WO2008157157A2 - Drought-resistant plants and method for producing the plants - Google Patents

Abstract

This application provides a recombinant expression cassette for expressing CPK4 or CPK11, two calcium-dependent protein kinases found in plants. Also provided are a transgenic plant with heightened ABA-sensitivity and drought-resistance, as well as a method for producing such plants.

Description

PROTEIN KINASE CPK4 AND CPKI l, DROUGHT-RESISTANT PLANTS AND METHOD FOR PRODUCING THE PLANTS

CROSS-REFERENCES TO RELATED APPLICATIONS

[0001] The present patent application claims benefit of priority to U.S. Provisional Patent Application No. 60/934,402, filed June 13, 2007, the contents of which are incorporated by reference in the entirety for all purposes.

FIELD OF THE INVENTION

[0002] This invention relates to methods and compositions for generating plants with altered abscisic acid (ABA) sensitivity.

BACKGROUND OF THE INVENTION

[0003] The phytohormone abscisic acid (ABA) regulates many agriculturally important stress and developmental responses throughout the life cycle of plants. In seeds, ABA is responsible for the acquisition of nutritive reserves, desiccation tolerance, maturation and dormancy (M. Koornneef et al., Plant Physiol. Biochem., 36:83 (1998); J. Leung & J. Giraudat, Annu. Rev. Plant. Physiol. Plant. MoI. Biol., 49: 199 (1998)). During vegetative growth, ABA is a central internal signal that triggers plant responses to various adverse environmental conditions including drought, salt stress and cold (M. Koornneef et al., Plant Physiol. Biochem., 36:83 (1998); J. Leung & J. Giraudat, Annu. Rev. Plant. Physiol. Plant. MoI. Biol., 49: 199 (1998)). A rapid response mediated by ABA is stomatal closure in response to drought (J. Leung & J. Giraudat, Annu. Rev. Plant. Physiol. Plant. MoI. Biol., 49: 199 (1998); E. A. C. MacRobbie, Philos. Trans. R Soc. Lond. B Biol. Sci., 353: 1475 (1998); J. M. Ward et al., Plant Cell, 7:833 (1995)). Stomata on the leaf surface are formed by pairs of guard cells whose turgor regulates stomatal pore apertures (E. A. C. MacRobbie, Philos. Trans. R Soc. Lond. B Biol. Sci., 353: 1475 (1998); J. M. Ward et al., Plant Cell, 7:833 (1995)). ABA induces stomatal closure by triggering cytosolic calcium ([Ca ²⁺ _cyt) increases which regulate ion channels in guard cells (E. A. C. MacRobbie, Philos. Trans. R Soc. Lond. B Biol. Sci., 353: 1475 (1998); J. M. Ward et al., Plant Cell, 7:833 (1995)). This response is vital for plants to limit transpirational water loss during periods of drought.

[0004] It has been demonstrated that calcium play a central role in mediating ABA signal transduction, but many of the Ca²⁺-binding sensory proteins as the components of ABA signaling pathway remain to be characterized. Whereas many biochemical approaches show functions of calcium-dependent protein kinases (CDPKs) in ABA signal transduction, molecular genetic evidence via gene disruption has been lacking to unequivocally link defined CDPK genes with ABA-regulated biological functions at the whole-plant level. The present inventors provide additional information relating to Ca²⁺-dependent protein kinases and their role in ABA signaling. This invention was in part described in Zhu et al., Plant Cell. 2007 Oct; 19(10):3019-36.

BRIEF SUMMARY OF THE INVENTION [0005] The present inventors have discovered, for the first time, that plant calcium- dependent protein kinases CPK4 and CPKl 1 are involved in signal transduction mediated by abscisic acid (ABA), a phytohormone that plays a role in plant physiology. Because ABA is involved in regulating the opening of stomatal aperture, an important mechanism for a plant to adjust transpirational water loss in response to changes in water availability in the environment, this discovery provides a method for increasing drought tolerance in a plant, as well as other stress tolerance related to ABA-sensitivity. This method involves expressing a CPK4 or CPKl 1 protein in a plant, for instance, by introducing a recombinant expression vector comprising a heterologous promoter and a polynucleotide sequence encoding the CPK4 or CPKl 1 protein into the plant. The heterologous promoter and the CPK 4 or CPK 11 -coding sequence being operably linked in the expression vector, the CPK4 or CPKl 1 protein is therefore expressed in the plant and confers enhanced ABA sensitivity to the plant. The CPK4 or CPKl 1 protein suitable for use in this method is one having an amino acid sequence with substantial identity to one of the exemplary CPK sequences set forth in SEQ ID NOs:2, 4, 6, 8, 10, 12, 14, and 16. In some cases, a CPK protein suitable for use in the claimed method comprises a consensus sequence between SEQ ID NO:2 and SEQ ID NO:4 (shown in Figure 10, where conserved residues are presented in shaded segments and the non-conserved positions can be occupied by any amino acids), or comprises a sequence segment that has a substantial identity to the consensus sequence or includes one or more conservatively modified variants in the consensus sequence.

[0006] The present invention provides methods of enhancing ABA sensitivity in a plant. In some embodiments, the methods comprise introducing a recombinant expression cassette into a plant, wherein the expression cassette comprises a promoter operably linked to a polynucleotide encoding a CPK4 or CPKl 1, wherein the promoter is heterologous to the polynucleotide, wherein the plant has increased ABA sensitivity compared to an otherwise identical plant lacking the expression cassette. In some embodiments, the plant has improved drought tolerance compared to an otherwise identical plant lacking the expression cassette

[0007] In some embodiments, the calcium-dependent protein kinase has an amino acid sequence at least 50%, 60%, 70%, 75%, 80%, 90%, 94%, or 95% identical to SEQ ID NO:2 or SEQ ID NO:4. In some cases, the percentage sequence identity to SEQ ID NO:2 or SEQ ID NO: 4 is even higher than 95%, e.g., reaching 100%.

[0008] In some embodiments, the promoter is constitutive. In some embodiments, the promoter is inducible. In some embodiments, the promoter is tissue-specific. In other embodiments, the promoter directs expression in guard cells, for example is guard cell specific. In yet some other embodiments, the promoter is a drought-induced promoter.

[0009] In some embodiments, the methods comprise generating a plurality of plants comprising the introduced expression cassette, and screening the plants for enhanced ABA sensitivity compared to an otherwise identical plant lacking the expression cassette.

[0010] The present invention also provides methods of decreasing ABA sensitivity in a plant. In some embodiments, the methods comprise introducing an recombinant expression cassette into a plant, wherein the expression cassette comprises a promoter operably linked to a polynucleotide comprising at least 20 nucleotides complementary or identical to a contiguous sequence in an mRNA encoding a CKP4 or CKPl 1 in the plant, wherein the promoter is heterologous to the polynucleotide, thereby reducing expression of the CPK4 or CPKl 1 in the plant, wherein the plant has reduced ABA sensitivity compared to an otherwise identical plant lacking the expression cassette. The CPK4 or CPKl 1 protein suitable for use in this method is one having an amino acid sequence with substantial identity to one of the exemplary CPK sequences set forth in SEQ ID NOs:2, 4, 6, 8, 10, 12, 14, and 16. In some cases, a CPK protein suitable for use in the claimed method comprises a consensus sequence between SEQ ID NO:2 and SEQ ID NO:4 (shown in Figure 10, where conserved positions are presented in shaded segments, and the non-conserved positions can be occupied by any amino acids), or comprises a sequence segment that has a substantial identity to the consensus sequence or includes one or more conservatively modified variants in the consensus sequence. [0011] In some embodiments, the polynucleotide comprises at least 50 nucleotides complementary or identical to a contiguous sequence in a cDNA encoding a CKP4 or CKPl 1 in the plant. In some embodiments, the polynucleotide comprises at least 200 nucleotides complementary or identical to a contiguous sequence in a cDNA encoding a CKP4 or CKPl 1 in the plant.

[0012] In some embodiments, the CKP has an amino acid sequence at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 94%, or 95% identical to SEQ ID NO:2 or SEQ ID NO:4. In some cases, the percentage sequence identity is higher than 95% and can reach 100%. In some embodiments, the polynucleotide comprises at least 20, 50, 100, or 200 nucleotides complementary or identical to a contiguous sequence of any one of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, and 15. In some cases, the polynucleotide comprises at least 20, 50, 100, or 200 nucleotides complementary or identical to a contiguous sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, or 15, particularly SEQ ID NO: l or 3.

[0013] In some embodiments, the promoter directs expression of the polynucleotide to abscission zones of the plant.

[0014] The present invention also provides for recombinant expression cassettes comprising a promoter operably linked to a polynucleotide encoding the CPK4 or CPKl 1 protein, wherein the promoter is heterologous to the polynucleotide, and wherein introduction of the expression cassette into a plant results in enhanced abscisic acid sensitivity in the plant compared to an otherwise identical plant lacking the expression cassette.

[0015] In some embodiments, introduction of the expression cassette into a plant results in improved drought tolerance in the plant compared to an otherwise identical plant lacking the expression cassette

[0016] In some embodiments, the CPK4 or CPKl 1 has an amino acid sequence at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 94%, or 95% identical to SEQ ID NO:2 or SEQ ID NO:4. In some cases, the percentage sequence identity is higher than 95% and can reach 100%. Generally, the CPK4 or CPKl 1 protein is one having an amino acid sequence with substantial identity to one of the exemplary CPK sequences set forth in SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, and 16. In some cases, a CPK protein encoded by the expression cassette comprises a consensus sequence between SEQ ID NO: 2 and SEQ ID NO: 4 (shown in Figure 10, where conserved positions are presented in shaded segments, and the non-conserved positions can be occupied by any amino acids), or comprises a sequence segment that has a substantial identity to the consensus sequence or includes one or more conservatively modified variants in the consensus sequence. [0017] In some embodiments, the promoter is constitutive. In some embodiments, the promoter is inducible. In some embodiments, the promoter is tissue-specific. In some embodiments, the promoter directs expression in guard cells. In other embodiments, the promoter is a drought-induced promoter. [0018] The present invention also provides for transgenic plants comprising a recombinant expression cassette, wherein the expression cassette comprises a promoter operably linked to a polynucleotide encoding a CPK4 or CPKl 1, wherein the promoter is heterologous to the polynucleotide, and wherein the plant has enhanced abscisic acid sensitivity compared to an otherwise identical plant lacking the expression cassette. [0019] In some embodiments, the plant has improved drought tolerance compared to an otherwise identical plant lacking the expression cassette.

[0020] In some embodiments, the CPK4 or CPKl 1 has an amino acid sequence at least 80%, 85%, 90%, 94%, or 95% identical to SEQ ID NO:2 or SEQ ID NO:4. In some cases, the percentage sequence identity is even higher and can reach 100%. Generally, the CPK4 or CPKl 1 protein is one having an amino acid sequence with substantial identity to one of the exemplary CPK sequences set forth in SEQ ID NOs:2, 4, 6, 8, 10, 12, 14, and 16. In some cases, a CPK protein encoded by the expression cassette comprises a consensus sequence between SEQ ID NO:2 and SEQ ID NO:4 (shown in Figure 10, where conserved positions are presented in shaded segments, and the non-conserved positions can be occupied by any amino acids), or comprises a sequence segment that has a substantial identity to the consensus sequence or includes one or more conservatively modified variants in the consensus sequence.

[0021] In some embodiments, wherein the promoter is constitutive. In some embodiments, the promoter is inducible. In some embodiments, the promoter is tissue-specific. In some embodiments, the promoter directs expression in guard cells. In other embodiments, the promoter is a drought-induced promoter.

[0022] The invention also provides for any plant part from the transgenic plants of the invention. Examples of such plant parts include, but are not limited to: seeds, flowers, leafs and fruits.

DEFINITIONS

[0023] "CPK4" and "CPKIl" are two calcium-dependent protein kinases found in plants such as Arabidopsis thaliana (see GenBank Accession No. NM_117025 and NM_103271). Homologous CPKs can be from a variety of other plant species, such as potato, maize, grape, fava bean, soybean, and tobacco. In some embodiments, the interspecies homolog of CPK4 or CPKl 1 protein has an amino acid sequence substantially identical (i.e., at least 50% identical, in some cases at 70%, 75%, 80%, 85%, 90%, 94%, 95% or greater identity) to SEQ ID NO:2 or SEQ ID NO:4.

[0024] The term "nucleic acid" or "polynucleotide" refers to a single or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases read from the 5' to the 3' end, or an analog thereof.

[0025] The term "promoter," as used herein, refers to a polynucleotide sequence capable of driving transcription of a coding sequence in a cell. Thus, promoters used in the polynucleotide constructs of the invention include cis-acting transcriptional control elements and regulatory sequences that are involved in regulating or modulating the timing and/or rate of transcription of a gene. For example, a promoter can be a cis-acting transcriptional control element, including an enhancer, a promoter, a transcription terminator, an origin of replication, a chromosomal integration sequence, 5' and 3' untranslated regions, or an intronic sequence, which are involved in transcriptional regulation. These cis-acting sequences typically interact with proteins or other biomolecules to carry out (turn on/off, regulate, modulate, etc.) gene transcription. A "plant promoter" is a promoter capable of initiating transcription in plant cells. A "constitutive promoter" is one that is capable of initiating transcription in nearly all tissue types, whereas a "tissue-specific promoter" initiates transcription only in one or a few particular tissue types. A "drought-induced promoter" is a promoter that initiates transcription in a plant or plant cells while under stress from lack of water.

[0026] The term "plant" includes whole plants, shoot vegetative organs and/or structures (e.g., leaves, stems and tubers), roots, flowers and floral organs (e.g., bracts, sepals, petals, stamens, carpels, anthers), ovules (including egg and central cells), seed (including zygote, embryo, endosperm, and seed coat), fruit (e.g., the mature ovary), seedlings, plant tissue (e.g., vascular tissue, ground tissue, and the like), cells (e.g., guard cells, egg cells, trichomes and the like), and progeny of same. The class of plants that can be used in the method of the invention is generally as broad as the class of higher and lower plants amenable to transformation techniques, including angiosperms (monocotyledonous and dicotyledonous plants), gymnosperms, ferns, and multicellular algae. It includes plants of a variety of ploidy levels, including aneuploid, polyploid, diploid, haploid, and hemizygous. [0027] A polynucleotide sequence is "heterologous" to an organism or a second polynucleotide sequence if it originates from a foreign species, or, if from the same species, is modified from its original form. For example, when a promoter is said to be operably linked to a heterologous coding sequence, it means that the coding sequence is derived from one species whereas the promoter sequence is derived another, different species; or, if both are derived from the same species, the coding sequence is not naturally associated with the promoter (e.g. , is a genetically engineered coding sequence, e.g. , from a different gene in the same species, or an allele from a different ecotype or variety).

[0028] A polynucleotide "exogenous" to an individual plant is a polynucleotide which is introduced into the plant by any means other than by a sexual cross. Examples of means by which this can be accomplished are described below, and include Agrobactenum-mediated transformation, biolistic methods, electroporation, and the like. Such a plant containing the exogenous nucleic acid is referred to here as a Ti (e.g., in Arαbidopsis by vacuum infiltration) or Ro (for plants regenerated from transformed cells in vitro) generation transgenic plant. [0029] As used herein, the term "transgenic" describes a non-naturally occurring plant that contains a genome modified by man, wherein the plant includes in its genome an exogenous nucleic acid molecule, which can be derived from the same or a different plant species. The exogenous nucleic acid molecule can be a gene regulatory element such as a promoter, enhancer, or other regulatory element, or can contain a coding sequence, which can be linked to a heterologous gene regulatory element. Transgenic plants that arise from sexual cross or by selfing are descendants of such a plant.

[0030] An "expression cassette" refers to a nucleic acid construct that, when introduced into a host cell, results in transcription and/or translation of an RNA or polypeptide, respectively. Antisense or sense constructs that are not or cannot be translated are expressly included by this definition. In the case of both expression of transgenes and suppression of endogenous genes (e.g., by antisense, or sense suppression) one of skill will recognize that the inserted polynucleotide sequence need not be identical, but may be only "substantially identical" to a sequence of the gene from which it was derived. As explained below, these substantially identical variants are specifically covered by reference to a specific nucleic acid sequence.

[0031] "Increased" or "enhanced" CPK4 or CPKl 1 expression or activity refers to an augmented change in the protein's expression or activity. Examples of such increased activity or expression include the following: CPK expression or activity is increased above the level of that in wild-type, non-transgenic control plants (i.e., the quantity of CPK activity or expression of the CPK gene is increased). CPK expression or activity is present in an organ, tissue, or cell where it is not normally detected in wild-type, non-transgenic control plants (i.e. , CPK expression or activity is increased within certain tissue types). CPK expression or activity is increased when its expression or activity is present in an organ, tissue or cell for a longer period than in a wild-type, non-transgenic controls (i.e., duration of CPK expression or activity is increased).

[0032] Two nucleic acid sequences or polypeptides are said to be "identical" if the sequence of nucleotides or amino acid residues, respectively, in the two sequences is the same when aligned for maximum correspondence as described below. The terms "identical" or percent "identity," in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same, when compared and aligned for maximum correspondence over a comparison window, as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. When percentage of sequence identity is used in reference to proteins or peptides, it is recognized that residue positions that are not identical often differ by conservative amino acid substitutions, where amino acids residues are substituted for other amino acid residues with similar chemical properties (e.g. , charge or hydrophobicity) and therefore do not change the functional properties of the molecule. Where sequences differ in conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well known to those of skill in the art. Typically this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing the percentage sequence identity. Thus, for example, where an identical amino acid is given a score of 1 and a non-conservative substitution is given a score of zero, a conservative substitution is given a score between zero and 1. The scoring of conservative substitutions is calculated according to, e.g., the algorithm of Meyers & Miller, Computer Applic. Biol. Sa. 4: 11-17 (1988) e.g., as implemented in the program PC/GENE (Intelligenetics, Mountain View, California, USA).

[0033] The phrase "substantially identical," used in the context of two nucleic acids or polypeptides, refers to a sequence that has at least 25% sequence identity with a reference sequence. Alternatively, percent identity can be any integer from 25% to 100%. More preferred embodiments include at least: 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 94%, 95%, or 99%, compared to a reference sequence using the programs described herein; preferably BLAST using standard parameters, as described below. This definition also refers to the complement of a test sequence, when the test sequence has substantial identity to a reference sequence.

[0034] For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.

[0035] A "comparison window", as used herein, includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of from 20 to 600, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Methods of alignment of sequences for comparison are well-known in the art. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. MoI. Biol. 48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Nat 7. Acad. Sa. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by manual alignment and visual inspection. [0036] Examples of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (199O) J. MoI. Biol. 215: 403-410 and Altschul έtf α/. (1977) Nucleic Acids Res. 25: 3389-3402, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (NCBI) web site. The algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al, supra). These initial neighborhood word hits acts as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a word size (W) of 28, an expectation (E) of 10, M=I, N=-2, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a word size (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sa. USA 89: 10915 (1989)).

[0037] The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc. Natl. Acad. Sci. USA 90:5873-5787 (1993)). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.01, more preferably less than about 10^"5, and most preferably less than about 10^"20.

[0038] "Conservatively modified variants" applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, conservatively modified variants refers to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are "silent variations," which are one species of conservatively modified variations. Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of the nucleic acid. One of skill will recognize that each codon in a nucleic acid (except AUG, which is ordinarily the only codon for methionine) can be modified to yield a functionally identical molecule. Accordingly, each silent variation of a nucleic acid which encodes a polypeptide is implicit in each described sequence.

[0039] As to amino acid sequences, one of skill will recognize that individual substitutions, in a nucleic acid, peptide, polypeptide, or protein sequence which alters a single amino acid or a small percentage of amino acids in the encoded sequence is a "conservatively modified variant" where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art.

[0040] The following six groups each contain amino acids that are conservative substitutions for one another:

1) Alanine (A), Serine (S), Threonine (T); 2) Aspartic acid (D), Glutamic acid (E);

3) Asparagine (N), Glutamine (Q);

4) Arginine (R), Lysine (K);

5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and

6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W). (see, e.g., Creighton, Proteins (1984)).

[0041] As used herein, the term "drought-resistance" or "drought-tolerance," including any of their variations, refers to the ability of a plant to recover from periods of drought stress (i.e., little or no water for a period of days). Typically, the drought stress will be at least 5 days and can be as long as 18 to 20 days or more, depending on, for example, the plant species.

BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1. Molecular Analysis of T-DNA Insertion Mutants and CPK4- and CPKl 1 -Transgenic Lines. (A) T-DNA insertion site in cpk4-l (Col ecotype, SALK_081860 from ABRC). Tandem T-DNA of two copies was inserted into the genome in an inverted fashion at the same locus, which generates an 11-bp deletion from -67 to -57 bp 5 '-upstream of the translation start codon (ATG). Boxes and lines represent exons and introns, respectively (figure not drown to the scale). The locations of the primers for identification of the mutants are indicated by arrows. LB and RB represent the left and right border of T-DNA insertion, respectively; LBaI represents left border primer for T-DNA; LP2 and RP2, left and right genomic primers for CPK4 gene, respectively; and T-DNAl and T-DNA2, first and second copy of the inserted T-DNAs, respectively, noting that the two copies were inserted in an inverted manner. (B) T-DNA insertion sites in cpkll-1 (Col ecotype, SALK_023086, ABRC) and cpkll-2 (Col ecotype, SALK_054495, ABRC). Tandem T-DNA of two copies was inserted into the genome for the cpkll-1 mutant in an inverted fashion at the same locus, which generates a 34-bp deletion from -120 to -87 bp 5'-upstream of the translation start codon (ATG). A single copy of T-DNA was inserted for the cpkll-2 mutant, generating a 39- bp deletion from 320 to 358 bp downstream of the translation start codon (ATG). LPl, LP3 represent two left genomic primers for CPKIl gene; RBaI, right border primer for T-DNA; RPl, right genomic primer for CPKIl gene. Other abbreviations are the same as described in (A). (C) RT-PCR analysis OΪCPK4 (indicated by CP K4) and CPKl 1 (CPKIl) expression in wild-type Col and homozygous mutants cpk4-l, cpkll-1 and cpkll-2 and double mutants cpk4-lcpkll-l and cpk4-l cpkll-2. Actιn2/8 primers served as control. (D) Immunoblotting analysis with anti-CPKl l^c serum, which recognizes both CPKl 1 and AtCP4, in the total proteins (20 μg for each line) extracted from leaves in wild-type Col and the CPK4- overexpressing line 12 (4OE 12) and CPA77-overexpressing line 2 (11OE2). Relative band intensities, normalized relative to the intensity of Col, are indicated by numbers in box below the bands. Tubulin was taken as a control. (E) Real-time PCR and immunoblotting analysis of CPK4 and CPKl 1 during early stages before and after germination. Immunoblotting was done with anti-CPKl l^c serum in the total proteins extracted from the leaves of the seedlings grown in the MS-medium from 1 to 10 days after stratification in homozygous mutants cpk4- 1 (possessing CPKl 1) and cpkll-2 (possessing CPK4). Relative band intensities, normalized relative to the intensity with the seedling 3 d after stratification, are indicated by numbers in box below the bands. Tubulin was taken as a control. For the real-time PCR analysis for each gene, the assays were repeated three times with the independent biological experiments. The value obtained from the seedlings 3 d after stratification was taken as 100%, and all the other values were normalized relative to this value. Each value for real-time PCR is the mean ± SE of three independent biological determinations.

[0043] Figure 2. ABA Stimulates both CPK4 and CPKl 1. (A) and (B) ABA treatment enhances both protein amounts (A) and enzymatic activities (B) of CPK4 and CPKl 1, which depends on ABA dose and displays a time course. In the ABA -dose assays, germinating seeds were transferred, 48 h after stratification, to the MS-medium containing (±)ABA (0, 0.5, 1, 2, 5 μM), and ten-day-old seedlings were used for preparation of total proteins. The CPK4 plus CPKl 1 were immuno-detected with the anti-CPK4^c serum in the total proteins from Col plants (left panel in (A), indicated by 'CPK4+CPK11 in Col'), and the CPK4 with the anti-CPK4^c serum in the total proteins from the cpkll-2 mutant (left panel in (A), indicated by 'CPK4 in cpkll-2'), and the CPKl 1 with the anti-CPKl l^c serum in the total proteins from the cpk4-l mutant ((left panel in (A), indicated by 'CPKl 1 in cpk4-V). A 20- μg portion of the total proteins was used in each line for this immunoblotting. The in-gel histone-phosphorylating activity was assayed in the pure CPK4 protein obtained by immunoprecipitation with the anti-CPK4^c serum from the total proteins of the cpkll-2 mutant (left panel in (B), indicated by 'CPK4 m cpkll-V), and in the pure CPKl 1 with the anti-CPKl l^c serum form the total proteins of the cpk4-l mutant (left panel in (B), indicated by 'CPKl 1 in cpk4-V). A 50-μg portion of the total proteins was used in each line for the immunoprecipitation. In the time-course assays, the three-week-old seedlings of the cpkll-2 and cpk4-l mutants were sprayed with 50 μM (±)ABA solution and the leaves were harvested for preparing total proteins at the indicated time after the treatment (0, 30, 60, 120, 300 min). The immunoblotting was done as described above for CPK4 in the total proteins of the cpkll-2 mutant (right panel in (A), indicated by 'CPK4 in cpkll-2') and for CPKl 1 in the total proteins of the cpk4-l mutant (right panel in (A), indicated by 'CPKl 1 in cpk4-V). The in-gel histone-phosphorylating activity was assayed as described above in the immunoprecipitated CPK4 protein from the cpkll-2 mutant (right panel in (B), indicated by 'CPK4 in cpkll-2'), and in the immunoprecipitated CPKl 1 from the cpk4-l mutant (right panel in (B), indicated by 'CPKl 1 in cpk4-V). The assays described in the left panels of (A) and (B) were done with the same total proteins, and those in the right panels with another batch of the same total proteins. Tubulin was taken as a loading control. In the case of the immunoprecipitation, immunoblotting for tubulin was done with the total proteins prior to the immunoprecipitation. Relative band intensities, normalized relative to the corresponding intensity with 0 μM ABA or at 0 min time point, are indicated by numbers in box below the bands. The experiments were biologically repeated three times with the similar results.

[0044] Figure 3. Loss-of-Function Mutation in CP K4 or CPKIl Gene Results in ABA- Insensitive Phenotypes, and Overexpression of the Two CDPK Genes Leads to ABA- Hypersensitive Phenotypes, in ABA-Induced Inhibition of Seed Germination and Seedling Growth. (A) Seed germination. The germination rates were recorded in the MS-medium supplemented with 0 μM (top panel), 0.5 μM (middle panel), or 3 μM (bottom panel) (±)ABA during a period from 24 h to 72 h after stratification for wild-type Col, cpk4-l, cpkll-1 and cpkll-2 mutants, cpk4-lcpkll-l and cpk4-lcpkll-2 double mutants, mutant complementation lines 35S::CPK4/cpk4-l and 35 S: :CPK11/cpkl 1-2, and two lines overexpressing CP K4 (4OE 12) or CPKIl (11OE2). Each value is the mean ± SE of three biological determinations. (B) Seedling growth 1O d after transfer from ABA-free MS- medium to the MS-medium supplemented with different concentrations of (±)ABA for the plants as mentioned in (A). The transfer of seedlings from ABA-free medium to ABA- containing medium was done 48 h after stratification. (C) The data of primary root growth for the same lines as mentioned in (B) in the medium containing 0, 1, 5, 10, 20 or 40 μM ABA. Each value is the mean ± SE of at least 50 seedlings. (D) Postgermination growth in the MS-medium containing 0.8 μM (±)ABA 16 d after stratification for the plants as mentioned in (B). Seeds were planted in the ABA-containing medium and the postgermination growth was directly investigated 16 d after stratification without transferring the seedlings. (E) Lateral root growth in the MS-medium containing 1 μM (±)ABA 1O d after transfer from the ABA-free medium for the plants as mentioned in (B). The transfer of seedlings from ABA-free medium to ABA-containing medium was done 4 d after stratification. Top panel, status of lateral root growth. Bottom panel, statistics of lateral root growth with white columns indicating ABA-free-treatment and hatched columns ABA- treatment. Each value in the bottom panel is the mean ± SE of at least 50 seedlings.

[0045] Figure 4. Loss-of-Function Mutation in CP K4 or CPKl 1 Gene Results in NaCl- Insensitive Phenotypes in NaCl-Induced Inhibition of Seed Germination and Decreases Tolerance of Seedlings to Salt Stress. (A) Seed germination. Germination rates were recorded at 48 h, 60 h and 72 h in the MS-medium supplemented with different concentrations of NaCl from 0 mM to 200 mM for wild-type Col, cpk4-l and cpkll-2 mutants, cpk4-lcpkll-2 double mutant, and two lines overexpressing CPK4 (4OE12) or CPKIl (11OE2). Each value is the mean ± SE of three biological determinations. (B), (C) Tolerance of seedlings to salt stress. The status of seedling growth was recorded 7 d after transfer of the 4-days-old seedlings from the medium containing 170 (B) or 200 (C) mM NaCl. A map is presented in (D) for the distribution of wild-type Col, cpk4-l and cpkll-2 mutants, cpk4-lcpkll-2 double mutant, and two lines overexpressing CPK4 (4OE12) or CPKIl (11OE2) in the panels (B) and (C). The entire experiment was replicated three times with similar results.

[0046] Figure 5. Loss-of-Function Mutation in CPK4 or CPKIl Gene Decreases, but Overexpression of the Two CDPK Genes Enhances, Stomata-Responsiveness to ABA and Ability of Preserving Water in Leaves. (A) ABA-induced stomatal closure (top panel) and inhibition of stomatal opening (bottom panel) for wild-type Col, cpk4-l and cpkll-2 mutants, cpk4-lcpkll-2 double mutant, and a line overexpressing CPKIl (11OE2). Values are the means ± SE from three independent experiments; n = 60 apertures per experiment. (B) Water loss rates during a 6 h-period from the detached leaves of wild-type Col, cpk4-l, cpkll-1 and cpkll-2 mutants, cpk4-lcpkll-l and cpk4-lcpkll-2 double mutants, mutant complementation lines 35S::CPK4/cpk4-l and 35 S: :CPK11/cpkl 1-1 , and two lines overexpressing CP K4 (4OE 12) or CPKIl (11OE2). Values are the means ± SE of five individual plants per genotype. The entire experiment was replicated five times with similar results. (C) Survival rate for wild-type and different mutant lines as mentioned in (B). Drought was imposed on the three-week-old plants by withholding water until the lethal effects was observed on the knockout mutant plants, then the plants were re-watered and survival rate was recorded one week later. Values are the means ± SE from three independent experiments; n = 50 plants per line for each experiment. (D), (E) Whole plant status in the water loss assays. For assaying water loss from whole plants of the different lines as mentioned in (B), intact plants were well-watered (Control) or drought stressed by withholding water (Drought) for 15 d (D), or for 18 d for assaying water loss of the two lines overexpressing CPK4 (4OE 12) or CPKIl (11OE2) in comparison with wild-type Col (E). The entire experiment was replicated three times with similar results. [0047] Figure 6. Two Protein Kinases CPK4 and CPKl 1 Phosphorylate both ABFl and ABF4. The three-week-old seedlings of the different genotypes were sprayed with 0 μM or 50 μM (±)ABA solution, and were sampled 1 h after the spraying. The quantity of the total proteins, prepared from leaves and used in each lane of the following assays was 50 μg. Tubulin was used as a protein loading control. (A), (B) Mapping of protein kinases phosphorylating ABFl (A) and ABF4 (B). The recombinant ABFl or ABF4 (0.5 mg/mL) were embedded in the separating polyacrylamide SDS gel. Total proteins from wide-type 'Col' and cpk4-l cpkll-2 double mutant were separated on the gel and assayed to in-gel phosphorylate the two substrates. At the same time after electrophoresis, the gels harboring the total proteins from the ABA-free-treated wild-type plants (other gels than those for phosphorylation) were used to detect immuno-signals with anti-CPK4^c serum to provide a reference for the position of the CPK4/CPK11 proteins in the lanes of phosphorylation ('58 kD CPK4/CPK11'). '-ABA' and '+ABA' indicate the treatments with 0 μM or 50 μM (±)ABA, respectively. The assays were repeated three times with the same results. (C), (D) Phosphorylation of ABFl (C) or ABF4 (D) by CPK4 and CPKl 1. The mixed proteins of two kinases ('CPK4 + CPKl 1 in Col') were obtained by immunoprecipitation with anti-CPK4^c serum from the total proteins of wild-type Col, and the pure CPKl 1 ('CPKl 1 in cpk4-F) and CPK 4 ('CPK4 in cpkl 1-2') were immuno-precipitated with the anti-CPKl l^c serum from the total proteins of cpk4-l mutant and with anti-CPK4^c serum from the total proteins of cpkl 1-2 mutant, respectively. The total proteins from the double mutant cpk4-lcpkll-2 were also immuno-precipitated with anti-CPK4^c serum for obtaining 'background in cpk4-l cpkl 1-2' as a negative control to show the absence of activity other than CPK4/11 in these assays. The ABFl and AB F4 were in-gel phosphorylated by the immuno-precipitated proteins as described in (A) and (B). Top panels (columns) represent the relative band intensities of the phosphorylated ABFl or ABF4 shown in middle panels, normalized relative to the corresponding intensity of the wild-type Col with 0 μM-(±)ABA treatment (100%). Values are the means ± SE from three biological independent experiments. Immunoblotting for tubulin (bottom panels) was done with the total proteins prior to the immunoprecipitation. Symbols '-' and '+' indicate the treatments with 0 μM and 50 μM (±)ABA, respectively.

[0048] Figure 7. Expression of ABA-Responsive Genes in the CPK4- and CPKl 1 -Loss- of-Function Mutants and Overexpressing lines. Expression of ABA-responsive genes was assayed by real-time PCR in the leaves of wild-type Col, cpk4-l and cpkl 1-2 mutants, cpk4- 1 cpkl 1-2 double mutant, and two lines overexpressing CPK4 (4OE12) or CPKIl (11OE2). - ABA, ABA-free treatment; + ABA, 50 μM (±)ABA treatment. The expression levels were presented as relative units with the levels of ABA -treated Col leaves being taken as 100 %. Each value is the mean ± SE of three independent biological determinations.

[0049] Figure 8. Identification of T-DNA Insertion for cpk4-l , cpkl 1 -1 and cpkl 1 -2 Mutations in the Arabidopsis Genome by PCR Analysis. The left (LBaI) and right (RBaI) border primers for T-DNA insertion, the left (LPl, LP3) and right (RPl) genomic primers for CPKIl gene, and the left (LP2) and right (RP2) genomic primers for CPK4 gene, are presented in Table 3. (A) The genomic sequences spanning the potential inserted T-DNA region between LPl and RPl (CPKIl) or between LP2 and RP2 (CP K4) for Col are intact, whereas disrupted in cpkl 1-1, cpkl 1-2 or cpk4-l mutants. However, the sequences of the T- DNA insertion between LBaI and RPl in both cpkl 1-1 and cpkl 1-2 mutants or between LBaI and RP2 in cpk4-l mutant are detected, but not in the wild-type Col. (B) The sequences of the T-DNA insertion between LP3 and LBaI in cpkl 1-1, and between LPl and RBaI in cpkl 1-2, as well as between LP2-LBal in cpk4-l were also detected. These results show the occurrence of the T-DNA insertion in the CPK4 gene in the cpk4-l mutant and in the CPKIl gene in the cpkll-1 and cpkll-2 mutants, and indicate that one single copy T- DNA is present in the cpkll-2 mutant, but tandem T-DNAs were inserted in an inverted manner into the genome for the cpk4-l and cpkll-1 mutants. Mr, molecular markers. [0050] Figure 9. Southern-Blot Analysis for the T-DNA Insertion in cpk4-l, cpkll-1 and cpkll-2 Mutants. A 10-μg portion of Arabidopsis genomic DNA isolated from the cpk4-l, cpkll-1 and cpkll-2 mutants was digested with EcorRl plus Pstl and Hindlϊl, respectively, electrophoresed in a 0.8% agarose gel, and transferred onto a nylon membrane. The membranes were hybridized with the ³²P -labelled specific probe for the T-DNA (see METHODS). The results indicate that one single copy of the T-DNA was inserted into the genome for the cpkll-2 mutant, and tandem T-DNA of two copies was inserted into the genome for the cpk4-l and cpkll-1 mutants (for tandem T-DNA insertion, see also the results of sequencing of the T-DNA flanking sequences in Table 3).

[0051] Figure 10. Alignment of Deduced Amino Acid Sequences of CPK4 and CPKl 1. Identical amino acid residues are indicated by white letters on a black background. Gaps, indicated by points (.), were introduced to maximize alignment. The two CPKs share high sequence identity (94%). The C-terminal fragment of CPK4 from amino acid 386 to 501 (indicated by top line) was used to produce anti-CPK4^c serum, and the C-terminal fragment of CPKl 1 from amino acid 387 to 495 (indicated by bottom line) was used to produce anti- CPKl l^c serum.

[0052] Figure 11. Subcellular Localization of CPK4 and CPKl 1. Expression of CPK4:GFP (top panel) and CPKl 1:GFP (bottom panel) fusion proteins in the root cells of Arabidopsis transgenic plants. The fusion proteins of both CDPKs are present in cytoplasm and nucleus, shown by the CPK4:GFP and CPKl 1:GFP fluorescence images (left panels) under laser-scanning confocal microscope. The right panels show the corresponding bright field. For generation of the transgenic CPK4. GFP- and CPKl 1 :GFP-expressing lines, see METHODS.

[0053] Figure 12. Expression of CPK4 and CPKl 1 in Different Tissues and during Different Periods. (A) Immunoblotting analysis with anti-CPKl l^c serum in the total proteins extracted from different tissues in wild-type Col and homozygous mutants cpk4-l, cpkll-1 and cpkll-2 and double mutants cpk4-l cpkll-1 and cpk4-l cpkll-2. Tubulin was taken as a loading control. (B) Immunoblotting analysis with anti-CPKl l^c serum in the total proteins extracted from leaves during different growth periods in wild-type Col and homozygous mutants cpk4-l and cpkll-2. Tubulin was taken as a loading control. Because the anti- CPK4^C or anti-CPKl l^c serum is able to recognize both CPKl 1 and CPK4 (see METHODS), the immuno-signal detected by either of the antisera in wild-type Col is CPK4 plus CPKl 1; and in the knockout mutant cpk4-l presents CPKl 1, and in the cpkll-1 and cpkll-2, CPK4.

[0054] Figure 13. ABA Concentrations in the Different Mutants. Three -week-old plants of the mutants cpk4-l, cpkll-2 and cpk4-l cpkll-2, CPK4- and CPA77-overexpressors (4OE 12 and 11OE2, respectively) and wild-type Col were subjected to drought treatment (withholding water for 1 d, 5 d and 1O d, respectively), and the rosette leaves from these plants were used to assay ABA concentrations by ELISA method as described in Chen et al. (2006) Plant Physiol 140, 302-310.

[0055] Figure 14. Enzymatic Characterization of CPK4 and CPKl 1. (A) Ca²⁺-dependent electrophoretic mobility shift of CPK4 (left panel) and CPKl 1 (right panel) in the assay of in- gel autophosphorylation activity. The CPK4 protein was obtained by immunoprecipitation in the total proteins prepared from the three-week-old seedling of the cpkll-2 mutant with the anti-CPK4^c serum, and the CPKl 1 protein from the total proteins of the cpk4-l mutant with the anti-CPKl l^c serum. Ca²⁺ or EGTA was added to the immunoprecipitated proteins dissolved in SDS-PAGE sample buffer. After SDS-PAGE, the in-gel phosphorylation assay was done in the presence of Ca²⁺. - and + indicate the absence of Ca²⁺ (in the presence of EGTA) and presence of Ca²⁺ in the SDS-PAGE buffer, respectively. (B) Inhibition of the histone-phosphorylating activity of the CPK4 and CPKl 1 by CaM antagonists or kinase inhibitors. The CPK4 protein from cpkll-2 mutant (panel above) and CPKl 1 from cpk4-l mutant (panel below) were prepared as described above in (A) by immunoprecipitation. CaM (form bovine brain, Sigma) was used at 5 μM; TFP, W7 and W5 at 250 μM, and K252a at 10 μM. These reagents were added, respectively, to the phosphorylation reaction medium (buffer B as described in METHODS) for a preincubation and a subsequent reaction incubation for ³²P -labeling to the kinase substrate histone. - and + indicate the absence and presence of Ca²⁺ in the reaction buffer, respectively. The gels phosphorylated in the different reaction media were grouped to detect the phosphorylated histone bands by autoradiography. DETAILED DESCRIPTION I. Introduction

[0056] The phytohormone abscisic acid (ABA) regulates many processes of plant growth and development such as seed maturation and germination, seedling growth, flowering, and stomatal movement, and is a central hormone to control plant adaptation to various environmental challenges including drought, salt and cold stresses (reviewed in Koornneef et al. (1998) Plant Physiol Biochem 36, 83-89; Leung and Giraudat, (1998) Ann Rev Plant Physiol Plant MoI Biol 49, 199-222; and Finkelstein and Rock(2002) Abscisic acid biosynthesis and signaling. In The Arabidopsis Book, CR. Somerville and E.M. Meyerowitz, eds (Rockville, MD: American Society of Plant Biologists)). Numerous cellular components that modulate ABA responses (reviewed in Finkelstein et al., (2002) Plant Cell 14 (suppl.), S15-S45; Himmelbach et al., (2003) Curr Opin Plant Biol 6, 470-479; and Fan et al., (2004) Curr Opin Plant Biol 7, 537-546), and three ABA receptors, FCA controlling flowering time (Razem et al., (2006) Nature 439, 290-294) and ABAR and GCR2 regulating seed germination, seedling growth and stomatal movement (Shen et al., (2006) Nature 443, 823- 826; Liu et al., (2007) Science 315, 1712-1715), have been identified, which leads to considerable progress in understanding the ABA signaling pathway. However, the ABA signaling network is largely unknown, and many signaling components remain to be discovered. [0057] Calcium plays an essential role in plant cell signaling (Hepler, (2005) Plant Cell 17, 2142-2155), and has been shown to be an important second messenger involved in ABA signal transduction (reviewed in Finkelstein et al., 2002, supra; Himmelbach et al., 2003, supra; and Fan et al., 2004, supra). Calcium signaling is modulated by specific calcium signatures, i.e., the specific patterns of variations in the amplitude, duration, location and frequency of cytosolic free Ca²⁺-spikes in response to different stimuli. These specific calcium signatures are recognized by different calcium sensors to transduce specific calcium- mediating signals into downstream events (Sanders et al., (1999) Plant Cell 11, 691-706; Harmon et al., (2000) Trends Plant Sci 5, 154-159; Rudd and Franklin-Tong, (2001) New Phytologist 151, 7-33). Plants have several classes of calcium sensor proteins, including calmodulin (CaM) and CaM-related proteins (Zielinski, (1998) Ann Rev Plant Physiol Plant MoI Biol 49, 697-725; Snedden and Fromm, (2001) New Phytol 151, 35-66; Luan et al., (2002) Plant Cell 14 (suppl.), S389-S400), calcineurin B-like (CBL) proteins (Luan et al., 2002, id), and calcium-dependent protein kinases (CDPKs) (Harmon et al., (2001) New Phytol 151, 175-183; Cheng et al., (2002) Plant Physiol 129, 469-485). A CBL-interacting protein kinase CIPK 15 interacts with two calcium-modulated protein phosphatases (PPs) 2C ABIl and ABI2 (Guo et al., (2002) Dev Cell, 3, 233-244), both of which are the most characterized negative regulators of ABA signaling (Leung et al., (1994) Science 264, 1448- 1452; Meyer et al., (1994) Science 264, 1452-1455; Leung et al., (1997) Plant Cell 9, 759- 771; Sheen, (1998) Proc Natl Acad Sa USA 95, 975-980; Gosti et al., (1999) Plant Cell 11, 1897-1909; Merlot et al., (2001) Plant J25, 295-303). CIPK15 and its homolog CIPK3 and CBL9 negatively regulate ABA signaling (Guo et al., 2002, supra; Kim et al., (2003) Plant Cell 15, 411-423; Pandey et al., (2004) Plant Cell 16, 1912-1924), possibly by acting as Ca²⁺- sensors upstream of the PP2Cs ABIl and ABI2 (Pandey et al., 2004, id) when forming a protein complex for perceiving calcium signal (Allen et al., (1999) Plant Cell 11, 1785- 1798). Additionally, an AP2 transcription factor AtERF7 that negatively regulates ABA response was shown to be a kinase substrate of CIPK15 (Song et al., (2005) Plant Cell 17, 2384-2396), suggesting that CIPK 15 may regulate ABA signaling more directly by phosphorylating transcription factor and modulating gene expression.

[0058] CDPKs are the best characterized calcium sensor in plants, which are structurally Ser/Thr protein kinases and have an N-terminal kinase domain joined to a C-terminal CaM- like domain via a junction region that serves to stabilize and maintain kinase in an auto- inhibited state (Harper et al., (1991) Science 252, 951-954; Harper et al., (1994) Biochemistry 33, 7267-7277; Harmon et al., 2001, supra; Cheng et al., 2002, supra). CDPKs are encoded by a large multigene family with possible redundancy and/or diversity in their functions (Harmon et al., 2001, supra; Cheng et al., 2002, supra). Growing evidence indicates that CDPKs regulate many aspects in plant growth and development as well as plant adaptation to biotic and abiotic stresses (Bachmann et al., (1995) Plant Physiol 108, 1083-1091; Bachmann et al., (1996) Plant Cell 8, 505-517; McMichael et al., (1995) Plant Physiol. 108, 1077-1082; Pei et al., (1996) EMBO J. 15, 6564-6574; Sheen, (1996) Science 21 A, 1900-1902; Li et al., (1998) Plant Physiol. 116, 785-795; Sugiyama et al., (2000) J. Phycol. 36, 1145-1152; Romeis, et al., (2001) EMBO J20, 5556-5567; Hrabak et al., (2003) Plant Physiol. 132, 666- 680; Shao and Harmon, (2003) Plant MoI. Biol. 53, 731-740; McCubbin et al., (2004) Plant J. 39: 206-218; Choi et al., (2005) Plant Physiol. 139, 1750-1761; Ivashuta et al., (2005) Plant Cell 17, 2911-2921; Mori et al., (2006) Plos Biol. 4, 1749-1761). In plant hormone signaling, CDPKs are believed to be important regulators to be involved in various signaling pathways (Cheng et al., 2002, supra; Ludwig et al., (2004) J Exp Bot 55, 181-188). The constitutively ectopic expression of two Arabidopsis CDPKs CPK10/CDPK1 (Arabidopsis gene identifier number Atlg74740) and CPK30/CDPKla (AtIg 18890) in maize leaf protoplasts activated a stress- and ABA-inducible promoter, showing the connection of CDPKs to ABA signaling pathway (Sheen, 1996, supra). An Arabidopsis CDPK, CPK32, was shown to interact with an ABA-induced transcription factor ABF4, and constitutive overexpression of CPK32 resulted in ABA-hypersensitive phenotypes in ABA-induced inhibition of seed germination (Choi et al., 2005, supra). However, molecular genetic evidence via gene disruption has been scarce to unequivocally link defined CDPK genes with ABA-regulated biological functions such as seed maturation and germination, seedling growth, stomatal movement, and plant stress tolerance. To our knowledge, only have the Arabidopsis CDPKs CPK3 and CPK6 recently been identified through gene knockout mutation as players in ABA-regulated stomatal signaling, but the ABA-induced phenotypes in seed germination or postgermination growth were not observed in the loss-of-function mutants of these two CDPK genes, and alteration in plant tolerance to environmental stresses associated with ABA signaling possibly due to the gene disruption of the two CDPKs was not reported (Mori et al., 2006, supra). The Arabidopsis CDPK gene family includes 34 members (Cheng et al., 2002, supra). Redundancies in the functions of CDPK genes have been believed to hamper genetically functional analysis of these CDPKs.

[0059] The present inventors previously identified an ABA-stimulated CDPK, ACPKl, from grape berry, which may be involved in ABA signaling (Yu et al., (2006) Plant Physiol 140, 558-579; Yu et al., (2007) Plant MoI. BwI. 64, 531-538). Further studies have since been conducted in Arabidopsis to explore the biological functions of the two closest homologues of ACPKl, CPK4 and CPKl 1 in ABA signaling pathways. Here the inventors report that CPK4 and CPKl 1 are positive regulators in the CDPK/calcium-mediated ABA signaling processes involving seed germination, seedling growth, guard cell regulation, and plant tolerance to salt stress, which provide clear, inplanta genetic, evidence for the modulation of CDPK/calcium in ABA signal transduction at the whole -plant level. Accordingly, when ABA sensitivity is increased by overexpressing CPK4 or CPKI l, desirable characteristics in plants such as increased stress (e.g., drought) tolerance and delayed seed germination are achieved. II. Calcium-dependent Protein Kinases CPK4 and CPKIl

[0060] A wide variety of CPK4 and CPKl 1 polypeptide sequences are known in the art and can be used according to the methods and compositions of the invention. A list of some known CPK4 and CPKI l homologs from various species is provided in Table 1.

Table 1

[0061] The present invention provides for use of the above proteins and/or nucleic acid sequences, or sequences substantially identical (e.g., 50%, 70%, 75%, 78%, 80%, 85%, 90%, 94%, 95%, 98% identical) to those listed above in the methods and compositions (e.g., expression cassettes, plants, etc.) of the present invention. In situations where variants of the above sequences are desired, it can be useful to generate sequence alignments to identify conserved amino acid or motifs (i.e., where alteration in sequences may alter protein function) and regions where variation occurs in alignment of sequences (i.e., where variation of sequence is not likely to significantly affect protein activity).

[0062] As shown in Table 2, these known plant CPK homolog polypeptide sequences are at least about 50% identical to the Arabidopsis sequences (SEQ ID NO:2 and 4), most having at least 70% or 75% sequence identity.

Table 2

[0063] The isolation of a polynucleotide sequence encoding a plant CPK4 or CPKl 1 (e.g., from plants where CPK4 or CPKl 1 sequences have not yet been identified) may be accomplished by a number of techniques. For instance, oligonucleotide probes based on the CPK4 or CPKl 1 coding sequences disclosed (e.g., as listed in Table 1) here can be used to identify the desired CPK4 or CPKl 1 gene in a cDNA or genomic DNA library. To construct genomic libraries, large segments of genomic DNA are generated by random fragmentation, e.g., using restriction endonucleases, and are ligated with vector DNA to form concatemers that can be packaged into the appropriate vector. To prepare a cDNA library, mRNA is isolated from the desired tissue, such as a leaf from a particular plant species, and a cDNA library containing the gene transcript of interest is prepared from the mRNA. Alternatively, cDNA may be prepared from mRNA extracted from other tissues in which CPK4 or CPKl 1 gene is expressed.

[0064] The cDNA or genomic library can then be screened using a probe based upon the sequence of a CPK4 or CPKI l gene disclosed here (e.g., as listed in Table 1). Probes may be used to hybridize with genomic DNA or cDNA sequences to isolate homologous genes in the same or different plant species. Alternatively, antibodies raised against an polypeptide can be used to screen an mRNA expression library.

[0065] Alternatively, the nucleic acids encoding a CPK4 or CPKl 1 can be amplified from nucleic acid samples using amplification techniques. For instance, polymerase chain reaction (PCR) technology can be used to amplify the coding sequences of a CPK4 or CPKl 1 directly from genomic DNA, from cDNA, from genomic libraries or cDNA libraries. PCR and other in vitro amplification methods may also be useful, for example, to clone polynucleotide sequences encoding a CPK4 or CPKl 1 to be expressed, to make nucleic acids to use as probes for detecting the presence of the desired mRNA in samples, for nucleic acid sequencing, or for other purposes. For a general overview of PCR see PCR Protocols: A

Guide to Methods and Applications . (Innis, M, Gelfand, D., Sninsky, J. and White, T., eds.), Academic Press, San Diego (1990). Appropriate primers and probes for identifying sequences from plant tissues are generated from comparisons of the sequences provided here with other related genes.

[0066] The advancement in studies of plant genomes also permits a person of skill in the art to quickly determine the CPK4 or CPKl 1 coding sequence for a selected plant. For instance, the partial or entire genome of a number of plants has been sequenced and open reading frames identified. By a routine BLAST search, one can immediately identify the coding sequence for a CPK4 or CPKl 1 in various plants.

III. Use of CPK4 or CPKIl nucleic acids of the invention [0067] The invention provides methods of modulating ABA sensitivity in a plant by altering CPK4 or CPKl 1 expression or activity, for example, by introducing into a plant a recombinant expression cassette comprising a regulatory element (e.g., a promoter) operably linked to a CPK4 or CPKl 1 polynucleotide, i.e. , a nucleic acid encoding a CPK4 or CPKl 1 or a sequence comprising a portion of the sequence of a CPK4 or CPKl 1 mRNA or complement thereof. [0068] In some embodiments, the methods of the invention comprise increasing and/or ectopically expressing a CPK4 or CPKl 1 polypeptides in a plant. Such embodiments are useful for increasing ABA sensitivity of a plant, and resulting in, for example, improved stress (e.g., drought) tolerance and/or delayed seed germination (to avoid pre-mature germination, for example as can occur in humid environments or due to other exposure to moisture). For stress tolerance, promoters can be selected that are generally constitutive and are expressed in most plant tissues, or can be leaf or root specific. To affect seed germination, promoters are generally used that result in expression in seed or, in some embodiments, floral organs or embryos.

[0069] In some embodiments, the methods of the invention comprise decreasing endogenous CPK4 or CPKl 1 expression in plant, thereby decreasing ABA sensitivity in the plant. Such methods can involve, for example, mutagenesis (e.g., chemical, radiation, transposon or other mutagenesis) of CPK4 or CPKl 1 sequences in a plant to reduce CPK4 or CPKl 1 expression or activity, or introduction of a polynucleotide substantially identical to at least a portion of a CPK4 or CPKI l cDNA sequence or a complement thereof (e.g., an "RNAi construct") to reduce CPK4 or CPKl 1 expression. Decreased (or increased) CPK4 or CPKl 1 expression can be used to control the development of abscission zones in leaf petioles and thereby control leaf loss, i.e., delay leaf loss if expression is decreased and speed leaf loss if expression is increased in abscission zones leaf.

A. Increasing CPK4 or CPKl 1 expression or activity

[0070] Isolated sequences prepared as described herein can also be used to prepare expression cassettes that enhance or increase CPK4 or CPKl 1 gene expression. Where overexpression of a gene is desired, the desired gene from a different species may be used to decrease potential sense suppression effects.

[0071] Any of a number of means well known in the art can be used to increase CPK4 or CPKl 1 activity in plants. Any organ or plant part can be targeted, such as shoot vegetative organs/structures (e.g. leaves, stems and tubers), roots, flowers and floral organs/structures (e.g. bracts, sepals, petals, stamens, carpels, anthers and ovules), seed (including embryo, endosperm, and seed coat), fruit, abscission zone, etc. Alternatively, one or several CPK4 or CPKl 1 genes can be expressed constitutively (e.g., using the CaMV 35S promoter or other constitutive promoter). [0072] One of skill will recognize that the polypeptides encoded by the genes of the invention, like other proteins, have different domains which perform different functions. Thus, the overexpressed or ectopically expressed polynucleotide sequences need not be full length, so long as the desired functional domain of the protein is expressed. Alternatively, or in addition, active CPK4 or CPKl 1 proteins can be expressed as fusions, without necessarily significantly altering CPK4 or CPKl 1 activity. Examples of fusion partners include, but are not limited to, poly-His or other tag sequences.

B. Decreasing CPK4 or CPKl 1 expression or activity

[0073] A number of methods can be used to inhibit gene expression in plants. For instance, antisense technology can be conveniently used. To accomplish this, a nucleic acid segment from the desired gene is cloned and operably linked to a promoter such that the antisense strand of RNA will be transcribed. The expression cassette is then transformed into plants and the antisense strand of RNA is produced. In plant cells, it has been suggested that antisense RNA inhibits gene expression by preventing the accumulation of mRNA which encodes the enzyme of interest, see, e.g., Sheehy et al., Proc. Nat. Acad. Sa. USA, 85:8805-8809 (1988); Pnueli et al, The Plant Cell 6: 175-186 (1994); and Hiatt et al, U.S. Patent No. 4,801,340. [0074] The antisense nucleic acid sequence transformed into plants will be substantially identical to at least a portion of the endogenous gene or genes to be repressed. The sequence, however, does not have to be perfectly identical to inhibit expression. Thus, an antisense or sense nucleic acid molecule encoding only a portion of CPK4 or CPKl 1, or a portion of the CPK4 or CPKl 1 cDNA, can be useful for producing a plant in which CPK4 or CPKl 1 expression is suppressed. The vectors of the present invention can be designed such that the inhibitory effect applies to other proteins within a family of genes exhibiting homology or substantial homology to the target gene.

[0075] For antisense suppression, the introduced sequence also need not be full length relative to either the primary transcription product or fully processed mRNA. Generally, higher homology can be used to compensate for the use of a shorter sequence. Furthermore, the introduced sequence need not have the same intron or exon pattern, and homology of non- coding segments may be equally effective. For example, a sequence of between about 30 or 40 nucleotides can be used, and in some embodiments, about full length nucleotides should be used, though a sequence of at least about 20, 50 100, 200, or 500 nucleotides substantially identical to SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, or 15, or an endogenous CPK4 or CPKl 1 mRNA or cDNA can be used.

[0076] Catalytic RNA molecules or ribozymes can also be used to inhibit expression of CPK4 or CPKl 1 genes. It is possible to design ribozymes that specifically pair with virtually any target RNA and cleave the phosphodiester backbone at a specific location, thereby functionally inactivating the target RNA. In carrying out this cleavage, the ribozyme is not itself altered, and is thus capable of recycling and cleaving other molecules, making it a true enzyme. The inclusion of ribozyme sequences within antisense RNAs confers RNA-cleaving activity upon them, thereby increasing the activity of the constructs. [0077] A number of classes of ribozymes have been identified. One class of ribozymes is derived from a number of small circular RNAs that are capable of self-cleavage and replication in plants. The RNAs replicate either alone (viroid RNAs) or with a helper virus (satellite RNAs). Examples include RNAs from avocado sunblotch viroid and the satellite RNAs from tobacco ringspot virus, lucerne transient streak virus, velvet tobacco mottle virus, solanum nodiflorum mottle virus and subterranean clover mottle virus. The design and use of target RNA-specific ribozymes is described in Haseloff et al. Nature, 334:585-591 (1988). [0078] Another method of suppression is sense suppression (also known as co- suppression). Introduction of expression cassettes in which a nucleic acid is configured in the sense orientation with respect to the promoter has been shown to be an effective means by which to block the transcription of target genes. For an example of the use of this method to modulate expression of endogenous genes see, Napoli et al, The Plant Cell 2:279-289

(1990); Flavell, Proc. Natl. Acad. Sa., USA 91:3490-3496 (1994); Kooter and MoI, Current Opm. Biol. 4: 166-171 (1993); and U.S. Patents Nos. 5,034,323, 5,231,020, and 5,283,184.

[0079] Generally, where inhibition of expression is desired, some transcription of the introduced sequence occurs. The effect may occur where the introduced sequence contains no coding sequence per se, but only intron or untranslated sequences homologous to sequences present in the primary transcript of the endogenous sequence. The introduced sequence generally will be substantially identical to the endogenous sequence intended to be repressed. This minimal identity will typically be greater than about 65%, but a higher identity can exert a more effective repression of expression of the endogenous sequences. In some embodiments, sequences with substantially greater identity are used, e.g. , at least about 80, at least about 95%, or as high as 100% identity are used. As with antisense regulation, the effect can be designed and tested to apply to any other proteins within a similar family of genes exhibiting homology or substantial homology.

[0080] For sense suppression, the introduced sequence in the expression cassette, needing less than absolute identity, also need not be full length, relative to either the primary transcription product or fully processed mRNA. This may be preferred to avoid concurrent production of some plants that are overexpressers. A higher identity in a shorter than full length sequence compensates for a longer, less identical sequence. Furthermore, the introduced sequence need not have the same intron or exon pattern, and identity of non- coding segments will be equally effective. Normally, a sequence of the size ranges noted above for antisense regulation is used, i.e., 30-40, or at least about 20, 50, 100, 200, 500 or more nucleotides substantially identical to SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, or 15, or an endogenous CPK4 or CPKl 1 mRNA or cDNA can be used.

[0081] Endogenous gene expression may also be suppressed by means of RNA interference (RNAi) (and indeed co-suppression can be considered a type of RNAi), which uses a double- stranded RNA having a sequence identical or similar to the sequence of the target gene. RNAi is the phenomenon in which when a double-stranded RNA having a sequence identical or similar to that of the target gene is introduced into a cell, the expressions of both the inserted exogenous gene and target endogenous gene are suppressed. The double-stranded RNA may be formed from two separate complementary RNAs or may be a single RNA with internally complementary sequences that form a double-stranded RNA. Although complete details of the mechanism of RNAi are still unknown, it is considered that the introduced double-stranded RNA is initially cleaved into small fragments, which then serve as indexes of the target gene in some manner, thereby degrading the target gene. RNAi is known to be also effective in plants (see, e.g., Chuang, C. F. & Meyerowitz, E. M., Proc. Natl. Acad. Sa. USA 97: 4985 (2000); Waterhouse et al., Proc. Natl. Acad. Sa. USA 95: 13959-13964 (1998); Tabara et al. Science 282:430-431 (1998); Matthew, Comp Funct Genom 5: 240-244 (2004); Lu, et at, Nucleic Acids Research 32(21):el71 (2004)). For example, to achieve suppression of the expression of a DNA encoding a protein using RNAi, a double-stranded RNA having the sequence of a DNA encoding the protein, or a substantially similar sequence thereof (including those engineered not to translate the protein) or fragment thereof, is introduced into a plant of interest. The resulting plants may then be screened for a phenotype associated with the target protein and/or by monitoring steady-state RNA levels for transcripts encoding the protein. Although the genes used for RNAi need not be completely identical to the target gene, they may be at least 70%, 80%, 90%, 95% or more identical to the target gene sequence. See, e.g., U.S. Patent Application Publication No. 2004/0029283. The constructs encoding an RNA molecule with a stem-loop structure, which is unrelated to the target gene and positioned distally to a sequence specific for the gene of interest, may also be used to inhibit target gene expression. See, e.g., U.S. Patent Application Publication No. 2003/0221211.

[0082] The RNAi polynucleotides can encompass the full-length target RNA or may correspond to a fragment of the target RNA. In some cases, the fragment will have fewer than 100, 200, 300, 400, 500 600, 700, 800, 900 or 1,000 nucleotides corresponding to the target sequence. In addition, in some embodiments, these fragments are at least, e.g., 50, 100, 150, 200, or more nucleotides in length. In some cases, fragments for use in RNAi will be at least substantially similar to regions of a target protein that do not occur in other proteins in the organism or may be selected to have as little similarity to other organism transcripts as possible, e.g., selected by comparison to sequences in analyzing publicly-available sequence databases.

[0083] Expression vectors that continually express siRNA in transiently- and stably- transfected have been engineered to express small hairpin RNAs, which get processed in vivo into siRNAs molecules capable of carrying out gene-specific silencing (Brummelkamp et at, Science 296:550-553 (2002), and Paddison, et at, Genes & Dev. 16:948-958 (2002)). Post- transcriptional gene silencing by double -stranded RNA is discussed in further detail by Hammond et al. Nature Rev Gen 2: 110-119 (2001), Fire et at Nature 391: 806-811 (1998) and Timmons and Fire Nature 395: 854 (1998).

[0084] One of skill in the art will recognize that using technology based on specific nucleotide sequences (e.g., antisense or sense suppression technology), families of homologous genes can be suppressed with a single sense or antisense transcript. For instance, if a sense or antisense transcript is designed to have a sequence that is conserved among a family of genes, then multiple members of a gene family can be suppressed.

Conversely, if the goal is to only suppress one member of a homologous gene family, then the sense or antisense transcript should be targeted to sequences with the most variance between family members.

[0085] Another means of inhibiting CPK4 or CPKl 1 function in a plant is by creation of dominant negative mutations. In this approach, non-functional, mutant CPK4 or CPKl 1 polypeptides, which retain the ability to interact with proteins upstream or downstream from the wild-type CPK4 or CPKl 1 in the ABA signaling pathway, are introduced into a plant. A dominant negative construct also can be used to suppress CPK4 or CPKl 1 expression in a plant. A dominant negative construct useful in the invention generally contains a portion of the complete CPK4 or CPKl 1 coding sequence sufficient, for example, for DNA -binding or for a protein-protein interaction such as a homodimeric or heterodimeric protein-protein interaction but lacking the transcriptional activity of the wild-type protein.

IV. Recombinant Expression Vector

[0086] Once the coding or cDNA sequence for CPK4 or CPKl 1 is obtained, it can also be used to prepare an expression cassette for expressing the CPK4 or CPKl 1 protein in a transgenic plant, directed by a heterologous promoter. Increased expression of CPK4 or CPKl 1 polynucleotide is useful, for example, to produce plants with enhanced drought- resistance. Alternatively, as described above, expression vectors can also be used to express CPK4 or CPKl 1 polynucleotides and variants thereof that inhibit endogenous CPK4 or CPKI l expression.

[0087] Any of a number of means well known in the art can be used to increase or decrease CPK4 or CPKl 1 activity or expression in plants. Any organ can be targeted, such as shoot vegetative organs/structures (e.g. leaves, stems and tubers), roots, flowers and floral organs/structures (e.g. bracts, sepals, petals, stamens, carpels, anthers and ovules), seed (including embryo, endosperm, and seed coat) and fruit. Alternatively, the CPK4 or CPKl 1 gene can be expressed constitutively (e.g., using the CaMV 35 S promoter). [0088] To use CPK4 or CPKl 1 coding or cDNA sequences in the above techniques, recombinant DNA vectors suitable for transformation of plant cells are prepared. Techniques for transforming a wide variety of higher plant species are well known and described in the technical and scientific literature. See, e.g., Weising et al. Ann. Rev. Genet. 22:421-477 (1988). A DNA sequence coding for the CPK4 or CPKl 1 polypeptide preferably will be combined with transcriptional and translational initiation regulatory sequences which will direct the transcription of the sequence from the gene in the intended tissues of the transformed plant.

[0089] For example, a plant promoter fragment may be employed to direct expression of the CPK4 or CPKl 1 gene in all tissues of a regenerated plant. Such promoters are referred to herein as "constitutive" promoters and are active under most environmental conditions and states of development or cell differentiation. Examples of constitutive promoters include the cauliflower mosaic virus (CaMV) 35 S transcription initiation region, the 1'- or T- promoter derived from T-DNA of Agrobacterium tumafaciens, and other transcription initiation regions from various plant genes known to those of skill. [0090] Alternatively, the plant promoter may direct expression of the CPK4 or CPKl 1 protein in a specific tissue (tissue-specific promoters) or may be otherwise under more precise environmental control (inducible promoters). Examples of tissue-specific promoters under developmental control include promoters that initiate transcription only in certain tissues, such as leaves or guard cells (including but not limited to those described in WO/2005/085449; U.S. Patent No. 6,653,535; Li et al, Sa China C Life Sci. 2005

Apr;48(2): 181-6; Husebye, et al., Plant Physiol, April 2002, Vol. 128, pp. 1180-1188; and Plesch, et al, Gene, Volume 249, Number 1, 16 May 2000, pp. 83-89(7)). Examples of environmental conditions that may affect transcription by inducible promoters include anaerobic conditions, elevated temperature, or the presence of light. [0091] If proper protein expression is desired, a polyadenylation region at the 3'-end of the coding region should be included. The polyadenylation region can be derived from the natural gene, from a variety of other plant genes, or from T-DNA. [0092] The vector comprising the sequences (e.g., promoters or CPK4 or CPKl 1 coding regions) will typically comprise a marker gene that confers a selectable phenotype on plant cells. For example, the marker may encode biocide resistance, particularly antibiotic resistance, such as resistance to kanamycin, G418, bleomycin, hygromycin, or herbicide resistance, such as resistance to chlorosluforon or Basta.

[0093] In some embodiments, the CPK4 or CPKl 1 nucleic acid sequence is expressed recombinantly in plant cells to enhance and increase levels of total CPK4 or CPKl 1 polypeptide. A variety of different expression constructs, such as expression cassettes and vectors suitable for transformation of plant cells can be prepared. Techniques for transforming a wide variety of higher plant species are well known and described in the technical and scientific literature. See, e.g., Weising et al. Ann. Rev. Genet. 22:421-477 (1988). A DNA sequence coding for a CPK4 or CPKl 1 protein can be combined with cis- acting (promoter) and trans-acting (enhancer) transcriptional regulatory sequences to direct the timing, tissue type and levels of transcription in the intended tissues of the transformed plant. Translational control elements can also be used.

[0094] The invention provides a CPK4 or CPKl 1 nucleic acid operably linked to a promoter that, in some embodiments, is capable of driving the transcription of the CPK4 or CPKl 1 coding sequence in plants. The promoter can be, e.g. , derived from plant or viral sources. The promoter can be, e.g., constitutively active, inducible, or tissue specific. In construction of recombinant expression cassettes, vectors, transgenics, of the invention, a different promoters can be chosen and employed to differentially direct gene expression, e.g., in some or all tissues of a plant or animal.

A. Constitutive Promoters [0095] A promoter fragment can be employed to direct expression of a CPK4 or CPKl 1 nucleic acid in all transformed cells or tissues, e.g., as those of a regenerated plant. The term "constitutive regulatory element" means a regulatory element that confers a level of expression upon an operatively linked nucleic molecule that is relatively independent of the cell or tissue type in which the constitutive regulatory element is expressed. A constitutive regulatory element that is expressed in a plant generally is widely expressed in a large number of cell and tissue types. Promoters that drive expression continuously under physiological conditions are referred to as "constitutive" promoters and are active under most environmental conditions and states of development or cell differentiation. [0096] A variety of constitutive regulatory elements useful for ectopic expression in a transgenic plant are well known in the art. The cauliflower mosaic virus 35 S (CaMV 35S) promoter, for example, is a well-characterized constitutive regulatory element that produces a high level of expression in all plant tissues (Odell et al, Nature 313:810-812 (1985)). The CaMV 35 S promoter can be particularly useful due to its activity in numerous diverse plant species (Benfey and Chua, Science 250:959-966 (1990); Futterer et al, Physiol. Plant 79: 154 (1990); Odell et al., supra, 1985). A tandem 35S promoter, in which the intrinsic promoter element has been duplicated, confers higher expression levels in comparison to the unmodified 35S promoter (Kay et al., Science 236: 1299 (1987)). Other useful constitutive regulatory elements include, for example, the cauliflower mosaic virus 19S promoter; the

Figwort mosaic virus promoter; and the nopaline synthase (nos) gene promoter (Singer et al. , Plant MoI. Biol. 14:433 (1990); An, Plant Physiol. 81:86 (1986)).

[0097] Additional constitutive regulatory elements including those for efficient expression in monocots also are known in the art, for example, the pEmu promoter and promoters based on the rice Actin-1 5' region (hast et al., Theor. Appl. Genet. 81:581 (1991); Mcelroy et al., MoI. Gen. Genet. 231: 150 (1991); Mcelroy et al, Plant Cell 2: 163 (1990)). Chimeric regulatory elements, which combine elements from different genes, also can be useful for ectopically expressing a nucleic acid molecule encoding a CPK4 or CPKl 1 protein (Comai et al., Plant MoI. Biol. 15:373 (1990)). [0098] Other examples of constitutive promoters include the 1'- or 2'- promoter derived from T-DNA of Agrobacterium tumafaciens (see, e.g., Mengiste (1997) supra; O'Grady (1995) Plant MoI. Biol. 29:99-108); actin promoters, such as the Arabidopsis actin gene promoter (see, e.g., Huang (1997) Plant MoI. Biol. 1997 33: 125-139); alcohol dehydrogenase (Adh) gene promoters (see, e.g., Millar (1996) Plant MoI. Biol. 31:897 ^r-904); ACTIl from Arabidopsis (Huang et al. Plant MoI. Biol. 33 : 125 - 139 ( 1996)), Cat 3 from Arabidopsis (GenBank No. U43147, Zhong et al. , MoI. Gen. Genet. 251 : 196-203 (1996)), the gene encoding stearoyl-acyl carrier protein desaturase from Brassica napus (Genbank No. X74782, Solocombe et al. Plant Physiol. 104: 1167-1176 (1994)), GPcI from maize (GenBank No. X15596, Martinez et al. J. MoI. Biol 208:551-565 (1989)), Gpc2 from maize (GenBank No. U45855, Manjunath et al, Plant MoI. Biol 33:97-112 (1997)), other transcription initiation regions from various plant genes known to those of skill. See also Holtorf Plant MoI Biol 29:637-646 (1995). B. Inducible Promoters

[0099] Alternatively, a plant promoter may direct expression of the CPK4 or CPKl 1 gene under the influence of changing environmental conditions or developmental conditions. Examples of environmental conditions that may effect transcription by inducible promoters include anaerobic conditions, elevated temperature, drought, or the presence of light. Such promoters are referred to herein as "inducible" promoters. For example, the invention can incorporate drought-specific promoter such as the drought-inducible promoter of maize (Busk (1997) supra); or alternatively the cold, drought, and high salt inducible promoter from potato (Kirch (1997) Plant Mot Biol. 33:897-909). [0100] Alternatively, plant promoters which are inducible upon exposure to plant hormones, such as auxins, are used to express the CPK4 or CPKl 1 gene. For example, the invention can use the auxin -response elements El promoter fragment (AuxREs) in the soybean (Glycine max L.) (Liu (1997) Plant Physiol. 115:397-407); the auxin-responsive Arabidopsis GST6 promoter (also responsive to salicylic acid and hydrogen peroxide) (Chen (1996) Plant J. 10: 955-966); the auxin-inducible parC promoter from tobacco (Sakai (1996) 37:906-913); a plant biotin response element (Streit (1997) MoI. Plant Microbe Interact. 10:933-937); and, the promoter responsive to the stress hormone abscisic acid (Sheen (1996) Science 274: 1900-1902).

[0101] Plant promoters inducible upon exposure to chemicals reagents that may be applied to the plant, such as herbicides or antibiotics, are also useful for expressing the CPK4 or CPKl 1 gene. For example, the maize In2-2 promoter, activated by benzenesulfonamide herbicide safeners, can be used (De Veylder (1997) Plant Cell Physiol. 38:568-577); application of different herbicide safeners induces distinct gene expression patterns, including expression in the root, hydathodes, and the shoot apical meristem. A CPK4 or CPKl 1 coding sequence can also be under the control of, e.g. , a tetracycline -inducible promoter, e.g. , as described with transgenic tobacco plants containing the Avena sativa L. (oat) arginine decarboxylase gene (Masgrau (1997) Plant J. 11:465-473); or, a salicylic acid-responsive element (Stange (1997) Plant J. 11: 1315-1324; Uknes et al., Plant Cell 5: 159-169 (1993); Bi et a\., Plant J. 8:235-245 (1995)). [0102] Examples of useful inducible regulatory elements include copper-inducible regulatory elements (Mett et al., Proc. Natl. Acad. Sci. USA 90:4567-4571 (1993); Furst et at, Cell 55:705-717 (1988)); tetracycline and chlor-tetracycline-inducible regulatory elements (Gatz et at, Plant J. 2:397-404 (1992); Roder et al. , MoI. Gen. Genet. 243:32-38 (1994); Gatz, Meth. Cell BwI. 50:411-424 (1995)); ecdysone inducible regulatory elements (Christopherson et αl, Proc. Nαtl. Acαd. Sci. USA 89:6314-6318 (1992); Kreutzweiser et al, Ecotoxicol. Environ. Safety 28: 14-24 (1994)); heat shock inducible regulatory elements (Takahashi et al, Plant Physiol . 99:383-390 (1992); Yabe et al, Plant Cell Physiol. 35: 1207- 1219 (1994); Veda et al., MoI. Gen. Genet. 250:533-539 (1996)); and lac operon elements, which are used in combination with a constitutively expressed lac repressor to confer, for example, IPTG-inducible expression (Wilde et al, EMBO J. 11: 1251-1259 (1992)). An inducible regulatory element useful in the transgenic plants of the invention also can be, for example, a nitrate-inducible promoter derived from the spinach nitrite reductase gene (Back et al , Plant MoI. Biol. 17:9 (1991)) or a light-inducible promoter, such as that associated with the small subunit of RuBP carboxylase or the LHCP gene families (Feinbaum et al, MoI Gen. Genet. 226:449 (1991); Lam and Chua, Science 248:471 (1990)).

C. Tissue-Specific Promoters

[0103] Alternatively, the plant promoter may direct expression of the CPK4 or CPKl 1 gene in a specific tissue (tissue-specific promoters). Tissue specific promoters are transcriptional control elements that are only active in particular cells or tissues at specific times during plant development, such as in vegetative tissues or reproductive tissues.

[0104] Examples of tissue-specific promoters under developmental control include promoters that initiate transcription only (or primarily only) in certain tissues, such as vegetative tissues, e.g., roots or leaves, or reproductive tissues, such as fruit, ovules, seeds, pollen, pistols, flowers, or any embryonic tissue. Reproductive tissue-specific promoters may be, e.g., ovule-specific, embryo-specific, endosperm-specific, integument-specific, seed and seed coat-specific, pollen-specific, petal-specific, sepal-specific, or some combination thereof. [0105] Other tissue-specific promoters include seed promoters. Suitable seed-specific promoters are derived from the following genes: MACl from maize (Sheridan (1996) Genetics 142: 1009-1020); Cat3 from maize (GenBank No. L05934, Abler (1993) Plant MoI. Biol. 22: 10131-1038); vιvparous-1 from Arabidopsis (Genbank No. U93215); atmycl from Arabidopsis (Urao (1996) Plant MoI. Biol. 32:571-57; Conceicao (1994) Plant 5:493-505); napA from Brassica napus (GenBank No. J02798, Josefsson (1987) JBL 26: 12196-1301); and the napin gene family from Brassica napus (Sjodahl (1995) Planta 197:264-271). [0106] A variety of promoters specifically active in vegetative tissues, such as leaves, stems, roots and tubers, can also be used to express the CPK4 or CPKl 1 gene. For example, promoters controlling patatin, the major storage protein of the potato tuber, can be used, see, e.g., Kim (1994) Plant MoI. Biol. 26:603-615; Martin (1997) Plant J. 11:53-62. The ORF13 promoter from Agrobacterium rhizogenes that exhibits high activity in roots can also be used (Hansen (1997) MoI. Gen. Genet. 254:337-343. Other useful vegetative tissue-specific promoters include: the tarin promoter of the gene encoding a globulin from a major taro (Colocasia esculenta L. Schott) corm protein family, tarin (Bezerra (1995) Plant MoI. Biol. 28: 137-144); the curculin promoter active during taro corm development (de Castro (1992) Plant Cell 4: 1549-1559) and the promoter for the tobacco root-specific gene TobRB7, whose expression is localized to root meristem and immature central cylinder regions (Yamamoto (1991) Plant Cell 3:371-382).

[0107] Leaf-specific promoters, such as the ribulose biphosphate carboxylase (RBCS) promoters can be used. For example, the tomato RBCSl, RBCS2 and RBCS3A genes are expressed in leaves and light-grown seedlings, only RBCSl and RBCS2 are expressed in developing tomato fruits (Meier (1997) FEBS Lett. 415:91-95). A ribulose bisphosphate carboxylase promoters expressed almost exclusively in mesophyll cells in leaf blades and leaf sheaths at high levels, described by Matsuoka (1994) /7α«^J. 6:311-319, can be used. Another leaf-specific promoter is the light harvesting chlorophyll a/b binding protein gene promoter, see, e.g., Shiina (1997) Plant Physiol. 115:477-483; Casal (1998) Plant Physiol.

116: 1533-1538. The Arabidopsis thaliana myb-related gene promoter (Atmyb5) described by Li (1996) FEBS Lett. 379: 117-121, is leaf-specific. The Atmyb5 promoter is expressed in developing leaf trichomes, stipules, and epidermal cells on the margins of young rosette and cauline leaves, and in immature seeds. Atmyb5 mRNA appears between fertilization and the 16 cell stage of embryo development and persists beyond the heart stage. A leaf promoter identified in maize by Busk (1997) Plant J. 11: 1285-1295, can also be used.

[0108] Another class of useful vegetative tissue-specific promoters are meristematic (root tip and shoot apex) promoters. For example, the "SHOOTMERISTEMLESS" and "SCARECROW" promoters, which are active in the developing shoot or root apical meristems, described by Di Laurenzio (1996) Cell 86:423-433; and, Long (1996) Nature 379:66-69; can be used. Another useful promoter is that which controls the expression of 3-hydroxy-3- methylglutaryl coenzyme A reductase HMG2 gene, whose expression is restricted to meristematic and floral (secretory zone of the stigma, mature pollen grains, gynoecium vascular tissue, and fertilized ovules) tissues (see, e.g., Enjuto (1995) Plant Cell. 7:517-527). Also useful are knl-related genes from maize and other species that show meristem-specific expression, see, e.g., Granger (1996) Plant MoI. Biol. 31:373-378; Kerstetter (1994) Plant Cell 6: 1877-1887; Hake (1995) Philos. Trans. R. Soc. Lond. B. Biol. Sa. 350:45-51. Another example is the Arabidopsis thahana KNATl promoter (see, e.g., Lincoln (1994) Plant Cell 6: 1859-1876).

[0109] One of skill will recognize that a tissue-specific promoter may drive expression of operably linked sequences in tissues other than the target tissue. Thus, as used herein a tissue- specific promoter is one that drives expression preferentially in the target tissue, but may also lead to some expression in other tissues as well.

[0110] In another embodiment, the CPK4 or CPKl 1 gene is expressed through a transposable element. This allows for constitutive, yet periodic and infrequent expression of the constitutively active polypeptide. The invention also provides for use of tissue-specific promoters derived from viruses including, e.g., the tobamovirus subgenomic promoter (Kumagai (1995) Proc. Natl. Acad. Sa. USA 92: 1679-1683; the rice tungro bacilliform virus (RTBV), which replicates only in phloem cells in infected rice plants, with its promoter which drives strong phloem-specific reporter gene expression; the cassava vein mosaic virus (CVMV) promoter, with highest activity in vascular elements, in leaf mesophyll cells, and in root tips (Verdaguer (1996) Plant MoI. BwI. 31: 1129-1139).

IV. Production of Transgenic Plants

[0111] As detailed herein, the present invention provides for transgenic plants comprising recombinant expression cassettes either for expressing CPK4 or CPKl 1 proteins in a plant or for inhibiting or reducing endogenous CPK4 or CPKl 1 expression. Thus, in some embodiments, a transgenic plant is generated that contains a complete or partial sequence of an endogenous CPK4 or CPKl 1 encoding polynucleotide, either for increasing or reducing CPK4 or CPKl 1 expression and activity. In some embodiments, a transgenic plant is generated that contains a complete or partial sequence of a polynucleotide that is substantially identical to an endogenous CPK4 or CPKl 1 encoding polynucleotide, either for increasing or reducing CPK4 or CPKl 1 expression and activity. In some embodiments, a transgenic plant is generated that contains a complete or partial sequence of a polynucleotide that is from a species other than the species of the transgenic plant. It should be recognized that transgenic plants encompass the plant or plant cell in which the expression cassette is introduced as well as progeny of such plants or plant cells that contain the expression cassette, including the progeny that have the expression cassette stably integrated in a chromosome.

[0112] A recombinant expression vector comprising a CPK4 or CPKl 1 coding sequence driven by a heterologous promoter may be introduced into the genome of the desired plant host by a variety of conventional techniques. For example, the DNA construct may be introduced directly into the genomic DNA of the plant cell using techniques such as electroporation and microinjection of plant cell protoplasts, or the DNA construct can be introduced directly to plant tissue using ballistic methods, such as DNA particle bombardment. Alternatively, the DNA construct may be combined with suitable T-DNA flanking regions and introduced into a conventional Agrobacterium tumefaciens host vector. The virulence functions of the Agrobacterium tumefaciens host will direct the insertion of the construct and adjacent marker into the plant cell DNA when the cell is infected by the bacteria. While transient expression of CPK4 or CPKl 1 is encompassed by the invention, generally expression of construction of the invention will be from insertion of expression cassettes into the plant genome, e.g., such that at least some plant offspring also contain the integrated expression cassette.

[0113] Microinjection techniques are also useful for this purpose. These techniques are well known in the art and thoroughly described in the literature. The introduction of DNA constructs using polyethylene glycol precipitation is described in Paszkowski et al. Embo J. 3:2717-2722 (1984). Electroporation techniques are described in Fromm et al. Proc. Natl.

Acad. Sci. USA 82:5824 (1985). Ballistic transformation techniques are described in Klein et al. Nature 327:70-73 (1987).

[0114] Agrobacterium tumefaciens-mediated transformation techniques, including disarming and use of binary vectors, are well described in the scientific literature. See, for example, Horsch et αl. Science 233:496-498 (1984), and Fraley et αl. Proc. Nαtl. Acαd. Sci. USA 80:4803 (1983).

[0115] Transformed plant cells derived by any of the above transformation techniques can be cultured to regenerate a whole plant that possesses the transformed genotype and thus the desired phenotype such as enhanced drought-resistance. Such regeneration techniques rely on manipulation of certain phytohormones in a tissue culture growth medium, typically relying on a biocide and/or herbicide marker which has been introduced together with the desired nucleotide sequences. Plant regeneration from cultured protoplasts is described in Evans et al. , Protoplasts Isolation and Culture, Handbook of Plant Cell Culture, pp. 124-176, MacMillilan Publishing Company, New York, 1983; and Binding, Regeneration of Plants, Plant Protoplasts , pp. 21-73, CRC Press, Boca Raton, 1985. Regeneration can also be obtained from plant callus, explants, organs, or parts thereof. Such regeneration techniques are described generally in Klee et al. Ann. Rev. of Plant Phys. 38:467-486 (1987).

[0116] One of skill will recognize that after the expression cassette is stably incorporated in transgenic plants and confirmed to be operable, it can be introduced into other plants by sexual crossing. Any of a number of standard breeding techniques can be used, depending upon the species to be crossed. [0117] The expression cassettes of the invention can be used to confer drought-resistance on essentially any plant. Thus, the invention has use over a broad range of plants, including species from the genera Asparagus, Atropa, Avena, Brassica, Citrus, Citrullus, Capsicum, Cucumis, Cucurbita, Daucus, Fragana, Glycine, Gossypium, Hehanthus, Heterocallis, Hordeum, Hyoscyamus, Lactuca, Linum, Lolium, Lycopersicon, Malus, Manihot, Majorana, Medicago, Nicotiana, Oryza, Panieum, Pannesetum, Per sea, Pisum, Pyrus, Prunus,

Raphanus, Secale, Senecio, Sinapis, Solanum, Sorghum, Tngonella, Tnticum, Vitis, Vigna, and, Zea. In some embodiments, the plant is selected from the group consisting of rice, maize, wheat, soybeans, cotton, canola, and alfalfa. In some embodiments, the plant is an ornamental plant. In some embodiment, the plant is a vegetable- or fruit-producing plant. [0118] In some embodiments, the methods of the invention are used to confer drought- resistance on turf grasses. A number of turf grasses are known to those of skill in the art. For example, fescue, Festuca spp. (e.g., F. arundinacea, F. rubra, F. ovina var. dunuscula, and F. ovina) can be used. Other grasses include Kentucky bluegrass Poa pratensis and creeping bentgrass Agrostis palustns. [0119] Those of skill will recognize that a number of plant species can be used as models to predict the phenotypic effects of transgene expression in other plants. For example, it is well recognized that both tobacco (Nicotiana) and Arabidopsis plants are useful models of transgene expression, particularly in other dicots.

[0120] The plants of the invention have either enhanced or reduced abscisic acid sensitivity compared to plants are otherwise identical except for expression of CPK4 or CPKl 1. Abscisic acid sensitivity can be monitored by observing or measuring any phenotype mediated by ABA. Those of skill in the art will recognize that ABA is a well-studied plant hormone and that ABA mediates many changes in characteristics, any of which can be monitored to determined whether ABA sensitivity has been modulated. In some embodiments, modulated ABA sensitivity is manifested by altered timing of seed germination or altered stress (e.g., drought) tolerance. [0121] Drought resistance can assayed according to any of a number of well-known techniques. For example, plants can be grown under conditions in which less than optimum water is provided to the plant. Drought-resistance can be determined by any of a number of standard measures including turgor pressure, growth, yield, and the like. In some embodiments, the methods described in the Example section, below can be conveniently used.

EXAMPLES

[0122] The following examples are offered to illustrate, but not to limit the claimed invention.

[0123] The present inventors discovered that ABA stimulated two homologous calcium- dependent protein kinases in Arabidopsis, CPK4 and CPKl 1. Loss-of-function mutations cpk4-l in CPK4 gene and cpkll-1 and cpkll-2 in CPKIl gene resulted in pleiotropically ABA-insensitive phenotypes in seed germination, seedling growth, and stomatal movement, and led to salt-insensitivity in seed germination and decreased tolerance of seedlings to salt stress. Double mutations cpk4-lcpkll-l and cpk4-lcpkll-2 in the two CDPK genes had stronger ABA- and salt-responsive phenotypes than the single mutations did. The CPK4- or CPA77-overexpressing plants showed generally inverse ABA-related phenotypes compared to those of the loss-of-function mutants. The expression levels of many ABA-responsive genes were altered in the loss-of-function mutants and overexpression lines. The CPK4 and CPKl 1 kinases both phosphorylated two ABA-responsive transcription factors ABFl and ABF4 in vitro, indicating that the two kinases regulate ABA signaling through these transcription factors. These data provide clear, inplanta genetic, evidence for the involvement of CDPK/calcium in ABA signaling at the whole-plant level, and demonstrate that CPK4 and CPKl 1 are two important positive regulators in the CDPK/calcium-mediated ABA signaling pathways. [0124] Disruption of Arabidopsis calcium-dependent protein kinase CPK4 and CPKl 1 genes reduces, but over-expression of the two kinase genes increases, ABA sensitivity in all major ABA responses and plant ability to conserve water. The two kinases have two transcription factors ABFl and ABF4 as their substrates. So, CPK4 and CPKl 1 are positive regulators in the CDPK/calcium-mediated ABA signaling pathways.

RESULTS

Identification of T-DNA Insertion Mutants and Overexpression Lines and Expression Profile of CPK4 and CPKIl

[0125] The present inventors isolated, from the pool of T-DNA insertion mutants in the Arabidopsis Biological Resource Center (ABRC), a mutant cpk4-l in CPK4 gene (SALK_081860) and two different mutant lines cpkll-1 (SALK_023086) and cpkll-2 (SALK_054495) in CPKIl gene. The cpk4-l mutant harbors a tandem-two-copy T-DNA insertion in 5' untranslated region (UTR) upstream of exon 1 of the CPK4 gene (Figures IA, 8, and 9, Table 3). The tandem T-DNAs were inserted into the genome in an inverted fashion at the same locus, which generates an 11-bp deletion from -67 to -57 bp 5'-upstream of the translation start codon (Figures IA, 8, and 9, Table 3). The cpkll-1 mutant also harbors a tandem-two-copy T-DNA insertion in an inverted fashion at the same locus in 5 ' UTR upstream of exon 1 of the CPKIl gene, generating a 34-bp deletion from -120 to -87 bp 5'- upstream of the translation start codon (Figures IB, 8, and 9, Table 3). A single copy of T- DNA was inserted into the genome for the cpkll-2 mutant, generating a 39-bp deletion from 320 to 358 bp downstream of the translation start codon (Figures IB, 8, and 9, Table 3). The genetic background for all the mutants is ecotype Columbia (Col). The three insertions were identified by PCR analysis of the Arabidopsis genome (Figures IA, IB, and 8), by sequencing of the genomic PCR products (Table 3) and also by genomic DNA blot analysis which helped to determine the number of T-DNA inserts (Figure 9). In addition to these assays, tandem T-DNA insertion at the same genomic locus in the cpk4-l and cpkll-1 mutants was supported by genetic segregation analysis. The segregation assay for the nptll gene was performed by selecting for growth on medium containing kanamycin (50 μg/mL) with seeds from heterozygous cpk4-l and cpkll-1 mutants. The ratio of the resistant to sensitive plants was approximately 3: 1. Also, the inventors obtained 30 plants (1/16) of the homologous cpk4-lcpkll-l double mutants from a population of 512 F2 plants when crossing the cpk4-l with cpkll-1 single mutant. These results demonstrated that the T-DNAs have segregated as one locus. The cpk4-l and cpkll-1 are single-locus T-DNA insertion mutants.

[0126] To confirm that cpk4-l, cpkll-1 and cpkll-2 are transcript-null mutants, RT-PCR analysis was performed with RNA isolated from wild-type and mutant plants. The results showed that the three mutants did not yield their corresponding RT-PCR products under the growth conditions where wild-type plants produced normally CPK4 and CPKIl mRNA (Figure 1C). However, the transcription of CPK4 gene was not affected in the cpkll-1 and cpkll-2 mutants, and that of CPKIl gene was not affected either in the cpk4-l mutant (Figure 1C).

[0127] The CPK4 and CPKl 1 share high identity (94%) in their amino acid sequences even in generally the most variable N- or C-terminus (Figure 10), and both proteins localize in cytoplasm and nucleus (Dammann et al., (2003) Plant Physiol 132, 1840-1848; Milla et al., (2006) FEBS Letters 580, 904-911; see also Figure 11). It is difficult to generate antiserum specific to distinguish the two proteins one from another because of their high amino acid sequence identity. The inventors produced two antisera against the most variable C-terminal fragments of CPK4 (CPK4^C) and CPKl 1 (CPKl l^c), respectively (see METHODS and Figure 10), anyone of which is able to recognize both CPK4 and CPKl 1 (data not shown). Using either anti-CPK4^c or anti-CPKl l^c serum, the inventors detected immuno-signals in all the T- DNA insertion mutants, and the signals in the cpk4-l mutant are CPKl 1, whereas those in the cpkll-1 and cpkll-2 mutants are CPK4 (Figures IE and 12A). This is consistent with above- mentioned RT-PCR assays (Figure 1C). The cpk4-lcpkll-l and cpk4-lcpkll-2 double mutants, obtained by crossing, were shown to have neither mRNA of the two genes in their total mRNA (Figure 1C) nor immuno-signal of the two proteins in their total proteins (Figure 12A), revealing that both genes are disrupted from the double mutants, and also indicating that the two antisera are specific to CPK4 and CPKl 1. The disruption of any of the two kinase genes does not affect the ABA biosynthesis when plants are grown under no-stressful conditions or under drought (Figure 13) or salt stress (data not shown). It should be noted that the two allelic T-DNA insertion lines cpkll-1 and cpkll-2 in CPKIl gene, as well as two double mutants cpk4-lcpkll-l and cpk4-l cpkll-2, show similar phenotypes in response to ABA or stress treatments. Thus, it is shown here the results of cpkll-2 as a representative of two mutants cpkll-1 and cpkll-2, and the results of cpk4-l cpkll-2 as a representative of the two double mutants in some cases.

[0128] CPK4- and CPA77-overexpressing lines were also created under the control of CaMV 35 S promoter. Ten lines were obtained, and their phenotypes related to ABA and stress tolerance were similar. Only CPAT4-overexpression line 12 (4OE 12) and CPKIl- overexpression line 2 (11OE2) are shown as examples herein. Immunoblotting assays showed that the levels of CPK4 or CPKl 1 protein significantly increased in these overexpression lines (Figure ID).

[0129] Available data at the Genevestigator site (genevestigator.ethz.ch) show that both CPK4 and CPKIl genes are expressed in different plant organs. Consistently with this, it has been shown that the mRNA and proteins of both kinases are present in all the organs tested (Figure 12A). Their expression could be detected in the germinating seeds. The expression levels increased rapidly during the first three days after stratification, kept relatively stable thereafter for more than 10 days, and increased once again from about 20 days after germination (Figures IE and 12B).

ABA Stimulates Both CPK4 and CPKIl Kinases

[0130] The enzymatic properties of CPK4 and CPKl 1 proteins were first analyzed. Ca²⁺- binding proteins such as CDPKs migrate in gels at different rates in the Ca -bound versus Ca²⁺-free state (Roberts and Harmon (1992) Ann Rev Plant Physiol Plant MoI BwI 43, 375- 414). To investigate this phenomenon, Ca²⁺ or EGTA was added to the protein sample just before electrophoresis and then the in gel phosphorylation was analyzed in the presence of Ca²⁺. The assays of in-gel autophosphorylation of both kinase showed a clear mobility shift when the kinase migrated in the presence of Ca²⁺ (Figure 14A). The in-gel histone- phosphorylating activity of both kinases was shown to be dependent on the presence of Ca²⁺ (Figure 14B). The effects of the CaM antagonists N-(6-aminohexyl)-5-chloro-l-naphthalene sulfonamide (W7) and trifluoperazine (TFP) and inhibitor of serine/threonine protein kinases K252a on CPK4 and CPKl 1 kinases were also analyzed. The calcium-dependent in-gel histone-phosphorylating activity of both kinases could be inhibited by W7, TFP and K252a (Figure 14B). By contrast, CaM and N-(6-aminohexyl)-l -naphthalene sulfonamide (W5, an inactive analogue of W7) had no apparent effect on phosphorylating activity of the two kinases (Figure 14B). These results indicate that CPK4 and CPKl 1 possess enzymatic properties of a typical CDPK.

[0131] It was further tested whether the two CDPKs are stimulated by ABA, and found that the mRΝA levels of CPK4 and CPKIl genes were not significantly altered by ABA treatments. However, ABA treatments significantly increased the protein levels of both CPK4 and CPKl 1, and also correspondingly enhanced their histone-phosphorylating activities (Figure 2A and 2B). The ABA-stimulating effects were dependent on the ABA dose used, in which ABA was most effective at around 1 μM concentration, and higher concentrations of ABA showed reduced effects (Figure 2A and 2B), which is likely physiologically explainable, because the endogenous levels of ABA due to the exogenously- applied ABA at about 1 μM may mimic the elevated ABA levels during stressful conditions (Finkelstein and Rock, 2002, supra), but a higher level over the physiological concentrations may be harmful to optimization of the response. The ABA-stimulating effects were also shown to be transient, with a maximum stimulation at 60 to 120 min after ABA treatments (Figure 2A and 2B).

Disruption of CPK4 and CPKIl Reduces ABA- and Salt-Responsiveness in Seed Germination [0132] The seeds of all the mutants including T-DNA insertion mutants and transgenic overexpression lines of CPK4 and CPKIl germinated normally as the wild-type seeds did in the ABA-free medium (0 μM ABA, Figure 3A). However, in the media supplemented with 0.5 μM or 3 μM ABA, the germination rates of the T-DNA insertion mutant seeds were significantly higher than those of the wild-type seeds (Figure 3A). On the contrary, the germination of the CPK4- and CPKl 1 -overexpression seeds was significantly more inhibited by ABA than that of the wild-type seeds (Figure 3A). The double mutants cpk4-lcpkll-l and cpk4-lcpkll-2 resulted in significantly more intense ABA-insensitive phenotypes in ABA-induced inhibition of seed germination (Figure 3A). Similarly to the responses to ABA, in the media containing different concentrations of NaCl ranging from 50 mM to 200 mM, the cpk4-l, cpkll-1 and cpkll-2 mutant seeds germinated significantly faster than those of wild-type seeds with more apparent phenotypes in cpk4-l seeds but weaker phenotypes in cpkll-2 (and also cpkll-1, data not shown) (Figure 4A). The cpk4-l cpkll-1 and cpk4- 1 cpkll-2 double mutants had significantly stronger NaCl-insensitive phenotypes in NaCl- induced inhibition of seed germination than the cpk4-l, cpkll-1 and cpkll-2 single mutants (Figure 4A). The CPK4- and CPKl 1 -overexpression, however, did not significantly alter the NaCl-related phenotypes in seed germination (Figure 4A).

[0133] The transgenic expression of CPK4 cDNA in the cpk4-l mutant and CPKl 1 cDNA in the cpkll-1 and cpkll-2 mutants under the control of CaMV 35S prompter rescued the ABA- and salt-insensitive phenotypes in their seeds (Figure 3A), showing that the phenotypes oϊcpk4-l, cpkll-1 and cpkll-2 are indeed caused by defects in the CPK4 or CPKIl gene. Disruption of CPK4 and CPKIl Reduces, but Overexpression of the Genes Enhances, ABA-Sensitivity in Seedling Growth

[0134] There were no significant differences found in seedling growth in ABA-free media among the different genotypes (Figure 3B and 3C, 0 μM ABA). Two approaches were used to assess the effects OΪCPK4 and CPKIl genes on the response of seedling growth to ABA. One was that the germinating seeds were transferred 48 h after stratification from the ABA- free MS-medium to ABA-containing MS-medium to investigate the response of growth to ABA (Figure 3B and 3C), and another was that the seeds were directly planted in ABA- containing MS-medium to investigate the response of seedling growth to ABA after germination (Figure 3D). The results obtained with these two approaches were similar. The seedlings of all the T-DNA insertion mutants grew significantly better than those of wild-type Col in the ABA-containing medium, while the growth of the CP K4- and CPKIl- overexpression seedlings was significantly more reduced by ABA treatment than that of the wild-type seedlings (Figure 3B-3D). In the assays with the 48 h-transferred seedlings to ABA-containing medium, the effects of ABA on seedling growth were more apparent when the applied ABA concentrations were higher than 5 μM, and the growth of the transgenic overexpression seedlings was completely inhibited in the media containing more than 10 μM ABA, while the seedling of wild-type Col and T-DNA insertion mutants still grew more or less (Figure 3B and 3C). It should be noted that the phenotypes in ABA-responsive seedling growth were easily observed if the seedlings were transferred to the ABA-containing medium less than 48 h after stratification, but the phenotypes were less apparent when the transfer was done more than 48 h after stratification. The same phenomenon was also observed in the ABA receptor ABAR-regulated seedling growth (Shen et al., 2006, supra), which may be associated with mechanisms like the postgermination developmental arrest checkpoint mediated by temporal expression ofABI5 (Lopez-Molina et al., (2001) Proc. Natl. Acad. Sa. USA 98, 4782-4787). Double disruption of two CDPK genes CPK4 and CPKIl in the cpk4- lcpkll-1 and cpk4-lcpkll-2 mutants resulted in significantly more intense ABA-insensitive phenotypes in ABA-induced arrest of seedling growth (Figure 3B-3D). It is noteworthy, however, that the phenotypes associated with the postgermination growth are relatively weak, especially at the ABA concentrations lower than 10 μM (Figure 3 and 3C).

[0135] The effects of CPK4 and CPKIl genes on lateral root growth were also tested in relation to ABA. The results showed that, in the ABA-containing medium, the total length of lateral roots of the cpk4-l, cpkll-1 and cpkll-2 mutants tended to increase, and that of the double mutants cpk4-lcpkll-l and cpk4-lcpkll-2 increased significantly, in comparison with that of wild-type plants (Figure 3E). In contrast to this, overexpression 0ΪCPK4 or CPKIl gene significantly reduced the total length of lateral roots in the presence of ABA compared to wild-type Col (Figure 3E).

Disruption of CPK4 and CPKIl Leads to Salt-Hypersensitivity in Seedling Growth

[0136] The response of seedling growth to NaCl was further investigated by transferring 4- days-old seedling from the NaCl-free medium to NaCl-containing medium. There was no significant difference observed in growth status among the seedlings of various genotypes in the media containing up to 150 mM NaCl (data not shown). However, NaCl at 170 mM began to blench the seedlings of cpk4-l, cpkll-1 and cpkll-2 mutants, as well as cpk4- lcpkll-1 and cpk4-l cpkll-2 double mutants, and at 200 mM NaCl, all the seedlings of the cpk4-l mutant and cpk4-l cpkll-1 and cpk4-l cpkll-2 double mutants were completely blenched (Figure 4B and 4C). The seedlings of the cpkll-1 and cpkll-2 mutants were shown to be less damaged by NaCl at above 170 mM concentrations compared with those of the cpk4-l mutant and the two double mutants (Figure 4B and 4C).

[0137] The CPK4- and CPKl 1 -overexpression did not significantly alter the NaCl-related phenotypes in seedling growth (Figure 4B and 4C). The transgenic complementation lines of the cpk4-l, cpkll-1 or cpkll-2 mutants rescued the NaCl-related phenotypes.

Disruption of CPK4 and CPKIl Reduces, but Overexpression of the Genes Enhances, ABA- Sensitivity in Stomata and Capacity to Conserve Water

[0138] Loss-of-function mutation in CPK4 or CPKIl gene resulted in ABA -insensitive phenotypes, but overexpression OΪCPK4 or CPKIl gene led to ABA-hypersensitive phenotypes, in ABA-induced promotion of stomatal closure (Figure 5A, panel above) and inhibition of stomatal opening (Figure 5 A, panel below). The detached leaves of the loss-of- function mutants cpk4-l, cpkll-1 and cpkll-2 lost more water under dehydration conditions, while CPK4- and CPK77-overexpressing plants lost less water than the detached leaves of the wild-type plants (Figure 5B). This may be due to the alteration in ABA-sensitivity of stomatal closure of these genotypes (Figure 5A). Furthermore, obvious differences in the capacity to conserve water at the whole-plant level were observed among these genotypes: when drought stress was imposed on plants, cpk4-l, cpkll-1 and cpkll-2 mutants showed lower (Figure 5C and 5D), but the CPK4 and CPKl 1 -overexpression lines presented higher (Figure 5C and 5E), capacity to conserve their water, than wild-type plants. In the well- watered conditions, however, there was not difference found in their growth status among these genotypes (Figure 5 D and 5E).

[0139] Double mutants cpk4-lcpkll-l and cpk4-lcpkll-2 showed stronger ABA- insensitive phenotypes in ABA-induced promotion of stomatal closure (Figure 5 A, panel above) and inhibition of stomatal opening (Figure 5A, panel below), and lost more water from both their detached leaves (Figure 5B) and whole plants (Figure 5C and 5D), in comparison with the single mutants cpk4-l, cpkll-1 or cpkll-2.

[0140] The transgenic complementation lines of the cpk4-l, cpkll-1 or cpkll-2 mutants rescued the ABA-insensitive phenotypes in their stomata, and regained a level of water loss rates from detached leaves (Figure 5B) and an ability of preserving their water at the whole plant level under water deficit (Figure 5D) comparable to wild-type plants, which shows that the phenotypes of cpk4-l, cpkll-1 and cpkll-2 results indeed from disruption of the CPK4 or CPKIl gene.

CPK4 and CPKIl Kinases Phosphorylate ABA-Responsive Transcription Factors ABFl and ABF4 in Vitro

[0141] The ABA-responsive transcription factors ABFl, ABF2 (AREBl), ABF3 and ABF4 (AREB2) (Choi et al, (2000) JBwI Chem 275, 1723-1730; Uno et al., (2000) Proc Natl Acad Sa USA 97, 11632-11637) were previously reported to be phosphorylated by upstream protein kinases to mediate ABA signaling (Uno et al., 2000, supra; Furihata et al., (2006) Proc Natl Acad Sa USA 103, 1988-1993; Fujii et al., (2007) Plant Cell 19, 484-494). To analyze if the ABFs are also involved in CPK4- and CPKl 1 -mediated ABA signaling, the inventors mapped protein kinases that could phosphorylate two ABA-responsive transcription factors ABFl and ABF4 through ABFl- or ABF4-in-gel phosphorylation by total proteins from wild-type Col or double mutant cpk4-l cpkll-2. ABFl was phosphorylated apparently by the kinase(s) of sole molecular mass of 58 kD, but ABF4 by kinases of two molecular masses of 58 and 67 kD in the absence of exogenous ABA treatment (Figure 6A and 6B). In the presence of exogenous ABA, however, both ABFl- and ABF4-phosphorylating kinases displayed two additional bands of 42 and 44 kD (Figure 6A and 6B). These 42-kD and 44- kD phosphorylating activities are consistent with previous reports of SnRK activities on the ABFs (Uno et al., 2000, supra; Furihata et al., 2006, supra; Fujii et al., 2007, supra).

Immunoblotting assays indicated that CPK4 and CPKl 1 run at 58 kD (Figure 6 A and 6B), showing that the 58-kD phosphorylating activities may be due to CPK4 and CPKl 1. Both the ABFl- and ABF4-phosphorylating activity of the 58-kD kinase(s), apparently stimulated by ABA, was clearly reduced, but not disappeared, in the cpk4-lcpkll-2 double mutant (Figure 6A and 6B), indicating that the most portions of 58-kD kinase(s) are CPK4 and CPKl 1, but other kinase (s) with the same molecular mass exist to phosphorylate the ABFl and AB F4. Further experiments showed that ABFl and ABF4 were phosphorylated in vitro by the immuno-precipitated natural proteins of both CPK4 and CPKl 1, and this phosphorylation was significantly stimulated by ABA treatment (Figure 6C and 6D). The ABFl- and ABF4-phosphorylating activities of CPK4- and CPKl 1 were completely abolished by double mutation in CPK4 and CPKIl genes (Figure 6C and 6D). Taken together, these data demonstrate that CPK4 and CPKl 1, having ABA-inducible kinase activity (Figures 2 and 6), play an important role in phosphorylating and activating ABFl and ABF4, and also possibly other ABFs. It is noteworthy, however, that these two transcription factors can be phosphorylated by multiple kinases, and other 58-kD kinase(s) than CPK4 and CPKl 1 are also involved in this phosphorylation event (Figure 6A and 6B).

Disruption or Overexpression of CPK4 and CPKIl Alter Expression of Some ABA- Responsive Genes

[0142] Expression of some ABA-inducible genes ABFs (ABFl , ABF2 or AREBl , ABF 3, ABF4 or AREB2; Choi et al., 2000, supra; Uno et al., 2000, supra), ABIl (Leung et al., 1994, supra; Meyer et al. 1994, supra; Gosti et al., 1999, supra), ABI2 (Leung et al., 1997, supra), ABB (Giraudat et al., (1992) Plant Cell 4, 1251-1261), ABI4 (Finkelstein et al., (1998) Plant Cell 10, 1043-1054), ABI5 (Finkelstein and Lynch, (2000) Plant Cell 12, 599-609), RD29A (Yamaguchi-Shinozaki and Shinozaki, (1994) Plant Cell 6, 251-264), RABl 8 (Lang and Palva, (1992) Plant MoI. Biol. 20, 951-962), KINl and KIN2 (Kurkela and Borg-Franck, (1992) Plant MoI. Biol. 19, 689-692), ERDlO (Kiyosue et al., (1994) Plant Cell Physiol. 35, 225-231), and MYB2 andMYC2 (Abe et al., (2003) Plant Cell 15, 63-78) were tested in the T-DNA insertion mutants and transgenic overexpression lines. As reported previously, the expression of all these ABA-responsive genes was strongly stimulated by ABA except for ABI4 (Figure 7). Disruption OΪCPK4 or CPKIl gene down-regulated expression of ABFl, ABF2, ABF4, ABI4, ABI5, RD29A, RAB18, KINl, KIN2 and ERDlO, and double disruption of the two CDPK genes had stronger inhibiting effects on expression of these ABA-responsive genes, which was true both in the absence or presence of the ABA treatments (Figure 7), except for ERDlO whose expression, assayed in the absence of the ABA treatments, was not significantly reduced in cpkll-2 mutant (and cpkll-1, data not shown), or less reduced in cpk4-lcpkll-2 mutant (and cpk4-lcpkll-l, data not shown) (Figure 7). Overexpression of CPK4 or CPKIl gene amplified the ABA-induced stimulating effects on these genes except for ERDlO (Figure 7). However, disruption or overexpression of CPK4 or CPKIl gene did not affect expression of ABIl, ABI2, ABI3, MYB2 and MYC2 genes except for the cpk4-l mutant whose ABA-stimulated expression level of MYB2 gene was down-regulated, and for cpk4-lcpkll-2 double mutant whose expression level of ABIl was significantly increased in the absence of ABA treatment (Figure 7). Overexpression 0ΪCPK4 and CPKIl significantly enhanced the expression level ofABF3, but loss-of-function mutations in the CDPK genes did not show any effects (Figure 7).

DISCUSSION

CPK4 and CPKIl Are Two Positive Regulators in CDPK/Ca2+-Mediated ABA

Signaling

[0143] The present experiment showed that two closely homologous CDPKs in

Arabidopsis, CPK4 and CPKl 1, are ABA -inducible and regulate positively ABA signal transduction pleiotropically in seed germination, seedling growth and stomatal movement (Figures 2-5), though the ABA -related phenotypes in seedling growth are relatively weak (Figure 3). Additionally, as regulators of ABA signaling, CPK4 and CPKl 1 are required for plants to respond to salt stress (Figures 4), an environmental stress to which plant responses are most closely associated with the functions of ABA (Zhu, (2002) Annu. Rev. Plant Biol. 53, 247-273; Shinozaki et al, (2003) Curr. Opm. Plant BwI. 6, 410-417). Whereas current evidence suggests that redundancies in CDPK genes make major obstacles to identify their biological functions through genetic approaches (Harmon et al., 2000, 2001, supra; Hrabak et al., 2003, supra; Choi et al., 2005, supra; Mori et al., 2006, supra), and only in regulation of stomatal aperture have the ABA-responsive phenotypes been detected by loss-of-function mutation in CPK3 and CPK6 genes (Mori et al., 2006, supra), our experiments showed relatively strong, pleiotropic, ABA- and salt-responsive phenotypes that resulted from disruption or overexpression of CPK4 or CPKIl genes (Figures 3-5), revealing that the two CDPKs are important regulators in CDPK/Ca²⁺-mediated ABA signaling pathways. The CPK4 and CPKl 1 kinases are structurally highly similar (Figure 10), have the similar expression profile (Figures 1 and 12), both localize in cytoplasm and nucleus (Figure 14), and phosphorylate the same transcription factors ABFl and AB F4 (Figure 6), suggesting that the two kinases may function redundantly in the same pathway. However, it is noteworthy that the double mutations in the two kinase genes resulted in stronger consequences in ABA-, and partly in salt-, responsive phenotypes than the single mutations (Figures 3-5). This synergistic effect in the phenotypes of the double mutants in response to ABA or salt treatments is suggestive of these kinases to be involved in different pathways. Although these suggestions may appear to be contradictory, both circumstances can be possible if these kinases have additional targets to ABFl and ABF4 and these still unknown targets may be different for CPK4 and CPKl 1. Nevertheless, the instant findings, by identifying two CDPKs as important regulators in ABA signaling pathways, provide genetically unequivocal evidence for the involvement of CDPK/Ca ⁺ in ABA signal transduction at the whole-plant level in seed germination, seedling growth, stomatal movement and plant response to salt- stress.

[0144] It has been known that ABA regulates plant adaptation to water deficit and salt stress mainly through its functions in regulating water balance and osmotic stress/cellular dehydration tolerance. Whereas the role in water balance is mainly through guard cell regulation, the latter role has to do with induction of genes that encode dehydration tolerance proteins in nearly all cells (Zhu, 2002, supra; Shinozaki et al., 2003, supra). The two CDPKs CPK4 and CPKI l mediate ABA signaling to regulate stomatal apertures (Figure 5), which is likely to be mainly responsible to their function in conserving water under water-deficit conditions (Figure 5). The two kinases regulate also some stress tolerance-related genes (Figure 7), suggesting that they may also function at the level of cellular dehydration tolerance.

[0145] The salt-induced ABA accumulation is a well-known consequence of salt stress, which results in inhibition of seed germination and is required for tolerance of seedling growth to salt (Zhu, 2002, supra; Shinozaki et al., 2003, supra). The CPK4- and CPKl 1- dependent salt tolerance of seedling growth (Figure 4) reveals the indispensability of the two CDPK genes for plant tolerance to salt stress, which may be ascribed to the functions of the two kinases to regulate ABA signaling. The same phenomenon was also observed in ABA signaling mutants such as abil (Achard et al., (2006) Science 311, 91-94). It is noteworthy, however, that the CPK4 kinase plays more important role than the CPKl 1 does in plant response to salt, which was shown by stronger salt-responsive phenotypes in the cpk4-l mutant (Figure 4). Furthermore, overexpression of CPK4 and CPKIl did not alter significantly plant response to salt stress (Figure 4). These phenotypes differ from those related to ABA where both kinases appear to have comparable effects on ABA- responsiveness (Figures 3 and 5) and the overexpression of the two kinase genes enhanced plant capacity to conserve water (Figure 5). This divergence between the ABA- and salt- responsiveness suggests the possible additional involvement of the two kinases in pathways that may diverge at some point between the response to salt and to ABA.

Role of CPK4 and CPKIl in Mediating ABA Signal Transduction [0146] The CPK4 and CPKl 1 kinases localize both in cytoplasm and nucleus (Dammann et al., supra; Milla et al., 2006a, supra; Figure 9). This double localization in cells appears to facilitate their functions in both early and delayed responses of cells to ABA (Zhu, 2002, supra). For example, the cytoplasm-located CPK4 and CPKl 1 would more easily mediate quick response by sensing Ca²⁺ signal and phosphorylating downstream messengers already in place, such as guard cell regulation, while the nuclear-CPK4 and CPKl 1 would be able to more easily phosphorylate nuclear-localized regulators such as transcription factors present there to mediate gene expression.

[0147] What are the downstream targets of the CPK4 and CPKl 1 kinases to relay ABA signaling? Several ABA/stress signaling regulators including ABA/stress-responsive transcription factors have been shown to be modulated at posttranslational level by changing their phosphorylation states (Li et al., 1998, supra; Guo et al., 2002, supra; Johnson et al., (2002) Plant Physiol 130, 837-846; Mustilli et al., (2002) Plant Cell 14, 3089-3099; Yoshida et al., (2002) Plant Cell Physiol. 43, 1473-1483;Yoshida et al. (2006) J Biol Chem 281, 5310- 5318; Zhu, 2002, supra; Shinozaki et al., 2003; Choi et al., 2005, supra; Song et al., 2005, supra; Milla et al., 2006a, supra; Furihata et al., 2006, supra; Fujii et al., 2007, supra). Among ABA-responsive transcription factors, ABFs transcription factors, including 4 members of basic leucine zipper protein family, are better defined (Choi et al., 2000, supra; Uno et al., 2000, supra; Kang et al., (2002) Plant Cell 14, 343-357; Fujita et al., 2005, 2006, supra; Fujii et al., 2007, supra). The present inventors show that two ABA-responsive transcription factors ABF 1 (Choi et al., 2000, supra) and ABF4 (AREB2) (Choi et al., 2000, supra; Uno et al., 2000, supra) were phosphorylated in vitro by both CPK4 and CPKl 1 (Figure 6), but an ABA-responsive APETALA2 domain transcription factor AB 14 (Finkelstein et al., 1998, supra) was not (data not shown), suggesting that the two ABFs may be downstream targets of both kinases. Additionally, the inventors showed that ABFl and ABF4 were also phosphorylated by other, multiple, kinases than CPK4 and CPKl 1 (Figure 6). These findings suggest that multiple kinases may have common substrates in ABA signaling pathways. Consistently, a recent report showed that ABFl was also the phosphorylation target of two SnRK, SnRK2.2 and SnRK2.3, which positively regulate ABA signaling (Fujii et al., 2007, supra). ABF4, as well as other ABFs, ABF2 and ABF3, were previously shown to play important roles in ABA-mediated drought tolerance (Kang et al., 2002, supra; Fujita et al., 2005, supra). Taken together, the CPK4 and CPKl 1 kinases may regulate ABA signaling at least partly through the functions of their potential targets ABFl and ABF4.

[0148] As regards guard cell regulation, it is noteworthy that stomatal aperture may be regulated by a complex cooperation of, among other regulators, numerous protein kinases including CPK3, CPK6 (Mori et al., 2006, supra) and other kinases such as SNFl- RELATED PROTEIN KINASE (SnRK) 2.6 (OSTl) (Mustilli et al., 2002, supra; Yoshida et al., 2002, 2006, supra). CPK4 and CPKl 1 belong to the same subgroup of CDPKs as CPK6 (Hraback et al, 2003, supra), suggesting that these three CDPKs may possibly function in close cooperation in regulating stomatal aperture. The SnRK2.6 interacts with ABIl to regulate stomatal closure (Yoshida et al., 2006, supra), while CPK4 and CPKl 1 may regulate stomatal aperture through phosphorylating ABFl or ABF4. ABFs transcription factors bind the ABA-responsive G-box motif (Choi et al., 2000, supra; Uno et al., 2000, supra) of which the core ACGT consensus sequence is found in the promoter regions of many ABA-regulated genes including all the 16 genes tested in the present study (Figure 7), and thus may regulate expression of the CPK4- and CPKl 1-downstream target genes to induce ABA-related physiological responses including stomatal regulation. Finally, it is noteworthy that CPKl 1 was also previously reported to interacts with AtDi 19, a zinc-finger protein, and to phosphorylate it in vitro (Milla et al., 2006a, supra), and^Z)z79-related genes were stimulated by drought and salt stresses (Milla et al., (2006b) Plant MoI. Biol. 61, 13-30). This suggests that CPKl 1, possibly as well as CPK4, might be involved in ABA signaling or regulation of plant tolerance to stresses through a complex signaling network.

METHODS

Screening of Loss-of-Function Mutants

[0149] T-DNA insertion lines in the Arabidopsis thahana CPK4 gene (Arabidopsis genomic locus tag: At4g09570, CPK4) and CPKIl gene (Atlg35670, CPKIl) in Columbia ecotype were obtained from the SaIk Institute (website signal.salk.edu) through the Arabidopsis Biological Resource Center (ABRC). The screening for the knockout mutants was done following the recommended procedures. Briefly, for the T-DNA insertion in CPKIl gene, the mutant lines were genotyped by amplifying the genomic DNA with the left genomic primer 1 (LPl) or left genomic primer 3 (LP3) and right genomic primer 1 (RPl), and for the T-DNA insertion in CPK4 gene, the mutant lines were genotyped with the left genomic PCR primer 2 (LP2) and right genomic primer 2 (RP2). These genomic primers were used together with a T-DNA left border primer (LBaI) and a right border primer (RBaI) to constitute specific primer pairs for genotyping the T-DNA insertion lines (see Figure IA and IB). The sequences for these primers are presented in Table 3. The T-DNA insertion in the mutants was identified by PCR and DNA gel-blot analysis, and the exact position was determined by sequencing. The present inventors identified a homozygous T- DNA insertion allele, SALK_081860, in the 5'-UTR of the CPK4 gene, designated cpk4-l, and two homozygous T-DNA insertion alleles, SALK_023086 in the 5'-UTR and SALK_054495 in the 1st exon of the CPKIl gene, designated cpkll-1 and cpkll-2, respectively. For the cpk4-l mutant, the PCR products could be generated with both the primer pair LBaI -RP2 and LP2-LBal (Figures IA and 8), but could not with the primer pair LP2-RBal (data not shown), indicating that tandem T-DNAs were inserted into the genome in an inverted fashion at the same locus, which was supported by DNA-gel blot analysis that detected a two-copy T-DNA insertion (Figure 9). Sequencing assay showed that the T-DNA insertion generates a DNA-fragment deletion in the T-DNA insertion site (see RESULTS section). For the cpkll-1 mutant, the PCR products could be generated with both the primer pair LBaI-RPl and LP3-LBal (Figures IB and 8), but were not found with the primer pair LP3-RBal (data not shown), indicating that, like the cpk4-l mutant, tandem T-DNA insertion was present for the cpkll-1 mutant in an inverted fashion at the same locus, which also was supported by DNA-gel blot analysis that detected a two-copy T-DNA insertion (Figure 9). Also, the T-DNA insertion generates a DNA-fragment deletion in the T-DNA insertion site (see RESULTS section). For the cpkll-2 mutant, analysis of PCR, sequencing and DNA-gel blot all showed that a single copy of T-DNA was inserted into the genome (Figures IB, 8, and 9), and the T-DNA insertion results also in a DNA-fragment deletion in the T-DNA insertion site (see RESULTS section). The cpk4-l cpkll-1 and cpk4-lcpkll-2 double mutants were constructed by crossing, and their genotypes were confirmed by PCR-based genotyping.

Mutant Complementation and Generation of Transgenic Plants

[0150] To create transgenic plant lines overexpressing CPK4 or CPKIl gene or expressing these two genes in the knockout mutants, the open reading frame (ORF) for the CPK4 gene was isolated by polymerase chain reaction (PCR) using the forward primer 5'- GCTCTAGAATGGAGAAACCAAACCCTAG-S' and reverse primer 5'- CGGGATCC TTACTTTGGTGAATCATCAGA-S '; and the ORF for the CPKIl gene was isolated using the forward primer 5'- GCTCTAGA ATGGAGACGAAGCCAAACCCTAG-3 ' and reverse primer 5'- CGGGATCC TCAGTCATC AGATTTTTCACCA -3'. The ORF (1506 bp) of CPK4 and the ORF (1488 bp) of CPKIl were inserted, respectively, into the pCAMBIA- 1300-221 vector (website cambia.org/daisy/cambia/materials/vectors/SδS.html) by Xba I and BamH I sites under the control of a constitutive cauliflower mosaic virus (CaMV) 35 S promoter. These constructs were all verified by sequencing and introduced into the GV3101 strain of Agrobactermm tumefaciens. The constructions were transformed, by floral infiltration as described previously (Clough and Bent, (1998) Plant J. 16, 735-743), into plants of the wild-type Columbia for generating the CPK4- and CFAT77-overexpressing lines, or into cpk4-l and cpkll-1 and cpkll-2 mutant plants for assays of complementation. Transgenic plants were selected by hygromycin resistance and confirmed by PCR. The homozygous T3 seeds of the transgenic plants were used for further analysis.

Growth Conditions

[0151] Plants were grown in a growth chamber at 20-21 ⁰C on Murashige-Skoog (MS) medium at about 80 μmol photons m^"2 s^"1 or in compost soil at about 120 μmol photons m^"2 s^"1 over a 16-h photoperiod at 22⁰C

Phenotype Analysis [0152] Phenotype analysis was done essentially as previously described (Shen et al., 2006, supra). For germination assay, approximately 100 seeds each from wild types (Columbia ) and mutants or transgenic mutants were planted in triplicate on MS medium (Sigma, product#, M5524; full-strength MS). The medium contained 3% sucrose and 0.8% agar (pH 5.7) and supplemented with or without different concentrations of (±)-ABA or NaCl. The seeds were incubated at 4⁰C for 3 days before being placed 22⁰C under light conditions, and germination (emergence of radicals) was scored at the indicated times.

[0153] For seedling growth experiment, seeds were germinated after stratification on common MS medium and 48 h later transferred to MS medium supplemented with different concentrations of ABA in the vertical position. Seedling growth was investigated 10 days after the transfer, and the length of primary roots was measured using a ruler. Seedling growth was also assessed by directly planting the seeds in ABA-containing MS-medium to investigate the response of seedling growth to ABA after germination. [0154] Lateral root growth assays were performed according to the protocol of Xiong et al. (2006, Plant Physiol. 142, 1065-1074) with some modifications. Four-day-old seedlings were individually transferred with a pair of forceps to the treatment medium consisting of the basal salts along with 4% sucrose solidified with 1.2% agar (catalog no. A-296, Sigma). The basal salts included 1.0 mM CaCl₂, 0.5 mM MgSO₄, 0.4 mM KH₂PO₄, 6.0 mM KNO₃, and 7.0 mM NH₄NO₃. Micronutrients were added at full strength (1 * that used in the MS medium) and the pH was adjusted to 5.7 with KOH, and 1.0 μM ABA was added to the medium after autoclaving. After growing for 10 d on the treatment medium, seedlings were photographed with a digital camera. The length of lateral roots was measured using a ruler. The total length of lateral roots of each individual plant was calculated and the means for each line was used as an index to measure lateral root growth.

[0155] For seedling growth in salt, seeds of wild-type, cpk4-l, cpkll-2, cpk4-lcpkll-2 and transgenic plants were surface-sterilized, stratified at 4°C for 3 days to obtain uniform germination, and sown on common MS media without salt. Seedlings were allowed to grow for four days with the plates in a vertical orientation at 22⁰C under light conditions. Then seedlings were transferred to MS medium (full-strength MS, 3% sucrose, pH 5.7) containing 1.2% agar and different salt concentrations (0, 100, 150, 170 or 200 mM NaCl) in the vertical position using forceps. The status of seedling growth was recorded 7 days after the transfer.

[0156] For drought treatment, plants were grown on soil until they were 3 weeks old, and then drought was imposed by withdrawing irrigation for one-half of the plants until the lethal effects was observed on most of these plants, whereas the other half were grown under a standard irrigation regime as a control.

[0157] For water loss assay, rosette leaves were detached from their roots, placed on filter paper, and left on the lab bench. The loss in fresh weight was monitored at the indicated times.

[0158] For stomatal aperture assays, leaves were floated in the buffer containing 50 mM KCl and 10 mM Mes-Tris (pH 6.15) under a halogen cold-light source (Colo-Parmer) at 200 μmol m^"2 sec^"1 for 2 hr followed by addition of different concentrations of (±)-ABA. Apertures were recorded on epidermal strips after 2 h of further incubation to estimate ABA- induced closure. To study inhibition of opening, leaves were floated on the same buffer in the dark for 2 hr before they were transferred to the cold-light for 2 h in the presence of ABA, and then apertures were determined. Production of Anti-CPK4 and Anti-CPKll Sera

[0159] A fragment of CPK4 cDNA corresponding C-terminal 116 amino acids (from 386 to 501) was isolated using forward primer 5'-

CCGGAATTCATGGCTTGCAC AGAGTTTGGTCT-S' and reverse primer 5'- ACGCGTCGACTTACTTTGGTGAATCATCAGA-3 ' , and a fragment of CPKl 1 cDNA corresponding C-terminal 109 amino acids (from 387 to 495) was isolated using forward primer 5 '-CCGGAATTCATGGCTTGCACAGAGTTTGGTCT-S ' and reverse primer 5'- ACGCGTCGACTCAGTCATCAGATTTTTCACCA-3'. They were expressed in E. cob BL21 (DE3) as glutathione S-transferase- (GST-) CPK4^C and GST-CPKI l⁰ fusion proteins. The affinity-purified fusion protein was used for standard immunization protocols in rabbit. The antisera were affinity-purified. Each antiserum, anti-CPK4 or anti-CPKl 1 serum, was shown to recognize both CPK4 and CPKl 1, which is because the C-terminus of the two CDPKs shares high sequence identity. However, the two antisera do not cross-react with any other proteins. In the most cases, one of the two antisera was used to detect CPK4 or CPKl 1.

Extraction of Proteins and Protein Determination

[0160] Total protein extracts were obtained from Arabidopsis plants by grinding whole seedlings or leaf tissue first in liquid nitrogen and then on ice for 3 h in one volume of the extraction buffer. The extraction buffer consists of 50 mM Tris-HCl, pH 7.6, 100 mM NaCl, 0.5% Triton X-100, 10 mM dithiothreitol (DTT), 1 mM phenylmethylsulfonyl fluoride, 5 μg mL^"1 antipain, 5 μg mL^"1 aprotinin, and 5 μg mL^"1 leupeptin. Lysates were cleared of debris by centrifugation at 12,00Og for 30 min at 4 ⁰C .

[0161] Protein concentrations were determined by the method of Bradford (1976, Anal Biochem 72, 248-254) with bovine serum albumin (BSA) as a standard. Fifty micrograms of total proteins were used for each extract for protein concentration determination.

Gel Electrophoresis and Immunoblotting

[0162] SDS-PAGE was carried out according to the method of Laemmli (1970, Nature 227, 680-685). The protein samples (20 μg) were boiled for 2 min before analyzed on a 12% SDS-polyacrylamide gel. Immunoblotting was done essentially as described by Yu et al. (2006, supra). After SDS-PAGE, the proteins on gels were electrophoretically transferred to nitrocellulose membranes (0.45 μm, Amersham Pharmacia). The membranes were blocked for 2 h at room temperature with 3% (w/v) bovine serum albumin (BSA) and 0.05% (v/v) Tween 20 in a Tris-buffered saline (TBS) containing 10 mM Tris-HCl (pH 7.5) and 150 mM NaCl, and then were incubated with gentle shaking for 2 h at room temperature in the rabbit polyclonal antibodies anti-CPK4^c (1:3000) or anti-CPKl l^c serum (1: 1000) diluted in the blocking buffer. After being washed three times for 10 min each in the TBS containing 0.05% (v/v) Tween 20, the membranes were incubated with the alkaline phosphatase- conjugated antibody raised in goat against rabbit IgG (diluted 1 : 1000 in the blocking buffer) at room temperature for 1 h, and then washed three times for 10 min each with 50 mM Tris- HCl (pH 7.5) buffer containing 150 mM NaCl and 0.1% (v/v) Tween 20. Protein bands were visualized by incubation in the colour-development solution using a 5-bromo-4-chloro-3- indolyl-phosphate/nitroblue tetrazolium substrate system according to the manufacturer's protocol. Protein band intensity was estimated by densitometric scans using a digital imaging system and analyzed with QuantityOne software (BioRad). Tubulin, immuno-detected with anti-rat-tubulin serum (Sigma), was used as a loading control.

Immunoprecipitation

[0163] Immunoprecipitation was done essentially as described by Yu et al. (2006, supra). The total proteins (50 μg) were resuspended in 0.5 mL immunoprecipitation buffer containing 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EGTA, 1 mM Na₃VO₄, 1 mM NaF, 10 mM β-glycerophosphate, 1 mM phenylmethylsulfonyl fluoride, 5 μg mL^"1 antipain, 5 μg mL^"1 aprotinin, 5 μg mL^"1 leupeptin, and 0.5% Triton X-100. The mixture was incubated with either the purified anti-CPK4^c or anti-CPKl l^c serum (about 3 μg protein) or the same amount of preimmune serum protein (as a control) at 4⁰C for 2 h. Then 25 μL protein A- agarose suspension was added to the mixture, and the mixture was incubated further for 2 h. Following a brief centrifugation, the immunoprecipitated proteins, after three washes with the immunoprecipitation buffer, were used for the assays of immunoblotting or kinase activity.

In-GeI Kinase and Autophosphorylation Assays [0164] The in-gel kinase activity assay of proteins was done essentially as described by Yu et al. (2006, supra). After SDS-PAGE as described above but with the separating polyacrylamide SDS gel that was polymerized in the presence of 0.5 mg mL^"1 histone III-S as a substrate for kinases, the gels were washed twice with 50 mM Tris-HCl, pH 8.0, containing 20% (v/v) 2-propanol for 1 h per wash, and then with buffer A composed of 50 mM Tris- HCl, pH 8.0, 5 mM 2-mercaptoethanol, and 0.1 mM EDTA for 1 h at room temperature. Proteins in the gels were denatured by incubating the gels in buffer A containing 6 M guanidine hydrochloride for two incubations of 1 h each at room temperature. Proteins were then renatured using buffer A containing 0.05% (v/v) Tween 20 for six incubations of 3 h each at 4°C. After preincubation at room temperature for 30 min with buffer B composed of

40 mM HEPES-NaOH, pH 7.5, 10 mM MgCl₂, 0.45 mM EGTA (l mM in the Ca²⁺-free medium), and 2 mM DTT in the absence or presence of 0.55 mM CaCl₂, the gels were incubated with buffer B containing 50 μM ATP and 10 μCi/mL [r³²-P]-ATP (3,000 Ci/mmol; Amersham Pharmacia) for 1 h at room temperature. The gels were then washed extensively with 5% trichloroacetic acid and 1% sodium pyrophosphate until radioactivity in the used wash solution was barely detectable. The gels were then stained with Coomassie Brilliant Blue R-250 (Amersham Pharmacia Biotech Ltd, Buckinghamshire, UK). After destaining, the gels were air dried between two sheets of cellophane, and the histone III-S in gel phosphorylated by CDPK was detected by autoradiography after exposition of the dried gels to Kodak X-Omat AR film for 5 to 7 d at -20⁰C. Films were scanned using a digital imaging system and radioactivity was quantified with QuantityOne software (BioRad).

[0165] For Ca²⁺-dependent electrophoretic mobility shift of the kinases in the in-gel autophosphorylating activity assays, Ca²⁺ or EGTA to a final concentration of 2 mM was added to the immunoprecipitated proteins dissolved in SDS-PAGE sample buffer. The in-gel autophosphorylation assay of CDPK was performed as described above, except that the separating gel was polymerized in the absence of substrate.

Preparation of ABFl and ABF4 Proteins and Phosphorylation Assay [0166] To prepare recombinant ABFl and AB F4, their coding regions were prepared by PCR. For ABFl, the primers used were 5'-

CGGGATCCGATGGGTACTCACATTGATATC-3' (forward primer) and 5'- CCCAAGCTTTTACCACGGACCGGTAAGGGTTC-3' (reverse primer). For ABF4, the primers were 5 '-GGAATTCTATGGGAACTCACATCAATTTCAAC-S ' (forward primer) and 5 ' -CCGCTCGAGTCACCATGGTCCGGTTAATGTCCT-S ' (reverse primer) . The PCR product was digested with BamH I and Hind III (ABFl) or EcoR I and Sal I (ABF4) and subcloned into pET-48 b(+) vector (Novagen) for the production of His fusion protein using Escherichia coli BL21(DE3) cells (Novagen). The cell lysate was applied to the nickel- nitrilotriacetic acid agarose column (Qiagen) and processed according to the manufacturer's instruction. The purified proteins were dialyzed with 10 mM Tris-HCl, pH 7.5, for 16 h at 4⁰C and stored at -80⁰C in working aliquots. Phosphorylation of His-ABFl and His-ABF4 was carried out as described above, except when separating the immuno-precipitated proteins on a SDS-PAGE gel that contained 0.5 mg/mL His-tagged ABFl or AB F4 as potential substrates of the protein kinases.

Reverse Transcriptase-Mediated PCR and Real-Time PCR Analysis

[0167] Reverse transcriptase (RT)-mediated PCR analysis was performed to analyze the expression 0ΪCPK4 and CPKIl genes. Total RNA was isolated from leaves of three-week- old Arabidopsis seedlings with the RNasy Plant Mini Kit (Qiagen, Valencia, CA) supplemented with an on-column DNA digestion (Qiagen RNase-Free DNase set) according to the manufacturer's instructions, and then the RNA sample was reverse transcribed with the Superscript II RT kit (Invitrogen, Carlsbad, CA) in 25 μL volume at 42 ⁰C for Ih. PCR was conducted at linearity phase of the exponential reaction for each gene. The gene-specific primer pairs were: for CPK4: forward primer 5'- GAGAAACCAAACCCTAGAAGACC -3' and reverse primer 5'- CAGGTGC AACATAATACGGAC -3', and for CPKl 1: forward primer 5 '-CCCTAGACGTCCTTCAAACACA-S ' and reverse primer 5'- CTCTGGTGCAACATAGTACGG-3'. Actin gene (At5g09810) expression level was used as a quantitative control.

[0168] To assay the expression levels of CPK4 and CPKIl genes after ABA treatment, quantitative real-time PCR analysis was performed with the RNA samples isolated from three-week-old seedlings harvested at the indicated times after 50 μM ABA treatments (mixed isomers; Sigma, St. Louis, MO). Total RNA isolation and reverse transcription were done as described above for RT-PCR. PCR amplification was performed with primers specific for CPK4 or CPKIl genes: for CPK4 forward 5'- TCTGTGACACTCCTCTTGATGAC-3' and reverse 5'- GCTCATCTACAAAAGTGGAAACG-3'; for CPKIl forward 5'- CGAAGAAGAACCAACAAAAAACC-3' and reverse 5'- GCCATACATCTTCGTAATCCTCG-3 ' . Amplification ofACTIN2/8 (forward primer 5 ' - GGTAACATTGTGCTCAGTGGTGG-S' and reverse primer 5'-

AACGACCTTAATCTTCATGCTGC-3') genes was used as an internal control (Charrier et al., 2002, Plant Physiol 130, 577-590). The suitability of the oligonucleotide sequences in term of efficiency of annealing was evaluated in advance using the Primer 5.0 program. The cDNA was amplified using SYBR Premix Ex Taq™ (TaKaRa) using a DNA Engine Opticon 2 thermal cycler (MJ Research, Watertown, MA) in 10 μL volume with the following program: 1 cycle of 95 ⁰C, 10 s; and 40 cycles of 94⁰C, 5s; 58.5⁰C, 20s; 72⁰C, 20s. The amplification of the target genes was monitored every cycle by SYBR-Green fluorescence. The Ct (threshold cycle), defined as the PCR cycle at which a statistically significant increase of reporter fluorescence was first detected, was used as a measure for the starting copy numbers of the target gene. Relative quantitation of the target gene expression level was performed using the comparative Ct method. Three technical replicates were performed for each experiment.

[0169] To assay the expression of ABA-responsive genes, real-time PCR analysis was done with the RNA samples isolated from three-week-old seedlings harvested 5 h after the treatments with or without 50 μM ABA, except for ABB of which the expression was assayed with the seedlings grown in the ABA-free MS-medium for 4 days and then transferred to the ABA-free (a control) or 50 μM ABA-containing medium for 5 days. Total RNA isolation and reverse transcription were done as described above. PCR amplification was performed with oligonucleotides specific for various ABA-responsive genes: RD29A (At5g52310) forward 5ΑTCACTTGGCTCCACTGTTGTTC-3' and reverse 5'- ACAAAACACACATAAACATCCAAAGT-3'; MYB 2 (At2g47190) forward 5'- TGCTCGTTGGAACCACATCG-3 ' and reverse 5 ' -ACCACCTATTGCCCCAAAGAGA-3 ' ; MYC2 (Atlg32640) forward 5 '-TCATACGACGGTTGCCAGAA-S ' and reverse 5'- AGCAACGTTTACAAGCTTTGATTG-3'; RAB 18 (At5g66400) forward 5'- CAGCAGCAGTATGACGAGTA-3' and reverse 5'-CAGTTCCAAAGCCTTCAGTC-3'; KINl (At5gl5960) forward 5 '-ACCAACAAGAATGCCTTCCA-S ' and reverse 5'- CCGCATCCGATACACTCTTT-3 ' ; KIN2 (At5g 15970) forward 5 ' -

ACCAACAAGAATGCCTTCCA-3' and reverse 5 '-ACTGCCGCATCCGATATACT-S '; ERDlO (Atlg20450) forward 5 '-TCTCTGAACCAGAGTCGTTT-S ' and reverse 5'- CTTCTTCTCACCGTCTTCAC-3'; Λ£/7 (At4g26080) forward 5- AGAGTGTGCCTTTGTATGGTTTTA-3' and reverse 5'- CATCCTCTCTCTACAATAGTTCGCT-S'; ABI2 (At5g57050) forward 5'- GATGGAAGATTCTGTCTCA ACGATT-3' and reverse 5'- GTTTCTCCTTCACTATCTCCTCCG-3'; Λ£/3 (At3g24650) forward 5'-

TCC ATTAGACAGCAGTCAAGGTTT-S' and reverse 5'- GGTGTCAAAGAACTCGTTGCTATC-3'^5/4 (At2g40220) forward 5'- GGGCAGGAACAAGGAGGAAGTG-3 ' and reverse 5 ' -

ACGGCGGTGGATGAGTTATTGAT-S'; ABI5 (At2g36270) forward 5'- CAATAAGAGAGGGATAGCGAACGAG-S' and reverse 5'- CGTCCATTGCTGTCTCCTCCA-3'. ABFl (Atlg49720) forward 5'- TCAACAACTTAGGCGGCGATAC-3' and reverse 5'- GCAACCGAAGATGTAGTAGTCA-3'; ABF2 (Atlg45249) forward 5'- TTGGGGAATGAGCCACCAGGAG-3' and reverse 5'- GACCCAAAATCTTTCCCTACAC-3'; y45« (At4g34000) forward 5'- CTTTGTTGATGGTGTGAGTGAG-3 ' and reverse 5 ' -GTGTTTCCACTATTACCATTGC- 3'; ABF 4 (At3g 19290) forward 5 '-AACAACTTAGGAGGTGGTGGTC-S ' and reverse 5'- CTTCAGGAGTTCATCCATGTTC-3'. Amplification OΪACTIN2/8 genes was used as an internal control, and real-time quantitative PCR experimental procedures were performed as described above. Three technical replicates were performed for each experiment. [0170] For all the above quantitative real-time PCR analysis, the assays were repeated three times along with three independent repetitions of the biological experiments, and the means of the three biological experiments were calculated for estimating gene expression.

Southern-Blot Analysis

[0171] Genomic DNA was extracted from 4-week-old cpk4-l or cpkll-1 or cpkll-2 plants using the method of Doyle and Doyle (1990, Focus 12, 13-15). Ten micrograms of DNA was digested to completion with EcoRl plus Pstl, and Hmdlll restriction enzymes, electrophoresed through 0.8% agarose, and blotted onto nylon membranes (Hybond-N⁺, Amersham Pharmacia Biotech). The specific probe was produced as follows: the 597-bp specific sequence of T-DNA was amplified using the genomic DNA of cpk4-l by forward primer 5 ' -TCAGAAGAACTCGTC AAGAAGG -3 ' , and reverse primer 5 ' -

CTATCGTGGCTGGCCACGACG-S'; and then the PCR product was gel purified and radiolabeled with ³²P by a random primer labeling kit (Takara). DNA gel blot hybridization was performed at 65⁰C for 24 h using hybridization solution (200 mM sodium phosphate buffer, pH 7.2, 1 mM EDTA, pH 8.0, 50% formamide, 10% BSA, and 7% SDS) with ³²P- labeled specific probes. Then the membranes were washed at 65⁰C in 5 * SSC and 0.5% SDS, 1 x SSC and 0.5% SDS, and 0.1 x SSC and 0.5% SDS for 30 min sequentially. The copy of T-DNA insertion was detected by autoradiography after exposition of the membranes to Kodak X-Omat AR film for one week at -7O⁰C.

[0172] The probe sequence was: TCAGAAGAACTCGTCAAGAAGGCGATAGAAGGCGATGCGCTGCGAATCGGGAGC GGCGATACCGTAAAGCACGAGGAAGCGGTCAGCCCATTCGCCGCCAAGCTCTTC AGCAATATCACGGGTAGCCAACGCTATGTCCTGATAGCGGTCCGCCACACCCAG CCGGCCACAGTCGATGAATCCAGAAAAGCGGCCATTTTCCACCATGATATTCGGC AAGCAGGCATCGCCATGGGTCACGACGAGATCATCGCCGTCGGGCATGCGCGCC TTGAGCCTGGCGAACAGTTCGGCTGGCGCGAGCCCCTGATGCTCTTCGTCCAGAT CATCCTGATCGACAAGACCGGCTTCCATCCGAGTACGTGCTCGCTCGATGCGATG TTTCGCTTGGTGGTCGAATGGGCAGGTAGCCGGATCAAGCGTATGCAGCCGCCGC ATTGCATCAGCCATGATGGATACTTTCTCGGCAGGAGCAAGGTGAGATGACAGG AGATCCTGCCCCGGCACTTCGCCCAATAGCAGCCAGTCCCTTCCCGCTTCAGTGA CAACGTCGAGCACAGCTGCGCAAGGAACGCCCGTCGTGGCCAGCCACGATAG

Subcellular Localization of CPK4 and CPKIl [0173] For subcellular localization of CPK4 and CPK 11 , the full-length ORF of CPK4 was PCR-amplified by using forward primer 5 '-CCGCTCGAGATGGAGAAACCAAACCCTAG -3' and reverse primer 5'- CGGGATCCCGCTTTGGTGAATCATCAGATTTAG-3', and the full-length ORF of CPKIl was PCR-amplified by using forward primer 5'- CCGCTCGAGATGGAGACGAAGCCAAACCCTAG-3' and reverse primer 5'- CGGGATCCCGGTCATC AGATTTTTCACCATC-3 ' . The PCR products were then fused to the upstream of the enhanced GFP (Cormack et al., (1996) Gene 173, 33-38) atthe ^ΛoI (5'- end) / BamΑ I (3 '-end) sites in the CaMV 35S-EGFP-Ocs 3'- vector (p-EZS-NL vector, Dr. Ehrhardt, deepgreen.stanford.edu), respectively. The full-length CPK4 cDNA with GFP sequence at C-terminal was then amplified by PCR using p-EZS-NL-CPK4-EGFP vector as the template using the forward primer 5 ' -GCTCTAGAATGGAGAAACCAAACCCTAG-S ' and reverse primer 5 '-TCCCCCGGGTTACTTGTACAGCTCGTCCATGC-S' . The full- length CPKIl cDNA with GFP sequence at C-terminal was amplified by PCR using p-EZS- NL-CPKl 1-EGFP vector as the template using the forward primer 5'- GCTCTAGAATGGAGACGAAGCCAAACCCTAG-3' and reverse primer 5'- TCCCCCGGGTTACTTGTACAGCTCGTCCATGC-3 ' . The resulting PCR product was digested with Xba I and Sma I, subcloned into pCAMBIA- 1300-221 vector under the control of CaMV 35 S promoter. Finally, each vector was sequenced to confirm that the fusion was in-frame and without PCR-induced mistakes. These constructions were then transformed into Agrobacterium strain GV3101 and introduced into plants of wild-type Columbia by the floral dip method as previously described (Clough and Bent (1998) Plant J. 16, 735-743), respectively. The homozygous T3 seeds of the transgenic plants were used for assays of subcellular localization using a confocal laser scanning microscope (Bio-Rad MRC 1024) (see Figure 11). ABA Measurement

[0174] Rosette leaves were excised from 3 -week-old mutant and wild-type plants grown under drought treatment (withholding water for 1 d, 5 d and 1O d, respectively). ABA contents in tissues were measured by ELISA method as described previously (Chen et al. , (2006) Plant Physiol 140, 302-310).

[0175] It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All patents, patent applications, and other publications cited in this application, including published amino acid or polynucleotide sequences, are incorporated by reference in the entirety for all purposes.

Table 3 Analysis of T-DNA Insertion into Arabidopsis Genome for Identification of the cpk4 and cpkll Knockout Mutants. Start codon (ATG) is underlined.

1- Identification of the T-DNA insertion site and the possible sequence deletion due to the T-DNA insertion in the cpkll-1 mutant The primers used for identification of the cpkll-1 mutation: Left border primer (LBaI): 5'-GGTTCACGTAGTGGGCCATC-3' Right genomic primer 1 (RPl): 5 '-AAACCAATTAGGCGATGAACC-S ' Left genomic primer 3 (LP3): 5 '-TGGGATGAAAACACACAAGCGG-S '

The deleted genomic sequence (bold and underlined letters, nt -120 to -87, 34 bp deleted) due to the insertion of a tandem -two-copy T-DNA into this site in a inverted fashion in the cpkll-1 mutant:

CAAAGAAAAAGTCTGTTTATCATCTTCTTCTTCTTCAAATCGAGATCGAAGAAGA

ACCAACAAAAAACCAAAAATG

The presence of the PCR products obtained with both the primer pair LBaI-RPl and LP3- LBaI, together with the fact that the PCR products could not been found (data not shown) when PCR was performed with the primer pair LP3-RBal (see below for the RBaI sequence), indicate that tandem T-DNAs were inserted into the genome for the cpkll-1 mutant in an inverted fashion at the same locus, and the T-DNA insertion generates a 34-bp deletion from -120 to -87 bp 5'-upstream of the CPKl 1 translation start codon. Southern blot analysis further indicates that a tandem T-DNA of two copies was inserted at the locus (see Figure 9).

2- Identification of the T-DNA insertion site and the possible sequence deletion due to the T-DNA insertion in the cpkll-2 mutant

The primers used for identification of the cpkll-2 mutation:

Left border primer (LBaI) and right genomic primer 1 (RPl) are the same as those mentioned above for cpkll-1 mutant identification.

Left genomic primer 1 (LPl): 5'-. V - :. . .. I ^■■ . ■ . . .. .. ; ^';^'■• ^: V" : . -3' Right border primer (RBaI): 5 '-GTTTCTGACGTATGTGCTTAGC-3 '

The deleted genomic sequence (bold and underlined letters, nt 320 to 358, 39 bp deleted) due to the insertion of a single-copy T-DNA into this site in the cpkll-2 mutant: \ I GGAGACGAAGCCAAACCCTAGACGTCCTTCAAACACAGTTCTACCATATCAA ACACCACGATTAAGAGATCATTACCTΓCTGGGAAAAAAGCTAGGCCAAGGCCAA TTTGGAACAACCTATCTCTGCACAGAGAAATCAACCTCCGCTAATTACGCCTGCA AATCGATCCCGAAGCGAAAGCTCGTGTGTCGCGAGGATTACGAAGATGTATGGC GTGAGATTCAGATCATGCATCATCTCTCTGAGCATCCAAATGTTGTTAGGATCAA AGGGACTTATGAAGATTCGGTGTTTGTTCATATTGTTATGGAGGTTT^XYAΛCΛIX

The results indicate that a single copy of T-DNA was inserted into the genome for the cpkll- 2 mutant, and the T-DNA insertion generates a 39-bp deletion from 320 to 358 bp downstream of the CPKl 1 translation start codon.

3- Identification of the T-DNA insertion site and the possible sequence deletion due to the T-DNA insertion in the cpk4-l mutant

The primers used for identification of the cpk4-l mutation: Left border primer (LBaI): 5'-GGTTCACGTAGTGGGCCATC-3' Right genomic primer 2 (RP2) : 5 ' -GCTTAGCATCATCACTGGGAC-3 ' Left genomic primer 2 (LP2): 5 '-AATCCGACTTACTTTGGTTAGAA-S '

The deleted genomic sequence (bold and underlined letters, nt -67 to -57, 11 bp deleted) due to the insertion of a tandem-two-copy T-DNA into this site in a inverted fashion in the cpk4-l mutant: AACTTC£XA £i;ABlLO^CTCCTCCTCCTTTGATAAACACCAAAAAAAGGCAGAG ACTTTCGAAATCAAGAACA VRI

The presence of the PCR products obtained with both the primer pair LBal-RP2 and LP2- LBaI, together with the fact that the PCR products could not been generated (data not shown) when PCR was performed with the primer pair LP2-RBal, indicate that tandem T-DNAs were inserted into the genome for the cpk4-l mutant in an inverted fashion at the same locus, and the T-DNA insertion generates an 11-bp deletion from -67 to -57 bp 5 '-upstream of the CPK4 translation start codon. Southern blot analysis further indicates that a tandem T-DNA of two copies was inserted at the locus (see Figure 9). SEQUENCE LISTING

SEQ ID NO: 1

Arabidopsis CPK4 cDNA sequence, Genbank Accession No. NM_117025

ATGGAGAAACCAAACCCTAGAAGACCCTCAAACAGTGTTCTTCCATACGAAACACCAAGATT AAGAGATCACTATCTCCTCGGCAAAAAGCTAGGCCAAGGCCAATTTGGAACAACCTATCTCT

GTACAGAGAAATCATCATCAGCTAATTACGCTTGCAAATCAATCCCAAAACGTAAGCTTGTA

TTAAAGCTACAGACTTTGGTTTGTCTGTCTTCTACAAGCCAGGGCAGTATCTGTATGATGTA

GTTGGAAGTCCGTATTATGTTGCACCTGAGGTTCTGAAGAAATGTTATGGACCAGAGATAGA

CGTGTGGAGCGCCGGTGTTATCTTGTACATCTTACTAAGTGGGGTTCCTCCTTTTTGGGCAG

GAAGGAATTGTTCAAAATGATAGATACAGACAACAGTGGAACAATCACCTTTGAAGAGCTTA AAGCAGGTCTAAAGAGAGTTGGATCTGAATTGATGGAATCAGAAATCAAGTCTCTTATGGAT GCGGCGGATATAGACAACAGTGGGACAATAGACTACGGTGAATTCCTAGCAGCGACATTACA TATAAACAAGATGGAGAGAGAAGAGAACTTGGTGGTTGCGTTTTCATACTTTGATAAAGATG GTAGCGGTTATATCACCATTGACGAGCTTCAACAAGCCTGCACAGAGTTTGGTCTCTGTGAC

GATGATTCACCAAAGTAA

SEQ ID NO:2

Arabidopsis CPK4 amino acid sequence, Genbank Accession No. NP_192695.1

MEKPNPRRPSNSVLPYETPRLRDHYLLGKKLGQGQFGTTYLCTEKSSSANYACKSIPKRKLV CREDYEDVWREIQIMHHLSEHPNVVRIKGTYEDSVFVHIVMEVCEGGELFDRIVSKGCFSER EAAKLIKTILGVVEACHSLGVMHRDLKPENFLFDSPSDDAKLKATDFGLSVFYKPGQYLYDV VGSPYYVAPEVLKKCYGPEIDVWSAGVILYILLSGVPPFWAETESGIFRQILQGKIDFKSDP WPTISEGAKDLIYKMLDRSPKKRISAHEALCHPWIVDEHAAPDKPLDPAVLSRLKQFSQMNK IKKMALRVIAERLSEEEIGGLKELFKMIDTDNSGTITFEELKAGLKRVGSELMESEIKSLMD AADIDNSGTIDYGEFLAATLHINKMEREENLVVAFSYFDKDGSGYITIDELQQACTEFGLCD TPLDDMIKEIDLDNDGKIDFSEFTAMMKKGDGVGRSRTMRNNLNFNIAEAFGVEDTSSTAKS DDSPK SEQ ID NO: 3

Arabidopsis CPKl 1 cDNA sequence, Genbank Accession No. NM_103271

ATGGAGACGAAGCCAAACCCTAGACGTCCTTCAAACACAGTTCTACCATATCAAACACCACG

CGTGAAGCTGTCAAGCTTATTAAGACGATTCTTGGTGTTGTTGAGGCTTGTCATTCTCTTGG TGTTATGCATAGAGATCTCAAACCTGAGAATTTCTTGTTTGATAGTCCTAAAGATGATGCTA

AGCTTAAGGCTACCGATTTTGGTTTGTCTGTCTTCTATAAGCCAGGACAATATTTATATGAC

CACCAGACAAGCCTCTTGATCCAGCAGTCTTATCTCGTCTAAAGCAGTTTTCTCAAATGAAT

AAGATTAAGAAAATGGCATTACGGGTAATTGCTGAGAGACTTTCAGAGGAAGAAATTGGAGG

TCTGAAGGAATTGTTCAAGATGATAGACACAGACAACAGCGGAACGATTACTTTTGAAGAGC TCAAAGCGGGTTTGAAGAGAGTCGGATCTGAACTGATGGAATCAGAAATCAAGTCTCTCATG

CTCGGAGTTTACAGCAATGATGAGGAAAGGAGATGGAGTTGGGAGAAGCAGAACCATGATGA AGAACTTGAACTTCAACATTGCTGATGCTTTTGGAGTTGATGGTGAAAAATCTGATGACTGA

SEQ ID NO: 4

Arabidopsis CPKl 1 amino acid sequence, Genbank Accession No. NP_174807.1 METKPNPRRPSNTVLPYQTPRLRDHYLLGKKLGQGQFGTTYLCTEKSTSANYACKSIPKRKL VCREDYEDVWREIQIMHHLSEHPNVVRIKGTYEDSVFVHIVMEVCEGGELFDRIVSKGHFSE REAVKLIKTILGVVEACHSLGVMHRDLKPENFLFDSPKDDAKLKATDFGLSVFYKPGQYLYD VVGSPYYVAPEVLKKCYGPEIDVWSAGVILYILLSGVPPFWAETESGIFRQILQGKLDFKSD PWPTISEAAKDLIYKMLERSPKKRISAHEALCHPWIVDEQAAPDKPLDPAVLSRLKQFSQMN KIKKMALRVIAERLSEEEIGGLKELFKMIDTDNSGTITFEELKAGLKRVGSELMESEIKSLM DAADIDNSGTIDYGEFLAATLHMNKMEREENLVAAFSYFDKDGSGYITIDELQSACTEFGLC

SEQ ID NO: 5 Vicia faba (fava bean) calcium-dependent protein kinase 1 (CPKl) cDNA, GenBank Accession No. AY753552

ATCTGAGCATCCTAATGTTGTCAGGATCCATGGGACTTATGAGGATTCGGTTTCTGTTCATT TGGTTATGGAGCTTTGTGAAGGTGGTGAGTTGTTTGATAGGATTGTGAACAAGGGGCATTAT AGTGAGAGGGAAGCTGCTAAGCTGATTAGGACTATTGTCGAGGTTGTGGAAAATTGTCATTC TCTTGGTGTTATGCATAGGGACCTTAAGCCGGAGAATTTTTTGTTTGATACTGTTGAGGAAG

GAATCCGAAAACTAGGTTTACAGCTCACCAAGTGCTATGTCATCCATGGATTGTCGATGATA

GCATTGCACCAGACAAACCTCTTGATTCTGCTGTTTTATCTCGCTTGAAGCAGTTCTCTGCA

ATGAATAAACTTAAAAAGATGGCTTTACGTGTTATTGCGGAGAGGCTGTCTGAGGAAGAAAT

TTGGATGATATCCATATTGATGAAATGGTCAAGGAAATTGATCAAGACAACGATGGACAAAT AGATTATGGAGAATTTGCTGCTATGATGAGAAAAGGCAATGGCGGGATGGGAAGACGAACCA TGACAAGCCGACTCAATTTCAGAAATGCTCTCGGAATCATAGGCAATGAATCCAATTAAATT ATTGTCCCTCAAATAACTAATTGGTGATAAAATTTTTGGACGAGGAATAAACTATGGTATCT TTCAATATTATCAGTAACATGATCAAATTTTGTCTTCGTATGCAGACCCAAAAATGAGAAGC CAAAATCACACCATTGTGATACTTGGAGATAAAAAAAAAAAAAAAAA

SEQ ID NO: 6

Vicia faba (fava bean) calcium-dependent protein kinase 1 (CPKl) amino acid sequence, GenBank Accession No. AAV28169.1 MSNSNNPPPPKPTWVLPYITENIRELYTLGRKLGQGQFGTTYLCTHNPTGKTYACKSIPKKK LLCKEDYDDVWREIQIMHHLSEHPNVVRIHGTYEDSVSVHLVMELCEGGELFDRIVNKGHYS EREAAKLIRTIVEVVENCHSLGVMHRDLKPENFLFDTVEEDAVLKTTDFGLSAFYKPGEIFS DVVGSPYYVAPEVLHKHYGPEADVWSAGVILYILLSGVPPFWAETEIGIFKQILQGRLDFQS EPWPGISDSAKDLIRKMLDRNPKTRFTAHQVLCHPWIVDDSIAPDKPLDSAVLSRLKQFSAM NKLKKMALRVIAERLSEEEIGGLKELFKMLDADSSGTITLDELKEGLKRVGSELMESEIKDL

DDIHIDEMVKEIDQDNDGQIDYGEFAAMMRKGNGGMGRRTMTSRLNFRNALGIIGNESN

SEQ ID NO: 7

Vitis labrusca x Vitis vinifera (grape) calcium-dependent protein kinase (VCPKl) cDNA, GenBank Accession Number AY394009

AGAGAGAGAGTCTTCCGTTCTCCATCCTTTCCTCGACCGGAGAGAGAAGGGCTTCTAGGATT TGCCCATTTCCCCCAGAAATGAAGAAATCGTCCGCAGGAGCACCATCAAAACCCACAAAACC

ATGAGGACCCCGTGTTTGTGCATTTGGTCATGGAGTTGTGTGAGGGAGGTGAGCTTTTTGAT TCTTGTTTGATACCACTGCTGAGGATGCTGCTCTCAAGGCCACTGATTTTGGGTTGTCTGTT TTCTATAAGCCAGGTGAAACCTTTTCTGATGTAGTTGGGAGTCCCTACTATGTTGCACCAGA GGTGTTGTGCAAGCATTATGGACCTGAAGCAGATGTTTGGAGTGCGGGAGTTATTTTGTATA TCTTACTAAGCGGGGTTCCACCTTTTTGGGCAGAGACTGAAACAGGAATCTTCCGTCAGATA

ACAATAGTGGAACAATAACGTTTGATGAATTGAAAGATGGCTTAAAAAGAGTGGGCTCCGAA

CTAATGGAGTCTGAAATCAGGGATCTCATGAATGCAGCTGACATTGACAACAGTGGAACAAT

AGATTATGGGGAATTCCTTGCTGCTACTGTGCACTTGAATAAGTTGGAGAGGGAGGAAAATT

AATGTGGAAAGAAACCAATGGGCTGAAACTCAAATATTAACTTGTAGCTGGTTTCGGCATTA ATTTGATCGTTTTGTTAAAAAAAAAAAAAAAAAAAAAAAA

SEQ ID NO: 8

Vitis labrusca x Vitis vinifera (grape) calcium-dependent protein kinase (VCPKl) amino acid sequence, GenBank Accession No. AAR28766.1

MKKSSAGAPSKPTKPAWVLPYKTQDLRTLYTIGQKLGQGQFGTTFLCTDKATGHNYACKSIP KRKLFCKEDYDDVWREIQIMHHLSEHPNVVRIRGTYEDPVFVHLVMELCEGGELFDRIVQRG

TFSDVVGSPYYVAPEVLCKHYGPEADVWSAGVILYILLSGVPPFWAETETGIFRQILQGKLD FESEPWPCISETAKELLRKMLDRNPKKRLTAHEVLSHPWVVDDRMAPDKPLDSAVLSRLKQF SAMNKLKKMALRVIAEGLSEEEIGGLRELFKMIDTDNSGTITFDELKDGLKRVGSELMESEI RDLMNAADIDNSGTIDYGEFLAATVHLNKLEREENLVSAFSFFDKDKSGYITIDELQQACKE FGLSEAHLDDMIKEIDQDNDGQIDYGEFAAMMRKGNGGIGRRTMRNNLNLGDVLGIPDMRLT N SEQ ID NO:9

Solanum tuberosum (potato) RiCDPK2 calcium dependent protein kinase cDNA, GenBank Accession No. AB051809

TCTCCATTAACATGGAACCAAAACCAGCAACTGAGCCCAAGAAATCATCTGTTTGGGTTCTT CCTTACAAGACTCAAAGCCTTCAGAGTCTTTACACAATAGGCAAAAAGTTAGGCCAAGGTCA ATTTGGAACTACACATCTTTGTATAGAAAAATCAAGTGGCAATCTTTACGCTTGTAAGACAA TACCCAAAAAGAAACTGATCTGTAAAGAAGATTATGAGGATGTTTGGAAAGAGATTCAAATA ATGCATCATTTATCTGAACACCCAAATGTGGTAAGAATAAAGGGTACTTATGAAGATGCATT

GTGAAACATTTTCAGATGTTGTTGGAAGTCCTTATTATGTTGCCCCAGAGGTTTTATGCAAG TAGATCTTGAATCTGAACCTTGGCCTGGAATTTCAGATAGTGCCAAGGATTTGATACGTAAA ATTCTTGATAGGAATCCAAAGAGGAGGTTAACTGCCCATGAAGTTTTGTGCCATCCATGGAT TGTGGATGACACAGTGGCTCCTGATAAACCTCTTGACTCTGCAGTTCTTTCACGCCTCAAGC AGTTCTCAGCAATGAACAAACTAAAGAAAATGGCTTTACGTGTGATTGCCGAGAGGCTTTCA

AAGAATTTGGTCTTAGCGAGCTCAATCTTGATGAAATTATTAAAGATATTGATCAAGATAAT

GATGGACAGATAGACTATAAGGAATTTTCTGCAATGATGAGGAAAGGCACAGGTGGAGCCGT

TGGAAGGAGGACCATAAGAAACAATTTGAATTTAGGAGAAGCACTAGGACTCGTACAGAGTG

CCAAGGTTAGAACTTTAGACTTTGACACCAGTAACAAAGAGCAATTTTCTTCCAAAAT

SEQ ID NO: 10

Solanum tuberosum (potato) RiCDPK2 calcium dependent protein kinase amino acid sequence, GenBank Accession No. BAB63464.1

MEPKPATEPKKSSVWVLPYKTQSLQSLYTIGKKLGQGQFGTTHLCIEKSSGNLYACKTIPKK KLICKEDYEDVWKEIQIMHHLSEHPNVVRIKGTYEDALYVHIVMELCAGGELFDRIVEKGHY SEREAAKLIKTIVGVVEACHSLGVMHRDLKPENFLFLSSDEDAALKATDFGLSVFYKPGETF SDVVGSPYYVAPEVLCKHYGHESDVWSAGVILYILLSGVPPFWAETDMGIFRQILRGKLDLE SEPWPGISDSAKDLIRKILDRNPKRRLTAHEVLCHPWIVDDTVAPDKPLDSAVLSRLKQFSA

LMDAADIDNNGTIDYGEFIAATVHLNKLEREENLLSAFSYFDKDGSGYITIEELQQACKEFG LSELNLDEIIKDIDQDNDGQIDYKEFSAMMRKGTGGAVGRRTIRNNLNLGEALGLVQSEEIL

SEQ ID NO: 11

Zea mays (maize) calcium-dependent protein kinase ZmCPKl 1 cDNA, GenBank Accession Number NM 001112282

CCGGGAGGACTACGAGGACGTCTACCGCGAGATCCAGATCATGCACCACCTCTCCGAGCACC CCAACGTCGTCCGCATCCGCGGCGCCTACGAGGACGCGCTCTTCGTGCACATCGTCATGGAG CTCTGCGCCGGCGGCGAGCTCTTCGACCGCATCGTCGCCAAGGGCCACTACAGTGAGCGCGC GGCTGCGAAGCTCATCAAGACCATTGTCGGGGTCGTGGAGGGATGTCACTCGCTCGGCGTCA TGCACCGGGACCTCAAGCCGGAGAATTTTCTCTTTGCCAGCACCGCCGAGGAAGCCCCACTC

CAAGAAGATGGCATTAAGGGTTATTGCTGAAAGCCTGTCTGAGGAAGAGATTGGAGGTCTCA AGGAGTTGTTCAAGATGATTGATACTGACAGTAGTGGGACTATAACATTTGATGAGCTGAAA GATGGCTTGAAAAGGGTAGGCTCTGAATTAACAGAGAACGAAATCCAGGCTCTAATGGAAGC AGCTGATATTGATAACAGCGGAACCATCGACTACGGCGAATTCATTGCAGCTACGTTGCACA

GCCTTGTCAGGTACATTCCCATGGCATCTGAAGTTTTTGGATGCGTTGTCGATCTGCTGGCC TATTCTGAGATGTTGAGTGGTTATCCTGTGGTATTACTAAGATGCTGGACAATTTTCGTAGT GCTGGTGGTATCAGCTAGAGAAGAGGGATGGCTGGCACAATGTGGCTACTATCGCCAACTGC TT

SEQ ID NO: 12

Zea mays (maize) calcium-dependent protein kinase ZmCPKl 1 amino acid sequence, GenBank Accession No. NP_001105752.1 MQPDPSGNANAKTKLPQLVTAPAPSSGRPASVLPYKTANVRDHYRIGKKLGQGQFGTTYQCV GKADGAEYACKSIPKRKLLCREDYEDVYREIQIMHHLSEHPNVVRIRGAYEDALFVHIVMEL CAGGELFDRIVAKGHYSERAAAKLIKTIVGVVEGCHSLGVMHRDLKPENFLFASTAEEAPLK ATDFGLSMFYKPGDKFSDVVGSPYYVAPEVLQKCYGPEADVWSAGVILYILLCGVPPFWAET EAGIFRQILRGKLDFESEPWPSISDSAKDLVCNMLTRDPKKRFSAHEVLCHAWIVDDAVAPD KPIDSAVLSRLKHFSAMNKLKKMALRVIAESLSEEEIGGLKELFKMIDTDSSGTITFDELKD GLKRVGSELTENEIQALMEAADIDNSGTIDYGEFIAATLHMNKLEREENLVSAFSFFDKDGS GFITIDELSQACREFGLDDLHLEDMIKDVDQNNDGQIDYSEFTAMMRKGNAGATGRRTMRNS LHLNLGELLNPSKT

SEQ ID NO: 13

Nicotiana tabacum (common tobacco) calcium-dependent protein kinase 3 cDNA, GenBank Accession Number AJ344155

TCTTGTTGATTTCTTGGGACATTATAGAGCTCTATGAAAAGAAAGATCTTAGTGTAGGAAGG

AACTACAACAAGGGGTAGTGTGTACGCACACCTTACGAGTTTAGGAAGGAACTATAGGAACA

AATGGGGAACACTTGTGTAGGACCAAGCATTTCTAAAAATGGGATCTTTCAATCAGTTTCAG CAGCAATGTGGCGATCCCGGTCGCCCGATGACACTGCTTCCACCACTAATGGTGAAAGTGCT

TTGCTAAGAGGAAGTTGTTAACAGATGATGATGTGGAAGATGTTAGAAGGGAAGTACAGATA GGGGGCACTATACAGAAAGAAAAGCAGCTGAGCTTACTAGGACTATTGTTGGAGTTGTAGAA GCTTGTCATTCTCTTGGTGTCATGCATCGTGATCTTAAGCCTGAAAATTTTCTCTTTGTTGA TCAGAAGGAGGATTCACTTCTCAAAGCAATTGACTTTGGGTTATCGATATTCTTCAAACCAG GCGACAGATTTACTGATGTTGTTGGCAGTCCATATTATGTTGCACCAGAAGTTCTCCGAAAA

AATTTTCTGCAATGAACAAGCTCAAGAAAATGGCTTTGAGAGTCATTGCTGAAAGCCTATCC

GAAGAAGAAATTGCTGGTCTTAAAGAAATGTTCAAGATGATAGACACAGACAATAGCGGTCA

AATAACTTTTGAGGAGCTCAAAGATGGGTTAAAACGATTTGGCTCTAATCTGAAGGAGTCCG

TGGTGGCAAGAAAGGTCTAGAGCATAGTTTCAGCATTGGATTCAGAGAAGCAGTAAAACTAT AGAGAGCCTTGAAGAGGAATTTTTTTCTTCCTTAGTGTTTGTCTATTATTTTCTTGTGGAAA TTTTTCTGTTACTAGGAGTGTATAGTAATTAGCTAGTGCTTGCTAAACAGCATCTTTGTTTT CCAGTGCACTTTTCAATTCTTTCCAATTTTGTGCAATTTTTCATTTAATTATCTTTTATCTG GCAGTTATACCCTACTGAGTAACTTGTTTAACATGGTAGTAACAACATTCAACTTCATCCTG

AGATAAAAAAAAAAAAAAA

SEQ ID NO: 14

Nicotiana tabacum (common tobacco) calcium-dependent protein kinase 3 amino acid sequence, GenBank Accession No. CAC82999.1

MGNTCVGPSISKNGIFQSVSAAMWRSRSPDDTASTTNGESARIETPISVKEPDSPLPVQEPP EQMTMPKSEKKEEEKEQPKKPKKPAEMKRVSSAGLRTDSVLQKKTGNLKEFFSIGKKLGQGQ FGTTFKCVEKATGKEYACKSIAKRKLLTDDDVEDVRREVQIMHHLAGHPHVISIKGAYEDAV AVHVVMEFCAGGELFDRIIQRGHYTERKAAELTRTIVGVVEACHSLGVMHRDLKPENFLFVD QKEDSLLKAIDFGLSIFFKPGDRFTDVVGSPYYVAPEVLRKRYGPEADVWSAGVIIYILLSG VPPFWAENEQGIFEQVLHGDLDFTSDPWPSISEDAKDLMRRMLVRDPRRRLTAHEVLCHPWV QVDGVAPDKPLDSAVLSRMKQFSAMNKLKKMALRVIAESLSEEEIAGLKEMFKMIDTDNSGQ ITFEELKDGLKRFGSNLKESEIYDLMQAADVDNSGTIDYGEFIAATLHMNKIERQDHLFAAF CYFDKDGSGYITADELQQACEEFGIGDVRMEEMIREADQDNDGRIDYNEFVAMMQKGNPVLG GGKKGLEHSFSIGFREAVKL

SEQ ID NO: 15

Glycine max (soybean) seed calcium dependent protein kinase β cDNA, GenBank Accession

No. AY247755

GGACCACTACGTTCTGGGGAAGAAGCTGGGGCAAGGGCAATTCGGGACGACGTACCTGTGCA AAACGTTGTCCAGATACAAGGCACGTACGAGGATTCCGTGTTCGTGCACCTTGTCATGGAAC TATGTGCCGGCGGGGAGCTTTTCGACAGGATCATTCAGAAGGGGCATTACAGCGAGAGAGAG GCTGCCAAGTTGATAAAGACCATTGTTGGGGTGGTGGAGGCGTGCCACTCTCTTGGGGTCAT GCATAGGGATCTCAAGCCTGAGAATTTCTTGTTTGATACCCCTGGCGAAGATGCCCAGATGA

AAGAATTTCTGCTCATGAAGTTTTATGTAACCCTTGGGTTGTTGATGACATTGCACCTGACA

AACCTCTGGACTCTGCTGTTTTGACACGCCTAAAGCATTTCTCAGCAATGAATAAACTTAAG

AAGATGGCATTACGGGTCATAGCAGAGAGGCTTTCAGAGGAAGAAATAGGTGGATTGAAAGA

CTGGATGAGATGATCAAAGAGATTGATCAAGATAATGATGGGAGGATTGATTATGCGGAGTT TGCAGCAATGATGAAAAAGGGTGATCCAAATATGGGTAGAAGCAGAACCATGAAAGGCAATT TGAACTTCAATATTGCAGATGCATTTGGAATGAAAGACTCTTCTTGA

SEQ ID NO: 16 Glycine max (soybean) seed calcium dependent protein kinase β amino acid sequence, GenBank Accession Number AAP03013.1

MQKHGFASKRNVLPYQTARLRDHYVLGKKLGQGQFGTTYLCTHKVTGKLYACKSIPKRKLMC QEDYDDVWREIQIMHHLSEHPNVVQIQGTYEDSVFVHLVMELCAGGELFDRIIQKGHYSERE AAKLIKTIVGVVEACHSLGVMHRDLKPENFLFDTPGEDAQMKATDFGLSVFYKPGQAFHDVV GSPYYVAPEVLCKQYGPEVDVWSAGVILYILLSGVPPFWAETEAGIFRQILNGDLDFVSEPW PSISENAKELVKQMLDRDPKKRISAHEVLCNPWVVDDIAPDKPLDSAVLTRLKHFSAMNKLK KMALRVIAERLSEEEIGGLKELFKMIDTDNSGTITFEELKEGLKSVGSNLMESEIKSLMEAA DIDNNGSIDYGEFLAATLHLNKMEREENLVAAFAYFDKDGSGYITIDELQQACKDFSLGDVH LDEMIKEIDQDNDGRIDYAEFAAMMKKGDPNMGRSRTMKGNLNFNIADAFGMKDSS

Claims

WHAT IS CLAIMED IS:

1. A method of enhancing abscisic acid sensitivity in a plant, the method comprising introducing a recombinant expression cassette into a plant, wherein the expression cassette comprises a promoter operably linked to a polynucleotide encoding a CPK4 or CPKl 1 protein, wherein the promoter is heterologous to the polynucleotide, wherein the plant has increased abscisic acid sensitivity compared to an otherwise identical plant lacking the expression cassette.

2. The method of claim 1, wherein the plant has improved drought tolerance compared to an otherwise identical plant without the recombinant expression cassette.

3. The method of claim 1, wherein the CPK4 or CPKl 1 protein has an amino acid sequence at least 50% identical to SEQ ID NO:2, 4, 6, 8, 10, 12, 14, or 16.

4. The method of claim 3, wherein the CPK4 or CPKl 1 protein has an amino acid sequence at least 70% identical to SEQ ID NO:2 or 4.

5. The method of claim 1, wherein the polynucleotide comprises one of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, and 15.

6 . The method of claim 1, wherein the promoter is constitutive.

7 . The method of claim 1, wherein the promoter is inducible.

8. The method of claim 1, wherein the promoter is tissue-specific.

9. The method of claim 1, wherein the promoter directs expression in guard cells.

10. The method of claim 1, wherein the promoter is drought-inducib Ie.

11. The method of claim 1, comprising generating a plurality of plants comprising the introduced expression cassette, and screening the plants for abscisic acid sensitivity compared to an otherwise identical plant lacking the expression cassette.

12. A method of decreasing abscisic acid sensitivity in a plant, the method comprising introducing an recombinant expression cassette into a plant, wherein the expression cassette comprises a promoter operably linked to a polynucleotide comprising at least 20 nucleotides complementary or identical to a contiguous sequence in a cDNA encoding an endogenous CPK4 or CPKl 1 protein in the plant, wherein the promoter is heterologous to the polynucleotide, thereby reducing expression of the CPK4 or CPKl 1 protein in the plant, wherein the plant has reduced abscisic acid sensitivity compared to an otherwise identical plant lacking the expression cassette.

13. The method of claim 12, wherein the polynucleotide comprises at least 50 nucleotides complementary or identical to a contiguous sequence in a cDNA encoding the CPK4 or CPKl 1 protein in the plant.

14. The method of claim 12, wherein the polynucleotide comprises at least 200 nucleotides complementary or identical to a contiguous sequence in a cDNA encoding the CPK4 or CPK 11 protein in the plant.

15. The method of claim 12, wherein the CPK4 or CPKl 1 protein has an amino acid sequence at least 50% identical to SEQ ID NO:2, 4, 6, 8, 10, 12, 14, or 16.

16. The method of claim 15, wherein the CPK4 or CPKl 1 protein has an amino acid sequence at least 70% identical to SEQ ID NO: 2 or 4.

17. The method of claim 12, wherein the polynucleotide comprises one of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, and 15.

18. The method of claim 12, wherein the promoter directs expression of the polynucleotide to abscission zones of the plant.

19. A recombinant expression cassette comprising a promoter operably linked to a polynucleotide encoding a CPK4 or CPKl 1 protein, wherein the promoter is heterologous to the polynucleotide, and wherein introduction of the expression cassette into a plant results in enhanced abscisic acid sensitivity in the plant compared to an otherwise identical plant lacking the expression cassette.

20. The recombinant expression cassette of claim 19, wherein introduction of the expression cassette into a plant results in improved drought tolerance in the plant compared to an otherwise identical plant lacking the expression cassette

21. The recombinant expression cassette of claim 19, wherein the CPK4 or CPKl 1 protein has an amino acid sequence at least 50% identical to SEQ ID NO:2, 4, 6, 8, 10, 12, 14, or 16.

22. The recombinant expression cassette of claim 21, wherein the CPK4 or CPKl 1 protein has an amino acid sequence at least 70% identical to SEQ ID NO: 2 or 4.

23. The recombinant expression cassette of claim 19, wherein the polynucleotide comprises one of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, and 15.

24. The recombinant expression cassette of claim 19, wherein the promoter is constitutive.

25. The recombinant expression cassette of claim 19, wherein the promoter is inducible.

26. The recombinant expression cassette of claim 19, wherein the promoter is tissue-specific.

27. The recombinant expression cassette of claim 19, wherein the promoter directs expression in guard cells.

28. The recombinant expression cassette of claim 19, wherein the promoter is drought-inducible.

29. A transgenic plant comprising a recombinant expression cassette, wherein the expression cassette comprises a promoter operably linked to a polynucleotide encoding a CPK4 or CPKl 1 protein, wherein the promoter is heterologous to the polynucleotide, and wherein the plant has enhanced abscisic acid sensitivity compared to an otherwise identical plant lacking the expression cassette.

30. The plant of claim 29, wherein the plant has improved drought tolerance compared to an otherwise identical plant lacking the expression cassette

31. The plant of claim 29, wherein the CPK4 or CPKI l protein has an amino acid sequence at least 50% identical to SEQ ID NO:2, 4, 6, 8, 10, 12, 14, or 16.

32. The plant of claim 31, wherein the CPK4 or CPKl 1 protein has an amino acid sequence at least 70% identical to SEQ ID NO:2 or 4.

33. The plant of claim 29, wherein the polynucleotide comprises one of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, and 15.

34. The plant of claim 29, wherein the promoter is constitutive.

35. The plant of claim 29, wherein the promoter is inducible.

36. The plant of claim 29, wherein the promoter is tissue-specific.

37. The plant of claim 29, wherein the promoter directs expression in guard cells.

38. The plant of claim 29, wherein the promoter is drought-inducible.

39. A seed, flower, leaf or fruit from the plant of claim 29.