Changes in the activity of antioxidant enzyme superoxide dismutase in Crepis capillaris plants after the impact of UV-B and ozone

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1 SCIENTIFIC WORKS OF THE LITHUANIAN INSTITUTE OF HORTICULTURE AND LITHUANIAN UNIVERSITY OF AGRICULTURE. SODININKYSTĖ IR DARŽININKYSTĖ (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 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 Introduction. Unfavourable environmental factors, like UVB and ozone, are usually related with each other. Incident UVB radiation (in particular, the waveband 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 209

2 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 ). 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

3 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 211

4 (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. 212

5 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. References Gauta Parengta spausdinti Ballaré C. L Stress under the sun: spotlight on ultraviolet-b responses. Plant Physiology, 132: Beyer W. F., Fridovich I Assaying for superoxide dismutase activity: some large cobsequences of minor changes in conditions. Analytical Biochemistry, 161: Bradford M. N A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry, 72: Caldwell M. M, Teramura A. T, Tevini M The changing solar ultraviolet climate and the ecological consequences for higher plants. Trends in Ecology and Evolution, Koch J. R., Robert A. et al Ozone sensitivity in hybrid poplar correlates with insensitivity to both salicylic acid and jasmonic acid. The role of programmed cell death in lesion formation. Plant Physiology, 123: Mazza C. A., Battista D, Zima A. M., Szwarcberg-Bracchitta M., Giordano C. V., Acevedo A., Scopel A. L., Ballare C. L The effects of solar UV-B radiation on the growth and yield of barley are accompanied by increased DNA damage and antioxidant responses. Plant Cell and Environment, 22: Mittler R Abiotic stress, the field environment and stress combination. Trends in Plant Science, 1: Pasqualini S., Piccioni C Ozone-induced cell death in tobacco cultivar bel W3 plants. The Role of programmed cell death in lesion formation. Plant Physiology, 33(3): Rančelienė V., Vyšniauskienė R., Šlekytė K., Radžiūnaitė-Paukštienė A Kompleksinis UVB ir ozono poveikis žaliajai kreisvei (Crepis capillaris (L.) Wallr.). Sodininkystė ir daržininkystė, 25(2): Rozema J., Boelen P., Solheim B Zielke M. Buskens A., Doorenbosch M., Fijn R., Herder J., Callaghan T., Björn L. O., Jones D. G., Broekman R., Blokker P., Poll W Stratospheric ozone depletion: high arctic tundra plant growth on Svalbard is not affected by enhanced UV-b after 7 years of UV-B supplementation in the field. Plant Ecology, 182: Vyšniauskienė R., Rančelienė V., Radžiūnaitė-Paukštienė A., Spalinskas R The UV-B impact upon the enzyme of antioxidant system superoxide dismutase (SOD) of potato somatic hybrids. Biologija, 18(2):

6 12. Yun-Hee Kim, Soon Lim, Sim-Hee Han, Jae-Cheon Lee, Wan-Keun Song, Jae-Wook Bang, Suk-Yoon Kwon, Haeng-Soon Lee, Sang-Soo Kwak Differential expression of 10 sweet potato peroxidase in response to dioxide, ozone, and ultraviolet radiation. Plant Physiology and Biochemistry, 45: SODININKYSTĖ IR DARŽININKYSTĖ. MOKSLO DARBAI (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