Changes in the activity of antioxidant enzyme superoxide dismutase ...

SCIENTIFIC WORKS OF THE LITHUANIAN INSTITUTE OF
HORTICULTURE AND LITHUANIAN UNIVERSITY OF
AGRICULTURE. SODININKYSTĖ IR DARŽININKYSTĖ. 2008. 27(2).
Changes in the activity of antioxidant enzyme
superoxide dismutase in Crepis capillaris plants
after the impact of UV-B and ozone
Regina Vyšniauskienė, Vida Rančelienė
Institute of Botany, Žaliųjų ežerų str. 49, Vilnius LT-08406, Lithuania
E-mail: regina.vysniauskiene@botanika.lt
The aim of the work was to determine the changes of antioxidant enzyme superoxide
dismutase (SOD) activity in model plant Crepis capillaris after the impact of UV-B and ozone.
Comparison of the SOD activity in Crepis capillaris leaves after the effect of small adaptational
doses of UV-B and ozone with the control revealed that after UV-B (3 kJm -2 ) the SOD activity
increases 1.4 times and after the impact of ozone the SOD increases by 1.94 times. After larger
UV-B (9 kJm -2 ) doses the SOD activity increases even 2 times. Still three times higher ozone
dose already inhibits the SOD activity but does not reach the level of the control.
Investigations of the impact of adaptation on repeated impact of UV-B and ozone showed
that SOD activity to Crepis capillaris plants is similar and increases by 1.74 and 1.98 times
comparing with the control. Even when plants are adapted to one factor and influenced by the
other (cross adaptation), the SOD activity remains similar. The research showed that adaptation
by small UV-B and ozone doses to one and the other factor influences the increase of SOD activity
in plants. The increased SOD activity after UV-B irradiation and ozone should be considered
as adaptational response of a plant to oxidative stress caused by unfavourable factors.
Key words: Crepis capillaris, ozone, UVB, adaptation, superoxide dismutase,
EC 1.15. 1.1.
Introduction. Unfavourable environmental factors, like UVB and ozone, are
usually related with each other. Incident UVB radiation (in particular, the waveband
297–310 nm) is increasing due to the reduction in the stratospheric ozone concentration
(Caldwell et al., 1989). Environmental stress factors are known to cause oxidative
stress. Stress caused by these abiotic factors induces the excess of free radicals. Part
of free radicals cause changes in macromolecules, such as DNA and proteins. The
other part acts as a signal triggering the protective antioxidant mechanisms of plants
(Ballarй, 2003). Enzyme system is one of such mechanisms. Such antioxidative
enzymes as superoxide dismutase (SOD), catalase (CAT), peroxidase (POD) detoxify
the excess of free radicals (Mazza et al., 1999). Selection of test-plants characterizing
the contamination is essential while investigating the impact of natural factors upon
ambient environment. Harmful factors are usually investigated separately, and complex
mechanism of the impact of both factors is still under investigated.
Aim of the work was to determine changes in the activity of antioxidative enzyme
superoxide dismutase (SOD) in leaves of Crepis capillaris after complex impact of
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UVB and ozone and to answer the question whether enzyme of antioxidative stress
SOD participates in protective mechanisms of plant.
Object, methods and conditions. Crepis capillaris (L.) Wallr. plants grown for 2
weeks in a phytotron at the Laboratory of Cell Engineering of the Institute of Botany
and later transferred into the phytotron of the Lithuanian Institute of Horticulture
were used. Growth regime – 16/8 hour photoperiod; 21/16 °C day/night temperature.
The plants were divided into groups of unequal size: control (K) – unaffected plants;
plants affected by ozone or UVB, i.e. affected for 5 days by low adaptive doses: or
3 kJ m -2 d -1 (UVB), or 120 µg m -3 ozone (O 3
), respectively. During the second stage of
the research the first portion of plants was divided into 3 groups: control – unaffected
plants (K + K) affected plants by threefold dose (without adaptation): UVB (9 kJ m -2 d -1 )
or ozone (360 µg m -3 ), marked (K + UV) and (K + O 3
), respectively. Second group of
plants adapted by UVB or ozone was also affected by the same threefold doses only in
various combinations: combining the first impact with the second one: UVB + UVB;
O 3
+ O 3
; UVB + O 3
; O 3
+ UVB. During the second stage of the research exposition
to UVB or ozone was 7 days.
Activity of superoxide dismutase (SOD, EC 1.15. 1.1). Leaf material (1g) was
grounded with extraction buffer (2 ml), consisting of 1mM EDTA, 0.1 % Triton
X-100 in 0.05M Na-K phosphate buffer pH 7.8, with mortar and pestle at 4 °C. The
homogenates were centrifuged for 15 min at 12,000 Ч g (4 °C), and supernatants were
used as crude extract for soluble protein quantification according to Bradford (1976)
with bovine serum albumin as the standard.
Total SOD activity was assayed by the inhibition of the photochemical reduction
of nitro blue tetrazolium (NBT) according to modified method of Beyer and Fridovich
(1987). The reaction mixture (2.2 ml) consisted of 50 mM (Na-K) phosphate (pH 7.8)
and 20 µl of leaves extracts. Each extract was assayed twice with three replications
and measured by spectrophotometer at 560 nm.
The statistical data analysis was carried out by packet of statistical analysis tools
of MS Exel 2002 (Microsoft Corporation) program.
Results. It has been determined that in the course of adaptation, after 5 days
treatment (i. e. first measurement) by low UVB (3 kJ m -2 d -1 ) or ozone (120 µg m -3 )
doses the concentration of soluble proteins decreased comparing with the control (K).
The decrease was particularly significant after ozone treatment (table).
Table. Protein content changes after UVB and ozone treatment in leaves of Crepis
capillaris plants. Protein, mg g -1 F. w.; F. w. – fresh weight.
Lentelė. Baltymo kiekio pokyčiai po UV ir ozono poveikių Crepis capillaris augalų
lapuose, mg g -1
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After of 7 days direct treatment of plants with threefold UVB (9 kJm -2 ) and ozone
(360 µg m -3 ) doses increased the content of proteins in comparison with the control
(K + K) (i. e. second measurement). It is rather difficult to compare these data because
control plants differ by their age. Differences between the controls K and K + K
could be predetermined by the fact that in leaves of control (K + K) plants, which are
older, than in the control (K). Investigation of the impact of adaptation upon repeated
treatment with UVB and ozone (table) revealed no significant impact of adaptation
with UVB or ozone on the protein content.
However, comparison of the SOD activity in Crepis capillaris leaves after the
impact of small adaptation doses of UVB and ozone with the control revealed that
after UVB treatment (3 kJm -2 ) the SOD activity increases to 1.4 times and after the
impact of ozone the SOD increases to 1.94 times. After higher UVB (9 kJ m -1 ) dose
SOD activity doubles, but after threefold O 3
dose SOD activity is lower but does not
reach the level of the control.
Fig. Complex action of UVB and ozone on SOD activity in Crepis capillaris
plants adaptation. Exposition of 5 days in 3 kJ m -2 d -1 UVB or 120 µg m -3 O 3
environment. Treatment without adaptation – exposition of 7 days in 9 kJ m -2 d -1
to UV or 360 µg m -3 O 3
; K and K + K – control plants, grown in the same
conditions without any treatment.
Pav. Kompleksinis UVB ir ozono poveikis Crepis capillaris SOD aktyvumui.
Adaptacija. 5 dienų ekspozicija 3 kJ m -2 d -1 UVB ar 120 µg m -3 ozono aplinkoje. Poveikis be
adaptacijos – septynių dienų ekspozicija 9 kJ m -2 d -1 UV ar 360 µg m -3 O 3
aplinkoje;
K ir K + K – kontroliniai augalai, auginti tomis pačiomis sąlygomis be poveikių.
Investigations of the influence of adaptation upon repeated UVB and ozone
treatment (UV + UV and O 3
+ O 3
) showed that SOD activity in Crepis capillaris
plants is similar and increases up to 1.74 and 1.98 times comparing with the control.
Even after adaptation of a plant to one factor and later under treatment with the other
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(UV + O 3
; O 3
+ UV) SOD activity remains similar (Fig.). The research showed that
adaptation by low UVB and ozone doses towards one of the other factor influences
the increase of SOD activity in plants.
Discussion. Abiotic stress condition weakens plants; they become more susceptible
to pathogens, and this leads to extensive losses to of agricultural crop worldwide.
Usually the impact of environmental factors is not individual but combined. Therefore,
under conditions of climate changes plants have to adapt to simultaneous impact of
several factors: drought and heat; cold stress and drought combined with high light
conditions. For example, during heat and high ozone stresses plants open leaf stomata
for transpiration to avoid overheating, but thus the way for more abundant passing of
ozone into leaf intercellular ducts is opened, otherwise stomata would close. Therefore
in case of complex stress plants might require conflicting or antagonistic responses
(Pasqualini et al., 2003; Mittler, 2006).
The response type depends upon the plant genotype. Cultivated plants are more
susceptible to stresses than wild plants, which are better adapted to climate changes.
Even the slightest changes of climate factors could be relevant for agricultural plants.
Previous investigations have shown that even the lowest UVB (2 kJ m -1 ) and ozone
doses reduce the leaf size of a model plant Crepis capillaris; it is particularly true in
case of ozone (Rančelienė et al., 2006). Ozone, as a strong oxidant, or its secondary
derivatives, such as ROS (Reactive Oxygen Species) frequently cause leaf necroses.
It is known that viruses also cause necroses of plant leaves. In case of hypersensitivity
reaction necrotic spots, localizing the virus spread, form on the injured areas of leaves.
There is an opinion that ozone also triggers a hypersensitive response (Koch et al.,
2000). Therefore, necroses indicate the death of some cells irrespective of the stress
factor causing them: viruses, bacteria, UVB, ozone, cold and drought. So the damages
indicate the so-called programmed cell death (Pasqualini et al., 2002). But response
and defense genes are simultaneously induced, and they determine triggering of plant
defence mechanism to antioxidative response (Yun-Hee Kim et al., 2007). Antioxidative
enzyme systems, especially SOD enzyme, actively participates in this process. Our
research showed that after preadaptation by low doses the repeated treatment by other
factor doubly increases the activity of antioxidative SOD enzyme. It demonstrates that
plants adapted to one factor are frequently more tolerant to other factors. It confirms
the results of our earlier researches performed with cold-resistant interspecific potato
hybrids treated with UVB (Vyšniauskienė et al., 2006). However, results of other 7-year
long researches performed with plants growing in tundra showed that under conditions
of stratospheric ozone depletion and by enhanced UVB, the supplementation of UVB
in field produced no negative effect upon growth parameters (Rozema et al., 2006). It
demonstrates that in the course of time plants become UVB-tolerant.
Conclusions. 1. Increased activity of plant enzymes, such as SOD, after the UVB
and ozone irradiation was assessed as adaptational response of plant towards oxidative
stress caused by harmful factors. 2. Plants adapted to one factor are frequently more
tolerant to the impact of other factors.
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Acknowledgements. This research was supported by the Lithuanian State Science
and Studies Foundation programme “APLIKOM”. The authors gratefully acknowledge
to the Lithuanian Institute of Horticulture for availability to perform our experiments
in growth chambers of Institute.
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SODININKYSTĖ IR DARŽININKYSTĖ. MOKSLO DARBAI. 2008. 27(2).
Antioksidacinio fermento superoksido dismutazės aktyvumo
pokyčiai Crepis capillaris augaluose po UVB ir ozono poveikio
R. Vyšniauskienė, V. Rančelienė
Santrauka
Darbo tikslas buvo nustatyti antioksidantinio fermento superoksido dismutazės (SOD)
aktyvumo pokyčius modelinio augalo Crepis capillaris augalams po UVB ir ozono poveikių.
Lyginant SOD aktyvumą Crepis capillaris lapuose po poveikio mažomis –adaptuojančiomis
UVB ir ozono dozėmis su kontrole, gauta, kad po UVB (3 kJ m -1 ) SOD aktyvumas pakyla 1,4
kartus, o po ozono poveikio SOD padidėja 1,94 karto. Po didesnės UVB (9 kJ m -1 ) dozės SOD
aktyvumas pakyla net 2 kartus, tačiau po trigubos O 3
dozės jau SOD aktyvumas mažesnis, tačiau
nesiekia kontrolės lygio. Tiriant adaptacijos poveikį pakartotiniam UVB ir ozono paveikiui
gavome, kad SOD aktyvumas Crepis capillaris augalams yra panašus ir lyginant su kontrole
padidėja 1,74 ir 1,98 karto. Tačiau ir adaptavus augalus vienam veiksniui, o poveikus augalus
kitu veiksniu (kryžminė adaptacija), SOD aktyvumas išlieka panašus. Tyrimai parodė, kad
mažų UVB ir ozono dozių adaptacija tiek tam pačiam, tiek ir kitam veiksniui, turi poveikį SOD
aktyvumo padidėjimui augaluose. Padidėjęs SOD aktyvumas po UVB spinduliuotės ir ozono
vertintinas kaip augalo adaptacinis atsakas į nepalankių veiksnių sukeltа oksidacinį stresą.
Reikšminiai žodžiai: Crepis capillaris, ozonas, UVB, adaptacija, superoksido dismutazė,
EC 1.15. 1.1.
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