Journal of Plant Nutrition

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Journal of Plant Nutrition

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Manganese Supply and pH Influence Growth, Carboxylate Exudation and Peroxidase Activity of Ryegrass and White Clover

To cite this Article: , 'Manganese Supply and pH Influence Growth, Carboxylate Exudation and Peroxidase Activity of Ryegrass and White Clover', Journal of Plant Nutrition, 30:2, 253 - 270 xxxx:journal To link to this article: DOI: 10.1080/01904160601118034 URL: http://dx.doi.org/10.1080/01904160601118034

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Journal of Plant Nutrition, 30: 253–270, 2007 Copyright © Taylor & Francis Group, LLC ISSN: 0190-4167 print / 1532-4087 online DOI: 10.1080/01904160601118034

Manganese Supply and pH Influence Growth, Carboxylate Exudation and Peroxidase Activity of Ryegrass and White Clover Anal´ı Rosas,1 Zed Rengel,2 and Mar´ıa de la Luz Mora3 1

Doctorado en Ciencias de Recursos Naturales, Universidad de La Frontera, Temuco, Chile 2 Soil Science and Plant Nutrition, School of Earth and Geographical Sciences, The University of Western Australia, Crawley, Australia 3 Departamento de Ciencias Qu´ımicas, Universidad de La Frontera, Temuco, Chile

ABSTRACT The effects of differential manganese (Mn) supply (0 to 355 μM) and pH (4.8 and 6.0) on dry weight (DW), tissue concentrations of Mn, exudation of carboxylates, and the peroxidase activity were studied in ryegrass (Lolium perenne L.) and white clover (Trifolium repens L.) grown in nutrient solution. In both plant species, the increase in Mn supply caused a significant reduction in DW due to severe Mn toxicity, especially at pH 4.8. The critical toxicity concentration of Mn in shoots was 421 mg kg−1 for ryegrass and 283 mg kg−1 for white clover. For both plant species, an increase in Mn supply levels stimulated the exudation of carboxylates and the activity of peroxidase, which was related to stress conditions. The highest amount of carboxylates was exuded at pH 4.8. There was no clear effect of carboxylates on the complexation of Mn2+ . Keywords: carboxylate exudation, manganese toxicity, peroxidase, ryegrass, white clover

INTRODUCTION Manganese (Mn) is a plant micronutrient that, depending on its content in soil and factors controlling its availability (e.g. pH), can be toxic to plants (Rengel, 2000). Meat and dairy production in southern Chile is grassland based, with the Received 22 December 2005; accepted 7 April 2006. Address correspondence to Zed Rengel, Soil Science and Plant Nutrition, School of Earth and Geographical Sciences, The University of Western Australia, 35 Stirling Highway, Crawley WA 6009, Australia. E-mail: [email protected] 253


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predominant use of forage species like ryegrass and white clover. Large areas of pastures are developed on acidic soils (Mora et al., 2002). Soil chemical factors that diminish the crop production in acid soils include Al and Mn toxicities, which result in reduction of the plant growth as well as the quality of pasture (Mora et al., 2004). The critical shoot concentration for Mn toxicity depends on plant species and genotypes as well as the environmental conditions, such as temperature and pH (Sparrow and Uren, 1987). The Mn concentration in pastures of southern Chile ranges between 50 and 1,000 mg kg−1 (Data Bases of Soil Service Laboratory of La Frontera University). However, information about the critical concentration of Mn in individual grassland species of southern Chile is limited. The availability of Mn is strongly dependent on soil pH (Rengel 2000). When the soil pH drops below 5.5, Mn toxicity may be evident, whereas above pH 6.5 Mn deficiency may occur (Ducic and Polle, 2005). The biochemical responses of higher plants to toxicity of heavy metals are very complex. Several strategies have been suggested to overcome this stress, including complexation of metal ions, reduced influx of metals and enhanced production of antioxidants that detoxify reactive oxidative species produced in response to toxic metals (Schützendübel and Polle, 2002). Many studies have shown that the oxidative damage in plants occurs under either Mn deficiency or Mn toxicity (González et al., 1998; Rengel, 1997). Manganese is involved in (i) the oxygen radical production via its involvement in the photosynthetic pathway, and (ii) the oxygen radical detoxification through its role in the superoxide dismutase activity. Superoxide dismutase and catalase enzymes form hydrogen peroxide (H2 O2 ), which can be detoxified by the enzyme peroxidase (EC 1.11.1.7). Peroxidase has an important role in the antioxidative response of plant cells to different types of stress, particularly caused by metals (Van Gronsveld and Clijters, 1994). However, the influence of Mn concentration in plant tissues on the peroxidase activity in ryegrass and white clover is unknown. Plants can influence the solubility and the speciation of metals in the rhizosphere through the exudation of chelators (e.g. organic acid anions) and the changes in the rhizosphere pH (Reichman, 2002; Rengel, 2002). The role of organic acid anions in the Al detoxification and P acquisition in the rhizosphere has been extensively studied (Delhaize et al., 1993; Jones, 1998; Miyasaka et al., 1991; Neumann and Römheld, 1999; Otani et al., 1996; Rengel, 2002; Taylor and Foy, 1985; Zheng et al., 1998). Nevertheless, there are no reports regarding the effect of Mn toxicity on carboxylate release by roots of ryegrass and white clover under varying pH. The objective of this study was to evaluate the effect of increasing Mn supply and differential pH on plant growth, exudation of carboxylates, and the peroxidase activity in perennial ryegrass and white clover.


Physiological Responses to Mn Toxicity in Pasture Plants

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MATERIALS AND METHODS Plant Material and Growth Conditions Seeds of perennial ryegrass (Lolium perenne L.) cv. Nui and white clover (Trifolium repens L.) cv. Huia were germinated on filter paper moistened with deionized water. Five-day-old seedlings were transplanted to 1 L pots containing aerated nutrient solution (16 seedlings per pot). The nutrient solutions were selected according to the nutrient requirements of ryegrass and white clover. For ryegrass, the solution proposed by Taylor and Foy (1985) was chosen because it has been used in studies concerning Al tolerance in wheat, with a Mn level of 2.4 μM. For white clover, the solution proposed by Lee et al. (1984) was selected because it has been used specifically for white clover in studies of Al tolerance, with a Mn level of 5.3 μM. Solutions were replaced every three to four days, and the pH was adjusted daily with diluted HCl or NaOH. Two pH levels (4.8 and 6.0) were tested in a factorial design with six Mn treatments (0, 2.4, 24, 59, 178, and 355 μM for ryegrass, and 0, 5.3, 24, 59, 178, and 355 μM for white clover) applied as MnSO4 . Each treatment was replicated four times. Plants were grown in a growth chamber with a 16 h light period, at 20◦ C and 60–80% relative humidity. After 40 days, plants were harvested to measure growth parameters such as shoot and root dry weight and to determine phosphorus (P), Mn, and iron (Fe) concentrations in shoots and roots. Dry Weight and Chemical Analysis of Plants Shoots were dried in a forced-air oven for 2 days at 65◦ C. After dry weight determination, samples were dry-ashed in a muffle furnace at 500◦ C for 8 hours and digested with 2 M HCl. Manganese and Fe were extracted as described by Sadzawka et al. (2004), and the concentration was determined by atomic absorption spectrophotometry. Phosphorus concentration was measured by the molybdo-vanadate method (A.O.A.C., 1975). Collection and Analysis of Root Exudates Exudates were collected 15 days after the beginning of treatments according to the method of Neumann and Römheld (1999; 2000). Roots of intact plants, which have been treated with 0 to 355 μM Mn, were submerged for 2 h in aerated deionized water, and the resulting solution containing exudates was stored at −20◦ C for analysis. The collection time of 2 h was selected because degradation of organic acid anions by microorganisms is intensified after 2 hours (Jones and Darrah, 1994; Jones et al., 1996). In order to quantify the concentration of organic acid anions (oxalate, malate, citrate, and succinate) root exudates were lyophilized, the residue


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re-dissolved in 300–500 μL of deionized water and used for HPLC analysis. Separation was achieved on a 250 × 4 mm reversed phase column (Merck, LiChrospher 100 RP-18, 5-μm particle size). Sample solutions (20 μL) were injected into the column, and 200 mM ortho-phosphoric acid solution (pH 2.1) was used for isocratic elution with a flow rate of 1 mL min−1 and UV detection at 210 nm. A preliminary study, with standard organic anion compounds, indicated that recovery of the organic anions was about 98%. A modified GEOCHEM computer program (Parker et al., 1987), version 2.0, was used to evaluate the Mn speciation and the formation of carboxylateMn complexes in the nutrient solutions. The total amount of organic acid anions exuded in 1 L of nutrient solution was considered for chemical speciation (16 plants per treatment). Enzyme Assay The peroxidase activity in shoots and roots was measured 15 days after the beginning of treatments. Peroxidase was extracted according to the modified procedure of Adorada et al. (2000). Root and shoot samples were homogenized in 50 mM Tris-HCl buffer and 1 mM dithiotreitol (DTT) at pH 7.5. Homogenates were filtered and centrifuged at 1,500 g for 15 min. The resulting supernatant was used to assay activity or was stored at −20◦ C for analysis. Prior to the peroxidase assay, plant extracts were pre-incubated for 1 min with Triton X100 [0.65% (v/v)]. The analysis was performed at 4◦ C. Peroxidase activity was determined using the guaiacol oxidation method in a 3-mL reaction mixture containing 50 mM acetate buffer (pH 5.0), 10 mM guaiacol, 10 mM H2 O2, and 100 μL enzyme extract. The increase in absorbance due to the formation of tetraguaiacol (ε 26.6 mM−1 cm−1 ) at 470 nm was measured by a spectrophotometer. The activity was expressed in units (U) per g fresh weight. One unit equals the amount of substrate (μmol) transformed by the enzyme in one minute. Statistics The experimental design was a randomized complete block with four replications. Before statistical analysis, data were logarithmically transformed and analyzed by ANOVA. Means were compared using Tukey Test (P ≤ 0.05). RESULTS AND DISCUSSION Concentration of Mn in Plant Tissues and Plant Growth Concentration of Mn in shoots and roots (Table 1) was highly related to Mn activity in nutrient solution (Table 2). The concentration of Mn was greater in



257

Table 1 Mn concentrations (mg kg−1 ) in shoots and roots of ryegrass and white clover grown in nutrient solution with different pH and Mn treatments Treatments pH 6.0 .

4.8

Ryegrass

White clover

Mn (μM)

Shoot

Root

Shoot

Root

0 2.4 5.3 24 59 178 355 0 2.4 5.3 24 59 178 355

68 h 148 g n.d. 954 e 1355 d 2851 ab 2238 c 31 i 180 g n.d. 723 f 1277 d 3148 a 2357 bc

22 g 39 g n.d. 1006 f 3297 bc 5447 b 6302 a 106 g 177 g n.d. 1115 f 1561 e 3244 c 2408 d

59 h n.d. 163 g 378 e 723 c 1521 b 2120 a 53 h n.d. 187 f 475 d 742 c 1653 b 2050 a

77 j n.d. 198 i 1197 f 1665 e 3225 c 6164 b 151 h n.d. 417 g 1270 f 2781d 6044 b 7481a

Means with the same letter in a column are not significantly different (P ≤ 0.05) based on Tukey test. n.d.: not determined.

the roots than in the shoots for both species. There was no significant difference in the Mn concentration in shoots at the two pH levels, whereas the Mn concentration in roots was higher at pH 4.8 than 6.0. Fifteen days after the beginning of the Mn treatments, visible differences were observed in the growth of ryegrass and white clover, and these differences intensified gradually during the experiment. The plants showed symptoms that varied from deficiency to severe toxicity, depending on the Mn activity in solution. Plants were small at the highest supply of Mn (355 μM), with necrotic leaves and short roots. The toxicity symptoms were manifested as chlorosis in the ryegrass and as reddish borders on clover leaves. Dry weight of roots and shoots decreased significantly with increasing Mn supply from 24 to 355 μM in nutrient solution, and the detrimental effect of the Mn concentration was greater at pH 4.8 than 6.0 (Figures 1 and 2). The critical tissue concentrations of Mn (defined as those associated with 10% growth reduction) were 421 mg Mn kg−1 for ryegrass and 283 mg Mn kg−1 for white clover. The critical Mn toxic concentration for ryegrass was estimated by MacNicol and Beckett (1985) at 500 mg Mn kg−1 , which is slightly greater than the threshold obtained in this experiment. However, the critical Mn toxic concentrations reported by Smith et al. (1983) for white clover (670 mg kg−1 ) and ryegrass (1110 mg kg−1 ) were more than twice the thresholds for Mn

258

Mn2+ Mn-SO4 Mn-Cl Mn-EDTA Mn-carboxylates

Mn2+ Mn-SO4 Mn-Cl Mn-EDTA Mn-carboxylates

84.5 8.23 0.07 0.14 7.08

5.3

89.4 1.63 0.07 0.08 8.83

2.4

83.9 8.18 0.07 0.19 7.64

24

88.9 1.61 0.07 0.07 9.30

24

82.7 8.13 0.07 0.24 8.89

59

88.5 1.60 0.07 0.08 9.56

59

83.4 8.07 0.07 0.17 8.31

178

87.9 1.57 0.07 0.10 9.88

178

pH 4.8 Mn treatment (μM) 2.4

24

5.3

24

Nutrient solution for white clover 85.12 73.8 72.2 7.59 7.25 7.16 0.07 0.06 0.06 0.17 7.56 7.42 7.018 11.13 13.17

355

Nutrient solution for ryegrass 88.8 87.6 87.8 1.55 1.59 1.59 0.07 0.07 0.07 0.09 1.15 0.91 8.82 9.62 10.08

355

76.1 7.52 0.06 7.48 8.81

59

86.9 1.57 0.07 1.07 10.24

59

76.4 7.45 0.06 6.65 15.2

178

88.8 1.57 0.07 0.79 9.47

178

pH 6.0 Mn treatment (μM)

76.2 7.26 0.06 5.72 10.77

355

88.5 1.54 0.07 0.58 9.21

355

Table 2 Percent distribution of Mn species in nutrient solutions for ryegrass and white clover with different levels of pH and Mn. Calculated by GEOCHEM


259



Figure 1. Interaction effects of Mn and pH on dry weight of shoot and roots of ryegrass grown in nutrient solution. Means with common letters are not significantly different at P ≤ 0.05 according to Tukey test.

toxicity calculated in the present study, which can be attributed to differences in both initial Mn supply and cultivars studied in the two reports. Above 24 μM of Mn in the nutrient solution, DW and Mn concentrations in shoots were higher in ryegrass than in white clover because legumes are more susceptible to Mn toxicity than grasses (Whitehead, 2000). This was demonstrated by the higher critical Mn toxic concentration in shoots of ryegrass compared with white clover. When no Mn was added, the plants showed a reduction of approximately 15% in DW of shoots compared with treatments of 2.4 μM Mn for ryegrass

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Figure 2. Effects of interaction between Mn and pH on dry weight of shoot and roots of white clover grown in nutrient solution. Means with common letters are not significantly different at P ≤ 0.05 according to Tukey test.

and 5.3 μM Mn for white clover. The internal Mn requirement was 136 mg Mn kg−1 for ryegrass and 145 mg Mn kg−1 for white clover shoots for 95% of the maximum growth (Figure 3). Jones et al. (1991) indicated that the Mn requirement is satisfied at tissue levels of 20–60 mg kg−1 for ryegrass and 25– 100 mg kg−1 for white clover. A decrease in the DW when plants were grown without Mn additions (Figures 1 and 2) likely occurred as a consequence of Mn deficiency because shoot concentration of Mn represented about 50% and

261



Figure 3. Relationship between Mn concentration in ryegrass and white clover shoots and dry weight.

40% of the internal Mn requirement calculated for ryegrass and white clover, respectively (Figure 3). The influence of pH on plant DW can be partly explained by differences in the Mn speciation among the nutrient solutions used for ryegrass and white clover (Table 2). Thus, the percentage of free Mn2+ in the nutrient solution used for ryegrass was similar at pH 4.8 and 6.0, and only slight differences were observed in shoot DW at the two pH levels. However, for white clover solutions, the lower percentage of free Mn2+ and the higher plant DW was observed at pH 6.0 than 4.8 as a consequence of higher Mn complexation by EDTA at pH 6.0 than 4.8 (Table 2). The differences in Mn speciation among


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the studied nutrient solutions can be attributed to the greater formation of MnSO4 and Mn-EDTA complexes in the nutrient solution for white clover because this solution has higher concentrations of SO2− 4 and EDTA compared with the nutrient solution for ryegrass. The higher content of P and Fe were measured in ryegrass and white clover at pH 6.0 than 4.8 (data not shown). This difference may be another contributing factor to explain the differences in DW obtained at different pH treatments.

Effect of the Mn and pH Levels on the Quantity and Type of Exuded Carboxylates Both ryegrass and white clover exuded oxalate in higher amounts than other carboxylates (Figures 4 and 5). The exudation rates of oxalate ranged between 0.37 and 3.3 μmol g−1 h−1 . In ryegrass, the Mn additions increased the exudation of carboxylates significantly (P ≤ 0.05). The roots exuded the highest amount of carboxylates at pH 4.8 (Figures 4 and 5). At the highest Mn rate (355 μM; pH 4.8), the increase was approximately 4-fold (oxalate), 3-fold (citrate), 7-fold (malate), and 2-fold (succinate) compared with the treatments 2.4 μM Mn for ryegrass and 5.3 μM Mn for white clover. At pH 6.0 the same tendency was observed, but the increase in exuded carboxylates was about 2-fold when Mn was increased to 355 μM. In white clover, the roots exuded greater quantity of oxalate and citrate at pH 4.8 than 6.0 (Figure 5). Compared with treatments of 2.4 μM Mn for ryegrass and 5.3 μM Mn for white clover, there was an increase in the exudation of oxalate, citrate, and malate of about 1-, 2-, and 4-fold, respectively, at 355 μM Mn in the nutrient solution (pH 4.8). Increasing Mn supply to 355 μM at pH 6.0 resulted in an increase of about 2-fold for oxalate, malate, and succinate, with no increase for citrate. The increase in carboxylate exudation especially that of citrate and oxalate, has been previously observed in conditions of Al toxicity in different plant species and genotypes as a strategy of preventing the excess metal uptake (Delhaize et al., 1993; Miyasaka et al., 1991; Zheng et al., 1998; Ma et al., 2001). Organic acid anions (carboxylates) are chelating substances (Reichman, 2002) that form strong complexes with heavy metals (Horst et al., 1999). For both plant species tested in the present study, an increase was observed in the formation of carboxylate-Mn complexes as a consequence of both the increase in Mn supply and the increase in the carboxylate exudation (Tables 2 and 3). According to the chemical speciation of organic acid anions (GEOCHEM), the percentage of oxalate-Mn and citrate-Mn complexes in solution increased between 100- and 140-fold (ryegrass) and between 17- and 140-fold (white clover) as the Mn supply levels increased in the nutrient solutions (Table 3). Although in the study described here an increase in the percentage of carboxylate-Mn complexes was observed in response to the increase in Mn

263



Figure 4. Carboxylates exuded by ryegrass roots growing in nutrient solution with different Mn and pH levels. Different letters denote significant differences (P ≤ 0.05) according to Tukey test.

concentration in the nutrient solution (Table 3), there was no clear effect of the carboxylates on the complexation of Mn2+ (Table 2). The low ability of the carboxylates to complex Mn2+ can be attributed to: (i) the high percent of oxalate-Ca (77–92%) and citrate-Ca (37–72%) complexes formed as consequence of a higher availability of Ca2+ than Mn2+ in solution [even though the Ca stability constants for oxalate-Mn and -Ca (log K OMnx = 3.9 and log K Ox = 3.0) Ca Mn and citrate-Mn and -Ca (log K Cit = 3.2 and log K Cit = 3.5) are similar] and (ii) succinate and malate remained mainly as protonated and free forms in the nutrient solution as shown in Table 3. Nevertheless, Hoffland et al. (2004) and Nogueira et al. (2004) demonstrated that oxalate and citrate bound Mn2+

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264

Figure 5. Carboxylates exuded by white clover growing in nutrient solution with different Mn and pH levels. Different letters denote significant differences (P ≤ 0.05) according to Tukey test.

strongly, thus decreasing the activity of free Mn2+ in the soil solution and consequently the uptake by mycorrhizal plants. Therefore, studies in the soil-plant system with different genotypes are required to clarify whether exudation of carboxylates is indeed a plant strategy for overcoming Mn toxicity. Additionally, a significant increase in the exudation of carboxylates was observed in white clover when no Mn was added at pH 6.0 (Figures 4 and 5), because carboxylate exudation has been implicated in the tolerance to Mn deficiency as shown by Gherardi and Rengel (2004) in experiments with lucerne.

265

0.21 0.26 0.01 0.07 46.6 83.5 98.0 88.4

Citrate-Mn Oxalate-Mn Succinate-Mn Malate-Mn Citrate-Ca Oxalate-Ca∗ Succinate-H+ /free Malate-H+ /free

Complexed and solid.

5.3

Complexes

∗

0.12 0.08 0 0.03 47.2 90.4 98.0 88.4

2.4

Citrate-Mn Oxalate-Mn Succinate-Mn Malate-Mn Citrate-Ca Oxalate-Ca * Succinate-H+ /free Malate-H+ /free

Complexes

1.00 1.06 0.05 0.31 42.1 84.7 98.0 88.4

24

1.15 0.72 0.05 0.32 46.2 90.1 98.0 88.2

24

2.57 1.62 0.12 0.74 41.0 89.5 98.1 88.7

59

2.76 1.99 0.13 0.79 46.3 87.7 97.9 87.7

59

pH 4.8 2.4

355

5.3

Nutrient solution for ryegrass Mn treatment (μM) 14.75 0.20 10.41 0.09 0.78 0.02 4.61 0.07 40.7 69.3 80.7 90.4 97.3 92.7 84.4 77.0

355

24

1.93 0.96 0.19 0.72 66.0 88.7 93.0 76.3

24

Nutrient solution for white clover Mn treatment (μM) 7.02 11.33 0.18 1.88 4.98 4.34 0.11 0.65 0.37 0.74 0.02 0.16 2.23 4.25 0.06 0.62 37.4 44.14 72.0 69.2 85.8 91.8 86.8 91.3 97.8 96.67 93.1 93.3 87.2 81.7 78.1 79.0

178

8.05 5.68 0.39 2.34 44.4 84.6 97.6 86.4

178

4.71 1.47 0.40 1.58 67.3 91.5 93.2 78.1

59

4.59 2.25 0.45 1.75 65.9 87.8 92.2 75.7

59

pH 6.0

12.94 4.46 1.20 4.62 61.5 88.5 92.4 75.6

178

17.61 7.99 1.38 5.15 56.1 80.0 91.2 72.5

178

22.25 10.43 2.36 8.74 55.2 81.3 91.1 71.8

355

20.60 12.95 2.71 9.79 49.5 77.7 90.1 69.2

355

Table 3 Percent of carboxylates complexed with Mn and Ca in nutrient solution for ryegrass and white clover with different levels of pH and Mn. Calculated by GEOCHEM



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Effect of Mn and pH on Peroxidase Activity In ryegrass and white clover, the peroxidase activity of shoots increased in response to low or high concentrations of Mn at pH 6.0 and 4.8 (Figure 6). Similar results have been reported for soybean (Leidi et al., 1986) and rice (Horiguchi, 1988). In plants suffering Mn deficiency or toxicity, cell metabolism cannot efficiently control excess formation of oxygen radicals and oxidative damage occurs (Tanaka et al., 1995; Yu and Rengel, 1999). In the present study,

Figure 6. Shoot peroxidase activity of ryegrass and white clover grown with different Mn supply at pH 6.0 and 4.8. Different letters denote significant differences (P ≤ 0.05) according to Tukey test.



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peroxidase activity increased under severe Mn toxicity (355 μM Mn), indicating oxidative stress. Measuring the peroxidase activity is particularly useful for an early detection of plant stress, and it has been proposed as a possible biological indicator for diagnosing micronutrient imbalances (Moreno et al., 2000). The effects of Mn supply levels on peroxidase activity (Figure 6 ) and on plant growth (Figures 1 and 2) clearly show that deficiency or toxicity of Mn leads to a decrease in plant growth as a consequence of elevated oxidative stress (see also González et al., 1998). The activity of peroxidase was greater in white clover than ryegrass, suggesting only a minor oxidative stress in ryegrass due to Mn toxicity. Ryegrass was more tolerant to Mn toxicity than white clover.

ACKNOWLEDGEMENTS This work was supported by the Andes Foundation N◦ C 13755–28 and the Fondecyt N◦ 1020934 and MECESUP FRO 0309 Grants.

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