EP1417315A2 - Pflanzliche ionenkanäle und verfahren - Google Patents

Pflanzliche ionenkanäle und verfahren

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Publication number
EP1417315A2
EP1417315A2 EP02768333A EP02768333A EP1417315A2 EP 1417315 A2 EP1417315 A2 EP 1417315A2 EP 02768333 A EP02768333 A EP 02768333A EP 02768333 A EP02768333 A EP 02768333A EP 1417315 A2 EP1417315 A2 EP 1417315A2
Authority
EP
European Patent Office
Prior art keywords
nucleotide sequence
plant
amino acid
set forth
seq
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP02768333A
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English (en)
French (fr)
Inventor
Alan M. Kinnersley
Frank J. Turano
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Emerald Bioagriculture Corp
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Emerald Bioagriculture Corp
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Publication date
Application filed by Emerald Bioagriculture Corp filed Critical Emerald Bioagriculture Corp
Publication of EP1417315A2 publication Critical patent/EP1417315A2/de
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
    • Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants

Definitions

  • GABA The amino acid ⁇ -aminobutyric acid
  • GABA is the major neurotransmitter in the mammalian central nervous system.
  • Such neurotransmitters generally function in regulating the conductance of ions across neuronal membranes, typically in regulating influx of ions into a cell.
  • GABA is considered an inhibitory neurotransmitter that acts to inhibit synaptic transmission in both vertebrate and invertebrate nervous systems.
  • glutamate is an excitatory neurotransmitter that depolarizes the postsynaptic membrane and acts to promote synaptic transmission. Both GABA and glutamate affect synaptic transmission by binding to their respective receptors, also known as ligand-gated ion channels.
  • GABA A GABA A
  • GABA B GABA B
  • GABAc GABA receptors
  • GABA receptors have been implicated in mediating anxiety, seizures, cognitive function, addictive disorders, sleep disorders and other disorders of the central nervous system.
  • GABA receptors are the target of many pharmaceutical preparations that act on the central nervous system, including barbiturates and benzodiazepines, and thus have therapeutic value.
  • compounds that affect the function of insect GABA receptors are commercially useful as insecticides. GABA receptors have been found in insects and in the animal kingdom.
  • GABA has been shown to exert certain beneficial effects on plants.
  • GABA has been shown to increase plant growth and productivity as shown in U.S. Patent No. 5,439,873 to Kinnersley.
  • beneficial effects have been increased when GABA is applied to plants along with a readily metabolized source of carbon, such as succinic acid (U.S. Patent No. 5,604,177).
  • GABA has been found to increase fertilizer efficiency when administered with glutamic acid as described in U.S. Patent No. 5,840,656 to Kinnersley et al.
  • the present invention relates to the new discovery that plants respond to compounds known to act on animal mitochondrial GABA receptor proteins, and the related discovery that plants express receptor proteins that respond to these compounds, hi this regard, the invention provides nucleotide sequences that have been discovered in plants that are expected to encode benzodiazepine or benzodiazepine-like receptor proteins having significant sensitivity to benzodiazepines. Based upon the data presented herein, such proteins are expected to function as modulators of GABA action and, in particular, as ion channels, such as ligand-gated ion channels. Furthermore, the proteins are expected to participate in stress-related physiological response of plants, and incorporation of nucleic acid molecules encoding the proteins into a plant is expected to enhance the plant's ability to withstand stresses. Accordingly, the present invention provides purified plant proteins, including recombinant proteins, nucleotide sequences encoding the proteins and methods of using the nucleotide sequences and proteins.
  • a method includes introducing into a plant cell a nucleic acid molecule encoding a plant protein described herein.
  • methods of treating a plant include providing a plant having an introduced nucleotide sequence encoding a plant protein described herein and treating the plant with an effective amount of GABA.
  • the plant is treated with a composition including GABA and a GABA agonist or is treated only with a GABA antagonist or GABA agonist.
  • a plant is treated with agonists or antagonists of animal benzodiazepine receptors and including agonists or antagonists of peripheral benzodiazepine receptors in animals.
  • methods of regulating plant metabolism include utilizing antisense DNA or RNA to reduce formation of a plant protein or RNA transcript, such as an mRNA transcript.
  • the method includes introducing into a plant cell an antisense nucleic acid molecule having a nucleotide sequence that is complementary to a coding nucleotide sequence described herein, or a portion thereof.
  • the antisense nucleic acid molecule includes a nucleotide sequence complementary to an RNA sequence, preferably a mRNA sequence, transcribed from a sequence described herein.
  • the antisense nucleotide sequence hybridizes to nucleic acid, including either the template strand or the RNA transcript, of the plant to reduce formation of a plant protein described herein.
  • methods of identifying potential plant receptors include hybridizing to plant nucleic acid a probe having a nucleotide sequence encoding the proteins described herein or a portion thereof.
  • methods of expressing plant proteins described herein are provided.
  • a method includes introducing into a host cell a nucleotide sequence encoding a plant receptor protein as described herein and culturing under conditions to achieve expression of the receptor protein.
  • isolated nucleic acid molecules, including recombinant nucleic acid molecules are provided that include nucleotide sequences encoding plant proteins as described herein.
  • Plant host cells and transgenic plants are also provided that include nucleotide sequences encoding a plant protein described herein.
  • the molecules, plant cells and transgenic plants further may include a foreign promoter sequence operably linked to a terminal 5' end of the plant nucleotide sequences described herein.
  • FIG. 1 depicts a schematic showing the proposed roles of GABA in plant stress responses (hypothetical pathways by which GABA may function as a cellular barometer and transducer of environmental stress signals).
  • FIG. 2 depicts a graph showing the effect of cyclosporin A on GABA- mediated growth promotion in duckweed as more fully described in Example 1.
  • FIG. 3 depicts a graph showing the effect of spermine on GABA-mediated growth promotion in duckweed as more fully described in Example 1.
  • FIG. 4 depicts a graph showing the effect of quinine on GABA-mediated growth promotion in duckweed as more fully described in Example 1.
  • FIG. 5 depicts a graph showing the effect of diazepam and PK11195 (isoquinoline carboxamide) on GABA-mediated growth promotion in duckweed as more fully described in Example 1.
  • the present invention relates to the discovery that plants respond to compounds that are known to act on animal mitochondrial GABA receptor proteins, and the related discovery that plants express receptor proteins that respond to these compounds.
  • the invention further relates to the discovery of a nucleotide sequence in Arabidopsis thaliana that is expected to encode a plant benzodiazepine, and/or benzodiazepine-like, receptor protein (hereinafter referred to collectively as "receptor protein").
  • the invention also relates to nucleotide sequences that encode analogous receptor proteins in other species and that exhibit similar functionality and have sequence identity to the exemplary Arabidopsis thaliana sequences set forth herein. Accordingly, the present invention provides purified receptor proteins and isolated nucleic acid molecules comprising nucleotide sequences encoding plant receptor proteins.
  • Recombinant nucleic acid molecules, plant host cells and transgenic plants are also provided that include the nucleotide sequences encoding the plant receptor proteins.
  • methods of expressing a receptor protein, and methods of using the nucleotide and amino acid sequences described herein are also provided.
  • purified plant benzodiazepine or benzodiazepine-like receptor proteins are provided. While it is not intended that the invention be limited by any theory whereby it achieves its advantageous result, it is believed that plant receptor proteins described herein function as ion channel proteins, such as ligand-gated ion-channel proteins in plants, and therefore have the ability to regulate cellular ion influx and/or transport ions within a cell.
  • Candidate ions whose entry may be regulated include anions, such as chloride and cations, such as calcium, sodium, and potassium.
  • the receptors may, for example, release calcium ions from intracellular stores into the cytosol.
  • receptor proteins are provided that are substantially pure. As used herein, “substantially pure” is intended to mean that the receptor proteins are at least about 95% free from other proteins with which they naturally occur.
  • an Arabidopsis thaliana receptor protein in accordance with the invention has the amino acid sequence as set forth in SEQ ID NO:2.
  • SEQ ID NO:2 the amino acid sequence set forth in SEQ ID NO:2.
  • Skilled artisans will recognize that, through the process of mutation and or evolution, polypeptides of different lengths and having differing constituents, e.g., with amino acid insertions, substitutions, deletions, and the like, may arise that are related to, or sufficiently similar to, a sequence set forth herein by virtue of amino acid sequence homology and advantageous functionality as described herein.
  • benzodiazepine receptor protein and "benzodiazepine-like receptor protein” are used herein to refer generally to a protein having the features described herein, one example of which is a polypeptide having the amino acid sequence set forth in SEQ ID NO: 2. Further included within this definition, and in the scope of the invention, are variants of the polypeptide which have the structural features and exhibit the functionality described herein.
  • a variant of the protein described herein is expected to be functionally similar to that set forth in SEQ ID NO: 2, for example, if it includes amino acids which are conserved among a variety of plant species or if it includes non- conserved amino acids which exist at a given location in another plant species that expresses a protein as described herein.
  • Another manner in which similarity may exist between two amino acid sequences is where a given amino acid of one group (such as a non-polar amino acid, an uncharged polar amino acid, a charged polar acidic amino acid or a charged polar basic amino acid) is substituted with another amino acid from the same amino acid group.
  • a given amino acid of one group such as a non-polar amino acid, an uncharged polar amino acid, a charged polar acidic amino acid or a charged polar basic amino acid
  • the uncharged polar amino acid serine may commonly be substituted with the uncharged polar amino acid threonine in a polypeptide without substantially altering the functionality of the polypeptide.
  • Whether a given substitution will affect the functionality of the enzyme may be determined without undue experimentation using synthetic techniques and screening assays known in the art, including screens employing methods set forth in the Examples below.
  • the invention provides amino acid sequences that have at least about 60% identity to the amino acid sequence set forth in SEQ ID NO: 2 and that exhibit similar functionality as the amino acid sequence set forth in SEQ ID NO: 2.
  • the invention provides a receptor protein having an amino acid sequence that has at least about 70% identity to the amino acid sequence set forth in SEQ ID NO: 2 and that exhibits similar functionality as the amino acid sequence set forth in SEQ ID NO: 2.
  • the invention provides a receptor protein having an amino acid sequence that has at least about 80% identity to the amino acid sequence set forth in SEQ ID NO: 2 and that exhibits similar functionality as the amino acid sequence set forth in SEQ ID NO: 2.
  • the invention provides a receptor protein having an amino acid sequence that has at least about 90% identity to the amino acid sequence set forth in SEQ ID NO: 2 and that exhibits similar functionality as the amino acid sequence set forth in SEQ ID NO: 2.
  • Percent identity may be determined, for example, by comparing sequence information using the MacNector computer program, version 6.0.1, available from Oxford Molecular Group, Inc. (Beaverton, OR). Briefly, the Mac Vector program defines identity as the number of identical aligned symbols (i.e., nucleotides or amino acids), divided by the total number of symbols in the shorter of the two sequences. The program may be used to determine percent identity over the entire length of the proteins being compared.
  • isolated nucleic acid molecules are provided that encode a protein as described herein.
  • the invention provides a nucleotide sequence, originally isolated from Arabidopsis thaliana, as set forth in SEQ ID NO: 1.
  • an isolated nucleic acid molecule that has a nucleotide sequence encoding a protein having an amino acid sequence having at least about 60% identity to the amino acid sequence set forth in SEQ ID NO: 2 and that exhibits similar functionality as the amino acid sequence set forth in SEQ ID NO: 2.
  • the invention provides an isolated nucleic acid molecule that has a nucleotide sequence encoding a protein having an amino acid sequence having at least about 70% identity to the amino acid sequence set forth in SEQ ID NO: 2 and that exhibits similar functionality as the amino acid sequence set forth in SEQ ID NO: 2.
  • the invention provides an isolated nucleic acid molecule that has a nucleotide sequence encoding a protein having an amino acid sequence having at least about 80% identity to the amino acid sequence set forth in SEQ ID NO: 2 and that exhibits similar functionality as the amino acid sequence set forth in SEQ ID NO: 2.
  • the invention provides an isolated nucleic acid molecule that has a nucleotide sequence encoding a protein having an amino acid sequence having at least about 90% identity to the amino acid sequence set forth in SEQ ID NO: 2 and that exhibits similar functionality as the amino acid sequence set forth in SEQ ID NO: 2.
  • isolated nucleic acid is intended to refer to nucleic acid that is not in its native environment.
  • this term refers to nucleic acid that is separated from other contaminants that naturally accompany it, such as proteins, lipids and other nucleic acid sequences.
  • the term includes nucleic acid that has been removed or purified from its naturally occurring environment or clone library, and further includes recombinant or cloned nucleic acid isolates and chemically synthesized nucleic acid.
  • nucleotide sequence is intended to refer to a natural or synthetic linear and sequential anay of nucleotides and/or nucleosides, including deoxyribonucleic acid, ribonucleic acid, and derivatives thereof.
  • encoding and coding refer to the process by which a nucleotide sequence, through the mechanisms of transcription and translation, provides the information to a cell from which a series of amino acids can be assembled into a specific amino acid sequence to produce a functional polypeptide, such as, for example, an active enzyme or other protein that has a specific function.
  • the process of encoding a specific amino acid sequence may involve DNA sequences having one or more base changes (i.e., insertions, deletions, substitutions) that do not cause a change in the encoded amino acid, or which involve base changes which may alter one or more amino acids, but do not eliminate the functional properties of the polypeptide encoded by the DNA sequence. It is therefore understood that the invention encompasses more than the specific exemplary nucleotide sequence set forth in SEQ ID NO: 1. For example, nucleic acid sequences encoding variant amino acid sequences, as discussed above, are within the scope of the invention.
  • Modifications to a sequence such as deletions, insertions, or substitutions in the sequence, which produce "silent" changes that do not substantially affect the functional properties of the resulting polypeptide molecule are expressly contemplated by the present invention.
  • alterations in a nucleotide sequence which reflect the degeneracy of the genetic code, or which result in the production of a chemically equivalent amino acid at a given site, are contemplated.
  • a codon for the amino acid alanine, a hydrophobic amino acid may be substituted by a codon encoding another less hydrophobic residue, such as glycine, or a more hydrophobic residue, such as valine, leucine, or isoleucine.
  • Nucleotide changes which result in alteration of the N-terminal and C- terminal portions of the encoded polypeptide molecule would also not generally be expected to alter the activity of the polypeptide. In some cases, it may in fact be desirable to make mutations in the sequence in order to study the effect of alteration on the biological activity of the polypeptide. Each of the proposed modifications is well within the routine skill in the art.
  • the present invention provides a nucleotide sequence that has substantial similarity to the entire sequence set forth in SEQ ID NO: 1, and variants described herein.
  • the term "substantial similarity" is used herein with respect to a nucleotide sequence to designate that the nucleotide sequence has a sequence sufficiently similar to a reference nucleotide sequence that it will hybridize therewith under moderately stringent conditions. This method of determining similarity is well known in the art to which the invention pertains. Briefly, moderately stringent conditions are defined in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed. Vol. 1, pp.
  • 5X SSC sodium chloride/sodium citrate solution
  • SDS sodium dodecyl sulfate
  • EDTA ethylene diammetetraacetic acid
  • nucleotide sequences having selected percent identities to specified regions of the nucleotide sequence set forth in SEQ ID NO: 1 are provided.
  • nucleotide sequences are provided that have at least about 50% identity to a nucleotide sequence of substantial length within the nucleotide set forth in SEQ ID NO: 1.
  • the invention provides a nucleotide sequence that has at least about 60% identity to a nucleotide sequence of substantial length within the nucleotide set forth in SEQ JJD NO: 1.
  • the invention provides a nucleotide sequence that has at least about 70% identity to a nucleotide sequence of substantial length within the nucleotide set forth in SEQ ID NO: 1.
  • the invention provides a nucleotide sequence that has at least about 80% identity to a nucleotide sequence of substantial length within the nucleotide set forth in SEQ JD NO: 1. In another embodiment, the invention provides a nucleotide sequence that has at least about 90% identity to a nucleotide sequence of substantial length within the nucleotide set forth in SEQ JD NO: 1.
  • substantially length refers to a length of at least about 50 nucleotides. In another embodiment, the substantial length is a length of at least about 100 nucleotides. In another embodiment, the substantial length is a length of at least about 200 nucleotides. In another embodiment, the substantial length is a length of at least about 300 nucleotides. In another embodiment, the substantial length is a length of at least about 400 nucleotides. In another embodiment, the substantial length is a length of at least about 500 nucleotides. In another embodiment, the substantial length is the entire sequence set forth in SEQ ID NO: 1. The percent identity may be determined, for example, by comparing sequence information using the MacNector program, as described above with reference to amino acid identity.
  • a further requirement of a nucleotide sequence in accordance with the invention is that it encodes a protein that functions as described herein.
  • a suitable D ⁇ A sequence in accordance with the invention may be obtained by cloning techniques using cD ⁇ A or genomic libraries of Arabidopsis thaliana or other species, which are available commercially or which may be constructed using standard methods known in the art.
  • Suitable nucleotide sequences may be isolated from D ⁇ A libraries obtained from a wide variety of species by means of nucleic acid hybridization or polymerase chain reaction (PCR) procedures, using as probes or primers nucleotide sequences selected in accordance with the invention, such as that set forth in SEQ JD NO: 1 , nucleotide sequences having substantial similarity thereto, or portions thereof.
  • the nucleotide sequences provided herein are cDNA sequences.
  • nucleic acid sequences encoding a plant protein described herein may be constructed by recombinant DNA technology, for example, by cutting or splicing nucleic acids using restriction enzymes and DNA ligase.
  • nucleic acid sequences may be constructed using chemical synthesis, such as solid-phase phosphoramidate technology, or PCR. PCR may also be used to increase the quantity of nucleic acid produced.
  • the particular nucleic acid sequence is of a length which makes chemical synthesis of the entire length impractical, the sequence may be broken up into smaller segments which may be synthesized and ligated together to form the entire desired sequence by methods known in the art.
  • nucleic acid molecules that includes a nucleotide sequence as described herein.
  • the protein encoded by the nucleotide sequence has the amino acid sequence set forth in SEQ JD NO:2, or variants thereof as described above.
  • the vector may be a T-DNA vector.
  • Representative T-DNA vector systems are discussed in the following publications: An et al., (1986) EMBO J. 4:277; Henera- Estrella et al., (1983) EMBO J. 2:987; Henera-Estrella et al., (1985) in Plant Genetic Engineering, New York: Cambridge University Press, p. 63.
  • the desired recombinant vector may be constructed by ligating DNA linker sequences to the 5' and 3' ends of the desired nucleotide insert, cleaving the insert with a restriction enzyme that specifically recognizes sequences present in the linker sequences and the desired vector, cleaving the vector with the same restriction enzyme, mixing the cleaved vector with the cleaved insert and using DNA ligase to incorporate the insert into the vector as known in the art.
  • the vectors may include other nucleotide sequences, such as those encoding selectable markers, including those for antibiotic resistance or color selection.
  • the vectors also preferably include a promoter nucleotide sequence.
  • the desired nucleic acid insert is preferably operably linked to the promoter.
  • a nucleic acid is "operably linked" to another nucleic acid sequence, such as a promoter sequence, when it is placed in a specific functional relationship with the other nucleic acid sequence.
  • the functional relationship between a promoter and a desired nucleic acid insert typically involves the nucleic acid and the promoter sequences being contiguous such that transcription of the nucleic acid sequence will be facilitated.
  • Two nucleic acid sequences are further said to be operably linked if the nature of the linkage between the two sequences does not (1) result in the introduction of a frame-shift-mutation; (2) interfere with the ability of the promoter region sequence to direct the transcription of the desired nucleotide sequence, or (3) interfere with the ability of the desired nucleotide sequence to be transcribed by the promoter sequence region.
  • the promoter element is generally upstream (i.e., at the 5' end) of the nucleic acid insert coding sequence.
  • a wide variety of promoters are known in the art, including cell-specific promoters, inducible promoters, and constitutive promoters. Such promoters that direct transcription in plants cells may be used.
  • the promoters may be of viral, bacterial or eukaryotic origin, including those from plants and plant viruses.
  • the promoter may be of viral origin, including a cauliflower mosaic virus promoter (CaMN), such as CaMV 35S or 19S, a figwort mosaic virus promoter (FMN 35S), or the coat protein promoter of tobacco mosaic virus (TMN).
  • CaMN cauliflower mosaic virus promoter
  • FMN 35S figwort mosaic virus promoter
  • TNN tobacco mosaic virus
  • the promoter may further be, for example, a promoter for the small subunit of ribulose-l,3-diphosphate carboxylase.
  • Promoters of bacterial origin include the octopine synthase promoter, the nopaline synthase promoter and other promoters derived from native Ti plasmids as discussed in Henera-Estrella et al., Nature, 303:209-213 (1983).
  • the promoter may further be one that responds to various forms of environmental stresses, or other stimuli.
  • the promoter may be one induced by abiotic stresses such as wounding, cold, dessication, ultraviolet-B [van Der Krol et al. (1999) Plant Physiol. 121:1153-1162], heat shock [Shinmyo et al., (1998) Biotechnol. Bioeng. 58:329-332] or other heat stress, drought stress or water stress.
  • the promoter may further be one induced by biotic stresses including pathogen stress, such as stress induced by a virus [Sohal et al. (1999) Plant Mol. Biol. 41:75-87] or fungi [Eulgem (1999) EMBO. J.
  • the promoters may further be selected such that they require activation by other elements known in the art, so that production of the protein encoded by the nucleic acid sequence insert may be regulated as desired.
  • the promoter is a foreign promoter.
  • a "foreign promoter” is defined herein to mean a promoter other than the native, or natural, promoter that promotes transcription of a length of DNA.
  • the vectors may further include other regulatory elements, such as enhancer sequences, which cooperate with the promoter to achieve transcription of the nucleic acid insert coding sequence.
  • enhancer sequences which cooperate with the promoter to achieve transcription of the nucleic acid insert coding sequence.
  • enhancer is meant nucleotide sequence elements that can stimulate promoter activity in a cell, such as a plant host cell.
  • the vectors may further include 3' regulatory sequence elements known in the art, such as those, for example, that increase the stability of the RNA transcribed.
  • the vectors may include another nucleotide sequence insert that encodes a peptide or polypeptide used as a tag to aid in purification of the desired protein encoded by the desired nucleotide sequence or that encodes another functional protein.
  • the additional nucleotide sequence can be positioned in the vector such that a fusion, or chimeric, protein is obtained.
  • a protein described herein may be produced having at its C-terminal end linker amino acids, as known in the art, joined to the other protein that acts as a tag. After purification procedures known to the skilled artisan, the additional amino acid sequence is cleaved with an appropriate enzyme. The protein may then be isolated from the other proteins, or fragments thereof, by methods known in the art.
  • a vector in another embodiment, includes a second nucleotide sequence that encodes another functional protein, such as, for example, a plant GAD enzyme, as described in the inventors' copending U.S. patent application, Serial No.
  • plants can be transformed in accordance with the invention with two different vectors, one including a DNA construct for expression of a GAD enzyme, by way of example, and the other for expression of a plant receptor protein as described herein. It is expected that overexpression of a GAD enzyme and a receptor protein in a plant will result in a plant with excellent features, such as, for example, enhanced stress resistance.
  • inventive recombinant vectors may be used to transform a host cell. Accordingly, methods of transforming a cell or a plant are provided that include introducing into a plant cell a nucleic acid molecule having an inventive nucleotide sequence.
  • methods of transforming a cell or a plant are well known in the art, and may be found in references including, for example, Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Springs Laboratory, Cold Springs Harbor, New York (1982) and Current Protocols in Molecular Biology, John Wiley and Sons, edited by Ausubel et al. (1988). Plant gene transfer techniques may also be found in references including Fromm et al., (1985) Proc. Natl. Acad. Sci.
  • a host cell that includes the inventive recombinant vectors described above.
  • host cells may be used in the invention, including prokaryotic and eukaryotic host cells.
  • Prefened host cells are eukaryotic and are further preferably plant cells, such as, for example, those derived from monocotyledons, such as duckweed, corn, turf (including rye grass, Bermuda grass, Blue grass, Fescue), dicotyledons, including lettuce, cereals such as wheat, crucifers (such as rapeseed, radishes and cabbage), solanaceae (including green peppers, potatoes and tomatoes), and legumes such as soybeans and bush beans.
  • the host cells may be cultured as known in the art to produce a transgenic plant.
  • a transformed plant can be made, for example, by transforming a cell, tissue or organ from a host plant with an inventive nucleic acid molecule; selecting a transformed cell, cell callus, somatic embryo, or seed which contains the nucleic acid molecule; regenerating a whole plant from the selected transformed cell, cell callus, somatic embryo, or seed; and selecting a regenerated whole plant that expresses the nucleotide sequence.
  • nucleotide sequences described above, or portions thereof are used as probes to locate other, similar nucleotide sequences that may encode other benzodiazepine or benzodiazepine-like receptors.
  • General methods for screening for selected nucleotide sequences in a DNA or RNA sample are known to the art. For example, DNA may be isolated from selected plants, treated with various restrictions enzymes and analyzed by Southern blotting techniques utilizing a radioactively or fluorescently-labeled probe of interest. RNA fragments may be similarly analyzed by Northern blotting techniques. Alternatively, commercially available cDNA or genomic libraries may be screened.
  • a nucleic acid molecule used as a probe has a nucleotide sequence having at least about 60% identity to a nucleotide sequence having a length of about 25 to about 100 nucleotides within the nucleotide sequence set forth in SEQ JD NO:l.
  • a nucleic acid molecule used as a probe has a nucleotide sequence having at least about 60% identity to a nucleotide sequence having a length of about 25 to about 400 nucleotides within the nucleotide sequence set forth in SEQ JD NO:l.
  • a nucleic acid molecule used as a probe has a nucleotide sequence having at least about 60% identity to a nucleotide sequence having a length of about 25 to about 500 nucleotides within the nucleotide sequence set forth in SEQ JD NO: 1.
  • the probe has a nucleotide sequence having at least about 60% identity to the entire length of nucleotides set forth in SEQ JD NO: 1.
  • the probe has a nucleotide sequence having at least about 70% identity to the length of nucleotides indicated directly above.
  • the probe has a nucleotide sequence having at least about 80% identity to the length of nucleotides indicated directly above.
  • the probe has a nucleotide sequence having at least about 90% identity to the length of nucleotides indicated directly above.
  • the probe may be radioactively labeled at its 5 'end, for example, with polynucleotide kinase and P and hybridized to the isolated nucleic acid fragments.
  • a method includes providing a plant having an introduced nucleic acid molecule described herein, wherein the plant expresses the encoded receptor protein, and treating the plant with an effective amount of GABA.
  • Such treating of the plant is expected to advantageously stimulate growth of the plant, as well as provide other beneficial results, including reducing the effects of plant stress.
  • transgenic plants are prepared as described above and treated with an effective amount of GABA.
  • effective amount refers to an amount of GABA that will provide one or more advantages to the plant, such as, for example, stimulation of plant growth and/or reduction of plant stress. The amount may vary depending upon a wide variety of factors, including, for example, the particular advantage provided to the plant, the number of introduced nucleotide sequences expressed, the type of plant, the number of plants treated and the environmental conditions.
  • plants are treated with about 1 ppm to about 24,000 ppm GABA [about 0.013 oz/acre (oz/A) to about 20 lbs/ A] [about 0.93 g/hectare (g/ha) to about 22 kg/ha].
  • plants are treated with about 1 ppm to about 12,000 ppm GABA [about 0.013 oz/A to about 10 lbs/A] [about 0.93 g/ha to about 11 kg/ha]. In another embodiment, plants are treated with about 1 ppm to about 7,500 ppm
  • GABA [about 0.013 oz/A to about 6.3 lbs/ A] [about 0.93 g/ha to about 7.1 kg/ha].
  • plants are treated with about 1 ppm to about 5,000 ppm GABA [about 0.013 oz/A to about 4.2 lbs/A] [about 0.93 g/ha to about 4.8 kg/ha].
  • concentrations of about 1 ppm to about 5,000 ppm as described in U.S. Patent No. 5,439,873 to Kinnersley, may be advantageously employed.
  • concentrations of GABA of from about 1 ppm to about 2,500 ppm [about 0.013 oz/A to about 2.1 lbs/A] [about 0.93 g/ha to about 2.4 kg/ha] may be advantageously employed.
  • the application rates in brackets above are derived for a treatment utilizing a standard volume of 100 gallons of the specified solutions dispersed over 1 acre.
  • the plant in addition to being treated with GABA, may also be treated with a composition that includes GABA and a GABA agonist.
  • a composition that includes GABA and a GABA agonist.
  • plants may be treated with baclofen as well as other GABA agonists known to the art, including, for example, cis-4-aminopent-2-enoic acid (CACA), imidazole-4-acetic acid (IAA) and 4,5,6,7 -tetrahydroisoxazolo[5,4- c]pyridin-3-ol (THJP).
  • Plants may also be treated with only a GABA antagonist, such as picrotoxin or bicuculline, or only a GABA agonist to regulate plant metabolism as desired.
  • the plants may also be treated only with an agonist or antagonist of a benzodiazepine receptor, such as an animal peripheral benzodiazepine receptor.
  • a benzodiazepine receptor such as an animal peripheral benzodiazepine receptor.
  • Such compounds include quinine and spermine, and other benzodiazepine receptor antagonists and agonists described herein.
  • GABA, the GABA agonists or antagonists and other agonists and antagonists described herein are typically applied to the foliage of the plant but may also be administered as a soil drench.
  • the compounds and compositions may be applied to the aqueous solution in which the plants are grown.
  • the compositions are further preferably applied by spraying.
  • the compounds and compositions may also be applied as a seed treatment.
  • GABA GABA
  • GABA agonists or GABA antagonists and other agonists and antagonists described herein are preferably combined with a canier medium as known in the art.
  • the compounds and compositions may, for example, be combined with water, such as tap water or with distilled water to which has been added selected minerals.
  • the compositions of the present invention may be applied as a solid. In such a form, the solid is preferably applied to the soil.
  • the compositions may further include agricultural additives or formulation aids known to those skilled in the art. Such additives or aids may be used to ensure that the compositions disperse well in a spray tank, stick to or penetrate plant surfaces (particularly leaf or other foliage surfaces) as well as provide other benefits to the plant.
  • surfactants, dispersants, humectants, and binders may be used to disperse the compounds or compositions described herein in a spray tank as well as to allow the compound or compositions to adhere to and/or penetrate the plant surfaces.
  • Methods of regulating plant metabolism are also provided by the present invention. Regulation of plant metabolism may include positively or negatively affecting nutrient utilization, such as nitrogen-assimilation, plant growth, plant productivity and the plant's resistance to the effects of plant stress.
  • an inventive method that may negatively affect plant productivity includes introducing into a plant cell an antisense nucleotide sequence having a sequence complementary to a coding nucleotide sequence provided herein.
  • this invention also provides strategies for manipulating a gene involved in plant receptor protein production and thus is an invaluable tool for further research of cellular stress and/or developmental processes.
  • manipulation of a plant receptor protein gene can provide quantitative information on the role of GAB A-related processes on metabolic fluxes, nutrient utilization and storage, cellular differentiation, growth, senescence, and signaling.
  • Such manipulation also provides a method for increasing crop productivity through enhancing crop resistance to biotic and abiotic stresses. Crop quality and yield is improved by increasing tolerance to a variety of environmental stresses, including disease, which cause a decrease in photosynthetic and nitrogen efficiency of crop plants resulting in decreased yields.
  • the invention provides an antisense nucleotide sequence that is complementary to a nucleotide sequence having at least about 50% identity to a length of nucleotides within the nucleotide sequence set forth in SEQ JD NO: 1. In another embodiment, the invention provides an antisense nucleotide sequence that is complementary to a nucleotide sequence having at least about 60% identity to a length of nucleotides within the nucleotide sequence set forth in SEQ JD NO: 1. In another embodiment, the invention provides an antisense nucleotide sequence that is complementary to a nucleotide sequence having at least about 70% identity to a length of nucleotides within the nucleotide sequence set forth in SEQ JD NO: 1.
  • the invention provides an antisense nucleotide sequence that is complementary to a nucleotide sequence having at least about 80% identity to a length of nucleotides within the nucleotide sequence set forth in SEQ JD NO: 1. In another embodiment, the invention provides an antisense nucleotide sequence that is complementary to a nucleotide sequence having at least about 90% identity to a length of nucleotides within the nucleotide sequence set forth in SEQ ID NO: 1.
  • the antisense nucleotide has a length of about 30 to about 100 nucleotides. In another embodiment, the antisense nucleotide has a length of about 30 to about 200 nucleotides. In another embodiment, the antisense nucleotide has a length of about 30 to about 300 nucleotides. In another embodiment, the antisense nucleotide has a length of and about 30 to about 400 nucleotides. In another embodiment, the antisense nucleotide sequence is as long as the entire length of the nucleotide sequence set forth in SEQ ID NO: 1.
  • the antisense nucleotide sequence may hybridize to the template strand, which serves as the strand from which RNA is produced, so that transcription will be reduced.
  • the antisense nucleotide sequence may be complementary to, and therefore hybridize to, the RNA sequence, such as the mRNA sequence, transcribed from the nucleotide sequences described herein, so that translation of the mRNA sequence to express the encoded protein will be reduced.
  • the antisense nucleotide sequence may be either DNA or RNA.
  • Prefened antisense oligonucleotides are complementary to the coding region of a particular polynucleotide, although the sequences may in addition bind to selected sequences in a non-coding region. In further prefened forms of the invention, the antisense oligonucleotides will bind to nucleotides adjacent to the ATG initiation codon.
  • a plant may be mutated by methods known to the skilled artisan, including chemical methods and DNA-insertion activation-tagged mutagenesis.
  • a method includes introducing into a plant cell a nucleic acid molecule having a nucleotide sequence encoding a plant protein as described herein.
  • the method includes providing a nucleotide sequence described above, or variants thereof, that encodes a protein described herein, and introducing the nucleotide sequence into a host cell, as described above.
  • the desired nucleotide sequence may be advantageously incorporated into a vector to form a recombinant vector.
  • the recombinant vector may then be introduced into a host cell according to known procedures in the art. Such host cells are then cultured under conditions, well known to the skilled artisan, effective to achieve expression of the plant protein.
  • the protein may then be purified using conventional techniques.
  • the target plant is selected from the group consisting of duckweed, rice, wheat, barley, rye, corn, Bermuda grass, Blue grass, fescue, rapeseed, potato, canot, sweet potato, bean, pea, chicory, lettuce, cabbage, cauliflower, broccoli, turnip, radish, spinach, asparagus, onion, garlic, eggplant, pepper, celery, squash, pumpkin, zucchini, cucumber, apple, pear, quince, melon, plum, cheny, peach, nectarine, apricot, strawbeny, grape, raspberry, blackbeny, pineapple, avocado, papaya, mango, banana, soybean, bush beans, tobacco, tomato, green pepper, sorghum and sugarcane.
  • Benzodiazepine receptors are sensitive to the agonist diazepam and the antagonists PK11195 (isoquinoline carboxamide), spermine, quinine and cyclosporin A.
  • Duckweed (Lemna Minor L) was grown following the general procedure described by Kinnersley (U.S. Patent No. 4,813,997) except that the culture media was Solu-Spray 20-20-20 fertilizer dissolved in tap water at 1 g/1 and the pH was adjusted to 5.5 as discussed in U.S. Patent No. 5,439,873 to Kinnersley.
  • Duckweed was treated with, independently, the indicated concentrations of GABA and either cyclosporin A, spermine, quinine, diazepam or PK11195.
  • Cyclosporin A is an immunosuppressant and has been shown to be the most potent pharmacological inhibitor of the PTP in animal mitochondria.
  • the inhibitory activity of cyclosporin has been attributed to binding to mitochondrial cyclophilin in the mitochondrial inner membrane.
  • 3 ⁇ M cyclosporin A significantly inhibited plant growth in cultures containing 10 mM GABA. Relative to the respective controls, inhibition of GABA-mediated growth by cyclosporin A (FIG. 2), spermine (FIG.
  • PK11195 is a diagnostic ligand of the peripheral benzodiazepine receptor, which is associated with the PTP in animal mitochondria. PK11195 blocked GABA- mediated growth response at 50 ⁇ M (FIG. 5). Table 1. Effect of PKl 1195 on dry weight of duckweed
  • Table 2 Effect of pharmacological agents on activity of the mitochondrial permeability transition pore (PTP) and peripheral benzodiazepine receptor (PBR) in animals and on GABA-mediated growth activity in Lemna.
  • PTP mitochondrial permeability transition pore
  • PBR peripheral benzodiazepine receptor
  • Arabidopsis thaliana (L.) Heynh. Ecotype Columbia (Col-0) seeds can be obtained from the Arabidopsis Biological Resource Center (Ohio State University, Columbus, OH).
  • Arabidopsis seedlings are grown under aseptic conditions in flasks containing MS media [Murashige and Skook, Physiol. Plant 15:485 (1962)] on a rotary shaker (150 rpm). Two-day-old seedlings are collected for total RNA isolation. Total RNA are isolated as described in Turano, F.J. et al.(1992) Plant Physiol. 100:374.
  • Primer 3'EcoPBR is used to synthesize a first strand cDNA from 1 ⁇ g of poly (A + )RNA isolated from two-day-old plants following the manufacturers instructions.
  • One-fifth of the first strand cDNA synthesis is used as a template in a gene amplification reaction with both primers, 5'EcoPBR and 3'EcoPBR.
  • the components Prior to the amplification, the components are incubated at 95°C for 4 minutes.
  • the gene amplification reaction is conducted at 94°C for 1 minute, 68°C for 1 minute and 72°C for 2 minutes, for 30 cycles followed by a 5 minute, 72°C extension.
  • Genomic DNA is isolated from leaves of 24 day old Arabidopsis as described in Turano, F.J.
  • a transgenic plant that overexpresses a plant receptor protein, or one that overexpresses an antisense receptor protein is made as follows.
  • the entire (591 base pairs) open reading frame for the sense (over-expression) or antisense (under- expression) of the receptor protein, or the portions thereof as small as about 25 base pairs (for antisense or RNAi only) is cloned into a plant transformation vector, such as pBI121(Clonetech, Palo Alto, CA) using PCR, RT-PCR or conventional cloning methods to make antisense constructs.
  • RT-PCR For the RT-PCR, a 5' RACE system (Life Technologies, Rockville, MD, USA) or a simpler reverse transcriptase (RT) based system, is used to identify a full-length cDNA clone.
  • Primer 3'EcoPBR is used to synthesize first strand cDNA from 1 ⁇ g from poly (A + ) RNA isolated from 2 day old plants following the manufacturer's instructions.
  • One fifth of the first strand cDNA synthesis is used as a template in a gene amplification reaction with both primers, 5'EcoPBR and 3'EcoPBR. Prior to the amplification, the components are incubated at 95°C for 2 min.
  • the gene amplification reaction is conducted at 94°C for 1 min, 58°C for 1 min and 72°C for 2 min, for 30 cycles followed by a 5 min 72°C extension.
  • the genomic DNA or cDNA fragments are cloned into plant transformation vectors in a sense (forward) or anti sense (backwards) direction, depending on the desired result.
  • the vectors may contain constitutive promoters such as CaMV 35S promoter and the nopaline synthase terminator, or other promoters described herein and known to the art.
  • the vectors may be modified to include promoters that can be induced by biotic [Sohal et al.,(1999) Plant Mol. Biol. 41:75-87] or abiotic stresses [Ngai et al., (1997) Plant J. 12:1021-1034; van Der Krol et al., (1999) Plant Physiol.
  • the binary vector construct Upon completion of cloning, the binary vector construct is transfened into a disarmed strain of Agrobacterium tumefaciens, such as EHA105, and subsequently into Arabidopsis (Ws ecotype) using the vacuum infiltration method [Bechtold, N. and Bouchez, D. (1995) In planta Agrobacterium-mediatc ⁇ transformation of adult Arabidopsis thaliana plants by vacuum infiltration. In Gene Transfer to Plants. I. Potrykus and G. Spangenberg Eds. Springer-Nerlag, Heidelberg, pp. 19-23] with one modification (i.e., the addition of 0.02% (v/v) Silwet to the infiltration media). Seeds collected from the transformed plants are germinated and selected for kanamycin resistance.
  • Agrobacterium tumefaciens such as EHA105

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