BACKGROUND
aluminium  toxicity is one of the major concerns for crop productivity in acidic soil, accounting for more than 50% of the global arable land
<cit> .
al is one of the highly abundant elements in the earth crust and under low ph conditions , it is solubilised in the soil in a toxic ionic form that inhibits root elongation.
the primary response of plants to al toxicity is the rapid inhibition of root growth, particularly in the root apex by blocking the process of cell division along with cell elongation, and subsequently inefficient absorption of nutrients and water from soil, resulting in the reduction of plant growth and overall productivity
<cit> .
plant species differ in the level of al resistance because they evolved different mechanisms to overcome the selective pressure of al toxicity imposed under acid soils
<cit> .
these mechanisms can be broadly divided into two categories: al resistance and al tolerance.
the al resistance mechanism is often based on the al exclusion from the root apex or the external detoxification of al in the rhizosphere through the release of organic acid anions  from the roots limiting al uptake
<cit> .
on the other hand, the tolerance mechanism may involve the entrance of al through the roots and its relocation  or internal detoxification through al chelating with organic acids
<cit> .
these mechanisms have been validated at the molecular level, particularly with the functional characterization of the major genes such as almt  and mate  in bread wheat
<cit>  and sorghum
<cit> , respectively.
in addition, genes encoding transporters have been identified through mutational analysis, especially abc transporters type  in arabidopsis <cit>  and bacterial-type abc transporters  in rice
<cit> .
recently, a plasma membrane localized transporter nrat <dig>  was also found to be associated with al tolerance particularly to trivalent form of al in rice
<cit> .
these genes are essential for al resistance, and seem to be a part of the pathway involved in the secondary level of protection through uptake and redistribution of al to less sensitive tissues in plants, although their actual function in al tolerance mechanism in plant is still unclear.
there is strong evidence supporting the important role of regulatory genes  in plant tolerance to abiotic stresses
<cit> .
recently, the zinc finger transcription factors, stop <dig>  and art <dig>  have been identified in al sensitive mutants of arabidopsis and rice, respectively
<cit> .
transcriptome analysis under al stress revealed that  <dig> and  <dig> genes were down-regulated in the arabidopsis stop <dig> <cit>  and rice art <dig> mutants
<cit> , respectively.
interestingly, the major genes related to al tolerance, particularly almt <dig> and mate <dig> were regulated by these transcription factors
<cit> .
recently, the contribution of art <dig> locus to the variation for al tolerance in rice has also been identified in qtl analysis
<cit> .
the limited impact of single functional genes in plant stress tolerance has been associated with the polygenic nature of such traits.
thus, the identification and characterization of key regulatory genes that act as master regulators controlling entire response networks would be the most promising and sustainable approach to modify complex traits in plants as they coordinate the expression of many target genes
<cit> .
wheat is one of the most important natural allopolyploid species, as it is not only directly or indirectly contributing in the food supply for nearly half of the worlds’ population but also can serve as a model plant for other economically important polyploid crop species.
it is considered as one of the sensitive crop to al stress among cereals.
bread wheat, with hexaploid nature comprised from three genomes  are organized in seven homoeologous groups, each homoeologous group has individual gene in triplicate form .
consequently, it is of great interest to reveal how the expressions of homoeologues genes are regulated in hexaploid wheat because theoretically, all the three homoeologues of a gene are assumed to be uniformly expressed.
in the previous decade, several studies have been conducted in order to identify the molecular markers  linked to al tolerance through qtl mapping and genome-wide association analyses
<cit> .
so far only two candidate genes almt <dig> and mate <dig> for al tolerance in wheat have been identified and also mapped on chromosome 4dl using chinese spring deletion lines and 4bl through qtl mapping, respectively
<cit> .
recently, the three homoeologues of tamate <dig> have been cloned
<cit> , although, no information is available on the expression of respective homoeologues of these candidate genes for al tolerance in hexaploid wheat.
therefore, in order to improve al tolerance in bread wheat, identification of three homoeologues of the candidate gene is a promising strategy that could be utilized to develop functional markers for genomic assisted breeding programme in wheat.
herein, we report on the identification, physical localization, and molecular characterization of a novel transcription factor tastop <dig> homoeologues genes in bread wheat for al and proton  tolerance.
RESULTS
cloning and structure of tastop1
in order to clone the stop <dig> in wheat, primers from wheat est showing highest similarity with arabidopsis thaliana stop <dig> were used to amplify the tastop <dig> in bread wheat genotype barbela 7/72/ <dig>  further, 5’ and 3’ utr ends of tastop <dig> were amplified using race as described in material and methods.
tastop <dig> was amplified in six different bread wheat genotypes and its multiple alignments suggested the mixed amplification from distinct wheat genomes .
moreover, the comparison of the tastop <dig> cdna sequence with genomic sequence revealed that tastop <dig> gene does not contain introns.
the coding region of tastop <dig>  from genome a has  <dig> bp length and is differentiated from genomes b  and d  by having a 6-bp deletion .
tastop1-b and tastop1-d are differentiated by several snps in the respective open reading frame  [additional file
1].
tastop1-d in bread wheat genotype barbela 7/72/ <dig> has a full-length cdna of  <dig> bp, containing a coding region of  <dig> bp that encodes a polypeptide of  <dig> amino acids .
tastop1-d has a molecular weight of  <dig>  kd, with a  <dig>  isoelectric point .
interproscan function domain analysis suggests that tastop <dig> belongs to the cys2his <dig> zinc finger family protein.
subcellular prediction analysis indicated that tastop <dig> protein is localized in the nucleus.
phylogenetic studies for stop <dig> gene among plant species
deduced amino acid sequence of tastop <dig> consists of  <dig>  and  <dig>  amino acids in bread wheat and the phylogenetic relationship with other stop-like proteins from  <dig> different plant species was studied based on their full length sequences.
phylogenetic analysis of stop like proteins clearly formed two distinct clusters which implied that stop <dig>  was completely distinct from stop <dig>  in arabidopsis.
furthermore, stop <dig> and stop <dig> protein from monocotyledons can be clearly distinguished from eudicots .
tastop <dig> homoeologues from bread wheat showed high similarity with stop <dig> from barley  and brachypodium distachium with identities of  <dig> and 87%, respectively.
noticeably, the phylogenetic relationship of tastop <dig> homoeologoues particularly tastop1-a and tastop1-d from bread wheat  displayed the maximum similarity with stop <dig> like proteins from triticum urartu  and aegilops tauschii , respectively .
sequence alignment of stop <dig> protein from bread wheat with its homologous from cereals  and arabidopsis, shows that tastop <dig> encodes a putative cys2his <dig> zinc finger protein containing four potential zinc finger domains.
three zinc finger domains  are predicted as the cys2his <dig> type, whereas zf <dig> is predicted as the cys2his-cys or the cys2his <dig> type .
furthermore, sequence alignment revealed that stop-like proteins share highly conserved regions in the zf domains.
in addition, the stop <dig> protein also showed greater sequence conservation in the c-terminus than in the n-terminus among family members.
the 6-bp deletion in tastop1-a resulted in a loss of two amino acids  at position  <dig> without changing the reading frame [additional file
2].
we also observed in barbela 7/72/ <dig>  two snps at position  <dig>  and  <dig>  which substituted an asn  by a ser  and asp  by asn  in tastop1-b than in both tastop1-a and tastop1-d homoeologues, respectively [additional file
1].
multiple alignment of stop <dig> protein revealed that the snp at position  <dig> is located in the zinc finger domain  of tastop <dig> homoeologues .
tastop <dig> localization on distinct wheat genomes
for localization of tastop <dig> on homoeologous chromosomes, locus specific primer pairs were designed [additional file
3] and tastop <dig> homoeologues were amplified in a series of nullitetrasomic lines of chinese spring wheat along with chinese spring as a positive control.
on the basis of the presence or absence of pcr products visualized in agarose gels, we observed that tastop <dig> genes are located on homoeologous chromosomes 3a, 3b and 3d, and named tastop1-a, tastop1-b and tastop1-d, respectively .
furthermore, ditelosomic lines of chinese spring for homoeologous group  <dig> chromosomes were also used to assign the tastop <dig> on chromosomal arms and confirmed that tastop1-a, tastop1-b and tastop1-d genes are located on the long arm of homoeologous chromosomes 3a, 3b and 3d, respectively .
in order to further validate these results, physical chromosomal localization of tastop <dig> was performed using tyramide signal amplification fish  technique in the root-tip metaphase chromosome spreads of bread wheat genotype barbela 7/72/ <dig>  for this purpose, a probe was developed by pcr amplification of tastop <dig> from genomic dna of barbela 7/72/ <dig>  fish with the tastop <dig> specific probe on metaphase chromosomes depicted hybridization signals of tastop <dig> on both chromatids of the long arms of three chromosomes  and simultaneous re-probing of chromosome preparations with gaa-  and pas <dig>  identified homoeologous group  <dig> with positive signals on chromosomes 3al, 3bl and 3dl .
some background was also observed but it was distinguished from true hybridization signals because they usually appear as dots without any pattern and not in both chromatids of the same chromosome.
only those signals having the same size and appearance on the same position of both chromatids of one specific chromosome were analyzed as true-positive hybridization signals.
tastop <dig> transactivation activity
the nuclear localization of tastop <dig> protein was predicted by wolf-psort programme
<cit> .
tastop <dig> is a putative transcription factor of the cys2his2-type zinc fingers family.
the three homoeologues of tastop <dig> showed similar genomic structure in bread wheat [additional file
2].
therefore, we only proceed to evaluate the transactivation potential of tastop <dig> located on genome a .
to this aim, we performed a modified yeast one-hybrid assay
<cit>  to evaluate the ability of the lexa-tastop1-a fusion protein to activate the lexa-driven expression of the lacz gene in a heterologous yeast system, the results of which are illustrated in figure
<dig>  it is shown that lexa-tastop1-a has the potential to transactivate lacz expression either in the presence or absence of al, further corroborating its potential role as a transcription factor.
expression profile of three tastop <dig> homoeologues in bread wheat
to understand the homoeologue specific expression of tastop <dig> in bread wheat, we measured the temporal expression of tastop1-a, tastop1-b and tastop1-d by real-time qpcr in the root and shoot tissues of two bread wheat genotypes  grown under al stress .
the 18s rna expression was used as an internal control.
we noticed a biased transcription of homoeologues of tastop <dig> gene in the root and shoot tissue of these diverse bread wheat genotypes .
the transcript levels of homoeologue tastop1-a under control as well as al treatment were significantly higher than those of tastop1-b and tastop1-d in root and shoot tissues of both genotypes  whereas expression of homoeologue tastop1-d was only two-fold higher than tastop1-b in the root tissues of both genotypes .
a slight induction  followed by return to basal levels of the homoeologue tastop1-a transcripts was observed in the root tissues of barbela 7/72/ <dig>  whereas in the anahuac  under al stress it was observed a quite stable transcript expression .
the expression of tastop1-b was also almost unaltered in the roots of both genotypes under al stress.
similarly, tastop1-d was also constitutively expressed under al stress in the roots of both genotypes except a rapid  and significant repression in the al sensitive genotype anahuac .
nevertheless, a quite stable transcript expression of tastop <dig> homoeologues was observed in the shoot of both genotypes except homoeologue tastop1-a .
interestingly, and similarly to roots, the expression of tastop1-a in the shoots of both genotypes was also slightly induced under al stress, however, its up-regulation was noticed only after  <dig> h and return to its basal level .
it is worth noting that the transcript levels of tastop1-b of al tolerant genotype barbela 7/72/ <dig> are lower in the shoot tissues, compared to the sensitive genotype anahuac .
moreover, to evaluate the transcript expression of tastop <dig> homoeologues under protons  stress, we also performed the relative quantification of homoeologue specific expression of tastop <dig> transcript in root tissues of both genotypes under different levels of ph .
a slight gradual decrease in the transcript levels of tastop1-a homoeologue was observed in the roots of barbela 7/72/ <dig> under protons  stress, but a quite stable expression was observed in genotype anahuac .
furthermore, considerable up-regulation of the homoeologue tastop1-b and slight repression of the homoeologue tastop1-d transcript level was also observed under low ph  in the roots of genotype anahuac and barbela 7/72/ <dig>  respectively .
discussion
so far only almt <dig> and mate genes have been described as responsible for most of the genotypic variation for al tolerance and are considered as major genes for al resistance in bread wheat
<cit> .
these genes have been identified in wheat
<cit>  and also in the model plant arabidopsis
<cit>  and major cereals such as barley
<cit> , maize
<cit> , sorghum
<cit>  and rye
<cit> .
the mechanisms underlying al-tolerance in plant species have yet to be fully elucidated, as it seems that al targets multiple cellular sites such as cell walls, plasma membranes, and cellular processes, like signal transduction pathways and homeostasis mechanisms
<cit> .
recent evidence suggests that regulatory genes  play a key role in al detoxification at different cellular levels
<cit> .
in the present study, a novel transcription factor gene named tastop <dig> was cloned from an al tolerant genotype barbela 7/72/ <dig>  which was derived from portuguese bread wheat landrace barbela through single seed descent method
<cit> .
tastop <dig> belongs to a member of cys2his <dig> zinc finger family proteins that contains four potential zinc finger domains and has highly conserved regions in the zinc finger  domains .
like other c2h2-type zinc finger proteins it contains more than one zinc finger motif with highly conserved amino acid sequence for dna binding
<cit> .
phylogenetic analysis clearly differentiated the stop <dig> like proteins from monocots in a group  and also suggested that genome a and b are more distant from genome d [additional file
2], as genome d was incorporated recently in comparison to the a and b genomes in bread wheat
<cit> .
in addition, our phylogenetic analysis results also support previous findings at molecular level that the a and d genomes in bread wheat were derived from t. urartu and a. tauschii, respectively [additional file
2]
<cit> .
in past, the nullitetrasomic and ditelosomic lines of chinese spring wheat have been extensively used in classical and molecular genetic studies for the identification of loci associated with numerous traits including al tolerance
<cit> .
thus, gene mapping has practical implications in plant genetics and breeding, and the determination of its physical localization is even more precise as it enables us to confirm the exact position of a gene on a chromosome.
pcr based mapping of tastop <dig> using nullitetrasomic and ditellosomic lines of chinese spring wheat revealed that tastop <dig> is located on the long arm of wheat homoeologous group  <dig> chromosomes .
in situ hybridization is not only the most direct method for physical localization of genes in chromosomes, but also it is a valuable tool for the identification of copy numbers of a gene in species having a highly complex genome such as the bread wheat.
due to its hexaploid nature, most genes can be found in triplicate with one copy on each genome
<cit> .
tyramide signal amplification fish  technique has been successfully used for the identification of low copy number dna sequences in wheat
<cit> .
however, for the successful localization of a gene using the fish technique, the minimum length of the probe size seems to be crucial.
recently, rd <dig> gene has been localized in bread wheat with a probe size of  <dig> kb
<cit> .
physical localization of tastop <dig> by tyr-fish technique not only confirmed the results obtained from pcr based mapping, but also indicated that tastop <dig> could be a single copy gene localized on the long arm of homoeologous group  <dig> chromosomes .
in bread wheat, physical localization of single copy gene glu- <dig> and rd <dig> with fish technique has been demonstrated on long and short arms of the homoeologous group  <dig> chromosomes, respectively
<cit> .
in the present work, we were able for the first time to localize a gene with a probe size smaller than  <dig>  kb demonstrating that the fish technique can be used to simultaneously anchor homoeologous chromosomes with  <dig>  kb probes even in bread wheat.
subcellular prediction of tastop <dig> protein in the nucleus is in agreement with data previously reported
<cit> .
zinc finger motifs are thought to recognize and bind to target dna sequences, but they are not required for transcriptional activity
<cit> .
our results clearly exhibited the transactivation potential of tastop1-a at least in yeast .
in present investigation, we observed highly similar genomic structure of tastop <dig> genes [additional files
<dig> and
2], therefore, it seems that tastop <dig> transactivation function is constitutive and may not depend on the presence/absence of al.
the snps observed among tastop <dig> homoeologues showed minor changes in respective amino acids of putative proteins which could alter the secondary structures by influencing the folding of these proteins .
therefore, the three homoeologues of the same locus in bread wheat share high sequence similarity that could illustrate the flexibility of a polyploidy species in which due to the mutation in one homoeologue may be compensated for by the homoeologue
<cit> .
allopolyploidy plays an important role in plant evolution that arises with the merging of two or more genomes into single nucleus which may contribute either equally or disproportionately.
however, recent molecular findings confirmed the asymmetric genomic expression pattern in natural and synthetic allopolyploid plant species, but the predominant transcript expression of one genome over the other genome vary from gene to gene
<cit> .
in hexaploid wheat, a uniform level of expression for all the three homoeologues has been reported for approximately 20% of the unigene loci
<cit> , whereas 20-29% genes did not express at least one homoeoallele
<cit> .
thus, the relative contribution of the three homoeologues of tastop <dig> gene at transcript expression level was determined under al and proton  stresses in two bread wheat genotypes showing contrasting behaviour for al toxicity.
transcript expression profiling of tastop <dig> homoeologues in root and shoot tissues identified the predominance of tastop1-a homoeologue followed by tastop1-d over tastop1-b in root and only predominance of tastop1-a in shoot tissues of both genotypes under control and stress  conditions .
similarly, among the three homoeologues, the higher expression of one homoeologue of a mad box transcription factor and spa gene have also been observed in bread wheat
<cit> .
although, the presence of cis elements within the 5' untranslated region of a gene is unusual, it is not aberrant or abnormal
<cit> .
surprisingly, sequence analysis of 5' utr of tastop <dig> genes differentiated the tastop1-a homoeologue from tastop1-b and tastop1-d due to the presence of a pyrimidine-rich stretch and absence of light responsive element [additional file
4].
in tomato, the deletion of the 5' utr containing pyrimidine-rich stretch from the hmg <dig> promoter reduced the level of hmg <dig> gene expression by a factor of 10
<cit> .
therefore, among the three homogeologues in bread wheat, the up- or down-regulation of the expression of specific homoeologue could be the result from either the dominancy of ancestral diploid donor parent or early polyploidization-cis-regulatory variation.
interestingly, the time-dependent al-responsive expression of tastop <dig> homoeologues observed in the root tissues of two bread wheat showing contrasting phenotypes for al toxicity suggests a putative role for tastop <dig> in al resistance .
al responsive stop <dig> expression has also been reported in arabidopsis <cit> , alfalfa
<cit>  and common bean
<cit> .
similarly to arabidopsis, the transcript expression of homoeologues of tastop <dig> in the roots of diverse bread wheat genotypes was also modulated in response to proton stress
<cit> .
it is noticeable that genotype barbela 7/72/ <dig> is highly resistant to al toxicity compared with anahuac, but in the absence of al under low ph we did not observe significant differences between these genotypes for root growth [additional file
5].
it is worth noticing that the three homoeologues of tastop <dig> have similar genomic structures, but showed a different transcript expression in response to al and proton  stress.
these results may reveal that the homoeologues of tastop <dig> are differentially contributing to al or proton  tolerance in bread wheat.
further work is in progress in order to definitely establish if these genes play a significant role in aluminium tolerance.
CONCLUSIONS
bread wheat has wide genotypic variation for al resistance
<cit>  and the role of several chromosomes such as chromosome arms 2dl, 3dl, 4bl, 4dl, 6al, 7as and chromosome 7d, in al tolerance has been revealed in classical genetic studies through chromosome manipulation in wheat
<cit> .
furthermore, several qtl associated with al tolerance in bread wheat have also been reported by many researchers
<cit> .
contrarily to arabidopsis and rice, due to the paucity of a priori candidate genes in wheat only two major qtl located on chromosome 4dl and 4bl have yet been elucidated at molecular level, showing the co-segregation with candidate genes taalmt <dig> homoeologue  and tamate <dig> homoeologue , respectively
<cit> .
the classical studies using chromosomal manipulation as well as recent qtl mapping and genome-wide association analysis have also detected loci associated with al resistance on homoeologous group  <dig> chromosomes  in bread wheat
<cit> .
recently, the role of a zinc finger transcription factor art <dig> identified through mutational analysis in rice has also been shown in natural variation of al tolerance in rice, earlier which was suggested that it was not involved in al tolerance
<cit> .
in the present investigation, we cloned and characterized the novel candidate genes tastop <dig> in bread wheat.
the homoeologues of tastop <dig> exhibited similar genomic structures, but showed biased transcript expression and different response to al and proton  toxicity.
furthermore, tastop1-b homoeologue from al tolerant genotypes barbela 7/72/ <dig> and viloso mole not only showed highest similarity but also contain the same snp located in zf <dig> domain than al sensitive genotypes anahuac, chinese spring and saloio [additional file
2]
<cit> .
finally, in order to correlate with al tolerance, it would be very interesting either to functionally characterize or further verify the role of tastop <dig> because gene underlying the qtl on homoeologous group  <dig> chromosomes particularly 3bl has not been so far identified in this important cereal.

 aluminium toxicity is one of the major concerns for crop productivity in acidic soil, accounting for more than 50% of the global arable land. al is one of the highly abundant elements in the earth crust and under low ph conditions, it is solubilised in the soil in a toxic ionic form that inhibits root elongation. the primary response of plants to al toxicity is the rapid inhibition of root growth, particularly in the root apex by blocking the process of cell division along with cell elongation, and subsequently inefficient absorption of nutrients and water from soil, resulting in the reduction of plant growth and overall productivity. plant species differ in the level of al resistance because they evolved different mechanisms to overcome the selective pressure of al toxicity imposed under acid soils. these mechanisms can be broadly divided into two categories: al resistance and al tolerance. the al resistance mechanism is often based on the al exclusion from the root apex or the external detoxification of al in the rhizosphere through the release of organic acid anions from the roots limiting al uptake. on the other hand, the tolerance mechanism may involve the entrance of al through the roots and its relocation or internal detoxification through al chelating with organic acids. these mechanisms have been validated at the molecular level, particularly with the functional characterization of the major genes such as almt and mate in bread wheat and sorghum, respectively. in addition, genes encoding transporters have been identified through mutational analysis, especially abc transporters type in arabidopsis and bacterial-type abc transporters in rice. recently, a plasma membrane localized transporter nrat was also found to be associated with al tolerance particularly to trivalent form of al in rice. these genes are essential for al resistance, and seem to be a part of the pathway involved in the secondary level of protection through uptake and redistribution of al to less sensitive tissues in plants, although their actual function in al tolerance mechanism in plant is still unclear. there is strong evidence supporting the important role of regulatory genes in plant tolerance to abiotic stresses. recently, the zinc finger transcription factors, stop and art have been identified in al sensitive mutants of arabidopsis and rice, respectively. transcriptome analysis under al stress revealed that and genes were down-regulated in the arabidopsis stop and rice art mutants, respectively. interestingly, the major genes related to al tolerance, particularly almt and mate were regulated by these transcription factors. recently, the contribution of art locus to the variation for al tolerance in rice has also been identified in qtl analysis. the limited impact of single functional genes in plant stress tolerance has been associated with the polygenic nature of such traits. thus, the identification and characterization of key regulatory genes that act as master regulators controlling entire response networks would be the most promising and sustainable approach to modify complex traits in plants as they coordinate the expression of many target genes. wheat is one of the most important natural allopolyploid species, as it is not only directly or indirectly contributing in the food supply for nearly half of the worlds’ population but also can serve as a model plant for other economically important polyploid crop species. it is considered as one of the sensitive crop to al stress among cereals. bread wheat, with hexaploid nature comprised from three genomes are organized in seven homoeologous groups, each homoeologous group has individual gene in triplicate form. consequently, it is of great interest to reveal how the expressions of homoeologues genes are regulated in hexaploid wheat because theoretically, all the three homoeologues of a gene are assumed to be uniformly expressed. in the previous decade, several studies have been conducted in order to identify the molecular markers linked to al tolerance through qtl mapping and genome-wide association analyses. so far only two candidate genes almt and mate for al tolerance in wheat have been identified and also mapped on chromosome 4dl using chinese spring deletion lines and 4bl through qtl mapping, respectively. recently, the three homoeologues of tamate have been cloned, although, no information is available on the expression of respective homoeologues of these candidate genes for al tolerance in hexaploid wheat. therefore, in order to improve al tolerance in bread wheat, identification of three homoeologues of the candidate gene is a promising strategy that could be utilized to develop functional markers for genomic assisted breeding programme in wheat. herein, we report on the identification, physical localization, and molecular characterization of a novel transcription factor tastop homoeologues genes in bread wheat for al and proton tolerance.  cloning and structure of tastop1 in order to clone the stop in wheat, primers from wheat est showing highest similarity with arabidopsis thaliana stop were used to amplify the tastop in bread wheat genotype barbela 7/72/ further, 5’ and 3’ utr ends of tastop were amplified using race as described in material and methods. tastop was amplified in six different bread wheat genotypes and its multiple alignments suggested the mixed amplification from distinct wheat genomes. moreover, the comparison of the tastop cdna sequence with genomic sequence revealed that tastop gene does not contain introns. the coding region of tastop from genome a has bp length and is differentiated from genomes b and d by having a 6-bp deletion. tastop1-b and tastop1-d are differentiated by several snps in the respective open reading frame [additional file 1]. tastop1-d in bread wheat genotype barbela 7/72/ has a full-length cdna of bp, containing a coding region of bp that encodes a polypeptide of amino acids. tastop1-d has a molecular weight of kd, with a isoelectric point. interproscan function domain analysis suggests that tastop belongs to the cys2his zinc finger family protein. subcellular prediction analysis indicated that tastop protein is localized in the nucleus. phylogenetic studies for stop gene among plant species deduced amino acid sequence of tastop consists of and amino acids in bread wheat and the phylogenetic relationship with other stop-like proteins from different plant species was studied based on their full length sequences. phylogenetic analysis of stop like proteins clearly formed two distinct clusters which implied that stop was completely distinct from stop in arabidopsis. furthermore, stop and stop protein from monocotyledons can be clearly distinguished from eudicots. tastop homoeologues from bread wheat showed high similarity with stop from barley and brachypodium distachium with identities of and 87%, respectively. noticeably, the phylogenetic relationship of tastop homoeologoues particularly tastop1-a and tastop1-d from bread wheat displayed the maximum similarity with stop like proteins from triticum urartu and aegilops tauschii, respectively. sequence alignment of stop protein from bread wheat with its homologous from cereals and arabidopsis, shows that tastop encodes a putative cys2his zinc finger protein containing four potential zinc finger domains. three zinc finger domains are predicted as the cys2his type, whereas zf is predicted as the cys2his-cys or the cys2his type. furthermore, sequence alignment revealed that stop-like proteins share highly conserved regions in the zf domains. in addition, the stop protein also showed greater sequence conservation in the c-terminus than in the n-terminus among family members. the 6-bp deletion in tastop1-a resulted in a loss of two amino acids at position without changing the reading frame [additional file 2]. we also observed in barbela 7/72/ two snps at position and which substituted an asn by a ser and asp by asn in tastop1-b than in both tastop1-a and tastop1-d homoeologues, respectively [additional file 1]. multiple alignment of stop protein revealed that the snp at position is located in the zinc finger domain of tastop homoeologues. tastop localization on distinct wheat genomes for localization of tastop on homoeologous chromosomes, locus specific primer pairs were designed [additional file 3] and tastop homoeologues were amplified in a series of nullitetrasomic lines of chinese spring wheat along with chinese spring as a positive control. on the basis of the presence or absence of pcr products visualized in agarose gels, we observed that tastop genes are located on homoeologous chromosomes 3a, 3b and 3d, and named tastop1-a, tastop1-b and tastop1-d, respectively. furthermore, ditelosomic lines of chinese spring for homoeologous group chromosomes were also used to assign the tastop on chromosomal arms and confirmed that tastop1-a, tastop1-b and tastop1-d genes are located on the long arm of homoeologous chromosomes 3a, 3b and 3d, respectively. in order to further validate these results, physical chromosomal localization of tastop was performed using tyramide signal amplification fish technique in the root-tip metaphase chromosome spreads of bread wheat genotype barbela 7/72/ for this purpose, a probe was developed by pcr amplification of tastop from genomic dna of barbela 7/72/ fish with the tastop specific probe on metaphase chromosomes depicted hybridization signals of tastop on both chromatids of the long arms of three chromosomes and simultaneous re-probing of chromosome preparations with gaa- and pas identified homoeologous group with positive signals on chromosomes 3al, 3bl and 3dl. some background was also observed but it was distinguished from true hybridization signals because they usually appear as dots without any pattern and not in both chromatids of the same chromosome. only those signals having the same size and appearance on the same position of both chromatids of one specific chromosome were analyzed as true-positive hybridization signals. tastop transactivation activity the nuclear localization of tastop protein was predicted by wolf-psort programme. tastop is a putative transcription factor of the cys2his2-type zinc fingers family. the three homoeologues of tastop showed similar genomic structure in bread wheat [additional file 2]. therefore, we only proceed to evaluate the transactivation potential of tastop located on genome a. to this aim, we performed a modified yeast one-hybrid assay to evaluate the ability of the lexa-tastop1-a fusion protein to activate the lexa-driven expression of the lacz gene in a heterologous yeast system, the results of which are illustrated in figure  it is shown that lexa-tastop1-a has the potential to transactivate lacz expression either in the presence or absence of al, further corroborating its potential role as a transcription factor. expression profile of three tastop homoeologues in bread wheat to understand the homoeologue specific expression of tastop in bread wheat, we measured the temporal expression of tastop1-a, tastop1-b and tastop1-d by real-time qpcr in the root and shoot tissues of two bread wheat genotypes grown under al stress. the 18s rna expression was used as an internal control. we noticed a biased transcription of homoeologues of tastop gene in the root and shoot tissue of these diverse bread wheat genotypes. the transcript levels of homoeologue tastop1-a under control as well as al treatment were significantly higher than those of tastop1-b and tastop1-d in root and shoot tissues of both genotypes whereas expression of homoeologue tastop1-d was only two-fold higher than tastop1-b in the root tissues of both genotypes. a slight induction followed by return to basal levels of the homoeologue tastop1-a transcripts was observed in the root tissues of barbela 7/72/ whereas in the anahuac under al stress it was observed a quite stable transcript expression. the expression of tastop1-b was also almost unaltered in the roots of both genotypes under al stress. similarly, tastop1-d was also constitutively expressed under al stress in the roots of both genotypes except a rapid and significant repression in the al sensitive genotype anahuac. nevertheless, a quite stable transcript expression of tastop homoeologues was observed in the shoot of both genotypes except homoeologue tastop1-a. interestingly, and similarly to roots, the expression of tastop1-a in the shoots of both genotypes was also slightly induced under al stress, however, its up-regulation was noticed only after h and return to its basal level. it is worth noting that the transcript levels of tastop1-b of al tolerant genotype barbela 7/72/ are lower in the shoot tissues, compared to the sensitive genotype anahuac. moreover, to evaluate the transcript expression of tastop homoeologues under protons stress, we also performed the relative quantification of homoeologue specific expression of tastop transcript in root tissues of both genotypes under different levels of ph. a slight gradual decrease in the transcript levels of tastop1-a homoeologue was observed in the roots of barbela 7/72/ under protons stress, but a quite stable expression was observed in genotype anahuac. furthermore, considerable up-regulation of the homoeologue tastop1-b and slight repression of the homoeologue tastop1-d transcript level was also observed under low ph in the roots of genotype anahuac and barbela 7/72/ respectively. discussion so far only almt and mate genes have been described as responsible for most of the genotypic variation for al tolerance and are considered as major genes for al resistance in bread wheat. these genes have been identified in wheat and also in the model plant arabidopsis and major cereals such as barley, maize, sorghum and rye. the mechanisms underlying al-tolerance in plant species have yet to be fully elucidated, as it seems that al targets multiple cellular sites such as cell walls, plasma membranes, and cellular processes, like signal transduction pathways and homeostasis mechanisms. recent evidence suggests that regulatory genes play a key role in al detoxification at different cellular levels. in the present study, a novel transcription factor gene named tastop was cloned from an al tolerant genotype barbela 7/72/ which was derived from portuguese bread wheat landrace barbela through single seed descent method. tastop belongs to a member of cys2his zinc finger family proteins that contains four potential zinc finger domains and has highly conserved regions in the zinc finger domains. like other c2h2-type zinc finger proteins it contains more than one zinc finger motif with highly conserved amino acid sequence for dna binding. phylogenetic analysis clearly differentiated the stop like proteins from monocots in a group and also suggested that genome a and b are more distant from genome d [additional file 2], as genome d was incorporated recently in comparison to the a and b genomes in bread wheat. in addition, our phylogenetic analysis results also support previous findings at molecular level that the a and d genomes in bread wheat were derived from t. urartu and a. tauschii, respectively [additional file 2]. in past, the nullitetrasomic and ditelosomic lines of chinese spring wheat have been extensively used in classical and molecular genetic studies for the identification of loci associated with numerous traits including al tolerance. thus, gene mapping has practical implications in plant genetics and breeding, and the determination of its physical localization is even more precise as it enables us to confirm the exact position of a gene on a chromosome. pcr based mapping of tastop using nullitetrasomic and ditellosomic lines of chinese spring wheat revealed that tastop is located on the long arm of wheat homoeologous group chromosomes. in situ hybridization is not only the most direct method for physical localization of genes in chromosomes, but also it is a valuable tool for the identification of copy numbers of a gene in species having a highly complex genome such as the bread wheat. due to its hexaploid nature, most genes can be found in triplicate with one copy on each genome. tyramide signal amplification fish technique has been successfully used for the identification of low copy number dna sequences in wheat. however, for the successful localization of a gene using the fish technique, the minimum length of the probe size seems to be crucial. recently, rd gene has been localized in bread wheat with a probe size of kb. physical localization of tastop by tyr-fish technique not only confirmed the results obtained from pcr based mapping, but also indicated that tastop could be a single copy gene localized on the long arm of homoeologous group chromosomes. in bread wheat, physical localization of single copy gene glu- and rd with fish technique has been demonstrated on long and short arms of the homoeologous group chromosomes, respectively. in the present work, we were able for the first time to localize a gene with a probe size smaller than kb demonstrating that the fish technique can be used to simultaneously anchor homoeologous chromosomes with kb probes even in bread wheat. subcellular prediction of tastop protein in the nucleus is in agreement with data previously reported. zinc finger motifs are thought to recognize and bind to target dna sequences, but they are not required for transcriptional activity. our results clearly exhibited the transactivation potential of tastop1-a at least in yeast. in present investigation, we observed highly similar genomic structure of tastop genes [additional files and 2], therefore, it seems that tastop transactivation function is constitutive and may not depend on the presence/absence of al. the snps observed among tastop homoeologues showed minor changes in respective amino acids of putative proteins which could alter the secondary structures by influencing the folding of these proteins. therefore, the three homoeologues of the same locus in bread wheat share high sequence similarity that could illustrate the flexibility of a polyploidy species in which due to the mutation in one homoeologue may be compensated for by the homoeologue. allopolyploidy plays an important role in plant evolution that arises with the merging of two or more genomes into single nucleus which may contribute either equally or disproportionately. however, recent molecular findings confirmed the asymmetric genomic expression pattern in natural and synthetic allopolyploid plant species, but the predominant transcript expression of one genome over the other genome vary from gene to gene. in hexaploid wheat, a uniform level of expression for all the three homoeologues has been reported for approximately 20% of the unigene loci, whereas 20-29% genes did not express at least one homoeoallele. thus, the relative contribution of the three homoeologues of tastop gene at transcript expression level was determined under al and proton stresses in two bread wheat genotypes showing contrasting behaviour for al toxicity. transcript expression profiling of tastop homoeologues in root and shoot tissues identified the predominance of tastop1-a homoeologue followed by tastop1-d over tastop1-b in root and only predominance of tastop1-a in shoot tissues of both genotypes under control and stress conditions. similarly, among the three homoeologues, the higher expression of one homoeologue of a mad box transcription factor and spa gene have also been observed in bread wheat. although, the presence of cis elements within the 5' untranslated region of a gene is unusual, it is not aberrant or abnormal. surprisingly, sequence analysis of 5' utr of tastop genes differentiated the tastop1-a homoeologue from tastop1-b and tastop1-d due to the presence of a pyrimidine-rich stretch and absence of light responsive element [additional file 4]. in tomato, the deletion of the 5' utr containing pyrimidine-rich stretch from the hmg promoter reduced the level of hmg gene expression by a factor of 10. therefore, among the three homogeologues in bread wheat, the up- or down-regulation of the expression of specific homoeologue could be the result from either the dominancy of ancestral diploid donor parent or early polyploidization-cis-regulatory variation. interestingly, the time-dependent al-responsive expression of tastop homoeologues observed in the root tissues of two bread wheat showing contrasting phenotypes for al toxicity suggests a putative role for tastop in al resistance. al responsive stop expression has also been reported in arabidopsis, alfalfa and common bean. similarly to arabidopsis, the transcript expression of homoeologues of tastop in the roots of diverse bread wheat genotypes was also modulated in response to proton stress. it is noticeable that genotype barbela 7/72/ is highly resistant to al toxicity compared with anahuac, but in the absence of al under low ph we did not observe significant differences between these genotypes for root growth [additional file 5]. it is worth noticing that the three homoeologues of tastop have similar genomic structures, but showed a different transcript expression in response to al and proton stress. these results may reveal that the homoeologues of tastop are differentially contributing to al or proton tolerance in bread wheat. further work is in progress in order to definitely establish if these genes play a significant role in aluminium tolerance.  bread wheat has wide genotypic variation for al resistance and the role of several chromosomes such as chromosome arms 2dl, 3dl, 4bl, 4dl, 6al, 7as and chromosome 7d, in al tolerance has been revealed in classical genetic studies through chromosome manipulation in wheat. furthermore, several qtl associated with al tolerance in bread wheat have also been reported by many researchers. contrarily to arabidopsis and rice, due to the paucity of a priori candidate genes in wheat only two major qtl located on chromosome 4dl and 4bl have yet been elucidated at molecular level, showing the co-segregation with candidate genes taalmt homoeologue and tamate homoeologue, respectively. the classical studies using chromosomal manipulation as well as recent qtl mapping and genome-wide association analysis have also detected loci associated with al resistance on homoeologous group chromosomes in bread wheat. recently, the role of a zinc finger transcription factor art identified through mutational analysis in rice has also been shown in natural variation of al tolerance in rice, earlier which was suggested that it was not involved in al tolerance. in the present investigation, we cloned and characterized the novel candidate genes tastop in bread wheat. the homoeologues of tastop exhibited similar genomic structures, but showed biased transcript expression and different response to al and proton toxicity. furthermore, tastop1-b homoeologue from al tolerant genotypes barbela 7/72/ and viloso mole not only showed highest similarity but also contain the same snp located in zf domain than al sensitive genotypes anahuac, chinese spring and saloio [additional file 2]. finally, in order to correlate with al tolerance, it would be very interesting either to functionally characterize or further verify the role of tastop because gene underlying the qtl on homoeologous group chromosomes particularly 3bl has not been so far identified in this important cereal. 
physical localization of tastop results indicated the presence of a single copy of tastop on homoeologous group chromosomes in bread wheat. these results indicate that tastop homoeologues may differentially contribute under al or proton toxicity in bread wheat. tastop showed four potential zinc finger domains and the homoeologue tastop1-a exhibited transactivation activity in yeast. expression profiling of tastop transcripts identified the predominance of homoeologue tastop1-a followed by tastop1-d over tastop1-b in root and only predominance of tastop1-a in shoot tissues of two diverse bread wheat genotypes. being a trait governed by multiple genes, the identification and characterization of novel transcription factors regulating the expression of entire response networks is a very promising approach. the three homoeologues of tastop have similar genomic structures, but showed biased transcript expression and different response to al and proton toxicity. therefore, the aim of the present study was to clone, localize, and characterize the tastop gene, which belongs to the zinc finger family transcription factor, at molecular level in bread wheat. aluminium toxicity is considered to be one of the major constraints affecting crop productivity on acid soils. tastop loci were cloned and localized on the long arm of homoeologous group chromosomes in bread wheat. al and proton stress appeared to slightly modulate the transcript of tastop homoeologues expression in both genotypes of bread wheat. finally, the localization of tastop on long arm of homoeologous group chromosomes and the previously reported major loci associated with al resistance at chromosome 3bl, through qtl and genome wide association mapping studies suggests that tastop could be a potential candidate gene for genomic assisted breeding for al tolerance in bread wheat.
 aluminium toxicity is considered to be one of the major constraints affecting crop productivity on acid soils. being a trait governed by multiple genes, the identification and characterization of novel transcription factors regulating the expression of entire response networks is a very promising approach. therefore, the aim of the present study was to clone, localize, and characterize the tastop gene, which belongs to the zinc finger family transcription factor, at molecular level in bread wheat.  tastop loci were cloned and localized on the long arm of homoeologous group chromosomes in bread wheat. tastop showed four potential zinc finger domains and the homoeologue tastop1-a exhibited transactivation activity in yeast. expression profiling of tastop transcripts identified the predominance of homoeologue tastop1-a followed by tastop1-d over tastop1-b in root and only predominance of tastop1-a in shoot tissues of two diverse bread wheat genotypes. al and proton stress appeared to slightly modulate the transcript of tastop homoeologues expression in both genotypes of bread wheat.  physical localization of tastop results indicated the presence of a single copy of tastop on homoeologous group chromosomes in bread wheat. the three homoeologues of tastop have similar genomic structures, but showed biased transcript expression and different response to al and proton toxicity. these results indicate that tastop homoeologues may differentially contribute under al or proton toxicity in bread wheat. moreover, it seems that tastop1-a transactivation potential is constitutive and may not depend on the presence/absence of al at least in yeast. finally, the localization of tastop on long arm of homoeologous group chromosomes and the previously reported major loci associated with al resistance at chromosome 3bl, through qtl and genome wide association mapping studies suggests that tastop could be a potential candidate gene for genomic assisted breeding for al tolerance in bread wheat. aluminiumtastop1triticum aestivum lin situ hybridizationtranscription factortransactivationhomoeologueph 