AU2016202733B2 - Transgenic plants with enhanced growth characteristics - Google Patents

Abstract

TRANSGENIC PLANTS WITH ENHANCED GROWTH CHARACTERISTICS Abstract The invention relates to transgenic plants exhibiting dramatically enhanced growth rates, greater seed and fruit/pod yields, earlier and more productive flowering, more efficient nitrogen utilization, increased tolerance to high salt conditions, and increased biomass yields. Transgenic plants engineered to overexpress both glutamine phenylpyruvate transaminase (GPT), and glutamine synthetase (GS) are provided. The GPT +GS double-transgenic plants consistently exhibit enhanced growth characteristics, with TO generation lines showing an increase in biomass over wild type counterparts of between 50% and 300%. Generations that result from sexual crosses and/or selfing typically perform even better, with some of the double-transgenic plants achieving an astounding four-fold biomass increase over wild type plants.

Description

2016202733 28 Apr 2016

TRANSGENIC PLANTS WITH ENHANCED GROWTH CHARACTERISTICS

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER 5 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-AC52-06MA25396, awarded by the United States Department of Energy to Los Alamos National Security, LLC. 10 The government has certain rights in this invention.

RELATED APPLICATIONS

This application claims priority to United States Provisional Application No. 61/190.520 'filed August 29.2008. 15

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. 20 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 25 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. 30

Genetic engineering has and continues to play an increasingly important yet controversial rale 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 ere 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 substantia! and is on the rise, with approximately 250 million acres planted with transgenic plants in 2006, 2016202733 28 Apr 2016 5

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 1 {) 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 15 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 20 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 25 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 CO2 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 (RuBisGo) 30 catalyzes the combination of CO2 with ribulose bisphosphate to produce 3-phosphogiycerate, 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), 2 in a reaction catalyzed by the enzyme phosphoeno! pyruvate carboxylase. The acids are transferred to bundle sheath ceils, where they are decarboxylated to release COa, which Is then combined with ribuiese bisphosphate in the same reaction employed by C-3 plants. 2016202733 28 Apr 2016 5

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) 10 which catalyzes the combination of ammonia with glutamate to form glutamine. GS piays 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 (Miflln and Habash, 2002, Journal of Experimental Botany, Vol. 53, No. 370, pp. 979-987), GS also reassimiiates ammonia released as a result of 15 photorespiration and the breakdown of proteins and nitrogen transport compounds. GS enzymes may be divided into two general classes, one representing the cytoplasmic form (GST) and the other representing the plastidic (i.e., chioropiastic) form (GS2). 20 Previous work has demonstrated that increased expression levels of GS1 result in increased leveis 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 leveis of GS expression and GS activity in leaf tissue, increased growth under 25 nitrogen starvation, but no effect on growth under optimal nitrogen fertilization conditions (Fuentes et at., 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 leveis of GS transcript, and GS polypeptide which assembled into active enzyme, but did not report phenotypic 30 effects on growth (Temple et al., 1993, Molecular and Genera! Genetics 236: 315-325). Corruzi et al. have reported that transgenic tobacco overexpresslng a pea cytosolic GS1 transgene under the control of the CaMV S35 promoter show increased GS activity, Increased cytosolic GS protein, and improved growth 3 characteristics (U.S. Patent No, 6,107,547), Unkefer et a!, have more recently reported that transgenic tobacco plants overexpresslng the A/fa/fa GS1 in foliar tissues, which had been screened for Increased leaf-to-root GS activity following genetic segregation by seffsng to achieve increased GS1 transgene copy number, 5 were found to produce increased 2~hydroxy-5-oxopraline 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). 2016202733 28 Apr 2016 10 Unkefer et al. have further described the use of 2-hydroxy-S-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 at. disclose that Increased concentrations of 2-hydroxy-5-oxoproiine in toiler tissues (relative to root tissues) triggers a cascade of events that result in increased plant growth characteristics. 15 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-oxoproiine directly to the foliar portions of the plant and over-expressing glutamine synthetase preferentially in ieaf tissues. 20 A number of transaminase and hydrofyase 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, 25 the biochemical synthesis of 2-hydroxy-5-oxoproline has been known but has been poorly characterized. Moreover, the function of 2-hydroxy-5-oxopro!ine in plants and the significance of its poo! size (tissue concentration) are unknown. Finally, the art provides no specific guidance as to precisely what transaminase(s) or hydroiase(s) may exist and/or be active in catalyzing the synthesis of 2- 30 hydroxy-5-oxoproiine in plants, and no such plant transaminases have been reported, isolated or characterized. 4

SUMMARY OF THE INVENTION 2016202733 28 Apr 2016

The invention relates to transgenic plants exhibiting dramatically enhanced growth rates, greater seed and fruit/pod yields, earlier and more productive flowering, 5 more efficient nitrogen utilization, increased tolerance to high salt conditions, and increased biomass yields, in one embodiment, transgenic plants engineered to over-express both glutamine phenylpyruvate transaminase (GPT) and giutamine synthetase (GS) are provided, The GPT+GS doubie-transgenic plants of the invention consistently exhibit enhanced growth characteristics, with TO generation 10 Sines showing an increase in biomass over wiid type counterparts of between 50% and 300%, Generations that resuit from sexual crosses and/or seiftng typically perform even better, with some of the doubie-transgenic plants achieving an astounding four-fold biomass increase over wild type plants. Similarly, flower and fruit or pod yields are also tremendously improved, with TO generation lines 15 typically showing 50% to 70% increases over their wiid type counterparts, and in some cases showing a 100% increase. Transgenic plants exhibiting such enhanced growth phenotypic characteristics have been successfully generated across a spectrum of individual plant species, using various transformation methodologies, different expression vectors and promoters, and heteroiogous and 20 homologous transgene sequences from a variety of species, as exemplified by the numerous working examples provided herein. This invention, therefore, provides a fundamental break-though technology that has the potential to transform virtually ail areas of agriculture. 25 Applicants have identified the enzyme giutamine phenyipyruvate transaminase (GPT) as a catalyst of 2 -h y drox y-5-oxoproline (2-oxoglutaramate) synthesis in plants. 2-oxogiutaramate 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. The invention provides isolated nucleic 30 acid molecules encoding GPT, and discloses the novei finding that the encoded enzyme is directly involved in the synthesis of 2~}iydroxy-5~oxopro!ine. This aspect of the invention is exemplified herein by the disclosure of GPT polynucleotides encoding GPTS from several species, including Arabidopsis, 5

Grape, Rice, Soybean, Barley, Bamboo and a non-plant homolog from Zebra fish, most of which have been expressed as recombinant GPTs and confirmed as having GPT activity. 2016202733 07 Mar 2017

The invention further provides transgenic plants which express both a GPT transgene and a GS transgene. The expression of these two transgenes in such "doubletransgene" plants results in a substantially increased rate of carbon dioxide fixation and an extremely potent growth enhancing effect, as these plants exhibit very significantly and sometimes tremendously enhanced growth rates and flower/fruit/pod/seed yields. Methods for the generation of such growth-enhanced transgenic plants are provided.

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 and GS 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.

Herein disclosed is a transgenic plant comprising a GPT transgene and a GS transgene, wherein said GPT transgene and said GS transgene are operably linked to a plant promoter. The GS transgene may be a GS1 transgene. The GPT transgene may encode a polypeptide having an amino acid sequence selected from the group consisting of (a) SEQ ID NO: 2; SEQ ID NO: 9; SEQ ID NO: 15. SEQ IO NO: 19, SEQ ID NO: 21. SEQ ID NO: 24, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34. SEQ ID NO: 35 and SEQ ID NO: 36, and (b) an amino acid sequence that is at least 75% identical to any one of SEQ ID NO: 2; SEQ ID NO: 9; SEQ ID NO: 15, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 24, SEQ ID NO: 30, SEQ ID NO: 31. SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35 and SEQ ID NO: 36 and have GPT activity. The GS transgene may encode a polypeptide having an amino acid sequence selected form the group consisting of (a) SEQ ID NO: 4 and SEQ ID NO: 7 from residue 11, and (b) an amino acid sequence that is at least 75% identical to SEQ ID NO: 4 or SEQ ID NO: 7. 6

Thus, according to an embodiment of the present invention, there is provided a transgenic plant comprising a glutamine phenylpyruvate transaminase (GPT) transgene and a glutamine synthetase (GS) transgene, wherein each of said GPT transgene and said GS transgene is operably linked to a plant promoter, and wherein said GPT transgene encodes a polypeptide having GPT activity and wherein said GS transgene encodes a polypeptide having GS activity. 2016202733 07 Mar 2017

In some embodiments, the GPT and GS transgenes are 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 and a GS transgene, as well as a seed of any generation of the transgenic plants of the invention, wherein said seed comprises said GPT transgene and said GS 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 and/or GS activity, and/or increased levels of 2-oxoglutaramate. In some embodiments, the transgenic plants of the invention display increased nitrogen use efficiency or increased tolerance to salt or saline conditions.

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.

Thus, according to another embodiment of the present invention, there is provided a method for increasing growth characteristics of a plant relative to an analogous wild type or untransformed plant, comprising: a. introducing a glutamine phenylpyruvate transaminase (GPT) transgene into a plurality of plants, wherein the GPT transgene encodes a polypeptide having GPT catalytic activity; b. introducing a glutamine synthetase (GS transgene) into the plurality of plants or progeny thereof, wherein the GS transgene encodes a polypeptide having GS catalytic activity; 7 c. expressing the GPT transgene and the GS transgene in the plurality of plants or the progeny thereof; and 2016202733 07 Mar 2017 d. selecting a plant having an increased growth characteristic relative to an analogous wildtype or untransformed plant of the same species.

According to another embodiment of the present invention, there is provided a method for increasing growth characteristics of a plant relative to an analogous wild type or untransformed plant, comprising: a. generating a plurality of transgenic plants by: i. introducing a glutamine phenylpyruvate transaminase (GPT) transgene into a plurality of plants and introducing a glutamine synthetase (GS) transgene into the plurality of plants or progeny thereof, or ii. introducing a GS transgene into the plurality of plants and introducing a GPT transgene into the plurality of plants or progeny thereof; wherein said GPT transgene encodes a polypeptide having GPT activity and wherein said GS transgene encodes a polypeptide having GS activity; b. expressing the GS transgene and the GPT transgene in the plurality of plants or the progeny thereof; and, c. selecting a plant having an increased growth characteristic relative to an analogous wild type or untransformed plant of the same species.

According to another embodiment of the present invention, there is provided a method of increasing growth characteristics of a plant relative to an analogous wild type or untransformed plant, comprising: a. introducing a glutamine phenylpyruvate transaminase (GPT) transgene into a first plurality of plant cells and generating a first plurality of transgenic plants from said first plurality of plant cells, wherein said GPT transgene encodes a polypeptide having GPT activity; b. introducing a glutamine synthetase (GS) transgene into a second plurality of plant cells and generating a second plurality of transgenic plants from said second plurality of plant cells, wherein said GS transgene encodes a polypeptide having GS activity; c. selecting a first plant from the first plurality of transgenic plants or the progeny thereof, said plant comprising the GPT transgene; and 7a d. selecting a second plant from the second plurality of transgenic plants or the progeny thereof, said second plant comprising the GS transgene; and 2016202733 07 Mar 2017 e. crossing the first and second plants to produce a plurality of double transgenic plants, said double transgenic plants having increased production of 2-oxo-glutaramate and at least one increased growth characteristic relative to an analogous wild type or untransformed plant of the same species.

According to another embodiment of the present invention, there is provided a method of increasing growth characteristics of a plant relative to an analogous wild type or untransformed plant, comprising: a. generating a transgenic plant by: i. introducing a glutamine phenylpyruvate transaminase (GPT) transgene into a plant and introducing a glutamine synthetase (GS) transgene into the plant or progeny thereof, or ii. introducing a GS transgene into a plant and introducing a GPT transgene into the plant or progeny thereof; wherein said GPT transgene encodes a polypeptide having GPT activity and wherein said GS transgene encodes a polypeptide having GS activity; and b. expressing the GS transgene and the GPT transgene in the plant or the progeny thereof.

According to another embodiment of the present invention, there is provided a method of increasing growth characteristics of a plant relative to an analogous wild type or untransformed plant, comprising: a. introducing a glutamine phenylpyruvate transaminase (GPT) transgene into a plant, wherein the GPT transgene encodes a polypeptide having GPT catalytic activity; b. introducing a glutamine synthetase (GS transgene) into the plant or progeny thereof, wherein the GS transgene encodes a polypeptide having GS catalytic activity; and c. expressing the GPT transgene and the GS transgene in the plant or the progeny thereof.

Transgenic plants produced by methods according to the invention, as described above, as well as progeny thereof, parts thereof, and seed of any generation of said plants are also hereby provided. 7b

BRIEF DESCRIPTION OF THE DRAWINGS 2016202733 28 Apr 2016

The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. 5 FIG. 1, Nitrogen assimilation and 2~oxog!utaramate biosynthesis: schematic of metabolic pathway. FIG, 2. Photograph showing comparison of transgenic tobacco plants over-10 expressing either GS1 or GPT, compared to wild type tobacco plant. From left to right: wild type plant, Alfalfa GS1 transgene, Arabidopsis GPT transgene. See

Examples 3 and 5, infra. FIG. 3. Photograph showing comparison of transgenic Micro-Tom tomato plants 15 over-expressing either GS1 or GPT, compared to wild type tomato plant. From left to right: wild type plant, Alfalfa GS1 transgene, Arabidopsis GPT transgene.

See Examples 4 and 8, infra. FIG., 4, Photographs showing comparisons of leaf sizes between wild type and 20 GS1 or GPT transgenic tobacco plants. A: Comparison between leaves from GS1 transgenic tobacco (bottom leaf) and wild type (top leaf). S: Comparison between leaves from GPT transgenic tobacco (bottom leaf) and wild type (top leaf). 25 FIG. 5,. Photographs showing comparisons of transgenic tobacco plants generated from various crosses between GS1 and GPT transgenic tobacco fines with wild type and single transgene plants, A-C: Cross 2, 3 and 7, respectively. See Example 7, infra. 30 FIG, 6. Photographs showing comparisons of leaf sizes between wild type and crosses between GS1 and GPT transgenic tobacco plants. A: Comparison between leaves from GSXGPT Cross 3 (bottom leaf) and wild type (top leaf). S: Comparison between leaves from GSXGPT Cross 7 (bottom leaf) and vviid type (top leaf). See Example 7, infra. 8 2016202733 28 Apr 2016 FIG, 7. Photograph of transgenic pepper plant (right) and wild type control pepper plant (left), showing larger pepper fruit yield in the transgenic plant relative to the wild type control plant. See Example 8, infra. 5 FIG. 8. Transgenic bean plants compared to wild type control bean plants (several transgenic lines expressing Arabidopsis GPT and GS transgenes). Upper Left: plant heights on various days; Upper right: flower bud numbers; Lower left: flower numbers; Lower right: bean pod numbers. Wiidtype is the control, and 10 Sines 2A. 4A and 58 are all transgenic plant lines. See Example 9, infra.

FIG. 9, Photograph of transgenic bean plant (right) and wild type control bean plant (felt), showing increased growth in the transgenic plant relative to the wiid type centre! piant Transgenic line expressing Arabidopsis GPT and GS 15 transgenes. See Example 9, infra. FIG. 10. Transgenic bean plants pods, flowers and flower buds compared to wild type control bean plants (transgenic Sine expressing grape GPT and Arabidopsis GS transgenes). See Example 10, infra. 20 FIG. 11. Photograph of transgenic bean plant (right) and wiid type control bean plant (left), showing increased growth in the transgenic plant relative to the wild type control plant. Transgenic line expressing Grape GPT and Arabidopsis GS transgenes. See Example 10, infra. 25 FIG. 12. Transgenic Cowpea Line A plants compared to wiid type contra! Cowpea plants (transgenic line expressing Arabidopsis GPT and GS transgenes), showing that the transgenic plants grow faster and flower and set pods sooner than wild type control plants. (A) Relative height and longest leaf measurements as of May 30 21, (8) Relative trifolate leafs and flower buds as of June 18, (C) Relative numbers of flowers, flower buds and pea pods as of June 22. See Example 11, infra, 9 FIG. 13. Photograph of transgenic Cowpea Line A plant (right) and wild type control Cowpea plant (left), showing increased growth in the transgenic plant relative to the wild type control plant. Transgenic line expressing Arabidopsis GPT and GS transgenes. See Example 11, infra, 2016202733 28 Apr 2016 5 FIG. 14. Transgenic Cowpea Line G plants compared to wild type control Cowpea plants (transgenic fine expressing Grape GPT and Arabidopsis GS transgenes), showing that the transgenic plants grow faster and flower and set pods sooner than wild type control plants. (A) plant heights, (B) flowers and pea pod numbers, 10 (C) leaf bud and trifolate numbers. See Example 12, infra. FIG. 15. Photograph of transgenic Cowpea Line G plant (right) and wiid type controi Cowpea plant (left), showing Increased growth in the transgenic plant relative to the wild type control plant. Transgenic line expressing Grape GPT and 15 Arabidopsis GS transgenes. See Example 12, infra. FIG. 18. Photograph of transgenic Cantaloupe plant (right) and wild type controi Cantaloupe plant (left), showing increased growth in the transgenic piant relative to the wild type control piant. Transgenic line expressing Arabidopsis GPT and 20 GS transgenes. See Example 14, infra. FIG. 17. Photograph of transgenic Pumpkin plants (right) and wild type control Pumpkin plants (left), showing increased growth in the transgenic plants relative to the wild type control plants. Transgenic lines expressing Arabidopsis GPT and 25 GS transgenes. See·. Example 15, infra. FIG. 18. Photograph of transgenic Arabidopsis plants (right) and wild type control Arabidopsis plants (left), showing increased growth in the transgenic plants relative to the wild type control plants. Transgenic lines expressing Arabidopsis 30 GPT and GS transgenes. See Example 16, infra. FIG. 19. Transgenic tomato plants expressing Arabidopsis GPT and GS transgenes compared to controi tomato plants. (A) Photograph of transgenic 10 tomato plant leaves (right) vs. wild type control leaves (left) showing larger leaves in the transgenic plant. (B) Photograph of transgenic tomato plants (right) and wild type control plants (left), showing increased growth in the transgenic plants relative to the wild type control plants. See Example 17, infra. 2016202733 28 Apr 2016 5 FIG. 20. Photograph of transgenic Camelina plant (right) and wild type control Gamelina plant (left), showing increased growth in the transgenic plant relative to the wild type control plant. Transgenic line expressing Arabidopsts GPT and GS transgenes. See Example 18, infra. 10

DETAILED DESCRIPTION OF THE INVENTION

DEFINITIONS 15 Unless otherwise defined, ail 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 20 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,, 25 Molecular Cloning: A Laboratory Manual 3rd. edition (2001) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Current Protocols in Molecular Biology (Ausbef et a!., eds., John Wiley & Sons, Inc. 2001; Transgenic Plants: Methods and Protocols (Leandro Pena, ed., Humana Press, 1st edition, 2004); and, Agrohacterium Protocols (Wan, ed., Humana Press, 2nd edition, 2006). As 30 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 35 polymers thereof (“polynucleotides") in either single- or double-stranded form. 11

Unless specifically limited, the term "polynucleotide” encompasses nucleic· adds 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 5 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. Specificaily, 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 10 deoxyinosine residues (Batzer et a!,s 1991, Nucleic Acid Res, 19: 5081; Ghtsuka et ai., 1985 3, Biol. Chem. 260: 2605-2608; and Cassol et ai.t 1992; Rossoiini et at, 1994, Mol. Cell. Probes 8: 91-98), The term nucleic add is used interchangeably with gene, eDNA, and mRNA encoded by a gene, 2016202733 28 Apr 2016 15 The term ’’promoter" refers to an array of nucleic acid control sequences that direct transcription of an operabiy 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 H type promoter, a TATA element. A promoter also optionally 20 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 "operabiy linked” 25 refers to a functional linkage between a nucielc 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, 30 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 poiymers in which one or more amino acid residue is an artificial chemical mimetic 12 of a corresponding naturally occurring amino acid, as well as to naturaiiy occurring amino acid polymers and non-naturaliy occurring amino acid polymers. 2016202733 28 Apr 2016

The term “amino acid,! refers to naturally occurring and synthetic amino acids, as 5 well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino adds. Naturaiiy occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, γ-carboxygiutamate, and O-phosphoserine, Amino acid anaiogs refers to compounds that have the same basic chemical structure as 10 a naturaiiy 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, norleuctne, methionine sulfoxide, methionine methyl suifonium. Such anaiogs have modified R groups (e.g,, norleucine) or modified peptide backbones, but retain the same basic chsmicai structure as a naturaiiy occurring amino acid. Amino acid 15 mimetics refers to chemical compounds that have a structure that is different from the genera! chemical structure of an amino acid, but that functions in a manner similar to a naturaiiy occurring amino acid.

Amino acids may be referred to herein by either their commonly known three letter 20 symbols or by the one-ietter symbols recommended by the SUPAC-iUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted Single-Setter codes.

The term "plant" includes whole plants, plant organs (e.g., leaves, stems, flowers, 25 roots, etc,), seeds and plant ceils 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 gymnosperrrts. St includes plants of a variety of pfoidy levels, including polyploid, diploid, hapioid 30 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 13 gene which encodes a polypeptide involved in catalyzing the synthesis of 2-oxoglu tana mate, 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 5 encodes a ΘΡΤ protein. The term "targeting sequence” refers to the amino terminal part of a protein which directs the protein into a subceiiuiar compartment of a celi, such as a chloroplast in a plant ceil. GPT polynucleotides are further defined by their ability to hybridize under defined conditions to the GPT polynucleotides specifically disclosed herein, or to PGR products derived 10 therefrom, 2016202733 28 Apr 2016

A “GPT transgene” is a nucleic acid molecule comprising a GPT polynucleotide which is exogenous to transgenic plant, or pfant embryo, organ or seed, harboring the nucleic acid molecule* or which is exogenous to an ancestor plant, or plant 15 embryo, organ or seed thereof, of a transgenic plant harboring the GPT polynucleotide.

The terms GS polynucleotide" and “GS nucleic acid” are used interchangeably herein, and refer to a full length or partial length polynucleotide sequence of a 20 gene which encodes a giutamine synthetase protein, and includes polynucleotides containing both translated (ceding) and un-transiated sequences, as well as the complements thereof. The term ‘‘GS coding sequence” refers to the part of the gene which is transcribed and encodes a GS protein. The terms "GS1 polynucleotide" and “GS1 nucleic acid" are used interchangeably herein, and refer 25 to a full length or partial length polynucleotide sequence of a gene which encodes a giutamine synthetase isofdrm 1 protein, and includes polynucleotides containing both translated (coding) and un-transiated sequences, as well as the complements thereof. The term “GS1 coding sequence” refers to the part of the gene which is transcribed and encodes a GS1 protein, 30 A “GS transgene” is a nucleic acid molecuie comprising a GS 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 14 embryo, organ or seed thereof, of a transgenic plant harboring the GPT polynucleotide. A “GS1 transgene" is a nucleic acid molecule comprising a GS1 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 5 plant, or plant embryo, organ or seed thereof, of a transgenic plant harboring the GPT polynucleotide. 2016202733 28 Apr 2016

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

Partial length GPT polynucleotides include polynucleotide sequences encoding N-or C-terminai truncations of GPT, mature GPT (without targeting sequence) as well as sequences encoding domains of GPT. Exemplary GPT polynucleotides 15 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 20 transformed ceils 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 25 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 30 polypeptides that are either mutants of wiid 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. 15 2016202733 28 Apr 2016

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 5 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 iarge number of functionally identical nucleic acids encode any given protein. For instance, the codons GCA, GCC, GCG and GCU ait encode the amino acid alanine. Thus, at every position where 1() 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 15 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 yiefd a functionally identical molecule. Accordingly, each silent variation of a nucleic acid which encodes a polypeptide is implicit in each described sequence. 20

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 25 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. 30

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); 16 5) isoieucine (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)). 2016202733 28 Apr 2016 5 Macromoiecular 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 at, Molecular Biology of the Cell (3rd ed.t 1994) and Cantor and Sohimmel, Biophysical Chemistry Part i: The Conformation of Biological Macromoiecui&s (1980). “Primary structure” refers to the amino add 10 sequence of a particular peptide, “Secondary structure” refers to iocaiiy 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 15 as stretches of p-sheet and «-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 noncovaient association of independent tertiary units, Anisotropic terms are also known as energy terms. 20 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 25 use in another application or already in use in the new appiication/miiieu. 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 wouid interfere with diagnostic or therapeutic uses for the antibody, and may Include enzymes, hormones, and 30 other proteinaceous or non-protelnaceous solutes. In preferred embodiments, the antibody wifi 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 17 amino acid sequence by use of a spinning cup sequenator, or (3} to homogeneity by SDS-PAGE under reducing or nonreducing conditions using Coomassie biue or, preferably, stiver stain, isolated antibody includes the antibody to situ within recombinant ceiis since at ieast one component of the antibody's natural 5 environment will not be present. Ordinarily, however, isolated antibody will be prepared by at least one purification step. 2016202733 28 Apr 2016

The term “heterologous” when used with reference to portions of a nucleic acid indicates that fee nucleic acid comprises two or more subsequences that are not 10 found in the same relationship to each other in nature. For instance, a nucleic acid is typically recombinantiy 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 IS fee protein comprises two or more subsequences feat 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 adds or polypeptide sequences, refer to two or more sequences or subsequences 20 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 25 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 fee test sequence has substantial Identity to 30 a reference sequence.

When percentage of sequence identity is used in reference to poiypeptides, it is recognized that residue positions that are not identical often differ by conservative 18 amino acid substitutions, where amino acids residues are substituted for other amino acid residues with similar chemical properties (e.g,, charge or hydrophobtdiy) and therefore do not change the functional properties of the polypeptide. Where sequences differ in conservative substitutions, the percent 5 sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. 2016202733 28 Apr 2016

For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison JO 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 15 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 SO to about 200, more usually about 100 to about 20 150 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. Optima! alignment of sequences for comparison can be conducted, e.g., by the iocal homology algorithm of Smith &amp; Waterman, 1981, Adv, Appi. Math, 2:482, by 25 the homology alignment algorithm of Needieman &amp; Wunsch, 1970, J, Mol. Biot. 48:443, by the search for similarity method of Pearson &amp; Lipman, 1988, Proc. Natl. 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 30 by manual alignment and visual inspection (see, e.g., Current Protocols in Molecular Biology (Ausube! et at., eds. 1995 supplement}}. 19 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 Aitschu! etal.« 1977, Nuc. Acids Res, 25:3389-3402 and Alisehul et al„ 1990, J. Mol. Biol. 215:403-410, respectively. BLAST 5 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 10 length W in the query sequence, which either match or satisfy some positivevalued threshold score I when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et ai, supra}. These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are 15 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 20 score. Extension of the word hits in each direction are halted when: the cumulative alignment score fails 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 25 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. Sc/. USA 30 89:10915 (1989)) alignments (B) of 50, expectation (E) of 10, M-5, N--4, and a 2016202733 28 Apr 2016 comparison of both strands. 20

The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin &amp; Aftschul, 1993, Proc. Natl. Acad, Sci. USA 90:5873-5787), One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N}J, which provides an indication of the probability by 5 which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence If the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0,2, more preferably less than about 0.01, and most preferably iess than about 0,001. 2016202733 28 Apr 2016 10 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 acid, but to no other sequences. Stringent conditions are sequence-dependent and wifi be different in different circumstances. Longer sequences hybridize specifically at higher temperatures. An extensive guide to the 15 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 5-10: C. lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength pH. Low 20 stringency conditions are generally selected to be about 15-3CTC. 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 25 conditions will be those in which the salt concentration is less than about 1,0M sodium ton, 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 30 destabilizing agents such as fornrcamide. For selective or specific hybridization, a positive signal is at least two times background, preferably 10 times background hybridization. 21

Nucleic acids that do not hybridize to each other under stringent conditions are stiil 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, 5 the nucleic acids typically hybridize under moderately stringent hybridization conditions. 2016202733 28 Apr 2016

Genomic DNA or cDNA comprising GPT polynucleotides may be identified in standard Southern blots under stringent conditions using the GPT polynucleotide 10 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 37*C, 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 80X, for 20 minutes, or equivalent conditions. A positive hybridization is at least twice is 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 20 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 biot, 25 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, 30 pod 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 synthetase (GS) and glutamine phenyipyruvate transaminase (GPT). in an exemplary embodiment, single-transgene parental lines carrying either a GPT or 22 GS1 transgene coding sequence are generated, preferably selfed until homozygous for the fransgene, then crossed to generate progeny plants containing both transgenes, 2016202733 28 Apr 2016 5 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 GS and GPT enzyme capacity into the plant, which serves to mediate increased synthesis of 2-oxogiutaramate, which in turn signals metabolic gene expression, resulting in increased plant growth and the 10 enhancement other agronomic characteristics. 2-oxogiutaramate 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 plvotai role in the coordination of the carbon and nitrogen metabolism systems (Lancien et ai„ 2000, Enzyme Redundancy and the Importance of 2-Oxogiuiatate in Higher Rants 15 Ammonium Assimilation, Plant Physiol. 123; 817-824). See, also, the schematic of the 2-oxogiotaramate 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 20 (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-oxogiutaramate (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 25 enhanced CQ2 fixation rates and increased growth characteristics (Example 3, infra).

Applicants’ previous work demonstrated that over-expression of Alfalfa GS1 gene under the control of a strong constitutive promoter results in transgenic tobacco 30 plants with higher levels of GS activity in the leaves. These plants outgrow their wild-type counterparts, fix CO2 faster, contain increased concentrations of total protein, as well as increased concentrations of glutamine and 2-oxoglutaramate, and show increased rates of uptake of nitrate through their roots. 23 2016202733 28 Apr 2016

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 CO2 fixation, and increased levels of total 5 protein, glutamine and 2~oxogiutaramate. 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 10 Example 4, infra).

In one particular embodiment, exemplified herein by way of Examples 3, 5 and 7, infra, a first set of parental single-transgene tobacco plant lines carrying the Alfalfa GS1 gene, including 5' and 3’ untranslated regions, were generated using 15 Agrobacierium mediated gene transformation, under selective pressure, together with screening for the fastest growing phenotype, and seifing to transgene/phenotype homozygosity (see Example 5, infra). A second set of parental single-transgene tobacco plant lines carrying the full length coding sequence of Arabidopsis GPT were generated in the same manner (Example 3, 20 infra), High growth rate performing plants from each of the parental lines were then sexually crossed to yield progeny lines (Example 7, infra).

The resulting progeny from multiple crosses of Arabidopsis GS1 and GPT transgenic tobacco plants produce far better and quite surprising increases in 25 growth rates over the single-transgene parental Sines as well as wildtype plants. FIG. 5 shows photographs of double-transgene progeny from single-transgene GS1 X GPT plant crosses, relative to wild type and single-transgene parental plants. FIG. 6 shows photographs comparing leaf sizes of doubie-transgene progeny and wild type plants. Experimentally observed growth rates in these 30 double transgenic plants ranged between 200% and 300% over wild-type piants (Example 7, infra). Moreover, total biomass levels Increased substantially in the double-transgene plants, with whole plant fresh weights typically being about two to three times the wild-type plant weights. Similarly, seed yields showed similar 24 increases in the doubie-transgene plants, with seed pod production typically two to three times the wild type average, and overall seed yields exceeding wild-type plant yields by 300-400%. 2016202733 28 Apr 2016 5 In addition to the transgenic tobacco plants referenced above, various other species of transgenic plants comprising GPT and GS transgenes are specifically exemplified herein. As exemplified herein, transgenic plants showing enhanced growth characteristics have been generated in two species of Tomato (see Examples 4 and 17), Pepper (Example 8), Beans (Examples 9 and 10), Cowpea 10 (Examples 11 and 12), Alfalfa (Example 13), Cantaloupe (Example 14), Pumpkin (Example 15), Arabidopsis (Example 16) and Camiiena (Example 18). These transgenic plants of the invention 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 15 thereof, and via sexuai crosses of single transgene plants, as exemplified herein. Different GPT and GS transgenes were successfully employed in generating the transgenic plants of the Invention, as exemplified herein.

The invention also provides methods of generating a transgenic plant having 20 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 25 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 piant cell one or more nucleic acid constructs or expression cassettes comprising nucleic acid molecules encoding a 30 GPT transgene and an GS transgene, under the control of one or more suitable promoters (and, optionally, other regulatory elements) capable of driving the 25 expression of the transgenes, so as to yield a plant cell transformed thereby, and obtaining a transgenic plant which expresses the GPT and GS transgenes. 2016202733 28 Apr 2016

Based on the results disclosed herein, It is dear that any number of GPT and GS 5 polynucleotides may be used to generate the transgenic plants of the invention. Both GS1 and 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., Dania rerio GPT). With respect to GPT, numerous GPT polynucleotides derived from different species have been 10 shown to be active and useful as GPT transgenes. Similarly, different GS polynucleotides may be used, including without limitation any plant GS1 encoding polynucleotide that generates GS activity in a host ceil transformed with an expressible GS1 construct, 15 in a specific embodiment, the GPT transgene is a GPT polynucleotide encoding ah Arabidopsis derived GPT, such as the GPT of SEQ ID NO: 2, SEQ ID NO: 21 and SEQ ID NO: 30, and the GS transgene is a GS polynucleotide encoding an Alfalfa derived GS1 (i,e>, SEQ ID NO: 4) or an Arabidopsis derived GS1 (SEQ ID NO: 7), The GPT transgene may be encoded by the nucleotide sequence of SEQ 20 SD NO: 1; a nucleotide sequence having at least 75% and more preferably at least 80% identity to SEQ SD NO; 1, and encoding a poiypeptide having GPT activity; a nucleotide sequence encoding the polypeptide of SEQ !D NO: 2, or a polypeptide having at feast 75% and more preferably at least 80% sequence identity thereto which has GPT activity; and a nucleotide sequence encoding the polypeptide of 25 SEQ ID NO: 2 truncated at its amino terminus by between 30 to 58 amino acid residues, or a polypeptide having at least 75% and more preferably at least 80% sequence identity thereto which has GPT activity. The GS1 transgene may be encoded by the polynucleotide of SEQ ID NO: 3 or SEQ ID NO: 8 or a nucleotide sequence having at least 75% and more preferably at least 80% identity to SEQ 30 ID NO: 3 or SEQ ID NO: 6, and encoding a polypeptide having GPT .activity; and a nucleotide sequence encoding the poiypeptide of SEQ ID NO: 4 or 7, or a polypeptide having at least 75% and more preferably at least 80% sequence identity thereto which has GS activity. 26

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: 9 and SEQ ID NO: 31, and the GS transgene is a GS1 poiynueleotide. The GPT transgene may be encoded by the nucieotide sequence of SEQ ID NO: 8; a 5 nucieotide sequence having at ieast 75% and more preferably at feast 80% identity to SEQ iD NO: 8, and encoding a polypeptide having GPT activity: a nucieotide sequence encoding the polypeptide of SEQ ID NO: 9 or SEQ ID NO: 2016202733 28 Apr 2016 31, or a polypeptide having at least 75% and more preferably at ieast 80% sequence identity thereto which has GPT activity. 10

In yet another specific embodiment, the GPT transgene is a GPT poiynucieotide encoding a Rice derived GPT, such as the Rice GPTs of SEQ iD NO: 11 and SEQ ID NO: 32, and the GS transgene is a GS1 poiynucieotide. The GPT transgene may be encoded by the nucieotide sequence of SEQ iD NO: 10; a 15 nucieotide sequence having at ieast 75% and more preferably at ieast 80% identity to SEQ ID NO: 10, and encoding a polypeptide having GPT activity; a nucleotide sequence encoding the polypeptide of SEQ iD NO: 11 or SEQ iD NO: 32, or a polypeptide having at least 75% and more preferably at ieast 80% sequence identity thereto which has GPT activity. 20

In yet another specific embodiment, the GPT transgene is a GPT poiynucieotide encoding a Soybean derived GPT, such as the Soybean GPTs of SEQ iD NO: 13, SEQ IS NO: 33 or SEQ ID NO: 33 with a further isoleucine at the N-terminus of the sequence, and the GS transgene is a GS1 poiynucieotide. The GPT 25 transgene may be encoded by the nucieotide sequence of SEQ iD NO; 12; a nucieotide sequence having at ieast 75% and more preferably at least 80% identity to SEQ iD NO: 12, and encoding a polypeptide having GPT activity; a nucieotide sequence encoding the polypeptide of SEQ ID NO. 13 or SEQ ID NO: 33 or SEQ ID NO: 33 with a further Isoleucine at the N-terminus of the sequence, 30 or a poiypeptide having at least 75% and more preferably at ieast 80% sequence identity thereto which has GPT activity. 27

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: 15 and SEQ ID NO: 34, and the GS transgene is a GS1 polynucleotide. The GPT transgene may be encoded by the nucleotide sequence of SEQ ID NO: 14; a 5 nucleotide sequence having at least 75% and more preferably at least 80% identity to SEQ ID NO: 14, and encoding a polypeptide having GPT activity; a nucleotide sequence encoding the polypeptide of SEQ ID NO: 15 or SEQ ID NO:34, or a polypeptide having at least 75% and more preferably at least 80% sequence identity thereto which has GPT activity. 2016202733 28 Apr 2016 10 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: 17 and SEQ ID NO: 35, and the GS transgene is a GS1 polynucleotide. The GPT transgene may be encoded by the nucleotide sequence of SEQ ID NO: 16; a nucleotide sequence having at least 75% and more preferably at least 80% identity 15 to SEQ ID NO: 16; and encoding a polypeptide having GPT activity; a nucleotide sequence encoding the polypeptide of SEQ ID NO: 17 or SEQ ID NO: 35, or a polypeptide having at least 75% and more preferably at least 80% sequence identify thereto which has GPT activity.

In yet another specific embodiment, the GPT transgene is a GPT polynucleotide 20 encoding a Bamboo derived GPT, such as the Bamboo GPT of SEQ ID NO: 36, and the GS transgene is a GS1 polynucleotide. The GPT transgene may be encoded by a nucleotide sequence encoding the polypeptide of SEQ ID NO: 36, or a polypeptide having at least 75% and more preferably at least 80% sequence identity thereto which has GPT activity. 25 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., E. coli (see Examples 20-23), in a transient in planta expression system (see Example 19), or in a transgenic plant 30 (see Examples 1-18). 28

TRANSGENE CONSTRUCTS/EXPRESSiON VECTORS 2016202733 28 Apr 2016

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 5 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 add constructs carrying the transgene(s) of interest may be introduced into a plant celi or cells using a number of methods if) known in the art, including but not limited to electroporation, ONA 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 ceil, the nucleic acid construct may direct the expression of the incorporated transgene(s) (i.e., GPT), either in a 15 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 cel! has been successfully transformed, it may be cultivated to regenerate a transgenic plant, 20 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 25 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. 30 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 celi, the transgene (s) (i.e., GPT coding sequence) operably linked to the 29 promoter, preferably a suitable transcription termination sequence (i.e,, nopaiine synthetic enzyme gene terminator) operably linked to the transgene, and typically other elements useful for controiSing the expression of the transgene, as well as one or more selectable marker genes suitable for selecting the desired 5 transgenic product {i.e,, antibiotic resistance genes). 2016202733 28 Apr 2016

As Agrobacterium tumefaciens is the primary transformation system used to generate transgenic plants, there are numerous vectors designed for Agrobaeierium transformation. For stable transformation, Agrobacterium systems 10 utilize “binary” vectors that permit plasmid manipulation In both £ coli and Agrobacterium, and typically contain one or more selectable markers to recover transformed plants (Heilens et ai., 2000, Technical focus: A guide to Agrobacterium binary Ti vectors, Trends Riant Sci 5:446-451), Binary vectors for use in Agrobacterium transformation systems typically- comprise the borders of T-15 DMA, multiple cloning sites, replication functions for Escherichia cofi 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 at., 2006, 20 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 iimitation virB, virE, and virG (Vain et ai„ 2004, The effect of additional virulence genes on transformation efficiency, transgene integration and expression in rice plants using the pGreen/pSoup dual binary 25 vector system. Transgenic Res. 13:593-603; Srivaianakui et ai., 2000, Additional virulence genes influence transgene expression: transgene copy number, integration pattern and expression. X Plant Physiol. 157, 685-690; Park et ai, 2000, Shorter T-DNA or additional virulence genes improve Agrohacterium-mediated transformation Theor, Appi. Genet. 101, 1015-1020; Jin et ai., 1987, 30 Genes responsible for the supervirulence phenotype of Agrobacterium tumefaciens A281. J. Bacterioi 169:4417--4425). 30 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 and the RuBisCo promoter are employed. A number of expression vectors which utilize the CaMV 35$ and RuBsCo promoter are known and/or 5 commercially available and/or derivable using ordinary skill in the art, 2016202733 28 Apr 2016

PLANT PROMOTERS

The term ‘promoter’ is used to designate a region in the genome sequence 10 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 15 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. 20 A large number of promoters which are functional in plants are known in the art. In constructing· GPT and GS transgene constructs, the selected promoters) may be constitutive, no'n-spqcific promoters such as the Cauliflower Mosaic Virus 35S ribosomal promoter (CaMV 35S promoter), which is widely employed for the 25 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 nopaiine synthase promoter, the alcohol dehydrogenase promoter and the sucrose synthase promoter. 30 Alternatively, in some embodiments, it may be desirable to select a promoter based upon the desired plant ceils to be transformed by the transgene construct, the desired expression level of the transgene, the desired tissue or subceliular compartment for transgene expression, the developmental stage targeted, and 31 the like. 2016202733 28 Apr 2016

For example, when expression in photasynthetic tissues and compartments is desired, a promoter of the ribuiose bssphosphate carboxyiase (RuBisCo) gene 5 may be employed, in the Examples which follow, expressible nucleic acid constructs comprising GPT and GS1 transgenes under the control of a tomato RuBisCo promoter were prepared and used in the generation of transgenic plants or to assay for GPT activity in ptanta or in E, coil, if) 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 iectin (Vodkin et ai., 1983, Cell 34:1023-31; Undstrom et at,, 1990, Developmental Genetics 11:160-167), com alcohol 15 dehydrogenase 1 (Vogel et ai, 1989, J, Ceil. Biochem, (Suppl 0) 13:Part D; Dennis et ai,, 1984, Nucl. Acids Res., 12(9): 3983-4000), corn light harvesting complex (Simpson, 1986, Science, 233: 34-38; Bansa! et a!., 1992, Proc. Natl. Acad, Sci. USA, 89: 3654-3658), corn heat shock protein (Odell et ai., 1985, Nature, 313: 810-812; Rochester et al., 1986, EMBO J,, 5: 451-458), pea smai! 20 subunit RuBP carboxylase (Poutsen 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 piasmid nopaiine synthase (Langridge et ai., 1989, Proc, Natl. Acad. Sci. USA, 86: 3219-3223), petunia chaicone isomerase (Van Tunen et ai., 1988, EMBG J. 7(5): 1257-1263), bean giycine rich 25 protein 1 (Keiier et al, 1989, EMBO J. 8(5): 1309-1314), truncated CaMV 35s (Odei! et al, 1985, supra), potato paiatin (Wen2fer et ai., 1989, Plant Mol Biol 12: 41-50), root ceii (Conkiing et ai., 1990, Plant Physio!, 93: 1203-1211), maize zein (Reina et al, 1990, Nucf. Acids Res. 18(21): 6426; Kriz et al, 1987, Mol Gen. Genet. 207(1): 90-98; Wandeit and Feix, 1889, Nuc. Acids Res, 17(6): 2354; 30 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), a~tubuiin (Carpenter etal, 1992, Plant Ceil 4(5): 557-571; Uribe et al, 1998, Plant Mol. Biol. 37(6): 1069-1078), cab (Sullivan, etal, 1989, Mol Gen. Genet. 215(3): 32 431-440), PEPCase {Hudspeth and Gruia. 1989, Plant Mol. Biol. 12: 579-589), R gene complex (Chandler et aL, 1989, The Plant Cel! 1: 1175-1183), ehaicone synthase (Franken et PL, 1991, EMBO J. 10(9): 2605-2612) and glutamine synthetase promoters (U.S, Pat. Ho. 5,391,725; Edwards et at, 1990, Proc. Hath 5 Acad, Sci. USA 87: 3459-3463; Brears et ai„ 1991, Plant J. 1 (2): 235-244). 2016202733 28 Apr 2016

In addition to constitutive promoters, various inducible promoter sequences may be employed in oases where it is desirable to regulate transgene expression as the transgenic plant regenerates, matures, flowers, etc. Examples of such 10 inducible promoters include promoters of heat shock genes, protection responding genes (be., phenylalanine ammonia iyase; see, for example Sevan et al.s 1989, EMBO J, 8(7): 899-906), wound responding genes (i.e., celi wall protein genes), chemically inducible genes (i.e., nitrate reductase, chitinase) and dark inducibie genes (i.e., asparagine synthetase; see, for example U.S. Patent No. 5,256,558). 15 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-bi$phosphate carboxylase (rhcS) (see, for example, Tobin and Silverthorne, 1985, Annu. Rev. Plant Physiol. 36: 569-593; Dean etal., 1989, Annu. Rev. Plant Physiol. 40:415-439.). 20

Other inducible promoters include A8A- and turgor-inducible promoters, the auxin-binding protein gene promoter (Schwob et aL* 1993, Plant J. 4(3): 423-432), the UDP glucose fiavonold glycosyl-transferase gene promoter (Ralston et aL, 1988, Genetics 119(1): 185-197); the MPi proteinase inhibitor promoter (Cordero 25 et aL, 1994, Plant J, 6(2): 141-150), the glyceraldehyde-3-phosphate dehydrogenase gene promoter (Kohler et aL, 1995, Plant Mol. Biol. 29(B): 1293-1298; Quigley et a!., 1989, J, Mot·. 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 etal., 1990, supra). 30

For a review of plant promoters used in plant transgenic plant technology, see Fotenza et al, 2004, In Vitro Cell DeveL Biol - Plant, 40(1): 1-22. For a review of 33 2016202733 28 Apr 2016 synthetic plant promoter engineering, see, for example, Venter, M., 2007, Trends Plant Sci., 12(3): 118-124,

GLUTAMINE PHENYLPYRUVATE TRANSAMINASE fGFTY TRANSGENE

The present invention discloses for the first time that plants contain a glutamine phenyl pyruvate transaminase (GPT) enzyme which is directly functional in the synthesis of the signal metabolite 2-hydroxy~5-oxaproSine. Until now, no plant transaminase with a defined function has been described. Applicants have 10 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, increased glutamine synthetase activity in foliar tissue, and faster 0¾ fixation rates. 15

In the practice of the invention, the GPT gene functions as one of at least two transgenes incorporated into the transgenic plants of the invention, the other being: the glutamine sythetase gene (see infra). 20 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-S-oxoproilne. 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 25 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. 30 When individually compared (by BLAST alignment) to the Arabidopsis mature protein sequence provided in SEG iD NO: 30, the following sequence identities and homoiogies (BLAST “positives15, including similar amino acids) were obtained for the following mature GPT protein sequences: 2016202733 28 Apr 2016 [SEQ ID] ORIGIN %IDENTITY %POSITIVE [31] Grape 84 93 [32] Rice 83 91 [33] Soybean 83 93 5 [34] Barley 82 91 [35] Zebra fish 83 92 [36] Bamboo 81 90 Corn 79 90 Castor 84 93 10 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 15 with the mature Arabidopsis GPT of SEQ ID NO: 30, 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 20 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 25 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, such as the GPT activity assay described in Example 7. 35 2016202733 28 Apr 2016

Any plant GPT with 2-oxoglutaramate synthesis activity may be used to transform plant ceils 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 5 extend beyond plants, as evidenced by the close homology between various plant GPT proteins and the putative Zebra fish GPT homolog. Therefore, various piant 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 10 characteristics as well (i.e., nee giutamine transaminase over-expressed in transgenic rice plants), although it is possible that regulation within a homologous cel! may attenuate the expression of the transgene in some fashion that may not be operable in a heterologous cell. 15 GLUTAMINE SYNTHETASE (GS) TRANSGENE:

In the practice of the invention, the glutamine synthetase (GS) gene functions as one of at least two transgenes incorporated into the transgenic plants of the 20 invention {GPT being the other of the two).

Giutamine synthetase plays a key role in nitrogen metabolism in plants, as well as in animals and bacteria. The GS enzyme catalyzes the addition of ammonium to glutamate to synthesize glutamine in an ATP-dependent reaction. GS enzymes 25 from assorted species show highly conserved amino acid residues considered to be important for active site function, indicating that GS enzymes function similarly {for review, see Eisenberg et at, Bsochimica et Biophysics Acta, 1477:122 145, 2000). 30 GS is distributed in different subceliular locations (chloropiast and cytoplasm) and is found in various piant tissues, including leaf, root, shoot, seeds and fruits. There are two major isoforms of plant GS: the cystoiic isoform (G.S1) and the ptastidic {chloroplastic) isoform (GS2). GS2 is principally found in leaf tissue and functions in the assimilation of ammonia produced by photorespiration or by 38 nitrate reduction. GS1 is mainly found in ieaf and root tissue, typicaily exists in a number of different isoforms in higher plants, and functions to assimilate ammonia produced by ali other physioiogicai processes (Coruzzi, 1991, Riant Science 74: 145-155; McGrati and Coruzzi, 1991, Plant J. 1(3); 275-280; Lam et a!., 1996, 5 Ann. Rev. Plant Physiol. Plant Mol. Biol, 47: 569-593; Stitt, 1999, Curr. Op. Plant Biol. 2: 178-186; Oliveira et at., 2001, Brazilian J. Med. Biol. Res. 34: 567-575). Multiple GS genes are associated with a complex promoter repertoire which enable the expression of GS in an organ and tissue specific manner, as well as in an environmental factor-dependent manner. 2016202733 28 Apr 2016 10

Plant glutamine synthetase consists of eight subunits, and the native enzyme in plants has a molecular mass ranging from 320 to 380 kD, each subunit having a molecular mass of between 38 and 45 kD. The GS1 genes of several plants, especially legumes, have been cloned and sequenced (Tischer et al., 1986, Mol 15 Gen Genet, 203: 221-229; Gebhardt et al,, 1986, EMBO J. 5: 1429-1435; Tingey et al., 1987, EMBO J. 6: 1-9; Tingey et al, 1988, J Biol Chem. 263: 9651-9657; Bennett et al, 1989, Plant Mo! Biol. 12; 553-565; Boron and Legockt, 1993, Gene 136: 95-102; Roche et al, 1993, Plant Mol Biol 22: 971-983; Marsofier et al, 1995, Plant Mo! Biol 27: 1-15; Temple et al., 1995, Mo! Plant-Microbe interact. 8: 20 218-227). All have been found to be encoded by nuclear genes (for review, see,

Morey et al, 2002, Plant Physiol 128(1): 182-193),

Chioroplastic GS2 appears to be encoded by a single gene, whsie various cystoloic GS1 isoforms are encoded within multigene families (Tingey et al, 1987, 25 supra: Sakamoto et al, 1989, Plant Mol Biol. 13: 611-814; Brears et al, 1991, supra; Li et al, 1993, Plant Mol Biol, 23:401-407; Dubois et al,, 1996, Riant Mol, Biol,, 31:803-817; Lam et al, 1996, supra), GS1 multigene families appear to encode different subunits which may combine to form homo- or hetero-octamers, and the different members show a unique expression pattern suggesting that the 30 gene members are differentially regulated, which may relate to the various functional roles of glutamine synthetase plays in overall nitrogen metabolism (Gebhardt et al, 1986, supra; Tingey et al, 1987, supra; Bennett et al, 1989, supra; Walker and Coruzzi, 1989, supra; Peterman and Goodman, 1991, Moi Gen 37

Genet 1991:330:145-154.; Marsolier etal, 1995, supra: Temple et at., 1995« supra; Dubois etal, 1998, supra). 2016202733 28 Apr 2016

In one embodiment, a GS1 gene coding sequence is employed to generate GS 5 transgene constructs, in particular embodiments, further described in the Examples, infra, the Alfalfa or Arabidopsis GS1 gene coding sequence is used to generate a transgene construct that may be used to generate a transgenic plant expressing the GS1 transgene. As an example, such a construct may be used to transform Agrobacteria, The transformed Agrobacteria are then used to generate If) To transgenic plants. Example 5 demonstrates the generation of To GS1 transgenic tobacco plants using this approach. Similarly, Examples 6 and 17 demonstrates the generation of To GSt transgenic tomato plants, Example 8 demonstrates the generation of To GS1 transgenic pepper plants, Examples 9 and 10 demonstrate the generation of Τΰ GS1 transgenic bean piants, Examples 11 15 and 12 demonstrate the generation of To QS1 transgenic cowpea plants. Example 13 demonstrates the generation of TQ GS1 transgenic alfalfa piants, Example 14 demonstrates the generation of T0 GS1 transgenic cantaloupe plants, Example 15 demonstrates the generation of T0 GS1 transgenic pumpkin piants, Example 16 demonstrates the generation of To GS1 transgenic Arabidopsis piants, and 20 Example 18 demonstrates the generation of To GS1 transgenic Cantaloupe plants,

TRANSCRIPTIONS 25 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 potyadenyiation of the mRNA transcript. Suitable transcription terminators are those which are known to function in piants, including without limitation, the nopallne synthase (NGS) and octopine synthase (OCS) genes of 30 Agrobacterium iumefaciens, the T7 transcript from the octopine synthase gene, the 3’ end of the protease inhibitor i or il genes from potato or tomato, the GaMV 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 nopaiine synthase transcription 38 terminator is employed. 2016202733 28 Apr 2016 5 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 ceils, such as genes which impart resistance to an antibiotic {such 10 as kanarnyein, gentamycih, anamycin, hygromycin and hygromycinB) or resistance to a herbicide (such as sulfonylurea, gulfosinate, phosphinothricin and giyphosate). Screenabie markers include, for example, genes encoding β-giucuronidase (Jefferson, 1987, Riant Mol. Biol. Rep 5: 387-405), genes encoding iuesferase (Ow et al., 1986, Science 234: 856-859) and various genes encoding 15 proteins involved in the production or control of anthocyanin pigments {See, for example, U.S, Patent 6,573,432). The E call 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, spectrophotometnc, various hisiochemtcai methods). 20 Moreover, there is essentially no detectable glucuronidase In most higher plant species. TRANSFORMATION METHODOLOGIES AND SYSTEMS: 25 Various methods for introducing the transgene expression vector constructs of the invention into a plant or plant cel! are well known to those skied in the art, and any capable of transforming the target plant or plant cell may be utilized.

Agmbacterium-mediated transformation is perhaps the most common method 30 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, Agrobactenum Protocols, Wan, ed., Humana Press, 2nd edition, 2008). Agrobactenum tumefaciens is a Gram negative soil bacteria that causes tumors (Crown Gall disease) in a great many dicot species, via the 39 insertion of a smali segment of tumor-inducing DNA (“T~DNA\ transfer DNA') into the pfant 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 5 ends of the T-DNA. The T-DNA can be physically separated from the remainder of the Ti-piasmid, creating a 'binary vector* system. 2016202733 28 Apr 2016

Agrobacierium transformation may be used for stably transforming dieots, monocots, and cells thereof (Rogers et a)., 1986, Methods EnzymoL, 118: 62?» 10 841; Hemalsteen et a!,, 1984, EMBO J., 3: 3039-3041; Hoykass-Van Slogteren et at., 1984, Nature, 311: 763-764; Grimsley et ai., 1987, Nature 325: 167-1679; Boulton et a!., 1989, Rant Mot. Biol 12: 31-40; Gould et a!„ 1991, Plant Physiol. 95: 426-434). Various methods for introducing DNA into Agrobacteria are known, including electroporation, freeze/thaw methods, and triparentai mating. The most 15 efficient method of placing foreign DNA into Agrobacterium is via electroporation (Wise et al„ 2006, Three Methods for the Introduction of Foreign DNA into Agrobacierium, Methods in Molecular Biology, vol. 343: Agrobacierium 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, 20 Agrobacterium-me&amp;ated transformation may be used to obtain transient expression of a transgene via the transcriptional competency of unincorporated transgene construct molecules (Helens etal, 2005, Plant Methods 1:13). A large number of Agrobacterium transformation vectors and methods have been 25 described (Karimi et at·., 2002, Trends Plant Set. 7(5): 193-5), and many such vectors may be obtained commercially (for example, Inyitrogen), In addition, a growing number of “open-source” Agrobacierium transformation vectors are available (for example, pCambia vectors; Gambia, Canberra, Australia). See, also, subsection herein on TRANSGENE CONSTRUCTS, supra. In a specific 30 embodiment described further in the Examples, a pMON316-based vector was used in the leaf disc transformation system of Borsch et. al. (Horsch et 31.,1995, Science 227:1229-1231) to generate growth enhanced transgenic tobacco and tomato plants. 40 2016202733 28 Apr 2016

Other commonly used transformation methods that may be employed in generating the transgenic plants of the invention include without limitation micropfojectiie bombardment, or biolistic transformation methods, protoplast 5 transformation of naked DNA by calcium, polyethylene glycol (PEC) or electroporation (Paszkowski et ai., 1984, EMBO J. 3: 2727-2722; Potrykus at a!., 1985, Moi. Gen, Genet 199: 189-177; Fromm et ai,, 1985, Proc. Nat. Acad. Sci. USA 82; 5824-5828; Shimamoto et ai., 1989, Nature, 338; 274-276. 19 Biolistic transformation involves injecting millions of DNA-ooated 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 Klkkert et ai., 2005, Stable Transformation of Plant 15 Cells by Particle Bombardment/Bloiistics, in: Methods in Molecular Biology, vol. 286; Transgenic Plants; Methods and Protocols, Ed. L Peha, Humana Press Inc., Totowa, NJ).

Electroporation is a technique that utilizes short, high-intensity electric fields to 20 permeabiiize 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 Peha, Humana Press Inc., Totowa, NJ, pp. 79-90; Fromm et a!.,1987, Electroporation of DNA and RNA into plant protoplasts, in 25 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, 256-284; Bates, 1994, Genetic transformation of plants by protoplast electroporation. Mol. Biotech. 2: 135-145; 30 Dillon et ai,, 1998, Electroporation-mediated DNA transfer to plant protoplasts and intact plant tissues for transient gene expression assays, in Cell Bioiogy, Vol. 4, ed., Ceiis, Academic Press, London, UK, pp. 92-99). The technique operates by creating aqueous pores in the bacteria! membrane, which are of sufficiently large 41 size to 'allow DMA molecules (and other macromofecules) to enter the cell, where the transgehe expression construct (as T-DNA) may be stably incorporated into plant genomic DMA, leading to the generation of transformed cells that can subsequently be regenerated into transgenic plants, 2016202733 28 Apr 2016 5

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 a!„ 2006, Arabidopsis thaliana Floral Dip Transformation Method, Methods Mol Biol, voi. If) 343: Agrobacterium Protocols, 2/e, volume 1; Ed., Wang, Humana Press inc., Totowa,· NJ, pp, 87-103; Clough and Bent, 1998, Floral dip: a simplified method for Agrobacierium-mediated transformation of Arabidopsis thaliana, Plant J, 18: 735-743), However, with the exception of Arabidopsis, these methods have not been widely used across a broad spectrum of different plant species. Briefly, 15 floral dip transformation is accomplished by dipping or spraying flowering plants in with an appropriate strain of Agrobacterium tumefaciens. Seeds collected from these T0 plants are then germinated under selection to identify transgenic Ti individuals. Example 16 demonstrated floral dip inoculation of Arabidopsis to generate transgenic Arabidopsis plants. 20

Other transformation methods include those in which the developing seeds or seedlings of plants are transformed using vectors such as Agrobacterium vectors. For example, as exemplified in Example 8, such vectors may be used to transform developing seeds by injecting a suspension or mixture of the vector (i.e„ 25 Agrobacteria) directly into the seed cavity of developing pods (i.e., pepper pods, bean pods, pea pods and the like). Seedlings may be transformed as described for Alfalfa in Example 13, Germinating seeds may be transformed as described for Cameiina in Example 18. Intra-fruit methods, in which the vector is injected into fruit or developing fruit, may be used as described for Cantaloupe melons in 30 Example 14 and pumpkins in Example 15.

Still other transformation methods include those in which the flower structure is targeted for vector inoculation, such as the flower inoculation methods described 42 2016202733 28 Apr 2016 for beans in Examples 9 and 10, peas in Examples 11 and 12 and tomatoes in Example 17,

The foregoing plant transformation methodologies may be used to introduce 5 transgenes into a number of different plant ceils and tissues, including without limitation, whole plants, tissue and organ explants including chloropfasts, flowering tissues and cells, protoplasts, meristem ceils, 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 Id regenerated transgenic plant may be generated. Callus is initiated from tissue sources including, but not limited to, immature embryos, seedling apical mertstems, mlorospores and the like. Cells capable of proliferating as callus are also recipient ceils for genetic transformation. 15 Methods of regenerating Individual plants from transformed plant ceils, tissues or organs are known and are described for numerous plant species.

As an illustration, transformed piantlets (derived from transformed cells or tissues) are cultured in a root-permissive growth medium supplemented with the selective 20 agent used in the transformation strategy (i.e., and antibiotic such as kanamycin). Once rooted, transformed piantlets 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 25 tomato plants.

To transgenic plants may be used to generate subsequent generations (e.g., Ti, T2, etc.) by selling of primary or secondary transformants, or by sexual crossing of primary or secondary transformants with other piants (transformed or 30 untransformed). For example, as described in Example 7, infra, individual plants over expressing the Aifaffa GS1 gene and outperforming wlldtype piants were crossed with individual plants over-expressing the Arabidopsis GPT gene and outperforming wiidtype plants, by simple sexual crossing using manual pollen transfer. Reciprocal crosses were made such that each plant served as the male in a set of crosses and each plant served as the female in a second set of crosses. During the mature plant growth stage, the plants are typically examined for growth phenotype, CO2 fixation rate, etc. (see following subsection). 2016202733 28 Apr 2016 5 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 10 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 15 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 20 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 25 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 30 the RNA transcription products, and protein gel electrophoresis, Western blotting, 44 immunoprecspitation, enzyme immunoassay, and the like for identifying the protein encoded and expressed by the transgene- 2016202733 28 Apr 2016 in another approach, expression levels of genes, proteins and/or metabolic 5 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-oxogiutaramate may be used to screen for desirable transformants, as exemplified in the Examples, Similarly, increased levels of GPT and/or GS activity may be assayed, as exemplified in the 10 Examples.

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 15 with the fastest growth rates, the highest seed yields, etc,, particularly when identifying plants for subsequent selfmg, 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 20 sizes, plant sizes, increased flowering, time to flowering, overall protein content (in seeds, fruits, piant 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 25 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 characteristic(s) 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 30 superiority of the transgenic plant In respect of any particular phenotypic characteristic. 45

Preferably, initial transformants are selected and then used to generate Ti 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 5 the desired traits and setting those individuals. As exemplified herein, transgenic plant lines propagated through at least one sexual generation (Tobacco, Arabidopsis, Tomato) demonstrated higher transgene product activities compared to lines that did not have the benefit of sexual reproduction and the concomitant increase in transgene copy number. 2016202733 28 Apr 2016 10 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 15 plants created by single transformation events which introduce multiple copies of a given transgene or multiple transgenes. Various common breeding methods are well known to those skilled in the art (see, e.g., Breeding Methods for Cultivar Development, Wilcox J. ed., American Society of Agronomy, Madison Wis. (1987)).

In a another aspect, the invention provides transgenic plants characterized by 20 increased nitrogen use efficiency. Nitrogen use efficiency may be expressed as plant yield per given amount of nitrogen. In the Examples provided herein, the transgene and control plants all received the same nutrient solutions in the same amounts. The transgenic plants were consistently characterized by higher yields, and thus have higher nitrogen use efficiencies. 25 In yet another aspect, the invention provides transgenic plants and seeds thereof with increased tolerance to high salt growth conditions. This aspect of the invention is exemplified by Example 24, which describes the germination of transgenic tobacco plant seeds in very high salt conditions (200 mM NaCI). While counterpart wild type tobacco seeds germinated at a rate of only about 10%, on 46 average, the transgenic tobacco seeds achieved nearly the same rate of germination obtained under no sait conditions for both transgenic and wild type seeds, or about 92%. 2016202733 28 Apr 2016

5 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. 10 EXAMPLE 1: ISLOATiON OF ARABfDOPSIS 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 15 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-oxogiutaramate. The human protein, glutamine transaminase K (E.C. 2.6.1.64) (also referred in the literature as cysteine conjugate δ -lyase, kyneurenine aminotransferase, glutamine phenylpyruvate transaminase, and other names), 20 had been shown to be involved in processing of cysteine conjugates of haiogenated xenobiotics (Perry et ai., 1995, FE8S 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. Nevertheless, the potential involvement of this protein in the synthesis of 2-25 oxoglutaramate was of interest

Using the protein sequence of human cysteine conjugate E-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 30 Arabidopsts gene locus at At1q77670, sharing approximately 36% sequence homology/identity across aligned regions. 47

The full coding region of the gene was then amplified from an Arabidopsis cDNA library (Stnatagene) with the following primer pair: 2016202733 28 Apr 2016 5-CCCATCGATGTACC T GGACATAAATGGT GIG AT G~3’ 5 5 - GATGGTACCTCAGACTTTTCTCTTAAGCTTCTGCTTC-3’

These primers were designed to incorporate Ola i tATCGAT) and Κρη I (GGTACC) restriction sties to facilitate subsequent subdoning into expression vectors for generating transgenic plants. Takara ExTaq DNA polymerase enzyme If) was used for high fidelity PCR using the following conditions: initial denaturing 94G for 4 minutes, 30 cycles of 94C 30 second, annealing at 55G for 30 seconds, extension at 72C for 90 seconds, with a final extension of 720 for 7 minutes. The amplification product was digested with Cla ! and Κρη I restriction enzymes, isolated from an agarose gel electrophoresis and ligated into vector pMon316 15 (Rogers, et. at. 1987 Methods in Enzyrnoiogy 153:253-277) which contains the cauliflower mosaic virus (CaMV, also GMV) 35S constitutive promoter and the nopaline synthase (NOS) 3' terminator. The ligation was transformed into DH5« ceils and transformants sequenced to verify the insert. 20 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 chioropiast signal sequence. EXAMPLE 2: PRODUCTION OF BIOLOGICALLY ACTIVE RECOMBINANT 25 ARABiDOPStS GLUTAMINE PHENYL PYRUVATE TRANSAMINASE (GPTJ:

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 £ coil, purified, and assayed for its ability to synthesize 30 2-oxoglutaramate using a standard method, N.MB.. Assay, for, g^xogiutaramate:

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 giutamine, 100

4S mM giyoxylate and 200 microM pyrldoxal 5’-phosphate. The reaction mixture without added test protein was used as a control. Test and control reaction mixtures were incubated at 37X for 20 hours, and then clarified by centrifugation to remove precipitated material. Supernatants were tested for the presence and 5 amount of 2-oxoglutaramate using ,3C 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 giyoxylate) diminish in abundance. The cyclic 2-oxoglutaramate gives rise to a distinctive signal allowing it to be readily distinguished from the open chain glutamine 10 precursor. 2016202733 28 Apr 2016 HPLC Assay for 2-oxoalutaramate:

An alternative assay for GPT activity uses HPLC to determine 2-oxoglutaramate production, following a modification of Calderon et at,, 1985, J Bacteriol 181(2); 15 807-809, 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) Mercaptoethanoi, Tissue samples from the test material (i.e., plant tissue) am added to the extraction buffer at approximately a 1/3 ratio (w/v), incubated for 30 minutes at 37‘;C> and stopped with 200ul of 20% TCA. After about 5 minutes, the assay 20 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 M h2S04, a flow rate of approximately 0.2 mi/min, at 40 C. Injection volume is approximately 20 ui, and retention time between about 38 and 39 minutes. Detection is achieved with 210nm UV light. 25

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 30 synthesis of 2-oxogiutaramate in plants. Accordingly, the test protein was designated Arahidopsis glutamine phenylpyruvate transaminase, or “GPT”. 49

The nucleotide sequence of the Arabidopsis GPT coding sequence is shown in the Table of Sequences, SEG ID NO, 1, The translated amino add sequence of the GPT protein is shown in SEG ID NO. 2. 2016202733 28 Apr 2016 5 EXAMPLE 3: CREATION OF TRANSGENIC TOBACCO PLANTS OVEREXPRESSING ARABtDOPSIS&amp;PTi 10 Briefly, the plant expression vector pMon316-PjU was constructed as follows. The isolated cDNA encoding Arabidopsis GPT (Example 1} was cloned into the Clai-Kprtl. polytinker site of the pMON316 vector, which places the GPT gene under the control of the constitutive cauliflower mosaic virus (CaMV) 3SS promoter and the nopaline synthase (NOS) transcriptional terminator, A 15 kanamycin resistance gene was included to provide a selectable marker.

Agrobacfer/pm-iyiediated Plant Transformations; pMON-PJU and a control vector pMon316 (without inserted DMA) were transferred to Agrobactenum tumefaciens strain pTSTT37ASE using a standard 20 electroporation method (McCormac et al,, 1998, Molecular Biotechnology 9:155-159), followed by plating on LB plates containing the antibiotics spectinomydn (1Ό0 micro gm / ml) and kanamycin (50 micro gm / mi). Antibiotic resistant colonies of Agrobacterium were examined by PCR to assure that they contained plasmid. 25

Nicotians tabacum cv. Xanthi plants were transformed with pMGN-PJU transformed Agrobacieria using the Seat disc transformation system of Horseh et. at. (Horseh et al„1993, Science 227:1229-1231), Briefly, sterile leaf disks were inoculated and cultured for 2 days, then transferred to selective MS media 30 containing 100 pg/ml kanamycin and 500 pg/ml ciafaran. 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 calfus on Murashige &amp; Skoog 35 (M&amp;S) media from which the transformant plantiets emerged. These piantlets 50 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 ptaniiets were then transferred to soil and allowed to grow to maturity and upon flowering the plants were seifed and the resultant seeds were 5 harvested. During the growth stage the plants had been examined for growth phenotype and the C02 fixation rate was measured for many of the young transgenic plants. 2016202733 28 Apr 2016

Production of T1 and T2 Generation GPT Transgenic Plants: 10 Seeds harvested form the T0 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 resuit of normal genetic segregation of the gene) 15 and more than half of the remaining seeds were removed because of demonstrated sensitivity (even mild) to the kanamycin.

The surviving plants (Ti generation) were thriving and these plants were then selfed to produce seeds for the T2 generation. Seeds from the Ti generation were 20 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 Hoagiande 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 micromoles per meter squared per second. They were 23 harvested 14 days after being transferred: to the sand culture.

Characterization of GPT Transgenic Rants:

Harvested transgenic plants (both GPT transgenes and vector control transgenes) were analyzed for giutamine sythetase activity in root and leaf, whole plant fresh 30 weight, total protein in root and Seat, and C02 fixation rate (Knight et at., 1988, Plant Phystoi. 88: 333). Non-transformed, wild-type A. tumefadens piants were also analyzed across the same parameters in order to establish a baseline control.. 51

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, see Example 5). Across all parameters evaluated, the GPT transgenic tobacco plants showed; 2016202733 28 Apr 2016 5 enhanced growth characteristics, in particular, the GPT transgenic plants exhibited a greater than 50% increase in the rate of C02 fixation, and a greater than two-foid 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.

Id 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 Ambidopsts GPT transgene achieve significantly enhanced growth and C02 fixation rates.

15 Table I

Protein mg/gram fresh weight Leaf Root Wild type - control 8.3 2.3 Line PN1-8 a second control 8.9 2.98 Line PN9-9 13.7 3.2 Glutamine Synthetase activity, micromoies/min/mq protein Wild type {Ratio of leaf: root * 4.1:1) 4.3 1.1 PN1-8 (Ratio of leaf: root = 4.2:1) 5.2 1.3 PN9-9 {Ratio of leaf: root = 10.8:1} 10.5 0.97 Whole Plant Fresh Weight, g Wild type 21.7 PN1-8 26.1 PN9-9 33.1 COj Fixation Rate, umo!e/m2/sec Wlid type 8.4 PN1-8 8,9 PN9-9 12.9

Gate ~ average of three plants

Wild type ~ Control plants; not regenerated of transformed. PN1 lines were produced by regeneration after transformation using a construct without inserted gene. 20 A control against the processes of regeneration and transformation, PN 9 lines were produced by regeneration after transformation using a construct with the Ambidcpsis OPT gene. 52 2016202733 28 Apr 2016 EXAMPLE 4: GENERATION OF TRANSGENIC TOMATO PLANTS CARRYING ARABIDOPSIS GPT TRANSGENE:

Transgenic Lycopersicon esculentum (Micro-Tom Tomato) plants carrying the 5 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 Tabie ii. The transgenic plants showed significant enhancement of growth rate, flowering, and seed yield in relation to wild type control plants, in 10 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 piant compared to a wild type plant is presented in FIG. 3 (together with GS1 transgenic tomato plants, see Example 8).

15 TABLE II

Growth Characteristics Wiidtype Tomato GPT Transgenic Tomato Stem height, cm 6.5 18,12,11 major stems Stems 1 3 major, 0 other Buds 2 16 Flowers 8 12 Fruit 0 3 EXAMPLE 5: GENERATION OF TRANSGENIC TOBACCO PLANTS 20 OVEREXPRESSING ALFALFA GS1:

Generation of Plant Expression Vector 0GSI II;

Transgenic tobacco plants overexpressing the Alfalfa GS1 gene were generated as previously described (Temple et al., 1993, Mol. Gen. Genetics 236: 315*325). 25 Briefly, the piant expression vector pGSHI was constructed by inserting the entire coding sequence together with extensive regions of both the 5’ and 3’ untranslated regions of the Alfalfa GSi gene [SEQ ID NO: 3] (DasSarma at aL 1986, Science, Vo! 232, Issue 4755, 1242*1244) into pMON316 (Rogers et ai., 1987, supra), placing the transgene under the control of the constitutive 30 cauliflower mosaic virus (CaMV) 35S promoter and the nopaiine synthase (NOS) transcriptionai terminator, A kanamyein resistance gene was included to provide a selectable marker. 2016202733 28 Apr 2016 5 pGS111 was transferred to Agrobacterium iumefaciem strain pTiTT37ASE using triparental mating as described (Rogers et al., 1987, supra; Unkefer et al., U.S, Patent No, 6,555,500), Nicotians tabacum cv. Xanthi plants were transformed with pGS111 transformed Agrobacteria using the leaf disc transformation system of Horsch et. at {Horsch et 81,1995, Science 227:1229-1231). Transformants 10 were selected and regenerated on MS medium containing IGOpg/ml kanamyein. Shoots were rooted.on the same medium (with kanamyein, absent hormones) and transferred to potting soilperiitewermicuSite (3:1:1), grown to maturity, and'allowed to seif. Seeds were harvested from this T0 generation, and subsequence generations produced by selling and continuing selection with kanamyein. The 15 best growth performers were used to yield a T3 for crossing with the best performing GPT over-expressing tines identified as described in Example 3. A photograph of the GS1 transgenic plant compared to a wild type control plant is shown in FIG. 2 (together with GPT transgenic tobacco plant, see Example 3) 20 EXAMPLE 6: GENERATION OF TRANSGENIC TOMATO PLANTS CARRYING ALFALFA GS1 TRANSGENE:

Transgenic Lycopersicon esculentum (Micro-Tom Tomato) plants carrying the 25 Alfalfa GS1 transgene were generated using the vector described in Example 5 and a transformation protocol essentially as described (Sun et al,, 2006. Plant Cell Physiol 46(3} 426-31). To transgenic tomato plants were generated and grown to maturity. Initial growth characteristic data of the GPT transgenic tomato plants is presented in Table Hi. The transgenic plants showed significant enhancement of 30 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 6S1 transgenic tomato plant compared to a wild type plant is presented in FIG. 3 (together with GPT transgenic tomato piant, see Example 4). 35 54 TABLE Hf

Growth Characteristics Wifdtype Tomato GS1 Transgenic Tomato Stem height, cm 6.5 16,7, 5 major stems Stems 1 3 major, 3 med, 1 sm Buds 2 2 Flowers 8 13 Fruit 0 4 2016202733 28 Apr 2016 5 EXAMPLE 7: GENERATION OF DOUBLE TRANSGENIC TOBACCO PLANTS CARRYING GS1 AND GPT TRANSGENES:

In an effort to determine whether the combination of GS1 and GPT transgenes in a single transgenic plant might improve the extent to which growth and other 10 agronomic characteristics may be enhanced, a number of sexual crosses between high producing lines of the single transgene (GS1 or GPT) transgenic plants were carried out. The results obtained are dramatic, as these crosses repeatedly generated progeny plants having surprising and heretofore unknown increases in growth rates, biomass yield, and seed production. 15

Materials and Methods:

Single-transgene, transgenic tobacco plants overexpressing GPT or GS1 were generated as described in Examples 3 and 4, respectively. Several of fastest growing T2 generation GPT transgenic plant lines were crossed with the fastest 20 growing T3 generation GS1 transgenic plant lines using reciprocal crosses. The progeny were then selected on kanamycin containing M&amp;S media as described in Example 3, and their growth, flowering and seed yields examined.

Tissue extractions for GPT and GS activities: GPT activity was extracted from 25 fresh plant tissue after grinding in cold 100 mM Tris-HCI, pH 7.6, containing 1 mm ethylenediaminetetraacetic, 200 mM pyridoxai phosphate and 6 mM mercaptoethanol in a ratio of 3 ml per gram of tissue. The extract was clarified by centrifugation and used in the assay. GS activity was extracted from fresh plant tissue after grinding in cold 50 mM Imidazole, pH 7.5 containing 10 mM Mg€l2s 30 and 12.5 mM mercaptoethanol in a ratio of 3 mi per gram of tissue. The extract 55 was clarified by centrifugation and used In the assay. GPT activity was assayed as described in Calderon and Mora, 1985, Journal Bacteriology 161:807-809, GS activity was measured as described in Shapiro and Stadtmahn, 1970, Methods in Enzymoiogy 17A: 910-922, Both assays involve an Incubation with substrates 5 and cofactor at the proper pH. Detection was by HPLC. 2016202733 28 Apr 2016

Results:

The resuits are presented in two ways, First, specific growth characteristics are 10 tabulated in Tables iV.A and IV. S (biomass, seed yields, growth rate, GS activity, GPT activity, 2-oxoglutaramate activity, etc). Second, photographs of progeny plants and their leaves are shown in comparison to singie-transgene and wild type plants and leaves are presented in FIG. 5 and FIG. 6, which show much larger whole plants, larger leaves, and earlier and/or more abundant flowering in 15 comparison to the parental singie-transgene plants and wild type control plants.

Referring to Tabie IV.A, double-transgene progeny plants form these crosses showed tremendous increases totai biomass (fresh weight), with fresh weights ranging from 45-89 grams per individual progeny plant, compared to a range of 20 only 19-24 grams per individual wild type piant, representing on average, about a two- to three-fold increase over wiid type plants, and representing at the high end, an astounding four-fold increase in biomass over wild type plants. Taking the 24 individual doubie-transgene progeny plants evaluated, the average individual plant biomass was about 2,75 times that of the average wild type control piant 25 Four of the progeny fines showed approximately 2,5 fold greater average per piant fresh weights, while two lines showed over three-fold greater fresh weights In comparison to wild type plants.

In comparison to the singie-transgene parental lines, the doubie-transgene 30 progeny plants also showed far more than an additive growth enhancement. Whereas GPT singie-transgene lines show as much as about a 50% increase over wild type biomass, and GS1 singie-transgene lines as much as a 66% increase, progeny plants averaged almost a 200% increase over wild type plants. 56

Similarly, the double transgene progeny plants flowered earlier and more prolificalfy than either the wild type or single transgene parental lines, and produced a fer greater number of seed pods as well as total number of seeds per plant. Referring again to Table IV A on average, the double-transgene progeny 5 produced over twice the number of seed pods produced by wild type plants, with two of the high producer piants generating over three times the number of seed pods compared to wild type. Total seed yield in progeny plants, measured on a per plant weight basis, ranged from about double to nearly quadruple the number produced in wild type piants. 2016202733 28 Apr 2016 57 2016202733 28 Apr 2016 S-112,983

TABLE IV.A PLANT UNE FRESH WEIGHT α/whole plant SEED POOS #pods/plant SEED YIELD g/plant LEAF GS ACTIVITY ROOT L/R RATIO Wild Type Tobacco Wild type 1 18.73 26 0.967 Wild type 2 24.33 24 1.07 Wild type 3 23.6 32 0.9 Wild type 4 18.95 32 1.125 WT Average 21.4025 28.5 12155 7.75 1.45 524 Cross 1 X1L1a x PA9-9ff 1 59.21 62 2.7811 2 65.71 56 3 55.36 72 4 46.8 56 Cross 1 Average Compared to WT 56.77 +265% 61.5 +216% +274% 14.98 +193% 1.05 •28% 1427 +267% Cross 2 PA9-2 x L9 1 70.83 61 1.76 2 49.17 58 3.12 3 5023 90 NA 4 45.77 Cross 2 Average Compared to WT 54 +252% 582 +205% 2ΔΔ +240% 16.32 +211% 121 +125% 9.02 +169% Cross 3 PA9-9ff xL1a 1 89.1 77 3.687 2 78.18 3 58.34 4 61.79 Cross 3 Average Compared to WT 7125 +336% 77 (one plant) +270% 3.678 (one plant) +362% 15.92 +205% 128 -5% 1124 +216% 58 2016202733 28 Apr 2016 PLANT LINE FRESH WEIGHT g/whole plant SEED PODS #pods/plant SEED YIELD g/plant LEAF GS ACTIVITY ROOT L/R RATIO Cross 5 PA9-10aa x L1a 1 65.34 45 2.947 2 53.28 64 3.3314 3 49.85 42 1.5667 4 44.63 42 2.5013 Cross 5 Average Compared to WT 53.275 +244% 48.25 +169% 2.86928 +283% 13.03 +168% 1.8 7.24 Cross 6 PA9-17b x L1a 1 56.7 64 2.492 2 55.05 66 2.162 3 51.51 59 1.8572 4 45.38 72 4.742 Cross 6 Average Compared to WT 52.16 +244% 65.25 +229% 2.8133 +277% 14.114.7 52 1.1.1124 13.29 Cross 7 PA9-20aa x L1b 1 76.26 67 2.0535 2 66.27 42 1.505 3 72.26 72 2.3914 4 63.91 91 2.87 Cross 7 Average Compared to WT 69.675 +326% 68 +239% 2.204975 +217% 14.12 1.24 11.39 Control PA9-9ff 1 32.18 N/A 2 32.64 N/A 3 34.67 N/A 4 25.18 N/A Average Compared to WT 31.17 +148% N/A 11.57 1.14 10.15 59 2016202733 28 Apr 2016 PLANT LINE FRESH WEIGHT g/whole plant SEED PODS #pods/plant SEED YIELD g/plant LEAF GS ACTIVITY ROOT UR RATIO Control GS L1a 1 41.74 N/A 2 36.24 N/A 3 33.8 N/A 4 30.48 N/A Average Compared to WT 35.57 +166% N/A 13.15 1.23 10.69 60 2016202733 28 Apr 2016

Table IV.B shows growth rate, biomass and yield, and biochemical characteristics of Line XX (Line 3 further selfed) compared to the single transgene line expressing GS1 and wild type control tobacco. All parameters are greatly increased in the double transgenic plant (Line XX). Notably, 2-oxoglutaramate activity was almost 17-fold 5 higher, and seed yield and foliar biomass was three-fold higher, in Line XX plants versus control plants.

TABLE IV.B

Plant Type Specific Growth Rate mg/g/d Foliar Biomass FWt, g Fruit/ Flowers /Buds Seed Yield g GS Activity umol/ min/gFW t GPT Activity nmol/h /gFWt 2- oxoglu- taramate nmol/gF Wt Trans Gene Assay Wildtype, avg 228 21.40 28.5 1.02 7.75 16.9 68.9 No Line 1 GS 269 35.57 NM NM 11.6 NM 414 Yes Line XX 339 59.71 62.9 2.94 16.3 243.9 1,153.6 Yes NM Not Measured 1 10 EXAMPLE 8: GENERATION OF DOUBLE TRANSGENIC PEPPER PLANTS CARRYING GS1 AND GPT TRANSGENES: IS In this example, Big Jim chili pepper plants (New Mexico varietal) were transformed with the Arabidopsis GPT full length coding sequence of SEQ ID NO: 1 under the control of the CMV 35S promoter, and the Arabidopsis GS1 coding sequence of SEQ ID NO: 6 under the control of the RuBisCo promoter, using Agrobacterium-mediated transfer to seed pods. After 3 days, seeds were harvested and used to generate TO 20 plants and screened for transformants. The resulting double-transgenic plants showed higher pod yields, faster growth rates, and greater biomass yields in comparison to the control plants. 61 2016202733 28 Apr 2016

Materials and Methods:

Solanaceae Capisicum Pepper plants (“Big Jim” varietal) were transformed with the Arabidopsis GPT full length coding sequence of SEQ ID NO: 1 under the control of 5 the CMV 358 promoter within the expression vector pMON (see Example 3), and the Arabidopsis GS1 coding sequence of SEQ ID NO: 6 under the control of the RuBisCo promoter within the expression vector pCambia 1201 (Tomato rubisco rbeS3C promoter: Kyozulka et al., 1993, Plant Physiol. 103: 991-1000; SEQ ID NO: 22; vector construct of SEQ ID NO: 6), using Agrobacterium-mediated transfer to seed 10 pods.

For this and all subsequent examples, the Cambia 1201 or 1305.1 vectors were constructed according to standard cloning methods (Sambrook et aL, 1989, supra, Saikl et a!., 1988, Science 239: 487-491). The vector is supplied with a 358 CaMV 15 promoter; that promoter was replaced with RcbS-3C promoter from tomato to control the expression of the target gene. The Cambia 1201 vectors contain bacterial chlorophenicol and plant hygromycin resistance selectable marker genes. The Cambia 1305.1 vectors contain bacteria! chlorophenicol and hygromycin resistance selectable marker genes. 20

The transgene expression vectors pMON (GPT transgene) and pCambia 1201 (GS transgene) were transferred to separate Agrobacterium tumefaciens strain LBA4404 cultures using a standard electroporation method (McCormac et al., 1998, Molecular Biotechnology 9:155-159). Transformed Agrobacterium were selected on media 25 containing 50 pg/ml of either streptamycin for pMON constructs or chloroamphenicol for the Cambia constructs. Transformed Agrofeacterium cells were grown in LB culture media containing 25 pg/ml of antibiotic for 36 hours. At the end of the 36 hr growth period cells were collected by centrifugation and cells from each transformation were resuspended in 100 ml LB broth without antibiotic. 30 62 2016202733 28 Apr 2016

Pepper plants were then transformed with a mixture of the resulting Agrabacierium cell suspensions using a transformation protocol In which the Agrobacterium is injected directly into the seed cavity of developing pods. Briefly, developing pods were injected with the 200 mi mixture In order to inoculate immature seeds with the 5 Agrobacteria essentially as described (Wang and Waterhouse, 1997, Plant Mol. Biol. Reporter 15: 209-215). In order to induce Agrobacteria virulence and improve transformation efficiencies, 10 pg/mi acetosyringonone was added to the Agrobacteria cultures prior to pod inoculations (see, Sheikholeslam and Weeks, 1988, Plant Mol. Biol. 8: 291-298). 10

Using a syringe, pods were injected with a liberai quantity of the Agrobacterium vector mixture, and left to incubate for about 3 days. Seeds were then harvested and germinated, and developing plants observed for phenotypic characteristics including growth and antibiotic resistance. Plants carrying the transgenes were green, whereas 15 untransformed plants showed signs of chlorosis in leaf tips. Vigorous growing transformants were grown and compared to wild type pepper plants grown under identical conditions. 20 Results:

The results are presented in FIG. 7 and Table V. FIG. 7 shows a photograph of a GPT+GS double transgenic pepper plant compared to a controi plant grown for the same time under identical conditions. This photograph shows tremendous pepper 25 yield in the transgenic iine compared to the control plant

Table V presents biomass yield and GS activity, as weii as transgene genotyping, in the transgenic lines compared to the wild type control. Referring to Table V, double-transgene progeny plants showed tremendous increases total biomass (fresh 30 weight), with fresh weights, ranging from 393 - 882 grams per individual transgenic plant, compared to an average of 328 grams per wild type plant Transgenic line AS 63 2016202733 28 Apr 2016 produced more than twice the total biomass of the controls. Moreover, pepper yields in the transgenic lines were greatly improved over wild type plants, and were 50% greater than control plants (on average). Notably, one of the transgene lines produced twice as many peppers as the control plant average. 5

TABLE V: TRANSGENIC PEPPER GROWTH/BIOMASS AND REPRODUCTION

Planttype Biomass, i Foliar Fresh Wtg Yield Peppers, g DWt GS activity Umoies/min /q FWt Transgene Presence Assay Wildtype, avg 323.2 83.7 1.09 Negative Line A2 457.3 184.2 1.57 GPT - Yes i Line A5 661.7 148.1 1.8 GPT - Yes Line B1 493.4 141.0 1.3 GPT-Yes Line B4 393.1 136.0 1.6 GPT-Yes Line C1 509.4 152.9 1.55 GPT-Yes FWi Fresh Weight; DWt Dry Weight EXAMPLE 9: GENERATION OF DOUBLE TRANSGENIC BEAN PLANTS CARRYING ARABIDOPSJS GS1 AND GPT TRANSGENES: 15 In this example, yellow wax bean plants (Phaseolus vulgaris) were transformed with the Arabidopsis GPT full length coding sequence of SEG ID NO: 1 under the control of the GMV 35S promoter within the expression vector pCambia 1201, and the Arabidopsis GS1 coding sequence of SEQ ID NO: 6 under the control of the RuBisCo promoter within the expression vector pCambia 1201, using Agrobacterium-mediated 20 transfer into flowers.

Materials and Methods:

The transgene expression vectors pCambia 1201-GPT (vector construct of SEG ID 25 NO: 27) and pCambia 1201-GS (vector construct of SEQ ID NO: 6) were transferred to separate Agrobacterium tumefaciens strain L8A44Q4 cultures using a standard 64 2016202733 28 Apr 2016 electroporation method (McCormac et ai.( 1998, Molecular Biotechnology 9:155-159), Transformed Agrobacterium were selected on media containing 50 pg/mf of chloroamphenlcol. Transformed Agrobacterium cells were grown In LB culture media containing 25 pg/ml of antibiotic for 36 hours. At the end of the 36 hr growth period 5 cells were collected by centrifugation and ceils from each transformation were resuspended in 100 ml LB broth without antibiotic.

Bean plants were then transformed with a mixture of the resulting Agrobacterium cell suspensions using a transformation protocol in which the Agrobacteria is injected 10 directly into the flower structure (Yasseem, 2009, Plant Mol. Biol. Reporter 27: 20-28). In order to induce Agrobacteria virulence and improve transformation efficiencies, TO pg/ml acetosyringonone was added to the Agrobacteria cultures prior to flower inoculation. Briefly, once flowers bloomed, the outer structure encapsulating the reproductive organs was gently opened with forceps in order to 15 permit the introduction of the Agrobacteria mixture, which was added to the flower structure sufficient to flood the anthers.

Plants were grown until bean pods developed, and seeds were harvested and used to generate transgenic plants. Transgenic plants were then grown together with 20 control bean plants under identical conditions, photographed and phenotypically characterized. Growth rates were measured for both transgenic and control plants, in this and ail examples, Glutamine synthetase (GS) activity was assayed according to the methods in Shapiro and Stadtmann, 1970, Methods in Enzymoiogy 17A; 910-922; and, Glutamine phenyipyruvate transaminase {GPT) activity was assayed 25 according to the methods in Calderon et ai., 1985, J. Bacterioi. 161: 807-809. See details in Example 7:, Methods, supra,

Results: 30 The results are presented in FIG, 8, FIG. 9 and Table VI. 65 2016202733 28 Apr 2016 FIG. 8 shows GPT+GS transgenic bean line A growth rate data relative to controi plants, including plant heights on various days into cultivation, as well as numbers of flower buds, flowers, and bean pods. These data show that the GPT+GS double 5 transgenic bean plants outgrew their counterpart control plants. The transgenic plants grew taller, flowered earlier and produced more flower buds and flowers, and developed bean pods and produced more bean pods that the wild type control plants.

TABLE V!; TRANSGENIC BEANS LINE A

Plant Type Bean Pod Yield FWt, g GPT Activity nmoles/h/gF Wf GS Activity umoles/min /gFWt Antibiotic Resistance Wildtype, avg 126.6 101.9 25.2 Negative 2A 211,5 NM NM + 4A 207,7 NM NM 5B 205J 984.7 101.3 + WT Wildtype; FWt Fresh Weight; N'M Not Measured

Table VI presents bean pod yield, GPT and GS activity, as wei! as antibiotic 15 resistance status, in the transgenic lines compared to the wild type control (average of several robust control plants; control plants that did not grow well were excluded from the analyses). Referring to Table VI, double-transgene progeny plants showed substantial bean pod biomass increases (fresh pod weight) in comparison to the control plants, with bean pod biomass yields consistently above 200 grams per 20 individual transgenic plant, compared to an average of 127 grams per wild type plant, representing an over 60% increase in pod yield in the double transgene lines relative to controi plant(s). 66 2016202733 28 Apr 2016

Lastly, FIG. 9 shows a photograph of a GPT+GS double transgenic bean plant compared to a control plant grown for the same time under identical conditions, showing increased growth in the transgenic plant. 5 EXAMPLE 10: GENERATION OF DOUBLE TRANSGENIC BEAN PLANTS CARRYING ARABIDOPS1S GS1 AND GRAPE GPT TRANSGENES: in this example, yellow wax bean plants (Phaseolus vulgaris) were transformed with 10 the Grape GPT full length coding sequence of SEQ ID NO: S under the control of the RuBisCo promoter within the expression vector pCambia 1305.1, and the Arabidopsis GS1 coding sequence of SEQ ID NO; 6 under the control of the RuBisCo promoter within the expression vector pCambia 1201, using Agrobacterium-mediated transfer into developing pods, 15

Materials and Methods:

The transgene expression vectors pCambia 1201-GPT(grape) (vector construct of SEQ ID NO: 8) and pCambia 1201-GS (vector construct of SEQ (D NO: 6) were 20 transferred to separate Agrobacterium tumefaciens strain LBA4404 cultures using a standard electroporation method (McCormac et al,, 1998, Molecular Biotechnology 9:153-159). Transformed Agrobacterium were selected on media containing 50 pg/mi of chloroamphenicol. Transformed Agrobacterium ceils were grown in LB culture media containing 25 pg/ml of antibiotic for 36 hours. At the end of the 36 hr 25 growth period cells were collected by centrifugation and cells from each transformation were resuspended in 100 ml LB broth without antibiotic.

Bean plants were then transformed with a mixture of the resulting Agrobacterium cell suspensions using a transformation protocol in which the Agrabacteria is injected 30 directly into the flower structure. In order to induce Agrobacteria virulence and improve transformation efficiencies, 10 pg/ml acetosyringonone was added to the 67 2016202733 28 Apr 2016

Agrobacteria cultures prior to flower inoculation. Briefly, once flowers bloomed, the outer structure encapsulating the reproductive organs was gently opened with forceps in order to permit the introduction of the Agrobacteria mixture, which was added to the flower structure sufficient to flood the anthers. 5

Plants were grown until bean pods developed, and seeds were harvested and used to generate transgenic plants. Transgenic plants were then grown together with control bean plants under identical conditions, photographed and phenotypically characterized. Growth rates were measured for both transgenic and control plants. 10

Results:

The results are presented in FIG. 10, FIG. 11 and Table VII. 15 FIG. 10 shows GPT+GS transgenic bean line G growth rate data relative to control plants, specifically including numbers of flower buds, flowers, and bean pods. These data show that the GPT+GS double transgenic bean plants outgrew their counterpart control plants. Notably, the transgenic plants produced substantially more bean pods that the wild type control plants. 20

TABLE Vi!: TRANSGENIC BEANS LINE G: POD YIELDS

Plant Type Bean Pod Yield FWt, g Antibiotic Resistance Wild type, avg 157.9 Negative G1 200.5 G2 178.3 -+ WT Wildtype; FWt Fresh Weight; NM Not Measured 25

Table Vii presents bean pod yield and antibiotic resistance status, in the transgenic lines compared to the wild type control (average of several robust control plants; control plants that did not grow well were excluded from the analyses). Referring to Table VII, double-transgene progeny plants showed substantial bean pod biomass 68 2016202733 28 Apr 2016 increases (fresh pod weight) in comparison to the control plants, with bean pod biomass yields of 200.5 (line G1) and 178 grams (line G2) per individual transgenic plant, compared to an average of 158 grams per individual wild type plant, representing approximately a 27% increase in pod yield in the double transgene lines 5 relative to control plants,

Lastly, FIG, 11 shows a photograph of a GPT+GS double transgenic bean plant compared to a control plant grown for the same time under identical conditions. The transgenic plant shows substantially increased size and biomass, larger leaves and a 10 more mature flowering compared to the control plant, EXAMPLE 11: GENERATION OF DOUBLE TRANSGENIC COWPEA PLANTS CARRYING ARABIDOPSIS GS1 AND GPT TRANSGENES: 15 in this example, common Cowpea plants were transformed with the Arabidopsis GPT full length coding sequence of SEG ID NO: 1 under the control of the CMV 35S promoter within the expression vector pIVION, and the Arabidopsis GS1 coding sequence of SEQ ID NO: 8 under the control of the RuBIsCo promoter within the expression vector pCambia 1201, using Agrobacterium-mediated transfer into 20 flowers. Materials and methods were as in Example 9, supra.

Results:

The results are presented in FIGS, 12 and 13, and Table VI, FIG, 12 shows relative 25 growth rates for the GPT+GS transgenic Cowpea line A and wild type control Cowpea at several intervals during cultivation, including (FIG, 12A) height and longest leaf measurements, (FIG. 12B) trffbiate leafs and flower buds, and (FIG. 12C) flowers, flower buds and pea pods. These data show that the GPT+GS double transgenic Cowpea plants outgrew their counterpart control plants, The transgenic 69 2016202733 28 Apr 2016 TABLE Viii: TRANSGENIC GOWPEA LINE A Plant Type Pea Pod Yield, FWt, g :i GPT Activity :: nmoles/h/gF Wt GS Activity umol/min/gF Wt Antibiotic Resistance Wiidtype, avg 74.7 44,4 28.3 Negative 4A 112.8 NM 41.3 "f 8B 113.8 736,2 54.9 WT Wiidtype, FWt Fresh Weight; NM Not Measured plants grew faster and taller, had longer leaves, and set flowers and pods sooner than wild type control plants.

Table VIII presents pea pod yield, GPT and GS activity, as well as antibiotic resistance status, in the transgenic lines compared to the wiid type control (average of several robust control plants; control plants that did not grow well were excluded 10 from the analyses). Referring to Table VIII, double-transgene progeny plants showed substantia! pea pod biomass increases (fresh pod weight) in comparison to the control plants, with average transgenic plant pea pod biomass yields nearly 52% greater than the yields measured in control p!ant(s). 15 Lastly, FIG. 13 shows a photograph of a GPT+GS double transgenic bean plant compared to a control plant grown for the same time under identical conditions, showing increased biomass and pod yield in the transgenic plant relative to the wiid type control plant. 20 EXAMPLE 12: GENERATION OF DOUBLE TRANSGENIC COWPEA PLANTS CARRYING ARABIDOPSIS GS1 AND GRAPE GPT TRANSGENES: in this example, common Gowpea plants were transformed with the Grape GPT full 25 length coding sequence of SEG ID NO: 8 under the control of the RuBisCo promoter within the expression vector pCambia 1305,1 (vector construct of SEQ ID NO: 8),

TO 2016202733 28 Apr 2016 and the Arabidopsis GS1 coding sequence of SEQ ID NO: 6 under the control of the RuBisCo promoter within the expression vector pCambia 1201 (vector construct of SEG ID NO: 6), using Agrobacterium-mediated transfer into flowers. Materials and methods were as in Example 11, supra. 5

Results:

The results are presented in FIGS. 14 and 15, and Table IX. 10 FIG. 14 shows relative growth rates for the GPT+GS transgenic Gowpea line G and wild type control Cowpea. These data show that the transgenic plants are consistently higher (FIG. 14A), produce substantially more flowers, flower buds and pea pods (FIG. 148), and develop trifoiates and leaf buds faster (FIG. 14C).

15 TABLE ΪΧ: TRANSGENIC COWPEA LINE G 1 Plant Type Pod Yield, FWt, g GPT Activity nmoles/h/gF WT GS Activity i umoi/min/gF i Wt Antibiotic Resistance ( Wildtype, avg 59.7 44.4 26.7 Negative G9 102.0 555.6 34.5 WT Wi.ldtype; FWt Fresh Weight;NM Not Measured

Table IX presents pea pod yield, GPT and GS activity, as well as antibiotic resistance 20 status, in the transgenic lines compared to the wild type control (average of several robust control plants; control plants that did not grow well were excluded from the analyses). Referring to Table IX, double-transgene progeny plants showed substantial pea pod biomass increases (fresh pod weight) in comparison to the control plants, with average pea pod biomass yields 70% greater in the transgenic 25 plants compared to control plant(s). 71 2016202733 28 Apr 2016

Lastly, FIG. 15 shows a. photograph of a GPT+GS double transgenic pea plant compared to a control plant grown for the same time under identical conditions, showing Increased height, biomass and leaf size in the transgenic plant relative to the wild type control plant. 5 EXAMPLE 13: GENERATION OF DOUBLE TRANSGENIC ALFALFA PLANTS CARRYING ARA8IDOPS.IS GS1 AND OPT TRANSGENES: I0 In .this, example, Alfalfa plants (Medicago sativa, var Ladak) were transformed with the Arabidopsis GPT full length coding sequence of SEQ ID NO: 1 under the control of the CMV 35S promoter within the expression vector pMON316 (see Example 3, supra), and the Arabidopsis GS1 coding sequence of SEQ ID NO: 6 under the control of the RuBisCo promoter within the expression vector pCamhsa 1201 (vector .15 construct of SEQ ID NO: 6), using Agrobacterium-medsated transfer into seedling plants. Agrobacterium vectors and mixtures were prepared for seedling inoculations as described in Example 11, supra.

Seedling inocuiations: 20 When Alfalfa seedlings were still less than about 1/2 inch tail, they were soaked in paper toweling that had been flooded with the Agrobacteria mixture containing both transgene constructs. The seedlings were left in the paper toweling for two to three days, removed and then planted in potting soil- Resulting TO and controi plants were then grown for the first 30 days in a growth chamber, thereafter cultivated in a 25 greenhouse, and then harvested 42 days after sprouting. At this point, only the transgenic Alfalfa line displayed flowers, as the wild type plants only displayed immature flower buds. The plants were characterized as to flowering status and total biomass. 30 72 2016202733 28 Apr 2016

Results:

The results are presented In Table X, The data shows that the transgenic Alfalfa plants grew faster, flowered sooner, and yielded on average about a 62% biomass 5 increase relative to the control plants.

TABLE X: TRANSGENIC ALFALFA VS, CONTROL ; Plant Type ! Biomass at Sacrifice, g ! Flowering Stage | Wildtype, avg 6.03 i Small defined buds No buds swelling, i No flowers Transgene #5 10.38 i 4 Open flowers T ransgene #11 9.03 Flower buds swelling T ransgene #13 9.95 Flower buds swelling EXAMPLE 14: GENERATION OF DOUBLE TRANSGENIC CANTALOUPE PLANTS CARRYING ARABIDOPSIS GS1 AND GPT TRANSGENES: 15

In this example, Cantaloupe plants {Cucumis melo var common) were transformed with the Arabidopsis GPT full length coding sequence of SEG ID NO: 1 under the control of the CMV 35S promoter within the expression vector pMON316 (see Example 3, supra), and the Arabidopsis G61 coding sequence of SEG ID NO; 6 20 under the control of the RuBisCo promoter within the expression vector pCambia 1201 (vector construct of SEG ID N.O: 6), using Agrobacterium-mediated transfer via injection into developing melons. Agrobacterium vectors and mixtures were prepared for intra-melon inoculations as described in Example 8, supra. Inoculations into developing melons were carried out essentially as described in Example 8. The 25 plants were characterized as to flowering status and total biomass relative to control melon plants grown under identical conditions. 73 2016202733 28 Apr 2016 TABLE XI: TRANGENIC CANTALOUPE VERSUS CONTROL Plant Type Biomass Foliar FWt, g Flowers / Flower ; Buds at Sacrifice Antibiotic Resistance Wildtype, avg 22.8 0/5 Negative Line 1 37.0 3/21 Line 2 35.0 2/30 + Une 3 37.1 3/27 "f" Line 4 40.6 5/26 Line 5 35.7 4/30 + FWt Fresh Weight

The results are presented in FIG. 16 and Table XL Referring to Table X!, the transgenic plants showed substantial foliar plant biomass increases in comparison to the control plants, with an average increase in biomass of 63%. Moreover, a 5 tremendous increase in flower and flower bud yields was observed in all five transgenic lines. Control plants displayed no flowers and only 5 buds at sacrifice, on average, in sharp contrast, the transgenic plants displayed between 2 and 5 flowers per plant, and between 21 and 3G flower buds, per plant, indicating a substantially higher growth rate and flower yield. Increased flower yield would be expected to 10 translate Into correspondingly higher melon yields in the transgenic plants. Referring to FIG. 16 (a photograph comparing transgenic Cantaloupe plants to control Cantaloupe plants), the transgenic Cantaloupe plants show dramatically increased height, overall biomass and flowering status relative to the control plants. 15 20 EXAMPLE 15: GENERATION OF DOUBLE TRANSGENIC PUMPKIN PLANTS CARRYING ARABIDOPSIS GS1 AND GPT TRANSGENES: 25 in this example, common Pumpkin plants {Cucurbits maxima) were transformed with the Arabidopsis GPT full length coding sequence of SEQ ID NO: 1 under the control 74 2016202733 28 Apr 2016 of the GMV 35S promoter within the expression vector pMGN31S (see Example 3, supra), and the Arabkfopsis GS1 coding sequence of SEQ ID NO: 6 under the control of the RuSisCo promoter within the expression vector pOambia 1201 (vector construct of SEQ ID NO: 6), using Agrobacterium-mediated transfer via injection into 5 developing pumpkins, essentially as described in Example 14, supra. The transgenic and control pumpkin plants were grown under identical conditions until the emergence of flower buds in the control plants, then ail plants were characterized as to flowering status and total biomass. 10 The results are presented in FIG. 17 and Table XII, Referring to Table Xil, the transgenic plants showed substantial foliar plant biomass increases in comparison to the control plants, with an increase in average biomass yield of 67% over control plants. Moreover, an increase in flower bud yields was observed in four of the five transgenic lines in comparison to control Control plants displayed only 4 buds at 15 sacrifice (average). In contrast, four transgenic plant lines displayed between 8 and 15 flowers buds per plant, representing a two-to nearly four-fold yield increase.

TABLE XII: TRANGENIC PUMPKIN VERSUS CONTROL 20

Plant Type Biomass Foliar FWt, g Wildtype, avg 47,7 Line 1 (Photo) 82.3 Line 2 74.3 Line 3 80.3 Line 4 (Photo) 77.8 Line 5 84.5

Flower Buds at ! Antibiotic Sacrifice Resistance 4.2 Negative 8 8 9 4 + 15 +- FWt Fresh Weight; Referring to FIG. 17 (a photograph comparing transgenic pumpkin plants to control plants), the transgenic pumpkin plants show substantially increased plant size, overall biomass and leaf sizes and numbers relative to the control plants. 25 75 2016202733 28 Apr 2016 EXAMPLE 18: GENERATION OF DOUBLE TRANSGENIC ARABIDOPSIS PLANTS CARRYING ARABIDOPSIS GS1 AND GPT TRANSGENES:

In this example, Arabidopsis (haliana plants were transformed with the truncated 5 Arabidopsis GPT coding sequence of SEQ ID NO: 18 under the control of the CMV 35S promoter within the expression vector pMON318 (see Example 3, supra), and transgenic plants thereafter transformed with the Arabidopsis GS1 coding sequence of SEG ID NO: 6 under the control of the RuBisCo promoter within the expression vector pCambia 1201 (vector construct of SEG ID NO: 6), using Agrobacterium-10 mediated "floral dip” transfer as described (Harrison et al., 2006, Plant Methods 2:19-23; Clough and Bent, 1998, Plant J. 16:735-743). Agrobactehum vectors pMON316 carrying GPT and pCambia 1201 carrying GS1 were prepared as described in Examples 3 and 11, respectively. 15 Transformation of two different cultures of Agrobacterium with either a pMon 316 + Arabidopsis GTP construct or with a Cambia 1201 + Arabidopsis GS construct was done by electroporation using the method of Weigel and Glazebrook 2002. The transformed Agrobacterium were then grown under antibiotic selection, collected by centrifugation resuspended in LB broth with antibiotic and used in the floral dip of 20 Arabidopsis inflorescence. Floral dipped Arabidopsis plants were taken to maturity and seif-fertilized and seeds were collected. Seeds from twice dipped plants were first geminated on a media containing 20ug/m! of kanamycin and by following regular selection procedures surviving seedlings were transferred to media containing 20 ug of hygromydn, Plants (3) surviving the selection process on both antibiotics were 25 self-fertilized and seeds were collected. Seeds from the II generation were germinated on MS media containing 20 ug/mS of hygromydn and surviving seedlings were taken to maturity, self-fertilized and seeds collected. This seed population the T2 generation was then used for subsequent growth studies. 30 The results are presented in FIG. 18 and Table XIII. Referring to Table XIII, which shows data from 6 wild type and 8 transgenic Arabidopsis plants (averaged), the 76 2016202733 28 Apr 2016 transgenic plants displayed increased levels of both GPT and GS activity. GPT activity was over twenty-fold higher than the control plants. Moreover, the transgenic plant fresh foliar weight average was well over four-fold that of the wild type control plant average. A photograph of young transgene Arabidopsis plants in comparison 5 to wild type control Arabidopsis plants grown under identical conditions is shown in FIG. 18, and reveals a consistent and very significant increase in transgenic plants relative to the control plants.

TABLE XIII: TRANSGENIC ARABIDOPSIS VERSUS CONTROL i Plant type \ Biomass, g i; Fresh foliar wt GPT Activity nmol/h/gFWt GS Activity ; umol/min/gF wt Antibiotic Resistance i ; Wildtype, avg 0.246 18,4 7,0 Negative ; Transgene 1.106 395.6 182 Positive 10 EXAMPLE 17; GENERATION OF TRANSGENIC TOMATO PLANTS CARRYING ARABIDOPSIS GPT AND GS1 TRANSGENES: 15

In this example, tomato plants (Solatium lycopersicon, “money Maker” variety) were transformed with the Arabidopsis GPT full length coding sequence of SEQ ID NO; 1 under the control of the CMV 35S promoter within the expression vector pMON316 (see Example 3, supra), and the Arabidopsis GS1 coding sequence of SEQ iD NO: 8 20 under the control of the RuBisCo: promoter Within the expression vector pCambia 1201 (vector construct of SEQ ID NO: 6), Single transgene (GPT) transgenic tomato plants were generated and grown to flowering essentialiy as described in Example 4. The Arabidopsis GS1 transgene was then introduced into the singfe-transgene TO plants using Agrobactenum-mediated transfer via injection directly into flowers (as 25 described in Example 8). The transgenic and control tomato plants were grown under identical conditions and characterized as to growth phenotype characteristics. Resulting TO double-transgene plants were then grown to maturity, photographed along with control tomato plants, and phenotypically characterized. 77 2016202733 28 Apr 2016

The results are presented in FIG. 19 and in Table IXX. Referring to Table IXX, double-transgene tomato plants showed substantial foliar plant biomass increases in comparison to the control plants, with an increase in average biomass yield of 45% over control. Moreover, as much as a 70% increase in tomato fruit yield was 5 observed in the transgenic lines compared to control plants (e,g„ 51 tomatoes harvested from Line 4G, versus and average of approximately 30 tomatoes from control plants). A much higher level of GPT activity was observed in the transgenic plants (e.g,, line 4C displaying an approximately 32-fold higher GPT activity in comparison to the average GPT activity measured in control plants), GS activity was 10 also higher in the transgenic plants relative to control plants (almost double in Line 4C).

With respect to growth phenotype, and referring to FIG. 19, the transgenic tomato plants displayed substantially larger leaves compared to control plants (FIG 19A). In 15 addition, it can be seen that the transgenic tomato plants were substantially larger, taller and of a greater overall biomass (see FIG. 198). 20

TABLE IXX: TRANSGENIC TOMATO GROWTH AND REPRODUCTION

Plant Type Biomass Foliar FWt, 9 Total Tomatoes Harvested until Sacrifice GPT Activity nmoles/h /gFWt GS Activity umcles/mi n /gFWt Transgene Presence Assay Wildtype, 891 30.2 287 14.27 Negative ; Line 6C 1288 43 9181 18.3 Ί·' Line 4C 1146 51 1718 26.4 + 25 78 2016202733 28 Apr 2016 EXAMPLE 18; GENERATION OF TRANSGENIC CAMILENA PLANTS CARRYING ARABIDOPSIS GPT AND GS1 TRANSGENES: in this example, Cameiina plants (Cameiina saliva, Var MT 303) were transformed 5 with the Arabidopsis GPT full length coding sequence of SEG ID NO: 1 under the control of the RuBisCo promoter within the expression vector pCambia 1201 and the Arabidopsis GS1 coding sequence of SEG ID NO: 6 under the control of the RuBisCo promoter within the expression vector pCambia 1201, using Agrobacterium-mediated transfer into germinating seeds according to the method described in Chee et a!., 10 1989, Plant Physiol. 91: 1212-1218. Agrobacterium vectors and mixtures were prepared for seed inoculations as described in Example 11, supra.

Transgenic and control Cameiina plants were grown under identical conditions (30 days in a growth chamber and then moved to greenhouse cultivation) for 39 days, 15 and characterized as to biomass, growth characteristics and flowering stage.

The results are presented in Table XX and FIG. 20. Referring to Table XX, it can be seen that total biomass in the transgenic plants was, on average, almost double control plant biomass. Canopy diameter was also significantly improved in the 20 transgenic plants. FiG, 20 shows a photograph of transgenic Cameiina compared to control. The transgenic plant is noticeably larger and displays more advanced flowering status.

TABLE XX: TRANSGENIC CAM ELINA VERSUS CONTROL

Plant Type Height / Canopy Diameter, inches Biomass .........................9.......................... Flowering Stage Wildtype, avg 14/4 8.35 Partial flowering Transgene C-1 15.5/5 16.54 Full flowering Transgene C-3 14/7 14.80 initial flowering 79 2016202733 28 Apr 2016

EXAMPLE 19: ACTIVITY OF BARLEY OPT TRANSGENE IN PLANTA

In this example, the putative coding sequence for Barley GPT was isolated and expressed from a transgene construct using an in plania transient expression assay. 5 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 10 example is provided in SEQ ID NO: 14, and the encoded GPT protein amino acid sequence is presented in SEQ ID NO: 15.

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

The transient tobacco leaf expression assay consisted of injecting a suspension of 20 transformed Agrobacterium (1.5-2.0 QD 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. 25 GPT activity in the inoculated leaf tissue (1217 nanomoles/gFWt/h) was three-fold the level measured In the control plant leaf tissue (407 nanomoies/gFWt/h), indicating that the Hordeum GPT construct can direct the expression of functional GPT in a transgenic plant. 80 2016202733 28 Apr 2016

EXAMPLE 20: ISOLATION AND EXPRESSION OF RECOMBINANT RICE OPT GENE CODING SEQUENCE AND ANALYSIS OF BIOLOGICAL ACTIVITY

In this example, the putative coding sequence for rice GPT was isolated and 5 expressed in £ colt. Bioiogicaily 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 £ coil: 10 The rice (Oryza sativia) GPT coding sequence was determined and synthesized, inserted into a PET28 vector, and expressed in £ coil Briefly, E coil cells were transformed with the expression vector and transformants grown overnight in LB broth diluted and grown to OD 0.4, expression induced with isopropyi-B-D-thiogalactoside (0.4 micromolar}, grown for 3 hr and harvested. A total of 25 X 106 15 cells were then assayed for biological activity using the NMR assay, below. Untransformed, wild type £ coif cells were assayed as a control. An additional control used E eoli ceils transformed with an empty vector.

The DNA sequence of the rice GPT coding sequence used in this example is 20 provided in SEQ ID NO: 10, and the encoded GPT protein amino acid sequence is presented in SEQ ID NO: 11. HPLC Assay for 2~oxoolutaramate: HPLC was used to determine 2~oxoglutaramate production in GPT-overexpressing £ 25 coli cells, following a modification of Calderon et al, 1985, J Bacterio! 161(2): SOT-BOO. Briefly, a modified extraction buffer consisting of 25 mM Tris-HC! pH 8.5, 1 mM EDTA, 20 μΜ Pyridoxai phosphate, 10 mM Cysteine, and -1.5% (v/v) Mercaptoethanoi was used. Samples (lysate from £ coil ceils, 25 X 106 cells) were added to the extraction buffer at approximately a 1/3 ratio (w/v), incubated for 30 30 minutes at 373C, and stopped with 200ui of 20% TCA. After about 5 minutes, the 81 2016202733 28 Apr 2016 assay mixture is centrifuged and the supernatant used to quantify 2-oxoglutaramate by HPLC, using an IDN-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 mi/min, at 40CC. Injection volume Is approximately 20 id, 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 Arabldoplsis full length sequence expresses a GPT with 2-oxoglutaramate synthesis activity. Briefly, authentic 2-oxogiutarmate (structure confirmed with NMR) 10 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 IS the disappearance of the substrate glutamine and showing that there was a 1:1 molar stoechiometry between glutamine consumed to 2-oxoglutaramte 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 20 shows that the production of the 2-oxogiutaramate was dependent upon the substrate glutamine.

Results: 25 Expression of the rice GPT coding sequence of SEQ ID NO: TO 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. coll cells overexpressing the recombinant rice GPT, compared to only 0,02 nanomoles of 2-oxogiutaramate activity in control E. coll cells, an 86-fold 30 activity level increase over control. 82 2016202733 28 Apr 2016

EXAMPLE 21: ISOLATION ANO-EXPRESSION OF RECOMBINANT SOYBEAN OFT GENE CODING SEQUENCE AND ANALYSIS OF BIOLOGICAL ACTIVITY

In this example, the putative coding sequence for soybean GPT was isolated and 5 expressed in E. colL Biologically active recombinant soybean GPT was produced, and catalyzed the increased synthesis of 2- oxogiutaramate, as confirmed by HPLC.

Materials and Methods: 10 Soybean GPT coding sequence and expression in E. 'colt

The soybean (Glycine max) GPT coding sequence was determined and synthesized, inserted into a PET28 vector, and expressed in E, coii. Briefly, E. coii cells were transformed with the expression vector and transformants grown overnight in LB broth diluted and grown to OD 0.4, expression induced with isopropyi-B-D-.15 thiogatactoside (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 £. coii cells were assayed as a control. An additional control used E coii cells transformed with an empty vector. 20 The DNA sequence of the soybean GPT coding sequence used in this example is provided in SEG ID NO: 12, and the encoded GPT protein amino acid sequence is presented in SEG ID NO: 13. HPLC Assay for 2-oxoalutaramate: 25 HPLC was used to determine 2-oxogiutaramafe production in GPT-overexpressing £ coii ceils, as described in Example 20, supra.

Results: 30 Expression of the soybean GPT coding sequence of SEQ ID NO: 12 resulted In the over-expression of recombinant GPT protein having 2~oxogiutaramate synthesis- 33 2016202733 28 Apr 2016 catalyzing bioactivity. Specifically, 31.9 nanomoles of 2-oxogiutaramate activity was observed in the £. colt cells overexpressing the recombinant soybean GPT, compared to only 0.02 nanomoles of .2-oxoglutaratnate activity in control E coll cells, a nearly 1,600-fold activity level increase over control. 5

EXAMPLE 22; ISOLATION AND EXPRESSION OF RECOMBINANT ZEBRA FISH GPT GENE CODING SEQUENCE AND ANALYSIS OF BIOLOGICAL ACTIVITY 10 In this example, the putative coding sequence for Zebra fish GPT was isolated and expressed in E coil Biologically active recombinant Zebra fish GPT was produced, and catalyzed the increased synthesis of 2- cxogSutaramate, as confirmed by NMR.

Materials and Methods: 15

Zebra fish GPT coding sequence and expression in £. colt The Zebra fish (D&amp;nio rer/b) GPT coding sequence was determined and synthesized, inserted into a PET28 vector, and expressed in £. coll Briefly, E coil ceils were transformed with the expression vector and transformants grown overnight in LB 20 broth diluted and grown to OD 0..4, expression induced with isopropyi-B-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 £. coli cells were assayed as a control. An additional control used E coli cells transformed with an empty vector. 25

The DMA sequence of the Zebra fish GPT coding sequence used in this example is provided in $EG ID NO: 16, and the encoded OPT protein amino add sequence is presented in $EG ID NO: 17. 30 84 2016202733 28 Apr 2016 HFLO Assay for 2-oxoalutaramate; HPLC was used to determine 2-oxoglutaramate production in GPT-overexpressing E. coli cells, as described in Example 20, supra. 5 Results:

Expression of the Zebra fish GPT coding sequence of SEQ ID NO: 16 resulted in the over-expression of recombinant GPT protein having 2-oxogiutaramate synthesis-eataiyzing bioactivity. Specifically, 28,6 nanomoles of 2-oxogiutaramate activity was 10 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 23: GENERATION AND EXPRESSION OF RECOMBINANT 15 TRUNCATED ARABIDOPSIS GPT GENE CODING SEQUENCES AND 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 20 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-terminai amino acid residues, were successfully expressed and showed biological activity in catalyzing 25 the increased synthesis of 2- oxoglutaramate, as confirmed by NMR.

Materials and Methods:

Truncated Arabidopsis GPT coding sequences and expression in E. coli: 30 The DNA coding sequence of a truncation of the Arabidopsis thaiiana GPT coding sequence of SEQ ID NO: 1 was designed, synthesized, inserted into a PET28 vector, 85 2016202733 28 Apr 2016 and expressed in E coti. The DMA sequence of the truncated Arabidopsis GPT coding sequence used in this example is provided in SEQ ID MO: 20 (-45 AA construct}, and the corresponding truncated GPT protein amino acid sequence is provided in SEG ID NO: 21. Briefly, E. coii cells were transformed with the 5 expression vector and transformants grown overnight in LB broth diluted and grown to GD 0.4, expression induced with isopropyl-B-D-thiogalactoside (0.4 micromolar), grown for 3 hr and harvested. A iota! of 25 X 106 cells were then assayed for biological activity using HPLC as described in Example 20, Untransformed, wild type £. coii cells were assayed as a control. An additional control used E coii cells I Q transformed with an empty vector.

Expression of the truncated -45 Arabidopsis GPT coding sequence of SEG ID NO: 20 resulted in the over-expression of biologically active recombinant GPT protein .(2-Qxogiutaramate synthesis-catalyzing bioactivity), Specifically, 16.1 nanomoles of 2-15 oxoglutaramate activity was observed in the £ coii ceils overexpressing the truncated -45 GPT, compared to only 0.02 nanomoles of 2-oxoglutaramate activity in control E coii ceils, a more than 800-fold activity level increase over control. For comparison, the fuil length Arabidopsis gene coding sequence expressed in the same &amp; coii assay generated 2.8 nanomoles of 2-oxogfutaramate activity, or roughly 20 less than one-fifth the activity observed from the truncated recombinant GPT protein.

EXAMPLE 24: GPT + GS TRANSGENIC TOBACCO SEED GERMINATION TOLERATES HIGH SALT CONCENTRATIONS 25

In this example, seeds form the double transgene tobacco line XX-3 (Gross 3 in Table 4, see Example 7) were tested in a seed germination assay designed to evaluate tolerance to high salt concentrations. 30 86 2016202733 28 Apr 2016

Materials and Methods:

Tobacco seeds tom the wild type and XX-3. populations were surfaced sterilized (5% bleach solution for 5 minutes followed by a 10% ethanol wash for 3 minutes) and 5 rinsed with sterile distilled water. The surface sterilized seeds were then spread on fyiurashige and Skoog media (10% agarose) without sucrose and containing either 0 or 200 nmM NaCl The seeds were allowed to germinate in darkness for 2 days followed by 6 days under a 16:8 photoperiod at 24C. On day eight the rate of germination was determined by measuring the percentage of seeds from the control 1.0 or transgene plants that had germinated.

Results:

The resuits are tabulated in Table XXI beiow, The rate of germination of the 15 transgenic plant line seeds under zero salt conditions was the same as observed with wild type controf plant seeds, in stark contrast, the germination rate of the transgenic plant fine seeds under very high sait conditions far exceeded the rate seen in wild type control seeds. Whereas over 81 % of the transgenic plant seeds had germinated under the high salt conditions, only about 9% of the wild type control plant seeds had 20 germinated by the same time point. These data indicate that the transgenic seeds are capable of germinating very well under high sait concentrations, an important trait for plant growth in areas of increasingly high water and/or soil salinity, 25 TABLE XXI:

TRANSGENIC TOBACCO PLANTS GERMINATE AND TOLERATE HIGH SALT

Plant type Control (0 mM Nad) Test (200 mM NaCi)a % Germination % Germination Wi id type 92, 87, 94 .-9, ΪΪ, 8 Transgene iine XX-3 92, 91, 94 84, 82, 78 87 2016202733 28 Apr 2016

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, 5 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 10 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. 88

Claims (38)

1. CLAIMS:

   1. A transgenic plant comprising a glutamine phenylpyruvate transaminase (GPT) transgene and a glutamine synthetase (GS) transgene, wherein each of said GPT transgene and said GS transgene is operably linked to a plant promoter, and wherein said GPT transgene encodes a polypeptide having GPT activity and wherein said GS transgene encodes a polypeptide having GS activity.
2. 2. The transgenic plant of claim 1, wherein the GS transgene is a GS1 transgene.
3. 3. The transgenic plant of claim 1 or 2, wherein said GPT transgene encodes a polypeptide having at least 80% sequence identity to any one of SEQ ID NO: 2; SEQ ID NO: 9; SEQ ID NO: 11; SEQ ID NO: 13; and SEQ ID NO: 15.
4. 4. The transgenic plant of claim 1 or 2, wherein said GPT transgene encodes a polypeptide having at least 80% sequence identity to SEQ ID NO: 19 or SEQ ID NO: 21.
5. 5. The transgenic plant of claim 3, wherein the GS transgene encodes a polypeptide having at least 85% sequence identity to SEQ ID NO: 4 or SEQ ID NO: 7.
6. 6. The transgenic plant according to any one of claims 1-5, wherein the GPT and GS transgenes are incorporated into the genome of the plant.
7. 7. The transgenic plant of claim 6, wherein the transgenic plant is a monocotyledonous plant.
8. 8. The transgenic plant of claim 6, wherein the transgenic plant is a dicotyledonous plant.
9. 9. The transgenic plant according to any one of claims 1-8, wherein said GPT transgene is operably linked to a plant promoter having preferred expression in photosynthetic plant tissues.
10. 10. The transgenic plant according to any one of claims 1-9, wherein said GS transgene is operably linked to a plant promoter having preferred expression in photosynthetic plant tissues.
11. 11. The transgenic plant according to any one of claims 1-8 and 10, wherein said GPT transgene includes a plant promoter, wherein the plant promoter is a cauliflower mosaic virus 35S ribosomal promoter.
12. 12. The transgenic plant according to any one of claims 1-11 or a progeny thereof, wherein the transgenic plant or the progeny thereof has an increased leaf-to-root ratio of 2-oxo-glutaramate in comparison to an analogous wild type or untransformed plant of the same species.
13. 13. The transgenic plant according to any one of claims 1 -12 or a progeny thereof, wherein the transgenic plant or the progeny thereof has an increased leaf-to-root ratio of GPT activity in comparison to an analogous wild type or untransformed plant of the same species.
14. 14. The transgenic plant according to any one of claims 1-13 or a progeny thereof, wherein the transgenic plant or the progeny thereof has an increased leaf-to-root ratio of GS activity in comparison to an analogous wild type or untransformed plant of the same species.
15. 15. The transgenic plant of any one of claims 1-14 which displays an increased growth rate, increased biomass yield, increased seed yield, increased flower or flower bud yield, increased fruit or pod yield, larger leaves, increased GPT activity, increased GS activity, increased 2-oxoglutaramate levels, increased nitrogen use efficiency, increased tolerance to salt or saline conditions, or any combination thereof when compared to an analogous wild-type or untransformed plant of the same species.
16. 16. A progeny of any generation of the transgenic plant of any one of claims 1-15, wherein said progeny comprises said GPT transgene and said GS transgene.
17. 17. A seed of any generation of the transgenic plant of any one of claims 1 -15, wherein said seed comprises said GPT transgene and said GS transgene.
18. 18. A method for increasing growth characteristics of a plant relative to an analogous wild type or untransformed plant, comprising: a. introducing a glutamine phenylpyruvate transaminase (GPT) transgene into a plurality of plants, wherein the GPT transgene encodes a polypeptide having GPT catalytic activity; b. introducing a glutamine synthetase (GS transgene) into the plurality of plants or progeny thereof, wherein the GS transgene encodes a polypeptide having GS catalytic activity; c. expressing the GPT transgene and the GS transgene in the plurality of plants or the progeny thereof; and d. selecting a plant having an increased growth characteristic relative to an analogous wildtype or untransformed plant of the same species.
19. 19. A method for increasing growth characteristics of a plant relative to an analogous wild type or untransformed plant, comprising: a. generating a plurality of transgenic plants by: i. introducing a glutamine phenylpyruvate transaminase (GPT) transgene into a plurality of plants and introducing a glutamine synthetase (GS) transgene into the plurality of plants or progeny thereof, or ii. introducing a GS transgene into the plurality of plants and introducing a GPT transgene into the plurality of plants or progeny thereof; wherein said GPT transgene encodes a polypeptide having GPT activity and wherein said GS transgene encodes a polypeptide having GS activity; b. expressing the GS transgene and the GPT transgene in the plurality of plants or the progeny thereof; and, c. selecting a plant having an increased growth characteristic relative to an analogous wild type or untransformed plant of the same species.
20. 20. A method of increasing growth characteristics of a plant relative to an analogous wild type or untransformed plant, comprising: a. introducing a glutamine phenylpyruvate transaminase (GPT) transgene into a first plurality of plant cells and generating a first plurality of transgenic plants from said first plurality of plant cells, wherein said GPT transgene encodes a polypeptide having GPT activity; b. introducing a glutamine synthetase (GS) transgene into a second plurality of plant cells and generating a second plurality of transgenic plants from said second plurality of plant cells, wherein said GS transgene encodes a polypeptide having GS activity; c. selecting a first plant from the first plurality of transgenic plants or the progeny thereof, said plant comprising the GPT transgene; and d. selecting a second plant from the second plurality of transgenic plants or the progeny thereof, said second plant comprising the GS transgene; and e. crossing the first and second plants to produce a plurality of double transgenic plants, said double transgenic plants having increased production of 2-oxo-glutaramate and at least one increased growth characteristic relative to an analogous wild type or untransformed plant of the same species.
21. 21. The method according to any one of claims 18 to 20, wherein the increased growth characteristic is selected from the group consisting of increased growth rate, increased biomass, earlier flowering, earlier budding, increased plant height, increased flowering, increased budding, larger leaves, increased fruit or pod yield, increased seed yield, increased GPT activity, increased GS activity, increased nitrogen use efficiency, and increased tolerance to salt or saline conditions when compared to an analogous wild-type or untransformed plant of the same species .
22. 22. A method of increasing growth characteristics of a plant relative to an analogous wild type or untransformed plant, comprising: a. generating a transgenic plant by: i. introducing a glutamine phenylpyruvate transaminase (GPT) transgene into a plant and introducing a glutamine synthetase (GS) transgene into the plant or progeny thereof, or ii. introducing a GS transgene into a plant and introducing a GPT transgene into the plant or progeny thereof; wherein said GPT transgene encodes a polypeptide having GPT activity and wherein said GS transgene encodes a polypeptide having GS activity; and b. expressing the GS transgene and the GPT transgene in the plant or the progeny thereof.
23. 23. A method of increasing growth characteristics of a plant relative to an analogous wild type or untransformed plant, comprising: a. introducing a glutamine phenylpyruvate transaminase (GPT) transgene into a plant, wherein the GPT transgene encodes a polypeptide having GPT catalytic activity; b. introducing a glutamine synthetase (GS transgene) into the plant or progeny thereof, wherein the GS transgene encodes a polypeptide having GS catalytic activity; and c. expressing the GPT transgene and the GS transgene in the plant or the progeny thereof.
24. 24. The method according to any one of claims 18-23, wherein said GPT transgene encodes a polypeptide having at least 80% sequence identity to any one of SEQ ID NO: 2; SEQ ID NO: 9; SEQ ID NO: 11; SEQ ID NO: 13; and SEQ ID NO: 15, and having GPT catalytic activity.
25. 25. The method according to any one of claims 18-23, wherein said GPT transgene encodes a polypeptide having at least 80% sequence identity to SEQ ID NO: 19 or SEQ ID NO: 21, and having GPT catalytic activity.
26. 26. The method according to any one of claims 18-25, wherein the GS transgene encodes a polypeptide having at least 85% sequence identity to SEQ ID NO: 4 or SEQ ID NO: 7, and GS catalytic activity.
27. 27. The method according to any one of claims 18-26, wherein the GPT and GS transgenes are incorporated into the genome of the plant.
28. 28. The method according to any one of claims 18-27, wherein said GPT transgene is operably linked to a plant promoter having preferred expression in photosynthetic plant tissues.
29. 29. The method according to any one of claims 18-28, wherein said GS transgene is operably linked to a plant promoter having preferred expression in photosynthetic plant tissues.
30. 30. The method according to any one of claims 18-27 and 29, wherein said GPT transgene includes a plant promoter, wherein the plant promoter is a cauliflower mosaic virus 35S ribosomal promoter.
31. 31. The method according to any one of claims 18-30, wherein the transgenic plant or the progeny thereof has an increased leaf-to-root ratio of 2-oxo-glutaramate in comparison to an analogous wild type or untransformed plant of the same species.
32. 32. The method according to any one of claims 18-31, wherein the transgenic plant or the progeny thereof has an increased leaf-to-root ratio of GPT activity in comparison to an analogous wild type or untransformed plant of the same species.
33. 33. The method according to any one of claims 22-32, wherein the transgenic plant or the progeny thereof has an increased leaf-to-root ratio of GS activity in comparison to an analogous wild type or untransformed plant of the same species.
34. 34. The method according to any one of claims 18-23, wherein said plant displays an increased growth rate, increased biomass yield, increased seed yield, increased flower or flower bud yield, increased fruit or pod yield, larger leaves, increased GPT activity, increased GS activity, increased 2-oxoglutaramate levels, increased nitrogen use efficiency, increased tolerance to salt or saline conditions, or any combination thereof when compared to an analogous wild-type or untransformed plant of the same species.
35. 35. The method according to any one of claims 18-34, further comprising harvesting seeds from said plant and selecting a seed that demonstrates increased germination in high salt conditions.
36. 36. The method according to claim 35, further comprising propagating a plant from the seed so selected and harvesting a seed therefrom.
37. 37. A progeny of any generation of a transgenic plant generated by a method according to any one of claims 18-34, wherein said progeny comprises said GPT transgene and said GS transgene.
38. 38. A seed of any generation of a transgenic plant generated by a method according to any one of claims 18-34, wherein said seed comprises said GPT transgene and said GS transgene.