Spermidine priming promotes germination of deteriorated seeds and reduced salt stressed damage in rice seedlings

Deterioration of seeds is an important problem leading to low germination and uneven crop establishment causing poor crop growth and productivity. Soil salinity is another factor limiting crop cultivation. Spermidine (Spd) is a compound widely reported for diminishing adverse effects of salt stress in plants. In this study, the effects of Spd priming on normal and deteriorated rice seeds were investigated under salt stress during germination and young seedling stages. Rice seeds were primed with H2O and Spd solutions for 24 h before germination. All rice seeds were grown in Petri dishes containing 5 ml of deionized water with 0 (control) or 150 mM NaCl (salt treatment) for 1-10 d. The results showed that priming groups had higher germination percentage (GP) and germination index (GI) in both normal and deteriorated seeds. Moreover, deteriorated seeds primed with 0.5, 1.0 and 1.5 mM Spd showed significantly higher GP and GI than H2Oprimed and non-primed seeds. During early hours of hydration, Spd-primed seeds produced reactive oxygen species (ROS) faster and at higher level than non-priming and H2O priming groups in both control and saline conditions. Earlier accumulation of ROS was associated with more rapid germination. In young seedlings, salinity stress caused a marked decrease in growth and increased membrane damage indicated by higher malondialdehyde (MDA) and electrolyte leakage (EL). Conversely, Spd priming increased growth and reduced membrane damage of rice seedlings established from normal and deteriorated seeds under salt stress. The finding suggested that Spd priming can effectively improve germination of deteriorated seeds and enhance seedling growth under control and salt stress conditions.


Introduction
Salinity causes a limitation of plant growth, development and production of crops around the globe. In Thailand, salinity is one of serious problems in agricultural areas and productivity of rice which is a very important economic crop of the country. Rice is sensitive to salt stress especially in early seedling stage. Salt stress induces osmotic stress as a result of water deficit followed by ion toxicity effect resulting from excessive uptake and accumulation of sodium and chloride ions leading to oxidative stress due to high production of reactive oxygen species (ROS) (Munns and Tester, 2008). Generally, ROS such as hydrogen peroxide (H2O2), 2 superoxide anion (O2 •-) and hydroxyl radical (OH • ) are by-products produced from normal metabolism processes in chloroplasts, mitochondria and peroxisomes.
In seeds, ROS are produced throughout the life of the seeds from embryogenesis process to the end of germination . Mostly, ROS in seeds especially, O2 •are produced in mitochondria and subsequently dismutated to H2O2. Several types of enzymes, such as NADPH oxidases, amine oxidases, polyamine oxidases, oxalate oxidases, and a large family of class III peroxidases are contributed to production of O2 •and H2O2 You and Chan, 2015). It has been reported that high ROS production during seed imbibition associated with high NADPH oxidase which occurred in the embryo and in aleurone cells promoted radicle protrusion during seed germination (Ishibashi et al., 2015).
On the other hand, excessive ROS are harmful and easy to interact with other essential molecules resulted in cellular damage. The most damaging process caused by ROS is lipid peroxidation which resulted in membrane damage. Many compounds such as malondialdehyde (MDA) increase significantly from the reaction between polyunsaturated precursors and ROS (Gill and Tuteja, 2010a). Numerous reports from many plant species presented that under salt stress plants accumulated higher H2O2, malondialdehyde (MDA; an indicator for lipid peroxidation) and electrolyte leakage (EL; an indicator of membrane damage) compared to non-stressed plants (Demidchik et al., 2014;Esfandiari and Gohari, 2017). However, beneficial role of optimum level of ROS has been pointed out. ROS are believed to play a critical role as signal transduction molecules under several stresses (Miller et al., 2010). Low level ROS such as H2O2 has been reported in controlling the specific biological/physiological processes cascades (such as cell cycle and plant responses to biotic and abiotic stresses) (Sofo et al., 2015). In addition, ROS can participate in transduction of stress signals from chloroplasts to nucleus. ROS trigger protein kinases/protein phosphatases leading to redox hemostasis (Kreslavski et al., 2012).
Seed priming is considered an alternative way for enhancing seed germination and seedling growth. This method is able to enhance stress tolerance (Chen and Arora, 2013). There are several chemical solutions used as priming agent. For example, rice seeds primed with beta-amino butyric acid produced seedlings with enhanced growth, higher chlorophyll content and low MDA level under non-stressed and stressed (NaCl and polyethylene glycol; PEG) conditions when compared to those of non-priming group (Jisha and Puthur, 2013). Priming rice seed with PEG could increase plant growth parameters and photosynthetic pigments as compared to seeds without PEG priming under nano-ZnO stress. The results showed that priming rice seeds with PEG could alleviate the toxicity of nano-ZnO stress and improved the cell structures of leaf and roots in rice (Salah et al., 2015). Spermidine (Spd), one of polyamines derivatives, is phytohormone-like aliphatic amine natural compounds with aliphatic nitrogen structure (Gill and Tuteja, 2010b). Exogenous Spd and Spd priming have been reported to improve abiotic stress tolerance in plants. Spraying Spd on rice leaves could alleviate oxidative damage by reducing O2 •production and H2O2 content under submergence stress (Liu et al., 2015). Spd priming of seeds also induced better expression of genes related to salt stress tolerance such as ion transporter and osmolyte synthesis resulting in up-regulation of several metabolic pathways to defend salt stress .
In Thailand, farmers commonly store rice seeds for use as seed source for the following growing seasons. Inappropriate storage condition in high humidity and temperature lead to seed deterioration, thus, contributing to reduced seed germination, seeding establishment and growth, and finally decrease in rice yield. Deteriorated seeds may lose their viability, vigour, and quality (Jyoti and Malik, 2013). Thus, to solve problems due to both salinity and deteriorated seeds, Spd was used as priming agent for normal and deteriorated seeds to investigate its role in promoting seed germination and mitigate salt stress effects.

Seeds preparation
Thai rice elite cultivar 'Khao Dawk Mali 105' (KDML105) was used in this study (KDML105 rice seeds was provided by Khon Kaen Rice Research Center, Department of Rice, Thailand). Seeds were harvested in November 2016 and stored for 4 months at room temperature before being used in the experiment. Seeds were divided into 2 groups; normal healthy and deteriorated seeds. Deteriorated seeds were prepared following the method modified from Chhetri (2009) by incubating normal seeds under 100% RH at 60 °C for 48 h and airdried.

Seed priming
Seeds were surface-sterilized for 10 min in 2% sodium hypochlorite and washed three times with distilled water. Seeds were soaked with H2O and in different concentrations of Spd (0.5, 1.0 and 1.5 mM) for 24 h, and then washed three times with distilled water. After that, immersed seeds were dehydrated by airdrying until seed moisture content reduced to the same value as that of seeds before priming. Seeds without soaking with H2O or Spd were used as control (non-priming). Ten primed seeds were transferred to sterile filter paper in Petri dishes (90-mm diameter) containing 5 ml of sterilized deionized water (control) or 150 mM NaCl (salt treatment). The concentration of NaCl used in this study followed that by Theerakulpisut et al. (2016). Seeds were incubated in darkness at 25 °C. Each treatment was replicated three times.

Germination test and growth measurement
Seeds were considered germinated when the radicle was at least 5 mm long. Germination of seeds was observed daily until 10 d to calculate GP and GI according to Hussain et al. (2015). GP was the ratio between the number of seeds germinated and the total number of seeds, and expressed as percentage. GI was calculated as (no. of germinated seeds/days of first count) + … + …. + …. (no. of germinated seeds/days of first count). Root and shoot lenght, and biomass of seedlings were measured at 10 d after germination. For dry weight, root and shoot were dried in a hot-air oven at 70 °C for 2 d until the dry weight was stabilized. Meanwhile, roots and shoots were collected from each group of experiment and stored at -20 °C for further analysis.
Histochemical localization of O2 •-Localization of O2 •was done following the method of Chen et al. (2016) with some modifications.

Electrolyte leakage (EL) and MDA content
To determine EL, seedling samples were cut (0.1 g) and placed in closed vials containing 10 ml of deionized distilled water. The electrical conductivity of the solution was measured using the conductivity meter (Gondo, PL-700PC, Taipei) after incubating at 25 °C for 24 h in the dark (EL1). Then, the solution with leaf pieces was boiled (100 °C) for 15 min. After cooling, electrical conductivity of the solution was measured (EL2). EL (%) was estimated as: (EL1/EL2) × 100. For MDA, seedling samples (0.1 g) were used to determine MDA content according to the modified method of Heath and Packer (1968). Briefly, 0.1 g of shoot samples was homogenized with 5 mL 0.1% trichloroacetic acid (TCA), followed by centrifugation at 10,000 g for 20 min. The supernatant (0.5 ml) was mixed with 0.5 mL 0.5% thiobarbituric acid (TBA) in 20% TCA. The reaction mixture was incubated at 95 °C for 20 min. The reaction mixture was then incubated on ice to stop the reaction then centrifuge at 10,000 g for 5 min. Absorbance was read at 532 and 600 nm using a spectrophotometer. MDA content was calculated using extinction coefficient at 155 mM cm -1 .

Statistical analysis
Statistical analysis was done using SPSS ver.19. Duncan's multiple range test (DMRT) (p ≤ 0.05) was used to determine means of significant difference among the treatments.

Effects of Spd priming on seed germination
Priming normal seeds with both H2O and Spd showed better GP and GI compared the non-priming group under NaCl stress (14% increase in GP in H2O and 0.5 mM Spd priming groups and 25% increase in 1.0 and 1.5 mM Spd priming groups). Moreover, GI under NaCl stress of Spd priming groups increased over 50%. For deteriorated seeds, only Spd priming groups enhanced GP by 22-30% under normal and 14 -25% in salt treatment conditions compared to non-priming groups. GI of deteriorated seeds treated with Spd (0.5, 1.0 and 1.5 mM) was significantly increased by 28, 33 and 50%, respectively under normal condition and 18, 32 and 52%, respectively under NaCl stress (Table 1). Effects of Spd priming on seedling growth Root length of normal seedlings produced from H2O and Spd priming were significantly longer than those from non-primed seeds under both normal and salt-stress conditions. When germinated under normal condition, root length of seedlings grown from seeds primed with H2O, and 0.5, 1.0 and 1.5 mM Spd showed 35, 38, 43 and 48% increase compared to root length of seedlings from non-primed seeds. Under NaCl, root length of all priming treatments were longer than non-priming (50, 66, 72 and 81% longer) group. In deteriorated seeds germinated under control and NaCl stress conditions, priming with H2O and Spd also significantly increased root length compared to non-priming groups. Under normal condition, H2O priming increased 31% of root length while Spd priming at 0.5, 1.0 and 1.5 mM increased 32, 44 and 50%, respectively. When deteriorated seeds were germinated under NaCl stress, Spd priming groups promoted longer root length (14, 31 and 43% increase in 0.5, 1.0 and 1.5 mM, respectively) compared to non-priming group ( Figure 1A). For shoot length, shoot length of H2O priming group was significantly higher than non-priming group under control condition (24% increase). In contrast, for seeds germinated under NaCl stress, H2O priming did not show beneficial effects to reduce NaCl-induced growth inhibition in both normal and deteriorated seeds. Normal seeds primed with Spd in all concentrations and germinated under both control and salt treatment conditions showed longer shoot length than non-priming and H2O-priming groups. Priming deteriorated seeds with Spd also promoted shoot length in both control and salt treatment conditions. Deteriorated seeds previously primed with Spd produced seedlings with markedly longer shoot length under control condition (21, 33 and 34% increase in 0.5, 1.0 and 1.5 mM, respectively) and under NaCl stress (58, 62 and 67% increase in 0.5, 1.0 and 1.5 mM, respectively) compared to non-priming groups ( Figure 1B).
Priming normal seeds with H2O and Spd caused an increase in root dry weight of seedlings under nonstressed condition (increased: 28, 49, 51 and 64% in H2O, 0.5 mM Spd, 1.0 mM Spd and 1.5 mM Spd groups, respectively). In contrast, for deteriorated seeds, only high concentration of Spd (1.0 and 1.5 mM Spd) was able to enhance root dry weight (26 and 39% increase). When stressed with NaCl, normal seeds primed with 1.0 and 1.5 mM Spd developed into seedlings having 41 and 35% higher root dry weight compared to non-priming group, while 56 and 69% increase were observed for deteriorated seeds (Figure 2A).
No significant difference in shoot dry weight was found between seedlings grown under normal condition from non-primed and H2O-primed normal seeds whereas Spd priming resulted in significantly increased shoot dry weight. When exposed to NaCl, priming normal seeds with H2O enhanced seedling shoot dry weight (27% increase) compared to those from non-primed seeds. Seeds primed with Spd also produced seedlings with higher shoot dry weight than non-primed seeds. The amounts of increase in shoot dry weight were 39, 54 and 57% for priming with 0.5, 1.0 and 1.5 mM Spd, respectively. For deteriorated seeds, priming with H2O was able to enhance shoot dry weight only under control condition (increased 14%). Priming with Spd resulted in increased shoot dry weight (seeds treated with highest Spd concentration produced seedlings with highest shoot dry weight). Under salt stress, priming with 0.5 mM Spd showed slightly increased shoot dry weight compared to non-priming and H2O priming groups. However, this change was not statistically significant. Deteriorated seeds treated with 1.0 and 1.5 mM Spd resulted in markedly increased seedling shoot dry weight (increased 38 and 64%, respectively) compared to non-priming group ( Figure 2B).

Effects of Spd priming on ROS (O2 •-) production
For histological localization of O2 •productions, normal and deteriorated dehusked seeds were stained with NBT. At 12h, it was found that all normal seeds in all priming and non-priming treatments were strongly stained with NBT under control condition. When seeds were germinated under NaCl stress, seeds primed with Spd showed stronger color of NBT staining than seed primed with H2O while no color development was observed in non-primed seeds. In deteriorated seeds, moderate intensity of NBT staining was found in seeds primed with Spd in both normal and salt stress conditions. For H2O priming, faint staining could be seen in seeds incubated under control condition, in contrast to seed germinated under salt treatment which showed no staining. For non-primed seeds, both seeds grown under normal and NaCl stress showed no NBT staining ( Figure 3A).  At 24 h, NBT staining was observed in embryos of all groups of normal seeds incubated under non-stress treatments. It was clearly seen that the strongest staining was found in Spd-primed seeds followed by H2Oprimed and non-primed seeds. In deteriorated seeds, high intensity of NBT staining was seen in seeds primed with Spd in both control and salt stress conditions. Under control condition, embryos of seedlings grown from Spd-primed seeds were more darkly stained than those from H2O priming and non-priming groups. Under NaCl condition, no NBT staining was observed in seeds primed with H2O priming and non-priming groups ( Figure 3B).
At 36 h, normal seeds primed with Spd and H2O imbibed under control condition developed coleoptile and radicle in contrast to seeds from non-priming group. Stronger color of NBT staining was observed in radicle while no staining appeared in the coleoptile. When seeds were exposed to salt stress, the highest intensity of NBT stain was found in embryos of seeds primed with Spd whereas faint stain was observed in embryos of seeds primed with H2O and non-primed seeds. For deteriorated seeds incubated under control condition, embryos were stained with NBT in all treatments with strongest color in the Spd-primed seed. Visible coleoptiles started to develop from seeds primed with Spd under both control and NaCl stress. Conversely, non-primed seeds incubated with NaCl showed no staining ( Figure 4A).
At 48 h, radicle protrusion and coleoptile emergence were found in normal seeds primed with H2O and Spd under non-stressed condition. It was clearly seen that radicles were well stained with NBT but coleoptiles were not. When seeds were incubated with NaCl, the elongation of radicle and coleoptile only occurred in the Spd-primed seed. For deteriorated seeds grown under non-stressed condition, seedlings with radicles and coleoptiles were observed in Spd and H2O priming groups whereas no coleoptile emergence was found in nonpriming group. When seeds were germinated under NaCl, radicle (stained) and coleoptile (unstained) began to emerge only from seeds primed with Spd ( Figure 4B).

Effects of Spd priming on membrane damage
At 3 d, seedlings from normal seeds primed with H2O or Spd showed significantly lower EL (decreased by 26% in both groups) compared to seedlings from non-primed seeds under control, and decreased by 21% in H2O priming and 24% in Spd priming groups under NaCl stress conditions. For deteriorated seeds in both conditions, seedlings from seeds primed with Spd showed the lowest EL followed by those primed with H2O. In comparison with the non-priming group, Spd priming resulted in 51 and 31% reduction in EL under control and stress conditions, respectively, while H2O priming showed 38 and 27% reduction ( Figure 5A). At 10 d, priming with H2O did not affect EL in seedlings from normal seeds germinated under normal and salt stress conditions. EL levels were considerably lower in seedlings produced from seeds primed with Spd (31 and 45% reduction, respectively) compared to seedlings from non-primed seeds. For deteriorated seeds, no significant differences in EL were noticed between seedlings of H2O priming and non-priming groups under non-stressed condition. However, significant differences were found under NaCl stress (EL of seedlings of H2O priming group was 9% lower). EL decreased markedly in seedlings grown from seeds primed with Spd compared to other groups in both normal and salt stress conditions. It was found that Spd priming caused 36 and 32% lower in seedling EL compared to non-priming groups under control and NaCl stress, respectively ( Figure 5B).
MDA is an indicator of lipid peroxidation caused by oxidative stress. At 3 d, normal seeds primed with both H2O and Spd produced seedlings with lower MDA content under control condition (decreased by 28 and 78%, respectively compared with seedlings from non-primed seeds). Spd priming resulted in significant decline (15%) in MDA under salt stress compared to non-priming group. In contrast, differences in seedling MDA content were not significant among non-priming, H2O priming and Spd priming treatments in deteriorated seeds under salt stress ( Figure 5C). At 10 d under non-stressed and salt stress conditions, it was noted that MDA content of seedlings from normal seeds treated with Spd was lowest (decreased by 54 and 66%, respectively) compared to non-priming groups. For deteriorated seeds, Spd priming seeds produced seedlings having 41 and 83% lower MDA than H2O priming and non-priming groups under non-stressed 8 condition. Similarly, under NaCl stress, seedlings from Spd-primed seeds had 48 and 79% lower MDA than those of H2O priming and non-priming groups ( Figure 5D). Discussion It is known that deteriorated seeds have poor germination rate and seedling establishment. Not only seed physiology factor, environmental factors such as salinity or dehydration also exacerbate seed germination and seedling growth. This research aimed to study the effects of priming normal and deteriorated KDML105 seeds with Spd on germination and physiological changes of seedlings under salt stress. Salt stress limits seed germination and seedling growth. In addition, deteriorated seeds lost their ability to germinate. Salt stress causes more negative effects on seed germination and seedling growth in seeds or seedling established from deteriorated seeds than normal seeds (Table 1). Priming normal seeds with H2O or Spd resulted in better seed germination and seedling growth when seed germinated under control condition. Similarly, priming normal seed with H2O or Spd was able to improve seed germination under salt stress (Table 1). These results indicated that seed priming (either H2O or Spd) gave beneficial effects to promote germination. Seed hydropriming was reported to improve germination velocity and uniformity of Chenopodium quinoa and Amaranthus caudatus, resulted in high GP, high GI and reduced mean germination time under salinity (Moreno et al., 2018). Seed priming also promoted seedling growth under salt stress with Spd as the more effective priming agent than water (Figure 1 and Figure 2). For deteriorated seeds, H2O priming did not promote seed germination in both non-stressed and salt-stressed treatments which are different from Spd (Table 1). H2O priming was beneficial for seedling growth only under control condition while influence of Spd priming resulted in increased seedling growth under both conditions (Figure 1 and Figure 2). It is clearly seen that Spd is better priming agent than H2O not only for reducing adverse effects of salt stress but also superiority for stimulating germination of deteriorated seed and enhancing seedling growth. Similar to the finding of Chunthaburee et al. (2014) who reported that Spd priming improved growth of rice seedlings when plants were exposed to NaCl stress. Application of Spd has been demonstrated to ameliorate salinity stress effects on many plants such as rice, Kentucky blue grass and Zoysia grass (Roychoudhury et al., 2011;Puyang et al., 2015;Li et al., 2016).
Salinity stress elevates cellular ROS production. Balance between ROS and antioxidative defence system is important for reducing cellular damage and maintaining required ROS level essential for cell signalling (Munns and Tester, 2008). Histochemical localization of ROS by staining with NBT to detect O2 •-(for localization of H2O2 see Supplementary File) found that H2O2 and O2 •were generated faster in normal seeds primed with Spd than other groups during 12-24 h after hydration under both control and NaCl stress ( Figure  3). This ROS detection could be associated with faster germination of Spd-primed seeds as suggested by several reports. Huang et al. (2017) presented that the mechanism underlying the effects of Spd priming on acceleration of seed germination was probably related to Spd induction of high rate of H2O2 generation in the radicle via Spd oxidation. Moreover, Chen et al. (2016) proposed that polyamine oxidase-induced H2O2 production during seed imbibition was involved in promoting seed germination. Ishibashi et al. (2015) suggested that H2O2 (or other ROS molecules) could play a role as oxidative signalling and stimulate radicle protrusion process. Similarly, early generation of ROS of Spd-primed deteriorated seeds was coincided with earlier germination (Figure 3 and Figure 4; Supplementary File). It is believed that ROS was involved in endosperm weakening leading to emergence of coleoptile and radicle (Gomes and Garcia, 2013). Some seeds in H2O priming and non-priming also produced ROS in the early stage of hydration under salt stress (Figure 3 and Figure 4; Supplementary File). However, those seeds were unable to germinate or germinate slower. These may be because excessive ROS level was accumulated leading to inhibition of seed germination process. Although ROS are key factor stimulating seed germination, when seeds are germinated under abiotic stress excessive ROS can become harmful molecules (Kumar et al., 2015).
Under several types of abiotic stresses including salt, EL and MDA occur inevitably by oxidative stress.
Less membrane damage contributes to salt tolerance ability (Wu et al., 2017). Seedlings of normal and deteriorated seeds produced via Spd priming showed less effects of oxidative damage while H2O priming was able to mitigate membrane damage at only the beginning period of seedling establishment (3-d-old seedlings). However, priming with Spd or H2O effectively reduced salt stress effects in young seedlings as compared with non-priming suggesting that priming seeds could induce stress tolerance ( Figure 5). Parvin et al. (2014) reported that during salinity stress, application of Spd showed protective roles in reducing the intensity of oxidative stress. Many studies have demonstrated that priming seeds with Spd resulted in salt stress tolerance by alleviating stress injury caused by oxidative damages to near-normal condition (Puyang et al., 2015;Zhang et al., 2016;. Overall, decreasing salt stress injury in seedlings was more pronounced when seeds were primed with Spd than H2O.

Conclusions
Priming seeds with H2O or Spd resulted in better seed germination compared to non-priming. The result showed that 1.5 mM Spd was the best concentration for priming to promote seed germination and seedling growth. During salt treatment, Spd priming resulted in increasing seed germination, improving seedling growth and alleviating oxidative stress in seedlings. In addition, Spd promoted germination of deteriorated seeds, increased seedling growth and decreased membrane damage in both non-stressed and saltstressed conditions. Thus, priming seeds with Spd stimulated seed acclimation leading to promote seed germination and reduced salt stressed damages in rice seedlings under salt stress.