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Biologia 68/2: 223—230, 2013 Section Botany DOI: 10.2478/s11756-013-0005-9 Cadmium effects on mineral nutrition of the Cd-hyperaccumulator Pfaffia glomerata Marcelo Pedrosa Gomes 1,3 *, Teresa Cristina Lara Lanza Sá e Melo Marques 2 & Angela Maria Soares 3 1 Université du Québec `a Montréal, Institut des Sciences de l’environnement, Succ. Centre-Ville, C.P. 8888,H3C 3P8, Montréal, Québec, Canada; e-mail: [email protected] 2 Universidade Federal de Lavras, Departamento de Ciˆ encias do Solo, C.P. 3037, 37200-000, Lavras, MG, Brazil 3 Universidade Federal de Lavras, Departamento de Biologia, Setor de Fisiologia Vegetal, C.P. 3037, 37200-000, Lavras, MG, Brazil; e-mail: author: [email protected] Abstract: A plant’s ability to survive in a stressful environment is correlated with its nutritional status, which can be affected by cadmium (Cd) uptake. The present study evaluated the influence of Cd on the concentrations and distributions of nutrients in the roots and shoots of the Cd-hyperaccumulator Pfaffia glomerata (Sprengel) Pedersen. Plantlets were cultivated in nutrient solutions containing increasing Cd concentrations during 20 days under greenhouse conditions, and the concentrations of Cd and essential macro- (N, P, K, Ca, Mg and S) and micro- (Zn, Fe, Mn, Cu) elements in the roots and shoots were subsequently determined. Cd did not affect the plant biomass production. Cd accumulation was found to be higher in roots than in shoots, and influenced the distribution of macro and micro elements in those plants. Despite the high phytotoxicity of this element, our results indicated the existence of Cd-tolerance mechanisms in both nutrient uptake and distribution processes that enabled these plants to survive in Cd-contaminated sites. Key words: phytotoxicity; tolerance mechanisms; translocation; symptoms Introduction The heavy metal cadmium (Cd) is highly toxic to hu- mans and all other living organisms and it has no known biological function in aquatic or terrestrial organisms (Chen et al. 2007). Recent advances in industry and agriculture have led to increasing Cd levels in agricul- tural soil environments (Sarwar et al. 2010). Anthro- pogenic sources including phosphate fertilizers, waste water, Cd-contaminated sewage sludge and manures, metal industries, urban traffic, and cement industries have been associated with increasing environmental lev- els of Cd and its accumulation in plants due to its high mobility in the soil (Alloway & Steinnes, 1999; Yang et al. 2004). Heavy metals cannot be biodegraded and thus can only be extracted from contaminated sites (Ghnaya et al. 2005). Common physic-chemical techniques used to remediate soils or sediments polluted by metals are usu- ally expensive and unsuitable for situations affecting extensive areas (Dary et al. 2010), but biotechnological approaches have received a great deal of attention in recent years. Phytoremediation (the use of plants for metal reclamation) has emerged as an environmentally friendly proposal and may be the most cost-effective treatment for metal-polluted soils, especially in cases of extensive contamination (Dary et al. 2010). A strong indicator of the potential of a given plant for use in phytoremediation programs is its natural occurrence in contaminated areas. Natural occurrences of species of the genus Pfaffia (Amaranthaceae) in heavy metal con- taminated soil have been reported, and these species have been observed growing quite abundantly in a soil with 90 and 1,450 mg kg -1 of Cd and Zn respectively (Carneiro et al. 2002). These species can also accumu- late Cd to concentrations higher than 100 mg kg -1 and is considered as Cd hyperaccumulator (Carneiro et al. 2002). P. glomerata are perennial subshrubs or shrubs usually found growing at edges of woods or rivers and in the Brazilian rupestrian field (Nascimento et al. 2007). From their tuberous roots, several economically im- portant compounds have been isolated and identified (De Paris et al. 2000) which poses this species as has high commercial pharmaceutical value (Montanari et al. 1999). Due to the intense predatory exploitation of natural sources of this species, its cultivation have been increasingly stimulated (Montanari Jr. 1999), so that the use of P. glomerata in environmental recuper- ation programs could represent an interesting strategy * Corresponding author c 2013 Institute of Botany, Slovak Academy of Sciences

Cadmium effects on mineral nutrition of the Cd-hyperaccumulator Pfaffia glomerata

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Biologia 68/2: 223—230, 2013Section BotanyDOI: 10.2478/s11756-013-0005-9

Cadmium effects on mineral nutrition of the Cd-hyperaccumulatorPfaffia glomerata

Marcelo Pedrosa Gomes1,3*, Teresa Cristina Lara Lanza Sá e Melo Marques2

& Angela Maria Soares3

1Université du Québec a Montréal, Institut des Sciences de l’environnement, Succ. Centre-Ville, C.P. 8888, H3C 3P8,Montréal, Québec, Canada; e-mail: [email protected] Federal de Lavras, Departamento de Ciencias do Solo, C.P. 3037, 37200-000, Lavras, MG, Brazil3Universidade Federal de Lavras, Departamento de Biologia, Setor de Fisiologia Vegetal, C.P. 3037, 37200-000, Lavras,MG, Brazil; e-mail: author: [email protected]

Abstract: A plant’s ability to survive in a stressful environment is correlated with its nutritional status, which can beaffected by cadmium (Cd) uptake. The present study evaluated the influence of Cd on the concentrations and distributionsof nutrients in the roots and shoots of the Cd-hyperaccumulator Pfaffia glomerata (Sprengel) Pedersen. Plantlets werecultivated in nutrient solutions containing increasing Cd concentrations during 20 days under greenhouse conditions, andthe concentrations of Cd and essential macro- (N, P, K, Ca, Mg and S) and micro- (Zn, Fe, Mn, Cu) elements in the rootsand shoots were subsequently determined. Cd did not affect the plant biomass production. Cd accumulation was found tobe higher in roots than in shoots, and influenced the distribution of macro and micro elements in those plants. Despite thehigh phytotoxicity of this element, our results indicated the existence of Cd-tolerance mechanisms in both nutrient uptakeand distribution processes that enabled these plants to survive in Cd-contaminated sites.

Key words: phytotoxicity; tolerance mechanisms; translocation; symptoms

Introduction

The heavy metal cadmium (Cd) is highly toxic to hu-mans and all other living organisms and it has no knownbiological function in aquatic or terrestrial organisms(Chen et al. 2007). Recent advances in industry andagriculture have led to increasing Cd levels in agricul-tural soil environments (Sarwar et al. 2010). Anthro-pogenic sources including phosphate fertilizers, wastewater, Cd-contaminated sewage sludge and manures,metal industries, urban traffic, and cement industrieshave been associated with increasing environmental lev-els of Cd and its accumulation in plants due to its highmobility in the soil (Alloway & Steinnes, 1999; Yang etal. 2004).Heavy metals cannot be biodegraded and thus can

only be extracted from contaminated sites (Ghnaya etal. 2005). Common physic-chemical techniques used toremediate soils or sediments polluted by metals are usu-ally expensive and unsuitable for situations affectingextensive areas (Dary et al. 2010), but biotechnologicalapproaches have received a great deal of attention inrecent years. Phytoremediation (the use of plants formetal reclamation) has emerged as an environmentallyfriendly proposal and may be the most cost-effective

treatment for metal-polluted soils, especially in casesof extensive contamination (Dary et al. 2010). A strongindicator of the potential of a given plant for use inphytoremediation programs is its natural occurrence incontaminated areas. Natural occurrences of species ofthe genus Pfaffia (Amaranthaceae) in heavy metal con-taminated soil have been reported, and these specieshave been observed growing quite abundantly in a soilwith 90 and 1,450 mg kg−1 of Cd and Zn respectively(Carneiro et al. 2002). These species can also accumu-late Cd to concentrations higher than 100 mg kg−1 andis considered as Cd hyperaccumulator (Carneiro et al.2002).P. glomerata are perennial subshrubs or shrubs

usually found growing at edges of woods or rivers and inthe Brazilian rupestrian field (Nascimento et al. 2007).From their tuberous roots, several economically im-portant compounds have been isolated and identified(De Paris et al. 2000) which poses this species as hashigh commercial pharmaceutical value (Montanari etal. 1999). Due to the intense predatory exploitationof natural sources of this species, its cultivation havebeen increasingly stimulated (Montanari Jr. 1999), sothat the use of P. glomerata in environmental recuper-ation programs could represent an interesting strategy

* Corresponding author

c©2013 Institute of Botany, Slovak Academy of Sciences

224 M.P. Gomes et al.

for both environmental recover and economic develop-ment.Growth reductions in plants in response to rising

levels of Cd clearly indicates its ecophysiological im-pact of this heavy metal (Monni et al. 2001), and re-ductions in assimilation capacity, water balance distur-bances, and structural changes have been reported inCd-exposed plants (Barceló & Poschenreider 1990; Luxet al. 2004; Marques et al. 2000; Wójcik et al. 2005).Negative effect of Cd on photosynthetic processes havebeen attributed to the ability of this metal to inter-fere with chlorophyll synthesis (Horváth et al. 1996;Prasad 1995), chloroplast organization (Sandalio et al.2001), and photosynthetic electron transport by inhibit-ing water-splitting enzymes located at the oxidizingsite of photosystem II (PSII) (Van Assche & Clijsters,1990). The presence of Cd in the growth media may alsocause nutritional disturbances directly competing withnutrient uptake and/or altering essential element distri-butions and other metabolic processes (Gothberg et al.2004; Gussarson et al. 1996; Paiva et al. 2004; Sarwaret al. 2010). Plant responses to Cd contamination arevariable and species-specific (Paiva et al. 2004; Vate-hová et al. 2012), and the present study investigatedthe effects of different Cd doses in nutrient solution onthe macro- and micronutrient content of P. glomerata.

Material and methods

Plant growthP. glomerata seeds were germinated in Styrofoam trays ina thermostat-controlled and darkened chamber (70% rela-tive humidity and 25◦C). Forty-five-day-old seedlings havingsimilar sizes and weights were transferred to plastic beakers(6 L capacity, two plants per beaker) filled with Clark’s nu-trient solution (Clark 1975). After an initial growth periodof 15 days, Cd was added (as CdSO4) at different concen-trations (0, 15, 25, 45 and 90 µmol) to the culture mediaof plants selected for their apparent vigor. The nutrient so-lutions were continuously aerated and renewed weekly. ThepH of the medium was checked and adjusted on a daily basisto 5.5 ± 0.1. After 20 days of Cd-treatment, the plants wereharvested and divided into root and shoot fractions, placedin paper bags and dried in an air circulation oven at 70◦Cto a constant weight to determine biomass production.

Chemical analysesPlant samples (0.1 g) were digested in 5 ml of a strong acidsolution (HNO3/HClO4, 3:1, v/v) (Van Assche & Clijsters,1990) and their Ca, Cd, Cu, K, Fe, Mg, Mn and Zn concen-trations were determined using a flame atomic absorptionspectrometer (Perkin-Elmer Analyst 400, Norwalk, CT). Ni-trogen was determinate by titration after Kjeldahl diges-tion; sulfur by turbidimetry (Malavolta et al. 1989); andphosphorous concentrations in solution were measured bycolorimetry following the phosphorus-molybdate method.

Statistical analysesThe results are expressed as the averages of five replicates.The data were statistically evaluated using analysis of vari-ance run on the SAS software program (SAS Institute Ins.,1996). Regression and correlation analyses were also per-formed to test for relationships between the variables.

Fig. 1. Total Dry weight (DW) production and Root/ShootDW ratio of P. glomerata plants cultivated in nutritive solutionamended with 0, 15, 25, 45 and 90 µmol Cd. Vertical bars repre-sent standard errors (n = 5).

Results and discussion

Plant growth and visual symptomsTotal dry weight (DW) production and root/shoot DWratios were not statistically affected by Cd doses in so-lution (Fig. 1). Biomass reduction is a typical symptomof metal phytotoxicity (Barceló & Poschenreider 1990),although this negative effect was not seen in our study.The maintenance of biomass production, even whenexposed to Cd levels considered phytotoxic (Kabata-Pendias & Adriano 1995), is an indicator of the Cd-tolerance of P. glomerata. Furthermore, this species oc-curs naturally in soils with high levels of this metal,such as in mining areas, and it has been shown to betolerant of heavy metal stress (Carneiro et al. 2002;Skrebsky et al. 2008).Although dry matter production was not affected,

plants growing in the highest Cd doses in solutionshowed symptoms of heavy metal phytotoxicity. At theend of the experimental period the plants were wiltedand had yellowed leaves and blackened roots. Similar re-sults were seen with Eucalyptus urophylla and E. mac-ulate, both of which are considered to be Cd-tolerant at90 µmol Cd (Soares et al. 2005); and visual symptomsof toxicity occurred at doses of 25 µmol Cd (Lagriffoulet al. 1998) in maize plants. According to Breckle &Kahle (1992), these symptoms may be associated withmultiple deficiencies of several nutrients essential to theformation, expansion, and operation of chloroplasts andwith Cd-phytotoxic effects on extensibility or synthesisof cell wall materials (Barceló & Poschenrieder 1992).

Cadmium contentThe Cd content of root and shoots increased as the Cddoses in the nutrient solution were increased (Fig. 2).Regression analysis was used to establish the relation-ship between the Cd contents of the roots (r2 = 0.99)and shoots (r2 = 0.93) and Cd additions to the nutrient

Pfaffia glomerata Cd-phytoremediation ability 225

Fig. 2. Cd contents of P. glomerata plants cultivated in nutritivesolution amended with 0, 15, 25, 45 and 90 µmol Cd. Each pointis the mean of five measurements.

solution (Fig. 2). The Cd content of roots was consis-tently higher than in the shoots, regardless of the Cddoses in solution, and they showed sharp increases asCd doses increased (Fig. 2). In contrast, the Cd con-tent of shoots showed only small increases as Cd dosesincreased (Fig. 2). P. glomerata plants increased theirnatural Cd-absorption with increasing doses of Cd innutrient solution, but allocated most of the ions to theirroot systems. In spite of the high Cd content in theroots, they did not show growth restrictions – suggest-ing that this species is heavy metal-tolerant. One ofthe mechanisms associated with heavy metal tolerancein plants involves decreasing metal translocation to theshoots (Shi & Cai 2009). Plants can thus avoid neg-ative growth effects by reducing heavy metal interfer-ence of photosynthetic processes (including chlorophyllsynthesis, chloroplast organization, and PSII activity)(Horváth et al. 1996; Prasad 1995; Sandalio et al. 2001).

Macronutrient contentsNitrogenNitrogen (N) is an essential macronutrient and an im-portant component of many structural, genetic, andmetabolic compounds in plants (Hassan et al. 2005).N content was higher in shoots (33.66 to 49.25 g kg−1

DW) than in roots (21.66 to 27.75 g kg−1 DW) for allCd treatments (Fig. 3). The higher N content seen inthe shoots than in the roots was probably related to themaintenance of protein synthesis, electron transferencein photosynthesis, and respiration processes (Wieden-hoeft 2006). Additionally, increases in Cd accumulationin the shoots would lead to increased N contents thereas N is required to synthesis Cd-detoxifying chelatormolecules such as glutathione and phytochelatins (Go-jon & Gaymard 2010). A positive correlation (r = 0.999,P = 0.01) was seen for shoot N content and shoot dryweigh production with higher Cd treatments, indicat-ing the contribution of N to the maintenance of biomassincreases in these treated plants. No statistical differ-

ences (P > 0.05) were seen between root and shoot Ncontents of Cd treated plants in relation to controls, re-gardless of the Cd doses used. Previous reports aboutthe relationship between Cd and N accumulation inplants were contradictory. While Mitchel et al. (2000)noted that N and Cd accumulation were positively asso-ciated, Bhandal & Kuar (1992) reported the oppositeresults. Cd concentrations have been found to be re-lated to decreases in nitrate reductase (NR) activity inplants, leading to decreases in photosynthetic rates andchlorophyll contents (Campbell 1999; Hernandez et al.1997), or to increases in enzymatic break down inducedby active oxygen species generated during their expo-sure to stress (Hassan et al. 2008). Our data, however,indicates that Cd had little or no influence on N nutri-tion in P. glomerata.PhosphorousPhosphate is an important element for energy transferand protein metabolism in plants (Marschener 1995),and decreases in phosphorous (P) contents in responseto Cd have been reported (Paiva et al. 2000; Paiva etal. 2004; Soares et al. 2005; Yang et al. 1996). Root Pcontent did not statistically differ (P > 0.05) amongthe different Cd concentrations, except for the control,and the P content was greater in shoots (1.62 to 4.00g kg−1 DW) than roots (1.72 to 2.28 g kg−1 DW) inall Cd treated plants (Fig. 3). Shoot P contents werehigher in Cd treated plants, which could be directlyrelated to their greater Cd contents, and phosphorousmay be accumulated to promote plant growth levelsthat can neutralize the toxic effect of Cd by dilution(Sarwar et al. 2010). Phosphorous is involved in glu-tathione (GSH) biosynthesis (May et al. 1998) (a com-pound that is considered a precursor of phytochelatin[PC] synthesis) (Sarwar et al. 2010), and phytochelatinsare known to compartmentalize Cd into vacuoles byforming Cd/PC complexes (Salt & Rauser 1995). Ad-ditionally, the higher P contents seen in shoots when Cdwas present in large amounts was apparently related tothe proliferation of antioxidant systems (i.e., superoxidedismutase, ascorbate peroxidase and catalase) that canmitigate oxidative stress and prevent membrane dam-age (Wang et al. 2009). We saw a positive correlationbetween P and N in roots (r = 0.976, P = 0.05) andshoots (r = 0.971, P = 0.05) at higher Cd exposures, sothat N as well as P can apparently partly alleviate Cdtoxicity (Sarwar et al. 2010) – and the mobilization ofthese elements to the shoots represents a Cd-tolerancemechanism in P. glomerata.SulfurSulfur (S) is an important component of amino acidsand is a biologically ubiquitous element (Wiedenhoeft2006); it is also closely linked with phytochelatinmetabolism involved in heavy metal-tolerance (Hartley-Whitaker et al. 2001). Glutathione (a sulfur-containingtripeptide thiol with the formula γ-glutamate-cysteine-glycinesulfur) is a precursor of phytochelatins (Rosen2002) and is a very important antioxidant involved incellular defenses against toxicants (Scott et al. 1993).We did not find statistical differences (P > 0.05) in root

226 M.P. Gomes et al.

Fig. 3. Macronutrient contents of P. glomerata plants cultivated in nutritive solution amended with 0, 15, 25, 45 and 90 µmol Cd.Vertical bars represent standard errors (n = 5).

S contents among the different Cd treatments (Fig. 3). Scontents were greater in shoots that in roots in plantsexposed to 15 and 45 than 90 µmol Cd. Changes inN supply affect S demands of plants, and vice versa(Matula 2004). Sulfur is an important structural com-ponent of many co-enzymes and prosthetic groups, suchas the ferredoxins necessary for N assimilation (Sar-war et al. 2010). As can be seen in Fig. 3, shoot Ncontent was lower in plants exposed to 90 mmol Cd,and the positive correlation (r = 0.965, P = 0.05)found between shoot N and S contents could explaintheir lower S content. Gomes et al. (2011) reporteddecreases in the S translocation indexes of Salix hum-boldtiana plants grown in soils contaminated by heavymetals. According to these authors, these transloca-tion indexes can explain the differences in S content inthe shoots, as heavy metals appear to affect S translo-

cation from the roots to the shoots but not S up-take.PotassiumThe potassium (K) contents of roots were lower inplants exposed to 25 and 45 µmol Cd than in controlplants, while shoot K content was higher in the 25 µmolCd treatment than under control conditions (Fig. 3).This data indicates that K is translocated from rootsto shoots at 25 to 45 µmol Cd as Cd accumulates inshoots, leading to lower K contents in the roots. A neg-ative correlation (r = -0.950, P = 0.05) was in fact seenbetween root K and shoot Cd in 45 µmol Cd treatmentplants. The increase seen in the K translocated indexcould also explain the greater shoot K content observedin 25 µmol Cd treated plants. According to Mengel &Kirkby (1987), K is preferentially transported to theshoots and has close relationships with protein synthe-

Pfaffia glomerata Cd-phytoremediation ability 227

Fig. 4. Micronutrient contents of P. glomerata plants cultivated in nutritive solution amended with 0, 15, 25, 45 and 90 µmol Cd.Vertical bars represent standard errors (n = 5).

sis, cytokinin supplies and plant growth, and also servesas a dominant cation for counterbalancing anions inplants (Marschner 1995). Disturbances of K nutritionwith Cd exposure have been reported in several stud-ies (Ghnaya et al. 2005; Zornoza et al. 2002). Changesin shoot translocation of K and other nutrients havealso been attributed to vascular system alterations andreductions in the numbers and diameters of xylem ele-ments (Ouzounidou et al. 1994).Calcium and MagnesiumNo statistical differences between root and shoot cal-cium (Ca) and magnesium (Mg) contents (P > 0.05)were observed at different Cd concentrations, withshoot concentrations being consistently higher than inroots (Fig. 3). Maintenance of Ca and Mg uptake anddistribution may be a tolerance strategy of P. glom-erata plants. Studies have shown that Ca can aid inalleviating Cd toxicity and that Cd competes for Cachannels in plants (Clemens et al. 1998; Nelson 1986).The alleviation of Cd toxicity by Ca may be due tothe fact that plasma membrane surfaces are usuallynegatively charged, and high concentrations of Ca2+

would tend to neutralize them and thus minimize thetoxic effect of Cd; likewise, high Ca concentrations nearion channels might decrease the influx of Cd (Sarwaret al. 2010). Heavy metals are known to interfere inchlorophyll biosynthesis (Cagno et al. 1999; Chugh &Sawhney 1999; Horváth et al. 1996), and as magnesiumserves as the central atom of the chlorophyll moleculeand as a co-factor in many enzymes activating phos-phorylation processes (Tu & Ma 2005), maintenance of

uptake and Mg transport to shoots may help ensurethe maintenance of chlorophyll biosynthesis and avoidfurther damage to the photosynthetic system.

Micronutrient contentsZincZinc (Zn) is an important micronutrient essential forplant growth and development as a component of en-zyme structures and regulatory transcriptional proteins(Valle & Falchuk, 1993) Zn content was observed tobe higher in roots (79.14 to 102.90 mg kg−1 DW)than in shoots (39.24 to 63.60 mg kg−1 DW) in allCd treatments (Fig. 4). Zn is important in alleviatingCd-phytotoxicity as this heavy metal induces rapid freeradical synthesis (ROS) (Ercal et al. 2001; Kawano etal. 2001) that can be reduced by Zn (Aravind et al.2009). As such, the maintenance of higher Zn levelsin roots (where Cd was preferably allocated) will con-tribute to minimizing toxic Cd effects.As reported above, Zn content was lower in the

shoots of all Cd-treated plantlets as compared to con-trol plants (Fig. 4). Phosphorous-zinc interactions havebeen described in the literature (Cakmak & Marschner1986; Webb & Loneragan 1988) and, according to Cak-mak & Marschener (1986), Zn deficiencies in leaves en-hance P uptake rates by roots and its translocation tothe shoots. As such, the greater shoot P contents ofCd-treated plants would be associated with their lowershoot Zn contents. The negative correlation observedbetween Zn and P contents in the shoots of Cd-treatedplants (data not showed), especially at 25 mmol Cd L−1

228 M.P. Gomes et al.

(r = −0.990, P = 0.01), would explain the greater shootP content in these plants (4.00 mg kg−1 DW).IronIron (Fe) is an important plant modulator of re-dox potentials and a component of two major groupsof proteins – heme and Fe-S proteins (Romheld &Nikolic 2007). Fe contents were higher in roots (1,228.31to 2,454.75 mg kg−1 DW) than shoots (61.94 to116.68 mg kg−1 DW) in all Cd treatments, althoughroot Fe contents did not statically differ (P > 0.05)between Cd-treated and control plants (except at 25µmol Cd) (Fig. 4). Fe contents were lower in Cd-treatedshoots than in control plants (Fig. 4) however, Shao etal. (2007) showed that Cd-induced oxidative stress inrice is alleviated by Fe. Fe is an integral cofactor of an-tioxidant enzymes such as catalase and ascorbate per-oxidase (Sharma et al. 2004). As such, the maintenanceof high levels of Fe in the roots when Cd concentrationswere highest can increase the activity of these enzymes,and would be an important defense mechanism againstROS generated by Cd-stress (Wang et al. 2013). Ourdata suggests that Cd did not affect Fe uptake by P.glomerata roots, and that the differences seen in shootFe contents could be explained by Cd interference inthe translocation of this micronutrients to the shoots(as was reported by Sandalio et al. 2001). Cd can in-hibit the translocation of Fe into the aerial portion of aplant by different mechanisms, such as the radial move-ment of Fe in the roots, Fe loading into xylem vessels,or its absorption by the shoots (Sandalio et al. 2001).Fe translocation is also dependent on the productionof phytochelatins that have variable affinities for dif-ferent metals, and Cd could affect Fe translocation tothe shoots (Sandalio et al. 2001). The high Cd contentsseen in roots may lead to increased productions of phy-tochelatins that could sequester Fe as well as Cd in theroots.ManganeseManganese (Mn) is involved in many biochemical func-tions, as an activator of the enzymes involved in respira-tion, in redox reactions within the photosynthetic elec-tron transport system, in the Hill reactions in chloro-plasts, in amino acid and lignin synthesis, and in regu-lating hormone concentrations (Humphries et al. 2007).Mn concentrations were higher in shoots (73.20 to78.89 mg kg−1 DW) than roots (33.63 to 19.86 mg kg−1

DW) with all Cd-treatments (Fig. 4). Mn translocationto shoots may represent a tolerance mechanism thatalleviates Cd toxicity effects on the photosynthetic ap-paratus, as Cd can replace the central Mg2+ ion in thechlorophyll structure and Mn can partially restore thesedamaged chloroplast structures (Baszynski et al. 1980).Mn content was lower in Cd-treated than in the rootsand shoots of control plants as the availability of Mnis known to be partly decreased by Cd (Sarwar et al.2010). According to Baszynski et al. (1980), Cd and Mncompete for the same membrane transporters, whichmay have contributed to the lower Mn contents seen inCd-treated plants. A positive correlation (r = 0.979, P=0.05) between root Mn and shoot N contents was seen

at 90 µmol Cd. According to Balang et al. (2006), Mnis related to NO−

3 uptake, so that root Mn content mayimprove N nutrition under conditions of Cd toxicity andtherefore reduce Cd-phytotoxicity effects.CopperCopper (Cu) contents were higher in roots (18.10 to42.63 mg kg−1 DW) than shoots (6.81 to 12.37 mg kg−1

DW) under all Cd treatments, and root Cu contentswere greater in Cd-treated than in control plants (ex-cept at 15 µmol Cd) (Fig. 4). We noted a decrease inCu content in the shoots in relation to control plants,but this was only statistically significant at 15 µmol Cd(Fig. 4). According to Obata & Umebayashi (1997),Cd increases Cu uptake but restricts its transport toshoots, which was confirmed in the present study.In conclusion, we observed that Cd did not influ-

ence the growth of Pfaffia glomerata seedlings. Despitethe high phytotoxicity of this element, our results in-dicated the existence of Cd-tolerance mechanisms inboth nutrient uptake and distribution processes thatenabled these plants to survive in Cd-contaminatedsites. Plants exposed to very high Cd concentrations,however, demonstrated marked symptoms of intoxica-tion that may be related to the developmental age ofplants or to other tolerance mechanisms – aspects thatwill require more detailed investigations.

Acknowledgements

Authors are grateful to the Fundacao de Amparo a Pesquisado Estado de Minas Gerais (FAPEMIG) for financial sup-port.

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Received May 11, 2012Accepted September 20, 2012