Efficacy of KNO3, SiO2 and SA priming for improving emergence, seedling growth and antioxidant enzymes of rice (Oryza sativa), under drought

Rice is an important staple crop produced and consumed worldwide. However, poor seed emergence is one of the main impediments to obtaining higher yield of rice especially in hot and dry ecosystems of the world that are ravaged by drought. Therefore, this study was carried out to evaluate the effects of potassium nitrate (KNO3), salicylic acid (SA) and silicon dioxide (SiO2) priming in improving emergence, seedling growth, biochemical attributes and antioxidant activities of FARO44 rice under drought conditions. Rice seedlings primed with 2.5% and 5% KNO3, 3% and 3.5% SiO2, and 1 mM and 2.5 mM SA were subjected to three drought levels of low, moderate and severe under the greenhouse. Seed emergence, seedling growth, biochemical attributes and antioxidant activities were thereafter evaluated. Seed priming experiments were laid in a completely randomized design with five replicates per treatment. The results found that rice seedlings responded differently to different priming treatments. However, all primed rice seedlings had significantly (P ≤ 0.05) improved emergence percentage (72–92%), seedling growth, seedling vigor, seedling fresh and dry biomass and shorter emergence time compared with controls. Likewise, total soluble protein content, activities of catalase, ascorbate peroxidase and superoxide dismutase, carbohydrate, soluble sugar and total chlorophyll contents of rice seedlings were increased by more than two-folds by seed priming compared with control. Salicylic acid showed less effect in increasing emergence, seedling growth, antioxidant activities and biochemical attributes of rice. Thus, this study established that seed priming with KNO3 (2.5% and 5%) and SiO2 (3% and 3.5%) were more effective in improving emergence, seedling growth, biochemical attributes and antioxidant activities of FARO44. Thus, priming of FARO44 rice with this chemical is recommended for fast emergence, seedling growth and drought resistance in dry ecosystems.

Seed priming improved seedling growth performance of rice under drought. Seed priming, drought and their interaction had significant (P ≤ 0.05) effects on seedling length, plumule and root length of FARO44 rice seedlings as presented in Table 2 Under all drought levels, plumule length of FARO44 rice seedlings was increased by KNO 3 , SiO 2 and SA priming compared with control. Moreover, KNO 3 , SiO 2 and SA primed FARO44 rice seedlings recorded significantly increased root length compared with control under all drought levels. However, under severe drought, 3.5% SiO 2 priming showed no significant effect on plumule length of rice seedlings compared with control.
Seed priming enhanced seedling biomass and vigor of rice under drought. Seed priming, drought and their interaction had significant (P ≤ 0.05) effects on seedling fresh and dry biomass, seedling vigor index I (SVI I) and seedling vigor index II (SVI II) of FARO44 rice as presented in Table 3. At mild drought, KNO 3 and 3% SiO 2 primed FARO44 rice seedlings had significantly higher seedling fresh biomass compared with control. However, 3.5% SiO 2 and SA priming had no significant effects on fresh biomass of FARO44 rice seedlings compared with control. The maximum fresh biomass (630 mg) was recorded by 5% KNO 3 primed rice seedlings under mild drought. At moderate drought, all primed FARO44 rice seedlings had improved fresh biomass compared with control. FARO44 rice seedlings primed with 5% KNO 3 (408 mg) had the highest fresh biomass under moderate drought. At severe drought, except 3.5% SiO 2 , all other priming treatments significantly increased fresh biomass of FARO44 rice seedlings compared with control. Maximum fresh biomass (347.97 mg) was recorded by 2.5% KNO 3 primed FARO44 rice seedlings under severe drought. At mild drought, except 1 mM SA, dry biomass of FARO44 rice seedlings was enhanced by all priming treatments compared with control. FARO44 rice seedlings primed with 3% SiO 2 had the highest fresh biomass of 90.84 mg compared with other primed rice seedlings. At moderate and severe drought, dry biomass of FARO44 rice seedlings was significantly improved by KNO 3 , SiO 2 and SA priming compared with control. However, 3.5% SiO 2 priming had no significant effect on dry biomass of FARO44 rice seedlings at severe drought compared with control.
Across all drought levels, KNO 3 , SiO 2 and SA priming enhanced SVI I of FARO44 rice compared with control. However, at severe drought, 3.5% SiO 2 primed rice seedlings were not significantly different from control. At mild and moderate drought, SVI II of FARO44 rice seedlings were significantly improved by KNO 3 , SiO 2 Table 1. Emergence attributes of KNO 3 , SiO 2 and SA primed rice under drought conditions. Mean values ± SE in the same column followed with similar letters are not significantly different according to DMRT (P ≤ 0.05); SE Standard error of the mean, EP Emergence percentage, EI Emergence index, MET Mean emergence time, *Significant at 5% level of probability, **Significant at 1% level of probability, ns not significant. Priming improved total soluble protein content, catalase, ascorbate peroxidase and superoxide dismutase activities of rice seedlings under drought. Seed priming had significant (P ≤ 0.05) effects on total soluble protein content, catalase (CAT), ascorbate peroxidase (APX) and superoxide dismutase (SOD) activities of FARO44 rice seedlings. However, drought and its interaction with priming had significant effects on total soluble protein content of FARO44 rice seedlings, but not on CAT, APX and SOD activities of FARO44 rice seedlings as shown in Table 4. Except 1 mM SA primed rice seedlings under moderate drought, total soluble protein content of FARO44 rice seedlings was enhanced by priming treatments under all drought levels compared with control. Under all drought levels, KNO 3 , SiO 2 and SA priming increased CAT activities of FARO44 rice seedlings compared with control. Priming with 3.5% SiO 2 showed more effect in increasing CAT activities of FARO44 rice seedlings than other priming treatments. At mild drought, only SiO 2 primed FARO44 rice seedlings had higher APX activities, KNO 3 and SA priming showed no significant effects in increasing APX activities of rice seedlings compared with control. At moderate drought, except 5% KNO 3 and 2.5 mM SA primed rice seedlings, other primed FARO44 rice seedlings had significantly increased APX activities compared with control. At severe drought, only FARO44 rice seedlings primed with 3% and 3.5% SiO 2 and 1 mM SA had significantly increased APX activities, other primed rice seedlings were not significantly different from control. Under all drought levels, except KNO 3 , SOD activities of rice seedlings were significantly increased by SiO 2 and SA priming. Priming with SA showed more effect in increasing SOD activities of FARO44 rice seedlings than other priming treatments.
Seed priming enhanced carbohydrate, total soluble sugar, total chlorophyll contents and decreased malondialdehyde content of rice seedlings under drought. Seed priming, drought and their interaction had significant (P ≤ 0.05) effects on carbohydrate, total soluble sugar, total chlorophyll and malondialdehyde contents of FARO44 rice seedlings as shown in Table 5. Under all drought levels, except SiO 2 priming, carbohydrate content of FARO44 rice seedlings was significantly improved by KNO 3 and SA priming compared with control. At mild drought, except 3.5% SiO 2 , total soluble sugar content of FARO44 rice seedlings was significantly enhanced by 2.5% and 5% KNO 3 , 3% SiO 2 and 1 mM and 2.5 mM SA priming compared with control. At moderate drought, KNO 3 , SiO 2 and SA significantly increased total soluble sugar content of FARO44 Table 2. Seedling growth performance of rice primed with KNO 3 , SiO 2 and SA under drought conditions. Mean values ± SE in the same column followed with similar letters are not significantly different according to DMRT (P ≤ 0.05); SE Standard error of the mean, *Significant at 5% level of probability, **Significant at 1% level of probability, ns Not significant. www.nature.com/scientificreports/ rice seedlings compared with control. At severe drought, except SA, total soluble sugar content of FARO44 rice seedlings was markedly increased by KNO 3 and SiO 2 priming compared with control. Priming with KNO 3 showed more effect in increasing total soluble sugar content of FARO44 rice seedlings than other priming treatments. Under all drought levels, total chlorophyll content of FARO44 rice seedlings was significantly increased by KNO 3 , SiO 2 and SA priming compared to non-primed rice seedlings. Under all drought levels, malondialdehyde (MDA) content of FARO44 rice seedlings was significantly decreased by KNO 3 , SiO 2 and SA priming compared with control. Malondialdehyde causes lipid peroxidation and oxidative stress in rice seedlings.

Discussion
This study investigated the influence of KNO 3 , SiO 2 and SA seed priming to improve emergence, seedling growth, biochemical attributes and antioxidant activities of rice seedlings under drought. It is well known that rice production in arid and semi-arid ecosystems of the world are being ravaged by drought under a changing climate. Drought affects germination, seedling emergence and yield of rice 35 . This study found that KNO 3 , SiO 2 and SA priming substantially improved emergence percentage, emergence index and decreased mean emergence time of FARO44 rice under drought. Improved emergence attributes of rice might be related to increased cell division and elongations, water imbibition by seeds, repair and synthesis of DNA and RNA, increased activities of reserve mobilizing enzymes such as acid phosphatase, dehydrogenase, α-amylase and β-amylase in primed seeds. Primed rice seedlings had increased activities of catalase, ascorbate peroxidase and superoxide dismutase activities and low lipid peroxidation which are important factors in plant for deficit water resistance. Priming of seeds stimulate pre-germination processes in rice, wheat and maize that improved faster germination and seedling emergence 36 .
Many germination processes such as increased water imbibition, cell division and hydrolytic enzymes in rice were stimulated by priming which triggered faster germination as well as establishment of seedlings under stressful conditions 37,38 . Previous results by Yuan-yuan et al. 39 , found that rice priming with water and polyethylene glycol under drought had considerably increased emergence percent, emergence index and decreased emergence time. Zhang et al. 9 reported that sorghum primed with polyethylene glycol grown under drought showed improved emergence percentage, emergence index and vigour index. Khaliq et al. 40 found that rice priming with selenium substantially increased emergence, emergence index and decreased mean emergence time. Tabatabaei et al. 41 reported that SA priming of sorghum substantially increased percent emergence, emergence index and decreased emergence time under drought. www.nature.com/scientificreports/ This study found that KNO 3 , SiO 2 and SA priming substantially increased seedling growth of rice under drought. Enhancement of rice seedling growth might be due to increased cell division and elongation and activation of ROS scavenging enzymes in primed seeds. Potassium nitrate, silicon dioxide and SA stimulated rice seedling growth by serving as nutrients and initiators of essential emergence and growth processes in seedlings. Previously, Zhang et al. 9 found that sorghum priming with polyethylene glycol increased shoot length, root length and seedling length under drought. Khaliq et al. 40 reported that selenium priming of rice considerably improved shoot and root length, and seedling dry biomass. Tabassum et al. 22 found that wheat priming with CaCl 2 and hydropriming enhanced plant height, leaf area, panicle length and grain yield under terminal drought.
Seedling biomass and vigour of FARO44 rice seedlings grown under drought were improved by KNO 3 , SiO 2 and SA priming. Improved biomass and vigour of rice were as a results of increased cell elongation and division, nucleic acid synthesis and repair in primed seeds. Similar results were reported by Farooq et al. 19 that SA priming of rice improved growth, seedling fresh and dry weights under drought. Selenium primed rice and PEG primed sorghum showed substantially enhanced seedling dry biomass and vigor index under drought 9,40 . Javed et al. 5 reported that two rice cultivars primed with 0.75% KNO 3 recorded significantly increased seedling growth, seedling biomass and vigour. Previously, Wang et al. 38 reported that salicylic acid and selenium primed rice seedling under chilling stress recorded increased germination, shoot and root fresh biomass, shoot and root length.
The present study found that total soluble protein content, antioxidant activities of catalase, ascorbate peroxidase and superoxide dismutase of FARO44 rice seedlings were improved by KNO 3 , SiO 2 and SA priming under drought. Improvement of protein content and antioxidant activities in primed rice seedlings indicated enhanced tolerance to drought stress. Increased antioxidant activities in rice seedlings justified the increased seedling emergence and seedling growth of rice under drought. Increased protein content and antioxidant activities in rice seedlings were associated with the potentials of KNO 3 , SiO 2 and SA priming for activating antioxidant machinery, synthesis of protein and nucleic acids. Under water deficit conditions, plants developed antioxidant machinery that scavenge excess ROS which impair lipid, protein and nucleic acids thereby causing lipid peroxidation and oxidative stress 42 . Under drought, plants accumulate soluble solutes and increase activities of antioxidants that scavenge ROS that cause oxidative stress and lipid peroxidation 35 . Corroborating these findings, Ahmad et al. 43 reported that maize priming with salicylic acid and ascorbic acid improved catalase and peroxidase activities with consequent increase in germination, shoot and root fresh weight and dry weight under drought. Higher activities of these enzymes in primed maize seedlings under drought suggested their roles in minimizing harmful effects of drought on plant growth and development. Abdel-Latef and Tran 21 reported that silicon priming Table 4. Total soluble protein content, CAT, APX and SOD activities of rice seedlings primed with KNO 3 , SiO 2 and SA under drought conditions. Mean values ± SE in the same column followed with similar letters are not significantly different according to DMRT (P ≤ 0.05); SE Standard error of the mean, CAT Catalase, APX Ascorbate peroxidase, SOD Superoxide dismutase, *Significant at 5% level of probability, **Significant at 1% level of probability, ns Not significant. www.nature.com/scientificreports/ of maize under alkalinity stress markedly recorded increased in total soluble protein, CAT, POD and SOD activities. Agreeing these results, earlier study has found that rice primed with salicylic acid and ascorbic acid under drought stress exhibited increased activities of CAT and APX 41 . Previously, Zhang et al. 9 reported that PEG primed sorghum grown under drought stress showed substantially improved CAT, SOD, APX and POD activities. Rice seedlings primed with selenium and SA under drought stress were found to have enhanced total soluble protein, GPX (glutathione peroxidase), CAT, APX, and SOD activities 19,40 . Hussain et al. 44 reported that two rice cultivars primed with SA and Se grown under low temperature stress recorded substantially higher activities of CAT, POD, SOD and glutathione. This study found that priming with KNO 3 , SiO 2 and SA substantially enhanced carbohydrate, total soluble sugar and total chlorophyll contents and decreased malondialdehyde content in FARO44 rice seedlings under drought. Enhanced carbohydrate, soluble sugar and total chlorophyll contents in rice seedlings were essential for osmotic adjustment and increased photosynthetic activities under drought conditions. Increased carbohydrate, soluble sugar and total chlorophyll contents in rice seedlings justified their increased emergence, growth and seedling vigour under drought. Low malondialdehyde content in primed rice seedlings suggested defense from lipid peroxidation and oxidative stress. Active accumulation of soluble solutes such as carbohydrate, soluble sugar, glycine betaine and proline is a key tolerance strategy shown by plants under drought 45 . Corroborating these results, Zhang et al. 9 found that sorghum primed with PEG grown under drought stress has significantly higher total soluble sugar and chlorophyll contents and reduced malondialdehyde content. Previous results of Parveen et al. 33 found that two maize varieties primed with 4 mM and 6 mM Si recorded increased emergence, seedling growth, activities of POD, SOD, CAT and APX, and low malondialdehyde content and increased content of glycine betaine, proline and soluble sugar. Khaliq et al. 40 reported that rice seedlings primed with selenium had markedly improved soluble sugar and total chlorophyll content. Abdel-Latef and Tran 21 found that Si primed maize grown under alkalinity stress showed substantially increased total chlorophyll and soluble sugar contents. Wang et al. 38 reported that rice seedlings primed with Se and SA under chilling stress showed higher total soluble sugar content. Jisha and Puthur 46 found that three rice cultivars osmoprimed with NaCl and grown under drought and salt stresses had increased carbohydrate and total chlorophyll contents. Table 5. Effects of KNO 3 , SiO 2 and SA priming on carbohydrate, total soluble sugar, chlorophyll and MDA contents of rice seedlings under drought conditions. Mean values ± SE in the same column followed with similar letters are not significantly different according to DMRT (P ≤ 0.05); SE Standard error of the mean, DMRT Duncan's multiple range test, *Significant at 5% level of probability, **Significant at 1% level of probability, ns Not significant, MDA Malondialdehyde.

Conclusions
Priming with concentrations of 2.5% and 5% KNO 3 , 3% and 3.5% SiO 2 and 1 mM and 2.5 mM SA responded differently in improving emergence, seedling growth, biochemical attributes and antioxidant activities of FARO44 rice seedlings grown under drought conditions. Increased emergence, seedling growth, biochemical attributes and antioxidant activities of rice seedlings indicated increased tolerance to drought. Seed pre-soaking with 2.5% and 5% KNO 3 and 3% and 3.5% SiO 2 were found to be more effective in improving emergence, seedling growth, biochemical attributes and antioxidant enzyme activities of rice seedlings than SA priming. Increased emergence and seedling growth of primed rice might be associated with the potentials of KNO 3 and SiO 2 priming in stimulating pre-germination metabolic events such as increased water imbibition, cell division and elongation, repair of damaged nucleic acids, activation of reserve mobilizing enzymes and antioxidant machinery within the seeds that eventually enhanced emergence, growth and vigour. The findings of this study justified the reliability of seed priming, an easy and affordable technique to be adopted by farmers in dry regions of the world for improving emergence, seedling establishment and growth under drought conditions.

Methods
FARO44 rice seeds (Oryza sativa L.) were obtained from Badeggi Rice Research Institute in north-central Nigeria.
It is a rice variety with a long grain produced from a hybridization between Taiwan Indica and African local rice 47 . It is high yielding and matures in 110-120 days. It can be produced under rain-fed and irrigation agriculture 47,48 .
The seed initial moisture level was 10.9% while the dried seed moisture was 8.97% on the basis of dry weight.
Pre-optimization of priming chemical concentrations and duration. To obtain reliable results, preliminary priming studies were performed by soaking rice seeds in varying concentrations of KNO 3 , SiO 2 and SA with different durations prior to obtaining the effective priming duration (8 h) and priming chemical concentrations 44 . The effective concentrations used for this study are: KNO 3 (2.5% and 5% w/v), SiO 2 (3% and 3.5%) and SA (1 mM and 2.5 mM). These selections were on the bases of germination and seedling growth performances.
Seed priming treatments. Prior to seed pre-soaking treatments, the different concentrations of SiO 2 , KNO 3 and SA were prepared and kept in a fridge. Viable quality rice seeds were surface sterilized in 0.5% sodium hypochlorite (v/v) for 10 min to suppresses microbial growth and rinsed thoroughly with distilled water. Rice seeds were separately soaked in solutions of KNO 3 (2.5% and 5% w/v), SiO 2 (3% and 3.5% w/v) and SA (1 mM and 2.5 mM) for 8 h, and the systems were kept in the dark laboratory growth room at 25 ± 2 °C, relative humidity of 50-70% and a photoperiod of 12 h light/12 h dark. The ratio of 1:5 (w/v) seed weight to solution volume was maintained 49 . The seeds were dried back to their near-original weight of 10.1% at 25 °C temperature for 48 h prior to germination tests 50 .

Rice emergence and drought stress induction experiment.
The emergence experiments were carried out in the Greenhouse of the Department of Biology, University Putra Malaysia (3.000384° N, 101.705545° E), Selangor. To assess the emergence and seedling growth of rice under drought conditions, pot experiments were conducted. Plastic pots (24 cm × 20 cm) were filled with 2 kg silty-loamy soil. The soil used for the experiment had organic matter content (0.94%), saturation percentage (0.94%), electrical conductivity (1.02 dSm −1 ), total nitrogen content (0.07%), potassium content (167.00 ppm), phosphorus content (6.00 ppm) and chloride ion content (8.30 mmol L −1 ). Ten (n = 10) rice seeds primed with 2% and 5% KNO 3 ; 3% and 3.5% SiO 2 ; 1 mM and 2.5 mM SA and unprimed seeds (control) were separately sown in each of the pots. After sowing the seeds, the pots were watered daily and maintained at 100% field capacity (FC) (well-watered) for thirty days prior to imposition of drought stress. Three levels of drought stress were imposed to the rice seedlings by limited watering, these include: mild drought (75% FC), moderate drought (50% FC) and severe drought (25% FC) for twenty-one days 2,4 . All priming experiments were laid in a completely randomized design (CRD) with five replicates. All the pots were placed in the greenhouse with 12:12 h light/night duration, day and night temperatures of 35 °C and 27 °C. Records of seed emergence were taken daily until all seeds emerged, a seed was considered emerged if the radicle was about 2 mm long 51 . Seedlings were harvested after 51 days and the following emergence and seedling growth parameters from six randomly selected seedlings from each replicates were measured 52 : Emergence index defined as the total number of seeds that emerge daily was calculated as 53 : Mean emergence time (MET) was evaluated using MET = Dn n , where n stands for the number of seeds that emerged on day D and D is the number of days counted from the start of emergence 54  www.nature.com/scientificreports/ Biochemical analyses and Antioxidant enzymes assays. Total soluble protein content of KNO 3 , SiO 2 and SA primed rice seedlings was determined according to the slightly modified method of Bradford 56 , while Bovine serum albumin (BSA) was used as a standard. Catalase activity of KNO 3 , SiO 2 and SA primed rice seedlings was evaluated according to the method of Zhang et al. 9 . The activity of CAT was expressed in U/mg −1 protein. One unit of CAT activity was defined as the change in absorbance of 0.01 units per minute.
Ascorbate peroxidase activity of KNO 3 , SiO 2 and SA primed rice seedlings was evaluated according to the method described by Nakano and Asada 57 by determining the absorbance decrease of oxidized ascorbate after every 15 s for 1 min at wavelength of 290 nm (ε = 2.8 mM −1 cm −1 ). The activity of APX was expressed in U/ mg −1 protein.
Superoxide dismutase activity of KNO 3 , SiO 2 and SA primed rice seedlings was determined according to the method described by Zheng et al. 3 . The decrease of NBT was evaluated by reading the absorbance change at 560 nm with a spectrophotometer (Model-Hitachi U-1900, Tokyo, Japan), and the activity of SOD was expressed as U/mg −1 FW.
Extraction and determination of total chlorophyll content of KNO 3 , SiO 2 and SA primed rice seedlings were performed according to the method described by Lichtenthaler and Wellburn 58 . The total chlorophyll content was calculated with the formula: Total Chlorophyll = C a + C b + C x + c . C a = 13.95(A 665 ) − 6.88(A 649 ); C b = 24.96(A 649 ) − 7.32(A 665 ); C x + c = (1000A 470 − 2.05C a − 114.8C b )/245; Where, C a = chlorophyll a, C b = chlorophyll b and C x+c = carotenoid.
Total carbohydrate content of KNO 3 , SiO 2 and SA primed rice seedlings was determined according to the method described by Nielsen 59 . Total carbohydrate content was determined from a standard curve prepared with mg/ml of D-glucose solution.
Total soluble sugar of KNO 3 , SiO 2 and SA primed rice seedlings was determined according to the method described by Watanabe 60 . The total soluble sugar content was calculated from a linear equation based on a standard curve produced from d-glucose. The absorbance of the reaction mixture was read at 620 nm with a spectrophotometer (Model-Hitachi U-1900, Tokyo, Japan).
Lipid peroxidation of KNO 3 , SiO 2 and SA primed rice seedlings was determined by referring to the malondialdehyde (MDA) content and was measured according to the slightly modified method of 61 . First, fresh leaf sample weighing 250 mg was frozen in liquid nitrogen. The frozen leaves were ground in chilled mortar placed on ice in 3 millilitre of trichloroacetic acid (TCA). The mixture was then centrifuged at 13,000 g for 7 min. at 4 °C. The supernatant (2 ml) was mixed with 2 ml of 0.67% thiobarbituric acid. The mixture was then heated in a water-bath set at 100 °C for 30 min. and the reaction was swiftly terminated by cooling in ice. The mixture was centrifuged again for 12,000 g for 10 min. for the suspended particles to settle. The absorbance of the supernatant collected was read at 532 and 600 nm. The mixture of 0.025% thiobarbituric acid in 10% trichloroacetic acid was used as a blank sample. The content of MDA (µmolmg −1 FW) was computed with the formula: MDA = (A532-A600)/ε, ε is the extinction coefficient (155 mM −1 cm −1 ).

Statistical analyses.
Prior to statistical analyses, normality of all the data was checked with the Kolmogorov-Smirnov test. Two-way analyses of variance were performed to compare the effects of seed priming and drought on emergence, seedling growth, biochemical attributes and antioxidant activities of FARO44 rice (ANOVA) with an SPSS (window version 24). Significant differences of means were separated using the Duncan's Multiple Range test (P ≤ 0.05) 55 .

Data availability
The data supporting the findings of this study will be obtained from the corresponding author upon request.