heavy metals in white lupin: uptake, root-to-shoot transfer and redistribution within the plant

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Research

Blackwell Publishing Ltd

Heavy metals in white lupin: uptake, root-to-shoot

transfer and redistribution within the plant

Valérie Page

1

, Laure Weisskopf

2

and Urs Feller

1

1

Institute of Plant Sciences, University of Bern, Altenbergrain 21, 3013 Bern, Switzerland;

2

Laboratory of Molecular Plant Physiology, Institute of Plant Biology,

University of Zürich, Zollikerstrasse 107, 8008 Zürich, Switzerland

Summary

• The translocation of manganese (Mn), nickel (Ni), cobalt (Co), zinc (Zn) andcadmium (Cd) in white lupin (

Lupinus albus

cv. Amiga) was compared consideringroot-to-shoot transport, and redistribution in the root system and in the shoot, aswell as the content at different stages of cluster roots and in other roots.• To investigate the redistribution of these heavy metals, lupin plants were labelledvia the root for 24 h with radionuclides and subsequently grown hydroponically forseveral weeks.•

54

Mn,

63

Ni and

65

Zn were transported via the xylem to the shoot.

63

Ni and

65

Znwere redistributed afterwards via the phloem from older to younger leaves, while

54

Mn remained in the oldest leaves. A strong retention in the root was observed for

57

Co and

109

Cd.• Cluster roots contained higher concentrations of all heavy metals than nonclusterroots. Concentrations were generally higher at the beginning of cluster rootdevelopment (juvenile and immature stages). Mature cluster roots also containedhigh levels of

54

Mn and

57

Co, but only reduced concentrations of

63

Ni,

65

Zn and

109

Cd.

Key words:

cluster roots, heavy metals,

Lupinus albus

, phloem, redistribution,transport, xylem.

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© The Authors (2006). Journal compilation ©

New Phytologist

(2006)

doi

: 10.1111/j.1469-8137.2006.01756.x

Author for correspondence:

Urs Feller Tel: +41 31 631 49 58 Fax: +41 31 631 49 42 Email: [email protected]

Received:

19 December 2005

Accepted:

20 March 2006

Introduction

Plants have a natural ability to extract elements from soil andto distribute them between roots and shoot depending onthe biological processes in which the element is involved(Ximénez-Embún

et al

., 2002). In addition to the uptake ofnutrients, toxic compounds such as heavy metals can also betaken up by the plants. Heavy metals are defined as metalswith a density > 5.0 g cm

−

3

(Seaward & Richardson, 1990).Some heavy metals also play a role in plant metabolism, andcan be considered nutrients. This is the case for manganese(Mn), zinc (Zn) and nickel (Ni), which are involved in majorfunctions (Welch, 1995). Manganese has been shown to playa role in enzyme activation, biological redox systems (e.g.electron transport reactions in photosynthesis) or detoxifi-cation of oxygen free radicals; while Zn is involved in

membrane integrity, enzyme activation and gene expression.Nickel is needed for urea metabolism, iron absorption andnitrogen (N) fixation (Welch, 1995). There is no evidencethat cobalt (Co) has any direct role in the metabolism ofhigher plants in general (Marschner, 1995), but it has beendemonstrated to be required for N

2

fixation in legumes andin root nodules of nonlegumes (Ahmed & Evans, 1960;Marschner, 1995). In contrast, some heavy metals such ascadmium (Cd) are not needed by the plant, but are also takenup (Römer

et al

., 2000, 2002; Ximénez-Embún

et al

., 2002;Zornoza

et al

., 2002). Both kinds of heavy metal, nutrientsand pollutants, can accumulate in excess in the plant to levelsundesirably high for human or animal nutrition, and mayeven become toxic to plants at a certain concentration(William

et al

., 2000). Thus uptake of heavy metals by theroots, their transport in the different parts of the root system,

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their release to the shoot and further redistribution within theshoot are important processes for (i) redistribution of heavymetals in the plant/soil system; (ii) supply of shoot parts withnutrients; and (iii) quality of harvested plant parts.

The transport and mobility of heavy metals have beenstudied in plants including bean and wheat. In bean, Bukovac& Wittwer (1957) determined the absorption, transportand mobility of radionuclides applied on the leaves. Theyobserved that these radioactive isotopes had a differentmobility in the phloem. Schmidke & Stephan (1995)concluded that the metal micronutrients Mn, Zn, Co andiron (Fe) were supplied by phloem transport, and that thistransport was linked to the presence of nicotianamine.Takahashi

et al

. (2003) reported that nicotianamine isimportant for the regulation of metal transfer within cells, inaddition to its role in long-distance transport. More informationconcerning the distribution of Zn, Mn, Ni, Co and Cd isavailable for wheat. For example, Zn can be transportedrapidly in the phloem (Herren & Feller, 1994, 1996; Pearson

et al

., 1995; Haslett

et al

., 2001; Erenoglu

et al

., 2002; Page &Feller, 2005; Riesen & Feller, 2005), while Cd, which ischemically very similar to Zn (Chesworth, 1991) and thusmay be transported in plants by similar pathways (Grant

et al

., 1998), has a mobility different from Zn in wheat (Page& Feller, 2005; Riesen & Feller, 2005). Varieties of cropplants may differ considerably in the symplastic redistributionof Zn (Hajiboland

et al

., 2001; Erenoglu

et al

., 2002).Comparing two rice varieties differing in Zn efficiency, thetransport of Zn from older (source) to younger (sink) leaveshas been found to be more rapid under Zn deficiency thanunder adequate Zn supply, and higher in the Zn-efficient thanin the Zn-inefficient genotype (Hajiboland

et al

., 2001).Erenoglu

et al

. (2002) reported that differences in the expressionof Zn efficiency were not related to the redistribution offoliar-applied Zn within wheat plants. Therefore caution isrecommended when generalizing results obtained with aparticular species or genotype. Manganese may be translocatedas a free divalent cation in the xylem from roots to shoot(Marschner, 1995). On the other hand, redistribution of Mnin the phloem is very limited (El-Baz

et al

., 1990; Page & Feller,2005; Riesen & Feller, 2005), and may depend on the plantspecies and developmental stage (Herren & Feller, 1994). Thedistribution of heavy metals may depend on the availability ofother elements. An accumulation of Mn at the penetrationposition of

Erysyphe graminis

on wheat leaves was observedonly in plants supplied with silicate, but not in comparableplants grown without silicate (Leusch & Buchenauer, 1988).These results suggest that, at least under certain conditions,silicon influences the redistribution of Mn in wheat. Nickeland Co were considered to have an intermediate phloemmobility (Marschner, 1995), but recent studies in wheatdemonstrated that the mobility of Ni in the phloem is ratherhigh compared with Co (Page & Feller, 2005; Riesen & Feller,2005). In legumes, Ni and Mo are redistributed efficiently

from vegetative plant parts to maturing seeds, while theaccumulation of these two micronutrients in the ears ofcereals is less pronounced (Horak, 1985).

In the studies cited above, it was demonstrated that heavymetals have a different mobility in plants. It remains aninteresting challenge to elucidate in more detail the acquisitionand redistribution of various heavy metals in a plant formingcluster roots (e.g. white lupin). Lupin differs in its root anatomyfrom cereals and other plants mentioned above. Four recentreports on Cd uptake by white lupin are available in theliterature (Römer

et al

., 2000, 2002; Ximénez-Embún

et al

.,2002; Zornoza

et al

., 2002). Ximénez-Embún

et al

. (2002)investigated the tolerance, uptake and accumulation of Cd inwhite lupin grown on contaminated sand, and found that Cdconcentration was significantly higher in roots than in shoots.Zornoza

et al

. (2002) demonstrated that lupin roots have ahigh capacity of Cd retention by cell walls and complexationby thiol groups. Römer

et al

. (2000, 2002) compared themobilization and uptake of Cd by white lupin, blue lupin andryegrass on two soil types. These authors observed that the Cdcontent in shoots was 28 times lower for white lupin than forryegrass, while blue lupin had an intermediate Cd contentin the shoot. They suggested that the higher secretion ofcarboxylates into the rhizosphere by lupins might modify Cdspeciation in the soil solution, and that the consequence wasa lower Cd uptake, observed in both types of soils. Thesecretion of carboxylates may contribute to the solubilizationof iron and Mn in the soil and, as a consequence, lead to highercontents of these metals in lupin plants (Dinkelaker

et al

.,1995 and references therein).

Apart from the results mentioned above, only limitedinformation is available on heavy metal translocation in whitelupin, despite the fact that this plant is often used as a modelfor the study of nutrient uptake because of its capacity toproduce cluster roots and acquire phosphorus (P) from soilswhere it is sparsely available (Purnell, 1960; Dinkelaker

et al

.,1995; Neumann & Martinoia, 2002; Lamont, 2003). Thesecluster roots are the main site of phosphate acquisition in theroot system of white lupin, thanks to their large surface andtheir secretion activity. White lupin cluster roots secrete largeamounts of carboxylates, mainly citrate and malate, as wellas protons, phenolic compounds and phosphatases. In whitelupin, the carboxylate secretion and acidification of the rhizo-sphere have been studied intensively for many years (Gardner

et al

., 1982a, 1982b, 1983; Johnson

et al

., 1994; Neumann

et al

., 1999, 2000; Massonneau

et al

., 2001; Penaloza

et al

.,2002; Shane

et al

., 2003), and it has been shown that thesesecretion processes are related to the developmental stagesof cluster roots. Massonneau

et al

. (2001) defined fourdevelopmental stages of white lupin cluster roots. At the juvenilestage, roots are still growing and small amount of malate issecreted. Several days later, roots reach the immature stage,characterized by fully grown roots secreting reduced amountsof carboxylates and high quantities of phenolic compounds


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(Weisskopf

et al

., 2006). The mature stage is when mostcarboxylate secretion occurs and acidification takes place. Atthe senescent stage, secretion of carboxylates and phenoliccompounds is much reduced. It is very likely that theseinduced changes, mediated by the massive secretion ofprotons and carboxylates into the rhizosphere of cluster rootsand especially into the rhizosphere of mature cluster roots,will affect not only the availability of phosphate, but also thesolubility and uptake of heavy metals (Dinkelaker

et al

.,1995). Moreover, white lupin is the only cluster-rootedspecies of agricultural importance. Because of its very efficientP-acquisition strategy, as well as its ability to fix N in symbioticassociation with

Bradyrhizobiae

(Raza

et al

., 2001), whitelupin is a very promising crop in a world of nonrenewableresources, as it is able to acquire both N and phosphate withreduced application of external fertilizers. Thus more preciseknowledge about the heavy metal uptake and transfer tothe plant parts used as fodder or human food would be veryuseful from the agronomic point of view.

The secretion of citrate from mature cluster roots isimportant for the solubilization of phosphate and heavymetals in soil. Such solubilization processes are not relevantin hydroponic culture. However, such an experimental setupcan be used to test whether mature cluster roots are alsocharacterized by high acquisition rates for solubilized heavymetals from the medium, and particularly by a higherretention of heavy metals in vacuoles of cluster roots, thusresulting in a low root-to-shoot translocation.

The aim of the present study was to characterize thedistribution of five heavy metals in the roots and shoots ofwhite lupin after labelling a single root in hydroponic culture.The acquisition of heavy metals in the different developmentalstages of white lupin cluster roots was also analysed. Weinvestigated three essential heavy metals (Mn, Ni and Zn); theconditionally (for N

2

fixation) required Co; and a nonessentialheavy metal, Cd, which is a pollutant found in manycontaminated soils (Sauvé

et al

., 2000; Lugon-Moulin

et al

.,2004). White lupin was chosen for this study because ofthe well known physiology of its roots and the potentialagronomic application of this research.

Materials and Methods

Transport of heavy metals in white lupins

Seeds of white lupin (

Lupinus albus

L. cv. Amiga, Suedwest-deutsche Saatzucht, Rastatt, Germany) were washed in 1%NaOCl for 15 min, rinsed three times with deionized water,incubated overnight in aerated deionized water, andgerminated in the dark (for 4 d) and in the light (for 1 d) in aquadratic Petri dish prepared with five sheets of filter paper(Whatman 3MM) moistened with 0.2 m

M

CaSO

4

. Lupinseeds were placed in between the filter papers (four papersbelow and one above the seeds). The Petri dish was arranged

with a slope of 30

°

. After 5 d germination the root was placedfor 24 h on a 10-ml tube containing 10 ml nutrient solutionwith radioactive heavy metals. The nutrient solution used inthese experiments contained 1.5 m

M

KH

2

PO

4

, 0.76 m

M

MgSO

4

, 0.34 m

M

Ca(NO

3

)

2

, 0.22 m

M

KNO

3

, 7.5 µ

M

Fe(added as Sequestren), 0.25 µ

M

MnCl

2

, 1.23 µ

M

H

3

BO

3

,0.04 µ

M

ZnSO

4

, 0.05 µ

M

Na

2

MoO

4

, 0.012 µ

M

Ni(NO

3

)

2

,0.025 µ

M

CuSO

4

(Hildbrand

et al

., 1994, four timesdiluted). For preparation of the nutrient solution without P,KH

2

PO

4

was eliminated from the nutrient solution. Toinvestigate the distribution of radioactive heavy metals inwhole plants by gamma or beta spectrometry, the plants werelabelled with nutrient solution containing a mixture of

54

Mn(6.0 kBq l

−

1

),

63

Ni (739.4 kBq l

−

1

) and

57

Co (2.9 kBq l

−

1

)(first set of plants); or a mixture of

65

Zn (24.4 kBq l

−

1

) and

109

Cd (79.2 kBq l

−

1) (second set of plants). With gammaspectrometry it is possible to measure more than one isotopeat the same time: 65Zn and 109Cd, which have different gammaenergies, can be measured at the same time. To diminish thenumber of samples, 63Ni (beta energy), which is not detectedby the gamma counter, was added to the couple 54Mn and57Co (which also have different gamma energies). Afterwards,63Ni was detected using a beta counter. For autoradiography(separate experiments), plants were labelled separately eitherwith 54Mn (274.1 kBq l−1), 63Ni (261.1 kBq l−1), 57Co(274.1 kBq l−1), 65Zn (274.1 kBq l−1) or 109Cd (274.1 kBq l−1).After labelling, roots were washed (dipped three times sequen-tially in 100 ml nutrient solution) to remove radioactivesolutes from the root surface, and placed for 2 h in nutrientsolution with 0.1% Congo red to allow identification of theroot part initially labelled with heavy metals after furtherelongation of the root. Then the roots were washed threetimes to remove excessive dye and incubated for 1 h onnutrient solution. Thereafter the seedlings were transferred toaerated nutrient solution. Four labelled plants and twoidentically treated but initially unlabelled control plants wereplaced together on pots with 1 l nutrient solution. The fourreplicate plants from the same pot were sampled and analysedseparately. Plants were grown at a temperature of 21°Cduring the night and 25°C during the day, 65% humidity.The photoperiod was 14 h light (100 µmol photons m−2 s−1

from four Philips TLD 36 W/25 and two Osram Fluora L36 W/77 fluorescent lamps, measured 20 cm above theculture pot); 10 h night. The nutrient solution was changedevery week.

For analysis of the distribution of heavy metals in whitelupins labelled with radionuclides at the seedling stage (after5 d germination), plants were harvested at different timepoints after the labelling phase (0, 1, 4, 8, 12, 20 and 28 dafter labelling, four plants per time point). Then the plantswere dissected into the labelled part of the main root (stainedred); lateral roots outgrowing from the labelled part of themain root; the apical part of the main root including its lateralroots; the hypocotyl; the cotyledons; the stem; and leaf 1

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(oldest) to leaf 12 (youngest). The different plant parts weredried at room temperature and then weighed. Plant parts wereanalysed simultaneously at the end of the experiment. Theradioactivity of 54Mn, 57Co, 65Zn and 109Cd was detectedin a gamma counter (1480 Wizard 3′, Wallac Oy, Turku,Finland). For 63Ni measurement, plant parts were ashedafterwards at 550°C for 8 h. After cooling, the ash wasdissolved in 1 ml 20 mM citric acid, mixed, and 200 µl weretransferred to Ready Caps· (Beckman Instruments, Fullerton,CA, USA) and dried at < 65°C for 4 h. The radioactivity of 63Niwas measured in a liquid scintillation counter (beta counter,Betamatic V, Kontron Instruments, Zurich, Switzerland).Interferences from other radionuclides presented in thesamples (54Mn and 57Co) were corrected. For autoradiography,the harvested plants were placed on paper, protected withbaking paper and three sheets of typewriter paper, and

dried by placing a metal plate heated to 220°C on the top for30 s. At the end of the experiment, all plants were exposedsimultaneously to X-ray film (Fuji medical X-ray film, superRX) for 3 months (54Mn, 57Co, 65Zn and 109Cd); for 63Ni alonger exposure time of 6 months was needed.

The dry matter values for the different plant parts areshown for one set of plants (labelled with 54Mn, 63Ni and57Co) in Fig. 1. These values were very similar to the other setof plants (data not shown). Comparing the dry matter valuesof plant parts and their radionuclide content allows us todistinguish the absence of heavy metals because an organ wasnot yet present; and the absence of heavy metals because theywere not transported into an existing organ.

To investigate the transport of heavy metals in olderplants, 6-wk-old plants (germinated and grown following themethod mentioned above, but not yet labelled with radioactive

Fig. 1 Time course of 54Mn, 63Ni, 57Co, 65Zn and 109Cd in white lupins (Lupinus albus) labelled at the seedling stage. Plants were collected at different time points (0, 1, 4, 8, 12, 20 and 28 d after the labelling phase) and dissected into: labelled part of main root; lateral roots outgrowing from labelled part of main root; apical part of main root (including its lateral roots); hypocotyl; cotyledons; stem; and leaf 1 (oldest leaf) to leaf 12 (youngest leaf). Three subsequent leaves were combined for the analyses. The dry matter and contents of 54Mn, 63Ni and 57Co were analysed in one set of plants, while 65Zn and 109Cd contents were quantified in another set. Counts per minute (cpm) and dry matter (mg) are expressed per plant part as means and standard errors of four replicates.

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Research 333

heavy metals) were placed for 24 h on radiolabelled nutrientsolution containing a mixture of 54Mn (0.17 kBq l−1), 63Ni(0.016 kBq l−1) and 57Co (0.61 kBq l−1). One plant wasplaced on each pot with 1 l radiolabelled nutrient solution.Four plants were collected immediately after the labellingphase (day 0), and four other plants were transferred tononlabelled nutrient solution for seven other days (day 7)and harvested afterwards. For this experiment, plants weredissected into root system, hypocotyl, stem and leaf 1 (oldest)to leaf 30 (youngest). On plants of this age, cotyledons andleaves 1–3 were often missing as a consequence of senescenceand abscission. At the end of the experiment, the radioactivitypresent in the plant parts was analysed using a gamma or abeta counter as described above.

Heavy metal contents in cortex, vascular cylinder and different cluster root stages of 6-wk-old plants

Plants were germinated following the method mentionedabove, but not yet labelled with radioactive heavy metals.Then plants were grown on nutrient solution withoutphosphate to induce the formation of cluster roots. One plantwas placed on each pot with 1 l nutrient solution. After 5 wk,cluster roots appeared in the roots. When the plants were 6 wkold they were placed on nutrient solution without phos-phate containing a mixture of 54Mn (0.16 kBq l−1), 63Ni(0.46 kBq l−1) and 57Co (0.70 kBq l−1); or a mixture of 65Zn(0.017 kBq l−1) and 109Cd (0.13 kBq l−1) for labelling. Oneplant was placed on each pot with 1 l radiolabelled nutrientsolution. Four plants were labelled with 54Mn, 57Co and 63Ni,while four other plants were labelled with 109Cd and 65Zn.After labelling for 24 h, the root system was dipped threetimes sequentially in 1 l nutrient solution to remove radio-active solutes from the root surface, then immersed in asolution of bromocresol purple (0.04% w/v) to differentiatethe developmental stages of cluster roots. The pH of thesolution was adjusted to 6.5 so that both acidification (yellow)and alkalinization (purple) could be observed. After 20 mincontact with the root system, colour changes became visible.The root system was dissected into different parts, such as apexand juvenile, immature, mature, senescent and nonclusterroots according to the different cluster root developmentalstages described by Massonneau et al. (2001). The shoot wasalso collected. A small section (1 cm) of the main root wascollected (immediately above the first lateral roots) anddissected afterwards. After a longitudinal cut, the vascularcylinder and cortex could easily be separated with surgicaltools. The plant parts were weighed and radioactivity wascounted using a gamma or a beta counter.

Solubility of radionuclides accumulated in roots

The solubility of heavy metals within plant tissues wasaddressed by a sequential extraction experiment. Lupin plants

were germinated and seedlings (5 d old) were labelled for 24 hwith a mixture of 54Mn (0.29 kBq l−1) and 57Co (1.33 kBq l−1),or with 109Cd (0.12 kBq l−1) alone. After labelling withradionuclides, roots were dipped three times sequentiallyin 100 ml nutrient solution to remove the radioactive solutesfrom their surface, and incubated 10 min in nutrientsolution. Then plants were collected and a 1-cm section wascut in the main root and dissected into cortex and vascularcylinder. The two root parts were placed overnight in waterfor initial counting in a gamma counter. Then a sequentialextraction with two different buffer systems was performed totest solubility at pH 5.4 (20 mM sodium acetate buffer) andat pH 8 (20 mM Tris–HCl). In a first step, plant parts wereplaced in tubes containing the buffer, homogenized with aPolytron mixer (20 s middle speed; 5 s high speed) andincubated for at least 30 min. After centrifugation (5 min,16 000 g ), pellets were extracted in the original buffer supple-mented with 500 mM NaCl. After centrifugation, pellets wereextracted again in the original buffer supplemented with10 mM Na2EDTA. The supernatants of each step and thepellets of the final step (pellet 3) were analysed by gammaspectrometry. The radionuclide content in the water initiallyused was added to that of supernatant 1 (first column inTable 1). Three replicates were analysed for each buffer andeach radionuclide.

Statistical analyses

Values of heavy metal content are means of three or fourreplicates. Differences in heavy metal content between cortexand vascular tissues were tested for statistical significanceusing Student’s t-test (P < 0.05, n = 3 or 4). For the differentparts of cluster roots, ANOVA was performed and leastsignificant differences were calculated (STATISTIX for Windowsver. 1.0, Analytical Software, Tallahassee FL, USA; rejectionlevel 0.05, n = 3 or 4).

Results

Time courses for distribution of heavy metals in whole plants

The time courses for 54Mn, 63Ni, 57Co, 65Zn and 109Cd differedin whole plants labelled via the main root at the seedling stage(Fig. 1). The 54Mn and 63Ni contents decreased rapidly in thelabelled part of the main root, while 65Zn, 109Cd and 57Cowere released more slowly. At the end of the experiment(day 28), only small amounts of 54Mn (6.5% of total) and63Ni (18.6%) could still be found in the initially labelled partof the root, while higher percentages were found for 109Cd(75%), 57Co (55%) and 65Zn (33%).

A release from the labelled root zone to other parts ofthe root system occurred for some heavy metals (Fig. 1). Forinstance, 15% of the total content of 57Co was found in the


Research334

lateral roots of the labelled part of the main root. In contrast,54Mn and 63Ni were transferred more rapidly to the shootthan 57Co. 54Mn transiently accumulated in the hypocotyl(day 1) before moving into leaves 1–3 (day 4). A small quantityof 54Mn also reached leaves 4–6. Afterwards, no major changesin 54Mn distribution were detected until the final harvest(day 28). The transient accumulation of 63Ni in the hypocotylwas less pronounced than for 54Mn, as well as accumulationin leaves 1–3. But in contrast to 54Mn, the content of 63Nidiminished in leaves 1–3 to reach leaves 4–6, and later alsoleaves 7–9 and 10–12. This indicates that 63Ni was retrans-located from the older leaves to newly expanding leaves. Similartranslocation properties were observed for 65Zn, whichdecreased in the oldest leaves and increased in the upper(youngest) leaves.

The three radionuclides 63Ni (rapidly released from rootsto shoot and retranslocated from older to younger leaves);54Mn (rapidly released from roots to shoot but not retrans-located within the shoot); and 57Co (retained in roots) behavedvery differently in young plants (Fig. 1). The transport ofthese radionuclides was also investigated in older plants witha more complex root system. Figure 2 shows the release of54Mn, 63Ni and 57Co from roots to shoot in 6-wk-old lupinplants. Immediately after the labelling phase (day 0), 54Mnwas present in a large amount in the root system, hypocotyland stem. Seven days later (day 7) almost all 54Mn had movedto the youngest fully expanded leaves (leaves 16–18) as well asto the other leaves, to a lesser extent. A similar pattern wasobserved for 63Ni, but with slower transfer from roots to shoot(after 1 wk 36% remained in roots, compared with 3%

Table 1 Solubility of heavy metals in cortex and vascular cylinder of white lupin (Lupinus albus)

Radionuclide Root partBuffer Supernatant 1

Buffer + 500 mM NaCl Supernatant 2

Buffer + 10 mM Na2EDTA

Supernatant 3 Pellet 3

Sodium acetate buffer (20 mM, pH 5.4)54Mn Cortex 45 ± 23 44 ± 19 3 ± 3 8 ± 5

Vascular cylinder 94 ± 1 2 ± 2 2 ± 2 2 ± 257Co Cortex 69 ± 9 16 ± 3 4 ± 2 11 ± 5

Vascular cylinder 43 ± 10 12 ± 7 21 ± 7 23 ± 5109Cd Cortex 40 ± 5 36 ± 15 14 ± 7 10 ± 9

Vascular cylinder 38 ± 18 19 ± 9 34 ± 17 9 ± 9

Tris–HCl buffer (20 mM, pH 8)54Mn Cortex 41 ± 9 54 ± 5 2 ± 2 3 ± 2

Vascular cylinder 55 ± 9 24 ± 17 21 ± 14 0 ± 057Co Cortex 69 ± 11 7 ± 4 5 ± 4 19 ± 4

Vascular cylinder 44 ± 5 34 ± 17 6 ± 3 16 ± 12109Cd Cortex 45 ± 4 33 ± 7 9 ± 5 13 ± 9

Vascular cylinder 56 ± 23 24 ± 24 20 ± 18 0 ± 0

A 1-cm segment of the main root of white lupins was collected and dissected into cortex and vascular cylinder. Solubility was tested at pH 5.4 (20 mM sodium acetate buffer) and pH 8 (20 mM Tris–HCl buffer). In the first step, the heavy metals were extracted in the original buffer; after centrifugation, pellets were extracted in the original buffer supplemented with 500 mM NaCl. After centrifugation, pellets were extracted again in the original buffer supplemented with 10 mM Na2EDTA. Supernatants of each step and pellets of the final step (pellet 3) were analysed by gamma spectrometry. Means and standard errors of three replicates are shown for relative radionuclide contents in the percentage of total label in the sample (100% = supernatant 1 + supernatant 2 + supernatant 3 + pellet 3).

Fig. 2 Distribution of 54Mn, 63Ni and 57Co in white lupins (Lupinus albus) labelled after 6 wk growth. Plants were collected immediately after the labelling phase (day 0) or 7 d later (day 7). Plants were dissected into: root system; hypocotyl; stem; and leaf 1 (oldest leaf) to leaf 30 (youngest leaf). Three subsequent leaves were combined for the analyses. Means and standard errors of four replicates are shown for the radionuclide contents in counts per minute (cpm).


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for 54Mn) and with a more even distribution between thedifferent leaves. Finally, 57Co showed extreme behaviour withpractically no transfer to the shoot.

Distribution of heavy metals in whole plants of lupin as revealed by autoradiography

Autoradiography is a suitable technique for visualizing andlocalizing in detail the labelled heavy metals in different plantparts. Figure 3 shows autoradiographs of lupin seedlings 4 dafter labelling (for 24 h) via the main root with 54Mn, 63Ni,57Co, 65Zn or 109Cd. These pictures clearly show the differentdistribution of the five radionuclides analysed in whole lupinplants. While 54Mn and 63Ni were already transferred to theshoot, most of 57Co, 65Zn and 109Cd were still found in theroot system 4 d after labelling. This method allowed us todetermine more precisely where heavy metals were storedin the leaves. Interestingly, 54Mn moved to the shoot andaccumulated at the very periphery of the oldest leaves(Fig. 3a, d). Autoradiography was also prepared for roots andshoot of older plants labelled with 54Mn, 63Ni, 57Co, 65Znand 109Cd. The distribution of 54Mn, 57Co, 65Zn and 109Cdin roots and shoot of 4-wk-old white lupins (20 d afterlabelling phase) is shown in Fig. 4. For 63Ni, the signal was toolow and the distribution could not be seen. Plants werelabelled simultaneously and in the same stage as those used forthe quantitative analyses (Fig. 1) and autoradiographyanalyses (Fig. 3). In agreement with the quantitative results

presented in Fig. 1, at day 20 54Mn was mainly present inleaves 1 and 2 (the oldest leaves) and in smaller quantities inleaves 3 and 4, while no signal was detected in the youngestleaves (leaves 6–8). Again, 54Mn accumulated at the peripheryof the oldest leaves. 57Co was also present in larger amountsin the oldest leaves (1 and 2) but, in contrast to 54Mn, 57Cowas distributed more homogeneously in the leaf lamina andalso reached the youngest leaves. The autoradiograph of lupinlabelled with 65Zn showed that this heavy metal was presentin higher quantities in the youngest leaves (leaves 5–8). Therewas a strong accumulation of 65Zn in the newly formed(expanding) leaf 8. In contrast to the three other radionuclides,the small amount of 109Cd that reached the shoot wasdistributed evenly among the different leaves. Moreover, theautoradiographs showed that 109Cd was concentrated in thecentral vein of the leaflets.

Distribution of heavy metals in cluster roots of white lupin

To investigate the accumulation of heavy metals in the rootsystem more precisely, the distribution of heavy metals in thedifferent stages of cluster roots was analysed. Figure 5 showsthe partitioning of 54Mn, 63Ni, 57Co, 65Zn and 109Cd in thedifferent cluster root stages, as percentages of radionuclidesper plant part (Fig. 5a); and the content of these heavy metalsin the different cluster root stages, measured in counts perminute per root FW (Fig. 5b). The content of heavy metals in

Fig. 3 Distribution of 54Mn, 63Ni, 57Co, 65Zn and 109Cd in roots and shoot of young (10-d-old) white lupins (Lupinus albus). (a–c,e,f) show autoradiographs of plants 4 d after labelling via the main root with 54Mn, 63Ni, 57Co, 65Zn and 109Cd, respectively. (d) Magnified leaf of a plant labelled with 54Mn. Bars, 1 cm.


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Fig. 4 Distribution of 54Mn, 57Co, 65Zn and 109Cd in shoot and roots of 4-wk-old white lupin (Lupinus albus). (a,c,e,g) Autoradiographs of the shoot of white lupins 20 d after labelling via the main root with 54Mn, 57Co, 65Zn and 109Cd, respectively. (b,d,f,h) Autoradiographs of the corresponding root system. Leaves are numbered from 1 (oldest) to 8 (youngest). Bars, 1 cm.

Fig. 5 Content of heavy metals in different parts of cluster roots of 6-wk-old white lupins (Lupinus albus). Cluster roots of lupin plants were dissected into apex and juvenile (A + J); immature (I); mature (M); and senescent (Se). Noncluster roots (NCR) were also analysed. Contents of 54Mn, 63Ni, 57Co, 65Zn and 109Cd in the different parts of the root system are shown as percentage of the total content in the plant in (a). The content of heavy metals in counts per minute (cpm) per mg FW of root parts is shown in (b). Means and standard errors of three or four replicates are shown. Significant differences between various root types (a,b) were calculated for each radionuclide separately, indicated by different characters (P < 0.05, n = 3 or 4).


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different parts of the root system varied. For all heavy metalsexcept 63Ni, the noncluster roots contained the highest part ofthe heavy metals absorbed, because of their dominance in theroot system (Fig. 5a). While the contribution of cluster rootsto heavy metal sequestration was very minor for 54Mn, it wasmore significant for 63Ni, 57Co, 65Zn and 109Cd. Most of theheavy metals were found in senescent cluster roots, with a minorcontribution of mature cluster roots in the case of 63Ni and109Cd. When taking into account the differences in abundanceof each root type within the entire root system, and thusconsidering the amounts of heavy metals per FW root, thesituation was quite different (Fig. 5b): for all radionuclides,cluster roots showed a much higher content of heavy metalsthan noncluster roots. Highest heavy metal concentrations werefound in young and immature cluster roots, while senescentcluster roots behaved like noncluster roots and accumulatedonly small amounts of heavy metals. Mature cluster roots didnot accumulate all heavy metals tested in the same way: while54Mn and 57Co were present in high amounts (similar to thelevels observed in young and immature cluster roots), 63Ni,65Zn and 109Cd levels in mature cluster roots were notsignificantly higher than in senescent or noncluster roots.

Localization of heavy metals within roots

The results presented above and in Figs 1–4 indicated that ahigh percentage of some radionuclides was retained for severalweeks in the labelled part of the main root (57Co, 109Cd), whileother radionuclides were released more rapidly to the shoot(54Mn, 63Ni). The question of localization of heavy metalsstaying in the root at the tissue level was addressed by separat-ing root cortex from vascular tissue in a 1-cm segment of the

main root, segment collected immediately above the first lateralroots (Fig. 6). More than 80% of 57Co and of 109Cd was presentin the cortex, while 54Mn and 65Zn were present in similarquantities in the two parts of the root. A high percentage(> 80%) of 63Ni was detected in the vascular cylinder.

Solubility of radionuclides

As the solubility of heavy metals is a key factor in theirmovement and distribution within plant tissues, this aspectwas also analysed. Three heavy metals were tested: Mn(rapidly released to the first leaves); and Cd and Co (retainedin the main root). In general, a high percentage of allradionuclides was soluble in the two buffers used (Table 1).Most of the heavy metals were present in the supernatantsafter the first and second steps of solubilization. Surprisingly,109Cd was also very soluble in both buffers and < 15% waspresent in the final pellet. 57Co showed the strongest insolubility,as revealed by the highest percentage in the last pellet, after thethree subsequent extraction steps. These measurements referto the solubility of the heavy metals regardless of theirsubcellular distribution in intact roots, as compartmentationwas completely destroyed during extraction. Accumulation incertain compartments may be important for the low root-to-shoot translocation of 57Co and 109Cd.

Discussion

Distribution of heavy metals in white lupin

In this study, the redistribution of five heavy metals in theroots and shoot of white lupin was characterized. 54Mn, 63Ni,

Fig. 6 Contents of different radionuclides in cortex and vascular cylinder of the main root of white lupins (Lupinus albus). Contents of 54Mn, 63Ni and 57Co, 65Zn and 109Cd in cortex and vascular cylinder are shown as percentage of the total content in the root segment. Standard errors of four replicates are shown for the relative radionuclide contents. Significant differences between radionuclides are indicated by different characters (P < 0.05, n = 3 or 4).


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57Co, 65Zn and 109Cd behaved differently with regard toroot-to-shoot transfer and redistribution within the shoot androot system. 65Zn and 63Ni were probably transported via thexylem to transpiring leaves and then retranslocated to newlyformed leaves. These findings suggest that 65Zn and 63Ni weretransported via the phloem from the older leaves to younger,expanding leaves. As Zn and Ni are micronutrients (Welch,1995), it makes sense that they move from roots to shoot inthe xylem to reach the leaves. The rapid retranslocation of65Zn and 63Ni from old to young leaves via the phloemcontributes to the supply of rapidly growing plant parts withthese micronutrients. In contrast to Zn and Ni, Mn (which isalso a micronutrient; Welch, 1995) was transported only fromthe labelled part of the main root to the first leaves, andaccumulated in these leaves. This distribution pattern suggeststhat 54Mn is rapidly released from roots into the xylem andreaches photosynthetically active leaves via the transpirationstream, and that this radionuclide is not (or only in minorquantities) redistributed afterwards via the phloem to otherleaves. This is in agreement with experiments performed withother plant species, such as maize and horse bean (El-Bazet al., 1990); and wheat (Page & Feller, 2005; Riesen & Feller,2005). Solubilization of Mn in the soil by the secretion ofcarboxylates from cluster roots, and the rapid translocation ofMn from roots to shoot, may cause an accumulation of Mnin lupin leaves under certain conditions (Dinkelaker et al.,1995). The low mobility of 54Mn within the shoot may beexplained by a restricted loading of soluble Mn into thephloem, or by insolubilization in the leaves. These possi-bilities remain to be investigated in future studies. It must bealso considered whether Mn may be translocated to someextent from stems and petioles via the phloem to maturingseeds (Hannam et al., 1985).

Cadmium showed a specific behaviour in the sense thatmost of it stayed in the root system. This is in agreement withthe results of Römer et al. (2000, 2002); Ximénez-Embúnet al. (2002); Zornoza et al. (2002). One can imagine that Cd,a nonessential and pollutant heavy metal found in differentconcentrations in soils (Sauvé et al., 2000; Lugon-Moulinet al., 2004), is recognized as a toxic compound at the rootlevel and, as it is not needed in the shoot, the plant sequestersit in the roots to avoid damage to the shoot. Cadmium isknown to have several toxic effects on plants (Sanità di Toppi& Gabbrielli, 1999 and references therein). Cadmiumsequestration in the cell wall, as already shown by Zornozaet al. (2002), or in the vacuoles of root cells may explain thehigh quantity of this radionuclide found in the root throughoutthis experiment. As judged from the solubility of 109Cd inextracts of cortex and vascular cylinder (Table 1), it can besupposed that 109Cd is sequestered in a soluble form (e.g. inthe vacuole of root cells). The root-to-shoot long-distancetransport of 109Cd in white lupin was not similar to thatobserved in wheat (Page & Feller, 2005): 109Cd was shown tobe transported from roots to shoot in wheat, and was also found

in the grain (Herren & Feller, 1997; Riesen & Feller, 2005).By contrast, as 109Cd stayed in the root system of lupin and,as reported by Grant et al. (1998), restricting Cd transport fromroots to shoot reduces the Cd concentration in grains muchmore than in leaves, lupin seeds should contain only very lowlevels of Cd, which is an advantage for seed consumption.

The behaviour of 57Co was very similar to that of 109Cd.Cobalt is also known to have toxic effects in plants (Palit et al.,1994), and it is suggested that Co is also recognized as a toxiccompound at the root level and sequestered in roots to avoiddamage to the shoot. Moreover, Co is required by legumesand nodulating nonlegumes for N2 fixation (Palit et al., 1994;Marschner, 1995). Even if, in our experiments, N fixation didnot take place because of a sufficient nitrate supply, Co maybe recognized by lupin as a micronutrient useful in the roots,where N fixation is normally taking place, and thus stay in theroot system (labelled part and distribution in lateral roots andapical part as shown in Fig. 1) rather than being translocatedto shoots as 54Mn, 65Zn or 63Ni.

The results obtained with autoradiographs confirmed theresults of gamma and beta counting (Figs 3, 4) and providedmore detail on the levels of heavy metals in the differentparts of individual leaves. 54Mn accumulated strongly atthe periphery of older leaves (fully expanded with a hightranspiration rate). Interestingly, a similar pattern of heavymetals at the edge of the leaf was observed for 109Cd in Thlaspicaerulescens (Cosio et al., 2005). These authors asked whether109Cd allocation at the edge of older leaves might be explainedby transport of 109Cd with the transpiration stream andexcretion of 109Cd in excess with guttation. The guttationfluid may serve to excrete various elements, such as potassium,magnesium and calcium, as shown in sunflower (Tanner &Beevers, 2001). The pattern of guttation drops can be foundon the entire leaf surface, as in tobacco, potato and bean;or at the edge or tips of the leaf, as in mustard, barley andcucumber (Kormarnytsky et al., 2000). It is therefore possiblethat guttation could be a way for white lupin to excrete excessMn, but this question requires further investigation. Incontrast to the findings of Cosio et al. (2005) in T. caerulescens,no Cd accumulation was observed in our experiments at theperiphery of the leaves. Very little 109Cd was detected in theshoot compared with the root. Nevertheless, this smallquantity of 109Cd was present in all parts of the leaf area. Thishomogeneous distribution may be explained by the uptake of109Cd into mesophyll or epidermal cells. A large amount of109Cd was also detected in the major leaf veins (leaf 4, Fig. 4g).It remains open whether the 109Cd detected in the veins wasloaded into the phloem or was present in other tissues.

Distribution of heavy metals in cluster and noncluster roots

Considering the root system as a whole, because of the largemajority of noncluster roots over cluster roots at the time of


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the experiment, noncluster roots had the largest contributionto heavy metal root content (Fig. 5a). When looking at thespecific acquisition rates of heavy metals by the different roottypes and stages, it was clear that cluster roots were moreefficient than noncluster roots in terms of heavy metalaccumulation per FW unit (Fig. 5b). This may be caused bythe very densely branched and ramified structure of clusterroots, and the consequently higher uptake surface. Secretionof carboxylates and protons did not appear to be correlatedwith heavy metal uptake, as mature cluster roots, wheresecretion of the highest amounts of carboxylates as well asacidification occur, did not show higher uptake than the otherstages of cluster roots. This is consistent with previous resultsof Römer et al. (2002), who showed that plant uptake was notenhanced despite a higher excretion of citrate in P deficiencycausing an increased Cd concentration in the soil solution.This suggests that, even if citrate and acidification affect Cdsolubility, some mechanisms must enable the plant to limitroot uptake of this toxic heavy metal. Interestingly, high heavymetal acquisition coincided with the stages where the highestsecretion of phenolics takes place (juvenile and immaturestages; Weisskopf et al., 2006). Previous reports have studiedthe impact of phenolics on heavy metal tolerance: foraluminium in maize (Kidd et al., 2001); and copper in alfalfa(Parry & Edwards, 1994) and white lupin (Jung et al., 2003).However, little information is available about the possible roleof phenolics in tolerance to the heavy metals used in our study.

The results reported here were obtained from hydroponiccultures allowing direct access to the various parts of the rootsystem. However, this system differs considerably from soilcultures (high phosphate concentration in the nutrientsolution; no solubilization of heavy metals from soil particles).Root activities may affect soil properties and the solubility ofheavy metals locally. The release of chelators, carboxylatesor protons, as well as an increased reductive potential, mayaffect the solubilization and acquisition of these elements(Moraghan, 1979; Dinkelaker et al., 1995). Strong gradientscan be generated by the local activities of cluster roots in soil,but not in hydroponic culture.

It is possible that a reallocation of the heavy metals withinthe different stages of cluster roots and within the differentplant parts occurred during the time of treatment (24 h). Thedistribution of heavy metals in old lupin plants collected imme-diately after a 24-h labelling period support this assumption(Fig. 2). These results, showing the presence of 54Mn and 63Niin the shoot, lead to the conclusion that some of these heavymetals might be reallocated within the labelling phase.

Localization and solubility of heavy metals in main root

After a 24-h labelling period, a high percentage of 63Ni waspresent in the vascular cylinder of the main root, while < 20%was in the cortex (Fig. 6). This distribution is in agreementwith the good mobility of 63Ni in xylem and phloem. In

contrast to 63Ni, 80% of 57Co and 109Cd was located in thecortex. The poor transfer of these radionuclides to the vascularcylinder may partially explain the very low transfer to theshoot. These heavy metals might be insolubilized (e.g. in cellwall components) or accumulated in soluble form in specialcompartments (e.g. vacuoles). The solubility of 57Co and109Cd in extracts of cortex and vascular cylinder (Table 1)shows that most of these heavy metals were present in thesupernatants after the first and second steps of solubilization,indicating a good solubility of these elements. Thus it appearslikely that the compartmentalization of soluble 57Co and109Cd, and not the formation of insoluble forms, was theprimary cause for retention in the root system. The root-to-shoot translocation of Cd can be more rapid in other plantsspecies (Page & Feller, 2005). Therefore caution is recommendedwhen generalizing these results obtained with white lupin.

In conclusion, the study presented here showed that thefive heavy metals tested differed in their distribution patternsand mobility in white lupin. While most of the 57Co and109Cd remained in the root system, the other radionuclideswere transported from roots to shoot. 63Ni and 65Zn wereredistributed via the phloem from the oldest to the youngestleaves. 54Mn accumulated in the first leaves, especially in theedge of the leaflets. Regarding the acquisition of heavy metalsby the different stages of cluster roots, the levels were highestin young and immature cluster roots, where large amounts ofphenolics are secreted; mature cluster roots accumulated lessheavy metal, despite the high secretion of carboxylates andprotons. Our results showed that white lupin may absorbheavy metals in the roots and transfer some of them to theshoot. This knowledge may be useful in agriculture in thecontext of the quality of harvested plant parts. As most Cdstays in the root, we can assume that, in general, it will notaccumulate to high levels in the seeds – the part of white lupinused for animal and human nutrition. Hydroponic cultureproved a useful system for precise monitoring of heavy metaldistribution within the plant. However, the situation may bequite different when studying soil-grown plants, where heavymetals are attached to soil particles and might be less availablethan in a nutrient solution. A study in microcosms with soil-grown plants would help to come closer to natural conditionsand would constitute the next step towards a better understand-ing of the real field situation.

Acknowledgements

This project was founded by the National Centre ofCompetence in Research (NCCR) Plant Survival researchprogramme of the Swiss National Science Foundation.

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heavy metals in white lupin: uptake, root-to-shoot transfer and redistribution within the plant

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