this phenomenon is common on soils which  have developed on naturally enriched rock substrates .
another source of soil pollution can be attributed to human activities , including mining , metal and ore processing , and industrial and agricultural activities ( padmavathiamma and li , 2007 ) . during recent years
several plant species have been identified that extract , tolerate and/or hyperaccumulate pollutants from soils in their above - ground tissues ( shah and nongkynrih , 2007 ; verbruggen et  al .
the accumulation of metals and the final concentration in tissues often exceed one hundred - times the concentration of common plant species ( maestri et  al . , 2010 ) .
major disadvantages of these species ( e.g. thlaspi caerulescens , arabidopsis halleri ) are their slow growth , low biomass production and short life cycle .
therefore , researchers over the world are interested in how to make the process of phytoextraction more  effective ( zhao and mcgrath , 2009 ) .
one of the possible solutions is  the utilization of fast growing woody plant species with  high  biomass production and high genetic variability ( pulford and watson , 2003 ) .
several studies about different woody species have been published in recent years dealing with their uptake , translocation and accumulation capacity , as well as tolerance to various metals .
trees from the salicaceae family , especially the genera salix  and populus , might be suitable candidates for these purposes ( e.g. greger and landberg , 1999 ; sebastiani et  al . , 2004 ; vyslouilov et  al . , 2006 ; dos santos utmazian and wenzel , 2007 ;
, 2007 ; wieshammer et  al . , 2007 ; jensen et  al . , 2009 ; vollenweider et  al . ,
, salix caprea represents a suitable candidate for remediation because of fast colonization of mineral substrates , high biomass production and landscape restoration .
variability in the uptake capacity of several elements has been  reported between populations or clones of the same species , sometimes originating from different environmental conditions .
metallicolous and non - metallicolous populations of the heavy metal tolerant plant t. caerulescens have shown different responses to soil cd and zn contamination ( escarr et  al . , 2000 ; lombi et  al . , 2000 ; jimnez - ambriz et  al . , 2007
( 2009 ) compared characteristics important for phytoextraction such as metal tolerance , translocation and accumulation of cd in poplar hybrids ( populus x canadensis , p. x deltoides , p. x generosa , p. x nigra , p. x alba , p. x trichocarpa ) and willow ( s.  alba ) clones .
a similar study was published by dos santos utmazian and wenzel ( 2007 ) on six different willows ( s. babylonica , s.  caprea , s.  dasyclados , s.  matsudana x alba , s.  purpurea , s.  smithiana ) and two poplar species ( populus tremula , p.  nigra ) and discovered that s.  smithiana leaves accumulated the highest cd concentration .
differences in the tolerance and accumulation capacity of cd , cu and zn between clones of five salix species ( s. viminalis , s.  dasyclados , s.  daphnoides , s.  purpurea and s.  triandra ) grown in metal polluted and unpolluted areas were compared by landberg and greger ( 1996 ) .
clones from polluted areas accumulated more metals in roots while the transport to shoots was decreased when compared with clones from unpolluted areas .
however , these authors found no  difference in the concentration of metals in stems between these two groups of salix species .
( 2010 ) analyzed 170 s. caprea isolates of four metal polluted and three uncontaminated middle european sites to  reveal potential selective effects of long - term heavy metal contaminations on the genetic structure and zn / cd accumulation capacity .
they found differentiation of metallicolous and non - metallicolous s.  caprea populations based on phenotypic characteristics and nuclear microsatellite ( ssr ) markers ( puschenreiter et  al . , 2010 ) .
the root is the first plant part to encounter soil heavy metal pollution ; therefore the molecular uptake mechanisms at the root 
soil interface are the subject of intensive study in hyperaccumulating species ( lombi et  al . , 2000 ;
concerning the uptake and translocation of heavy metals , there is very limited knowledge about their relation to root tissue organization and development in the woody plants used for phytoremediation .
( 2004 ) described differences in the anatomy and apoplastic barrier development of adventitious roots of willows ( s. viminalis , s.  viminalis x schwerinii , s.  daphnoides ) with contrasting characteristics of cd accumulation and cd sensitivity .
they show that both the casparian strips and suberin lamellae developed more distantly from root tips in high cd than in low accumulating and translocating cuttings
. however , the influence of cd and/or zn on the development of root tissues of genetically distinct s.  caprea plants with different zn and cd translocation capabilities has not been evaluated ( puschenreiter et  al . , 2010 ) .
therefore the present study focuses on anatomical differences and metal accumulation of root tissues of cuttings from s. caprea plants originating from polluted and uncontaminated areas .
the development of apoplastic barriers affecting radial transport of elements across the root ( casparian strips and suberin lamellae ) , changes in tissue proportions , and metal localization within root tissues were compared .
the aim of this work was to get a better understanding of the involvement of root tissues in the uptake , translocation and accumulation of cd and zn in a woody species promising for phytoextraction , s.  caprea .
all experiments were performed with two genetically distinct s. caprea isolates which differed in the ability to tolerate and accumulate zn and cd in above - ground tissues in perlite cultures . the term  isolate
 refers to an individual plant collected in a specific area as part of a group of 2025 individuals representing the population at this site ( puschenreiter et  al . , 2010 ) . isolate kh21 originated from an old mining area ( kutn hora , czech republic )
isolate f20 originated from a non - polluted control area ( forchtenstein , austria ) ; it accumulated less zn and cd than kh21 but had a higher biomass production in perlite - based hydroponics ( puschenreiter et  al . , 2010 ) .
all cuttings of both s.  caprea isolates used in our experiments were obtained from a stock cutting cultivated in non - polluted control soil at the ages ( vienna , austria ) .
green cuttings of approx . 10  cm length were rooted in a mixture of perlite and coarse sand ( 20:80 ) in a greenhouse ( ages and boku , vienna , austria ) .
rooting was initiated in tap water for 60 days at 24/18  c day / night , a 12  h photoperiod , 60% relative humidity and 200  m  m  s par . for the effect of zn and cd exposure on root anatomy and the distribution of minerals in root tissues , each pre - rooted cutting with new shoot of 1520  cm length and 2  5 adventitious roots of an average total length of 250  mm
the plants were watered twice a week with a nutrient solution containing ( in m ) 1000 ca(no3)2 , 500 mg(so4)2 , 50 kh2po4 , 100 kcl , 5 h3bo3 , 0.2 h24mo7n6o24 , 10 mnso4 , 2.5 cuso4 , 0.25 niso4 , 2.5 znso4 and 50 fe ( iii)-eddha ( ethylenediamine - di(o - hydroxyphenylacetic acid ) ) .
solution ph was adjusted to 6.0 with 1  mm mes as potassium salt ( shen et  al . , 1997 ) .
four different treatments were used : 1 ) standard nutrient solution , 2 ) nutrient solution with 0.5  mg
cd / l in the form of cdno34 h2o ( cd ) , 3 ) nutrient solution with 5  mg / l zn in the form of znso47 h2o ( zn ) , and 4 ) nutrient solution with 0.5  mg cd / l and 5  mg / l zn ( cd  +  zn ) .
plants were cultivated for 14 weeks . for the analysis of the development of casparian strips and suberin lamellae , rooted cuttings
were transferred to 0.5  l pots and watered twice a week for three months with the solutions described above .
plants for apoplastic barrier analysis were cultivated for 6 weeks . for the analysis of zn and cd concentration in below- and above - ground tissues , plant material was divided into roots , shoots and leaves and washed with distilled water before drying at 80  c .
after recording the dw , samples were ground in a metal - free mill ( ika  werke , mf 10 ) and digested in a mixture of hno3 ( puriss .
( 4:1 ,  v / v ) using an automated heating block ( digester dk 42/26 , velp scientifica , milano , italy ) .
zinc and cd concentrations were measured with a flame atomic absorption spectrometer ( aas , perkin elmer 2100 ) . for quality assurance , six biological replicate samples , blanks and standardized reference materials
were included in all analyses . to determine if anatomical changes of the main root tissues correlate with the different origin and metal translocation behavior of the two s.  caprea isolates
, the areas of different tissues were quantified on transverse roots sections at a distance of 1.21.5  cm from the apex .
root cells at this distance are already developed and well differentiated and this part of the root mostly contributes to the uptake of elements .
approximately 2  m thick semi - thin sections were prepared on microtome microm hm36 and stained with toluidine blue and basic fuchsine according to lux ( 1981 ) .
sections from eight different roots were analyzed with a zeiss axioskop 2 +  microscope ( zeiss , germany ) , equipped with olympus dp72 camera .
the tissue areas ( total root area , area of the central cylinder , area of xylem elements ) were analyzed with the image analysis software lucia g 4.80 ( lim , czech republic ) .
to determine the developmental point where casparian strips and suberin lamellae start to differentiate in roots , and if the developmental program of casparian strips and suberin lamellae differentiation varies between isolates from an unpolluted ( f20 ) and a heavy metal polluted site ( kh21 ) , six different roots from each isolate and each treatment were stained with fluorescent dyes for casparian strips and suberin lamellae visualization .
free hand sections were made at regular distances from the apex to the base of the root ( in 1  mm distance for the first 10  mm from the root apex and later in 1  cm distance for the rest of the root ) .
casparian strips were visualized by staining with 0.2% berberine hemisulphate and post - staining with 0.1% toluidine blue .
suberin lamellae were stained with 0.2% fluorol yellow 088 according to brundrett et  al .
all sections were observed with a zeiss axioskop 2 + epifluorescence microscope ( zeiss , germany ) . approximately 2  mm long root segments were cut from the basal part of roots and immediately frozen in liquid nitrogen according to markhart and luchli ( 1982 ) and mccully et  al .
dried segments were coated with carbon and fixed on aluminum stubs covered with a carbon sticker .
surface conductivity was increased by coating with carbon ; sputter coating was stopped as soon as the carbon film showed a golden to bluish color , resulting in a uniform thickness of the coating ( approx .
60  nm ) . cross sections of root tissues and the distribution of eight elements ( zn , cd , ca , k , s , si , mg , na ) were observed and analyzed with the phillips xl 20 scanning electron microscope equipped with an edx analyzer .
all samples were observed at 1.200 magnification at 30  kv , working distance 12.0  mm .
data were collected for 100 live seconds and areas of 50    50  m were analyzed . as uneven surfaces may heavily distort edx measurements ( goldstein , 2003 ) , only flat parts of the samples were selected for analysis .
the element distribution was analyzed from three different roots of each treatment . in total , 24 different roots were investigated by measuring thirty different spots on each root .
raw data were processed with the genesis spectrum 5.11 ( edax , usa ) and all the values are expressed as weight% of total analyzed elements .
zinc and cd were selected because of the main interest of this paper and the other investigated elements share chemical and physical similarities with zn and cd or are important macro- and micronutrients and beneficial elements .
significances according to student 's t - tests and pearson correlations were calculated using the statgraphics ( statgraphics centurion xv v. 15.2.05 , statpoint , inc . ) and excel ( microsoft office 2003 ) programs .
the principal component analysis was performed in simca - p 11 ( umetrics , ume , sweden ) on log - transformed weight% data of all analyzed elements ( zn , cd , ca , k , s , si , mg , na ) .
loadings plots are based on a correlation matrix , scores scatter plots on a covariance matrix .
ellipses in scores scatter plots represent the 95% confidence interval according to hotelling 's t distribution .
all experiments have been performed on six biological replicates / clones with an adequate number of repetitions ( see above ) for statistical analysis .
this was observed in zn and also in the combined cd  +  zn treatment ( fig . 
similarly , the zn concentration was not different in leaves and roots of both isolates when grown in control conditions or excess cd ( fig . 
however , significantly more cd was detected in leaves of isolate kh21 from the polluted site compared to isolate f20 from the control site .
the same tendency was observed when plants were treated with both metals ( cd  +  zn ) together , but the difference was not significant ( fig . 
2b , d ) start to  differentiate , the exo- and endodermis of roots were stained with  fluorescent dyes . in the roots of both
a striking difference between isolates f20 and kh21 could be detected in the casparian strips development already in control conditions ( fig . 
2e , f ; table  s1 ) ; in f20 , the casparian strips differentiated first in the endodermis and later in the exodermis .
in contrast , the casparian strip development in kh21 followed an opposite pattern in these two tissues and differentiated first in the exodermis and later in the endodermis .
another difference was in the starting point of the casparian strip development from the root apex ; in f20 the zone of exo- and endodermal casparian strip development was between 2.5 and 3.5  mm from the root apex while in kh21 this distance was shorter , between 1.0 and 2.0  mm from the root apex ( fig . 
the formation of casparian strips in the exodermis and endodermis of roots after exposure to zn or / and cd also varied between the isolates ; while the casparian strips differentiated in the exo- and endodermis closer to the apex in f20 roots exposed to zn or cd , this was not the case in kh21 .
exposure to zn or cd , as well as the combined treatment , had the same effect indicating that the  development of apoplastic barriers relates to the presence of these metals .
the unresponsiveness of kh21 could indicate a limit in the root apex beyond which the differentiation can not take place .
similar to the casparian strips , the suberin lamellae developed differently in exo- and endodermis of the two isolates . for both isolates , the suberin lamellae form earlier in the exodermis than in the endodermis .
however , the distance of suberin lamellae development from the root apex was different between the two isolates . while the exodermal suberin lamellae of f20 started approximately 15  mm from the apex , the distance was only 10  mm in kh21 .
the endodermal suberin lamellae were detectable 30  mm from the root apex in f20 under control conditions , while in kh21 the distance was only 15  mm ( fig . 
the exodermal suberin lamellae started at around 10  mm from the root apex in both isolates .
metal treatment affected the development of the endodermal suberin lamellae in f20 and kh21 in an opposite manner . while  for f20 the onset of endodermal suberin lamellae differentiation shifted towards the root apex in roots exposed to heavy metals , in kh21 the onset of endodermal suberin lamellae development was at about 25  mm and shifted further from the apex in roots exposed to heavy metals .
interestingly , the same effects were observed in zn , cd , and the combined treatment ( fig . 
if anatomical changes of the main root tissues correlate with the different origin and metal translocation behavior of the two s.  caprea isolates , the areas of different tissues were quantified on transverse roots sections .
3a ) , vascular bundles ( fig .  3b ) and xylem elements ( fig . 
3c ) were significantly larger in f20 than kh21 under control conditions , indicating that all tissues contribute to the thicker roots of f20 .
while in f20 a significant decrease was evident for the total root area , the area of vascular bundles and the area of xylem elements after metal exposure , only the total area of kh21 roots decreased in the zn and cd  +  zn treatments ( fig . 
the area of the vascular bundles remained constant while the area of xylem elements slightly increased with zn or cd treatment , but decreased if zn and cd were offered together ( fig . 
three different zones could be clearly distinguished : the central part of the root consists of secondary xylem ( fig . 
the outer part of the root , here called outer bark or rhytidome ( fig . 
4a , b ) , is highly heterogeneous and includes secondary ( periderm ) and primary vascular bundles , the endodermis , the primary cortex and the exodermis .
the distribution of zn , cd , ca , k , s , si , mg and na in this zone was analyzed by edx ( fig .  4b , table  s2 ) .
to determine which parameter ( tissues , treatment and/or isolate ) affected the variation in the edx data most , the weight% values were subjected to a principal component analysis ( pca ) with all elements as variables .
the first principal component ( pc1 ) ( 45.5% of the total variance ) was interpreted as  responsiveness to metal treatment  .
the main descriptors of pc2 are cd and si , correlating oppositely with this axis ( fig . 
1b ) along with its generally higher si weight% compared to kh21 ( fig . 
the third pc , contributing 11.5% of the total variance , was most influenced by zn ( fig . 
the scores scatter plots show that tissue - specific clusters form along all three pcs ( fig . 
pc3 is able to separate the effects of the cd from the zn and zn / cd treatments ( fig . 
the pc1/pc3 and pc2/pc3 plot  also shows that the cd treatment largely overlaps with the xylem while the zn treatment correlates with the bark values and the cd / zn treatment with the phloem ( fig . 
s1a  f ) . in this way a weak separation can be visualized in the zn and cd  +  zn treatment of plot pc1/pc2 .
pca of all measured elements revealed a grouping in treatment- , isolate- and tissue - specific clusters .
but since the pca can not visualize the effect of the individual elements , we analyzed their weight% distribution relative to the tissues and the isolates separately ( fig . 
7 ) . as already noticeable in the pca the weight% of zn was highest in the peripheral bark zone in both isolates
however , an isolate - specific response was revealed since kh21 had less weight% of zn in the combined treatment while f20 had an increased zn weight% .
cd had a slightly higher weight% in the xylem tissues except in f20 upon the combined zn  +  cd treatment where the cd weight% was increased in peripheral bark zone .
this different pattern of the cd distribution is consistent with the finding that the isolate from the unpolluted site ( f20 ) accumulated less cd in the above - ground tissues ( fig .  1 ) . for calcium ( ca ) ,
a descending trend was observed from the peripheral bark zone towards the xylem in both isolates .
however , the isolates differed with a higher weight% distribution in f20 upon cd exposure and a dramatic increase in the peripheral bark zone for f20 upon the combined treatment with zn  +  cd .
pearson correlation analyses showed that the distribution of ca and zn is positively correlated and stronger for zn uptake in f20 than in kh21 isolate ( fig . 
addition of cd even increased this correlation in root tissues of f20 ( fig . 
the zn / ca correlation is lost in the presence of cd ( fig . 
weight% distributions had a tendency of descending from the peripheral bark zone to the xylem in kh21 while in f20 this tendency was not detectable in zn treatment .
a similar general gradient was detected for sodium ( na ) but the weight% levels were different between the isolates for the zn and the cd treatment ( fig .  7 ) .
the weight% of potassium ( k ) was generally highest in the secondary phloem and was increased upon cd treatment in both isolates and in f20 upon cd  +  zn treatment ( fig . 
furthermore , a positive correlation existed between k and cd but not zn ( fig . 
s2e , f ) . in both isolates the silicon ( si ) weight% was highest in the peripheral bark zone ( fig . 
7 ) , but with a clear difference between the isolates . upon cd or zn treatment the values of si
however , this effect was lost upon exposure of cd  +  zn together .
sulfur ( s ) distribution was elevated in the phloem and most prominent in kh21 upon zn treatment . while for most treatments and in both isolates s was higher in the peripheral bark zone than in the xylem , this tendency was exaggerated in f20 upon zn  +  cd and shifted upon zn treatment ( fig . 
, the analyses of the individual elements in different tissues and isolates showed that their distributions are altered upon cd , zn and cd  +  zn treatment in most cases in an isolate - specific manner .
to better understand uptake and accumulation of elements into shoots , knowledge of root anatomy and physiology is essential .
elements are transported radially from the rhizodermis through the apoplast or symplast across the cortex to the xylem and the shoot .
uptake is controlled by apoplastic barriers in the endo- and exodermis ( white , 2001 ; ma and peterson , 2003 ; baxter et  al . , 2009 ; schreiber , 2010 ) .
first , a mixture of lignin and suberin is deposited into the radial and transversal cell walls ( casparian strips ) , which is usually followed by lamellar suberin deposition on the inner surface of exo- and endodermal cell walls ( suberin lamellae ) and , in some species , also by secondary thickening of the walls ( von guttenberg , 1968 ) .
the development of these apoplastic barriers is variable and often differs between plant species and environmental conditions ( zimmerman and steudle , 1998 ; seago et  al .
, 1999 ; enstone and peterson , 2005 ; redjala et  al . , 2011 ) .
we observed that the initiation of the casparian strip and suberin lamellae formation varied between two s.  caprea isolates  originating from different environments .
the kh21 isolate , collected from metal polluted soil and previously characterized by high cd and zn accumulation capacities ( puschenreiter et  al . , 2010 ) , develops casparian strips and suberin lamellae closer to the root apex compared to the genetically distinct f20 isolate which originated from unpolluted soil and showed phenotypic differences to kh21 .
furthermore , f20 first developed casparian strips in endodermis and later in exodermis , oppositely to kh21 .
similarly , differences in the development of apoplastic barriers between isolates of s. viminalis differing in metal tolerance and accumulation were also observed by lux et  al .
therefore we propose that apoplastic barrier development is an adaptive trait and can vary between different isolates of the same species .
similarly , we have observed that anatomical reactions to heavy metal exposure differed between isolates . in f20 , exposure to cd , zn or cd  +  zn resulted in earlier development of both barriers while the development in kh21 was not influenced by metal exposure .
the behavior of f20 is consistent with several studies showing that roots exposed to cd develop earlier apoplastic barriers ( schreiber et  al . , 1999 ; martinka and lux , 2004 ; zelko and lux , 2004 ; vaculk et  al . , 2009 ) .
accelerated apoplastic barrier development was also induced by higher salinity ( reinhardt and rost , 1995 ; karahara et  al . , 2004 ) .
thus the typical reaction is to minimize the uptake of pollutants by developing the exo- and endodermis closer to the root apex .
this is consistent with the finding that the concentration of cd in leaves was lower in f20 than in kh21 . on the other hand ,
delayed development of the endodermal suberin lamellae of kh21 upon exposure to heavy metals could be the reason of the higher accumulation capacity of cd in shoots .
however , the understanding of the delay in apoplastic barriers development after metal stress in isolate kh21 originating from metal polluted soil remains still poorly understood .
changes in root tissue areas might also be the consequence of continuous exposure to metal polluted soils .
( 2004 ) showed that willow clones characterized by high tolerance to cd had a higher proportion of epi- , exo- and endodermal tissues when compared with the sensitive clones , which had a higher proportion of mid - cortex .
we found that kh21 produced thinner roots with a smaller area of vascular bundles and xylem elements when compared with f20 .
however , when the same isolates were exposed to elevated metal concentrations , the reaction of roots was different . in roots of f20
the total area and areas of central cylinder and xylem elements decreased in comparison to control conditions .
we suppose that the differences in the root anatomy of the salix isolates treated with metals reflect an adaptive predisposition and depend on the different natural origins .
cd - induced differences in the proportions of specific root tissues ( rhizodermis  cortex  vascular bundles ) or in the size and shape of individual cells have been reviewed recently by lux et  al .
( 2011 ) . described syndromes of cd toxicity on roots are a decrease of root number , length and dry mass as well as enlargement of the root diameter .
( 2003 ) reported that roots of s.  alba and populus x euramericana became shorter and thicker upon cd treatment .
however , the reactions of root tissues to cd mostly differ among plant species and used cd concentrations ( e.g. lux et  al . , 2011 ) .
conversely , cd , zn and cd  +  zn treatment in our experiments decreased the total area of the s. caprea roots in f20 .
the total root area of kh21 was unaffected when cd was applied , although after zn and zn  +  cd application the total area of root decreased .
( 1992 ) reported that the root diameter of bean ( phaseolus vulgaris ) remained unaffected , although the number of cortical parenchyma cells decreased when cd was applied .
( 1993 ) did not find any changes in the root diameter , length or surface area in two different maize inbred lines exposed to cd .
we  expected that unresponsiveness of kh21 tissues to elevated cd concentration probably related with higher cd accumulation capacity .
this pattern of root tissue development probably disappeared when plants were exposed to zn or cd  +  zn .
( 2005 ) found that the total area of the root , the diameter of the vascular bundles , the number of xylem strands and the area they occupied in stele were higher in coffee plants grown without zn in the medium than in plants exposed to zn . a similar decrease of tissue areas after zn application
was observed in our experiments in f20 while kh21 differed only in the total root area .
balanced zn concentration and distribution is essential for optimal growth and function of metabolic processes ( broadley et  al . , 2007 ) .
( 2009 ) for poplar , macfarlane and burchett ( 2000 ) for gray mangrove and brunner et  al .
( 2008 ) for spruce exposed to elevated zn , in our experiments most of the zn accumulated in the peripheral tissues of the secondary thickened root .
therefore we assume that the peripheral bark zone of secondary thickened roots serves as an effective barrier for radial zn transport to vascular bundles .
freeze drying of the samples prevented leakage of elements during fixation , allowing us to detect tissue - specific distribution of elements with low abundance such as cd ( fig . 
in contrast to zn , most of the cd was detected in the xylem in both isolates treated with cd .
however , when both metals cd  +  zn were applied to kh21 , no significant differences in the cd distribution between the peripheral zone and vascular bundles were observed .
( 2008 ) found that in poplar roots exposed to higher doses of cd this element accumulated predominantly in cells surrounding the central cylinder .
nevertheless , in the combined cd  +  zn treatment , double values of cd in each investigated tissue of f20 were detected when compared with kh21 . together with the observations that the cd weight% in kh21 tissues is lower as in f20 ( table  s2 ) we assume that kh21 is more efficiently translocating cd to the shoot , especially when excess of zn is present .
these differences in cd localization within root tissues probably relate with the uptake and translocation of cd and zn , and might reflect the situation on the site of kh21 's origin in natural conditions , which is adapted to a soil containing up to 84  mg total cd  kg and 8.6  g total zn  kg ( puschenreiter et  al .
the levels of zn and ca are positively correlated in both isolates when treated with excess zn . in the combined cd  +  zn treatment , this correlation is lost only in kh21 .
cadmium is among others transported into the root via ca uptake and translocation pathways ( lux et  al . , 2011 ) .
kh21 accumulates more cd in the shoot than f20 , therefore it is possible that higher amounts of cd are taken up via ca transport mechanisms .
cadmium competes with ca uptake , which might explain the lower ca concentration in the combined zn  +  cd treatment .
furthermore , a positive correlation exists between k and cd , but not zn , distribution in root tissues .
it was recently found that k supplement alleviated cd toxicity in soybean ( shamsi et  al .
, 2010 ) , which might be also one possible tolerance mechanism in s.  caprea .
silicon repressed root - to - shoot translocation of cd in brassica chinensis and increased cd tolerance ( song et  al . ,
consistently , f20 , which translocates less cd into shoots , accumulates significantly more si than kh21 in all root tissues .
proposed mechanisms which reduce translocation to the shoot are si co - precipitation with cd and zn in roots or physical blockage of metal flow across cell walls ( neumann and zur nieden , 2001 ; shi et  al . , 2005 ) .
edx analyses show that cd and zn interfere differently with the element uptake systems in s.  caprea , and that for some elements this effect is isolate - specific .
variation in the organization of root tissues , development of apoplastic barriers and the distribution pattern of zn and cd and other elements in s.  caprea isolates originating from polluted and unpolluted sites support the hypothesis that these features are results of environmental adaptation . understanding the molecular basis of these adaptive traits
might help to increase the tolerance and heavy metal accumulation capacities of s.  caprea for phytoremediation technologies .
 the understanding of the influence of toxic elements on root anatomy and element distribution is still limited . 
 this study describes anatomical responses , metal accumulation and element distribution of rooted cuttings of salix caprea after exposure to cd and/or zn . 
 differences in the development of apoplastic barriers and tissue organization in roots between two distinct s.  caprea isolates with divergent cd uptake and accumulation capacities in leaves might reflect an adaptive predisposition based on different natural origins . 
 energy - dispersive x - ray spectroscopy ( edx ) revealed that cd and zn interfered with the distribution of elements in a tissue- and isolate - specific manner . 
 zinc , ca , mg , na and si were enriched in the peripheral bark , k and s in the phloem and cd in both vascular tissues . 
 si levels were lower in the superior cd translocator . since the cuttings originated from stocks isolated from polluted and unpolluted sites we probably uncovered different strategies against toxic elements 
 . 