IJERD – International Journal of Environmental and Rural Development (2013) 4-2

Research articleerd

The Biochemical Substances in Plants on Salt Affected
Area in Northeast Thailand, Bamnet Narong District,
Chaiyaphum Province, Thailand
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
Received 15 December 2012

Accepted 10 June 2013(*Corresponding Author)

Abstract This study was aimed to examine biochemical substances and their relationships
in plants growing on salt affected area. The study took place in a very high salinity in
Bamnetnarong District, Chaiyaphum Province between the rainy season (August 2011)
and the dry season (April 2012). Two groups of plants were analyzed; halophytes and salt
tolerant species. The halophytes consisted of Azima sarmentosa, Maytenus mekongensis,
and Pluchea indica whereas the salt tolerance plant is Combretum quadrangulare. The
results of the biochemical substances analyzes and showed that in the dry season all theplants produced more the contents of proline, hydrogen peroxide, peroxidase activity andmalondiadehyde, and the percentage of electrolyte leakage than in the rainy season.
Additionally, in both two seasons, the plants in the halophyte group produced substancesless than in the salt tolerance plant. The best self-adjustment mechanism was discovered in
A. sarmentosa, which produced a small and stable quantity of substances even though theelectrical conductivity rose up.
Keywords important substances, halophytes, salt-tolerant species, salt affected area

INTRODUCTION
Salt stress leads to a series of morphological, physiological, biochemical and molecule changes thatadversely affect plant growth and productivity. In glycophytes, plant growth and development are
generally limited by salinity. The most of the word’s crop species are glycophytes, and they do not
grow under high soil salinity (Abbaspour, 2012). Salt tolerance is complex genetically andphysiologically. Tolerance often shows the characteristics of a multigenic trait, with quantitative
trait loci (QTLs) associated with tolerance identified in barley, citrus, rice, and tomato and with ion
trans-port under saline conditions in barley, citrus and rice (Flowers, 2004).
When plants are subjected to salt stresses, active oxygen species (AOS) are generated inresponse to stress condition. AOS include superoxide (O2-), hydroxyl radicals (OH-), hydrogenperoxide (H2O2) and singlet oxygen (1O2) are metabolic by products of plant cell (Abbaspour,2012). It is already known that these cytotoxic active oxygen species, which are also generated
during metabolic processes in the mitochondria and peroxisomes (Khan and Panda, 2008).
Under stresses, plants possess several antioxidant enzyme systems to protect their cell form
the negative effects of AOS, such as peroxidase (POX), catalase (CAT), superoxide dismutase(SOD), glutathione reductase (GR), and ascorbate peroxidase (Sevengor et al., 2011). Peroxidases© ISERD
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catalyze the subsequent breakdown of H2O2 water and oxygen Catalase is involved in scavenging
H2O2 generated during the photorespiration and β-oxidative of fatty acids. Super oxide dismutase
can convert O2- to H2O2 and then H2O2 is removed by ascorbate peroxidase and glutathione
reductase in the ascorbate-glutathione cycle. (Lu et al., 2006). Moreover, plants accumulatecompatible osmolytes such as proline, glycine betaine and sugar alcohols, when they are exposed todrought or salinity stress (Yoshiba et al., 1997). Verdoy et al. (2006) considered in higher plant,
proline is synthesized form both glutamic acid and ornithine. Nanjo et al. (1999) reported to manyorganisms, including higher plants, accumulate free proline in response to osmotic stress.
Salt tolerance is a complex phenomenon, but the specific mechanisms of salt tolerance in eachplants species, can grow in high soil salinity in Northeast Thailand is poorly defined. The
detrimental effects of high saline soil salinity on biochemical substances such as the contents ofproline, hydrogen peroxide, peroxidase activity and malondiadehyde, and the percentage ofelectrolyte leakage in both season (rainy and dry season) and their relationship in each plant grown
were determined.
METHODOLOGY
Study Area
The study took place in Bamnetnarong District, Chaiyaphum Province between the rainy season(August 2011) and the dry season (April 2012).

Fig. 1 The study area in Bamnetnarong District, Chaiyaphum Province between
the rainy season (August 2011) and the dry season (April 2012)
Plant and soil samples
Plants and soil samples were collected by stratified sampling method between the dry season (April2012) and the rainy season (August 2011). Two groups of dominate plants in the site study such as
the 4 halophytic plants (namely, Azima sarmentosa, Maytenus mekongensis, Pluchea indica) andthe salt tolerance plant (namely, Combretum quadrangulare) were selected.
Soil was sampled form root zone of each plant at width of 20 cm and at the depth of 15 cm
Electrical conductivity (EC) and soil pH were determined in 1:5 (soil to water solution) by using a
conductivity meter and a pH meter, respectively.
Electrolyte leakage (EL) was determined according to the method of Dionisio-Sese and Tobita(1998). The 0.1 g of fresh leaf samples was cut into 5 mm length and placed in test tubes
containing 10 ml of redistilled water. The tubes were incubated in a water bath at 32 °C for 2 h andthe initial electrical conductivity of the medium (EC1). The samples were autoclaved at 121 °C for
20 min to release all the electrolytes and the final electrical conductivity (EC 2) measured. The
percentage of EL was calculated by using the formula: EL (%) = (EC1/EC2) x100
Malondialdehyde (MDA) content was measured according to the method of Stewart andBewley (1980). The 0.1 g of fresh leaf samples was homogenized under ice-cold (liquid nitrogen)was extracted with 50 mM phosphate buffer (pH 7.0) and centrifuged at 12,000 rpm at 4 °C for 30min. As much as 1 ml of supernatant was then vortexed with 1 ml of 0.5% (w/v) thiobarbituric acid

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solution containing 20% (w/v) trichloroacetic acid. The mixture was heated at 95°C for 25 min.
The sample was cooled on ice for 10 min and centrifugated at 10,000 x g for 10 min. After
subtracting the non-specific absorbance at 532 and 600 nm, the MDA content was determined by
its extinction coefficient of 155 mM-1cm-1 and the concentration was express as mM g-1 FW.
Proline content was determined according to the procedures of Bates et al. (1973). Leaf
sample 0.1 g was homogenized in 5 ml of 3% (w/v) sulfosalicylic acid and the homogrnate wasfiltrated through Whatman No.2. One ml of the filtrate was pipette laced in a 10 ml test tube andreacted with 1 ml of acid ninhydrin mixture and placed in a water bath at 100 °C for 1 h. The
reaction was stopped on ice bath. The mixture was extracted with 2 ml. of toluene and stirred for 15sec. The toluene phase containing the chromophores was aspired, warmed to room temperature,and the absorbance was read at 520 nm. The proline content was determined using from a standard
curve and expressed as ug g-1FW.
Hydrogen peroxide content in the leaves was determined according to the methods 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. The homogenate 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. The absorbance of supernatant was measured
at 390 nm. The content of H2O2 was determined using a standard curve and the concentration wasexpressed as mole g-1 FW.
Peroxidase enzyme activity was determined using the guaiacol oxidation method (Chance andMaehly, 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 at470 nm within 30s (linear phase) after H2O2 was added. One unit of peroxidase activity wasexpressed as A470 min-1 mg protein-1
The protein was extracted by the method of Bradford (1976) using the Bio-Rad assay kit (BioRad Laboratories, USA) with bovine serum albumin (BSA) as standard.
ABC

DFig. 2 Leaf of dominant plants in Bamnetnarong District, Chaiyaphum Province
Azima sarmentosa (A) Maytenus mekongensis (B), Pluchea indica (C), Combretum quadrangulare (D)RESULTS AND DISCUSSION
Electrical conductivity and pH of soil
The EC in the rainy season and the dry season in the halophytes were ranged from 1-22 and 36-147
dS/m respectively, while in salt tolerance plant was ranged from 0.4-3.0 and 28-76 dS/m (Fig.1 AC). During the rainy season the electrical conductivity was lower than in the dry season. In therainy season, the rain will wash off salt of the surface. Soil pH in the rainy season and the dry
season in the halophytes were ranged from 5.35-8.38 and 4.56-8.11, respectively, while in salt
tolerance plant was ranged from 6.00-7.61 and 5.96-6.82 (data not shown).
Effect of soil salinity on electrolyte leakage
The effect of soil salinity on membrane integrity was monitor means of EL test in fresh samples.
The amount of EL out of the cells was assessed indirect by conductometric measurements. EL of© ISERD
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the halophytes in the rainy season and in the dry season was in the range of 11-36% and 24-55%,respectively, whereas EL of salt tolerance plant (C. quadrangulare) was in the range of 15-24%
and 21-45%, respectively (Fig. 3A-C). In both season, EL increased with increasing soil salinity
level. Similar changes in the level of lipid peroxidation and electrolyte leakage in response to NaCl
stress have been reported by several authors. Dhindsa et al. (1981) reported that there are similarchanges in the level of lipid peroxidation and in electrolyte leakage in tobacco leaves. Lutts et al.
(1996) also found a significant positive correlation between MDA and EL.
Rainy season
Dry season

Fig. 3 The effect of soil salinity on electrolyte leakage in leaves of 4 plants
Azima sarmentosa (A), Maytenus mekongensis (B), Pluchea indica (C), Combretum quadrangulare (D),Vertical bars represent mean + S.E. (n = 5)

Effect of soil salinity on lipid peroxidation
A product of lipid peroxidation in the leaves of 4 plants was assessed as the content of MDA. In therainy season, the content of MDA in the halophytes ranging from 0.3-4 mM/gFWx10-3 and in thesalt tolerance group produced MDA ranging from 1-5 mM/gFWx10-3 is more than in the halophyte
group. The dry season in the salt tolerance group produced MDA more than in the halophyte group.
In the dry season salt tolerance group produced MDA more than the rainy season. Due to the higher
electrical conductivity of the soil affect the oxidation increases as shown in Fig. 4.
These result is support by previous reports such as by Khan and Panda (2006) studied theeffect of NaCl stress (50, 100 and 150 mmol l-1). They found that the salt-tolerant Lunishreeshowed higher MDA than the salt-sensitive Begunbitchi. Indicating a higher rate of MDA inBegunbitchi due to salt stress MDA with the increase in salt stress.

Fig. 4 The effect of soil salinity on malondialdehyde content (mM gFWx10-3) in leaves of4 plants
Azima sarmentosa (A), Maytenus mekongensis (B), Pluchea indica (C), Combretum quadrangulare (D),Vertical bars represent mean + S.E. (n = 5)

Effect of soil salinity on proline
Proline is an osmoprotectant that has been show to accumulate in plants in response to salt stress.
Earlier studies form this showed that there is accumulation of proline in 4 plant species. In
concurrence with this (Fig. 5), our results show that in the rainy season, proline content in thehalophyte group and in the salt tolerance group were similar ranging from 1-15 µg g-1 FW.
Whereas, in the dry season, the salt tolerance plant accumulate proline more than in the halophyte
group. The results are in accordance to other findings such as by Lin and Kao (1996) studied theeffect of NaCl stress (0, 50 and 150 mM) on root rice accumulation of proline when theconcentration of the solution increases.

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IJERD – International Journal of Environmental and Rural Development (2013) 4-2Fig. 5 The effect of soil salinity on proline content (µg/gFW) in leaves of 4 plants
Azima sarmentosa (A), Maytenus mekongensis (B), Pluchea indica (C), Combretum quadrangulare (D),Vertical bars represent mean + S.E. (n = 5)

Effect of soil salinity on peroxidase enzyme activity
The present results on increase in the peroxidase enzyme activity are shown in Fig. 6. Peroxidase
enzyme activity in the rainy season plants in the halophyte group with peroxidase enzyme activity
ranged from 0.1-1470 min mg g-1 protein. Plants in the salt tolerance group ranged from 0.1-4
470 min mg g-1 protein. In dry season plants in the halophyte group with peroxidase enzyme
activity ranged from 0.1-4 470 min mg g-1 protein and salt tolerance plants ranged from 0.1-4
470 min mg g-1 protein. When plants are under stress to reduce stress by creating antioxidants,antioxidant enzymes to prevent damage from ROS to the plant cell such as catalase, glutathione
reductase, superoxide dismutase and ascorbate peroxidase when compared with the controlledplant.

Fig. 6 The effect of soil salinity on peroxidase enzyme activity (Δ470min mg-1protein) in leavesof 4 plants
Azima sarmentosa (A), Maytenus mekongensis (B), Pluchea indica (C), Combretum quadrangulare (D), Verticalbars represent mean + S.E. (n = 5)

Effect of soil salinity on hydrogen peroxideFig. 7 The effect of soil salinity on hydrogen peroxide content (µmol g-1FW) in leavesof 4 plants
Azima sarmentosa (A), Maytenus mekongensis (B), Pluchea indica (C), Combretum quadrangulare (D), Verticalbars represent mean + S.E. (n = 5)

Many authors has been reported that reactive oxygen species, including superoxide and hydrogenperoxide, are elevated with increased salinity, due to the imbalance in the production anddestruction of reactive oxygen species (Uchida et al., 2002; Vaidyanathan et al., 2003) Fig. 7 showthat the hydrogen peroxide level was increased with increasing salinity in this similar in 4 plantspecies. This result of hydrogen peroxide in the dry season than in the rainy season and halophytes© ISERD131

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group increased hydrogen peroxide than the salt tolerant species of soil salinity was increased. This
result supported the previous report of Vaidyanathan et al. (2003), who studied the effect of NaClstress (100-300 mM) on two rice cultivars differing in salt tolerance. They found that the salttolerant Pokkali showed higher activity of catalase and lower levels of H2O2 than the salt-sensitive
Pusa Basmati 1.
CONCLUSION
In summary, with increasing soil salinity, all plants showed a remarkable increase in the percentageof EL, the contents of proline, MDA, hydrogen peroxide, and peroxidase enzyme activity. Theability of salt tolerance in each plant depend on it's ability to produce biochemical substances, helps
that it can be grow and survive in different soil salinity level. The results showed that A.
sarmentosa is the best one that can be grown on high soil salinity upto 147 dS/m, while the otherplants can grow in soil salinity not over than 102 dS/m. One of the characteristics of thick andglossy leaves of A. sarmentosa can be reduce water loss and maintain water status. In the future A.
sarmentosa may be used to improve saline soil structure and used as a renewable energy such as
wood for firewood.
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.
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