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WO2008157157A2 - Protéine kinase cpk4 et cpk11, des plantes résistant à la sécheresse et procédé de production - Google Patents

Protéine kinase cpk4 et cpk11, des plantes résistant à la sécheresse et procédé de production Download PDF

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WO2008157157A2
WO2008157157A2 PCT/US2008/066495 US2008066495W WO2008157157A2 WO 2008157157 A2 WO2008157157 A2 WO 2008157157A2 US 2008066495 W US2008066495 W US 2008066495W WO 2008157157 A2 WO2008157157 A2 WO 2008157157A2
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plant
cpk4
cpkl
promoter
aba
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WO2008157157A3 (fr
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Da-Peng Zhang
Sai-Yong Zhu
Xiang-Chun Yu
Xiao-Jing Wang
Xiao-Fang Wang
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D-HELIX
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8273—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for drought, cold, salt resistance

Definitions

  • This invention relates to methods and compositions for generating plants with altered abscisic acid (ABA) sensitivity.
  • ABA abscisic acid
  • ABA phytohormone abscisic acid
  • 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)).
  • 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.
  • 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.
  • 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.
  • 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.
  • the present invention provides methods of enhancing ABA sensitivity in a plant.
  • 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.
  • the plant has improved drought tolerance compared to an otherwise identical plant lacking the expression cassette
  • 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%.
  • 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.
  • 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.
  • the present invention also provides methods of decreasing ABA sensitivity in a plant.
  • 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.
  • 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.
  • 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.
  • 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%.
  • 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.
  • the promoter directs expression of the polynucleotide to abscission zones of the plant.
  • 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.
  • 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
  • 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%.
  • 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.
  • 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.
  • 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.
  • 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.
  • the plant has improved drought tolerance compared to an otherwise identical plant lacking the expression cassette.
  • 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%.
  • 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.
  • 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.
  • 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.
  • the invention also provides for any plant part from the transgenic plants of the invention.
  • plant parts include, but are not limited to: seeds, flowers, leafs and fruits.
  • 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.
  • 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.
  • promoter refers to a polynucleotide sequence capable of driving transcription of a coding sequence in a cell.
  • 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.
  • 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.
  • 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.
  • 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.
  • shoot vegetative organs and/or structures e.g., leaves, stems and tubers
  • roots 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
  • 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.
  • 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.
  • a promoter 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).
  • 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.
  • a plant containing the exogenous nucleic acid is referred to here as a Ti (e.g., in Ar ⁇ bidopsis by vacuum infiltration) or Ro (for plants regenerated from transformed cells in vitro) generation transgenic plant.
  • 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.
  • 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.
  • CPK expression or activity refers to an augmented change in the protein's expression or activity.
  • 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).
  • 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.
  • sequence identity 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.
  • 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).
  • 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.
  • 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.
  • sequence comparison typically one sequence acts as a reference sequence, to which test sequences are compared.
  • 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.
  • sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.
  • 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.
  • HSPs high scoring sequence pairs
  • 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).
  • M forward score for a pair of matching residues; always >0
  • N penalty score for mismatching residues; always ⁇ 0.
  • 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 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)).
  • 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.
  • P(N) the smallest sum probability
  • 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 .
  • Constantly 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.
  • 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.
  • each codon in a nucleic acid except AUG, which is ordinarily the only codon for methionine
  • each silent variation of a nucleic acid which encodes a polypeptide is implicit in each described sequence.
  • 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.
  • 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.
  • FIG. 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 O ⁇ CPK4 (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. Act ⁇ n2/8 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 ⁇ g 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.
  • 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.
  • FIG. 1 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.
  • germinating seeds were transferred, 48 h after stratification, to the MS-medium containing ( ⁇ )ABA (0, 0.5, 1, 2, 5 ⁇ M), 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- ⁇ g 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- ⁇ g portion of the total proteins was used in each line for the immunoprecipitation.
  • 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.
  • immunoblotting for tubulin was done with the total proteins prior to the immunoprecipitation. Relative band intensities, normalized relative to the corresponding intensity with 0 ⁇ M 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.
  • 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 ⁇ M (top panel), 0.5 ⁇ M (middle panel), or 3 ⁇ M (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 ⁇ M ABA. Each value is the mean ⁇ SE of at least 50 seedlings.
  • D Postgermination growth in the MS-medium containing 0.8 ⁇ M ( ⁇ )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.
  • 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.
  • FIG. 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.
  • Each value is the mean ⁇ SE of three biological determinations.
  • 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.
  • 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.
  • 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) 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 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.
  • 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').
  • 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 ⁇ M-( ⁇ )ABA treatment (100%).
  • FIG. 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 ⁇ M ( ⁇ )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.
  • FIG. 8 Identification of T-DNA Insertion for cpk4-l , cpkl 1 -1 and cpkl 1 -2 Mutations in the Arabidopsis Genome by PCR Analysis.
  • FIG. 9 Southern-Blot Analysis for the T-DNA Insertion in cpk4-l, cpkll-1 and cpkll-2 Mutants.
  • a 10- ⁇ g portion of Arabidopsis genomic DNA isolated from the cpk4-l, cpkll-1 and cpkll-2 mutants was digested with EcorRl plus Pstl and Hindl ⁇ l, respectively, electrophoresed in a 0.8% agarose gel, and transferred onto a nylon membrane. The membranes were hybridized with the 32 P -labelled specific probe for the T-DNA (see METHODS).
  • FIG. 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.
  • FIG. 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.
  • FIG. 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.
  • 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.
  • FIG. 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.
  • FIG. 14 Enzymatic Characterization of CPK4 and CPKl 1.
  • A Ca 2+ -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.
  • Ca 2+ or EGTA was added to the immunoprecipitated proteins dissolved in SDS-PAGE sample buffer.
  • CaM (form bovine brain, Sigma) was used at 5 ⁇ M; TFP, W7 and W5 at 250 ⁇ M, and K252a at 10 ⁇ M. 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 32 P -labeling to the kinase substrate histone. - and + indicate the absence and presence of Ca 2+ 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
  • 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 Ca 2+ -spikes in response to different stimuli.
  • CaM calcium sensor proteins
  • CaM-related proteins CaM-related proteins
  • 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
  • CBL calcineurin B-like proteins
  • CDPKs calcium-dependent protein kinases
  • 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).
  • PPs calcium-modulated protein phosphatases
  • 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 Ca 2+ - 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).
  • CIPK15 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.
  • 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.
  • 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 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.
  • 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
  • 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.
  • 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.
  • 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).
  • 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.
  • a polynucleotide sequence encoding a plant CPK4 or CPKl 1 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.
  • 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.
  • cDNA may be prepared from mRNA extracted from other tissues in which CPK4 or CPKl 1 gene is expressed.
  • 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.
  • the nucleic acids encoding a CPK4 or CPKl 1 can be amplified from nucleic acid samples using amplification techniques.
  • 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.
  • PCR Protocols A
  • 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.
  • a regulatory element e.g., a promoter
  • 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).
  • stress tolerance promoters can be selected that are generally constitutive and are expressed in most plant tissues, or can be leaf or root specific.
  • promoters are generally used that result in expression in seed or, in some embodiments, floral organs or embryos.
  • 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.
  • mutagenesis e.g., chemical, radiation, transposon or other mutagenesis
  • 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"
  • 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.
  • 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.
  • 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.
  • shoot vegetative organs/structures e.g. leaves, stems and tubers
  • roots e.g. bracts, sepals, petals, stamens, carpels, anthers and ovules
  • seed including embryo, endosperm, and seed coat
  • fruit abscission zone
  • abscission zone etc.
  • one or several CPK4 or CPKl 1 genes can be expressed constitutively (e.g., using the CaMV 35S promote
  • polypeptides encoded by the genes of the invention like other proteins, have different domains which perform different functions.
  • the overexpressed or ectopically expressed polynucleotide sequences need not be full length, so long as the desired functional domain of the protein is expressed.
  • active CPK4 or CPKl 1 proteins can be expressed as fusions, without necessarily significantly altering CPK4 or CPKl 1 activity.
  • fusion partners include, but are not limited to, poly-His or other tag sequences.
  • a number of methods can be used to inhibit gene expression in plants.
  • antisense technology can be conveniently used.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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).
  • 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.
  • 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.
  • 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.
  • RNAi RNA interference
  • co-suppression can be considered a type of RNAi
  • 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.
  • 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.
  • RNAi 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.
  • 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.
  • the RNAi polynucleotides can encompass the full-length target RNA or may correspond to a fragment of the target RNA.
  • the fragment will have fewer than 100, 200, 300, 400, 500 600, 700, 800, 900 or 1,000 nucleotides corresponding to the target sequence.
  • these fragments are at least, e.g., 50, 100, 150, 200, or more nucleotides in length.
  • 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.
  • 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).
  • the sense or antisense transcript should be targeted to sequences with the most variance between family members.
  • CPK4 or CPKl 1 function in a plant is by creation of dominant negative mutations.
  • 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.
  • the coding or cDNA sequence for CPK4 or CPKl 1 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.
  • expression vectors can also be used to express CPK4 or CPKl 1 polynucleotides and variants thereof that inhibit endogenous CPK4 or CPKI l expression.
  • 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.
  • the CPK4 or CPKl 1 gene can be expressed constitutively (e.g., using the CaMV 35 S promoter).
  • CPK4 or CPKl 1 coding or cDNA sequences 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.
  • 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.
  • 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.
  • 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).
  • 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
  • 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.
  • the vector comprising the sequences will typically comprise a marker gene that confers a selectable phenotype on plant cells.
  • 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.
  • 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.
  • transcriptional control elements can also be used.
  • 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.
  • 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 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.
  • CaMV 35S cauliflower mosaic virus 35 S
  • 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)).
  • 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)).
  • 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.
  • a plant promoter may direct expression of the CPK4 or CPKl 1 gene under the influence of changing environmental conditions or developmental conditions.
  • environmental conditions that may effect transcription by inducible promoters include anaerobic conditions, elevated temperature, drought, or the presence of light.
  • inducible promoters are referred to herein as "inducible" promoters.
  • 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).
  • plant promoters which are inducible upon exposure to plant hormones are used to express the CPK4 or CPKl 1 gene.
  • 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).
  • 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.
  • 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)).
  • useful inducible regulatory elements include copper-inducible regulatory elements (Mett et al., Proc. Natl. Acad. Sci.
  • 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)).
  • a nitrate-inducible promoter derived from the spinach nitrite reductase gene
  • a light-inducible promoter such as that associated with the small subunit of RuBP carboxylase or the LHCP gene families
  • 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.
  • 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.
  • 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); v ⁇ vparous-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.
  • 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.
  • 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.
  • Leaf-specific promoters such as the ribulose biphosphate carboxylase (RBCS) promoters can be used.
  • RBCS ribulose biphosphate carboxylase
  • 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) /7 ⁇ « ⁇ 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.
  • 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.
  • Another class of useful vegetative tissue-specific promoters are meristematic (root tip and shoot apex) promoters.
  • 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).
  • tissue-specific promoter may drive expression of operably linked sequences in tissues other than the target tissue.
  • 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.
  • 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.
  • RTBV rice tungro bacilliform virus
  • CVMV cassava vein mosaic virus
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • Agrobacterium tumefaciens-mediated transformation techniques including disarming and use of binary vectors, are well described in the scientific literature. See, for example, Horsch et ⁇ l. Science 233:496-498 (1984), and Fraley et ⁇ l. Proc. N ⁇ tl. Ac ⁇ d. Sci. USA 80:4803 (1983).
  • 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.
  • 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).
  • 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.
  • 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,
  • the plant is selected from the group consisting of rice, maize, wheat, soybeans, cotton, canola, and alfalfa.
  • the plant is an ornamental plant.
  • the plant is a vegetable- or fruit-producing plant.
  • 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.
  • 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.
  • 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.
  • 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.
  • the CPK4 and CPKl 1 both phosphorylated two ABA-responsive transcription factors ABFl and ABF4 in vitro, indicating that the two kinases regulate ABA signaling through these transcription factors.
  • 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).
  • 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).
  • 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).
  • 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 ⁇ g/mL) with seeds from heterozygous cpk4-l and cpkll-1 mutants.
  • the ratio of the resistant to sensitive plants was approximately 3: 1.
  • 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.
  • 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).
  • CPK4 C CPK4
  • CPKl 1 CPKl 1
  • Figures IE and 12A 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).
  • allelic T-DNA insertion lines cpkll-1 and cpkll-2 in CPKIl gene show similar phenotypes in response to ABA or stress treatments.
  • results of cpkll-2 as a representative of two mutants cpkll-1 and cpkll-2
  • results of cpk4-l cpkll-2 as a representative of the two double mutants in some cases.
  • 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).
  • the ABA-stimulating effects were dependent on the ABA dose used, in which ABA was most effective at around 1 ⁇ M 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 ⁇ M 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).
  • 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 ⁇ M ( Figure 3 and 3C).
  • 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.
  • CPK4 and CPKIl Kinases Phosphorylate ABA-Responsive Transcription Factors ABFl and ABF4 in Vitro
  • 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).
  • 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.
  • CPK4 and CPKIl are Two Positive Regulators in CDPK/Ca2+-Mediated ABA
  • 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).
  • 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.
  • 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.
  • 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).
  • the cytoplasm-located CPK4 and CPKl 1 would more easily mediate quick response by sensing Ca 2+ 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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).
  • LP2 left genomic primer 1
  • RP2 right genomic primer 2
  • 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).
  • LBaI T-DNA left border primer
  • RBaI right border primer
  • 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.
  • 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).
  • 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).
  • cpkll-2 mutant 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.
  • 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'.
  • PCR polymerase chain reaction
  • 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/S ⁇ S.html) by Xba I and BamH I sites under the control of a constitutive cauliflower mosaic virus (CaMV) 35 S promoter.
  • CaMV cauliflower mosaic virus
  • Transgenic plants were selected by hygromycin resistance and confirmed by PCR. The homozygous T3 seeds of the transgenic plants were used for further analysis.
  • Plants were grown in a growth chamber at 20-21 0 C on Murashige-Skoog (MS) medium at about 80 ⁇ mol photons m "2 s "1 or in compost soil at about 120 ⁇ mol photons m "2 s "1 over a 16-h photoperiod at 22 0 C
  • MS Murashige-Skoog
  • 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 4 0 C for 3 days before being placed 22 0 C under light conditions, and germination (emergence of radicals) was scored at the indicated times.
  • 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.
  • Lateral root growth assays were performed according to the protocol of Xiong et al. (2006, Plant Physiol. 142, 1065-1074) with some modifications.
  • the basal salts included 1.0 mM CaCl 2 , 0.5 mM MgSO 4 , 0.4 mM KH 2 PO 4 , 6.0 mM KNO 3 , and 7.0 mM NH 4 NO 3 .
  • 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 ⁇ M 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.
  • 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 22 0 C 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.
  • MS medium full-strength MS, 3% sucrose, pH 5.7
  • 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.
  • 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.
  • 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 ⁇ g mL "1 antipain, 5 ⁇ g mL "1 aprotinin, and 5 ⁇ g mL "1 leupeptin. Lysates were cleared of debris by centrifugation at 12,00Og for 30 min at 4 0 C .
  • 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.
  • SDS-PAGE was carried out according to the method of Laemmli (1970, Nature 227, 680-685). The protein samples (20 ⁇ g) 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 ⁇ m, 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.
  • BSA bovine serum albumin
  • Tween 20 Tris-buffered saline
  • 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 was done essentially as described by Yu et al. (2006, supra). The total proteins (50 ⁇ g) were resuspended in 0.5 mL immunoprecipitation buffer containing 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EGTA, 1 mM Na 3 VO 4 , 1 mM NaF, 10 mM ⁇ -glycerophosphate, 1 mM phenylmethylsulfonyl fluoride, 5 ⁇ g mL "1 antipain, 5 ⁇ g mL "1 aprotinin, 5 ⁇ g 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 ⁇ g protein) or the same amount of preimmune serum protein (as a control) at 4 0 C for 2 h. Then 25 ⁇ L 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.
  • 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
  • the gels were incubated with buffer B containing 50 ⁇ M ATP and 10 ⁇ Ci/mL [r 32 -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 -20 0 C. Films were scanned using a digital imaging system and radioactivity was quantified with QuantityOne software (BioRad).
  • 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 4 0 C and stored at -80 0 C 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.
  • RNA sample was reverse transcriptase (RT)-mediated PCR analysis was performed to analyze the expression 0 ⁇ CPK4 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 ⁇ L volume at 42 0 C 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.
  • RNA samples isolated from three-week-old seedlings harvested at the indicated times after 50 ⁇ M 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 TaqTM (TaKaRa) using a DNA Engine Opticon 2 thermal cycler (MJ Research, Watertown, MA) in 10 ⁇ L volume with the following program: 1 cycle of 95 0 C, 10 s; and 40 cycles of 94 0 C, 5s; 58.5 0 C, 20s; 72 0 C, 20s.
  • the amplification of the target genes was monitored every cycle by SYBR-Green fluorescence.
  • the Ct threshold cycle
  • the Ct 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.
  • 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 5 ⁇ TCACTTGGCTCCACTGTTGTTC-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
  • ABFl 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 O ⁇ ACTIN2/8 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.
  • 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 ' -
  • DNA gel blot hybridization was performed at 65 0 C 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 32 P- labeled specific probes. Then the membranes were washed at 65 0 C 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 -7O 0 C.
  • the probe sequence was: TCAGAAGAACTCGTCAAGAAGGCGATAGAAGGCGATGCGCTGCGAATCGGGAGC GGCGATACCGTAAAGCACGAGGAAGCGGTCAGCCCATTCGCCGCCAAGCTCTTC AGCAATATCACGGGTAGCCAACGCTATGTCCTGATAGCGGTCCGCCACACCCAG CCGGCCACAGTCGATGAATCCAGAAAAGCGGCCATTTTCCACCATGATATTCGGC AAGCAGGCATCGCCATGGGTCACGACGAGATCATCGCCGTCGGGCATGCGCGCC TTGAGCCTGGCGAACAGTTCGGCTGGCGCGAGCCCCTGATGCTCTTCGTCCAGAT CATCCTGATCGACAAGACCGGCTTCCATCCGAGTACGTGCTCGCTCGATGCGATG TTTCGCTTGGTGGTCGAATGGGCAGGTAGCCGGATCAAGCGTATGCAGCCGCCGC ATTGCATCAGCCATGATGAAATACTTTCTCGGCAGGAGCAAGGTGAGATGACAGG A
  • PCR products were then fused to the upstream of the enhanced GFP (Cormack et al., (1996) Gene 173, 33-38) atthe ⁇ oI (5'- end) / Bam ⁇ 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.
  • 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).
  • 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
  • Left border primer (LBaI) and right genomic primer 1 (RPl) are the same as those mentioned above for cpkll-1 mutant identification.
  • 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 ACACCACGATTAAGAGATCATTACCT ⁇ CTGGGAAAAAAGCTAGGCCAAGGCCAA TTTGGAACAACCTATCTCTGCACAGAGAAATCAACCTCCGCTAATTACGCCTGCA AATCGATCCCGAAGCGAAAGCTCGTGTGTCGCGAGGATTACGAAGATGTATGGC GTGAGATTCAGATCATGCATCATCTCTCTGAGCATCCAAATGTTGTTAGGATCAA AGGGACTTATGAAGATTCGGTGTTTGTTCATATTGTTATGGAGGTTT ⁇ XYA ⁇ C ⁇ IX
  • 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.
  • 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
  • Vicia faba (fava bean) calcium-dependent protein kinase 1 (CPKl) amino acid sequence GenBank Accession No. AAV28169.1 MSNSNNPPPPKPTWVLPYITENIRELYTLGRKLGQGQFGTTYLCTHNPTGKTYACKSIPKKK LLCKEDYDDVWREIQIMHHLSEHPNVVRIHGTYEDSVSVHLVMELCEGGELFDRIVNKGHYS EREAAKLIRTIVEVVENCHSLGVMHRDLKPENFLFDTVEEDAVLKTTDFGLSAFYKPGEIFS DVVGSPYYVAPEVLHKHYGPEADVWSAGVILYILLSGVPPFWAETEIGIFKQILQGRLDFQS EPWPGISDSAKDLIRKMLDRNPKTRFTAHQVLCHPWIVDDSIAPDKPLDSAVLSRLKQFSAM NKLKKMALRVIAERLSEEEIGGLKELFKMLDADSSGTITLDELKEGLKRVGSELMESEIKDL
  • Vitis labrusca x Vitis vinifera (grape) calcium-dependent protein kinase (VCPKl) cDNA, GenBank Accession Number AY394009
  • Vitis labrusca x Vitis vinifera (grape) calcium-dependent protein kinase (VCPKl) amino acid sequence, GenBank Accession No. AAR28766.1
  • Zea mays calcium-dependent protein kinase ZmCPKl 1 amino acid sequence, GenBank Accession No. NP_001105752.1 MQPDPSGNANAKTKLPQLVTAPAPSSGRPASVLPYKTANVRDHYRIGKKLGQGQFGTTYQCV GKADGAEYACKSIPKRKLLCREDYEDVYREIQIMHHLSEHPNVVRIRGAYEDALFVHIVMEL CAGGELFDRIVAKGHYSERAAAKLIKTIVGVVEGCHSLGVMHRDLKPENFLFASTAEEAPLK ATDFGLSMFYKPGDKFSDVVGSPYYVAPEVLQKCYGPEADVWSAGVILYILLCGVPPFWAET EAGIFRQILRGKLDFESEPWPSISDSAKDLVCNMLTRDPKKRFSAHEVLCHAWIVDDAVAPD KPIDSAVLSRLKHFSAMNKLKKMALRVIAESLSEEEIGGLKELFKMIDTDSSGTITFDELKD GLKR
  • Nicotiana tabacum common tobacco calcium-dependent protein kinase 3 cDNA, GenBank Accession Number AJ344155
  • Nicotiana tabacum (common tobacco) calcium-dependent protein kinase 3 amino acid sequence, GenBank Accession No. CAC82999.1
  • Glycine max (soybean) seed calcium dependent protein kinase ⁇ cDNA, GenBank Accession

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Abstract

L'invention concerne une cassette d'expression recombinante pour exprimer CPK4 ou CPK11, deux protéines kinases dépendant du calcium qu'on trouve dans des plantes. Une plante transgénique avec une sensibilité à l'ABA et une résistance à la sécheresse renforcées, ainsi qu'un procédé de production de telles plantes sont également décrits.
PCT/US2008/066495 2007-06-13 2008-06-11 Protéine kinase cpk4 et cpk11, des plantes résistant à la sécheresse et procédé de production Ceased WO2008157157A2 (fr)

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CN106244568A (zh) * 2016-08-26 2016-12-21 中国农业大学 一种钙依赖型蛋白激酶cpk32及其编码基因和应用
CN110904111A (zh) * 2019-12-19 2020-03-24 西南大学 一种靶向敲除FcMYC2基因的sgRNA序列、CRISPR/Cas9载体及其应用
CN110938126A (zh) * 2019-12-19 2020-03-31 西南大学 柑橘FcMYC2基因及其编码蛋白在调控柑橘精油合成中的应用
CN113046375A (zh) * 2021-05-19 2021-06-29 新疆农业科学院园艺作物研究所 SpCPK33基因及其编码蛋白在调控番茄耐旱性中的应用
CN117448359A (zh) * 2023-11-03 2024-01-26 四川农业大学 CircCDPK6基因在调控植物耐旱性中的应用
CN119614595A (zh) * 2024-09-04 2025-03-14 华南农业大学 拟南芥cpk18基因在提高植物铜胁迫抗性中的应用和方法

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CN106244568A (zh) * 2016-08-26 2016-12-21 中国农业大学 一种钙依赖型蛋白激酶cpk32及其编码基因和应用
CN106244568B (zh) * 2016-08-26 2019-12-24 中国农业大学 一种钙依赖型蛋白激酶cpk32及其编码基因和应用
CN110904111A (zh) * 2019-12-19 2020-03-24 西南大学 一种靶向敲除FcMYC2基因的sgRNA序列、CRISPR/Cas9载体及其应用
CN110938126A (zh) * 2019-12-19 2020-03-31 西南大学 柑橘FcMYC2基因及其编码蛋白在调控柑橘精油合成中的应用
CN110938126B (zh) * 2019-12-19 2021-06-11 西南大学 柑橘FcMYC2基因及其编码蛋白在调控柑橘精油合成中的应用
CN113046375A (zh) * 2021-05-19 2021-06-29 新疆农业科学院园艺作物研究所 SpCPK33基因及其编码蛋白在调控番茄耐旱性中的应用
CN113046375B (zh) * 2021-05-19 2023-06-27 新疆农业科学院园艺作物研究所 SpCPK33基因及其编码蛋白在调控番茄耐旱性中的应用
CN117448359A (zh) * 2023-11-03 2024-01-26 四川农业大学 CircCDPK6基因在调控植物耐旱性中的应用
CN119614595A (zh) * 2024-09-04 2025-03-14 华南农业大学 拟南芥cpk18基因在提高植物铜胁迫抗性中的应用和方法

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