BACKGROUND
polychlorinated dibenzo-p-dioxins  and polychlorinated dibenzofurans , collectively termed dioxins, are the most toxic group of persistent organic pollutants .
composed of two aromatic rings linked via one  or two atom of oxygen  and one to eight related chlorine atoms, these halogenated chemicals are structurally very stable and extremely hydrophobic.
therefore, dioxins can persist in the environment and bioaccumulate at the top of food chain  <cit> .
humans may become exposed to dioxins mainly through food and less by inhalation or dermal contact.
<dig> , <dig> -tetrachlorodibenzo-p-dioxin , with a toxic equivalency factor  of  <dig> , is the most toxic congener of dioxins.
consequently, tcdd was used as a good candidate for investigations of the physiological and toxicological effects of this class of chemicals .
in mammals, dioxins essentially accumulate in fats because of their high lipophilicity.
for example, they reached maximal levels in liver adipose lipids and in milk lipid droplets  <cit> .
in contrast low levels of these xenobiotics were measured in brain tissue  <cit> .
the affinity of dioxins to lipids seems to be modulated by the biochemical nature of the particular lipids concerned.
the accumulation of dioxins was observed to be highest in the lipid fraction composed of triglycerides than in those composed of phospholipids  <cit> .
also, it is well known that tcdd seriously affects lipid metabolism in exposed mammals.
for example, exposure to tcdd increases membrane lipid oxidation and phospholipase  activity, which in turn could increase the pool of free arachidonic acid   <cit> .
moreover, exposure to tcdd may target aa metabolism downstream of pla <dig> by inducing the enzymes which metabolize such fatty acids, the cytochrome p <dig>  the cyclooxygenase and probably the lipoxygenase pathways  <cit> .
at the bottom of the food chain, plants are increasingly and persistently exposed to pcdd/fs.
such xenobiotics cannot be used for nutrition or as a source of energy, but are nevertheless taken up and accumulated in plant tissues.
pcdd/fs-bioaccumulation in plant may has a serious impact on plant health, but also can contribute to bio-transmission of these xenobiotics to the top of food chain.
these concerns led research efforts to focus on the biological capacity of plants to uptake contaminants from the soil via their roots and then translocate them into upper parts for storage, a mechanism called phytoextraction  <cit> .
due to their high hydrophobicity and low mobility, uptake of dioxins may not be readily accomplished by a passive diffusion in plants  <cit> .
there are however, a very limited number of reports about the capacity of a few plants to uptake dioxins from the environment.
for example, it has been documented that a variety of zucchini plant  accumulated various dioxin congeners and that their accumulation in roots depended on their hydrophobicity  <cit> .
uptake by plants of polychlorinated biphenyl , few of them known also as dioxin-like compounds, has been more commonly reported.
it has been found that some plant species, such as solidago canadensis, vicia cracca, chrysanthemum sp., and polygonumpersicaria sp., specifically transmitted pcbs into aerial parts and they are known as pcbs accumulators  <cit> .
in common with animals, plant lipids and their metabolites, mainly those derived from c18-unsaturated fatty acids are involved in many biological functions enabling plant to overcome biotic and abiotic stress including environmental pollutants  <cit> .
in contrast, the biological connections between dioxins and plant lipids remain largely unknown.
we recently described that tcdd administration to arabidopsis plant caused phytotoxicity effects including a decrease in seed germination, in fresh weight and in chlorophyll content, but it induced the formation of lateral roots.
additionally, the uptake of tcdd by arabidopsis provoked an enhanced level of hydrogen peroxide h2o <dig> and a massive stimulation of anti-oxidative enzyme activities  <cit> .
in the current study, three main issues were therefore addressed: i) determination of the accumulation and translocation of tcdd in the tissues of arabidopsis during a whole growth cycle.
ii) effect of tcdd on the chronology of principal growth stages of arabidopsis and its consequent impact on plant yield.
iii) as tcdd has a high affinity to lipids, modifications in fatty acids content and their peroxidation in arabidopsis tissues and seeds after exposure to tcdd were demonstrated.
findings from this work will contribute to understand how plants respond to dioxins in the environment, a question which is of great importance.
RESULTS
tcdd is up-taken and accumulated in arabidopsis tissues
hr/gc-ms diagrams presented in additional file  <dig> show the presence of a single peak, corresponding to the tcdd , in the organic extracts from the root of 30-days old arabidopsis plants grown in the presence of various concentrations of tcdd  <dig>   <dig> and 100 ng l− <dig> .
similarly, the tcdd peak was also detected in the extracts from the shoot of the same plants .
compared to the standard curve of commercial tcdd , the content of tcdd in plant tissues paralleled its initial concentration in the media.
plants roots exposed to  <dig>   <dig> and 100 ng l− <dig> of tcdd contained  <dig>  ±  <dig> ,  <dig>  ±  <dig>  and  <dig>  ±  <dig>  pg g− <dig> fw tcdd, respectively , while tcdd-content in the shoots did not exceeded  <dig>  ±  <dig> ,  <dig>  ±  <dig>  and  <dig>  ±  <dig>  pg g− <dig> fw.
the recovery of tcdd from the spiked samples was approximately 97 % .
these results indicate that tcdd is taken up by the roots and subsequently translocated into the aerial parts of arabidopsis.fig.
<dig> detection and quantification of tcdd in arabidopsis tissues by hr/gc-ms. plants were grown for 30-days in glass tubes containing ms media supplemented with various concentrations of tcdd  <dig>   <dig>   <dig> and 100 ng l− <dig>  in each treatment, shoots and roots of plant were taken and subjected separately to the extraction and analysis of tcdd.
concentration of tcdd in plant tissues was expressed as pg g− <dig> fresh weight.
inset, a representative hr/gc-ms diagram indicating the peak of tcdd  in the plant extract.
three measurements were done for three individual plants.
data are mean values ± sd
tcdd uptake depends on the growth stage of arabidopsis
to determine a possible variation in the plant capacity to uptake the tcdd throughout the plant life cycle, we quantified the content of tcdd in arabidopsis shoot and root at different stages of development, corresponding to  <dig>   <dig>   <dig>   <dig> and 60 days after tcdd administration.
as shown in fig.
2a, we started to detect the tcdd on day  <dig> in the shoot of arabidopsis with its content reaching 6 ±  <dig> , 23 ±  <dig>  and 26 ±  <dig>  pg g− <dig> fw.
tcdd content had doubled on day  <dig> and tripled on day  <dig>  then reached a plateau on day  <dig> post tcdd-exposure.
similarly, tcdd found to be detected in arabidopsis root on day  <dig> and its accumulation tended to peak 36 days after treatment .
globally, the tcdd accumulation tended to be superposed in the shoots and roots of arabidopsis throughout the plant life cycle.
these data indicate that the tcdd uptake capacity of arabidopsis varies throughout its life cycle and reaches its maximum on day  <dig> fig.
<dig> differential accumulation of tcdd in various tissues of arabidopsis as a function of the growth stage.
arabidopsis seeds were sown directly on tcdd-supplemented ms media.
tcdd content was quantified in the shoot  and in the root  of arabidopsis by hr/gc-ms at the indicated times.
tcdd content was then quantified in the leaves, stem, flowers, siliques and seeds of 60-days old plants grown in the presence of  <dig>   <dig> and 100 ng l− <dig> tcdd .
three measurements were taken for three individual plants at indicate times.
values are mean ± sd .
fw: fresh weight
tcdd is accumulated mainly in leaves and seeds of arabidopsis
we next evaluated the accumulation of tcdd in the upper parts of the arabidopsis plant: i.e.
leaves, stem, flowers, siliques and seeds.
leaves were the most active accumulators of tcdd, followed by seeds and siliques .
leaves accumulated tcdd at levels  <dig> to  <dig> times higher than stems and  <dig> to  <dig> times higher than flowers.
thus it seems that tcdd accumulates preferentially in leaves and seeds.
tcdd shifts up the chronology of principal growth stages of arabidopsis
we previously reported that tcdd had an inhibiting effect on the seed germination of arabidopsis and affected the biomass and morphology of survival plants  <cit> .
here, we investigated the chronological effect of tcdd on the life cycle of the arabidopsis plant including principal growth stages: i.e.
germination, leaf formation, flowering and siliques ripening.
compared with controls, completion of the growth stages of tcdd-treated plants were delayed  <dig>   <dig> and 30 days when they were exposed to  <dig>   <dig> and 100 ng l− <dig> tcdd, respectively .
the stages of leaf formation and siliques ripening were especially affected with completion delayed  <dig> and 18 days at the highest dose of tcdd .
therefore the subsequent delay in flowering time  was marked.
the duration of germination and flowering stages were not influenced by tcdd.
altogether, these data indicate that tcdd slows down the arabidopsis growth cycle mainly by affecting leaf development and siliques maturation.fig.
<dig> chronological effect of tcdd on the principal growth stages of arabidopsis plant.
a. a representative image of plants growth in the presence or absence of tcdd under in vitro conditions.
seeds were directly sown into ms-tubes and left for germination as described before.
image was taken on day  <dig> after sowing.
b. presentation idea is inspired from boyes et al.,  <cit> .
plants were grown in the presence of tcdd with the indicated doses.
start-point and end-point for each stage of arabidopsis development including germination, leaves formation, flowering and siliques ripening were determined according to boyes et al.
<cit> .
measurements for each stage were taken for six individual plants.
data are mean values ± sd
tcdd affects both yield and oil content of arabidopsis seeds
the effect of tcdd on the productivity of arabidopsis was addressed.
seeds produced from tcdd-exposed plants were qualitatively and quantitatively examined.
first, siliques number per plant decreased markedly from  <dig> siliques in non-exposed plants to  <dig>   <dig> and  <dig> siliques in tcdd-exposed plants at the indicated doses, as shown in fig.
4a.
moreover, the mean weight of arabidopsis seeds was seriously affected as a function of tcdd administration.
the mean weight per plant was significantly reduced from 98 mg in control plants to  <dig>   <dig> and 61 mg in tcdd-exposed plants at indicated concentrations, respectively .
next, we examined the oil content in the harvested seeds as a relationship of tcdd-exposure.
figure 4b shows that seeds lost approximately  <dig>   <dig> and 54 % of their total oil as a result of tcdd treatment with the indicated concentrations.
furthermore, the vitality of seeds produced from the tcdd-treated plants have decreased by half compared with those produced from non-treated plants .
these results indicate that exposure to tcdd yields plants with fewer seeds of a reduced oil content and a low vitality.fig.
<dig> effect of tcdd on seed yield of arabidopsis.
a. the final number of siliques per plant was determined after the completion of flower production.
yield is reported as the desiccated mass  of seed produced per plant.
b. seed oil content is expressed as percentage of seed dry weight.
c. vitality of seeds produced from tcdd-treated plants compared with seeds of non-treated plants.
ten seeds were sown on ms-plate and left for germination as described before.
three measurements were taken for three individual plants.
data are mean values ± sd .
dw: dry weight.
three independent biological experiments were analyzed.
statistical significance of the data was evaluated by anova analysis.
asterisks indicate significant differences between treatment and control: *p <  <dig>  ; **p <  <dig>
tcdd affects lipid metabolism and induces a high level of lipid peroxidation in arabidopsis
tcdd is known to affect lipids in mammals  <cit> .
we investigated whether it has the same effect in plants by analyzing the content of the most abundant unsaturated fatty acids i.e.
oleic , linoleic  and linolenic  in post 36-day tcdd-exposed plants .
tcdd exposure led to a decrease in the content of all these fatty acids with linolenic acid being the most affected.
compared with control, the content of c18: <dig> declined  <dig>  fold in plant tissues after exposure to 50 ng l− <dig>  while the contents of c18: <dig> and c18: <dig> were reduced  <dig>  and  <dig>  fold, respectively.
these results suggest that exposure to tcdd provokes a net reduction of the c18-unsaturated fa content in arabidopsis tissues.
moreover, it is well known that the exposure of plants to xenobiotic leads to lipid peroxidation  <cit> .
to test this possibility, we measured the levels of total lipid peroxides over time in whole arabidopsis plants after treatment with tcdd.
the data in fig.
5b shows that lipid peroxides increased progressively and significantly in tcdd-exposed plants aged between  <dig> and 36 days.
lipid peroxidation considerably declined in these plants on days  <dig> and  <dig> but remained slightly higher in the same tissues of control plants .
these data suggest that exposure to tcdd induces lipid peroxidation in arabidopsis tissues.fig.
<dig> lipid peroxidation as response to tcdd-exposure.
a. content of major c18-fatty acids  in arabidopsis shoot on day  <dig> after exposure to tcdd at indicated doses.
b. total hydroperoxides produced by untreated and tcdd-treated arabidopsis tissues was monitored using fox- <dig> assay at various stage of development.
three independent plants were examined at each concentration of tcdd.
three measurements were taken per extract.
data are mean values ± sd .
fw: fresh weight.
statistical significance of the data was evaluated by anova analysis.
asterisks indicate significant differences in lipid peroxides according to the plant age compared to germination stage : *p <  <dig>  ; **p <  <dig>
tcdd-induced hydroperoxides are essentially derived from loxs pathways
unsaturated fatty acid hydroperoxides can be formed either chemically  <cit>  or enzymatically under the action of α-dioxygenases   <cit>  and lipoxygenases   <cit> .
arabidopsis contains six genes encode loxs  <cit>  and at least two genes encode α-doxs  <cit> .
transcriptional analysis of loxs genes showed an up-regulation of lox <dig>  lox <dig>  lox <dig> and lox <dig> in whole plants aged 36 days after exposure to tcdd, whereas the expression levels of lox <dig> and lox <dig> were not affected .
the expression of lox <dig>  lox <dig>  lox <dig> and lox <dig> was significantly increased in treated plants for 36 days compared to control and reached about  <dig>   <dig>   <dig> and  <dig>  fold, respectively .
accordingly, the hydroperoxides deriving from c18: <dig> and c18: <dig>  under 9-lox and 13-lox catalysis, accumulated and reached their maximum on day  <dig> then declined on days  <dig> and  <dig>  the accumulation of hydroperoxide derivatives from c18: <dig> were higher than those of c18: <dig>  the highest being 9-hpod  followed by 13-hpod  then 13-hpot  and 9-hpot  as shown in fig.
6c, d, e, f, g and h. these results taken together suggest that the exposure of arabidopsis to a high dose of tcdd leads to the accumulation of fatty acid hydroperoxides probably resulting from the up-regulation of lox genes expression.fig.
<dig> accumulation of lox-derived fatty acids hydroperoxides in tcdd-exposed arabidopsis.
a. transcriptional fold changes of lipoxyganse encoding genes  in whole plants after exposure to tcdd  for  <dig>   <dig>   <dig>   <dig>   <dig> and 60 days.
for each gene, the transcript level was estimated by qrt-pcr as described in methods.
b. quantification of lox genes expression in arabidopsis tissues after treatment with indicated concentrations of tcdd for 36 days.
three measurements were taken in three cdnas prepared from three individual plants for each tcdd-treatment.
different lowercase letters indicate significant differences in the expression for each lox gene according to various concentrations of tcdd and control: a
p <  <dig>  ; b
p <  <dig>  .
asterisks indicate significant differences between the expression of loxs genes according to each treatment: *p <  <dig>  ; **p <  <dig>  .
c and d. uv-hplc-separation of 9- or 13-hpod and 9- or 13-hpot extracted from tcdd-exposed arabidopsis tissues at different stages of development.
e, f, g and h. the four major hydroperoxide fatty acids  of arabidopsis were extracted and quantified as described in methods.
for each hydroperoxide, three measurements were taken in three individual plants.
data are mean values ± sd .
fw: fresh weight.
statistical significance of the data was evaluated by anova analysis and duncan’s multiple range test.
asterisks indicate significant differences in lipid peroxides according to the plant age compared to germination stage : *p <  <dig>  ; **p <  <dig>
fig.
<dig> representative schema for tcdd accumulation and its subsequent physiological and biochemical effects on arabidopsis.
tcdd is up-taken by the root and accumulated into the upper parts especially in the leaves and the mature seeds.
the salient biological features affected by tcdd-exposed plant are: flowering time, siliques repining and seeds yield.
polyunsaturated fatty acids  and their metabolism are seriously alerted by tcdd.
thus, the role of lipid metabolism as a response to tcdd-exposure in plant merits a particular focus
discussion
we have recently reported that the immediate uptake of  <dig> , <dig> -tcdd by arabidopsis yielded various phytotoxicological effects  <cit> .
herein, we have shown that the accumulation and translocation of tcdd in arabidopsis plant depended on the growth stage.
moreover, tcdd-exposed arabidopsis plants were affected in their lipid metabolism, flowered late and produced less seeds than non-exposed plants.
these results are summarized in fig.
<dig>
using hr/gc-ms analysis, we showed that tcdd-accumulation in arabidopsis plants was ordered in a tissues-specific manner, mainly in the root and less in the shoot.
these estimations confirm our previous data  <cit>  and come in line with previous reports on the natural ability of various plant species to take up dioxins/furans from their environment .
from the upper parts, we observed that the highest levels of tcdd were found in leaves and mature seeds of arabidopsis.
the accumulation of tcdd in seeds can be explained by the high affinity of tcdd toward lipids that are abundantly present in brassicaceae seeds  <cit> .
indeed, it is well known that dioxins essentially bioaccumulate in animal fats because of their high lipophilicity  <cit> .
accordingly, dioxin and dioxin-like compounds were often detected in rape and olive oils  <cit> .
moreover, the sorption of hydrophobic organic compounds  by lipid bodies of rapeseeds as a hoc-removal strategy is well documented  <cit> .
similarly, the high concentration of tcdd in leaves might result from the high affinity of tcdd for the lipid-membrane of chloroplasts, mitochondria and peroxisomes.
in this context, it is known that the major xenobiotic-oxidations catalyzed by enzymatic systems take place in the endoplasmic reticulum and in the membrane of chloroplasts and peroxisomes  <cit> .
accordingly, data published from proteomic analyses carried out on whole tissue/organ preparations of arabidopsis revealed a total of  <dig> environmental stress responding proteins.
most of them were located in chloroplast, mitochondria and peroxisome  <cit> .
thus, accumulation of dioxins in vegetative tissues and in seeds may reflect their biological fate in plants and suggest a possible tissue-specific mechanism for accumulation and subsequent detoxification of tcdd in plants.
a point of a particular importance is the mechanism responsible of translocation of tcdd in plant.
although the molecular and biochemical mechanisms involved in reception, translocation, genes activation and enzymes metabolism of tcdd are well documented in mammals, no data however are available to date about these mechanisms in plants.
as many of xenobiotics including dioxins are highly lipophilic, they can accumulate to toxic levels in the plant tissues, unless effective means of detoxification are present.
in the case of tcdd, one of the most active detoxification systems in plants can probably disposed of this xenobiotic by two sequential processes: a chemical transformation and a subsequent compartmentation  <cit> .
the chemical transformation of lipophilic xenobiotics in plants is of two types: phase i, the activation reactions and phase ii, the conjugation reactions.
the primary function of the phase i is to create reactive sites in the xenobiotic by the addition of functional groups  which make it more hydrophilic and prepare it therefore for phase ii reactions, the conjugation to glutathione  <cit> .
after having achieved these two protective phases, such xenobiotics are transported and accumulated in apoplastic cell walls or central vacuoles in plant cells.
biochemical, molecular, and genetic evidences have been reported on the functions of a handful of atp-binding cassette and multidrug and toxic compound extrusion family transporters engaged in transport of organic xenobiotics  <cit> .
from them, p-glycoprotein, identified in plants  <cit>  as a vacuolar glutathione-conjugate transporter, has some attractions to be involved in tcdd transportation.
another possible and potential transporter of tcdd can be a protein, called mrp , which is recognized as a glutathione-conjugate transporter  <cit> .
however, the nature and the mechanism of tcdd transportation is still yet uncharacterized.
a chronological effect on the principle growth stages has been also observed.
regardless of the level of exposure, our results showed that the tcdd-content was maximal on day  <dig> post-administration.
at this developmental stage, arabidopsis possesses a complete rosette growth and an initial emergence of inflorescence.
the high level of tcdd found in plant tissues at this stage of growth could be explained by the optimal development and extension of the lateral roots of arabidopsis which seemed to be induced by tcdd  <cit> .
moreover, the biomass of vegetative tissues reached its maximum in this stage  <cit> .
absorption of tcdd delayed flowering time  through presently undetermined mechanisms.
one possible mode of action of tcdd might be linked to its up-regulation of various myb transcription factor genes in the root of arabidopsis  <cit> .
flowering time is in part regulated through the transcriptional regulation of flowering locus t .
interestingly, it was recently reported that a myb transcription factor early flowering myb protein  plays an important role in directly repressing flt expression in the root and leaf vasculature under normal conditions  <cit> .
following this hypothesis, the activating of myb factors by tcdd would repress flt and thus delays flowering.
alternatively, the strong decline of c18: <dig> might indicate a role of pufas in response to tcdd.
in particular, tcdd might act through oxidized lipids.
we observed here that the amount of lipid peroxides tripled 36 days after exposure to tcdd, this effect seems similar to the previous increase level of lipid peroxides in suspensions of tobacco cells when submitted to dioxins exposure  <cit> .
lipid oxidation is derived from chemical and enzymatic processes  <cit> .
however the parallel rise in transcripts levels of 9-lox and 13-lox genes with the accumulations of 9- and 13-ooh deriving from linolei  ic acids suggests that the arabidopsis response to tcdd occurred predominantly via lox pathway.
the involvement of the lox pathway in responses of plants to tress-induced senescence  <cit>  and heavy metals tolerance  <cit>  has been reported.
the lox pathway is also involved during the regulation of lateral root development and flowering  <cit> .
in particular, transition from vegetative growth to flowering in arabidopsis was associated with the accumulation of 13-hpot, resulting from the oxidation of linolenic acid by lox  <cit> .
it is tempting to hypothesize that the delay of accumulation of 13-hoo-fa in tcdd-treated plants might postpone flowering time.
in addition oxylipins deriving from the reduction of 13-ooh-fa might play a role in the control of flowering transition.
indeed, overexpression of the caleosin/peroxygenase rd <dig> that catalyzes the formation of oh-fa led to early flowering whereas plants deprived of this caleosin flowered later than the control  <cit> .
CONCLUSIONS
in conclusion, as fig.
<dig> summarized, the current work highlights a side of toxicological effects related to the administration of  <dig> , <dig> -tcdd on the arabidopsis plant.
in a tissue-specific manner, the highest tcdd levels were detected in rosette leaves and mature seeds and affected lipid metabolism.
similarly to animals, plants may accumulate tcdd in their lipids by involving few of the fa-metabolizing enzymes for sculpting a new oxylipins “signature” typified to plant tcdd-tolerance.
together, our results will contribute to a better understanding of the mechanisms adopted by plants in response to dioxin contamination, and therefore, these potential strategies protect the plants as well as their environment.

 polychlorinated dibenzo-p-dioxins and polychlorinated dibenzofurans, collectively termed dioxins, are the most toxic group of persistent organic pollutants. composed of two aromatic rings linked via one or two atom of oxygen and one to eight related chlorine atoms, these halogenated chemicals are structurally very stable and extremely hydrophobic. therefore, dioxins can persist in the environment and bioaccumulate at the top of food chain. humans may become exposed to dioxins mainly through food and less by inhalation or dermal contact., -tetrachlorodibenzo-p-dioxin, with a toxic equivalency factor of, is the most toxic congener of dioxins. consequently, tcdd was used as a good candidate for investigations of the physiological and toxicological effects of this class of chemicals. in mammals, dioxins essentially accumulate in fats because of their high lipophilicity. for example, they reached maximal levels in liver adipose lipids and in milk lipid droplets. in contrast low levels of these xenobiotics were measured in brain tissue. the affinity of dioxins to lipids seems to be modulated by the biochemical nature of the particular lipids concerned. the accumulation of dioxins was observed to be highest in the lipid fraction composed of triglycerides than in those composed of phospholipids. also, it is well known that tcdd seriously affects lipid metabolism in exposed mammals. for example, exposure to tcdd increases membrane lipid oxidation and phospholipase activity, which in turn could increase the pool of free arachidonic acid. moreover, exposure to tcdd may target aa metabolism downstream of pla by inducing the enzymes which metabolize such fatty acids, the cytochrome p the cyclooxygenase and probably the lipoxygenase pathways. at the bottom of the food chain, plants are increasingly and persistently exposed to pcdd/fs. such xenobiotics cannot be used for nutrition or as a source of energy, but are nevertheless taken up and accumulated in plant tissues. pcdd/fs-bioaccumulation in plant may has a serious impact on plant health, but also can contribute to bio-transmission of these xenobiotics to the top of food chain. these concerns led research efforts to focus on the biological capacity of plants to uptake contaminants from the soil via their roots and then translocate them into upper parts for storage, a mechanism called phytoextraction. due to their high hydrophobicity and low mobility, uptake of dioxins may not be readily accomplished by a passive diffusion in plants. there are however, a very limited number of reports about the capacity of a few plants to uptake dioxins from the environment. for example, it has been documented that a variety of zucchini plant accumulated various dioxin congeners and that their accumulation in roots depended on their hydrophobicity. uptake by plants of polychlorinated biphenyl, few of them known also as dioxin-like compounds, has been more commonly reported. it has been found that some plant species, such as solidago canadensis, vicia cracca, chrysanthemum sp., and polygonumpersicaria sp., specifically transmitted pcbs into aerial parts and they are known as pcbs accumulators. in common with animals, plant lipids and their metabolites, mainly those derived from c18-unsaturated fatty acids are involved in many biological functions enabling plant to overcome biotic and abiotic stress including environmental pollutants. in contrast, the biological connections between dioxins and plant lipids remain largely unknown. we recently described that tcdd administration to arabidopsis plant caused phytotoxicity effects including a decrease in seed germination, in fresh weight and in chlorophyll content, but it induced the formation of lateral roots. additionally, the uptake of tcdd by arabidopsis provoked an enhanced level of hydrogen peroxide h2o and a massive stimulation of anti-oxidative enzyme activities. in the current study, three main issues were therefore addressed: i) determination of the accumulation and translocation of tcdd in the tissues of arabidopsis during a whole growth cycle. ii) effect of tcdd on the chronology of principal growth stages of arabidopsis and its consequent impact on plant yield. iii) as tcdd has a high affinity to lipids, modifications in fatty acids content and their peroxidation in arabidopsis tissues and seeds after exposure to tcdd were demonstrated. findings from this work will contribute to understand how plants respond to dioxins in the environment, a question which is of great importance.  tcdd is up-taken and accumulated in arabidopsis tissues hr/gc-ms diagrams presented in additional file  show the presence of a single peak, corresponding to the tcdd, in the organic extracts from the root of 30-days old arabidopsis plants grown in the presence of various concentrations of tcdd and 100 ng l−. similarly, the tcdd peak was also detected in the extracts from the shoot of the same plants. compared to the standard curve of commercial tcdd, the content of tcdd in plant tissues paralleled its initial concentration in the media. plants roots exposed to and 100 ng l− of tcdd contained  ± ,  ±  and  ±   pg g− fw tcdd, respectively, while tcdd-content in the shoots did not exceeded  ± ,  ±  and  ±   pg g− fw. the recovery of tcdd from the spiked samples was approximately 97 %. these results indicate that tcdd is taken up by the roots and subsequently translocated into the aerial parts of arabidopsis.fig. detection and quantification of tcdd in arabidopsis tissues by hr/gc-ms. plants were grown for 30-days in glass tubes containing ms media supplemented with various concentrations of tcdd and 100 ng l− in each treatment, shoots and roots of plant were taken and subjected separately to the extraction and analysis of tcdd. concentration of tcdd in plant tissues was expressed as pg g− fresh weight. inset, a representative hr/gc-ms diagram indicating the peak of tcdd in the plant extract. three measurements were done for three individual plants. data are mean values ± sd tcdd uptake depends on the growth stage of arabidopsis to determine a possible variation in the plant capacity to uptake the tcdd throughout the plant life cycle, we quantified the content of tcdd in arabidopsis shoot and root at different stages of development, corresponding to and 60 days after tcdd administration. as shown in fig. 2a, we started to detect the tcdd on day in the shoot of arabidopsis with its content reaching 6 ± , 23 ±  and 26 ±   pg g− fw. tcdd content had doubled on day and tripled on day then reached a plateau on day post tcdd-exposure. similarly, tcdd found to be detected in arabidopsis root on day and its accumulation tended to peak 36 days after treatment. globally, the tcdd accumulation tended to be superposed in the shoots and roots of arabidopsis throughout the plant life cycle. these data indicate that the tcdd uptake capacity of arabidopsis varies throughout its life cycle and reaches its maximum on day fig. differential accumulation of tcdd in various tissues of arabidopsis as a function of the growth stage. arabidopsis seeds were sown directly on tcdd-supplemented ms media. tcdd content was quantified in the shoot and in the root of arabidopsis by hr/gc-ms at the indicated times. tcdd content was then quantified in the leaves, stem, flowers, siliques and seeds of 60-days old plants grown in the presence of and 100 ng l− tcdd. three measurements were taken for three individual plants at indicate times. values are mean ± sd. fw: fresh weight tcdd is accumulated mainly in leaves and seeds of arabidopsis we next evaluated the accumulation of tcdd in the upper parts of the arabidopsis plant: i.e. leaves, stem, flowers, siliques and seeds. leaves were the most active accumulators of tcdd, followed by seeds and siliques. leaves accumulated tcdd at levels to times higher than stems and to times higher than flowers. thus it seems that tcdd accumulates preferentially in leaves and seeds. tcdd shifts up the chronology of principal growth stages of arabidopsis we previously reported that tcdd had an inhibiting effect on the seed germination of arabidopsis and affected the biomass and morphology of survival plants. here, we investigated the chronological effect of tcdd on the life cycle of the arabidopsis plant including principal growth stages: i.e. germination, leaf formation, flowering and siliques ripening. compared with controls, completion of the growth stages of tcdd-treated plants were delayed and 30 days when they were exposed to and 100 ng l− tcdd, respectively. the stages of leaf formation and siliques ripening were especially affected with completion delayed and 18 days at the highest dose of tcdd. therefore the subsequent delay in flowering time was marked. the duration of germination and flowering stages were not influenced by tcdd. altogether, these data indicate that tcdd slows down the arabidopsis growth cycle mainly by affecting leaf development and siliques maturation.fig. chronological effect of tcdd on the principal growth stages of arabidopsis plant. a. a representative image of plants growth in the presence or absence of tcdd under in vitro conditions. seeds were directly sown into ms-tubes and left for germination as described before. image was taken on day after sowing. b. presentation idea is inspired from boyes et al.,. plants were grown in the presence of tcdd with the indicated doses. start-point and end-point for each stage of arabidopsis development including germination, leaves formation, flowering and siliques ripening were determined according to boyes et al.. measurements for each stage were taken for six individual plants. data are mean values ± sd tcdd affects both yield and oil content of arabidopsis seeds the effect of tcdd on the productivity of arabidopsis was addressed. seeds produced from tcdd-exposed plants were qualitatively and quantitatively examined. first, siliques number per plant decreased markedly from siliques in non-exposed plants to and siliques in tcdd-exposed plants at the indicated doses, as shown in fig. 4a. moreover, the mean weight of arabidopsis seeds was seriously affected as a function of tcdd administration. the mean weight per plant was significantly reduced from 98 mg in control plants to and 61 mg in tcdd-exposed plants at indicated concentrations, respectively. next, we examined the oil content in the harvested seeds as a relationship of tcdd-exposure. figure 4b shows that seeds lost approximately and 54 % of their total oil as a result of tcdd treatment with the indicated concentrations. furthermore, the vitality of seeds produced from the tcdd-treated plants have decreased by half compared with those produced from non-treated plants. these results indicate that exposure to tcdd yields plants with fewer seeds of a reduced oil content and a low vitality.fig. effect of tcdd on seed yield of arabidopsis. a. the final number of siliques per plant was determined after the completion of flower production. yield is reported as the desiccated mass of seed produced per plant. b. seed oil content is expressed as percentage of seed dry weight. c. vitality of seeds produced from tcdd-treated plants compared with seeds of non-treated plants. ten seeds were sown on ms-plate and left for germination as described before. three measurements were taken for three individual plants. data are mean values ± sd. dw: dry weight. three independent biological experiments were analyzed. statistical significance of the data was evaluated by anova analysis. asterisks indicate significant differences between treatment and control: *p < ; **p <  tcdd affects lipid metabolism and induces a high level of lipid peroxidation in arabidopsis tcdd is known to affect lipids in mammals. we investigated whether it has the same effect in plants by analyzing the content of the most abundant unsaturated fatty acids i.e. oleic, linoleic and linolenic in post 36-day tcdd-exposed plants. tcdd exposure led to a decrease in the content of all these fatty acids with linolenic acid being the most affected. compared with control, the content of c18: declined fold in plant tissues after exposure to 50 ng l− while the contents of c18: and c18: were reduced and fold, respectively. these results suggest that exposure to tcdd provokes a net reduction of the c18-unsaturated fa content in arabidopsis tissues. moreover, it is well known that the exposure of plants to xenobiotic leads to lipid peroxidation. to test this possibility, we measured the levels of total lipid peroxides over time in whole arabidopsis plants after treatment with tcdd. the data in fig. 5b shows that lipid peroxides increased progressively and significantly in tcdd-exposed plants aged between and 36 days. lipid peroxidation considerably declined in these plants on days and but remained slightly higher in the same tissues of control plants. these data suggest that exposure to tcdd induces lipid peroxidation in arabidopsis tissues.fig. lipid peroxidation as response to tcdd-exposure. a. content of major c18-fatty acids in arabidopsis shoot on day after exposure to tcdd at indicated doses. b. total hydroperoxides produced by untreated and tcdd-treated arabidopsis tissues was monitored using fox- assay at various stage of development. three independent plants were examined at each concentration of tcdd. three measurements were taken per extract. data are mean values ± sd. fw: fresh weight. statistical significance of the data was evaluated by anova analysis. asterisks indicate significant differences in lipid peroxides according to the plant age compared to germination stage: *p < ; **p <  tcdd-induced hydroperoxides are essentially derived from loxs pathways unsaturated fatty acid hydroperoxides can be formed either chemically or enzymatically under the action of α-dioxygenases and lipoxygenases. arabidopsis contains six genes encode loxs and at least two genes encode α-doxs. transcriptional analysis of loxs genes showed an up-regulation of lox lox lox and lox in whole plants aged 36 days after exposure to tcdd, whereas the expression levels of lox and lox were not affected. the expression of lox lox lox and lox was significantly increased in treated plants for 36 days compared to control and reached about and fold, respectively. accordingly, the hydroperoxides deriving from c18: and c18: under 9-lox and 13-lox catalysis, accumulated and reached their maximum on day then declined on days and the accumulation of hydroperoxide derivatives from c18: were higher than those of c18: the highest being 9-hpod followed by 13-hpod then 13-hpot and 9-hpot as shown in fig. 6c, d, e, f, g and h. these results taken together suggest that the exposure of arabidopsis to a high dose of tcdd leads to the accumulation of fatty acid hydroperoxides probably resulting from the up-regulation of lox genes expression.fig. accumulation of lox-derived fatty acids hydroperoxides in tcdd-exposed arabidopsis. a. transcriptional fold changes of lipoxyganse encoding genes in whole plants after exposure to tcdd for and 60 days. for each gene, the transcript level was estimated by qrt-pcr as described in methods. b. quantification of lox genes expression in arabidopsis tissues after treatment with indicated concentrations of tcdd for 36 days. three measurements were taken in three cdnas prepared from three individual plants for each tcdd-treatment. different lowercase letters indicate significant differences in the expression for each lox gene according to various concentrations of tcdd and control: a p < ; b p < . asterisks indicate significant differences between the expression of loxs genes according to each treatment: *p < ; **p < . c and d. uv-hplc-separation of 9- or 13-hpod and 9- or 13-hpot extracted from tcdd-exposed arabidopsis tissues at different stages of development. e, f, g and h. the four major hydroperoxide fatty acids of arabidopsis were extracted and quantified as described in methods. for each hydroperoxide, three measurements were taken in three individual plants. data are mean values ± sd. fw: fresh weight. statistical significance of the data was evaluated by anova analysis and duncan’s multiple range test. asterisks indicate significant differences in lipid peroxides according to the plant age compared to germination stage: *p < ; **p <  fig. representative schema for tcdd accumulation and its subsequent physiological and biochemical effects on arabidopsis. tcdd is up-taken by the root and accumulated into the upper parts especially in the leaves and the mature seeds. the salient biological features affected by tcdd-exposed plant are: flowering time, siliques repining and seeds yield. polyunsaturated fatty acids and their metabolism are seriously alerted by tcdd. thus, the role of lipid metabolism as a response to tcdd-exposure in plant merits a particular focus discussion we have recently reported that the immediate uptake of, -tcdd by arabidopsis yielded various phytotoxicological effects. herein, we have shown that the accumulation and translocation of tcdd in arabidopsis plant depended on the growth stage. moreover, tcdd-exposed arabidopsis plants were affected in their lipid metabolism, flowered late and produced less seeds than non-exposed plants. these results are summarized in fig.  using hr/gc-ms analysis, we showed that tcdd-accumulation in arabidopsis plants was ordered in a tissues-specific manner, mainly in the root and less in the shoot. these estimations confirm our previous data and come in line with previous reports on the natural ability of various plant species to take up dioxins/furans from their environment. from the upper parts, we observed that the highest levels of tcdd were found in leaves and mature seeds of arabidopsis. the accumulation of tcdd in seeds can be explained by the high affinity of tcdd toward lipids that are abundantly present in brassicaceae seeds. indeed, it is well known that dioxins essentially bioaccumulate in animal fats because of their high lipophilicity. accordingly, dioxin and dioxin-like compounds were often detected in rape and olive oils. moreover, the sorption of hydrophobic organic compounds by lipid bodies of rapeseeds as a hoc-removal strategy is well documented. similarly, the high concentration of tcdd in leaves might result from the high affinity of tcdd for the lipid-membrane of chloroplasts, mitochondria and peroxisomes. in this context, it is known that the major xenobiotic-oxidations catalyzed by enzymatic systems take place in the endoplasmic reticulum and in the membrane of chloroplasts and peroxisomes. accordingly, data published from proteomic analyses carried out on whole tissue/organ preparations of arabidopsis revealed a total of environmental stress responding proteins. most of them were located in chloroplast, mitochondria and peroxisome. thus, accumulation of dioxins in vegetative tissues and in seeds may reflect their biological fate in plants and suggest a possible tissue-specific mechanism for accumulation and subsequent detoxification of tcdd in plants. a point of a particular importance is the mechanism responsible of translocation of tcdd in plant. although the molecular and biochemical mechanisms involved in reception, translocation, genes activation and enzymes metabolism of tcdd are well documented in mammals, no data however are available to date about these mechanisms in plants. as many of xenobiotics including dioxins are highly lipophilic, they can accumulate to toxic levels in the plant tissues, unless effective means of detoxification are present. in the case of tcdd, one of the most active detoxification systems in plants can probably disposed of this xenobiotic by two sequential processes: a chemical transformation and a subsequent compartmentation. the chemical transformation of lipophilic xenobiotics in plants is of two types: phase i, the activation reactions and phase ii, the conjugation reactions. the primary function of the phase i is to create reactive sites in the xenobiotic by the addition of functional groups which make it more hydrophilic and prepare it therefore for phase ii reactions, the conjugation to glutathione. after having achieved these two protective phases, such xenobiotics are transported and accumulated in apoplastic cell walls or central vacuoles in plant cells. biochemical, molecular, and genetic evidences have been reported on the functions of a handful of atp-binding cassette and multidrug and toxic compound extrusion family transporters engaged in transport of organic xenobiotics. from them, p-glycoprotein, identified in plants as a vacuolar glutathione-conjugate transporter, has some attractions to be involved in tcdd transportation. another possible and potential transporter of tcdd can be a protein, called mrp, which is recognized as a glutathione-conjugate transporter. however, the nature and the mechanism of tcdd transportation is still yet uncharacterized. a chronological effect on the principle growth stages has been also observed. regardless of the level of exposure, our results showed that the tcdd-content was maximal on day post-administration. at this developmental stage, arabidopsis possesses a complete rosette growth and an initial emergence of inflorescence. the high level of tcdd found in plant tissues at this stage of growth could be explained by the optimal development and extension of the lateral roots of arabidopsis which seemed to be induced by tcdd. moreover, the biomass of vegetative tissues reached its maximum in this stage. absorption of tcdd delayed flowering time through presently undetermined mechanisms. one possible mode of action of tcdd might be linked to its up-regulation of various myb transcription factor genes in the root of arabidopsis. flowering time is in part regulated through the transcriptional regulation of flowering locus t. interestingly, it was recently reported that a myb transcription factor early flowering myb protein plays an important role in directly repressing flt expression in the root and leaf vasculature under normal conditions. following this hypothesis, the activating of myb factors by tcdd would repress flt and thus delays flowering. alternatively, the strong decline of c18: might indicate a role of pufas in response to tcdd. in particular, tcdd might act through oxidized lipids. we observed here that the amount of lipid peroxides tripled 36 days after exposure to tcdd, this effect seems similar to the previous increase level of lipid peroxides in suspensions of tobacco cells when submitted to dioxins exposure. lipid oxidation is derived from chemical and enzymatic processes. however the parallel rise in transcripts levels of 9-lox and 13-lox genes with the accumulations of 9- and 13-ooh deriving from linolei ic acids suggests that the arabidopsis response to tcdd occurred predominantly via lox pathway. the involvement of the lox pathway in responses of plants to tress-induced senescence and heavy metals tolerance has been reported. the lox pathway is also involved during the regulation of lateral root development and flowering. in particular, transition from vegetative growth to flowering in arabidopsis was associated with the accumulation of 13-hpot, resulting from the oxidation of linolenic acid by lox. it is tempting to hypothesize that the delay of accumulation of 13-hoo-fa in tcdd-treated plants might postpone flowering time. in addition oxylipins deriving from the reduction of 13-ooh-fa might play a role in the control of flowering transition. indeed, overexpression of the caleosin/peroxygenase rd that catalyzes the formation of oh-fa led to early flowering whereas plants deprived of this caleosin flowered later than the control.  in conclusion, as fig.  summarized, the current work highlights a side of toxicological effects related to the administration of, -tcdd on the arabidopsis plant. in a tissue-specific manner, the highest tcdd levels were detected in rosette leaves and mature seeds and affected lipid metabolism. similarly to animals, plants may accumulate tcdd in their lipids by involving few of the fa-metabolizing enzymes for sculpting a new oxylipins “signature” typified to plant tcdd-tolerance. together, our results will contribute to a better understanding of the mechanisms adopted by plants in response to dioxin contamination, and therefore, these potential strategies protect the plants as well as their environment. 
simultaneously, tcdd induced the expression of 9-lox and 13-lox genes and the formation of their corresponding hydroperoxides, 9- and 13-hpod as well as 9- or 13-hpot, derived from linoleic and linolenic acids, respectively. using a high resolution gas chromatography/mass spectrometry, we demonstrated that arabidopsis plant uptakes tcdd by the roots and accumulates it in the vegetative parts in a tissue-specific manner. tcdd mainly accumulated in rosette leaves and mature seeds and less in stem, flowers and immature siliques. due to the lack of data on dioxin/plant research, this study sets out to determine few responsive reactions adopted by arabidopsis plant towards, -tetrachlorodibenzo-p-dioxin, the most toxic congener of dioxins. the current work highlights a side of toxicological effects resulting in the administration of, -tcdd on the arabidopsis plant. together, our results uncover novel responses of arabidopsis to dioxin, possibly emerging to overcome its toxicity. their biotransmission through the food chain constitutes a potential risk for human health. plants as principal actors in the food chain can play a determinant role in removing dioxins from the environment. dioxins are one of the most toxic groups of persistent organic pollutants. moreover, we observed that plants exposed to high doses of tcdd exhibited a delay in flowering and yielded fewer seeds of a reduced oil content with a low vitality. a particular focus on the plant fatty acid metabolism showed that tcdd caused a significant reduction in c18-unsaturated fatty acid level in plant tissues. similarly to animals, it seems that plants may accumulate tcdd in their lipids by involving few of the fa-metabolizing enzymes for sculpting a specific oxylipins “signature” typified to plant tcdd-tolerance.
 dioxins are one of the most toxic groups of persistent organic pollutants. their biotransmission through the food chain constitutes a potential risk for human health. plants as principal actors in the food chain can play a determinant role in removing dioxins from the environment. due to the lack of data on dioxin/plant research, this study sets out to determine few responsive reactions adopted by arabidopsis plant towards, -tetrachlorodibenzo-p-dioxin, the most toxic congener of dioxins.  using a high resolution gas chromatography/mass spectrometry, we demonstrated that arabidopsis plant uptakes tcdd by the roots and accumulates it in the vegetative parts in a tissue-specific manner. tcdd mainly accumulated in rosette leaves and mature seeds and less in stem, flowers and immature siliques. moreover, we observed that plants exposed to high doses of tcdd exhibited a delay in flowering and yielded fewer seeds of a reduced oil content with a low vitality. a particular focus on the plant fatty acid metabolism showed that tcdd caused a significant reduction in c18-unsaturated fatty acid level in plant tissues. simultaneously, tcdd induced the expression of 9-lox and 13-lox genes and the formation of their corresponding hydroperoxides, 9- and 13-hpod as well as 9- or 13-hpot, derived from linoleic and linolenic acids, respectively.  the current work highlights a side of toxicological effects resulting in the administration of, -tcdd on the arabidopsis plant. similarly to animals, it seems that plants may accumulate tcdd in their lipids by involving few of the fa-metabolizing enzymes for sculpting a specific oxylipins “signature” typified to plant tcdd-tolerance. together, our results uncover novel responses of arabidopsis to dioxin, possibly emerging to overcome its toxicity. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. issue-copyright-statement© the author 2015 