УДК 581.1

BIOCHEMICAL RESPONSES OF THIOUREA IN AMELIORATING HIGH TEMPERATURE STRESS BY ENHANCING ANTIOXIDANT DEFENSE SYSTEM

IN WHEAT 1

© 2015 B. Asthir*, R. Thapar*, N. S. Bains**, M. Farooq***

*Department of Biochemistry, Punjab Agricultural University, Ludhiana, Punjab, India

**Department of Plant Breeding and Genetics, Punjab Agricultural University, Ludhiana, Punjab, India

***Department of Agronomy, University of Agriculture, Faisalabad, Pakistan

Received April 21, 2014

  1. In this study, effect of exogenously applied thiourea (TU, 6.6 mM) and dithiothreitol (DTT, 0.07 mM) as seed pretreatment or as foliar spray (at 90 days after sowing) in two wheat (Triticum aestivum L.) cvs. C 306 (heat tolerant) and PBW 343 (heat susceptible) at seedling and reproductive stages under high temperature (HT) stress was investigated. The heat tolerant cv. C 306 exhibited much lower membrane injury index (MII), thiobarbituric acid reactive substances (TBARs) and H2O2 contents, but such effect was not accompanied by higher activities of the enzymes involved in the reactive oxygen species scavenging system during both developmental stages. Application of TU under HT reduced MII and TBARs and H2O2 contents, but as a rule did not affect activities of antioxidant enzymes. Therefore pretreatment by TU/DTT no doubt improved the resistance against oxidative stress through increase in membrane stability parameters but their effect on antioxidant enzymes was not apparent under the prevailing conditions of the experiment.

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1 This text was submitted by the authors in English.

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Abbreviations: APX  ascorbate peroxidase; CAT  catalase; DAS  days after sowing; DPA  days post anthesis; GPX  guaiacol peroxidase; GR  glutathione reductase; HT  high temperature; MII  injury index; SOD  superoxide dismutase; TBARs  thiobarbituric acid reactive substances; TU  thiourea; NBT  nitro blue tetrazolium.

Corresponding author: B. Asthir. Department of Biochemistry, Punjab Agricultural University, Ludhiana-141004, Punjab, India; fax: 0091 (161) 2400945; e-mail: b.asthir@rediffmail.com

Keywords: Triticum aestivum  antioxidant enzymes  membrane injury  lipid peroxidation  thiourea  high temperature

 

INTRODUCTION

Heat stress is one of the important abiotic stresses hindering productivity in many areas in the world. It threatens plant growth and development and may lead to drastic reduction in economic yield of plants [1]. As a result, many studies have been emphasized on the mechanism of heat stress. High temperature (HT) induces numerous biochemical changes leading to the production of ROS such as lipid peroxide, singlet oxygen and superoxide radicals, hydrogen peroxide (H2O2) and hydroxyl radicals [2]. ROS production is controlled by enzymatic system such as superoxide dismutase (SOD; EC 1.15.1.1), ascorbate peroxidase (APX; EC 1.11.1.7), and catalase (CAT; EC 1.11.1.6) [3] and antioxidants (ascorbate, tocopherol). A number of studies have demonstrated the effectiveness of ROS scavenging mechanisms that play an important role in protecting plants against HT stress [4, 5]. High temperature tolerant genotypes possess superior expression and levels of antioxidant system. Consequently, development of heat-tolerant cultivars is of major concern in wheat breeding programme. However, the physiological and biochemical basis of HT tolerance remains poorly understood.

The plant stress tolerance can be improved by various means, the most important of which include the exogenous use of stress alleviating agents. For instance, application of thiols not only enhances crop productivity, but also do not lead to any kind of metabolic imbalances. Thiols are selected as they are well-known to maintain the redox state (–SH/SS-ratio) of the cell and its proper functioning under stress conditions [6]. Thiols play a crucial role in influencing metabolic reactions in plants under stress conditions [7]. Since, the redox states of thiols improves stress tolerance, it is quite possible that seed pretreatment with external thiols might result in switching on some metabolic processes in order to combat oxidative stress. Improvement in plant growth and development under different stresses due to application of thiourea (TU) has been observed in crops like maize [8], wheat [911], pearl millet [12], cluster bean [13], and Brassica [14]. Our work published earlier [11] emphasized the role of TU under HT stress on photosynthetic tissue (flag leaf) which improved wheat performance by enhancing membrane stability, antioxidant potential, and yield components.

In the present study, we have reported the effect of TU/DTT at early stage of seedling (root, shoot) and during reproductive stage (grains) to see how two different stages are affected under the influence of HT.

 

MATERIALS AND METHODS

Plant material and growth conditions. Seeds of two wheat (Triticum aestivum L.) cvs. C 306 (tolerant) and PBW 343 (susceptible) were obtained from the Department of Plant Breeding and Genetic, Punjab Agricultural University, Ludhiana, Punjab, India. Seeds were surface sterilized with 1% HgCl2 for 1 min, rinsed thoroughly with distilled water and imbibed in distilled water, thiourea (TU, 6.6 mM) or dithiothreitol (DTT, 0.07 mM) for 6 h and germinated at 25C (normal) and 32C (high temperature, HT) in Petri dishes (9.0 cm) on double layer Whatman 1 filter paper moistened with 5 mL of distilled water. Twenty seedlings were used in each experiment and each experiment was repeated in triplicates. A seed was considered to have germinated when its radical emerged at least 5 mm.

The experiment was divided in four sets. One set of seeds was grown at 25 ± 1°C in continuous dark conditions in Petri plates containing germination paper moistened with 4 mL of distilled water. Second set was maintained to a continuous HT (32 ± 1°C) in an incubator under dark. Third set of seeds were soaked for 6 h in TU or DTT and then raised at 25 ± 1°C in continuous dark conditions, and fourth set of seedlings were soaked in TU or DTT as depicted in set three but maintained at continuous HT (32 ± 1°C) conditions. Uniform sized wheat seedlings were sampled after sixth day of germination, and the measurements were performed on root and shoot in triplicates.

For studies on developing grains, two genotypes PBW 343 and C 306 pretreated with TU (6.6 mM), i.e seed soaking at the time of sowing, followed by foliar spray at 90 days after sowing (DAS) were raised under normal (16 Nov 2010) and late planting (11 Dec 2010) conditions in plots and the mean temperature during grain development for two sowing period varied between 46C. Each plot consisted of 4 rows of 1 m each. Row to row spacing was maintained at 23 cm and the material was sown in three replications. There were three replications for each determination and the developing grains were collected at 7, 14, 21, 28, and 35 days post anthesis (DPA) for metabolite determination and enzyme analysis.

Determination of membrane injury index (MII). 0.5 g fresh tissue was excised and washed with distilled water to remove adhering electrolytes. The tissue was then immersed in test tubes containing 20 mL of distilled water. After 24 h the MII was estimated by conductivity meter at 25°C. The sample was then boiled for 30 min and conductivity was measured again. Membrane injury index was calculated as a ratio of electrical conductivity before and after boiling and was expressed in percentage.

Determination of lipid peroxidation. Lipid peroxidtion was determined as content of thiobarbituric acid reactive substance (TBARs) content with the formation of colored complexes of thiobarbituric acid in acid medium, and the content of TBARs (MDA) was calculated with ε = 155/(mM cm) [15].

Determination of hydrogen peroxide content. Tissue (0.3 g fr wt) was homogenized with 3 mL of 1% (w/v) TCA. The homogenates were centrifuged at 10 000 g (4°C) for 10 min. Subsequently, 0.75 mL of the supernatants were added to 0.75 mL of 10 mM K-phosphate buffer (pH 7.0) and 1.5 mL of 1 M KI. H2O2 content of the supernatant was evaluated by comparison of the absorbance values at 390 nm to a standard calibration curve in the range of 10 to 200 nmol [16].

Enzyme extraction and assays. Root, shoot, and developing grains (1 g) were homogenized with 3 mL of ice-cold 50 mM phosphate buffer, pH 7.0, for ascorbate peroxidase (APX), guaiacol peroxidase (GPX), glutathione reductase (GR), superoxide dismutase (SOD), catalase (CAT). The homogenate was centrifuged at 10 000 g for 20 min at 4C and clear supernatant was used for assaying enzymes activities.

APX activity was determined by measuring the decrease in absorbance at 280 nm due to ascorbate oxidation (ε = 2.8/(mM cm)) in a reaction mixture containing 50 mM phosphate buffer (pH 7.0), 0.5 mM ascorbic acid, 0.1 mM EDTA for three min and the enzyme activity was expressed as μmol ascorbate oxidized/(min mg protein) [17].

GPX activity was assayed by measuring the increase in absorbance at 470 nm (ε = 26.6/(mM cm)) due to guaiacol oxidation for 3 min. The assay mixture contained 50 mM phosphate buffer (pH 7.0), 0.1 mM EDTA, 10 mM guaiacol and 10 mM H2O2 and specific activity was expressed as μmol tetraguaiacol formed/(min mg protein) [18].

GR activity was assayed by measuring OD of the reaction mixture at 340 nm (ε = 6.2/(mM/cm)) in a reaction mixture containing 50 mM TrisHCl buffer (pH 7.5), 0.1 mM EDTA, 3 mM MgCl2 and 0.15 mM NADPH. Specific activity was expressed as μmol NADPH oxidized/(min mg protein) [19].

SOD activity was assayed following the inhibition of the photochemical reduction of nitro blue tetrazolium (NBT). The reaction mixture contained 50 mM Na-phosphate buffer (pH 7.8), 0.1 mM EDTA, 14.3 mM methionine, 82.5 μM NBT and 2.2 μM riboflavin. The reaction was initiated by placing the test tubes under 15 W fluorescent lamps and was terminated after 10 min by removing the reaction tubes from the light source. Non-illuminated and illuminated reactions without enzyme extract served as calibration standards and were measured at 560 nm. One unit of SOD was defined as the amount of enzyme that produced a 50% inhibition of NBT reduction under assay conditions [20].

CAT activity was measured following the decomposition of H2O2 at 240 nm (ε = 39.4/(mM cm)) in a reaction mixture containing 50 mM phosphate buffer (pH 7.0), 15 mM H2O2 for 2 min. One CAT unit was defined as the enzyme amount that decomposes μmol H2O2/(min mg protein) [18].

Statistical analysis. The values were statistically analyzed by multifactor ANOVA (Statgraphics plus v. 2.1). Values are presented as a means ± standard deviation (n = 3 or more). Statistically significant differences among values at the significance level p < 0.01 are indicated by different letters.

 

RESULTS AND DISCUSSION

Membrane thermostability parameters are vital components for assessing stability and cell homeostasis against any kind of stresses. Lipid peroxidation is commonly taken as stress indicator along with other specific assays like membrane injury and H2O2 that act as a signaling molecule [15]. High temperature caused significant increase in MII, TBARs (MDA) and H2O2 contents in shoots and roots of tolerant (C 306) and susceptible (PBW 343) cultivars, but the overall damage was more in roots as compared to shoots (fig. 1) indicating that shoots are better equipped to combat oxidative stress. A similar decrease in membrane stability parameter was observed in shoots of maize seedling when subjected to HT stress [21]. Tolerant cultivar had lower MII, TBARs and H2O2 contents over susceptible one indicating less severe oxidative damage in these cultivars (fig. 1) which suggests that some wheat genotypes are better equipped with HT tolerance mechanism such as osmoregulation, antioxidant defense system, and heat shock protein [1]. Since application of TU/DTT led to a decrease in MII, TBARs and H2O2 content, it is quite probable that sulphydryl compounds ameliorates HT stress by a mechanism involving membrane repair. Protection of membranes from lipid peroxidation could involve direct binding of sulphydryl groups to membrane phospholipids and thus stabilizing cell walls under stress conditions [16]. Thiourea has earlier been reported to bring down the level of ROS and MDA content in salt stressed mustard seedling [22].

Activities of antioxidant enzymes in root and shoot of two wheat cvs. PBW 343 and C 306 under HT and TU/DTT conditions were determined (fig. 2). There was no significant changes in the activities of APX, GPX, CAT, GR, and SOD in shoot under HT in heat susceptible cv. PBW343 (fig. 2). Respectively, activities of these enzymes were not influenced under HT and TU/DTT application (fig. 2). The APX, CAT, and SOD activities increased in shoot of tolerant C 306 cultivar under HT (32C) as compared to control (25C) (figs. 2a, 2c, 2e). While these activities remained unaffected under TU/DTT application. Contrary to shoots, HT treatment led to dramatic decline of APX, GPX, CAT, GR, and SOD activities in roots of both tested cultivars as compared to control (fig. 2). Application of TU/DTT did not stimulate CAT, GR, and SOD activities under HT stress in roots of both tested cultivar (figs. 2c, 2d, 2e), but increase of APX and GPX activities by TU/DTT in roots were apparent. Given the reducing power of thiols and the importance of cysteine, –SH groups, and disulphide in protein structure and function [23], the activity of some antioxidant proteins seems to be modulated by DTT and TU.

Though, our findings of this study are not in parallel with the trend reported in the literature [2426] but variable changes of antioxidant enzymes under HT in certain plant organs were described. For instance, altered pattern of SOD activity under different temperature regimes was observed in wheat genotypes [27], which was related to reduced transcript levels of genes encoding SOD in tobacco plants [28]. Peroxidase activities decreased in response to HT ranging from 3050°C in six varieties of lentil (Lens culinaris Medik.) [29]. The observed decline in CAT activity has also been reported in wheat and some other plants species [30, 31]. In this study, APX and GR activities were high in roots of susceptible cultivar (PBW 343) under control and stressed condition, which indicates that APX and GR activities alone did not specify the overall scavenging ability efficient enough to combat HT stress as TBARs and H2O2 content were high in this cultivar (fig. 1). Roots in general had higher activities of antioxidant enzymes except for SOD that was more in shoot.

Tolerance to HT stress is associated with an increase in antioxidant enzymes activity [25]. However, under stress the extent of ROS production exceeds the antioxidant defense capability of the cell, resulting in cellular damages. In developing grains, there was some increase in TBARs content and MII in response to late planting in susceptible cultivar (fig. 3) which can be attributed to damaging effect of HT. Membrane disruption may alter water, ion and organic solute movement, photosynthesis, and respiration [25]. However, MII increased only up to 21 DPA and thereafter decreased towards grain maturity. As a rule thiourea pretreatment decreased ion leakage in grains and thereby improved cell membrane integrity. Lipid peroxidation in form of TBARs (MDA content) decreased throughout grain development. APX activity increased up to 21 DPA, and thereafter was decreased towards grain maturity under both normal and late planting. TU pretreatment increased APX activity in all cases studied (fig. 4). CAT activity was not differed between normal and late planting and was not improved with TU in developing grain of both tested cultivars. Pertinently, effect of TU was not apparent in case of GPX, SOD, and GR at most of the developmental stages of studied grains, which indicated that TU ameliorate HT stress conditions under the prevailing experimental conditions by increasing APX activity.

In crux, we infer that although TU/DTT no doubt improved the resistance against oxidative stress through increase in membrane stability parameters but their effect on antioxidant enzymes was not apparent under the prevailing conditions of the experiment. Therefore, a detailed experimental analysis using different temperature regimes is required.

 

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FIGURE CAPTIONS

 

Fig. 1. Effect of TU, DTT, and high temperature (32C) on MII (a), TBARs (b), and H2O2 (c) contents in shoot and root of two wheat cvs. PBW 343 and C 306.

1  25C, 2  32C. Bars represent ± SD of three independent experiments. Different lower case letters denote significant differences among values (p < 0.01). Significant differences between cvs represented as a; between TU and DTT as b; between temp as c.

 

Fig. 2. Effect of TU, DTT, and high temperature (32C) on activities of APX (a), GPX (b), CAT (c), GR (d), and SOD (e) in shoot and root of two wheat cvs. PBW 343 and C 306.

1  25C, 2  32C. Bars represent ± SD of three independent experiments. Different lower case letters denote significant differences among values (p < 0.01). Significant differences between cvs represented as a; between TU and DTT as b; between temp as c.

 

Fig. 3. Effect of TU on MII (a) and TBARs content (b) in the developing grains (days post anthesis represent on abscissa) of two wheat cvs. PBW 343 and C 306 under normal sown and late sown conditions.

1  control, 2  thiourea. Bars represent ± SD of three independent experiments. Different lower case letters denote significant differences among values (p < 0.01). Significant differences between cvs represented as a; between TU and DTT as b; between temp as c.

 

Fig. 4. Effect of TU on activities of APX (a), GPX (b), CAT (c), GR (d), and SOD (e) in the developing grains (days post anthesis represent on abscissa) of two wheat cvs. PBW 343 and C 306 under normal sown and late sown conditions.

1  control, 2  thiourea. Bars represent ± SD of three independent experiments. Different lower case letters denote significant differences among values (p < 0.01). Significant differences between cvs represented as a; between TU and DTT as b; between temp as c.