Analele tiinifice ale Universitii Alexandru Ioan Cuza, Seciunea Genetic i Biologie Molecular, TOM XIV, 2013

SOME ASPECTS OF ANTIOXIDANT DEFENSE MECHANISMS IN WOOD SPECIES EXPOSED TO ANTHROPIC POLLUTION IN SUCEAVA COUNTY
MARIUS VIOREL ONICIUC1, ELENA TUTU1, COJOCARU SABINA IOANA1, ELENA CIORNEA1*
Keywords: pollution, sulphur, copper, barite, catalase, peroxidase, angiosperms, gymnosperms. Abstract: Increased production of reactive oxygen species in plant tissues caused by unfavorable environmental conditions is early response to different stresses and may provide cells with resistance against their formation. The subject of this paper is determination of catalase and peroxidase levels, components of the antioxidant defense mechanism, in various types of woody plants in order to study the effect of pollution by sulphur and copper exploitation in mining areas on the antioxidant enzymes activity in the leaf material taken from different Gymnosperm species as Picea abies L.Karst., Larix decidua Mill. and Angyosperms like Salix ssp alba L., Populus tremula, Betula verrucosa Ehrh. i Fagus sylvatica L. For this purpose, the measurement of catalase activity was performed using the Sinha method (Artenie Vl. et al., 2008), the determination of peroxidase level was carried out on the basis of orthodianisidine method (Cojocaru D.C., 2009) and the determination of soluble proteins on Bradford method (Cojocaru et al.,2009). The results obtained lead to the conclusion that both catalase and peroxidase are effective biomarkers of pollution with sulfur, copper ores and barite but especially the acclimatization of species studied in conditions of chronic exposure.

INTRODUCTION
Pollution impact on woody plants is due to the generation of reactive oxygen species and induction of socalled oxidative stress (Schtzendbel and Polle, 2002); their counteracting beeing made by the various enzymatic and non-enzymatic antioxidant systems (Smirnoff, 1995, Navarri-Izzoand and Rascio, 2010, Singh Gill and Tuteja, 2010), the beginning of stress in plants requiring a reorganization of cellular metabolism in this ensemble, for their acclimatization to stress. The incipient stages of the response (the so-called alarm stage), involve rapid induction on the specific signaling paths to stress and to a strong oxidative stress, while the later stages (the acclimatization stage), are associated with the de novo biosynthesis of proteins with protective functions against stress (chaperones, antioxidant enzymes) and other compounds (carotenoids, tocopherols, osmoprotectans -proline), followed by the followed by the activation of degrading processes of these protective compounds and a stabilizations of the new cell homeostasis during the recovery period (Kosova et al., 2011). Starting from the hypothesis that a significant increase of enzymatic production integrated in foliar defensive system in some genotypes of wood plant population may result from increase of respective varieties resistance to anthropogenic pollution factors, we can conclude therefore, that these enzymes can be used as biomarkers of oxidative stress, and also in evaluating the degree of acclimatization of the species in mining areas studied.

MATERIAL AND METHODS

The investigations were performed on leaf material collected in May 2011 from Gymnosperms species of the conifers family such as Picea abies L.Karst., Larix decidua ssp Mill. and Angiosperms such as Salix alba L., Populus tremula and Betula verrucosa Ehrh. under the influence of pollution (Climani and Tarnia), and from species located in the control areas considered unpolluted Rduti, Suceava County. The determination of the catalase activity was made using the spectrophotometric Sinha method (Artenie et al., 2007), the peroxidase activity was detected by the Gudkova, L. and Degtiari, G. method (Artenie et al., 2007) with ortho-dianisidine, while the total soluble proteins were dosed using the Bradford method (Cojocaru et al., 2009). For each biochemical determination were used tree parallel samples, the presented data representing the arithmetic mean of the obtained results.

37

Marius Viorel Oniciuc et al Some aspects of antioxidant defense mechanisms in wood species exposed to anthropic pollution in Suceava County

RESULTS AND DISCUSSION The results regarding the catalase and peroxidase specific activity to Gymnosperms and Angiosperms species from polluted and unpolluted areas of Suceava are presented graphically in Figures 1-2. Some studies in the literature highlight the fact that the catalase activity in needles peroxisomes of Picea abies decreases with age, being higher in the growth conditions of plants in the presence of ozone (Morral et al., 1990), and can also be concluded that, in terms of pollution of air and soil with sulfur, the catalase activity can be used as a biomarker, higher than the one found in Picea abies specimens grown in unpolluted areas (15.731 UC/mg proteins detected in Climani Mountains, compared to 7.001 UC/mg proteins recorded in the needles taken from Rduti). A quantitative approach on the data obtained about the antioxidant defense in leaf tissues of Populus tremula specimens of Climani Mountains, shows a three times higher activity of catalase (17.182 UC/mg proteins) compared to those provided by the foliar limb of the species harvested from the control zone (5.054 UC/mg proteins). According to other investigations, Populus sp. is one of the species in which catalase shows a high sensitivity to pollution (Stobrawa and Lorenc-Pluciska, 2007), the decreased fluid regime potentiating the formation of superoxide radicals, which results in a sustained activity of the superoxidedismutase and implicitly, in a catalase activity that prevents the accumulation of hydrogen peroxide.
30

2 1,8

25

1,6 1,4 PU/mg protein 1,2 1 0,8 0,6 0,4 0,2

CU/mg protein

20

15

10

0 Picea abies Populus tremula Calimani Salix spp alba Betula verucosa Larix decidua

0 Picea abies Populus tremula Calimani Salix spp alba Betula verucosa Radauti Larix decidua

Tarnita

Radauti

Tarnita

Fig.1.The specific activity of catalase in Gymnosperm and Angiosperm species in polluted and unpolluted areas of Suceava County

Fig.2.The specific activity of peroxidase in Gymnosperms and Angiosperms species in polluted and unpolluted areas of Suceava County

The existence of a strong oxidative stress in this area is confirmed by the very high activity of peroxidase (1.822 UP/mg proteins in samples from Climani), which indicates the high sensitivity of this species to pollutants. Moreover in the leaf device of Populus tremula, the oxidoreductase activity was the highest among all species in the areas monitored in our studies (fig. 2), which allows us to use it as a biomarker in the investigations of this genus. Salix alba is considered to be a resistant species, moderately affected by gaseous pollutants and is commonly used to restore damaged ecosystems (Pulford and Dickinson, 2005) together with Populus sp. (Komives and Gullner, 2006, Olejniczak et al., 2012). In the leaves of these species, this antiperoxidative enzyme catalase, had registered a dominant level compared to that of other studied species (29.192 UC/mg proteins).

38

Analele tiinifice ale Universitii Alexandru Ioan Cuza, Seciunea Genetic i Biologie Molecular, TOM XIV, 2013

Immediately after this value, Betula verrucosa confirms the fact that hydrogen peroxide acts as signal molecule under abiotic stress factors, which together with the excess sulfur present in the environment, forces a successful acclimatization with bioremediation effect at this species 28.222 UC/mg proteins. Larix decidua, in its turn, is known in the literature as being a species that shows a high susceptibility to SO2 pollution, its sensitivity becoming extreme as the plant ages (Davis and Wilhour, 1976). From our findings, the catalase activity in this species of conifers was higher than that of Picea abies (16.642 UC/mg proteins), which proves the high antioxidant adaptability of Larix decidua, confirming a higher resistance than other conifers to stress induced by anthropogenic sulfur, since the contamination deposits on the permanently green needles of the coniferous trees and not on larch (Lines, 1984 cited by Chalupa, 1991). The activity of copper and barite ore processing works of Tarnia, even though stopped in 2007, focused on copper ores exploited trough underground mining (dominant minerals in copper zinc ores are: chalcocite, chalcopyrite, blende (ZnS), pyrite (FeS2), pyrotine (FeS), and baritine. The impact the two sections activity was felt in the same area, with the same receptors of the noxious pollutants, having the same waste facilities. The dominant pollutants are the polymetallic particulate matters, sulfur oxides, nitrogen oxides and hydrogen sulfide, The analyzes for copper and zinc indicators shows constant exceeds for these parameters, while the zinc indicator shows a steady increase after the closing of the mining operations (Ionce, 2010). The synergistic action of a toxic metal with a key metal for the cellular activity has as a consequence the reduction of the cellular damage, while the combinations of metals and phytochelatin toxicity can act against the excess toxicity of the elements found in the environment (Bertrand and Poirier, 2005, Grill et al., 2007). This could explain, probably, why at the analytic approach of the catalase activity on the foliar samples taken from different wood species located in the polluted areas from the Tarnita region, the values obtained after their quantitative measuring, the level of the oxidative stress at these species is smaller than that of other mining areas (Climani), having medium values, some of them quite similar to those found in the control area (Rduti). The effective defense capabilities of plants to the negative action of the oxidative stress inducted by the presence of metallous and non-metallous (baritine) particles in the environment is different. In Larix decidua species was registered the existence of a maximum level of catalase activity 19.62 UC/mg proteins, while in the leaves of birch (Betula verrucosa) a relatively high value was detected -18.35 UC /mg proteins. The accumulation of hydrogen peroxide in the leaf material of Populus tremula induces an increase in catalase activity to a level of 15.966 UC/mg proteins, and in Salix alba was detected the presence of a relatively high oxidative stress in the presence of iron-contaminated soil (Wahsha et al., 2011), the species in question being considered more tolerant toenvironmental pollution with pyrite, which recommends it as a useful in the phytoremediation of contaminated sites. Minimal level of activity of the oxidoreductase in Picea abies 8.262 UC/mg protein probably is due to processes in the rhizosphere (Vamerali et al., 2009), the absorption of iron being made in the roots, metallothioneins, ferritin and other chellators being capable to create a protection against damage induced by excess iron, copper, zinc and barite. In interpreting of the one should take into account the fact that, it is possible that under stressful conditions, in the leaves of species with low resistant to anthropogenic pollution might be a misbalance balance between the production of reactive oxygen species and antioxidative defense enzyme activity. This assumption relies on the fact that the production of antioxidant in its turn affected by stressful conditions (Bowler et al., 1992), for in some cases enzyme activity

39

Marius Viorel Oniciuc et al Some aspects of antioxidant defense mechanisms in wood species exposed to anthropic pollution in Suceava County

rise and in others decreases, facts that depend on the intensity of stress factors and chronic pollution, of the receptivity of plant organism, individual development stage, the climatic conditions and, especially, the genetic background. The great versatility of peroxidase is its predominant characteristic, and, therefore, there is no major physiological process that would function without this enzyme, that has a wide range of isoenzymes and large physiological implications: auxins oxidation (Tognetti et al., 2012), involvmnent in the ethylene biosynthesis (Gaspar et al., 1982), lignin metabolism (Diaz et al., 2001, Chen et al., 2002, Rodrguez Dorantes and Guerrero Ziga, 2012), the hydroxylation of proline to hydroxyproline (Ishikawa et al., 2006), the mechanisms of plant resistance (Cheng, 2003). Consequently, in the interpretation of the data obtained in regard to peroxidase activity in the leaf samples collected from species of Picea abies, Salix alba, Larix decidua, Betula verrucosa and Populus tremula in areas with a different nature of pollution (metalliferous, nonmetalliferous, radioactive) or from places considered to have no mining residual wastes, one should take into account all these aspects, and not only the participation of this oxidoreductase to antioxidant defense. In concordance with the data from the literature, the peroxidase is considered to be an indicator of anthropogenic sulfur accumulation in the environment (Keler, 1976, Horsman and Welburn, 1977 quoted by Khan and Malhotra, 1982), the activity of this oxidoreductase being higher in the aging leaf tissues of Betula and Pinus sp. compared to the younger ones, their possible role in their senescence being attributed to peroxidase, because it has the ability to oxidize indolil-acetic acid, a growth hormone and, in agreement with other studies, the senescence is proportional directly to the accumulation of hydrogen peroxide at cells. The chronic exposure to anthropogenic sulfur was followed by a strong induction of the peroxidase activity at Populus tremula, the foliar material sampled from the Climani Mountains showing a quantitative level of de 1.822 UP/ mg proteins. It is known SO2 is capable of altering the biochemistry of sulfur in cells and to rise the internal basin of the active osmotic material, affecting its metabolism. At certain plants, the start of the growth of the peroxidase activity caused by high quantities of manmade sulfur at a certain exposure can coincide with the apparition of the foliar necrosis, despite the fact that a close bio-monitoring of certain Angiosperm species had shown that the activity of the oxidoreductase can be an early biological marker capable of giving an alert before the apparition of visible symptoms, when sometimes it can be too late (Tripathi and Gautam, 2007). In this case, given the chronic exposure to pollutant, it is very likely that the high activity of the enzyme can be due to an adaptive mechanism of the plant to stressful conditions that has a well-regulated homeostasis. The poplar is well known as being an excellent choice for bio-recovery, as it offers multiple cycles of decontamination (Bittsnszky et al., 2005). The samples taken from Salix spp. alba individuals from the Climani Mountains, showed in their turn a high activity of the peroxidase 1.736 UP/mg proteins, which is nothing unusual considering the fact that normally, the activity of the peroxidase rises with together with the growing quantity of sulfur of the plants exposed to chronic pollution, an eventual tolerance phenomenon in this could be related to the activity of the SOD and of peroxidase in the vegetal cell (Jager et al., 1985, Agrawal et al., 1986 cited by Abrol and Ahmad, 2003). In regard to the activity of this peroxidase in the foliar material sampled from Betula verrucosa and Larix decidua, the results are somehow surprising, both species showing enzymatic activity values close to those registered in the control area (0.258 UP/mg proteins for

40

Analele tiinifice ale Universitii Alexandru Ioan Cuza, Seciunea Genetic i Biologie Molecular, TOM XIV, 2013

birch, respectively 0.146 UP/mg proteins for larch). The investigations made on Larix deciduas showed that a growth of the peroxidase on the course of the growing season is in close connection with the synthesis of new izoenzymes of the same oxidoreductase (Grill et al., 1980). Taking into account the fact that the intervention of the peroxidase takes place in the condition in which the quantity of H2O2 is small and that the activity of the enzyme is much more diminished in the condition of the acidification of the reaction environment, it cannot be excluded the valability of this explanation for the obtain results, even more if we account for the high activity of the catalase at this species, this oxidoreductase acting complementary with the peroxidase. Analyzing the samples taken from the wood species from Tarnia it can be observed that the greatest sensibility for the accumulation of the hydrogen peroxide in the vegetal cell under the pressure of the metals found in the environment was found at Populus tremula - 1.754 UP/mg proteines, Salix alba -1.58 UP/mg proteins and Picea abies - 1.446 UP/mg proteins. In the Rdui area, considered to be non-polluted, the accumulation of small hydrogen peroxide quantities was different from one species to another, having a higher intensity at Populus tremula (1.79 UP/mg proteins), Picea abies (1.693 UP/mg proteins) i Salix alba (0,676 UP/mg proteins). CONCLUSIONS In this stage of our investigations, proposing a so-called scale of resistance for Angiosperm and Gymnosperm species to the aggression of anthropic pollution has a rather quasi guide character. A biochemical monitoring on long term is necessary in order to follow the effects of chronic exposure to heavy metals and barite, but also the supplementation of test that can offer a larger picture in regard to the implication of contaminating factors on the foliar mechanisms of woodland plants. REFERENCES
Abrol, Y.P. and Ahmad, A. (2003): Sulphur in plants, Springer, page 285. Artenie, Vl., Ungureanu E., Negura, A.M. (2008): Metode de investigare a metabolismului glucidic i lipidic manual de lucrri practice, Editura Pim, Iai. Bertrand, M. and Poirier, I. (2005): Photosynthetic organisms and excess of metals, Photosynthetica, vol.43, no.3, pages 345-353. Bittsnszky A, Kmives T, Gullner G, Gyulai G, Kiss J, Heszky L, Radimszky L, Rennenberg H. (2005): Ability of transgenic poplars with elevated glutathione content to tolerate zinc (2+) stress, Environ Int., vol. 31, no. 2: 251-254. Bowler, C.,Van Montagu, M., Inze, D. (1992): Superoxide dismutase and stress tolerance. Annu. Rev. Plant. Physiol., 43: 83 116. Chalupta, V. (1991): Larch (Larix decidua Mill. )in Trees III, Biotechnology Agriculture and Forestry 16 (ed. Bajaj, Y.P.S.), Springer, pages 446-447. Chen, E.L., Chen, Y.A., Chen, L.M. and Liu, Z.H. (2002): Effect of copper on peroxidase activity and lignin content in Raphanus sativus. Plant Physiology and Biochemistry, 40: 439-444. Cheng, S. (2003): Effects of heavy metals on plants and resistance mechanisms, Environ. Sci. & Pollut. Res., vol. 10, no. 4, pages 2 5 6 - 2 6 4. Cojocaru D. C., Toma O., Sabina Ioana Cojocaru, Elena Ciornea (2009): Practicum de biochimia proteinelor i acizilor nucleici, Ed. Tehnopress, Iai, ISBN 978-973-702-640-8, 261 p. Davis, D.D. and Wilhour, R.G. (1976): Susceptibility of woody plants to sulphur dioxide and photochemical oxidants:a literature review, U.S. Environmental Protection Agency, Office of Research and Development, Corvallis Environmental Research Laboratory, pages 34, 41.

41

Marius Viorel Oniciuc et al Some aspects of antioxidant defense mechanisms in wood species exposed to anthropic pollution in Suceava County Diaz, J., Bernal, A., Pomar, F., Merino, F. (2001): Induction of shikimate dehydrogenase 862 and peroxidase in pepper (Capsicum annuum L.) seedlings in response to copper stress and its relation to lignification. Plant Science, vol. 161, pages 179-188. Gaspar, T., Penel, Cl., Thorpe, T. and Greppin, H. (1982): Peroxidases. A survey of their biochemical and physiological roles in higher plants, pages 44-48. Grill, D., Esterbauer, H. and Birkner, M. (1980): Untersuchungen fiber die Peroxidaseaktivit/it in L/irchennadeln. Beitr. Bol. Pflanzen, 55: 67-76. Grill, E., Mishra, S., Srivastava, S. and Tripathi, R.D. (2007): Role of phytochelatins in phytoremediation of heavy metals, Environmental Bioremediation Technologies, pages 101-146. Ionce, A. (2010): Impactul sistemic al activitii de preparare a substanelor minerale utile n judeul Suceava, Tez de doctorat. Ishikawa, T., Dowdle, J., Smirnoff, N. (2006): Progress in manipulating ascorbic acid biosynthesis and accumulation in plants. Physiologia Plantarum, vol.126: 343355. Khan, A.A. and Malhotra, S.S. (1982): Peroxidase activity as a indicator of SO2 injury in Jack Pine and White Birch, Biochem.Physiol.Phlanzen., vol. 177: 648-650. Komives, T. and Gullner, G. (2006): Dendroremediation: The Use of Trees in Cleaning up Polluted Soils, Phytoremediation Rhizoremediation, pages 23-31. Kosova, K., Vtmvs, P., Prasil, I.T. and Renaut, J. (2011): Plant proteome changes under abiotic stress Contribution of proteomics studies to understanding plant stress response, Journal of Proteomics, 74 (8): 13011322. Morr, D.J., Selldn, G., Ojanper,K., Sandelius, A.S., Egger, A., Morr, D.M., Chalko, C.M. and Chalko, R.A. (1990): Peroxisome proliferation in Norway spruce induced by ozone, Protoplasma, 155 (1-3): 58-65. Navarri-Izzoand, F. and Rascio, N. (2010): Heavy Metal Pollution Damage and Defense Strategies in Plants, in Handbook of Plant and Crop Stress, Third Edition, Edited by Mohammad Pessarakli, CRC Press 2010, pages 635674. Olejniczak, A., Cyganiuk, A., Kuciska, A. and ukaszewicz, J.P. (2012): Energetic Willow (Salix viminalis) Unconventional Applications, in Sustainable Growth and Applications in Renewable Energy Sources, pages 181-208. Pulford, I.D. and Dickinson, M.N. (2005): Phytoremediation technologies using trees - in Trace Elements in the Environment (Eds. Prassad, M.N.V. and Naidu, R.), CRC Press, New York, pages. 375-395. Rodrguez Dorantes, A. and Guerrero Ziga, L.A. (2012): Phenoloxidases activity in root system and their importance in the phytoremediation of organic contaminants, Journal of Environmental Chemistry and Ecotoxicology, vol. 4, no.3, pages. 35-40. Schtzendbel, A. and Polle A. (2002): Plant responses to abiotic stresses: Heavy metal-induced oxidative stress and protection by mycorrhization. J. Exp Bot., 53: 1351-65. Singh Gill, S. and Tuteja, N. (2010): Reactive oxygen species and antioxidant machinery in abiotic stress tolerance in crop plants, Plant Physiology and Biochemistry, 48: 909-930. Smirnoff, N. (1995): Antioxidant systems and plant response to the environment. In:Environment and plant Metabolism (Smirnoff, N. ed.). Bios Scientific Publishers, pages 217-243. Stobrawa, C. and Lorenc-Pluciska, G. (2007): Changes in antioxidant enzyme activity in the fine roots of black poplar (Populus nigra L.) and cottonwood (Populus deltoides Bartr. ex Marsch) in a heavy-metal-polluted environment, Plant and Soil, vol. 298, no. 1-2, pages 57-68. Tognetti, V.B., Mhlenbock, P. and Van Breusegem, F. (2012): Stress homeostasis the redox and auxin perspective, Plant, Cell & Environment, Special Issue: Special Issue on Redox Signaling, vol. 35, Issue 2, pages 321333. Tripathi, A.K. and Gautam, M. (2007): Biochemical parameters of plants as indicators of air pollution, J. Environ. Biol., vol. 28, no.1, pages 127-32. Vamerali, T., Bandiera, M., Coletto, L., Zanetti, F., Dickinson, N.M. and Mosca, G. (2009): Phytoremediation trials on metal- and arsenic-contaminated pyrite wastes (Torviscosa, Italy), Environ Pollut. 157(3): 887-94. Wahsha, M., Bini, C., Fontana, S., Washa, A. and Zilioli, D. (2011): Toxicity assessment of contaminated soils from a mining area in Northeast Italy by using lipid peroxidation assay, Journal of Geochemical Exploration, 113: 112117 1) Alexandru Ioan Cuza University of Jassy, Romania * ciornea@uaic.ro

42