Research Note
Effect of Hydrogen Peroxide on Physiological Quality and Germination of Aged Pumpkin Seeds under Drought Stress
Condition
Hossein Reza Rouhi
1, Mohammad Hasan Vafaei
1,*, Maryam Saman
2, and Ali Abbasi Surki
31Bu-Ali Sina University, Faculty of Agriculture, Department of Agronomy and Plant Breeding, Hamedan, Islamic Republic of Iran
2Payame Noor University, Department of Agricultural Sciences, Tehran, Islamic Republic of Iran
3Shahrekord University, Faculty of Agriculture, Department of Agronomy, Shahrekord, Islamic Republic of Iran
*Author for correspondence; email: [email protected]; Phone: +98(81)34425401; Fax: +98(81)34424192 Received: 26 April 2019/ Revised: 21 November 2020/ Accepted: 04 December 2020
The physiological quality of seeds decreases during their storage under unfavourable conditions. These deteriorated seeds exhibit a poorer performance, especially under stress conditions. A factorial experiment was conducted to investigate the effects of H2O2 on the germination of aged pumpkin seeds under drought stress. Hydroprimed seeds and also the seeds pre-treated with 100, 200, and 400 µM H2O2 were exposed to drought (0, -0.2, -0.4, and -0.6 MPa applied using PEG). Under drought conditions, seeds primed with H2O2
showed improved germination percentages and rates, higher vigour index, greater plumule and radicle lengths, enhanced soluble sugars and protein contents, and more antioxidant enzymes activities e.g.
catalase, ascorbate peroxidase, and superoxide dismutase in comparison to untreated controls. However, mean germination time and malondialdehyde (MDA) content total soluble proteins decreased in primed seeds. Those primed with 100 µM H2O2 showed the highest germination rate, vigour index, soluble sugars, and total soluble protein by 93, 46, 76, and 51% respectively at a drought level of -0.6 MPa. H2O2 at 100 µM applied to deteriorated total soluble proteins pumpkin seeds reduced of the negative effects of deterioration, especially under drought stress.
Keywords: enzyme, deterioration, drought, pumpkin, storage
Abbreviations: ABA – abscisic acid, APX – ascorbate peroxidase, BSA –bovine serum albumin, CAT – catalase, FGP – final germination percentage, GR –germination rate, H2O2 – hydrogenperoxide, LSD – light significant difference, MDA – malondialdehyde, MGT – mean germination time, MPa – mega Pascal, mRNA – messenger RNA, NBT – nitrobluetetrazolium, PEG – polyethylene glycol, PL – Plumule length, RL– Radicle length, ROS – reactive
oxygen species, SOD – superoxide dismutase, SS – soluble sugars, TBARS – thiobarbituric acid reactive substances, TSP – total soluble protein, VI – vigour index
INTRODUCTION
Pumpkin (Cucurbita pepo L.) is an important medicinal plant in the pharmaceutical industry because of its unique oil content (30–50%) and compounds such as phytosterols, flavonoids, fatty acids, vitamin A and tocopherol (Sedghi et al. 2008). Rapid oxidation of unsaturated fatty acids in oily seeds reduces their storability, due to accelerated deterioration (Jyoti and Malik 2013) that affects physiological seed quality,
viability and vigour (Hou et al. 2014). Drought stress is one of the most important abiotic stresses that significantly reduces germination, growth, and yield of crops (Jisha et al. 2013). Drought causes oxidative stress in plant cells and increases reactive oxygen species (ROS) leading to damage of cellular organs, proteins, lipids, carbohydrates, nucleic acids, and cell membranes, and may ultimately lead to cell death (Sharma et al. 2012).
Therefore, poor vigour and weak seedling establishment
under drought stress are big challenges in deteriorated oilseed crops. Hydrogen peroxide (H2O2) is a signalling molecule that is naturally produced in chloroplasts, mitochondria, and peroxisomes in response to environmental stimuli, and has a complex role in maintaining cell homeostasis and integrity (Cerny et al.
2018).
Hydrogen peroxide is capable of activating genes that are directly responsive to biotic and abiotic stresses and it can induce a wide range of molecular, biochemical, and physiological responses as a mediator in signalling pathways (Moller and Sweetlove, 2010; Qiao et al. 2014).
During germination, H2O2 may affect hormone balance in seeds via increasing GA or reducing ABA content, promoting lateral roots and somatic embryogenesis, protein turnover and/or facilitated electron transfer, (Jeevan Kumar et al. 2015). Lu et al. (2009) reported an increase in antioxidant compounds in Bermuda grass (Cynodon dactylon) as a result of ABA application, due to induction of catalase and superoxide dismutase genes by H2O2. Barba-Espin et al. (2011) showed that H2O2, as a signalling molecule, plays a crucial role in pea (Pisum sativum) seed germination because of its specific effects on hormone content, the proteome and transcription of genes. Hydrogen peroxide treatments protect plants against many stresses. Application of H2O2 improves growth and development of cotton (Gossypium hirsutum) under drought stress conditions (Santhy et al.
2014). He et al. (2009) reported that priming of wheat (Triticum aestivum) seeds with H2O2 improves seed germination and vigour indices under drought conditions and oxidative stress. The authors also show that H2O2 acts as a secondary messenger in cell signaling pathways and increases tolerance to drought stress due to an increase in defence proteins, transcription factors and antioxidant enzymes. Barba-Espin et al. (2012) showed that pre-treatment of pea seeds with H2O2 improves germination percentage, seedling length and fresh weight. This study is to determine the (1) effects of H2O2 on germination, vigour indices and antioxidant capacity of aged pumpkin seeds under drought stress, and (2) to evaluate the role of H2O2 in recovery and enhancement of aged pumpkin seeds.
MATERIALS AND METHODS
The experiment was conducted in the Laboratory of Seed Science and Technology, Faculty of Agriculture, Bu-Ali Sina University, Iran, as a factorial in completely randomized design with three replications. Nonprimed pumpkin seeds (control group) and different concentrations of H2O2 include 0 (hydroprimed), 100, 200
and 400 µM were one factor that was used and four water potentials (0, -0.2, -0.4, and -0.6 MPa, using polyethylene glycol 6000) were applied as the second factor.
Seed materials and treatments. Pumpkin (Cucurbita pepo L.
var. styriaca) seeds were obtained from a certified seed supplier company in Iran (Hamedan province) in July 2017. The seeds were surface sterilized with sodium hypochlorite 1% (v/v) for 5 min and then washed with distilled water. Initial seed viability was determined based on the standard germination test of ISTA (2007).
Subsequently, the seeds were aged by placing them on metal meshed sheets above water in a closed container at 40°C for 48 h. The relative humidity in the container was 100% (Delouche and Baskin 1973). Then, seeds were soaked in H2O2 solutions for 18 h at 25°C. After priming, seeds were removed and washed with tap water and then rinsed three times in distilled water. Seeds were air dried to their original moisture content at room temperature. Finally, the following characteristics were determined:
Standard germination test. Aged seeds were tested for germination using three replications using the Between- Paper (BP) method for 8 days at 25°C (ISTA 2007).
Emergence of the 2 mm radicle was used as the germination criteria:
Final germination percentage (FGP) = (Ni/N)×100, where Ni is total number of seeds finally germinated, N=
total number of seeds.
Mean germination time (MGT) = ∑(Dn/n) (Ellis and Roberts 1981).
Where n is the number of germinated seeds on day D and D is the number of days from the beginning.
Germination rate (GR) = 1/MGT.
Vigour index (VI) = SL × GP/100 (Sepehri and Rouhi 2016).
Biochemical measurements. After 8 days (end of experiment), seedlings were used to determine biochemical parameters.
Soluble sugars. These were determined by the anthrone method (Irigoyen et al. 1992) using glucose for standardization.
Total soluble proteins. Determined by the Bradford method (Bradford 1976) using bovine serum albumin (BSA) as standard.
Malondialdehyde content. Lipid peroxidation was measured as in Cavalcanti et al. (2004) using the thiobarbituric acid
test and measuring malondialdehyde content (MDA).
0.1 g of fresh sample was homogenized in 3 ml 1% (w/v)
TCA at 5C. The homogenate was centrifuged at 14000 × g for 15 min and 1 ml supernatant was added
to 3 ml 20% (w/v) TCA containing 0.5% (w/v) thiobarbituric acid (TBA). The mixture was incubated at 95C for 30 min and the reaction was stopped by quickly placing in an ice bath. The cooled mixture was centrifuged at 12000 × g for 8 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-1cm-1.
Catalase (CAT) activity. Activity of this enzyme (extinction coefficient = 39.4 mM cm-1) was measured spectrophotometrically (Cary 100 UV-Vis., Australia) (Cakmak and Horst 1991) based on H2O2 consumption at 240 nm and recorded as enzyme units (1 μmol of H2O2 decomposed per minute) per mg protein.
Superoxide dismutase (SOD) activity. This was measured spectrophotometrically (Cary 100 UV-Vis., Australia) based on SOD ability to inhibit Nitro Blue Tetrazolium (NBT) reduction at 560 nm. An enzyme unit (one unit of SOD was defined as the amount of enzyme activity that was able to inhibit the photo reduction of NBT to blue formazan by 50%) per mg protein was recorded (Giannopolitis and Ries 1977).
Ascorbate peroxidase (APX) activity. Activity of APX (excitation coefficient = 2.8 mM cm-1) was spectrophotometerically (Cary 100 UV-Vis., Australia) determined based on the amount of ascorbate oxidation in 290 nm. An enzyme unit (1 μmol of ascorbate oxidized per minute) was recorded per mg protein (Nakano and Asada 1981).
Statistical Analysis. Data were analysed as a factorial experiment in a completely randomized design using SAS 9.1 software. Germination percentage data were arcsine transformed to normalize them for analysis, and means comparison was done by least significant difference (LSD) at 5% probability.
RESULTS AND DISCUSSION
Final Germination Percentage (FGP)
In non-drought conditions, the highest germination was observed in aged seeds primed with 100 μM H2O2 and the lowest germination in the controls, indicating the recovery of damaged seeds during hydropriming and/or application of H2O2. There was no significant difference between the values obtained from hydroprimed seeds
and those primed with 100 μM H2O2 (Table 1). Drought stress caused a significant reduction in germination percentage of all seeds. This trend was intensified at the higher levels of stress, except for seeds primed in 100 and 200 µM H2O2 (Table 1). Under stress conditions, the highest germination was obtained from seeds primed with 100μM H2O2, which did not significantly differ from that of seeds primed with 200 μM H2O2 or hydroprimed seeds. However, the germination of seeds primed with 400 μM H2O2 was the same as non-treated seeds. In other words, the higher (more than 200 μM) H2O2 concentrations inhibited recovery of aged pumpkin seeds (Table 1). Decrease in viability and physiological quality of aged seeds especially under stress conditions such as drought have been previously reported (Fu et al.
2015; Sepehri and Rouhi, 2016). While H2O2 acts as a mediator in signalling pathways of plant cells, an increase in germination percentage of aged seeds caused by application of H2O2, may be due to increased molecular and biochemical responses at the cell that result in inducing the synthesis of germination stimulating hormones (Jeevan Kumar et al. 2015; Cerny et al. 2018). Similarly, Barba-Espin et al. (2012) suggested that improvement of germination percentage of pea seeds by H2O2 might be due to its role in stimulating the signalling pathway of ethylene. The authors mentioned that H2O2 increased the concentration of the ethylene precursor (1-aminocyclopropane carboxylic acid) and consequently germination percentage was increased under stress conditions. Moreover, Yadav et al. (2011) stated that pre-treatment of pepper (Capsicum annum) seeds with H2O2 can alleviate damages caused by oxidative stress under stress conditions and increase the germination percentage due to activation of the antioxidant system.
Mean Germination Time (MGT)
In non-stress conditions, maximum MGT was observed for non-primed aged seeds and those seeds primed with 400 μM H2O2, followed by hydroprimed, while H2O2 concentrations of 200 and 100 μM were not significantly different (Table 1). The lowest MGT was obtained for the seeds treated with 200 and 100 μM H2O2 while the non- primed and the seeds treated with 400 μM H2O2 exhibited higher MGT especially in severe drought conditions (Table 1). Studies have shown that H2O2 induces specific changes in the carbonylation pattern and synthesize some effective glycolytic enzymes in pentose-phosphate cycle and indirectly leads to increased NADPH production, consequently accelerates seed germination. However, lower amounts of these enzymes may cause to structures and organelle damages
Table 1. Mean comparison of hydrogen peroxide (H2O2) priming effect on germination characteristics of aged pumpkin seeds under drought stress conditions.
Priming
Treatments Drought Stress
(MPa) Germination
Percentage (%) Mean Germination
Time (days) Germination Rate
(1day-1) Plumule Length
(cm) Radicle Length
(cm) Vigour Index
H2O2 (100 µM) 0 74.33 a 4.12 l 0.244 a 3.93 b 2.26 b 4.61 b
-0.2 37.66 c 7.75 j 0.129 de 2.50 e 1.13 c 1.37 e
-0.4 34.33 cde 8.35 hi 0.119 ef 2.13 efg 0.80 c-h 1.00 fgh
-0.6 29.00 fgh 9.20 g 0.108 fg 1.73 g-j 0.63 gh 0.68 hij
0 70.33 a 4.20 l 0.237 ab 3.62bc 2.20 b 4.10 c
H2O2 (200 µM) -0.2 35.66 cd 7.81 ij 0.128 de 2.33 ef 1.06 cde 1.21 ef
-0.4 32.33 d-g 8.41 h 0.119 ef 2.10 e-h 0.73 e-h 0.91 f-i
-0.6 27.33 ghi 9.26 g 0.108 fg 1.63 hij 0.66 fgh 0.62 ij
0 45.33 b 5.58 k 0.179 c 3.40 cd 2.10 b 2.50 d
H2O2 (400 µM) -0.2 27.33 ghi 9.51 fg 0.105 gh 2.16 efg 1.00 c-f 0.87 f-i
-0.4 23.33 ij 10.31 cde 0.097 ghi 1.90 g-j 0.66 fgh 0.60 ij
-0.6 17.66 k 10.85 bc 0.092 ij 1.53 ij 0.50 h 0.35 j
0 69.33 a 4.41 l 0.227 b 4.91 a 4.00 a 6.19 a
Hydro-primed -0.2 32.66 c-f 9.50 fg 0.105 gh 2.30 ef 1.10 cd 1.11 efg
-0.4 29.33 efg 10.20 de 0.098 ghi 2.00 f-i 0.96 c-g 0.86 f-i
-0.6 24.00 hi 10.66 bcd 0.093 hij 1.70 g-j 0.76 d-h 0.59 ij
Nonprimed controls 0 47.00 b 7.30 j 0.137 d 3.10 d 1.93 b 2.36 d
-0.2 28.66 fgh 10.06 ef 0.099 ghi 2.00 f-i 0.96 c-g 0.85 ghi
-0.4 24.00 hi 11.06 b 0.090 ij 1.76 g-j 0.66 fgh 0.58 ij
-0.6 18.33 jk 11.76 a 0.084 j 1.43 j 0.50 h 0.35 j
In each column means followed by the same letter are not significantly different at the P < 0.01 level, H2O2: Hydrogen peroxide,MPa: MegaPascal.
(Barba-Espin et al. 2012; Qiao et al. 2014). Manish et al.
(2010) have reported that application of 15 and 20 mM H2O2 reduced the MGT of Indian mustard (Brassica juncea) seeds under drought stress conditions. MGT may be increased under stress conditions due to the inability of seeds to absorb enough water, since enzyme activation and consequently the breakdown of the polysaccharides requires a high amount of water. Enzyme activation and increased amounts of soluble mono and disaccharides in primed seeds caused a lot of water absorption and also faster germination (Sepehri and Rouhi 2016).
Germination Rate (GR)
The germination rates of aged seeds treated with 100 and 200 μM H2O2, hydroprimed and 400 μM H2O2 increased by 77, 73, 66, and 31%, respectively, compared to the control (Table 1). Drought stress significantly reduced germination rate (Table 1). At -0.2 MPa, the highest germination rates were detected for the seeds primed with 100 and 200 μM H2O2, similar to seeds tested at -0.4 and -0.6 MPa. Germination rate is one of
the parameters strongly influenced by adverse environmental conditions and could be as an effective indicator for evaluating beneficial or harmful effects of environmental factors (Jisha et al. 2013). Santhy et al.
(2014) reported that pre-treatments of cotton (Gossypiumhirsutum) seeds with 80 mM H2O2 improved the rate of germination. Also, the germination rate of corn (Zea mays) seed increased with 140 mM H2O2 under drought stress (Ashraf et al. 2015). Rehman et al. (2015) found that reduced germination percentages and rates of aged corn seeds were related to decreased activity of α-amylase.
Barba-Espin et al. (2012) proposed that the improved germination rate of pea seeds treated with H2O2, may be due to induced synthesis of proteins associated with the signalling pathway of plant hormones such as gibberellin and ethylene, which increase α-amylase activities leading polysaccharide degradation thereby accelerating the transfer of soluble carbohydrates to growing embryo. In this regard, Barba-Espin et al. (2011) reported that an increase in content of ethylene increased rates of germination in pea seeds.
Plumule Length (PL)
At normal conditions, pre-treatment of aged seeds with 100, 200, and 400 μM H2O2 and hydropriming increased the plumule length of seedlings by 27, 17, 68, and 14%, respectively, compared to controls. Plumule elongation decreased especially at higher drought levels (Table 1).
This could be attributed to lack of food transfer from the cotyledons to the embryo or lower secretion of hormones and also low activity of germination enzymes that consequently disrupt stem growth (Sepehri and Rouhi 2016). H2O2 is promotes endosperm weakening, inhibiting abscisic acid (ABA) synthesis and stimulate ethylene and cytokinin synthesis, which reduce damages occurred during seed ageing (deterioration) and stimulate cell division (Cerny et al. 2018). Müller et al.
(2009) also reported that a variety of ROS, such as H2O2 and hydroxide radicals, are able to weaken cell wall structure and thus facilitate cell division.
Radicle Length (RL)
Hydropriming in non-stressed seeds improved radicle length in deteriorated seeds compared to other treatments (Table 1). Hydrogen peroxide had no obvious effect on the growth of pumpkin radicles under drought stress. There was no significant difference between H2O2 treatments and control treatments. At higher drought levels, radicle length of all seeds decreased, but this decline was more apparent for non-primed than primed seeds. Several researchers have reported the improvement of radicle growth of seeds treated with H2O2 under unstressed and stressed conditions (Amjad et al. 2003; Snathe et al. 2014). Here, application of H2O2 reduced the adverse effects especially at 100μM (Table 1), but not at high concentrations. In this regard, some researchers have reported that ROS especially H2O2 played a dual role in the signalling pathways of cells. At low concentrations, act as a messenger signal and increase tolerance to environmental stresses, while at high concentrations may cause to programmed cell death (Hancock et. al. 2006; Barba-Espin et al. 2010: Jeevan Kumar et al. 2015; Cerny et al. 2018).
Vigour Index (VI)
Drought stress decreased the vigour index. Higher vigour was obtained in hydroprimed seeds and those pre-treated with H2O2, which was significantly different from controls. However, there was no significant difference between primed droughted and control seeds, but hydrogen peroxide-treated seeds produced vigorous seedlings under both normal and stress conditions (Table 1). Santhy et al. (2014) showed also that pre- treatment of cotton seeds with 80 mM H2O2 improved
vigour index. In addition, researchers have stated that application of exogenous H2O2, as a seed pre-treatment, increased the rate of storage protein transfer to the growing embryos, thereby increasing seed vigour and seedling establishment due to changes in the pattern of carbonylation in the cells (Job et al. 2005; Tanou et al.
2009; Cerny et al. 2018).
Soluble Sugars (SS)
Aged seeds primed with 100 μM contained higher amounts of soluble sugars than seeds from other treatments. Soluble sugars did not differ between seeds that were hydroprimed and pre-treated with hydrogen peroxide at 200 μM, but these values were higher than those for non-primed seeds and those treated with 400 μM of H2O2 (Table 2). Under non-stressed conditions, the lowest amount of soluble sugars was in non-primed seeds. With increasing drought, the amount of soluble sugars in primed and non-primed seeds decreased, but the decline was less in primed than non-primed seeds.
The highest amount of soluble sugars in all drought levels was in seeds primed with 100 μM hydrogen peroxide, which was not statistically different from other priming treatments or from non-primed seeds (Table 2).
It seems that during seed deterioration, the cell membrane integrity is reduced and its permeability is strongly affected, resulting in leakage of membrane electrolytes and soluble sugars. Jyoti and Malik (2013) explained that oligosaccharides which accounted for membrane integrity such as raffinose severely damaged with increased deterioration. Raffinose is a protective carbohydrate for membrane lipid and proteins.
Furthermore, a decrease in α-amylase activity in deteriorated seeds decreases the breakdown of starch into soluble sugars, which are necessary for embryo growth (Bailly 2004). An increase in activity of amylase and dehydrogenase enzymes has been found in primed seeds (Andoh and Kobata 2002). Activity of these enzymes leads to an increase in soluble sugar content and in their transfer to the embryo (Andoh and Kobata 2002; Bailly 2004). It has been reported that hydrogen peroxide prevents the synthesis and transfer of ABA to the embryo and activates the synthesis of an ethylene precursor and gibberellin, which accelerates the germination process (Cerny et al. 2018). Regarding synergic effects of ethylene with gibberellin, auxin and cytokinin, hydrogen peroxide can increase sugar contents indirectly via induction of gibberellin synthesis and thus activity of amylase.
Table 2. Mean comparison of hydrogen peroxide (H2O2) priming effect on biochemical characteristics of aged pumpkin seed under drought stress conditions.
Priming
Treatments Drought Stress
(MPa) Soluble Sugars (mggdw-1)
Soluble Proteins (mggfw-1)
Malon- dialdehyde
Content (nmolgfw-1)
Catalase Units (mgpr-1)
Superoxidedis- mutase Units
(mgpr-1)
Ascorbate Peroxidase Units (mgpr-1)
H2O2 (100 µM)
0 55.44 a 10.08 ab 21.78 l 0.271 a 28.67 a 0.420 a
-0.2 10.16 e 3.33 e 53.77 i 0.243 ab 19.85 f 0.310 b
-0.4 8.29 ef 2.66 g 57.23 gh 0.220 bc 18.54 gh 0.299 b
-0.6 6.52 f-i 1.74 hi 61.02 ef 0.183 e-h 17.23 jkl 0.276 c
H2O2 (200 µM)
0 52.86 b 10.42 a 22.48 kl 0.258 a 27.74 b 0.407 a
-0.2 8.26 ef 3.44 e 56.25 hi 0.211 cde 19.21 fg 0.277 c
-0.4 6.52 f-i 2.75 fg 59.87 fg 0.197 c-g 17.98 hij 0.266 cd
-0.6 4.95 ij 1.80 h 63.46 de 0.167 hij 16.68 lmn 0.243 ef
H2O2
(400 µM)
0 20.57 c 6.34 c 44.28 j 0.213 cd 22.15 d 0.301 b
-0.2 7.50 fg 1.35 hij 63.10 de 0.182 f-h 19.29 fg 0.273 c
-0.4 5.39 g-j 1.26 ij 68.55 bc 0.171 g-j 17.54 ijk 0.256 cde
-0.6 4.30 ij 1.17 j 75.82 a 0.145 j 16.28 mno 0.225 f
Hydro-primed
0 51.57 b 9.70 b 25.03 k 0.249 ab 26.42 c 0.404 a
-0.2 8.06 ef 3.20 ef 61.81 ef 0.190 d-h 18.30 hi 0.259 cde
-0.4 6.53 f-i 2.56 g 65.79 cd 0.179 f-i 17.12 kl 0.246 def
-0.6 4.51 ij 1.67 hij 70.13 b 0.153 ij 15.88 no 0.226 f
Nonprimed controls
0 18.05 d 5.76 d 45.24 j 0.205 c-f 20.90 e 0.271 c
-0.2 7.26 fgh 1.32 hij 63.27 de 0.176 ghi 18.24 hi 0.245 def
-0.4 5.06 hij 1.25 ij 68.68 bc 0.168 hij 16.75 klm 0.227 f
-0.6 3.80 j 1.19 j 74.72 a 0.145 j 15.84 o 0.198 g
Total Soluble Proteins (TSP)
Under non-stressed conditions, there was no significant difference between aged seed protein content in seeds primed in100 and 200 μM H2O2, or in hydroprimed seeds (Table 2). The amounts of soluble protein decreased due to high drought stress. The highest amount of soluble proteins was observed for seeds primed with 100 μM H2O2, regardless of drought level (Table 2). A reduction in protein content in aged seeds may be due to denaturation or structural damage of proteins by free oxygen radicals. Seed priming minimizes these processes through increase mRNA synthesis and consequently synthesis of new proteins (Kibinza et al. 2011; Sepehri and Rouhi 2016). Tale Ahmad and Haddad (2011) reported that amounts of soluble protein in wheat decreased by increase the drought time period, which attributed to decomposition of soluble proteins during drought conditions. Structural damage of chromosomes, RNA and DNA are the molecular changes that occur in deteriorated seeds (Jyoti and Malik 2013). Damages to protein synthesis can occur in both transcriptional or
translation phases. Researchers have reported that transcription elements such as tRNA, enzymes and ribosomes may be injured during seed deterioration.
These damages may occur due to free radicals, or activation of special enzymes such as ribonuclease (Varier et al. 2010). It has been reported that soluble proteins are more susceptible to oxidative stress than membrane and insoluble species (Kibinza et al. 2011; Fu et al. 2015). Evidence suggests that H2O2 plays a key role in regulating protein signalling pathways by controlling the cell cycle and repairing damaged parts of the ribosomes. Since protein synthesis occurs in ribosomes, their repair as a result of H2O2 will increases the synthesis of proteins (Barba-Espin et al. 2011).
Malondialdehyde (MDA)
MDA content increased with drought stress intensity.
The lowest amount was in seeds treated with 100 and 200μM H2O2. Maximum levels of MDA were observed for untreated seeds and the seeds primed with 400 μM H2O2 (Table 2). Seeds primed with 400μM H2O2 had
highest levels of MDA in normal and stress conditions and those primed with 200 and 100 μM H2O2 showed the lowest ones. Priming of seeds with 100μM H2O2 reduced MDA content by 15, 17 and 18% at drought levels of -0.2, -0.4, and -0.6 MPa, respectively, while 200 μM H2O2 decreased it by 11, 13, and 15% (Table 2).
Malondialdehyde is an indicator of lipid peroxidation and is positively correlated with membrane leakage (Sepehri and Rouhi 2016). Jyoti and Malik (2013) reported a significant decrease in protein, total carbohydrates and lipid content of deteriorated seeds, accompanied by higher free fatty acids, H2O2 and reactive oxygen species.
Unsaturated fatty acids of cell membranes will be susceptible to free radicals and reactive oxygen species leading to lipid peroxidation which can produce MDA.
Malondialdehyde is a product of linoleic acid peroxidation and has the ability to damage membrane proteins by cross-linking (Varier et al. 2010; Sepehri and Rouhi 2016). Santhy et al. (2014) stated that pre-treatment of cotton seeds with 80 mM of H2O2 is an effective method to reduce MDA content under oxidative stress conditions. Reduced peroxidation of the membrane will decrease MDA contents and also the leakage of electrolytes from the seeds.
Catalase Activity (CAT)
Mean comparisons showed that at normal conditions, activity of this enzyme was higher in all priming treatments (with the exception of 400 μM H2O2). There was no significant difference between hydroprimed seeds and those treated with 100 and 200μm H2O2 (Table 2). Under drought conditions, the lowest activity of CAT was observed in control seeds, which did not significantly differ from that of seeds primed with 400 μM H2O2 or hydroprimed seeds. The highest activity of CAT was observed in seeds primed with 100 and 200μM H2O2 at all levels of drought stress, with the exception of -0.2 MPa. A decrease in CAT activity has been reported as a result of seed deterioration. Formation of CAT subunits occurs in the cytoplasm, while its synthesis occurs in the peroxisome (Kibinza et al. 2011; Sepehri and Rouhi 2016). Due to cytoplasmic leakage through the membrane and organelle injuries, the CAT formation may not be completed (Jyoti and Malik 2013). Seed priming is able to compensate some of the cell damage via synthesis and repair mechanisms of proteins, reduction of oxidative stress and also increase in production of antioxidant enzymes (Kibinza et al. 2011;
Jisha et al. 2013; Xia et al. 2015; Sepehri and Rouhi, 2016).
Application of H2O2 in seed provides the critical signals for genes encoding CAT. Synthesis of this enzyme is accompanied by repair of cellular structures and O2
release as a result of H2O2 decomposition with CAT, which improve mitochondrial activity. However, H2O2 at high concentrations can play a destructive role as a reactive oxygen species, inducing irreparable damage to membranes or even cellular organelles such as peroxisomes and prevent the synthesis and activity of CAT (Bailly et al. 2008; Barba-Espin et al. 2010). Kibinza et al. (2011) reported an increase in CAT activity of deteriorated wheat seeds during priming. Santhy et al.
(2014) also reported improvements in CAT enzyme activity in H2O2 treated cotton seeds.
Superoxide Dismutase Activity (SOD)
At normal conditions, activity of SOD at 100 μM H2O2 was significantly higher than other treatments. H2O2 increased the activity of the SOD enzyme by 37% in treated seeds versus non-primed seeds, as 200 μM H2O2, hydropriming and 400μM H2O2 increased SOD activity by 33, 26 and 6%, respectively (Table 2). The activity of SOD in both treated and untreated seeds decreased with drought intensity (Table 2). Except for 400 μM H2O2, effects of H2O2 levels on the activity of this enzyme was greater than hydropriming. At drought conditions, SOD activity was decreased when H2O2 concentration increased (Table 2). Although the enzyme activity of treated seeds was improved under drought stress, it was significantly higher at normal conditions. As seed was deteriorated, probably severe injuries occur for organelles and consequently cytoplasmic membrane and SOD activity decreased. It should be noted that mitochondria is one of the organelles that may damage during deterioration, which is a place for SOD production in addition to its respiratory function and energy production. Xia et al. (2015) proposed that scavenging of reactive oxygen species by SOD enzyme in mitochondria accounts for the main cause of enzymatic activity improvement in oat (Avena sativa) seeds. He et al. (2009) also found that pre-treatment of wheat seeds at concentrations of 20, 40, 60, 80 and 120 mM H2O2 under drought stress (-0.5 MPa) increased the activity of superoxide dismutase, catalase and ascorbate peroxidase, which minimized the oxidative stress induced by drought stress. They also reported that 80 mM H2O2 was the most effective concentration for wheat seeds.
Ascorbate Peroxidase Activity (APX)
At normal conditions (non-stressed), concentrations of 100 and 200 μM H2O2, hydropriming and 400 μM H2O2 increased the APX activity by 55, 50, 49 and 11%, respectively, compared to the control. However, no significant difference was found between 100 and 200 μM of H2O2 and hydroprimed seeds (Table 2). At -0.2 MPa,
seed treated with 100 μM H2O2 showed the highest activity and significantly differed from the other treatments, especially the control. A same trends were observed for -0.4 and -0.6 MPa. The enzyme activity was improved at 400 μM H2O2 treatment at all drought levels compare to hydropriming. It was observed that APX activity was increased by 11, 13, and 14% at drought levels of -0.2, -0.4, and -0.6 MPa, respectively (Table 2).
Ascorbate peroxidase has at least five different isoforms and is found in thylakoids (tAPX), glyoxysome membrane (gmAPX), stroma (sAPX) and cytosol (cAPX).
Therefore, it is able to scavenge free radicals. Barba-Espin et al. (2010) who investigated the effects of H2O2 on APX activity in pea seeds stated that H2O2 induced mRNAs which encode the enzyme in stroma and cytosol, resulting in increased activity. They also reported that induction of the stromal form was higher than the cytosolic form. Other studies indicate that priming of wheat, corn, and cotton seeds with H2O2 improves the activity of APX and CAT enzymes (He et al. 2009;
Ahmad et al. 2012; Santhy et al. 2014).
CONCLUSION
Drought stress decreased the performance of aged seeds of pumpkin, resulting in weak seedlings. Lipid peroxidation is the main cause of cell deterioration in seeds, the resultant production of ROS, which will degrade molecules and important cellular structures. The production of MDA in aged seeds suggests that peroxidation of lipids occurs, which is decreased in H2O2, -treated seeds, especially at 100 μM. Free radicals may cause mitochondrial damage, inactivation of enzymes and membrane and genetic damage that affect germination and vigour. Reduction of germination and quality traits of aged pumpkin seeds was partially remedied by hydropriming or H2O2 pre-treatments.
Hydrogen peroxide plays dual roles in cellular activity, depending on its concentration. H2O2 in higher concentrations causes damage to cellular organelles, plasma membranes and enzymes essential for germination, while in optimal concentrations it stimulates germination via preventing the synthesis and transfer of ABA, excitation of ethylene or changes in the carbonylation of proteins. Adverse effects of deterioration and drought stress on pumpkin seeds were eliminated by application of H2O2, in particular as a result of increased activity of CAT and APX enzymes.
Mean comparison between priming treatments (hydropriming and various concentrations of H2O2) showed that 100 μM H2O2 had significant positive effects on deteriorated pumpkin seeds. Finally, application of 100 μM H2O2 via priming is recommended to reduce the
negative effects of deterioration of pumpkin seeds, especially during drought stress.
ACKNOWLEGEMENT
Special thanks to Professor Derek Bewley for editing this article as well as Dr. Mojtaba Karimi for helping to write the article.
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