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Plant Growth Regul (2006) 49:113–118 DOI 10.1007/s10725-006-9000-2

ORIGINAL PAPER

A simple colorimetric method for determination of hydrogen peroxide in plant tissues Biyan Zhou Æ Jihua Wang Æ Zhenfei Guo Æ Huoquan Tan Æ Xiaochuan Zhu

Received: 19 January 2006 / Accepted: 10 July 2006 / Published online: 29 September 2006 Ó Springer Science+Business Media B.V. 2006

Abstract A simple colorimetric method for determination of hydrogen peroxide in plant materials is described. The method is based on hydrogen peroxide producing a stable red product in reaction with 4-aminoantipyrine and phenol in the presence of peroxidase. Plant tissues was ground with trichloroacetic acid (5% w/v) and extracts were adjusted to pH 8.4 with ammonia solution. Activated charcoal was added to the homogenate to remove pigments, antioxidants and other interfering substances. The colorimetric reagent (pH 5.6) consisted of 4-aminoantipyrine, phenol, and peroxidase. With this method, we have determined the hydrogen peroxide concentration in leaves of eight species which ranged from 0.2 to 0.8 lmol g–1 FW. Changes in hydrogen peroxide concentration of Stylosanthes guianensis in response to heat stress are also analyzed using this method. Keywords 4-aminoantipyrine Æ Hydrogen peroxide Æ Peroxidase Æ Plant tissue B. Zhou Æ J. Wang Æ Z. Guo (&) Æ H. Tan College of Life Science, South China Agricultural University, Guangzhou 510642, China e-mail: [email protected] B. Zhou Æ X. Zhu Physiological Laboratory for South China Fruits, College of Horticulture, South China Agricultural University, Guangzhou 510642, China

Introduction Hydrogen peroxide (H2O2) is one of the major reactive oxygen species (ROS) in plant tissues. It is produced in chloroplasts and mitochondria via electron transport, where oxygen is reduced to superoxide which is further dismuted into H2O2 spontaneously or catalyzed by superoxide dismutase (Asada 1999; Moller 2001). H2O2 production in plant cells is also catalyzed by glycollate oxidase in peroxisomes (Noctor et al. 2002), membrane bound NADPH oxidase (Jiang and Zhang 2003) and oxalate oxidase (Hu et al. 2003). When plants are subject to environment stress, it accumulates and leads to oxidative damage (Asada 1999). Accumulating evidence suggests that H2O2 is a key signaling molecule involved in plant response to both biotic and abiotic stresses, such as pathogen attacks, extreme temperatures, drought, excessive radiation, ozone and wounding (Neill et al. 2002; Foyer et al. 1997; Prasad et al. 1994; Orozco-Ca´rdenas et al. 2001; Wohlgemuth et al. 2002). Therefore it is frequently important to determine hydrogen peroxide concentration in plant tissues. There are several analytical methods for determination of H2O2, including spectrophotometric (Patterson et al. 1984), chemiluminescent (Pe´rez and Rubio 2006; Warm and Laties 1982) and fluorometric (Genfa and Dasgupta 1992). Patterson’s spectrophotometric method has been

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used for estimation of H2O2 in plant extracts (Aroca et al. 2003; Vaidyanathan et al. 2003). However, we failed to detect any H2O2 in Stylosanthes guianensis, an important pasture legume, by using Patterson’s spectrophotometric method, suggesting that some substances in plant tissues might interfere with the assay. This has led us to find a novel method suitable for this pasture legume as well as other plant materials. Glucose oxidase/peroxidase system is widely used for determination of glucose in blood (Trinder 1969). In this reaction, glucose is converted to gluconic acid and H2O2, catalyzed by glucose oxidase. H2O2 is subsequently reacted with 4-aminoantipyrine and phenol which produces a stable red product in the presence of peroxidase. The objective of this study is to establish an easy and rapid protocol for determination of H2O2 in plant tissues using this colorimetric approach. Materials and methods Plant materials and chemicals Rice (Oryza sativa L.), Stylosanthes guianensis, alfalfa (Medicaga sativa L.) and tobacco (Nicotiana tabacum L.) seedlings were grown in a greenhouse as described (Huang and Guo 2005; Zhou et al. 2005). Litchi (Litchi chinensis Sonn.), longan (Dimocarpus longana Lour.), banana (Musa acuminata Colla (AAA)) and taro (Colocasia esculenta) were grown in the field at the South China Agriculture University. Heat stress treatment was applied to Stylosanthes guianensis when moved into a growth chamber for a 12 h photoperiod (160 lmol m–2 s–1 PPFD) at 38°C. In each case fully expanded second leaves were selected for H2O2 determination. Catalase and 4-aminoantipyrine were purchased from Sigma. Peroxidase was obtained from Bo Ao Biochemical Company (Shanghai, China). All other chemicals were of analytic grade obtained from Guangzhou Chemical Factory (China). Standardization of hydrogen peroxide Hydrogen peroxide was standardized as described by Aebi (1974) and Patterson et al. (1984). H2O2

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(30%, w/v) was diluted to approximately 10 mM in 50 mM phosphate buffer (pH 7.0). The diluted H2O2 solution was standardized by its absorbance at 240 nm, by measuring against blanks from which H2O2 had been removed by addition of excessive catalase. Absorbance due to the addition of catalase was corrected with a blank of catalase alone in the buffer. The molar absorptivity of hydrogen peroxide at 240 nm and pH 7.0 was 40 M–1 cm–1. Determination of H2O2 Hydrogen peroxide was extracted from plant tissues as described by Patterson et al. (1984). Fresh leaves (0.5 g) were frozen in liquid nitrogen and ground to powder in a mortar with pestle, together with 5 ml of 5% TCA and 0.15 g activated charcoal. The mixture was centrifuged at 10,000g for 20 min at 4°C. The supernatant was adjusted to pH 8.4 with 17 M ammonia solution and then filtered. The filtrate was divided into aliquots of 1 ml. To one of these, the blank was added 8 lg of catalase and then kept at room temperatures for 10 min. To both aliquots with and without catalase, 1 ml of colorimetric reagent was added. The reaction solution was incubated for 10 min at 30°C. Absorbance at 505 nm was determined spectrophotometrically (Model UV-2010, Hitachi, Japan). The colorimetric reagent contained 10 mg of 4-aminoantipyrine, 10 mg of phenol, 5 mg of peroxidase (150 U mg–1), dissolved in 50 ml of 100 mM acetic acid buffer (pH 5.6).

Results and discussion An absorbance spectrum of the colorimetric products was measured from 450 nm to 600 nm and revealed a peak at 505 nm (Fig. 1), suggesting the optimum colorimetric wavelength for the red reaction product was 505 nm. In order to establish the optimum amount of colorimetric reagent used in the assay medium, five concentrations of reaction reagents were tested and the result are shown in Table 1. The absorbance of colorimetric products increased with the increase of the concentration of colorimetric reagent. Treatment A and B had a similar absorbance.

Plant Growth Regul (2006) 49:113–118

115

Absorbance

0.25 0.20 0.15 0.10 0.05 0.00

400 420 440 460 480 500 520 540 560 580 600

Wavelength (nm)

Fig. 1 Absorbance spectrum of colorimetric reaction product. 5% (w/v) TCA containing 100 lM H2O2 was adjusted to pH 8.4 with 17 M ammonia solution and mixed by equal volume of colorimetric reagent. Absorbance of the reaction product at wavelengths from 450 nm to 600 nm was measured by spectrophotometer

Treatment C had 3.7% lower absorbance than that of Treatment B, but had 10.8% or 26.4% higher absorbance than Treatment D or Treatment E, respectively, although the concentration of colorimetric reagent increased significantly from Treatment E to Treatment C. Therefore, the concentration of colorimetric reagent in Treatment C should be sufficient to initiate the colorimetric reaction with H2O2. For determination of the stability of reaction product, absorbance at 505 nm of the product was recorded immediately after mixing 100 lM of H2O2 solution with the colorimetric reagent. The absorbance showed a slight decline at the first 5 min and maintained stability at least to 20 min (data not shown). Absorbance of the reaction Table 1 Effect of 5 concentrations of colorimetric reagent on the absorbance of the reaction product at 505 nm Treatment

Absorbance at 505 nm

A B C D E

0.167 0.161 0.155 0.140 0.123

± ± ± ± ±

0.001 0.001 0.001 0.002 0.00

H2O2 solution (60 lM) was mixed by equal volumes of colorimetric reagent. 50 ml of colorimetric reagents contained 40 mg 4-aminoantipyrine, 40 mg phenol and 20 mg peroxidase in Treatment A; 20 mg 4-aminoantipyri ne, 20 mg phenol and 10 mg peroxidase in Treatment B; 10 mg 4-aminoantipyrine, 10 mg phenol and 5 mg peroxidase in Treatment C; 5 mg 4-aminoantipyrine, 5 mg phenol and 2.5 mg peroxidase in Treatment D, and 2.5 mg 4-aminoantipyrine, 2.5 mg phenol and 1.25 mg peroxidase in Treatment E. Values are means ± SE (n = 3)

product could be consistently measured within 10 to 20 min after initiating the reaction. Colorimetric reaction was affected by pH. Absorbance of the reaction product increased with the increase in the pH of the filtrate obtained from rice leaf extracts (Fig. 2). TCA, the acidic extraction solution, inhibited the action of both catalase and peroxidases in plant tissues (Patterson et al. 1984), but the pH of the extract should be adjusted to above 8.0 to give an adequate coloration. Although the reaction solution had the highest absorbance at pH 9.0, the blank which was added catalase had deep color at pH 9.0, but colorless at pH 8.4; pH 8.4 was therefore adopted for future H2O2 assay. The effect of catalase quantity on the color of the blank was tested. The absorbance of the reaction product decreased with the increase in catalase quantity. 7 lg of catalase produced zero absorbance, suggesting this quantity of enzyme was sufficient to remove all H2O2 (Table 2). Thus 8 lg catalase was used as the standard blank assay. In order to determine if the above colorimetric reaction was effective for quantification of H2O2, known standard concentrations of H2O2 were assayed. Absorbance of the colorimetric products showed a highly linear positive correlation to a wide range of H2O2 concentrations (Fig. 3). For 0.1

Absorbance at 505 nm

0.30

y = 0.0023x2 - 0.0131x + 0.0155 R2 = 0.9577

0.08 0.06 0.04 0.02

1 0 2

3

4

5

6

7

8

9

10

Fig. 2 Effects of the adjusted pH of extracts from rice on absorbance of the reaction product. Fresh leaves of rice (Oryza sativa L.) were frozen in liquid nitrogen and ground in a mortar and pestle in 60 ml of 5% TCA. Activated charcoal was added to the homogenate and mixed thoroughly. The mixture was centrifuged. The supernatant was divided into aliquots of 3 ml and adjusted pH value from 3.6 to 9.0 with 17 M ammonia solution. Then the adjusted extract was filtered. The filtrate was colored by colorimetric reagent. Values represent the means of 3 replicates and bars indicate SE

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Table 2 Absorbance of colorimetric reaction product by the H2O2 solution added different amount of catalase Absorbance at 505 nm

0 1 3 5 7 8

0.175 0.079 0.028 0.011 0 0

0.008 0.004 0.001 0.001

y = 0.0023x + 0.1059 R2 = 0.9991

0.3 0.25 0.2 0.15 0.1 0.05

5% TCA containing 60 lM H2O2 was adjusted to pH 8.4 with 17 M ammonia solution. The adjusted TCA solution without H2O2 was served as a blank. The solution was divided into aliquots of 1 ml. 0, 1, 3, 5, 7 and 8 lg of catalase was added respectively to the aliquots and kept at room temperatures for 10 min, after which 1 ml of colorimetric reagent was added to the solution. Absorbance was determined at 505 nm. Values are means ± SE (n = 3) 1.2

0

B

0.4 0.35

Absorbance at 505 nm Ab

± ± ± ±

0.4 0.35

Absorbance at 505

Amount of catalase (lg)

A

y = 0.0018x + 0.126 R2 = 0.9997

0.3 0.25 0.2 0.15

B

0.1 0.05

Absorbance at 505 nm

1

0

y = 0.0027x - 0.0029 R2 = 0.9997

0

20

40

60

80

100

120

0.8

Conc Concentrations of hydrogen peroxide (µM) 0.6 0.4 0.2 0 0

50

100

150

200

250

300

350

400

Concentrations of hydrogen peroxide (µM)

Fig. 3 Standard curve of absorbance vs. hydrogen peroxide concentration. Values represent the means of 3 replicates and bars indicate SE

quantification of H2O2 in most of the plant material used here, we used a calibration range of 0–100 lM of H2O2. When H2O2 was added to the extracts of rice and litchi leaves after adjusted to pH 8.4 prior to quantification of H2O2, the absorbance of the colorimetric products was also highly linearly correlated to H2O2 concentration (Fig. 4). Ascorbic acid and reduced glutathione are important antioxidants and can scavenge H2O2 via ascorbate-peroxidase or react directly with H2O2 (Noctor and Foyer 1998). They can also be extracted from plant tissues in 5% TCA (Law et al. 1983; Zhou et al. 2005). The effect of ascorbic acid (2.5 lM) on the determination of H2O2 was to suppress absorbance by 52%. 10 lM of ascorbic acid was sufficient to make the colorimetric products undetectable (Table 3). This shows that

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Fig. 4 Absorbance of the colorimetric products vs. added hydrogen peroxide concentration in the extract of rice (A) and litchi leaves (B). Fresh leaves (2 g) were frozen in liquid nitrogen and ground in a mortar and pestle in 20 ml of 5% TCA. Activated charcoal was added to the homogenate and mixed thoroughly. The mixture was centrifuged. The supernatant was adjusted to pH 8.4 with 17 M ammonia solution. Then the adjusted extract was filtered. Hydrogen peroxide was added to the filtrate. The filtrate with 0–100 lM hydrogen peroxide was colored by colorimetric reagent. Values represent the means of 3 replicates and bars indicate SE

Table 3 Absorbance of colorimetric reaction product by the H2O2 solution in different concentration of ascorbic acid Ascorbic acid concentration (lM) Absorbance at 505 nm 0 2.5 4 10 12.5

0.175 ± 0.002 0.085 ± 0.003 0.056 ± 0.002 0 0

5% TCA containing 60 lM H2O2 was adjusted to pH 8.4 with 17 M ammonia solution. Then ascorbic acid was added to the solution to reach 2.5 lM, 5 lM, 10 lM and 12.5 lM, respectively and then mixed with colorimetric reagent. Absorbance of the colorimetric product was determined at 505 nm. Values are means ± SE (n = 3)

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Table 4 Recovery of H2O2 over a range from 60 to 120 lM after addition of activated charcoal H2O2 (lM)

Recovery (%)

60 120 180

68.6 ± 10 66.7 ± 1 60.4 ± 1

the quantification of H2O2, it is important to use a standard amount of activated charcoal. H2O2 concentration in leaves ranged from 0.2 to 0.8 lmol g–1 FW in eight plant species grown in greenhouse or field (Table 5), this is in consistence with estimated by other methods (Patterson et al. 1984; Vaidyanathan et al. 2003; Li et al. 2000; Fadzillah et al. 1996; Srivalli et al. 2003). H2O2 is known to accumulate in cells when plants are subject to environmental stresses (Fadzillah et al. 1996). H2O2 concentration in Stylosanthes guianensis leaves at 3 days of 28°C remained relatively constant, while at 38°C it had risen by heat stress (Fig. 5). 0.6

H 2O 2 concentration (µmol g -1 FW)

ascorbic acid in plant tissues extract interfered with H2O2 determination, and would lead to an under estimate of H2O2 concentration. Antioxidants, such as ascorbic acid and reduced glutathione, need to be removed from the assay medium. Activated charcoal was used by Patterson et al. (1984) during extraction of H2O2 to remove pigments and other interfering substances in plant extracts. Addition of activated charcoal into a solution of ascorbic acid (0.2 mM) or into alfalfa extracts produced assays in which ascorbic acid was undetectable, suggesting that ascorbic acid was completely removed by activated charcoal. However, some H2O2 was also removed by activated charcoal. H2O2 over a range from 60 to 120 lM was measured for its absorbance after addition of activated charcoal. A similar recovery of H2O2 solution (60–68%) with the addition of activated charcoal was observed (Table 4). To minimize experimental errors between samples in

0.5

28 ˚C

0.4

38 ˚C

0.3 0.2 0.1 0 0

1

2

3

4

5

6

7

8

Days after heat treatment

H2O2 was dissolved in 5% TCA and determined after it was mixed with activated charcoal (0.03 g per ml solution). Recovery was calculated by the ratio of H2O2 content after mixed with activated charcoal to that before mixed. Values are means ± SE (n = 3)

Fig. 5 Hydrogen peroxide content as affected by heat stress in Stylosanthes guianensis. Plants of 8-week old Stylosanthes guianensis were transferred into a growth chambers with a 12 h photoperiod at 160 lmol m–2 s–1 photosynthetic photon flux density at 38°C for heat stress treatment or at 28°C as control. Values represent the means of 3 replicates and bars indicate SE

Table 5 H2O2 concentrations in leaves of various plant species Species

H2O2 concentration (lmol g–1 FW)

Reference

Notes

Stylosanthes guianensis Litchi (Litchi chinensis Sonn.) Longan (Dimocarpus longana Lour.) Banana (Musa acuminata Colla (AAA)) Rice (Oryza sativa L.) Rice (Oryza sativa L.) Taro (Colocasia esculenta) Alfalfa (Medicaga sativa L.) Tobacco (Nicotiana tabacum L.) Wheat (Triticum aestivum L.) Passionfruit (Passiflora edulis) Mungbean (Vigna radiate) Spinach (Spinacia olearacea)

0.29 0.64 0.52 0.72 0.80 0.26 0.38 0.63 0.55 0.58 0.15 0.64 0.54

This study This study This study This study This study Li et al. (2000) This study This study This study Li et al. (2000) Patterson et al. (1984) Patterson et al. (1984) Patterson et al. (1984)

Fully expanded Fully expanded Fully expanded Fully expanded Fully expanded Young leaves Fully expanded Fully expanded Fully expanded Old leaves Fully expanded Fully expanded Fully expanded

± ± ± ± ± ± ± ± ± ± ± ± ±

0.05 0.06 0.05 0.05 0.06 0.02 0.03 0.04 0.01 0.05 0.01 0.05 0.04

leaves leaves leaves leaves leaves leaves leaves leaves leaves leaves leaves

Values are means ± SE (n = 3)

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In summary, a simple spectrophotometric method for determination of H2O2 in plant tissues was established. H2O2 was extracted from plant tissues according to Patterson’s published method, to this extract was added activated charcoal to remove interfering substances including pigments and soluble antioxidants, the colorimetric products were determined spectrophotometrically in the presence of 4-aminoantipyrine and phenol catalyzed by peroxidase. Acknowledgements The project was funded by grants from Guangdong Science and Technology Projects (2003C201018), Guangdong Provincial Natural Science Foundation (04105978, 5300846), and National Natural Science Foundation (30571283).

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