Composition and Properties of Hydrogen Peroxide Decomposing Systems in Extracellular and Total Extracts from Needles of Norway Spruce ( Picea abies L . , Karst . 4 "

Hydrogen peroxide (H202) scavenging systems of spruce (Picea abies) needles were investigated in both extracts obtained from the extracellular space and extracts of total needles. As assessed by the lack of activity of symplastic marker enzymes, the extracellular washing fluid was free from intracellular contaminations. In the extracellular washing fluid ascorbate, glutathione, cysteine, and high specific activities of guaiacol peroxidases were observed. Guaiacol peroxidases in the extracellular washing fluid and needle homogenates had the same catalytic properties, i.e. temperature optimum at 500C, pH optimum in the range of pH 5 to 6 and low affinity for guaiacol (apparent Km = 40 millimolar) and H202 (apparent Km = 1-3 millimolar). Needle homogenates contained ascorbate peroxidase, dehydroascorbate reductase, monodehydroascorbate reductase, glutathione reductase, and catalase, but not glutathione peroxidase activity. None of these activities was detected in the extracellular washing fluid. Ascorbate and glutathione related enzymes were freeze sensitive; ascorbate peroxidase was labile in the absence of ascorbate. The significance of extracellular antioxidants for the detoxification of injurious oxygen species is discussed. Plants are exposed to hydrogen peroxide (H202) from both external and internal sources. In the environment, H202 is photochemically produced in the atmosphere and is found in concentrations up to 100 AM in rain, clouds, and fog (15). On the cellular level, H202 is generated as a metabolite ofenzymic reactions and during photosynthesis (24). Because H202 is toxic at low concentrations (10 AsM; 24), its accumulation in living tissues should be prevented. For this purpose cells have a variety of defense strategies including antioxidants and detoxifying enzymes. The antioxidants ascorbate and glutathione participate in both enzymic and nonenzymic H202 degradation (28). Enzymes dealing directly with H202 are catalases and peroxidases. Peroxidases decompose H202 by oxidation of cosubThis study was financially supported by a grant of the Bayerisches Staatsministerium fur Landesentwicklung und Umweltfragen. 2 Present address: Department of Biochemistry, University College of Science, 35 Ballygunge Circular Rd., Calcutta 700109, India. 3Present address: TU Munchen, Institut fur Botanik und Mikrobiologie, Arcisstr. 21, D-8000 Munchen 2, Federal Republic of Germany. strates such as phenolic compounds or antioxidants (1 1). The 'unspecific' peroxidases in plant extracts are most commonly investigated with the artificial cosubstrate guaiacol and were found to be ubiquitous (1 1). Peroxidases using glutathione as a cosubstrate have only rarely been identified in plants (7), but peroxidases specific for ascorbate were frequently observed (12, 16). Ascorbate peroxidases are thought to be of particular importance in chloroplasts which are lacking catalase. They catalyze the first step of the ascorbate dependent H202 scavenging pathway: ascorbate + H202 ascorbate peroxidase dehydroascorbate (and/or monodehydroascorbate) + 2 H20 monodehydroascorbate + 2 NADH monodehydroascorbate reductase ascorbate (1)

Plants are exposed to hydrogen peroxide (H202) from both external and internal sources.In the environment, H202 is photochemically produced in the atmosphere and is found in concentrations up to 100 AM in rain, clouds, and fog (15).On the cellular level, H202 is generated as a metabolite ofenzymic reactions and during photosynthesis (24).Because H202 is toxic at low concentrations (10 AsM; 24), its accumulation in living tissues should be prevented.For this purpose cells have a variety of defense strategies including antioxidants and detoxifying enzymes.
The antioxidants ascorbate and glutathione participate in both enzymic and nonenzymic H202 degradation (28).En- zymes dealing directly with H202 are catalases and peroxidases.Peroxidases decompose H202 by oxidation of cosub- This study was financially supported by a grant of the Bayerisches Staatsministerium fur Landesentwicklung und Umweltfragen. 2Present address: Department of Biochemistry, University College of Science, 35 Ballygunge Circular Rd., Calcutta 700109, India.
strates such as phenolic compounds or antioxidants (1 1).The 'unspecific' peroxidases in plant extracts are most commonly investigated with the artificial cosubstrate guaiacol and were found to be ubiquitous (1 1).Peroxidases using glutathione as a cosubstrate have only rarely been identified in plants (7), but peroxidases specific for ascorbate were frequently ob- served (12,16).Ascorbate peroxidases are thought to be of particular importance in chloroplasts which are lacking cata- lase.They catalyze the first step of the ascorbate dependent H202 scavenging pathway: ascorbate + H202 ascorbate peroxidase dehydroascorbate (and/or monodehydroascorbate) + 2 H20 monodehydroascorbate + 2 NADH monodehydroascorbate reductase ascorbate + 2 NAD+ dehydroascorbate + 2 GSH dehydroascorbate reductasS )1.ascorbate + GSSG (2b) GSSG + 2 NADPH glutathione reductase 2 GSH + 2 NADP+ (3) The complete set of enzymes of this pathway has been loca- lized in the cytoplasm (14) and in chloroplasts (10).In the apoplastic space peroxidases have been found that are capable of both production and use of H202 (11).The apoplastic peroxide concentration is not only affected by the activities of these enzymes, but also by air pollutants giving rise to H202 in aqueous phases such as 03 (13) or by the deposition of atmospheric H202.Since high concentrations of H202 can induce lipid peroxidation and cell injury, it has been suggested that H202 consuming processes in the apoplastic space pro- vide an important defense against oxidative damage and, thus, might be essential for determining the H202 resistance of plants (3,23).To date, this hypothesis is supported by only few experimental results: Washing fluids from the extracellu- lar space (EWF4) of Sedum and pinto bean leaves showed ascorbate peroxidase activity (3, 18).EWFs from spruce and Sedum contained ascorbate (4,5).The levels of ascorbate and ascorbate peroxidase increased in response to ozone (3)(4)(5).Levels of antioxidants and enzymes related to H202 scavenging are particularly important in needles ofconifers, which persist for many vegetation periods.In spruce levels of an- tioxidants and unspecific peroxidases, dehydroascorbate re- ductase, and glutathione reductase have been determined under various experimental, developmental, and environ- mental conditions (4,8,9,28).However, neither the complete set of H202 detoxification enzymes, nor the occurrence of these systems in the apoplastic and symplastic space has been determined in spruce.The objective of this study was to characterize the composition and properties of H202 scaveng- ing systems in both fluids obtained from the apoplastic space and total extracts of spruce needles.
The media for the extraction of ascorbate peroxidase, de- hydroascorbate reductase, and monodehydroascorbate reduc- tase contained additionally 5 mM ascorbate.When ascorbate peroxidase activity was investigated in the EWF, the infiltra- tion media contained 5 mM ascorbate and the EWF was collected in a tube containing 50 ,L of a buffered ascorbic acid solution (20 mM KH2PO4/K2HPO4 [pH 7.0], 5 mM ascorbate).

Determination of Enzymic Activities
All assays were performed at 25°C and had a final volume of 1 mL.All measurements were performed in triplicates.
Catalase (EC 1.11.1.6)activity was determined as H202 consumption measured as the decrease in absorbance at 240 nm according to the method of Aebi (1).The assay contained

Plants
The experiments were performed with needles collected in the morning from approximately 100 to 140-year-old, healthy spruce trees (Picea abies L., Karst.).The trees are growing in a mountain region of the Northern Alps (750-850 m above sea level) under clean air conditions with mean concentrations of about 2 nL/L So2, 4 nL/L NO,, and 30 nL/L 03 during the vegetation period (R Sladkovic, K Munzert, personal communication).

Extraction of Enzymes
Extracts of spruce needles for the determination of enzyme activities were prepared as described previously (20).To de- termine the activities of dehydroascorbate reductase and monodehydroascorbate reductase, interfering compounds had to be removed.For this purpose, solid (NH4)2SO4 was slowly added to crude extracts up to 35% saturation.After centrifugation (10 min, 3200g), the pigments and Triton X- 100, which formed an upper layer, were removed.The super- natant was brought up to 80% saturation with (NH4)2SO4 and the proteins were pelleted by centrifugation (20 min, 20000g).The pellet was taken up in 2.5 mL of a buffer containing 50 mM Mes/KOH (pH 6.5).Washing fluids from the apoplastic space were obtained by vacuum infiltration using a modifi- cation of the method of Rathmell and Sequeira (22) as elab- orated by Pfanz (personal communication).Five grams of freshly cut needles were washed with distilled water and infiltrated for periods of 30 s at -70 kPa in 30 mL of an infiltration solution (50 mm Mes/KOH [pH 6.0]; 40 mM KCI, 2 mM CaCl2, 0.1% PVP-10).The needles were thoroughly blotted, filled into a centrifuge tube (90 x 25 mm) with a perforated bottom and placed over an Eppendorf tube.The extracellular washing fluid was collected by centrifugation (10 min, 3200g).
Before the determination of the enzymic activities, the extracts were desalted over Sephadex G-25 columns (PD-10 or NAP-5, Pharmacia) which had been equilibrated with Mes/ KOH (pH 6.5) ([NH4]2SO4-fractions and EWFs) or with 100 without and (b) with a pretreatment with boric acid.The retention time of the fluorescent derivative of dehydroascorbate was 4.0 ± 0.1 min and the peak area corresponded to 56 pmol ascorbate.
Table I.Low Mol Wt Components in Extracellular Washing Fluids and Total Extracts from Spruce Needles Each experiment was performed with current year's needles collected at the end of September from six trees.Samples were individually analyzed in three parallels as described under "Materials and Methods."The standard deviation of the individual experiments was less than 5%.Values in the table are average values of six experiments (± SD).Under the assumption of homogenous distributions, the antioxidant concentrations were calculated using the liquid volumes of needles (541 jiL/g fresh weight i 17 ,uL/g fresh weight) and EWFs (24 ML/g fresh weight ± 13 AL/g fresh weight).

Extraction of Antioxidants
Extracts for the determination of the content ofglutathione and ascorbate in the needles were prepared as described previously (25).EWF was obtained from 5 g of needles by vacuum infiltration as described by Castillo et al. (4).During the centrifugation (10 min, 3200g) the extracellular washing fluid was collected in tubes containing 100 ,L 0.1 N HCl.

Quantification of Thiolcompounds
After reduction of the samples and derivatization of the thiol groups with monobromobimanes, the contents of total glutathione, cysteine and y-glutamylcysteine were determined in needle extracts and EWFs.The fluorescent derivates were separated by HPLC (17) as adapted for spruce extracts (25).

Quantification of Ascorbate
The total ascorbate contents (oxidized + reduced) in needle extracts and EWFs were determined by a modification of the HPLC method of Speek et al. (29).This method is based on the complete oxidation of ascorbate to dehydroascorbate with ascorbate oxidase.Dehydroascorbate is derivatized with o- phenyldiamine and. the reaction product is detected as a fluorescent compound.If dehydroascorbate is blocked by boric acid before the derivatization, unspecific background fluorescence can be assessed.
Extracts were diluted 10-fold with 0.2 M Na acetate buffer (pH 6.2).Aliquots of 100 ML of this dilution were incubated for 15 min at 37°C with 10 ,L ascorbate oxidase (1 mg/mL, Boehringer) and vigorously mixed twice during the incubation period to achieve complete oxidation of the ascorbate.Then 100 ,uL of a solution containing 3.7 M Na acetate was added to the samples.In control samples the solution contained additionally 3% (w/v) boric acid).The mixture was incubated for 30 min in the dark at room temperature.Then 740 ML of H20 and 50 MuL of a freshly prepared o-phenyldiamine solu- tion (1 mg/mL ethanol, Sigma) was added and the mixture was incubated for 30 min at room temperature.The samples were stable in the dark for at least 24 h.Samples of 50 ML were loaded (Waters, 712 WISP autosampler) on a reversedphase column (ODS-Hypersil, 125 x 4.6 mm, 5 Mm, refill, Bischoff) and separated by HPLC under isocratic conditions with a flow rate of 1 mL/min (Waters pump 51 0) of an eluent containing 20% (v/v) methanol, 80 mM K2HPO4 (pH 7.8).The fluorescence was detected at an emission wavelength of 450 nm with an excitation wavelength of 350 nm (Fluores-cence HPLC monitor Shimadzu, RF535).Figure 1 shows a chromatogram of a spruce extract.The recovery of ascorbate standards added to spruce extracts was 76 ± 15%.External ascorbate standards which were treated as the samples accom- panied every experiment.All determinations were performed in triplicates.

Basic Parameters
The protein content was determined in the desalted samples with the BCA reagent kit from Pierce.BSA was used as standard.To obtain the liquid volume of the needles, the dry weight to fresh weight ratio was determined after incubation of fresh needles (0.5 g) for 72 h at 80°C in a drying oven.

Properties of H202-Scavening Enzymes in Spruce Needles
Some general properties with respect to extraction, assay, and storage conditions of the ascorbate and glutathione re- lated enzymes and guaiacol peroxidases were explored.The activities of ascorbate peroxidase, dehydroascorbate reduc- tase, monodehydroascorbate reductase, and glutathione re- ductase were freeze-sensitive.Storage of crude needle extracts at -20°C as well as storage of the needles in liquid nitrogen or at -80°C led to a complete loss of the ascorbate peroxidase activity and to partial losses in the range of 50 to 80% of the other enzymic activities.By contrast, the activities of the guaiacol peroxidases obtained from needles and the extracel- lular space were only slightly freeze sensitive.The losses of specific activity were in the range of 10 to 20%, regardless of whether extracts or needles were kept frozen (-80°C).
The losses of enzyme activities during the extraction pro- cedure were assessed for commercially available enzymes.The recovery of guaiacol peroxidase (horseradish peroxidase, Sigma) in spruce extracts was 80%.The recovery of glutathione reductase (yeast glutathione reductase, Boehringer) was 55 to 65%.Inclusion of thiol protecting agents (glutathione, mercaptoethanol) did not improve the recovery ofglutathione reductase.Since ascorbate related enzymes were not commer- cially available, it was not possible to determine recoveries for ascorbate peroxidase, dehydroascorbate reductase, and mon- odehydroascorbate reductase.
We observed that the activity of ascorbate peroxidase was labile in the absence of ascorbate.Therefore, ascorbate at a concentration of 5 mm was included throughout the preparation and processing ofthe extracts.Crude extracts contained approximately 30% higher ascorbate peroxidase activities than extracts after gel fitration in the presence of ascorbate.This might indicate either an inactivation despite the presence of ascorbate or the loss of a low mol wt component with ascorbate peroxidase activity similar to that detected in pea extracts (12).The interference of catalase with the ascorbate peroxidase assay was in the range of 10% and was avoided by addition of aminotriazole (500 gM) to the assay mixture.
EDTA which was originally included in ascorbate peroxidase assays, was found to increase the nonenzymic blanks, but did not affect the enzyme catalyzed rates and, therefore, was omitted.

Antioxidants in Extracellular Washing Fluids and Total Extracts of Spruce Needles
The amounts of washing fluids (EWF) obtained after vac- uum infiltration from the extracellular spaces of spruce needles were generally small.In the present set ofexperiments 24 IuL (+ 13 AL) were obtained per g fresh weight.To deter- mine in these small volumes both the contents of ascorbate and glutathione, highly sensitive detection methods were needed.
For the determination of thiol compounds an HPLC method sensitive up to 1 pmol was available (17, 25).An HPLC method of similar sensitivity relying on the detection of a fluorescent derivative of dehydroascorbate ( 29) was adapted to determine total ascorbate contents in spruce ex- tracts.Under our experimental conditions the detection limit was 3 pmol.Figure 2. Dependency of the guaiacol peroxidase activities on the pH of the assay.The activities of the guaiacol peroxidases from needle extracts (0), from EWF (A), and from needle extracts obtained after extraction of the EWF (*) were determined between pH 4 and pH 8.Under standard conditions, 17, 400, and 17 nkat, respectively, were determined for the three sets of extracts.In the pH range 4 to 6, the pH-values were adjusted using a buffer system of citrate/ NaH2PO4 (0.032-0.052M).In the pH range 5 to 8, the pH-value was adjusted using a buffer system of KH2PO4/K2HPO4 (0.05 M).The experiments were repeated three times.The bars indicate standard deviation.
compound was prevented by blocking dehydroascorbate with boric acid, background fluorescence was detected (Fig. lb).Unspecific interferences with other compounds did not occur.
Table I shows that ascorbate, glutathione, and cysteine were identified in EWFs.'y-Glutamylcysteine which was present in spruce needles only in small amounts, was not detected in EWFs.Although the concentrations ofascorbate, glutathione, and cysteine in the EWFs were considerable (1.1 mM, 6.5 AM, and 0.5 Mm, respectively), the relative portions of these com- pounds as compared to the amounts present in total needles were fairly low (0.28, 0.07, and 0.07%; cf.Table I).This raised the question as to whether the occurrence of ascorbate, glutathione, or cysteine was caused by unspecific leakage.Leakage of cytoplasmatic and chloroplastic marker proteins was not observed (see below), but leakage of low mol wt compo- nents cannot unequivocally be ruled out.However, it appears unlikely that membrane injury and unspecific release of sym- plastic components will lead to a relative enrichment of ascorbate in the EWF (ascorbate:glutathione:cysteine = 2000:12:1) as compared to the relative occurrence of ascor- bate, glutathione, and cysteine in total needle extracts (540:14:1).Whether glutathione and cysteine are natural con- stituents of the apoplastic space or indicate contamination remains unknown.
Enzymes Related to the H202 Metabolism in Extracellular Washing Fluids and in Total Extracts of Spruce Needles The activities of H202 scavenging enzymes were investigated in EWFs and total extracts of spruce needles.The contamination of the EWF with cellular components was assessed (Table II).
Catalase activities as determined by H202 consumption (1) were observed in needle extracts and at a relative portion of 0.02% of the total activity also in the EWF (Table II).Since the H202 consumption in the EWF was not blocked in the presence of the specific inhibitor of catalase, aminotriazole, it is likely that the observed H202 degradation was due to the catalatic properties of peroxidases rather than to catalase itself (1 1).This observation indicates that contaminations of the EWF with cellular enzymes might even be lower than 0.02% of the activity found in total needle extracts.This conclusion was supported by the observation that the activities ofglucose-6-phosphate dehydrogenase and glyceraldehyde-3-phosphate dehydrogenase which are considered as marker enzymes for the cytoplasmic and chloroplastic space, respectively, were not detected in the EWFs (Table II).For instance, a relative contamination of the EWF with 0.02% of the total glucose-6-phosphate dehydrogenase activity would account for 0.22 pkat/g fresh weight and, thus, be 40-fold higher than the detection limit for glucose-6-phosphate dehydrogenase (Table II).
Peroxidase activities with guaiacol as cosubstrate were observed in both extracts of total needles and EWFs (Table II).In the present set of experiments 2.1% of the total activity was extracted with the EWF.The specific peroxidase activity of the EWF was 8.85 kat/kg protein and, thus, by factor of 66 higher than the specific peroxidase activity in extracts from total needles (0.13 kat/kg protein).However, in experiments conducted with needles from different stands, these figures were found to be extremely variable: The relative portions of the peroxidase activities in the EWFs were ranging from 0.4 to 7% of total peroxidase activities extracted from needles.The specific peroxidase activities in the EWFs were higher by factors in the range between 10 and 400 than in needle extracts.
Glutathione peroxidase activity was not detected in extracts of spruce needles prepared after three different protocols: standard procedure as under "Materials and Methods," stand- ard procedure, but all media contained 5 mm GSH, extraction in phosphate buffer (100 mm, pH 7.0) containing 5 mM EDTA, 10 mm DTE, 1% soluble PVP, 1% Tween 80, 2% casein.Neither crude nor gel filtrated extracts showed gluta- thione peroxidase activity.
Ascorbate peroxidase activity and the subsequent series of enzymic activities regenerating reduced ascorbate, namely dehydroascorbate reductase, monodehydroascorbate reduc- tase, and glutathione reductase were found in needle extracts (Table II).However, none of these activities was detected in EWFs (Table II).Under the present assay conditions the relative enzymic activities for ascorbate peroxidase: dehy- droascorbate reductase:monodehydroascorbate reduc- tase:glutathione reductase were 23:6:2:1.
Characteristics of Gualacol Peroxidase Activities in Extracellular Washing Fluids and Totals Extracts of Spruce Needles Among the enzymic activities investigated in this paper, only guaiacol peroxidases were present in both total extracts of spruce and EWFs.To determine whether extra-and intra- cellular peroxidases had similar features, the catalytic prop- erties ofguaiacol peroxidases in EWFs, in total needle extracts, and in extracts obtained from needles after the extraction of EWF were characterized.

pH-Dependence
The peroxidase activities in EWFs, total, and residual needle extracts had the same, relatively broad pH-optimum between pH 5 and pH 6 (Fig. 2).At pH 7 the peroxidase activity was approximately 50% and at pH 8 only 10% of its maximum value.The enzyme activity was not affected by different buffers (citrate, phosphate) or by variation of the ionic strength of the assay in the range between 20 and 200 mm buffer concentration.

Temperature Dependence
The optimum assay temperature for guaiacol peroxidases in the three sets of extracts, EWF, total, and residual needle extract was 50°C (Fig. 3).At 80°C the peroxidase activities were completely lost.In the physiologically important temperature range between 5°C and 35°C, 25 to 70% of the maximum activity were observed and an activation energy of 27 kJ/M was calculated from the Arrhenius plot (inset, Fig. 3) (26).

Kinetic Constants
To investigate the effect of the substrate concentration on the guaiacol peroxidase activity for the three different sets of extracts, i.e.EWF, total, and residual needle extract, the concentration of H202 was varied between 0.5 and 250 mm and guaiacol was kept at a constant concentration of 40 mM.Vice versa, the concentration of guaiacol was varied between 1 and 160 mM and H202 was kept at a constant concentration is Plot Figure 3. Dependency of the guaiacol peroxidase activities on the assay temperature.The activities of the guaiacol peroxidases from total (0) and residual needle extracts (*) and from EWF (A) were determined at assay temperatures  Under standard conditions the assays contained the following guaiacol peroxidases activities: 0.26, 0.24, and 5.4 nkat (total, resid- ual needle extract, and EWF, respectively).The experiments were repeated three times.The standard deviation was less than 5%.The figures in the table are average values (± SD) calculated from the results obtained with three independent sets of needles.Further explanations are given in the text.

DISCUSSION Defense Enzymes
In the present study we investigated the distribution and some properties of enzymes related to the detoxification of hydrogen peroxide in needles from field growing spruce trees.Glutathione peroxidase was not found in spruce needle ex- tracts.Guaiacol peroxidases were found in total needle ex- tracts and with 66-fold higher specific activity in apoplastic washing fluids.In both sets of extracts, the catalytic features of guaiacol peroxidases were similar with regard to substrate affinities, temperature, and pH-optima.Since conifer leaves have apoplastic pH values of about 5.5 (19), the results (Fig. 2) suggest that extracellular peroxidase are likely to operate at their pH optimum, while peroxidases present in compart- ments with neutral or slightly alkaline pH values might be inhibited up to 50 or 90%.
The major difference between peroxidases in the EWF and total peroxidases was the lack of ascorbate peroxidase activity in the extracellular space.This was surprising, because (a) ascorbate was present in the EWFs of spruce needles (4; and this paper, Table I) and (b) extracellular peroxidases ofSedum and pinto bean leaves could use ascorbate as a substrate (3,  18).Since the extracellular ascorbate peroxidases of Sedum and pinto bean were obtained by similar extraction procedures (3, 18), it appears unlikely that spruce ascorbate peroxidase remained associated with the cell walls during the extraction.
In spruce needle extracts the activities of the complete set of enzymes necessary to operate the ascorbate-glutathione cycle were found.All enzymes investigated in conifers so far showed high seasonal variations (8, 9).Therefore, the present results give only a 'snapshot' of the enzymic activities, typical for the period at the end of September.However, similar relative enzyme activities as observed in spruce extracts have also been observed in other plant species (6, 14, 27) and, therefore, might indicate some general feature.Whether or not glutathione reductase which has the lowest rates in the assay, does constitute the rate limiting step remains unknown, because the enzymic activities as determined under optimized substrate and pH-conditions, do not necessarily reflect their in vivo activities.

Antioxidants
The amounts of extracellular ascorbate found in needles of field-growing spruce trees (4.6 ,g/g fresh weight, Table II) were approximately twofold higher than the amounts found in a previous study with spruce saplings (1.2-2.8 ,ug/fresh weight [4]).To our knowledge, glutathione and also cysteine have not been identified in EWFs before.In EWFs of pinto beans, total sulfhydryl groups have been determined (18) and were approximately, by a factor of 6, higher than the gluta- thione and cysteine contents found in the EWFs of spruce in this study.Under the assumption that glutathione and cys- teine indicated leakage and that the subcellular distribution of glutathione and ascorbate was similar, unspecific leakage of ascorbate accounted for a maximum of 25% of the total amount of ascorbate present in EWFs.
Detoxifying enzymes dealing with ascorbate or glutathione such as ascorbate peroxidase, dehydroascorbate reductase, monodehydroascorbate reductase, and glutathione reductase have not been detected in the EWF of spruce needles.Therefore, it might be speculated that extracellular ascorbate is involved in nonenzymic defense reactions.Since regenerating systems for ascorbate were apparently lacking in the EWF, the suggested antioxidative function of extracellular ascorbate would require an efficient ascorbate/dehydroascorbate trans- locator in the plasmalemma membrane.

Figure 1 .
Figure1.Chromatographic detection of total ascorbate in extracts of spruce needles.The experimental conditions are described under "Materials and Methods."The chromatograms were recorded(a) Figure la shows a typical chromatogram of a spruce needle extract.The fluorescent derivative of dehy- droascorbate was peaking at 4.00 min.Ifthe formation of this

3.5 3. 7 from 5
to 80°C.Under standard conditions the 1/T 10' (KW') activities were 1, 4, and 147 nkat, respectively.The experiments were repeated three times with extracts obtained from different sets of needles.The bars indicate standard deviation.The inset is an Arrhenius plot of the mean values of the data presented in this figure in the temperature range 5 to 350C.temperature(CC)

Table II .
Activities of H202 Detoxification Enzymes and Subcellular Marker Enzymes in Extracellular Washing Fluids and Total Extracts of Spruce Needles Experimental conditions were as in TableI(± SD).
Not detected

Table Ill .
Kinetic Parameters of the Guaiacol Peroxidases in Assays Performed with EWF, Total, and Residual Needle Extracts