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860
* For correspondence.
Oxidation Communications 38, No 2, 860–868 (2015)
Overall ecology
ACTIVITY OF A CATALASE ENZYME IN PLANTS FROM
THE BURNED AREAS OF THE VIDLIC MOUNTAIN BEECH
FOREST
M. S. MАRKOVICa*, B. S. ILICb, D. L. MILADINOVICb,
S. M. STAMENKOVICa, R. TRAJKOVICc, V. P. STАNKOV-JOVАNOVICd,
G. T. DJELICe
aDepartment of Biology and Ecology, Faculty of Science and Mathematics,
University of Nis, 18 000 Nis, Serbia
E-mail: marijam@pmf.ni.ac.rs
bDepartment of Pharmacy, Faculty of Medicine, University of Nis, 18 000 Nis,
Serbia
cDepartment of Biology, Faculty of Science and Mathematics, University of
Kosovska Mitrovica, 38 220 Kosovska Mitrovica, Serbia
dDepartment of Chemistry, Faculty of Science and Mathematics, University of Nis,
18 000 Nis, Serbia
eFaculty of Science and Mathematics, University of Kragujevac, Institute of
Biology and Ecology, 34 000 Kragujevac, Serbia
ABSTRACT
In 2007, a catastrophic re on the Vidlic Mountain in south-east Serbia occurred.
It burned down nearly 1000 ha of forest. Study of biochemical and physiological
parameters in plants which inhabit post re areas and their comparison with control
is of essential importance in estimating the impact of re on plant characteristics and
potential applications. After the re pioneer and indigenous plants from habitats af-
fected by re have a characteristic metabolism.
In this comparative study, the activity of enzyme catalase (ЕC 1.11.1.6, H2O2:
H2O2 oxidoreductase) was determined in plant species from a habitat affected by re
and the same plant species from the forest which had not been affected by re as a
control. Assessment of enzyme activity was carried out on the root, leaves and ow-
ers of plant species Geranium macrorrhizum, Doronicum columnae, Aegopodium
podagraria, Fagus moesiaca, Tussilago farfara, Glechoma hirsuta, Chelidonium
majus and Primula veris. The rst group of plant samples used for determination was
obtained from the habitat that was affected by re two years ago. Catalase activity was
861
measured using the gasometric method and the values obtained for this activity were
expressed as ml of O2. The presented results show a signicant increase in catalase
activity in individuals from habitats affected by re in relation to the control group.
Increased catalase activity is a consequence of oxidative stress caused by chemical
changes in soil that were generated by re.
Keywords: enzyme catalase, forest affected by re, Vidlic Mountain, oxidative stress.
AIMS AND BACKGROUND
From the year 2003 to 2007, there were 579 wild res registered in Serbia. The largest
number of these res (370) was recorded in the summer of 2007 (Ref. 1), including the
re on the Vidlic Mountain. The vegetation of the forest, rocks, shrubs and grassland
formations burned in the re. After the re was completely put out, it was estimated
that more than 2500 ha of low vegetation, scrublands and forests burned down2. Ap-
proximately 1000 ha of beech forests were burned.
Numerous studies examined the effect of re on the physiological functions
of different plants. Plants have different defense response mechanism to stress that
occurs as a result of re. After a forest re, the temperature and amount of light in-
crease, which causes the change in the anatomical and physiological characteristics
of plants3. Knapp et al.4 discovered that plants on the burnt area are thicker and have
wider leaves, higher specic leaf weight and a higher density of stoma. The increased
thickness of leaves means an increase of the mesophyll relative to the entire surface
of the leaf and leads to an increased CO2 usage and degree of photosynthesis5. Fleck
et al.6,7 indicated a higher level of photosynthesis in the leaves of the Quercus ilex
after the re.
From the areas that were burned and also the areas that were not affected by the
re from the Vidlic Mountain was determined the content of: organic acids8, heavy
metals in soil and plant samples9, chloroplast pigments10. In order to assess what kind
of impact does re have on the antioxidant properties of plants, plant species extracts
from the burnt and the non-affected areas were tested11,12. The impact of re on the
antimicrobial and antioxidant activities of some plant species was examined13. The
activity of the enzyme catalase was determined in root and shoot system plant parts of
the species Geranium macrorrhizum of the burnt and non-affected areas14. Moreover,
an examination and comparison of the chemical composition and antioxidant activity
of the essential oil species Ajuga chamaepytis from the burnt and non-affected areas
of Vidlic was made15.
The high intensity light and UV-B radiation causes oxidative stress and damage
in plants16. These conditions can be achieved in the areas where the vegetation was
completely burned by the re. The functioning of the antioxidant system and the
degree of oxidative damage can be evaluated successfully on the basis of determin-
ing the biochemical parameters – indicators of the oxidative stress17,18. One of the
indicators of oxidative stress in plants is the activity of the enzyme catalase. Changes
862
in the biotic and abiotic factors at the burnt area cause morphological-physiological
changes in plants. As a result of a group of changes in their environment, plants alter
their metabolism. First of all, an acceleration of the plant metabolism occurs which
hence leads to an acceleration of the enzymatic activity. Signicant changes were re-
corded because concentrations of some enzymes in plants increased. These enzymes,
whose concentrations signicantly increase in stressful conditions, are catalase and
peroxidase. These two enzymes remove free radicals whose concentration in a cell
increases in stressful conditions19. Increased concentrations of the enzyme catalase
indicate oxidative stress in plants that grow on burnt areas. Catalase (EC 1.11.1.6,
H2O2: H2O2 oxidoreductase) is one of the most powerful enzymes known. The reac-
tions which this enzyme catalyses are essential for plant life. Catalase breaks down
the toxic hydrogen peroxide into water and molecular oxygen. By using hydrogen
peroxide, which breaks down into water and molecular oxygen, the catalase further
oxidises toxic molecules which include phenol, formic acid, formaldehyde and alcohol.
Numerous authors have found a point of comparison between the activity of the
enzyme catalase and the content of chlorophyll20–22. They have noticed that the catalase
activity is several times higher in etiolated seedlings and leaves with chlorosis than
in green leaves. They interpret the similar behaviour of catalase and chlorophyll as
having closely linked ways of biosynthesis, which was shown in results obtained by
Mikhlin and Mutuskin23.
EXPERIMENTAL
Determination of plant material for analysis was performed by using the key for the
regional ora24,25.
Plant material for determining the activity of the enzyme catalase was collected
based on the conditions of the area in the spring of rst and second year after the re
in the burnt area of the beech forest on the Vazganica location. Plants collected at the
same time from a nearby area not affected by the re were used as the control plant
group. Herbarium samples of the analysed plants were placed in the herbarium of
University of Belgrade, Faculty of Biology (BEOU) and their voucher numbers are
given in Table 1. Immediately after being picked, plant samples were put into liquid
nitrogen in which they were transported and then put into a freezer kept at –20ºС
where they were stored until the analysis. Before the analysis, the root and shoot
system plant parts were separated and cut into little pieces.
Beech samples (Fagus moesiaca), besides being collected and stored in a freezer
until analysis like all of the other samples, were amassed the rst year after the re.
Newly sprouted seeds, along with the soil from the burnt area, were taken to a labora-
tory where the seedlings were kept in a plastic container for several weeks until the
analysis of the catalase enzyme activity.
863
Table 1. Inventory numbers of plants and the coordinates of the location where the plants were collected for determining the activity of catalase enzyme
Inventory
number
Plant species Location Habitat Date Coordinates Elevation (m)
16422 Fagus moesiaca (K. Maly) C z e c z. Vazganica burnt area of
beech forest
29.04.2008 43°10′36.6″ N
22°43′35.4″ E 1120
16423 Aegopodium podagraria L. Vazganica burnt area of
beech forest
22.06.2008 43°10′37.2″ N
22°43′29.5″ E 1140
16424 Glechoma hirsuta W a l d s t. & K i t. Vazganica burnt area of
beech forest
11.05.2008 43°10′43.2″ N
22°43′30.4″ E 1110
16425 Chelidonium majus L. Vazganica burnt area of
beech forest
21.06.2008 43°10′39.1″ N
22°42′34.1″ E 1115
16426 Doronicum columnae T e n. Vazganica burnt area of
beech forest
29.04.2008 43°10′37.0″ N
22°42′28.8″ E 1080
16427 Tussilago farfara L. Vazganica burnt area of
beech forest
30.03.2008 43°10′41.2″ N
22°42′37.1″ E 1070
16428 Primula veris L. Crni vrh rocky surface 28.04.2008 43°10′22.6″ N
22°39′19.6″ E 840
16431 Geranium macrorrhizum L. Vazganica burnt area of
beech forest
31.05.2008 43°10′41.1″ N
22°42′49.1″ E 1190
864
The catalase enzyme activity was determined by a gasometric method26. This
method was based on assessing the amount of oxygen being released after reacting
with H2O2 which was added to the plant extract that contained catalase.
First, 0.5–1 g of plant material were measured. Afterwards, the material was
extracted in a crucible with 0.5 g of CaCO3 and 20 ml of distilled water which was
added gradually. After that, the extract was transferred into an Erlenmeyer ask with
a port on the side and using the apparatus the activity of the enzyme catalase was
measured. The Erlenmeyer ask was sealed with a stopper which was pierced with a
needle with an attached syringe. The syringe contained 5 ml of 3% H2O2. The H2O2
was injected in the Erlenmeyer ask at the beginning of the experiment.
The content was stirred for a minute and after waiting 1 min stirred again for one
minute. After these 3 min, the amount of freed oxygen could be read on the burette
scale. Afterwards, the recalculation for 1 g of material was made.
Each analysis was repeated three times and the average value was calculated.
Catalase activity was expressed indirectly through the freed oxygen volume.
RESULTS AND DUSCUSSION
The stress in the living environment can extremely inhibit the enzymes, directly or
indirectly through physiological and biochemical processes, as well as in terms of
enzyme activation which will catalyse their decomposition. A group of enzymes,
which includes catalase and peroxidase, is important for the physiology of resistance
and endotoxicology27. Hydrogen peroxide is the substrate on which these enzymes
act, which is indicated by the fact that their presence in the medium encourages in-
creased activities of these enzymes – substrate induction28,29. The hydrogen peroxide
forms during the course of various metabolic processes as a reduced form of oxygen
and can cause a number of metabolic changes in the tissues of the plant. Because of
the high toxicity to living cells, it is necessary to remove or degrade it. The activity
of the enzyme catalase breaks it down into products that are not harmful to plants27.
Catalase activity was measured in root and shoot system parts of plants from the
burnt areas in the beech forests of the Vidlic Mountain and from the closest beech
forest area not affected by the re which represent a control group. The given results
show that the catalase activity is different in certain plant species and shows that the
root and shoot system plant parts of the tested plants are uneven (Tables 2 and 3).
It was noticed that, in the rst year after the re, the activity of the enzyme catalase
in the species Geranium macrorrhizum, Aegopodium podagraria and Fagus moesiaca
increased both in the root and shoot system plant parts of the burnt area (experimental
group) compared to the non-affected surface (control group), while only the catalase
activity in the root system of the species Doronicum columnae and Tussilago farfara
increased in relation to the control group (Table 2). Therefore, the activity of catalase
in all underground parts of the plants increased the rst year after the re.
865
Table 2. Activity of enzyme catalase in the root and shoot system plant parts on the not burned area and
the burnt area of Vidlic Mountain, the rst year after the re, converted to 1 g of fresh matter
Plant species Control group (not
burned area) (ml O2)
Experimental group
(burnt area) (ml O2)
Geranium macrorrhizum, shoot system plant part 6.19 6.68
Geranium macrorrhizum, rhyzome with roots 6.35 7.75
Doronicum columnae, ower heads 33.17 26.5
Doronicum columnae, stems – 13.77
Doronicum columnae, leaves – 19.69
Doronicum columnae, rhyzome with roots 12.35 21.13
Aegopodium podagraria, shoot system plant part 8.18 8.61
Aegopodium podagraria, rhyzome with roots 6.69 8.97
Fagus moesiaca, shoot system plant part under
laboratory conditions
13.76 19.16
Fagus moesiaca, root under laboratory conditions 6.80 9.09
Fagus moesiaca, shoot system plant parts 9.66 9.84
Fagus moesiaca, roots 11.37 11.70
Tussilago farfara, shoot system plant parts 20.10 17.78
Tussilago farfara, rhyzome with roots 15.95 17.44
In the second year after the re, the activity of the enzyme catalase in the species
Geranium macrorrhizum and Primula veris increased in both the shoot and root system
parts of the experimental group, compared to the control group; in the Doronicum
columnae, the catalase activity increased in the underground parts of the plant, stems
and leaves of the experimental group in relation to the control group, and it decreased
in the ower heads; in Chelidonium majus, the catalase activity was higher in the
underground parts, leaves and stems in the experimental group than in the control
plants, but it only decreased in relation to the control plants; in the Glechoma hirsuta
species, the catalase activity was higher in the control than in the experimental plants
(Table 3). The results show that the activity of the enzyme catalase has generally in-
creased in plants from the burnt area of the beech forest compared to the plants from
the non-affected area and it was almost always higher in the underground parts of
the plants in relation to above ground parts. This can be explained by the fact that the
root system plant parts are in direct contact with the chemical substances of the soil,
which contains plenty of ash and has a different, qualitatively and quantitatively new
chemical composition in relation to the land unaffected by the re. Concentrations
of compounds that are harmful to the plant increase in the soil and, consequently, in
the underground parts of the plants in the burnt area of the beech forest, because it
occurs during the metabolism of the plants under stressful conditions which occur
after the re. The catalase breaks down harmful compounds into harmless products,
and they are found in a greater amount in the root system than in the shoot system of
the experimental plants from the area affected by the re, with the exception of the
Glechoma hirsutа. This herbaceous plant has creeping sprouts that have thin wiry
866
roots, which do not penetrate deep into the soil and has a small absorption surface.
Therefore, it can be assumed that, in the burnt area, it accumulates less material harm-
ful to the plant, meaning that the catalase activity does not increase in relation to the
same plant from the non-affected area.
Table 3. Activity of enzyme catalase in the root and shoot system plant parts on the not burned area and
the burnt area of the Vidlic Mountain, the second year after the re, converted to 1 g of fresh matter
Plant species Control group
(not burned area)
(ml O2)
Experimental
group (burnt
area) (ml O2)
Geranium macrorrhizum, owers 12.12 17.17
Geranium macrorrhizum, leaves 7.90 10.89
Geranium macrorrhizum, rhyzome with roots 8.56 11.36
Doronicum columnae, ower heads 54.62 49.21
Doronicum columnae, stems 29.72 31.95
Doronicum columnae, leaves 37.99 48.92
Doronicum columnae, rhyzome with roots 19.95 26.61
Glechoma hirsuta, shoot system plant parts in bloom 30.44 23.54
Glechoma hirsuta, creeping sprouts with roots 24.56 15.96
Chelidonium majus, owers 33.15 36.60
Chelidonium majus, leaves 11.22 12.19
Chelidonium majus, stems 41.02 40.01
Chelidonium majus, roots 10.36 11.33
Primula veris, leaves 27.84 38.59
Primula veris, owers with ower stems 11.86 21.59
Primula veris, rhyzome with roots 10.06 10.68
Activity of the enzyme catalase in root and shoot system plant parts varies is
uneven and varies among species. Increased or decreased activity of catalase depends
on the morphological-anatomical structure of the plant leaves and roots, as well as a
rich and diverse chemical composition, which is genetically determined and specie
specic27. An increased catalase activity is a result of oxidative stress, which is caused
by chemical changes in the soil inuenced by re14. Increasing the activity of the en-
zyme catalase in plants at the re site is a metabolic form of breaking down harmful
compounds, i.e. detoxication, which is a high-quality mechanism of resistance that
responds to the changed conditions in the environment after the re.
CONCLUSIONS
Plants that grow on the burnt areas of the Vidlic Mountain are highly adaptive plants,
which have a characteristic metabolism and survival mechanisms. The changed condi-
tions after the re require an anatomical, physiological and biochemical adaptations
from plants. The ability of plants to adapt to stressful conditions is crucial for their
867
survival. Different plants respond differently to stress depending on different genetic
backgrounds, different phenophases and different morph-anatomic characteristics of
plants. An increased concentration of the enzyme catalase indicates oxidative stress
in plants which grow in the burnt areas. Catalase breaks down harmful products of
the plant metabolism at the burnt area into harmless compounds. On the basis of these
results, we can conclude that the catalase is involved in the plant protective defense
mechanisms against the toxic effects of the active forms of oxygen, which are formed
during a reciprocal action of the harmful products of metabolism after the re, with
cell membranes and membranes of cell organelles.
ACKNOWLEDGEMENTS
This research was supported by the Ministry of Education, Science and Technological
Development of the Republic of Serbia (Grant No OI 171025).
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Revised 13 November 2014
