WO2001002541A2 - Compositions et procedes de regulation de la fermeture induite par l'acide abscisique de stomates de vegetaux - Google Patents

Compositions et procedes de regulation de la fermeture induite par l'acide abscisique de stomates de vegetaux Download PDF

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Publication number
WO2001002541A2
WO2001002541A2 PCT/US2000/018014 US0018014W WO0102541A2 WO 2001002541 A2 WO2001002541 A2 WO 2001002541A2 US 0018014 W US0018014 W US 0018014W WO 0102541 A2 WO0102541 A2 WO 0102541A2
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aapk
plant
nucleic acid
sequence
acid molecule
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PCT/US2000/018014
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WO2001002541A3 (fr
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Sarah M. Assmann
Jiaxu Li
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The Penn State Research Foundation
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    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1205—Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
    • 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
    • C12N15/8291—Hormone-influenced development
    • C12N15/8293—Abscisic acid [ABA]

Definitions

  • the present invention relates to the field of molecular biology of plants.
  • Transpirational loss of water by evaporation occurs mainly through the pores, called stomata, primarily located in the lower epidermis of the leaves.
  • stomata primarily located in the lower epidermis of the leaves.
  • Each stoma is surrounded by two guard cells, which control the opening and closure of the stomata by their relative turgor pressure.
  • the cell wall properties of guard cells allow them to deform such that when the guard cells develop turgor pressure, the stoma is opened, but when the guard cells lose turgor, the stoma closes.
  • the rate of evaporation of water from the air spaces of the leaf to the outside air depends on the water potential gradient between the leaf and the outside air.
  • Environmental factors which directly influence the aperture of the plant's stomata affect its transpiration rate. Such factors include light conditions, relative humidity of the air, temperature, water status of the plant, CO 2 concentration, relative concentration of certain ions, and concentration of abscis
  • Abscisic acid is a multifunctional phytohormone involved in a variety of important protective functions including bud dormancy, seed dormancy and/or maturation, abscission of leaves and fruits, and response to a wide variety of biological stressors (e.g. cold, heat, salinity, and drought). It is also responsible for regulating stomatal closure by a mechanism independent of CO 2 concentration.
  • ABA is synthesized rapidly in response to water stress m plants, and is stored in the guard cells. Du ⁇ ng drought, ABA alteration of guard cell ion transport promotes stomatal closure and also prevents stomatal opening, thus reducing transpirational water loss.
  • the hormone sets off a variety of biological messages that require or include a protein phopsphorylation cascade.
  • a protein phopsphorylation cascade One member of this cascade was identified in guard cells of Viciafaba as an ABA-activated, calcium-independent protein kinase. (Li & Assmann, Plant Cell 8: 2359-2368, 1996; Mo ⁇ & Muto, Plant Physiol. 113: 833-839, 1997).
  • the kinase was identified by SDS polyacrylamide gel electrophoresis as a 48 kDa protein, but was not further isolated or characterized. It exhibited ABA-activated autophosphorylation and kinase activity.
  • Stomata simultaneously regulate both the transpiration of water and the exchange of gases for photosynthesis. Open stomata allow for maximum gas exchange rate so that photosynthetic reactions may proceed more quickly, however under these conditions, water loss will be maximal. On the other hand, closed stomata minimize transpirational water loss but also substantially reduce photosynthetic reaction rates. Paradoxically, the plant undergoes a continual trade-off between maximizing CO 2 uptake for carbon fixation, and minimizing desiccating water loss. Thus, the ability to control stomatal opening and closure could be of tremendous agronomic significance. Several studies in the literature provide examples of the benefits of selecting for increased stomatal conductance under certain conditions.
  • the plant hormone abscisic acid causes stomatal closure du ⁇ ng pe ⁇ ods of reduced water availability by reducing the ion and water content of the pair of guard cells that flanks each stoma.
  • ABA abscisic acid
  • ABA response is protective; always somewhat limiting to water loss, but thus unavoidably, also limiting to CO 2 uptake.
  • guard cells respond to in the control of stomatal aperture, for example, light and decreased intracellular concentrations of CO 2 drive stomatal opening, and conversely, darkness and high CO 2 concentrations drive stomatal closure.
  • stomatal aperture for example, light and decreased intracellular concentrations of CO 2 drive stomatal opening, and conversely, darkness and high CO 2 concentrations drive stomatal closure.
  • the grower would still want the stomata to close in response to darkness, because in darkness there is no photosynthesis anyway, and open stomata during the night would simply waste irrigation water and thus money.
  • novel mutants and genes that encode altered ABA- mediation of transpirational water loss will broaden the range of options for growers. It would be particularly advantageous to isolate mutants or genes involved in altered ABA-mediation of transpiration without spontaneously occurring abnormal responses to other roles of ABA or abnormal responses to factors such as light levels and concentrations of CO 2 . Novel regulatory mutants are likely to have distinct induction of unique subsets of genes. The isolation of mutants will yield the critical gene(s) involved with altered ABA-mediation of transpiration, which can be used to transgenically transfer the novel trait to other species.
  • AAPK novel nucleic acid molecule
  • ABA abscisic acid
  • the invention further provides transgenic plants and mutants having modified ABA-mediated stomatal closure.
  • ABA-mediated stomatal closure is modified in a manner that is independent of CO 2 - and light-mediated responses of transpiration, as measured by changes in stomatal aperture.
  • AAPK ABA-activated protein kinase
  • An exemplary AAPK-encoding nucleic acid molecule of the invention is that of Vicia faba, a food crop of major importance in the Middle East. Also exemplified are homologs of the gene in Arabidopsis thaliana. The invention further provides homologs of the exemplified AAPK, having a level of nucleotide sequence or amino acid sequence identity with the exemplified AAPK nucleic acids or encoded AAPK proteins, specifically at certain regions of the coding sequence, that clearly distinguish the homologs as AAPK homologs, as opposed to other kinases.
  • these homologs comprise nucleotide or amino acid sequences at least 60%, preferably 67%, more preferably 70% and even more preferably 80% identical to the Viciafaba and Arabidopsis AAPK nucleic acid and AAPK amino acid sequences set forth herein..
  • disrupted gene product of the AAPK gene comprises lost or reduced activity of the AAPK protein. Reduction in amount or activity of AAPK in plants results in decreased sensitivity of the plants to ABA- induced stomatal closure, but does not affect the plants' s sensitivity to dark- or CO 2 - induced stomatal closure.
  • an oligonucleotide molecule of at least 15 nucleotides in length, preferably at least 20 nucleotides in length, and most preferably at least 30 nucleotides in length, that is identical in sequence to a portion of an AAPK nucleic acid is provided.
  • the invention provides a nucleic acid molecule of at least 15, preferably 20, and most preferably 30 or more nucleotides in length, that is identical to or complementary to a consecutive 15, 20 or 30 nucleotide portion, respectively, of the sequence set out in one of SEQ ID NOS: 1, 3 or 6.
  • an isolated polypeptide produced by expression of a nucleic acid molecule of the invention is provided. Also featured are antibodies immunologically specific for such a polypeptide.
  • a vector for transforming a plant cell, comprising a nucleic acid molecule of the invention is provided. Also featured are plant cells transformed with the vector, and intact fertile plants regenerated from the plant cells. It will be appreciated by persons of skill in the art, that various portions of such genetically altered plants are also encompassed by the present invention. These include, but are not limited to, roots, modified roots (e.g., tubers), stems, leaves, flowers, fruits and seeds, and components thereof, e.g., extracts or oils.
  • a genetically altered plant which possesses decreased sensitivity to ABA-induced stomatal closure as compared with an equivalent but unaltered plant.
  • These genetically altered plants contain an AAPK that is largely nonfunctional or absent.
  • the plant is produced by subjecting a population of plants to mutagenesis and selecting a mutagenized plant wherein the AAPK is largely nonfunctional or absent.
  • the plant is produced by transforming cells of the plant with a transgene that causes the plant's endogenous AAPK to become largely nonfunctional or absent, and regenerating the plant from the transformed cell.
  • expression of the transgene is inducible.
  • a genetically altered plant possessing increased sensitivity to ABA-induced stomatal closure as compared with an equivalent but unaltered plant is provided.
  • Plants of this type contain an AAPK that is increased in amount or activity as compared with the unaltered plant.
  • these plants are produced by subjecting a population of plants to mutagenesis and selecting a mutagenized plant wherein the AAPK is largely nonfunctional or absent.
  • the plants are produced by transforming cells of the plant with a transgene that causes the plant's endogenous
  • AAPK to become largely nonfunctional or absent, and regenerating the plant from the transformed cells.
  • expression of the transgene is inducible.
  • Another aspect of the invention features a method to increase transpiration in a plant.
  • the method comprises reducing or preventing function of an AAPK in guard cells of the plant, thereby reducing sensitivity of the plant to ABA- induced stomatal closure, resulting in the increased transpiration.
  • a method is provided to decrease transpiration in a plant, comprising increasing function of AAPK in guard cells of the plant, thereby increasing sensitivity of the plant to ABA- induced stomatal closure, resulting in decreased transpiration.
  • FIG. 1 Alignment of the deduced AAPK amino acid sequence with those of homologous protein kinases.
  • GenBank accession numbers for the nucleic acid molecules encoding the displayed amino acid sequences are: AAPK (AF186020), Arabidopsis Atpk (L05562, S71172), tobacco WAPK (AF032465), soybean SPK-4 (L38855), rice REK (AB002109), ice plant MK9 (Z26846), and wheat PKABAl (M94726).
  • AAPK is SEQ ID NO:2 (encoded by SEQ ID NO:l); Atpk is SEQ ID NO:4 (encoded by SEQ ID NO:3); WAPK is SEQ ID NO: 11; SPK-4 is SEQ ID NO: 12; REK is SEQ ID NO: 13; MK9 is SEQ ID NO: 14; PKABAl is SEQ ID NO: 10
  • the sequence shown between the Query and the Subject sequences shows the consensus sequence. A letter indicates identity, a '+' indicates a similarity, while a blank space indicates the two sequences are different at that residue.
  • Figure 3 Alignment of the deduced amino acid sequence of AAPK from Viciafaba (SEQ ID NO:2) with the deduced amino acid sequence from a gene encoding a homologous protein kinase from Arabidopsis thaliana (SEQ ID NO:7).
  • Figure 4 Alignment of the deduced amino acid sequence of AAPK from Viciafaba (SEQ ID NO:2) with the amino acid sequence of a homolog, Protein Kinase SPK-2, from Arabidopsis thaliana (SEQ ID NO:8).
  • AAPK AAPK
  • AAPK AAPK
  • null mutant the term “null mutant” or
  • “loss-of-function mutant” is used to designate an organism or genomic DNA sequence with a mutation that causes the product of the gene of interest to be non-functional or largely absent. Such mutations may occur in the coding and/or regulatory regions of the gene of interest, and may be changes of individual residues, or insertions or deletions of regions of nucleic acids. These mutations may also occur in the coding and/or regulatory regions of other genes which may regulate or control the gene of interest and or its encoded gene product so as to cause said gene product to be nonfunctional or largely absent.
  • pGFP green fluorescent protein
  • pAAPK-GFP refers to constructs made from the green fluorescent protein (GFP) expression vector, pGFP, which allows cells transformed with the pGFP to express the readily-detected green fluorescent protein.
  • pAAPK-GFP refers to a pGFP expression vector with the sequence encoding AAPK inserted upstream of and in-frame with the sequence encoding the GFP, such that both proteins can be expressed in cells transformed with this construct.
  • pAAPK(K43A)-GFP refers to a pGFP expression vector with the sequence encoding an AAPK, modified such that the lysine residue at position 43 in the ATP binding site is changed to an alanine, inserted upstream of and in-frame with the sequence encoding the GFP, such that both proteins can be expressed in cells transformed with this construct.
  • isolated nucleic acid refers to a DNA molecule that is separated from sequences with which it is immediately contiguous (in the 5' and 3' directions) in the naturally-occurring genome of the organism from which it was derived.
  • the "isolated nucleic acid” may comprise a DNA molecule inserted into a vector, such as a plasmid or virus vector, or integrated into the genomic DNA of a procaryote or eukaryote.
  • An “isolated nucleic acid molecule” may also comprise a cDNA molecule or a synthetic DNA molecule.
  • isolated nucleic acid primarily refers to an RNA molecule encoded by an isolated DNA molecule as defined above.
  • the term may refer to an RNA molecule that has been sufficiently separated from RNA molecules with which it would be associated in its natural state (i.e., in cells or tissues), such that it exists in a "substantially pure” form .
  • Nucleic acid sequences and amino acid sequences can be compared using computer programs that align the similar sequences of the nucleic or amino acids thus define the differences.
  • the BLAST programs NCBI
  • parameters used therein are employed to align nucleotide and amino acid sequences.
  • equivalent alignments and similarity/identity assessments can be obtained through the use of any standard alignment software.
  • the DNAstar system (Madison, WI) may be used to align sequence fragments of genomic or other DNA sequences.
  • nucleic acid or amino acid sequences having sequence variation that do not materially affect the nature of the protein (i.e. the structure, stability characteristics and/or biological activity of the protein).
  • nucleic acid sequences the term “substantially the same” is intended to refer to the coding region and to conserved sequences governing expression, and refers primarily to degenerate codons encoding the same amino acid, or alternate codons encoding conservative substitute amino acids in the encoded polypeptide.
  • amino acid sequences refers generally to conservative substitutions and/or variations in regions of the polypeptide not involved in determination of structure or function.
  • percent identical refers to the percent of the amino acids of the subject amino acid sequence that have been matched to identical amino acids in the compared amino acid sequence by a sequence analysis program.
  • Percent similar refers to the percent of the amino acids of the subject amino acid sequence that have been matched to identical or conserved amino acids. conserved amino acids are those which differ in structure but are similar in physical properties such that the exchange of one for another would not appreciably change the tertiary structure of the resulting protein. Conservative substitutions are defined by Taylor (1986, J. Theor. Biol. 119:205).
  • percent identical refers to the percent of the nucleotides of the subject nucleic acid sequence that have been matched to identical nucleotides by a sequence analysis program.
  • isolated protein or “isolated and purified protein” is sometimes used herein. This term refers primarily to a protein produced by expression of an isolated nucleic acid molecule of the invention. Alternatively, this term may refer to a protein which has been sufficiently separated from other proteins with which it would naturally be associated, so as to exist in "substantially pure” form.
  • isolated or isolated and purified also refers to its separation or removal from a chromatography column matrix or a gel, such as a polyacrylamide gel. That is, a polypeptide that has been separated by chromatography or polyacrylamide gel electrophoresis, but is not eluted from the matrix or gel, is not considered “isolated” or “isolated and purified”.
  • the terms “immunologically specific” or “specific” refer to antibodies that bind to one or more epitopes of a protein of interest, but which do not substantially recognize and bind other molecules in a sample containing a mixed population of anti genie biological molecules.
  • the term “specifically hybridizing” refers to the association between two single-stranded nucleic acid molecules of sufficiently complementary sequence to permit such hybridization under pre-determined conditions generally used in the art (sometimes termed “substantially complementary”).
  • the term refers to hybridization of an oligonucleotide with a substantially complementary sequence contained within a single- stranded DNA or RNA molecule, to the substantial exclusion of hybridization of the oligonucleotide with single-stranded nucleic acids of non-complementary sequence.
  • a “coding sequence” or “coding region” refers to a nucleic acid molecule having sequence information necessary to produce a gene product, when the sequence is expressed.
  • operably linked means that the regulatory sequences necessary for expression of the coding sequence are placed in a nucleic acid molecule in the appropriate positions relative to the coding sequence so as to enable expression of the coding sequence.
  • This same definition is sometimes applied to the arrangement of other transcription control elements (e.g. enhancers) in an expression vector.
  • Transcriptional and translational control sequences are DNA regulatory sequences, such as promoters, enhancers, polyadenylation signals, terminators, and the like, that provide for the expression of a coding sequence in a host cell.
  • promoter refers generally to transcriptional regulatory regions of a gene, which may be found at the 5' or 3' side of the coding region, or within the coding region, or within introns.
  • a promoter is a DNA regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3' direction) coding sequence.
  • the typical 5 'promoter sequence is bounded at its 3' terminus by the transcription initiation site and extends upstream (5' direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background.
  • a transcription initiation site (conveniently defined by mapping with nuclease SI), as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase.
  • a “vector” is a replicon, such as plasmid, phage, cosmid, or virus to which another nucleic acid segment may be operably inserted so as to bring about the replication or expression of the segment.
  • nucleic acid construct or "DNA construct” is sometimes used to refer to a coding sequence or sequences operably linked to appropriate regulatory sequences and inserted into a vector for transforming a cell. This term may be used interchangeably with the term "transforming DNA”.
  • a nucleic acid construct may contain a coding sequence for a gene product of interest, along with a selectable marker gene and/or a reporter gene.
  • selectable marker gene refers to a gene encoding a product that, when expressed, confers a selectable phenotype such as antibiotic resistance on a transformed cell.
  • reporter gene refers to a gene that encodes a product which is easily detectable by standard methods, either directly or indirectly.
  • a “heterologous" region of a nucleic acid construct is an identifiable segment (or segments) of the nucleic acid molecule within a larger molecule that is not found in association with the larger molecule in nature.
  • the gene when the heterologous region encodes a mammalian gene, the gene will usually be flanked by DNA that does not flank the mammalian genomic DNA in the genome of the source organism.
  • a heterologous region is a construct where the coding sequence itself is not found in nature (e.g., a cDNA where the genomic coding sequence contains introns, or synthetic sequences having codons different than the native gene). Allelic variations or naturally-occurring mutational events do not give rise to a heterologous region of DNA as defined herein.
  • the term "DNA construct", as defined above, is also used to refer to a heterologous region, particularly one constructed for use in transformation of a cell.
  • a cell has been "transformed” or “transfected” by exogenous or heterologous DNA when such DNA has been introduced inside the cell.
  • the transforming DNA may or may not be integrated (covalently linked) into the genome of the cell.
  • the transforming DNA may be maintained on an episomal element such as a plasmid.
  • a stably transformed cell is one in which the transforming DNA has become integrated into a chromosome so that it is inherited by daughter cells through chromosome replication. This stability is demonstrated by the ability of the eukaryotic cell to establish cell lines or clones comprised of a population of daughter cells containing the transforming DNA.
  • a "clone” is a population of cells derived from a single cell or common ancestor by mitosis.
  • a "cell line” is a clone of a primary cell that is capable of stable growth in vitro for many generations.
  • an isolated nucleic acid molecule that encodes a novel regulator of ABA-mediated stomata aperture control.
  • This nucleic acid molecule is referred to herein as AAPK ("ABA- activated protein kinase").
  • AAPK ABA- activated protein kinase
  • Its manner of regulating ABA-mediated stomata aperture control is novel and interesting.
  • the functional product of the gene is eliminated or specifically altered in a plant, the plant exhibits decreased sensitivity to ABA- induced stomatal closure, but without changing the plant's responses to stomatal closure induced by darkness or CO 2 .
  • the loss of function of AAPK had no effect on ABA-mediated inhibition of stomatal opening. While not intending to limit the present invention by describing one possible mechanism of action of AAPK, it may be that AAPK functions as a negative regulator of ABA-mediated stomatal aperture control, and resultant transpiration and gas exchange.
  • AAPK was selected as an important target for cloning firstly because its ABA-activated phosphorylation activity is specific to guard cells. Secondly, AAPK was selected because it was not activated by other stoma-closing factors such as CO 2 or darkness, making it a potential guard cell-specific, ABA response regulator of stomatal closure. Furthermore, since maintaining control of transpirational water loss and gas exchange is of a vital and fundamental nature to plants, AAPK is likely to be a highly conserved function among all plant species.
  • the AAPK cDNA was isolated from a Viciafaba cDNA library by using degenerate primers created based on peptide sequence data.
  • the degenerate primers were used in reverse transcriptase - PCR to generate a 310 bp probe from guard cell RNA.
  • the probe was then used to screen a V. faba guard cell cDNA library. Based on analysis of the probes, the AAPK gene product appears to be a significant protein kinase in the guard cells, inasmuch as other protein kinases were not identified despite the fact that the probe was homologous to domains common to other protein kinase family members.
  • PKABAl SEQ ID NO: 10
  • AAPK AAPK also possesses these conserved kinase domains.
  • AAPK protein sequence contains high similarity to a large number of protein kinases, as revealed, for example, by the alignment of plant protein kinases shown in Figure 1.
  • the functional specialization that allows these kinases to operate in specific signal transduction pathways lies both in the kinase domain and non-kinase domains.
  • the Viciafaba AAPK kinase protein (SEQ ID NO:2) displays significant similarity to PKABAl (SEQ ID NO: 10). While the similarity is highest in the putative kinase domains, there are several regions where the two proteins are less different from one another.
  • PKABA is expressed from an ABA-induced transcript, but it has not been shown to possess the ABA-activated protein kinase activity of AAPK, suggesting that it plays a different role.
  • PKABAl (SEQ ID NO:10). These peptides were: (1) PLMHDSDRYDF (SEQ ID NO: 15), corresponding to residues 5-15 of SEQ ID NO:2 at the amino terminus; and (2) PADLVNENLMDNQFEEPDQ (SEQ D NO; 16), corresponding to residues 275-293 of SEQ ID NO:2 near the carboxyl terminus. Screening with either of these peptides corroborated the physical and database screening using the complete sequence, identifying each the aforementioned Arabidopsis proteins. This screening also revealed a fourth homolog, identified in the database as Protein Kinase SPK-2, Accession No. S56718 (SEQ ID NO:8). It is possible that this Arabidopsis protein is the same as the predicted protein from clone L05561. Although the AAPK cDNA clone from Viciafaba, and homologs from
  • Arabidopsis are described and exemplified herein, this invention is intended to encompass nucleic acid sequences and proteins from other plants that are sufficiently similar to be used instead of the Viciafaba or Arabidopsis AAPK nucleic acids and proteins for the purposes described below. These include, but are not limited to, allelic variants and natural mutants of AAPK, which are likely to be found in different varieties of Viciafaba, as well as homologs of AAPK from different species of plants.
  • this invention provides an isolated AAPK nucleic acid molecule having at least about 50% (preferably 60%, more preferably 70% and even more preferably over 80%) sequence identity in the coding regions with the nucleotide sequence set forth as SEQ ID NOs: 1, 3, 5, 7 or 9 (and, most preferably, specifically comprising the coding regions of SEQ ID NOs:l, 3, or 6
  • This invention also provides isolated polypeptide products of the open reading frames of AAPK, having at least about 60% (preferably 70%, 75%, 80% or greater) sequence identity with the amino acid sequences of SEQ ID NOS: 2, 4, 5, 7 or 9..
  • Such plant species include dicotyledenous and monocotyledenous flowering plants, as well as any other plant that possesses stomata.
  • AAPKs from agronomically or horticulturally important plant species, including maize, wheat, rye, oats, barley, rice, sorghum, soy and other beans, alfalfa, sunflower, canola, lawn and turfgrasses, tobacco, aster, zinnia, chrysanthemum, beet, carrot, cruciferous vegetables, cucumber, grape, pea, potato, rutabaga, tomato, tomatillo and tumip, to name a few.
  • AAPK nucleic acid molecules of the invention include DNA, RNA, and fragments thereof which may be single- or double-stranded.
  • this invention provides oligonucleotides (sense or antisense strands of DNA or RNA) having sequences capable of hybridizing with at least one sequence of a nucleic acid molecule encoding the protein of the present invention. Such oligonucleotides are useful as probes for detecting AAPK genes or transcripts.
  • the present invention is drawn to artificially created mutants, produced by in vitro mutagenesis or isolated from mutagenized plants, as described in greater detail below.
  • mutant AAPK nucleic acids and their encoded proteins are integral to practicing the methods of the invention, which involve regulating ABA-mediated stomatal closure in plants.
  • the present invention further encompasses genetically modified plants having altered transpiration and gas exchange characteristics due to the down-regulation or up- regulation of AAPK in those plants.
  • AAPK nucleic acid molecules of the invention may be prepared by two general methods: (1) they may be synthesized from appropriate nucleotide triphosphates, or (2) they may be isolated from biological sources. Both methods utilize protocols well known in the art. The availability of nucleotide sequence information, such SEQ ID NOS: 1, 3 and 6, enables preparation of an isolated nucleic acid molecule of the invention by polynucleotide synthesis. Synthetic oligonucleotides may be prepared by the phosphoramadite method employed in the Applied Biosystems 38 A DNA Synthesizer or similar devices.
  • the resultant construct may be purified according to methods known in the art, such as high performance liquid chromatography (HPLC).
  • Long, double-stranded polynucleotides such as a DNA molecule of the present invention, must be synthesized in stages, due to the size limitations inherent in current oligonucleotide synthetic methods.
  • a long double-stranded molecule may be synthesized as several smaller segments of appropriate complementarity.
  • Complementary segments thus produced may be annealed such that each segment possesses appropriate cohesive termini for attachment of an adjacent segment. Adjacent segments may be ligated by annealing cohesive termini in the presence of DNA ligase to construct an entire long double-stranded molecule.
  • a synthetic DNA molecule so constructed may then be cloned and amplified in an appropriate vector.
  • Modified (i.e., "mutant") nucleic acid molecules of the invention also may be synthesized as described above. In this embodiment, the desired alteration is simply programmed into the synthetic scheme. In another embodiment, an unaltered synthetic nucleic acid molecule is manufactured, and subsequently altered by site- directed mutagenesis.
  • Nucleic acid molecules encoding the AAPK protein may be isolated from V. faba, Arabidopsis or any other plant of interest using methods well known in the art. It will be appreciated that such methods may be used to screen libraries of mutant plants as well as wild-type plants. In order to isolate AAPK-encoding nucleic acids from plants other than V.
  • oligonucleotides designed to match the nucleic acids encoding the V. faba or Arabidopsis AAPK protein may be used with cDNA or genomic libraries from the desired species. If the AAPK gene from a species is desired, the genomic library is screened. Alternately, if the protein coding sequence is of particular interest, the cDNA library is screened. In positions of degeneracy, where more than one nucleic acid residue could be used to encode the appropriate amino acid residue, all the appropriate nucleic acids residues may be incorporated to create a mixed oligonucleotide population, or a neutral base such as inosine may be used. Such degenerate libraries also may be customized for the codon preference of the plant species to be screened. The strategy of oligonucleotide design is well known in the art (see Ausbel et al., Sambrook et al.).
  • known AAPK sequences may be used in "data mining” to screen databases for homologous sequences, as is well known in the art and exemplified herein.
  • PCR (polymerase chain reaction) primers may be designed by the above method to encode a portion a known AAPK protein, and these primers used to amplify nucleic acids from isolated cDNA or genomic DNA.
  • the oligonucleotides used to isolate AAPK nucleic acids are designed to encode sequences conserved among AAPKs, but not between AAPK and other kinases (e.g., the PKABAl protein kinase family), as described above.
  • nucleic acids having the appropriate sequence homology with a known AAPK nucleic acid molecule may be identified by using hybridization and washing conditions of appropriate stringency.
  • hybridizations may be performed, according to the method of Sambrook et al. (1989, supra), using a hybridization solution comprising: 5X SSC, 5X Denhardt's reagent, 1.0% SDS, 100 ⁇ g/ml denatured, fragmented salmon sperm DNA, 0.05% sodium pyrophosphate and up to 50% formamide. Hybridization is carried out at 37- 42°C for at least six hours.
  • filters are washed as follows: (1) 5 minutes at room temperature in 2X SSC and 1% SDS; (2) 15 minutes at room temperature in 2X SSC and 0.1% SDS; (3) 30 minutes- 1 hour at 37°C in IX SSC and 1% SDS; (4) 2 hours at 42-65° in IX SSC and 1% SDS, changing the solution every 30 minutes.
  • T m 81.5EC + 16.6Log [Na+] + 0.41(% G+C) - 0.63 (% formamide) - 600/#bp in duplex
  • the T m is 57°C.
  • the T m of a DNA duplex decreases by 1 - 1.5°C with every 1% decrease in homology.
  • targets with greater than about 75% sequence identity would be observed using a hybridization temperature of 42°C.
  • the hybridization is at 37°C and the final wash is at 42°C, in a more preferred embodiment the hybridization is at 42°C and the final wash is at 50°C, and in a most preferred embodiment the hybridization is at 42°C and final wash is at 65°C, with the above hybridization and wash solutions.
  • Nucleic acids of the present invention may be maintained as DNA in any convenient cloning vector.
  • clones are maintained in plasmid cloning/expression vector, such as pBluescript (Stratagene, La Jolla, CA), which is propagated in a suitable E. coli host cell.
  • AAPK polypeptides may be prepared in a variety of ways, according to known methods.
  • the availability of nucleic acid molecules encoding the polypeptides enables synthesis of the proteins by known methods, or production of the proteins using in vitro expression methods known in the art.
  • a cDNA or gene may be cloned into an appropriate in vitro transcription vector, such a pSP64 or pSP65 for in vitro transcription, followed by cell-free translation in a suitable cell-free translation system, such as wheat germ or rabbit reticulocytes.
  • in vitro transcription and translation systems are commercially available, e.g., from Promega Biotech, Madison, Wisconsin or BRL, Rockville, Maryland.
  • the pCllE in vitro translation system (Novagen) also may be utilized.
  • larger quantities of the proteins may be produced by expression in a suitable procaryotic or eucaryotic system.
  • a DNA molecule such as the coding portion of SEQ ID NOS: 1, 3 or 6, or appropriate complementary sequences, may be inserted into a plasmid vector adapted for expression in a bacterial cell (such as E. coli) or a yeast cell (such as Saccharomyces cerevisiae), or into a baculovirus vector for expression in an insect cell.
  • a plasmid vector adapted for expression in a bacterial cell (such as E. coli) or a yeast cell (such as Saccharomyces cerevisiae), or into a baculovirus vector for expression in an insect cell.
  • Such vectors comprise the regulatory elements necessary for expression of the
  • AAPK polypeptides produced by gene expression in a recombinant procaryotic or eucyarotic system may be purified according to methods known in the art.
  • a commercially available expression secretion system can be used, whereby the recombinant protein is expressed and thereafter secreted from the host cell, to be easily purified from the surrounding medium.
  • an alternative approach involves purifying the recombinant protein by affinity separation, such as by immunological interaction with antibodies that bind specifically to the recombinant protein. Such methods are commonly used by skilled practitioners.
  • AAPK proteins prepared by the aforementioned methods, may be analyzed according to standard procedures. Methods for analyzing the functional activity of kinases are well known to persons skilled in the art. Alternatively, the function of the kinase in stomatal closure may be analyzed, as described in greater detail below and in Example 1.
  • the present invention also provides antibodies that are immunologically specific to the AAPK of the invention.
  • Polyclonal antibodies may be prepared according to standard methods.
  • monoclonal antibodies are prepared, which are specific to various epitopes of the protein.
  • Monoclonal antibodies may be prepared according to general methods of Kohler and Milstein, following standard protocols.
  • Polyclonal or monoclonal antibodies that are immunologically specific for the AAPK can be utilized for identifying and purifying AAPK from V. faba and other species.
  • antibodies may be utilized for affinity separation of proteins for which they are specific or to quantify the protein.
  • Antibodies may also be used to immunoprecipitate proteins from a sample containing a mixture of proteins and other biological molecules.
  • Example 1 describes a synthetic mutant, AAPK(K43A) in Viciafaba, which displays insensitivity to ABA-induce stomatal closure due to the loss of function of AAPK.
  • Any plant may be transgenically engineered to display a similar phenotype. This approach is particularly appropriate to plants with high ploidy numbers, including but not limited to wheat.
  • These synthetic null mutant are created by a expressing a mutant form of the AAPK protein to create a "dominant negative effect". While not limiting the invention to any one mechanism, this mutant AAPK protein competes with wild-type AAPK protein for interacting proteins in the transgenic plant, or poisons an AAPK multimeric complex. By over-producing the mutant form of the protein, the signaling pathway used by the wild-type AAPK protein can be effectively blocked. Examples of this type of "dominant negative” effect are well known for both insect and vertebrate systems (Radke et al, 1997, Genetics 145:163-171; Kolch et al., 1991, Nature 349:426-428).
  • the mutant protein is produced by mutating the coding sequence of AAPK corresponding to residues in the active site.
  • the coding sequence corresponding to the lysine residue at position 43 is mutated to code for a different, preferably non-similar, amino acid residue, for example, alanine.
  • a second kind of synthetic null mutant can be created by inhibiting the translation of the AAPK mRNA by "post-transcriptional gene silencing".
  • the AAPK gene from the species targeted for down-regulation, or a fragment thereof, may be utilized to control the production of the encoded protein.
  • Full-length antisense molecules or antisense oligonucleotides are used that are targeted to specific regions of the AAPAT-encoded RNA that are critical for translation.
  • the use of antisense molecules to decrease expression levels of a pre-determined gene is known in the art.
  • Antisense molecules may be provided in situ by transforming plant cells with a DNA construct which, upon transcription, produces the antisense RNA sequences. Such constructs can be designed to produce full-length or partial antisense sequences.
  • This gene silencing effect can be enhanced by transgenically over-producing both sense and antisense RNA of the gene coding sequence so that a high amount of dsRNA is produced (for example see Waterhouse et al., 1998, PNAS 95: 13959-13964).
  • part or all of the AAPK coding sequence antisense strand is expressed by a transgene.
  • hybridizing sense and antisense strands of part or all of the AAPK coding sequence are transgenically expressed.
  • a third type of synthetic null mutant can also be created by the technique of "co-suppression". Plant cells are transformed with a copy of the endogenous gene targeted for repression. In many cases, this results in the complete repression of the native gene as well as the transgene.
  • the AAPK gene from the plant species of interest is isolated and used to transform cells of that same species.
  • Transgenic plants can also be created that have enhanced AAPK activity. This can be accomplished by transforming plant cells with a transgene that expresses part or all of the AAPK coding sequence, or a sequence that encodes the either the AAPK protein or a protein functionally similar to it.
  • the complete AAPK coding sequence is transgenically over-expressed.
  • the coding sequence corresponding to the kinase domain of AAPK is over-expressed.
  • Transgenic plants with one of the transgenes mentioned above can be generated using standard plant transformation methods known to those skilled in the art. These include, but are not limited to, Agrobacte ⁇ um vectors, polyethylene glycol treatment of protoplasts, biolistic DNA delivery, UV laser microbeam, gemini virus vectors, calcium phosphate treatment of protoplasts, electroporation of isolated protoplasts, agitation of cell suspensions in solution with microbeads coated with the transforming DNA, agitation of cell suspension in solution with silicon fibers coated with transforming DNA, direct DNA uptake, liposome-mediated DNA uptake, and the like. Such methods have been published in the art.
  • Agrobacterium vectors are often used to transform dicot species.
  • Agrobacterium binary vectors include, but are not limited to, BLN19 (Bevan, 1984) and derivatives thereof, the pBI vector series (Jefferson et al., 1987), and binary vectors pGA482 and pGA492 (An, 1986)
  • biolistic bombardment with particles coated with transforming DNA and silicon fibers coated with transforming DNA are often useful for nuclear transformation.
  • DNA constructs for transforming a selected plant comprise a coding sequence of interest operably linked to appropriate 5' (e.g., promoters and translational regulatory sequences) and 3' regulatory sequences (e.g., terminators).
  • the coding region is placed under a powerful constitutive promoter, such as the Cauliflower Mosaic Virus (CaMV) 35S promoter or the figwort mosaic virus 35S promoter.
  • CaMV Cauliflower Mosaic Virus
  • Other constitutive promoters contemplated for use in the present invention include, but are not limited to: T-DNA mannopine synthetase, nopaline synthase (NOS) and octopine synthase (OCS) promoters.
  • Transgenic plants expressing a sense or antisense AAPK coding sequence under an inducible promoter are also contemplated to be within the scope of the present invention.
  • Inducible plant promoters include the tetracycline repressor/operator controlled promoter, the heat shock gene promoters, stress (e.g., wounding)-induced promoters, defense responsive gene promoters (e.g. phenylalanine ammonia lyase genes), wound induced gene promoters (e.g.
  • hydroxyproline rich cell wall protein genes hydroxyproline rich cell wall protein genes
  • chemically-inducible gene promoters e.g., nitrate reductase genes, glucanase genes, chitinase genes, etc.
  • dark-inducible gene promoters e.g., asparagine synthetase gene
  • Tissue specific and development-specific promoters are also contemplated for use in the present invention. Examples of these included, but are not limited to: the ribulose bisphosphate carboxylase (RuBisCo) small subunit gene promoters or chlorophyll a/b binding protein (CAB) gene promoters for expression in photosynthetic tissue; the various seed storage protein gene promoters for expression in seeds; and the root-specific glutamine synthetase gene promoters where expression in roots is desired.
  • RuBisCo ribulose bisphosphate carboxylase
  • CAB chlorophyll a/b binding protein
  • AAPK gene of the preferred embodiments taught with this invention are specifically expressed in guard cells, this in no way limits the application of this invention to any specific tissue or development phase, but rather represents only the particular embodiments taught herein.
  • the coding region is also operably linked to an appropriate 3' regulatory sequence.
  • the nopaline synthetase polyadenylation region NOS
  • Other useful 3' regulatory regions include, but are not limited to the octopine (OCS) polyadenylation region.
  • the selected coding region under control of a constitutive or inducible promoter as described above, is linked to a nuclear drug resistance marker, such as kanamycin resistance.
  • a nuclear drug resistance marker such as kanamycin resistance.
  • Other useful selectable marker systems include, but are not limited to: other genes that confer antibiotic resistances (e.g., resistance to hygromycin or bialaphos) or herbicide resistance (e.g., resistance to sulfonylurea, phosphinothricin, or glyphosate).
  • Plants are transformed and thereafter screened for one or more properties, including the lack of AAPK protein, AAPK mRNA, or altered stomatal aperture responses to ABA treatment. It should be recognized that the amount of expression, as well as the tissue-specific pattern of expression of the transgenes in transformed plants can vary depending on the position of their insertion into the nuclear genome. Such positional effects are well known in the art. For this reason, several nuclear transformants should be regenerated and tested for expression of the transgene. Transgenic plants that exhibit one or more of the aforementioned desirable phenotypes can be used for plant breeding, or directly in agricultural or horticultural applications.
  • Plants containing one transgene may also be crossed with plants containing a complementary transgene in order to produce plants with enhanced or combined phenotypes.
  • An alternative to the transgenic approach described above is the screening of populations of plant mutants of a variety of species, from which AAPK mutants can be isolated. Such populations can be made by chemical mutagenesis, radiation mutagenesis, and transposon or T-DNA insertion, as is well known in the art.
  • the mutants would be null mutants having a phenotype comprising reduced or substantially absent stomatal closure in response to abscisic acid, but no reduction in stomatal closure response to darkness or CO 2 .
  • mutant are screened for the phenotypes related to overproduction of the AAPK gene product and/or increased sensitivity to ABA- induced stomatal closure.
  • the nucleic acids of the invention can be used to isolate or create
  • oligonucleotide primers can be designed to screen lines for insertions in the AAPK gene. Plants with transposon or T-DNA insertions in the AAPK gene are very likely to have lost the function of the gene product. Through breeding, a plant line may then be developed that is homozygous for the non-functional copy or the altered copy of the AAPK gene.
  • the PCR primers for this purpose are designed so that large portions of the coding sequence the AAPK gene are specifically amplified using the sequence of the AAPK gene from the species to be probed (see Baumann et al., 1998, Theor. Appl. Genet. 97:729-734).
  • AAP ⁇ T-like mutants can be isolated from mutant populations using the distinctive phenotype characterized in accordance with the present invention. This approach is particularly appropriate in plants with low ploidy numbers where the phenotype of a recessive mutation is more easily detected.
  • the population of plants would be exposed to abscisic acid (ABA) or analogs of the hormone. Plants would then be screened for phenotype of the AAPK mutants: the reduced stomatal closure in response to applied ABA, without a reduction in stomatal response to darkness or CO 2 . That the phenotype is caused by an AAPK mutation is then established by molecular means well known in the art.
  • any of the aforementioned transformation or mutagenesis techniques may be applied to any selected plant species.
  • Such species include, but are not limited to, agronomically important crop plants such as maize, wheat, rice, rye, oats, barley, soy and other beans, sorghum, sunflower, canola, tobacco and alfalfa; vegetable and fruit crop plants such as beet, carrot, cruciferous vegetables, cucumber, grape, pea, potato, rutabaga, tomato, tomatillo and turnip; and horticulturally important plants such as aster, begonia, chrysanthemum, clover, lawn and turf grasses, mint and other herbs, and zinnia.
  • AAPK nucleic acids may be used for a variety of purposes in accordance with the present invention. DNA, RNA, or fragments thereof, may be used as probes to detect the presence and/or expression of AAPK genes. Methods in which AAPK nucleic acids may be utilized as probes for such assays include, but are not limited to: (1) in situ hybridization; (2) Southern hybridization (3) Northern hybridization; and (4) assorted amplification reactions such as polymerase chain reactions (PCR).
  • PCR polymerase chain reactions
  • AAPK nucleic acids of the invention may also be utilized as probes to identify related genes from other plant species.
  • hybridization stringencies may be adjusted to allow hybridization of nucleic acid probes with complementary sequences of varying degrees of homology.
  • AAPK nucleic acids may be used to advantage to produce large quantities of substantially pure AAPK, or selected portions thereof.
  • the AAPK nucleic acids can be used to identify and isolate other putative members of this novel ABA-mediated stomatal aperture control signal cascade in vivo.
  • a yeast two hybrid system can be used to identify proteins that physically interact with the AAPK protein, as well as isolate their nucleic acids.
  • the sequence encoding the protein of interest is operably linked to the sequence encoding half of a activator protein.
  • This construct is used to transform a yeast cell library which has been transformed with DNA constructs that contain the coding sequence for the other half of the activator protein operably linked to a random coding sequence from the organism of interest.
  • the protein made by the random coding sequence from the library interacts with the protein of interest, the two halves of the activator protein are physically associated and form a functional unit that activates the reporter gene.
  • all or part of the AAPK coding sequence may be operably linked to the coding sequence of the first half of the activator, and the library of random coding sequences may be constructed with cDNA from V.
  • activator protein/reporter genes are customarily used in the yeast two hybrid system.
  • the bacterial repressor LexA DNA-binding domain and the Gal4 transcription activation domain fusion proteins associate to activate the LacZ reporter gene (see Clark et al., 1998, PNAS 95:5401-5406). Kits for the two hybrid system are also commercially available from Clontech, Palo Alto CA, among others.
  • interaction cloning is used identify proteins that physically interact with the AAPK protein and to isolate the nucleic acids encoding them.
  • a cDNA expression library is screened for proteins that interact with the AAPK catalytic domain, or other selected domains that might be involved in protein-protein interactions. This is done using a filter binding assay and a labeled peptide comprising the putative interacting site. Positive clones are then purified, amplified if necessary, and characterized.
  • AAPK proteins of the present invention can be used to identify molecules with binding affinity for AAPK, which are likely to be novel participants in this resistance pathway.
  • the known protein is allowed to form a physical interaction with the unknown binding molecule(s), often in a heterogenous solution of proteins.
  • the known protein in complex with associated molecules is then isolated, and the nature of the associated protein(s) and/or other molecules is determined.
  • AAPK may also be generated as part of a fusion protein with one or more other proteins, for example with a green fluorescent protein (GFP).
  • GFP green fluorescent protein
  • Such fusion products may have utility from either or each part of the fusion molecule. For example, the easy detection of their presence is provided by the GFP moiety, while the specific kinase activity is retained by the AAPK moiety. Additionally they may allow convenient use of commercially available antibodies specific to the fused portion (e.g antiGFP antibodies are readily available.)
  • Antibodies that are immunologically specific for AAPK may be utilized in affinity chromatography to isolate the AAPK protein, to identitfy or quantify the AAPK protein utilizing techniques such as western blotting and ELISA, or to immuno- precipitate AAPK from a sample containing a mixture of proteins and other biological materials.
  • the immuno-precipitation of AAPK is particularly advantageous when utilized to isolate affinity binding complexes of AAPK, as described above.
  • Mutants and Transgenic Plants The AAPK mutants of the invention display altered sensitivity to ABA-induced stomatal closure, and therefore can be used to improve crop and horticultural plant species.
  • the AAPK mutants taught in this invention are particularly valuable in that the mutation is very specific.
  • mutants will therefore be particularly useful in crop and horticultural varieties in which reduction of moisture content is important.
  • crops include but are not limited to cereal grains such as corn, wheat, rye, oats, barley, and rice, soybeans and other beans, as well as other products such as hay and commercial seed.
  • failure to adequately dry the crop due to weather or other conditions results in substantial losses.
  • dried fruits such as raisins and prunes, nuts, coffee, tea, cocoa, and many ornamental goods
  • the produce needs to be dried immediately after harvest prior and to use.
  • the mutants of this invention may be of tremendous value to growers who could accelerate or control the rate of crop drying.
  • the AAPK mutants exhibit a decreased induction by ABA of normal stomatal closure. They therefore have influence over transpirational water loss in plants. It is therefore contemplated that in addition to the specific applications mentioned heretofore, these mutants will have myriad applications to other important plant problems especially in irrigated crops or other crops where water and yield are delicately balanced. It is also trivial to one skilled in the art to extend this invention to the production of mutants with increased sensitivity to ABA-induced stomatal closure. These mutants are useful for a variety agronomic purposes. It is clear that such mutants would keep the stomatal aperture small, and would therefore experience reduced transpirational water loss. Such mutants can be used to help enhance tolerance to water stress or drought conditions.
  • Such mutants could be the result of active site changes or modifications which allow them to respond to lower concentrations of ABA, or they could be the result of mutations in genes in a common regulatory pathway. Such mutants could also be the result of overexpression of the AAPK gene product via a transgenic modification such that expression of AAPK is driven by an inducible promoter, a strong, highly active constitutive promoter, or by increasing the copy number of the gene in the plant.
  • AAPK mutants of the invention can be used to identify and isolate additional members of this ABA-regulation of transpiration pathway.
  • transgenic plants of the invention are particularly useful in conferring the AAPK phenotype to many different plant species.
  • a host of plant species with enhanced disease resistance can be easily made, to be used as breeding lines or directly in commercial operations.
  • Such plants can have uses as crop species, or for ornamental use.
  • a plant that has had functional AAPK transgenically depleted will exhibit the same altered sensitivity to ABA-induced stomatal closure as AAPK mutants. It is therefore contemplated that transgenic AAP -phenotype plants will be used with in the same aforementioned manner as the AAPK mutants.
  • a transgenic approach is advantageous because it allows AAP ⁇ T-phenotype plants to be created quickly, without time-consuming mutant generation, selection, and back-crossing.
  • a plant that has had functional AAPK increased may have enhanced sensitivity to ABA-induced stomatal closure compared to wild-type plants. Plants with enhanced sensitivity to ABA-induced stomatal closure will be extremely valuable to agriculture and horticulture by allowing plants to better tolerate periods of restricted water or drought. Additionally, such mutants may provide the advantage of allowing produce to retain water as long as possible. For many fruits, vegetables and flowers, including cut flowers, it would be advantageous to minimize water loss during the harvest, transport and distribution. Retail customers too would benefit from the extended shelf-life of such fruits, vegetables and flowers which would remain fresher for longer periods of time.
  • Protein Kinase-Encoding cDNA from Guard Cells This example describes the cloning and characterization of a Vicia faba complementary DNA, AAPK, encoding a guard cell-specific ABA-activated serine-threonine protein kinase (AAPK).
  • AAPK Guard cell protoplasts (2 X 10 6 ; 99.6% pure) were prepared from Vicia faba. Protoplasts were treated with either darkness, ABA or elevated CO 2 concentrations prior to protein isolation. Protoplast proteins were extracted and subjected to 2-dimensional gel electrophoresis. Separation was via 12% SDS- polyacrylamide gel electrophoresis (SDS-PAGE). Kinase autophosphorylation activity of subsequently renatured proteins was detected by established methods..
  • the AAPK protein was excised from the 2-D gels (first dimension, nondenaturing PAGE) after digestion with trypsin.
  • the AAPK peptides generated by trypsin digestion were subjected to peptide sequencing by tandem mass spectrometry on a Finnigan LCQ quadrupole ion trap mass spectrometer.
  • AAPK ABA-Activated Protein Kinase
  • the PCR product was labelled with ⁇ 32 P ⁇ dCTP.
  • This 32 P-labelled probe was then used to screen a V. faba guard cell cDNA library in ⁇ -Zap II. A full length cDNA of the appropriate size to encode the AAPK was obtained. The cDNA was sequenced in both directions.
  • Northern analysis was performed by standard methods. The probe was the 3 P-labelled BglU-Csp45I fragment of the AAPK cDNA clone. This probe includes the sequence encoding the relatively unique AAPK amino terminal region and part of the 3' untranslated region of the cDNA.
  • AAPK Functional Analysis of the AAPK gene. Functional analysis of the AAPK gene product was complicated by the observation that ABA-activation of the AAPK does not occur in vitro. Prior treatment of intact guard cells with ABA was required to elicit active AAPK upon extraction. In light of this apparent requirement for an intact cellular signal system, DNA constructs were created to facilitate the analysis of expression and activity of AAPK. Creation of AAPK mutants and hybrid expression DNA constructs. A construct encoding a green fluorescent protein (GFP)-tagged AAPK was made.
  • GFP green fluorescent protein
  • pAAPK-GFP This construct, pAAPK-GFP, was created by amplifying the AAPK coding sequence from the AAPK cDNA and inserting the amplified coding sequence downstream of the 35S promoter and upstream of, and in-frame with, the GFP coding sequence in the GFP expression vector, pGFP.
  • the amplification was performed via PCR with the primers 5'-GAATCTCCACTACGACGCCGTTTACTTCCC-3' (SEQ ED NO: 19) and 5'-CCGTGCAACCATGGATATGGCATATACAAT-3' (SEQ ID NO:20). Ncol was used for digestion and insertion.
  • pAAPK(K43A)-GFP Another DNA construct, pAAPK(K43A)-GFP was created.
  • This construct contained a site-directed mutation of AAPK, such that the coding sequence for a highly conserved lysine residue (Lys 43 ), believed to be in the ATP-binding site of the kinase AAPK, was specifically modified to encode an alanine residue instead of the lysine.
  • Lys 43 highly conserved lysine residue
  • Such mutations have been shown to yield kinases with reduced or absent catalytic activity.
  • Analysis of AAPK mutants and hybrid expression DNA constructs for ABA-activated protein kinase activity 1.5 X 10 7 V.
  • faba guard cell protoplasts were transfected with either the vector, pGFP, or the constructs, pAAPK-GFP or pAAPK(K43A)-GFP by polyethylene glycol (PEG)-mediated DNA transfer. After uptake and expression, protoplasts were lysed and recombinant proetin was immunoprecipitated with anti-GFP peptide antibodies (Clontech) and protein A- Sepharose CL-4B (Amersham Pharmacia Biotech). Immunoprecipitated proteins were assayed for kinase activity using histone IJJ-S (Sigma) as substrate.
  • PEG polyethylene glycol
  • V. faba leaves were biolistically transformed with the pGFP, pAAPK-GFP, or pAAPK(K43A)-GFP constructs.
  • the V. faba leaves were bombarded with gold particles (Bio-Rad) coated with one of the DNA constructs. Bombardment was via a particle delivery system 1000/He (Bio-Rad) as described. (J. Marc et al., 1998, Plant Cell 10:1927).
  • Bath solution contained 40 mM CaCl 2 , 2 mM MgCl and 10 mM MES-Tris pH5.6. Osmolalities were adjusted with sorbitol to 500 mosmol/kg (in the pipette) or 470 mosmol/kg (in the bath).
  • Protein kinase inhibitor K-252a (Calbiochem) was prepared as a stock solution at 2 mM in dimethyl sulfoxide (DMSO).
  • AAPK Viciafaba ABA-Activated Protein Kinase
  • AAPK was identified as a 48 kDa ABA-dependent and Ca 2+ - independent autophosphorylation spot with the in-gel kinase assay.
  • the peptide fragment sequence information obtained is provided in Figure 1. Two sequenced
  • AAPK peptides had similarity to the protein kinase ABA1 (PKABAl) protein kinase subfamily in subdomains I and VIb. PKABAl is transcriptionally up-regulated by ABA.
  • PKABAl protein kinase ABA1
  • AAPK ABA- Activated Protein Kinase
  • nucleotide sequence of the full length AAPK cDNA and the amino acid sequence of the deduced AAPK protein are given in Figure 1.
  • the deduced AAPK amino acid sequence shows the greatest homology to the PKABAl subfamily. However, the predicted sequence also has unique regions.
  • AAPK AAPK appears to be a single copy gene based on results from Southern analysis; however, further analysis may reveal the presence of more than one copy.
  • AAPK activity could be an indication that an intact cellular signal or cascade is required. Any discussion or explanation offered here is intended to provide clarity and is not intended to limit the invention in any way to one theory or avenue as to the mechanism or application.
  • ABA-mediated stomatal aperture regulation and anion channel activation in AAPK mutants were identified by their green fluorescence. Transformation with pAAPK(K43A)-GFP eliminated ABA- induced stomatal closure, but had no effect on stomatal closure induced by CO 2 or darkness. Transformation with wild-type AAPK via the pAAPK-GFP vector had no measurable effect on either ABA-induced stomatal closure nor on ABA-inhibition of stomatal opening. In V. faba guard cells, ABA activated slow anion channels.
  • the K43A mutant kinase competes with the activity of native AAPK in a dominant negative fashion.
  • the kinase inhibitor K-252a inhibits (i) native AAPK activity, (ii) ABA-induced stomatal closure, and (iii) ABA regulation of anion channels in untransformed cells, implying that the channels are indeed normally regulated by AAPK.
  • an AAPK may mediate ABA-induced anion channel activation and stomatal closure through a phosphorylation event, while ABU opposes ABA action through a dephosphorylation event.
  • ABA inhibition of stomatal opening ( Table 1 ).
  • ABA inhibition of stomatal opening and ABA promotion of stomatal closure may, therefore, employ different signaling cascades.
  • ABA activation of anion channels may not be required for ABA inhibition of stomatal opening.
  • loss of ABA-stimulated stomatal closure in plants transformed with mutant AAPK under control of an inducible promoter should allow accelerated and controlled desiccation of crops that are dried before harvest or distribution. Basal levels of ABA remain even in irrigated crops; under these conditions, inhibition of AAPK activity should alleviate stomatal limitation of CO 2 uptake, and thus accelerate growth or increase yield.
  • the probe was the Nco I - Bgl II fragment (393 base pairs) of V. faba AAPK cDNA.
  • the gel-purified Nco I - Bgl ⁇ fragment of AAPK cDNA was labeled by 32P-dCTP. This probe corresponds to sequences encoding the region from the aspartic acid residue (position 2, SEQ ID NO:2) to the arginine residue (position 132, SEQ ID NO:2) of the AAPK protein.
  • Nylon membranes containing the library were prehybridized with 5x SSC, 5x Denhardt's solution, 1% SDS, and 0.2% nonfat milk at 60 C for 2 hours and then hybridized in the same solution with the labeled probe at 60 C overnight.
  • the membranes were washed with 2x SSC and 0.1% SDS twice for 15 minutes at 60 C, once in 2x SSC and 0.1% SDS, and once in 0.5X SSC and 0.1% SDS.
  • the membranes were exposed to X-ray films and positive plaques were identified by autoradiography.
  • the positive clones were subcloned into pCR BlueScript vector and then analyzed by DNA sequencing and compared to known sequences to look for matches.
  • NCBI National Center for Biological Information

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Abstract

L'invention se rapporte à un nouveau gène, AAPK. La perte de fonctionnalité de la protéine codée par AAPK est associée à une sensibilité réduite à la fermeture stomatale induite par l'acide abscisique dans les végétaux. L'invention se rapporte également à des plantes transgéniques et à des mutants présentant une sensibilité accrue à la transpiration à médiation assurée par l'acide abscisique et d'autres caractéristiques agronomiques souhaitables. La régulation de la transpiration que permet la présente invention est différente de celle de mécanismes précédemment décrits pour contrôler la transpiration des végétaux.
PCT/US2000/018014 1999-07-01 2000-06-29 Compositions et procedes de regulation de la fermeture induite par l'acide abscisique de stomates de vegetaux WO2001002541A2 (fr)

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WO2002090547A1 (fr) * 2001-05-07 2002-11-14 Agriculture Victoria Services Pty Ltd Modification du developpement de plantes et de semences ainsi que des reponses de plantes a des contraintes et a des stimuli
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EP1539996A1 (fr) * 2002-07-02 2005-06-15 The Australian National University Procede de production de plantes presentant une efficacite de transpiration amelioree et plantes obtenues au moyen de ce procede
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Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001045492A2 (fr) * 1999-12-22 2001-06-28 Basf Plant Science Gmbh Proteines de proteine kinase liees au stress et procedes d'utilisation dans les plantes
WO2001045492A3 (fr) * 1999-12-22 2002-08-01 Basf Plant Science Gmbh Proteines de proteine kinase liees au stress et procedes d'utilisation dans les plantes
US7223903B2 (en) 1999-12-22 2007-05-29 Da Costa E Silva Oswaldo Protein kinase stress-related proteins and methods of use in plants
US6867351B2 (en) 2000-04-07 2005-03-15 Basf Plant Science Gmbh Protein kinase stress-related proteins and methods of use in plants
US7179962B2 (en) 2000-04-07 2007-02-20 Basf Plant Science Gmbh Protein kinase stress-related proteins and methods of use in plants
WO2002090547A1 (fr) * 2001-05-07 2002-11-14 Agriculture Victoria Services Pty Ltd Modification du developpement de plantes et de semences ainsi que des reponses de plantes a des contraintes et a des stimuli
AU2002252825B2 (en) * 2001-05-07 2008-01-24 Agriculture Victoria Services Pty Ltd Modification of plant and seed development and plant responses to stresses and stimuli
US7176026B2 (en) 2001-11-09 2007-02-13 Basf Plant Science Gmbh Protein kinase stress-related polypeptides and methods of use in plants
EP1539996A1 (fr) * 2002-07-02 2005-06-15 The Australian National University Procede de production de plantes presentant une efficacite de transpiration amelioree et plantes obtenues au moyen de ce procede
EP1539996A4 (fr) * 2002-07-02 2006-11-15 Univ Australian Procede de production de plantes presentant une efficacite de transpiration amelioree et plantes obtenues au moyen de ce procede
WO2006008271A1 (fr) * 2004-07-16 2006-01-26 Cropdesign N.V. Plantes aux caracteristiques de croissance ameliorees et procedes d'obtention correspondants
AU2005263730B2 (en) * 2004-07-16 2011-04-28 Cropdesign N.V. Plants having improved growth characteristics and method for making the same
US20130167263A1 (en) * 2007-10-30 2013-06-27 Monsanto Technology Llc Nucleic acid molecules and other molecules associated with plants and uses thereof

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