ANTIOXIDANT ENZYMES IN THE NEEDLES OF DIFFERENT OMORIKA LINES

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 external 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-dismutase (EC 1.15.1.1)in six half-sib lines of omorika.This is the first study of these antioxidant enzymes in omorika.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 indicator 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 rb 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 genetic variability, it is necessary to evaluate and understand genetic variability of the biochemical parameters used in biomonitoring.Our results obtained on the example of 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 continuous 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 experiments: A ("borealis") -branching similar to the branching in spruce, broad tree crown; B ("semidichotomus") -characterized by a spontaneously appearing double treetop, 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") -oneand two-year branches downardly oriented.

Determination of enzyme activities
All reagents and substances used in the experiments were analytical grade and obtained from Sigma (Germany) and Fluka (Germany).
Extraction of the enzymes was performed on ice by grinding frozen needles to pieces and 1 min homogenization in the ice-cold medium in a 1:5 ratio (w/v) using an Ultra-Turrax homogenizer.The homogenate was squeezed through eight gauze layers and centrifuged at 12000 x g and 4 o C for 10 min.The supernatant was used for enzyme activity measurements.Soluble SOD was extracted using 0.1 M TRIS-HCl buffer (pH 7.6), containing 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 contained 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 spectrophotometer in a total volume 3 ml.That of SOD was determined by measuring the percent of SOD-induced inhibition 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 guaiacol 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 H 2 O 2 and 10 μl of enzyme extract.The turnover of guaiacol was monitored at 470 nm and the reaction rate calculated from an extinction coefficient for guaiacol of 25.5 mM -1 cm -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 determined 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 H 2 O 2 .The rate of O 2 release was calculated as the amount of O 2 released per min, and CAT activity was expressed as the amount of H 2 O 2 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) 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 H 2 O 2 in Na-acetate buffer (pH 5.5) for 10 min at 25 o C. Polyphenol 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 25 o c.

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 concentration 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 significantly 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 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 isoenzymes were detected, with pI values between 3 and 4. When guaiacol was used as the substrate, two basic isoenzymes 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.The obtained results show that omorika lines A3 and B5 have the highest activity of the studied antioxidant 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).However, 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 electrophoretic patterns show that variation of activity among the lines cannot be attributed to variation in the isoenzyme pattern.
The obtained results show variation of enzyme activities within particular phenogroups, i.e., their dependence 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 compared 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 experiments.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.Enzyme activities constitute a fine indication of pollution stress in plants, since they are sensitive to low pollutant 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 taken into consideration when planning and designing programs for monitoring of tree and forest damage.ензими заштите од оксидационих оштећења у различитим линијама оморике јелена богдановић 1 , тања дучић 1 , н. милосавић 2 , з. вујчић 3 , мирјана шијачић 4 , в. исајев 4 и ксенија радотић1 Needles were collected in 2002 and immediately stored in liquid nitrogen until the experiments.The plants used for the experiments were healthy, without any exogenous 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).

Fig. 1 .
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.

Fig. 2 .
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.
dIScuSSION A common feature of POD, CAT, and SOD is their response to an increased level of organic and oxygen free radicals and related H 2 O 2 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 nd 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 removing 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) possibly mediated by their products, such as oxidized quinones, 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).