RESPONSE OF THE ANTIOXIDATIVE MACHINERY TO SALINITY STRESS IN NICOTIANA TABACUM PLANTS AND LEAF DISC

1
RESPONSE OF THE ANTIOXIDATIVE MACHINERY TO
SALINITY STRESS IN NICOTIANA TABACUM PLANTS AND
LEAF DISCS 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
Catalase specific activity in control Z232
plants was 2-fold higher compared to control wild
type plants (Fig. 6A). Furthermore, catalase
specific activity increased 1.25-fold compared to
controls in Z232 plants, in response to severe
stress, in contrast to wild type plants where it
exhibited a 1.4-fold increase under moderate
stress (200mM NaCl) and decreased under severe
stress (400mM NaCl). In addition isoenzymic
analysis revealed that the first and third
isoenzymes are responsible for the detoxification
of H2O2 under NaCl stress (Fig. 6B) and
immunodetection of catalase indicated induction
of the a-subunit in salt stressed plants (Fig.
6C). Figure 6. Catalase specific
activity (A), isoenzymic analysis (B) and
immunoreactive protein (C) in salt-treated wild
type and transgenic Z232 tobacco plants. APO
specific activity in Z232 plants was lower than
in wild type plants as expected in a plant with
increased catalase specific activity (Fig. 7). In
wild type plants a 1.5-fold increase was measured
under moderate NaCl stress (200mM), while the
specific activity decreased under severe stress.
The specific activity in Z232 plants exhibited no
significant change. Figure 7. Ascorbate
peroxidase specific activity in salt-treated wild
type and Z232 transgenic tobacco
plants. CONCLUSIONS Overall, in salt treated
tobacco leaf discs both O2- and H2O2 were
detected intracellularly, and only O2-
extracellularly. In wild type tobacco plants the
highest ROS titers were induced by 200 mM NaCl
which were lower at higher (400 mM) salt
concntrations. In both cases the generated ROS
were sufficient to induce the expression of SOD
and catalase, respectively which paralleled the
pattern of ROS increases. In genetically modified
plants overexpressing CAT1 (Z232) no ROS were
detected and the specific activity of SOD and APO
was significantly lower than that of the wild
type. The specific activity of catalase in
genetically moified plants was greater in all
salt treatments. REFERENCES Alexandrou D.
(2000) Modificaiton of catalase (CAT1) expression
in transgenic tobacco plants. University of
Crete, Biology Departrment, Plant Physiology and
Biochemistry Laboratory Asada K., (1999) Annu.
Rev. Plant Physiol. Plant Mol. Biol. 50
601-639. Bowler C., Van Camp W., Van Montagu M.,
Inze D., (1994) Crit. Rev. Plant Sci.
13199-218. Holden M., (1965) Chemistry and
Biochemistry of Plant Pigments. Academic Press,
London. Leung J, Bouvier-Durand M, Morris PC,
Guerrier D, Chedfor F, Giraudat J, (1994) Science
2641448-1452. Louise F, Zelitch BI, Havir EA
(1998) Manipulation of Catalase Levels Produces
Altered Photosynthesis in Transgenic Tobacco
Plants. Plant Physiol. 116 259269 Mittler R
(2002) Oxidative stress, antioxidants and stress
tolerance. Trends Plant Sci. 7 405-410 Noji M,
Saito M, Nakamura M, Aono M, Saji H, Saito K.,
(2001) Plant Physiol 126973-980. Papadakis AK,
Roubelakis-Angelakis KA (1999) Plant Physiol 121
197-245. Papadakis AK, Siminis CI,
Roubelakis-Angelakis KA (2001) Plant Physiol 126
434-444. Papadakis A.K., Roubelakis-Angelakis
K.A., (2002) Plant Physiol. Biochem. (in
press). Perez FJ, Villegas D, Mejia N (2002)
Ascorbic acid and flavonoid-peroxidase reaction
as a detoxifying system of H2O2 in grapevine
leaves. Phytochemistry 60 573-580 Sehmer L.,
Alauri S.B., Dizengremel P., (1995) J. Plant
Physiol. 147144-151. Siminis CI, Kanellis AK,
Roubelakis-Angelakis KA (1994) Plant Physiol 105
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INTRODUCTION Reactive oxygen species (ROS), and
more specifically superoxide anion (O2.-), and
hydrogen peroxide (H2O2) are harmful molecules
that are continuously produced in higher plant
cells during normal aerobic metabolism. Exposure
of plants to extreme environmental conditions
often results in generation of ROS and subsequent
oxidative stress (Asada, 1999). Toxic ROS 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 ROS
(Asada, 1999 Bowler, 1994). The main antioxidant
enzymes are the superoxide dismutases (EC
1.15.1.1, SOD), which dismutate O2.- and produce
H2O2 in turn, H2O2 is eliminated, catalases (EC
1.11.1.6, CAT), and also by ascorbate peroxidase
(APO EC 1.11.1.11) which is one of 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 the correlation of salt
stress and ROS generation in tobacco plants and
leaf discs, and the respective response of the
antioxidant machinery, specifically SOD, CAT, and
APO in wild type and transgenic tobacco
plants.   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 addition
genetically modified Z232 plants overexpressing
CAT1 were used, as well as the wild type
Nicotiana tabacum cv. Petit Havana (SRI), which
the Z232 genotype derived from. Salt Treatments
Twelve discs with diameter of 10 mm excised from
leaves of tobacco plants were immersed in
solutions containing various concentrations of
NaCl for 24 h under continuous light (100
µmole/m2 s) at 250.5oC. Plants were grown at
250.5oC and a 16/8h photoperiod, in perlite, and
fed with modified 20 MS (Murashige and Skoog,
1962) with no sucrose or hormones, supplemented
with various concentrations of NaCl. O2.- and
H2O2 Detection Assays The generation of O2.- and
H2O2 from tobacco leaves and leaf discs was
determined by the chemiluminescence assay of
lucigenin and luminol, respectively (Papadakis
and Roubelakis-Angelakis, 1999). Protein
Extraction and Enzyme Assays Total leaf proteins
extraction and determination of SOD, catalase,
and APO specific activity, activity staining and
immunodetection were performed as summarized by
Papadakis et al., 2001. RESULTS AND
DISCUSSION Nicotiana tabacum cv. Xanthi 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.-
levels increased up to 400 mM NaCl where the
levels were 3-fold higher compared to untreated
leaf, while H2O2 levels increased up to 40 mM
NaCl, where the levels were approximately
5.2-fold higher compared to untreated leaf.
Extracellular O2.- level was higher in all salt
treatments at 400 mM NaCl it was 17-fold greater
than in the untreated leaf discs. On the other
hand, extracellular H2O2 was not detected.
  Figure 1. Extra- (A) and intra-cellular
(B) O2.- (red) and H2O2 (blue) levels in
salt-treated tobacco leaf discs. SOD specific
activity slightly increased in salt-treated leaf
discs (Fig. 2A). 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 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 (Papadakis et al, 2001). In
salt-treated leaf discs the content of
cytoplasmic and chloroplastic SOD isoenzymes
increased (Fig. 2B). Figure 2. SOD
specific activity (A) and isoenzymic analysis (B)
in salt- treated tobacco leaf discs.
Catalase, a major antioxidant defense
enzyme that removes H2O2 with high efficiency,
was also studied in salt-treated leaf discs.
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. On the
other hand, CAT activity decreased at high salt
concentrations (above 50 mM) (Figs 3A,
3B). Figure 3. Catalase
specific activity (A), isoenzymic analysis (B)
and immunodetection (C) in salt-treated tobacco
leaf discs.  Transgenic plants In order to
investigate whether the antioxidative mechanism
may offer protection against salinity stress,
transgenic plants expressing cytosolic CAT1 were
developed using the Z232 plasmid, which was a
kind contribution from the laboratory of Dr.
Zelitch and is described in the relevant
publication (Louise et al., 1998). The
transgenic plants, genotype Z232, carrying the
plasmid were constructed in our laboratory
(Alexandrou, 2000). The Z232 plants, which
exhibit increased CAT specific activity, were
stressed with a range of NaCl concentrations. ROS
were not detectable in the leaves of Z232 plants
in contrast to wild type plants which exhibited
increased levels of O2- and H2O2 under moderate
stress of 100 and 200 mM respectively, but
decreased when subjected to more severe stress
(Fig. 4). The ROS concentration under moderate
stress was 1.3-fold higher compared to untreated
plants. Figure 4. O2- (A) and
H2O2 (B) levels in salt treated SRI and Z232
plants The specific activity of SOD increased
1.5-fold compared to control in wild type plants
at 200 mM NaCl (Fig. 5A). More severe stress
resulted in a decrease of specific activity. The
specific activity of SOD in Z232 plants was the
same for untreated plants and remained almost
unchanged for all NaCl concentrations.
Furthermore the isoenzymic analysis revealed that
the cytoplasmic Cu/ZnSOD and the chloroplastic
FeSOD were primarily responsible for the
detoxification of O2- produced under salt stress
(Fig. 5B). Figure 5. SOD
specific activity (A) and isoenzymic analysis (B)
in salt- treated wild type and Z232 transgenic
tobacco plants.