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Cadmium Accumulation in SunflowerPlants Influenced by ArbuscularMycorrhizaSara Adrián López de Andrade a , Adriana Parada Dias da Silveira a ,Renato Atílio Jorge b & Mônica Ferreira de Abreu aa Instituto Agronômico-IAC , Centro de Pesquisa e Desenvolvimentode Solos e Recursos Ambientais , Campinas, SP, Brazilb Universidade Estadual de Campinas-UNICAMP, Departamento deFísico-Química, Instituto de Química , Campinas, SP, BrazilPublished online: 04 Apr 2008.

To cite this article: Sara Adrián López de Andrade , Adriana Parada Dias da Silveira , RenatoAtílio Jorge & Mônica Ferreira de Abreu (2008) Cadmium Accumulation in Sunflower PlantsInfluenced by Arbuscular Mycorrhiza, International Journal of Phytoremediation, 10:1, 1-13, DOI:10.1080/15226510701827002

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International Journal of Phytoremediation, 10:1–13, 2008Copyright C© Taylor & Francis Group, LLCISSN: 1522-6514 print / 1549-7879 onlineDOI: 10.1080/15226510701827002

CADMIUM ACCUMULATION IN SUNFLOWER PLANTSINFLUENCED BY ARBUSCULAR MYCORRHIZA

Sara Adrian Lopez de Andrade,1 Adriana Parada Dias daSilveira,1 Renato Atılio Jorge,2 and Monica Ferreira de Abreu1

1Instituto Agronomico-IAC, Centro de Pesquisa e Desenvolvimento de Solos eRecursos Ambientais, Campinas, SP, Brazil2Universidade Estadual de Campinas-UNICAMP, Departamento deFısico-Quımica, Instituto de Quımica, Campinas, SP, Brazil

In order to investigate the cadmium (Cd) accumulation patterns and possible alleviationof Cd stress by mycorrhization, sunflower plants (Helianthus annuus L.) were grown inthe presence or absence of Cd (20 µmol L−1) and inoculated or not inoculated with thearbuscular mycorrhizal fungus (AMF) Glomus intraradices. No visual symptoms of Cdphytotoxicity were observed; nevertheless, in non-mycorrhizal plants the presence of Cddecreased plant growth. The addition of Cd had no significant effect on either mycorrhizalcolonization or the amount of extra-radical mycelia that was produced by the AMF. Cdaccumulated mainly in roots; only 22% of the total Cd absorbed was translocated to theshoots, where it accumulated to an average of 228 mg Cd kg−1. Although the shoot-to-rootratio of Cd was similar in both the AMF inoculated and non-inoculated plants, the totalabsorbed Cd was 23% higher in mycorrhizal plants. Cd concentration in AMF extra-radicalmycelium was 728 µg g−1 dry weight. Despite the greater absorption of Cd, mycorrhizalplants showed higher photosynthetic pigment concentrations and shoot P contents. Cd alsoinfluenced mineral nutrition, leading to decreased Ca and Cu shoot concentrations; N, Feand Cu shoot contents; and increased S and K shoot concentrations. Cd induced guaiacolperoxidase activity in roots in both mycorrhizal and non-mycorrhizal plants, but this increasewas much more accentuated in non-mycorrhizal roots. In conclusion, sunflower plantsassociated with G. intraradices were less sensitive to Cd stress than non-mycorrhizal plants.Mycorrhizal sunflowers showed enhanced Cd accumulation and some tolerance to excessiveCd concentrations in plant tissues.

KEY WORDS: cadmium accumulation, heavy metals, mycorrhiza, nutrient uptake, peroxi-dase, phytoextraction

INTRODUCTION

Cadmium (Cd) is a widespread trace element found in many natural and agriculturalenvironments, resulting mainly from human activities (Wagner, 1993). This non-essentialmetal is readily taken up by the root system and can be accumulated in plant tissues,inhibiting plant growth and development (Hernandez and Cooke, 1997). Cd is usuallyretained in the roots but small amounts are translocated to the shoots (Benavides, Gallego

Address correspondence to Adriana Parada Dias da Silveira, Instituto Agronomico-IAC, Centro de Pesquisae Desenvolvimento de Solos e Recursos Ambientais, C.P.-28, CEP 13012-970, Campinas, SP, Brazil. E-mail:apdsil@iac.sp.gov.br

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2 S. A. LOPEZ DE ANDRADE ET AL.

and Tomaro, 2005). It is known that Cd damages the photosynthetic apparatus and mayaffect chlorophyll contents (Krupa and Baszynski, 1995). Another toxic effect causedby excessive concentrations of Cd involves oxidative stress, which is related to lipidperoxidation of cellular membranes (Verma and Dubey, 2003). Excess concentrations ofCd may trigger an increased production of the reactive oxygen species (ROS) that affectimportant biomolecules (Benavides et al., 2005). To combat oxidative damage, plants havean antioxidant defence system comprised of enzymes (catalases, peroxidases, superoxidedismutases) and metabolites that neutralize and scavenge the ROS (Gratao et al., 2005).

However, there are significant differences in plant tolerance to Cd and some speciesare able to tolerate higher concentrations of Cd in their tissues than others. Plant mechanismsthat avoid Cd toxicity, such as immobilization, exclusion, and synthesis of phytochelatins,have been comprehensively reviewed by Sanita di Toppi and Gabrielli (1999). Arbuscularmycorrhiza (AM) is not usually considered when dealing with a plant’s metal-tolerancemechanisms, yet its role in ameliorating the effects of heavy metal toxicity in the host planthas been reported (Andrade et al., 2003, 2004, 2005, Davies et al., 2002; Rivera-Becerrilet al., 2002; Shetty, Hetrick, and Schwab, 1995). In mycorrhizal symbiosis, increasesor reductions of metal content in the host plant have been observed depending on thegrowth conditions, as well as the fungi and plant species involved (Joner and Leyval, 1997;Weissenhorn and Leyval, 1995). The possibility of increasing metal accumulation in planttissues, particularly in the shoot, has potential for phytoextraction purposes (Citterio et al.,2005; Gohre and Paskowski, 2006). Arbuscular mycorrhizal fungi (AMF) can alleviatemetal toxicity by enhancing nutrient supply, improving water relations (Meharg andCairney, 2000), or by metal sequestration in fungal structures, such as intra- and extra-radicalhyphae (Joner, Briones, and Leyval, 2000). Other possible metal-tolerance mechanisms inthe AM symbiosis include dilution of the metal ions by increased root or shoot growth,increased metal:phosphorus ratio, exclusion by precipitation of polyphosphate granules,and compartmentalization into plastids (Kaldorf et al., 1999; Shetty, Hetrick and Schwab,1995). There is some evidence that mycorrhizal plants can present significant differencesin the response of the antioxidative system to heavy metal toxicity (Schutzendubel andPolle, 2002). In relation to phytochelatin production, Turnau, Kottke, and Oberwinkler(1993) observed high concentrations of N and S, as well as Cd, in the vacuoles of an AMFassociated with Pteridium aquilinum collected from Cd-contaminated soil, suggesting theexistence of thiol-binding peptides in this class of fungi. The presence of metal-bindingpeptides in AM plants was reported by Galli, Schuepp, and Brunold (1995): they found anincrease in thiol compounds related to the mycorrhizal status in response to Cu stress inmaize plants.

The purpose of this investigation was to evaluate how the mycorrhization of sunflowerplants by the AMF Glomus intraradices alleviates the effect of Cd stress and to comparethe Cd accumulation patterns in mycorrhizal and non-mycorrhizal sunflower plants.

MATERIALS AND METHODS

Experimental Design

A greenhouse experiment was conducted using a 2 × 2 factorial scheme andcompletely randomized design, with 10 replications. The treatments used were the absenceor presence of 20 µmol L−1 of Cd in the nutrient solution and the inoculation or notwith the AMF Glomus intraradices (Schenk and Smith). Treatments were denominated as:

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Cd ACCUMULATION IN SUNFLOWER PLANTS 3

control (grown in the absence of Cd and G. intraradices), M (grown in the absence of Cdbut inoculated with G. intraradices), Cd (grown in the presence of Cd but not inoculatedwith G. intraradices), and M+Cd (grown in the presence of Cd and inoculated withG. intraradices).

AMF Inoculum

The AMF used was G. intraradices (IAC-43), obtained from the AMF collectionmaintained by the Instituto Agronomico (IAC), Campinas, Brazil, and propagated on stockcultures with Brachiaria brizantha Stapf for 6 months. The original spores arose froma non-contaminated soil. Colonized root fragments, mycelium, and a sand–soil mixturecontaining spores were used as inoculum. Each pot received approximately 2700 spores atthe time of sowing. The non-mycorrhizal treatments received washings of the soil-inoculummixture filtered through Whatman no 42 filter paper.

Pot Culture Experiment

Two liters of sterilized ground silica (2–3 mm) were placed in 2.79-L plastic pots.Sunflower (Helianthus annuus L. cv. ‘IAC Uruguai’) seeds were surface-sterilized with 1:3(v/v) of 2.5% sodium hypochlorite solution for 10 min. Six seeds were sown per pot and,after emergence, seedlings were thinned to one plant per pot. Plants were cultivated in ahydroponic system with silica and irrigated with complete nutrient solution: N-NO3 154.6;N-NH4 19.5; S-SO4 18.7; Ca 151.2; K 70.9; Mg 18.8; P 10; B 0.53; Fe 1.99; Mn 0.97; Cu0.076; Zn 0.3; Mo 0.15 mg L−1 (Furlani and Furlani, 1988). Distilled water was suppliedon alternate days. Plants undergoing Cd treatment received nutrient solution containing20 µmol L−1 of Cd supplied as Cd(NO3)2. The nutrient solution added to non-Cd treatedplants was supplied with N to compensate for the amount of nitrate added as Cd(NO3)2.Speciation calculations using Visual MINTEQ ver. 2.23 (Gustafsson, 2003) indicated that86% of the Cd in solution was free Cd2+ of the ion. The total volume of nutrient solutionadded to each pot during the experiment was 3.62 L, which corresponded to the 8.137 mgof Cd added cumulatively in Cd-treated plants. The plants were allowed to grow for 8 wkuntil harvest at the beginning of the flowering stage. Day and night temperatures rangedbetween 29 and 16◦C, respectively, with a photoperiod of 12 h.

Analytical Methods

At harvest, leaves, stems, and roots were separated. The leaf area was immediatelymeasured with a LiCor 3100 area meter. Shoots and roots were washed. Root fresh weightwas recorded and subsamples were: 1) stored in 50% ethanol for mycorrhizal colonizationdetermination, 2) stored in liquid nitrogen until use in enzymatic assays, or 3) dried andground. After drying at 60◦C, shoots were weighed and ground. The contents of P, K, Ca,Mg, S, Cu, Fe, Mn, Zn, and Cd in the plant tissues were determined by inductively coupledplasma-optical emission spectroscopy (ICP-OES; Jobin Yvon, JY50P Longjumeau, France)after HNO3-HClO4 digestion. Total-N in the digest was determined by Kjeldahl analysis(Bremner, 1965).

Chlorophyll (Chl) a, b and carotenoids were extracted with 100% dimethylsulfoxidefrom the youngest fully-expanded leaf 1 wk before harvest and was measured spectropho-tometrically (Hitachi U-2000, Tokyo, Japan) directly in the extracts (Lichtenhaler and

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4 S. A. LOPEZ DE ANDRADE ET AL.

Welburn, 1983). Mycorrhizal colonization was evaluated by the grid-line intersect technique(Giovannetti and Mosse, 1980) by first clearing the roots with KOH and staining withtrypan blue. The length of the extra-radical mycelium (ERM) of AMF in the substratewas estimated according to Boddington et al. (1999), with modifications. The ERM wasextracted by wet sieving 20 g of substrate. The extracted ERM was stained with 0.05%trypan blue in lactoglycerol and its total length was assessed under a light microscope(125× magnification). Substrate with and without AMF inoculation was submitted to theprocedure and only hyphae with characteristics and morphology of AMF were considered.The absence of AMF hyphae was verified in the non-inoculated treatment. Sixty-fourfields were counted and the results expressed as meters per gram of dry substrate. In orderto quantify Cd in the extra-radical mycelia of AMF, hyphae extraction was performed byflotation based on Joner et al. (2000). At harvest, 2 L of the substrate of each pot was washedseveral times in a large bucket with running water and the washing solution was passedthrough a 0.45-mm mesh and then collected in dark flasks. The mycelium suspension wascleaned by flotation and its purity was observed under a light microscope. The suspensionwas vacuum filtered using a nitrocellulose filter, collected, dried at 40◦C for 36 h, andweighed. Dry mycelium was hydrolyzed with 2 mL of 65% w/w HNO3 and four dropsof 70% w/w HClO4 and Cd concentration in the mycelium determined by flame atomicabsorption spectrometry (F-AAS; Perkin Elmer, 5000, Norwalk, CT, USA).

Fractionation of the root cytoplasm and cell wall for determination of Cd content ineach fraction was performed according to Inouhe et al. (1994) with modifications. Rootsfrozen in liquid nitrogen (0.5–1.0 g fresh weight) were homogenized using a mortar andpestle in 20 mmol L−1 Tris-HCl buffer (pH 7.8). The homogenates were centrifuged twiceat 1500 g for 10 min and at 10000 g for another 10 min. The resulting supernatant wascollected and represented the cytoplasmic fraction. The precipitate in the tube was collected,dried at 37–40◦C, and represented the cell wall fraction. Cd concentration in the cytoplasmicfraction was determined directly by F-AAS. The dried cell wall fraction was hydrolyzedwith 2 mL of 65% HNO3 for 20 min at 100◦C. Four drops of 70% w/w HClO4 were addedto the hydrolyzates and further hydrolyzed for 15 min at 100◦C. After this process, threedrops of 30% H2O2 were added. The mixture was maintained at 100◦C for 15 min andfinally diluted to 5 mL with deionized water. The Cd content was determined by F-AAS.

Guaiacol peroxidase (GPX) (EC 1.11.1.7) activity was assayed spectrophotometri-cally using a diode array spectrophotometer (Hewlett Packard, 8452 A, Palo Alto, CA,USA) according to Boscolo, Menossi, and Jorge (2003). Roots frozen in liquid nitrogen(0.1–0.05 g) were washed three times in deionized water and homogenized in 50 mmol L−1

phosphate buffer (KH2PO4/K2HPO4, pH 6.8). Homogenates were centrifuged at 10000g for 8 min and the supernatant was used immediately to determine peroxidase activity.The reaction mixture contained 500 µmol L−1 phosphate buffer, 8 mmol L−1 guaiacol,and 8 mmol L−1 H2O2 and protein extract. The increase in absorbance was recorded at470 nm (extinction coefficient, ε = 26.6 mmol L−1 cm−1). Catalase (CAT) (EC 1.11.1.6)activity was determined by monitoring the disappearance of H2O2 through the decrease inabsorbance at 240 nm (ε = 39.4 mmol L−1 cm−1). Total soluble proteins in each extractwere determined using the Bio-Rad protein assay (Bradford, 1976). Determinations ofenzyme activity were performed in duplicate using five roots per treatment.

The nutrient and Cd translocation indexes were calculated as the percentage of thetotal amount absorbed that was translocated to the shoots. Cd transfer index (TF) wascalculated as the ratio of Cd concentration in the plant tissues (shoot or root) to that inthe solution. All data were processed by an analysis of variance. Significant treatment

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Cd ACCUMULATION IN SUNFLOWER PLANTS 5

Table 1 Shoot dry weight (SDW), leaf area, root fresh weight (RFW), mycorrhizal colonization (Myc.Colon.) of sunflower plants and extra-radical mycelium length (ERM), as influenced by inoculation withGlomus intraradices and Cd addition (20 µmol L−1)

TreatmentSDW

GLeaf Area

cm2RFW

gMyc. Colon.

%ERMmg−1

Control 3.85 Ba 367 Ba 13.16 Aa 0 0M 4.05 Aa 464 Aa 12.34 Aa 35.88 a 0.98 aCd 3.01 Bb 345 Ba 13.56 Aa 0 0M + Cd 3.49 Ab 395 Ab 12.72 Aa 30.8 a 0.92 a

Control: grown in the absence of Cd and G. intraradices, M: grown in the absence of Cd but inoculatedwith G. intraradices, Cd: grown in the presence of Cd but not inoculated with G. intraradices and M+Cd:grown in the presence of Cd and inoculated with G. intraradices. Means with the same letter are notsignificantly different (p ≤ 0.05) by the Tukey test. Upper case letters are used for comparisons of treatmentswith and without mycorrhizal inoculation for each Cd concentration, and lower case letters for comparisonsbetween Cd treatments.

effects were determined by the Tukey test (α = 0.05). Data expressed as a percentage weretransformed to arcsin-square root values prior to statistical analysis.

RESULTS

The effects of Cd and AMF inoculation on growth, expressed as shoot dry weight(SDW), leaf area, and root fresh weight (RFW), are shown in Table 1. No visual symptomsof Cd phytotoxicity were observed. Nevertheless, in non-mycorrhizal plants (control and Cdtreatments), the presence of Cd resulted in a 22% and 11% decrease in SDW and leaf area,respectively. Mycorrhizal plants (M and M+Cd treatments) showed a 10% higher SDWand 17% higher leaf area than non-mycorrhizal plants. Root growth was not affected by Cdaddition or AMF inoculation. Inoculated plants had about 33% of the root length colonizedbut no mycorrhizal structures were found in non-inoculated plants. The Cd addition had nosignificant effect on mycorrhizal colonization nor on the amount of ERM produced by theAMF.

Both Cd addition and AMF inoculation influenced the contents of photosyntheticpigments in sunflower plants (Table 2). In general, Cd caused a significant reduction inthe contents of Chl a, Chl b, and carotenoids in non-mycorrhizal plants. The Chl a/b ratiowas not influenced by Cd or mycorrhizae. Mycorrhizal plants showed higher chlorophyllcontents only in the presence of Cd. The reduction in Chl a contents in mycorrhizal plantswas approximately 12%, whereas in non-mycorrhizal plants the decrease reached 26%, ascompared to treatments without Cd addition.

Cd ions accumulated mainly in roots, with only 22% of the total Cd absorbedtranslocated to the shoots (IT) (Table 3). Cd-treated sunflower plants contained an averageof 228 mg Cd kg−1 in shoots. Although the ratio of Cd in shoots to roots was similar inboth AMF inoculated and non-inoculated plants, the total absorbed Cd was 23% higher inmycorrhizal plants. The concentration of Cd found in roots and shoots of mycorrhizal plantswas 23% and 34% higher, respectively, than in non-mycorrhizal plants. The distribution ofCd in the root cell wall fraction was approximately 80% with the remaining 20% in thecytoplasm (Table 3). The Cd concentration found in AMF extra-radical mycelium was of728 µg g−1 dry weight.

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6 S. A. LOPEZ DE ANDRADE ET AL.

Table 2 Chlorophyll a, Chl b, Chl a+b, xanthophyll and carotenoid (C x+c) contents and Chl a: Chl b ratioas influenced by inoculation with Glomus intraradices and Cd addition (20 µmol L−1)

Treatment Chl a Chl bChl a + b

µg mL−1 extract C x + c R a/b

Control 11.46 Aa 2.41 Aa 13.88 Aa 3.23 Aa 4.72 AaM 12.24 Aa 2.55 Aa 14.80 Aa 3.47 Aa 4.79 AaCd 8.44 Bb 1.78 Bb 10.22 Bb 2.47 Bb 4.77 AaM+Cd 10.62 Aa 2.15 Aa 12.78 Aa 3.02 Aa 4.99 Aa

Control: grown in the absence of Cd and G. intraradices, M: grown in the absence of Cd but inoculatedwith G. intraradices, Cd: grown in the presence of Cd but not inoculated with G. intraradices and M+Cd:grown in the presence of Cd and inoculated with G. intraradices. Means with the same letter are notsignificantly different (p ≤ 0.05) by the Tukey test. Upper case letters are used for comparisons of treatmentswith and without mycorrhizal inoculation for each Cd concentration, and lower case letters for comparisonsbetween Cd treatments.

The Cd addition influenced plant mineral nutrition, decreasing Ca and Cu concentra-tions as well as N, Cu, and Fe contents in shoots. In non-mycorrhizal plants, the Cd additiondiminished shoot N content by 22% and its concentration in roots by 27% (Table 4). Bycontrast, the presence of Cd in the nutrient solution led to an increase of 20% and 9% inS and K concentrations in plant shoots, respectively, in relation to non-Cd-treated plants.Zn concentrations in shoots and roots were higher in non-mycorrhizal plants and showed adifferent response to the Cd addition as compared to mycorrhizal plants. Shoot Zn contentwas diminished by the Cd addition in mycorrhizal plants and increased in non-mycorrhizalones.

AMF inoculation influenced plant mineral nutrition. In general, shoot P concentrationwas 18% higher in mycorrhizal plants than in non-mycorrhizal ones. A 24% higher P contentwas found in mycorrhizal plants, even in the presence of Cd (Table 4). Mn concentrationand content in shoots were significantly lower in mycorrhizal plants, whereas S, N, K,Ca, Mg, and Cu concentrations in shoots were not influenced by AMF inoculation. Thetranslocation index (defined as the percentage of the total amount of nutrient absorbed andtranslocated to the shoots) of N, K, P, and Ca was generally higher in mycorrhizal plantseven in the presence of Cd (Table 5).

The Cd addition caused a 26% and 96% increase in GPX activity of mycorrhizaland non-mycorhizal roots, respectively, when compared to non-Cd-treated roots (Figure1). The GPX activity was highly and positively correlated with Cd concentration in shoots(R2 = 0.717; p < 0.001). GPX activities of mycorrhizal and non-mycorrhizal roots weresimilar in the absence of Cd. Root GPX activity was negatively correlated with SDW (R2

= −0.558, p < 0.05) and with Chl a and Chl b contents in leaves (R2 = −0.702; p < 0.01).No CAT activity was observed in sunflower roots (data not shown).

DISCUSSION

The amount of Cd that was added to each plant was 8.14 mg, an amount considered tobe of environmental relevance and in the range found in Cd-polluted soils (Sanita di Toppiand Gabbrielli, 1999). Plant growth and development, which involves the coordination ofmany physiological processes, can readily reflect the stress that heavy metals may cause inplants. As already established by others for some mobile elements such as Cd (Pulford andDickinson, 2006), no visual symptoms of phytotoxicity were observed in Cd-treated plants,

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Tabl

e3

Cd

conc

entr

atio

nin

shoo

tsan

dro

ots,

tota

lC

dab

sorb

ed,s

hoot

Cd

cont

ent,

tran

sloc

atio

nin

dex

(TI)

,sho

otto

root

Cd

conc

entr

atio

nra

tioan

dC

dtr

ansf

erin

dex

from

nutr

ient

solu

tion

toth

esh

oot(

TF s

hoot

)an

dto

the

root

s(T

F roo

t)in

sunfl

ower

plan

tsas

influ

ence

dby

inoc

ulat

ion

with

Glo

mus

intr

arad

ices

and

Cd

addi

tion

(20

µm

olL

−1)

Tre

atm

ent

Cd

shoo

tm

gkg

−1C

dro

otm

gkg

−1C

dab

sorb

edm

gkg

−1C

dco

nten

tµ

gpl

ant−

1C

dT

IC

dsh

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root

TF s

hoot

TF r

oot

Cd

cell

wal

lm

gg−

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Cd

cyto

plas

m

Con

trol

1015

-31

--

--

--

M7.

814

-32

--

--

--

Cd

204

B71

4B

918

B67

4B

22.3

A0.

28A

90B

318

B22

.5A

5.13

AM

+Cd

252

A88

5A

1137

A90

5A

22.7

A0.

28A

114

A39

4A

17.9

A4.

88A

Con

trol

:gr

own

inth

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senc

eof

Cd

and

G.

intr

arad

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,M

:gr

own

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Cd

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ofC

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ated

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ntra

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ces

and

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own

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esen

ceof

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and

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ulat

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ithG

.int

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dice

s.M

eans

with

the

sam

ele

tter

are

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igni

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tlydi

ffer

ent(

p≤

0.05

)by

the

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st(A

,Bco

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nm

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ents

).

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8 S. A. LOPEZ DE ANDRADE ET AL.

Table 4 Shoot nutrient concentrations and contents and root nutrient concentrations as influenced by inoculationwith Glomus intraradices and Cd addition (20 µmol L−1)

(g kg−1) (mg kg−1)

Shoot concentrations N P K S Ca Mg Cu Fe Mn Zn

TreatmentsControl 24.0 0.7 15.4 2.0 27.0 4.58 8.88 67 323 56M 23.9 1.0 15.0 2.2 31.2 5.16 9.62 79 228 55Cd 21.0 0.8 16.0 2.5 25.4 5.26 4.90 67 331 78M+Cd 23.7 0.9 17.1 2.5 23.3 4.96 5.12 71 288 50

SignificanceAMF NS 0.007 NS NS NS NS NS NS 0.03 0.0005Cd NS NS 0.02 0.001 0.002 NS 0.00002 NS NS 0.01AMF × Cd NS NS NS NS 0.02 0.004 NS NS NS 0.001LSD 3.19 0.14 1.60 0.31 3.91 0.40 1.67 15.7 9.55

(mg plant−1) (µg plant−1)

Shoot contents N P K S Ca Mg Cu Fe Mn Zn

TreatmentsControl 89.8 2.9 59.3 7.8 101 4.5 34.1 254 1145 217M 91.3 3.8 57.9 8.3 113 5.1 33.4 302 825 210Cd 69.3 2.9 62.7 8.7 90 5.2 16.1 222 1091 257M+Cd 82.2 3.4 52.7 8.4 82 4.9 18.0 271 978 178

SignificanceAMF NS 0.0006 NS NS NS NS NS 0.004 0.03 0.0007Cd 0.007 NS NS NS 0.003 NS 0.00001 0.04 NS NSAMF × Cd NS NS 0.02 NS NS 0.004 NS NS NS 0.002LSD 14.6 0.46 6.89 1.18 18.5 2.33 6.06 43.6 280 29.14

(g kg−1) (mg kg−1)

Root concentrations N P K S Ca Mg Cu Fe Mn Zn

TreatmentsControl 14.8 1.26 8.6 1.9 8.9 2.5 17 376 125 42M 12.9 1.18 5.4 1.6 6.7 1.8 15 1102 112 46Cd 10.8 1.04 8.0 1.5 7.0 1.8 14 527 109 52M+Cd 12.9 1.22 6.2 1.8 6.9 2.0 16 1394 132 32

SignificanceAMF NS NS 0.00001 NS NS NS NS 0.0001 NS 0.01Cd 0.02 NS NS NS NS NS NS NS NS NSAMF x Cd 0.03 NS 0.01 0.0504 NS 0.02 NS NS NS 0.001LSD 2.55 0.26 0.88 0.35 1.76 0.52 3.53 391 45.3 9.05

but they did show reduced growth (Table 1). By contrast, it is known that the physiologyof plants is strongly influenced by AMF and that plant growth promotion by these fungihas been frequently observed in conditions of excess metal concentrations (Andrade et al.,2003, 2004; Greipsson and Hovsepyan, 2004; Rivera-Becerril et al., 2002). As expected,the mycorrhizal condition positively influenced plant development yielding higher shootdry weights, leaf area, and photosynthetic pigment content than in non-mycorrhizal plants.The fact that mycorrhizal fungus colonization and the amount of ERM produced by theAMF were not affected by the Cd addition to the solution allowed us to compare Cd effectsin plants with very similar colonization rates and amounts of ERM. Thus, we propose thatthe reduction of plant growth in Cd-treated mycorrhizal plants was due to a direct toxic

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Table 5 Nutrient translocation index as influenced by inoculation with Glomus intraradices and Cd addition(20 µmol L−1)

Treatments N K P Ca Mg Cu Fe Mn Zn S

Control 59 Ba 64 Ba 38 Bb 75Ba 64 Bb 30 Ba 14 Aa 72 Aa 55 Aa 50 BbM 63 Aa 66 Aa 44 Aa 78 Aa 74 Aa 24 Aa 11 Aa 75 Aa 60 Aa 62 AaCd 66 Aa 74 Aa 46 Aa 82 Aa 73 Aa 38 Aa 7 Ba 67 Aa 56 Aa 57 AaM+Cd 65 Aa 73 Aa 47 Aa 77 Ab 71 Aa 24 Ab 5 Ba 68 Ba 61 Aa 60 Aa

Control: grown in the absence of Cd and G. intraradices, M: grown in the absence of Cd but inoculatedwith G. intraradices, Cd: grown in the presence of Cd but not inoculated with G. intraradices and M+Cd:grown in the presence of Cd and inoculated with G. intraradices. Means with the same letter are not significantlydifferent (p ≤ 0.05) by the Tukey test. Upper case letters are used for comparisons of treatments with andwithout mycorrhizal inoculation for each Cd concentration, and lower case letters for comparisons between Cdtreatments.

effect on the physiology of the plant rather than to reduced growth promotion caused bylower colonization rates. Other authors (Rivera-Becerril et al., 2002) also reported a lackof effect of relevant levels of Cd on mycorrhizal formation.

The observations of Cd accumulation indicate that sunflower plants may attain levelsof 228 mg Cd kg−1 SDW. Although sunflower roots acted as a barrier for Cd translocation,retaining most of the Cd absorbed, a quarter of the total absorbed metal was translocatedto the shoots (Table 3). Davies et al. (2002) observed the capacity of sunflower plants forchromium phytoextraction. AMF may either reduce or increase metal absorption, dependingon the plant and associated AMF species, metal concentration, and growth conditions(Weinserhorn and Leyval, 1995). Nevertheless, in this study, the Cd shoot:root accumulationratio was similar in mycorrhizal and non-mycorrhizal plants and the total absorbed Cd washigher in AMF-colonized plants. We calculated the transfer factor (TF) in order to quantifythe ability of the plant to accumulate Cd with respect to the concentration of this metalin the medium (Elkhatib, Thabet, and Mahdy, 2001). The TF values obtained indicatethat sunflower plants had a great ability to accumulate Cd in the shoots, in agreementwith Elkhatib et al. (2001). AMF-colonized plants had a greater TF than non-colonizedplants (Table 3), indicating that mycorrhizal plants had greater Cd accumulating capacity.Enhanced Cd absorption in mycorrhizal plants was also observed for other plant and AMFspecies (Hutchinson et al., 2004; Rivera-Becerril et al., 2002; Weinserhorn and Leyval,1995). It is known that roots can accumulate Cd in the apoplast by ionic interactions withcomponents of the cell wall and that part of the metal can be complexed by phytochelatinsand sequestered in the cytoplasm and deposited in the vacuole (Cohen et al., 1998). Inthis study, sunflower roots, independent of their mycorrhizal condition, accumulated Cd2+

preferentially in the cell wall fraction (80%), a result comparable to that observed byInouhe et al. (1994). Cd binding to components of the cell walls is one of the mechanismswhereby plants may reduce Cd concentration in the shoots. In addition, the immobilizationof metals in the AMF mycelium has been proposed as a “filter” during metal uptake (Joneret al., 2000). The Cd concentration found in mycelium of the AMF was similar to thatin roots and confirms the high adsorption capability of the AMF mycelia for Cd (Joneret al., 2000). Nonetheless, in this study mycorrhizal plants had greater root and shoot Cdconcentrations, and no differences in the Cd content of the cell wall fraction was observedbetween mycorrhizal and non-mycorrhizal roots, indicating that AMF was not particularly

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efficient in avoiding translocation of Cd to the shoots and did not prevent excess Cd inaboveground tissues.

The Cd addition to the nutrient solution reduced the contents of photosyntheticpigments in sunflower leaves (Table 2). By contrast, the Chl a/b ratio remained constant inboth the absence and presence of the metal, indicating that neither of the chlorophylls waspreferentially affected by Cd addition. Chlorophyll has been shown to be one of the targetsof Cd toxicity leading to a reduction in its content (Vassilev et al., 2002). This reduction isdue to the inhibition of its biosynthesis by Cd ions and has been proposed to be responsible,in part, for the growth reduction caused by this metal (Bazzaz, Rolfe, and Carlson, 1992).Nevertheless, mycorrhizal plants showed higher photosynthetic pigment contents only inthe presence of Cd and, in general, were less affected by Cd ions than non-mycorrhizalplants.

Disturbances in the uptake and distribution of nutrients in plants have also beencorrelated with Cd toxicity. Cd may affect the content of polyvalent cations throughantagonistic processes involving metal competition for binding sites or transporters(Gussarson et al., 1996). In this study, the Cd addition decreased Ca and Cu shootconcentrations and the N, Fe, and Cu shoot contents (Table 4). Cd caused a reductionin N and Mg concentration in the roots and an increase in Mg content in the shootsof non-mycorrhizal plants. By contrast, S and K shoot concentrations increased in thepresence of the metal. The increase in S content in plant tissues in Cd-treated plants hasbeen observed previously (Nocito et al., 2002). An increase in sulfate uptake as a directeffect of Cd accumulation has been proposed as an adaptive response to support S demandfor phytochelatin biosynthesis (Nocito et al., 2002). AMF inoculation influenced plantmineral content in sunflower plants (Table 4). Mycorrhizal plants showed higher shoot Pconcentration and content in both the presence and absence of Cd. The translocation indexof N, K, P, and Ca was generally higher in mycorrhizal plants, even in the presence ofCd (Table 5) and the higher TI for nutrient found in mycorrhizal sunflower plants may berelated to the greater plant development (Table 1).

Figure 1 Guaiacol peroxidase (GPX) ativity and total protein concentrations as influenced by inoculation withGlomus intraradices and Cd addition (20 µmol L−1). Control: grown in the absence of Cd and G. intraradices,M: grown in the absence of Cd but inoculated with G. intraradices, Cd: grown in the presence of Cd but notinoculated with G. intraradices and M+Cd: grown in the presence of Cd and inoculated with G. intraradices.Means with the same letter are not significantly different (p ≤ 0.05) by the Tukey test. Upper case letters are usedfor comparisons of treatments with and without mycorrhizal inoculation for each Cd concentration, and lowercase letters for comparisons between Cd treatments.

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GPX, together with superoxide dismutase and glutathione reductase, is considered tobe one of the key enzymes involved in cell protection against ROS and, although Cd doesnot generate ROS directly, it generates oxidative stress that interferes with the antioxidantdefence system (Gratao et al., 2005). In this study, GPX activity in mycorrhizal andnon-mycorrhizal roots was similar in the absence of Cd (Figure 1). However, GPX activitywas induced in plant roots as a response to Cd concentration in the nutrient solution,suggesting that this enzyme, and possibly other important antioxidant enzymes, acted as adefence mechanism to resist Cd-induced oxidative damage in sunflower roots. The increasein its activity due to the presence of Cd was much more accentuated in non-mycorrhizalthan in mycorrhizal roots (Figure 1). From these results, it can be inferred that mycorrhizalplants were less stressed than non-mycorrhizal ones and therefor better supported thestressful levels of Cd in the medium. CAT activity could not be detected in sunflowerroots, indicating the absence of significant amounts of this enzyme, an observation alsoreported for barley roots (Hegedus, Erdei and Gaber, 2001). In the present investigation, thetotal soluble protein content was higher in mycorrhizal roots, maintaining a similar proteincontent in the presence and absence of Cd. On the other hand, in non-mycorrhizal rootsthe content of soluble proteins was increased by the Cd addition. This increase may berelated to a higher synthesis of phytochelatins since it has been observed that Cd inducesprogressively the synthesis of water-soluble proteins with an amino acid compositionvery similar to that found in phytochelatins (Leita et al., 1993). Gianinazzi-Pearson andGianinazzi (1995), on comparing different studies, observed higher protein concentrationsin extracts of mycorrhizal roots, most with an unknown function.

In conclusion, mycorrhizal sunflower plants, in spite of their higher Cd accumulation,exhibited better growth than non-mycorrhizal plants, showing certain tolerance to excessconcentrations of Cd in the plant tissues. This was reflected by the higher levels ofphotosynthetic pigments and P in shoots and the lower activity of guaiacol peroxidase inroots. Therefore, sunflower plants associated with this AMF were less sensitive to Cd stressthan non-associated plants. Ongoing research will help elucidate the role of mycorrhiza inmetal sequestration and alleviation of heavy metal stress.

ACKNOWLEDGEMENTS

The authors would like to recognize CNPq for the fellowship awarded to the firstauthor and Miss Rosana Gierts Goncalves for technical support.

REFERENCES

Andrade, S.A.L., Abreu, C.A., de Abreu, M.F., and Silveira, A.P.D. 2003. Interacao de chumbo, dasaturacao por bases do solo e de micorriza arbuscular no crescimento e nutricao mineral dasoja. Rev. Bras. Ci. Solo. 27, 945–954.

Andrade, S.A.L., Abreu, C.A., de Abreu, M.F., and Silveira, A.P.D. 2004. Influence of lead additionon arbuscular mycorrhiza and Rhizobium symbioses under soybean plants. Appl. Soil Ecol. 26,123–131.

Andrade, S.A.L., de Abreu, M.F., Jorge, R.A., and Silveira, A.P.D. 2005. Cadmium effect on theassociation of jackbean (Canavalia ensiformis) and arbuscular mycorrhizal fungi. ScientiaAgricola 62, 389–394.

Baker, A.M.J., McGrath, S.P., Reeves, R.D., and Smith, J.A.C. 2000. Metal hyperaccumulator plants:A review of the ecology and the physiology of a biochemical resource for phytoremediation of

Dow

nloa

ded

by [

Uni

vers

ity o

f R

egin

a] a

t 11:

00 0

3 Se

ptem

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12 S. A. LOPEZ DE ANDRADE ET AL.

metal polluted soil. In: Phytoremediation of Contaminated Soil and Water, pp. 85–107. (Terry,N. and Banuelos, G., Eds.). Boca Raton, Florida, Lewis.

Bazzaz, F.A., Rolfe, G.L., and Carlson, R.W. 1992. Effect of cadmium on photosynthesis andtranspiration of excised leaves of corn and sunflower. Physiol. Plant. 32, 373–377.

Benavides, M.P., Gallego, S.M., and Tomaro, M.L. 2005. Cadmium toxicity in plants. Braz. J. PlantPhysiol. 17, 21–34.

Boddington, C.L., Bassett, E.E., Dodd, J.C., and Jakobsen, I. 1999. Comparison of techniques forthe extraction and quantification of extra-radical mycelium of arbuscular mycorrhizal fungi insoils. Soil Biol. Biochem. 31, 479–482.

Boscolo, P.R.S., Menossi, M., and Jorge, R.A. 2003. Aluminium-induced oxidative stress in maize.Phytochem. 62, 181–189.

Bradford, N.M. 1976. A rapid and sensitive methods for the quantification of microgram quantitiesof protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254.

Bremner, J.M. 1965. Total nitrogen. In: Methods of Soil Analysis, pp. 1149–1178. (Black, C.A., Ed.).Madison, American Society of Agronomy.

Citterio, S., Prato, N., Fumagalli, P., Aina, R., Massa, N., Santagostino, A., Sgorbati, S., and Berta,G. 2005. The arbuscular mycorrhizal fungus Glomus mosseae induces growth and metalaccumulation changes in Cannabis sativa L. Chemosphere 59, 21–29.

Cohen, C.K., Fox, T.C., Garvin, D.F., and Kochian, L.V. 1998. The role of iron-deficiencystress responses in stimulating heavy-metal transport in plants. Plant Physiol. 116, 1063–1072.

Davies, Jr. F.T., Puryear, J.D., Newton, R.J., Egilla, J.N., and Saraiva Grossi, J.A. 2002. Mycorrhizalfungi increase chromium uptake by sunflower plants: influence on tissue mineral concentration,growth and gas exchange. J. Plant Nutr. 25, 1389–2407.

Elkhatib, E.A., Thabet, A.G., and Mahdy, A.M. 2001. Phytoremediation of cadmium contaminatedsoils: role of organic complexing agents in cadmium phytoextraction. Land Contam. Reclam.9, 359–366.

Furlani, A.M.C. and Furlani, P.R., 1988. Composicao e pH de solucoes nutritivas para estudosfisiologicos e selecao de plantas em condicoes nutricionais adversas. Campinas: InstitutoAgronomico, 34p. (Boletim Tecnico).

Galli, U., Schuepp, H., and Brunold, C. 1995. Thiols of Cu-treated maize plants inoculated with thearbuscular mycorrhizal fungus Glomus intraradices. Physiol Plant. 94, 247–253.

Giovannetti, M.E. and Mosse, B. 1980. An evaluation of techniques for measuring vesicular-arbuscular mycorrrhizal infection in roots. New Phytol. 84, 482–500.

Gianinazzi-Pearson, V. and Gianinazzi, S. 1995. Proteins and proteins activities in endomycorrhizalsymbioses. In: Mycorrhiza, pp. 251–266. (Varma, and Hock,, Eds.). Berlin Springer-Verlag.

Gohre, V. and Paskowski, U. 2006. Contribution of the arbuscular mycorhizal symbiosis to heavymetal phytoremediation. Planta 223, 1115–1122.

Gratao, P.L., Polle, A., Lea, P.J., and Azevedo, R.A. 2005. Making the life of heavy-stressed plantsa little easier. Funct. Plant Biol. 32, 481–494.

Greipsson, S. and Hovsepyan, A. 2004. Effect of arbuscular mycorrhizal fungi on phytoextraction bycorn (Zea mays) of lead-contaminated soil. International. J. Phytoremediation 6, 305–321.

Gussarson, M., Asp, H., Adalsteinsson, S., and Jensen, P. 1996. Enhancement of cadmium effectson growth and nutrient composition of birch (Betula pendula) by buthionine sulphoximine(BSO). J. Exp. Bot. 47, 211–215.

Gustafsson, J.P., 2003. Visual Minteq ver. 2.15 http://www.lwr.kth.se/english/OurSoftware/vminteq>

Hergedus, A., Erdei, S., and Gabor, H. 2001. Comparative studies of H2O2 detoxifying enzymes ingreen and greening barley seedlings under cadmium stress. Plant Sci. 160, 1085–1093.

Hernandez, L.E. and Cooke, D.T. 1997. Modifications of root plasma membrane lipid compositionof cadmium treated Pisum sativum. J. Exp. Bot. 48, 1375–1381.

Dow

nloa

ded

by [

Uni

vers

ity o

f R

egin

a] a

t 11:

00 0

3 Se

ptem

ber

2014

Cd ACCUMULATION IN SUNFLOWER PLANTS 13

Hutchinson, J.J., Young, S.D., Black, C.R., and West, H.M. 2004. Determining radio-labile soilcadmium by arbuscular mycorrhizal hyphae using isotopic dilution in a compartmented-potsystem. New Phytol. 164, 477–484.

Inouhe, M., Ninomiya, S., Tohoyama, H., Joho, M., and Murayama, T. 1994. Different characteristicsof roots in cadmium-tolerance and Cd-binding complex formation between mono anddicotyledonous plants. J. Plant Res. 107, 201–207.

Joner, E.J., Briones, R., and Leyval, C. 2000. Metal-binding capacity of arbuscular mycorrhizalmycelium. Plant Soil 226, 227–234.

Joner, E.J. and Leyval, C. 1997. Uptake of 109Cd by roots and hyphae of a Glomus mosseae/Trifoliumsubterraneum mycorrhiza from soil amended with high and low concentrations of cadmium.New Phytol. 135, 353–360.

Kaldorf, M., Kuhn, A.J., Schroder, W.H., Hildebrandt, U., and Bothe, H. 1999. Selective elementdeposits in maize colonized by a heavy metal tolerance conferring arbuscular mycorrhizalfungus. J. Plant Physiol. 154, 718–728.

Krupa, Z. and Baszynski, T. 1995. Some aspects of heavy metals toxicity towards photosyntheticapparatus: direct and indirect effects on light and dark reactions. Acta Physiol. Plant 7, 55–64.

Leita, L., Nobili, M., Mondini, C., and Baca Garcia, M.T. 1993. Response of Leguminosae to cadmiumexposure. J. Plant Nutr. 16, 2001–2012.

Lichtenthaler, H.K. and Wellburn, A. 1983. Determination of total carotenoids and chlorophylls aand b of leaf extracts in different solvents. Biochem. Soc. Trans. 603, 591–592.

Meharg, A.A. and Cairney, J.W.G. 2000. Co-evolution of mycorrhiza symbionts and their host tometal contaminated enviroments. Adv. Ecol. Res. 30, 69–112.

Nocito, F.F., Pirovano, L., Cocucci, M., and Sacchi, A.G. 2002. Cadmium-induced sulfate uptake inmaize roots. Plant Physiol. 129, 1872–1879.

Pulford, I.D. and Dickinson, N.M. 2006. Phytoremediation technologies using trees. In: Traceelements in the environment: biogeochemistry, biotechnology, and bioremediation, pp. 383–404. (Prassad, M.N.V., Sajwan, K.S.S., and Naidu, R., Eds.). Boca Raton, Florida,

Rivera-Becerril, F., Calantzis, C., Turnau, K., Caussanel, J.P., Belismov, A.A., Gianinazzi, S.,Strasser, J.R., and Gianinazzi-Pearson, V. 2002. Cadmium accumulation and buffering ofcadmium-induced stress by arbuscular mycorrhiza in three Pisum Sativum L. genotypes. J. Exp.Bot. 371, 1177–1185.

Sanita di Toppi, L. and Gabrielli, R. 1999. Response to cadmium in higher plants. Environ. Exp. Bot.41, 105–130.

Schutzendubel, A. and Polle, A. 2002. Plant responses to abiotic stresses: heavy metal-inducedoxidative stress and protection by mycorrhization. J. Exp. Bot. 372, 1351–1365.

Shetty, K.G., Hetrick, B.A.D., and Schwab, A.P. 1995. Effects of mycorrhizae and fertilizeramendments on zinc tolerance of plants. Environ. Pollution 88, 307–314.

Turnau, K., Kottke, I., and Oberwinkler, F. 1993. Element localization in mycorrhizal roots ofPteridium aquilinum L. Kuhn collected from experimental plots treated with cadmium dust.New Phytol. 123, 313–324.

Vassilev, A., Lidon, C.F., Matos, M.C., Ramalho, J.C., and Yordanov, I. 2002. Photosyntheticperformance and content of some nutrients in cadmium and copper treated barley plants.J. Plant Nutr. 25, 2343–2360.

Verma, S. and Dubey, R.S. 2003. Lead toxicity induces lipid peroxidation and alters the activities ofantioxidant enzymes in growing rice plants. Plant Sci. 164, 645–655.

Wagner, G.J. 1993. Accumulation of cadmium in crop plants and its consequences to human health.Adv. Agron. 51, 173–212.

Weissenhorn, I. and Leyval, C. 1995. Root colonization of maize by a Cd-sensitive and a Cd-tolerantGlomus mosseae and cadmium uptake in sand culture. Plant Soil 175, 233–238.

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