IJERD – International Journal of Environmental and Rural Development (2012) 3-1

Research article
erd

Hydrogen Peroxide and Peroxidase Activity as Potential
Indicators on Adaptability of Plants to Salinity StressCondition
NARARAT POODEETIP
Faculty of Science, Khon Kaen University, Khon Kaen, Thailand

KANLAYA KONG-NGERN*
Faculty of Science, Khon Kaen University, Khon Kaen, Thailand
Email: kkanla@kku.ac.thSAMANG HOMCHUEN
Faculty of Science, Khon Kaen University, Khon Kaen, Thailand

BUPHA TOPARKNGAM
Faculty of Agriculture, Khon Kaen University, Khon Kaen, Thailand

VIDHAYA TRELO-GES
Faculty of Agriculture, Khon Kaen University, Khon Kaen, Thailand
Received 15 December 2011

Accepted 12 March 2012(*: Corresponding Author)

Abstract The purpose of this study is to investigate whether hydrogen peroxide content aswell as peroxidase enzyme activity in the leaves of the cultivars can be used asindicatorsof the adaptability of plants to salinity stress conditions. For this purpose, leaves of 6
cultivated plant species: Cassaia siamea, Acacia auriculiformis, Acacia magium,
Pithecellobium dulce, Combretum quadrangulare and Albizia lebbeck were collected frompreviously high salinity areas where the salinity of soil have been improved by plantcultivation, and the relationships between soil characteristics, plant species and the content
of hydrogen peroxide and peroxidase enzyme activity were examined. The results showthat C. quadrangulare has low hydrogen peroxide content and high peroxidase enzyme
activity compared to other plants at the same electrical conductivity (0.16 dS m-1),
suggesting that this plant has less stress than the others. In other words, C. quadrangulare
can adapt to high salinity stress conditions better than the others. A.magium andA.auriculiformi are the second best. Based on the present results, C. quadrangulare is thebest promising species for growing on high salinity areas.
Keywords salt affected area, hydrogen peroxide, peroxidase enzyme activity

INTRODUCTION
Soil salinity is one of the most significant abiotic stress factors that decrease productivity. High salt
concentration in soil or in water affects plant growth, nutrient uptake and metabolism through thedecrease in the amount of water available for plants, through ion imbalance or disturbances in ion
homeostasis by disturbance of essential intracellular ion concentrations, and through ion toxicity
due to excessive Na+ or Cl- uptake (Greenway and Munns, 1980; Gossett et al., 1994; Zhu, 2001;
Parida et al., 2004). An excess of exchangeable Na ion is harmful to plants, principally because it
induces undesirable physical and chemical conditions of the soil. In addition, Na influences soilstructure, resulting in a decrease of water infiltration and gas exchangeability. Salinity affects thegrowth of plants by decreasing the availability of water to the roots due to the osmotic effect ofexternal salt and by toxic effects of excessive salt accumulation within the plant. Drastic changes inⒸ ISERD85

IJERD – International Journal of Environmental and Rural Development (2012) 3-1

ion and water homeostasis lead to molecular damage, growth arrest and even death (Munns, 1993;
Munns et al., 1995).
There is now conclusive evidence that production of reactive oxygen species (ROS) isenhanced in plants in response to different environmental stress conditions such as salinity, drought,
water logging, temperature extremes, high light intensity, herbicide treatment and mineral nutrientdeficiency. The most stable ROS is hydrogen peroxide (H2O2), which is highly reactive and cancause serious oxidative damages to membrane lipids leading to loss of membrane integrity andincrease in electrolyte leakage and cell death. ROS were considered to be toxic by-products ofaerobic metabolism (Wise and Naylor, 1987; Mittova et al., 2000; Zhu, 2001; Mittova et al., 2002;
Slesak et al., 2008).
Salt tolerant plants maintain their growth and have many mechanisms to maintain ionichomeostasis, osmotic homeostasis and detoxification pathways (Dubey, 1994; Zhu, 2001; Paridaand Das, 2004). It is known that plants containing high concentration of antioxidants show
considerable resistance to the oxidative damage caused by   free radicals (Ashraf and Harris, 2004;
Parida and Das, 2004; Slesak et al., 2008).
Several authors have discussed the effect of salt stress on the amount of H2O2. One mechanismthat underlies the tolerance of plants to salt stresses is the ability of plants to detoxify ROS andscavenging systems by antioxidative pathways. The salt-tolerant plant presented lower increase in
the amounts of ROS and antioxidant enzymes than the salt-sensitive plants. Therefore, theincrement in ROS such as H2O2 content and antioxidant enzymes activity in leaves depended upon
the levels of salt tolerance (Dubey, 1994; Zhu, 2001; Parida and Das, 2004; Slesak et al., 2007).
Chen et al. (1993) and Duand (1997) proposed that peroxidase and catalase are two major systems
for the enzymatic removal of H2O2 in plants. Until now, the information about the biochemicalparameters of each plant that can grow on high salinity soil is insufficient. Therefore, in this study,
we use H2O2 and peroxidase enzyme as potential indicators to detect the ability of plants to growon high salinity areas.
METHODOLOGY
Soil and plant samples were collected in May, 2010 at the area near Akkrasathsoonthorn Reservoir,
Somsanuk Village, Borabue District, Mahasarakam Province, where the high salinity of soil has
been improved by tree plantation during the past 2 years. The study area at the beginning wasclassified into high salinity (6.23 dS m-1) and very high salinity (18.9 dS m-1) areas. After 2 years oftree plantation, the salinity of both areas decreased to slight salinity (< 1 dS m-1).
Electrical conductivity and pH of soil were measured by using a conductivity meter and a pH
meter, respectively, based on a soil-water ratio of 1:5 by weighing10 g of soil in a beaker. The soil
sample was left for 30 minutes to measure the electrical conductivity and for an hour to measure
pH value.
Hydrogen peroxide content in the leaves was determined according to the method of Sergiev
et al. (1997). Leaf tissues (0.5 g) were homogenized with 5 ml of 0.1% (w/v) trichloroacetic acid(TCA) on an ice bath. Crude extract was centrifuged at 12,000 x g for 15 min and 0.5 ml of thesupernatant was transferred to a 15 ml test tube. The supernatant was added with 0.5 ml of 10 mM
potassium phosphate buffer (pH 7.0), 1 ml of 1 M KI, and mixed by vortexing briefly. Theabsorbance of mixture was read at 390 nm. A mixture without the supernatant served as a blank.
The content of H2O2 was calculated from a standard curve and the concentration was expressed asmole g-1 fresh weight.
Peroxidase activity was determined using the guaiacol oxidation method (Chance and Maehly,1955) in a 3 ml reaction mixture containing 10 mM phosphate buffer (pH 6.4), 8 mM guaiacol,
100-200 l crude extract and 2.75 mM H2O2. The increase in absorbance was recorded at 470 nm
within 30 s (linear phase) after H2O2 was added. One unit of peroxidase activity was expressed as
A470 min-1 mg protein-1.

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Soluble protein content of the crude extract was determined according to the method ofBradford (1976) using the Bio-Rad assay kit (Bio-Rad Laboratories, USA) with bovine serum
albumin (BSA) as a calibration standard.
RESULTS AND DISCUSSION
In the present study, the H2O2 content and the peroxidase activity in the leaves of 6 plant species
harvested from various salinity areas were measured. With increasing EC value in soil, salinity had
a significant effect onthe amounts of H2O2 and peroxidase enzyme activity in all 6 cultivated plant
species. The data show that the leaf H2O2 contents in 6 cultivated plant species were increased anddifferent (Fig. 1).

Fig. 1 The effects of salinity of the soil on hydrogen peroxide content and peroxidase
activity in leaves of 6 plants.

Combretum quadrangulare (A), Pithecellobium dulce(B), Albizia lebbeck (C), Acacia auriculiformis (D),Cassaia siamea (E), Acacia magium (F)With the increase of EC value in soil, the relation between salt tolerant plants and the amountof H2O2 and peroxidase enzyme activity under salt stress can be classified into 2 groups: group I(less responsive to salt stress than group II) consisting of 4 plant species: C. quadrangulare, A.
auriculiformis, C. siamea, and A. magium, that showed a slight increase in both the amount of H2O2
and the peroxidase activity; and group II (more responsive to salt stress than group I) consisting ofP. dulce and Albizia lebbeck, that showed increase in both H2O2 content and peroxidase activity inleaves. The result indicated that C. quadrangulare is the most tolerant plant based on its ability toⒸ ISERD87

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maintain the lowest H2O2 content value (ranging from 0.2 - 0.3 mol/g FW) when compared theothers. Besides, it showed the lowest peroxidase enzyme activity (ranging from 1-3 units min-1 mg1protein). Thus, the ability of salt tolerance in C. quadrangulare did not depend on the peroxidase
activity. This means it may be equipped with some mechanisms which help protect it from H2O2.
These results support previous reports such as Fadzilla et al. (1997) that described an increaseof H2O2 production, occurred gradually in response to salt stress in rice plants. In addition, Uchidaet al. (2002) reported that the most tolerant cultivar (Pokkali) had a lower level of H 2O2 than thesalt-sensitive Pusa Basmati 1. Also, Vaidyanathan et al. (2003), studied the effect of NaCl stress(100-300 mM) on two rice cultivars differing in salt tolerance. They found that the salt-tolerant
Pokkali showed higher activity of catalase and lower levels of H2O2 than the salt-sensitive Pusa
Basmati 1.
Moreover, in the group I, C. siamea also showed a slight increase in both the amount of H2O2
(ranging from 0.2 - 0.5 mol/g FW) and the peroxidase activity (ranging from 2 - 4 units min-1 mg-1protein) with increasing EC value in soil. In contrast, in A. magium, the H2O2 content and theperoxidase activity increased with salinity levels, but the peroxidase activity decreased at 0.16 dS
m-1 and 0.93 dS m-1values. The result indicated that it has low efficiency to produce peroxidase
activity when it grows on high salinity areas. While, in A. auriculiformis, though the H2O2 content
and the peroxidase activity increased with salinity levels, the H2O2 content decreased at 0.16 dS
m-1. This means that A. auriculiformis has high efficiency to produce antioxidant enzyme todetoxify H2O2. Also, when comparing to the others in group I, A. auriculiformis is the best

species for growing on high salinity areas.
Lesstolerant plant

Albizia lebbeck
Pithecellobium dulce
Acacia auriculiformis
Cassaia siamea
Combretum quadrangulareMoretolerant plant

Acacia magium

H2O2(µmol g-1FW)

Peroxidase activity(Δ470min  mg-1protein)

Fig. 2 The effects of salinity of the soil (0.16 dS m-1) on hydrogen peroxide content andperoxidase activity in leaves of 6 plants
When comparing the amount of both parameters at the same electrical conductivity (0.16 dSm ) in the leaves of 6 plant species (as shown in Fig.2), the amount of H2O2 and peroxidase
enzyme activity may be depending upon the levels of salinity in soil and also depending on theplant species.
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CONCLUSION
The results presented here revealed that salt affected both the content of hydrogen peroxide and theactivity of peroxidase in the cultivated plant species. This study confirms that with increasing ofEC value in soil, halophytes do not accumulate H2O2 uniformly. Both biochemical parameters may
be used as potential indicators to detect the ability of plants to survive and grow in their respective
areas. This study also indicated that hydrogen peroxide content and peroxidase activity were clearly
related to the ability of survival in each plant and the ability to detoxify H2O2, respectively. Insummary, the accumulation of both important substances in plants could be used as an index toⒸ ISERD88

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indicate the salt tolerance of plant and the best promising species to improve saline soil and toserve as plant energy was C. quadrangulare.
ACKNOWLEDGEMENTS
This work was supported by the research group of Soil Problem in Northeast Thailand, and theresearch group of Integrated Studies on Ecology and Classification of Saline Soils for Sustainable
Agriculture and Environment, Thailand.
We wish to acknowledge the support of the Khon Kaen University Publication Clinic,
Research and Technology Transfer Affairs, Khon Kaen University.
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