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First published online February 17, 2006; 10.1104/pp.105.070474 Plant Physiology 140:1451-1463 (2006) © 2006 American Society of Plant Biologists The Role of Hydrogen Peroxide-Producing and Hydrogen Peroxide-Consuming Peroxidases in the Leaf Apoplast of Cowpea in Manganese Tolerance1,[W]Institute of Plant Nutrition (M.M.F.-C., H.F., W.J.H.) and Department of Applied Genetics (H.-P.B.), University of Hannover, 30419 Hannover, Germany
The apoplast is considered the leaf compartment decisive for manganese (Mn) toxicity and tolerance in cowpea (Vigna unguiculata). Particularly apoplastic peroxidases (PODs) were proposed to be key enzymes in Mn toxicity-induced processes. The presented work focuses on the characterization of the role of hydrogen peroxide (H2O2)-producing (NADH peroxidase) and H2O2-consuming peroxidase (guaiacol POD) in the apoplastic washing fluid (AWF) of leaves for early stages of Mn toxicity and genotypic differences in Mn tolerance of cowpea. Leaf AWF of the Mn-sensitive cultivar (cv) TVu 91 but not of the Mn-tolerant cv 1987 showed an increase of guaiacol-POD and NADH-peroxidase activities at elevated AWF Mn concentrations. two-dimensional resolutions of AWF proteins revealed that cv TVu 91 expressed more and additional proteins at high Mn treatment, whereas Mn-tolerant cv TVu 1987 remained nearly unaffected. In both cultivars, NADH-peroxidase activity and accompanied H2O2 formation rate in vitro were significantly affected by Mn2+, p-coumaric acid, and metabolites occurring in the AWF. The total phenol concentration in the AWF was indicative of advanced stages of Mn toxicity but was rather unrelated to early stages of Mn toxicity and genotypic differences in Mn tolerance. The NADH oxidation by AWF PODs was significantly delayed or enhanced in the presence of the protein-free AWF from cv TVu 1987 or cv TVu 91, respectively. High-performance liquid chromatography analysis of AWF indicates the presence of phenols in cv TVu 1987 not observed in cv TVu 91. We conclude from our studies that the H2O2-producing NADH peroxidase and its modulation by stimulating or inhibiting phenolic compounds in the leaf apoplast play a major role for Mn toxicity and Mn tolerance in cowpea.
Manganese (Mn) excess represents an important factor limiting growth and crop yields particularly on acid and insufficiently drained soils with low redox potential (Foy, 1984  ; Schlichting and Sparrow, 1988). The oxidation of MnII and phenolic compounds catalyzed by peroxidase (apoplastic peroxidase [POD]) in the leaf apoplast is considered as a key reaction leading to Mn toxicity symptoms and finally leaf injury in cowpea (Vigna unguiculata L. Walp.; Horst, 1988). This assumption is based mainly on two observations: (1) Mn toxicity symptoms (brown spots on leaves) occur in the cell wall and consist of oxidized Mn and oxidized phenolic compounds (Wissemeier and Horst, 1992), (2) Kenten and Mann (1950, 1956) observed a close relationship between POD-catalyzed phenol and Mn oxidation. The oxidation of MnII and phenols is probably mediated by the formation of highly reactive MnIII and phenoxyradicals, which are considered as phytotoxic agents (Horst, 1988; Horst et al., 1999). Particularly the simultaneous enhancement of POD activity and expression of Mn toxicity symptoms (brown spots) suggest a close relationship between PODs and the oxidation of Mn and phenolic compounds (Fecht-Christoffers et al., 2003a, 2003b). However, the activation of PODs by Mn excess in cotton (Gossypium hirsutum) was considered a secondary effect (Sirkar and Amin, 1974), and also from our own work in cowpea the enhanced apoplastic POD activity could be the reason for or the consequence of Mn-induced leaf injury.
Peroxidases are widely distributed in plant tissues with different functions associated with an apparent lack in substrate specificity (Campa, 1991
Plant species and cultivars within species show considerable differences in Mn tolerance. Several mechanisms of Mn tolerance have been proposed: (1) sequestration of Mn in the apoplast thus preventing Mn oxidation (Maier, 1997 In one of our recent studies, the emphasis was on the characterization of the Mn toxicity-induced alterations in the proteome of the leaf AWF of a Mn-sensitive cultivar. This work focuses on the characterization of genotypic differences in Mn tolerance with emphasis on the role of H2O2-producing (NADH peroxidase) POD and the apoplastic metabolites affecting the resulting H2O2 formation at early stages of Mn toxicity.
Mn Uptake and Formation of Visible Mn Toxicity Symptoms Plants of the Mn-sensitive and Mn-tolerant cowpea cv TVu 91 and cv TVu 1987, respectively, readily took up Mn, thus causing 10-fold higher Mn contents in the leaf tissue than those of control plants. Characteristic Mn toxicity symptoms (brown spots) were expressed on leaves of cv TVu 91, whereas cv TVu 1987 was unaffected (Fig. 1A ). In spite of similar Mn tissue contents, cv TVu 1987 showed significantly higher Mn concentration in the AWF than cv TVu 91 (Fig. 1B).
Effect of Mn Treatment on the Protein Composition in the AWF
Two-dimensional (2D) resolutions of AWF proteins of control leaves did not show markedly different protein patterns between the two cultivars (Supplemental Fig. 1). However, they indicate an enhanced release of proteins from the symplast into the AWF particularly in the acid (pH 3–4) and neutral (pH 6) range of the polyacrylamide gel in leaves of cv TVu 91 treated with an elevated Mn supply compared to control plants. However, the protein pattern of cv TVu 1987 was hardly affected by the Mn treatment. The 2D resolution from cv TVu 91 showed a high conformity to previously shown results. Therefore, strongly expressed proteins on the gels can be considered identical with proteins identified previously by nanoliquid chromatography-mass spectrometry (MS)/MS (Fecht-Christoffers et al., 2003a
Total (Fig. 2, A1 and B1 ) and specific (Fig. 2, A2 and B2) activities of guaiacol POD and particularly of NADH peroxidase in the AWF were much higher in Mn-sensitive cv TVu 91 than in Mn-tolerant cv TVu 1987. Mn treatment induced a significant increase of apoplastic NADH peroxidase only in cv TVu 91 (highly significant Mn/cultivar interaction; Fig. 2, B1 and B2). In contrast to the total activity the specific guaiacol-POD activity did not respond to Mn treatment. This was due to enhanced (P = 0.0558, comparison of means for TVu 91; data not shown) protein concentrations of the AWF in cv TVu 91 (Fig. 2C).
The Formation of H2O2 by Leaf-AWF NADH Peroxidase in Vitro Because NADH peroxidase was proposed to generate H2O2, the H2O2 formation by apoplastic (AWF) NADH peroxidase was measured in vitro. The rate of H2O2 formation was significantly lower in the leaf AWF of cv TVu 1987 than in the AWF of cv TVu 91 (Fig. 3 ). The H2O2 formation was significantly enhanced by Mn treatment in cv TVu 91, but not in cv TVu 1987.
Effect of Mn and p-Coumaric Acid on the Leaf-AWF NADH Peroxidase and H2O2 Formation in Vitro To get a better quantitative understanding of the role of Mn and phenols for the activity of leaf-AWF NADH peroxidase, Mn and p-coumaric acid concentrations were varied in the enzyme test. The NADH-oxidation rate by NADH peroxidase was significantly affected by the concentrations of p-coumaric acid and Mn (Fig. 4 ). Enzyme activities increased with increasing concentration of p-coumaric acid and increasing Mn concentrations up to 1.6 mM. The application of 16 mM Mn did not further enhance NADH-peroxidase activity significantly, and the highest Mn concentration applied (160 mM) strongly suppressed the NADH-oxidation rate. The AWF from plants treated with 50 µM Mn during cultivation showed generally higher NADH-peroxidase activities than control plants. This was observed in both cultivars. However, NADH-peroxidase activity was significantly lower in the Mn-tolerant cv TVu 1987 than in the Mn-sensitive cv TVu 91, particularly at the high p-coumaric acid concentration (significant cultivar x p-coumaric acid interaction).
In agreement with the NADH-peroxidase activity, the formation rate of H2O2 in vitro was similarly dependent on the presence of p-coumaric acid and Mn (Fig. 5 ). The highest H2O2 formation rate was measured in the presence of 1.6 mM p-coumaric acid and 16 mM Mn in cv TVu 91. Although the overall cultivar difference was not significant, the H2O2 formation was significantly lower in the Mn-tolerant cv 1987 at the high p-coumaric acid concentration (significant cultivar x p-coumaric acid interaction) in agreement with the higher NADH-peroxidase activity shown in Figure 4.
Effect of Apoplastic Water-Soluble Metabolites on the NADH-Peroxidase Activity in the AWF The differences in NADH-peroxidase activity in the AWF between the cultivars could be due to stimulating or inhibiting metabolites in the AWF. To investigate the influence such AWF metabolites have on the NADH-peroxidase activity (measurements were made in the presence of optimal Mn and p-coumaric acid concentrations), the purified apoplastic proteins from controls and Mn treatments of both cultivars were crosswise combined with protein-free AWF (AWF filtrate) or water. AWF filtrate alone and boiled AWF samples did not show any NADH-peroxidase activity (data not shown). The AWF filtrates added to the enzyme assay had differential effects on the kinetics of NADH oxidation (Fig. 6 ). In the presence of AWF filtrates from cv TVu 91 or water, the NADH oxidation by apoplastic enzymes of cv TVu 91 and cv TVu 1987 (controls and Mn treatments) started shortly after addition of NADH (lines a, b, and e in each plot). AWF filtrates from cv TVu 91 (lines a and b) accelerated the oxidation compared to water (line e). In the presence of AWF filtrates from cv TVu 1987 (lines c and d in each plot), NADH oxidation showed a lag time of 2 to 8 min. The longest lag time was observed in the combination of both proteins and filtrates of cv TVu 1987 treated with high Mn (plot D, line d). The combination of proteins from cv TVu 1987 and water (plots C and D, line e) also produced a lag time.
From the kinetics of the NADH oxidation, the duration of the lag time and the slope at maximum NADH-oxidation rate was submitted to statistical analysis (Fig. 7 ). The lag time (Fig. 7A) was significantly affected by the origin of AWF filtrates added to the test mixture, the origin of the AWF proteins, and the Mn status of the plants: AWF filtrates from leaves of cv TVu 91 reduced the lag time particularly in combination with AWF proteins from the same Mn-treated cultivar. Filtrates from TVu 1987 delayed the NADH oxidation especially of proteins from the same Mn-treated cultivar. No significant effect of the origin of the AWF filtrates on the maximum NADH-oxidation rate existed (Fig. 7B), but AWF proteins from cv TVu 91 generally, but more clearly when Mn treated, showed a significantly higher NADH-oxidation capacity than proteins from cv TVu 1987. This compares well with the NADH-oxidation capacity of the crude AWF shown in Figure 2.
Concentrations and Composition of Phenolic Compounds of the AWF In a kinetic experiment with the Mn-sensitive cv TVu 91 that allowed to vary the severity of Mn toxicity as expressed by the number of brown spots over a wide range [0–160 (cm2)–1], the apoplastic phenol concentration increased with the severity of Mn toxicity (Fig. 8 ). Only in leaves with clearly expressed Mn toxicity symptoms [>50 spots (cm2)–1] phenol concentrations in the AWF were significantly enhanced. The Mn-tolerant cv TVu 1987 did not show any brown spots under the same experimental conditions (data not shown).
In a further experiment the relationship between the AWF phenol concentrations and a wide range of Mn leaf-tissue contents were studied (Fig. 9A ). With increasing Mn contents the phenol concentrations increased in cv TVu 91 but not in cv TVu 1987. To elucidate primary rather than secondary lesions of Mn toxicity the cultivars differing in Mn sensitivity were additionally compared at much lower Mn toxicity stress than in the previous experiment [0–20 brown spots (cm2)–1; Fig. 9B]. Phenol concentrations in the AWF showed a considerable variability. They appeared not to be affected by the Mn tissue content up to 1.2 µmol (g fresh weight)–1, and there was no difference between the cultivars.
Since quantitative differences between Mn supplies and cultivars could not be measured, the phenols in the leaf AWF were separated using HPLC to study possible qualitative differences. The HPLC chromatograms revealed differences in the pattern of phenols not only between optimum and elevated Mn supply (compare Fig. 10A with Fig. 10B) but also between the cultivars at optimum (Fig. 10A) and elevated (Fig. 10B) supply. The qualitative difference between the cultivars was particularly marked: Compared to the Mn-sensitive cv TVu 91 the Mn-tolerant cv TVu 1987 showed at least four additional peaks independent of the Mn treatment. Since no similarities between absorption spectra of apoplastic and commercial available phenolic compounds existed, the identities of the phenolic compounds in the AWF remain unknown so far.
Mn Uptake and Development of Mn Toxicity Symptoms
Excess Mn supply during the cultivation of cowpea plants caused the formation of visible small, dark-brown spots on leaves, representing oxidized Mn and phenols (Wissemeier and Horst, 1992
In the Mn-sensitive cv TVu 91, development of toxicity symptoms is accompanied by an enhanced release of proteins into the leaf apoplast (Fecht-Christoffers et al., 2003a
Particularly basic (cationic) POD isoenzymes, present in young and developing tissues (MacAdam et al., 1992
In cowpea, the magnitude of the POD activity particularly in the leaf AWF is indicative of the severity of Mn toxicity (Fecht-Christoffers et al., 2003a In this study, the Mn treatment only slightly affected the total AWF guaiacol-POD activity (Fig. 2A1) in both cultivars, whereas the specific activity was unaffected (Fig. 2A2). This can be related to the mild expression of Mn toxicity in this experiment and confirms the above-made assumption that the guaiacol-POD activity is closely related to the formation of the brown depositions in the leaf apoplast. Guaiacol-POD activity was lower in the Mn-tolerant cv TVu 1987 compared to the Mn-sensitive cv TVu 91. This genotypic difference was even greater regarding the activity of the NADH peroxidase. Both total and specific activities were highly significantly increased by Mn treatment exclusively in the Mn-sensitive cv TVu 91 (Fig. 2, B1 and B2) and remained on a low level in the Mn-tolerant cv TVu 1987. This suggests that NADH-peroxidase activity in the leaf AWF is more closely related to early events of Mn toxicity than guaiacol POD and that the control of the NADH-peroxidase activity is important for genotypic Mn leaf-tissue tolerance.
The regulatory effect of phenolic compounds and Mn on NADH-peroxidase activity is widely documented (Yamazaki and Piette, 1963
The phenolic compounds had inconsistent effects on the functionality of PODs (Lee, 1977
The presented results support the assumed linkage between NADH-peroxidase activity and H2O2 formation. The formation rate of H2O2 in vitro was significantly increased by Mn treatment only in the Mn-sensitive cv TVu 91 (Fig. 3). The addition of the cofactors Mn and p-coumaric acid influenced both NADH-peroxidase activities (Fig. 4) and H2O2 formation rates (Fig. 5) in both cultivars, but stronger in TVu 91.
The development of Mn toxicity was attributed to an increased formation of H2O2 in the apoplast (Horst et al., 1999
In relation to the presumably significant effect of phenolic compounds on the functionality of PODs, phenol concentration and phenol composition in the AWF of cowpea leaves were investigated. In the Mn-sensitive cowpea cv TVu 91, total phenol concentrations of the leaf tissue (water and NaOH extracts; Maier, 1997 Not the quantity of total phenols but rather the composition of the phenols in the leaf apoplast appears to be important for the difference in Mn tolerance of the cowpea cv TVu 91 and cv TVu 1987. Such qualitative differences are indicated by the HPLC chromatograms shown in Figure 10A. Moreover, the cross combining of AWF proteins with AWF filtrates (Figs. 6 and 7) suggests that the metabolome of the apoplast is equally or even more important for the expression of Mn tolerance than the proteome. Therefore, an in-depth analysis of the metabolites present in the AWF is necessary. As already mentioned and shown above, particularly phenolic compounds affect the functionality of PODs. Genotypic differences in phenol compositions of the AWF might be important for the formation of H2O2 and, therefore, the oxidation of MnII and phenolic compounds (see discussion above). We conclude from our results that PODs in the leaf AWF of cowpea are capable of producing and consuming H2O2 in the apoplast. For the H2O2-producing NADH oxidase cycle, the interaction between PODs and phenolic compounds in the leaf apoplast is crucial for the development and avoidance of Mn toxicity. The further characterization of the phenol and protein composition of the apoplast is expected to give in-depth insights into the regulation of H2O2 production and consumption in the leaf apoplast and its importance for the expression of Mn toxicity and Mn tolerance in the leaves of cowpea.
Plant Material and Cultivation
Plants of cowpea (Vigna unguiculata L. Walp) cv TVu 91 and cv TVu 1987 differing in Mn leaf-tissue tolerance (Horst et al., 1999 After preculture for at least 14 d, the MnSO4 concentration in the nutrient solution was increased to 50 or 100 µM Mn for 1 to 6 d. Control plants received 0.2 µM Mn continuously. Plants were treated until distinct visible symptoms were detectable on leaves of the sensitive cultivar. All plants were harvested at the same day. The nutrient solution was changed two to three times a week to provide optimum mineral nutrition.
For the quantification of Mn toxicity symptoms, leaf discs (1.54 cm2) were cut out at the base, middle, and tip of the trifoliate leaf and incubated in ethanol for at least 3 d. Numbers of brown spots per leaf disc were counted. Density of brown spots per square centimeter was calculated.
AWF was extracted by a vacuum infiltration/centrifugation technique. Therefore, leaflets of second trifoliate leaves were cut from plants, weighted, and infiltrated with demineralized water. The pressure was reduced to 35 hPa (1 min) by using a water jet pump followed by slow relaxation for 2 min. Leaflets were removed from the water and dry blotted. Infiltrated leaflets were weighted again and AWF was recovered by centrifugation at 1,324g for 5 min at room temperature. There were no differences in the yield of AWF between cultivars and Mn treatments. According to the activity of malate dehydogenase in the AWF, the contamination of the AWF by cytosolic proteins was less than 1%.
Measurements were carried out according to Bradford (1976)
For the extraction of proteins from the AWF, the AWF of the second-oldest trifoliate leaves of all plants in each variant were pooled and equal volumes were used for the precipitation of the proteins by phenol and acetate/methanol (Fecht-Christoffers et al., 2003a
For separation of proteins by their pI, the IPGphor system (Amersham Pharmacia Biotech) was used. Proteins were resuspended in demineralized water and supplemented with a rehydration solution (8 M urea, 2% [w/v] CHAPS, 0.5% [v/v] carrier ampholyte mixture [IPG buffer; Amersham Pharmacia], 0.28% [w/v] dithiothreitol, and a trace of bromphenol blue). Focusing was carried out using Immobiline DryStrip gels (18 cm) with a nonlinear pH gradient (pH 3–10) under conditions outlined by Werhahn and Braun (2002)
The 2-D gels were stained with colloidal Coomassie Blue (Neuhoff et al., 1985
For the measurement of H2O2-consuming POD activity in the AWF, the oxidation of the artificial substrate guaiacol was determined spectrophotometrically at
For the measurement of NADH-peroxidase activity in the AWF, AWF samples were combined with 0.3 mM NADH, 1.6 mM p-coumaric acid, and 16 mM MnCl2. All components were dissolved in 100 mM sodium acetate buffer, pH 5. The activity was measured at
The detection of H2O2 was based on the peroxidase-catalyzed oxidation of guaiacol. AWF was used for the measurement of NADH-peroxidase activity (as described above). After at least 1 min detection of NADH oxidation (measurements were done in the presence of 16 mM MnCl2, 1.6 mM p-coumaric acid, and 0.3 mM NADH), 3 mM guaiacol (in 10 mM sodium phosphate buffer, pH 6) were added to the enzyme assay. Changes in absorption were monitored at
NADH peroxidase and H2O2 formation were determined as described above. However, the concentration of added p-coumaric acid was varied from 0 to 1.6 mM and of MnCl2 from 0 to 160 mM. Extinction coefficients were calculated at each p-coumaric acid concentration: for 0, 0.016, 0.16, and 1.6 mM p-coumaric acid
To verify the effect of apoplastic water-soluble nonenzyme compounds of the apoplast on the activity of NADH peroxidase, proteins of the AWF were separated from the AWF by using mini centrifuge filter with a molecular weight cutoff (MWCO) at 4 kD (Nalgene Nunc International). Sample volumes of 500 µL were centrifuged at 6,000g, 4°C, for 1 to 2 h. AWF filtrates were removed and protein concentrates were washed with double demineralized water at 4°C. Purified proteins were recovered by centrifugation at 3,000g, 4°C, and 10 min. Just before detection of enzyme activities, proteins and AWF filtrate of controls and Mn-treated plants from TVu 91 and TVu 1987 were combined. For measurement of enzyme activities, 1 µL of protein concentrate and 50 µL AWF filtrate or double demineralized water were mixed with 16 mM MnCl2, 1.6 mM p-coumaric acid, and 0.3 mM NADH.
AWF (50 µL) was combined with 350 µL double demineralized water and 50 µL Folin-Denis solution (Merck). After 3 min incubation, 100 µL saturated Na2CO3 solution was added. After 1 h incubation and centrifugation (1 min, 5,000g) absorption was measured at
For the determination of the phenol composition in the AWF, phenols were extracted from the AWF. AWF (3 mL) was hydrolyzed in the presence of 150 µL HCL (8 M) and 200 µL acetonitril (ACN). After 1 h incubation at 80°C, 50 µL of saturated NaHCO3 was added. Samples were loaded on Bankerbond spe octadecyl C18 columns (Mallinckrodt Baker), which were previously equilibrated two times with 1 mL methanol and 500 µL double demineralized water. After loading, columns were washed with double demineralized water and samples were eluted with 500 µL 80% ACN/20% H3PO4 (0.1% [w/v]). HPLC analysis was carried out with a HPLC-DAD system (BioTek Instruments GmbH); precolumn: Phenomenex AJ0-4286 C18; column: Phenomenex Luna 5µ C18(2) (Phenomenex). The mobile phase used was: (A) ACN, and (B) H3PO4 (0.1% [w/v]). The solvent gradient changed as follows: (1) time 0 to 9 min, 0% A/100% B, flow 1.5 mL min–1; (2) time 9 to 22 min, 15% A/85% B, flow 1.5 mL min–1; (3) time 22 to 25 min, 30% A/70% B, flow 1.5 mL min–1; (4) time 25 to 27 min, 100% A/0% B, flow 1.5 mL min–1; and (5) time 27 to 36 min, 0% A/100% B, 1.5 mL min–1. Absorptions were measured at
For the analysis of Mn in the bulk-leaf tissue, center ribs were cut out of the middle leaflets of trifoliate leaves and 0.5 to 1 g leaf tissue was dried at 65°C. Drying was followed by dry ashing (480°C, 8 h) and dissolving the ash in 6 N HCl with 1.5% (w/v) hydroxylammonium chloride and dilution (1:10) with double demineralized water. AWF was diluted 1:10, and HCl and hydroxylammonium chloride were added to give final concentration of 0.6 N HCl and 0.15% (w/v) hydroxylammoniumchloride. Measurements were carried out by optical emission spectrometry, inductive coupled plasma (Spektro Flame, Spectro.
Means and SDs were from independent replications within an experiment and calculated by Excel 2000 (Microsoft). The numbers of replications are noted in the figure legends. Regression analysis, multiple analysis of variance (F test), and comparisons of means (Tukey test) were carried out by SAS 8e (SAS Institute). Levels of significance are indicated in graphs by *, **, and *** for P < 0.05, 0.01, and 0.001. Means with different letters are significantly different at P < 0.05 (Tukey). Received August 25, 2005; returned for revision January 24, 2006; accepted February 6, 2006.
1 This work was supported by the Deutsche Forschungsgemeinschaft (Special Research Programme 717, "The apoplast of higher plants" and Ho 931/17–1).  The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Walter Johannes Horst (horst{at}pflern.uni-hannover.de).
[W] The online version of this article contains Web-only data. Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.105.070474. * Corresponding author; e-mail horst{at}pflern.uni-hannover.de; fax 49–511–762–3611.
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ABSTRACT
RESULTS
= 340 nm per min. AWF from controls and Mn treatments (50 µM for 4 d) from cowpea cv TVu 91 and cv TVu 1987 were divided into two fractions (purified proteins and AWF filtrate) using centrifugal concentrators with MWCO 5 kD. Protein concentrates (A, TVu 91 control; B, TVu 91 Mn; C, TVu 1987 control; and D, TVu 1987 Mn), and AWF-filtrates (a, TVu 91 control; b, TVu 91 Mn; c, TVu 1987 control; and d, TVu 1987 Mn) or water (e) were combined. For measurement of enzyme activities, 1 µL of protein concentrate and 50 µL AWF filtrate or double demineralized water were mixed with 16 mM MnCl2, 1.6 mM p-coumaric acid, and 0.3 mM NADH.
by MnII in vitro is significantly affected by the presence of several buffers and complexing agents. Aqueous Mn2+ proved to be a poor scavenger for ROS, whereas in the presence of orthophosphate, pyrophosphate, and a number of organic acids e.g. lactate, succinate, and malate the ROS scavenging activity of MnII increased (Archibald and Fridovich, 1982a
= 4.94, 4.68, 4.60, and 4.23 mM–1, respectively. 
