OXALATE OXIDASE AND NON-ENZYMATIC COMPOUNDS OF THE ANTIOXIDATIVE SYSTEM IN YOUNG SERBIAN SPRUCE PLANTS EXPOSED TO CADMIUM STRESS

We st�died changes in the concentrations of ascorbate and gl�tathione, composition of sol�ble phenolics, and activity of o�alate o�idase in 75-day-old Serbian spr�ce plants after e�pos�re to 5 μM and 5� μM cadmi�m for 6-48 h. The presence of O�O� activity in a conifer species is here demonstrated for the first time. Both Cd concentrations ind�ced a decrease of O�O� activity in treated plants in comparison with the control at all sampling dates. The concentrations of red�ced gl�tathione, its o�idized form, and red�ced ascorbate in the plants decreased d�ring 48-h treatment with cadmi�m. Among simple phenolics, only catechin increased significantly d�ring Cd treatment.


INTRODUCTION
Increased contents of heavy metals, especially nonessential metals like Cd, in soils affect physiological processes in plants.Cadmi�m, a highly to�ic envi-Cadmi�m, a highly to�ic environmental poll�tant with high to�icity to animals and plants, has no known f�nction in any live cells.It is released into the environment by traffic, metalworking ind�stries, mining, as a by-prod�ct of mineral fertilizers, and from other so�rces (N r i a g � and P a c y n a , 1988).One of the conse��ences of the One of the conse��ences of the presence of to�ic metals in plants is the formation of free radical species, which ca�se severe damage to different cell components (D e Vo s et al., 1992 , 1957, 1966).There are only a few st�dies on Serbian spr�ce (Š i l j a k -j a k o v l j e v et al., 2��2; T� c i ć� and S t o j k o v i ć� , 2��1) and its antio�idative systems (B o g d a n o v i ć� et al., 2��5, 2��6, 2��7).
The antio�idative system (AOS) in Serbian spr�ce is interesting beca�se it is more tolerant to air poll�tion and dro�ght compared to other conifers (� i l m a n and Wa t s o n , 1994; k r á l , 2��2) and beca�se trees of this species grow in a wide edaphic and altit�dinal range (3��-16�� m).Besides, there Besides, there is no evidence indicating that Cd ind�ces the same defense pathways in plants.We st�died some of the physiological defense reactions, e.g., changes in the concentrations of ascorbate and gl�tathione, composition of phenolics, and activity of o�alate o�idase, occ�rring in roots and needles of 75-day-of 75-dayold Serbian spr�ce plants after Cd e�pos�re for 6 after Cd e�pos�re for 6 for 6 -48 h.The plants were e�posed to 5 or 5� µM Cd The plants were e�posed to 5 or 5� µM Cd in hydroponics.The aim of this st�dy was to analyze The aim of this st�dy was to analyze the part of the AOS that is the main defense line in yo�ng Serbian spr�ce plants d�ring the first days of e�pos�re to the cadmi�m stress.More generally, this st�dy may deepen o�r knowledge abo�t the metal poll�tion resistance of yo�ng Serbian spr�ce trees.

Oxalate oxidase activity
�rozen roots (1�� mg) were powdered in li��id rozen roots (1�� mg) were powdered in li��id (1�� mg) were powdered in li��id were powdered in li��id nitrogen and mi�ed with 1.1 ml of �.25 M s�crose 1.1 ml of �.25 M s�crose in �.�5 M Tris-HCl, pH 7.2 (1:11 (w/v) ratio).The e�tract was s��eezed thro�gh �.5 mm nylon mesh and the filtrate centrif�ged at 4��� g for 1� min at 4°C.The pellet was washed twice with an e�traction medi�m containing 1% (w/v) Triton X-1�� and three times with the same medi�m witho�t Triton X-1��.After each wash, the pellet was collected by centrif�gation at 4���g for 1� min.The final pellet was considered to be the p�rified cell wall fraction and �sed for the e�periments.O�alate o�idase was detected spectrophotometrically as described by P � n d i r (1991).Containing �.9 ml of 4� mM Na s�ccinate (pH 3.2) and �.1 ml of the sample with cell wall, the assay mi�t�re (1 ml) was inc�bated for 1� min at 37ºC in the dark.The reaction was started by adding o�alic acid to a final concentration of 1� mM.After 5 min, �.5 ml of color reagent for H 2 O 2 meas�rement was added to stop the reaction, and color was allowed to develop for 3� min at room temperat�re.The color reagent consisted of 1�� mg of solid phenol, 5� mg of 4-aminophenazine, and 5 U • ml -1 of horseradish pero�idase in �.4 M Naphosphate b�ffer (pH 7).Absorbance of the sol�tion was meas�red at 52� nm and corrected for absorbance obtained when o�alic acid was omitted from the assay mi�t�re.Hydrogen pero�ide generated d�ring the reaction was determined by interpolation from a standard c�rve in the range of from �.�1 to �.25 µmol H 2 O 2 in 5� mM s�ccinate b�ffer.Enzyme activity e�pressed as the amo�nt of H 2 O 2 prod�ced per min and per mg fresh weight.

Extraction and Analysis of Antioxidants
�or the e�traction of water-sol�ble antio�idants, 1�� mg of frozen plant tiss�e (roots and needles) were powdered in li��id nitrogen, mi�ed with 1 ml of 2% (w/v) meta-phosphoric acid containing 1 mM EDTA and 1 mg of polyvinylpolypyrrolidone per mg of sample, and centrif�ged (2� min, 4°C, 3�,���g).The s�pernatant was �sed for analyses of antio�idants.Ascorbate was determined at 268 nm after separation by capillary electrophoresis as described by D a v e y et al. (1996).The system consisted of a high-performance capillary zone electrophoresis (model P/ACE 55�� HPCE) system (Beckman Instr�ments, ��llerton, CA) fitted with a UV-VIS diode array detector (P/ACE 55�� DAD, Beckman) and e��ipped with �OlD software (Beckman) for peak analysis.Injections were made for 5 s �nder hydrostatic (N 2 ) press�re (�.5 po�nds per s��are inch) into a 57-cm f�sed silica capillary at a constant temperat�re of 25°C and constant voltage of 25 kV.Separations were r�n for 1� min with 1�� mM borate b�ffer, pH 9, as a carrier electrolyte.The capillary was s�bse��ently conditioned for the ne�t r�n with �.1 M NaOH, water, and the carrier electrolyte.�or determination of total ascorbate, DHA was red�ced by dithiothreitol at pH 8.3 to 8.5 for 6� min at room temperat�re (A n d e r s o n et al., 1992).This was achieved by mi�ing 1�� µl of the sample with 15� µl of 6� mM dithiothreitol in 1 M 2-[N-cyclohe�ylamino] ethans�lfonic acid.The mi�t�re was analyzed as above and DHA was calc�lated by s�btracting ascorbate from total ascorbate.�l�tathione was determined after red�ction, derivatization with monobromobimanes, HPlC separation (Beckman System �old, M�nich, �ermany) on a C-18 col�mn, and detection with a fl�orescence detector (R�-55�, Shimadz�, D�isb�rg, �ermany) according to the method of S c h � p p and R e n n e n b e r g (1988).�SS� was determined in the same manner after removal of gl�tathione by alkylation with N-ethylmaleimide (� o r i n et al., 1966).�SH was calc�lated as the difference of gl�tathione and �SS�.The redo� state was defined as the �SS� content in percent of gl�tathione content.
Determination of sol�ble phenolics was performed by HPlC after isolation from 2�� mg of frozen needles gro�nded in li��id nitrogen and transfer to 5 ml of 5�% (v/v) methanol and water.After shaking and inc�bation for 6� min at 4�°C on an �ltrashaker, samples were centrif�ged 1� min at 25��g at 4°C.The s�pernatant was taken, while the pellet was washed with 2 ml of 5�% methanol.After inc�bation of 1� min at room temperat�re and centrif�gation, this s�pernatant was then combined with the first one.Reverse phase HPlC analysis Reverse phase HPlC analysis was carried o�t on the 11�� Hewlett Packard (Palo (Palo Alto, CA, USA) system with diode-array detector system with diode-array detector adj�sted to 21�, 28�, and 33� nm and reference signal at 6�� nm.Separations were performed Separations were performed were performed on a Waters Symmetry C-18 RP col�mn (125�4 mm) with 5-µm particle size and a corresponding precol�mn (Waters, Milford, MA, USA).Mobile (Waters, Milford, MA, USA).Mobile .Mobile phases were �.1% phosphoric acid (mobile phase A) and acetonitrile (mobile phase B).Acetonitrile (j.T. Baker, USA), methanol (Carbo Reagenti, Milan), and p.a. grade phosphoric acid were �sed.

RESUlTS AND DISCUSSION
Metals can ind�ce o�idative stress directly or indirectly, by interaction with biochemical redo� processes (Va n A s s c h e and C l i j s t e r s , 199�).One of the conse��ences of the presence of to�ic metals in cells is the formation of free radicals and activated o�ygen species (AOS), which ca�se severe damage to different cell components.
O�alate o�idase (O�O�) activity has been detected in a few plant species, for e�ample wheat (l a n e et al., 1986; H a m e l et al., 1998), sorgh�m (P�ndir, 1991), barley (Z h a n g et al., 1995; Z h o � et al., 1998;), and maize (V� l e t i ć� and H a d ž i -Ta š k o v i ć� , 2���).The presence of O�O� activity in a conifer species is here demonstrated for the first time.After e�pos�re of plants to both 5 µM and 5� µM cadmi�m, a decrease in O�O� activity was observed in comparison with the control at all sampling dates (�ig.2).There was a slight increase of O�O� acivity in treated plants with treatment d�ration.It seems that the decrease of enzyme activity was not Cd concentration-dependent, especially in short-time treatment.O�O� has been considered to be a candidate for prod�ction of e�tracell�lar H 2 O 2 (Z h a n g et al., 1995; l a n e et al., 1993; D � m a s et al., 1993), which is involved in the signal transd�ction events associated with defense responses to vario�s kinds of stress (l e v i n e et al., 1994).In this way, o�alate o�idase may play a role in reg�lation of the hypersensitive response (Z h o � et al., 1998).Additionally, increased transcription of a gene for germin-like o�alate o�idase in wheat and barley leaves following pathogen attack (Z h o � et al., 1998; H � r k m a n and Ta n a k a , 1996) and mod�lation of germin gene e�pression in wheat seedlings by heavy metal ions (B e r n a and B e r n i e r , 1999) s�ggested the involvement of this enzyme in the plant response to stress.On the other hand, it is known that metal to�icity depends on soil pH, organic matter, and the phosphate stat�s of the soil (D a s et al., 1997).Under conditions of low pH, cadmi�m appears to be absorbed passively (C � t l e r and R a i n s , 1974) and translocated freely (H e m p h i l l , 1972).In the cell, it can interfere with metabolism thro�gh competition for �ptake, inac-tivation of enzymes, and displacement of elements from f�nctional sites (D a s et al., 1997).O�r res�lts show a to�ic effect of Cd on O�O�, which implies that this enzyme prod�ces a lower amo�nt of H 2 O 2 in comparison with the control.This may affect f�rther reactions involved in the plant response to Cd stress.An increase of O�O� content and activity was fo�nd in plants e�posed to different treatments (Z h a n g et al., 1995; D e l i s l e et al., 2��1).O�r res�lts show that Cd treatment ca�sed a decrease of O�O� activity in yo�ng Serbian spr�ce plants.The slight temporary increase of O�O� acitivity after initial decline in treated plants may be d�e to partial recovery of the enzyme.
Decrease in concentration of both red�ced and o�idized gl�tathione was observed in plants treated with cadmi�m (�ig.3), the highest difference in relation to �ntreated plants being observed after 24-and 48-h treatment.There was no significant considerable difference of gl�tathione concentration between plants treated with 5 µM and 5� µM Cd.
Decrease of total ascorbate and increase of ascorbic acid (AA) concentration with the passage of in the control plants (�ig.4) are d�e to changes of this compo�nd d�ring growth and development of yo�ng plants.In plants treated for 6 h, there was no difference of ascorbic acid concentration in comparison with the control plants, while total ascorbate was increased, which indicates that the dehydroascorbate (DHA) concentration increased.In f�rther treatments, there was a considerable decrease of red�ced ascorbate in treated plants in comparison with �ntreated ones, especially after 12 and 48 ho�rs.However, a decrease of AA concentration was observed in relation to the control, being independent of cadmi�m concentration in the case of plants treated for 12 and 24 h.A decrease of AA concentration in the case of higher Cd concentration was observed in plants treated for 48 h.These res�lts show that both the gl�tathione and the ascorbate defense systems were damaged after short-term e�pos�re to cadmi�m, and the decrease was proportional to the d�ration of treatment.
Table 1 shows phenolic acids detected in yo�ng Serbian spr�ce plants.It is evident that the concen- The fact that cadmi�m treatment of yo�ng P. omorika plants for 48 ho�rs ind�ced a decrease in concentration of the antio�idant compo�nds ascorbate and gl�tathione and a parallel decline of O�O� activity demonstrates a general to�ic effect of this metal on the antio�idative capacity of these yo�ng plants.Moreover, since Cd red�ced the concentration of both ascorbate and gl�tathione, a dist�rbance  of the cell's redo� e��ilibri�m will probably occ�r as a conse��ence.An increase in phenol concentration was commonly observed after e�pos�re of plants to poll�ted environments (k a r o l e w s k i and � i e r t y c h 1995; � i e r t y c h and k a r o l e w s k i , 1993).This increase �nder poll�tion stress is probably connected with the higher energy re��irements of plants in s�ch conditions and conse��ent increase of respiration (To m a s z e w s k i , 1961).The fact that among sol�ble phenolics fo�nd in yo�ng Serbian spr�ce plants only catechin reacted significantly to cadmi�m stress with an increase in content co�ld mean that the protective mechanism of sol�ble phenolics is not yet activated in yo�ng plants after short-term stress.On the other hand, catechin has been considered to be a possible bioindicator of early stress in Scotch pine, since among many phenolics, it was only the content of catechin in needles that followed the dynamics of e�ternal poll�tion (H ä r t l i g and S c h � l z , 1998).
; H e n d r y and C r a w f o r d , 1994).Cd treatment Cd treatment affects the activities of antio�idative systems (AOS) (R a d o t i ć� et al., 2���) and especially �SH in dif-et al., 2���) and especially �SH in dif-and especially �SH in different organisms (S c h � t z e n d � b e l et al., 2��1; (S c h � t z e n d � b e l et al., 2��1; S c h � t z e n d � b e l et al., 2��1; S c h � t z e n d � b e l and P o l l e , 2��2).Antio�idative systems, consisting of several nonenzymatic and enzymatic mechanisms, are activated in the cell as a response to different types of damaging conditions (R a b e and C r e e b , 1979; H e n d r y and C r a w f o r d , 1994; H i p p e l i and E l s t n e r , 1996), partic�larly the presence of heavy metals (D e Vo s et al., 1992; � a l l e g o et al., 1996; S a n i t a d i To p p i and � a b r i e l l i , 1999).�l�tathione and ascorbate, the most important non-enzymatic components of AOS, in plants play a major part in their resistance mechanisms to e�ternal stress (N o c t o r and � o y e r , 1998) by reg�lating N o c t o r and � o y e r , 1998) by reg�lating ) by reg�lating redo� e��ilibri�m in the cell.�l�tathione is involved �l�tathione is involved to a considerable e�tent in deto�ification of heavy metals and �enobiotics and also has a role in gene activation and protection against o�idative stress (N o c t o r and � o y e r , 1998; S c h � t z e n d � b e l et al., 2��1).As an antio�idant, gl�tathione -together with ascorbate and antio�idative enzymes -controls the cell�lar concentrations of hydrogen pero�ide (H 2 O 2 ) and s�pero�ide radical (O 2 .-)(N o c t o r and � o y e r , 1998).Phenolic compo�nds also take part in defense mechanisms in plants (B r i g n o l a s et al., 1995; Ye e -M e i l e r , 1974) and are considered to be a bioindicator of environmental poll�tion.Breakdown of the antio�idative systems in plants s�bjected to metal stress has been taken as an indicator of seri-o�s plant damage ca�sed by a given concentration of the metal (Va n � r o s v e l d and C l i j s t e r s , 1994).There was direct infl�ence of Cd on the phenolic composition of Scotch pine meas�red in situ (S c h � t z e n d � b e l et al., 2��1).S c h � t z e n d � b e l et al., 2��1

Fig. 1 .
Fig. 1.Seventy-five-day-old Serbian spruce plants grown on a hydroponic solution.From left to right: untreated plants and plants treated with 5 μM and 50 μM CdSO4.tration of catechin is considerably higher than that of other phenolic species.Catechin was also fo�nd in significantly higher concentration in relation to other sol�ble phenolic compo�nds in the needles of older Serbian spr�ce trees (B o g d a n o v i ć� et al., 2��6).In yo�ng plants, only catechin content increased considerably d�ring Cd treatment (Table1).There was a slight increase in coniferyl alcohol and co�maric acid content.S c h � t z e n d � b e l and S c h � t z e n d � b e l and co-workers (2��1) fo�nd that Cd ca�sed acc�-m�lation of sol�ble phenolics in the cytosol, and this reaction was m�ch faster than lignification.Together with ascorbate, phenolics may contrib�te to H 2 O 2 destr�ction in the so-called phenol-co�pled
(S c h � t z e n d � b e l et al., 2��2).O�r st�dy shows that the meas�red O�r st�dy shows that the meas�red biochemical parameters indicate inj�ry to yo�ng plants at a time when symptoms of damage are not yet visible.Increase in the content of catechin in treated plants shows that this phenol is one of the first indicators of early stress in yo�ng Serbian spr�ce plants. ).

Table 1 .
Table 1.Individual soluble phenols in the extract of 75-day-old Serbian spruce plants: untreated plants and plants treated with 5 μM and 50 μM Cd for 6 and 48 h as measured by HPLC [μmol /g FW].