Plant, Cell and Environment

(2004)

27

, 1122–1134

1122

© 2004 Blackwell Publishing Ltd

Blackwell Science, LtdOxford, UKPCEPlant, Cell and Environment0016-8025Blackwell Science Ltd 2004? 2004

27911221134Original Article

Cd-induced accumulation of O

2

·

–

and H

2

O

2

in pea M. C. Romero-Puertas

et al.

Correspondence: Dr Luisa M. Sandalio. Fax: 34 958129600; e-mail:luisamaria.sandalio@eez.csic.es

Cadmium-induced subcellular accumulation of O

2·----

and H

2

O

2

in pea leaves

M. C. ROMERO-PUERTAS

1

, M. RODRÍGUEZ-SERRANO

1

, F. J. CORPAS

1

, M. GÓMEZ

2

, L. A. DEL RÍO

1

& L. M. SANDALIO

1

1

Departamento de Bioquímica, Biología Celular y Molecular de Plantas and

2

Departamento de Agroecología y Protección Vegetal, Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas, Apartado 419, E-18080 Granada, Spain

ABSTRACT

Cadmium is a toxic metal that produces disturbances inplant antioxidant defences giving rise to oxidative stress.The effect of this metal on H

2

O

2

and O

2·

-

production wasstudied in leaves from pea plants growth for 2 weeks with50

mmmm

M

Cd, by histochemistry with diaminobenzidine (DAB)and nitroblue tetrazolium (NBT), respectively. The subcel-lular localization of these reactive oxygen species (ROS)was studied by cytochemistry with CeCl

3

and Mn/DABstaining for H

2

O

2

and O

2·

-

, respectively, followed by elec-tron microscopy observation. In leaves from pea plantsgrown with 50

mmmm

M

CdCl

2

a rise of six times in the H

2

O

2

content took place in comparison with control plants, andthe accumulation of H

2

O

2

was observed mainly in theplasma membrane of transfer, mesophyll and epidermalcells, as well as in the tonoplast of bundle sheath cells. Inmesophyll cells a small accumulation of H

2

O

2

was observedin mitochondria and peroxisomes. Experiments with inhib-itors suggested that the main source of H

2

O

2

could be aNADPH oxidase. The subcellular localization of O

2·

-

pro-duction was demonstrated in the tonoplast of bundle sheathcells, and plasma membrane from mesophyll cells. The Cd-induced production of the ROS, H

2

O

2

and O

2·

-

, could beattributed to the phytotoxic effect of Cd, but lower levelsof ROS could function as signal molecules in the inductionof defence genes against Cd toxicity. Treatment of leavesfrom Cd-grown plants with different effectors and inhibi-tors showed that ROS production was regulated by differ-ent processes involving protein phosphatases, Ca

2+

channels, and cGMP.

Key-words

: cadmium; cytochemistry; histochemistry; oxida-tive stress; pea; reactive oxygen species; signalling.

Abbreviations

: ABA, abscisic acid; ASC, ascorbate; DAB,3,3

¢

-diaminobenzidine; DPI, diphenylene iodonium; NBT,nitroblue tetrazolium; ODQ, 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one; PCD, programmed cell death; ROS,reactive oxygen species; SA, salicylic acid; SOD, superox-

ide dismutase; TMP, tetramethylpiperidinooxy; TUNEL,TdT-mediated X-dUTP nick end labelling.

INTRODUCTION

Cadmium is a toxic trace pollutant for humans, animals andplants which enters the environment mainly from industrialprocesses and phosphate fertilizers and then is transferredto the food chain (Wagner 1993). Studies carried out indifferent plant species have revealed that Cd is stronglyphytotoxic and causes growth inhibition and even plantdeath. It is well established that different metabolic pro-cesses such as photosynthesis and cell respiration areaffected by the presence of Cd (Prasad 1995; Poschenrieder& Barceló 1999), although the mechanisms involved in itstoxicity are still not completely understood.

In previous works it was demonstrated that Cd caninduce oxidative stress in pea leaves, characterized by anaccumulation of lipid peroxides and oxidized proteins, anda reduction of catalase and SOD activity (Sandalio

et al

.2001; Romero-Puertas

et al

. 2002, 2003). In shoot and roottissues of pea plants treated with Cd an enhancement of thelipid peroxidation products was also reported (Lozano-Rodríguez

et al

. 1997; Romero-Puertas

et al

. 2003). In per-oxisomes purified from pea leaves, Cd produced anincrease of the H

2

O

2

content and imbalances in the activityof antioxidative enzymes (Romero-Puertas

et al

. 1999). Anenhancement of lipid peroxidation by Cd was found in

Phaseolus vulgaris

(Somashekaraiah, Padmaja & Prasad1992; Shaw 1995; Chaoui

et al

. 1997) and

Helianthusannuus

(Gallego, Benavides & Tomaro 1996). The declineof the enzymes catalase and superoxide dismutase whichscavenge H

2

O

2

and O

2

·

–

radicals, respectively, has beenassociated with Cd toxicity in

Phaseolus vulgaris

(Somashekaraiah

et al

. 1992; Chaoui

et al

. 1997),

Phaseolusaureus

(Shaw 1995),

Helianthus annuus

(Gallego

et al

.1996), and

Secale cereale

(Streb, Michael-Knauf & Feiera-bend 1993).

Reactive oxygen species (ROS), such as H

2

O

2

, O

2

·

–

radi-cals, hydroxyl radicals (·OH), and singlet oxygen, are sub-products of the aerobic metabolism and their productionoccurs in different cell compartments (Foyer, Lelandais &Kunert 1994; Bolwell

et al

. 1999; Halliwell & Gutteridge2000; del Río

et al

. 2002a). In plants under abiotic and

Cd-induced accumulation of O

2·–

and H

2

O

2

in pea

1123

© 2004 Blackwell Publishing Ltd,

Plant, Cell and Environment,

27,

1122–1134

biotic stress conditions, an overproduction of ROS takesplace which act as mediators of oxidative damage (Dat

et al

. 2000; del Río

et al

. 2002b). The overproduction andrapid accumulation of ROS, also called ‘oxidative burst’, isone of the earliest responses of incompatible interactionsbetween pathogens and plants (the hypersensitiveresponse), and is caused by the activation of a membrane-bound enzyme system similar to neutrophil NADPH oxi-dase and cell wall peroxidases (Bolwell

et al

. 1999). Theoxidative burst has also been associated with some abioticstresses such as temperature (Dat, Foyer & Scott 1998;López-Delgado

et al

. 1998; Dat

et al

. 2000), wounding(Orozco-Cárdenas & Ryan 1999) and ozone (Sharma

et al

.1996).

However, studies on plant defence against pathogens andozone treatment have revealed that H

2

O

2

can also have arole as signal molecule in triggering the induction ofdefence mechanisms against stress (Lamb & Dixon 1997;Schraudner

et al

. 1998). By using CeCl

3

techniques, the pro-duction of H

2

O

2

at subcellular level has been recently dem-onstrated in lettuce cells infected with

Pseudomonassyringae

(Bestwick

et al

. 1997; Bestwick, Brown & Mans-field 1998), barley leaves interacting with the powdery mil-dew fungus (Thordal-Christensen

et al

. 1997), and birch leafcells exposed to ozone (Pellinen, Palva & Kangasjärvi1999). A signalling role for H

2

O

2

has been proposed forABA-mediated stomatal closure (Pei

et al

. 2000) and auxin-regulated gravitropic responses (Joo, Bae & Lee 2001;Neill, Desikan & Hancock 2002). Apparently, the expres-sion of at least 1–2% of

Arabidopsi

s genes is dependent onH

2

O

2

, and some of them are antioxidant genes and othersencode proteins involved in signalling, such as calmodulin,protein kinases and transcription factors, and PCD (Desi-kan

et al

. 2001; Neill

et al

. 2002). Moreover, H

2

O

2

can alsoactivate Ca

2+

channels in plasma membranes (Murata

et al

.2001) and MAPK cascades (Joo

et al

. 2001; Neill

et al

.2002).

In this work, the production of ROS in leaves from peaplants grown with cadmium was investigated at subcellularlevel by cytochemistry and electron microscopy. The partic-ipation of ROS in damage induced by metal toxicity, andthe transduction pathways involved in ROS productionwere also studied.

MATERIALS AND METHODS

Plant material and growth conditions

Pea (

Pisum sativum

L., cv. Lincoln) plants were obtainedfrom Royal Sluis (Enkhuizen, Holland). Plants were grownin the greenhouse in aerated full-nutrient media under opti-mum conditions for a period of 14 d (Sandalio

et al

. 2001).Then, the media either remained unsupplemented (controlplants) or were supplemented with 50

m

M

CdCl

2

(Cd-treatedplants), and plants were grown for 14 d. In these conditionsthe plants experimented a reduction of growth and leaf sizebut their viability was not affected and the plants evenfructified. To carry out the experiments, in both control and

Cd-treated plants, leaves from the middle part of the plantwere selected to avoid the overlapping of senescence- andCd induced effects that takes place in the oldest leaves.

Transmission electron microscopy

H

2

O

2

cytochemistry

The method used for the localization of H

2

O

2

was based onthe generation of precipitates of cerium perhydroxides(Bestwick

et al

. 1997). Leaf pieces (approximately 1 mm

2

)were cut from tissue between the main veins and wereincubated in freshly prepared 5 m

M

CeCl

3

in 50 m

M

MOPSbuffer (pH 7.2) for 1 h. As controls, samples were incubatedwith: (1) 25

m

g

m

L

-

1

bovine liver catalase; and (2) withoutCeCl

3

(negative control). To check the effect of inhibitors,pieces of leaves were incubated separately for 30 min in50 m

M

MOPS buffer (pH 7.2), containing either 1 m

M

Na-azide and 10

m

M

diphenylene iodonium chloride (DPI), or1 m

M

3-amino-1,2,4-triazole, and tissues were transferredto CeCl

3

solutions supplemented with the inhibitors at thesame concentration. Leaf pieces were then fixed in 1.25%(v/v) glutaraldehyde and 1.25% (p/v) paraformaldehyde in50 m

M

Na-cacodylate buffer (pH 7.2) for 1 h. Tissues werewashed with the same buffer without fixatives, three timesfor 10 min, and post-fixed for 1 h in 1% (v/v) OsO

4

, andthen were dehydrated in a graded ethanol series (30–100%;v/v) and embedded in Spurr resin. Ultra-thin sections werestained with uranyl acetate and lead citrate and examinedin a Zeiss (Oberkochen, Germany) EM 10C transmissionelectron microscope.

O

2

·

–

cytochemistry

The method used to detect O

2

·

–

radicals was based on thegeneration of DAB precipitates in the presence of Mn

2+

ions and O

2

·

–

(Steinbeck

et al. 1993). This method is basedon the oxidation by O2

·– of Mn2+ to Mn3+, which then oxi-dizes 3,3¢-diaminobenzidine-HCl (DAB) to an insolubleosmiophilic polymer (Steinbeck et al. 1993). Leaf pieces(approximately 1 mm2) were incubated for 30 min in 0.1 MHEPES buffer (pH 7.2) containing 2.5 mM DAB, 0.5 mM

MnCl2 and 1 mM Na-azide. As controls, pieces were incu-bated in the absence of either DAB or MnCl2. The specific-ity of the reaction was checked by incubating the tissueswith 1 mM CuZn–SOD for 30 min before the treatment withDAB. The effect of 1 mM diethyldithiocarbamate, a CuZn–SOD inhibitor, was also studied by incubating the tissuesfor 30 min before DAB staining. Samples were fixed,embedded and stained as described above.

H2O2 localization in situ

Leaves from control and Cd-treated plants were excisedand immersed in a 1% solution of DAB in 10 mM MESbuffer (pH 6.5), vacuum-infiltrated for 5 min and then incu-bated at room temperature for 8 h in the absence of light.Leaves were illuminated until appearance of brown spots

1124 M. C. Romero-Puertas et al.

© 2004 Blackwell Publishing Ltd, Plant, Cell and Environment, 27, 1122–1134

characteristic of the reaction of DAB with H2O2. Leaveswere bleached by immersing in boiling ethanol to visualizethe brown spots. To verify the specificity of precipitates,before staining with DAB some leaves were immersed for2 h in solutions containing the H2O2 scavenger 1 mM ascor-bate (ASC). H2O2 deposits were quantified by scanningspots from leaf pictures and the number of pixels werequantified with the PHOTOSHOP 6.0 program (Adobe Sys-tems, San Jose, CH, USA). The results were expressed aspercentage of spots area versus total leaf area [(spot area/total leaf area) ¥ 100] to compensate for differences in leafsize.

O2·- localization in situ

Leaves from control and Cd-treated plants were excisedand immersed in a 0.1% solution of NBT in 50 mM K-phosphate buffer (pH 6.4), containing 10 mM Na-azide, andwere vacuum-infiltrated for 5–10 min and illuminated untilappearance of dark spots, characteristic of blue formazanprecipitates. Leaves were bleached by immersing in boilingethanol. As negative controls, before staining with NBTleaves were immersed in 1 mM tetramethylpiperidinooxy(TMP), a O2

·– scavenger, for 3 h. Superoxide deposits werequantified by scanning spots from leaf pictures as men-tioned above.

H2O2 determination in leaf extracts

The H2O2 concentration of crude extracts from pea leaveswas determined by spectrofluorometry, basically asdescribed Creissen et al. (1999). All operations were per-formed at 0–4 ∞C. Leaves (0.4 g) were homogenized in1.2 mL 25 mM HCl, and the crude extracts were filteredthrough two nylon layers, and the pigments were removedby mixing with 15 mg of charcoal (Sigma-Aldrich, St Louis,MO, USA). The pigment-containing charcoal was sepa-rated by centrifugation at 5000 g for 5 min, and the super-natants were clarified by filtration through a 0.22-mm filterunit. The pH of leaf extracts was adjusted to 7.0 with NaOHand these extracts were used to measure the H2O2 concen-tration. The reaction mixtures (3 mL) contained 50 mM

Hepes buffer, pH 7.6, 5 mM homovanillic acid, and 100 mLof sample. The reaction was started by adding 40 mM horse-radish peroxidase and the fluorescence produced was mea-sured in a spectrofluorophotometer Shimadzu (Kyoto,Japan) RF-540 at excitation and emission wavelengths of315 and 425 nm, respectively. The H2O2 concentration wasdetermined from a calibration curve of H2O2 (Merck,Darmstadt, Germany) in the range 0.5–20 mM.

Treatments with inhibitors and inducers

Treatments with 5 mM cantharidin (protein phosphataseinhibitor), 1 mM LaCl3 (Ca2+ channel blocker), 1 mM

salicylic acid (SA), 10 mM DPI (oxidase inhibitor), 200 mM

1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ, gua-nylate cyclase inhibitor), and 100 mM 8-Br-cGMP (cGMP

donor), were supplied in water solutions to excised pealeaves through the petioles and kept for 3–18 h at 25 ∞C.After incubation, leaves were either immersed in DAB orNBT solutions to localize H2O2 and O2

·– production, respec-tively. ROS deposits were quantified by scanning spotsfrom leaf pictures as mentioned above.

Detection of cell death

To determine changes in viability of cells by Cd treatment,excised leaves were infiltrated with a 0.25% (w/v) aqueoussolution of Evans Blue (Turner & Novacky 1974), throughthe petioles for 5 h. Then the leaves were decoloured inboiling ethanol to develop the blue precipitates, which werequantified by solubilization with 1% (w/v) SDS in 50% (v/v) methanol at 50 ∞C for 10 min, and the absorbance wasmeasured at 600 nm.

Cell death was also estimated by measuring ion leakagefrom leaf discs. Two leaf discs, 9 mm in diameter, werefloated, with the adaxial side up, on 2 mL of Milli-Q ultra-pure water for 3 h at room temperature. Following incuba-tion, the conductivity of the solution was measured, andthen was boiled at 100 ∞C for 10 min. When the solution wascooled, the conductivity was measured again and the resultswere expressed as relative conductivity [(conductivity afterincubation/conductivity after boiling) ¥ 100].

DNA extraction and fragmentation analysis

Genomic DNA of pea leaves from control and Cd-treatedplants was isolated using the DNeasy Plant Kit fromQIAGEN, following the recommendations of the manufac-turer. DNA fragmentation analysis was assayed in samplessubjected to 7.5% non-denaturing polyacrylamide gel elec-trophoresis (PAGE) in 89 mM Tris-boric acid buffer(pH 7.6), containing 2 mM ethylenediaminetetraacetic acid.Gels were stained with silver nitrate.

In situ detection of apoptotic cells

Leaf tissue from control and Cd-treated plants was cut intopieces (1 mm2) and fixed, dehydrated and embedded in LRWhite, as described by Sandalio et al. (1997). Sections weremounted on polylysine slides, incubated with proteinase K(20 mg mL-1 in 10 mM Tris-HCl, pH 7.5) for 15 min at roomtemperature, and rinsed twice with phosphate-bufferedsaline. Sections were labelled by the TUNEL method fol-lowing the instructions of the manufacturer (BoehringerMannheim, Germany) and fluorescence was observed witha Zeiss fluorescence microscope.

RESULTS

Cadmium induces overproduction of H2O2 in leaves

To determine if the growth of pea plants with 50 mM CdCl2

induced the H2O2 accumulation in leaves, a fluorimetric

Cd-induced accumulation of O2·– and H2O2 in pea 1125

© 2004 Blackwell Publishing Ltd, Plant, Cell and Environment, 27, 1122–1134

method was used. Leaves from plants grown in the pres-ence of cadmium contained a H2O2 concentration six timeshigher than control plants (Table 1). To verify in situ theaccumulation of H2O2 under cadmium treatment, a his-tochemical method with DAB that is based on the forma-tion by H2O2 of local brown spots in leaves, which werequantified by counting the pixel number of spots, was used(Fig. 1a). The H2O2 accumulation could be prevented byinfiltration with H2O2 scavengers such as ascorbate, whichdemonstrates the specificity of this reaction for H2O2, andwas abolished by infiltration with DPI, suggesting theinvolvement of a NADPH oxidase (Fig. 1b).

Table 1. Effect of cadmium treatment of pea plants on H2O2 accumulation in leaves

Cd 2+ (mM) H2O2 (mM)

0 0.23 ± 0.1350 1.50 ± 0.51

Leaves were homogenized, and H2O2 was assayed byspectrofluorometry as described in Materials and Methods. Valuesare mean ± SE of four different plants. Differences were significantat P < 0.05 using Duncan’s multiple range test.

Figure 1. Histochemical detection of H2O2 in pea leaves. Leaves were infiltrated with 0.1% (w/v) DAB for 8 h in the dark. (a) Leaves from control and Cd-treated plants. To visualize DAB deposits, leaves were decoloured in boiling ethanol (right leaves). Arrows indicate brown deposits of H2O2. (b) Effect of inhibitors and effectors on H2O2 accumulation in pea leaves from plants treated with 50 mM CdCl2. As control reaction, leaves were incubated with 1 mM ascorbate (Asc, H2O2 scavenger) before infiltrating with DAB. 10 mM DPI, 5 mM cantharidin (Cant, protein phosphatase inhibitor), 1 mM LaCl3 (calcium channel blocker), 1 mM SA, 200 mM ODQ (guanylate cyclase inhibitor), and 100 mM 8-Br-cGMP (cGMP donor), were supplied to excised leaves through the petioles and kept for 18 h at 25 ∞C, except for SA which was kept for 3 h. After incubation, leaves were stained with DAB. H2O2 deposits were quantified by measuring the number of pixel of spots by PHOTOSHOP 6.0 (Adobe Systems). Results are expressed as percentage of spot area, in pixels, versus total leaf area (in pixels). Figure is representative from five different experiments.

(a)

(b)

1126 M. C. Romero-Puertas et al.

© 2004 Blackwell Publishing Ltd, Plant, Cell and Environment, 27, 1122–1134

In order to get some insights into the signal transductionpathway involved in the plant response to cadmium, theeffect of different effectors on H2O2 accumulation in pealeaves grown with cadmium was studied (Fig. 1b). The infil-tration of leaves with cantharidin, an inhibitor of proteinphosphatases, produced a three-fold decrease in H2O2 accu-mulation, which suggests that protein dephosphorylation isinvolved in this process. The blockage of Ca2+ fluxes byLaCl3 brought about a 3.3-fold reduction in Cd-dependentH2O2 accumulation in pea leaves and this suggests theinvolvement of intracellular Ca2+ in the H2O2 productioninduced by cadmium (Fig. 1b).

The treatment of leaves with 8-Br-cGMP, which is a per-meable and biologically effective cGMP donor, retainedthe production of DAB precipitates, whereas ODQ, acGMP inhibitor, depressed it 9.0-fold (Fig. 1b). Salicylicacid brought about a 1.5-fold increase of DAB precipitatesafter 3 h treatment (Fig. 1b), but longer incubations pro-duced severe damage in leaves (data not shown).

Subcellular localization of H2O2

In pea leaves from Cd-treated plants, precipitates of elec-tron-dense cerium perhydroxides, indicating the presence

Figure 2. Localization of H2O2 accumulation in pea leaves by CeCl3-staining and transmission electron microscopy. (a), (c), (e), Pea leaf sections from plants grown in the presence of 50 mM CdCl2. (b), (d), (f), control plants. (a), (b), Transfer cells in vascular tissues. (c), (d), Bundle sheath cells. (e), (f), Epidermic cells. Abbreviations: ch, chloroplasts; m, mitochondria; cw, cell wall; p, peroxisome; v, vacuole. Arrows indicate CeCl3 precipitates. Bars represent 1 mm.

(a) (b)

(c) (d)

(e) (f)

Cd-induced accumulation of O2·– and H2O2 in pea 1127

© 2004 Blackwell Publishing Ltd, Plant, Cell and Environment, 27, 1122–1134

of H2O2, were located in four different types of cells (Figs 2& 3): (1) plasma membrane of transfer cells, associated withphloem sieve elements (Fig. 2a); (2) tonoplasts of bundlesheath cells (Fig. 2c); (3) plasma membrane of epidermalcells (Fig. 2e); and (4) plasma membrane of mesophyll cells(Fig. 3b). In mesophyll cells, Ce precipitates were alsoobserved in the outer membrane of mitochondria (Fig. 3c),and in the presence of aminotriazole, a catalase inhibitor,narrow bands of cerium precipitate were observed in themembrane of peroxisomes in those sites in close contactwith chloroplasts and tonoplasts (Fig. 3d). In control plantsonly few Ce precipitates were associated with transfer cellsin vascular tissues (Figs 2b, d & f and 3a). The specificity ofthe H2O2 reaction was checked using catalase as control. Inthe presence of catalase no staining was observed (Table 2)and this confirmed that the Ce-precipitates were derivedfrom H2O2. A negative control in the absence of Ce wasalso used (data not shown). To determine the source ofH2O2 production in Cd-treated plants, the staining withCeCl3 was examined in the presence of Na-azide, an inhib-itor of peroxidases, and DPI, an inhibitor of NADPH oxi-dase (Table 2). Results showed that only DPI inhibited theformation of cerium precipitates which indicates the possi-ble involvement of an oxidase in the H2O2 production.

Cadmium induces O2·- production in leaves

The production of O2·– in pea leaves was studied by infil-

trating leaves with NBT which is reduced by O2·– giving rise

to dark spots of blue formazan. The accumulation of O2·–

took place in pea leaves treated with CdCl2 (Fig. 4a) andwas prevented by infiltration with TMP, a superoxide radi-cal scavenger (Fig. 4b). The effect of different effectors andinhibitors on O2

·– accumulation in leaves from plants grownwith CdCl2 is shown in Fig. 4b. Results obtained were sim-ilar to those found for H2O2. The infiltration of leaves withcantharidin and LaCl3, inhibited the O2

·– accumulationinduced by Cd, which suggests the involvement of proteinphosphatases and changes in intracellular Ca2+ fluxes in theactivation of ROS production. The infiltration with 8-Br-cGMP also kept the production of NBT precipitateswhereas the inhibitor, ODQ, abolished the formation ofthese precipitates, which is indicative of the involvement of

Figure 3. Localization of H2O2 accumulation in mesophyll cells from pea leaves. (a), Cell from control plants; (b), (c), and (d), cells from Cd-treated plants. In (d) the tissue was treated with 1 mM aminotriazole before CeCl3 staining. Abbreviations as in Fig. 2. Arrows indicate CeCl3 precipitates. Bars represent 1 mm.

(a) (b)

(c) (d)

Table 2. Effect of inhibitors on H2O2 accumulation in leaf sec-tions from Cd-treated pea plants

Treatment H2O2 accumulation

DPI (10 mM) –NaN3 (1 mM) +Catalase (0.25 mg mL–1) –Aminotriazole (1 mM) +

Before staining with CeCl3, leaf pieces were incubated withdifferent inhibitors in 0.1 M HEPES buffer (pH 7.2) for 30 min.Results are expressed as presence (+) or absence (–) of H2O2-dependent cerium precipitates, and were obtained from fourdifferent experiments and three different grids.

1128 M. C. Romero-Puertas et al.

© 2004 Blackwell Publishing Ltd, Plant, Cell and Environment, 27, 1122–1134

cGMP in the induction of O2·– accumulation by Cd. Treat-

ment with SA produced a 1.1-fold increase in NBT precip-itates (Fig. 4b).

Subcellular localization of O2·- radicals

In leaves from plants exposed to Cd, O2·–-dependent DAB

precipitates were observed mainly in tonoplasts of bundlesheath cells close to the vascular tissue (Fig. 5b), whereasin control plants no precipitates were observed (Fig. 5a).The incubation of plant tissue with 1 mM diethyldithiocar-bamate, a CuZn–SOD inhibitor, also produced the appear-ance of precipitates in the plasma membrane of mesophyllcells, and these precipitates were associated with severelydamaged cells (Fig. 5c). The specificity of O2

·–-derived pre-cipitates was checked by incubating the tissue, before DAB

staining, with CuZn–SOD, which scavenges the O2·– radicals

(Fridovich 1986), and not including Mn or DAB in theincubation solution (data not shown). In the presence ofCuZn–SOD no DAB precipitates were observed.

Programmed cell death assays

The induction of programmed cell death (PCD) by cad-mium was studied using different approaches. Ion leakagehas been considered as a PCD marker and can easily bemeasured by changes in the conductivity of leaf discs. Toavoid differences in ion balance due to the treatment withcadmium, results were expressed as relative instead ofabsolute conductivity. Cadmium induced a 1.7-foldincrease in the conductivity, and this was reduced by infil-trating leaves with TMP (Fig. 6). The other signal transduc-

Figure 4. Histochemical detection of O2·– in pea leaves. Leaves were infiltrated with 0.1% (w/v) NBT for 30 min under light conditions.

(a), Leaves from control and Cd-treated plants. To visualize formazan deposits, leaves were decoloured in boiling ethanol. Arrows indicate blue deposits of O2

·– (b), Effect of inhibitors and effectors on O2·– accumulation in pea leaves from plants treated with 50 mM CdCl2. As

control reaction, leaves were incubated with 1 mM TMP (O2·– scavenger) before infiltrating with NBT. Treatment with 10 mM DPI, 5 mM

cantharidin (protein phosphatase inhibitor), 1 mM LaCl3 (calcium channel blocker), 1 mM SA, 200 mM ODQ (guanylate cyclase inhibitor), and 100 mM 8-Br-cGMP (cGMP donor), were supplied to excised leaves through the petioles and kept for 18 h at 25 ∞C, except for SA which was kept for 3 h. After incubation, leaves were stained with NBT. O2

·– deposits were quantified by measuring the number of pixel of spots by PHOTOSHOP 6.0 (Adobe Systems). Results are expressed as percentage of spot area, in number of pixels, versus total leaf area (in number of pixels). The figure is representative of five different experiments.

(a)

(b)

Cd-induced accumulation of O2·– and H2O2 in pea 1129

© 2004 Blackwell Publishing Ltd, Plant, Cell and Environment, 27, 1122–1134

tion modulators assayed slightly reduced the conductivityin Cd-treated leaves. In contrast, most of modulatorsassayed produced an increase of relative conductivity incontrol leaves (Fig. 6). The staining with the Evans Bluedye was also used as a marker of cell death. The resultsobtained after infiltrating leaves with this dye and decolour-ing with methanol are shown in Fig. 7. Plants treated withcadmium showed a higher capacity to fix the dye in com-parison with control plants, and the measurement of theEvans Blue extracted from leaves showed a four-foldincrease in plants treated with CdCl2 (Fig. 7). These resultsdemonstrated that cadmium induced damage in leaf cellswhich could produce cell death although it cannot be con-cluded that cadmium induces PCD.

Another PCD marker used in this study was the analysisof DNA fragment ladders by PAGE and silver staining.However, no ladders were observed either in control or inCd-treated plants, which is indicative of the absence ofPCD symptoms (data not shown). The analysis of DNAfragments was also carried out by fluorescence microscopyusing the TUNEL method. Semi-thin leaf sections preparedin LR White resin were assayed with TUNEL and observedwith a fluorescence microscope, but the presence of apop-totic nuclei with their characteristic green-yellow colourwas not observed, either in control or Cd-treated plants(results not shown).

DISCUSSION

The effect of growing pea plants with 50 mM CdCl2 on dif-ferent physiological parameters, has been previously stud-ied (Sandalio et al. 2001). Pea plants grown for 15 d with50 mM CdCl2 accumulated 52.77 mg Cd g-1 FW in leaves,which produced a significant inhibition of growth (2.7-foldreduction in dry weight and leaf area), as well as a reductionin the transpiration and photosynthesis rate, chlorophyllcontent and disturbances in the nutrient status of peaplants. After 15 d of treatment plants did not show visualtoxicity symptoms and even fructified, although theirgrowth (especially leaf size) was diminished in comparisonwith control plants (Sandalio et al. 2001). In these condi-tions it was demonstrated that oxidative stress was involvedin Cd toxicity in pea plants (Romero-Puertas et al. 1999,2002; Sandalio et al. 2001) and an oxidative burst inducedby Cd was also reported in tobacco cell cultures (Piqueraset al. 1999). However, in whole plants under heavy-metalstress conditions the subcellular localization of ROS hasnot been studied and little information is available on the

(a)

(b)

(c)

Figure 5. Localization of O2·– accumulation in pea leaves by Mn/

DAB staining and transmission electron microscopy. (a), Section of control pea leaves showing transfer cells and bundle sheath cells. (b) and (c), Sections of leaves from Cd-treated plants showing bundle sheath and mesophyll cells, respectively. In (c), tissue was treated with 1 mM diethyldithiocarbamate before Mn/DAB stain-ing. Abbreviations as in Fig. 2. bs, bundle sheath cells; tc, transfer cells. Arrows indicate Mn/DAB precipitates. Bars represent 1 mm.

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regulation of ROS production in those conditions. In thiswork, it was demonstrated that in pea plants Cd inducesROS production in different leaf cells and organelles, andsome of the processes involved in this response werestudied.

Cadmium induced a significant accumulation of H2O2 inpea leaves and this result was corroborated in whole leavesby histochemistry with DAB (Fig. 1a). Accumulation ofH2O2 has also been observed by histochemistry in Cd-exposed pine and pea roots (Schützendübel et al. 2001;Romero-Puertas et al. 2003). In contrast to biotic stress, inwhich necrotic zones can be visualized at the point ofpathogen infection, in Cd-stressed plants no toxicity symp-toms were observed, which makes the study of plantresponse at subcellular level more difficult. In randompieces of pea leaves stained with CeCl3, the accumulationof H2O2 was associated with secondary vascular tissue, epi-dermal, and spongy mesophyll cells (Figs 2 & 3). However,it must be taken into account that, at least, part of the H2O2

detected by cytochemical methods could be derived fromO2

·– radicals which, by either a SOD-catalysed reaction orspontaneously, dismutate into H2O2 and O2 (Fridovich1986). The Ce-precipitates observed in transfer cells fromvascular tissues are probably associated with lignificationprocesses that are very active in this tissue under physio-logical conditions (Olson & Varner 1993), and are alsoinduced under metal toxicity (Schützendübel et al. 2001;Delisle, Champoux & Houde 2001). In bundle sheath cellsCd treatment induced the accumulation of H2O2 and O2

·–

in the tonoplast surrounding the vacuole (Fig. 2c). The pro-duction of ROS in these cells could be due to a diminishedantioxidant capacity, such as has been reported in maizeplants (Kingston-Smith & Foyer 2000). However, in C3

plants, such as pea, no data about differential distributionof antioxidants in mesophyll cells are available.

Epidermal cells are also involved in the production ofH2O2 by Cd, being the Ce-precipitates localized in theplasma membrane (Fig. 2e). These cells could be more sus-ceptible to damage by Cd because the metal can be accu-mulated there driven by transpiration forces (Chardonnenset al. 1998). In this work, different attempts were made tostudy the accumulation of Cd by X-ray microanalysis, butno specific electron-dense precipitates were observed (datanot shown).

In mesophyll cells, deposition of H2O2-derived ceriumprecipitates was observed in plasma membrane but not intonoplasts. Some precipitates were also detected in mito-chondria and, in the presence of aminotriazole, a catalaseinhibitor, sharp narrow bands of precipitate were alsodetected in the membrane of peroxisomes, in those sites inclose contact with tonoplasts and chloroplasts (Fig. 3d). Thesource of H2O2 in the outer mitochondrial membrane couldbe a NADH oxidoreductase that can use cytosolic NADHto produce O2

·– radicals in the cytoplasm (Nohl 1994), whichquickly dismutates to H2O2. In peroxisomes, part of theH2O2 produced could be due to the H2O2-generating glyco-late oxidase and xanthine oxidase whose activities demon-strated an increase as a result of cadmium treatment(Romero-Puertas et al. 1999). However, in chloroplasts nei-ther Ce nor DAB precipitates were observed. Chloroplastsare one of the main sources of ROS under stress conditions(Foyer et al. 1994) and their photosynthesis rate is verysensitive to Cd (Greger & Ögren 1991; Sandalio et al. 2001).The absence of H2O2 or O2

·–-dependent precipitates in chlo-roplasts could be due to the capacity of these organelles toremove the ROS produced using their efficient antioxidant

Figure 6. Effect of CdCl2 on ion leakage in pea leaves. Ion leak-age was measured by incubating leaf discs in Milli-Q water for 3 h and measuring the conductivity of the solutions before and after boiling for 10 min. The results are expressed as relative conductiv-ity [(conductivity after incubation/conductivity after boiling) ¥ 100]. The effect of signal transduction modulators on ions leakage by Cd-treated plants was studied by infiltrating the different chemicals as described in Fig. 2 before incubating leaf discs. Dark columns represents control leaves and white columns cadmium-treated leaves. Values represent mean ±SE of four dif-ferent experiments.

HH 2O

Figure 7. Induction of cell death by cadmium treatment. Cell death was assayed by Evans Blue staining in whole leaves from control and cadmium-treated plants, and the dye bound to leaf cells was quantified by solubilization with 1% SDS in methanol at 50 ∞C and measuring the optical density of solutions at 600 nm. Data are means of three different replicates.

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systems (Foyer et al. 1994; Noctor & Foyer 1998). In thesame way, in birch leaves exposed to ozone, no ROS accu-mulation was detected in the chloroplasts, although H2O2

was detected in the plasma membrane, cell wall, mitochon-dria and peroxisomes (Pellinen et al. 1999).

The inhibition of CuZn–SOD by diethyldithiocarbamateoriginates the formation of O2

·–-dependent DAB precipi-tates in the plasma membrane. The source of H2O2 in mem-branes has been preliminarily identified using inhibitors ofthe NADPH oxidase (DPI) and peroxidase (azide). Ourresults suggest that a NADPH-oxidase or other flavin-con-taining oxidases are the main source of H2O2 in membranesbecause the cerium precipitate accumulation was pre-vented by incubation with DPI, whereas in the presence ofazide the precipitate was visible. Cadmium toxicity is alsoassociated with Zn deficiency (Sandalio et al. 2001) and thiselement is necessary for the NADPH-oxidase regulation inboth animal and plant membranes (Pinton, Cakmak &Marschner 1994). A Cd-induced reduction in Zn availabil-ity could stimulate O2

·– production in cell membranes. Oxi-dases and peroxidases have been involved in the oxidativeburst produced by pathogen infection (Allan & Fluhr 1997;Bolwell et al. 1999) and exposure to ozone (Pellinen et al.1999). In both situations, the accumulation of H2O2 takesplace normally in the cell wall or the outer side of theplasma membrane, whereas Cd induced the H2O2 accumu-lation mainly on the inner side of the plasma membraneand tonoplast. In the oxidative burst induced by pathogensand ozone, the activity of extracellular SOD and peroxi-dases was increased (Pellinen et al. 1999), but in Cd-treatedplants a reduction in both activities, especially SOD, wasfound (Sandalio et al. 2001). These results suggest the exist-ence of similarities in the ROS production induced by Cdand biotic factors, although there are differences in theplant response to Cd treatment.

Results obtained in pea plants with inhibitors or modu-lators of signal transduction processes showed that ROSproduction induced by Cd can be regulated by differentprocesses, some of which are common to the response ofplants to ozone toxicity and pathogen attack (Grant &Loake 2000; Langebartels et al. 2002; Vranová, Inzé & VanBreusegem 2002). The supply of cantharidin, a protein-phosphatase A2 and 1 type inhibitor, completely abolishedthe Cd-induced production of O2

·– and H2O2, whichsuggests that the earliest control point in ROS regulationis at the level of phosphorylation/dephosphorylationof proteins. Arabidopsis mutants defective in proteinphosphatases 2C (abi1–1) are not able to produce ABA-dependent ROS in guard cells (Murata et al. 2001). On theother hand, the blockage of Ca2+ channels by LaCl3 reducedthe production of ROS, which suggests the involvement ofchanges in Ca2+ fluxes through the plasma membrane inROS generation. Lanthanum was also reported to inhibitH2O2 production during PCD in tomato cells (De Jong et al.2002). Changes in cytosolic Ca2+ content are a commonresponse induced by different biotic and abiotic factors andthe magnitude, duration and localization of the perturba-tion in Ca2+ distribution are likely to determine the signal

specificity (Volotovski et al. 1998; Knight & Knight 2001).Cyclic GMP apparently is also involved in Cd-induced ROSproduction, because the inhibitor of guanylate cyclase,ODQ, suppressed its production. Cyclic GMP could induceROS production by causing a transient elevation of Ca2+

concentration, such as has been described by Volotovskiet al. (1998) in tobacco protoplasts.

Salicylic acid has been involved in plant responses tobiotic (Stange et al. 1997) and abiotic stresses, such as UV-B radiation (Sharma et al. 1996; Rao & Davies 2001), ortemperature (Dat et al. 1998). The infiltration of Cd-treatedpea leaves with SA for 3 h produced H2O2- and O2

·–-derivedDAB and NBT precipitates, respectively, which suggests aninduction of ROS, but longer exposures produced necroticeffects on leaves. Rao et al. (1997) have also observedsevere damage in Arabidopsis leaves exposed to SA forlong periods, with the damage being greater than thoseproduced by H2O2. The generation of H2O2 by SA could beexplained by its ability to inhibit catalase and peroxidaseactivity (Durner & Klessig 1995) or an activation of bothsuperoxide production and dismutase activity (Vuletic et al.2003).

During exposure of plants to Cd, symptoms of cell deathwere found by Evans Blue staining and also by measuringelectrolyte leakage in leaf discs. These two parameters havebeen considered as indexes on programmed cell death inplants, although they only give information about plasmamembrane integrity. The results obtained with different sig-nal transduction modulators on ion leakage showed that thecadmium-dependent increase of ion leakage was revertedby TMP, a O2

·– scavenger, suggesting the involvement of O2·–

in the loss of cell integrity. In contrast to Cd-treated leaves,control leaves showed an increase of relative conductivityafter infiltrating with the compounds assayed. This factcould be due to disturbances in cell metabolism and damageproduced by the different chemicals assayed in control,whereas these compounds could protect Cd-treated leavesby alleviating the damage induced by the metal. The higheruptake of Evans blue and the increase of ion leakage incadmium-exposed plants could be due to the oxidative dete-rioration and accelerated senescence induced by cadmium(Sandalio et al. 2001; Schützendübel et al. 2001; Romero-Puertas et al. 2002; McCarthy et al. 2002). This fact wouldexplain the absence of specific necrotic spots in leaves andthe scattered appearance of ROS in the tissue.

The analysis of nucleosomal chromatin fragmentation byboth electrophoresis in polyacrylamide gels and by fluores-cence microscopy using the TUNEL technique, did notshow any DNA degradation in leaves from plants exposedto cadmium. In spite of that, we can not exclude PCD inleaves of pea plants exposed to cadmium because smallamounts of degraded DNA could not be detected by themethods used in this work. In contrast, Fojtová & Kovarík(2000) reported that chronic exposure of tobacco cells tocadmium triggered programmed cell death, but theseauthors used Cd concentrations of 50–100 mM which weremore than 1000 times higher than that used in this work(50 mM CdCl2).

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The Cd-dependent ROS production reported in this workcan be the result of an imbalance between antioxidants andpro-oxidants as a consequence of cell damages induced bycadmium. Under Cd stress, the generation of ROS in mem-branes could be due to the long period of plant growth withthe metal. Cadmium has a strong affinity for membrane sur-faces (Fodor, Szabó-Nagy & Erdei 1995) and its accumula-tion could induce oxidative stress characterized by H2O2/O2

·–

production, lipid peroxidation and protein oxidation(Lozano-Rodríguez et al. 1997; Sandalio et al. 2001;Romero-Puertas et al. 2002). This oxidative stress could bea characteristic of Cd toxicity in plants, being responsible forsome of the physiological and cellular disturbances previ-ously described (Sandalio et al. 2001). However, the possi-bility of a signalling role for ROS under Cd toxicity cannotbe disregarded. Low levels of ROS can have a useful func-tion in the different signalling processes of plant cells (Datet al. 2000; del Río et al. 2002b). The extent of ROS gener-ation in different cells and organelles of pea leaves,described in this work, could determine the type of roleplayed by ROS in plants exposed to cadmium. The local-ization of Ce-precipitate bands in peroxisomal membranes,in close contact with other cell compartments, supports theidea that H2O2 produced in peroxisomes could act as a mes-senger molecule and in the communications among differentcell compartments (Corpas, Barroso & del Rio 2001). Anincrease in H2O2 in peroxisomes or any other cell compart-ment, could produce changes in the expression of proteinsinvolved in the response to cadmium toxicity. Thus, Romero-Puertas et al. (1999) have reported in pea leaf peroxisomesthat cadmium induced the activity and protein content ofenzymes involved in the ascorbate–glutathione cycle, andthe NADPH-dehydrogenases, as a defence mechanism ofcells against Cd-derived oxidative stress.

In preliminary results obtained in our laboratory withpea plants treated with cadmium, an increase in the tran-script levels of total catalase, and a reduction of those ofCuZn–SOD and glutathione reductase was found. On theother hand, an increase in the expression of heat shockproteins has also been observed (Rodríguez-Serrano,unpublished results). Research to determine the identity ofthe specific genes induced by cadmium and the signallingrole of ROS in the response of plants to abiotic stress, isunderway.

ACKNOWLEDGEMENTS

M.C.R.-P. and M.R.-S. acknowledge a PFPI fellowship fromthe Junta de Andalucía, and DGESIC (Ministry of Educa-tion and Culture, Spain), respectively. This work was sup-ported by grants PB98-0493–01 and BFI2002-04440-C02-01from the DGESIC, the European Union (contract HPRN-CT-2000–00094), and the Junta de Andalucía (ResearchGroup CVI 0192).

The electron microscopy assays were carried out at theCentre of Scientific Instrumentation of the University ofGranada.

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Received 4 March 2004; received in revised form 19 April 2004;accepted for publication 22 April 2004