Biologia 66/6: 1052—1059, 2011
Section Cellular and Molecular Biology
DOI: 10.2478/s11756-011-0120-4
Alleviation of ultraviolet-C-induced oxidative damage through
overexpression of cytosolic ascorbate peroxidase
Saurabh C. Saxena1, Pankaj K. Joshi1, Bernhard Grimm2 & Sandeep Arora3*
1
Department of Biochemistry, GB Pant University of Agriculture & Technology, Pantnagar – 263145, Uttarakhand, India
Department of Plant Physiology, Institute of Biology, Humboldt University, Berlin, Germany
3
Department of Molecular Biology & Genetic Engineering, GB Pant University of Agriculture & Technology, Pantnagar –
263145, Uttarakhand, India; e-mail: [email protected]
2
Abstract: Experiments were conducted to investigate the relationship between ultraviolet (UV) C-induced oxidative damage and the activity of ascorbate peroxidase (APX), using transgenic tobacco (Nicotiana tabacum L. cv. Petit Havana)
plants overexpressing cytosolic APX gene (apx1). Transgenic plants having 2.3 fold higher total APX activity, as compared
to the wild type plants, showed normal morphological characters. Exposure of 70-day-old plants to fixed intensity UV-C
radiation caused an increase in the malondialdehyde (MDA) content in wild type as well as transgenic plants. However,
the wild type plants showed significantly higher (p < 0.05) lipid peroxidation as compared to the transgenic plants. Higher
proline accumulation was recorded in transgenic plants as compared to the wild type plants, after 24 hours of UV-C exposure. Although the ascorbate content decreased continuously with increasing exposure to UV-C radiation, yet the wild type
plants exhibited higher ascorbate levels than the transgenic plants. A marked difference in H2 O2 content, between the wild
type and transgenic plants, was consistently observed up to 20 hours of UV-C exposure. A direct correlation of ascorbate,
MDA and H2 O2 levels was recorded with the extent of oxidative stress, signifying that these could be used as potential
bio-marker molecules for oxidative stress. The results clearly demonstrate that overexpression of cytosolic APX can protect
tobacco plants from UV-C-induced oxidative damage.
Key words: ascorbate peroxidase; Nicotiana tabacum; UV-C radiation; oxidative stress.
Abbreviations: APX, ascorbate peroxidase; MDA, malondialdehyde; ROS, reactive oxygen species; UV, ultraviolet.
Introduction
Ultraviolet (UV) radiation, reaching the earth’s surface has increased dramatically due to various anthropogenic activities. It has been the cause of a multitude
of physiological and biochemical perturbations in living organisms. The affects include retardation of plant
growth and alteration in metabolic processes such as
photosynthesis, respiration, etc. Excessive production
of reactive oxygen species (ROS) is one of the major consequences of exposure to such ionizing radiations. There are specific evidences suggesting that UV
radiation-induced damage is related to acceleration in
ROS generation (Zacchini & Agazio 2004), leading to
oxidative stress.
ROS are produced in response to various abiotic
stresses, including ionizing UV radiation. Most of the
time their levels are rigorously controlled and kept compartmentalized. But excessive and uncontrolled generation of ROS inactivates enzymes and damages important cellular components, apart from disturbing the
electron transport (Joshi et al. 2011). Thus, the deleterious effects of UV radiation on plant growth and development can be assessed by measuring the extent of free
radical-induced damage. Free radical-induced lipid peroxidation causes leakage of cellular electrolytes, thereby
damaging cellular integrity (Du & Jin 2000). Further,
the presence of an array of photosensitive pigments in
plants makes them prone to oxidative damage (Asada
1994; Blokhina et al. 2003).
During the course of evolution, plants have developed several mechanisms to protect themselves against
oxidative damage. The antioxidant molecules such as
ascorbate, glutathione and α-tocopherol and protective
enzymes like ascorbate peroxidase (APX), superoxide
dismutase and monodehydro ascorbate reductase, are
essential components of a plant’s anti-oxidative defence
system (Jaleel et at. 2009).
In plants exposed to ionizing radiation, hydrogen
peroxide is one of the most potent damaging molecules.
It is a strong oxidant and is known to be a potential
inhibitor of photosynthesis (Prasad et al. 2004). Exces-
* Corresponding author
c 2011 Institute of Molecular Biology, Slovak Academy of Sciences
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Role of APX under UV-C-induced oxidative damage
sive accumulation of H2 O2 is reported in many plants,
exposed to abiotic stresses (Jaleel et al. 2009). Catalase and various peroxidase enzymes have been implicated in the metabolism of this stress (Yang et al. 2009).
However, destruction of catalase under photo-oxidative
stress (Foyer et al. 1994) and the high diffusion potential of H2 O2 through cellular membranes further aggravate the crisis, caused by stress-induced H2 O2 accumulation (Joshi et al. 2011). Under these conditions
the possible protection provided by increased activities
of peroxidases, seems to be specific for photo-oxidative
stress. Computer simulation models have shown that
APX is one of the key enzymes for detoxifying H2 O2 in
plant cells (Polle 2001).
APX (EC 1.11.1.11) is a widely distributed antioxidant enzyme in plant cells (Wang et al. 1999). It
is a part of ascorbate-glutathione cycle, which utilizes
ascorbate as its specific electron donor to reduce H2 O2
with the concomitant generation of monodehydro ascorbate, a univalent oxidant of ascorbate. APX activity has
been shown to increase in response to a number of stress
conditions (Jaleel et al. 2009). Thus, APX in combination with an effective ascorbate-glutathione cycle can
prevent the toxic accumulation of H2 O2 in photosynthetic organisms, exposed to oxidative stress.
Transgenic plants with ectopically enhanced activity of APX are expected to have an improved tolerance against oxidative stress. Therefore, the present
experiments were conducted to evaluate the potential
of transgenic tobacco plants, overexpressing cytosolic
APX, in alleviating the UV-C-induced oxidative damage.
Material and methods
Plant material and growth conditions
Nicotiana tabacum (L.) cv. Petit Havana was previously
transformed with pea cytosolic APX (apx1) cDNA, using
Agrobacterium-mediated plant transformation (Pitcher et
al. 1994). The construct ptpApx was placed in Agrobacterium tumefaciens strain C58C1rifR (pGV2260), under the
control of CaMV-35S promoter. Wild type and transgenic
tobacco plants were used in the present experiments. Transgenic and wild type tobacco seeds were used to raise plants
under controlled conditions of temperature (26 ± 1 ◦C) and
relative humidity 70% with a 16h/8h day/night cycle.
Presence of apx1 cDNA construct in the transgenic
plants was confirmed through PCR using apx1 and nptII
gene specific primers. Total genomic DNA was isolated and
subjected to PCR analysis by amplifying the apx1 and nptII
genes. The apx1-specific primers (5’-GGCTGTTGAGAAG
TGCAG-3’ and 5’-CAGCAAAAGCGCAACGG-3’) and
nptII-specific primers (5’-AGATGGATTGCACGCAGG-3’
and 5’-AGCGGCGATACCGTAAAG-3’) were used as forward and reverse primers, respectively.
Seventy-day-old wild type and transgenic plants were
exposed to UV-C radiation (254 nm, 50 kJ/m2 ), provided by
30 W germicide fluorescent lamp (Osram Sylvania Danvers,
USA) for increasing intervals of time. Samples were taken at
regular intervals with increasing UV-C exposure time points
and analyzed for various stress-marker molecules.
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APX activity
For APX assay, 0.2 g leaves were homogenized in 2 mL of
100 mM sodium phosphate buffer (pH 7.0) containing 5 mM
ascorbate, 10% glycerol and 1 mM EDTA. Enzyme activity
was determined in 1 mL reaction mixture containing 50 mM
potassium phosphate buffer (pH 7.0), 0.1 mM ascorbate, 0.3
mM H2 O2 , and 50 µL of the enzyme extract. The decrease
in absorbance of the reaction mixture was recorded at 290
nm (ε = 2.8 mM−1 cm−1 ) (Chen & Asada 1989). Protein
concentration was determined according to Bradford (1976)
and the activity of APX was expressed as µmol/min per mg
of protein.
Ascorbate content
Ascorbate content was measured volumetrically (Thimmaiah 1999). One g of leaf sample was homogenized in 10 mL of
4% oxalic acid. The homogenized extract was centrifuged at
10,000 × g for 30 min. One mL of supernatant was mixed
with 2 mL of 4% oxalic acid. The resultant solution was
titrated against 2,6-dichlorophenol indophenol dye. Amount
of ascorbate in the sample was calculated by using a standard solution of 100 µM ascorbate.
Free proline content
Free proline was determined by the method of Bates et al.
(1973). One gram of leaf material was homogenized in 10
mL of 3% sulfosalicylic acid and the homogenate was centrifuged at 12,000 × g for 20 min at room temperature. Two
mL of supernatant was mixed with 2 mL of glacial acetic
acid and 2 mL of acid ninhydrin reagent. The contents were
incubated for one hour at 100 ◦C. After incubation the reaction was terminated in an ice bath. To each tube of reaction mixture, 4 mL of toluene was added to extract the
chromophore. The absorbance of chromophore-containing
toluene layer was measured at 520 nm. Concentration of
proline in the sample was computed from a standard curve
of L-proline. Results were expressed in µg of proline/g of
fresh weight.
Malondialdehyde (MDA) content
The procedure of Heath and Packer (1968) was followed for
measuring the MDA content. Leaf material was homogenized in 0.25% (w/v) 2-thiobarbituric acid prepared in 10%
(w/v) trichloroacetic acid. The homogenate was incubated
at 95 ◦C for 30 min and then centrifuged at 10,000 × g for 30
min. Absorbance of the supernatant was measured at 532
nm and 600 nm. Absorbance at 600 nm was subtracted from
the absorbance at 532 nm for non-specific absorbance. The
concentration of MDA was calculated by using an extinction
coefficient of 155 mM−1 cm−1 .
Hydrogen peroxide content
Hydrogen peroxide was measured spectrophotometrically
after reaction with potassium iodide (Alexieva et al. 2001).
Freshly harvested leaf material was homogenized in 0.1%
(w/v) trichloroacetic acid and centrifuged at 10,000 × g for
30 min at 4 ◦C. The reaction mixture consisted of 0.5 mL of
supernatant, 0.5 mL of 0.1 M potassium phosphate buffer
and 2 mL of 1 M KI reagent. The reaction was allowed
to develop for 1 hour in dark and absorbance of the resultant solution was measured at 390 nm. The concentration
of H2 O2 was calculated using a standard curve of H2 O2 .
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S.C. Saxena et al.
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Table 1. Growth parameters of 70-day-old Nicotiana tabacum plants.a
a
Growth parameters
Wild type plant
Seed germination
Shoot fresh weight/plant
Root fresh weight/plant
Area of largest leaf
Root length
Shoot length
Vigor index
94
850
25
0.30
6
4.6
± 4.5 %
± 40 mg
± 3 mg
± 0.02 cm2
± 0.4 cm
± 0.35 cm
996.4
Transgenic plant
96
1600
40
0.48
8
6.5
± 3.6 %
± 90 mg
± 3 mg
± 0.036 cm2
± 0.7 cm
± 0.4 cm
1392.0
Data plotted are an average of three independent experiments with two replicates ± SE (n = 3).
Fig. 1. PCR analysis of transgenic Nicotiana tabacum plants for presence of apx1 gene (a) and nptII gene (b), using specific primers.
Lanes: M, molecular weight marker (100 bp ladder); 1, positive control (PCR of plasmid); 2, negative control (PCR of genomic DNA
from wild type plants); 3, PCR of genomic DNA from transgenic line.
Statistical analysis
All experiments were carried out with three independent determinations, having two replicates each. Analysis of variance (ANOVA) and t-test were used for analyzing significant
differences between the two genotypes (p ≤ 0.05).
Results
Percent seed germination and growth of 70-day-old
transgenic plants, measured in terms of shoot and root
fresh weight, shoot and root length, leaf area and vigor
index was comparable with the wild type plants (Table 1). PCR analysis confirmed the presence of transgene in the transgenic plants (Fig. 1). The growth profile of transgenic plants was recorded to be better than
the wild type plants. Exposure of tobacco plants to increasing intervals of UV-C radiation induced a significant increase in total APX activity, in wild type as well
as transgenic plants. In absence of UV-C stress, transgenic plants recorded a 2.3 fold higher APX activity
over that of control plants (Fig. 2). Further the APX
activity in the leaves of transgenic plants was significantly higher than the wild type plants, at all UV-C
exposure time points.
Proline levels increased with an increase in the
duration of UV-C exposure, in wild type as well as
transgenic plants. However, at respective UV-C exposure time points, the accumulation of proline in transgenic plants was significantly higher than the wild type
plants. At 20 and 24 hours of UV-C exposure, leaves
of transgenic plants showed 59% and 101% higher proline accumulation over wild type plants, respectively
(Fig. 3). Interestingly, under control conditions also the
proline content was higher in transgenic plants than in
the wild type plants.
Under control conditions (0 hour exposure) no staUnauthenticated
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Role of APX under UV-C-induced oxidative damage
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Fig. 2. Ascorbate peroxidase activity in the leaves of wild type and transgenic Nicotiana tabacum plants exposed to UV-C radiation
for different intervals of time. Line above bars represents mean ± SE (n = 3). The treatments are significantly different (p ≤ 0.05)
between two genotypes.
Fig. 3. Proline content in the leaves of wild type and transgenic Nicotiana tabacum plants exposed to UV-C radiation for different
intervals of time. Line above bars represents mean ± SE (n = 3). The treatments are significantly different (p ≤ 0.05) between two
genotypes.
tistically significant difference in MDA content was
recorded between wild type and transgenic plants. However, at any other UV-C exposure time point, MDA
accumulation was higher in the wild types plants, as
compared to the transgenic plants. Exposure to 20 and
24 hours of UV-C radiation brought about an 8.4 and
10 fold increase in MDA contents in transgenic plants,
whereas under similar conditions the wild type plants
showed a 13.5 and 16 fold increase in MDA contents,
respectively (Fig. 4).
Ascorbate content, in the leaves of transgenic and
wild type plants, decreased significantly with an increase in the duration of UV-C exposure (Fig. 5). At all
levels of UV-C exposure, ascorbate content was lower in
the leaves of transgenic plants than in wild type plants.
At 0 and 24 hours of UV-C exposure the wild type
plants had 1.5 and 2.2 fold higher ascorbate content
than the transgenic plants, respectively.
Hydrogen peroxide content increased progressively
in the leaves of wild type as well as transgenic tobacco
plants, concomitant with an increase in UV-C exposure time. However, at any given time the transgenic
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S.C. Saxena et al.
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Fig. 4. Malondialdehyde content in the leaves of wild type and transgenic Nicotiana tabacum plants exposed to UV-C radiation for
different intervals of time. Line above bars represents mean ± SE (n = 3). The treatments are significantly different (p ≤ 0.05) between
two genotypes.
Fig. 5. Ascorbate content in the leaves of wild type and transgenic Nicotiana tabacum plants exposed to UV-C radiation for different
intervals of time. Line above bars represents mean ± SE (n = 3). The treatments are significantly different (p ≤ 0.05) between two
genotypes.
plants accumulated significantly lower levels of H2 O2 ,
as compared to the wild type plants. The difference in
the H2 O2 levels of transgenic and wild type plants was
more prominent at lower UV-C exposure time points,
than at higher exposure time points. After four hours
of UV-C exposure, transgenic plants accumulated 62%
lower H2 O2 than did the wild type plants; while at 24
hours of UV-C exposure transgenic plants accumulated
only 13% lower H2 O2 than the wild type (Fig. 6).
Discussion
Production of ROS is a common denominator in the
cellular response to several environmental stresses including UV-C radiation. However, the understanding
of the cellular mechanism(s) that mediates plant responses to UV stress is still inconclusive. Present results indicate that the imposition of UV stress leads to
a build up of ROS, resulting in initiation of oxidative
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Role of APX under UV-C-induced oxidative damage
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Fig. 6. Hydrogen peroxide content in the leaves of wild type and transgenic Nicotiana tabacum plants exposed to UV-C radiation
for different intervals of time. Line above bars represents mean ± SE (n = 3). The treatments are significantly different (p ≤ 0.05)
between two genotypes.
damage at the cellular level. This activates the cellular
defence machinery and leads to an increase in the activity of antioxidant enzymes like APX (Zacchini et al.
2004). Cellular protection machinery functions to interrupt the cascades of uncontrolled oxidation reactions.
Enhanced APX activity in plants is an adaptive
mechanism under photo-oxidative stress. Overexpression of cytosolic APX in tobacco plants improved their
tolerance to UV-C-induced oxidative stress. An increase
in APX activity in plants exposed to UV radiation has
been reported by Yannarelli et al. (2006). However,
plants possess only a limited ability to increase the activity of APX for combating oxidative stress. Overexpression of APX could easily augment this limited capacity of the plants to detoxify H2 O2 ; thereby strengthening the antioxidant defence system of the affected
plant. An increase in total APX activity under increasing levels of UV-C stress could result in improved protection against ROS by detoxifying H2 O2 . This might
function by preventing the downstream cascading reactions of H2 O2 in conjunction with superoxide radicals.
Exposure to high energy UV-C radiation induces
the production of singlet oxygen in plants that can also
cause oxidative damage in the host tissue. Proline has
been reported to quench singlet oxygen and hence acts
as an antioxidant compound under stress (Matysik et
al. 2002). Our results clearly demonstrate that the UVC-induced proline accumulation is positively correlated
with the UV-C exposure time. This implies that transgenic plants having higher proline accumulation possess
better tolerance to UV stress than the wild type plants.
Free radical generation is one of the initial cytochemical responses of plants exposed to ionizing radiations. These free radicals potentially initiate lipid
peroxidation, leading to extensive membrane damage
(Sreenivasulu et al. 2007). Du et al. (2003) have re-
ported an increase in MDA content in Taxus cuspidata
exposed to UV-C radiation. In our experiments, a linear
relationship was observed between the extent of UVC-induced oxidative damage and MDA accumulation.
Thus MDA content can be used as a qualitative index
to measure the free radical induced damage in living
tissues. In the present experiments transgenic plants
recorded better tolerance to UV-C-induced lipid peroxidation, as compared to the wild type plants. This
improved capacity of transgenic plants is attributed to
the increased APX activity, translating to lower intracellular levels of H2 O2 .
With increasing UV exposure the ascorbate concentration decreased continuously, indicating its greater
utilization under stress. Ascorbate is an important component of a plant’s anti-oxidative defence system and
plays a protective role against stress induced ROS
(Prasad et al. 2004). Ascorbate, apart from acting as a
direct free radical scavenger, also functions as a specific
substrate for APX. Among various H2 O2 -scavenging
enzymes, cytosolic APX occupies a prime position under stress, due to its higher affinity for H2 O2 (Wang
et al. 1999). Thus transgenic plants had lower ascorbate levels than the wild type plants, due to persistently higher APX activity. A lower ascorbate content
was recorded in Solanum lycopersicum under temperature and radiation stress (Rosales et al. 2006), signifying
higher utilization of ascorbate under stress.
Hydrogen peroxide formed primarily in the chloroplasts and mitochondria could potentially diffuse from
these organelles into the cytosol and is then scavenged
by APX located in the cytosol (Ishikawa et al. 1993;
Takeda et al. 1995). Zacchini et al. (2004) observed
an increase in hydrogen peroxide content in Nicotiana
tabacum calli, exposed to UV-C radiation. A continuous increase in H2 O2 levels with increasing UV-C exUnauthenticated
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posure signifies a continuous production of H2 O2 under
stress. The experimental data clearly reveal that even
transgenic plants have a limit to which they can detoxify H2 O2 . At higher photo-oxidative stress levels, (20
and 24 hours of UV exposure), even transgenic plants
suffer from the noxious ionizing effects of UV-C radiation. Thus after 20 hours of UV-C exposure, even the
ectopically strengthened antioxidant defence machinery
of the transgenic plants fails to counteract the cytotoxic
effects of H2 O2 over accumulation.
Davletova et al. (2005) have reported that lack of
apx1 activity results in the oxidation of chloroplastic
proteins and the collapse of entire chloroplastic H2 O2 scavenging system in Arabidopsis thaliana. These observations suggest that the activity of cytosolic APX might
also be important for chloroplast protection. It has also
been reported that chloroplasts are extremely sensitive
to external application of H2 O2 (Asada 2000) and the
chloroplastic APX isoforms are inactivated by increased
H2 O2 (Mano et al. 2001). These observations support
the improved tolerance of transgenic plants against UVC stress, as recorded in the present experiments.
Under limited-stress conditions, the inherent antioxidative defence system provides adequate protection
against stress-induced ROS. Our results indicate that
protracted exposure to UV-C radiation caused impairment of the defence machinery in plant tissues, triggered by an uncontrolled burst of ROS and an inadequate ambient increase in the activities of antioxidant
enzymes. This leads to photo-oxidative stress, causing
loss of function and tissue destruction in a programmed
fashion.
Thus a higher APX activity of transgenic plants
seems to be the main cause for improved growth characteristics and enhanced tolerance of the transgenic plants
to UV-C-induced oxidative stress. As ROS are a common denominator in plants exposed to a variety of environmental stresses, therefore strengthening of antioxidant defence pathway could be a viable strategy to
develop stress-tolerant crop plants.
Acknowledgements
Authors are thankful to Prof. Dirk Inze, Department of
Plant System Biology, University of Ghent, Belgium for providing the research material. The grant received from DBT,
Government of India and DAAD, Germany is thankfully
acknowledged.
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Received February 8, 2011
Accepted August 27, 2011
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