oniciuc, tutu, cojocaru, ciornea.pdf

7/27/2019 Oniciuc, Tutu, Cojocaru, Ciornea.pdf

1/6

Analele tiinifice ale Universitii Alexandru Ioan Cuza, Seciunea Genetici 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 TUTU

1,

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

verrucosaEhrh. 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 ortho-

dianisidine 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 so-

called 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 denovo 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 abiesL.Karst., Larix decidua ssp Mill. and Angiosperms such as Salix alba L., Populus

tremulaandBetula verrucosaEhrh. 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


2/6

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 abiesdecreases with age, being higher in the growth conditions of plantsin 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 proteinsdetected 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 ofPopulus tremulaspecimens 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 thespecies 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 topollution (Stobrawa and Lorenc-Pluciska, 2007), the decreased fluid regime potentiating the

formation of superoxide radicals, which results in a sustained activity of the superoxide-

dismutase and implicitly, in a catalase activity that prevents the accumulation of hydrogenperoxide.

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 highactivity 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 withPopulus 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).

0

5

10

15

20

25

30

CU/mgprotein

Picea abies Populus

tremula

Salix spp alba Betula

verucosa

Larix decidua

Calimani Tarnita Radauti

0

0,2

0,4

0,6

0,8

1

1,2

1,4

1,6

1,8

2

PU/mgprotein

Picea abies Populus

tremula

Salix spp alba Betula

verucosa

Larix decidua

Calimani Tarnita Radauti

38


3/6


Immediately after this value,Betula verrucosaconfirms the fact that hydrogen peroxideacts 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 SO2pollution, its sensitivity becoming extreme as the plantages (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 highantioxidant adaptability of Larix decidua, confirming a higher resistance than other conifers to

stress induced by anthropogenic sulfur, since the contamination deposits on the permanentlygreen 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 incopper 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 thepolymetallic 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 theenvironment (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 Tarnitaregion, 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, someof 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. InLarix deciduaspecies was registered the existence of a maximum level of catalaseactivity 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 ofPopulus tremulainduces

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 toleranttoenvironmental 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 8.262UC/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 beingcapable to create a protection against damage induced by excess iron, copper, zinc and barite.

In interpreting of the one should take into accountthe fact that, it is possible that understressful 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 inits turn affected by stressful conditions (Bowler et al., 1992), for in some cases enzyme activity

39


4/6



rise and in others decreases, facts that depend on the intensity of stress factors and chronicpollution, 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 rangeof 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, BetulaverrucosaandPopulus tremulain areas with a different nature of pollution (metalliferous, non-

metalliferous, 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 toantioxidant defense.

In concordance with the data from the literature, the peroxidase is considered to be anindicator 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, theirpossible 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, thesenescence is proportional directly to the accumulation of hydrogen peroxide at cells.

The chronic exposure to anthropogenic sulfur was followed by a strong induction of theperoxidase activity atPopulus tremula, the foliar material sampled from the Climani Mountains

showing a quantitative level of de 1.822 UP/ mg proteins. It is known SO2is capable of alteringthe 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 theapparition 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 biologicalmarker 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 tolerancephenomenon 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 Betulaverrucosa 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


5/6


birch, respectively 0.146 UP/mg proteins for larch). The investigations made onLarix deciduasshowed 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 diminishedin 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 activityof 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 thatthe 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 atPopulus tremula- 1.754 UP/mg

proteines, Salix alba -1.58 UP/mg proteins andPicea 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(0,676UP/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 guidecharacter.

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 largerpicture 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


6/6



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 inL/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,

Tezde 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 RenewableEnergy 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: 112-

117

1) Alexandru Ioan Cuza University of Jassy, Romania

* [email protected]

42

oniciuc, tutu, cojocaru, ciornea.pdf

Documents