Role of Abscisic Acid and Water Stress on the Activities of Antioxidant Enzymes in Wheat

Eight wheat varieties (Chinese Spring, Pavon, Gabo, Saleem-2000, Zamindar-04, Siren, NR-264 and Marvi) were compared for their response to exogenous application of abscisic acid (ABA), Water Stress (WS) and Control (C) during invitro condition. Their responses were studied in the form of seedlings growth and antioxidant enzymes. Exogenous application of ABA reflected ameliorating effect on catalase activity. Water stress treatment led to increase in levels of catalase except Pavon. Increased activity of antioxidant enzymes showed tolerance capacity under water stress. Correlation coefficient analysis reflected negative and significant relationship between total protein contents and peroxidase and catalase. Further, investigations are needed to enhance the understanding on the effect of different abiotic stresses and growth hormones during early seed development.


INTRODUCTION
Drought stress is among the most damaging abiotic stresses that affect agriculture.Due to current climate change scenario, plants suffer from erratic water stress (Zhang et al., 2006).Wheat is the main cereal crop of Pakistan and it is a prime food of the people.It contributes 14.4% to the value added in agriculture and 3.1% to GDP (Anonymous, 2010).Plant response to water stress include morphological and biochemical changes and later as water stress become more severe to functional damage and loss of plant parts (Sangtarash, 2010).Phytohormones are considered as main signals during stress conditions (Davies, 1995;Pospisilova et al., 2005).And almost all processes in the life of a plant are directly or indirectly influenced phytohormones (Pospisilova, 2003).Especially abscisic acid (ABA) has been considered to be one of the main hormone which triggers various acclimations processes under water stress conditions (Zhu, 2002).Under water stress condition exogenously applied ABA stimulated the synthesis of proteins in different species (Riccardi et al., 1998).ABA also expresses the gene encoding enzymes that participate in the repair of spontaneous protein damage (Mudgett and Clark, 1996).Moreover, ABA induced increase in the osmolyte might also help in stabilizing the proteins under water stress (Noiraud et al., 2001).Exogenous application of ABA has also been reported to significantly increase tolerance (Jiang and Zhang, 2004;Farooq and Bano, 2006;Iqbal et al., 2012;Khan, 2013).
Increasing evidence indicates that one mode of ABA action may be related to its role in the oxidative stress in plant cells.It has been documented that ABA can cause an increased generation of O - 2 (Jiang and Zhang, 2001) and H 2 O 2 (Guan et al., 2000;Jiang and Zhang, 2001;Zhang et al., 2001).To alleviate adverse effects of reactive oxygen species, plants have evolved an antioxidant defense system that include enzymes like superoxide dismutase, peroxidase and catalase (Agarwal and Pandey, 2004;Bano et al., 2012).
The objective of this study was an effort made to investigate the role of ABA under water stress and also interrelationship between antioxidant enzymes such as peroxidase and catalase in the seedlings of different wheat varieties exposed to water stress and ABA condition.

MATERIALS AND METHODS
Plant materials and stress treatments: A laboratory experiment was conducted in 2011; the experiment was carried out in a factorial on a randomized block design with three replications.Seeds of eight different wheat varieties (Chinese Spring, Pavon, Gabo, Saleem-2000, Zamindar-04, Siren, NR-264, Marvi) were used.Seeds were surface sterilized with 0.1% bleach solution, thoroughly washed three times with distilled water and were placed in petri plates containing sterilized filter paper.In each petri plate eight seeds were placed.Water was added in each petri plate at regular intervals of time.After one week seedlings were exposed to three different treatments i.e., Water Stress (WS), abscisic acid (ABA) and control (C).No water was added at water stress condition whereas 30 µM of ABA was applied under ABA condition and there was regular watering under control condition.After one week, treated and untreated seedlings were harvested and biochemical analysis was done.
Growth parameters: Shoot and root length was measured in cms and then average was calculated.Fresh weight of all seedlings was measured in gm with the help of electronic balance.Seedlings were crushed into pre chilled pestle and mortar containing 2 mL cold homogenizing buffer solutions (pH7.5) per gm fresh weight of material.The homogenate were centrifuge first at 4,000 rpm for 10 min and then 10,000 rpm for 15 min.The resultant supernatant was collected and used for biochemical analysis.

Biochemical analysis:
The protein content of determined follows the method of Lowry et al. (1951).Quantitative estimation of peroxidase was done as described by Everse et al. (1994), the assay mixture containing 10µl plant extract, 3.75 mL of 0.15 M phosphate buffer (pH5.6), 100 µL of 176 mM H 2 O 2 and the same amount of 0.1 M guaiacol as H 2 O 2 was left for 8 min and then the reaction mixture was shaken well after adjusting the total volume up to 10 mL with distilled water.The increase in absorbance due to guaiacol oxidation was recorded at 470 nm.Catalase was assayed by monitoring the decrease of absorbance.The reaction mixture consisted of 50 µL of enzyme, 2.5 mL of 50 mM potassium phosphate buffer (PH 7.6), 200 µL of 12.5 mM H 2 O 2 and 250 µL of distilled water.The absorbance of the mixture was recorded at 240nm.This method was described by Aebi (1984).
Statistical analysis: Analysis of variance, mean comparisons and correlation coefficient analysis were performed through computer program SPSS software version 11.0.

RESULTS AND DISCUSSION
Growth parameters: The mean root length of the wheat genotypes responded differently to treatments (Table 1).Zamindar-04 showed maximum root length (7.68 cm) whereas Marvi had a minimum root length (2.8 cm) under control condition.All genotypes showed reduction in root length except Marvi at ABA condition (Table 2).In this study the root length of wheat genotypes has affected by water stress.The less retardation of growth was found in Marvi among eight genotypes of wheat.Imposition of water deficit stress led to inhibition of growth in wheat seedlings.Maintenance of root growth during water deficit is beneficial to maintain adequate plant water supply (Pinheiro et al., 2004).However Gabo showed higher reduction under water stress.Baloch et al. (2012) reported that root length had a high percentage decrease due to osmotic stress.Marvi showed a consistent performance under ABA condition with respect of root length.The continued growth of roots in water stressed soil is particularly important to avoid the effect of water stress (Dhanda et al., 1995;Misra, 1990Misra, , 1994)).In general, root length of the drought sensitive genotypes was significantly reduced as compared to drought tolerant genotypes (Devi et al., 2012).The development of root system in water deficit conditions appears to be a very viable criterion to select for water stress because it takes the moisture from lower layers of soil and also obviously suffers from more than any other seedling trait (Baloch et al., 2012).Exogenous supplied ABA under water stress contributes to protective role/reactions aimed to decrease its injurious effect on growth and acceleration of growth resumption (Sakhabutdinova et al., 2003).
Total protein contents: Table 3 revealed nonsignificant differences among treatments whereas genotypes showed significant differences.Contrasting reports regarding the changes in protein contents are available in literature (Iqbal et al., 2010).Increasing water stress was found to cause a significant reduction in soluble protein content in moth beans (Garg et al., 2001) and vigna radiata L. (Farooq and Bano, 2006).The increase in total soluble proteins under water stress in some accessions and decrease in others is consistent with previous findings (Bensen et al., 1988;Riccardi et al., 1998;Ti-Da et al., 2006) water stress increased some soluble proteins and decreased others.

Peroxidase:
The specific activity of peroxidase enzyme non-significantly differed under different treatments and genotypes (Table 3).Oxidative damages in plant caused by biotic and abiotic stresses are usually reduced by enzymatic antioxidants (superoxide dismutase, ascorbate peroxidase, glutathione reductase and catalase and by non-enzymatic antioxidants (Ismail et al., 2005).The ability of antioxidant enzymes to scavenge ROS and reduce their damaging effects may confer drought resistance to plants.Yang et al. (2009) exhibited that as compared with 100% field capacity, at 25% field

Catalase:
The catalase activity of the treatments and genotypes differed significantly (Table 4).In the control condition, Pavon had a higher activity of catalase i.e., 4.10 U.mg -1 protein/min and Chinese Spring had a lesser catalase activity (0.95 U.mg -1 protein/min).All genotypes reflected enhanced catalase activity except Pavon that withstand their activity where no supplied of water.Exogenously ABA induced to wheat seedlings that most of the genotypes reflected increased activity but few had decreased than control.Yong et al. (2006) found that the rules of SOD, CAT and POX activity change are similar, which indicated that these three enzymes cooperated with each other during water deficits.However, reports on the effect of stresses on CAT activities vary (Salekjalali et al., 2012).Increased, decreased, or unchanged CAT activities under drought stress have been observed by Jiang and Huang (2001), Sobkowiak et al. (2004) and Salekjalali et al. (2012).Higher levels of antioxidant enzymes are related to drought tolerance in different plants ( Khanna-Chopra and Selote, 2007;Hameed et al., 2011;Bhagi et al., 2013).
Drought stress caused an increase in catalase activity in leaf tissues of both wheat cultivars (Valifard et al., 2012).Catalase plays a key role in detoxification of H 2 O 2 produced specially in chloroplasts under oxidative stresses (Blokhina et al., 2003).Our results agree with those reported by Ismail et al. (2005) who found higher catalase activity in drought tolerant bean plants as compared to drought sensitive.Similar results were reported by Valifard et al. (2012), Devi et al. (2012) and Khan (2013) in wheat cultivars.Increase in CAT activity has been shown to be related with increase in stress tolerance capacity (Kraus et al., 1995;Devi et al., 2012).
Correlation coefficient: Table 5 depicted correlation coefficient among different treatments and genotypes.Total protein content showed negative and significant correlation with peroxidase and catalase under control condition.Similar results were obtained under rest of the stresses.These results indicated that increase in protein content caused decrease in antioxidant enzymes.Peroxidase exhibited positive and significant correlation with catalase at irrigated, water stress and ABA condition.Positive correlation occurs between endogenous ABA and antioxidant activities of SOD and POD were reported by Bano et al. (2012).

CONCLUSION
Activities of antioxidant enzymes could be a useful tool for depicting drought tolerance of wheat, which could be useful to plant breeders for developing drought-tolerant cultivars.It could be suggested that ABA played a positive role for plant to improve its antioxidant defense mechanism in plants.Further studies are needed to confirm the role of antioxidant enzymes for understanding drought tolerance in a large number of genotypes.

Table 1 :
Mean squares for root length of wheat varieties among

Table 3 :
Mean squares for total protein content, POX and CAT among different treatments