BACKGROUND
arsenic  is a toxic metalloid found ubiquitously in the environment  <cit>  and is classified as a human carcinogen  <cit> .
currently, the us environmental protection agency declares arsenic as the highest priority hazardous substance found at contaminated sites in the united states .
naturally high levels of arsenic in drinking water have caused major human health problems in the united states, china, argentina, taiwan, and most notably in bangladesh and india where tens of millions of people have been affected  <cit> .
arsenic is highly toxic at low concentrations, therefore drinking water safety standards were lowered from  <dig> to  <dig> μg/l in the u.s.  <cit> .
plants typically encounter arsenic in the anionic forms of arsenate  and arsenite , both of which have different cytotoxic effects  <cit> .
as  reacts with the sulfhydryl groups of enzymes and proteins, thereby inhibiting cellular function and resulting in death  <cit> .
alternatively, as  is an analog of the macronutrient phosphate, so it competes with phosphate for uptake in the roots, as well as in the cytoplasm where it might disrupt metabolism by replacing phosphate in atp to form unstable adp-as  <cit> .
once taken up by the roots, arsenate is reduced to a more highly toxic species, arsenite, which is subsequently detoxified via soluble thiols such as glutathione and/or phytochelatins  and transported for vacuolar sequestration  <cit> .
pcs are low molecular weight thiolate peptides of the general structure n-gly  and are synthesized from glutathione by the constitutively present phytochelatin synthase  <cit> .
both arsenate and arsenite efficiently induce the production of pcs in plants  <cit> , however it is believed since arsenate has no affinity for the sulfhydryl groups in pcs, as  is reduced in the cytoplasm, resulting in as-pc complexes  <cit> .
glutathione and pcs have been reported to form as-tris-thiolate complexes in brassica juncea upon exposure to as  <cit> .
therefore, pc synthesis causes a depletion of cellular glutathione, resulting in a decreased capacity to quench reactive oxygen species   <cit> .
phytoremediation has emerged as an alternative technology for removing toxic metals from contaminated soils and groundwater.
the potential for phytoremediation to be an effective means of removing arsenic from contaminated sites has been demonstrated in hyperaccumulators of the pteris genus  <cit>  and may be enhanced by a better understanding of plant transcriptional responses to arsenic.
many plant studies have demonstrated the direct involvement of thiol-containing molecules  in arsenic detoxification, however more robust approaches  should help clarify how arsenic affects plant physiological processes on a global scale.
the goals of our study were to test our hypothesis that many genes would be differentially expressed in response to arsenate stress and to identify genes as putative players in as  detoxification using arabidopsis as a model.
in this paper, we investigate the transcriptional responses to as  in arabidopsis thaliana using oligonucleotide microarrays.
our results demonstrate that as  stress strongly induces cu/zn superoxide dismutase  activity, but represses the production of fe sods.
our microarray data also suggest the involvement of other antioxidant genes, various transcription factors, tonoplast proteins, and proteins associated with cell wall growth.
of particular interest, we report that as  stress represses numerous genes induced by pi starvation.
we discuss the physiological implications of these findings, and suggest new avenues for research of arsenic metabolism in plants.
RESULTS
root growth under as  stress
arsenate exposure resulted in reduced arabidopsis root growth and branching .
exposure of  <dig> μm as  resulted in significantly reduced arabidopsis root growth .
in addition to known relevant physiological data, the exposure study was used to determine suitable as  exposure for the microarray study.
we noted no seed germination effects with regard to arsenate treatments, notably at  <dig> μm, the arsenate concentration used for transcriptomics experiments.
gene ontology for genes affected by as
forty-six genes were induced by as  treatment  as indicated by microarray analysis.
the largest functional categories affected included unknown function, hydrolase, and antioxidant activity.
other functional categories affected by as  included genes with transferase, kinase, lyase, transporter, and binding activity .
alternatively,  <dig> genes were repressed by as  , with unknown function, hydrolase, and binding activity representing the largest categories.
genes with transporter, kinase, transferase, and transcriptional regulator activity were also repressed by as  .
differentially expressed as -induced and -repressed genes are listed below  and complete lists of all genes affected by as  stress are also included as additional files .
most interestingly, it was discovered that as  stress repressed transcription of many genes involved in the phosphate starvation response, and also repressed several transcriptional factors.
several genes involved in oxidative stress were also highly modulated in response to as.
rp, rank products; fc, fold change.
only genes upregulated above  <dig> -fold and meeting a significance criteria of p <  <dig>  and fdr of 1% are shown.
rank products analysis reveals that most significantly induced genes display the lowest rp value.
rp, rank products; fc, fold change.
only genes upregulated above - <dig> -fold and meeting a significance criteria of p <  <dig>  and fdr of 1% are shown.
rank products analysis reveals that most significantly repressed genes display the lowest rp value.
superoxide dismutases
sods represented the highest ranked of both significantly induced as well as repressed genes in response to as  stress , therefore these genes presented logical primary targets for the validation of our microarray data.
results demonstrated  <dig> -fold induction of a chloroplast cu/zn sod ,  <dig> -fold induction of a cu/zn sod , as well as a  <dig> -fold induction of an sod copper chaperone .
alternatively, fe sod  transcripts were downregulated in response to arsenic stress .
these findings were confirmed with quantitative rt-pcr  .
a microarray data
b rt-pcr data  arabidopsis actin was used as a reference gene
p values for rt-pcr data were <  <dig> .
based upon our observations of transcript-level changes in sod gene expression, we performed a nondenaturing page enzyme assay of superoxide dismutase activity  <cit>  to assess whether the observed specific changes in sod transcript correlated with enzyme activity.
this method enables the distinction between the three sod isoenzymes found in arabidopsis  by using inhibitors of specific sods.
gels were preincubated with kcn, which inhibits cuzn sod, as well as h2o <dig>  which inhibits both cuzn sod and fe sod.
mnsod is resistant to both inhibitors .
plants were harvested from control plates containing no arsenate and treated plates containing  <dig> μm arsenate at seven-, ten-, and thirteen days post-germination.
irrespective of harvest date, cuznsod activity was strongly induced by arsenate treatment, whereas fesod activity was repressed, and mnsod showed no change in activity, therefore providing sufficient evidence to confirm our microarray results.
transcription of other antioxidant genes  were indicated by our microarray experiment as affected by arsenate stress, however these genes were not included in our qrt-pcr validation.
transcription factors
our microarray experiment indicated that eight different genes encoding proteins with known transcription factor activity all displayed lower expression levels in as -stressed plants .
one of these transcription factors  encodes a member of the dreb subfamily a- <dig> of the erf/ap <dig> transcription factor family .
one other ap2-domain-containing transcription factor  that encodes a member of the erf  subfamily b- <dig> of the erf/ap <dig> transcription factor family was also repressed in response to as .
two zinc finger  genes  encoded a zat <dig> and a protein similar to zat <dig>  respectively.
also exhibiting lower expression in as -treated plants were three members of the wrky family of transcription factors , wrky <dig>  wrky <dig>  and wrky <dig>  respectively as well as one gene encoding nac domain containing protein  <dig>
as  represses genes involved in phosphate starvation response
a notable transcriptional trend is that as  stress results in repression of some genes involved in the pi starvation response.
of particular interest, a p-type cyclin  that was affected by as -stress shares significant homology to the pho <dig> gene from yeast.
we performed qrt-pcr for this gene and found that its expression was actually strongly repressed at both day  <dig> and day  <dig> .
three genes that were repressed by as  in this study have also been reported to be repressed by pi starvation  <cit> .
interestingly, the three highest ranking differentially expressed genes found to be strongly induced by pi starvation   <cit> , were also repressed by as  in our study.
these genes are of particular interest on account of their unknown function.
quantitative rt-pcr confirmed that transcription of these genes was strongly repressed at both  <dig> day and  <dig> day time points.
additionally, the qrt-pcr data indicate that these genes were more repressed at day  <dig> than at day  <dig> .
these data also corroborated our microarray experiments, which reflect global expression ratios at  <dig> days post germination .
sulfate assimilation
the role that thiol groups play in arsenic detoxification has been well characterized, therefore we expected to see induction of genes involved in sulfate assimilation and metabolism in response to arsenic stress.
ferredoxin , a key redox protein found in the chloroplast was as -induced.
expression levels for another gene involved in the sulfate reduction pathway, 5'-adenylylsulfate reductase   were also elevated in response to as  stress.
this enzyme catalyzes the reduction of aps to sulfite using glutathione as an electron donor.
although not involved in sulfate assimilation, the cysteine-rich metal-binding protein, metallothionein  1a  was also induced.
arabidopsis knockout mutants that were generated for class  <dig> mts accumulated significantly less aboveground as, cd, and zn, suggesting that class  <dig> mts may play a role in metal and metalloid ion translocation  <cit> .
genes involved in cell wall assembly, architecture, and growth
a wide range of genes encoding proteins involved in cell wall activities exhibit altered expression levels in response to as  .
peroxidases, which were indicated by microarray as affected by as  stress, are known to strengthen the cell wall in response to biotic stress via formation of lignin, extension cross-links, and dityrosine bonds  <cit> .
additionally, as  affected transcription of numerous xyloglucan endotransglucosylase/hydrolases  and glycosyl hydrolase genes , with the majority of these exhibiting lower expression in the presence of as .
discussion
arsenic and oxidative stress
superoxide dismutases
increasing evidence from mammalian studies demonstrates that ros are generated in response to exposure to inorganic forms of arsenic  <cit> .
the reduction of arsenic is linked with in vivo and in vitro ros production in mammalian cells  <cit> , but little is known about the mechanisms by which arsenic-induced ros generation occurs in plants.
it is believed that the reduction of as  to as , which is well documented in plants, results in the production of ros  <cit> .
however, this increase in ros may also be the result of either depletion of glutathione or inhibition of antioxidant enzymes.
plants have evolved both nonenzymatic antioxidants , as well as antioxidant enzymes  to manage the balance of ros in the cell.
sods represent a first line of defense by converting superoxide radicals to h <dig>  whereas catalases and peroxidases remove h2o <dig>  three classes of sods have been identified according to the active site metal cofactor: fesod, mnsod, and cu/znsod.
as  and as  were both shown to induce expression of glutathione s-transferases , catalases, and sods in zea mays  <cit> .
an increase in sod activity was correlated with an increase in as  treatment in holcus lanatus  <cit> .
higher levels of sod, catalase, and ascorbate peroxidase were observed in pteris vittata, an arsenic hyperaccumulator, than in arsenic-sensitive fern species pteris ensiformis and nephrolepsis exaltata  <cit> .
these researchers concluded that arsenic-induced increases in antioxidant enzymes levels may represent a secondary defensive mechanism against oxidative stress in pteris vitatta and correspond with its arsenic accumulation and lack of toxicity symptoms.
it was shown that pteris vittata sod, catalase, and peroxidase levels rose sharply in response to low levels of as , but leveled off at as  levels >  <dig> mg kg- <dig>  which was consistent with changes in biomass in the arsenic hyperaccumulator  <cit> .
although the strong induction of sods in response to as  stress was not surprising, the dramatically lower levels of fesods were unexpected.
we suggest the involvement of an nac domain-containing transcription factor to explain the observed decrease in fesod transcription based on our microarray results .
one group recently generated transgenic plants to overexpress three different arabidopsis nac transcription factors and identified nac-dependent genes using microarrays  <cit> .
not only was at4g <dig>  expression found to be nac-dependent, but transcription of other genes we have observed to be repressed by as  stress also appear to be dependent on nac-domain containing transcription factors.
we continue this discussion more thoroughly in the following section on transcription factors.
peroxidases
peroxidases are functionally diverse and participate in two major cycles: the hydroxylic cycle where peroxidases regulate h2o <dig> levels and release ros  and the peroxidative cycle where various substrates  are oxidized or polymerized.
their involvement in a broad range of physiological processes allows peroxidase expression in all plant organs from germination to early senescence, however they are predominantly expressed in the roots  <cit> .
it is not surprising that peroxidases seem to be affected by arsenate stress , especially in consideration of the elevated sod activity, which produces h2o <dig> as a product of superoxide radical dismutation.
transcription factors
our microarray data corroborate those of tran et al.
<cit> , suggesting the involvement of a different nac domain-containing transcription factor  in expression of fesod, as well as several other genes known to exhibit nac-dependent expression.
nac proteins comprises a large gene family  of plant-specific transcription factors that have roles in wide-ranging processes such as development, defense, and abiotic stress response  <cit> .
microarray experiments were carried out on nac-overexpression arabidopsis mutants to discover genes exhibiting dependence on nac transcription factors for transcription  <cit> .
we speculate that repression of nac <dig>  in as -stressed arabidopsis may be responsible for the observed repression of fesod , ferritin  <dig>  , xth <dig> , xth <dig> , erd <dig> atp-dependent clp protease atp-binding subunit , and a branched-chain amino acid amino transferase  <dig> , as these genes were reported as exhibiting nac-dependent expression  <cit> .
as  stress represses genes induced by pi deprivation
although phosphate is undoubtedly one of the most biologically important nutrients, its availability in soils is quite low.
therefore, plants have evolved mechanisms to maximize pi accessibility/availability, such as increased root hair growth, lateral root branching, and induction of phosphate transporters and phosphatases  <cit> .
certain phosphate starvation-induced genes have evolved to release phosphate from plasma membranes by hydrolyzing phospholipids under conditions of low pi availability, as phospholipids comprise a major pi pool in planta  <cit> .
conversion from phospholipids to galactolipids is one such strategy and can result from the activity of monogalactosyldiacylglycerol  synthase or digalactosyl diacylglycerol  synthase  <cit> .
arabidopsis plants expressing mgd <dig> and mgd <dig> promoter-gus fusion constructs showed that under pi starvation, mgd3::gus was expressed in apices of serrated edges  and in the lateral root branch  <cit> .
through investigation of arabidopsis mgdg synthase gene expression under pi starvation, these authors showed that global changes in plant membranes under pi deprivation are tightly regulated by pi signaling and that signal transduction through a pi-sensing mechanism is responsible for regulating mgdg synthase gene expression  <cit> .
we report here that the expression of mgd <dig>  is lower in as -treated arabidopsis at  <dig> days and  <dig> days .
therefore, it is conceivable that our observations may either reflect a pi/as  sensing mechanism or simply the lower number of lateral roots in as -stressed plants .
senescence related gene  <dig> , a glycerophosphoryl diester phosphor-diesterase, is believed to participate in processes similar to those of the mgdg synthase genes  <cit> srg <dig> had lower transcript abundance in as -treated plants in our microarray study  , as well as, in  <dig> day and  <dig> day as -treated plants .
a type  <dig> acid phosphatase  was also repressed in our as -treated plants as indicated by microarray  and was strongly repressed in our qrt-pcr validation experiments at both  <dig> day and  <dig> day time points .
in arabidopsis, acp <dig> has been shown to be induced by h2o <dig>  but not by paraquat or salicylic acid and is thought to be involved in both phosphate mobilization and in the metabolism of reactive oxygen species  <cit> .
in contrast, acp <dig> was strongly repressed by as  despite elevated sod levels, which generate h2o <dig>  therefore, further study is required to determine the specific cause of as -mediated acp <dig> repression.
recent investigations into the genome-scale transcriptional changes to phosphate deprivation in arabidopsis have elucidated a broad range of genes involved in phosphate metabolism  <cit> .
our microarray data suggested that many genes repressed by as  stress have been reported by others  <cit>  to be induced in response to pi deprivation in arabidopsis thaliana.
because as  behaves as a phosphate analog, it is likely that this observation can be explained by a saturation effect of the phosphate analog, as , thereby misleading metabolic and regulatory perception of the toxic metalloid as an abundant supply of pi.
however, arsenate likely disrupts critical biological processes that involve reversible phosphorylation, as well as pathways for phosphate signaling, but even under arsenate stress, arabidopsis accumulates much higher concentrations of as in the root than is translocated to the shoot.
in another study, when wild-type  arabidopsis plants were grown on  <dig> μm sodium arsenate for  <dig> weeks, low concentrations of arsenic were accumulated in the shoot, whereas high concentrations of arsenic were observed in roots  <cit> .
however, when the arsenate reductase homolog  was silenced, arsenate was translocated to the shoot at concentrations that classified as hyperaccumulation  <cit> .
nevertheless, the signaling mechanisms by which plants distinguish between as  and phosphate are unknown and other mechanisms of as detoxification and storage besides the well documented phytochelatin response  <cit>  may exist.
in order to confirm the observation that as  stress represses genes involved in phosphate starvation/acquisition, we performed qrt-pcr on some of the more interesting candidates .
we are particularly interested in elucidating pathways involved in as  signaling in plants.
the p-type cyclin  that was affected by as   shares significant homology to the pho <dig> gene from yeast.
cyclins bind and activate cyclin-dependent kinases, which play key roles in cell division via phosphorylation of critical substrates, such as the retinoblastoma protein, transcription factors, nuclear laminar proteins, and histones  <cit> .
interestingly, it was demonstrated that expression of this cyclin from arabidopsis restored the phosphate signaling pathway in a pho80-deficient yeast mutant, suggesting a putative key pi signaling role  <cit> .
protein kinases play crucial roles in signal transduction pathways in all eukaryotes  <cit> .
at3g <dig>  is one of two nearly identical kinase genes in arabidopsis that encode for proteins that share high sequence homology with the mammalian 40s ribosomal protein kinases s6k <dig> and s6k <dig>  <cit> .
atpk <dig> was demonstrated to be the functional plant homolog of mammalian p70s6k when ectopic expression of this gene specifically phosphorylated ribosomal protein s <dig> derived from either plant or animal  <cit> .
atpk <dig> has recently been implicated as a crucial nodal point in a network evolved for integrating stress signals with plant growth regulation  <cit> .
lower expression levels observed for atpk <dig> in as -treated plants, which was most severe at day  <dig> , lends us to conclude that as  stress may suppress plant growth through the downregulation of this growth-regulating kinase, possibly as a result of the chemical similarity between as  and phosphate.
alternatively, the downregulation of atpk <dig> may result from the more general stress responses imposed by the toxic metalloid .
our results are in agreement with the recently proposed ideas of catarecha et al.
<cit>  who studied an arabidopsis mutant that displayed enhanced arsenic accumulation.
these authors identified a pi transporter  mutant with a decreased rate of as  uptake and increased as  accumulation.
by comparing gene expression of the mutant with wild-type plants, it was shown that in arabidopsis, as  rapidly repressed genes involved in the pi starvation response and induced the expression of other as -responsive genes  <cit> .
interestingly, the repression of pi starvation genes was shown to be specific for as , whereas the as -induced genes were also induced by as .
a model resulted that suggests arsenic acts via two separate signaling pathways  <cit> .
because of the chemical similarity of as  and pi, as  fools the pi sensor, thus initiating the repression of the pi starvation response.
although our microarray experiments did not detect differential expression of any high-affinity pi transporter, which may be due to differences in experimental approach, catarecha et al.
<cit>  illustrated the high sensitivity of the pi transporter, pht1; <dig>  to as  and suggested that plants have evolved an as  sensing system whereby as  and pi signaling pathways oppose each other to protect the plant from arsenic toxicity.
based on our results, it is conceivable that the p-type cyclin  and atpk <dig>  may be involved in as  sensing, but further study is required to confirm this finding.
our comparison of as -repressed genes that have also been shown to be induced by pi deprivation elucidate some promising candidates for future studies.
for example, we are particularly interested in genes with unknown function that are strongly induced in both roots and leaves by pi starvation .
both at1g <dig> and at1g <dig> are described as phosphoric monoester hydrolases , but to our knowledge, these have not been studied in this regard.
most recently, a study described gene networks for the arabidopsis transcriptome based on the graphical gaussian model of global-scale transcriptional studies  <cit> .
in a constructed subnetwork of genes involved in phosphate starvation, srg <dig>  at1g <dig>  at3g <dig> , at2g <dig>  and at5g <dig> were all closely linked, suggesting their critical roles in phosphate metabolism in arabidopsis  <cit> .
based on their strong induction in response to pi starvation  <cit> , it is reasonable to conceive that at1g <dig> and at1g <dig> have evolved as pi scavengers for increasing pi availability.
we have confirmed the downregulation of these genes in response to as  at both  <dig> and  <dig> day time points .
in this study, at2g <dig> transcript levels were strongly repressed at  <dig> and  <dig> day time points, whereas at5g <dig> was repressed at day  <dig> and day  <dig> .
interestingly, at2g <dig> encodes for a putative retroelement pol polyprotein that has been reported as highly expressed in salt overly sensitive  arabidopsis mutants  <cit> .
because the function of these two pi starvation-induced genes is unknown, these putative gene candidates may provide opportunities for gaining insight into as /pi dynamics in arabidopsis thaliana.
the data presented here have led to the development of new hypotheses for future research.
the potential antagonistic effects of various arsenate and pi concentrations on the expression of the aforementioned genes in arabidopsis are poorly understood.
additionally, the efficiency of arsenate reduction and subsequent detoxification via phytochelatins or glutathione is poorly understood.
under conditions of arsenate stress , perhaps the cellular concentrations of arsenate surpass those that may be efficiently reduced by glutathione or arsenate reductase, thus allowing free arsenate to interfere with biological reactions that involve phosphate.
additionally, the selected arsenate concentration to employ in this study could have resulted in free arsenite after reduction in vivo that would likely have deleterious consequences.
therefore, it is reasonable to conceive that the observed transcriptional responses, as well as the impaired phenotype seen in this study, may be reflective of either arsenate, arsenite, or both.
CONCLUSIONS
our data show that in arabidopsis, cu/zn sods are strongly induced in response to as  stress, while fe sod expression is repressed.
we also demonstrate that as  stress results in the repression of genes involved in phosphate acquisition, redistribution, and phosphorylation, which supports a recent study  <cit>  that suggests as  and pi signaling pathways act in opposition to protect plant health.
although this study identifies some interesting targets for exploring as  metabolism, further studies using arabidopsis mutants with altered expression of these genes are necessary to elucidate their biological significance, as well as to clarify new pathways involved in arsenic signaling in plants.

 arsenic is a toxic metalloid found ubiquitously in the environment and is classified as a human carcinogen. currently, the us environmental protection agency declares arsenic as the highest priority hazardous substance found at contaminated sites in the united states. naturally high levels of arsenic in drinking water have caused major human health problems in the united states, china, argentina, taiwan, and most notably in bangladesh and india where tens of millions of people have been affected. arsenic is highly toxic at low concentrations, therefore drinking water safety standards were lowered from to μg/l in the u.s.. plants typically encounter arsenic in the anionic forms of arsenate and arsenite, both of which have different cytotoxic effects. as reacts with the sulfhydryl groups of enzymes and proteins, thereby inhibiting cellular function and resulting in death. alternatively, as is an analog of the macronutrient phosphate, so it competes with phosphate for uptake in the roots, as well as in the cytoplasm where it might disrupt metabolism by replacing phosphate in atp to form unstable adp-as. once taken up by the roots, arsenate is reduced to a more highly toxic species, arsenite, which is subsequently detoxified via soluble thiols such as glutathione and/or phytochelatins and transported for vacuolar sequestration. pcs are low molecular weight thiolate peptides of the general structure n-gly and are synthesized from glutathione by the constitutively present phytochelatin synthase. both arsenate and arsenite efficiently induce the production of pcs in plants, however it is believed since arsenate has no affinity for the sulfhydryl groups in pcs, as is reduced in the cytoplasm, resulting in as-pc complexes. glutathione and pcs have been reported to form as-tris-thiolate complexes in brassica juncea upon exposure to as. therefore, pc synthesis causes a depletion of cellular glutathione, resulting in a decreased capacity to quench reactive oxygen species. phytoremediation has emerged as an alternative technology for removing toxic metals from contaminated soils and groundwater. the potential for phytoremediation to be an effective means of removing arsenic from contaminated sites has been demonstrated in hyperaccumulators of the pteris genus and may be enhanced by a better understanding of plant transcriptional responses to arsenic. many plant studies have demonstrated the direct involvement of thiol-containing molecules in arsenic detoxification, however more robust approaches should help clarify how arsenic affects plant physiological processes on a global scale. the goals of our study were to test our hypothesis that many genes would be differentially expressed in response to arsenate stress and to identify genes as putative players in as detoxification using arabidopsis as a model. in this paper, we investigate the transcriptional responses to as in arabidopsis thaliana using oligonucleotide microarrays. our results demonstrate that as stress strongly induces cu/zn superoxide dismutase activity, but represses the production of fe sods. our microarray data also suggest the involvement of other antioxidant genes, various transcription factors, tonoplast proteins, and proteins associated with cell wall growth. of particular interest, we report that as stress represses numerous genes induced by pi starvation. we discuss the physiological implications of these findings, and suggest new avenues for research of arsenic metabolism in plants.  root growth under as stress arsenate exposure resulted in reduced arabidopsis root growth and branching. exposure of μm as resulted in significantly reduced arabidopsis root growth. in addition to known relevant physiological data, the exposure study was used to determine suitable as exposure for the microarray study. we noted no seed germination effects with regard to arsenate treatments, notably at μm, the arsenate concentration used for transcriptomics experiments. gene ontology for genes affected by as forty-six genes were induced by as treatment as indicated by microarray analysis. the largest functional categories affected included unknown function, hydrolase, and antioxidant activity. other functional categories affected by as included genes with transferase, kinase, lyase, transporter, and binding activity. alternatively, genes were repressed by as, with unknown function, hydrolase, and binding activity representing the largest categories. genes with transporter, kinase, transferase, and transcriptional regulator activity were also repressed by as. differentially expressed as -induced and -repressed genes are listed below and complete lists of all genes affected by as stress are also included as additional files. most interestingly, it was discovered that as stress repressed transcription of many genes involved in the phosphate starvation response, and also repressed several transcriptional factors. several genes involved in oxidative stress were also highly modulated in response to as. rp, rank products; fc, fold change. only genes upregulated above -fold and meeting a significance criteria of p < and fdr of 1% are shown. rank products analysis reveals that most significantly induced genes display the lowest rp value. rp, rank products; fc, fold change. only genes upregulated above - -fold and meeting a significance criteria of p < and fdr of 1% are shown. rank products analysis reveals that most significantly repressed genes display the lowest rp value. superoxide dismutases sods represented the highest ranked of both significantly induced as well as repressed genes in response to as stress, therefore these genes presented logical primary targets for the validation of our microarray data. results demonstrated -fold induction of a chloroplast cu/zn sod, -fold induction of a cu/zn sod, as well as a -fold induction of an sod copper chaperone. alternatively, fe sod transcripts were downregulated in response to arsenic stress. these findings were confirmed with quantitative rt-pcr. a microarray data b rt-pcr data arabidopsis actin was used as a reference gene p values for rt-pcr data were <. based upon our observations of transcript-level changes in sod gene expression, we performed a nondenaturing page enzyme assay of superoxide dismutase activity to assess whether the observed specific changes in sod transcript correlated with enzyme activity. this method enables the distinction between the three sod isoenzymes found in arabidopsis by using inhibitors of specific sods. gels were preincubated with kcn, which inhibits cuzn sod, as well as h2o which inhibits both cuzn sod and fe sod. mnsod is resistant to both inhibitors. plants were harvested from control plates containing no arsenate and treated plates containing μm arsenate at seven-, ten-, and thirteen days post-germination. irrespective of harvest date, cuznsod activity was strongly induced by arsenate treatment, whereas fesod activity was repressed, and mnsod showed no change in activity, therefore providing sufficient evidence to confirm our microarray results. transcription of other antioxidant genes were indicated by our microarray experiment as affected by arsenate stress, however these genes were not included in our qrt-pcr validation. transcription factors our microarray experiment indicated that eight different genes encoding proteins with known transcription factor activity all displayed lower expression levels in as -stressed plants. one of these transcription factors encodes a member of the dreb subfamily a- of the erf/ap transcription factor family. one other ap2-domain-containing transcription factor that encodes a member of the erf subfamily b- of the erf/ap transcription factor family was also repressed in response to as. two zinc finger genes encoded a zat and a protein similar to zat respectively. also exhibiting lower expression in as -treated plants were three members of the wrky family of transcription factors, wrky wrky and wrky respectively as well as one gene encoding nac domain containing protein as represses genes involved in phosphate starvation response a notable transcriptional trend is that as stress results in repression of some genes involved in the pi starvation response. of particular interest, a p-type cyclin that was affected by as -stress shares significant homology to the pho gene from yeast. we performed qrt-pcr for this gene and found that its expression was actually strongly repressed at both day and day. three genes that were repressed by as in this study have also been reported to be repressed by pi starvation. interestingly, the three highest ranking differentially expressed genes found to be strongly induced by pi starvation, were also repressed by as in our study. these genes are of particular interest on account of their unknown function. quantitative rt-pcr confirmed that transcription of these genes was strongly repressed at both day and day time points. additionally, the qrt-pcr data indicate that these genes were more repressed at day than at day. these data also corroborated our microarray experiments, which reflect global expression ratios at days post germination. sulfate assimilation the role that thiol groups play in arsenic detoxification has been well characterized, therefore we expected to see induction of genes involved in sulfate assimilation and metabolism in response to arsenic stress. ferredoxin, a key redox protein found in the chloroplast was as -induced. expression levels for another gene involved in the sulfate reduction pathway, 5'-adenylylsulfate reductase were also elevated in response to as stress. this enzyme catalyzes the reduction of aps to sulfite using glutathione as an electron donor. although not involved in sulfate assimilation, the cysteine-rich metal-binding protein, metallothionein 1a was also induced. arabidopsis knockout mutants that were generated for class mts accumulated significantly less aboveground as, cd, and zn, suggesting that class mts may play a role in metal and metalloid ion translocation. genes involved in cell wall assembly, architecture, and growth a wide range of genes encoding proteins involved in cell wall activities exhibit altered expression levels in response to as. peroxidases, which were indicated by microarray as affected by as stress, are known to strengthen the cell wall in response to biotic stress via formation of lignin, extension cross-links, and dityrosine bonds. additionally, as affected transcription of numerous xyloglucan endotransglucosylase/hydrolases and glycosyl hydrolase genes, with the majority of these exhibiting lower expression in the presence of as. discussion arsenic and oxidative stress superoxide dismutases increasing evidence from mammalian studies demonstrates that ros are generated in response to exposure to inorganic forms of arsenic. the reduction of arsenic is linked with in vivo and in vitro ros production in mammalian cells, but little is known about the mechanisms by which arsenic-induced ros generation occurs in plants. it is believed that the reduction of as to as, which is well documented in plants, results in the production of ros. however, this increase in ros may also be the result of either depletion of glutathione or inhibition of antioxidant enzymes. plants have evolved both nonenzymatic antioxidants, as well as antioxidant enzymes to manage the balance of ros in the cell. sods represent a first line of defense by converting superoxide radicals to h whereas catalases and peroxidases remove h2o three classes of sods have been identified according to the active site metal cofactor: fesod, mnsod, and cu/znsod. as and as were both shown to induce expression of glutathione s-transferases, catalases, and sods in zea mays. an increase in sod activity was correlated with an increase in as treatment in holcus lanatus. higher levels of sod, catalase, and ascorbate peroxidase were observed in pteris vittata, an arsenic hyperaccumulator, than in arsenic-sensitive fern species pteris ensiformis and nephrolepsis exaltata. these researchers concluded that arsenic-induced increases in antioxidant enzymes levels may represent a secondary defensive mechanism against oxidative stress in pteris vitatta and correspond with its arsenic accumulation and lack of toxicity symptoms. it was shown that pteris vittata sod, catalase, and peroxidase levels rose sharply in response to low levels of as, but leveled off at as levels > mg kg- which was consistent with changes in biomass in the arsenic hyperaccumulator. although the strong induction of sods in response to as stress was not surprising, the dramatically lower levels of fesods were unexpected. we suggest the involvement of an nac domain-containing transcription factor to explain the observed decrease in fesod transcription based on our microarray results. one group recently generated transgenic plants to overexpress three different arabidopsis nac transcription factors and identified nac-dependent genes using microarrays. not only was at4g expression found to be nac-dependent, but transcription of other genes we have observed to be repressed by as stress also appear to be dependent on nac-domain containing transcription factors. we continue this discussion more thoroughly in the following section on transcription factors. peroxidases peroxidases are functionally diverse and participate in two major cycles: the hydroxylic cycle where peroxidases regulate h2o levels and release ros and the peroxidative cycle where various substrates are oxidized or polymerized. their involvement in a broad range of physiological processes allows peroxidase expression in all plant organs from germination to early senescence, however they are predominantly expressed in the roots. it is not surprising that peroxidases seem to be affected by arsenate stress, especially in consideration of the elevated sod activity, which produces h2o as a product of superoxide radical dismutation. transcription factors our microarray data corroborate those of tran et al., suggesting the involvement of a different nac domain-containing transcription factor in expression of fesod, as well as several other genes known to exhibit nac-dependent expression. nac proteins comprises a large gene family of plant-specific transcription factors that have roles in wide-ranging processes such as development, defense, and abiotic stress response. microarray experiments were carried out on nac-overexpression arabidopsis mutants to discover genes exhibiting dependence on nac transcription factors for transcription. we speculate that repression of nac in as -stressed arabidopsis may be responsible for the observed repression of fesod, ferritin, xth, xth, erd atp-dependent clp protease atp-binding subunit, and a branched-chain amino acid amino transferase, as these genes were reported as exhibiting nac-dependent expression. as stress represses genes induced by pi deprivation although phosphate is undoubtedly one of the most biologically important nutrients, its availability in soils is quite low. therefore, plants have evolved mechanisms to maximize pi accessibility/availability, such as increased root hair growth, lateral root branching, and induction of phosphate transporters and phosphatases. certain phosphate starvation-induced genes have evolved to release phosphate from plasma membranes by hydrolyzing phospholipids under conditions of low pi availability, as phospholipids comprise a major pi pool in planta. conversion from phospholipids to galactolipids is one such strategy and can result from the activity of monogalactosyldiacylglycerol synthase or digalactosyl diacylglycerol synthase. arabidopsis plants expressing mgd and mgd promoter-gus fusion constructs showed that under pi starvation, mgd3::gus was expressed in apices of serrated edges and in the lateral root branch. through investigation of arabidopsis mgdg synthase gene expression under pi starvation, these authors showed that global changes in plant membranes under pi deprivation are tightly regulated by pi signaling and that signal transduction through a pi-sensing mechanism is responsible for regulating mgdg synthase gene expression. we report here that the expression of mgd is lower in as -treated arabidopsis at days and days. therefore, it is conceivable that our observations may either reflect a pi/as sensing mechanism or simply the lower number of lateral roots in as -stressed plants. senescence related gene, a glycerophosphoryl diester phosphor-diesterase, is believed to participate in processes similar to those of the mgdg synthase genes srg had lower transcript abundance in as -treated plants in our microarray study, as well as, in day and day as -treated plants. a type acid phosphatase was also repressed in our as -treated plants as indicated by microarray and was strongly repressed in our qrt-pcr validation experiments at both day and day time points. in arabidopsis, acp has been shown to be induced by h2o but not by paraquat or salicylic acid and is thought to be involved in both phosphate mobilization and in the metabolism of reactive oxygen species. in contrast, acp was strongly repressed by as despite elevated sod levels, which generate h2o therefore, further study is required to determine the specific cause of as -mediated acp repression. recent investigations into the genome-scale transcriptional changes to phosphate deprivation in arabidopsis have elucidated a broad range of genes involved in phosphate metabolism. our microarray data suggested that many genes repressed by as stress have been reported by others to be induced in response to pi deprivation in arabidopsis thaliana. because as behaves as a phosphate analog, it is likely that this observation can be explained by a saturation effect of the phosphate analog, as, thereby misleading metabolic and regulatory perception of the toxic metalloid as an abundant supply of pi. however, arsenate likely disrupts critical biological processes that involve reversible phosphorylation, as well as pathways for phosphate signaling, but even under arsenate stress, arabidopsis accumulates much higher concentrations of as in the root than is translocated to the shoot. in another study, when wild-type arabidopsis plants were grown on μm sodium arsenate for weeks, low concentrations of arsenic were accumulated in the shoot, whereas high concentrations of arsenic were observed in roots. however, when the arsenate reductase homolog was silenced, arsenate was translocated to the shoot at concentrations that classified as hyperaccumulation. nevertheless, the signaling mechanisms by which plants distinguish between as and phosphate are unknown and other mechanisms of as detoxification and storage besides the well documented phytochelatin response may exist. in order to confirm the observation that as stress represses genes involved in phosphate starvation/acquisition, we performed qrt-pcr on some of the more interesting candidates. we are particularly interested in elucidating pathways involved in as signaling in plants. the p-type cyclin that was affected by as shares significant homology to the pho gene from yeast. cyclins bind and activate cyclin-dependent kinases, which play key roles in cell division via phosphorylation of critical substrates, such as the retinoblastoma protein, transcription factors, nuclear laminar proteins, and histones. interestingly, it was demonstrated that expression of this cyclin from arabidopsis restored the phosphate signaling pathway in a pho80-deficient yeast mutant, suggesting a putative key pi signaling role. protein kinases play crucial roles in signal transduction pathways in all eukaryotes. at3g is one of two nearly identical kinase genes in arabidopsis that encode for proteins that share high sequence homology with the mammalian 40s ribosomal protein kinases s6k and s6k. atpk was demonstrated to be the functional plant homolog of mammalian p70s6k when ectopic expression of this gene specifically phosphorylated ribosomal protein s derived from either plant or animal. atpk has recently been implicated as a crucial nodal point in a network evolved for integrating stress signals with plant growth regulation. lower expression levels observed for atpk in as -treated plants, which was most severe at day, lends us to conclude that as stress may suppress plant growth through the downregulation of this growth-regulating kinase, possibly as a result of the chemical similarity between as and phosphate. alternatively, the downregulation of atpk may result from the more general stress responses imposed by the toxic metalloid. our results are in agreement with the recently proposed ideas of catarecha et al. who studied an arabidopsis mutant that displayed enhanced arsenic accumulation. these authors identified a pi transporter mutant with a decreased rate of as uptake and increased as accumulation. by comparing gene expression of the mutant with wild-type plants, it was shown that in arabidopsis, as rapidly repressed genes involved in the pi starvation response and induced the expression of other as -responsive genes. interestingly, the repression of pi starvation genes was shown to be specific for as, whereas the as -induced genes were also induced by as. a model resulted that suggests arsenic acts via two separate signaling pathways. because of the chemical similarity of as and pi, as fools the pi sensor, thus initiating the repression of the pi starvation response. although our microarray experiments did not detect differential expression of any high-affinity pi transporter, which may be due to differences in experimental approach, catarecha et al. illustrated the high sensitivity of the pi transporter, pht1; to as and suggested that plants have evolved an as sensing system whereby as and pi signaling pathways oppose each other to protect the plant from arsenic toxicity. based on our results, it is conceivable that the p-type cyclin and atpk may be involved in as sensing, but further study is required to confirm this finding. our comparison of as -repressed genes that have also been shown to be induced by pi deprivation elucidate some promising candidates for future studies. for example, we are particularly interested in genes with unknown function that are strongly induced in both roots and leaves by pi starvation. both at1g and at1g are described as phosphoric monoester hydrolases, but to our knowledge, these have not been studied in this regard. most recently, a study described gene networks for the arabidopsis transcriptome based on the graphical gaussian model of global-scale transcriptional studies. in a constructed subnetwork of genes involved in phosphate starvation, srg at1g at3g, at2g and at5g were all closely linked, suggesting their critical roles in phosphate metabolism in arabidopsis. based on their strong induction in response to pi starvation, it is reasonable to conceive that at1g and at1g have evolved as pi scavengers for increasing pi availability. we have confirmed the downregulation of these genes in response to as at both and day time points. in this study, at2g transcript levels were strongly repressed at and day time points, whereas at5g was repressed at day and day. interestingly, at2g encodes for a putative retroelement pol polyprotein that has been reported as highly expressed in salt overly sensitive arabidopsis mutants. because the function of these two pi starvation-induced genes is unknown, these putative gene candidates may provide opportunities for gaining insight into as /pi dynamics in arabidopsis thaliana. the data presented here have led to the development of new hypotheses for future research. the potential antagonistic effects of various arsenate and pi concentrations on the expression of the aforementioned genes in arabidopsis are poorly understood. additionally, the efficiency of arsenate reduction and subsequent detoxification via phytochelatins or glutathione is poorly understood. under conditions of arsenate stress, perhaps the cellular concentrations of arsenate surpass those that may be efficiently reduced by glutathione or arsenate reductase, thus allowing free arsenate to interfere with biological reactions that involve phosphate. additionally, the selected arsenate concentration to employ in this study could have resulted in free arsenite after reduction in vivo that would likely have deleterious consequences. therefore, it is reasonable to conceive that the observed transcriptional responses, as well as the impaired phenotype seen in this study, may be reflective of either arsenate, arsenite, or both.  our data show that in arabidopsis, cu/zn sods are strongly induced in response to as stress, while fe sod expression is repressed. we also demonstrate that as stress results in the repression of genes involved in phosphate acquisition, redistribution, and phosphorylation, which supports a recent study that suggests as and pi signaling pathways act in opposition to protect plant health. although this study identifies some interesting targets for exploring as metabolism, further studies using arabidopsis mutants with altered expression of these genes are necessary to elucidate their biological significance, as well as to clarify new pathways involved in arsenic signaling in plants. 
microarray data suggest that as induces genes involved in response to oxidative stress and represses transcription of genes induced by phosphate starvation. on the other hand, fe sods were strongly repressed in response to as stress. arsenate stress resulted in the repression of numerous genes known to be induced by phosphate starvation. most importantly, these data reveal that arsenate stress affects the expression of several genes with little or unknown biological functions, thereby providing new putative gene targets for future research. the microarray experiment revealed induction of chloroplast cu/zn superoxide dismutase, cu/zn sod, as well as an sod copper chaperone. whole genome oligonucleotide microarrays were employed to investigate the transcriptional responses of arabidopsis thaliana plants to as stress.
 arsenic is toxic to plants and a common environmental pollutant. there is a strong chemical similarity between arsenate and phosphate. whole genome oligonucleotide microarrays were employed to investigate the transcriptional responses of arabidopsis thaliana plants to as stress.  antioxidant-related genes play prominent roles in response to arsenate. the microarray experiment revealed induction of chloroplast cu/zn superoxide dismutase, cu/zn sod, as well as an sod copper chaperone. on the other hand, fe sods were strongly repressed in response to as stress. non-parametric rank product statistics were used to detect differentially expressed genes. arsenate stress resulted in the repression of numerous genes known to be induced by phosphate starvation. these observations were confirmed with qrt-pcr and sod activity assays.  microarray data suggest that as induces genes involved in response to oxidative stress and represses transcription of genes induced by phosphate starvation. this study implicates as as a phosphate mimic in the cell by repressing genes normally induced when available phosphate is scarce. most importantly, these data reveal that arsenate stress affects the expression of several genes with little or unknown biological functions, thereby providing new putative gene targets for future research. 