BACKGROUND
calcium signal transduction through calcium channels is a fundamental mechanism used by plants to sense and respond to endogenous and environmental stimuli  <cit> , including hormone responses  <cit> , plant–pathogen interaction  <cit> , development  <cit> , symbiosis  <cit> , salt stress  <cit> , light signaling  <cit> , and circadian rhythm  <cit> .
one of the potential pathways for the uptake of ca2+ ions in signal transduction is via cyclic nucleotide-gated ion channels   <cit> .
cngcs, functionally defined as ligand-gated protein channels, are nonselective cation channels, gated by the direct binding of cyclic nucleotides, and are present in both animals and plants system .
in animals, their biological roles and regulation have been well studied, however, plant cngcs have only been investigated much more recently, and corresponding regulatory mechanisms are just starting to be discovered  <cit> .
studies have revealed that cngcs may function as a pathway for ca2+ conduction into the cytosol as an early event during pathogen defense signaling cascades  <cit> , and plant response to other biotic and abiotic stresses  <cit> .
plant cngc ion channels were first identified in a screen for calmodulin  binding partners in barley  <cit> .
a large family of cngcs composed of  <dig> members have been identified in arabidopsis genome  <cit>  which are classified into four groups  and two sub-groups “iv-a and iv-b”  <cit> .
these cngcs are characterized by general structural resemblance to animal cngcs  <cit> , having a predicted structure of six transmembrane domains  with a pore domain  between s <dig> and s <dig>  c-terminal cyclic nucleotide-binding domain  and cam-binding domains   <cit> .
in the presence of ca2+, cam binds to the cngc at the αc helix of the cnbd and thus blocks channel gating by cnmps  <cit> .
cnbd being most conserved region of the cngc with a phosphate binding cassette  and a “hinge” region .
where, the pbc binds to the sugar and phosphate moieties of the cyclic nucleotide  ligand  <cit> , and the hinge region contributes to ligand binding efficacy and selectivity  <cit> .
in arabidopsis, cngc genes are differentially expressed in all tissues and their functions critically depend on the presence of camp and/or cgmp that function as signaling molecules .
physiological processes in which these signaling molecules are believed to be involved include various developmental processes, photo-morphogenesis and tolerance to salt stress  <cit> , gibberellic acid-induced signaling in barley  <cit>  and phytochrome signaling  <cit> .
approximately 5% of the oryza sativa genome appears to encode membrane transport proteins.
many of these proteins are classified and several hundred putative transporters have not yet been assigned to families, including the cngc gene family  <cit> .
the early investigations on rice genome, revealed a clue about the existence of putative cngc fragments, as suggested by bridges et al.
<cit>  and ramanjaneyulu et al.
<cit> .
however, no comprehensive study has yet been undertaken, which can lead to the proper identification, characterization and functional verification of the cngc family in rice.
taking privilege from the accessibility of recent release of entire genome sequences of rice and information available from a. thaliana, we performed a genome-wide identification of cngc family in rice in the present study.
we found that the rice genome contains  <dig> cngc full length genes which are classified into four major classes and two sub-groups as in arabidopsis.
evolutionary history reconstruction via comparison with cngcs from other plant species was accomplished.
furthermore, comprehensive analyses were performed to characterize these genes and protein sequence features e.g., domain, motif, phylogenetic relationships, gene structure, multiple alignment, cis-acting regulatory elements etc.
to verify their functions, we analyzed the expression pattern of rice cngc genes by quantitative pcr  in response to various hormonal, biotic and abiotic stresses.
the present work represents the first comprehensive study of cngc genes in rice and offers a solid base for further functional investigation of this important gene family in rice.
RESULTS
identification of cngc genes in rice genome
to obtain a complete overview of cngc gene family in rice, we conducted a genome-wide analysis by using various bioinformatics resources.
considering the fact that the arabidopsis cngc gene family is fully characterized, we used the amino acid  sequences of  <dig> arabidopsis cngc genes as the queries in blast searches and the keyword ‘cyclic nucleotide-gated ion channel’ in the putative function search tool of rice database .
seventeen non-redundant putative gene sequences were retrieved in rice with five genes  having potential alternative splice forms.
since plant cngcs, are characterized by the presence of cnbd, specifically phosphate binding cassettes and hinge region  <cit> , thus their presence in a protein sequence is a validation criteria for these genes.
the sequence searches of databases based on annotation are not completely accurate and could potentially miss some bonafide cngc sequences or inaccurately assign certain sequences as cngcs  <cit> .
therefore, the deduced protein sequences of all the  <dig> putative genes were further analyzed to confirm the presence of cnbd/cyclic nucleotide-monophosphate binding domain , cap family effector domain , rmlc-like jelly roll fold , and ion transport domains.
we found that loc_os06g <dig>  previously designated as “genomic cyclic nucleotide-gated ion channel 1”, was identified as “cyclic nucleotide-gated channel c ”, and was eradicated from analysis due to lack of above cited domains.
however, a  <dig> aa short fragment, loc_os06g <dig>  contained the cngc characteristic cnbd domain, with phosphate binding cassettes and hinge region as suggested by zelman et al.
<cit> , was recognized as bonafide cngc.
these identifications were purely based on the presence of primary cnbd domain, and the validation of the consensus motif derived by zelman et al.
<cit> .
we noticed that blast search of arabidopsis cngc also resulted into gene from potassium akt /kat channels  as homologs of cngcs, due to the presence of transmembrane region, cnbd and ion transport domains and additional ankyrin repeats  <cit> .
such unrelated genes were rejected from analysis.
finally,  <dig> full length cngc genes were confirmed to contain the specific domains in their proteins sequences, and hence were identified as cngc genes in rice .table  <dig>
domain architecture of
oscngc
gene family
1
group i
group ii
group iii
group iva
group ivb
<dig> <cit> : itp ,  <cit>  cnmp_bd ,  <cit>  is- cnmp_bd .
<cit>  cyclic nucleotide- monophosphate,  <cit>  cap_ed , tm ,  <cit>  crp ;  <cit>  ccer <dig> ,  <cit>  pln ,  <cit>  cnmp_bd ,  <cit>  cnmp_bd-like ,  <cit>  rmlc .
oscngcs chromosomal localization and diversification
chromosome localization analysis demonstrated that the oscngcs are located on eight rice chromosomes  .
to better reflect the orthologous relationship between the rice and arabidopsis cngc genes, the nomenclature of respective rice cngc genes was annotated according to their order in phylogenies, with os abbreviating o. sativa .figure  <dig>
genomic localization and duplication of cngc genes in rice chromosomes.
chromosome numbers are indicated at the top of each chromosome.
genes are marked in abbreviations i.e.
c stands for cngc; those present on duplicated segments of genome are connected by red lines and tandem duplicated genes are marked in blue.
arabidopsis has been reported to have  <dig> cngc genes   <cit> .
conversely, in rice that has a genome size nearly three times as that of arabidopsis   <cit> , the number of cngc genes was found to be only  <dig>  the reason for this could be the variable status of whole genome duplications in arabidopsis and rice   <cit> .
in this study, both segmental and tandem duplication events were investigated for elucidating the potential mechanism of evolution of oscngc gene family in rice.
analysis of pgdd revealed that three genes pairs, oscngc <dig> and oscngc <dig>  oscngc <dig> and oscngc <dig>  as well as oscngc <dig> and oscngc <dig> located on different chromosomes regions, might have been induced by segmental duplication, and could be assigned to rgap segmental duplication blocks .
in addition, one gene pair, oscngc <dig> and oscngc <dig> were located at the adjacent positions on chromosome  <dig> and separated by  <dig> genes only.
hence suggested, that these two genes were generated from tandem duplication event .
these results indicate that gene duplication not only expanded the number of genes in the rice cngc family but also increased its functional diversity .
gene structure and features of oscngc proteins
comparisons between genomic dna sequences with corresponding cdna sequences showed that coding sequences of oscngc genes are interrupted by introns.
introns may interrupt the reading frame of a gene between two consecutive codons , between the first and second nucleotide of a codon , or between the second and the third nucleotide   <cit> .
gene structural analysis showed that the rice cngcs had different gene structures as compared to arabidopsis cngcs, with a variable number of introns and lengths.
the numbers of introns in arabidopsis cngcs ranged from  <dig> to  <dig> introns, while rice cngc ranged from  <dig> to  <dig> introns.
unlike the arabidopsis cngcs, introns of all oscngcs belong to phase  <dig> and  <dig> , while, arabidopsis cngc <dig> contained, four phase  <dig> introns.
this suggests that all cngcs with introns did not ascend from a retrotransposition event, and the different numbers may be caused by the insertion and loss of introns during their evolution  <cit> .
the  <dig> oscngc proteins range in length from  <dig>  to  <dig>  amino acids , with an average of  <dig> aa.
expasy analysis showed that oscngc proteins vary greatly in isoelectric point  values  and molecular weights .
according to their instability index values, only  <dig>  of  <dig> oscngc proteins can be considered as stable with an instability index of < =  <dig>   <cit> .table  <dig>
predicted sequence features of oscngc proteins
oscngc1
oscngc2
oscngc3
oscngc4
oscngc5
oscngc6
oscngc7
oscngc8
oscngc9
oscngc10
oscngc11
oscngc12
oscngc13
oscngc14
oscngc15
oscngc16
mw , pi , camp- cgmp , casein , pkc , tyr , n-myr , n-glyc , p.m , cyt , c.t.m , m.i.m .
the subcellular localization of each oscngc was predicted by psort analysis.
our results evidenced that thirteen oscngcs  are predicted to be localized in plasma membrane, while the others i.e., oscngc <dig>  oscngc <dig> and oscngc <dig> to be in cytoplasm, chloroplast thylakoid membrane and mitochondrial inner membrane, respectively .
all relevant experiments in the past have indicated that plant cngc proteins are specifically localized to the plasma membrane  <cit> .
the presence of oscngc <dig> in cytoplasm is not clear, which needs further experimental elucidation.
arabidopsis cngc proteins, when analyzed revealed that  <dig> out of  <dig> cngc proteins were localized in plasma membrane, while the other  <dig> in chloroplast thylakoid membrane .
translated proteins are often subjected to post–translational modifications  to become functionally active.
ptms are the chemical modification of a protein after its translation, and have wide effects on broadening its range of functionality  <cit> .
cngc channels can be controlled by variations in the phosphorylation state catalyzed by ser/thr kinases  <cit>  and phosphatases  <cit> .
when analyzed in scanprosite, multiple putative phosphorylation sites were revealed in oscngc protein sequences, which may act as substrates for several kinases in the form of casein kinase ii, protein kinase c, tyrosine kinase and camp/cgmp kinases  <cit> .
francis and corbin  <cit>  demonstrated that camp/cgmp-dependent protein kinases are major intracellular receptors for these nucleotides , and the actions of these enzymes account for much of the cellular responses to increased levels of camp or cgmp.
as a special class of ptms, several proteins including cngcs could be covalently altered by a range of lipids, such as myristate and palmitate, a phenomenon known as lipidation  <cit> .
protein n-myristoylation can alter the lipophilicity of the target protein and facilitate its interaction with membranes thereby affecting its subcellular localization .
our bioinformatics analysis revealed that all oscngc proteins bear potential n-myristoylation/ n-glycosylation motif sites.
the maximum number of n-myristoylation sites is present in oscngc <dig> and oscngc <dig> , while oscngc <dig> contain highest number  of n-glycosylation sites.
in mammals, n-myristoylated proteins include protein kinases, phosphatases, guanine nucleotide-binding proteins , and ca2+-binding proteins, many of which participate in signal transduction pathways  <cit> .
similarly, n-glycosylation, post-translational modification that occur in many eukaryotic proteins exposed to the extracellular medium , is reported to play various roles, including facilitating the folding, trafficking and function of the protein as well as protecting it from an extracellular medium, which, in plants, is acidic and rich in proteases  <cit> .
in a recent study, meighan et al.
<cit>  demonstrated that extracellular ca2+ and zn2+-dependent proteases  increase the ligand sensitivity of rod and cone cng channels, and prolonged exposure of cng channels to mmp even caused the channels to become nonconductive due to mmp-dependent proteolysis.
they have further indicated that presence of glycosylation sites may defend cng channels from mmp-dependent proteolysis, and prevent the cngc channels to become nonconductive  <cit> .
therefore the ptms and other sequence-specific features such as n-myristoylation and n-glycosylation in rice cngcs are likely to be necessary for their proper functioning, localization and regulation .
motif composition analysis
a functional motif-based recognition of cngc proteins can contribute to understanding the evolutionary history of this gene family including possible role of genome and gene duplication events  <cit> .
motif search by the meme/mast identified  <dig> conserved motifs in rice cngc proteins .
among these, five motifs  were identified in rice, and found to be associated with functionally defined domains .
all the oscngc proteins share a conserved binding domain for cyclic nucleotides  in the central region, referred to as motif  <dig>  plant cngcs are believed to be activated by direct and reversible binding of nucleotides cgmp and camp to the cnbd, which allosterically causes the channel to open  <cit> .
in the presence of calcium, cam binds to the cambd and prevents the binding of cyclic nucleotides to the cnbd, and thereby stops the activation of cngcs by cyclic nucleotides  <cit> .
this competitive regulation by cyclic nucleotides and cams was predicted  <cit>  and experimentally verified for atcngc <dig>  <cit> .
the cam-binding site was recently mapped to an isoleucine glutamine  motif by fischer et al.
<cit> .
our results also identified iq as a functional motif  within cambd, downstream of the cnbd .
iq motifs have been investigated in detail in animal proteins, which often bind cam in a ca2+-dependent manner, while ca2+ induces the displacement of the cam ligand  <cit> .
fischer et al.
<cit>  showed that iq motif is conserved among plant cngcs, this motif enhances the variability of ca2+-dependent channel control mechanisms featuring the functional diversity within this multigene family.
however, in rice, iq motifs were present in less than 50% of the members of oscngc family, mainly in the members of group  <dig>   <dig> and  <dig>  , suggesting that other regulators may alter the channel opening and for gating control of plant cngcs which lack these motifs  <cit> .
other functional motifs denoted as motif  <dig>  and  <dig>  associated with prenyltransferase and squalene oxidase repeat, and ion transport, respectively, was found to be conserved in all oscngc proteins except oscngc <dig>  the functions of other motifs identified in oscngc proteins  <dig>  5– <dig>  9– <dig>  and  <dig> have not been reported in any plant or animal so far.
moreover, the sequence logo of three potential motifs, cnbd, iq and ion transport one can determine not only the consensus sequence but also the relative frequency of bases and the information content  at every position in a site or sequence.
the logo displays both significant residues and subtle sequence patterns .figure  <dig>
distribution of conserved motifs in rice cngc proteins identified using meme search tool.
schematic representation of motifs composition in oscngc protein sequences using meme motif search tool for each groups given separately.
each motif is represented by a number in colored box.
length of box does not correspond to length of motif.
order of the motifs corresponds to position of motifs in individual protein sequence.
we also performed meme analysis on  <dig> arabidopsis cngc proteins for comparison.
the diagram of atcngc motifs, their details and the sequence logo of functional motifs is given in additional files  <dig>   <dig>  and  <dig>  the numbers of motifs are designated similarly in both rice and arabidopsis cngc proteins, each corresponding to the position/location of the respective motif.
in arabidopsis, only three motifs  were identified representing the cnbd domain, and iq motif, where cnbd domain was represented by two motifs  interrupted by  <dig> non-functional motifs.
interestingly, motifs associated with ion transport, zinc finger, prenyltransferase and squalene oxidase repeat were absent in arabidopsis .
though, individual scan of atcngc proteins revealed the presence of ion transport domain in some members .
as mentioned earlier, an essential structural feature of plant cngcs is their cnbd, a direct binding site for camp/cgmp to modify the channel opening  <cit> .
the most conserved feature of cnbd domain is the pbc that makes direct contact with camp  <cit> , and the hinge region, essential for the capping of camp by the c-helix of the cnbd  <cit> .
no plant cngc-specific motifs had been reported until zelman et al.
<cit>  aligned the pbc and hinge regions for  <dig> atcngc proteins, from which the following consensus motif was derived: --x--x-g-x--ll-x- <cit> -x--e-x-f- <cit> ).
similarly we aligned the cnbd region of rice cngcs  and identified a putative pbc and a hinge .
we identified a conserved  glycine , acidic residue glutamate  followed by two aliphatic leucines  and aromatic tryptophan  inside the pbcs .
we also detected that the putative hinge also comprises a conserved  aromatic phenylalanine , aliphatic alanine  and leucines  .
a neutral residue, threonine  was also found to be conserved  near the hinge region.
we successively built a stringent motif: --x--x-g--ell-x-w-x-sa-x-t-x--af-x-l) that recognizes the  <dig> rice cngcs specifically.
the rice pbc and hinge regions differed from the conserved residues of arabidopsis.
for example, residues e, w and t within pbcs, and residues a and l in hinges were found be conserved at 100% in rice cngcs only.
the diversity of motif patterns between the rice and arabidopsis indicate the functional divergence across the species and individual groups.figure  <dig>
the rice cngc-specific motif spans the putative pbc and the hinge within the cnbd of the  <dig> oscngcs.
the diagram at the top represents three regions of plant cngcs: the six transmembrane domains , a cnbd containing a pbc and the hinge, and cambd.
the rice cngc-specific amino acid motif is shown below the cartoon.
in the square brackets “” are the amino acids allowed in this position of the motif, “x” represents any amino acid and the round brackets “()” indicate the number of amino acids.
below is the alignment of cnbd domains of  <dig> rice cngcs.
residues in white highlighted in blue indicate 100% identity among the  <dig> rice cngcs.
phylogenetic analysis
phylogenetic relationship between rice and arabidopsiscngc family genes
to determine the phylogenic relationship of cngc family between rice and arabidopsis, a maximum likelihood  phylogenetic tree was constructed using full-length amino acid sequences.
four groups, as described by mäser et al.
<cit>  were identified containing representative gene of both rice and arabidopsis.
all the arabidopsis cngc proteins were found to lie in groups similar to those identified previously  <cit> .
of the four groups, three groups  are monophyletic, while one group  is sub-divided into two distinct clades, named group iv-a and iv-b .
group i comprises three members from rice cngcs  and six from arabidopsis .
similarly, group ii contains three rice cngcs  and five atcngcs .
however, group iii embraces five in rice  and five in arabidopsis , thus form the largest group, with  <dig> members.
two cngcs from each rice  and arabidopsis  were assigned to group iv-a, while, three rice cngcs  and two arabidopsis cngcs  segregated into group iv-b .
a separate phylogenetic tree was also generated from conserved cnbd domains sequences of all the cngc proteins in rice and arabidopsis, which resulted into similar clustering pattern.figure  <dig>
phylogenetic tree of oscngc and atcngc proteins.
the multiple alignment was performed by clustalx program.
mega  <dig>  was used to create the maximum likelihood  under the jones-taylor-thornton  model.
the bootstrap values from  <dig> resampling are given at each node.
the rice cngc genes identified in this study are shown in red circles and arabidopsis cngcs are shown in blue squares.
rice cngc genes were designated according to their order in phylogeny.
during the evolution of a gene family, gene duplication plays important roles in generating new members and creating novel gene functions, hence increasing numbers and diversifying functions of genes within a gene family  <cit> .
the phylogenetic analysis between rice and arabidopsis following previously described model  <cit>  demonstrates the evolutionary trend of gene duplication in both taxa.
phylogenetic data provide a framework for making more appropriate intergenomic comparisons, by determining whether chromosomal duplications within taxa pre-date or post-date divergence among taxa  <cit> .
the current analysis shows that divergence of two taxa may have pre-dated the gene duplication events among them resulting in gene expansion within individual group  <cit>  .
this is further strengthened by our detailed phylogenetic analysis among all plant lineages given ahead.
the detailed phylogeny also points towards the duplication of cngc genes occurring after speciation and after divergence of angiosperms into mono and dicots.
interestingly atcngc <dig> and oscngc14; atcngc <dig> and oscngc <dig> are not in congruence with the pattern of other sister groups.
the existing anomaly between these taxa could not be reasoned out.
phylogenetic analysis of the cngcgenes in all plant lineages
for a better understanding of the evolutionary history of the cngc gene family, we comprehensively analyzed the phylogeny and evolution of the cngc gene family in all plant lineages.
we used the amino acid sequences of rice and arabidopsis cngcs to query the orthologdb, gramene, tair, congenie, ncbi, phytozome and rgap databases of plant species .
in order to obtain only specific cngc sequences, we removed the hits with exact duplicates , annotated fragments, as well as any unrelated gene like akt or kat family channels from subsequent analyses .
we identified  <dig> cngc-like sequences from  <dig> different species including green algae , bryophytes , lycophytes , gymnosperms , monocots  and dicots   <cit> , whose protein sequences harbored typical domains and motifs of cngc proteins, i.e.
cnbd , ion transport  and iq .
the copy number of cngc gene family ranged from  <dig> to  <dig> amongst the species, where, glycine max had the largest cngc gene family containing  <dig> genes .
however, it should be indicated that the single cngc in wild banana  may not reflect the true number of cngc genes in this species; rather it is due to the limited availability of its genomic information.
a maximum likelihood tree was generated using amino acid sequences of the deduced full-length peptides with jtt  model.
we used syg <dig> from saccharomyces cerevisiae as out-group  <cit> .
according to the tree’s topology, cngc gene family of all plant lineages clustered into four distinct groups  with significant bootstrap values .
group ii and group iv are subdivided into ii-a/ii-b and iv-a/iv-b.
all the cngcs from algae formed the basal lineage and clustered into same group , whereas those from the land plants grouped into several other groups , showing that the cngc family originated earlier than the separation of green algae and the ancestor of land plants.
among these four groups, only group ii was conserved in all land plants, showing that all cngc genes from land plants shared a mutual ancestor after the divergence from aquatic plants.
group ii-b was solely present in lower land plants including mosses  and lycophytes.
similarly, cngcs from group i expanded in both mono- and dicotyledonous angiosperms, while group ii-a and iii was prevalent in all embryophytes  .
group iv-a contain cngcs from vascular plants i.e., embryophytes and lyophytes.
these analyses revealed that all cngc genes from land plants originated from a common ancestor, earlier than the split between lower and higher land plants  <cit> , while the lineage specific expansion and divergence happened in higher land plants, particularly in dicots, after diversification from lower land plants, which lead to generation of group ii-b in basal land plant and group i-iv in higher land plants.
moreover, cngc genes from similar lineage, such as mosses, lycophytes, gymnosperms and angiosperms, inclined to be clustered together.
our interpretations are corroborated by the findings of previous studies  <cit> .figure  <dig>
the maximum-likelihood  phylogenetic tree of
cngc
family genes in  <dig> representative plant species.
the rooted maximum-likelihood  phylogenetic tree was inferred from the amino acid sequences alignment of full length proteins using mega  <dig> , under the jones-taylor-thornton  model.
the bootstrap values from  <dig> resampling, and cut-off values >70% are given at each node.
the node color specifies group i.e.
pink = i, light green = ii-a, aqua = ii-b, red = iii, blue = iv-a, and black = iv-b.
rice cngc genes are shown in blue color.
plant lineages are shown by shapes with different colors, i.e.
green circle = dicots, maroon diamond = monocots, green diamond = gymnosperms, aqua blue square = lycophytes, pink square = moss, and black triangles = green algae.
in silicoprediction of potential cis-acting regulatory elements
the cis-acting regulatory elements are important molecular switches involved in the transcriptional regulation of genes during biotic and abiotic stress responses, and may be induced through independent signal transduction pathways  <cit> .
the 5′ upstream noncoding sequences are not well conserved among the oscngc genes , signifying that these genes may be differentially expressed in response to various stimuli, even if they encode identical proteins  <cit> .
to obtain a preliminary clue about the regulatory function and the molecular mechanism of oscngc genes in transcriptional regulation, potential cis-acting regulatory elements in upstream  sequences of the cngc genes were analyzed.
results exhibited that oscngc gene promoter sequences contain numerous cis-acting regulatory elements regulatory sites for hormones, different biotic and abiotic factors .
twenty different cis-acting regulatory elements are regulated by aba; among them, myb recognition site  related to the drought- and aba-induced gene expression  <cit> , is conserved among  <dig> oscngc genes.
moreover, only  <dig> elements  are regulated by ethylene, two elements  are regulated by cytokinin .
the patterns of cis-acting regulatory elements differed significantly among the oscngc genes.
for example, the promoter of nine oscngcs  contain elements that may respond to all these hormones, biotic and abiotic stress, while others lack the elements responsive to ethylene and cytokinin .
comparatively, only three elements were found to be conserved and consistently present in the upstream region of all  <dig> oscngcs, i.e.
two abiotic stress signaling related elements, consensus gt- <dig> binding site  and myc recognition site , and wrky transcription factor .
the wrky transcription factor  is reported as binding site of rice wrky <dig>  a transcriptional repressor of the gibberellin signaling pathway  <cit> , and a core of tgac-containing w-box in wrky <dig>  which plays an important role in the regulation of early defense-response genes in rice.
the presence of most these cis-acting regulatory elements in oscngc gene promoters depicts their importance in regulation of this gene family .table  <dig>
total number of cis-acting regulatory elements in
oscngc
gene family
oscngcs
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
aba
auxin
ethylene
cytokinin
biotic
abiotic
expression analysis of oscngcs
plant cngc genes have been implicated in diverse aspects of plant growth and development, including responses to hormonal, abiotic and biotic stress  <cit> .
in past, many plant mutants of cngcs, and transgenic plants expressing full-length or mutant cngcs, were characterized to explore their role in plants  <cit> .
these researches mainly focused on arabidopsis cngcs and found that cngc mutants exhibit various phenotypes and multi-functional behaviors.
briefly, atcngc <dig> are probably involved in ca2+ uptake into plants  <cit> , atcngc <dig>   <dig>   <dig> and  <dig> in plant defense and disease resistance pathway  <cit> , atcngc <dig> in homeostasis  <cit> , and atcngc <dig>   <dig>   <dig>   <dig>   <dig>   <dig>  and  <dig> in plant development .
to obtain information about potential differential gene functions in rice, we employed bioinformatics and experimental approaches to assess the role of oscngcs in plant development, and expression patterns in response to various stress.
expression of oscngcsin development
the expression profile of rice leaves obtained from public database ricexpro exhibited that most of oscngc genes showed their maximum expression in late vegetative and early reproductive stage  during life cycle of rice in field conditions, while, oscngc <dig> had its highest expression level at early vegetative stage.
overall, highest expression was observed for oscngc <dig> at 118th day during late reproductive stage, while lowest expression was noted for oscngc <dig> at 20th day of rice growth in field conditions .
expression of oscngcs in rice seedlings
by using quantitative real-time pcr  analysis, the expression level of oscngc genes was investigated in leaves of the rice cultivar nipponbare .
the results indicated there were tremendous variations among the oscngc genes .
oscngc <dig> and  <dig> were highly expressed and had transcripts abundances of  <dig> and  <dig> times that of oscngc <dig>  while oscngc <dig> had the lowest transcripts abundances .figure  <dig>
expression profiles of oscngc genes in 4 weeks old rice leaves.
the expression patterns of oscngc genes under hormonal , biotic  and abiotic  stresses are depicted in figure.
gene expression was perforemd by qpcr, 4 h after each treatment and non-treated ck.
the y-axis indicates the relative experssion levels  of treated versus untreated  for each gene and x axis shows oscngc genes.
the error bars were calculated based on three biological repliactes using standard deviation.
*significant difference at p <  <dig> , **means very significant difference p <  <dig> .
response to hormonal stresses
to examine the expression pattern of oscngcs in response to different plant hormones, qpcr analysis were performed using the total rna extracted from leaves of the rice cultivar nipponbare , subjected to aba , eth , iaa  and kn .
primers used for rt-pcr analysis are listed in table  <dig> table  <dig>
list of
oscngc
and internal control genes primers used for qpcr gene expression
oscngc
1-f
oscngc
1-r
oscngc
2-f
oscngc
2-r
oscngc
3-f
oscngc
3-r
oscngc
4-f
oscngc
4-r
oscngc
5-f
oscngc
5-r
oscngc
6-f
oscngc
6-r
oscngc
7-f
oscngc
7-r
oscngc
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oscngc
8-r
oscngc
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oscngc
9-r
oscngc
10-f
oscngc
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oscngc
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oscngc
11-r
oscngc
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oscngc
12-r
oscngc
13-f
oscngc
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oscngc
14-f
oscngc
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oscngc
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oscngc
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oscngc
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oscngc
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18s rrna-f
18s rrna-r
ubq5-f
ubq5-r
the expression of several cngc genes in rice is regulated by plant hormones.
in general, more genes were up-regulated by aba  and iaa  as compared to kn  and eth .
in sharp contrast, twelve and  <dig> genes were significantly down-regulated by kn and eth, respectively, while none and  <dig> genes were down-regulated by aba and iaa, respectively .
the cngc genes belonging to different phylogenetic groups showed different expression levels from each other, depending on the treatment type, while the genes in the same group showed a similar expression pattern.
for example, the expression of oscngc <dig> and oscngc <dig>  was simultaneously up-regulated by all but eth treatments .
it has been proposed that the proteins classified in the same classes may have similar functions  <cit> .
similar conclusion has been drawn by ma et al.
<cit> , while studying the expression of the c3hc4-type ring finger gene family in rice.
response to biotic and abiotic stress
to determine oscngcs role in disease resistance, we quantified gene expression levels in 4 weeks old rice seedlings, inoculated with two phytopathogens, xanthomonas oryzae pv.
oryzae  and pseudomonas fuscovaginae, that cause a serious blight and bacterial sheath brown rot of rice, respectively.
the expression of the oscngc family genes at  <dig> hpi  are shown in figure  <dig>  the data depicted that large number of oscngc genes were significantly up-regulated after infection with each pathogen within 4 h. fourteen oscngcs were significantly up-regulated by p. fuscovaginae inoculation with oscngc <dig> being at the highest relative level of expression.
the expression of oscngc <dig> and  <dig> were not affected .
similarly  <dig> oscngcs were up-regulated by xoo, where the expression of oscngc <dig> and  <dig> increased by  <dig>  and  <dig> -folds respectively.
previous genetic approaches have identified three cngcs in arabidopsis  exhibited alterations in defense responses and reported to be involved in plant immunity  <cit> .
these two mutants are reported to have successfully managed the lower growth rates of p. syringae, accomplished by sustaining high levels of salicylic acid, leading to constitutive expression of pathogenesis-related  genes, and other defense responses  <cit> .
it is noteworthy to mention that atcngc <dig> &  <dig> are the closest homologue of the members of rice cngcs from group iv, which showed highest expression levels in response to p. fuscovaginae, suggesting that these oscngc genes are early responsive and sensitive to biotic stress, and their regulation plays an important role in plant defense.
similarly, the expression levels of the oscngc family genes under cold stress condition were also investigated.
the results indicated that  <dig> oscngcs were up-regulated under cold stress.
it was noted that the expression of oscngcs belonging to phylogenetic group i, ii and iii were significantly induced by cold stress, with fold range of 5– <dig> for group i , 3– <dig> for group ii , and 4– <dig> folds for group iii , respectively .
on other hand, oscngcs from group iv  showed relatively lower expression under cold stress.
overall, oscngc <dig> gene showed the highest expression level  in response to cold stress, while oscngc <dig> showed the lowest level expression  .
interestingly, six of  <dig> gene pairs located on duplicated chromosomal segments showed differential expression patterns, suggesting that most duplicated gene pairs were under the diverse transcriptional controls.
for example, oscngc <dig> and oscngc <dig> localized on duplicated segments exhibit similar expression patterns in response to both pathogenic and abiotic stresses , indicating their overlapping functions.
this finding was similar to previous results showing that the expression of duplicated genes frequently diverges compared to that of their ancestors, suggesting that duplication was a major reason for the enrichment of the functions of this family during the long course of evolution  <cit> .
it is proposed that a change in a duplicated locus of two genes might not exhibit the same morphological and/or physiological phenotypes.
however, when gene duplication occurs, some genes may retain their original functions and expression patterns  <cit> .
no close correlation was observed between cis-acting regulatory elements in upstream sequences of rice cngc genes and their qpcr expression levels.
it is probable that the differential response of genes to different stimuli is due to different upstream sequences and introns.
these results suggest that many of the oscngc genes play discrete roles in development, response to stress and plant defense related functions in rice.
however, further studies based on gene knockout techniques are required to properly identify and explore their functions.
CONCLUSIONS
there are  <dig> cngc genes in rice, which are classified into  <dig> groups  and two sub-groups ; this gene family appears to have expanded through both chromosomal segmentation and tandem duplications.
the cngcs from all plant lineages are also clustered into four groups, with group ii being conserved in all land plants.
all the oscngc protein sequences contain a cngc specific domain cnbd that comprises a pbc and a “hinge” region, featured by a stringent motif: --x--x-g--ell-x-w-x-sa-x-t-x--af-x-l).
various cis-acting regulatory elements were identified in the upstream sequences present on both positive and negative strands.
in addition, the genes transcripts significantly respond to multiple stimuli, as demonstrated by their expression patterns to exogenous hormonal , biotic  and abiotic  stresses.

 calcium signal transduction through calcium channels is a fundamental mechanism used by plants to sense and respond to endogenous and environmental stimuli, including hormone responses, plant–pathogen interaction, development, symbiosis, salt stress, light signaling, and circadian rhythm. one of the potential pathways for the uptake of ca2+ ions in signal transduction is via cyclic nucleotide-gated ion channels. cngcs, functionally defined as ligand-gated protein channels, are nonselective cation channels, gated by the direct binding of cyclic nucleotides, and are present in both animals and plants system. in animals, their biological roles and regulation have been well studied, however, plant cngcs have only been investigated much more recently, and corresponding regulatory mechanisms are just starting to be discovered. studies have revealed that cngcs may function as a pathway for ca2+ conduction into the cytosol as an early event during pathogen defense signaling cascades, and plant response to other biotic and abiotic stresses. plant cngc ion channels were first identified in a screen for calmodulin binding partners in barley. a large family of cngcs composed of members have been identified in arabidopsis genome which are classified into four groups and two sub-groups “iv-a and iv-b”. these cngcs are characterized by general structural resemblance to animal cngcs, having a predicted structure of six transmembrane domains with a pore domain between s and s c-terminal cyclic nucleotide-binding domain and cam-binding domains. in the presence of ca2+, cam binds to the cngc at the αc helix of the cnbd and thus blocks channel gating by cnmps. cnbd being most conserved region of the cngc with a phosphate binding cassette and a “hinge” region. where, the pbc binds to the sugar and phosphate moieties of the cyclic nucleotide ligand, and the hinge region contributes to ligand binding efficacy and selectivity. in arabidopsis, cngc genes are differentially expressed in all tissues and their functions critically depend on the presence of camp and/or cgmp that function as signaling molecules. physiological processes in which these signaling molecules are believed to be involved include various developmental processes, photo-morphogenesis and tolerance to salt stress, gibberellic acid-induced signaling in barley and phytochrome signaling. approximately 5% of the oryza sativa genome appears to encode membrane transport proteins. many of these proteins are classified and several hundred putative transporters have not yet been assigned to families, including the cngc gene family. the early investigations on rice genome, revealed a clue about the existence of putative cngc fragments, as suggested by bridges et al. and ramanjaneyulu et al.. however, no comprehensive study has yet been undertaken, which can lead to the proper identification, characterization and functional verification of the cngc family in rice. taking privilege from the accessibility of recent release of entire genome sequences of rice and information available from a. thaliana, we performed a genome-wide identification of cngc family in rice in the present study. we found that the rice genome contains cngc full length genes which are classified into four major classes and two sub-groups as in arabidopsis. evolutionary history reconstruction via comparison with cngcs from other plant species was accomplished. furthermore, comprehensive analyses were performed to characterize these genes and protein sequence features e.g., domain, motif, phylogenetic relationships, gene structure, multiple alignment, cis-acting regulatory elements etc. to verify their functions, we analyzed the expression pattern of rice cngc genes by quantitative pcr in response to various hormonal, biotic and abiotic stresses. the present work represents the first comprehensive study of cngc genes in rice and offers a solid base for further functional investigation of this important gene family in rice.  identification of cngc genes in rice genome to obtain a complete overview of cngc gene family in rice, we conducted a genome-wide analysis by using various bioinformatics resources. considering the fact that the arabidopsis cngc gene family is fully characterized, we used the amino acid sequences of arabidopsis cngc genes as the queries in blast searches and the keyword ‘cyclic nucleotide-gated ion channel’ in the putative function search tool of rice database. seventeen non-redundant putative gene sequences were retrieved in rice with five genes having potential alternative splice forms. since plant cngcs, are characterized by the presence of cnbd, specifically phosphate binding cassettes and hinge region, thus their presence in a protein sequence is a validation criteria for these genes. the sequence searches of databases based on annotation are not completely accurate and could potentially miss some bonafide cngc sequences or inaccurately assign certain sequences as cngcs. therefore, the deduced protein sequences of all the putative genes were further analyzed to confirm the presence of cnbd/cyclic nucleotide-monophosphate binding domain, cap family effector domain, rmlc-like jelly roll fold, and ion transport domains. we found that loc_os06g previously designated as “genomic cyclic nucleotide-gated ion channel 1”, was identified as “cyclic nucleotide-gated channel c ”, and was eradicated from analysis due to lack of above cited domains. however, a aa short fragment, loc_os06g contained the cngc characteristic cnbd domain, with phosphate binding cassettes and hinge region as suggested by zelman et al., was recognized as bonafide cngc. these identifications were purely based on the presence of primary cnbd domain, and the validation of the consensus motif derived by zelman et al.. we noticed that blast search of arabidopsis cngc also resulted into gene from potassium akt /kat channels as homologs of cngcs, due to the presence of transmembrane region, cnbd and ion transport domains and additional ankyrin repeats. such unrelated genes were rejected from analysis. finally, full length cngc genes were confirmed to contain the specific domains in their proteins sequences, and hence were identified as cngc genes in rice.table domain architecture of oscngc gene family 1 group i group ii group iii group iva group ivb: itp, cnmp_bd, is- cnmp_bd. cyclic nucleotide- monophosphate, cap_ed, tm, crp; ccer, pln, cnmp_bd, cnmp_bd-like, rmlc. oscngcs chromosomal localization and diversification chromosome localization analysis demonstrated that the oscngcs are located on eight rice chromosomes. to better reflect the orthologous relationship between the rice and arabidopsis cngc genes, the nomenclature of respective rice cngc genes was annotated according to their order in phylogenies, with os abbreviating o. sativa.figure genomic localization and duplication of cngc genes in rice chromosomes. chromosome numbers are indicated at the top of each chromosome. genes are marked in abbreviations i.e. c stands for cngc; those present on duplicated segments of genome are connected by red lines and tandem duplicated genes are marked in blue. arabidopsis has been reported to have cngc genes. conversely, in rice that has a genome size nearly three times as that of arabidopsis, the number of cngc genes was found to be only the reason for this could be the variable status of whole genome duplications in arabidopsis and rice. in this study, both segmental and tandem duplication events were investigated for elucidating the potential mechanism of evolution of oscngc gene family in rice. analysis of pgdd revealed that three genes pairs, oscngc and oscngc oscngc and oscngc as well as oscngc and oscngc located on different chromosomes regions, might have been induced by segmental duplication, and could be assigned to rgap segmental duplication blocks. in addition, one gene pair, oscngc and oscngc were located at the adjacent positions on chromosome and separated by genes only. hence suggested, that these two genes were generated from tandem duplication event. these results indicate that gene duplication not only expanded the number of genes in the rice cngc family but also increased its functional diversity. gene structure and features of oscngc proteins comparisons between genomic dna sequences with corresponding cdna sequences showed that coding sequences of oscngc genes are interrupted by introns. introns may interrupt the reading frame of a gene between two consecutive codons, between the first and second nucleotide of a codon, or between the second and the third nucleotide. gene structural analysis showed that the rice cngcs had different gene structures as compared to arabidopsis cngcs, with a variable number of introns and lengths. the numbers of introns in arabidopsis cngcs ranged from to introns, while rice cngc ranged from to introns. unlike the arabidopsis cngcs, introns of all oscngcs belong to phase and, while, arabidopsis cngc contained, four phase introns. this suggests that all cngcs with introns did not ascend from a retrotransposition event, and the different numbers may be caused by the insertion and loss of introns during their evolution. the oscngc proteins range in length from to amino acids, with an average of aa. expasy analysis showed that oscngc proteins vary greatly in isoelectric point values and molecular weights. according to their instability index values, only of oscngc proteins can be considered as stable with an instability index of < =.table predicted sequence features of oscngc proteins oscngc1 oscngc2 oscngc3 oscngc4 oscngc5 oscngc6 oscngc7 oscngc8 oscngc9 oscngc10 oscngc11 oscngc12 oscngc13 oscngc14 oscngc15 oscngc16 mw, pi, camp- cgmp, casein, pkc, tyr, n-myr, n-glyc, p.m, cyt, c.t.m, m.i.m. the subcellular localization of each oscngc was predicted by psort analysis. our results evidenced that thirteen oscngcs are predicted to be localized in plasma membrane, while the others i.e., oscngc oscngc and oscngc to be in cytoplasm, chloroplast thylakoid membrane and mitochondrial inner membrane, respectively. all relevant experiments in the past have indicated that plant cngc proteins are specifically localized to the plasma membrane. the presence of oscngc in cytoplasm is not clear, which needs further experimental elucidation. arabidopsis cngc proteins, when analyzed revealed that out of cngc proteins were localized in plasma membrane, while the other in chloroplast thylakoid membrane. translated proteins are often subjected to post–translational modifications to become functionally active. ptms are the chemical modification of a protein after its translation, and have wide effects on broadening its range of functionality. cngc channels can be controlled by variations in the phosphorylation state catalyzed by ser/thr kinases and phosphatases. when analyzed in scanprosite, multiple putative phosphorylation sites were revealed in oscngc protein sequences, which may act as substrates for several kinases in the form of casein kinase ii, protein kinase c, tyrosine kinase and camp/cgmp kinases. francis and corbin demonstrated that camp/cgmp-dependent protein kinases are major intracellular receptors for these nucleotides, and the actions of these enzymes account for much of the cellular responses to increased levels of camp or cgmp. as a special class of ptms, several proteins including cngcs could be covalently altered by a range of lipids, such as myristate and palmitate, a phenomenon known as lipidation. protein n-myristoylation can alter the lipophilicity of the target protein and facilitate its interaction with membranes thereby affecting its subcellular localization. our bioinformatics analysis revealed that all oscngc proteins bear potential n-myristoylation/ n-glycosylation motif sites. the maximum number of n-myristoylation sites is present in oscngc and oscngc, while oscngc contain highest number of n-glycosylation sites. in mammals, n-myristoylated proteins include protein kinases, phosphatases, guanine nucleotide-binding proteins, and ca2+-binding proteins, many of which participate in signal transduction pathways. similarly, n-glycosylation, post-translational modification that occur in many eukaryotic proteins exposed to the extracellular medium, is reported to play various roles, including facilitating the folding, trafficking and function of the protein as well as protecting it from an extracellular medium, which, in plants, is acidic and rich in proteases. in a recent study, meighan et al. demonstrated that extracellular ca2+ and zn2+-dependent proteases increase the ligand sensitivity of rod and cone cng channels, and prolonged exposure of cng channels to mmp even caused the channels to become nonconductive due to mmp-dependent proteolysis. they have further indicated that presence of glycosylation sites may defend cng channels from mmp-dependent proteolysis, and prevent the cngc channels to become nonconductive. therefore the ptms and other sequence-specific features such as n-myristoylation and n-glycosylation in rice cngcs are likely to be necessary for their proper functioning, localization and regulation. motif composition analysis a functional motif-based recognition of cngc proteins can contribute to understanding the evolutionary history of this gene family including possible role of genome and gene duplication events. motif search by the meme/mast identified conserved motifs in rice cngc proteins. among these, five motifs were identified in rice, and found to be associated with functionally defined domains. all the oscngc proteins share a conserved binding domain for cyclic nucleotides in the central region, referred to as motif plant cngcs are believed to be activated by direct and reversible binding of nucleotides cgmp and camp to the cnbd, which allosterically causes the channel to open. in the presence of calcium, cam binds to the cambd and prevents the binding of cyclic nucleotides to the cnbd, and thereby stops the activation of cngcs by cyclic nucleotides. this competitive regulation by cyclic nucleotides and cams was predicted and experimentally verified for atcngc. the cam-binding site was recently mapped to an isoleucine glutamine motif by fischer et al.. our results also identified iq as a functional motif within cambd, downstream of the cnbd. iq motifs have been investigated in detail in animal proteins, which often bind cam in a ca2+-dependent manner, while ca2+ induces the displacement of the cam ligand. fischer et al. showed that iq motif is conserved among plant cngcs, this motif enhances the variability of ca2+-dependent channel control mechanisms featuring the functional diversity within this multigene family. however, in rice, iq motifs were present in less than 50% of the members of oscngc family, mainly in the members of group and, suggesting that other regulators may alter the channel opening and for gating control of plant cngcs which lack these motifs. other functional motifs denoted as motif and associated with prenyltransferase and squalene oxidase repeat, and ion transport, respectively, was found to be conserved in all oscngc proteins except oscngc the functions of other motifs identified in oscngc proteins 5– 9– and have not been reported in any plant or animal so far. moreover, the sequence logo of three potential motifs, cnbd, iq and ion transport one can determine not only the consensus sequence but also the relative frequency of bases and the information content at every position in a site or sequence. the logo displays both significant residues and subtle sequence patterns.figure distribution of conserved motifs in rice cngc proteins identified using meme search tool. schematic representation of motifs composition in oscngc protein sequences using meme motif search tool for each groups given separately. each motif is represented by a number in colored box. length of box does not correspond to length of motif. order of the motifs corresponds to position of motifs in individual protein sequence. we also performed meme analysis on arabidopsis cngc proteins for comparison. the diagram of atcngc motifs, their details and the sequence logo of functional motifs is given in additional files and the numbers of motifs are designated similarly in both rice and arabidopsis cngc proteins, each corresponding to the position/location of the respective motif. in arabidopsis, only three motifs were identified representing the cnbd domain, and iq motif, where cnbd domain was represented by two motifs interrupted by non-functional motifs. interestingly, motifs associated with ion transport, zinc finger, prenyltransferase and squalene oxidase repeat were absent in arabidopsis. though, individual scan of atcngc proteins revealed the presence of ion transport domain in some members. as mentioned earlier, an essential structural feature of plant cngcs is their cnbd, a direct binding site for camp/cgmp to modify the channel opening. the most conserved feature of cnbd domain is the pbc that makes direct contact with camp, and the hinge region, essential for the capping of camp by the c-helix of the cnbd. no plant cngc-specific motifs had been reported until zelman et al. aligned the pbc and hinge regions for atcngc proteins, from which the following consensus motif was derived: --x--x-g-x--ll-x- -x--e-x-f- ). similarly we aligned the cnbd region of rice cngcs and identified a putative pbc and a hinge. we identified a conserved glycine, acidic residue glutamate followed by two aliphatic leucines and aromatic tryptophan inside the pbcs. we also detected that the putative hinge also comprises a conserved aromatic phenylalanine, aliphatic alanine and leucines. a neutral residue, threonine was also found to be conserved near the hinge region. we successively built a stringent motif: --x--x-g--ell-x-w-x-sa-x-t-x--af-x-l) that recognizes the rice cngcs specifically. the rice pbc and hinge regions differed from the conserved residues of arabidopsis. for example, residues e, w and t within pbcs, and residues a and l in hinges were found be conserved at 100% in rice cngcs only. the diversity of motif patterns between the rice and arabidopsis indicate the functional divergence across the species and individual groups.figure the rice cngc-specific motif spans the putative pbc and the hinge within the cnbd of the oscngcs. the diagram at the top represents three regions of plant cngcs: the six transmembrane domains, a cnbd containing a pbc and the hinge, and cambd. the rice cngc-specific amino acid motif is shown below the cartoon. in the square brackets “” are the amino acids allowed in this position of the motif, “x” represents any amino acid and the round brackets “()” indicate the number of amino acids. below is the alignment of cnbd domains of rice cngcs. residues in white highlighted in blue indicate 100% identity among the rice cngcs. phylogenetic analysis phylogenetic relationship between rice and arabidopsiscngc family genes to determine the phylogenic relationship of cngc family between rice and arabidopsis, a maximum likelihood phylogenetic tree was constructed using full-length amino acid sequences. four groups, as described by mäser et al. were identified containing representative gene of both rice and arabidopsis. all the arabidopsis cngc proteins were found to lie in groups similar to those identified previously. of the four groups, three groups are monophyletic, while one group is sub-divided into two distinct clades, named group iv-a and iv-b. group i comprises three members from rice cngcs and six from arabidopsis. similarly, group ii contains three rice cngcs and five atcngcs. however, group iii embraces five in rice and five in arabidopsis, thus form the largest group, with members. two cngcs from each rice and arabidopsis were assigned to group iv-a, while, three rice cngcs and two arabidopsis cngcs segregated into group iv-b. a separate phylogenetic tree was also generated from conserved cnbd domains sequences of all the cngc proteins in rice and arabidopsis, which resulted into similar clustering pattern.figure phylogenetic tree of oscngc and atcngc proteins. the multiple alignment was performed by clustalx program. mega was used to create the maximum likelihood under the jones-taylor-thornton model. the bootstrap values from resampling are given at each node. the rice cngc genes identified in this study are shown in red circles and arabidopsis cngcs are shown in blue squares. rice cngc genes were designated according to their order in phylogeny. during the evolution of a gene family, gene duplication plays important roles in generating new members and creating novel gene functions, hence increasing numbers and diversifying functions of genes within a gene family. the phylogenetic analysis between rice and arabidopsis following previously described model demonstrates the evolutionary trend of gene duplication in both taxa. phylogenetic data provide a framework for making more appropriate intergenomic comparisons, by determining whether chromosomal duplications within taxa pre-date or post-date divergence among taxa. the current analysis shows that divergence of two taxa may have pre-dated the gene duplication events among them resulting in gene expansion within individual group. this is further strengthened by our detailed phylogenetic analysis among all plant lineages given ahead. the detailed phylogeny also points towards the duplication of cngc genes occurring after speciation and after divergence of angiosperms into mono and dicots. interestingly atcngc and oscngc14; atcngc and oscngc are not in congruence with the pattern of other sister groups. the existing anomaly between these taxa could not be reasoned out. phylogenetic analysis of the cngcgenes in all plant lineages for a better understanding of the evolutionary history of the cngc gene family, we comprehensively analyzed the phylogeny and evolution of the cngc gene family in all plant lineages. we used the amino acid sequences of rice and arabidopsis cngcs to query the orthologdb, gramene, tair, congenie, ncbi, phytozome and rgap databases of plant species. in order to obtain only specific cngc sequences, we removed the hits with exact duplicates, annotated fragments, as well as any unrelated gene like akt or kat family channels from subsequent analyses. we identified cngc-like sequences from different species including green algae, bryophytes, lycophytes, gymnosperms, monocots and dicots, whose protein sequences harbored typical domains and motifs of cngc proteins, i.e. cnbd, ion transport and iq. the copy number of cngc gene family ranged from to amongst the species, where, glycine max had the largest cngc gene family containing genes. however, it should be indicated that the single cngc in wild banana may not reflect the true number of cngc genes in this species; rather it is due to the limited availability of its genomic information. a maximum likelihood tree was generated using amino acid sequences of the deduced full-length peptides with jtt model. we used syg from saccharomyces cerevisiae as out-group. according to the tree’s topology, cngc gene family of all plant lineages clustered into four distinct groups with significant bootstrap values. group ii and group iv are subdivided into ii-a/ii-b and iv-a/iv-b. all the cngcs from algae formed the basal lineage and clustered into same group, whereas those from the land plants grouped into several other groups, showing that the cngc family originated earlier than the separation of green algae and the ancestor of land plants. among these four groups, only group ii was conserved in all land plants, showing that all cngc genes from land plants shared a mutual ancestor after the divergence from aquatic plants. group ii-b was solely present in lower land plants including mosses and lycophytes. similarly, cngcs from group i expanded in both mono- and dicotyledonous angiosperms, while group ii-a and iii was prevalent in all embryophytes. group iv-a contain cngcs from vascular plants i.e., embryophytes and lyophytes. these analyses revealed that all cngc genes from land plants originated from a common ancestor, earlier than the split between lower and higher land plants, while the lineage specific expansion and divergence happened in higher land plants, particularly in dicots, after diversification from lower land plants, which lead to generation of group ii-b in basal land plant and group i-iv in higher land plants. moreover, cngc genes from similar lineage, such as mosses, lycophytes, gymnosperms and angiosperms, inclined to be clustered together. our interpretations are corroborated by the findings of previous studies.figure the maximum-likelihood phylogenetic tree of cngc family genes in representative plant species. the rooted maximum-likelihood phylogenetic tree was inferred from the amino acid sequences alignment of full length proteins using mega, under the jones-taylor-thornton model. the bootstrap values from resampling, and cut-off values >70% are given at each node. the node color specifies group i.e. pink = i, light green = ii-a, aqua = ii-b, red = iii, blue = iv-a, and black = iv-b. rice cngc genes are shown in blue color. plant lineages are shown by shapes with different colors, i.e. green circle = dicots, maroon diamond = monocots, green diamond = gymnosperms, aqua blue square = lycophytes, pink square = moss, and black triangles = green algae. in silicoprediction of potential cis-acting regulatory elements the cis-acting regulatory elements are important molecular switches involved in the transcriptional regulation of genes during biotic and abiotic stress responses, and may be induced through independent signal transduction pathways. the 5′ upstream noncoding sequences are not well conserved among the oscngc genes, signifying that these genes may be differentially expressed in response to various stimuli, even if they encode identical proteins. to obtain a preliminary clue about the regulatory function and the molecular mechanism of oscngc genes in transcriptional regulation, potential cis-acting regulatory elements in upstream sequences of the cngc genes were analyzed. results exhibited that oscngc gene promoter sequences contain numerous cis-acting regulatory elements regulatory sites for hormones, different biotic and abiotic factors. twenty different cis-acting regulatory elements are regulated by aba; among them, myb recognition site related to the drought- and aba-induced gene expression, is conserved among oscngc genes. moreover, only elements are regulated by ethylene, two elements are regulated by cytokinin. the patterns of cis-acting regulatory elements differed significantly among the oscngc genes. for example, the promoter of nine oscngcs contain elements that may respond to all these hormones, biotic and abiotic stress, while others lack the elements responsive to ethylene and cytokinin. comparatively, only three elements were found to be conserved and consistently present in the upstream region of all oscngcs, i.e. two abiotic stress signaling related elements, consensus gt- binding site and myc recognition site, and wrky transcription factor. the wrky transcription factor is reported as binding site of rice wrky a transcriptional repressor of the gibberellin signaling pathway, and a core of tgac-containing w-box in wrky which plays an important role in the regulation of early defense-response genes in rice. the presence of most these cis-acting regulatory elements in oscngc gene promoters depicts their importance in regulation of this gene family.table total number of cis-acting regulatory elements in oscngc gene family oscngcs 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 aba auxin ethylene cytokinin biotic abiotic expression analysis of oscngcs plant cngc genes have been implicated in diverse aspects of plant growth and development, including responses to hormonal, abiotic and biotic stress. in past, many plant mutants of cngcs, and transgenic plants expressing full-length or mutant cngcs, were characterized to explore their role in plants. these researches mainly focused on arabidopsis cngcs and found that cngc mutants exhibit various phenotypes and multi-functional behaviors. briefly, atcngc are probably involved in ca2+ uptake into plants, atcngc and in plant defense and disease resistance pathway, atcngc in homeostasis, and atcngc and in plant development. to obtain information about potential differential gene functions in rice, we employed bioinformatics and experimental approaches to assess the role of oscngcs in plant development, and expression patterns in response to various stress. expression of oscngcsin development the expression profile of rice leaves obtained from public database ricexpro exhibited that most of oscngc genes showed their maximum expression in late vegetative and early reproductive stage during life cycle of rice in field conditions, while, oscngc had its highest expression level at early vegetative stage. overall, highest expression was observed for oscngc at 118th day during late reproductive stage, while lowest expression was noted for oscngc at 20th day of rice growth in field conditions. expression of oscngcs in rice seedlings by using quantitative real-time pcr analysis, the expression level of oscngc genes was investigated in leaves of the rice cultivar nipponbare. the results indicated there were tremendous variations among the oscngc genes. oscngc and were highly expressed and had transcripts abundances of and times that of oscngc while oscngc had the lowest transcripts abundances.figure expression profiles of oscngc genes in 4 weeks old rice leaves. the expression patterns of oscngc genes under hormonal, biotic and abiotic stresses are depicted in figure. gene expression was perforemd by qpcr, 4 h after each treatment and non-treated ck. the y-axis indicates the relative experssion levels of treated versus untreated for each gene and x axis shows oscngc genes. the error bars were calculated based on three biological repliactes using standard deviation. *significant difference at p < , **means very significant difference p < . response to hormonal stresses to examine the expression pattern of oscngcs in response to different plant hormones, qpcr analysis were performed using the total rna extracted from leaves of the rice cultivar nipponbare, subjected to aba, eth, iaa and kn. primers used for rt-pcr analysis are listed in table  table list of oscngc and internal control genes primers used for qpcr gene expression oscngc 1-f oscngc 1-r oscngc 2-f oscngc 2-r oscngc 3-f oscngc 3-r oscngc 4-f oscngc 4-r oscngc 5-f oscngc 5-r oscngc 6-f oscngc 6-r oscngc 7-f oscngc 7-r oscngc 8-f oscngc 8-r oscngc 9-f oscngc 9-r oscngc 10-f oscngc 10-r oscngc 11-f oscngc 11-r oscngc 12-f oscngc 12-r oscngc 13-f oscngc 13-r oscngc 14-f oscngc 14-r oscngc 15-f oscngc 15-r oscngc 16-f oscngc 16-r 18s rrna-f 18s rrna-r ubq5-f ubq5-r the expression of several cngc genes in rice is regulated by plant hormones. in general, more genes were up-regulated by aba and iaa as compared to kn and eth. in sharp contrast, twelve and genes were significantly down-regulated by kn and eth, respectively, while none and genes were down-regulated by aba and iaa, respectively. the cngc genes belonging to different phylogenetic groups showed different expression levels from each other, depending on the treatment type, while the genes in the same group showed a similar expression pattern. for example, the expression of oscngc and oscngc was simultaneously up-regulated by all but eth treatments. it has been proposed that the proteins classified in the same classes may have similar functions. similar conclusion has been drawn by ma et al., while studying the expression of the c3hc4-type ring finger gene family in rice. response to biotic and abiotic stress to determine oscngcs role in disease resistance, we quantified gene expression levels in 4 weeks old rice seedlings, inoculated with two phytopathogens, xanthomonas oryzae pv. oryzae and pseudomonas fuscovaginae, that cause a serious blight and bacterial sheath brown rot of rice, respectively. the expression of the oscngc family genes at hpi are shown in figure  the data depicted that large number of oscngc genes were significantly up-regulated after infection with each pathogen within 4 h. fourteen oscngcs were significantly up-regulated by p. fuscovaginae inoculation with oscngc being at the highest relative level of expression. the expression of oscngc and were not affected. similarly oscngcs were up-regulated by xoo, where the expression of oscngc and increased by and -folds respectively. previous genetic approaches have identified three cngcs in arabidopsis exhibited alterations in defense responses and reported to be involved in plant immunity. these two mutants are reported to have successfully managed the lower growth rates of p. syringae, accomplished by sustaining high levels of salicylic acid, leading to constitutive expression of pathogenesis-related genes, and other defense responses. it is noteworthy to mention that atcngc & are the closest homologue of the members of rice cngcs from group iv, which showed highest expression levels in response to p. fuscovaginae, suggesting that these oscngc genes are early responsive and sensitive to biotic stress, and their regulation plays an important role in plant defense. similarly, the expression levels of the oscngc family genes under cold stress condition were also investigated. the results indicated that oscngcs were up-regulated under cold stress. it was noted that the expression of oscngcs belonging to phylogenetic group i, ii and iii were significantly induced by cold stress, with fold range of 5– for group i, 3– for group ii, and 4– folds for group iii, respectively. on other hand, oscngcs from group iv showed relatively lower expression under cold stress. overall, oscngc gene showed the highest expression level in response to cold stress, while oscngc showed the lowest level expression. interestingly, six of gene pairs located on duplicated chromosomal segments showed differential expression patterns, suggesting that most duplicated gene pairs were under the diverse transcriptional controls. for example, oscngc and oscngc localized on duplicated segments exhibit similar expression patterns in response to both pathogenic and abiotic stresses, indicating their overlapping functions. this finding was similar to previous results showing that the expression of duplicated genes frequently diverges compared to that of their ancestors, suggesting that duplication was a major reason for the enrichment of the functions of this family during the long course of evolution. it is proposed that a change in a duplicated locus of two genes might not exhibit the same morphological and/or physiological phenotypes. however, when gene duplication occurs, some genes may retain their original functions and expression patterns. no close correlation was observed between cis-acting regulatory elements in upstream sequences of rice cngc genes and their qpcr expression levels. it is probable that the differential response of genes to different stimuli is due to different upstream sequences and introns. these results suggest that many of the oscngc genes play discrete roles in development, response to stress and plant defense related functions in rice. however, further studies based on gene knockout techniques are required to properly identify and explore their functions.  there are cngc genes in rice, which are classified into groups and two sub-groups; this gene family appears to have expanded through both chromosomal segmentation and tandem duplications. the cngcs from all plant lineages are also clustered into four groups, with group ii being conserved in all land plants. all the oscngc protein sequences contain a cngc specific domain cnbd that comprises a pbc and a “hinge” region, featured by a stringent motif: --x--x-g--ell-x-w-x-sa-x-t-x--af-x-l). various cis-acting regulatory elements were identified in the upstream sequences present on both positive and negative strands. in addition, the genes transcripts significantly respond to multiple stimuli, as demonstrated by their expression patterns to exogenous hormonal, biotic and abiotic stresses. 
likewise, the cngcs from all plant lineages clustered into four groups, where group ii was conserved in all land plants. the various cis-acting regulatory elements in the upstream sequences may be responsible for responding to multiple stimuli, including hormonal, biotic and abiotic stresses. prediction of cis-acting regulatory elements in 5′ upstream sequences and expression analyses through quantitative qpcr demonstrated that oscngc genes were highly responsive to multiple stimuli including hormonal, biotic and abiotic stress. there are cngc genes in rice, which were probably expanded through chromosomal segmentation and tandem duplications and comprise a pbc and a “hinge” region in the cnbd domain, featured by a stringent motif. to date there has not been a genome-wide overview of the cngc gene family in any economically important crop, including rice. there is an urgent need for a thorough genome-wide analysis and experimental verification of this gene family in rice. gene duplication analysis revealed that both chromosomal segmentation and tandem duplications significantly contributed to the expansion of this gene family. cngcsricebiotic and abiotic stressbioinformaticsphylogenetic analysisresistanceissue-copyright-statement© the author 2014 in addition, oscngc proteins also contain various other functional motifs and post-translational modification sites. we successively built a stringent motif: --x--x-g- -ell-x-w-x-sa-x-t-x--af-x-l) that recognizes the rice cngcs specifically. they have been implicated in the uptake of both essential and toxic cations, ca2+ signaling, pathogen defense, and thermotolerance in plants.
 cyclic nucleotide-gated channels are ca2+-permeable cation transport channels, which are present in both animal and plant systems. they have been implicated in the uptake of both essential and toxic cations, ca2+ signaling, pathogen defense, and thermotolerance in plants. to date there has not been a genome-wide overview of the cngc gene family in any economically important crop, including rice. there is an urgent need for a thorough genome-wide analysis and experimental verification of this gene family in rice.  in this study, a total of full length rice cngc genes distributed on chromosomes 1– and were identified by employing comprehensive bioinformatics analyses. based on phylogeny, the family of oscngcs was classified into four major groups and two sub-groups. likewise, the cngcs from all plant lineages clustered into four groups, where group ii was conserved in all land plants. gene duplication analysis revealed that both chromosomal segmentation and tandem duplications significantly contributed to the expansion of this gene family. motif composition and protein sequence analysis revealed that the cngc specific domain “cyclic nucleotide-binding domain ” comprises a “phosphate binding cassette” and a “hinge” region that is highly conserved among the oscngcs. in addition, oscngc proteins also contain various other functional motifs and post-translational modification sites. we successively built a stringent motif: --x--x-g- -ell-x-w-x-sa-x-t-x--af-x-l) that recognizes the rice cngcs specifically. prediction of cis-acting regulatory elements in 5′ upstream sequences and expression analyses through quantitative qpcr demonstrated that oscngc genes were highly responsive to multiple stimuli including hormonal, biotic and abiotic stress.  there are cngc genes in rice, which were probably expanded through chromosomal segmentation and tandem duplications and comprise a pbc and a “hinge” region in the cnbd domain, featured by a stringent motif. the various cis-acting regulatory elements in the upstream sequences may be responsible for responding to multiple stimuli, including hormonal, biotic and abiotic stresses. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords cngcsricebiotic and abiotic stressbioinformaticsphylogenetic analysisresistanceissue-copyright-statement© the author 2014 