Cadmium-Induced Changes in Antioxidative Systems, Hydrogen Peroxide Content, and Differentiation in Scots Pine Roots

Cadmium-Induced Changes in Antioxidative Systems,Hydrogen Peroxide Content, and Differentiation in ScotsPine Roots1

Andres Schutzendubel, Peter Schwanz, Thomas Teichmann, Kristina Gross,Rosemarie Langenfeld-Heyser, Douglas L. Godbold, and Andrea Polle*

Forstbotanisches Institut, Abteilung I: Forstbotanik und Baumphysiologie, Georg-August-UniversitatGottingen, Busgenweg 2, 37077 Gottingen, Germany (A.S., P.S., T.T., K.G., R.L.-H., A.P.); and School ofAgricultural and Forest Sciences, University of Wales, Bangor, Gwynedd LL57 2UW, United Kingdom(D.L.G.)

To investigate whether Cd induces common plant defense pathways or unspecific necrosis, the temporal sequence ofphysiological reactions, including hydrogen peroxide (H2O2) production, changes in ascorbate-glutathione-related antiox-idant systems, secondary metabolism (peroxidases, phenolics, and lignification), and developmental changes, was charac-terized in roots of hydroponically grown Scots pine (Pinus sylvestris) seedlings. Cd (50 �m, 6 h) initially increased superoxidedismutase, inhibited the systems involved in H2O2 removal (glutathione/glutathione reductase, catalase [CAT], andascorbate peroxidase [APX]), and caused H2O2 accumulation. Elongation of the roots was completely inhibited within 12 h.After 24 h, glutathione reductase activities recovered to control levels; APX and CAT were stimulated by factors of 5.5 and1.5. Cell death was increased. After 48 h, nonspecific peroxidases and lignification were increased, and APX and CATactivities were decreased. Histochemical analysis showed that soluble phenolics accumulated in the cytosol of Cd-treatedroots but lignification was confined to newly formed protoxylem elements, which were found in the region of the root tipthat normally constitutes the elongation zone. Roots exposed to 5 �m Cd showed less pronounced responses and only a smalldecrease in the elongation rate. These results suggest that in cells challenged by Cd at concentrations exceeding thedetoxification capacity, H2O2 accumulated because of an imbalance of redox systems. This, in turn, may have triggered thedevelopmental program leading to xylogenesis. In conclusion, Cd did not cause necrotic injury in root tips but appeared toexpedite differentiation, thus leading to accelerated aging.

Cd is an important environmental pollutant withhigh toxicity to animals and plants. It is released intothe environment by traffic, metal-working industries,mining, as a by-product of mineral fertilizers, andfrom other sources (Nriagu and Pacyna, 1988). Theregulatory limit of Cd in agricultural soils is 100 mgkg�1 soil, but regionally this threshold is exceeded(Salt et al., 1998). Heavy metal toxicity is also animportant issue in reclamation of industrial sites. Cdaccumulation causes reductions in photosynthesis,diminishes water and nutrient uptake (Sanita diToppi and Gabbrielli, 1999), and results in visiblesymptoms of injury in plants such as chlorosis,growth inhibition, browning of root tips, and finallydeath (Kahle, 1993).

The question as to how Cd acts at the cellular leveland how plants may defend themselves against thispollutants is receiving increasing attention. It has

been shown that Cd induces the synthesis of phy-tochelatins (�-glutamyl-Cys [�-EC] peptides), whichbind metals in the cytosol and sequester them in thevacuole (Rauser, 1995; Mehra and Tripathi, 2000).The precursor for phytochelatin synthesis is glutathi-one, whose cellular level was decreased after Cdexposure (Rauser, 1995; Zenk, 1996; Xiang and Ol-iver, 1998). Exposure to sublethal Cd concentrationsresulted in the recovery of cellular glutathione con-centrations and was accompanied by increased �-ECsynthetase and glutathione synthetase mRNA tran-script levels (Xiang and Oliver, 1998).

Glutathione is the major non-protein thiol in plantsand has many functions in plant metabolism. It isinvolved in the detoxification of heavy metals andxenobiotics and plays a role in gene activation and inthe protection from oxidative stress (Lamoureux andRusness, 1989; Bergmann and Rennenberg, 1993;Noctor and Foyer, 1998). As an antioxidant glutathi-one together with ascorbate and antioxidative en-zymes, superoxide dismutases (SOD; EC 1.15.1.1),ascorbate peroxidases (APX; EC 1.11.1.11), and cata-lases (CAT; EC 1.11.1.6) controls the cellular concen-trations of hydrogen peroxide (H2O2) and O2

.� (Noc-tor and Foyer, 1998). Recycling of ascorbate and GSHis achieved by monodehydroascorbate radical reduc-

1 This work was supported by the European Community(project no. FAIR3–CT961377; Metal Tolerant EctomycorrhizalFungi: Selection, Characterisation, and Utilisation for Restorationof Polluted Forests).

* Corresponding author; e-mail [email protected]; fax 49–0–551–39–2705.

Article, publication date, and citation information can be foundat www.plantphysiol.org/cgi/doi/10.1104/pp.010318.

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tase (MDAR; EC 1.6.5.4.), dehydroascorbate reduc-tase (DAR; EC 1.8.5.1), and glutathione reductase(GR; EC 1.6.4.2).

Cd treatment affects the activities of antioxidativeenzymes, but contrasting results have been reported.For example, in leaves of Cd-exposed Helianthus an-nuus plants, the activities of ascorbate-glutathione-related defense enzymes were decreased (Gallego etal., 1996). Roots and leaves of Phaseolus vulgaris aswell as suspension cultures of tobacco (Nicotiana taba-cum) cells contained elevated APX activities after Cdexposure (Chaoui et al., 1997; Piqueras et al., 1999). InPhaseolus aureus seedlings, Cd induced elevated guaia-col peroxidase (POD) but decreased CAT activities(Shaw, 1995). Cd also caused lipid peroxidation, sug-gesting that the tissues suffered from oxidative stress(Shaw, 1995; Gallego et al., 1996; Lozano-Rodriguezet al., 1997; Chaoui et al., 1997). The involvement ofantioxidants in plant responses against Cd toxicity isunclear because Cd does not belong to the group oftransition metals like copper, iron, and zinc, whichmay induce oxidative stress via Fenton-type reac-tions. It is possible that the observed changes in theantioxidant systems occurred as a result of unspecificcellular degradation processes. However, anotherpossibility is that Cd triggers common defense path-ways in plants cells like other biotic or abiotic envi-ronmental stresses. A joint initial event of these path-ways is an accumulation of H2O2, which acts as asignaling molecule. In plant-pathogen interactions,H2O2 induces an orchestrated sequence of reactionsinvolving the activation of peroxidases, the stimula-tion of secondary metabolism, structural changessuch as lignin deposition, and eventually cell death(Alvarez and Lamb, 1997).

Because it is not known whether Cd induces com-mon plant defense pathways, we investigated thesequence of physiological reactions, including H2O2production, changes in ascorbate-glutathione-relatedantioxidant systems, secondary metabolism (peroxi-dases, phenolics, and lignification), developmentalchanges, and cell death, occurring in roots after Cdexposure. Scots pine (Pinus sylvestris) seedlings werechosen as a model system because forest ecosystemsare particularly vulnerable to heavy metal pollution.The seedlings were exposed to 5 or 50 �m Cd inhydroponics and used to study physiological defensereactions and anatomical changes in root tips.

RESULTS

Growth Responses and Cd Accumulation

After acclimation to fresh medium, pine roots grewat a constant rate of about 6.4 mm d�1 (Fig. 1).Addition of Cd immediately affected root elongation.Exposure to Cd at 5 and 50 �m caused reductions inthe growth rates of 20% and 90%, respectively, within12 h. At that time the root tips contained Cd at about150 and 1,000 �g g�1 dry weight (Fig. 2). The major

accumulation of Cd occurred during the first 24 hafter Cd exposure. Thereafter, neither roots exposedto Cd at 5 �m nor those exposed to Cd at 50 �mshowed further significant changes in their Cd con-centrations (Fig. 2). Roots exposed to the higher Cdconcentration completely stopped growth after 12 h,whereas those treated with the lower Cd concentra-tion continued to grow at a 20% diminished rate(Fig. 1).

Responses of Antioxidant Systems to Cd Exposure

In root tips of control seedlings, activities of anti-oxidative enzymes were determined over an experi-mental period of 96 h in parallel with measurementsin Cd-treated roots tips. In this period, enzyme ac-tivities of controls fluctuated only slightly aroundtheir means (Fig. 3). Enzymes involved in the re-moval of H2O2 and O2

.� were generally by one ortwo orders of magnitude higher than those involvedin the regeneration of antioxidants. DAR activitieswere not detected (Fig. 3).

Treatment with high Cd concentrations (50 �m)initially resulted in doubling of SOD activities (Fig.4A). However, this stimulation was only transient.After 12 h, SOD activities in roots treated with 50 �mCd were again similar to those found in controls anddecreased significantly at the end of the experiment.The rapid induction of SOD after 6 h in the 50 �m Cdtreatment was not observed for transcripts of Cu/Zn-SOD (data not shown). Analysis of SOD activities inthe presence of cyanide (5 mm) showed that Mn-and/or Fe-SOD activity contributed 18% of total SODactivity in controls and increased to 31% within 96 hafter exposure to 50 �m Cd (data not shown). Expo-

Figure 1. Growth of root tips of Scots pine seedlings in the absence(�, control) or presence (E, 5 �M; F, 50 �M) of Cd. n � 20 replicatesper sampling date (�SD). Asterisks indicates values that differ signif-icantly from the control at P � 0.05.

Schutzendubel et al.

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sure to 5 �m Cd had no significant effects on SODactivities (Fig. 4A).

In contrast to SOD, which was stimulated or hardlyaffected by Cd, CAT and APX activities were initiallysignificantly suppressed by Cd (Figs. 4B and 5A).After 24 h, the activities had recovered (CAT, 5 �mCd) or were strongly increased (APX and CAT at 50�m Cd; Figs. 4B and 5A). At later stages, the CAT andAPX dropped back to activities similar to those foundin controls.

“Total ascorbate,” defined as the sum of ascorbate� dehydroascorbate (DHA), was significantly in-creased after 12 h exposure of root tips to Cd at 5 and50 �m compared with controls (Fig. 5C). A depletionof “total ascorbate” was observed in root tips ofplants treated with the higher Cd concentration (Fig.5C), whereas “total ascorbate” increased in controls.The fraction of reduced ascorbate in Cd-exposedroots was initially increased (from 13% in controls to

40% in the presence of 50 �m Cd, 6 h), but thereafterdeclined rapidly and dropped below the detectionlimit after 24 h of Cd treatment. In controls, ascorbatealso decreased but was below the detection limit onlyafter 96 h (Fig. 5C). The activities of MDAR increased1.5- to 2-fold in response to both Cd treatments (Fig.5B), suggesting Cd caused an increased redox cyclingof ascorbate. DAR activities were generally below thedetection limit (Fig. 3), and effects of Cd on DARactivities were not detected (data not shown).

Cd at 50 �m caused a significant inhibition of GRactivity (�70%) after 6 and 12 h (Fig. 6A). Recoveryoccurred within 24 h and after 96 h the GR activitywas approximately 1.5-fold increased compared withcontrols. In the presence of 5 �m Cd, a significantinhibition of GR activity (�65%) was also observed.However, this suppression occurred delayed incomparison with roots treated with Cd at 50 �m;recovery to levels similar to those of controls wasfound after 96 h.

Upon exposure to 50 �m Cd, the glutathione pool(GSH � oxidized glutathione [GSSG]) was almostcompletely depleted within 6 h and the remainingglutathione was oxidized (GSSG; Fig. 6, B and C). InCd-treated roots, the increase in glutathione was ini-tially accelerated as compared with controls but didnot reach the high levels present in controls at 24 h(Fig. 6B). After 96 h, Cd-treated roots containednearly four times higher concentrations of glutathi-one than controls (Fig. 6B). Despite fluctuations inthe glutathione concentrations, the redox state of the

Figure 2. Cd content in root tips of Scots pine seedlings treated with5 �M Cd (E) or 50 �M Cd (F). One-centimeter-long root tips wereused for analysis (n � 3–4 replicates per date, �SD). The detectionlimit for Cd determination was 5 �g g�1 dry weight. Asterisks indi-cates values that differ significantly from the control at P � 0.05.

Figure 3. Activities of antioxidative enzymes in root tips of Scotspine seedlings. Root tips from control plants were harvested 6, 12,24, 48, and 96 h after transfer to fresh medium and were used todetermine antioxidative systems (n � 4 per sampling date). The barsindicate overall means from all sampling dates (n � 20, �SD). SOD*,SOD activity is indicated in units. Gray bars, Left y axis; black bars,right y axis. ND, Not detected.

Figure 4. Activity of SOD (A) and CAT (B) in root tips of Cd-treated(E, 5 �M; F, 50 �M) Scots pine seedlings. The activity was expressedrelative to the activity in control plants (�100%, � with dashed line).Each value is the mean of four individual replicates (�SD). Asterisksindicate values that differ significantly from the control at P � 0.05.

Cd-Induced Oxidative Stress and Differentiation

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glutathione pool remained relatively constant formost of the time accounting for 50% to 70% GSSG inroot tips of control plants. After recovery from theinitial stress in both Cd treatments, the fraction ofGSSG decreased below 20% after 96 h (Fig. 6C).

The effects of Cd on the precursors of glutathionesynthesis, �-EC and Cys, were also investigated (Fig.7, A and B). In roots tips of control seedlings, theconcentrations of these compounds were low (�-EC:1.26 � 0.61 nmol g�1 fresh weight and Cys: 14.3 �4.47 nmol g�1 fresh weight) accounting 0.2% and2.7% of the thiol pool. Both Cd concentrations causedsignificant decreases in �-EC within 6 h (not seen dueto the scaling of Fig. 7B). Thereafter, �-EC concentra-tions increased extremely, reaching 10- and 20-foldhigher concentrations in roots exposed for 12 h to Cdat 5 and 50 �m, respectively, than those present incontrols (Fig. 7B). Elevated �-EC concentrations were

maintained in Cd-treated roots until the end of theexperiment. Upon exposure to Cd, Cys concentra-tions also started to increase but with a delay of 12 has compared with �-EC (Fig. 7A). Maximum Cysconcentrations were found in roots after 24 h expo-sure to Cd at 50 �m; thereafter, the concentrationsdecreased but maintained elevated levels for 96 h(Fig. 7A).

To investigate whether the inhibition of antioxida-tive enzymes, observed after Cd exposure, was ac-companied by an increase in activated oxygen spe-cies, intact roots were stained histochemically for thepresence of H2O2 (Fig. 8). Roots exposed 6, 9, and12 h to 50 �m Cd showed significant accumulation ofH2O2 compared with unstressed controls. After 24 h,the staining was less pronounced. At later stages, Cdhad also caused browning of the roots tips, whichwould interfere with low H2O2 staining intensities.

Figure 5. Activity of APX (A), MDAR (B), and concentrations ofascorbate � DHA (C) in root tips of controls (�) and Cd-treated (E,5 �M; F, 50 �M) Scots pine seedlings. Enzyme activities were ex-pressed relative to the activity in control plants (�100%, dashedline). Each value is the mean of four individual replicates (�SD).Asterisks indicate values that differ significantly from the control atP � 0.05. Bars indicate redox state (%) � (ascorbate � 100)/(ascor-bate � DHA). Gray bar, Control, white bar, 5 �M Cd; black bar, 50�M Cd.

Figure 6. Activity of GR (A), concentration of glutathione (B), andredox state of the glutathione pool (C) in root tips of controls (�) andCd-treated (E, 5 �M; F, 50 �M) Scots pine seedlings. The activity wasexpressed relative to the activity in control plants (�100%, dashedline). Each value is the mean of four individual replicates (�SD).Asterisks indicate values that differ significantly from the control atP � 0.05.





Despite the initial depletion of antioxidative sys-tems and accumulation of H2O2 at 6 to 12 h after Cdexposure, these conditions did not result immedi-ately in cell death (Fig. 9). Only at later stages of Cdexposure after 24 h, when ascorbate was hardly de-tected, cell death was slightly and only transientlyincreased (Fig. 9).

Secondary Metabolism and Developmental Changes inResponse to Cd

Cd treatment initially had no effect on nonspecificperoxidase activities (POD), i.e. peroxidases reducingH2O2 by the oxidation of aromatic substrates likephenolics or monolignols (Fig. 10A). A significantinduction was found only after 48 h (Fig. 10A). Theinduction was less pronounced in roots of pine seed-lings exposed to Cd at 5 �m as compared with thosetreated with Cd at 50 �m (Fig. 10A). It is interestingto note that the Cd-induced stimulation in POD ac-tivities was delayed as compared with APX or CATactivities and that initial reductions in POD activitiesdid not occur (Fig. 10A). Soluble phenolics increasedimmediately after Cd exposure (Fig. 10B). The ele-vated levels were maintained in root tips exposed toCd at 50 �m but not in those exposed to 5 �m (Fig.10B). Histochemical analysis of roots tips showedthat these newly formed phenolics were localized inthe cytosol (Fig. 11E). The intense staining was notfound in controls (Fig. 11D).

In root tips of seedlings exposed to 5 �m Cd, thelignin concentration was unaffected as comparedwith controls (136 � 2.0 mg�1 dry weight, n � 16,�sd), whereas treatment with 50 �m Cd resulted insignificant increases in lignin (Fig. 10C). To find outthe structural basis of this increased lignification,cross sections were stained with toluidine blue (Fig.11, A–C). After 96 h of Cd exposure was complete,lignified protoxylem poles were found at a distanceof 6,000 �m from the tip (Fig. 11B), whereas this partof untreated roots did not show any xylem-like dif-ferentiation (Fig. 11A). Further analysis revealed thatfirst lignified protoxylem elements were detected al-ready at a distance of 1,500 �m from the root tips inplants exposed for 96 h to 50 �m Cd (Fig. 11C).

DISCUSSION

Detoxification of Cd

It is well known that Cd inhibits growth (Godboldand Huttermann, 1985; Arduini et al., 1996; Arisi etal., 2000) and affects glutathione metabolism (Rauser,1995; Zenk, 1996; Xiang and Oliver, 1998; Arisi et al.,2000). In the present study, we showed that inhibi-tion of root elongation was among the most sensitiveresponses to Cd exposure and faster than most of theother physiological reactions analyzed (Fig. 1) andpreceded cell death (Fig. 9). Complete inhibition ofroot elongation occurred within 12 h at Cd concen-trations in the root tip in the range between 112 and560 nmol Cd g�1 fresh weight as judged from the 5and 50 �m Cd treatments (Figs. 1 and 2). It is inter-esting that the root tips accumulated Cd only forabout 24 h and maintained relatively stable levelslater on, regardless of the concentration applied (Fig.2). This observation suggests that Cd uptake by roottips is counterbalanced by transport to leaves withinapproximately 24 h. This finding is important be-cause it shows that reactions occurring after 48 or96 h were not caused directly by changes in the Cdconcentrations of the tissue. This notion applies, forexample, for the 4- and 30-fold increases in glutathi-one and �-EC concentrations, respectively, occurringafter 96 h Cd exposure (Figs. 6B and 7B). It is unlikelythat these “late” thiol enhancements reflected a Cddefense mechanism. Arisi et al. (2000) also found thatelevated glutathione concentrations in transgenicpoplars overexpressing �-EC synthetase activitiesdid not protect from Cd toxicity. Perhaps the in-creases in thiols found in the present study at stageswhen Cd did not increase any longer were caused byenhanced sulfate assimilation. Such a response hasbeen reported for Brassica juncea exposed to Cd (Leeand Leustek, 1999).

Six hours after Cd exposure, a significant deple-tion of glutathione initially was observed (Fig. 6B).This is a common response to Cd caused by anincreased consumption of glutathione for phytoch-elatin production (Delhaize et al., 1989; Meuwly and

Figure 7. Concentrations of Cys (A) and �-EC (B) in root tips ofcontrols (�) and Cd-treated (E, 5 �M; F, 50 �M) Scots pine seedlings.Each value is the mean of four individual replicates (�SD). Asterisksindicate values that differ significantly from the control at P � 0.05.





Rauser, 1992; De Knecht et al., 1995; Schneider andBergmann, 1995; Noctor et al., 1998; Xiang and Ol-iver, 1998). Because the synthesis of glutathione isdemand driven, the low glutathione concentrationmight have triggered increased sulfur uptake andits own synthesis (May et al., 1998), thus resulting inelevated glutathione concentrations at later stages.

Cd Induces Oxidative Stress

Cd exposure initially resulted in severe oxidativestress because the low residual glutathione pool wascompletely oxidized (6 h, Fig. 6C) and H2O2 accumu-lated (Fig. 9). It is seemingly paradox that the redoxstate of ascorbate initially increased (Fig. 5C, 6 h)when H2O2 accumulated in the presence of 50 �m Cd.However, this is theoretically possible when APX isdecreased (Polle, 2001). Such a situation occurredapparently after 6 h of 50 �m Cd exposure (Fig. 5A).Ascorbate was then consumed and DHA accumu-lated, perhaps because of shortage of reductant tomaintain MDAR activities in vivo (Fig. 5B)

At the first glance, it may appear surprising thatCd, which is not a transition metal, may cause oxi-dative stress. However, Cd binds to thiol groups andthereby inactivates thiol-containing enzymes such asGR (Creissen and Mullineaux, 1995; Mullineaux andCreissen, 1997). The same inhibition mechanism maybe possible for APX being sensitive to thiol reagents(Chen and Asada, 1989, 1992). In fact, we found thatCd simultaneously inhibited the systems involved inH2O2 removal, i.e. GSH/GR, CAT, and APX, andresulted in elevated activities of SOD, an enzyme,whose product is, beside O2, H2O2 (Figs. 6, A and B,5A, and 4, A and B). Such a situation must inevitablylead to the accumulation of H2O2 (Polle, 2001).

Figure 8. H2O2 in roots of 6-week-old seedlings of Scots pine grown in liquid culture. Roots of control plants and rootstreated with 50 �M Cd were stained for H2O2 after exposure to Cd for the indicated times. Bar � 5 mm.

Figure 9. Cell death indicated as Evans blue uptake by root tips ofcontrols (�) and Cd-treated (E, 5 �M; F, 50 �M) Scots pine seedlings.Each value is the mean of four individual replicates (�SD) and wasexpressed relative to controls.





Both glutathione and H2O2 play roles as signals forthe regulation of stress enzymes (May et al., 1998;Noctor and Foyer, 1998). Accumulation of H2O2 is ageneral stress response, which has been observed inplants exposed to low temperature, heat, pathogens,and chilling (Doke et al., 1994; Levine et al., 1994;Mehdy, 1994; Prasad et al., 1994). H2O2 is a systemicsignal for the induction of APX (Karpinski et al.,1999). In callus cultures of rice (Oryza sativa) em-bryos, H2O2 transiently induced mRNA for cytosolicAPX (Morita et al., 1999). In the pine seedlings stud-ied here, H2O2 accumulation was followed withinfew hours by recovery and significant increases inAPX and CAT activities (Figs. 4B and 5A). GR activ-ities also recovered, but interestingly, this recoverywas faster in plants exposed to 50 �m than in thoseexposed to 5 �m Cd, suggesting that the decreasebelow a certain threshold of GR was necessary toinduce the restoration. Xiang and Oliver (1998) found

that the recovery was related to increased synthesisof GR protein because the transcript levels increasedafter Cd exposure. In contrast to APX gene expres-sion, which is activated by H2O2, GR was not stim-ulated by H2O2 but by jasmonic acid (Xiang andOliver, 1998). The different time courses for the in-duction of APX and GR activities observed here alsosupport the finding that these enzymes are regulatedby different stimuli.

The time courses of antioxidative responses afterCd exposure suggest that the following sequence ofevents may take place: Initially, Cd uptake leads to adepletion of glutathione and inhibits CAT, APX, andGR. This causes an accumulation of H2O2 and in-duces the synthesis of ascorbate and glutathione.H2O2 has been shown to act as a signaling moleculein the activation of cellular defenses including CATand APX (Prasad et al., 1994; Karpinski et al., 1999).We have not investigated whether H2O2 productionwas augmented by the stimulation of plasmamembrane-bound NADPH oxidases as in pathogendefense reactions (Doke et al., 1994; Alvarez andLamb, 1997). However, this is likely to occur becauseCd can induce an oxidative burst (Piqueras et al.,1999) and early pathogen defense reactions also in-volve elevated SOD activities (Doke et al., 1994) asobserved here (Fig. 4A). The pathogen-related SODinduction is caused by increases in extracellular SODactivity (Doke et al., 1994). We currently cannot ex-clude such a response in Cd-exposed plants since wecould not explain the increases in SOD by changes inthe gene expression of the cytosolic CuZn-SOD,which is a major SOD in unstressed pine roots.

Cd Triggers Secondary Metabolism and Differentiation

H2O2 is a signaling intermediate in programmedcell death (Alvarez and Lamb, 1997), which is trig-gered by pathogen-derived elicitors and occurs aspart of the normal developmental program of theplants, strictly controlled in committed cells duringxylogenesis (Teichmann, 2001). Fungal elicitors in-duce H2O2-mediated oxidative cross linking of cellwalls in processes not requiring transcription, trans-lation, or activation of secondary metabolism (Brad-ley et al., 1992). Addition of H2O2 leads to increasedmechanical strength and lowers the extensibility ofplant cell walls (Schopfer, 1996). Such a rapid H2O2-mediated rigidification of cell walls would explainthe fast abolishment of growth occurring in our studywithin 12 h, thus preceding the activation of peroxi-dases and lignification by more than 24 h (Fig. 10, Aand C). To our knowledge, the induction of the phe-nylpropanoid metabolism upon Cd exposure has notbeen reported before. The activation of this pathwayis also a typical event in pathogen defense and pro-grammed cell death. Our results suggest that Cd incells challenged by concentrations, which overridethe capacity for detoxification, may set off common

Figure 10. Activity of POD and concentrations of soluble phenolics(B) and lignin (C) in root tips (1.5 cm) of controls (�) and Cd-treated(E, 5 �M; F, 50 �M) Scots pine seedlings. The POD activity and ligninwere expressed relative to controls (�100%, dashed line). Each valueis the mean of four individual replicates (�SD). Asterisks indicatevalues that differ significantly from the control at P � 0.05.





plant defense pathways. In our system, the thresholdwas exceeded somewhere between 5 and 50 �m ofexternally applied Cd and was accompanied specifi-cally by depletion of GSH in combination with failureof H2O2-consuming systems and transient increase incell death (Fig. 9). Fojtova and Kovarik (2000) re-cently showed that Cd induced apoptotic changes insuspension cultures of tobacco cells. These changeswere characterized by DNA fragmentation that oc-curred delayed about 48 h after Cd addition (50–100�m). Once cells are committed to programmed celldeath, the process cannot be reversed. In contrast tonecrosis, programmed cell death is a strictly con-trolled process. In line with data on cell death (Fig. 9),we found no evidence for arbitrary injury in crosssections of roots after 96 h of Cd exposure (Fig. 11, Band E), which would have been expected for necroticreactions. However, Cd appeared to induce the nor-

mal developmental program leading to xylogenesisbecause the formation of “normal” protoxylem ele-ments was observed (Fig. 11B). Lignification is thefinal steøp in this process (Polle et al., 1997). How-ever, in contrast to normal development, the lignifiedxylem elements were found here at a distance fromthe root tip, which normally constitutes the elonga-tion zone.

The processes leading to lignification must be dis-tinguished from those leading to the production ofsoluble phenolics. Cd caused an accumulation of sol-uble phenolics in the cytosol. However, this reactionwas much faster than lignification (Fig. 10B) andspread over the whole cross section of the root (Fig.11E). From the differences in temporal and spatialresponse patterns, it is clear that different signals ordifferences in the perceptibility of signals must havecaused the rapid accumulation of phenolics on the

Figure 11. Localization of lignin and suberin (A, control; B and C, 50 �M Cd) and phenolics (D, control; E, 50 �M Cd) inroot tips of Scots pine seedlings. Ninety-six hours after exposure to Cd, cross sections were taken at distances from the roottip of 6 mm (A, B, D, and E) and 1.5 mm (C) and were stained with 1% (w/v) toluidine blue (A–C) or 0.1% (w/v)berberine-sulfate (D and E). Magnification, 20 � 12.5 (A, B, D, and E); 40 � 12.5 (C). Arrows indicate localization ofprotoxylem elements.





one hand and delayed lignification on the otherhand. However, which signals affected secondarymetabolism in this distinct way is still unknown.

A further question is why phenolics were increasedin response to Cd. Phenolics may contribute, togetherwith ascorbate, to H2O2 destruction in the so-calledphenol-coupled APX reaction (Polle et al., 1997), andthus protect from oxidative stress. However, this pro-tection was probably limited by low concentrationsof ascorbate present in roots (Fig. 5C). Whether phe-nolics have direct protective functions is unknownfor Cd, but in cultured tobacco cells, phenolics pro-tected from aluminum toxicity (Yamamoto et al.,1998).

Taken together, our results suggest that the inhibi-tion of antioxidative systems by Cd promotes H2O2production. This was probably the key event for theinhibition of elongation growth and might have setoff a sequence of reactions leading to cell death.However, the latter response occurred seeminglyonly in committed cells, i.e. in those in which thisprocess would normally occur. In conclusion, wesuggest that Cd does not cause unspecific necrosis ofroot cells but expedites developmental and differen-tiation processes leading to accelerated aging.

MATERIALS AND METHODS

Culture of Plants and Growth Conditions

Seeds of Scots pine (Pinus sylvestris) were surface steril-ized for 1 h in 30% (w/v) H2O2. After 3 d at 21°C indarkness, the seeds were germinated on sterile 1% (w/v)water-agar, pH 4.5, with a day/night regime of 16 h/8 h.The seedlings were grown at day/night conditions of23°C/21°C air temperature, 16-h/8-h day length underwhite light of 200 �mol m�2 s�1 photosynthetic photonflux (Osram L18 W/21 lamps, Munich). After 3 weeks, theseedlings were transferred to aerated nutrient solutionscontaining the following nutrient elements: 300 �mNH4NO3, 100 �m Na2SO4, 200 �m K2SO4, 60 �m MgSO4,130 �m CaSO4, 30 �m KH2PO4, 10 �m MnSO4, and 92 �mFeCl3; and 5 mL of a stock solution of micronutrients:0.1545 g L�1 H3BO3, 0.012 g L�1 NaMoO4, 0.0144 g L�1

ZnSO4, and 0.0125 g L�1 CuSO4 per liter of nutrient solu-tion. The pH was adjusted to 4.0. The solution was changedevery 3 d. After 2 weeks of acclimation, the seedlings weretreated with 5 or 50 �m CdSO4 for 96 h and sampled inregular intervals for analyses.

Root Length Measurements and Sampling

Two days before Cd exposure, roots were marked 5 mmbehind the tip with water-resistant ink. Root lengths weremeasured daily under a binocular (Stemi SV 8, Zeiss,Oberkochen, Germany) with a measuring ocular (ZeissCPL 10-fold). Measurements were performed on 20 plantsper treatment and date.

Root samples for analysis of enzymes activities, antioxi-dants, phenolics, and lignin were collected after 6, 12, 24,

48, and 96 h of Cd treatment. Each sample consisted of 2015-mm-long root tips. Activities of APX, MDAR, GR, andDHAR were measured in fresh extracts immediately afterharvest. For analyses of further biochemical parameters,root tips were kept frozen at �80°C.

Each treatment was replicated four times. Statisticalanalyses were performed with Statgraphics using ANOVAfollowed by a least-significance difference test (STN, St.Louis).

Cd Determination

Root tips were cut 10 mm behind the tip, washed 15 minin 5 mm CaCl for exchange of apoplastic Cd (Rauser, 1987),dried to a constant weight, and wet ashed at 170°C in pureHNO3 for 12 h (Feldmann, 1974). Cd was determined byinductively coupled plasma-atomic emission spectroscopywith a standard method.

Enzyme Assays and Protein Determination

Root tips were powdered in liquid nitrogen. The powder(150 mg) was extracted 15 min at 4°C in 5 mL of coldextraction buffer (100 mm potassium phosphate, pH 7.8,300 mg polyvinylpolypyrrolidone, 1% [v/v] Triton X-100,and 5 mm ascorbate). The extract was centrifuged (30 min,48,000g, 4°C) and the supernatant was passed through aSephadex G-25 column (PD-10 column, Pharmacia,Freiburg, Germany) and equilibrated with 100 mm potas-sium phosphate, pH 7.8. For stabilization of APX, the elu-tion buffer contained 5 mm ascorbate. The enzyme activi-ties were determined according to the following methods:SOD (McCord and Fridovich, 1969), CAT (Aebi, 1983), APX(Nakano and Asada, 1981), MDAR (Hossain et al., 1984),DAR (Dalton et al., 1986), GR (Foyer and Halliwell, 1976),and PODs (Putter, 1970; modified after Polle et al., 1990).Soluble protein was determined in enzyme extracts withbicinchoninic acid reagent (Pierce, Munich) using bovineserum albumin as the standard. All solutions used foranalytical and enzymatical investigations were preparedwith double-ionized and ultrafiltrated water (SeralpurDelta UV/UF, USF Seral, Ransbach-Baumbach, Germany).

Extraction and Analysis of Antioxidants

For the extraction of water soluble antioxidants, 60 to 100mg of frozen root tips were powdered in liquid nitrogen,mixed with 1 mL of 2% (w/v) meta-phosphoric acid con-taining 1 mm EDTA and 1 mg polyvinylpolypyrrolidoneper mg of sample, and centrifuged (20 min, 4°C, 30,000g).The supernatant was used for analyses of antioxidants.

Ascorbate was determined at 268 nm after separation bycapillary electrophoresis as described by Davey et al.(1996). The system consisted of a High-Performance Cap-illary Zone Electrophoresis (model P/ACE 5500 HPCE Sys-tem, Beckman Instruments, Fullerton, CA) fitted with aUV-VIS diode array detector (P/ACE 5500 DAD, Beckman)and equipped with GOLD software (Beckman) for peakanalysis. Injections were made for 5 s under hydrostatic





(N2) pressure (0.5 pound per square inch) into a 57-cmfused silica capillary at a constant temperature of 25°C anda constant voltage of �25 kV. Separations were run for 10min with 100 mm borate buffer, pH 9, as a carrier electro-lyte. The capillary was subsequently conditioned for thenext run with 0.1 m NaOH, water, and carrier electrolyte.The detection limit for ascorbate was 0.5 �g mL�1 underthe present experimental conditions.

For determination of “total ascorbate,” DHA was re-duced by dithiothreitol at pH 8.3 to 8.5 for 60 min at roomtemperature (Anderson et al., 1992). This was achieved bymixing 100 �L of sample with 150 �L of 60 mm dithiothre-itol in 1 m 2-[N-cyclohexylamino] ethansulfonic acid. Themixture was analyzed as above and DHA was calculatedby substracting ascorbate from “total ascorbate.”

Glutathione, �-EC, and Cys were determined after re-duction, derivatization with monobromobimanes, HPLCseparation (Beckman System Gold, Munich, Germany) on aC-18 column, and detection with a fluorescence detector(RF-550, Shimadzu, Duisburg, Germany) after the methodof Schupp and Rennenberg (1988). GSSG was determinedin the same manner after removal of glutathione by alky-lation with N-ethylmaleimide (Gorin et al., 1966) GSH wascalculated as the difference of glutathione and GSSG. Theredox state was defined as the GSSG content in percent ofglutathione content.

Measurement of Cell Death

Cell death, indicated as loss of plasma membrane integ-rity, was measured spectrophotometrically as Evans blueuptake (Baker and Mock, 1994). After Cd treatment, threeroot tips (1 cm) were incubated in Evans blue solution(0.025% [w/v] Evans blue in water) for 30 min. Afterwashing the roots for 15 min with water, the trapped Evansblue was released from the roots by homogenizing root tipswith a microhomogenizer in 800 �L of a measuring solu-tion (50% [v/v] MeOH and 1% [w/v] SDS). The homoge-nate was incubated for 15 min in a water bath at 50°C andcentrifuged at 14,000g for 15 min. The optical density of thesupernatant was determined at 600 nm and expressed onthe basis of fresh mass.

Determination of Phenolics and Lignin

Frozen root tips (about 100 mg) were ground in liquidnitrogen, transferred into 1 mL of 50% (v/v) MeOHMeOH/H2O/H2O, incubated for 30 min on a shaker, andcentrifuged at 48,000g. Five hundred microliters of extractwas used for the determination of free phenolic com-pounds with the Folin-Ciocalteus reagent (Pritchard et al.,1997). Tannic acid was used as a standard. The pellet wassubjected to several washing steps, modified after Strack etal. (1988): 2� 80% (v/v) MeOH/H2O, 1� water, 1� ace-tone, and hydrolyzed 1 h at 70°C with 1 m NaOH. Thehydrolyzed pellet was washed with 80% (v/v) MeOH/H2O (two times), distilled water and acetone, and dried.The resulting residue was used for lignin analysis with the

thioglycolic acid method after Bruce and West (1989) asmodified by Otter (1996).

Histochemical Staining of Roots for H2O2 and PhenolicCompounds

To detect H2O2, roots attached to the plants were stainedfor 30 to 45 min in KI/starch reagent (4% [w/v] starch and0.1 m KI, pH 5.0; modified after Olson and Varner, 1993).Stained roots were photographed under a binocular (StemiSV 8, Zeiss).

To analyze anatomical changes, roots were freeze driedand embedded for microscopy as described by Fritz (1989).Cross sections of roots (1 �m) were stained with 1% (w/v)toluidine blue in 0.1% (w/v) disodium tetraborate decahy-drate for light microscopy (Axioplan microscope, Zeiss) orstained with 0.1% (w/v) berberine-sulfate in water for UVmicroscopy (UV filter UV-G365, Zeiss). The sections werephotographed with a digital camera (Coolpix 990, Nikon,Tokyo).

ACKNOWLEDGMENTS

We are grateful to Claudia Rudolf and Karin Lange forexcellent technical assistance.

Received April 3, 2001; returned for revision June 23, 2001;accepted July 30, 2001.

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Cadmium-Induced Changes in Antioxidative Systems, Hydrogen Peroxide Content, and Differentiation in Scots Pine Roots