Rice seed priming with sodium selenate: Effects on germination, seedling growth, and biochemical attributes

The aim of this study was to determine the effects of sodium selenate (15, 30, 45, 60, 75, 90, and 105 mg kg−1) on the germination and seedling growth of Changnongjing 1 rice (Oryza sativa L.) at 25 °C and 30 °C. Low selenate concentrations induced shorter and more uniform germination periods than did ultrapure water at both temperatures. Seedlings primed with low selenate concentrations were superior to those primed with ultrapure water in terms of plant height, fresh weight, dry matter accumulation, and soluble carbohydrate and protein contents. Lower selenate concentrations (15–75 mg kg−1) induced higher chlorophyll and phenol contents in seedlings than did ultrapure water. Lower selenate concentrations also increased the superoxide dismutase (SOD), peroxidase (POX), catalase (CAT), and glutathione peroxidase (GPx) contents in seedlings and significantly decreased the stress-related malondialdehyde (MDA) content compared to ultrapure water. In conclusion, rice seedling germination and growth were promoted by priming with low selenate concentrations (15–75 mg kg−1) but inhibited by priming with high selenate concentrations (90–105 mg kg−1).

www.nature.com/scientificreports www.nature.com/scientificreports/ seed germination and seedling quality, sodium selenate is rarely used as a seed initiator. Sodium selenite is known to initiate and prime seeds 3 . In the present study, we assume that sodium selenate can also ensure the uniformity of germination. Our objective was to investigate the application of sodium selenate as a seed germination initiator at different temperatures.

Results
Emergence attributes. The various concentrations of sodium selenate at the two temperatures significantly affected rice seed germination characteristics (P ≤ 0.05; Table 1) and the time to start of emergence (TSE) (P < 0.05). Nevertheless, the relative sensitivity of the TSE to the various sodium selenate treatments differed between the two temperatures. The TSE was advanced by 2 d after treatment with 60 mg kg −1 sodium selenate at 25 °C and 45 mg kg −1 sodium selenate at 30 °C. After treatment with > 60 mg kg −1 sodium selenate, the TSE was significantly delayed at both temperatures. Sodium selenate significantly affected the final emergence percentage (FEP) on day 7 (P ≤ 0.05). The TSE did not significantly differ between the 25 °C and 30 °C treatments. On day 7, the germination potential increased with an increase in sodium selenate concentration from 15-45 mg kg −1 . At 45 mg kg −1 sodium selenate, the germination potential reached a maximum. When the concentration exceeded 75 mg kg −1 , the germination potential tended to decline. The same pattern was observed for the FEP. Sodium selenate concentration significantly affected the FEP (P ≤ 0.05), but there were no significant differences between 25 and 30 °C. However, at both temperatures, the FEP reached a maximum at 60 mg kg −1 sodium selenate. Therefore, low-concentration selenate treatments promoted Changnongjing 1 rice seed germination, whereas high-concentration selenate treatments inhibited it.
The various sodium selenate concentrations significantly affected the Changnongjing 1 germination indices (P ≤ 0.05) at both temperatures. The effects of sodium selenate concentration on Changnongjing 1 priming at 25 °C and 30 °C are shown in Table 2. Clearly, the E 50 varied significantly with sodium selenate concentration. Nevertheless, E 50 did not significantly vary with temperature. At both 25 °C and 30 °C, the E 50 decreased with increasing sodium selenate concentration from 15-75 mg kg −1 , reaching a minimum at 45 mg kg −1 . However, >75 mg kg −1 sodium selenate prolonged germination time by 50%. Temperature and sodium selenate concentration significantly affected the germination index. The germination index was highest at a temperature of 30 °C and a sodium selenate concentration of 45 mg kg −1 . At both temperatures, the germination index increased with an increase in sodium selenate concentration from 15-75 mg kg −1 but decreased at >90 mg kg −1 sodium selenate. The germination index represents the speed and uniformity of germination. After receiving 45 mg kg −1 sodium selenate, the Changnongjing 1 rice seeds germinated rapidly and uniformly at both 25 °C and 30 °C (Table 2). www.nature.com/scientificreports www.nature.com/scientificreports/ seedling growth. Sodium selenate also had a strong positive effect on rice seedling growth. Seedlings developed from seeds germinated with sodium selenate were of higher quality than those primed with water alone (Table 3). Sodium selenate significantly affected rice plant height (Table 3). When the selenate concentration was >45 mg kg −1 , the plant height began to decrease until 75 mg kg −1 . At this high dose, the plants were shorter than those derived from seeds initiated with plain water. At all selenate concentrations, the plants subjected to 25 °C were taller than those exposed to 30 °C. Sodium selenate significantly affected the shoot fresh weight (P ≤ 0.05) ( Table 3). Shoot fresh weights were greater in the 45, 60, and 75 mg kg −1 selenate treatments than in the water controls and lower in the 15, 90, and 105 mg kg −1 selenate treatments than in the controls. The maximum shoot fresh weight was obtained at 45 mg kg −1 sodium selenate. At 30 °C, the shoot fresh weights at all selenate concentrations except 105 mg kg −1 were greater than those in the water controls. The maximum shoot fresh weight was recorded at 45 mg kg −1 sodium selenate. The shoot fresh weights at 25 °C were significantly higher than those at 30 °C (P ≤ 0.05). Therefore, only certain sodium selenate concentrations and a temperature of 25 °C were conducive to rice seedling growth. Sodium selenate also significantly affected the shoot dry weight. Shoot dry matter accumulation in rice seedlings significantly varied with selenate concentration at 25 °C. Shoot dry matter accumulation increased with selenate concentration between 15 and 70 mg kg −1 , peaked at 45 mg kg −1 , and decreased from 75-105 mg kg −1 . In contrast, at 30 °C, the shoot dry matter accumulation did not significantly differ between any selenate doses and the water control. Nevertheless, the shoot dry matter increased with an increase in sodium selenate concentration from 15-60 mg kg −1 , peaked at 45 mg kg −1 , and decreased between 7-105 mg kg −1 . These patterns parallel those observed at 25 °C. Overall, the accumulation of shoot dry matter in rice seedlings did not significantly differ between 25 °C and 30 °C.
The effects of sodium selenate on rice seedling root development are shown in Table 4. Neither selenate concentration nor temperature significantly affected rice seedling root length. Selenate did, however, significantly affect root fresh weight (P ≤ 0.05). At 25 °C, the root fresh weights for all selenate treatments except 30 mg kg −1 were greater than those for the water control. The root fresh weight was highest in the 60 mg kg −1 treatment. At 30 °C, however, the root fresh weights for all selenate treatments except 15 mg kg −1 and 75 mg kg −1 surpassed those for the water control. At 30 °C, the maximum root fresh weight was measured in the 60 mg kg −1 sodium selenate treatment. The root fresh weights at 30 °C were significantly higher than those at 25 °C (P ≤ 0.05). Thus, rice seedling roots grew better at 30 °C. Sodium selenate significantly affected the rice root dry weight ( Table 3). The greatest root dry weights at 25 °C and 30 °C were recorded for the 60 mg kg −1 and 75 mg kg −1 sodium selenate treatments, respectively. The root dry weights also significantly differed between 25 °C and 30 °C.  www.nature.com/scientificreports www.nature.com/scientificreports/ Biochemical attributes. Sodium selenate concentration and temperature both significantly affected the total soluble carbohydrate content in rice seedlings (Fig. 1a). The total soluble carbohydrate content varied with selenate concentration (y 25 = −0.0015x 2 + 0.1072x + 15.746; y 30 = −0.0013x 2 + 0.0816x + 15.863). The maximum total soluble carbohydrate content was detected at 45 mg kg −1 selenate. The total soluble carbohydrate content was higher within the 15-60 mg kg −1 selenate concentration range and lower when the selenate concentration was >75 mg kg −1 than in the water control. Selenate also significantly affected the α-amylase activity in rice seedlings. The α-amylase activity varied quadratically with selenate concentration and temperature (y 25 = −0.0004x 2 + 0. 0306x + 8.5172; y 30 = −0.0005x 2 + 0.0339x + 8.5543) (Fig. 1b). The maximum α-amylase activity was observed at 45 mg kg −1 selenate. The α-amylase activity was higher within the 15-60 mg kg −1 selenate concentration range and lower when the selenate concentration was >75 mg kg −1 than in the water control.

Discussion
Selenium is not considered an essential trace element (micronutrient) for the growth and development of higher plants 15 . Nevertheless, this element has positive effects on many crops such as potato (Solanum tuberosum L.), ryegrass (Lolium perenne L.), lettuce (Lactuca sativa L.), and mustard (Brassica rapa L.) 7,16,17 . The present study showed that low concentrations of sodium selenate act as seed priming initiators and are beneficial for plants. Khaliq et al. 6 studied the effect of sodium selenite on Basmati rice germination and seedling growth. Moulick et al. 18 investigated the effects of selenium on the germination of rice seeds under heavy metal stress. In both cases, however, selenium was supplied in the form of sodium selenite instead of sodium selenate. In the present study, sodium selenate was used for seed priming. Low-concentration sodium selenate treatments had positive effects on seed germination mainly by optimizing germination characteristics such as the TSE, E 50 , MET, EI, and FEP. Selenate at 45-60 mg kg −1 significantly outperformed water and higher selenate concentrations as a seed germination initiator. In contrast, very high selenate levels inhibited germination. This finding corroborated those reported by Khaliq et al. 6 . Compared to the water control and the high-dose selenate treatments, low selenate concentrations significantly increased plant height, root length, and shoot and root biomass accumulation. Therefore, low selenate concentrations significantly affect plant growth ability. However, high selenate doses inhibited seedling growth and development, corroborating the data of Moulick et al. 18 . Low selenate concentrations are thus preferable for seed germination and seedling development 19 . The use of low levels of sodium selenate can increase the germination rate and uniformity in direct-seeded rice. Such levels of sodium selenate can also improve overall seedling quality. Relative to the water control, low selenate doses improved dry matter accumulation in rice seedlings. In contrast, high selenate concentrations actually inhibited seedling dry matter accumulation. This negative effect may be explained by the inhibitory influence of selenate on chlorophyll content and α-amylase activity. There is no evidence that sodium selenate directly participates in higher plant photosynthesis. Nevertheless, photosynthetic activity in rice is closely correlated with sodium selenate application 20 . Low selenate concentrations may promote photosynthesis 21,22 and increase the chlorophyll content 18 and α-amylase activity 3 in rice. High sodium selenate concentrations have the opposite effects. The present study shows that there is a quadratic relationship between soluble sugar/soluble protein content and selenate concentration. Similarly, Lyons et al. 16 found that low selenate concentrations increased photosynthesis. The total phenol content increased with selenate concentration, but the correlation was not significant at the early stages of seedling development. At >75 mg kg −1 www.nature.com/scientificreports www.nature.com/scientificreports/ selenate, the seedling phenol content significantly increased. The rice seedlings were abiotically stressed at these very high selenate concentrations. MDA is another important biochemical indicator of plant stress. MDA levels increase quadratically with selenate concentration. MDA was lowest at 45 mg kg −1 selenate. The MDA content was lower within the 15-75 mg kg −1 selenate concentration range and higher at >75 mg kg −1 selenate than in the water control. Since sodium selenate is also an antioxidant 23 , the reduced MDA content at low selenate concentrations may be correlated with selenate antioxidant activity. Relative to the water control, however, >75 mg kg −1 selenate increased the MDA content. Therefore, very high selenate levels may be phytotoxic, as the high seedling phenol content at these selenate doses can have negative impacts on rice seedlings; this finding is consistent with the result for phenol content. The changes in CAT, GPx, SOD, and POX activity were also consistent with selenate antioxidant activity. Peroxide and free radicals formed during seed germination and seedling development are scavenged and quenched by a series of antioxidant enzymes 24,25 . Khaliq et al. 6 reported similar observations using sodium selenite as a seed initiator.
As a seed germination initiator, 15-75 mg kg −1 sodium selenate can increase rice seed germination and enhance seedling growth. Low selenate doses may also increase starch, protein and sugar accumulation in the seedlings and improve their antioxidant capacity. The accumulation of sodium selenate in rice should also be assessed as a biofortification mechanism.

Methods
In this study, the rice (Oryza sativa L.) variety Changnongjing 1 (Yangtze University, Hubei, China) was used. The initial seed moisture content was 9.22%. Dry rice seeds were primed at two different temperatures (25 °C or 30 °C) in a solution of sodium selenate (Na 2 O 4 Se) at a 0, 15, 30, 45, 75, 90, or 105 mg kg −1 concentration. A two-factor, completely randomized design was used. In each treatment, 25 g of seeds was placed in a 200-mL conical flask containing 125 mL initiator solution 26 . The flasks were placed in an incubator [darkness, 25 ± 1 °C or 30 ± 1 °C, and 80% relative humidity]. The flasks were shaken once every 6 h 27 . After priming, the seeds were filtered through gauze, placed in distilled water for 20 min, rinsed five times with ultrapure water and set aside. Autoclavable glass Petri dishes were lined with double layers of filter paper, placed on a laboratory bench and left to air dry for 24 h 3 .
The Petri dishes were sterilized by autoclaving and left to cool. The seeds were surface sterilized by soaking for 15 Min in 5% sodium hypochlorite and washed with ultrapure water 3-5 times. Thereafter, 10 mL sterile water was aseptically pipetted into each Petri dish; then, 100 seeds were placed in each Petri dish, and each seed-soaking treatment was performed in triplicate. The Petri dishes were transferred into a phytotron (KRG-400BP, China) and incubated under 8,000 lx illumination for 14 h d −1 . No light was used for the remaining 10 h. The temperature was either 25 ± 1 °C or 30 ± 1 °C, and the relative humidity was 80% 28 . The Petri dishes were weighed daily and replenished with the required amount of selenate solution. www.nature.com/scientificreports www.nature.com/scientificreports/ Seed bioassay. Seedling emergence was counted daily using the method prescribed by the Association of Official Seed Analysts (AOSA) 29 until a constant count was reached. A seedling was scored as emerged when the hypocotyl length was ≥2 mm. The time to 50% emergence (E 50 ) was calculated according to the modified equation of Farooq et al. 30 : where N is the final number of emerged seeds; n i and n j are the cumulative number of emerged seeds counted at time t i and t j , respectively, where n i < N/2 < n j . The mean emergence time (MET) was calculated following Ellis and Roberts 31 : where n is the number of emerged seeds on day D, and D is the number of days counted from the beginning of emergence. The root and shoot lengths of five seedlings randomly selected from each experimental unit were measured 18 d after sowing (DAS). The roots and shoots were excised separately and oven dried at 70 °C for 48 h to obtain the dry biomass of each component. The two measurements were summed to derive the total seedling biomass.
Biochemical analyses. Lipid peroxidation in the treated and untreated rice seeds was determined from the malondialdehyde (MDA) content using the thiobarbituric acid method 33 . The α-amylase activity in ground rice seeds (1 g) was measured according to the technique of Suksoon and Jaehyeun 34 . The rate of maltose liberation from starch was calculated from the reduction in 3,5-dinitrosalicylic acid. Total soluble sugars and soluble proteins were quantified according to Dubois et al. 35 and Bradford 36 , respectively. The activities of superoxide dismutase (SOD) at 560 nm, CAT at 240 nm, POD at 470 nm, and GPx at 340 nm were determined according to Giannopolitis and Ries 37 , Dhindsa et al. 38 , Egley et al. 39 and Drotar et al. 40 , respectively. One unit of enzyme activity was defined as that required to reduce 50% of nitro blue tetrazolium chloride per minute (SOD), oxidize 1 μmol H 2 O 2 per minute (CAT), oxidize 1 μmol guaiacol per minute (POD), or catalyse the H 2 O 2 -induced oxidation of 1 μmol reduced glutathione at a pH of 7.0 and temperature of 25 °C (GPx). At 18 DAS, three randomly selected rice seedlings were harvested from each experimental unit. The total foliar soluble phenol content was determined from absorbance readings at 663 nm and 645 nm 41 . Photosynthetic pigments (chlorophyll a and b) were extracted in 80% v/v ice-cold acetone 42 and read at 663 and 645 nm wavelengths.
Statistical analyses. Data were analysed using the statistical software Statistix 8.1 (Analytical Software, Tallahassee, FL, USA), while differences among means were separated by using a least significant difference (LSD) test at the 5% probability level. Origin 8.1 (OriginLab Co., Northampton, MA, USA) was used for graphical representation.

Data Availability
The data used to support the findings of this study are available from the corresponding author upon request.