Antioxidant enzymes in the needles of different omorika lines

ANTIOXIDANT ENZYMES IN THE NEEDLES OF DIFFERENT OMORIKA LINES

Arch. Biol. Sci., Belgrade, 57 (4), 277-282, 2005.

JELENA BOGDANOVIĆ1, TANJA DUČIĆ1, N. MILOSAVIĆ2, Z. VUJČIĆ3, MIRJANA ŠIJAČIĆ4, V. ISAJEV4 and KSENIJA RADOTIĆ1

1Center for Multidisciplinary Studies, University of Belgrade, 11000 Belgrade, Serbia and Montenegro 2Institute of Chemistry, Technology and Metalurgy, Department of Chemistry, 11000 Belgrade, Serbia and Montenegro

3Faculty of Biochemistry, University of Belgrade, 11000 Belgrade, Serbia and Montenegro 4Faculty of Forestry, University of Belgrade, 11000 Belgrade, Serbia and Montenegro

Abstract - Picea omorika (Panč.) Purkyně (Serbian spruce) is a Balkan endemic coniferous species. We studied soluble peroxidase, catalase, polyphenol oxidase, and superoxide-dismutase activity in the needles of five omorika lines grown in a generative seed orchard. The peroxidase and polyphenol oxidase isoenzyme patterns were also investigated. Activity of the studied enzymes varied among different lines. The highest activity of peroxidase, catalase and polyphenol oxidase was found in the A3 (“borealis”) and B5 (“semidichotomous”) lines. Four acidic and two basic peroxidase isoenzymes and one polyphenol oxidase isoenzyme were detected. There was no variation in either the peroxidase or the polyphenol oxidase isoenzyme pattern among the different lines.

udc 582.47(497):581.1

INTROducTION

Picea omorika (Panč.) Purkyně (Serbian spruce) is a Balkan endemic coniferous species and Tertiary relict of the European flora. Omorika is naturally restricted to small communities occupying exclusively habitats along the middle and upper courses of the river Drina. It is more tolerant to air pollution and drought than are other conifers (G i l m a n and W a t s o n , 1994; K r á l , 2002). Cultivated omorika is used throughout Europe as a decorative species due to its elegant shape and pollution resistance.

Only a few studies on omorika have been published. Scanty data on glutathione reductase activity in omorika needles in comparison with other evergreen trees were reported by E s t e r b a u e r and G r i l l (1978). Data on omorika genome size and base composition have also been published (Š i l j a k and J a k o v l j e v et al. 2002).

In plants, antioxidant enzymes are known to share a considerable part of the mechanism of resistance to ex-ternal stress (R a b e and K r e e b , 1979). In the present

work, we studied the activities and isoenzyme profiles of guaiacol peroxidase (EC 1.11.1.7), catalase (EC 1.11.1.6), polyphenol oxidase (EC 1.10.3.1), and superoxide-dis-mutase (EC 1.15.1.1) in six half-sib lines of omorika. This is the first study of these antioxidant enzymes in omori-ka. Our aim was to see whether there is a difference in the activity of antioxidant enzymes among various omorika lines. Such study may contribute to the selection of the omorika genotypes with a more successful response to polluted and urban environments. More generally, such studies may help to expand the range of omorika. On the other hand, activities of antioxidant enzymes in trees have been reported in the literature and used as an in-dicator in monitoring pollution-induced forest damage (H e r m a n and S m i d t , 1994; K e l l e r , 1974; N o r -b y , 1989; S c h u l z and H ä r t l i g , 2001). Coniferous trees are specially interesting in this respect, since they indicate pollution over a longer period (M u l g r e w and W i l l i a m s , 2000). Because plant enzyme activities are subject not only to environmental factors, but also to ge-netic variability, it is necessary to evaluate and understand genetic variability of the biochemical parameters used in biomonitoring. Our results obtained on the example of

277

Key words: Peroxidase, pollution resistance, polyphenol oxidase, superoxide-dismutase

P. omorika represent a contribution to the understanding the effect of genetic variation on the studied enzymes in the context of their use in monitoring programs.

MATERIALS ANd METHOdS

Needles were obtained from 15-year-old omorika trees grown in a generative seed orchard in Godovik (43° 51’ N, 20° 02’ E, 400 m), Serbia. The generative seed orchard of omorika was planned on the basis of previous studies on collective and individual variability of contin-uous and discontinuous features of half-sib omorika lines (Š i j a č i ć and N i k o l i ć , 2001).

The following omorika lines were used in the exper-iments: A (“borealis”) – branching similar to the branch-ing in spruce, broad tree crown; B (“semidichotomus”) - characterized by a spontaneously appearing double tree-top, without visible biotic or abiotic causes; C (“serbica”) - type of branching characteristic of the typical habitat of omorika, narrow pyramidal crown; D (“argentea”) - one- and two-year needles upwardly oriented, giving the crown a silvery appearance; and E (“viminalis”) - one- and two-year branches downardly oriented.

Needles were collected in 2002 and immediately stored in liquid nitrogen until the experiments. The plants used for the experiments were healthy, without any exog-enous infection detected. Analyzed metal (Fe, Mn, Zn, Co, Cr, Pb, Ni, Cd) concentrations in the needles and soil were within the threshold values (K a b a t a -P e n d i a s and P e n d i a s , 1984; International Union of Biological Sciences, 1994).

Determination of enzyme activities

All reagents and substances used in the experiments were analytical grade and obtained from Sigma (Germa-ny) and Fluka (Germany).

Extraction of the enzymes was performed on ice by grinding frozen needles to pieces and 1 min homog-enization in the ice-cold medium in a 1:5 ratio (w/v) us-ing an Ultra-Turrax homogenizer. The homogenate was squeezed through eight gauze layers and centrifuged at 12000 x g and 4oC for 10 min. The supernatant was used for enzyme activity measurements. Soluble SOD was ex-tracted using 0.1 M TRIS-HCl buffer (pH 7.6), contain-ing 1 mM dithiothreitol and 1 mM EDTA. Extraction of soluble POD and PPO was performed using 0.1 M TRIS-

HCl buffer (pH 7.6), containing 1 mM dithiothreitol and 0.2 % Tween 80, while the CAT extraction buffer con-tained 1 % Tween 80. The homogenates were stirred on ice for 1 hour before centrifugation.

Activitie of SOD, POD, and PPO were determined spectrophotometrically using a Shimadzu UV-160 spec-trophotometer in a total volume 3 ml. That of SOD was de-termined by measuring the percent of SOD-induced inhi-bition of adrenalin autooxidation at alkaline pH (M i s r a and F r i d o v i c h , 1972). The reaction was monitored at 480 nm. Peroxidase activity was determined with guaia-col as a substrate (C h a n c e and M a e h l y , 1955). The assay mixture contained 50 mM acetate buffer (pH) 4.5, 92 mM guaiacol, 18 mM H2O2 and 10 μl of enzyme ex-tract. The turnover of guaiacol was monitored at 470 nm and the reaction rate calculated from an extinction coeffi-cient for guaiacol of 25.5 mM-1cm-1. Activity was of PPO determined using 40 mM catechol in 100 mM phosphate buffer (pH 6.5), and 0.1 ml of enzyme extract at 410 nm. One unit of PPO activity was defined as the change of absorbance in 0.001 min-1. Catalase activity was deter-mined using a Clark-type polarographic electrode at 29˚C

in a total volume 1 ml. The enzyme extract (20 μl) was added to 100 mM phosphate buffer (pH 7.5) and oxygen release was initiated by 12 μl of 1 M H2O2. The rate of O2 release was calculated as the amount of O2 released per min, and CAT activity was expressed as the amount of H2O2 consumed per min and g fresh weight.

Enzyme activities were referred to fresh weight of the samples. Protein concentration of the needle extracts was determined by the method of L o w r y et al. (1951)

JELENA BOGDANOVIĆ et al.278

Fig. 1. Total soluble protein concentration in the needles of different omorika lines.A2, A3, B5, C2, C4, D1, D6, E3: letters and corresponding numbers indicate the phenogroup and related mother genotype, respectively (for the characteristics of the phenogroups see Materials and Methods). *Statistically significant difference in comparison with the other omorika lines; **Statistically significant difference in comparison with E3 line. The bars indicate S.E.

with bovine serum albumin as the standard.

Isoelectrofocusing and native polyacrylamide gel electrophoresis of soluble enzymes

Soluble POD isoenzymes were separated in a pH gradient from 3 to 9 (using 5 % ampholite solution) on a 7.5 % polyacrylamide (PAA) gel. Native PAGE of PPO was performed using 4/10 % PAA gel (D a v i s , 1964). Peroxidase isoenzymes were stained on gel with 20 mM guaiacol or 0.6 mM 4-chloro-1-naphthol and 5 mM H2O2 in Na-acetate buffer (pH 5.5) for 10 min at 25oC. Poly-phenol oxidase isoenzymes on the native PAGE gels were stained with 5 mM L-DOPA in 0.1 M phosphate buffer (pH 6.5) for 1 hour at 25oc.

Statistics

Each experimental variant was represented by four trees, which were separately treated in four replicates each. Statistical analysis of the results was performed using the Mann-Whitney ranking test at a 0.05 level of

significance.

RESuLTS

There was no significant variation in protein con-centration between different omorika lines. An exception is the B5 line, where protein content was considerably lower than in the other lines (Fig. 1).

Guaiacol peroxidase activity was highest in the A3 line, followed by activity in the B5 line (Fig. 2a). The A2, D, C and E lines had relatively similar activity values that were lower that in the A3 and B5 lines. Significantly higher catalase activity was detected in the A3 line in comparison with all the other lines. The lowest activity was found in the D1 and D6 lines (Fig. 2b). There was no significant difference in CAT activity between D and E lines, with the exception of lower activity in the D1 line. Polyphenol oxidase activity in the B5 line was signifi-cantly higher than in all the other lines, while the lowest activity was recorded in the C2 line (Fig. 2c). Activity of SOD had relatively similar levels in all the lines, except

ANTIOXIDANT ENZYMES IN THE NEEDLES OF DIFFERENT OMORIKA LINES 279

Fig. 2. Activity of the soluble guaiacol peroxidase (a), catalase (b), polyphenol oxidase (c), and superoxide dismutase (d) in needles of different omorika lines. *Significantly higher activity in comparison with the omorika lines cited in parentheses. The bars indicate S.E.

A3, line where its activity was significantly lower than in the other lines (Fig. 2d).

The presence of acidic and basic POD isoenzymes in the IEF pattern depended on the substrate used for gel staining (Fig. 3a). When 4-chloro-naphthol was used as substrate, only acidic isoenzymes appeared, while in the case of guaiacol both acidic and basic isoenzymes were present. In the case of both substrates, four acidic isoen-zymes were detected, with pI values between 3 and 4. When guaiacol was used as the substrate, two basic iso-enzymes were present with pI values of 8.5 and 9.

In the case of PPO isoenzymes, the results of native PAGE are presented here, since it showed higher band resolution in comparison with IEF. One PPO band was detected (Fig. 3b) in all the omorika lines.

dIScuSSION

A common feature of POD, CAT, and SOD is their response to an increased level of organic and oxygen free radicals and related H2O2 increase (A n d e r s o n et al. 1995; B o w l e r et al. 1992; C a s t i l l o , 1986; S c a n -d a l i o s , 1993; V a n g r o s v e l d and C l i j s t e r s 1994) induced by environmental pollution. Besides re-moving hydrogen- and organic peroxides, POD has a role in the synthesis of cell wall polymer lignin, this function being also involved in the response to stress conditions (C a s t i l l o , 1986; S i e g e l and S i e g e l , 1986). It has been suggested that polyphenol oxidases are a component of defense responses (G e r d e r m a n et al. 2002) pos-sibly mediated by their products, such as oxidized qui-nones, some of which can inactivate pectolytic enzymes produced by pathogens (C o n s t a b e l et al. 2000) and exhibit antimicrobial activity (L e a t h a m et al. 1980).

The obtained results show that omorika lines A3 and B5 have the highest activity of the studied antioxi-dant enzymes (Fig. 2). On the other hand, total protein concentration showed relatively low variation between the lines (Fig. 1). Despite significant variation of enzyme activity among different lines, no difference in their POD and PPO isoenzyme patterns was detected (Fig. 3). How-ever, the IEF results show that acidic POD isoenzymes have higher affinity for 4-chloro-naphthol in comparison with the basic ones (Fig. 3a). The POD and PPO electro-phoretic patterns show that variation of activity among the lines cannot be attributed to variation in the isoen-zyme pattern.

The obtained results show variation of enzyme ac-tivities within particular phenogroups, i.e., their depen-dence on the mother genotype. Thus the A3 line exceeds the A2 line in POD, CAT and PPO activity, while the D10 line has higher POD and CAT activity in comparison with the D1 and D6 lines.

Unfortunately, the obtained results cannot be com-pared with previously published data on other coniferous species, since most previous experiments were performed on samples collected in the field from considerably older trees in comparison with the trees used in our experi-ments. Our results may contribute to the selection of omorika lines with a successful response to air pollution and urban conditions. Such genotypes may also help to expand the range of omorika.

JELENA BOGDANOVIĆ et al.280

Fig. 3. Isoelectrofocusing of the soluble peroxidase (a) and native electrophoresis of polyphenol oxidase (b) from needles of various omorika lines. Protein quantity applied to each lane: (a) in the case of staining with guaiacol and 4-chloro-1-naph-thol: 11 μg and 3 μg, respectively; (b) 16 μg.

ANTIOXIDANT ENZYMES IN THE NEEDLES OF DIFFERENT OMORIKA LINES 281

Enzyme activities constitute a fine indication of pol-lution stress in plants, since they are sensitive to low pol-lutant levels, where there are not yet visible symptoms. The presented results on the example of omorika show that genetic variation within a tree species should be tak-en into consideration when planning and designing pro-grams for monitoring of tree and forest damage.

Acknowledgements: Grant 1911 (Cellular response to pollution stress in trees. Possibility of application in biomonitoring of the environment) from the Minis-try of Science and Technology of the Republic of Serbia supported this study.

REFERENCES

Anderson, M.D., Tottempudi, K.P., Stewart, C.R. (1995). Changesin isozyme profiles of catalase, peroxidase, and glutathione reductase during acclimation to chilling in mesocotylsof maize seedlings. Plant Physiol. 109, 1247-1257.

Bowler, C., Van Montagu, M., Inzé, D. (1992) Superoxide dismutaseand stress tolerance. Annu. Rev. Plant Physiol. Plant Mol.Biol. 43, 83-116.

Castillo, F.J. (1986). Extracellular peroxidases as markers of stress?In: Molecular and Physiological Aspects of Plant Peroxidases,(Eds. H. Grepin, C. Penel and T. Gaspar), 419-426, Universityof Geneva, Geneva.

Chance, B., Maehly, A.C. (1955). Assay of catalases and peroxidases.In: Methods in Enzymology, 2, (Eds. S.P. Colowick and N.O.Kaplan), 765-775, Academic Press Publishers, New York.

Constabel, C.P., Lynn, Y., Patton, J.J., Christopher, M.E. (2000).Polyphenol oxidase from hybrid poplar. Cloning and expression in response to wounding and herbivory. Plant Physiol. 124, 285–296.

Davis, B.J. (1964). Disc electrophoresis-II: Method and application tohuman serum proteins. Ann. NY Acad. Sci. 121, 404-427.

Esterbauer, H., Grill, D. (1978). Seasonal variation of glutathione andglutahione reductase in needles of Picea abies. Plant Physiol. 61, 119-121.

Gerdemann, C., Eicken, C., Krebs, B. (2002). The crystal structureof catechol oxidase: New insight into the function of type-3copper proteins. Chem. Res. 35, 183-191.

Gilman, E.F., Watson, D.G. (1994). Picea omorika Serbian Spruce Fact Sheet ST-451, Environmental Horticulture Depart-ment, Florida Cooperative Extension Service, University of Florida.

Herman, F., Smidt, St. (1994). Results from the fields of integratedmonitoring, bioindicators, and indicator values for the charac-terization of the physiological condition of trees. Phyton 34, 169-192.

International Union of Biological Sciences. (1994). Element con-centration cadasters in ecosystems. Progress report, 25th Gen-eral Assembly, Paris.

Kabata-Pendias, A., Pendias, H. (1984). Trace elements in soils

and plants, CRC Press, Inc., Boca Raton, Florida, 1-440.

Keller, T. (1974). The use of peroxidase activity for monitoring and map-ping air pollution areas. Eur. J. For. Pathol. 4, 11-19.

Král, D. (2002). Assessing the growth of Picea omorika [Panč.] Purkyně in the Masaryk forest training forest enterprise at Křtiny. J. Forest Sci 48, 388-398.

Leatham, G.F., King, M., Stahmann, M.A. (1980). In vitro protein polymerization by quinones or free radicals generated by plant or fungal oxidative enzymes. Phytopathology 70, 1134-1140.

Lowry, O.H., Rosebrough, N.J., Farr, A.L, Randall, R.J. (1951). Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265-275.

Misra, H.P., Fridovich, I. (1972). The role of superoxide-anion in the autooxidation of epinephrine and a simple assay for super-oxide dismutase. J. Biol. Chem. 247, 3170-3175.

Mulgrew, A., Wiliams, P. (2000). Biomonitoring air quality using plants. Air Hygene Report No 10, WHO Collaborating Center for Air Quality Management and Air Pollution control.

Norby, R. J. (1989). Foliar nitrate reductase: a marker for assimilation of atmospheric nitrate oxides. In: Biologic Markers of Air-Pol-lution Stress and Damage in Forests, Project Report, 247-274, Committee on biologic markers of air-pollution damage in trees, Board on environmental studies and toxicology, Com-mission on life sciences and National research council, National Academy Press, Washington.

Rabe, R., Kreeb, K. H. (1979). Enzyme activities and chlorophyll and protein content in plants as indicators of air pollution. Environ. Pollut. 19, 119-136.

Scandalios, J.G. (1993). Oxygen stress and superoxide dismutases. Plant Physiol. 101, 7 12.

Schulz, H., Härtling, S. (2001). Biochemical parameters as biomarkersfor the early recognition of environmental pollution on Scotch pine trees. II. The antioxidative metabolites ascorbic acid, glutathione, α-tocopherol, and the enzymes superoxide dismutase and glutathione reductase. Z. Naturforsch. 56C, 767-780.

Siegel, S.M., Siegel, B.Z. (1986). Peroxidase activity and stress: a complex relationship. In: Molecular and Physiological As-pects of Plant Peroxidases, (Eds. H. Grepin, c. Penel and T. Gaspar), 427-431, University of Geneva, Geneva.

Šijačić-Nikolić, M. (2001). Analysis of the genetic potential of a Ser-bian spruce (Picea omorika (Panč.) Purkině) generative seed orchard by the controlled hybridization of half-sib lines. Doc-toral dissertation, Faculty of Forestry, Belgrade University, 1-66 (in Serbian).

Šiljak-Yakovlev, S., Cerbah, M., Coulaud, J., Stoian, V., Brown, S.C., Zoldos, V., Jelenić, S., Papes, D. (2002). Nuclear DNA con-tent, base composition, heterochromatin, and rDNA in Picea omorika and Picea abies. Theor. Appl. Genet. 104, 505-512.

Vangronsveld, J., Clijsters, H. (1994). Toxic effects of metals. In: Plants and the Chemical Elements-Biochemistry, Uptake, Tol-erance and Toxicity (Ed. M.E. Farago), 149-177, VCH, Weinheim.

JELENA BOGDANOVIĆ et al.282

ензими заштите од оксидационих оштећења у различитим линијама оморике

јелена богдановић1, тања дучић1, н. милосавић2, з. вујчић3, мирјана шијачић4, в. исајев4 и ксенија радотић1

1Центар за мултидисциплинарне студије Универзитета у Београду, 11000 београд, србија и Црна гора; 2Институт за хемију, технологију и металургију, 11000 београд, србија и Црна гора; 3Факултет за хемију, Универзитет у Београду, 11000 београд, србија и Црна гора; 4Шумарски факултет, Универзитет у Београду, 11000 београд, србија и Црна гора

Picea omorika (Panč.) Purkyně је балканска ендемска врста конифера. испитиване су активности ензима пероксидазе, каталазе, полифенол оксидазе и супероксид дисмутазе у четинама шест линија оморике гајених у генеративној семенској плантажи. изоензимски састав пероксидазе и полифенол оксидазе су такође проучавани. највише активности

пероксидазе, каталазе и полифенол оксидазе нађене су у линијама A3 (“borealis”) и B5 (“semidichotomus”). утврђена су четири кисела и два базна изоензима пероксидазе, као и један изоензим полифенол оксидазе није утврђена разлика у изоензимском профилу између различитих линија оморике.