Plant Science 165 (2003) 311
/319

www.elsevier.com/locate/plantsci

Selenium effects on oxidative stress in potato

Mervi Seppanen a,c,*, Marja Turakainen a, Helina Hartikainen b
a
Department of Applied Biology, University of Helsinki, P.O. Box 27, FIN-00014 Helsinki, Finland
b
Department of Applied Chemistry and Microbiology, University of Helsinki, P.O. Box 27, FIN-00014 Helsinki, Finland
c
Department of Natural Resource Sciences and Landscape Architecture, University of Maryland, 2125B Plant Sciences, College Park, MD 20742, USA

Received 24 December 2002; received in revised form 12 February 2003; accepted 19 February 2003

Abstract

Higher plants are considered not to require selenium (Se). However, it has recently been shown that Se increases the antioxidative
capacity and stress tolerance of lettuce (Lactuca sativa L.) and ryegrass (Lolium perenne L.). This research was undertaken to
investigate the antioxidative properties of Se during photooxidative stress in potato (Solanum tuberosum L.) and to determine the
defence mechanisms. Potato plants were exposed to 600 mmol/m2/s light intensity at low temperature (4 8C) or paraquat-mediated
oxidative stress. The stress responses were monitored by measuring chlorophyll content and following changes in chlorophyll
fluorescence and membrane ion leakage. Moreover, the effects of Se on the transcript levels of chloroplast CuZnSOD,
mitochondrial MnSOD, glutathione peroxidase (GPX), and psbA were analyzed using northern hybridization. Se supplementation
improved the recovery of chlorophyll content following light stress. After prolonged exposure to light, the reduction of Fv/Fm was
slightly lower compared with plants cultivated without additional Se. The photosynthesis of Se treated plants was somewhat more
tolerant of paraquat and the integrity of membranes was improved during oxidative stress. Se altered transcript accumulation of
chloroplast CuZnSOD and GPX but the MnSOD and psbA transcript levels were unaffected. The results suggest that Se is an
antioxidant or it activates protective mechanisms, which can alleviate oxidative stress in the chloroplasts.
# 2003 Elsevier Science Ireland Ltd. All rights reserved.

Keywords: Potato; Low temperature stress; Oxidative stress; Photoinhibition

1. Introduction balance between energy supply and demand is lost

Cultivated potato, Solanum tuberosum L., is a freez-
ing sensitive plant, which suffers from significant frost
damage in the field even at temperatures slightly below
0 8C [1]. Freezing nights are often followed by bright
sunlight in the morning and light is an important
component of frost injury in the field. Exposure to
high intensity light after freezing can significantly
increase the severity of freezing injury [1
/3]. Light stress
can promote an accumulation of active oxygen species
(AOS) in the chloroplasts in situations where the
antioxidant capacity to detoxify AOS is exceeded. At
low temperature the risk of over-excitation of photo-
synthesis and formation of AOS is increased as many
energy-requiring processes are slowed down and the
* Corresponding author. Tel.: /358-9-191-58356; fax: /358-9-191-
58463.
E-mail address: mervi.seppanen@helsinki.fi (M. Seppanen).
0168-9452/03/$ - see front matter # 2003 Elsevier Science Ireland Ltd. All rights reserved.
doi:10.1016/S0168-9452(03)00085-2
resulting in reduction of oxygen [4
/6]. Antioxidant
capacity increases during cold acclimation in several
plant species as an adaptive mechanism to low tempera-
ture there include freezing tolerant wild potato, S.
commersonii (Dun.) [7,8]. Enhanced antioxidant capa-
city is also evident during acclimation to photoinhibi-
tion, indicating the important role of AOS scavenging
capacity for preventing injury to the photosynthetic
apparatus [9].
Selenium (Se) is an essential micronutrient needed in
antioxidation and hormone balance in human and
animal cells [10
/12]. According to current thinking,
higher plants do not require Se. However, recent
findings on lettuce (Lactuca sativa L.) and ryegrass
(Lolium perenne L.) show that although Se is toxic at
high concentrations, it can exert beneficial effects on
plants at low concentrations. Se can increase the
tolerance of plants to UV-induced oxidative stress as
well as delay senescence and promote the growth of
312 M. Seppanen et al. / Plant Science 165 (2003) 311
/319
aging seedlings [13
/15]. The anti-aging effect was
related to decreased lipid peroxidation and enhanced
glutathione peroxidase (GPX) activity in Se-treated
plants. In plants grown under high light intensities, Se
counteracted senescence-related oxidative stress and
maintained green leaf color longer [15]. Although Se
containing GPX has not yet been identified in plants, Se
supplementation consistently increased GPX activity
[15]. Se also decreased the activity of superoxide
dismutase (SOD) and lowered the amount of tocopher-
ols in some cases. It was suggested that while Se
promotes H2O2 scavenging, by increasing GPX activity,
it also enhances spontaneous disproportion of super-
oxide radicals to H2O2 and thus decreases the need for
high SOD activity [15].
One approach to improve crop plant tolerance to
environmental stresses is to increase their antioxidant
capacity [16,17]. The most recent studies on maize (Zea
mays L.) have shown that elevated antioxidant levels
can protect the photosynthetic apparatus from oxidative
damage [18,19]. In potato, improved photosynthetic
capacity and productivity at low temperature could be
achieved by enhancing the detoxification of AOS. This
study was undertaken to determine whether Se con-
tributes to recovery of photosynthesis from prolonged
high light stress in potato. The working hypothesis was
that through its antioxidative effect Se can alleviate
oxidative stress in chloroplasts. The responses of potato
to Se supplementation were investigated by monitoring
chlorophyll fluorescence and the transcription of anti-
oxidative enzymes.
2. Materials and methods
2.1. Plant material and growth conditions
Potato plants (Solanum tuberosum L. cv. Kulta or
Satu) were grown in peat:sand (10:1) in a greenhouse at
18
/28 8C under a 16-h photoperiod of 200
/400 mmol/
m2/s. The daylight was supplemented with 400 W high-
pressure sodium lamps (Lucalox, LU 400/HO/T/40NG)
to provide a quantum flux density of 220 mmol/m2/s at
the upper leaf canopy. Plants were grown in plastic pots
(7.5 l) and fertilized with 1% NPK (14-5-21, Puutarhan
Tayslannos, Kemira Agro Ltd) once a week from the
surface of each pot. Two liters of nutrient solution were
applied the during 4-week-growth period. The total
amount of nutrients was 2.8 mg N, 1 mg P, 2 mg K, 1.4
Mg, 1.8 S and trace amounts of the micronutrients Fe,
B, Mn, Mo, Zn and Co. Plants were grown in 5 l pots
containing quartz sand (0.1
/0.6 mm, SP-Minerals Oy,
Nilsian kvartsi, Finland) in growth chambers (WEISS
Bio1300, Germany) at 20/15 8C day/night under an 18 h
photoperiod. The pots were fertilized with Hoagland
and Arnon solutions [20] and limed with 4 g dolomite
per pot (Saxo, Finland). The total amount of nutrients
was 2.8 mg N, 0.3 mg P, 1.6 mg K, 0.6 mg Ca, 1.8 mg S
and trace amounts of the micronutrients Mg, S, B, Mn,
Zn, Cu, Fe, Ni and Na.
2.2. Selenium treatment and analysis
The pots were treated with 0 (0 M), 0.5 ppm (6.3 mM)
and 2.0 ppm (25 mM) Se as selenite (supplied as H2SeO3,
Fluka Chemie AG product) or selenate (Na2SeO4 /
10H2O) solution. The final concentration was 0.07 mg
or 0.285 mg Se/l peat or 0.075 mg, 0.3 mg S/kg soil
(denoted Se-0.07 and Se-0.3, respectively), or without
added Se (Se-0). Each treatment was replicated four
times. During the experiment, 250 ml of Se solution was
added once a week to a total volume of 750 ml. For Se
analysis, about 30 fully expanded leaflets were collected
from each plant and were analyzed and dried at 60 8C
for 48 h. The Se concentration was determined using
electrothermal atomic absorption spectrometry accord-
ing to the method of Kumpulainen et al. [21]. An in-
house reference sample was included in every analytical
round.
2.3. Stress treatments and experimental design
The stress tests were carried out with leaf samples
collected from three individual 4-week-old plants. The
experiment on induction of oxidative stress at high light
intensity was done in a growth chamber (WEISS,
Bio2000) with leaf samples taken from potato plants
cultivated at 0, 0.07 and 0.3 mg Se/l peat. Leaf discs
(diameter 2 cm) were exposed to continuous light of
500
/600 mmol/m2/s for 4, 6, 8 or 20 h. For light
exposure, two leaf discs were put onto petri dishes on
a piece of wet towel and placed on crushed ice. At least
three individual plants were tested. The experiment was
carried out at 4 8C. Temperature and moisture condi-
tions were monitored throughout the experiment.
The paraquat-mediated oxidative stress was investi-
gated using leaves representing plants cultivated at 0,
0.07 mg and 0.3 mg Se/l peat. Paraquat herbicide causes
superoxide radical formation in chloroplasts during
light treatment. Superoxide radicals are then scavenged
by SODs and H2O2 is produced. In the chloroplast,
H2O2 is detoxified mainly by peroxidases, or it is
diffuses to other cellular compartments. When the
detoxification capacity of antioxidants is exceeded,
accumulated AOS can damage photosynthetic and
cellular membranes. Here the effect of paraquat on
chloroplast activity and cell membrane integrity was
evaluated with some modifications to the protocols
described by Kraus et al. and Iannelli et al. [22,23].
Two leaf discs (diameter 2 cm) from three plants were
placed in a sealed petri dish with 4 ml of 0, 1, 2, 5 or 10
mM paraquat (1,1?-dimethyl-4,4?-dipyridium dichloride,
 M. Seppanen et al. / Plant Science 165 (2003) 311
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Sigma) solution supplemented with 0.1% (v/v) Tween
20. At least three individual plants were tested. The
paraquat solution was vacuum infiltrated and the leaf
discs were put onto petri dishes. The leaf discs were
incubated in paraquat solution in darkness for 19 h
prior to exposure to continuous light (600 mmol/m2/s)
for 19 h. The effect of paraquat was evaluated by
monitoring changes in fluorescence kinetics or by using
ion leakage tests.
All stress treatments were performed on leaf discs
(two to three discs per plant) from three individual Se-0,
Se-0.07 or Se-0.3 plants. The plates carrying leaf discs
were arranged in the growth chamber in a randomized
block design with three replications.
2.4. Measurement of chlorophyll fluorescence and
membrane integrity
To study the effect of high intensity light or paraquat
on chloroplast activity, the ratio of variable (Fv) to
maximum (Fm) chlorophyll a fluorescence was recorded
with a fluorometer (Hansatech FMS-2, King Lynn,
UK), after 15-min dark adaptation. This was taken to
serve as a measure of photosynthetic efficiency [24].
Membrane integrity was measured by the ion-leakage
method described by [7]. After paraquat treatments, leaf
discs were transferred into scintillation vessels contain-
ing 15 ml of Milli-Q water and shaken for 1 h at 150 rpm
(Belly dancer, Stovall Life Science, USA). The conduc-
tivity of the water was measured with a conductometer
(Check Mate 90, Mettler
/Toledo AG, Schwerzenbach,
Switzerland).
Slow fluorescence kinetics were monitored with a
fluorometer (Hansatech FMS-2) in leaf discs exposed to
light at 200 or 900 mmol/m2/s for 60 s . The excitation
pressure on PSII was calculated as 1-qP, where qP is a
coefficient for photochemical quenching [25,26].
2.5. Chlorophyll determination
Three leaf discs (diameter 3 cm) were harvested from
the last fully expanded leaves of three individual plants,
frozen immediately in liquid N2 and stored at /70 8C.
Frozen leaf discs were ground under dim light in a
mortar containing liquid N2. Four milliliter of ice-cold
methanol (100%, v/v) was added to extract the pigments.
The samples were centrifuged in a pre-cooled centrifuge
(4 8C) at 3000 rpm for 10 min. Chlorophyll content was
analyzed using a spectrophotometer (Shimadzu, UV-
160A, Shimadzu Co., Japan) at 665.2, 652.4 and 470 nm
and the amounts of chlorophyll were calculated as
reported by Lichtenthaler [27].
313
2.6. SOD activity
Leaf samples were harvested and proteins extracted as
described by Seppanen and Fagerstedt [7]. The extract
was assayed for protein content [28], and the SOD
activity calculated as the amount of SOD units per mg
of total protein (units/mg protein). Total SOD activity
was measured spectrophotometrically by the method of
McCord and Fridovich [29]. SOD activity was expressed
as units, where one unit is defined as the amount of SOD
required to inhibit the rate of reduction of Cyt c by 50%.
2.7. RNA isolation and Northern-blot analysis
Total RNA of leaf samples was isolated using a Plant
RNeasy extraction kit (QIAGEN). Total RNA of 10 mg
was used for agarose formaldehyde gels and blotted
onto nylon transfer membranes (MagnaGraph, Micro-
separations) and hybridized according to general proce-
dures [30]. A 300 bp Acc 1-Hind III fragment of
chloroplastic CuZnSOD (pGSOD4, a gift from D.
Inze), a 1000 bp Pst 1 fragment of mitochondrial
MnSOD (pSOD1) [31,32], a 580 bp Arabidopsis EST
clone E8G1T7 coding for chloroplast GPX (AT000469,
AA042773) and a 1100 kb Eco R1-Xba 1 fragment of
psbA [33] were used as probes. The probes were labeled
with 32P by nick translation (Pharmacia or Ladder-
manTM labeling Kit, Tanaka Biomedicals). Membranes
were washed at 65 8C once with 2/SSC, 0.5% SDS for
30 min; once with 1 /SSC, 0.5% SDS for 30 min and
finally once with 0.5 /SSC, 0.1% SDS for 10 min. After
visualization of corresponding mRNA using a phos-
phoimager (Molecular Dynamics Storm System), the
probe was removed by stripping the membranes accord-
ing to the instructions provided by the supplier. Equal
loading was verified by rehybridizing membranes with a
probe corresponding to the soybean ribosomal gene
(rRNA). Two separate plants were analyzed by northern
hybridization.
2.8. Data analysis
Data was analyzed using an analysis of variance
(ANOVA) in the GLM procedure of SAS (the SAS
system 6.12, SAS Institute Inc. 1989
/1996, USA).
Significantly different means between treatments were
separated with Duncans multiple test.
3. Results
3.1. Chlorophyll degradation during light stress
The Se supplementation increased the Se concentra-
tion from 0.029/0.0 mg Se/kg DW (the mean of three
analysis9/S.D.) in control plants to 0.179/0.02 and
314 M. Seppanen et al. / Plant Science 165 (2003) 311
/319

Fig. 1. The effect of Se on chlorophyll degradation in light stressed potato leaves. Leaf discs of control (Se-0) and Se treated (Se-0.07, Se 0.3) potato
plants were exposed to light (600 mmol/m2/s) for 0 to 6 h as indicated in the figure legend. A. chlorophyll a (mg/cm2), B. chlorophyll a (mg/cm2), C.
total chlorophyll (mg/cm2) and D. chlorophyll a/b ratio. Data represent the mean values (9/S.E., n/3) from three leaf discs excised from three
individual plants.

0.869/0.09 mg Se/kg DW in Se-0.07 and Se-0.3 (mg Se/l 2
/6 h of light, Se supplemented plants (Se-0.07, Se-0.3)
peat) fertilized plants, respectively. The effect of Se on suffered from a slightly more significant reduction in Fv/
photooxidative stress tolerance in potato was studied by Fm than the control plants (Se-0). However, when the
monitoring the degree of chlorophyll degradation. The exposure time was extended to 20 h, the reduction in Fv/
light intensity of 600 mmol/m2/s significantly affected Fm was higher in Se-0 plants (17%) than in Se-0.07 or
chlorophylls. In leaf discs taken from control (Se-0) and Se-3 plants (14, 12.5%, respectively). This difference was
Se supplemented (Se-0.07, Se-0.3) plants subjected to 2 h not, however, statistically significant (P /0.5).
of light stress at low temperature (4 8C) chlorophyll a,
chlorophyll b and total chlorophyll contents decreased
in all treatments (Fig. 1a
/c) and the chlorophyll a/b
ratio decreased (Fig. 1d). The decrease in chlorophyll
contents was smaller in Se supplemented than in control
plants when leaf discs were exposed to high light for 6 h.
In Se treated plants, the chlorophyll content returned to
initial levels whereas in the control plants the recovery
was not complete.

3.2. Development of chlorophyll a fluorescence in high

light stressed plants

Chlorophyll fluorescence measurements were taken to

determine if Se supplementation could protect photo-
synthesis from photoinhibition. Reduction in the vari-
able to maximum fluorescence (Fv/Fm) was monitored
in leaves exposed to light for up to 20 h (Fig. 2). After
Fig. 2. The development of variable to maximum fluorescence (Fv/
Fm) upon exposure to light (600 mmol/m2/s). Leaf discs of control (Se-
0) and Se (Se-0.07, Se-0.3) treated plants were exposed to high light for
0
/20 h as indicated in the figure. Data represent the mean values (9/
S.E., n /3) from three leaf discs excised from three individual plants.
 M. Seppanen et al. / Plant Science 165 (2003) 311
/319
3.3. Effect of selenium on gene transcription
Se supplementation is known to alter transcript levels
of defense genes such as GPX in animal cells. In the
present study, total RNA was extracted from control
(Se-0) and Se supplemented (Se-0.07, Se-0.3) potato
plants before and after exposure to 20 h of high light
(Fig. 3). In the non-stressed Se-0.3 plants, an elevated
level of chloroplast CuZnSOD transcript was moni-
tored. In addition, a low but detectable level of GPX
transcript was detected. However, after light stress,
higher accumulation of CuZnSOD transcript was re-
corded in Se-0 and Se-0.07 plants compared with Se-0.3
plants. There were no significant changes in mitochon-
drial MnSOD or psbA transcript levels. The possibility
of elevated CuZnSOD transcript accumulation resulting
in changes in enzyme activity was studied by measuring
total SOD activity in non-stressed plants. Slightly higher
SOD activity was detected in Se treated plants (Table 1).
3.4. Selenium-induced changes in chlorophyll fluorescence
parameters
Slow fluorescence kinetics was monitored under two
light intensities (200 and 900 mmol/m2/s) to study
whether Se supplementation alters photosynthetic (qP)
or non-photosynthetic (qNP) electron transport or
Fig. 3. The effect of Se on the transcript levels of enzymatic
antioxidants and D1 protein in high light stressed potato. Leaf discs
of control (0) and Se (0.07, 0.3) treated plants were exposed to high
light for 0 (Before) or 20 h (20 h HL). Total RNA was isolated and 10
mg was loaded per lane. Hybridization was performed with chloroplast
CuZnSOD, mitochondrial MnSOD, GPX or PsbA gene probes. The
probe was removed and the membrane was rehybridized with soybean
ribosomal gene to show equal loading of the samples.
315
Table 1
Total SOD activity (units/mg protein) in Se treated potato plants
Treatment SOD
Se-0 15.39/0.41
Se-0.07 18.39/1.18
Se-0.3 19.59/1.00
Data represent means of three measurements9/S.E.
excitation pressure (1-qP) of photosynthesis (Fig. 4).
There were only moderate differences between the Se
treatments. At low light intensity (200 mmol/m2/s)
photosynthesis was slightly more reduced (1-qP) in Se
supplemented plants. These differences were not statis-
tically significant (P /0.5).
3.5. Tolerance to paraquat-mediated oxidative stress
Paraquat-mediated damage in photosynthesis was
demonstrated by changes in chlorophyll fluorescence
kinetics and ion leakage of the membranes. Treatment
with a very high concentration of paraquat (10 mM)
caused up to 100% reduction in Fv/Fm and almost a
complete loss of membrane properties (Fig. 4a and b).
Damage was more severe in the Se supplemented (Se-
0.07, Se-0.3) than in the control plants (Se-0). In
contrast, after treatment with 2 to 5 mM paraquat, the
smallest reduction of Fv/Fm was monitored in the Se-
0.3 plants (Fig. 5).
4. Discussion
4.1. Recovery after photooxidative stress
Se slightly improved the recovery of photosynthesis
from light stress. It probably alleviated oxidative stress
in chloroplasts, since chlorophyll content returned to the
initial level after a transient decrease at light only in the
Se treated plants. During the initial hours at light, the
higher chlorophyll content and thus, greater supply of
electrons for photosynthesis, may have lead to increased
photoinhibition in Se treated plants. However, after 20 h
a balance between energy demand and supply was
achieved and the reduction of Fv/Fm was smallest in
Se treated plants. The results show that Se can improve
adaptation to photoinhibitory light intensities. AOS,
which accumulate at high light stress in PSII, are excised
chlorophyll molecules and singlet oxygen species [5,34].
These molecules are mainly scavenged by carotenoids
and a-tocopherol [34]. Increased tolerance to high light
has been explained by increased capacity to dissipate
excess light non-radiatively to carotenoids [35,36]. In
these experiments qNP was not affected by Se treatment
in light stressed plants. This preliminary result at light
316 M. Seppanen et al. / Plant Science 165 (2003) 311
/319
Fig. 4. The effect of Se on (a) photochemical quenching (qP), (b) non-
photochemical quenching (qNP) and c) PSII excitation pressure (1-
qP). The fluorescence parameters were monitored from leaf discs
exposed to 200 or 900 mmol/m2/s light intensities. Values represent
means9/S.E., n/3.
discouraged us from continuing studies on altered non-
photochemical quenching in Se treated plants. Recent
studies with lettuce [37] show, however, total tocopher-
ols increase with plant development to meet the
increased need for antioxidants. Simultaneously, the
synthesis of a-tocopherol, a more active species, than g-
tocopherol is enhanced. Furthermore, total tocopherols
were shown to diminish during plant senescence, but the
added Se counteracted this process and tended to
maintain tocopherol concentration at a higher level
than in the control plants cultivated without Se. The
Fig. 5. The effect of Se on paraquat-mediated oxidative stress in
potato. Paraquat dependent A. reduction in variable to maximum
fluorescence (Fv/Fm) and B. increase of ion leakage (%). Fv/Fm data is
presented as a percentage of reduction in Fv/Fm compared with the
initial value. Values represent means9/S.E., n/3.
possibility that singlet oxygen is scavenged more effi-
ciently by tocopherol in Se treated plants during light
stress is currently under investigation.
The Se related improvement in tolerance to oxidative
stress was more moderate in the plants treated with
paraquat, a herbicide causing superoxide radical forma-
tion in chloroplasts. In the Se treated plants photo-
synthesis was slightly more protected against oxidative
stress at low paraquat concentrations, and the suscept-
ibility of their cell membranes was slightly altered. In
general, the antioxidative effect of Se is associated with
improved (GPX) enzyme activity [14,15,38] and thus,
with enhanced scavenging of hydrogen peroxide. How-
ever, as suggested [15], the spontaneous disproportion of
superoxide to H2O2 may explain the slightly improved
tolerance of photosynthesis. Moreover, there was a
moderate increase in total SOD activity in Se treated
plants, which may have enhanced the scavenging of
 M. Seppanen et al. / Plant Science 165 (2003) 311
/319
superoxide radicals. However, when plants were ex-
posed to extremely high concentrations of paraquat, the
damage was accelerated in Se treated plants. The current
understanding of the role of Se in plant metabolism does
not allow speculation on this observation.
4.2. Selenium alters transcript accumulation of
chloroplast CuZnSOD and GPX
Even though SOD is not a selenoenzyme like many
GPX, Se altered transcript accumulation of GPX and
chloroplast CuZnSOD but not of mitochondrial
MnSOD. Se deficiency causes loss of GPX mRNA in
animal cells and decreases enzyme activity, which can be
restored by Se supplementation [39]. The mechanisms of
Se regulation of selenoperoxidase mRNA are unknown,
but it has been suggested that Se status regulates the
stability of GPX mRNA [40]. Changes in SOD gene
expression are assumed to be governed by the subcel-
lular site at which oxidative stress is generated [16,31].
The redox state of glutathione is also thought to play a
role in the regulation of CuZnSOD genes [41]. Thus, if
transcript accumulation is used as an indicator of
subcellular AOS generation or changes in the redox
state, Se has an effect on chloroplast activities in non-
stressed plants. Studies by Pennanen et al. [42] have
indicated that Se increases starch accumulation in
chloroplasts, which is reflected as promoted plant
growth. Also, assimilation of Se, which requires redu-
cing power from NADPH and GSH, occurs in the
chloroplast and can theoretically alter the redox state of
chloroplasts [43]. Se is effectively utilized in protein
synthesis although it can accumulate also in inorganic
form [44]. In the present study, the allocation of
incorporated Se among various fractions was not
analyzed and the fraction of incorporated Se was not
measured. The positive effects of Se on the recovery of
potato from photooxidative and paraquat-generated
oxidative stress point to mechanisms, yet unknown,
which can protect chloroplasts during stress. Although
elevated transcript levels do not necessarily result in
increased enzyme activity [41,45], slightly increased
SOD activity in the Se treated plants was recorded.
Enhanced capacity to scavenge superoxide radicals may
be one of the mechanisms to alleviate oxidative stress in
the chloroplast. There are numerous reports showing
that H2O2 as well as other AOS are important signaling
molecules in plants [46]. An alternative explanation
could be that Se induces acclimation processes via
changes in H2O2 content.
Under light stress, CuZnSOD transcript accumulated
in control plants whereas the transcript decreased in the
Se treated plants. In general, the chloroplast CuZnSOD
is up-regulated in light and the transcript levels react to
long-term changes in photoproduced AOS [45]. This
kind of response was seen in the plants cultivated
317
without added Se. Further studies, including detailed
measurements of redox changes during light treatment,
are needed to understand why CuZnSOD transcript did
not accumulate in Se treated plants.
In contrast to chloroplasts, mitochondria are not
subject to significant accumulation of AOS during light
stress and, thus, no changes in mitochondrial MnSOD
were observed [45]. However, when Se was applied to
Trigonella foenum-graecum seedlings, the mitochondial
oxygen uptake was found to increase concomitantly
with enhanced mitochondrial SOD activity [47]. The
finding that the transcript level of MnSOD was un-
affected by Se application in these experiments does not
support the hypothesis of more active mitochondrial
reactions.
After prolonged exposure to light, the degree of
photoinhibition was lower in Se treated plants. Thus,
Se may have protected photosynthesis during light
stress. To study changes in the PSII reaction center in
more detail, the transcription of the gene coding for D1
protein, psbA , was followed. Despite improved recovery
from light stress, the transcript level of psbA remained
unaltered in Se treated plants. In general, the expression
of chloroplast-coded genes is regulated mainly at post-
transcriptional, translational level [48] although tran-
scriptional control is also reported [49
/53]. In Synecho-
cystis, the psbA gene is thought to be regulated at
transcriptional level by the redox state of electron
carriers between the plastoquinone pool and PSI [52]
or signals caused by accumulation of closed PSII
reaction centers [54]. In pea (Pisum sativum L.) and
wheat (Triticum aestivum L.), however, psbA transcript
levels were found to be relatively stable under changing
light and temperature conditions [53,55]. If psbA is
similarly regulated in potato as in Synechocystis by
signals from the photosynthesis, it is possible that the
alternations in PS redox state (1-qP), which were
recorded in this study, were not significant enough in
these experiments to affect psbA transcription.
Our studies indicate that Se has positive effects on
photooxidative stress tolerance in potato. The data
suggest that Se has antioxidant properties or activates
protective mechanisms that can alleviate oxidative stress
in the chloroplasts. We believe this to be the first time
that Se is reported to have synergistic effects on the
transcription of antioxidative enzymes such as CuZn-
SOD and GPX in plants. Further studies are required to
determine how Se mediates these responses.
Acknowledgements
The financial support of Wihuri Foundation and
University of Helsinki Foundation is gratefully ac-
knowledged. Salla Hartikainen, Eerika Karli, Lilia
Sarelainen and Anssi Vuorinen are acknowledged for
318 M. Seppanen et al. / Plant Science 165 (2003) 311
/319
excellent technical assistance and Maija Ylinen for
selenium analysis.
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