Introduction

Wheat (Triticum aestivum L.) is recognized as a staple food crop globally and it typically grown over 200 Mha throughout the world. India ranks second on the list of producers of wheat all over the world. The advent of green revolution witnessed a steady increase in wheat productivity which has been associated with genetic improvements in yield potential, resistance to diseases, adaptation to abiotic stresses (Reynolds and Borlaugh 2006) as well as better agronomic practices (Evenson & Gollin, 2003). Ever-increasing global demand for wheat and limited availability of land is placing pressure on breeding programs to provide elite cultivars that can adapt to a range of environments without compromising agronomic performance, grain quality, stress tolerance and disease resistance.

Crop cultivation in open fields is seen to be dependent on various biotic and abiotic factors. Abiotic stresses lead to high leakage of electrons towards oxygen during photosynthetic and respiratory processes leading to enhancement in Reactive oxygen species (ROS) generation (Asada, 1999). ROS are ubiquitous molecules produced as a consequence of normal cellular metabolism (Kotchoni, 2004). Several environmental factors such as cold, high light, ozone, drought, salt, pathogen and UV radiations can cause stress in plants and may lead to the over production of reactive oxygen species (ROS) in plants which are highly reactive and toxic and cause damage to proteins. This excessive accumulation of ROS necessitates the activation of additional defenses (Doke & Scandalios, 1997). The antioxidant defense machinery protects plants from oxidative stress damage. Plants possess very efficient enzymatic (Superoxide Dismutase, Ascorbate Peroxidase, Glutathione Reductase, Guaiacol Peroxidase and Catalase) and non-enzymatic (ascorbic acid, glutathione and alpha tocopherol) antioxidant defense systems to protect the plants from oxidative damage (Gill & Tuteja, 2010). Many of the genes are shared between abiotic and biotic stresses. This highlights the complexity of stress response and adaptation to plants (Mantri et al., 2012).

The ROS such as O2, H2O2 and OH∙ radicals can directly attack membrane lipids, inactivate metabolic enzymes, and damage the nucleic acid leading to cell death. It has been reported that high antioxidant enzymes in wheat are related with various kinds of abiotic and biotic stresses (Sairam et al., 1998; Valifard et al., 2012). Therefore, a genotype which has higher status of antioxidant enzymes could behave differently as compared to a genotype which has lower status of these enzymes. In plant cells chloroplast, mitochondria and peroxisomes are important intracellular generators of reactive oxygen species.

O2∙ is the primary ROS formed in the cell which initiates a cascade of reactions to generate “secondary” ROS, either directly or through enzyme or metal catalyzed processes depending on the cell type or cellular compartment (Valko et al., 2006). By comparison, O2∙ and H2O2 are weaker oxidizing agents. Under normal condition, the half-life of H2O2 is probably 1 ms, and other forms of ROS, including superoxide anion (O2∙), hydroxyl radicals (OH∙) and singlet oxygen (1O2), have very short half-life, about 2–4 µs (Gill & Tuteja, 2010). H2O2 plays a dual role in plants at low concentrations, it acts as a signal molecule involved in signaling tolerance to various biotic and abiotic stresses and at high concentrations it leads to programmed cell death (Quan et al., 2008). OH∙ is among the most highly reactive ROS known. In the presence of suitable transition metals, especially Fe, OH∙ can also be produced from O2∙ and H2O2 at neutral pH and ambient temperatures by the iron catalyzed, O2∙ driven Fenton reaction (Gill & Tuteja, 2010).

Reactive oxygen species produced as a result of various abiotic stresses needs to be scavenged for maintenance of normal growth. The primary scavenger is superoxide dismutase (SOD; EC 1.15.1.1), which converts O2∙ to H2O2. This toxic product of SOD reaction is eliminated by ascorbate peroxidase (APX; EC 1.11.1.11) in association with dehydro-ascorbate reductase (EC1.8.5.1) and glutathione reductase (GR; EC 1.6.4.2), the latter two help in regeneration of ascorbic acid (AA). H2O2 is also scavenged by catalase (EC 1.11.1.6), though the enzyme is less efficient than APX-GR system (Quan et al., 2008).

Drought poses critical environmental constraints to plant survival and crop productivity (Chaves et al., 2003). Dynamic changes in the antioxidative enzyme activities have been attributed as an important anti-drought mechanism to cope with oxidative stress during drought conditions (Shao et al., 2005). Better resistance and acclimation to drought is experimentally correlated with enhanced antioxidative protection (Khanna-Chopra & Selote, 2007). Similar to water deficient stress, reactive oxygen species are also produced during salinity stress, and are responsible for the damage to membranes and other essential macro-molecules such as photosynthetic pigments, proteins, DNA and lipids.

Esfandiari et al. (2007) observed that high SOD, CAT and GR activity in leaves of Sardari a normal growing wheat cultivar is associated with salt tolerance while the less SOD, CAT and GR activity in leaves of Alvand a normal growing wheat cultivar is associated with salt sensitive nature. Variability in enzymatic and non enzymatic antioxidants activity has been associated with salt tolerance. The activitites of antioxidant enzymes such as catalase, peroxidase, glutathione reductase and superoxide dismutase differently change in wheat genotypes (Huseynova et al., 2010). Kavir a normal growing wheat cultivar having high SOD, CAT and APX is associated with drought tolerance (Hasheminasab et al., 2012). Thus, the variability in enzymatic and non enzymatic antioxidants could be useful for breeding genotypes tolerant to a number of factors that can cause stress to the plant cultivation.

Material and methods

Plant material

Crop was raised in the fields of Punjab Agricultural University, Ludhiana, India. Activities of antioxidant enzymes and non-enzymatic antioxidants were determined in leaves after 45 days of sowing and in flag leaf at 5 days after anthesis. These crops were raised in the fields of Punjab Agricultural University, Ludhiana, India. Activities of antioxidant enzymes and non-enzymatic antioxidants were determined in leaves after 45 days of sowing and in flag leaf at 5 days after anthesis.

Extraction and estimation of antioxidative enzymes

Antioxidative enzymes assay

Enzymes were extracted from fresh plant tissues at 4 °C as described by Kaur et al. (2009). SOD, POX and GR activities were determined spectrophotometrically by the methods of Marklund and Marklund (1974), Shannon et al. (1966), respectively. APX and CAT activities were determined following the procedure of Nakano and Asada (1987) and Chance and Maehly (1955), respectively. Protein concentration was measured by the method of Lowry et al. (1951).

Ascorbate and H2O2 measurement

Ascorbate (Asc) measurement was based on the reduction of ferric to ferrous ion with ascorbate in acid solution followed by the formation of the pink complex between ferrous ion and bipyridyl that absorbs at 525 nm. Fresh tissue (0.1 g) was homogenized in 1.5 mL of 5% ice cold metaphosphoric acid and centrifuged at 10,000 g for 10 min. Supernatant was collected and used for the estimation of ascorbate according to Law et al. (1983). For H2O2 extraction, fresh tissue (0.3 g) was homogenized in 2.0 mL of ice-cold 10 mm sodium phosphate buffer (pH 7.0) and centrifuged at 10 000 g for 20 min. Supernatant was collected, and H2O2 content was estimated by reaction with 5% potassium dichromate and acetic acid (1: 3 V/V) as described by Sinha (1971).

Malondialdehde (MDA) content measurement

Malondialdehde (MDA) equivalent content as thiobarbituric acid reactive substances (TBARS) was measured as described by Ohkawa et al. (1979). Fresh tissue (0.2 g) was homogenized in 1.0 mL of 5% trichloroacetic acid (TCA) and centrifuged at 13,500 g for 15 min at room temperature. The supernatant of tissue was mixed with an equal volume of 20% (w/v) TCA containing 0.5% (w/v) thiobarbituric acid. The mixture was heated to 96 °C for 30 min, cooled quickly in ice and centrifuged at 9500 g for 10 min. The absorbance of the supernatant was measured at 532 nm. Correction of nonspecific turbidity was obtained by subtracting the absorbance value taken at 600 nm. The extinction coefficient used for this assay was 155 mm−1 cm−1.

Proline content measurement

Proline content was measured as described by Bates (1973). 100 mg of sample was homogenized with 4 mL of 3% aqueous sulfosalicylic acid and the homogenate was filtered through Whatman filter paper and filterate was used for proline estimation. To 2 mL of supernatant, 2 mL of acidic ninhydrin solution and 2 mL of glacial acetic acid were added. The tubes were kept in a water bath at 100 °C for 1 h. Thereafter, the reaction was terminated in an ice bath. The reaction mixture was extracted with 4 mL toluene, mixed vigorously with a test tube stirrer for 15–20 s. The chromophore containing upper toluene layer was aspirated from aqueous phase and absorbance was read at 520 nm using toluene as blank. The concentration of proline was determined using proline standards (0.02 to 0.1 µmol) run simultaneously.

DPPH radical scavenging activity

The free radical scavenging capacity of wheat extracts was determined using the stable 2, 2-diphenyl-1-picrylhydrazyl radical (DPPH.) as outlined by Blois (1958). The required tissue (100 mg) was homogenized in 2 mL of methanol and centrifuged at 10,000×g for 10 min. To 1 mL of supernatant, 3 mL of DPPH were added. After 30 min of incubation at room temperature in dark, the absorbance was measured at 517 nm.

Statistical analysis

The results are analyzed as means ± S.D (n ≥ 3). Tukey’s test (SPSS 16.0 software) was used to determine the difference between the genotypes (P ≤ 0.05).

Result and discussion

Variability of enzymatic and non enzymatic antioxidants was studied in forty different wheat genotypes at different stages of crop development.

Variability of enzymatic and non enzymatic antioxidants in leaves of wheat after 45 days of sowing

Tukey’s comparison indicated significant difference in superoxide dismutase (SOD) activity in the wheat cultivars (Table 1). Mean comparison of the cultivars showed that highest activity of SOD was observed in advanced breeding lines. Within advanced breeding lines wheat cultivars BW 4101and PBW 687 had maximum SOD activity. High specific activity of SOD was observed in PBW 687 and Australian cultivar Cook. High SOD activity in leaves of wheat has been related with salt tolerance (Kahrizi et al. 2012) and with drought stress tolerance (Hasheminasab et al., 2012). So wheat genotypes having high SOD activity could be tolerant to salt/drought stress. Minimum SOD activity was observed in PBW 502 and WH 542. High activity of SOD in normally growing seedlings has also been related with high temperature stress tolerance (Sairam et al., 1998). High activity of SOD therefore appears to be good trait for abiotic stress tolerance.

Table 1 Variability of enzymatic antioxidants in leaves of wheat after 45 days of sowing
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Peroxidase (POX) activity in wheat cultivar has been illustrated in Table 1. Almeselmani et al. (2006) observed that high POX activity in leaves was associated with high temperature tolerance. Mean comparison of the cultivars showed that highest activity of POX was observed in advanced breeding lines while the lowest POX activity was observed in commercial cultivars. Highest POX activity was observed in PBW 668 and BWL 83 whereas specific activity of POX was high in PBW 687 and Australian cultivar Cook. These cultivars may be associated with high temperature tolerance but validation in the field is necessary.

Activity of glutathione reductase (GR) is depicted in Table 1. Mean comparison of commercial, rain-fed and advanced breeding lines did not show significant differences among themselves. Minimum GR activity was observed in Australian cultivars. Within Australian cultivars Ruby and Binnu has the minimum GR activity. Valifard et al. (2012) observed that in the wheat high GR activity was related to drought tolerance. WH 1021, BWL 83 and BW 7197 have highest activity of GR hence they could be good candidate for studying drought tolerance in the field.

Mean comparison showed that highest catalase (CAT) activity was present in Australian cultivars while there were no significant differences between commercial and advanced breeding lines (Table 1). Advanced breeding line BW 6866 has highest CAT activity. Huseynova et al. (2010) reported that high CAT activity was associated with drought tolerance. However, the cultivars recommended for sowing under rain-fed condition have lower CAT activity in leaves.

Tukey’s comparison indicated significant difference in ascorbate peroxidase (APX) activity in the wheat cultivars (Table 1). Higher APX activity was observed in advanced breeding lines. Within advanced breeding lines BW 4101 and BWL 924 have the highest APX activity. Esfandiari et al. (2007) showed that Egypt 449 a wheat cultivar having high APX activity was related with drought tolerance hence BW 4101 and BWL 924 could be drought tolerant.

Variability was also observed in non enzymatic antioxidants in different wheat cultivars. DPPH (2, 2-diphenyl-1-picrylhydrazyl) radical scavenging activity showed variability in different wheat genotype (Table 2). Highest DPPH radical scavenging activity was observed in advanced breeding lines while the lowest DPPH radical scavenging activity was observed in Australian cultivars. Among advanced breeding lines BWL 73, BW 7197, RAJ 4134 and BWL 936 have high DPPH radical scavenging activity. High DPPH activity could be positive trait for scavenging superoxide radicals produced during abiotic stresses.

Table 2 Variability of non-enzymatic antioxidants in leaves of wheat after 45 days of sowing
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Sairam et al. (1998) observed that C 306 a wheat cultivar having less H2O2 content was tolerant to drought stress while HD 2329 a wheat cultivar having high H2O2 content was sensitive to drought stress. On this basis genotypes BWL 83, BWL 936, PBW 343, PBW 621 having less H2O2 content could be an ideal material for studying drought stress tolerance in the field. Lower H2O2 content was observed in advanced breeding lines while the highest H2O2 content was observed in rain-fed cultivars (Table 2).

Data on malondialdehyde (MDA) content in different wheat genotype has been presented in Table 2. Maximum MDA content was observed in Australian cultivars while the minimum MDA content was observed in commercial cultivars. Asharaf et al. (2010) observed that less MDA was associated with salt tolerance.

Mean comparison of proline content showed no significant variability between rainfed, Australian and advanced breeding lines (Table 2). Commercial cultivars have maximum proline content while the Australian cultivars have minimum proline content. BW 7296, WH 542 and PBW 621 have maximum proline content. Khan et al. (2009) observed that Lu-26s wheat cultivar having high proline content was associated with salt tolerance. Wheat cultivars BW 7296, WH 542 and PBW 621 have relatively high proline content.

Table 2 depicts ascorbic acid content in different wheat genotypes. Tuekey’s comparison showed significant variability between different wheat cultivars. Highest ascorbic acid content was observed in rainfed cultivars while the lowest ascorbic acid content was observed in commercial cultivars. Among rainfed cultivars PBW 596, PBW 175 and PBW 175 have high ascorbic acid content. DBW 17, PBW 550, DATATINE have low ascorbic acid content. High ascorbic acid content could be useful trait as an antioxidant. However, no reports are available in the literature showing correlation between leaf ascorbic acid content and abiotic stresses, though Mandhania et al. (2010) have reported high ascorbate and proline content in wheat seedlings and correlated it with salt stress tolerance.

After 45 days of sowing, the leaves showed maximum variability of 3.69 folds in APX with BW 4101 showing highest activity (Table 3). BW 4101 also has highest SOD activity. MDA showed maximum variability of 4.09 folds among non enzymatic antioxidants. Wheat genotypes BWL 83 and BWL 932 showed lowest H2O2 and MDA content (Table 4).

Table 3 Status in terms of highest/lowest ratio of enzymatic antioxidants in leaves after 45 days of sowing
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Table 4 Status in terms of highest/lowest ratio of non enzymatic antioxidants in leaves after 45 days of sowing
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Variability of enzymatic and non enzymatic antioxidants in wheat in flag leaf stage after 5 days of anthesis

Activity of Superoxide Dismutase (SOD) is depicted in Table 5. Mean comparison of SOD activity between commercial, Australian cultivars and advanced breeding lines did not show significant differences. However, Australian cultivars have high specific activity of SOD. Minimum SOD activity was observed in commercial cultivars. Within commercial cultivars PBW 502, WH 542 and PBW 621 have low SOD activity. Valifard et al. (2012) observed that in the wheat high SOD activity was related to drought tolerance. Rainfed genotype PBW 644, Advanced breeding line PBW 668 and BWL 73 and Australian cultivar Binnu have high SOD activity.

Table 5 Variability of enzymatic antioxidants in flag leaf stage after 5 days of anthesis
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Tukey’s comparison indicated significant difference in peroxidase (POX) activity in the wheat cultivars (Table 5). Mean comparison of the cultivars showed that highest activity of POX was observed in commercial cultivars. Within commercial cultivars wheat cultivars PBW 343 and PBW 502 have maximum POX activity. High POX activity in leaves of wheat has been related with salt tolerance (Kahrizi et al. 2012) and with drought stress (Hasheminasab et al., 2012). So wheat cultivar having high POX activity could be studied for tolerance to these stresses. Minimum POX activity was observed in Stretton and Binnu so these cultivars could be susceptible to salt stress. High activity of POX in the roots of normally growing wheat has also been related with drought stress (Csiszar et al., 2008).

Data on glutathione reductase (GR) activity in wheat cultivar has been given in Table 5. Almeselmani et al. (2006) observed that high GR activity in leaves was associated with high temperature tolerance. Mean comparison of the cultivars showed that highest activity of GR was observed in commercial cultivars while the lowest GR activity was observed in Australian cultivars. Highest GR activity was observed in PBW 343 and PBW 502 however, these cultivars have been recommended for timely sowing under irrigated condition.

Tukey’s comparison indicated significant difference in catalase (CAT) activity in the wheat cultivars (Table 5). High CAT activity was observed BWL 932, BWL 924 BWL 927 and Sunmist. High CAT activity could be responsible for reducing the H2O2 content of leaves. The genotype WH 542 has the high specific activity of CAT while the low specific activity was observed in BWL 936.

Mean comparison showed that ascorbate peroxidase (APX) activity was more in rainfed cultivars and less in Australian cultivars (Table 5). Tukey’s comparison showed significant difference among wheat cultivars. PBW 550 which is a commercial cultivar has the highest APX activity. Esfandiari et al. (2007) showed that Egypt 449 a wheat cultivar having high APX activity was related with drought tolerance. PBW 550 a high yielding cultivar can also be tried under water deficient condition.

High DPPH radical scavenging activity was observed in advanced breeding lines while the low DPPH radical scavenging activity was observed in Australian cultivars (Table 6). DBW 17, PBW 373, BWL 932, BWL 934 and DT BWL 0927 have high DPPH radical scavenging activity. Though no report could be found in literature showing correlation between abiotic stress and DPPH radical scavenging activity but better radical scavenging activity could be a desirable trait during different stress conditions.

Table 6 Variability of non-enzymatic antioxidants in flag leaf stage after 5 days of anthesis
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Data on hydrogen peroxide (H2O2) content in different wheat genotypes has been presented in Table 6. High H2O2 content was observed in rainfed cultivars while the low H2O2 content was observed in Australian cultivars. Sairam et al. (1998) observed that less H2O2 was associated with drought tolerance. Among Australian cultivars Binnu, Datatine and Carnamah have low H2O2 content hence these cultivars could be studied for tolerance to drought stress.

Asharf et al. (2010) observed that S-24 a wheat cultivar having less malondialdehyde (MDA) content was tolerant to salt stress while MH-97 a wheat cultivar having high MDA content was sensitive to drought stress. On this basis genotypes BWL 932, BWL 931, PBW 373, PBW 621 having less MDA content could be an ideal material for studying salt stress tolerance in the field. Lower MDA content was observed in rain-fed cultivars while the higher H2O2 content was observed in advanced breeding lines (Table 6).

Tuekey’s comparison showed significant variability between different wheat cultivars. Higher proline content was observed in rain-fed cultivars while the lower proline content was observed in Australian cultivars (Table 6). Among rainfed cultivars PBW 527 and PBW 175 have maximum proline content. Cook and Carnamah have minimum proline content. Proline could act as protective osmolyte during stress condition (Ashraf et al. 2010).

Mean comparison of ascorbic acid content showed no significant variability between rainfed, Australian, advanced breeding lines and commercial cultivars (Table 6). Commercial cultivars have maximum ascorbic acid content while the advanced breeding lines have minimum ascorbic acid content. PBW 343, PBW 550 and PBW 621 have maximum ascorbic acid content. High ascorbic acid content could be useful trait as an antioxidant. However reports linking ascorbic acid content with abiotic stresses are lacking in literature.

In the flag leaf maximum variability of 10.9 folds was observed with GR. Cultivar PBW 502 has the highest GR activity. This cultivar also showed maximum POX activity (Table 7). In non enzymatic antioxidants, variability in DPPH and ascorbic acid was more as compared to H2O2, MDA and proline (Table 8). Many of the Australian cultivars showed lower non enzymatic antioxidants.

Table 7 Status in terms of highest/lowest ratio of enzymatic antioxidants in flag leaf after 5 days of anthesis
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Table 8 Status in terms of highest/lowest ratio of non enzymatic antioxidants in flag leaf after 5 days of anthesis
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Variability of enzymatic and non enzymatic antioxidants in mature grains of wheat

Tukey’s comparison indicated significant difference in superoxide dismutase (SOD) activity in the mature grains of wheat cultivars (Table 9). Mean comparison of the cultivars showed that higher activity of SOD in Australian cultivars. Within Australian cultivars wheat cultivars Carnamah has maximum SOD activity. Minimum SOD activity was observed in BW 7197 and PBW 175.

Table 9 Variability of enzymatic antioxidants in mature grains of wheat
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Activity of glutathione reductase (GR) is depicted in Table 9. Minimum GR activity was observed in Australian cultivars. Within Australian cultivars Stretton and Binnu has minimum GR activity. DBW 17, WH 1021, PBW 175, PBW 527, BWL 73, BW 7296 and BWL 676 have higher activity of GR. Among four groups rain-fed cultivars have the highest GR activity.

Mean comparison of catalase (CAT) activity did not show significant difference among four groups. Maximum CAT activity was present in advanced breeding lines while the minimum CAT activity was present in rain-fed cultivars (Table 9). BWL 927 and BWL 934 have high CAT activity.

Tukey’s comparison indicated significant difference in ascorbate peroxidase (APX) activity in the wheat cultivars (Table 9). High APX activity was observed in rain-fed cultivars. Within advanced breeding lines BW 4101 and BWL 924 have highest APX activity. Esfandiari et al. (2007) showed that in the leaves of wheat cultivar having high APX activity was related with drought tolerance but no reports are available in the literature showing correlation between activity of APX in wheat grains and abiotic stress tolerance.

Mean comparison of DPPH did not show significant difference (Table 10). Data of non enzymatic antioxidants such as Proline, MDA and H2O2 showed the variability in their content in the mature grains of wheat. Tukey’s comparison indicated significant difference in Proline, MDA and H2O2 content (Table 10). Among the four groups rainfed cultivars have maximum Proline, MDA and H2O2 content.

Table 10 Variability of non-enzymatic antioxidants in mature grains of wheat
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Kumar (2007) reported that high activity of APX and high proline content along with activity of GR and APX might be related to drought stress tolerance. Devi (2008) proposed that high activity APX and GR in mature grains of wheat could be associated with drought tolerance. Grains of DBW 17 (commercial cultivar), PBW 175, PBW 527 and BW 7296 (rainfed cultivars) and PBW 676, BWL 934, BW 7296 (advanced breeding lines) have both high APX and GR activities. These advanced breeding lines can be tested in the field for drought tolerance.

In the Table 11, an attempt has been made to identify genotypes which could show tolerance to different abiotic stresses. On the basis of available information in the literature, high activity of antioxidant enzymes with low H2O2 and MDA content in tissues of normally growing wheat genotypes could help in tolerating different abiotic stresses (Kahrizi et al. 2012, Sairam et al. 1998, Hasheminasab et al. 2012, Valifard et al. 2012, Ashraf et al. 2010).

Table 11 Proposed genotypes for studying tolerance to different abiotic stresses on the basis of status of enzymatic and non enzymatic antioxidants
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A genotype if having three characters (high antioxidant enzymes like APX, GR, SOD, CAT, POX and low H2O2 and MDA content) out of proposed seven, it might be worth studying for tolerance towards different abiotic stresses. However, it is difficult to pin point the abiotic stress to which genotypes could be tolerant as of non specificity in the role of antioxidant enzymes to different kinds of stresses. Though PBW 550 has been recommended for cultivation under irrigated conditions, but because of high antioxidant activity in leaves during vegetative and flag leaf stage, it could be worthwhile to study the performance of this high yielding cultivar under different abiotic stresses like water deficit conditions (rainfed) and high temperature stress (late sowing). In study conducted over fifty genotypes, Kumar (2007) proposed high activity of APX and proline content of wheat grains could be related with drought tolerance. Devi (2008) proposed that high activity APX and GR in mature grains of wheat could be associated with drought tolerance. On this basis, nine genotypes have at least two of three characters in mature grains. Two advanced breeding lines namely PBW 676 and BWL 73 could be drought tolerant. However, a field study is necessary for validating this proposal.