BACKGROUND
jasmonic acid and its bioactivity derivatives are collectively known as jasmonates .
they play important roles in regulating the responses to biotic and abiotic stresses, such as herbivory, pathogen invasion, wounding, uv radiation and ozone stress in plants .
in addition, jas also control various developmental processes in plants, viz.
pollen maturation, anther dehiscence, embryo maturation, tendril coiling, tuber and trichome growth .
therefore, jas are regarded as moderators that mainly involved in regulating the defense reaction, inhibiting the photosynthesis and cell division, and keeping the balance between growth and defense in plants  <cit> .
in plants, ja signaling pathway is a very complex process involved in many genes or proteins  <cit> , and this pathway can be divided into three processes, including the biosynthesis and metabolism of signaling molecules, ja signal transduction and responses of downstream genes.
previous studies showed that ja signaling molecules, scfcoi <dig> receptor complex, jasmonate-zim  domain repressor and transcription activator myc <dig> participate together and interact with each other in ja signaling pathway .
in the plant cells with a lower-concentration of ja, the transcription of ja response genes is restrained owing to the combination between jaz protein and myc <dig>  under stimulation, ja is compounded and accumulated in plant cells.
high concentrations of ja can promote the combination between jaz proteins and scfcoi <dig>  and make jaz proteins be ubiquitinated.
then the ubiquitinated protein is alternatively degraded by 26s protease.
finally, the activity inhibition of transcription activator myc <dig> is removed.
expression of ja response genes is activated subsequently  <cit> .
it is known that jaz proteins have a crucial effect on the ja signaling pathway and they are the linkers of ja signaling transduction.
the jaz protein family is a member of tify transcription factor superfamily, and the family members have two conservative functional domains, tify  and jas  .
tify domain is usually consisted of  <dig> amino acids, which located in the n-terminus of jaz protein sequences, and its core sequence is tifxg  <cit> .
in ja signaling pathway, tify domain can mediate the interaction between jaz protein and its co-suppressor, a novel interactor of jaz , and then collectively restrict the ja signal transduction  <cit> .
jas domain is near the c-terminus of jaz protein sequences, and its sequence is extremely conserved among the members of jaz protein family, of which  <dig> amino acids are same or replaced in conservation.
its function is to mediate the direct combination between jaz protein and myc <dig> in ja signaling pathway and inhibit the transcriptional activity of myc <dig>  <cit> , and then further restrict the expression of ja response genes.
as the connector between myc <dig> and coi <dig>  jaz protein was firstly reported in  <dig>  <cit> .
afterwards, many studies about jaz proteins have been conducted.
to date,  <dig> and  <dig> jaz genes have been found in arabidopsis thaliana  <cit>  and rice  <cit> , respectively.
according to previous studies, the jaz gene family has plenty of members, but each has a different biological function.
for example, the protein encoded by osjaz <dig> is a regulator of ja signaling pathway and can regulate the salt tolerance of rice  <cit> .
the expression of ghjaz <dig> can be activated by gbwrky <dig>  so that it can strengthen the capacity for resistance against verticillium dahlia in cotton  <cit> .
overexpression of modified atjaz <dig>  is able to enhance host resistance to spodoptera exigua in arabidopsis  <cit> .
overexpressing the modified osjazs can cause the malformation of flower organ in rice  <cit> .
according to the above researches, jaz genes have important effects on regulating the adaptability to biotic and abiotic stresses and maintaining the normal development in plants.
wheat is one of the most important crops in the world.
the changes of climate and growing environments bring huge challenge to wheat production.
thus, use of molecular biology and genetics methods is an important approach for improving the stress tolerance and quality of wheat.
thermosensitive genic male sterile  lines, such as bs <dig>  are of particular significance in two-line hybrid system, which is more efficient in breeding  <cit> , because the fertility of tgms lines is strictly regulated by temperature .
hybrid seed could be produced under sterile condition , while tgms line itself could be reproduced under fertile condition  <cit> .
in our early research, we found that the anther dehiscence in bs <dig> is abnormal, and its pollen can not fully spill out.
this defective phenotype can be recovered via spraying meja in vitro  <cit> .
so, we assumed that this phenotype is closely related to the synthesis and regulatory pathway of ja.
based on the current wheat genome sequencing data, we firstly identified the tajaz gene family, and analyzed its architectural features, evolutionary history and expression patterns in anther tissues in tgms wheat line.
the results presented in this study were expected to enrich our knowledge of jaz gene family in wheat, and provide the theoretical basis and novel candidate genes for improving and creating the male sterility wheat lines.
RESULTS
identification of jaz repressor gene family in wheat
based on the latest genome data of wheat, a hidden markov model  search was carried out using the hmm profiles of the tify domain  and jas domain  as queries against the local protein database.
by retrieving the database, we detected  <dig> non-redundant sequences.
among these,  <dig> were removed because they lacked the typical tify or jas domain.
finally, we obtained  <dig> full-length protein sequences .
the corresponding coding sequences and genome sequences were simultaneously isolated.
the location of each tajaz gene was confirmed by aligning the wheat chromosome genomic sequences using blastn.
according to the information of phylogenetic relationship,  <dig> tajaz genes were clustered into  <dig> groups .
the genes from different wheat sub-genomes and in the same group were regarded as different copies of each member of the tajaz gene family.
thus, we obtained the tajaz gene family including  <dig> members designated from  <dig> to  <dig> according to the naming convention, and the copies of each member were distinguished by subjoining the wheat sub-genome symbols a, b or d.table  <dig>
jaz gene family in wheat
tajaz1-a
tajaz1-b
tajaz1-d
tajaz2-b
tajaz3-a
tajaz3-d
tajaz4-a
tajaz4-b
tajaz4-d
tajaz5-a
tajaz5-b
tajaz5-d
tajaz6-a
tajaz6-d
tajaz7-a
tajaz7-b
tajaz7-d
tajaz8-a
tajaz8-d
tajaz9-a
tajaz9-b
tajaz9-d
tajaz10-a
tajaz10-b
tajaz10-d
tajaz11-a
tajaz12-a
tajaz12-d
tajaz13-a
tajaz13-d1
tajaz13-d2
tajaz14-a
tajaz14-b
tajaz14-d
athe sequence name beginning with treas and taloc means download from the wheat genome ensembl database and urgl database, respectively
fig.
<dig> phylogenetic relationship and motif structure of wheat jaz proteins.
a the phylogenetic tree of tajaz proteins constructed from a complete alignment of  <dig> wheat jaz proteins using mega  <dig>  by the neighbor-joining method with  <dig> bootstrap replicates.
bootstrap scores are indicated on the nodes and the  <dig> members of tajaz, most of which contain duplicated genes, are indicated by yellow or pink block.
b domain distribution of tajaz proteins were investigated using the meme web server.
color blocks represent the position of motifs on corresponding proteins.
c the consensus sequence of tify and jas motif from wheat jaz proteins.
the relative position of each motif can be determined using the scale below
genome distribution of wheat jaz genes
among  <dig> wheat jaz genes,  <dig>   <dig> and  <dig> were distributed in wheat sub-genomes a, b and d, respectively .
none of tajaz gene copy was distributed on chromosomes 1a, 3a, 1b, 3b, 6b, 1d and 3d.
each of the  <dig> tajaz genes  had three copies on group  <dig>   <dig>   <dig> and  <dig> chromosomes, respectively .
four tajaz genes had two copies each including tajaz3-a/-d, tajaz6-a/-d, tajaz8-b/-d, and tajaz12-a/-d .
tajaz <dig> and tajaz <dig> had only one copy on chromosomes 2b and 6a, respectively .
tajaz <dig> also had three copies, and tajaz13-a was found on chromosome 7a, tajaz13-d <dig> and -d <dig> were detected on chromosome 7d .
in addition, chromosome 4d had the highest number of five tajaz gene copies, whereas other chromosomes contained no more than four copies .fig.
<dig> chromosome distribution of jaz gene family in wheat.
red dots on the chromosomes indicate the position of centromeres.
the red arrows next to gene names show the direction of transcription.
the position of each gene could be estimated using the left scale
structure analysis of wheat jaz genes, and jaz proteins
in order to obtain more insights about the gene structural evolution, the exon-intron organization of wheat jaz genes was raveled by aligning the predicted coding sequences  against the corresponding genomic sequences using the online service gsds.
in the tajaz gene family, the number of exons ranged from  <dig> to  <dig>  and the number of introns ranged from  <dig> to  <dig> .
among  <dig> tajaz genes containing two or three copies,  <dig>  had the same gene structures, whereas  <dig>  had one, two or four exons in each group.
in addition, tajaz <dig> and tajaz <dig> lacked intron .
overall, a highly similar gene structure was exhibited in the duplicated tajaz genes.fig.
<dig> exon-intron structures of tajaz genes.
exons are represented by blank boxes and introns by blank lines.
the sizes of exons and introns could be estimated using the scale below
the tify and jas domains are important for the repressor activity of jaz proteins in plants.
the tify and jas domains locate at the n-terminus and c-terminus of jaz proteins, respectively.
in order to understand the architecture of jaz proteins in wheat, we scanned these two domains using the meme web server.
as shown in fig.
1b and c, single tify and jas motifs were distributed in each tajaz protein and all tajaz proteins had tify motif at n-terminus while jas motif at c-terminus.
the results were in accordance with the previous studies  <cit> , indicating that the structures of jaz proteins were conserved.
moreover, the multi-sequence alignments of protein sequences revealed that the amino acid sequence “tify” was changed into “tvfy” in tajaz <dig>  “tmfy” in tajaz <dig>  “tlfy” in tajaz <dig>  “tlvy” in tajaz <dig> and “tlsf” in tajaz <dig> .
the same variations also existed in rice  <cit> , maize  <cit> , and b. distachyon  <cit> .
besides tify domain, the jas domain is also conserved.
in arabidopsis, jas domain contains 12- <dig> amino acids, and the sequences are basically coincident or conserved  <cit> .
but in wheat, the jas domain in some tajaz proteins, such as tajaz <dig>   <dig> and  <dig>  was inserted into a nuclear localization signal  sequence .
phylogenetic analysis of tajaz proteins
in order to understand the phylogeny of jaz proteins, n-j tree was built using mega  <dig>  software and the reliability was tested by bootstrap analysis for  <dig> replicates.
as shown in fig.
<dig>  all the jaz proteins derived from different plants were clustered into sub-groups g1-g <dig>  and the tajaz proteins were clustered into sub-groups g3-g <dig>  sub-group g <dig> had the maximal number of tajaz proteins, and  <dig> tajaz proteins  were clustered into this sub-group.
based on the order that the jaz proteins appeared in plants, those  <dig> sub-groups were divided into groups i  and ii  .
group i included sub-groups g <dig>  g <dig>  g <dig>  g <dig>  g <dig> and g <dig>  and group ii comprised sub-groups g <dig>  g <dig>  g <dig>  g <dig> and g <dig>  moreover, we found that the tajaz proteins were clustered into the same clades with some aetjaz, bdjaz or osjaz proteins.
for example, tajaz <dig> was clustered into the sub-group g <dig> with osjaz <dig>  tajaz <dig> was clustered into the sub-group g <dig> with bdjaz <dig>  and tajaz <dig> was clustered into the sub-group g <dig> with aetjaz <dig> .
these revealed that the tajaz proteins shared a high similarity with those in ae.
tauschii, b. distachyon and rice.fig.
<dig> phylogenetic relationship of jaz proteins among wheat and other species.
the full-length amino acid sequences of 34 t. aestivum, 6 s. moellendorffii,  <dig> a. thaliana,  <dig> o. sativa,  <dig> p. patens,  <dig> maize,  <dig> b. distachyon, 15 s. bicolor,  <dig> p. sitchensis,  <dig> ae.
tauschii and 7 h. brasiliensis genes were aligned by using clustalx and the phylogenetic tree was constructed using mega  <dig>  by the neighbor-joining method with  <dig> bootstrap replicates.
each tajaz protein is indicated by a red dot.
two major groups, group i and ii, are represented by the red and blue, respectively
adaptive evolution analysis of the tajaz gene family
to investigate which type of darwinian selection determined the process of tajaz gene divergence after duplication, the ka/ks substitution ratio was utilized to the coding sequences of  <dig> pairs of orthologs between wheat and b. distachyon jaz gene family .
according to the previous research, ka/ks ratio <  <dig> means purifying selection, ratio =  <dig> means neutral evolution and ratio >  <dig> means positive selection  <cit> .
as shown in table  <dig>  the ka/ks ratios of tajaz genes ranged from  <dig>  to  <dig> , suggesting that the tajaz gene family had undergone purifying selection in wheat.table  <dig> ka/ks ratio of the duplicated jaz genes in wheat using brachypodium distachyon as an outgroup
putative cis-acting regulatory elements in the promoter region of tajaz genes
to gain the information about the cis-acting regulatory elements of tajaz gene family, the putative promoter region sequence of each tajaz gene was analyzed.
in this study, promoter sequences were available for  <dig> of  <dig> tajaz genes, while only the promoter of tajaz <dig> was unavailable due to the limited genome data of wheat.
eight types of putative cis-acting regulatory elements were identified in tajaz genes, including ltr element involved in low-temperature responsiveness, tga element related to auxin-responsiveness, cgtca-motif involved in meja responsiveness, abre associated with aba signaling pathway, box-w <dig> for the response of fungal elicitor, gare-motif of a gibberellin responsive element regulating gibberellin responsive genes, mbs, a myb-binding sequence involved in regulation of drought-inducible genes and tca element regulating the sa related genes .
the type and number of cis-acting regulatory elements in the promoter region of each tajaz gene were discrepant .
these showed that the members of tajaz gene family might be able to respond to different abiotic stresses.
expression profiling of tajaz genes under abiotic stresses in tgms wheat line
in order to obtain the expression profiling of tajaz genes under plant hormones, low temperature , high salinity and drought treatments, qrt-pcr was performed to gain the relative expression pattern of each tajaz gene using the 14-day-old tgms wheat seedlings.
as shown in fig.
<dig>  the transcript profiling of tajaz genes showed up-regulation of tajaz  <dig>   <dig>   <dig>   <dig>   <dig>   <dig>   <dig>   <dig>   <dig> and  <dig> and down-regulation for tajaz <dig>   <dig>   <dig> and  <dig> in response to meja treatment.
aba treatment influenced the expression dynamics of tajaz genes.
except that tajaz <dig>   <dig> and  <dig> were inhibited, the expression levels of the remaining tajaz genes were up-regulated under aba treatment .
under ga treatment, the transcriptional levels of tajaz <dig> and  <dig> were increased, and tajaz <dig> and  <dig> were inhibited.
the expression patterns of other tajaz genes were similar, i.e., up- at 2–4 h or 2–8 h followed by down-regulated at 24 h .
under iaa treatment, the expression levels of tajaz <dig>   <dig> and  <dig> were up-regulated at time points 2 h, 4 h and 8 h; tajaz <dig> and  <dig> were rapidly up-regulated at 2 h, but down-regulated subsequently; tajaz <dig> was induced at each time point, but inhibited at 4 h. the expression levels of remaining tajaz genes were inhibited in different degree .
moreover, the expression analysis of tajaz genes showed that tajaz <dig>   <dig>   <dig>   <dig>   <dig> and  <dig> were up-regulated under sa treatment, and the rest of tajaz genes were down-regulated .
these revealed that all tajaz genes could respond to various plant hormones, and they might be involved in some complex signaling pathways.fig.
<dig> expression heat maps of  <dig> tajaz genes under  <dig> phytohormone , drought, salt and cold treatments in tgms wheat line.
qrt-pcr strategy was used to analyze the relative expression level of each tajaz gene.
the expression level of wheat actin was used as the internal control to standardize the rna samples for each reaction, and the expression at 0 h was set as  <dig> .
the data are from three biological replicates
under high salinity treatment, the expression levels of tajaz <dig>   <dig>   <dig> and  <dig> were up-regulated, while tajaz <dig> and  <dig> were up-regulated at 2–4 h post treatment, but down-regulated at 8 h subsequently .
the expression levels of tajaz <dig> were reduced at 2–8 h, and increased at 12–24 h. for drought stress, the expression analysis of tajaz genes showed that  <dig> genes  were up-regulated at 2–8 h or 2–12 h, whereas tajaz <dig>   <dig> and  <dig> were inhibited at each time point after treatment, and tajaz <dig>   <dig> and  <dig> were decreased firstly but increased subsequently .
in addition, except tajaz <dig>   <dig> and  <dig>  we found that tajaz genes could respond to low temperature , and the transcriptional levels of the rest of genes were obviously up-regulated.
ulteriorly, we found that the expression levels of tajaz <dig>   <dig>   <dig>   <dig>   <dig> and  <dig> were up-regulated at each time point .
these results indicated that the members of tajaz gene family were highly sensitive to high salinity, drought and low temperature treatments, and might be involved in some very complex regulation networks in tgms wheat line bs <dig>
expression patterns of tajaz genes during the whole heading stage
the tgms wheat line bs <dig> exhibits temperature-dependent sterility.
the anthers of bs <dig> are indehiscent in sterile environment   <cit> , and abnormal dehiscence in fertile environment  .
the anthers of the normal control wheat line jing <dig> can normally dehisce in fertile environment .
in order to explore the relationship between the expression patterns of tajaz genes and the abnormal anther dehiscence in tgms wheat line bs <dig>  the whole heading stage was divided into six periods, from the start of heading to pre-abloom stage .
qrt-pcr was used to analyze the relative expression levels of each tajaz gene in bs <dig> and jing <dig> anther tissues, which were collected from the fertile environment.fig.
<dig> expression patterns of tajaz genes during the whole heading stage.
a a, b the phenotype of anther dehiscence in conventional wheat line jing411; c, d the phenotype of anther dehiscence in tgms wheat line bs <dig>  scale bars: a, c = 4 mm, b = 2 mm, d = 1 mm.
b the schematic diagram of  <dig> stages during the whole heading period and the size of corresponding anther in each heading stage.
scale bars = 2 mm.
c the relative expression patterns of tajaz genes in each heading stage.
qrt-pcr was used to analyze the relative expression level of each tajaz gene.
f1–f <dig> represent  <dig> stages during the whole heading period.
the expression level of wheat actin was used as the internal control to standardize the rna samples for each reaction, and the expression in stage  <dig> was set as  <dig>  the data are from three biological replicates, and error bars represent the standard error
during the whole heading stage of bs <dig> and jing <dig>  the expression pattern of each tajaz gene was obviously fluctuant and regular .
except tajaz <dig>  the relative expression levels of the rest of tajaz genes were markedly increased at the stage  <dig> and stage  <dig>  and the corresponding expression patterns displayed some similarities .
at the heading stage  <dig> of bs <dig>  the transcriptional levels of  <dig> tajaz genes  were reduced compared with that in the heading stage  <dig>  whereas the expression levels of tajaz <dig>   <dig>   <dig>   <dig>   <dig> and  <dig> were increased .
at the heading stage of jing <dig>  the expression levels of tajaz <dig>   <dig>   <dig>   <dig>   <dig>  and  <dig> were inhibited, but the transcriptional levels of tajaz <dig>   <dig>   <dig>   <dig>   <dig>   <dig>   <dig> and  <dig> were induced .
interestingly, the expression patterns of tajaz <dig>   <dig> and  <dig> were discrepant at the heading stage  <dig> of bs <dig> and jing <dig>  but similar at the heading stages 1– <dig> .
these results revealed that the functions of tajaz genes were distinctly different, and tajaz <dig>   <dig> and  <dig> might play their conclusive roles in regulation of the degree of anther dehiscence at the heading stage  <dig> in wheat.
tissue-specific expression profiles of tajaz genes
the qrt-pcr was performed to investigate the tissue expression patterns of tajaz genes.
the expression profiles of tajaz genes were analyzed in different tissues of the tgms wheat line bs <dig> and the normal control jing <dig> at heading stage  <dig> .
as shown in fig.
<dig>  five tajaz genes  were expressed in root, stem, leave, stamen and pistil tissues, and they were expressed constitutively in bs <dig> and jing <dig>  among the rest of tajaz genes, tajaz <dig> and  <dig> were specifically expressed in root, tajaz <dig>  was observably highly expressed in leaf tissues, and tajaz <dig> and  <dig> were expressed specifically in glume .
the relative expression levels of tajaz <dig>   <dig> and  <dig> in stamen tissues were markedly higher than those in other tissues .
in addition, the expression level of tajaz <dig> in root tissues was as high as that in stamen, and none of tajaz genes was specifically expressed in stem or pistil tissues .fig.
<dig> real-time pcr analysis of tajaz genes in  <dig> wheat tissues in heading stage  <dig>  the expression level of wheat actin was used as the internal control to standardize the rna samples for each reaction, and the expression in the root was set as  <dig>  the data are from three biological replicates, and error bars represent the standard error
cloning of tajaz7-d, 8-d and 12-d and subcellular location
to investigate the reliability of identified tajaz genes, we used the specific primers to clone the orf sequences of tajaz7-d, 8-d and 12-d and confirmed these sequences by dna sequencing.
the results showed that the orf length and the nucleic acid constituent of tajaz7-d, 8-d and 12-d were in line with the data that identified from wheat genome database .
further, we confirmed the subcellular localization of tajaz7-d-gfp, tajaz8-d-gfp and tajaz12-d-gfp fusion proteins.
as expected, all three fusion proteins localized in nucleus in arabidopsis mesophyll protoplasts cells .
these revealed that the tajaz7-d, 8-d and 12-d were true genes, and directly participated in ja signaling pathway in wheat.
discussion
the structural characteristics of jaz transcription repressors family in bread wheat
to date, the jaz gene family has been reported in many plants, such as a. thaliana  <cit>  and h. brasiliensis  <cit> , b. distachyon  <cit>  and o. sativa  <cit> .
the expression patterns, gene and protein characteristics and the function of some jaz genes have already been unfolded.
however, the relative research on jaz gene family was still infrequent in wheat.
based on the latest draft of wheat genome data, we isolated a jaz gene family including  <dig> members from wheat .
in plants, the gene construction of jaz is manifold, and this multiformity is mainly reflected in the length and number of introns  <cit> .
the length of introns in tajaz gene family ranged from  <dig> to 2098 bp.
the shortest intron was only 54 bp in tajaz7-b, while the longest was 2098 bp in tajaz14-d .
previous study revealed big gaps in the length of introns in jaz gene family in many plants, ranging from  <dig> to 4422 bp  <cit> .
we found that the intron length in tajaz gene family was in line with this range.
in terms of the number of introns, the tajaz gene family members had 0– <dig> introns.
tajaz <dig> had the maximum  <dig> introns, whereas tajaz <dig> and  <dig> had no introns .
this result was consistent with the number of introns in the jaz genes of other plants  <cit> .
however, some discrepancies were found in the number of introns among different copies of the same tajaz gene.
for example, among the three copies of tajaz <dig>  tajaz4-a and -b had  <dig> introns each, whereas tajaz4-d had  <dig> introns.
tajaz14-a and -d had  <dig> introns each, whereas tajaz14-b had  <dig> introns.
the similar characteristics were also found in the copies of tajaz <dig> and  <dig> .
this characteristic of gene structure was proper in wheat, because wheat has sub-genomes a, b and d. overall, the results mentioned above suggested that the phenomenon of intron indels or intron lose occurred in tajaz genes during the long evolutionary process, resulting in various structures of tajaz genes.
generally, a typical jaz protein has one tify and one jas domain at its n- and c-terminal, respectively.
tify domain has two main functions, with one as a medium for the interaction of homomeric and heteromeric complexes formation, and the other as a medium for the interaction between jaz proteins and myc transcription factors  <cit> .
the other distinguishing feature of jaz proteins is the highly conserved jas domain located near the c-terminus  <cit> .
it is well known that jas domain participates in protein–protein interaction with both coi and transcription factors, such as myc <dig>  <cit> , and the nuclear localization signal  is included in this domain sequence  <cit> .
in the present study, all tajaz proteins contained one tify and one jas domain, but the amino acid composition of tify and jas domain was different.
the multiple sequence alignment showed that some amino acid substitutions existed in the core sequence of tify domain .
for example, the protein sequence “tify” was replaced with “tvfy” in tajaz <dig>  “tmfy” in tajaz <dig>  and “tlfy” in tajaz <dig>  this architectural feature is familiar in jaz proteins in many other plants  <cit> .
the protein sequences of jas domain were relatively conserved, and the sequence lengths were almost coincident in arabidopsis and gramineous rice and maize  <cit> .
but in wheat, the jas motif sequences of some jaz proteins, such as tajaz <dig>   <dig> and  <dig>  were inserted by a short and highly similar nls-like peptide, respectively .
this structural feature was rare in other plant jaz protein, and this specific structure was only available in wheat jaz proteins.
in conclusion, there were some similarities and otherness in the characteristics between tajaz gene family and other plant jaz gene families.
the results mentioned above suggested that the tajaz gene family might have multiple functions due to the complex structural characteristics as same as jaz gene families in many other plants.
the putative functions of tajaz gene family
in order to gain more insights into the function of tajaz genes, we analyzed the cis-acting regulatory elements composition and the expression patterns under various stresses.
cis-acting regulatory elements are important molecular switches involved in the regulation of gene transcription under abiotic or biotic stresses  <cit> .
in this study, we mainly detected the cis-acting regulatory elements that can respond to plant hormones  and stress tolerances .
due to the restriction of genomic sequencing data, we failed to gain the promoter region of tajaz <dig>  in wheat jaz gene promoter regions, the differences were mainly in the number and type of cis-acting regulatory elements.
for example, the promoter region of tajaz <dig> had  <dig> types of cis-acting regulatory elements, i.e.
abre , tga , box-w <dig>  and cgtca-motif , while tajaz <dig> only had  <dig> abre and  <dig> mbs elements .
the promoter sequences of some tajaz genes lacked some types of cis-acting regulatory elements, but the expression patterns revealed that all tajaz genes could respond to all  <dig> treatments.
for example, we found that all tajaz genes had no high salinity response-related cis-acting regulatory elements, but the relative expression levels of all tajaz genes were up- or down-regulated under high salinity treatment .
this indicated that the gene expression level under different treatments was not only dependent on the presence of relevant cis-acting regulatory elements, but also might be regulated by other physiological pathways in wheat.
to further investigate the biological functions of tajaz genes, we used different phytohormone to treat the wheat seedlings.
under meja treatment, the relative expression levels of tajaz <dig>   <dig>   <dig>   <dig>   <dig>   <dig>   <dig>   <dig> and  <dig> were obviously increased, while tajaz <dig>   <dig>   <dig>   <dig> and  <dig> were inhibited.
by combining the cis-acting regulatory elements analyzed results, we found that the promoter sequences of tajaz <dig>   <dig> and  <dig> lacked the meja-responsive elements.
thus, we assumed that tajaz <dig>   <dig>   <dig>   <dig>   <dig>   <dig>   <dig>   <dig>   <dig> and  <dig> were directly involved in ja signaling pathway in wheat, and tajaz <dig>   <dig> and  <dig> could also respond to meja treatment by other unknown regulative pathways.
likewise, for ga treatment, the relative expression levels of tajaz <dig> and  <dig> were depressed, and the rest of tajaz genes were up-regulated.
based on the composition of cis-acting regulatory elements in promoter sequences, only tajaz <dig>   <dig>   <dig> and  <dig> had gare element involved in ga response, but the tajaz genes lacking gare element could also respond to ga. for iaa treatment, only tajaz <dig>   <dig>   <dig>   <dig> and  <dig> had the tga-element, but all tajaz genes could obviously respond to it.
for sa treatment, only tajaz <dig>   <dig>   <dig>   <dig> and  <dig> had the tca-element, but the response was available in all genes.
for aba treatment only tajaz <dig>   <dig>   <dig>   <dig> and  <dig> had no abre element, but they were still sensitive to the presence of aba .
given this, we found that some tajaz genes could respond to various phytohormones, and they might be directly or indirectly involved in the phytohormone crosstalk.
it is clear that the phytohormone crosstalk is universally available in plants, and a very complex signaling regulative network, which constituted by many phytohormone crosstalk, plays a very important role in the regulation of growth and development in plants.
there is no doubt that ja signaling pathway plays a leading role in connecting different phytohormone signaling pathways  <cit> .
as a repressor in downstream of ja signaling, jaz proteins were irreplaceable, and they are the linkers among different crosstalks  <cit> .
by synthesizing the analytic results of the cis-acting regulatory elements composition and the expression patterns under abiotic stresses , we speculated that tajaz <dig>   <dig>   <dig> and  <dig> were involved in the ja-ga crosstalk, which is a crosstalk to promote plant growth and defense against pathogens.
moreover, the ja-ga crosstalk can also act synergistically during stamen development, and jaz proteins appear to play a significant role in this developmental function by interacting with the transcription factors myb <dig> and myb <dig>  which both required for ja- and ga-mediated stamen development and male fertility  <cit> .
therefore, we thought that tajaz <dig>   <dig>   <dig> and  <dig> had a close contact with the development of stamen in wheat.
the mechanism of ja-sa crosstalk in plant remains largely unknown, but the previous evidence has shown the effect of sa on ja signaling through direct or indirect regulation of the stabilization of jaz proteins  <cit> .
this crosstalk can protect plants from the biotic and abiotic stresses, especially the pathogen infection  <cit> .
here, we found that tajaz <dig>   <dig>   <dig>   <dig> and  <dig> had two types of cis-acting regulatory elements cgtca-motif and tca-element, and they were sensitive to ja and sa treatments .
thus, we thought that the functions of tajaz <dig>   <dig>   <dig>   <dig> and  <dig> were to enhance or inhibit the antiviral ability of plants.
iaa is a very important phytohormone and plays vital roles during the development of plants  <cit> .
the evidences revealed that the function of ja-iaa crosstalk was mainly embodied in regulating the root meristem activity and stem cell maintenance via antagonistic effect in plants .
based on the results of cis-acting regulatory element component and the expression patterns , we assumed that tajaz <dig>   <dig>   <dig>   <dig> and  <dig> were directly participated in the ja-iaa crosstalk, and they might be involved in the regulation of primary root growth in wheat.
aba is a “stress hormone” that can regulate growth, stress tolerance, seed germination and senescence in plants.
in ja-aba crosstalk, both synergetic and antagonistic interactions are well known, and it is possible that jaz proteins play important roles in regulating the ja-aba crosstalk  <cit> .
given this, except tajaz <dig>   <dig>   <dig>   <dig> and  <dig>  which lacked abre element, we speculated the rest of tajaz genes took part in the ja-aba crosstalk, and played a role in plant stress tolerance, such as high salinity, drought and low temperature stresses .
in addition, the analysis of phylogenetic relationship among the jaz genes can also reveal the putative function of them, because the homologous genes usually have similar biological functions  <cit> .
in this study, we found that tajaz <dig> was clustered with osjaz <dig> into the sub-group g <dig> .
it is clear that osjaz <dig> protein interacts with a basic helix-loop-helix protein, osbhlh <dig>  to regulate the drought tolerance in rice  <cit> .
here, we found  <dig> drought-inducible cis-acting regulatory elements mbs in the promoter sequence of tajaz <dig>  and the expression level of tajaz <dig> was also variational under drought stress.
thus, tajaz <dig> was likely participated in drought tolerance in wheat.
then, we found that tajaz <dig> was clustered with bdjaz <dig> into the sub-group g <dig> .
the expression levels of bdjaz <dig> are increased under salt, cold and heat treatments in b. distachyon  <cit> .
there were  <dig> low temperature related cis-acting regulatory elements ltr in the promoter region of tajaz <dig>  and the expression pattern of tajaz <dig> was similar with that of bdjaz <dig> under cold stress, so tajaz <dig> might be involved in cold response in wheat.
further, we found that tajaz <dig> shared a high similarity with bdjaz <dig> in sub-group g <dig> , and there were  <dig> mbs elements in its promoter sequence.
the expression patterns of tajaz <dig> and bdjaz <dig> were similar under drought stress   <cit> , indicating that tajaz <dig> was also involved in drought tolerance in wheat.
overall, the functions of many tajaz genes were overlapping, which needed to be studied minutely in the future.
the evolution analysis of jaz transcription repressors family
in the ancient terricolous plants, there are  <dig> and  <dig> members in the jaz gene family in p. patens and s. moellendorffii genome, respectively  <cit> .
in neonatal terricolous plants, there are  <dig> jaz genes in gymnospermous p. stichensis genome,  <dig> jaz genes in dicotyledonous a. thaliana genome,  <dig>   <dig> and  <dig> jaz genes in gramineous b. distachyon, s. bicolor, and o. sativa genomes, respectively  <cit> .
in the present study,  <dig> tajaz genes were identified from wheat genome , and this number was similar to those in gramineous plants, but obviously more than those in older plants p. patens and s. moellendorffii.
this result suggested that the number of jaz genes in higher plants undergone the expansion, and became stable subsequently.
based on the analysis of chromosome localization, we found that the chromosome distribution of tajaz genes was tufted.
for example, the copies of tajaz <dig>   <dig>   <dig> and  <dig> were densely distributed on the chromosomes 4a, 4b and 4d, respectively .
in addition, both tajaz <dig> and  <dig> were clustered into sub-group g <dig> , while tajaz <dig> and  <dig> were clustered into sub-groups g <dig> and g <dig> , respectively, indicating that tajaz <dig> and  <dig> undergone the event of tandem duplication, and maybe the event of divergence happened in the evolutionary process of tajaz genes.
moreover, we found that a pair of tandem tajaz genes, tajaz13-d <dig> and -d <dig>  shared a high similarity in their protein sequences , exhibiting that the duplication event of these two genes also happened.
given this, we thought that the gene tandem duplication was the main result leading to the augmentation in the number of tajaz gene.
based on the n-j phylogenetic tree, all jaz proteins from different plants were clustered into  <dig> subgroups .
the sub-groups g <dig>  g <dig>  g <dig>  g <dig>  g <dig> and g <dig> each had one smjaz protein, while none was present in the rest of sub-groups.
thus, we separated these  <dig> sub-groups into groups i  and ii  .
in group i, the sub-group g <dig> had only one jaz protein, smjaz <dig>  indicating that all jaz proteins in terrestrial plants originated from a common ancestor.
in sub-groups g <dig> and g <dig>  the jaz proteins from s.moellendorffii and other plants, such as t. aestivum, b. distachyon and a. thaliana, were clustered in these two clades, indicating that the differentiation of these jaz proteins might have predated the divergence between flowering plants and pteridophyte .
in sub-groups g <dig> and g <dig>  the jaz proteins from p. patens and other plants, such as a. thaliana, and o. sativa, were clustered in these two sub-groups, suggesting that the differentiation of those jaz proteins might have predated the divergence between bryophyte and tracheophyte .
in group ii, the jaz proteins from gymnospermous p. sitchensis and other angiosperm were included in g <dig>  suggesting that those jaz proteins might have predated the divergence between gymnosperms and angiosperms .
in addition, sub-groups g <dig>  g <dig> and g <dig> comprised some jaz proteins from monogenus and dicotyledonous plants, indicating that these jaz proteins appeared before the divergence between monogenus and dicotyledonous plants .
the sub-group g <dig> only included the jaz proteins from gramineous plants , suggesting that the differentiation of these jaz proteins ahead of the formation of gramineous plants.
for tajaz protein family, we found that all tajaz proteins were directly clustered with the jaz proteins from ae.
tauschii, b. distachyon, s. bicolor or o. sativa.
for example, tajaz <dig> was clustered with bdjaz <dig> into sub-group g4; tajaz <dig> was clustered with aetjaz <dig> into g6; tajaz <dig> was clustered with bdjaz <dig> and sbjaz <dig> into g7; and tajaz <dig> was clustered with bdjaz <dig> and aetjaz <dig>   <dig>   <dig>   <dig> and  <dig> .
this could be attributed to the fact that these  <dig> plant species are gramineous.
thus, we speculated that the differentiation of tajaz protein family occurred after the divergence between monocotyledon and dicotyledon , and ahead of the formation of gramineous plants .
in addition, the ka/ks ratio revealed that the tajaz protein family undergone a process of purifying selection , suggesting that the tajaz protein family tended to be stable during the long evolutionary process.
tajaz <dig>   <dig> and  <dig> were involved in the abnormal anther dehiscence
it is clear that ja, as a kind of important phytohormone, is widely involved in the regulation of anther dehiscence, filaments elongation and pollen fertility in plants  <cit> .
the ja biosynthesis and signaling pathways were important for the development of anther during late developmental stage  <cit> .
jaz proteins, as repressors in ja signaling pathway, inhibit the transcription of ja response genes  <cit> , and there is no doubt that jaz genes play an essential role in the ja-mediate regulation pathway of anther dehiscence in plants  <cit> .
the tgms wheat line bs <dig> is a temperature dependent variety and its fertility can convert under different environments  <cit> .
hybrid seed could be produced under sterile condition , while tgms line itself could be propagated under fertile condition  <cit> .
the anther dehiscence of conventional wheat line jing <dig> is normal under fertile condition , and the pollen can spill out from anthers smoothly.
in sterile environment, the anther of tgms wheat line bs <dig> is absolutely indehiscent  <cit> , resulting in the male sterility.
interestingly, we found that the anther of bs <dig> could not fully dehisce, and only the topmost part of anther could crack , leading to the pollen spilling out incompletely.
this abnormal phenotype was profitless for the seed multiplication of tgms wheat line bs <dig>
in order to explore the potential relationship between the expression patterns of tajaz genes and the phenomenon of abnormal anther dehiscence, we divided the heading stage into six periods, and the expression pattern of each tajaz gene was checked in the anther tissues of bs <dig> and jing <dig> .
as shown in fig.
6c, the expression patterns of all tajaz genes were highly similar from stage  <dig> to stage  <dig>  and the relative expression levels in stage  <dig> and stage  <dig> increased obviously.
given this, we speculated that all  <dig> tajaz genes played important roles in regulation of anther development at stages  <dig>   <dig>   <dig>   <dig> and  <dig>  further, we noticed that the relative expression levels of tajaz genes at stage  <dig> were distinguishing in the anther of bs <dig>  and the expression patterns were mainly divided into two types.
the first category included tajaz <dig>   <dig>   <dig>   <dig>   <dig>   <dig>   <dig> and  <dig>  and the expression levels of these genes were inhibited .
the second category included tajaz <dig>   <dig>   <dig>   <dig>   <dig> and  <dig>  and the expression levels of these genes were induced .
in the anther of jing <dig>  the expression patterns of tajaz <dig>   <dig>   <dig>   <dig>   <dig>   <dig>   <dig>   <dig>   <dig>   <dig> and  <dig> were as same as those in bs <dig> at the heading stage  <dig> .
it was obvious that the expression levels of tajaz <dig>   <dig> and  <dig> in the anther of jing <dig> were adverse to those in bs <dig> at stage  <dig>  for example, in the anther of bs <dig>  the expression level of tajaz <dig> was inhibited at stage  <dig>  exhibiting  <dig> times lower than that at stage  <dig> .
in contrast, the transcriptional level of tajaz <dig> increased at the heading stage  <dig> in the anther of jing <dig>  displaying  <dig> to  <dig> folds higher than that at stage  <dig> .
similar results were also found in the expression patterns of tajaz <dig> and  <dig>  these indicated that not all tajaz genes were involved in the regulation of anther dehiscence, and the genes with the same expression patterns in the anthers of jing <dig> and bs <dig> may not be involved in the regulation of the anther dehiscence.
moreover, the tissue-specific expression assay was performed to check the expression levels of tajaz genes in different tissues of bs <dig> and jing <dig> .
the relative expression levels of tajaz <dig>   <dig>   <dig>   <dig>   <dig> and  <dig> were obviously high in stamen tissues, but they all had high expression levels in other tissues .
therefore, we thought that the regulatory effect of these genes was constitutive.
the relative expression levels of tajaz <dig>   <dig> and  <dig> were markedly higher in stamen than that in other tissues, showing a high degree of expression specificity.
for most of tajaz genes, the tissue-specific expression patterns were consistent in the anther tissues of jing <dig> and bs <dig>  in addition, the subcellular localization showed that tajaz <dig>   <dig> and  <dig> were all located in nucleus .
based on the results mentioned above, we thought that tajaz <dig>   <dig> and  <dig> were directly participated in ja signaling pathway, and most likely to directly regulate the abnormal anther dehiscence.
thus, tajaz <dig>   <dig> and  <dig> were regarded as the candidate genes for the regulation of abnormal anther dehiscence in tgms wheat line.
the functions of tajaz <dig>   <dig> and  <dig> will be analyzed in our future works.
CONCLUSIONS
fourteen jaz family genes were identified from common wheat genome.
the structure analysis revealed that tajaz gene family had unique characteristic in protein structure.
jaz proteins from wheat and some other plants were classified into  <dig> different orthologous groups, showing that all jaz proteins from terrestrial plants derived from the same ancestor, and tajaz proteins shared high similarity with the jaz proteins from ae.
tauschii, b. distachyon, and o. sativa.
the ka/ks ratio of tajaz family genes were very low , suggesting that these genes had been under purifying selection in the evolutionary histories.
moreover, we found that wheat jaz genes showed differential tissue-specific expression patterns responsive to abiotic stresses.
tajaz <dig>   <dig> and  <dig> directly participated in ja signaling pathway, and closely involved in the regulation of the abnormal anther dehiscence in tgms wheat line.
in conclusion, these results enriched our knowledge of jaz gene family in plants, and provided novel candidate genes for improving the tgms wheat line in seed reproduction.

 jasmonic acid and its bioactivity derivatives are collectively known as jasmonates. they play important roles in regulating the responses to biotic and abiotic stresses, such as herbivory, pathogen invasion, wounding, uv radiation and ozone stress in plants. in addition, jas also control various developmental processes in plants, viz. pollen maturation, anther dehiscence, embryo maturation, tendril coiling, tuber and trichome growth. therefore, jas are regarded as moderators that mainly involved in regulating the defense reaction, inhibiting the photosynthesis and cell division, and keeping the balance between growth and defense in plants. in plants, ja signaling pathway is a very complex process involved in many genes or proteins, and this pathway can be divided into three processes, including the biosynthesis and metabolism of signaling molecules, ja signal transduction and responses of downstream genes. previous studies showed that ja signaling molecules, scfcoi receptor complex, jasmonate-zim domain repressor and transcription activator myc participate together and interact with each other in ja signaling pathway. in the plant cells with a lower-concentration of ja, the transcription of ja response genes is restrained owing to the combination between jaz protein and myc under stimulation, ja is compounded and accumulated in plant cells. high concentrations of ja can promote the combination between jaz proteins and scfcoi and make jaz proteins be ubiquitinated. then the ubiquitinated protein is alternatively degraded by 26s protease. finally, the activity inhibition of transcription activator myc is removed. expression of ja response genes is activated subsequently. it is known that jaz proteins have a crucial effect on the ja signaling pathway and they are the linkers of ja signaling transduction. the jaz protein family is a member of tify transcription factor superfamily, and the family members have two conservative functional domains, tify and jas. tify domain is usually consisted of amino acids, which located in the n-terminus of jaz protein sequences, and its core sequence is tifxg. in ja signaling pathway, tify domain can mediate the interaction between jaz protein and its co-suppressor, a novel interactor of jaz, and then collectively restrict the ja signal transduction. jas domain is near the c-terminus of jaz protein sequences, and its sequence is extremely conserved among the members of jaz protein family, of which amino acids are same or replaced in conservation. its function is to mediate the direct combination between jaz protein and myc in ja signaling pathway and inhibit the transcriptional activity of myc, and then further restrict the expression of ja response genes. as the connector between myc and coi jaz protein was firstly reported in. afterwards, many studies about jaz proteins have been conducted. to date, and jaz genes have been found in arabidopsis thaliana and rice, respectively. according to previous studies, the jaz gene family has plenty of members, but each has a different biological function. for example, the protein encoded by osjaz is a regulator of ja signaling pathway and can regulate the salt tolerance of rice. the expression of ghjaz can be activated by gbwrky so that it can strengthen the capacity for resistance against verticillium dahlia in cotton. overexpression of modified atjaz is able to enhance host resistance to spodoptera exigua in arabidopsis. overexpressing the modified osjazs can cause the malformation of flower organ in rice. according to the above researches, jaz genes have important effects on regulating the adaptability to biotic and abiotic stresses and maintaining the normal development in plants. wheat is one of the most important crops in the world. the changes of climate and growing environments bring huge challenge to wheat production. thus, use of molecular biology and genetics methods is an important approach for improving the stress tolerance and quality of wheat. thermosensitive genic male sterile lines, such as bs are of particular significance in two-line hybrid system, which is more efficient in breeding, because the fertility of tgms lines is strictly regulated by temperature. hybrid seed could be produced under sterile condition, while tgms line itself could be reproduced under fertile condition. in our early research, we found that the anther dehiscence in bs is abnormal, and its pollen can not fully spill out. this defective phenotype can be recovered via spraying meja in vitro. so, we assumed that this phenotype is closely related to the synthesis and regulatory pathway of ja. based on the current wheat genome sequencing data, we firstly identified the tajaz gene family, and analyzed its architectural features, evolutionary history and expression patterns in anther tissues in tgms wheat line. the results presented in this study were expected to enrich our knowledge of jaz gene family in wheat, and provide the theoretical basis and novel candidate genes for improving and creating the male sterility wheat lines.  identification of jaz repressor gene family in wheat based on the latest genome data of wheat, a hidden markov model search was carried out using the hmm profiles of the tify domain and jas domain as queries against the local protein database. by retrieving the database, we detected non-redundant sequences. among these, were removed because they lacked the typical tify or jas domain. finally, we obtained full-length protein sequences. the corresponding coding sequences and genome sequences were simultaneously isolated. the location of each tajaz gene was confirmed by aligning the wheat chromosome genomic sequences using blastn. according to the information of phylogenetic relationship, tajaz genes were clustered into groups. the genes from different wheat sub-genomes and in the same group were regarded as different copies of each member of the tajaz gene family. thus, we obtained the tajaz gene family including members designated from to according to the naming convention, and the copies of each member were distinguished by subjoining the wheat sub-genome symbols a, b or d.table jaz gene family in wheat tajaz1-a tajaz1-b tajaz1-d tajaz2-b tajaz3-a tajaz3-d tajaz4-a tajaz4-b tajaz4-d tajaz5-a tajaz5-b tajaz5-d tajaz6-a tajaz6-d tajaz7-a tajaz7-b tajaz7-d tajaz8-a tajaz8-d tajaz9-a tajaz9-b tajaz9-d tajaz10-a tajaz10-b tajaz10-d tajaz11-a tajaz12-a tajaz12-d tajaz13-a tajaz13-d1 tajaz13-d2 tajaz14-a tajaz14-b tajaz14-d athe sequence name beginning with treas and taloc means download from the wheat genome ensembl database and urgl database, respectively fig. phylogenetic relationship and motif structure of wheat jaz proteins. a the phylogenetic tree of tajaz proteins constructed from a complete alignment of wheat jaz proteins using mega by the neighbor-joining method with bootstrap replicates. bootstrap scores are indicated on the nodes and the members of tajaz, most of which contain duplicated genes, are indicated by yellow or pink block. b domain distribution of tajaz proteins were investigated using the meme web server. color blocks represent the position of motifs on corresponding proteins. c the consensus sequence of tify and jas motif from wheat jaz proteins. the relative position of each motif can be determined using the scale below genome distribution of wheat jaz genes among wheat jaz genes, and were distributed in wheat sub-genomes a, b and d, respectively. none of tajaz gene copy was distributed on chromosomes 1a, 3a, 1b, 3b, 6b, 1d and 3d. each of the tajaz genes had three copies on group and chromosomes, respectively. four tajaz genes had two copies each including tajaz3-a/-d, tajaz6-a/-d, tajaz8-b/-d, and tajaz12-a/-d. tajaz and tajaz had only one copy on chromosomes 2b and 6a, respectively. tajaz also had three copies, and tajaz13-a was found on chromosome 7a, tajaz13-d and -d were detected on chromosome 7d. in addition, chromosome 4d had the highest number of five tajaz gene copies, whereas other chromosomes contained no more than four copies.fig. chromosome distribution of jaz gene family in wheat. red dots on the chromosomes indicate the position of centromeres. the red arrows next to gene names show the direction of transcription. the position of each gene could be estimated using the left scale structure analysis of wheat jaz genes, and jaz proteins in order to obtain more insights about the gene structural evolution, the exon-intron organization of wheat jaz genes was raveled by aligning the predicted coding sequences against the corresponding genomic sequences using the online service gsds. in the tajaz gene family, the number of exons ranged from to and the number of introns ranged from to. among tajaz genes containing two or three copies, had the same gene structures, whereas had one, two or four exons in each group. in addition, tajaz and tajaz lacked intron. overall, a highly similar gene structure was exhibited in the duplicated tajaz genes.fig. exon-intron structures of tajaz genes. exons are represented by blank boxes and introns by blank lines. the sizes of exons and introns could be estimated using the scale below the tify and jas domains are important for the repressor activity of jaz proteins in plants. the tify and jas domains locate at the n-terminus and c-terminus of jaz proteins, respectively. in order to understand the architecture of jaz proteins in wheat, we scanned these two domains using the meme web server. as shown in fig. 1b and c, single tify and jas motifs were distributed in each tajaz protein and all tajaz proteins had tify motif at n-terminus while jas motif at c-terminus. the results were in accordance with the previous studies, indicating that the structures of jaz proteins were conserved. moreover, the multi-sequence alignments of protein sequences revealed that the amino acid sequence “tify” was changed into “tvfy” in tajaz “tmfy” in tajaz “tlfy” in tajaz “tlvy” in tajaz and “tlsf” in tajaz. the same variations also existed in rice, maize, and b. distachyon. besides tify domain, the jas domain is also conserved. in arabidopsis, jas domain contains 12- amino acids, and the sequences are basically coincident or conserved. but in wheat, the jas domain in some tajaz proteins, such as tajaz and was inserted into a nuclear localization signal sequence. phylogenetic analysis of tajaz proteins in order to understand the phylogeny of jaz proteins, n-j tree was built using mega software and the reliability was tested by bootstrap analysis for replicates. as shown in fig.  all the jaz proteins derived from different plants were clustered into sub-groups g1-g and the tajaz proteins were clustered into sub-groups g3-g sub-group g had the maximal number of tajaz proteins, and tajaz proteins were clustered into this sub-group. based on the order that the jaz proteins appeared in plants, those sub-groups were divided into groups i and ii. group i included sub-groups g g g g g and g and group ii comprised sub-groups g g g g and g moreover, we found that the tajaz proteins were clustered into the same clades with some aetjaz, bdjaz or osjaz proteins. for example, tajaz was clustered into the sub-group g with osjaz tajaz was clustered into the sub-group g with bdjaz and tajaz was clustered into the sub-group g with aetjaz. these revealed that the tajaz proteins shared a high similarity with those in ae. tauschii, b. distachyon and rice.fig. phylogenetic relationship of jaz proteins among wheat and other species. the full-length amino acid sequences of 34 t. aestivum, 6 s. moellendorffii, a. thaliana, o. sativa, p. patens, maize, b. distachyon, 15 s. bicolor, p. sitchensis, ae. tauschii and 7 h. brasiliensis genes were aligned by using clustalx and the phylogenetic tree was constructed using mega by the neighbor-joining method with bootstrap replicates. each tajaz protein is indicated by a red dot. two major groups, group i and ii, are represented by the red and blue, respectively adaptive evolution analysis of the tajaz gene family to investigate which type of darwinian selection determined the process of tajaz gene divergence after duplication, the ka/ks substitution ratio was utilized to the coding sequences of pairs of orthologs between wheat and b. distachyon jaz gene family. according to the previous research, ka/ks ratio <  means purifying selection, ratio =  means neutral evolution and ratio >  means positive selection. as shown in table  the ka/ks ratios of tajaz genes ranged from to, suggesting that the tajaz gene family had undergone purifying selection in wheat.table ka/ks ratio of the duplicated jaz genes in wheat using brachypodium distachyon as an outgroup putative cis-acting regulatory elements in the promoter region of tajaz genes to gain the information about the cis-acting regulatory elements of tajaz gene family, the putative promoter region sequence of each tajaz gene was analyzed. in this study, promoter sequences were available for of tajaz genes, while only the promoter of tajaz was unavailable due to the limited genome data of wheat. eight types of putative cis-acting regulatory elements were identified in tajaz genes, including ltr element involved in low-temperature responsiveness, tga element related to auxin-responsiveness, cgtca-motif involved in meja responsiveness, abre associated with aba signaling pathway, box-w for the response of fungal elicitor, gare-motif of a gibberellin responsive element regulating gibberellin responsive genes, mbs, a myb-binding sequence involved in regulation of drought-inducible genes and tca element regulating the sa related genes. the type and number of cis-acting regulatory elements in the promoter region of each tajaz gene were discrepant. these showed that the members of tajaz gene family might be able to respond to different abiotic stresses. expression profiling of tajaz genes under abiotic stresses in tgms wheat line in order to obtain the expression profiling of tajaz genes under plant hormones, low temperature, high salinity and drought treatments, qrt-pcr was performed to gain the relative expression pattern of each tajaz gene using the 14-day-old tgms wheat seedlings. as shown in fig.  the transcript profiling of tajaz genes showed up-regulation of tajaz and and down-regulation for tajaz and in response to meja treatment. aba treatment influenced the expression dynamics of tajaz genes. except that tajaz and were inhibited, the expression levels of the remaining tajaz genes were up-regulated under aba treatment. under ga treatment, the transcriptional levels of tajaz and were increased, and tajaz and were inhibited. the expression patterns of other tajaz genes were similar, i.e., up- at 2–4 h or 2–8 h followed by down-regulated at 24 h. under iaa treatment, the expression levels of tajaz and were up-regulated at time points 2 h, 4 h and 8 h; tajaz and were rapidly up-regulated at 2 h, but down-regulated subsequently; tajaz was induced at each time point, but inhibited at 4 h. the expression levels of remaining tajaz genes were inhibited in different degree. moreover, the expression analysis of tajaz genes showed that tajaz and were up-regulated under sa treatment, and the rest of tajaz genes were down-regulated. these revealed that all tajaz genes could respond to various plant hormones, and they might be involved in some complex signaling pathways.fig. expression heat maps of tajaz genes under phytohormone, drought, salt and cold treatments in tgms wheat line. qrt-pcr strategy was used to analyze the relative expression level of each tajaz gene. the expression level of wheat actin was used as the internal control to standardize the rna samples for each reaction, and the expression at 0 h was set as. the data are from three biological replicates under high salinity treatment, the expression levels of tajaz and were up-regulated, while tajaz and were up-regulated at 2–4 h post treatment, but down-regulated at 8 h subsequently. the expression levels of tajaz were reduced at 2–8 h, and increased at 12–24 h. for drought stress, the expression analysis of tajaz genes showed that genes were up-regulated at 2–8 h or 2–12 h, whereas tajaz and were inhibited at each time point after treatment, and tajaz and were decreased firstly but increased subsequently. in addition, except tajaz and we found that tajaz genes could respond to low temperature, and the transcriptional levels of the rest of genes were obviously up-regulated. ulteriorly, we found that the expression levels of tajaz and were up-regulated at each time point. these results indicated that the members of tajaz gene family were highly sensitive to high salinity, drought and low temperature treatments, and might be involved in some very complex regulation networks in tgms wheat line bs expression patterns of tajaz genes during the whole heading stage the tgms wheat line bs exhibits temperature-dependent sterility. the anthers of bs are indehiscent in sterile environment, and abnormal dehiscence in fertile environment. the anthers of the normal control wheat line jing can normally dehisce in fertile environment. in order to explore the relationship between the expression patterns of tajaz genes and the abnormal anther dehiscence in tgms wheat line bs the whole heading stage was divided into six periods, from the start of heading to pre-abloom stage. qrt-pcr was used to analyze the relative expression levels of each tajaz gene in bs and jing anther tissues, which were collected from the fertile environment.fig. expression patterns of tajaz genes during the whole heading stage. a a, b the phenotype of anther dehiscence in conventional wheat line jing411; c, d the phenotype of anther dehiscence in tgms wheat line bs scale bars: a, c = 4 mm, b = 2 mm, d = 1 mm. b the schematic diagram of stages during the whole heading period and the size of corresponding anther in each heading stage. scale bars = 2 mm. c the relative expression patterns of tajaz genes in each heading stage. qrt-pcr was used to analyze the relative expression level of each tajaz gene. f1–f represent stages during the whole heading period. the expression level of wheat actin was used as the internal control to standardize the rna samples for each reaction, and the expression in stage was set as the data are from three biological replicates, and error bars represent the standard error during the whole heading stage of bs and jing the expression pattern of each tajaz gene was obviously fluctuant and regular. except tajaz the relative expression levels of the rest of tajaz genes were markedly increased at the stage and stage and the corresponding expression patterns displayed some similarities. at the heading stage of bs the transcriptional levels of tajaz genes were reduced compared with that in the heading stage whereas the expression levels of tajaz and were increased. at the heading stage of jing the expression levels of tajaz and were inhibited, but the transcriptional levels of tajaz and were induced. interestingly, the expression patterns of tajaz and were discrepant at the heading stage of bs and jing but similar at the heading stages 1–. these results revealed that the functions of tajaz genes were distinctly different, and tajaz and might play their conclusive roles in regulation of the degree of anther dehiscence at the heading stage in wheat. tissue-specific expression profiles of tajaz genes the qrt-pcr was performed to investigate the tissue expression patterns of tajaz genes. the expression profiles of tajaz genes were analyzed in different tissues of the tgms wheat line bs and the normal control jing at heading stage. as shown in fig.  five tajaz genes were expressed in root, stem, leave, stamen and pistil tissues, and they were expressed constitutively in bs and jing among the rest of tajaz genes, tajaz and were specifically expressed in root, tajaz was observably highly expressed in leaf tissues, and tajaz and were expressed specifically in glume. the relative expression levels of tajaz and in stamen tissues were markedly higher than those in other tissues. in addition, the expression level of tajaz in root tissues was as high as that in stamen, and none of tajaz genes was specifically expressed in stem or pistil tissues.fig. real-time pcr analysis of tajaz genes in wheat tissues in heading stage the expression level of wheat actin was used as the internal control to standardize the rna samples for each reaction, and the expression in the root was set as the data are from three biological replicates, and error bars represent the standard error cloning of tajaz7-d, 8-d and 12-d and subcellular location to investigate the reliability of identified tajaz genes, we used the specific primers to clone the orf sequences of tajaz7-d, 8-d and 12-d and confirmed these sequences by dna sequencing. the results showed that the orf length and the nucleic acid constituent of tajaz7-d, 8-d and 12-d were in line with the data that identified from wheat genome database. further, we confirmed the subcellular localization of tajaz7-d-gfp, tajaz8-d-gfp and tajaz12-d-gfp fusion proteins. as expected, all three fusion proteins localized in nucleus in arabidopsis mesophyll protoplasts cells. these revealed that the tajaz7-d, 8-d and 12-d were true genes, and directly participated in ja signaling pathway in wheat. discussion the structural characteristics of jaz transcription repressors family in bread wheat to date, the jaz gene family has been reported in many plants, such as a. thaliana and h. brasiliensis, b. distachyon and o. sativa. the expression patterns, gene and protein characteristics and the function of some jaz genes have already been unfolded. however, the relative research on jaz gene family was still infrequent in wheat. based on the latest draft of wheat genome data, we isolated a jaz gene family including members from wheat. in plants, the gene construction of jaz is manifold, and this multiformity is mainly reflected in the length and number of introns. the length of introns in tajaz gene family ranged from to 2098 bp. the shortest intron was only 54 bp in tajaz7-b, while the longest was 2098 bp in tajaz14-d. previous study revealed big gaps in the length of introns in jaz gene family in many plants, ranging from to 4422 bp. we found that the intron length in tajaz gene family was in line with this range. in terms of the number of introns, the tajaz gene family members had 0– introns. tajaz had the maximum introns, whereas tajaz and had no introns. this result was consistent with the number of introns in the jaz genes of other plants. however, some discrepancies were found in the number of introns among different copies of the same tajaz gene. for example, among the three copies of tajaz tajaz4-a and -b had introns each, whereas tajaz4-d had introns. tajaz14-a and -d had introns each, whereas tajaz14-b had introns. the similar characteristics were also found in the copies of tajaz and. this characteristic of gene structure was proper in wheat, because wheat has sub-genomes a, b and d. overall, the results mentioned above suggested that the phenomenon of intron indels or intron lose occurred in tajaz genes during the long evolutionary process, resulting in various structures of tajaz genes. generally, a typical jaz protein has one tify and one jas domain at its n- and c-terminal, respectively. tify domain has two main functions, with one as a medium for the interaction of homomeric and heteromeric complexes formation, and the other as a medium for the interaction between jaz proteins and myc transcription factors. the other distinguishing feature of jaz proteins is the highly conserved jas domain located near the c-terminus. it is well known that jas domain participates in protein–protein interaction with both coi and transcription factors, such as myc, and the nuclear localization signal is included in this domain sequence. in the present study, all tajaz proteins contained one tify and one jas domain, but the amino acid composition of tify and jas domain was different. the multiple sequence alignment showed that some amino acid substitutions existed in the core sequence of tify domain. for example, the protein sequence “tify” was replaced with “tvfy” in tajaz “tmfy” in tajaz and “tlfy” in tajaz this architectural feature is familiar in jaz proteins in many other plants. the protein sequences of jas domain were relatively conserved, and the sequence lengths were almost coincident in arabidopsis and gramineous rice and maize. but in wheat, the jas motif sequences of some jaz proteins, such as tajaz and were inserted by a short and highly similar nls-like peptide, respectively. this structural feature was rare in other plant jaz protein, and this specific structure was only available in wheat jaz proteins. in conclusion, there were some similarities and otherness in the characteristics between tajaz gene family and other plant jaz gene families. the results mentioned above suggested that the tajaz gene family might have multiple functions due to the complex structural characteristics as same as jaz gene families in many other plants. the putative functions of tajaz gene family in order to gain more insights into the function of tajaz genes, we analyzed the cis-acting regulatory elements composition and the expression patterns under various stresses. cis-acting regulatory elements are important molecular switches involved in the regulation of gene transcription under abiotic or biotic stresses. in this study, we mainly detected the cis-acting regulatory elements that can respond to plant hormones and stress tolerances. due to the restriction of genomic sequencing data, we failed to gain the promoter region of tajaz in wheat jaz gene promoter regions, the differences were mainly in the number and type of cis-acting regulatory elements. for example, the promoter region of tajaz had types of cis-acting regulatory elements, i.e. abre, tga, box-w and cgtca-motif, while tajaz only had abre and mbs elements. the promoter sequences of some tajaz genes lacked some types of cis-acting regulatory elements, but the expression patterns revealed that all tajaz genes could respond to all treatments. for example, we found that all tajaz genes had no high salinity response-related cis-acting regulatory elements, but the relative expression levels of all tajaz genes were up- or down-regulated under high salinity treatment. this indicated that the gene expression level under different treatments was not only dependent on the presence of relevant cis-acting regulatory elements, but also might be regulated by other physiological pathways in wheat. to further investigate the biological functions of tajaz genes, we used different phytohormone to treat the wheat seedlings. under meja treatment, the relative expression levels of tajaz and were obviously increased, while tajaz and were inhibited. by combining the cis-acting regulatory elements analyzed results, we found that the promoter sequences of tajaz and lacked the meja-responsive elements. thus, we assumed that tajaz and were directly involved in ja signaling pathway in wheat, and tajaz and could also respond to meja treatment by other unknown regulative pathways. likewise, for ga treatment, the relative expression levels of tajaz and were depressed, and the rest of tajaz genes were up-regulated. based on the composition of cis-acting regulatory elements in promoter sequences, only tajaz and had gare element involved in ga response, but the tajaz genes lacking gare element could also respond to ga. for iaa treatment, only tajaz and had the tga-element, but all tajaz genes could obviously respond to it. for sa treatment, only tajaz and had the tca-element, but the response was available in all genes. for aba treatment only tajaz and had no abre element, but they were still sensitive to the presence of aba. given this, we found that some tajaz genes could respond to various phytohormones, and they might be directly or indirectly involved in the phytohormone crosstalk. it is clear that the phytohormone crosstalk is universally available in plants, and a very complex signaling regulative network, which constituted by many phytohormone crosstalk, plays a very important role in the regulation of growth and development in plants. there is no doubt that ja signaling pathway plays a leading role in connecting different phytohormone signaling pathways. as a repressor in downstream of ja signaling, jaz proteins were irreplaceable, and they are the linkers among different crosstalks. by synthesizing the analytic results of the cis-acting regulatory elements composition and the expression patterns under abiotic stresses, we speculated that tajaz and were involved in the ja-ga crosstalk, which is a crosstalk to promote plant growth and defense against pathogens. moreover, the ja-ga crosstalk can also act synergistically during stamen development, and jaz proteins appear to play a significant role in this developmental function by interacting with the transcription factors myb and myb which both required for ja- and ga-mediated stamen development and male fertility. therefore, we thought that tajaz and had a close contact with the development of stamen in wheat. the mechanism of ja-sa crosstalk in plant remains largely unknown, but the previous evidence has shown the effect of sa on ja signaling through direct or indirect regulation of the stabilization of jaz proteins. this crosstalk can protect plants from the biotic and abiotic stresses, especially the pathogen infection. here, we found that tajaz and had two types of cis-acting regulatory elements cgtca-motif and tca-element, and they were sensitive to ja and sa treatments. thus, we thought that the functions of tajaz and were to enhance or inhibit the antiviral ability of plants. iaa is a very important phytohormone and plays vital roles during the development of plants. the evidences revealed that the function of ja-iaa crosstalk was mainly embodied in regulating the root meristem activity and stem cell maintenance via antagonistic effect in plants. based on the results of cis-acting regulatory element component and the expression patterns, we assumed that tajaz and were directly participated in the ja-iaa crosstalk, and they might be involved in the regulation of primary root growth in wheat. aba is a “stress hormone” that can regulate growth, stress tolerance, seed germination and senescence in plants. in ja-aba crosstalk, both synergetic and antagonistic interactions are well known, and it is possible that jaz proteins play important roles in regulating the ja-aba crosstalk. given this, except tajaz and which lacked abre element, we speculated the rest of tajaz genes took part in the ja-aba crosstalk, and played a role in plant stress tolerance, such as high salinity, drought and low temperature stresses. in addition, the analysis of phylogenetic relationship among the jaz genes can also reveal the putative function of them, because the homologous genes usually have similar biological functions. in this study, we found that tajaz was clustered with osjaz into the sub-group g. it is clear that osjaz protein interacts with a basic helix-loop-helix protein, osbhlh to regulate the drought tolerance in rice. here, we found drought-inducible cis-acting regulatory elements mbs in the promoter sequence of tajaz and the expression level of tajaz was also variational under drought stress. thus, tajaz was likely participated in drought tolerance in wheat. then, we found that tajaz was clustered with bdjaz into the sub-group g. the expression levels of bdjaz are increased under salt, cold and heat treatments in b. distachyon. there were low temperature related cis-acting regulatory elements ltr in the promoter region of tajaz and the expression pattern of tajaz was similar with that of bdjaz under cold stress, so tajaz might be involved in cold response in wheat. further, we found that tajaz shared a high similarity with bdjaz in sub-group g, and there were mbs elements in its promoter sequence. the expression patterns of tajaz and bdjaz were similar under drought stress, indicating that tajaz was also involved in drought tolerance in wheat. overall, the functions of many tajaz genes were overlapping, which needed to be studied minutely in the future. the evolution analysis of jaz transcription repressors family in the ancient terricolous plants, there are and members in the jaz gene family in p. patens and s. moellendorffii genome, respectively. in neonatal terricolous plants, there are jaz genes in gymnospermous p. stichensis genome, jaz genes in dicotyledonous a. thaliana genome, and jaz genes in gramineous b. distachyon, s. bicolor, and o. sativa genomes, respectively. in the present study, tajaz genes were identified from wheat genome, and this number was similar to those in gramineous plants, but obviously more than those in older plants p. patens and s. moellendorffii. this result suggested that the number of jaz genes in higher plants undergone the expansion, and became stable subsequently. based on the analysis of chromosome localization, we found that the chromosome distribution of tajaz genes was tufted. for example, the copies of tajaz and were densely distributed on the chromosomes 4a, 4b and 4d, respectively. in addition, both tajaz and were clustered into sub-group g, while tajaz and were clustered into sub-groups g and g, respectively, indicating that tajaz and undergone the event of tandem duplication, and maybe the event of divergence happened in the evolutionary process of tajaz genes. moreover, we found that a pair of tandem tajaz genes, tajaz13-d and -d shared a high similarity in their protein sequences, exhibiting that the duplication event of these two genes also happened. given this, we thought that the gene tandem duplication was the main result leading to the augmentation in the number of tajaz gene. based on the n-j phylogenetic tree, all jaz proteins from different plants were clustered into subgroups. the sub-groups g g g g g and g each had one smjaz protein, while none was present in the rest of sub-groups. thus, we separated these sub-groups into groups i and ii. in group i, the sub-group g had only one jaz protein, smjaz indicating that all jaz proteins in terrestrial plants originated from a common ancestor. in sub-groups g and g the jaz proteins from s.moellendorffii and other plants, such as t. aestivum, b. distachyon and a. thaliana, were clustered in these two clades, indicating that the differentiation of these jaz proteins might have predated the divergence between flowering plants and pteridophyte. in sub-groups g and g the jaz proteins from p. patens and other plants, such as a. thaliana, and o. sativa, were clustered in these two sub-groups, suggesting that the differentiation of those jaz proteins might have predated the divergence between bryophyte and tracheophyte. in group ii, the jaz proteins from gymnospermous p. sitchensis and other angiosperm were included in g suggesting that those jaz proteins might have predated the divergence between gymnosperms and angiosperms. in addition, sub-groups g g and g comprised some jaz proteins from monogenus and dicotyledonous plants, indicating that these jaz proteins appeared before the divergence between monogenus and dicotyledonous plants. the sub-group g only included the jaz proteins from gramineous plants, suggesting that the differentiation of these jaz proteins ahead of the formation of gramineous plants. for tajaz protein family, we found that all tajaz proteins were directly clustered with the jaz proteins from ae. tauschii, b. distachyon, s. bicolor or o. sativa. for example, tajaz was clustered with bdjaz into sub-group g4; tajaz was clustered with aetjaz into g6; tajaz was clustered with bdjaz and sbjaz into g7; and tajaz was clustered with bdjaz and aetjaz and. this could be attributed to the fact that these plant species are gramineous. thus, we speculated that the differentiation of tajaz protein family occurred after the divergence between monocotyledon and dicotyledon, and ahead of the formation of gramineous plants. in addition, the ka/ks ratio revealed that the tajaz protein family undergone a process of purifying selection, suggesting that the tajaz protein family tended to be stable during the long evolutionary process. tajaz and were involved in the abnormal anther dehiscence it is clear that ja, as a kind of important phytohormone, is widely involved in the regulation of anther dehiscence, filaments elongation and pollen fertility in plants. the ja biosynthesis and signaling pathways were important for the development of anther during late developmental stage. jaz proteins, as repressors in ja signaling pathway, inhibit the transcription of ja response genes, and there is no doubt that jaz genes play an essential role in the ja-mediate regulation pathway of anther dehiscence in plants. the tgms wheat line bs is a temperature dependent variety and its fertility can convert under different environments. hybrid seed could be produced under sterile condition, while tgms line itself could be propagated under fertile condition. the anther dehiscence of conventional wheat line jing is normal under fertile condition, and the pollen can spill out from anthers smoothly. in sterile environment, the anther of tgms wheat line bs is absolutely indehiscent, resulting in the male sterility. interestingly, we found that the anther of bs could not fully dehisce, and only the topmost part of anther could crack, leading to the pollen spilling out incompletely. this abnormal phenotype was profitless for the seed multiplication of tgms wheat line bs in order to explore the potential relationship between the expression patterns of tajaz genes and the phenomenon of abnormal anther dehiscence, we divided the heading stage into six periods, and the expression pattern of each tajaz gene was checked in the anther tissues of bs and jing. as shown in fig. 6c, the expression patterns of all tajaz genes were highly similar from stage to stage and the relative expression levels in stage and stage increased obviously. given this, we speculated that all tajaz genes played important roles in regulation of anther development at stages and further, we noticed that the relative expression levels of tajaz genes at stage were distinguishing in the anther of bs and the expression patterns were mainly divided into two types. the first category included tajaz and and the expression levels of these genes were inhibited. the second category included tajaz and and the expression levels of these genes were induced. in the anther of jing the expression patterns of tajaz and were as same as those in bs at the heading stage. it was obvious that the expression levels of tajaz and in the anther of jing were adverse to those in bs at stage for example, in the anther of bs the expression level of tajaz was inhibited at stage exhibiting times lower than that at stage. in contrast, the transcriptional level of tajaz increased at the heading stage in the anther of jing displaying to folds higher than that at stage. similar results were also found in the expression patterns of tajaz and these indicated that not all tajaz genes were involved in the regulation of anther dehiscence, and the genes with the same expression patterns in the anthers of jing and bs may not be involved in the regulation of the anther dehiscence. moreover, the tissue-specific expression assay was performed to check the expression levels of tajaz genes in different tissues of bs and jing. the relative expression levels of tajaz and were obviously high in stamen tissues, but they all had high expression levels in other tissues. therefore, we thought that the regulatory effect of these genes was constitutive. the relative expression levels of tajaz and were markedly higher in stamen than that in other tissues, showing a high degree of expression specificity. for most of tajaz genes, the tissue-specific expression patterns were consistent in the anther tissues of jing and bs in addition, the subcellular localization showed that tajaz and were all located in nucleus. based on the results mentioned above, we thought that tajaz and were directly participated in ja signaling pathway, and most likely to directly regulate the abnormal anther dehiscence. thus, tajaz and were regarded as the candidate genes for the regulation of abnormal anther dehiscence in tgms wheat line. the functions of tajaz and will be analyzed in our future works.  fourteen jaz family genes were identified from common wheat genome. the structure analysis revealed that tajaz gene family had unique characteristic in protein structure. jaz proteins from wheat and some other plants were classified into different orthologous groups, showing that all jaz proteins from terrestrial plants derived from the same ancestor, and tajaz proteins shared high similarity with the jaz proteins from ae. tauschii, b. distachyon, and o. sativa. the ka/ks ratio of tajaz family genes were very low, suggesting that these genes had been under purifying selection in the evolutionary histories. moreover, we found that wheat jaz genes showed differential tissue-specific expression patterns responsive to abiotic stresses. tajaz and directly participated in ja signaling pathway, and closely involved in the regulation of the abnormal anther dehiscence in tgms wheat line. in conclusion, these results enriched our knowledge of jaz gene family in plants, and provided novel candidate genes for improving the tgms wheat line in seed reproduction. 
the jasmonate-zim domain repressor family proteins are jasmonate co-receptors and transcriptional repressor in jasmonic acid signaling pathway, and they play important roles in regulating the growth and development of plants. gene expression patterns obtained by quantitative real-time pcr revealed differential temporal and spatial regulation of tajaz genes under multifarious abiotic stress treatments of high salinity, drought, cold and phytohormone. in conclusion, the results of this study contributed some novel and detailed information about jaz gene family in wheat, and also provided potential candidate genes for improving the tgms wheat line. the ka/ks ratios of tajaz genes ranged from to, suggesting that the tajaz family had undergone purifying selection in wheat. this study analyzed the size and composition of the jaz gene family in wheat, and investigated stress responsive and differential tissue-specific expression profiles of each tajaz gene in tgms wheat line bs in addition, we isolated tajaz genes that would be more likely to be involved in the regulation of abnormal anther dehiscence in tgms wheat line. recently, more and more researches on jaz gene family are reported in many plants. by phylogenetic analysis, all jaz proteins from wheat and other plants were clustered into sub-groups, and tajaz proteins shared a high degree of similarity with some jaz proteins from aegliops tauschii, brachypodium distachyon and oryza sativa. among these, tajaz and were specifically expressed in the anther tissues of the thermosensitive genic male sterile wheat line bs and normal control wheat line jing compared with the gene expression patterns in the normal wheat line jing tajaz and had different expression patterns in abnormally dehiscent anthers of bs at the heading stage suggesting that specific up- or down-regulation of these genes might be associated with the abnormal anther dehiscence in tgms wheat line. structural analysis revealed that the tajaz proteins in wheat were as conserved as those in other plants, but had structural characteristics. anther dehiscencegene expressionjasmonate-zim domainphylogenetic analysisthermo sensitive genic male sterile national key research project2016yfd0101601zhao changping http://dx.doi.org/ /501100001809national natural science foundation of china31371699zhang liping national science and technology support plan2013bad04b012014bad01b09zhang liping natural science foundation of beijing 5131001zhang liping special project of science and technology innovation ability construction of baafskjcx20140405zhang liping shanxi province science foundation for youths2015021145qiao linyi issue-copyright-statement© the author 2017 fourteen jaz genes were identified in the wheat genome.
 the jasmonate-zim domain repressor family proteins are jasmonate co-receptors and transcriptional repressor in jasmonic acid signaling pathway, and they play important roles in regulating the growth and development of plants. recently, more and more researches on jaz gene family are reported in many plants. although the genome sequencing of common wheat and its relatives is complete, our knowledge about this gene family remains vacant.  fourteen jaz genes were identified in the wheat genome. structural analysis revealed that the tajaz proteins in wheat were as conserved as those in other plants, but had structural characteristics. by phylogenetic analysis, all jaz proteins from wheat and other plants were clustered into sub-groups, and tajaz proteins shared a high degree of similarity with some jaz proteins from aegliops tauschii, brachypodium distachyon and oryza sativa. the ka/ks ratios of tajaz genes ranged from to, suggesting that the tajaz family had undergone purifying selection in wheat. gene expression patterns obtained by quantitative real-time pcr revealed differential temporal and spatial regulation of tajaz genes under multifarious abiotic stress treatments of high salinity, drought, cold and phytohormone. among these, tajaz and were specifically expressed in the anther tissues of the thermosensitive genic male sterile wheat line bs and normal control wheat line jing compared with the gene expression patterns in the normal wheat line jing tajaz and had different expression patterns in abnormally dehiscent anthers of bs at the heading stage suggesting that specific up- or down-regulation of these genes might be associated with the abnormal anther dehiscence in tgms wheat line.  this study analyzed the size and composition of the jaz gene family in wheat, and investigated stress responsive and differential tissue-specific expression profiles of each tajaz gene in tgms wheat line bs in addition, we isolated tajaz genes that would be more likely to be involved in the regulation of abnormal anther dehiscence in tgms wheat line. in conclusion, the results of this study contributed some novel and detailed information about jaz gene family in wheat, and also provided potential candidate genes for improving the tgms wheat line. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords anther dehiscencegene expressionjasmonate-zim domainphylogenetic analysisthermo sensitive genic male sterile national key research project2016yfd0101601zhao changping http://dx.doi.org/ /501100001809national natural science foundation of china31371699zhang liping national science and technology support plan2013bad04b012014bad01b09zhang liping natural science foundation of beijing 5131001zhang liping special project of science and technology innovation ability construction of baafskjcx20140405zhang liping shanxi province science foundation for youths2015021145qiao linyi issue-copyright-statement© the author 2017 