World Journal of Agricultural Sciences 4 (4): 471-475, 2008 ISSN 1817-3047 © IDOSI Publications, 2008

Drought-Stress Induced Changes in the Expression of Acid Phosphatases in Drought Tolerant and Susceptible Cultivars of Wheat Arun Dev Sharma and Parminder Kaur Department of Biotechnology, Lyallpur Khalsa College, GT Road, Jallandhar-144001, Punjab, India Abstract: The variability in acid phosphatase activities was studied with an objective to find out its physiological role in response to drought stress. The effect of drought stress on acid P-ase activity in relationship with phosphorus was studied in drought tolerant (C-306) and drought sensitive (HD-2004) cultivars of wheat. Drought stress was imposed by withholding water irrigation to the growing seedlings in the net house conditions. The tissues (leaves and grains) were separated and acid P-ase activity, western blotting studies were carried out. After drought stress, a significant increase in acid P-ase activity was observed in leaves and grains of drought tolerant cv. C-306. However, no change was observed in drought sensitive cv. HD-2004. Immuno-blotting analysis also revealed similar type of induction of wPase-94 (band detected using antiserum in western blot analysis) expression under drought stress conditions. Imposition of drought stress caused significant decrease in phosphorous level (Pi) in both the varieties at all the stages of development. However, increased acid P-ase activity coupled with low Pi level in drought tolerant cv. 306, indicated variety specific key role of acid P-ases under drought stress in maintaining Pi level. Based upon these results, a possible physiological role of acid P-ases in wheat is discussed. Key words: Acid phosphatase activity

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INTRODUCTION

Wheat

and phosphorous uptake in bean [6] and barley [7]. However, a negative relationship was also observed between acid P-ases and phosphorous uptake under low phosphorous stress in wheat [8]. Szabo-Negy et al. [9] and Barrettt-Lennard et al. [8], indicating that phosphatase activities are independent of phosphate levels. Hence, role of acid P-ases against phosphorous stress is still a matter of conjuncture. In addition to act as a Pi scavenger, several possible physiological roles also have been attributed to supraoptimal level of acid P-ases such as: seed dormancy, embryo germination and cell wall regeneration [10, 11]. Although, some roles have been suggested for acid P-ases accumulation under low phosphorous stress, however, its physiological role in under drought stress has received far less attention. Therefore, this study was designed to examine the role of acid P-ases under drought stress in relationship with Pi in drought tolerant and sensitive cultivars of wheat. In contrast to drought sensitive cultivar, there was a strong increase in acid P-ase activity accompanied by wPase-94 expression under drought stress in drought tolerant cultivar, suggesting that acid P-ases may be playing some adaptive role under stress condition.

Acid phosphatases form a group of enzymes catalyzing hydrolysis of a variety of phosphate esters in the acidic environments. Acid P-ases are believed to increase orthophosphate (Pi) availability under phosphorous deficient conditions [1]. In most agricultural soils, organic P comprises 30-80% of the total P [2]. The largest fraction of organic P, approx 50%, is in the form of phytin and its derivatives [3]. For organic P sources in the soils to be used, they must be first hydrolyzed by acid phosphatases. Free soluble phosphate (Pi) play a vital role in many biological processes including photosynthesis, respiration, enzyme regulation, energy transfer, metabolic regulation, important structural constituent of biomolecules like phytin bodies in the ungerminated seeds, protein and nucleotide phosphorylation [4, 5]. Although, there are many controversial issues with acid P-ase accumulation and stress resistance, but, it is believed that high levels of acid P-ases can be beneficial to stressed plants [5]. Enhanced excretion of acid P-ases under phosphorous stress has been documented in a number of plants [3, 1]. A positive relation was reported between root acid P-ases

Corresponding Author: Dr. Arun Dev Sharma, Department of Biotechnology, Lyallpur Khalsa College, GT Road, Jallandhar-144001, Punjab, India

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MATERIALS AND METHODS

NaOH. The liberated p-nitrophenol (pNP) was determined at 410 nm and calibration curve of pNP prepared in the same conditions. One unit (U) of phosphatase is equivalent to the amount of enzyme liberating 1 µmole of product per min under assay conditions.

Plant material: Seeds of drought tolerant (C-306) and drought susceptible (HD-2004) cultivars of Triticum aestivum were procured from Guru Nanak Dev University, Amritsar, India. Plants were raised in soils in pots (one plant per pot) in the net house under natural conditions and tagged on the day of anthesis. Drought treatments were imposed by restricting irrigation. Control plants were irrigated as frequently as needed. Single water stress treatment at different stages of development was imposed (5-6 days prior to sampling) by withholding water supply while the control plants were watered daily. Leaf samples were collected at 30 and 60 days after sowing (DAS) while the developing grains were collected at 30, 38 and 42 days post anthesis (DPA). For biochemical analysis leaves from stressed and control plants harvested in triplicate at different stages of growth were frozen in -80°C until further analysis. Panicles from three pots, representing one replicate, were harvested in triplicate at 30, 38 and 42 DPA and stored in -80°C until further analysis. Relative water content (RWC) was measured using flag leaves (at vegetative phase) and grains (at reproductive stage) after imposing drought conditions. Immediately tissues were sealed with in plastic bag and quickly transferred to the laboratory. Fresh weights were determined with in 2 h after collection. Turgid weights were obtained after soaking the tissues in distilled water in test tubes for 16 to 18 h at 4°C under low light conditions. After soaking, tissues were quickly and carefully blotted dry with tissue paper in determination of turgid weight. Dry weights were obtained after oven drying the samples for 72 h at 70°C. RWC was calculated from given equation:

Extraction and assay of phosphorous (Pi): For determination of total soluble Pi, only fresh tissue samples were used, which were homogenized with 5 ml of 10% (v/v) HClO4 at 4ºC. After centrifugation at 5000,g at 4ºC, the supernatant was collected for analysis of Pi. The Pi content of the resultant soluble fraction was measured by the formation of a blue molybdenum complex as described by Tsvetkova and Georgiev [12]. Briefly, appropriate aliquots were mixed with 5 ml 0.1 M acetate buffer pH 4.0, 0.5 ml 1% (w/v) ammonium molybdate in 0.05 N H2 SO4, 0.5 ml 1% (w/v) Na-ascorbate. To avoid the delay in the conversion of the blue color of molybdatephosphoric complex, 1 mM CuSO 4.5H2O was added into the ascorbate solution. The blue color of the complex was obtained after 10 min and the absorption was determined using spectrophotometer at 620 nm. Western blot analysis: Western blotting analysis was carried out with antibodies (a gift from Dr. Carrol P. Vance) raised against a 70-kDa white lupin antiserum. After electrophoresis, proteins were electroblotted to a nitrocellulose membrane. Protein blots were reacted with anti-APase (1:500 dilution) and developed using an alkaline phosphatase-conjugated secondary antibody (1: 3000 dilution) and 5-bromo-4-chloro-3-indoyl phosphate p-toluidine salt/p-nitroblue tetrazolium chloride reagent systems [13]. RESULTS AND DISCUSSION

RWC(%) = fresh weight-dry weight/turgid weight-dry weight x 100

Phosphatase activity and wPase-94 expression under drought conditions in leaves and grains: Acid P-ases in plants are classes of enzymes that display considerable heterogeneity with regard to their kinetics and functions [14]. This complexity may contribute to conflicting reports regarding role of acid P-ases in phosphorous nutrition. Acid P-ases are reported to be induced under phosphorous (Pi) deficiency, in order to maintain certain level of Pi inside the cells [8, 15]. However, their precise role of phosphatases during drought stress is still not known. Our studies revealed a substantial enhancement in acid phosphatase activities in a temporal manner under drought stress. We used both chemical assay and western blotting analysis to determine the role of acid

Enzyme extraction: The enzyme was extracted from the tissues as described previously [10]. Briefly, the tissue was ground with mortar and pestle at 0-4°C using 50 mM sodium acetate buffer (pH 5.0). The homogenate was centrifuged at 8000,g for 15 min and the supernatant collected. Phosphatase activities were assayed by measuring the amount of p-nitrophenol produced. Phosphatase activities were measured spectrophotometrically at 410 nm in a final volume of 1 ml. The reaction mixture contained 300 µl of enzyme extract, 0.05 M buffer [Sodium acetate (pH5.0)], 0.1 M NaCl and 0.2 mg/ml BSA, with 5 mM para-nitrophenylphosphate (pNPP) as a substrate. The time of reaction was 10 min. The reaction was stopped by adding 1.5 ml of 0.25 M 472

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Fig. 1: The effect of drought stress on leaf RWC (A), Pase activity (B) and phosphorus content (C) of drought susceptible cv. HD-2004 (control: ; treatment: ) and tolerant cv. C-306 (control: ; treatment: ) of wheat under irrigated control and water stress conditions at different stages of development (DAS: days after sowing). Data shown are average ± SD of three replicates. d indicates significant difference vs. control at P= 0.05.

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Fig. 2: The effect of drought stress on seed RWC (A), P-ase activity (B) and phosphorus content (C) of drought tolerant cv. C-306 (control: ; treatment: ) and susceptible cv. HD-2004 (control: ; treatment: ) of wheat under irrigated control water stress conditions at different stages of development (DPA: days post anthesis). Data shown are average ± SD of three replicates.

P-ases against water stress. As compared to controls, in both the cultivars, a significant decrease in relative water content (RWC) was observed in both leaves and grains at different stages of growth, indicating that plants were under stress. With respect to control, a substantial increase in acid P-ase activity was observed in the droughted leaves of drought tolerant cv. C-306 at both 30 and 60 DAS. However, no significant change was observed in drought susceptible cv. HD-2004 (Fig. 1A, B). Western blot analysis of drought tolerant cv. C-306 also revealed a significant increase in wPase-94 expression under stress conditions at 30 and 60 DAS (Fig. 3A). However, no significant differences were observed in drought susceptible cv. HD-2004. Similarly, in grains, with respect to control, in drought tolerant cv- C-306, a dramatic increase in acid P-ase activity was observed at

30 and 38 DPA (Fig. 2A, B). However, in drought susceptible cv. HD-2004, no significant change in acid P-ase activity was observed. Immunoblotting analysis in the developing grains revealed that under stress conditions in drought tolerant cv. C-306, a drastic increase in wPase-94 expression was observed at all the stages. In contrast, no substantial change was observed in drought susceptible cv. HD-2004. Overall, results obtained suggest that the increase in acid P-ase activities and wPase-94 expression in both the tissues in drought tolerant cv. C-306, may be due to fact that under conditions of drought, delivery of phosphate (Pi) is 473

World J. Agric. Sci., 4 (4): 471-475, 2008

well documented morphological, physiological and biochemical changes. To understand, whether droughtinduced change in phosphatase activities in leaves and grains was by a low level of Pi, Pi deficiency, or independent of Pi, the effect of drought treatment on the level of Pi was determined at different developmental stages. As indicated in Fig. 1C, with comparison to control, the Pi level was drastically decreased at 30 and 60 DAS in both the cultivars under drought treatment. The enhanced leaf acid P-ase activity at both the stages (Fig. 1A) was directly coupled with decreased Pi level in drought tolerant cv. C-306. However, in drought sensitive cv. HD-2004, acid P-ase activity was independent of Pi level. Similarly, in the developing grains of drought tolerant cv. C-306, an increase in acid P-ase activity coupled with decrease in Pi level was observed at 30, 38 and 42 DPA (Fig. 2C). Increase in acid P-ase activities in inverse relationship with the low level of Pi has been demonstrated in numerous species and plant parts viz; wheat leaves and roots [8, 16], maize leaves [17], sorghum roots [18]. In contrast, in drought sensitive cv. HD-2004, no direct coupling was observed between acid P-ase activity and Pi level. Overall, these results suggest a dependence of the enzyme level on Pi availability as a signal for induction of acid P-ase activities in wheat in water stress. To conclude, it became apparent that in arid- and semi-arid areas of the world, the acid P-ases may be playing very important role under drought stress in order to sustain adverse environmental conditions in correlation with low phosphorus levels. The expression of higher acid P-ase activities in both tissues particularly in drought tolerant cultivar C-306, is suggestion of its global role in enhancing Pi availability and possibility recycling of organic Pi compounds. In addition, results provide valuable information to develop screening marker tools for selecting lines with tolerance to drought stress and phosphorus status, thus improving filed emergence and survival percentage of plants.

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Fig. 3: Western blot analysis of wPase-94 expression under drought stress in leafs (A) and seeds (B) of drought tolerant cv. C-306 and susceptible cv. HD-2004 of wheat under irrigated control (C) and water stress (T) conditions at different stages of development (DAS: days after sowing, DPA: days post anthesis). Numerical values as shown in the bottom of Panel A and B, indicates relative band intensities, which were determined using Gel Visualization, Documentation and Analysis system (Bio-Rad, USA). Numerical comparisons are only valid within panels and cannot be made between panels. Each lane loaded with 60µg of total soluble proteins was resolved on 12% SDS-PAGE and transferred to nitrocellulose membrane and probed with 70-kDa white lupin antiserum. impaired, thus, resulting in the activation of the cellular phosphatases that release soluble phosphate from its insoluble compounds inside or outside of the cells thereby modulate osmotic adjustment by free phosphate uptake mechanism. Olmos and Hellin [15] also observed that acid phosphatases are known to act under salt stress by maintaining a certain level of inorganic phosphate which can be co-transported with H+ along a gradient of proton motive force.

ACKNOWLEDGEMENT AD Sharma would like to thank DST, Govt. of India for providing financial assistance for the present study. We are also grateful to Dr. Carrol P. Vance, Dept of Agronomy and Plant Genetics, University of Minnesota, St. Paul, MN-55108, USA for the gift of lupin APase antiserum. We also thank Dr Prabhjeet Singh, Dept of Biotechnology for providing seeds of drought tolerant and susceptible cultivars of wheat.

Phosphatases in relationship with phosphorous (Pi) under drought stress: An integral part of the plant response to Pi deficiency is the induction of both extracellular and intercellular acid P-ases. Plants respond to Pi deficiency depending on persistence of stresses with coordinated adaptations on multiple levels comprising 474

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