Temperature stress induced antioxidative and biochemical changes in wheat ( Triticum aestivum L . ) cultivars

High temperature is one of the key abiotic stresses for hampering plant growth and yield. (Hasanuzzaman et al., 2012). The most favorable temperature for wheat cultivation is between 17°C and 23°C (Porter and Gawith, 1999). High temperature up to 47-48°C negatively affect wheat plant growth and development and adversely affects productivity in many parts of the world (Hameed et al., 2010). Exposure of plants to high temperature creates imbalance of physiological and biochemical activity which may have negative impact on water relations, photosynthesis, respiration and membrane stability due to changes in membrane fluidity and permeability (Mavi and Tupper, 2004) which can lead to cell damage and cell death resulting in calamitous break down in cellular organization (Schoffl et al., 1999). Like other abiotic stresses, heat stress results in production of excessive reactive oxygen species (ROS) such as superoxide radical (O), hydrogen peroxide (H 2 O 2 ), hydroxyl radical (OH), and singlet oxygen (O 2 ) causing disparity between anti and pro-oxidants (Wahid et al., 2007). The hydroxyl radical OH can damage plant proteins, different macromolecules, DNA and lipids, chlorophyll preventing growth and yield (Sairam and Tyagi, 2004). H 2 O 2 oxidizes thiol group of enzymes resulting in inactivation of enzymatic activity as well as at low concentration it acts as signaling molecule for different biotic and abiotic stresses, but at high concentration, it causes program cell death (Quan et al., 2008). ROS produced within plant tissue by heat stress damages membrane by lipid peroxidation forming minute hydrocarbon fragments, like malonaldehyde (MDA), one of the important indicators of oxidative stress. As a result Temperature stress induced antioxidative and biochemical changes in wheat (Triticum aestivum L.) cultivars


INTRODUCTION
High temperature is one of the key abiotic stresses for hampering plant growth and yield.(Hasanuzzaman et al., 2012).The most favorable temperature for wheat cultivation is between 17°C and 23°C (Porter and Gawith, 1999).High temperature up to 47-48°C negatively affect wheat plant growth and development and adversely affects productivity in many parts of the world (Hameed et al., 2010).Exposure of plants to high temperature creates imbalance of physiological and biochemical activity which may have negative impact on water relations, photosynthesis, respiration and membrane stability due to changes in membrane fluidity and permeability (Mavi and Tupper, 2004) which can lead to cell damage and cell death resulting in calamitous break down in cellular organization (Schoffl et al., 1999).
Like other abiotic stresses, heat stress results in production of excessive reactive oxygen species (ROS) such as superoxide radical (O2.− ), hydrogen peroxide (H 2 O 2 ), hydroxyl radical (OH − ), and singlet oxygen (1 O 2 ) causing disparity between anti and pro-oxidants (Wahid et al., 2007).The hydroxyl radical OH − can damage plant proteins, different macromolecules, DNA and lipids, chlorophyll preventing growth and yield (Sairam and Tyagi, 2004).H 2 O 2 oxidizes thiol group of enzymes resulting in inactivation of enzymatic activity as well as at low concentration it acts as signaling molecule for different biotic and abiotic stresses, but at high concentration, it causes program cell death (Quan et al., 2008).ROS produced within plant tissue by heat stress damages membrane by lipid peroxidation forming minute hydrocarbon fragments, like malonaldehyde (MDA), one of the important indicators of oxidative stress.As a result Temperature stress induced antioxidative and biochemical changes in wheat (Triticum aestivum L.) cultivars Jayanwita Sarkar 1 , Bishwanath Chakraborty 2 , Usha Chakraborty 1 * of increased lipid peroxidation phospholipids easily can cross lipid bilayer and causing enhancement of membrane leakage and inactivation of receptors, ion channels, and membrane-bound enzymes (Gill and Tuneja, 2010).
Production of ROS within plants tissues turns on different enzymatic and non-enzymatic signaling pathways.Antioxidative enzymes contributing to stress signaling such as peroxidase, catalase (CAT), ascorbate peroxidase (APOX), glutathione reductase, and superoxide reductase (Lee and Lee, 2000).Superoxide dismutases are the first enzyme to initiate ROS scavenging mechanism participate in dismuting of O  (Foyer and Noctor, 2003).Non-enzymatic antioxidants such as glutathione, tocopherol, ascorbic acid, and proline carotenoid play an important role in protecting the plant from stress-induced oxidative injury by performing photoprotective role and by inhibition of lipid peroxidation (Gill and Tuneja, 2010;Ashraf and Foolad, 2007).
Keeping in view the burning problem of global warming which may result into yield loss, negative impact on growth and photosynthetic ability, the present investigation was proposed to understand the antioxidative response of tolerant and susceptible cultivars of wheat to hightemperature stress with the main emphasis to study the role of antioxidative enzymes in imparting tolerance to wheat.

Plant Material and Experimental Conditions
Seeds of four locally available wheat (Triticum aestivum L.) cultivars, namely gayetri (GY), gandhari (GN), kedar (KD), and PBW343, were chosen and collected for experimental purposes.These locally available cultivars were selected for these experiments to compare responses of different cultivars subjected to high-temperature stress.The seeds were surface sterilized with 0.1% (w/v) HgCl 2 solution, washed with distilled water.Then, the seeds were transferred to earthen pots containing soil with an appropriate amount of compost.The transferred seedlings in the pots were then kept in growth chamber at a favorable temperature of 20-25°C. 1 month old seedlings were selected for heat stress.Pots containing 1 month old seedlings were exposed to different temperatures (25°C, 30°C, 35°C, and 40°C) for 6 h.Leaf samples from control and heat-stressed seedlings were collected and used for analysis.The fresh weight of seedlings was taken immediately after collecting to avoid any water loss from leaf samples of both control and treated seedlings.

Relative Water Content (RWC) and Heat Susceptibility Index (HSI)
RWC was calculated by determining the fresh weight, turgid weight, and dry weight of leaf samples under treatment and using formula given by Farooqui et al., 2000.( ) ( ) (Fresh weight -dry weight) RWC % 1 00 Fully turgid weight -dry weight = × HSI was calculated using variation in fresh weight and RWC (degree in percent decrease) using following formula (Hameed et al., 2010).
(Reduction in seedling FW % reduction in RWC) HSI 2 Calculated HSI values of different cultivars were compared to have an idea about degree of heat susceptibility of different cultivars.

Extraction and estimation of total sugar
Extraction of total sugar was done by following the method of Harborne (1973) by crushing of leaf samples in 95% ethanol, and the alcoholic fraction was evaporated on a boiling water bath, and the aqueous fraction was resuspended in distilled water which was then centrifuged at 5000 rpm for 10 min.The supernatant was collected for estimation of total sugar.Estimation of total sugar was done by anthrone reagent following the method of Plummer (1978).

Extraction and estimation of total chlorophyll
Chlorophyll was extracted following the method of Harborne (1973) by homogenizing leaf in 80% acetone.Estimation of chlorophyll was done by measuring the absorbance at 645 nm and 663 nm, respectively, in an ultraviolet (UV)-vis spectrophotometer and calculated using the formula as given by Arnon (1949).

Extraction and quantifi cation carotenoids
Method described by Lichtenthaler (1987) was pursued for carotenoids extraction and estimation.For this, leaf samples of both control and treated plants were crushed using 100% methanol in dark and extract was filtered.
Then, the absorbance of filtrate was taken at 663,645 and 480 nm, respectively, in the UV-vis spectrophotometer.

Extraction and estimation of free proline
Extraction of free proline from the leaves was performed following the method of Caverzan et al. (2012).0.5 g of leaf tissue was homogenized in 3% sulfosalicylic acid and filtered through a Whatman No. 1 filter paper.The supernatant was used for estimation.Quantification of proline was done by the mixing filtrate with 2 ml of acid ninhydrin reagent.After that, the mixture was kept for1 h in a boiling water bath and chilled rapidly.The reaction mixture was then shifted in separating funnel, and 5 ml of toluene was added and mixed vigorously.The lower colored layer was taken to measure absorbance at 520 nm in a colorimeter using toluene as blank and quantified from a standard curve of proline.

Quantifi cation of H 2 O 2
H 2 O 2 within leaf tissue was measured by following the method described by Jena and Choudhuri (1981).A definite quantity of the leaf samples was taken and crushed in 50 mM potassium phosphate buffer (pH 6.5).Crushed samples were centrifuged at 2,415 g for 25 min.Whole experiment was done in minimal light conditions (Chakraborty and Pradhan, 2011).The reaction was completed as an intense yellow color started developing.This was monitored at 410 nm spectrophotometrically.The levels of H 2 O 2 in the samples were determined by the use of extinction coefficient 0.28 μmol −1 cm −1 in the calculation.

Determination of lipid peroxidation
MDA is an indicator of lipid peroxidation within the plant.The presence of MDA was measured by following the method described by Heath and Packer (1968).It was measured by thiobarbiturate reaction where leaf tissue was homogenized in 0.1% (w/v) trichloroacetic acid.Absorbance of MDA content was then measured at 600 and 532 nm and MDA was quantified using extinction coefficient of 155 mM −1 cm −1 .

Extraction
Leaf samples were homogenized in 50 mM sodium phosphate buffer (For peroxidase and CAT [pH 6.8]; and ascorbate peroxidase (APOX) [pH 7.2]) and 100 mM potassium phosphate buffer pH 7.6 (For glutathione reductase and superoxide dismutase) using PVP under ice cold conditions.The homogenates were then centrifuged at 10,000 rpm for 10 min at 4°C.Supernatants were used as crude enzyme extracts.

Estimation of antioxidative enzymes
Glutathione reductase (GR,EC 1.6.4.2) GR activity was evaluated by quantifying the oxidation of NADPH at 340 nm as described by Lee and Lee (2000).The reaction mixture consisted of 1 ml of 0.1 M potassium buffer (pH 7.6), 0.2 ml of 0.1 M ethylenediaminetetraacetic acid, 0.1 ml of freshly prepared 6 mM glutathione, 0.2 ml of 0.1 mM NADPH, and 0.2 ml of enzyme extract.Change in absorbance was measured instantly at 340 nm in the UV-VIS spectrophotometer.
APOX, EC 1.11.1.11APOX activity was measured by quantifying oxidation of ascorbate in the UV-vis spectrophotometer following the method of Asada and Takahashi (1987).Enzyme activity was expressed as mmol ascorbate mg protein −1 min −1 .
Peroxidase (POX,EC 1.11.17)POX activity was assayed by monitoring the oxidation of o-dianisidine in the presence of H 2 O 2 at 460 nm following the method of Chakraborty et al. (1993).The change in absorbance was noted down immediately at specific intervals up to 3 min in the UV-vis spectrophotometer.The POX activity was expressed as mmol O-dianisidine mg protein −1 min −1 .
CAT (CAT,EC 1.11.1.6)CAT activity was assayed following the method of Chance and Maehly (1955) by estimating the decomposition of H 2 O 2 at 240 nm in the UV-VIS spectrophotometer.The enzyme activity was expressed as mol H 2 O 2 mg protein −1 min −1 .

Polyacrylamide gel electrophoresis (PAGE) analysis of peroxidase isozymes
PAGE was performed for isozyme analyses of peroxidase and CAT enzymes by following the method given by Davis (1964) and Reddy and Gasber (1971).

Statistical Analysis
Data were investigated statistically by the least significance test difference at P = 0.05 probability level.

RESULTS
It is necessary to recognize different biochemical and physiological changes proceeding within plant tissue to develop heat tolerant wheat cultivars.The objective of this study is to evaluate different biochemical parameters within four wheat cultivars subjected to temperatureinduced oxidative stress.Findings regarding tolerance response under heat stress in terms of antioxidative and biochemical response are given below.

Evaluation of Heat Susceptibility of Cultivars
Elevated temperature significantly decreased fresh weight of seedlings (P = 0.05) in all four wheat cultivars.Reduction in fresh weight ranges from 21.97% highest in PBW343 to 4.02% lowest in GN at 40°C.FW in the leaf of all the cultivars gradually reduced with elevated temperature stress (Figure 1).RWC change (Figure 2) was lesser in case of GN and KD at 40°C when compared to their respective controls (20.60% and 30.98%, respectively); however, the heat-induced reduction in RWC was much more in GY and PBW343 in respect to their controls (36.48% and 42.63%, respectively).HSI of GN,KD,GY,and PBW343 are 11.65,19.73,25.48,30.90,respectively.Maximum and minimum values were noticed in case of PBW343 and GN, making these two heat sensitive and heat tolerant cultivars, respectively, and the other two cultivars KD and GY were moderately heat tolerant and moderately heat sensitive among four cultivars (Table 1).

Lipid peroxidation and H 2 O 2 content
Temperature stress enhanced membrane peroxidation as reflected by the elevated level of MDA and H 2 0 2 (Table 2).In all four cultivars, MDA content gradually increased with elevated temperature.Accumulation of MDA with elevated temperature in relation to control was highest in heat sensitive PBW343 (3.55-fold higher than control) and minimum in heat tolerant GN (0.61-fold higher than control).
Similarly, maximum accumulation of H 2 O 2 was noticed within leaf samples of heat sensitive PBW343 and minimum in GN.

Antioxidative Response
Activities of antioxidative enzymes in both control and treated seedlings were analyzed.Among four antioxidative enzymes, APOX (Figure 3a) and glutathione reductase (Figure 3b) activity enhanced up to 30°C.Similarly, CAT activity (Figure 4a) gradually increased up to 35°C in GN, while it decreased after 30° in all other cultivars.Enhancement of CAT was maximum in case of GN (1.02-fold higher in relation to control) and minimum in PBW343 (0.07-fold higher in relation to control) (Figure 4a).The highest reduction at 40° was noticed in PBW343 and lowest in GN, which was in conformity with results from HSI.
On the other hand, peroxidase activity continued to increase with elevated temperature in all four cultivars in relation to control.The highest increase was noticed in GN at 40°C (3.58-fold), and lowest was obtained in PBW343 (0.78-fold) (Figure 4b).
Peroxidase isozyme analysis of four cultivars was performed.Isozyme analysis showed an increase in band number and color intensity with elevated temperature stress in all four cultivars.In heat tolerant cultivar, GN color of bands was much more intense than heat susceptible cultivars (Figure 5).Calculated on the basis of 3 replicate sets of 10 seedlings each.GN: Gandhari, KD: Kedar, GY: Gayetri, HSI: Heat susceptibility index

Carotenoid and Chlorophyll
Carotenoid (Figure 6a) enhanced significantly in all four cultivars at 30°C.At 30°C, carotenoid content was 50.01%, 29.50%, 53.48%, and 30.90 % higher than control in GN, KD, GY, PBW343, respectively.In case of GN and KD with further raising of temperature up to 35°C, there was slight increase in carotenoid content, on contrary in other two varieties raising temperature beyond 35°C reduced carotenoid content within leaf tissue.
6 h incubation at 30°C led to an increase in total chlorophyll in all cultivars.Further increase in temperature resulted  in a decline in total chlorophyll content in all cultivars (Figure 6b).

Proline and Total Sugar
Proline content in GN, KD, GY, and PBW343 was 1.87, 2.29, 1.77, and 1.68 times higher, respectively, in relation to control at 35°C (Figure 7a).Raising temperature from 35°C to 40°C resulted in decrease in accumulation of proline in leaf tissues of all four cultivars.There was sharp increase in total soluble sugar within leaf tissue of all four cultivars with gradual increase in temperature suggesting possible role of total soluble sugar in heatinduced oxidative stress regulation.The highest increase was noticed in KD at 40°C (1.34-fold) and lowest noticed in PBW343 (0.974-fold) in relation to control (Figure 7b).

DISCUSSION
High-temperature stress beyond certain limit irreversibly affects plant growth and development (Wahid, 2007).
Plants act in response to different abiotic stresses by shifting and changing concentration of metabolite related to different biochemical pathways so that they can acclimatize to unfavorable physiological changes (Kumar et al., 2012).Heat stress produces a significant reduction in fresh weight and RWC in aerial parts of plants (Rodríguez et al., 2015).In this study, FW and RWC gradually reduced with an increase in temperature in all four wheat cultivars and heat susceptibility index was calculated using these two parameters.Based on HSI, PBW343 was identified as more heat susceptible variety, and GN was most tolerant variety.Heat stress generates excessive ROS than optimal level resulting in enhanced lipid peroxidation and H 2 O 2 production in leaf tissue (Kipp and Boyle, 2013).H 2 O 2 is related to oxidative injury in plants (Ozden et al., 2009) and act as signaling molecule which upregulate genes related to scavenging of free radicals from cell via a number of antioxidant mechanisms including both enzymatic and non-enzymatic (Kumar et al., 2012).
In the present study, lipid peroxidation as indicated by MDA content and H 2 O 2 increased significantly in all cultivars, but augmentation was higher in case of heat susceptible lines than in heat tolerant lines.This result was in accordance with previous studies in wheat (Tatar and Gevrek, 2008) and in mung bean (Mansoor and Naqvi, 2013).Increased lipid peroxidation and H 2 O 2 level inactivate cell membrane enzymes and proteins, consequently antioxidative (enzymatic and non-enzymatic) machinery avert cell damage by regulating level of ROS (Ge et al., 2005).
Antioxidative enzymes such as POX, APX, GR, and CAT play important roles in the alleviation of ROS.
In the present study, exposure to higher temperature initially upregulated enzyme activity up to 35°C in all cultivars, indicating that higher temperatures could trigger antioxidative enzymes to scavenge H 2 O 2 and ROS (Gosavi, 2014).However, further increase in temperature revealed different activities.Peroxidase continued to increase significantly with temperature in all cultivars.Probable role of POX is to defend cells against oxidative damage of H 2 O 2 by decomposition through oxidation of phenolic and endiolic cosubstrates (Lin and Kao, 2002).Both APOX and GR are engaged in the ascorbate-glutathione pathway and work in combination for scavenging of the radicals.Maximum GR, APOX, and CAT activity was noticed at 30°C in all cultivars except CAT activity for GN, wherein the activity was found to be highest at 35°C.
Beyond 35°C, all these three enzyme activities (GR, APOX, and CAT) decreased gradually, but downregulation of enzyme activity was minimum in heat tolerant variety and maximum in heat susceptible variety making it prominent that plants cannot endure heat stress beyond certain threshold level.Chakraborty and Pradhan (2010) reported an initial increase of antioxidative enzyme activity in lentil subjected to high temperature up to 45°C.From these finding, it is clear that thermotolerant cultivars better ability to withstand damaging effect of H 2 O 2 and lipid peroxidation induced by higher temperature through antioxidative machinery.
Changes in photosynthetic elements and photosynthesis are good indicators of high temperature induced oxidative stress and degree of thermotolerance of the plant.High temperature limits photosynthesis and plant growth (Wahid et al., 2007).In the present investigation, chlorophyll content gradually reduced after an initial increase at 30°C in all cultivars except in relatively tolerant variety GN where it increased 35°C.Since membrane integrity is immediately affected by high temperature, chlorophyll structure damage may be one of the reasons for reduced photosynthesis (Mansoor and Naqvi, 2013).Similar results also found in several crops including maize and rice (Kumar et al., 2012).
Other than enzymes non-enzymatic antioxidant carotenoid and osmoprotectant proline plays crucial role in oxidative stress tolerance.In all the four cultivars, maximum accumulation of proline and carotenoid took place at 35°C.According to Sairam and Tyagi (2004), accumulation of proline and total sugar might contribute to heat tolerance.Increasing concentration of soluble sugar was reported in sugarcane (Wahid 2007).Carotenoids are considered to be the main 1 O 2 quenchers in chloroplasts.Carotenoids facilitate to defend the plants from oxidative injury by preventing the production of singlet oxygen (Havaux, 2013).Beyond 35°C, carotenoid and proline content continue to decrease.However, the percentage of reduction was higher in heat susceptible cultivars than heat tolerant varieties.

CONCLUSION
Outcomes from our study clearly indicate that high temperature was responsible for the bringing on oxidative stress as shown by reduction in fresh weight and RWC, increase in the MDA content (i.e., lipid peroxidation), H 2 O 2 accumulation, degradation of chlorophyll molecule, enhanced antioxidative responses as evident in the differential levels of antioxidative enzyme activities, accumulation of different non-enzymatic antioxidants carotenoid, and osmolytes such as soluble sugars.Out of the four cultivars taken for the study, GN and KD showed a much more distinct antioxidative mechanism after the induction of heat stress, and therefore, they seemed to be cosseted from the harmful damage caused by oxidative stress as a result of heat stress and increased severity of stress.Considering all the above data and comparing with HSI, it was found that among the four cultivars GN was able to withstand the heat stress more effectively than the other three cultivars, whereas PBW343 was more susceptible to heat stress than the other cultivars.

Figure 1 :Figure 2 :
Figure 1: Fresh weight of leaf of four wheat cultivars exposed to heat stress.Results are articulated as the mean±standard deviation.n=4.Least signifi cance test difference (P=0.05)within cultivars=162.9 and within treatments=155.50

Figure 6 :Figure 7 :
Figure 6: Changes in carotenoid (a) and total chlorophyll, (b) content in four cultivars after heat treatments.Results are expressed as mean±standard deviation.n=4.(a) Least signifi cance test difference (LSD) (P=0.05)within cultivars=0.02and within treatments=0.05,(b) LSD (P=0.05)within cultivars=0.614and within treatments=0.915a b 2.− radical to molecular O 2 and H 2 O 2 .Hydrogen peroxide is then converted to H 2 O and O 2 either by CAT and peroxidase or in ascorbate-glutathione cycle by APOX and glutathione reductase help to regenerate glutathione pool resulting detoxification of H 2 O 2