Interaction between reactive oxygen species and hormones during the breaking of embryo dormancy in Sorbus pohuashanensis by exogenous nitric oxide

The breaking of dormancy mediated by reactive nitrogen species (RNS) is related to the accumulation of reactive oxygen species (ROS) in germinating embryos but the underlying mechanism is unclear. The objectives of this study were: (1) to explore the relationship between RNS-mediated dormancy release and ROS accumulation in germinating embryos of Sorbus pohuashanensis; and, (2) to investigate the relationships among germination time, ROS metabolism, and endogenous hormone synthesis. We studied the effects of exogenous nitric oxide (NO) donor sodium nitroprusside (SNP), the NO scavenger (4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (PTIO), abscisic acid (ABA), the exogenous ethylene donor ethrel, and the ethylene receptor inhibitor 2,5-norbornadien (NBD) on embryo germination and seedling growth. Embryos were released from dormancy by pretreatment with NO or ethylene, which was related to increased ethylene biosynthesis and decreased ABA levels. Breaking of dormancy by SNP was related to increased levels of ethylene, hydrogen peroxide, and glutathione, increased activities of superoxide dismutase and glutathione peroxidase, and decreased levels of ABA, superoxide anions, and malondialdehyde. These effects of nitric oxide were especially significant in seedling hypocotyls and radicles. These results demonstrate that NO can break S. pohuashanensis embryo dormancy by inducing ethylene biosynthesis, and that this signalling pathway is closely related to ROS accumulation and the antioxidant defence response.


Introduction
Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are emerging as important regulators of plant development (germination, flowering, senescence) that function as secondary messenger in cooperation with classical phytohormones (Gniazdowska et al. 2010). Both ROS and RNS play signalling roles in alleviating seed dormancy, thereby promoting germination (Ishibashi et al. 2017). Hydrogen peroxide (H 2 O 2 ) also plays a positive role as a signalling molecule in the seed germination of several plant species such as Hordeum vulgare L. (Ishibashi et al. 2017), Vigna radiata (L.) R. Wilczek (Chaudhuri et al. 2013), and Oryza sativa L. cv. Hitomebore (Sasaki et al. 2015). The promoting effects of inorganic nitrates and other nitrogencontaining compounds on germination are thought to represent the release of seed dormancy via a process involving Abstract The breaking of dormancy mediated by reactive nitrogen species (RNS) is related to the accumulation of reactive oxygen species (ROS) in germinating embryos but the underlying mechanism is unclear. The objectives of this study were: (1) to explore the relationship between RNS-mediated dormancy release and ROS accumulation in germinating embryos of Sorbus pohuashanensis; and, (2) to investigate the relationships among germination time, ROS metabolism, and endogenous hormone synthesis. We studied the effects of exogenous nitric oxide (NO) donor sodium nitroprusside (SNP), the NO scavenger (4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (PTIO), abscisic acid (ABA), the exogenous ethylene donor ethrel, and the ethylene receptor inhibitor 2,5-norbornadien (NBD) on embryo germination and seedling growth. Embryos were released from dormancy by pretreatment with NO or nitric oxide (NO) (Beligni and Lamattina 2000;Bethke et al. 2004). Sodium nitroprusside (SNP) is an exogenous NO donor that promotes seed germination in many plant species (Beligni and Lamattina 2000;Bethke et al. 2004;Gniazdowska et al. 2010;Yang et al. 2013). Nitric oxide has a concentration-dependent effect on seed germination, i.e., a low concentration promotes germination whereas a high concentration inhibits it (Giba et al. 2007). Previous studies have focused on the mechanism by which exogenous H 2 O 2 stimulates germination in O. sativa (Sasaki et al. 2015) and H. vulgare (Ishibashi et al. 2017), and early seedling growth in V. radiata (Chaudhuri et al. 2013). However, few studies have focused on the role of NO and associated factors in seed dormancy and germination.
Mountain ash (Sorbus pohuashanensis (Hance) Hedl.), a deciduous species in the genus Sorbus (Maloideae, Rosaceae), is an economically valuable species for ornamental and medicinal purposes, and parts of the tree are edible (Yang and Shen 2011). Naturally growing S. pohuashanensis is mainly distributed in the north to northeast of China and also sporadically in northern Korea and eastern Russia (Yang et al. 2012a, b). It is a companion species that grows in small sporadic populations with a small population density. S. pohuashanensis is sensitive to high-intensity interspecific competition and has a poor update capability, for example, the number of seedlings is rare under natural conditions (Mala et al. 2009;Xu et al. 2010;Yang et al. 2012a, b). Because of its ornamental value, S. pohuashanensis has gradually gained more attention. Exploitation of the wild resource in the eastern part of north-eastern China has caused great damage, and a portion of the population is on the verge of disappearing.
In previous studies, we have analysed the physiological dormancy characteristics of S. pohuashanensis embryos (Yang and Shen 2011). It takes 105 days (seeds sealed and stored at low temperature) to 120 days (newly harvested mature seeds) to germinate after cold stratification at 2-5 °C (Yang et al. 2008a, b). During the process of cold stratification, the ABA level in the seed coat decreases significantly, and the relative levels of auxin, cytokinin, and gibberellin increase significantly in both seed coat and embryo (Yang et al. 2008a, b). In studies on the effects of various pre-treatments on germination, two-thirds (67%) of S. pohuashanensis seeds soaked in 4% KNO 3 for 48 h germinated under variable temperatures from 25 to 5 °C . Pre-treatment of S. pohuashanensis embryos with 5 mmol L -1 SNP for 3 h inhibited germination, but treatment with the NO scavenger (4-carboxyphenyl)-4,4,5,5tetramethylimidazoline-1-oxyl-3-oxide (PTIO) to remove excess cellular NO promoted embryo germination, and facilitated chlorophyll synthesis and the accumulation of reactive oxygen species (ROS) . A low concentration of exogenous NO (2 mmol L -1 ) was found to increase the proportion and growth rate of germinating S. pohuashanensis embryos (Yang et al. 2018). However, the mechanism by which exogenous NO releases S. pohuashanensis seeds from dormancy is unclear.
On the basis of the above research, we studied the effects of the exogenous NO donor SNP, the NO scavenger PTIO, abscisic acid (ABA), the exogenous ethylene donor ethrel, and the ethylene receptor inhibitor 2,5-norbornadiene (NBD) on the germination of S. pohuashanensis embryos, the level of endogenous hormones, and ROS metabolism using a laboratory-based Petri dish germination method. The findings could be used to promote germination of S. pohuashanensis seeds, which could foster the protection and expansion of S. pohuashanensis natural resources.

Experimental materials
S. pohuashanensis fruit were collected in late September at the Fenghuang mountain forest farm, Shanhetun Forestry Bureau in Wuchang City, Heilongjiang Province, China (east longitude 127° 28′ 18″-128° 14′ 36″, north latitude 44° 3′ 34″-44° 38′ 34″). Mature fruit harvested from a full-grown, healthy wild mature tree were kneaded by hand in water to remove the pericarp and pulp, seeds washed with water to remove any debris and air-dried at room temperature. Full dry seeds were stored in sealed plastic bags at 5 °C.

Seed pretreatment
Dormant seeds with full grain and uniform texture were selected (average weight per 1000 seeds, 1.95 g; average moisture content, 8.4%; average seed viability, 83.3%). Seeds were soaked in distilled water at 20 °C ± 5 °C for 48 h, further soaked in 0.2% (v/v) NaClO solution for 10-15 min, and rinsed with water. Seed coats were stripped on the ice (low temperature environment to avoid enzyme inactivation) and subsequent experiments were carried out with peeled embryos.

Embryo germination
The chemical reagents used for embryo treatment are shown in Table S1.

Pretreatment of embryos with SNP
Embryos were treated with SNP according to Bethke et al. (2006). A piece of filter paper was placed in a 500-mL beaker and dampened with 5 mL of 0.05 mol L -1 Hepes-KOH (pH 7.0). Ninety embryos were placed on the filter paper. A small beaker containing 5 mL freshly prepared SNP solution was placed in the 500-mL beaker, and the mouth sealed with plastic wrap (Fig. S1). Gaseous NO was released directly from the SNP solution and embryos exposed to the SNP vapour at 25 °C under 60 μmol m -2 s -1 light intensity for 3 h. After treatment, the embryos were washed with distilled water and placed on new filter paper wetted with distilled water. Controls were peeled embryos without SNP pretreatment. Control and pretreated embryos were germinated in 9-cm-diameter culture dishes at 25 °C under 60 μmol m -2 s -1 light intensity. The control and treatment experiments comprised three replicates.

Combined treatment of embryos with SNP and PTIO
After pretreatment with SNP, the embryos were placed on filter paper dampened with 3 mL 300 μmol L -1 PTIO solution. Control embryos were placed similarly. Germination conditions were the same as in pretreatment with SNP. Control and treatments experiments comprised three replicates.

Combined treatment of embryos with SNP and ABA
After pretreatment with SNP, the embryos were placed on filter paper dampened with 3 mL 3.0 μmol L -1 ABA solution. Control embryos were similarly placed on filter paper wetted with 3 mL of different ABA concentrations. Germination conditions were the same as in pretreatment with SNP. Control and treatments experiments comprised three replicates.

Combined treatment of embryos with SNP and NBD
After pretreatment with SNP, the embryos were placed on filter paper dampened with 3 mL 1 mmol L -1 NBD solution or with 3 mL 20 mmol L -1 ethrel. Control embryos were similarly placed on the filter paper wetted with 3 mL NBD or with ethrel solution. Germination conditions were the same as in pretreatment with SNP. Control and treatments experiments comprised three replicates.

Observing and recording germination
Germination was evident when the two cotyledons were green and the hypocotyl and radicle were elongated. Statistics were recorded for the time of germination initiation for the different treatments, germination percentage each day during the experiment, and cumulative germination percentage for different treatments.

Endogenous hormone assay
Embryos and seedlings cultured for 1-8 days were collected as test materials (0.5 g for each treatment) with three replicates. ABA level was assayed according to Ali-Rachedi et al. (2004) and ethylene level according to Gniazdowska et al. (2007).

Data statistics and analysis
Excel 2003 software (Microsoft) was used for data processing, and SPSS software (v17.0, SPSS Inc.) to analyze level differences of ROS, MDA, GSH and antioxidase, embryo germination percentage, radicle length, and ethylene production by ANOVA and Duncan's multiple comparison and correlation analysis. Graphs were plotted using Sigmaplot software (v12.5, SYSTAT). Data concerning percentages were arcsine-transformed before analysis. All data represent the means of three replicates ± SD for each treatment. The effect of each influencing factor was evaluated by variance analysis, and a significance test performed at P = 0.05. Mean values were compared using the least significant difference test (Duncan's) at the 5% level of significance.

Germination of S. pohuashanensis embryos
Treatment with exogenous chemicals significantly affected dormancy, germination, and growth of the embryos (Figs. 1a-i, 2a-f). Both the SNP and ethrel treatments significantly promoted embryo germination (Fig. 2a, c). By day 8, embryo germination percentage in the SNP and ethrel treatments was ˃ 75% (40% higher than in the controls), while those in the PTIO, NBD, and ABA treatments were only 43%, 29%, and 22%, respectively (19%, 45% and 58% lower than in the controls, respectively) (Fig. 2a, c, e). Pretreatment with SNP before the PTIO, NBD, or ABA treatments weakened but did not completely alleviate their inhibitory effects on embryo germination. The germination percentage in the SNP pretreatment was increased but it was still significantly lower than in the controls (Fig. 2a, c, e).
SNP and ethrel also promoted radicle growth of newly germinated S. pohuashanensis seedlings. By day 8, average length of radicles in the SNP and ethrel treatments were 22% and 33%, respectively, greater than in the controls (Fig. 2b,  d). PTIO, NBD, and ABA inhibited radicle growth with the average radicle length decreased by 52%, 86%, and 84%, respectively, compared to the controls (Fig. 2b, d, f). SNP pretreatment before treatments with PTIO, NBD, or ABA relieved the inhibitory effect of PTIO treatment on radicle growth (Fig. 2b), but the effects of NBD and ABA were still significant (Fig. 2d, f).

Changes in endogenous hormones during germination of S. pohuashanensis
In SNP-treated and ethrel-treated embryos, the levels of endogenous ethylene increased gradually from day 1-8 (by day 8, 37% and 42% greater, respectively, than the controls, Fig. 3a, b. Ethylene level was inhibited by treatments with PTIO, NBD, and ABA ( Fig. 3a-c). Pretreatment with SNP before the PTIO treatment weakened its inhibitory effect but the results were not statistically significant (data not shown). Treatment with SNP or ethrel decreased endogenous ABA levels. Pretreatment with SNP (combined SNP and ABA) reduced endogenous ABA to 0.3 μg g -1 DW from day 5 onward. Treatment with NBD or the combination of SNP and NBD did not significantly affect endogenous ABA in embryos (0.81 μg g -1 DW).

Analysis of endogenous ROS accumulation in S. pohuashanensis embryos
In each treatment, H 2 O 2 level increased during germination. Compared with the controls, SNP-treated embryos had significantly higher H 2 O 2 levels and PTIO-treated embryos significantly lower (Fig. 4a). The levels of superoxide anions in  SNP-treated embryos were lower than in the controls, while they were higher in PTIO-treated embryos (Fig. 4b).
The ROS levels in various organs responded differently to the treatments. The H 2 O 2 level in radicles significantly increased in SNP-treated seedlings and significantly decreased in PTIO-treated seedlings (0.63 μg g -1 mL −1 ), while the superoxide levels decreased significantly to 7.33 μg g -1 mL −1 and 13.33 μg g -1 mL −1 , respectively (Table 1).
Malondialdehyde (MDA) levels decreased in SNP-treated embryos but increased significantly in ones treated with PTIO (Fig. 5); SNP pretreatment decreased the effect of the PTIO. This pattern of change was also observed in radicles and both upper and lower cotyledons ( Table 1).

Effect of NO on antioxidant enzyme activities in S. pohuashanensis embryos
The activities of superoxide dismutase (SOD) and catalase (CAT), antioxidant enzymes that scavenge ROS in embryos, were increased significantly in SNP-treated embryos but decreased in PTIO-treated ones. SNP pretreatment alleviated the inhibitory effects of PTIO on SOD and CAT activities (Fig. 6a, b). The effects of these treatments on SOD and CAT activities were also detected in radicles and upper and lower cotyledons, with trends that were consistent with those detected in whole embryos (Table 2).
Compared to the controls, SNP-treated and PTIO-treated seedlings showed increased GSH levels in upper cotyledons, and significantly decreased GSH in lower cotyledons and radicles (Table 2). GSH-PX activity in all seedling organs increased significantly after SNP treatment and decreased significantly after PTIO treatment (Table 2). Similarly, SNP pretreatment before PTIO treatment weakened its effect on GSH level in lower cotyledons and GHS-PX activity in upper and lower cotyledons (Table 2).
Means followed by different lowercase letters are significantly different according to Duncan's -test (P < 0.05) (mean ± SD; n = 3).

Roles of NO and H 2 O 2 in seed dormancy release and germination
Seed dormancy is an adaptive mechanism of plants to environmental change (Holdsworth et al. 2008;Su et al. 2016). The ability to germinate is regulated by combination of environmental and endogenous signals with both synergistic and antagonistic effects (Arc et al. 2013). Some previous studies have focused on the relationship between hormonal regulation and the breaking of seed dormancy   . 6 Effects of SNP and NO scavenger on SOD and CAT activities of Sorbus pohuashanensis embryos and seedlings; a SOD activity; b CAT activity; Bars indicate means ± SE (n = 3), different letters indicate significant differences at p = 0.05 (Yang et al. 2009;Deng et al. 2016;Ma et al. 2018). There is experimental evidence that nitric oxide can break dormancy (Bethke et al. 2007;Basson-Bard et al. 2008;Arc et al. 2013), and that this is related to changes in reactive oxygen species in the seeds (Gniazdowska et al. 2010;Arc et al. 2013;Yang et al. 2013Yang et al. , 2018. In this study, we found that NO and ROS are involved in embryo germination and seedling development, consistent with the proposed role of NO as an important inducer of seed dormancy release (Arc et al. 2013).
In the present study, after 8 days of SNP treatment, germination of embryos was > 90% (Fig. 2a), and seedlings also showed the fastest radicle growth (Fig. 2b). However, germination and radicle elongation were significantly reduced after treatment with the NO scavenger PTIO (Fig. 2a,  b). SNP treatment increased H 2 O 2 concentration in S. pohuashanensis embryos (Fig. 4a). Moreover, variations in H 2 O 2 concentration were related to variations in germination percentage (96%). (Fig. S2). These results demonstrate that NO and ROS are involved in the release of embryo dormancy and subsequent germination, and in seedling development of S. pohuashanensis. This is consistent with results reported for embryos of other species such as apple (Malus domestica; Gniazdowska et al. 2010) and empress tree (Paulownia tomentosa (Thunb.) Steud.; Giba et al. 1998).
Germination is a complex physiological and biochemical process mainly controlled by the abscisic acid concentration or by the ratio (or balance) of ABA to GA (Nambara and Marion-Poll 2003;Pérez-Flores et al. 2003;Finch-Savage and Leubner-Metzger 2006). There are two mechanisms by which NO breaks S. pohuashanensis embryo dormancy and promotes germination. First, as a signal molecule, nitric oxide can promote ABA catabolism (Matakiadis et al. 2009;Arc et al. 2013) to release dormancy. ABA can significantly inhibit germination of S. pohuashanensis embryos (Fig. 2e). In Arabidopsis, NO upregulates the expression of CYP707A2 and promotes ABA catabolism to reduce its concentration in seeds, or reduce the sensitivity of seeds to ABA (Liu et al. 2009a). Secondly, NO may interact with H 2 O 2 to promote ABA catabolism, thereby breaking seed dormancy and promoting germination. Several lines of evidence support this; first, it was observed that exogenous SNP results in   Fig. 7 Effects of SNP and NO scavenger on GSH level and GSH-PX activity of Sorbus pohuashanensis embryos and seedlings; a GSH level; b GSH-PX activity; Bars indicate means ± SE (n = 3); letters indicate significant differences at p = 0.05 an increase in H 2 O 2 in S. pohuashanensis embryos (Fig. 4a). Increased H 2 O 2 levels promote the synthesis of endogenous NO, thus accelerating ABA catabolism (Bethke et al. 2007;Liu et al. 2009bLiu et al. , 2010a; secondly, with the participation of NO, H 2 O 2 can upregulate genes encoding ABA 8′-hydroxylase (e.g., CYP707A2) (Liu et al. 2010a, b), leading to a significant decrease in ABA in embryos; thirdly, H 2 O 2 can upregulate GA biosynthesis genes (e.g., GA3ox, GAw20ox) to increase biosynthesis and regulate the ratio of GA and ABA, which is also conducive to dormancy breaking and germination (Liu et al. 2010a, b).

Roles of SNP, H 2 O 2 , and antioxidant enzymes in seed dormancy release and germination
Our results show that the breaking of dormancy in S. pohuashanensis embryos is closely related to increased activities of antioxidant enzymes. Embryos treated with SNP accumulated H 2 O 2 (Fig. 4a). This did not damage embryo cells (i.e., the MDA level was relatively low, Fig. 5) but promoted germination (Fig. S2). This may be explained by the regulation of ROS homeostasis by NO (Corpas et al. 2011;Ahmad et al. 2019;Fig. 4b). As demonstrated in our study, a low concentration of NO not only increases GSH level (Fig. 7a), but also increases SOD, CAT (Fig. 6a, b) and GSH-PX activities (Fig. 7b). With increased concentrations of H 2 O 2 and superoxide (Fig. S3), the activities of SOD and CAT increase significantly to reduce ROS damage (Hu et al. 2007;Arc et al. 2013;Ishibashi et al. 2017). Previous studies have demonstrated that NO can react with reduced GSH or thiol groups, leading to the reversible formation of S-nitrosothiols (e.g., GSNO, S-nitrosylatedproteins). GSNO is a storage and transport form for NO in seeds (Catusse et al. 2008). NO-based modifications of antioxidant enzymes play a key role in the regulation of oxidative stress and in ROS homeostasis (Yang et al. 2015). Although our results show that antioxidant enzymes are involved in RNS-dependent dormancy release, the specific details of the NO-regulated ROS homeostasis mechanism need to be further studied.

Roles of SNP and hormones (ethylene) in seed dormancy release and germination
We also studied the interactions (crosstalk) between hormonal signals and NO-regulated ROS accumulation in seed dormancy and germination. Interestingly, in this study, exogenous ethylene as well as SNP broke dormancy and promoted germination of S. pohuashanensis embryos (Fig. 2c). Germination percent and radicle elongation were decreased by the ethylene receptor inhibitor NBD (Fig. 2c). Furthermore, we found that the NO signal led to an increase in H 2 O 2 , which was related to an increase in ethylene biosynthesis and a decrease in ABA during breaking of embryo dormancy. Ethylene decreased in embryos treated with the NO scavenger PTIO (Fig. 3a) and in those treated with the ethylene receptor inhibitor NBD (Fig. 3b). The ethylene level was positively correlated significantly with levels of H 2 O 2 and superoxide, and the levels of all three were positively correlated significantly with percent germination of S. pohuashanensis embryos (see Figs. S2 and S4). These results demonstrate that NO participates in the crosstalk between ABA and ethylene through ROS signals to regulate dormancy and germination of S. pohuashanensis embryos.
Our results indicate that this NO-mediated release of dormancy depends on ethylene biosynthesis and signalling, which counteract the effect of ABA. This conclusion is consistent with findings for Arabidopsis seeds (Arc et al. 2013). Previous studies have demonstrated that the NO-induced ABA decrease is correlated with the regulation of CYP707A2 transcription and (+)-ABA 8′-hydroxylase (encoded by CYP707A2) protein expression (Liu et al. 2009a;Ma et al. 2018). A recent study showed that the ethylene receptors ETR1, EIN4, and ETR2 control ABA signalling during Arabidopsis seed germination (Bakshi et al. 2018). Yu et al. (2019) reported that ethylene and ABA antagonistically regulate ascorbic acid biosynthesis and ROS accumulation via ETHYLENE-INSENSITIVE3 (EIN3) and ABA INSENSITIVE4 (ABI4), key factors in the ethylene and ABA signalling pathways, respectively. However, it is still largely unknown whether or how ethylene affects GA biosynthesis and signalling during seed dormancy and germination.

Conclusions
This study focused on the roles and correlations among reactive nitrogen species (RNS), reactive oxygen species (ROS), and plant hormones in seed dormancy. The results show that nitric oxide breaks seed dormancy of S. pohuashanensis and is closely related to ethylene biosynthesis and abscisic acid catabolism. This signalling pathway is closely related to ROS accumulation and the antioxidant defence response. These results shed new light on the positive role of NO, ROS, and their interactions with plant hormones in germination of S. pohuashanensis.

Conflict of interest
The authors declare that they have no conflict of interest.
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