Seed priming is a pre-sowing seed treatment that has been widely used by farmers and seed companies to improve seed germination performance (Paparella et al., 2015). The basic procedures of seed priming include the controlled seed hydration to induce various metabolic processes during the early phase of germination, and the drying of primed seeds to an initial moisture content before radicle emergence (Heydecker and Gibbins, 1978). In recent years, the effects of seed priming have been intensively studied in rice, maize, lettuce, tomato and other crops and the benefits of seed priming to increase seed germination, uniformity of seedling emergence, seedling vigor, and to enhance stress tolerance have been widely reported (Rehman et al., 2015; Nawaz et al., 2016; Imran et al., 2017; Petronilio et al., 2021; Lyalina et al., 2023). Meanwhile, rice (Oryza sativa L.) is the world’s second largest source of food and is grown on 163.2 million ha, with an average annual production of 740.9 million tons worldwide (FAOSTAT, 2014). The use of seed priming technique in modern rice production is critical to maintain or increase crop yield because the transition from the traditional manual rice establishment to simplified and mechanized methods require lower seed sowing rate and thus need higher seed vigor (Farooq et al., 2019; Li et al., 2022). The previous studies proved that seed priming was effective in promoting rice seed germination and seedling growth under chilling (Nie et al., 2020; Nie et al., 2022) drought (Zheng et al., 2016), and waterlogging stresses (Hussain et al., 2016). However, the rapid loss of seed viability during storage is a major limitation to the adoption of seed priming in rice production.

Previously, the fast deteriorations of primed-seeds during storage have been observed in rice and other plant species (Sano et al., 2017; Sano and Seo, 2019). Hussain et al. (2015) reported that the germination of primed seeds was significantly reduced after 15 days storage at room temperature. Similar results were also observed in many field crops such as oat, maize and rapeseeds (Wattanakulpakin et al., 2012; Xia et al., 2020; Malek et al., 2022). Meanwhile, the deterioration speed of primed seeds was varied under different storage condition. Wang et al. (2018) reported that increasing the storage temperature did not reduce the viability of primed rice seeds stored under vacuum conditions, whereas seeds stored in the absence of oxygen but with high RH showed reduced longevity. Moreover, the deterioration of primed seeds was suggested to affected by initial seed vigor, the priming-induced seed deterioration was more significant in high vigor seeds, but was much lower in low vigor seeds (Powell et al., 2000; Butler et al., 2009). In addition, the priming methods such as hydropriming, osmoriming and chemical priming were reported to greatly affected the deterioration of primed seeds during storage (Fabrissin et al., 2021). In rice seeds, however, few study has evaluated the differences in deterioration speed of primed rice seeds in response to different priming methods.

The physiological and molecular mechanisms underlying the deterioration of primed seeds have been studied in recent years. A study by Sano et al. (2017) has detected several quantitative trait loci (QTL) that were uniquely associated with primed-seed longevity, which indicated that there might independent mechanisms that controls primed seed deterioration. Several studies suggested the plant hormone signaling may regulate the seed coat permeability and then affected the longevity of primed seeds during storage (Zhou et al., 2020). In addition, the changes of several physiological events including starch metabolism, seed respiration and reactive oxygen species accumulation were suggested to be strongly associated with seed deterioration during storage (Bailly and Kranner, 2011; Jovanović et al., 2014; Wang et al., 2022), and the regulating effects of those metabolic events on seed deterioration and longevity have been well elucidated. Nevertheless, the physiological mechanism underlying the fast deterioration of primed rice seeds need to be further unrevealed.

The present study was conducted to evaluate the differences between two typical priming methods, viz., hydropriming and osmopriming with regards to seed deterioration speed, starch degradation, accumulation and scavenging of reactive oxygen species, and respiratory metabolism by using artificially accelerated aging methods (controlled deterioration test — CDT). The CDT is a classical seed aging method that has widely been used by researchers to study seed longevity within relative short duration by incubating seeds at high temperature and high relative humidity condition (Schwember and Bradford, 2010). The objectives of present study were to evaluate the effects of priming methods on the deterioration speed of rice seeds, and to unravel the physiological mechanisms underlying the differences in seed deterioration between priming methods.

The effects of seed priming on seed storability have been assessed in multiple crop species and the negative effects have been observed in most cases. However, little attention has been given to the difference in deterioration speed of primed seeds in a controlled and constant environment, which might be affected by cultivars and priming methods. In the present study, it was observed that PEG-primed rice seeds exhibited lower deterioration speed than hydro-primed rice seeds under controlled accelerated aging condition in terms of germination percentage and germination speed, These results suggest that manipulating the priming methods can potentially extend the storability of primed rice seeds. Previously, several studies have observed the regulatory effects of priming type, imbibition time, temperature and drying speed on seed longevity, which have been summarized and reviewed by Fabrissin et al. (2021). However, conflicting results exist between different studies. For instance, PEG-priming reduced the longevity of leek seeds (Dearman et al., 1987), but extended the storability of onion seeds (Dearman et al., 1986). The inconsistent findings may be attributed to variations in aging methods, priming protocols, and cultivars selections employed across various investigations. In rice seeds, Wang et al. (2018) found that the deterioration speed of hydro-primed seeds showed no difference with that of PEG-primed seeds under vacuum or low-temperature condition, but deteriorated faster under room temperature and 60%RH (Wang et al., 2018). This finding is consistent with that of Moradi and Younesi (2009) and with the present work that conducted under accelerated aging conditions.

Seed hydration is a crucial step in seed priming that affects the germination performance and storage of seeds after priming. It has been reported that prolonged hydration during priming had a deleterious effect on the subsequent germination of seeds after storage (Fabrissin et al., 2021). This is likely because prolonged hydration activated various metabolic events that are essential for seed germination (Bewley, 1997; Lemmens et al., 2019), but can be harmful for seed storage. In the present study, the seed hydration duration was consistent between the two priming methods. However, the water absorption rate was lower in PEG-priming compared to hydro-priming (unpublished data; Al-Karaki, 1998), which could have led to varied seed hydration levels and consequently different deterioration rate between the two priming methods. To further explore why hydro-primed seeds deteriorated faster than PEG-primed seeds, several indicators that related to the key metabolic events during seed deterioration or germination including starch degradation, respiration and ROS scavenging were determined. The results suggested that the starch degradation ability was highly correlated with the deterioration of primed rice seeds. Specifically, the α-amylase activity and total soluble sugar content in hydro-primed seeds decreased faster than those in PEG-primed seeds. The reduced α-amylase activity and total soluble sugar content in primed rice seeds during storage was also observed by Hussain et al. (2015). It was suggested that starch metabolism is the major driving force for rice seed germination and could support plants to grow faster under various environment (Murata et al., 1968). The starch degradation ability during rice seed germination is strongly associated with respiratory metabolism (Wang et al., 2016). The activation of α-amylase enzymes triggered an increase in respiration rate (Paul and Mukherji, 1972), while seed respiration provides essential energy for starch degradation and subsequent seed germination (Nie et al., 2020). These findings indicated that the decreased seed respiration observed in the present study may contribute to the reduced starch degradation ability in aged primed seeds. Moreover, the decline in starch degradation ability in deteriorated seeds may be related to the oxidative stress of seeds during storage (Wang et al., 2018). Membrane damage and solute leakage triggered by lipid peroxidation could inevitably impair starch metabolism that occurred in seeds embryo and starchy endosperms (Riewe et al., 2017; Wiebach et al., 2020), which led to a decrease in seed viability. The present study also found that the ROS accumulation and lipid peroxidation may be the key events that contribute to different deterioration speed of primed rice seeds. Although the H₂O₂ and O₂ ^- levels did not differ between hydro-primed seeds and PEG-primed seeds before accelerated aging, the improved antioxidant enzyme activity may indicate higher ROS generation level in hydro-primed rice seeds than in PEG-primed and non-primed ones. During storage, the antioxidant enzymes generally losses their function because the mobilization and diffusion of the reactive substrate are strictly limited in the glassy cytoplasm of dry seeds (Kranner et al., 2010; Kumar et al., 2015). However, the seed respiration accompanied with ROS production will not stop during storage, which ultimately lead to lipid peroxidation and seed death during storage (Zhang et al., 2021). In present study, we hypothesize that the faster deterioration of hydro-primed rice seeds might be attributed to faster ROS production and accumulation during accelerated aging, because the hydro-primed seeds exhibited significantly higher H₂O₂ and MDA content than PEG-primed seeds, and the antioxidant enzyme activity were greatly inhibited. This hypothesis might be partially proved by the NAC addition experiment, in which the seed deterioration, as well as the ROS accumulation and lipid peroxidation of hydro-primed seeds after aging was greatly inhibited by NAC. NAC is an important factor, as well as other nonenzymatic antioxidants, due to the fact that they have lower molecular masses, having greater mobility and quenching ability in regard to enzymatic antioxidants (Gerna et al., 2022). Nevertheless, the NAC content in primed rice seeds was not determined in present study, and the possible function of NAC that regulate the deterioration speed of primed rice seeds need to be further evaluated.

In the present study, elevated ROS levels in hydro-primed rice seeds might be attributed to differences in seed physiological status influenced by seed hydration. In the priming process, seeds are allowed to absorb water up to phase II of the three phases water absorption model (Bradford, 2017), during which several metabolic events that favors seed germination would be activated (Bradford, 2017). The research of Nie et al. (2020) observed the repair and biogenesis of mitochondria during priming. This restoration of mitochondrial function is accompanied by a rapid increase in ROS production (Farooq et al., 2021). Previous research has also shown a negative correlation between seed respiration and seed storability (Ferguson et al., 1990; Vertucci and Roos, 1990). In the present study, hydropriming resulted in increased seed oxygen consumption and ATP levels prior to aging. This effect might be attributed to the faster water absorption rate of hydro-priming compared to PEG-priming (Al-Karaki, 1998), which may trigger higher respiratory activity during a specific priming duration (24h in present study), and subsequently lead to accelerated ROS accumulation and lipid peroxidation. However, the present study only determined the respiration rate, ATP content and MDH activity, the mechanisms underlying why hydro-primed seeds exhibit higher respiration rate after priming remained to be unknown, the changes in respiratory metabolism, and their possible relationship with the deterioration of primed rice seeds needs to be explored in the future.

The hydroprimed rice seeds exhibited significantly faster deterioration speed than that of PEG-primed seeds in terms of germination speed and percentage. Additionally, α-amylase activity and total soluble sugar content in hydroprimed seeds reduced by 19.3% and 10.0% respectively after aging compared to PEG-primed seeds. Such effects were observed to be significantly associated with the elevated ROS accumulation and lipids peroxidation. The levels of superoxide anion, hydrogen peroxide, and malondialdehyde in hydroprimed seeds were 4.4%, 12.3%, and 13.7% higher than those in PEG-primed seeds after aging. This effect could be attributed to the augmented respiratory metabolism in hydroprimed seeds. Additionally, the simultaneous use of N-acetylcysteine with HP and PEG priming significantly inhibited the deterioration of primed rice seeds. These findings imply that the ability to eliminate reactive oxygen species may be the critical factor affecting the deterioration speed of primed rice seeds during storage. Furthermore, this study demonstrates the potential to lengthen the storage duration of primed rice seeds by adjusting the priming methods. However, the physiological and molecular mechanisms underlying why hydro-primed seeds exhibit a higher respiration rate and faster ROS accumulation than PEG-primed seeds remain unknown and require future exploration.

The original contributions presented in the study are included in the article/supplementary files, further inquiries can be directed to the corresponding author.

MR: Data curation, Formal Analysis, Investigation, Methodology, Writing – original draft. BT: Investigation, Writing – original draft. JX: Investigation, Writing – original draft. ZY: Investigation, Writing – original draft. HZ: Writing – review & editing. QT: Funding acquisition, Writing – review & editing. XZ: Funding acquisition, Writing – review & editing. WW: Conceptualization, Funding acquisition, Writing – original draft.