BACKGROUND
contamination of soil by agronomical and industrial activities, notably heavy metals, is a major problem for human health.
in the past years, decontamination by plants  has been the subject of intensive research.
some heavy metals such as copper, iron and zinc are oligo-elements essential for plant development, however they can become toxic at higher concentrations.
conversely, non-nutrient metals such as cadmium , lead and mercury are potentially toxic even at very low doses.
nonetheless, their toxicity varies between plant species.
for example, metal-tolerant plants are able to grow in highly contaminated soils.
mechanisms responsible for the uptake and storage of heavy metals in plants began to be understood  <cit> .
first after mobilization of metal ions from soils, uptake of heavy metals occurs into root cells through more or less specific channels and/or transporters  <cit> .
in a second phase occuring in the cytoplasm metal ions are associated with amino acids, organic acids, glutathione or longer glutathione-derived peptide, phytochelatins .
when plants are exposed to cd, an increase in pcs synthesis occurs and these pcs participate in the root to shoot translocation of cd  <cit> .
in a third phase, glutathione and pcs-cd complexes are excluded from the cytosol into vacuolar or extra-cellular compartments by various transporters, among which are abc transporters  <cit> .
the atp-binding cassette  superfamily is the largest family of transporters in living organisms, ranging from bacteria to humans  <cit> .
in humans, abc transporters have received considerable attention as their deficiency or mutations are associated with severe diseases such as cystic fibrosis and diabetes  <cit> .
these transporters are able to carry various substrates, including ions, carbohydrates, lipids, xenobiotics, drugs and heavy metals  <cit> .
in the arabidopsis genome,  <dig> genes encoding abc proteins have been identified  <cit> , but for most of them, their function and substrates are still unknown.
a number of abc transporters were recently characterized for auxin and chlorophyll catabolites transport  <cit> , pathogen and antibiotic resistance  <cit> , detoxification of heavy metals  <cit> , as well as for controlling water stress via anions and calcium channel regulation  <cit> .
fifteen members of the arabidopsis abc transporters belong to the multidrug resistance-associated protein  subfamily  <cit> .
mrp proteins display two hydrophobic domains  containing six membrane spans and two hydrophilic, cytosolic, nucleotide binding domains  which are organized in pairs.
in most of mrp proteins, an additional hydrophobic domain  is present at the n-terminal part of the transporter.
in most abc transporters, the binding and subsequent hydrolysis of atp at their nbd provides the energy required for substrate translocation across the membrane.
structurally, each nbd exhibits one 'walker a' and one 'walker b' motif which is endowed by all abc members, as well as by other atp-binding proteins, and a highly conserved c motif or abc transporter signature, being located between both walker sequences, which is specific to abc transporters.
until now, five members of this subclass  have been characterized and atmrp <dig>  atmrp <dig> and atmrp <dig> have been found to exhibit glutathione s-conjugate transport activity  <cit> .
in the case of atmrp <dig> and atmrp <dig>  an additive chlorophyll catabolites transport activity was reported  <cit> .
interestingly, atmrp <dig> is also able to complement the loss of ycf <dig>  which is an abc transporter involved in cd detoxification in yeast  <cit> .
in planta, atmrp <dig> is up-regulated by a cd treatment  <cit> , but the evidence that atmrp <dig> is a cd-transporter has not yet been obtained and to our knowledge there is no description of any atmrp <dig> mutant in the literature till now.
in addition, atmrp <dig> and atmrp <dig> are involved in the control of stomatal movements.
more precisely atmrp <dig> participates in the control of water loss via the regulation of anion and calcium channels  <cit> .
here, we report the expression pattern of atmrp <dig> which is part of a cluster of three mrp genes co-regulated by cd.
two t-dna insertion mutants were isolated, and an increased sensitivity to cd during early stages of development was observed in these two lines.
RESULTS
cdna isolation and protein organization
atmrp <dig>  was directly cloned by rt-pcr using mr06-notstart and mr06r-stopnot oligonucleotide primers  and a full-length cdna of  <dig> bp was obtained .
alignment of this cdna with the genomic sequence  from chromosome iii allowed us to deduce the genomic organization of the gene.
atmrp <dig> extends on a  <dig>  kb fragment and is spaced out into  <dig> exons.
.
this cdna was unstable in escherichia coli, requiring a growth of the bacteria at 30°c in order to avoid mutations.
two other members of the mrp sub-family, atmrp <dig> and atmrp <dig>  flank the atmrp <dig> gene at its 5'- and 3'-end, respectively.
all are oriented in the same transcription direction.
atmrp <dig> and atmrp <dig> are the closest related genes to atmrp <dig> and this cluster probably results from two successive gene duplications  <cit> .
mean percentage amino acid identities of atmrp <dig> compared to atmrp <dig> and atmrp <dig> were  <dig> % and  <dig> %, respectively.
the atmrp <dig> cdna contains an open reading frame, which encodes a 1466-aminoacids polypeptide with a predicted molecular weight of  <dig>  kda.
based on a kyte and doolittle hydropathy plot using protscale and depending on the software used for transmembrane spans prediction, atmrp <dig> exhibits  <dig>  to  <dig>  transmembrane helixes.
when using aramemnon,  <dig> different algorithms are compared and a consensus sequence is proposed with  <dig> transmembrane spans.
however, in this prediction, downstream from the first nucleotide-binding domain, the second half of the protein exhibits only  <dig> transmembrane helixes whereas  <dig> transmembrane spans are usually found.
hmmtop_v <dig>  as well as phobius, proposed a model with  <dig> transmembrane helixes.
taking into account the fact i) abc transporters should have an internal symmetry; ii) the two nbd should be accessible to the cytosol; iii) the two nbd should not overlap the transmembrane region, we consider that the most probable model is the one presented in figure 1b, with at least  <dig> transmembrane helixes, two-halves of  <dig> transmembrane helixes and a tmd <dig> of at least  <dig> transmembrane spans.
atmrp <dig> can be expressed in mammalian cells but not in yeast
in order to investigate the ability of atmrp <dig> to transport classical substrates of mrps proteins, heterologous expression of the cdna was realized in both yeast and mammalian cells .
egfp was fused at the c-terminal part of atmrp <dig> to localize its expression in both expression systems.
particular attention was dedicated to the integrity of plasmids due to the instability of atmrp <dig> in e. coli.
as shown in figure  <dig>  a weak expression of the full size transporter was observed in hek- <dig> cells.
in yeast, the plasmid was intact but the protein underwent a maturation step, leading to a truncated version of the transporter .
in these conditions, no complementation of the Δycf <dig> mutant by atmrp6-gfp was observed .
in hek- <dig> cells, atmrp6-gfp was fully translated  but its expression level was low due to a weak yield of transfection and cellular expression , compared for instance to the gfp control .
cell survival experiments conducted in the presence of exogenous cd in the culture medium did not allow us to distinguish vector-transformed cells from cells expressing atmrp <dig> .
atmrp <dig> promoter-gus fusion is essentially expressed in seedlings
atmrp <dig> gene expression was determined by rt-q-pcr in different tissues.
as shown in figure 3a, atmrp <dig> transcripts were principally detected in seedlings but at a very low level compared to the actin- <dig> gene.
expression was also found in roots, leaves and flowers but was absent from stems.
this data was confirmed by analysis of independent homozygous transgenic lines expressing the β-glucuronidase reporter gene under the control of two different promoter regions of atmrp <dig>  one corresponding to the intergenic region , the other corresponding to a  <dig>  kb promoter region overlapping the orf of atmrp <dig>  transgenic plants expressing both constructions exhibited the same expression pattern.
the gus reporter gene was observed in germinating seeds , in young seedlings essentially in cotyledons , in more developed seedlings at the base of leaves and in the apical meristem .
expression was also detected in lateral root primordia , restricted to pericycle cells, which are found opposite the xylem pole on the side where lateral roots initiate .
atmrp <dig> is up-regulated by h2o <dig> and cd exposure
in order to determine in which process atmrp <dig> could be involved, its expression level in response to numerous stresses was investigated by rt-q-pcr in arabidopsis plantlets.
a significant variation of atmrp <dig> expression level was observed after hydrogen peroxide treatment but not in response to hormones  or to salt or cold stress .
concomitantly by a transcriptomic analysis of genes regulated by cd  <cit> , we observed that atmrp <dig> was one of the most induced abc transporter genes.
such an up-regulation by cd was confirmed by rt-q-pcr, atmrp <dig> being up-regulated in roots after a 30-hr exposition to  <dig> μm cd .
isolation and characterization of atmrp <dig> knockout plants
in order to elucidate the function of atmrp <dig>  three t-dna insertion knockout lines  were isolated from the salk institute collection: atmrp <dig>  , atmrp <dig>   and atmrp <dig>  which are located downstream of the stop codon .
since no full-length mrna was detected in either atmrp <dig>  or atmrp <dig> , they were selected for further analysis.
amplification of the full messenger was obtained by rt-pcr in the case of the atmrp <dig>  mutant .
growth and development of wild type plants as well as t-dna ko plants  were similar when phenotypes were screened under various conditions such as sugar stress, oxydative stress , hormones , continuous light or darkness, or in the presence of calcium channels inhibitors known to interfere with cd entry into the plant .
in hydroponic conditions, wild type columbia ecotype , atmrp <dig>  and atmrp <dig>  ko mutant plants were exposed to  <dig> or  <dig> μm cdso <dig>  conditions that triggered an up-regulation of atmrp <dig> .
for all plant genotypes, similar cd contents were found by icp-aes analysis in roots and leaves as well as similar gsh, γ-ec and phytochelatin contents determined by hplc.
finally, all genotypes exhibited an equivalent resistance to cd in terms of root growth and development .
since the expression of atmrp <dig> was essentially pronounced in seedlings , investigation of cd effects was evaluated in atmrp <dig>  and atmrp <dig>  seedlings when seeds were directly sown on a cd-contaminated medium.
three weeks after germination, root elongation and ramification in the absence or presence of 1– <dig> μm cdso4were equivalent in all plant genotypes.
however, atmrp <dig>  seedlings were more affected than control plants, notably at shoot level .
in the absence of cd, the fresh weight of atmrp <dig> , atmrp <dig>  and wild type rosette-leaves from seedlings were similar .
conversely, after cd treatment, the fresh weight of atmrp <dig>  and atmrp <dig>  seedlings were significantly lower compared to wild-type  .
this reduction in fresh weight of the mutants was not accompanied by a change in cd, gsh, γ-ec or phytochelatin content.
thus, it can be concluded that invalidation of atmrp <dig> increases cd-sensitivity of seedlings.
the possibility of an eventual functional redundancy within the atmrp3/atmrp6/atmrp <dig> cluster was investigated.
since it had already been demonstrated that atmrp <dig> is induced by cd  <cit> , we examined comparatively in wild type plants the expression levels of the three mrp genes belonging to the cluster, together with atmrp <dig> as a control.
as shown in figure 5d, the expression of the three genes was up-regulated by cd in plant roots, whereas the expression level of atmrp <dig> remained unchanged.
the likely gene duplication at the basis of the atmrp3/atmrp6/atmrp <dig> cluster  <cit>  led us to investigate the expression level of atmrp <dig> and atmrp <dig> in the atmrp <dig>  mutant genetic background at the seedling stage of development.
whatever the presence or absence of cd, no significant difference in atmrp <dig> and atmrp <dig> expression levels was observed.
therefore, invalidation of atmrp <dig> was not correlated with an over-expression of atmrp <dig> or atmrp <dig>
discussion
abc transporters, especially from the mrp subfamily, are frequently involved in the detoxification of various xenobiotics, among which, heavy metals are found.
here, we tried to decipher the function of a previously uncharacterized a. thaliana gene, atmrp <dig>  which is flanked by two other mrps gene on chromosome iii, atmrp <dig> and atmrp <dig>
analysis of atmrp <dig> gene expression by rt-q-pcr as well as by promoter gus analysis, demonstrated that this gene is weakly expressed and has a restricted pattern of expression, mainly in germinating seeds and seedlings.
subcellular localization of atmrp <dig> in planta was attempted through two different approaches.
first, camv35s transgenic plants expressing atmrp6-gfp were generated.
strikingly, whereas empty vector and atmrp <dig> antisens plants were easily obtained, it was never the case for the sense construction, probably indicating a toxicity of this gene product under over-expressing conditions.
as an alternative way to address the localization of the transporter, mesophyll cell protoplasts were transfected with atmrp6-gfp by the classical polyethylene glycol method.
no fluorescence could be observed in these conditions whereas, in control cells expressing the gfp alone, fluorescence was detected in the cytoplasm and in the nucleus.
the subcellular localization of atmrp <dig> could not be determined however, our experiments highlighted the difficulties when working with this gene.
in addition, heterologous expression of transporters in yeast constitutes an elegant approach to screening for complementation of various mutants and also to perform flux experiments with radiolabelled compounds.
in the case of atmrp <dig>  no complementation of the Δycf <dig> mutant could be obtained in this study: atmrp <dig> being truncated .
we assume that this truncation of the protein was probably due to a toxicity of the transporter for the host.
the development of such host toxicity is also consistent with an almost systematic mutation of the corresponding plasmid that occurred in bacteria at 37°c.
when looking for an alternative expression system for atmrp <dig>  hek- <dig> cells were transfected.
as shown in figure 2b–c, atmrp <dig> expression was successfully obtained.
however, despite many efforts , the yield of expression was too weak to initiate any flux experiment.
results obtained in this study by rt-q-pcr  and within a previous transcriptomic analysis  <cit> , demonstrate that atmrp <dig> expression is up-regulated in roots within 30-hr by  <dig> μm cd.
interestingly, not only atmrp <dig>  but the three members of the gene cluster were also up-regulated by after cd exposition.
these results are in accordance with an enhanced level of both atmrp <dig> and atmrp <dig> transcripts, reported previously in cdna microarray experiments  <cit> .
it has already been reported that atmrp <dig> can be important in cd detoxification since its heterologous expression in the yeast strain deprived of ycf <dig> restores cd tolerance  <cit> .
however, in arabidopsis, despite the fact that cd-related induction of atmrp <dig> is correlated with cd uptake after a short metal exposure  <cit> , whether atmrp <dig> is involved in cd transport or in the detoxification of toxic compounds produced after the metal stress awaits future studies.
in the case of atmrp <dig>  very little data is available about its tissue expression  <cit>  and its function is still unknown.
a fourth gene, located upstream of the mrp cluster, is also up-regulated in roots by cd treatment: it encodes a mitochondrial-localized serine acetyl-transferase, sat <dig> or serat <dig>  .
this enzyme catalyzes the formation of o-acetyl-ser from l-ser and acetyl-coa, which is used in cysteine synthesis, an important component of glutathione.
expression of the bacterial enzyme in tobacco led to an increase in cysteine and glutathione contents  <cit> .
moreover, the high activity of sat is associated with nickel tolerance in thlaspi nickel hyper-accumulators  <cit>  suggesting a major role of sat in heavy metal resistance.
recently, expression of sat <dig> has been achieved in tobacco; however no experiments have been performed in relation to cd  <cit> .
all these results suggest that these four genes , oriented in the same transcription direction on chromosome iii, are members of a cd-responding cluster.
this hypothesis is also supported by the fact that all these genes are up-regulated by a cd treatment into the same organ  and in the same time scale .
identification of such cd-responsive elements would be useful in the context of phytoremediation strategies either to drive the expression of cadmium-transporter or reporter genes that might be used as biosensors of contaminated soils.
at the sight of the expression pattern of this gene , a phenotype was expected at root level in t-dna ko lines.
one cannot exclude that the neighboring mrp genes might complement the deletion of atmrp <dig>  for this reason, the expression levels of atmrp <dig> and atmrp <dig> were compared in wild type plants and in atmrp <dig> genetic backgrounds.
no significant difference in their expression levels was detected in the presence or in the absence of cadmium .
thus, it is possible that if a mechanism of gene compensation is taking place in atmrp <dig> ko plants, it involves other gene than atmrp <dig> and atmrp <dig> or that the basal levels of expression of atmrp3/ <dig> are sufficient to compensate for the absence of atmrp <dig>  alternatively, these two genes could be up-regulated in the few cells expressing atmrp <dig> in roots without significantly affecting their global root-expression level.
the screening of several dozen conditions to observe a phenotype for atmrp <dig> ko plants allowed us to show that, in the presence of cd, the deletion of atmrp <dig> has a small but significant impact on the development of primary leaves whereas roots elongation and ramification were unaffected.
this phenotype was lost in 3- to 5-week-old plant, probably because at this developmental stage, cd translocation from root to shoot is much lower, as already reported for atmrp <dig>  <cit> .
CONCLUSIONS
we have shown that atmrp <dig>  atmrp <dig> and atmrp <dig>  as well as sat <dig>  are part of a cd-regulated gene cluster.
the narrow expression profile of the atmrp <dig> gene in the plant, essentially during the first step of seedling development might explain the discrete phenotype observed in t-dna ko lines and is more consistent with a function of this transporter in plant growth/development rather than in cd detoxification.
if our results demonstrate that atmrp <dig> is part of a cluster involved in metal tolerance, and that invalidation of this gene leads to a higher susceptibility of young seedlings, the precise function of this transporter in the plant will remain to be determined.

 contamination of soil by agronomical and industrial activities, notably heavy metals, is a major problem for human health. in the past years, decontamination by plants has been the subject of intensive research. some heavy metals such as copper, iron and zinc are oligo-elements essential for plant development, however they can become toxic at higher concentrations. conversely, non-nutrient metals such as cadmium, lead and mercury are potentially toxic even at very low doses. nonetheless, their toxicity varies between plant species. for example, metal-tolerant plants are able to grow in highly contaminated soils. mechanisms responsible for the uptake and storage of heavy metals in plants began to be understood. first after mobilization of metal ions from soils, uptake of heavy metals occurs into root cells through more or less specific channels and/or transporters. in a second phase occuring in the cytoplasm metal ions are associated with amino acids, organic acids, glutathione or longer glutathione-derived peptide, phytochelatins. when plants are exposed to cd, an increase in pcs synthesis occurs and these pcs participate in the root to shoot translocation of cd. in a third phase, glutathione and pcs-cd complexes are excluded from the cytosol into vacuolar or extra-cellular compartments by various transporters, among which are abc transporters. the atp-binding cassette superfamily is the largest family of transporters in living organisms, ranging from bacteria to humans. in humans, abc transporters have received considerable attention as their deficiency or mutations are associated with severe diseases such as cystic fibrosis and diabetes. these transporters are able to carry various substrates, including ions, carbohydrates, lipids, xenobiotics, drugs and heavy metals. in the arabidopsis genome, genes encoding abc proteins have been identified, but for most of them, their function and substrates are still unknown. a number of abc transporters were recently characterized for auxin and chlorophyll catabolites transport, pathogen and antibiotic resistance, detoxification of heavy metals, as well as for controlling water stress via anions and calcium channel regulation. fifteen members of the arabidopsis abc transporters belong to the multidrug resistance-associated protein subfamily. mrp proteins display two hydrophobic domains containing six membrane spans and two hydrophilic, cytosolic, nucleotide binding domains which are organized in pairs. in most of mrp proteins, an additional hydrophobic domain is present at the n-terminal part of the transporter. in most abc transporters, the binding and subsequent hydrolysis of atp at their nbd provides the energy required for substrate translocation across the membrane. structurally, each nbd exhibits one'walker a' and one'walker b' motif which is endowed by all abc members, as well as by other atp-binding proteins, and a highly conserved c motif or abc transporter signature, being located between both walker sequences, which is specific to abc transporters. until now, five members of this subclass have been characterized and atmrp atmrp and atmrp have been found to exhibit glutathione s-conjugate transport activity. in the case of atmrp and atmrp an additive chlorophyll catabolites transport activity was reported. interestingly, atmrp is also able to complement the loss of ycf which is an abc transporter involved in cd detoxification in yeast. in planta, atmrp is up-regulated by a cd treatment, but the evidence that atmrp is a cd-transporter has not yet been obtained and to our knowledge there is no description of any atmrp mutant in the literature till now. in addition, atmrp and atmrp are involved in the control of stomatal movements. more precisely atmrp participates in the control of water loss via the regulation of anion and calcium channels. here, we report the expression pattern of atmrp which is part of a cluster of three mrp genes co-regulated by cd. two t-dna insertion mutants were isolated, and an increased sensitivity to cd during early stages of development was observed in these two lines.  cdna isolation and protein organization atmrp was directly cloned by rt-pcr using mr06-notstart and mr06r-stopnot oligonucleotide primers and a full-length cdna of bp was obtained. alignment of this cdna with the genomic sequence from chromosome iii allowed us to deduce the genomic organization of the gene. atmrp extends on a kb fragment and is spaced out into exons.. this cdna was unstable in escherichia coli, requiring a growth of the bacteria at 30°c in order to avoid mutations. two other members of the mrp sub-family, atmrp and atmrp flank the atmrp gene at its 5'- and 3'-end, respectively. all are oriented in the same transcription direction. atmrp and atmrp are the closest related genes to atmrp and this cluster probably results from two successive gene duplications. mean percentage amino acid identities of atmrp compared to atmrp and atmrp were % and %, respectively. the atmrp cdna contains an open reading frame, which encodes a 1466-aminoacids polypeptide with a predicted molecular weight of kda. based on a kyte and doolittle hydropathy plot using protscale and depending on the software used for transmembrane spans prediction, atmrp exhibits to transmembrane helixes. when using aramemnon, different algorithms are compared and a consensus sequence is proposed with transmembrane spans. however, in this prediction, downstream from the first nucleotide-binding domain, the second half of the protein exhibits only transmembrane helixes whereas transmembrane spans are usually found. hmmtop_v as well as phobius, proposed a model with transmembrane helixes. taking into account the fact i) abc transporters should have an internal symmetry; ii) the two nbd should be accessible to the cytosol; iii) the two nbd should not overlap the transmembrane region, we consider that the most probable model is the one presented in figure 1b, with at least transmembrane helixes, two-halves of transmembrane helixes and a tmd of at least transmembrane spans. atmrp can be expressed in mammalian cells but not in yeast in order to investigate the ability of atmrp to transport classical substrates of mrps proteins, heterologous expression of the cdna was realized in both yeast and mammalian cells. egfp was fused at the c-terminal part of atmrp to localize its expression in both expression systems. particular attention was dedicated to the integrity of plasmids due to the instability of atmrp in e. coli. as shown in figure a weak expression of the full size transporter was observed in hek- cells. in yeast, the plasmid was intact but the protein underwent a maturation step, leading to a truncated version of the transporter. in these conditions, no complementation of the Δycf mutant by atmrp6-gfp was observed. in hek- cells, atmrp6-gfp was fully translated but its expression level was low due to a weak yield of transfection and cellular expression, compared for instance to the gfp control. cell survival experiments conducted in the presence of exogenous cd in the culture medium did not allow us to distinguish vector-transformed cells from cells expressing atmrp. atmrp promoter-gus fusion is essentially expressed in seedlings atmrp gene expression was determined by rt-q-pcr in different tissues. as shown in figure 3a, atmrp transcripts were principally detected in seedlings but at a very low level compared to the actin- gene. expression was also found in roots, leaves and flowers but was absent from stems. this data was confirmed by analysis of independent homozygous transgenic lines expressing the β-glucuronidase reporter gene under the control of two different promoter regions of atmrp one corresponding to the intergenic region, the other corresponding to a kb promoter region overlapping the orf of atmrp transgenic plants expressing both constructions exhibited the same expression pattern. the gus reporter gene was observed in germinating seeds, in young seedlings essentially in cotyledons, in more developed seedlings at the base of leaves and in the apical meristem. expression was also detected in lateral root primordia, restricted to pericycle cells, which are found opposite the xylem pole on the side where lateral roots initiate. atmrp is up-regulated by h2o and cd exposure in order to determine in which process atmrp could be involved, its expression level in response to numerous stresses was investigated by rt-q-pcr in arabidopsis plantlets. a significant variation of atmrp expression level was observed after hydrogen peroxide treatment but not in response to hormones or to salt or cold stress. concomitantly by a transcriptomic analysis of genes regulated by cd, we observed that atmrp was one of the most induced abc transporter genes. such an up-regulation by cd was confirmed by rt-q-pcr, atmrp being up-regulated in roots after a 30-hr exposition to μm cd. isolation and characterization of atmrp knockout plants in order to elucidate the function of atmrp three t-dna insertion knockout lines were isolated from the salk institute collection: atmrp, atmrp and atmrp which are located downstream of the stop codon. since no full-length mrna was detected in either atmrp or atmrp, they were selected for further analysis. amplification of the full messenger was obtained by rt-pcr in the case of the atmrp mutant. growth and development of wild type plants as well as t-dna ko plants were similar when phenotypes were screened under various conditions such as sugar stress, oxydative stress, hormones, continuous light or darkness, or in the presence of calcium channels inhibitors known to interfere with cd entry into the plant. in hydroponic conditions, wild type columbia ecotype, atmrp and atmrp ko mutant plants were exposed to or μm cdso conditions that triggered an up-regulation of atmrp. for all plant genotypes, similar cd contents were found by icp-aes analysis in roots and leaves as well as similar gsh, γ-ec and phytochelatin contents determined by hplc. finally, all genotypes exhibited an equivalent resistance to cd in terms of root growth and development. since the expression of atmrp was essentially pronounced in seedlings, investigation of cd effects was evaluated in atmrp and atmrp seedlings when seeds were directly sown on a cd-contaminated medium. three weeks after germination, root elongation and ramification in the absence or presence of 1– μm cdso4were equivalent in all plant genotypes. however, atmrp seedlings were more affected than control plants, notably at shoot level. in the absence of cd, the fresh weight of atmrp, atmrp and wild type rosette-leaves from seedlings were similar. conversely, after cd treatment, the fresh weight of atmrp and atmrp seedlings were significantly lower compared to wild-type. this reduction in fresh weight of the mutants was not accompanied by a change in cd, gsh, γ-ec or phytochelatin content. thus, it can be concluded that invalidation of atmrp increases cd-sensitivity of seedlings. the possibility of an eventual functional redundancy within the atmrp3/atmrp6/atmrp cluster was investigated. since it had already been demonstrated that atmrp is induced by cd, we examined comparatively in wild type plants the expression levels of the three mrp genes belonging to the cluster, together with atmrp as a control. as shown in figure 5d, the expression of the three genes was up-regulated by cd in plant roots, whereas the expression level of atmrp remained unchanged. the likely gene duplication at the basis of the atmrp3/atmrp6/atmrp cluster led us to investigate the expression level of atmrp and atmrp in the atmrp mutant genetic background at the seedling stage of development. whatever the presence or absence of cd, no significant difference in atmrp and atmrp expression levels was observed. therefore, invalidation of atmrp was not correlated with an over-expression of atmrp or atmrp discussion abc transporters, especially from the mrp subfamily, are frequently involved in the detoxification of various xenobiotics, among which, heavy metals are found. here, we tried to decipher the function of a previously uncharacterized a. thaliana gene, atmrp which is flanked by two other mrps gene on chromosome iii, atmrp and atmrp analysis of atmrp gene expression by rt-q-pcr as well as by promoter gus analysis, demonstrated that this gene is weakly expressed and has a restricted pattern of expression, mainly in germinating seeds and seedlings. subcellular localization of atmrp in planta was attempted through two different approaches. first, camv35s transgenic plants expressing atmrp6-gfp were generated. strikingly, whereas empty vector and atmrp antisens plants were easily obtained, it was never the case for the sense construction, probably indicating a toxicity of this gene product under over-expressing conditions. as an alternative way to address the localization of the transporter, mesophyll cell protoplasts were transfected with atmrp6-gfp by the classical polyethylene glycol method. no fluorescence could be observed in these conditions whereas, in control cells expressing the gfp alone, fluorescence was detected in the cytoplasm and in the nucleus. the subcellular localization of atmrp could not be determined however, our experiments highlighted the difficulties when working with this gene. in addition, heterologous expression of transporters in yeast constitutes an elegant approach to screening for complementation of various mutants and also to perform flux experiments with radiolabelled compounds. in the case of atmrp no complementation of the Δycf mutant could be obtained in this study: atmrp being truncated. we assume that this truncation of the protein was probably due to a toxicity of the transporter for the host. the development of such host toxicity is also consistent with an almost systematic mutation of the corresponding plasmid that occurred in bacteria at 37°c. when looking for an alternative expression system for atmrp hek- cells were transfected. as shown in figure 2b–c, atmrp expression was successfully obtained. however, despite many efforts, the yield of expression was too weak to initiate any flux experiment. results obtained in this study by rt-q-pcr and within a previous transcriptomic analysis, demonstrate that atmrp expression is up-regulated in roots within 30-hr by μm cd. interestingly, not only atmrp but the three members of the gene cluster were also up-regulated by after cd exposition. these results are in accordance with an enhanced level of both atmrp and atmrp transcripts, reported previously in cdna microarray experiments. it has already been reported that atmrp can be important in cd detoxification since its heterologous expression in the yeast strain deprived of ycf restores cd tolerance. however, in arabidopsis, despite the fact that cd-related induction of atmrp is correlated with cd uptake after a short metal exposure, whether atmrp is involved in cd transport or in the detoxification of toxic compounds produced after the metal stress awaits future studies. in the case of atmrp very little data is available about its tissue expression and its function is still unknown. a fourth gene, located upstream of the mrp cluster, is also up-regulated in roots by cd treatment: it encodes a mitochondrial-localized serine acetyl-transferase, sat or serat. this enzyme catalyzes the formation of o-acetyl-ser from l-ser and acetyl-coa, which is used in cysteine synthesis, an important component of glutathione. expression of the bacterial enzyme in tobacco led to an increase in cysteine and glutathione contents. moreover, the high activity of sat is associated with nickel tolerance in thlaspi nickel hyper-accumulators suggesting a major role of sat in heavy metal resistance. recently, expression of sat has been achieved in tobacco; however no experiments have been performed in relation to cd. all these results suggest that these four genes, oriented in the same transcription direction on chromosome iii, are members of a cd-responding cluster. this hypothesis is also supported by the fact that all these genes are up-regulated by a cd treatment into the same organ and in the same time scale. identification of such cd-responsive elements would be useful in the context of phytoremediation strategies either to drive the expression of cadmium-transporter or reporter genes that might be used as biosensors of contaminated soils. at the sight of the expression pattern of this gene, a phenotype was expected at root level in t-dna ko lines. one cannot exclude that the neighboring mrp genes might complement the deletion of atmrp for this reason, the expression levels of atmrp and atmrp were compared in wild type plants and in atmrp genetic backgrounds. no significant difference in their expression levels was detected in the presence or in the absence of cadmium. thus, it is possible that if a mechanism of gene compensation is taking place in atmrp ko plants, it involves other gene than atmrp and atmrp or that the basal levels of expression of atmrp3/ are sufficient to compensate for the absence of atmrp alternatively, these two genes could be up-regulated in the few cells expressing atmrp in roots without significantly affecting their global root-expression level. the screening of several dozen conditions to observe a phenotype for atmrp ko plants allowed us to show that, in the presence of cd, the deletion of atmrp has a small but significant impact on the development of primary leaves whereas roots elongation and ramification were unaffected. this phenotype was lost in 3- to 5-week-old plant, probably because at this developmental stage, cd translocation from root to shoot is much lower, as already reported for atmrp.  we have shown that atmrp atmrp and atmrp as well as sat are part of a cd-regulated gene cluster. the narrow expression profile of the atmrp gene in the plant, essentially during the first step of seedling development might explain the discrete phenotype observed in t-dna ko lines and is more consistent with a function of this transporter in plant growth/development rather than in cd detoxification. if our results demonstrate that atmrp is part of a cluster involved in metal tolerance, and that invalidation of this gene leads to a higher susceptibility of young seedlings, the precise function of this transporter in the plant will remain to be determined. 
the position of atmrp on chromosome flanked by two other mrp genes, suggests that atmrp is part of a cluster involved in metal tolerance, although additional functions in planta cannot be discarded. this gene, located on chromosome is bordered by atmrp and atmrp using real-time quantitative pcr and the gus reporter gene, we found that this gene is essentially expressed during early seedling development, in the apical meristem and at initiation point of secondary roots, especially in xylem-opposite pericycle cells where lateral roots initiate. in arabidopsis thaliana, genes encoding abc transporters have been identified.
 abc proteins constitute one of the largest families of transporters found in all living organisms. in arabidopsis thaliana, genes encoding abc transporters have been identified. here, the characterization of one member of the mrp subclass, atmrp is described.  this gene, located on chromosome is bordered by atmrp and atmrp using real-time quantitative pcr and the gus reporter gene, we found that this gene is essentially expressed during early seedling development, in the apical meristem and at initiation point of secondary roots, especially in xylem-opposite pericycle cells where lateral roots initiate. the level of expression of atmrp in response to various stresses was explored and a significant up-regulation after cadmium treatment was detected. among the three t-dna insertion lines available from the salk institute library, two knock-out mutants, atmrp and atmrp were invalidated for the atmrp gene. in the presence of cd, development of leaves was more affected in the mutants than wild-type plants, whereas root elongation and ramification was comparable.  the position of atmrp on chromosome flanked by two other mrp genes, suggests that atmrp is part of a cluster involved in metal tolerance, although additional functions in planta cannot be discarded. 