Seed dormancy and germination are crucial stages in a plant’s life. In wild plant species, seed dormancy plays a key role in ensuring survival by blocking germination until conditions become favorable for the later stages of germination and growth of that species (Bewley, 1997). Unlike many wild plant species, cultivated crops such as barley (Hordeum vulgare L.) and wheat (Triticum aestivum L.) display weak grain dormancy at maturity due to selective breeding against dormancy for uniform and vigorous germination on industrial and agricultural fields. For malting barley, long dormancy increases costs and potential damage resulting from grain storage (Carn, 1980). In addition, cool, moist conditions in the field can disrupt seed dormancy and cause PHS, resulting in serious losses of grain yield and quality (Gubler et al., 2005; Rodríguez et al., 2015). Therefore, research aimed at understanding environmental and genetic controls of dormancy will assist in the development of new strategies for industrial utilization and the elimination of PHS worldwide in domesticated crops.

Plant hormones such as GAs and ABA play key roles in seed dormancy and germination. In particular, ABA is considered to be the key molecule in dormancy induction and maintenance (Finkelstein et al., 2008; Rodríguez et al., 2015; Shu et al., 2016). During seed development, embryonic ABA is required to impose lasting dormancy (Nambara and Marion-Poll, 2005), and de novo ABA synthesis in the embryo during imbibition ensures maintenance of dormancy (Kucera et al., 2005). Dormancy release by after-ripening is mediated by a decrease in ABA content in imbibed wheat and barley grains as a result of the coordinated promotion of ABA catabolism and repression of ABA biosynthesis genes (Millar et al., 2006; Jacobsen et al., 2013). In several species, the transcriptional regulation of nine-cis-epoxycarotenoid dioxygenase (NCED) and ABA-8′-hydroxylase (ABA8′OH) genes are considered key steps in this control mechanism (Nambara et al., 2010). In barley, HvNCED1 is particularly important in the regulation of primary dormancy in blue light (Gubler et al., 2008) or at high temperatures (Leymarie et al., 2008), whereas HvNCED2 expression has been implicated in the induction of primary dormancy (Chono et al., 2006) and the maintenance of secondary dormancy (Leymarie et al., 2008). The HvABA8′OH1 gene plays a major role in that ABA catabolism that is required to alleviate barley grain dormancy (Chono et al., 2006; Millar et al., 2006; Gubler et al., 2008). ABA functions are regulated, in part, by crosstalk with other hormones such as GA and their associated signaling networks. Application of GA, an antagonist of ABA, can break dormancy in cereals (Jacobsen et al., 2002; Tuttle et al., 2015).

Recently, it was reported that several signal molecules such as nitric oxide (NO) and ROS also regulate seed dormancy and germination (Ma et al., 2016). In barley, ROS break seed dormancy and promote germination (Fontaine et al., 1994; Ishibashi et al., 2010; Bahin et al., 2011). The relationships between ROS, seed dormancy, and germination have been described for many plant species, including Zinnia elegans, and sunflower (Ogawa and Iwabuchi, 2001; Oracz et al., 2007). Hydrogen peroxide (H₂O₂), which is a ROS, is regarded as a signaling hub for the regulation of seed dormancy and germination; the precise regulation of H₂O₂ accumulation by the cell antioxidant machinery is essential to achieve a balance between oxidative signaling that promotes germination and oxidative damage that prevents or delays germination (Wojtyla et al., 2016). These findings were clearly summarized and presented by Bailly et al. (2008) as the principle of the “oxidative window” for germination. According to this hypothesis, both lower and higher levels of ROS impair seed germination, which is only possible within a defined range of concentrations.

Recent evidence shows that the selective oxidation of proteins and mRNAs can act as a positive regulator of seed germination (Job et al., 2005; Oracz et al., 2007; Barba-Espín et al., 2010; Bazin et al., 2011). Bazin et al. (2011) showed that approximately 24 stored mRNAs undergo oxidation during sunflower (Helianthus annuus) after-ripening. ROS production during germination contributes to reserve mobilization through oxidative modifications of stored proteins; storage organs may then recognize these modifications as signals to mobilize reserves to the rapidly growing axis. Due to the abundance of available seed storage proteins, the oxidized forms of these proteins such as heat shock proteins and elongation factors can also be considered as actor of ROS signaling in seed germination (Job et al., 2005; Barba-Espín et al., 2010). Oracz et al. (2007) proposed a mechanism for seed dormancy release that involves a change in proteome oxidation resulting from the accumulation of ROS during the after-ripening phase.

The breaking of dormancy by ROS has also been reported in relation to plant hormone signaling in several seeds (El-Maarouf-Bouteau et al., 2013). Studies on phytohormone interactions in germinated seeds have shown that exogenously applied ABA inhibits ROS accumulation in barley (Ishibashi et al., 2012), rice (Oryza sativa; Ye et al., 2012), lettuce (Zhang et al., 2014), and sunflower (El-Maarouf-Bouteau et al., 2015). By contrast, the addition of GA enhances the production of ROS, mainly superoxide and H₂O₂, found in radish plants (Schopfer et al., 2001) and Arabidopsis (Liu et al., 2010; Lariguet et al., 2013). Bahin et al. (2011) suggested that exogenously applied H₂O₂ does not influence ABA biosynthesis and signaling but has a pronounced effect on GA signaling, resulting in the modulation of hormonal balance and in subsequent germination initiation. The modulation of phytohormone balance during germination by exogenously applied H₂O₂ is also a product of changes in H₂O₂ levels in seeds treated with GA and ABA. Exogenous H₂O₂ and NADPH oxidase inhibitor increased ABA catabolism by enhancing the expression of CYP707A genes, which encode ABA 8′-hydroxylases, and enhanced the expression of genes for GA synthesis in dormant Arabidopsis seeds (Liu et al., 2010) and non-dormant barley seeds (Ishibashi et al., 2015). In non-dormant barley seeds, H₂O₂ accumulation via superoxide produced by NADPH oxidases promote GA biosynthesis in embryos; the resulting GA induces and activates NADPH oxidases in aleurone cells, and the H₂O₂ accumulated by the NADPH oxidases induce α-amylase in these cells (Ishibashi et al., 2015). Therefore, it is likely that ROS is central molecule in the regulation of barley seed dormancy and germination through GA and ABA. However, the role of H₂O₂ interactions with phytohormones in the regulation of barley seed dormancy and germination is still open to debate. The mechanism of dormancy breaking by ROS needs to be examined in detail. In this study, we, therefore, focused on the balance between plant hormones and ROS in dormant and non-dormant seeds.

The present study demonstrates that the balance between ABA and ROS in the barley seed embryo is involved in the regulation of seed dormancy and germination. The first line of evidence supporting this conclusion is the relationship between seed dormancy and H₂O₂ level, because the degree of seed dormancy was dependent on the H₂O₂ levels in the embryo after imbibition (Figure 1 and Supplementary Figure S2). Additionally, germination rate of dormant seeds were promoted by H₂O₂ and suppressed by sodium ascorbate, while that of non-dormant seeds were suppressed by sodium ascorbate, but not promoted by H₂O₂ (Figure 2A and Supplementary Figure S3). There have been several reports about the relationship between ROS and seed dormancy and germination, many of which are focused on phytohormone signaling and ROS involvement (Oracz and Karpiński, 2016). H₂O₂ has been implicated in dormancy alleviation via its activation of GA signaling and synthesis rather than repression of ABA signaling (Bahin et al., 2011). In sunflower seed, however, ROS interact at the transcriptional level with the ABA signaling pathway (El-Maarouf-Bouteau et al., 2015). We previously reported that ROS produced by NADPH oxidase in the embryos of barley seeds promoted the catabolism of ABA (Ishibashi et al., 2015). In the present study, NADPH oxidase activity in the embryos of non-dormant seeds was higher than that in the embryos of dormant seeds (Figure 3E). In non-dormant seeds, ABA content in the embryos treated with ascorbate was higher than that in treated with distilled water, whereas, in dormant seeds, the ABA content in the embryos treated with H₂O₂ was lower than in treated with distilled water (Figures 2C,D). In addition, the regulation of ABA in the embryos of both dormant and non-dormant seeds could be attributed to the induction of HvABA8′-OH1 by ROS (Figures 2G,H). Two genes encoding ABA8′-OH have been identified in barley, and HvABA8′OH1 is predominantly expressed in the embryos of imbibing seeds (Millar et al., 2006). Of note, HvABA8′OH1 expression is closely associated with ABA level and germination capacity (Chono et al., 2006). Consistent with these facts, the knocking down of ABA8′OH1 leads to increased seed ABA levels and to enhanced dormancy (Gubler et al., 2008). ROS produced after imbibition is involved in the regulation of barley seed dormancy and germination through the catabolism of ABA; therefore, we focused on the mechanism of ROS production. It had been reported that ROS generated by NADPH oxidases regulate barley seed germination through GA/ABA metabolism and signaling in the embryo and aleurone cells (Ishibashi et al., 2015). In the present study, H₂O₂ content and NADPH oxidase activity in the embryos of dormant seeds were lower than those in the embryos of non-dormant seeds (Figures 1B,C, 3E). In the embryos of barley seeds, ABA suppressed the activity of NADPH oxidases (Ishibashi et al., 2015). The decrease in ABA content by catabolism in the embryo after imbibition is promoted by ROS production through NADPH oxidase. In Arabidopsis, ABA treatment induces the expression of NADPH oxidases (AtrbohD and AtrbohF) in guard cells (Kwak et al., 2003). The ROS enhanced by NADPH oxidase could result in enhanced ABA accumulation, whereas enhanced ABA could result in enhanced ROS production in guard cells, creating a positive feedback loop to mediate stomatal closure (Mittler and Blumwald, 2015). Our results indicate that in the embryo after imbibition, ROS and ABA act antagonistically to regulate seed dormancy and germination, although this suggestion is inconsistent with other reports.

In this study, we also examined the activity of the antioxidant enzymes SOD, CAT, and peroxidases (APX, GPX) that can scavenge ROS. The CAT activities in the embryos of the dormant seeds were significantly higher than those in the embryos of the non-dormant seeds (Figure 3D). These results suggest that the H₂O₂ contents in the embryos of dormant and non-dormant seeds depend on CAT activity. CAT is reported to play an important role in the seed germination process. CAT expression levels increase in germinating sunflower seeds prior to radicle protrusion, with a concomitant decrease in H₂O₂ content (Bailly, 2004). A similar increase in CAT activity has been described during the germination of maize, soybean, Arabidopsis, and sweet corn seeds (Puntarulo et al., 1991; Gallardo et al., 2001; Posmyk et al., 2001; Chiu et al., 2002). In dormant barley seeds, H₂O₂ treatment induced an increase in CAT activity that was associated with accumulation of the HvCAT2 transcript in the embryos (Bahin et al., 2011). Barley has two CAT isozymes, HvCAT1 and HvCAT2; however, HvCAT1 was not expressed in our embryos (data not shown). In the present study, the expression of HvCAT2 in the embryos of dormant seeds was significantly higher than in those of non-dormant seeds (Figure 4A). In nine cultivars that were at different stages of dormancy, HvCAT2 expression correlated with the germination index except for one cultivar (Supplementary Figure S6). In addition, precocious germination of isolated immature embryos was suppressed by the transient introduction of HvCAT2 driven by the maize (Zea mays) ubiquitin promoter (Figure 4E). These results indicated that CAT is involved in germination of barley seed after imbibition through the regulation of ROS.

The expression of HvCAT2 was suppressed by ABA (Figures 5A,B, also Bahin et al., 2011); therefore, we investigated the promoter sequence of HvCAT2. The promoter sequence included an ABA-responsive element (ABRE), an ABRE-related coupling element (CE), and RY repeat motifs (Supplementary Figure S5). Interestingly, the promoter of Sdr4, which regulates rice seed dormancy, has seven RY repeats, which are important for seed-specific gene expression and are the target of the VP1/ABI3 subfamily of B3 domain transcription factors (Baumlein et al., 1992), along with the ABRE and CE (Sugimoto et al., 2010). The dormancy state is characterized by the transcription of genes with large numbers of ABRE sequences to which transcription factors like ABI5 bind to regulate seed dormancy (Finch-Savage and Leubner-Metzger, 2006). In the present study, the expression of HvABI5 in the embryos was increased by ABA (Figure 5C), but not HvVP1 (data not shown). Moreover, we determined that recombinant HvABI5 binds to the HvCAT2 promoter including the ABRE (Figure 5E). These results suggest that the expression of HvCAT2 is regulated through HvABI5 induced by ABA.

Bahin et al. (2011) suggested that H₂O₂ plays a role in the alleviation of barley seed dormancy through the activation of GA signaling and/or biosynthesis rather than through the inhibition of ABA signaling. They found that exogenously applied H₂O₂ does not influence ABA biosynthesis and signaling but does have a pronounced effect on GA signaling, resulting in the modulation of hormonal balance and in subsequent germination initiation. However, by using dormant and non-dormant seeds in this study, we found that H₂O₂ regulated ABA content through HvABA8′-OH1-mediated catabolism of ABA (Figures 2C,D,G,H). Liu et al. (2010) proposed a hypothetical model that explains the interrelationships between H₂O₂ and NO in the regulation of Arabidopsis seed germination in terms of the joint actions of ABA and GA. According to this model, H₂O₂ can interrupt the dormancy of Arabidopsis seeds through two pathways. The first pathway relies on the enhancement of ABA catabolism and GA biosynthesis. The signaling molecule (NO) does not regulate GA biosynthesis directly but instead acts as a temporary signaling molecule involved in the H₂O₂ regulation of ABA catabolism. The second pathway assumes negative regulation of GA biosynthesis by ABA. We previously reported that ROS induced by NADPH oxidase regulate barley seed germination by promoting ABA catabolism and GA biosynthesis (Ishibashi et al., 2015). Our results in this study suggest that dormant seeds maintain high ABA contents, promoting HvCAT2 expression through ABI5 for H₂O₂ catabolism. In addition, ABA catabolism through HvABA8′OH1 was promoted by H₂O₂. Therefore, dormant seeds had low ROS contents with decreased HvABA8′OH1 expression, which maintained high ABA contents. Taken together, our findings suggest that the interrelationship between ABA and ROS may play an important role in seed dormancy and germination, as shown in Figure 6, although it should also be added that the mechanism might be included in seedling stage.

Conceived and designed the experiments: YI, TY, and MI-I. Performed the experiments: YI, SK, MS, NA, KK, RT, and GW. Analyzed the data: YI, NA, TY, and MI-I. Contributed reagents/materials/analysis tools: YI, SK, MS, NA, KK, RT, and GW. Wrote the paper: YI and MI-I.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.