# Patent

## Title
Plants having enhanced nitrogen use efficiency and methods of producing same

## Abstract

Methods are provided for enhancing yield and nitrogen use efficiency in plants, and to methods of increasing biomass and seed yield in plants grown under nitrogen limiting conditions using variants of enzymes involved in nitrogen assimilation or metabolism from non-plant organisms.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a 35 U.S.C. 371 National Phase of PCT Application Number PCT/EP2014/054171 filed Mar. 4, 2014, which claims priority to U.S. provisional application No. 61/773,770 filed Mar. 6, 2013, the disclosure of which is hereby incorporated by reference in its entirety.
JOINT RESEARCH AGREEMENT
Work disclosed herein was performed in accordance with the terms of a joint research agreement between Bayer CropScience AG, Monheim am Rhein, Germany (subsequently assigned to Bayer CropScience LP, Research Triangle Park, USA), The Governors of the University of Alberta, Alberta, Canada and Dr. Allen Good, Alberta, Canada.


FIELD OF INVENTION
The present invention relates to plants having enhanced yield and/or nitrogen utilization efficiency (NUE), to methods for enhancing yield and NUE in plants, and to methods of increasing biomass and seed yield in plants grown under nitrogen limiting conditions.
BACKGROUND OF THE INVENTION
The productivity of plants is limited by the three primary nutrients: nitrogen, phosphorous and potassium, in most natural and agricultural ecosystems. Generally nitrogen is the most important of the three limiting nutrients and the major components in fertilizers. Since nitrogen is usually the rate-limiting element in plant growth, most field crops have a fundamental dependence on inorganic nitrogenous fertilizer. The nitrogen source in fertilizer is usually ammonium nitrate, potassium nitrate, or urea (McAllister et al., 2012).
Increased nitrogen use efficiency by plants has a number of beneficial effects, for example, increased growth and yield when compared to conventional plants grown in nitrogen poor soils, and reduced requirement for the addition of nitrogenous fertilizers to crops (Good and Beatty, 2011a). Fertilizers account for a significant percentage of the costs associated with crop production, therefore using less fertilizer would reduce the producers' costs. A reduction in fertilizer application would also lessen the environmental damage resulting from extensive nitrogenous fertilizer use. Excess fertilizer application causes increased eutrophication, acid rain, soil acidification and the greenhouse effect. These environmental disasters cause further problems such as fish kills, loss of biodiversity, increased algal blooms, loss of arable land and accelerated global climate change, affecting the world population on both social and economic scales (Good and Beatty, 2011b).
Of the commercially grown plants, monocots (which include the main cereal crops) represent a large percentage of the crops grown in the world with approximately 217 million hectares of wheat and 158 million hectares of both maize and rice planted in 2007. Approximately half of the global calorie and protein requirement is derived from wheat, rice and maize. Rice is routinely used as a model crop for genetic and physiological studies in other monocot crops including maize, wheat, sugarcane, barley, sorghum, rye and grass. Rice has a small, diploid genome that is well conserved and syntenic across monocots (McAllister et al., 2013).
In the case of NUE plant engineering, a number of different genes have been evaluated for their role in increasing the efficacy of N uptake, utilization or remobilization in the plant. One way to improve the nitrogen use efficiency (NUE) of the cereal crops would be to improve the different components of NUE. NUE can be partitioned into N uptake efficiency (NUpE) and N utilization efficiency (NUtE; Good et al., 2004). NUtE can be further reduced to N assimilation and N remobilization. Therefore, increasing the efficiency of either N uptake or N utilization, could lead to an increase in NUE of the crop. There have been a number of single genes targeted as candidates for genetic engineering to try and increase the NUE of crop plants (reviewed in Good and Beatty, 2011a and McAllister et al., 2012). Many of these candidate genes are primary N uptake and assimilation genes such as nitrate and ammonia transporters, nitrate reductase, GS and GOGAT. Gene targets that have shown an NUE phenotype in a crop plant after bioengineering overexpression include genes that are not primary N assimilation genes, but instead are involved in N metabolism further downstream than GS (glutamine synthase) and GOGAT (Glutamine oxoglutarate aminotransferase) such as alanine aminotransferase (AlaAT), or are transcriptional regulators, such as Dof1 (Good et al., 2007; Shrawat et al., 2008; Yanagisawa et al., 2000; Yanigisawa et al., 2004). While some of these genes have shown some efficacy, in most cases the over-expression of these genes, under the specific promoter used, did not result in any significant increase in NUE or components of NUE (reviewed in Good and Beatty, 2011a and McAllister et al., 2012).
SUMMARY OF THE INVENTION
The present invention relates to plants having enhanced yield and/or nitrogen utilization efficiency (NUE) and methods of producing such plants. The present invention also relates to methods for enhancing yield and NUE in plants, and to methods of increasing biomass and seed yield in plants grown under nitrogen limiting conditions. This invention also relates to the use of AlaAT encoding polynucleotides from non-plant organisms in plants, and the use of different variants of AlaAT encoding polynucleotides from organisms such as Archaea and mammals in plants under control of a plant-expressible promoter.
It is an object of the invention to provide an improved plant having enhanced nitrogen efficiency.
According to the present invention there is provided a non-naturally occurring plant or plant part from a non-naturally occurring plant comprising, elevated levels of one or more different nitrogen utilization proteins, as described above from a plant or non-plant under control of a plant-expressible promoter.
The one or more nitrogen utilization proteins may be selected from aminotransferases, alanine aminotransferase (AlaAT) and aspartate aminotransferase. The one or more nitrogen utilization proteins may be variants of AlaAt encoding polynucleotides from non-plants, such as from Archaea and mammals.
The non-naturally occurring plant or plant part may be selected from corn, wheat, maize, rice, barley, canola, soybean, cotton, alfalfa, safflower, sugarcane, tomato and potato.
The present invention also provides a non-naturally occurring plant or plant part wherein the plant or plant part comprises a first nucleic acid encoding one or more non-naturally occurring AlaAT gene operably linked to a second nucleic acid comprising a promoter, such as, but not limited to, a tissue-specific promoter.
The present invention also pertains to a seed obtained from the non-naturally occurring plant as described above.
The present invention provides a method of generating a plant with increased nitrogen use efficiency comprising, introducing a nucleic acid encoding one or more nitrogen utilization protein, operably linked to a promoter, such as, but not limited to, a tissue-specific promoter, and producing the plant, the plant comprising elevated levels of one or more nitrogen utilization protein. The one or more nitrogen utilization protein may be selected from the group consisting of the aminotransferases. Preferably, the one or more nitrogen utilization proteins is alanine aminotransferase.
To generate monocot plants that are able to grow under suboptimal nutrient conditions, a monocot plant may be generated that uses nitrogen more efficiently. Such monocot plants are able to grow in soils that are poorer in nitrogen, as a result of being able to more efficiently use the nitrogen that is available, with no loss in yield. Additionally, such monocot plants may also demonstrate enhanced yield in soils that have normal nitrogen levels as well. Nitrogen use efficiency in plants is a result of two main subcomponents; N uptake efficiency and N utilization efficiency. A plant exhibiting a nitrogen use efficiency phenotype may have improvements in its ability to take up nitrogen from the soil, which is a desired trait in plants growing in lower nutrient-available soils. Or the NUE plant may have improvements in its ability to utilize the N that it has taken up so that the available N (whether it be low or high) is efficiently incorporated into the subcellular components (such as nucleic acids, proteins, storage etc.), translocated to the necessary tissues and remobilized at the correct developmental stage into seed. Or, another possibility is that the NUE plant has improvements in both N uptake and utilization. Any one of these possibilities would allow for an increased yield from a NUE crop grown in normal nitrogen conditions because those plants would be able to take up a non-limiting amount of nitrogen and be able to use the nitrogen to increase biomass and seed yield, either by increased number of seeds or an increase in seed weight, or both.
This present invention will allow the user to develop plants that have an environmental benefit in that they can maintain yield, while reducing the need for high levels of nutrient application. Alternatively, under high nutrient levels this invention would improve plant nutrient up-take allowing plants to extract more nutrients from their environment during times of nutrient abundance. Using the methods and compositions of the invention, plants may be improved for growth and development under environmental conditions usually unsuitable for growth of the plant. Furthermore, the methods and compositions of the invention permit the genetic engineering of a plant to alter one or more plant characteristics in only selected tissues of the plant.
The present invention provides a method for directing expression of a specific target gene or nucleotide sequence in a plant, including producing a plant from a transformed plant cell such that tissue-specific expression of a target gene or nucleotide sequence occurs within a selected tissue of the plant, wherein the transformed plant cell contains a target gene or nucleotide sequence for a novel variant to AlaAT.
The present invention further provides a method for increasing biomass of a plant growing under one or more environmentally adverse conditions comprising: transforming a plant with a novel target gene or nucleotide sequence in operative linkage with an OsANT1 or PBpr1 promoter element to produce a transformed plant, the target gene or nucleotide sequence encoding an enzyme involved in nitrogen assimilation or metabolism; and growing the transformed plant.
This invention also relates to the use of AlaAT encoding polynucleotides from non-plant organisms in plants, and the use of different variants of AlaAT encoding polynucleotides from other organisms, such as Archaea and mammals, in plants under control of a plant-expressible promoter.
Also embraced within the present invention is a method for increasing biomass of a plant growing under conditions of low nitrogen comprising: transforming a plant with a target gene or nucleotide sequence in operative linkage with an OsANT1 or PBpr1 promoter element to produce a transformed plant, the target gene or nucleotide sequence encoding an enzyme involved in nitrogen assimilation; and growing the transformed plant. Preferably, the target gene or nucleotide sequence encodes an enzyme that is a variant of AlaAT.
The present invention also pertains to a method for increasing the biomass of a plant growing under one or more environmentally adverse conditions comprising; transforming a plant with a gene or nucleotide sequence encoding AlaAT in operative linkage with a promoter element to produce a transformed plant; and growing the transformed plant.
The present invention is also directed to a method for increasing seed yield of a plant comprising: transforming the plant with a target gene or nucleotide sequence or nucleotide sequence in operative linkage with a specific promoter element, to produce a transformed plant, the target gene or nucleotide sequence or nucleotide sequence encoding an enzyme involved in nitrogen assimilation or metabolism; and growing the transformed plant. Preferably, the target gene or nucleotide sequence encodes an enzyme that is a variant of AlaAT.
Also embraced within the present invention is a method for increasing seed yield of a plant growing under conditions of high nitrogen comprising; transforming a plant with a target gene or nucleotide sequence or nucleotide sequence in operative linkage with an promoter element to produce a transformed plant, the target gene or nucleotide sequence or nucleotide sequence encoding an enzyme involved in nitrogen assimilation; and growing the transformed plant. Preferably, the target gene or nucleotide sequence encodes an enzyme that is a variant of AlaAT.
This summary of the invention does not necessarily describe all features of the invention.



BRIEF DESCRIPTION OF THE DRAWINGS
These and other features of the invention will become more apparent from the following description in which reference is made to the appended drawings wherein:
FIG. 1 outlines the components of the various constructs contemplated by the present invention. Each of these genetic constructs has a promoter operatively linked to a variant of AlaAT (including HvAlaAt, MmAlaAT1, MmAlaAT2 and PfAlaAt. These constructs are given the designation of Promoter::AlaATgene and terminated by a nos terminator. The selection marker is kanamycin (Kan) for plasmid selection and hygromycin (Hyg) for plant selection.
FIGS. 2A and 2B show the effects of specific constructs on flowering time in Arabidopsis thaliana. FIG. 2A represents the constructs introduced into the ecotype Columbia (COL background); and FIG. 2B represents the constructs introduced into the alaat1;2 double knockout line of Arabidopsis thaliana (alaat1;alaat2 Background).
FIGS. 3A and 3B show the effects of specific constructs introduced into the ecotype Columbia on average change in tap root growth (cm) over time (DAS=days after inbibition/starting) under two different conditions. FIG. 3A shows tap root growth under the condition of low light, 0% sucrose and 1 mM nitrate (NO3−); and FIG. 3B shows tap root growth under the condition of low light, 0.2% sucrose and 0.25 NO3−.
FIG. 4 shows Arabidopsis plants grown on modified 1⁄2 MS (Murashige and Skoog medium) supplemented with 2.5 mM alanine as the sole N source at 18 DAS. WT (COL) and AlaAT over-expressers (OE) were grown on the same plate and “blocks” are replicates that were moved accordingly. OE lines are a COL background. A: 35S::PfAlaAT; B: 35S::MmAlaAT; C: OsAnt1::HvAlaAT; D: 35S::PfAlaAT; E: 35S::MmAlaAT; F: OsAnt1::HvAlaAT.
FIG. 5 shows Arabidopsis plants grown on modified 1⁄2 MS supplemented with 2.5 mM alanine as the sole N source at 21 DAS. WT (COL) and AlaAT over-expressers (OE) were grown on the same plate. OE lines are a COL background over-expressing MmAlaAT1 (35S:MmAlaAT1 line 2-1-7).
FIG. 6 shows Arabidopsis plants grown on modified 1⁄2 MS supplemented with 2.5 mM glutamate as the sole N source. WT (COL) and AlaAT over-expressers (OE) were grown on the same plate. OE lines are a COL background over-expressing PfAlaAT (35S:PfAlaAT line 4-1).
FIG. 7 shows the change in tap root growth (cm) between 0-5 DAS of control (COL) and AlaAT over-expressers (OE) in COL background grown on modified 1⁄2 MS supplemented with 0.25 mM NO3− as the sole N source. A: OsAnt1::HvAlaAT; B: 35S::HvAlaAT; C: 35S::MmAlaAT2; D: 35S::MmAlaAT1; E: 35S::PfAlaAT.
FIG. 8 shows the change in tap root growth (cm) between 5-8 DAS of control (COL) and AlaAT over-expressers (OE) in COL background grown on modified 1⁄2 MS supplemented with 0.25 mM NO3− as the sole N source. A: OsAnt1::HvAlaAT; B: 35S::HvAlaAT; C: 35S::MmAlaAT2; D: 35S::MmAlaAT1; E: 35S::PfAlaAT.
FIG. 9 shows the change in tap root growth (cm) between 0-5 DAS of control (COL) and AlaAT over-expressers (OE) in COL background grown on modified 1⁄2 MS supplemented with 2 mM NO3− as the sole N source. A: OsAnt1::HvAlaAT; B: 35S::HvAlaAT; C: 35S::MmAlaAT2; D: 35S::MmAlaAT1; E: 35S::PfAlaAT.
FIG. 10 shows the change in tap root growth (cm) between 5-8 DAS of control (COL) and AlaAT over-expressers (OE) in COL background grown on modified 1⁄2 MS supplemented with 2 mM NO3− as the sole N source. A: OsAnt1::HvAlaAT; B: 35S::HvAlaAT; C: 35S::MmAlaAT2; D: 35S::MmAlaAT1; E: 35S::PfAlaAT.
FIG. 11 shows SEQ ID NO:1, which is the nucleotide sequence of barley (Hordeum vulgare) AlaAT used herein.
FIG. 12 shows SEQ ID NO:2, which is the amino acid sequence of barley (Hordeum vulgare) AlaAT used herein.
FIG. 13 shows SEQ ID NO:3, which is the nucleotide sequence of mouse (Mus musculus) AlaAT1 used herein.
FIG. 14 shows SEQ ID NO:4, which is the nucleotide sequence of Pyrococcus furiosus AlaAT used herein.
FIG. 15 SEQ ID NO:5, which is the nucleotide sequence of the pPBpr1 promoter element used herein.
FIG. 16 shows SEQ ID NO:6, which is the nucleotide sequence of the maize Ubiquitin 1 promoter elements, plus its first intron used herein.
FIG. 17 shows amino acid sequence comparisons of various alanine aminotransfereases (AlaAT's). Saccharomyces cerevisea AlaAT1 (SEQ ID NO:7); Saccharomyces cerevisea AlaAT2 (SEQ ID NO:8); Arabidopsis thaliana AlaAT1 (SEQ ID NO: 9); Arabidopsis thaliana AlaAT2 (SEQ ID NO: 10); Medicago truncatula AlaAT (SEQ ID NO: 11); Arabidospsis thaliana GGT1 (SEQ ID NO: 12); Arabidospsis thaliana GGT2 (SEQ ID NO: 13); Pyrococcus furiosus AlaAT (SEQ ID NO: 14); Mus musculus AlaAT1 (SEQ ID NO: 15); Mus musculus AlaAT2 (SEQ ID NO: 16); Homo sapiens AlaAT1 (SEQ ID NO: 17); Homo sapiens AlaAT2 (SEQ ID NO: 18).
FIG. 18 shows average biomass and average seed weight from T1 transgenic Oryza sativa plant lines (11 lines with each 6 plants selected by biomass and seed weight). Hatched bars: average biomass; solid bars: average seed weight.



DETAILED DESCRIPTION
The present invention relates to monocot plants having enhanced yield and/or nitrogen utilization efficiency (NUE), to methods of producing such plants, to methods for enhancing yield and NUE in monocot plants, and to methods of increasing biomass and seed yield in monocot plants grown under nitrogen limiting conditions. This invention also relates to variants of AlaAT and their operative linkage with different promoters, for example, without limitation, the cauliflower mosaic virus promoter CaMV 35S and tissue specific promoters (such as OsANT1 and PBpr1; Shrawat et al., 2008; Lock, 2011). The present invention also relates to the use of AlaAT encoding polynucleotides from non-plant organisms in plants, and the use of different variants of AlaAT encoding polynucleotides from organisms such as Archaea and mammals in plants under control of a plant-expressible promoter.
The following description is of a preferred embodiment.
The headings provided are not meant to be limiting of the various embodiments of the invention. Terms such as “comprises”, “comprising”, “comprise”, “includes”, “including” and “include” are not meant to be limiting. In addition, the use of the singular includes the plural, and “or” means “and/or” unless otherwise stated. Unless otherwise defined herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.
The language “tissue-specific expression of a target nucleotide sequence” is known in the art and includes the expression of a target nucleotide sequences in only selected tissues; although the target nucleotide sequence may be present in multiple tissues, it is expressed in only a subset of those tissues. Such selective expression may be due to the influence of one or more regulatory genetic elements, for example but not limited to promoter elements, repressor elements, enhancer elements, or other regulatory factors that may interact with DNA, RNA.
The language “target gene” or “target nucleotide sequence” is art-recognized, and includes any nucleotide sequence which is desirably expressed in one or more selected plant tissues. Examples of target genes or nucleotide sequences which may advantageously be utilized in conjunction with the methods of the invention include genes or nucleotide sequences involved in nitrogen assimilation and/or utilization, genes or nucleotide sequences involved in stress resistance or disease and pest resistance, and genes or nucleotide sequences involved in nutrient uptake and utilization. Such genes or nucleotide sequences are well known to one of skill in the art.
The language “plant” is art-recognized, and includes any monocotyledenous or dicotyledenous plant. Examples of plants for use in the invention include but are not limited to canola, barley, maize, rice, tobacco, soybean, cotton, alfalfa, tomato, wheat, potato, Arabidopsis thaliana and certain tree genera, including conifers and Populus species.
The language “non-plant” includes any other organisms that are not plants, such as, but not limited to, microorganisms (for example, without limitation, bacteria, yeast, fungi, algae, Archaea, virus), mammals and non-mammals (for example, without limitation, arthropods, birds, reptiles, amphibians and fish).
The language “operative linkage” is art-recognized, and includes the placement of a target nucleotide sequence relative to a nucleic acid regulatory sequence such that the expression of the target nucleotide sequence is controlled by the regulatory sequence. This regulatory sequence can have a positive effect (increase) on the expression of the target gene or nucleotide sequence (e.g., the regulatory sequence is a promoter or an enhancer element), or the regulatory sequence can reduce the expression of the target gene or nucleotide sequence (e.g., the regulatory sequence is a repressor element). The regulatory sequence may be physically located 5′ or 3′ of the target gene or nucleotide sequence, may be within the coding sequence of the target gene or nucleotide sequence, or may be contained on an intron within the target gene or nucleotide sequence.
The language “nulls” is art-recognized and includes a plant that has undergone tissue culturing but does not carry a transgene or selectable marker.
Production of plants which express one or more target genes or nucleotide sequences under a plant-expressible promoter are described herein. The invention further provides seeds containing one or more target genes or nucleotide sequences under the control of a plant-expressible promoter, such as a tissue-specific promoter element that specifically directs the tissue-specific expression of the gene or nucleotide sequence. By the methods of the invention, it is possible to produce plants having one or more desired traits or properties in selected tissues; e.g., to alter specifically the genetic and/or physiological properties of the fruit or the roots of the plant. The invention further provides methods of producing plants having root and leaf-specific expression of one or more desired nucleotide sequences, using the OsANT1 and PBpr1 promoter elements (Shrawat et al., 2008; Lock, 2011).
The methods of the invention for the production of plants involve the operative linkage of one or target genes or nucleotide sequences to a genetic regulatory element, such as, but not limited to, a promoter. As is known in the art, promoters are nucleic acid sequences that allow for regulation of transcription of a gene or nucleotide sequence. Promoters can allow for constitutive expression, such as the well known cauliflower mosaic virus promoter CaMV 35S, inducible expression, such as the stress inducible promoter rd29A (Pino et al., 2007), tissue-specific expression, such as the root-specific OsANT1 promoter (Good et al., US 2009/0288224), and developmentally specific expression, such as the senescence induced IPT promoters (Ma 2008). Promoters can also be weak or strong, suggesting that whenever or wherever they are induced, they will allow for expression of the attached gene or nucleotide sequence at varying levels.
The methods of the invention for the production of plants having tissue-specific expression of one or more target genes or nucleotide sequences are accomplished through the use of a genetic regulatory element which directs the tissue-specific expression of the target gene(s). This regulatory element may be either negative or positive in activity: a plant tissue-specific promoter or enhancer element permits the expression of the target gene or nucleotide sequence(s) in one or more specific tissues, whereas a plant tissue-specific repressor suppresses the expression of the target genes or nucleotide sequences in one or more specific tissues, while expression in the other tissue(s) continues unabated. For the purposes of the present invention, it will be understood that promoter sequences constitute the preferred genetic regulatory elements of the invention.
It will be understood by one skilled in the art that modifications may be made to the promoters used in the methods and constructs of the invention to improve or modulate the activity of the promoter. Multiple copies of a selected promoter may be operatively linked to a single target gene or nucleotide sequence to thereby alter the expression level of the linked gene or nucleotide sequence, or a selected promoter may be operatively linked to one or more target genes or nucleotide sequences such that the expression of each target gene or nucleotide sequence is coordinately regulated. A promoter may be of any size appropriate to permit the tissue-specific functioning of the promoter. A promoter may be modified (e.g., by mutagenesis, deletion, insertion, or truncation) to alter the degree to which the operatively linked gene or nucleotide sequence is expressed in the selected tissue, or to alter the specificity of tissue expression directed by the promoter. Further, the placement of the promoter relative to the operatively linked target gene or nucleotide sequence may be modulated (e.g., moved further away or closer together) to attain a desired level of promoter-directed expression.
A target gene or nucleotide sequence of the invention may be any gene or nucleotide sequence which is desirably expressed in a plant, including the expression in a tissue-specific manner in a plant. General classes of target genes or nucleotide sequences that may be employed in the methods and constructs of the invention include one or more genes or nucleotide sequences encoding plant structural proteins, genes or nucleotide sequences encoding proteins involved in the transport and/or uptake of nutrients, genes or nucleotide sequences encoding enzymes and proteins involved in nutrient utilization, genes or nucleotide sequences encoding proteins involved in stimulation or continuation of plant growth. Further, the target gene or nucleotide sequence may be a nucleotide sequence which, when transcribed, is antisense to a native sequence, the transcription and translation of which is desired to be suppressed.
For example, which is not to be considered limiting in any manner, the genes or nucleotide sequences of interest are those encoding enzymes in the assimilation and/or metabolism of nitrogen. The genes or nucleotide sequences of interest may include genes or nucleotide sequences which encode proteins involved in assimilating ammonia into amino acids or use the formed amino acids in biosynthetic reactions, that is, “nitrogen utilization proteins”. Example of nitrogen utilization proteins include, but are not limited to, nitrate ammonium and amino acid transporters, glutamine synthetase (GS), asparagine synthetase (AS), glutamate synthase (also known as glutamate 2:oxogluturate amino transferase and GOGAT), asparaginase (ANS), glutamate dehydrogenase (GDH), aspartate aminotransferase (AspAT) and alanine aminotransferase (AlaAT) and those genes or nucleotide sequences which may be involved in Nutrient Use Efficiency (NUE) as described by Beatty et al. (2009), and in U.S. Pat. No. 7,589,257 (which is incorporated herein by reference).
The target gene or nucleotide sequence may be a gene or nucleotide sequence naturally expressed in a selected plant (endogenous), or it may be not naturally expressed in the selected plant (non-endogenous). The gene or nucleotide sequence may originate from a plant or from a non-plant, including, without limitation, viral, bacterial, Archaea or animal sources. Preferably, the gene or nucleotide sequence is heterologous to the promoter to which it is linked, in that it is not linked to an unmodified, inducible promoter to which the gene or nucleotide sequence is naturally linked. Thus, as used herein, the term “heterologous” with respect to a nucleic acid or DNA refers to a nucleic acid or DNA which is operably linked to a nucleic acid to which it is not naturally linked in nature. For example, a promoter is said to be heterologous with respect to a coding region, when that promoter is not naturally linked to that coding region.
As used herein, the term “endogenous” with respect to a nucleic acid molecule or a polypeptide in an organism refers to a nucleic acid molecule or a polypeptide having a nucleotide sequence or respectively an amino acid sequence, which is naturally occurring within cells of that organism. Nucleic acid molecules or polypeptides do not become endogenous to a particular organism because that organism or cells of that organism have been modified using, for example, recombinant DNA techniques to artificially contain such nucleic acid molecule or polypeptide. Therefore, as used herein, a “non-endogenous” nucleic acid molecule or polypeptide is a nucleic acid molecule or polypeptide that is not naturally occurring with cells of an organism, and may have been artificially introduced.
It is therefore contemplated herein that the target gene or nucleotide sequence be a non-plant, non-endogenous AlaAT for expression in a plant, such as SEQ ID NOs: 1, 3 or 4 and Mus musculus AlaAT2, which is known in the art (see for example McAllister et.al, 2013; which is incorporated herein by reference). The target gene or nucleotide sequence can be any non-plant nucleotide sequence having an nucleic acid sequence similarity of approximately 70% to the nucleic acid sequence of barley (Hordeum vulgare) AlaAT (SEQ ID NO:1), mouse (Mus musculus) AlaAT (SEQ IDNO:3), Mus musculus AlaAT2, (McAllister et.al, 2013), or Pyrococcus furiosus AlaAT (SEQ ID NO:4), or any amount between 70% and 100% amino acid sequence, for example, but not limited, to 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% nucleic acid sequence similarity to the nucleic acid sequence of barley (Hordeum vulgare) AlaAT, mouse (Mus musculus) AlaAT, Mus musculus AlaAT2, (McAllister et.al, 2013), or Pyrococcus furiosus AlaAT, or any amount therebetween.
Sequence identity or sequence similarity may be determined using a nucleotide sequence comparison program, such as that provided within DNASIS (for example, using, but not limited to, the following parameters: GAP penalty 5, # of top diagonals 5, fixed GAP penalty 10, k-tuple 2, floating gap 10, and window size 5). However, other methods of alignment of sequences for comparison are well-known in the art for example the algorithms of Smith & Waterman (1981, Adv. Appl. Math. 2:482), Needleman & Wunsch (J. Mol. Biol. 48:443, 1970), Pearson & Lipman (1988, Proc. Nat'l. Acad. Sci. USA 85:2444), and by computerized implementations of these algorithms (e.g. GAP, BESTFIT, FASTA, and BLAST), or by manual alignment and visual inspection.
Also included are nucleotide sequences that hybridize under stringent hybridization conditions to SEQ ID NO:1; SEQ ID NO:3; SEQ ID NO:4, or Mus musculus AlaAT2, (McAllister et.al, 2013). The present invention also includes a nucleotide sequence that hybridizes under stringent hybridization conditions to a compliment of SEQ ID NO:1; SEQ ID NO:3; SEQ ID NO:4, or Mus musculus AlaAT2, (McAllister et.al, 2013). These nucleotide sequences that hybridize to SEQ ID 1, 3, 4, or Mus musculus AlaAT2, (McAllister et.al, 2013), a complement of SEQ ID 1, 3, 4, or Mus musculus AlaAT2, (McAllister et.al, 2013), encode a protein that exhibits AlaAT activity.
Hybridization under stringent hybridization conditions is known in the art (see for example Current Protocols in Molecular Biology, Ausubel et al., eds. 1995 and supplements; Maniatis et al., in Molecular Cloning (A Laboratory Manual), Cold Spring Harbor Laboratory, 1982; Sambrook and Russell, in Molecular Cloning: A Laboratory Manual, 3rd edition 2001; each of which is incorporated herein by reference). An example of one such stringent hybridization conditions may be about 16-20 hours hybridization in 4×SSC at 65° C., followed by washing in 0.1×SSC at 65° C. for an hour, or 2 washes in 0.1×SSC at 65° C. each for 20 or 30 minutes. Alternatively, an exemplary stringent hybridization condition could be overnight (16-20 hours) in 50% formamide, 4×SSC at 42° C., followed by washing in 0.1×SSC at 65° C. for an hour, or 2 washes in 0.1×SSC at 65° C. each for 20 or 30 minutes, or overnight (16-20 hours), or hybridization in Church aqueous phosphate buffer (7% SDS; 0.5M NaPO4 buffer pH 7.2; 10 mM EDTA) at 65° C., with 2 washes either at 50° C. in 0.1×SSC, 0.1% SDS for 20 or 30 minutes each, or 2 washes at 65° C. in 2×SSC, 0.1% SDS for 20 or 30 minutes each.
In addition, the target gene or nucleotide sequence can be any non-plant nucleotide sequence having an amino acid sequence similarity of approximately 80% to the amino acid sequence of barley (Hordeum vulgare) AlaAT, mouse (Mus musculus) AlaAT or Pyrococcus furiosus AlaAT, or any amount between 80% and 100% amino acid sequence, for example, but not limited, to 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% amino acid sequence similarity to the amino acid sequence of barley (Hordeum vulgare) AlaAT, mouse (Mus musculus) AlaAT or Pyrococcus furiosus AlaAT, or any amount therebetween (see FIG. 17). For example, the following sequences may be used:

















mRNA NCBI
Protein NCBI


Species
Gene
Reference
Reference





H. sapiens
GPT
NM_005309.2
NP_005300.1


M. mulatta
GPT
XM_001093616.2
XP_001093616.1


C. lupus
GPT
XM_847258.2
XP_852351.2


B. taurus
GPT
NM_001083740.1
NP_001077209.1


M. musculus
Gpt
NM_182805.2
NP_877957.1


R. norvegicus
Gpt
NM_031039.1
NP_112301.1


D. rerio
gpt21
NM_001142774.1
NP_001136246.1


D. rerio
LOC100148522
XM_001919861.2
XP_001919896.1


S. cerevisiae
ALT1
NM_001181976.1
NP_013190.1


K. lactis
KLLA0F19162g
XM_455940.1
XP_455940.1


S. pombe
SPBC582.08
NM_001021084.1
NP_595176.1


318829
MGG_06503
XM_369988.2
XP_369988.2


N. crassa
NCU03973
XM_952519.2
XP_957612.1


A. thaliana
ALAAT2
NM_105892.4
NP_565040.2


A. thaliana
AlaAT1
NM_101591.5
NP_173173.3


O. sativa
Os10g0390500
NM_001071039.1
NP_001064504.1







Genbank
NCBI


Species
Protein
Accession No.
Reference





Thermococcus
Alanine
AEK72704.1



sp.
aminotransferase




Thermococcus
Aspartate
EEB72915.1
YP_002581272.1


sp.
aminotransferase




Thermococcus
Aspartate
ADT83250.1
YP_004070473.1


barophilus
aminotransferase




Thermococcus
Alanine

ZP_11216073.1


zilligii
aminotransferase




Thermococcus
aminotransferase 
AFL94660.1
YP_006424454.1


sp.
2




Thermococcus
Alanine
ACS33579.1
YP_002959443.1


gammatolerans
aminotransferase




Thermococcus
Alanine
ACJ15716.1
YP_002306613.1


onnurineus
aminotransferase




Thermococcus
Alanine
ACS90484.1
YP_002994833.1


sibiricus
aminotransferase




Thermococcus
alanine
BAD85283.1
YP_183507.1


kodakarensis
aminotransferase




Thermococcus
Alanine
AAK98527.1
ZP_09730551.1


litoralis
aminotransferase




Pyrococcus
Alanine
AEH24862.1
YP_004624134.1


yayanosii
aminotransferase




Pyrococcus sp.
alanine
AEC52815.1
YP_004424819.1



aminotransferase




Pyrococcus sp.
alanine
AFK22834.1
YP_006354909.1



aminotransferase




Pyrococcus
alanine

NP_143210.1


horikoshii
aminotransferase




Pyrococcus
alanine

NP_126507.1


abyssi
aminotransferase




Pyrococcus
Chains A, B, 

1XI9_A


furiosus
C and D alanine





aminotransferase









The target gene or nucleotide sequence may be, but not necessarily, modified as required. For example, the gene or nucleotide sequence may be modified to be transcribable and translatable in a plant system; for example, the gene or nucleotide sequence can be modified such that it contains all of the necessary poly-adenylation sequences, start sites and termination sites which allow the coding sequence to be transcribed into messenger ribonucleic acid (mRNA) and the mRNA to be translated into a functional protein in the selected plant system. Further, the target gene or nucleotide sequence may be modified such that its codon usage is more similar to that of native genes or nucleotide sequences or nucleotide sequences of the selected plant. Such target gene or nucleotide sequence modifications and the methods by which they may be made are well known in the art.
The methods and genetic constructs disclosed herein may be used to produce a plant or a plant part of any species capable of utilizing the promoter such that the transgenic plant, or non-natural plant, has tissue-specific expression of one or more desired genes or nucleotide sequences. Both monocotyledenous and dicotyledenous plants are amenable to such alteration. The invention is intended to be particularly applicable to, for example, crop plants (especially those of the genus Oryza), ornamental plants, and trees (particularly conifers and the genus Populus). Particularly suitable plants for the practice of the present invention include, but are not limited to, canola, barley, sugar cane, corn, canola, tobacco, soybean, cotton, alfalfa, tomato, wheat, potato, aspen, cottonwood, Arabidopsis thaliana, conifers and poplar, or parts of any of these plants, for example, roots, root tips, leaves, stems, flowers, apical buds, meristematic tissues, and the like.
The transgenic plants (non-natural plants), plant parts, and seeds produced according to the present invention may be further useful in breeding programs for the production of plant species having more than one desired trait. For example two transgenic plants of the invention each having expression of a desired transgene in differing plant tissues may be crossed to result in progeny transgenic plants having tissue-specific expression of both transgenes; or two transgenic plants of the invention each having expression of a different desired transgene in the same plant tissue may be crossed to result in progeny transgenic plants having tissue-specific expression of both transgenes. In this fashion it is possible to produce transgenic plants having a combination of desirable traits in selected tissue(s) of the plant.
Furthermore, it will be understood by one skilled in the art that different species of plants may be more or less amenable to genetic manipulation in general, and that, therefore, it may be advantageous to first transform a related species of the desired plant by the methods and with the constructs of the invention and to subsequently introduce the tissue-specific expression of the target gene or nucleotide sequence into the desired plant species by cross-breeding techniques. Such techniques and appropriately related plant species are well known to one skilled in the art.
Plant cells or protoplasts that have been transformed with the gene constructs of the present invention can be regenerated into differentiated plants using standard nutrient media supplemented with shoot-inducing or root-inducing hormone, using methods known to those skilled in the art (see, for example, Shahin, E. A. U.S. Pat. No. 4,634,674 and references therein, incorporated herein by reference in their entirety). Seeds may additionally be harvested from such transgenic plants using methods well known in the art and further used to re-grow the transgenic plants and hybrids of the invention.
Uses of the Invention
The methods and constructs of the invention allow the production of plants and seeds having expression of one or more desired genes or nucleotide sequences in one or more selected tissues of the plant. Thus, the methods and constructs of the invention permit the production of plants having one or more desired traits limited to selected plant tissues, thereby enabling the targeting of a trait to the tissue to which it is best suited, or avoiding the expression of a desirable gene or nucleotide sequence in a tissue where its effects are unwanted. There are a wide variety of specific applications of the invention, including, but not limited to, the production of plants having increased yield, stress tolerance, having improved nutrient uptake and/or utilization, having improved nutrient content and/or yields of desired compounds. Specific applications of the invention are further described below.
One application of the invention is in the production of plants better able to thrive on nutrient-poor soils. It is well known in the art that certain plant species, particularly crop plants, deplete the soil of nutrients necessary to sustain growth, such as nitrogen, phosphate, and potassium. In order to replenish the lacking nutrients, it is necessary either to fertilize the soil (an expensive and environmentally damaging procedure) or to cultivate plants known to deposit the depleted nutrient into the soil (e.g., clover or soybean in the case of nitrogen depletion), which may be crops that are less profitable or nutritive and therefore less desirable to grow. Frequent fertilizing to maintain optimal nutrients in the soil is costly in terms of agriculture outputs such as labour and fuel therefore crop plants are usually in a state of either nutrient abundance or depletion. Another application of the invention is in the production of plants better able to capture and utilize nitrogen when it is present in either an adequate or abundant supply in the soil before nitrogen loss due to leaching, volatilization or microbial degradation occurs. The methods of the invention permit the targeted expression of genes or nucleotide sequences involved in nutrient uptake (e.g., transport molecules) to those tissues in which the uptake occurs (e.g., the root or root hairs) to thereby improve the ability of the plant to absorb the nutrient from the environment. The invention may also be used to produce plants which express heterologous nutrient utilization nucleotide sequences, or optimized (for example, optimized for plant expression) native nutrient utilization nucleotide sequences, in selected tissues (e.g., the root or leaves) that permit more efficient use of the nutrient, such that less of the nutrient is required for the normal growth and functioning of the plant. Further, it is possible, using the methods of the invention, to express genes or nucleotide sequences involved in the use and uptake of nutrients not normally used by the plant in those plant tissues which are directly exposed to the different nutrient (e.g., root and leaf). In this fashion, plants which are able to grow and thrive on different nutrient sources (e.g., different nitrogen sources) may be produced.
Development of Novel Cereal Crops:
As described below, Oryza sativa (rice) plants may be transformed with novel gene contracts such that the plants ectopically express a nucleotide sequence encoding an enzyme involved in nitrogen assimilation or metabolism, for example, but not limited to, alanine dehydrogenase, glutamine synthetase, asparagine synthetase, glutamate synthase, asparaginase, glutamate dehydrogenase, aspartate aminotransferase, alanine aminotransferase, and those nucleotide sequences which may be involved in Nutrient Use Efficiency described by Beatty et al. (2009), and U.S. Pat. No. 7,589,257. The present invention contemplates transformation of Oryza sativa with a novel gene construct, as described above, operatively linked to a tissue-specific promoter. The transformed plant is grown under laboratory conditions to determine the beneficial effects of tissue-specific expression on plant growth and yield under controlled growth conditions.
As indicated in Example 4 below, transgenic plants expressing a gene or nucleotide sequence encoding an enzyme involved in nitrogen assimilation or metabolism under the control of a tissue-specific promoter (pPBpr1) exhibited higher biomass and seed weight than those plants expressing a gene or nucleotide sequence expressed under a constitutive promoter (maize ubiquitin 1 promoter, ubi-1) in the presence of an adequate supply of nitrogen.
Transgenic Oryza plants ectopically expressing a gene or nucleotide sequence encoding an enzyme involved in nitrogen assimilation or metabolism under the control of a tissue-specific promoter (pPBpr1) are also capable of optimising the utilization of available nitrogen under a range of environmental conditions thereby resulting in an increase in plant biomass, seed weight or a combination thereof.
Therefore, the present invention provides a method for increasing seed yield of a plant comprising; transforming the plant with a target gene or nucleotide sequence in operative linkage with a promoter element, to produce a transformed plant, the target gene or nucleotide sequence encoding an enzyme involved in nitrogen assimilation or metabolism; and growing the transformed plant.
The above description is not intended to limit the claimed invention in any manner, furthermore, the discussed combination of features might not be absolutely necessary for the inventive solution.
The present invention will be further illustrated in the following examples. In order to assess whether enzyme isoforms of AlaAT have different kinetics and if different isoforms favor an NUE phenotype, AlaAT from mouse and Pyrococcus were tested.
EXAMPLES
Materials and Methods
Expression Vector Construction and Arabidopsis thaliana Transformation
The alanine aminotransferase enzymes assayed were chosen based on differences in their amino acid sequence as described by McAllister et al. 2012 (see also FIG. 17) and the availability of a cloned gene. Hordeum vulgare AlaAT (GenBank accession no. Z26322) (HvAlaAT) cDNA, described by Muench and Good (1994), as well as the pCAMBIA 1300 vector containing HvAlaAT driven by an OsANT1 promoter (OsANT1:HvAlaAT) and OsANT1 promoter driving β-glucuronidase (Shrawat et al., 2008) were obtained from Ashok Shrawat, University of Alberta. Mouse (Mus musculus) AlaAT1 (MmAlaAT1) (GenBank accession no. NP_877957) and mouse (Mus musculus) AlaAT2 (MmAlaAT2) (GenBank accession no. NP_776291) were both obtained from Rong ze Yang, University of Maryland (Yang et al., 2009). Pyrococcus furiosus AlaAT (PfAlaAT) (GenBank accession no. NP_579226) was amplified from ATCC gDNA (DSM 3638). Restriction enzyme cut sites were incorporated at the 5 and 3 prime ends of each cDNA using primers specific to each AlaAT as outlined in detail in McAllister et al., 2012. HvAlaAT construction of the AlaAT plasmids was done as outlined in McAllister et al., 2012. The nucleotide sequences of barley (Hordeum vulgare) AlaAT, mouse (Mus musculus) AlaAT1, and Pyrococcus furiosus AlaAT used herein are provided as FIGS. 11, 13 and 14, respectively (SEQ ID NOs: 1, 3 and 4, respectively). Mouse (Mus musculus) AlaAT2 is as previously described. The final AlaAT constructs for transformation into Arabidopsis thaliana, the initial AlaAT source, the promoter used to drive AlaAT expression in vivo, as well as the binary vectors utilized for transformation are summarized in Table 1.







TABLE 1







List of promoters, genes and sequences used to build the specific


expression vectors for transformation into Arabidopsis thaliana.
















Ter-
Vector


Vector


Accession
mi-
Back-


Name
Promoter
Gene
#
nator
bone





Arabi-
2XCaMV35S
HvAlaAT
Z26322
nos
binary


dopsis




vector







pMDC32


Arabi-
2XCaMV35S
Mm1AlaAT
NP_877957
nos
binary


dopsis




vector







pMDC32


Arabi-
2XCaMV35S
Mm2AlaAT
NP_776291
nos
binary


dopsis




vector







pMDC32


Arabi-
2XCaMV35S
PfAlaAT
NP_579226
nos
binary


dopsis




vector







pMDC32


Arabi-
OsAnt1
HvAlaAT
Z26322
nos
binary


dopsis




vector







pMDC32









Binary vectors were transformed into Agrobacterium tumefaciens strain GU3101 and selected using kanamycin. Agrobacterium cultures (OD600 0.3-0.7) were then used to transform Arabidopsis thaliana plants, both COL (ecotype Columbia background) and alaat1;2 knockout backgrounds, using floral dip (alaat1;2 knockout background is explained in detail in Miyashita, 2008). Transformed plants were selected by sowing seeds on hygromycin (Hyg) and using a protocol modified from Harrison et al. (2006), with plants left covered at 4° C. for 4 days and at room temperature for 3 days; PCR was carried out on all primary transgenics with primers specific to the AlaAT insert: for HvAlaAT, 5′-GAGGTTCTTGCCCTTTGTGA-3′ (SEQ ID No: 19) and 5′-TTCAGCTCGTTGCAAGTAA-3′ (SEQ ID No: 20); for MmAlaAT1, 5′-CCAGAGGATGCCAAGAGAAG-3′ (SEQ ID No: 21) and 5′-GCTCCGTGAGTTTAGCCTTG-3 (SEQ ID No: 22)′; for MmAlaAT2, 5′-GCAGGCTTGTGGTGGAAA-3′ (SEQ ID No: 23) and 5′-GCACTTTCTTAAAGGAGTGGAATC-3′ (SEQ ID No: 24); for PfAlaAT, 5′-GCGCTCTACGACAAAAAGACACTTGA-3′ (SEQ ID No: 25) and 5′-CGTTAGTCCTGCTATAGCTGCGAATT-3′ (SEQ ID No: 26). T3 seed was sown on Hyg media to select for homozygous lines; PCR was used to verify the presences of the specific AlaAT insertions. Three independent Arabidopsis insertion lines for each AlaAT construct, in both COL and alaat1;2 knockout backgrounds, were selected for and used for future analyses.
Plate Assays
Sterilized seeds were stratified in 0.15% (w/v) agar for approximately 48 hrs; sterilized seeds were sown onto square 100×100×15 mm petri plates containing modified 1⁄2 MS media (0.5% (w/v) sucrose and 0.8% (w/v) agar) with 0 mM KNO3−, 2 mM KNO3−, 0.25 mM KNO3−, 2.5 mM alanine or 2.5 mM glutamate as the sole nitrogen source. Seeds from both control lines, COL and alaat1;2, as well as three independent insertion lines each of OsANT1:HvAlaAT, CaMV35S:PfAlaAT, CaMV35S:HvAlaAT, CaMV35S:MmAlaAT1 and CaMV35S:MmAlaAT2, in both a COL and an alaat1;2 knockout background, were used for analysis on 0 mM, 0.25 mM and 2 mM KNO3− plates.
Transgenic plants containing OsANT1:HvAlaAT, CaMV35S:PfAlaAT and CaMV35S:MmAlaAT1, in both COL and alaat1;2 knockout backgrounds, were used for analysis on 2.5 mM glutamate and alanine. Plants were sown horizontally across square petri plates approximately 2 cm from the top of each plate; 6 plants were sown per plate. Control plants and transgenics were sown on the same plates in an alternating fashion. Three independent insertion lines for each AlaAT were assayed in quadruplicate along with control plants. Plants were grown vertically at 21° C., 60% humidity and a lighting cycle of 16 light/8 dark. Chambers were blocked for variations in lighting conditions, resulting in four blocks, with final lighting blocks containing a maximum difference of 20% across a single block with an average light intensity of 170 μE m−2 sec−1. Plates were moved within lighting blocks daily and monitored for changes in growth and development. Changes in vertical tap root length were measure (cm) between 0-5 after sowing (DAS), 5-8 DAS and 8-12 DAS for plants grown on 2 mM and 0.25 mM KNO3−. The vertical growth of tap roots of plants grown on 2.5 mM glutamate and alanine was measured (cm) between 0-5, 5-8, 8-12, 12-15, 18 and 21 DAS for changes in root length. Changes in tap root lengths over these time periods were then used to analyze the rate of tap root growth between transgenics and controls. At 18 DAS or 21 DAS (earlier if plants were senescing or had grown too large for plates) tap root lengths were measured (cm) on all 1⁄2 MS plates (top of hypocotyl to root tip).
1⁄2 MS plates were also prepared varying nitrogen and carbon amounts. Modified 1⁄2 MS (0.8% (w/v) agar) containing 1 mM KNO3− and 0% (w/v) sucrose was used to grow transgenic plants containing OsANT1:HvAlaAT, CaMV35S:PfAlaAT and CaMV35S:MmAlaAT1, in both COL and alaat1;2 knockout backgrounds, as well as control lines, in two different lighting conditions:  ̃170 μE m−2 sec−1 (high) and  ̃100 μE m−2 sec−1 (low) (see FIG. 3A). Modified 1⁄2 MS containing 0.25 mM KNO3− and 0.2% (w/v) sucrose was also used to grow all of the above lines in a low light condition ( ̃100 μE m−2 sec−1) (see FIG. 3B). Changes in vertical tap root length were measured (cm) between 0-5 DAS, 5-8 DAS, 8-12 DAS and 12-15 DAS for all plates (top of hypocotyl to root tip) to assess the rate of tap root growth between transgenic and control lines. Plating, blocking and data collection, as well as chamber conditions for the growth of plants on these plates, was as outlined previously.
Phenotypic similarity between individual plant lines expressing the same AlaAT construct in all plating conditions was assessed via one-way ANOVA (α=0.05, P value<0.05). Final tap root lengths of control plants and transgenic lines were compared using two-way ANOVA (P value<0.05), analyzing the genotype of plants and the lighting block plants were grown in.
Analysis of AlaAT Primary Structure
The enzymes with AlaAT activity that were obtained for expression studies and kinetic analysis and the amino acid sequences were compared using ClustalW software (full primary sequence comparison is provided in McAllister et al. 2012 (see FIG. 17).
Construction of Binary Vectors and Agrobacterium Mediated Transformation
The sequences of the genes using in the cloning are described in FIGS. 11 and 13-16 (SEQ ID NOs: 1 and 3-7, respectively) and the components for the gene constructs are provided in FIG. 1. PBpr1 promoter (SEQ ID NO: 5) was selected for tissue-specific expression in rice (Oryza sativa c.v. Nipponbare (NB)) and designed to be cloned using GeneArt (Invitrogen, Life Technologies, Carlsbad, Calif., USA). The constitutive promoter, maize Ubiquitin 1 promoter (SEQ ID NO: 6) was selected for expression in rice (Oryza sativa c.v. Nipponbare (NB)). The HvAlaAT cDNA (SEQ ID NO: 1) was introduced into pCAMBIA1300.
The constructs were transformed separately into Agrobacterium tumefaciens strain EHA105 by the freeze thaw method (Weigel and Glazebrook, 2002). The transformation of Arabidopsis thaliana is as described above.
Rice callus (Oryza sativa c.v. Nipponbare (NB)) was transformed with constructs, using an Agrobacterium transformation system developed in our laboratory (Shrawat and Good, 2011). The final AlaAT constructs for transformation into Oryza sativa c.v. Nipponbare (NB), the initial AlaAT source, the promoter used to drive AlaAT expression in vivo, as well as the binary vectors utilized for transformation are summarized in Table 2.







TABLE 2







List of promoters, genes and sequences used to build the specific


expression vectors for transformation into Oryza sativa.












Vector


Accession
Termi-
Vector


Name
Promoter
Gene
#
nator
Backbone





pGPMn
PBpr1
Mm1AlaAT
NP_877957
nos
pGPro2


pGPPn
PBpr1
PfAlaAT
NP_579226
nos
pGPro2


pGUMn
UBi1
Mm1AlaAT
NP_877957
nos
pGPro2


pGUPn
UBi2
PfAlaAT
NP_579226
nos
pGPro2









EXAMPLE 1
Novel AlaAT gene constructs (CaMV35S::MmAlaAT1;CaMV35S::PfAlaAT; and OsAnt1::HvAlaAT1) were introduced into either the alaat1;2 double knock out (DNK) (FIG. 2B) or the normal genetic background (COL background) (FIG. 2A).
The novel AlaAT gene constructs reduced the time to flowering in the transgenic Arabidopsis thaliana. 
Furthermore, the introduction of a novel AlaAT gene into either the normal genetic background or the DNK background, under two different conditions (as described above) increases the rate of root growth of the transgenic plant, and this rate changes as the plants get older (see FIGS. 3A and 3B). FIG. 3A illustrates the rate of tap root growth (cm) over 0-5, 5-8, 8-12 and 12-15 DAS with no sucrose added to the medium and 1 mM nitrate as the nitrogen source. FIG. 3B 3A illustrates the rate of tap root growth (cm) over 0-5, 5-8, 8-12 and 12-15 DAS with 0.2% sucrose added to the medium and 0.25 mM nitrate as the nitrogen source. The comparison between the two conditions suggests that the level of carbon availability or carbon/nitrogen ratio may have some impact on the rate of root development.
The effect of the novel AlaAT gene constructs (CaMV35S::MmAlaAT1; CaMV35S::PfAlaAT; and OsAnt1::HvAlaAT1) introduced into the normal genetic background (COL background) on root growth in Arabidopsis plants was determined at 18 DAS. As noted above, these plants were grown on 1⁄2 MS with 2.5 mM alanine or 2.5 mM glutamate as the only nitrogen source. The CaMV35S:PfAlaAT and CaMV35S:MmAlaAT grew significantly larger than the COL control; and both of them grew better than the OsANT1 promoter driving HvAlaAT. COL and AlaAT over-expressers (OE) were grown on the same plate and blocks are replicates that were moved accordingly.
EXAMPLE 2
In FIG. 5, wild-type Arabidopsis plants and transgenic Arabidopsis plants over-expressing CaMV35S::MMAlaAT1 were grown on modified 1⁄2 MS supplemented with 2.5 mM alanine as the sole nitrogen source. WT and AlaAT over-expressers (OE) were grown on the same plate. OE lines are a COL background over-expressing MmAlaAT1 (35S:MmAlaAT1 line 2-1-7).
In FIG. 6, wild-type Arabidopsis plants and transgenic Arabidopsis plants over-expressing CaMV35S::PfAlaAT were grown on modified 1⁄2 MS supplemented with 2.5 mM glutamate as the sole nitrogen source. WT and AlaAT over-expressers (OE) were grown on the same plate. OE lines are a COL background over-expressing PfAlaAT (35S:PfAlaAT line 4-1-2).
FIGS. 5 and 6 demonstrate that when a novel AlaAT gene is driven by the correct promoter, it has a significant effect on root growth, root length and overall root branching.
EXAMPLE 3
In FIG. 7, wild-type Arabidopsis plants and transgenic Arabidopsis plants over-expressing novel AlaAT genes (OsAnt1::HvAlaAT; CaMV35S::HvAlaAT; CaMV35S::MMAlaAT1; CaMV35S::MMAlaAT2; and CaMV35S::PfAlaAT) in a COL background were grown on modified 1⁄2 MS supplemented with 0.25 mM KNO3− as the sole nitrogen source. The change in tap root growth (cm) between 0-5 DAS was measured.
In FIG. 8, wild-type Arabidopsis plants and transgenic Arabidopsis plants over-expressing novel AlaAT genes (OsAnt1::HvAlaAT; CaMV35S::HvAlaAT; CaMV35S::MMAlaAT1; CaMV35S::MMAlaAT2; and CaMV35S::PfAlaAT) in a COL background were grown on modified 1⁄2 MS supplemented with 0.25 mM KNO3− as the sole nitrogen source. The change in tap root growth (cm) between 5-8 DAS was measured.
In FIG. 9, wild-type Arabidopsis plants and transgenic Arabidopsis plants over-expressing novel AlaAT genes (OsAnt1::HvAlaAT; CaMV35S::HvAlaAT; CaMV35S::MMAlaAT1; CaMV35S::MMAlaAT2; and CaMV35S::PfAlaAT) in a COL background were grown on modified 1⁄2 MS supplemented with 2 mM KNO3− as the sole nitrogen source. The change in tap root growth (cm) between 0-5 DAS was measured.
In FIG. 10, wild-type Arabidopsis plants and transgenic Arabidopsis plants over-expressing novel AlaAT genes (OsAnt1::HvAlaAT; CaMV35S::HvAlaAT; CaMV35S::MMAlaAT1; CaMV35S::MMAlaAT2; and CaMV35S::PfAlaAT) in a COL background were grown on modified 1⁄2 MS supplemented with 2 mM KNO3− as the sole nitrogen source. The change in tap root growth (cm) between 5-10 DAS was measured.
The experiments carried out for FIGS. 7 through 10 demonstrate that when a novel AlaAT gene is driven by the correct promoter, the significant effect on root growth, and root length occurs at a very early developmental stage.
EXAMPLE 4: TRANSGENIC RICE
Heterozygous T0 Seed Yield and Biomass Preliminary Screens
Transgenic rice (Oryza sativa) containing the specific genetic constructs above and outlined in FIG. 1 were produced using an Agrobacterium mediated approach, as described above. Leaf tissue was collected from two week old plants to determine if they were transgenic by means of a genomic DNA polymerase chain reaction (PCR) analysis using primers specific to the hygromycin resistance gene.
It was examined whether the amount of available nitrogen would have an effect on plant biomass and seed weight on plants expressing PBpr1::AlaAT grown under laboratory conditions when compared to similar plants that ectopically expressed the target nucleotide sequence under a constitutive promoter (maize ubiquitin 1).
Seed weights and biomass of the transgenic plants grown under laboratory conditions were examined at the dates provided below in Table 3. At the T0 stage, several of the transgenic plants expressing AlaAT in a tissue-specific manner had higher above ground biomass and seed weight compared to transgenic plants expressing AlaAT in a constitute manner (Table 3).







TABLE 3







Biomass and seed weight for transgenic Oryza sativa plants


expressing AlaAT in a tissue-specific or constitutive manner.
















bio-
seed





prod
mass
wt


plant #
seeding date
flowering date
tillers
(g)
(g)















pGPMn1/1-1
12 Jun. 2012
28 Aug. 2012
10
31.5
10.88


pGPMn1/1-2
12 Jun. 2012
17 Aug. 2012
9
25.73
10.06


pGPMn1/1-3
12 Jun. 2012
19 Aug. 2012
10
27.6
5.67


pGPMn1/1-4
12 Jun. 2012
19 Aug. 2012
8
30.26
11.52


pGPMn1/1-5
12 Jun. 2012
27 Aug. 2012
14
31.62
10.11


pGPMn1/1-6
12 Jun. 2012
18 Aug. 2012
13
34.64
8.26


pGPMn1/1-7
12 Jun. 2012
17 Aug. 2012
10
33.98
11.20


pGPMn1/1-8
12 Jun. 2012
23 Aug. 2012
9
31.78
12.16


pGPMn1/1-9
12 Jun. 2012
19 Aug. 2012
12
33.41
10.14


pGPMn1/1-10
12 Jun. 2012
17 Aug. 2012
10
29.43
9.66


pGPMn1/1-11
12 Jun. 2012
18 Aug. 2012
12
33.6
8.95


pGPMn1/1-12
12 Jun. 2012
23 Aug. 2012
10
29.28
9.30


pGPMn1/1-13
12 Jun. 2012
31 Aug. 2012
13
30.86
11.79


pGPMn1/1-14
12 Jun. 2012
21 Sep. 2012
10
28.51
9.98


pGPMn1/1-15
12 Jun. 2012
21 Sep. 2012
15
33.38
12.45


pGPMn1/1-16
25 Jun. 2012
2 Sep. 2012
13
28.78
5.91


pGPMn1/1-17
25 Jun. 2012
4 Sep. 2012
13
26.14
5.95


pGPMn1/1-20
25 Jun. 2012
9 Sep. 2012
16
36.15
14.88


pGPMn1/1-21
25 Jun. 2012
9 Sep. 2012
13
29.01
5.20


pGPPn2/1-2
12 Jun. 2012
20 Aug. 2012
10
32.07
12.99


pGPPn2/1-3
12 Jun. 2012
4 Sep. 2012
7
26.35
7.06


pGPPn2/1-4
12 Jun. 2012
28 Aug. 2012
8
24.56
8.29


pGPPn2/1-5
12 Jun. 2012
28 Aug. 2012
7
24.61
8.06


pGPPn2/1-6
12 Jun. 2012
20 Aug. 2012
9
27.35
9.69


pGPPn2/1-7
25 Jun. 2012
30 Sep. 2012
9
26.3
3.70


pGPPn2/1-8
25 Jun. 2012
4 Sep. 2012
13
36.19
12.32


pGPPn2/1-9
25 Jun. 2012
28 Aug. 2012
15
34.72
11.84


pGPPn2/2-1
28 Aug. 2012
15 Nov. 2012
14
35.34
5.66


pGPPn2/2-2
28 Aug. 2012
6 Nov. 2012
21
40.48
7.38


pGPPn2/2-3
11 Sep. 2012
15 Nov. 2012
14
34.17
13.24


pGPPn2/2-4
11 Sep. 2012
19 Nov. 2012
13
32.28
7.92


pGPPn2/2-5
11 Sep. 2012
23 Nov. 2012
11
38.3
11.38


pGPPn2/2-6
11 Sep. 2012
23 Nov. 2012
14
28.16
8.61


pGPPn2/2-7
11 Sep. 2012
28 Nov. 2012
9
28.4
9.94


pGPPn2/2-8
11 Sep. 2012
23 Nov. 2012
10
33.29
9.40


pGUMn3/1-1
10 Sep. 2012
19 Nov. 2012
11
27.36
6.14


pGUMn3/1-3
14 Sep. 2012
26 Nov. 2012
12
34.5
6.70


pGUMn3/1-4
14 Sep. 2012
26 Nov. 2012
13
32.94
8.43


pGUMn3/1-6
14 Sep. 2012
30 Nov. 2012
20
30.08
4.07


pGUMn3/1-7
14 Sep. 2012
6 Dec. 2012
18
30.61
3.03


pGUMn3/1-8
14 Sep. 2012
14 Nov. 2012
9
21.1
7.98


pGUPn4/1-1
10 Sep. 2012
18 Nov. 2012
11
22.71
3.28


pGUPn4/1-2
10 Sep. 2012
19 Nov. 2012
7
22.05
3.69


pGUPn4/1-4
11 Sep. 2012
12 Nov. 2012
8
18.54
3.31


pGUPn4/1-5
11 Sep. 2012
13 Nov. 2012
10
29.62
11.08


pGUPn4/1-6
11 Sep. 2012
19 Nov. 2012
9
29.8
12.74


pGUPn4/1-7
14 Sep. 2012
21 Dec. 2012
9
20.8
3.05


pGUPn4/1-8
14 Sep. 2012
25 Nov. 2012
6
20.93
2.07


pGUPn4/1-9
14 Sep. 2012
30 Nov. 2012
16
33.09
2.88


pGUPn4/1-10
14 Sep. 2012
30 Nov. 2012
13
31.06
7.68


pGUPn4/1-11
14 Sep. 2012
19 Nov. 2012
12
24.93
3.20


pGUPn4/1-12
14 Sep. 2012
19 Nov. 2012
7
20.1
3.10


pGUPn4/1-13
14 Sep. 2012
18 Nov. 2012
15
33.12
5.47









Thus, transgenic plants expressing a gene or nucleotide sequence encoding an enzyme involved in nitrogen assimilation or metabolism under tissue-specific promoter exhibited higher biomass and seed weight than plants expressing the same gene or nucleotide sequence under a constitutive promoter, in the presence of an adequate supply of nitrogen.
This indicates that transgenic Oryza sativa plants ectopically expressing an enzyme involved in nitrogen assimilation or metabolism are capable of optimising the utilization of available nitrogen under a range of environmental conditions thereby resulting in an increase in plant biomass, seed yield or a combination thereof.
T1 plant lines were obtained and average biomass and seed-weight was determined for 6 plants per transgenic plant lines. The results are shown in FIG. 18.
Collectively the above results indicate that transgenic plants ectopically expressing an enzyme involved in nitrogen assimilation or metabolism are capable of optimising the utilization of available nitrogen under a range of environmental conditions thereby resulting in an increase in plant biomass, seed weight, root tap growth, root growth and root length or a combination thereof.
The present invention has been described with regard to one or more embodiments. However, it will be apparent to persons skilled in the art that a number of variations and modifications can be made without departing from the scope of the invention as defined in the claims
All citations are hereby incorporated by reference.
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## Claims

1. A transgenic plant, plant cell, or plant part comprising a polynucleotide encoding an alanine amino transferase (AlaT) protein from a non-plant organism gene operably linked to a PBpr1 promoter which comprises the nucleotide sequence of SEQ ID No. 5, wherein the polynucleotide comprises a nucleotide sequence encoding a protein having at least 80% amino acid identity to the amino acid sequence of SEQ ID No. 14 or SEQ ID No. 15 or SEQ ID No. 16.

2. The transgenic plant, plant cell, or plant part according to claim 1, wherein the plant is corn, wheat, maize, rice, barley, canola, soybean, cotton, alfalfa, safflower, tomato or potato.

3. A seed obtained from the transgenic plant according to claim 1, comprising said polynucleotide operably linked to said PBpr1 promoter.

4. The transgenic plant, plant cell, or plant part according to claim 1, wherein the plant cell is a rice plant cell, a barley plant cell, a wheat plant cell or a maize plant cell.

5. The transgenic plant, plant cell, or plant part according to claim 1, wherein the plant cell is a rice plant cell.

6. The transgenic plant, plant cell, or plant part according to claim 1, wherein the gene encoding the AlaAT protein comprises a nucleotide sequence having 95% identity to SEQ ID No. 3 or 4.

7. The transgenic plant, plant cell, or plant part according to claim 6, wherein the plant cell is a rice plant cell, a barley plant cell, a wheat plant cell or a maize plant cell.

8. A genetic construct comprising a polynucleotide encoding an alanine amino transferase (AlaT) protein from a non-plant organism operably linked to a PBpr1 promoter which comprises the nucleotide sequence of SEQ ID No. 5, wherein the polynucleotide comprises a nucleotide sequence encoding a protein having at least 80% amino acid identity to the amino acid sequence of SEQ ID No. 14 or SEQ ID No. 15 or SEQ ID No. 16.

9. The genetic construct according to claim 8, wherein the polynucleotide comprises a nucleotide sequence having 95% identity to SEQ ID No. 3 or 4.

10. A method of generating a plant having increased biomass, increased nitrogen use efficiency or increased seed yield comprising:
transforming a plant cell with the genetic construct according to claim 9, and
growing the transformed plant cell to produce a plant that expresses the protein, thereby generating a plant having increased biomass, increased nitrogen use efficiency or increased seed yield, wherein the increased biomass, increased nitrogen use efficiency or increased seed yield is relative to the biomass, nitrogen use efficiency or seed yield of a wild-type plant grown under identical conditions.

11. A method of generating a plant having increased nitrogen use efficiency, increased biomass or increased seed yield comprising:
transforming a plant cell with the genetic construct according to claim 8, and
growing the transformed plant cell to produce a plant that expresses the protein, thereby generating a plant having increased nitrogen use efficiency, increased biomass or increased seed yield, wherein the increased nitrogen use efficiency, increased biomass or increased seed yield is relative to the nitrogen use efficiency, biomass or seed yield respectively of a wild-type plant grown under identical conditions.

12. The method according to claim 11, wherein the plant is corn, wheat, maize, rice, barley, canola, soybean, cotton, alfalfa, safflower, sugarcane, tomato or potato.

13. A method for producing a plant having increased nitrogen use efficiency, increased biomass, increased seed yield, earlier development time, or a combination thereof, comprising:
growing a plant from a plant, plant part or seed, comprising the genetic construct according to claim 8, thereby producing a plant having increased nitrogen uptake, increased biomass, increased seed yield, earlier development time, or a combination thereof, wherein the increased nitrogen uptake, increased biomass and increased seed yield is relative to the nitrogen uptake, biomass and seed yield of a wild-type plant grown under identical conditions.

14. The method of claim 13, further comprising providing a fertilizer to the plant or the habitat of the plant.

15. The method of claim 14, wherein the fertilizer is a nitrogen containing fertilizer.