Cold plasma treatment enhances oilseed rape seed germination under drought stress

Effects of cold plasma treatment on seed germination, seedling growth, antioxidant enzymes, lipid peroxidation levels and osmotic-adjustment products of oilseed rape under drought stress were investigated in a drought-sensitive (Zhongshuang 7) and drought-tolerant cultivar (Zhongshuang 11). Results showed that, under drought stress, cold plasma treatment significantly improved the germination rate by 6.25% in Zhongshuang 7, and 4.44% in Zhongshuang 11. Seedling growth characteristics, including shoot and root dry weights, shoot and root lengths, and lateral root number, significantly increased after cold plasma treatment. The apparent contact angle was reduced by 30.38% in Zhongshuang 7 and 16.91% in Zhongshuang 11. Cold plasma treatment markedly raised superoxide dismutase and catalase activities by 17.71% and 16.52% in Zhongshuang 7, and by 13.00% and 13.21% in Zhongshuang 11. Moreover, cold plasma treatment significantly increased the soluble sugar and protein contents, but reduced the malondialdehyde content in seedlings. Our results suggested that cold plasma treatment improved oilseed rape drought tolerance by improving antioxidant enzyme activities, increasing osmotic-adjustment products, and reducing lipid peroxidation, especially in the drought-sensitive cultivar (Zhongshuang 7). Thus, cold plasma treatment can be used in an ameliorative way to improve germination and protect oilseed rape seedlings against damage caused by drought stress.

conditions, using radio frequency or microwave sources 15 . The cold plasma contains different oxygen radicals, charged particles, ions and UV light, which response for the stimulating effects on seeds 16 . The number of potential applications of cold plasma in agriculture has grown significantly in recent years. Cold plasma treatment is thought to be a fast, economic and pollution-free method to improve seed performance, plant growth and ultimately plant production 17,18 . This treatment plays a crucial role in a broad spectrum of plant development and physiological processes in plants, including the promotion of seed germination and seedling growth 19,20 , activation of photosynthesis 21,22 , regulation of carbon and nitrogen metabolism 23,24 . Plasma treatments are thought to enhance the ability of plants to cope with biotic and abiotic stress, such as drought stress 25 and disease stress 26 . Plasma treatment has been found to promote seedling growth, activities of SOD and POD, increase proline concentration of wheat under drought stress 25 . Jiang et al. also reported that cold plasma treatment increase activities of POD, polyphenol oxidase (PPO; EC 1.10.3.1) of tomato under disease stress 26 .
However, little information is available on the evaluation of cold plasma effect on oilseed rape responses under drought stress. The objectives of the current study were (1) to investigate the influences of cold plasma treatment on seed germination, seedling growth, antioxidant enzyme activities and osmolytes content of oilseed rape under drought stress; and (2) to explore how the regulatory mechanisms underlying oilseed rape resistance to drought stress are altered by cold plasma treatment.

Results
Seed germination. Positive influence of cold plasma treatment was recorded on seed germination of Zhongshuang 7 and Zhongshuang 11 under drought stress. Germination improvement was significantly more pronounced in Zhongshuang 7 than in Zhongshuang 11. Under drought stress, the germination rate, germination index and vigor index of Zhongshuang 7 were significantly reduced by 13.98% (p < 0.001), 28.14% (p < 0.001) and 29.91% (p = 0.003), while those of Zhongshuang 11 were significantly reduced by 8.89% (p = 0.008), 23.73% (p < 0.001) and 4.80%, respectively, compared with the well-watered seeds ( Table 1). The cold plasma treatment significantly increased the germination rate, germination index and vigor index by 6.25% (p = 0.004), 6.89% (p = 0.003) and 29.59% (p = 0.007) in Zhongshuang 7 and 4.44% (p = 0.015), 4.77% (p = 0.013) and 19.64% (p = 0.001) in Zhongshuang 11, respectively, compared to the drought-stressed seeds. The germination data was well fitted by the Richards' function ( Fig. 1). The value of Me in the plasma treated seeds of both cultivars were lower than those in the non-treated seeds under drought stress, which indicated that the speed of germination was enhanced by the cold plasma treatment under drought stress (Table 2). No significant differences in the parameters Vi, Qu and Sk in Zhongshuang 7 and Zhongshuang 11 were found among the non-treated and plasma treated seeds under drought stress.
Seed apparent contact angle. The apparent contact angle is shown in Fig. 2. Compared with the non-treated seeds, cold plasma treatment significantly decreased the apparent contact angle by 30.38% (p = 0.012) in Zhongshuang 7, and 16.91% (p = 0.024) in Zhongshuang 11, respectively.    Table 3. Effect of drought stress and cold plasma treatment on seedling growth of oilseed rape. WW: well watered; WW + Plasma: well watered + plasma; DS: drought stress; DS + Plasma: drought stress + plasma. Data were means ± SE of three replications. Different lowercase letters denote statistical differences between treatments groups at the 5% level according to Duncan's test. MDA content. MDA contents in the seedlings of Zhongshuang 7 and Zhongshuang 11 were markedly increased under drought stress (Fig. 5). The MDA content in plasma-treated Zhongshuang 7 was significantly reduced by 28.85% (p = 0.003), and that was reduced by 13.08% (p = 0.046) in Zhongshuang 11, respectively, compared with those of drought-stressed seedlings. The reduction in Zhongshuang 7 seedling MDA content after pre-treatment with cold plasma was significantly higher than that of Zhongshuang 11.
Soluble sugar content. Soluble sugar contents in the seedlings of both cultivars were significantly increased in all treatments under drought stress (Fig. 6). Cold plasma treatment markedly increased the soluble sugar content in Zhongshuang 7, but had no obvious influence on Zhongshuang 11, under well watered conditions. After pre-treated with cold plasma, the soluble sugar content was significantly raised by 13.21% (p = 0.002) in Zhongshuang 7, while that was raised by 18.58% (p = 0.001) in Zhongshuang 11, respectively, compared with the drought-stressed seedlings.  Soluble protein content. Effect of the plasma treatment on soluble protein content was similar to its effect on soluble sugar content (Fig. 7). The soluble protein contents in the seedlings of both cultivars were markedly raised in all treatments under drought stress. Zhongshuang 7 showed greater improvement in soluble protein content than Zhongshuang 11. After pre-treated with cold plasma, the soluble protein contents in Zhongshuang 7 and Zhongshuang 11 were significantly increased by 10.90% (p = 0.034) and 6.04% (p = 0.046), respectively, compared to the drought-stressed seedlings.

Discussion
In the present study, seed germination of oilseed rape was adversely affected by drought stress, but this was significantly ameliorated by the cold plasma treatment, especially for the drought-sensitive cultivar, Zhongshuang 7 (Table 1). This result is consistent with the findings of Selcuk et al. who found that plasma treatment significantly increased tomato seed germination 27 . Šerá et al. showed that poppy seed germination was also significantly increased by cold plasma treatment 28 . Richards' function was used to fit the germination process of seed 29,30 . In our study, germination of oilseed rape seed was well followed to the Richards' function ( Fig. 1), the speed of germination in Zhongshuang 7 and Zhongshuang 11 were reduced by drought stress, and those were effectively promoted by the cold plasma treatment ( Table 2). It is thought that cold plasma treatment improves absorptive ability, which might contribute to increased seed imbibitions, and therefore germination under drought stress 31,32 .  It is known that the growth status of plants affects their resistance to stress because improved growth can enhance plant resistance to stress 33 . Previous studies have suggested that plasma treatment significantly promotes poppy 28 , and tomato seedling growth 34 . This study has demonstrated that cold plasma treatment markedly increased seedling growth indices, i.e. shoot and root dry weights, shoot and root lengths, and lateral root number, which were significantly reduced by drought stress. Furthermore, cold plasma pre-treatment effect was pronounced on the drought-sensitive cultivar than on the drought-tolerant cultivar (Table 3). A study has shown that the cold plasma treatment significantly increased the dry weight of tomatoes under disease stress 26 . Plasma treatment also improved the growth of maize seedlings under cold stress 35 . Our results indicated that the improvement in resistance to drought stress in the oilseed rape seedlings pre-treated with cold plasma was partly due to their improved growth.
There is a direct association between wettability and germination of seed. The wettability of seed can be reflected by apparent contact angle, and with strong wettability seeds can absorb more water to stimulate seed germination under drought stress 30,36 . In the present study, cold plasma treatment significantly decreased the apparent contact angle of oilseed rape seed (Fig. 2). In agreement with this result, Li et al. who demonstrated that cold plasma treatment decreased the apparent contact angle of soybean seed 19 . Bormashenko et al. also found that cold radiofrequency air plasma treatment decreased the apparent contact angle of lentil and wheat seeds 32 . This suggested that the wettability of seed was improved by cold plasma treatment, which was important to improve seed germination by increasing water uptake of seed under drought stress.
Plants have naturally developed a number of physiological and biochemical strategies to adapt to drought stress. Drought stress usually induces the accumulation of ROS, which cause oxidative damage to plants. In contrast, plants can raise the efficiency of the antioxidant system activity, which improves their resistance to drought stress by increasing the levels of antioxidant enzymes, such as SOD, POD and CAT, and non-enzymatic compounds 10,37 . Plasma treatment plays an important role in regulating water balance by modulating antioxidant enzymes 35 . Plasma treatment stimulated SOD and POD activity in tomato seedlings 23,26 . In our study, the cold plasma treatment significantly increased the SOD and CAT activities in oilseed rape seedlings under drought stress (Figs 3 and 4). This implicates that the cold plasma treatment plays an important role to reduce oxidative damage and helps to maintain normal physiological metabolic activities, leading to improved oilseed rape seedling growth under drought stress.
The overproduction of ROS, induced by drought stress, leads to oxidative stress damage to protein and membrane lipids 38 . MDA is the product of membrane peroxidation and has been used as a direct indicator of lipid peroxidation and membrane damage 39,40 . The present study demonstrated that under drought stress conditions, the cold plasma treatment reduced membrane lipid peroxidation damage by increasing antioxidant enzyme activities, thus significantly reducing the accumulation of MDA induced by drought stress. Moreover, the cold plasma treatment effect was more pronounced on the drought-sensitive cultivar (Zhongshuang 7) than on the drought-tolerant cultivar (Zhongshuang 11) (Fig. 5). This result agreed with Yin et al. who reported that plasma treatment reduced MDA content in tomato seedlings 34 . Our results suggested that the cold plasma treatment can partially increase oilseed rape seedling growth by preventing the oxidative damage generated by drought stress.
Soluble sugar and protein contents are strongly correlated to improved stress tolerance in plants. The accumulation of soluble sugars and soluble proteins maintains plant turgidity, enhances absorptive ability, and protects membranes and macromolecules during drought stress 41 3). Different lowercase letters denote statistical differences between treatment groups at the 5% level according to Duncan's test.
significantly increased the soluble sugar and protein contents in soybean seedlings 19 . Chen et al. showed that the soluble sugar and protein contents in brown rice also increased when it was treated with cold plasma 21 . Our data indicated that the cold plasma treatment significantly increased the accumulation of soluble sugars and proteins in oilseed rape seedlings under drought stress, and the increase was greater in Zhongshuang 7 than in Zhongshuang 11 (Figs 6 and 7). Under drought stress, cold plasma treatment might maintain a favorable potential gradient for water uptake into the seedlings and increase energy supply to the plant through the increased accumulation of soluble sugars and soluble proteins. Hence, the cold plasma treatment would have alleviated the negative effects of drought stress on oilseed rape seedling growth.
Finally, it is concluded that cold plasma treatments alleviate drought stress damage in oilseed rape. The seed germination and seedling growth improvement was due to the cold plasma treatment via improvement in wettability of seed, antioxidant enzymes activities, soluble sugar and protein contents, and reduced lipid peroxidation-linked membrane deterioration. Thus, cold plasma treatment can be used in an ameliorative way to protect the oilseed rape seedlings against the damage caused by drought stress. However, further studies are needed to investigate the effects of cold plasma on oilseed rape growth and yield under drought stress.

Materials and Methods
Experimental apparatus. Experiment was performed in the commercial computer-controlled plasma treatment apparatus HD-2N, which was consisted of a vacuum device, cold plasma generator and transmission device and inlet/outlet hopper. The core of the cold plasma processing system is the cold plasma generator. The device is composed of two parallel plates and has a metal suspension shell. The area between the plates and the metal shell is filled with insulating materials. Seeds receive the non-ionizing radiation treatment when they are in the cavity between the two polar plates. Capacitive coupled plasma (CCP) was generated by radio frequency discharge. The apparatus is illustrated in Fig. 8. Treatment conditions. Healthy and uniform seeds were selected and exposed to inductive helium plasma discharge under the following parameters: the plasma frequency was 13.56 MHz, the power was 100 W, the pressure was 150 Pa and the volume of the discharge chamber was 1200 mm × 180 mm × 20 mm. The time span of irradiation was 15 s and the temperature of the discharge, measured by a thermistor, was about 25 °C. Meanwhile, control seeds were exposed to the same vacuum and helium flux as the treated seeds, but did not receive the plasma treatment. The above seedling measurements and content analyses were carried out approximately one day after the seeds had been treated with cold plasma. Academy of Sciences, Nanjing, China (118°46′ E, 32°03′ N), between September, 2012 and December, 2013. Water limitation was applied using 15% (w/v) PEG 6000 (polyethylene glycol, Sigma). There were four treatments: (1) WW -well watered; (2) WW + Plasma -well watered + plasma; (3) DS -drought stress (15% PEG); (4) DS + Plasma -drought stress + plasma. The seeds were germinated in 9 cm petri dishes on two layers of filter paper. A total of 100 seeds were placed on each filter paper, and moistened with either 10 ml of PEG solution or 10 ml distilled water for the DS and WW treatments, respectively. The filter papers were replaced every 2 days to keep them completely moist. The seeds were incubated in a 25 °C light incubator. The seeds were considered to be germinated when the radicals were half the seed length. The germination percentage was recorded every 24 h for 7 days. Root length, shoot length, SOD and CAT activities, and soluble sugar and protein contents were measured on the 7 th day after germination. Seedlings were separated into shoots and roots for dry weight measurement. The experiment was a completely randomized design with three replicates. where G t represents the number of germinated seeds on t day, and D t represents germination days.
( ) Vigor index Germination index total length 3 The ratio cumulative germination was calculated as in Šerá et al. 43 . The Richards' function and population parameters Vi -final germination rate, index of viability; Me -median germination time, demonstrate the germination time of seed; Qu -dispersion, uniformity of seedling growth; and Sk -skewness, the composition of a population, were calculated according to Hara 44 .
The Richards' function Y t was calculated according to the following equation: Determination of SOD and CAT activity. SOD activity was assayed according to the nitroblue tetrazolium method, and CAT activity was determined by monitoring the enzyme-catalyzed decomposition of hydrogen peroxide (H 2 O 2 ) by potassium permanganate 45 .
Determination of MDA, soluble sugar and protein contents. The MDA content was determined according to Health et al. 46 . The soluble sugar content was determined by the phenol sulfuric acid method 47 , and the soluble protein content was assayed using Bradford's method 48 .
Statistical analysis. All data are presented as the mean value ± standard error (SE) of three replicates. Analyses were performed using the SPSS statistical software package (Version 16.0) and the variance (p < 0.05) of the data was analyzed by one-way ANOVA (Duncan's test).