Glutathione in reduced form GSH the

Glutathione in reduced form (GSH, the tripeptide, γ-glutamyl cysteinyl glycine) is a non-enzymatic low-molecular-weight antioxidant necessary for the maintenance of a cellular redox state (Noctor et al., 2012). At the stage of the transition from germination to seedling growth the process of cell division depends on increasing GSH content (Diaz-Vivancos et al., 2010). In orthodox seeds at the phase of early germination, this tripeptide is the most important antioxidant (Tommasi et al., 2001). Contrary, overaccumulation of GSSG is toxic and results in loss of viability. Glutathione half-cell reduction potential (EGSSG/2GSH) correlates with cell viability and can be used as a valuable marker to predict seed germination potential (Kranner et al., 2006). Glutathione reductase (GR, EC 1.6.4.2) is NADPH dependent enzyme responsible for reduction of GSSG. Using transgenic Arabidopsis lines overexpressing GSNOR or with antisense GSNOR it was confirmed that redox homeostasis is also under GSNOR regulation. Roots of plants of tested lines were characterised by a lower intracellular GSH level than observed in wild type (Espunya et al., 2006). Our previous results demonstrated a pivotal role of RNS in release of apple AZ 628 dormancy (Bogatek and Gniazdowska, 2006; Gniazdowska et al., 2007, 2010a, 2010b). Nevertheless, the main intracellular source and regulation of availability of these molecules at the beginning of germination sensu stricto remain unclear and need further investigation. The aim of this work was to demonstrate experimental evidence for GSNO occurrence and variation in its level depending on dormancy status of the embryos. Our study was focused on determination of GSNOR protein level, catalytic activity and gene transcription in axes of dormant and non-dormant embryos after initial 24 h of water uptake (imbibition). We linked GSNO level and GSNOR activity to the total glutathione pool as well as GSH/GSSG ratio. Previous data indicated that apple embryos dormancy release stimulated by NO fumigation was accompanied by increased GR activity, preventing GSSG overaccumulation (Krasuska and Gniazdowska, 2012). Therefore, in the current research we studied GR activity and transcription in axes of non-dormant embryos isolated from stratified seeds.
Material and methods
Results
Discussion GSNOR activity is strictly linked to GSNO concentration. Data concerning the presence and concentration of GSNO in seeds during dormancy release and germination are unique. We have shown variations of GSNO level in axes of dormant and non-dormant apple embryos (Fig. 3). Higher level of GSNO in axes of non-dormant embryos correlates well with lower GSNOR activity (Fig. 2C). Our results are in agreement with those obtained for unstressed pepper (Capsicum annuum L.) plants. The lowest GSNOR activity in roots was linked to higher GSNO content (Airaki et al., 2011). Barroso et al. (2006) proposed that GSNO may induce GSNOR activity. However, our data indicate that during germination probably occurs other regulation of GSNOR activity since in axes of apple embryos a higher GSNO level is accompanied by a lower enzyme catalytic action. GSH an abundant and stable metabolite plays a pivotal function not only as an antioxidant but participates in posttranslational modifications of proteins such as glutathionylation (Noctor et al., 2012). Increased GSSG level leads to decrease of GSH:GSSG ratio, resulting in more oxidised cellular redox state. Total glutathione pool and GSH content was higher in axes of non-dormant apple embryos (Table 1). Enhanced expression of genes coding gamma-glutamylcysteine synthetase and glutathione synthetase was observed after application of NO donors (Innocenti et al., 2007), suggesting impact of high NO level on regulation of glutathione synthesis. Moreover, in axes of dormant embryos around 12% of the total glutathione content was calculated for GSSG comparing to only 6% of GSSG from non-dormant plant material. High GSH:GSSG ratio in non-dormant tissue and lack of differences in GSSG concentration in axes of dormant and non-dormant embryos may be due to high GR activity in axes of stratified seeds (Table 2). The increase of total glutathione pool was observed in lupine (Lupinus luteus L.) embryonic axes before the onset of germination while afterwards a decrease of the glutathione content was noticed (Garnczarska and Wojtyla, 2008). Decline of the total glutathione was characteristic for barley embryos during first hours of post imbibition, which most probably was linked to intensive S-nitrosation of GSH. Nevertheless, the authors observed an increase of glutathione reduction after more than 24 h of post imbibition (Ma et al., 2016). Concentration of total glutathione and particularly GSH shows a close correlation with seed germination ability (Fontaine et al., 1995; Nagel et al., 2014). Nagel et al. (2014) demonstrated that artificial ageing and long-term seed storage leading to decline of viability and low germination rate, resulted in declining levels of total glutathione, GSH and increasing EGSSG/2GSH value. In axes of both dormant and non-dormant embryos EGSSG/2GSH was below −250 mV (Table 1), typical for seeds of high viability and high germination rate (Kranner et al., 2006; Nagel et al., 2014). Our results are in agreement with the idea of Kranner et al. (2006) suggesting that changes of GSH or % of GSSG and particularly EGSSG/2GSH are universal markers for seed viability and germination.