WO2013152317A2 - Nitrite transporter and methods of using the same - Google Patents

Abstract

The invention generally relates to a nitrite transporter and methods of using the same.

Description

NITRITE TRANSPORTER AND METHODS OF USING THE SAME

Related Applications

This application claims the benefit, under 35 U.S.C. § 119(e), of U.S. Provisional Application Serial No. 61/621,032, filed on April 6, 2012, the disclosure of which is hereby incorporated herein by reference in its entirety.

Field of the Invention

The invention generally relates to a nitrite transporter and methods of using the same.

Background of the Invention

Nitrogen is limiting for many plants. For example, nitrogen is limiting for maize growth globally and estimates suggest that only 50% of nitrogen fertilizer is taken up by maize roots, with the remainder leached or volatilized ^{2> 49-5} \ Plants have evolved multiple strategies to cope with wide variation in concentrations of soil nitrate and ammonium ^" . For example, at low external nitrogen, plants employ high affinity nitrogen transporters, while low affinity transporters are used when external nitrogen is high \ To allow for fine-tuned control, plants have evolved transporter paralogs that perform similar functions but in different tissues, to facilitate nitrogen uptake from soil, xylem loading from roots for transport to shoot tissues, unloading in shoot organs and storage in vacuoles ⁴. For example, the Arabidopsis genome encodes at least 67 nitrate transporters, including 53 Nrtl genes, 7 Nrt2 genes and 7 AtClc (chloride channel) genes ⁴' ⁵. Nitrogen demand also changes throughout plant development ⁶. Part of this changing plant demand is met by scavenging nitrogen from senescing tissues, requiring additional intra-plant nitrogen transport ¹. Plants also coordinate nitrogen assimilation with energy availability, as the conversion of nitrate to ammonium alone consumes 12-26% of the primary photosynthetic reductant ⁸. In maize (Zea mays L.), significant gaps remain in characterizing the regulation of nitrogen uptake and assimilation genes in different tissues and at different stages of development.

With respect to nitrogen transporters in maize, at least two genes encoding low affinity nitrate transporters (ZmNrtl. l, ZmNrtl.2) and three genes encoding high affinity nitrate transporters (ZmNrt2. \, ZmNnrt2,2, ZmNnrt2.3) have been reported ^" . NRT2 requires interaction with co-transporter NAR2 (NRT3) proteins to be functionally active ^" . The maize genome encodes at least two NAR2-encoding genes {ZmNar2.1, ZmNar2.2) ⁹. In higher plants, a chloroplast-localized nitrite transporter (CsNitrl-L) was reported in cucumber, with a functional ortholog in Arabidopsis but none have been reported in maize. The genomes of higher plants have been reported to encode up to 14 ammonium transporter paralogs (AMT1 family), with the highest affinity transporters (AMT1;1 ; AMT1 ;3; AMT1;5) typically localized to root hairs and outer root cells and the lower affinity transporter(s) (AMT1;4) located in the root endodermis ⁷. In Arabidopsis, at least five gene families have been reported to transport amino acids and peptides, some of which also transport inorganic nitrogen ⁷.

With respect to nitrogen assimilation, higher plants directly assimilate ammonium into glutamate . By contrast, nitrate is first converted to nitrite in the cytoplasm by nitrate reductase (NR), followed by reduction to ammonia in plastids by nitrite reductase (NiR) ¹. Ammonium is fixed onto glutamate to form glutamine by glutamine synthetase (GS; Gin gene family), of which a plastidic isoform (GS2) and a cytosolic isoform (GS1) exist. A single gene in maize encodes GS2 (Gln2) whereas at least five genes encode GS1 (Glnl-1 to Glnl-5), which are differentially expressed during development ¹⁷' ¹⁸. Glutamine can also react with 2-oxoglutarate to form two molecules of glutamate via glutamine 2-oxoglutarate amino transferase, also called glutamate synthase or GOGAT ¹⁹. Plants have two types of GOGAT enzymes, NADH-GOGAT and Fd-GOGAT, which use NADH and ferredoxin as electron donors, respectively ¹⁹. Different GOGAT paralogs show constitutive or tissue specific gene expression in plants, including in maize ¹⁸' ¹⁹. Following nitrogen assimilation, glutamine and glutamate, other amino acids including asparagine and aspartate, and inorganic nitrogen, are transported by vascular tissues to growing organs ¹ , Much less information has been reported on whether plant developmental phases have any importance in categorizing root development. In order to improve nitrogen utilization, more information and a better understanding of nitrogen uptake and assimilation genes is needed.

The present invention addresses previous shortcomings in the art by providing a nitrite transporter and methods of using the same.

Summary of the Invention

One aspect of the present invention comprises an isolated nucleic acid comprising a nucleotide sequence selected from the group consisting of: (a) a nucleotide sequence of SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:5; (b) a nucleotide sequence that encodes a polypeptide having an amino acid sequence of SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, or SEQ ID NO: 10; (c) a nucleotide sequence that encodes a polypeptide having at least 90% identity to the amino acid sequence of SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, or SEQ ID NO: 10; (d) a nucleotide sequence that encodes a fragment of a polypeptide having an amino acid sequence of SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, or SEQ ID NO:10, wherein the fragment comprises at least 15 consecutive amino acids of SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, or SEQ ID NO: 10; (e) a nucleotide sequence that encodes a naturally occurring allelic variant of a polypeptide having an amino acid sequence of SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, or SEQ ID NO: 10; (f) a nucleotide sequence that hybridizes to the complete complement of the nucleotide sequences of any one of (a) to (e) under stringent conditions comprising a wash stringency of 50% Formamide with 5x Denhardt's solution, 0.5% SDS and lx SSPE at 42°C; (g) a degenerate nucleotide sequence of any one of (a) to (f) as a result of the genetic code; and/or (h) a nucleotide sequence having at least 90% sequence identity to the nucleotide sequences of any one of (a) to (g).

A second aspect of the present invention comprises an isolated polypeptide comprising an amino acid sequence selected from the group consisting of: (a) SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, or SEQ ID NO:10; (b) a fragment of at least 15 consecutive amino acids of SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, or SEQ ID NO: 10; (c) a naturally occurring allelic variant of a polypeptide having an amino acid sequence of SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, or SEQ ID NO: 10; and/or (d) an amino acid sequence having at least 90% sequence identity to the amino acid sequences of any one of (a) to (c).

The foregoing and other aspects of the present invention will now be described in more detail with respect to other embodiments described herein. It should be appreciated that the invention can be embodied in different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.

Detailed Description of the Invention

The present invention will now be described more fully hereinafter. This invention may, however, be embodied in different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art. Unless the context indicates otherwise, it is specifically intended that the various features of the invention described herein can be used in any combination. Moreover, the present invention also contemplates that in some embodiments of the invention, any feature or combination of features set forth herein can be excluded or omitted. To illustrate, if the specification states that a complex comprises components A, B and C, it is specifically intended that any of A, B or C, or a combination thereof, can be omitted and disclaimed.

The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the present application and relevant art and should not be interpreted in an idealized or overly formal sense unless expressly so defined herein. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. In the event of conflicting terminology, the present specification is controlling.

As used in the description of the invention and the appended claims, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.

Also as used herein, "and/or" refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative ("or").

As used herein, the transitional phrase "consisting essentially of (and grammatical variants) is to be interpreted as encompassing the recited materials or steps "and those that do not materially affect the basic and novel characteristic(s)" of the claimed invention. Thus, the term "consisting essentially of as used herein should not be interpreted as equivalent to "comprising."

The term "about," as used herein when referring to a measurable value such as an amount, concentration, time period and the like, is meant to encompass variations of ± 20%, ± 10%, ± 5%, ± 1%, ± 0.5%, or even ± 0.1% of the specified value as well as the specified value. A range provided herein for a measureable value may include any other range and/or individual value therein.

"Nitrite transporter" as used herein refers to an isolated polypeptide that aids, regulates, facilitates, transports, and/or the like, the movement of nitrite (e.g., protonated and/or non-protonated) into, out of, and/or within a plant cell, plant part, and/or plant.

"Nitrite transporter activity" as used herein refers to an isolated polypeptide that has one or more functions and/or characteristics of a nitrite transporter as described herein.

"Yield" as used herein refers to the production of a commercially and/or agriculturally important plant, plant biomass (e.g., dry biomass), plant part (e.g., roots, tubers, seed, leaves, fruit, flowers), plant material (e.g., an extract) and/or other product produced by the plant (e.g., a recombinant polypeptide). In some embodiments of the present invention, "increased yield" is assessed in terms of an increase in plant growth (e.g., height and/or width) or an increase in the rate of plant growth. In some embodiments of the present invention, "increased yield" is assessed in terms of an increase in the amount of a fruit, seed/grain, or other harvestable product produced from a plant.

"Increased yield" may be determined using a yield assay that compares the yield (e.g., plant growth, rate of plant growth, and/or amount of a harvestable product) produced from a stably transformed plant comprising a vector or expression cassette comprising an isolated nucleic acid of the present invention (e.g., SEQ ID NO: l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, and/or SEQ ID NO: 5) and the yield from a control plant that does not comprise the vector or expression cassette comprising an isolated nucleic acid of the present invention. For example, in some embodiments, increased yield may be determined by comparing the amount of a fruit, seed/grain, or other harvestable product produced from a stably transformed plant comprising a vector or expression cassette comprising an isolated nucleic acid of the present invention with the amount of a fruit, seed/grain, or other harvestable product produced from a control plant that does not comprise the vector or expression cassette comprising an isolated nucleic acid of the present invention.

The term "modulate" (and grammatical variations) refers to an increase or decrease.

As used herein, the terms "increase," "increases," "increased," "increasing" and similar terms indicate an elevation of at least about 5%, 10%, 25%, 50%, 75%, 100%, 150%, 200%, 300%, 400%, 500% or more.

As used herein, the terms "reduce," "reduces," "reduced," "reduction" and similar terms refer to a decrease of at least about 5%, 10%, 25%, 35%, 50%, 75%, 80%, 85%, 90%, 95%, 97%) or more. In particular embodiments, the reduction results in no or essentially no (i.e., an insignificant amount, e.g., less than about 10% or even 5%) detectable activity or amount.

A "promoter" is a nucleotide sequence that controls or regulates the transcription of a nucleotide sequence (i.e., a coding sequence) that is operatively associated with the promoter. The coding sequence may encode a polypeptide and/or a functional RNA. Typically, a "promoter" refers to a nucleotide sequence that contains a binding site for RNA polymerase II and directs the initiation of transcription. In general, promoters are found 5', or upstream, relative to the start of the coding region of the corresponding coding sequence. The promoter region may comprise other elements that act as regulators of gene expression. These include a TATA box consensus sequence, and often a CAAT box consensus sequence (Breathnach and Chambon, (1981) Annu. Rev. Biochem. 50:349). In plants, the CAAT box may be substituted by the AGGA box (Messing et al, (1983) in Genetic Engineering of Plants, T. Kosuge, C. Meredith and A. Hollaender (eds.), Plenum Press, pp. 211-227).

By "operably linked" or "operably associated" as used herein, it is meant that the indicated elements are functionally related to each other, and are also generally physically related. For example, a promoter is operatively linked or operably associated to a coding sequence (e.g., nucleotide sequence of interest) if it controls the transcription of the sequence. Thus, the term "operatively linked" or "operably associated" as used herein, refers to nucleotide sequences on a single nucleic acid molecule that are functionally associated. Those skilled in the art will appreciate that the control sequences (e.g., promoter) need not be contiguous with the coding sequence, as long as they functions to direct the expression thereof. Thus, for example, intervening untranslated, yet transcribed, sequences can be present between a promoter and a coding sequence, and the promoter sequence can still be considered "operably linked" to the coding sequence.

By the term "express," "expressing" or "expression" (or other grammatical variants) of a nucleic acid coding sequence, it is meant that the sequence is transcribed. In particular embodiments, the terms "express," "expressing" or "expression" (or other grammatical variants) can refer to both transcription and translation to produce an encoded polypeptide.

"Wild-type" nucleotide sequence or amino acid sequence refers to a naturally occurring ("native") or endogenous nucleotide sequence (including a cDNA corresponding thereto) or amino acid sequence.

The terms "nucleic acid," "polynucleotide" and "nucleotide sequence" are used interchangeably herein unless the context indicates otherwise. These terms encompass both RNA and DNA, including cDNA, genomic DNA, partially or completely synthetic (e.g., chemically synthesized) RNA and DNA, and chimeras of RNA and DNA. The nucleic acid, polynucleotide or nucleotide sequence may be double- stranded or single-stranded, and further may be synthesized using nucleotide analogs or derivatives (e.g., inosine or phosphorothioate nucleotides). Such nucleotides can be used, for example, to prepare nucleic acids, polynucleotides and nucleotide sequences that have altered base-pairing abilities or increased resistance to nucleases. The present invention further provides a nucleic acid, polynucleotide or nucleotide sequence that is the complement (which can be either a full complement or a partial complement) of a nucleic acid, polynucleotide or nucleotide sequence of the invention. Nucleotide sequences are presented herein by single strand only, in the 5' to 3' direction, from left to right, unless specifically indicated otherwise. Nucleotides and amino acids are represented herein in the manner recommended by the IUPAC-IUB Biochemical Nomenclature Commission, or (for amino acids) by either the one-letter code, or the three letter code, both in accordance with 37 CFR § 1.822 and established usage.

The nucleic acids and polynucleotides of the invention are optionally isolated. An "isolated" nucleic acid molecule or polynucleotide is a nucleic acid molecule or polynucleotide that, by the hand of man, exists apart from its native environment and is therefore not a product of nature. An isolated nucleic acid molecule or isolated polynucleotide may exist in a purified form or may exist in a non-native environment such as, for example, a recombinant host cell. Thus, for example, the term "isolated" means that it is separated from the chromosome and/or cell in which it naturally occurs. A nucleic acid or polynucleotide is also isolated if it is separated from the chromosome and/or cell in which it naturally occurs and is then inserted into a genetic context, a chromosome, a chromosome location, and/or a cell in which it does not naturally occur. The recombinant nucleic acid molecules and polynucleotides of the invention can be considered to be "isolated."

Further, an "isolated" nucleic acid or polynucleotide can be a nucleotide sequence (e.g., DNA or RNA) that is not immediately contiguous with nucleotide sequences with which it is immediately contiguous (one on the 5' end and one on the 3' end) in the naturally occurring genome of the organism from which it is derived. The "isolated" nucleic acid or polynucleotide can exist in a cell (e.g., a plant cell), optionally stably incorporated into the genome. According to this embodiment, the "isolated" nucleic acid or polynucleotide can be foreign to the cell/organism into which it is introduced, or it can be native to the cell/organism, but exist in a recombinant form (e.g., as a chimeric nucleic acid or polynucleotide) and/or can be an additional copy of an endogenous nucleic acid or polynucleotide. Thus, an "isolated nucleic acid molecule" or "isolated polynucleotide" can also include a nucleotide sequence derived from and inserted into the same natural, original cell type, but which is present in a non-natural state, e.g., present in a different copy number, in a different genetic context and/or under the control of different regulatory sequences than that found in the native state of the nucleic acid molecule or polynucleotide.

In representative embodiments, the "isolated" nucleic acid or polynucleotide is substantially free of cellular material (including naturally associated proteins such as histones, transcription factors, and the like), viral material, and/or culture medium (when produced by recombinant DNA techniques), or chemical precursors or other chemicals (when chemically synthesized). Optionally, in representative embodiments, the isolated nucleic acid or polynucleotide is at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99%, or more pure.

As used herein, the term "recombinant" nucleic acid, polynucleotide or nucleotide sequence refers to a nucleic acid, polynucleotide or nucleotide sequence that has been constructed, altered, rearranged and/or modified by genetic engineering techniques. The term "recombinant" does not refer to alterations that result from naturally occurring events, such as spontaneous mutations, or from non-spontaneous mutagenesis.

A "vector" is any nucleic acid molecule for the cloning of and/or transfer of a nucleic acid into a cell. A vector may be a replicon to which another nucleotide sequence may be attached to allow for replication of the attached nucleotide sequence. A "replicon" can be any genetic element (e.g., plasmid, phage, cosmid, chromosome, viral genome) that functions as an autonomous unit of nucleic acid replication in the cell, i.e., capable of nucleic acid replication under its own control. The term "vector" includes both viral and nonviral (e.g., plasmid) nucleic acid molecules for introducing a nucleic acid into a cell in vitro, ex vivo, and/or in vivo, and is optionally an expression vector. A large number of vectors known in the art may be used to manipulate, deliver and express polynucleotides. Vectors may be engineered to contain sequences encoding selectable markers that provide for the selection of cells that contain the vector and/or have integrated some or all of the nucleic acid of the vector into the cellular genome. Such markers allow identification and/or selection of host cells that incorporate and express the proteins encoded by the marker. A "recombinant" vector refers to a viral or non-viral vector that comprises one or more nucleotide sequences of interest (e.g., transgenes), e.g., two, three, four, five or more nucleotide sequences of interest.

Viral vectors have been used in a wide variety of gene delivery applications in cells, as well as living animal subjects. Plant viral vectors that can be used include, but are not limited to, geminivirus vectors. Non-viral vectors include, but are not limited to, plasmids, liposomes, electrically charged lipids (cytofectins), nucleic acid-protein complexes, and biopolymers. In addition to a nucleic acid of interest, a vector may also comprise one or more regulatory regions, and/or selectable markers useful in selecting, measuring, and monitoring nucleic acid transfer results (e.g., delivery to specific tissues, duration of expression, etc.).

Two nucleotide sequences are said to be "substantially identical" to each other when they share at least about 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or even 100% sequence identity.

Two amino acid sequences are said to be "substantially identical" or "substantially similar" to each other when they share at least about 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%o, 99% or even 100%o sequence identity or similarity, respectively.

As used herein "sequence identity" refers to the extent to which two optimally aligned polynucleotide or polypeptide sequences are invariant throughout a window of alignment of components, e.g., nucleotides or amino acids.

As used herein "sequence similarity" is similar to sequence identity (as described herein), but permits the substitution of conserved amino acids (e.g., amino acids whose side chains have similar structural and/or biochemical properties), which are well-known in the art.

As is known in the art, a number of different programs can be used to identify whether a nucleic acid has sequence identity or an amino acid sequence has sequence identity or similarity to a known sequence. Sequence identity or similarity may be determined using standard techniques known in the art, including, but not limited to, the local sequence identity algorithm of Smith & Waterman, Adv. Appl. Math. 2, 482 (1981), by the sequence identity alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48,443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Natl. Acad. Sci. USA 85,2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, WI), the Best Fit sequence program described by Devereux et al, Nucl. Acid Res. 12, 387-395 (1984), preferably using the default settings, or by inspection.

An example of a useful algorithm is PILEUP. PILEUP creates a multiple sequence alignment from a group of related sequences using progressive, pairwise alignments. It can also plot a tree showing the clustering relationships used to create the alignment. PILEUP uses a simplification of the progressive alignment method of Feng & Doolittle, J. Mol. Evol. 35, 351-360 (1987); the method is similar to that described by Higgins & Sharp, CABIOS 5, 151-153 (1989).

Another example of a useful algorithm is the BLAST algorithm, described in Altschul et al, J. Mol Biol. 215, 403-410, (1990) and Karlin et al, Proc. Natl Acad. Sci. USA 90, 5873-5787 (1993). A particularly useful BLAST program is the WU-BLAST-2 program which was obtained from Altschul et al, Methods in Enzymology, 266, 460-480 (1996); http://blast.wustl/edu/blast/ README.html. WU-BLAST-2 uses several search parameters, which are preferably set to the default values. The parameters are dynamic values and are established by the program itself depending upon the composition of the particular sequence and composition of the particular database against which the sequence of interest is being searched; however, the values may be adjusted to increase sensitivity.

An additional useful algorithm is gapped BLAST as reported by Altschul et al Nucleic Acids Res. 25, 3389-3402 (1997).

The CLUSTAL program can also be used to determine sequence similarity. This algorithm is described by Higgins et al. (1988) Gene 73:237; Higgins et al. (1989) CABIOS 5:151-153; Corpet et al (1988) Nucleic Acids Res. 16: 10881-90; Huang et al (1992) CABIOS 8: 155-65; and Pearson et al. (1994) Meth. Mol. Biol 24: 307-331.

The alignment may include the introduction of gaps in the sequences to be aligned. In addition, for sequences which contain either more or fewer nucleotides than the nucleic acids disclosed herein, it is understood that in one embodiment, the percentage of sequence identity will be determined based on the number of identical nucleotides acids in relation to the total number of nucleotide bases. Thus, for example, sequence identity of sequences shorter than a sequence specifically disclosed herein, will be determined using the number of nucleotide bases in the shorter sequence, in one embodiment. In percent identity calculations relative weight is not assigned to various manifestations of sequence variation, such as, insertions, deletions, substitutions, etc.

Two nucleotide sequences can also be considered to be substantially identical when the two sequences hybridize to each other under stringent conditions. A nonlimiting example of "stringent" hybridization conditions include conditions represented by a wash stringency of 50% Formamide with 5x Denhardt's solution, 0.5% SDS and lx SSPE at 42°C. "Stringent hybridization conditions" and "stringent hybridization wash conditions" in the context of nucleic acid hybridization experiments such as Southern and Northern . hybridizations are sequence dependent, and are different under different environmental parameters. An extensive guide to the hybridization of nucleic acids is found in Tijssen Laboratory Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic Acid Probes part I chapter 2 "Overview of principles of hybridization and the strategy of nucleic acid probe assays" Elsevier, New York (1993). In some representative embodiments, two nucleotide sequences considered to be substantially identical hybridize to each other under highly stringent conditions. Generally, highly stringent hybridization and wash conditions are selected to be about 5°C lower than the thermal melting point (T_m) for the specific sequence at a defined ionic strength and pH.

As used herein, the term "polypeptide" encompasses both peptides and proteins (including fusion proteins), unless indicated otherwise.

The polypeptides of the invention are optionally "isolated." An "isolated" polypeptide is a polypeptide that, by the hand of man, exists apart from its native environment and is therefore not a product of nature. An isolated polypeptide may exist in a purified form or may exist in a non-native environment such as, for example, a recombinant host cell. The recombinant polypeptides of the invention can be considered to be "isolated."

In representative embodiments, an "isolated" polypeptide means a polypeptide that is separated or substantially free from at least some of the other components of the naturally occurring organism or virus, for example, the cell or viral structural components or other polypeptides or nucleic acids commonly found associated with the polypeptide. In particular embodiments, the "isolated" polypeptide is at least about 1%, 5%, 10%, 25%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or more pure (w/w). In other embodiments, an "isolated" polypeptide indicates that at least about a 5-fold, 10-fold, 25-fold, 100-fold, 1000-fold, 10,000-fold, or more enrichment of the protein (w/w) is achieved as compared with the starting material. In representative embodiments, the isolated polypeptide is a recombinant polypeptide produced using recombinant nucleic acid techniques.

A "biologically active" polypeptide is one that substantially retains at least one biological activity normally associated with the wild-type polypeptide. In particular embodiments, the "biologically active" polypeptide substantially retains all of the biological activities possessed by the unmodified {e.g., native) sequence. By "substantially retains" biological activity, it is meant that the polypeptide retains at least about 50%, 60%, 75%, 85%, 90%, 95%, 97%, 98%, 99%, or more, of the biological activity of the native polypeptide (and can even have a higher level of activity than the native polypeptide).

"Introducing" in the context of a plant cell, plant tissue, plant part and/or plant means contacting a nucleic acid molecule with the plant cell, plant tissue, plant part, and/or plant in such a manner that the nucleic acid molecule gains access to the interior of the plant cell or a cell of the plant tissue, plant part or plant. Where more than one nucleic acid molecule is to be introduced, these nucleic acid molecules can be assembled as part of a single polynucleotide or nucleic acid construct, or as separate polynucleotide or nucleic acid constructs, and can be located on the same or different nucleic acid constructs. Accordingly, these polynucleotides can be introduced into plant cells in a single transformation event, in separate transformation events, or, e.g., as part of a breeding protocol.

The term "transformation" as used herein refers to the introduction of a heterologous and/or isolated nucleic acid into a cell. Transformation of a cell may be stable or transient. Thus, a transgenic plant cell, plant tissue, plant part and/or plant of the invention can be stably transformed or transiently transformed.

"Transient transformation" in the context of a polynucleotide means that a polynucleotide is introduced into the cell and does not integrate into the genome of the cell.

As used herein, "stably introducing," "stably introduced," "stable transformation" or "stably transformed" (and similar terms) in the context of a polynucleotide introduced into a cell, means that the introduced polynucleotide is stably integrated into the genome of the cell (e.g., into a chromosome or as a stable-extra-chromosomal element). As such, the integrated polynucleotide is capable of being inherited by progeny cells and plants.

Transient transformation may be detected by, for example, an enzyme-linked immunosorbent assay (ELISA) or Western blot, which can detect the presence of a peptide or polypeptide encoded by one or more transgene introduced into an organism. Stable transformation of a cell can be detected by, for example, a Southern blot hybridization assay of genomic DNA of the cell with nucleic acid sequences which specifically hybridize with a nucleotide sequence of a transgene introduced into an organism (e.g., a plant). Stable transformation of a cell can be detected by, for example, a Northern blot hybridization assay of RNA of the cell with nucleic acid sequences which specifically hybridize with a nucleotide sequence of a transgene introduced into a plant or other organism. Stable transformation of a cell can also be detected by, e.g., a polymerase chain reaction (PCR) or other amplification reactions as are well known in the art, employing specific primer sequences that hybridize with target sequence(s) of a transgene, resulting in amplification of the transgene sequence, which can be detected according to standard methods Transformation can also be detected by direct sequencing and/or hybridization protocols well known in the art.

"Genome" as used herein includes the nuclear and/or plastid genome, and therefore includes integration of a polynucleotide into, for example, the chloroplast genome. Stable transformation as used herein can also refer to a polynucleotide that is maintained extrachromosomally, for example, as a minichromosome.

As used herein, the terms "transformed" and "transgenic" refer to any plant, plant cell, plant tissue (including callus), or plant part that contains all or part of at least one recombinant or isolated nucleic acid, polynucleotide or nucleotide sequence. In representative embodiments, the recombinant or isolated nucleic acid, polynucleotide or nucleotide sequence is stably integrated into the genome of the plant {e.g., into a chromosome or as a stable extra-chromosomal element), so that it is passed on to subsequent generations of the cell or plant.

The term "plant part," as used herein, includes but is not limited to reproductive tissues (e.g., petals, sepals, stamens, pistils, receptacles, anthers, pollen, flowers, fruits, flower bud, ovules, seeds, embryos, nuts, kernels, ears, cobs and husks); vegetative tissues (e.g., petioles, stems, roots, root hairs, root tips, pith, coleoptiles, stalks, shoots, branches, bark, apical meristem, axillary bud, cotyledon, hypocotyls, and leaves); vascular tissues (e.g., phloem and xylem); specialized cells such as epidermal cells, parenchyma cells, chollenchyma cells, schlerenchyma cells, stomates, guard cells, cuticle, mesophyll cells; callus tissue; and cuttings. The term "plant part" also includes plant cells, including plant cells that are intact in plants and/or parts of plants, plant protoplasts, plant tissues, plant organs plant cell tissue cultures, plant calli, plant clumps, and the like. As used herein, "shoot" refers to the above ground parts including the leaves and stems.

The term "tissue culture" encompasses cultures of tissue, cells, protoplasts and callus.

As used herein, "plant cell" refers to a structural and physiological unit of the plant, which typically comprise a cell wall but also includes protoplasts. A plant cell of the present invention can be in the form of an isolated single cell or can be a cultured cell or can be a part of a higher-organized unit such as, for example, a plant tissue (including callus) or a plant organ.

Any plant (or groupings of plants, for example, into a genus or higher order classification) can be employed in practicing the present invention including angiosperms or gymnosperms, monocots or dicots. In certain embodiments, the plant is a monocot.

Exemplary plants include, but are not limited to, corn (Zea mays), canola (Brassica napus, Brassica rapa ssp.), alfalfa (Medicago saliva), rice (Oryza sativa, including without limitation Indica and/or Japonica varieties), rape (Brassica napus), rye (Secale cereale), sorghum (Sorghum bicolor, Sorghum vulgare), sunflower (Helianthus annus), wheat (Triticum aestivum), soybean (Glycine max), tobacco (Nicotiana tobacum), potato (Solanum tuberosum), peanuts (Arachis hypogaea), cotton (Gossypium hirsutum), sweet potato (Ipomoea batatus), cassava (Manihot esculenta), coffee (Cofea spp.), coconut (Cocos nucifera), pineapple (Ananas comosus), citrus trees (Citrus spp.), cocoa (Theobroma cacao), tea (Camellia sinensis), banana (Musa spp.), avocado (Persea americana), fig (Ficus casica), guava (Psidium guajava), mango (Mangifera indica), olive (Olea europaea), papaya (Carica papaya), cashew (Anacardium occidentale), macadamia (Macadamia integrifolia), almond (Prunus amygdalus), sugar beets (Beta vulgaris), apple (Malus pumila), blackberry (Rubus), strawberry (Fragaria), walnut (Juglans regia), grape (Vitis vinifera), apricot (Prunus armeniaca), cherry (Prunus), peach (Prunus persica), plum (Prunus domestica), pear (Pyrus communis), watermelon (Citrullus vulgaris), duckweed (Lemna), oats (Avena sativa), barley (Hordium vulgare), vegetables, ornamentals, conifers, and turfgrasses (e.g., for ornamental, recreational or forage purposes), and biomass grasses (e.g., switchgrass and miscanthus). In certain embodiments, the plant is maize. In other embodiments, the plant is rice. In some embodiments, the plant is wheat.

Vegetables include, but are not limited to, Solanaceous species (e.g., tomatoes; Lycopersicon esculentum), lettuce (e.g., Lactuea sativa), carrots (Caucus carota), cauliflower (Brassica oleracea), celery (apium graveolens), eggplant (Solanum melongena), asparagus (Asparagus officinalis), ochra (Abelmoschus esculentus), green beans (Phaseolus vulgaris), lima beans (Phaseolus limensis), peas (Lathyrus spp.), members of the genus Cucurbita such as Hubbard squash (C. Hubbard), Butternut squash (C. moschata), Zucchini (C. pepo), Crookneck squash (C. crookneck), C. argyrosperma , C. argyrosperma ssp sororia, C. digitata, C. ecuadorensis, C. foetidissima, C. lundelliana, and C. martinezii, and members of the genus Cucumis such as cucumber (Cucumis sativus), cantaloupe (C. cantalupensis), and musk melon (C. melo).

Ornamentals include, but are not limited to, azalea (Rhododendron spp.), hydrangea (Macrophylla hydrangea), hibiscus (Hibiscus rosasanensis), roses (Rosa spp.), tulips (Tulipa spp.), daffodils (Narcissus spp.), petunias (Petunia hybrida), carnation (dianthus caryophyllus), poinsettia (Euphorbia pulcherima), and chiysanthemum.

Conifers, which may be employed in practicing the present invention, include, for example, pines such as loblolly pine (Pinus taeda), slash pine (Pinus elliotii), ponderosa pine (Pinus ponderosa), lodgepole pine (Pinus contorta), and Monterey pine (Pinus radiata); Douglas-fir (Pseudotsuga menziesii); Western hemlock (Tsuga canadensis); Sitka spruce (Picea glauca); redwood (Sequoia sempervirens); true firs such as silver fir (Abies amabilis) and balsam fir (Abies balsamea); and cedars such as Western red cedar (Thuja plicata) and Alaska yellow-cedar (Chamaecyparis nootkatensis).

Turfgrass include, but are not limited to, zoysiagrasses, bentgrasses, fescue grasses, bluegrasses, St. Augustinegrasses, bermudagrasses, bufallograsses, ryegrasses, and orchardgrasses.

Also included are plants that serve primarily as laboratory models, e.g., Arabidopsis.

According to one aspect of the present invention, an isolated nucleic acid of the present invention is provided comprising, consisting essentially of, or consisting of a nucleotide sequence selected from the group consisting of: (a) a nucleotide sequence of SEQ ID NO: l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:5; (b) a nucleotide sequence that encodes a polypeptide having an amino acid sequence of SEQ ID NO: 6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, or SEQ ID NO:10; (c) a nucleotide sequence that encodes a polypeptide having at least 90% identity to the amino acid sequence of SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, or SEQ ID NO:10; (d) a nucleotide sequence that encodes a fragment of a polypeptide having an amino acid sequence of SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, or SEQ ID NO:10, wherein the fragment comprises at least about 10, 25, 50, 75, 100, 150,125, 175 or more consecutive amino acids of SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, or SEQ ID NO: 10 and wherein the fragment has nitrite transporter activity; (e) a nucleotide sequence that encodes a naturally occurring allelic variant of a polypeptide having an amino acid sequence of SEQ ID NO: 6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, or SEQ ID NO:10; (f) a nucleotide sequence that hybridizes to the complete complement of the nucleotide sequences of any one of (a) to (e) under stringent conditions comprising a wash stringency of 50% Formamide with 5x Denhardt's solution, 0.5% SDS and lx SSPE at 42°C; (g) a degenerate nucleotide sequence of any one of (a) to (f) as a result of the genetic code; and/or (h) a nucleotide sequence having at least about 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity to the nucleotide sequences of any one of (a) to (g).

In some embodiments of the present invention, an isolated nucleic acid of the present invention does not comprise a nucleotide sequence that encodes a naturally occurring allelic variant of a polypeptide having an amino acid sequence of SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, or SEQ ID NO: 10. In other embodiments of the present invention, an isolated nucleic acid of the present invention does not comprise a nucleotide sequence that encodes a naturally occurring allelic variant that is less than 60% sequence identity to a polypeptide having an amino acid sequence of SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, or SEQ ID NO: 10. In some embodiments of the present invention, an isolated nucleic acid of the present invention does not comprise a nucleotide sequence that encodes a naturally occurring allelic variant that is less than 55% sequence identity to a polypeptide having an amino acid sequence of SEQ ID NO: 6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, or SEQ ID NO: 10. In other embodiments of the present invention, an isolated nucleic acid of the present invention does not comprise a nucleotide sequence that encodes a naturally occurring allelic variant comprising UniProtKB/Swiss-Prot accession number Q96400.

In some embodiments of the present invention, an isolated nucleic acid of the present invention is operably associated with a promoter. In particular embodiments of the present invention, the promoter comprises one or more nucleotide sequences comprising, consisting essentially of, or consisting of: SEQ ID NO:l l, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO:14, SEQ ID NO: 15, SEQ ID NO:16, SEQ ID NO: 17, SEQ ID NO:18, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, and any combination thereof. In other embodiments of the present invention, the promoter can comprise one or more promoters that drive the expression of a nitrate reductase.

A "plant promoter" is a promoter capable of initiating transcription in a plant cell. Exemplary plant promoters include, but are not limited to, those that are obtained from plants, plant viruses and/or bacteria which comprise genes expressed in plant cells such as Agrobacterium or Rhizobium. Further examples include plant promoters that preferentially initiate transcription in certain tissues, such as leaves, roots, seeds, fibres, xylem vessels, tracheids, and/or sclerenchyma. Such promoters are referred to as "tissue preferred." A "cell type" specific promoter primarily drives expression in certain cell types in one or more organs, for example, vascular cells in roots or leaves. An "inducible" or "regulatable" promoter is a promoter that is under environmental control. Examples of environmental conditions that may affect transcription by inducible promoters include, but are not limited to, anaerobic conditions and/or the presence of light. Another type of promoter is a developmentally regulated promoter, for example, a promoter that drives expression during pollen development. Tissue preferred, cell type specific, developmentally regulated, and inducible promoters constitute the class of "non-constitutive" promoters. A "constitutive" promoter is a promoter that is active under most environmental conditions. Any suitable promoter sequence may be used with a nucleic acid construct of the present invention. According to some embodiments of the invention, the promoter is a constitutive promoter. In some embodiments, the promoter is a tissue-specific promoter. In certain embodiments, the promoter is an abiotic stress-inducible promoter and/or a nitrate reductase gene promoter.

Suitable constitutive promoters include, for example, CaMV 35S promoter (Odell et al, Nature 313:810-812, 1985); Arabidopsis At6669 promoter (see PCT Publication No. WO04081173A2); maize Ubi 1 (Christensen et al, Plant Sol. Biol. 18:675-689, 1992); rice actin (McElroy et al, Plant Cell 2: 163-171, 1990); pEMU (Last et al, Theor. Appl. Genet. 81 :581-588, 1991); CaMV 19S (Nilsson et al., Physiol. Plant 100:456-462, 1997); GOS2 (de Pater et al, Plant J November; 2(6):837-44, 1992); ubiquitin (Christensen et al., Plant Mol. Biol. 18: 675-689, 1992); Rice cyclophilin (Bucholz et al, Plant Mol Biol. 25(5):837-43,

1994) ; Maize H3 histone (Lepetit et al, Mol. Gen. Genet. 231 : 276-285, 1992); Actin 2 (An et al., Plant J. 10(1);107-121, 1996), constitutive root tip CT2 promoter (see PCT application No. IL/2005/000627), and Synthetic Super MAS (Ni et al, The Plant Journal 7: 661-76,

1995) , the disclosures of the references cited herein are incorporated herein for the portions relevant to this paragraph. Other constitutive promoters include those described in U.S. Pat. Nos. 5,659,026; 5,608,149; 5,608,144; 5,604,121; 5,569,597: 5,466,785; 5,399,680; 5,268,463; and 5,608,142, the disclosures of which are incorporated herein for the portions relevant to this paragraph.

Suitable tissue-specific promoters include, but are not limited to, leaf-specific promoters such as, for example, those described by Yamamoto et al., Plant J. 12:255-265, 1997; Kwon et al, Plant Physiol. 105:357-67, 1994; Yamamoto et al., Plant Cell Physiol. 35:773-778, 1994; Gotor et al., Plant J. 3:509-18, 1993; Orozco et al., Plant Mol. Biol. 23:1129-1138, 1993; and Matsuoka et al, Proc. Natl. Acad. Sci. USA 90:9586-9590, 1993; seed-preferred promoters such as, for example, seed-preferred promoters from seed specific genes (Simon, et al., Plant Mol. Biol. 5. 191, 1985; Scofield, et al., J. Biol. Chem. 262: 12202, 1987; Baszczynski, et al, Plant Mol. Biol. 14: 633, 1990), Brazil Nut albumin (Pearson' et al, Plant Mol. Biol. 18: 235-245, 1992), legumin (Ellis, et al. Plant Mol. Biol. 10: 203-214, 1988), glutelin (rice) (Takaiwa, et al, Mol. Gen. Genet. 208: 15-22, 1986; Takaiwa, et al, FEBS Letts. 221 : 43-47, 1987), zein (Matzke et al, Plant Mol Biol, 143)323- 32 1990), napA (Stalberg, et al, Planta 199: 515-519, 1996), wheat SPA (Albanietal, Plant Cell, 9: 171-184, 1997), sunflower oleosin (Cummins, etal., Plant Mol. Biol. 19: 873-876, 1992)], endosperm specific promoters [e.g., wheat LMW and HMW, glutenin-1 (Mol Gen Genet 216:81-90, 1989; NAR 17:461-2), wheat a, b and g gliadins (EMB03: 1409-15, 1984), barley ltrl promoter, barley Bl, C, D hordein (Theor Appl Gen 98:1253-62, 1999; Plant J 4:343-55, 1993; Mol Gen Genet 250:750-60, 1996), barley DOF (Mena et al, The Plant Journal, 116(1): 53-62, 1998), Biz2 (EP99106056.7), synthetic promoter (Vicente-Carbajosa et al., Plant J. 13: 629-640, 1998), rice prolamin NRP33, rice -globulin Glb-1 (Wu et al., Plant Cell Physiology 39(8) 885-889, 1998), rice alpha-globulin REB/OHP-1 ( akase et al. Plant Mol. Biol. 33: 513-S22, 1997), rice ADP-glucose PP (Trans Res 6:157-68, 1997), maize ESR gene family (Plant J 12:235-46, 1997), sorgum gamma-kafirin (PMB 32: 1029-35, 1996)]; embryo specific promoters [e.g., rice OSH1 (Sato et al, Proc. Nati. Acad. Sci. USA, 93: 8117-8122), KNOX (Postma-Haarsma of al, Plant Mol. Biol. 39:257-71, 1999), rice oleosin (Wu et at, J. Biochem., 123:386, 1998)]; and flower-specific promoters [e.g., AtPRP4, chalene synthase (chsA) (Van der Meer, et al., Plant Mol. Biol. 15, 95-109, 1990), LAT52 (Twell et al., Mol. Gen Genet. 217:240-245; 1989), apetala-3].

Suitable abiotic stress-inducible promoters include, but are not limited to, salt- inducible promoters such as RD29A (Yamaguchi-Shinozalei et al., Mol. Gen. Genet. 236:331-340, 1993); drought-inducible promoters such as maize rabl7 gene promoter (Pla et. al, Plant Mol. Biol. 21 :259-266, 1993), maize rab28 gene promoter (Busk et. al., Plant J. 11 :1285-1295, 1997) and maize Ivr2 gene promoter (Pelleschi et. al, Plant Mol. Biol. 39:373-380, 1999); heat-inducible promoters such as heat tomato hsp80-promoter from tomato (U.S. Pat. No. 5,187,267).

In some embodiments, a light inducible and/or light regulated promoter may be used. Light inducible promoters control transcription of a gene or coding region upon exposure to light. In some embodiments of the invention, the promoters described contain one or more motifs selected from a BOXIIPCCHS motif, CIACADIANLELHC motif, GT1 CONSENSUS motif, IBOX motif, IBOXCORE motif, IBOXCORENT motif, INRNTPSADB motif, LRENPCABE motif, SORLIP1AT motif, SORLIP2AT, SORLIP5AT motif, and any combination thereof. Light regulated promoters may drive the expression of native genes for photosystem I, photosystem II, or Calvin Cycle proteins. For example, the amino acid sequences for Hordeum vulgare Photosystem I reaction center subunit psaD with Swiss-Prot ID P36213.1, the Hordeum vulgare Photosystem I reaction center subunit psaK with Swiss- Prot ID P36886.1 (formerly Swiss-Prot ID A48527), the Pisum sativum light harvesting protein of photosystem I LHCA3 with Genbank ID AAA84545.1, and the Hordeum vulgare chlorophyll a/b-binding protein precursor LHCA4 with Genbank ID AAF90200.1 may be used in a tBLASTn search of a rice genome database to find rice genes. Further exemplary rice genes are available from public rice genome databases. Exemplary light regulated promoters may include those described in International publication number WO 2012/061585. In certain embodiments, an isolated nucleic acid of the present invention (e.g., SEQ ID NO: l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, and/or SEQ ID NO:5) can be operably associated with a light inducible and/or light regulated promoter.

In certain embodiments of the present invention, the promoter comprises one or more nucleotide sequences comprising, consisting essentially of, or consisting of: SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, and/or one or more promoters that drive the expression of nitrate reductase. Exemplary nitrate reductase gene promoters include, but are not limited to, NIA1 gene (e.g., nnrl,nnr2, and/or nnr3), NIA2 gene, AtNRTl .l, AtNRT2.1, and any combination thereof.

In some embodiments, the nitrite transporter may be targeted to an organelle, such as the chloroplast. Various mechanisms for targeting gene products are known to exist in plants and the sequences controlling the functioning of these mechanisms have been characterized. For example, the targeting of gene products to the chloroplast is controlled by a signal sequence found at the amino terminal end of various polypeptides that is cleaved during chloroplast import to yield the mature polypeptides (see e.g., Comai et al, (1988) J Biol Chem 263:15104-15109). These signal sequences can be fused to heterologous gene products to affect the import of heterologous products into the chloroplast (Van den Broeck et al, (1985) Nature 313:358-363). DNA encoding for appropriate signal sequences can be isolated from the 5' end of the cDNAs encoding the ribulose-l,5-bisphosphate carboxylase/oxygenase (RUBISCO) polypeptide, the chlorophyll a/b binding (CAB) polypeptide, the 5-enol-pyruvyl shikimate-3 -phosphate (EPSP) synthase enzyme, the GS2 polypeptide and many other polypeptides which are known to be chloroplast localized. See also, the section entitled "Expression With Chloroplast Targeting" in Example 37 of U.S. Patent No. 5,639,949, the disclosure of which is herein incorporated by reference for the portions relevant to this paragraph. In some embodiments, an isolated nucleic acid of the present invention (e.g., SEQ ID NO: l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, and/or SEQ ID NO: 5) can be operably linked to a targeting and/or signaling sequence that targets the isolated nucleic acid to a chloroplast in a plant cell. Such targeting and/or signaling sequences are well known in the art.

In other embodiments, gene products may be localized to other organelles such as the mitochondrion and the peroxisome (e.g. Unger et al, 1989). The cDNAs encoding these products can also be manipulated to effect the targeting of heterologous gene products to these organelles. Examples of such sequences are the nuclear-encoded ATPases and specific aspartate amino transferase isoforms for mitochondria. Targeting cellular polypeptide bodies has been disclosed by Rogers et al, (1985) Proc. Natl Acad. Sci. USA 82:6512-6516.

In addition, sequences have been characterized that control the targeting of gene products to other cell compartments. Amino terminal sequences are responsible for targeting to the endoplasmic reticulum (ER), the apoplast, and extracellular secretion from aleurone cells (Koehler & Ho, (1990) Plant Cell 2:769-783). Additionally, amino terminal sequences in conjunction with carboxy terminal sequences are responsible for vacuolar targeting of gene products (Shinshi et al, (1990) Plant Mol Biol 14:357-368).

By the fusion of the appropriate targeting sequences disclosed above to transgene sequences of interest it is possible to direct the transgene product to any organelle or cell compartment. For chloroplast targeting, for example, the chloroplast signal sequence from the RUBISCO gene, the CAB gene, the EPSP synthase gene, or the GS2 gene can be fused in frame to the amino terminal ATG of the transgene. The signal sequence selected can include the known cleavage site, and the fusion constructed can take into account any amino acids after the cleavage site that are required for cleavage. In some cases, this requirement can be fulfilled by the addition of a small number of amino acids between the cleavage site and the transgene ATG or, alternatively, replacement of some amino acids within the transgene sequence. Fusions constructed for chloroplast import can be tested for efficacy of chloroplast uptake by in vitro translation of in vitro transcribed constructions followed by in vitro chloroplast uptake. These construction techniques are well known in the art and are equally applicable to mitochondria and peroxisomes.

The above-disclosed mechanisms for cellular targeting can be utilized not only in conjunction with their cognate promoters, but also in conjunction with heterologous promoters so as to effect a specific cell-targeting goal under the transcriptional regulation of a promoter that has an expression pattern different from that of the promoter from which the targeting signal derives.

Any suitable method can be used to prepare a vector or expression cassette comprising an isolated nucleic acid of the present invention, the nucleic acid optionally being associated with a promoter. Methods for designing constructs and vectors are well known in the art and include those described by J. Sambrook, et al, Molecular Cloning: A Laboratory Manual, 3d Ed., Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press (2001); by T.J. Silhavy, M.L. Berman, and L.W. Enquist, Experiments with Gene Fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1984) and by Ausubel, F.M. et al, Current Protocols in Molecular Biology, New York, John Wiley and Sons Inc., (1988), Reiter, et al., Methods in Arabidopsis Research, World Scientific Press (1992), and Schultz et al., Plant Molecular Biology Manual, Kluwer Academic Publishers (1998). Many vectors are available for transformation using Agrobacterium tumefaciens . These vectors typically carry at least one T-DNA border sequence and include vectors such as pBIN19 (Bevan, Nucl. Acids Res. (1984)). For the construction of vectors useful in Agrobacterium transformation, see, for example, US Patent Application Publication No. 2006/0260011, which is herein incorporated by reference in its entirety. The choice of vector depends largely on the preferred selection for the species being transformed. For the construction of such vectors, see, for example, US Application No. 20060260011, which is herein incorporated by reference in its entirety.

In order to enhance nitrate assimilation, expression cassettes for plants can comprise light regulated or nitrate inducible promoters operably linked to an isolated nucleic acid of the present invention. For example, monocot light regulated promoters include promoters described in WO 2012/061585, which is herein incorporated by reference in its entirety. A dicot light regulated promoter may include small subunit of ribulose-l,5-bisphosphate- carboxylase promoter from tomato and soybean as described by Gittins, et. al. (2000) Planta 210:232-240 or a light-inducible promoter derived from a myxobacterium described in European Patent No. 310619, both of which are herein incorporated by reference. Possible nitrate inducible promoters can include the spinach nitrite reductase gene promoter described in Back, et. al. (1991) Plant Molecular Biology 17:9-18; the maize nitrite reductase; CHL1 (AtNRTl) derived from Arabidopsis thaliana (Cell, Vol. 72, pp. 705-713, 1993; The Plant Cell, Vol. 8, pp. 2183-2191, 1996); NTL1 (AtNRTl :2) derived from Arabidopsis thaliana (The Plant Cell, Vol. 11, pp. 1381-1392, 1999); OsNRTl derived from rice (Plant Physiol, Vol. 122, pp. 379-388, 2000); BnNRTl :2 derived from rapeseed (J. Biol. Chem., Vol. 273, pp. 1201, 1998); and LeNRTl derived from tomato (Proc. Natl. Acad. Sci. USA, Vol. 93, pp. 8139-8144, 1996); and CHL1 (The Plant Cell, Vol. 11, pp. 865-874, 1999).

In addition, a nitrite transporter gene may be targeted to the chloroplast. Therefore, the nitrite transporter gene may be operably linked to a chloroplast targeting sequence, such as those described in U.S. Patent Publication No. 2011/023179 and hereby incorporated by reference.

Further, the isolated nucleic acid and/or vector can be expressed in a plant cell, plant part, and/or plant using methods known to those of skill in the art. In particular embodiments of the present invention, an isolated nucleic acid is incorporated into the genome of a cell. In other embodiments of the present invention, a stably transformed plant cell, plant part, and/or plant is produced. In particular embodiments, the methods provide for the expression of an isolated polypeptide of the present invention in a plant cell, plant part, and/or plant. Exemplary methods include, but are not limited to, those described in U.S. Patent Application Publication Nos. 2009/0005296 and 2011/0061132, which are incorporated by reference herein in their entirety.

One or more assays may be used to determine and/or test gene function in a plant cell, plant part, and/or plant of the present invention. Any assay known to those of skill in the art may be used to determine and/or test gene function in a plant cell, plant part, and/or plant of the present invention. For example, the expression of a nitrite transporter of the present invention may be determined by performing a yield assay. Any suitable yield assay known to those of skill in the ait may be used. For example, a yield assay may be performed to determine if a stably transformed plant comprising a heterologous gene comprising a nucleic acid of the present invention (e.g., SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, and/or SEQ ID NO:5) provides an increased yield compared to the yield from a control plant that does not comprise the heterologous gene. Further exemplary assays for determining and/or testing gene function in a plant cell, plant part, and/or plant of the present invention include, but are not limited to, assays that measure the amount of nitrite and/or nitrate in a plant cell, plant part, and/or plant, such as the assays described in Sugiura et al., Plant and Cell Physiology, 2007; 48:1022-35, which is incorporated herein by reference for the portions relevant to this paragraph.

In other embodiments of the present invention, an isolated polypeptide of the present invention is provided comprising, consisting essentially of, or consisting of an amino acid sequence selected from the group consisting of: (a) SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, or SEQ ID NO: 10; (b) a fragment of at least about 10, 25, 50, 75, 100, 150,125, 175 or more consecutive amino acids of SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, or SEQ ID NO:10, wherein the fragment has nitrite transporter activity; (c) a naturally occurring allelic variant of a polypeptide having an amino acid sequence of SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, or SEQ ID NO:10; and/or (d) an amino acid sequence having at least about 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity to the amino acid sequences of any one of (a) to (c).

In particular embodiments of the present invention, an isolated nucleic acid of the present invention and/or an isolated polypeptide of the present invention comprises, consists essentially of, or consists of a nitrite transporter. In certain embodiments of the present invention, an isolated nucleic acid of the present invention and/or an isolated polypeptide of the present invention comprises, consists essentially of, or consists of a biologically active nitrite transporter.

A nitrite transporter of the present invention can be a high-affinity nitrite transporter or a low-affinity nitrite transporter. A high affinity nitrite transporter can act and/or be expressed when nitrite concentration is low, such as less than about 1 mM, and a low affinity nitrite transporter can act and/or be expressed when nitrite concentration is high, such as more than about 1 mM. A nitrite transporter of the present invention can be bifunctional (i.e., the nitrite transporter can transport, regulate, and the like, the movement of more than one, such as 2, 3, 4, or more, compounds and/or ions, such as, but not limited to nitrate, ammonium, formate, and any combination thereof). In certain embodiments of the present invention, a nitrite transporter comprises a nitrite/nitrate transporter. In other embodiments of the present invention, a nitrite transporter comprises a nitrite/formate transporter.

A nitrite transporter of the present invention can be located partially or fully in a plant cell wall, cell membrane or organelle membrane. In some embodiments of the present invention, a nitrite transporter of the present invention can be located partially or fully in a chloroplast envelope membrane (e.g., an inner and/or outer envelope membrane). Alternatively, a nitrite transporter of the present invention can be located in the cytosol, a plastid, and/or an organelle of a plant cell. In some embodiments of the present invention, the function of a nitrite transporter of the present invention is to acquire nitrite from the cytosol during biochemical reduction of nitrate. A nitrite transporter of the present invention can be constitutive and/or induced by nitrite. A nitrite transporter of the present invention can be repressed by other sources of nitrogen, such as, but not limited to, nitrate, ammonium, etc.

In some embodiments of the present invention, a nitrite transporter regulates nitrite availability in a plant cell, plant part, and/or plant. "Regulate," "regulation," "regulating" and grammatical variations thereof, as used herein in reference to the function of a nitrite transporter, refer to the partial or full control over the available nitrite in a plant cell, plant part, and/or plant, such as, but not limited to, partial or full control over the movement of nitrite within a plant cell, plant part, and/or plant. A nitrite transporter of the present invention can regulate the available nitrite by increasing or decreasing the conversion of nitrate to nitrite.

A nitrite transporter of the present invention can aid and/or participate (directly or indirectly) in the conversion of nitrate to nitrite. For example, a nitrite transporter of the present invention can induce and/or inhibit (directly or indirectly, such as, but not limited to releasing a inducing or inhibiting compound or ion) the conversion of nitrate to nitrite. In some embodiments of the present invention, a nitrite transporter can regulate nitrite availability by signally directly and/or indirectly for an increase or decrease in nitrate conversion to nitrite, such as, but not limited to by activating a peptide or protein or releasing a signaling compound or ion.

A nitrite transporter of the present invention can, in some embodiments, regulate nitrite availability in a plant cell, plant part, and/or plant by regulating nitrite fluctuation in a plant cell, plant part, and/or plant. "Fluctuation" as used herein refers to the movement of nitrite into, out of, and/or within a plant, plant part, and/or plant cell. For example, fluctuation can refer to the movement of nitrite from outside the plant to inside the plant, from one plant part to another plant part, from outside a plant cell to inside the plant cell, from the cytosol of a plant cell to inside an organelle of the plant cell, and vice versa. In some embodiments of the present invention, a nitrite transporter of the present invention can inhibit nitrite transport in a plant cell, plant part, and/or plant as nitrite can be toxic to a plant cell, plant part, and/or plant.

In particular embodiments of the present invention, a nitrite transporter can regulate nitrite efflux in a plant cell. "Efflux" as used herein refers to the removal and/or transport of nitrite from inside a plant cell (e.g., the cytosol) to outside the plant cell. A nitrite transporter of the present invention can regulate nitrite efflux by transporting nitrite outside the plant cell. In some embodiments of the present invention, a nitrite transporter can transport nitrite out of a plant cell in response to the concentration of nitrite in the plant cell and/or in response to the concentration of another ion (e.g., chloride, ammonium, nitrate, etc.) and/or a compound (e.g., carbon dioxide, etc.) in a plant cell.

In other embodiments of the present invention, a nitrite transporter can regulate the amount of nitrite entering an organelle of a plant cell. In particular embodiments of the present invention, a nitrite transporter regulates the amount of nitrite entering a chloroplast. A nitrite transporter of the present invention can allow and/or aid in the transport of nitrite into an organelle, such as, but not limited to a chloroplast (e.g., into the chloroplast stroma), and/or a nitrite transporter of the present invention can block or inhibit nitrite transport into an organelle.

Accordingly, by regulating nitrite availability in a plant cell, plant part, and/or plant, a nitrite transporter of the present invention can, in some embodiments, provide for a decrease in intracellular (i.e., cytosol) accumulation of nitrite compared to the amount of nitrite present in the cytosol of a control, wherein the control does not express an isolated nucleic acid of the present invention or an isolated polypeptide of the present invention. In other embodiments of the present invention, a nitrite transporter of the present invention can provide for an increase in the amount of nitrite present in the chloroplast compared to the amount of nitrite present in the chloroplast of a control, wherein the control does not express an isolated nucleic acid of the present invention or an isolated polypeptide of the present invention.

Another aspect of the present invention provides a method of using an isolated nucleic acid or vector of the present invention, the method comprising, consisting essentially of, or consisting of: transforming a plant, plant part, and/or plant cell with an isolated nucleic acid of the present invention or a vector of the present invention. In some embodiments, the plant is a monocot and/or the plant part and/or plant cell is derived from a monocot. In some embodiments, the plant is maize and/or the plant part and/or plant cell is derived from maize. In particular embodiments of the present invention, the method further comprises expressing the polypeptide encoded by the nucleic acid of the present invention or the nucleic acid in a vector of the present invention.

In some embodiments of the present invention, a method of modulating the amount of a nitrite transporter in a plant, plant part, and/or plant cell is provided, the method comprising, consisting essentially of, or consisting of: transforming a plant, plant part, and/or plant cell with an isolated nucleic acid of the present invention or a vector of the present invention, wherein the polypeptide encoded by the nucleic acid of the present invention or the nucleic acid in a vector of the present invention comprises a nitrite transporter, thereby modulating the amount of the nitrite transporter in a plant, plant part and/or plant cell. In some embodiments, the plant is a monocot and/or the plant part and/or plant cell is derived from a monocot. In some embodiments, the plant is maize and/or the plant part and/or plant cell is derived from maize.

Modulating the amount of a nitrite transporter of the present invention in a plant, plant part and/or plant cell can result in increased amounts of the nitrite transporter in a plant, plant part, and/or plant cell compared to the amount of a wild-type (i.e., native) nitrite transporter in a plant, plant part, and/or plant cell. Thus, a nitrite transporter of the present invention can be over-expressed in a plant cell, plant part, and/or plant compared to the amount of a wild- type nitrite transporter in a plant, plant part, and/or plant cell. Expression of a nitrite transporter of the present invention can be measured by any suitable method, such as, but not limited to, the methods described in U.S. Patent Application Publication No. 2011/0061132, the contents of which are incorporated herein in their entirety. According to some embodiments of the present invention, expression of a nitrite transporter of the present invention can be increased in a particular plant cell, plant part, or plant. For example, in some embodiments of the present invention, expression of a nitrite transporter of the present invention can be increased in roots and/or root hairs. In other embodiments of the present invention, expression of a nitrite transporter of the present invention can be increased during a particular developmental, stage such as, but not limited to, an early developmental stage and/or flowering stage. In particular embodiments of the present invention, the amount of a nitrite transporter of the present invention can be increased in juvenile roots. In other embodiments of the present invention, the amount of a nitrite transporter of the present invention can be increased in a plant, plant part and/or plant cell expressing nitrate reductase.

In other embodiments of the present invention, a method of regulating nitrite fluctuation in a plant, plant part and/or plant cell is provided, the method comprising, consisting essentially of, or consisting of: transforming a plant, plant part and/or plant cell with an isolated nucleic acid of the present invention or a vector of the present invention, wherein the encoded polypeptide regulates nitrite fluctuation. In some embodiments, the plant is a monocot and/or the plant part and/or plant cell is derived from a monocot. In some embodiments, the plant is maize and/or the plant part and/or plant cell is derived from maize. In certain embodiments of the present invention, the method of regulating nitrite fluctuation comprises regulating the amount of nitrite entering a chloroplast in a plant, plant part and/or plant cell, wherein the encoded polypeptide regulates the amount of nitrite entering a chloroplast. In other embodiments of the present invention, the method of regulating nitrite fluctuation comprises regulating nitrite efflux in a plant, plant part and/or plant cell, wherein the encoded polypeptide regulates efflux of nitrite.

In a further embodiment of the present invention, a method of decreasing intracellular accumulation of nitrite in a plant, plant part and/or plant cell is provided, the method comprising: transforming a plant, plant part and/or plant cell with an isolated nucleic acid of the present invention or a vector of the present invention, wherein the encoded polypeptide decreases intracellular accumulation of nitrite. In some embodiments, the plant is a monocot and/or the plant part and/or plant cell is derived from a monocot. In some embodiments, the plant is maize and/or the plant part and/or plant cell is derived from maize.

According to some embodiments of the present invention, a method of increasing nitrite transport into a chloroplast in a plant, plant part and/or plant cell is provided, the method comprising, consisting essentially of, or consisting of: transforming a plant, plant part and/or plant cell with an isolated nucleic acid of the present invention or a vector of the present invention, wherein the encoded polypeptide increases nitrite transport into a chloroplast. In some embodiments, the plant is a monocot and/or the plant part and/or plant cell is derived from a monocot. In some embodiments, the plant is maize and/or the plant part and/or plant cell is derived from maize.

In other embodiments of the present invention, a method of increasing a plant's yield is provided, the method comprising, consisting essentially of, or consisting of: transforming a plant, plant part and/or plant cell with an isolated nucleic acid of the present invention or a vector of the present invention, wherein the encoded polypeptide increases a plant's yield compared to the yield of a plant that does not comprise the isolated nucleic acid of the present invention or vector of the present invention. In some embodiments, the plant is a monocot and/or the plant part and/or plant cell is derived from a monocot. In some embodiments, the plant is maize and/or the plant part and/or plant cell is derived from maize.

A further aspect of the present invention provides a method of increasing nitrite availability and/or nitrite utilization efficiency in a plant, plant part, and/or plant cell, the method comprising, consisting essentially of, or consisting of: transforming a plant, plant part and/or plant cell with an isolated nucleic acid of the present invention or a vector of the present invention, wherein the encoded polypeptide increases nitrite availability and/or nitrite utilization efficiency in a plant, plant part, and/or plant cell compared to the nitrite availability and/or nitrite utilization efficiency in a plant, plant part, and/or plant cell that does not comprise the isolated nucleic acid of the present invention or vector of the present invention. In some embodiments, the plant is a monocot and/or the plant part and/or plant cell is derived from a monocot. In some embodiments, the plant is maize and/or the plant part and/or plant cell is derived from maize.

In particular embodiments of the present invention, the transforming step in a method of the present invention comprises stably transforming a plant cell and regenerating a stably transformed plant from the stably transformed plant cell. Optionally, the methods of the present invention can further comprise obtaining a progeny plant derived from the stably transformed plant, wherein the progeny plant comprises in its genome the isolated nucleic acid.

The present invention is explained in greater detail in the following non-limiting Examples. Examples

Example 1

Characterization of the global expression pattern of nitrogen transport and assimilation genes during maize development was performed. A custom Affymetrix 82K maize array was used to analyze the expression of 65 nitrogen uptake and assimilation probes in 50 maize tissues from seedling emergence to 31 days after pollination. The expression of orthologs of nitrite transporters ( iTRl) was discovered in early and late-stage leaves. Nitrite transporters have not previously been reported in maize. In addition, the results suggest that both maize leaves and roots exhibit distinguishable expression clusters of nitrogen related genes corresponding to juvenile, adult and reproductive phases.

Results

Tissue and developmental stage selective expression.

Plant development above ground has been divided into distinct developmental phases based on characteristics including leaf shape, surface wax, the presence of trichomes and underlying genetic networks ²⁰' ²¹. In maize, the juvenile vegetative phase (V) is from seedling emergence (Ve) to the formation of leaves 4 to 6 (V2 stage), which is followed by the adult vegetative phase (here >V5). Flowering initiates the reproductive phase (R), which includes seed formation, the latter divided into stages based on the number of days after pollination (DAP).

RNA was collected from 50 tissues from the seedling emergence stage to 31 days after pollination (DAP; Table 1) and hybridized onto customized maize Affymetrix 82K Unigene arrays 22. A total of 65 nitrogen uptake and assimilation array probes were analyzed. The expression patterns from all 50 tissue samples were summarized using a heatmap of the relative expression of nitrogen-related genes across vegetative and reproductive tissues from the seedling emergence stage to 31 days after pollination (DAP). In reproductive tissues, the highest nitrogen-related expression was observed in anthers. Interestingly, probes corresponding to nitrite transporters (NiTRl) 16, a gene class not previously reported in maize, were expressed. In vegetative tissues, four nitrogen-related gene expression clusters were detected using a heatmap of the relative expression of nitrogen- related genes in root and leaf tissues at different developmental stages, which were as follows: Ve leaf: coleoptile tissue; VI leaf: leaves 1 and 2; V2 leaf: actively growing leaf 4; V5 leaf: actively growing leaf 8, 15 cm from the tip; R1-R31 leaf: second leaf above top ear, 15 cm from the tip; VI -V5 sroot: seminal root from vegetative stages; V2-V5 nsroot: nodal root (crown root) from vegetative stages; R1-R24 nroot: nodal root (crown root) from reproductive stages, where V stands for vegetative pre-flowering stage; R stands for reproductive post-flowering stage; and VI -V5 stages refer to the number of visible stem-leaf nodes. Cluster 1 probes showed juvenile-selective expression, with peak expression in early juvenile stage roots (Ve-V2) and vegetative stage leaves (Ve-V5). Cluster 1 probes matched genes encoding a high affinity transporter NRT2.1, the low affinity transporter NRT1.1, and ammonium transporters. Cluster 2 probes were leaf-selective and more highly expressed in vegetative stage leaves (juvenile and adult: Ve-V5) than post-flowering leaves (R1-R31). Cluster 2 probes matched low affinity nitrate transporters including NRT1.5, nitrite transporter NiTRl, and nitrate reductases (NR1, NR2). Cluster 3 probes were root selective and were expressed throughout development (juvenile to post-flowering). Cluster 3 probes matched genes encoding glutamine synthetases including Gln4/Glnl-4 and Gln5/Glnl-5 as well as several glutamate synthases. Also included in Cluster 3 were ammonium transporters, the low affinity transporter NRT 1.1, and the companion protein (NAR2.1) of the high affinity nitrate transporter complex. Finally, Cluster 4 probes were leaf selective and showed the most consistent expression in older leaves at later stages of development (adult to post flowering: V5-R31). Several Cluster 4 probes matched genes encoding nitrate reductase (NR1) and nitrate transporters, as well as one NAR2 paralog and the nitrite transporter NiTRl.

Table 1: Detailed description of developmental stages and tissues sampled.

V7 tassel 12 top ear shoot

V8-V9 tassel 13-14 tassel 12-14 cm

V8-V9 ear 13-14 top ear 3~5mm

V10-V11 tassel 15-16 top 10cm of tassel (~20cm)

V10-V11 ear 15-16 top ear 1-1.5cm

V13-V15 tassel 15-16 spikelet of tassel (~22cm)

V13-V15 ear 15-16 top ear 3 -3.5 cm

V15-V16 floret 15-16 top ear(5cm) floret

V15-V16 cob 15-16 top ear (5cm)cob

V15-V16 silk 15-16 top ear (5cm)silk

V15-V16 tassel 15-16 spikelet of tassel (top 10cm)

VT anthers 15-16 anther

Rl ovule 15-16 Rl -ovule of top ear

Rl cob 15-16 Rl-cob of top ear

Rl silk 15-16 Rl -silk of top ear

Rl husk 15-16 Rl-most inner husk of top ear

Rl leaf 15-16 Rl-15cm tip of 2nd leaf above top ear

Rl nodal root 15-16 Rl -adult root

Rl stalk 15-16 Rl-15cm stalk below tassel

5DAP ovule 15-16 ovule of top ear

5DAP cob 15-16 cob of top ear

10DAP embryo 15-16 embryo of top ear

10DAP endosperm 15-16 endosperm of top ear

10DAP leaf 15-16 15 cm tip of 2nd leaf above top ear

17DAP embryo 15-16 embryo of top ear

17DAP endosperm 15-16 endosperm of top ear

17DAP pericarp 15-16 pericarp of top ear

17DAP leaf 15-16 15cm tip of 2nd leaf above top ear

24DAP leaf 15-16 15cm tip of 2nd leaf above top ear

24DAP nodal root 15-16 root

24DAP pericarp 15-16 pericarp of top ear

24DAP embryo 15-16 embryo of top ear

24DAP endosperm 15-16 endosperm of top ear

31DAP leaf 15-16 15cm tip of 2nd leaf above top ear

31DAP embryo 15-16 embryo of top ear

Nitrite transporter expression.

The expression of probes corresponding to maize ortholog(s) of a nitrite transporter (NiTR) is reported, a gene class not previously been reported in maize. A gene encoding NiTR (Narl) was initially reported in plants in Chlamydomonas chloroplasts ⁴⁸. In higher plants, an NiTR gene was first reported in cucumber (CsNitrl-L), along with a functional ortholog tested in Arabidopsis (Atlg68570) where a Icnockout mutation showed a five-fold increase in nitrite accumulation in leaves ¹⁶. The cucumber NiTR protein was localized to the inner envelope membrane of chloroplasts, where it was hypothesized to load nitrite from the cytoplasm into the stroma of the chloroplast during nitrate assimilation . Consistent with chloroplast localization, transcripts of the maize NiTR(s) orthologs were detected in the two leaf-selective expression clusters (Clusters 2 and 4) with additional strong expression in the husk leaves surrounding the cob but not in the root-selective clusters.

Materials and Methods

Plant growth and tissue harvest.

Syngenta hybrid SRG150 seeds were grown in a greenhouse, using the following conditions: 16 h light (about 600 μιηοΐ m^"2 s^"1) at 28°C, 8 h dark at 23°C, and 50% relative humidity. Plants were grown semi-hydroponically in pots containing Turface^® clay, watered with a modified Hoagland's solution containing: 0.4 g/L 28-14-14 fertilizer, 0.4 g/L 15-15-30 fertilizer, 0.2 g/L NH4N03, 0.4 g/L of MgS04^»7H20 and 0.03 g/L of micronutrient mix (S, Co, Cu, Fe, Mn, Mo and Zn). Three biological replicates per tissue/stage were harvested, always at about 11 am.

Microarray analysis.

RNA isolation and microarray analysis was previously described in Wagner F, Radelof U. "Performance of different small sample RNA amplification techniques for hybridization on Affymetrix GeneChips" Journal of Biotechnology 2007; 129:628-34 and Bi et al. (2007) BMC Genomics 8:281. RNA was isolated from 50 tissues/stages (three biological replicates) and hybridized onto customized maize Affymetrix 82K Unigene arrays ²². Array expression was normalized using the RMA method ⁵² from Bioconductor ⁵³, and analyzed for tissue selective gene expression using the Intersection Union Test, also named IUT54 using R coding modified from ppw.kuleuven.be/okp/software/BayesianIUT/ ⁵⁵. Multiple testing was corrected using the Sidak's adjustement, equivalente to the Bonferroni's correction ⁵⁶. To compare juvenile tissues (Ve-V2 stage leaf/root) to adult tissue (V5 stage), a linear model was fitted to the data using the Limma Package from Bioconductor ⁵¹ , adjusted using the empirical Bayesian method, and corrected for multiple testing using the Benjamini- Hochberg method ⁵⁸, with the p-value set at 0.05.

Annotation and clustering of nitrogen related genes.

Clustering was conducted using K-means clustering ⁵⁹. Array probes corresponding to nitrogen-related genes were retrieved using three methods. First, as the probes were designed from the maize Unigene set, the corresponding original Genbank^® sequence were used to retrieve matches, using nucleotide BLAST against the B73 maize genome (MaizeSequence.org, release 4a.53). Probe sets with no expression (relative expression < 100) in any of the 150 microarray experiments were removed from the annotation (26,989 probe sets). Each probe set was composed of 16 probes of 25 nucleotides each. If 75% of the probes in the probe set (12/16) matched the same gene model, the probe set was identified as a match for that gene. If only 12% - <75% of the probes in the probe set (1/16-11/16) matched the same gene model, the probes were considered as a partial match for that gene. A probe was required to have 85% sequence identity with the gene model to be considered as a valid match of this gene. A total of 33,664 probe sets matching to unique gene models were mapped using these steps. Exonerate alignment 60 was used to annotate an additional 9,919 probes. Nucleotide BLAST was not successful in identifying EST matches because of the biases created by gene model issues related to gene-calling software (GeneBuilder or FGENESH). The remaining 12,089 probe sets on the array showed expression, however did not map to the maize genome. After the elimination of the probe sets with either no expression, cross-hybridizing or redundant probe sets, there were 22,787 high-quality annotated probes. The array probe sequences were then re-screened for matches with EST sequences from NCBI using BLAST. Finally, nitrogen uptake and assimilation keywords were used to search gene annotations and protein domains in the B73 maize genome from MaizeSequence.org, and the search results were each matched to a Genbank^® protein with the highest homology with the microarray probe set.

References

1. Crawford NM, Glass ADM. Molecular and physiological aspects of nitrate uptake in plants. Trends in Plant Science 1998; 3:389-95.

2. Kant S, Bi Y-M, Rothstein SJ. Understanding plant response to nitrogen limitation for the improvement of crop nitrogen use efficiency. Journal of Experimental Botany 2011; 62:1499- 509.

3. Gaudin ACM, McClymont SA, Holmes BM, Lyons E, Raizada MN. Novel temporal, fine- scale and growth variation phenotypes in roots of adult-stage maize (Zea mays L.) in response to low nitrogen stress. Plant, Cell & Environment 2011; 34:2122-37.

4. Dechorgnat J, Nguyen CT, Armengaud P, Jossier M, Diatloff E, Filleur S, et al. From the soil to the seeds: the long journey of nitrate in plants. Journal of Experimental Botany 2011; 62:1349-59.

5. Forde BG. Nitrate transporters in plants: structure, function and regulation. Biochimica et Biophysica Acta (BBA) - Biomembranes 2000; 1465:219-35.

6. Peng Y, Niu J, Peng Z, Zhang F, Li C. Shoot growth potential drives N uptake in maize plants and correlates with root growth in the soil. Field Crops Research 2010; 115:85-93. 7. Masclaux-Daubresse C, Daniel- Vedele F, Dechorgnat J, Chardon F, Gaufichon L, Suzuki A. Nitrogen uptake, assimilation and remobilization in plants: challenges for sustainable and productive agriculture. Annals of Botany 2010; 105:1141-57.

8. Noctor G, Foyer CH. A re-evaluation of the ATP :NADPH budget during C3 photosynthesis: a contribution from nitrate assimilation and its associated respiratory activity? Journal of Experimental Botany 1998; 49:1895-908.

9. Gaudin ACM, McClymont SA, Raizada MN. The Nitrogen Adaptation Strategy of the Wild Teosinte Ancestor of Modern Maize, Zea mays subsp. parviglumis. Crop Science 2011; 51 :in Press.

10. Quaggiotti S, Ruperti B, Borsa P, Destro T, Malagoli M. Expression of a putative high-affinity N03- transporter and of an H+-ATPase in relation to whole plant nitrate transport physiology in two maize genotypes differently responsive to low nitrogen availability. Journal of Experimental Botany 2003; 54: 1023-31.

11. Quaggiotti S, Ruperti B, Pizzeghello D, Francioso O, Tugnoli V, Nardi S. Effect of low molecular size humic substances on nitrate uptake and expression of genes involved in nitrate transport in maize (Zea mays L.). Journal of Experimental Botany 2004; 55:803-13.

12. Schnable PS, Ware D, Fulton RS, Stein JC, Wei F, Pasternak S, et al. The B73 maize genome: complexity, diversity, and dynamics. Science 2009; 326:1112-5.

13. Orsel M, S. F, V. F, F. DV. Nitrate transport in plant: Which gene and which control? Journal of Experimental Botany 2002; 53:825-33.

14. Yong Z, Kotur Z, Glass ADM. Characterization of an intact two-component high-affinity nitrate transporter from Arabidopsis roots. Plant J 2010; 63:739-48.

15. Okamoto M, Kumar A, Li W, Wang Y, Siddiqi MY, Crawford NM, et al. High -affinity nitrate transport in roots of Arabidopsis depends on expression of the NAR2-like gene AtNRT3.1. Plant Physiol 2006; 140: 1036-46.

16. Sugiura M, Georgescu MN, Takahashi M. A Nitrite Transporter Associated with Nitrite Uptake by Higher Plant Chloroplasts. Plant and Cell Physiology 2007; 48:1022-35.

17. Martin A, Lee J, Kichey T, Gerentes D, Zivy M, Tatout C, et al. Two Cytosolic Glutamine Synthetase Isoforms of Maize Are Specifically Involved in the Control of Grain Production. The Plant Cell 2006; 18:3252-74.

18. Sakakibara H, Kawabata S, Takahashi H, Hase T, Sugiyama T. Molecular Cloning of the Family of Glutamine Synthetase Genes from Maize: Expression of Genes for Glutamine Synthetase and Ferredoxin-Dependent Glutamate Synthase in Photosynthetic and Non- Photo synthetic Tissues. Plant and Cell Physiology 1992; 33:49-58.

19. Suzuki A, Knaff DB. Glutamate synthase: structural, mechanistic and regulatory properties, and role in the amino acid metabolism. Photosynthesis Research 2005; 83:191- 217.

20. Poethig RS. Phase Change and the Regulation of Shoot Morphogenesis in Plants. Science 1990; 250:923-30.

21. Poethig RS. The Past, Present, and Future of Vegetative Phase Change. Plant Physiology 2010; 154:541-4.

22. Wagner F, Radelof U. Performance of different small sample RNA amplification techniques for hybridization on Affymetrix GeneChips. Journal of Biotechnology 2007; 129:628-34.

23. Konishi M, Yanagisawa S. Identification of a nitrate-responsive cis-element in the Arabidopsis NIR1 promoter defines the presence of multiple cis-regulatory elements for nitrogen response. The Plant Journal 2010; 63:269-82.

24. Ishige F, Takaichi M, Foster R, Chua N-H, Oeda K. A G-box motif (GCCACGTGCC) tetramer confers high-level constitutive expression in dicot and monocot plants. The Plant Journal 1999; 18:443-8. 25. Wang R, Guan P, Chen M, Xing X, Zhang Y, Crawford N. Multiple Regulatory Elements in the Arabidopsis NIA1 Promoter Act Synergistically to Form a Nitrate Enhancer. Plant Physiology 2010.

26. Park SH, Koh SS, Chun JH, Hwang HJ, Kang HS. Nrgl Is a Transcriptional Repressor for Glucose Repression of STA1 Gene Expression inSaccharomyces cerevisiae. Molecular and Cellular Biology 1999; 19:2044-50.

27. Zhou H, Winston F. NRG1 is required for glucose repression of the SUC2 and GAL genes of Saccharomyces cerevisiae. BMC Genetics 2001 ; 2:5.

28. Kim M, Ahn J-W, Song K, Paek K-H, Pai H-S. Forkhead-associated Domains of the Tobacco NtFHAl Transcription Activator and the Yeast Fhll Forkhead Transcription Factor Are Functionally Conserved. Journal of Biological Chemistry 2002; 277:38781-90.

29. Babiychuk E, Kushnir S, Belles-Boix E, Van Montagu M, Inze D. Arabidopsis thaliana NADPH Oxidoreductase Homologs Confer Tolerance of Yeasts toward the Thiol-oxidizing Drug Diamide. Journal of Biological Chemistry 1995; 270:26224-31.

30. Carmel-Harel O, Stearman R, Gasch AP, Botstein D, Brown PO, Storz G. Role of thioredoxin reductase in the Yaplp-dependent response to oxidative stress in Saccharomyces cerevisiae. Molecular Microbiology 2001; 39:595-605.

31. Moye-Rowley WS, Harshman KD, Parker CS. Yeast YAP1 encodes a novel form of the jun family of transcriptional activator proteins. Genes & Development 1989; 3:283-92.

32. Yokoyama H, Mizunuma M, Okamoto M, Yamamoto J, Hirata D, Miyakawa T. Involvement of calcineurin-dependent degradation of Yaplp in Ca2+-induced G2 cell-cycle regulation in Saccharomyces cerevisiae. EMBO Rep 2006; 7:519-24.

33. Hermann-Le Denmat S, Werner M, Sentenac A, Thuriaux P. Suppression of yeast RNA polymerase III mutations by FHL1, a gene coding for a fork head protein involved in rRNA processing. Molecular and Cellular Biology 1994; 14:2905-13.

34. Jonczyk M, Sobkowiak A, Siedlecki P, Biecek P, Trzcinska-Danielewicz J, Tiuryn J, et al. Rhythmic Diel Pattern of Gene Expression in Juvenile Maize Leaf. PLoS ONE 2011 ; 6:e23628.

35. Li P, Ponnala L, Gandotra N, Wang L, Si Y, Tausta SL, et al. The developmental dynamics of the maize leaf transcriptome. Nat Genet 2010; 42: 1060-7.

36. Strable J, Borsuk L, Nettleton D, Schnable PS, Irish EE. Microarray analysis of vegetative phase change in maize. The Plant Journal 2008; 56: 1045-57.

37. Sekhon RS, Lin H, Childs KL, Hansey CN, Buell CR, de Leon N, et al. Genome-wide atlas of transcription during maize development. The Plant Journal 2011; 66:553-63.

38. Krapp A, Berthome R, Orsel M, Mercey-Boutet S, Yu A, Castaings L, et al. Arabidopsis Roots and Shoots Show Distinct Temporal Adaptation Patterns toward Nitrogen Starvation. Plant Physiology 2011 ; 157: 1255-82.

39. Scheible W-R, Lauerer M, Schulze E-D, Caboche M, Stitt M. Accumulation of nitrate in the shoot acts as a signal to regulate shoot-root allocation in tobacco†. The Plant Journal 1997; 11 :671-91.

40. Irizarry RA, Bolstad BM, Collin F, Cope LM, Hobbs B, Speed TP. Summaries of Affymetrix GeneChip probe level data. Nucl Acids Res 2003; 31 :el5-.

41. Hwang YS, Karrer EE, Thomas BR, Chen L, Rodriguez RL. Three cis-elements required for rice oc-amylase Amy3D expression during sugar starvation. Plant Molecular Biology 1998; 36:331-41.

42. Toledo-Ortiz G, Huq E, Quail PH. The Arabidopsis Basic/Helix-Loop-Helix Transcription Factor Family. The Plant Cell Online 2003; 15:1749-70.

43. Ramon M, Rolland F, Sheen J. Sugar Sensing and Signaling. The Arabidopsis Book 2008:e0117.

44. Jang JC, Sheen J. Sugar sensing in higher plants. Trends in Plant Science 1997; 2:208-14. 45. Lejay L, Tillard P, Lepetit M, Olive Francesc D, Filleur S, Daniel- Vedele F, et al. Molecular and functional regulation of two N03- uptake systems by N- and C-status of Arabidopsis plants. The Plant Journal 1999; 18:509-19.

46. Dobrenel T, Marchive C, Sormani R, Moreau M, Mozzo M, Montane M-H, et al. Regulation of plant growth and metabolism by the TOR kinase. Biochemical Society Transactions 2011; 39:477-81.

47. Diaz-Troya S, Perez-Perez ME, Florencio FJ, Crespo JL. The role of TOR in autophagy regulation from yeast to plants and mammals. Autophagy 2008; 4:851-65.

48. Rexach J, Fernandez E, Galvan A. The Chlamydomonas reinhardtii Narl Gene Encodes a Chloroplast Membrane Protein Involved in Nitrite Transport. The Plant Cell Online 2000; 12: 1441-54.

49. Gallais A, Hirel B. An approach to the genetics of nitrogen use efficiency in maize. Journal of Experimental Botany 2004; 55:295-306.

50. Hirel B, Le Gouis J, Ney B, Gallais A. The challenge of improving nitrogen use efficiency in crop plants: towards a more central role for genetic variability and quantitative genetics within integrated approaches. Journal of Experimental Botany 2007; 58:2369-87.

51. Eickhout B, Bouwman AF, van Zeijts H. The role of nitrogen in world food production and environmental sustainability. Agriculture, Ecosystems &amp; Environment 2006; 116:4- 14.

52. Irizarry RA, Bolstad BM, Collin F, Cope LM, Hobbs B, Speed TP. Summaries of Affymetrix GeneChip probe level data. Nucleic Acids Research 2003; 31 :el5.

53. Gentleman R, Carey V, Bates D, Bolstad B, Dettling M, Dudoit S, et al. Bioconductor: open software development for computational biology and bioinformatics. Genome Biology 2004; 5:R80.

54. Berger RL, Hsu JC. Bioequivalence trials, intersection-union tests and equivalence confidence sets. Statistical Science 1996; 11 :283-319.

55. Van Deun K, Hoijtink H, Thorrez L, Van Lommel L, Schuit F, Van Mechelen I. Testing the hypothesis of tissue selectivity: the intersection-union test and a Bayesian approach. Bioinformatics 2009; 25:2588-94.

56. Sidak Z. Rectangular Confidence Regions for the Means of Multivariate Normal Distributions. Journal of the American Statistical Association 1967; 62:626-33.

57. Smyth G. limma: Linear Models for Microarray Data. In: Gentleman R, Carey VJ, Huber W, Irizarry RA, Dudoit S, eds. Bioinformatics and Computational Biology Solutions Using R and Bioconductor: Springer New York, 2005:397-420.

58. Benjamini Y, Hochberg Y. Controlling the False Discovery Rate: A Practical and Powerful Approach to Multiple Testing. Journal of the Royal Statistical Society Series B 1995; 57:289-300.

59. Hartigan JA, Wong MA. Algorithm AS 136: A K-Means Clustering Algorithm. Journal of the Royal Statistical Society Series C (Applied Statistics) 1979; 28: 100-8.

60. Slater G, Birney E. Automated generation of heuristics for biological sequence comparison. BMC Bioinformatics 2005; 6:31.

61. Liseron-Monfils CV, Ashlock D, McNicholas PD, Fauteux F, Stromvik M, Raizada MN. Promzea: A pipeline for discovery of regulatory motifs in maize (Zea mays L.) and its application to the anthocyanin biosynthetic pathway. 2011 :In preparation.

62. Pavesi G, Zambelli F, Pesole G. WeederH: an algorithm for finding conserved regulatory motifs and regions in homologous sequences. BMC Bioinformatics 2007; 8:46.

63. Bailey TL, Elkan C. Fitting a mixture model by expectation maximization to discover motifs in biopolymers. Proceedings of the Second International Conference on Intelligent Systems for Molecular Biology. Menlo Park, California: AAAI Press, 1994:28-36. 64. Liu X, Brutlag D, Liu J. BioProspector: discovering conserved DNA motifs in upstream regulatory regions of co-expressed genes. In: Altman RB, Dunker AK, Hunter L, Klein TE, eds. Pacific Symposium on Biocomputing 2001, 2001 :127-38.

65. Biilow L, Engelmann S, Schindler M, Hehl R. AthaMap, integrating transcriptional and post-transcriptional data. Nucleic Acids Research 2009; 37:D983-D6.

66. Higo K, Ugawa Y, Iwamoto M, Korenaga T. Plant cis-acting regulatory DNA elements (PLACE) database: 1999. Nucleic Acids Research 1999; 27:297-300.

67. Mahony S, Benos PV. STAMP: a web tool for exploring DNA-binding motif similarities. Nucleic Acids Research 2007:253-8.

Example 2

SEQ ID NO:6 to SEQ ID NO: 10 were analyzed using InterProScan and TMHMM2.0. Domains identified are listed in Table 2 below. Domains identified by InterProScan are described in the databases found in The European Bioinformatics Institute (EBI) which is part of European Molecular Biology Laboratory (EMBL). TMHMM predicted regions occurring outside a membrane, forming a transmembrane helix, and/or occurring inside a membrane.

Table 2: Domains indentified using InterProScan and TMHMM2.0.

SEQ ID NO:6

FT TOPO DOMA 1 93 Potential inside TMHMM2.0

FT REGION 1 586 superfamily SSF103473 MFS general substrate

FT transporter 9.3e-29 14-Mar-2012

FT REGION 5 588 HMMPanther PTHR11654:SF20 NITRATE

FT TRANSPORTER (NRTl) 6.6e-239 14-Mar-2012

FT REGION 5 588 HMMPanther PTHR11654 OLIGOPEPTIDE

FT TRANSPORTER-RELATED 6.6e-239 IPR000109

FT TGF-beta receptor, type I/II extracellular

FT region 14-Mar-2012

FT REGION 82 91 Seg seg seg NA 14-Mar-2012

FT REGION 93 513 HMMPfam PF00854 PTR2 3.7e-128 IPR000109

FT TGF-beta receptor, type VII extracellular

FT region 14-Mar-2012

FT TRANSMEM 94 116 Potential TMhelix TMHMM2.0

FT REGION 101 1 12 Seg seg seg NA 14-Mar-2012

FT TOPO DOMA 117 135 Potential outside TMHMM2.0

FT REGION 121 133 Seg seg seg NA 14-Mar-2012

FT TRANSMEM 136 158 Potential TMhelix TMHMM2.0

FT REGION 137 157 Seg seg seg NA 14-Mar-2012

FT TOPO DOMA 159 193 Potential inside TMHMM2.0

FT REGION 176 193 Seg seg seg NA 14-Mar-2012

FT TRANSMEM 194 216 Potential TMhelix TMHMM2.0

FT REGION 204 210 Seg seg seg NA 14-Mar-2012

FT TOPO DOMA 217 225 Potential outside TMHMM2.0

FT TRANSMEM 226 248 Potential TMhelix TMHMM2.0

FT TOPO DOMA 249 378 Potential inside TMHMM2.0

FT TRANSMEM 379 401 Potential TMhelix TMHMM2.0

FT TOPO DOMA 402 420 Potential outside TMHMM2.0

FT TRANSMEM 421 443 Potential TMhelix TMHMM2.0

FT TOPO DOMA 444 454 Potential inside TMHMM2.0

FT TRANSMEM 455 477 Potential TMhelix TMHMM2.0

FT TOPO DOMA 478 504 Potential outside TMHMM2.0

FT REGION 503 514 Seg seg seg NA 14-Mar-2012

FT TRANSMEM 505 527 Potential TMhelix TMHMM2.0

FT TOPO DOMA 528 550 Potential inside TMHMM2.0

FT TRANSMEM 551 573 Potential TMhelix TMHMM2.0

FT TOPO DOMA 574 602 Potential outside TMHMM2.0

SQ SEQUENCE 602 AA

SEQ ID NO:7

FT TOPO DOMA 1 341 Potential outside TMHMM2.0

FT REGION 15 26 Seg seg seg NA 14-Mar-2012

FT REGION 37 59 Seg seg seg NA 14-Mar-2012

FT REGION 62 89 Seg seg seg NA 14-Mar-2012

FT REGION 90 131 Seg seg seg NA 14-Mar-2012

FT REGION 133 214 Seg seg seg NA 14-Mar-2012

FT REGION 230 263 Seg seg seg NA 14-Mar-2012 SEQ ID NO:8

FT TOPO DOMA 1 28 Potential outside TMHMM2.0

FT REGION 1 153 HMMPanther PTHR11654:SF20 NITRATE

FT TRANSPORTER (NRTl) 2.4e-61 14-Mar-2012

FT REGION 1 153 HMMPanther PTHR11654 OLIGOPEPTIDE

FT TRANSPORTER-RELATED 2.4e-61 IPR000109

FT TGF-beta receptor, type I/II extracellular

FT region 14-Mar-2012

FT REGION 1 80 HMMPfam PF00854 PTR2 2.6e-19 IPR000109

FT TGF-beta receptor, type I/II extracellular

FT region 14-Mar-2012

FT TRANSMEM 29 51 Potential TMhelix TMHMM2.0

FT REGION 34 145 superfamily SSF103473 MFS general substrate

FT transporter 1.3e-09 14-Mar-2012

FT TOPO DOMA 52 71 Potential inside TMHMM2.0

FT TRANSMEM 72 94 Potential TMhelix TMHMM2.0

FT TOPO DOMA 95 116 Potential outside TMHMM2.0

FT TRANSMEM 117 136 Potential TMhelix TMHMM2.0

FT TOPO DOMA 137 171 Potential inside TMHMM2.0

SEQ ID N0:9

FT TOPO DOMA 1 187 Potential inside TMHMM2.0

FT REGION 1 571 superfamily SSF103473 MFS general substrate

FT transporter 2.8e-28 14-Mar-2012

FT REGION 9 24 Seg seg seg NA 14-Mar-2012

FT REGION 24 579 HMMPanther PTHRl 1654:SF20 NITRATE

FT TRANSPORTER (NRTl) 9.6e-235 14-Mar-2012

FT REGION 24 579 HMMPanther PTHRl 1654 OLIGOPEPTIDE

FT TRANSPORTER-RELATED 9.6e-235 IPR000109

FT TGF-beta receptor, type I/II extracellular

FT region 14-Mar-2012

FT REGION 97 506 HMMPfam PF00854 PTR2 7.7e-118 IPR000109

FT TGF-beta receptor, type I/II extracellular

FT region 14-Mar-2012

FT TRANSMEM 188 210 Potential TMhelix TMHMM2.0

FT TOPO DOMA 211 219 Potential outside TMHMM2.0

FT TRANSMEM 220 242 Potential TMhelix TMHMM2.0

FT TOPO DOMA 243 333 Potential inside TMHMM2.0

FT TRANSMEM 334 353 Potential TMhelix TMHMM2.0

FT TOPO DOMA 354 372 Potential outside TMHMM2.0

FT TRANSMEM 373 395 Potential TMhelix TMHMM2.0

FT TOPO DOMA 396 418 Potential inside TMHMM2.0

FT TRANSMEM 419 436 Potential TMhelix TMHMM2.0

FT TOPO DOMA 437 455 Potential outside TMHMM2.0

FT TRANSMEM 456 478 Potential TMhelix TMHMM2.0

FT TOPO DOMA 479 498 Potential inside TMHMM2.0

FT TRANSMEM 499 521 Potential TMhelix TMHMM2.0

FT TOPO DOMA 522 542 Potential outside TMHMM2.0

FT TRANSMEM 543 562 Potential TMhelix TMHMM2.0

FT TOPO DOMA 563 597 Potential inside TMHMM2.0

SQ SEQUENCE 597 AA SEQ ID NO: 10

FT TOPO DOMA 1 71 Potential outside TMHMM2.0

FT REGION 1 240 HMMPfam PF00854 PTR2 1.9e-06 IPR000109

FT TGF-beta receptor, type I/II extracellular

FT region 14-Mar-2012

FT REGION 31 316 HMMPanther PTHR11654:SF20 NITRATE

FT TRANSPORTER (NRT1) 2.6e-124 14-Mar-2012

FT REGION 31 316 HMMPanther PTHR11654 OLIGOPEPTIDE

FT TRANSPORTER-RELATED 2.6e-124 IPR000109

FT TGF-beta receptor, type I/II extracellular

FT region 14-Mar-2012

FT TRANSMEM 72 94 Potential TMhelix TMHMM2.0

FT TOPO DOMA 95 105 Potential inside TMHMM2.0

FT TRANSMEM 106 128 Potential TMhelix TMHMM2.0

FT REGION 112 313 superfamily SSF103473 MFS general substrate

FT transporter 5.3e-08 14-Mar-2012

FT TOPO DOMA 129 147 Potential outside TMHMM2.0

FT TRANSMEM 148 170 Potential TMhelix TMHMM2.0

FT TOPO DOMA 171 189 Potential inside TMHMM2.0

FT TRANSMEM 190 212 Potential TMhelix TMHMM2.0

FT TOPO DOMA 213 231 Potential outside TMHMM2.0

FT TRANSMEM 232 254 Potential TMhelix TMHMM2.0

FT REGION 246 262 Seg seg seg NA 14-Mar-2012

FT TOPO DOMA 255 277 Potential inside TMHMM2.0

FT TRANSMEM 278 300 Potential TMhelix TMHMM2.0

FT TOPO DOMA 301 332 Potential outside TMHMM2.0

References

1. E. L.L. Sonnhammer, G. von Heijne, and A. Krogh. "A hidden Markov model for predicting transmembrane helices in protein sequences."

2. In J. Glasgow, T. Littlejohn, F. Major, R. Lathrop, D. Sankoff, and C. Sensen, editors, Proceedings of the Sixth International Conference on Intelligent Systems for Molecular Biology, pages 175-182, Menlo Park, CA, 1998. AAAI Press.

3. Zdobnov E.M. and Apweiler R. "InterProScan - an integration platform for the signature- recognition methods in lnterPro." Bioinformatics, 2001 , 17(9): 847-8.

Example 3

Constructs comprising one or more isolated nucleic acids of the present invention (e.g., SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, and/or SEQ ID NO:5) as described herein will be used for Agrobacterium-mediated maize transformation. Transformation of immature maize embryos will be performed essentially as described in Negrottoet al., 2000, Plant Cell Reports 19: 798-803. For this example, all media constituents will be essentially as described in Negrottoet al., supra. However, various media constituents known in the art may be substituted. The genes used for transformation will be ligated into a vector suitable for maize transformation. Vectors used in this example will contain the phosphomannoseisomerase (PMI) gene for selection of transgenic lines (Negrottoet ah, supra), as well as the selectable marker phosphinothricin acetyl transferase (PAT) (U.S. Patent No. 5,637,489). Briefly, Agrobacterium strain LBA4404 (pSBl) containing a plant transformation plasmid will be grown on YEP (yeast extract (5 g/L), peptone (lOg/L), NaCl (5g/L), 15g/l agar, pH 6.8) solid medium for 2 - 4 days at 28°C. Approximately 0.8 X 10⁹ Agrobacterium will be suspended in LS-inf media supplemented with 100 μΜ As (Negrottoet al, supra). Bacteria will be pre- induced in this medium for 30-60 minutes.

Immature embryos from A188 or other suitable genotype will be excised from 8 - 12 day old ears into liquid LS-inf + 100 μΜ As. Embryos will be rinsed once with fresh infection medium. Agrobacterium solution will then be added and embryos will be vortexed for 30 seconds and allowed to settle with the bacteria for 5 minutes. The embryos will then be transferred, scutellum side up to LSAs medium and cultured in the dark for two to three days. Subsequently, between 20 and 25 embryos per petri plate will be transferred to LSDc medium supplemented with cefotaxime (250 mg/1) and silver nitrate (1.6 mg/1) and cultured in the dark for 28°C for 10 days.

Immature embryos, producing embryogenic callus will be transferred to LSD1M0.5S medium. The cultures will be selected on this medium for about 6 weeks with a subculture step at about 3 weeks. Surviving calli will be transferred to Regl medium supplemented with mannose. Following culturing in the light (16 hour light/ 8 hour dark regiment), green tissues will then be transferred to Reg2 medium without growth regulators and incubated for about 1-2 weeks. Plantlets will be transferred to Magenta GA-7 boxes (Magenta Corp, Chicago 111.) containing Reg3 medium and grown in the light.

Plants will be assayed for PMI, at least one candidate gene of the present invention {e.g., SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, and/or SEQ ID NO:5) and vector backbone by TaqMan. Plants that are positive for PMI and the at least one candidate gene marker, and negative for vector backbone will be transferred to the greenhouse. Expression for all trait expression cassettes will be assayed by qRT-PCR. Fertile, single copy events will be identified and maintained.

Example 4

Constructs comprising one or more isolated nucleic acids of the present invention (e.g., SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, and/or SEQ ID NO:5) operably linked to either a light regulated promoter or a nitrate inducible promoter, such as a spinach nitrate inducible promoter, and a chloroplast targeting sequence will be created. Agwbacterium-medi&ted transformation will be used to generate transgenic plants. Positively transformed plants will be selected using the phosphomannose isomerase (PMI) test (Negrotto et al. PLANT CELL REP. 19:798 (2000)).

Example 5

Transgenic Arabidopsis plants comprising an expression cassette comprising an isolated nucleic acid of the present invention operably linked to either a light inducible or nitrate inducible promoter will be generated by Agrobacterium-mediated transformation (Bechtold, N., Ellis, J. & Pelletier, G. (1993) C R Acad Sci 316, 1194-1199). Transgenic plants will be selected on kanamycin containing medium. The plants will then be selected for self pollination. Transgenic lines of the T3 generation homozygous for the transgene will be used for further analysis. The expression levels of the nitrite transporter in the transgenic lines will be determined by real-time RT-PCR.

Example 6

The transformed plants will be tested to understand the growth rate under defined conditions in which nitrogen limits growth. The Rockwool system will be employed (Hirai et al., 1995 Plant Cell Physiol 36, 1331-1339) with three defining conditions: one where growth is maximal; one where nitrogen limits growth to 70-75% maximal growth levels; and one where there is a more severe limitation to 30-35% maximal growth levels. The nitrogen limitation acts as a 'stress' with the amount of 'stress' easily varied by altering the concentration of nitrate. The physiological "nitrogen status" is measured by measuring nitrate, chlorophyll (which is often used as a reflection of nitrogen status under field conditions (see, e.g., Fox RH et al 2001 Agron J. 93, 590-597; Minotti PL et al 1994 Hort Science 29, 1497-1550), amino acid levels, and nitrate reductase and glutamine synthetase activities in order to give a baseline in which to assess studies on mutant lines.

The foregoing is illustrative of the present invention, and is not to be construed as limiting thereof. The invention is defined by the following claims, with equivalents of the claims to be included therein. All publications, patent applications, patents, patent publications, and other references cited herein are incorporated by reference in their entireties for the teachings relevant to the sentence and/or paragraph in which the reference is presented.

Claims

THAT WHICH IS CLAIMED IS:

1. An isolated nucleic acid comprising a nucleotide sequence selected from the group consisting of:

(a) a nucleotide sequence of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:5;

(b) a nucleotide sequence that encodes a polypeptide having an amino acid

sequence of SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, or SEQ ID NO: 10;

(c) a nucleotide sequence that encodes a polypeptide having at least 90% identity to the amino acid sequence of SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, or SEQ ID NO: 10;

(d) a nucleotide sequence that encodes a fragment of a polypeptide having an amino acid sequence of SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, or SEQ ID NO: 10, wherein the fragment comprises at least 15 consecutive amino acids of SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, or SEQ ID NO: 10;

(e) a nucleotide sequence that encodes a naturally occurring allelic variant of a polypeptide having an amino acid sequence of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO:8, SEQ ID NO:9, or SEQ ID NO: 10;

(f) a nucleotide sequence that hybridizes to the complete complement of the

nucleotide sequences of any one of (a) to (e) under stringent conditions comprising a wash stringency of 50% Formamide with 5x Denhardt's solution, 0.5% SDS and lx SSPE at 42°C;

(g) a degenerate nucleotide sequence of any one of (a) to (f) as a result of the genetic code; and/or

(h) a nucleotide sequence having at least 90% sequence identity to the nucleotide sequences of any one of (a) to (g).

2. The isolated nucleic acid of claim 1, wherein the nucleotide sequence is operably associated with a promoter.

3. The isolated nucleic acid of claim 2, wherein the promoter comprises one or more nucleotide sequences selected from the group consisting of: SEQ ID NO: l 1, SEQ ID NO:12, SEQ ID NO:13, SEQ ID N0:14, SEQ ID N0:15, SEQ ID N0:16, SEQ ID N0:17, SEQ ID N0: 18, SEQ ID N0: 19, SEQ ID NO:20, SEQ ID N0:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, and/or SEQ ID N0:31.

4. The isolated nucleic acid of claim 2, wherein the promoter comprises one or more promoters that drive the expression of nitrate reductase.

5. The isolated nucleic acid of any one of claims 1-4, wherein the polypeptide comprises a nitrite transporter.

6. A vector comprising the isolated nucleic acid of any one of claims 1-5.

7. A cell comprising the isolated nucleic acid of any one of claims 1-5 or the vector of claim 6.

8. The cell of claim 7, wherein the cell is a plant cell.

9. The plant cell of claim 8, wherein the isolated nucleic acid or vector is stably incorporated into the genome of the cell.

10. The plant cell of claim 8, wherein the plant cell is derived from a monocot.

11. The plant cell of claim 8, wherein the plant cell is derived from maize.

12. A plant part comprising the plant cell of claim 8 or claim 9.

13. The plant part of claim 12, wherein the plant part is derived from a monocot.

14. The plant part of claim 12, wherein the plant part is derived from maize.

15. A transgenic plant comprising the plant cell of claim 8 or claim 9.

16. A stably transformed plant comprising the isolated nucleic acid of any one of claims 1-5 or the vector of claim 6 incorporated in its genome.

17. The transgenic plant of claim 15 or the stably transformed plant of claim 16, wherein the plant is a monocot.

18. The transgenic plant of claim 15 or the stably transformed plant of claim 16, wherein monocot is maize.

19. A product harvested from the plant of any one of claims 15-18.

20. A processed product produced from the harvested product of claim 19.

21. A crop comprising a plurality of the plant of any one of claims 15-18.

22. A seed comprising the isolated nucleic acid of any one of claims 1-5 or the vector of claim 6 stably incorporated in its genome.

23. An isolated polypeptide comprising an amino acid sequence selected from the group consisting of:

(a) SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, or SEQ ID

NO:10;

(b) a fragment of at least 15 consecutive amino acids of SEQ ID NO: 6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, or SEQ ID NO:10;

(c) a naturally occurring allelic variant of a polypeptide having an amino acid sequence of SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, or SEQ ID NO: 10; and/or

(d) an amino acid sequence having at least 90% sequence identity to the amino acid sequences of any one of (a) to (c).

24. A method of modulating the amount of a nitrite transporter in a plant, plant part or plant cell, the method comprising:

transforming a plant, plant part or plant cell with the isolated nucleic acid of any of claims 2-5 or the vector of claim 6, wherein the encoded polypeptide comprises a nitrite transporter, thereby modulating the amount of the nitrite transporter in a plant, plant part or plant cell.

25. The method of claim 24, wherein the amount of the nitrite transporter is increased.

26. The method of claim 25, wherein the amount of the nitrite transporter is increased in plants, plant parts or plant cells expressing nitrate reductase.

27. The method of any one of claims 25-26, wherein the amount of the nitrite transporter is increased in juvenile roots.

28. A method of regulating nitrite fluctuation in a plant, plant part or plant cell, the method comprising:

transforming a plant, plant part or plant cell with the isolated nucleic acid of any of claims 2-5 or the vector of claim 6,

wherein the encoded polypeptide regulates nitrite fluctuation.

29. A method of regulating the amount of nitrite entering a chloroplast in a plant, plant part or plant cell, the method comprising:

transforming a plant, plant part or plant cell with the isolated nucleic acid of any of claims 2-5 or the vector of claim 6,

wherein the encoded polypeptide regulates the amount of nitrite entering a chloroplast.

30. A method of regulating nitrite efflux in a plant, plant part or plant cell, the method comprising:

transforming a plant, plant part or plant cell with the isolated nucleic acid of any of claims 2-5 or the vector of claim 6,

wherein the encoded polypeptide regulates efflux of nitrite.

31. A method of decreasing intracellular accumulation of nitrite in a plant, plant part or plant cell, the method comprising: transforming a plant, plant part or plant cell with the isolated nucleic acid of any of claims 2-5 or the vector of claim 6,

wherein the encoded polypeptide decreases intracellular accumulation of nitrite.

32. A method of increasing nitrite transport into a chloroplast in a plant, plant part or plant cell, the method comprising:

transforming a plant, plant part or plant cell with the isolated nucleic acid of any of claims 2-5 or the vector of claim 6,

wherein the encoded polypeptide increases nitrite transport into a chloroplast.

33. A method of increasing a plant's yield, the method comprising:

(a) stably transforming a plant cell with the isolated nucleic acid of any of claims 2-5 or the vector of claim 6,

(b) regenerating a stably transformed plant from the stably transformed plant cell of (a),

wherein the encoded polypeptide increases a plant's yield compared to the yield of a plant that does not comprise the isolated nucleic acid or vector.

34. The method of any one of claims 24-32, wherein the transforming step comprises stably transforming a plant cell and the method further comprises regenerating a stably transformed plant from the stably transformed plant cell.

35. The method of any one of claims 33-34, further comprising obtaining a progeny plant derived from the stably transformed plant, wherein the progeny plant comprises in its genome the isolated nucleic acid.

36. The method of any one of claims 24-35, wherein the promoter comprises one or more nucleotide sequences selected from the group consisting of: SEQ ID NO:l 1, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, and/or SEQ ID NO:31.

37. The method of any one of claims 24-35, wherein the promoter comprises one or more promoters that drive the expression of nitrate reductase.

38. The method of any one of claims 24-37, wherein the plant is a monocot or the plant part or the plant cell is derived from a monocot.

39. The method of any one of claims 24-37, wherein the plant is maize or the plant part or the plant cell is derived from maize.

40. A stably transformed plant produced by the method of any one of claims 33-

34.

41. The stably transformed plant of claim 40, wherein the plant is a monocot.

42. The stably transformed plant of claim 40, wherein the plant is maize.

43. A seed produced from the transgenic plant of any one of claims 15- 18 or 40- 42 wherein the seed comprises the isolated nucleic acid stably incorporated in its genome.