Evaluation of oxidative stress tolerance in maize (Zea mays L.) seedlings in response to drought

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1 Indian Journal of Biochemistry & Biophysics Vol. 48, February 2011, pp Evaluation of oxidative stress tolerance in maize (Zea mays L.) seedlings in response to drought Vishal Chugh, Narinder Kaur and Anil K Gupta* Department of Biochemistry, Punjab Agricultural University, Ludhiana , Punjab, India Received 15 June 2010; revised 29 November 2010 Seedlings of selected six genotypes of maize (Zea mays L.) differing in their drought sensitivity (LM5 and Parkash drought-tolerant and PMH2, JH3459, Paras and LM14 as drought-sensitive) were exposed to 72 h drought stress at two leaf stage. Alterations in their antioxidant pools combined with activities of enzymes involved in defense against oxidative stress were investigated in leaves. Activities of some reactive oxygen species (ROS)-scavenging enzymes, catalase (CAT) and ascorbate peroxidase (APX) were enhanced in tolerant genotypes in response to drought stress. Superoxide dismutase (SOD) activity was significantly decreased in sensitive genotypes, but remained unchanged in tolerant genotypes under stress. Peroxidase (POX) activity was significantly induced in tolerant, as well as sensitive genotypes. Imposition of stress led to increase in H 2 O 2 and malondialdehyde (MDA, a marker for lipid peroxidation) content in sensitive genotypes, while in tolerant genotypes no change was observed. Significant increase in glutathione content was observed in sensitive genotypes. Ascorbic acid pool was induced in both tolerant and sensitive genotypes, but induction was more pronounced in tolerant genotypes. Significant activation of antioxidative defence mechanisms correlated with drought-induced oxidative stress tolerance was the characteristic of the drought tolerant genotypes. These studies provide a mechanism for drought tolerance in maize seedlings. Keywords: Zea mays L., Maize, Drought, Antioxidant system, Lipid peroxidation Maize (Zea mays L.) is the third most important food crop grown worldwide. Various environmental stresses adversely affect the growth and productivity of this crop. Abiotic stresses are the primary cause of crop loss worldwide reducing average yields for most major crop plants, including maize by more than 50% 1. Among the abiotic stresses, drought stress is the most important. Considering ongoing climatic changes caused principally by global warming, the pressure on crop production in water-limited environments is expected to increase. It is estimated that by 2050 food production will need to increase by at least 50% to match the projected population growth 2. One way for improving maize production is to elucidate the mechanism of drought tolerance which is important to construct drought-resistant genotypes. Of the numerous biochemical responses to drought stress 3, the antioxidative response is one of the most important 4. This is because reactive oxygen species (ROS), which are produced in large amounts under drought conditions can cause a serious damage to Corresponding author anilkgupta@sify.com Fax: ; Tel: (M) plant cell 5,6. To cope with the high reactivity of ROS, plants possess antioxidative mechanisms, including superoxide dismutase (SOD), ascorbate peroxidase (APX), catalase (CAT) and peroxidase (POX). Activities of these enzymes are induced by oxidative injury caused by biotic and abiotic factors 4. It is generally known that superoxide radicals originated in electron transport chains due to excess energy are dismutated to oxygen and hydrogen peroxide (H 2 O 2 ) by SOD, whereas APX and CAT are responsible for the detoxification of H 2 O 2. Low molecular weight antioxidants, such as ascorbic acid (ASA) and glutathione also play important role in quenching ROS. Ascorbic acid is essential in cell physiology as a quencher of ROS and donor of electrons for APXmediated H 2 O 2 detoxification 7. Glutathione acts as an antioxidant in several ways and is utilized in cell defense mechanisms 8. For instance, in ascorbateglutathione cycle, reduced glutathione (GSH) is utilized to reduce dehydroascorbate (DHA), both enzymatically as well as non-enzymatically and itself oxidized to GSSG 9. Over the past several years, there has been a great interest in research investigating the antioxidant defence system in plant leaves, as leaf is the principle

2 48 INDIAN J. BIOCHEM. BIOPHYS. VOL. 48, FEBRUARY 2011 plant part which acts as one of the early sensor for the drought stress. It has been demonstrated that the antioxidative response is correlated with the tolerance of individual crop cultivars to drought 10. For example, dehydration during pre- and post-flowering stages of maize crop increases the activities of various antioxidant enzymes 11. Differential response of antioxidant enzymes in maize seedlings during salt stress has also been reported 12. Maize seed oil quality has been related with phenolics and carotenoids under water deficit conditions 13. Water deficit stress induces an increased photosynthetic activity in maize seedlings tolerant to water deficit conditions, indicating that stress tolerant mechanism exists at seedling stage in maize 14. These studies prompted us to look in to changes in the activities of SOD, CAT, POX, APX and GR after the exposure of drought treatment as well as the effect of stress on H 2 O 2, glutathione, ascorbic acid and malondialdehyde (MDA) content in the leaves of six maize genotypes differing in their susceptibility to drought stress during seedling stage, in order to evaluate the oxidative stress and the response of the protective systems to drought stress. Materials and Methods Screening and plant material The germplasm of maize was provided by the Maize Section, Department of Plant Breeding and Genetics, Punjab Agricultural University, Ludhiana. The seeds were surface sterilized with 0.1% mercuric chloride for 5 min and then washed with doubledistilled water before use. Experiments were conducted in plant growth chamber under controlled temperature of 30 ± 2 o C in the dark (12 h) and continuous illumination (12 h) of 40 µmol m -2 s -1 photosynthetically active radiation (PAR). For screening of the germplasm, disposable plastic cups (250 cm 3 ) were used to grow maize seedlings. Cups were filled with 220 cm 3 of its volume with mixture of farmyard manure and siphoned soil, and filled cups were then placed in plastic trays ( cm). After 7 days of normal growth, when second leaf was fully expanded, the seedlings were subjected to drought condition which was induced by withholding the irrigation in the cups till seedling death. This screening experiment was repeated three-times and on the basis of survival of the seedlings under stress the genotypes were categorized as tolerant and sensitive. Six genotypes (LM5, Parkash, PMH2, JH3459, Paras and LM14) were selected for further study, out of which LM5 and Parkash were found tolerant and PMH2, JH3459, Paras and LM14 were found sensitive to drought stress. Appearance of drought tolerant and sensitive seedlings after drought treatment is shown in Fig. 1. For further experiments, these six genotypes were grown by the above method for 7 days and thereafter 72 h drought treatment was given by withholding irrigation. On the other hand, control plants were provided by normal moisture throughout the experiment. After 72 h drought treatment, the leaves were used for extraction and assay of enzymes. Extraction and assay of enzyme activities All extractions were conducted in three replicates at 4 C. For enzyme extraction, seedlings from three different cups were taken separately. Each cup constituted one independent replicate. SOD, POX and GR were extracted by homogenizing the samples in 0.1 M phosphate buffer (ph 7.5) containing 1% polyvinylpyrrolidone (PVP), 1 mm EDTA and 10 mm β-mercaptoethanol. CAT and APX were extracted with 0.05 M phosphate buffer (ph 7.5) containing 1% PVP 15. Both the homogenates were centrifuged at g for 20 min and the supernatant was used for assaying above mentioned enzymes. All enzymes were assayed at 30 C. Components of enzyme assay system, except the enzyme were preincubated at 30 C for 20 min before starting the reaction. Activity of APX was assayed by taking 1 ml of 50 mm sodium phosphate buffer (ph 7.0), 0.8 ml of 0.5 mm ascorbic acid, 0.2 ml of enzyme extract and Fig. 1 Effect of drought stress (5 days) on growth of maize seedlings of tolerant and sensitive maize genotypes

3 CHUGH et al.: OXIDATIVE STRESS TOLERANCE IN MAIZE SEEDLINGS TO DROUGHT 49 1 ml of H 2 O 2 solution in total volume of 3 ml 16. Absorbance was recorded at 290 nm at an interval of 30 s up to 3 min. Extinction coefficient of monodehydroascorbic acid (MDAA) was 2.8 mm -1 cm -1. APX activity was expressed as nmoles of MDAA formed min -1 g -1 of FW. Activity of CAT was determined by taking 1.8 ml of 50 mm sodium phosphate buffer (ph 7.5) to which 0.2 ml of enzyme extract was added. The reaction was initiated by adding 1 ml H 2 O 2 and utilization of H 2 O 2 was recorded at an interval of 30 s for 3 min by measuring the decrease in absorbance at 240 nm 17. Extinction coefficient for H 2 O 2 was mm -1 cm -1. CAT activity was expressed as µmoles of H 2 O 2 decomposed min -1 g -1 of FW. Assay system of SOD contained 1.4 ml of 100 mm Tris HCl buffer (ph 8.2), 0.5 ml of 6 mm EDTA, 1 ml of 6 mm pyrogallol solution and 0.1 ml of enzyme extract 18. Change in absorbance was recorded at 420 nm after an interval of 30 s up to 3 min. A unit of enzyme activity was expressed as the amount of enzyme causing 50% inhibition of auto-oxidation of pyrogallol observed in blank. Assay of GR contained 0.2 ml of 200 mm potassium phosphate buffer (ph 7.5), 0.1 ml MgCl 2 (1.5 mm), 0.1 ml EDTA (0.2 mm), 0.2 ml NADPH (0.025 mm), 0.2 ml of enzyme extract, followed by 0.2 ml of oxidized glutathione (0.25 mm) in a quartz cuvette 19. Decrease in absorbance at 340 nm after an interval of 30 s up to 3 min was recorded. The molar extinction coefficient for NADPH was 6.22 mm ¹ cm ¹. GR activity was expressed as nmoles of NADP + formed min ¹g ¹ of FW. Assay system of POX contained 3 ml of 0.05 M guaiacol in 100 mm phosphate buffer (ph 6.5), 0.1 ml of enzyme extract and 0.1 ml of 0.8 M H 2 O The reaction mixture without H 2 O 2 was taken as a blank. The reaction was initiated by adding H 2 O 2 and rate of change in absorbance was recorded at 470 nm for 3 min at an interval of 30 s. POX activity was expressed as change in absorbance min ¹g ¹ of FW. Protein content of all the enzyme extracts was determined by the method of Lowry et al 21. Estimation of antioxidants Tissue (0.3 g) was homogenized with 2 ml of icecold 50 mm sodium phosphate buffer (ph 7.0) using liquid nitrogen. Homogenate was centrifuged at g for 20 min and supernatant was collected and H 2 O 2 content was estimated 22. For estimation of reduced ascorbic acid, tissue (0.1 g) was crushed in 1.5 ml of 5% ice-cold metaphosphoric acid, and then centrifuged at g for 10 min. Content of ascorbic acid was estimated in the supernatant 23. For glutathione estimation, tissue (0.1 g) was ground and homogenized in 2 ml of 5% sulphosalicylic acid and mixture was centrifuged at g for 15 min. Glutathione was estimated using 5,5 -dithiobis-2- nitrobenzoic acid (DTNB), NADPH and glutathione reductase 24. Malondialdehyde (MDA) was extracted and estimated by using a thiobarbituric acid reaction 25. Results Antioxidant enzymes status An increase in APX activity was observed in LM5 and Parkash (tolerant genotypes) in response to drought stress, whereas activity of APX declined in sensitive genotypes (PMH2, JH3459, Paras and LM14) under stress (Fig. 2A). Drought stress stimulated the CAT activity by 46% and 14% in LM5 and Parkash genotypes, whereas a significant decrease of 37%, 29%, 15% and 24% was observed in PMH2, JH3459, Paras and LM14, respectively (Fig. 2B). SOD activity either slightly increased (in LM5 by 22%) or remained unchanged (in Parkash) in tolerant genotypes under drought stress (Fig. 2C). Among sensitive genotypes, PMH2 and JH3459 were most severely affected and drought stress led to significant inhibition of SOD activity in leaf tissues of both the genotypes. Other genotypes namely Paras and LM14 were comparatively less affected and showed a decline of 48% and 15% in leaf SOD activity. Drought stress did not cause any substantial change in GR activity in tolerant and sensitive genotypes (Fig. 2D). An increase in POX activity was observed in the leaves of tolerant genotypes under drought stress. LM5 genotype showed greater upregulation in POX activity (68%) than Parkash (25%). With exception to LM14, sensitive genotypes showed comparatively higher increase in leaf POX activity under drought stress (Fig. 2E). For example, 4-fold upregulation in JH3459 and 8-fold in leaves of Paras was observed under drought stress. One of the reasons for such large upregulation of POX was the low status of this enzyme in control seedlings of sensitive genotypes. In spite of such upregulation the activity of POX in these sensitive genotypes did not increase over that of tolerant genotypes under stress.

4 50 INDIAN J. BIOCHEM. BIOPHYS. VOL. 48, FEBRUARY 2011 Fig 2 Changes in APX (A), CAT (B), SOD (C), GR (D) and POX (E) activities [Vertical bars show SD from mean of three replicates. a- significant at 1% level as compared to control. (Student s t-test)] H 2 O 2 and MDA content Drought stress enhanced the H 2 O 2 content in leaf tissues of most of the sensitive genotypes, whereas in tolerant genotypes it remained relatively unchanged. Among sensitive genotypes, an increase of 93%, 44% and 33% was observed for JH3459, Paras and LM14 genotypes, respectively (Fig. 3A). However, in PMH2, it was comparable to that of control. No change in MDA content was observed in the tolerant genotypes under drought stress, but the stress led to substantial accumulation of MDA in leaves of sensitive genotypes (Fig. 3B). Glutathione and ASA content Glutathione (GSH) content in leaves of tolerant genotypes remained almost unchanged under the

5 CHUGH et al.: OXIDATIVE STRESS TOLERANCE IN MAIZE SEEDLINGS TO DROUGHT 51 Fig. 3 Changes in H 2 O 2 (A), MDA (B), GSH (C) and ASA (D) content [Vertical bars show SD from mean of three replicates. a- significant at 1% level as compared to control. (Student s t-test)] influence of drought stress. An increase of 93%, 50% and 91% in GSH content was observed in sensitive genotypes, PMH2, JH3459 and LM14 genotypes, respectively except for Paras, which showed unaltered GSH content under drought stress conditions (Fig. 3C). Ascorbic acid content was higher in the tolerant genotypes. Drought stress caused 104% increase in ascorbic acid content of leaves of Parkash genotype, whereas only a moderate increase was observed in LM5 genotype (Fig. 3D). Ascorbic acid content of sensitive genotypes was also influenced in the same manner and significant increase was observed in all the genotypes. Among sensitive genotypes, maximum increase (195%) was observed in LM14 genotype. Discussion Tolerant genotypes showed moderate increase in APX activity due to drought stress, however, most of the sensitive genotypes showed fall in APX activity. LM5 and Parkash were also characterized by the higher activity of APX under stress conditions (stress tolerant genotypes Fig. 2A). An increase in APX activity in leaves of drought tolerant wheat cultivar, whereas no change or slight decrease in enzyme activity in sensitive cultivar has been reported 26. Increase in APX activity of leaves of tolerant genotypes might be an adaptive response of maize seedlings to higher amounts of ROS generated under water deficit conditions. Seedlings of the tolerant genotypes under drought stress also showed an increase of CAT activity, another H 2 O 2 detoxifying enzyme, whereas in the drought sensitive genotype, decrease in CAT activity was observed (Fig. 2B). Induction in CAT activity under water deficit stress in drought tolerant genotypes shows inbuilt capacity of these genotypes to induce CAT when required, whereas none of the sensitive genotypes showed this kind of behavior. CAT activity is associated with scavenging H 2 O 2 and an increase in its activity is related with increase in stress tolerance 27,28. Role of CAT in averting the cellular damage under unfavorable conditions like water stress has been suggested 29. An increase in CAT

6 52 INDIAN J. BIOCHEM. BIOPHYS. VOL. 48, FEBRUARY 2011 activity under drought stress of different crops has been reported 10,30,31. Substantial decrease in SOD activity of sensitive genotypes under drought stress was observed, however, tolerant genotypes maintained their activity levels equal to controls (Fig. 2C). Maintaining the SOD status under drought stress might protect plants from oxidative injury and possibly would not favour accumulation of O However, varied responses of SOD activity to water stress have been reported. It has been observed that water stress does not influence SOD activity in Sorghum and wheat and increase SOD activity in maize under stress 32,33. A higher level of SOD in drought tolerant maize inbreds, as compared to drought intolerant ones has been reported previously 34. GR activity was not significantly altered under drought stress (Fig. 2D). Drought stress does not significantly change GR activity in leaf and petiole tissues in Ctenanthe setosa versus the control 35. A small, but non-significant increase in GR activity in ten grass species under water deficit stress has been reported 36. Besides APX and CAT, H 2 O 2 can be scavenged by POX, another enzyme of antioxidant defense system. Peroxidases are often the first enzymes, the activities of which are altered under stress conditions. In the present investigation, drought stress induced the POX activity in leaves of tolerant as well as sensitive genotypes. POX activity was higher in control seedlings of tolerant genotypes in comparison to that of sensitive genotypes (Fig. 2E). An increase in POX activity in drought tolerant as well as sensitive maize genotypes at seedling stage under osmotic stress has been reported 10. An enhanced POX activity due to de novo synthesis has been reported in rice seedlings under anoxia 37 and low temperature 38. As observed in the present study, increase in POX activity in leaves of drought tolerant and sensitive maize cultivars has also been reported earlier under water deficit stress 39. In general, it appeared that activation of certain antioxidant enzymes in drought tolerant genotypes helped to overcome unfavorable environmental conditions induced by drought. Tolerant genotypes did not accumulate more H 2 O 2 under drought stress, indicating less severe oxidative damage. Almost unchanged H 2 O 2 content in tolerant genotypes under drought conditions might be due to higher APX, CAT and SOD activities in the leaf tissues of these genotypes. Accumulation of higher H 2 O 2 content in leaf tissue of sensitive genotype of maize, as compared to tolerant ones under drought stress induced by polyethylene glycol has been reported 40. Higher H 2 O 2 content in the leaves of drought sensitive maize seedlings under drought stress as compared to drought tolerant maize seedlings has been observed 14. It might be assumed that in LM5 and Parkash, H 2 O 2 detoxification was effectively managed by combined action of APX, CAT and POX, as compared to sensitive genotypes in which only POX activity was induced. This might be the reason for sensitive behaviour of these genotypes under drought conditions. The content of MDA is often used as an indicator of lipid peroxidation in plant tissues, resulting from an oxidative stress induced by various abiotic stresses. Recently, an increase in MDA content under drought stress has been reported in leaves of drought sensitive genotype of maize at seedling stage, whereas no change is observed in tolerant genotypes 14. In present study, no change was observed in MDA content of leaves of tolerant genotypes under drought stress (Fig. 3B), indicating that LM5 and Parkash genotypes were better equipped with efficient free radical quenching system than sensitive genotypes that offered protection against oxidative stress. The unchanged lipid peroxidation seemed to be a characteristic of tolerant plants that cope with environmental stresses like drought. Drought stress can either increase or decrease GSH content 41,42. GSH content was unaffected by drought stress in the leaves of tolerant genotypes, while higher GSH content was accumulated in sensitive genotypes than in tolerant ones (Fig. 3C). Earlier similar results have been reported in different crop plants 28,31,43. As an antioxidant, ascorbic acid has an important role in protecting against oxidative stress. It eliminates ROS through multiple mechanisms and also maintains membrane-bound antioxidant α-tocopherol in the reduced state and indirectly eliminates H 2 O 2 through activity of APX. Ascorbic acid content was higher in the leaves of tolerant genotypes. An increase in ascorbic acid content in leaf and stem tissues of cowpea under drought stress has suggested it to be a necessary response for efficient destruction of O 2 -. under stress conditions 28. It may be concluded that drought tolerance of genotypes LM5 and Parkash was linked to the effective antioxidative defence mechanism i.e. cooperation of enzymes ability to quench H 2 O 2 and

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