Enhancement of Seed Vigor Performance in Aged Groundnut (Arachis

PHILIPP AGRIC SCIENTIST Vol. 99 No. 4, 339-347 December 2016

ISSN 0031-7454

Enhancement of Seed Vigor Performance in Aged Groundnut (Arachis hypogaea L.) Seeds by Sodium Nitroprusside under Drought Stress Ali Sepehri* and Hossein Reza Rouhi Bu-Ali Sina University, Faculty of Agriculture, Department of Agronomy and Plant Breeding, Hamedan, Islamic Republic of Iran * Author for correspondence; e-mail: [email protected]; Tel: +98(81)34425401; Fax: +98(81)34424192 The effect of sodium nitroprusside (nitric oxide donor) on enhancement of aged groundnut (Arachis hypogaea L.) seeds was investigated under drought stress. Seeds were primed in different solutions containing 0, 50, 100 or 150 µM sodium nitroprusside at 25 °C for 18 h and then subjected to 0, −0.4, −0.6, and −0.8 MPa drought stress for 10 d. Priming of aged seeds at different drought levels improved final germination percentage, germination rate, vigor index, seedling length, soluble sugars, total soluble proteins and the activities of antioxidant enzymes (catalase, superoxide dismutase, and ascorbate peroxidase) while mean germination time, electrical conductivity, malondialdehyde and hydrogen peroxide content decreased. Seed priming with 150 µM sodium nitroprusside at −0.8 MPa increased germination rate, seedling length, activity of catalase, superoxide dismutase and ascorbate peroxidase to 38.62%, 66.66%, 39.14%, 14.43%, and 42.51%, respectively. These results suggest that seed priming with sodium nitroprusside is effective in improving germination and seedling vigor of aged groundnut seeds through alleviating oxidative stress caused by drought stress.

Key Words: drought stress, groundnut, oxidative stress, sodium nitroprusside Abbreviations: ABA – abscisic acid, APX – ascorbate peroxidase, CAT – catalase, EC – electrical conductivity, EDTA – ethylenediamine tetraacetic acid, FGP – final germination percentage, GR – germination rate, H2O2 – hydrogen peroxide, MDA – malondialdehyde, MGT – mean germination time, MPa – Mega Pascal, mRNA – messenger RNA, NBT – nitroblue tetrazolium, NO – nitric oxide, PEG – polyethylene glycol, ROS – reactive oxygen species, SL – seedling length, SNP – sodium nitroprusside, SOD – superoxide dismutase, SS – soluble sugars, TBARS – thiobarbituric acid reactive substances, TSP – total soluble protein, VI – vigor index

INTRODUCTION Groundnut (Arachis hypogaea L.) is one of the most important legume (Fabaceae) oilseed crops but its seeds deteriorate rapidly during storage resulting in loss of viability and vigor (Nautiyal 2009). Seed deterioration results in a decrease in germination percentages and rates as well as a decrease in percentage of seedling emergence (Mohammadi et al. 2011). Hence, poor vigor and weak seedling establishment are big challenges for regeneration and propagation of deteriorated oilseed crops. One of the consequences of seed deterioration and/or exposure to environmental stress is the formation of maximum amounts of reactive oxygen species (ROS) including free radicals (Dong et al. 2014; Fu et al. 2015). Antioxidant enzymes such as superoxide dismutase and catalase are considered to be the main protective components engaged in the removal of free radicals and activated oxygen species (Sarvajeet Singh and Tuteja 2010; Dong et al. 2014). Interestingly, priming repairs damage of aged seeds or seeds exposed to abiotic stresses, and also improves germination performance (Jisha et al. 2013). The benefits of priming include an 339

increase in germination rate, consistent seedling emergence under a broad range of environments and improved seedling vigor and growth (McDonald 1999; Jisha et al. 2013). Some studies have reported positive effects of priming on deteriorated seeds of different species (Jisha et al. 2013). Nitric oxide (NO) is an important bioactive molecule that is involved in the regulation of various biochemical and physiological processes in plants (Sirova et al. 2011). It has an important role in seed germination and interplays with plant hormones in response to stressful conditions. The antioxidant role of NO has been reported in many animal and plant models (Sirova et al. 2011). NO antioxidant can scavenge the superoxide radical and the lipids derived from lipid peroxidation. Moreover, NO can activate cellular antioxidant enzymes (Sirova et al. 2011; Qiao et al. 2014). Seed priming with sodium nitroprusside (SNP) can increase germination and respiration rates as well as ATP synthesis. Also, NO can decrease ROS accumulation in seeds exposed to abiotic stress via improvement of the antioxidant system (Zheng et al. 2009). Rice seeds primed with SNP increased gene expression resulting in aquaporin synthesis, which improved water uptake

The Philippine Agricultural Scientist Vol. 99 No. 4 (December 2016)

Sodium Nitroprusside on Seed Vigor of Aged Groundnut Seeds

Ali Sepehri and Hossein Reza Rouhi

capacity during imbibition and promoted cell growth and development (Maurel et al. 2008). There is no available information on the influence of redox priming with SNP on the repair of deteriorated groundnut seed under drought stress. This study was therefore conducted to investigate the ameliorative effects of seed priming with SNP on the germination parameters of aged groundnut seeds under drought stress.

Morphological Measurements Germination percentage was recorded every day up to the end of the experiment. Mean germination time (MGT) was calculated by Ellis and Roberts’s formula (1981):

MATERIALS AND METHODS This study was carried out at the Department of Agronomy and Plant Breeding, Faculty of Agriculture, Bu-Ali Sina University, Iran. Groundnut seeds cv. NC2 (North Carolina 2) were obtained from AstanehAshrafieh (Gilan Province, located in northern Iran). Accelerated ageing was carried out at 40 ± 1 °C and 96–100% relative humidity for 96 h (Delouche and Baskin 1973). Accelerated ageing duration was determined by conducting a preliminary experiment. The optimal priming duration was determined in preliminary experiments (data not shown). Seeds were primed in sodium nitroprusside (nitric oxide donor) solutions at concentrations of 0 (distilled water), 50, 100 and 150 µM at 25 ± 1 °C. To make these concentrations, a stock of 1 M solution was prepared (297.95 g reached to 1000 mL with distilled water). Then 50, 100 and 150 µM SNP were made by diluting the stock solution based on the equation where C1= stock concentration, V1 = stock volume, C2 = final desired/working concentration and V2 is the final/ desired volume to make. After 18 h, seeds were removed from the priming solutions, surface-dried and allowed to dry back to their original moisture content at room temperature (about 25 ± 2 °C) for 24 h. Non-primed seeds were used as the control. To simulate drought stress under laboratory conditions, groundnut seeds were immersed in polyethylene glycol 6000 (PEG 6000) at osmotic potentials of 0, −0.4, −0.6, and −0.8 MPa solutions at 25 °C for 10 d under dark conditions (Michel and Kaufmann 1973): Ψs = - (1.18 × 10-2) C - (1.18 × 10-4) C2 + (2.6 × 10-4) CT + (8.39 × 10-7) C2T where Ψs is the osmotic potential (MPa), C is the PEG concentration (g-1 L) and T is room temperature (degrees Celsius). Seeds were aged with accelerated ageing test, then primed with aqueous solution of sodium nitroprusside and put into the simulated drought stress levels by PEG 6000. Seeds were placed in 15 cm Petri dishes between two layers of paper moistened with 15 mL of distilled water and incubated at 25 ± 1 °C for 10 d. Seeds were considered to be germinated when the radicle was 2 mm long (ISTA 2007).

where n is the number of seeds which were germinated on day D and D is the number of days counted from the beginning of germination. Germination rate (GR) was calculated by reversing the MGT formula: The seedling vigor index was calculated as follows (Rahnama-Ghahfarokhi and Tavakkol-Afshari 2007):

where VI is vigor index, SL is the mean of seedling length (cm) and FGP is final germination percentage. Physiological Measurements Electrical conductivity test. The electrical conductivity (EC) test, which was done according to the method of Hampton and TeKrony (1995). Four samples of sound looking, unbroken seeds were weighed, and then each sample was placed in 250 mL of distilled water in 500-mL beakers. The beakers were then sealed and kept at a controlled temperature of 25 °C for 24 h. The electrical conductivity of the seed leachates was then measured using an EC-meter (CyberScan PC 510). Electrical conductivity was expressed per gram of seed weight asµS cm-1 g-1 for each sample. Soluble sugars assay. Soluble sugars content was measured based on the anthrone method (Irigoyen et al. 1992). Total soluble proteins. For protein extraction, about 0.5 g of groundnut tissue was ground with liquid nitrogen and then resuspended in extraction buffer (50 mM TrisHCl, pH 7.4, 1 mM EDTA, 1 mM dithiothreitol, 1 mM leupeptin, 1 mM pepstatin, and 1 mM phenylmethylsulfonyl fluoride). After centrifugation at 12000 × g for 30 min at 4 °C, the protein content in the supernatant was determined according to the method of Bradford (1976) with bovine serum albumin (BSA) as standard. Determination of Lipid Peroxidation (Malonyldialdehyde Content) Lipid peroxidation was determined in terms of thiobarbituric acid-reactive substances (TBARS) concentration based on the method of Cavalcanti et al. (2004). Fresh sample (300 mg) was homogenized in 3 mL of 1.0% (w/v) trichloroacetic acid (TCA) at 5 °C. The homogenate was centrifuged at 12000 × g for 20 min and 1 mL of the supernatant was added to 3 mL of 20% TCA containing 0.5% (w/v) thiobarbituric acid


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(TBA). The mixture was incubated at 95 °C for 30 min and the reaction was stopped by quickly placing ice bath. The cooled mixture was centrifuged at 11000 × g for 10 min, and the absorbance of the supernatant at 532 and 600 nm was read. After subtracting the non-specific absorbance at 600 nm, the TBARS concentration was determined by its extinction coefficient of 155 mM -1 cm1 .

ascorbate, 0.1 mM H2O2 and 100 µL of enzyme extract. The level of activity was calculated with an extinction coefficient (2.8 mM -1 cm -1) and expressed in (1 µmol of ascorbate oxidized per minute) per mg protein.

Determination of Hydrogen Peroxide (H2O2) The H2O2 accumulation was measured according to Velikova et al. (2000). Fresh sample (500 mg) was homogenized in ice bath with 5 mL of 0.1% (w/v) TCA. The homogenate was centrifuged at 12000 × g for 15 min and 0.5 mL of the supernatant was added to 0.5 mL 10 mM potassium phosphate buffer (pH 7.0) and 1 mL 1 M KI. The absorbancy of the supernatant was read at 390 nm. Enzyme Assay Ground tissues (0.5 g) were mixed with 1 mL of 100 mM potassium phosphate buffer (pH 7.8), and centrifuged at 15000 × g for 30 min at 4 °C. The supernatant was collected and used for enzyme assay. Activity of the enzymes was then determined spectrophotometrically (Cary 100 UV-Vis., Australia). Catalase (CAT, 1.11.1.6) activity was assayed by the method of Cakmak and Horst (1991). Frozen tissues were homogenized in a mortar and pestle with 3 mL of ice-cold extraction buffer (25 mM sodium phosphate; pH 7.8). The reaction mixture contained 100 µL of crude enzyme extract, 500 µL of 10 mM H2O2 and 1400 µL of 25 mM sodium phosphate buffer. The decrease in the absorbance at 240 nm was recorded. The CAT activity was calculated with an extinction coefficient (39.4 mM 1 cm -1) and was expressed in units (1 µmol of H2O2 decomposed per minute) per mg protein. Superoxide dismutase (SOD, 1.15.1.1) activity was assayed according to the method of Giannopolitis and Ries (1977). The reaction mixture contained 100 µL of 1 µM riboflavin, 100 µL of 12 mM L-methionine, 100 µL of 0.1 mM EDTA (pH 7.8), 100 µL of 50 mM Na2CO3 (pH 10.2), 100 µL of 75 µM nitroblue tetrazolium (NBT), 2300 µL of 25 mM sodium phosphate buffer (pH 6.8) and 200 µL of crude enzyme extract with a final volume of 3 mL. SOD activity was assayed by measuring the ability of the enzyme extract to inhibit the photoreduction of NBT. Glass test tubes containing the mixture were illuminated with a fluorescent lamp (120 W), and identical tubes that were not illuminated served as blanks. After illumination for 15 min, the absorbance was measured at 560 nm. The SOD activity of the extract was expressed in U mg-1 protein. One unit of SOD (U) was defined as the amount of enzyme activity that was able to inhibit the photoreduction of NBT to blue formazan by 50%. Ascorbate peroxidase (APX, EC 1.11.1.11) activity was determined by monitoring the decrease in absorbance at 290 nm from ascorbate oxidation (Nakano and Asada 1981). The assay mixture (3 mL) contained 50 mM of potassium phosphate buffer (pH 7.0), 0.5 mM 341

Statistical Analysis A 5 × 4 factorial experiment based on completely randomized design with four replications was used. Data for germination percentage were arcsine transformed before analysis of variance. Statistical analysis was carried out using SAS 9.2 software. Mean comparison was performed using least significant difference (LSD) test at 5% probability level.

RESULTS AND DISCUSSION Final Germination Percentage (FGP) Final germination percentage improved at all levels of seed priming in non-stress conditions (Table 1). FGP in primed and non-primed seeds were decreased significantly by increasing intensity of drought stress, but the decrease was lower in primed seeds (Table 1). The highest FGP was obtained from 150 µM SNP under all levels of drought stress. There was no significant difference between 100 µM SNP and hydropriming or between hydropriming, 50 µM SNP and non-primed seeds under low level of drought. At −0.6 MPa, a significant difference was not observed between 100 and 150 µM levels SNP as well as between hydropriming and 50 and 100 µM SNP, while seeds primed with 150 µM SNP exhibited higher germination when they were exposed to high level of drought. Fu et al. (2015) reported that decreases in seed viability and quality are two symptoms of ageing/deterioration, especially when seeds are exposed to stresses such as drought. Since NO is a mediator in the cell stress signal pathway, an increase in FGP of aged seeds primed with SNP could be the result of molecular and biochemical responses increasing in the cell that induce the synthesis of germinationstimulating hormones such as gibberellin and ethylene (Varier et al. 2010; Sirova et al. 2011). Arc et al. (2013) found that germination percentages increased as a result of seed priming with SNP, and they suggested that it was because of the role of NO in abscisic acid (ABA) catabolism and the promotion of ethylene signal pathway. Hayat et al. (2014) stated that germination percentages of tomato seeds primed with SNP increased via β-Dglucanase activation and promotion of the gibberellin biosynthesis pathway. Mean Germination Time (MGT) Under non-stress conditions, the ranking of MGT from highest to lowest was non-primed seeds (7.19 d), hydropriming (4.21 d) and redox priming with 50 (3.92 d), 100 (2.79 d), 150 µM SNP (2.00 d) (Table 1). MGT increased with an increase in drought stress, and the pattern was more observable in primed than in nonprimed seeds. Maximum MGT was recorded from nonprimed seeds at all levels of drought stress and the lowest




Table 1. Mean comparison of sodium nitroprusside (SNP) priming effect on morphological characteristics of aged groundnut seed under drought stress conditions. Drought Final Mean Germination Seedling Electrical Priming Vigor stress Germination Germination Rate Length Conductivity Treatments Index (MPa) Percentage Time (d) (d-1) (cm) (µS cm-1 g-1) 0

-0.4

-0.6

-0.8 LSD

Non-prime Hydropriming 50 µM SNP 100 µM SNP 150 µM SNP Non-prime Hydropriming 50 µM SNP 100 µM SNP 150 µM SNP Non-prime Hydropriming 50 µM SNP 100 µM SNP 150 µM SNP Non-prime Hydropriming 50 µM SNP 100 µM SNP 150 µM SNP

47.00 ± 1.15 73.00 ± 0.57 61.50 ± 1.45 70.66 ± 0.88 77.50 ± 1.20 28.66 ± 0.88 34.00 ± 0.57 32.66 ± 1.20 38.00 ± 1.15 44.33 ± 1.85 24.00 ± 1.15 30.33 ± 0.88 27.00 ± 1.00 34.33 ± 0.88 37.66 ± 0.33 18.33 ± 1.20 25.33 ± 0.88 22.00 ± 2.08 28.66 ± 0.66 35.33 ± 0.88 4.28

7.20 ± 0.01 4.22 ± 0.41 3.91 ± 0.20 2.79 ± 0.02 2.00 ± 0.02 10.03 ± 0.03 9.36 ± 0.06 9.31 ± 0.34 7.95 ± 0.02 7.01 ± 0.01 10.98 ± 0.15 10.10 ± 0.05 10.11 ± 0.19 8.55 ± 0.02 7.80 ± 0.02 11.78 ± 0.06 10.75 ± 0.12 10.65 ± 0.22 9.40 ± 0.02 8.50 ± 0.02 0.59

0.139 ± 0.00 0.242 ± 0.0228 0.258 ± 0.0144 0.357 ± 0.037 0.500 ± 0.0072 0.099 ± 0.0003 0.107 ± 0.0010 0.107 ± 0.0038 0.126 ± 0.0005 0.142 ± 0.003 0.091 ± 0.0015 0.099 ± 0.0005 0.099 ± 0.0020 0.117 ± 0.0005 0.128 ± 0.005 0.084 ± 0.0006 0.093 ± 0.0010 0.094 ± 0.0020 0.106 ± 0.0003 0.117 ± 0.003 0.02

5.00 ± 0.05 8.76 ± 0.08 8.10 ± 0.08 8.73 ± 0.14 9.40 ± 0.10 2.96 ± 0.11 3.43 ± 0.28 3.16 ± 0.12 3.73 ± 0.24 4.06 ± 0.08 2.40 ± 0.10 3.01 ± 0.07 2.63 ± 0.16 3.20 ± 0.17 3.50 ± 0.05 1.90 ± 0.12 2.41 ± 0.07 2.13 ± 0.03 2.76 ± 0.20 3.16 ± 0.08 0.52

2.34 ± 0.039 6.39 ± 0.018 4.96 ± 0.069 6.17 ± 0.066 7.30 ± 0.155 0.85 ± 0.030 1.17 ± 0.116 1.03 ± 0.038 1.41 ± 0.048 1.80 ± 0.043 0.57 ± 0.008 0.91 ± 0.038 0.71 ± 0.061 1.10 ± 0.080 1.31 ± 0.013 0.34 ± 0.031 0.61 ± 0.020 0.47 ± 0.047 0.79 ± 0.062 1.12 ± 0.051 0.23

26.85 ± 0.25 20.12 ± 0.34 18.32 ± 0.15 17.73 ± 0.28 15.63 ± 0.37 32.24 ± 0.98 26.74 ± 0.04 26.46 ± 0.37 23.93 ± 0.10 21.69 ± 0.67 33.26 ± 0.55 29.70 ± 0.15 28.29 ± 0.64 25.06 ± 0.41 23.03 ± 0.22 34.04 ± 0.52 31.15 ± 0.66 28.96 ± 0.37 27.12 ± 0.39 24.08 ± 0.16 1.71

Each value indicates treatment mean ± S.E. SNP – sodium nitroprusside, MPa – Mega pascal

MGTs were detected in 150, 100, 50 µM SNP and hydropriming, respectively (Table 1). Studies have shown that NO causes synthesis of glycolytic enzymes that are effective in triggering the pentose phosphate pathway, via specific changes in the carbonilation pattern (Barba-Espin et al. 2012; Qiao et al. 2014). NO also indirectly results in the production of increased amounts of nicotinamide adenine dinucleotide phosphate (NADPH), and germination rates are accelerated causing a decrease in MGT. Liu et al. (2009) reported that the decrease in MGT as a result of redox priming with SNP rapidly decreased seed ABA content; this corresponds to an increase in expression of cyp707a2. Inability of seeds to take up water can be one reason for an increase in MGT with an increase in intensity of stress due to lack of activation of effective enzymes for breaking of polysaccharides. Primed seeds take up water more effectively than non-primed seeds, and thus they germinate faster (Varier et al. 2010). Germination Rate (GR) The highest GR was recorded in 150, 100 and 50 µM SNP and hydropriming. In non-stress conditions, it increased to 5.2, 3.14, 1.71, 1.49-fold, respectively, compared with the non-primed seeds (Table 1). GR was significantly diminished by drought stress. At −0.4 MPa, no significant difference was observed between 100 and 150 µM SNP or between 50 and 100 µM SNP, hydropriming and non-primed seeds (Table 1). Similar patterns were seen at −0.6 and −0.8 MPa. Germination rate is one of the parameters that can effectively be influenced by unfavorable conditions; thus, it can be used as an effective index for evaluation of useful and harmful environmental factors (Jisha et al. 2013). Piterkova et al. (2012) reported that under drought stress, redox priming with SNP improved GR of tomato seeds compared with

that of non-primed ones. Rehman et al. (2015) claimed that the decrease in germination rate and percentage of aged corn seeds was the result of a reduction in αamylase activity. Sirova et al. (2011) suggested that improvement of GR as a result of NO priming was due to induction of proteins synthesis by the growth hormone signal pathway such as gibberellin and ethylene. It seems that improvement of the GR of primed seeds is the result of an increase of gibberellin synthesis. Thus an increase in gibberellin content increases α-amylase enzyme activity resulting in increased polysaccharide degradation, which accelerates soluble carbohydrates transfer to the growing embryo. Seedling Length (SL) Drought stress decreased SL in primed and non-primed seeds. Seed priming enhanced SL compared with nonprimed under both stress and non-stress conditions (Table 1). Priming with 150 µM SNP increased SL more than the other treatments did. SL reduction in drought stress can be related to nutrition decrease or lack of transport from cotyledon to embryo. In addition, reduced water uptake in stress conditions causes a decrease in hormone secretion and germination enzyme activity resulting in seedling growth disorders (Kafi et al. 2009). Since one of the effects of NO is ABA catabolism and hormone synthesis stimulation such as ethylene and gibberellin, it seems that enhancement of seedling cell division in primed seeds under stress condition is a result in an increase in cellular gibberellin concentration. This hormone can reduce the resultant damage of deterioration in cells and stimulate cell division of seedlings (Li et al. 2013). SNP prevents fatty acid oxidation and preserves plant cells, thereby decreasing negative stress on SL by reducing lipoxygenase enzyme which is produced under oxidative stress (He et al. 2014). Improvement of millet


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seedling length was reported as a result of seed priming with 200 µM SNP (Sarath et al. 2007). Vigor Index (VI) The vigor index markedly diminished under drought stress (Table 1). At all drought treatments, no significant difference was detected between priming treatments and non-primed except for 150 µM SNP, which was higher compared with the other treatments. VI is one of the important morphological parameters used to evaluate seed germination under stress conditions. Germination percentage and seedling length decreased with increasing stress intensity and pattern had an influence on VI. Li et al. (2013) also found that priming wheat seeds with 100 µM SNP improved the vigor index. Electrical Conductivity (EC) The highest EC was obtained in non-primed and hydroprimed seeds (Table 1). With increasing drought intensity, EC increased in primed and non-primed seeds, but seed priming especially with 150 µM SNP diminished those effects. Maximum EC was for nonprimed seeds at −0.8 and −0.6 MPa, and it was not significantly different from that of hydroprimed seeds (Table 1). Exposure of aged seeds to stress conditions intensifies damage to cell membranes because of oxidative stress, thereby resulting in ion leakage from membranes and subsequently an increase in EC of seeds. Seed priming can repair the damage to membranes via DNA and new messenger RNA (mRNA) synthesis as well as re-expression of genes (Varier et al. 2010). When damaged membranes are repaired, EC is reduced. Liu et al. (2013) suggested that SNP is an effective treatment for reduction of EC in cotton seeds under oxidative stress. These authors suggested that NO enhances activity of antioxidative enzymes and increases membrane integrity via scavenging ROS. They also reported that NO prevents leakage of cellular content by increasing osmotic adjustment. Jyoti and Malik (2013) claimed that some causes of increased EC in aged seeds are due to existence of cracks in the plasmalema and its separation from the cell wall, and tearing of the endoplasmic reticulum in the absence of polyribosomes and dictosomes. Jisha et al. (2013) reported that dehydrogenase activity has a key role in counteracting the negative effects of membrane peroxidation during seed priming. Reduction of membrane peroxidation can result in a decrease in malondialdehyde content as well as electrolyte leakage.

Soluble Sugars (SS) The highest amount of SS was detected in primed and non-primed seeds under non-stress conditions (Fig. 1). Soluble sugars in all aged groundnut seeds decreased severely with increasing drought stress, although it was lower in primed than in non-primed seeds (Fig. 1). Seeds primed with 150 µM SNP had significantly the highest SS under drought condition. It seems that membrane integrity was severely affected, thus leakage of membrane electrolytes and soluble sugars occurred. Jyoti and Malik (2013) reported that with an increase in 343

Fig 1. Effect of priming treatments on soluble sugars of aged groundnut seeds under different drought stress levels. The values are means ± SE.

deterioration time, oligosaccharides which accounted for membrane stability are lost. Moreover, reduction of αamylase activity in deteriorated seeds reduced starchy storage degradation to monosaccharide and disaccharide, which are necessary for the nutrition of the growing embryo (Bailly 2004). Amylase and dehydrogenase activities have been shown to increase in primed seeds (Andoh and Kobata 2002), which would result in an increase in amount of soluble sugars that can be transferred to the embryo (Andoh and Kobata 2002; Bailly 2004). NO can increase monosaccharide and disaccharide contents indirectly via induction of gibberellin synthesis and subsequent amylase activity. Also, it has been found that NO can enhance β-amylase in wheat via breaking disulphidic bonds, and as a result, there was an increase in rate of starch degradation, and subsequently, an increase in the production of monosaccharides and disaccharides (Wu et al. 2011). Total Soluble Proteins (TSP) In non-stressed conditions, there were no significant differences between 50 and 100 µM SNP, while TSP amount in 150 µM SNP was higher than for the other treatments (Fig. 2). Under stress conditions, total soluble proteins decreased with an increase in drought intensity and the lowest amount was detected in non-primed seeds (Fig. 2). Priming significantly affected total soluble proteins and the highest soluble proteins were obtained from seeds that were primed in 100 and 150 µM SNP. It is clear that protein content decreased during the ageing process due to denaturation and irreversible damage to protein structure as a result of free radical invasion. Seed priming can minimize this damage via mRNA synthesis and subsequent synthesis of new protein (Tabatabaei 2013). Also, damage to chromosome structure, RNA and DNA are regarded as some of the molecular changes in aged seeds (Jyoti and Malik 2013). Protein synthesis system can be damaged in transcription and translation stages. Many researchers reported damage of the transcription components such as enzymes related to transfer RNA (tRNA) transcription and ribosomes during seed deterioration. Such damage can be caused by free radicals or some enzymes activities like ribonuclease (Varier et al. 2010). Soluble amino acids such as




Fig. 2. Effect of priming treatments on total soluble proteins of aged groundnut seeds under different drought stress levels. The values are means ± SE.

Fig.

3.

Effect of priming treatments on malondialdehyde content of aged groundnut seeds under different drought stress levels. The values are means ± SE.

cysteine, histidine, tryptophan and phenylalanine are more susceptible than insoluble and membrane types under oxidative stress (Fu et al. 2015). Asadi Sanam et al. (2015) suggested that using SNP can increase soluble protein under oxidative stress. They claimed that the reason could be increasing synthesis and inhibition of protein denaturation as a result of SNP use. Malondialdehyde (MDA) In non-stress conditions, the maximum amount of MDA was found in non-primed seeds (Fig. 3). MDA was elevated by an increase in drought intensity. Abbasi et al. (2014) reported an increase in MDA content in common vetch (Vicia sativa) as a result of drought stress intensity. Seeds primed with 150 µM SNP had the lowest MDA content under all stress treatments. No significant difference was observed between MDA amounts from 50 µM SNP priming and hydropriming at all drought levels. Dong et al. (2014) stated that seed priming can cause MDA content to be reduced in aged welsh onion seeds. MDA is one of the indicators of membrane peroxidation and has a positive correlation with membrane compound leakage. In this study, evidence is provided that reduction of MDA by 150 µM SNP priming is simultaneous with a decrease in membrane peroxidation and electrolyte leakage and the resultant possible damages. Hydrogen Peroxide (H2O2) Significant differences in H2O2 accumulation were not observed between 50 µM SNP and hydropriming under non-stress treatments. In this condition, the maximum amount of H2O2 was produced by non-primed seeds, while the minimum was found in seeds primed with 150 µM SNP (Fig. 4). H2O2 accumulation increased in primed seeds with an increase in drought stress. At the lowest drought levels, maximum H 2 O 2 accumulation was detected in non-primed seeds that it did not differ significantly with hydropriming and 50 µM SNP priming. At all drought stress levels, the lowest accumulation of H2O2 was obtained when 150 µM SNP was used in the priming solution. A similar pattern was observed in −0.6 and −0.8 MPa drought treatments (Fig. 4). Sirova et al. (2011) reported that application of NO

Fig. 4. Effect of priming treatments on hydrogen peroxide accumulation of aged groundnut seeds under different drought stress levels. The values are means ± SE.

donors such as SNP resulted in decreased H2O2 and MDA content by increasing antioxidant enzymes levels. H2O2 is one of the non-radical ROS which is produced under oxidative stress and can damage cell organelles, protein, lipids, carbohydrates, nucleic acid and cell membranes resulting in programmed cell death (Barba-Espin et al. 2012). Several studies showed that there is a direct association between seed viability, vigor and the accumulation of H2O2 and MDA (Barba-Espin et al. 2012; Dong et al. 2014). Catalase (CAT) Under non-stress treatments, CAT activity was enhanced to 19%, 29%, 46%, and 55% by using hydropriming and 50, 100 and 150 µM SNP compared with non-primed, respectively (Fig. 5). Transcription and expression of CAT encoding genes are induced by using SNP (Qiao et al. 2014). In the present study, the priming treatment alleviated the reduction of CAT activity at all drought treatments. At −0.4 MPa, the lowest CAT activity was observed in non-primed seeds which did not differ significantly from seeds primed with 50 µM SNP or those in the hydropriming treatment. The maximum CAT activity was recorded in seeds primed with 150 µM SNP,


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Fig. 5. Effect of priming treatments on catalase activity of aged groundnut seeds under different drought stress levels. The values are means ± SE.

Fig. 6. Effect of priming treatments on superoxide dismutase activity of aged groundnut seeds under different drought stress levels. The values are means ± SE.

which differed significantly from the other treatments. A similar pattern was detected with −0.6 and −0.8 MPa drought stress. It is known that seed priming can compensate for some of the changes via synthesis and repair of seed protein structures and reduction of intensity of oxidative stress, partly mediated by improvement of antioxidant enzyme activity (Jisha et al. 2013; Xia et al. 2015). CAT synthesis repairs cell structures by releasing oxygen which is the result of the decomposition of H2O2. It improves mitochondrial activities and subsequent respiration rate and ATP synthesis increases (Sirova et al. 2011).

Ascorbate Peroxidase (APX) Under non-stress conditions, maximum APX activity was observed in 150 µM SNP, hydropriming, 100 and 50 µM SNP, and activity was increased to 50%, 45%, 32% and 29% respectively, compared with non-primed treatment (Fig. 7). At all drought stress levels, priming with 150 µM SNP enhanced APX activity, which was significantly different from that of the other treatments. Zheng et al. (2009) reported that NO can increase transcription and activity of APX by having an effect on thiol-containing enzyme under drought and salt stresses. Also, Sheokand et al. (2010) claimed that activity enhancement of APX in the presence of SNP is the result of an increase in encoding the induction of mRNAs

Superoxide Dismutase (SOD) In non-stress conditions, maximum SOD activity was recorded in 100 µM SNP, which was significantly different from that of the other treatments and nonprimed seeds. This treatment enhanced SOD activity by 54% compared with that of non-primed seeds (Fig. 6). In the above condition, the priming treatments including 150 µM SNP, hydropriming and 50 µM SNP increased SOD activity to 42%, 40%, and 38%, respectively, compared with non-primed seeds. An increase in intensity of stress reduced SOD activity in primed and non-primed seeds at different drought levels (Fig. 6). In stress conditions, 100 µM SNP also showed higher activity compared with the other treatments and non-primed seeds at all drought levels. The protective effect of NO, which is the result of using SNP under oxidative stress, can be due to repair of mitochondria and induction of SOD activity. Xia et al. (2015) claimed that the main factor of enzyme activity improvement in oat (Avena sativa) seeds is ROS scavenged by SOD enzyme in the mitochondria. Li et al. (2013) found that wheat seeds primed with 100 µM SNP under oxidative stress could increase SOD activity and minimize cold-resultant oxidative stress. Dong et al. (2014) also reported improved SOD activity in aged welsh onion seed as a result of priming.

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Fig. 7. Effect of priming treatments on ascorbate peroxidase activity of aged groundnut seeds under different drought stress levels. The values are means ± SE.

CONCLUSION Generally, our results demonstrated that drought stress had a negative effect on morphological and physiological germination parameters of aged groundnut seeds. By increasing the intensity of stress, a reduction was observed in FGP, GR, VI, SL, SS, TSP, as well as CAT, SOD and APX activities, while an increase was recorded



for MGT, EC, MDA and H2O2. After aged groundnut seeds were exposed to priming solutions, aged seeds responded positively to priming treatments under both stress and non-stress conditions. SNP improved FGP and VI of seedling that may be due to scavenging of reactive oxygen species and/or free radicals by antioxidant activities system such as CAT, SOD and APX. The decrease in H2O2, MDA and EC in aged seeds indicated that oxidative stress and lipid peroxidation that occurred under drought stress were alleviated by SNP priming, especially at 150 µM concentration. It is clear that in lipid peroxidation, radical and non-radical ROS can directly and indirectly cause mitochondrial damage, inactivation of effective enzymes in germination, and membrane disorders. In this study, SNP mitigated stress intensity mainly by reduction of lipid peroxidation and H2O2. Seed pretreatment with 150 µM SNP solution increased tolerance to subsequent drought stress and reduced MGT resulting in higher GR and VI. Therefore, SNP as an exogenous plant alleviator and protectant against oxidative stress could be used to prevent negative effects of drought stress in aged groundnut seeds.

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