Ⅰ. INTRODUCTION
Oilseed rape (Brassica napus L.) is an important oil crop that is widely cultivated around the world. However, its growth and productivity are severely affected by various environmental stresses including salt stress, which reduces seed germination (Kumar et al., 2020). Salinity conditions during seed imbibition increase the osmotic potential and inhibit water absorption, which is a critical process for germination, seedling establishment, and overall plant growth (Bentsink, 2018). Salinity can be harmful to seed germination by causing toxicity to the embryo due to elevated levels of sodium and chloride ions. It while also disrupts the normal cellular processes of plants and causing damage to cellular components through the accumulation of reactive oxygen species (ROS) induced by oxidative stress (Daszkowska-Golec, 2011;Goud and Kachole, 2011).
Hydrogen sulfide (H2S) is a gas that occurs naturally in plants. H2S functions as a signaling molecule modulating various plant processes such as root organogenesis, seed germination, stomata movement, transpiration, and senescence (Liu et al., 2019;Zhang et al., 2020;Zhou et al., 2020). In recent years, there has been increasing interest in investigating the application of NaHS, a donor of H2S, to mature plants can improve their ability to endure different abiotic stresses, including drought (Zhou et al., 2020), extreme temperature (Pan et al, 2020), and high salt (Kaya et al., 2020). H2S has been shown to mitigate the negative impact of salt stress on plant growth and development by reducing ROS accumulation and increasing antioxidant activity (Shen et al., 2013;Guo, 2018), positively affect on seed germination under salt stress. In addition, salt-stressed rice plants in H2S application display higher antioxidants such as ascorbate, glutathione, and redox potential (Mostofa et al., 2015). Although H2S has been reported to involve plant resistance mechanisms, the mechanisms of NaHS application to improve germination in oilseed rape under salt stress have not been fully elucidated.
Therefore, in the present study, the protein content, amylase and protease activity, and H2O2 accumulation were carried out to unveil the effects of H2S pretreatment on oxidative stress induced by salt stress during seed germination of oilseed rape. The study will provide insights into the role of H2S in regulating oxidative stress and improving seed germination under salt stress conditions, which could contribute to developing more salt-tolerant crops and sustainable agriculture practices.
Ⅱ. MATERIALS AND METHODS
1. Seed material and treatments
The seeds of oilseed rape (Brassica napus L. cv. Mosa) were sterilized with 70% EtOH for 3 min and washed with distilled water. The seeds were primarily divided into two groups: one group was immersed in water, and the other group was pre-treated with 500μM NaHS for 12 hours. The seeds were then allocated into Petri dish (9 cm in diameter and 1.2 cm in depth) and placed in an incubator at 28℃. To investigate the inhibitory effect of salt stress on seed germination, the water-treated group was divided into two subgroups: water (control) and 100 mM NaCl solution (NaCl group). The NaHS-pretreated group were also divided into two subgroups: one was watered with normal water (NaHS group), and the other was watered with 100 mM NaCl (NaHS+NaCl group). Samples were collected at 0, 12, 48, and 96 hours for analysis.
2. Determination of seed germination percentage and root length
The germination percentage of the seeds of oilseed rape was calculated as described previously (Samani et al., 2013). Germination percentage = S/T × 100 (S is the number of germinated seeds and T is the total number of seeds). The seeds were considered as germinated when the sprout’s length reached or exceeded the half length of the seeds. Root length was measured using a ruler and expressed in millimeters.
3. Determination of H2O2 content
For H2O2 determination, a 100 mg seed was extracted with potassium phosphate (50 mM, pH 6.8) solution. The extracted solution was combined (1:1) with 0.1% titanium chloride in 20% (v/v) H2SO4 and centrifuged at 10,000 × g for 5 min. The absorbance of the supernatant was recorded at 410 nm. The H2O2 level was calculated by using an extinction coefficient of 0.28 μmol-1 cm-1.
4. Determination of protein content, and amylase and protease activity
For measurement of protein content, seeds were extracted with 50 mM K-phosphate buffer (pH 7.8) and incubated for 30 min. Then the sample was centrifuged 12,000 × g for 10 min at 4℃. The supernatant was added (1:1) with Bradford solution, and the absorbance was measured at 595 nm.
For amylase activity checking, the seeds were crushed using homogenization in a 100 mM sodium acetate buffer (pH 5.5) and centrifuged at 12,000 × g for 10 min under 4℃. Then the supernatant was added with 1% soluble starch and incubated at 37℃ for 5 min. After incubation, DNS color reagent was added and left at boiling temperature for 5 min. The solution was kept at room temperature until cool down. Then absorbance was taken at 540 nm.
The protease was assayed according to the method described by Beyene (2006) with modifications. The 100 mg seeds were homogenized with of extraction medium (50 mM Tris-HCl, pH 9). The homogenate was centrifuged at 12,000 × g for 10 min at 4℃. The supernatant was added with 2% (m/v) azocasein which was dissolved in 50 mM Tris-HCl buffer (pH 9.0) and incubated at 37℃ for 30 min. An equal volume of ice-cold 10% (v/v) trichloroacetic acid (TCA) was added to the mixture solution.Uncleavedin was precipitated at 4℃, then was centrifuged at 12,000 × g for 10 min. The supernatant was added with 1 M NaOH, and the absorbance was taken at 440 nm.
5. Statistical analysis
The current experiment was conducted with a completely randomized design of three replications for each sampling day. Duncan's multiple range test was used to compare the means of separate replicates for each treatment day. The differences in letters were considered statistically significant at p<0.05. For statistical analysis, SAS 9.1.3 was used (SAS Institute Inc. in Cary, NC, United States).
Ⅲ. RESULTS
1. H2S pretreatment effects on the seed germination of oilseed rape under salt stress
Seed germination was prominently inhibited by NaCl (100 mM) treatment (Table 1 and Fig. 1). Noticeably, salinity (NaCl) causes a delayed emergence of both shoot and root, compared with control plants (Table 1). NaHS pretreatment improved seed germination and root development compared to the other treatments. NaHS pretreatment alleviated the reduction of seed germination and root growth along with NaCl.
2. H2S pretreatment effects on H2O2 contents during seed germination under salt stress
H2O2 content in control was gradually decreased during seed germination. H2O2 content was prominently increased with NaCl treatment along with restricted seed germination (Figs. 1 and 2) at 48 h and 96 h. However, NaCl along with NaHS pretreatment showed a lower H2O2 content at 48 h compared to NaCl treatment and a similar to control at 96 h (Fig. 2).
3. Effects of NaHS pretreatment on the activities of amylase and protease, and protein content during seed germination under salt stress
Amylase and protease activities were remarkably increased in control and NaHS-pretreated plants under normal conditions. It was slightly or not increased in NaCl-treated plants but largely enhanced by NaHS pretreatment (Fig. 3A and B). Protease activity was inversely consistent with protein content during seed germination. Protein content in control and NaHS-pretreated plants gradually decreased under normal conditions as seeds were germinated (Fig. 3C). NaCl treatment maintained a higher level of protein content but less in NaHS-pretreated plants.
Ⅳ. Discussion
Numerous studies have reported the positive effects of H2S on seed germination rate, seedling growth, and root length. Nevertheless, the underlying mechanism of H2S to promote seed germination is still poorly elucidated. In the present study, NaHS was used as an H2S donor. We confirmed that NaHS pretreatment alleviated the reduction of seed germination induced by salt stress.
In this study, salt stress caused by NaCl treatment significantly reduced seed germination and H2O2 accumulation (Fig. 1A and Table 1). These findings are consistent with other observations that high concentration of salt not only has toxic effects on specific ions but also decreases the water potential in the medium, making it difficult for germinating seeds to absorb water, ultimately leading to a reduction in germination (Tanyolac et al., 2007). However, NaHS-pretreated seeds were able to offset the inhibitory impact of salt stress on the germination process (Fig. 1A and Table 1) as well as H2O2 production (Fig. 2), indicating the positive effects of NaHS pretreatment on salt stress. The alterations in H2O2 levels that H2S induced at lower concentrations may be linked to the activation of antioxidant systems that are relyon H2S. Similar to our findings, salt stress response of H2S were previously reported in alfalfa (Lai et al., 2014) and rice (Mostofa et al., 2015), with the increase of superoxide dismutase, glutathione reductase, and peroxidase activities. It has been suggested that H2S triggers antioxidants by increasing glutathione and ascorbate levels, thereby increasing redox potential (Mostofa et al., 2015). These results indicate that H2S plays a crucial role in the mechanisms that enable plants to withstand salt stress-induced oxidative stress during seed germination.
It has been reported that storage metabolites such as starch and proteins in the aleurone layers of seeds are hydrolyzed by amylase and protease enzymes to provide nutrients for seedling growth and development (Rahman et al., 2007). Previous physiological and biochemical studies have revealed that hydrolyzing starch increases metabolizable sugars by amylase promoted by gibberellin (Beck and Ziegler, 1989;Gulber et al., 1995). In this study, amylase and protease activities were gradually increased in germinating seeds (Fig. 3A and B), accompanied by a decrease in protein content (Fig. 3C) which provide amino acids to the seedlings before the onset of autotrophic growth (Zafar et al., 2005). Salt stress maintained protein content at a high level, concomitant with lower protease activity, leading to a delay in germination and seedling growth. However, NaHS pretreatment enhanced amylase and protease activities along with a decrease in protein content under salt stress. A similar pattern of changes was observed in barley aleurone layers of seeds in the presence of H2S (Zhang et al., 2015). The results suggest that NaHS-induced increase of amylase and protease activity alleviates the seed germination delay induced by salt stress.
In conclusion, NaHS pretreatment could improve the germination of oilseed rape seeds exposed to salt stress. NaHS pretreatment significantly promoted oilseed rape seed germination and root growth, increased the activities of amylase and protease, restored the plasma membrane integrity in root tips damaged by osmotic stress, and reduced the accumulation of H2O2 in the presence of salt stress. The findings suggest that H2S may serve as a novel antioxidant signal during oxidative stress, a function not previously observed in oilseed rape seeds.
