth-enhanced transgenic plants in a variety of heterologous plant species. In addition, GPT transgenes expressed in a homologous plant would be expected to result in the desired enhanced-growth characteristics as well (i.e., rice glutamine transaminase over-expressed in transgenic rice plants), although it is possible that regulation within a homologous cell may attenuate the expression of the transgene in some fashion that may not be operable in a heterologous cell. TRANSCRIPTION TERMINATORS:

In preferred embodiments, a 3’ transcription termination sequence is incorporated downstream of the transgene in order to direct the termination of transcription and permit correct polyadenylation of the mRNA transcript. Suitable transcription terminators are those which are known to function in plants, including without limitation, the nopaline synthase (NOS) and octopine synthase (OCS) genes of

Agrobacterium tumafaciens, the 77 transcript from the octopine synthase gene, the 3’ end of the protease inhibitor I or II genes from potato or tomato, the CaMV 35S terminator, the tml terminator and the pea rbcS E9 terminator. In addition, a gene's native transcription terminator may be used. In specific embodiments, described by way of the Examples, infra, the nopaline synthase transcription terminator is employed. SELECTABLE MARKERS:

Selectable markers are typically included in transgene expression vectors in order to provide a means for selecting transformants. While various types of markers are available, various negative selection markers are typically utilized, including those which confer resistance to a selection agent that inhibits or kills untransformed cells, such as genes which impart resistance to an antibiotic (such as kanamycin, gentamycin, anamycin, hygromycin and hygromycinB) or resistance to a herbicide (such as sulfonylurea, gulfosinate, phosphinothricin and glyphosate). Screenable markers include, for example, genes encoding β-glucuronidase (Jefferson, 1987, Plant Mol. Biol. Rep 5: 387-405), genes encoding luciferase (Ow et al., 1986, Science 234: 856-859) and various genes encoding proteins involved in the production or control of anthocyanin pigments (See, for example, U.S. Patent 6,573,432). The E coli glucuronidase gene (gus, gusA or uidA) has become a widely used selection marker in plant transgenics, largely because of the glucuronidase enzyme's stability, high sensitivity and ease of detection (e.g., fluorometric, spectrophotometric, various histochemical methods). Moreover, there is essentially no detectable glucuronidase in most higher plant species. TRANSFORMATION METHODOLOGIES AND SYSTEMS:

Various methods for introducing the transgene expression vector constructs of the invention into a plant or plant cell are well known to those skilled in the art, and any capable of transforming the target plant or plant cell may be utilized.

Agrobacterium-mediated transformation is perhaps the most common method utilized in plant transgenics, and protocols for Agrobacterium-mediated transformation of a large number of plants are extensively described in the literature (see, for example, Agrobacterium Protocols, Wan, ed.p Humana Press, 2nd edition, 2006). Agrobacterium tumefaciens is a Gram negative soil bacteria that causes tumors (Crown Gall disease) in a great many dicot species, via the insertion of a small segment of tumor-inducing DNA ( “T-DNA”, 'transfer DNA') into the plant cell, which is incorporated at a semi-random location into the plant genome, and which eventually may become stably incorporated there. Directly repeated DNA sequences, called T-DNA borders, define the left and the right ends of the T-DNA. The T-DNA can be physically separated from the remainder of the Ti-plasmid, creating a 'binary vector· system.

Agrobacterium transformation may be used for stably transforming dicots, monocots, and cells thereof (Rogers et al., 1986, Methods Enzymol., 118: 627-641; Hernalsteen et al., 1984, EMBO J., 3: 3039-3041; Hoykass-Van Slogteren et al., 1984, Nature, 311: 763-764; Grimsley et al., 1987, Nature 325: 167-1679; Boulton et al., 1989, Plant Mol. Biol. 12: 31-40; Gould et al., 1991, Plant Physiol. 95: 426-434). Various methods for introducing DNA into Agrobacteria are known, including electroporation, freeze/thaw methods, and triparental mating. The most efficient method of placing foreign DNA into Agrobacterium is via electroporation (Wise et al., 2006, Three Methods for the Introduction of Foreign DNA into Agrobacterium, Methods in Molecular Biology, vol. 343: Agrobacterium Protocols, 2/e, volume 1; Ed., Wang, Humana Press Inc., Totowa, NJ, pp. 43-53). In addition, given that a large percentage of T-DNAs do not integrate, Agrobacterium-mediated transformation may be used to obtain transient expression of a transgene via the transcriptional competency of unincorporated transgene construct molecules (Helens et al., 2005, Plant Methods 1:13). A large number of Agrobacterium transformation vectors and methods have been described (Karimi et al., 2002, Trends Plant Sci. 7(5): 193-5), and many such vectors may be obtained commercially (for example, Invitrogen). In addition, a growing number of “open-source” Agrobacterium transformation vectors are available (for example, pCambla vectors; Cambia, Canberra, Australia). See, also, subsection herein on TRANSGENE CONSTRUCTS, supra. In a specific embodiment described further in the Examples, a pMON316-based vector was used in the leaf disc transformation system of Horsch et. al. (Horsch et al.,1995, Science 227:1229-1231) to generate growth enhanced transgenic tobacco and tomato plants.

Other oommonly used transformation methods that may be employed in generating the transgenic plants of the invention include without limitation microprojectile bombardment, or biolistic transformation methods, protoplast transformation of naked DNA by calcium, polyethylene glycol (PEG) or electroporation (Paszkowski et al., 1984, EMBO J. 3: 2727-2722; Potrykus et al., 1985, Mol. Gen. Genet. 199: 169-177; Fromm et al., 1985, Proc. Nat. Acad. Sci. USA 82:5824-5828; Shimamoto et al.. 1989, Nature, 338: 274-276.

Biolistic transformation involves injecting millions of DNA-coated metal particles into target cells or tissues using a biolistic device (or “gene gun”), several kinds of which are available commercially; once inside the cell, the DNA elutes off the particles and a portion may be stably incorporated into one or more of the cell’s chromosomes (for review, see Kikkert et al., 2005, Stable Transformation of Plant Cells by Particle Bombardmentf&amp;olistics, in: Methods in Molecular Biology, vol. 286: Transgenic Plants: Methods and Protocols, Ed. L. Peri a, Humana Press Inc., Totowa, NJ).

Electroporation is a technique that utilizes short, high-intensity electric fields to permeabilize reversibly the lipid bilayers of cell membranes (see, for example, Fisk and Dandekar, 2005, Introduction and Expression of Transgenes in Plant Protoplasts, in: Methods in Molecular Biology, vol. 286: Transgenic Plants: Methods and Protocols, Ed. L. Pefia, Humana Press Inc., Totowa. NJ, pp. 79-90; Fromm et al.,1987, Electroporation of DNA and RNA into plant protoplasts, in Methods in Enzymology, Vol. 153, Wu and Grossman, eds., Academic Press, London, UK, pp. 351-366; Joersbo and Brunstedt, 1991, Electroporation: mechanism and transient expression, stable transformation and biological effects in plant protoplasts. Physiol. Plant. 81, 256-264; Bates, 1994, Genetic transformation of plants by protoplast electroporation. Mol. Biotech. 2: 135-145; Dillen et al., 1998, Electroporation-mediated DNA transfer to plant protoplasts and intact plant tissues for transient gene expression assays, in Cell Biology, Vol. 4, ed., Celis, Academic Press, London, UK, pp. 92-99). The technique operates by creating aqueous pores in the bacterial membrane, which are of sufficiently large size to allow DNA molecules (and other macromolecules) to enter the cell, where the transgene expression construct (as T-DNA) may be stably incorporated into plant genomic DNA, leading to the generation of transformed cells that can subsequently be regenerated into transgenic plants.

Newer transformation methods include so-called "floral dip” methods, which offer the promise of simplicity, without requiring plant tissue culture, as is the case with all other commonly used transformation methodologies (Bent et al., 2006, Arabidopsis thaliana Floral Dip Transformation Method, Methods Mol Biol, vol. 343: Agrobacterium Protocols, 2ie, volume 1; Ed., Wang, Humana Press Inc., Totowa, NJ, pp. 87-103; Clough and Bent, 1998, Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana, Plant J. 16: 735-743). However, with the exception of Arabidopsis, these methods have not been widely used across a broad spectrum Of different plant species. Briefly, floral dip transformation is accomplished by dipping or spraying flowering plants in with an appropriate strain of Agrobacterium tumefaciens. Seeds collected from these To plants are then germinated under selection to identify transgenic Ti individuals. Example 16 demonstrated floral dip inoculation of Arabidopsis to generate transgenic Arabidopsis plants.

Other transformation methods include those in which the developing seeds or seedlings of plants are transformed using vectors such as Agrobacterium vectors. For example, such vectors may be used to transform developing seeds by injecting a suspension or mixture of the vector (i.e., Agrobacteria) directly into the seed cavity of developing pods (Wang and Waterhouse, 1997, Plant Mol. Biol. Reporter 15: 209-215). Seedlings may be transformed as described in Yasseem, 2009, Plant Mol. Biol. Reporter 27: 20-28. Germinating seeds may be transformed as described in Chee et al., 1989, Plant Pysiol. 91: 1212-1218. Intra-fruit methods, in which the vector is injected into fruit or developing fruit, may be also be used. Still other transformation methods include those in which the flower structure is targeted for vector inoculation, such as the flower inoculation methods.

The foregoing plant transformation methodologies may be used to introduce transgenes into a number of different plant cells and tissues, including without limitation, whole plants, tissue and organ explants including chloroplasts, flowering tissues and cells, protoplasts, meristem cells, callus, immature embryos and gametic cells such as microspores, pollen, sperm and egg cells, tissue cultured cells of any of the foregoing, any other cells from which a fertile regenerated transgenic plant may be generated. Callus is initiated from tissue sources including, but not limited to, immature embryos, seedling apical meristems, microspores and the like. Cells capable of proliferating as callus are also recipient cells for genetic transformation.

Methods of regenerating individual plants from transformed plant cells, tissues or organs are known and are described for numerous plant species.

As an illustration, transformed plantlets (derived from transformed cells or tissues) are cultured in a root-permissive growth medium supplemented with the selective agent used in the transformation strategy (i.e., and antibiotic such as kanamycin). Once rooted, transformed plantlets are then transferred to soil and allowed to grow to maturity. Upon flowering, the mature plants are preferably selfed (self-fertilized), and the resultant seeds harvested and used to grow subsequent generations. Examples 3-6 describe the regeneration of transgenic tobacco and tomato plants.

To transgenic plants may be used to generate subsequent generations (e.g., Ti, T2, etc.) by selfing of primary or secondary transformants, or by sexual crossing of primary or secondary transformants with other plants (transformed or untransformed). SELECTION OF GROWTH-ENHANCED TRANSGENIC PLANTS:

Transgenic plants may be selected, screened and characterized using standard methodologies. The preferred transgenic plants of the invention will exhibit one or more phenotypic characteristics indicative of enhanced growth and/or other desirable agronomic properties. Transgenic plants are typically regenerated under selective pressure in order to select transformants prior to creating subsequent transgenic plant generations. In addition, the selective pressure used may be employed beyond T0 generations in order to ensure the presence of the desired transgene expression construct or cassette. T0 transformed plant cells, calli, tissues or plants may be identified and isolated by selecting or screening for the genetic composition of and/or the phenotypic characteristics encoded by marker genes contained in the transgene expression construct used for the transformation. For example, selection may be conducted by growing potentially-transformed plants, tissues or cells in a growth medium containing a repressive amount of antibiotic or herbicide to which the transforming genetic construct can impart resistance. Further, the transformed plant cells, tissues and plants can be identified by screening for the activity of marker genes (such as β-glucuronidase) which may be present in the transgene expression construct.

Various physical and biochemical methods may be employed for identifying plants containing the desired transgene expression construct, as is well known. Examples of such methods include Southern blot analysis or various nucleic acid amplification methods (i.e., PCR) for identifying the transgene, transgene expression construct or elements thereof; Northern blotting, S1 RNase protection, reverse transcriptase PCR (RT-PCR) amplification for detecting and determining the RNA transcription products; and protein gel electrophoresis, Western blotting, immunoprecipitation, enzyme immunoassay, and the like for identifying the protein encoded and expressed by the transgene.

In another approach, expression levels of genes, proteins and/or metabolic compounds that are know to be modulated by transgene expression in the target plant may be used to identify transformants. In one embodiment of the present invention, increased levels of the signal metabolite 2-oxoglutaramate may be used to screen for desirable transformants.

Ultimately, the transformed plants of the invention may be screened for enhanced growth and/or other desirable agronomic characteristics. Indeed, some degree of phenotypic screening is generally desirable in order to identify transformed lines with the fastest growth rates, the highest seed yields, etc., particularly when identifying plants for subsequent selfing, cross-breeding and back-crossing. Various parameters may be used for this purpose, including without limitation, growth rates, total fresh weights, dry weights, seed and fruit yields (number, weight), seed and/or seed pod sizes, seed pod yields (e.g., number, weight), leaf sizes, plant sizes, increased flowering, time to flowering, overall protein content (in seeds, fruits, plant tissues), specific protein content (i.e., GS), nitrogen content, free amino acid, and specific metabolic compound levels (i.e., 2-oxoglutaramate). Generally, these phenotypic measurements are compared with those obtained from a parental identical or analogous plant line, an untransformed identical or analogous plant, or an identical or analogous wild-type plant (i.e., a normal or parental plant). Preferably, and at least initially, the measurement of the chosen phenotypic characteristics) in the target transgenic plant is done in parallel with measurement of the same characteristic(s) in a normal or parental plant. Typically, multiple plants are used to establish the phenotypic desirability and/or superiority of the transgenic plant in respect of any particular phenotypic characteristic.

Preferably, initial transformants are selected and then used to generate T-ι and subsequent generations by selfing (self-fertilization), until the transgene genotype breeds true (i.e., the plant is homozygous for the transgene). In practice, this is accomplished by selfing for 3 or 4 generations, screening at each generation for the desired traits and selfing those individuals.

Stable transgenic lines may be crossed and back-crossed to create varieties with any number of desired traits, including those with stacked transgenes, multiple copies of a transgene, etc. Additionally, stable transgenic plants may be further modified genetically, by transforming such plants with further transgenes or additional copies of the parental transgene. Also contemplated are transgenic plants created by single transformation events which introduce multiple copies of a given transgene or multiple transgenes. Various common breeding methods are well know to those skilled in the art (see, e.g., Breeding Methods for Cultivar Development, Wilcox J. ed., American Society of Agronomy, Madison Wis. (1987)).

EXAMPLES

Various aspects of the invention are further described and illustrated by way of the several examples which follow, none of which are intended to limit the scope of the invention. EXAMPLE 1: ISLOATION OF ARABIDOPSIS GLUAMINE PHENYLPYRUVATE TRANSAMINASE (GPT) GENE:

In an attempt to locate a plant enzyme that is directly involved in the synthesis of the signal metabolite 2-oxoglutaramate, applicants hypothesized that the putative plant enzyme might bear some degree of structural relationship to a human protein that had been characterized as being involved in the synthesis of 2-oxoglutaramate. The human protein, glutamine transaminase K (E.C. 2.6.1.64) (also referred in the literature as cysteine conjugate β -lyase, kyneurenine aminotransferase, glutamine phenylpyruvate transaminase, and other names), had been shown to be involved in processing of cysteine conjugates of halogenated xenobiotics (Perry et al., 1995, FEBS Letters 360:277-280). Rather than having an activity involved in nitrogen metabolism, however, human cysteine conjugate β-lyase has a detoxifying activity in humans, and in animals (ref). Nevertheless, the potential involvement of this protein in the synthesis of 2-oxoglutaramate was of interest.

Using the protein sequence of human cysteine conjugate β-lyase, a search against the TIGR Arabidopsis plant database of protein sequences identified one potentially related sequence, a polypeptide encoded by a partial sequence at the Arabidopsis gene locus at At1q77670, sharing approximately 36% sequence homology/identity across aligned regions.

The full coding region of the gene was then amplified from an Arabidopsis cDNA library (Stratagene) with the following primer pair: 5-CCC ATCGATGTAC C TGGAC AT AAAT G GTGT GATG-3’ 5’- GATGGTACCTCAG ACTTTT CTCTTAAGCTTCTGCTTC-3’

These primers were designed to incorporate Cla I fATCGATI and Κρη I (GGTACC1 restriction sites to facilitate subsequent subcloning into expression vectors for generating transgenic plants. Takara ExTaq DNA polymerase enzyme was used for high fidelity PCR using the following conditions: initial denaturing 94C for 4 minutes, 30 cycles of 94C 30 second, annealing at 55C for 30 seconds, extension at 72C for 90 seconds, with a final extension of 72C for 7 minutes. The amplification product was digested with Cla I and Κρη I restriction enzymes, isolated from an agarose gel electrophoresis and ligated into vector pMon316 (Rogers, et. al. 1987 Methods in Enzymology 153:253-277) which contains the cauliflower mosaic virus (CaMV) 35S constitutive promoter and the nopaline synthase (NOS) 3’ terminator. The ligation was transformed into DH5a cells and transformants sequenced to verify the insert. A 1.3 kb cDNA was isolated and sequenced, and found to encode a full length protein of 440 amino acids in length, including a putative chloroplast signal sequence. EXAMPLE 2: PRODUCTION OF BIOLOGICALLY ACTIVE ARABIDOPSIS GLUTAMINE PHENYLPYRUVATE TRANSAMINASE:

To test whether the protein encoded by the cDNA isolated as described in Example 1, supra, is capable of catalyzing the synthesis of 2- oxoglutaramate, the cDNA was expressed in E. coli, purified, and assayed for its ability to synthesize 2-oxoglutaramate using a standard method. NMR Assay for 2-oxoQlutaramate

Briefly, the resulting purified protein was added to a reaction mixture containing 150 mM Tris-HCI, pH 8.5, 1 mM beta mercaptoethanol, 200 mM glutamine, 100 mM glyoxylate and 200 microM pyridoxal 5’-phosphate. The reaction mixture without added test protein was used as a control. Test and control reaction mixtures were incubated at .37°C for 20 hours, and then clarified by centrifugation to remove precipitated material. Supernatants were tested for the presence and amount of 2-oxoglutaramate using 13C NMR with authentic chemically synthesized 2-oxoglutaramate as a reference. The products of the reaction are 2-oxoglutaramate and glycine, while the substrates (glutamine and glyoxylate) diminish in abundance. The cyclic 2-oxoglutaramate gives rise to a distinctive signal allowing it to be readily distinguished from the open chain glutamine precursor. HPLC Assay for 2-oxoalutaramate

An alternative assay for GPT activity uses HPLC to determine 2-oxoglutaramate production, following a modification of Calderon et al., 1985, J Bacteriol 161(2): 807-809. Briefly, a modified extraction buffer consisting of 25 mM Tris-HCI pH 8.5, 1 mM EDTA, 20 μΜ FAD, 10 mM Cysteine, and -1.5% (v/v) Mercaptoethanol. Tissue samples from the test material (i.e., plant tissue) are added to the extraction buffer at approximately a 1/3 ratio (w/v), incubated· for 30 minutes at 37°C, and stopped with 200μΙ of 20% TCA. After about 5 minutes, the assay mixture is centrifuged and the supernatant used to quantify 2-oxoglutaramate by HPLC, using an ION-300 7.8mm ID X 30 cm L column, with a mobile phase in 0.01 N h2S04, a flow rate of approximately 0.2 ml/min, at 40°C. Injection volume is approximately 20 μΙ, and retention time between about 38 and 39 minutes. Detection is achieved with 210nm UV light.

Results Using NMR Assay:

This experiment revealed that the test protein was able to catalyze the synthesis of 2- oxoglutaramate. Therefore, these data indicate that the isolated cDNA encodes a glutamine phenylpyruvate transaminase that is directly involved in the synthesis of 2-oxoglutaramate in plants. Accordingly, the test protein was designated Arabidopsis glutamine phenylpyruvate transaminase, or “GPT.

The nucleotide sequence of the Arabidopsis GPT coding sequence is shown in the Table of Sequences, SEQ ID NO. 1. The translated amino acid sequence of the GPT protein is shown in SEQ ID NO. 2. EXAMPLE 3: CREATION OF TRANSGENIC TOBACCO PLANTS OVEREXPRESSING ARABIDOPSIS GPT:

Generation of Plant Expression Vector pMON-PJU:

Briefly, the plant expression vector pMon316-PJU was constructed as follows. The isolated cDNA encoding Arabidopsis GPT (Example 1) was cloned into the Clal-Kpnl polylinker site of the pMON316 vector, which places the GPT gene under the control of the constitutive cauliflower mosaic virus (CaMV) 35S promoter and the nopaline synthase (NOS) transcriptional terminator. A kanamycin resistance gene was included to provide a selectable marker.

Aarobacterium-Mediated Plant Transformations: pMON-PJU and a control vector pMon316 (without inserted DNA) were transferred to Agrobacterium tumefacians strain pTiTT37ASE using a standard electroporation method (McCormac et al., 1998, Molecular Biotechnology 9:155-159), followed by plating on LB plates containing the antibiotics spectinomycin (100 micro gm / ml) and kanamycin (50 micro gm / ml). Antibiotic resistant colonies of Agrobacterium were examined by PCR td assure that they contained plasmid.

Nicotiana tabacum cv. Xanthi plants were transformed with pMON-PJU transformed Agrobacteria using the leaf disc transformation system of Horsch et. al. (Horsch et al.,1995, Science 227:1229-1231). Briefly, sterile leaf disks were inoculated and cultured for 2 days, then transferred to selective MS media containing 100 pg/ml kanamycin and 500 pg/ml clafaran. Transformants were confirmed by their ability to form roots in the selective media.

Generation of GPT Transgenic Tobacco Plants:

Sterile leaf segments were allowed to develop callus on Murashige &amp; Skoog (M&amp;S) media from which the transformant plantlets emerged. These plantlets were then transferred to the rooting-permissive selection medium (M&amp;S medium with kanamycin as the selection agent). The healthy, and now rooted, transformed tobacco plantlets were then transferred to soil and allowed to grow to maturity and upon flowering the plants were selfed and the resultant seeds were harvested. During the growth stage the plants had been examined for growth phenotype and the CO2 fixation rate was measured for many of the young transgenic plants.

Production of T1 and T2 Generation GPT Transgenic Plants:

Seeds harvested form the To generation of the transgenic tobacco plants were germinated on M&amp;S media containing kanamycin (100 mg / L) to enrich for the transgene. At least one fourth of the seeds did not germinate on this media (kanamycin is expected to inhibit germination of the seeds without resistance that would have been produced as a result of normal genetic segregation of the gene) and more than half of the remaining seeds were removed because of demonstrated sensitivity (even mild) to the kanamycin.

The surviving plants (T1 generation) were thriving and these plants were then selfed to produce seeds for the T2 generation. Seeds from the T1 generation were germinated on MS media supplemented for the transformant lines with kanamycin (10mg/liter). After 14 days they were transferred to sand and provided quarter strength Hoagland’s nutrient solution supplemented with 25 mM potassium nitrate. They were allowed to grow at 24°C with a photoperiod of 16 h light and 8 hr dark with a light intensity of 900 micomoles per meter squared per second. They were harvested 14 days after being transferred to the sand culture.

Characterization of GPT Transgenic Plants:

Harvested transgenic plants (both GPT transgenes and vector control transgenes) were analyzed for glutamine sythetase activity in root and leaf, whole plant fresh weight, total protein in root and leaf, and CO2 fixation rate (Knight et al., 1988,

Plant Physiol. 88: 333). Non-transformed, wild-type A. tumefaciens plants were also analyzed across the same parameters in order to establish a baseline control.

Growth characteristic results are tabulated below in Table I. Additionally, a photograph of the GPT transgenic plant compared to a wild type control plant is shown in FIG. 2 (together with GS1 transgenic tobacco plant). Across all parameters evaluated, the GPT transgenic tobacco plants showed enhanced growth characteristics. In particular, the GPT transgenic plants exhibited a greater than 50% increase in the rate of CO2 fixation, and a greater than two-fold increase in glutamine synthetase activity in leaf tissue, relative to wild type control plants. In addition, the leaf-to-root GS ratio increased by almost three-fold in the transaminase transgenic plants relative to wild type control. Fresh weight and total protein quantity also increased in the transgenic plants, by about 50% and 80% (leaf), respectively, relative to the wild type control. These data demonstrate that tobacco plants overexpressing the Arabidopsis GPT transgene achieve significantly enhanced growth and CO2 fixation rates.

Table i

Data = average of three plants

Wild type - Control plants; not regenerated or transformed. PN1 lines were produced by regeneration after transformation using a construct without inserted gene. A control against the processes of regeneration and transformation. PN 8 lines were produced by regeneration after transformation using a construct with the Arabidopsis GPT gene. EXAMPLE 4: GENERATION OF TRANSGENIC TOMATO PLANTS CARRYING ARABIDOPSIS GPT TRANSGENE:

Transgenic Lycopersicon esculentum (Micro-Tom Tomato) plants carrying the Arabidopsis GPT transgene were generated using the vectors and methods described in Example 3. To transgenic tomato plants were generated and grown to maturity. Initial growth characteristic data of the GPT transgenic tomato plants is presented in Table II. The transgenic plants showed significant enhancement of growth rate, flowering, and seed yield in relation to wild type control plants. In addition, the transgenic plants developed multiple main stems, whereas wild type plants developed with a single main stem. A photograph of a GPT transgenic tomato plant compared to a wild type plant is presented in FIG. 3.

TABLE II

EXAMPLE 5: ACTIVITY OF BARLEY GPT TRANSGENE IN PLANTA

In this example, the putative coding sequence for Barley GPT was isolated and expressed from a transgene construct using an in planta transient expression assay. Biologically active recombinant Barley GPT was produced, and catalyzed the increased synthesis of 2- oxoglutaramate, as confirmed by HPLC.

The Barley (Hordeum vulgare) GPT coding sequence was determined and synthesized. The DNA sequence of the Barley GPT coding sequence used in this example is provided in SEQ ID NO: 9, and the encoded GPT protein amino acid sequence is presented in SEQ ID NO: 10.

The coding sequence for Barley GPT was inserted into the 1305.1 cambia vector, and transferred to Agrobacterium tumefaciens strain LBA404 using a standard electroporation method (McCormac et al., 1998, Molecular Biotechnology 9:155-159), followed by plating on LB plates containing hygromycin (50 micro gm / ml). Antibiotic resistant colonies of Agrobacterium were selected for analysis.

The transient tobacco leaf expression assay consisted of injecting a suspension of transformed Agrobacterium (1.5-2.0 OD 650) into rapidly growing tobacco leaves. Intradermal injections were made in a grid across the leaf surface to assure that a significant amount of the leaf surface would be exposed to the Agrobacterium. The plant was then allowed to grow for 3-5 days when the tissue was extracted as described for all other tissue extractions and the GPT activity measured. GPT activity in the inoculated leaf tissue (1217 nanomoles/gFWt/h) was three-fold the level measured in the control plant leaf tissue (407 nanomoles/gFWt/h), indicating that the Hordeum GPT construct can direct the expression of functional GPT in a transgenic plant.

EXAMPLE 6: ISOLATION AND EXPRESSION OF RECOMBINANT RICE GPT GENE CODING SEQUENCE AND ANALYSIS OF BIOLOGICAL ACTIVITY

In this example, the putative coding sequence for rice GPT was isolated and expressed in E. coli. Biologically active recombinant rice GPT was produced, and catalyzed the increased synthesis of 2- oxoglutaramate, as confirmed by HPLC.

Materials and Methods:

Rice GPT coding sequence and expression in E. coli:

The rice (Oryza sativia) GPT coding sequence was determined and synthesized, inserted into a PET28 vector, and expressed in E, coli. Briefly, E. coli cells were transformed with the expression vector and transformants grown overnight in LB broth diluted and grown to OD 0.4, expression induced with isopropyl-B-D-thiogalactoside (0.4 micromolar), grown for 3 hr and harvested. A total of 25 X 106 ceils were then assayed for biological activity using the NMR assay, below. Untransformed, wild type E. coli cells were assayed as a control. An additional control used E coli cells transformed with an empty vector.

The DNA sequence of the rice GPT coding sequence used in this example is provided in SEQ ID NO: 5, and the encoded GPT protein amino acid sequence is presented in SEQ ID NO: 6. HPLC Assay for 2-oxoalutaramate: HPLC was used to determine 2-oxoglutaramate production in GPT-overexpressing E. coli cells, following a modification of Calderon et al., 1985, J Bacteriol 161(2): 807-809. Briefly, a modified extraction buffer consisting of 25 mM Tris-HCI pH 8.5, 1 mM EDTA, 20 μΜ Pyridoxal phosphate, 10 mM Cysteine, and ~1.5% (v/v) Mercaptoethanol was used. Samples (lysate from E. coli cells, 25 X 106 cells) were added to the extraction buffer at approximately a 1/3 ratio (w/v), incubated for 30 minutes at 37°C, and stopped with 200μΙ of 20% TCA. After about 5 minutes, the assay mixture is centrifuged and the supernatant used to quantify 2-oxoglutaramate by HPLC, using an ION-300 7.8mm ID X 30 cm L column, with a mobile phase in 0.01 N H2S04, a flow rate of approximately 0.2 ml/min, at 40°C. Injection volume is approximately 20 pi, and retention time between about 38 and 39 minutes. Detection is achieved with 210nm UV light. NMR analysis comparison with authentic 2-oxoglutaramate was used to establish that the Arabidopisis full length sequence expresses a GPT with 2-oxoglutaramate synthesis activity. Briefly, authentic 2-oxoglutarmate (structure confirmed with NMR) made by chemical synthesis to validate the HPLC assay, above, by confirming that the product of the assay (molecule synthesized in response to the expressed GPT) and the authentic 2-oxoglutaramate elute at the same retention time. In addition, when mixed together the assay product and the authentic compound elute as a single peak. Furthermore, the validation of the HPLC assay also included monitoring the disappearance of the substrate glutamine and showing that there was a 1:1 molar stoichiometry between glutamine consumed to 2-oxoglutaramate produced. The assay procedure always included two controls, one without the enzyme added and one without the glutamine added. The first shows that the production of the 2-oxoglutaramate was dependent upon having the enzyme present, and the second shows that the production of the 2-oxoglutaramate was dependent upon the substrate glutamine.

Results:

Expression of the rice GPT coding sequence of SEQ ID NO: 5 resulted in the over-expression of recombinant GPT protein having 2-oxoglutaramate synthesis-catalyzing bioactivity. Specifically, 1.72 nanomoles of 2-oxoglutaramate activity was observed in the E. coli cells overexpressing the recombinant rice GPT, compared to only 0.02 nanomoles of 2-oxoglutaramate activity in control E. coli cells, an 86-fold activity level increase over control.

EXAMPLE 7: ISOLATION AND EXPRESSION OF RECOMBINANT SOYBEAN GPT GENE CODING SEQUENCE AND ANALYSIS OF BIOLOGICAL ACTIVITY

In this example, the putative coding sequence for soybean GPT was isolated and expressed in E. coli. Biologically active recombinant soybean GPT was produced, and catalyzed the increased synthesis of 2-oxoglutaramate, as confirmed by HPLC.

Materials and Methods:

Soybean GPT coding sequence and expression in E. coli:

The soybean (Glycine max) GPT coding sequence was determined and synthesized, inserted into a PET28 vector, and expressed in E. coli. Briefly, E. coli cells were transformed with the expression vector and transformants grown overnight in LB broth diluted and grown to OD 0.4, expression induced with isopropyl-P-D-thiogalactoside (0.4 micromolar), grown for 3 hr and harvested. A total of 25 X 106 cells were then assayed for biological activity using the NMR assay, below. Untransformed, wild type E. coli cells were assayed as a control. An additional control used £. coli cells transformed with an empty vector.

The DNA sequence of the soybean GPT coding sequence used in this example is provided in SEQ ID NO: 7, and the encoded GPT protein amino acid sequence is presented in SEQ ID NO: 8. HPLC Assay for 2-oxoalutaramate: HPLC was used to determine 2-oxoglutaramate production in GPT-overexpressing E. coli cells, as described in Example 20, supra.

Results:

Expression of the soybean GPT coding sequence of SEQ ID NO: 7 resulted in the over-expression of recombinant GPT protein having 2-oxoglutaramate synthesis-catalyzing bioactivity. Specifically, 31.9 nanomoles of 2-oxoglutaramate activity was observed in the E. coli cells overexpressing the recombinant soybean GPT, compared to only 0.02 nanomoles of 2-oxoglutaramate activity in control E. coli cells, a nearly 1,600-fold activity level increase over control.

EXAMPLE 8: ISOLATION AND EXPRESSION OF RECOMBINANT ZEBRA FISH GPT GENE CODING SEQUENCE AND ANALYSIS OF BIOLOGICAL ACTIVITY

In this example, the putative coding sequence for Zebra fish GPT was isolated and expressed in E. coli. Biologically active recombinant Zebra fish GPT was produced, and catalyzed the increased synthesis of 2-oxoglutaramate, as confirmed by NMR.

Materials and Methods:

Zebra fish GPT coding sequence and expression in E coli:

The Zebra fish (Danio rerio) GPT coding sequence was determined and synthesized, inserted into a PET28 vector, and expressed in E. coli. Briefly, E. coli cells were transformed with the expression vector and transformants grown overnight in LB broth diluted and grown to OD 0.4, expression induced with isopropyl-3-D-thiogalactoside (0.4 micromolar), grown for 3 hr and harvested. A total of 25 X 106 cells were then assayed for biological activity using the NMR assay, below. Untransformed, wild type E. coli cells were assayed as a control. An additional control used E. coli cells transformed with an empty vector.

The DNA sequence of the Zebra fish GPT coding sequence used in this example is provided in SEQ ID NO: 11, and the encoded GPT protein amino acid sequence is presented in SEQ ID NO: 12. 5 HPLC Assay for 2-oxoalutaramate: HPLC was used to determine 2-oxoglutaramate production in GPT-overexpressing E. coli cells, as described in Example 20, supra.

Results:

Expression of the Zebra fish GPT coding sequence of SEQ ID NO: 11 resulted in the over-expression of recombinant GPT protein having 2-oxoglutaramate synthesis-catalyzing bioactivity. Specifically, 28.6 nanomoles of 2-oxoglutaramate activity was observed in the E. coli cells overexpressing the recombinant Zebra fish GPT, compared to only 0.02 nanomoles of 2-oxoglutaramate activity in control E. coli cells, a more than 1,400-fold activity level increase over control.

EXAMPLE 9: GENERATION AND EXPRESSION OF RECOMBINANT TRUNCATED ARABIDOPSIS GPT GENE CODING SEQUENCES AND 0 ANALYSIS OF BIOLOGICAL ACTIVITY

In this example, two different truncations of the Arabidopsis GPT coding sequence were designed and expressed in E. coli, in order to evaluate the activity of GPT proteins in which the putative chloroplast signal peptide is absent or truncated. Recombinant truncated GPT proteins corresponding to the full length Arabidopsis GPT amino acid sequence SEQ ID NO: 1, truncated to delete either the first 30 amino-terminal amino acid residues, or the first 45 amino-terminal amino acid residues, were successfully expressed and showed biological activity in catalyzing the increased synthesis of 2-oxoglutaramate, as confirmed by NMR.

Materials and Methods:

Truncated Arabidopsis GPT coding sequences and expression in E. coli:

The DNA coding sequence of a truncation of the Arabidopsis thaliana GPT coding sequence of SEQ ID NO: 2 was designed, synthesized, inserted into a PET28 vector, and expressed in E. coli. The DNA sequence of the truncated Arabidopsis GPT coding sequence used in this example is provided in SEQ ID NO: 15 (-45 AA construct), and the corresponding truncated GPT protein amino acid sequence is provided in SEQ ID NO: 16. Briefly, E. coli cells were transformed with the expression vector and transformants grown overnight in LB broth diluted and grown to OD 0.4, expression induced with isopropyl-p-D-thiogalactoside (0.4 micromolar), grown for 3 hr and harvested. A total of 25 X 106 cells were then assayed for biological activity using HPLC as described in

Example 20. Untransformed, wild type £ coli cells were assayed as a control. An additional control used £ coli cells transformed with an empty vector.

Expression of the truncated -45 Arabidopsis GPT coding sequence of SEQ ID NO: 15 resulted in the over-expression of biologically active recombinant GPT protein (2-oxoglutaramate synthesis-catalyzing bioactivity). Specifically, 16.1 nanomoles of 2-oxoglutaramate activity was observed in the £ coli cells overexpressing the truncated -45 GPT, compared to only 0.02 nanomoles of 2-oxoglutaramate activity in control £ coli cells, a more than 800-fold activity level increase over control. For comparison, the full length Arabidopsis gene coding sequence expressed in the same £ coli assay generated 2.8 nanomoles of 2-oxoglutaramate activity, or roughly less than one-fifth the activity observed from the truncated recombinant GPT protein.

All publications, patents, and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference.

The present invention is not to be limited in scope by the embodiments disclosed herein, which are intended as single illustrations of individual aspects of the invention, and any which are functionally equivalent are within the scope of the invention. Various modifications to the models and methods of the invention, in addition to those described herein, will become apparent to those skilled in the art from the foregoing description and teachings, and are similarly intended to fall within the scope of the invention. Such modifications or other embodiments can be practiced without departing from the true scope and spirit of the invention. TABLE OF SEQUENCES: SEQ ID NO: 1 Arabidopsis glutamine phenylpyruvate transaminase DNA coding sequence:

AT GT ACCT GG ACATAAAT G GTGT GAT GAT CAAACAGTTT AG CTT CAAAGCCT C T CTT CTCCCATT CT CTT CT AATTT CCG AC AAAGCTCCGCC AAAAT CCAT CGTC CT AT CGG AGCCACCAT GACCACAGTTT CGACT CAGAACG AGT CT ACT CAAAA ACCCGT CCAGGTGGCGAAGAGATTAGAGAAGTTCAAGACTACTATTTT CACT CAAATGAGCATATTGGCAGTTAAACATGGAGCGATCAATTTAGGCCAAGGCT TT CCCAATTT CGACGGT CCT GATTTT GTT AAAGAAGCTGCG AT CCAAGCT ATT AAAG AT G GTAAAAACC AGTATG CTCGT GG ATACG G C ATT CCTC AG CT C AACT CT GCTATAGCT GCGCGGTTT CGT G AAG AT ACGGGT CTT GTT GTT GAT CCT G A G AAAG AAGTTACT GTTACAT CTG GTT GC AC AG AAG CCAT AG CTGCAG CTATG TTG G GTTTAATAAACCCT G GT GAT G AAGT C ATT CT CTTT GC ACCGTTTTATG A TT CCTAT GAAGCAACACT CT CTATGGCT GGTGCT AAAGT AAAAGG AAT CACTT TACGT CCACCGGACTT CT CCAT CCCTTTGG AAG AGCTTAAAGCTGCGGTAAC T AACAAGACT CGAGCCAT CCTTATGAACACT CCGCACAACCCGACCGGGAA GAT GTT CACTAGGGAGGAGCTT GAAACCATT GCAT CT CT CTGCATT GAAAAC GAT GT GCTT GT GTT CT CGG AT GAAGT ATACG ATAAGCTTGCGTTT G AAATGG AT CACATTT CT ATAGCTT CT CTT CCCGGTAT GT AT G AAAG AACT GT GACCAT G AATTCCCTGGGAAAGACTTTCTCTTTAACCGGATGGAAGATCGGCTGGGCGA TTGCGCCGCCT CAT CT GACTT GGGG AGTT CGACAAGCACACT CTTACCT CAC ATTCGCCACATCAACACCAGCACAATGGGCAGCCGTTGCAGCTCTCAAGGC ACC AG AGT CTT ACTT CAAAG AG CT G AAAAG AG ATT ACAAT GT GAAAAAGG AG ACT CT GGTT AAGGGTTT GAAGGAAGT CGGATTT ACAGT GTT CCCAT CGAGCG G G ACTTACTTT GTGGTTGCTG AT CACACT CCATTT G G AAT GG AG AACG AT GT TGCTTT CTGT G AGT AT CTT ATT G AAG AAGTT GGG GT CGTT GCG AT CCCAACG AGCGT CTTTTAT CT GAAT CCAGAAGAAGGGAAGAATTTGGTT AGGTTTGCGTT CT GT AAAG ACG AAG AG ACGTT G CGT GGTGCAATT GAG AG GAT G AAG CAG AA GCTT AAG AG AAAAGT CT G A SEQ ID NO: 2 Arabidopsis GPT amino acid sequence

MYLDINGVMIKQFSFKASLLPFSSNFRQSSAKIHRPIGATMTTVSTQNESTQKPV

QVAKRLEKFKTTIFTQMSILAVKHGAINLGQGFPNFDGPDFVKEAAIQAIKDGKNQ

YARGYGIPQLNSAIAARFREDTGLWDPEKEVTVTSGCTEAIAAAMLGLINPGDE

VILFAPFYDSYEATLSMAGAKVKGITLRPPDFSIPLEELKAAVTNKTRAILMNTPHN

PTGKMFTREELETIASLCIENDVLVFSDEVYDKLAFEMDHISIASLPGMYERTVTM

NSLGKTFSLTGWKIGWAIAPPHLTWGVRQAHSYLTFATSTPAQWAAVAALKAPE

SYFKELKRDYNVKKETLVKGLKEVGFTVFPSSGTYFWADHTPFGMENDVAFCE

YLIEEVGWAIPTSVFYLNPEEGKNLVRFAFCKDEETLRGAIERMKQKLKRKV SEQ ID NO: 3 Grape GPT DNA sequence

Showing Cambia 1305.1 with (3' end of) rbcS3C+Vitis (Grape). Bold ATG is the start site, parentheses are the catl intron and the underlined actagt is the spel cloning site used to splice in the Hordeum gene.

AAAAAAG AAAAAAAAAAC ATAT CTT GTTT GTC AG TAT GGGAAG TTT GAG AT AA GGACGAGTGAGGGGTTAAAATTCAGTGGCCATTGATTTTGTAATGCCAAGAA CCAC AAAAT CC AAT GGTTACC ATT CCT GTAAG AT G AGGTTT GCTAACT CTTTT T GT CCGTT AG AT AGG AAGCCTTATCACTATATATACAAGGCGTCCTAATAACC T CTT AGTAACCAATT ATTT C AG C ACCATGGT AG AT CT GAGG (GT AAATTT CTAG TTTTT CTC CTT C ΑΤΊΓΤΤ CTTG GTT AG G AC CCTTTT CT CTTTTT ATTTTTTT G AG C TTTG ATCTTT CTTT AAACT G ATCT ATTTTTTAATT GATT G GTT ATG GTGT AAAT A TTACATAGCTTTAACTGATAATCTGATTACTTTATTTCGTGTGTCTATGATGAT GAT GAT AGTT AC AG)AACCG ACGAAC7AGTAT GCAGCT CT CT CAAT GT ACCT G GACATT CCCAGAGTT GCTT AAAAGACCAG CCTTTTT AAGG AGGAGTATT GATA GTATTT CG AGTAGAAGTAGGTCCAGCTCCAAGT ATCCAT CTTT CAT GGCGTC CGCATCAACGGTCTCCGCTCCAAATACGGAGGCTGAGCAGACCCATAACCC CCCT CAACCT CT ACAGGTTGC AAAGCGCTT GG AG AAATT CAAAAC AAC AATC TTT ACTCAAAT GAGCATGCTTGCCAT CAAACATGG AGCAAT AAACCTTGGCCA AGGGTTTCCCAACTTTGAT GGTCCTGAGTTT GT CAAAGAAGCAGCAATT CAA GCCATTAAGGATGGGAAAAACCAATATGCTCGTGGATATGGAGTTCCTGATC T C AACT CTGCT GTT GCT GATAG ATT C AAG AAG GAT AC AGG ACT CGTGGTGG A CCCCG AGAAGGAAGTTACT GTT ACTT CT GGAT GTACAGAAGCAATT GCTGCT ACTATGCTAGGCTT GATAAAT CCTGGTG AT G AGGT GAT CCT CTTT GCT CCATT TTAT GATT CCTAT GAAGCCACT CTAT CC ATGGCTGGT GCCCAAATAAAAT CCA T CACTTTACGT CCT CCGG ATTTT GCT GT GCCCAT GGAT G AGCT CAAGT CT GC AAT CT C AAAG AATACCCGT GCAAT CCTTATAAACACT CCCCATAACCCC ACAG GAAAGAT GTT CACAAGGGAGG AACT G AAT GT GATT GC AT CCCT CT GCATT G A GAATGAT GTGTT GGTGTTTACT GAT GAAGTTTACGACAAGTT GGCTTTCGAAA TGG AT C AC ATTT CCAT GGCTT CT CTT CCT GGGATGTACG AGAGG ACCGT GAC T AT G AATT CCTT AGG G AAAACTTT CT CCCTG ACT G G AT GG AAGATT GGTTGG ACAGTAGCTCCCCCACACCTGACATGGGGAGTGAGGCAAGCCCACTCATTC CT CACGTTT GCT ACCTGCACCCCAAT GCAAT GGGCAGCTGCAACAGCCCTC CGGGCCCCAG ACTCTT ACT AT GAAGAGCT AAAGAGAGATTACAGT GCAAAGA AGGCAATCCTGGTGGAGGGATTGAAGGCTGTCGGTTTCAGGGTATACCCAT CAAGTGGGACCTATTTTGTGGTGGTGGATCACACCCCATTTGGGTTGAAAGA CG AT ATT GCGTTTT GT G AGT AT CTGAT C AAG G AAGTT GG GGTGGTAG CAATT CCG AC AAGCGTTTT CTACTTAC ACCCAG A AG AT G G AAAG AACCTT GT GAGGT TT ACCTT CT GT AAAG ACG AGGG AACT CT GAGAGGT GC AGTT G AAAGG AT GAA GGAG AAACT GAAGCCT AAACAATAGGGGCACGT GA SEQ ID NO: 4 Grape GPT amino acid sequence

MVDLRNRRTSMQLSQCTWTFPELLKRPAFLRRSIDSISSRSRSSSKYPSFMASA

STVSAPNTEAEQTHNPPQPLQVAKRLEKFKTTIFTQMSMLAIKHGAINLGQGFPN

FDGPEFVKEAAIQAIKDGKNQYARGYGVPDLNSAVADRFKKDTGLWDPEKEVT

VTSGCTEAIAATMLGLINPGDEVILFAPFYDSYEATLSMAGAQIKSITLRPPDFAVP

MDELKSAISKNTRAILINTPHNPTGKMFTREELNVIASLCIENDVLVFTDEVYDKLA

FEMDHISMASLPGMYERTVTMNSLGKTFSLTGWKIGWTVAPPHLTWGVRQAHS

FLTFATCTPMQWAAATALRAPDSYYEELKRDYSAKKAILVEGLKAVGFRVYPSS

GTYFVWDHTPFGLKDDIAFCEYLIKEVGWAIPTSVFYLHPEDGKNLVRFTFCKD

EGTLRAAVERMKEKLKPKQ SEQ ID NO: 5 Rice GPT DNA sequence

Rice GPT codon optimized for E. coli expression; untranslated sequences shown in lower case atgtggATGAACCTGGCAGGCTTTCTGGCMCCCCGGCAACCGCAACCGCAAC CCGT CAT G AAAT GCCGCTG AACCCGAGC AGCAGCGCGAGCTTT CT GCT GAG CAGCCT GCGT CGT AGCCT G GT GGCG AGCCT GCGT AAAGCGAGCCCGGCAG CAGCAGCAGCACTGAGCCCGATGGCAAGCGCAAGCACCGTGGCAGCAGAA AACGGTGCAGCAAAAGCAGCAGCAGAAAAACAGCAGCAGCAGCCGGTGCA GGT G GCGAAACGT CT GGAAAAATTT AAAACCACCATTTTTACCC AG AT G AGC AT GCT GGCGATT AAACATGGCGCG ATT AACCT GGGCCAGGGCTTT CC GAACTTT GAT GGCCCGGATTTT GT G AAAG AAGCGGCGATT CAGGCG ATT AAC GCGGGCAAAAACCAGT AT GCGCGT GGCTAT GGCGT GCCGGAACT GAACAGC GCGATTGCGGAACGTTTTCTGAAAGATAGCGGCCTGCAGGTGGATCCGGAA AAAGAAGT GACCGT GACCAGCGGCT GCACCGAAGCGATT GCGGCGACCATT CTGGGCCTGATTAACCCGGGCGATGAAGTGATTCTGTTTGCGCCGTTTTATG ATAGCTATGAAGCGACCCTGAGCATGGCGGGCGCGAACGTGAAAGCGATTA CCCT GCGTCCGCCGGATTTT AGCGTGCCGCT GGAAGAACTG AAAGCGGCCG T GAGCAAAAACACCCGT GCGATTAT GATTAACACCCCGCAT AACCCGACCGG CAAAAT GTTT ACCCGT G AAGAACT GG AATTT ATT GCG ACCCT GTGCAAAG AA AACGAT GT GCTGCT GTTT GCGGAT GAAGT GTAT GATAAACT GGCGTTT G AAG CGGAT CATATT AGCATGGCGAGCATT CCGGGCAT GTAT GAACGT ACCGT GAC CATGAACAGCCTGGGCAAAACCTTTAGCCT GACCGGCT GGAAAATT GGCT G GGCGATTGCGCCGCCGCATCTGACCTGGGGCGTGCGTCAGGCACATAGCTT TCTGACCTTTGCAACCTGCACCCCGATGCAGGCAGCCGCCGCAGCAGCACT GCGTGCACCGGAT AGCT ATT ATGAAGAACT GCGTCGTGATT ATGGCGCGAAA AAAGCGCTGCTGGT GAACGGCCT GAAAGAT GCGGGCTTT ATT GTGTATCCG AGC AG CG GC ACCT ATTTT GTG ATG GTGG AT CAT ACCCCGTTT G G CTTT GAT A ACGAT ATT GAATTTT GCGAAT AT CT GATTCGT GAAGTGGGCGTGGT GGCG AT T CCGCCGAGCGT GTTTT ATCTGAACCCGG AAG AT GGCAAAAACCT GGT GCG TTTTACCTTTTGCAAAGATGAT GAAACCCTGCGT GCGGCGGT GGAACGTAT G AAAACCAAACT GCGT AAAAAAAAGCTT gcggccgcactcgagcaccaccaccaccaccactg a SEQ ID NO: 6 Rice GPT amino acid sequence

Includes amino terminal amino acids MW for cloning and His tag sequences from pet28 vector in italics.

/WIVMNLAGFLATPATATATRHEMPLNPSSSASFLLSSLRRSLVASLRKASPAAAA

ALSPMASASTVAAENGAAKAAAEKQQQQPVQVAKRLEKFKTTIFTQMSMLAIKH

GAINLGQGFPNFDGPDFVKEAAIQAINAGKNQYARGYGVPELNSAIAERFLKDSG

LQVDPEKEVTVTSGCTEAIAATILGLINPGDEVILFAPFYDSYEATLSMAGANVKAI

TLRPPDFSVPLEELKAAVSKNTRAIMINTPHNPTGKMFTREELEFIATLCKENDVL

LFADEVYDKLAFEADHISMASIPGMYERTVTMNSLGKTFSLTGWKIGWAIAPPHL

TWGVRQAHSFLTFATCTPMQAAAAAALRAPDSYYEELRRDYGAKKALLVNGLK

DAGFIVYPSSGTYFVMVDHTPFGFDNDIEFCEYLIREVGVVAIPPSVFYLNPEDG

KNLVRFTFCKDDETLRAAVERMKTKLRKKKMAALEHWWHHH SEQ ID NO: 7 Soybean GPT DNA sequence TOPO 151D WITH SOYBEAN for E coli expression From starting codon. Vector sequences are italicized

AT GCATCATCACCATCACCATGGTAAGCCTATCCCTAACCCTCTCCTCGGTC TCGATTCTACGGAAAACCTGTATTTTCAGGGAATTGATCCCTTCACCGCGAAA CGTCT GGAAAAATTTCAGACCACCATTTTTACCCAGATGAGCCT GCTGGCGA TTAAACATGGCGCGATTAACCTGGGCCAGGGCTTTCCGAACTTTGATGGCCC GGAATTT GT GAAAGAAGCGGCGATT CAGGCGATTCGT GAT GGCAAAAACCA GTAT GCGCGT GGCTAT GGCGT GCCGGAT CT GAACATTGCGATT GCGG AACG TTTT AAAAAAG AT ACCGGCCT GGT GGT GGAT CCGGAAAAAG AAATT ACCGT G ACCAGCGGCT GCACCGAAGCGATT GCGGCGACCAT GATT GGCCT GATTAAC CCGGGCG AT G AAGT GATTAT GTTT GCGCCGTTTTAT GATAGCTAT GAAGCG A CCCT GAGCATGGCGGGCGCG AAAGT GAAAGGCATTACCCT GCGT CCGCCG GATTTTGCGGT GCCGCT GG AAG AACT GAAAAGC ACC ATT AGCAAAAACACCC GT GCG ATT CT GATTAACACCCCGCAT AACCCGACCGGCAAAAT GTTT ACCCG T G AAG AACT G AACT GCATT GCGAGCCT GTGC ATT G AAAACG AT GT GCTGGT G TTT ACCGATG AAGTGT ATGATAAACT GGCGTTTGATATGGAACATATTAGCAT GGCGAGCCTGCCGGGCATGTTTGAACGTACCGTGACCCTGAACAGCCTGGG CAAAACCTTTAGCCTGACCGGCTGGAAAATTGGCTGGGCGATTGCGCCGCC GCATCTGAGCTGGGGCGTGCGTCAGGCGCATGCGTTTCTGACCTTTGCAAC CGCACAT CCGTTTC AGT GCGC AGC AGCAGCAGCACTGCGT GCACCGGAT AG CT ATT AT GTGG AACT G AAACGT GATT AT AT GGCGAAACGT GCGATTCT GATTG AAGGCCTGAAAGCGGTGGGCTTTAAAGTGTTTCCGAGCAGCGGCACCTATTT T GT GGTGGTGGATCAT ACCCCGTTT GGCCT GGAAAACGAT GT GGCGTTTT GC GAAT AT CT GGT GAAAGAAGTGGGCGTGGTGGCG ATT CCGACCAGCGTGTTT T AT CT G AACCCGGAAGAAGGC AAAAACCT GGT G CGTTTT ACCTTTT G CAAAG ATGAAGAAACCATTCGTAGCGCGGTGGAACGTATGAAAGCGAAACTGCGTAA AGTCGACTAA SEQ ID NO: 8 Soybean GPT amino acid sequence Translated protein product, vector sequences italicized

MHHHHHHGKPIPNPLLGLDSTENLYFQGIDPFTAKRLEK.FQTT\FTQMSLLA\KHG

AINLGQGFPNFDGPEFVKEAAIQAIRDGKNQYARGYGVPDLNIAIAERFKKDTGL

WDPEKEITVTSGCTEAIAATMIGLINPGDEVIMFAPFYDSYEATLSMAGAKVKGIT

LRPPDFAVPLEELKSTISKNTRAILINTPHNPTGKMFTREELNCIASLCIENDVLVF

TDEVYDKLAFDMEHISMASLPGMFERTVTLNSLGKTFSLTGWKIGWAIAPPHLS

WGVRQAHAFLTFATAHPFQCAAAAALRAPDSYYVELKRDYMAKRAILIEGLKAV

GFKVFPSSGTYFVWDHTPFGLENDVAFCEYLVKEVGWAIPTSVFYLNPEEGK

NLVRFTFCKDEETIRSAVERMKAKLRKVD SEQ ID NO: 9 Barley GPT DNA sequence Coding sequence from start with intron removed

A TGGT AGAT CT G AGGAACCGACGAA CTAG ΓΑΤ GGCAT CCGCCCCCGCCT CC GCCTCCGCGGCCCTCTCCACCGCCGCCCCCGCCGACAACGGGGCCGCCAA GCCCACGGAGCAGCGGCCGGT ACAGGT GGCT AAGCG ATT GGAGAAGTT CA

AAACAACAATTTTCACACAGATGAGCATGCTCGCAGTGAAGCATGGAGCAAT AAACCTT GGACAGGGGTTTCCCAATTTT GATGGCCCT GACTTTGT CAAAGAT GCT GCTATT GAGGCT AT CAAAGCT GGAAAGAAT CAGTATGCAAGAGGATAT G GT GTG CCT G AATT G AACT C AG CT GTT GCT G AG AG ATTT CT C AAGG AC AGT GG ATT GCACAT CGAT CCTGAT AAGG AAGTTACT GTTACAT CTGGGT GC ACAG AA GCAATAGCT GCAACGAT ATT GGGT CT GAT CAACCCT GGGGAT GAAGT CAT AC T GTTT GCT CCATTCTAT GATT CTTAT GAGGCT ACACT GT CCAT GGCT GGT GCG AATGTCAAAGCCATTACACTCCGCCCTCCGGACTTTGCAGTCCCTCTTGAAG AGCT AAAGGCT GCAGT CT CGAAGAAT ACC AGAGC AATAAT GATTAAT ACACC TCACAACCCTACCGGGAAAATGTTCACAAGGGAGGAACTTGAGTTCATTGCT GAT CT CT GCAAGGAAAAT G ACGT GTT GCT CTTTGCCGATGAGGT CTACG AC A AGCTGGCGTTT GAGGCGGAT CACATAT CAAT GGCTT CTATT CCT GGCAT GTA T GAGAGGACCGTCACT AT GAACTCCCT GGGGAAGACGTT CT CCTTGACCGG ATGGAAGATCGGCT GGGCGAT AGCACCACCGCACCT GACAT GGGGCGTAAG GCAGGCACACT CCTTCCT CACATT CGCCACCT CCACGCCGAT GCAAT C AGCA GCGGCGGCGGCCCTGAGAGCACCGGACAGCTACTTTGAGGAGCTGAAGAG GGACTACGGCGCAAAGAAAGCGCTGCTGGTGGACGGGCTCAAGGCGGCGG GCTT CAT CGT CT ACCCTT CGAGCGGAACCT ACTT CAT CAT GGT CGACCACAC CCCGTT CGGGTT CG ACAACG ACGT CG AGTT CT GCG AGT ACTT GATCCGCGA GGTCGGCGT CGT GGCCAT CCCGCCAAGCGTGTT CT ACCT GAACCCGG AGGA CGGGAAGAACCTGGTGAGGTTCACCTTCTGCAAGGACGACGACACGCTAAG GGCGGCGGTGGACAGGAT GAAGGCCAAGCT CAGGAAGAAAT GA SEQ ID NO: 10 Barley GPT amino acid sequence Translated sequence from start site (intron removed)

MVDLRNRRTSMASAPASASAALSTAAPADNGAAKPTEQRPVQVAKRLEKFKTTI

FTQMSMLAVKHGAINLGQGFPNFDGPDFVKDAAIEAIKAGKNQYARGYGVPELN

SAVAERFLKDSGLHIDPDKEVTVTSGCTEAIAATILGLINPGDEVILFAPFYDSYEA

TLSMAGANVKAITLRPPDFAVPLEELKAAVSKNTRAIMINTPHNPTGKMFTREEL

EFIADLCKENDVLLFADEVYDKLAFEADHISMASIPGMYERTVTMNSLGKTFSLT

GWKIGWAIAPPHLTWGVRQAHSFLTFATSTPMQSAAAAALRAPDSYFEELKRDY

GAKKALLVDGLKAAGFIVYPSSGTYFIMVDHTPFGFDNDVEFCEYLIREVGWAIP PSVFYLNPEDGKNLVRFTFCKDDDTLRAAVDRMKAKLRKK ’ SEQ ID NO: 11 Zebra fish GPT DNA sequence

Danio rerio sequence designed for expression in E coli. Bold, italicized nucleotides added for cloning or from pET28b vector.

A TGTCCGTGGCGAAACGT CT GGAAAAATTT AAAACCACCATTTTTACCCAG AT GAGCATGCTGGCGATTAAACATGGCGCGATTAACCTGGGCCAGGGCTTTCC GAACTTTGATGGCCCGGATTTTGTGAAAGAAGCGGCGATTCAGGCGATTCGT GAT GGCAACAACC AGT AT GCGCGTGGCTATGGCGT GCCGG ATCT GAACATT GCGATTAGCGAACGTTATAAAAAAGATACCGGCCTGGCGGTGGATCCGGAA AAAGAAATTACCGT G ACCAGCGGCT GCACCGAAGCGATT GCGGCGACCGT G CTGGGCCTGATTAACCCGGGCGATGAAGTGATTGTGTTTGCGCCGTTTTATG AT AGCTAT G AAGCG ACCCT G AGCAT GGCGGGCGCGAAAGT GAAAGGCATT A CCCT GCGT CCGCCGGATTTTGCGCT GCCG ATT GAAG AACT GAAAAGC ACC A TTAGCAAAAACACCCGTGCGATTCTGCTGAACACCCCGCATAACCCGACCG

GCAAAAT GTTT ACCCCGGAAGAACT GAACACCATT GCG AGCCT GT GCATT GA

AAACG AT GT G CTGGT GTTTAG CG AT G AAGT GTAT GATAAACT GG CGTTT GAT

ATGGAACATATTAGCATTGCGAGCCTGCCGGGCATGTTTGAACGTACCGTGA

CCATGAACAGCCTGGGCAAAACCTTTAGCCTGACCGGCTGGAAAATTGGCT

GGGCGATTGCGCCGCCGCATCTGACCTGGGGCGTGCGTCAGGCGCATGCG

TTTCTGACCTTTGCAACCAGCAACCCGATGCAGTGGGCAGCAGCAGTGGCA

CT GCGT GCACCGG AT AGCTATTATACCGAACT G AAACGT G ATTATATGGCG A

AACGTAGCATTCTGGTGGAAGGCCTGAAAGCGGTGGGCTTTAAAGTGTTTCC

G AGCAGCGGCACCTΑΓΠΤ GT GGTGGT GGAT CATACCCCGTTT GGCCAT GAA

AACGAT ATT GCGTTTTGCGAATAT CTGGTGAAAGAAGTGGGCGT GGT GGCGA

TTCCGACCAGCGTGTTTTATCTGAACCCGGAAGAAGGCAAAAACCTGGTGCG

TTTTACCTTTTGCAAAGATGAAGGCACCCTGCGTGCGGCGGTGGATCGTATG

AAAGAAAAACTGCGTAAAGTCGACAAGCTTGCGGCCGCACTCGAGCACCAC

CACCACCACCACTGA SEQ ID NO: 12 Zebra fish GPT amino acid sequence

Amino acid sequence of Danio rerio cloned and expressed in E. coli (bold, italicized amino acids are added from vector/ cloning and His tag on C-terminus)

MSVAKRLEKFKTTIFTQMSMLAIKHGAINLGQGFPNFDGPDFVKEAAIQAIRDGN

NQYARGYGVPDLNIAISERYKKDTGLAVDPEKEITVTSGCTEAIAATVLGLINPGD

EVIVFAPFYDSYEATLSMAGAKVKGITLRPPDFALPIEELKSTISKNTRAILLNTPH

NPTGKMFTPEELNTIASLCIENDVLVFSDEVYDKLAFDMEHISIASLPGMFERTVT

MNSLGKTFSLTGWKIGWAIAPPHLTWGVRQAHAFLTFATSNPMQWAAAVALRA

PDSYYTELKRDYMAKRSILVEGLKAVGFKVFPSSGTYFVWDHTPFGHENDIAFC

EYLVKEVGWAIPTSVFYLNPEEGKNLVRFTFCKDEGTLRAAVDRMKEKLRKVD KLAAALEHHHHHH- SEQ ID NO: 13 Arabidopsis truncated GPT -30 construct DNA sequence Arabidopsis GPT with 30 amino acids removed from the targeting sequence.

ATGGCCAAAATCC AT CGTCCT AT CGG AGCCACCAT GACCACAGTTT CG ACT C AG AACG AGT CT ACT CAAAAACCCGT CCAGGT GGCG AAG AG ATTAGAG AAGTT CAAGACTACTATIITCACT CAAAT GAGCATATT GGCAGTTAAACAT GGAGCGA TCAATTTAGGCCAAGGCTTT CCCAATTT CGACGGT CCT GATTTT GTT AAAGAA GCTGCG AT CCAAG CTATTAAAG AT GGTAAAAACC AGTATG CTCGT GGATACG GCATT CCT CAGCT CAACT CTGCTATAGCT GCGCGGTTT CGT GAAGATACGGG T CTT GTT GTT G ATCCT GAG AAAG AAGTTACT GTTAC AT CTG GTTG C ACAG AAG CCAT AGCT GCAGCTAT GTT GGGTTTAATAAACCCT GGT GAT GAAGT CATT CT C TTT GCACCGTTTTAT GATT CCTAT GAAGCAACACT CT CTATGGCTGGTGCTAA AGTAAAAGGAAT CACTTTACGTCCACCGGACTT CT CCAT CCCTTTGGAAG AG CTT AAAGCT GCGGT AACTAACAAG ACT CG AG CCAT CCTT AT G AACACT CCGC ACAACCCGACCGGGAAG AT GTT CACT AGGGAGG AGCTT GAAACCATTGCAT CT CT CT GCATT GAAAACG AT GT GCTT GTGTTCTCG G AT G AAGTAT ACG AT AAG CTT GCGTTT G AAATGGAT CACATTT CT AT AGCTT CT CTT CCCGGTATGTATG A AAGAACT GT GACCAT G AATT CCCT GGGAAAGACTTT CTCTTT AACCGGATGG AAGATCGGCTGGGCGATTGCGCCGCCTCATCTGACTTGGGGAGTTCGACAA GCACACT CTT ACCTCACATT CGCCACATCAACACCAGCACAATGGGCAGCCG

TT GCAGCT CTCAAGGCACCAGAGTCTT ACTT CAAAGAGCTGAAAAGAG ATT A CAATGTGAAAAAGGAGACTCTGGTTAAGGGTTTGAAGGAAGTCGGATTTACA GT GTT CCCAT CGAGCGGG ACTTACTTT GTGGTT GCT GAT CACACT CC ATTT G GAAT GG AGAACGAT GTTGCTTT CT GT GAGTAT CTT ATT G AAGAAGTT GGGGT CGTT GCG AT CCC AACGAGCGT CTTTTAT CT GAAT CCAGAAG AAGGG AAGAAT TT GGTT AGGTTT GCGTT CT GT AAAG ACG AAGAGACGTT GC GT GGTGCAATT G AGAGG ATG AAGCAGAAGCTT AAGAG AAAAGT CT GA SEQ ID NO: 14 Arabidopsis truncated GPT -30 construct amino acid sequence

MAKIHRPIGATMTTVSTQNESTQKPVQVAKRLEKFKTTIFTQMSILAVKHGAINLG

QGFPNFDGPDFVKEAAIQAIKDGKNQYARGYGIPQLNSAIAARFREDTGLWDPE

KEVTVTSGCTEAIAAAMLGLINPGDEVILFAPFYDSYEATLSMAGAKVKGITLRPP

DFSIPLEELKAAVTNKTRAILMNTPHNPTGKMFTREELETIASLCIENDVLVFSDEV

YDKLAFEMDHISIASLPGMYERTVTMNSLGKTFSLTGWKIGWAIAPPHLTWGVR

QAHSYLTFATSTPAQWAAVAALKAPESYFKELKRDYNVKKETLVKGLKEVGFTV

FPSSGTYFWADHTPFGMENDVAFCEYLIEEVGWAIPTSVFYLNPEEGKNLVRF

AFCKDEETLRGAIERMKQKLKRKV SEQ ID NO: 15: Arabidopsis truncated GPT -45 construct DNA sequence Arabidopsis GPT with 45 residues in the targeting sequence removed,

ATGGCGACT CAGAACGAGT CTACTCAAAAACCCGT CCAGGTGGCGAAGAGA TTAGAGAAGTT CAAGACT ACTATTTT C ACTCAAAT GAGCAT ATT GGC AGTT AA ACATGGAGCGATCAATTTAGGCCAAGGCTTTCCCAATTTCGACGGTCCTGAT TTTGTTAAAGAAGCTGCGATCCAAGCTATTAAAGATGGTAAAAACCAGTATGC T CGT GG ATACGGCATTCCT CAGCT CAACT CTGCTATAGCT GCGCGGTTT CGT G AAGATACGGGT CTTGTTGTT GAT CCT GAG AAAG AAGTT ACT GTTAC ATCTG GTTGCACAGAAGCCATAGCTGCAGCTATGTTGGGTTTAATAAACCCTGGTGA T G AAGT CATT CT CTTT GC AC CGTTTTAT GATT CCTAT G AAGC AAC ACT CTCTAT GGCTGGT GCTAAAGT AAAAGGAAT C ACTTT ACGT CCACCGG ACTT CT C CAT C CCTTTGGAAGAGCTTAAAGCTGCGGTAACTAACAAGACTCGAGCCATCCTTA TGAACACTCCGCACAACCCGACCGGGAAGATGTTCACTAGGGAGGAGCTTG AAACCATTGCATCTCTCTGCATT GAAAACGATGTGCTTGTGTTCTCGGAT GAA GTATACGAT AAGCTT GCGTTT G AAATGG AT CACATTT CT ATAGCTT CT CTT CC CGGTATGTAT GAAAG AACTGT GACCAT GAATT CCCT GGGAAAG ACTTT CT CTT TAACCGGATGGAAGATCGGCTGGGCGATTGCGCCGCCTCATCTGACTTGGG G AGTT CG AC AAGC AC ACT CTT ACCT CACATT CGCCACAT C AAC ACC AGC AC A

Atgggcagccgttgcagctctcaaggcaccagagtcttacttcaaagagct

G AAAAGAG ATT AC AAT GT GAAAAAGG AGACT CTGGTTAAG GGTTT GAAGG AA GTCGGATTTACAGTGTTCCCATCGAGCGGGACTTACTTTGTGGTTGCTGATC ACACTCCATTT GG AAT GGAG AACGAT GTT GCTTT CT GT GAGT AT CTT ATT GAA GAAGTT GGGGT CGTTGCGATCCCAACGAGCGT CTTTTAT CTG AATCCAGAAG AAGGGAAG AATTTGGTTAGGTTT GCGTT CT GTAAAGACGAAGAGACGTT GCG TGGT GCAATT GAGAGGAT GAAGCAGAAGCTT AAGAGAAAAGTCT G A SEQ ID NO: 16: Arabidopsis truncated GPT -45 construct amino acid sequence

MATQNESTQKPVQVAKRLEKFKTTIFTQMSILAVKHGAINLGQGFPNFDGPDFVK

EAAIQAIKDGKNQYARGYGIPQLNSAIAARFREDTGLWDPEKEVTVTSGCTEAIA

AAMLGLINPGDEVILFAPFYDSYEATLSMAGAKVKGITLRPPDFSIPLEELKAAVT

NKTRAILMNTPHNPTGKMFTREELETIASLCIENDVLVFSDEVYDKLAFEMDHISI

ASLPGMYERTVTMNSLGKTFSLTGWKIGWAIAPPHLTWGVRQAHSYLTFATSTP

AQWAAVAALKAPESYFKELKRDYNVKKETLVKGLKEVGFTVFPSSGTYFWADH

TPFGMENDVAFCEYLIEEVGWAIPTSVFYLNPEEGKNLVRFAFCKDEETLRGAI

ERMKQKLKRKV SEQ ID NO: 17: Tomato Rubisco promoter TOMATO RuBisCo rbcS3C promoter sequence from Kpnl to Ncol

GGTA CCGTTT G AAT CCT CCTTAAAGTTTTT CT CT GG AG AAACTGTAGTAATTT T ACTTT GTT GT GTT CCCTT CAT CTTTT G AATT AATGGCATTT GTTTT AATACT AA TCT GCTT CT G AAACTT GTAAT GT AT GT AT AT C AGTTT CTT AT AATTT AT CC AAG T AAT AT CTT CCATT CTCTAT GCAATT GCCTGCAT AAGCT CGACAAAAGAGT AC AT CAACCCCT CCT CCTCT GG ACT ACT CT AGCTAAACTT G AATTTCCCCTT AAG ATTAT G AAATT GATATATCCTTAAC AAACG ACT CCTT CT GTT GG AAAAT GT AGT ACTT GT CTTT CTT CTTTT GGGTATATAT AGTTTATATAC ACC AT ACT AT GT AC A AC AT CC AAGTAG AGT G AAATGG AT AC AT GTACAAG ACTT ATTT GATT GATT G A T G ACTT G AGTT GCCTT AGG AGTAAC AAATTCTTAGGT CAATAAAT CGTT GATT T GAAATT AAT CT CT CT GT CTT AG ACAGAT AGGAATT AT G ACTT CCAAT GGT CC AGAAAGCAAAGTTCGCACTGAGGGTATACTTGGAATTGAGACTTGCACAGGT CCAG AAACCAAAGTT CCCAT CG AGCT CT AAAAT C AC AT CTTTG GAAT GAAATT CAATT AG AG AT AAGTTGCTT CAT AGCATAGGT AAAAT G G AAG AT GT GAAGTAA CCT GCAAT AAT CAGT GAAAT GACATTAAT ACACTAAAT ACTT CATAT GT AATT A T CCTTTCC AGGTT AAC AAT ACT CT ATAAAGTAAG AATT AT C AG AAAT GG G CTC AT CAAACTTTT GT ACT ATGTATTT CAT AT AAGG AAGTAT AACT AT AC AT AAGTG T AT AC AC AACTTT ATT CCTATTTT GTAAAGGT GGAG AGACT GTTTT CG ATG GA T CTAAAG CAATATGTCT AT AAAAT G C ATT GATAT AAT AATTAT CT GAG AAAAT C CAGAATT GGCGTTGGATT ATTT C AGCCAAAT AGAAGTTT GT ACCAT ACTT GTT GATT CCTT CT AAGTT AAGGT GAAGTATCATT CAT AAACAGTTTTCCCCAAAGT ACT ACT C ACCAAGTTT CCCTTT GT AG AATTAAC AGTT C AAAT ATATG G CG C AG AAATT ACT CT AT GCCCAAAACCAAACG AG AAAG AAACAAAAT ACAGGGGTT G CAGACTTT ATTTTCGT GTT AGGGTGT GTTTTTT CAT GT AATTAAT CAAAAAATA TT AT GACAAAAACATTT AT AC AT ATTTTT ACT CAACACT CT GGGTAT CAGGGTG GGTT GT GTTCG ACAAT CAAT AT GGAAAGG AAGTATTTT CCTTATTTTTTT AGTT AAT ATTTT C AGTT AT ACC AAAC AT ACCTTGTG AT ATT ATTTTT AAAAAT G AAAAA CTCGT C AG AAAG AAAAAGCAAAAGCAAC AAAAAAATT GCAAGT ATTTTTT AAA AAAG AAAAAAAAAACAT ATCTT GTTT GT CAGTATGGGAAGTTT GAG AT AAGG A CGAGT G AGGGGTT AAAATT CAGT GGCCATT G ATTTT GT AATGCCAAGAACC A C AAAAT CCAAT GGTT ACC ATT CCT GTAAG AT G AGGTTT GCT AACT CTTTTT GT CCGTT AGAT AGG AAGCCTTAT CACTATAT AT AC AAGGCGT CCT AAT AACCT CT T AGTAACCAATT ATTT CAGCACCA TGG SEQ ID NO: 18: Bamboo GPT DNA sequence

AT GGCCT CCGCGGCCGT CT CCACCGTCGCCACCGCCGCCGACGGCGT CGC GAAGCCG ACGGAGAAGCAGCCGGT ACAGGT CGCAAAGCGTTT GGAAAAGTT T AAG ACAAC AATTTT C ACACAG AT G AGC AT GCTT GCCAT CAAGCATGG AGCA AT AAACCT CG GCCAGGGCTTT CCGAATTTT GATGGCCCT G ACTTT GT GAAAG AAGCTGCTATTCAAGCTATCAATGCTGGGAAGAATCAGTATGCAAGAGGATA T GGT GTGCCT G AACT GAACTCGGCT GTT GCT G AAAGGTTCCT GAAGGACAGT GGCTTGCAAGTCG AT CCCGAG AAGGAAGTTACT GTCACATCTGGGT GCACG GAAGCGATAGCTGCAACGATATTGGGTCTTATCAACCCTGGCGATGAAGTGA T CTT GTTTGCT CCATT CT AT GATTCAT ACGAGGCT ACGCTGT CG AT GGCTGGT GCCAATGTAAAAGCC ATTACT CTCCGT CCT CCAGATTTTGCAGTCCCT CTT GA GG AGCT AAAGGCCACAGTCT CTAAGAACACCAG AGCGAT AATGAT AAACACA CCACACAATCCTACT GGGAAAATGTTTT CTAGGGAAGAACTTGAATT CATT GC T ACT CT CTGCAAG AAAAAT GAT GTGTT GCTTTTT G CT GAT G AGGTCTAT G AC A AGTT GG C ATTT G AGGC AG AT CAT AT AT C AAT GGCTT CT ATTCCT GGCAT GTAT GAG AGG ACT GT GACT AT G AACT CTCT GGGG AAGACATT CTCTCT AAC AGG AT GGAAGATCGGTTGGGCAATAGCACCACCACACCTGACATGGGGTGTAAGGC AGGCACACT CATT CCTCACATTT GCCACCT GCACACCAAT GCAATCGGCGGC GGCGGCGGCTCTTAGAGCACCAGATAGCTACTATGGGGAGCTGAAGAGGGA TTACGGTGCAAAG AAAGCGATACT AGT CGACGGACT CAAGGCT GCAGGTTTT ATTGTTTACCCTTCAAGTGGAACATACTTTGTCATGGTCGATCACACCCCGTT T GGTTT CGACAAT GAT ATT G AGTT CTGCGAGTATTT GAT CCGCG AAGT CGGT GTT GT CGCC AT ACCACCAAGCGTATTTTAT CTCAACCCT G AGG ATGGGAAG A ACTT GGT GAGGTT CACCTT CT GCAAGGAT GAT GATACGCT G AGAGCCGCAGT T GAGAGGAT GAAGACAAAGCT CAGGAAAAAAT GA SEQ ID NO: 19: Bamboo GPT amino acid sequence

MASAAVSTVATAADGVAKPTEKQPVQVAKRLEKFKTTIFTQMSMLAIKHGAINLG

QGFPNFDGPDFVKEAAIQAINAGKNQYARGYGVPELNSAVAERFLKDSGLQVDP

EKEVTVTSGCTEAIAATILGLINPGDEVILFAPFYDSYEATLSMAGANVKAITLRPP

DFAVPLEELKATVSKNTRAIMINTPHNPTGKMFSREELEFIATLCKKNDVLLFADE

WDKLAFEADHISMASIPGMYERTVTMNSLGKTFSLTGWKIGWAIAPPHLTWGV

RQAHSFLTFATCTPMQSAAAAALRAPDSYYGELKRDYGAKKAILVDGLKAAGFIV

YPSSGTYFVMVDHTPFGFDNDIEFCEYLIREVGWAIPPSVFYLNPEDGKNLVRF

TFCKDDDTLRAAVERM KTKLRKK SEQ ID NO: 20: 1305.1+rbcS3C promoter + catl intron with rice GPT gene.

Cambial 305.1 with (3' end of) rbcS3C+rice GPT. Underlined ATG is start site, parentheses are the catl intron and the underlined actagt is the spel cloning site used to splice in the rice gene.

AAAAAAG AAAAAAAAAAC ATAT CTT GTTT GTC AGTAT GGGAAGTTT GAG AT AA GG ACG AGT GAGGGGTTAAAATT CAGT GGCCATT GATTTTGT AAT GCCAAG AA CCACAAAATCCAAT GGTT ACCATT CCTGTAAGAT GAGGTTT GCT AACTCTTTT T GT CCGTT AGATAGG AAGCCTTATCACTATATATACAAGGCGTCCTAAT AACC T CTTAGTAACCAATTATTT C AGCACCA TGGTAGAT CT GAGGf GT AAATTT CTAG TTTTT CT CCTT CATTTT CTTGGTT AGGACCCTTTT CT CTTTTTATTTTTTT G AGC

TTTG AT CTTT CTTTAAACT GAT CTATTTTTTAATT GATT GGTT AT GGTGT AAAT A TTACATAG CTTTAACT GATAAT CT GATTACTTT ATTT CGTGTGTCTAT GAT GAT GAT GAT AGTT ACAG)AACCGACG AACTAGTAT G AAT CTGGCCGGCTTTCT CG CCACGCCCGCGACCGCGACCGCGACGCGGCATGAGATGCCGTTAAATCCC TCCTCCT CCGCCT CCTTCCTCCT CT CCT CGCT CCGCCGCT CGCTCGT CGCGT CGCTCCGGAAGGCCTCGCCGGCGGCGGCCGCGGCGCTCTCCCCCATGGC CTCCGCGTCCACCGTCGCCGCCGAGAACGGCGCCGCCAAGGCGGCGGCG GAGAAGCAGCAGCAGCAGCCTGTGCAGGTTGCAAAGCGGTTGGAAAAGTTT AAG ACGACC ATTTT CACACAGAT GAGT ATGCTT GCCAT CAAGCATGGAGCAA T AAACCTT GG CC AG G GTTTT CCG AATTT CG ATGGCCCT G ACTTT GT AAAAG A GGCTGCTATTCAAGCTATCAATGCTGGGAAGAATCAGTACGCAAGAGGATAT GGTGTGCCT GAACT GAACT CAGCTATTGCT GAAAGATT CCT GAAGGACAGCG GACT GCAAGT CGAT CCGGAGAAGG AAGTT ACT GT CACATCT GGATGCACAG AAG CT AT AG CT GC AAC AATTTTAG G TCTAATTAAT CCAGGCG AT G AAGT G ATA TT GTTTGCTCCATT CT AT GATT CAT AT GAGGCTACCCT GTCAAT GGCT GGT GC CAACGT AAAAGCCATT ACT CT CCGT CCTCCAGATTTTTCAGTCCCTCTTGAAG AGCT AAAGGCT GCAGT CT CGAAGAACACCAG AGCT ATTAT GATAAACACCCC GCACAAT CCT ACT GGGAAAATGTTTACAAGGGAAGAACTTGAGTTTATTGCC ACTCTCTGCAAGGAAAATGATGTGCTGCTTTTTGCTGATGAGGTCTACGACA AGTTAGCTTTTGAGGCAGATCATATATCAATGGCTTCTATTCCTGGCATGTAT G AG AGG ACCGT G ACCAT GAACT CT CTT GGG AAGACATT CT CT CTT ACAGG AT GG AAG ATCG GTT GGGCAAT CGCACCGCCACACCT G ACAT GGGGT GT AAGGC AGGC ACACT CATT CCT CACGTTT GCGACCT GCACACCAAT GCAAGC AGCT GC AGCTGCAGCTCTGAGAGCACCAGATAGCTACTATGAGGAACTGAGGAGGGA TTATGG AGCTAAGAAGGCATT GCTAGT CAACGGACT CAAGGATGCAGGTTT C ATT GTCTAT CCTT CAAGTGG AACATACTTCGT CATGGT CGACCACACCCCATT T GGTTTCG ACAATGATATT G AGTT CTGCGAGTATTT GATT CGCGAAGT CGGT GTTGTCGCCATACCACCTAGTGTATTTTATCTCAACCCTGAGGATGGGAAGA ACTTGGT GAGGTTCACCTTTT GCAAGGATGAT GAGACGCTGAGAGCCGCGG TT G AGAGGAT G AAG ACAAAGCT CAGGAAAAAAT GA SEQ ID NO: 21: HORDEUM GPT SEQUENCE IN VECTOR Cambial 305.1 with (3’ end of) rbcS3C+hordeum IDI4. Underlined ATG is start site, parentheses are the catl intron and the underlined actagt is the spel cloning site used to splice in the hordeum gene.

AAAAAAG AAAAAAAAAAC ATAT CTT GTTT GT CAGTAT GGGAAGTTT GAG ATAA GG ACG AGT GAGGGGTTAAAATT CAGTGGCCATTGATTTT GT AATGCCAAGAA CCACAAAAT CCAATGGTTACCATT CCT GT AAGAT G AGGTTT GCT AACT CTTTT TGTCCGTTAGATAGGAAGCCTTATCACTATATATACAAGGCGTCCTAATAACC T CTT AGT AACCAATT ATTT CAGCACCA TGGT AGATCT GAGGfGT AAATTTCT AG ΤΊΓΤΤΤ CTCCTT C ATTTT CTTG GTT AG GACCCTTTT CT CTTTTT ATTTTTTT GAG C TTT GAT CTTT CTTT AAACT GAT CT ATTTTTTAATT GATT GGTT AT GGTGTAAAT A TT ACAT AGCTTT AACT GAT AAT CT GATTACTTTATTT CGTGTGTCT AT GATG AT GATGATAGTTACAG1AACCGACGAACTAGTATGGCATCCGCCCCCGCCTCCG CCT CCGCGGCCCT CTCCACCGCCGCCCCCGCCGACAACGGGGCCGCCAAG CCC ACGGAGCAGCGGCCGGT ACAGGTGGCT AAGCGATT GGAG AAGTT CAAA ACAAC AATTTT CACACAGAT GAGCATGCT CGCAGT GAAGCATGGAGCAAT AA

ACCTT GGACAGGGGTTTCCC ΑΑΤΠΤ GAT GGCCCT GACTTT GT CAAAG ATGC TGCT ATT GAGGCTAT CAAAGCT GGAAAG AAT CAGTAT G CAAG AGG AT ATGGT GTGCCT G AATT G AACT CAGCT GTT GCT G AGAG ATTT CT CAAGGACAGTGG AT T GCAC AT CGAT CCT GAT AAGG AAGTT ACTGTT AC ATCTGGGTGCAC AGAAGC AAT AGCT GCAACGAT ATTGGGTCTGAT CAACCCTGGGGAT GAAGTC AT ACT G TTT GCT CCATT CT AT GATT CTT AT GAGGCTAC ACT GTCCATGGCTGGTGC G AA T GT CAAAGCCATT ACACTCCGCCCTCCGGACTTT GCAGT CCCTCTT GAAG AG CTAAAGGCTGCAGTCTCGAAGAATACCAGAGCAATAATGATTAATACACCTC ACAACCCTACCGGGAAAATGTTCACAAGGGAGGAACTTGAGTTCATTGCTGA TCTCTG C AAGG AAAAT G ACGT GTTGCT CTTTGCCG AT G AGGT CT ACG AC AAG CTGGCGTTTGAGGCGGATCACATATCAATGGCTTCTATTCCTGGCATGTATG AGAGGACCGTCACTATGAACTCCCTGGGGAAGACGTTCTCCTTGACCGGAT GGAAGATCGGCTGGGCGATAGCACCACCGCACCTGACATGGGGCGTAAGG CAGGCACACTCCTTCCTCACATTCGCCACCTCCACGCCGATGCAATCAGCAG CGGCGGCGGCCCT GAGAGCACCGGACAGCT ACTTT GAGGAGCT GAAGAGG GACTACGGCGCAAAGAAAGCGCTGCTGGTGGACGGGCTCAAGGCGGCGGG CTT CAT CGTCT ACCCTT CGAGCGG AACCT ACTT CAT CATGGT CGACCACACC CCGTT CGGGTTCG ACAACGACGT CG AGTT CT GCGAGTACTT GAT CCGCGAG GTCGGCGTCGTGGCCATCCCGCCAAGCGTGTTCTACCTGAACCCGGAGGAC GGGAAGAACCT GGT GAGGTT CACCTTCT GCAAGGACG ACG AC ACGCT AAGG GCGGCGGTGGACAGGAT G AAGGCCAAGCT CAGG AAG AAAT GATT G AGGG G CG CACGTGTGA SEQ ID NO: 22 Cambia 1201 + Arabidopsis GPT sequence (35S promoter from CaMV in italics)

CATGGAGTCAAAGATTCAAATAGAGGAGCTAACAGAACTCGCCGTAAAGACT GGCGAACAGTTCATACAGAGTCTCTTACGACTCAATGACAAGAAGAAAATCT TCGTCAA CA TGGTG GA GCACGA CA CACTTGTC TA C TCCAAAAA TA TCAAA GA TACAGTCTCAGAAGACCAAAGGGCAATTGAGACTTTTCAACAAAGGGTAATA TCCGGAAACCTCCTCGGATTCCATTGCCCAGCTATCTGTCACTTTATTGTGAA GA TAGTGGAAAAGGAAGGTGGCTCCTACAAA TGCCATCA TTGCGA TAAAGGA AAGGCCATCGTTGAAGATGCCTCTGCCGACAGTGGTCCCAAAGATGGACCC CCACCCACGAGGAGCATCGTGGAAAAAGAAGACGTTCCAACCACGTCTTCA AAGCAAGTGGATTGATGTGATATCTCCACTGACGTAAGGGATGACGCACAAT CCCA CTATCCTTCG CAA GACCCTTCCTC TA TA TAAGGAAGTTCA TTTCA TTTG GAGAGAACACGGGGGACTCTTGACCATGJACCJGGACATAAATGGTGTGAJ GATCAAACAGTTTAGCTTCAAAGCCTCTCTTCTCCCATTCTCTTCTAATTTCC GACAAAGCT CCGCC AAAAT CCAT CGT CCT AT CGG AGCCACC AT G ACCACAGT TT CGACT CAGAACGAGTCTACTCAAAAACCCGT CCAGGTGGCGAAGAGATT A GAG AAGTT CAAG ACT ACT ATTTT CACT C AAAT G AGC AT ATT G G C AGTT AAAC A TGGAGCGATCAATTTAGGCCAAGGCTTTCCCAATTTCGACGGTCCTGATTTT GTT AAAG AAGCT GCG AT CCAAGCT ATT AAAG ATGGT AAAAACC AGT ATGCTC GTGGATACGGCATTCCTCAGCTCAACTCTGCTATAGCTGCGCGGTTTCGTGA AG AT ACGG GTCTT GTT GTT GATCCT G AG AAAGAAGTT ACTGTTAC AT CTG GTT GCACAGAAGCC AT AGCT GCAGCT AT GTT GGGTTT AAT AAACCCT GGT GAT G A AGTCATTCTCTTTGCACCGTTTTATGATTCCTATGAAGCAACACTCTCTATGG · CT GGT GCT AAAGTAAAAGG AAT CACTTTACGT CCACCGG ACTT CT CCAT CCC TTT G G AAG AGCTT AAAGCT GCGGT AACT AAC AAG ACT CG AGCC AT C CTTATG

AACACTCCGCACAACCCG ACCGGGAAGATGTT CACT AGGGAGGAGCTT GAA ACCATT GCAT CT CT CTGCATT GAAAACGAT GT GCTT GT GTT CTCGGAT GAAGT ATACG ATAAGCTT G CGTTT G AAAT GG AT C AC ATTT CT AT AG CTTCT CTTCCCG GTAT GT AT G AAAG AACT GT G ACCAT GAATT CCCT GGGAAAG ACTTT CT CTTT A ACCGGATGGAAGATCGGCTGGGCGATTGCGCCGCCTCATCTGACTTGGGGA GTT CGACAAGCACACT CTTACCTCACATT CGCCACATCAACACCAGCACAAT GGGCAGCCGTTGCAGCTCTCAAGGCACCAGAGTCTTACTTCAAAGAGCTGA AAAG AG ATT AC AAT GT G AAAAAGG AG ACT CT GGTT AAGGGTTT G AAGG AAGT CGGATTT ACAGT GTT CCC AT CG AGCG GG ACTT ACTTT GT GGTT GCTG AT C AC ACTCC ATTTGG AAT GGAG AACG AT GTT GCTTTCT GT GAGT AT CTT ATTG AAG A AGTTGGGGTCGTTGCGATCCCAACGAGCGTCTTTTATCTGAATCCAGAAGAA GGG AAG AATTT GGTTAGGTTT GCGTTCTGTAAAG ACG AAG AG ACGTTGCGT G GTGCAATT GAGAGGAT GAAGCAGAAGCTTAAGAGAAAAGT CT G A SEQ ID NO: 23 Cambia p1305.1 with (3’ end of) rbcS3C+Arabidopsis GPT. Underlined ATG is start site, parentheses are the catl intron and the underlined actagt is thespei cloning site used to splice in the Arabidopsis gene.

AAAAAAGAAAAAAAAAAC AT AT CTT GTTT GT C AGTAT GGGAAGTTT GAG AT AA GGACGAGT GAGGGGTTAAAATT CAGT GGCCATT GATTTT GTAAT GCCAAGAA CCACAAAAT CCAAT GGTT ACCATT CCT GTAAGAT G AGGTTT GCTAACT CTTTT T GT CCGTT AGAT AGG AAGCCTTAT CACTATAT AT AC AAGGCGT CCT AATAACC T CTTAGTAACCAATT ATTT CAGCACCATOGT AGAT CT G AGG(GT AAATTT CTAG ΤΤΊΓΤΤ CTCCTT C ATTTT CTTG GTTAGG ACCCTTTT CT CTTTTTATTTTTTT G AG C TTT GAT CTTT CTTT AAACT GAT CT ATTTTTT AATTG ATTGGTT ATG GTGT AAAT A TT AC ATAG CTTT AACT GAT AAT CT GATT ACTTT ATTT C GTGTGTCT ATG ATG AT GAT GAT AGTT AC AG1AACCG AC GAA CTAG TAT GTACCT G G AC AT AAATGGT GT GAT GAT CAAACAGTTTAGCTT CAAAGCCT CTCTT CT CCCATT CTCTT CT AATTT CCG AC AAAGCT CCGCCAAAATCCAT CGTCCTATCGGAGCCACCAT G ACCACA GTTT CG ACT CAGAACG AGT CTACTCAAAAACCCGT CCAGGT GGCGAAGAG AT TAG AG AAGTT CAAGACT ACT ATTTT CACT CAAAT G AGCATATTGGC AGTTAAA CATGGAGCGATCAATTTAGGCCAAGGCTTTCCCAATTTCGACGGTCCTGATT TTGTT AAAG AAGCT GCG ATCC AAGCT ATT AAAG ATGGTAAAAACCAGTAT GCT CGTGGATACGGCATTCCTCAGCTCAACTCTGCTATAGCTGCGCGGTTTCGTG AAGAT ACGGGTCTT GTT GTT G ATCCTG AGAAAGAAGTT ACT GTTACATCT GGT T GCACAGAAGCCATAGCTGCAGCTAT GTTGGGTTTAATAAACCCT GGTG ATG AAGT C ATT CTCTTT GC ACC GTTTTAT GATT CCTATGAAGC AAC ACT CTCTATG GCTGGTGCTAAAGTAAAAGGAATCACTTTACGTCCACCGGACTTCTCCATCC CTTT GG AAGAGCTT AAAGCTGCGGT AACT AACAAG ACT CGAGCCAT CCTT AT GAACACT CCGCACAACCCGACCGGGAAGAT GTTCACTAGGGAGGAGCTT GA AACCATTGCATCTCTCTGCATTGAAAACGATGTGCTTGTGTTCTCGGATGAAG TATACG ATAAGCTT GCGTTT GAAAT GGAT CACATTT CTATAGCTT CT CTT CCC GGTATGTAT GAAAGAACT GTG ACC AT GAATT CCCTGGGAAAGACTTT CT CTTT AACCGGATGGAAGATCGGCTGGGCGATTGCGCCGCCTCATCTGACTTGGGG AGTTCGACAAGCACACTCTT ACCT CACATT CGCCACAT CAAC ACCAGC ACAA T GGGCAGCCGTT GC AGCT CTCAAGGC ACCAG AGT CTTACTT CAAAGAGCT G AAAAG AGATT ACAAT GT GAAAAAGGAGACTCT GGTTAAGGGTTT GAAGG AAG T CGGATTT AC AGTGTTCCCATCG AGCGGGACTTACTTT GTGGTT GCT GAT CA C ACTCC ATTT G G AAT GGAG AACG AT GTT GCTTT CT GT GAGT AT CTT ATT G AAG

AAGTT GGGGTCGTT GCGAT CCC AACGAGCGT CTTTT AT CT G AAT CCAGAAG A AGGG AAG AATTT GGTTAGGTTT GCGTT CTGTAAAG ACGAAGAG ACGTT GCGT GGTGCAATT G AGAGG AT G AAGCAG AAGCTTAAGAGAAAAGT CT GA SEQ ID NO: 24 Arabidpsis GPT coding sequence (mature protein, no targeting sequence)

GT GGCGAAGAG ATT AGAG AAGTT C AAG ACT ACT ATTTT CACT C AAAT GAGCAT ATT G GC AGTT AAAC AT GGAGCG AT C AATTTAGGCC AAGG CTTT CCCAATTT C G ACGGT CCT GATTTT GTT AAAGAAG CTGCGAT CC AAG CT ATT AAAG AT G GTA AAAACCAGT AT GCT CGTGGATACGGCATTCCTCAGCTCAACTCT GCT AT AGC TGCGCGGTTTCGTGAAGATACGGGTCTTGTTGTTGATCCTGAGAAAGAAGTT ACT GTTACAT CT GGTTGCACAG AAGCCATAGCT GCAGCTAT GTT GG GTTT AA TAAACCCTGGT GAT GAAGTCATTCT CTTT GC ACCGTTTTAT GATT CCTAT GAA GCAAC ACTCT CTAT GGCT GGT GCTAAAGTAAAAGG AAT CACTTTACGT CCAC CGG ACTT CT CCATCCCTTT GGAAGAGCTTAAAGCT GCGGTAACTAACAAG AC TCGAGCCATCCTTAT GAACACTCCGCACAACCCGACCGGGAAGAT GTT CACT AGGG AGGAGCTT GAAACCATTGC AT CTCTCT GCATT GAAAACGAT GT GCTT G T GTT CTCGGAT GAAGTATACGATAAGCTTGCGTTT GAAAT GG ATCACATTT CT ATAGCTTCTCTT CCCGGT ATGTAT GAAAGAACT GT GACCAT GAATTCCCT GG GAAAG ACTTT CTCTTTAACCGGATGGAAGAT CGGCTGGGCGATT GCGCCGC ctcatctgacttggggagttcgacaagcacactcttacctcacattcgCcac

ATCAACACCAGCACAAT GGGCAGCCGTT GCAGCT CT CAAGGCACCAGAGT C TTACTTCAAAGAGCTGAAAAGAGATTACAATGTGAAAAAGGAGACTCTGGTTA AGGGTTTGAAGGAAGTCGGATTTACAGTGTTCCCATCGAGCGGGACTTACTT T GT GGTT GCT GAT CAC ACT CC ATTTGG AAT GGAG AACG AT GTT GCTTT CTGT GAGT AT CTT ATT G AAG AAGTT GGGGT CGTTGCG AT CCCAACGAGCGT CTTTT AT CT G AAT CCAGAAG AAGGGAAGAATTTGGTT AG GTTT GCGTT CTGT AAAG A CGAAGAGACGTT GCGT GGT GCAATT GAGAGG AT GAAGCAG AAGCTT AAG AG AAAAGTCTGA SEQ ID NO: 25 Arabidpsis GPT amino acid sequence (mature protein, no targeting sequence)

VAKRLEKFKTTIFTQMSILAVKHGAINLGQGFPNFDGPDFVKEAAIQAIKDGKNQY

ARGYGIPQLNSAIAARFREDTGLWDPEKEVTVTSGCTEAIAAAMLGLINPGDEVI

LFAPFYDSYEATLSMAGAKVKGITLRPPDFSIPLEELKAAVTNKTRAILMNTPHNP

TGKMFTREELETIASLCIENDVLVFSDEVYDKLAFEMDHISIASLPGMYERTVTMN

SLGKTFSLTGWKIGWAIAPPHLTWGVRQAHSYLTFATSTPAQWAAVAALKAPES

YFKELKRDYNVKKETLVKGLKEVGFTVFPSSGTYFWADHTPFGMENDVAFCEY

LIEEVGWAIPTSVFYLNPEEGKNLVRFAFCKDEETLRGAIERMKQKLKRKV SEQ ID NO: 26 Grape GPT amino acid sequence (mature protein, no targeting sequence)

VAKRLEKFKTTIFTQMSMLAIKHGAINLGQGFPNFDGPEFVKEAAIQAIKDGKNQY

ARGYGVPDLNSAVADRFKKDTGLWDPEKEVTVTSGCTEAIAATMLGLINPGDE

VILFAPFYDSYEATLSMAGAQIKSITLRPPDFAVPMDELKSAISKNTRAILINTPHN PTGKMFTREELNVIASLCIENDVLVFTDEVYDKLAFEMDHISMASLPGMYERTVT MNSLGKTFSLT GWK1GWTVAPPHLTWGVRQAHSFLTFAT CTPMQWAAAT ALRA PDSYYEELKRDYSAKKAILVEGLKAVGFRVYPSSGTYFVWDHTPFGLKDDIAFC EYLIKEVGWAIPTSVFYLHPEDGKNLVRFTFCKDEGTLRAAVERMKEKLKPKQ SEQ ID NO: 27 Rice GPT amino acid sequence (mature protein, no targeting sequence)

VAKRLEKFKTTIFTQMSMLAIKHGAINLGQGFPNFDGPDFVKEAAIQAINAGKNQ

YARGYGVPELNSAIAERFLKDSGLQVDPEKEVTVTSGCTEAIAATILGLINPGDEV

ILFAPFYDSYEATLSMAGANVKAITLRPPDFSVPLEELKAAVSKNTRAIMINTPHN

PTGKMFTREELEFIATLCKENDVLLFADEVYDKLAFEADHISMASIPGMYERTVT

MNSLGKTFSLTGWKIGWAIAPPHLTWGVRQAHSFLTFATCTPMQAAAAAALRAP

DSYYEELRRDYGAKKALLVNGLKDAGFIVYPSSGTYFVMVDHTPFGFDNDIEFC

EYLIREVGWAIPPSVFYLNPEDGKNLVRFTFCKDDETLRAAVERMKTKLRKK SEQ ID NO: 28 Soybean GPT amino acid sequence (-1 mature protein, no targeting sequence)

AKRLEKFQTTIFTQMSLLAIKHGAINLGQGFPNFDGPEFVKEAAIQAIRDGKNQYA

RGYGVPDLNIAIAERFKKDTGLWDPEKEITVTSGCTEAIAATMIGLINPGDEVIMF

APFYDSYEATLSMAGAKVKGITLRPPDFAVPLEELKSTISKNTRAILINTPHNPTG

KMFTREELNCIASICIENDVLVFTDEVYDKLAFDMEHISMASLPGMFERTVTLNS

LGKTFSLTGWKIGWAIAPPHLSWGVRQAHAFLTFATAHPFQCAAAAALRAPDSY

YVELKRDYMAKRAILIEGLKAVGFKVFPSSGTYFVWDHTPFGLENDVAFCEYLV

KEVGWAIPTSVFYLNPEEGKNLVRFTFCKDEETIRSAVERMKAKLRKVD SEQ ID NO: 29 Barley GPT amino acid sequence (mature protein, no targeting sequence)

VAKRLEKFKTTIFTQMSMLAVKHGAINLGQGFPNFDGPDFVKDAAIEAIKAGKNQ

YARGYGVPELNSAVAERFLKDSGLHIDPDKEVTVTSGCTEAIAATILGLINPGDEV

ILFAPFYDSYEATLSMAGANVKAITLRPPDFAVPLEELKAAVSKNTRAIMINTPHN

PTGKMFTREELEFIADLCKENDVLLFADEVYDKLAFEADHISMASIPGMYERTVT

MNSLGKTFSLTGWKIGWAIAPPHLTWGVRQAHSFLTFATSTPMQSAAAAALRAP

DSYFEELKRD YGAKKALLVDG LKAAG FI WPSSGTYFIMVDHTPFG FDN DVEFCE

YLIREVGWAIPPSVFYLNPEDGKNLVRFTFCKDDDTLRAAVDRMKAKLRKK SEQ ID NO: 30 Zebra fish GPT amino acid sequence (mature protein, no targeting sequence)

VAKRLEKFKTTIFTQMSMLAIKHGAINLGQGFPNFDGPDFVKEAAIQAIRDGNNQ

YARGYGVPDLNIAISERYKKDTGLAVDPEKEITVTSGCTEAIAATVLGLINPGDEVI

VFAPFYDSYEATLSMAGAKVKGITLRPPDFALPIEELKSTISKNTRAILLNTPHNPT

GKMFTPEELNTIASLCIENDVLVFSDEVYDKLAFDMEHISIASLPGMFERTVTMNS

LGKTFSLTGWKIGWAIAPPHLTWGVRQAHAFLTFATSNPMQWAMVALRAPDS

YYTELKRDYMAKRSILVEGLKAVGFKVFPSSGTYFWVDHTPFGHENDIAFCEYL

VKEVGWAIPTSVFYLNPEEGKNLVRFTFCKDEGTLRAAVDRMKEKLRK SEQ ID NO: 31 Bamboo GPT amino acid sequence (mature protein, no targeting sequence)

VAKRLEKFKTTIFTQMSMLAIKHGAINLGQGFPNFDGPDFVKEAAIQAINAGKNQ

YARGYGVPELNSAVAERFLKDSGLQVDPEKEVTVTSGCTEAIAATILGLINPGDE

VILFAPFYDSYEATLSMAGANVKAITLRPPDFAVPLEELKATVSKNTRAIMINTPH

NPTGKMFSREELEFIATLCKKNDVLLFADEVYDKLAFEADHISMASIPGMYERTV

TMNSLGKTFSLTGWKIGWAIAPPHLTWGVRQAHSFLTFATCTPMQSAAAAALRA

PDSYYGELKRDYGAKKAILVDGLKAAGFIWPSSGTYFVMVDHTPFGFDNDIEFC

EYLIREVGWAIPPSVFYLNPEDGKNLVRFTFCKDDDTLRAAVERMKTKLRKK

Claims (24)

1. Claims:

   1. A method for generating and selecting transgenic plants having increased production of 2-oxo-glutaramate by transforming plants with transgenes that encode enzymes in the synthesis pathway for 2-oxo-glutaramate to thereby improve growth characteristics of the transgenic plant relative to an analogous wild type or untransformed plant, comprising: (a) . generating a plurality of transgenic plants by transforming a plurality of plants with a glutamine phenylpyruvate transaminase (GPT) transgene that encodes a GPT polypeptide having GPT catalytic activity; (b) . expressing the GPT transgene in the plurality of transgenic of plants or the progeny thereof, wherein GPT polypeptides are overexpressed in at least one of the plurality of transgenic of plants or said progeny relative to an analogous wild type or untransformed plant of the same species; and (c) . selecting a transgenic plant from said plurality of transgenic plants or said progeny that has increased production of 2-oxo-glutaramate relative to a wild type or untransformed plant of the same species.
2. 2. A method for generating and selecting transgenic plants having increased production of 2-oxo-glutaramate by transforming plant cells with transgenes that encode enzymes in the synthesis pathway for 2-oxo-glutaramate to thereby improve growth characteristics of the transgenic plants relative to an analogous wild type or untransformed plant, comprising: (a) . transforming a plurality of plant cells with a glutamine phenylpyruvate transaminase (GPT) transgene that encodes a polypeptide having GPT catalytic activity; (b) . generating a plurality of transgenic plants from the transformed plant cells; (c) . expressing the GPT transgene in the plurality of transgenic plants or the progeny thereof, wherein GPT polypeptides are overexpressed in the plurality of transgenic of plants or the progeny thereof relative to an analogous wild type or untransformed plant of the same species; (d) . selecting a transgenic plant from said plurality of transgenic plants or said progeny having increased production of 2-oxo-glutaramate relative to an analogous wild type or untransformed plant of the same species.
3. 3. A method for generating transgenic plants having increased production of 2-oxo-glutaramate by transforming plants with transgenes that encode enzymes in the synthesis pathway for 2-oxo-glutaramate to thereby improve growth characteristics of the transgenic plants relative to an analogous wild type or untransformed plant, comprising: (a) . generating a transgenic plant having a glutamine phenylpyruvate transaminase (GPT) transgene, wherein the GPT transgene is linked to a plant promoter and the GPT transgene is an exogenous GPT gene from a plant; and (b) . expressing the GPT transgene in the transgenic plant or the progeny thereof, wherein the polypeptide formed by the expression of the GPT transgene has GPT catalytic activity, and wherein said transgenic plant and the progeny thereof produce more 2-oxo-glutaramate relative to an analogous wild type or untransformed plant of the same species.
4. 4. A method of for generating and selecting transgenic plants having increased production of 2-oxo-glutaramate by transforming plants with transgenes that encode enzymes in the synthesis pathway for 2-oxo-glutaramate to thereby improve growth characteristics of the transgenic plants relative to an analogous wild type or untransformed plant, comprising: (a) . generating a plurality of transgenic plants by transforming a plurality of plants with a GPT transgene; (b) . expressing the GPT transgene in the plurality of transgenic plants or the progeny thereof, wherein GPT polypeptides are overexpressed in the plurality of transgenic of plants or the progeny thereof relative to a wild type or untransformed plant of the same species; and, (c) . selecting at least one transgenic plant having an increased biomass yield relative to a wild type or untransformed plant of the same species.
5. 5. The method according to claim 4, wherein the transgenic plant displays at least one increased growth characteristic in addition to increased biomass yield, selected from the following group: leaf-to-root ratio of glutamine synthetase (GS) activity, increased GPT activity, increased leaf-to-root ratio of protein per fresh weight, increased CO2 fixation, increased stem height, increased fruit yield, increased flowering, and increased budding when compared to an analogous wild-type or untransformed plant.
6. 6. The method according to any one of claims 1-3, wherein the transgenic plant displays at least one increased growth characteristic selected from the following group: leaf-to-root ratio of glutamine synthetase (GS) activity, increased GPT activity, increased leaf-to-root ratio of protein per fresh weight, increased biomass yield, increased CO2 fixation, increased stem height, increased fruit yield, increased flowering, and increased budding when compared to an analogous wild-type or untransformed plant.
7. 7. The method according to any one of claims 1-2 and 4-6, wherein the GPT transgene is an exogenous GPT gene from a plant.
8. 8. The method according to any one of claims 1-2 and 4-7, wherein the GPT transgene includes a plant promoter.
9. 9. The method according to any one of claims 1-8, wherein the plant promoter has preferred expression in photosynthetic plant tissues.
10. 10. The method according to any one of claims 1-8, wherein the plant promoter is a CaMV 35S promoter.
11. 11. The method according to any one of claims 1-10, wherein the GPT transgene encodes a polypeptide having an amino acid sequence selected from the group consisting of: (a) . SEQ ID NO: 2, SEQ ID NO: 2 truncated at its amino terminus by between 30 to 56 amino acid residues, SEQ ID NO: 14, SEQ ID NO: 16 and SEQ ID NO: 25, and (b) . an amino acid sequence that has at least 80% sequence identity to any one of SEQ ID NO: 2, SEQ ID NO: 14, SEQ ID NO: 16, and SEQ ID NO: 25, and which has GPT catalytic activity.
12. 12. The method according to any one of claims 1-10, wherein the GPT transgene encodes a polypeptide having an amino acid sequence selected from the group consisting of: (a). SEQ ID NO: 2, SEQ ID NO: 2 truncated at its amino terminus by between 30 to 56 amino acid residues, SEQ ID NO: 14, SEQ ID NO: 16 and SEQ ID NO: 25, and (b). an amino acid sequence that has at least 90% sequence identity to any one of SEQ ID NO: 2, SEQ ID NO: 2 truncated at its amino terminus by between 30 to 56 amino acid residues, SEQ ID NO: 14, SEQ ID NO: 16 or SEQ ID NO: 25, and which has GPT catalytic activity.
13. 13. The method according to any one of claims 1-10, wherein the GPT transgene encodes a polypeptide having an amino acid sequence selected from the group consisting of: (a) SEQ ID NO: 2, SEQ ID NO: 2 truncated at its amino terminus by between 30 to 56 amino acid residues, SEQ ID NO: 14, SEQ ID NO: 16 and SEQ ID NO: 25, and (b) . an amino acid sequence that has at least 95% sequence identity to any one of SEQ ID NO: 2, SEQ ID NO: 2 truncated at its amino terminus by between 30 to 56 amino acid residues, SEQ ID NO: 14, SEQ ID NO: 16 or SEQ ID NO: 25, and which has GPT catalytic activity.
14. 14. The method according to any one of claims 1-10, wherein said GPT transgene encodes a GPT polypeptide having an amino acid sequence that has at least 80% sequence identity to SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, or SEQ ID NO: 14, and GPT catalytic activity.
15. 15. The method according to any one of claims 1-10, wherein said GPT transgene encodes a GPT polypeptide having an amino acid sequence that has at least 85% sequence identity to SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, or SEQ ID NO: 14, and GPT catalytic activity.
16. 16. The method according to any one of claims 1-10, wherein said GPT transgene encodes a GPT polypeptide having an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, or SEQ ID NO: 14, and GPT catalytic activity.
17. 17. The method according to any one of claims 1-10, wherein said GPT transgene encodes a GPT polypeptide having an amino acid sequence that has at least 95% sequence identity to SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, or SEQ ID NO: 14, and GPT catalytic activity.
18. 18. The method according to any one of claims 1-17, further comprising harvesting seeds from said transgenic plant and selecting a seed that demonstrates increased germination in high salt conditions.
19. 19. The method according to claim 18, further comprising propagating a plant from the seed so selected and harvesting a seed therefrom.
20. 20. The method according to any one of claims 1-19, wherein the GPT transgene is incorporated into the genome of the plant.
21. 21. The method according to any one of claims 1-20, wherein the plurality of transgenic plants are monocotyledonous plants.
22. 22. The method according to any one of claims 1-20, wherein the plurality of transgenic plants are dicotyledonous plants.
23. 23. A progeny of any generation of a transgenic plant generated and selected by the method of any of claims 1-22, wherein said progeny comprises said GPT transgene.
24. 24. A seed of any generation of a transgenic plant generated and selected by the method according to any one of claims 1-17 and 20-23, wherein said seed comprises said GPT transgene. Los Alamos National Security, LLC University of Maine System Board of Trustees Patent Attorneys for the Applicant/Nominated Person SPRUSON &amp; FERGUSON