ROLE OF SUPEROXIDE DISMUTASE AND CATALASE IN VARIOUS ABIOTIC CONDITIONS CAUSING OXIDATIVE STRESS IN

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ROLE OF SUPEROXIDE DISMUTASE AND CATALASE IN
VARIOUS ABIOTIC CONDITIONS CAUSING OXIDATIVE
STRESS IN PLANTS Ioannis D. Delis, Efthimios A.
Andronis, Anastasia K. Papadakis, and Kalliopi
A. Roubelakis Angelakis Department of Biology,
University of Crete, P.O. Box 2208, 714 09
Heraklion, Crete, Greece

• H2O2 treatment
• Leaf discs from tobacco plants were also treated
  with a range of exogenous H2O2 concentrations
  (0100 mM) under constant light. The specific
  activity of SOD remained unchanged in treatments
  with low concentrations of H2O2 (up to 25 mM) and
  decreased at high concentrations (Fig 3),
  accompanied by reduction of the cytoplasmic
  isoenzyme (Fig. 6), probably due to its
  inhibition by H2O2. Catalase specific activity
  increased at low concentrations of H2O2 (2.55
  mM), concomitant with accumulation of "catalatic"
  isoezymes (Fig 5), as in salt treatments. At high
  concentrations (above 50 mM) catalase activity
  decreased.
•  
• Figure 5. Activity staining of SOD (A) and
  catalase (B) in H2O2 -treated tobacco leaf
  discs.
• Furthermore, treatment with exogenous H2O2
  caused severe membrane damage, which led to
  intense electrolyte leakage at 100 mM H2O2 there
  was an 2.2-fold increase compared to untreated
  leaf discs (Fig. 6).

INTRODUCTION Active oxygen species (AOS), and
more specifically superoxide anion radical
(O2-.), hydrogen peroxide (H2O2) and hydroxyl
radicals (.OH) are harmful molecules that are
continuously produced in higher plant cells
during normal aerobic metabolism. Exposure of
plants to extreme environmental conditions or
pathogen infections often results in generation
of AOS and subsequent oxidative stress (Asada,
1999). Toxic AOS levels initiate
self-perpetuating peroxidation of lipids, damage
nucleic acids and proteins and ultimately cause
cellular dysfunction and cell death. Living
organisms cope with oxidative stress by a range
of antioxidant strategies in addition to a
direct radical-scavenging activity performed by
chemical species, a chain of enzymic reactions
co-operate for the removal of the AOS (Asada,
1999 Bowler, 1994). The main antioxidant enzymes
are the superoxide dismutases (EC 1.15.1.1, SOD),
which are considered to act in the first line of
defense system, as they dismutate O2.- and
produce H2O2 in turn, H2O2 is eliminated by
peroxidases (EC 1.11.1.7, POX), catalases (EC
1.11.1.6, CAT), and also by the enzymes of
Halliwell-Asada cycle. Salt stress can affect
several physiological phenomena and key metabolic
pathways in plants and may give rise to oxidative
stress (Sehmer et al., 1995). Salinity damage to
plants has been attributed to a combination of
factors including mainly osmotic stress and
accumulation of toxic ions. In this work, we are
attempting to reveal any potential effect of salt
stress on AOS generation in tobacco leaf discs,
and the possible induction of the antioxidant
mechanism, specifically SOD and catalase.
Furthermore, we have correlated exogenously
applied H2O2 to the induction of SOD and
catalase.   MATERIALS AND METHODS Plant
Material Discs from fully expanded, but not
senescent, leaves of glasshouse grown Nicotiana
tabacum L. cv Xanthi plants, were used in all
experiments. Salt and H2O2 Treatments Twelve
discs with diameter of 10 mm excised from leaves
of tobacco plants were immersed in solutions
containing various concentrations of NaCl or H2O2
for 24 h or 14h respectively under continuous
light (100 µmole/m2 s) at 250.5 oC. O2.- and
H2O2 Detection Assays The generation of O2.- and
H2O2 from tobacco leaf discs was determined by
the chemiluminescence assay of lucigenin and
luminol, respectively (Papadakis and
Roubelakis-Angelakis, 1999). Protein Extraction
and Enzyme Assays Total proteins were extracted
from leaf tissue according to Siminis et al.
(1994). Determination of SOD and catalase
specific activity and activity staining were
performed as summarized by Siminis et al., 1994
and Papadakis et al., 2001. Determination of
Chlorophyll and Ion Leakage The extraction of
chlorophyll from the leaf discs was performed
according to Holden (1965) and the values of
remaining chlorophyll content were determined as
described by Noji et al. (2001). Ion leakage was
measured as conductivity of the medium. RESULTS
AND DISCUSSION Salt treatment Leaf discs from
tobacco plants treated with a range of
concentrations of NaCl exhibited increased levels
of O2-. intra- and extracellularly, compared to
untreated ones (Fig. 1). Intracellular O2-. and
H2O2 levels increased up to 40 mM NaCl, where the
levels were respectively 1.4- and 12-fold higher
compared to untreated leaf. In higher
concentrations, both AOS levels decreased.
Extracellular O2-. level was higher in all salt
treatments at 200 mM NaCl it was 11.7-fold
greater than in the untreated leaf discs. On the
other hand, H2O2 was not detected at all
extracellularly. exogenously applied H2O2 to the
induction of SOD and catalase.   F
igure 1. Intra- and extracellular O2.- and H2O2
levels in salt-treated tobacco leaf discs.
The photosynthetic activity in plants can be a
target for salt stress (Leung et al., 1994).
Treatment of tobacco leaf discs with NaCl caused
severe bleaching of chlorophylls under constant
light (Fig. 2). aT 100 mM NaCl, total chlorophyll
content decreased by 70 compared to untreated
tobacco leaf discs. Figure 2.
Chlorophyll content in salt-treated tobacco leaf
discs. SOD specific activity slightly
increased in salt-treated leaf discs (Fig. 3).
The contribution of individual SOD isoenzymes to
total SOD activity was determined by performing
SOD assays directly on protein extracts separated
in non-denaturating gels. Three bands of SOD
activity were detected in tobacco leaf tissue in
a previous study (Papadakis et al, 2001), we have
shown that the upper band represents a
mitochondrial MnSOD, the band with higher
mobility represents the cytosolic Cu/ZnSOD
isoenzyme and the lowest band represents a
chloroplastic FeSOD. In salt-treated leaf discs
the content of cytoplasmic and chloroplastic SOD
isoenzymes increased (Fig. 4A).
Figure 3. SOD and catalase
specific activity in salt- and H2O2- treated
tobacco leaf discs. Catalase, a major
antioxidant defense enzyme that removes H2O2 with
high efficiency, was also studied in salt-treated
leaf discs. The multiple catalase isoforms from
both species were immunologically related.
Previous studies in our lab showed that tobacco
catalase isoenzymes consisted of two subunits, a-
and ß, with molecular mass of 57 and 56 kD,
respectively The cathodic and anodic catalase
isoenzymes consisted exclusively of subunits a-
and ?-, respectively the de novo accumulation of
the catalase ?-subunit gave rise to the less
catalatic, acidic isoenzymes, whereas the
catalatic, basic isoenzymes derived from the de
novo accumulation of ?-subunit (Siminis et al.,
1994). The specific activity of catalase
increased at low concentrations of NaCl (1050 mM
with a concomitant induction of isoenzymes
exhibiting higher catalatic activity (Figs 3, 4).
On the other hand, CAT activity decreased at high
salt concentrations (above 50 mM).
Figure 4. Activity staining of SOD
(A) and catalase (B) in salt-treated tobacco
leaf discs.

Units mg-1 prot.
150
100
50
30
20
10
0
0.0
2.5
5.0
7.5
10.0
50
100
mM H
O
2
2