Embed Size (px)
Citation preview
Environmental Pollution 145 (2007) 161e170www.elsevier.com/locate/envpol
Improved phytoaccumulation of cadmium by genetically modifiedtobacco plants (Nicotiana tabacum L.). Physiological and biochemical
response of the transformants to cadmium toxicity
N. Gorinova a,*, M. Nedkovska a, E. Todorovska a, L. Simova-Stoilova b, Z. Stoyanova b,K. Georgieva b, K. Demirevska-Kepova b, A. Atanassov a, R. Herzig c
a AgroBioInstitute, 8 Dragan Tzankov Blvd., 1164 Sofia, Bulgariab Institute of Plant Physiology, Bulgarian Academy of Sciences, 1113 Sofia, Bulgaria
c PhytotechdFoundation PT-F, Quartiergasse 12, CH 3013 Bern, Switzerland
Received 30 November 2005; received in revised form 14 February 2006; accepted 22 March 2006
Genetic transformation of Nicotiana tabacum L. by metallothionein gene improved phytoaccumulation of cadmium.
Abstract
The response of tobacco plants (Nicotiana tabacum L.)dnon-transformed and transformed with a metallothionein gene MThis from Silenevulgaris L.dto increase cadmium supply in the nutrient solution was compared. The transgenic plants accumulated significantly more Cd bothin the roots and the leaves. Visual toxicity symptoms and disturbance in water balance were correlated with Cd tissue content. Treatment with300 mM CdCl2 resulted in inhibition of photosynthesis and mobilization of the ascorbate-glutathione cycle. Treatment with 500 mM CdCl2 led toirreversible damage of photosynthesis and oxidative stress. An appearance of a new peroxidase isoform and changes in the leaf polypeptidepattern were observed at the highest Cd concentration. The level of non-protein thiols gradually increased following the Cd treatment bothin transgenic and non-transformed plants.� 2006 Elsevier Ltd. All rights reserved.
Keywords: Tobacco; Metallothionein; Genetic transformation; Cadmium phytotoxicity; Oxidative stress
1. Introduction
Heavy metal pollution of soils and waters is a very seriousenvironmental problem with potentially harmful consequences
Abbreviations: APX, ascorbate peroxidase; ASC, ascorbic acid; CAT, cat-
alase; Cd, cadmium; DW, dry weight; Fo, minimum chlorophyll fluorescence;
Fm, maximum chlorophyll fluorescence; FW, fresh weight; GPX, guaiacol per-
oxidase; LNU, proportion of excitation light not used for photochemistry; LS,
large subunit of Rubisco; MDA, malondialdehyde; MM, molecular mass; MT,
metallothionein; PSII, photosystem II; qN, non-photochemical quenching; qP,
photochemical quenching; Rfd, fluorescence decline ratio; RBP, Rubisco bind-
ing protein; ROS, reactive oxygen species; SOD, superoxide dismutase; SS,
small subunit of Rubisco.
* Corresponding author. Tel.: þ359 2 963 5409; fax: þ359 2 963 5408.
E-mail address: [email protected] (N. Gorinova).
0269-7491/$ - see front matter � 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.envpol.2006.03.025
for agriculture and human health. The modern agriculturalpractices and the industrial activities have polluted soils withcadmium, copper, lead and zinc, in areas that are favorablefor crop production in terms of climatic conditions(Nedkovska and Atanassov, 1998). Plant metal extraction isone of the effective and promising phytoremediation technol-ogies because it can be carried out in situ, thus minimizing thecosts and human exposure (McGrath et al., 2001; Pilon-Smitsand Pilon, 2002; Schnoor et al., 1995; Vassilev et al., 2002).Nevertheless, the potential application of wild-type hyperaccu-mulators, such as Thlaspi caerulescens L., in soil remediationis limited by low plant productivity (Cunningham and Ow,1996). The ideal plants for phytoextraction should possessthe ability to tolerate and accumulate high levels ofheavy metals in their harvestable parts, while producing high
162 N. Gorinova et al. / Environmental Pollution 145 (2007) 161e170
biomass. Screening has been made recently to identify cropspecies that carry these properties (Kumar et al., 1995; Laszlo,1999). Nicotiana tabacum L. could become one of the mostpromising crops for phytoextraction since it is considered tobe a Cd accumulator (Isermann et al., 1983; Davis, 1984).
Today the use of genetically engineered plants allows todouble or triple the metal accumulation capacity especiallyfor Cd and Cu by overproduction of metallochelating mole-culesdcitrate, phytochelatins, metallothioneins (MTs) andothers (Clemens et al., 2002; Nedkovska and Atanassov,1998; Pilon-Smits and Pilon, 2002). The introduction of MTgenes to improve the plant ability to tolerate heavy metalshas been demonstrated (de Borne et al., 1998; Macek et al.,2002; Pan et al., 1994; Suh et al., 1998). The Ti-plasmid me-diated genetic transformation of MT genes in plants provideda valuable method of generating metal tolerant varieties thatmay be useful for reclamation of waste lands and mine soils.Metallothioneins are different classes of low-molecular-weightcysteine-rich heavy metal chelating proteins, which take partin heavy metal detoxification and homeostasis (Cobbett andGoldsbrough, 2002; Rauser, 1999). Recently it was shownthat a stress-induced MT had additional antioxidant properties(Akashi et al., 2004). The response of transgenic plants toheavy metal accumulation is rather complex and still notwell understood.
Cadmium is one of the most toxic non-essential elementswith high mobility and is a potential target for phytoremedia-tion by transgenic plants (Macek et al., 2002). However, de-tailed analyses are necessary concerning the biochemicalresponse of transgenic plants to Cd toxicity, especially howthey tolerate the high internal Cd levels and how they copewith Cd-generated oxidative stress. Cadmium directly or indi-rectly inhibits main physiological processes, such as photo-synthesis, water relations, gas exchange and respiration, anddisturbs plant mineral nutrition (Seregin and Ivanov, 2001;Van Assche and Clijsters, 1990). The photosynthetic appara-tus appears to be especially sensitive to Cd toxicity despitethe very low Cd content (about 1% of the total leaf Cd)found in the chloroplasts (Krupa, 1999; Seregin and Ivanov,2001). The primary targets of Cd toxicity are PSII and theenzymatic phase of photosynthesis, particularly ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco; EC 4.1.1.39)(Krupa, 1999). The development of oxidative stress underCd toxicity is well documented. It varies depending on theplant species, the age, the duration and the severity of thetreatment and seems to be related to the Cd content inthe metabolically active compartments, e.g. cytoplasm(Iannelli et al., 2002; Sandalio et al., 2001; Singh and Tewari,2003; Vitoria et al., 2001; Wu et al., 2003). Reactive oxygenspecies (ROS) under Cd toxicity are generated indirectlytrough disturbances in the electron-transport chains, activa-tion of lipoxygenase and alteration in the structure or inhibi-tion of the antioxidative metalloenzymes (Sandalio et al.,2001). As ROS are formed in situ, the antioxidant protectionin plant cells is also highly compartmentalized (Mittler,2002). Superoxide dismutases (SOD; EC 1.15.1.1) catalyzethe dismutation of O2
$� (generated mainly by the electron
transport chains) to the less reactive H2O2. The regulationof the H2O2 level in chloroplasts, mitochondria, cytosol andthe apoplast is mainly achieved by the enzymes and metabo-lites of the ascorbateeglutathione cycle. H2O2 is also re-moved by catalases (CAT; EC 1.11.1.6) localized inperoxisomes, and various peroxidases localized in vacuoles,the cell walls and the cytosol (Mittler, 2002). The balance be-tween SOD and ascorbate peroxidase (APX; EC 1.11.1.11),CAT and GPX activities may be crucial for maintaining thesteady-state level of O2
$� and H2O2 and for prevention of the for-mation of the highly cytotoxic OH$. The imbalance in the anti-oxidative protection leads to accumulation of H2O2, formationof OH$ and to oxidative damage to lipids and proteins.
As to many other stresses, the cell responses to Cd are pri-marily non-specific and focus on maintaining the cell homeo-stasis. They include mobilization of the antioxidativeprotection, changes in the chemical composition of the cellwalls and switching on the synthesis of metal-binding phyto-chelatins and metallothioneins in the cytoplasm (Seregin andIvanov, 2001). We could not find detailed studies on the anti-oxidative protection in tobacco plants under cadmium toxicity.However, the biochemical understanding of plant metal accu-mulation affected by genetic manipulations could lead to newinsights into some fundamental aspects of plant physiologyand biochemistry.
The aim of the present study was to evaluate the effect ofmetallothionein gene expression on the ability of tobaccoplants to accumulate cadmium ions and its influence on theplant growth, photosynthesis and antioxidant defense system.
2. Material and methods
2.1. Plant material for transformation
Seeds of the tobacco somaclonal variety NBZn 7-51 F1, kindly provided by
Dr. Rolf Herzig (Switzerland), were used for the production of in vitro plants
for genetic transformation experiments. After surface sterilization the seeds
were germinated in medium containing mineral salts and vitamins (Murashige
and Skoog, 1962) plus 3% (w/v) sucrose and solidified with 0.7% agar. The
test tubes were incubated for 3 weeks at 24 � 1 �C. Seedlings were transferred
to glass vessels containing 20 ml of this medium and were grown under
140 mmol m�2 s�1 PAR at plant level, at 16/8 h (light/dark) photoperiod and
50e60% relative air humidity. In vitro tobacco plants were micropropagated
at regular intervals.
2.2. Genetic transformation experiments
For the genetic transformation experiments, leaves from tobacco plants
grown in vitro (4e6 leaf stage) were used. The transformation was performed
with a construct carrying the plant metallothionein gene MT from Silene vul-
garis L. (Van Hoof et al., 2001), made and kindly provided by Professor
Sirpa Karenlampi and her group (Fig. 1). The GUS-gene in the binary vector,
pCAMBIA 2301 was replaced by MThis-gene.
The Agrobacterium leaf disk transformation method was applied (Horsch
et al., 1985). It was used Agrobacterium tumefaciens strain LBA 4404. The se-
lectable marker was kanamycin.
The selection method for the transformants was the following: After co-
cultivation with A. tumefaciens LBA 4404, leaf explants were selected on MS
medium (Murashige and Skoog, 1962) supplemented with 100 mg L�1 kana-
mycin and 300 mg L�1 claforan. The transformed explants were subcultured
every 3 weeks on a fresh medium containing 100 mg L�1 kanamycin and
163N. Gorinova et al. / Environmental Pollution 145 (2007) 161e170
300 mg L�1 claforan. The obtained regenerants were transferred for rooting
on MS basal medium including vitamins and 100 mg L�1 kanamycin.
The obtained putative transformants (4e6 leaf stage) were removed from
agar media, transplanted into soil and kept at a high humidity for 2e3 weeks
under artificial light for acclimatization.
2.2.1. Molecular analysis for confirmation of the
presence of the MT gene in tobacco transformants
2.2.1.1. PCR analyses. Genomic DNA of the transformants was isolated ac-
cording to Dellaporta et al. (1983) with some modifications, including RNaseI
treatment before the second chloroform extraction. The PCR reaction was car-
ried out in a total volume of 30 ml containing 125e150 ng DNA, 0.2 mM
dNTPs, 1� PCR buffer (1.5 mM MgCl2), 1.5 U Taq polymerase (Amersham)
and 12 pmol primers. The following PCR program was applied: denaturation
at 94 �C for 4 min, 35 cycles (94 �C for 30 s, 56 �C for 30 s, 72 �C for 40 s)
and 72 �C for 5 min. About a half of each PCR product (17 ml) was electro-
phoresed on 1.5% agarose gel in TAE-buffer.
In the PCR analysis the primers C876 (F) and C880 (R) were used, pro-
vided by Professor Sirpa Karenlampi’s group. Their sequences are the follow-
ing: C876: 50-GCG GAA TTC GAT GTC GTG CTG TAA TGG AA-30; C880:
50-CGG CTC GAG CTC ATT TGC AAG TGC AAG GG-30
2.3. Cadmium treatment and sampling
Clone 35 was used for all the analyses performed. This clone showed very
good regeneration potential in comparison with the other clones. It was
multiplied by micropropagation to receive plant material sufficient for the
presented studies. Southern blot analysis showed the presence of one copy
of MThis gene in the clone (data not shown).
After acclimatization the plants were grown at 25 � 1 �C temperature,
50e60% relative humidity and 16/8 h (light/dark) photoperiod under
260 mmol m�2 s�1 PAR in nutrient solution according to Zhurbickij (1968),
supplemented with increasing concentrations of CdCl2 (0 mM, 100 mM,
300 mM and 500 mM). The medium was aerated every day and changed every
2 days. Samples were taken 5 days after the beginning of the treatment when
the final age of the plants was 55 days. All of enzyme analyses were performed
on mixed leaf samples from the fully developed fourth to sixth leaf (the middle
part of the canopy), which were stored in liquid N2 until extraction and the
other analyses were made with fresh leaf material.
Fig. 1. The vector used for genetic transformation of tobacco NBZn 7-51 F1
line.
2.4. Determination of dry weight and cadmium content
Dry weight (DW) per gram fresh weight (FW) was determined by weight
out the roots, the leaves and the stems. For determination of cadmium content
plant leaves were washed with distilled water. Plant roots were washed several
times with distilled water to remove nutrient solution absorbed to the root sur-
face. Dry plant material (0.1 g) from leaves and roots were ashed at 450 �C.
The dried residue was brought to a standard volume with 20% HCl. The cad-
mium content was determined directly by atomic absorption spectrometry (PE
400, PerkineElmer with graphite furnace).
2.5. Chlorophyll fluorescence
Chlorophyll fluorescence emission from the upper leaf surface was mea-
sured with a pulse amplitude modulation fluorometer (PAM 101-103, Heinz
Walz GmbH, Effeltrich, Germany) at room temperature as described by
Schreiber et al. (1986) after 20 min dark adaptation. The initial fluorescence
yield (Fo) in weak, modulated light [0.075 mmol m�2 s�1 photosynthetic photon
flux density (PPFD)] and maximum total fluorescence yield (Fm) emitted dur-
ing a saturating white light pulse (1 s, over 3500 mmol m�2 s�1 PPFD by
Schott KL 1500 light source, Heinz Walz GmbH) were determined from
a leaf disk (1 cm diameter). The leaf disk was then illuminated with continu-
ous red light (125 mmol m�2 s�1 PPFD). Short pulses of white light (at 20-s
intervals) on the background of a red light were used to obtain the fluorescence
intensity, Fm0, with all PSII reaction centers closed in any light-adapted state.
The induction kinetics were recorded and analyzed with the program FIP 4.3
written by Tyystjarvi and Karunen (1990). Photochemical (qP) and non-
photochemical quenching (qN) were calculating according to Van Kooten
and Snel (1990): qP ¼ (Fs � Fo0)/(Fm
0 � Fo0); qN ¼ (Fm � Fm
0)/(Fm � Fo0).
2.6. Oxygen evolution
Oxygen evolution rate was determined using a leaf disk electrode (Type
LD2/2, Hansatech, U.K.). It was measured at 800 mmol m�2 s�1 PPFD at sat-
urating CO2 concentration (provided by a carbonate/bicarbonate buffer).
2.7. SDSePAGE of soluble leaf proteins
For electrophoretic analyses the leaves samples were homogenized (1:5 w/v)
at 4 �C with ice cold 100 mM TriseHCl buffer (pH 8) containing: 20 mM
MgCl2, 10 mM NaHCO3, 1 mM EDTA, 2 mM phenylmethylsulfonyl fluoride
(PMSF), 12.5% glycerol (v/v), 20 mM b-mercaptoethanol, 3% Polyclar (w/v).
After incubation at 4 �C for 30 min, the homogenate was centrifuged at
13,000 � g for 20 min. The leaf total soluble proteins were separated by 12%
SDSePAGE (Laemmli, 1970). Equal amounts of 30 mg protein per lane were
loaded.
2.8. Enzyme assays
For enzyme analyses, 0.5 g of frozen leaf samples were ground and ex-
tracted (1:10 w/v) as previously described (Demirevska-Kepova et al.,
2004). The extracts were desalted on Sephadex-G 25 mini-columns. The activ-
ities were determined using a Shimadzu spectrophotometer. SOD activity was
measured at 560 nm based on the inhibition of the photochemical reduction of
nitroblue tetrazolium (Beauchamp and Fridovich, 1971). One unit of SOD was
defined as the quantity of enzyme required to inhibit the reduction of NBT by
50%. CAT activity was assayed following H2O2 decomposition at 240 nm
(3 ¼ 0.0394 mM�1 cm�1) according to Aebi (1984). GPX activity was assayed
according to McRae and Thompson (1983). The formation of the reaction
product tetraguaiacohinone (3 ¼ 26 mM�1 cm�1) was registered at 420 nm.
APX activity was determined according to Gonzalez et al. (1998) following
the decrease in the absorbance at 290 nm due to enzymatic oxidation of ascor-
bic acid by H2O2 (3 ¼ 2.8 mM�1 cm�1). Correction for non-enzymatic oxida-
tion of ASC was made.
164 N. Gorinova et al. / Environmental Pollution 145 (2007) 161e170
2.9. Isoenzyme staining
In gel staining methods were used after native 7.5% PAGE (for CAT and
GPX) and 10% native PAGE (for APX and SOD) at 4 �C, loading 30 mg protein
per lane. SOD activities were visualized and SOD types were differentiated ac-
cording to Gonzalez et al. (1998) by pre-stain incubation with 5 mM H2O2. CAT
isoenzymes were stained according to Woodbury et al. (1971). GPX isoenzymes
were separated and revealed according to Hart et al. (1971). APX isoenzymes
were analyzed as described by Mittler and Zilinskas (1993).
2.10. Antioxidant compounds quantification
Non-protein SH groups were determined by the Edreva and Hadjiiska
(1984). The analyses were made with fresh leaf material. After grinding with
2.5% sulfosalicilic acid the extracts were incubated in 0.4 M TriseHCl buffer,
pH 7.8, in 0.02 M EDTA and Ellman’s reagent (5,5 dithiobis-2-nitrobenzoic
acid). The absorbance of the reaction product (2-nitro-5-benzoic acid) was reg-
istered at 412 nm using 3 ¼ 13,600 M�1 cm�1. The ascorbate pool (reduced-
ASC and total ASC) was assayed according to the protocol of Hodges et al.
(1996) on the basis of the reduction of Fe3þ to Fe2þ by ascorbate and complex-
ation of Fe2þwith a,a0-dipyridyl, resulting in a pink color. The ASC content was
quantified using a standard curve.
2.11. Other assays
Lipid peroxidation was estimated spectrophotometrically according to the
improved thiobarbituric acid reactive substances assay (Hodges et al., 1999).
Protein carbonylation was assayed at 366 nm according to Reznick and Packer
(1994) using 2,4-dinitrophenylhydrazine. The carbonyl content was calculated
from 3 ¼ 22,000 M�1 cm�1. Corrections for protein loss during washings
were made. Hydrogen peroxidewas measured spectrophotometrically after reac-
tion with KI as previously described (Demirevska-Kepova et al., 2004). The
amount of H2O2 was calculated using a standard curve. Leaf pigments were ex-
tracted with 80% acetone and estimated according to Arnon (1949). Total soluble
protein content was determined by the method of Bradford (1976) using bovine
serum albumin as a standard. Rubisco quantity was determined in leaf tissues by
ELISA as previously described (Metodiev and Demirevska-Kepova, 1992).
2.12. Statistical analysis
The results were based on three replicates from two independent experi-
ments at least. The data were analyzed by one-way ANOVA inserted in the
graphic program Origin. Asterisks were used to identify the levels of signifi-
cance in the differences between non-modified and transgenic plants at each
Cd treatment on the figures: *p < 0.05, **p < 0.01 and ***p < 0.001. Statis-
tics was also performed to compare the Cd treatment to controls and to discuss
the obtained results. These data are not shown on the figures.
3. Results
3.1. Confirmation of the transgenicity of thetransformants
The results from molecular analysis of the transformantsshowed a PCR-positive signal for most tested transformants(Fig. 2). Integration of the transgene is shown in Fig. 2. Oneclone, N 35, which showed a positive PCR result, was chosenfor further studies.
3.2. Cd accumulation and toxicity symptoms
Cadmium concentrations in the roots and leaves of tobaccoplants after short-term exposure of 5 days of Cd are presented
in Fig. 3. The transgenic plants revealed a better ability for bothCd uptake and accumulation in roots and leaves (1.5e2 timesmore Cd) compared to the non-transformed tobacco plants.This improved metal uptake characteristic was pronouncedfor relatively high Cd exposure of 300e500 mM CdCl2.
Whereas in transformants the concentrations of metals inthe roots reached an upper threshold when treated with300 mM CdCl2, the accumulation in the leaves was increasingwhen treated with 500 mM CdCl2. In non-transformed plantsthe roots absorbed most Cd at 500 mM CdCl2, while in theleaves the accumulation of Cd reached a plateau at 300 mMCdCl2. At 300 and 500 mM CdCl2 visual symptoms of Cd tox-icity were observed in both non-transformed and transgenic to-bacco plants (root browning, leaf spot chlorosis, wilting andtip necrosis in older leaves). The transgenic leaves weremore affected at the highest Cd concentration, which couldbe explained with the higher internal Cd content as a resultof enhanced uptake.
3.3. Growth parameters and leaf pigment contents
The root and shoot length did not change after treatment.The data of DW per g FW in tobacco leaves, stems and roots
300 bp200 bp 257 bp
1 2 3 4 5 6 7 8 9 10 11 12
Fig. 2. PCR analysis of putative MThis transformants of tobacco NBZn 7-51
line with specific primers C876 (F) and C880 (R) corresponding to Silene vul-
garis metallothionein cDNA (237 bp) and a part of the body of binary vector
pCambia 2301 (20 bp). The total length of the amplified fragment is 257 bp.
Lanes: 1, 100 bp marker; 2, NBZn 7-51 control; 3, clone 13; 4, clone 31; 5,
clone 35; 6, clone 37; 7, clone 52; 8, clone 61; 9, clone 77; 10, clone 80;
11, clone 88; 12, plasmid MThis/pCambia 2301.
Fig. 3. Cd accumulation in leaves (left) and roots (right) of transgenic (open
symbols) and non-transformed (closed symbols) tobacco plants. On the ab-
scissa: Cd concentration in the nutrient solution. Means and standard devia-
tions of at least three replicates are shown. Significant differences between
transgenic and non-transformed plants in each treatment at the p < 0.05 (*),
p < 0.01 (**) and p < 0.001 (***) level are indicated.
165N. Gorinova et al. / Environmental Pollution 145 (2007) 161e170
as well as leaf pigment content are presented in Fig. 4. The risein DW per g FW in leaf and stem reflected a disturbance of theplant water balance after the Cd treatment. The transgenicplants treated with 500 mM CdCl2 had more wilted leavescompared to the non-transgenic ones. The chlorophyll a andb concentrations diminished with increasing Cd concentrationswhile the level of carotenoids did not change significantly. Inboth the transgenic and non-transformed plants the develop-ment of toxicity symptoms at 300 mM CdCl2 was not accom-panied by a change in the chlorophyll a/b ratio, which was2.7e2.8. At the 500 mM CdCl2-treatment this ratio droppedto 2.2 both in the transgenic and the non-transformed plants.
3.4. Chlorophyll fluorescence
The primary photochemical activity of PSII, estimated bythe ratio of Fv/Fm started to decrease as a result of the300 mM CdCl2-treatment and it was significantly reduced at500 mM CdCl2 (Fig. 5).
The rate of whole chain electron transport [VPSII ¼(Fm
0 � Fs)/Fm0] and the efficiency of excitation capture by
open PSII reaction centers (Fv0/Fm
0) decreased with increasingCd-concentrations, even more than the Fv/Fm values. Thevalue of fluorescence decline ratio Rfd [(Fm � Fs)/Fs] wasbelow 1 after treatment with 500 mM CdCl2, indicating severedamage of the photosynthetic apparatus of tobacco plants(Fig. 5). The photochemical quenching (qP) decreased with
Fig. 5. Effect of Cd treatment on the maximum quantum efficiency of PSII
(Fv/Fm), the actual quantum yield of PSII electron transport in the light-
adapted state (FPSII), the efficiency of excitation energy capture by ‘‘open’’
PSII reaction centers (Fv0/Fm
0) and the fluorescence decreased ratio (Rfd) in
non-transformed (white columns) and transgenic tobacco plants (dark col-
umns). Means and standard deviations of at least three replicates are shown.
Fig. 4. DW per g FW accumulation and leaf pigments in non-transformed (left) and transgenic (right) tobacco plants treated with increasing concentrations of Cd in
the nutrient solution. Data of DW per g FW are presented in leaves (white columns), stems (striped columns) and roots (squared columns). Data of pigments are
presented as follows: chlorophyll a (gray columns), chlorophyll b (horizontally striped) and carotenoids (striped columns). Means and standard deviations of at
least three replicates are shown. Significant differences between transgenic and non-transformed tobacco plants in each treatment at the p < 0.05 (*), p < 0.01 (**)
and p < 0.001 (***) level are indicated.
166 N. Gorinova et al. / Environmental Pollution 145 (2007) 161e170
increasing Cd concentrations while non-photochemicalquenching (qN) and the proportion of excitation light notused for photochemistry (LNU) increased (Fig. 6). We foundthat the Cd treatment reduced the rate of photosyntheticoxygen evolution more than the mechanisms of PSII photo-chemistry. The comparison of the results obtained for trans-genic and non-transformed tobacco plants clearly showedthat Cd stress affected them in a similar way.
3.5. Leaf total soluble protein and Rubisco quantity
The content of leaf total soluble protein on FW basis didnot change significantly in the transgenic and non-transformedplants (Fig. 7). The Rubisco quantity was not affected by thetransformation and was stable in the transformed plants treatedwith 100 and 300 mM CdCl2, but under the highest Cd toxicity(500 mM CdCl2) it diminished to 49.75% and to 89.27% fortransgenic and non-transformed plants, respectively, whencompared with control plants (0 mM CdCl2) (Fig. 7).
3.6. Changes in leaf protein pattern
Considerable changes in the leaf polypeptide pattern afterSDS electrophoresis were detected only in the variants treatedwith 500 mM CdCl2. The changes were more expressed inthe transgenic plants (Fig. 8). The highest Cd concentrationprovoked Rubisco LS and SS diminution along with theenhancement of some bands with MM between 20 and43 kDa (approximately 23 and 34 kDa, respectively). Theintensity of the band at the position corresponding with RBPdiminished substantially.
Fig. 6. Effect of Cd treatment on the photochemical quenching (qP), non-pho-
tochemical quenching (qN), the fraction of the light energy which was not
used for photochemistry (LNU) and the oxygen evolution rate) in non-trans-
formed (white columns) and transgenic tobacco plants (dark columns). Means
and standard deviations of at least three replicates are shown.
3.7. Response of antioxidant enzymes to Cdaccumulation
The isoenzyme activities and profiles of some main en-zymes involved in the antioxidative protection (SOD, APX,GPX, CAT) were analyzed in the leaves of Cd-treated tobaccoplants. The total SOD activity gradually enhanced with theincrease of Cd concentrations (Fig. 9).
After electrophoretic separation and activity staining,(Fig. 10), five bands of SOD isoforms according our compar-ative analyses (data are not shown) were revealed: oneMnSOD (mitochondrial), one FeSOD (chloroplastic), and
Fig. 7. Effect of Cd concentration on the level of the leaf total soluble protein
and Rubisco quantity in non-transformed (white columns) and transgenic (dark
columns) tobacco plants. Means and standard deviations of at least 3 replicates
are shown. Significant differences between transgenic and non-transformed to-
bacco plants in each treatment at the p < 0.05 (*), p < 0.01 (**) and p < 0.001
(***) level are indicated.
94 kDa67 kDa----RBP--------------RLS
43 kDa
30 kDa
20 kDa
14 kDa-----RSS
1 2 3 4 5 6 7 8 M
Fig. 8. Effect of Cd on the pattern of soluble proteins in non-transformed (1e4)
and transgenic (5e8) tobacco plants. Extracts were analyzed by SDSePAGE
(12%). Treatments: 1, 5, control (without Cd); 2, 6, 100 mM CdCl2; 3, 7,
300 mM CdCl2; 4, 8, 500 mM CdCl2. The polypeptides were visualized by
Coomassie blue staining. Each lane was loaded with 50 ml leaf extract. The
kDa values in the figure indicate the position of molecular mass standards.
167N. Gorinova et al. / Environmental Pollution 145 (2007) 161e170
three Cu/ZnSOD isoformsdone chloroplastic (Cu/ZnSOD II)and two cytosolic (Cu/ZnSOD I and Cu/ZnSOD III). Cu/ZnSOD I and II were the major isoforms.
The treatment with 500 mM CdCl2 raised the activities ofCu/ZnSODs. Typically, the Cu/ZnSOD from transgenic plantsreacted more intensively to the metal toxicity. The MnSODand FeSOD isoforms were not apparently changed followingthe Cd treatment. The total APX activity enhanced following300 mM and 500 mM CdCl2 treatments without visible changesin the isoenzyme pattern (APX 1, APX 2 and APX 3). Com-pared with non-transformed plants the control transgenicplants had lower APX activity, however, the increase of theactivity was higher and the difference became insignificantwhen treated with 300 and 500 mM CdCl2, respectively. Inthe non-treated plants GPX activity was higher in transgenicplants and tended to decrease following 100 and 300 mM
Fig. 9. Effects of Cd concentration on the activities of superoxide dismutase
(SOD), catalase (CAT), guaiacol peroxidase (GPX) and ascorbate peroxidase
(APX) in non-transformed (white columns) and transgenic (dark columns) to-
bacco plants. Units were defined as: the quantity of enzyme required to inhibit
the reduction of NBT by 50% per 1 min for SOD, nmol H2O2 decomposed per
1 min for CAT, mmol tetraguaiacohinone produced per min for GPX, nmol
ascorbate degraded per min for APX. Means and standard deviations of at least
three replicates are shown. Significant differences between transgenic and non-
transformed tobacco plants in each treatment at the p < 0.05 (*), p < 0.01 (**)
and p < 0.001 (***) level are indicated.
CdCl2 treatment. Contrary to this, GPX activity sharply in-creased (7 to 11 times) at the 500 mM CdCl2 treatment andwas higher in transgenic plants when compared with thenon-transgenic ones. The appearance of a new GPX isoformwas revealed at 500 mM CdCl2 treatment both in the non-modified and in the transgenic plants (GPX X). The totalCAT activity diminished after treatment with 300 mM CdCl2.The 500 mM CdCl2 treatment caused some increase in CAT ac-tivity without, however, reaching the level of the untreatedplants. Isoenzyme staining revealed one dominating peroxi-somal isoform of CAT in leaves. At the 300 mM and 500 mMCdCl2 treatments its electrophoretic mobility was changed.
3.8. Low molecular compounds influenced by Cd toxicity
The low-molecular-weight antioxidative protectors werealso mobilized as a result of tobacco plants treatment withCd (Fig. 11). The leaf ascorbate pool strongly increased atthe 300 mM and 500 mM CdCl2 treatments without significantchanges in the percentage of reduced ASC. The non-proteinthiol content (basically glutathione, phytochelatins andother SH compounds) was significantly higher in the trans-genic plants and rose even at 100 mM CdCl2 treatment butreached a plateau at 300e500 mM CdCl2. The leaves of the
MnSOD
Cu/Zn SOD I
FeSOD
Cu/Zn SOD II
APX 1
APX 2
APX 3
GPX1
GPX 2
GPX 3
CAT
GPX X
1 2 3 4 5 6 7 8
1 2 3 4 5 6 7 8
Fig. 10. Effects of Cd concentration on isoenzyme patterns of superoxide
dismutase (SOD), ascorbate peroxidase (APX), guaiacol peroxidase (GPX)
and catalase (CAT) in non-transformed (1e4) and transgenic (5e8) tobacco
plants. Leaf extracts were analyzed by native PAGE (10% gels for SOD and
APX isoenzymes and 7.5% gels for CAT and GPX isoenzymes). The lanes
for the activity staining were loaded with 50 ml leaf extract of the following
samples: 1, 5, control (without Cd); 2, 6, 100 mM CdCl2; 3, 300 mM CdCl2;
4, 500 mM CdCl2.
168 N. Gorinova et al. / Environmental Pollution 145 (2007) 161e170
Fig. 11. Contents of H2O2, malondialdehyde, carbonyl groups in proteins, total ascorbate, reduced ASC and non-protein thiol groups in leaf extracts of non-trans-
formed (white columns) and transgenic (dark columns) tobacco plants, grown on elevated Cd concentrations. Means and standard deviations of at least three rep-
licates are shown. Significant differences between transgenic and nontransformed tobacco plants in each treatment at the p < 0.05 (*), p < 0.01 (**) and p < 0.001
(***) level are indicated.
non-transformed plants contained two times less non-proteinthiols in comparison to the transgenic ones. Nevertheless,the content of non-protein thiols also rose sharply to valuescomparable to those in transgenic plants at the highest Cdtreatment.
3.9. Hydrogen peroxide level and oxidativedamage of lipids and proteins
The accumulation of H2O2 and the oxidative damage inlipids and proteins as indices for the development of oxidativestress under Cd toxicity are presented in Fig. 11. The leafH2O2 level was slightly lower in the control non-transformedplants compared to the modified ones. However, in both casesthe tendency was the same: a small rise at 100 mM CdCl2,a drop to the level of the untreated plants at 300 mM CdCl2and an abrupt approx. 4-fold increase at 500 mM CdCl2. Themalondialdehyde (MDA) content as a marker of oxidativedamage of lipids increased following treatment with 300 and500 mM CdCl2, whereas the level of protein carbonyl groupsas a marker for oxidative damage of proteins did not changesignificantly.
4. Discussion
The new somaclonal tobacco variety NBZn 7-51 F1, ob-tained from in vitro breeding, is characterized with enhancedyield, metal tolerance, Zn and Cd extraction and enhanced re-sistance against Cu stress and a linear accumulation of Cd asa function of the external metal concentration (Guadagnini,
2000; Herzig et al., submitted for publication). The genetictransformation with the metallothionein gene MTs fromSilene vulgaris L. aimed at a further increase of the metal up-take capacity of this variety, as tested on short term experi-ments on hydroponics spiked with relatively highconcentration of Cd.
A very important result of the genetic transformation ofNBZn 7-51 F1 tobacco plants with plant MT gene was the es-sentially improved accumulation of Cd in the leaves and roots,without affecting the Cd distribution between roots and shootsin comparison to the non-transformed plants of the samesomaclonal variety. It could be expected that both enhancedtolerance and accumulation of Cd were related to the overpro-duction of metallothioneins (MTs). Similar changes in thephotosynthetic apparatus, leaf proteins and antioxidative de-fense system were observed in transgenic and non-transformedplants under Cd toxicity. The toxicity symptoms were rathercorrelated with the Cd concentration in the leaves. The poolof non-protein thiols gradually increased with increasing Cduptake. This result most probably reflected the inductionof phytochelatin synthesis as a general mechanism of Cddetoxification in plants (Cobbett and Goldsbrough, 2002;Rauser, 1999).
At 100 mM CdCl2 treatment the studied parameters did notchange, except for the slight increase in the H2O2 level, whichwas most probably a result of the enhanced SOD and dimin-ished GPX activities and was consistent with the signalingrole of H2O2 (Neill et al., 2002). No inhibition of the photo-synthesis and no signs of oxidative stress were observed.Both the transgenic and the non-transformed tobacco plants
169N. Gorinova et al. / Environmental Pollution 145 (2007) 161e170
adapted efficiently to this Cd supply. Nevertheless, the trans-genic plants accumulated a two times higher amount of Cdthan the non-transformed ones both in roots and shoots.
At elevated stress of 300 mM CdCl2 resulted in inhibitionof photosynthesis, which was evidenced by the decreasedPSII efficiency, the reduced rate of photosynthetic oxygenevolution, the diminished chlorophyll content, Rubisco quan-tity and CAT activity. The decrease in the CAT activity wasprobably connected with photosynthetic inhibition, as theH2O2 level was comparable to that of the controls withoutany Cd treatment. The increased SOD and APX activitiesand the enlarged ASC pool without changes in the level ofreduced ASC indicated a mobilization of the ascorbate-glutathione cycle at this Cd treatment. A similar protectiverole of the ascorbate-glutathione cycle against Cd-inducedoxidative stress has been observed in radish, barley andArabidopsis thaliana (Skorzynska-Polit et al., 2003/4; Vitoriaet al., 2001; Wu et al., 2003) but not in other species like pea(Sandalio et al., 2001). The inhibition of photosynthesis andthe mobilization of the antioxidative protection could be re-lated to the appearance of visual toxicity symptoms in Cd-treated plants.
At a more severe Cd stress (500 mM CdCl2) an irreversibledamage of the photosynthesis (vitality index below 1) could beobserved, as well as a strong inhibition of the quantum yield ofPSII, a change in the chlorophyll a/b ratio, a sharp rise in GPXactivity and H2O2 accumulation. Hence, the highest Cd con-centration led to the development of oxidative stress in thetransgenic as well as in the non-transformed tobacco plants.The peroxidase induction is considered as a general responseof plants to heavy metal toxicity and has been correlated tothe tissue metal level (Van Assche and Clijsters, 1990). Thehigh peroxidase activity induction and the appearance ofa new GPX isoform were related to the marked leaf tissuedamage at the highest Cd concentration. The revealing ofnew isoforms of GPX has been reported in some species underCd toxicity and it is probably linked to the protection fromoxidative stress (Milone et al., 2003; Tahlil et al., 1999). Wesuggest that the accumulation of the O2P2 was in connectionwith the sharp rise of GPX activity and occurred in the cytosol.The increase in the MDA content indicated that 300 and500 mM CdCl2 damaged to the membrane structures. Both therise of MDA, as an index of lipid peroxidation, and of proteincarbonyl groups, as a result of damage by hydroxyl radicals,under Cd toxicity have been well documented for some plantspecies (Sandalio et al., 2001; Wu et al., 2003). We did notnote an increased carbonylation of proteins under Cd toxicityin tobacco. However, we observed a marked change in theleaf polypeptide pattern, particularly a diminution of some pro-tein bands. It can be supposed that either the formation of highlytoxic OH$ is quenched efficiently, or the carbonylated proteinsare quickly degraded and not stored. This presumes a conservedcapacity for proteolytic degradation under Cd toxicity. Changesin the polypeptide pattern following heavy metal treatment ofrice leaf discs have been observed by Hajduch et al. (2001).
In conclusion, the cadmium content in the transgenic plantsexpressing MT gene is higher than in the non-transformed
plants. The established similar impact of the toxic Cd concen-tration on photosynthesis, leaf proteins and the anti-oxidativeprotection in the leaves of both the non-transformed and thetransgenic plants in spite of the higher cadmium concentrationin the transgenic plants. Such transgenic plants are not moresensitive then non-transformed ones and after positively pass-ing pot experiments on metal contaminated soil could beused in phytoextraction of Cd for elevated levels of Cdcontamination.
Acknowledgements
This work was supported by a grant from PHYTAC Project,Contract No: QLK3-CT-2001-00429 in 5FP. The authors aregrateful to B. Juperlieva-Mateeva, A. Kostadinova and Z. Kru-mova for their excellent technical assistance, and to ErikaNehnevajova and Sara Bangerter from Switzerland for theircorrections in English.
References
Aebi, H., 1984. Catalase in vitro. In: Willam, B.J. (Ed.), Methods in Enzymol-
ogy. Academic Press, New York, pp. 121e126.
Akashi, K., Nishimura, N., Ishida, Y., Yokota, A., 2004. Potent hydroxyl rad-
ical-scavenging activity of drought-induced type-2 metallothionein in wild
watermelon. Biochemical and Biophysical Research Communications 323,
72e78.
Arnon, D.I., 1949. Copper enzymes in isolated chloroplasts: polyphenoloxi-
dase in Beta vulgaris. Plant Physiology 24, 1e15.
Beauchamp, Ch, Fridovich, Y., 1971. Superoxide dismutase: improved assays
and an assay applicable to acrylamide gels. Analytical Biochemistry 44,
276e287.
Bradford, M.M., 1976. A rapid and sensitive method for the quantification of
microgram quantities of proteins utilizing the principle of protein-dye
binding. Analytical Biochemistry 72, 248e254.
de Borne, F.D., Elmayan, T., de Roton, C., Hys, L., Tepfer, M., 1998. Cad-
mium partitioning in transgenic tobacco plants expressing a mammalian
metallothionein gene. Molecular Breeding 4, 83e90.
Clemens, S., Palmgren, M.G., Kramer, U., 2002. A long way ahead: under-
standing and engineering plant metal accumulation. Trends in Plant Sci-
ence 7, 309e314.
Cobbett, Ch., Goldsbrough, P., 2002. Phytochelatins and metallothioneins: role
in heavy metal detoxification and homeostasis. Annual Review of Plant
Biology 53, 159e182.
Cunningham, S., Ow, D.W., 1996. Promises and prospects of phytoremedia-
tion. Plant Physiology 110, 715e719.
Davis, R.D., 1984. Cadmium-complex environmental problem. Experentia 40,
117e126.
Dellaporta, S., Wood, J., Hicks, J., 1983. A plant DNA minipreparation: ver-
sion II. Plant Molecular Biology Reporter 1, 19e22.
Demirevska-Kepova, K., Simova-Stoilova, L., Stoyanova, Z., Holzer, R.,
Feller, U., 2004. Biochemical changes in barley plants after excessive sup-
ply of copper and manganese. Environmental Experimental Botany 52,
253e266.
Edreva, A., Hadjiiska, E., 1984. About the determination of sulfhydryl (thiol)
group content in plant material (in Bulgarian). Bulgarian Journal of Plant
Physiology 10, 73e83.
Gonzalez, A., Steffen, K.L., Lynch, J.P., 1998. Light and excess manganese.
Implications for oxidative stress in common bean. Plant Physiology 118,
493e504.
Guadagnini, M., 2000. In-vitro breeding for metal-accumulation in two
tobacco (Niccotiana tabacum) cultivars, in: Inaugural dissertation N
1288, 1e100.
170 N. Gorinova et al. / Environmental Pollution 145 (2007) 161e170
Hajduch, M., Rakwall, R., Agraval, G.K., Yonekura, M., Pretova, A., 2001.
High-resolution two-dimensional electrophoresis separation of proteins
from metal-stressed rice (Oryza sativa L.) leaves: drastic reductions/frag-
mentation of ribulose-1,5-bisphosphate carboxylase/oxygenase and induc-
tion of stress-related proteins. Electrophoresis 22, 2824e2831.
Hart, M.A., Tyson, H., Bloomberg, B., 1971. Measurement of activity of per-
oxidase isoenzymes in flax. Canadian Journal of Botany 49, 2129e2137.
Herzig, R., Rehnert, A., Guadagnini, M., Erismann, K.H. Comparative screen-
ing and assessment of the phytoextraction efficiency of biotechnically im-
proved tobacco clones using a spiked soil with elevated concentrations of
plant available metals (Cd, Zn and Cu)dPart 1. Journal of Soil and Sed-
iments, submitted for publication.
Hodges, D.M., Andrews, Ch.J., Johnson, D.A., Hamilton, R.I., 1996. Antiox-
idant compound responses to chilling stress in differentially sensitive
inbred maize lines. Physiologia Plantarum 98, 685e692.
Hodges, D.M., de Long, J.M., Forney, Ch.F., Prange, R.K., 1999. Improving
the thiobarbituric acid-reactive-substances assay for estimating lipid per-
oxidation in plant tissues containing anthocyanin and other interfering
compounds. Planta 207, 604e611.
Horsch, R.B., Fry, J.E., Hoffman, N.L., Eichholtz, D., Rogers, S.G.,
Fraley, R.T., 1985. A simple and general methods for transferring genes
into plants. Science 227, 1229e1231.
Iannelli, M.A., Pietrini, F., Fiore, L., Petrilli, L., Massacci, A., 2002. Antiox-
idant response to cadmium in Phragmites australis plants. Plant Physiol-
ogy and Biochemistry 40, 977e982.
Isermann, K., Karch, P., Schmidt, J.A., 1983. Cadmium-gehalte des erntegutes
verschiedener sorten mehrer kulturpflanzen bei Anbau auf stark mit cad-
mium belastetem, neutralem Lehmboden. Landwirtschung und Forschung
36, 283e294.
Krupa, Z., 1999. Cadmium against higher plant photosynthesisda variety of
effects and where do they possibly come from? Zeitschrift Naturforschung
54c, 723e729.
Kumar, N., Dushenkov, V., Motto, H., Raskin, I., 1995. Phytoextraction: the
use of plants to remove heavy metals from soils. Environmental Science
and Technology 9, 1232e1238.
Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of
the head of bacteriophage T4. Nature 227, 680e685.
Laszlo, S., 1999. Heavy metal phytoextraction capacity of several agricultural
crop plant species. Proceedings of the 5th International Conference on Bio-
geochemistry of trace elements, Vienna.
Macek, T., Mackova, M., Pavlikova, D., Szakova, J., Truksa, M., Singh
Cundy, A., Kotrba, P., Yancey, N., Scouten, W.H., 2002. Accumulation
of cadmium by transgenic tobacco. Acta Biotechnologica 22, 101e106.
McGrath, S.P., Zhao, F.J., Lombi, E., 2001. Plant and rhizosphere processes
involved in phytoremediation of metal-contaminated soils. Plant and Soil
232, 207e214.
McRae, D.G., Thompson, J.E., 1983. Senescence-dependent changes in super-
oxide anion production by illuminated chloroplasts from bean leaves.
Planta 158, 185e193.
Metodiev, M., Demirevska-Kepova, K., 1992. Rubisco quantitation in different
barley varieties by enzyme-linked immunosorbent assay. Journal of Exper-
imental Botany 43, 155e158.
Milone, M.T., Sgherri, C., Clijsters, H., Navai-Izzo, F., 2003. Antioxidative re-
sponses of wheat treated with realistic concentration of cadmium. Environ-
mental and Experimental Botany 50, 265e276.
Mittler, R., 2002. Oxidative stress, antioxidants and stress tolerance. Trends in
Plant Science 7, 405e410.
Mittler, R., Zilinskas, B., 1993. Detection of ascorbate peroxidase activity in
native gels by inhibition of the ascorbate-dependent reduction of nitroblue
tetrazolium. Analytical. Biochemistry 212, 540e546.
Murashige, T., Skoog, F., 1962. A revised medium for rapid growth and bio-
assays with tobacco tissue culture. Physiologia Plantarum 15, 473e497.
Nedkovska, M., Atanassov, A.I., 1998. Metallothionein genes and expression
for heavy metal resistance. Biotechnology and Biotechnological Equip-
ment 12, 11e16.
Neill, S.J., Desikan, R., Clarke, A., Hurst, R.D., Hancock, J.T., 2002. Hydro-
gen peroxide and nitric oxide as signalling molecules in plants. Journal of
Experimental Botany 53, 1237e1247.
Pan, A.H., Yang, M.Z., Tie, F., Li, L.G., Chen, Z.L., Ru, B., 1994. Expression
of mouse metallothionein-I gene confers cadmium resistance in transgenic
tobacco plants. Plant Molecular Biology 24, 341e351.
Pilon-Smits, E., Pilon, M., 2002. Phytoremediation of metals using transgenic
plants. Critical Review in Plant Sciences 21, 439e456.
Rauser, W.E., 1999. Structure and function of metal chelators produced by
plants. Cell Biochemistry and Biophysics 31, 19e48.
Reznick, A.Z., Packer, L., 1994. Oxidative damage to proteins: spectrophoto-
metric method for carbonyl assay. In: Packer, L. (Ed.), Methods in Enzy-
mology, 233. Academic Press, San Diego, CA, pp. 357e363.
Sandalio, L.M., Dalurco, H.C., Gomez, M., Romero-Puertas, M.C., del
Rio, L.A., 2001. Journal of Experimental Botany 52, 2115e2126.
Schnoor, J.L., Light, L.A., McCutchbon, S.C., Woles, N.L., Carreira, L.H.,
1995. Phytoremediation of organic and nutrient contaminants. Environ-
mental Science and Technology 29, A318eA323.
Schreiber, U., Schliwa, U., Bilger, W., 1986. Continuous recording of photo-
chemical and non-photochemical chlorophyll fluorescence quenching
with a new type of modulation fluorometer. Photosynthesis Research 10,
51e62.
Seregin, I.V., Ivanov, V.B., 2001. Physiological aspects of cadmium and lead
toxic effects on higher plants. Russian Journal of Plant Physiology 48,
523e544.
Singh, P.K., Tewari, R.K., 2003. Cadmium toxicity induced changes in plant
water relations and oxidative metabolism of Brassica juncea L. plants.
Journal of Environmental Biology 24, 107e112.
Skorzynska-Polit, E., Drazkiewicz, M., Krupa, Z., 2003/4. The activity of the
antioxidative system in cadmium-treated Arabidopsis thaliana. Biologia
Plantarum 47, 71e78.
Suh, M.C., Choi, D., Liu, J.R., 1998. Cadmium resistance in transgenic
tobacco plants expressing the Nicotiana glutinosa L. metallothionein-like
gene. Molecules and Cells 8, 678e684.
Tahlil, N., Rada, A., Baaziz, M., Morel, J.L., El-Meray, M., El-Aatmani, M.,
1999. Quantitative and qualitative changes in peroxidase of Cucurbita
pepo cultivars stressed with heavy metals. Biologia Plantarum 42,
85e89.
Tyystjarvi, E., Karunen, J., 1990. A microcomputer program and fast analog to
digital converter card for the analysis of fluorescence induction transients.
Photosynthetic Research 26, 127e132.
Van Assche, F., Clijsters, C., 1990. Effects of metals on enzyme activity in
plants. Plant Cell and Environment 13, 195e206.
Van Hoof, N.A.L.M., Hassinen, V.H., Hakvoort, H.W.J., Ballintijn, K.F.,
Schat, H., Verkleij, J.A.C., Ernst, W.H.O., Karenlampi, S.O.,
Tervahauta, A.I., 2001. Enhanced copper tolerance in Silene vulgaris
(Moench) garcke populations from copper mines is associated with in-
creased transcript levels of a 2b-type metallothionein gene. Plant Physiology
126, 1519e1526.
Van Kooten, O., Snel, J., 1990. The use of chlorophyll fluorescence nomen-
clature in plant stress physiology. Photosynthesis Research 25,
147e150.
Vassilev, A., Vangronsveld, J., Yordanov, I., 2002. Cadmium phytoextraction:
present state, biological backgrounds and research needs. Bulgarian Jour-
nal of Plant Physiology 28, 68e95.
Vitoria, A.P., Lea, P.J., Azevedo, R.A., 2001. Antioxidant response to cad-
mium in radish tissues. Phytochemistry 57, 701e710.
Woodbury, W., Spencer, A.K., Stahmann, M.A., 1971. An improved procedure
using ferricyanide for detecting catalase isozymes. Analytical Biochemis-
try 44, 301e305.
Wu, F., Zhang, G., Dominy, P., 2003. Four barley genotypes respond differ-
ently to cadmium: lipid peroxidation and activities of antioxidant capacity.
Environmental and Experimental Botany 50, 67e78.
Zhurbickij, Z.I., 1968. Theory and Practice of the Vegetative Method(in Rus-
sian). Nauka, Moskow.
