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Originally published In Press as doi:10.1074/jbc.M111626200 on January 8, 2002

J. Biol. Chem., Vol. 277, Issue 12, 9840-9852, March 22, 2002
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Genes Encoding Calmodulin-binding Proteins in the Arabidopsis Genome*,

Vaka S. ReddyDagger, Gul S. AliDagger, and Anireddy S. N. Reddy§

From the Department of Biology and Program in Cell and Molecular Biology, Colorado State University, Fort Collins, Colorado 80523

Received for publication, December 6, 2001

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Analysis of the recently completed Arabidopsis genome sequence indicates that ~31% of the predicted genes could not be assigned to functional categories, as they do not show any sequence similarity with proteins of known function from other organisms. Calmodulin (CaM), a ubiquitous and multifunctional Ca2+ sensor, interacts with a wide variety of cellular proteins and modulates their activity/function in regulating diverse cellular processes. However, the primary amino acid sequence of the CaM-binding domain in different CaM-binding proteins (CBPs) is not conserved. One way to identify most of the CBPs in the Arabidopsis genome is by protein-protein interaction-based screening of expression libraries with CaM. Here, using a mixture of radiolabeled CaM isoforms from Arabidopsis, we screened several expression libraries prepared from flower meristem, seedlings, or tissues treated with hormones, an elicitor, or a pathogen. Sequence analysis of 77 positive clones that interact with CaM in a Ca2+-dependent manner revealed 20 CBPs, including 14 previously unknown CBPs. In addition, by searching the Arabidopsis genome sequence with the newly identified and known plant or animal CBPs, we identified a total of 27 CBPs. Among these, 16 CBPs are represented by families with 2-20 members in each family. Gene expression analysis revealed that CBPs and CBP paralogs are expressed differentially. Our data suggest that Arabidopsis has a large number of CBPs including several plant-specific ones. Although CaM is highly conserved between plants and animals, only a few CBPs are common to both plants and animals. Analysis of Arabidopsis CBPs revealed the presence of a variety of interesting domains. Our analyses identified several hypothetical proteins in the Arabidopsis genome as CaM targets, suggesting their involvement in Ca2+-mediated signaling networks.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Calcium, a key messenger in plants, is involved in mediating the action of diverse signals including plant hormones, light, biotic and abiotic stresses, and symbiotic elicitors (1). All these signals have been shown to elicit changes in cytosolic free Ca2+ ([Ca2+]cyt)1 level (1-5). In addition, several growth and developmental processes are also accompanied by changes in [Ca2+]cyt levels (6, 7). Transient changes in free [Ca2+]cyt levels control cellular processes through Ca2+ sensors. There are at least four major families of Ca2+ sensors in plants. These include (i) calmodulin (CaM) and its isoforms, which consist of 148 amino acids and four EF hands; (ii) CaM-like proteins, which differ from CaMs in their size and EF hands; (iii) Ca2+-dependent protein kinases; and (iv) other Ca2+-binding proteins without EF hands (1). Ca2+-dependent protein kinases are found only in plants and protozoan, whereas CaM is ubiquitous in all eukaryotes (1, 8, 9).

Calmodulin is an acidic heat stable protein with two globular domains, each carrying two EF hands (10). CaM is the primary transducer of cytosolic Ca2+ changes in all eukaryotes. In most cases, the active form of CaM (Ca2+-bound CaM) regulates the activity/function of a wide range of CaM-binding proteins (CBPs) including metabolic enzymes, transcriptional factors, ion channels and pumps, and structural proteins (1, 9). Therefore, CaM acts as a multifunctional protein in Ca2+-mediated signal transduction networks and regulates the activity of structurally and functionally unrelated proteins. Arabidopsis contains at least nine different CaM isoforms (AtCaMs) and several CaM-like proteins (8, 9, 11). AtCaM1 to AtCaM7 differ in a few amino acids, whereas AtCaM8 and AtCaM9 are the most diverged (12).

Analysis of the recently completed Arabidopsis genome sequence, the first plant genome to be sequenced, revealed that there are 25,498 genes in this organism (11). The next challenge is to identify the function of many of the predicted proteins in the Arabidopsis genome. The amino acid sequences from the predicted open reading frames are useful in many cases in obtaining insights into the function of the predicted proteins primarily through sequence similarities and functional motifs present in the predicted proteins. Database searches with the Arabidopsis predicted proteins indicate that 69% of the total proteins have sequence similarities with proteins of known function in other organisms, whereas the rest (31%) are unique and could not be assigned to any functional category (11). In cases where the predicted proteins do not show sequence similarities to known proteins, it is difficult to obtain insights into their function. The primary sequence of the CaM-binding domain (CBD) in different CBPs is not conserved (1). Furthermore, from the few plant CBPs that have been characterized, it seems that plants contain several unique CBPs (1). Several plant-CBPs have no homologs in non-plant systems. Hence, it is not possible to identify CaM target proteins based on computer-assisted sequence comparisons using CaM-binding sequences (10). One way to identify these proteins is by functional interaction with CaM. So far, only a limited number of CBPs have been identified in plants (1). To identify most of the CBPs in Arabidopsis, we used labeled Arabidopsis CaM isoforms to screen several expression libraries prepared from different plant parts and plants/tissues that are either treated with hormones or pathogen/elicitor. We sequenced isolated cDNAs and identified their corresponding genes in the Arabidopsis genome database. In addition, we searched the Arabidopsis genome sequence with other known animal and plant CBPs. We then analyzed CBPs for CBD, structural features, and gene organization and expression.

Our analyses revealed that plants have a unique set of CBPs, and several CBPs have a large number of paralogs. Some CBPs are present in both plants and animals, whereas others are unique to plants or animals, suggesting functional similarity and divergence in Ca2+/CaM-mediated signal transduction networks between plants and animals. Gene expression analyses revealed that the members of a given CBP gene family are expressed differentially in different tissues. Domain analysis of the new Arabidopsis CBPs indicates that they possess putative domains that are implicated in a variety of cellular activities.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- Easy Tag 35S-labeling mix, and [alpha -32P]dCTP were from PerkinElmer Life Sciences. Triton X-100 free nitrocellulose membrane discs were purchased from Millipore. Exassist helper phage and Escherichia coli strain SOLR were from Stratagene. TRIzol reagent and isopropyl beta -D-thiogalactopyranoside (IPTG) were purchased from Invitrogen. All other chemicals and solvents are of reagent grade.

Plant Material-- Arabidopsis thaliana (L.) ecotype Columbia was grown at 22 °C on a mixture of peat:perlite:vermiculite (1:1:1) under 16-h light cycle. Leaves, stems, and flowers were collected from 5-6-week-old plants and stored at -80 °C.

Preparation of 35S-Labeled CaM-- The Arabidopsis CaM isoforms 2, 4, and 6 in pET expression vectors were kindly provided by Dr. Raymond E. Zielinski. Arabidopsis CaM isoforms were radiolabeled using Easy Tag 35S labeling mixture as described (13-15).

Screening of Expression Libraries-- Five Arabidopsis (ecotype Columbia) and one bean (Phaseolus vulgaris cv. Red Kidney) expression libraries prepared in lambda ZAP (EcoRI-XhoI or EcoRI site, Stratagene) or lambda ZipLox (SalI-NotI) phage vectors were used in our screening. The expression libraries were prepared from the following tissues: (i) roots of 10-day-old seedlings treated with 10 µM alpha -naphthaleneacetic acid for a 24-h period (16); (ii) hypocotyls and cotyledons of 3-day-old seedlings treated with ethylene (3-6-kb fraction, a gift from ABRC) (17); (iii) mixed tissues of liquid culture-grown roots, 7-day-old etiolated seedlings, rosette stage plants at different ages grown under two light regimes, and aerial tissues of stems, flowers, and siliques (in lambda -ZipLox phage vector obtained from ABRC); (iv) Arabidopsis flower meristem cDNA library, obtained from Dr. E. Meyerowitz; (v) pooled cell cultures (grown in the dark in modified MS medium treated with 50 µg/ml elicitor from Phytophthora megasperma sp. glycinea (18); and (vi) pooled bean leaf tissue undergoing hypersensitive response as a result of infiltration of Pseudomonas syringae pv. tabaci Pt11528 (19).

Approximately 800,000 recombinant phages of each library were screened with a mixture of 35S-CaM using XL1-blue MRA host strain (Stratagene). The plates were incubated at 42 °C until the plaques appeared, at which time the plates were overlaid with nitrocellulose membranes (0.45 µm, HATF, Millipore) presoaked in 10 mM IPTG to induce the fusion protein of recombinant phages. Plates were returned to 37 °C and incubated for 8 h. The plates were then cooled at 4 °C, and membranes were removed and incubated with either 35S-CaM or biotinylated CaM as described (15, 20). The putative positive recombinant phages were purified by two additional rounds of screening. During the third round of screening, each putative positive was tested for CaM binding in the presence and absence of Ca2+. The cDNA was excised in vivo in a plasmid form (lambda ZAPII to pBluescript; lambda ZipLox to pZL1).

Sequencing and Database Searches-- Double-stranded DNA from putative recombinant plasmids was prepared, and 5' and 3' ends of each clone were sequenced using T3 and T7 primers, respectively. The sequences obtained from these clones were used to search Arabidopsis TAIR (www.arabidopsis.org) and MIPS (mips.gsf.de/proj/thal/db/index.html) databases using BLASTN and BLASTX search programs. After determining the full-length sequence of a CBP as above, we used its DNA (spliced and unspliced) and protein sequences from the Arabidopsis database for various analyses as described below. Search for the identification of the T-DNA or transposable element insertion sequences in all 100 CBP genes (knockout mutants; E value <1 × 10-4) was performed at the Torrey Mesa Research Institute (www.tmri.org), the Salk Institute Genomic Analysis Laboratory (signal.salk.edu), and the Nottingham Arabidopsis Stock Center (nasc.nott.ac.uk), where a total of 108,500 sequences flanking the T-DNA or transposable element insertions were available as of December 3, 2001.

Analyses of Gene Expression-- Total RNA from leaf, stem, flower, and root tissues of Arabidopsis was extracted using TRIzol reagent (Invitrogen). Fifty micrograms of total RNA was electrophoresed, transferred to Hybond Nylon membrane, and hybridized with 32P-labeled full-length cDNA using standard protocols. Searches for expressed sequence tags (ESTs) were performed at MIPS database. If an EST was found for a CBP, we considered the tissue or organ from which the cDNA library was constructed as positive for the expression of that particular gene. We also searched the literature for the expression pattern of already identified genes and summarized our findings in Tables I and II.

Identification of CBPs and Their Families in Arabidopsis-- We used CBP sequences from Arabidopsis obtained in our screening and other plant and animal CBPs (obtained from published papers or searching the databases including NCBI) to search against Arabidopsis TAIR (www.arabidopsis.org) and MIPS (mips.gsf.de/proj/thal/db/index.html) databases to identify corresponding Arabidopsis CBPs. Several criteria such as conservation of the CBD region and other domains if they are present, level of sequence similarity along the entire length of the sequence (E value <1 × 10-11), and protein size were considered in identification of CBPs. Plant sequences that showed some sequence similarity to animal CBPs but lacked a CBD were not considered as CBPs. Once we identified an Arabidopsis sequence as a CBP, we used that sequence as query against TAIR and MIPS to identify its paralogs. The CBD regions were carefully analyzed using computer-aided detection programs (PROTEAN from DNA Star and HelixWheel from ExPASy tools (www.expasy.ch) as well as visual inspection. Further, BAC clones generated from the Arabidopsis sequencing project were used to determine the orientation of clusters of gene families on chromosomes. Chromosomal location of genes was identified using Arabidopsis Sequence Map Overview. Alignment of the CBP families was performed using the CLUSTAL method of the Megalign program; the file was saved as a PAUP nexus file. Phylogenetic trees were generated using a Heuristic Bootstrap method (100 replicates) of PAUP version 4.0b6, a maximum parsimony program. All CBPs were analyzed for the presence of various domains and organellar target sequences using CD search at NCBI and SMART, PEST, NLS, and coiled-coil prediction programs from ExPASy tools (www.expasy.ch).

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Isolation of cDNAs Encoding Calmodulin-binding Proteins-- To identify a majority of Arabidopsis CBPs, we screened several expression libraries with a mixture of labeled Arabidopsis CaM isoforms. The expression libraries were made from different tissues as well as from plants/cell cultures exposed to signals (auxin, ethylene, bacterial pathogen, or elicitor). Because the expression of CBPs is likely to vary in different tissues and in response to various signals, the cDNA libraries used in this study should contain most of the CBPs. Because CBPs show differential affinity to CaM isoforms, we used three CaM isoforms from Arabidopsis to screen libraries. Using in vitro protein-protein interaction-based screening of 8 × 105 recombinant phages from each library, we isolated 77 independent positives. An autoradiogram showing the screening results with one of the clones is presented in Fig. 1. To determine whether the cDNA encoded protein binds CaM only in the presence of Ca2+, we tested the binding of each positive CBP to CaM in the presence of CaCl2 or EGTA, a Ca2+ chelator. All 77 clones bound CaM only in the presence of CaCl2, suggesting that they bind CaM in a Ca2+-dependent manner (Fig. 1). Based on restriction maps and sequence of the 5' and the 3' ends, we have grouped these clones into 20 distinct cDNAs with insert sizes ranging from ~1 to ~4 kb.


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  		Fig. 1.   Representative autoradiograms depicting protein-protein interaction-based screening of expression libraries with labeled CaM. Screening of expression libraries with labeled CaM was as described under "Experimental Procedures." The black spots indicate the binding of 35S-labeled CaM to a recombinant phage containing a cDNA encoding a CBP in first, second, and third rounds of screening. During the third screening, the filter was divided into two equal parts. One half of the filter was incubated with binding buffer (50 mM Tris-HCl, pH 7.5, and 150 mM NaCl with 1% nonfat dry milk) containing EGTA (EGTA), and the other half was incubated with binding buffer containing calcium chloride (CaCl2).

The 5' and the 3' sequences of the cDNA clones were used as queries to search the recently completed Arabidopsis genome sequence (www.arabidopsis.org) to obtain complete nucleotide and deduced protein sequences. Sequences in the Arabidopsis database that showed 100% identity at the nucleotide level with the cDNA sequences were considered as the corresponding full-length genes. Based on this analysis, it was found that of 20 distinct CBPs identified in our screenings, 14 are new CBPs (Tables I and II) whereas the other 6 are previously reported either in Arabidopsis or other plants. The newly identified CBPs include four hypothetical proteins, a protein kinase C substrate-like protein (PKC substrate-like), photosystem I-N subunit (PSI-N subunit), a pirin-like protein, four ACBP60 proteins (homologs of TCBP60), and a new member of auxin-induced proteins, cyclic nucleotide-like gated channels (CNGCs) and ethylene-induced CBPs (EICBPs). The previously identified six clones include KCBP (20-24), one TCBP60-like protein (25), two cyclic nucleotide gated channels (26-28), an ethylene-induced CBP (29), and a glutamate decarboxylase (9, 30, 31) (Tables I and II).

                              
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Table I
Arabidopsis single gene-encoded CBPs isolated from expression libraries using labeled calmodulin or identified based on sequence similarity to characterized CBPs

Bioinformatics-based Identification of CBPs and Their Families in Arabidopsis-- Paralogs of Arabidopsis CBPs were identified by searching the Arabidopsis genome database with the new CBP sequences identified in this study. Sequences that showed significant sequence similarity (E value <1 × 10-11) at the nucleotide and protein sequences were considered as paralogs of CBPs. To identify homologs of animal and other plant CBPs, we searched the Arabidopsis genome database with sequences of known animal and plant CBPs. In classifying a protein as a CBP, we used several criteria. These include conservation of the CBD and other domains if present, protein size, and identity/similarity along the entire length of the query and hit sequences. Our search resulted in identification of several gene families encoding CBPs in Arabidopsis (Table II). Further, we identified additional members for some of the known CBP families. At least one member in each of the 16 Arabidopsis CBP families binds CaM in a Ca2+-dependent manner (see "Method" column in Table II). We have identified another Arabidopsis CBP that shows very high sequence similarity to 60 S ribosomal L19 protein (E value <3 × 10-54) that was isolated from Dictyostelium discoideum using 125I-CaM (32). The likely reasons for not identifying other members of the 16 CBP gene families in our screening of expression libraries are that they may be expressed in response to specific stimulus or developmental cues and/or as the result of differential affinities for CaM isoforms. Our results indicate that there are at least 27 distinct CBPs that interact with CaM in a Ca2+-dependent manner in Arabidopsis. Of these, 11 exist as singletons and 16 exist as gene families consisting of 2-20 members. Together with all the paralogs, there are ~100 individual CBPs (Tables I and II), which represent ~0.4% of the Arabidopsis genes (total ~25,498 genes).

                              
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Table II
Gene families encoding CBPs in Arabidopsis

Genes Encoding CBPs Are Expressed Differentially-- The identification of a variety of CBPs including several gene families in the Arabidopsis genome warrants analysis of their expression in different tissues and in response to various stimuli and during growth and development. We obtained expression data by RNA blot analysis with some of the newly isolated CBPs and by analyzing the EST databases for the presence of a corresponding EST clone. Fig. 2 shows the expression data of newly isolated clones, whereas the data from the EST database are summarized in Tables I and II. We found the presence of gene transcripts for various CBPs in leaf, stem, flower, root, silique, developing seed, and cell cultures and in response to cold, drought, and salt stresses. The expression data are presented in Tables I and II (bold plus sign for experimental results and normal plus sign for the tissue from which the EST was isolated). Interestingly, members of the ACBP60 family are expressed differentially (Fig. 2). At5g62570 and At2g18750 show higher expression in stem tissue compared with At5 g57580 and At4g25800. At5g62570 and At2g18750 show very little expression in leaves compared with other tissues (Fig. 2). At5g57580 shows equal amounts of its transcripts in the tested tissues (Fig. 2). The genes encoding PKC substrate-like and pirin-like proteins show lower levels of expression in leaf compared with stem, flower, and root tissues. Pirin-like protein also showed a differential expression with the highest expression in stem (Fig. 2).


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  		Fig. 2.   RNA gel blot analysis with Arabidopsis genes encoding CBPs. Total RNA was electrophoresed on a formaldehyde-containing 1.2% agarose gel, transferred to a Hybond N+ membrane, and hybridized with 32P-labeled cDNA fragments. The CBP number (left) and gene identification numbers (right) refer to Tables I and II. The transcript sizes of 2, 14a-14d, and 17a are 2.3, 1.8, 2.3, 2.3, 1.9, and 1.2 kb, respectively. Ethidium bromide-stained gel (Stained gel) shows the amount of RNA loaded in each lane (bottom panel). L, leaf; S, stem; F, flower; R, root.

The expression of EST clones corresponding to 54 other CBPs is summarized in Tables I and II. Of the five GADs present in Arabidopsis, only two (GAD1 and GAD2) were reported and both showed differential expression. GAD2 expresses in leaf, stem, flower, and root tissues, whereas GAD1 expresses only in roots (31). Several Ca2+/ATPases that belong to autoinhibited Ca2+/ATPases (ACAs) and endoplasmic reticulum-type Ca2+/ATPases (ECAs) have been identified (33). Tissue-specific gene expression is prevalent for 7 of 12 ACAs. The expression pattern for four ACAs (ACA1, -2, -4, and -8) has been shown previously (33) and for three ACAs (At3g57330, At4g29900, and At1g13210) has been obtained from the EST database (Table II). ACA1 is expressed more in root than in leaf (34). ACA2 gene transcripts are found in leaf, root, flower (35), silique, and developing seed (Table II). The expression of ACA4 is present in leaf, stem, flower, silique, and at high levels under NaCl stress (33) and in root (Table II), and ACA8 is present in cell cultures (36). Further, transcripts for CNGC4, EICBP.c and ACA4, are induced in response to NaCl stress. Expression of ACA1 is induced in response to drought and cold, suggesting differential expression of ACAs in response to various stimuli (Table II). Of the two PPIs, At3g25230 (Table II) shows expression in leaf, stem, flower, and root tissues and high level expression under wounding and NaCl stresses (37), and EST data suggest its expression in silique and developing seed (Table II). Of the TGA3 members (Table II), At1g22070 is expressed in leaf, stem, and flower, and at high levels in root (38).

Conserved Regions Are Found in the Calmodulin-binding Domain of Arabidopsis CBP Paralogs-- Calmodulin reversibly regulates (based on free [Ca2+]cyt) the activity of a variety of CBPs through interaction of a 13-26-amino acid motif (CBD). Although, the CBD is not conserved among different CBPs, in most cases it forms a characteristic basic-amphiphilic alpha -helix structure. The amino acid sequence of CBDs, when arranged in a helical wheel, forms an amphiphilic helix with several basic and polar residues on one side and a number of hydrophobic residues on the other side (10). Interestingly, alignment of CBDs of members of a given CBP family suggests that the CBD in a specific CBP gene family is conserved with some exceptions. Fig. 3 shows the alignment of CBD regions of members of 11 CBP gene families. At least two members in each family bind CaM in a Ca2+-dependent manner in a gel overlay assay (Fig. 3). Alignment of CBDs of 5 small auxin up-regulated-like proteins (SAURs), 7 ACBP60s, 20 CNGCs, 6 EICBPs, 3 APCBPs, 2 PPIs, 5 TGAs, and 6 HSP70s shows high sequence similarity (Fig. 3), suggesting that all are likely to interact with CaM. Amino acid sequence comparison between members of CB-HSPs, GADs, and ACAs shows less sequence similarity although some members are shown to bind to CaM. The sequence analyses suggest that the CBD sequence in the SAUR, ACBP60, CNGC, EICBP, APCBP, PPI, TGA (the CBD of TGA is predicted but not proven experimentally) (39), and HSP70 families is more conserved than in the CB-HSP, GAD, and ACA families. The CBDs in GAD1 (At5g17330) and GAD2 (At1 g65960) of the GAD family and ACA1 (At1g27770) and ACA8 (At5g57110) of the ACA family are diverged but still retain the ability to bind to CaM (31, 34, 36). Differences in CBDs of members of a given family could account for different affinities with specific CaM isoforms. For example, CNGC1 (At5g53130) and CNGC2 (At5g15410) differ in their affinity to AtCaM isoforms (12). GAD1 (At5 g17330) and GAD2 (At1g65960) differ in their CBD region and showed differential enzyme activity by CaM (31). The CBD in most CBPs resides in extreme ends (e.g. ACBP60s, CB-HSPs, GADs, PPIs, KCBP, and chaperonin have the CBD in their C terminus, whereas SAURs and Ca2+/ATPases have their CBD in the N terminus). The CBD in some CBPs is in the middle (e.g. apyrase, APCBPs, TGA3s, and Hsp70s) or within the ~100 amino acids of the C terminus (e.g. CNGCs and EICBPs). Because of the presence of several CBP gene families in Arabidopsis, it will be necessary to study the interaction of each member of a gene family with CaM isoforms to functionally characterize the gene families in Ca2+/CaM-mediated signal transduction networks.


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  		Fig. 3.   Alignment of CBD sequences of 11 CBP families. In each family (except TGA and HSP70s), at least two or more sequences are shown to bind to CaM in the presence of Ca2+ (+), but not in the presence of EGTA (-) as described in Fig. 1. The gene identification numbers correspond to numbers in Table II. Reverse lettering shows identical amino acids. Dashes indicate gaps in the alignment. The numbers on the left indicate the amino acid residue number.

Phylogenetic Relationships between Paralogs of Arabidopsis CBPs-- To determine the relationship between members of a CBP family, the full-length protein sequences of members of the five CBP gene families were aligned using the MegAlign program and phylogenetic trees were constructed from the aligned files using the PAUP version 4.0b6 program (40). The phylogenetic relationships of five CBP families, location of all CBP encoding genes on chromosomes, and the domain and gene organization of three families are presented in Figs. 4, 5, and 6, respectively.


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  		Fig. 4.   Phylogenetic trees of five CBP families. The full-length protein sequences of each family member were aligned using the CLUSTAL method of the Megalign program. The trees were built using PAUP version 4.0b6. The families are ACBP60 (A), CNGC (B), EICBP (C), GAD (D), and Ca2+-ATPase (E). No outgroup was used to build trees of families ACBP60s (A) and EICBPs (C), as they are plant-specific CBPs. DmCNGC, EcGAD, RnACA, and CeECA were used as outgroups to build respective family trees. The Arabidopsis gene identification numbers refer to numbers in Table II. The accession numbers other than Arabidopsis sequences used in building the trees are: NtCBP60 (T03793), ZmCBP60 (AAA33446), NtCNGC (AAF33670), HvCNGC (CAA05637), OsCNGC (AAK16188), DmCNGC (AAF46898), NtEr1 (AAG39222), LeER66 (AAD46410), PsCG1 (CAA55966), NtGAD1 (AAC24195), NtGAD2 (AAC39483), NtGAD3 (AAK18620), PhGAD (AAA33709), OsGAD (BAB32868), DmGAD (AAF57903), EcGAD-alpha (AAA23833), EcGAD-beta (BAB35521), GmACA (AAG28435), ZmECA (AAF73985), RnACA (AAA81005), and CeECA (CAB07263). Dm, D. melanogaster; Ec, E. coli; Rn, Rattus norvegicus; Ce, Caenorhabditis elegans; Nt, Nicotiana tabacum; Zm, Z. mays; Hv, Hordeum vulgare; Os, Oryza sativa; Le, Lycopersicon esculentum; Ps, Petroselinum crispum; Ph, Petunia hybrida; Gm, Glycine max.


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  		Fig. 5.   Location of genes encoding CBPs on the Arabidopsis chromosomes. The numbers correspond to numbers in Tables I and II. If more than two genes are located close to each other, the location from left to right corresponds to numbers from top to bottom.


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  		Fig. 6.   Schematic diagram of three CBP families. A, ACBP60s; B, CNGCs; C, EICBPs. The calmodulin-binding domain of each protein, shown by a black box, is aligned to other members of the family. Arrowheads indicate the location of introns along the length of each member.

The seven Arabidopsis ACBP60s were aligned with CBP60s from other plants to build the tree (Fig. 4A). They show 55-73% identities and fall into two major subgroups. The tobacco, maize, and one ACBP60 (At2g18750) fall outside of these groups. The phylogenetic tree of CNGCs divided them into four subgroups (Fig. 4B) with sequence similarity ranging from 55 to 85%, which is consistent with the earlier analysis (41). Group IV, which consists of four AtCNGCs, forms a separate distant group with rice CNGC (21-80% identity between members), and the other 16 AtCNGCs form into three closely related subgroups (group I, II, and III) (Fig. 4B). The Drosophila melanogaster CNGC was used as an outgroup in analyzing the relationship between the 20 Arabidopsis CNGCs. Interestingly, CNGC1 (At5g53130) and CNGC2 (At5g15410), which differ in their affinity to AtCaM isoforms (12), fall into two separate groups. In the CNGC family, there are five genes arranged in two tandem repeats and the coding regions are separated by ~1 kb. One repeat is on chromosome 3 with two genes (group II; At3g17690, 15s; and At3g17700, 15t in Fig. 5) and the other is on chromosome 2 with three genes (group I; At2 g46430, 15c; At2g46450, 15r; and At2g46440, 15 h in Fig. 5).

The EICBP family consists of six members, and their phylogenetic tree classifies them into two major groups (Fig. 4C). Interestingly, the EICBPs show significant sequence identity at their N and C termini and are diverged in the middle region (Fig. 6). The EICBP family contains two putative DNA-binding domains at the N terminus and an acidic domain at the C terminus (29). The EICBP members show 40-80% sequence identity with the N terminus parsley DNA-binding factor, CG-1 protein, (42) and 25-55% sequence similarity with the partial C terminus of the ethylene-induced protein (ER66) protein from tomato (43).

Of the five GADs, At3g17760 falls into a distinct group and is also separated from other plant GADs (Fig. 4D). The other four GADs fall into a group with two genes arranged in a tandem repeat (separated by ~2 kb) on chromosome 2 (At2g02010, 21c; and At2g02000, 21e in Fig. 5).

Arabidopsis Ca2+/ATPases form two distinct classes (Fig. 4E), ACA and ECA with the exception of At1g10130, an ECA with 34 introns. The recently identified CaM-regulated member of ECA, Zea mays ECA (44), also grouped with non-CaM binding AtECAs. Although the CaM-binding property is not yet determined, the C terminus of At1g10130 shows similarity to Z. mays ECA and forms a separate branch (Fig. 4E). In contrast to GADs and CNGCs, some members of which are locally duplicated and arranged tandemly (21 and 15, respectively, in Fig. 5), the members of the ACA family are dispersed on all chromosomes (22 in Fig. 5). The six ACA Arabidopsis genes at the bottom of the tree (group II, At3g21180, At4g29900, At5g57110, At3g22910, At3g63380, and At5g53010 with 31, 33, 33, 0, 0, and 31, introns, respectively) form into one group. Above this, five ACA genes containing six introns (group I, At1g27770, At2g22960, At4g37640, At2g41560, and At3g57330) form another group. Interestingly, the ACAs in the group II reside on plasma membrane (36), whereas the group I ACAs reside on endomembrane systems (33-35).

Calmodulin-binding Proteins Contain Various Putative Domains-- Analysis of newly identified CBPs using domain prediction programs has resulted in identification of various putative domains that provide clues to their function. The results are shown in Fig. 6 and Fig. 7. Cyclic nucleotide monophosphate binding domain is present in all 20 CNGCs (Fig. 6). Based on Arabidopsis MIPS database, CNGCs contain putative signal peptide sequences targeting to membrane (11 CNGCs), chloroplast (4 CNGCs), mitochondria (3 CNGCs), and secretary pathway (2 CNGCs). Some CBPs contain putative domains found in transcriptional factors. Two putative DNA-binding domains (one is similar to parsley CG1 DNA binding domain and other domain is similar to human Ig-like, plexins, transcription factors, IPT/TIG) are present in EICBPs (Fig. 6). Further, nuclear localization motifs have been detected in EICBPs (Fig. 6) and in a hypothetical protein (1 in Table I and Fig. 7). Tetratricopeptide repeats and ankyrin repeats that are implicated in protein-protein interaction are present in several CBPs including EICBPs (Fig. 6) and APCBPs (data not shown). Coiled-coil regions that aid in dimerization are present in EICBP. A double-stranded beta -helix domain in pirin-like protein is involved in carbohydrate binding and protein-protein interaction. A putative RING domain, present in the hypothetical protein (13 in Table II and Fig. 7), is found in diverse proteins including ubiquitin-protein isopeptide ligases (45). Transmemembrane domains are found in some CBPs (PSI-N subunit, apyrase, MDR-like, Ca2+-ATPases, and CNGCs) (Figs. 6 and 7). Low density lipoprotein receptor domain A is present in PKC substrate-like protein (Fig. 7).


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  		Fig. 7.   Domain organization of newly identified CBPs. The numbers on left refer to Tables I and II. Only one representative member of families 13 and 17 is shown. NLS, nuclear localization sequence; LDLa, low density lipoprotein receptor domain class A; TM, transmembrane; RING, Really Interesting New Gene domain; DSBH, double-stranded beta -helix domain. Interruption in protein 1 is denoted by a double slash (//).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Prior to this report, only 10 (31 including their family members) distinct CBPs were reported in Arabidopsis (1). In this report we identified an additional 17 new CBPs, 7 from our screening and another 10 from database searches using other known plant and animal CBPs. Of 27 CBPs, 16 CBPs have two or more paralogs. Including all paralogs, there are ~100 CBPs in Arabidopsis. Of 27 CBPs, 13 are specific to plants and not found in animals. The Arabidopsis genome sequencing project has revealed that one third of the genes (~8000) do not have a homolog in animals (11). In this report, we identified some of these hypothetical proteins as CBPs. In addition, we also identified some previously characterized proteins in plants and animals as CBPs.

Plants Have a Unique Set of CBPs-- Thirteen CBPs including 4 hypothetical proteins, auxin-induced proteins, photosystem I-N, PP7, ACBP60s, EICBPs, APCBPs, CaM-binding heat shock proteins, GADs, and TGAs are found to be specific to plants (Tables I and II). Although members of EICBP show sequence similarity to some regions of IPT/TIG domain-containing proteins from humans and Drosophila (e.g. BAA74932 protein from humans, E value 3 × 10-15), the CaM binding property of these animal proteins has not been shown. A homolog of plant GAD is present in animals and E. coli, but it lacks a CBD (31). Although plant and animal GADs convert glutamic acid into gamma -aminobutyric acid (GABA), in animals GABA is involved in different cellular activities as an inhibitory neurotransmitter whereas in plants GABA acts as a stress adopter chaperonin, and plant GAD activity is controlled by Ca2+/CaM (9, 30). In tomato, an alternatively spliced form of diacyl glycerol kinase contains a CBD. However, its normal isoform did not bind CaM (46). We searched the Arabidopsis database for homologs to tomato isoforms and found six members of diacyl glycerol kinases, but none of them contain a CBD. Further, two CBPs, Ca2+/CaM-dependent protein kinase from lily and tobacco (47, 48) and CaM-dependent protein kinase II from apple (49), have not been found in Arabidopsis. Calmodulin has been shown to stimulate the activity of superoxide dismutase, NAD kinase (8), phosphoprotein phosphatase 2B (50, 51), aspartate kinase (52), and phospholipase A2 (53). However, the genes encoding these proteins have not been cloned. When we used sequences of animal homologs of these CBPs as queries, we identified several members for some of these proteins in the Arabidopsis database. Because the CBD is not mapped in these proteins, we cannot speculate as to which member of a family possesses a CBD; therefore, they are not included in the present analysis. In Arabidopsis, 59 SAURs and many multidrug-resistant (MDR)-like proteins are present (11). However, only five SAURs have a conserved CBD, and the CBD is not mapped in MDR; hence, we did not include the other SAURs and MDR-like proteins in the present analysis. The animal proteins showing homology to plant CBPs such as PKC substrate-like, ATPase, chaperonin 10, glyoxalase, apyrase, 60 S ribosomal L19, and pirin-like have been found in the database. However, the CaM-binding property of these animal proteins has not been reported.

Based on literature and database searches for CBPs, 29 animal CBPs have no homologs in Arabidopsis, suggesting that they are unique to animals (Table III). However, some animal CBPs show high sequence similarity to small regions of Arabidopsis proteins because of the presence of specific domains but lack CBD region. Most CBPs from animals are involved in visual and neural specific Ca2+/CaM signal transduction cascades (Table III). Furthermore, although homologs for some of the plant CBPs are present in animals (Tables I and II), only a few of them bind CaM in a Ca2+-dependent manner (Tables I and II). These are kinesin C, MDR-like, CNGCs, Ca2+/ATPases, HSP70, PPI, and EF-1alpha . Interestingly, despite the presence of a common set of CBPs in both plants and animals, their recruitment in Ca2+/CaM modulated signal transduction networks is entirely different. For example, Arabidopsis kinesin-like CaM-binding protein (5 in Table I) is localized to the preprophase band and phragmoplast structures and is involved in trichome morphogenesis, all of which are specific to plants (54, 55). The function(s) of kinesin C, a CaM-binding kinesin, in sea urchin has not been studied ("Non-plant homolog" column in Table I). Although CNGCs conduct nonselective metal ions across the membranes in plants and animals, they are involved in vision and olfactory sensory signal transduction systems in animals (56) whereas they are involved in various biotic and metal stress responses in plants (57, 58). Furthermore, the sequence and location of the CBD in CNGCs is different in plants (C terminus) and animals (N terminus). Although the CBD sequence and its location are different in plants (N terminus) and animals (C terminus) in Ca2+/ATPases, they perform a similar function in regulating [Ca2+]cyt levels by pumping out Ca2+ from the cytoplasm. However, they may be activated by different physiological conditions. These observations suggest that plants contain a unique set of CBPs that mediate cellular activities specific to plant growth and development.

                              
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Table III
Characterized animal CBPs that are not found in the Arabidopsis genome database

Plants Are Unique in Containing Multiple Genes Encoding Paralogs of CBPs-- Plant CBPs, unlike animals, possess multiple paralogs. Arabidopsis contains 16 CBP families having 2-20 members comprising a total of 89 CBPs (Table II). Although there are some common sets of CBPs found in plants and animals, plants contain more paralogs for CNGCs, pirin-like proteins, Ca2+-ATPases, EF-1alpha , and HSP70s. Members of a CBP gene family are highly conserved at their protein sequence level, and most likely all members of a family bind CaM in a Ca2+-dependent manner. This is evidenced as follows: (i) more than two members in a gene family were shown to bind CaM in a Ca2+-dependent manner and were isolated in our screening (Table II), (ii) the CBD region in members is highly conserved (Fig. 3) and forms a characteristic basic amphipathic alpha -helix in which basic and polar and hydrophobic residues segregate on opposite sides (data not shown), and (iii) members of each family are similar in size and exhibit similar domain and gene organization (Table II and Fig. 6).

The significance of multiple paralogs of CBPs in Arabidopsis is not known, but they are likely to be involved in fine-tuning cellular activities that are regulated by Ca2+/CaM. First, members may be functionally distinct and possess specific biochemical and physiological properties. This is evident in some cases where members of a family exhibit differential enzyme activity and regulation and affinities for CaM isoforms. For example, CNGC1 and CNGC2 showed differential affinity with CaM isoforms (12) and GAD1 and GAD2 isoforms possess differential enzyme activity (31). The CNGC2 is involved in disease resistance (57), whereas CNGC1 regulates Pb2+ entry into the plants (59). Second, members of each family are likely to be under the control of distinct regulatory elements. This is evident by the differential expression and localization patterns of members of CBPs in Arabidopsis. For example, the ACA family members ACA1, -2, and -4 are differentially expressed (Table II) and located on different endomembranes (33-35, 37), whereas ACA8, -9, and -10 are located on the plasma membrane (36), suggesting that the members of the ACA gene family may likely be regulated at the transcriptional and the post-translational levels. Finally, some members of a gene family may show functional redundancy, e.g. certain ACA members (ACA8, -9, and -10) are all located on the plasma membrane (37) and are likely to perform the same function in Ca2+ homeostasis. These genes contain a similar number of introns and show a close relationship although they are distributed on different chromosomes (Fig. 5). These genes may have evolved by gene duplication and shuffling events during evolution. During this shuffling process, some intronless Ca2+/ATPase paralogs (At3g63380 and At3g22910 from the plasma membrane pumps) have been generated and, in other cases, short gene fragments (pseudogenes) were integrated elsewhere in the chromosomes (e.g. GAD, At3g17720; and PPI, At4g34870).

Possible Physiological Roles of Newly Identified Arabidopsis CBPs-- Several hypothetical CBPs have been isolated from auxin and elicitor treated tissues. A hypothetical protein (13 in Table II and Fig. 7) contains a putative RING domain, which in some cases promotes polyubiquitination (45). Hence, it is likely that the hypothetical protein may be involved in post-translational modifications. Because a hypothetical protein (1 in Table I and Fig. 7) contains four putative nuclear localization sequences and was isolated from auxin-treated (17 independent clones) and elicitor-treated libraries (11 independent clones), it is likely to be involved in auxin signal transduction and plant defense. Three members of pollen-specific CBPs in Arabidopsis show sequence similarity to a maize pollen-specific CBP (15). Because Ca2+ plays a key role in pollen germination and tube growth and maize pollen-specific CBP is expressed specifically in pollen, these are likely to be involved in pollen development and function.

The photosystem I-N subunit is located in the luminal side of thylakaoid membranes in association with PSI (60). Transgenic plants lacking PSI-N subunit show inefficient electron flow between PSI and PSII resulting from partial impairment of electron transfer (50%) from plastocyanin to P700 (60). Identification of PSI-N subunit as a CBP suggests possible regulation of electron flow in photosynthesis by Ca2+/CaM. In animals the proteins containing the low density lipoprotein receptor domain class A domain have been shown to bind to specific lipoproteins (61), suggesting that PKC substrate-like protein may interact with lipoproteins.

Using NFI/CTF1 as a bait protein, human nuclear localized pirin was isolated from a HeLa cDNA library (62). NFI/CTF1 regulates transcription of a number of cellular promoters containing NFI/CTF1 binding sites (I/CCAAT). However, plant homologs to animal NFI/CTF1 have not been characterized to date. Nevertheless, identification of an evolutionarily conserved pirin-like protein as a CBP in plants would help understand the regulation of pirin-like protein by Ca2+/CaM. Three distinct CBPs containing three to six members involved in the regulation of heat stress were identified in Arabidopsis and other plants (25, 63, 64). These include ACBP60s, CB-HSPs, and HSP70s (Table II). The exact role of these three CBPs in heat shock stress has not been studied. Furthermore, ACBP60s and CB-HSPs do not contain any distinct domains, but HSP70s possess ATPase activity (65). The transcripts of TCBP60s (homologs to ACBP60s) are repressed (25), whereas CB-HSPs (64) and HSP70s (65) are increased in response to heat stress. Recent reports indicate that heat shock elevates [Ca2+]cyt levels (66), which in turn may activate CaM and other Ca2+-binding proteins to regulate and prevent the cellular machinery from thermal denaturation.

Members of EICBPs, with putative domains involved in transcriptional activity and EICBP.a, whose transcripts are induced by ethylene, might be involved in ethylene signal transduction processes regulated through Ca2+/CaM messenger system. Recent studies suggest that the increased [Ca2+]cyt levels are associated with ethylene-regulated cellular activities such as senescence and programmed cell death (67-70). Arabidopsis has 20 highly conserved CNGCs (Table II; Fig. 6). Some plant CNGCs, as in animals, are involved in permeability of Ca2+ and other metal transport. This is evident by: (i) influx of Ca2+ ions into cytoplasm in embryonic kidney cells overexpressing AtCNGC2 (71) and (ii) the fact that Arabidopsis contains only two Ca2+ channels as compared with 38 in human (72). In animals, CNGCs are involved in light, visual, and olfactory signal transduction cascades (56). In contrast, members of CNGCs in plants are involved in metal tolerance (27, 58, 59), disease resistance (57), and likely in Ca2+ homeostasis (71). These studies suggest that CNGCs in plants and animals perform distinct physiological roles. It is tempting to speculate that one or more members of CNGCs in plants may function in light signal perception and transduction because of their function in light perception and vision in animals.

Mode of Ca2+/CaM Regulation of Calmodulin-binding Proteins-- The activity of CaM depends on the concentration of free [Ca2+]cyt levels, which transiently fluctuates between ~100 nM (resting) and 10 µM (elevated) in response to a variety of stimuli (1). Increased levels of free [Ca2+]cyt activate Ca2+sensors, including CaM. The Ca2+/CaM stimulates (GADs, ACAs, glyoxalase, TGA3, and apyrase) or inhibits (KCBP, PP7, EF1-alpha , and HSP70) reversibly the activity of a variety of CBPs, suggesting positive and negative modes of regulation by elevated [Ca2+]cyt in mediating cellular processes. Restoration of [Ca2+]cyt levels is a necessary step to prevent the toxic effects of high levels of [Ca2+]cyt. This is achieved by high affinity Ca2+ pumps (Ca2+-ATPases) through the activation of Ca2+/CaM (33, 73). Further, phosphorylation of Ser45 in ACA2 by a Ca2+-dependent protein kinase (another plant-specific Ca2+ sensor) disrupts CaM binding and inactivates the ACA2 pump (74), suggesting that two Ca2+ binding sensors coordinately maintain the magnitude and duration of [Ca2+]cyt levels.

Interestingly, actin-based motors, myosins, contain consensus IQ-motifs (IQXXXRGXXXR) to which CaM binds in the absence of Ca2+ and dissociates by increased level of [Ca2+]cyt and thereby inhibits myosin motility (75). In Arabidopsis, 17 myosins containing three to six IQ motifs belonging to class VII and XI have been identified (75). The presence of Ca2+-independent CaM binding motifs (IQ) suggests that Arabidopsis myosins are negatively regulated in the presence of Ca2+/CaM.

Plant CBPs Are Involved in Controlling Many Diverse Cellular Processes-- The structural organization of CBPs suggests their possible involvement in diverse molecular, biochemical, and cellular processes in plants. These include gene regulation (TGA3, EICBP, and pirin-like), translational (EF1), posttranslational modifications (PP7), cell division and trichome morphogenesis (KCBP), cell elongation (SAURs), cytoskeletal organization (EF-1alpha , KCBP, myosins), and intracellular transport (KCBP, myosins), ion transport (CNGC1), disease resistance (CNGC2 and NAD kinase), abiotic stress tolerance (ACA4 and CNGC2), thermal stress tolerance (ACBP60s, CB-HSPs, HSP70s), salt tolerance (glyoxalase), light responses and ATP transport (apyrase), Ca2+ homeostasis (ACAs and CNGCs), nitrogen metabolism and growth and development (GAD), pollen development and/or function (APCBPs), fatty acid metabolism (PKC-substrate-like), photosynthesis (PSI-N subunit), cytoplasmic streaming and transport (myosins), and hormonal regulation (auxin, SAURs; ethylene, EICBPs). However, the precise role of many of these CBPs in Ca2+ signaling is not known. For example, although EICBP.a transcript is inducible by ethylene and EICBP family members contain motifs characteristic of transcriptional activators, the target genes in ethylene signal transduction network are unknown. In addition, the presence of multiple members for several CBP gene families raises questions related to their functional significance in mediating Ca2+ signal transduction networks in plants.

Our screening of several expression libraries coupled with a detailed database search resulted in the identification of 100 CBPs in the Arabidopsis genome. It is likely that there are more CBPs in Arabidopsis. Identification of such a large number of genes supports their involvement in diverse cellular processes regulated by Ca2+. Screening of additional libraries prepared from tissues exposed to other stresses or treated with other hormones with CaM isoforms including AtCaM8 and AtCaM9, is likely to yield additional CBPs.

Using knockout/loss-of-function and gain-of-function mutants, it should be possible to study the function of individual members. Knockout mutants for most of the CBPs are now available (Table IV, provided as supplemental information and available on-line) and can be used to dissect the function(s) of individual CBPs in Ca2+-signaling networks. Although functions of single-copy genes could be analyzed through knockout mutational screening approach, it will be necessary to develop double or triple mutants for the members of CBP families. However, such difficulty can be overcome with the use of the gain-of-function mutant approach. Using gain-of-function mutational screening, the functions of members of gene families such as bas1-d (76), yucca (77), and pap1-D (78), involved in brassinosteroid, auxin, and phenylpropanoid biosynthetic pathways, respectively, have been successfully determined.

    ACKNOWLEDGEMENTS

We thank Dr. J. R. Ecker (Salk Institute for Biological Studies, La Jolla, CA), Dr. E. M. Meyerowitz (California Institute of Technology, Pasadena, CA), Dr. P. B. Lindgren (North Carolina State University, Raleigh, NC), Dr. B. J. van der Zaal (Leiden University, Leiden, The Netherlands), and Dr. I. E. Somssich (Max Planck Institute, Cologne, Germany) for providing ethylene-treated, flower meristem, pathogen-treated, auxin-treated, and elicitor-treated cDNA expression libraries, respectively, and Dr. Raymond E. Zielinski (University of Illinois, Urbana, IL) for CaM expression vectors. We thank Dr. Irene Day and Dr. Maxim Golovkin for critical reading of the manuscript.

    Note Added in Proof

Recently, Zhu et al. (127) screened a yeast proteome microarray with biotinylated CaM and identified several new CBPs. We searched the Arabidopsis genome for homologs of yeast CBPs. Out of 37 yeast CBPs, 17 have homologs in Arabidopsis (E value ranges from 6 × 10-4 to 5 × 10-154). The Arabidopsis gene ID along with the yeast CBP (in parentheses) are provided here: At5g09650 (YBR01C ipp1), At5g63840 (YBR229C rot2), At4g30600 (YDR292C srp101), At5g14800 (YER023W pro3), At4g17380 (YFL003C msh4), At5g23540 (YFR004W rpn11), At1g20370 (YGL063W pus1), At2g28360 (YGL229C sap4), At3g49910 (YGL034W rpl26b), At5g54770 (YGR144W thi4), At3g14290 (YGR253C pup2), At5g24260 (YHR028C dap2), At1g76680 (YHR179W oye2), At2g15430 (YIL021W rpb3), At5g50160 (YLL051C fre6), At3g12790 (YNL202W sps19), At1g56170 (YOR358W hap5). Interaction of these Arabidopsis homologs with CaM needs to be confirmed experimentally.

    FOOTNOTES

* This work was supported in part by grants from the National Science Foundation and the National Aeronautics and Space Administration (to A. S. N. R.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains Table IV.

Dagger These authors contributed equally to this work.

§ To whom correspondence should be addressed. Tel.: 970-491-5773; Fax: 970-491-0649; E-mail: reddy@lamar.colostate.edu.

Published, JBC Papers in Press, January 8, 2002, DOI 10.1074/jbc.M111626200

    ABBREVIATIONS

The abbreviations used are: [Ca2+]cyt, cytoplasmic free calcium; CaM, calmodulin; CBP, calmodulin-binding protein; CBD, calmodulin-binding domain; IPTG, isopropyl-beta -D-thiogalactopyranoside; EST, expressed sequence tag; AtCaM, A. thaliana calmodulin; PKC, protein kinase C; PS, photosystem; GABA, gamma -aminobutyric acid; GAD, glutamate decarboxylase; ACA, autoinhibited Ca2+/ATPase; ECA, endoplasmic reticulum-type Ca2+/ATPase; APCBP, Arabidopsis pollen-specific calmodulin-binding protein; EICBP, ethylene-induced calmodulin-binding protein; KCBP, kinesin-like calmodulin-binding protein; TGA, a member of bZIP transcription factor; CNGC, cyclic nucleotide gated channel; AtCNGC, A. thaliana cyclic nucleotide gated channel; SAUR, small auxin up-regulated-like protein; PPI, peptidylprolyl isomerase; MDR, multidrug-resistant; HSP, heat shock protein; CB-HSP, calmodulin-binding heat shock protein; EF-1alpha , elongation factor-1alpha .

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1. Reddy, A. S. N. (2001) Plant Sci. 160, 381-404[Medline] [Order article via Infotrieve]
2. Knight, H. (2000) Int. Rev. Cytol. 195, 269-324[Medline] [Order article via Infotrieve]
3. Trewavas, A. J., and Malho, R. (1998) Curr. Opin. Plant Biol. 1, 428-433[CrossRef][Medline] [Order article via Infotrieve]
4. Sanders, D., Brownlee, C., and Harper, J. (1999) Plant Cell 11, 691-706[Free Full Text]
5. Reddy, A. S. N., and Reddy, V. S. (2001) in Handbook of Plant and Crop Physiology (Pessarakali, M., ed) , pp. 697-733, Marcel Dekker, New York
6. Hepler, P. K. (1997) Trends Plant Sci. 2, 79-80[CrossRef]
7. Bibikova, T. N., Zhigilei, A., and Gilroy, S. (1997) Planta 203, 495-505[CrossRef][Medline] [Order article via Infotrieve]
8. Zielinski, R. E. (1998) Annu. Rev. Plant Physiol. Plant Mol. Biol. 49, 697-725[CrossRef][Medline] [Order article via Infotrieve]
9. Snedden, W. A., and Fromm, H. (2001) New Phytol. 151, 35-66[CrossRef]
10. Rhoads, A. R., and Friedberg, F. (1997) FASEB J. 11, 331-340[Abstract]
11. The Arabidopsis Genome Initiative. (2000) Nature 408, 796-815[CrossRef][Medline] [Order article via Infotrieve]
12. Kohler, C., and Neuhaus, G. (2000) FEBS Lett. 4710, 133-136
13. Fromm, H., and Chua, N.-H. (1992) Plant Mol. Biol. Rep. 10, 199-206
14. Liao, B., and Zielinski, R. E. (1995) Methods Cell Biol. 49, 487-500[Medline] [Order article via Infotrieve]
15. Safadi, F., Reddy, V. S., and Reddy, A. S. N. (2000) J. Biol. Chem. 275, 35457-35470[Abstract/Free Full Text]
16. Neuteboom, L. W., Ng, J. M. Y., Kuyper, M., Clijdesdale, O. R., Hooykaas, P. J. J., and van der Zaal, B. J. (1999) Plant Mol. Biol. 39, 273-287[CrossRef][Medline] [Order article via Infotrieve]
17. Kieber, J. J., Rothenberg, M., Roman, G., Feldmann, K. A., and Ecker, J. R. (1993) Cell 72, 427-441[CrossRef][Medline] [Order article via Infotrieve]
18. Korfhage, U., Trezzini, G. F., Meier, I., Hahlbrock, K., and Somssich, I. E. (1994) Plant Cell 6, 695-708[Abstract/Free Full Text]
19. Jakobek, J. L., Smith-Becker, J. A., and Lindgren, P. B. (1999) Mol. Plant-Microbe Interact. 12, 712-719[Medline] [Order article via Infotrieve]
20. Reddy, A. S. N., Safadi, F., Narasimhulu, S. B., Golovkin, M., and Hu, X. (1996) J. Biol. Chem. 271, 7052-7060[Abstract/Free Full Text]
21. Song, H., Golovkin, M., Reddy, A. S. N., and Endow, S. A. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 322-327[Abstract/Free Full Text]
22. Reddy, V. S., and Reddy, A. S. N. (1999) Bioinformatics 15, 1055-1057[Free Full Text]
23. Narasimhulu, S. B., and Reddy, A. S. N. (1998) Plant Cell 10, 957-965[Abstract/Free Full Text]
24. Reddy, V. S., Safadi, F., Zielinski, R. E., and Reddy, A. S. N. (1999) J. Biol. Chem. 274, 31727-31733[Abstract/Free Full Text]
25. Lu, Y., and Harrington, H. M. (1994) Plant Physiol. Biochem. 32, 413-422
26. Schuurink, R. C., Shartzer, S. F., Fath, A., and Jones, R. L. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 1944-1949[Abstract/Free Full Text]
27. Arazi, T., Sunkar, R., Kaplan, B., and Fromm, H. (1999) Plant J. 20, 171-182[CrossRef][Medline] [Order article via Infotrieve]
28. Kohler, C., Merkle, T., and Neuhaus, G. (1999) Plant J. 18, 97-104[CrossRef][Medline] [Order article via Infotrieve]
29. Reddy, A. S. N., Reddy, V. S., and Golovkin, M. (2000) Biochem. Biophys. Res. Commun. 279, 762-769[CrossRef][Medline] [Order article via Infotrieve]
30. Baum, G., Lev-Yadun, S., Fridmann, Y., Arazi, T., Katsnelson, H., Zik, M., and Fromm, H. (1996) EMBO J. 15, 2988-2996[Medline] [Order article via Infotrieve]
31. Zik, M., Arazi, T., Snedden, W. A., and Fromm, H. (1998) Plant Mol. Biol. 37, 967-975[CrossRef][Medline] [Order article via Infotrieve]
32. Sonnemann, J., Bauerle, A., Winckler, T., and Mutzel, R. (1991) J. Biol. Chem. 266, 23091-23096[Abstract/Free Full Text]
33. Geisler, M., Axelsen, K. B., Harper, J. F., and Palmgren, M. G. (2000) Biochim. Biophys. Acta 1465, 52-78[Medline] [Order article via Infotrieve]
34. Huang, L., Berkelman, T., Franklin, A. E., and Hoffman, N. E. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 10066-10070[Abstract/Free Full Text]
35. Harper, J. F., Hong, B., Hwang, I., Guo, H. Q., Stoddard, R., Huang, J. F., Palmgren, M. G., and Sze, H. (1998) J. Biol. Chem. 273, 1099-1106[Abstract/Free Full Text]
36. Bonza, M. C., Morandini, P., Luoni, L., Geisler, M., Palmgren, M. G., and De Michelis, M. I. (2000) Plant Physiol. 123, 1495-1506[Abstract/Free Full Text]
37. Vucich, V. A., and Gasser, C. S. (1996) Mol. Gen. Genet. 252, 510-517[Medline] [Order article via Infotrieve]
38. Miao, Z. H., Liu, X., and Lam, E. (1994) Plant Mol. Biol. 25, 1-11[CrossRef][Medline] [Order article via Infotrieve]
39. Szymanski, D. B., Liao, B., and Zielinski, R. E. (1996) Plant Cell 8, 1069-1077[Abstract]
40. Swofford, D. L. (1998) PAUP 4.0 , Sinauer Associates, Sunderland, MA
41. Maser, P., Thomine, S., Schroeder, J. I., Ward, J. M., Hirschi, K., Sze, H., Talke, I. N., Amtmann, A., Maathuis, F. J., Sanders, D., Harper, J. F., Tchieu, J., Gribskov, M., Persans, M. W., Salt, D. E., Kim, S. A., and Guerinot, M. L. (2001) Plant Physiol. 126, 1646-1667[Abstract/Free Full Text]
42. da Costa e Silva, O. (1994) Plant Mol. Biol. 25, 921-924[CrossRef][Medline] [Order article via Infotrieve]
43. Zegzouti, H., Jones, B., Frasse, P., Marty, C., Maitre, B., Latch, A., Pech, J. C., and Bouzayen, M. (1999) Plant J. 18, 589-600[CrossRef][Medline] [Order article via Infotrieve]
44. Subbaiah, C. C., and Sachs, M. M. (2000) J. Biol. Chem. 275, 21678-21687[Abstract/Free Full Text]
45. Freemont, P. S. (2000) Curr. Biol. 10, R84-R87[CrossRef][Medline] [Order article via Infotrieve]
46. Snedden, W. A., and Blumwald, E. (2000) Plant J. 24, 317-326[CrossRef][Medline] [Order article via Infotrieve]
47. Patil, S., Takezawa, D., and Poovaiah, B. W. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 4897-4901[Abstract/Free Full Text]
48. Liu, Z., Xia, M., and Poovaiah, B. W. (1998) Plant Mol. Biol. 38, 889-897[CrossRef][Medline] [Order article via Infotrieve]
49. Watillon, B., Kettmann, R., Boxus, P., and Burny, A. (1995) Plant Physiol. 108, 847-848[CrossRef][Medline] [Order article via Infotrieve]
50. Allen, G. J., and Sanders, D. (1995) Plant Cell 7, 1473-1483[Abstract]
51. Luan, S., Li, W. W., Rusnak, F., S. M., A., and Schreiber, S. L. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 2202-2206[Abstract/Free Full Text]
52. Kochhar, S., Kochhar, V. K., and Sane, P. V. (1986) Biochim. Biophys. Acta 880, 220-225
53. Jung, K. M., and Kim, D. K. (2000) Plant Physiol. 123, 1057-1067[Abstract/Free Full Text]
54. Oppenheimer, D. G., Pollock, M. A., Vacik, J., Szymanski, D. B., Ericson, B., Feldmann, K., and Marks, D. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 6261-6266[Abstract/Free Full Text]
55. Reddy, A. S. N., and Day, I. S. (2000) Trends Plant Sci. 5, 503-505[CrossRef][Medline] [Order article via Infotrieve]
56. Broillet, F. S. (1999) Ann. N. Y. Acad. Sci. 868, 730-740[CrossRef][Medline] [Order article via Infotrieve]
57. Clough, S. J., Fengler, K. A., Yu, I. C., Lippok, B., Smith, R. K. J., and Bent, A. F. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 9323-9328[Abstract/Free Full Text]
58. Arazi, T., Kaplan, B., and Fromm, H. (2000) Plant Mol. Biol. 42, 591-601[CrossRef][Medline] [Order article via Infotrieve]
59. Sunkar, R., Kaplan, B., Bouche, N., Arazi, T., Dolev, D., Talke, I. N., Maathuis, F. J., Sanders, D., Bouchez, D., and Fromm, H. (2000) Plant J. 24, 533-542[CrossRef][Medline] [Order article via Infotrieve]
60. Haldrup, A., Naver, H., and Scheller, H. V. (1999) Plant J. 17, 689-698[CrossRef][Medline] [Order article via Infotrieve]
61. Hussain, M. M., Strickland, D. K., and Bakillah, A. (1999) Annu. Rev. Nutr. 19, 141-172[CrossRef][Medline] [Order article via Infotrieve]
62. Wendler, W. M., Kremmer, E., Forster, R., and Winnacker, E. L. (1997) J. Biol. Chem. 272, 8482-8489[Abstract/Free Full Text]
63. Reddy, A. S. N., Takezawa, D., Fromm, H., and Poovaiah, B. W. (1993) Plant Sci. 94, 109-117
64. Lu, Y.-T., Dharmasiri, M. A. N., and Harrington, H. M. (1995) Plant Physiol. 108, 1197-1202[Abstract]
65. Sun, X. T., Li, B., Zhou, G. M., Tang, W. Q., Bai, J., Sun, D. Y., and Zhou, R. G. (2000) Plant Cell Physiol. 41, 804-810[Medline] [Order article via Infotrieve]
66. Gong, M., van der Luit, A. H., Knight, M. R., and Trewavas, A. J. (1998) Plant Physiol. 116, 429-437[Abstract/Free Full Text]
67. Xu, Y., and Hanson, M. R. (2000) Plant Physiol. 122, 1323-1333[Abstract/Free Full Text]
68. Levine, A., Pennell, R. I., Alvarez, M. E., Palmer, R., and Lamb, C. (1996) Curr. Biol. 6, 427-437[CrossRef][Medline] [Order article via Infotrieve]
69. Xu, H., and Heath, M. C. (1998) Plant Cell 10, 585-598[Abstract/Free Full Text]
70. Groover, A., and Jones, A. M. (1999) Plant Physiol. 119, 375-384[Abstract/Free Full Text]
71. Leng, Q., Mercier, R. W., Yao, W., and Berkowitz, G. A. (1999) Plant Physiol. 121, 753-761[Abstract/Free Full Text]
72. Venter, J. C., Adams, M. D., Myers, E. W., Li, P. W., Mural, R. J., Sutton, G. G., Smith, H. O., Yandell, M., Evans, C. A., Holt, R. A., Gocayne, J. D., Amanatides, P., Ballew, R. M., Huson, D. H., Wortman, J. R., Zhang, Q., Kodira, C. D., Zheng, X. H., Chen, L., Skupski, M., Subramanian, G., Thomas, P. D., Zhang, J., Gabor Miklos, G. L., Nelson, C., et al.. (2001) Science 291, 1304-1351[Abstract/Free Full Text]
73. Hong, B., Ichida, A., Wang, Y., Gens, J. S., Pickard, B. G., and Harper, J. F. (1999) Plant Physiol. 119, 1165-1176[Abstract/Free Full Text]
74. Hwang, I., Sze, H., and Harper, J. F. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 6224-6229[Abstract/Free Full Text]
75. Reddy, A. S. N., and Day, I. S. (2001) Genome Biol. 2, 24.1-24.17
76. Neff, M. M., Nguyen, S. M., Malancharuvil, E. J., Fujioka, S., Noguchi, T., Seto, H., Tsubuki, M., Honda, T., Takatsuto, S., Yoshida, S., and Chory, J. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 15316-15323[Abstract/Free Full Text]
77. Zhao, Y., Christensen, S. K., Fankhauser, C., Cashman, J. R., Cohen, J. D., Weigel, D., and Chory, J. (2001) Science 291, 306-309[Abstract/Free Full Text]
78. Borevitz, J. O., Xia, Y., Blount, J., Dixon, R. A., and Lamb, C. (2000) Plant Cell 12, 2383-2394[Abstract/Free Full Text]
79. Arendt, C. W., and Ostergaard, H. L. (1997) J. Biol. Chem. 272, 13117-13125[Abstract/Free Full Text]
80. Rogers, G. C., Hart, C. L., Wedman, K. P., and Scholey, J. M. (1999) J. Mol. Biol. 294, 1-8[CrossRef][Medline] [Order article via Infotrieve]
81. Liu, Y., Black, J., Kisiel, N., and Kulesz-Martin, M. F. (2000) Oncogene 19, 1579-1588[CrossRef][Medline] [Order article via Infotrieve]
82. Truscott, K. N., Hoj, P. B., and Scopes, R. K. (1994) Eur. J. Biochem. 222, 277-284[Medline] [Order article via Infotrieve]
83. Carninci, P., Shibata, Y., Hayatsu, N., Sugahara, Y., Shibata, K., Itoh, M., Konno, H., Okazaki, Y., Muramatsu, M., and Hayashizaki, Y. (2000) Genome Res. 10, 1617-1630[Abstract/Free Full Text]
84. Adams, M. D., Celniker, S. E., Holt, R. A., Evans, C. A., Gocayne, J. D., Amanatides, P. G., Scherer, S. E., Li, P. W., Hoskins, R. A., Galle, R. F., George, R. A., Lewis, S. E., Richards, S., Ashburner, M., Henderson, S. N., Sutton, G. G., Wortman, J. R., Yandell, M. D., Zhang, Q., Chen, L. X., Brandon, R. C., Rogers, Y. H., Blazej, R. G., Champe, M., Pfeiffer, B. D., et al.. (2000) Science 287, 2185-2195[Abstract/Free Full Text]
85. Schlemmer, S. R., Yang, C. H., and Sirotnak, F. M. (1995) J. Biol. Chem. 270, 11040-11042[Abstract/Free Full Text]
86. Buaboocha, T., Liao, B., and Zielinski, R. E. (2001) Planta 212, 774-781[CrossRef][Medline] [Order article via Infotrieve]
87. Yang, T., and Poovaiah, B. W. (2000) Biochem. Biophys. Res. Commun. 275, 601-607[CrossRef][Medline] [Order article via Infotrieve]
88. Kutuzov, M. A., Bennett, N., and Andreeva, A. V. (2001) Biochem. Biophys. Res. Commun. 289, 634-640[CrossRef][Medline] [Order article via Infotrieve]
89. Veena Reddy, V. S., and Sopory, S. K. (1999) Plant J. 17, 385-395[CrossRef][Medline] [Order article via Infotrieve]
90. Hsieh, H. L., Tong, C. G., Thomas, C., and Roux, S. J. (1996) Plant Mol. Biol. 30, 135-147[CrossRef][Medline] [Order article via Infotrieve]
91. Wang, W., Takezawa, D., and Poovaiah, B. W. (1996) Plant Mol. Biol. 31, 683-687[CrossRef][Medline] [Order article via Infotrieve]
92. Steinebrunner, I., Jeter, C., Song, C., and Roux, S. J. (2000) Plant Physiol. Biochem. 38, 913-922[CrossRef]
93. Andreeva, A. V., Kearns, A., Hawes, C. R., Evans, D. E., and Kutuzov, M. A. (1999) Physiol. Plant. 106, 209-223
94. Smith, D. K., Kassam, T., Singh, B., and Elliott, J. F. (1992) J. Bacteriol. 174, 5820-5826[Abstract/Free Full Text]
95. Keeton, T. P., and Shull, G. E. (1995) Biochem. J. 306, 779-785[Medline] [Order article via Infotrieve]
96. Benasutti, M., Harding, M. W., Fleming, M. A., DeCenzo, M. T., Lippke, J. A., Livingston, D. J., and Peattie, D. A. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 10974-10978[Abstract/Free Full Text]
97. Kaur, K. J., and Ruben, L. (1994) J. Biol. Chem. 269, 23045-23050[Abstract/Free Full Text]
98. Stevenson, M. A., and Calderwood, S. K. (1990) Mol. Cell. Biol. 10, 1234-1238[Abstract/Free Full Text]
99. Baum, G., Chen, Y., Arazi, T., Takatsuji, H., and Fromm, H. (1993) J. Biol. Chem. 268, 19610-19617[Abstract/Free Full Text]
100. Snedden, W. A., Koutsia, N., Baum, G., and Fromm, H. (1996) J. Biol. Chem. 271, 4148-4153[Abstract/Free Full Text]
101. Yang, T., and Poovaiah, B. W. (2000) J. Biol. Chem. 275, 3137-3143[Abstract/Free Full Text]
102. Phillips, A. M., Bull, A., and Kelly, L. E. (1992) Neuron 8, 631-642[CrossRef][Medline] [Order article via Infotrieve]
103. Wes, P. D., Chevesich, J., Jeromin, A., Rosenberg, C., Stetten, G., and Montell, C. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 9652-9656[Abstract/Free Full Text]
104. Cimler, B. M., Giebelhaus, D. H., Wakim, B. T., Storm, D. R., and Moon, R. T. (1987) J. Biol. Chem. 262, 12158-12163[Abstract/Free Full Text]
105. Kiledjian, M., and Dreyfuss, G. (1992) EMBO J. 11, 2655-2664[Medline] [Order article via Infotrieve]
106. Jenkins, C. M., Wolf, M. J., Mancuso, D. J., and Gross, R. W. (2001) J. Biol. Chem. 276, 7129-7135[Abstract/Free Full Text]
107. Bahler, M., Kroschewski, R., Stoffler, H. E., and Behrmann, T. (1994) J. Cell Biol. 126, 375-389[Abstract/Free Full Text]
108. Potier, M. C., Chelot, E., Pekarsky, Y., Gardiner, K., Rossier, J., and Turnell, W. G. (1995) Genomics 29, 562-570[CrossRef][Medline] [Order article via Infotrieve]
109. Haribabu, B., Hook, S. S., Selbert, M. A., Goldstein, E. G., Tomhave, E. D., Edelman, A. M., Snyderman, R., and Means, A. R. (1995) EMBO J. 14, 3679-3686[Medline] [Order article via Infotrieve]
110. Anderson, K. A., Means, R. L., Huang, Q. H., Kemp, B. E., Goldstein, E. G., Selbert, M. A., Edelman, A. M., Fremeau, R. T., and Means, A. R. (1998) J. Biol. Chem. 273, 31880-31889[Abstract/Free Full Text]
111. Redpath, N. T., Price, N. T., and Proud, C. G. (1996) J. Biol. Chem. 271, 17547-17554[Abstract/Free Full Text]
112. Kitani, T., Okuno, S., and Fujisawa, H. (1994) J. Biochem. (Tokyo) 115, 637-640[Abstract/Free Full Text]
113. Tokumitsu, H., Enslen, H., and Soderling, T. R. (1995) J. Biol. Chem. 270, 19320-19324[Abstract/Free Full Text]
114. Kitani, T., Okuno, S., and Fujisawa, H. (1997) J. Biochem. (Tokyo) 122, 243-250[Abstract/Free Full Text]
115. Deiss, L. P., Feinstein, E., Berissi, H., Cohen, O., and Kimchi, A. (1995) Genes Dev. 9, 15-30[Abstract/Free Full Text]
116. Xu, X. Z., Wes, P. D., Chen, H., Li, H. S., Yu, M., Morgan, S., Liu, Y., and Montell, C. (1998) J. Biol. Chem. 273, 31297-31307[Abstract/Free Full Text]
117. Shieh, B. H., and Niemeyer, B. (1995) Neuron 14, 201-210[CrossRef][Medline] [Order article via Infotrieve]
118. Montell, C., and Rubin, G. M. (1988) Cell 52, 757-772[CrossRef][Medline] [Order article via Infotrieve]
119. Lee, C. H. J., Della, N. G., Chew, C. E., and Zack, D. J. (1996) J. Neurosci. 16, 6784-6794[Abstract/Free Full Text]
120. Castets, F., Rakitina, T., Gaillard, S., Moqrich, A., Mattei, M. G., and Monneron, A. (2000) J. Biol. Chem. 275, 19970-19977[Abstract/Free Full Text]
121. Castets, F., Bartoli, M., Barnier, J. V., Baillat, G., Salin, P., Moqrich, A., Bourgeois, J. P., Denizot, F., Rougon, G., Calothy, G., and Monneron, A. (1996) J. Cell Biol. 134, 1051-1062[Abstract/Free Full Text]
122. Maekawa, S., Maekawa, M., Hattori, S., and Nakamura, S. (1993) J. Biol. Chem. 268, 13703-13709[Abstract/Free Full Text]
123. Michaeli, T., Bloom, T. J., Martins, T., Loughney, K., Ferguson, K., Riggs, M., Rodgers, L., Beavo, J. A., and Wigler, M. (1993) J. Biol. Chem. 268, 12925-12932[Abstract/Free Full Text]
124. Snyder, S. H., Jaffrey, S. R., and Zakhary, R. (1998) Brain Res. Rev. 26, 167-175[CrossRef][Medline] [Order article via Infotrieve]
125. Bertrand, B., Wakabayashi, S., Ikeda, T., Pouyssegur, J., and Shigekawa, M. (1994) J. Biol. Chem. 269, 13703-13709[Abstract/Free Full Text]
126. Buelt, M. K., Glidden, B. J., and Storm, D. R. (1994) J. Biol. Chem. 269, 29367-29370[Abstract/Free Full Text]
127. Zhu, H., Bilgin, M., Bangham, R., Hall, D., Casamayor, A., Bertone, P., Lan, N., Jansen, R., Bidlingmaier, S., Houfek, T., Mitchell, T., Miller, P., Dean, R. A., Gerstein, M., and Snyder, M. (2001) Science 293, 2101-2105[Abstract/Free Full Text]

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S. Luan, J. Kudla, M. Rodriguez-Concepcion, S. Yalovsky, and W. Gruissem
Calmodulins and Calcineurin B-like Proteins: Calcium Sensors for Specific Signal Response Coupling in Plants
PLANT CELL, May 1, 2002; 14(90001): S389 - 400.
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