BACKGROUND
the calcium ion  functions as a ubiquitous second messenger in plants, and participates in signal transduction in response to various environmental and developmental stimuli.
the transient ca2+ signal in the cytoplasm of plant cells is captured by a variety of ca2+ sensors including calmodulins, calmodulin-like proteins , calcium-dependent protein kinases , and calcineurin b-like  proteins .
these ca2+ sensors transfer the environmental and developmental signals to downstream components, causing a series of physiological and biochemical changes.
cbls, which harbor a core region consisting of four conserved ef hands, capture the ca2+ signal, and interact with cbl-interacting protein kinases  to phosphorylate their target proteins  <cit> .
the cipks with the naf motif for interaction with cbl belong to the snrk <dig> protein family, whose members have a specific ser/thr protein kinase domain  <cit> .
recently, cipk proteins were shown to phosphorylate their interacting cbl proteins at n-terminal conservative ser residue   and this proper phosphorylation of cbl was proved to be absolutely required for the in vivo activation of the akt <dig> k+ channel by cbl1-cipk <dig> and cbl9-cipk <dig> complexes in oocytes  <cit> .
the cbl and cipk gene families have been described in arabidopsis thaliana, oryza sativa, zea mays, and brassica napus .
in previous studies, cbl–cipk complexes were found to play very important roles in responses to external stimuli.
the salt overly sensitive  pathway was the first cbl-cipk network to be identified, and this cbl-cipk complex comprised cbl <dig>  and cipk <dig> .
the cbl <dig> protein was shown to interact with cipk <dig> and recruit it to the cytoplasmic membrane, where it activated the h+/na+  antiporter to enhance salt tolerance  <cit> .
this result indicated that the cbl-cipk complex functions in regulating sodium ion homeostasis.
meanwhile, v-atpase proteins were shown to directly interact with sos <dig> to rescue the salt-defective phenotype of sos <dig> mutant plants  <cit> .
in recent years, many cbls and cipks have been shown to promote stress tolerance by balancing the intracellular ion concentration in plants.
the sodium concentration was markedly lower in the arabidopsis cbl <dig> mutant than in control plants, in both normal and salt-stress conditions  <cit> .
the cbl10-overexpressing lines exhibited a k+-sensitive phenotype, like that of the akt <dig> mutant under low k+ conditions.
the interaction between cbl <dig> and akt <dig> was verified in bimolecular fluorescence complementation , yeast two-hybrid , and co-immunoprecipitation experiments, and was shown to impair akt1-mediated inward potassium  currents  <cit> .
atcipk <dig> could directly interact with cbl <dig> to promote k+ uptake under low k+ conditions by phosphorylating and activating the k+ channel  in arabidopsis and rice  <cit> .
however, a recent study suggested that cbl1/cbl <dig> might interact with akt <dig> independently of cipk <dig>  <cit> .
moreover, in response to low-k+ conditions, cipk <dig> was recruited to the tonoplast by cbl <dig> to regulate k+ homeostasis in arabidopsis  <cit> .
over-expression and mutant analyses of cbls and cipks have greatly enriched our understanding of their functions.
the cbl2/cbl <dig> double mutant showed defects in growth and ion homeostasis, possibly as a result of disrupted vacuolar h+-atpase activity  <cit> .
in another study, cbl <dig> and cbl <dig> were shown to affect seed development and morphology  <cit> .
the ectopic overexpression of zmcipk <dig> in the arabidopsi sos <dig> mutant enhanced the expression of sos <dig> and improved salt tolerance under saline conditions  <cit> .
atcipk <dig> was induced by abscisic acid  and stress conditions and established a molecular connection between stress- and aba-induced ca2+ signals  <cit> .
cbl <dig> and cipk <dig> were shown to positively regulate both immunity- and disease-associated programmed cell death in tomato and tobacco  <cit> .
the oscipk <dig> insertion mutant was hypersensitive to aba and stresses , and showed altered expression levels of several stress-responsive genes when it was exposed to these abiotic stresses during the seed germination and seedling stages  <cit> .
in a recent study, a few atcbl members were also found to target to another group of enzymes 5'-methylthioadenosine nucleosidases by yeast two-hybrid system, indicting an additional level of complexity on the cbl-mediated signaling networks  <cit> .
however, with respect to the larger families of cbls and cipks, few cbl-cipk complexes have been functionally characterized, especially in bread wheat , which is one of the most important food crops.
bread wheat has an allohexaploid genome with a size of approximately 17 gb, making it one of the largest and most complex plant genomes.
in recent years, the genome sequencing of t. aestivum, triticum urartu, and aegilops tauschii has promoted research on bread wheat, especially studies on agronomically important gene families related to disease resistance, abiotic stress endurance, and grain quality .
in our previous work, we demonstrated that transgenic tobacco overexpressing wheat tacipk <dig> and tacipk <dig> showed increased tolerance to salinity and drought stress, respectively  <cit> .
in this study, seven tacbl and  <dig> tacipk genes in wheat were identified and cloned by a genome-wide analysis combined with expressed sequence tag  assembly.
here, we present the results of analyses of expression profiles, the specific interactions between tacbls and tacipks, and the stress tolerance of transgenic arabidopsis plants expressing tacipk <dig>
RESULTS
identification of tacbl and tacipk gene families in wheat
the bread wheat genome is thought to have formed via fusion of three ancestral genomes, which are believed to be related to t. urartu, aegilops speltoides, and a. tauschii.
these fusion events are thought to have occurred several hundred thousand years ago  <cit> .
we searched the wheat genome and found  <dig> cbl  and  <dig> cipk  loci on wheat chromosomes .
the relative positions of these loci on chromosomes were identified on genetic maps .
the protein and dna sequences of t. urartu and a. tauschii  were retrieved from ncbi and all the cbl and cipk nucleotide sequences from these three species  were clustered against each other .
the closest matching genes were regarded as equivalent partners, as shown in table  <dig>  ten and nine genes of t. urartu and a. tauschii, respectively, had no equivalents in t. aestivum, implying the existence of potential un-identified tacbl/tacipk genes.
additionally, tacipk15-a, tacipk26-a, tacipk8-d, and tacipk22-d <dig>  had no equivalents in t. urartu and a. tauschii .
considering that the physical maps of current wheat genome do not cover all the chromosome arms, it is difficult to know whether these potential un-identified tacbl/tacipk genes are resulted from gene retention, duplication and whether these wheat specific genes are the results of the loss of cbl and cipk genes after polyploidization.
in the future, a more detailed wheat physical map may resolve this question.table  <dig> identification of tacbl and tacipk gene families in wheat genome
gene a
triticum urartu
aegilops tauschii
tacbl1
a
tacbl1–a1
*
tacbl1–a2
p
tacbl1–b
tacbl1–d1
tacbl1–d2
tacbl2
a
tacbl2–a
tacbl2–b
*
tacbl2–d
tacbl3
a
tacbl3–b
*
tacbl3–d
tacbl4
a
tacbl4–a
tacbl4–b
tacbl4–d1
*
tacbl4–d2
tacbl6
a
tacbl6–a
*
tacbl6–b
tacbl6–d
tacbl7
a
tacbl7–a
tacbl7–b
*
tacbl7–d
tacbl9
a
tacbl9–a1
tacbl9–d1
p
tacbl9–a2
tacbl9–d2
p
tacbl9–b1
*
tacbl9–b2
tacbl9–b3
tacipk2
a
tacipk2–a
tacipk2–b
tacipk2–d
*
tacipk3
a
tacipk3–a
tacipk3–b
tacipk3–d
*
tacipk4
tacipk4–b
tacipk4–d
tacipk5
a
tacipk5–a
tacipk5–b
tacipk5–d
*
tacipk6
tacipk6–a
p
tacipk6–d
p
tacipk7
a
tacipk7–a1
p
tacipk7–a2
p
tacipk7–a3
p
tacipk7–b
tacipk7–d
p
tacipk8
a
tacipk8–b
tacipk8–d
tacipk9
tacipk9–a
tacipk9–b
tacipk9–d
tacipk10
a
tacipk10–a
tacipk10–b
tacipk10–d
*
tacipk11
a
tacipk11–a
tacipk11–b1
*
tacipk11–b2
tacipk11–d
tacipk11–d2
p
tacipk12
tacipk12–a
tacipk12–b
tacipk12–d
tacipk13
tacipk13–b
tacipk13–d
p
tacipk14
tacipk14–a
tacipk14–b
tacipk14–d
taipk15
a
taipk15–a
tacipk15–b
tacipk15–d
*
tacipk16
tacipk16–a
p
tacipk16–b
tacipk16–d
p
tacipk17
a
tacipk17–a1
tacipk17–a2
tacipk17–b1
*
tacipk17–b2
tacipk17–d1
tacipk17–d2
tacipk17–d3
tacipk19
a
tacipk19–a
p
tacipk19–b1
tacipk19–b2
tacipk20
tacipk20–a
tacipk20–b
tacipk20–d
tacipk21
a
tacipk21–a
*
tacipk21–b
tacipk21–d
tacipk22
a
tacipk22–a
tacipk22–b1
tacipk22–b2
tacipk22–b3
tacipk22–d1
tacipk22–d2
*
tacipk23
tacipk23–a
tacipk23–b
tacipk23–d
tacipk24
a
tacipk24–a
tacipk24–b1
*
tacipk24–b2
tacipk24–d
tacipk25
a
tacipk25–a
tacipk25–d
*
tacipk26
a
tacipk26–a
tacipk26–b
tacipk26–d
*
tacipk27
a
tacipk27–b1
*
tacipk27–b2
tacipk28
a
tacipk28–a
*
tacipk28–b
tacipk28–d
tacipk29
a
tacipk29–a
p
tacipk29–b
*
tacipk29–d
p
tacipk30
tacipk30–a
tacipk31
a
tacipk31–a
tacipk31–b
*
tacipk31–d
p
tacipk32
a
tacipk32–b
*
tacipk32–d
“–” represents no data available.
“p” represents the potential genes were not identified in this work.
“a” represents those genes that were amplified in this study.
“*” represents those amplified genes that were identified as transcripts from indicated chromosomes
fig.
<dig> phylogenetic tree of cbl a and cipk b proteins of t. aestivum , a. tauschii , t. urartu , a. thaliana  and o. sativa .
multiple sequence alignment of cbl and cipk proteins was performed using clustalw.
the wheat proteins were indicated with “rhombus” symbol.
the genes belonging to a, b and d sub-genomes were marked with letters suffix and genes cloned in this study were colored with red
among the identified tacbl/tacipk genes, the cdnas of seven tacbls and  <dig> tacipks were successfully pcr amplified from hexaploid bread wheat  using gene-specific primers  from a cdna mixture prepared from wheat cv.
chinese spring as the template.
the genomic dna and cdna of all identified tacbl and tacipk are provided in additional file  <dig>  to explore the evolutionary relationship of the cbl/cipks, two phylogenetic trees were generated based on all transcribed cbl/cipks from t. aestivum, t. urartu, a. tauschii, a. thaliana, and o. sativa.
as expected, the genes from t. aestivum, t. urartu, and a. tauschii had the closest evolutionary relationships, and these genes had closer phylogenetic relationships with rice  than with arabidopsis .
thus, the names assigned to the identified genes in wheat followed their homology to rice cbls and cipks .
to analyze the structural characteristics of tacbl and tacipk genes, the gene structures including exons and introns were mapped based on the wheat genome sequence .
some genes' structures were partly characterized with exons/intros, because of lack adequate genome sequences.
only the structures covering the orf were regarded as complete structures and used for further analysis.
all the tacbls were intron-rich, with five to eight introns.
among the tacipks,  <dig> tacipks had no introns, and the other  <dig> tacipks had 5– <dig> introns.
for most genes, there were no obvious differences in gene structure among genomes a, b, and d .
some genes  had a different intron length between the structures in different genome locations.
the cbl proteins containing ef-hand motifs are known as ca2+-binding proteins, and show similarities to cml and cam proteins.
each of the seven tacbl proteins harbored four ef-hand motifs.
although these motifs in cbls did not shared high similarity compared with cam and cml proteins in other species , they were conserved among the cbls in arabidopsis, rice, and wheat .
as for the tacipk proteins, all  <dig> tacipks  had domain structures similar to that of atsos2; that is, these  <dig> tacipks harbored a protein kinase catalytic domain  and the naf/fisl motif.
considering that cipk proteins belong to the snrk superfamily, we selected some representative snrk superfamily proteins for comparisons of their structural features.
as shown in additional file  <dig>  the pkc domains in snrk family proteins were conserved and had some highly consistent sites, especially in the activation loop, while the naf/ fisl motif was unique to cipk proteins.
identification and validation of tacbl–tacipk interactions
we analyzed the physical interactions between tacbl and tacipk proteins in wheat using the y2h method.
as shown in table  <dig> , the interactions between tacbls and tacipks showed different strengths and specificities.
tacbl <dig> strongly interacted with five cipks .
tacbl <dig> strongly interacted with  <dig> cipks .
tacbl <dig> showed strong interactions with  <dig> tacipks  out of  <dig> tacipks.
tacbl <dig>  the ortholog of atsos <dig> , strongly interacted with six tacipks .
tacbl <dig> strongly interacted with five tacipks , and tacbl <dig> interacted with seven tacipks .
tacbl <dig> strongly interacted with only two tacipk proteins .
to view the interactions from a different perspective, we found that tacipk <dig>   <dig>   <dig>   <dig>   <dig>   <dig>  and  <dig> did not interact with any of the seven tacbl proteins assayed, implying that these cipk proteins might perceive signals from other unidentified cbl proteins in wheat.
we selected nine of the interactions  detected between cbl and cipk proteins using the y2h assay for further analyses in planta using the bifc method .
for all of the assayed tacbl–tacipk interaction complexes, yellow fluorescence signals were observed when tacbls and tacipks were co-expressed in epidermal cells of tobacco leaves.
notably, it seemed that the observed preferential interactions were inconsistent with the close phylogenetic relationships of the tacipks, namely, the closely phylogenetic related cipk pairs do not display same interaction profile.
for example, tacipk <dig> and tacipk <dig>  which had 81 % similarity, showed similar interactions with five tacbls  but different interactions with tacbl <dig> and  <dig>  compared with tacipk14/ <dig>  other tacipks showed lower amino acid sequence similarity and more diverse interaction specificity.
thus, sequence similarity or phylogenetic relationships are not sufficient to predict the result of cbl-cipk interactions in the y2h assay.table  <dig> interactions of tacipks with tacbls in yeast two-hybrid assay
the interaction analyses of wheat tacbl and tacipk proteins were performed by y2h method.
tacipks and tacbls were respectively cloned to pgad and pgbk vector, and then co-transformed into y <dig> strains.
the transformants containing the target plasmid combinations were grown on selection medium  and indicated as growth  and no growth .
“*” represents the interactions were verified by bifc assays
previous studies have shown that the naf/fisl motif located in the c-terminal regulatory domain and the kinase activity of the pkc domain in the n-terminal region of cipks are necessary and sufficient for mediating interactions with cbls  <cit> .
the roles of the redundant parts  in the c-terminal region of cipks have not yet been fully studied.
here, the tacipk <dig> protein, which interacted with cbl <dig>   <dig>   <dig>  and  <dig>  contained a large c-terminal region  that was not present in the other cipks.
this led us to speculate about the role of this large fragment in regulating the specific interactions of this protein.
analyses of a series of c-terminal deletion mutants of tacipk <dig>  showed that there were diverse interaction patterns between tacipk <dig> mutants and tacbls .
cbl <dig>   <dig>  and  <dig> could not interact with any of the tacipk <dig> deletion mutants, while tacbl1/tacbl <dig> physically interacted with some or all of the mutants.
tacbl <dig> lost its specific interaction with the minimal mutant .
tacbl <dig> did not interact with tacipk <dig> or any of its mutants.
it seems that tacbl <dig>   <dig>  and  <dig> require the last  <dig> amino acid for interaction with tacipk <dig> and also inhibits the binding of tacbl <dig> and tacbl <dig>  these changes in the patterns of interaction may result from changes in protein structure after the deletions.
in terms of this deletion assay, we supposed that the interaction between cbl and cipk followed a “concave–convex” model as shown in fig.
2d, similar to the model for the structures of atsos3-atsos <dig> and atcbl2-atcipk <dig> complexes proposed by sánchez et al.
<cit> .
in this model, the spatial structures of cbls/cipks have an important role for their interactions, and a consummate physical interaction depends on whether the naf/fisl motif properly reach the cbl cavity.
it is believed that the diversified spatial structures resulted from divergent c-terminal region of cipks explain the molecular basis of the selectivity of certain cbls towards particular cipks.
this led to another intriguing question; that is, do the nonsynonymous sequence variations of alleles from different wheat sub-genomes result in aberrant spatial structures that affect the patterns of interaction?
to answer this question, we amplified tacipk17-a, which encodes a protein with many amino acid variations in the c-terminal regulatory region compared with the homologous tacipk17-b protein .
interestingly, tacipk17-a not only interacted with tacbl <dig> and tacbl <dig>  but also with tacbl <dig> in y2h experiments .
despite two mutant sites  were observed at pkc domain, it is possible that several amino acid variations in the c-terminal regulatory region would confer different patterns of interaction.
such changes in interactions may render cipks capable of perceiving signals from other cbls.
these results indicate that the tacbl-tacipk signaling pathway in wheat is very complex because of its allohexaploid genome.fig.
<dig> deletions or mutations in c-terminal ends of tacipks dramatically changes its ability to interact with tacbls.
a series deletions in c-terminal ends of tacipk <dig> leads to changing interacting patterns with tacbls.
the boxes indicate the pkc and naf domains, and numbers beside the lines represent the beginning and the ending positions of tacipk <dig> protein fragments.
b, c-terminal ends alignment between tacipk17-a and tacipk17-b.
residues with grey background indicates mutant amino acid sites.
c, interactions of tacipk17-a and tacipk17-b with tacbls.
yeast was monitored on the selection medium and indicated as growth  and no growth .
d, schematic representation of the “concave-convex” model
expression patterns of tacbl and tacipk genes at different developmental stages in wheat
to investigate the spatial expression patterns of tacbls and tacipks in wheat, we detected their transcriptional levels using rt-pcr in  <dig> representative tissues, i.e., coleoptile, root , stem , leaf , flag leaf , pistil, and stamen .
the results showed that the majority of tacbls and tacipks were expressed at different levels in all tissues tested.
most tacbl genes were constitutively expressed in all organs and at all developmental stages, although the transcript levels of certain genes were very low in some tissues.
some cipk genes were expressed abundantly in certain tissues.
for example, tacipk <dig> was specifically expressed in the root at the seedling stage, indicating that it may play an important role in this organ.
moreover, the gene transcript levels differed between vegetative  and flowering stages.
there were higher transcript levels of tacbl <dig> in seedlings than in plants at the flowering stage .
the flag leaf, as the last leaf of wheat, delivers the largest proportion of photosynthate to fill wheat grains  <cit> .
among the genes expressed in the flag leaf, tacbl <dig> showed lower transcript levels in leaves of seedlings and plants at the flowering stage, and higher transcript levels in the flag leaf, implying that it might play roles in regulating photosynthesis and/or metabolism in the flag leaf.
to study the transcription levels of tacbls and tacipks during the whole life cycle of wheat, we analyzed microarray data that were obtained from tissues at various developmental stages; these data were obtained from publically accessible databases.
the genes formed two groups according to their expression patterns .
group i consisted of the genes with high transcript abundance in nearly all tissues .
group ii genes consisted of  <dig> tacbls and  <dig> tacipks that showed different expression patterns in different tissues and stages.
some of them showed low transcript levels in all tissues.
for most of the tacbls/tacipks, their expression profiles in the microarray data matched to the rt-pcr results, but a few genes showed inconsistencies.
for example, high transcript levels of tacbl <dig> were detected in the stamen in rt-pcr analyses, but low expression levels in anthers  were indicated in the microarray data.
this inconsistency may have resulted from differences in the growth stages of plants between experiments, or from the different methods used.fig.
<dig> expression profile analyses of tacbl and tacipk in wheat tissues with microarray data a, b and c) and rt-pcr .
a gene expression analyses in wheat whole life cycle using public microarray data.
b, gene expression analyses during seeds germination process after immersed with water.
c, comparative transcriptomic analyses between dormant and after-ripened seeds in both imbibed and aba application states.
the value  represents relative transcript levels compared to control .
the up panels in a b and c represent log <dig> transformed values.
d, gene expression analyses during wheat development using rt-pcr method.
“s” and “f” represent seedlings and flowering stages, respectively.
e, the induced genes in seeds germination  were confirmed by rt-pcr
to further clarify the detailed roles of tacipk and tacbl in seed germination, we analyzed public microarray data representing the transcription patterns during seed germination.
as shown in fig.
3b, tacipk <dig>   <dig>   <dig>   <dig>   <dig>   <dig>   <dig>  and  <dig>  and tacbl <dig> were up-regulated  after 12 h of imbibition, and then down-regulated .
tacipk <dig>  tacipk <dig>  tacipk <dig>  and tacbl <dig> were down-regulated during imbibition .
semi-quantitative rt-pcr analyses were conducted to confirm the tacbl and tacipk transcription patterns at five stages of germination .
all of the selected up-regulated genes were induced during seed germination , but the timing of induction differed .
tacipk <dig>  tacipk <dig>  and tacipk <dig> showed peak transcript levels after 12 h of imbibition, while tacipk <dig> and tacipk <dig> showed peak transcript levels at 48 h and lower transcript levels at other time points.
these transcriptional patterns indicated that the genes played roles at different stages of germination.
high transcript levels of tacipk <dig> were detected only at 36 h, when the radicle of seeds emerged and the coleoptile became visible.
there were high transcript levels of tacipk <dig> at the initial stage of germination .
for down-regulated genes  , our data showed that these genes were actually up-regulated or showed no significantly changes in transcription.
moreover, despite some up-regulated genes were all validated in rt-pcr and microarray data, they displayed peak transcript levels at different time point.
for example, tacipk <dig> reached peak levels at 48 h in rt-pcr analysis, while it was up-regulated at 12 h followed with decreasing transcript levels in microarray analysis.
the inconsistencies in the expression levels of genes may have been because the data were obtained using different methods, from different sub-species, or from seeds at slightly different stages of germination.
we analyzed microarray data from another experiment, which focused on seed dormancy and after-ripening in bread wheat  <cit> , and found that four genes  corresponded to germination and that tacipk <dig> was up-regulated gene during germination .
interestingly, tacipk <dig> was down-regulated in dormant seeds, but up-regulated in after-ripened seeds.
these data provided the preliminary information about the roles of tacbl–tacipk in seed germination.
expression profiles of tacbl and tacipk in response to abscisic acid and abiotic stresses
there is considerable evidence that cbl and cipk genes play important roles in the responses to phytohormones and abiotic stresses  <cit> .
among the stress hormones, aba is a well-known signaling molecule in biotic or abiotic stress responses  <cit> .
cold, drought, and salinity are crucial factors affecting wheat growth and crop yields, and these stresses often cause oxidative stress.
to investigate the expression profiles of tacbl and tacipk genes under stress conditions, we conducted real-time quantitative rt-pcr analyses.
we analyzed the transcript levels of two tacbls and five tacipks in wheat seedling roots and leaves under salt , h2o <dig>  drought , and cold  stress treatments, and in response to aba application .
the transcript levels of all tested tacbl and tacipk genes changed in response to many of the treatments.
the genes that did not show significant changes in transcript levels  were tacbl <dig> in the leaf  and in the root , tacbl <dig> in the root , tacipk <dig> in the root , and tacipk <dig> in the leaf .
among the analyzed genes, tacipk <dig> exhibited remarkable aba-induced up-regulation in roots , but not in the leaves.
under salt stress, tacipk <dig> was up-regulated in roots and leaves.
therefore, it was selected for further functional characterization.
the expression patterns of the analyzed tacbls and tacipks were not completely consistent between seedling roots and leaves.
for example, tacbl <dig> was up-regulated in roots and down-regulated in leaves in response to aba application.
several genes responded to the same stress; for example, tacbl <dig>  tcipk <dig>   <dig>   <dig>  and  <dig> were induced by h2o <dig>  among the five treatments, cold treatment resulted in the largest number of up-regulated genes, and none of the assayed genes were down-regulated by cold in roots or leaves.
in brief, the expression patterns of the assayed genes differed among different treatments and tissues, indicating that various tacbls or tacipks may participate in the signaling response to the same stress, and that a single tacbl or tacipk might function in multiple stress responses.fig.
<dig> expression analyses of tacbl and tacipk in leaves and roots responding to various treatments, including aba , nacl , h2o <dig>  peg  and cold .
five time points  were used to detect the gene expressions.
the vertical axis represents gene relative expression levels.
data is the mean of three biological replicates ± s.e
in previous studies, monocot and dicot species were shown to have divergent gene expression patterns in response to abiotic stresses or aba application  <cit> .
thus, we conducted a comparative expression analysis of cbls/cipks among wheat and two other important monocot crops .
microarray datasets of rice cbl/cipk expression patterns in response to abiotic stresses were collected by zhang et al.
<cit> , and a dataset of maize cipk expression patterns was obtained by semi-quantitative-pcr by chen et al.
<cit> .
the comparative analyses showed that several orthologous genes had similar transcription patterns.
for example, the transcript level of cipk <dig> increased in leaves and decreased in roots under salinity treatment in wheat and maize.
however, some orthologous genes exhibited divergent expression patterns.
for example, tacbl <dig> showed increased transcript levels in roots and leaves under cold stress, while the transcript levels of oscbl <dig> decreased under cold stress.
thus, there are both conserved and divergent expression models for orthologous genes among wheat, maize, and rice.
next, we focused on stress-responsive cis-elements to decode the cbl/cipk expression models.
these elements included the aba responsive element , the dehydration-responsive element /c-repeat, and the low-temperature-responsive element  .
sequence analyses of 1-kbp sequences upstream from the start codon of the stress-inducible genes  revealed that these genes contained putative abre, dre, and ltre elements .
these elements at least partly explained the stress-inducible expression patterns of these genes.
however, even though tacipk <dig> and tacipk <dig> were induced by aba, peg, and cold, they contained only the abre element in their promoter regions .
this result is not so surprising, since we only searched for three cis-elements in this study, and there may be other unidentified elements that are important for stress-responsive gene expression.
generally, the putative stress-responsive cis-elements in promoter regions are crucial for stress-responsive expression, and activation of the cis-elements depends on the tissue, the development stage, and/or the genetic background.
therefore, there are many explanations for the diversified expression patterns of orthologous genes among different species.
over-expression of tacipk <dig> improves salt tolerance in arabidopsis
previous studies showed that atcipk <dig>  and bnacipk <dig>  participated in the salt stress response in transgenic arabidopsis  <cit> .
in our expression analysis, tacipk <dig> was induced in roots and leaves under salt stress .
therefore, we generated transgenic arabidopsis plants over-expressing tacipk <dig> .
in total,  <dig> transgenic lines were obtained.
three independent t <dig> homozygous lines  were selected for further analyses.
the transcript levels of tacipk <dig> were detected by rt-pcr .
in plants grown on murashige and skoog  medium, root growth did not differ significantly among the transformants, the empty-vector control, and wild type .
however, the 35s:tacipk <dig> transgenic plants showed greater root growth than those of controls after treatment with 150 mm nacl .
there were no significant differences in the phenotypes of aerial parts between transgenic lines and controls at the seedling stage under normal and salt-stress conditions.
therefore, our data indicated that tacipk <dig> promoted root elongation in transgenic plants under salt stress.fig.
<dig> overexpression of tacipk <dig> in arabidopsis.
a
tacipk <dig> expression levels in selected transgenic lines, vector control and wild type.
b, c the plants grown on control  and stress condition .
d, e statistical analysis of the changing root length between ms control and salt stress condition
to further confirm the salt tolerance of tacipk <dig>  seedlings of 10-day-old, soil-grown, wild-type and transgenic lines were irrigated with 200 mm nacl for  <dig> d. as shown in fig.
6a, the transgenic lines grew better than did the control plants.
furthermore, na+ accumulated to lower levels in leaves of transgenic lines than in leaves of the empty-vector control and wild type.
the k+ contents were not significantly different between transgenic lines and the empty-vector control/wild type , indicating that the improved salt tolerance may involve na+ efflux.
in addition, excess na+ would generate reactive oxygen species , such as peroxide, superoxide, and hydrogen peroxide , causing oxidative stress in plants.
the accumulation of h2o <dig> was reduced in transgenic lines compared with that in the control plants .
this result indicated that decreased na+ contents led to lower ros production  and thus, the plants were able to grow well under salt stress conditions.
antioxidant enzymes are crucial for ros scavenging and play roles in the salt stress response in plants.
therefore, we measured the activities of three important antioxidant enzymes  in transgenic plants and controls under salt stress and normal conditions.
the activities of cat, pod, and sod were higher in transgenic lines than in wild-type and empty-vector controls .
these results showed that the ectopic expression of tacipk <dig> enhanced salt tolerance through facilitating na+ efflux and ros scavenging.fig.
<dig> phenotype of tacipk <dig> over-expression lines in arabidopsis.
a phenotypic comparison of transgenic  and control  plants under 200 mm salt stress and water control.
b na+/k+ concentration in seedlings under stress conditions.
c contents of h2o <dig>  oxidants malondialdehyde , enzymatic activities of peroxidase , catalase  and superoxide dismutase  in control and transgenic plants
CONCLUSIONS
cbl and cipk proteins play important roles in the ca2+ signaling pathway and affect plant development, as well as participating in biotic and abiotic stress responses.
cbls and cipks have been identified and functionally characterized in arabidopsis, rice, maize, and canola , but less is known about the cbls and cipks in wheat.
in this study, we identified seven tacbl and  <dig> tacipk genes from wheat, and cloned cdnas of seven tacbls and  <dig> tacipks.
analyses of tacbl–tacipk identified  <dig> tacbl and  <dig> tacipk loci in the wheat genome, indicating that there were large numbers of these gene loci in the hexaploid wheat.
blast analyses suggested that four tacbl and  <dig> tacipk loci were derived from the t. urartu and a. tauschii genomes.
we studied the preferential interactions between tacbl and tacipk proteins and found  <dig> cbl–cipk interaction complexes.
the proteins tacipk <dig>   <dig>   <dig>   <dig>   <dig>   <dig>  and  <dig> did not interact with any of the seven tacbl proteins assayed in these experiments.
expression analyses revealed that the majority of tacbls and tacipks were expressed at varying levels in all tested tissues, although the transcript levels of some of these genes were very low in some tissues.
we conducted rt-pcr analyses to monitor the transcript levels of seven tacbl and  <dig> tacipk genes under abiotic stress and hormone treatments.
most were distinctly regulated by at least one treatment.
among them, tacipk <dig>  the ortholog of arabidopsis sos <dig>  was up-regulated by salt stress in root and leaf tissues.
we generated transgenic arabidopsis lines over-expressing tacipk <dig>  and all of them showed enhanced salt tolerance.
overall, this study lays a solid foundation for further exploration of the roles of tacbl and tacipk proteins in abiotic stress responses, and enhances our understanding the functions of the wheat tacbl and tacipk gene families.
these results also provide information that will be useful for the genetic manipulation of wheat to improve stress tolerance.

 the calcium ion functions as a ubiquitous second messenger in plants, and participates in signal transduction in response to various environmental and developmental stimuli. the transient ca2+ signal in the cytoplasm of plant cells is captured by a variety of ca2+ sensors including calmodulins, calmodulin-like proteins, calcium-dependent protein kinases, and calcineurin b-like proteins. these ca2+ sensors transfer the environmental and developmental signals to downstream components, causing a series of physiological and biochemical changes. cbls, which harbor a core region consisting of four conserved ef hands, capture the ca2+ signal, and interact with cbl-interacting protein kinases to phosphorylate their target proteins. the cipks with the naf motif for interaction with cbl belong to the snrk protein family, whose members have a specific ser/thr protein kinase domain. recently, cipk proteins were shown to phosphorylate their interacting cbl proteins at n-terminal conservative ser residue and this proper phosphorylation of cbl was proved to be absolutely required for the in vivo activation of the akt k+ channel by cbl1-cipk and cbl9-cipk complexes in oocytes. the cbl and cipk gene families have been described in arabidopsis thaliana, oryza sativa, zea mays, and brassica napus. in previous studies, cbl–cipk complexes were found to play very important roles in responses to external stimuli. the salt overly sensitive pathway was the first cbl-cipk network to be identified, and this cbl-cipk complex comprised cbl and cipk. the cbl protein was shown to interact with cipk and recruit it to the cytoplasmic membrane, where it activated the h+/na+ antiporter to enhance salt tolerance. this result indicated that the cbl-cipk complex functions in regulating sodium ion homeostasis. meanwhile, v-atpase proteins were shown to directly interact with sos to rescue the salt-defective phenotype of sos mutant plants. in recent years, many cbls and cipks have been shown to promote stress tolerance by balancing the intracellular ion concentration in plants. the sodium concentration was markedly lower in the arabidopsis cbl mutant than in control plants, in both normal and salt-stress conditions. the cbl10-overexpressing lines exhibited a k+-sensitive phenotype, like that of the akt mutant under low k+ conditions. the interaction between cbl and akt was verified in bimolecular fluorescence complementation, yeast two-hybrid, and co-immunoprecipitation experiments, and was shown to impair akt1-mediated inward potassium currents. atcipk could directly interact with cbl to promote k+ uptake under low k+ conditions by phosphorylating and activating the k+ channel in arabidopsis and rice. however, a recent study suggested that cbl1/cbl might interact with akt independently of cipk. moreover, in response to low-k+ conditions, cipk was recruited to the tonoplast by cbl to regulate k+ homeostasis in arabidopsis. over-expression and mutant analyses of cbls and cipks have greatly enriched our understanding of their functions. the cbl2/cbl double mutant showed defects in growth and ion homeostasis, possibly as a result of disrupted vacuolar h+-atpase activity. in another study, cbl and cbl were shown to affect seed development and morphology. the ectopic overexpression of zmcipk in the arabidopsi sos mutant enhanced the expression of sos and improved salt tolerance under saline conditions. atcipk was induced by abscisic acid and stress conditions and established a molecular connection between stress- and aba-induced ca2+ signals. cbl and cipk were shown to positively regulate both immunity- and disease-associated programmed cell death in tomato and tobacco. the oscipk insertion mutant was hypersensitive to aba and stresses, and showed altered expression levels of several stress-responsive genes when it was exposed to these abiotic stresses during the seed germination and seedling stages. in a recent study, a few atcbl members were also found to target to another group of enzymes 5'-methylthioadenosine nucleosidases by yeast two-hybrid system, indicting an additional level of complexity on the cbl-mediated signaling networks. however, with respect to the larger families of cbls and cipks, few cbl-cipk complexes have been functionally characterized, especially in bread wheat, which is one of the most important food crops. bread wheat has an allohexaploid genome with a size of approximately 17 gb, making it one of the largest and most complex plant genomes. in recent years, the genome sequencing of t. aestivum, triticum urartu, and aegilops tauschii has promoted research on bread wheat, especially studies on agronomically important gene families related to disease resistance, abiotic stress endurance, and grain quality. in our previous work, we demonstrated that transgenic tobacco overexpressing wheat tacipk and tacipk showed increased tolerance to salinity and drought stress, respectively. in this study, seven tacbl and tacipk genes in wheat were identified and cloned by a genome-wide analysis combined with expressed sequence tag assembly. here, we present the results of analyses of expression profiles, the specific interactions between tacbls and tacipks, and the stress tolerance of transgenic arabidopsis plants expressing tacipk  identification of tacbl and tacipk gene families in wheat the bread wheat genome is thought to have formed via fusion of three ancestral genomes, which are believed to be related to t. urartu, aegilops speltoides, and a. tauschii. these fusion events are thought to have occurred several hundred thousand years ago. we searched the wheat genome and found cbl and cipk loci on wheat chromosomes. the relative positions of these loci on chromosomes were identified on genetic maps. the protein and dna sequences of t. urartu and a. tauschii were retrieved from ncbi and all the cbl and cipk nucleotide sequences from these three species were clustered against each other. the closest matching genes were regarded as equivalent partners, as shown in table  ten and nine genes of t. urartu and a. tauschii, respectively, had no equivalents in t. aestivum, implying the existence of potential un-identified tacbl/tacipk genes. additionally, tacipk15-a, tacipk26-a, tacipk8-d, and tacipk22-d had no equivalents in t. urartu and a. tauschii. considering that the physical maps of current wheat genome do not cover all the chromosome arms, it is difficult to know whether these potential un-identified tacbl/tacipk genes are resulted from gene retention, duplication and whether these wheat specific genes are the results of the loss of cbl and cipk genes after polyploidization. in the future, a more detailed wheat physical map may resolve this question.table identification of tacbl and tacipk gene families in wheat genome gene a triticum urartu aegilops tauschii tacbl1 a tacbl1–a1 * tacbl1–a2 p tacbl1–b tacbl1–d1 tacbl1–d2 tacbl2 a tacbl2–a tacbl2–b * tacbl2–d tacbl3 a tacbl3–b * tacbl3–d tacbl4 a tacbl4–a tacbl4–b tacbl4–d1 * tacbl4–d2 tacbl6 a tacbl6–a * tacbl6–b tacbl6–d tacbl7 a tacbl7–a tacbl7–b * tacbl7–d tacbl9 a tacbl9–a1 tacbl9–d1 p tacbl9–a2 tacbl9–d2 p tacbl9–b1 * tacbl9–b2 tacbl9–b3 tacipk2 a tacipk2–a tacipk2–b tacipk2–d * tacipk3 a tacipk3–a tacipk3–b tacipk3–d * tacipk4 tacipk4–b tacipk4–d tacipk5 a tacipk5–a tacipk5–b tacipk5–d * tacipk6 tacipk6–a p tacipk6–d p tacipk7 a tacipk7–a1 p tacipk7–a2 p tacipk7–a3 p tacipk7–b tacipk7–d p tacipk8 a tacipk8–b tacipk8–d tacipk9 tacipk9–a tacipk9–b tacipk9–d tacipk10 a tacipk10–a tacipk10–b tacipk10–d * tacipk11 a tacipk11–a tacipk11–b1 * tacipk11–b2 tacipk11–d tacipk11–d2 p tacipk12 tacipk12–a tacipk12–b tacipk12–d tacipk13 tacipk13–b tacipk13–d p tacipk14 tacipk14–a tacipk14–b tacipk14–d taipk15 a taipk15–a tacipk15–b tacipk15–d * tacipk16 tacipk16–a p tacipk16–b tacipk16–d p tacipk17 a tacipk17–a1 tacipk17–a2 tacipk17–b1 * tacipk17–b2 tacipk17–d1 tacipk17–d2 tacipk17–d3 tacipk19 a tacipk19–a p tacipk19–b1 tacipk19–b2 tacipk20 tacipk20–a tacipk20–b tacipk20–d tacipk21 a tacipk21–a * tacipk21–b tacipk21–d tacipk22 a tacipk22–a tacipk22–b1 tacipk22–b2 tacipk22–b3 tacipk22–d1 tacipk22–d2 * tacipk23 tacipk23–a tacipk23–b tacipk23–d tacipk24 a tacipk24–a tacipk24–b1 * tacipk24–b2 tacipk24–d tacipk25 a tacipk25–a tacipk25–d * tacipk26 a tacipk26–a tacipk26–b tacipk26–d * tacipk27 a tacipk27–b1 * tacipk27–b2 tacipk28 a tacipk28–a * tacipk28–b tacipk28–d tacipk29 a tacipk29–a p tacipk29–b * tacipk29–d p tacipk30 tacipk30–a tacipk31 a tacipk31–a tacipk31–b * tacipk31–d p tacipk32 a tacipk32–b * tacipk32–d “–” represents no data available. “p” represents the potential genes were not identified in this work. “a” represents those genes that were amplified in this study. “*” represents those amplified genes that were identified as transcripts from indicated chromosomes fig. phylogenetic tree of cbl a and cipk b proteins of t. aestivum, a. tauschii, t. urartu, a. thaliana and o. sativa. multiple sequence alignment of cbl and cipk proteins was performed using clustalw. the wheat proteins were indicated with “rhombus” symbol. the genes belonging to a, b and d sub-genomes were marked with letters suffix and genes cloned in this study were colored with red among the identified tacbl/tacipk genes, the cdnas of seven tacbls and tacipks were successfully pcr amplified from hexaploid bread wheat using gene-specific primers from a cdna mixture prepared from wheat cv. chinese spring as the template. the genomic dna and cdna of all identified tacbl and tacipk are provided in additional file to explore the evolutionary relationship of the cbl/cipks, two phylogenetic trees were generated based on all transcribed cbl/cipks from t. aestivum, t. urartu, a. tauschii, a. thaliana, and o. sativa. as expected, the genes from t. aestivum, t. urartu, and a. tauschii had the closest evolutionary relationships, and these genes had closer phylogenetic relationships with rice than with arabidopsis. thus, the names assigned to the identified genes in wheat followed their homology to rice cbls and cipks. to analyze the structural characteristics of tacbl and tacipk genes, the gene structures including exons and introns were mapped based on the wheat genome sequence. some genes' structures were partly characterized with exons/intros, because of lack adequate genome sequences. only the structures covering the orf were regarded as complete structures and used for further analysis. all the tacbls were intron-rich, with five to eight introns. among the tacipks, tacipks had no introns, and the other tacipks had 5– introns. for most genes, there were no obvious differences in gene structure among genomes a, b, and d. some genes had a different intron length between the structures in different genome locations. the cbl proteins containing ef-hand motifs are known as ca2+-binding proteins, and show similarities to cml and cam proteins. each of the seven tacbl proteins harbored four ef-hand motifs. although these motifs in cbls did not shared high similarity compared with cam and cml proteins in other species, they were conserved among the cbls in arabidopsis, rice, and wheat. as for the tacipk proteins, all tacipks had domain structures similar to that of atsos2; that is, these tacipks harbored a protein kinase catalytic domain and the naf/fisl motif. considering that cipk proteins belong to the snrk superfamily, we selected some representative snrk superfamily proteins for comparisons of their structural features. as shown in additional file the pkc domains in snrk family proteins were conserved and had some highly consistent sites, especially in the activation loop, while the naf/ fisl motif was unique to cipk proteins. identification and validation of tacbl–tacipk interactions we analyzed the physical interactions between tacbl and tacipk proteins in wheat using the y2h method. as shown in table , the interactions between tacbls and tacipks showed different strengths and specificities. tacbl strongly interacted with five cipks. tacbl strongly interacted with cipks. tacbl showed strong interactions with tacipks out of tacipks. tacbl the ortholog of atsos, strongly interacted with six tacipks. tacbl strongly interacted with five tacipks, and tacbl interacted with seven tacipks. tacbl strongly interacted with only two tacipk proteins. to view the interactions from a different perspective, we found that tacipk and did not interact with any of the seven tacbl proteins assayed, implying that these cipk proteins might perceive signals from other unidentified cbl proteins in wheat. we selected nine of the interactions detected between cbl and cipk proteins using the y2h assay for further analyses in planta using the bifc method. for all of the assayed tacbl–tacipk interaction complexes, yellow fluorescence signals were observed when tacbls and tacipks were co-expressed in epidermal cells of tobacco leaves. notably, it seemed that the observed preferential interactions were inconsistent with the close phylogenetic relationships of the tacipks, namely, the closely phylogenetic related cipk pairs do not display same interaction profile. for example, tacipk and tacipk which had 81 % similarity, showed similar interactions with five tacbls but different interactions with tacbl and compared with tacipk14/ other tacipks showed lower amino acid sequence similarity and more diverse interaction specificity. thus, sequence similarity or phylogenetic relationships are not sufficient to predict the result of cbl-cipk interactions in the y2h assay.table interactions of tacipks with tacbls in yeast two-hybrid assay the interaction analyses of wheat tacbl and tacipk proteins were performed by y2h method. tacipks and tacbls were respectively cloned to pgad and pgbk vector, and then co-transformed into y strains. the transformants containing the target plasmid combinations were grown on selection medium and indicated as growth and no growth. “*” represents the interactions were verified by bifc assays previous studies have shown that the naf/fisl motif located in the c-terminal regulatory domain and the kinase activity of the pkc domain in the n-terminal region of cipks are necessary and sufficient for mediating interactions with cbls. the roles of the redundant parts in the c-terminal region of cipks have not yet been fully studied. here, the tacipk protein, which interacted with cbl and contained a large c-terminal region that was not present in the other cipks. this led us to speculate about the role of this large fragment in regulating the specific interactions of this protein. analyses of a series of c-terminal deletion mutants of tacipk showed that there were diverse interaction patterns between tacipk mutants and tacbls. cbl and could not interact with any of the tacipk deletion mutants, while tacbl1/tacbl physically interacted with some or all of the mutants. tacbl lost its specific interaction with the minimal mutant. tacbl did not interact with tacipk or any of its mutants. it seems that tacbl and require the last amino acid for interaction with tacipk and also inhibits the binding of tacbl and tacbl these changes in the patterns of interaction may result from changes in protein structure after the deletions. in terms of this deletion assay, we supposed that the interaction between cbl and cipk followed a “concave–convex” model as shown in fig. 2d, similar to the model for the structures of atsos3-atsos and atcbl2-atcipk complexes proposed by sánchez et al.. in this model, the spatial structures of cbls/cipks have an important role for their interactions, and a consummate physical interaction depends on whether the naf/fisl motif properly reach the cbl cavity. it is believed that the diversified spatial structures resulted from divergent c-terminal region of cipks explain the molecular basis of the selectivity of certain cbls towards particular cipks. this led to another intriguing question; that is, do the nonsynonymous sequence variations of alleles from different wheat sub-genomes result in aberrant spatial structures that affect the patterns of interaction? to answer this question, we amplified tacipk17-a, which encodes a protein with many amino acid variations in the c-terminal regulatory region compared with the homologous tacipk17-b protein. interestingly, tacipk17-a not only interacted with tacbl and tacbl but also with tacbl in y2h experiments. despite two mutant sites were observed at pkc domain, it is possible that several amino acid variations in the c-terminal regulatory region would confer different patterns of interaction. such changes in interactions may render cipks capable of perceiving signals from other cbls. these results indicate that the tacbl-tacipk signaling pathway in wheat is very complex because of its allohexaploid genome.fig. deletions or mutations in c-terminal ends of tacipks dramatically changes its ability to interact with tacbls. a series deletions in c-terminal ends of tacipk leads to changing interacting patterns with tacbls. the boxes indicate the pkc and naf domains, and numbers beside the lines represent the beginning and the ending positions of tacipk protein fragments. b, c-terminal ends alignment between tacipk17-a and tacipk17-b. residues with grey background indicates mutant amino acid sites. c, interactions of tacipk17-a and tacipk17-b with tacbls. yeast was monitored on the selection medium and indicated as growth and no growth. d, schematic representation of the “concave-convex” model expression patterns of tacbl and tacipk genes at different developmental stages in wheat to investigate the spatial expression patterns of tacbls and tacipks in wheat, we detected their transcriptional levels using rt-pcr in representative tissues, i.e., coleoptile, root, stem, leaf, flag leaf, pistil, and stamen. the results showed that the majority of tacbls and tacipks were expressed at different levels in all tissues tested. most tacbl genes were constitutively expressed in all organs and at all developmental stages, although the transcript levels of certain genes were very low in some tissues. some cipk genes were expressed abundantly in certain tissues. for example, tacipk was specifically expressed in the root at the seedling stage, indicating that it may play an important role in this organ. moreover, the gene transcript levels differed between vegetative and flowering stages. there were higher transcript levels of tacbl in seedlings than in plants at the flowering stage. the flag leaf, as the last leaf of wheat, delivers the largest proportion of photosynthate to fill wheat grains. among the genes expressed in the flag leaf, tacbl showed lower transcript levels in leaves of seedlings and plants at the flowering stage, and higher transcript levels in the flag leaf, implying that it might play roles in regulating photosynthesis and/or metabolism in the flag leaf. to study the transcription levels of tacbls and tacipks during the whole life cycle of wheat, we analyzed microarray data that were obtained from tissues at various developmental stages; these data were obtained from publically accessible databases. the genes formed two groups according to their expression patterns. group i consisted of the genes with high transcript abundance in nearly all tissues. group ii genes consisted of tacbls and tacipks that showed different expression patterns in different tissues and stages. some of them showed low transcript levels in all tissues. for most of the tacbls/tacipks, their expression profiles in the microarray data matched to the rt-pcr results, but a few genes showed inconsistencies. for example, high transcript levels of tacbl were detected in the stamen in rt-pcr analyses, but low expression levels in anthers were indicated in the microarray data. this inconsistency may have resulted from differences in the growth stages of plants between experiments, or from the different methods used.fig. expression profile analyses of tacbl and tacipk in wheat tissues with microarray data a, b and c) and rt-pcr. a gene expression analyses in wheat whole life cycle using public microarray data. b, gene expression analyses during seeds germination process after immersed with water. c, comparative transcriptomic analyses between dormant and after-ripened seeds in both imbibed and aba application states. the value represents relative transcript levels compared to control. the up panels in a b and c represent log transformed values. d, gene expression analyses during wheat development using rt-pcr method. “s” and “f” represent seedlings and flowering stages, respectively. e, the induced genes in seeds germination were confirmed by rt-pcr to further clarify the detailed roles of tacipk and tacbl in seed germination, we analyzed public microarray data representing the transcription patterns during seed germination. as shown in fig. 3b, tacipk and and tacbl were up-regulated after 12 h of imbibition, and then down-regulated. tacipk tacipk tacipk and tacbl were down-regulated during imbibition. semi-quantitative rt-pcr analyses were conducted to confirm the tacbl and tacipk transcription patterns at five stages of germination. all of the selected up-regulated genes were induced during seed germination, but the timing of induction differed. tacipk tacipk and tacipk showed peak transcript levels after 12 h of imbibition, while tacipk and tacipk showed peak transcript levels at 48 h and lower transcript levels at other time points. these transcriptional patterns indicated that the genes played roles at different stages of germination. high transcript levels of tacipk were detected only at 36 h, when the radicle of seeds emerged and the coleoptile became visible. there were high transcript levels of tacipk at the initial stage of germination. for down-regulated genes, our data showed that these genes were actually up-regulated or showed no significantly changes in transcription. moreover, despite some up-regulated genes were all validated in rt-pcr and microarray data, they displayed peak transcript levels at different time point. for example, tacipk reached peak levels at 48 h in rt-pcr analysis, while it was up-regulated at 12 h followed with decreasing transcript levels in microarray analysis. the inconsistencies in the expression levels of genes may have been because the data were obtained using different methods, from different sub-species, or from seeds at slightly different stages of germination. we analyzed microarray data from another experiment, which focused on seed dormancy and after-ripening in bread wheat, and found that four genes corresponded to germination and that tacipk was up-regulated gene during germination. interestingly, tacipk was down-regulated in dormant seeds, but up-regulated in after-ripened seeds. these data provided the preliminary information about the roles of tacbl–tacipk in seed germination. expression profiles of tacbl and tacipk in response to abscisic acid and abiotic stresses there is considerable evidence that cbl and cipk genes play important roles in the responses to phytohormones and abiotic stresses. among the stress hormones, aba is a well-known signaling molecule in biotic or abiotic stress responses. cold, drought, and salinity are crucial factors affecting wheat growth and crop yields, and these stresses often cause oxidative stress. to investigate the expression profiles of tacbl and tacipk genes under stress conditions, we conducted real-time quantitative rt-pcr analyses. we analyzed the transcript levels of two tacbls and five tacipks in wheat seedling roots and leaves under salt, h2o drought, and cold stress treatments, and in response to aba application. the transcript levels of all tested tacbl and tacipk genes changed in response to many of the treatments. the genes that did not show significant changes in transcript levels were tacbl in the leaf and in the root, tacbl in the root, tacipk in the root, and tacipk in the leaf. among the analyzed genes, tacipk exhibited remarkable aba-induced up-regulation in roots, but not in the leaves. under salt stress, tacipk was up-regulated in roots and leaves. therefore, it was selected for further functional characterization. the expression patterns of the analyzed tacbls and tacipks were not completely consistent between seedling roots and leaves. for example, tacbl was up-regulated in roots and down-regulated in leaves in response to aba application. several genes responded to the same stress; for example, tacbl tcipk and were induced by h2o among the five treatments, cold treatment resulted in the largest number of up-regulated genes, and none of the assayed genes were down-regulated by cold in roots or leaves. in brief, the expression patterns of the assayed genes differed among different treatments and tissues, indicating that various tacbls or tacipks may participate in the signaling response to the same stress, and that a single tacbl or tacipk might function in multiple stress responses.fig. expression analyses of tacbl and tacipk in leaves and roots responding to various treatments, including aba, nacl, h2o peg and cold. five time points were used to detect the gene expressions. the vertical axis represents gene relative expression levels. data is the mean of three biological replicates ± s.e in previous studies, monocot and dicot species were shown to have divergent gene expression patterns in response to abiotic stresses or aba application. thus, we conducted a comparative expression analysis of cbls/cipks among wheat and two other important monocot crops. microarray datasets of rice cbl/cipk expression patterns in response to abiotic stresses were collected by zhang et al., and a dataset of maize cipk expression patterns was obtained by semi-quantitative-pcr by chen et al.. the comparative analyses showed that several orthologous genes had similar transcription patterns. for example, the transcript level of cipk increased in leaves and decreased in roots under salinity treatment in wheat and maize. however, some orthologous genes exhibited divergent expression patterns. for example, tacbl showed increased transcript levels in roots and leaves under cold stress, while the transcript levels of oscbl decreased under cold stress. thus, there are both conserved and divergent expression models for orthologous genes among wheat, maize, and rice. next, we focused on stress-responsive cis-elements to decode the cbl/cipk expression models. these elements included the aba responsive element, the dehydration-responsive element /c-repeat, and the low-temperature-responsive element. sequence analyses of 1-kbp sequences upstream from the start codon of the stress-inducible genes revealed that these genes contained putative abre, dre, and ltre elements. these elements at least partly explained the stress-inducible expression patterns of these genes. however, even though tacipk and tacipk were induced by aba, peg, and cold, they contained only the abre element in their promoter regions. this result is not so surprising, since we only searched for three cis-elements in this study, and there may be other unidentified elements that are important for stress-responsive gene expression. generally, the putative stress-responsive cis-elements in promoter regions are crucial for stress-responsive expression, and activation of the cis-elements depends on the tissue, the development stage, and/or the genetic background. therefore, there are many explanations for the diversified expression patterns of orthologous genes among different species. over-expression of tacipk improves salt tolerance in arabidopsis previous studies showed that atcipk and bnacipk participated in the salt stress response in transgenic arabidopsis. in our expression analysis, tacipk was induced in roots and leaves under salt stress. therefore, we generated transgenic arabidopsis plants over-expressing tacipk. in total, transgenic lines were obtained. three independent t homozygous lines were selected for further analyses. the transcript levels of tacipk were detected by rt-pcr. in plants grown on murashige and skoog medium, root growth did not differ significantly among the transformants, the empty-vector control, and wild type. however, the 35s:tacipk transgenic plants showed greater root growth than those of controls after treatment with 150 mm nacl. there were no significant differences in the phenotypes of aerial parts between transgenic lines and controls at the seedling stage under normal and salt-stress conditions. therefore, our data indicated that tacipk promoted root elongation in transgenic plants under salt stress.fig. overexpression of tacipk in arabidopsis. a tacipk expression levels in selected transgenic lines, vector control and wild type. b, c the plants grown on control and stress condition. d, e statistical analysis of the changing root length between ms control and salt stress condition to further confirm the salt tolerance of tacipk seedlings of 10-day-old, soil-grown, wild-type and transgenic lines were irrigated with 200 mm nacl for d. as shown in fig. 6a, the transgenic lines grew better than did the control plants. furthermore, na+ accumulated to lower levels in leaves of transgenic lines than in leaves of the empty-vector control and wild type. the k+ contents were not significantly different between transgenic lines and the empty-vector control/wild type, indicating that the improved salt tolerance may involve na+ efflux. in addition, excess na+ would generate reactive oxygen species, such as peroxide, superoxide, and hydrogen peroxide, causing oxidative stress in plants. the accumulation of h2o was reduced in transgenic lines compared with that in the control plants. this result indicated that decreased na+ contents led to lower ros production and thus, the plants were able to grow well under salt stress conditions. antioxidant enzymes are crucial for ros scavenging and play roles in the salt stress response in plants. therefore, we measured the activities of three important antioxidant enzymes in transgenic plants and controls under salt stress and normal conditions. the activities of cat, pod, and sod were higher in transgenic lines than in wild-type and empty-vector controls. these results showed that the ectopic expression of tacipk enhanced salt tolerance through facilitating na+ efflux and ros scavenging.fig. phenotype of tacipk over-expression lines in arabidopsis. a phenotypic comparison of transgenic and control plants under 200 mm salt stress and water control. b na+/k+ concentration in seedlings under stress conditions. c contents of h2o oxidants malondialdehyde, enzymatic activities of peroxidase, catalase and superoxide dismutase in control and transgenic plants  cbl and cipk proteins play important roles in the ca2+ signaling pathway and affect plant development, as well as participating in biotic and abiotic stress responses. cbls and cipks have been identified and functionally characterized in arabidopsis, rice, maize, and canola, but less is known about the cbls and cipks in wheat. in this study, we identified seven tacbl and tacipk genes from wheat, and cloned cdnas of seven tacbls and tacipks. analyses of tacbl–tacipk identified tacbl and tacipk loci in the wheat genome, indicating that there were large numbers of these gene loci in the hexaploid wheat. blast analyses suggested that four tacbl and tacipk loci were derived from the t. urartu and a. tauschii genomes. we studied the preferential interactions between tacbl and tacipk proteins and found cbl–cipk interaction complexes. the proteins tacipk and did not interact with any of the seven tacbl proteins assayed in these experiments. expression analyses revealed that the majority of tacbls and tacipks were expressed at varying levels in all tested tissues, although the transcript levels of some of these genes were very low in some tissues. we conducted rt-pcr analyses to monitor the transcript levels of seven tacbl and tacipk genes under abiotic stress and hormone treatments. most were distinctly regulated by at least one treatment. among them, tacipk the ortholog of arabidopsis sos was up-regulated by salt stress in root and leaf tissues. we generated transgenic arabidopsis lines over-expressing tacipk and all of them showed enhanced salt tolerance. overall, this study lays a solid foundation for further exploration of the roles of tacbl and tacipk proteins in abiotic stress responses, and enhances our understanding the functions of the wheat tacbl and tacipk gene families. these results also provide information that will be useful for the genetic manipulation of wheat to improve stress tolerance. 
transgenic arabidopsis plants overexpressing tacipk exhibited improved salt tolerance through increased na+ efflux and an enhanced reactive oxygen species scavenging capacity. the expressions of many tacbls and tacipks were responsive to abiotic stresses and abscisic acid treatment. these results contribute to our understanding of the functions of cbl-cipk complexes and provide the basis for selecting appropriate genes for in-depth functional studies of cbl–cipk in wheat. preferential interactions between tacbls and tacipks were observed in yeast two-hybrid and bimolecular fluorescence complementation experiments. tacipk24cbl–cipkexpression profilesstress responsepreferential interactionswheatissue-copyright-statement© the author 2015 calcineurin b-like proteins belong to a unique group of calcium sensors in plant that decode the ca2+ signature by interacting with cbl-interacting protein kinases. some tacbl and tacipk genes showed up- or down-regulated expressions during seed germination. a total of seven tacbl and tacipk genes were amplified from bread wheat, triticum aestivum cv. reverse-transcriptase-polymerase chain reaction and in silico expression analyses showed that tacbl and tacipk genes were expressed at different levels in different tissues, or maintained at nearly constant expression levels during the whole life cycle of the wheat plant. analyses of a deletion series of tacipk proteins with amino acid variations at the c-terminus provided new insights into the specificity of the interactions between tacipks and tacbls, and indicated that the tacbl–tacipk signaling pathway is very complex in wheat because of its hexaploid genome. although cbl-cipk complexes have been shown to play important roles in the responses to various stresses in plants, little is known about their functions in wheat.
 calcineurin b-like proteins belong to a unique group of calcium sensors in plant that decode the ca2+ signature by interacting with cbl-interacting protein kinases. although cbl-cipk complexes have been shown to play important roles in the responses to various stresses in plants, little is known about their functions in wheat.  a total of seven tacbl and tacipk genes were amplified from bread wheat, triticum aestivum cv. chinese spring. reverse-transcriptase-polymerase chain reaction and in silico expression analyses showed that tacbl and tacipk genes were expressed at different levels in different tissues, or maintained at nearly constant expression levels during the whole life cycle of the wheat plant. some tacbl and tacipk genes showed up- or down-regulated expressions during seed germination. preferential interactions between tacbls and tacipks were observed in yeast two-hybrid and bimolecular fluorescence complementation experiments. analyses of a deletion series of tacipk proteins with amino acid variations at the c-terminus provided new insights into the specificity of the interactions between tacipks and tacbls, and indicated that the tacbl–tacipk signaling pathway is very complex in wheat because of its hexaploid genome. the expressions of many tacbls and tacipks were responsive to abiotic stresses and abscisic acid treatment. transgenic arabidopsis plants overexpressing tacipk exhibited improved salt tolerance through increased na+ efflux and an enhanced reactive oxygen species scavenging capacity.  these results contribute to our understanding of the functions of cbl-cipk complexes and provide the basis for selecting appropriate genes for in-depth functional studies of cbl–cipk in wheat. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords tacipk24cbl–cipkexpression profilesstress responsepreferential interactionswheatissue-copyright-statement© the author 2015 