INCREASED TOLERANCE TO OXIDATIVE STRESS IN TRANSGENIC PLANTS

1
INCREASED TOLERANCE TO OXIDATIVE STRESS IN
TRANSGENIC PLANTS THAT OVEREXPRESS CATALASE
 Photini V. Mylona1, Ioannis D. Delis1,
Athanasios S. Tsaftaris2 and Kalliopi A.
Roubelakis-Angelakis1 1Department of Biology,
University of Crete, 71409 Heraklion, Crete,
Greece 2Department of Genetics and Plant
Breeding, Aristotle University of Thessaloniki,
54006 Thessaloniki, Greece
INTRODUCTION Aerobic organisms gain significant
energetic advantage by using molecular oxygen as
the terminal oxidant in respiration. However,
they can be severely damaged by partially reduced
oxygen species, which are produced through normal
or aberrant metabolic processes, or as a
consequence of various environmental stresses.
The toxic effects of reactive oxygen species
(ROS) -termed oxidative stress- are circumvented
by a combination of enzymic and non-enzymic
mechanisms that can reduce oxidative stress by
converting ROS to harmless compounds. Among the
enzymes involved in the defense against oxidative
stress, catalase is a candidate enzyme of great
expectations, as it catalyzes the conversion of
H2O2 to oxygen and water at an extremely rapid
rate. Several abiotic stress conditions have
been proposed to generate oxidative stress in
plants (Foyer and Mullineaux, 1994). Herbicide
and salt stress can affect several physiological
phenomena and key metabolic pathways in plants
(Borsani et al., 2001). In particular, the
redox-cycling methyl viologen intercepts
electrons from various electron transport chains,
thereby reducing oxygen to syperoxide (Dodge,
1994). Most of the generated superoxide is
dispropotionated to H2O2, spontaneously or
enzymically by the action of superoxide dismutase
(Bowler et al., 1992). The combined action of
methyl viologen and light induces oxidative
stress in chloroplasts, since photosystem I is
thought to be the primary site of electron
capture by methyl viologen (Fujii et al., 1990).
Leaf discs from transgenic tobacco plants that
constitutively express the Cat1 gene in
antisenese orientation showed increased levels of
light-mediated damage upon treatment with methyl
viologen (Willekens et al., 1997). Furthermore,
the photosynthetic activity in plants can be a
target for salt stress (Leung et al., 1994).
Additionally, both ionic and osmotic stress
imposed by salinity have been proposed to induce
ROS generation in chloroplasts resulting in
inhibition of photosynthesis (Price and Hendry,
1991). We have used tobacco as a model system to
study the possible involvement of catalase as
protection against ROS formed in/or diffused
through cytoplasm in certain conditions of biotic
or abiotic stress. Thus, an enhancement of the
antioxidant capacity in the cytoplasm was
attempted. For this purpose, transgenic tobacco
genotypes expressing the tobacco Cat1 gene in the
cytosol have been constructed, by deleting the
peroxisome target motif (PTM). T1 progeny was
analyzed for catalase specific activity in
various developmental stages. Among them, a few
transgenic tobaccos, with increased specific
activities compared to wild type plants, were
examined to various abiotic factors inducing
oxidative stress, such as NaCl and methyl
viologen. MATERIALS AND METHODS Plant material
pBI.PVM1 are transgenic lines of N. tobaccum cv.
Petit Havana SR1, over-expressing
cytoplasm-targeted catalase. pBI.PVM1 contains
the mutated tobacco Cat1 gene, lacking the
transit peptide sequence which directs the
catalase enzyme to peroxisome (Mylona,
unpublished data). T1 progeny homozygous for the
transgene locus was used for analysis. Seedlings
were germinated on MS solid medium (Murashige and
Skoog, 1962) under low irradiation (PAR, 400-700
nm) and, after to soil, grown under low light
condition (80-100 µmole/m/s, 16h light/8h dark
cycle, 25.5 0.5 oC). Table 1. Catalase
specific activity of T1 progeny homozygous for
the mutated tobacco Cat I gene. SR1
Non-transformed tobacco plants (cv
SR1).
Immunodetection of catalase The procedure was
described by Towbin et al. (1979). The antibody
against tobacco CAT1 was produced in our
laboratory. Treatment of leaf discs with methyl
viologen Twelve discs with diameter of 10 mm
excised from leaves of 3-month-old tobacco plants
were floated in the solution that contained 5 µM
methyl viologen. Ion leakage of the leaf disc was
measured as conductivity of the medium under
light (100µmole/m s) at 250.5 oC for 26
h. Treatment of leaf discs with NaCl Twelve
discs with diameter of 10 mm excised from leaves
of 3-month-old tobacco plants were floated in the
solution that contained 400mM NaCl for 2 days
under continuous light (100µmole/m s) at 250.5
oC. Determination of chlorophyll The content of
chlorophyll in the leaf discs was determined
according to Holden (1965) and the values of
remaining chlorophyll content were determined as
described by Noji et al. (2001). Lipid
peroxidation Determination of malondialdehyde
(MDA) was used as an index of lipid peroxidation
in leaf discs treated with methyl viologen and
NaCl (Draper and Hadley, 1990). Figure 1.
Western blot analysis of T1 progeny homozygous
for the mutated tobacco Cat I gene. SR1e and SR1d
Non-transformed tobacco plants (cv SR1).
RESULTS AND DISCUSSION Catalase activity and
western blot analysis in T1 progeny T1 progeny
was analyzed for catalase specific activity in
various developmental stages. Plants with
different genotype were numbered as 1, 2, 4, etc
each genotype was represented 5 times (a, b, c, d
and e). Among them, several plants exhibited
increased catalase specific activity from 1.2- to
more than 2-fold compared to non-transformed SR1
plants (Table 1). In particular, the plants A5a,
A5c, 11a, 11b, 5d, which displayed the highest
catalase activity, were used to perform
experiments with abiotic factors that induce
oxidative stress. Catalase specific activity was
markedly increased at all the developmental
stages in these plants (data not show). However,
there were a few plants exhibiting lower catalase
activity compared to non-transformed SR1 plants.
These plants may represent examples at which the
insertion of a transgene produced co-suppression
(Flavell, 1994). Immunodetection of CAT1
protein on nitrocellulose membranes revealed that
the activity values acquired from the enzymic
assay were in accordance with the amount of the
protein present in leaf extracts. Plants selected
for increased catalase activity (A5c, A5a, 11b,
11a and 5d) also displayed a high content of
catalase protein, as shown in Fig. 1. Effect of
salt stress on T1 progeny To investigate the
possible role of overexpressed CAT1 in salt
tolerance, leaf discs from non-transformed SR1
and Cat1 mutated transgenic tobacco plants were
floated in a solution that contained 400mM NaCl
and placed under low light conditions. After 2
days ofNaCl treatment, chlorophyll bleaching was
observed in non-transformed SR1 leaf discs,
whereas leaf discs from transgenic plants
exhibited a relative tolerance. This was
confirmed by determining the remaining
chlorophyll content of each leaf disc after the
NaCl treatment (Fig. 2A). The wild type SR1
plants showed almost 50 reduction of chlorophyll
as compared with transgenics (A5c, 5d, 11b).
Monitoring changes in lipid peroxidation can
also assess oxidative damage, induced by NaCl. To
determine at what extent NaCl imposed oxidative
damage in wild type SR1 and transgenic tobacco
plants, we monitored changes in lipid
peroxidation by measuring the MDA level at 400mM
NaCl under low light conditions (Fig. 2B).
Transgenic plants exhibited decreased MDA levels
from 2-to almost 8-fold compared to wild type SR1
plants. A B Figure 2. Effects of NaCl
on leaf discs of transgenic tobacco. A, The
percentage of remaining chlorophyll content per
leaf discc after 400mM NaCl. B, Lipid
peroxidation after treatment with 400mM NaCl. MDA
levels were determined as described in Material
and Methods. SR1 Non-transformed tobacco (cv
SR1).
Effect of methyl viologen on T1 progeny Ion
leakage of Cat1 mutated and wild type SR1 leaf
discs challenged with 5µM methyl viologen under
low light conditions was studied. Leaf discs of
Cat1 mutated plants showed decreased ion leakage
after 6 hours of methyl viologen treatment (Fig.
3). This demonstrates that over-expression of
CAT1 leads to enhanced tolerance to oxidative
stress generated at the plasma membrane or in the
chloroplasts. However, ion leakage from Cat1
mutated leaf discs increased after 12 h of methyl
viologen treatment, suggesting that
over-expressed CAT1 cannot protect transgenic
tobacco against oxidative stress following
exposure to methyl viologen for several
hours. Figure 3. Ion leakage of Cat1 and
wild type SR1 leaf discs as a time-course methyl
viologen treatment. SR1 Non-transformed tobacco
(cv SR1). Monitoring changes in chlorophyll
content also assessed the oxidative damage
generated by methyl viologen. In particular, the
remaining chlorophyll content was determined in
each leaf disc after 26 h of methyl viologen
treatment (Fig. 4A). The Cat1 mutated plants
displayed 4 to 5 times higher percentage of
residual chlorophyll as compared to the wild type
SR1. Another parameter to determine the extent
of oxidative damage imposed by methyl viologen in
wild type SR1 and transgenic tobacco plants, was
to monitor changes in lipid peroxidation by
measuring the MDA after 26 h of methyl viologen
treatment (Fig. 4B). The results showed that MDA
level were 1.5- to 3-fold lower in leaf discs of
transgenic plants compared to wild type
SR1. A B Figure 4. Effects of methyl
viologen on leaf discs of transgenic tobacco. A,
The percentage of remaining chlorophyll content
per leaf discc after 5 µM methyl viologen. B,
Lipid peroxidation after treatment with 5 µM
methyl viologen MDA levels were determined as
described in Material and Methods. SR1
Non-transformed tobacco (cv SR1). REFERENCES Borsa
ni O, Valpuesta V, Botella MA. (2001), Plant
Physiol 126 1024- 1030 Bowler C, VanMontagu M,
Inze D (1992), Annu Rev Plant Physiol Plant Mol
Biol 43 83-116 Dodge AD (1994), In CH Foyer and
PM Mullineaux, eds, Causes of Photooxidative
Stress and Amelioration of Defence System in
Plants.CRC Press, Boca Raton, FL Draper H. H,
Hadley M (1990), Methods of Enzymology 186
421-431 Flavell RB (1994), Proc Natl Acad Sci USA
91 3490-3496 Foyer CH, Mullineaux PM (1994),
Causes of Photooxidative Stress and Amelioration
of Defence System in Plants. CRC Press, Boca
Raton, FL Fujii T, Yokoyama E, Inoue K, Sakurai
H (1990), Biochimica Biophysica Acta 1015
41-48 Holden (1965), In Chemistry and
Biochemistry of Plant Pigments. Academic Press,
London. Leung J, Bouvier-Durand M, Morris PC,
Guerrier D, Chedfor F, Giraudat J (1994), Science
264 1448-1452 Murashige T, Skoog F (1962),
Physiol Plant 15 473-497 Noji M, Saito M,
Nakamura M, Aono M, Saji H, Saito K. (2001),
Plant Physiol 126 973-980 Price AH, Hendry GAF
(1991), Plant Cell Environ 14 477-484 Towbin H,
Staehelin T, Gordon J (1979), Proc Natl Acad Sci
USA 76 4350-4354 Willekens H, Chamnongpol S,
Davey M, Schraudner M, Langebartels C, Van
Montagu M, Inze D, Van Camp W (1997), EMBO J 16
4806-4816