Dormancy is a physiological state when viable seeds do

Dormancy is a physiological state when viable seeds do not germinate. Seed stratification, a commonly used technique, is used for dormancy removal and can be performed in moisture for an experimentally revealed time at warm or cold temperatures. All conditions depends on plant species (Dębska et al., 2013; Lewak, 2011). Cold stratification of apple seeds (90 days at 5 °C) remarkably enhances germination rate of isolated embryos (Dębska et al., 2013; Lewak, 2011). These embryos imbibed for 24 h in distilled water are characterised by longer embryonic axes comparing to the control (dormant apple embryos isolated from seeds imbibed for 24 h) (Fig. 1). Based on previously obtained data, dormancy release during cold stratification is accompanied by endogenous increase of NO and ROS (mainly H2O2) level (Dębska et al., 2013; Lewak, 2011). Regulation of seed germination by RNS includes a cross-talk with polyamines (Krasuska et al., 2013, 2017b) and depends on protein modification e.g. nitration, carbonylation and/or S-nitrosation (Krasuska et al., 2014, 2016; Sen, 2010). In contrast, the maintenance of seed dormancy can be achieved by application of NO scavengers (Gniazdowska et al., 2007). Naturally occurring NO scavengers are class 1 plant hemoglobin (Igamberdiev et al., 2006) and GSNOR as both lower free “NO pool” (Ma et al., 2016). Thus we assumed that activity of GSNOR regulates the strength of apple embryos dormancy. We indicated that GSNOR protein level was similar in axes of dormant and non-dormant apple embryos (Fig. 2A), though its activity was twice lower in non-dormant embryos comparing to the control (Fig. 2C). Lowering activity of this enzymatic NO scavenger goes together with transient increase of RNS level which occurs during AH 6809 dormancy release (Dębska et al., 2013; Gniazdowska et al., 2010c; Krasuska et al., 2013). Opposite, during first hours post imbibition of non-dormant barley (Hordeum vulgare L.) embryos besides high NO level, stimulation of GSNOR activity was noticed (Ma et al., 2016). Thus it is the most probably that GSNOR activity depends on plant species, developmental stage and physiological conditions. Ma et al. (2016) demonstrated relatively constant expression of GSNOR and they assumed that alteration in enzyme activity could be attributed to the posttranscriptional regulation. In embryonic axes of non-dormant (stratified) apple seeds we observed higher GSNOR transcript level (Fig. 2B). Taking together our results concerning GSNOR protein and transcript level as well as enzyme activity we think that besides posttranscriptional there is also posttranslational regulation. As demonstrated by Xu et al. (2013) GSNOR from Arabidopsis has most of the ex-zinc cysteines inaccessible to solvent but three of these amino acids that are positionally conserved are solvent accessible. These residues could serve as sites of posttranslational regulation by glutathionylation or S-nitrosation (Xu et al., 2013). Increased NO level in apple embryos during dormancy release possibly could be engaged in GSNOR activity regulation (a negative feedback loop) including S-nitrosation. Contrary to the nitration, this modification is reversed, thus if necessary the activation of GSNOR might occur. Just recently other possibility of GSNOR activity regulation was described as induction of selective autophagy by S-nitrosation at Cys-10 in Arabidopsis during hypoxia (Zhan et al., 2018). The authors demonstrated that GSNOR1 S-nitrosation initiated the protein conformational modification exposing its AUTOPHAGY-RELATED8 (ATG8)-interacting motif (AIM) to autophagy machinery. Kovacs et al. (2016) proposed other, ROS-dependent regulation of GSNOR activity. They demonstrated the inhibition of GSNOR enzymatic activity as a result of in vitro treatment with H2O2 and in vivo paraquat application. The authors suggested that this regulation is related to oxidative modifications of Zn2+-coordinating Cys-47 and Cys-177. Initiation of germination of apple embryos is also linked to increased ROS generation leading to temporal oxidative stress (Krasuska and Gniazdowska, 2012). Thus, we believe that ROS transient increase may acts as regulator of NO metabolism via e.g. modulation of GSNOR activity.