BACKGROUND
aluminum is considered as the most limiting factor for plant productivity in acidic soils.
it is estimated that over 50% of the world's potential arable land surface is composed of acid soils mostly distributed in developing countries  <cit> .
al tolerance is second to drought tolerance for its importance as agronomic trait for worldwide crop production.
the root apex is considered the first target of al toxicity and the reduction in root biomass leads to poor uptake of water and nutrients  <cit> .
at the cellular level, al toxicity results from a broad spectrum of deleterious effects caused by the al3+ ion, which is considered the most toxic species of al for both plant and animal cells under low ph conditions  <cit> .
it was proposed that the toxic effect of al can be reduced by chelating al in the rhizosphere with organic anions.
exudation of a variety of organic anions such as malate, citrate, or oxalate upon exposure to al have been reported  <cit>  and al tolerance of several species can be enhanced by increasing organic acid biosynthesis  <cit> .
overexpression of the al-inducible malate transporter improves al tolerance in barley  <cit>  while overexpression of the sbmate protein, a putative citrate transporter, improves al tolerance in arabidopsis and wheat  <cit> .
the release of phosphate was also associated with al tolerance, indicating that multiple mechanisms are involved in wheat  <cit> .
in arabidopsis, the major al tolerance quantitative trait loci  was proposed to be proximal to the atalmt <dig> malate transporter and to involve additional genes  <cit> .
fine mapping indicated that al tolerance is more closely associated with a chromosome location that is distal to atalmt by  <dig> to  <dig> kbp indicating that other gene products may regulate or complement its activity  <cit> .
this regulation may be controlled at the transcriptional, translational, post-translational and enzymatic levels.
the association of organic acid release and al tolerance is not universal as several al-sensitive plants were found to secrete large amounts of organic acids  <cit> .
these results suggest that organic acid excretion, per se, is not a tolerance  mechanism, but a consequence of biochemical reactions required for al tolerance.
in arabidopsis, there was no induction of atalmt <dig> by al3+ in the stop <dig> arabidopsis mutant suggesting that the stop <dig> zinc finger protein is required to activate almt <dig> transcription  <cit> .
the als <dig> gene encodes an abc transporter-like protein that is required for al tolerance/resistance and may function to redistribute accumulated al away from sensitive tissues to protect growing roots from al toxicity  <cit> .
several genes up-regulated during al exposure have been identified using various molecular approaches  <cit> .
overexpression of some of these genes in transgenic plants has resulted in modest improvement of al tolerance, suggesting that they alleviate part of the toxicity caused by al  <cit> .
in rice, al tolerance appears to be a complex multigenic trait that involves all twelve chromosomes.
however, fewer loci were reported to be involved in other grasses  <cit> .
two major loci on chromosome 4dl and 6a, and additional loci with additive effects are involved in al tolerance of the wheat cultivar atlas <dig>  <cit> .
the availability of near-isogenic lines derived from the cultivar atlas <dig> having similar qtls for al tolerance provides a useful tool to identify genes associated with al stress and tolerance  <cit> .
in this study, several genes associated with al stress and tolerance were identified using transcriptome analyses.
the putative functions of identified genes in several biochemical pathways are discussed in relation to stress responses and the maintenance of root growth.
RESULTS
to identify genes associated with al stress and tolerance in wheat, a large scale expression profiling study was initiated using the affymetrix genechip® wheat genome array which allows the screening of  <dig> transcripts.
to better discriminate between genes that are associated with al stress and tolerance, four different wheat lines  were analyzed.
in addition to the well studied tolerant wheat atlas <dig> and the sensitive wheat bounty used in our previous study  <cit> , we used two near isogenic lines  derived from a cross between atlas <dig> and the sensitive cultivar century  <cit> .
the sensitive nil ok91g <dig>  has a high degree of genetic similarity  with the tolerant nil ok91g <dig> .
the major difference between tolerant and sensitive plants is the ability to maintain growth under high al concentrations.
the tolerant cultivars used in this study are able to grow for several days  = 50%) in the presence of  <dig> μm al .
the sensitive cultivars are unable to grow at this high al concentration and an rgi of 50% is obtained in the presence of  <dig> μm al.
we have previously shown that the stress-associated genes are expressed at comparable levels in the sensitive and tolerant cultivars after  <dig> hours when they are exposed to al concentrations resulting in a similar rgi  <cit> .
the microarray experiments were thus designed to identify genes that are differentially expressed after  <dig> hours of al exposure at concentrations resulting in 50% rgi for all lines used.
microarray analyses
the number of al-regulated genes  is over  <dig> when cultivars are analyzed separately.
however, only  <dig> genes are differentially expressed in all four cultivars.
anova analyses with stringent p values retained  <dig> of these genes as highly significantly regulated .
among these genes,  <dig> were previously identified as up-regulated by al .
we randomly selected  <dig> of the stress candidate genes for qrt-pcr validation .
two of them  are strongly over-expressed in response to al, one  was previously shown to be up-regulated by al and the fourth one  was represented by  <dig> different genes .
gene number/
a: the response type is based on previous publications.
b: according to affymetrix gene chip® wheat genome array description.
the probesetids are presented in decreasing order of differential expression .
the last  <dig> entries are down-regulated genes.
for more details on gene expression level, see additional file  <dig>  genes subsequently tested by qrt pcr are in bold.
c: annotations were made based on affymetrix gene annotation complemented with blast results using the public representative id provided with the affymetrix gene chip® wheat genome array.
d: the fold change represents the mean ratio of gene expression in all cultivars exposed to al /the controls not treated with al.
as found for stress-associated genes, microarray analyses allowed the identification of more than  <dig> genes potentially associated with al tolerance when selection was performed using only one pair of wheat lines  but this number was reduced to  <dig> common genes when both pairs were considered.
this result suggests that a large number of genes are associated with cultivar specific responses and only a limited number of them are associated with al tolerance.
this list was reduced to  <dig> genes using anova analyses with stringent p values .
these genes were subdivided into constitutively or differentially expressed genes based on the difference in expression between al-treated and non-treated control plants.
table 2a lists the  <dig> constitutively expressed genes classified in decreasing order of expression .
the most strongly expressed gene is the previously identified al malate transporter almt- <dig>  <cit> .
we selected two of these genes for qrt-pcr validation .
table 2b lists the  <dig> genes that are regulated by al and differentially expressed between the two tolerant and the two sensitive cultivars.
four of these genes were selected for validation by qrt-pcr .
f-box associated domain ;
a: the gene are numbered consecutively to table  <dig> to simplify presentation in the text and figure  <dig>  the response type is based on previous publications.
b: according to affymetrix gene chip® wheat genome array description.
the probesetids are presented in decreasing order of differential expression .
the last  <dig> entries in table 2a are more expressed in sensitive cultivars.
the last entry in table 2b is a down-regulated gene in the tolerant cultivars.
for more details on gene expression level, see additional file  <dig>  genes subsequently tested by qrt pcr are in bold.
c: annotations were made based on affymetrix gene annotation complemented with blast results using the public representative id provided with the affymetrix gene chip® wheat genome array.
d: the fold change represents the mean ratio of gene expression in the two tolerant cultivar/the two sensitive cultivar .
quantitative rt-pcr validation of microarray data
we selected ten genes for validation of the microarray data by qrt-pcr .
four different al concentrations  were used to determine the effect on gene expression.
four new replicates of each al exposure conditions using all wheat lines were prepared.
the values indicated in each histogram column represent the expression levels relative to the first column  .
the level of expression of the tested genes under control conditions is generally similar in the tolerant and sensitive cultivars.
genes were classified as associated with al tolerance when there was a significant difference in expression in both tolerant cultivars  compared to their respective sensitive counterparts .
these conditions allow the detection of either 1) al-regulated genes that may be expressed differentially in tolerant and sensitive lines, or 2) genes not regulated by al  but showing constitutive difference in expression between tolerant and sensitive lines.
once classified as associated with stress or tolerance, gene regulation by al was confirmed by comparing the al-treated and non-treated controls.
qrt-pcr, analyses confirmed that three  of the four stress candidate genes are stress-associated while the 4th gene  is more closely associated with al tolerance.
among the six selected tolerance candidates genes, four were confirmed as associated with al tolerance  while the last two genes , were not clearly linked to either stress or tolerance.
the correlation between gene expression data obtained by microarray and qrt-pcr is presented in fig.
<dig>  al-regulated genes are shown in fig.
2a for the cultivars atlas <dig> and bounty.
the fold change in gene expression  obtained using microarray experiments compared to the fold change obtained using qrt-pcr gives a correlation coefficient of  <dig> .
a similar analysis was performed to compare the change in gene expression between the tolerant and sensitive cultivars  calculated from the microarray or qrt-pcr data .
in this case, a correlation coefficient of  <dig>  was obtained between the two experimental methods.
these correlation coefficients are very good considering that microarray data are semi-quantitative and subject to error for multigene families where different transcripts could hybridize to similar probes on the array.
qrt-pcr data are more specific since the amplification of single transcripts are confirmed by melting curves and gel analyses.
the ta.
<dig> .s1_x_at was initially selected as a stress-associated gene by microarray profiling  while it was reclassified as associated with al tolerance using qrt-pcr .
this may be related to the presence of multiple oxalate oxidase genes in wheat.
based on these results, we conclude that most of the genes identified in table  <dig> and table  <dig> are associated with al stress and tolerance.
however, real-time pcr analyses will be required to validate the other genes that were identified using microarrays.
discussion
several al-associated qtls with major and minor contributions were reported, indicating that al tolerance is a multigenic trait.
a large scale transcriptome analysis was initiated to identify some gene components of this multigenic trait.
the genes that are differentially expressed between tolerant and sensitive cultivars are of particular interest since the tolerant cultivars are exposed to al concentrations that are ten times higher, yet they are still growing at the same rate as the sensitive cultivars.
this design allows the distinction of genes that are regulated in response to stress from those that are associated with al tolerance.
transcriptome analyses allowed the identification of several genes with known function that could improve our understanding of the biochemical processes and pathways involved in al stress and tolerance.
as indicated in the result section,  <dig> of the  <dig> genes associated with the al stress response were identified in previous studies indicating that the genes associated with al stress are appropriately identified using this approach.
signal transduction
we have previously shown that al inhibits a redox reaction associated with root growth and al tolerance  <cit> .
the perturbation of this redox reaction by al could lead to the accumulation of different reactive oxygen species which can stimulate a redox signalling pathway and increase the expression of antioxidant enzymes  <cit> .
the accumulation of reactive oxygen species during al exposure was observed in maize, soybean and wheat  <cit> .
al was also shown to affect mitochondrial functions leading to ros production  <cit> .
table  <dig> shows that the genes encoding wci- <dig> , was- <dig>  and the ef-hand ca2+-binding protein  are up-regulated during al exposure.
in arabidopsis, the wci- <dig> and was- <dig> genes were shown to be induced via a map kinase pathway associated with the pathogen response  <cit> .
in wheat seedlings, the wci- <dig> gene was shown to be induced by pathogen infection  <cit>  and mediated by the ef-hand ca2+-binding protein ccd <dig>  <cit> .
these results suggest that a map kinase pathway is stimulated by al in wheat roots as observed in cell suspension cultures of coffea arabica l.  <cit> .
three different protein kinase-like genes  are up-regulated during al exposure suggesting that different signal transduction pathways may be activated.
we identified two other potential signalling genes associated with al tolerance.
the first one encodes a putative c2h <dig> zinc finger transcription factor  that has a  <dig> aa region sharing 50% identity with the stop <dig> c2h <dig> zinc finger protein required for al3+ induction of almt <dig> in arabidopsis  <cit> .
the other gene encodes an f-box containing protein , which is member of a family known for its involvement in the controlled degradation of target proteins.
an arabidopsis f-box protein homologous to a bacterial redox sensor was proposed to be involved in the cell wall receptor-like associated kinase  signalling pathway  <cit> .
since wak <dig> was shown to be transiently overexpressed during al exposure and associated with al tolerance  <cit> , the reduced expression of an f-box protein may stimulate this pathway by allowing a stronger expression of wak <dig> in tolerant cultivars.
the tir <dig> gene is another f-box protein in arabidopsis that interacts with different proteins in the scftir <dig> complex to mediate responses to auxin  <cit> .
these two genes  require further characterization and functional analysis to understand their roles in the response to al.
management of al-associated stresses and maintenance of energy supply
the identification of stress-regulated genes provide new tools to reduce al stress, as shown by the ectopic overexpression of some al stress-regulated genes  <cit> .
among the  <dig> candidate genes regulated by al stress , several could play a role in alleviating phosphate deficiency and provide energy to fight oxidative stress.
nutrient deficiency, and especially phosphate, occurs in the presence of al due to the precipitation of al-phosphate  <cit> .
under phosphate deficiency, several pathways are hindered due to the reduced availability of atp and related nucleoside phosphates.
these imbalances lead to the induction of alternative pathways of glycolysis to maintain energy and carbon skeletons for key metabolic processes  <cit> .
the fructose- <dig> -bisphosphatase  associated with al tolerance  is normally inhibited by phosphate but is stimulated under low phosphate availability .
this activity produces fructose-6-p and liberates pi.
the glycolytic alternative enzyme pyrophosphate phosphofructokinase is stimulated by low phosphate and can resynthesise fructose- <dig> -bisphosphate and liberate more pi.
this apparently futile cycle is useful to conserve atp and recycle pi that is readily available in the pyrophosphate molecule  <cit> .
furthermore, since sucrose is the most abundant photosynthate available in the phloem sap, fructose  is readily available and will enter the glycolytic pathway at the level of fructose-6-p thereby maintaining a stable supply of this metabolite.
the high availability of fructose-6-p  can provide sufficient metabolites to support both the pentose phosphate pathway and organic acid synthesis .
al-tolerant rye was found to maintain a higher content of glucose-6-phosphate than al-sensitive wheat in response to al exposure  <cit> .
this metabolite is an important precursor of the pentose phosphate pathway  as the first enzyme) and is an important source of nadph production.
the importance of energy metabolism in modulating al tolerance is exemplified in pseudomonas fluorescens where g6pdh was shown to play a pivotal role in enhancing nadph production and protecting against ros toxicity  <cit> .
the up-regulation of pyruvate dehydrogenase  and alternative oxidase  supports the involvement of an alternative pathway to maintain energy production   <cit> .
members of the alternative oxidase gene family were found to be highly responsive to oxidative stress and to reduce mitochondrial ros production while maintaining nadh supply  <cit> .
in al tolerant plants, a higher nadh supply  would provide reducing power to regenerate ascorbate and glutathione to fight oxidative stress .
exudation of organic acids has received much attention as an al tolerance  mechanism.
the secretion of malate or other organic acids  can chelate al in the rhizosphere and has been associated with al tolerance in several species  <cit> .
enzymes involved in malate synthesis  were shown to be stimulated during al stress  <cit> .
these enzymes are also stimulated during pi deficiency where malate is known to mobilize external pi  <cit> .
these two actions are complementary since the chelation of al will also free pi from precipitated al-phosphate in the rhizosphere.
some of the genes identified in this study may complement the almt <dig> malate transporter to improve al exclusion.
an interesting possibility is that organic acids, such as malate or oxalate, could form intracellular al-bound complexes that are transported outside the cell .
it is possible that the almt <dig> malate transporter  associated with al tolerance can transport free malate but that it would require a co-factor to transport the larger al-bound malate complex.
oxalate is another organic acid known to be a strong al chelator associated with al tolerance in taro  <cit> .
this organic acid is another potential molecule that could form an al complex transported out of the cell by an anion transporter such as almt <dig> or the abc transporter  that is up-regulated during al exposure.
several proteins encoded by genes that are up-regulated during al exposure have a high content of cysteine .
the high amount of thiol groups in these proteins may help protect enzymes containing thiol sensitive sites by providing new targets of oxidation.
the presence of  <dig> cysteine residues in the small protein  encoded by gene #  <dig> may explain why this gene is associated with al tolerance .
the up-regulation of four different gsts , containing cysteine residues in their active sites, indicate that these enzyme may be inactivated by oxidative stress.
the synthesis of new enzyme molecules during al exposure may partially compensate for gst inactivation.
on the other hand, two of the four gsts are associated with al tolerance .
these gsts may help to reduce oxidative stress as suggested for parb, a tobacco gst isoform  <cit> .
maintenance of root growth
the ability to maintain root elongation under al stress may require complementary mechanisms such as al sequestering  and increased expression of genes involved in detoxification or root growth.
we identified several genes that could play a role in maintaining root growth.
different enzymes such as sugar hydrolases and transferases may participate in cell wall remodelling/loosening in the elongation zone .
during the cell wall loosening process, ascorbate provides an electron to reduce cu2+ to cu+   <cit> , which participates in the fenton reaction with h2o <dig> to ultimately produce •oh radicals.
the non-enzymatic cleavage of cell wall polysaccharides thus requires the presence of three basic molecules: ascorbate, cu+ <dig> and h2o <dig>   <cit> .
the enzymatic reactions required to maintain/regenerate ascorbate and provide h2o <dig> are essential components to maintain root growth and are likely associated with al tolerance.
the redox activity previously shown to be associated with al tolerance  <cit>  may represent the transfer of electrons required to regenerate the apoplasmic ascorbate.
the great avidity of al3+ for electrons  could prevent the regeneration of apoplasmic ascorbate and inhibit root growth.
oxalate oxidase may help maintain the production of h2o <dig> for the fenton reaction .
however, h2o <dig> could become a substrate for cell wall peroxidases which are not inhibited when ascorbate levels are reduced  <cit> .
the inhibition of peroxidase activity was associated with al tolerance  <cit>  and may help maintain h2o <dig> levels needed for the non-enzymatic wall loosening process.
in our study, three peroxidases  are up-regulated and could be related to the reduced growth rate under our experimental conditions.
transcripts encoding blue copper binding proteins   is possibly up-regulated to stimulate the cu2+-dependent fenton reaction needed for cell wall loosening.
when apoplasmic ascorbate loses an electron, it is transformed into monodehydroascorbate/dehydroascorbate  and new electrons are needed to regenerate ascorbate and avoid degradation into oxalate .
the up-regulation of an ascorbate dependent oxidoreductase  could be associated with the regeneration of apoplasmic ascorbate .
impaired ascorbate metabolism was proposed to be involved in the reduction of root growth in squash roots exposed to al  <cit>  while a higher level of ascorbate and gluthathione was shown to be associated with al tolerance in tobacco  <cit> .
furthermore, feeding with d-glucose or l-galactono-γ-lactone to enhance ascorbate levels was able to improve al tolerance in rice  <cit> .
these results indicate that maintaining a high ascorbate level is an essential aspect of al tolerance.
gene #  <dig> annotated as an arabinonolactone oxidase is homologous to galactonolactone oxidase involved in ascorbate synthesis .
this last gene participates in the regeneration of cytoplasmic ascorbate while providing additional nadh .
the arabinonolactone oxidase is known to synthesize erythroascorbate in yeast.
interestingly, the overexpression of the yeast arabinonolactone oxidase enzyme in rice improves al tolerance  <cit> .
CONCLUSIONS
genome wide expression profiling and qrt-pcr using different wheat cultivars subjected to controlled stress treatments allowed the identification of several new genes associated with al stress and tolerance.
several genes associated with al tolerance could play an important role in maintaining the energy balance and al exclusion.
the maintenance of ascorbate homeostasis is proposed to be a key element to sustain elongation growth.
the availability of genes associated with al tolerance provides new tools for qtl analyses and for breeding programs aimed at improving al tolerance of cultivated crops.

 aluminum is considered as the most limiting factor for plant productivity in acidic soils. it is estimated that over 50% of the world's potential arable land surface is composed of acid soils mostly distributed in developing countries. al tolerance is second to drought tolerance for its importance as agronomic trait for worldwide crop production. the root apex is considered the first target of al toxicity and the reduction in root biomass leads to poor uptake of water and nutrients. at the cellular level, al toxicity results from a broad spectrum of deleterious effects caused by the al3+ ion, which is considered the most toxic species of al for both plant and animal cells under low ph conditions. it was proposed that the toxic effect of al can be reduced by chelating al in the rhizosphere with organic anions. exudation of a variety of organic anions such as malate, citrate, or oxalate upon exposure to al have been reported and al tolerance of several species can be enhanced by increasing organic acid biosynthesis. overexpression of the al-inducible malate transporter improves al tolerance in barley while overexpression of the sbmate protein, a putative citrate transporter, improves al tolerance in arabidopsis and wheat. the release of phosphate was also associated with al tolerance, indicating that multiple mechanisms are involved in wheat. in arabidopsis, the major al tolerance quantitative trait loci was proposed to be proximal to the atalmt malate transporter and to involve additional genes. fine mapping indicated that al tolerance is more closely associated with a chromosome location that is distal to atalmt by to kbp indicating that other gene products may regulate or complement its activity. this regulation may be controlled at the transcriptional, translational, post-translational and enzymatic levels. the association of organic acid release and al tolerance is not universal as several al-sensitive plants were found to secrete large amounts of organic acids. these results suggest that organic acid excretion, per se, is not a tolerance mechanism, but a consequence of biochemical reactions required for al tolerance. in arabidopsis, there was no induction of atalmt by al3+ in the stop arabidopsis mutant suggesting that the stop zinc finger protein is required to activate almt transcription. the als gene encodes an abc transporter-like protein that is required for al tolerance/resistance and may function to redistribute accumulated al away from sensitive tissues to protect growing roots from al toxicity. several genes up-regulated during al exposure have been identified using various molecular approaches. overexpression of some of these genes in transgenic plants has resulted in modest improvement of al tolerance, suggesting that they alleviate part of the toxicity caused by al. in rice, al tolerance appears to be a complex multigenic trait that involves all twelve chromosomes. however, fewer loci were reported to be involved in other grasses. two major loci on chromosome 4dl and 6a, and additional loci with additive effects are involved in al tolerance of the wheat cultivar atlas. the availability of near-isogenic lines derived from the cultivar atlas having similar qtls for al tolerance provides a useful tool to identify genes associated with al stress and tolerance. in this study, several genes associated with al stress and tolerance were identified using transcriptome analyses. the putative functions of identified genes in several biochemical pathways are discussed in relation to stress responses and the maintenance of root growth.  to identify genes associated with al stress and tolerance in wheat, a large scale expression profiling study was initiated using the affymetrix genechip® wheat genome array which allows the screening of transcripts. to better discriminate between genes that are associated with al stress and tolerance, four different wheat lines were analyzed. in addition to the well studied tolerant wheat atlas and the sensitive wheat bounty used in our previous study, we used two near isogenic lines derived from a cross between atlas and the sensitive cultivar century. the sensitive nil ok91g has a high degree of genetic similarity with the tolerant nil ok91g. the major difference between tolerant and sensitive plants is the ability to maintain growth under high al concentrations. the tolerant cultivars used in this study are able to grow for several days = 50%) in the presence of μm al. the sensitive cultivars are unable to grow at this high al concentration and an rgi of 50% is obtained in the presence of μm al. we have previously shown that the stress-associated genes are expressed at comparable levels in the sensitive and tolerant cultivars after hours when they are exposed to al concentrations resulting in a similar rgi. the microarray experiments were thus designed to identify genes that are differentially expressed after hours of al exposure at concentrations resulting in 50% rgi for all lines used. microarray analyses the number of al-regulated genes is over when cultivars are analyzed separately. however, only genes are differentially expressed in all four cultivars. anova analyses with stringent p values retained of these genes as highly significantly regulated. among these genes, were previously identified as up-regulated by al. we randomly selected of the stress candidate genes for qrt-pcr validation. two of them are strongly over-expressed in response to al, one was previously shown to be up-regulated by al and the fourth one was represented by different genes. gene number/ a: the response type is based on previous publications. b: according to affymetrix gene chip® wheat genome array description. the probesetids are presented in decreasing order of differential expression. the last entries are down-regulated genes. for more details on gene expression level, see additional file genes subsequently tested by qrt pcr are in bold. c: annotations were made based on affymetrix gene annotation complemented with blast results using the public representative id provided with the affymetrix gene chip® wheat genome array. d: the fold change represents the mean ratio of gene expression in all cultivars exposed to al /the controls not treated with al. as found for stress-associated genes, microarray analyses allowed the identification of more than genes potentially associated with al tolerance when selection was performed using only one pair of wheat lines but this number was reduced to common genes when both pairs were considered. this result suggests that a large number of genes are associated with cultivar specific responses and only a limited number of them are associated with al tolerance. this list was reduced to genes using anova analyses with stringent p values. these genes were subdivided into constitutively or differentially expressed genes based on the difference in expression between al-treated and non-treated control plants. table 2a lists the constitutively expressed genes classified in decreasing order of expression. the most strongly expressed gene is the previously identified al malate transporter almt-. we selected two of these genes for qrt-pcr validation. table 2b lists the genes that are regulated by al and differentially expressed between the two tolerant and the two sensitive cultivars. four of these genes were selected for validation by qrt-pcr. f-box associated domain; a: the gene are numbered consecutively to table to simplify presentation in the text and figure the response type is based on previous publications. b: according to affymetrix gene chip® wheat genome array description. the probesetids are presented in decreasing order of differential expression. the last entries in table 2a are more expressed in sensitive cultivars. the last entry in table 2b is a down-regulated gene in the tolerant cultivars. for more details on gene expression level, see additional file genes subsequently tested by qrt pcr are in bold. c: annotations were made based on affymetrix gene annotation complemented with blast results using the public representative id provided with the affymetrix gene chip® wheat genome array. d: the fold change represents the mean ratio of gene expression in the two tolerant cultivar/the two sensitive cultivar. quantitative rt-pcr validation of microarray data we selected ten genes for validation of the microarray data by qrt-pcr. four different al concentrations were used to determine the effect on gene expression. four new replicates of each al exposure conditions using all wheat lines were prepared. the values indicated in each histogram column represent the expression levels relative to the first column. the level of expression of the tested genes under control conditions is generally similar in the tolerant and sensitive cultivars. genes were classified as associated with al tolerance when there was a significant difference in expression in both tolerant cultivars compared to their respective sensitive counterparts. these conditions allow the detection of either 1) al-regulated genes that may be expressed differentially in tolerant and sensitive lines, or 2) genes not regulated by al but showing constitutive difference in expression between tolerant and sensitive lines. once classified as associated with stress or tolerance, gene regulation by al was confirmed by comparing the al-treated and non-treated controls. qrt-pcr, analyses confirmed that three of the four stress candidate genes are stress-associated while the 4th gene is more closely associated with al tolerance. among the six selected tolerance candidates genes, four were confirmed as associated with al tolerance while the last two genes, were not clearly linked to either stress or tolerance. the correlation between gene expression data obtained by microarray and qrt-pcr is presented in fig. al-regulated genes are shown in fig. 2a for the cultivars atlas and bounty. the fold change in gene expression obtained using microarray experiments compared to the fold change obtained using qrt-pcr gives a correlation coefficient of. a similar analysis was performed to compare the change in gene expression between the tolerant and sensitive cultivars calculated from the microarray or qrt-pcr data. in this case, a correlation coefficient of was obtained between the two experimental methods. these correlation coefficients are very good considering that microarray data are semi-quantitative and subject to error for multigene families where different transcripts could hybridize to similar probes on the array. qrt-pcr data are more specific since the amplification of single transcripts are confirmed by melting curves and gel analyses. the ta..s1_x_at was initially selected as a stress-associated gene by microarray profiling while it was reclassified as associated with al tolerance using qrt-pcr. this may be related to the presence of multiple oxalate oxidase genes in wheat. based on these results, we conclude that most of the genes identified in table and table are associated with al stress and tolerance. however, real-time pcr analyses will be required to validate the other genes that were identified using microarrays. discussion several al-associated qtls with major and minor contributions were reported, indicating that al tolerance is a multigenic trait. a large scale transcriptome analysis was initiated to identify some gene components of this multigenic trait. the genes that are differentially expressed between tolerant and sensitive cultivars are of particular interest since the tolerant cultivars are exposed to al concentrations that are ten times higher, yet they are still growing at the same rate as the sensitive cultivars. this design allows the distinction of genes that are regulated in response to stress from those that are associated with al tolerance. transcriptome analyses allowed the identification of several genes with known function that could improve our understanding of the biochemical processes and pathways involved in al stress and tolerance. as indicated in the result section, of the genes associated with the al stress response were identified in previous studies indicating that the genes associated with al stress are appropriately identified using this approach. signal transduction we have previously shown that al inhibits a redox reaction associated with root growth and al tolerance. the perturbation of this redox reaction by al could lead to the accumulation of different reactive oxygen species which can stimulate a redox signalling pathway and increase the expression of antioxidant enzymes. the accumulation of reactive oxygen species during al exposure was observed in maize, soybean and wheat. al was also shown to affect mitochondrial functions leading to ros production. table shows that the genes encoding wci-, was- and the ef-hand ca2+-binding protein are up-regulated during al exposure. in arabidopsis, the wci- and was- genes were shown to be induced via a map kinase pathway associated with the pathogen response. in wheat seedlings, the wci- gene was shown to be induced by pathogen infection and mediated by the ef-hand ca2+-binding protein ccd. these results suggest that a map kinase pathway is stimulated by al in wheat roots as observed in cell suspension cultures of coffea arabica l.. three different protein kinase-like genes are up-regulated during al exposure suggesting that different signal transduction pathways may be activated. we identified two other potential signalling genes associated with al tolerance. the first one encodes a putative c2h zinc finger transcription factor that has a aa region sharing 50% identity with the stop c2h zinc finger protein required for al3+ induction of almt in arabidopsis. the other gene encodes an f-box containing protein, which is member of a family known for its involvement in the controlled degradation of target proteins. an arabidopsis f-box protein homologous to a bacterial redox sensor was proposed to be involved in the cell wall receptor-like associated kinase signalling pathway. since wak was shown to be transiently overexpressed during al exposure and associated with al tolerance, the reduced expression of an f-box protein may stimulate this pathway by allowing a stronger expression of wak in tolerant cultivars. the tir gene is another f-box protein in arabidopsis that interacts with different proteins in the scftir complex to mediate responses to auxin. these two genes require further characterization and functional analysis to understand their roles in the response to al. management of al-associated stresses and maintenance of energy supply the identification of stress-regulated genes provide new tools to reduce al stress, as shown by the ectopic overexpression of some al stress-regulated genes. among the candidate genes regulated by al stress, several could play a role in alleviating phosphate deficiency and provide energy to fight oxidative stress. nutrient deficiency, and especially phosphate, occurs in the presence of al due to the precipitation of al-phosphate. under phosphate deficiency, several pathways are hindered due to the reduced availability of atp and related nucleoside phosphates. these imbalances lead to the induction of alternative pathways of glycolysis to maintain energy and carbon skeletons for key metabolic processes. the fructose- -bisphosphatase associated with al tolerance is normally inhibited by phosphate but is stimulated under low phosphate availability. this activity produces fructose-6-p and liberates pi. the glycolytic alternative enzyme pyrophosphate phosphofructokinase is stimulated by low phosphate and can resynthesise fructose- -bisphosphate and liberate more pi. this apparently futile cycle is useful to conserve atp and recycle pi that is readily available in the pyrophosphate molecule. furthermore, since sucrose is the most abundant photosynthate available in the phloem sap, fructose is readily available and will enter the glycolytic pathway at the level of fructose-6-p thereby maintaining a stable supply of this metabolite. the high availability of fructose-6-p can provide sufficient metabolites to support both the pentose phosphate pathway and organic acid synthesis. al-tolerant rye was found to maintain a higher content of glucose-6-phosphate than al-sensitive wheat in response to al exposure. this metabolite is an important precursor of the pentose phosphate pathway as the first enzyme) and is an important source of nadph production. the importance of energy metabolism in modulating al tolerance is exemplified in pseudomonas fluorescens where g6pdh was shown to play a pivotal role in enhancing nadph production and protecting against ros toxicity. the up-regulation of pyruvate dehydrogenase and alternative oxidase supports the involvement of an alternative pathway to maintain energy production. members of the alternative oxidase gene family were found to be highly responsive to oxidative stress and to reduce mitochondrial ros production while maintaining nadh supply. in al tolerant plants, a higher nadh supply would provide reducing power to regenerate ascorbate and glutathione to fight oxidative stress. exudation of organic acids has received much attention as an al tolerance mechanism. the secretion of malate or other organic acids can chelate al in the rhizosphere and has been associated with al tolerance in several species. enzymes involved in malate synthesis were shown to be stimulated during al stress. these enzymes are also stimulated during pi deficiency where malate is known to mobilize external pi. these two actions are complementary since the chelation of al will also free pi from precipitated al-phosphate in the rhizosphere. some of the genes identified in this study may complement the almt malate transporter to improve al exclusion. an interesting possibility is that organic acids, such as malate or oxalate, could form intracellular al-bound complexes that are transported outside the cell. it is possible that the almt malate transporter associated with al tolerance can transport free malate but that it would require a co-factor to transport the larger al-bound malate complex. oxalate is another organic acid known to be a strong al chelator associated with al tolerance in taro. this organic acid is another potential molecule that could form an al complex transported out of the cell by an anion transporter such as almt or the abc transporter that is up-regulated during al exposure. several proteins encoded by genes that are up-regulated during al exposure have a high content of cysteine. the high amount of thiol groups in these proteins may help protect enzymes containing thiol sensitive sites by providing new targets of oxidation. the presence of cysteine residues in the small protein encoded by gene # may explain why this gene is associated with al tolerance. the up-regulation of four different gsts, containing cysteine residues in their active sites, indicate that these enzyme may be inactivated by oxidative stress. the synthesis of new enzyme molecules during al exposure may partially compensate for gst inactivation. on the other hand, two of the four gsts are associated with al tolerance. these gsts may help to reduce oxidative stress as suggested for parb, a tobacco gst isoform. maintenance of root growth the ability to maintain root elongation under al stress may require complementary mechanisms such as al sequestering and increased expression of genes involved in detoxification or root growth. we identified several genes that could play a role in maintaining root growth. different enzymes such as sugar hydrolases and transferases may participate in cell wall remodelling/loosening in the elongation zone. during the cell wall loosening process, ascorbate provides an electron to reduce cu2+ to cu+, which participates in the fenton reaction with h2o to ultimately produce •oh radicals. the non-enzymatic cleavage of cell wall polysaccharides thus requires the presence of three basic molecules: ascorbate, cu+ and h2o. the enzymatic reactions required to maintain/regenerate ascorbate and provide h2o are essential components to maintain root growth and are likely associated with al tolerance. the redox activity previously shown to be associated with al tolerance may represent the transfer of electrons required to regenerate the apoplasmic ascorbate. the great avidity of al3+ for electrons could prevent the regeneration of apoplasmic ascorbate and inhibit root growth. oxalate oxidase may help maintain the production of h2o for the fenton reaction. however, h2o could become a substrate for cell wall peroxidases which are not inhibited when ascorbate levels are reduced. the inhibition of peroxidase activity was associated with al tolerance and may help maintain h2o levels needed for the non-enzymatic wall loosening process. in our study, three peroxidases are up-regulated and could be related to the reduced growth rate under our experimental conditions. transcripts encoding blue copper binding proteins is possibly up-regulated to stimulate the cu2+-dependent fenton reaction needed for cell wall loosening. when apoplasmic ascorbate loses an electron, it is transformed into monodehydroascorbate/dehydroascorbate and new electrons are needed to regenerate ascorbate and avoid degradation into oxalate. the up-regulation of an ascorbate dependent oxidoreductase could be associated with the regeneration of apoplasmic ascorbate. impaired ascorbate metabolism was proposed to be involved in the reduction of root growth in squash roots exposed to al while a higher level of ascorbate and gluthathione was shown to be associated with al tolerance in tobacco. furthermore, feeding with d-glucose or l-galactono-γ-lactone to enhance ascorbate levels was able to improve al tolerance in rice. these results indicate that maintaining a high ascorbate level is an essential aspect of al tolerance. gene # annotated as an arabinonolactone oxidase is homologous to galactonolactone oxidase involved in ascorbate synthesis. this last gene participates in the regeneration of cytoplasmic ascorbate while providing additional nadh. the arabinonolactone oxidase is known to synthesize erythroascorbate in yeast. interestingly, the overexpression of the yeast arabinonolactone oxidase enzyme in rice improves al tolerance.  genome wide expression profiling and qrt-pcr using different wheat cultivars subjected to controlled stress treatments allowed the identification of several new genes associated with al stress and tolerance. several genes associated with al tolerance could play an important role in maintaining the energy balance and al exclusion. the maintenance of ascorbate homeostasis is proposed to be a key element to sustain elongation growth. the availability of genes associated with al tolerance provides new tools for qtl analyses and for breeding programs aimed at improving al tolerance of cultivated crops. 
to identify genes associated with al stress and tolerance, transcriptome analyses of four different wheat lines that differ in their response to al were performed. the stress-associated genes include important enzymes such as pyruvate dehydrogenase, alternative oxidase, and galactonolactone oxidase, abc transporter and ascorbate oxido-reducatase. aluminum is considered the most limiting factor for plant productivity in acidic soils, which cover large areas of the world's potential arable lands. in this survey, we identified stress- and tolerance-associated genes that may be involved in the detoxification of al and reactive oxygen species. the inhibition of root growth is recognized as the primary effect of al toxicity. alternative pathways could help maintain the supply of important metabolites needed for al tolerance and root growth.
 aluminum is considered the most limiting factor for plant productivity in acidic soils, which cover large areas of the world's potential arable lands. the inhibition of root growth is recognized as the primary effect of al toxicity. to identify genes associated with al stress and tolerance, transcriptome analyses of four different wheat lines that differ in their response to al were performed.  microarray expression profiling revealed that candidate genes are associated with al stress and are associated with tolerance. the stress-associated genes include important enzymes such as pyruvate dehydrogenase, alternative oxidase, and galactonolactone oxidase, abc transporter and ascorbate oxido-reducatase. the al tolerance-associated genes include the almt- malate transporter, glutathione s-transferase, germin/oxalate oxidase, fructose -bisphosphatase, cysteine-rich proteins, cytochrome p monooxygenase, cellulose synthase, zinc finger transcription factor, disease resistance response protein and f-box containing domain protein.  in this survey, we identified stress- and tolerance-associated genes that may be involved in the detoxification of al and reactive oxygen species. alternative pathways could help maintain the supply of important metabolites needed for al tolerance and root growth. the al tolerance-associated genes may be key factors that regulate these pathways. 