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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.
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Marius Viorel Oniciuc et al Some aspects of antioxidant defense mechanisms in wood species exposed to anthropic
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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
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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
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Marius Viorel Oniciuc et al Some aspects of antioxidant defense mechanisms in wood species exposed to anthropic
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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
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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.
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1) Alexandru Ioan Cuza University of Jassy, Romania
* ciornea@uaic.ro
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