BACKGROUND
plant cation diffusion facilitator  family members have been suggested to be involved in metal ion transport, and implicated in metal resistance in plants  <cit> .
however, the physiological role of these transporters is not well understood.
atmtp <dig>  from arabidopsis thaliana was the first member of the cdf family to be characterized in plants  <cit> .
when ectopically over expressed in a. thaliana atmtp <dig> confers enhanced resistance to zn2+, and increased zn2+ accumulation in roots  <cit> .
enhanced zn2+ resistance and accumulation was attributed to increased vacuolar sequestration of zn2+.
more recent evidence has established that atmtp <dig> is predominantly localized at the tonoplast membrane in both root and shoot tissue in a. thaliana  <cit> .
consistent with a role in zn2+ transport into the vacuole, reduction in expression of atmtp <dig> leads to increased sensitivity to zn2+, but not co2+, cd2+, ni2+ or mn2+  <cit> .
reduced expression of atmtp <dig> also leads to decreased accumulation of zn2+ in shoots  <cit> .
reconstruction of atmtp <dig> in proteoliposomes, and expression of the atmtp <dig> cdna in xenopus oocytes has provided direct evidence that atmtp <dig> is competent to transport zn2+ but not cd2+ or co2+  <cit> .
to date all studies have found that atmtp <dig> expression is not modulated by exposure to elevated zn2+, cd2+, co2+, cu2+, fe2+ or mn2+  <cit> .
expression of atmtp <dig> in a. thaliana occurs throughout the plant, though transcript levels are higher in roots than shoots in young seedlings, and expression is higher in the inflorescences  <cit> .
however, little is known about tissue-specific expression patterns of atmtp <dig>  such information is critical if we are to integrate the currently available data into a model describing how mtp <dig> functions within the physiological context of the whole plant.
homologues of atmtp <dig> have been found in other plant species including the hyperaccumulators thlaspi goesingense  <cit> , thlaspi caerulescens  <cit>  and arabidopsis halleri  <cit> .
it has been suggested that the constitutively higher shoot expression of mtp <dig> in these hyperaccumulators is involved in metal hyperaccumulation  <cit> , and recent genetic evidence supports this hypothesis  <cit> .
however, the role of the mtp <dig> protein in hyperaccumulation is still unknown.
in the zn2+ hyperaccumulator a. halleri ahmtp <dig> appears to be localized at the tonoplast membrane when transiently expressed as an ahmtp1::gfp fusion in a. thaliana protoplasts  <cit> .
similar localization has also been observed for an mtp <dig> homologue from poplar  <cit> .
however, in a similar experiment with tgmtp <dig> from the zn2+/ni2+ hyperaccumulator t. goesingense the gfp fusion protein was found to localize to the plasma membrane when transiently expressed in a. thaliana protoplasts derived from shoot tissue  <cit> .
resolution of this interesting difference awaits further comparative studies.
brassica juncea is an amphidiploid plant, resulting from the hybridization of the crop brassicas brassica nigra and brassica campestris .
it contains the conserved genomes of both of its diploid parents  <cit> , and is self compatible, unlike other crop brassica species.
due to its rapid growth and large biomass b. juncea has been considered as a possible plant for use in the phytoextraction process, for the removal of pollutant metals from soils by their accumulation into harvestable above ground biomass  <cit> .
the relatedness of b. juncea to numerous hyperaccumulators in the brassicaceae family, and its ability to be self fertilized makes b. juncea a potentially good recipient organisms for the bioengineering of a practical phytoextraction plant using genetic material derived from natural hyperaccumulator species  <cit> .
however, such promise has yet to be realized.
brassica juncea have been shown to accumulate heavy metals, though b. juncea is not a hyperaccumulator  <cit> .
however, there are no reports available that attribute a specific role of bjmtp <dig> in metal accumulation in b. juncea.
here we report that both ni2+ and cd2+ induce transcriptional activation of bjmtp <dig> in whole seedlings, as well as root and shoot tissue of mature plants, where as zn2+ has very little effect on bjmtp <dig> expression.
we establish that a  <dig> bp dna fragment upstream of the bjmtp <dig> translational start site is sufficient for this regulation, and that expression in roots is specifically localized to the xylem parenchyma cells.
this study is the first to identify transcriptional regulation and tissue localization of bjmtp <dig>  however, further work is needed to understand the functional role of bjmtp <dig> in b. juncea's response to ni2+ and cd2+.
RESULTS
mtp <dig> sequences from b. juncea and its parents
using atmtp <dig> primers mtp <dig> homologues were amplified from b. juncea and its two parents b. nigra and b. campestris .
all brassica sequences showed 80% similarity to atmtp <dig>  and all were devoid of introns similar to atmtp <dig>  brassica nigra and b. campestris mtp <dig> showed 97% and 95% similarity to the b. juncea mtp <dig> sequence, respectively.
a phylogenetic analysis of plant mtp <dig> sequences revealed that all brassica mtp <dig> sequences fall into a monophyletic clade, with mtp <dig> from b. juncea being equally related to mtp <dig> from both its parents .
isolation of the dna sequence 5' of bjmtp <dig>  revealed that the next gene upstream of bjmtp <dig> is 88% similar to the a. thaliana gene locus at2g <dig>  at2g <dig> is the next upstream gene to atmtp <dig> in a. thaliana.
such results show that synteny is conserved within the region of mtp <dig> between b.juncea and a.thaliana, and provides further strong evidence that the mtp sequence identified in b. juncea is the b. juncea homologue of atmtp <dig>
isolation and characterization of bjmtp <dig> promoter
a two-step genome walking technique was used to isolate dna 5' of the b. juncea mtp <dig> translational start site.
as a first step a  <dig>  bp pcr product was amplified from the drai genomic dna library and cloned.
to isolate additional upstream sequence further genome walking was performed using the pvuii genomic dna library giving a new  <dig>  bp pcr product.
sequence analysis of this  <dig>  bp dna fragment indicated it contains not only the remaining sequence 5' of bjmtp <dig> but also partial sequence of the next gene upstream of bjmtp <dig> which showed 88% similarity to the a. thaliana gene locus at2g <dig> .
the  <dig>  bp and  <dig>  bp dna fragments were compared and a contiguous  <dig>  bp fragment determined.
the complete 5' upstream region , as well as the  <dig>  bp fragment, were amplified from the b. juncea genomic dna and the pcr products used for all further analysis.
a  <dig> bp dna fragment 5' of bjmtp <dig> translational start site was predicted to contain the majority of the regulatory elements within the complete  <dig>  bp upstream region, and was therefore also amplified and used for further experiments.
transcriptional activation of bjmtp <dig> by various metal ions
total rna was isolated from dark grown seven-day-old b. juncea seedlings which had been treated with either  <dig> μm cd2+,  <dig> μm ni2+ or  <dig> μm zn2+ for  <dig> h, treatments that also produced maximal gus activity in transgenic plants expressing a gus report gene .
these three metals were chosen for this study based on the fact that the hyper accumulators that are known to have constitutively elevated mtp <dig> expression hyperaccumulator cd, ni or zn.
bjmtp <dig> mrna were quantified using real time quantitative rt-pcr  and normalized to bjactin <dig> as an internal control.
steady-state levels of bjmtp <dig> mrna were found to be increased after exposure of seedlings to cd2+ or ni2+ when compared to the level of expression in the untreated control plants .
cd2+ and ni2+ treatment caused a  <dig>  and  <dig>  fold increase in bjmtp <dig> transcript levels, respectively, compared to untreated control seedlings.
conversely, zn2+ treated seedlings showed only a minor  <dig> -fold increase in bjmtp <dig> mrna.
analysis of the transcriptional competency of the bjmtp <dig> promoter region
a  <dig> bp dna fragment, originating upstream of the bjmtp <dig> translational start site, was constructed as a transcriptional fusion with the gusplus reporter gene .
five-day-old dark grown seedlings stably transformed with pbjmtp1::gusplus were treated with varying concentrations of ni2+, cd2+ and zn2+ for  <dig> h and gus activity measured .
gus activity was observed to peak at  <dig> μm ni2+,  <dig> μm cd2+ and  <dig> μm zn2+, with increases in gus activity of  <dig> ,  <dig>  and  <dig> -fold, respectively .
the metal concentrations used in this assay spanned a range from moderately to severally toxic, with the highest concentrations of each metal causing complete loss of turger in the seedlings.
to assess the level of toxicity at the end of this  <dig> h assay rates of k+ leakage from the seedlings were measured.
all metal treatments caused an increase in k+ leakage peaking at  <dig> μm cd2+, and  <dig> μm ni2+ and zn2+, after which leakage rates dropped and this was associated with a lose of both seedling turger and gus activity .
histochemical gus staining of both cd2+ and ni2+ treated seedlings demonstrated that the gus protein product of the pbjmtp1::gusplus construct is localized mainly in the roots, showing strong expression at the root-shoot transition zone .
in the untreated transgenic seedlings, there was no observable gus staining .
plants stably transformed with pbjmtp1::gusplus were also grown in hydroponic culture for four weeks and transferred to medium of the same composition with the addition of  <dig> μm ni2+.
root and shoot samples were taken over a  <dig> h time course and both ni2+ accumulation and gus activity measured.
ni2+ accumulated in both roots and shoots almost linearly with time, with roots accumulating ni2+ at approximately  <dig> times the rate of the shoots .
in root samples, gus enzyme activity increased linearly as the ni2+ exposure time increased, with a  <dig>  fold increase in gus activity after  <dig> h ni2+ treatment compared to basal expression , or to untransformed control plants .
in shoots, the overall gus activity was less than in the roots.
interestingly, in shoots gus activity only transiently increased after  <dig> h exposure, compared to untransformed plants, after which gus activity decreased to levels observed at  <dig> h exposure , even though ni2+ accumulation in shoots continued .
similar results for both root and shoot expression were also obtained with plants stably transformed with pbjmtp1::eyfp  , and pbjmtp1::mrfp <dig>  .
a comparison of total tissue ni2+ accumulation and gus activity revealed a strong positive correlation between the level of ni2+ accumulation and gus activity in roots .
in order to test whether the induction process is reversible, after  <dig> h ni2+ treatment plants were transferred to nutrient solution lacking ni2+.
after a further  <dig> h recovery gus activity in roots was observed to return to that observed at  <dig> h exposure .
histochemical gus analysis of roots from four-week old plants, after  <dig> h ni2+ treatment, demonstrated clear gus expression throughout the main root .
after  <dig> h ni2+ treatment gus expression was not detected in the stem.
gus expression was, however, observed in the vasculature of the leaves  and in the vascular tissues of the anthers and the stigma  after  <dig> hr ni2+ exposure.
the regulatory competency of the  <dig> bp,  <dig>  bp and  <dig>  bp dna sequences upstream of the bjmtp <dig> translations start site were compared to establish if further metal regulatory elements exist upstream of the  <dig> bp fragment.
the transcriptional activity of these dna fragments was assessed after construction and transformation of b. juncea with transcriptional fusions with the monomeric red fluorescent protein  as a reporter gene.
hydroponically grown four-week-old b. juncea plants transformed with the mrfp <dig> reporter constructs were exposed to either  <dig> μm cd2+,  <dig> μm ni2+ or  <dig> μm zn2+ in aerated  <dig> × hoaglands solution.
root and shoot samples were collected over a  <dig> h time-course and mrfp <dig> expression quantified using a luminescence spectrometer.
as a control for metal related effects on the in vivo stability of mrfp <dig> pcamv35s:: mrfp <dig> transformed plants were also generated and treated in a similar manner.
in roots isolated from ni2+ treated plants, mrfp <dig> accumulation increased linearly with time of ni2+ exposure, and the kinetics of mrfp <dig> accumulation were equivalent regardless of the size of the promoter construct used to drive mrfp <dig> expression .
all three promoter constructs produced higher mrfp <dig> accumulation after  <dig> h ni2+ exposure than observed with the camv35s promoter.
plants were removed from the ni2+ containing medium after  <dig> h exposure and allowed to recover for a further  <dig> h. during this recovery period, mrfp <dig> accumulation decreased linearly with time in a similar manner regardless of the size of the promoter region .
in leaves from ni2+ exposed plants, all three promoter constructs drove a similar transient increase in mrfp <dig> accumulation that peaked at  <dig> h ni2+ treatment, after which they declined at a similar rate to the response observed for the promoter gus construct.
mrfp <dig> expression in plants transformed with pcamv35s::mrfp <dig> was constant at all time points and tissues after ni2+ exposure.
similar mrfp <dig> expression was also obtained when plants were exposed to  <dig> μm cd2+ for  <dig> h and allowed to recover from cd2+ exposure for a further  <dig> h. mrfp <dig> accumulated rapidly in roots after cd2+ exposure to levels equivalent to expression driven by pcamv35s .
removal of plants from the cd2+ containing nutrient solution caused a rapid drop in mrfp <dig> accumulation, with mrfp <dig> levels returning to those observed prior to cd2+ exposure, after  <dig> h recovery .
again, all three-promoter sizes gave similar responses.
cd2+ treatment also produced a transient accumulation of mrfp <dig> in shoots after  <dig> h exposure , as observed during ni2+ exposure .
unlike ni2+ and cd2+ exposure, zn2+ treatment produced no significant alteration in mrfp <dig> accumulation in either root or shoot tissue .
elemental analysis of the nutrient solutions during the ni2+, cd2+ and zn2+ experiments confirmed that concentrations of ni2+, cd2+ and zn2+ did not vary significantly in the solution during the course of the experiments .
response of the  <dig> bp promoter element to other abiotic stresses
the  <dig> bp promoter region, which was found to be sufficient for expression in response to cd2+ and ni2+, was also tested for its ability to activate transcription when seedlings were exposed to other abiotic stresses including cold, heat, nacl and h2o <dig>  five-day-old b. juncea seedlings stably transformed with p  bjmtp1::gusplus were exposed to a cold  or heat  shock, and also  <dig> mm nacl, or h2o <dig>  after which gus activity was assayed.
there were no significant increases in gus activity compared to the untreated seedlings for any of the treatments .
five-day-old dark grown seedlings stably transformed with pbjmtp1::gusplus were incubated at 4°c for  <dig> h , 37° c for  <dig> h , <dig> mm nacl  and  <dig> μm h2o <dig>  and gus activity measured.
data represents the average  of three independent replicate samples.
putative regulatory elements in the bjmtp <dig> promoter region
a search for putative regulatory dna elements within the  <dig> bp promoter region of bjmtp <dig> was performed using the place  <cit>  and plantcare databases  <cit>  .
dna regulatory motifs such as the ccaat boxes, abre , bzip, g box, and several pathogen responsive elements such as w box, eire, sebf motif, gcc box, g box coupler and myb core elements were found.
however, no known metal regulatory elements such as mres which are known to mediate zn2+ and cd2+ specific transcriptional activation of metallothionine genes in mammals were found  <cit> .
considering that atmtp <dig> appears not to be regulated by cd2+  <cit>  and bjmtp <dig> is , we compared the  <dig> bp bjmtp <dig> promoter region with a similar region of the a. thaliana atmtp <dig> sequence upstream of the transcriptional start site .
even though both promoter regions had many known regulatory elements in common, namely bzip, g box, w box, eire box and myb core element, the bjmtp <dig> promoter region also has several unique regulatory elements, namely abre and pathogen related sebf motif, gcc box and g box coupler elements .
the core sequence of the metal regulatory element  found in mammals , is present in the full  <dig> bp upstream region of bjmtp <dig> with a single base pair difference .
however, because this motif is not present in the  <dig> bp promoter region sufficient for cd2+ and ni2+ regulation , this mre is unlikely to play a role in the cd2+ and ni2+ transcriptional regulation of bjmtp <dig>
localization of bjmtp <dig> expression to root xylem parenchyma
our results demonstrate that a  <dig> bp region upstream of the translational start site of bjmtp <dig> is sufficient to drive strong root expression of gus and mrfp <dig> in response to cd2+ or ni2+ treatment .
however, in order to help understand the function of bjmtp <dig>  it is also important to identify which root tissues are expressing bjmtp <dig> during the response to cd2+ or ni2+ exposure.
sections from the main root and intact lateral roots of four-week-old b. juncea plants expressing mrfp <dig> in response to ni2+ were analyzed by epifluorescence and light microscope .
sections were taken from the top of the main root as well as  <dig>  cm from the root tip, and the morphology of the roots examined .
the main root has clearly undergone secondary growth.
the epidermis found in the primary roots has been replaced in 4-week-old roots by periderm produced by the cork cambium.
within the periderm lies the pericycle, and the cambial zone which gives rise to the secondary phloem and secondary xylem.
the secondary xylem parenchyma, produced by vascular cambium, was observed surrounding the secondary xylem vessels.
the presence of diarch xylem at the center of the root section is a characteristic of the brassicaceae family .
sections taken  <dig>  cm from the tip of the main root were observed to have similar root architecture to sections taken from the top of the root.
after exposure to ni2+ mrfp <dig> fluorescence was observed to be specifically localized in the secondary xylem parenchyma tissues that surround the secondary xylem .
fluorescence from mrfp <dig> was also present in the periderm  but no significant fluorescence was observed in other root tissues.
in the lateral roots, mrfp <dig> fluorescence was clearly evident in the vascular region .
after ni2+ exposure no fluorescence was observed in roots from plants identified as null segregants for the reporter construct even after longer  exposures, compared to the shorter  exposure for ni2+-treated transgenic plants .
cd2+ treated roots gave an equivalent expression pattern to that of ni2+ treated plants .
in both ni2+ and cd2+ treated roots there was no difference in the pattern of mrfp <dig> accumulation between sections taken from the top of the main root and its tip.
furthermore, mrfp <dig> expression was localized in the secondary xylem parenchyma tissues when expressed from the  <dig>   <dig>  or  <dig>  bp promoter regions.
the strong accumulation of mrfp <dig> observed in the vascular tissue of intact lateral roots  is also consistent with the secondary xylem parenchyma localization observed in the root cross-sections.
cross-sections of roots prepared from ni2+ exposed plants transformed with the gusplus reporter gene also revealed strong gus expression in the secondary xylem parenchyma surrounding the secondary xylem , similar to that observed for mrfp <dig> expression .
secondary xylem parenchyma localization was also observed in plants expressing a eyfp reporter driven by the  <dig> bp promoter region .
discussion
here we establish that mrna levels of bjmtp <dig> in b. juncea are transcriptionally regulated in response to cd2+ and ni2+, with maximal expression in the root xylem parenchyma cells.
we identify a  <dig> bp dna fragment, within the total  <dig>  bp dna sequence upstream of the bjmtp <dig> translational start site, which is sufficient for this transcriptional regulation, which appears specific to stress induced by cd2+ and ni2+.
bjmtp <dig> promoter activity in response to ni2+, cd2+ and zn2+ was assessed at concentrations that span a similar range of toxicities from mild to serve.
over these similar ranges of metal-induced stress only ni2+ and cd2+ were observed to elicit a transcriptional response from the bjmtp <dig> promoter, with zn2+ induced stress having no significant effect.
other forms of abiotic stress including cold, heat or nacl also produced no significant transcriptional response.
transcriptional activation of bjmtp <dig> promoter by cd2+ and ni2+ is also not an indirect response to oxidative stress, since this promoter region is not activated by direct exposure to h2o <dig>
our results are consistent with the observation in a. thaliana that the homologue atmtp <dig> is also not regulated by zn2+  <cit> .
however, our observation that bjmtp <dig> expression is transcriptionally regulated by cd2+ and ni2+ is not consistent with that observed in a. thaliana, where atmtp <dig> mrna levels are unaffected by cd2+  <cit> .
our results suggests that rather than being a house-keeping gene involved in zn2+ homeostasis as has been suggested in a. thaliana, in b. juncea bjmtp <dig> may be involved in the dynamic regulation of zn homeostasis as part of the plant's response to cd2+ and ni2+ induced stress.
we emphasize that our data does not suggest that the function of bjmtp <dig> is to transport cd2+ or ni2+, for which we have no evidence.
however, increased expression of bjmtp <dig> under cd2+ or ni2+ stress may be required to adjust zn homeostasis as a response to the stress imposed by exposure to cd2+ or ni2+.
this is supported by our observation that expression of bjmtp <dig> promoter is dynamically regulated in response to these metals, rapidly increasing after exposure to cd2+ and ni2+, and rapidly returning back to basal levels after these metals are removed from the growth medium.
though expression of the bjmtp <dig> promoter in response to cd2+ and ni2+ is highest in roots, expression was also observed in the vascular tissue of leaves, anthers and in the stigma.
expression of bjmtp <dig> in these tissues suggests that the bjmtp <dig> protein may also be involved in the plants response to cd2+ and ni2+ stress in leaves and inflorescences.
though transcriptional activation of the  <dig> bp promoter region in roots by cd2+ or ni2+ occurs nearly in a linear fashion with time of exposure and accumulation, shoot activation appears transient.
after  <dig> h of metal exposure transcription of bjmtp <dig> reaches a maximum and then declines to baseline expression after  <dig> h. such transient expression is intriguing considering that over the same time frame ni2+ accumulates linearly in shoots.
the different expression patterns of bjmtp <dig> promoter in roots and shoots imply that this protein plays different roles in the plant's coordinated response to cd2+ and ni2+ exposure, though what these roles are remain unclear at this time.
in five-day-old seedlings of b. juncea expression of bjmtp <dig> promoter in response to cd2+ and ni2+ occurs throughout the seedlings, though is especially localized to the root and shoot-root transition zone.
though the reasons for stronger expression bjmtp <dig> promoter in the shoot-root transition zone are not known, this type of expression pattern has been observed previously for several proteins, including naphthylphthalmic acid  associated amino peptidases, glutathione s-transferase atgstf <dig>  the auxin transporter pin <dig>  and pgp <dig> an abc type transporter  <cit> .
when compared to a. thaliana, the unique elements present in the bjmtp <dig> promoter region, including abre, sebf motif, gcc box and g box coupler elements all have known functions in aba signaling  <cit>  or pathogen related stress  <cit> .
however, none of these promoter elements have been established to play a role in signaling cd2+ or ni2+ stress.
the elements responsible for regulation of bjmtp <dig> expression in response to cd2+ and ni2+ remain to be identified.
this study is the first to identify transcriptional regulation and tissue localization of bjmtp <dig>  however, further work is needed to understand the functional role of bjmtp <dig> in b. juncea's response to ni2+ and cd2+.
CONCLUSIONS
here we conclude that a  <dig> bp dna fragment upstream of the bjmtp <dig> translational start site is sufficient for the specific activation by ni2+ and cd2+ of bjmtp <dig> expression primarily in roots.
activation of expression by both metals in roots is primarily localized to the xylem parenchyma cells.
this study is the first to identify specific ni2+ and cd2+ transcriptional regulation and tissue localization of bjmtp <dig> and supports the conclusion that bjmtp <dig> is involved in the response of b. juncea to ni2+ and cd2+ exposure.

 plant cation diffusion facilitator family members have been suggested to be involved in metal ion transport, and implicated in metal resistance in plants. however, the physiological role of these transporters is not well understood. atmtp from arabidopsis thaliana was the first member of the cdf family to be characterized in plants. when ectopically over expressed in a. thaliana atmtp confers enhanced resistance to zn2+, and increased zn2+ accumulation in roots. enhanced zn2+ resistance and accumulation was attributed to increased vacuolar sequestration of zn2+. more recent evidence has established that atmtp is predominantly localized at the tonoplast membrane in both root and shoot tissue in a. thaliana. consistent with a role in zn2+ transport into the vacuole, reduction in expression of atmtp leads to increased sensitivity to zn2+, but not co2+, cd2+, ni2+ or mn2+. reduced expression of atmtp also leads to decreased accumulation of zn2+ in shoots. reconstruction of atmtp in proteoliposomes, and expression of the atmtp cdna in xenopus oocytes has provided direct evidence that atmtp is competent to transport zn2+ but not cd2+ or co2+. to date all studies have found that atmtp expression is not modulated by exposure to elevated zn2+, cd2+, co2+, cu2+, fe2+ or mn2+. expression of atmtp in a. thaliana occurs throughout the plant, though transcript levels are higher in roots than shoots in young seedlings, and expression is higher in the inflorescences. however, little is known about tissue-specific expression patterns of atmtp such information is critical if we are to integrate the currently available data into a model describing how mtp functions within the physiological context of the whole plant. homologues of atmtp have been found in other plant species including the hyperaccumulators thlaspi goesingense, thlaspi caerulescens and arabidopsis halleri. it has been suggested that the constitutively higher shoot expression of mtp in these hyperaccumulators is involved in metal hyperaccumulation, and recent genetic evidence supports this hypothesis. however, the role of the mtp protein in hyperaccumulation is still unknown. in the zn2+ hyperaccumulator a. halleri ahmtp appears to be localized at the tonoplast membrane when transiently expressed as an ahmtp1::gfp fusion in a. thaliana protoplasts. similar localization has also been observed for an mtp homologue from poplar. however, in a similar experiment with tgmtp from the zn2+/ni2+ hyperaccumulator t. goesingense the gfp fusion protein was found to localize to the plasma membrane when transiently expressed in a. thaliana protoplasts derived from shoot tissue. resolution of this interesting difference awaits further comparative studies. brassica juncea is an amphidiploid plant, resulting from the hybridization of the crop brassicas brassica nigra and brassica campestris. it contains the conserved genomes of both of its diploid parents, and is self compatible, unlike other crop brassica species. due to its rapid growth and large biomass b. juncea has been considered as a possible plant for use in the phytoextraction process, for the removal of pollutant metals from soils by their accumulation into harvestable above ground biomass. the relatedness of b. juncea to numerous hyperaccumulators in the brassicaceae family, and its ability to be self fertilized makes b. juncea a potentially good recipient organisms for the bioengineering of a practical phytoextraction plant using genetic material derived from natural hyperaccumulator species. however, such promise has yet to be realized. brassica juncea have been shown to accumulate heavy metals, though b. juncea is not a hyperaccumulator. however, there are no reports available that attribute a specific role of bjmtp in metal accumulation in b. juncea. here we report that both ni2+ and cd2+ induce transcriptional activation of bjmtp in whole seedlings, as well as root and shoot tissue of mature plants, where as zn2+ has very little effect on bjmtp expression. we establish that a bp dna fragment upstream of the bjmtp translational start site is sufficient for this regulation, and that expression in roots is specifically localized to the xylem parenchyma cells. this study is the first to identify transcriptional regulation and tissue localization of bjmtp however, further work is needed to understand the functional role of bjmtp in b. juncea's response to ni2+ and cd2+.  mtp sequences from b. juncea and its parents using atmtp primers mtp homologues were amplified from b. juncea and its two parents b. nigra and b. campestris. all brassica sequences showed 80% similarity to atmtp and all were devoid of introns similar to atmtp brassica nigra and b. campestris mtp showed 97% and 95% similarity to the b. juncea mtp sequence, respectively. a phylogenetic analysis of plant mtp sequences revealed that all brassica mtp sequences fall into a monophyletic clade, with mtp from b. juncea being equally related to mtp from both its parents. isolation of the dna sequence 5' of bjmtp revealed that the next gene upstream of bjmtp is 88% similar to the a. thaliana gene locus at2g at2g is the next upstream gene to atmtp in a. thaliana. such results show that synteny is conserved within the region of mtp between b.juncea and a.thaliana, and provides further strong evidence that the mtp sequence identified in b. juncea is the b. juncea homologue of atmtp isolation and characterization of bjmtp promoter a two-step genome walking technique was used to isolate dna 5' of the b. juncea mtp translational start site. as a first step a bp pcr product was amplified from the drai genomic dna library and cloned. to isolate additional upstream sequence further genome walking was performed using the pvuii genomic dna library giving a new bp pcr product. sequence analysis of this bp dna fragment indicated it contains not only the remaining sequence 5' of bjmtp but also partial sequence of the next gene upstream of bjmtp which showed 88% similarity to the a. thaliana gene locus at2g. the bp and bp dna fragments were compared and a contiguous bp fragment determined. the complete 5' upstream region, as well as the bp fragment, were amplified from the b. juncea genomic dna and the pcr products used for all further analysis. a bp dna fragment 5' of bjmtp translational start site was predicted to contain the majority of the regulatory elements within the complete bp upstream region, and was therefore also amplified and used for further experiments. transcriptional activation of bjmtp by various metal ions total rna was isolated from dark grown seven-day-old b. juncea seedlings which had been treated with either μm cd2+, μm ni2+ or μm zn2+ for h, treatments that also produced maximal gus activity in transgenic plants expressing a gus report gene. these three metals were chosen for this study based on the fact that the hyper accumulators that are known to have constitutively elevated mtp expression hyperaccumulator cd, ni or zn. bjmtp mrna were quantified using real time quantitative rt-pcr and normalized to bjactin as an internal control. steady-state levels of bjmtp mrna were found to be increased after exposure of seedlings to cd2+ or ni2+ when compared to the level of expression in the untreated control plants. cd2+ and ni2+ treatment caused a and fold increase in bjmtp transcript levels, respectively, compared to untreated control seedlings. conversely, zn2+ treated seedlings showed only a minor -fold increase in bjmtp mrna. analysis of the transcriptional competency of the bjmtp promoter region a bp dna fragment, originating upstream of the bjmtp translational start site, was constructed as a transcriptional fusion with the gusplus reporter gene. five-day-old dark grown seedlings stably transformed with pbjmtp1::gusplus were treated with varying concentrations of ni2+, cd2+ and zn2+ for h and gus activity measured. gus activity was observed to peak at μm ni2+, μm cd2+ and μm zn2+, with increases in gus activity of, and -fold, respectively. the metal concentrations used in this assay spanned a range from moderately to severally toxic, with the highest concentrations of each metal causing complete loss of turger in the seedlings. to assess the level of toxicity at the end of this h assay rates of k+ leakage from the seedlings were measured. all metal treatments caused an increase in k+ leakage peaking at μm cd2+, and μm ni2+ and zn2+, after which leakage rates dropped and this was associated with a lose of both seedling turger and gus activity. histochemical gus staining of both cd2+ and ni2+ treated seedlings demonstrated that the gus protein product of the pbjmtp1::gusplus construct is localized mainly in the roots, showing strong expression at the root-shoot transition zone. in the untreated transgenic seedlings, there was no observable gus staining. plants stably transformed with pbjmtp1::gusplus were also grown in hydroponic culture for four weeks and transferred to medium of the same composition with the addition of μm ni2+. root and shoot samples were taken over a h time course and both ni2+ accumulation and gus activity measured. ni2+ accumulated in both roots and shoots almost linearly with time, with roots accumulating ni2+ at approximately times the rate of the shoots. in root samples, gus enzyme activity increased linearly as the ni2+ exposure time increased, with a fold increase in gus activity after h ni2+ treatment compared to basal expression, or to untransformed control plants. in shoots, the overall gus activity was less than in the roots. interestingly, in shoots gus activity only transiently increased after h exposure, compared to untransformed plants, after which gus activity decreased to levels observed at h exposure, even though ni2+ accumulation in shoots continued. similar results for both root and shoot expression were also obtained with plants stably transformed with pbjmtp1::eyfp, and pbjmtp1::mrfp. a comparison of total tissue ni2+ accumulation and gus activity revealed a strong positive correlation between the level of ni2+ accumulation and gus activity in roots. in order to test whether the induction process is reversible, after h ni2+ treatment plants were transferred to nutrient solution lacking ni2+. after a further h recovery gus activity in roots was observed to return to that observed at h exposure. histochemical gus analysis of roots from four-week old plants, after h ni2+ treatment, demonstrated clear gus expression throughout the main root. after h ni2+ treatment gus expression was not detected in the stem. gus expression was, however, observed in the vasculature of the leaves and in the vascular tissues of the anthers and the stigma after hr ni2+ exposure. the regulatory competency of the bp, bp and bp dna sequences upstream of the bjmtp translations start site were compared to establish if further metal regulatory elements exist upstream of the bp fragment. the transcriptional activity of these dna fragments was assessed after construction and transformation of b. juncea with transcriptional fusions with the monomeric red fluorescent protein as a reporter gene. hydroponically grown four-week-old b. juncea plants transformed with the mrfp reporter constructs were exposed to either μm cd2+, μm ni2+ or μm zn2+ in aerated × hoaglands solution. root and shoot samples were collected over a h time-course and mrfp expression quantified using a luminescence spectrometer. as a control for metal related effects on the in vivo stability of mrfp pcamv35s:: mrfp transformed plants were also generated and treated in a similar manner. in roots isolated from ni2+ treated plants, mrfp accumulation increased linearly with time of ni2+ exposure, and the kinetics of mrfp accumulation were equivalent regardless of the size of the promoter construct used to drive mrfp expression. all three promoter constructs produced higher mrfp accumulation after h ni2+ exposure than observed with the camv35s promoter. plants were removed from the ni2+ containing medium after h exposure and allowed to recover for a further h. during this recovery period, mrfp accumulation decreased linearly with time in a similar manner regardless of the size of the promoter region. in leaves from ni2+ exposed plants, all three promoter constructs drove a similar transient increase in mrfp accumulation that peaked at h ni2+ treatment, after which they declined at a similar rate to the response observed for the promoter gus construct. mrfp expression in plants transformed with pcamv35s::mrfp was constant at all time points and tissues after ni2+ exposure. similar mrfp expression was also obtained when plants were exposed to μm cd2+ for h and allowed to recover from cd2+ exposure for a further h. mrfp accumulated rapidly in roots after cd2+ exposure to levels equivalent to expression driven by pcamv35s. removal of plants from the cd2+ containing nutrient solution caused a rapid drop in mrfp accumulation, with mrfp levels returning to those observed prior to cd2+ exposure, after h recovery. again, all three-promoter sizes gave similar responses. cd2+ treatment also produced a transient accumulation of mrfp in shoots after h exposure, as observed during ni2+ exposure. unlike ni2+ and cd2+ exposure, zn2+ treatment produced no significant alteration in mrfp accumulation in either root or shoot tissue. elemental analysis of the nutrient solutions during the ni2+, cd2+ and zn2+ experiments confirmed that concentrations of ni2+, cd2+ and zn2+ did not vary significantly in the solution during the course of the experiments. response of the bp promoter element to other abiotic stresses the bp promoter region, which was found to be sufficient for expression in response to cd2+ and ni2+, was also tested for its ability to activate transcription when seedlings were exposed to other abiotic stresses including cold, heat, nacl and h2o five-day-old b. juncea seedlings stably transformed with p bjmtp1::gusplus were exposed to a cold or heat shock, and also mm nacl, or h2o after which gus activity was assayed. there were no significant increases in gus activity compared to the untreated seedlings for any of the treatments. five-day-old dark grown seedlings stably transformed with pbjmtp1::gusplus were incubated at 4°c for h, 37° c for h, mm nacl and μm h2o and gus activity measured. data represents the average of three independent replicate samples. putative regulatory elements in the bjmtp promoter region a search for putative regulatory dna elements within the bp promoter region of bjmtp was performed using the place and plantcare databases. dna regulatory motifs such as the ccaat boxes, abre, bzip, g box, and several pathogen responsive elements such as w box, eire, sebf motif, gcc box, g box coupler and myb core elements were found. however, no known metal regulatory elements such as mres which are known to mediate zn2+ and cd2+ specific transcriptional activation of metallothionine genes in mammals were found. considering that atmtp appears not to be regulated by cd2+ and bjmtp is, we compared the bp bjmtp promoter region with a similar region of the a. thaliana atmtp sequence upstream of the transcriptional start site. even though both promoter regions had many known regulatory elements in common, namely bzip, g box, w box, eire box and myb core element, the bjmtp promoter region also has several unique regulatory elements, namely abre and pathogen related sebf motif, gcc box and g box coupler elements. the core sequence of the metal regulatory element found in mammals, is present in the full bp upstream region of bjmtp with a single base pair difference. however, because this motif is not present in the bp promoter region sufficient for cd2+ and ni2+ regulation, this mre is unlikely to play a role in the cd2+ and ni2+ transcriptional regulation of bjmtp localization of bjmtp expression to root xylem parenchyma our results demonstrate that a bp region upstream of the translational start site of bjmtp is sufficient to drive strong root expression of gus and mrfp in response to cd2+ or ni2+ treatment. however, in order to help understand the function of bjmtp it is also important to identify which root tissues are expressing bjmtp during the response to cd2+ or ni2+ exposure. sections from the main root and intact lateral roots of four-week-old b. juncea plants expressing mrfp in response to ni2+ were analyzed by epifluorescence and light microscope. sections were taken from the top of the main root as well as cm from the root tip, and the morphology of the roots examined. the main root has clearly undergone secondary growth. the epidermis found in the primary roots has been replaced in 4-week-old roots by periderm produced by the cork cambium. within the periderm lies the pericycle, and the cambial zone which gives rise to the secondary phloem and secondary xylem. the secondary xylem parenchyma, produced by vascular cambium, was observed surrounding the secondary xylem vessels. the presence of diarch xylem at the center of the root section is a characteristic of the brassicaceae family. sections taken cm from the tip of the main root were observed to have similar root architecture to sections taken from the top of the root. after exposure to ni2+ mrfp fluorescence was observed to be specifically localized in the secondary xylem parenchyma tissues that surround the secondary xylem. fluorescence from mrfp was also present in the periderm but no significant fluorescence was observed in other root tissues. in the lateral roots, mrfp fluorescence was clearly evident in the vascular region. after ni2+ exposure no fluorescence was observed in roots from plants identified as null segregants for the reporter construct even after longer exposures, compared to the shorter exposure for ni2+-treated transgenic plants. cd2+ treated roots gave an equivalent expression pattern to that of ni2+ treated plants. in both ni2+ and cd2+ treated roots there was no difference in the pattern of mrfp accumulation between sections taken from the top of the main root and its tip. furthermore, mrfp expression was localized in the secondary xylem parenchyma tissues when expressed from the or bp promoter regions. the strong accumulation of mrfp observed in the vascular tissue of intact lateral roots is also consistent with the secondary xylem parenchyma localization observed in the root cross-sections. cross-sections of roots prepared from ni2+ exposed plants transformed with the gusplus reporter gene also revealed strong gus expression in the secondary xylem parenchyma surrounding the secondary xylem, similar to that observed for mrfp expression. secondary xylem parenchyma localization was also observed in plants expressing a eyfp reporter driven by the bp promoter region. discussion here we establish that mrna levels of bjmtp in b. juncea are transcriptionally regulated in response to cd2+ and ni2+, with maximal expression in the root xylem parenchyma cells. we identify a bp dna fragment, within the total bp dna sequence upstream of the bjmtp translational start site, which is sufficient for this transcriptional regulation, which appears specific to stress induced by cd2+ and ni2+. bjmtp promoter activity in response to ni2+, cd2+ and zn2+ was assessed at concentrations that span a similar range of toxicities from mild to serve. over these similar ranges of metal-induced stress only ni2+ and cd2+ were observed to elicit a transcriptional response from the bjmtp promoter, with zn2+ induced stress having no significant effect. other forms of abiotic stress including cold, heat or nacl also produced no significant transcriptional response. transcriptional activation of bjmtp promoter by cd2+ and ni2+ is also not an indirect response to oxidative stress, since this promoter region is not activated by direct exposure to h2o our results are consistent with the observation in a. thaliana that the homologue atmtp is also not regulated by zn2+. however, our observation that bjmtp expression is transcriptionally regulated by cd2+ and ni2+ is not consistent with that observed in a. thaliana, where atmtp mrna levels are unaffected by cd2+. our results suggests that rather than being a house-keeping gene involved in zn2+ homeostasis as has been suggested in a. thaliana, in b. juncea bjmtp may be involved in the dynamic regulation of zn homeostasis as part of the plant's response to cd2+ and ni2+ induced stress. we emphasize that our data does not suggest that the function of bjmtp is to transport cd2+ or ni2+, for which we have no evidence. however, increased expression of bjmtp under cd2+ or ni2+ stress may be required to adjust zn homeostasis as a response to the stress imposed by exposure to cd2+ or ni2+. this is supported by our observation that expression of bjmtp promoter is dynamically regulated in response to these metals, rapidly increasing after exposure to cd2+ and ni2+, and rapidly returning back to basal levels after these metals are removed from the growth medium. though expression of the bjmtp promoter in response to cd2+ and ni2+ is highest in roots, expression was also observed in the vascular tissue of leaves, anthers and in the stigma. expression of bjmtp in these tissues suggests that the bjmtp protein may also be involved in the plants response to cd2+ and ni2+ stress in leaves and inflorescences. though transcriptional activation of the bp promoter region in roots by cd2+ or ni2+ occurs nearly in a linear fashion with time of exposure and accumulation, shoot activation appears transient. after h of metal exposure transcription of bjmtp reaches a maximum and then declines to baseline expression after h. such transient expression is intriguing considering that over the same time frame ni2+ accumulates linearly in shoots. the different expression patterns of bjmtp promoter in roots and shoots imply that this protein plays different roles in the plant's coordinated response to cd2+ and ni2+ exposure, though what these roles are remain unclear at this time. in five-day-old seedlings of b. juncea expression of bjmtp promoter in response to cd2+ and ni2+ occurs throughout the seedlings, though is especially localized to the root and shoot-root transition zone. though the reasons for stronger expression bjmtp promoter in the shoot-root transition zone are not known, this type of expression pattern has been observed previously for several proteins, including naphthylphthalmic acid associated amino peptidases, glutathione s-transferase atgstf the auxin transporter pin and pgp an abc type transporter. when compared to a. thaliana, the unique elements present in the bjmtp promoter region, including abre, sebf motif, gcc box and g box coupler elements all have known functions in aba signaling or pathogen related stress. however, none of these promoter elements have been established to play a role in signaling cd2+ or ni2+ stress. the elements responsible for regulation of bjmtp expression in response to cd2+ and ni2+ remain to be identified. this study is the first to identify transcriptional regulation and tissue localization of bjmtp however, further work is needed to understand the functional role of bjmtp in b. juncea's response to ni2+ and cd2+.  here we conclude that a bp dna fragment upstream of the bjmtp translational start site is sufficient for the specific activation by ni2+ and cd2+ of bjmtp expression primarily in roots. activation of expression by both metals in roots is primarily localized to the xylem parenchyma cells. this study is the first to identify specific ni2+ and cd2+ transcriptional regulation and tissue localization of bjmtp and supports the conclusion that bjmtp is involved in the response of b. juncea to ni2+ and cd2+ exposure. 
activation of expression by both metals in roots is primarily localized to the xylem parenchyma cells. gus expression in ni2+ treated seedlings was localized in roots, particularly in the root-shoot transition zone. in shoots gus expression was prominently present in the vasculature of leaves, and floral parts. our studies establish that a bp dna fragment upstream of the bjmtp translational start site is sufficient for the specific activation by ni2+ and cd2+ of bjmtp expression primarily in roots. this study is the first to identify specific ni2+ and cd2+ transcriptional regulation and tissue localization of bjmtp here we present data on the transcriptional regulation and localization of expression of the homologous gene bjmtp in brassica juncea. our goal in this study is to determine the transcriptional regulation of mtp in order to start to better understanding the physiological role of mtp in b. juncea. though b. juncea lacks the ability to hyperaccumulate metals, its relatively high biomass, rapid growth and relatedness to true metal hyperaccumulating plants makes it a promising starting point for the development of plants for phytoremediation. steady-state mrna levels of bjmtp were found to be enhanced,, and -fold in five-day-old b. juncea seedlings after exposure to ni2+, cd2+ or zn2+, respectively. this was also reflected in enhanced gus activity in b. juncea seedlings transformed with bjmtp promoter::gusplus after exposure to these metals over a similar range of toxicities from mild to severe. however, no increase in gus activity was observed after exposure of seedlings to cold or heat stress, nacl or hydrogen peroxide. in four- week- old transgenic plants bjmtp promoter activity also primarily increased in roots in response to ni2+ or cd2+ in plants transformed with either gus or mrfp as reporter genes, and expression was localized to the secondary xylem parenchyma.
 metal hyperaccumulators, including various thlaspi species, constitutively express the putative metal transporter mtp to high levels in shoots. here we present data on the transcriptional regulation and localization of expression of the homologous gene bjmtp in brassica juncea. though b. juncea lacks the ability to hyperaccumulate metals, its relatively high biomass, rapid growth and relatedness to true metal hyperaccumulating plants makes it a promising starting point for the development of plants for phytoremediation. our goal in this study is to determine the transcriptional regulation of mtp in order to start to better understanding the physiological role of mtp in b. juncea.  steady-state mrna levels of bjmtp were found to be enhanced,, and -fold in five-day-old b. juncea seedlings after exposure to ni2+, cd2+ or zn2+, respectively. this was also reflected in enhanced gus activity in b. juncea seedlings transformed with bjmtp promoter::gusplus after exposure to these metals over a similar range of toxicities from mild to severe. however, no increase in gus activity was observed after exposure of seedlings to cold or heat stress, nacl or hydrogen peroxide. gus expression in ni2+ treated seedlings was localized in roots, particularly in the root-shoot transition zone. in four- week- old transgenic plants bjmtp promoter activity also primarily increased in roots in response to ni2+ or cd2+ in plants transformed with either gus or mrfp as reporter genes, and expression was localized to the secondary xylem parenchyma. in leaves, bjmtp promoter activity in response to ni2+ or cd2+ spiked after h then decreased. in shoots gus expression was prominently present in the vasculature of leaves, and floral parts.  our studies establish that a bp dna fragment upstream of the bjmtp translational start site is sufficient for the specific activation by ni2+ and cd2+ of bjmtp expression primarily in roots. activation of expression by both metals in roots is primarily localized to the xylem parenchyma cells. this study is the first to identify specific ni2+ and cd2+ transcriptional regulation and tissue localization of bjmtp 