ANTIOXIDANT ACTIVITIES IN INDICA RICE (Oryza sativa L.) SEEDLINGS DURING SALINITY TREATMENT Sutee Chutipaijit, 1, * Kanokporn Sompornpailin 1,2

Transcription

1 K_K4 1 ANTIOXIDANT ACTIVITIES IN INDICA RICE (Oryza sativa L.) SEEDLINGS DURING SALINITY TREATMENT Sutee Chutipaijit, 1, * Kanokporn Sompornpailin 1,2 1 College of Nanotechnology, King Mongkut s Institute of Technology Ladkrabang, Bangkok 152, Thailand 2 Thailand Center of Excellence in Physics, CHE, Ministry of Education, Bangkok 14, Thailand * kchsuthe@kmitl.ac.th Abstract: The responses of the total antioxidant activities of indica rice to salinity were investigated in 3 tolerant varieties (KDML15, SY, and KD) and 4 sensitive varieties (RD15, KS, KK and PTT1). The relative growth rate and ion leakage content of salt-tolerant varieties were slightly affected by exposed to 1 mm NaCl for 2 and 4 days but those of saltsensitive varieties were significantly affected. The determination of total antioxidant activities by ABTS and DPPH assays in rice grown under salinity presented that salt- tolerant varieties contained higher total antioxidant activities than those of salt-sensitive varieties. The results indicated that higher capacities of the total antioxidant activities under stress were associated with tolerance to salinity in indica rice seedlings. Introduction: Salinity is the most significant factor restricting plant growth and productivity in the agricultural fields. 1 Plants are subjected to salt stress which can cause both osmotic and ionic toxicity effects on plant cells. These effects are known to induce secondary stress in plants by forming reactive oxygen species (ROSs) such as superoxide (O - 2 ), hydrogen peroxide (H 2 O 2 ), hydroxyl radicals ( OH) and singlet oxygen ( 1 O 2 ). The disturbance in equilibrium between the production of ROSs by various abiotic stresses and the scavenging of ROSs leads to enhance the levels of ROSs. ROSs may initiate the destruction of oxidative processes such as lipid peroxidation, chlorophyll bleaching, protein oxidation, and damage to nucleic acids. However, enzymatic antioxidants such as superoxide dismutase (SOD), glutathione reductase (GR), catalase and peroxidase, and/or non-enzymatic antioxidants such as ascorbic acid, glutathione, α-tocopherol, flavonoids and carotenoids play a role in scavenging ROSs. 2 Therefore, an alteration of the activities of both antioxidant groups may be an important factor in the tolerance of various plants to abiotic stresses including salinity treatment. The correlation between abiotic stress tolerance and enzymatic/non-enzymatic antioxidant capacities has been studied in various plant species. 3,4 Rice is one of the important crops worldwide, and is also considered to be a model plant for monocot plants. The aim of this research was investigated the relationship of growth rate and ion leakage tolerance and the total antioxidant activities (ABTS and DPPH assay) in indica rice seedlings grown in salinity. Methodology: Seeds of seven rice varieties, Khao Dawk Mali 15 (KDML15), Sangyod (SY), Khaodang (KD), Rice Department 15 (RD15), Kum Sakon Nakhon (KS), Kum Khon Kaen (KK) and Pathum Thani 1 (PTT1), were disinfected with 7% ethanol for 3 min, soaked with 5% commercial beach (5.25% sodium hypochlorite) for 4 min, 3% commercial beach for 3 min and then washed thoroughly with distilled water. After the sterilization, seeds were placed onto NB medium 5 containing 3 gl -1 sucrose and 8 gl -1 agar. They were germinated under 25±2 C air temperature, 85-9% relative humidity and 6±5 µmol m -2 s -1 photosynthetic photon flux with 16 h photoperiod. The seedlings were grown in this condition until 7 days. The rice seedlings were grown in NB-liquid medium under photoautotrophic condition. 6 After 7 days; seedlings were treated with NB-liquid medium

2 K_K4 2 containing 1 mm NaCl and maintained for 2 and 4 days in this condition in comparing to their seedlings grown in NB medium without NaCl. The relative growth rate were determined using the method described by Noggle and Fritz (1976). 7 The relative ion leakages were performed according to the method of Yan and co-worker (1996). 8 The rice seedlings were ground by liquid nitrogen. The powder was extracted with acidic methanol/water (3:2) and the extraction was thoroughly shaken at room temperature for 2 h. After that, chloroform was added for the chlorophyll elimination. The homogenates were centrifuged at 1, rpm at 4 C for 1 min. The supernatants were immediately measured for the total antioxidant activities. The determinations of the total antioxidant activities by ABTS assay were based on the method of Re et al. (1999). 9 The ABTS + scavenging activities were expressed on the fresh weight (FW) basis as µm Trolox equivalent g -1 FW. The DPPH assay was based on the method of Brand-Williams et al. (1995). 1 The half maximal inhibitory concentration (IC 5 ) value was calculated which denotes the volume of extract required to scavenge 5% of DPPH free radicals. A completely randomized design was used with five replicates per treatment (n=5). The data were presented the mean values ±standard deviation (S.D.). Results, Discussion and Conclusion: The effects of salinity on seven varieties of indica rice were determined the growth rate and ion leakage of their seedlings by comparing to control untreated treatment. In Figure 1 and 2 presented that addition of 1 mm NaCl in NB basal medium significantly retarded the growth rate and enhanced ion leakage in all rice varieties. The growth rate retardations of KDML15, SY and KD varieties induced by NaCl treatment were 4-18% which less affected than that of RD15, KS, KK and PTT1 varieties (5-55%). Furthermore, the relative ion leakages of the KDML15, SY and KD seedlings were 15-11% lower than those of RD15, KS, KK and PTT1 seedlings, which were %, compared to their non-nacl treated controls (Figure 2). These results indicated that the seedlings of KDML15, SY and KD varieties showed a high degree of tolerance to NaClmediated stress at 2 and 4 days, as compared to the seedlings of RD15, KS, KK and PTT1 varieties. A reduction in growth rates and increase of ion leakage contents in the NaCl stressed-seedlings have previously been observed in thyme 11, barley 12 and rapeseed. 13 Relative growth rate (day -1 ) KDML15 SY KD RD15 KS KK PTT1 Figure 1. Effect of salinity (1 mm NaCl) on relative growth rate of rice seedlings under photoautotropic condition for 2 and 4 days. Values are mean ± S.D. of five replicates.

3 K_K4 3 The total antioxidant activities are represented by a series of antioxidant molecules that the plant uses against ROS formation. 14 ABTS and DPPH assays were used to provide information on the total antioxidant activities of abiotic stressed-seedlings. In this research, these assays showed significantly high antioxidant activities under the treatment with 1 mm NaCl as compared to their non-nacl treated controls (Figure 3A and B). The total antioxidant activities of the non-stressed and stressed seedling extracts were equivalent to µm Trolox g -1 FW. In our study showed that the extractions from KDML15, SY and KD stressed-seedlings trend to increase higher ABTS + radical scavenging activity (62-15%), than the RD15, KS, KK and PTT1 stressed-seedlings extracts (18-36%). Relative ion leakage (%) KDML15 SY KD RD15 KS KK PTT1 NaCl 2 days NaCl 4 days Figure 2. Effect of salinity (1 mm NaCl) on relative ion leakage of rice seedlings under photoautotropic condition for 2 and 4 days. Values are mean ± S.D. of five replicates. The antioxidant extraction of stressed-seedlings was performed the DPPH assay. This result had the similar trend activities according to the ABTS assay. The IC 5 values of DPPH assay ranged from µl g -1 FW. The IC 5 values of KDML15, SY and KD stressedseedling extracts were decrease by 13-17% while the RD15, KS, KK and PTT1 stressedseedling extracts were decrease by 3-5% as compared with their extracts of non-stressed controls. The extractions of KDML15, SY and KD stressed-seedlings were found to have the higher antioxidant activities in both ABTS and DPPH assays than the RD15, KS, KK and PTT1 stressed-seedling extracts. The results of these radical scavenging assays (ABTS and DPPH assays) indicated that the high-performance extracts might prevent reactive radical species from damaging such biomolecules as lipoproteins, polyunsaturated fatty acids, DNA, 15, 16 amino acids, proteins, and sugars in susceptible biological systems. In conclusion, this study showed that 1 mm NaCl concentration in the culture medium can provoke oxidative damage in seedlings of indica rice. In salt stress condition, the relative growth rates of salt-tolerant varieties were higher than those of salt-sensitive varieties and also the ion leakages of plant cell were lower in salt-tolerant seedlings than in saltsensitive seedlings. The high increase rates of the total antioxidant activities in salt-tolerant varieties were observed under salinity condition. The data also suggested that the total antioxidant activities could protect against oxidative stress by salinity in plant cell. We found that KDML15, SY and KD varieties were tolerance to the administered salt stress, protecting itself from oxidative damage by high increasing the total antioxidant activities in stressed-seedlings. These factors could be used as biochemical parameters for screening of salt tolerance in indica rice varieties or the other plant species, when selecting salt tolerant varieties for breeding in the future.

4 K_K4 4 6 um Trolox g -1 FW A KDML15 SY KD RD15 KS KK PTT1 IC 5 (ul) B KDML15 SY KD RD15 KS KK PTT1 Figure 3. Effect of salinity (1 mm NaCl) on the total antioxidant activities using ABTS (A) and DPPH (B) assays of rice seedlings under photoautotropic condition for 2 and 4 days. Values are mean ± S.D. of five replicates. References: 1. Mansour MMF, Salama KHA, Ali FZM, Abou Hadid AF. Gen Appl Plant Physiol 25;31: Hussain TM, Chandrasekhar T, Hazara M, Sultan Z, Saleh BK, Gopal GR. Biotechnol Mol Biol Rev 28;3: Gracia NAT, Iribarne C, Palma F, Lluch C. Plant Physiol Biochem 27;45: Hoque MA, Banu MNA, Nakamura Y, Shimoishi Y, Murata Y. J Plant Physiol 28;165: Li L, Qu R, De Kochko A, Frauquet C, Beachy RN. Plant Cell Rep 1993;12: Chutipaijit S, Cha-um S, Sompornpailin K. Aus J Crop Sci 211;5: Noggle GR, Fritz GJ. New Jersey: Prentice-Hall, Yan B, Dai Q, Liu X, Huang S, Wang Z. Plant Soil 1996;179:

5 K_K Re R, Pellegrini N, Proteggente A, Pannala A, Yang M, Rice-Evans C. Free Rad Biol Med 1999;26: Brand-Williams W, Cuvelier ME, Berset C. Lebensmittel Wiss Technol 1995;28: Najafian S, Khoshkhui M, Tavallali V, Saharkhiz M.J. Aus J Basic Appl Sci 29;3: Yang CW, Xu HH, Wang LL, Liu J, Shi DC, Wang DL. Photosynthetica 29;47: Farhoudi R. Adv Environ Biol 211;5: Dominguez-Perles R, Martinez-Ballesta MC, Riquelme F, Carvajal M, Garcia- Viguera C, Moreno DA. J Sci Food Agric 211;91: Arnao MB, Cano A, Acosta M. Food Chem 21;73: Falleh H, Jalleli I, Ksouri R, Boulaaba M, Guyot S, Magné C, Abdelly C. Plant Physiol Biochem 212;52:1-8. Acknowledgements: This research was kindly supported by grant No. KREF1541 from King Mongkut s Institute of Technology Ladkrabang Research Fund. The authors are grateful to Pathum Thani Rice Research Center, Udon Thani and Roi Et Rice Seed Center, for providing rice seeds. Keywords: salinity, rice, antioxidant, ABTS, DPPH