BACKGROUND
calcium , a universal second messenger in eukaryotes, mediates stimulus–response coupling in the regulation of diverse cellular functions  <cit> .
various extracellular stimuli elicit specific calcium signatures that can be recognized by different calcium sensors.
the three main classes of ca2+ sensors identified in plants are: cams  and cam-like proteins, cbls , and cdpks  .
the cdpks, also termed cpks, consist of a variable n-terminal domain, a conserved serine/threonine kinase domain, an auto-inhibitory junction region, and a c-terminal regulatory cam-like domain  <cit> .
unlike the other ca2+ sensors, cdpks have both ca2+ sensing and responding activities due to their unique, cam-like domain and protein kinase domain, which convert upstream ca2+ signals into downstream phosphorylation events and cellular responses  <cit> .
genome-wide analysis, together with comparative genomics, provides an effective way to understand the structures and functions of members of a gene family, using the insights gained from evolutionary relationships and experimental data.
the original, systematic report on the cdpk gene family in arabidopsis thaliana identified  <dig> cdpk gene family members  <cit> , and was followed by research in rice   <cit>  and wheat   <cit> .
recently, genome-wide analyses of the cdpk gene family have been reported in maize   <cit>  and poplar   <cit> .
meanwhile, more and more investigations of cdpk genes have also involved horticultural plants, such as alfalfa  <cit> , potato  <cit> , strawberry  <cit> , and tomato  <cit> .
work in arabidopsis showed that cdpks function in immune and stress signaling, growth and development, and hormone responses.
atcpks play vital parts in immune signaling pathways; for example, atcpk <dig> activates nadph oxidase, resulting in an oxidative burst  <cit> , and phosphorylates pal  resulting in accumulation of salicylic acid   <cit> .
atcpk4/5/6/ <dig> phosphorylates a specific subgroup of wrky transcription factors, wrky8/28/ <dig>  which participate in nlr-dependent restriction of pathogen growth.
in addition, atcpk <dig> activates rboh to induce a reactive oxygen species  burst  <cit> .
cpks also function in the response to abiotic stress.
for example, atcpks help to enhance drought tolerance by responding to abscisic acid , leading to induction of expression of genes such as atcpk4/ <dig>  <cit> , and stomatal closure via induction of atcpk3/ <dig>  <cit> .
atcpk4/ <dig>  <cit>  and atcpk <dig>  <cit>  trigger plant salt tolerance via controlling of k+ channels.
in hormone signaling, studies have systematically examined aba  <cit> , and a few studies have examined meja  <cit> , sa  <cit> , and ethylene  <cit> .
the rich sequence and functional information from arabidopsis enables us to extrapolate the functions of the orthologous genes in other species.
grapevine is one of the most important fruit crops in the world.
however, most cultivated grapevine varieties  are susceptible to many pathogens and are sensitive to abiotic stresses.
by contrast, chinese wild grapevine  accession baihe-35- <dig> has demonstrated resistance to multiple diseases and to various environmental stresses  <cit> .
previous examination of the cdpk gene family in vitis vinifera identified  <dig> members  <cit> , and in vitis amurensis isolated  <dig> members  <cit> .
however, prior work focused on evolutionary relationships with only a few transcriptional analyses in some tissues and developmental stages.
in this study, we employed bioinformatics and publicly available data to identify and analyse grape cdpk genes on a genome-wide scale in vitis pseudoreticulata, identifying two more members.
furthermore, we measured the expression of the cdpk genes  in chinese wild grapevine vitis pseudoreticulata accession baihe-35- <dig> in response to various biotic and abiotic stresses as well as multiple phytohormone treatments.
in addition, we showed that the vpcdpks have different subcellular localizations when transiently expressed in arabidopsis mesophyll protoplasts.
more significantly, comparing arabidopsis and grapevine cdpk gene structures, evolution, and experimental data provides insights on the functions of vpcdpks.
our results provide a set of potential candidate cdpk genes for future genetic modification of pathogen resistance and stress tolerance in grapevine.
RESULTS
characteristics of grape cdpk genes
we identified cdpk genes by searching the pfam database and obtained the hmm  profiles of protein kinase domain pkinase  and ef-hand domain ef-hand_ <dig> .
then we used blast-p to search the 12x grapevine  genome using pkinase and ef-hand hmm profiles.
we also performed blast-p searches at ncbi using full-length amino acid of putative grape cdpk genes.
after that, we identified a bunch of candidates .
by removing incomplete gene sequences, transcripts of the same genes, and redundant sequences, we identified nineteen non-redundant cdpk genes in the grape genome .
among those with alternative splice variants, we selected the longest variant for further analysis.
the sequences of the nineteen grape cdpks were submitted to interpro and smart databases to confirm their pkinase domains and ef-hand domains.
previous work identified seventeen cdpk genes in grapevine  <cit> .
the seventeen genes were named based on their distributions and relative linear orders on the respective chromosomes.
on this basis, we added two members  and named them vvcdpk <dig> and vvcdpk <dig> according to their chromosomal locations.
the nineteen putative cdpk genes mapped on eleven grape chromosomes .
among them, eight chromosomes possess one cdpk gene, three possess two cdpk genes, and chr.
<dig> possesses five cdpk genes.table  <dig> characteristics of grape cdpk genes
f
vvcdpk1
vpcdpk1
vvcdpk2
vpcdpk2
vvcdpk3
vpcdpk3
vvcdpk4
vpcdpk4
vvcdpk5
vpcdpk5
vvcdpk6
vpcdpk6
vvcdpk7
vpcdpk7
vvcdpk8
vpcdpk8
vvcdpk9
vpcdpk9
vvcdpk10
vpcdpk10
vvcdpk11
vpcdpk11
vvcdpk12
vpcdpk12
vvcdpk13
vpcdpk13
vvcdpk14
vpcdpk14
vvcdpk15
vpcdpk15
vvcdpk16
vpcdpk16
vvcdpk17
vpcdpk17
vvcdpk18
vpcdpk18
vvcdpk19
vpcdpk19
a ids are available in the grape genome browser
b first eight amino acids at the n-terminal of the corresponding protein.
the amino acids underlined indicate putative palmitoylation sites
c the myristoylation sites were predicted by the myristoylator program .
nd, not determined
d the palmitoylation sites were predicted by css-palm  <dig>
e number of ef-hands were predicted by interpro
f homologous cdpk genes in chinese wild grapevine vitis pseudoreticulata
g sequence identity calculated with amino acid sequences using vector nti
fig.
<dig> chromosomal distribution of cdpk genes in the grape genome.
the chromosome number is shown at the top of each chromosome.
the positions of the grape cdpk genes are marked by blue lines on the chromosomes.
the black lines and blocks indicate the coding sequence of the grape genome.
the green lines show tandem duplications and the red lines show segmental duplications
characteristics of the nineteen cdpk genes are shown in table  <dig>  the lengths of the gene sequences vary widely, from  <dig>  to  <dig>  kb, but the numbers of predicted amino acids are relatively similar for most genes, around  <dig> to  <dig>  except vvcdpk <dig> with  <dig> amino acids.
fifteen of the nineteen cdpks are predicted to be palmitoylated and eight of the nineteen cdpks are predicted to be myristoylated.
the predicted proteins for six cdpk genes have both n-terminal myristoylation sites and palmitoylation sites.
nine cdpk genes encode proteins with only palmitoylation sites and two  have only myristoylation sites.
besides these, vvcdpk <dig> and vvcdpk <dig> have neither myristoylation nor palmitoylation sites.
typical cdpks have four ef-hands, but not necessarily  <cit> .
the seventeen previously identified cdpks were predicted to each have four ef-hands.
in addition, the two new members, vvcdpk <dig> and vvcdpk <dig> were predicted to have two ef-hands and three ef-hands, respectively.
grape cdpk genes possess typical domains but complicated intron–exon organization
the predicted protein sequences of the nineteen grape cdpks contain the four typical cdpk domains, including the n-terminal variable domain, the protein kinase domain, the junction domain, and the calmodulin-like domain .
the intron-exon organization can indicate the evolutionary relationships within multi-gene families  <cit> .
as shown in fig.
<dig>  grape cdpk genes can be divided into four groups  on the basis of the arabidopsis classification  <cit> .
most of the grape cdpk genes have seven or eight exons, six or seven introns, and clear intron phase patterns.
group i members have seven exons, except vvcdpk <dig> with ten exons, and their intron phases, with respect to the open reading frame, occur in the same pattern, with type- <dig> intron phases at the first three positions from the beginning and type- <dig> intron phases at the last three positions.
all of the group ii members contain eight exons and also share the same intron phase pattern.
compared with the group i members, group ii members have an additional exon.
group iii can be divided into two subgroups.
subgroup  <dig> shares the same intron-exon organization as most of the group i members, such as vvcdpk <dig> and vvcdpk <dig>  but subgroup  <dig> members, such as vvcdpk <dig>  vvcdpk <dig>  and vvcdpk <dig>  have one more exon at the first position and one more type- <dig> phase intron.
compared with the other three groups, group iv has a complicated intron-exon organization with differing numbers of exons and intron phases.
clustering the intron-exon structures of the nineteen cdpk genes by an unrooted phylogenetic tree suggests a connection between intron-exon structures and evolutionary relationships.fig.
<dig> phylogenetic relationships and intron-exon organization of grape cdpk genes.
the unrooted phylogenetic tree was constructed using the full-length protein sequences of nineteen grape cdpk genes by the neighbor-joining method with  <dig>  bootstrap replicates.
the four subgroups are marked by square boxes and numbered with roman numerals.
the sizes of the exons are proportional to their sequence lengths
gene duplication and synteny analysis of grape cdpk genes
gene duplication and divergence are important in gene family expansion  <cit>  and in the evolution of novel functions  <cit> .
grapevine has undergone whole-genome duplications during its evolutionary history  <cit> .
to examine the effect of duplications on the cdpk gene family, we obtained tandem duplication and segmental duplication gene pairs from pgdd  and visualized them using circos.
in this study, we identified two segmental duplication pairs of grape cdpk genes   but did not find tandem duplication events among the grape cdpk genes.fig.
<dig> segmental duplication of grape cdpk genes and synteny analysis of grape and arabidopsis cdpk genes.
chromosomes of v. vinifera and arabidopsis are shown in different colors and in circular form.
the approximate positions of the atcdpk and vvcdpk genes are marked with a short red line on the circle.
colored curves denote the syntenic relationships between grape and arabidopsis cdpk genes
to further explore the origin and evolution of grape cdpk genes, we investigated the syntenic relationship between grapevine and arabidopsis.
the synteny analysis showed that grape cdpk genes can be divided into two types , vvcdpk4-at2g <dig> , vvcdpk5-at5g <dig> , vvcdpk10-at3g <dig> , vvcdpk15-at1g <dig> , and vvcdpk17-at3g <dig> .
the second type has a single grape cdpk gene that corresponds to multiple arabidopsis genes, including vvcdpk3-at2g <dig> /at4g <dig> / at5g <dig> , vvcdpk11-at2g <dig> / at3g <dig> , vvcdpk12-at1g <dig> / at3g <dig> / at4g <dig> / at4g <dig> , and vvcdpk14-at1g <dig> / at1g <dig> .
these results provide insights that will assist in prediction of the functions of grape cdpks.
phylogenetic analysis of the grape cdpk genes
to investigate the evolutionary relationships and functional associations, we constructed a neighbor-joining tree using the full-length amino acid sequences of cdpks from grape, arabidopsis, rice, maize, and poplar .
the phylogenetic analysis indicated that nineteen vvcdpks can be divided into four groups.
of the four groups, three groups appear to be well-defined, except group iv, according to the distributions of branches.
most of the cdpk genes from monocots  clustered into one sub-branch, as did the cdpk genes from the eudicots .
also, grape cdpk genes clustered more often with poplar cdpk genes than with arabidopsis cdpk genes.
for the four groups of vvcdpks, group i contains six vvcdpk genes.
five of the six genes have homologs in arabidopsis or poplar, or both, except vvcdpk <dig>  which clustered with zmcpk <dig> and oscpk <dig>  group ii contains five grape cdpk genes and clustered into two subgroups.
among these genes, vvcdpk <dig> and vvcdpk <dig> show high sequence similarity with each other.
group iii contains five grape cdpk genes and group iv, the smallest group, contains three grape cdpk genes.
these phylogenetic relationships suggest evolutionary conservation of the basal architecture of the cdpk family.fig.
<dig> phylogenetic analysis of grape cdpk genes.
the full-length amino acid sequences of cdpk genes from grape , arabidopsis , populus trichocarpa , rice  and maize  were aligned by clustalx and the phylogenetic tree was constructed using the neighbor-joining method with  <dig> bootstrap replicates by mega  <dig> .
the four subgroups are marked with distinct colors and denoted by roman numerals
subcellular localization of grape cdpks
most cdpks possess either n-myristoylation sites, s-palmitoylation sites, or both.
these acylation sites are believed to be involved in targeting to membranes  <cit> .
because those acylation sites are predicted to be present in grape cdpks , to determine if grape cdpks localize to membranes, we cloned six vpcdpks from chinese wild vitis pseudoreticulata accession baihe-35- <dig> and assessed the subcellular localizations of the encoded vpcdpks by transient expression assays in arabidopsis protoplasts, using translational fusions to gfp.
as shown in fig.
<dig>  five genes  expressed vpcdpk-gfp fusion proteins in transformed arabidopsis protoplasts.
vpcdpk3-gfp, vpcdpk10-gfp, and vpcdpk11-gfp only localized on the plasma membrane.
vpcdpk5-gfp localized on the plasma membrane and in the nucleus, and vpcdpk2-gfp localized in the nucleus and cytosol.fig.
<dig> subcellular localization of five vpcdpks.
the selected cdpk genes were cloned from chinese wild grape  and used to construct camv35s::cdpks–gfp vectors in which gfp was fused at the c terminus.
the five vpcdpk-gfp fusion proteins  as well as gfp as the control, were transiently expressed in col- <dig> arabidopsis protoplasts and observed by fluorescence microscopy.
the merged pictures include the green fluorescence channel  and the chloroplast autofluorescence channel .
the corresponding bright field images are shown on the right.
bar = 5 μm
unlike the other cdpks, vpcdpk <dig> localized to four places :  vpcdpk <dig> localized on some kind of plastids that could not be identified.
considering the closest gene in arabidopsis is atcpk <dig>  which localizes to lipid bodies and peroxisomes  <cit> , the plastids that vpcdpk9-gfp localized in may well include lipid bodies and peroxisomes.
vpcdpk9-gfp localized to the biomembrane system, most likely on the endoplasmic reticulum , as well as on vesicles.
vpcdpk9-gfp also showed extra fluorescence in the cytosol.
we also detected vpcdpk9-gfp in the nucleus.
the complexity of vpcdpk <dig> subcellular localization suggests its functional diversity and variety.fig.
<dig> subcellular localization of vpcdpk <dig>  the roman numerals  represent the corresponding four subcellular localization patterns of vpcdpk <dig>  vpcdpk <dig> was cloned from chinese wild grape  and used to construct camv35s::vpcdpk9-gfp vectors in which gfp was fused at the c terminus.
the vpcdpk9-gfp fusion protein, as well as gfp as a control, was transiently expressed in col- <dig> arabidopsis protoplasts and observed by fluorescence microscopy.
the merged pictures include the green fluorescence channel  and the chloroplast autofluorescence channel .
the corresponding bright field images are shown on the right.
bar = 5 μm
expression of grape cdpk genes in chinese wild grape
increasingly evidence shows that plant cdpks function in responses to biotic and abiotic stress and signal transduction  <cit> .
to further test how grape cdpk genes respond to various stresses, we measured the expression of grape cdpk genes in chinese wild grape  accession baihe-35- <dig>
biotic stress
cdpks act as vital sensors and responders in immune signaling  <cit> .
in this study, we used rt-qpcr to perform a time course analysis of the transcript levels of vpcdpks following inoculation of chinese wild grape vitis pseudoreticulata accession baihe-35- <dig> with erysiphe necator, the causal agent of grapevine powdery mildew .
five of the grape cdpk genes were up-regulated, including vpcdpk <dig>  vpcdpk <dig>  vpcdpk <dig>  vpcdpk <dig>  and vpcdpk <dig>  with transcript levels that increased up to  <dig> -fold  but differed in response time and degree.
for example, vpcdpk <dig> transcript rapidly increased in abundance and reached a peak of  <dig> -fold at 96 h post inoculation  .
similarly, vpcdpk <dig> transcript also rapidly increased and remained at high levels .
vpcdpk <dig>  and vpcdpk <dig>  shared similar expression patterns, with a slow increase in transcript levels to a peak and gradual plateau.
vpcdpk <dig>  transcript abundance increased to nearly  <dig> -fold, nearly instantly at  <dig> hpi, and remained at high levels.
by contrast, several cdpk genes were down-regulated.
for instance, vpcdpk <dig> and vpcdpk <dig>  transcript levels decreased to  <dig> - to  <dig> -fold.
these results demonstrate that several cdpk genes responded to powdery mildew inoculation in chinese wild grapevine v. pseudoreticulata accession baihe-35- <dig>  suggesting their vital roles in immune signaling and responses.fig.
<dig> expression of cdpk genes in chinese wild grape  during powdery mildew infection.
expression was measured by reverse transcription, followed by real-time, quantitative pcr, is indicated as fold-change of experimental treatments relative to control samples, and is visualized as heatmaps  and histogram .
grape actin <dig>  was used as an internal control.
the experiments were repeated three times and gave consistent results.
mean values and sds were obtained from three biological and three technical replicates.
expression profile of vpcdpk gene family under powdery mildew infection.
the color scale represents log <dig> expression values, with red indicating increased transcript abundance and green indicating decreased transcript abundance.
detailed expression levels of four vpcdpk genes that significantly up-regulated during powdery mildew infection.
the data were showed as mean value ± sd.
* and ** represent statistically significant  or highly significant , respectively
abiotic stress
previous work showed that cdpks participate in abiotic stress responses, including responses to salt and osmotic stress  <cit>  and to temperature stress  <cit> .
in this study, we used nacl treatment, and incubation at low or high temperature  to understand how vpcdpks responded to these abiotic stresses on the transcriptional level.
our expression data show consistency with the expression of cdpk genes in vitis vinifera  <cit>  and vitis amurensis  <cit> .
as shown in fig.
<dig> , almost all nineteen cdpk genes were up- or down-regulated to at least one abiotic stress treatment, but some responded rather slightly.
as a whole, the vpcdpk genes responded to nacl treatment much more strongly than to temperature stress .
vpcdpk <dig> and vpcdpk <dig> transcript abundance increased in all three treatments, but none of the cdpk genes showed continuously low transcript levels in all treatments.fig.
<dig> expression of cdpk genes in chinese wild grape  under salt and temperature stress treatments.
expression was measured by reverse transcription, followed by real-time, quantitative pcr, is indicated as fold-change of experimental treatments relative to control samples, and is visualized as heatmaps a and histogram b. grape actin <dig>  was used as an internal control.
the experiments were repeated three times and gave consistent results.
mean values and sds were obtained from three biological and three technical replicates.
a expression profile of vpcdpk gene family under nacl, 4 °c, and 42 °c treatments.
the color scale represents log <dig> expression values, with red indicating increased transcript abundance and green indicating decreased transcript abundance.
b detailed expression levels of four vpcdpk genes showed unusual expression patterns under nacl, 4 °c, and 42 °c treatments.
the data were showed as mean value ± sd.
* and ** represent statistically significant  or highly significant , respectively
after nacl treatment , twelve of the nineteen cdpk genes were up-regulated, and six genes  responded strongly with transcript levels increasing to  <dig> - to 16-fold higher than the control sample .
vpcdpk <dig>  the most rapidly responding gene, reached a peak of nearly 10-fold at  <dig>  h post treatment  and rapidly decreased to  <dig> -fold at  <dig> hpt, before its transcript levels increased from  <dig> -fold to  <dig> -fold at 2 h to  <dig> hpt .
the homologous gene of vpcdpk <dig> in vitis amurensis, vacpk <dig>  was also highly up-regulated under nacl treatment  <cit> .
in addition, vpcdpk <dig> showed a peak of  <dig> -fold at  <dig> hpt and increased transcript abundance of more than  <dig> -fold from  <dig> to  <dig> hpt , similar with the expression pattern of its homologs, vacpk <dig>  <cit>  and vvcpk <dig>  <cit> .
we noted that the margins of treated leaves became dry from the outside to the inside and the plants had dry-dead leaves after  <dig> hpt, which might relate to the rapid down-regulation of vpcdpk <dig> at  <dig> hpt.
by contrast, vpcdpk <dig>  and vpcdpk <dig>  transcript abundance decreased after  <dig> hpt, indicating their probable negative regulatory roles.
the cdpk genes that responded rapidly and strongly most likely participate in the nacl stress response.
for 4 °c treatments , only vpcdpk <dig>  vpcdpk <dig> and vpcdpk <dig>  showed up-regulation .
vpcdpk <dig> was rapidly up-regulated, reached a peak of  <dig> -fold at  <dig> hpt, and then declined to lower than  <dig> - fold .
moreover, vpcdpk <dig> had a peak of  <dig> -fold at  <dig> hpt but then fluctuated around  <dig> -fold during the rest of the treatment time .
the vpcdpk genes with decreased transcript abundance might not participate in the signaling response to cold stress but their low activities might result in pervasive down-regulation of transcription.
however, vpcdpk <dig>  was an exception, as its transcript levels decreased rapidly at  <dig> hpt and remained at a steady low level.
intriguingly, vacpk <dig>  as the homolog of vpcdpk <dig> in vitis amurensis, showed significant up-regulation under 10 °c treatment  <cit> .
we also measured the transcript levels of vpcdpk genes in response to 42 °c treatment.
we found that five cdpk genes  significantly responded to 42 °c treatment ; of these, vpcdpk <dig>  had a strong response at  <dig> hpt, with its transcript abundance increasing to  <dig> -fold and remaining at a high level.
the transcript abundance of vpcdpk <dig>  and vpcdpk <dig>  increased, similar to vpcdpk <dig> , although vpcdpk <dig> transcripts were almost undetectable at  <dig> hpt.
in particular, vpcdpk <dig>  responded to 42 °c treatment rapidly and intensely, reaching a peak of more than  <dig> -fold and then returning to nearly normal transcript levels at  <dig> hpt.
taken together, our data showed that expression of vpcdpks responded to low and high temperatures  suggest that the vpcdpks may play key roles in the response to temperature stress.
hormone treatment
plant hormones such as aba, sa, meja, and ethylene have well-established roles in modulating plant signaling networks  <cit> .
in this study, hormone treatments resulted in a wide variety of changes in the transcript levels of vpcdpk genes .fig.
<dig> expression of cdpk genes in chinese wild grape  in response to treatment with plant hormones.
expression was measured by reverse transcription, followed by real-time, quantitative pcr, is indicated as fold-change of experimental treatments relative to control samples, and is visualized as heatmaps  and histogram .
grape actin <dig>  was used as an internal control.
the experiments were repeated three times and gave consistent results.
mean values and sds were obtained from three biological and three technical replicates.
a expression profile of vpcdpk gene family under aba, sa, meja, and ethylene  treatments.
the color scale represents log <dig> expression values, with red indicating increased transcript abundance and green indicating decreased transcript abundance.
b detailed expression levels of four vpcdpk genes showed unusual expression patterns under aba, sa, meja, and ethylene  treatments.
the data were showed as mean value ± sd.
* and ** represent statistically significant  or highly significant , respectively
an increasing body of evidence has shown that cdpks regulate aba-mediated signal transduction in plants  <cit> .
our experimental data suggest that  <dig> of the  <dig> vpcdpk genes respond to aba treatment .
among them, eight vpcdpk genes  showed significant up-regulation in response to aba .
however, vpcdpk <dig>  transcript levels rapidly increased up to  <dig> -fold at  <dig> hpt and then decreased to lower than  <dig> -fold at  <dig> hpt.
interestingly, the majority of the responsive cdpk genes showed a similar pattern, with their transcript abundance increasing rapidly to a peak at  <dig> hpt and remained at high levels until  <dig> hpt.
similar to the aba response,  <dig> of the  <dig> vpcdpk genes also showed positive regulation under sa treatment .
six genes  were significantly up-regulated , exhibiting a steady or gradual increase from  <dig> to  <dig> or  <dig> to  <dig> hpt.
interestingly, the expression of vpcdpk <dig>  vpcdpk <dig>  vpcdpk <dig>  and vpcdpk <dig> was also induced after inoculation with powdery mildew pathogen .
for instance, vpcdpk <dig> showed positive regulation by inoculation of powdery mildew and peaked  at  <dig> hpi , while under sa treatment, vpcdpk <dig> was also up-regulated and peaked  at  <dig> hpt .
the expression pattern after sa treatment and powdery mildew inoculation, suggests the vital roles of grape cdpk genes in immune signaling.
in contrast with aba and sa, the vpcdpks showed more limited responses to meja treatment.
a total of ten genes  were up-regulated; and among them, only three vpcdpk genes  showed distinct up-regulation  .
in particular, their transcript abundance peaked at  <dig> or  <dig> hpt, except for vpcdpk <dig> , which strongly and rapidly responded to the treatment from  <dig>  to  <dig> hpt with meja and peaked at  <dig>  hpt.
that might indicate its functional significance in the meja signaling pathway.
the expression changes caused by ethylene treatment  were distinct from those caused by the other hormone treatments.
for ethylene treatment, only a handful of vpcdpk genes  were up-regulated , while more genes showed down-regulation.
for example, two up-regulated genes, vpcdpk <dig>  and vpcdpk <dig> , their transcript abundance increased rapidly at  <dig>  hpt, but then increased only slightly or returned to normal transcript levels, respectively.
chinese wild grapevine baihe-35- <dig> seems to be very sensitive to ethylene treatment and almost all the leaves of baihe-35- <dig> plants fell at  <dig> hpt, so we had to use the fallen leaves for rna extraction.
vpcdpk <dig>  vpcdpk <dig>  vpcdpk <dig>  vpcdpk <dig>  and vpcdpk <dig> had decreased transcript abundance lower than  <dig> - fold during the treatment time, suggesting their potential negative regulatory functions in plant responses to ethylene signaling.
discussion
identification of grape cdpk genes
previously, chen  <cit>  investigated the cdpk gene family in grapevine and identified  <dig> members, which were predicted to have the typical cdpk gene structure with a ser/thr protein kinase domain and four ef-hands.
considering the complexity and variability in the calmodulin-like domain, we performed blast-p searches in ncbi using the putative grape cdpk genes predicted by pkinase  and ef-hand_ <dig>  hmm profiles, and identified the previously discovered  <dig> cdpks and two more members .
compared with arabidopsis cdpks  <cit>  and rice cdpks  <cit> , we found that gsvivt <dig>  possesses a similar domain organization to atcpk <dig> and oscpk <dig>  which are predicted to have no ef-hand with default parameters in pfam but are predicted to have ef-hands in prosite and interpro.
in addition, the phylogenetic tree showing that gsvivt <dig>  and oscpk <dig> are homologous genes provides more evidence .
therefore, we considered gsvivt <dig>  as members of the grape cdpk gene family and the same with gsvivt <dig> .
it is worth to mention that a homolog of vvcdpk <dig> in vitis amurensis, named as vacpk <dig>  has been identified as a cdpk gene  <cit> .
both of these two genes  are same in gene length and phylogenetic relationship.
consider that, vpcdpk <dig> can also be identified as a cdpk gene.
these two new members of the grape cdpk gene family were designated vvcdpk <dig> and vvcdpk <dig> on the basis of their chromosomal locations.
structural characteristics of grape cdpk genes
the structural conservation and divergence of grape cdpk genes led to gene family expansion and functional conservation or differentiation.
structural characteristics such as acylation sites and intron-exon structures show the details of gene family expansion and divergence.
most of cdpks possess acylation sites, including n-myristoylation sites and s-palmitoylation sites, which are generally believed to function in membrane targeting  <cit> .
in arabidopsis,  <dig> atcpks have both myristoylation and palmitoylation sites and all of them target exclusively to membranes, especially to the plasma membrane, although several have not been determined  <cit> .
among our identified nineteen grape cdpk genes, six contain both myristoylation and palmitoylation sites, nine contain only palmitoylation sites, two contain only myristoylation sites, and two do not contain any acylation site .
we tested the subcellular localization of six vpcdpk proteins.
vpcdpk <dig>  vpcdpk <dig>  vpcdpk <dig>  and vpcdpk <dig> have palmitoylation sites while vpcdpk <dig> has a myristoylation site  and they all localized on the plasma membrane , consistent with results in arabidopsis  <cit> .
three pivotal mechanisms contribute to gene family evolution and expansion: exon/intron gain or loss, exonization/ pseudo-exonization, and insertion /deletion  <cit> .
previous work reported that grape cdpk genes have a single origin and can be dated back to green algae, before plants colonized the land  <cit> .
for the four groups in fig.
<dig>  group iv was the earliest one that expanded from the evolutionary branch  <cit> .
as a result, it has the longest evolutionary history, leading to complex intron-exon organization.
both group ii and group iii originated from group i in evolutionary history, and show similar structural divergence.
all of the members of group ii contain one more exon and a phase- <dig> intron at the sixth position, indicating that there was an intron insertion in the last exon, which contributed to the generation of group ii and expansion of the vvcdpk family.
group iii can be separated into two subgroups in evolutionary history, as reflected in their intron-exon structures.
one subgroup was similar to group i in intron-exon structures and the other one contains one more exon and a phase- <dig> intron at the first position, suggesting that there was an intron insertion in the first exon.
our result is consistent with the previous work that can be reflected in the origin and evolutionary history of vvcpks  <cit> .
the intron-exon divergence was closely related to the evolutionary relationship of the grape cdpk family and might result in functional diversity.
evolutionary relationships of grape cdpk genes
to study the evolutionary relationships among different vvcdpk genes and the history of the vvcdpk gene family, as well as to further study their gene function, we investigated gene duplication events, syntenic regions, and phylogenetic relationships among the vvcdpk genes.
segmental duplications and tandem duplications are the main mechanisms leading to gene family expansion  <cit> .
these processes may lead to functional redundancy, sub-functionalization and neo-functionalization.
no tandem duplication involving vvcdpk genes was discovered in the grapevine genome, but two segmental duplications were found, vvcdpk5-vvcdpk <dig> and vvcdpk12-vvcdpk <dig> .
previously, chen identified three segmental duplications  <cit> .
except for vvcdpk5-vvcdpk <dig> and vvcdpk12-vvcdpk <dig>  they also identified triplet-duplicated genes, vvcpk8-vvcpk9-vvcpk <dig>  however, the method they used was not clearly presented, so we cannot follow their method.
anyway, both of chen and us indeed identified the two duplicated gene pairs, vvcdpk5-vvcdpk <dig> and vvcdpk12-vvcdpk <dig>  these two duplicated gene pairs are quite similar in gene length, acylation sites, and intron-exon organization .
furthermore, they possess the closest phylogenetic relationship among grape cdpk genes .
however, despite the fact vvcdpk <dig> and vvcdpk <dig> are phylogenetically closest among cdpks from other species, these two genes have different syntenic genes in arabidopsis , which means the duplication events happened before the evolutionary divergence of arabidopsis and grapevine.
both vvcdpk <dig> and vvcdpk <dig> showed closer phylogenetic relationships with arabidopsis cdpk genes than that with each other, indicating that this duplication event might also have happened before the divergence of arabidopsis and grapevine.
from what has been discussed above, vvcdpk5-vvcdpk <dig> and vvcdpk12-vvcdpk <dig> likely have different functions but still possess potential functional connections and similarities.
comparative genomics approaches structure genomes into syntenic blocks that exhibit conserved features across the genomes  <cit> .
the synteny analysis provides evolutionary and functional connections between grape and arabidopsis syntenic genes.
furthermore, a large number of syntenic relationships suggest that some of the grape cdpk genes arose before the divergence of the arabidopsis and grapevine lineages.
ten grape cdpk genes were found to have syntenic relationships with arabidopsis genes  all of these vvcdpk genes show close phylogenetic relationships with the corresponding atcdpk genes , suggesting their potential functional similarities.
interestingly, all of the grape cdpk genes in group iii  have syntenic genes in arabidopsis, suggesting that group iii cdpk genes are relatively conserved over evolutionary history.
analysis of the phylogenetic tree revealed that cdpk homologs among several monocots and eudicots clustered into four distinct groups, which correspond to the clades formerly identified in green plants  <cit> .
previously, hamel  <cit>  examined the cdpk families from green algae to land plants to demonstrate that cdpk families are conserved among land plants, whereas cdpks from green algae have continued to evolve independently.
supporting this, our data suggest that cdpks from monocots and eudicots cluster separately  and evolutionary relationships of the four clades are relatively conserved.
the previous work  <cit>  also constructed a phylogenetic tree that gives the consistent results and conclusions.
functional prediction of grape cdpk genes by comparison with arabidopsis
comparative genomics provides an effective way to understand the structure and function of genomes by translating knowledge gained from model species to the species of interest.
combining synteny analysis with phylogenetic analysis provides new insights for further investigating the functions of the grape cdpk genes by comparing orthologous genes between two species, in this case, between grapevine and arabidopsis.
our study provided substantial evidence to help predict the functional conservation or divergence of cdpk genes between grapevine and arabidopsis.
chinese wild grape  accession baihe-35- <dig>  compared with the sequenced vitis vinifera, reported to have remarkable resistance to both biotic and abiotic stress  <cit> .
in addition, vitis amurensis is also a wild grapevine species with a high level of resistance to multiple stresses  <cit> .
our expression profile of vpcdpk genes showed consistency with that reported in vitis vinifera  <cit>  and vitis amurensis  <cit> .
for example, the homologous cdpk genes, vpcdpk3/vvcpk3/vacpk <dig> and vpcdpk17/vvcdpk17/vacpk <dig> showed distinct up-regulation under salt stress treatment  <cit>  .
however, there are also some differences on genes expression, e.g., vacpks respond to cold stress much significantly than vpcdpks, probably because of vitis amurensis shows strong capacity of cold-resistance.
two duplicated gene pairs, vpcdpk5/vpcdpk <dig> and vpcdpk12/vpcdpk <dig>  as discussed above, provide experimental evidence to support our predictions and views.
vpcdpk <dig> and vpcdpk <dig> each have particular syntenic genes in arabidopsis showing a complicated evolutionary history.
in addition, the experimental data showed that these two genes have distinct expression patterns under a range of treatments.
for example, vpcdpk <dig> was obviously up-regulated under hormone treatments , while vpcdpk <dig> was only slightly up-regulated under aba treatment .
also, for abiotic stress treatments, vpcdpk <dig> was down-regulated under nacl and 42 °c treatments, but vpcdpk <dig> transcripts remained at constant levels .
furthermore, subcellular localization analysis demonstrated that vpcdpk5-gfp localized on plasma membrane and in the nucleus, but vpcdpk11-gfp only localized on plasma membrane .
the aforementioned information indicated that vpcdpk <dig> and vpcdpk <dig> might well have undergone neo-functionalization.
moreover, vvcdpk <dig> in v. vinifera cv.
corvina expressed in almost all of the organs, whereas vvcdpk <dig> only expressed in pollen  <cit> .
however, we tested vpcdpk <dig> in the chinese wild grape v. pseudoreticulata responded to hormone treatments  in leaves, suggesting that vpcdpk <dig> was not only transcribed in pollen.
for vpcdpk <dig>  its low expression under multiple treatments  and high expression in almost all tissues  <cit>  indicates that its function most likely relates to housekeeping genes.
the other duplicated gene pair, vpcdpk <dig> and vpcdpk <dig> were quite different.
the transcript levels of these two genes showed similar tendencies under abiotic stress, aba and sa treatments , however, vpcdpk <dig> seemed to respond much strongly than vpcdpk <dig>  based on chen  <cit> , vvcdpk <dig> had high transcript levels in almost all tissues while vvcdpk <dig> had low transcript levels.
these data suggest that the duplicated gene pair vpcdpk12/vpcdpk <dig> probably have undergone sub-functionalization and vpcdpk <dig> tends to be non-functional.
it is worth noting that vpcdpk <dig> is unique in the grape cdpk family.
for one thing, it showed an unusual expression pattern under various treatments, being strongly up-regulated, except for 4 °c treatment .
its transcript abundance increased to a high level during most or all of the treatment .
subcellular localization also demonstrated its considerable differences with the other grape cdpks we examined.
the other vpcdpks have only one pattern of localization , while vpcdpk <dig> shows four patterns, including in some kind of plastids, in biomembranes, in the cytosol, or in the nucleus .
testing why vpcdpk <dig> has four patterns of localization, and the relationship between its localization and function, will require more experimental data.
the genes that are phylogenetically close to vpcdpk <dig> in arabidopsis are atcpk <dig> and atcpk <dig> .
vpcdpk <dig> has structural and functional commonalities with atcpk <dig>  for example, atcpk <dig> expression is rapidly induced by fungal elicitors and loss-of-function mutants of atcpk <dig> exhibit higher susceptibility to pathogen infection  <cit> .
ectopic expression of atcpk <dig> enhanced nadph oxidase activity and the oxidative burst in tomato protoplasts  <cit> .
cold-stress can also induce atcpk <dig> transcripts via phosphoprotein signals  <cit> .
our data also showed that vpcdpk <dig> not only responded to powdery mildew infection and abiotic stress, but also responded to hormone treatments .
these results are consistent with the expression profile of atcpk <dig>  future work will be directed toward identification of potential defense components that may be directly or indirectly regulated by vpcdpk <dig> in grapevine baihe-35- <dig>
vpcdpk <dig>  whose arabidopsis homolog is atcpk <dig>  is found in the chromosomal region syntenic with atcpk <dig>  and plays positive regulatory roles in various treatments .
results on atcpk <dig> show high consistency with our results on vpcdpk <dig>   atcpk <dig> was reported to localize to the plasma membrane and the nucleus  <cit> , consistent with our data showing that vpcdpk <dig> also localized to the plasma membrane and nucleus .
atcpk <dig> can localize to the nucleus where it interacts with and phosphorylates wrky <dig>   <dig>  and  <dig> to activate defense genes  <cit> , while vpcdpk <dig> also localized in the nucleus , suggesting it has similar functions with atcpk <dig>   atcpk <dig> also phosphorylates rbohd in vivo, resulting in h2o <dig> production and leading to cell death  <cit> , involving in immune signaling.
vpcdpk <dig> responded to powdery mildew inoculation with up-regulated transcript levels , indicating its potential function in immune signaling and that vpcdpk <dig> most likely participates in the analogous pathways to those mentioned above for arabidopsis.
these structural and locational similarities provide indications of functional consistency.
vpcdpk <dig> has a syntenic gene in arabidopsis, atcpk <dig>  further analysis provides valuable comparisons of these two genes and helps predict the function of vpcdpk <dig>  in our study, vpcdpk <dig> localized on the plasma membrane , and atcpk <dig> was also reported to localize on the plasma membrane  <cit> .
vpcdpk <dig> expression did not respond to stress treatments but was distinctly up-regulated in response to aba treatment .
atcpk <dig> specifically inhibits kat <dig> and kat <dig> shaker channels  <cit> , which participate in aba-induced stomatal movements by affecting cdpk phosphorylation  <cit> .
these insights indicate that vpcdpk <dig> plays important roles in aba- and ca2+-mediated stomatal regulation.
another intriguing gene, vpcdpk <dig>  is phylogenetically close to atcpk <dig> and atcpk <dig> , which are activated by aba and phosphorylate the c-terminus of acs <dig> in ethylene biosynthesis  <cit> .
meanwhile, atcpk <dig> and atcpk <dig> can phosphorylate the aba-responsive transcription factors abf <dig> and abf <dig> and lead to salt insensitivity in seed germination and decrease tolerance of seedlings to salt stress  <cit> .
sustained atcpk <dig> and atcpk <dig> activation directly phosphorylated wrky transcription factors involved in immune signaling  <cit> .
expression patterns suggest that vpcdpk <dig> responded to powdery mildew inoculation as well as nacl, aba, and sa treatments , showing high functional consistency with atcpk <dig> and atcpk <dig>  this evidence indicates that vpcdpk <dig> might participate in similar pathways as atcpk <dig> and atcpk <dig> in arabidopsis.
in addition to stress, cdpk genes also play important roles in growth and development process.
on this regard, plant hormones are mainly responsible.
for instance, several plant hormones may play central roles in the control of ripening in the grape berry  <cit> .
as a support, kühn et al.
<cit>  investigated the aminocyclopropane-1-carboxylate  synthases genes in first developed grape berries, which pathway is related to ethylene signaling.
for cdpk genes, several genes including atcpk <dig>  <cit> , ntcdpk <dig>  <cit>  and lecdpk <dig>  <cit>  were reported to phosphorylate acc synthases to trigger ethylene biosynthesis and accumulation.
coincidence is that vpcdpk <dig> and vpcdpk <dig>  as homologs of ntcdpk <dig> and lecdpk <dig>  are positively regulated under ethylene treatment, indicating their possible functions involved in ethylene signaling.
however, cdpks functions in plants growth and development process still need more experimental evidence to deeply understand their biological roles and the pathways they involved in.
CONCLUSIONS
so far, little systematic analysis of the cdpk family has been reported in grapevine, and the functions of most cdpk genes remain unclear.
however, accumulating evidence indicates that cdpks play important roles in response to a broad variety of abiotic and biotic stresses and biological processes.
in this paper, genome-wide identification, evolutionary, and expression analyses were carried out to provide a framework for further analysis of grape cdpk genes in defining their biological functions and pathways during stress responses as well as growth and development.
expression profiles showed grape cdpk genes respond to various stresses and hormone treatments; moreover, analysis of cdpk subcellular localization gave evidence as to their functions.
comparisons of the grape and arabidopsis genomes and expression profiles provide novel insights into the functions of less well-studied genes according to their better-understood homologs.
by prediction and experimental data, we speculated that cdpk gene family might participate in responses to pathogen, cold, heat and salt stress, and the related biological processes might covering the regulation of gene expression, control of the ion channel, regulation of the enzyme activity, mediation of the cross-talk between signaling pathways and so on.
these observations may lay the foundation for future functional analysis of grape cdpk genes to unravel their biological roles.

 calcium, a universal second messenger in eukaryotes, mediates stimulus–response coupling in the regulation of diverse cellular functions. various extracellular stimuli elicit specific calcium signatures that can be recognized by different calcium sensors. the three main classes of ca2+ sensors identified in plants are: cams and cam-like proteins, cbls, and cdpks. the cdpks, also termed cpks, consist of a variable n-terminal domain, a conserved serine/threonine kinase domain, an auto-inhibitory junction region, and a c-terminal regulatory cam-like domain. unlike the other ca2+ sensors, cdpks have both ca2+ sensing and responding activities due to their unique, cam-like domain and protein kinase domain, which convert upstream ca2+ signals into downstream phosphorylation events and cellular responses. genome-wide analysis, together with comparative genomics, provides an effective way to understand the structures and functions of members of a gene family, using the insights gained from evolutionary relationships and experimental data. the original, systematic report on the cdpk gene family in arabidopsis thaliana identified cdpk gene family members, and was followed by research in rice and wheat. recently, genome-wide analyses of the cdpk gene family have been reported in maize and poplar. meanwhile, more and more investigations of cdpk genes have also involved horticultural plants, such as alfalfa, potato, strawberry, and tomato. work in arabidopsis showed that cdpks function in immune and stress signaling, growth and development, and hormone responses. atcpks play vital parts in immune signaling pathways; for example, atcpk activates nadph oxidase, resulting in an oxidative burst, and phosphorylates pal resulting in accumulation of salicylic acid. atcpk4/5/6/ phosphorylates a specific subgroup of wrky transcription factors, wrky8/28/ which participate in nlr-dependent restriction of pathogen growth. in addition, atcpk activates rboh to induce a reactive oxygen species burst. cpks also function in the response to abiotic stress. for example, atcpks help to enhance drought tolerance by responding to abscisic acid, leading to induction of expression of genes such as atcpk4/, and stomatal closure via induction of atcpk3/. atcpk4/ and atcpk trigger plant salt tolerance via controlling of k+ channels. in hormone signaling, studies have systematically examined aba, and a few studies have examined meja, sa, and ethylene. the rich sequence and functional information from arabidopsis enables us to extrapolate the functions of the orthologous genes in other species. grapevine is one of the most important fruit crops in the world. however, most cultivated grapevine varieties are susceptible to many pathogens and are sensitive to abiotic stresses. by contrast, chinese wild grapevine accession baihe-35- has demonstrated resistance to multiple diseases and to various environmental stresses. previous examination of the cdpk gene family in vitis vinifera identified members, and in vitis amurensis isolated members. however, prior work focused on evolutionary relationships with only a few transcriptional analyses in some tissues and developmental stages. in this study, we employed bioinformatics and publicly available data to identify and analyse grape cdpk genes on a genome-wide scale in vitis pseudoreticulata, identifying two more members. furthermore, we measured the expression of the cdpk genes in chinese wild grapevine vitis pseudoreticulata accession baihe-35- in response to various biotic and abiotic stresses as well as multiple phytohormone treatments. in addition, we showed that the vpcdpks have different subcellular localizations when transiently expressed in arabidopsis mesophyll protoplasts. more significantly, comparing arabidopsis and grapevine cdpk gene structures, evolution, and experimental data provides insights on the functions of vpcdpks. our results provide a set of potential candidate cdpk genes for future genetic modification of pathogen resistance and stress tolerance in grapevine.  characteristics of grape cdpk genes we identified cdpk genes by searching the pfam database and obtained the hmm profiles of protein kinase domain pkinase and ef-hand domain ef-hand_. then we used blast-p to search the 12x grapevine genome using pkinase and ef-hand hmm profiles. we also performed blast-p searches at ncbi using full-length amino acid of putative grape cdpk genes. after that, we identified a bunch of candidates. by removing incomplete gene sequences, transcripts of the same genes, and redundant sequences, we identified nineteen non-redundant cdpk genes in the grape genome. among those with alternative splice variants, we selected the longest variant for further analysis. the sequences of the nineteen grape cdpks were submitted to interpro and smart databases to confirm their pkinase domains and ef-hand domains. previous work identified seventeen cdpk genes in grapevine. the seventeen genes were named based on their distributions and relative linear orders on the respective chromosomes. on this basis, we added two members and named them vvcdpk and vvcdpk according to their chromosomal locations. the nineteen putative cdpk genes mapped on eleven grape chromosomes. among them, eight chromosomes possess one cdpk gene, three possess two cdpk genes, and chr. possesses five cdpk genes.table characteristics of grape cdpk genes f vvcdpk1 vpcdpk1 vvcdpk2 vpcdpk2 vvcdpk3 vpcdpk3 vvcdpk4 vpcdpk4 vvcdpk5 vpcdpk5 vvcdpk6 vpcdpk6 vvcdpk7 vpcdpk7 vvcdpk8 vpcdpk8 vvcdpk9 vpcdpk9 vvcdpk10 vpcdpk10 vvcdpk11 vpcdpk11 vvcdpk12 vpcdpk12 vvcdpk13 vpcdpk13 vvcdpk14 vpcdpk14 vvcdpk15 vpcdpk15 vvcdpk16 vpcdpk16 vvcdpk17 vpcdpk17 vvcdpk18 vpcdpk18 vvcdpk19 vpcdpk19 a ids are available in the grape genome browser b first eight amino acids at the n-terminal of the corresponding protein. the amino acids underlined indicate putative palmitoylation sites c the myristoylation sites were predicted by the myristoylator program. nd, not determined d the palmitoylation sites were predicted by css-palm e number of ef-hands were predicted by interpro f homologous cdpk genes in chinese wild grapevine vitis pseudoreticulata g sequence identity calculated with amino acid sequences using vector nti fig. chromosomal distribution of cdpk genes in the grape genome. the chromosome number is shown at the top of each chromosome. the positions of the grape cdpk genes are marked by blue lines on the chromosomes. the black lines and blocks indicate the coding sequence of the grape genome. the green lines show tandem duplications and the red lines show segmental duplications characteristics of the nineteen cdpk genes are shown in table  the lengths of the gene sequences vary widely, from to  kb, but the numbers of predicted amino acids are relatively similar for most genes, around to except vvcdpk with amino acids. fifteen of the nineteen cdpks are predicted to be palmitoylated and eight of the nineteen cdpks are predicted to be myristoylated. the predicted proteins for six cdpk genes have both n-terminal myristoylation sites and palmitoylation sites. nine cdpk genes encode proteins with only palmitoylation sites and two have only myristoylation sites. besides these, vvcdpk and vvcdpk have neither myristoylation nor palmitoylation sites. typical cdpks have four ef-hands, but not necessarily. the seventeen previously identified cdpks were predicted to each have four ef-hands. in addition, the two new members, vvcdpk and vvcdpk were predicted to have two ef-hands and three ef-hands, respectively. grape cdpk genes possess typical domains but complicated intron–exon organization the predicted protein sequences of the nineteen grape cdpks contain the four typical cdpk domains, including the n-terminal variable domain, the protein kinase domain, the junction domain, and the calmodulin-like domain. the intron-exon organization can indicate the evolutionary relationships within multi-gene families. as shown in fig.  grape cdpk genes can be divided into four groups on the basis of the arabidopsis classification. most of the grape cdpk genes have seven or eight exons, six or seven introns, and clear intron phase patterns. group i members have seven exons, except vvcdpk with ten exons, and their intron phases, with respect to the open reading frame, occur in the same pattern, with type- intron phases at the first three positions from the beginning and type- intron phases at the last three positions. all of the group ii members contain eight exons and also share the same intron phase pattern. compared with the group i members, group ii members have an additional exon. group iii can be divided into two subgroups. subgroup shares the same intron-exon organization as most of the group i members, such as vvcdpk and vvcdpk but subgroup members, such as vvcdpk vvcdpk and vvcdpk have one more exon at the first position and one more type- phase intron. compared with the other three groups, group iv has a complicated intron-exon organization with differing numbers of exons and intron phases. clustering the intron-exon structures of the nineteen cdpk genes by an unrooted phylogenetic tree suggests a connection between intron-exon structures and evolutionary relationships.fig. phylogenetic relationships and intron-exon organization of grape cdpk genes. the unrooted phylogenetic tree was constructed using the full-length protein sequences of nineteen grape cdpk genes by the neighbor-joining method with bootstrap replicates. the four subgroups are marked by square boxes and numbered with roman numerals. the sizes of the exons are proportional to their sequence lengths gene duplication and synteny analysis of grape cdpk genes gene duplication and divergence are important in gene family expansion and in the evolution of novel functions. grapevine has undergone whole-genome duplications during its evolutionary history. to examine the effect of duplications on the cdpk gene family, we obtained tandem duplication and segmental duplication gene pairs from pgdd and visualized them using circos. in this study, we identified two segmental duplication pairs of grape cdpk genes but did not find tandem duplication events among the grape cdpk genes.fig. segmental duplication of grape cdpk genes and synteny analysis of grape and arabidopsis cdpk genes. chromosomes of v. vinifera and arabidopsis are shown in different colors and in circular form. the approximate positions of the atcdpk and vvcdpk genes are marked with a short red line on the circle. colored curves denote the syntenic relationships between grape and arabidopsis cdpk genes to further explore the origin and evolution of grape cdpk genes, we investigated the syntenic relationship between grapevine and arabidopsis. the synteny analysis showed that grape cdpk genes can be divided into two types, vvcdpk4-at2g, vvcdpk5-at5g, vvcdpk10-at3g, vvcdpk15-at1g, and vvcdpk17-at3g. the second type has a single grape cdpk gene that corresponds to multiple arabidopsis genes, including vvcdpk3-at2g /at4g / at5g, vvcdpk11-at2g / at3g, vvcdpk12-at1g / at3g / at4g / at4g, and vvcdpk14-at1g / at1g. these results provide insights that will assist in prediction of the functions of grape cdpks. phylogenetic analysis of the grape cdpk genes to investigate the evolutionary relationships and functional associations, we constructed a neighbor-joining tree using the full-length amino acid sequences of cdpks from grape, arabidopsis, rice, maize, and poplar. the phylogenetic analysis indicated that nineteen vvcdpks can be divided into four groups. of the four groups, three groups appear to be well-defined, except group iv, according to the distributions of branches. most of the cdpk genes from monocots clustered into one sub-branch, as did the cdpk genes from the eudicots. also, grape cdpk genes clustered more often with poplar cdpk genes than with arabidopsis cdpk genes. for the four groups of vvcdpks, group i contains six vvcdpk genes. five of the six genes have homologs in arabidopsis or poplar, or both, except vvcdpk which clustered with zmcpk and oscpk group ii contains five grape cdpk genes and clustered into two subgroups. among these genes, vvcdpk and vvcdpk show high sequence similarity with each other. group iii contains five grape cdpk genes and group iv, the smallest group, contains three grape cdpk genes. these phylogenetic relationships suggest evolutionary conservation of the basal architecture of the cdpk family.fig. phylogenetic analysis of grape cdpk genes. the full-length amino acid sequences of cdpk genes from grape, arabidopsis, populus trichocarpa, rice and maize were aligned by clustalx and the phylogenetic tree was constructed using the neighbor-joining method with bootstrap replicates by mega. the four subgroups are marked with distinct colors and denoted by roman numerals subcellular localization of grape cdpks most cdpks possess either n-myristoylation sites, s-palmitoylation sites, or both. these acylation sites are believed to be involved in targeting to membranes. because those acylation sites are predicted to be present in grape cdpks, to determine if grape cdpks localize to membranes, we cloned six vpcdpks from chinese wild vitis pseudoreticulata accession baihe-35- and assessed the subcellular localizations of the encoded vpcdpks by transient expression assays in arabidopsis protoplasts, using translational fusions to gfp. as shown in fig.  five genes expressed vpcdpk-gfp fusion proteins in transformed arabidopsis protoplasts. vpcdpk3-gfp, vpcdpk10-gfp, and vpcdpk11-gfp only localized on the plasma membrane. vpcdpk5-gfp localized on the plasma membrane and in the nucleus, and vpcdpk2-gfp localized in the nucleus and cytosol.fig. subcellular localization of five vpcdpks. the selected cdpk genes were cloned from chinese wild grape and used to construct camv35s::cdpks–gfp vectors in which gfp was fused at the c terminus. the five vpcdpk-gfp fusion proteins as well as gfp as the control, were transiently expressed in col- arabidopsis protoplasts and observed by fluorescence microscopy. the merged pictures include the green fluorescence channel and the chloroplast autofluorescence channel. the corresponding bright field images are shown on the right. bar = 5 μm unlike the other cdpks, vpcdpk localized to four places: vpcdpk localized on some kind of plastids that could not be identified. considering the closest gene in arabidopsis is atcpk which localizes to lipid bodies and peroxisomes, the plastids that vpcdpk9-gfp localized in may well include lipid bodies and peroxisomes. vpcdpk9-gfp localized to the biomembrane system, most likely on the endoplasmic reticulum, as well as on vesicles. vpcdpk9-gfp also showed extra fluorescence in the cytosol. we also detected vpcdpk9-gfp in the nucleus. the complexity of vpcdpk subcellular localization suggests its functional diversity and variety.fig. subcellular localization of vpcdpk the roman numerals represent the corresponding four subcellular localization patterns of vpcdpk vpcdpk was cloned from chinese wild grape and used to construct camv35s::vpcdpk9-gfp vectors in which gfp was fused at the c terminus. the vpcdpk9-gfp fusion protein, as well as gfp as a control, was transiently expressed in col- arabidopsis protoplasts and observed by fluorescence microscopy. the merged pictures include the green fluorescence channel and the chloroplast autofluorescence channel. the corresponding bright field images are shown on the right. bar = 5 μm expression of grape cdpk genes in chinese wild grape increasingly evidence shows that plant cdpks function in responses to biotic and abiotic stress and signal transduction. to further test how grape cdpk genes respond to various stresses, we measured the expression of grape cdpk genes in chinese wild grape accession baihe-35- biotic stress cdpks act as vital sensors and responders in immune signaling. in this study, we used rt-qpcr to perform a time course analysis of the transcript levels of vpcdpks following inoculation of chinese wild grape vitis pseudoreticulata accession baihe-35- with erysiphe necator, the causal agent of grapevine powdery mildew. five of the grape cdpk genes were up-regulated, including vpcdpk vpcdpk vpcdpk vpcdpk and vpcdpk with transcript levels that increased up to -fold but differed in response time and degree. for example, vpcdpk transcript rapidly increased in abundance and reached a peak of -fold at 96 h post inoculation. similarly, vpcdpk transcript also rapidly increased and remained at high levels. vpcdpk and vpcdpk shared similar expression patterns, with a slow increase in transcript levels to a peak and gradual plateau. vpcdpk transcript abundance increased to nearly -fold, nearly instantly at hpi, and remained at high levels. by contrast, several cdpk genes were down-regulated. for instance, vpcdpk and vpcdpk transcript levels decreased to - to -fold. these results demonstrate that several cdpk genes responded to powdery mildew inoculation in chinese wild grapevine v. pseudoreticulata accession baihe-35- suggesting their vital roles in immune signaling and responses.fig. expression of cdpk genes in chinese wild grape during powdery mildew infection. expression was measured by reverse transcription, followed by real-time, quantitative pcr, is indicated as fold-change of experimental treatments relative to control samples, and is visualized as heatmaps and histogram. grape actin was used as an internal control. the experiments were repeated three times and gave consistent results. mean values and sds were obtained from three biological and three technical replicates. expression profile of vpcdpk gene family under powdery mildew infection. the color scale represents log expression values, with red indicating increased transcript abundance and green indicating decreased transcript abundance. detailed expression levels of four vpcdpk genes that significantly up-regulated during powdery mildew infection. the data were showed as mean value ± sd. * and ** represent statistically significant or highly significant, respectively abiotic stress previous work showed that cdpks participate in abiotic stress responses, including responses to salt and osmotic stress and to temperature stress. in this study, we used nacl treatment, and incubation at low or high temperature to understand how vpcdpks responded to these abiotic stresses on the transcriptional level. our expression data show consistency with the expression of cdpk genes in vitis vinifera and vitis amurensis. as shown in fig. , almost all nineteen cdpk genes were up- or down-regulated to at least one abiotic stress treatment, but some responded rather slightly. as a whole, the vpcdpk genes responded to nacl treatment much more strongly than to temperature stress. vpcdpk and vpcdpk transcript abundance increased in all three treatments, but none of the cdpk genes showed continuously low transcript levels in all treatments.fig. expression of cdpk genes in chinese wild grape under salt and temperature stress treatments. expression was measured by reverse transcription, followed by real-time, quantitative pcr, is indicated as fold-change of experimental treatments relative to control samples, and is visualized as heatmaps a and histogram b. grape actin was used as an internal control. the experiments were repeated three times and gave consistent results. mean values and sds were obtained from three biological and three technical replicates. a expression profile of vpcdpk gene family under nacl, 4 °c, and 42 °c treatments. the color scale represents log expression values, with red indicating increased transcript abundance and green indicating decreased transcript abundance. b detailed expression levels of four vpcdpk genes showed unusual expression patterns under nacl, 4 °c, and 42 °c treatments. the data were showed as mean value ± sd. * and ** represent statistically significant or highly significant, respectively after nacl treatment, twelve of the nineteen cdpk genes were up-regulated, and six genes responded strongly with transcript levels increasing to - to 16-fold higher than the control sample. vpcdpk the most rapidly responding gene, reached a peak of nearly 10-fold at  h post treatment and rapidly decreased to -fold at hpt, before its transcript levels increased from -fold to -fold at 2 h to hpt. the homologous gene of vpcdpk in vitis amurensis, vacpk was also highly up-regulated under nacl treatment. in addition, vpcdpk showed a peak of -fold at hpt and increased transcript abundance of more than -fold from to hpt, similar with the expression pattern of its homologs, vacpk and vvcpk. we noted that the margins of treated leaves became dry from the outside to the inside and the plants had dry-dead leaves after hpt, which might relate to the rapid down-regulation of vpcdpk at hpt. by contrast, vpcdpk and vpcdpk transcript abundance decreased after hpt, indicating their probable negative regulatory roles. the cdpk genes that responded rapidly and strongly most likely participate in the nacl stress response. for 4 °c treatments, only vpcdpk vpcdpk and vpcdpk showed up-regulation. vpcdpk was rapidly up-regulated, reached a peak of -fold at hpt, and then declined to lower than - fold. moreover, vpcdpk had a peak of -fold at hpt but then fluctuated around -fold during the rest of the treatment time. the vpcdpk genes with decreased transcript abundance might not participate in the signaling response to cold stress but their low activities might result in pervasive down-regulation of transcription. however, vpcdpk was an exception, as its transcript levels decreased rapidly at hpt and remained at a steady low level. intriguingly, vacpk as the homolog of vpcdpk in vitis amurensis, showed significant up-regulation under 10 °c treatment. we also measured the transcript levels of vpcdpk genes in response to 42 °c treatment. we found that five cdpk genes significantly responded to 42 °c treatment; of these, vpcdpk had a strong response at hpt, with its transcript abundance increasing to -fold and remaining at a high level. the transcript abundance of vpcdpk and vpcdpk increased, similar to vpcdpk, although vpcdpk transcripts were almost undetectable at hpt. in particular, vpcdpk responded to 42 °c treatment rapidly and intensely, reaching a peak of more than -fold and then returning to nearly normal transcript levels at hpt. taken together, our data showed that expression of vpcdpks responded to low and high temperatures suggest that the vpcdpks may play key roles in the response to temperature stress. hormone treatment plant hormones such as aba, sa, meja, and ethylene have well-established roles in modulating plant signaling networks. in this study, hormone treatments resulted in a wide variety of changes in the transcript levels of vpcdpk genes.fig. expression of cdpk genes in chinese wild grape in response to treatment with plant hormones. expression was measured by reverse transcription, followed by real-time, quantitative pcr, is indicated as fold-change of experimental treatments relative to control samples, and is visualized as heatmaps and histogram. grape actin was used as an internal control. the experiments were repeated three times and gave consistent results. mean values and sds were obtained from three biological and three technical replicates. a expression profile of vpcdpk gene family under aba, sa, meja, and ethylene treatments. the color scale represents log expression values, with red indicating increased transcript abundance and green indicating decreased transcript abundance. b detailed expression levels of four vpcdpk genes showed unusual expression patterns under aba, sa, meja, and ethylene treatments. the data were showed as mean value ± sd. * and ** represent statistically significant or highly significant, respectively an increasing body of evidence has shown that cdpks regulate aba-mediated signal transduction in plants. our experimental data suggest that of the vpcdpk genes respond to aba treatment. among them, eight vpcdpk genes showed significant up-regulation in response to aba. however, vpcdpk transcript levels rapidly increased up to -fold at hpt and then decreased to lower than -fold at hpt. interestingly, the majority of the responsive cdpk genes showed a similar pattern, with their transcript abundance increasing rapidly to a peak at hpt and remained at high levels until hpt. similar to the aba response, of the vpcdpk genes also showed positive regulation under sa treatment. six genes were significantly up-regulated, exhibiting a steady or gradual increase from to or to hpt. interestingly, the expression of vpcdpk vpcdpk vpcdpk and vpcdpk was also induced after inoculation with powdery mildew pathogen. for instance, vpcdpk showed positive regulation by inoculation of powdery mildew and peaked at hpi, while under sa treatment, vpcdpk was also up-regulated and peaked at hpt. the expression pattern after sa treatment and powdery mildew inoculation, suggests the vital roles of grape cdpk genes in immune signaling. in contrast with aba and sa, the vpcdpks showed more limited responses to meja treatment. a total of ten genes were up-regulated; and among them, only three vpcdpk genes showed distinct up-regulation. in particular, their transcript abundance peaked at or hpt, except for vpcdpk, which strongly and rapidly responded to the treatment from to hpt with meja and peaked at hpt. that might indicate its functional significance in the meja signaling pathway. the expression changes caused by ethylene treatment were distinct from those caused by the other hormone treatments. for ethylene treatment, only a handful of vpcdpk genes were up-regulated, while more genes showed down-regulation. for example, two up-regulated genes, vpcdpk and vpcdpk, their transcript abundance increased rapidly at hpt, but then increased only slightly or returned to normal transcript levels, respectively. chinese wild grapevine baihe-35- seems to be very sensitive to ethylene treatment and almost all the leaves of baihe-35- plants fell at hpt, so we had to use the fallen leaves for rna extraction. vpcdpk vpcdpk vpcdpk vpcdpk and vpcdpk had decreased transcript abundance lower than - fold during the treatment time, suggesting their potential negative regulatory functions in plant responses to ethylene signaling. discussion identification of grape cdpk genes previously, chen investigated the cdpk gene family in grapevine and identified members, which were predicted to have the typical cdpk gene structure with a ser/thr protein kinase domain and four ef-hands. considering the complexity and variability in the calmodulin-like domain, we performed blast-p searches in ncbi using the putative grape cdpk genes predicted by pkinase and ef-hand_ hmm profiles, and identified the previously discovered cdpks and two more members. compared with arabidopsis cdpks and rice cdpks, we found that gsvivt possesses a similar domain organization to atcpk and oscpk which are predicted to have no ef-hand with default parameters in pfam but are predicted to have ef-hands in prosite and interpro. in addition, the phylogenetic tree showing that gsvivt and oscpk are homologous genes provides more evidence. therefore, we considered gsvivt as members of the grape cdpk gene family and the same with gsvivt. it is worth to mention that a homolog of vvcdpk in vitis amurensis, named as vacpk has been identified as a cdpk gene. both of these two genes are same in gene length and phylogenetic relationship. consider that, vpcdpk can also be identified as a cdpk gene. these two new members of the grape cdpk gene family were designated vvcdpk and vvcdpk on the basis of their chromosomal locations. structural characteristics of grape cdpk genes the structural conservation and divergence of grape cdpk genes led to gene family expansion and functional conservation or differentiation. structural characteristics such as acylation sites and intron-exon structures show the details of gene family expansion and divergence. most of cdpks possess acylation sites, including n-myristoylation sites and s-palmitoylation sites, which are generally believed to function in membrane targeting. in arabidopsis, atcpks have both myristoylation and palmitoylation sites and all of them target exclusively to membranes, especially to the plasma membrane, although several have not been determined. among our identified nineteen grape cdpk genes, six contain both myristoylation and palmitoylation sites, nine contain only palmitoylation sites, two contain only myristoylation sites, and two do not contain any acylation site. we tested the subcellular localization of six vpcdpk proteins. vpcdpk vpcdpk vpcdpk and vpcdpk have palmitoylation sites while vpcdpk has a myristoylation site and they all localized on the plasma membrane, consistent with results in arabidopsis. three pivotal mechanisms contribute to gene family evolution and expansion: exon/intron gain or loss, exonization/ pseudo-exonization, and insertion /deletion. previous work reported that grape cdpk genes have a single origin and can be dated back to green algae, before plants colonized the land. for the four groups in fig.  group iv was the earliest one that expanded from the evolutionary branch. as a result, it has the longest evolutionary history, leading to complex intron-exon organization. both group ii and group iii originated from group i in evolutionary history, and show similar structural divergence. all of the members of group ii contain one more exon and a phase- intron at the sixth position, indicating that there was an intron insertion in the last exon, which contributed to the generation of group ii and expansion of the vvcdpk family. group iii can be separated into two subgroups in evolutionary history, as reflected in their intron-exon structures. one subgroup was similar to group i in intron-exon structures and the other one contains one more exon and a phase- intron at the first position, suggesting that there was an intron insertion in the first exon. our result is consistent with the previous work that can be reflected in the origin and evolutionary history of vvcpks. the intron-exon divergence was closely related to the evolutionary relationship of the grape cdpk family and might result in functional diversity. evolutionary relationships of grape cdpk genes to study the evolutionary relationships among different vvcdpk genes and the history of the vvcdpk gene family, as well as to further study their gene function, we investigated gene duplication events, syntenic regions, and phylogenetic relationships among the vvcdpk genes. segmental duplications and tandem duplications are the main mechanisms leading to gene family expansion. these processes may lead to functional redundancy, sub-functionalization and neo-functionalization. no tandem duplication involving vvcdpk genes was discovered in the grapevine genome, but two segmental duplications were found, vvcdpk5-vvcdpk and vvcdpk12-vvcdpk. previously, chen identified three segmental duplications. except for vvcdpk5-vvcdpk and vvcdpk12-vvcdpk they also identified triplet-duplicated genes, vvcpk8-vvcpk9-vvcpk however, the method they used was not clearly presented, so we cannot follow their method. anyway, both of chen and us indeed identified the two duplicated gene pairs, vvcdpk5-vvcdpk and vvcdpk12-vvcdpk these two duplicated gene pairs are quite similar in gene length, acylation sites, and intron-exon organization. furthermore, they possess the closest phylogenetic relationship among grape cdpk genes. however, despite the fact vvcdpk and vvcdpk are phylogenetically closest among cdpks from other species, these two genes have different syntenic genes in arabidopsis, which means the duplication events happened before the evolutionary divergence of arabidopsis and grapevine. both vvcdpk and vvcdpk showed closer phylogenetic relationships with arabidopsis cdpk genes than that with each other, indicating that this duplication event might also have happened before the divergence of arabidopsis and grapevine. from what has been discussed above, vvcdpk5-vvcdpk and vvcdpk12-vvcdpk likely have different functions but still possess potential functional connections and similarities. comparative genomics approaches structure genomes into syntenic blocks that exhibit conserved features across the genomes. the synteny analysis provides evolutionary and functional connections between grape and arabidopsis syntenic genes. furthermore, a large number of syntenic relationships suggest that some of the grape cdpk genes arose before the divergence of the arabidopsis and grapevine lineages. ten grape cdpk genes were found to have syntenic relationships with arabidopsis genes all of these vvcdpk genes show close phylogenetic relationships with the corresponding atcdpk genes, suggesting their potential functional similarities. interestingly, all of the grape cdpk genes in group iii have syntenic genes in arabidopsis, suggesting that group iii cdpk genes are relatively conserved over evolutionary history. analysis of the phylogenetic tree revealed that cdpk homologs among several monocots and eudicots clustered into four distinct groups, which correspond to the clades formerly identified in green plants. previously, hamel examined the cdpk families from green algae to land plants to demonstrate that cdpk families are conserved among land plants, whereas cdpks from green algae have continued to evolve independently. supporting this, our data suggest that cdpks from monocots and eudicots cluster separately and evolutionary relationships of the four clades are relatively conserved. the previous work also constructed a phylogenetic tree that gives the consistent results and conclusions. functional prediction of grape cdpk genes by comparison with arabidopsis comparative genomics provides an effective way to understand the structure and function of genomes by translating knowledge gained from model species to the species of interest. combining synteny analysis with phylogenetic analysis provides new insights for further investigating the functions of the grape cdpk genes by comparing orthologous genes between two species, in this case, between grapevine and arabidopsis. our study provided substantial evidence to help predict the functional conservation or divergence of cdpk genes between grapevine and arabidopsis. chinese wild grape accession baihe-35- compared with the sequenced vitis vinifera, reported to have remarkable resistance to both biotic and abiotic stress. in addition, vitis amurensis is also a wild grapevine species with a high level of resistance to multiple stresses. our expression profile of vpcdpk genes showed consistency with that reported in vitis vinifera and vitis amurensis. for example, the homologous cdpk genes, vpcdpk3/vvcpk3/vacpk and vpcdpk17/vvcdpk17/vacpk showed distinct up-regulation under salt stress treatment. however, there are also some differences on genes expression, e.g., vacpks respond to cold stress much significantly than vpcdpks, probably because of vitis amurensis shows strong capacity of cold-resistance. two duplicated gene pairs, vpcdpk5/vpcdpk and vpcdpk12/vpcdpk as discussed above, provide experimental evidence to support our predictions and views. vpcdpk and vpcdpk each have particular syntenic genes in arabidopsis showing a complicated evolutionary history. in addition, the experimental data showed that these two genes have distinct expression patterns under a range of treatments. for example, vpcdpk was obviously up-regulated under hormone treatments, while vpcdpk was only slightly up-regulated under aba treatment. also, for abiotic stress treatments, vpcdpk was down-regulated under nacl and 42 °c treatments, but vpcdpk transcripts remained at constant levels. furthermore, subcellular localization analysis demonstrated that vpcdpk5-gfp localized on plasma membrane and in the nucleus, but vpcdpk11-gfp only localized on plasma membrane. the aforementioned information indicated that vpcdpk and vpcdpk might well have undergone neo-functionalization. moreover, vvcdpk in v. vinifera cv. corvina expressed in almost all of the organs, whereas vvcdpk only expressed in pollen. however, we tested vpcdpk in the chinese wild grape v. pseudoreticulata responded to hormone treatments in leaves, suggesting that vpcdpk was not only transcribed in pollen. for vpcdpk its low expression under multiple treatments and high expression in almost all tissues indicates that its function most likely relates to housekeeping genes. the other duplicated gene pair, vpcdpk and vpcdpk were quite different. the transcript levels of these two genes showed similar tendencies under abiotic stress, aba and sa treatments, however, vpcdpk seemed to respond much strongly than vpcdpk based on chen, vvcdpk had high transcript levels in almost all tissues while vvcdpk had low transcript levels. these data suggest that the duplicated gene pair vpcdpk12/vpcdpk probably have undergone sub-functionalization and vpcdpk tends to be non-functional. it is worth noting that vpcdpk is unique in the grape cdpk family. for one thing, it showed an unusual expression pattern under various treatments, being strongly up-regulated, except for 4 °c treatment. its transcript abundance increased to a high level during most or all of the treatment. subcellular localization also demonstrated its considerable differences with the other grape cdpks we examined. the other vpcdpks have only one pattern of localization, while vpcdpk shows four patterns, including in some kind of plastids, in biomembranes, in the cytosol, or in the nucleus. testing why vpcdpk has four patterns of localization, and the relationship between its localization and function, will require more experimental data. the genes that are phylogenetically close to vpcdpk in arabidopsis are atcpk and atcpk. vpcdpk has structural and functional commonalities with atcpk for example, atcpk expression is rapidly induced by fungal elicitors and loss-of-function mutants of atcpk exhibit higher susceptibility to pathogen infection. ectopic expression of atcpk enhanced nadph oxidase activity and the oxidative burst in tomato protoplasts. cold-stress can also induce atcpk transcripts via phosphoprotein signals. our data also showed that vpcdpk not only responded to powdery mildew infection and abiotic stress, but also responded to hormone treatments. these results are consistent with the expression profile of atcpk future work will be directed toward identification of potential defense components that may be directly or indirectly regulated by vpcdpk in grapevine baihe-35- vpcdpk whose arabidopsis homolog is atcpk is found in the chromosomal region syntenic with atcpk and plays positive regulatory roles in various treatments. results on atcpk show high consistency with our results on vpcdpk atcpk was reported to localize to the plasma membrane and the nucleus, consistent with our data showing that vpcdpk also localized to the plasma membrane and nucleus. atcpk can localize to the nucleus where it interacts with and phosphorylates wrky and to activate defense genes, while vpcdpk also localized in the nucleus, suggesting it has similar functions with atcpk atcpk also phosphorylates rbohd in vivo, resulting in h2o production and leading to cell death, involving in immune signaling. vpcdpk responded to powdery mildew inoculation with up-regulated transcript levels, indicating its potential function in immune signaling and that vpcdpk most likely participates in the analogous pathways to those mentioned above for arabidopsis. these structural and locational similarities provide indications of functional consistency. vpcdpk has a syntenic gene in arabidopsis, atcpk further analysis provides valuable comparisons of these two genes and helps predict the function of vpcdpk in our study, vpcdpk localized on the plasma membrane, and atcpk was also reported to localize on the plasma membrane. vpcdpk expression did not respond to stress treatments but was distinctly up-regulated in response to aba treatment. atcpk specifically inhibits kat and kat shaker channels, which participate in aba-induced stomatal movements by affecting cdpk phosphorylation. these insights indicate that vpcdpk plays important roles in aba- and ca2+-mediated stomatal regulation. another intriguing gene, vpcdpk is phylogenetically close to atcpk and atcpk, which are activated by aba and phosphorylate the c-terminus of acs in ethylene biosynthesis. meanwhile, atcpk and atcpk can phosphorylate the aba-responsive transcription factors abf and abf and lead to salt insensitivity in seed germination and decrease tolerance of seedlings to salt stress. sustained atcpk and atcpk activation directly phosphorylated wrky transcription factors involved in immune signaling. expression patterns suggest that vpcdpk responded to powdery mildew inoculation as well as nacl, aba, and sa treatments, showing high functional consistency with atcpk and atcpk this evidence indicates that vpcdpk might participate in similar pathways as atcpk and atcpk in arabidopsis. in addition to stress, cdpk genes also play important roles in growth and development process. on this regard, plant hormones are mainly responsible. for instance, several plant hormones may play central roles in the control of ripening in the grape berry. as a support, kühn et al. investigated the aminocyclopropane-1-carboxylate synthases genes in first developed grape berries, which pathway is related to ethylene signaling. for cdpk genes, several genes including atcpk, ntcdpk and lecdpk were reported to phosphorylate acc synthases to trigger ethylene biosynthesis and accumulation. coincidence is that vpcdpk and vpcdpk as homologs of ntcdpk and lecdpk are positively regulated under ethylene treatment, indicating their possible functions involved in ethylene signaling. however, cdpks functions in plants growth and development process still need more experimental evidence to deeply understand their biological roles and the pathways they involved in.  so far, little systematic analysis of the cdpk family has been reported in grapevine, and the functions of most cdpk genes remain unclear. however, accumulating evidence indicates that cdpks play important roles in response to a broad variety of abiotic and biotic stresses and biological processes. in this paper, genome-wide identification, evolutionary, and expression analyses were carried out to provide a framework for further analysis of grape cdpk genes in defining their biological functions and pathways during stress responses as well as growth and development. expression profiles showed grape cdpk genes respond to various stresses and hormone treatments; moreover, analysis of cdpk subcellular localization gave evidence as to their functions. comparisons of the grape and arabidopsis genomes and expression profiles provide novel insights into the functions of less well-studied genes according to their better-understood homologs. by prediction and experimental data, we speculated that cdpk gene family might participate in responses to pathogen, cold, heat and salt stress, and the related biological processes might covering the regulation of gene expression, control of the ion channel, regulation of the enzyme activity, mediation of the cross-talk between signaling pathways and so on. these observations may lay the foundation for future functional analysis of grape cdpk genes to unravel their biological roles. 
the expression profiles derived from reverse transcription and quantitative pcr suggested that a large number of vpcdpks responded to various stimuli on the transcriptional level, indicating their versatile roles in the responses to biotic and abiotic stresses. segmentally duplicated grape cdpk genes showed high structural conservation and contributed to gene family expansion. calcium-dependent protein kinases play vital roles in plant growth and development, biotic and abiotic stress responses, and hormone signaling. additional comparisons between grape and arabidopsis thaliana demonstrated that several grape cdpk genes occured in the corresponding syntenic blocks of arabidopsis, suggesting that these genes arose before the divergence of grapevine and arabidopsis. moreover, we examined the subcellular localization of vpcdpks by transiently expressing six vpcdpk-gfp fusion proteins in arabidopsis mesophyll protoplasts; this revealed high variability consistent with potential functional differences. little is known about the cdpk gene family in grapevine. furthermore, we examined the expression of the corresponding nineteen homologous cdpk genes in the chinese wild grape under various conditions, including biotic stress, abiotic stress, and hormone treatments. comparison of the structures of grape cdpk genes allowed us to examine their functional conservation and differentiation. phylogenetic analysis divided the grape cdpk genes into four groups. taken as a whole, our data provide significant insights into the evolution and function of grape cdpks and a framework for future investigation of grape cdpk genes. grapevine cdpksynteny analysisphylogenetic treesubcellular localizationexpression profilesissue-copyright-statement© the author 2015 in this study, we performed a genome-wide analysis of the 12x grape genome and identified nineteen cdpk genes.
 calcium-dependent protein kinases play vital roles in plant growth and development, biotic and abiotic stress responses, and hormone signaling. little is known about the cdpk gene family in grapevine.  in this study, we performed a genome-wide analysis of the 12x grape genome and identified nineteen cdpk genes. comparison of the structures of grape cdpk genes allowed us to examine their functional conservation and differentiation. segmentally duplicated grape cdpk genes showed high structural conservation and contributed to gene family expansion. additional comparisons between grape and arabidopsis thaliana demonstrated that several grape cdpk genes occured in the corresponding syntenic blocks of arabidopsis, suggesting that these genes arose before the divergence of grapevine and arabidopsis. phylogenetic analysis divided the grape cdpk genes into four groups. furthermore, we examined the expression of the corresponding nineteen homologous cdpk genes in the chinese wild grape under various conditions, including biotic stress, abiotic stress, and hormone treatments. the expression profiles derived from reverse transcription and quantitative pcr suggested that a large number of vpcdpks responded to various stimuli on the transcriptional level, indicating their versatile roles in the responses to biotic and abiotic stresses. moreover, we examined the subcellular localization of vpcdpks by transiently expressing six vpcdpk-gfp fusion proteins in arabidopsis mesophyll protoplasts; this revealed high variability consistent with potential functional differences.  taken as a whole, our data provide significant insights into the evolution and function of grape cdpks and a framework for future investigation of grape cdpk genes. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords grapevine cdpksynteny analysisphylogenetic treesubcellular localizationexpression profilesissue-copyright-statement© the author 2015 