Antioxidant defence system and oxidative damages in Rice seedlings under heat stress

Impact of climate change showed heat stress most severely affecting crop growth and yield worldwide. In current study, oxidative damages and response of antioxidant enzymes were compared in 4 rice cultivars (IR-6, IR-8, K-95 and Sada Hayat). These varieties were exposed to heat (42±2 oC) stress at seedling stage. After 72 h, stressed plants transferred to normal temperature for recovery treatments. Oxidative stress damages were quantified through analysis of hydrogen peroxide (H2O2) and malondialdehyde (MDA) content. Effect of heat stress on antioxidant enzymes activities were determined by quantitative analysis of ascorbate peroxidase (APX) and catalase (CAT) enzymes. With increasing oxidative stress significant proline accumulation was detected in rice cultivars and K-95 had maximum proline among the group. Significant increment of MDA and hydrogen peroxide (H2O2) content was found in as compared to others. CAT and APX showed significantly declined activity in Sada Hayat and IR-8 under heat stress condition however K-95 showed maximum activity. These findings suggesting that (i) K-95 is comparatively thermotolerant cultivar (ii) thermo-tolerance in rice is closely related to efficient antioxidant enzyme defence system.


Introduction
Abiotic and biotic stresses are major challenges for sustainable agriculture that posing severe threat to maximum yield through limiting plant growth and development [1].Specifically, abiotic stress is the primary cause of crop yield loss worldwide [2].Yield of major economically important crops like wheat, maize and rice are affected by heat stress [3].It is reported that for a 1 °C increase in daily mean temperature from threshold nearly 10% rice yield decreased [4].Some specific effects are reported for each stress factor on plants but oxidative stress is common in all stress conditions due to over production and accumulation of reactive oxygen species (ROS).Over production of ROS are considered as deleterious and lethal to living systems because these are responsible of carbohydrates, lipids, proteins and deoxyribonucleic acid peroxidation leading cell necrosis.Consequently, higher level of free radicles causes a cellular oxidation though a chain reaction.In addition to the oxidative stress related production [5].
Plants have developed an antioxidant defence system and they play key role in detoxifying and balancing ROS in cell [6].Enzymatic antioxidants including superoxide dismutase (SOD), ascorbate peroxidase (APX), catalase (CAT) and peroxidase (POX) [7,8].Among these antioxidant enzymes, SOD is considered as first defence line because it detoxifies superoxide which is the first reactive oxygen species, generate in cell during stress condition [9].DNA, Protein and lipids are at high risk due to increased production and accumulation of free radicals under oxidative stress [10,11]

Plant materials, growth conditions, and stress treatments
The seeds of rice (Oryza sativa L.) cultivars (IR-6, IR-8, K-95 and Sada Hayat) were collected from Rice Research Institute, Dokri, (Pakistan).After surface sterilization, rice seeds were allowed to germinate in pots.Seedlings grown in a controlled growth chamber (28 ± 2 °C) with 16 h light (400 μmol m -2 s -1 ) photoperiods and 8 h dark.For irrigation Hoagland's solution (half strength) was used [20].Then plants (20day-old seedlings), growing under normal temperature was considered as control (C), were subjected to heat stress (HS) by gradually increasing the temperature and maintained at 42± 2 °C.Sampling were carried out after 24, 48 and 72 h of heat stress.After 72 h, stressed plants transferred to normal (28 ± 2 °C) temperature for recovery treatments (RC).For control treatment, rice seedlings allowed to grow in the same way and watered with properly in addition of Hoagland's solution supplement.Before investigation of physiological attributes, seedlings were harvested, shoot lengths, fresh and dry weights of shoots were observed.Analysis of MDA and hydrogen peroxide content were determined followed by total quantification of catalase (CAT) and ascorbate peroxidase (APX) enzymes.

Growth parameters
To show that heat stress effects on rice growth visually, total shoot lengths were recorded followed by photography of samples.Fresh shoot and dry weights were measured as weight of shoot tissue.Shoot and root samples were taken from rice seedlings treated with control, heat stress and recovery conditions, for analysis of fresh weight (FW) at 30 days of rice growth.For dry weights (DW) analysis rice seedling samples were kept in oven at 60 °C for 24 h.

Hydrogen peroxide (H2O2) content determination
The production level of H2O2 was measured spectrophotometrically according to Jessup [21].H2O2 was extracted by homogenizing 0.1g fresh leaves in 1ml of 0.1% TCA.The homogenate was centrifuged at 12000 rpm for 15 minutes.To quantify H2O2 concentration, 0.5 ml of potassium phosphate (10 mM) and 1 ml of potassium iodide (1M) was mixed with supernatant.Supernatant yellow color intensity was measured 390 nm using UVspectrophotometer.Extinction coefficient (0.28 µmole-1 cm -1 ) was used to calculate concentration of H2O2 in controlled and heat stressed rice seedlings and expressed as µmole g 1 FW.

Determination of malondialdehyde (MDA) content
Strength of lipid peroxidation was measured by quantification of total melondialdehyde (MDA) content generated by heat stress treatments for the evaluation of cell membrane damages, according to Ohkawa [22].Leaf tissues (0.1 g) was homogenized in 1 ml of 5% trichloroacetic acid (TCA).The homogenates subjected was then centrifugation for 15 at 12,000 rpm.Supernatant was mixed with freshly prepared thiobarbituric acid (TBA, 0.5%) in tricholoroacitic acid (TCA, 20%) and incubated for 30 min at 95 °C into new eppendorf tube followed by centrifugation at 10,000 rpm.Correction for un-specific turbidity during measurement of MDA content was performed by subtracting the absorbance of 600 nm and absorbance of 532 nm.Results were expressed in terms of nmol MDA g -1 FW after calculation of MDA contents using extinction coefficient (155 mM -1 cm -1 ).

Proline content determination
Bates [23] method was used for analysis of amount of free proline.For this purpose, 0.1 g leaf tissue was homogenized in 3% sulphosalicyclic acid followed by centrifugation at 1200 rpm for 10 minutes.Homogenized tissue (1 ml) was incubated with acid ninhydrin (2ml) and glacial acetic acid (1 ml) in a test tube incubated for 1 h at 100 °C.The reaction was stop by place the sample on ice.Toluene (2 ml) was used to extract the reaction mixture by mixed vigorously followed by incubation at room temperature (30 min) until two phase separation was observed.The chromospheres (upper phase) was used to quantify free proline at 520 nm using toluene for a blank.

Determination of enzyme activities
For antioxidant enzyme analysis, leaf samples were homogenized followed by centrifugation at 13,000 rpm.Supernatant of each sample were used for enzyme activity measurements after centrifugation (20 min) of homogenate at 12,000 g at 4 °C.Bradford method [24] was used to determine total protein concentration in leaf extracts Bovine serum albumin (BSA) was used as standard.Ascorbate peroxidase (APX) enzyme quantification was carried out according to Wang [25] protocol.Suspension solution (1 ml) having Tris-HCl (pH 7.2, 50 mM), PVP (2%), EDTA (1 mM) and 2 mM of ascorbate mixed with samples containing 100 μg of protein.For initiation of the reaction, assay medium was mixed with potassium phosphate buffer (50 mM, pH 6.6), ascorbate (0.25 mM) and H2O2 (1 mM).The absorbance of ascorbate was measured at 290 nm for 90s. 1 unit of APX was defined if 0.001 change in absorbance of ascorbate occurred in per minute.Catalase (CAT) activity was measured by using Chance and Maehly, [26] method.Extracted samples having protein (100 μg) were mixed in Tris-HCl (1 ml, 50 mM) solution at pH 7.8.Potassium phosphate (50 Mm, pH 7.0) buffer and H2O2 (10 mM) was mixed with assay medium.The decline in absorbance of H2O2 was monitored at 240 nm for 90 s. 1 unit of CAT was defined as 0.001 change in hydrogen peroxide absorbance per minute.

Data analysis
All analyses were done using a completely randomized design with 4 replicates (n = 4).All data were subjected to one-way analyses of variance (ANOVA).Duncan's multiple range test was used to separate statistically different means at p < 0.01 and p < 0.05.

Growth parameters
Result analysis revealed that with increase in intensity of heat stress treatment, fresh weights of shoots of all rice cultivars decreased but difference in degree of decline in shoot weight observed.Similarly, dry weight of heat treated rice seedling's shoots decreased significantly (p ≤ 0.05) in all cultivars with increase in duration of thermal stress while increment found upon recovery treatments (Table 1).Throughout the experiment no significant effect of thermal stress on the shoot length of rice seedlings were observed although plants showed diverse shoot length within the group may be due to difference in metabolic activities, growth regulators and genetic factors.Each value represents the mean of 3 replicates (n = 3) and standard deviation of mean (±SD).Duncan's multiple range test showed significant differences in means (±SD) between control and heat-treated plants at P < 0.05 .

Hydrogen peroxide, melondialdehyde and proline contents
H2O2 is a reactive compound produced due to scavenging of superoxide radicals by superoxide dismutase (SOD) enzyme.Lipid peroxidation and membrane injury is a sign of higher accumulation of H2O2 leading death of plants and yield loss [27].Linear grew in content of H2O2 in all cultivars were observed with increasing level of heat stress condition however maximum (p ≤ 0.01) generation and accumulation of H2O2 content were found in Sada Hayat as compared to others (Figure 1).In the present study, K-95 cultivars presented lowest values of H2O2 accumulation under thermal stress, indicated that K-95 has efficient free radical quenching system at a cellular level that offered maximum protection against oxidative stress damages.Oxidative stress leading cytotoxic damage of protein, DNA and lipid observed in plants subjected to environmental stress conditions because reactive oxygen species (ROS) production overcomes antioxidant defence system capacity [28].MDA quantification in order to figure out level of lipid peroxidation has been considered as a convenient biomarker due to its reaction with thiobarbituric acid (TBA) forming coloured chromogen [29].Minimal increment in content of MDA was observed in "K-95", seedlings of "Sada Hayat" (Figure 2).The MDA content was found to be significantly highest in Sada Hayat and and lowest in K-95.Minimum values of MDA exhibited by K-95 suggest that it has developed an efficient and effective ROS scavenging system and therefore, showed better performance.Compared to control plants, heat treated rice seedlings had increase level of MDA which shows that rise in temperature beyond optimum for growth could cause peroxidation of membrane lipids making cell porous consequently cell sap leakage leading cell shrink and death [30].In many plant species proline accumulations under stressful conditions has been positively correlated with tolerance mechanism [16].The cultivars showed significant increment patterns for proline content in shoot tissues in relation to severity of high temperature stress (Figure 3).Other researchers have reported that stress-tolerant cultivars exhibited higher accumulation of free proline opposed to stress-sensitive varieties [31].The accumulation of free proline under harsh environmental condition as an advantageous mechanism of living system to protect membrane lipids, proteins and key cellular components insuring maintenance of cell homeostasis when relative water content (RWC) decreases [32].Decrease in RWC with minimum MDA suggest that increased accumulation of proline insuring cell membrane integrity.In various plants (cucumber, sunflower, chickpea and tomato), increase in free proline accumulation have been reported under oxidative stress [33-37], which is considered important adaptive and protection mechanism to cope with consequences of heat stress.In addition of proline measurement, analysis of chlorophyll fluorescence can be carried out in order to elucidate effect of thermal stress on photosynthetic apparatus [38].

Conclusion
From the findings, it is concluded that rice cultivar "K-95" comparatively performed better under heat stress condition showing thermotolerant ability with minimal oxidative damages and improved antioxidant system and maximum accumulation of osmolyte (Proline) followed by "IR-6," while "Sada Hayat" showed heat susceptibility with maximum oxidative damages and compromised antioxidant system having week capability of osmolyte accumulation for maintaining cell homeostasis under harsh condition.

8, K-95 and Sada Hayat subjected with control (C), heat stress (T24, T48 and T72) and recovery (R24, R48 and R72) treatments at early growth stag
MDA amounts were stable as compared to the controls.According to our results, two cultivars (K-95 and IR-6) were thermotolerant, whereas K-95 appeared to be less affected biochemically and physiologically, rendering K-95 thermotolerant followed by IR-6 while Sada Hayat and IR-8 were thermo-susceptible.The rice cultivar Sada Hayat had more damages as compared to IR-8.