J. Plant Physiol. 161. 691 – 699 (2004)
http://www.elsevier.de/jplhp
Auxin autotrophic tobacco callus tissues resist oxidative stress: the
importance of glutathione S-transferase and glutathione peroxidase
activities in auxin heterotrophic and autotrophic calli
Jolán Csiszár1 *, Margit Szabó1, László Erdei1, László Márton2, Ferenc Horváth1, Irma Tari1
1
Department of Plant Physiology, Faculty of Science, University of Szeged, P.O. Box 654, H-6701 Szeged, Hungary
2
Department of Biological Sciences, University of South Carolina, Columbia, SC29208, USA
Received February 3, 2003 · Accepted July 17, 2003
Summary
Auxin autotrophic and heterotrophic tobacco callus lines were grown on MS medium with or without
100 mmol/L NaCl and growth and some of the stress-related activities, such as GPX, SOD, CAT, GST,
GSH-PX, as well as the concentration of ethylene and H2O2, were measured and compared with
each other. The auxin autotrophic calli grew slower, however, on the NaCl-containing medium the
growth rate was higher than that of the heterotrophic cultures after two weeks of culturing. The stressrelated ethylene production was lower in the autotrophic cultures and, contrary to the heterotrophic
tissues, its level did not change significantly upon NaCl treatment. The guaiacol peroxidase (GPX)
activities were higher in the autotrophic tissues in all cell fractions regardless of the presence of
NaCl. Treated with NaCl, the GPX activities elevated in the soluble and covalently-bound fractions in
the heterotrophic calli, but were not further increased in the autotrophic line. SOD and CAT activities
were higher in the heterotrophic tissues, and were increased further by 100 mmol/L NaCl treatment.
The GST and GSH-PX activities were higher in the autotrophic line, which might explain their
enhanced stress tolerance. In the autotrophic tissues, the elevated antioxidant activities led to
reduced levels of H2O2 and malondialdehyde; under mild NaCl stress, these levels decreased further. The lower growth rate and the effective protection against NaCl stress-induced oxidative damage of the autotrophic line can be explained by the cell wall-bound peroxidase and GSH-PX activities
in the auxin autotrophic tissues. Their maintained growth rate indicates that the autotropic cultures
were more resistant to exogenous H2O2.
Key words: Auxin-dependent and independent calli – ethylene – glutathione peroxidase – oxidative
stress resistance – scavenging enzymes
Abbreviations: ALA = δ-aminolevulinic acid. – AOS = active oxygen species. – APX = ascorbate peroxidase. – BHT = butylhydroxytoluene. – CAT = catalase. – CDNB = 1-chloro-2,4-dinitrobenzene. –
DTNB = 5,5′-dithio-bis(2-nitrobenzoic acid). – HRP = horseradish peroxidase. – HVA = 4-hydroxy-3methoxyphenylacetic acid or homovanillic acid. – IAA = indole-3-acetic acid. – GPX = guaiacol peroxidase. – GR = glutathione reductase. – GSH = reduced glutathione. – GSH-PX = glutathione peroxidase. – GST = glutathione S-transferase. – MDA = malondialdehyde. – MS medium = MurashigeSkoog medium. – NBT = nitro blue tetrazolium. – SOD = superoxide dismutase. – TCA = trichloroacetic acid
* E-mail corresponding author: [email protected]
0176-1617/04/161/06-691 $ 30.00/0
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Introduction
Auxin-independent autotrophic and auxin-dependent heterotrophic lines of tobacco calli may differ, not only in their indoleacetic acid (IAA) synthetizing abilities and in their sensitivities to exogenous auxins (Nakajima et al. 1979, Szabó et al.
1981, Michalchuk and Druart 1999), but also in their stress tolerance. Levels of low molecular weight antioxidants (e.g.,
glutathione or ascorbate) and activities of antioxidant enzymes, such as superoxide dismutase (SOD), catalase
(CAT), ascorbate peroxidase (APX), guaiacol peroxidase
(GPX), and glutathione reductase (GR), generally increase in
plants under stress conditions and correlate well with the enhanced tolerance (Foyer et al. 1997). Peroxidases, such as
those participating in lignin biosynthesis or the degradation
of IAA, have long been identified in plants (García-Florenciano et al. 1991, Quiroga et al. 2000). Peroxidases in this
group utilize a wide range of electron donors and can be referred to as GPXs (guaiacol is widely applied for their assay).
Plant peroxidases are able to catalyze a variety of different
reactions and may participate in the scavenging of active
oxygen species (AOS) because they can utilize and eliminate
H2O2. Examples include APX and glutathione peroxidase
(GSH-PX) (Roxas et al. 2000, Noctor et al. 2002, Shigeoka et
al. 2002). Identification of GSH-PX activity in plants, the isolation of plant genes with sequence homology to the animal
analogues, and isolation and characterization of the protein
product has proven that the enzymes existing in plants belong to the phospholipid hydroperoxide GSH-PX family. The
function of phospholipid hydroperoxide GSH-PXs is the reduction of alkyl hydroperoxides, such as fatty acid hydroperoxides (Eshdat et al. 1997). Some glutathione S-transferases
(GSTs) also exhibit GSH-PX activity. GSTs are ubiquitous enzymes that catalyze the addition of reduced glutathione
(GSH) to various hydrophobic electrophilic substrates, which
tags them for vacuolar sequestration. GSTs protect against
environmental stress and disease by detoxifying reactive
products generated by oxidative stress. GSTs with a high affinity for auxins and cytokinins have also been suggested to
contribute to hormone homeostasis (Marrs 1996, Edwards et
al. 2000).
Hormone autotrophic or habituated callus tissues have
been observed to exhibit substantial changes, not only in hormone metabolism, but also in primary metabolic pathways.
Bisbis et al. (1999) found that sugarbeet calli gradually lost
their peroxidase activities and their organogenic capacity
during neoplastic progression of cultures. They found that the
reduction of the synthesis of aminolevulinic acid (ALA), an obligate intermediate of tetrapyrrole biosynthesis, through reduction of the plastidial Beale pathway, might be responsible
for the substantial decrease of the heme-containing compounds. Hemoprotein synthesis of autotrophic cultures could
additionally be decreased by inhibiting ALA-dehydratase via
a disturbance of the phenol metabolism. Besides peroxidases, the heme-containing CAT, which removes H2O2 in peroxi-
somes and mitochondria, was also inhibited in sugarbeet
habituated tissues (Bisbis et al. 1999).
Auxin- or cytokinin-independent callus cultures can be
characterized by low ethylene production (Köves and Szabó
1987, Hagège et al. 1994) and increased polyamine levels
(Hagège et al. 1990, Kevers et al. 1999). As ethylene plays a
role in the induction of the biosynthesis or the secretion of
peroxidases into the apoplast (Ishige et al. 1993, Ito et al.
1994), the decreased ethylene production may be involved in
the reduced activity of peroxidases or their decreased secretion into the apoplast in habituated cultures.
In this paper we investigate the growth and the enzymatic
antioxidant defence mechanisms of heterotrophic and auxin
autotrophic tobacco cultures treated with 100 mmol/L NaCl in
order to reveal whether the possible decrease of heme biosynthesis and the subsequently reduced peroxidase or CAT
activities could negatively affect the acclimatization processes to salt stress, which causes oxidative damage to the
cells.
Materials and Methods
Plant material
The callus cultures originated from protoplasts of Nicotiana tabacum
SR1 plants (Csiszár et al. 2001). The auxin-requiring cultures were
grown on solid MS medium (Murashige and Skoog 1962) containing
2 µmol/L kinetin, 17.5 µmol/L IAA and 0.45 µmol/L 2,4-D, and the auxin
autotrophic cultures were transferred onto the same medium without
auxin. Each Petri dish contained 25 inocula. The weight of one inoculum was approximately 20 mg. The salt treatment was carried out
by adding 100 mmol/L NaCl to the culture media. The H2O2 was
added aseptically just before the medium became solid, and the inocula were plated immediately. The cultures were kept in a growth
chamber at 25 ˚C, under 8.4 W m – 2 warm white fluorescent light
(Tungsram F29 lamps, Hungary) and were analyzed over a 3-week
period.
Ethylene measurements
The ethylene production of calli of different ages was measured as
described earlier (Tari and Mihalik 1998). Callus tissues (0.5 g) were
kept in closed tubes for 1 h. The ethylene-containing samples were
withdrawn from the gas phase above the plant material and were analyzed in a gas chromatograph equipped with a flame ionization detector.
Na + contents
Two-week-old callus tissues were dried at 70 ˚C for 72 h. Na + concentrations were analyzed after wet digestion of the dry material in a mixture of HNO3 and 30 % H2O2 (5 : 4; v/v) at 200 ˚C for 3 h; the samples
were measured with a Hitachi Z-8200 Zeeman polarized atomic absorption spectrophotometer.
Auxin autotrophic calli and oxidative stress
Activity measurements of antioxidant enzymes
The enzyme activities were determined two weeks after the transfer
onto the NaCl-containing medium. Two grams of callus tissue was homogenized on ice in 4 mL extraction buffer (50 mmol/L phosphate buffer pH 7.0, containing 1 mmol/L EDTA, 1 mmol/L phenylmethylsulfonyl
fluoride and 1 % polyvinyl-polypirrolidone). The homogenate was filtered through two layers of cheese-cloth and centrifuged for 25 min at
15,000 g at 4 ˚C. The supernatant was used for enzyme activity assays. In the case of SOD, CAT, GPX, and GR, refinding experiments
were made for the control samples with standard enzymes. The percent of recovery was 92.3 ( ± 3.72) for SOD (from horseradish, Sigma),
78.38 ( ± 7.58) for catalase (from bovine liver, Sigma), 56.62 ( ± 5.51)
for GPX (from horseradish, Reanal), and 75.43 ( ± 6.18) for GR (Type II
from wheat, Sigma).
SOD (EC 1.15.1.1) activity was determined by measuring the ability
of the enzyme to inhibit the photochemical reduction of nitro blue
tetrazolium (NBT) in the presence of riboflavin in light (Dhindsa et al.
1981). One unit (U) of SOD was calculated as the amount causing a
50 % inhibition of NBT reduction in light. The enzyme activity was expressed in terms of specific activity (U mg –1 protein). CAT (EC
1.11.1.6) activity was determined by the decomposition of H2O2 and
was measured spectrophotometrically by following the decrease in
absorbance at 240 nm (Upadhyaya et al. 1985). One U = the amount
of H2O2 (in µmol) decomposed in 1min. GPX (EC 1.11.1.7) activity was
determined by monitoring the increase in absorbance at 470 nm during the oxidation of guaiacol (Upadhyaya et al. 1985). The amount of
enzyme producing 1 µmol min –1 of oxidized guaiacol was defined as
1U. The soluble, cell wall-bound and covalently bound fractions were
prepared according to Hendricks et al. (1985), with a slight modification for preparation of the ionically bound fraction with 1mol/L KCl. GR
(EC 1.6.4.2) activity was determined by measuring the absorbance increment at 412 nm when 5,5′-dithio-bis(2-nitrobenzoic acid) (DTNB)
was reduced by GSH, generated from glutathione disulfide (GSSG)
(Smith et al. 1988). The specific activity was calculated as the amount
of reduced DTNB, in µmol min –1 protein mg –1, ε420 = 13.6 mmol/
L –1 cm –1. GST (EC 2.5.1.18) activity was determined spectrophotometrically by using an artificial substrate, 1-chloro-2,4-dinitrobenzene
(CDNB), according to Habig et al. (1974). Reactions were initiated by
the addition of CDNB, and the increase in A340 was determined. One
U is the amount of enzyme producing 1 µmol conjugated product in
1 min, ε340 = 9.6 mmol/L –1 cm –1. GSH-PX (EC 1.11.1.9) activity was
measured by the method of Awasthi et al. (1975), with cumene hydroperoxide as a substrate. The reaction mixture contained 4 mmol/L
GSH, 0.2 mmol/L NADPH, 0.05 U of GR (Type II from wheat, Sigma),
100 µL enzyme extract, and 0.5 mmol/L substrate in phosphate buffer
(0.1 mol/L, pH 7.0) in a total volume of 1 mL. The decrease of NADPH
was followed by measuring the absorbance at 340 nm. The nonspecific NADPH decrease was corrected for by using additional measurements without substrate, ε340 = 6.22 mmol/L –1 cm –1. One U = µmol converted NADPH min –1. The protein contents of the extracts were determined by the method of Bradford (1976).
H2O2 and malondialdehyde (MDA) assays
H2O2 concentration was measured by the fluorometric method of
Asada et al. (1974) with some modifications published by Hideg et al.
(2003). A half gram (0.5 g) callus was frozen in liquid N2, and homogenized with 0.5 mL of 0.1 mol/L phosphate buffer, pH 7.0. After centrifugation, 25 µL of the extract was added to 3 mL of reaction mixture
693
containing 0.5 mmol/L homovanillic acid (HVA) and 0.25 U horseradish
peroxidase (HRP). The fluorescence yield due to H2O2 was determined in each sample by the increase in fluorescence intensity, using
excitation and emission wavelengths of 295 and 415 nm, respectively.
During the calculation of the H2O2 content, the results of the recovery
experiments were taken into consideration. MDA formation was assayed by using the thiobarbituric acid method (Ederli et al. 1997). A
half gram (0.5 g) callus was homogenized with 5 mL of 0.1% trichloroacetic acid (TCA). To avoid further lipid peroxidation, 500 µL of 4 %
butylhydroxytoluene (BHT) was added to the extract. After centrifugation at 15,000 g for 25 min, 1 mL of supernatant was mixed with 4 mL
0.5 % thiobarbituric acid in 20 % TCA and the mixture was incubated
in boiling water for 30 min. The absorbance was read at 532 nm and
adjusted for nonspecific absorbance at 600 nm. MDA concentration
was estimated by using an extinction coefficient of 155 mmol/L –1 cm –1.
Statistical analysis
The means ± SD were calculated from the data of at least three separate experiments. Differences between treatment means were deter-
Figure 1. Growth (A) and Na + concentration (B) of 2-week-old tobacco
callus cultures on MS medium containing 17.5 µmol/L IAA and 0.45
µmol/L 2,4-D for auxin heterotrophic calli, or no exogenous auxins in
the case of autotrophic tissues, and on the same media supplied with
100 mmol/L NaCl. Means denoted by different letters indicate a significant difference between the treatments at P < 0.05 according to Duncan’s test.
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Jolán Csiszár et al.
mined by Duncan’s multiple range test. Columns denoted by the
same letters did not differ significantly at a probability level of P < 0.05.
Results
The growth rate, Na + content, and ethylene release of
heterotrophic and autotrophic calli under NaCl stress
The growth of auxin heterotrophic calli was compared with
that of the autotrophic line of the same tissues two weeks
after the transfer onto MS medium containing 100 mmol/L
NaCl. At this age the calli were in their exponential growth
phase and the difference between the growth rates of the two
lines was maximal (Köves and Szabó 1987, and unpublished
results). The habituated cultures grew slowly even on NaCl
free media, but in the presence of NaCl the basic growth rate
did not drop as much as for the heterotrophic line (Fig. 1 A).
Determination of the Na + concentrations of the 2-week-old
calli revealed that the Na + uptake of the autotrophic tissues
was not inhibited, because their Na + concentration in the
presence of 100 mmol/L NaCl was slightly higher than that of
the heterotrophic line (Fig. 1B).
Figure 2. Ethylene evolution of auxin heterotrophic (A) and autotrophic calli (B) growing on MS medium containing 100 mmol/L NaCl.
The data are means of one representative experiment in at least 4
replicates. Note the different vertical scales in A and B.
Figure 3. Guaiacol peroxidase activities in the auxin heterotrophic
and autotrophic tobacco calli after 2 weeks of NaCl teatment. A: GPX
activity of the soluble fraction; B: ionically bound peroxidase activity;
C: activity of the covalently bound peroxidases. Means denoted by
the same letters were not significantly different (P < 0.05, Duncan
test).
Auxin autotrophic calli and oxidative stress
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Figure 5. H2O2 concentration of auxin heterotrophic and autotrophic
tissues growing on MS medium supplied with 100 mmol/L NaCl for two
weeks. Means denoted by different letters indicate a significant difference (P < 0.05, Duncan test).
Figure 4. SOD (A) and CAT (B) activities of the 2-week-old auxin heterotrophic and autotrophic cultures during NaCl stress. Means
denoted by different letters indicate a significant difference between
the treatments (P < 0.05, Duncan test).
The ethylene production of the two lines followed the pattern observed earlier for auxin-dependent and auxin autotrophic cultures. The heterotrophic tissues evolved much more
ethylene with an early maximum on an auxin-containing culture medium and the cells increased the ethylene production
in the presence of the salt. The ethylene formation was
20 times less in the autotrophic cultures, and its level did not
change significantly upon the NaCl treatment (Fig. 2).
The effect of salt stress on the activities of the
antioxidative enzymes and H2O2 concentrations of
heterotrophic and autotrophic tissues
Comparison of the GPX activities in the heterotrophic and
autotrophic calli revealed approximately two-fold higher activities in the autotrophic cultures in the soluble and ionically
bound fractions, and the fraction bound covalently to the cell
walls also exhibited a slight increase in peroxidative capacities (Figs. 3 A, B, C). The NaCl stress did not elevate the peroxidative capacity in the autotrophic line, but did increase the
activity in the soluble and covalently bound fractions in the
heterotrophic tissues. The H2O2-producing SOD activity was
about two-times higher in the heterotrophic calli than in the
autotrophic cells, and the enzyme activity was slightly, but not
significantly, further enhanced by 100 mmol/L NaCl after two
weeks of culturing (Fig. 4 A). In the absence of NaCl, an extremely low CAT activity was detected in the autotrophic tissues, but this did not reach the level of the heterotrophic cells
during salt stress (Fig. 4 B).
Despite the low CAT activity in the presence or absence of
NaCl in the autotrophic tissues, the outcome of the enhanced
antioxidant capacity was a reduced H2O2 level. After two
weeks the salt treatment had decreased the H2O2 contents of
both cultures (Fig. 5).
The activity of GR, which reduces GSSG to GSH, was
higher in the auxin-requiring cultures, but in response to a
NaCl stress the activity increased only in the heterotrophic
line (Fig. 6 A).
Interestingly, the GST and especially the GSH-PX activities
were higher in the autotrophic calli, which essentially contribute to the elevated stress tolerance of habituated tissues
(Figs. 6 B, C). While the auxin autotrophic line enhanced the
GSH-PX activity during salt stress, the enzyme activity remained exceptionally low in the heterotrophic cultures. The
level of MDA, a lipid peroxidation product in the tissues, was
less in the autotrophic line and, similar to the level of H2O2,
the amount of this product decreased further on NaCl treatment (Fig. 7), suggesting a membrane-protecting role of glutathione peroxidases during salt stress.
Growth of calli in the presence of exogenous H2O2
In order to reveal the sensitivities of the two lines to exogenous H2O2, the calli were transferred onto fresh MS medium
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Jolán Csiszár et al.
Figure 7. MDA level in the 2-week-old auxin heterotrophic and autotrophic tobacco cultures after 100 mmol/L NaCl treatment. Means
denoted by different letters indicate a significant difference between
the treatments (P < 0.05, Duncan test).
Figure 8. Growth of 2-week-old auxin heterotrophic and autotrophic
tobacco callus cultures in the presence of different H2O2 concentrations. The data are means ± SD of four measurements.
containing 0.1–10 mmol/L H2O2. The growth of the auxin heterotrophic calli decreased significantly at 1 mmol/L H2O2, while
the autotrophic line was inhibited only by 10 mmol/L H2O2
(Fig. 8). This proves that the autotrophic calli really do posses
a more effective H2O2-scavenging mechanism.
Figure 6. GR (A), GST (B) and GSH-PX (C) activities of auxin heterotrophic and autotrophic calli after 2 weeks of salt treatment. Means
denoted by the same letters were not significantly different (P < 0.05,
Duncan test).
Discussion
The data presented here suggest that tobacco calli that have
passed through several steps of neoplastic progression and
Auxin autotrophic calli and oxidative stress
have become habituated are very resistant to NaCl stress
treatment. During the habituation process, the cells regain
their auxin-synthetizing capacity and they are able to grow on
an auxin-free culture medium. The phenotype of our cultures
is slightly different from other habituated cultures, such as
those from fully auxin- and cytokinin habituated sugarbeet cell
lines, which involve very friable and achlorophyllous tissues
and, according to Bisbis et al. (1999), can be considered true
cancer cells. Our auxin autotrophic calli are compact and may
contain even more chlorophyll than the heterotrophic cells. It
seems that in this line tetrapyrrole synthesis is not inhibited. To
decide whether this is due to a maintained ALA accumulation
or to the synthesis of cinnamic acid derivatives, instead of
ALA-dehydratase-inhibiting benzoic acid derivatives in the
autotrophic calli, requires further investigations.
The heterotrophic line exhibited a high ethylene production
with an early maximum after subculturing, which may be due
to the exogenous growth regulators (auxins and cytokinins) in
the growth medium. This ethylene release was further enhanced by the NaCl treatment.
Ethylene, as a stress hormone, induces an acclimatization
process including the activation of defence mechanisms at a
transcriptional level or may activate those processes, such as
the generation of AOS including H2O2, which result in increased peroxidase activities (Ishige et al. 1993, Ievins et al.
1995). The steady-state activity of GPX (based on the steadystate hormonal and environmental factors) were even higher
in the 2-week-old auxin autotrophic line, but the tissues did
not enhance the peroxidative capacity significantly in response to 100 mmol/L NaCl stress, which may be due to the
lower ethylene production of these cells in the absence or
presence of NaCl than in the heterotrophic calli.
One of the most important roles of peroxidases in plant
cells is to catalyze the cross-linking of cell wall components
(Hatfield et al. 1999) and, in this manner, to restrict the expansion of the cells (Andrews et al. 2002). The differences in cell
wall-bound peroxidase activity may be responsible for the
slower growths of the autotrophic line. The covalently bound
peroxidase activity increased considerably in the presence of
100 mmol/L NaCl in the heterotrophic tissues, but not in the
autotrophic cells. The elevated enzyme activity may explain
why the auxin autotrophic line grew slowly, but the growth inhibition caused by NaCl was smaller in the autotrophic than in
the heterotrophic tissues.
A crucial difference was found between the heterotrophic
and autotrophic cultures in the CAT activity. The cells are supplied with reduced sugar exogenously and their photosynthetic activity is highly suppressed (Cséplő and Medgyesy
1986). Hence, there is no need for the detoxification of H2O2,
which is derived as a by-product of the photosynthetic electron transport and photorespiration. The auxin-requiring tissues increased the CAT activity during the NaCl stress and
this activation contributed considerably to the effective elimination of H2O2. However, the auxin autotrophic cultures must
activate other pathways to scavenge H2O2.
697
The GSTs play an important role in the detoxification mechanism by catalyzing the GSH conjugation of toxic substances
under stress. GST-encoding genes are selectively expressed
during abiotic or biotic stress, but some isoenzymes are involved in conjugating natural plant products and can mediate
isomerase reactions or function as carrier proteins. GST isoenzymes are also capable of acting as GSH-PX and reduce
organic hydroperoxides to their monohydroxy derivatives by
using GSH (Edwards et al. 2000). Some isoenzymes of GSTs
have been shown to exhibit considerable GSH-PX activity
with linolenic acid hydroperoxide as substrate (Gronwald and
Plaisance 1998). Plant GSH-PXs are induced by different
stresses, and transgenic tobacco plants overproducing a
GST gene with GSH-PX activity exhibit significant stress tolerance (Roxas et al. 2000).
Genes encoding GSTs are induced by several factors, including ethylene, IAA, and H2O2 (Marrs 1996). Callus cultures
grow on a medium containing exogenous IAA and 2,4-D;
these hormones may induce the transcription of genes encoding GSTs, which, in turn, can influence the level of active
auxin hormones, by conjugation for example (Takahashi et al.
1989, Ulmasov et al. 1994, Bianchi et al. 2002). GSTs, and especially the isoenzymes with GSH-PX activity, can scavenge
lipid hydroperoxides and maintain membrane integrity under
different stress conditions. Higher GST and GSH-PX activities
may provide some advantages for the growth of cells in the
presence of exogenous hormones, and especially in the
presence of the synthetic auxin 2,4-D and/or auxin-induced
oxidative stress. Our previous results indicated that the GST
activity of the auxin heterotrophic tissues is inhibited by ethylene. When the binding of ethylene to the receptor was
blocked with norbornadiene, the total GST activity increased,
whereas in the autotrophic calli, norbornadiene did not influence the extractable GST activity (Csiszár et al. 2001). The
lower ethylene production of autotrophic cells on an auxinfree culture medium could result in the enhanced GST and
GSH-PX activities of these tissues.
We found a high GSH-PX activity in our autotrophic line,
but not in the auxin-requiring cells. This seems to prove a
very effective acclimation mechanism. Despite the lower
SOD, CAT, and GR activities, the growth of the autotrophic
calli was not inhibited significantly in the presence of 100
mmol/L NaCl as compared with the autotrophic control, and
their H2O2 and MDA contents were lower than those in the
heterotrophic tissues. The growth of calli measured via the
fresh mass demonstrated that the autotrophic line was the
more resistant to exogenous H2O2. These are the first data todate indicating that an auxin autotrophic plant culture that has
passed through a certain degree of neoplastic transformation
exhibits a lower H2O2 sensitivity than an auxin heterotrophic
line.
In contrast with the considerable activities of the AOSscavenging SOD and CAT enzymes in the auxin heterotrophic
calli, it is the peroxidases and mainly the GSH-PX activity in
the autotrophic tissues that can ensure protection against
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Jolán Csiszár et al.
NaCl stress and presumably other stress-induced oxidative
damage.
Gronwald JW, Plaisance KL (1998) Isolation and characterization of
glutathione S-transferase isozymes from Sorghum. Plant Physiol
117: 877– 892
Acknowledgement. This work was supported by National Scientific
Foundation OTKA grants T 030259 and T 038392.
Habig WH, Pabst MJ, Jakoby WB (1974) Glutathione S-transferases.
The first enzymatic step in mercapturic acid formation. J Biol Chem
246: 7130–7139
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