Environmental Pollution 145 (2007) 161e170

www.elsevier.com/locate/envpol

Improved phytoaccumulation of cadmium by genetically modified

tobacco 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, Bulgaria
b
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 Silene
vulgaris L.dto increase cadmium supply in the nutrient solution was compared. The transgenic plants accumulated significantly more Cd both
in the roots and the leaves. Visual toxicity symptoms and disturbance in water balance were correlated with Cd tissue content. Treatment with
300 mM CdCl2 resulted in inhibition of photosynthesis and mobilization of the ascorbate-glutathione cycle. Treatment with 500 mM CdCl2 led to
irreversible damage of photosynthesis and oxidative stress. An appearance of a new peroxidase isoform and changes in the leaf polypeptide
pattern were observed at the highest Cd concentration. The level of non-protein thiols gradually increased following the Cd treatment both
in transgenic and non-transformed plants.
Ó 2006 Elsevier Ltd. All rights reserved.

Keywords: Tobacco; Metallothionein; Genetic transformation; Cadmium phytotoxicity; Oxidative stress

1. Introduction for agriculture and human health. The modern agricultural

practices and the industrial activities have polluted soils with
Heavy metal pollution of soils and waters is a very serious cadmium, copper, lead and zinc, in areas that are favorable
environmental problem with potentially harmful consequences for crop production in terms of climatic conditions
(Nedkovska and Atanassov, 1998). Plant metal extraction is
one of the effective and promising phytoremediation technol-
Abbreviations: APX, ascorbate peroxidase; ASC, ascorbic acid; CAT, cat- ogies because it can be carried out in situ, thus minimizing the
alase; Cd, cadmium; DW, dry weight; Fo, minimum chlorophyll fluorescence;
Fm, maximum chlorophyll fluorescence; FW, fresh weight; GPX, guaiacol per-
costs and human exposure (McGrath et al., 2001; Pilon-Smits
oxidase; LNU, proportion of excitation light not used for photochemistry; LS, and Pilon, 2002; Schnoor et al., 1995; Vassilev et al., 2002).
large subunit of Rubisco; MDA, malondialdehyde; MM, molecular mass; MT, Nevertheless, the potential application of wild-type hyperaccu-
metallothionein; PSII, photosystem II; qN, non-photochemical quenching; qP, mulators, such as Thlaspi caerulescens L., in soil remediation
photochemical quenching; Rfd, fluorescence decline ratio; RBP, Rubisco bind- is limited by low plant productivity (Cunningham and Ow,
ing protein; ROS, reactive oxygen species; SOD, superoxide dismutase; SS,
small subunit of Rubisco.
1996). The ideal plants for phytoextraction should possess
* Corresponding author. Tel.: þ359 2 963 5409; fax: þ359 2 963 5408. the ability to tolerate and accumulate high levels of
E-mail address: noraig60@yahoo.co.uk (N. Gorinova). heavy metals in their harvestable parts, while producing high

0269-7491/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.envpol.2006.03.025
162 N. Gorinova et al. / Environmental Pollution 145 (2007) 161e170

biomass. Screening has been made recently to identify crop transport chains) to the less reactive H2O2. The regulation
species that carry these properties (Kumar et al., 1995; Laszlo, of the H2O2 level in chloroplasts, mitochondria, cytosol and
1999). Nicotiana tabacum L. could become one of the most the apoplast is mainly achieved by the enzymes and metabo-
promising crops for phytoextraction since it is considered to lites of the ascorbateeglutathione cycle. H2O2 is also re-
be a Cd accumulator (Isermann et al., 1983; Davis, 1984). moved by catalases (CAT; EC 1.11.1.6) localized in
Today the use of genetically engineered plants allows to peroxisomes, and various peroxidases localized in vacuoles,
double or triple the metal accumulation capacity especially the cell walls and the cytosol (Mittler, 2002). The balance be-
for Cd and Cu by overproduction of metallochelating mole- tween SOD and ascorbate peroxidase (APX; EC 1.11.1.11),
culesdcitrate, phytochelatins, metallothioneins (MTs) and CAT and GPX activities may be crucial for maintaining the
others (Clemens et al., 2002; Nedkovska and Atanassov, steady-state level of O$
2 and H2O2 and for prevention of the for-
1998; Pilon-Smits and Pilon, 2002). The introduction of MT mation of the highly cytotoxic OH$. The imbalance in the anti-
genes to improve the plant ability to tolerate heavy metals oxidative protection leads to accumulation of H2O2, formation
has been demonstrated (de Borne et al., 1998; Macek et al., of OH$ and to oxidative damage to lipids and proteins.
2002; Pan et al., 1994; Suh et al., 1998). The Ti-plasmid me- As to many other stresses, the cell responses to Cd are pri-
diated genetic transformation of MT genes in plants provided marily non-specific and focus on maintaining the cell homeo-
a valuable method of generating metal tolerant varieties that stasis. They include mobilization of the antioxidative
may be useful for reclamation of waste lands and mine soils. protection, changes in the chemical composition of the cell
Metallothioneins are different classes of low-molecular-weight walls and switching on the synthesis of metal-binding phyto-
cysteine-rich heavy metal chelating proteins, which take part chelatins and metallothioneins in the cytoplasm (Seregin and
in heavy metal detoxification and homeostasis (Cobbett and Ivanov, 2001). We could not find detailed studies on the anti-
Goldsbrough, 2002; Rauser, 1999). Recently it was shown oxidative protection in tobacco plants under cadmium toxicity.
that a stress-induced MT had additional antioxidant properties However, the biochemical understanding of plant metal accu-
(Akashi et al., 2004). The response of transgenic plants to mulation affected by genetic manipulations could lead to new
heavy metal accumulation is rather complex and still not insights into some fundamental aspects of plant physiology
well understood. and biochemistry.
Cadmium is one of the most toxic non-essential elements The aim of the present study was to evaluate the effect of
with high mobility and is a potential target for phytoremedia- metallothionein gene expression on the ability of tobacco
tion by transgenic plants (Macek et al., 2002). However, de- plants to accumulate cadmium ions and its influence on the
tailed analyses are necessary concerning the biochemical plant growth, photosynthesis and antioxidant defense system.
response of transgenic plants to Cd toxicity, especially how
they tolerate the high internal Cd levels and how they cope 2. Material and methods
with Cd-generated oxidative stress. Cadmium directly or indi-
rectly inhibits main physiological processes, such as photo- 2.1. Plant material for transformation
synthesis, water relations, gas exchange and respiration, and
disturbs plant mineral nutrition (Seregin and Ivanov, 2001; 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
Van Assche and Clijsters, 1990). The photosynthetic appara-
for genetic transformation experiments. After surface sterilization the seeds
tus appears to be especially sensitive to Cd toxicity despite were germinated in medium containing mineral salts and vitamins (Murashige
the very low Cd content (about 1% of the total leaf Cd) and Skoog, 1962) plus 3% (w/v) sucrose and solidified with 0.7% agar. The
found in the chloroplasts (Krupa, 1999; Seregin and Ivanov, test tubes were incubated for 3 weeks at 24  1  C. Seedlings were transferred
2001). The primary targets of Cd toxicity are PSII and the to glass vessels containing 20 ml of this medium and were grown under
140 mmol m2 s1 PAR at plant level, at 16/8 h (light/dark) photoperiod and
enzymatic phase of photosynthesis, particularly ribulose-1,
50e60% relative air humidity. In vitro tobacco plants were micropropagated
5-bisphosphate carboxylase/oxygenase (Rubisco; EC 4.1.1.39) at regular intervals.
(Krupa, 1999). The development of oxidative stress under
Cd toxicity is well documented. It varies depending on the
2.2. Genetic transformation experiments
plant species, the age, the duration and the severity of the
treatment and seems to be related to the Cd content in For the genetic transformation experiments, leaves from tobacco plants
the metabolically active compartments, e.g. cytoplasm grown in vitro (4e6 leaf stage) were used. The transformation was performed
(Iannelli et al., 2002; Sandalio et al., 2001; Singh and Tewari, with a construct carrying the plant metallothionein gene MT from Silene vul-
2003; Vitoria et al., 2001; Wu et al., 2003). Reactive oxygen 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,
species (ROS) under Cd toxicity are generated indirectly pCAMBIA 2301 was replaced by MThis-gene.
trough disturbances in the electron-transport chains, activa- The Agrobacterium leaf disk transformation method was applied (Horsch
tion of lipoxygenase and alteration in the structure or inhibi- et al., 1985). It was used Agrobacterium tumefaciens strain LBA 4404. The se-
tion of the antioxidative metalloenzymes (Sandalio et al., lectable marker was kanamycin.
2001). As ROS are formed in situ, the antioxidant protection The selection method for the transformants was the following: After co-
cultivation with A. tumefaciens LBA 4404, leaf explants were selected on MS
in plant cells is also highly compartmentalized (Mittler, medium (Murashige and Skoog, 1962) supplemented with 100 mg L1 kana-
2002). Superoxide dismutases (SOD; EC 1.15.1.1) catalyze mycin and 300 mg L1 claforan. The transformed explants were subcultured
the dismutation of O$ 2 (generated mainly by the electron every 3 weeks on a fresh medium containing 100 mg L1 kanamycin and
 N. Gorinova et al. / Environmental Pollution 145 (2007) 161e170 163

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 m2 s1 photosynthetic photon
flux density (PPFD)] and maximum total fluorescence yield (Fm) emitted dur-
ing a saturating white light pulse (1 s, over 3500 mmol m2 s1 PPFD by
Schott KL 1500 light source, Heinz Walz GmbH) were determined from
Fig. 1. The vector used for genetic transformation of tobacco NBZn 7-51 F1 a leaf disk (1 cm diameter). The leaf disk was then illuminated with continu-
line. ous red light (125 mmol m2 s1 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
300 mg L1 claforan. The obtained regenerants were transferred for rooting
written by Tyystjarvi and Karunen (1990). Photochemical (qP) and non-
on MS basal medium including vitamins and 100 mg L1 kanamycin.
photochemical quenching (qN) were calculating according to Van Kooten
The obtained putative transformants (4e6 leaf stage) were removed from
and Snel (1990): qP ¼ (Fs  Fo0 )/(Fm0  Fo0 ); qN ¼ (Fm  Fm0 )/(Fm  Fo0 ).
agar media, transplanted into soil and kept at a high humidity for 2e3 weeks
under artificial light for acclimatization.
2.6. Oxygen evolution
2.2.1. Molecular analysis for confirmation of the
presence of the MT gene in tobacco transformants Oxygen evolution rate was determined using a leaf disk electrode (Type
LD2/2, Hansatech, U.K.). It was measured at 800 mmol m2 s1 PPFD at sat-
2.2.1.1. PCR analyses. Genomic DNA of the transformants was isolated ac- urating CO2 concentration (provided by a carbonate/bicarbonate buffer).
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 2.7. SDSePAGE of soluble leaf proteins
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 For electrophoretic analyses the leaves samples were homogenized (1:5 w/v)
at 94  C for 4 min, 35 cycles (94  C for 30 s, 56  C for 30 s, 72  C for 40 s) at 4  C with ice cold 100 mM TriseHCl buffer (pH 8) containing: 20 mM
and 72  C for 5 min. About a half of each PCR product (17 ml) was electro- MgCl2, 10 mM NaHCO3, 1 mM EDTA, 2 mM phenylmethylsulfonyl fluoride
phoresed on 1.5% agarose gel in TAE-buffer. (PMSF), 12.5% glycerol (v/v), 20 mM b-mercaptoethanol, 3% Polyclar (w/v).
In the PCR analysis the primers C876 (F) and C880 (R) were used, pro- After incubation at 4  C for 30 min, the homogenate was centrifuged at
vided by Professor Sirpa Karenlampi’s group. Their sequences are the follow- 13,000  g for 20 min. The leaf total soluble proteins were separated by 12%
ing: C876: 50 -GCG GAA TTC GAT GTC GTG CTG TAA TGG AA-30 ; C880: SDSePAGE (Laemmli, 1970). Equal amounts of 30 mg protein per lane were
50 -CGG CTC GAG CTC ATT TGC AAG TGC AAG GG-30 loaded.

2.3. Cadmium treatment and sampling 2.8. Enzyme assays

Clone 35 was used for all the analyses performed. This clone showed very For enzyme analyses, 0.5 g of frozen leaf samples were ground and ex-
good regeneration potential in comparison with the other clones. It was tracted (1:10 w/v) as previously described (Demirevska-Kepova et al.,
multiplied by micropropagation to receive plant material sufficient for the 2004). The extracts were desalted on Sephadex-G 25 mini-columns. The activ-
presented studies. Southern blot analysis showed the presence of one copy ities were determined using a Shimadzu spectrophotometer. SOD activity was
of MThis gene in the clone (data not shown). measured at 560 nm based on the inhibition of the photochemical reduction of
After acclimatization the plants were grown at 25  1  C temperature, nitroblue tetrazolium (Beauchamp and Fridovich, 1971). One unit of SOD was
50e60% relative humidity and 16/8 h (light/dark) photoperiod under defined as the quantity of enzyme required to inhibit the reduction of NBT by
260 mmol m2 s1 PAR in nutrient solution according to Zhurbickij (1968), 50%. CAT activity was assayed following H2O2 decomposition at 240 nm
supplemented with increasing concentrations of CdCl2 (0 mM, 100 mM, (3 ¼ 0.0394 mM1 cm1) according to Aebi (1984). GPX activity was assayed
300 mM and 500 mM). The medium was aerated every day and changed every according to McRae and Thompson (1983). The formation of the reaction
2 days. Samples were taken 5 days after the beginning of the treatment when product tetraguaiacohinone (3 ¼ 26 mM1 cm1) was registered at 420 nm.
the final age of the plants was 55 days. All of enzyme analyses were performed APX activity was determined according to González et al. (1998) following
on mixed leaf samples from the fully developed fourth to sixth leaf (the middle the decrease in the absorbance at 290 nm due to enzymatic oxidation of ascor-
part of the canopy), which were stored in liquid N2 until extraction and the bic acid by H2O2 (3 ¼ 2.8 mM1 cm1). Correction for non-enzymatic oxida-
other analyses were made with fresh leaf material. tion of ASC was made.
164 N. Gorinova et al. / Environmental Pollution 145 (2007) 161e170

2.9. Isoenzyme staining 1 2 3 4 5 6 7 8 9 10 11 12

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 González 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). 300 bp
200 bp 257 bp

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 Fig. 2. PCR analysis of putative MThis transformants of tobacco NBZn 7-51
2.5% sulfosalicilic acid the extracts were incubated in 0.4 M TriseHCl buffer, line with specific primers C876 (F) and C880 (R) corresponding to Silene vul-
pH 7.8, in 0.02 M EDTA and Ellman’s reagent (5,5 dithiobis-2-nitrobenzoic garis metallothionein cDNA (237 bp) and a part of the body of binary vector
acid). The absorbance of the reaction product (2-nitro-5-benzoic acid) was reg- pCambia 2301 (20 bp). The total length of the amplified fragment is 257 bp.
istered at 412 nm using 3 ¼ 13,600 M1 cm1. The ascorbate pool (reduced- Lanes: 1, 100 bp marker; 2, NBZn 7-51 control; 3, clone 13; 4, clone 31; 5,
ASC and total ASC) was assayed according to the protocol of Hodges et al. clone 35; 6, clone 37; 7, clone 52; 8, clone 61; 9, clone 77; 10, clone 80;
(1996) on the basis of the reduction of Fe3þ to Fe2þ by ascorbate and complex- 11, clone 88; 12, plasmid MThis/pCambia 2301.
ation of Fe2þ with a,a0 -dipyridyl, resulting in a pink color. The ASC content was
quantified using a standard curve. in Fig. 3. The transgenic plants revealed a better ability for both
Cd uptake and accumulation in roots and leaves (1.5e2 times
2.11. Other assays more Cd) compared to the non-transformed tobacco plants.
This improved metal uptake characteristic was pronounced
Lipid peroxidation was estimated spectrophotometrically according to the for relatively high Cd exposure of 300e500 mM CdCl2.
improved thiobarbituric acid reactive substances assay (Hodges et al., 1999). Whereas in transformants the concentrations of metals in
Protein carbonylation was assayed at 366 nm according to Reznick and Packer the roots reached an upper threshold when treated with
(1994) using 2,4-dinitrophenylhydrazine. The carbonyl content was calculated
300 mM CdCl2, the accumulation in the leaves was increasing
from 3 ¼ 22,000 M1 cm1. Corrections for protein loss during washings
were made. Hydrogen peroxide was measured spectrophotometrically after reac- when treated with 500 mM CdCl2. In non-transformed plants
tion with KI as previously described (Demirevska-Kepova et al., 2004). The the roots absorbed most Cd at 500 mM CdCl2, while in the
amount of H2O2 was calculated using a standard curve. Leaf pigments were ex- leaves the accumulation of Cd reached a plateau at 300 mM
tracted with 80% acetone and estimated according to Arnon (1949). Total soluble CdCl2. At 300 and 500 mM CdCl2 visual symptoms of Cd tox-
protein content was determined by the method of Bradford (1976) using bovine
icity were observed in both non-transformed and transgenic to-
serum albumin as a standard. Rubisco quantity was determined in leaf tissues by
ELISA as previously described (Metodiev and Demirevska-Kepova, 1992). bacco plants (root browning, leaf spot chlorosis, wilting and
tip necrosis in older leaves). The transgenic leaves were
2.12. Statistical analysis more affected at the highest Cd concentration, which could
be explained with the higher internal Cd content as a result
The results were based on three replicates from two independent experi- of enhanced uptake.
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 3.3. Growth parameters and leaf pigment contents
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 root and shoot length did not change after treatment.
the obtained results. These data are not shown on the figures. The data of DW per g FW in tobacco leaves, stems and roots

3. Results

3.1. Confirmation of the transgenicity of the

transformants

The results from molecular analysis of the transformants

showed a PCR-positive signal for most tested transformants
(Fig. 2). Integration of the transgene is shown in Fig. 2. One
clone, N 35, which showed a positive PCR result, was chosen
for further studies.
Fig. 3. Cd accumulation in leaves (left) and roots (right) of transgenic (open
3.2. Cd accumulation and toxicity symptoms 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
Cadmium concentrations in the roots and leaves of tobacco transgenic and non-transformed plants in each treatment at the p < 0.05 (*),
plants after short-term exposure of 5 days of Cd are presented p < 0.01 (**) and p < 0.001 (***) level are indicated.
 N. Gorinova et al. / Environmental Pollution 145 (2007) 161e170 165

as well as leaf pigment content are presented in Fig. 4. The rise

in DW per g FW in leaf and stem reflected a disturbance of the
plant water balance after the Cd treatment. The transgenic
plants treated with 500 mM CdCl2 had more wilted leaves
compared to the non-transgenic ones. The chlorophyll a and
b concentrations diminished with increasing Cd concentrations
while the level of carotenoids did not change significantly. In
both 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 was
2.7e2.8. At the 500 mM CdCl2-treatment this ratio dropped
to 2.2 both in the transgenic and the non-transformed plants.

3.4. Chlorophyll fluorescence

The primary photochemical activity of PSII, estimated by

the ratio of Fv/Fm started to decrease as a result of the
300 mM CdCl2-treatment and it was significantly reduced at
500 mM CdCl2 (Fig. 5).
The rate of whole chain electron transport [VPSII ¼
(Fm0  Fs)/Fm0 ] and the efficiency of excitation capture by
Fig. 5. Effect of Cd treatment on the maximum quantum efficiency of PSII
open PSII reaction centers (Fv0 /Fm0 ) decreased with increasing (Fv/Fm), the actual quantum yield of PSII electron transport in the light-
Cd-concentrations, even more than the Fv/Fm values. The adapted state (FPSII), the efficiency of excitation energy capture by ‘‘open’’
value of fluorescence decline ratio Rfd [(Fm  Fs)/Fs] was PSII reaction centers (Fv0 /Fm0 ) and the fluorescence decreased ratio (Rfd) in
below 1 after treatment with 500 mM CdCl2, indicating severe non-transformed (white columns) and transgenic tobacco plants (dark col-
damage of the photosynthetic apparatus of tobacco plants umns). Means and standard deviations of at least three replicates are shown.
(Fig. 5). The photochemical quenching (qP) decreased with

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-photochemical

quenching (qN) and the proportion of excitation light not
used for photochemistry (LNU) increased (Fig. 6). We found
that the Cd treatment reduced the rate of photosynthetic
oxygen evolution more than the mechanisms of PSII photo-
chemistry. The comparison of the results obtained for trans-
genic and non-transformed tobacco plants clearly showed
that 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 did

not change significantly in the transgenic and non-transformed
plants (Fig. 7). The Rubisco quantity was not affected by the
transformation and was stable in the transformed plants treated
with 100 and 300 mM CdCl2, but under the highest Cd toxicity
(500 mM CdCl2) it diminished to 49.75% and to 89.27% for
transgenic and non-transformed plants, respectively, when Fig. 7. Effect of Cd concentration on the level of the leaf total soluble protein
compared with control plants (0 mM CdCl2) (Fig. 7). 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-
3.6. Changes in leaf protein pattern bacco plants in each treatment at the p < 0.05 (*), p < 0.01 (**) and p < 0.001
(***) level are indicated.
Considerable changes in the leaf polypeptide pattern after
SDS electrophoresis were detected only in the variants treated 3.7. Response of antioxidant enzymes to Cd
with 500 mM CdCl2. The changes were more expressed in accumulation
the transgenic plants (Fig. 8). The highest Cd concentration
provoked Rubisco LS and SS diminution along with the The isoenzyme activities and profiles of some main en-
enhancement of some bands with MM between 20 and zymes involved in the antioxidative protection (SOD, APX,
43 kDa (approximately 23 and 34 kDa, respectively). The GPX, CAT) were analyzed in the leaves of Cd-treated tobacco
intensity of the band at the position corresponding with RBP plants. The total SOD activity gradually enhanced with the
diminished substantially. increase 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: one
MnSOD (mitochondrial), one FeSOD (chloroplastic), and

1 2 3 4 5 6 7 8 M

94 kDa
67 kDa----RBP
--------------RLS
43 kDa

30 kDa

20 kDa
14 kDa-----RSS

Fig. 8. Effect of Cd on the pattern of soluble proteins in non-transformed (1e4)

Fig. 6. Effect of Cd treatment on the photochemical quenching (qP), non-pho- and transgenic (5e8) tobacco plants. Extracts were analyzed by SDSePAGE
tochemical quenching (qN), the fraction of the light energy which was not (12%). Treatments: 1, 5, control (without Cd); 2, 6, 100 mM CdCl2; 3, 7,
used for photochemistry (LNU) and the oxygen evolution rate) in non-trans- 300 mM CdCl2; 4, 8, 500 mM CdCl2. The polypeptides were visualized by
formed (white columns) and transgenic tobacco plants (dark columns). Means Coomassie blue staining. Each lane was loaded with 50 ml leaf extract. The
and standard deviations of at least three replicates are shown. kDa values in the figure indicate the position of molecular mass standards.
 N. Gorinova et al. / Environmental Pollution 145 (2007) 161e170 167

MnSOD

FeSOD
Cu/Zn SOD I
Cu/Zn SOD II

1 2 3 4 5 6 7 8

APX 1

APX 2
APX 3

GPX1

GPX 2
GPX X
GPX 3

CAT

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.
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- CdCl2 treatment. Contrary to this, GPX activity sharply in-
bacco plants. Units were defined as: the quantity of enzyme required to inhibit creased (7 to 11 times) at the 500 mM CdCl2 treatment and
the reduction of NBT by 50% per 1 min for SOD, nmol H2O2 decomposed per was higher in transgenic plants when compared with the
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
non-transgenic ones. The appearance of a new GPX isoform
three replicates are shown. Significant differences between transgenic and non- was revealed at 500 mM CdCl2 treatment both in the non-
transformed tobacco plants in each treatment at the p < 0.05 (*), p < 0.01 (**) modified and in the transgenic plants (GPX X). The total
and p < 0.001 (***) level are indicated. CAT activity diminished after treatment with 300 mM CdCl2.
The 500 mM CdCl2 treatment caused some increase in CAT ac-
three Cu/ZnSOD isoformsdone chloroplastic (Cu/ZnSOD II) tivity without, however, reaching the level of the untreated
and two cytosolic (Cu/ZnSOD I and Cu/ZnSOD III). Cu/ plants. Isoenzyme staining revealed one dominating peroxi-
ZnSOD I and II were the major isoforms. somal isoform of CAT in leaves. At the 300 mM and 500 mM
The treatment with 500 mM CdCl2 raised the activities of CdCl2 treatments its electrophoretic mobility was changed.
Cu/ZnSODs. Typically, the Cu/ZnSOD from transgenic plants
reacted more intensively to the metal toxicity. The MnSOD 3.8. Low molecular compounds influenced by Cd toxicity
and FeSOD isoforms were not apparently changed following
the Cd treatment. The total APX activity enhanced following The low-molecular-weight antioxidative protectors were
300 mM and 500 mM CdCl2 treatments without visible changes also mobilized as a result of tobacco plants treatment with
in the isoenzyme pattern (APX 1, APX 2 and APX 3). Com- Cd (Fig. 11). The leaf ascorbate pool strongly increased at
pared with non-transformed plants the control transgenic the 300 mM and 500 mM CdCl2 treatments without significant
plants had lower APX activity, however, the increase of the changes in the percentage of reduced ASC. The non-protein
activity was higher and the difference became insignificant thiol content (basically glutathione, phytochelatins and
when treated with 300 and 500 mM CdCl2, respectively. In other SH compounds) was significantly higher in the trans-
the non-treated plants GPX activity was higher in transgenic genic plants and rose even at 100 mM CdCl2 treatment but
plants and tended to decrease following 100 and 300 mM reached a plateau at 300e500 mM CdCl2. The leaves of the
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-protein 2000; Herzig et al., submitted for publication). The genetic
thiols in comparison to the transgenic ones. Nevertheless, transformation with the metallothionein gene MTs from
the content of non-protein thiols also rose sharply to values Silene vulgaris L. aimed at a further increase of the metal up-
comparable to those in transgenic plants at the highest Cd take capacity of this variety, as tested on short term experi-
treatment. ments on hydroponics spiked with relatively high
concentration of Cd.
3.9. Hydrogen peroxide level and oxidative A very important result of the genetic transformation of
damage of lipids and proteins NBZn 7-51 F1 tobacco plants with plant MT gene was the es-
sentially improved accumulation of Cd in the leaves and roots,
The accumulation of H2O2 and the oxidative damage in without affecting the Cd distribution between roots and shoots
lipids and proteins as indices for the development of oxidative in comparison to the non-transformed plants of the same
stress under Cd toxicity are presented in Fig. 11. The leaf somaclonal variety. It could be expected that both enhanced
H2O2 level was slightly lower in the control non-transformed tolerance and accumulation of Cd were related to the overpro-
plants compared to the modified ones. However, in both cases duction of metallothioneins (MTs). Similar changes in the
the tendency was the same: a small rise at 100 mM CdCl2, photosynthetic apparatus, leaf proteins and antioxidative de-
a drop to the level of the untreated plants at 300 mM CdCl2 fense system were observed in transgenic and non-transformed
and an abrupt approx. 4-fold increase at 500 mM CdCl2. The plants under Cd toxicity. The toxicity symptoms were rather
malondialdehyde (MDA) content as a marker of oxidative correlated with the Cd concentration in the leaves. The pool
damage of lipids increased following treatment with 300 and of non-protein thiols gradually increased with increasing Cd
500 mM CdCl2, whereas the level of protein carbonyl groups uptake. This result most probably reflected the induction
as a marker for oxidative damage of proteins did not change of phytochelatin synthesis as a general mechanism of Cd
significantly. detoxification in plants (Cobbett and Goldsbrough, 2002;
Rauser, 1999).
4. Discussion At 100 mM CdCl2 treatment the studied parameters did not
change, except for the slight increase in the H2O2 level, which
The new somaclonal tobacco variety NBZn 7-51 F1, ob- was most probably a result of the enhanced SOD and dimin-
tained from in vitro breeding, is characterized with enhanced ished GPX activities and was consistent with the signaling
yield, metal tolerance, Zn and Cd extraction and enhanced re- role of H2O2 (Neill et al., 2002). No inhibition of the photo-
sistance against Cu stress and a linear accumulation of Cd as synthesis and no signs of oxidative stress were observed.
a function of the external metal concentration (Guadagnini, Both the transgenic and the non-transformed tobacco plants
 N. Gorinova et al. / Environmental Pollution 145 (2007) 161e170 169

adapted efficiently to this Cd supply. Nevertheless, the trans- plants. The established similar impact of the toxic Cd concen-
genic plants accumulated a two times higher amount of Cd tration on photosynthesis, leaf proteins and the anti-oxidative
than the non-transformed ones both in roots and shoots. protection in the leaves of both the non-transformed and the
At elevated stress of 300 mM CdCl2 resulted in inhibition transgenic plants in spite of the higher cadmium concentration
of photosynthesis, which was evidenced by the decreased in the transgenic plants. Such transgenic plants are not more
PSII efficiency, the reduced rate of photosynthetic oxygen sensitive then non-transformed ones and after positively pass-
evolution, the diminished chlorophyll content, Rubisco quan- ing pot experiments on metal contaminated soil could be
tity and CAT activity. The decrease in the CAT activity was used in phytoextraction of Cd for elevated levels of Cd
probably connected with photosynthetic inhibition, as the contamination.
H2O2 level was comparable to that of the controls without
any Cd treatment. The increased SOD and APX activities
Acknowledgements
and the enlarged ASC pool without changes in the level of
reduced ASC indicated a mobilization of the ascorbate-
This work was supported by a grant from PHYTAC Project,
glutathione cycle at this Cd treatment. A similar protective
Contract No: QLK3-CT-2001-00429 in 5FP. The authors are
role of the ascorbate-glutathione cycle against Cd-induced
grateful to B. Juperlieva-Mateeva, A. Kostadinova and Z. Kru-
oxidative stress has been observed in radish, barley and
mova for their excellent technical assistance, and to Erika
Arabidopsis thaliana (Skorzynska-Polit et al., 2003/4; Vitoria
Nehnevajova and Sara Bangerter from Switzerland for their
et al., 2001; Wu et al., 2003) but not in other species like pea
corrections in English.
(Sandalio et al., 2001). The inhibition of photosynthesis and
the mobilization of the antioxidative protection could be re-
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