Potential of antioxidant enzymes in depicting

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Department of Biochemistry, Punjab Agricultural University, Ludhiana 141004, Punjab, India. Received ..... resistance to photo inhibition in poplar36. Increased ...
Indian Journal of Biochemistry & Biophysics Vol. 49, August 2011, pp. 257-265

Potential of antioxidant enzymes in depicting drought tolerance of wheat (Triticum aestivum L.) Rachana Devi, Narinder Kaur and Anil Kumar Gupta* Department of Biochemistry, Punjab Agricultural University, Ludhiana 141004, Punjab, India Received 21 February 2012; revised 14 June 2012 In an effort to determine the biochemical markers for identifying genotypes before sowing for drought tolerance, changes in activities of antioxidant enzymes were determined in the seedlings of five drought-tolerant and five drought-sensitive wheat (Triticum aestivum L.) genotypes, each with different genetic background growing under normal and water deficit conditions induced by 6% mannitol. In comparison with non-stressed seedlings, the catalase (CAT) activity was upregulated by more than 50% in the roots of water-stressed seedlings in drought-tolerant genotypes. Water deficit stress also led to the upregulation of ascorbate peroxidase (APX) in the endosperms and glutathione reductase (GR), CAT and peroxidase (POD) in the shoots of stressed seedlings in drought-tolerant genotypes. Superoxide dismutase (SOD) activity was very low in roots and shoots and showed non-significant increase under water-stress in tolerant genotypes. Out of five specified enzyme activities (CAT in roots and shoots, APX in endosperms, GR and POD in shoots), if any three are upregulated in the specified tissues under water deficit conditions, the genotype is likely to be drought-tolerant. Wheat seedlings with low GR and APX activities and high POD activity in shoots with a low ratio of GR activity of shoot to root of non-stressed seedlings are likely to perform better under rainfed conditions. The observed data showed that status of antioxidant enzymes could provide a meaningful tool for depicting drought tolerance of a wheat genotype. Keywords: Antioxidant enzymes, Wheat, Seedling growth, Drought, Triticum aestivum L.

Abiotic stresses, such as drought, soil salinity and extreme temperatures adversely affect the productivity and quality of economically important crops throughout the world. Drought stress is one of the major limitations to crop productivity. In India, water deficit stress limits crop production in about 67% of net sown area. Wheat yields are reduced by 50-90% of their irrigated potential by drought on at least 60 million hectare in the developing world1. Improving drought tolerance and productivity is one of the most difficult tasks for cereal breeders. The difficulty arises from the diverse strategies adopted by the plants themselves to combat drought stress, depending on the timing, severity and stage of crop growth2,3. During optimal conditions, the balance between reactive oxygen species (ROS) formation and consumption is tightly controlled by antioxidant enzymes and redox metabolites4,5. These include superoxide dismutase (SOD), catalase (CAT), _______________ *Corresponding author E-mail: [email protected] Fax: 91-161-2400945; Tel: 09872452820 (M) Abbreviations: APX, ascorbate peroxidase; CAT, catalase; GR, glutathione reductase; POD, peroxidase; ROS, reactive oxygen species; SOD, superoxide dismutase.

ascorbate peroxidase (APX), peroxidase (POD), glutathione reductase (GR) and redox metabolites such as ascorbic acid and glutathione. Most environmental stresses, including drought induce enhanced production of ROS6-9. Antioxidant enzymes are important components in the mechanism of drought and desiccation tolerance10-14. Relationship between antioxidants levels and stress tolerance has been demonstrated in transgenic plants15. Drought/abiotic stress tolerance is dependent on multiple genes and antioxidant enzymes constitute an important group involved in abiotic stress tolerance. For depicting drought tolerance of wheat, we need to test a number of genotypes in the field. This involves lot of money, time and man power. There are various reports in the literature on upregulation of antioxidant enzymes under abiotic stresses in a number of crops16-19, but studies are lacking wherein status of antioxidant enzymes in the seedlings could depict drought tolerance of wheat crop. In this study, we have investigated the status of antioxidant enzymes under normal and water deficit conditions in seedlings of five drought-tolerant and droughtsensitive genotypes cultivated in India in order to see the possibility of using antioxidant enzymes as a

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possible indicator of drought tolerance. Some peculiar characters have been obtained based on the profile of antioxidative enzymes during seedling growth which could help in further depicting drought tolerance nature of wheat (Triticum aestivum L.) genotypes. Materials and Methods Plant material and growth conditions

Ten wheat (Triticum aestivum L.) genotypes comprising of five drought-tolerant (C306, NI5439, PBW175, PBW299 and PBW396) and five water deficit-sensitive genotypes (HD2329, HUW234, LOK1, PBW343 and WL711) were used in the study. Drought-tolerant genotypes are being cultivated in different parts of India under rain-fed conditions, whereas sensitive genotypes are sown under irrigated conditions. These genotypes were released in different years and have different genetic background (Table 1). Wheat seeds were dipped in 0.1% HgCl2 for 5 min and washed thoroughly with sterilized water under aseptic conditions. Seeds were germinated in the conical flasks containing 0.9% agar. Water deficit stress was induced by adding 6% mannitol. The flasks were kept in an incubator at 25ºC in the dark. Data for the root growth were taken at 7 day of growth. Samples of seedlings (roots and shoots at days 4 and 7 and endosperms at days 1, 4 and 7) were taken for enzymatic studies. Mean of the enzyme activity data obtained at different days was obtained and presented. Each experiment was replicated three-times and activities were determined in duplicate enzyme extracts. The values presented were mean of 12-18 recordings. Extraction and assay of enzymes

The enzymes were extracted at 4ºC in triplicate from roots, shoots and endosperms (500 mg) by crushing the tissues in a pre-chilled pestle and mortar with 3 ml of extraction buffer20. Each sample was Recommended cultivation conditions Rain-fed Rain-fed Rain-fed Rain-fed Rain-fed Irrigation Irrigation Irrigation Irrigation Irrigation

Genotype C 306 NI 5439 PBW 175 PBW 299 PBW 396 HD2329 HUW 234 LOK 1 PBW 343 WL 711

taken from a representative sample of at least three seedlings. All the activities were determined at 30ºC. Catalase (CAT, EC 1.11.1.6) activity was determined by the procedure described previously21. Assay mixture consisted of 30 mM potasium phosphate buffer (pH 7.5) and enzyme extract. The reaction was initiated by adding 13 mM H2O2 and utilization of H2O2 was recorded at intervals of 30 s for 3 min by measuring the decrease in absorbance at 240 nm. The activity was expressed as µmoles of H2O2 decomposed min-1g-1 tissue. Ascorbate peroxidase (APX, EC 1.11.1.1) was assayed according to the method of Nakano and Asada22. The reaction mixture consisted of 30 mM sodium phosphate buffer (pH 7.0), 0.13 mM ascorbic acid, enzyme extract and 13 mM H2O2 solution. The enzyme activity was measured as decrease of absorbance at 290 nm. Extinction coefficient of monodehydro ascorbic acid is 2.8 mM-1 cm-1. APX activity was expressed as nmole of monodehydro ascorbic acid formed min-1 g-1 tissue. Glutathione reductase (GR, EC 1.6.4.2) activity was assayed according to the previously described method23. Assay system consisted of 40 mM potassium phosphate buffer (pH 7.5), 0.02 mM EDTA, 0.15 mM MgCl2, 0.1 mM NADPH and enzyme extract along with 0.4 mM glutathione. The enzyme activity was estimated as decrease of absorbance at 340 nm after an interval of 30 s up to 3 min. GR activity was expressed as nmoles of NADP+ formed min-1 g-1 tissue. Peroxidase (POD, EC 1.11.1.7) activity was measured as described by Shannon et al24. The reaction mixture consisted of 50 mM guaiacol in 100 mM phosphate buffer (pH 6.5) and enzyme extract along with 25 mM H2O2. The reaction mixture without H2O2 was considered as a blank. The reaction was initiated by adding H2O2 and rate of change in absorbance was recorded at 470 nm for 3 min at an

Table 1___Pedigree and origin of 10 wheat genotypes used Pedigree RGN/CSK 3//2* C591/3/C 217/N 14//C 281 REPM 80/3* NP 710 HD2160/4/JN/GAGE/JN/KAL/3/V18/C273 BB/KAL//WL711/PBW65 CN067/MFD//NON’S’/3/SERI82 HD2252/UP262 HUW 12/SPRW//HUW12 S308/S331 ND/VG1944//KAL/BB/3/YCO “S”/4/VEE5 S308/CHR/KAL

Year of release 1965 1973 1988 1991 1996 1983 1985 1981 1995 1979

Origin Punjab, India Maharashtra, India Punjab, India Punjab, India Punjab, India New Delhi, India UP, India Gujarat, India Punjab, India Punjab, India

DEVI et al: POTENTIAL OF ANTIOXIDANT ENZYMES IN DROUGHT TOLERANCE

interval of 30 s. The activity was expressed as change in absorbance min-1 g-1 tissue. Superoxide dismutase (SOD, EC 1.15.1.1) activity was determined by the method of Marklund and Marklund25. Assay system consisted of 50 mM Tris HCl buffer (pH 8.2), 1 mM EDTA, 2 mM pyrogallol solution and enzyme extract. Absorbance was recorded at 420 nm after an interval of 30 s upto 3 min. One unit of enzyme activity was defined as the amount of enzyme causing 50% inhibition of auto-oxidation of pyrogallol observed in blank.

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Maximum CAT activity was observed in the shoots, followed by roots and endosperms of wheat seedlings both under control and stressed conditions. Of five genotypes recommended for cultivation under rainfed conditions, four (NI5439, PBW175, PBW299 and PBW396) showed more than 50% increase in CAT activity in roots under water deficit conditions, whereas such increase was observed only in one cultivar (HUW234) sensitive to water deficit conditions. Average increase in CAT activity in the roots of tolerant genotypes under water deficit conditions was more than 60% as compared to control seedlings. This increase was only 15% in the roots of sensitive genotypes under water deficit conditions. In shoots, three tolerant genotypes showed 40% induction in CAT activity under water deficit conditions, whereas no such induction was observed in sensitive genotypes (Table 3). No specific generalization could be made for CAT activity in endosperm of drought-tolerant and sensitive genotypes. Maximum APX activity was in the shoots, followed by roots and endosperms in the control as well as in the stressed seedlings. In all the genotypes, except PBW343 water deficit caused either no effect or upregulated the APX activity in roots. However, in shoots, all the tolerant genotypes, except PBW396 had low APX activity in control seedlings. In sensitive genotypes, only PBW343 had low APX activity in shoots. The average APX activity in the shoots of sensitive genotypes of control seedlings was about 52% higher, as compared to that observed in five tolerant genotypes. Low APX in shoots could be one of the indicators for drought tolerance capability. The average upregulation of APX (28%) in shoots of tolerant genotypes was higher as compared to only 6% in sensitive genotypes under water deficit conditions. However, individual variation in different

Results Effect of 6% mannitol on root growth

Imposition of water deficit stress induced by 6% mannitol led to reduction in the root length of all ten cultivars used in the study. The mean root length of control seedlings of five drought-tolerant genotypes was 13.1 cm as compared to 11.0 cm of the genotypes requiring assured irrigation. Application of water deficit caused on an average about 35% reduction in root length of drought-tolerant as compared to 44% in sensitive genotypes. However, individual variation like drought-sensitive HD 2329 showing only 37% reduction in root length under water deficit conditions was also observed (Table 2). Effect of water deficit stress on antioxidant enzymes

Activities of antioxidative enzymes in the roots, shoots and endosperms of wheat seedlings were determined at different days of seedling growth. Because of ten genotypes used in the present study and to minimize the effects of differential growth rates of the seedlings due to diverse genetic background, the average of activities observed in endosperm at day 1, 4 and 7 and in roots and shoots at day 4 and 7 were presented.

Table 2___Effect of water stress induced by 6% mannitol on root growth (cm) at 7 day of seedling growth [Values are mean + SD of at least 20 seedlings] Drought-tolerant hexaploid genotypes

Drought-sensitive hexaploid genotypes

C306

NI 5439

PBW 175

PBW 299

PBW 396

Mean length

HD 2329

HUW 234

LOK 1

PBW 343

WL 711

Mean Length

Control

11.3 +1.1

15.1 +1.4

11.0 +0.8

15.4 +0.8

12.7 +1.2

13.1

10.5 +1.9

9.0 +0.6

10.3 +0.4

12.6 +0.6

12.8 +0.2

11.0

6% Mannitol

7.4 +0.2*

10.6 +0.9*

6.8 +0.8*

9.9 +1.2*

7.7 +0.4*

8.5

6.6 +0.7*

5.0 +0.6*

5.1 +1.1*

6.6 +0.2*

7.3 +0.1*

6.1

Reduction in root length (%)

34.5

29.8

38.2

35.7

39.4

35.5

37.1

44.4

50.5

47.6

42.9

44.5

*

Differences significant in comparison with respective control at ≤ 0.05 (student’s t-test)

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Table 3___Effect of water stress induced by 6% mannitol on average CAT activity in the roots, shoots and endosperms of different wheat genotypes during seedling growth Control

Root 6% Mannitol

% Change

Control

Shoot 6% Mannitol

% Change

Control

228.2 84.7 87.8 110.2 158.5 133.9b

279.3 141.4 173.6 214.6 276.9 217.2a

22.3 66.9 97.7 94.7 74.7 62.2

445.9 188.1 444.3 287.6 357.3 344.6

476.3 273.3 154.6 399.9 552.2 371.3

6.8 45.3 -65.2 39.0 54.5 7.8

36.5 36.5 48.4 66.1 65.9 50.7

Genotype

Drought tolerant

C306 NI5439 PBW175 PBW299 PBW396 Mean*

Endosperms 6% % Change Mannitol 46.3 33.9 35.7 90.3 98.7 61.0

27.4 -7.1 -26.2 36.6 49.8 16.9

Drought sensitive

HD2329 139.5 123.5 -11.4 367.2 317.4 -13.5 52.9 34.0 -35.7 HUW234 79.6 126.7 59.1 311.8 280.0 -10.1 31.2 26.8 -14.1 LOK1 160.3 165.3 3.1 316.2 304.5 -3.7 16.5 27.4 66.1 PBW343 128.7 165.1 28.2 287.0 262.3 -8.6 81.4 104.0 27.8 WL711 166.2 199.0 19.7 428.5 532.8 24.3 86.5 138.1 -59.7 15.6 342.1 339.4 -0.8 53.7 66.1 18.8 Mean** 134.9 155.9c Differences significant in comparison with a to b; c to a at ≤ 0.05 (student’s t-test). CAT activity is represented as µmoles of H2O2 decomposed min -1 g-1 FW. Data have been calculated by taking the mean of enzyme activities at days 4 and 7 for roots and shoots, and at days 1, 4 and 7 for endosperms. The values are mean of 12 to 18 recordings obtained at different days in three replicate experiments. *Mean of five drought-tolerant genotypes. **Mean of five drought sensitive genotypes. Table 4___Effect of water stress induced by 6% mannitol on average APX activity in the roots, shoots and endosperms of different wheat genotypes during seedling growth. Root

Genotype Control Drought tolerant

C306 NI5439 PBW175 PBW299 PBW396 Mean*

1927 1046 419 769 1679 1168b

6% Mannitol 2487 1155 555 787 2039 1405a

Shoot % Change

Control

29.0 10.4 32.4 2.3 21.4 20.3

1818 1516 928 1563 2680 1701d

Endosperms

6% Mannitol % Change 3013 1590 791 1645 3899 2188

65.7 4.8 -14.7 5.2 45.4 28.6

Control 170 241 198 205 121 187f

6% Mannitol 386 254 296 296 129 272e

% Change 127.0 5.1 49.5 44.3 6.6 45.5

Drought sensitive

HD2329 1780 2043 14.7 3399 3675 8.1 197 200 1.5 HUW234 1907 2785 46.0 2528 3469 37.2 501 495 -1.1 LOK1 1656 1751 5.7 3275 2630 -19.6 285 269 -5.6 PBW343 622 411 -33.9 1212 1232 1.6 215 289 34.4 WL711 1043 1278 22.5 2572 2830 10.0 100 122 22.0 2767 6.5 260 275 5.8 Mean** 1402 1654 18.0 2597c Differences significant in comparison with a to b; c to d; e to f at ≤ 0.05 (student’s t-test) APX activity is represented as nmoles of monodehydro ascorbic acid formed min -1 g-1 FW. Data have been calculated by taking the mean of enzyme activities at days 4 and 7 for roots and shoots, and at days 1, 4 and 7 for endosperms. The values are mean of 12 to 18 recordings obtained at different days in three replicate experiments. *Mean of five drought-tolerant genotypes. **Mean of five drought sensitive genotypes.

genotypes was significant and no generalization on behaviour of APX activity could be made under water deficit conditions. In the endosperms of water deficit stressed seedlings, out of 5 drought-tolerant genotypes, three (C306, PBW175 and PBW299) showed about 40% or more induction of APX activity as compared to endosperms of control seedlings. None of the sensitive genotypes showed such large upregulation of APX in endosperms under water deficit conditions (Table 4). GR activity was significantly higher in the roots and shoots of wheat seedlings as compared to the endosperms under both normal and stressed

conditions. Imposition of water stress led to an increase in GR activity in the roots and shoots of most of the genotypes, however, no such trend was obtained in the endosperms. All the drought-tolerant genotypes showed lower GR activity (at least by 30%) in the shoots of control seedlings as compared to average GR activity in sensitive genotypes. Three drought-tolerant genotypes (C306, NI5439 and PBW175) showed 30% less GR activity in the endosperms of control seedlings, as compared to average GR activity in sensitive genotypes. An upregulation of GR activity was observed in the shoots (by about 40%) of all drought-tolerant

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Table 5___Effect of water stress induced by 6% mannitol on average GR activity in the roots, shoots and endosperms of different wheat genotypes during seedling growth. Genotype Control Drought tolerant

C306 NI5439 PBW175 PBW299 PBW396 Mean*

183.6 341.1 454.4 399.8 461.7 368b

Root 6% % Change Mannitol 325.9 77.5 345.6 1.3 510.1 12.2 528.9 32.2 520.3 12.6 446a 21.2

Control 286.7 655.6 592.8 713.0 650.5 580d

Shoot 6% % Change Mannitol 504.7 88.5 1045.5 59.4 1643.5 177.2 1377.3 93.1 972.3 49.4 1109c 91.2

Control 80.7 127.2 114.3 150.9 140.5 123h

Endosperms 6% % Change Mannitol 102.9 27.5 123.1 -3.2 112.3 -1.7 185.5 22.9 124.7 -11.2 130j 5.7

Drought sensitive

HD2329 432.3 558.4 29.1 878.5 1778.8 102.4 200.6 211.2 5.2 HUW234 412.8 582.0 40.9 934.6 1154.2 23.5 206.2 183.3 -11.1 LOK1 323.2 393.3 21.6 841.6 1236.3 46.9 144.1 170.5 18.3 PBW343 469.2 477.5 1.7 1384.7 1332.4 -3.7 225.6 210.6 -6.6 WL711 485.2 455.5 -6.1 1257.0 1452.4 15.5 164.8 156.1 -5.5 Mean** 425 493 16.0 1059f 1391e 31.4 188g 186i 1.1 Differences significant in comparison with a to b; c to d; e to f; f to d; g to h; i to j at ≤ 0.05 (student’s t-test). GR activity is represented as nmoles of NADP+ formed min-1 g-1 FW. Data have been calculated by taking the mean of enzyme activities at days 4 and 7 for roots and shoots, and at days 1, 4 and 7 for endosperms. The values are mean of 12 to 18 recordings obtained at different days in three replicate experiments. *Mean of five drought-tolerant genotypes. **Mean of five drought sensitive genotypes Table 6___Effect of water stress induced by 6% mannitol on average POD activity in the roots, shoots and endosperms of different wheat genotypes during seedling growth Root

Genotype Control Drought tolerant

C306 NI5439 PBW175 PBW299 PBW396 Mean*

510.8 383.7 370.9 183.5 274.2 345b

6% Mannitol 680.0 603.6 447.2 248.9 346.4 465a

Shoot %Change

Control

33.2 57.3 20.6 35.7 26.4 34.8

212.9 160.5 156.5 55.3 81.7 133d

6% Mannitol 383.6 246.6 442.9 127.7 112.5 263c

Endosperms %Change

Control

80.2 53.7 183.1 130.9 37.8 97.7

116.6 25.0 1.7 5.8 13.2 33

6% Mannitol 142.6 51.4 1.9 4.9 12.9 43

% Change 22.3 105.6 8.8 15.1 -1.7 30.3

Drought sensitive

HD2329 119.3 238.1 99.7 39.9 116.7 192.2 24.1 12.6 -47.5 HUW234 131.5 208.6 58.7 66.9 93.1 39.1 13.9 15.9 14.3 LOK1 216.9 279.0 28.7 121.2 114.3 -5.7 6.7 5.6 -16.4 PBW343 235.6 274.5 16.5 125.7 111.2 -11.5 4.6 4.3 -8.6 WL711 419.1 385.7 -7.9 135.0 134.6 -0.3 16.3 6.9 -57.6 Mean** 225 277e 23.1 98 114f 16.3 13 9 -30.8 Differences significant in comparison with a to b; c to d; e to a; f to c at ≤ 0.05 (student’s t-test) POD activity is represented as change in absorbance min -1 g-1 FW. Data have been calculated by taking the mean of enzyme activities at days 4 and 7 for roots and shoots, and at days 1, 4 and 7 for endosperms. The values are mean of 12 to 18 recordings obtained at different days in three replicate experiments. * Mean of five drought-tolerant genotypes. **Mean of five drought sensitive genotypes.

genotypes under water deficit conditions. However, such upregulation was also observed in two sensitive genotypes i.e., HD2329 and LOK1 (Table 5). Maximum POD activity was in the roots, followed by shoots and endosperms under both non-stressed and stressed conditions. In general, POD activity increased in the roots and shoots of stressed seedlings, except for roots of drought-sensitive genotype (WL 711) and shoots of three drought-sensitive genotypes LOK1, WL 711 and PBW 343. However, no significant pattern was observed in the endosperms

of different wheat seedlings. In all drought-tolerant genotypes, except PBW396 water deficit led to an upregulation of POD activity in the shoots by more than 50%. However, only one sensitive genotype (HD2329) showed 50% induction of POD activity in shoots of stressed seedlings (Table 6). Out of five drought-tolerant genotypes, C306, NI5439 and PBW175 showed at least 100% higher POD activity in the stressed shoots, as compared to the average POD activity of sensitive genotypes. However, in the roots, three drought-tolerant genotypes (C306,

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NI5439 and PBW175) had at least 50% higher POD activity in the control seedlings, as compared to corresponding average POD activity of sensitive genotypes. The average POD activity in roots and shoots of tolerant genotypes was at least 35% higher as compared to sensitive genotypes (Table 6). SOD activity was negligible in roots and shoots and low in endosperms. Water deficit led to small

increase of SOD activity in endosperms of tolerant genotypes. However, such increase in activity was observed only in one sensitive genotypes HD 2329 (Table 7). Important differences in activities of antioxidant enzymes in different tissues of ten genotypes under water deficit and control conditions have been summarized in Table 8. Discussion Imposition of water deficit stress led to inhibition of growth in wheat seedlings. In general, root length of the drought-sensitive genotypes was significantly reduced as compared to drought-tolerant genotypes. Maintenance of root growth during water deficit is beneficial to maintain adequate plant water supply26. More than 50% increase in CAT activity in roots of 4 tolerant genotypes as compared to only 1 sensitive genotype under water deficit conditions with almost similar response of this enzyme in shoots (Table 3) indicated an important role of CAT in drought tolerance. Though there was not much difference in the mean CAT activity in roots of tolerant and sensitive genotypes, but tolerant genotypes had significantly higher capacity to upregulate CAT under water deficit conditions and this property appeared to one of the determining factors towards drought tolerance. Increase in CAT activity has been shown to be related with increase in stress tolerance capacity27. CAT does not require any additional reductant for scavenging H2O2, and is efficient in removing high level of H2O2 in tissues28.

Table 7___Effect of water stress induced by 6% mannitol on average SOD activity in the endosperms of different wheat genotypes during seedling growth Genotype Control Drought tolerant

Endosperms 6% Mannitol % Change

C306

37.5

42.9

14.4

NI5439 PBW175 PBW299 PBW396 Mean*

67.0 20.8 61.9 43.1 46.1

73.6 26.5 73.4 49.2 53.1

9.9 27.4 18.5 14.1 15.3

HD2329

23.9

35.7

49.4

HUW234 LOK1 PBW343 WL711 Mean**

41.6 76.6 73.3 59.8 55.1

49.7 73.6 72.5 50.1 56.3

19.5 -3.9 -1.2 -16.2 2.2

Drought sensitive

SOD activity is represented as number of units min-1 g-1 FW. One unit corresponds to amount of enzyme required for 50% inhibition of autooxidation of pyrogallol. Data have been calculated by taking the mean of activities at days 1, 4 and 7 in the endosperms. *Mean of five drought-tolerant genotypes. **Mean of five drought sensitive genotypes.

Table 8___Overall summary for the effect of water stress induced by 6% mannitol on antioxidant enzymes in different wheat genotypes during seedling growth Genotype

Stressed seedlings a

Drought-tolerant

Drought-sensitive

C306 NI5439 PBW175 PBW299 PBW396 HD2329 HUW234 LOK1 PBW343 WL711

+ + + +

b + + +

c

d

+ + + + +

+ + + +

+

+

Control seedlings e

f

+ + +

+ + +

g

h

+ + +

+ + + + +

+ + + +

a) CAT induction in roots by more than 50% under stress ; b) CAT induction in shoots by more than 30% under stress; c) GR induction in shoots by more than 40% under stress ; d) 50% induction of POD in shoots under stress ; e) APX induction in endosperms by more than 40% under stress ; f) Low APX in shoots of control seedlings (at least 25% less in comparison with average APX in the shoots of control seedlings of sensitive genotypes) ; g) High POD in roots of control seedlings (at least 50% higher as compared to average POD of roots of control seedlings of sensitive genotypes) and h) Low GR in shoots of control seedlings (at least 30% less as compared to average GR activity in the shoots of control seedlings). + Sign indicates that the character identified is present.

DEVI et al: POTENTIAL OF ANTIOXIDANT ENZYMES IN DROUGHT TOLERANCE

APX scavenges H2O2 and uses ascorbate as an electron donor in plants. Water stress-induced stimulation of APX activity was more pronounced in the shoots and endosperms of drought-tolerant genotypes than the sensitive ones (Table 4). The overexpression of a gene for APX during drought can provide protection against excessive photorespiratory H2O2 production29. Upregulation of APX in wheat roots13 and leaves of drought-tolerant wheat genotype under water deficit conditions has been reported30. Increased APX activity is also reported in the roots of lentil seedlings under water deficit conditions31. Increase in APX activity might be an adaptive response of wheat seedlings to higher amounts of ROS generated under water deficit conditions32. Wheat genotypes with better drought tolerance than others maintain higher antioxidant enzyme activities, resulting in lower oxidative damage. This behavior is dependent on genetic potential of genotypes33. GR has an important role in maintaining adequate GSH/GSSG ratio in favour of GSH. GR activity increased in the roots and shoots of stressed seedlings in all wheat genotypes, except in the roots of WL711 (drought-sensitive) and shoots of PBW343 (droughtsensitive). However, water deficit-induced upregulation of GR activity was significantly higher in the shoots of drought-tolerant genotypes. Earlier, efficient function of GR has also been reported in drought-tolerant C306 wheat genotype30. Increased GR activity is also reported in the water-stressed maize seedlings34. Drought stress is also shown to cause an increase in GR activity in the roots of Ctenanthe setosa35. Overexpression of GR has been reported to increase the antioxidant capacity and resistance to photo inhibition in poplar36. Increased GR activity might contribute for increased GSH/GSSG ratio, which could be essential for drought resistance of plants. The elevated level of antioxidants under abiotic stress could play an important role in preventing the stress-induced accumulation of ROS4. An interesting observation was found in average GR ratio in shoots and roots of control seedlings. In five tolerant genotypes, this ratio varied from 1.3 to 1.9, whereas in sensitive genotypes, the average GR ratio of shoots to roots varied from 2.0 to 2.9 (calculated from data presented in Table 5). A lower GR ratio of shoot to root in control seedlings might indicate drought-tolerant nature of a genotype and could be an important marker of drought tolerance in wheat.

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PODs are often the first antioxidative enzymes the activities of which are altered under stress. In the present study, water deficit stress-induced increase in POD activity was more pronounced, especially in shoots of drought-tolerant genotypes than in sensitive ones (Table 6). In wheat, higher POD in leaves of C 306 in comparison with sensitive genotypes has been reported13. Hameed et al33 have also reported that H2O2 scavenging system is more actively involved in detoxification of oxidative stress induced by water deficit in wheat. Higher POD activity in the roots and shoots of control seedlings with significant upregulation in the shoots under water deficit conditions of drought-tolerant genotypes appeared to be related with drought tolerance capacity of wheat genotype. Enhanced POD activity due to its de novo synthesis has been observed in rice seedlings under anoxia37 and low temperature stress38. Water deficit stress-induced increase in POD activities was also more pronounced in drought-tolerant Sorghum cultivars39. SOD activity was higher in the endosperms of drought-tolerant genotypes under water deficit conditions, but no significant differences between drought-tolerant and susceptible genotypes were obtained (Table 7). There are reports indicating increased SOD activity in wheat21, maize40 and Cassia spp41 under water deficit conditions. However, reports also indicate that water deficit stress does not influence SOD activity in wheat3 and Sorghum42. Thus, it appears that the effect of water deficit on SOD might be tissue and genotype-specific. There are reports in the literature on differential expression of antioxidant genes in drought-tolerant and sensitive wheat genotypes under water deficit and chilling stress conditions43,44. For example, in two wheat genotypes having difference in their tolerance to water deficit conditions, relative transcript levels of APX and GR have shown different transcriptional changes under reduced irrigation. Increased expression level of a cytosolic and a chloroplastic GR has been detected only in the tolerant wheat cultivar43. Possibly, a robust transcription of ascorbate-based detoxification machinery might help in preventing the adverse effect of reduced irrigation. Under chilling stress also, in near isogenic lines of wheat, upregulation of GR has been reported44. In rice also, cytosolic APX has shown increased expression level under oxidative stress45. APX gene has been shown to improve drought tolerance in tobacco46. Most of the

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genes directly related to antioxidant responses are induced in drought-tolerant cultivar of potato, while they are repressed or induced to a lesser degree in sensitive genotype under drought stress47. Therefore, possibly the changes in the activities of antioxidant enzymes in the present study might be due to differential expression of corresponding genes. Antioxidant enzymes, such as SOD, CAT, APX, POD and GR are known to reduce the level of superoxide and H2O2 in plants9. Activity of APX and GR has been reported to increase in salt-tolerant genotypes under NaCl stress48. Free radical scavenging systems are important components of drought and desiccation tolerance10,11,49. Significant induction of CAT, GR and POD in shoots, CAT in roots and APX in endosperms under water deficit conditions is associated with tolerant nature of wheat genotype to water deficit conditions. Presence of at least three characters out of five proposed above indicated drought tolerance nature of a genotype. Drought tolerance nature can also be identified by low APX and GR activity in shoots and high POD activity in roots of control seedlings (Table 8). By combining the observations of antioxidant enzymes in both control and stressed seedlings, one can more accurately predict the drought tolerance nature of wheat genotype. Most of the contemporary studies on water deficit stress have been conducted on one drought-tolerant and one drought-sensitive genotypes. However, we used ten genotypes with different response to drought, and the study showed the potential of using antioxidant enzymes for depicting drought tolerance from the laboratory data obtained with seedlings. Thus, status of antioxidant enzymes could be a very useful tool for depicting drought tolerance of wheat, which could be useful to plant breeders for developing drought-tolerant cultivars. However, further studies are needed to confirm the role of antioxidant enzymes for depicting drought tolerance in a large number of genotypes. Acknowledgement Thanks are due to Dr R G Saini of Department of Plant Breeding and Genetics of Punjab Agricultural University, Ludhiana for providing the wheat germplasm.

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