Intra-specific variation in Ni tolerance, accumulation and translocation patterns in the Ni-hyperaccumulator Alyssum lesbiacum

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Intra-specific variation in Ni tolerance, accumulation and translocationpatterns in the Ni-hyperaccumulator Alyssum lesbiacum

G.C. Adamidis a,⇑, M. Aloupi b, E. Kazakou c, P.G. Dimitrakopoulos a

a Biodiversity Conservation Laboratory, Department of Environment, University of the Aegean, 81100 Mytilene, Lesbos, Greeceb Water and Air Quality Laboratory, Department of Environment, University of the Aegean, 81100 Mytilene, Lesbos, Greecec Montpellier SupAgro, UMRCentre d’Ecologie Fonctionnelle et Evolutive, CNRS UMR 5175, 1919 Route de Mende, 34293 Montpellier, France

h i g h l i g h t s

� A hydroponic experiment performed using the Ni hyperaccumulator Alyssum lesbiacum.� Different populations show significant variation in Ni tolerance and accumulation.� A. lesbiacum populations differed in Ni translocation from roots to shoots.� Seed Ni concentration was significantly correlated to shoot Ni accumulation.� There was a significant positive relationship between tolerance and accumulation.

a r t i c l e i n f o

Article history:Received 24 March 2013Received in revised form 16 September2013Accepted 26 September 2013Available online 30 October 2013

Keywords:Hydroponic experimentHyperaccumulationMicro-edaphic endemicSeed Ni concentrationPhytoremediationAlyssum lesbiacum

a b s t r a c t

A hydroponic experiment was conducted to investigate inter-population variation in Ni tolerance, accu-mulation and translocation patterns in Alyssum lesbiacum. The in vitro results were compared to field data(soil bioavailable and leaf Ni concentrations) so as to examine any potential relationship between hydro-ponic and natural conditions. Seeds from the four major existing populations of A. lesbiacum were usedfor the cultivation of plantlets in solution cultures with incrementally increasing Ni concentrations (rang-ing from 0 to 250 lmol L�1 NiSO4). Ni accumulation and tolerance of shoots and roots, along with initialseed Ni concentration for each population were measured. The ratio of root or shoot length of plantletsgrown in NiSO4 solutions to root or shoot lengths of plantlets grown in the control solution was used astolerance index. For the range of metal concentrations used, A. lesbiacum presented significant inter-pop-ulation variation in Ni tolerance, accumulation and translocation patterns. Initial seed Ni concentrationwas positively correlated to shoot Ni accumulation. A significant positive relationship between toleranceand accumulation was demonstrated. Initial seed Ni concentration along with physiological differences inxylem loading and Ni translocation of each population, appear to be the determining factors of the sig-nificant inter-population variation in Ni tolerance and accumulation. Our results highlight the inter-pop-ulation variation in Ni tolerance and accumulation patterns in the Ni-hyperaccumulator A. lesbiacum andgive support to the suggestion that the selection of metal hyperaccumulator species with enhanced phy-toremediation efficiency should be considered at the population level.

� 2013 Elsevier Ltd. All rights reserved.

1. Introduction

Hyperaccumulators are plants that can accumulate exception-ally high amounts of metals in their above-ground biomass; metalconcentrations in their dry mass are up to 100 times higher than innormal plants (Reeves and Baker, 2000). Presently over 450 Ni-hyperaccumulator species have been recorded (van der Ent et al.,2013), corresponding to about 2% of serpentine species worldwide.Although Ni is the most commonly hyperaccumulated metal

(Baker and Brooks, 1989), the rarity of the Ni-hyperaccumulationtrait in nature is apparent. The property of hyperaccumulationhas acquired much interest not only for its biochemical and phys-iological uniqueness (Kazakou et al., 2008) but also for the poten-tial use of the hyperaccumulator species in the remediation ofheavy metal-polluted soils and in phytomining technology (Cha-ney et al., 2005, 2007).

Alyssum (Brassicaceae) is the genus with the greatest number ofhyperaccumulator species (Baker and Brooks, 1989). Among them,Alyssum lesbiacum is a well-known Ni-hyperaccumulator (Brookset al., 1979; Reeves et al., 1997; Kazakou et al., 2010) endemic tothe serpentine soils of Lesbos Island Greece (Strid and Tan, 2002).

0045-6535/$ - see front matter � 2013 Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.chemosphere.2013.09.106

⇑ Corresponding author. Tel.: +30 22510 36299.E-mail address: [email protected] (G.C. Adamidis).

Chemosphere 95 (2014) 496–502

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It is one of the few Ni-hyperaccumulators for which significant pro-gress has been made in identifying the mechanism of Ni uptake andhyperaccumulation (Krämer et al., 1996; Kerkeb and Krämer, 2003;Ingle et al., 2005) and the cellular compartmentation of Ni in its tis-sues (Krämer et al., 1997; Küpper et al., 2001; Smart et al., 2007).Furthermore, it has been proposed that its phytoextractingefficiency is not significantly reduced even in soils marginally con-taminated with polycyclic aromatic hydrocarbons (PAHs) (Singeret al., 2007). In phytoremediation technology the ability of a speciesto tolerate and hyperaccumulate metals is more important than thetrait of producing high biomass yields (Chaney et al., 1997).

Recently, Kazakou et al. (2010) demonstrated that different pop-ulations of A. lesbiacum living in their natural habitats present dif-ferences in Ni-hyperaccumulation related to soil Ni concentration.Intra-specific variation in Ni-hyperaccumulation has been also pre-sented for other Ni-hyperaccumulating species (e.g. Alyssum murale(Massoura et al., 2004; Bani et al., 2009, 2010) and Alyssum bertolo-nii (Galardi et al., 2007a)). However, A. lesbiacum (Kazakou et al.,2010) and A. bertolonii (Galardi et al., 2007a) are the first ‘micro-edaphic’ endemic Ni-hyperaccumulating species (i.e. endemicspecies with populations that diverge in their ability to hyperaccu-mulate metals; Galardi et al., 2007a) for which the micro-edaphicfactors are being studied in detail. This inter-population differenti-ation in Ni-hyperaccumulation showed by both species is compara-ble or, in some cases, higher to the inter-specific differences shownamong the various hyperaccumulator species of the genus Alyssum(Kazakou et al., 2010). The previous observation supports the pro-posal that studies towards the selection of species with the highestphytoextraction ability should be conducted at the population level(Kazakou et al., 2010). In this context, the most efficient populationscould be used both directly as phytoremediation crops themselves(Bani et al., 2009) and indirectly for the improvement of plant traitsthrough selective breeding or as sources of genes for improvementof other remediation crops (Pollard et al., 2002).

The assessment of patterns of variation in hyperaccumulationand tolerance (sensu Baker, 1987) may enable us to elucidate therelationship between these two phenomena. The exact relation-ship between metal tolerance and hyperaccumulation has notbeen decoded and it is not yet known if this relationship is sim-ilar for all metals and/or species (reviewed by Pollard et al.(2002)). In general, tolerance is regarded as an adaptive trait inresponse to high soil metal concentrations (Pollard et al., 2002;Agrawal et al., 2012). The relationship between tolerance andhyperaccumulation has been mostly addressed for metallicolousand non-metallicolous populations (Wu et al., 2009; Mohtadiet al., 2012). However, the existence of micro-edaphic, endemicmetal hyperaccumulators offers ideal model plant species forinvestigating the micro-evolution of inter-population differencesin tolerance and hyperaccumulation due to adaptation in mi-cro-edaphic conditions. Furthermore, as metals affect seed ger-mination and seedling growth (reviewed by Kranner andColville (2011)), the seed Ni concentration could be another fac-tor influencing the plant Ni concentration (Reeves and Baker,1984). However, the hypothesis that the accumulation capacityof the different populations of a metal hyperaccumulating spe-cies is related to initial seed Ni concentration has not beentested so far.

The present study aimed to evaluate the inter-population var-iation in Ni tolerance, accumulation and translocation patternsamong the four populations of A. lesbiacum. Moreover it aimedto investigate any significant relationship between initial seedNi concentration and Ni accumulation, to determine if any sig-nificant relationship between tolerance and accumulation exists,and to reveal any significant relationship between these twophenomena and soil bioavailable Ni and/or Ni hyperaccumula-tion from a previous field survey.

2. Materials and methods

2.1. Hydroponic culture

Seeds were collected randomly from more than 50 individualsin each of the four populations of A. lesbiacum giving coverage ofthe altitudinal and geographic ranges shown by this species on Les-bos. The four distant localities chosen support the only four largepopulations of A. lesbiacum (LO, Loutra; VA, Vatera; OL, Olymposand AM, Ampeliko) and are described in detail in Kazakou et al.(2010) and in Adamidis et al. (in press). Seeds were germinatedfor 4 d on floating trays in vessels containing a nutrient solution,pH 5.5 ± 0.1 with the following composition: KNO3 60 lmol L�1,Ca(NO3)2�4H2O 30 lmol L�1, NH4H2PO4 10 lmol L�1, MgSO4�7H2O20 lmol L�1, FeSO4�7H2O 0.18 lmol L�1, tartaric acid 0.9 lmol L�1,H3BO3 4.6 lmol L�1, MnCl2�4H2O 0.92 lmol L�1, CuSO4�5H2O0.03 lmol L�1, ZnSO4�7H2O 0.077 lmol L�1, and H2MoO4

0.06 lmol L�1 (Barzanti et al., 2011). A metal chelator was not in-cluded in the medium since Kerkeb and Krämer (2003) showedthat Ni+2 is taken up by the roots of A. lesbiacum as the free aqueouscation, independent of the chelators and histidine presence. Aftergermination, the trays with seedlings were placed in vessels withcontinuously aerated fresh nutrient solutions spiked with NiSO4

solution to form increasing Ni concentrations (0-control-, 50,100, 175 and 250 lmol L�1 NiSO4). Ni concentration range was inline with that of other hydroponic cultures studying Alyssum spe-cies (e.g. Galardi et al. (2007b), Ghasemi and Ghaderian (2009),Ghasemi et al. (2009)). Four replicates were used for each popula-tion-by-treatment combination and the total number of vesselsused was 80. After 12 d of growth, the floating trays were removedfrom the hydroponic solutions and the seedlings harvested andprepared for the appropriate measurements. The experiment wasperformed in a glasshouse with temperatures ranging from 18 to23 �C between night and day.

2.2. Ni tolerance

After harvest the root and shoot lengths of at least 15 plantletsfrom each treatment were used as a measurement of the Ni toxiceffect on the different populations (Baker and Walker, 1989). Tocompare the four populations we used the same tolerance index(TI) as that proposed by Galardi et al. (2007b), namely the ratioof root or shoot length of plantlets grown on NiSO4 solutions tothe root or shoot lengths of plantlets grown on the control solution.

2.3. Ni accumulation

The harvested plantlets were rinsed with distilled water andwashed carefully with 10 � 103 lmol L�1 CaCl2 at 4 �C for 10 minto remove any adsorbed metals (Gonnelli et al., 2001). The plantletswere separated into roots and shoots, pre-frozen at�20 �C and thenfreeze-dried for 48 h in a LabconcoFreeZone 4.5 laboratory appara-tus, at �40 �C collector temperature under <5 mBar vacuum. Thiswas followed by pulverization of the freeze-dried samples in a lab-oratory mixer-mill. The pulverized samples were digested withconc. HNO3 in a Mars Xpress system (CEM), according to the USEPA’s method 3051A (2007). Ni determination was performed byFlame Atomic Absorption Spectrometry (Perkin–Elmer 5100ZL).Ni concentrations in plant tissues were calculated on a dry weightbasis. For simplicity the term Ni accumulation is used for describingboth shoot and root Ni concentrations, although we acknowledgethat the term Ni accumulation is used in case of Ni storage and thatroot Ni concentration is constituted by an unknown combination ofboth stored and transited Ni (Coinchelin et al., 2012).

G.C. Adamidis et al. / Chemosphere 95 (2014) 496–502 497


2.4. Analysis of seeds

For the determination of the metal concentration of the A. les-biacum seeds, approximately 100 seeds per population were com-bined and ground in the sample mill to form five compositesamples for each population. After pulverization the same analyti-cal procedure as for plant material was followed.

A detailed description of the analytical quality assurance andcontrol is provided at the Supplementary material (S1).

2.5. Soil bioavailable Ni

The bioavailable Ni fraction in soil was determined in selectedsamples from the four localities of origin of the A. lesbiacum popu-lations, by means of the single extraction with EDTA described byQuevauviller et al., (1998), which has been used by the Measure-ments and Testing Programme (formerly BCR) of the EuropeanCommission for the preparation of a reference material certifiedfor extractable metal contents in soil.

2.6. Data analysis

Two-way ANOVAs were performed to examine the main effectsof ‘population’ and a) of ‘Ni treatment’ on shoot and root Ni concen-tration (Ni accumulation) and length (plant growth), (b) of root Niconcentration on shoot Ni concentration (Ni translocation) and (c)of shoot and root Ni concentration on shoot and root tolerance in-dex (Ni tolerance). Standardized major axis (hereafter SMA) analy-sis (Warton et al., 2006) was used to further investigate whether theeffect of Ni treatment on Ni accumulation, plant growth, Ni translo-cation and Ni tolerance is consistent across the different popula-tions. For this purpose, the relationships ‘Shoot Ni vs Nitreatment’ and ‘Root Ni vs Ni treatment’ (describing shoot and rootNi accumulation respectively), ‘Shoot length vs Ni treatment’ and‘Root length vs Ni treatment’ (describing the effect of Ni treatmenton shoot and root growth respectively), ‘Shoot Ni vs Root Ni’(describing Ni translocation), ‘Shoot TI vs Shoot Ni’ and ‘Root TI vsRoot Ni’ (describing the effect of shoot and root nickel concentra-tion to shoot and root Ni tolerance respectively) were quantifiedand compared across the different populations (abbreviations ofvariables are given in Table S1). SMA was used to test whether thesebivariate relationships differed in slope (via a permutation test) andwhen no significant differentiation emerged, differences in inter-cept (elevation) or position along a common slope were tested.

In order to evaluate any potential relationship between Ni tol-erance and accumulation, we also quantified across different pop-ulations the relationships ‘Shoot TI vs Ni treatment’ and ‘Root TI vsNi treatment’, describing the effect of Ni concentration in the treat-ment solution on Ni tolerance. We then calculated the Pearson cor-relation coefficient between the slopes of the previousrelationships and the relationships describing the effect of plantnickel concentration to Ni tolerance (‘Shoot TI vs Shoot Ni’ and‘Root TI vs Root Ni’). According to Galardi et al. (2007b), any poten-tial correlation between the slopes of these relationships wouldindicate a significant correlation between tolerance to externaland to internal Ni concentration and thus a significant relationshipbetween Ni tolerance and accumulation.

Finally, correlation analysis was applied for the investigation ofthe relationship between the parameters studied in this experi-ment (e.g. initial seed Ni concentration, Ni accumulation, Ni toler-ance) and both the Ni concentrations in leaf of A. lesbiacum studiedin Kazakou et al. (2010) and bioavailable Ni concentration in thesoils of origin. The statistical software SMATR (http://www.bio.m-q.edu.au/ecology/SMATR/index.html) was used for the SMA analy-sis (Warton et al., 2006), and SPSS 19 for all others.

3. Results


The ANOVA revealed significant effects of both ‘Ni treatment’and ‘Population’ on shoot Ni concentration (P < 0.05; Table S2;see Table S3 for mean values ± 1 SE). Although the SMA analysisshowed a consistent slope, a significant elevation (intercept) differ-entiation across the four populations for the ‘Shoot Ni vs Ni treat-ment’ relationship emerged (Table S4), with the AM populationpresenting a significantly higher intercept relative to the threeother populations (Table S5; Fig. 1a).

Significant effects of ‘Ni treatment’ and ‘Population’ alsoemerged for root Ni accumulation (P < 0.05; Table S2; seeTable S3 for mean values ± 1 SE) while further investigation bySMA analysis revealed significant differentiations in slopes acrossthe different populations, for the ‘Root Ni vs Ni treatment’ relation-ship (Table S4). OL population presented a significantly lower sloperelative to AM and LO populations while VA population did notpresent any significant differentiation with any of the three otherpopulations (Table S5; Fig. 1b).

3.2. Root to shoot Ni translocation

Significant effects of ‘Root Ni’ and ‘Population’ were revealed on‘Shoot Ni’ (P < 0.05; Table S2). SMA analysis indicated significantshifts in slopes across the four populations for the ‘Shoot Ni vs RootNi’ relationship (Table S4). OL population presented a significantlyhigher slope relative to AM and LO populations while VA popula-tion showed the second highest slope presenting a significant dif-ferentiation from LO population (Table S5; Fig. 1c).

3.3. Initial seed Ni concentration effect on Ni accumulation

Seeds from AM population presented higher Ni concentrations(mean concentration ± S.E: (4851 ± 291 mg kg�1) in relation to OL(4321 ± 238 mg kg�1), LO (4304 ± 245 mg kg�1) and VA(3914 ± 231 mg kg�1)). There was no significant correlation be-tween seed Ni concentration and either leaf Ni concentration ofA. lesbiacum in the field or Ni accumulation factor, calculated asmean leaf Ni concentration/mean soil Ni concentration (P > 0.05).Moreover, initial seed Ni concentration was not correlated withbioavailable Ni concentration in the soils of origin (P = 0.08). Ahighly significant positive relationship did emerge between the ini-tial seed Ni concentration and the intercepts of the regression linesbetween shoot Ni accumulation and ‘Ni treatment’ for each popu-lation (r = 0.956; P < 0.05). No significant relationship was revealedfor roots (P > 0.05).

3.4. Ni influence on plant growth

Although the ANOVA did not show any significant effect of ‘Nitreatment’ on shoot length (P > 0.05; Table S2), significant differ-ences in mean values of shoot lengths (Table S4) among the fourpopulations (P < 0.05; Table S2) of A. lesbiacum were detected.SMA analysis indicated that the relationship ‘Shoot length vs Nitreatment’ (Table S4) was very weak in explaining the existing var-iance (Table S5) across the four populations.

On the other hand, root length (Table S6) was significantly af-fected by both ‘Ni treatment’ and ‘Population’ (P < 0.05;Table S2). The relationship ‘Root length vs Ni treatment’ presenteda significant slope differentiation across the four populations(Table S4), with LO and VA populations presenting the highest neg-ative slopes and OL population the lowest relative to all otherspopulations (Table S5; Fig. 1d).

498 G.C. Adamidis et al. / Chemosphere 95 (2014) 496–502


Fig. 1. Variation in the examined relationships across different populations. In all panels, LO population is shown by solid circles (d) and the dotted line (. . .), VA population byopen circles (s) and the short-dash line (- -), OL population by solid triangles (.) and the long-dash lines (� �) and AM population by open triangles (D) and the solid line (–).TI, tolerance index. Only the significant relationships are shown.



3.5. Tolerance and accumulation relationship

ANOVA revealed a significant effect of ‘Population’ (P < 0.05;Table S2) on ‘Shoot TI’ but the main effect of ‘Shoot Ni’ on ‘ShootTI’ was not significant (P > 0.05; Table S2). The relationship be-tween ‘Shoot TI’ and ‘Shoot Ni’ was significant only for AM popula-tion (Table S5; Fig. 1e). Similar results were revealed for the ‘ShootTI vs Ni treatment’ relationship, describing the effect of Ni treat-ment on shoot Ni tolerance (Tables S4 and S5).

On the other hand, significant differences of ‘Root TI’ acrosspopulations and root Ni concentrations emerged (P < 0.05;Table S2) and significant negative ‘Root TI vs Root Ni’ relationshipswere revealed for all populations (Table S5). The four populationspresented significant differentiation in slopes (Table S4), with OLand VA populations presenting the highest negative slopes andLO population the lowest relative to all others (Table S5; Fig. 1f).Similar results also revealed for the relationship describing the ef-fect of Ni treatment on root Ni tolerance (‘Root TI vs Ni treatment’)(Tables S4 and S5).

A significant positive correlation emerged between the slopes ofthe relationships ‘Root TI vs Ni treatment’ and ‘Root TI vs Root Ni’(r = 0.99; P < 0.01), indicating a positive relationship between tol-erance to external and to internal Ni concentration for the rootsof A. lesbiacum.

3.6. Tolerance, accumulation and their relation with Nihyperaccumulation in the field and bioavailable Ni concentration insoils of origin

All the parameters related to Ni tolerance, accumulation andtranslocation studied in this work along with Ni hyperaccumula-tion in the field and bioavailable Ni concentration in soils of originwere used as factors in the analysis investigating for potential rela-tions. There were no significant relations of shoot/root Ni accumu-lation and tolerance with either leaf Ni concentration of A.lesbiacum in the field or with bioavailable Ni concentration in thesoils of origin (P > 0.05).

4. Discussion


Although the four populations of A. lesbiacum showed equal effi-ciency, i.e. no significant differentiation in slopes of regressionlines, in accumulating Ni from the solutions in their shoots, signif-icant inter-population differentiations were revealed for shoot Niaccumulation. In particular, the AM population presented signifi-cantly higher shoot Ni concentration in comparison to all otherpopulations (significantly higher intercept, Table S5). On the otherhand the four populations showed significant differentiation inslopes of the ‘Root Ni vs Ni treatment’ relationship indicating thesignificant differentiation in their efficiency on accumulating Niin their roots. AM and LO populations presented the highest effi-ciency of root Ni accumulation, differing significantly only withOL population where the lowest efficiency was demonstrated(Table S5). Galardi et al. (2007b) showed that the patterns of rootand shoot Ni accumulation correlated across nine populations ofA. bertolonii, implying that the populations with high shoot Niaccumulation capacity also have high Ni accumulation in roots.In this study AM population followed the same pattern demon-strating the highest values of nickel concentration in both shootsand roots, but a similar consistent ranking has not appeared forthe three other populations. A significant differentiation in Cdaccumulation between two populations of Thlaspi (Noccaea) cae-rulescens was related to the existence of a high-affinity Cd uptake

mechanism which was active only in one of these populations(Lombi et al., 2001). However, in case of Ni uptake, so far, thereis no evidence that such mechanism exists. On the contrary, Redj-ala et al. (2010) suggested that maize and the Ni hyperaccumulat-ing species Leptoplax emarginata, regardless of their contrasting Niaccumulation capacity, may share a similar Ni transport system.

4.2. Root-to-shoot Ni translocation

Significant positive relationships were revealed between shootand root Ni concentration for the four populations studied. In thisstudy, although the four populations showed equal efficiency inaccumulating Ni in their shoots, at the same time, they showed sig-nificant differences in Ni translocation from roots to shoots. OLpopulation presented a significantly higher slope for the relation-ship ‘Shoot Ni vs Root Ni’ in comparison to AM and LO populationsindicating that for the same increase in root Ni concentrationtranslocated a significantly higher amount of Ni in the shoots. Inaccordance to our results, nine populations of A. bertolonii had sig-nificant but small differences in their shoot/root Ni concentrationquotient (Galardi et al., 2007b). Similarly to A. bertolonii (Galardiet al., 2007b), A. lesbiacum did not show any tendency of loweringthe shoot/root Ni concentration quotient as the Ni treatmentraised, at least for the range of Ni concentration used in this exper-iment and thus the saturation pattern found in the root-to-shoottransport system of T. caerulescens (Assunção et al., 2003) wasnot detected. However, cultivation period is an important factor(Coinchelin et al., 2012) determining the root-to-shoot transloca-tion patterns.

4.3. Initial seed Ni concentration effect

The four populations of A. lesbiacum showed equal efficiency inaccumulating Ni in their shoots based on the slopes of the regres-sion lines. However, the starting point of the accumulation process(see the intercepts in Fig. 1a) was found to be responsible for theoverall significant inter-population variation (Tables S4 and S5).For each population, the starting point of the accumulation processappeared to be strictly related to its initial seed Ni concentration.Thus, there is evidence that the initial seed Ni concentration mayserve as a determining factor of the significant inter-populationvariation in Ni accumulation of A. lesbiacum. No significant correla-tion emerged between initial seed Ni concentration and either leafNi concentration or Ni accumulation factor of A. lesbiacum in thefield. Moreover, there was no significant correlation between initialseed Ni concentration and bioavailable Ni concentration in thesoils of origin. However, Murakami et al. (2008) found that soybeanseed Cd content resembled the trend followed by the studied par-ent soil and Vogel-Mikuš et al. (2007) showed that seed Cd hyper-accumulation in Thlaspi (Noaccea) praecox, may enhance thesurvival of the plantlets growing on Cd-rich soils. Further studiesare necessary to test whether high levels of seed Ni influence thesurvival of A. lesbiacum plantlets in Ni-rich soils. In addition, theinvestigation of the spatial distribution of Ni in the seeds may shedsome light on the mechanism behind the high levels of seed Ni in A.lesbiacum.

4.4. Ni influence on plant growth

Ni concentration in the treatment solution did not affect shootgrowth (Table S2). As the shoot response is a secondary effect, con-trolled by internal transport and sequestering processes, it seemsthat a wider concentration range should be used so as to observesignificant variation of shoot lengths with Ni treatment.

On the other hand, significant inter-population differences wererevealed in root growth. OL population showed the lowest negative



slope for the ‘Root length vs Ni treatment’ relationship in compar-ison to the three other populations. This result indicates that the Niconcentration in the treatment solution inhibited less the rootelongation of the plantlets originating from OL population. How-ever, the other three populations always had higher root lengthsin comparison to the OL population (Fig. 1d).

The fact that roots appeared more sensitive to external Ni thanthe shoots is not unexpected as the primary effects of the increasedmetal concentrations are expected to be manifested on the organthat has the first contact with the metal (Meyer et al., 2010). In-tra-specific variation in the degree of Ni effects on plant growthhas also been demonstrated for the Ni hyperaccumulator A. bertol-onii (Galardi et al., 2007b).

4.5. Ni tolerance

Tolerance indices (TI) also confirmed that the roots were moresensitive to both plant Ni concentration and Ni treatment. Onlyin the case of the AM population was there a significant decreaseof shoot tolerance index due to both Ni concentration in the shoots(Fig. 1e) and in the treatment solution (Table S5). On the contrary,root Ni concentration decreased the root tolerance index of all pop-ulations (Table S5, Fig. 1f), with LO population presenting the low-est decrease and thus the highest root tolerance.

Galardi et al. (2007b) also showed that roots were more sensi-tive in Ni toxicity than the shoots in A. bertolonii, despite the highertotal Ni concentration of the shoots, and they assumed that a great-er quantity of cytosolic free Ni in roots may be responsible for theirhigher sensitivity to Ni toxicity. On the other hand, a limited effectof Cd (Keller et al., 2006) and Ni (Assunção et al., 2003) has beenfound on the roots of T. caerulescens and it was attributed to thelower metal concentrations in the roots in relation to the shoots.Conclusively, although A. lesbiacum presents a species-wide toler-ance pattern like numerous other hyperaccumulators of the genusAlyssum in the Mediterranean (Brooks et al., 1979; Pollard et al.,2002), it seems that tolerance is a variable trait across its differentpopulations probably due to inter-population variation in parts ofthe uptake and translocation processes.

Our results demonstrated a significant positive relationship be-tween tolerance to the root Ni concentration and tolerance to Niconcentration in the treatment solution and thus revealed a signif-icant relationship between tolerance and accumulation for roots ofA. lesbiacum. Galardi et al. (2007b) also found a positive relation-ship between tolerance and accumulation for shoots of A. bertolo-nii, based on a positive linear relationship between shoottolerance to the amount of accumulated Ni and to external Ni con-centration. So far the investigation of the relationship between me-tal tolerance and accumulation has not excluded the possibility ofthe existence of different relationship types between various spe-cies and/or metals (reviewed by Pollard et al. (2002)). Neverthe-less, metal antagonism (Kazakou et al., 2008) may be asignificant factor influencing the metal tolerance and accumulationrelationship (e.g. Ca interference in Ni toxicity (Gabbrielli and Pan-dolfini, 1984; Bani et al., 2009)).

4.6. Tolerance, accumulation and their relation with Nihyperaccumulation in the field and bioavailable Ni concentration insoils of origin

Kazakou et al. (2010) in a field survey across the four popula-tions of A. lesbiacum showed that Ni hyperaccumulation wasclearly associated with soil Ni concentration. Nevertheless, neitherNi hyperaccumulation in the field nor bioavailable Ni concentra-tion in soils of origin were found to be correlated with Ni tolerance,accumulation and translocation patterns, measured during thishydroponic experiment. However, the lack of significant correla-

tions could be related to the small number of the different existingpopulations.

Finally, although tolerance is considered a result of adaptiveevolution in response to soil metal concentrations (Pollard et al.,2002), in consistence with Galardi et al. (2007b), the most tolerantpopulation of A. lesbiacum (LO population) seems not to be locatedon soils with the highest total soil Ni concentrations (i.e. soils ofthe AM population, see Kazakou et al. (2010)). However, AM pop-ulation was the second most tolerant A. lesbiacum population, pre-senting simultaneously the highest shoot and root Ni accumulationand demonstrating the highest leaf Ni concentration in a previousfield study (Kazakou et al., 2010). Thus, although field trials areessential, it is suggested that the AM population would probablybe the most efficient among the populations of the Ni hyperaccu-mulator A. lesbiacum for potential use in phytoremediationtechnologies.

5. Conclusions

A. lesbiacum populations show significant variation in Ni toler-ance and accumulation under hydroponic conditions that is not re-lated to the variation in the Ni status of the soils of origin. Inter-population variation in Ni accumulation seems to be a result oftwo combined effects: (a) the inter-population differentiation inthe initial seed Ni concentration which practically could be inter-preted as a different starting point of the accumulation processfor each population, and (b) the physiological differences in xylemloading and Ni translocation across the populations of A. lesbiacum.In addition, a strong positive relationship was revealed betweentolerance and accumulation for A. lesbiacum. Our results give sup-port to the suggestion that the selection of metal hyperaccumula-tor species with enhanced phytoremediation efficiency should beconsidered at the population level. Although field trials on realcontaminated soils are essential, in the case of the Ni hyperaccu-mulator A. lesbiacum the AM population would be the first choicefor potential use in phytoremediation. A better understanding ofthe mechanisms underlying inter-population variation in metaltolerance and accumulation would provide great insights in theuse of metal hyperaccumulator plants in phytoremediationtechnologies.

Acknowledgments

We thank A.J.M. Baker and R.D. Reeves for their valuable com-ments and for correcting the English text. This research has beenco-financed by the European Union (European Social Fund – ESF)and Greek national funds through the Operational Program ‘‘Edu-cation and Lifelong Learning’’ of the National Strategic ReferenceFramework (NSRF) – Research Funding Program: Heracleitus II.Investing in knowledge society through the European Social Fund.

Appendix A. Supplementary material

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.chemosphere.2013.09.106.

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Intra-specific variation in Ni tolerance, accumulation and translocation patterns in the Ni-hyperaccumulator Alyssum lesbiacum

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