Journal of Experimental Botany, Vol. 52, No. 355, pp. 215-221,
February 2001
© 2001 Oxford University Press
Original Papers |
Auxin and heat shock activation of a novel member of the calmodulin like domain protein kinase gene family in cultured alfalfa cells
1 Institute of Plant Biology, Biological Research Center, Hungarian Academy of Sciences H-6701 Szeged, POB 521, Hungary
2 BAY ZOLTAN Foundation for Applied Research, Institute for Biotechnology H-6701 Szeged, POB 2337, Hungary
Received 12 June 2000; Accepted 5 September 2000
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Abstract |
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A calmodulin like domain protein kinase (CPK) homologue was identified in alfalfa and termed MsCPK3. The full-length sequence of cDNA encoded a 535 amino acid polypeptide with a molecular weight of 60.2 kDa. The deduced amino acid sequence showed all the conserved motifs that define other members of this kinase family, such as serine-threonine kinase domain, a junction region and four potential Ca2+-binding EF sites. The recombinant MsCPK3 protein purified from E. coli was activated by Ca2+and inhibited by calmodulin antagonist (W-7) in in vitro phosphorylation assays. The expression of MsCPK3 gene increased in the early phase of the 2,4-D induced alfalfa somatic embryogenesis. Heat shock also activated this gene while kinetin, ABA and NaCl treatment did not result in MsCPK3 mRNA accumulation. The data presented suggest that the new alfalfa CPK differs in stress responses from the previously described homologues and in its potential involvement in hormone and stress-activated reprogramming of developmental pathways during somatic embryogenesis.
Key words: Medicago sativa, CPK, stress, 2,4-D, phosphorylation, somatic embryogenesis.
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Introduction |
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Formation of embryos from somatic plant cells is a unique, plant-specific developmental pathway that also provides an efficient way for plant regeneration in in vitro cultures. The molecular bases of this metabolic and developmental switch are poorly understood despite extensive research with different tissue culture systems (Sharma and Thorpe, 1995
). The large body of previous tissue culture experiments emphasized the pivotal role of auxin-activated cell division in induction of an embryogenic state in cultured somatic cells (reviewed by Dudits et al., 1991). A growing number of experimental observations demonstrated the involvement of stress responses during somatic embryogenesis (Dudits et al., 1995), like activation of the heat shock system in alfalfa (Györgyey et al., 1991). Interestingly, several abscisic acid activated genes such as annexin, ferritin and aldose reductase were identified in a cDNA library from alfalfa cells exposed to embryogenic induction (Deák et al., 1995).
Both auxin and abscisic acid signalling pathways are dependent on Ca2+-mediated events (Saunders, 1990; reviewed by Poovaiah and Reddy, 1993). Therefore, it is postulated that the Ca2+-regulated kinase genes may also respond to hormonal and stress signals during embryogenic induction. In particular, the class of CAM-like domain protein kinases (CPKs) may be considered as potential components in these activation events. This unique class of plant specific kinases are encoded by a multigene family (reviewed by Roberts and Harmon, 1992; Roberts, 1993; Harmon et al., 2000). So far 29 members have been found in Arabidopsis but the estimated number is 40 in this single species (Hong et al., 1996; Hrabak et al., 1996; Harmon et al., 2000). In addition to plants, CPK genes have also been cloned from protozoa (Zhao et al., 1993). Notably, the CPK genes are absent from the yeast and nematode genomes.
A number of putative endogenous CPKs substrates have been identified by biochemical approaches that suggest regulatory roles in gene expression, metabolism, cytoskeleton dynamics, and extracellular plant signalling (Harmon et al., 2000). The plant CPKs have been associated with plasma membrane (Schaller et al., 1992), cytoskeleton (Putman-Evans et al., 1989) and chromatin (Roberts and Harmon, 1992). The multiple subcellular localization also suggests that CPKs have diverse functions. Despite the cloning and characterization of many CPK genes, the function of these proteins is still largely a mystery. The crucial role of the CPK in signal transduction pathways controlling cell growth and germination has been shown in maize pollen by inhibition of the CPK with calmodulin antagonists and antisense oligonucleotides (Estruch et al., 1994). Involvement of CPKs in stress signal transduction has been concluded from studies on a constitutively active mutant of ATCDPK1 gene. This mutation activated the stress-inducible HVA1 promoter, while mutation in the kinase domain abolished this effect. These findings indicated the importance of the ATCDPK1 in transmission of stress signals (Sheen, 1996). Different environmental stresses have been reported to affect the activity of CPK genes. The mRNA level of two Arabidopsis CPK genes increased in plants subjected to salt or drought stresses (Urao et al., 1994). In mung bean, mechanical strain, salt, cycloheximide, and auxin treatment could induce the CPK gene expression (Botella et al., 1996). NtCDPK1 mRNA accumulation was stimulated by Ca2+, wounding, auxin, cytokinin, gibberellin, and ABA (Yoon et al., 1999).
In this paper the cloning of an alfalfa cDNA that encodes a novel member of the CAM-like protein kinase family is reported. The Ca2+-dependent activity and calmodulin inhibitor sensitivity of the kinase is demonstrated by using the recombinant enzyme expressed in bacteria. As shown by Northern hybridization data the corresponding gene is activated by a high concentration of auxin (2,4-D) or heat treatment. The data presented outline new aspects of molecular mechanisms of somatic embryogenesis that are equally based on auxin-and stress-related Ca2+-signalling pathways.
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Materials and methods |
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Cloning of alfalfa CPK cDNA homologues
RNA prepared from Medicago sativa RA3 microcallus suspension (MCS), treated with 10 mg l-1 2,4-D for embryo induction, was used to construct a cDNA library in
-ZAP II vector (STRATAGENE). Another cDNA library prepared from M. sativa RA3 somatic embryos was kindly provided by Dr H Hirt (Vienna). Both libraries were screened with a partial carrot CPK cDNA (kindly provided by Dr JH Choi; Suen and Choi, 1991) and several clones were isolated. The longest cDNA clone containing a full length CPK gene with an open reading frame of 1841 nucleotide, was analysed. The nucleotide sequence of the cDNA was determined by the dideoxynucleotide-chain-termination method (Sanger et al., 1977) as double stranded DNA using the Sequenase kit (US Biochemicals). DNA sequence analysis was carried out using PCGENE. DataBank searching was performed by using the BLAST system (Altschul et al., 1990).
Other cDNA clones used in the present studies
To obtain a probe for an alfalfa calmodulin, PCR primers were synthesized based on the published sequence (Barnett and Long, 1990) and the EcoRI-NsiI region of the cDNA between nucleotides 250–747 was amplified. The resulting PCR fragment was cloned into the EcoRI-NsiI site of pGEM-7 vector (Promega) and its identity was confirmed by sequencing. The cDNA clone of an alfalfa small heat shock Mshsp18-1 gene was kindly provided by Dr J Györgyey (Györgyey et al., 1991). The soybean ß-tubulin gene was kindly provided by Dr E Lam (Rutgers University, NJ) and histone H3 cDNA (Kapros et al., 1991).
Plant material, 2,4-D and stress treatments
Medicago sativa RA3 MCS was maintained on SH medium (Savouré et al., 1995) in the presence of 1 mg l-1 
-naphthalene acetic acid and 0.2 mg l-1 kinetin. Embryogenic 2,4-D treatment was applied as described earlier (Dudits et al., 1991; Györgyey et al., 1991). After 2 d of subculture 300 ml of liquid cell suspensions were exposed to 10 mg ml-1 2,4-D treatment and after 1 h the cells were washed twice and transferred into hormone-free medium. Samples were taken from the same culture for 3 d and subsequently the microcalli were plated on hormone-free agar medium where somatic embryos developed within 3–4 weeks.
Heat shock treatment was applied by distributing the same Medicago sativa RA3 liquid culture at the beginning of the experiment into 100 ml flasks. 15 ml cell suspension was diluted with 15 ml 45 °C media and cultured further at 37 °C for 30 min. Control material was diluted with the same medium at room temperature. At least two independent hormone and heat induction experiments were carried out for gene expression analysis.
RNA preparation and Northern analysis
The total RNA was extracted according to the freezing phenol method (Maes and Messens, 1992) with slight modification to scale it down to the volume of Eppendorf tubes. Frozen tissue of 0.1–0.3 g was homogenized in liquid nitrogen in a small mortar with 0.5 ml of phenol (equilibrated to pH 4.9 with 3 M K-acetate). After diluting with 0.5 ml 1% SDS the samples were incubated for 15 min at 65 °C, then centrifuged for 10 min in an Eppendorf centrifuge at 4 °C. The supernatant was extracted with phenol/chloroform (1:1, v:v) twice, and precipitated with 
volume of 8 M LiCl overnight at 4 °C. The precipitates were dissolved in 100 µl ribonuclease free water, and a second LiCl precipitation was applied for safe elimination of DNA contamination. 20 µg total RNA was run on 1% formaldehyde gel, containing 0.01% EtBr. Transfer of RNAs to Amersham Hybond N filters was carried out with the classic upward technique, and the ribosomal RNA pattern of the filters were examined under UV to verify the efficiency of transfer, and the quality of loaded RNA.
Radioactive labelling and hybridization was carried out according to standard protocols supplied by the company (Amersham).
Cloning of MsCPK3 into bacterial expression vectors and purification of the recombinant protein
The cDNA coding for the full length MsCPK3 protein was cloned into pFLAG-ATS vector (IBI) in order to tag the N-terminal region of the protein with the eight amino acid FLAG antigen determinant. First, the 5' non-coding region of the cDNA containing three stop codons was eliminated using a specific PCR oligonucleotide. The oligonucleotide synthesized corresponded to 17 nucleotides of the MsCPK3 sequence beginning from the first methionine, carrying an additional six nucleotides, creating the XhoI site (5'-CCCTCGAGATGGGGTGTTTGTTAAGTAA-3'). The second oligonucleotide used for the PCR reaction corresponded to the MsCPK3 sequence from nucleotide 486–506 (5'-TTGATAAGCTCCTTTAAATTCAACAATATT-3'), covering the internal DraI site. The 0.46 kb PCR product was digested with the XhoI-DraI and ligated with the second 1.37 kb DraI-KpnI fragment of the cDNA in one step into the XhoI-KpnI site of pFLAG-ATS vector. The DNA sequence of the created pFLAG-MsCPK3 construct was verified and contained the full-length cDNA without 5'- non-coding sequence. The purification of the FLAG tagged protein was performed from a crude extract of E. coli and was not efficient as most of the fusion product was recovered in inclusion bodies.
To obtain the purified protein in the soluble fraction, the GEX expression system (Pharmacia) was used. The MsCPK3 cDNA with the attached FLAG tagging was amplified by PCR from the pFLAG-MsCPK3 plasmid. One of the oligonucleotides corresponded to the FLAG sequence, carrying an extra BamHI site: 5'-CGGATCCATGGACTACAAGGACGAGGA-3', while the other oligonucleotide corresponded to the C-24 region in the pFLAG-ATS vector. The resulting PCR product was treated with the Klenow fragment, then digested with the BamHI and was cloned into the BamHI-SmaI sites of pGEX-4T1 vector. The identity of the construct was confirmed by sequencing. The purification of the recombinant protein from bacteria by GST column was carried out according to the Pharmacia manual. The FLAG-MsCPK3 protein was detected by Western analysis using anti-FLAG M1 and M2 monoclonal antibodies according to the manufacturer's protocol (IBI).
Kinase assay
Protein kinase activity was determined by in vitro detecting the incorporation 32P from (
32P)ATP. The standard assay mixture (30 µl) for the in vitro phosphorylation assay contained 25 mM TRIS (pH 7.5), 10 mM MgCl2, 50 mM NaF, 0.5 mM CaCl2, 1 mg ml-1 histone type III-S (Sigma) or dephosphorylated ß-casein (Sigma) as substrates, 10 µM ATP, 5 µCi (32P) ATP, and 0.4 µg purified GST-MsCPK3 fusion protein. The Ca2+-free mixture contained 5 mM EGTA instead of CaCl2. The effect of calmodulin inhibitor was studied by addition of 10 µM, 100 µM and 500 µM of W-7 (N-(6-aminohexyl)-5-chloro-1-naphthalenesulphonamide) to the reaction mixtures. The kinase reactions were started by the addition of CPK and stopped after 30 min at room temperature with 7.5 µl 5xSDS sample buffer. The samples were analysed by 10% SDS-PAGE and autoradiography.
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Results |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Cloning and sequence analysis of an alfalfa CPK cDNA
The alfalfa cDNA library prepared from the RNA of 2,4-D treated Medicago sativa RA3 microcallus suspension was screened with the carrot CPK probe (kindly provided by Dr Choi). The longest cDNA clone, named MsCPK3, is composed of 1841 nucleotides with an open reading frame that corresponded to a 535 amino acid long polypeptide (Fig. 1
). Sequence analysis showed that the deduced polypeptide carried a serine-threonine kinase domain, a regulatory junction domain and four Ca2+-binding EF-hands. Comparison with other sequences available in GenBank revealed that the most related genes are soybean GmCDPK (Lee et al., 1997), tobacco NtCDPK1 (Yoon et al., 1999), OsCPK2 from rice (Breviario et al., 1995), Arabidopsis AtCDPK9, and maize ZmCDPK9. Like several other members of CPK family, at the N-terminus MsCPK3 contains the motif MGxxxS that defines a consensus sequence for a potential N-myristoylation site. The first 80 amino acids of the N-terminal region of MsCPK3 share no homology with other sequences.
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Purification of the recombinant protein and the in vitro kinase assays
To study the characteristics of MsCPK3, the FLAG-tagged MsCPK3 protein was expressed in bacterial cells. Altough the presence of the proper FLAG-MsCPK3 protein of approximately 60.2 kDa could be detected by immunoblot analysis (data not shown), the great majority of the recombinant protein formed inclusion bodies.
To obtain sufficient purified protein for biochemical characterization, the GST expression system was also applied. The pGST-MsCPK3 plasmid construct contained the glutathione S-transferase sequence joined to the 5' coding region of FLAG tagged MsCPK3. The gluthatione agarose affinity purified recombinant protein was analysed by SDS-PAGE and the 86 kDa size of the purified protein corresponded to molecular weight of GST (26 kDa) and FLAG-MsCPK3 (60.2 kDa). Immunodetection with anti-FLAG antibodies confirmed that the protein obtained is the recombinant GST-MsCPK3 (Fig. 2).
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Substrate specificity of the CPK was tested by using histone and casein as substrates. Histone showed a higher phosphorylation level and was used for further experiments. Kinase activity of the recombinant protein was induced in vitro by Ca2+ (Fig. 3A
). A CAM antagonist, W-7, was applied at concentrations from 10 µM to 500 µM in the in vitro phosphorylation assays. Both the phosphorylation of the histone and autophosphorylation were inhibited in the presence of this compound (Fig. 3B). These results showed that the analysed CPK homologue exhibited a Ca2+-dependent kinase activity and it could be inhibited by calmodulin antagonist W-7.
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Treatment of alfalfa callus cells with high concentration of 2,4-D or heat shock caused a transient MsCPK3 transcript accumulation
For characterization of the expression pattern of MsCPK3 gene during somatic embryogenesis, RA3 microcallus suspensions of Medicago sativa were treated with 10 mg l-1 2,4-D for 1 h. RNA samples were collected at 30 min, 1, 2, 4, 8, 24, 48, and 72 h after embryogenic induction (Fig. 4A). The steady-state mRNA level of MsCPK3 was analysed using the 5' region of the MsCPK3cDNA as a gene-specific probe. Considerable MsCPK3mRNA accumulated when the cells were exposed to auxin treatment. This prompt response declined after 4 h of auxin shock in hormone-free medium. The expression of a small heat shock protein Mshsp18-1 gene, was also induced, though the peak in the mRNA level appeared later, in samples from the 4 h and 8 h cultures. The level of calmodulin mRNA was almost constitutive during the analysed period.
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To investigate the effect of other environmental stresses on MsCPK3 mRNA accumulation, heat shock was applied for 30 min at 37 °C. As shown in (Fig. 2B
), this treatment could also transiently activate the expression of the MsCPK3 gene, with an expression peak at 30 min after induction. As expected, the small heat shock protein gene responded immediately to the treatment. The calmodulin mRNA was slightly decreased by the heat shock during the first 2 h, but then it was restored. Other treatments of alfalfa MCS such as application of a lower amount of 2,4-D (1 mg ml-1), kinetin (1 mg ml-1), abscisic acid (150 µM) or salt (100 mM NaCl) did not significantly affect the expression of the MsCPK3 gene (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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In this study cDNA libraries constructed from somatic embryos and 2,4-D treated microcallus suspension were screened and a full-length clone of putative alfalfa CPK, designated MsCPK3 was isolated. Although partial sequences of two alfalfa CPKs have been reported (Monroy and Dhindsa, 1995
) the MsCPK3 is the first full length CPK representative from this species. The deduced amino acid sequence of MsCPK3 depicts a protein with a typical structure of CPKs. The comparison with other sequences available in GenBank (Fig. 1) showed very strong homology to several plant CPK proteins. These data are in agreement with previous findings that certain CPK variants in monocots and dicots are more closely related than the different members of the gene family within the same species. The amino acid sequences of these CPKs are almost the same in the kinase, regulatory and EF-hands domains while the N terminal region shares no homology.
Previous reports showed that many CPKs are associated with membranes (Schaller et al., 1992). A possible mechanism for targeting the proteins to the membranes is the N-myristoylation. Recently, the in vitro myristoylation of zucchini CPK was demonstrated (Ellard-Ivey et al., 1999). The presence of the N-terminal MGxxxS MsCPK3 motif defines a possible N-myristoylation site, suggesting that MsCPK3 may be targeted to a membrane compartment.
In attempts to prove the Ca2+ dependency of the isolated alfalfa kinase a recombinant protein was purified. The GST-MsCPK3 expressed in E. coli required Ca2+ for its activity in vitro. The data presented suggest that MsCPK3 can function as Ca2+-dependent kinase in vivo as well. The calmodulin antagonist W-7 is widely used to study the role of calmodulin in different signalling pathways (Hidaka et al., 1981; Lam et al., 1989). Sensitivity of MsCPK3 to this inhibitor indicates that calmodulin homologue Ca2+-binding motifs are required for the kinase activity. These results further support that MsCPK3 can be an active component of Ca2+-dependent signal transduction in alfalfa. Similarly to previously studied CPK from alfalfa, this recombinant kinase exhibited autophosphorylation activity (Bögre et al., 1988). This function might contribute to the amplification of the signal.
Changes in gene expression frequently reflect the biological function of the genes in different processes. In this work the expression of MsCPK3 was induced by high concentration of 2,4-D treatment that was previously shown to induce somatic embryogenesis (Dudits et al., 1991). The kinetics of MsCPK3 transcript accumulation showed a rapid and transient response that was frequently observed with stress-related genes. Indeed the high concentration of this synthetic auxin can also activate the expression of the classic small heat shock protein Mshsp18-1. The overall alarm of stress responses was further shown by the synthesis of mRNA from the heat shock transcription factor gene (HSF) in the same cells. The HSF transcript level followed a similar pattern to MsCPK3 mRNA accumulation (D Dudits et al., unpublished results). Application of a lower amount of 2,4-D (1 mg l-1), which still efficiently promoted cell division in callus tissues, but does not induce organized embryogenic structures in RA3 culture, did not cause a significant increase in the MsCPK3 expression (data not shown). This suggests that the mRNA peak of MsCPK3 observed during embryogenic 2,4-D induction may primarily be related to the stress effects of this compound. Induction of Mshsp18-1 and an alfalfa heat shock transcription factor support the observation that various stresses might have significance during the early phase of embryogenic induction in Medicago sativa cells. However, 2,4-D induced somatic embryogenesis accompanied by increasing division activity of the cells, so the possibility can not be ruled out that the reactivation of cell cycle by 2,4-D can also be linked to the mRNA increase of MsCPK3. The relation between calcium signalling and cell division as well the potential role of CPKs have been discussed previously (Dudits et al., 1998).
Many CPK genes have been reported to be activated in response to environmental stresses. Thus, the alfalfa MSCK1 mRNA level increased during cold acclimation (Monroy and Dhindsa, 1995). Rice OsCPK2 was shown to be regulated by light and anoxic treatment (Breviario et al., 1995; Frattini et al., 1999), while VrCDPK1 was induced by mechanical strain, IAA, salt and cycloheximide treatment (Botella et al., 1996). Maize ZmCDPK1 expression was induced by low-temperature stress and cycloheximide (Berberich and Kusano, 1997). Activation by cycloheximide has been shown earlier for several CPKs involved in signal transduction pathways (Mahadevan and Edwards, 1991). Two Arabidopsis CPKs, ATCDPK1 and ATCDPK2, were induced in response to low temperature, salt and drought stress (Urao et al., 1994). In the work presented here the effect of several stress treatments (heat shock, high salt, low amount of 2,4-D, ABA, and kinetin) were studied. When heat shock was applied, MsCPK3 and Mshsp18-1 genes showed similar activation, though Mshsp18-1 responded earlier by accumulating the mRNA within minutes (Fig. 4B). Interestingly, the expression of CPKs described in other plant species was not induced by heat shock. Other stress treatments resulting in activation of the CPKs from different species (Urao et al., 1994; Botella et al., 1996; Yoon et al., 1999) had no effect on MsCPK3 expression under the conditions analysed in the present work. These differences in response to various external factors suggest that the alfalfa CPK might represent a novel type of these kinases, but it cannot be ruled out that large divergence in responses of various CPK genes originate from the use of different experimental systems.
Somatic embryogenesis involves initiation of an ontogenic programme in cultured plant cells and is strongly dependent on the reprogramming of gene expression required for hormone-induced cell division and differentiation. The present studies are concentrated on the early phases of embryogenic induction in Medicago cell suspensions, when the somatic cells become committed towards the completion of an embryo differentiation pathway. It was observed that the high 2,4-D application, altering the cell division pattern, also initiates the expression of MsCPK3 and several stress-related genes. Taken together these data suggest that MsCPK3 may be involved in signal transduction of auxin or/and stress-activated processes of developmental reprogramming in plant cells during somatic embryogenesis and cell division. Further studies are required to understand whether the occurrence of stress is a coincidence of the treatment, or whether it could be beneficial for the reprogramming of cells for differentiation.
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Acknowledgments |
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We thank Dr Jung H Choi (School of Biology, Georgia Institute of Technology, Atlanta) for providing the carrot cDNA of CPK and Dr Heribert Hirt (University of Vienna) for providing an alfalfa cDNA library.
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Notes |
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3 To whom correspondence should be addressed. Fax: +36 62 433 434. E-mail: Dudits{at}nucleus.szbk.u\|[hyphen]\|szeged.hu EMBL/GenBank Data Library accession number: X96723.
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N. Imin, M. Nizamidin, D. Daniher, K. E. Nolan, R. J. Rose, and B. G. Rolfe Proteomic Analysis of Somatic Embryogenesis in Medicago truncatula. Explant Cultures Grown under 6-Benzylaminopurine and 1-Naphthaleneacetic Acid Treatments Plant Physiology, April 1, 2005; 137(4): 1250 - 1260. [Abstract] [Full Text] [PDF]
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J. Leclercq, B. Ranty, M.-T. Sanchez-Ballesta, Z. Li, B. Jones, A. Jauneau, J.-C. Pech, A. Latche, R. Ranjeva, and M. Bouzayen Molecular and biochemical characterization of LeCRK1, a ripening-associated tomato CDPK-related kinase J. Exp. Bot., January 1, 2005; 56(409): 25 - 35. [Abstract] [Full Text] [PDF]
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A. A. Ludwig, T. Romeis, and J. D. G. Jones CDPK-mediated signalling pathways: specificity and cross-talk J. Exp. Bot., January 2, 2004; 55(395): 181 - 188. [Abstract] [Full Text] [PDF]
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E. M. Hrabak, C. W.M. Chan, M. Gribskov, J. F. Harper, J. H. Choi, N. Halford, J. Kudla, S. Luan, H. G. Nimmo, M. R. Sussman, et al. The Arabidopsis CDPK-SnRK Superfamily of Protein Kinases Plant Physiology, June 1, 2003; 132(2): 666 - 680. [Abstract] [Full Text] [PDF]
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B. Charrier, A. Champion, Y. Henry, and M. Kreis Expression Profiling of the Whole Arabidopsis Shaggy-Like Kinase Multigene Family by Real-Time Reverse Transcriptase-Polymerase Chain Reaction Plant Physiology, October 1, 2002; 130(2): 577 - 590. [Abstract] [Full Text] [PDF]
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T. P. Pasternak, E. Prinsen, F. Ayaydin, P. Miskolczi, G. Potters, H. Asard, H. A. Van Onckelen, D. Dudits, and A. Feher The Role of Auxin, pH, and Stress in the Activation of Embryogenic Cell Division in Leaf Protoplast-Derived Cells of Alfalfa Plant Physiology, August 1, 2002; 129(4): 1807 - 1819. [Abstract] [Full Text] [PDF]
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S.-H. Cheng, M. R. Willmann, H.-C. Chen, and J. Sheen Calcium Signaling through Protein Kinases. The Arabidopsis Calcium-Dependent Protein Kinase Gene Family Plant Physiology, June 1, 2002; 129(2): 469 - 485. [Abstract] [Full Text] [PDF]
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