WO2004013295A2 - Plant rna binding protein, encoding nucleic acids, and methods of use - Google Patents

Abstract

A plant RNA binding protein and nucleic acid encoding it are disclosed. The RNA binding protein is a substrate for phosphorylation by protein kinase. In guard cells, the ABA-activated protein kinase (AAPK) phosphorylates the RNA binding protein resulting in an increased binding affinity for mRNA, particularly of stress-regulated mRNA. Vectors and transgenic plants containing the nucleic acid molecules are also disclosed.

Description

PLANT RNA BINDING PROTEIN, ENCODING NUCLEIC ACIDS, AND METHODS

OF USE

[0001] STATEMENT OF GOVERNMENT INTEREST

[0002] Pursuant to 35 U.S.C. §200 et seq., it is acknowledged that the U.S. Government has certain rights in the invention described herein, which was made in part with funds from the National Science Foundation (MCB 98-74438 and MCB 00-86315).

[0003] CROSS-REFERENCE TO RELATED APPLICATIONS

[0004] This claims benefit of U.S. Provisional Application No. 60/400,549, filed August 1, 2002, the entirety of which is incorporated by reference herein.

[0005] FIELD OF THE INVENTION

[0006] This invention relates to abscisic acid (ABA) signaling in plants and more particularly to a novel RNA-binding protein whose mRNA binding is altered after its phosphorylation.

[0007] BACKGROUND OF THE INVENTION

[0008] Where referenced herein, patents and publications are incorporated by reference in their entireties.

[0009] In addition to the many environmental stresses plants face, they undergo a continual trade-off between maximizing CO₂ uptake for carbon fixation, and minimizing desiccating water loss. Thus, the ability to control stomatal opening and closure, for example, in response to stressors, is of tremendous agronomic significance.

[0010] Guard cells regulate the apertures of microscopic stomatal pores on the leaf epidermis through which plants take up CO and give off O₂ and water vapor. In response to environmental conditions, appropriate regulation of stomatal aperture is achieved by osmotic swelling and shrinking of guard cells, and is vital for plant productivity and drought resistance.

[0011] Environmental factors which influence the aperture of the plant's stomata include light conditions, relative humidity of the air, temperature, water status of the plant, CO₂ concentration, relative concentration of certain ions, and abscisic acid (ABA) concentration. [0012] Abscisic acid (ABA), is a phytohormone that mediates plant responses to stresses, including for example, cold, heat, salinity, synthetic and natural chemical agents, viral, fungal and bacterial pathogens, and drought. ABA is multifunctional; it is involved in a variety of important plant protective functions including bud dormancy, seed dormancy and/or maturation, abscission of leaves and fruits, in addition to its role in the response to biological stressors. It is also responsible for regulating stomatal closure by a mechanism independent of CO₂ concentration. ABA is synthesized rapidly in response to water stress in plants, and is stored in the guard cells. At the biochemical level, it is believed that the hormone initiates a variety of biological messages that require or include a protein phosphorylation cascade.

[0013] ABA has been shown to activate AAPK ("ABA-activated protein kinase"), a guard cell-specific kinase, previously shown to regulate plasma membrane ion channels. In earlier work, the present inventors employed de novo sequencing by tandem mass spectrometry to obtain peptide sequence information allowing the cloning of the DNA encoding the AAPK protein from Viciafaba, a food crop of major importance in the Middle East. Li et al, Science: 287: 300-303 (2000); see also commonly owned co-pending PCT Publication WO 01/02541. Expression of a dominant negative form of AAPK in guard cells prevented ABA-activation of anion channels as well as stomatal closure, implicating AAPK in rapid ABA signaling events in these specialized cells.

[0014] In addition to ABA-mediated responses, protein kinases are involved in stress signaling in plant, as well as animal, systems. Other stress responses are known. Dehydrins are stress-upregulated proteins thought to play a role as cellular protectants in response to drought and other stressors. They are ubiquitous stress-related proteins thought to improve enzyme and membrane stability under stress conditions. In addition, transcripts for dehydrins have been demonstrated to increase in abundance in ABA-treated Viciafaba guard cells.

[0015] Heterogenous nuclear RNA binding proteins (rrnRNPs) comprise a varied class of proteins involved in transcriptional, post-transcriptional, and translational control of gene expression. In particular, hnRNP A B RNA binding proteins are involved in alternative pre- mRNA splicing, telomere biogenesis, and mRNA trafficking. Many eukaryotic proteins that bind single-stranded RNA contain one or more RNA recognition motifs (RRMs), comprised of a loosely conserved region of about 90 amino acids. Stress-regulation of mammalian hnRNPs is a relatively new field (although insulin-regulation of hnRNP target affinity has been reported).

[0016] Little is known about the functional roles of plant hnRNPs. Assumptions based on sequence homology are the primary source of information available. One exception is UBPl hnRNP of Nicotiana plumbaginifolia, which has been shown to enhance the splicing of inefficiently processed introns. Mutations in other, non-hnRNP, types of RNA binding proteins have been shown to affect flowering and hormone sensitivity. One plant hnRNP, MAI 6 from maize, has been shown, by in vivo labeling with P-orthophosphate, to be phosphorylated, but its phosphorylation status had no effect on its subsequent ribohomopolymer affinity. Plant Mol. Biol., 29: 797-807 (1995).

[0017] ABA has long been known as a transcriptional regulator, and as a post- translational regulator of cellular and long-distance signaling in plants. Cloned DNAs encoding proteins that bind ribohomopolymers, and whose transcription is upregulated in response to ABA, have been identified in several species, however, the functions of these proteins remain unknown. However, recent reports also demonstrate altered plant sensitivity to ABA following mutation of a double-stranded RNA-binding protein, an Sm-like protein, and an mRNA cap protein.

[0018] Although an increased ABA sensitivity of plants with mutated RNA-binding proteins of certain types has been noted, there is a need for more specific understanding and control of stress-regulated hnRNP binding to specific target RNAs in plants. The art would be advanced through the isolation and characterization of particular genes and proteins involved in such stress regulated pathways.

[0019] SUMMARY OF THE INVENTION

[0020] Generally, the present invention provides, in several of its many aspects, an isolated nucleic acid molecule encoding a plant phosphorylation-regulated RNA binding protein, the phosphorylation of which is regulated by the plant hormone abscisic acid (ABA). Also provided in other embodiments is the ABA-regulated RNA binding protein encoded by the nucleic acid molecule. Methods for the use of the isolated nucleic acids and proteins are also provided herein.

[0021] In particular, the present invention, in one of its aspects, provides an isolated nucleic acid molecule encoding a plant RNA binding protein which is regulated by phosphorylation by a protein kinase; wherein the RNA binding protein comprises an RNA recognition motif selected from the group consisting of SEQ ID NOs: 22, 23, and 24, and wherein the RNA binding protein further comprises one or more sequences from the group consisting of SEQ ID NOs:25, 26, 27, 28, 29 and 30. These structural elements describe the nucleic acid molecules of the invention, relative to those encoding other plant proteins or those encoding nonplant RNA binding proteins. In particular embodiments, identifying structural elements preferred in the encoded protein include one or more of SEQ LD NOs: 26, 27, 29 and [0022] The encoded plant RNA binding proteins of particular interest, when expressed inplanta, are substrates for protein kinases. They are also substrates for such phosphorylation in vitro, and assays are available for measuring the phosphorylation in vitro. Certain preferred RNA binding proteins are substrates for ABA-regulated protein kinases. Other preferred RNA- binding proteins are substrates for particular ABA-regulated kinases, ABA-activated protein kinase (AAPK).

[0023] The phosphorylation-regulated RNA binding proteins encoded by the nucleic acid of the various embodiments exhibit altered ability to interact with RNA depending on the phosphorylation state. In certain embodiments, the altered interaction with RNA is a change in the binding affinity for a particular RNA, or RNA sequence, and the degree of expression of protein from a particular mRNA is altered, for example, expression may be enhanced or suppressed as a result of altered interaction with a particular mRNA. In an exemplary embodiment, the binding affinity for RNA increases upon phosphorylation of the RNA binding protein, with a concomitant increase in protein expression.

[0024] The invention provides isolated nucleic acid encoding plant RNA binding proteins as discussed above, and preferred are those wherein the plant is from a genus selected from the group of Vicia, Arabidopsis, Oryza, Lycopersicon, Solanum and Medicago. In more preferred embodiments, the plant is Viciafaba, Arabidopsis thaliana, Oryza sativa, Lycopersicon esculentum, Solanum tuberosum or Medicago sativa.

[0025] The invention also provides isolated nucleic acids encoding a plant RNA binding protein wherein the RNA recognition motif comprises SEQ ID NOs:23 or 24. In a more preferred embodiment, the RNA recognition motif is SEQ ID NO:24. The isolated nucleic acid molecule comprising SEQ ID NO:24 further comprises both SEQ ID NO:27 and SEQ ID NO:30 in more highly preferred embodiments.

[0026] The RNA binding protein encoded by the isolated nucleic acid molecules of the invention is preferably between about 400 to about 525 amino acids in length, in addition to having one or more of the properties as described above. The plant RNA binding protein having this length and these described properties includes all of the plant RNA binding protein which have been discovered to date, as well as others which will meet these criteria. In various embodiments the protein will have at least about 30% identity with the amino acid sequence, SEQ ID NO:2. More preferably the sequence will have at least about 40% identity. Even more preferable are sequences with at least about 50% identity or greater. In a particular embodiment, the invention provides an isolated nucleic acid molecule encoding an phosphorylation-regulated RNA binding protein which is phosphorylated by an ABA-activated protein kinase wherein the affinity of the encoded RNA binding protein for RNA increases upon phosphorylation, the nucleic acid molecule consisting of: (a) any of SEQ LD NOs:l, 3, 4, 6, 7, 9, 10, 12, 13, 14, 16, 18, and 20; (b) a sequence encoding a protein having an amino acid sequence of SEQ ID NO: 2, 5, 8, 11, 15, 17, 19, or 21; (c) a sequence encoding a protein having a sequence at least 50% identical to SEQ ID NO: 2; and (d) the complement of a sequence that hybridizes with any of SEQ ID NOs: 1, 3, 4, 6, 7, 9, 10, 12, 13, 14, 16, 18 and 20 under conditions comprising hybridization at 37-42°C in a solution comprising: 5X SSC, 5X Denhardt's reagent, 1.0% SDS, 100 μg/ml denatured, fragmented salmon sperm DNA, 0.05% sodium pyrophosphate and up to 50% formamide, and washing at 42-65° in IX SSC and 1% SDS.

[0027] In certain preferred embodiments, the nucleic ,acid is expressed in various host cells. In one preferred embodiment, the invention provides an isolated nucleic acid encoding a plant RNA binding, which when expressed in a plant, yields an RNA binding protein localized to the nucleus of a cell. More preferred are embodiments wherein the expressed RNA binding protein is further localized to nuclear speckles. Still more highly preferred embodiments include expressed RNA binding proteins which are localized to a nucleus and upon ABA treatment, are further localized to nuclear speckles as part of a subnuclear reorganization. The subnuclear reorganization preferably occurs rapidly upon phosphorylation and involves dynamic regulation of proteins in the cell, particularly in the presence of one or stimuli, for example various stressors, or a molecular stimulus in the form of a plant hormone.

[0028] In another of its aspects, the invention provides vectors comprising the isolated nucleic acid molecules of the invention. In particular embodiments, the vector is an expression vector that may be a plasmid, cosmid, baculovirus, bacmid, bacterial vector, yeast or other fungal vector, or viral vector.

[0029] In one embodiment, the vector contains a nucleic acid molecule encoding the RNA binding protein, the coding sequence operably linked to a constitutive promoter. In another, it is operably linked to an inducible promoter.

[0030] Host cells transformed with the vectors of the invention are also provided. These may be plant cells, bacterial cells, fungal cells, insect cells or mammalian cells. In one embodiment, the cell is a plant cell that may be obtained from any plant. Preferred plants are those of agronomic importance or scientific utility including food and feed crops, as well as ornamental plants, including, but not limited to, alfalfa, Arabidopsis, aster, barley, beans, beet, begonia, canola, carrot, chrysanthemum, clover, cotton, cucumber, delphinium, eggplant, fava bean, legumes, lettuce, maize, medicago, oats, pea, peanut, pepper, potato, rye, rice, safflower, sorghum, soybean, sugar beet, sunflower, tobacco, tomato, tomatillo, turfgrasses, wheat, and zinnia.

[0031] The invention, in another aspect, provides the vector wherein it is adapted for expression in the host cell. Such vectors are well-known in the art. Of particular interest are vectors adapted for expression in cells wherein the cells are plant cells. Again, the components for such vectors are known in the art and understood by the skilled artisan. Plant cells and transgenic plants comprising the described vector are also provided.

[0032] In one of its aspects, the invention provides a protein produced by the expression of the nucleic acids described above. Means for expressing proteins from the nucleic acids which encode them are well known to the skilled artisan. The protein may be expressed in any organism capable of expressing foreign proteins. Also provided herein are antibodies specific for the protein so expressed. Techniques for generating antibodies specific for epitopes of expressed proteins are routine in the art. Purification and uses for such antibodies are also routinely understood in the art.

[0033] In another presently preferred aspect, the invention provides an isolated nucleic acid molecule encoding a plant RNA binding protein wherein the binding of RNA by the encoded RNA binding protein is AB A-mediated, and further wherein accumulation of a transcript for an mRNA encoding the plant RNA binding protein is not necessarily mediated by ABA, wherein the encoded RNA binding protein comprises sequence SEQ ID NOs:31 or 32, and further comprises sequence SEQ ID NOs:33 or 34.

[0034] In another of its aspects, the invention also provides oligonucleotides having at least 15 consecutive nucleotides identical in sequence to a consecutive nucleotide sequence of the isolated nucleic acid molecule of the invention. Preferred are oligonucleotides wherein the isolated nucleic acid molecule is SEQ LD NOs: 1, 3, 4, 6, 7, 9, 10, 12, 13, 14, 16, 18, or 20.

[0035] In another of its aspects, the invention provides a genetically altered plant having altered response to ABA as compared with a genetically unaltered control plant, comprising an AB A-mediated phosphorylation-regulated RNA binding protein that is substantially nonfunctional or absent. In such a plant, the RNA binding protein may be completely absent, decreased in amount, decreased in activity (so as to be substantially biologically nonfunctional as compared to a control plant), or completely inactive (so as to be substantially incapable of, for example binding RNA, or being phosphorylated). The invention provides in a particular embodiment, the above plant produced by mutagenesis of the plant followed by a selection process, for example by screening for the desired product with an in vitro assay or other analysis. [0036] In another embodiment, the plant is produced by introducing a transgene into a plant cell wherein the transgene results in the plant cell's endogenous AB A-mediated, phosphorylation-regulated RNA binding protein becoming substantially nonfunctional or absent, and regenerating a plant from the cell containing the transgene. In preferred embodiments, the transgene disrupts the gene encoding the RNA binding protein, while in others the transgene is inducible. Inducible trangenes which result in RNA interference (RNAi), or in the presence of an antisense strand or double-stranded RNA effective for reducing the expression of the RNA binding protein are provided.

[0037] The invention, in another aspect, also provides a genetically altered plant having altered response to ABA as compared with an unaltered control plant, comprising an ABA- regulated RNA binding protein that is increased in amount or activity as compared with the control plant. In such a plant, the increase in amount can be through any means, for example through additional copies of the encoding gene, through increased transcription or translation, or through longer half-life or decreased turnover of the protein, or the mRNA encoding it, in the plant resulting in increased accumulation. The increase in activity can occur through any means, including such as by selecting for a mutant with altered phosphorylation properties or altered binding properties. The increase in activity can also result from altering other components in the pathway leading to or from the phosphorylation of the RNA binding protein, for example, in guard cells, this may be accomplished by altering the amount or activity of the kinase, AAPK. In other cell types, different kinases are expected to be involved in the phosphorylation of the RNA binding protein.

[0038] In certain embodiments, the genetically altered plant is produced by mutagenizing a population of plants and selecting a mutagenized plant with increased amount or activity of the RNA binding protein. The skilled artisan will appreciate that screening methods are readily adapted from the in vitro assays available.

[0039] The present invention also provides, in one aspect, a method for improving a plant's ABA-regulated response to a stressor comprising one or more of (a) altering the amount or activity of one or more phosphorylation-regulated RNA binding proteins in the plant; (b) altering the amount or activity of an ABA-activated protein kinase with respect to the phosphorylation-regulated RNA binding protein; and (c) altering a gene sequence encoding a transcript such that the transcript will be bound with altered affinity by the phosphorylation regulated RNA binding protein upon phosphorylation, thereby improving the plant's response to the stressor. Such a method combines the above described features to provide a wide range of means to utilizes the knowledge provided herein to its fullest extent. By describing the pathway of ABA activating the protein kinase, followed by recognition and phosphorylation of the RNA binding protein, resulting in mcreased binding affinity for particular transcripts, the skilled artisan is now provided a full understanding of what is needed to manipulate each of these steps to practice the method.

[0040] Preferred methods are practiced wherein the protein kinase is regulated by a hormone, and more particularly where the hormone is ABA. More preferred are methods wherein the protein kinase is ABA-activated protein kinase (AAPK) and the improved response is in the guard cells.

[0041] A method to alter the expression or activity of a stress-related protein in a plant, comprising altering the amount or activity of a phosphorylation-regulated RNA binding protein is also provided. A plant made by the method described herein has an altered expression or activity of a stress-related protein resulting from altering the amount or activity of the RNA binding protein, or of another related component as discussed above. Dehydrin biosynthesis is one example of a protein which is made in plants in response to stress. The RNA binding protein clearly binds this RNA with greater affinity upon phosphorylation of the RNA binding protein. Those of skill in the art will appreciate that the particular RNA sequences which are recognized and bound by the RNA binding protein can be determined. Such sequences may be useful for selectively designing other proteins which will have transcripts which are preferentially bound by the RNA binding protein upon phosphorylation, and thus it is now possible using the method of the present invention to design genes encoding proteins which will be preferentially expressed during stress. This method enhances what is already possible to do using, for example, stress- induced promoters for increasing expression by increasing the numbers of transcript.

[0042] In another of its aspects, the invention provides a method to alter ABA sensitivity in a plant, comprising increasing an amount or activity of AB A-mediated phosphorylation-regulated RNA binding protein in a plant, thereby altering sensitivity of the plant to ABA. In addition to broadly providing a method to manipulate ABA mediated stress responses in plant, it is equally possible to alter the plant's sensitivity to ABA according to the methods taught herein. For example, mutant plants may be selected wherein the RNA binding protein is more susceptible to phosphorylation by a protein kinase thus making the plant more sensitive to a given amount of ABA. Alternatively, a modified protein kinase, which is more susceptible to ABA may become activated and phosphorylate the RNA binding protein also tending to increase the plant's sensitivity to ABA. The method can likewise be used to decrease a plant's sensitivity to ABA by selecting for plants with RNA binding proteins which are less susceptible to phosphorylation, have attenuated binding affinity changes upon phosphorylation, or by selecting plants with ABA-activated kinase which require greater concentrations of ABA to become activated, or even selecting mutants, including negative dominant mutants of the protein kinase. The invention also provides the method wherein a transgene is used. Such a transgene can be used to either increase or decrease sensitivity to ABA, for example to provide an additional copy of the protein kinase or the RNA binding protein on a strong constitutive or selectively inducible promoter, or alternative to provide an antisense strand to the coding strand for either the protein kinase or the RNA binding protein. A transgene, or any foreign, for example transposons, T-DNA and the like, can also be used to knock out the protein kinase or the RNA binding protein to reduce or eliminate expression. Fertile plants made by the method are also provided as these are most useful for agronomic or research purposes.

[0043] The invention also provides in another aspect an isolated plant protein having RNA binding properties, wherein the protein is of a length of between about 400 and 525 amino acids, and further wherein the RNA binding properties are regulated by phosphorylation of the protein. The protein provided is novel, particularly for example, because phosphorylation- regulation of the type described herein is unique. The protein is found widely but presently preferred are those wherein the plant is from a genus selected from the group of Vicia, Arabidopsis, Oryza, Lycopersicon, Solanum and Medicago. In more preferred embodiments the plant is Viciafaba, Arabidopsis thaliana, Oryza sativa, Lycopersicon esculentum, Solanum tuberosum or Medicago sativa.

[0044] In preferred embodiments, the phosphorylation regulates RNA target discrimination. Preferably, the phosphorylation increases a binding affinity for RNA, most particularly, the phosphorylation results in the preferential binding of certain mRNA over other mRNA present locally in a cell. In a preferred embodiment, the mRNA encodes a stress-induced protein. In one embodiment the protein encoded is dehydrin, synthesized, or upregulated, for example, in response to water stress and other stress conditions.

[0045] In preferred embodiments, the phosphorylation of the plant protein is abscisic acid (ABA)-mediated. In this regard, the expression of the plant protein is not itself regulated or mediated by ABA, but rather the phosphorylation of the protein is mediated by ABA. Preferably, the ABA-mediation is via an ABA-activated protein kinase. In certain preferred embodiments, the protein kinase is ABA-activated protein kinase (AAPK). This is particularly true in guard cells.

[0046] The protein of the invention also comprises an RNA recognition motif. In particular, the RNA recognition motif corresponds, for example, to amino acids 151 and 228 of SEQ LD NO:2. such sequences are known in the art and resemble other RNA recognition motifs. More generally, the plant proteins of the invention have RNA recognition motif-like sequences, such as those of SEQ ID NO:22. Also preferred are those of SEQ ID NOs: 23, and 24.

[0047] In another preferred embodiment, the protein of the invention having the RNA recognition motif-like sequence of SEQ ID NO:22, 23 or 24, has less than about 40% identity in the RNA recognition motif-like sequence with a human hn RNA binding protein A/B. Also preferred are protein with less than about 39% to less than about 35% identity across this region. Proteins with less than about 35% identity but more than about 25% identity, for example those with 33% identity are also preferred.

[0048] In other preferred embodiments, the protein has the RNP2 and RNP 1 -like sequences of SEQ ID NOs: 31 or 32, and SEQ ID NOs:33 or 34 respectively. In yet other preferred embodiments, the protein further comprises one or more of the sequences 25, 26, 27, 28, 29 and 30. Most preferable are proteins comprising the RNA recognition motif-like sequence of SEQ ID NO:22, 23 or 24 and further comprising one each of SEQ ID NOs:25, 26, or 27, and SEQ ID NOs:28, 29, or 30. Still more highly preferred are proteins wherein the RNA recognition motif-like sequence is SEQ LD NO:22, and the protein further comprises SEQ ID NO:25 and SEQ LD NO:28.

[0049] Proteins with greater than about 45% identity to the region corresponding to amino acids 150 to 231 of SEQ DD NO:2 are preferred. More highly preferred are those proteins with greater than 50%, 55%, 60%, 65% idneitity across this region. Still more highly preferred are those proteins with at least about 75 - 80%, 81%, 82%, 83%, 84%, 85%, or more identity to this region.

[0050] Also preferred are proteins with greater than about 40% identity to the region corresponding to amino acids 246 - 331 of SEQ ID NO:2. More preferred are those proteins having at least about 45% -50% identity in this region. More highly preferred are proteins with at least about 60-65%, to at least about 70% identity in this region. Still more highly preferred are those proteins with greater than about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, or 79% identity in this region. Even more highly preferred are those proteins with greater than about 80% identity in this subsequence, for example 81%, 82% and the like.

[0051] The proteins of the invention also preferably have a sequence which corresponds to amino acids 416-435 of SEQ ID NO:2. Preferably, the proteins have greater than about 25%o identity in this region. More preferable are those proteins with at least about 30% identity in this region. Still more preferable are proteins with greater than about 50% identity in this region, and those with about at least 50-75% identity. Even more preferable are those proteins with greater than about 75% identity in this region, for example 80-90% identity is highly preferred in this region.

[0052] In another embodiment, the protein of the invention further comprises one or more domains consisting of greater than about 50% of a single amino acid. RNA binding proteins often encompass one or more auxiliary domains having a large proportion of a single amino acid. In other preferred embodiments, the domains contain at least about 60%, 65%, 70%), 75%o, or 78% of the single amino acid. Presently preferred are proteins with domains wherein the amino acid is glutamic acid for at least one of the one or more domains. While the domains abundant in an amino acid may be of any length, presently preferred are proteins wherein the at least one domain is at least about 20 or more amino acids in length. Also preferred are domains of at least about 25 or 30 amino acids in length.

[0053] Other features and advantages of the present invention will be understood by the drawings, detailed description and examples set forth herein.

[0054] BRIEF DESCRIPTION OF THE DRAWINGS

[0055] Figure 1.: AKIPl encodes a single-stranded RNA-binding protein.

[0056] The deduced amino acid sequence of AKLPl cDNA aligned (Clustal method) with several putative single-stranded RNA-binding proteins. A.t CAC00749 and O.s BAA90354 are from Arabidopsis thaliana and Oryza sativa sequencing projects; respectively. H.s. hnRNP A B and M.m AUF1 are from human (Homo sapiens) and mouse (Mus musculus) having GenBank Accession Nos. S17563 and NP-031542, respectively. Identities are in boxed in black shading and gaps are indicated with dashes. The numbers on the right refer to amino acid positions. RNP1 (SEQ ID NOs:31 or 31 ) and RNP2 (SEQ TD NOs:33 or 34 ) sequences are indicated.

[0057] Figure 2. AKIPl specifically interacts with AAPK in the yeast two-hybrid system.

[0058] Positive interactions between pairs are indicated by growth on media lacking histidine (Panel A, left), or adenine (Panel B, middle), and by the expression of β-galactosidase (Panel C, right). AAPK-CD and AAPK-CT represent the N terminal region including the entire catalytic domain of AAPK (residues 1-267 of the 349 amino acids) and the C-terminal region including the acidic region of AAPK (residues 263-349), respectively. Yeast SNF1 and SNF4 are interacting positive controls. [0059] Figure 3. AKIPl and AAPK localization in guard cells.

[0060] Panel a) AAPK-GFP localizes in the nucleus and cytoplasm.

[0061] Panel b) Brightfield image of a stomate showing the nucleus of left guard cell with gold particles evident.

[0062] Panel c) Fluorescence image corresponding to b) showing diffuse nuclear localization of AKLP1-GFP (no ABA treatment).

[0063] Representative guard cell nucleus expressing AKLPl-GFP at 0 and 20 min following incubation in

[0064] Panel d) control buffer (n = 19) or

[0065] Panel e) 50 μM ABA (n = 14).

[0066] Panel f) Number of AKL l-GFP labeled speckles following treatment with (n = 14) or without (n •= 19) 50 μM ABA. At 20 min. +ABA differs from Control at P < 0.05 (ANONA). Scale bar = 10 μm.

[0067] Figure 4. Phosphorylation of AKIPl by ABA-activated AAPK regulates AKIPl binding to dehydrin mRΝA.

[0068] Panel a) AAPK phosphorylates recombinant AKIPl in an ABA-dependent manner.

[0069] In vitro phosphorylation assay was performed as described.

[0070] Panel b) ABA-dependent phosphorylation of AKIPl occurs in vivo.

[0071] AKIPl was immunoprecipitated from ³²P-labeled guard cell protoplasts using AKIPl -specific antibodies. ABA was added at 25 μM with 0.25% DMSO as vehicle; DMSO alone had no effect.

[0072] Panel c) ABA treatment does not alter AKIPl protein abundance.

[0073] AKIPl was immunoprecipitated from guard cell protoplasts and detected by immunoblot. ABA treatment was as in b).

[0074] Figure 5.: Interaction of AAPK-phosphorylated AKIPl with dehydrin transcript.

[0075] Panel a) AAPK-phosphorylated AKIPl binds dehydrin mRΝA. [0076] RNA binding assay was performed as described in Methods. A ~300 bp dehydrin PCR product was amplified from RNAs bound to AAPK-phosphorylated AKTPl (lane 6, arrowhead). Molecular weight markers are shown on the right.

[0077] Panel b) AAPK-phosphorylated AKIPl specifically interacts with sense dehydrin RNA.

[0078] ³²P-labeled sense dehydrin RNA was incubated with AKIP 1 -GST previously treated with inactive AAPK (lane 1), active AAPK (lanes 2), or active CDPK (lane 3). Alternatively, AKTPl treated with active AAPK was incubated with ³²P-labeled sense dehydrin RNA (lane 4), or first incubated with an excess of unlabelled (competitor) sense dehydrin then with ³²P-labeled sense dehydrin RNA (lane 5), or with unrelated ³²P-labeled RNA (non- competitor) from the RNA synthesis kit (lane 6), or with ³²P-labeled antisense dehydrin RNA (lane 7). Binding of ³²P-labeled RNA to AKIPl was visualized by autoradiography.

[0079] Figure 6.: ABA Upregulates dehydrin transcript in Viciafaba [0080] Total RNA was isolated from purified V. faba guard cells treated with (Lane 1) or without (Lane 2) 25 μM ABA for 15 minutes. Dehydrin mRNA level was detected bynothern blot analysis using a V. faba dehydrin gene fragment as a probe. The top panel (Panel A) shows the northern blot and the dehydrin transcript (denoted by arrowhead). The bottom panel (Panel B) shows equal loading of RNA on the corresponding agarose gel.

[0081] Figure 7.: AKIPl is expressed in a wide variety of tissues.

[0082] Reverse transcriptase polymerase chain reaction (RT-PCR) data showing AKTPl mRNA expression in guard cell protoplasts (GCP) (lane 1), leaf (lane 2), root (lane 3) and stem (lane 4) tissues. Arrowhead denotes the position of AKIPl.

[0083] Figure 8.: AKIPl protein sequence homologs [0084] Alignment of AKIP 1 protein sequence homologs (AKIP 1 -like proteins, AKLP1LP) from various higher plants.

[0085] DETAILED DESCRIPTION

[0086] Definitions:

[0087] Various terms relating to the biological molecules and other aspects of the present invention are used throughout the specification and claims. These terms should be given the broadest reasonable meaning wherever possible except as specified herein. In interpreting the appended claims and the specification the definitions provided herein are to be used with the specific terms where the broadest meaning cannot be used or is not meaningful.

[0088] As used herein, "phosphorylation-regulated RNA binding protein" refers to a RNA binding protein that is a substrate for a kinase, typically a protein kinase, and is capable of being phosphorylated under biological conditions by such a kinase. In response to being phosphorylated, some property of the RNA binding protein, including, but not limited to affinity for RNA is altered. Specific phosphorylation-regulated RNA binding proteins are sometimes referred herein as AKIPl or AKIPl-like proteins (AKIPllp or AKIPl-lp).

[0089] As used herein, "abscisic acid" often abbreviated "ABA" refers to a compound which acts as a plant hormone inplanta and includes salts and derivatives which so act. It is understood that as used herein, concentrations or amounts of ABA which are reflective of those that are biologically relevant are most desirable, recognizing that local concentrations of ABA in a cell are difficult to accurately assess. Thus a wide range of ABA concentrations or amounts are contemplated to be useful in the various embodiments of the invention. Exogenously provided ABA for plants, in vitro assays and the like, use concentrations of ABA as practiced in the art if not specifically noted.

[0090] As used herein, "ABA-regulated" or "ABA-mediated" refer to biological phenomena that are affected in some way, directly or indirectly, by the presence or absence of ABA in one of its biologically compatible forms at a biologically relevant concentration. The ABA-mediated phosphorylation-regulated RNA binding protein is indirectly mediated by ABA through an ABA-activated protein kinase. "ABA-activated", as used herein implies a more direct effect of ABA on the subject modified by the term. A specific ABA-mediated phosphorylation-regulated RNA binding protein from Viciafaba is sometimes referred to herein as "AKIPl".

[0091] As used herein, "altered" means changed in some way. Where used with respect to binding, altered can refer to changes in various binding properties, including, but not limited to affinity, avidity, specificity, recognition sequence, and the like. Where used with respect to sequences, the term can refer to changes as are recognized in the art, for example insertions, deletions and substitutions, as well as modifications.

[0092] As used herein, "RNA recognition motif refers to art-described protein motifs of particular sequences. Also used herein is the closely related "RNA recognition motif-like sequence" which are closely related by include additional sequence which more descriptively distinguishes the RNA binding proteins of the instant invention from the prior art. [0093] "Isolated" means altered "by the hand of man" from the natural state. If a composition or substance^~occurs in nature, it has been "isolated" if it has been changed or removed from its original environment, or both. For example, a polynucleotide or a polypeptide naturally present in a living plant or animal is not "isolated," but the same polynucleotide or polypeptide separated from the coexisting materials of its natural state is "isolated", as the term is employed herein.

[0094] "Polynucleotide", primarily referred to herein as "nucleic acid molecule", generally refers to any polyribonucleotide or polydeoxribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. "Polynucleotides" include, without limitation single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions. In addition, "polynucleotide" refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA. The term polynucleotide also includes DNAs or RNAs containing one or more modified bases and DNAs or RNAs with backbones modified for stability or for other reasons. "Modified" bases include, for example, tritylated bases and unusual bases such as inosine. A variety of modifications can be made to DNA and RNA; thus, "polynucleotide" embraces chemically, enzymatically or metabolically modified forms of polynucleotides as typically found in nature, as well as the chemical forms of DNA and RNA characteristic of viruses and cells. "Polynucleotide" also embraces relatively short polynucleotides, often referred to as oligonucleotides.

[0095] "Protein" refers to any peptide or polypeptide comprising two or more amino acids joined to each other by peptide bonds or modified peptide bonds, i.e., peptide isosteres. Sometimes used interchangeably with "polypeptide," which refers to both short chains, commonly referred to as peptides, oligopeptides or oligomers, and to longer chains, generally referred to as proteins. Polypeptides and proteins may contain amino acids other than the 20 gene-encoded amino acids. Both include amino acid sequences modified either by natural processes, such as posttranslational processing, or by chemical modification techniques which are well known in the art. Such modifications are well described in basic texts and in more detailed monographs, as well as in a voluminous research literature. Modifications can occur anywhere in such a sequence, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini. It will be appreciated that the same type of modification may be present in the same or varying degrees at several sites in a given polypeptide. Also, a given polypeptide may contain many types of modifications. Polypeptides may be branched as a result of ubiquitination, and they may be cyclic, with or without branching. Cyclic, branched and branched cyclic polypeptides may result from natural posttranslational processes or may be made by synthetic methods. Modifications include acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cystine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination. See, for instance, Proteins - Structure and Molecular Properties, 2nd Ed., T. E. Creighton, W. H. Freeman and Company, New York, 1993 and Wold, F., Posttranslational Protein Modifications: Perspectives and Prospects, pgs. 1-12 in Posttranslational Covalent Modification of Proteins, B. C. Johnson, Ed., Academic Press, New York, 1983; Seifter et al., "Analysis for Protein Modifications and Nonprotein Cofactors", Meth Enzymol (1990) 182:626-646 and Rattan et al., "Protein Synthesis: Posttranslational Modifications and Aging", Ann NY Acad Sci (1992) 663:48-62.

[0096] "Variant" as the term is used herein, is a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide respectively, but retains essential properties. A typical variant of a polynucleotide differs in nucleotide sequence from another, reference polynucleotide. Changes in the nucleotide sequence of the variant may or may not alter the amino acid sequence of a polypeptide encoded by the reference polynucleotide. Nucleotide changes may result in amino acid substitutions, additions, deletions, fusions and truncations in the polypeptide encoded by the reference sequence, as discussed below. A typical variant of a polypeptide differs in amino acid sequence from another, reference polypeptide. Generally, differences are limited so that the sequences of the reference polypeptide and the variant are closely similar overall and, in many regions, identical. A variant and reference polypeptide may differ in amino acid sequence by one or more substitutions, additions, deletions in any combination. A substituted or inserted amino acid residue may or may not be one encoded by the genetic code. A variant of a polynucleotide or polypeptide may be naturally occurring, such as an allelic variant, or it may be a variant that is not known to occur naturally. Non-naturally occurring variants of polynucleotides and polypeptides may be made by mutagenesis techniques or by direct synthesis. [0097] In reference to mutant plants, the terms "null mutant" or "loss-of-function mutant" are used to designate an organism or genomic DNA sequence with a mutation that causes the product of the RNA binding protein-encoding gene to be substantially non-functional or largely absent. Such mutations may occur in the coding and/or regulatory regions of the gene, 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 RNA binding protein-encoding gene and/or the RNA binding protein, so as to cause the protein itself to be substantially non-functional or largely absent.

[0098] The term "substantially the same" refers to nucleic acid or amino acid sequences having sequence variations that do not materially affect the nature of the protein (i.e. the structure, stability characteristics, substrate specificity and/or biological activity of the protein). With particular reference to nucleic acid sequences, the term "substantially the same" is intended to refer to the coding region and to conserved sequences governing expression, and includes degenerate codons encoding the same amino acid, or alternate codons encoding conservative substitute amino acids in the encoded polypeptide. With reference to amino acid sequences, the term "substantially the same" refers generally to conservative substitutions and/or variations in regions of the polypeptide not involved in determination of structure or function.

[0099] The terms "percent identical" and "percent similar" are also used herein in comparisons among amino acid and nucleic acid sequences. When referring to amino acid sequences, "identity" or "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 in Taylor (1986, J. Theor. Biol. 119:205). When referring to nucleic acid molecules, "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.

[0100] "Identity" and "similarity" can be readily calculated by known methods. Nucleic acid sequences and amino acid sequences can be compared using computer programs that align the similar sequences of the nucleic or amino acids and thus define the differences. In preferred methodologies, the BLAST programs (NCBI) and parameters used therein are employed, and the DNAstar system (Madison, WI) is used to align sequence fragments of genomic DNA sequences. However, equivalent alignments and similarity/identity assessments can be obtained through the use of any standard alignment software. For instance, the GCG Wisconsin Package version 9.1, available from the Genetics Computer Group in Madison, Wisconsin, and the default parameters used (gap creation penalty= 12, gap extension penalty=4) by that program may also be used to compare sequence identity and similarity.

[0101] "Antibodies" as used herein includes polyclonal and monoclonal antibodies, chimeric, single chain, and humanized antibodies, as well as Fab fragments, including the products of an Fab or other immunoglobulin expression library. With respect to antibodies, the term, "specific" or "immunologically specific" refers 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 antigenic biological molecules.

[0102] The term "substantially pure" refers to a preparation comprising at least 50-60% by weight the compound of interest (e.g., nucleic acid, ohgonucleotide, protein, etc.). More preferably, the preparation comprises at least 75% by weight, and most preferably 90-99% by weight, the compound of interest. Purity is measured by methods appropriate for the compound of interest (e.g. chromatographic methods, agarose or polyacrylamide gel electrophoresis, HPLC analysis, and the like).

[0103] With respect to single-stranded nucleic acid 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"). In particular, the term refers to hybridization of an ohgonucleotide with a substantially complementary sequence contained within a single-stranded DNA or RNA molecule, to the substantial exclusion of hybridization of the ohgonucleotide with single-stranded nucleic acids of non-complementary sequence.

[0104] 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.

[0105] The term "operably linked" or "operably inserted" 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 other transcription control elements (e.g. enhancers) in an expression vector. [0106] 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.

[0107] The terms "promoter", "promoter region" or "promoter sequence" refer 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. Typically, 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. Within the promoter sequence is 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.

[0108] 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.

[0109] The term "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" or "transgene". Such 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.

[0110] The term "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.

[0111] The term "reporter gene" refers to a gene that encodes a product which is easily detectable by standard methods, either directly or indirectly.

[0112] 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. Thus, 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. In another example, 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.

[0113] 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. In prokaryotes, yeast, and mammalian cells for example, the transforming DNA may be maintained on an episomal element such as a plasmid. With respect to eukaryotic cells, 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 transfoπmng 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.

[0114] As used herein, the term "localized", for example when referring to nuclear "localized" refers to an abundance or majority of a molecule detectable in a particular location, although some portion of that molecule may be detectable in other locations, including nonnuclear locations.

[0115] Description

[0116] In accordance with the present invention, a phosphorylation-regulated RNA binding protein has been identified from plants. Of particular relevance is that the phosphorylation of this phosphorylation-regulated RNA protein alters its RNA binding properties; specifically, phosphorylation increases its binding affinity for certain mRNA molecules. Also of particular interest is that the phosphorylation of the RNA binding protein is mediated by a hormone, ABA, through its action on a ABA-activated protein kinase. The phosphorylation-regulated RNA binding protein has been named AKTPl and is a member of a class of AKTPl -like proteins which has now been identified in plants in accordance with the invention. The genes encoding the proteins have also been identified and are provided herein.

[0117] Accordingly, one aspect of the present invention relates to a nucleic acid molecule encoding this ABA-mediated phosphorylation-regulated RNA binding protein, AKTPl. An exemplary nucleic acid molecule of the invention is that of Viciafaba. Also exemplified are homologs of the gene in Arabidopsis, Oryza, Lycopersicon, Solanum, and Medicago. The invention further provides homologs of the exemplified AKIPl, having a level of nucleotide sequence or amino acid sequence identity with the exemplified AKIPl nucleic acids or encoded AKIPl proteins, specifically at certain regions of the coding sequence, that clearly distinguish the homologs as AKTPl homologs, as opposed to other types of RNA-binding proteins.

[0118] As summarized above, the invention features an isolated nucleic acid molecule encoding a plant RNA binding protein which is regulated by phosphorylation by a protein kinase; wherein the RNA binding protein comprises an RNA recognition motif selected from the group consisting of SEQ ID NOs: 22, 23, and 24, and wherein the RNA binding protein further comprises one or more sequences from the group consisting of SEQ ID NOs:25, 26, 27, 28, 29 and 30. The skilled artisan will appreciate that the class of nucleic acid molecules provided includes plant phosphorylation-regulated RNA binding proteins, but does not include those of mammalian origin, such as the human or mouse hnRNP A B proteins. The nucleic acid molecule comprises identifying subsequences, which represent consensus sequences derived from the phosphorylation-regulated RNA binding proteins from plants. These structural elements serve to describe fully the nucleic acids of the invention, particularly relative to those encoding other proteins or encoding nonplant RNA binding proteins. In particular embodiments, identifying structural elements in the encoded protein include SEQ LD NOs: 26, 27, 29 and 30.

[0119] The encoded plant RNA binding proteins are substrates for phosphorylation by protein kinases. They are also substrates for such phosphorylation in vitro; such assays are known in the art and readily available for measuring the phosphorylation of the encoded protein in vitro. Certain preferred RNA binding proteins are substrates for ABA-regulated protein kinases. Other preferred RNA-binding proteins are substrates for particular ABA-regulated kinases, for example, ABA-activated protein kinase (AAPK) of guard cells. Because AAPK is thus far only known to be expressed in guard cells, while the phosphorylation-regulated RNA binding proteins, such as AKLPl and the like are expressed in tissues throughout the plant, phosphorylation by ABA-activated protein kinase is considered exemplary but not exclusive of the means by which the encoded protein is phosphorylated.

[0120] The phosphorylation-regulated RNA binding proteins encoded by the nucleic acid of the various embodiments exhibit altered ability to interact with RNA depending on the phosphorylation state. It is to be appreciated that while a preferred altered interaction with RNA is a change in the binding affinity for a particular RNA, or RNA sequence, and the degree of expression of protein from a particular mRNA is altered, for example, expression may be enhanced or suppressed as a result of altered interaction with a particular mRNA, other alteration are contemplated herein. In an exemplary embodiment, the binding affinity for RNA increases upon phosphorylation of the RNA binding protein, with a concomitant increase in protein expression. In other embodiments changes in the preferred binding sequence, avidity or other parameters of binding occur on phosphorylation. In still others, binding of particular transcripts decreases upon phosphorylation to ensure sufficient expression of proteins required by the cell under stress conditions.

[0121] The invention provides isolated nucleic acid encoding plant RNA binding proteins as discussed above, including but not limited to those wherein the plant is from a genus selected from the group of Vicia, Arabidopsis, Oryza, Lycopersicon, Solanum and Medicago. In certain embodiments the plant is Viciafaba, Arabidopsis thaliana, Oryza sativa, Lycopersicon esculentum, Solanum tuberosum or Medicago sativa. Most exemplary is the nucleic acid molecule encoding the ABA-mediated phosphorylation-regulated RNA binding protein from V. faba.

[0122] Isolated nucleic acid molecules encoding a plant RNA binding protein wherein the RNA recognition motif comprises SEQ ID NOs:23 or 24 are also provided herein. In more preferred embodiments, the RNA recognition motif is SEQ ID NO:24. The isolated nucleic acid molecule comprising SEQ JD NO:24 further comprises both SEQ ID NO:27 and SEQ ID NO:30 in more highly preferred embodiments.

[0123] The RNA binding protein encoded by the isolated nucleic acid molecules of the invention is preferably between about 400 to about 525 amino acids in length, in addition to having one or more of the properties as described above. Longer encoded polypeptides are also contemplated, particularly where they encode a chimeric protein, for example a RNA binding protein attached to or combined with a green fluorescent protein. Such proteins have been formed and are useful for the study of the localization of the encoded protein, and particular for the study of nuclear and subnuclear reorganization events. For example, such fusion proteins, and others are useful for the study of the formation of nuclear speckles and other cellular foci.

[0124] The class of plant RNA binding proteins having the length of about 400 to about 525 amino acids and the other described elements includes all of the plant RNA binding protein which have been discovered to date, as well as others, as yet unknown, which will meet these criteria. In a particular embodiment, the invention provides an isolated nucleic acid molecule encoding an phosphorylation-regulated RNA binding protein which is phosphorylated by an ABA-activated protein kinase wherein the affinity of the encoded RNA binding protein for RNA increases upon phosphorylation consisting of: (a) any of SEQ ID NOs:l, 3, 4, 6, 7, 9, 10, 12, 13, 14, 16, 18, and 20; (b) a sequence encoding a protein having an amino acid sequence of SEQ ID NO: 2, 5, 8, 11, 15, 17, 19, or 21; (c) a sequence encoding a protein having a sequence at least 50% identical to SEQ ID NO: 2; and (d) the complement of a sequence that hybridizes with any of SEQ ID NOs: 1, 3, 4, 6, 7, 9, 10, 12, 13, 14, 16, 18 and 20 under conditions comprising hybridization at 37-42°C in a solution comprising: 5X SSC, 5X Denhardt's reagent, 1.0% SDS, 100 μg/ml denatured, fragmented salmon sperm DNA, 0.05% sodium pyrophosphate and up to 50% formamide, and washing at 42-65° in IX SSC and 1% SDS. Other conditions of stringency may also be used in accordance with the present invention to obtain and identify members of the class of claimed nucleic acids, h particular, nucleic acids having the appropriate level of sequence homology with part or all of the coding the AKIPl -like protein-encoding polynucleotides may be identified by using hybridization and washing conditions of appropriate stringency. Since the AKIPl -like proteins are similar to each other in sequence yet different from the human and mouse sequences, a preferred nucleic acid segment for hybridization is the RNA recognition motif-like sequence SEQ LD NO:22.

[0125] As a typical illustration, hybridizations may be performed, according to the method of Sambrook et al., using a hybridization solution comprising: 5X SSC, 5X Denhardt's reagent, 1.0% SDS, 100 μg/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. Following hybridization, 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 2X SSC and 0.1% SDS; (4) 2 hours at 45-55°C in 2X SSC and 0.1% SDS, changing the solution every 30 minutes.

[0126] One common formula for calculating the stringency conditions required to achieve hybridization between nucleic acid molecules of a specified sequence homology (Sambrook et al., 1989):

[0127] T_m = 81.5°C + 16.6Log [Na+] + 0.41(% G+C) - 0.63 (% formamide) - 600/#bp in duplex

[0128] As an illustration of the above formula, using [Na+] = [0.368] and 50% formamide, with GC content of 42% and an average probe size of 200 bases, the Tm is 57°C. The T_m of a DNA duplex decreases by 1 - 1.5 °C with every 1% decrease in homology. Thus, targets with greater than about 75%> sequence identity would be observed using a hybridization temperature of 42°C. In one embodiment, the hybridization is at 37°C and the final wash is at 42°C; in another embodiment the hybridization is at 42°C and the final wash is at 50°C; and in yet another embodiment the hybridization is at 42°C and final wash is at 65°C, with the above hybridization and wash solutions. Conditions of high stringency include hybridization at 42°C in the above hybridization solution and a final wash at 65°C in 0.1X SSC and 0.1% SDS for 10 minutes.

[0129] Nucleic acids of the present invention may be maintained as DNA in any convenient cloning vector. In a preferred embodiment, clones are maintained in plasmid cloning/expression vector, such as pGEM-T (Promega Biotech, Madison, WI) or pBluescript (Stratagene, La Jolla, CA), either of which is propagated in a suitable E. coli host cell.

[0130] In certain preferred embodiments, the nucleic acid is expressed in various host cells, including plant cells. In one preferred embodiment, the invention provides a isolated nucleic acid encoding a plant RNA binding protein, which when expressed in a plant, yields an RNA binding protein localized to the nucleus of a cell. Thus certain targeting information may be present to properly direct the protein within the subcellular protein traffic. Also preferred are embodiments wherein the expressed RNA binding protein is further localized to nuclear speckles, or the like. The skilled artisan will appreciate that the terminology for subnuclear foci may not be fully developed and will further appreciate that localization of the protein to subnuclear foci is preferred regardless of the terminology describing the sublocation. Still more highly preferred embodiments include expressed RNA binding proteins which are localized to a nucleus and upon phosphorylation of the RNA binding protein, are further localized to nuclear speckles as part of a subnuclear reorganization. The subnuclear reorganization preferably occurs rapidly upon phosphorylation and involves dynamic regulation of proteins in the cell, particularly in the presence of one or more stimuli, for example a stressor, or a molecular stimulus in the form of a plant hormone, either endogenous or exogenous. It is also contemplated that as ABA is capable of inducing these nuclear reorganizations, other signals or compounds may also be involved in such changes either before, after or independent of ABA's action.

[0131] In another of its aspects, the invention provides vectors comprising the isolated nucleic acid molecules of the invention. In particular embodiments, the vector is an expression vector that may be a plasmid, cosmid, baculovirus, bacmid, bacterial artificial chromosome (BAC), yeast artificial chromosome (YAC), or other bacterial vector, yeast or other fungal vector, or viral vector.

[0132] In one embodiment, the vector contains a nucleic acid molecule encoding the RNA binding protein, the coding sequence operably linked to a constitutive promoter. In another, it is operably linked to an inducible promoter. [0133] Host cells transformed with the vectors of the invention are also provided. These may be plant cells, bacterial cells, fungal cells, insect cells or mammalian cells. In one embodiment, the cell is a plant cell that may be obtained from any plant. Preferred plants are those of agronomic importance or scientific utility including food and feed crops, as well as ornamental plants including but not limited to alfalfa, Arabidopsis, aster, barley, beans, beet, begonia, canola, carrot, chrysanthemum, clover, cotton, cucumber, delphinium, eggplant, fava bean, legumes, lettuce, maize, medicago, oats, pea, peanut, pepper, potato, rye, rice, safflower, sorghum, soybean, sugar beet, sunflower, tobacco, tomato, tomatillo, turfgrasses, wheat, and zinnia.

[0134] The invention, in another aspect, provides the vector wherein it is adapted for expression in the host cell. Such vectors are well-known in the art. Of particular interest are vectors adapted fro expression in cells wherein the cells are plant cells. Again the components for such vectors are known in the art and understood by the skilled artisan. Plant cells and transgenic plants comprising the described vector are also provided.

[0135] In one of its aspects, the invention provides a protein produced by the expression of the nucleic acids described above. Means for expressing proteins from the nucleic acids which encode them are well known to the skilled artisan. The protein may be expressed in any organism capable of expressing foreign proteins. Also provided herein are antibodies specific for the protein so expressed. Techniques for generating antibodies specific for epitopes of expressed proteins are routine in the art.

[0136] In another presently preferred aspect, the invention provides an isolated nucleic acid molecule encoding a plant RNA binding protein wherein the binding of RNA by the encoded RNA binding protein is ABA-mediated, and further wherein accumulation of a transcript for an mRNA encoding the plant RNA binding protein is not mediated by ABA, wherein the encoded RNA binding protein comprises sequence SEQ ID NOs:31 or 32, and further comprises sequence SEQ ID NOs:33 or 34.

[0137] In another of its aspects, the invention also provides oligonucleotides having at least 15 consecutive nucleotides identical in sequence to a consecutive nucleotide sequence of the isolated nucleic acid molecule of the invention. Thus, 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 of the present invention. Such oligonucleotides are useful as probes for detecting phosphorylation-regulated RNA binding protein-encoding genes or mRNA in test samples of plant tissue, e.g. by PCR amplification, or for the positive or negative regulation of expression of these encoding genes at or before translation of the mRNA into proteins. Methods in which the oligonucleotides or polynucleotides 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) and ligase chain reaction (LCR).

[0138] Preferred are oligonucleotides wherein the isolated nucleic acid molecule is SEQ ID NOs: 1, 3, 4, 6, 7, 9, 10, 12, 13, 14, 16, 18, or 20.

[0139] In another of its aspects, the invention provides a genetically altered plant having altered response to ABA as compared with a genetically unaltered control plant, comprising an ABA-mediated phosphorylation-regulated RNA binding protein that is substantially nonfunctional or absent. In such a plant, the RNA binding protein may be completely absent, decreased in amount, decreased in activity (so as to be substantially biologically nonfunctional as compared to a control plant), or completely inactive (so as to be substantially incapable of, for example binding RNA, or being phosphorylated). The invention provides in a particular embodiment, the above plant produced by mutagenesis of the plant followed by a selection process, for example by screening for the desired product with an in vitro assay or other analysis. Preferred plants are those of agronomic importance or scientific utility including food and feed crops, as well as ornamental plants including but not limited to alfalfa, Arabidopsis, aster, barley, beans, beet, begonia, canola, carrot, chrysanthemum, clover, cotton, cucumber, delphinium, eggplant, fava bean, legumes, lettuce, maize, medicago, oats, pea, peanut, pepper, potato, rye, rice, safflower, sorghum, soybean, sugar beet, sunflower, tobacco, tomato, tomatillo, turfgrasses, wheat, and zinnia.

[0140] In another embodiment, the plant is produced by introducing a transgene into a plant cell wherein the transgene results in the plant cell's endogenous ABA-mediated, phosphorylation-regulated RNA binding protein becoming substantially nonfunctional or absent, and regenerating a plant from the cell containing the transgene. In preferred embodiments, the transgene disrupts the gene encoding the RNA binding protein, while in others the transgene is inducible. Inducible trangenes which result in the presence of an antisense strand effective for reducing the expression of the RNA binding protein are provided.

[0141] Transgenic plants can be generated using standard plant transformation methods known to those skilled in the art. These include, but are not limited to, Agrobacterium vectors, polyethylene glycol treatment of protoplasts, biolistic DNA delivery, UV laser microbeam, gemini virus vectors or other plant viral 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 transfomiing DNA, direct DNA uptake, liposome-mediated DNA uptake, and the like. Such methods have been published in the art. See, e.g., Methods for Plant Molecular Biology (Weissbach & Weissbach, eds., 1988); Methods in Plant Molecular Biology (Schuler & Zielinski, eds., 1989); Plant Molecular Biology Manual (Gelvin, Schilperoort, Verma, eds., 1993); and Methods in Plant Molecular Biology - A Laboratory Manual (Maliga, Klessig, Cashmore, Gruissem & Varner, eds., 1994).

[0142] The method of transformation depends upon the plant to be transformed. Agrobacterium vectors are often used to transform dicot species. Agrobacterium binary vectors include, but are not limited to, BIN19 and derivatives thereof, the pBI vector series, and binary- vectors pGA482 and pGA492 . For transformation of monocot species, biolistic bombardment with particles coated with transforming DNA and silicon fibers coated with transforming DNA are often useful for nuclear transformation. Alternatively, Agrobacterium "superbinary" vectors have been used successfully for the transformation of rice, maize and various other monocot species.

[0143] 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). In a preferred embodiment, a RNA binding protein coding sequence under control of its own 5' and 3' regulatory elements is utilized.

[0144] In an alternative embodiment, the coding region of the gene is placed under a powerful constitutive promoter, such as the Cauliflower Mosaic Virus (CaMV) 35S promoter or the figwort mosaic virus 35S promoter. Other constitutive promoters contemplated for use in the present invention include, but are not limited to: T-DNA mannopine synthetase, nopaline synthase and octopine synthase promoters. In other embodiments, a strong monocot promoter is used, for example, the maize ubiquitin promoter, the rice actin promoter or the rice tubulin promoter (Jeon et al., Plant Physiology. 123: 1005-14, 2000).

[0145] Transgenic plants expressing AKL 1 -like RNA binding protein coding sequences 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), chemically-inducible gene promoters (e.g., nitrate reductase genes, glucanase genes, chitinase genes, etc.) and dark- inducible gene promoters (e.g., asparagine synthetase gene) to name a few. Stress-indiuced promoters are well-known in the art and are preferred for certain embodiments for use herein.

[0146] Tissue specific and development-specific promoters are also contemplated for use in the present invention. Examples of these include, but are not limited to: the ribulose bisphosphate carboxylase (RuBisCo) small subunit gene promoters or chlorophyll ab 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.

[0147] In another embodiment, a phosphorylation-regulated RNA-binding protein coding region is operably linked to a heterologous promoter that is either generally stress inducible (i.e. inducible upon challenge by a broad range of stressors) or inducible by drought or other specific stressor.

[0148] The coding region is also operably linked to an appropriate 3' regulatory sequence. In a preferred embodiment, the nopaline synthetase polyadenylation region is used. Other useful 3' regulatory regions include, but are not limited to the octopine polyadenylation region.

[0149] Using an Agrobacterium binary vector system for transformation, the selected coding region, under control of appropriate regulatory elements, is linked to 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 or herbicide resistances (e.g., resistance to hygromycin or bialaphos) or herbicide resistance (e.g., resistance to sulfonylurea, phosphinothricin, or glyphosate).

[0150] Plants are transformed and thereafter screened for one or more properties, including the presence of AKIPl -like protein, AKIPl -like protein-encoding mRNA, in vitro phosphorylation assays, for example using antibodies to AKTPl -like protein, or altered sensitivity to ABA, or various stressors. 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.

[0151] The invention, in another aspect, also provides a genetically altered plant having altered response to ABA as compared with an unaltered control plant, comprising an ABA- regulated RNA binding protein that is increased in amount or activity as compared with the control plant. Preferred plants are those of agronomic importance or scientific utility including food and feed crops, as well as ornamental plants including but not limited to alfalfa, Arabidopsis, aster, barley, beans, beet, begonia, canola, carrot, chrysanthemum, clover, cotton, cucumber, delphinium, eggplant, fava bean, legumes, lettuce, maize, medicago, oats, pea, peanut, pepper, potato, rye, rice, safflower, sorghum, soybean, sugar beet, sunflower, tobacco, tomato, tomatillo, turfgrasses, wheat, and zinnia. In such a plant, the increase in amount can be through any means, for example through additional copies of the encoding gene, through increased transcription or translation, or through longer half-life or decreased turnover of the protein in the plant resulting in increased accumulation. The increase in activity can occur through any means, including such as by selecting for a mutant with altered phosphorylation properties or altered binding properties. The increase in activity can also result from altering other components in the pathway leading to or from the phosphorylation of the RNA binding protein, for example, in guard cells, this may be accomplished by altering the amount or activity of the kinase, AAPK. In other cell types, different kinases are expected to be involved in the phosphorylation of the RNA binding protein. These kinases may be directly or indirectly activated by ABA, or by stress.

[0152] In certain embodiments, the genetically altered plant is produced by mutagenizing a population of plants and selecting a mutagenized plant with increased amount or activity of the RNA binding protein. The skilled artisan will appreciate that screening methods are readily adapted from the in vitro assays available.

[0153] The present invention also provides, in one aspect, a method for improving a plant's ABA-regulated response to a stressor comprising one or more of (a) altering the amount or activity of one or more phosphorylation-regulated RNA binding proteins in the plant; (b) altering the amount or activity of an ABA-activated protein kinase with respect to the phosphorylation-regulated RNA binding protein; and (c) altering a gene sequence encoding a transcript such that the transcript will be bound with altered affinity by the phosphorylation regulated RNA binding protein upon phosphorylation, thereby improving the plant's response to the stressor. Such a method combines the above described features to provide a wide range of means to utilizes the knowledge provided herein to its fullest extent. By describing the pathway of ABA activating the protein kinase, followed by recognition and phosphorylation of the RNA binding protein, resulting in increased binding affinity for particular transcripts, the skilled artisan is now provided a full understanding of what is needed to manipulate each of these steps to practice the method.

[0154] Preferred methods are practiced wherein the protein kinase is regulated by a hormone, and more particularly where the hormone is ABA. More preferred are methods wherein the protein kinase is ABA-activated protein kinase (AAPK) and the improved response is in the guard cells.

[0155] A method to alter the expression or activity of a stress-related protein in a plant, comprising altering the amount or activity of a phosphorylation-regulated RNA binding protein is also provided. A plant made by the method described herein has an altered expression or activity of a stress-related protein resulting from altering the amount or activity of the RNA binding protein, or of another related component as discussed above. Dehydrin biosynthesis is one example of a protein which is made in plants in response to stress. The RNA binding protein clearly binds this RNA with greater affinity upon phosphorylation of the RNA binding protein. Those of skill in the art will appreciate that the particular RNA sequences which are recognized and bound by the RNA binding protein can be determined. Such sequences may be useful for selectively designing other proteins which will have transcripts which are preferentially bound by the RNA binding protein upon phosphorylation, and thus it is now possible using the method of the resent invention to design genes encoding proteins which will be preferentially expressed during stress - because the transcripts are preferentially recognized in the nucleus during, for example, stress-induced nuclear reorganization. This method enhances what is already possible to do using, for example, stress-induced promoters for increasing expression by increasing the numbers of transcript.

[0156] In another of its aspects, the invention provides a method to alter ABA sensitivity in a plant, comprising increasing an amount or activity of ABA-mediated phosphorylation-regulated RNA binding protein in a plant, thereby altering sensitivity of the plant to ABA. In addition to broadly providing a method to manipulate ABA mediated stress responses in plant, it is equally possible to alter the plant's sensitivity to ABA according to the methods taught herein. For example, mutant plants may be selected wherein the RNA binding protein is more susceptible to phosphorylation by a protein kinase thus making the plant more sensitive to a given amount of ABA. Alternatively, a modified protein kinase, which is more susceptible to ABA may become activated and phosphorylate the RNA binding protein also tending to increase the plant's sensitivity to ABA. The method can likewise be used to decrease a plant's sensitivity to ABA by selecting for plants with RNA binding proteins which are less susceptible to phosphorylation, have attenuated binding affinity changes upon phosphorylation, or by selecting plants with ABA-activated kinase which require greater concentrations of ABA to become activated, or even selecting mutants, including negative dominant mutants of the protein kinase. The invention also provides the method wherein a transgene is used. Such a transgene can be used to either increase or decrease sensitivity to ABA, for example to provide an additional copy of the protein kinase or the RNA binding protein on a strong constitutive or selectively inducible promoter, or alternative to provide an antisense strand to the coding strand for either the protein kinase or the RNA binding protein. A transgene can also be used to knock out the protein kinase or the RNA binding protein to reduce or eliminate expression. Fertile plants made by the method are also provided as these are most useful for agronomic or research purposes.

[0157] The invention also provides in another aspect an isolated plant protein having RNA binding properties, wherein the protein is of a length of between about 400 and 525 amino acids, and further wherein the RNA binding properties are regulated by phosphorylation of the protein. The protein provided is novel, particularly for example, because phosphorylation- regulation of the type described herein is unique. The protein is found widely, including but not limited to the genera Vicia, Arabidopsis, Oryza, Lycopersicon, Solanum and Medicago. In more preferred embodiments the plant is Viciafaba, Arabidopsis thaliana, Oryza sativa, Lycopersicon esculentum, Solanum tuberosum or Medicago sativa.

[0158] In preferred embodiments, the phosphorylation regulates RNA target discrimination. Preferably, the phosphorylation increases a binding affinity for RNA, most particularly, the phosphorylation results in the preferential binding of certain mRNA over other mRNA present locally in a cell. In a preferred embodiment, the mRNA encodes a stress-induced protein. In one embodiment the protein encoded is dehydrin, synthesized, or upregulated, for example, in response to water stress and other stress conditions.

[0159] In preferred embodiments, the phosphorylation of the plant protein is abscisic acid (ABA)-mediated. In this regard, the expression of the plant protein is not itself regulated or mediated by ABA, but rather the phosphorylation of the protein is mediated by ABA. Preferably, the ABA-mediation is via an ABA-activated protein kinase. In certain preferred embodiments, the protein kinase is ABA-activated protein kinase (AAPK). This is particularly true in guard cells.

[0160] The protein of the invention also comprises an RNA recognition motif. In particular, the RNA recognition motif corresponds, for example, to amino acids 151 and 228 of SEQ LD NO:2. such sequences are known in the art and resemble other RNA recognition motifs. More generally, the plant proteins of the invention have RNA recognition motif-like sequences, such as those of SEQ LD NO:22. Also preferred are those of SEQ ID NOs: 23, and 24.

[0161] In another preferred embodiment, the protein of the invention having the RNA recognition motif-like sequence of SEQ LD NO:22, 23 or 24, has less than about 40% identity in the RNA recognition motif-like sequence with a human hn RNA binding protein A/B. Also preferred are protein with less than about 39% to less than about 35% identity across this region. Proteins with less than about 35% identity but more than about 25% identity, for example those with 33% identity are also preferred.

[0162] In other preferred embodiments, the protein has the RNP2 and RNP 1 -like sequences of SEQ LD NOs: 31 or 32, and SEQ ID NOs:33 or 34 respectively. In yet other preferred embodiments, the protein further comprises one or more of the sequences 25, 26, 27, 28, 29 and 30. Most preferable are proteins comprising the RNA recognition motif-like sequence of SEQ ID NO:22, 23 or 24 and further comprising one each of SEQ ID NOs:25, 26, or 27, and SEQ ID NOs:28, 29, or 30. Still more highly preferred are proteins wherein the RNA recognition motif-like sequence is SEQ ID NO:22, and the protein further comprises SEQ ID NO:25 and SEQ ID NO:28.

[0163] Proteins with greater than about 45% identity to the region corresponding to amino acids 150 to 231 of SEQ ID NO:2 are preferred. More highly preferred are those proteins with greater than 50%, 55%, 60%, 65% identity across this region. Still more highly preferred are those proteins with at least about 75 - 80%, 81%, 82%, 83%, 84%, 85%, or more identity to this region.

[0164] Also preferred are proteins with greater than about 40% identity to the region corresponding to amino acids 246 - 331 of SEQ ID NO:2. More preferred are those proteins having at least about 45% -50% identity in this region. More highly preferred are proteins with at least about 60-65%, to at least about 70% identity in this region. Still more highly preferred are those proteins with greater than about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, or 79% identity in this region. Even more highly preferred are those proteins with greater than about 80% identity in this subsequence, for example 81%, 82% and so on, incrementally.

[0165] The proteins of the invention also preferably have a sequence which corresponds to amino acids 416-435 of SEQ ID NO:2. Preferably, the proteins have greater than about 25% identity in this region. More preferable are those proteins with at least about 30% identity in this region. Still more preferable are proteins with greater than about 50% identity in this region, and those with about at least 50-75% identity. Even more preferable are those proteins with greater than about 75% identity in this region, for example 80-90% identity is highly preferred in this region.

[0166] In another embodiment, the protein of the invention comprises one or more domains consisting of greater than about 50% of a single amino acid. RNA binding proteins often encompass one or more auxiliary domains having a large proportion of a single amino acid. In other preferred embodiments, the domains contain at least about 60%, 65%, 70%, 75%>, or 78% of the single amino acid. Presently preferred are proteins with domains wherein the amino acid is glutamic acid for at least one of the one or more domains. While the domains abundant in an amino acid may be of any length, presently preferred are proteins wherein the at least one domain is at least about 20 or more amino acids in length. Also preferred are domains of at least about 25 or 30 amino acids in length.

[0167] The following example is provided to describe certain aspects of the invention in greater detail. It is intended to illustrate those aspects, not to limit those aspects, or the invention.

[0168] Example [0169] Results

[0170] The inventors previously employed de novo sequencing by tandem mass spectrometry to obtain peptide sequence information allowing cloning of a gene encoding AAPK, an ABA-activated guard cell-specific kinase from Viciafaba, a food crop of major importance in the Middle East. Li et al, Science, 287: 300-303 (2000); see WO 01/02541. Expression of a dominant negative form of AAPK in guard cells prevented ABA-activation of anion channels and stomatal closure, implicating AAPK in rapid ABA signaling events in this specialized cell type.

[0171] To identify AAPK interaction partners, the inventors screened a V. faba guard cell cDNA expression library with radiolabelled AAPK and thereby identified "AAPK- Interacting Protein 1" (AKIPl). Sequence comparison indicates that AKLPl is most similar to l nRNP A/B type single-stranded RNA-binding proteins (Fig. 1).

[0172] Yeast two hybrid analysis provided an in vivo confirmation of the AAPK- AKIPl interaction and indicated that the N terminal region of AAPK including the entire catalytic domain interacts with AKLPl (Fig. 2). Ribonucleotide homopolymer binding assays using AKLPl purified from a yeast expression system confirmed AKIPl as an RNA binding protein (data not shown).

[0173] Physiologically relevant interacting proteins exhibit at least overlapping cellular localization. AAPK-GFP showed diffuse localization in both the cytoplasm and the nucleus (Fig. 3 a), as confirmed by confocal optical sectioning and by fluorescence microscopy on isolated nuclei (data not shown). The bi-partite distribution of AAPK in guard cells suggests that AAPK plays multiple regulatory roles, presumably by interacting with locale-specific downstream elements. AKLPl-GFP fusion protein showed nuclear localization (Fig. 3) characterized initially by a primarily diffuse nuclear labeling pattern (Fig. 3c). ABA treatment caused a rapid and dramatic increase in the number of AKLPl -GFP-labelled foci, indicating that ABA induces relocalization into and/or retention of AKLPl in nuclear speckles (Fig. 3d-f). Neither AAPK-GFP nor cGFP showed this response (data not shown). Hormonally-induced partitioning of an hnRNP into nuclear speckles is believed to not have been previously reported.

[0174] To determine whether AKT l is a substrate for AAPK, in vitro and in vivo phosphorylation assays were performed. As shown in Figure 4a, recombinant AKIPl can be phosphorylated in vitro by AAPK isolated from guard cells treated with ABA (active AAPK) but not by AAPK from untreated guard cells (inactive AAPK), consistent with previous observations of the ABA-dependence of AAPK activation. ABA-dependent phosphorylation of AKLPl occurs in vivo in guard cells: phosphorylated AKIPl was recovered by immunoprecipitation from intact guard cell protoplasts labeled in vivo with ³²P-orthophosphate and treated for 15 min. with ABA (Fig. 4b). ABA treatment did not alter AKIPl abundance (Fig. 4c). Collectively, these results demonstrate that AKIPl is indeed a substrate of AAPK.

[0175] To further assess the functional significance of AKIPl phosphorylation by AAPK, RNA targets of AKIPl were sought. Dehydrins are ubiquitous stress-upregulated proteins thought to improve enzyme and membrane stability under stress conditions. In addition, dehydrins have been demonstrated to increase in transcript abundance in ABA-treated Viciafaba guard cells. Accordingly, the inventors amplified by 3' end amplification (TPEA) (ref. 13) guard cell mRNA bound to AKLPl and then performed PCR using dehydrin-specific primers. As illustrated in Figure 5 a and confirmed by sequence analysis of the PCR product, dehydrin could be amplified from the mRNA population bound to AKIP 1 , but only if AKIP 1 had been phosphorylated by pretreatment with ABA-activated AAPK (Fig. 5a lanes 5 vs. 6). Thus, AKLPl phosphorylation by AAPK increased AKLPl affinity for a particular transcript.

[0176] Dehydrin transcript association with AKIPl was also only observed when the poly (A) RNA target population was isolated from guard cells initially treated with ABA (Fig. 5a, lanes 3 vs. 6). This initial ABA treatment increased dehydrin transcript abundance, likely increasing the number of dehydrin transcripts bound to AKIPl and thereby facilitating amplification.

[0177] To confirm direct interaction between AKLP 1 and dehydrin transcript, a LTV cross-linking RNA binding analysis was performed using ³²P-labelled dehydrin mRNA transcribed in vitro. AKIPl binding of the radiolabelled dehydrin RNA was indeed observed (Fig. 5b). Consistent with Fig. 5a, this binding was dependent on AKLPl phosphorylation by ABA-activated AAPK. Binding of labeled sense dehydrin mRNA was competed with cold dehydrin mRNA, demonstrating the specific nature of the dehydrin mRNA- AKL l interaction. Incubation of AKIPl with active CDPK ("Calcium-Dependent Protein Kinase"), also purified from guard cells, did not suffice to allow dehydrin transcript association with AKLPl, indicating kinase specificity of the AKIPl modification. Further, no RNA binding by AKIPl was observed with dehydrin RNA transcribed in the antisense direction, nor with RNA transcribed from positive control template provided with the Riboprobe in vitro transciption kit (Promega), confirming that AKTPl does not indiscriminately bind RNA species. Collectively, these results are consistent with the following sequence:

1) ABA activates AAPK;

2) AAPK phosphorylates AKIPl;

3) as a result, AKIPl shows a phosphorylation-induced increase in affinity for dehydrin mRNA concomitant with increased localization in nuclear speckles.

[0178] The observation that phosphorylation alters affinity of a plant hnRNP-type RNA binding protein for a specific mRNA is novel. Cloned DNAs encoding proteins that bind ribohomopolymers and whose transcription is upregulated in response to ABA have been identified in several species, however the functions of these proteins remain unknown. In contrast, neither AKIPl nor AAPK transcript abundance is ABA-regulated (data not shown).

[0179] Abiotic stresses are established modulators of mRNA stability in plants; the present data suggest the involvement of hnRNPs in this phenomenon. The ABA-mediated nuclear localization pattern of AKIPl in distinct foci is consistent with a role for this protein in RNA metabolism, since nuclear speckles of mammalian systems are implicated in splicing component storage, prespliceosome assembly, and possibly active RNA processing. linRNPS usually interact with multiple RNA partners. Following whole-plant treatment with ABA, numerous transcripts show an increase in steady-state levels. These or other transcripts comprise potential binding targets for these ABA-mediated phosphorylation-regulated RNA binding proteins. While AAPK gene expression appears to be guard cell specific, AKLPl is also expressed in leaves, stems, and roots (Fig. 7), meaning that other protein kinases can interact with phosphorylation-regulated RNA binding proteins, including AKLPl, and that other cell- or organ-specific kinases also may differentially regulate AKIPl throughout the plant.

[0180] ABA has long been known as a transcriptional regulator, and as a posttranslational regulator of cellular and long-distance signaling in plants. However, recent reports also demonstrate altered plant sensitivity to ABA following mutation of a double-stranded RNA- binding protein, an Sm-like protein, and an mRNA cap protein. The present description of the linRNP-like protein AKIPl complements these studies, and taken together with them suggests a new paradigm involving ABA-regulation of post-transcriptional RNA metabolism. The novel observations that ABA functions via protein kinases such as AAPK to induce rapid changes in nuclear architecture and AKLPl target affinity are consistent with this suggestion.

[0181] Methods

[0182] Interaction cloning and yeast two-hybrid analysis

[0183] AAPK protein was labelled with [ SJmethionine using an in vitro transcription /translation system (Novagen) and used to probe a Viciafaba guard cell cDNA expression library for AAPK-interacting clones. The coding sequences of AKIPl and AAPK cDNAs were cloned into pAS2-l and pACT2 (Clontech), respectively. Interaction of AAPK and AKIPl was examined by yeast two-hybrid analysis using strain PJ69-4A.

[0184] AKIPl subcellular localization

[0185] Viciafaba leaves were biolistically transfonned with AAPK-GFP or AKLPl- GFP plasmid (agrobacterium-mediated transformation may also be employed). Expression of AAPK-GFP or AKLPl-GFP in guard cells was examined by fluorescence microscopy. Fluorescence images of AKLPl-GFP distribution were acquired with a CCD camera (Photometries). The number of AKIPl -GFP labeled speckles was quantified from images taken at 0 and 20 min. following incubation in 10 mM Mes, 50 mM KC1, pH 6.2, with or without 50 μM ABA.

[0186] Production and purification of fusion protein

[0187] AKIPl was cloned into the pESP-3 vector (Stratagene) with AKIPl upstream of the FLAG tag and GST sequences and was confirmed by DNA sequencing. AKLPl -GST fusion protein expressed in Schizosaccharomyces pombe was affinity-purified using glutathione- sepharose columns. Purified AKIPl -GST fusion protein was recognized specifically by FLAG antibody (Stratagene) using immunoblot analysis (data not shown).

[0188] In vitro phosphorylation assay

[0189] Purified AKTPl -GST or GST was subjected to in vitro phosphorylation assays using ABA-activated or inactive AAPK as previously described for AAPK phosphorylation of liistone. Li et al, Plant Cell, 8, 2359-2368 (1996). Because AAPK cannot be activated in vitro by ABA, isolated guard cell protoplasts were treated with ABA, and native guard cell AAPK (or CDPK) then was gel-purified following in gel renaturation.

[0190] In vivo phosphorylation assay

[0191] An approximately 70 kDa protein affinity-purified from E. coli expressing an AKIPl -HIS ₆ construct (Promega) was in gel digested with trypsin. Resulting peptides were analyzed by tandem mass spectrometry (Gygi et al, Mol. Cell Biol, 19, 1720-1730 (1999)) on an LCQ-DΕCA mass spectrometer (ThennoFinnigan). AKLPl identity was unambiguously confirmed by matching of 5 peptides using the Sequest algorithm. AKIPl antibodies were then obtained from rabbit using recombinant AKLPl as antigen. In immunoblots of guard cell proteins, these antibodies specifically recognized AKIPl protein of approximately 75 kDa; bands were not detected with preimmune serum (data not shown).

[0192] Guard cell protoplast suspensions (1.0 mg protein ml^"1) were incubated with ³²P- orthophosphate (0.25 mCi mg protein^"1) under background red light for 80 min. at 24°C, then ABA was added to the suspension at 25 μM. Immunoprecipitation and in vivo phosphorylation assays were performed as described by Kinoshita et al, ΕMBO J., 18, 5548-5558 (1999).

[0193] mRNA binding assay

[0194] Approximately 3 μg of poly (A) RNA was isolated from about 2.5 x 10⁸ purified guard cells untreated or treated with 25 μM ABA as described by Li, et al. Purified AKIPl -GST or GST (10 μg) was treated with active or inactive AAPK and then incubated with GST affinity resin (Stratagene) equilibrated in 50 mM Tris-HCl (pH 7.4), 150 mM NaCl and 5 mM MgCl₂ for 30 min at 4°C. After washing 3 times to remove any unbound proteins, immobilized AKIPl- GST or immobilized GST was incubated with poly (A) RNA (-0.4 μg) at 30°C for 10 min. The resin was washed with the above buffer 4 times to remove unbound RNAs. Bound RNAs were then extracted from the resin as described by Moz et al, J. Biol. Chem., 274, 25266-25272 (1999).

[0195] A three prime end amplification method allowing the assay of specific mRNAs was employed. Dixon et al, Nucleic Acids Res., 26, 4426-4431 (1998). Recovered RNAs were reverse-transcribed with an anchored oligo primer [5'-CGC TCG AGT GCA GAA TTC TTT TTT TTT TTT TTT T(A,C,G)-3'] (SΕQ ID NO:35) and second-strand cDNA was synthesized using the primer [5'-GCC GCT CGA GTG CAG AT TCN NNN NCG AGA-3'] (SΕQ ID NO:36). cDNAs were further amplified using the following primers: [5'-CGC TCG AGT GCA GAA TTC-3'] (SEQ TD NO:37) and [5'-GCC GCT CGA GTG CAG AT TCN NNN NCG AGA- 3'] (SEQ ID NO:38). Primers were designed based on Dixon et al. with modification to minimize the likelihood of inadvertently favoring the amplification of any specific known plant DNA sequence(s). The resulting cDNAs were further amplified by PCR using primers: 5'-AAC AGG GTA CGG TGG AAG TG-3' and 5'-ATC CTC CAG TAG CAG GAA GC-3', which correspond to nucleotide positions 395 to 675 of dehydrin DHN1 (GenBank Accession No. X63061). The resultant PCR product was cloned into TOPO TA cloning vector (Invitrogen) and sequenced.

[0196] UV cross-linking assay

[0197] The dehydrin PCR product was subcloned into pGEM-3Z vector (Promega). After the vector was linearized with Sma I, dehydrin RNA was synthesized using an in vitro transcription system (Promega) in the presence of [α-³²P]rCTP according to Promega's protocol. Unincorporated nucleotides were removed by a NucTrap probe purification column (Stratagene). Purified AKLPl -GST or GST (2 μg) previously treated with active AAPK or CDPK was incubated with ³²P-labeled dehydrin RNA for 30 min at room temperature. UV cross-linking and detection of RNA-bound proteins were performed as described by Moz et al..

[0198] While certain preferred embodiments of the present invention have been described and specifically exemplified above, it is not intended that the invention be limited to such embodiments. Various modifications may be made to the invention without departing from the scope and spirit thereof as set forth in the appended claims.

Claims

What is Claimed:

1. An isolated nucleic acid molecule encoding a plant RNA binding protein, wherein the protein comprises an RNA recognition motif selected from the group consisting of SEQ ID NOs: 22, 23, and 24, and further comprises one or more sequences from the group consisting of SEQ ID NOs:25, 26, 27, 28, 29 and 30.

2. The isolated nucleic acid molecule of claim 1 wherein the one or more sequences are selected from the group consisting of SEQ ID NOs: 26, 27, 29, and 30.

3. The isolated nucleic acid molecule of claim 1 wherein the encoded protein is a substrate for phosphorylation by an ABA-activated protein kinase.

4. The isolated nucleic acid molecule of claim 3 wherein the ABA-activated protein kinase is AAPK.

5. The isolated nucleic acid molecule of claim 3 wherein the encoded protein's ability to interact with RNA is altered upon phosphorylation.

6. The isolated nucleic acid molecule of claim 5 wherein a binding affinity for RNA increases upon phosphorylation.

7. The isolated nucleic acid molecule of claim 1 wherein the plant is from a genus selected from the group consisting of Vicia, Arabidopsis, Oryza, Lycopersicon, Solanum, and Medicago.

8. The isolated nucleic acid molecule of claim 7 wherein the plant is Viciafaba, Arabidopsis thaliana, Oryza sativa, Lycopersicon esculentum, Solanum tuberosum, or Medicago sativa.

9. The isolated nucleic acid molecule of claim 1 wherein the RNA recognition motif comprises SEQ ID NOs:23 or 24.

10. The isolated nucleic acid molecule of claim 9 wherein the RNA recognition motif is SEQ ID NO:24.

11. The isolated nucleotide of claim 10 wherein the encoded protein comprises each of SEQ LD NOs:27, and 30.

12. The isolated nucleic acid molecule of claim 6 which encodes a protein of about 400 to about 525 amino acids in length.

13. The isolated nucleic acid molecule of claim 12 selected from the group consisting of:

(a) any of SEQ ID NOs:l, 3, 4, 6, 7, 9, 10, 12, 13, 14, 16, 18, and 20;

(b) a sequence encoding a protein having an amino acid sequence of SEQ LD NO: 2,

5, 8, 11, 15, 17, 19, or 21;

c) a sequence encoding a protein having a sequence at least 50% identical to SEQ LD NO: 2; and

d) the complement of a sequence that hybridizes with any of SEQ ID NOS : 1, 3, 4,

6, 7, 9, 10, 12, 13, 14, 16, 18 and 20 under conditions comprising hybridization at 37-42°C in a solution comprising: 5X SSC, 5X Denhardt's reagent, 1.0% SDS, 100 μg/ml denatured, fragmented salmon sperm DNA, 0.05% sodium pyrophosphate and up to 50% formamide, and washing at 42-65° in IX SSC and 1% SDS.

14. The isolated nucleic acid molecule of claim 1, which when expressed in a plant, yields an RNA binding protein localized to the nucleus of cells.

15. The isolated nucleic acid molecule of claim 14 in which the RNA binding protein is further localized to nuclear speckles upon phosphorylation.

16. A vector comprising the isolated nucleic acid molecule of claim 1.

17. The vector of claim 16 in a host cell.

18. The vector of claim 16 adapted for expression of the nucleic acid molecule in a host cell.

19. The vector of claim 18 wherein the cell is a plant cell.

20. A plant cell comprising the vector of claim 19.

21. A transgenic plant comprising the vector of claim 19.

22. A protein produced by the expression of the nucleic acid molecule of claim 1.

23. A protein produced by the expression of the nucleic acid molecule of claim 13.

24. An antibody specific for the protein of claim 22.

25. An isolated nucleic acid molecule encoding a plant RNA binding protein wherein the binding of RNA by the encoded RNA binding protein is ABA-mediated, and further wherein accumulation of a transcript for an mRNA encoding the plant RNA binding protein is not mediated by ABA, wherein the encoded RNA binding protein comprises sequence SEQ ID NOs:31 or 32, and further comprises sequence SEQ LD NOs:33 or 34.

26. The isolated nucleic acid molecule of claim 25 selected from the group consisting of:

(a) any of SEQ TD NOs:l, 3, 4, 6, 7, 9, 10, 12, 13, 14, 16, 18, and 20;

(b) a sequence encoding a protein having an amino acid sequence of SEQ ID NO: 2, 5, 8, 11, 15, 17, 19, or 21;

c) a sequence encoding a protein having a sequence at least 30% identical to SEQ LD NO: 2; and

d) the complement of a sequence that hybridizes with any of SEQ LD NOS: 1, 3, 4, 6, 1, 9, 10, 12, 13, 14, 16, 18 and 20 under conditions comprising hybridization at 37-42°C in a solution comprising: 5X SSC, 5X Denhardt's reagent, 1.0% SDS, 100 μg/ml denatured, fragmented salmon sperm DNA, 0.05% sodium pyrophosphate and up to 50% formamide, and washing at 42-65° in IX SSC and 1% SDS.

27. An ohgonucleotide having at least 15 consecutive nucleotides identical in sequence to a consecutive 15 nucleotide sequence of the isolated nucleic acid molecule of claim 26.

28. The ohgonucleotide of claim 27 wherein the isolated nucleic acid molecule is SEQ ID NOs:l, 3, 4, 6, 7, 9, 10, 12, 13, 14, 16, 18, or 20.

29. A genetically altered plant having altered response to ABA as compared with an unaltered control plant, comprising an ABA-mediated phosphorylation-regulated RNA binding protein that is substantially nonfunctional or absent.

30. The genetically altered plant of claim 29, produced by subjecting a population of plants to mutagenesis and selecting a mutagenized plant wherein the ABA-mediated phosphorylation- regulated RNA binding protein is substantially nonfunctional or absent.

31. The genetically altered plant of claim 29, produced by introducing into a plant cell a transgene that results in the plant cell's endogenous ABA-mediated phosphorylation-regulated RNA binding protein activity becoming substantially nonfunctional or absent, and regenerating a plant from the cell containing the transgene.

32. The genetically altered plant of claim 31 , wherein the transgene disrupts the gene encoding the endogenous ABA-mediated phosphorylation-regulated RNA binding protein.

33. The genetically altered plant of claim 31 ; wherein the transgene is inducible.

34. The genetically altered plant of claim 33, wherein expression of the transgene produces an antisense strand of the nucleic acid molecule of claim 26 effective for reducing expression of the ABA-mediated phosphorylation-regulated RNA binding protein.

35. A genetically altered plant having altered response to ABA as compared with an unaltered control plant, comprising an ABA-mediated phosphorylation-regulated RNA binding protein that is increased in amount or activity as compared with the control plant.

36. The genetically altered plant of claim 35, produced by subjecting a population of plants to mutagenesis and selecting a mutagenized plant wherein the ABA-mediated phosphorylation- regulated RNA binding protein is increased in amount or activity compared to a control plant.

37. A method for improving a plant's ABA-regulated response to a stressor comprising one or more of: (a) altering the amount or activity of one or more phosphorylation-regulated RNA binding proteins in the plant;

(b) altering the amount or activity of an ABA-activated protein kinase with respect to the phosphorylation-regulated RNA binding protein; and

(c) altering a gene sequence encoding a transcript such that the transcript will be bound with altered affinity by the phosphorylation regulated RNA binding protein upon phosphorylation;

thereby improving the plant's ABA-regulated response to the stressor.

38. The method of claim 37 wherein the protein kinase is regulated by a hormone.

39. The method of claim 38 wherein the hormone is ABA.

40. The method of claim 39 wherein the protein kinase is abscisic acid-activated protein kinase (AAPK).

41. A method to alter the expression or activity of a stress-related protein in a plant, comprising altering the amount or activity of a phosphorylation-regulated RNA binding protein.

42. A method to alter ABA sensitivity in a plant, comprising increasing an amount or activity of ABA-mediated phosphorylation-regulated RNA binding protein in a plant, thereby altering sensitivity of the plant to ABA.

43. The method of claim 42, wherein the amount or activity of the ABA-mediated phosphorylation regulated RNA binding protein is increased by the addition of at least one transgene to the plant genome.

44. A fertile plant produced by the method of claim 43.

45. An isolated plant protein having RNA binding properties, wherein the protein is of a length of between about 400 and 525 amino acids, and further wherein the RNA binding properties are regulated by phosphorylation of the protein.

46. The protein of claim 45 wherein the phosphorylation regulates RNA target discrimination.

47. The protein of claim 46 wherein the phosphorylation increases a binding affinity for RNA.

48. The protein of claim 47 wherein the RNA is mRNA.

49. The protein of claim 48 wherein the mRNA encodes a stress-induced protein.

50. The protein of claim 45 wherein the phosphorylation is abscisic acid (ABA)-mediated.

51. The protein of claim 50 wherein the AB A-mediation is via an ABA-activated protein kinase.

52. The protein of claim 51 wherein the protein kinase is ABA-activated protein kinase (AAPK).

53. The protein of claim 45 further comprising an RNA recognition motif like sequence of SEQ ID NO:22.

54. The protein of claim 53 with less than about 40% identity in the RNA recognition motif like sequence with a human hn RNA binding protein A/B.

55. The protein of claim 53 further comprising one or more domains consisting of greater than about 50% of a single amino acid.

56. The protein of claim 54 wherein the amino acid is glutamic acid for at least one of the one or more domains.

57. The protein of claim 56 wherein the at least one domain is at least about 25 or more amino acids in length.